Protein Separations’ Handbook Collection Antibody Purification
The Recombinant Protein Handbook
GST Gene Fusion System
Handbook
Protein Amplification and Simple Purification
Handbook
Protein Purification
Ion Exchange Chromatography
Affinity Chromatography
Principles and Methods
Principles and Methods
Hydrophobic Interaction Chromatography
PRINCIPLES AND METHODS
Handbook
18-1037-46
18-1142-75
18-1157-58
18-1132-29
18-1114-21
18-1022-29
Edition AB
Edition AB
Edition AA
Edition AB
Edition AA
Edition AC
Antibody Purification
The Recombinant Protein Handbook
GST Gene Fusion System
Protein Purification
Ion Exchange Chromatography
Reversed Phase Chromatography
Microcarrier cell culture
Percoll
Ficoll-Paque PLUS
Principles and Methods
Principles and Methods
principles & methods
Methodology and Applications
For in vitro isolation of lymphocytes
18-1134-16 Edition AB
Edition AI
Gel Filtration
Reversed Phase Chromatography
Updated July 2002 – Keep updated with new updates of the included handbooks.
18-1140-62 Edition AA
18-1152-69 18-1115-69
Edition AB
Edition AC
Microcarrier Cell Culture
Percoll
Edition AB
Affinity Chromatography
Gel Filtration
18-1022-18
18-1020-90
Ficoll-Paque PLUS
Hydrophobic Interaction Chromatography
Choose a handbook by clicking on it
Hydrophobic Interaction Chromatography
PRINCIPLES
AND
METHODS
Back to Collection
18-1020-90 Edition AB 1
to order: Asia Pacific Tel: +852 2811 8693 Fax: +852 2811 5251 Australasia Tel: +61 2 9894 5152 Fax: +61 2 9899 7511 Austria Tel: 01 576 0616 20 Fax: 01 576 0616 2 7 Belgium Tel: 0800 73 888 Fax: 03 272 1637 Canada Tel: 1 800 463 5800 Fax: 1 800 567 1008 Central, East, South East Europe Tel: +43 1 982 3826 Fax: +43 1 985 8327 Denmark Tel: 45 16 2400 Fax: 45 16 2424 Finland Tel: 09 512 3940 Fax: 09 512 1710 France Tel: 0169 35 67 00 Fax: 0169 41 9677 Germany Tel: 0761 4903 401 Fax: 0761 4903 405 Italy Tel: 02 27322 1 Fax: 02 27302 212 Japan Tel: 81 3 5331 9336 Fax: 81 3 5331 9370 Latin America Tel: +55 11 3667 5700 Fax: +55 11 3667 87 99 Middle East and Africa Tel: +30 (1) 96 00 687 Fax: +30 (1) 96 00 693 Netherlands Tel: 0165 580 410 Fax: 0165 580 401 Norway Tel: 2318 5800 Fax: 2318 6800 Portugal Tel: 01 417 7035 Fax: 01 417 3184 Russian Federation Tel: +7 (095) 232 0250,956 1137 Fax: +7 (095) 230 6377 South East Asia Tel: 60 3 724 2080 Fax: 60 3 724 2090 Spain Tel: 93 594 49 50 Fax: 93 594 49 55 Sweden Tel: 018 612 19 00 Fax: 018 612 19 10 Switzerland Tel: 01 802 81 50 Fax: 01 802 81 5 1 UK Tel: 0800 616 928 Fax: 0800 616 927 USA Tel: +1 800 526 3593 Fax: +1 877 295 8102
Sephadex, Sepharose, Superose, BioProcess, FPLC, SMART, Mono Q, SOURCE, HiTrap, HiLoad, BPG, Superloop, Superdex, Ultroscan, PhastSystem, and RESOURCE are trademarks of Amersham Pharmacia Biotech Limited or its subsidiaries. Amersham is a trademark of Nycomed Amersham plc. Pharmacia and Drop Design are trademarks of Pharmacia & Upjohn. Amersham Pharmacia Biotech AB Björkgatan 30, SE-751 84 Uppsala, Sweden. Amersham Pharmacia Biotech UK Limited Amersham Place, Little Chalfont, Buckinghamshire HP7 9NA, England. Amersham Pharmacia Biotech Inc 800 Centennial Avenue, PO Box 1327, Piscataway, NJ 08855 USA. Amersham Pharmacia Biotech Europe GmbH Munzinger Strasse 9, D-79111 Freiburg, Germany. Amersham Pharmacia Biotech K.K. Sanken Building, 3-25-1, Hyakunincho, Shinjuku-ku, Tokyo 169-0073, Japan. All goods and services are sold subject to the terms and conditions of sale of the company within the Amersham Pharmacia Biotech group that supplies them. A copy of these terms and conditions is available on request. © Amersham Pharmacia Biotech AB 2000 – All rights reserved.
Hydrophobic Interaction Chromatography PRINCIPLES AND METHODS
ISBN 91-970490-4-2
Foreword Many biotechnologists began their careers in chromatography reading Gel Filtration: Theory and Practice. First published in 1966, this monograph has had over 250,000 copies printed in five languages. It was soon followed by another helpful monograph from Amersham Pharmacia Biotech on ion exchange. About 15 years ago, Affinity Chromatography: Principles and Methods was published describing the emergence of this powerful separating method for macromolecules. In some ways this monograph series has defined the critical methods in the field at the time of publication and has been both good business and a public service for over 25 years. With the rise of the modern biotechnology industry and its requirement for highly purified pharmaceutical proteins, a further emphasis has been placed on entire processes with respect to their economy, capacity and resultant product quality. Often the extent of separation power required is defined by the need to resolve the product not only from the background impurities derived from the fermentation but also from degradation products and analogues of the drug itself. For many cases, hydrophobic interaction chromatography (HIC) is an ideal separation method. In my experience, HIC is finding dramatically increased use both in laboratory and production processes. Since the molecular mechanism of HIC relies on unique structural features, it serves as an orthogonal method to ion exchange, gel filtration and affinity chromatography. It is very generic, yet capable of powerful resolution. Usually media have high capacity and are economical and stable. Adsorption takes place in high salt and desorption in low salt concentrations. These special properties make HIC very useful in whole processes for bridging or transitioning between other steps in addition to the separation which is effected. This book can serve as an excellent introduction to the subject of HIC for those new to this method of separation. More experienced chromatographers can also benefit from the useful review. Topics include the molecular mechanism of separation by HIC in contrast to reversed phase chromatography, a helpful section on strategies for rapid method development, as well as a wide selection of examples. Practical aspects such as packing, use and sanitization of columns are discussed. There are many tricks, techniques and insights to be gained in a complete reading. I recommend it be read and kept handy on your personal book shelf and I predict that you will find HIC a surprisingly helpful technique both alone and especially in combination with other modes of separation.
Stuart E. Builder So. San Francisco January 15, 1993
Contents 1. Introduction to HIC................................................... 9 2. Principles of HIC.................................................... 11 Theory .............................................................................11 HIC vs RPC .....................................................................12 Factors affecting HIC .......................................................13 Type of ligand .......................................................13 Degree of substitution ..........................................14 Type of base matrix ..............................................14 Type and concentration of salt .............................15 Effect of pH ...........................................................16 Effect of temperature ............................................17 Additives ...............................................................18
3. Product Guide ........................................................ 19 BioProcess Media ...........................................................20 Base matrices .......................................................20 Coupling ...............................................................21 Chemical stability .................................................21 Physical stability ...................................................22 Binding capacity ...................................................22 Phenyl Sepharose 6 Fast Flow (low sub) and Phenyl Sepharose 6 Fast Flow (high sub) .......................23 Butyl Sepharose 4 Fast Flow ...........................................24 Phenyl Sepharose High Performance .............................25 Custom Designed HIC Media ..........................................26 HIC Media Test Kit ..........................................................26
Contents Phenyl Sepharose CL-4B and Octyl Sepharose CL-4B ...................................................27 Phenyl Superose and Alkyl Superose ..............................27
4. Experimental Design ............................................. 29 Hydrophobicity of proteins .............................................29 Multivariate mapping ......................................................29 Strategic considerations .................................................30 Choice of HIC media .......................................................31 General considerations .........................................31 Screening experiments .........................................32 Optimizing a HIC step .....................................................39 The solute .............................................................39 The solvent ...........................................................41 Elution ..................................................................42 Sample load and flow rate ....................................45 Regeneration ........................................................45 Process considerations ...................................................46 Method optimization in process ............................... chromatography ...................................................46 Scaleability ...........................................................49 Regulatory considerations ....................................50
5. Experimental Technique ........................................ 53 Choice of column ............................................................53 Column dimensions..............................................53 Packing the column ........................................................53 Packing Sepharose Fast Flow based HIC gels ......54 Packing Phenyl Sepharose High Performance .....55 Packing Sepharose CL-4B based HIC gels ...........55
Contents Use of an adaptor .................................................55 Checking the packed bed ......................................56 Prepacked HIC media ...........................................58 Sample preparation .........................................................59 Sample composition .............................................59 Sample volume .....................................................59 Sample viscosity ...................................................60 Particle content .....................................................60 Sample application ..........................................................61 Sample reservoir ..................................................61 Sample applicators ...............................................61 Sample loops with valves LV-4 or SRV-4 .............62 Sample loops or Superloop with valves V-7 or MV-7 ...............................................62 Batch separation .............................................................63 Cleaning, sanitization and sterilization procedures .........63 Storage of gels and columns ..........................................65 Prevention of microbial growth ............................65 Antimicrobial agents .............................................65 Storage of unused media ......................................67 Storage of used media ..........................................67 Storage of packed columns ..................................67 Process considerations ...................................................68 Selecting a column ...............................................68 Aspects of column design ....................................69 Packing large scale columns ................................71 Scale-up ...............................................................74
Contents 6. Applications ........................................................... 77 Preparative and analytical HIC applications in the research laboratory ...........................77 HIC in combination with ammonium sulphate precipitation ..........................................................77 HIC in combination with ion exchange chromatography ...................................................78 HIC in combination with gel filtration ................... 80 HIC as a ”single step” purification technique ........81 Analysis of conformational changes with HIC....... 84 Other HIC application areas in the research laboratory .............................................................84 Preparative, large scale applications ............................... 85 Purification of a monoclonal antibody for clinical studies of passive immunotherapy of HIV-1 ...................................................85 Purification of recombinant human Epidermal Growth Factor (h-EGF) from yeast ....... 87 Purification of a monoclonal antibody for in vitro diagnostic use .......................................... 90 Purification of a recombinant Pseudomonas aeruginosa exotoxin, produced in E. Coli ..............92
7. References ............................................................ 97 Order from................................................................ 102
10
1 Introduction to HIC In a classical paper published in 1948 and entitled: ‘‘Adsorption Separation by Salting Out’’, Tiselius [1] laid down the foundation for a separation method which is now popularly known as hydrophobic interaction chromatography (HIC). He noted that, ‘‘...proteins and other substances which are precipitated at high concentrations of neutral salts (salting out), often are adsorbed quite strongly already in salt solutions of lower concentration than is required for their precipitation, and that some adsorbents which in salt-free solutions show no or only slight affinity for proteins, at moderately high salt concentrations become excellent adsorbents”. Since then, great strides have been made in developing almost ideal stationary phases for chromatography (such as cellulose, cross-linked dextran (Sephadex™), cross-linked agarose (Sepharose™ CL, Sepharose High Performance and Sepharose Fast Flow), and in developing coupling methods for immobilizing ligands of choice [2,3] to such matrices. It was a combination of these two events which, in the beginning of 1970's, led to the synthesis of a variety of hydrophobic adsorbents for biopolymer separations based on this previously rarely exploited principle. The first attempt at synthesizing such adsorbents was made by Yon [4] followed by Er-el et al. [5], Hofstee [6] and Shaltiel & Er-el [7]. Characteristically, these early adsorbents showed a mixed ionic-hydrophobic character [8]. Despite this, Halperin et al. [9] claimed that protein binding to such adsorbents was predominantly of a hydrophobic character. Porath et al. [10] and Hjertén et al. [11] later synthesized charge-free hydrophobic adsorbents and demonstrated that the binding of proteins was enhanced by high concentrations of neutral salts, as previously observed by Tiselius [1], and that elution of the bound proteins was achieved simply by washing the column with salt-free buffer or by decreasing the polarity of the eluent [6, 10, 11]. Amersham Pharmacia Biotech was first in producing commercial HIC adsorbents (Phenyl and Octyl Sepharose CL-4B [12]) of the charge-free type and has continuously followed this up with new developments in agarose matrix design by introducing new stable HIC media based on Superose™, Sepharose Fast Flow and Sepharose High Performance, meeting various demands on chromatographic productivity, selectivity and efficiency.
11
The commercial availability of well-characterized HIC adsorbents opened new possibilities for purifying a variety of biomolecules such as serum proteins [12, 13], membrane-bound proteins [14], nuclear proteins [15], receptors [16], cells [17], and recombinant proteins [18, 19] in research and industrial laboratories. These adsorbents were also used for the reversible immobilization of enzymes [20] and liposomes [21]. The principle for protein adsorption to HIC media is complementary to ion exchange chromatography and gel filtration. HIC is even sensitive enough to be influenced by non-polar groups normally buried within the tertiary structure of proteins but exposed if the polypeptide chain is incorrectly folded or damaged (e.g. by proteases). This sensitivity can be useful for separating the pure native protein from other forms. Altogether this makes HIC a versatile liquid chromatography technique, being a logical part of any rational purification strategy, often in combination with ion exchange chromatography and gel filtration. HIC has also found use as an analytical tool to detect protein conformational changes. HIC requires a minimum of sample pre-treatment and can thus be used effectively in combination with traditional protein precipitation techniques. Protein binding to HIC adsorbents is promoted by moderately high concentrations of anti-chaotropic salts, which also have a stabilizing influence on protein structure. Elution is achieved by a linear or stepwise decrease in the concentration of salt in the adsorption buffer. Recoveries are often very satisfactory. A number of mechanisms have been proposed for HIC over the years and factors that affect the binding of proteins to such adsorbents have been investigated. These aspects will be briefly outlined in this handbook. Greater emphasis has been given to practical considerations on how to make optimal use of Amersham Pharmacia Biotech range of HIC products.
12
2 Principles of HIC Theory The discussions that follow in this chapter will be limited to the non-charged type of HIC adsorbents. The many theories that have been proposed for HIC are essentially based upon those derived for interactions between hydrophobic solutes and water (22,23), but none of them has enjoyed universal acceptance. What is common to all is the central role played by the structure-forming salts and the effects they exert on the individual components (i.e., solute, solvent and adsorbent) of the chromatographic system to bring about the binding of solute to adsorbent. In view of this, Porath (24) proposed ‘‘salt-promoted adsorption’’ as a general concept for HIC and other types of solute-adsorbent interactions occuring in the presence of moderately high concentrations of neutral salts. Hofstee (6) and later Shaltiel (7) proposed ‘‘hydrophobic chromatography’’ with the implicit assumption that the mode of interaction between proteins and the immobilized hydrophobic ligands is similar to the self association of small aliphatic organic molecules in water. Porath et al. (10) suggested a salting-out effect in hydrophobic adsorption, thus extending the earlier observations of Tiselius (1). They also suggested that ‘‘. . .the driving force is the entropy gain arising from structure changes in the water surrounding the interacting hydrophobic groups’’. This concept was later extended and formalized by Hjertén (25) who based his theory on the well known thermodynamic relationship: DG = DH - TDS. He proposed that the displacement of the ordered water molecules surrounding the hydrophobic ligands and the proteins leads to an increase in entropy (DS) resulting in a negative value for the change in free energy (DG) of the system. This implies that the hydrophobic ligand-protein interaction is thermodynamically favourable, as is illustrated in Fig. 1. An alternative theory is based on the parallelism between the effect of neutral salts in salting out (precipitation) and HIC (26,27). According to Melander and Horvath (27), hydrophobic interaction is accounted for by increase in the surface tension of water arising from the structure – forming salts dissolved in it. In fact, a combination of these two mechanisms seems to be an obvious extension and has been exploited long
13
L
+
H
S
L H
S +
P P=Polymer matrix S=Solute molecule L=Ligand attached to polymer matrix H=Hydrophobic patch on surface of solute molecule W=Water molecules in the bulk solution
W
Fig. 1. Close to the surface of the hydrophobic ligand and solute (L and H), the water molecules are more highly ordered than in the bulk water and appear to ‘‘shield off’’ the hydrophobic ligand and solute molecules. Added salt interacts strongly with the water molecules leaving less water available for the ‘‘shielding off’’ effect, which is the driving force for L and H to interact with each other.
before HIC adsorbents were synthesized (28). Finally, Srinivasan and Ruckenstein (29) have proposed that HIC is due to van der Waals attraction forces between proteins and immobilized ligands. The basis for this theory is that the van der Waals attraction forces between protein and ligand increase as the ordered structure of water increases in the presence of salting out salts.
HIC vs RPC In theory, HIC and reverse-phase chromatography (RPC) are closely related LC techniques. Both are based upon interactions between solvent-accessible non-polar groups (hydrophobic patches) on the surface of biomolecules and the hydrophobic ligands (alkyl or aryl groups) covalently attached to the gel matrix. In practice, however, they are different. Adsorbents for RPC are more highly substituted with hydrophobic ligands than HIC adsorbents. The degree of substitution of HIC adsorbents is usually in the range of 10–50 mmoles/ ml gel of C2–C8 alkyl or simple aryl ligands, compared with several hundred mmoles/ml gel of C4–C18 alkyl ligands usually used for RPC adsorbents. Consequently, protein binding to RPC adsorbents is usually very strong, which requires the use of non-polar solvents for their elution. RPC has found extensive applications in analytical and preparative separations of mainly peptides and low molecular weight proteins that are stable in aqueous-organic solvents. In summary, HIC is an alternative way of exploiting the hydrophobic properties of proteins, working in a more polar and less denaturing environment. Compared with RPC, the polarity of the complete system of HIC is increased by decreased ligand density on the stationary phase and by adding salt to the mobile phase.
14
Factors affecting HIC The main parameters to consider when selecting HIC media and optimizing separation processes on HIC media are: • Ligand type and degree of substitution • Type of base matrix • Type and concentration of salt • pH • Temperature • Additives
Type of ligand
Fig. 2. The effect of alkyl chain length and degree of substitution on binding capacity in HIC. In Fig. 2A it is assumed that the degree of substitution is the same for each alkyl chain length shown.
Binding capacity (mg protein/ml gel)
The type of immobilized ligand (alkyl or aryl) determines primarily the protein adsorption selectivity of the HIC adsorbent (6,7,30). In general, straight chain alkyl (hydrocarbon) ligands show ‘‘pure’’ hydrophobic character while aryl ligands show a mixed mode behaviour where both aromatic and hydrophobic interactions are possible (30). It is also established that, at a constant degree of substitution, the protein binding capacities of HIC adsorbents increase with increased alkyl chain length (Fig. 2A) (30,31). The charged type HIC adsorbents (6,7) show an additional mode of interaction, which will not be discussed here. The choice between alkyl or aryl ligands is empirical and must be established by screening experiments for each individual separation problem.
B
A
C4
C6
C8
n-Alkyl chain length
10
20
30
Degree of substitution (µmol ligand/ml gel)
15
HIC media shown in Fig. 3 are all based on the glycidyl ether coupling procedure, which produces gels that are charge free and that should thus only have hydrophobic interactions with proteins. The phenyl group shown in Fig. 3-C also has a potential for þ-þ interactions. The glycidyl-ether coupling technique will introduce a short spacer but the effect of this will be very limited since the short hydrophobic chain is ‘‘neutralized’’ with the hydrophilic OH-group.
–
OH
A
–O–CH2–CH–CH2–O–(CH2)3–CH3 Butyl Sepharose 4 Fast Flow
Fig. 3. Different hydrophobic ligands coupled to cross-linked agarose matrices.
–
OH B
–O–CH2–CH–CH2–O–(CH2)7–CH3 Octyl Sepharose CL-4B
–
OH C
–O–CH2–CH–CH2–O– Phenyl Superose Phenyl Sepharose High Performance Phenyl Sepharose CL-4B Phenyl Sepharose 6 Fast Flow (low sub) Phenyl Sepharose 6 Fast Flow (high sub)
–
OH D
–O–CH2–CH–CH2–O–CH2–C(CH3)3 Alkyl Superose
Degree of substitution The protein binding capacities of HIC adsorbents increase with increased degree of substitution of immobilized ligand. At a sufficiently high degree of ligand substitution, the apparent binding capacity of the adsorbent remains constant (plateau is reached) but the strength of the interaction increases (31–33, 35) (Fig. 2B). Solutes bound under such circumstances are difficult to elute due to multi-point attachment (34).
Type of base matrix It is important not to overlook the contribution of the base matrix. The two most widely used types of support are strongly hydrophilic carbohydrates, e.g. cross-linked agarose, or synthetic copolymer materials. The selectivity of a copolymer support will not be exactly the same as for an agarose based support substituted with the same type of ligand. To achieve the same type of results on an agarose-based matrix as on a copolymer support, it may be necessary to modify adsorption and elution conditions.
16
Type and concentration of salt The addition of various structure-forming (‘‘salting out’’) salts to the equilibration buffer and sample solution promotes ligand-protein interactions in HIC (10, 12, 36, 65, 66). As the concentration of such salts is increased, the amount of proteins bound also increases almost linearly up to a specific salt concentration and continues to increase in an exponential manner at still higher concentrations.
Fig. 4. Protein binding capacity on Phenyl Sepharose High Performance as a function of salt concentration in the column equilibration buffer (Work from Amersham Pharmacia Biotech, Uppsala, Sweden).
Protein capacity mg/ml packed bed
This latter phenomenon is demonstrated in Fig. 4 where total binding capacity of Phenyl Sepharose High Performance for a-chymotrypsinogen and RNAse was examined at gradually increasing salt concentrations.
80
α-chymotrypsinogen RNA se
60
40
20
1
2
3
4
Initial salt concentration M (NH4)2 SO4
In this experiment, the column was first equilibrated with buffer containing varying concentrations of salt as indicated in the Figure. The sample was dissolved in buffer including this initial salt concentration prior to application to the column. However, in those experiments where the protein begins to precipitate at high salt concentration (1.3 M and 2.3 M ammonium sulphate for a-chymotrypsinogen and RNAse respectively) the sample was dissolved at a slightly lower salt concentration. The samples were loaded on the column until breakthrough could be observed at the column outlet. Then start buffer with initial salt concentration was run through the column until UV-absorption in the eluent returned to the baseline. Finally, the bound proteins were eluted with a decreasing salt gradient. A significant increase in adsorption capacity can be seen when the salt concentration is increased above the precipitation point.
17
This phenomenon is probably due to the precipitation of proteins on the column. It has a concomitant negative effect on the selectivity of the HIC adsorbent.
t
The effects of salts in HIC can be accounted for by reference to the Hofmeister series for the precipitation of proteins or for their positive influence in increasing the molal surface tension of water (for extensive review, see refs. 27,29). These effects are summarized in Tables 1 and 2.
Increasing precipitation (‘‘salting -out’’) effect
Anions: PO43–, SO42–, CH3 • COO–, Cl–, Br–, NO3–, CLO4–, I–, SCN– Cations: NH4+, Rb+, K+, Na+, Cs+, Li+, Mg2+, Ca2+, Ba2+
Na2SO4>K2SO4>(NH4)2SO4>Na2HPO4>NaCl>LiCl. . . >KSCN
t
Increasing chaotropic (‘‘salting-in’’) effect
Table 1. The Hofmeister series on the effect of some anions and cations in precipitating proteins.
Table 2. Relative effects of some salts on the molal surface tension of water.
In both instances, sodium, potassium or ammonium sulphates produce relatively higher ‘‘salting-out’’ (precipitation) or molal surface tension increment effects. It is also these salts that effectively promote ligand-protein interactions in HIC. Most of the bound proteins are effectively desorbed by simply washing the HIC adsorbent with water or dilute buffer solutions at near neutral pH.
Effect of pH The effect of pH in HIC is also not straightforward. In general, an increase in pH weakens hydrophobic interactions (10,41), probably as a result of increased titration of charged groups, thereby leading to an increase in the hydrophilicity of the proteins. On the other hand, a decrease in pH results in an apparent increase in hydrophobic interactions. Thus, proteins which do not bind to a HIC adsorbent at neutral pH bind at acidic pH (9). Hjertén et al. (42) found that the retention of proteins changed more drastically at pH values above 8.5 and/or below 5 than in the range pH 5–8.5 (Fig 5). These findings suggest that pH is an important separation parameter in the optimization of hydrophobic interaction chromatography and it is advisable to check the applicability of these observations to the particular separation problem at hand.
18
Fig. 5. The pH dependence of the interaction between proteins and an octyl agarose gel expressed as Ve/VT (Ve is the elution volume of the different proteins and VT is the elution volume of a non-retarded solute). Elution was by a negative linear gradient of salt. The model proteins used were STI=soy trypsin inhibitor, A=human serum albumin, L=lysozyme, T=transferrin, E=enolase, O=ovalbumin, R=ribonuclease, ETI=egg trypsin inhibitor and C=cytochrome c. (Reproduced with permission, from ref. 42).
Effect of temperature Based on theories developed for the interaction of hydrophobic solutes in water (22,37), Hjertén (38) proposed that the binding of proteins to HIC adsorbents is entropy driven [ ÐG = (ÐH-TÐS) ~ -TÐS], which implies that the interaction increases with an increase in temperature. Experimental evidence to this effect has been presented by Hjertén (25) and Jennissen (34). It is interesting to note that the van der Waals attraction forces, which operate in hydrophobic interactions (29), also increase with increase in temperature (39). However, an opposite effect was reported by Visser & Strating (40) indicating that the role of temperature in HIC is of a complex nature. This apparent discrepancy is probably due to the differential effects exerted by temperature on the conformational state of different proteins and their solubilities in aqueous solutions. In practical terms, one should thus be aware that a downstream purification process developed at room temperature might not be reproduced in the cold room, or vice versa. 19
Additives Low concentrations of water-miscible alcohols, detergents and aqueous solutions of chaotropic (‘‘salting-in’’) salts result in a weakening of the protein-ligand interactions in HIC leading to the desorption of the bound solutes. The non-polar parts of alcohols and detergents compete effectively with the bound proteins for the adsorption sites on the HIC media resulting in the displacement of the latter. Chaotropic salts affect the ordered structure of water and/or that of the bound proteins. Both types of additives also decrease the surface tension of water (see Table 3) thus weakening the hydrophobic interactions to give a subsequent dissociation of the ligand-solute complex. Although additives can be used in the elution buffer to affect selectivity during desorption, there is a risk that proteins could be denatured or inactivated by exposure to high concentrations of such chemicals. However, additives can be very effective in cleaning up HIC columns that have strongly hydrophobic proteins bound to the gel medium.
Solvent Water Ethylene glycol Dimethyl Sulphoxide Dimethyl Formamide n-propanol
20
Viscosity (centipoise) 0.89 16.90 1.96 0.796 2.00
Dielectric constant 78.3 40.7 46.7 36.71 20.33
Surface tension (dynes/cm) 72.00 46.70 43.54 36.76 23.71
Table 3. Physical properties of some solvents used in HIC (data at 25 oC).
3 Product Guide Amersham Pharmacia Biotech manufactures a wide range of HIC media suitable for analytical, small scale preparative and process scale applications. The HIC product range is summarized in Table 4.
a e di ™ s M e from c e srformacnacle-upctohnical o r e s P n p gh ll te ices
BioQualityrcah throkued bypfuort serv . ac ea up res tion. B tory s rmacia ha ula duc pro nd reg from P a
B
dia ™ Me m fro e ss ance p to al rocn perfogrmh scalell-utechnicices P a u io rv y fu b se ality thro Qu earch acked pport . u res tion. B tory s rmacia ha ula duc pro nd reg from P a
Table 4. HIC products available from Amersham Pharmacia Biotech.
Phenyl Sepharose 6 Fast Flow (low sub) Phenyl Sepharose 6 Fast Flow (high sub) Butyl Sepharose 4 Fast Flow Octyl Sepharose 4 Fast Flow*
Suitable for all initial and intermediate step purifications. Available in laboratory pack sizes and bulk quantities.
Phenyl Sepharose High Performance
Suitable for all high resolution purifications. Available in laboratory pack sizes, bulk quantities and as prepacked columns.
Phenyl Sepharose CL-4B Octyl Sepharose CL-4B
Traditional medium for all applications. Available in laboratory pack sizes and bulk quantities.
Alkyl Superose and Phenyl Superose
For analytical and small scale preparative applications. Available as prepacked columns.
HIC Media Test Kit
For screening different types of ligands and for method development work at small scale. Five different HIC media as prepacked 1 ml columns.
* Octyl Sepharose 4 Fast Flow is currently (December 1992) only available as a CDM product (see p. 17), but will later be available as a standard catalogue product.
21
Pr
a e dmi ™ fro s M s nce -up to al e a oc orm le nic
h a perf gh sc ll tec ices an u by fu erv ality thro ts Qu earch acked ppor . u res tion. B tory s rmacia ha ula duc pro nd reg from P a
Bio
BioProcess Media BioProcess™ Media form a full range of separation media especially designed to meet the demands of today’s industrial production of biomolecules.
Productive:
High flow rates, high capacity and high recovery lead to good process economy.
Validated:
Manufactured according to fully validated process with strict quality standards and complete documentation.
Scaleable:
Work equally well in laboratory and pilot production systems as well as in industrial operation.
Cleanable:
Very high chemical stability enables thorough cleaning and sanitization treatments that reduce the risk of contamination of the end product and increase the media lifetime.
Documented:
Regulatory Support Files give full details of approval support data such as performance, stability (including leakage data), extractable compounds and analytical methods. A Regulatory Support File is an invaluable starting point, especially for pharmaceutical process validations.
Guaranteed supply: Large production capacity and guaranteed future supply.
Base matrices The BioProcess HIC media range is based on the highly cross-linked beaded agarose matrices Sepharose Fast Flow and Sepharose High Performance. Their macrostructures containing polysaccharide chains arranged in bundles (Fig. 6) are further strengthened by different degrees of inter-chain cross-linking. The resulting macroporous structures combine good capacities for molecules up to 4x106 (6% agarose) and 2.7x107 (4% agarose) in molecular mass with excellent flow properties and high physical and chemical stability. Fig. 6. Structure of cross-linked agarose gels.
22
All Sepharose based matrices have virtually no non-specific adsorption properties and are also resistant to microbial degradation due to the presence of the unusual sugar 3,6-anhydro-L-galactose.
Coupling The HIC ligands are coupled to the monosaccharide units by stable ether linkages. The structures of the coupled ligands are shown in Fig. 3.
Chemical stability BioProcess HIC Media are stable in all commonly used aqueous buffers and solvents in the pH range 2-14. When these media were challenged by storage for 7 days at 40oC in the solutions listed in Table 5, no significant change in chromatographic function was seen. Of special interest is their stability in alkaline solutions, as cleaning and sanitization with NaOH solutions are preferred in process applications. The functional stability and recommended pH ranges are summarized in Table 6. The ligand leakage of BioProcess HIC Media at different pH values has been tested and generally found to be extremely low (43). The pH range 2–14 can be used for cleaning-in-place (CIP) and sanitization-in-place (SIP), see ‘‘Cleaning, sanitization and sterilization procedures’’, page 63. BioProcess HIC Media are stable at high temperatures and can be sterilized by autoclaving at 120oC for 20 min. Table 5. Chemical stability test of BioProcess HIC Media. Tested media
Test solutions 1 M NaOH
1 M acetic acid
1 mM HCL
3M (NH4)2SO4
70% ethanol
30% isopropanol 6 M GuHCl 8 M Urea
Phenyl Sepharose 6 Fast Flow (low sub)
X
(n. t.)
(n. t.)
X
X
X
X
X
Phenyl Sepharose 6 Fast Flow (high sub)
X
(n. t.)
(n. t.)
X
X
X
X
X
Butyl Sepharose 4 Fast Flow
X
(n. t.)
X
(n. t.)
X
X
X
(n. t.)
Phenyl Sepharose High Performance
X
X
(n. t.)
(n. t.)
X
X
X
X
X = Functionally stable when tested for 7 days at +40°C (n. t.) = Not tested
Table 6. Stability and recommended pH ranges for BioProcess HIC Media.
Long term stability and recommended working pH range: Short term stability and recommended CIP and SIP pH range: Recommended long term storage:
3–13 2–14 0.01 M NaOH or 20% ethanol.
23
Physical stability The highly cross-linked structures of Sepharose Fast Flow and Sepharose High Performance matrices are physically stable resulting in very good flow properties. This is illustrated by the pressure-flow rate curves for Phenyl Sepharose 6 Fast Flow shown in Fig. 7. In columns with 5 cm inner diameter and a bed height of 15 cm, flow rates up to 500 cm/h can be used without exceeding a back pressure of 1 bar. The optimal working flow rate during elution is normally 50–150 cm/h but during equilibration, regeneration, and also often during sample application, higher flow rates of 200– 300 cm/h can be used. These higher flow rates reduce cycle times. Fig. 7. Typical pressure/flow rate curves for Phenyl Sepharose 6 Fast Flow (low sub) and Phenyl Sepharose 6 Fast Flow (high sub) in an XK 50/30 Column, bed height 15 cm, mobile phase 0.1 M NaCl. (Work from Amersham Pharmacia Biotech, Uppsala, Sweden).
Flow rate (cm/h) 700 600 500 400 high sub low sub
300 200 100 0.0
0.2
0.4
0.6
0.8
1.0
1.2
Pressure (bar)
Binding capacity One of the major features of BioProcess HIC Media is the high binding capacity, which results in high throughput and productivity even at relatively low salt concentrations. Fig. 8 shows the total dynamic binding capacities of human serum albumin and human IgG at different concentrations of ammonium sulphate as determined by frontal analysis. Phenyl Sepharose 6 Fast Flow (high sub) showed the highest capacities for both hIgG and HSA. Phenyl Sepharose High Performance had higher capacity for hIgG compared with HSA while Butyl Sepharose 4 Fast Flow showed the reverse, indicating the difference in selectivity. The protein recoveries when eluting with low salt buffer were all 80% or more. The dynamic binding capacity will decrease with increasing linear flow rates. This is especially important to consider when optimizing initial separation steps where large volumes need to be processed. Productivity may be higher at high flow rates even though the binding capacity is decreased.
24
Adsorption capacity (mg h IgG/ml gel) Adsorption capacity (mg HSA/ml gel)
Fig. 8. Total adsorption capacities of Phenyl and Butyl Sepharose media for human IgG and HSA as a function of the concentration of ammonium sulphate in the equilibration buffer. 1=Phenyl Sepharose 6 Fast Flow (high sub), 2=Phenyl Sepharose High Performance, 3=Phenyl Sepharose 6 Fast Flow (low sub), 4=Butyl Sepharose 4 Fast Flow. (Work from Amersham Pharmacia Biotech, Uppsala, Sweden).
30
hIgG
1 ▲
20 2 ▲
10
3 4
0.39
0.45 0.57 0.68 Concn. of ammonium sulphate (M) ▲
HSA 30 1 ▲
20
4 ▲
10
BioQualityrcah throkued bypfuort serv . ac ea up res tion. B tory s rmacia ha ula duc pro nd reg from P a
23
▲
0.6
a e di ™ s M e from c e srformacnacle-upctohnical o r P n pe gh s ll te ices
▲
0.9 1.2 1.5 Concn. of ammonium sulphate (M)
Phenyl Sepharose 6 Fast Flow (low sub) Phenyl Sepharose 6 Fast Flow (high sub)
Phenyl Sepharose 6 Fast Flow (low sub) and Phenyl Sepharose 6 Fast Flow (high sub) are based on highly cross-linked 6% agarose with phenyl ligands coupled via stable ether linkages. The media characteristics are summarized in Table 7. Phenyl Sepharose 6 Fast Flow (low sub) and Phenyl Sepharose 6 Fast Flow (high sub) were initially developed and tested in cooperation with leading pharmaceutical manufacturers. They are ideal for initial or intermediate step purification of proteins Table 7. Characteristics of Phenyl Sepharose 6 Fast Flow (low sub) and Phenyl Sepharose 6 Fast Flow (high sub).
Bead structure Mean particle size Particle size range Degree of substitution
cross-linked agarose, 6%, spherical 90 µm 45–165 µm approx. 20 (low sub) and 40 (high sub) µmol phenyl groups/ml gel
Further information is available in Data File 2040 (Code No. 18-1020-53).
25
and peptides with a low to medium degree of hydrophobicity. The availability of two degrees of substitution increases the possibility of finding the best selectivity and capacity for a given application. Phenyl Sepharose 6 Fast Flow (high sub) has been used as an effective capture step in methods for the purification of recombinant human Epidermal Growth Factor (h-EGF) and recombinant Pseudomonas aeruginosa exotoxin. These applications are presented in chapter 6, pages 87 and 92 respectively.
Product availability Phenyl Sepharose 6 Fast Flow (low sub) and Phenyl Sepharose 6 Fast Flow (high sub) are supplied as suspensions in 20% ethanol in packs of 200 ml, 1 litre and 5 litres.
a e dmi ™ fro s M s nce -up to al e a oc orm le nic
Pr perf h sca ll tech es BioQualityrcahn throkuegd bypfuort servic . ac ea up res tion. B tory s rmacia ha ula duc pro nd reg from P a
Butyl Sepharose 4 Fast Flow
Butyl Sepharose 4 Fast Flow is based on highly cross-linked 4% agarose with butyl ligands coupled via stable ether linkages. The characteristics of this medium are summarized in Table 8.
Bead structure Mean particle size Particle size range Degree of substitution
cross-linked agarose, 4%, spherical 90 µm 45–165 µm approx. 50 µmol butyl groups/ml gel
Table 8. Characteristics of Butyl Sepharose 4 Fast Flow.
Further information is available in Data File 3300 (Code No. 18-1020-70).
Butyl Sepharose 4 Fast Flow was initially developed and tested in cooperation with leading pharmaceutical manufacturers. It is intended for the initial or intermediate step purification of proteins and peptides with a low to medium degree of hydrophobicity and often works efficiently with rather low salt concentrations. For the butyl ligand, the mechanism of adsorption and desorption is different than for the phenyl ligand, which gives a difference in selectivity. This was illustrated in an application where recombinant human Annexin V, expressed in E. coli, was purified using HIC after an initial capture step on a cation exchanger. A comparison of the chromatograms in Fig. 9 shows that the elution position of Annexin V and the main impurities interchanged when changing from Butyl Sepharose 4 Fast Flow to Phenyl Sepharose 6 Fast Flow (high sub).
Product availability Butyl Sepharose 4 Fast Flow is supplied as suspension in 20% ethanol in packs of 200 ml, 500 ml and 5 litres. 26
A280 nm
Medium: Butyl Sepharose 4 Fast Flow Column: XK 16/20 Buffer A: 20 mM sodium phosphate pH 7.0 + 1.0 M ammonium sulphate Buffer B: 20 mM sodium phosphate pH 7.0 Sample: Partially purified Annexin V expressed in E. Coli Sample volume: 5 ml Flow rate: 100 cm/h Gradient: 0–100% B, 10 column volumes
A280 nm
Medium: Phenyl Sepharose 6 Fast Flow (high sub.) Column: XK 16/20 Buffer A: 20 mM sodium phosphate pH 7.0 + 1.0 M ammonium sulphate Buffer B: 20 mM sodium phosphate pH 7.0 Sample: Partially purified Annexin V expressed in E. Coli Sample volume: 5 ml Flow rate: 100 cm/h Gradient: 0–100% B, 10 column volumes
Annexin V
Annexin V
0
60
Time (min)
0
60
Time (min)
Fig. 9. Purification of Annexin V on Butyl Sepharose 4 Fast Flow and Phenyl Sepharose 6 Fast Flow (high sub). (Work from Amersham Pharmacia Biotech, Uppsala, Sweden).
dia ™ Me m ss ance -ufrop to al e c m ro rfor cale chnic
pe h s ll te P es BioQualityrcahn throkuegd bypfuort servic . ac ea up res tion. B tory s rmacia ha ula duc pro nd reg from P a
Phenyl Sepharose High Performance
Phenyl Sepharose High Performance is based on very highly crossed-linked 6% agarose with phenyl ligands coupled via stable ether linkages. The characteristics of this medium are summarized in Table 9. Table 9. Characteristics of Phenyl Sepharose High Performance.
Bead structure Mean particle size Particle size range Degree of substitution
cross-linked agarose, 6%, spherical 34 µm 24–44 µm approx. 25 µmol phenyl/ml gel
Further information is available in Data File 2050 (Code No. 18-1020-56).
Phenyl Sepharose High Performance is ideal for laboratory and process scale intermediate step purifications where high resolution is needed. The separation of slightly modified variants, clipped forms etc., of a recombinant protein from the native protein is a typical application example. It has also proven to be very efficient for the purification of monoclonal antibodies. Two large scale applications on monoclonal 27
antibodies, one for the purification of anti-gp120, which is in clinical trials for treatment of AIDS, the other for the purification of an antibody used in diagnostic tests, are presented in Chapter 6, pages 85 and 90 respectively. Product availability Phenyl Sepharose High Performance is supplied as a suspension in 20% ethanol in packs of 75 ml, 1 litre and 5 litres and prepacked in HiLoad™ 16/10 and 26/10 columns.
Custom Designed HIC Media Custom Designed Media (CDM) meet the needs of specific industrial process separations where chromatography media from our standard range are not suitable. CDM can be made to meet BioProcess Media specifications if required. The CDM group at Amersham Pharmacia Biotech works in close collaboration with the customer to design, manufacture, test and deliver media for specialized separation requirements. Several CDM products are also available to the general market. Some HIC media first produced as Custom Designed Media have proven so successful that they have subsequently been introduced as standard products, e.g. Phenyl Sepharose 6 Fast Flow (low sub), Phenyl Sepharose 6 Fast Flow (high sub) and Butyl Sepharose 4 Fast Flow. Product availability Please contact your local Amersham Pharmacia Biotech representative for further details of CDM products and services.
HIC Media Test Kit HIC Media Test Kit consists of five ready-to-use 1 ml plastic columns for screening different types of ligands and for method development work at small scale. The kit contains the following HIC media: • Phenyl Sepharose High Performance • Phenyl Sepharose 6 Fast Flow (low sub) • Phenyl Sepharose 6 Fast Flow (high sub) • Butyl Sepharose 4 Fast Flow • Octyl Sepharose 4 Fast Flow Product availability Please contact your local Amersham Pharmacia Biotech representative for further information. 28
Phenyl Sepharose CL-4B and Octyl Sepharose CL-4B Phenyl Sepharose CL-4B and Octyl Sepharose CL-4B are produced in large quantities with high and consistent quality. Their performance has been demonstrated in hundreds of applications and they have been approved by regulatory authorities for use in many pharmaceutical production processes. Phenyl Sepharose CL-4B and Octyl Sepharose CL-4B are based on cross-linked 4% agarose matrices with ligands coupled via stable ether linkages. The media characteristics are summarized in Table 10.
Table 10. Characteristics of Phenyl Sepharose CL-4B and Octyl Sepharose CL-4B.
Bead structure Mean particle size Paricle size range Degree of substitution
cross-linked agarose, 4%, spherical 90 µm 45-165 µm approx. 40 µmol phenyl or octyl groups/ml gel
Phenyl Sepharose CL-4B and Octyl Sepharose CL-4B are stable in all commonly used aqueous buffers. Long term stability and recommended working pH range is 3–12. 1 M NaOH can be used for short term exposure in cleaning and sanitization procedures, see ‘‘Cleaning, sanitization and sterilization procedures’’, page 63. Short term stability and recommended CIP and SIP pH range is 2–14. Maximum flow rate for a laboratory-scale column with an internal diameter of up to 5 cm and a bed height of up to 15 cm is 150 cm/h.
Product availability Phenyl Sepharose CL-4B and Octyl Sepharose CL-4B are supplied as suspensions in 20% ethanol in packs of 50 ml, 200 ml and 10 litres.
Phenyl Superose and Alkyl Superose Phenyl Superose and Alkyl Superose are media for high performance HIC, available in prepacked columns for use in FPLC™, SMART™ System (Phenyl Superose only) or HPLC systems. Phenyl and neopentyl groups respectively are attached to the matrix via a stable ether linkage. The characteristics of these media and columns are summarized in Table 11.
29
Bead structure Mean particle size Column sizes
cross-linked agarose, 12%, spherical 13 µm 5x50 mm (HR 5/5) 10x100 mm (HR 10/10) 1.6x50 mm (Phenyl Superose, PC 1.6/5 for SMART System)
Table 11. Characteristics of Phenyl Superose and Alkyl Superose.
Further information is available in Data File for prepacked HR columns (Code No. 18-1009-26) and in Data File for prepacked PC columns (Code No. 18-1009-02).
Phenyl Superose and Alkyl Superose are stable in all commonly used aqueous buffers. Long term stability and recommended working pH range is 2–13. 1 M NaOH can be used for cleaning and sanitization, see ‘‘Cleaning, sanitization and sterilization procedures’’, page 63. Short term stability and recommended CIP and SIP pH range is 2–14. The columns are typically used in laboratory scale protein purification schemes or as an analytical tool, as a complement to e.g. ion exchange chromatography and gel filtration. Examples of applications are shown in chapter 6, page 79–83. Suitable protein loads are in the mg range (HR columns) or, for micropurification, in the ngµg range (Phenyl Superose, PC 1.6/5). Alkyl Superose is less hydrophobic than Phenyl Superose and is therefore particularly suitable for high performance HIC with retained biological activities of labile proteins and of proteins which bind very tightly to media with higher hydrophobicities.
30
4 Experimental Design This chapter will deal with experimental methods of HIC which are applicable in the majority of cases. Since the factors which influence HIC are numerous, the relevant chromatographic parameters that lead to the selective purification of the protein(s) of interest should be optimized on a case to case basis.
Hydrophobicity of proteins It is estimated that as much as 40–50% of the accessible surface area of proteins is non-polar (44, 45). These areas are responsible for the binding of proteins to HIC adsorbents in the presence of moderate to high concentrations of salting-out salts. The strength of this salt-promoted interaction may be predicted from the close relationship between precipitation data for proteins and their relative retention on HIC adsorbents (27). Since such retention data are not readily available for the large majority of proteins, they must be established from case to case for the protein(s) of interest in a biological sample.
Multivariate mapping This is a useful method for: i. Characterizing hydrophobic media on the basis of their selectivity (46). ii. Choosing the most suitable medium for the optimum resolution of two closely related proteins. iii. Determining the adsorption behaviour of proteins on HIC media and thereby establishing a ‘‘practical hydrophobic scale’’ for the proteins in question.
31
The results obtained in our laboratories (46) suggest that: i. The adsorption selectivity of Octyl Sepharose CL-4B is related to the fraction of hydrophobic amino acids in the model proteins examined. ii. The retention of proteins on alkyl Superose and Pyridine sulphide-Sepharose 6 Fast Flow is proportional to a parameter best described as ‘‘absence of surface charge’’ on the sample molecules. iii. The phenyl- and butyl-based media separated proteins according to a combination of the above two mechanisms. iv. Different hydrophobicity coefficients co-variate with the retention data established for the various hydrophobic media examined. Multivariate analysis thus opens new possibilities in the design of HIC and other chromatography-based separations by using a minimum number of experimental data.
Strategic considerations One of the most important aspects of developing a complete purification scheme is to keep the number of unit operations to a minimum. A logical approach to reach the highest possible purity with the smallest number of individual chromatographic steps is to combine techniques based on different principles and thus exploit different surface properties of the substances to be separated. However, the sequence in which the chosen techniques are used must be carefully planned. In many applications HIC is useful especially in combination with techniques such as ion exchange chromatography and gel filtration. As an example, hydrophobic interaction chromatography is a logical choice when the sample already has a high ionic strength. The conductivity of most biological starting materials is typically in the range of 15–30 mS/cm, which makes HIC an attractive alternative to ion exchange chromatography (IEX) in the first step of a downstream purification scheme. High conductivity in the starting material will reduce the binding capacity of ion exchange media and some type of conditioning such as desalting, diafiltration or dilution has to be included before an ion exchange step. In contrast, the only conditioning needed if HIC is used, is to add enough salt to promote the proper binding to the medium. Used in the first step HIC, like IEX and other adsorption techniques, will serve as an effective means of concentrating a dilute sample. Other typical points in a purification scheme where HIC fits in naturally are after an ammonium sulphate precipitation, which often comes in the beginning of a downstream process, and after an ion exchange step where the sample is eluted with a rather high ionic strength. The further addition of salt that might be needed to retard the components in a desired way on the HIC medium is thus a very simple linking step. In a similar way, a sample eluted from a HIC step in a low ionic strength buffer can often be directly applied to an ion exchange column without an extra dialysis or desalting step.
32
Choice of HIC media The type of immobilized ligand, the degree of substitution and the type and concentration of salt and pH used during the adsorption stage have a profound effect on the overall performance (i.e., selectivity and capacity) of a HIC medium [see Chapter 2]. Moreover, the type of matrix used and the coupling chemistry can also influence to a variable degree the binding and elution behaviour of many proteins. The practical implications of these effects are that different HIC media must be compared much more rigorously than ion exchange or affinity media, especially when the HIC step is part of a downstream purification process intended for an industrial scale operation.
General considerations i. The HIC medium should bind the protein of interest at a reasonably low concentration of salt. This is often dependent on the type of salt chosen, e.g. up to four times higher concentration of NaCl might be necessary to obtain a binding effect comparable to that obtained with ammonium or sodium sulphate. The salt concentration should be below the concentration that causes precipitation of different proteins in the crude feed stock. 1 M ammonium sulphate is a good starting point for screening experiments. If the substance does not bind in e.g. 1 M ammonium sulphate, then choose a more hydrophobic medium. The right choice of a suitable HIC medium can often lead to a lower consumption of salt in the binding buffer. This in turn has a direct bearing on the economic and environmental aspects of the purification process, especially for large-scale HIC applications. ii. The bound protein should be eluted from the column with salt-free buffer and with high recovery (75% or higher). If non-polar solvents are required for its elution, try a less hydrophobic medium. iii. The pH of the start buffer and the type of salt to use are both parameters that can be exploited to maximize selectivity during the adsorption phase. This is done by checking the adsorption properties of the media at different pH-values and with different types of salts during the screening of different ligands. iv. Since hydrophobic interaction is dependent on temperature, it is important that method development work is performed at the intended final working temperature.
33
Screening experiments This section outlines a general procedure for performing HIC screening experiments where emphasis is laid on optimizing selectivity by proper choice of HIC medium and by roughly defining the most critical experimental parameters. It also presents some typical elution profiles that could be obtained in a variety of situations followed by relevant discussions of the results and recommendations for further experimental work. i. Pack the media in suitable columns according to our packing recommendations (a bed volume of 1–10 ml is adequate) or use the HIC Media Test Kit from Amersham Pharmacia Biotech. The HIC Media Test Kit consists of five 1 ml plastic columns prepacked with BioProcess HIC media. For more information about the HIC Media Test Kit, see Chapter 3, Selection Guide. ii. Equilibrate the column with 2 bed volumes of the equilibration Buffer A (50 mM sodium phosphate, 1.0 M ammonium sulphate, pH 7.0). Use a constant flow rate throughout (e.g. 100 cm/h). iii. Apply a suitable amount of sample, also containing 1.0 M ammonium sulphate (pH adjusted to 7.0), to the column and wash with 2–3 column volumes of Buffer A, or until the UV-trace of the effluent returns to near baseline. iv. Elute the bound fraction using a linear and descending salt gradient from 0 to 100% Buffer B (50 mM sodium phosphate buffer, pH 7.0). A total gradient volume of 10 bed volumes is usually sufficient.
34
Evaluation of results Figs. 10 to 15 show some typical elution profiles that could be obtained from screening experiments. The shaded area shows the elution position of the protein of interest. Each chromatogram is accompanied by a general discussion of the results and suggestions for further experiments to optimize the separation of the protein of interest.
Rel. Abs
Fig. 10.
Elution volume
Result:
Product is eluted early in gradient. Resolution is not satisfactory.
Discussion:
Not much can be gained in this situation by changing salt concentration. Decreasing the salt concentration will decrease the binding capacity of the protein of interest and might even lead to its elution together with the unbound fraction. Increasing the salt concentration might lead to the co-adsorption of unwanted impurities and thereby lead to a decrease in the selectivity of the adsorbent for the protein of interest. Changing the pH of the equilibration buffer might result in stronger binding and higher selectivity for the protein of interest. The effect of pH is variable for different proteins and usually a lowering of the pH leads to increased binding of proteins. Increasing the pH usually leads to a decreased binding of proteins, which, in this particular case, might result in the elution of the protein of interest together with the unbound fraction.
Next step:
Repeat the experiment at a lower and a higher pH. If no improvement in selectivity is obtained – TRY A MEDIUM WITH A DIFFERENT LIGAND or, if available, A MEDIUM WITH A HIGHER DEGREE OF LIGAND SUBSTITUTION. 35
Rel. Abs
Fig. 11.
Elution volume
36
Result:
Product is eluted near the end of the gradient. Resolution is not satisfactory
Discussion:
A decrease of the salt concentration will weaken the strength of binding resulting in the earlier elution of the protein of interest. It may also have a positive effect on selectivity since more of the less hydrophobic substances will be eluted together with the unbound fraction. However, the effect of this approach on the resolution is marginal since the contaminants are eluted very close to the protein of interest, both before and after. Changing the pH of the equilibration buffer may have a positive effect on resolution and should be tried.
Next step:
Repeat the experiment at a higher and a lower pH of the equilibration buffer. If no improvement in resolution is obtained – TRY A MEDIUM WITH A DIFFERENT LIGAND or, if available, A MEDIUM WITH A LOWER DEGREE OF LIGAND SUBSTITUTION.
Rel. Abs
Fig. 12.
Elution volume
Result:
Product is eluted in the middle of the gradient. Resolution is not satisfactory.
Discussion:
Changing the concentration of salt in the equilibration buffer will have a limited effect on resolution. However, a change of pH of the equilibration buffer (both lower and higher pH values) might have a favourable effect.
Next step:
Repeat the experiment at a higher and a lower pH value. If no improvement in resolution is obtained – TRY A MEDIUM WITH A DIFFERENT LIGAND or, if available, A MEDIUM WITH A HIGHER DEGREE OF LIGAND SUBSTITUTION.
37
Rel. Abs
Fig. 13.
Elution volume
Result:
Product is eluted early in gradient. Resolution is satisfactory.
Discussion:
In principle, this can be a good choice of medium. However, the fact that the protein of interest is eluted very early in the gradient indicates that the binding capacity may be low. This might be compensated for, if necessary, by a moderate increase of the salt in the equilibration buffer. This in turn may lead to a decrease in the selectivity of the adsorbent since some of the unbound proteins might be adsorbed together with the protein of interest. Another negative effect of increased salt concentration may be a decrease in resolution caused by the increase in gradient slope if the total gradient volume, or the cycle time, is kept constant. Increased salt concentration will also give increased costs which may be of importance if the HIC step is to be a part of a manufacturing process. Finally, not much can be gained by changing the pH of the equilibration buffer since the resolution obtained was considered to be satisfactory.
Next step:
Continue with method development as outlined under ‘‘Optimizing a HIC step’’. If low binding capacity is a problem and problems with increased salt concentration as outlined above are encountered – TRY A MEDIUM WITH A DIFFERENT LIGAND or, if available, A MEDIUM WITH A HIGHER DEGREE OF LIGAND SUBSTITUTION.
38
Rel. Abs
Fig. 14.
Elution volume
Result:
Product is eluted near the end of the gradient. Resolution is satisfactory.
Discussion:
This can also be a good choice of medium. Decreasing the concentration of salt in the equilibration buffer will give earlier elution of the protein of interest, reduced cycle time and decreased cost for salt. A disadvantage in this situation might be that some of the most hydrophobic contaminating substances bind so strongly that some organic solvent or chaotropic agent has to be used for their removal. Not much can be gained by changing pH since the selectivity is already good.
Next step:
Continue with method development as outlined under ‘‘Optimizing a HIC step’’. If problems with very strong binding of hydrophobic contaminants are encountered – TRY A MEDIUM WITH A DIFFERENT LIGAND or, if available, A MEDIUMWITH A LOWER DEGREE OF LIGAND SUBSTITUTION.
39
Rel. Abs
Fig. 15.
Elution volume
Result:
Product is eluted in the middle of the gradient. Resolution is satisfactory.
Discussion:
The choice of ligand is very good and there is less risk of strong binding of the most hydrophobic contaminants.
Next step:
Continue with method development as outlined under ‘‘Optimizing a HIC step’’.
The examples presented above do not cover two extreme cases that may arise, i.e. the situation in which the protein of interest is either not bound to the HIC medium or that it binds so strongly that it is difficult to elute it without using denaturing solvents. In both instances, one should try to use a different HIC medium or use another medium which operates on a different separation principle. In some of the examples above it is assumed that resolution is inadequate. The requirements for resolution in any particular chromatographic step must be stipulated on a case-by-case basis. What sometimes seems to be fairly bad resolution can often be good enough if it is an initial capture step where the main objective is reduction of volume, removal of critical contaminants and preparation for higher resolution chromatography.
40
Optimizing a HIC step The main purpose of optimizing a chromatographic step is to reach the pre-defined purity level with highest possible recovery by choosing the most suitable combination of the critical chromatographic parameters. In process applications there is also a need to reach the highest possible throughput. The screening experiments outlined previously will mainly help in establishing the most suitable medium to use. The sections below will deal with some important guidelines for optimizing the critical operational parameters which affect the maximum utilization of the HIC step. These parameters include: type of buffer salt, salt concentration, buffer pH, temperature, bed height, flow rate, gradient shape and gradient slope.
The solute As in other adsorption chromatography techniques, the way HIC is used depends on the size of the solute molecule. Small molecules such as small peptides interact with the medium by single point attachment. Their migration velocity depends directly on the binding constant of a single bond and can vary over a wide interval depending on the ionic strength of the mobile phase. Larger molecules such as proteins and nucleic acids interact with the medium by multi-point attachment. Their migration velocity depends on the sum of several bonds. Thus their velocity is extremely low at all ionic strengths over a certain value. The protein is more or less stuck to the column. Below this ionic strength, the protein is practically not retarded at all (47). The interval of eluting strength where a large molecule is partly retarded on the column is thus much smaller than for a small molecule. This means that purifying large molecules on HIC is a typical on-off technique where the difference in retention for the molecules to be separated can be substantial at any specific ionic strength. In other words, separation of large molecules on HIC is a high selectivity technique. The separation should be optimized by manipulating the parameters affecting the selectivity of the system, i.e. optimizing the chemistry of the system by means of salt concentration, type of salt, pH, gradient slopes or stepwise elution schemes. By effecting relatively small changes in selectivity, large changes in resolution can occur. When purifying small molecules on the other hand, the selectivity of the system is usually much lower and the requirements for purity might not be met by working on the selectivity alone. The efficiency parameters such as bed height, bead size, theoretical plates, linear flow rate and sample volume may also have to be optimized. In this handbook however, the focus will be on large molecules such as proteins and large peptides.
41
In conclusion, when purifying large molecules such as proteins, relatively short columns can be used if the selectivity of the adsorbent is exploited in an optimal way. The linear flow rate should, if required, be sufficiently reduced in order to optimize the kinetics of the adsorption and desorption process. This can also be further enhanced by choosing a smaller bead size. Smaller beads will also provide the necessary increace in efficiency when more difficult separation problems are encountered.
The solvent This is one of the most important parameters to have a significant influence on the binding capacity and selectivity of a HIC medium. In general, the adsorption process is often more selective than the desorption process and it is therefore important to optimize the starting ‘‘binding’’ buffer conditions with respect to critical parameters such as pH, type of salt, concentration of salt and temperature. The combination of salt and pH can be manipulated to give optimum selectivity during purification by HIC. Optimal conditions differ from application to application and are best established by running linear gradients and varying the parameters in a controlled way (for example by using Factorial design). Changes of temperature and pH are sometimes restricted by the stability of the substance of interest or by system constraints etc. but may often be of interest to evaluate. The Hofmeister series (Table 1) gives important guidelines in choosing the type of salt to use. The most efficient salts are normally ammonium sulphate and sodium sulphate but also ‘‘weaker’’ salts such as sodium chloride should be considered. In an ideal situation, the correct choice of salt and salt concentration will result in the selective binding of the protein of interest while the majority of the impurities pass through the column unretarded. If the protein of interest binds weakly to the column, an alternative approach is to choose the starting buffer conditions which will result in the maximum binding of a large proportion of the contaminating proteins but allowing the protein of interest to pass through unretarded. An extension of this strategy is to increase the salt concentration in the unbound fraction to such an extent that the protein of interest binds to the same column in a second run while most of the impurities pass through the column unretarded. The effect of varying the concentration of salt in the binding buffer on the purification of a monoclonal antibody (IgG1) from mouse ascites fluid is shown in Fig. 16. The column of Alkyl Superose was equilibrated with varying concentrations of ammonium sulphate (2 M to 0.8 M) and its selectivity for the IgG1 investigated. The results show that high selectivity for IgG1 is obtained using 1 M ammonium sulphate in the binding buffer. It should be pointed out that the higher the salt concentration in the equilibration buffer, the greater the risk that some of the proteins in the sample will precipitate. Since such precipitates can clog tubings and column filters, the sample must be filtered or centrifuged. This extra step can be avoided by equilibrating the sample in a lower salt concentration than is required for its precipitation and then applying it to a column which is equilibrated with a higher salt concentration (48). Some of the proteins will precipitate on the column (zone precipitation) but they redissolve upon reduction of the salt concentration during stepwise or gradient elution. 42
a)
b)
IgG
IgG
Albumin Albumin
c)
d)
IgG
IgG
Fig. 16. The effect of starting conditions in HIC. Sample, 100 µl anti-CEA MAB (-IgG1) from mouse ascites fluid in 0.8 M (NH4)2SO4 (corresponding to 20 µl ascites); column. Alkyl Superose HR 5/5; flow rate, 0.5 ml min -1; buffer A, 0.1 M sodium phosphate, pH 7.0, (NH4)2SO4). (a) Sample applied in 2 M (NH4)2SO4: both albumin and IgG are absorbed. (b) Sample applied in 1.5 M (NH4)2SO4: less albumin binds and IgG elutes earlier in the gradient. (c) Sample applied in 1.0 M (NH4)2SO4: albumin does not bind and, therefore, the column has a greater capacity for binding IgG. (d) Sample applied in 0.8 M (NH4)2SO4: albumin does not bind; IgG is retarded, but elutes in a broad peak. (Work from Amersham Pharmacia Biotech, Uppsala, Sweden).
43
0.25
I
2.0
conc. (NH4 )2 SO4 (M)
b A 280 nm 0.5
I
I
2.0
conc. (NH4 ) 2 SO4 (M)
a A 280 nm 0.5
0
I
I
20
40
I
60 Time (min)
0 0
I
I
I
20
40
60
0 Time (min)
Fig. 17. The effect of loading conditions in HIC. Column, Alkyl Superose HR 5/5; flow rate, 0.5 ml min-1; buffer A, 0.1 M sodium phosphate, pH 7.0, 2 M (NH4)2SO4. (a) Sample (500 µl anti-CEA MAB (IgG1) from mouse ascites fluid in 0.9 M (NH4)2SO4 (corresponding to 115 µl ascites) applied in one injection. (b) Sample as (a) applied in five 100 µl injections with 1.3 ml 2.0 M (NH4)2SO4 after each portion. (Work from Amersham Pharmacia Biotech, Uppsala, Sweden).
When sample is applied at a salt concentration lower than that used for equilibration of the column, the sample volume becomes important. This is demonstrated in Fig. 17. When a 500 ml sample of ascites fluid was applied to a 1 ml column of Alkyl Superose, albumin, the weakest interacting substance, started to elute during sample application (Fig. 17 a). Dividing the sample into portions, e.g. five 100 ml samples and adding equilibration buffer (1.3 ml) after each sample application to enhance the hydrophobic interaction prevented early elution of albumin (Fig. 17 b).
Elution This can be achieved by: i. A linear or step-wise decrease of the concentration of salt. ii. Adding various proportions of organic solvents to the elution buffer (see Chapter 2) provided that the protein of interest is stable upon exposure to such solvents. These additives decrease the polarity or surface tension of the eluent resulting in a reduction in the binding strength and the elution of the bound proteins from the column. Usually, 40% ethylene glycol or 30% iso-propanol, dissolved in salt-free buffer, is used. In some applications, it can be advantageous to linearly increase the concentration of such additives as the salt concentration of the elution buffer is simultaneously decreased by a linear gradient. The latter procedure can sometimes lead to increased resolution of the bound proteins. 44
iii. Adding neutral detergents (usually 1%) to the elution buffer. However, some detergents are bound so strongly that they are difficult to wash out completely with common organic solvents (e.g. ethanol). In the worst case, this might lead to a decrease in the capacity of the HIC medium for subsequent applications. These additives must therefore be used with care. The preferred method of elution is a linear or step-wise decrease of the salt concentation in the elution buffer. Some typical examples are presented below.
Gradient elution Simple linear gradients are the first choice for screening experiments, but when more experience is at hand it might be advantageous to make a gradient more shallow in areas where resolution is inadequate. Consequently, areas where resolution is good can be covered by a steep gradient (Fig. 18). Such complex gradients offer maximum flexibility in terms of combining resolution with speed during the same separation.
Rel. Abs
Rel. Abs
By increasing the total gradient volume (i.e. decreasing gradient slope) of a linear gradient, resolution will be improved in all parts of the chromatogram (Fig. 19). This is usually not the best approach in preparative mode where the prime issue is not to resolve as many peaks as possible but to separate the compound of interest from the rest of the compounds in the feed material. Increased gradient volume will also give increased cycle time and the separated fractions will also be more diluted.
Elution volume
Elution volume
Fig. 18. Effect of a complex gradient on resolution.
45
Rel. Abs
Rel. Abs
Elution volume
Elution volume
Rel. Abs
Rel. Abs
Fig. 19. Effect of gradient slope on resolution.
Elution volume
Fig. 20. Switching from a continuous gradient to step-wise elution.
46
Elution volume
Step-wise elution Step-wise elution is often preferred in large scale preparative applications since it is technically more simple and reproducible than gradient elution. Step-wise elution can sometimes be advantageous also in small scale applications since the compound of interest can be eluted in a more concentrated form if the eluting strength of the buffer can be kept high enough without causing co-elution of more strongly bound compounds. The principle of step-wise elution is to increase resolution in the area where the peak of interest elutes. Fig. 20 illustrates how a three step increase in eluting strength can be used to obtain maximum resolution of the fraction of interest (shaded peak). In the first step, the strength and the volume of the elution buffer is optimized to elute all compounds binding less strongly to the gel than the compound of interest. The elution strength and volume of buffer should be large enough to elute these contaminating weaker binding substances, but it must not exceed that level where the peak of interest starts to co-elute with the contaminating compounds. In the second step the elution strength is increased to the point where the compound of interest elutes. The elution strength should be large enough to elute the compound of interest without excessive dilution, but must be kept below the level where the more strongly bound contaminating compounds start to co-elute. In the final step, the elution strength is further increased to elute all of the remaining contaminating compounds. This step can be a very short one with high elution strength. When step-wise elution is applied, one has to keep in mind the danger of getting artefact peaks when a subsequent step is administered too early after a tailing peak. For this reason it is recommended to use continuous gradients in the initial experi-ments to characterize the sample and its chromatographic behaviour.
Sample load and flow rate The through-put of the method can be increased by increasing sample load and flow rate. However, this has to be traded off against decreased resolution (efficiency). The effects of sample load and flow rate are further discussed below under ‘‘Process considerations’’.
Regeneration After each cycle, bound substances must be washed out from the column to restore the original function of the medium. HIC adsorbents can normally be regenerated by washing with distilled water after each run. To prevent a slow build up of contaminants on the column over time, more rigorous cleaning protocols may have to be applied on a regular basis. (See page 63, ‘‘Cleaning, sanitization and sterilization procedures’’).
47
Process considerations In contrast to analytical chromatography or small scale preparative chromatography in research and development, process chromatography is used as part of a manufacturing process. Method development work has to focus on purifying the product of interest to the highest yield and the required purity as quickly, cheaply and easily as possible, i. e. to find the conditions that give the highest possible productivity (amount of product produced per volume of media and unit time) and process economy.
Method optimization in process chromatography Firstly, selectivity for the substance of interest is maximized by choosing the proper type of media, pH, type of salt, salt concentration and temperature, as has already been outlined above. In HIC, as for most other adsorption techniques, there are then basically two alternative routes to follow: i. If HIC is used in an intermediate or final step where the need for resolution is high in order to meet purity requirements for the final product, the resolution is maximized by working on the eluting conditions such as gradient shape, gradient slope or concentration and volume of steps in a step-wise elution procedure. Resolution should be the highest possible while still keeping separation time reasonably short and avoiding excessive dilution of eluted product. From this point, flow rate and sample load are optimized to find highest possible productivity where resolution is still high enough to meet the predefined purity requirements. In HIC, as in ion exchange chromatography, sample load, flow rate and gradient volume are interrelated. Increased flow rate will give a decrease in resolution, but this decrease will not be very significant at high sample loadings. This means that under process conditions, where maximum sample load is applied to achieve maximum throughput, the flow rate is limited primarily by the rigidity of chromatography media and by system constraints. The effect on resolution of increased gradient volume is usually more significant than the effect of flow rate. This means that when increasing gradient volume to increase resolution, flow rate can also be increased accordingly to compensate for loss in separation speed. The result is an increase in resolution that may be traded off for increased sample loading and thereby increased productivity. In other words, in process chromatography the best result will be obtained by using the maximum flow with the gradient volume that provides the best resolution, which is demonstrated in Fig. 21 for a model experiment on Phenyl Sepharose High Performance. The flow rate in each experiment is shown in the bottom square. The largest increase in resolution was seen when going diagonally from A to C, i.e. increasing gradient volume at a constant flow rate. Even when going from AD to BC, i.e. increasing gradient volume and flow rate in the same order and thereby keeping a 48
Fig. 21. The effect of gradient volume and flow rate on resolution and cycle time. (Work from Amersham Pharmacia Biotech, Uppsala, Sweden.)
Rs 6 5 4
tim
3 2 0
e (m
0.8
00
in) 1
0.4
200 0.0
300
3
0
1 l) × m ( . vol
B
A
200
200 100
100 50
200
50 100
100 cm/h
C
50
25 25 12.5
D
constant separation time, an increase in resolution could be observed. Going diagonally from D to B, i.e. increasing the gradient volume 2-fold while increasing the flow rate 4-fold, demonstrates how the separation time can be cut without losing resolution. As the resolution increases with increased gradient volume, however, dilution of each peak occurs, which also has to be taken into consideration. ii. If HIC, on the other hand, is used as an initial product capture step where the major concern is to remove critical contaminants and reduce volume, selectivity during desorption is not a prime issue. After having washed out the non-bound substances, the compound of interest is eluted with a single-step procedure. In this mode, the entire bed volume is utilized for sample binding and the prime consideration when optimizing for highest possible productivity is to find the highest possible sample load over the shortest possible sample application time with acceptable loss in yield. In this situation, more emphasis should be given to the binding strength of the compound of interest than to selectivity during sample application. This means that the salt concentration during sample application should not be too low since this will have a negative effect on dynamic binding capacity. Note also that less hydrophobic contaminating substances will not have any dramatic effect on the binding capacity for the compound of interest since they will be displaced by the latter. 49
C/Co 1
Fig. 22. Breakthrough curves for determination of dynamic capacity.
a)
Equilibrium bed capacity
Adsorbate not adsorbed by bed
0
Time
C/Co 1
b)
‘‘useful’’ capacity
0
Time
The dynamic binding capacity for the protein of interest should be determined by frontal analysis using real process feedstock. PAGE, ELISA or other appropriate techniques are used for the determination of the breakthrough profile of the actual protein (Fig. 22). The curve in Fig. 22 a) and b) shows the ratio of the concentration of product at the outlet of the bed (C) to the concentration of product at the inlet (Co) as more and more sample is applied to the column. When the ratio has risen to 1, the bed is at equilibrium with the inlet stream and no further adsorbtion occurs. Fig. 22 a)
Here the equilibrium bed capacity is reached only after a considerable amount of adsorbate has passed through the bed without being adsorbed.
Fig. 22 b) This shows the ‘‘useful’’ binding capacity of the bed if the loading is terminated when breakthrough of the protein of interest is detected. The actual loading capacity may have to be reduced even further to compensate for the unbound fraction still being present in the void volume of the column when breakthrough is detected. Since the dynamic capacity of a chromatographic adsorbent is a function of the linear flow rate used during sample application, sample loading capacity must be checked at different flow rates to reveal the optimum level that gives highest productivity without excessive leakage of product at the column outlet. 50
Significant increase in flow rate during sample application will always give a decrease in dynamic binding capacity. Even if dynamic capacity will be significantly lower at a higher flow rate it can still be advantageous from a productivity point of view to use a higher flow rate. To process a specific batch size, the process can be run in a cycling mode with a lower sample loading at a higher flow rate. The lower sample loading capacity per cycle may be compensated for by the decrease in cycle time caused by decreased sample volume and increased flow rate. The usefulness of this approach depends on how many cycles have to be run for a specific batch size and on how large the sample application time is in relation to the rest of the cycle time.
Scaleability Scaling-up a chromatographic process is discussed in Chapter 5, ‘‘Experimental Technique’’. Scaling-up is usually not a major concern if scaleability has been considered from the very beginning and built into the process during the method development stage. One important aspect of scaleability common to all types of chromatography is selection of chromatographic media. Important properties of media that should be considered during the initial media screening phase are physical and chemical stability. Physical stability (rigidity) is important in reaching the same high flow rates in the large production column as were achieved in the small column during method development. When column diameter is increased, the support from the column wall is decreased and if the media are not rigid enough bed compression will occur. This compression will increase back pressure and reduce flow rate. Chemical stability of the media is important for applying efficient regeneration, cleaning-in-place and sanitization-in-place protocols. Cleaning and sanitization are a vital part of any chromatographic process.They assure product integrity and maximize media life time. Often harsh chemicals, e.g. 1 M NaOH, are used in such procedures and the chromatographic media have to withstand exposure to such conditions without their chromatographic properties being adversely affected. Recommendations for cleaning, sanitization, and sterilization procedures are given in Chapter 5, ‘‘Experimental Technique’’. Another aspect of media selection and scaleability is bead size. Smaller beads give less peak broadening (higher efficiency) due to decreased diffusion distances (reducing non-equilibrium zone broadening) and decrease of eddy diffusion. However, smaller beads also give increased backpressure and more problems with fouling of the chromatographic bed, especially when crude feed material is applied to the column in the first chromatographic step.
51
As has been discussed earlier, HIC is a high selectivity technique where efficiency usually is of minor importance for the resolution achieved. In consequence, a moderate bead size (e.g. 90 µm) should always be used in initial steps. In intermediate and final steps, smaller beads (e.g. 34 µm) can be used if requirements for resolution cannot be met by selectivity alone. In addition, the type of salt used, salt concentration and gradient volume are all important aspects of scaleability which have to be considered early in the method development stage. Different types of salt, as well as the amount of salt consumed in the process, offer different degrees of environmental and waste disposal problems, and will also affect the overall cost of the process. Such problems can be overcome by minimizing initial salt concentration as discussed earlier in this chapter under ‘‘Choice of HIC media’’ and ‘‘Optimizing a HIC step’’. The effect of excessive gradient volumes on the consumption of salt should also be considered. The waste water treatment cost varies between different countries but according to Swedish conditions, where legislation in this area is very rigorous, the cost for ammonium sulphate (1 M) is approximately 300 USD per 1000 l and for sodium suphate (1 M) approximately 200 USD per 1000 l (December 1992). Sodium chloride (4 M) has to be diluted 50 times before it can be fed into the municipal sewage system.
Regulatory considerations Regulatory considerations are often as critical to the successful development and implementation of a chromatographic process as the purification scheme itself. This is particularly true when producing biologicals to be used as therapeutics. Licensing authorities look upon chromatography media as raw material used in the process. New batches of chromatography media have to be placed in quarantine and can be released for production only when they have been tested and found to be in compliance with established acceptance criteria. Generally, identity tests also have to be performed on each new lot to be brought into production. When selecting media for development of a production process, it is therefore of utmost importance that documentation needed to set up analytical test procedures is available from the media vendor. Part of the testing needed for acceptance of new batches of media, such as particle size distribution, total capacity, flow properties and microbial contamination, is normally provided by the vendor through a Certificate of Analysis. Some of the tests may have to be repeated as part of the acceptance routines at the production site and analytical methods and identity tests provided by the vendor can then be of great help. Besides the documentation needed for setting up acceptance criteria, extensive documentation about the chemical stability of the medium is also needed to define optimal conditions for regeneration, cleaning, sanitization and storage.
52
Another important aspect that has to be investigated during process development is whether any extractable compounds or leakage products from the medium can be potential contaminants in the end product. To be able to test for absence of such compounds, information should be available from the vendor on possible extractable compounds and leakage products and on the kind of methods to use to quantify these compounds in the column eluate. In this connection, it must be made clear that there is no such thing as ‘‘leakage-free’’ chromatography media. Whether leakage in the eluent stream will be detected or not is solely a question of the detection limit of the analytical method used. The leakage levels in product stream that may be accepted by different licensing authorities will be stipulated on a case-by-case basis depending on the application area of the final product, the form of administration, the life time dosage and the toxicity of the leakage product. A regulatory concern specific for HIC is the different additives such as chaotropic substances, organic solvents and detergents that can be used to modulate the separation behaviour. Such additives also may have to be proved absent from the final product.
53
54
5 Experimental Technique Choice of column The material of a chromatographic column should be chosen to prevent denaturation of labile biological substances and minimize non-specific binding to exposed surfaces. The nets or frits used to retain the media should be easily exchangeable to restore column performance whenever contamination and/or blockage in the column occurs. It is also important that all dead volumes, i.e. the volume of the distribution system and tubing, is kept to a minimum to prevent band spreading through dilution or remixing. The pressure specification of the column has to match the back pressure generated in the packed bed when run at optimal flow rate. This is particularly important when using high performance media with small bead sizes. Amersham Pharmacia Biotech has developed a series of standard laboratory chromatography columns (XK columns) suitable for HIC. Further information on the full range of laboratory chromatography columns can be found in the Amersham Pharmacia Biotech catalogue which is available upon request.
Column dimensions As for most adsorptive, high selectivity techniques, HIC is normally carried out in short columns. A typical HIC column is packed to a bed height of 5-15 cm. Once the separation parameters have been determined, scale-up is easily achieved by increasing the column diameter.
Packing the column As with any other chromatographic technique, packing is a very important step in a HIC experiment. A poorly packed column gives rise to poor and uneven flow, zone broadening, and loss of resolution. Packing a HIC column with a modern, highly crosslinked agarose-based gel such as Sepharose Fast Flow is however easier than packing a gel filtration column since the bed height required is much smaller. 55
Packing Sepharose Fast Flow based HIC gels Preparation of the gel The gel is supplied pre-swollen in 20% ethanol. Prepare a slurry by decanting the 20% ethanol solution and replace it with packing solution in a ratio of 50–70% settled gel to 50–30% packing solution. The packing solution should not contain agents which significantly increase the viscosity. Distilled water or a low ionic strength buffer are suitable packing solutions.
Packing 1. Equilibrate all materials to the temperature at which the chromatography will be performed. 2. De-gas the gel slurry to minimize the risk of air bubbles in the packed bed. 3. Eliminate air from the column dead spaces by flushing the end pieces with packing solution (or 20% ethanol). Make sure no air has been trapped under the column net. Close the column outlet. Leave a few centimeters of packing solution remaining in the column. 4. Pour the slurry into the column in one continuous motion. Pouring the slurry down a glass rod held against the wall of the column will minimize the introduction of air bubbles. 5. Immediately fill the remainder of the column with packing solution, mount the column top piece onto the column and connect the column to a pump. 6. Open the bottom outlet of the column and set the pump to run at the desired flow rate. Ideally, Sepharose 6 Fast Flow matrices are packed at a constant pressure of 0.15 MPa (1.5 bar) and Sepharose 4 Fast Flow matrices at a constant pressure of 0.10 MPa (1.0 bar). If the packing equipment does not include a pressure gauge, use a packing flow rate of 400 cm/h (15 cm bed height, 25°C, low viscosity buffer). If the recommended pressure or flow rate cannot be obtained, use the maximum flow rate the pump can deliver. This should also give a reasonably well-packed bed. Note: Do not exceed 70% of the packing flow rate in subsequent chromatographic procedures. 7. Maintain the packing flow rate for 3 bed volumes after a constant bed height is reached. 8. After packing is completed, the level of the packed bed is marked on the column tube before the pump is stopped. Next, the outlet is closed, the pump is stopped and the inlet tubing is disconnected from the pump. Then the adaptor O-ring is slackened and the adaptor is lowered down until it reaches the surface of the packed bed. The O-ring is then tightened sufficiently for the adaptor to slide when pushed. Finally, the adaptor is lowered down until it is 3 mm below the mark on the column tube.
56
Packing Phenyl Sepharose High Performance Preparation of the gel The gel is prepared in the same way as has been described previously for Sepharose Fast Flow based HIC gels.
Packing Sepharose High Performance media are packed by a two-step technique using a low settling flow rate in the first step and then compressing the bed with a high constant back pressure in the second step. STEP 1: Pack with a flow rate of 10–30 cm/h for 20–60 min or until the packed bed has reached a constant height. STEP 2: Lower the adaptor to approximately 1 cm above the surface of the bed. Increase the flow rate until a pressure of 5.0 bar is reached and maintain this pressure for 30–60 minutes. Points 1–5 and point 8 in the packing instruction for Sepharose Fast Flow based HIC gels also apply to the packing of Sepharose High Performance based gels. Note:
There could be some resistance from the packed bed when pushing the adaptor down the last 3 mm, but it is important to fix the adaptor at this level. The packing procedure described above has been developed for the XK 16 and XK 26 columns.
Packing Sepharose CL-4B based HIC gels Sepharose CL-4B based HIC gels are packed using a procedure similar to the method for Sepharose Fast Flow media. The maximum flow rate for a laboratory scale column with an internal diameter of up to 5 cm and a bed height of up to 15 cm is 150 cm/h. Do not exceed a maximum back pressure of 0.04 MPa (0.4 bar).
Use of an adaptor If an adaptor has not been used during column packing it should be fitted as follows: 1.
After the gel has been packed as described above, close the column outlet and remove the top piece from the column. Carefully fill the rest of the column with buffer to form an upward meniscus at the top.
2.
Slacken the adaptor tightening mechanism and insert the adaptor at an angle into the column, ensuring that no air is trapped under the net.
57
3.
Adjust the tightening mechanism to give a sliding seal between the column wall and the O-ring. Screw the adaptor end-piece on to the column.
4.
Make all tubing connections at this stage. There must be a bubble-free liquid connection between the column and the pump and the column and the sample application system.
5.
Slide the plunger slowly down the column so that the air above the net and in the capillary tubing is displaced by eluent. Valves on the inlet side of the column should be turned in all directions during this step to ensure that all air is removed.
6.
Lock the adaptor in position with the tightening mechanism, open the column outlet and start the eluent flow. Pass eluent through the column at the packing flow rate until the gel bed is stable.
7.
Mark the column tube at the level of the packed bed before the pump is stopped.
8.
Close the outlet, stop the pump and disconnect the inlet tubing from the pump.
9.
Slacken the adaptor O-ring and push the adaptor down until it reaches the surface of the packed bed.
10. Tighten the O-ring so the adaptor is still able to slide and push the adaptor down until it is 3–5 mm below the mark on the column tube.
Checking the packed bed Testing the bed is easily done by injecting a test substance on the column and calculating the number of theoretical plates (N) or the height equivalent to a theoretical plate (HETP). Choose a test substance which shows no interaction with the media and which has a low molecular weight to give full access to the interior of the beads. Acetone at a concentration of 1% (v/v) can be used with all kinds of chromatographic media and is easily detected by UV-absorption. Keep the sample volume small to have a narrow zone when the sample enters the top of the column. For optimal results, the sample volume should be - 0.5% of the column volume for a column packed with a medium of approximately 30 µm bead diameter, and - 2% of the column volume for a column packed with a medium of approximately 100 µm bead diameter. Keep the linear flow rate low to reduce that part of the zone spreading which is an effect of non-equilibrium at the front and rear of the zone. For 30 µm media the flow rate should be between 30–60 cm/h and for 100 µm media, 15–30 cm/h. Use the following equations to calculate the number of theoretical plates (N) and HETP.
N = 5.54
58
( ) Ve
W1/2
2
HETP =
L N
where, Ve is the volume eluted from the start of sample application to the peak maximum and W1/2 is the peak width measured as the width of the recorded peak at half of the peak height (see Fig. 23). L is the height of the packed bed. Measurements of Ve and W1/2 can be taken in distance (mm) or volume (ml). The plate count will be the same as the resulting ratio is dimensionless. The unit of measurement should be the most convenient available but both parameters must be expressed in the same units. Fig. 23.
Ve
W1/2
h 1/2 h
As a general rule of thumb, a good HETP value is about two to three times the mean bead diameter of the gel being packed. For a 90 mm particle packing, this means an HETP value of 0.018–0.027 cm. Another useful parameter for testing the packed bed is the Asymmetry factor (Af). b Af = a where, a = 1st half peak width at 10% of peak height b = 2nd half peak width at 10% at peak height (see Fig. 24)
59
Fig. 24.
Ve
h
a b 10% h
Af should be as close as possible to 1. A reasonable Af value for a short column such as a HIC column is 0.80–1.80. (For longer gel filtration columns it will probably fall within 0.70–1.30). An extensive leading edge is usually a sign of the gel being packed too tightly and extensive tailing is usually a sign of the gel being packed too loosely.
Prepacked HIC Media Sepharose High Performance and Superose based HIC media are available in prepacked HiLoad, HR or PC columns. (See Product Guide, Chapter 3). After connecting the column to the chromatography system, column preparation simply consists of washing out the 20% ethanol solution with start buffer and bringing the column to equilibrium. Details of the installation and use of these columns are available in their respective instructions.
60
Sample preparation Sample composition HIC requires a minimum of sample preparation work. Since adsorption is carried out at high salt concentration, it is not necessary to change the buffer of a sample before applying it to a HIC column. The only action to be taken is to add sufficient salt and adjust the pH if necessary to ensure that the component of interest binds. If the salt is added in solid form, some precipitation may occur due to high local salt concentration. This can be avoided if the salt is added as a high concentration stock solution. If chaotropic agents such as guanidine hydrochloride and urea are present in the start material, they have to be removed prior to sample application since their influence on binding to the HIC medium will be opposite to that of the salt used for promoting hydrophobic interaction. Sometimes lipids or other very hydrophobic substances are present in the sample. These may interact very strongly with a HIC column, blocking capacity and being very difficult to remove from the column after the purification cycle. In such cases, using a slightly less hydrophobic column as a pre-column can prove to be very efficient in removing such substances before the sample enters the actual chromatographic column. The pre-column should be chosen to bind the most hydrophobic material and allow the substance of interest to pass through under equilibration conditions.
Sample volume HIC is an adsorption technique and starting conditions are normally chosen so that all important substances are adsorbed at the top of the bed. As such, sample mass applied is of far greater importance than the sample volume. This means that large volumes of dilute solutions, such as cell culture supernatants, can be applied directly to a HIC column without prior concentration. HIC thus serves as a useful means of concentrating a sample, in addition to fractionating it. However, sample volume becomes important when the salt concentration in the sample is lower than in the buffer used for equilibration. This can be the case if the salt concentration in the sample has to be decreased to avoid precipitation as has been discussed in Chapter 4, ‘‘Experimental Design’’. When a large sample volume is applied under such conditions, the weakest interacting substances may start to elute during sample application. This can be avoided by dividing the sample into portions and adding equilibration buffer between each sample application to enhance the hydrophobic interaction and prevent early elution (see Fig. 17).
61
Sample viscosity The viscosity may limit the quantity of sample that can be applied to a column. A high sample viscosity causes instability of the zone and an irregular flow pattern. High viscosity can also create problems with high back pressure, in particular if the medium used has a small bead size, e.g. 10 or 34 µm. A rule of thumb is to use 4 cP (centipoise) as the maximum sample viscosity. This corresponds to a protein concentration of approximately 5% in water. Approximate relative viscosities can be quickly estimated by comparing emptying times from a pipette. If the sample is too viscous due to high solute concentration, it can be diluted with start buffer. High viscosity due to nucleic acid contaminants can be alleviated by precipitation by forming an aggregate with a poly-cationic macromolecule such as polyethyleneimine or protamine sulphate. Nucleic acid viscosity can also be reduced by digestion with endonucleases. Such additives may however be less attractive in an industrial process since they will have to be proven absent from the final product.
Particle content In all forms of chromatography, good resolution and maximum column life time depend on the sample being free from particulate matter. It is important that ‘‘dirty’’ samples are cleaned by filtration or centrifugation before being applied to the column. This requirement is particularly crucial when working with small particle media, e.g. 10 or 34 µm bead size. The filter required for sample preparation depends on the particle size of the HIC matrix which will be used. Samples to be separated on a 90 µm medium can be filtered using a 1 µm filter. For 34 µm and 10 µm media, samples should be filtered through a 0.45 µm filter. When sterile filtration or extra clean samples are required, a 0.22 µm filter is appropriate. Samples should be clear after filtration and free from visible contamination. If turbid solutions are injected onto the column, the column lifetime, resolution and capacity can be reduced. Centrifugation at 10 000 g for 15 minutes can also be used to prepare samples. This is not the ideal method of sample preparation but may be appropriate if samples are of very small volume or adsorb non-specifically to filters.
62
Sample application Sample reservoir (Fig. 25) Samples can be applied by connecting a sample reservoir (e.g. RK or R) to the valves LV-3 and LV-4 or SRV-3 and SRV-4. With this method, the sample is allowed to run onto the column by gravity. Small samples can be applied via a syringe using the valve as a syringe holder.
Sample applicators (Fig. 26) Sample applicators SA-5 and SA-50 are reservoirs which, when used in combination with a suitable valve e.g. SRV-4, allow the sample to be applied via a closed sample loop system using a pump. Sample can be introduced into the sample applicator as a layer below the eluent using a syringe and needle. As well as their large capacity (up to 5 ml for the SA-5 and 45 ml for the SA-50) the sample applicator offers the additional advantage of serving as a bubble trap.
Fig. 26. Sample application using a SA-5 in a sample loop system.
Fig. 25. Sample application using a reservoir.
RK 16/26
LV-3 From pump
XK 16/40
AK 16
63
Sample loops with valves LV-4 or SRV-4 (Fig. 27) This method is convenient for applying small samples. By using the same sample loop, very reproducible sample volumes can be applied, although exact knowledge of the applied volume requires calibration of the capillary tubing loop.
Sample loops or Superloop with valves V-7 or MV-7 (Fig. 28). This method is used for sample application when using high performance columns and other columns in FPLC System or BioPilot™ System. Superloop is a unique sample application device from which a sample of any volume up to the capacity of the Superloop™ (10, 50 or 150 ml) can be applied to a column without tailing. A movable seal separates the sample from the eluent. As eluent is pumped into the Superloop, the sample moves ahead of the seal and onto the column. When nearly all the sample has been applied, eluent flows round the seal to wash the remainder of the sample quantitatively onto the column. Superloop should be used for applying sample volumes larger than 1 ml. Fig. 27. Sample application with a sample loop and two SRV-4 valves.
Fig. 28. Seven-port valves, V-7 and MV-7 have three operating positions which make sample application and changing eluents particularly convenient.
64
Batch separation For an initial capturing step on a HIC medium in process scale, a batch separation procedure can sometimes be worthwhile considering instead of a more traditional column separation procedure. Although the resolution of batch separation is lower than in column chromatography, it may offer advantages in particular cases. When very large sample volumes with low protein concentration have to be processed, the sample application time on a column can be very long and filtration of such a large sample can also be rather difficult to perform. Binding the sample in batch mode will be much quicker and there will be no need to remove particulate matter. A batch procedure can also be an attractive approach if high sample viscosity generates high back pressure in a column procedure or if high back pressure is generated by contaminants such as lipids which may cause severe fouling and clogging of the column. When working with HIC in batch mode starting conditions should be selected in the same way as in column chromatography, i.e. to bind the substance of interest but to prevent as many contaminants as possible from binding. When starting conditions have been selected, the amount of adsorbent needed and the time to reach equilibrium should be determined at small scale in a beaker. Adsorbent is added to the sample and stirred until binding is complete. The gel slurry should not be stirred too rigorously since this will generate fines. Then the gel is allowed to settle and filtered by suction until the adsorbent is damp but not dry. The sedimented adsorbent is washed with buffer to remove non-adsorbed unwanted material. Then elution buffer is added (1–2 times the volume of the sedimented gel) and stirred until desorption is complete, which can take up to 30 minutes or more. Finally, suction is used to filter the buffer containing the desolved product of interest from the adsorbent. The gel can also be packed in a column after the washing step and be eluted stepwise in the same way as during normal column chromatography. Resolution will however be lower for such a combined batch and column procedure compared with a normal column procedure, since the sample is bound uniformly throughout the gel slurry and the subsequent chromatographic bed. At process scale, the complete procedure of adsorption, washing and desorption is most conveniently carried out in a batch application tank equipped with a stirrer and an outlet with a filter at the bottom of the tank.
Cleaning, sanitization and sterilization procedures Cleaning-in-place (CIP) is the removal from the purification system of very tightly bound, precipitated or denatured substances generated in previous purification cycles. In some applications, substances such as lipids or denatured proteins may remain in the column bed instead being eluted by the regeneration procedure. If such contaminants 65
accumulate on the column for a number of purification cycles, they may affect the chromatographic properties of the column. If the fouling is severe, it may also block the column, increasing back pressure and reducing flow rate. A specific CIP protocol should be designed according to the type of contaminants that are known to be present in the feed stream. NaOH is a very efficient cleaning agent that can be used for solubilizing irreversibly precipitated protein and lipid material and in HIC it can be effectively combined with solvent or detergent based cleaning methods. Sanitization is the inactivation of microbial populations. When a packed column is washed with a sanitizing agent, the risk of contaminating the purified product with viable microorganisms is reduced. The most commonly used sanitization method in chromatography today is to wash the column with NaOH. NaOH has a very good sanitizing effect and also has the additional advantage of cleaning the column. Sterilization, which is not synonymous with sanitization, is the destruction or elimination of all forms of microbial life in the system. Suggested protocols for cleaning-in-place (CIP), santization-in-place (SIP) and sterilization that can be applied to the full range of HIC products outlined in Chapter 3, ‘‘Product Guide’’, are summarized in Table 12. The CIP protocols should be used as guidelines to formulate a cleaning protocol specific for the raw material to be applied. The frequency of use will depend on the raw material applied to the column but it is recommended to use a CIP procedure at least every 5 cycles during normal use. Depending on the nature of the contaminants, different protocols may have to be used in combination. If fouling is severe the protocols may have to be further optimized. During CIP the flow direction through the column should be reversed. Table 12. Suggested CIP, SIP and sterilization protocols for HIC media from Amersham Pharmacia Biotech.
Purpose
Procedure
Removal of precipitated proteins
4 bed volumes of 0.5 –1.0 M NaOH at 40 cm/h followed by 2–3 bed volumes of water
Removal of strongly bound hydrophobic proteins, lipoproteins and lipids
4–10 bed volumes of up to 70% ethanol or 30% isopropanol followed by 3–4 bed volumes of water. (Removal of 20% ethanol from Phenyl Sepharose Fast Flow (high sub) is shown in Fig. 29) or 1–2 bed volumes of 0.5% non-ionic detergent (e.g. in 1 M acetic acid) followed by 5 bed volumes of 70% ethanol, to remove the detergent, and 3–4 bed volumes of water
Sanitization
0.5–1.0 M NaOH with a contact time of 30–60 min
Sterilization
autoclave the medium at 120oC for 20 min.
Note: Detergents should be used with care since they work as displacers and may sometimes bind so hard to the gel that it affects the binding capacity during subsequent purification cycles. 66
Fig. 29. Removal of 20% ethanol from Phenyl Sepharose 6 Fast Flow (high sub) in an HR 10/10 Column, bed volume 8 ml; mobile phase H2O; flow rate 1 ml/min. (Work from Amersham Pharmacia Biotech, Uppsala, Sweden).
Ethanol conc. (ppm) 100 000
10 000 1 000
100 10
1 0
2
4
6
8
10
12
Column volumes
Storage of gels and columns Prevention of microbial growth Steps should always be taken to prevent bacterial growth in columns during storage. Microbial growth can seriously interfere with the chromatographic properties of the column and contaminate the purified product with microorganisms and endotoxins or other pyrogenic material. During storage, an antimicrobial agent should always be added to the chromatographic media. Antimicrobial agents may be eluted from columns before chromatographic runs or they may be present in the eluent during chromatography. Antimicrobial agents which interact with sample substances must be avoided if they are to be used in eluents, otherwise any agent which does not interact with the gel may be used. Some of the more commonly used antimicrobial agents are described below.
Antimicrobial agents Sodium hydroxide Sodium hydroxide, 0.01 M, is an effective bacteriostatic agent and is, besides 20% ethanol, the main recommendation for storage of HIC media from Amersham Pharmacia Biotech. At higher concentrations (0.5–1.0 M) it is an effective sanitizer for contaminated columns. For the most frequent contaminants in chromatographic systems, such as gram-negative bacteria, a good bactericidal effect is reached even at such low concentrations as 0.01 M NaOH.
67
NaOH is a widely accepted agent for maintaining chromatographic columns and systems since it not only gives efficient sanitization but also effectively destroys endotoxin (LPS) and solubilizes precipitated and denatured substances that have accumulated on the column. An additional advantage is the lack of toxicity as a contaminant in the end product.
Ethanol 20% Chromatography media from Amersham Pharmacia Biotech are supplied as a suspension containing 20% ethanol. 20% ethanol can also be used as an alternative to NaOH for storing chromatography media under bacteriostatic conditions.
Chlorhexidine ™
Chlorhexidine (e.g. Hibitane ) is a very efficient bacteriostatic agent that inhibits the growth of many bacteria at a concentration of 0.002%. The effect against fungi is less pronounced, but the growth of many types can be inhibited by concentrations between 0.01% and 0.1%. Hibitane is incompatible with only a very few substances. Precipitation may occur on storage of Hibitane in solutions with appreciable concentrations of chloride or sulphate ions.
Phenyl mercuric salts 1 Phenyl mercuric salts (acetate, nitrate, borate) are most efficient as bacteriostatics in weakly alkaline solutions. Concentrations recommended are from 0.001% to 0.01%.
Thimerosal 1 Thimerosal (ethylmercuric thiosalicylate e.g. Merthiolate™) is a bacteriostatic most efficient in weakly acidic solutions. Concentrations recommended are from 0.005% to 0.01%. It is bound to and inactivated by substances containing thiol groups.
Trichlorobutanol Trichlorobutanol (e.g. Chloretone™) is another bacteriostatic showing highest efficiency in weakly acidic solutions. Concentrations recommended are from 0.01% to 0.05%.
1
68
The use of mercury containing antimicrobial agents is on the decline because of their toxicity. When used in a manufacturing process they may have to be proved absent in the end product.
Sodium azide Sodium azide is a very widely used bacteriostatic agent giving a high bacteriostatic effect at a concentration of 0.02%–0.05%. Note: The use of sodium azide is discouraged in many countries since it forms explosive insoluble salts with heavy metals and it is believed to be a mutagen.
Storage of unused media Unused media should be stored in closed containers at a temperature of +4oC to +25oC. Note that it is important that the media are not allowed to freeze as the structure of the beads may be disrupted by ice crystals. This disruption will generate fines.
Storage of used media Used media should be stored at a temperature of +4oC to +8oC in the presence of a suitable bacteriostatic agent, e.g. 0.01 M NaOH or 20% ethanol. Note that it is important that the media are not allowed to freeze as the structure of the beads may be disrupted by ice crystals. This disruption will generate fines.
Storage of packed columns Packed columns should be stored at a temperature of +4oC to +8oC in the presence of a suitable bacterostatic agent, e.g. 0.01 M NaOH or 20% ethanol. For long-term storage, the packed column should be thoroughly cleaned (CIP) before equilibration with the storage solution. Recycling the storage solution through the column or flushing the column once a week with fresh storage solution is recommended to prevent bacterial growth.
69
Process considerations Selecting a column When a chromatographic step is being developed to be a part of a manufacturing process and the time has come for scaling-up, the next crucial step in ensuring a reliable product quality and maximum production economy is the decision about which column to use. Large scale columns offered by Amersham Pharmacia Biotech are described in the ‘‘Process Column Selection Guide’’, which is available upon request. Different demands are put on a column for production compared with one used for the inital R&D and scale-up experiments. Flexibility, which is needed in R&D and scale up, is achieved by using a column with a movable adaptor. In production, consistency in performance and safety of the end product are the main concerns. Here the column packing has to be reproducible, materials of construction have to be well characterised for leakage and the design mechanically stable. A number of criteria have to be considered. These criteria are more dependent on the scale of operation than on the media and are thus very similar in their importance for HIC, ion exchange, gel filtration and affinity chromatography. Their ranking and importance change when moving through a chromatographic process (Fig. 30).
Start material Purification stage
t
Capturing
t Intermediate purification
Demands High flow rate, Large volume, CIP
t t
Polishing
t
Low dead volume, Low flow rate, Resolution, CIP, SIP
t
Pure Product Fig. 30. In the initial capturing, handling large volumes at high flow rates is important. When moving towards the final steps, usually gel filtration, the demand for high resolution and thus low dead volume becomes more and more important. As noted earlier, HIC is mainly used for intermediate purification but can also be applied as an initial capturing step.
70
Aspects of column design Flow distribution system The single most important factor in process column construction is that the packed column will give a low and consistent HETP value of the same order as previously established in the small scale column during method development. To achieve this, the flow distribution system has to be designed to make flow distribution as even as possible at the column inlet and outlet. Technically, the construction can vary but all columns showing an even flow distribution have a radial pressure drop that is negligible in relation to the axial pressure drop at the inlet (Fig. 31). The simplest design to assist radial distribution consists of a course mesh net positioned between the column end piece and the finer mesh net retaining the bed. The course mesh net acts to provide channels for radial distribution. Single or multiple inlet/ /outlet ports are used depending on the column diameter. Depth filters have a disadvantage compared with nets since the relatively large filter surface can become blocked due to adherence of molecules in the feed to the filter material. In continuous production situations, this drawback of depth filters might create serious problems. Fig. 31. Radial and axial back pressure in a column distribution system.
71
Material resistance and durability Wetted components of the column must be constructed from materials having high chemical resistance towards harsh chemicals such as 1 M NaOH, which is frequently used in regular cleaning-in-place (CIP) and sanitization procedures. Very large columns have to be constructed from stainless steel. Occasionally, a normal stainless steel column might not be compatible with some chemicals used. For example, even common NaCl buffers at mildly acidic conditions can cause corrision problems. In this case a fluoroplastic coated stainless steel construction is recommended. When using a stainless steel column in a HIC step, it is exposed to high salt concentrations. The steel type ASTM 316 L (DIN 1.4435, SS 2353), which is normally used in stainless steel constructions, can stand 0.5 M NaCl but only if the pH is kept at pH 6 or above. Sanitary design The possibility of effectively cleaning and sanitizing a packed column also depends on the smoothness of the wetted surfaces. Smooth surfaces hinder bacterial attachment and facilitate cleaning. The total column design, including the absence of threaded fittings, is important in eliminating dead volume in the column. Minimizing dead volumes will minimize trapping and growth of microorganisms and thus facilitate cleaning and sanitization of all wetted parts of the column. Columns constructed from calibrated borosilicate glass allow the use of thin Orings in the adaptor end-plate, which gives a minimum of dead volume in the column. Borosilicate glass will also provide a smooth and durable surface, minimizing bacterial attachment and facilitating the cleaning of the column. A plastic column is usually less expensive, but most plastics do not meet pharmaceutical industry requirements for chemical resistance, hygienic design and in-line cleaning. They might, however, be well suited for scale-up experiments. Pressure vessel safety When working with HIC, the pressure is usually kept low i.e. about 1 bar, but the volumes handled and the size of a process column can mean that it should be regarded as a pressure vessel. The design has then to conform to local regulations to be approved for use. Also, to facilitate a final regulatory approval, the whole column has to be produced under strict documentation routines where materials used and modifications made can be traced. Ergonomics Finally, for easy handling of a process column, it becomes important to have a column which is constructed in a stable way, which is easy to pack and to keep clean. If the column has an adaptor, it should be easy to move and lock in its new position. Valves should be easy to reach and the whole column should be possible to take apart for cleaning. Keeping all the above factors in mind will facilitate choosing the correct column for the specified need. 72
Packing large scale columns Column configuration Process columns with a moveable adaptor are essentially packed in the same way as laboratory columns with adaptors. In essence, this means that the gel slurry is compressed by a flowing liquid until the bed height has stabilized, at which point the flow is stopped and the adaptor is lowered onto the gel surface and secured in place. Large scale columns are, however, frequently supplied with fixed end pieces. This calls for a different packing technique. Some kind of extension tube has to be fitted on top of the column as a reservoir for the gel slurry. When the bed has been packed and settled at the join between the extension tube and the column, the extension tube is removed and the top column lid is secured in place.
Pressurized systems (pressure packing) Columns with moveable adaptors are packed in a pressurized system with a constant packing flow rate or a constant packing pressure. If constant flow rate is used, the pressure drop over the column will increase during packing as result of increased flow restriction from the packed bed. If constant pressure is used, the flow rate will be high in the beginning but decrease during packing for the same reason as above. Moderately sized columns with fixed end pieces can also be packed in a pressurized system. For such columns, two column tubes are fitted together to store the complete slurry volume before packing is started. When the bed has settled at the join between the two tubes, the upper tube is emptied of liquid by suction through the packed bed. At this stage the system is no longer pressurized but the bed is still kept compressed in the lower column tube by the liquid flow during the suction phase. When the upper tube is emptied the flow is stopped, the tube is quickly removed and the top lid is put in place and secured.
Non-pressurized systems (suction packing) Columns with fixed end pieces, such as the BioProcess Stainless Steel Columns from Amersham Pharmacia Biotech, are packed in a non-pressurized system by sucking packing solution through the column. Pressure packing of such columns would require very heavy packing tubes, which would be impractical to work with. In suction packing, the pump is connected to the column outlet and the excess packing solution in the gel slurry is pumped out at a predetermined flow rate. Immediately after the last part of the slurry has been sucked into the packed bed (when the surface starts to be dry), the flow is stopped, the packing device is quickly removed and the column lid is put in place and secured. Very simple packing devices can be used to store the gel slurry since the system is not pressurized. More detailed packing instructions are to be found in the instruction manual accompanying each column.
73
Packing flow rate Irrespective of the packing technique, the most important parameter for an optimally packed bed is the linear packing flow rate (velocity of flow through the column). In general, the higher the flow rate the better the performance of the packed bed, as long as the flow rate does not cause extensive bed compression, which can lead to channelling and an irregular flow pattern through the bed. Modern rigid media such as Sepharose Fast Flow and Sepharose High Performance withstand very high flow rates. In such cases, the pressure specification of the column rather than the separation medium, often sets the upper flow rate limit. However, with less rigidly cross-linked media such as Sepharose and Sepharose CL, the bed is often compressed and maximum flow rate reached, before the pressure limit of the column is reached. Irrespective of gel type, bed compression is more pronounced in large diameter columns since support given by the inner column wall to the gel bed is reduced. Since the optimum packing flow rate and pressure is dependent on gel type, gel batch, gel quantity, temperature, packing solution and equipment, it must be determined empirically for each individual system by establishing a pressure/flow curve for each specific column/gel set up. A pressure/flow curve is established in the following way: 1) Prepare the column in exactly the same way as for column packing 2) Pump buffer through the column at a low flow rate (e.g. 30% of the expected maximum flow rate). Record the flow rate and pressure when the bed is packed and the pressure has stabilized. 3) Slowly increase the flow rate in small steps and record the flow rate and pressure at each step after the pressure has stabilized. 4) Continue to increase the flow rate like this until the flow rate levels off at a plateau, indicating bed compression, or until the pressure reaches the pressure specification of the column. 5) Plot pressure against flow rate as indicated in Fig. 32. The optimal packing flow rate is the maximum flow rate, i.e. where the flow rate starts levelling off on the pressure/ flow curve. If no plateau is reached, use the flow rate at the maximum pressure specification of the column. Alternatively, use a constant pressure packing technique where the packing pressure is the pressure where flow rate starts levelling off, or if no plateau is reached, the specified maximum pressure for the column used. The packed column should not be run at more than 70% of the final flow rate reached during packing. This precaution prevents further bed compression when, for instance, a viscous sample is applied.
74
Linear flow rate cm/h
Fig. 32. Establishing a pressure/flow rate curve.
700
Packing flow rate
600 500 400 300 200 100 1
2
3 Pressure bar
Packing buffer The composition of the packing buffer can sometimes be critical for the performance of the packed bed. A packing solution frequently used is 0.1 M NaCl. For HIC media supplied by Amersham Pharmacia Biotech, water is equally effective.
Packing Sepharose CL and Sepharose Fast Flow based media The first task when packing Sepharose CL and Sepharose Fast Flow based media in large scale columns is to determine the optimal packing flow rate (constant flow rate packing) or optimal packing pressure (constant pressure packing), by establishing a pressure/flow curve as outlined opposite. For constant flow rate packing, the bed is packed by pumping packing solution through the column at the predetermined flow rate. The flow rate is checked at regular intervals and adjusted continuously since the increase in flow resistance from the packed bed will continuously reduce flow rate. The back pressure should be recorded with a manometer connected between the pump and the column to assure that maximum pressure for the gel or the column is not exceeded. For a constant pressure packing technique, the packing solution is applied through the column at a constant pressure by using a pump and a manometer, or by using a pressure vessel, to deliver the flow through the column continuously at a preset back pressure. The packing technique will differ in detail depending on which type of column is used. Detailed packing instructions for each type of column are included in the instruction manuals for large scale columns supplied by Amersham Pharmacia Biotech. Packing Phenyl Sepharose High Performance in
75
BioProcess Glass Columns Packing Phenyl Sepharose High Performance in BioProcess Glass Columns (BPG™) is performed with a two-step technique similar to that recommended for laboratory columns. The gel is packed with a low flow rate in the first step and then compressed with a high constant back pressure in the second step. * STEP 1: Pack the gel at a constant flow rate of 20 cm/h for 60 minutes. STEP 2: Lower the adaptor to approximately 1 cm above the surface of the bed. Increase the flow rate until a pressure of 3.0 bar is reached. Maintain this pressure for 30 minutes. This packing technique has been developed to give maximum column efficiency and bed stability when packing Phenyl Sepharose High Performance in BPG Columns.
Scale-up Once scaleability has been considered from start and built into the process during the method development work (see Chapter 4, ‘‘Experimental design’’), scaling up a chromatographic step is usually a straight-forward process. One important aspect of scaleability is the physical stability of the chromatography media. Scaling up to a larger diameter column means that most of the bed support from the friction against the column wall is lost. This loss can give increased bed compression and poorer flow/pressure characteristics. Using a highly cross-linked, rigid matrix during the method design work will ensure that the large scale column can be run at the same linear flow rate as the small scale column, without problems with increased back-pressure. Another important aspect of scaleability is the bead size. If a small bead (e.g. 10 µm) has been used during method development work in small scale, it is usually necessary to switch to a larger bead when scaling up to optimize throughput and reduce operating costs. In such cases, some re-optimization work has to be performed at the laboratory scale before the process can be scaled up.
* The recommendation for the first step is for BPG 100 and BPG 200 columns. For BPG 300 columns, a constant pressure of 0.5 bar should be applied for 30 minutes.
76
Some general guide-lines for scaling-up are outlined in Table 13. Table 13. Scale up guidelines. Maintain
Increase
Check system factors
Bed height Linear flow rate Sample concentration Gradient volume/bed volume
Sample load Volumetric flow rate Column diameter
Distribution system Wall effects Extra column zone spreading
Increasing the bed volume by increasing the column diameter and increasing volumetric flow and sample load accordingly, will ensure the same cycle time as in the laboratory scale method development. The column bed height, linear flow rate, sample concentration and ratio of sample to gel, all optimized on a laboratory scale, will be kept the same. If a gradient is used for elution, the ratio of gradient volume to bed volume will remain constant and, therefore, the time required for the gradient to develop and the effect on resolution, will remain the same on the larger column. The same principle is applied for the volume of each step in a step gradient. Different system factors may affect performance after scale up. If the large scale column has a less efficient flow distribution system, peak broadening may occur due to increased axial dispersion in the bed and extra zone spreading in the end pieces. This will cause extra dilution of the product fraction or even loss of resolution. Depending on the rigidity of the media, the loss of wall support in a large scale column will have a smaller or greater impact on bed compression, with accompanying deterioration of the flow/pressure properties of the packed bed. The effect of bed compression can be checked by running a pressure/flow rate curve such as outlined under ‘‘Packing large scale columns’’. Zone spreading can also be caused by non-column factors such as increased internal volumes of pumps, valves and monitoring cells and different lengths and diameters of pipes or tubing. If all the above aspects of scaling up are taken into consideration, chromatographic variability is normally not a big issue when scaling-up. Non-chromatographic factors may have a more significant effect on performance. These factors include: changes in sample composition and concentration that often occur as the fermentation scale increases, precipitation in the biological feedstock due to longer holding times when large volumes must be handled, non-reproducibility of the buffer quality due to inadequate equipment for consistently preparing large quantities of buffer solutions, and microbial growth in feed-stock or buffers due to increased handling and longer holding times. The effects of these kinds of variabilities should be checked by challenging the chromatographic process during method development by running it under ‘‘worstcase’’ situations.
77
78
6 Applications Preparative and analytical HIC applications in the research laboratory HIC is widely used in protein purification in the research laboratory as a complement to other techniques that separate according to other parameters such as charge (ion exchange chromatography), size (gel filtration) or biospecific recognition (affinity chromatography). The outcome of a protein purification procedure is obviously dependent on the choice of separation equipment and techniques. The order in which the different techniques are combined is also of great importance. This chapter, emphasises different possibilities to combine HIC with other separation techniques in laboratory scale protein purification schemes. Analytical separations by HIC are also discussed.
HIC in combination with ammonium sulphate precipitation When ammonium sulphate precipitation has been used early in a protein purification procedure to precipitate out contaminants, HIC is ideal as the next step. The protein of interest is present in the supernatant at a high ammonium sulphate concentration, and the sample can thus be directly applied to the HIC column. Purification occurs concomitant with a reduction in volume.
Crude purification of human autotaxin HIC was used for initial purification of autotaxin, a human 125K protein which stimulates tumour cell motility (49). The supernatant from ammonium sulphate precipitation of concentrated cell culture medium was applied directly to a Phenyl Sepharose CL-4B column (Fig. 33). Elution was achieved with a double linear gradient with decreasing ammonium sulphate and increasing ethylene glycol. Autotaxin was then further purified to homogeneity using affinity chromatography, gel filtration and anion exchange chromatography.
79
Column: Sample:
Buffer A: Buffer B: Flow rate: Detection:
Fig. 33. HIC purification of human autotaxin from the supernatant from ammonium sulphate precipitation of conditioned melanoma cell culture medium (reproduced with permission, from ref. 49).
Phenyl Sepharose CL-4B, 200 ml Supernatant from ammonium sulphate precipitation (1.2 M) of concentrated conditioned media (corresponding to 200 l of conditioned media) from A2058 melanoma cells 50 mM Tris, pH 7.5, 5% (v/v) methanol, 1.2 M ammonium sulphate 50 mM Tris, pH 7.5, 5% (v/v) methanol, 50% (v/v) ethylene glycol 1 ml/min A280 (solid curve) and motility (circles).
Chemotaxis was quantitated with a 2202 Ultroscan laser densitometer.
30
2.0
100
50
15 10
OD280 (—)
75 20
Gradient (% B) (–––)
Motility (density units)
25
25 5 0 0
200
400
600
800
0 1000 1200 Time (min)
0
HIC in combination with ion exchange chromatography HIC is often an excellent choice subsequent to ion exchange in a protein purification procedure. Both techniques have an extremely broad applicability, and are complementary (i.e. separation according to hydrophobicity and charge, respectively). Furthermore, material eluted with a salt gradient in an ion exchange separation requires a minimum of sample treatment before application to a HIC column. Usually sample treatment is limited to the addition of salt.
Purification of recombinant HIV reverse transcriptase Recombinant HIV reverse transcriptase, expressed as a 66K/51K heterodimer in E. coli, was purified using a multi-step procedure involving ion exchange, ammonium sulphate precipitation and HIC (50). The second chromatography step was anion exchange with DEAE Sepharose CL-6B. Pooled active material was diluted with 3 M 80
Fig. 34. Purification of recombinant HIV reverse transcriptase, expressed in E. coli, using HIC (reproduced with permission, from ref. 50).
Column: Sample:
HiLoad 16/10 Phenyl Sepharose High Performance 400 ml (10 mg protein), obtained from ion exchange on DEAE Sepharose CL-6B, diluted to 600 ml with 3 M ammonium sulphate. Buffer A: 10 mM Tris-HCl, pH 8.0, 1 M ammonium sulphate, 10% glycerol, 1 mM DTT Buffer B: 10 mM Tris-HCl, pH 8.0, 10% glycerol, 1 mM DTT Flow rate: 3 ml/min Detection: A280 (solid line) and reverse transcriptase activity (black area). The broken line is the programmed gradient. A280 nm 0.5
217 650
233 700
250 750
267 (min) 800 (ml)
ammonium sulphate and applied to a column containing Phenyl Sepharose High Performance (Fig. 34). The HIC step was used both as a purification step and a concentration step and it reduced the volume 15 fold (from 600 to 40 ml). The final purification step was anion exchange with FPLC™ using a Mono Q™ column.
Purification of mammalian transcription factors Transcription factors are present at extremely low levels in mammalian cell nuclei. A purification scheme was developed for µg amounts of six different transcription factors from an extract of 1012 HeLa cells (51). The complementary selectivities of HIC and ion exchange are well illustrated in this scheme. Transcription factors IIF and IIH co-purified in anion exchange (two different DEAE columns) and cation exchange (Phosphocellulose and Mono™ S), but were excellently separated on Phenyl Superose HR 10/10 (Fig. 35) with FPLC System. Further purification of the factors involved other chromatography steps. Final micropurification of transcription factor IIE was done on Phenyl Superose™ PC 1.6/5 with SMART™ System. 81
Column: Sample:
Phenyl Superose HR 10/10 HeLa cell extract purified on phosphocellulose, 2 x DEAE columns, Mono S HR 10/10 Buffer A: 20 mM Tris-HCl, pH 7.9, 0.1 mM EDTA, 20% glycerol, 10 mM b-mercaptoethanol, 0.2 mM phenylmethylsulfonyl fluoride, 1.4 M ammonium sulphate Buffer B: A without ammonium sulphate Detection: Triangles represent transcription factor IIF activity, and open circles represent transcription factor IIH activity in specific transcription
Fig. 35. HIC separation of mammalian transcription factors IIH and IIF (reproduced with permission, from ref. 51).
0.5
1,0
0.3
0,75
0.2
0,50
0,25
0.1
5
10 15 20 25 30 35 40 45 50 55 60 Fraction Number
Ammonium Sulphate – – –
1.0
0.4
α
1.5
Protein (mg/ml)
(α - 32P) -UMP Incorporated (pmol)
11 2.0
0
Micropurification of a GTPase activating protein Final micropurification of a GTPase activating protein, GAP-3, from bovine brain was done with micropreparative columns for HIC and anion exchange chromatography (52). Prior purification steps were anion exchange chromatography, dye affinity chromatography, ammonium sulphate precipitation, gel filtration, hydroxyapatite, HIC and anion exchange. GAP-3 containing fractions from the latter step (Mono Q HR 5/5) were applied to the Phenyl Superose PC 1.6/5 column (Fig. 36). Active material from HIC was then applied to a second micropreparative column (Mono Q PC 1.6/5), and subjected to N-terminal sequence analysis. The overall recovery was 50 µg GAP-3 from 1.6 kg of brain tissue, which corresponds to a purification factor of approximately 18000.
HIC in combination with gel filtration A major advantage with adsorption chromatography is the possibility to achieve a decrease in sample volume concomitant with an increase in purity. In a purification scheme, HIC and other adsorption chromatography techniques are therefore frequently used prior to gel filtration, in which sample volume is limited.
82
Phenyl Superose PC 1.6/5 Partly purified GAP-3 from bovine brain homogenate, active material from Mono Q HR 5/5 Buffer A: 25 mM Tris-HCl, pH 7.5, 1 mM DTT, 1 mM EDTA, 1.7 M ammonium sulphate Buffer B: 25 mM Tris-HCl, pH 7.5, 1 mM DTT, 1 mM EDTA Flow rate: 50 µl/min Detection: A280 (upper curve) and GAP-3 activity (32P-GTP hydrolysis; lower curve)
Absorbance (280 nm)
Column: Sample:
0.1 AU
POOL
CPM ( X 105 )
8
6
4
2
0
10
20
30
40
50
Retention time (min)
Fig. 36. Micropurification of GAP-3 (reproduced with permission, from ref. 52).
Human pituitary prolactin was purified on a Phenyl Sepharose CL-4B column (53) (Fig. 37). Elution was achieved stepwise, with 50% ethylene glycol. The sample volume was reduced in HIC from 300 ml to 45 ml, and the recovery of activity was 95%. The sample was applied to a gel filtration column (Sephadex G-100) for further purification.
HIC as a ‘‘single step’’ purification technique In general, ‘‘single step’’ purification of a protein to homogeneity from a complex biological sample requires the use of highly specific affinity techniques. If general techniques are chosen, e.g. HIC, ion exchange and gel filtration, they usually have to be combined to obtain a homogeneous product. In some instances, however, a single chromatography step with a general technique may be sufficient to give a pure product.
83
0.002 M glycine-NaOH, pH 9.8/50% ethylene-glycol
A280
10
0.2 M glycine-NaOH, pH 9.8
0.02 M glycine-NaOH, pH 9.8
20
15
Fig. 37. Chromatography on Phenyl Sepharose CL-4B of a prolactin preparation. The hatched area represents the prolactincontaining fractions. (reproduced with permission, from ref. 53.)
5
0
20
40
60
80
100
120
Fraction number
Column: Sample:
Alkyl Superose HR 5/5 100 µl mouse ascites containing monoclonal IgG1 (a) or IgG2a (b) + 100 µl buffer A, centrifuged and filtered Buffer A: 0.1 M phosphate, pH 7.0, 2.0 M amminium sulphate Buffer B: 0.1 M phosphate, pH 7.0 Detection: A280. Proteins were identified by SDS-PAGE with PhastSystem. The programmed gradient (FPLC System) is also shown. A280 nm
A280 nm
a)
b)
0.5
0.5
lgG1 lgG2a
Alb Trans
0
84
20
Alb Trans
40 60 Time (min)
0
20
40 60 Time (min)
Fig. 38. ‘‘Single step’’ HIC purification of monoclonal antibodies from ascites. (Work by Amersham Pharmacia Biotech).
Phenyl Sepharose High Performance, 5x10 mm. a) 60 µg methylamine treated, inactive (‘‘fast’’) a2-macroglobulin b) 60 µg active (‘‘slow’’) a2-macroglobulin c) a + b Buffer A: 20 mM sodium phosphate, pH 7.2, 1.0 M sodium sulphate Buffer B: 0.25 mM sodium phosphate, pH 7.2 Flow rate: 0.5 ml/min Detection: A280. The programmed gradient (FPLC System) is also shown. ”Slow” and ”fast” a2-macroglobulins were identified by native PAGE with PhastSystem. A280
a)
0.04 AU
‘‘fast’’
Column: Samples:
0
[Na2SO4]
A280
1.0 M
0.04 AU
[Na2SO4] 1.0 M
c)
0.0 0
20 min
0
10 ml
‘‘slow’’
b) 0.04 AU
1.0 M
0.0 0 0
20 min 10 ml
0
0.0 0 0
20 min 10 ml
Fig. 39. Separation of conformational isomers of a2-macroglobulin using HIC. (Work by Amersham Pharmacia Biotech).
Small scale purification of monoclonal antibodies Mouse monoclonal antibodies were purified from ascites on a milligram scale. IgG was the main protein in the sample and a single chromatography step with Alkyl Superose HR 5/5 was sufficient to obtain homogeneous IgG (Fig. 38). Purity was checked by SDS-PAGE and silver staining. Both IgG antibodies were well separated from the main contaminants albumin and transferrin. The two antibodies were eluted at clearly different positions (Fig. 38 a, b) in the gradient however, indicating that separation conditions may have to be modified for different antibodies. 85
20
10
1
5
0
0 0
5
10
1
Nonidet P - 40 (mM)
Protein (mg/ml)
15
2
Lauryl Maltoside (mM)
2
Fig. 40. Exchange of lauryl maltoside for protein-bound Nonidet P-40. (reproduced with permission, from ref. 54).
0
15
Fraction Number
Analysis of conformational changes with HIC A conformational change in a protein leads to a change in physico-chemical surface properties, e.g. hydrophobicity, of the molecule. Such changes can be exploited using HIC, offering exciting possibilities both in analytical and preparative applications.
Separation of conformational isomers of a2-macroglobulin with HIC
a2-macroglobulin, a tetrameric 720K plasma protein, undergoes a major, irreversible conformational change (without peptide bond cleavage) on treatment with methylamine. The two conformational isomers are referred to as ‘‘slow’’ and ‘‘fast’’ a2-macroglobulin, respectively, referring to their different mobilities in native PAGE. The two conformational isomers were clearly separated by HIC, using Phenyl Sepharose High Performance (Fig. 39). Ammonium sulphate was avoided, since ammonia mimicks the action of methylamine on the protein. Other HIC application areas in the research laboratory HIC using Phenyl Sepharose CL-4B has been used for exchange of protein-bound detergent (54) (Fig. 40). Octyl Sepharose CL-4B has been used for the separation of different forms of dermatan sulphate proteoglycans (55). HIC of nucleic acids, viruses and cells has also been described (17).
86
Preparative, large scale applications. When chromatographic techniques are developed to be part of a manufacturing process for a pharmaceutical or a diagnostic for commercial application, they have to comply with special requirements. As well as meeting demands for productivity and overall economy, the processes also have to fulfil different regulatory requirements regarding final product safety. Regulatory authority requirements are based on the concern that infectious, pyrogenic, immunogenic or tumourigenic agents may be present in the end product. This section shows some large scale applications that demonstrate how HIC can be integrated into a logical sequence of chromatographic steps intended for a large scale downstream process.
Purification of a monoclonal antibody for clinical studies of passive immunotherapy of HIV-1. In Sweden, scientists at the National Bacteriological Laboratory (SBL), Department of Virology, in collaboration with Amersham Pharmacia Biotech , have succeeded in developing a purification procedure for large scale production of a monoclonal IgG1 (anti-gp120) intended for intravenous use for clinical studies on AIDS (56). The specification for the purification was that the Mab could be used for in vivo therapy, which required steps to reduce endotoxins and DNA. Phenyl Sepharose High Performance was selected for the initial step in a three step procedure that gave a product purity of 99% and an overall yield of 61% (Fig. 41). Phenyl Sepharose High Performance is known to be a good choice for initial purification of monoclonal antibodies. In this case it was compared with an alternative technique consisting of desalting on Sephadex G-25 Super Fine followed by ion exchange chromatography on S Sepharose High Performance. The HIC technique gave superior resolution - no albumin was detected by SDS-PAGE. The second step on S Sepharose High Performance was included to reduce endotoxins by binding the Mab while allowing the endotoxins to pass unretarded through the column. In fact, it was the final gel filtration step on Superdex™ 200 prep grade that turned out to be the most efficient step for reduction of endotoxins in this case, but the cation step served as a concentration step, meeting the specification for protein concentration in the final product. The microbiological contamination as well as the DNA and endotoxin levels were all judged to be within the specifications for parenteral use in clinical trials. All chromatographic runs were performed on BioPilot System. The start material was hybridoma supernatant and the concentration of mouse IgG1(anti-gp 120), was 0.61 mg/ml. The method development work was performed on Pharmacia XK columns. Loading capacity for Phenyl Sepharose High Performance was tested by analysing the flow through fractions during sample application by SDS-PAGE and ELISA. 87
Start material 10 l supernatant Acusyst hollow fibre 10 l 0.61 mg/ml MAb
Fig. 41. Purification scheme for a large scale purification of mouse IgG1 anti-gp120. (Two cycles were run, i.e. a total of 20 l of Mab supernatant).
t Step 1
Phenyl Sepharose High Performance BPG 100/500 10 cm bed height
Process time: 3h
t
1.4 l
t
Dilution pH-adjustment pH 5.0 4.5 mS/cm 3.9 l
t
Step 2
S Sepharose High Performance BioPilot Column 60/100 10 cm bed height
Process time: 1.4 h
t
0.4 l
t Step 3
Superdex 200 prep grade BPG 100/950 60 cm bed height
Process time: 2h
t
0.72 l
Product: 3.7 g SDS page purity: 99% Overall yield: 61%
Dynamic binding capacity was 9.1 mg/ml separation medium at a flow rate of 90 cm/h. As a safety measure only 75% of maximum loading was used. The Mab was eluted with a single step of low salt buffer. The chromatogram and SDS-PAGE showed good resolution and high concentration of the Mab (Fig. 42 a). For large scale purification, 20 litres of supernatant were divided into 2 lots each containing an equal quantity of Mab. BioProcess Glass Columns were used for the first step on Phenyl Sepharose High Performance and for the final gel filtration step. A BioPilot Column was used for the intermediate step on S Sepharose High Performance. Both resolution and yield (Fig. 42b) were equally good as the small scale run.
88
Column: Gel: Sample: Sample volume: Sample load: Flow rate: Buffer A:
XK 16/20 Phenyl Sepharose High Performance, 10 cm bed height. Hybridoma cell culture supernatant; mouse IgG1 anti-gp120.
222 ml 6.8 mg Mab/ml gel 90 cm/h (3 ml/min) 10 mM potassium phosphate pH 7.0 + 0.5 M ammonium sulphate Buffer B: 10 mM potassium phosphate pH 7.0 Productivity: 2.32 mg Mab/ml gel . h
Column: Gel: Sample: Sample volume: Sample load: Flow rate: Buffer A:
2 x 10 l 7.77 mg Mab/ml gel 90 cm/ h (7.1 l/h) 10 mM potassium phosphate pH 7.0 + 0.5 M ammonium sulphate Buffer B: 10 mM potassium phosphate pH 7.0 Productivity: 3.34 mg Mab/ml gel . h
%
% a)
100
b)
50
50
0
0
0
BPG 100/500 Phenyl Sepharose High Performance, 10 cm bed height Hybridoma cell culture supernatant; mouse IgG1 anti-gp120.
50
100
Time (min)
0
50
100
150 Time (min)
Fig. 42. Laboratory and production scale purification of mouse IgG1 anti-gp120 on Phenyl Sepharose High Performance (Work from Amersham Pharmacia Biotech, Uppsala, Sweden).
Purification of recombinant human Epidermal Growth Factor (h-EGF) from yeast. A chromatographic downstream process has been developed for the purification of human Epidermal Growth Factor (h-EGF) expressed as an extracellular product by Saccharomyces cerevisiae (57). Phenyl Sepharose 6 Fast Flow (high sub) was selected for an initial capture step. This was followed by an intermediate anion exchange step on Q Sepharose High Performance and a final polishing gel filtration step on Superdex 75 prep grade. This three step procedure gave a product purity of 99% as determined by RPCHPLC, and an overall yield of 73% (Fig. 43). 89
Fig. 43. Purification scheme for large scale purification of h-EGF from yeast cell culture supernatant.
Process description Yeast cell culture supernatant
t
t
Centrifugation or 5 mm filtration Ammonium sulphate to 0.5 M
t
Phenyl Sepharose 6 Fast Flow (high sub)
t
Dilution 1/10 0.02 M Tris pH 7.6
t
Q Sepharose High Performance
t
Superdex 75 p.g.
HIC media scouting for initial step Media characteristics for EGF purification Phenyl Sepharose High Performance • Very high selectivity • Very high binding capacity • Higher backpressure than for Fast Flow media
Phenyl Sepharose 6 Fast Flow (low sub) • EGF comes in wash with binding buffer • Low selectivity for EGF
Phenyl Sepharose 6 Fast Flow (high sub) • Very high selectivity • High binding capacity • High throughput
Butyl Sepharose 4 Fast Flow • Was not possible to elute with low salt buffer • Binds too hard Fig. 44. HIC media screening experiments for the initial capture step. (Work from Amersham Pharmacia Biotech, Uppsala, Sweden).
90
Initial media screening experiments for the capture step were performed on four different HIC media. Phenyl Sepharose 6 Fast Flow (high sub) was selected due to its high selectivity for EGF, high binding capacity and high throughput (Fig. 44). A cation exchanger (S Sepharose Fast Flow) was also evaluated during this screening phase but was found to have lower selectivity for EGF than Phenyl Sepharose 6 Fast Flow (high sub). A high resolution anion exchanger, Q Sepharose High Performance, was selected for intermediate purification in order to reach a high degree of purity in the second step (> 96%). To achieve a high final purity by separating polymers and unwanted buffer salts from the EGF product, gel filtration on Superdex 75 prep grade was selected for final polishing. The start material was clarified cell culture supernatant supplied by Chiron-Cetus Corp., Emeryville, USA. Concentration of EGF in the start material was 0.018 mg/ml and the overall protein content was 63 mg/ml. The small scale development work was performed on BioPilot System and XK columns. The product was eluted with a single step procedure which gave adequate purification and high product concentration (Fig. 45). The large scale purification work was performed on BioProcess System and BioProcess Glass Columns. For the capture step, 80 l of feed material was applied to
Column: Gel:
Sample: Sample volume: Sample load: Flow rate: Buffer A:
Buffer B:
XK 16/20 Phenyl Sepharose 6 Fast Flow (high sub), 10 cm bed height Yeast supernatant. Ammonium sulphate added to 0.5 M
A280 nm Buffer A
3.00
450 ml 0.41 mg h-EGF/ml media 300 cm/h; 10 ml/min (loading) 60 cm/h; 2 ml/min (elution) 20 mM sodium phosphate pH 7.0 + 0.5 M ammonium sulphate 20 mM sodium phosphate pH 7.0
Purification time: 1.5 h
2.00
1.00
Buffer B
0.00 0
50
Time (min)
Fig. 45. Laboratory scale purification of h-EGF on Phenyl Sepharose 6 Fast Flow (high sub). (Work from Amersham Pharmacia Biotech, Uppsala, Sweden).
91
Column: Gel:
BPG 300/500 Phenyl Sepharose 6 Fast Flow (high sub), 10 cm bed height Sample: Yeast supernatant. Ammonium sulphate added to 0.5 M Sample volume: 80 l Sample load: 0.36 mg h-EGF/ml media Flow rate: 300 cm/h; 212 l/h (loading) 60 cm/h; 42 l/h (elution) Buffer A: 20 mM sodium phosphate pH 7.0 + 0.5 M ammonium sulphate Buffer B: 20 mM sodium phosphate pH 7.0 Purification time: 1.5 h A280
Fig 46. Production scale purification of h-EGF on Phenyl Sepharose 6 Fast Flow (high sub). (Work from Amersham Pharmacia Biotech, Uppsala, Sweden).
ms/cm 100
3.0
2.0 50 1.0
0.0 50
100
Volume (liter)
a BPG 300/500 column with a bed volume of 7.1 l (Fig. 46). No dilution or recovery losses were seen when scaling up from XK columns on BioPilot System to BPG columns on BioProcess System.
Purification of a monoclonal antibody for in vitro diagnostic use. A single step purification technique for the large scale purification of a monoclonal antibody using HIC has been developed (58). Purification was performed on Phenyl Sepharose High Performance to a product purity of > 95% and a yield of 78%. If a gel filtration polishing step on Superdex 200 prep grade was added on to the HIC step, a final purity of >99% was achieved (Fig. 47). The start material, from a hollow fibre bioreactor, was mouse hybridoma cell culture supernatant containing monoclonal IgG1 anti-IgE. Mab concentration, determined by nephelometry, was 0.63 mg/ml.
92
Fig. 47. Purification scheme for the large scale purification of mouse IgG1, anti-IgE.
Process design Cell culture
t
Filtration Through filter paper Addition of (NH4)2SO4 to optimized concentration
t
Hydrophobic Interaction Chromatography Phenyl Sepharose High Performance
t
Concentration Membrane concentration or Ion exchange chromatography
t
Gel Filtration Chromatography Superdex 200 prep grade
The small scale development work was performed on a HiLoad 16/10 Phenyl Sepharose High Performance column (Fig. 48). The Mab bound very strongly to the gel while most of the fetal calf serum proteins passed through unretarded. Different salt concentrations in the start buffer were tested and 0.5 M ammonium sulphate was selected since this showed the highest binding selectivity for the Mab. At higher salt concentrations, the IgG fraction was slightly contaminated with serum albumin. Dynamic binding capacity was determined to be 4.5 mg Mab/ml gel.
Column:
HiLoad 16/10 Phenyl Sepharose High Performance, 10 cm bed height Sample: Hybridoma cell culture supernatant; mouse IgG1 anti-IgE. Ammonium sulphate added to 0.5 M. Sample volume: 130 ml Sample load: 4.5 mg Mab/ml gel Flow rate: 100 cm/h (3.3 ml/min) Buffer A: 20 mM potassium phosphate, pH 7.0 + 0.5 M ammonium sulphate Buffer B: 20 mM potassium phosphate, pH 7.0 Gradient: 0-100% B; 10 column volumes
A280 nm .40 .30 .20 .10 .00 50
50
100
150 Time (min)
Fig. 48. Laboratory scale purification of mouse IgG1, anti-IgE, on Phenyl Sepharose High Performance. (Work from Amersham Pharmacia Biotech, Uppsala, Sweden).
93
Column: Gel:
BioPilot Column 35/100 Phenyl Sepharose High Performance, 10 cm bed height Sample: Hybridoma cell culture supernatant; mouse IgG1, anti-IgE. Ammonium sulphate added to 0.5 M. Sample volume: 735 ml Sample load: 4.5 mg Mab/ml gel Flow rate: 100 cm/h (16.7 ml/min) Buffer A: 20 mM potassium phosphate, pH 7.0 + 0.5 M ammonium sulphate Buffer B: 20 mM potassium phosphate pH 7.0 Gradient: 0–100 % B; 10 column volumes
Fig. 49. Production scale purification of mouse IgG1, anti-IgE, on Phenyl Sepharose High Performance. (Work from Amersham Pharmacia Biotech, Uppsala, Sweden).
A280 nm .50 .40 .30 .20 .10 .00 0
50
100 Time (min)
The large scale purification was performed on BioPilot System and BioPilot Column Phenyl Sepharose High Performance. The process was first scaled up to a BioPilot Column 35/100 with a total column volume of 100 ml (Fig. 49) and later to a BioPilot Column 60/100 with a total column volume of 300 ml. No difference in performance between the two columns was seen. An IgG preparation, highly homogeneous by electrophoretic criteria, was obtained in a single chromatographic step. One process cycle yielded over 1 g of IgG from 2.2 litres of culture medium. This corresponds to a capacity of 400 g per year, the productivity of the bioreactor being the limiting step.
Purification of a recombinant Pseudomonas aeruginosa exotoxin produced in E. coli. An optimized purification process for a recombinant Pseudomonas aeruginosa exotoxin produced in the periplasm of E. coli has been developed (59). The scheme resulted in high recovery of a homogeneous exotoxin with reduced levels of DNA, endotoxins, and other contaminants (Table 14). 94
Table 14.
Step
Medium
Column
Purity %
Sample
DNA content (pg)*
Endotoxin content (EU)*
12 000
1
DEAE Sepharose Fast Flow
XK 16/20 Bed height 10 cm
³25
1 500
2.4 x 106
2
Phenyl Sepharose Fast Flow (high sub)
XK 16/20 Bed height 15 cm
³65
118
1.2 x 106
3
Q Sepharose High Performance
XK 16/20 Bed height 10 cm
>99
8
24
Superdex 75 prep grade
XK 16/70 Bed height 60 cm
100
4
5
1
DEAE Sepharose Fast Flow
BPG 100/500 Bed height 10 cm
³25
820
2.4 x 106
2
Phenyl Sepharose Fast Flow (high sub)
BPG 100/500 Bed height 15 cm
³63
118
8.0 x 105
3
Q Sepharose High Performance
BPG 100/500 Bed height 10 cm
>99
6
10
Superdex 75 prep grade
XK 16/70 Bed height 60 cm
100
N. D.**
6
4
4
* = per mg of protein ** = Not Determined
The protein is a well characterized (60–63) cytotoxic agent that acts by irreversibly inhibiting protein synthesis (ADP ribosylation of elongation factor 2). By conjugating the exotoxin to the monoclonal antibody B3, which binds to a carbohydrate epitope present on the surface of many cancer cells (64), an immunotoxin is produced. This type of immunotoxin can then be used as a therapeutic agent for targeted treatment of cancer. LysPE38, a genetically modified P. aeruginosa exotoxin, was purified to support an Investigational New Drug filing with the FDA. The purification strategy involved the extraction of the toxin from the periplasm, followed by clarification and chromatographic purification. During the method development stages, both anion exchange and HIC were evaluated. By using HIC early in the process, the only sample pretreatment necessary was the addition of a suitable amount of solid ammonium sulphate for binding. Phenyl Sepharose Fast Flow (high sub) was a suitable medium as it demonstrated high selectivity for LysPE38 at a relatively low concentration of ammonium sulphate (0.5 M), an important economic consideration in large scale HIC applications.
95
Subsequent ion exchange and gel filtration steps were used, but the level of DNA in the purified exotoxin fraction remained high. To remove the DNA from the sample, a DEAE Sepharose Fast Flow anion exchange step was added prior to HIC. The goal was to capture as much DNA as possible at a conductivity high enough to prevent the protein from binding. The DEAE Sepharose Fast Flow was used to remove DNA at a high conductivity so that the subsequent Phenyl Sepharose Fast Flow (high sub) with higher selectivity for LysPE38 could be used as the capture step. The small scale development work was performed on XK columns. For the HIC step, the salt concentration in start buffer was 0.5 M ammonium sulphate. The column was first eluted with a 5 column volume gradient from 0.5 M ammonium sulphate to 0 M ammonium sulphate (buffer B). Another 5 column volumes of buffer B was used to elute the exotoxin (Fig. 50).
Column: Gel:
XK 16/20 Phenyl Sepharose Fast Flow (high sub), 15 cm bed height Sample: Flow-through from the DEAE Sepharose Fast Flow step. Ammonium sulphate added to 0.5 M Sample volume: 120 ml Flow rate: 60 cm/h (2 ml/min) Buffer A: 20 mM Tris™; 1 mM EDTA, pH 7.5 + 1 M ammonium sulphate Buffer B: 20 mM Tris; 1 mM EDTA, pH 7.5 Gradient: 50–100% B, 5 column volumes, followed by 5 column volumes of buffer B to elute the exotoxin %
40.0
30.0 Exotoxin 20.0
10.0
0.0 0.
96
100.
200.
min
Fig. 50. Laboratory scale purification of a recombinant Pseudomonas aeruginosa exotoxin on Phenyl Sepharose Fast Flow (high sub). (Work by National Institute of Health (NIH), Bethesda, U.S.A., in collaboration with Amersham Pharmacia Biotech, Uppsala, Sweden).
The process was scaled up to BioProcess Glass Columns (BPG 100/500), with the exception of the final gel filtration step. The HIC step was run under the same conditions as employed at small scale and no difference in performance was seen (Fig. 51). A highly homogeneous LysPE38 was obtained with very low content of DNA and endotoxin.
Fig. 51. Production scale purification of a recombinant Pseudomonas aeruginosa exotoxin on Phenyl Sepharose Fast Flow (high sub). (Work by National Institute of Health (NIH), Bethesda, U.S.A., in collaboration with Amersham Pharmacia Biotech, Uppsala, Sweden).
Column: Gel:
BPG 100/500 Phenyl Sepharose Fast Flow (high sub), 15 cm bed height Sample: Flow-through from DEAE Sepharose Fast Flow step. Ammonium sulphate added to 0.5 M Sample volume: 5.0 l Flow rate: 60 cm/h (4.7 l/h) Buffer A: 20 mM Tris; 1 mM EDTA, pH 7.5 + 1 M ammonium sulphate Buffer B: 20 mM Tris; 1 mM EDTA, pH 7.5 Gradient: 50–100% B, 5 column volumes, followed by 5 column volumes of buffer B to elute the exotoxin. %
50.0 40.0 Exotoxin 30.0 20.0 10.0 0.0 100.
200.
min
97
98
7 References 1.
Adsorption separation by salting out. Arkiv för Kemi, Mineralogi Geologi 26B (1948) 1–5, Tiselius, A.
2.
Chemical coupling of peptides and proteins to polysaccharides by means of cyanogen halides. Nature 214 (1967) 1302–1304, Axén, R., Porath, J., Ernback, S.
3.
Preparation of adsorbents for biospecific affinity chromatography. I. Attachment of amino groupcontaining ligands to insoluble polymers by means of bifunctional oxiranes. J. Chromatog. 90 (1974) 87–98, Sundberg, L., Porath, J.
4.
Chromatography of lipophilic proteins on adsorbents containing mixed hydrophobic and ionic groups. Biochem. J. 126 (1972) 765–767, Yon, R.J.
5.
Hydrocarbon-coated Sepharoses. Use in the purification of glycogen phosphorylase. Biochem. Biophys. Res. Commun. 49 (1972) 383-390, Er-el, Z., Zaidenzaig, Y., Shaltiel, S.
6.
Hydrophobic affinity chromatography of proteins. Anal. Biochem. 52 (1973) 430–448, Hofstee, B.H.J.
7.
Hydrophobic chromatography: Use for purification of glycogen synthetase. Proc. Nat. Acad. Sci. USA 70 (1973) 778–781, Shaltiel, S., Er-el, Z.
8.
On the mode of adsorption of proteins to ”hydrophobic columns”. Biochem. Biophys. Res. Commun. 72 (1976) 108–113, Wilchek, M., Miron, T.
9.
Hydrophobic chromatography on homologous series of alkyl agaroses. A comparison of charged and electrically neutral column materials. J. Chromatog. 215 (1981) 211–228, Halperin, G., Breitenbach, M., Tauber-Finkelstein, M., Shaltiel, S.
10. Salting-out in amphiphilic gels as a new approach to hydrophobic adsorption. Nature 245 (1973) 465–466, Porath, J., Sundberg, L., Fornstedt, N., Olson, I. 11. Hydrophobic interaction chromatography. The synthesis and the use of some alkyl and aryl derivatives of agarose. J. Chromatog. 101 (1974) 281–288, Hjertén, S., Rosengren, J., Påhlman, S. 12. Hydrophobic interaction chromatography on Phenyl- and Octyl-Sepharose CL-4B. in: Chromatography of synthetic and biological macromolecules. Roger, E. Ed., Ellis Horwood Ltd., Chichester, England, 1978. Janson, J-C., Låås, T. 13. Hydrophobic interaction chromatography of serum proteins on Phenyl-Sepharose CL-4B. J. Chromatog. 242 (1982) 385–388, Hrkal, Z., Rejnkova, J. 14. Proteins of the kidney microvillar membrane. The amphipathic form of dipeptidyl peptidase IV. Biochem. J. 179 (1979) 379-395, McNair, R.D., Kenny, A.J.
99
15. Nuclear proteins. VI. Fractionation of chromosomal non-histone proteins using hydrophobic chromatography. Biochim. Biophys. Acta 563 (1979) 253–260, Comings, D.E., Miguel, A.G., Lesser, H.H. 16. Hydrophobic interaction chromatography as a tool in insulin receptor study. Proc. 2nd. Intl. Insulin Symp. (1980) 243–250, Kuehn, L., Meyer, H., Reinauer, H. 17. Hydrophobic interaction chromatography of proteins, nucleic acids, viruses and cells on non-charged amphiphilic gels, in: Methods of Biochemical Analysis (D. Glick, ed.), John Wiley & Sons, Inc., 1981, pp. 89–108. Hjertén, S. 18. Hydrophobic adsorbants for the isolation and purification of biosynthetic human growth hormone from crude fermentation mixtures. J. Chromatog. 361 (1986) 209–216, Lefort, S., Ferrara, P. 19. Purification of recombinant hepatitis B surface antigen produced by transformed Chinese hamster ovary (CHO) cell line grown in culture. Bioseparation 1 (1991) 397–408, Belew, M., Yafang, M., Bin, L., Berglöf, J., Janson, J-C. 20. Utilization of hydrophobic interaction for the formation of an enzyme reactor bed. Biotechnology & Bioengineering 17 (1975) 613–616, Caldwell, K.D., Axén, R., Porath, J. 21. Immobilization of phospholipid vesicles on alkyl derivatives of agarose gel beads. Biochim. Biophys. Acta 924 (1987) 185–192, Sandberg, M., Lundahl, P., Greijer, E., Belew, M. 22. The hydrophobic effect: formation of micelles and biological membranes. Tanfor, C., John Wiley & sons, New York, 1973. 23. Proteins, structure and molecular properties. Creighton, T.E., W.E. Freeman, New York, 1984. 24. Salt-promoted adsorption: recent developments. J. Chromatog. 376 (1986) 331–341, Porath, J. 25. Fractionation of proteins by hydrophobic interaction chromatography, with reference to serum proteins. Proceedings Intl. Workshop on Technology for Protein Separation & Improvement of Blood Plasma Fractionation. Reston, Virginia, 1977, 410–421, Hjertén, S. 26. Fractionation of proteins by fractional interfacial salting out on unsubstituted agarose gels. Biochem. Biophys. Res. Comm. 70 (1976) 1009–1013, von der Haar, F. 27. Salt effects on hydrophobic interactions in precipitation and chromatography of proteins: an interpretation of the lyotropic series. Arch. Biochem. Biophys. 183 (1977) 200–215, Melander, W., Horvath, C. 28. Solubility chromatography of serum proteins. II. Partial purification of the second component of guinea pig complement by solubility chromatography in concentrated ammonium sulphate solutions. J. Chromatog. 40 (1969) 53–61, Hoffmann, L.G., McGivern, P.W. 29. Role of physical forces in hydrophobic interaction chromatography. Separation & Purification Methods 9 (1980) 267–370, Srinivasan, R., Ruckenstein, E. 30. Non-ionic adsorption chromatography of proteins. J. Chromatog. 159 (1978) 57–69, Hofstee, B.H.J, Otillio, N.F. 31. General aspects of hydrophobic chromatography. Adsorption and elution characteristics of some skeletal muscle enzymes. Biochemistry 14 (1975) 754–760, Jennissen, H.P., Heilmeyer, I.M.G. 32. Hydrophobic interaction chromatography on non-charged Sepharose derivatives. Binding of a model protein, related to ionic strength, hydrophobicity of the substituent, and degree of substitution (determined by NMR). Biochim. Biophys. Acta 412 (1975) 51–61, Rosengren, J., Påhlman, S., Glad, M., Hjertén, S. 33. Agar derivatives for chromatography, electrophoresis & gel-bound enzymes. IV. Benzylated dibromopropanol cross-linked Sepharose as an amphophilic gel for hydrophobic salting out chromatography of enzymes with special emphasis on denaturing risks. J. Chromatog. 111 (1975) 373–387, Låås, T.
100
34. Multivalent interaction chromatography as exemplified by the adsorption and desorption of skeletal muscle enzymes on hydrophobic alkyl-ligands. J. Chromatog. 159 (1978) 71–83, Jennissen, H.P. 35. Synthesis of new hydrophobic adsorbents based on homologous series of uncharged alkyl sulphide agarose derivatives. J. Chromatog. 321 (1985) 305–317, Maisano, F., Belew, M., Porath, J. 36. Hydrophobic interaction chromatography on uncharged Sepharose derivatives. Effects of neutral salts on the adsorption of proteins. J. Chromatog. 131 (1977) 99–108, Påhlman, S., Rosengren, J., Hjertén, S. 37. Displacement of water and its control of biochemical reactions. Levin, S., Academic Press, New York, 1974. 38. Hydrophobic interaction chromatography of proteins on neutral adsorbents, in: Methods of protein separation, vol. 2. Catsimpoolas, N., Ed., Plenum Publishing Corporatin, New York, 1976. Hjertén, S. 39. Temperature-dependent van der Waals forces. Biophys. J. 10 (1970) 664–674, Parsegian, V.A., Ninham, B.W. 40. Separation of lipoamide dehydrogenase isoenzymes by affinity chromatography. Biochim. Biophys. Acta 384 (1975) 69–80, Visser, J., Strating, M. 41. Some general aspects of hydrophobic interaction chromatography. J. Chromatog. 87 (1973) 325– 331, Hjertén, S. 42. Gradient and isocratic High Performance Hydrophobic Interaction Chromatography of proteins on agarose columns. J. Chromatog. 359 (1986) 99–109, Hjertén, S., Yao, K., Eriksson, K.-O., Johansson, B. 43. Determination of the leakage from Phenyl Sepharose CL-4B, Phenyl Sepharose Fast Flow and Phenyl Superose in bulk and column experiments. J. Chromatog. 403 (1987) 85-98, Johansson, B.-L., Hellberg, U., Wennberg, O. 44. Comparison of molecular structures of proteins: Helix content; distribution of apolar residues. Arch. Biochem. Biophys. 138 (1970) 704–706, Klotz, I.M. 45. The interpretation of protein structures: estimation of static accessibility. J. Mol. Biol. 55 (1971) 397–400, Lee, B., Richards, F.M. 46. Characterization of hydrophobic interaction and Hydrophobic Interaction Chromatography media by multivariate analysis. J. Chromatog. 599 (1992) 131–136, Kårsnäs, P., Lindblom, T. 47. Differences in retention behavior between small and large molecules in Ion Exchange Chromatography and Reversed Phase Chromatography. Anal. Biochem. 142 (1984) 134–139, Ekström, B., Jacobson, G. 48. Electron-donor-acceptor chromatography (EDAC) for biomolecules in aqueous solutions, in: Protein recognition of immobilized ligands, Alan R. Liss, Inc., (1989) pp. 101–122, Porath, J. 49. Identification, purification and partial sequence analysis of Autotaxin, a novel motility-stimulating protein. J. Biol. Chem. 267 (1992) 2524–2529, Stracke, M.L., Krutzsch, H.C., Unsworth, E.J., Årestad, A., Cioce, V., Schiffmann, E., Liotta, L.A. 50. Expression, Purification and Crystallization of the HIV-1 Reverse Transeriptase (RT). AIDS Res. Hum. Retrovir. 6 (1990) 1297–1303, Unge, T., Ahola, H., Bhikhabhai, R., Bäckbro, K., Lövgren, S., Fenyö, E.M., Honigman, A., Panet, A., Gronowitz, G.S., Strandberg, B. 51. Factors involved in specific transcription by mammalian RNA polymerase II. Identification and characterization of Factor II H. J. Biol. Chem. 267 (1992) 2786–2793, Flores, O., Lu, H., Reinberg, D.
101
52. A multidimensional HPLC strategy for the purification of proteins and peptides for micro-sequence analysis: - The role of micropreparative ion exchange columns. Poster presented at the 10th International Symposium of Proteins, Peptides and Polynucleotides, Wiesbaden, Germany, October 1990, Nice, E., Fabri, L., Burgess, A., Simpson, R., Hellman, V., Andersson, K. 53. Isolation of Human Pituitary Prolactin. Biochim. Biophys. Acta 588 (1979) 368–379, Roos, P., Nyberg, F., Wide, L. 54. Phenyl-Sepharose-mediated Detergent-Exchange Chromatography: Its application to exchange of detergents bound to membrane proteins. Biochemistry 23 (1984) 6121–6126, Robinson, N.C., Wiginton, D., Talbert, L. 55. Characterization of the dermatan sulphate proteoglycans, DS-PGI and DS-PGII, from bovine articular cartilage and skin isolated by Octyl Sepharose chromatography. J. Biol. Chem. 264 (1989) 2876–2884, Choi, H.U., Johnson, T.L., Pal, S., Tang, L.-H., Rosenberg L., Neame, P.J. 56. Production and purification of murine monoclonal antibodies directed against HIV-1, for use in passive immunotherapy of HIV-1. Poster presented at Biotech 92; International Symposium; New Generation of Monoclonal Antibodies in Diagnosis and Therapy, Genoa, Italy, April 1992, Gilljam, G., Hinkula, J., Daniels, A.I., Wahren, B. 57. Development and a scale up study of a chromatographic downstream process for the purification of recombinant EGF. Poster presented at Ninth International Biotechnology Symposium, Crystal City, Virginia, USA, August 1992, Daniels, A.I., Pettersson, N.T. 58. Characterization of Phenyl Sepharose High Performance – a new 34 µm medium for Hydrophobic Interaction Chromatography. Poster presented at 32nd International Union of Pure and Applied Chemistry (IUPAC) Congress, Stockholm, Sweden, August 1989, Daniels, A.I., Weitman, A., Westin, G., Söderström, L. 59. Production of clinical grade recombinant Exotoxin A from E. Coli. Presented at Recovery of Biological Products VI, An Engineering Foundation Conference, Interlaken, Switzerland, September 1992, Tsai, A.M., Kaufman, J.B., Shiloach, J., Gallo, M., Fass, S. 60. Recombinant toxins for cancer treatment. Science 254 (1991) 1173–1177, Pastan, I., FitzGerald, D. 61. Structure of exotoxin A of Pseudomonas aeruginosa at 3.0-Ångström resolution. Proc. Natl. Acad. Sci. USA 83 (1986) 1320–1324, Allured, V.S., Collier, R.J., Carroll, S.F., McKay, D.B. 62. Functional domains of Pseudomonas exotoxin identified by deletion analysis of the gene expressed in E. Coli. Cell 48 (1987) 129–136, Hwang, J., FitzGerald, D.J., Adhya, S., Pastan, I. 63. Functional analysis of domains II, Ib, and III of Pseudomonas exotoxin. J. Biol. Chem. 264 (1989) 14256–14261, Siegall, C.B., Chaudhary, V.K., FitzGerald, D.J., Pastan, I. 64. Anti-tumor activities of immunotoxins made of monoclonal antibody B3 and various forms of Pseudomonas exotoxin. Proc. Natl. Acad. Sci. USA 88 (1991) 3358-3362, Pai, L.H., Batra, J.K., FitzGerald, D.J., Willingham, M.C., Pastan, I. 65. Solvent modulation in Hydrophobic Interaction Chromatography. Biotechnol. Appl. Biochem. 13 (1991) 151–172, Arakawa T., Narhi, L.O. 66. Hydrophobic Interaction Chromatography in alkaline pH. Anal. Biochem. 182 (1989) 266–270, Narhi, L.O., Kita, Y., Arakawa, T.
102
Ordering information
104
Product/Bulk media
Pack size
Phenyl Sepharose 6 Fast Flow (low sub)
200 ml 1l 5l
17-0965-05 17-0965 03 17-0965-04
Phenyl Sepharose 6 Fast Flow (high sub)
200 ml 1l 5l
17-0973-05 17-0973-03 17-0973-04
Butyl Sepharose 4 Fast Flow
200 ml 500 ml 5l
17-0980-01 17-0980-02 17-0980-04
Octyl Sepharose 4 Fast Flow
25 ml 200 ml 500 ml 1l 5l
17-0946-10 17-0946-02 17-0946-05 17-0946-03 17-0946-04
Phenyl Sepharose High Performance
75 ml 1l 5l
17-1082-01 17-1082-03 17-1082-04
Phenyl Sepharose CL-4B
50 ml 200 ml 10 l
17-0810-02 17-0810-01 17-0810-05
Octyl Sepharose CL-4B
50 ml 200 ml 10 l
17-0790-02 17-0790-01 17-0790-05
SOURCE™ 15ETH
50 ml 200 ml 1l
17-0146-01 17-0146-02 17-0146-04
SOURCE 15ISO
50 ml 200 ml 1l
17-0148-01 17-0148-02 17-0148-04
SOURCEPHE
50 ml 200 ml 1l 5l
17-0147-01 17-0147-02 17-0147-04 17-0147-05
Product/Prepacked columns
Bed volume
HiTrap™ HIC test kit HiLoad™ 16/10 Phenyl Sepharose HP HiLoad 26/10 Phenyl Sepharose HP Alkyl Superose HR 5/5 Alkyl Superose HR 10/10 Phenyl Superose HR 5/5 Phenyl Superose HR 10/10 Phenyl Superose PC 1.6/5 RESOURCE™ 15ETH RESOURCE 15ISO RESOURCE 15PHE RESOURCE HIC Test kit RESOURCE 15PHE PE 4.6/100
5 x 1 ml 20 ml 53 ml 1 ml 8 ml 1 ml 8 ml 0.1 ml 1 ml 1 ml 1 ml
Code No.
Code No. 17-1349-01 17-1085-01 17-1086-01 17-0586-01 17-0587-01 17-0519-01 17-0530-01 17-0772-01 17-1184-01 17-1185-01 17-1186-01 17-1187-01 17-1171-01
ISBN 91-970490-4-2
105
Production: RAK Design AB
Gel Filtration Handbook – Principles and Methods
Gel Filtration Principles and Methods
www.chromatography.amershambiosciences.com Back to Collection 18-1022-18 Edition AI
Handbooks from Amersham Biosciences
Antibody Purification ÄKTA, FPLC, PlusOne, HiLoad , HiTrap, HiPrep, Hybond, ECL, ECL Plus, BioProcess, MabSelect, Sephacryl, Sephadex, Superose, Sepharose, Superdex and Drop Design are trademarks of Amersham Biosciences Limited.
Handbook 18-1037-46
Amersham and Amersham Biosciences are trademarks of Amersham plc.
The Recombinant Protein Handbook
Gel Filtration
Coomassie is a trademark of ICI plc.
Protein Amplification and Simple Purification 18-1142-75
Principles and Methods 18-1022-18
MicroSpin is a trademark of Lida Manufacturing Corp. Triton is a registered trademark of Union Carbide Chemicals and Plastics Co.
Percoll
Tween is a registered trademark of ICI Americas, Inc.
Methodology and Applications 18-1115-69
Eppendorf and Multipipette are trademarks of Eppendorf-Netheler-Hinz GmbH.
Ficoll-Paque Plus
All goods and services are sold subject to the terms and conditions of sale of the company within the Amersham Biosciences group that supplies them. A copy of these terms and conditions is available on request. © Amersham Biosciences AB 2002 – All rights reserved.
Protein Purification
Reversed Phase Chromatography
Handbook 18-1132-29
Principles and Methods 18-1134-16
Ion Exchange Chromatography
Expanded Bed Adsorption
Principles and Methods 18-1114-21
Principles and Methods 18-1124-26
Affinity Chromatography
Chromatofocusing
Principles and Methods 18-1022-29
with Polybuffer and PBE 18-1009-07
Hydrophobic Interaction Chromatography
Microcarrier cell culture
using immobilized pH gradients
Principles and Methods 18-1020-90
Principles and Methods 18-1140-62
Principles and Methods 80-6429-60
For in vitro isolation of lymphocytes 18-1152-69
GST Gene Fusion System Handbook 18-1157-58
BIACORE is a trademark of Biacore AB.
Amersham Biosciences AB, Björkgatan 30, SE-751 84 Uppsala, Sweden Amersham Biosciences UK Limited, Amersham Place, Little Chalfont, Buckinghamshire HP7 9NA, England Biosciences Corp., 800 Centennial Avenue, PO Box 1327, Piscataway NJ 08855, USA Amersham Biosciences Europe GmbH, Munzinger Strasse 9, D-79111 Freiburg, Germany
2-D Electrophoresis
Amersham Biosciences K.K., Sanken Bldg. 3-25-1, Hyakunincho Amersham Shinjuku-ku, Tokyo 169-0073, Japan.
Gel filtration Principles and Methods
1
Contents Introduction ............................................................................................................. 5 Symbols ................................................................................................................................................... 6 Common abbreviations .............................................................................................................................. 7
Chapter 1 Gel filtration in practice ........................................................................................... 9 Introduction ................................................................................................................ 9 Separation by gel filtration ........................................................................................... 9 Resolution in gel filtration .......................................................................................... 13 Media selection ......................................................................................................... 16 Sample preparation ................................................................................................... 20 Buffer composition and preparation ............................................................................. 21 Denaturing (chaotropic) agents and detergents ........................................................................................... 22
Column and media preparation ................................................................................... 23 Sample application .................................................................................................... 23 Elution and flow rates ................................................................................................ 24 Method development for high resolution fractionation ................................................... 26 Care of gel filtration media ......................................................................................... 27 Equipment selection .................................................................................................. 27 Scaling up ................................................................................................................ 27 BioProcess Media for large-scale production ................................................................ 29 Troubleshooting ......................................................................................................... 29
Chapter 2 Gel filtration media ................................................................................................ 35 Components of gel filtration media .............................................................................. 35 Superdex: first choice for high resolution, short run times and high recovery .................... 36 Separation options ................................................................................................................................... 38 Separation examples ................................................................................................................................ 39 Performing a separation ............................................................................................................................ 42 Cleaning ................................................................................................................................................. 43 Media characteristics ............................................................................................................................... 44 Chemical stability .................................................................................................................................... 44 Storage ................................................................................................................................................... 44
Sephacryl: fast, high recovery separations at laboratory and industrial scale .................... 45 Separation options ................................................................................................................................... 48 Separation examples ................................................................................................................................ 48 Performing a separation ............................................................................................................................ 49 Cleaning ................................................................................................................................................. 50 Media characteristics ............................................................................................................................... 51 Chemical stability .................................................................................................................................... 51 Storage ................................................................................................................................................... 51
Superose: broad fractionation range, but not suitable for industrial scale separations ....... 52
2
Separation options ................................................................................................................................... 53 Separation examples ................................................................................................................................ 54 Performing a separation ............................................................................................................................ 54 Cleaning ................................................................................................................................................. 55 Media characteristics ............................................................................................................................... 56 Chemical stability .................................................................................................................................... 56 Storage ................................................................................................................................................... 56
Sephadex: rapid group separation of high and low molecular weight substances, such as desalting, buffer exchange and sample clean up ............................................... 57 Separation options ................................................................................................................................... 59 Separation examples ................................................................................................................................ 61 Performing a separation ............................................................................................................................ 61 Scale up and processing large sample volumes ........................................................................................... 66 Media characteristics ............................................................................................................................... 68 Column Packing ...................................................................................................................................... 68 Cleaning ................................................................................................................................................. 68 Chemical stability .................................................................................................................................... 68 Storage ................................................................................................................................................... 69
Chapter 3 Gel filtration in theory ............................................................................................ 71 Defining the process .................................................................................................. 71 Selectivity curves and media selection ......................................................................... 74 Resolution ................................................................................................................ 75
Chapter 4 Molecular weight determination and molecular weight distribution analysis ............. 79 Chapter 5 Sephadex LH-20 .................................................................................................... 81 Separation options ................................................................................................................................... 82 Separation examples ................................................................................................................................ 82 Packing a column .................................................................................................................................... 83 Performing a separation ............................................................................................................................ 84 Cleaning ................................................................................................................................................. 84 Medium characteristics ............................................................................................................................ 84 Chemical stability .................................................................................................................................... 85 Storage ................................................................................................................................................... 85 Transferring Sephadex LH-20 between organic solvents ............................................................................... 85
Chapter 6 Gel filtration in a Purification Strategy (CIPP) ........................................................... 87 Applying CIPP ............................................................................................................ 87 Selection and combination of purification techniques ................................................... 88 Gel filtration as a polishing step ................................................................................................................ 91
3
Appendix 1 Column packing and preparation ............................................................................ 93 Columns for packing gel filtration media ...................................................................... 93 Checking column efficiency ........................................................................................ 95 Column packing for high resolution fractionation using Superdex prep grade and Sephacryl High Resolution ......................................................................................... 95 Column packing for group separations using Sephadex .................................................. 98
Appendix 2 Sephadex and Darcy's law .................................................................................... 102 Appendix 3 Sample preparation .............................................................................................. 103 Sample stability ..................................................................................................................................... 103 Sample clarification ............................................................................................................................... 104
Specific sample preparation steps ............................................................................. 105 Removal of lipoproteins ............................................................................................ 108 Removal of phenol red ............................................................................................. 108 Removal of low molecular weight contaminants .......................................................... 108
Appendix 4 Selection of purification equipment ...................................................................... 109 Appendix 5 Converting from linear flow (cm/hour) to volumetric flow rates (ml/min) and vice versa ........................................................................ 110 Appendix 6 Conversion data: proteins, column pressures ........................................................ 111 Appendix 7 Table of amino acids ............................................................................................ 112 Appendix 8 Analytical assays during purification .................................................................... 114 Appendix 9 Storage of biological samples .............................................................................. 116 Additional reading and reference material ............................................................ 117 Ordering information ............................................................................................ 118
4
Introduction Biomolecules are purified using chromatography techniques that separate them according to differences in their specific properties, as shown in Figure 1. Property
Technique
Size
Gel filtration (GF), also called size exclusion
Charge
Ion exchange chromatography (IEX)
Hydrophobicity
Hydrophobic interaction chromatography (HIC) Reversed phase chromatography (RPC)
Biorecognition (ligand specificity)
Affinity chromatography (AC)
Gel filtration
Hydrophobic interaction
Ion exchange
Affinity
Reversed phase
Fig. 1. Separation principles in chromatography purification.
For more than forty years since the introduction of Sephadex™, gel filtration has played a key role in the purification of enzymes, polysaccharides, nucleic acids, proteins and other biological macromolecules. Gel filtration is the simplest and mildest of all the chromatography techniques and separates molecules on the basis of differences in size. The technique can be applied in two distinct ways: 1. Group separations: the components of a sample are separated into two major groups according to size range. A group separation can be used to remove high or low molecular weight contaminants (such as phenol red from culture fluids) or to desalt and exchange buffers. 2. High resolution fractionation of biomolecules: the components of a sample are separated according to differences in their molecular size. High resolution fractionation can be used to isolate one or more components, to separate monomers from aggregates, to determine molecular weight or to perform a molecular weight distribution analysis. Gel filtration can also be used to facilitate the refolding of denatured proteins by careful control of changing buffer conditions.
5
Gel filtration is a robust technique that is well suited to handling biomolecules that are sensitive to changes in pH, concentration of metal ions or co-factors and harsh environmental conditions. Separations can be performed in the presence of essential ions or cofactors, detergents, urea, guanidine hydrochloride, at high or low ionic strength, at 37 °C or in the cold room according to the requirements of the experiment. This handbook describes the use of gel filtration for the purification and separation of biomolecules, with a focus on practical information for obtaining the best results. The media available, selection criteria and examples with detailed instructions for the most common applications are included, as well as the theoretical principles behind the technique. The first step towards a successful separation is to select the correct medium and this handbook focuses on the most up-to-date gel filtration media and prepacked columns. The biocompatibility, stability and utility of gel filtration media from Amersham Biosciences have made these products the standard choice in practically every laboratory using the technique. A wide variety of prepacked columns and ready to use media is available. The illustration on the inside cover shows the range of handbooks from Amersham Biosciences that have been produced to ensure that chromatography and other separation techniques are used easily and effectively at any scale, in any laboratory and for any application.
Symbols this symbol indicates general advice which can improve procedures or provide recommendations for action under specific situations. this symbol denotes advice which should be regarded as mandatory and gives a warning when special care should be taken. this symbol highlights troubleshooting advice to help analyse and resolve difficulties that may occur. chemicals, buffers and equipment. experimental protocol.
6
Common abbreviations In chromatography GF: gel filtration (sometimes referred to as SEC: size exclusion chromatography) IEX: ion exchange chromatography (also seen as IEC) AC: affinity chromatography RPC: reverse phase chromatography HIC: hydrophobic interaction chromatography CIPP: Capture, Intermediate Purification and Polishing MPa: megapascals psi: pounds per square inch SDS: sodium dodecyl sulphate CIP: cleaning in place A280nm, A214nm: UV absorbance at specified wavelength Mr: relative molecular weight N: column efficiency expressed as theoretical plates per meter Ve: elution volume is measured from the chromatogram and relates to the molecular size of the molecule. Vo: void volume is the elution volume of molecules that are excluded from the gel filtration medium because they are larger than the largest pores in the matrix and pass straight through the packed bed Vt: total column volume is equivalent to the volume of the packed bed (also referred to as CV) Rs: resolution, the degree of separation between peaks Kav and logMr: partition coefficient and log molecular weight, terms used when defining the selectivity of a gel filtration medium In product names HMW: high molecular weight LMW: low molecular weight HR: high resolution pg: prep grade PC: precision column SR: solvent resistant
7
8
Chapter 1 Gel filtration in practice Introduction Gel filtration separates molecules according to differences in size as they pass through a gel filtration medium packed in a column. Unlike ion exchange or affinity chromatography, molecules do not bind to the chromatography medium so buffer composition does not directly affect resolution (the degree of separation between peaks). Consequently, a significant advantage of gel filtration is that conditions can be varied to suit the type of sample or the requirements for further purification, analysis or storage without altering the separation. Gel filtration is well suited for biomolecules that may be sensitive to changes in pH, concentration of metal ions or co-factors and harsh environmental conditions. Separations can be performed in the presence of essential ions or cofactors, detergents, urea, guanidine hydrochloride, at high or low ionic strength, at 37 °C or in the cold room according to the requirements of the experiment. Purified proteins can be collected in any chosen buffer. This chapter provides general guidelines applicable to any gel filtration separation. A key step towards successful separation is to select the correct medium, so selection guides for the most up-to-date gel filtration media and prepacked columns are included. Other application examples and product-specific information are found in Chapter 2.
Separation by gel filtration
Interacting with medium
Low molecular weight
Sample injection
High molecular weight
Absorbance
Intermediate molecular weight
To perform a separation, gel filtration medium is packed into a column to form a packed bed. The medium is a porous matrix in the form of spherical particles that have been chosen for their chemical and physical stability, and inertness (lack of reactivity and adsorptive properties). The packed bed is equilibrated with buffer which fills the pores of the matrix and the space in between the particles. The liquid inside the pores is sometimes referred to as the stationary phase and this liquid is in equilibrium with the liquid outside the particles, referred to as the mobile phase. It should be noted that samples are eluted isocratically, i.e. there is no need to use different buffers during the separation. However, a wash step using the running buffer is usually included at the end of a separation to facilitate the removal of any molecules that may have been retained on the column and to prepare the column for a new run. Figure 2 shows the most common terms used to describe the separation and Figure 3 illustrates the separation process of gel filtration.
Vt
Vo Ve
Void volume Vo
Total column volume Vt
Vt – Vo
Vt – Vo
Fig. 2. Common terms in gel filtration.
9
1. Spherical particles of gel filtration medium are packed into a column.
2. Sample is applied to the column.
3. Buffer (mobile phase) and sample move through the column. Molecules diffuse in and out of the pores of the matrix (also described as partitioning of the sample between the mobile phase and the stationary phase). Smaller molecules move further into the matrix and so stay longer on the column.
4. As buffer passes continuously through the column, molecules that are larger than the pores of the matrix are unable to diffuse into the pores and pass through the column. Smaller molecules diffuse into the pores and are delayed in their passage down the column.
Diffusion Diffusion out of the pores Buffer
Buffer
Ve Vt – Vo
10
Interacting with medium Vt
Vo
Fig. 3. Process of gel filtration.
Low molecular weight
Sample injection
5. Large molecules leave the column first followed by smaller molecules in order of their size. The entire separation process takes place as one total column volume (equivalent to the volume of the packed bed) of buffer passes through the gel filtration medium.
High molecular weight
Absorbance
Intermediate molecular weight
Diffusion into the pores
Group separation Gel filtration is used in group separation mode to remove small molecules from a group of larger molecules and as a fast, simple solution for buffer exchange. Small molecules such as excess salt (desalting) or free labels are easily separated. Samples can be prepared for storage or for other chromatography techniques and assays. Gel filtration in group separation mode is often used in protein purification schemes for desalting and buffer exchange. For further details refer to Chapter 2, page 57 and the Protein Purification Handbook from Amersham Biosciences. Sephadex G-10, G-25 and G-50 are used for group separations. Large sample volumes up to 30% of the total column volume (packed bed) can be applied at high flow rates using broad, short columns. Figure 4 shows the elution profile (chromatogram) of a typical group separation. Large molecules are eluted in or just after the void volume, Vo as they pass through the column at the same speed as the flow of buffer. For a well packed column the void volume is equivalent to approximately 30% of the total column volume. Small molecules such as salts that have full access to the pores move down the column, but do not separate from each other. These molecules usually elute just before one total column volume, Vt, of buffer has passed through the column. In this case the proteins are detected by monitoring their UV absorbance, usually at A280nm, and the salts are detected by monitoring the conductivity of the buffer. A 280 nm UV 280 nm Conductivity 0.15 (His)6 protein
Sample:
0.10
Column: Buffer:
Salt
0.05
(His)6 protein eluted from HiTrap™ Chelating HP with sodium phosphate 20 mM, sodium chloride 0.5 M, imidazole 0.5 M, pH 7.4 HiTrap Desalting 5 ml Sodium phosphate 20 mM, sodium chloride 0.15 M, pH 7.0
void volume Vo, total column volume Vt Inject Vt
Vo 0 0
1
2 min
Fig. 4. Typical chromatogram of a group separation. The UV (protein) and conductivity (salt) traces enable pooling of the desalted fractions and facilitate optimization of the separation.
Refer to Chapter 2, page 57 for detailed information on group separation of high and low molecular weight substances, i.e. desalting, buffer exchange and sample clean up using Sephadex. Refer to Chapter 3 for detailed information on the theory of gel filtration.
11
High resolution fractionation Gel filtration is used in fractionation mode to separate multiple components in a sample on the basis of differences in their size. The goal may be to isolate one or more of the components, to determine molecular weight, or to analyze the molecular weight distribution in the sample (refer to Chapter 4 for details of molecular weight determination and distribution analysis). The best results for high resolution fractionation will be achieved with samples that originally contain few components or with samples that have been partially purified by other chromatography techniques (in order to eliminate proteins of similar size that are not of interest). High resolution fractionation by gel filtration is well suited for the final polishing step in a purification scheme. Monomers can be separated from aggregates (difficult to achieve by any other technique) and samples can be transferred to a suitable buffer for assay or storage. Gel filtration can be used directly after any of the chromatography techniques such as ion exchange, chromatofocusing, hydrophobic interaction or affinity since the components from any elution buffer will not affect the final separation. For further details on using gel filtration in a purification strategy, refer to Chapter 6 and the Protein Purification Handbook from Amersham Biosciences. Figure 5 shows the theoretical elution profile (chromatogram) of a high resolution fractionation. Molecules that do not enter the matrix are eluted in the void volume, Vo as they pass directly through the column at the same speed as the flow of buffer. For a well packed column the void volume is equivalent to approximately 30% of the total column volume (packed bed). Molecules with partial access to the pores of the matrix elute from the column in order of decreasing size. Small molecules such as salts that have full access to the pores move down the column, but do not separate from each other. These molecules usually elute just before one total column volume, Vt, of buffer has passed through the column. high molecular weight
Absorbance
low molecular weight
sample injection volume
void volume Vo total column volume Vt
intermediate molecular weight equilibration Vt
Vo
1 cv Column Volumes (cv)
Fig. 5. Theoretical chromatogram of a high resolution fractionation (UV absorbance).
12
Resolution in gel filtration Many factors influence the final resolution (the degree of separation between peaks of a gel filtration separation): sample volume, the ratio of sample volume to column volume, column dimensions, particle size, particle size distribution, packing density, pore size of the particles, flow rate, and viscosity of the sample and buffer. The molecular weight range over which a gel filtration medium can separate molecules is referred to as the selectivity of the medium (see selection guide for gel giltration media on page 18). Resolution is a function of the selectivity of the medium and the efficiency of that medium to produce narrow peaks (minimal peak broadening), as illustrated in Figure 6. The success of gel filtration depends primarily on choosing conditions that give sufficient selectivity and counteract peak broadening effects during the separation.
high efficiency
low efficiency
Fig. 6. Dependence of resolution on selectivity and the counteraction of peak broadening.
After selecting a gel filtration medium with the correct selectivity, sample volume and column dimensions become two of the most critical parameters that will affect the resolution of the separation.
13
Sample volume and column dimensions Sample volumes are expressed as a percentage of the total column volume (packed bed). Using smaller sample volumes helps to avoid overlap if closely spaced peaks are eluted. Figure 7 illustrates how sample volume can influence a high resolution fractionation. 1)
25 µl,1.0 ml/min (76 cm/h) A 280 nm 0.25
Vo
Vt
0.20 0.15 0.10 0.05
0.00 0.0
5.0
10.0
15.0
20.0
25.0 min
250 µl,1.0 ml/min (76 cm/h)
2)
Column: Superdex™ 200 HR 10/30 (Vt: 24 ml) Sample: Mr Conc. (mg/ml) Thyroglobulin 669 000 3 Ferritin 440 000 0.7 IgG 150 000 3 Transferrin 81 000 3 Ovalbumin 43 000 3 Myoglobin 17 600 2 Vitamin B12 1 355 0.5 Sample concentration: 15.2 mg/ml Sample volumes: 1) 25 µl (0.1% × Vt) 2) 250 µl (1% × Vt) 3) 1000 µl (4.2% × Vt) Buffer: 0.05 M sodium phosphate, 0.15 M NaCl, pH 7.0 Flow: 1.0 ml/min (76.4 cm/h)
A 280 nm Vo
Vt
0.15
0.10
0.05
0.00 0.0
3)
5.0
10.0
15.0
20.0
25.0 min
1 000 µl,1.0 ml/min (76 cm/h) A 280 nm Vo
Vt
0.10
0.05
0.00 0.0
5.0
10.0
15.0
20.0
25.0 min
Fig. 7. Influence of sample volume on resolution.
For group separations sample volumes up to 30% of the total column volume can be applied. For high resolution fractionation a sample volume from 0.5–4% of the total column volume is recommended, depending on the type of medium used. For most applications the sample volume should not exceed 2% to achieve maximum resolution. Depending on the nature of the specific sample, it may be possible to load larger sample volumes, particularly if the peaks of interest are well resolved. This can only be determined by experimentation. 14
For analytical separations and separations of complex samples, start with a sample volume of 0.5% of the total column volume. Sample volumes less than 0.5% do not normally improve resolution. To increase the capacity of a gel filtration separation samples can be concentrated. Avoid concentrations above 70 mg/ml protein as viscosity effects may interfere with the separation. Sample dilution is inevitable because diffusion occurs as sample passes through the column. In order to minimize sample dilution use a maximum sample volume that gives the resolution required between the peaks of interest.
Resolution, R s
The ratio of sample volume to column volume influences resolution, as shown in Figures 8a and 8b, where higher sample volume to column volume ratios give lower resolution. Column volumes are normally selected according to the sample volumes to be processed. However, since larger sample volumes may require significantly larger column volumes, there may be occasions when it is more beneficial to repeat the separation several times on a smaller column and pool the fractions of interest or to concentrate the sample (see Appendix 3 on sample preparation).
1.5
Column: Buffer: Sample:
1.0
Sample concentration: Flow:
HiLoad™ 16/60 Superdex 200 prep grade 50 mM NaPO 4, 0.1 M NaCl, pH 7.2 Solution of transferrin (Mr 81 000) and IgG (Mr 160 000) by equal weight 8 mg/ml 1 ml/min (30 cm/h)
0.5
0 0
4 5 2 3 Sample volume (% of column volume)
1
Fig. 8a. Influence of sample volume on the resolution of transferrin and IgG on prepacked HiLoad 16/60 Superdex 200 prep grade.
HiPrepTM 16/60 16
Columns:
HiPrep 16/60 Sephacryl™ S-100 HR HiPrep 16/60 Sephacryl S-200 HR HiPrep 16/60 Sephacryl S-300 HR Buffer: 50 mM NaPO 4, 0.15 M NaCl, 0.02% NaN3, pH 7.0 Sample: Ovalbumin Flow: 0.66 ml/min (20 cm/h) Protein load: 8 mg
14 S-300
Resolution, R s
12 10
S-200 8 6
S-100
4 2 0 0
1
2
3
4 5 Sample volume, ml
Fig. 8b. Influence of sample volume on the resolution of ovalbumin and IgG on different prepacked columns of HiPrep 16/60 Sephacryl High Resolution.
15
The height of the packed bed affects both resolution and the time taken for elution. The resolution in gel filtration increases as the square root of bed height. Doubling the bed height gives an increase in resolution equivalent to È2 = 1.4 (40%). For high resolution fractionation long columns will give the best results and a bed height between 30–60 cm should be satisfactory. Sufficient bed height together with a low flow rate allows time for all 'intermediate' molecules to diffuse in and out of the matrix pores and give sufficient resolution. If a very long column is judged to be necessary, the effective bed height can be increased by using columns, containing the same media, coupled in series. Refer to Chapter 3 for detailed information on the theory of gel filtration.
Media selection Chromatography media for gel filtration are made from porous matrices chosen for their inertness and chemical and physical stability. The size of the pores within a particle and the particle size distribution are carefully controlled to produce a variety of media with different selectivities. Today's gel filtration media cover a molecular weight range from 100 to 80 000 000, from peptides to very large proteins and protein complexes. The selectivity of a gel filtration medium depends solely on its pore size distribution and is described by a selectivity curve. Gel filtration media are supplied with information on their selectivity, as shown for Superdex in Figure 9. The curve has been obtained by plotting a partition coefficient Kav against the log of the molecular weight for a set of standard proteins (see Chapter 3 Gel filtration in theory for calculation of Kav). K av 1.00
Superdex peptide 0.75 Superdex 75 Superdex 200 Superdex 30 prep grade Superdex 75 prep grade
0.50
Superdex 200 prep grade
Fig. 9. Selectivity curves for Superdex.
16
1000000
100000
10000
1000
100
10
0.25
Log Mr
Selectivity curves are usually quite linear over the range Kav = 0.1 to Kav = 0.7 and it is this part of the curve that is used to determine the fractionation range of a gel filtration medium (Figure 10).
1.0
0.7
Kav
Exclusion limit 0.1 Log Mr Fractionation range
Fig. 10. Defining fractionation range and exclusion limit from a selectivity curve.
The fractionation range defines the range of molecular weights that have partial access to the pores of the matrix, i.e. molecules within this range should be separable by high resolution fractionation. The exclusion limit for a gel filtration medium, also determined from the selectivity curve, indicates the size of the molecules that are excluded from the pores of the matrix and therefore elute in the void volume. The steeper the selectivity curve, the higher the resolution that can be achieved. When choosing an appropriate medium, consider two main factors: 1. The aim of the experiment (high resolution fractionation or group separation). 2. The molecular weights of the target proteins and contaminants to be separated. The final scale of purification should also be considered. Figure 11 on the next page gives a step by step guide to media selection.
17
Superdex Upper - medium pressure systems High recovery High stability High selectivity
High selectivity (0.1–600 kD) Wide Mr range ( 1–5 000 kD)
Preparative (0.5–5 000 kD)
Preparative & analytical (0.1–5 000 kD) Preparative /Macro fractionation (1–500 000 kD)
2 11 3
4
5
6
1 2
4 5
High selectivity (0.5–600 kD) Wide M r range ( 1–5 000 kD)
7
14 13 12
7
6
3
200
10
9
75
8
Peptide 10 6
10 5
10 4
10 3
Superose TM
Analytical (0.1–5 000 kD)
1. Thyroglobulin 2. Ferritin 3. Aldolase 4. Albumin 5. Ovalbumin 6. Chymotrypsinogen A 7. Ribonuclease A 8. Cytochrome C 9. Aprotinin 10. Gastrin I 11. Substance P 12. (Gly) 6 13. (Gly)3 14. Gly
1
10 2 Mr (approx)
1. Thyroglobulin 2. Ferritin 3. Aldolase 4. Albumin 5. Ovalbumin 6. Chymotrypsinogen A 7. Ribonuclease A
1 2
Medium pressure systems High recovery Wide M r fractionation range
4 3
5 6 7
2
1
3 4
6 7 5
Superose 6 Superose 12
10 6
10 5
10 4 Mr (approx)
Sephacryl 1 2
Lower - medium pressure systems Macromolecule separation Product line covering wide fractionation range
1
2 5 34
1 2
3
3
1. Thyroglobulin 2. Ferritin 3. Aldolase 4. Albumin 5. Ovalbumin 6. Chymotrypsinogen A 7. Ribonuclease A
67
45
S - 300
6 4 5
6
S - 200
7
S - 100 10 6
10 5
10 4
Mr (approx)
Fractionation NaCl
Group separation Desalting
Sephadex
BSA
Desalting <70% organic solvents
Group separation
0
10
20
30
>70% organic solvents
40
50
Time (seconds)
O
1
2
1.
Sephadex LH
CH 3
O
C O
Sta
250 mg
2. CH 3 C HN
254mg
re he
C O
Separation in (nonpolar) organic solvents
rt
C OH NH
68
Time (h)
Fig. 11. Gel filtration media selection guide.
Superdex is the first choice for high resolution, short run times and high recovery. Sephacryl is suitable for fast, high recovery separations at laboratory and industrial scale. Superose offers a broad fractionation range, but is not suitable for large scale or industrial scale separations. After deciding upon Superdex, Sephacryl or Superose, select the medium with the fractionation range that covers the molecular weight values of interest in your sample. In cases where two media have a similar fractionation range: select the medium with the steepest selectivity curve for best resolution of all components in the sample. When you are interested in a specific component, select the medium where the log molecular weight of the target component falls in the middle of the selectivity curve. Sephadex is ideal for rapid group separations such as desalting and buffer exchange. Sephadex is used at laboratory and production scale, before, between or after other chromatography purification steps. 18
OH
Fractionation range (globular proteins) Peptides Semi-preparative
Small proteins
Analytical separation
Polynucleotides Proteins DNA-fragment
Preparative separation
Semi-preparative Analytical separation Preparative separation
Superdex 75 prep grade Superdex 200 prep grade
Large proteins
Macro molecules
10
6
10
7
10
8
Superose 12 prep grade
Sephacryl S-100 HR Sephacryl S-200 HR Sephacryl S-300 HR
Sephacryl S-500 HR
Small particles Virus
Sephacryl S-1000 SF
Low molecular steroids
5
Superose 6 prep grade
Sephacryl S-400 HR
Proteins
10
Superose 12
Purification of macromolecules
Small peptides
4
Superose 6
Fractionation of macromolecules
Peptides/small proteins
10
Superdex 200
Proteins DNA-fragment
Proteins
3
High resolution
Superdex 30 prep grade
Intermediate fractionation range Wide fractionation range Intermediate fractionation range
10
Superdex 75
Small proteins Polynucleotides
Wide fractionation range
2
Superdex Peptide
Peptides
Small proteins Proteins
10
Sephadex Sephadex Sephadex Sephadex Sephadex
G-10 G-25 G-25 G-25 G-50
Exclusion limit SF F M F
Exclusion limit Exclusion limit
Sephadex LH-20
Terpenoids, lipids and peptides
• Sephadex G-25 is recommended for the majority of group separations involving globular proteins. This medium is excellent for removing salt and other small contaminants away from molecules that are greater than Mr 5 000. • Sephadex G-10 is well suited for the separation of biomolecules such as peptides (Mr >700) from smaller molecules (Mr >100). • Sephadex G-50 is suitable for the separation of molecules Mr >30 000 from molecules Mr <1 500 such as labeled protein or DNA from free label. For group separations select gel filtration media so that high molecular weight molecules are eluted at the void volume, with minimum peak broadening or dilution and minimum time on the column. The lowest molecular weight substances should appear by the time one column volume of buffer has passed through the column.
19
Sample preparation Correct sample preparation is extremely important for gel filtration. Simple steps to clarify a sample before applying it to a column will avoid the risk of blockage, reduce the need for stringent washing procedures and extend the life of the packed chromatography medium. Samples must be clear and free from particulate matter, particularly when working with bead sizes of 34 µm or less. Appendix 3 contains an overview of sample preparation techniques. For small sample volumes a syringe-tip filter of cellulose acetate or PVDF can be sufficient. Sample buffer composition The pH, ionic strength and composition of the sample buffer will not significantly affect resolution as long as these parameters do not alter the size or stability of the proteins to be separated and are not outside the stability range of the gel filtration medium. The sample does not have to be in exactly the same buffer as that used to equilibrate and run through the column. Sample is exchanged into the running buffer during the separation, an added benefit of gel filtration. Sample concentration and viscosity Gel filtration is independent of sample mass, and hence sample concentration, as can be seen in Figure 12. Hence high resolution can be maintained despite high sample concentration and, with the appropriate medium, high flow rates.
Medium: Column: Column volume: Sample: Sample volume: Buffer: Flow:
Superdex 200 prep grade XK 16/70 140 ml Solution of transferrin (Mr 81 000) and IgG (Mr 160 000) by equal weight 0.8% × Vt 0.05 sodium phosphate, 0.1 M sodium chloride, pH 7.2 1 ml/min (30 cm/h)
0
24
48
72
96
120
155
mg/ml sample
0.0
0.2
0.4
0.6
0.8
1.0
1.2
mg sample/ml packed bed
Resolution, R S
1.5
1.0
0.5
0.0
Fig. 12. Influence of sample concentration on the resolution of transferrin and IgG on Superdex 200 prep grade.
20
However, the solubility or the viscosity of the sample may limit the concentration that can be used. A critical variable is the viscosity of the sample relative to the running buffer, as shown by the change in elution profiles of haemoglobin and NaCl at different sample viscosities in Figure 13.
Concentration
Too high sample viscosity causes instability of the separation and an irregular flow pattern. This leads to very broad and skewed peaks and back pressure can increase.
A
B
C
Elution Volume
Fig. 13. Elution diagrams obtained when haemoglobin (blue) and NaCl (red) were separated. Experimental conditions were identical except that the viscosities were altered by the addition of increasing amounts of dextran. A deterioration of the separation becomes apparent. (A lower flow rate will not improve the separation.)
Samples should generally not exceed 70 mg/ml protein. Dilute viscous samples, but note sample volume (refer to page 14 for more information on the importance of sample volume). Remember that viscosity varies with temperature. Sample volume Sample volume is one of the most important parameters in gel filtration. Refer to page 14 for more information.
Buffer composition and preparation Buffer composition does not directly influence the resolution obtained in gel filtration since the separation should depend only on the sizes of the different molecules. The most important consideration is the effect of buffer composition on the shape or biological activity of the molecules of interest. For example, the pH and ionic strength of the buffer and the presence of denaturing agents or detergents can cause conformational changes, dissociation of proteins into subunits, dissociation of enzymes and cofactors, or dissociation of hormones and carrier proteins. Select a buffer and pH that are compatible with protein stability and activity and in which the product of interest should be collected. Use a buffer concentration that is sufficient to 21
maintain buffering capacity and constant pH. Use up to 0.15 M NaCl to avoid non-specific ionic interactions with the matrix (shown by delays in peak elution). Note that some proteins may precipitate in low ionic strength solutions. Use volatile buffers such as ammonium acetate, ammonium bicarbonate or ethylenediamine acetate if the separated product is to be lyophilized. Use high quality water and chemicals. Solutions should be filtered through 0.45 µm or 0.22 µm filters. It is essential to degas buffers before any gel filtration separation as air bubbles can significantly affect performance. Buffers will be degassed if they are filtered under vacuum. When working with a new sample try these conditions first: 0.05 M sodium phosphate, 0.15 M NaCl, pH 7.0 or select the buffer into which the product should be eluted for the next step such as further purification, analysis or storage. Avoid extreme changes in pH or other conditions that may cause inactivation or even precipitation. If the sample precipitates in a gel filtration column, the column will be blocked, possibly irreversibly, and the sample may be lost.
Denaturing (chaotropic) agents and detergents Denaturing agents such as guanidine hydrochloride or urea can be used for initial solubilization of a sample and in gel filtration buffers in order to maintain solubility of the sample. However, because they will denature the protein, they should be avoided unless denaturation is required. Modern media such as Superdex, Sephacryl and Superose are in general more suitable than classical media such as Sepharose™ or Sephadex for working under dissociating or denaturing conditions or at extreme pH values. Detergents are useful as solubilizing agents for proteins with low aqueous solubility such as membrane components and will not affect the separation. If denaturing agents or detergents are necessary to maintain the solubility of the sample, they should be present in both the running buffer and the sample buffer. Note that high concentrations of detergent will increase the viscosity of the buffer so that lower flow rates may be necessary to avoid over-pressuring the column packing. If proteins that have been solubilized in a denaturant or detergent are seen to precipitate, elute later than expected or be poorly resolved during gel filtration, add a suitable concentration of the denaturing agent or detergent to the running buffer. Urea or guanidine hydrochloride are very useful for molecular weight determination. The presence of these denaturing agents in the running buffer maintains proteins and polypeptides in an extended configuration. For accurate molecular weight determination the calibration standards must also be run in the same buffer. Note that selectivity curves are usually determined using globular proteins so they do not reflect the behavior of denatured samples. Gel filtration can be used to exchange a protein solubilized initially in, for example SDS, into a more gentle detergent such as Triton™ X-100 without losing solubility. 22
Column and media preparation To perform a separation, gel filtration medium is packed into a column 30–60 cm in height for high resolution fractionation and up to 10 cm in height for group separations. The volume of the packed bed is determined by the sample volumes that will be applied. Efficient column packing is essential, particularly for high resolution fractionation. The efficiency of a packed column defines its ability to produce narrow symmetrical peaks during elution. Column efficiency is particularly important in gel filtration in which separation takes place as only a single column volume of buffer passes through the column. The uniformity of the packed bed and the particles influences the uniformity of the flow through the column and hence affects the shape and eventual peak width. Gel filtration media with high uniformity (lower particle size distribution) facilitate the elution of molecules in narrow peaks. Refer to Chapter 3 Gel filtration in theory and Appendix 1 for further information on column efficiency and column packing. Efficiency can be improved by decreasing the particle size of a medium. However, using a smaller particle size may increase back pressure so that flow rate needs to be decreased, lengthening the run time. Using prepacked columns is highly recommended to ensure the best performance and reproducible results. An evenly packed column ensures that, as the sample passes down the column, the component peaks are not unnecessarily broadened. Uneven packing causes peak broadening and high resolution results become impossible. Allow buffers, media or prepacked columns to reach the same temperature before beginning preparation. Rapid changes in temperature, for example removing packed columns from a cold room and applying buffer at room temperature, can cause air bubbles in the packing and affect the separation. Storage solutions and preservatives should be washed away thoroughly before using any gel filtration medium. Equilibrate the column with 1–2 column volumes of buffer before starting a separation.
Sample application The choice of sample application method depends largely on the volume to be applied and on the equipment available. Ensure that the sample is not diluted on the way to the column and that the top of the column bed is not disturbed during sample application. Samples can be applied automatically or manually. Apply samples directly to the column via a chromatography system, a peristaltic pump or a syringe. The choice of equipment depends largely on the size of column, the type of gel filtration medium and the sample volume. For example, a chromatography system will be required for a Superdex column whereas a syringe can be used with small prepacked columns such as HiTrap Desalting. Note that samples are applied by gravity feed to prepacked columns such as PD-10 Desalting.
23
Elution and flow rates Samples are eluted isocratically from a gel filtration column, using a single buffer system. After sample application the entire separation takes place as one column volume of buffer (equivalent to the volume of the packed bed) passes through the column. Use flow rates that allow time for molecules to diffuse in and out of the matrix (partitioning between the mobile phase and the stationary phase) in order to achieve a separation. The goal for any separation is to achieve the highest possible resolution in the shortest possible time. Figures 14a, 14b and 14c show that resolution decreases as flow rate increases and each separation must be optimized to provide the best balance between these two parameters. Put simply, maximum resolution is obtained with a long column and a low flow rate whereas the fastest run is obtained with a short column and a high flow rate. Suitable flow rates for high resolution fractionation or group separation are usually supplied with each product. The advantage of a higher flow rate (and consequently a faster separation) may outweigh the loss of resolution in the separation. Column: Superdex 200 HR 10/30 (Vt: 24 ml) Sample: Mr Conc. (mg/ml) Thyroglobulin 669 000 3 Ferritin 440 000 0.7 IgG 150 000 3 Transferrin 81 000 3 Ovalbumin 43 000 3 Myoglobin 17 600 2 Vitamin B12 1 355 0.5 Total sample concentration: 15.2 mg/ml Buffer: 0.05 M sodium phosphate, 0.15 M NaCl, pH 7.0 Flow: 1) 0.25 ml/min (19.1 cm/h) 2) 1.0 ml/min (76.4 cm/h)
1)
A 280 nm
2)
25 µl, 0.25 ml/min (19 cm/h)
0.30
Vo
Vt
A 280 nm
25 µl, 1.0 ml/min (76 cm/h) Vo
0.25
Vt
0.25 0.20 0.20 0.15 0.15 0.10
0.10
0.05
0.05 0.00 0.0
25.0
50.0
75.0
Fig. 14a. Influence of flow rate on resolution.
24
100.0 min
0.00 0.0
5.0
10.0
15.0
20.0
25.0 min
2.0
1.5 Resolution, R s
a b
1.0
Column: Buffer: Sample : Sample volume: Sample concentration:
HiLoad 16/60 Superdex 30 prep grade 50 mM sodium acetate, 0.1 M NaCl, pH 5.0 IGF-1 containing monomers and dimers 1 ml (0.8% × Vt) a) 1.25 mg/ml b) 5 mg/ml
0.5
0 0
20
40
60
80 Flow, cm/h
Fig. 14b. Resolution between two different concentrations of IGF-1 containing monomers and dimers at different flow rates.
HiPrep 16/60
3
Resolution, R s
Columns:
HiPrep 16/10 Sephacryl S-100 HR HiPrep 16/10 Sephacryl S-200 HR HiPrep 16/10 Sephacryl S-300 HR Buffer: 50 mM NaPO4, 0.15 M NaCl, 0.02% NaN3, pH 7.0 Sample: IgG, ovalbumin, cytochrome C, 1:2:1 Sample volume: 2.4 ml (2% × Vt) Total sample load: 8 mg
2 S-100 S-200 1
S-300
0 0.0
0.2
0.4
0.6
0.8
1.0 1.2 Flow, ml/min
Fig. 14c. Resolution between IgG, ovalbumin and cytochrome C at different flow rates.
If peaks are well separated at a low flow rate, increase the flow rate or shorten the column to save time. Alternatively, increase the sample volume and benefit from a higher capacity without significant loss of resolution. For group separations such as desalting, monitor the elution of protein at A280 and follow the elution of the salt peak using a conductivity monitor. Adjust flow rate and sample volume to balance speed of separation against an acceptable level of salt in the final sample. Recommended flow rates are given in the instructions supplied with each product. Flow rate is measured in simple volume terms, e.g. ml/min, but when comparing results between columns of different sizes it is useful to use the linear flow, cm/hour (see Appendix 5). Results obtained at the same linear flow on different size columns will be comparable as far as the effects of flow rate are concerned. Selecting a smaller particle size of the same medium (if available) can also help to achieve the the correct balance between flow rate and resolution. Smaller particles of the same medium can increase column efficiency, improve resolution and may allow the use of higher flow rates. However, smaller particles can also result in increased back pressure and this factor may become restrictive if the intention is to scale up the separation. 25
Include a wash step at the end of a run to facilitate the removal of any molecules that may have been retained on the column, to prevent cross-contamination and to prepare the column for a new separation. Controlling flow Accurate, reproducible control of the flow rate is not only essential for good resolution, but also for reliability in routine preparative work and repeated experiments. A pump is used to control liquid flow for most gel filtration separations, although gravity feed has been used in the past (see Appendix 1). Use a pump within a chromatography system (rather than a peristaltic pump or gravity feed) to fully utilize the high rigidity and excellent flow properties of Sephacryl, Superose or Superdex for high resolution fractionation. Always pump the buffer onto a column (rather than drawing the buffer through the column with the pump below). This reduces the risk of bubble formation as a result of suction. If you have packed the column yourself, always use a flow rate for separation that is less than the flow rate used for column packing. Use a syringe or pump with small prepacked columns such as HiTrap Desalting or gravity feed with PD-10 Desalting for group separations of small sample volumes. Gel filtration columns must not run dry. Ensure that there is sufficient buffer for long, unattended runs or that the pump is programmed to stop the flow after a suitable time. Columns that run dry must be repacked since the packed bed has been destroyed. Reversing flow through a gel filtration column should only be considered under cases of severe contamination. There is a risk that reversing the flow may cause channeling through the packed bed leading to poor resolution, loss of efficiency and the need to repack the column. Professionally packed columns are less likely to be affected, but extreme care must be taken.
Method development for high resolution fractionation Steps are given in order of priority. 1. Select the medium that will give the best resolution of the target protein(s), see media selection guide, page 18. 2. To ensure reproducibility and high resolution, select a prepacked column that is best suited to the volume of sample that needs to be processed (see Chapter 2 for details of prepacked columns containing Superdex, Sephacryl or Superose). 3. Select the highest flow rate that maintains resolution and minimizes separation time. Check recommended flow rates supplied in the instructions for the specific medium and column. 4. Determine the maximum sample volume that can be loaded without reducing resolution. Higher flow rates and viscous buffers yield higher operating pressures (remember that buffer viscosity increases when running at +4 °C). Check the maximum operating pressure of the packed column and set the upper pressure limit on the chromatography system accordingly.
26
If greater resolution is required, increase the bed height by connecting two columns containing the same medium in series. Alternatively, try a medium with the same or similar fractionation range, but with a smaller particle size. To process larger sample volumes, see scaling up on this page.
Care of gel filtration media When a gel filtration medium has been in use for some time, it may be necessary to remove precipitated proteins or other contaminants that can build up. The need for cleaning may show itself as the appearance of a colored band at top of the column, as a space between the upper adaptor and the bed surface, as a loss in resolution or as a significant increase in back pressure. Detailed cleaning procedures for each gel filtration medium are given in Chapter 2. In all cases, prevention is better than cure and routine cleaning is recommended. If an increase in back pressure is observed, either on the pressure monitor or by seeing the surface of the medium move downwards, check that the problem is actually caused by the column before starting the cleaning procedure. Disconnect one piece of equipment at a time (starting at the fraction collector), start the pump and check the pressure after each piece is disconnected. A dirty on-line filter is a common cause of increased back pressure. Check back pressure at the same stage during each run, since the value can vary within a run during sample injection or when changing to a different buffer. • Always use filtered buffers and samples to reduce the need for additional column maintenance. See Appendix 3 for further details on sample preparation. • Always use well degassed buffers to avoid the formation of air bubbles in the packed column during a run. • Buffers, prepacked columns and samples should be kept at the same temperature to prevent air bubbles forming in the column. • Filter cleaning solutions before use and always re-equilibrate the column with 2–3 column volumes of buffer before the next separation.
Equipment selection Appendix 4 provides a guide to the selection of systems recommended for gel filtration separation.
Scaling up After establishing a high resolution or group separation on a small column, it may be preferred to pack a larger column in order to process larger sample volumes in a single step. General guidelines for scaling up are shown below. Maintain
Increase
Bed height
Column diameter
Linear flow rate
Volumetric flow rate
Sample composition
Sample volume
27
When scaling up a gel filtration column, follow the points below: 1. Optimize the separation at small scale (see method development page 26). 2. Maintain the sample volume: column volume ratio and the sample concentration. 3. Increase the column volume by increasing the cross sectional area of the column. 4. Maintain the bed height. 5. Run the separation at the same linear flow rate as used on the smaller column (see Appendix 5). Refer to Appendix 1 for column selection and column packing. Different equipment factors may affect performance after scale-up. If the large scale column has a less efficient flow distribution system, or the large scale system introduces large dead volumes, peak broadening may occur. This will cause extra dilution of the product fraction or even loss of resolution if the application is sensitive to variations in efficiency. For certain media, e.g. Superdex, Superose or Sephadex, it is usually recommended to select a larger particle size. For high resolution fractionation, pack a small column containing the larger particles and repeat the separation to facilitate any optimization that may be needed to achieve the same resolution on the larger column. Scaling up on Sephadex G-25, even to production scale, is a straightforward and wellestablished process. Well known examples of commercial applications include buffer exchange in processes for removing endotoxins from albumin, and preparative steps during the production of vaccines. Figure 15 shows an example of a large scale buffer exchange step used during the production of albumin and IgG from human plasma. Column: Sample:
Eluent: Flow:
1 2
1 2
1 2
1 2
BPSS 400/600, 75 l 10 l human plasma 1: sample application 2: eluent application 0.025 M sodium acetate 240 l/h
1 2 A 280 Conductivity
Elution volume
Fig. 15. Chromatogram of the buffer exchange step on Sephadex G-25 Coarse during production of albumin and IgG from human plasma.
28
BioProcess Media for large-scale production Specific BioProcess™ Media have been designed for each chromatography stage in a process from Capture to Polishing. Large capacity production integrated with clear ordering and delivery routines ensure that BioProcess media are available in the right quantity, at the right place and at the right time. Amersham Biosciences can assure future supplies of BioProcess Media, making them a safe investment for long-term production. The media are produced following validated methods and tested under strict control to fulfill high performance specifications. A certificate of analysis is available with each order. Regulatory Support Files (RSF) contain details of performance, stability, extractable compounds and analytical methods. The essential information in these files gives an invaluable starting point for process validation, as well as providing support for submissions to regulatory authorities. Using BioProcess Media for every stage results in an easily validated process. High flow rate, high capacity and high recovery contribute to the overall economy of an industrial process. All BioProcess Media have chemical stability to allow efficient cleaning and sanitization procedures. Packing methods are established for a wide range of scales and compatible large-scale columns and equipment are available.
Troubleshooting This section focuses on practical problems that may occur when running a chromatography column. The diagrams indicate how a chromatogram may deviate from ideal behavior during a gel filtration separation. The following pages contain suggestions of possible causes and their remedies. Highly acidic or basic substances at low ionic strength or aromatic materials may behave differently during gel filtration, interacting with the matrix. For some applications this can be an advantage. For example aromatic peptides and other substances that differ only slightly in molecular weight can be separated on Sephadex. However this is not a true gel filtration separation.
29
Satisfactory separation Well resolved, symmetrical peaks.
Vo
Vt
Poor resolution Review factors affecting resolution (see page 13), including media selection, particle size, sample volume: column volume and flow rate.
Vo
Vt
Leading peaks Asymmetric peaks: sample elutes before void volume indicates channelling in column bed. Leading peaks can also be due to overpacking of column (packed at too high pressure or flow rate). Column may need to be repacked. Vo
Tailing peaks Asymmetric peaks: sample application uneven. Check top of column if possible. Ensure medium is evenly packed and that sample is applied without disturbing the packed bed. Tailing peaks can also be due to underpacking of column (packed at too low pressure or flow rate). Vo
Vo
30
Vt
Late elution Peaks seen after one column volume of buffer has passed through the column. Always include a wash step between runs to ensure removal of late eluting molecules. Molecules may be binding non-specifically to gel filtration medium. If the interaction is ionic in nature, increasing the concentration of sodium chloride (up to 150 mM) may help. If the interaction is hydrophobic in nature, reducing salt concentration, increasing pH or adding a detergent or organic solvent may help.
Situation
Cause
Remedy
Peak of interest is poorly resolved from other major peaks.
Sample volume is too high or sample has been incorrectly applied.
Decrease sample volume and apply carefully. Check bed surface and top filter for possible contamination.
Sample is too viscous.
Dilute with buffer, but check maximum sample volume. Maintain protein concentration below 70 mg/ml.
Sample filtered incorrectly.
Reequilibrate column, filter sample and repeat.
Column not mounted vertically.
Adjust column position. Column may need to be repacked.
Column is poorly packed.
Check column efficiency (see Appendix 1). Repack if needed. Use prepacked columns.
Column is dirty.
Clean and reequilibrate.
Incorrect medium.
Check selectivity curve. Check for adsorption effects. Consider effects of denaturing agents or detergents if present.
Large dead volumes.
Minimize dead volumes in tubings and connections.
Column too short.
See Appendix 1 for recommended bed heights.
Flow rate too high.
Check recommended flow rates. Reduce flow.
Uneven temperature.
Use a column with a water jacket.
Sample volume is different from previous runs.
Keep sample volume constant. Resolution is dependent on sample volume.
Ionic interactions between protein and matrix.
Maintain ionic strength of buffers above 0.05 M (preferably include 0.15 M NaCl).
Hydrophobic interactions between protein and matrix.
Reduce salt concentration to minimize hydrophobic interaction. Increase pH. Add suitable detergent or organic solvent, e.g. 5% isopropanol.
Sample has not been filtered properly.
Clean the column, filter the sample and repeat.
Sample has changed during storage.
Prepare fresh samples.
Column is not equilibrated sufficiently.
Repeat or prolong the equilibration step.
Proteins or lipids have precipitated on the column.
Clean the column or use a new column.
Column is overloaded with sample.
Decrease the sample load.
Microbial growth has occurred in the column.
Microbial growth rarely occurs in columns during use. To prevent infection of packed columns, store in 20% ethanol when possible.
Protein has changed during storage.
Prepare fresh samples.
Ionic interactions between protein and matrix.
Maintain ionic strength of buffers above 0.05 M (preferably include 0.15 M NaCl).
Hydrophobic interactions between protein and matrix.
Reduce salt concentration to minimize hydrophobic interaction. Add suitable detergent or organic solvent, e.g. 5% isopropanol.
Precipitation of protein in the column filter and/ or at the top of the bed.
If possible, clean the column, exchange or clean the filter or use a new column.
Protein elutes later than expected or even after running a total column volume.
Hydrophobic and/or ionic interactions between protein and matrix.
Reduce salt concentration to minimize hydrophobic interaction. Increase pH. Add suitable detergent or organic solvent e.g. 5% isopropanol. Increase salt concentration (up to 150 mM) to minimize ionic interaction.
Peaks elute late and are very broad.
Column is dirty.
Clean and reequilibrate.
Protein elutes earlier than expected (before the void volume)
Channelling in the column.
Repack column using a thinner slurry of medium. Avoid introduction of air bubbles.
Protein does not elute as expected.
Molecular weight or shape is not as expected.
31
Situation
Cause
Remedy
Leading or very rounded peaks in chromatogram.
Column overloaded.
Decrease sample load and repeat.
Tailing peaks.
Column is 'under' packed.
Check column efficiency (see Appendix 1). Repack using a higher flow rate. Use prepacked columns.
Leading peaks.
Column is 'over' packed.
Check column efficiency (see Appendix 1). Repack using a slower flow rate. Use prepacked columns.
Medium/beads appears in eluent.
Bed support end piece is loose or broken.
Replace or tighten.
Column operated at too high pressure.
Do not exceed recommended operating pressure for medium or column.
Protein may be unstable or inactive in the buffer.
Determine the pH and salt stability of the protein.
Enzyme separated from co-factor or similar.
Test by pooling aliquots from the fractions and repeating the assay.
Protein may have been degraded by proteases.
Add protease inhibitors to the sample and buffers to prevent proteolytic digestion. Run sample through a medium such as Benzamidine 4 Fast Flow (high sub) to remove trypsin-like serine proteases.
Adsorption to filter during sample preparation.
Use another type of filter.
Sample precipitates.
May be caused by removal of salts or unsuitable buffer conditions.
Hydrophobic proteins.
Use denaturing agents, polarity reducing agents or detergents.
Non-specific adsorption.
Reduce salt concentration to minimize hydrophobic interaction. Increase pH. Add suitable detergent or organic solvent e.g. 5% isopropanol. If necessary, add 10% ethylene glycol to running buffer.
Sample absorbs poorly at chosen wavelength.
Check absorbance range on monitor. If possible, use a different wavelength, e.g. 214 nm instead of 280 nm.
Low recovery of activity, but normal recovery of protein.
Lower yield than expected.
Peaks too small.
Excessive band broadening.
Check column packing. Repack if necessary.
More sample is recovered than expected.
Protein is co-eluting with other substances.
Optimize conditions to improve resolution. Check buffer conditions used for assay before and after the run. Check selection of medium.
More activity is recovered than was applied to the column.
Different assay conditions have been used before and after the chromatography step.
Use the same assay conditions for all the assays in the purification scheme.
Removal of inhibitors during separation. Reduced or poor flow through the column.
32
Presence of lipoproteins or protein aggregates.
Remove lipoproteins and aggregates during sample preparation (see Appendix 3).
Protein precipitation in the column caused by removal of stabilizing agents during separation.
Modify the eluent to maintain stability.
Blocked column filter.
If possible, replace the filter or use a new column. Always filter samples and buffer before use.
Blocked end-piece or adaptor or tubing.
If possible, remove and clean or use a new column.
Precipitated proteins.
Clean the column using recommended methods or use a new column.
Bed compressed.
If possible repack the column or use a new column.
Microbial growth.
Microbial growth rarely occurs in columns during use, but, to prevent infection of packed columns, store in 20% ethanol when possible.
Medium not fully swollen (Sephadex).
See Appendix 1 for reswelling conditions.
Situation
Back pressure increases during a run or during successive runs.
Air bubbles in the bed.
Cause
Remedy
Fines (Sephadex).
Decant fines before column packing. Avoid using magnetic stirrers that can break the particles.
Turbid sample.
Improve sample preparation (see Appendix 3). Improve sample solubility by the addition of ethylene glycol, detergents or organic solvents.
Precipitation of protein in the column filter and/or at the top of the bed.
Clean using recommended methods. If possible, exchange or clean filter or use a new column. Include any additives that were used for initial sample solubilization in the running buffer.
Column packed or stored at cool temperature and then warmed up.
Remove small bubbles by passing degassed buffer through the column. Take special care if buffers are used after storage in a fridge or cold-room. Do not allow column to warm up due to sunshine or heating system. Repack column, if possible (see Appendix 1).
Buffers not properly degassed.
Buffers must be degassed thoroughly.
Cracks in the bed.
Large air leak in column.
Check all connections for leaks. Repack the column if possible (see Appendix 1).
Distorted bands as sample runs into the bed.
Air bubble at the top of the column or in the inlet adaptor.
If possible, re-install the adaptor taking care to avoid air bubbles.
Particles in buffer or sample.
Filter or centrifuge the sample. Protect buffers from dust.
Blocked or damaged net in upper adaptor.
If possible, dismantle the adaptor, clean or replace the net. Keep particles out of samples and eluents.
Column poorly packed.
Suspension too thick or too thin. Bed packed at a temperature different from run. Bed insufficiently packed (too low packing pressure, too short equilibration). Column packed at too high pressure.
Distorted bands as sample passes down the bed.
33
Fig. 16. Time-line for a gel filtration separation on Sephadex G-200 compared to the latest Superdex 200 prep grade (which has replaced Sephadex G-200) and a prepacked HiLoad 16/60 Superdex prep grade column.
34
Packing Degas
4 5
10
Packing with adaptor
15
2 3
Result after: 3.3 h
Result Sample application Equilibration (1.5 × Vt )
0
Time (hours)
HiLoad 16/60 Superdex 200 prep grade
Packing
Washing gel slurry
Result Sample application Check the packed bed Equilibration (1.5 × Vt ) Packing with adaptor
21
Equilibration (1.5 × Vt )
Result after: 6.3 h
Superdex 200 prep grade XK 16/60 1.5 5 Time 0.5 4 (hours) 0 2 6
Swelling
0
Sephadex G-200 XK 16/60 30
Check the packed bed
36
40
Sample application
46
50
Time (hours)
Result after: 56 h
Result
56
Chapter 2 Gel filtration media Components of gel filtration media The development of gel filtration media has been driven by the need to achieve the highest flow rates while retaining the highest resolution. As a sample passes through a gel filtration column the separation or resolution of the different components is affected by several parameters: flow rate, particle size, particle size distribution, packing density, porosity of the particle and viscosity of the mobile phase. Attempts to optimize each parameter has led to the development of a series of gel filtration media. The earliest gel filtration matrices were formed by cross-linking polymers to form a threedimensional network, for example Sephadex is formed by cross-linking dextran. Controlling the degree of cross-linking and particle size made it possible to produce a broad range of media, each one having a high selectivity over a narrow range of molecular weight values. However, to increase the speed of a separation the medium must withstand higher flow rates and so alternative polymers such as agarose were investigated. This resulted in gel filtration media based on Sepharose and, later, the more highly cross-linked Superose. Matrices based on agarose are, in general, more porous than those based on dextran so that, although the speed of a separation could be increased there was less selectivity when compared to Sephadex. The porosity of Sepharose makes it highly suitable for binding techniques such as affinity chromatography where the high porosity facilitates a high binding capacity. A major advance in gel filtration technology occurred when composite gels could be prepared by grafting a second polymer onto a pre-formed matrix, for example Sephacryl (cross-linking allyl dextran with N,N'-methylene bisacrylamide) and the most recent, Superdex. In the case of Superdex, with the dextran chains covalently bonded to a highly cross-linked agarose matrix, it has been possible to create a range of media with the same high selectivity as Sephadex, but with the mechanical strength of a highly cross-linked agarose-based matrix. The selectivity curves and pressure-flow relationship curves shown for Superose, Sephacryl and Superdex in the following sections show how the performance of gel filtration media has developed. Figure 16 clearly illustrates the significant increase in speed without loss of performance that has been achieved with the development of Superdex. For Superose and Superdex, manipulation of the particle size (Superose prep grade or Superdex prep grade) is also used to allow higher flow rates when running larger columns.
35
Superdex: first choice for high resolution, short run times and high recovery 10 2
10 3
10 4
10 5
Superdex Peptide Superdex 75 Superdex 200 Superdex 30 prep grade Superdex 75 prep grade Superdex 200 prep grade
Fig. 17. Fractionation ranges for Superdex.
Fig. 18. Prepacked HiLoad Superdex prep grade columns.
From laboratory to process scale applications, Superdex is the first choice for a high resolution fractionation with short run times and good recovery. The success of Superdex is clearly demonstrated by the hundreds of scientific publications in which the use of Superdex has been described. Reference lists highlighting the use of the prepacked columns HiLoad Superdex 200 prep grade, HiLoad Superdex 75 prep grade and HiLoad Superdex 30 prep grade are available at www.chromatography.amershambiosciences.com. Selectivity curves and pressure-flow relationship curves for Superdex are shown in Figures 19a and 19b. A typical linear flow is up to 75 cm/h. K av 1.00
Superdex peptide 0.75 Superdex 75 Superdex 200 Superdex 30 prep grade Superdex 75 prep grade
0.50
Superdex 200 prep grade
1000000
100000
10000
1000
100
10
0.25
Log Mr
Fig. 19a. Selectivity curves for Superdex (13 µm) and Superdex prep grade (34 µm) media.
36
Pressure (bar)
4
3
0 /6 26
0
/6
16
2
1
20
40
60
80
100 120 140 Flow rate (cm/h)
Fig. 19b. Pressure drop as a function of flow rate for HiLoad columns packed with Superdex prep grade. Bed height approximately 60 cm in distilled water at +25 °C. To calculate volumetric flow rate, multiply linear flow by crosssectional area of column (2 cm2 for XK 16, 5.3 cm2 for XK 26).See Appendix 5 for more information about flow rate calculations.
Superdex is a composite medium based on highly cross-linked porous agarose particles to which dextran has been covalently bonded, as illustrated in Figure 20. The result is media with high physical and chemical stability, due mainly to the highly cross-linked agarose matrix, and excellent gel filtration properties determined mainly by the dextran chains. The mechanical rigidity of Superdex allows even relatively viscous eluents, such as 8 M urea, to be run at practical flow rates. The media can withstand high flow rates during equilibration or cleaning thereby shortening overall cycle times. This stability makes Superdex prep grade very suitable for use in industrial processes where high flow rates and fast, effective cleaningin-place protocols are required. Under normal chromatography conditions non-specific interactions between proteins and Superdex are negligible when using buffers with ionic strengths in the range 0.15 M to 1.5 M.
Crosslinked Agarose Dextran
Fig. 20. In Superdex the dextran chains are covalently linked to a highly cross-linked agarose matrix. The figure shows a schematic of a section through a Superdex particle.
37
Separation options Superdex is produced in two different mean particle sizes (13 µm and 34 µm) and four different selectivities (Superdex Peptide, Superdex 30, Superdex 75 and Superdex 200). • Use the 13 µm particles of Superdex Peptide, Superdex 75 and Superdex 200 in prepacked columns for highest resolution analytical separations with smaller sample volumes. • Use the 34 µm particles of Superdex prep grade (available in prepacked columns or as loose media) for larger scale applications. Product †
Fractionation range, Mr (globular proteins)
Sample loading capacity
Superdex Peptide PC 3.2/30
1×102–7×103
25–250 µl
1.8 MPa, 18 bar, 260 psi
<0.15 ml/min
Superdex Peptide HR 10/30
1×102–7×103
25–250 µl
1.8 MPa, 18 bar, 260 psi
<1.2 ml/min
HiLoad 16/60 Superdex 30 pg*
<1×104
<5 ml
0.3 MPa, 3 bar, 42 psi
<1.6 ml/min
HiLoad 26/60 Superdex 30 pg*
<1×104
<13 ml
0.3 MPa, 3 bar, 42 psi
<4.4 ml/min
Superdex 30 pg*
<1×104
0.5–4% of total column volume
0.5 MPa, 5 bar, 70 psi
10–50 cm/h
Superdex 75 PC 3.2/30
3×103–7×104
<50 µl
2.4 MPa, 24 bar, 350 psi
<0.1 ml/min
Superdex 75 HR 10/30
3×103–7×104
25–250 µl
1.8 MPa, 18 bar, 260 psi
<1.5 ml/min
HiLoad 16/60 Superdex 75 pg*
3×103–7×104
<5 ml
0.3 MPa, 3 bar, 42 psi
<1.6 ml/min
HiLoad 26/60 Superdex 75 pg*
3×103–7×104
<13 ml
0.3 MPa, 3 bar, 42 psi
<4.4 ml/min
Superdex 75 pg*
3×103–7×104
0.5–4% of total column volume
0.5 MPa, 5 bar, 70 psi
10–50 cm/h
Superdex 200 PC 3.2/30
1×104–6×105
<50 µl
1.5 MPa, 15 bar, 220 psi
<0.1 ml/min
Superdex 200 HR 10/30
1×104–6×105
25–250 µl
1.5 MPa, 15 bar, 220 psi
0.25–0.75 ml/min
HiLoad 16/60 Superdex 200 pg* 1×104–6×105
<5 ml
0.3 MPa, 3 bar, 42 psi
<1.6 ml/min
HiLoad 26/60 Superdex 200 pg* 1×104–6×105
<13 ml
0.3 MPa, 3 bar, 42 psi
<4.4 ml/min
0.5–4% of total column volume
0.5 MPa, 5 bar, 70 psi
10–50 cm/h
Superdex 200 pg*
1×104–6×105
Maximum operating back pressure ‡
Recommended operating flow ¶
* prep grade. †
HR and PC columns are packed with Superdex and HiLoad columns are packed with Superdex prep grade.
For maximum resolution apply as small a sample volume as possible, but note that sample volumes less than 0.5% do not normally improve resolution.
‡
¶
See Appendix 5 to convert linear flow (cm/hour) to volumetric flow rates (ml/min) and vice versa.
Start with Superdex 200 when the molecular weight of the protein of interest is unknown. Superdex 200 or Superdex 200 prep grade (pg) are especially suitable for the separation of monoclonal antibodies from dimers and from contaminants of lower molecular weight, for example albumin and transferrin. Start with Superdex Peptide or Superdex 30 prep grade for separations of peptides, oligonucleotides and small proteins below Mr 10 000. Exposure to temperatures outside the range +4 °C to +40 °C will destroy the efficiency of the packed bed and the column will need to be re-packed.
38
Separation examples The figures below illustrate examples of separations performed on Superdex Peptide, Superdex 75 and Superdex 200. a)
Sample:
b) mV
A 214 nm 0.12
5000
Sample volume: Column:
4000
Eluent:
Mr 4
1 2 3 4 5 6 7
0.10 2
3
0.08
Cytochrome C Aprotinin Gastrin I Substance P (Gly) 6 (Gly) 3 Gly
12 500 6 500 2 126 1 348 360 189 75
Flow: 3000
1 7 5
100 µl Two Superdex Peptide HR 10/30 in series, effective bed height 60 cm 30% acetonitrile, 0.1% TFA in water 0.6 ml/min
A - Tetramer B - Trimer C - Dimer D - Monomer
6
0.06
Synthetic cyclic peptide (polymers)
2000
0.04
1000
0.02
V0
Vt
0 0
5
10
15
1
20 25 Volume (ml)
5
30 A
45 B
60
C D
75 90 Time (min)
Fig. 21. a) Separation of standard peptides on Superdex Peptide HR 10/30. b) Separation of peptide polymers (monomer Mr ~1 000). The fractions indicated were analyzed by off-line mass spectrometry. Courtesy of K. Walhagen, Ferring Research Institute, Sweden.
2
A 280 nm
7
Column: Sample:
0.30 Vt
Vo
0.20 1
3
Superdex 200 HR 10/30 1. Thyroglobulin (Mr 669 000), 3.0 mg/ml 2. Ferritin (M r 440 000), 0.7 mg/ml 3. Human IgG (M r 150 000), 3.0 mg/ml 4. Human transferrin (Mr 81 000), 3.0 mg/ml 5. Ovalbumin (M r 43 000), 3.0 mg/ml 6. Myoglobin (Mr 17 600), 2.0 mg/ml 7. Vitamin B12 (M r 1 355), 0.5 mg/ml
Total sample amount: 0.38 mg Sample volume: 25 ml Buffer: 0.05 M sodium phosphate, 0.15 M NaCl, pH 7.0 Flow: 0.25 ml/min (19 cm/h)
4 5 6
0.10
0 0
5.0
10.0
15.0
20.0
25.0 Volume (ml)
Fig. 22. Separation of standard proteins on Superdex 200 HR 10/30.
39
A 280 nm Monomer
0.05
Column: Sample:
Superdex 75 HR 10/30 A specially prepared sample of rhGH in distilled water Concentration: 1.5 mg/ml Sample volume: 50 µl (75 µg rhGH) Buffer: 0.05 M sodium phosphate, 0.1 M Na2SO4, pH 7.3 Flow: 1 ml/min (76 cm/h)
0.025
Oligomer
Dimer 10
20
Time (min)
VO
Vt
Fig. 23. Separation of growth hormone oligomers on Superdex 75 HR 10/30.
b)
a) A 280 nm
A 280 nm
Vt
Vo
Vo
Vt
Column: a) Superdex 200 HR 10/30 b) HiLoad 16/60 Superdex 200 prep grade Bed volumes: a) 24 ml b) 122 ml Sample: Mouse monoclonal IgG1 10 × concentrated cell culture supernatant Sample volume: a) 200 µl, 0.8% × Vt b) 1.0 ml, 0.8% × Vt Buffer: 50 mM NaH2PO4, 0.15 M NaCl, pH 7.0 Flow: a) 1.0 ml/min (76 cm/h) b) 0.5 ml/min (15 cm/h)
0.60 0.60
IgG1 0.40
IgG 1
0.40
0.20
0.20
5.0
15.0
25.0
ml
50
150
250
ml
Fig. 24. Scale up (five times) of a mouse monoclonal IgG1 purification from Superdex 200 HR 10/30 (a) onto HiLoad 16/60 Superdex 200 prep grade (b).
Absorbance 0.005
TFA
Column: HiLoad 16/60 Superdex 30 prep grade Sample: 50 ml mix of five synthetic peptides in 1% TFA 1. Mr 3 894 2. Mr 3 134 3. Mr 2 365 4. Mr 1 596 5. Mr 827 Buffer: 20 mM Tris-HCl, 0.25 M NaCI, pH 8.5 Flow: 1 ml/min (30 cm/h)
1 2 3 4
0
80
160
240
5
320 Volume (ml)
Fig. 25. Separation of test substances on HiLoad 16/60 Superdex 30 prep grade.
40
Columns:
a) HiLoad 16/60 Superdex 75 prep grade b) HiLoad 16/60 Superdex 200 prep grade 1. Myoglobin 1.5 mg/ml, Mr 17 000 2. Ovalbumin 4 mg/ml, Mr 43 000 3. Albumin 5 mg/ml, Mr 67 000 4. IgG 0.2 mg/ml, Mr 158 000 5. Ferritin 0.24 mg/ml, Mr 440 000 Sample volume: 0.5 ml Buffer: 0.05 M phosphate buffer, 0.15 M NaCl, 0.01% sodium azide, pH 7.0 Flow: 1.5 ml/min (45 cm/h)
Sample:
b)
a) A 280 nm
A 280 nm
HiLoad 16/60 Superdex 75 prep grade
4 and 5
0.2
3
HiLoad 16/60 Superdex 200 prep grade
0.2
2 3
2
0.1
0.1
5
1
1 Vo
4
0
0 0
10
20
30
40
50
0
60 70 Time (min)
10
20
30
40
50
60 70 Time (min)
Fig. 26. Comparison of the selectivity of Superdex 75 prep grade and Superdex 200 prep grade for model proteins. Superdex 75 prep grade (a) gives excellent resolution of the three proteins in the Mr range 17 000 to 67 000 while the two largest proteins elute together in the void volume. Superdex 200 prep grade (b) resolves the two largest proteins completely. The three smaller proteins are not resolved quite as well as the larger ones or as in (a). The void volume (Vo) peak at 28 minutes in (b) is caused by protein aggregates.
Columns: Column volumes, Vt:
HiLoad Superdex 200 prep grade
a) ≈ 120 ml (16/60) b) ≈ 320 ml (26/60) Sample: Mouse monoclonal cell supernatant, IgG2b incl. 1% Fetal Calf Serum Sample Concentration ≈ 40× pretreatment: Sample volume: a) 1.2 ml (1% × Vt) b) 3.2 ml (1% × Vt) Buffer: 50 mM NaH2PO4, 0.15 NaCI, pH 7.0 Flow: a) 1.6 ml/min (50 cm/h) b) 4.4 ml/min (50 cm/h) (max recommended flow rates) b)
a)
A 280 nm
A 280 nm
HiLoad 16/60 Superdex 200 prep grade
0.60
IgG2b
IgG2b
0.40
0.40
0.20
0.20
0.00
0.00 0
20.0
40.0
HiLoad 26/60 Superdex 200 prep grade
0.60
60.0
80.0 Time (min)
0
20.0
40.0
60.0
80.0 Time (min)
Fig. 27. Purification of mouse monoclonal IgG2b from cell supernatant using a) HiLoad 16/60 Superdex 200 prep grade, column volume 120 ml and b) HiLoad 26/60 Superdex 200 prep grade, column volume 320 ml. Almost identical separations are the result, even using prepacked columns of different sizes.
41
Protein refolding After solubilization, recombinant proteins must be properly refolded to regain function. Denaturing agents must be removed to allow refolding of the protein and formation of the correct intramolecular associations. Critical parameters during refolding include pH, presence of thiol reagents, the speed of denaturant removal, and the relative concentrations of host proteins and recombinant protein. The table below compares the advantages and disadvantages of the alternative methods for refolding of recombinant proteins. Different proteins require different conditions for successful refolding. Gel filtration provides an alternative method that can be tried if on-column refolding during affinity purification is not possible. For further details about on-column refolding using affinity chromatography refer to The Recombinant Protein Handbook, Amplification and Simple Purification, available from Amersham Biosciences and Febs Letters 345 (1994) 125–130. Refolding technique
Advantages/Disadvantages
Step dialysis
Takes several days. Uses large volumes of buffer.
Dilution into near neutral pH
Dilutes the protein of interest.
On-column gel filtration
Slower than on-column refolding by affinity chromatography. Requires a second column to be run. Only small volumes can be processed per column.
On-column affinity chromatography
Fast and simple. No sample volume limitations.
Performing a separation Buffer: 0.05 M sodium phosphate, 0.15 M NaCl, pH 7 or select the buffer in which the sample should be stored or solubilized for the next step.
Use 0.15 M NaCl, or a buffer with equivalent ionic strength, to avoid pH dependent nonionic interactions with the matrix. At very low ionic strength, the presence of a small number of negatively charged groups on the medium may cause retardation of basic proteins. The sample should be fully dissolved. Centrifuge or filter to remove particulate material (see Appendix 3). Always use degassed buffers and maintain a constant temperature during the run to avoid introducing air into the column. Set an appropriate pressure limit on the chromatography system to avoid damage to the column packing. 1. For first time use, or after long term storage, equilibrate the column with 1 column volume of buffer, but containing 0.05 M NaCl at 30 cm/h (0.4 ml/min for HR 10/30, 1 ml/min for XK 16/60 or 2.6 ml/min for XK 26/60). 2. Equilibrate with 2 column volumes of buffer containing 0.15 M NaCl at 50 cm/h (0.65 ml/min for HR 10/30, 1.6 ml/min for XK 16/60 or 4.3 ml/min for XK 26/60). 3. Reduce linear flow to 30 cm/h. Apply a sample volume equivalent to 0.5–4% of the column volume (up to 0.25 ml for HR 10/30, 1.2 ml for XK 16/60 or 3.2 ml for XK 26/60). Note that the smaller the sample volume the better the resolution. 4. Elute with 1 column volume of buffer. 5. Before applying a new sample, re-equilibrate column with 1 column volume of buffer at 50 cm/h and until the baseline monitored at A280 is stable.
42
Column performance should be checked at regular intervals by determining the theoretical plate number per meter and peak symmetry. Prepacked columns are supplied with recommended values. See page 95 for how to check column efficiency. See page 26 for advice on optimizing the separation. Exposure to temperatures outside the range +4 °C to +40 °C will destroy the efficiency of a packed bed and the column will need to be re-packed.
Cleaning 1. Wash with 1 column volume of 0.5 M NaOH at a flow of 25 cm/h (0.33 ml/min for HR 10/30, 0.8 ml/min for XK 16/60 or 2.2 ml/min for XK 26/60) to remove most non-specifically adsorbed proteins. 2. Wash with 1 column volume of distilled water at 25 cm/hr. 3. Re-equilibrate with 2 column volumes of buffer at a flow of 50 cm/hr (0.4 ml/min for HR 10/30, 1.6 ml/min XK 16/60 or 4.3 ml/min for XK 26/60) or until the baseline monitored at A280 and the pH of the eluent are stable. Further equilibration may be necessary if the buffer contains detergent.
Routine cleaning after every 10–20 separations is recommended, but the frequency of cleaning will also depend on the nature of the samples being applied. To remove severe contamination 1. Reverse the flow and wash at a linear flow of 25 cm/h at room temperature. 2. Wash with 4 column volumes of 1 M NaOH (to remove hydrophobic proteins or lipoproteins) followed by 4 column volumes of distilled water. 3. Wash with 0.5 column volume of 30% isopropanol (to remove lipids and very hydrophobic proteins), followed by 2 column volumes of distilled water. 4. Equilibrate the column with at least 5 column volumes of buffer, or until the baseline monitored at A280 and the pH of the eluent are stable, before beginning a new separation. For extreme cases of contamination, check the instructions supplied with the product.
Reversing flow through a gel filtration column should only be considered under cases of severe contamination. There is a risk that reversing the flow may cause channeling through the packed bed leading to poor resolution, loss of efficiency and the need to repack the column. Professionally packed columns are less likely to be affected, but extreme care must be taken.
43
Media characteristics Composition: Superdex is formed from dextran bound covalently to highly cross-linked agarose. Product
Efficiency: theoretical plates per meter (prepacked columns only)
pH stability*
Particle size
Mean particle size
Superdex Peptide
>30 000 m-1
Long term: 1–14 Short term: 1–14
13–15 µm
13 µm
Superdex 75
>30 000 m-1
Long term: 3–12 Short term: 1–14
13–15 µm
13 µm
Superdex 200
>30 000 m-1
Long term: 3–12 Short term: 1–14
13–15 µm
13 µm
Superdex 30 prep grade
>13 000 m-1
Long term: 3–12 Short term: 1–14
22–44 µm
34 µm
Superdex 75 prep grade
>13 000 m-1
Long term: 3–12 Short term: 1–14
22–44 µm
34 µm
Superdex 200 prep grade
>13 000 m-1
Long term: 3–12 Short term: 1–14
22–44 µm
34 µm
*Long term pH stability refers to the pH interval where the medium is stable over a long period of time without adverse side effects on its chromatography performance. Short term pH stability refers to the pH interval for regeneration, cleaning-in-place and sanitization procedures. All ranges are estimates based on the experience and knowledge within Amersham Biosciences.
Chemical stability Superdex is stable in all commonly used aqueous buffers, pH 3–12, and additives such as detergents (1% SDS), denaturing agents (8 M urea or 6 M guanidine hydrochloride). The following solutions can be used for cleaning: up to 30% acetonitrile, up to 1 M sodium hydroxide, up to 70% ethanol (Superdex 30 prep grade), up to 24% ethanol (Superdex 75 prep grade and Superdex 200 prep grade), up to 1 M acetic acid, up to 30% isopropanol or up to 0.1 M HCl (Superdex 30 prep grade).
Storage Store unused media +4 °C to +25 °C in 20% ethanol. Do not freeze. Columns can be left connected to a chromatography system with a low flow rate (0.01 ml/min) of buffer passing through the column to prevent bacterial growth or the introduction of air into the column which would destroy the packing. For long term storage, wash with 4 column volumes of distilled water followed by 4 column volumes of 20% ethanol. Store at +4 °C to +25 °C. Degas the ethanol/water mixture thoroughly and use a low flow rate, checking the back pressure as the column equilibrates. Avoid changes in temperature which may cause air bubbles in the packing.
44
Sephacryl: fast, high recovery separations at laboratory and industrial scale Sephacryl High Resolution (HR) media provide a useful alternative to Superdex prep grade for applications that require a slightly broader fractionation range, as shown in Figure 28. High chemical stability and tolerance of high flow rates make Sephacryl well suited for industrial use. 10
2
10
3
10
4
10
5
10
6
10
7
10
8
Sephacryl S-100 HR Sephacryl S-200 HR Sephacryl S-300 HR Sephacryl S-400 HR Sephacryl S-500 HR Sephacryl S-1000 SF
Fig. 28. Fractionation ranges for Sephacryl High Resolution (HR).
Fig. 29. Sephacryl is available as loose media and in prepacked columns.
Reference lists highlighting the use of HiPrep Sephacryl S-300 HR, HiPrep Sephacryl S-200 HR and HiPrep Sephacryl S-300 HR are available at www.chromatography.amershambiosciences.com.
45
Figure 30 shows a comparison of the different selectivities of Sephacryl High Resolution. Typical selectivity and pressure-flow relationship curves for Sephacryl are shown in Figures 31a and 31b. A 280 nm
a) HiPrep 16/60 Sephacryl S-100 HR
0.3
BSA IgG
Cytochrome C
0.2
Cytidine b-lactoglobulin
0.1
20 A 280 nm
40
60
80
100
Column: a) HiPrep 16/60 Sephacryl S-100 HR b) HiPrep 16/60 Sephacryl S-200 HR c) HiPrep 16/60 Sephacryl S-300 HR Sample: 500 µl of a mixture containing IgG (Mr 160 000), BSA (Mr 67 000), b-lactoglobulin (M r 35 000), cytochrome C (Mr 12 400) and cytidine (Mr 240) Buffer: 0.05 M sodium phosphate, 0.15 M NaCl, pH 7.0 Flow: 0.8 ml/min (24 cm/h)
120 Vol (ml)
b) HiPrep 16/60 Sephacryl S-200 HR
0.3 BSA 0.2
Cytochrome C
IgG
Cytidine b-lactoglobulin
0.1
20 A 280 nm
40
60
80
100
120 Vol (ml)
c) HiPrep 16/60 Sephacryl S-300 HR BSA
0.2
Cytochrome C
Cytidine b-lactoglobulin
IgG 0.1
20
40
60
80
100
120 Vol (ml)
Fig. 30. Comparison of the selectivity of HiPrep 16/60 Sephacryl S-100 HR, HiPrep 16/60 Sephacryl S-200 HR, and HiPrep 16/60 Sephacryl S-300 HR columns.
K av
Globular proteins*
K av
0.8
0.8
0
50
S-
0.8
Dextran standards*
Proteins**
K av
00
0.6
HR
5
6
10 10 10 Molecular weight, M r
0.4
0.4
0.2
0.2
4
5
6
10 10 10 Molecular weight, M r
* In 0.05 M phosphate buffer, 0.15 M NaCl, pH 7.0. ** In 6 M guanidine hydrochloride.
Fig. 31a. Selectivity curves for Sephacryl High Resolution media.
46
HR
HR
0.2
4
00
0
4 S-
40 0.6
HR
S-4
S-
HR 00 HR 00 S-2 HR 00 S-1
0.4
S-3
0.6
4
5
6
10 10 10 Molecular weight, Mr
a)
b) MPa*
MPa*
Column diameter 1.6 cm
0.3
Column diameter 2.6 cm
0.3
R
0H
0.2
0 S-2
S-3
0
0.2
R 0H
S-
S-1
S-500
00
S-3
HR 00
HR
S-1
R 00 H
0.1
2
00
0.1
HR
HR
HR S-400 0 HR S-50
S-400 HR 25
75
50
100
25
75
50
Linear flow rate cm.h -1
100
Linear flow rate cm.h -1 *0.1 MPa = 100 cm H20 = 1 bar = 14.2 psi
Fig. 31b. Pressure drop as a function of flow rate for Sephacryl High Resolution. Bed height approximately 60 cm, distilled water, temperature +25 °C. To calculate the volumetric flow rate, multiply the linear flow by the cross-sectional area of the column (2 cm2 for XK 16 or 5.3 cm2 for XK 26).
Sephacryl High Resolution (HR) is a composite medium prepared by covalently cross-linking allyl dextran with N,N'-methylene bisacrylamide to form a hydrophilic matrix of high mechanical strength, illustrated in Figure 32. The porosity of the medium, determined by the dextran component, has been controlled to yield five different selectivities. The mechanical rigidity of Sephacryl HR allows even relatively viscous eluents, such as 8 M urea, to be run at practical flow rates. Under normal chromatography conditions (A280, 0.05 M phosphate, 0.15 M NaCl, pH 7.0) Sephacryl S-100 HR gave yields of at least 96% of the following substances: Blue Dextran 2000, ferritin, catalase, aldolase, BSA, ovalbumin, b-lactoglobulin A+B, chymotrypsinogen A, myoglobin, lysozyme, ribonuclease A and cytochrome C. An ionic strength of at least 0.15 M is recommended for best results. CH2 NH
CH
C=O CH2
n
O
CH2
CH
OH
O
CH
2
H
O=C CH2
O
HN
CH OH
C=O
O
CH
NH
2
O
OH
O
H
CH2 NH
O O
C=O CH2
CH2
CH
CH2 CH
CH2
HO
OH
CH2
O
HO
OH
O
CH2
OH
O
O
O
O
2
OH
OH
CH O H
CH
OH
O CH2
O
HO
OH
O CH2
O
Fig. 32. Partial structure of Sephacryl High Resolution.
47
Separation options Product †
Fractionation range, Mr (globular proteins)
Sample loading capacity
Maximum operating back pressure
HiPrep 16/60 Sephacryl S-100 HR*
1×103–1×105
<5 ml
0.15 MPa, 5 bar, 21 psi
0.5 ml/min
HiPrep 26/60 Sephacryl S-100 HR*
1×103–1×105
<13 ml
0.15 MPa, 5 bar, 21 psi
1.3 ml/min
Sephacryl S-100 HR*
1×103–1×105
0.5–4% of total column volume
0.2 MPa, 2 bar, 28 psi
10–35 cm/h
HiPrep 16/60 Sephacryl S-200 HR*
5×103–2.5×105
<5 ml
0.15 MPa, 5 bar, 21 psi
0.5 ml/min
HiPrep 26/60 Sephacryl S-200 HR*
5×103–2.5×105
<13 ml
0.15 MPa, 5 bar, 21 psi
1.3 ml/min
Sephacryl S-200 HR*
5×103–2.5×105
0.5–4% of total column volume
0.2 MPa, 2 bar, 28 psi
10–35 cm/h
HiPrep 16/60 Sephacryl S-300 HR*
1×104–1.5×106
<5 ml
0.15 MPa, 5 bar, 21 psi
0.5 ml/min
HiPrep 26/60 Sephacryl S-300 HR*
1×104–1.5×106
<13 ml
0.15 MPa, 5 bar, 21 psi
1.3 ml/min
Sephacryl S-300 HR*
1×104–1.5×106
0.5–4% of total column volume
0.2 MPa, 2 bar, 28 psi
10–35 cm/h
Sephacryl S-400 HR*
2×104–8×106
0.5–4% of total column volume
0.2 MPa, 2 bar, 28 psi
10–35 cm/h
Sephacryl S-500 HR*
–
0.5–4% of total column volume
0.2 MPa, 2 bar, 28 psi
10–35 cm/h
Sephacryl S-1000 SF (Superfine)
–
0.5–4% of total column volume
not determined
2–30 cm/h
†
Recommended operating flow ‡
* High Resolution. For maximum resolution apply as small a sample volume as possible, but note that sample volumes less than 0.5% do not normally improve resolution.
†
‡
See Appendix 5 to convert linear flow (cm/hour) to volumetric flow rates (ml/min) and vice versa.
Separation examples A 280 nm
Column: HiPrep 26/60 Sephacryl S-100 HR Sample: 1 ml of a mixture containing bovine insulin chain A (Mr 2 532) and chain B (Mr 3 496), 0.5 mg/ml of each Buffer: 0.05 M sodium phosphate, 0.15 M NaCl, pH 7.0 Flow: 2.0 ml/min (22 cm/h)
0.03 Chain A 0.02 Chain B 0.01
0.00
0
40
80
120
160
200
240 Vol. (ml)
Fig. 33. Separation of insulin chains on HiPrep 26/60 Sephacryl S-100 HR.
48
A 280 nm
1
2
Immunoglobulin
Sample: IgG fraction from previous ion exchange step (sample volume equivalent to 1% of Vt. (V t =column volume) Column: BP 113/120 containing Sephacryl S-200 HR, bed height 100 cm Buffer: 0.05 M Tris-HCl, 0.15 NaCl, pH 7.5 Flow: 7.5 cm/h
Elution volume
Fig. 34. Purification of monoclonal antibodies on Sephacryl S-200 HR. Inset shows analysis by gradient SDS-PAGE of the immunoglobulin pool. Lane 1, native sample; lane 2, sample reduced with 2-mercaptoethanol.
A 280 nm 1. Large phospoholipid vesicles (LPLV) 2. Small phospholipid vesicles (SPLV) 0.6
1 2
0.4
0.2
50
100
150
Medium: Sephacryl S-400 HR Sample: Integral membrane proteins prepared from human erythrocytes solubilized in 0.1 M phosphate, 100 mM SDS, 1 mM EDTA, 1 mM DTE, pH 7.4 Sample volume: 2 ml (2 mg/ml) Column: K 26/70, packed bed 2.6 x 61 cm Buffer: 0.1 M phosphate, 50 mM SDS, 1 mM EDTA, 1 mM DTE, pH 7.4 Flow: 1 ml/min (11 cm/h)
200 250 Elution volume (ml)
Fig. 35. Gel filtration on Sephacryl S-400 HR quickly separates phospholipid vesicles (liposomes) into large (LPLV) and small (SPLV) phospholipid vesicles. (Data provided by E. Greijer and P. Lundahl, Dept. of Biochemistry, Biomedical Centre, University of Uppsala, Sweden.)
Performing a separation Buffer: 0.05 M sodium phosphate, 0.15 M NaCl, pH 7.2 or select the buffer in which the sample should be stored or solubilized for the next step.
Use 0.15 M NaCl, or a buffer with equivalent ionic strength, to avoid pH dependent nonionic interactions with the matrix. At very low ionic strength, the presence of a small number of negatively charged groups may cause retardation of basic proteins and exclusion of acidic proteins. The sample should be fully dissolved. Centrifuge or filter to remove particulate material (see Appendix 3). Always use degassed buffers and maintain a constant temperature during the run to avoid introducing air into the column. Set an appropriate pressure limit on the chromatography system to avoid damage to the column packing.
49
1. For first time use, or after long term storage, equilibrate the column with at least 0.5 column volume of distilled water at 15 cm/h (0.5 ml/min for 16/60 column or 1.3 ml/min for 26/60). 2. Equilibrate with 2 column volumes of buffer at 30 cm/h (1.0 ml/min for 16/60 column or 2.6 ml/min for 26/60). 3. Reduce flow to 15 cm/h and, for best resolution, apply a sample volume equivalent to 1% of the column volume (1.2 ml for 16/60 column or 3.2 ml for 26/60). Sample volumes between 0.5–4% can be applied. 4. Elute with 1 column volume of buffer. 5. Before applying a new sample re-equilibrate column with 1 column volume of buffer at 30 cm/h until the baseline monitored at A280 is stable.
Column performance should be checked at regular intervals by determining the theoretical plate number per meter and peak symmetry. Prepacked columns are supplied with recommended values. See page 95 on how to check column efficiency. See page 26 for advice on optimizing the separation. Exposure to temperatures outside the range +4 °C to +40 °C will destroy the efficiency of a packed bed and the column will need to be re-packed.
Cleaning 1. Wash with 0.5 column volume of 0.2 M NaOH at a flow of 15 cm/h (0.5 ml/min for column 16/60 or 1.3 ml/min for 26/60) to remove most non-specifically adsorbed proteins. 2. Re-equilibrate immediately with 2 column volumes of buffer or until the baseline monitored at A280 and the pH of the eluent are stable. Further equilibration may be necessary if the buffer contains detergent.
Routine cleaning after every 10–20 separations is recommended, but the frequency of cleaning will also depend on the nature of the samples being applied. If required Sephacryl High Resolution may be autoclaved repeatedly at +121 °C, pH 7 for 30 minutes without significantly affecting its chromatography properties. The medium must be removed from a column before autoclaving as certain column components cannot tolerate such high temperatures. To remove severe contamination Reverse the flow and wash at a flow rate of 10 cm/h (0.3 ml/min for column 16/60 or 0.8 ml/min for 26/60) at room temperature using the following solutions: 1. Wash with 0.25 column volumes of 0.5 M NaOH (to remove hydrophobic proteins or lipoproteins) followed by 4 column volumes of distilled water. 2. Wash with 0.5 column volume of 30% isopropanol (to remove lipids and very hydrophobic proteins), followed by 2 column volumes of distilled water. For extreme cases of contamination, check the instructions supplied with the product.
Reversing flow through a gel filtration column should only be considered under cases of severe contamination. There is a risk that reversing the flow may cause channeling through the packed bed leading to poor resolution, loss of efficiency and the need to repack the column. Professionally packed columns are less likely to be affected, but extreme care must be taken.
50
Media characteristics Composition: Sephacryl is a composite medium prepared by covalently cross-linking allyl dextran with N,N'-methylene bisacrylamide to form a hydrophilic matrix of high mechanical strength. The porosity of the medium is determined by the dextran component. Product
Efficiency: theoretical plates per meter (prepacked columns only)
pH stability
Sephacryl S-100 HR*
>5 000 m-1
Sephacryl S-200 HR*
†
Particle size
Mean particle size
Long term: 3–11 Short term: 2–13
25–75 µm
47 µm
>5 000 m-1
Long term: 3–11 Short term: 2–13
25–75 µm
47 µm
Sephacryl S-300 HR*
>5 000 m-1
Long term: 3–11 Short term: 2–13
25–75 µm
47 µm
Sephacryl S-400 HR*
‡
Long term: 3–11 Short term: 2–13
25–75 µm
47 µm
Sephacryl S-500 HR*
‡
Long term: 3–11 Short term: 2–13
25–75 µm
47 µm
Sephacryl S-1000 SF (Superfine)
‡
Long term: 3–11 Short term: 2–13
40–105 µm
65 µm
* High Resolution. † Long term pH stability refers to the pH interval where the medium is stable over a long period of time without adverse side effects on its chromatography performance. Short term pH stability refers to the pH interval for regeneration, cleaning-in-place and sanitization procedures. All ranges are estimates based on the experience and knowledge within Amersham Biosciences. ‡
Efficiencies of 9 000 m-1 have been achieved, but depend significantly on how well the column is packed.
Chemical stability Sephacryl High Resolution is stable in all commonly used aqueous buffers and additives such as detergents (1% SDS), denaturing agents (8 M urea or 6 M guanidine hydrochloride). The medium is also stable in 30% acetonitrile, 0.5 M sodium hydroxide, up to 24% ethanol, up to 1 M acetic acid and up to 30% isopropanol.
Storage Store unused media +4 °C to +25 °C in 20% ethanol. Do not freeze. Columns can be left connected to a chromatography system with a low flow rate (0.01 ml/min) of buffer passing through the column to prevent bacterial growth or the introduction of air into the column which would destroy the packing. For long term storage, wash with 4 column volumes of distilled water followed by 4 column volumes of 20% ethanol. Store at +4 °C to +25 °C. Degas the ethanol/water mixture thoroughly and use a low flow rate, checking the back pressure as the column equilibrates. Avoid changes in temperature which may cause air bubbles in the packing.
51
Superose: broad fractionation range, but not suitable for industrial scale separations 10 2
10 3
10 4
10 5
10 6
Superose 6 Superose 12 Superose 6 prep grade Superose 12 prep grade
Fig. 36. Fractionation ranges of Superose.
Superose is a medium with high physical and chemical stability based on highly cross-linked porous agarose particles. Typical fractionation ranges for Superose are shown in Figure 36. The mechanical rigidity of Superose allows even relatively viscous eluents, such as 8 M urea, to be run at practical flow rates. Under normal chromatography conditions non-specific interactions between proteins and Superose are negligible when using buffers with ionic strengths in the range 0.15 M to 1.5 M. Some hydrophobic interactions have been noted, particularly for compounds such as smaller hydrophobic and/or aromatic peptides, membrane proteins and/or lipoproteins which may elute later than predicted. However, in some applications, these interactions can be an advantage for increasing the resolution of the separation. Typical selectivity curves for Superose are shown in Figure 37.
Kav 1.0
Superose 6 prep grade 0.8
0.6
Superose 12 0.4
Superose 6
0.2
Superose 12 prep grade 0 10
3
10
4
10
Fig. 37. Selectivity curves of Superose for globular proteins.
52
5
10
6
Mr
Figure 38 gives a comparison of the different selectivities of Superose 6 and Superose 12 prepacked columns. Column: Superose 6 HR 10/30 Sample: 100 µl solution containing: 1. Thyroglobulin (Mr 669 000), 5 mg/ml 2. Ferritin (M r 440 000), 0.3 mg/ml 3. Bovine serum albumin (Mr 67 000), 8 mg/ml 4. Ribonuclease A (M r 13 700), 5 mg/ml 5. Glycyl tyrosin (Mr 238), 0.6 mg/ml Buffer: 0.05 M phosphate buffer, 0.15 M NaCl, pH 7.0 Flow: 0.4 ml/min
Column: Superose 12 HR 10/30 Sample: 100 µl solution containing: 1. IgG (Mr 160 000), 2.5 mg/ml 2. BSA (Mr 67 000), 8 mg/ml 3. b-lactoglobulin (Mr 35 000), 2.5 mg/ml 4. Cytochrome C (Mr 12 400), 1 mg/ml 5. Vitamin B12 (Mr 1 355), 0.1 mg/ml 6. Cytidine (Mr 243), 0.1 mg/ml Buffer: 0.05 M phosphate buffer, 0.15 M NaCl, pH 7.0 Flow: 0.4 ml/min
a)
b)
A 280 nm
A 280 nm 3
0.5
0.5
2 6 1
1
3
5
4
4
5
2 0.25
0.25
30
60 Time (min)
30
60 Time (min)
Fig. 38. a) Standard proteins separated on Superose 6 HR 10/30, Mr range: 5 000–5 000 000. b) Standard proteins separated on Superose 12 HR 10/30, Mr range: 1 000–300 000.
Separation options Superose is produced in different particle sizes (11 µm, 13 µm and 30 µm) and with two different selectivities (Superose 6 and Superose 12). Use 11 µm or 13 µm particles for analytical separations and 30 µm particles for preparative separations. Product*
Fractionation range, Mr (globular proteins)
Sample loading capacity*
Maximum operating back pressure
Recommended operating flow †
Superose 6 PC 3.2/30
5×103–5×106
Superose 6 HR 10/30
5×103–5×106
200 µl
1.2 MPa, 12 bar, 175 psi
<0.1 ml/min
25–250 µl
1.5 MPa,15 bar, 220 psi
Superose 6 prep grade
0.3–0.5 ml/min
5×103–5×106
0.5–4% of total column volume
0.4 MPa, 4 bar, 58 psi
<40 cm/h
Superose 12 PC 3.2/30
1×103–3×106
200 µl
2.4 MPa,24 bar, 350 psi
<0.1 ml/min
Superose 12 HR 10/30
1×103–3×106
25–250 µl
3 MPa, 30 bar, 435 psi
0.5–1.0 ml/min
Superose 12 prep grade
1×103–3×106
0.5–4% of total column volume
0.7 MPa, 7 bar, 101 psi
<40 cm/h
* For maximum resolution apply as small a sample volume as possible, but note that sample volumes less than 0.5% do not normally improve resolution. †
See Appendix 5 to convert linear flow (cm/hour) to volumetric flow rates (ml/min) and vice versa.
53
Separation examples A 280 nm 0.025
Column: Sample: Buffer: Flow:
0.020
Superose 12 PC 3.2/30 0.75 µl human tears 0.02 M sodium phosphate, 0.5 M NaCl, pH 5.3 40 µl/min
0.015 0.010 0.005 0.000 0
20
40
60
Time (min)
Fig. 39. Microfractionation of 0.75 µl of human tears.
Column: 2× Superose 6 HR 10/30 in series Sample: 10 µg Hae III cleaved pBR 322 Buffer: 0.05 M Tris-HCl, 1 mM EDTA, pH 8.0 Flow: 0.1 ml/min
Column: Sample: Buffer: Flow:
A 254 nm
A 254 nm 0.05
Superose 6 HR 10/30 fX-174 RF DNA-Hae III digest, 10 µg 0.05 M Tris-HCl, pH 8.0 0.4 ml/min
0.05
434 458 504 540 587
1353 1078 872 603
267 192 184 234 213
310 281 271
123 124
234 104 89 80 64 57
194 51 21 18
118 11 7
2
3
4
5
Time (h)
30
72
60 Time (min)
Fig. 40. Separation of DNA fragments on Superose 6 HR 10/30. Peak figures correspond to number of base pairs.
Performing a separation Buffer: 0.05 M sodium phosphate, 0.15 M NaCl, pH 7 or select the buffer in which the sample should be stored or solubilized for the next step.
Use 0.15 M NaCl, or a buffer with equivalent ionic strength, to avoid pH dependent nonionic interactions with the matrix. At very low ionic strength, the presence of a small number of negatively charged groups may cause retardation of basic proteins and exclusion of acidic proteins.
54
The sample should be fully dissolved. Centrifuge or filter to remove particulate material (see Appendix 3). Always use degassed buffers and maintain a constant temperature during the run to avoid introducing air into the column. Set an appropriate pressure limit on the chromatography system to avoid damage to the column packing. Column performance should be checked at regular intervals by determining the theoretical plate number per meter and peak symmetry. Prepacked columns are supplied with recommended values. See page 96 for how to check column efficiency. See page 26 for advice on optimizing the separation. Exposure to temperatures outside the range +4 °C to +40 °C will destroy the efficiency of a packed bed and the column will need to be re-packed.
Cleaning 1. Wash with 1 column volume 0.5 M NaOH at 40 cm/h (0.5 ml/min for HR 10/30 columns). 2. Rinse immediately with 1 column volume of distilled water or buffer at 40 cm/h. 3. Continue to re-equilibrate with 2 column volumes of buffer or until the baseline and the eluent pH are stable. For extreme cases of contamination, check the instructions supplied with the product.
In special cases only, it may be necessary to change the bottom filter or to remove and discard the top 2–3 mm of the gel. These operations must be carried out extremely carefully to avoid serious loss of resolution. Reversing flow through a gel filtration column should only be considered under cases of severe contamination. There is a risk that reversing the flow may cause channeling through the packed bed leading to poor resolution, loss of efficiency and the need to repack the column. Professionally packed columns are less likely to be affected, but extreme care must be taken. Superose prep grade may be autoclaved repeatedly at +121 °C, pH 7 for 30 minutes without significantly affecting its chromatography properties. The medium must be removed from a column before autoclaving as certain column components cannot tolerate such high temperatures.
55
Media characteristics Composition: Superose is formed from highly cross-linked agarose. Superose prep grade shows less tendency towards hydrophobic interactions than Superose in prepacked columns. Superose 6 shows less tendency towards hydrophobic interactions than Superose 12. Product
Efficiency: theoretical plates per meter* (prepacked columns only)
pH stability
Superose 6
>30 000 m-1
Superose 6 prep grade
†
Particle size
Mean particle size
Long term: 3–12 Short term: 1–14
11–15 µm
13 µm
*
Long term: 3–12 Short term: 1–14
20–40 µm
30 µm
Superose 12
>40 000 m-1
Long term: 3–12 Short term: 1–14
9–13 µm
11 µm
Superose 12 prep grade
*
Long term: 3–12 Short term: 1–14
20–40 µm
30 µm
* A minimum column efficiency of 10 000 m-1 should be expected for a well packed column. Long term pH stability refers to the pH interval where the medium is stable over a long period of time without adverse side effects on its chromatography performance. Short term pH stability refers to the pH interval for regeneration, cleaning-in-place and sanitization procedures. All ranges are estimates based on the experience and knowledge within Amersham Biosciences.
†
Chemical stability Stable in all commonly used aqueous buffers and additives such as detergents (1% SDS), denaturing agents (8 M urea or 6 M guanidine hydrochloride) and 30% acetonitrile.
Storage Store unused media +4 °C to +25 °C in 20% ethanol. Do not freeze. Columns can be left connected to a chromatography system with a low flow rate (0.01 ml/min) of buffer passing through the column to prevent bacterial growth or the introduction of air into the column which would destroy the packing. For long term storage, wash with 2 column volumes of distilled water followed by 2 column volumes of 20% ethanol. Store at +4 °C to +25 °C. Degas the ethanol/water mixture thoroughly and use a low flow rate, checking the back pressure as the column equilibrates. Avoid changes in temperature which may cause air bubbles in the packing.
56
Sephadex: rapid group separation of high and low molecular weight substances, such as desalting, buffer exchange and sample clean up Sephadex is prepared by cross-linking dextran with epichlorohydrin, illustrated in Figure 41. CH2 HC H O H C OH H C O CH2 C HC OH C H O H H OH C OH H C O CH2 C HC H HO C O H H C OH O H C O C OH C CH 2
HC
H
OH
OH
CH2 H O
CH2
HO C H C OH C O C H
HC H HC O H C OH H C O CH2 C O C H OH CH2 HC
O
O
CH2
H C H O H C OH H C O C HO C H
OH
OH
CH2 OH
Fig. 41. Partial structure of Sephadex.
The different types of Sephadex vary in their degree of cross-linking and hence in their degree of swelling and selectivity for specific molecular sizes, as shown later on page 68 Media characteristics. • Sephadex G-10 is well suited for the separation of biomolecules such as peptides (Mr >700) from smaller molecules (Mr <100). • Sephadex G-50 is suitable for the separation of molecules Mr >30 000 from molecules Mr <1 500 such as labeled protein or DNA from unconjugated dyes. The medium is often used to remove small nucleotides from longer chain nucleic acids. • Sephadex G-25 is recommended for the majority of group separations involving globular proteins. These media are excellent for removing salt and other small contaminants away from molecules that are greater than Mr 5 000. Using different particle sizes enables columns to be packed according to application requirements, see below. The particle size determines the flow rates and the maximum sample volumes that can be applied. For example, smaller particles give higher column efficiency (narrow, symmetrical peaks), but may need to be run more slowly as they create higher operating pressures. Sephadex G-25
Application
Superfine
For highest column efficiency (highest resolution), but operating pressures increase
Fine
For laboratory scale separations
Coarse and Medium
Use when a high flow rate at a low operating pressure is essential, e.g. large scale
Coarse
For batch procedures
57
b
a
Fig. 42. Prepacked columns: a) HiPrep 26/10 Desalting, b) HiTrap Desalting 5 ml.
Dialysis is frequently mentioned in the literature as a technique to remove salt or other small molecules and to change the buffer composition of a sample. However, dialysis is generally a very slow technique, requiring large volumes of buffer. During handling or as a result of proteolytic breakdown or non-specific binding to the dialysis membranes, there is a risk of losing material. A simpler and much faster technique is to use a desalting column, packed with Sephadex G-25, for group separation between high and low molecular weight substances. Proteins are separated from salts and other small molecules. In a fast, single step, the sample is desalted, transferred into a new buffer and low molecular weight materials are removed. The high speed and high volume capacity of the separation enables even large sample volumes to be processed rapidly and efficiently. Sample volumes up to 30% of the total volume of the desalting column can be applied and separated at much higher flow rates than those used for high resolution fractionation, as illustrated in Figure 43. a)
RS
HiPrep 26/10 Desalting Flow: 9 ml/min (100 cm/h)
3.5
b)
RS
HiPrep 26/10 Desalting Sample volume: 15 ml
3.5
3
3
2.5
2.5
2
2
1.5
1.5
1
1
0.5
0.5
0
0
0
2
4
6
8
10
12 14 16 18 Sample volume (ml)
0
5
10
15
20
Fig. 43. a) Influence of sample volume on resolution. b) Influence of flow rate on resolution.
58
25
30 35 Flow (ml/min)
Desalting columns are used not only to remove low molecular weight contaminants such as salt, but also for buffer exchange before and after different chromatography techniques and for the rapid removal of reagents to terminate a reaction. Examples of group separations include: • removal of phenol red from culture fluids prior to anion exchange chromatography or nucleic acid preparations • removal of unincorporated nucleotides during DNA sequencing • removal of free low molecular weight labels • termination of reactions between macromolecules and low molecular weight reactants • removal of products, cofactors or inhibitors from enzymes • removal of unreacted radiolabels such as [a-32P] ATP from nucleic acid labeling reactions
Separation options For group separations the medium should be selected so that the high molecular weight molecules are eluted at the void volume with minimum peak broadening or dilution and minimum time on the column. The lowest molecular weight substances should appear by the time one column volume of buffer has passed through the column. Product
Exclusion limit
HiTrap Desalting 5 ml >5×103 (Sephadex G-25 Superfine) HiPrep 26/10 Desalting (Sephadex G-25 Fine)
Sample loading capacity
Sample elution volume (for maximum sample loading)
Maximum operating back pressure (MPa, bar, psi)
Recommended operating flow
0.25–1.5 ml
1.0–2.0 ml
0.3 MPa, 3 bar, 42 psi
1–10 ml/min
>5×103
2.5–15 ml
7.5–20 ml
0.15 MPa, 1.5 bar, 22 psi
9–31 ml/min
PD-10 >5×103 (Sephadex G-25 Medium)
1.5–2.5 ml
2.5–3.5 ml
Run under gravity
Run under gravity Run under gravity
NICK*
>3×104
<0.1 ml
0.4 ml
Run under gravity
MicroSpin™ G-25†
>5×103
10–100 µl
–
Centrifuge
Centrifuge
NAP-5*
>5×103
<0.5 ml
1.0 ml
Run under gravity
Run under gravity
NAP-10*
>5×103
<1.0 ml
1.5 ml
Run under gravity
Run under gravity
NAP-25*
>5×103
<2.5 ml
3.5 ml
Run under gravity
Run under gravity
Sephadex G-25 Superfine
>5×103
n.a.
n.a.
Darcy's law applies‡ applies‡
Darcy's law
Sephadex G-25 Fine
>5×103
n.a.
n.a.
Darcy's law applies‡
Darcy's law applies‡
Sephadex G-25 Medium
>5×103
n.a.
n.a.
Darcy's law applies‡
Darcy's law applies‡
‡
Darcy's law applies‡
Sephadex G-25 Coarse
>5×10
3
n.a.
n.a.
Darcy's law applies
Sephadex G-50 Fine
>3×104
n.a.
n.a.
Darcy's law applies‡
Darcy's law applies‡
Sephadex G-10
>700
n.a.
n.a.
Darcy's law applies‡
Darcy's law applies‡
* NICK columns are packed with Sephadex G-50 Fine DNA Grade and NAP columns are packed with Sephadex G-25 Medium DNA Grade. † A range of MicroSpin columns is available for desalting of proteins and purification of labeled DNA fragments and PCR products. Refer to the BioDirectory catalogue from Amersham Biosciences for further details. ‡ In practice this means that the pressure/flow considerations that must be made when using other gel filtration media do not apply to Sephadex. Doubling the flow rate doubles the column pressure. See Appendix 2 for an explanation of Darcy's law.
59
For convenience and reliable performance, use prepacked Sephadex columns such as HiTrap Desalting 5 ml and HiPrep 26/10 Desalting. Reference lists highlighting the use of HiPrep 26/10 Desalting and HiTrap Desalting are available at www.chromatography.amershambiosciences.com. Always use disposable columns if there is a risk of biological or radioactive contamination or when any possibility of carryover between samples is unacceptable. The type of equipment available and the sample volume to be processed also govern the choice of prepacked column, as shown in Figure 44.
Fig. 44. Selecting prepacked columns for desalting and buffer exchange.
60
Separation examples A 280 nm
Sample: UV 280 nm Conductivity
Column: Buffer:
0.15 (His)6 protein
(His)6 protein eluted from HiTrap Chelating HP with sodium phosphate 20 mM, sodium chloride 0.5 M, imidazole 0.5 M, pH 7.4 HiTrap Desalting 5 ml Sodium phosphate 20 mM, sodium chloride 0.15 M, pH 7.0
0.10
Salt
0.05 Inject Vo
Vt
0 0
1
2 min
Fig. 45. Desalting a (His)6 fusion protein using HiTrap Desalting 5 ml on ÄKTA™prime. The UV (protein) and conductivity (salt) traces enable pooling of the desalted fractions and facilitate optimization of the separation.
A 280 nm
NHS
Column: Sample:
HiPrep 26/10 Desalting 2 mg/ml BSA, 0.07 mg/ml N-Hydroxysuccinimide (NHS) in 50 mM sodium phosphate, 0.15 M NaCl, pH 7.0. Filtered through a 0.45 µm filter Sample volume: 13 ml Buffer: 50 mM sodium phosphate, 0.15 M NaCl, pH 7.0 Flow: 31 ml/min (350 cm/h)
BSA
0.0
1.0
2.0
Time (min)
Fig. 46. Reproducible removal of N-Hydroxysuccinimide from bovine serum albumin.
Performing a separation Desalting and buffer exchange can take less than 5 minutes per sample with greater than 95% recovery for most proteins. To prevent possible ionic interactions the presence of a low salt concentration (25 mM NaCl) is recommended during desalting and in the final sample buffer. Volatile buffers such as 100 mM ammonium acetate or 100 mM ammonium hydrogen carbonate can be used if it is necessary to avoid the presence of NaCl. The sample should be fully dissolved. Centrifuge or filter to remove particulate material (see Appendix 3). Always use degassed buffers to avoid introducing air into the column. Sample concentration up to 70 mg/ml protein should not influence the separation when using normal aqueous buffers.
61
If possible use a chromatography system with a UV and a conductivity monitor to facilitate optimization of the sample loading. The elution of the protein peak at A280 and the appearance of the salt peak can be followed exactly and different separations can be easily compared, as shown in Figure 47. If conductivity cannot be monitored and recovery of completely desalted sample is the major requirement, apply sample volumes of between 15 and 20% of the total column volume.
A 280 nm
Conductivity (mS/cm)
0.25
A 280
Conductivity
0.20
10.0
0.15 0.10 5.0 0.05 0.00 0.0
1.0
2.0
Fig. 47. Buffer exchange of mouse plasma on HiPrep 26/10 Desalting.
62
Time (min)
Alternative 1: Using a HiTrap column with a syringe The maximum recommended sample volume is 1.5 ml. The table below shows the effect of reducing the sample volume applied to the column. Table 1. Recommended sample and elution volumes using a syringe or Multipipette™ with HiTrap Desalting 5 ml Sample load
Add buffer
Elute and collect
Yield %
0.25 ml
1.25 ml
1.0 ml
> 95
0.0
4.0
0.50 ml
1.0 ml
1.5 ml
> 95
< 0.1
3.0
1.00 ml
0.5 ml
2.0 ml
> 95
< 0.2
2.0
1.50 ml
0 ml
2.0 ml
> 95
< 0.2
1.3
Step 3
Step 4
Remaining salt %
Dilution factor
Step 6
1. Fill the syringe with buffer. Unscrew the stop plug at the top of the column. To avoid introducing air into the column, connect the column "drop to drop" to the syringe (via the adapter provided). 2. Remove the twist-off end. 3. Wash the column with 25 ml buffer at 5 ml/min to completely remove the 20% ethanol (supplied as storage buffer). If air is trapped in the column, wash with degassed buffer until the air disappears. Air bubbles introduced onto the column by accident during sample application do not influence the separation. 4. Apply the sample using a 2–5 ml syringe at a flow rate between 1–10 ml/min. Discard the liquid eluted from the column. 5. If the sample volume is less than 1.5 ml, change to buffer and proceed with the injection until a total of 1.5 ml has been eluted. Discard the eluted liquid. 6. Elute the protein with the appropriate volume selected from Table 1. Collect the desalted protein in the volume indicated.
Note: 5 ml/min corresponds to approximately 120 drops/min when using a HiTrap 5 ml column.
A simple peristaltic pump can also be used to apply sample and buffers.
63
Alternative 2: Simple desalting with ÄKTAprime ÄKTAprime contains pre-programmed templates for individual HiTrap Desalting 5 ml and HiPrep 26/10 Desalting columns.
Buffer Preparation Prepare at least 500 ml of the required buffer 1. Follow the instructions supplied on the ÄKTAprime cue card to connect the column and load the system with buffer. 2. Select the Application Template. 3. Start the method. 4. Enter the sample volume and press OK.
Alternative 3: Desalting on a gravity-feed PD-10 column Buffer Preparation 1. Remove top cap and pour off the excess liquid. 2. Cut off the bottom tip. 3. Place column in the Desalting Workmate supplied onto the plastic tray and equilibrate with 25 ml buffer. Discard the eluent. 4. Add a total sample volume of 2.5 ml. If the sample volume is less than 2.5 ml, add buffer to reach a final volume of 2.5 ml. Discard the eluent. 5. Add 3.5 ml buffer to elute high molecular weight components and collect the eluent.
Using the standard procedure described above protein yield is typically greater than 95% with less than 4% salt (low molecular weight) contamination. The dilution factor is 1:4. Sephadex G-10 can be packed into empty PD-10 columns and run in the same manner as PD-10 Desalting columns. Optimization of desalting 1. When possible select a prepacked column that is best suited to the volume of sample that needs to be desalted (see Separation Options). For the majority of separations the instructions supplied ensure satisfactory results and very little optimization should be necessary. 2. Ensure that buffer conditions are optimal for the separation. 3. Select the highest flow rate recommended. Figure 48 shows an example of the influence of flow rate on group separation. 4. Determine the maximum sample volume that can be loaded. Figure 49 shows an example of the influence of sample volume on group separation.
64
Column: Sample: Buffer: Sample volume: Flow:
HiTrap Desalting 5 ml Bovine serum albumin, 2 mg/ml in 0.5 M NaCl, 0.05 M sodium phosphate, pH 7.0 0.05 M sodium phosphate, 0.15 M NaCl, pH 7.0 0.8 ml 1.7, 3.3, 6.7, 10.0, 13.3, 16.7, 20.0 ml/min
A 280 nm 3.3 ml/min
0.30
6.7 10.0 13.3
Conductivity (mS/cm) 75 1.2 1.0 Relative resolution
0.40
0.20
0.8 0.6 0.4 0.2
0.10
0.0
BSA
10 ml/min
0
NaCl
20
0
0.00 0
2
4
8 ml
6
Fig. 48. Influence of flow rate on separation using a HiTrap Desalting column.
Column: Sample: Buffer: Sample volume: Flow:
HiTrap Desalting 5 ml Bovine serum albumin, 2 mg/ml in 0.5 M NaCl, 0.05 M sodium phosphate, pH 7.0 0.05 M sodium phosphate, 0.15 M NaCl, pH 7.0 0.8, 1.3, 1.7, 2.2 ml 5 ml/min Volume collected: 1.5 + × ml
A 280 nm 0.40
Conductivity (mS/cm) 75
% NaCl contamination
2.2 ml 0.30 1.7 ml 1.3 ml
0.20
0.8 ml
0.8 ml sample
30
1.3 ml sample 1.7 ml sample 2.2 ml sample
20
10
0.10 2.0
2.5 3.0 Volume collected: 1.5 + × ml
3.0
0
0.00 0
2
4
6
8
ml
Fig. 49. Influence of sample volume on separation using a HiTrap Desalting column.
As the sample volume increases (up to a maximum of 30% of the total column volume) the dilution factor decreases and there may be a slight increase in the amount of salt remaining in the sample after elution. Table 1 on page 63 illustrates this effect when using a HiTrap Desalting 5 ml column. Sample volumes up to 30% of the total column volume give a separation with minimal sample dilution. Larger sample volumes can be applied, but resolution will be reduced.
65
Scale up and processing large sample volumes Connecting columns in series increases the effective column volume and so increases sample loading capacity. Table 2 shows the sample loading capacities and dilution factors when using prepacked desalting columns alone or in series, see also Figure 50 for HiTrap application examples. Table 2. Selection guide for desalting/buffer exchange columns Column
Loaded volume (ml)
Eluted volume (ml)
Dilution factor
Operation
HiPrep 26/10 Desalting
10 15 (max)
10–15 15–20
1–1.5 1–1.3
pump pump
2 x HiPrep 26/10 Desalting
30 (max)
30–40
1–1.3
pump
3 x HiPrep 26/10 Desalting
45 (max)
45–55
1–1.2
pump
4 x HiPrep 26/10 Desalting
60 (max)
60–70
1–1.2
HiTrap Desalting
0.25 0.5 1.0 1.5 (max)
1.0 1.5 2.0 2.0
4 3 2 1.3
pump syringe/pump syringe/pump syringe/pump syringe/pump
2 x HiTrap Desalting
3.0
4–5
1.3–1.7
syringe/pump
3 x HiTrap Desalting
4.5 (max)
6–7
1.3–1.7
syringe/pump
PD-10 Desalting columns
1.5 2.0 2.5 (max)
3.5 3.5 3.5
2.3 1.7 1.4
gravity gravity gravity
Increasing sample loading capacity from 1.5 ml up to 7.5 ml HiTrap Desalting 1 × 5 ml
Column: Sample:
HiTrap Desalting, 1 × 5 ml, 3 × 5 ml, 5 × 5 ml 2 mg/ml BSA in 50 mM sodium phosphate, 0.5 M sodium chloride, pH 7.0
A 280 nm
Conductivity (mS/cm) BSA
A
NaCl
0.40
Sample volume: Buffer:
28% × Vt (1.4, 4.3 and 7.1 ml respectively) 50 mM sodium phosphate, 0.15 M sodium chloride, pH 7.0 5 ml/min
Flow:
50
0.30 40 0.20 30 0.10 20
0.00 0
2.0
HiTrap Desalting 3 × 5 ml in series A 280 nm B
6.0
ml
HiTrap Desalting 5 × 5 ml in series
Conductivity (mS/cm) BSA
4.0
Conductivity (mS/cm)
A 280 nm BSA
NaCl
NaCl
C
0.40
50
0.30
0.40
50
0.30 40
0.20
40 0.20
30 0.10 20
0.00 0
5.0
10.0
15.0
20.0
ml
Fig. 50. Scale up using HiTrap columns connected in series.
66
30 0.10 20
0.00 0
10.0
20.0
30.0
ml
Increasing sample loading capacity from 15 ml up to 60 ml Connect HiPrep 26/10 Desalting columns in series, e.g. 2 columns: sample volume 30 ml, 4 columns: sample volume 60 ml, as shown in Figure 51. Even with four columns in series, high flow rates can be maintained without causing back pressure difficulties so that up to 60 ml of sample can be processed in 20–30 minutes.
Fig. 51. Four HiPrep 26/10 Desalting columns connected in series.
For sample volumes greater than 60 ml Select a suitable particle size of Sephadex G-25, rehydrate and pack into a short, wide column to facilitate high flow rates and rapid recovery of desalted materials. See Appendix 1 for details on column packing. The particle size determines the flow rates and sample volumes that can be applied, as shown in Figure 52. 100
200
cm/h flow velocity (linear flow rate)
% of column volume
maximum flow rate
maximum sample volume
Superfine
Fine
Medium
Coarse
increasing particle size
Fig. 52. Sephadex G-25: recommended sample volumes and flow rates vary with particle size.
• Use Superfine grade with a bed height of approximately 15 cm when requiring the highest efficiencies. • Use Fine grade with an approximate bed height of 15 cm for laboratory scale separations. • Use Coarse and Medium grades for preparative processes where a high flow rate at a low operating pressure is essential. Pack in a column less than 50 cm in bed height. The Coarse grade is suitable for batch procedures.
67
Media characteristics Sephadex is prepared by cross-linking dextran with epichlorohydrin. Variations in the degree of cross linking create the different Sephadex media and influence their degree of swelling and their selectivity for specific molecular sizes. Product
Fractionation range, Mr (globular proteins)
pH stability*
Bed volume ml/g dry Sephadex
Maximum operating flow
Particle size, wet
Sephadex G-10
<7×102
Long term: 2–13 Short term: 2–13
Sephadex G-25 Coarse
1×103–5×103
Long term: 2–13 Short term: 2–13
2–3
Darcy's law†
55–165 µm
4–6
Darcy's law†
170–520 µm
Sephadex G-25 Medium
1×103–5×103
Long term: 2–13 Short term: 2–13
4–6
Darcy's law†
85–260 µm
Sephadex G-25 Fine
1×103–5×103
Long term: 2–13 Short term: 2–13
4–6
Darcy's law†
35–140 µm
Sephadex G-25 Superfine
1×103–5×103
Long term: 2–13 Short term: 2–13
4–6
Darcy's law†
17–70 µm
Sephadex G-50 Fine
1×103–3×104
Long term: 2–10 Short term: 2–13
9–11
Darcy's law†
40–160 µm
* Long term pH stability refers to the pH interval where the medium is stable over a long period of time without adverse side effects on its chromatography performance. Short term pH stability refers to the pH interval for regeneration, cleaning-in-place and sanitization procedures. All ranges are estimates based on the experience and knowledge within Amersham Biosciences. In practice this means that the pressure/flow considerations that must be made when using other gel filtration media do not apply to Sephadex. Doubling flow rate will double column pressure. See Appendix 2 for an explanation of Darcy's Law.
†
Column Packing See Appendix 1.
Cleaning PD-10, NAP, NICK and HiTrap Desalting columns are disposable, but, depending on the type of sample and if cross-contamination is not a concern, they can be re-used a few times. For HiPrep 26/10 Desalting columns proceed as follows: 1. Wash the column with 2 column volumes of 0.2 M sodium hydroxide or a solution of a non ionic detergent (typically 0.1–0.5% Triton X-100 dissolved in distilled water or 0.1 M acetic acid) at a flow rate of 10 ml/min. Ensure that the pressure drop does not exceed 0.15 MPa (1.5 bar, 22 psi). 2. Wash the column with 5 column volumes of distilled water at a flow rate of 15 ml/min. 3. Before use, re-equilibrate the column with at least 5 column volumes of buffer until the UV base line and pH are stable.
To remove precipitated proteins and peptides, fill the column with 1 mg pepsin/ml in 0.1 M acetic acid, 0.5 M NaCl and leave at room temperature overnight or 1 hour at +37 ºC. Repeat the normal cleaning procedure above.
Chemical stability Sephadex is stable in all commonly used aqueous buffers and additives such as ionic and non-ionic detergents, denaturing agents (8 M urea or 6 M guanidine hydrochloride). The media are stable in short chain alcohols such as ethanol, methanol and propanol, but concentrations above 25% should not normally be used. Note that Sephadex shrinks in alcohol solutions. 68
Storage Store unused media +4 °C to +25 °C in 20% ethanol. Do not freeze. Wash used media with 2 column volumes of distilled water followed by 2 column volumes of 20% ethanol. Store at +4 °C to +25 °C. Alternatively, wash with 2 column volumes of distilled water followed by 2 column volumes 0.01 M NaOH. Sodium hydroxide solution is bacteriostatic, easily disposed of and does not shrink the medium. Degas the ethanol/water mixture thoroughly and use a low flow rate, checking the back pressure as the column equilibrates. Avoid changes in temperature which may cause air bubbles in the packing.
69
70
Chapter 3 Gel filtration in theory Defining the process Results from gel filtration are usually expressed as an elution profile or chromatogram that shows the variation in concentration (typically in terms of UV absorbance at A280nm) of sample components as they elute from the column in order of their molecular size (Figure 53). Molecules that do not enter the matrix are eluted in the void volume, Vo as they pass directly through the column at the same speed as the flow of buffer. Molecules with partial access to the pores of the matrix elute from the column in order of decreasing size. Molecules with full access to the pores move down the column, but do not separate from each other. These molecules usually elute just before one total column volume, Vt, of buffer has passed through the column.
high molecular weight
Absorbance
low molecular weight
sample injection volume
void volume Vo total column volume Vt
intermediate molecular weight equilibration Vt
Vo
1 cv Column Volumes (cv)
Fig. 53. Theoretical chromatogram of a high resolution fractionation.
The behavior of each component can be expressed in terms of its elution volume, Ve, determined by direct measurement from the chromatogram. As shown in Figure 54 there are three different ways of measuring Ve, dependent on the volume of sample applied to the column. A. When very small samples are applied (small enough to be neglected compared to the elution volume), take the position of the peak maximum in the elution diagram as Ve. B. If the sample volume cannot be neglected compared with the elution volume, measure the elution volume from half the sample volume to the position of the peak maximum. C. When very large sample volumes are used (giving a plateau region in the elution curve), take the volume eluted from the start of sample application to the inflexion point (or half height) of the rising part of the elution peak as Ve.
71
A 280 nm A
A. Sample size negligible compared with volume of packed bed.
Ve B
B. Sample size not negligible compared with volume of packed bed.
Ve C
C. Sample giving plateau elution curve. inflexion point
Ve
Elution volume
Fig. 54. Measurement of elution volume, Ve.
Since symmetrical peaks are common in gel filtration, elution volumes are easily determined. However, Ve does not completely define the behavior of the sample since Ve will vary with the total volume of the packed bed (Vt) and the way in which the column has been packed. The elution of a sample is best characterized by a distribution coefficient (Kd) derived as follows: The volume of the mobile phase (buffer) is equal to the void volume, Vo, i.e. the elution volume of molecules that remain in the buffer because they are larger than the largest pores in the matrix and pass straight through the packed bed. In a well packed column the void volume is approximately 30% of the total column. The volume of the stationary phase, Vs, is equal to Vi, the volume of buffer inside the matrix which is available to very small molecules, i.e. the elution volume of molecules that distribute freely between the mobile and stationary phases minus the void volume. Since, in practice, Vs or Vi are difficult to determine, it is more convenient to substitute the term (Vt – Vo). The estimated volume of the stationary phase will therefore include the volume of solid material which forms the matrix. Kd represents the fraction of the stationary phase that is available for diffusion of a given molecular species. The stationary phase Vs can be can be substituted by the term (Vt – Vo) in order to obtain a value Kav. Kav = (Ve – Vo)/(Vt – Vo)
72
Void volume Vo
Total column volume Vt
Vt – Vo
Fig. 55. Diagrammatic representation of Vt and Vo. Note that Vt – Vo will include the volume of the solid material which forms the matrix (Fischer, L. Laboratory Techniques in Biochemistry and Molecular Biology. Vol. 1 part II. An Introduction to Gel Chromatography. North Holland Publishing Company, Amsterdam. Reproduced by kind permission of the author and publisher).
Low molecular weight
Intermediate molecular weight
Sample injection
High molecular weight
Absorbance
Interacting with medium
Since (Vt – Vo) includes the volume of the matrix that is inaccessible to all solute molecules, Kav is not a true partition coefficient. However, for a given medium there is a constant ratio of Kav:Kd which is independent of the nature of the molecule or its concentration. Kav is easily determined and, like Kd, defines sample behavior independently of the column dimensions and packing. Other methods of normalizing data give values which vary depending upon how well the column is packed. The approximate relationships between some of these terms are shown in Figure 56.
Vt
Vo Ve
Kav =
Ve– Vo Vt – Vo
Kd =
Ve– Vo = Vt – Vo – Vgel matrix
Vt – Vo Vi 0.5
1.0
1
2
3
0
0.5
1.0
0
0.5
1.0
Ve– Vo Vi
Ve / Vt
Kav Kd
Fig. 56. Relationship between several expressions used for normalizing elution behavior.
73
Selectivity curves and media selection The partition coefficient Kav is related to the size of a molecule. Molecules of similar shape and density demonstrate a sigmoidal relationship between their Kav values and the logarithms of their molecular weights (Mr). Over a considerable range there is a linear relationship between Kav and log Mr. The selectivity of a gel filtration medium depends solely on its pore size distribution and is described by a selectivity curve. By plotting Kav against the log of the molecular weight for a set of standard proteins, selectivity curves are created for each gel filtration medium, as shown in Figure 57.
K av 0.8
Superdex 75 prep grade and Superdex 200 prep grade
0.6
Dextrans
Superdex 30 prep grade
0.4
g 0p 20 pg 75
K av 0.7 0.6
0.2 0.5
PEG Peptides
0.4
10 0.3
K av 0.8
0.2
10
5
10
7
Mr
10
7
Mr
Globular proteins 0
0.4
pg
75
10 000 Mr
6
20
1000
10
Superdex 75 prep grade and Superdex 200 prep grade
0.6
0.1
300
4
pg
0.2
10
4
10
5
10
6
Fig. 57. Selectivity curves for Superdex 30 prep grade, Superdex 75 prep grade and Superdex 200 prep grade.
Gel filtration media should be selected so that the high molecular weight molecules are eluted at the void volume (Kav = 0) with minimum peak broadening or dilution and minimum time on the column. The lowest molecular weight substances should be eluted near Vt (Kav = 1). Under ideal conditions, no molecules can be eluted with a Kav greater than 1 or less than 0. If the Kav is greater than 1, molecules have bound non-specifically to the medium. If Kav is less than 0 after calibration then there is channeling in the chromatography bed and the column must be repacked.
74
Resolution Final resolution, the degree of separation between peaks, is influenced by many factors: the ratio of sample volume to column volume, flow rate, column dimensions, particle size, particle size distribution, packing density, porosity of the particle and viscosity of the mobile phase. The success of gel filtration depends primarily on choosing conditions that give sufficient selectivity and counteract peak broadening effects during the separation. Resolution (Rs) is defined by the following expression: Rs =
Vr2 – Vr1 (W1 + W2) 2
Vr1 and Vr2 are the elution volumes for two adjacent peaks measured at the center of the peak. W1 and W2 are the respective peak widths.
(Vr2 – Vr1) represents the distance between the peaks and 1/2 (W1 + W2) the mean peak width of the two peaks as shown in Figure 58. ( Vr2 – Vr1 )
W1
W2
Fig. 58. Parameters used to define resolution (Rs).
Resolution is a function of the selectivity of the medium and the efficiency of that medium to produce narrow peaks (minimal peak broadening) as illustrated in Figure 59.
high efficiency
low efficiency
Fig. 59. Dependence of resolution on selectivity and the counteraction of peak broadening.
75
The efficiency of a packed column defines its ability to produce narrow symmetrical peaks during elution. Refer to Appendix 1 for column packing and preparation and determination of column efficiency. Efficiency is particularly important in gel filtration in which separation takes place as only a single column volume of buffer passes through the column. Efficiency is defined in terms of theoretical plates per meter (N): N = 5.54 (Ve/W1/2)2 × 1000/L where Ve = peak elution (retention) volume W1/2 = peak width at half peak height L = bed height (mm) Ve and W1/2 are in same units Efficiency can be improved by decreasing the particle size of the medium. However, using a smaller particle size may create an increase in back pressure so that flow rate needs to be decreased, lengthening the run time. The uniformity of the packed bed and the particles influences the uniformity of the flow through the column and hence affects the shape and eventual peak width. Gel filtration media with high uniformity (lower particle size distribution) facilitate the elution of molecules in narrow peaks. Gel filtration media with smaller particle sizes facilitate diffusion of sample molecules in and out of the particles by reducing the time to achieve equilibrium between mobile and stationary phases and so improve resolution by reducing peak width. Sample dilution is inevitable because sample passes through the column and diffusion occurs. In order to minimize sample dilution a maximum sample volume is used within the limits set by the separation distance i.e. the resolution required between the peaks of interest. The sample can be regarded as a zone passing down the column. Figure 60 shows how, if no zone broadening occurs, the maximum sample volume could be as great as the separation volume (VSep): VSep = VeB – VeA However, due to eddy diffusion, non-equilibrium between the stationary phase and the buffer, and longitudinal diffusion in the bed, the zones will always be broadened. Therefore the sample volume must always be smaller than the separation volume.
76
Concentration
A
A
B
Ve
B Ve
Elution volume
Vsep
Fig. 60. Elution curves for different sample sizes. The top diagram corresponds to the application of a small sample. The center diagram corresponds to the maximum sample volume that gives complete separation if there is no zone broadening. The bottom diagram corresponds to the maximum sample volume to obtain complete separation in the conditions of the experiment. The shaded areas correspond to the elution profiles that would be obtained if there was no zone broadening.
77
78
Chapter 4 Molecular weight determination and molecular weight distribution analysis Unlike electrophoretic techniques, gel filtration provides a means of determining the molecular weight or size (Stokes radius) of native or denatured proteins under a wide variety of conditions of pH, ionic strength and temperature, free from the constraints imposed by the charge state of the molecules. In order to understand and follow the procedures outlined, it is important to have read Chapter 3 Gel filtration in theory. For molecular weight determination, several theoretical models have been proposed to describe the behavior of solutes during gel filtration. Most models assume that the partition of solute molecules between the particles and surrounding liquid is an entirely steric effect. However, in practice a homologous series of compounds demonstrate a sigmodial relationship between their various elution volume parameters and the logarithm of their molecular weights. Thus molecular weight determination by gel filtration can be made by comparing an elution volume parameter, such as Kav of the substance of interest, with the values obtained for several known calibration standards. A calibration curve is prepared by measuring the elution volumes of several standards, calculating their corresponding Kav values (or similar parameter), and plotting their Kav values versus the logarithm of their molecular weight. The molecular weight of an unknown substance can be determined from the calibration curve once its Kav value is calculated from its measured elution volume. Various elution parameters, such as Ve, Ve/Vo, Kd, and Kav have been used in the literature for the preparation of calibration curves but the use of Kav is recommended since: 1) it is less sensitive to errors which may be introduced as a result of variations in column preparation and column dimensions, 2) it does not require the unreliable determination of the internal volume (Vi) as is required with Kd. For accurate determination of molecular weight, the calibration standards must have the same relationship between molecular weight and molecular size as the substance of interest. Calibration Kits from Amersham Biosciences provide well-characterized, globular protein standards for protein molecular weight determination. The Low Molecular Weight Gel Filtration Calibration Kit contains 4 individually lyophilized proteins with molecular weights in the range 13 700–67 000 and Blue Dextran 2000. The High Molecular Weight Gel Filtration Calibration Kit contains 4 individually lyophilized proteins with molecular weights in the range 158 000–669 000 and Blue Dextran 2000. Many of the parameters important for a successful molecular weight determination are the same as for any high resolution fractionation: Use a medium with the correct fractionation range for the molecules of interest. The expected molecular weight values should fall in the linear part of the selectivity curve (see gel filtration selection guide page 18). Use a prepacked column whenever possible. Homemade columns must be packed very carefully (see Appendix 1).
79
Use freshly prepared calibration standards, selected so that the expected molecular weight values are covered by the entire calibration range. Always filter Blue Dextran before use. Apply samples in a volume less than 2% of the total column volume. Use the same buffer for the separation of calibrants and sample, for example 50 mM sodium phosphate, 0.15 M NaCl, pH 7. Use the recommended flow rate for the prepacked column or medium selected. If the molecular weight is unknown, use a medium with a wide fractionation range such as Sephacryl HR. This is also recommended for molecular weight distribution analysis and for polymeric materials such as dextrans and polyethylene glycols. Performing a molecular weight determination in the presence of urea, guanidine hydrochloride or SDS transforms polypeptides and proteins to a random coil configuration and so reduces structural differences. Differences will be seen in the resulting molecular weight values when compared to values acquired under non-denaturing conditions. Deviation from a Kav:log Mr calibration curve may occur if the molecule of interest does not have the same molecular shape as the standards. Performing a molecular weight determination 1. If using a self-packed column, prepare a fresh, filtered solution of Blue Dextran 2000 (1.0 mg/ml) in the running buffer. Apply Blue Dextran to the column, using a volume <2% of the total column volume (Vt) to determine the void volume (Vo), and to check the column packing. 2. Dissolve the selected calibration proteins in the running buffer (at concentrations recommended by the manufacturer). Allow a few minutes for dissolution, stirring gently. Do not heat or mix vigorously. If necessary, filter the calibration solution. 3. Apply the calibration solution to the column, in a volume <2% of the total column volume (Vt). 4. Determine the elution volumes (Ve) for the standards by measuring the volume of the eluent from the point of application to the centre of the elution peak. 5. Calculate the Kav values for the standards and prepare a calibration curve of Kav versus the logarithm of their molecular weights, as follows: Kav =
Ve – Vo Vt – Vo
where Ve = elution volume for the protein Vo = column void volume = elution volume for Blue Dextran 2000 Vt = total bed volume On semilogarithmic graph paper, plot the Kav value for each protein standard (on the linear scale) against the corresponding molecular weight (on the logarithmic scale). Draw the straight line which best fits the points on the graph. Alternatively, use a statistics package to calculate the regression line. 6. Apply the sample in a volume <2% of the total column volume (Vt) and determine the elution volume (Ve) of the molecule of interest. 7. Calculate the corresponding Kav for the component of interest and determine its molecular weight from the calibration curve.
A calibrated column can be used for extended periods as long as the column is kept in good condition and not allowed to dry out, eliminating the need to set up a separate experiment for each determination.
80
Chapter 5 Sephadex LH-20 Sephadex LH-20 is specifically designed for the separation and purification of natural products that require the presence of organic solvents to maintain their solubility, including molecules such as steroids, terpenoids, lipids and low molecular weight peptides (up to 35 amino acid residues). Compounds are usually separated by a form of liquid/liquid partitioning or absorption chromatography. Sephadex LH-20 can have a very high selectivity for aromatic compounds in certain solvents and can be used at analytical or industrial scale for the preparation of closely related species. Sephadex LH-20 is made from hydroxypropylated dextran beads that have been crosslinked to yield a polysaccharide network. The medium can be swollen in water and organic solvents. The partial structure of Sephadex LH-20 is shown in Figure 61.
O O HO
HO
O O O
HO O HO O HO HO
O
O O O
O
O OH
O O
HO O HO
O
O HO
HO
O
O O
HO
O O
Fig. 61. Partial structure of Sephadex LH-20.
Sephadex LH-20 is suitable for an initial purification before polishing by ion exchange or reversed phase chromatography, or for a final polishing step, for example during the preparation of diastereoisomers. Depending upon the chosen solvents, Sephadex LH-20 can also separate components by partitioning between the matrix and the organic solvent. Sephadex LH-20 exhibits both hydrophilic and hydrophobic properties, the combination of which can offer unique chromatography selectivity for certain applications. For more detailed information on gel filtration in organic solvents refer to Preparative Gel Chromatography on Sephadex LH-20 by H. Henke, available from Amersham Biosciences. Sephadex has been used for gel filtration in organic solvents, for example dimethylformamide may be used with Sephadex G-10 and mixtures of water with the shorter chain alcohols may be used with Sephadex G-10, G-25 and G-50. 81
Separation options Product
Fractionation range (globular proteins)
Sample loading capacity*
Maximum operating back pressure
Maximum operating flow
Sephadex LH-20
< 5 × 103 (exclusion limit will depend on the solvent)
2% of column volume
Solvent dependent
12 cm/min (720 cm/hr) (bed height 14 cm, 15 MPa back pressure)
* If Sephadex LH-20 is used in adsorption mode then the sample volume is unlimited until reaching the point of column saturation.
Separation examples An HIV-1 reverse transcriptase inhibitor has been isolated from Phyllanthus niruri, a natural medicine that has been used for many years to combat edema and jaundice. The active component that inhibits HIV-1 reverse transcriptase has been identified as repandusinic acid A monosodium salt, a small tannin-like molecule. The structure of the free acid is shown in Figure 62. HO
OH HO
HO
HO
OH O
OH
6
O
O O
5
O HOOC 4'
H
5'
OH 1
3
OH
2
OH O HO
7'
6'
O O
O
4
6''
5''
OH
3'
4''
H
1''
OH
2'
H
HOOC
2''
1'
3''
O O
Fig. 62. Structure of free acid form of repandusinic acid A.
Table 3 shows the recovery of active inhibitor from an analytical separation on Sephadex LH-20. Table 3. Summary of data for the isolation of repandusinic acid A from P. niruri Purification step
Yield (mg)
ID50* (µg/ml)
Specific activity (×102 IU/mg)
Total activity (×103 IU) †
H2O extract
6 600
50
4
2 640
MeOH insoluble
2 500
20
10
2 500
Sephadex LH-20 Fr. 4–11‡
247
3.0–3.6
56–67
1 616
Cellulose Fr. 1 Fr. 2 Fr. 3 Fr. 4 Fr. 5
189 24 18 9 14
7.8 5.0 2.4 3.4 1.8
26 40 83 58 111
484 96 150 52 156
RA (pure substance)
5.9
0.3
668
394
* ID50 indicates the effectiveness of inhibitors expressed as concentrations which cause 50% inhibition of HIV-1-RT. Crude HIV-1-RT was used in this experiment. IU are arbitrary inhibitory activity units obtained by dividing the total weight of the fraction at each step by the weight of each fraction required to achieve 50% inhibition of [3H]dTTP incorporation into the polymer in the HIV-1-RT assay.
†
‡
82
Fractions 4–10 and Fraction 11 were combined because both fractions had the inhibitory activity.
Figure 63 shows Sephadex LH-20 used at a preparative scale for the separation of 2-acetamidobenzoic acid and 4-acetamidobenzoic acid. In this separation the hydrophilicity and hydrophobicity of the medium provide a unique chromatography selectivity resulting in high resolution of closely related species. The molecules differ only by the position of the acetamide moiety on the benzene ring. RI
1
2 O
1.
C
NH
OH
C
Column: Sample: Eluent: Flow rate: Detection: Yield:
CH 3
Sephadex LH-20, 2.5 × 200 cm Mixture of 2- and 4-acetamidobenzoic acid Acetone 8 ml/min Refractive Index 250 mg 2-acetamidobenzoic acid 254 mg 4-acetamidobenzoic acid
O
CH 3
6
C O
254 mg
250 mg
2.
8
HN
C
OH
O
Time (h)
Fig. 63. Separation of 2- and 4-acetamidobenzoic acid on Sephadex LH-20.
Packing a column Sephadex LH-20 should be packed in a solvent resistant column selected from Table 4 according to the column volume required for the separation. Table 4. Solvent resistant columns (*SR 10/50J includes a borosilicate glass jacket. Jackets are not available for other SR columns. All SR columns are supplied with two adaptors for top and bottom assembly) Column
Volume (ml)
Bed height (cm)
SR 10/50
16–39
20–50
SR 10/50J*
16–39
20–50
SR 25/45
73–220
15–45
SR 25/100
343–490
70–100
Simple steps to clarify a sample before applying it to a column will avoid the risk of blockage, reduce the need for stringent washing procedures and extend the life of the chromatography medium. Filter or centrifuge all solvents and samples before use. 1. Refer to Table 5, page 85, to calculate the amount of dry medium required as the extent of swelling depends upon the solvent system. Swell Sephadex LH-20 for at least 3 hours at room temperature in the solvent to be used for the separation. 2. Prepare a slurry 75:25 settled medium:solvent and decant any fine particles of medium. 3. Equilibrate all materials to room temperature. 4. Resuspend and pour the slurry into the column in one continuous step (using a glass rod will help to eliminate air bubbles). 5. Fill the column reservoir to the top with solvent. Seal, attach to a pump and open the column outlet. 6. Pack at 300 cm/h until the bed has reached a constant height. Stop the flow, empty and remove the packing reservoir.
83
7. Carefully fill the column with solvent and insert a wetted adaptor into the column. Ensure no air bubbles are trapped under the net and adjust the adaptor O-ring to give a sliding seal against the column wall. 8. Connect all tubings, ensuring that there are no air bubbles in the flow path. 9. Slowly slide down the adaptor so that any air in the tubings is displaced by solvent and lock the adaptor into position on the surface of the medium. 10. Open the column outlet and continue packing until the packed bed is stable and a final adjustment of the top adaptor can be made.
In solvents such as chloroform Sephadex LH-20 is less dense than the solvent and the medium will float. Pour the medium into the column and drain until the second adaptor can be inserted. Lock the adaptor in position at the surface of the medium and direct the flow of chloroform upwards. The bed will be packed against the top adaptor and the lower adaptor can be pushed slowly upwards towards the lower surface of the medium. Close the column outlet when moving the adaptor to avoid compressing the bed.
Performing a separation Start at a linear flow of 1 cm/h to check resolution. The lower the flow, the better the resolution. 1. Equilibrate the column with at least 2 column volumes of the solvent until a stable baseline is achieved. 2. Apply a sample volume equivalent to 1–2% of the total column volume. 3. Elute in 1 column volume. Re-equilibration is not needed between runs with the same solvent.
Cleaning Wash the column with 2–3 column volumes of the solvent, followed by re-equilibration in a new solvent if changing the separation conditions.
Medium characteristics Sephadex LH-20
pH stability
Bed volume ml/g dry Sephadex LH-20
Particle size
Working: 2–11 *Short term: 2–13
Depends on solvent, see table 5
Dry: 18–111 µm Wet: depends on solvent
* Short term pH stability refers to the pH interval for regeneration, cleaning-in-place and sanitization procedures. All ranges are estimates based on the experience and knowledge within Amersham Biosciences.
The wet particle size for Sephadex LH-20 varies according to the solvent used for swelling, as shown in Table 5.
84
Table 5. Approximate values for packed bed volumes of Sephadex LH-20 swollen in different solvents Solvent
Approx. bed volume (ml/g dry Sephadex LH-20)
Dimethyl sulfoxide
4.4–4.6
Pyridine
4.2–4.4
Water
4.0–4.4
Dimethylformamide
4.0–4.4
Methanol
3.9–4.3
Saline
3.8–4.2
Ethylene dichloride
3.8–4.1
Chloroform *
3.8–4.1
Propanol
3.7–4.0
†
3.6 –3.9
Ethanol
Isobutanol
3.6–3.9
Formamide
3.6–3.9
Methylene dichloride
3.6–3.9
Butanol
3.5–3.8
Isopropanol
3.3–3.6
Tetrahydrofuran
3.3–3.6
Dioxane
3.2–3.5
Acetone
2.4–2.6
Acetonitrile ‡
2.2–2.4
Carbon tetrachloride Benzene
‡
Ethyl acetate Toluene
‡
1.8–2.2 1.6–2.0
‡
‡
1.6–1.8 1.5 –1.6
* Containing 1% ethanol. †
Containing 1% benzene.
‡
Solvents that give a bed volume of less than 2.5 ml/g dry Sephadex LH-20 are not generally useful.
Chemical stability Sephadex LH-20 is stable in most aqueous and organic solvent systems. The medium is not stable below pH 2.0 or in strong oxidizing agents.
Storage Store dry at +4 °C to +25 °C. Store packed columns and used medium at +4 °C to +8 °C in the presence of a bacteriostatic agent.
Transferring Sephadex LH-20 between organic solvents Transfer Sephadex LH-20 from an aqueous solution to the organic solvent by moving through a graded series of solvent mixtures. This will ensure efficient replacement of the water by the required solvent. To transfer from aqueous solution or organic solvent (100% A) to a new organic solvent (100% B), proceed as follows: transfer to 70% A:30% B then to 30% A:70% B and finally to 100% B. If A and B are not mutually miscible, make the transfer via an intermediate solvent, for example from water to chloroform via acetone, as shown in Figure 64.
85
Water
Ethanol Dimethylformamide Dioxane Dimethyl sulfoxide
Acetone
Chloroform, dichloroethane, dichloromethane, tetrahydrofuran, n-heptane, ethyl acetate, toluene Fig. 64. Suggested routes for changing to organic solvents. 1. Transfer the required amount of medium to a sintered glass Buchner funnel and remove the excess aqueous solution by gentle suction. 2. Add the next solvent and resuspend the medium by stirring gently. 3. Suck off the excess solvent and resuspend in the same solvent. 4. Repeat the process with the next solvent in the series. Perform at least two resuspensions for each change of solvent conditions until the final solvent composition is reached. 5. Pack the medium into solvent resistant SR 10/50, SR 25/45 or SR 25/100 columns.
86
Chapter 6 Gel filtration in a Purification Strategy (CIPP) For a high degree of purity, or when a suitable ligand is unavailable for a single step affinity purification, an efficient multi-step process must be developed using the purification strategy of Capture, Intermediate Purification and Polishing (CIPP). CIPP is used in both the pharmaceutical industry and in the research laboratory to ensure faster method development, a shorter time to pure product and good economy. This chapter gives a brief overview of this approach, which is recommended for any multi-step protein purification. The Protein Purification Handbook from Amersham Biosciences is an ideal guide for planning efficient and effective protein purification strategies and for the selection of the correct medium for each step and scale of purification. As shown in Figure 65, an important first step for any purification is correct sample preparation and this is covered in more detail in Appendix 3. Gel filtration is often used for desalting and buffer exchange during sample preparation (using Sephadex G-25), when samples volumes up to 30% of the total column volume can be applied.
Purity
In high resolution mode, gel filtration is ideal for the final polishing steps in a purification when sample volumes have been reduced (sample volume significantly influences speed and resolution in gel filtration). Samples are eluted isocratically (single buffer, no gradient) and buffer conditions can be varied to suit the sample type or the requirements for subsequent purification, analysis or storage, since buffer composition does not directly affect resolution.
Polishing Achieve final high level purity
Intermediate purification Capture Preparation, extraction, clarification
Remove bulk impurities
Isolate, concentrate and stabilize
Step Fig. 65. Preparation and CIPP.
Applying CIPP Imagine the purification has three phases: Capture, Intermediate Purification and Polishing. Assign a specific objective to each step within the purification process. The purification problem associated with a particular step will depend greatly upon the properties of the starting material. Thus, the objective of a purification step will vary according to its position in the process.
87
In the capture phase the objectives are to isolate, concentrate and stabilize the target product. The product should be concentrated and transferred to an environment that will conserve potency/activity. During the intermediate purification phase the objectives are to remove most of the bulk impurities, such as other proteins and nucleic acids, endotoxins and viruses. In the polishing phase most impurities have already been removed except for trace amounts or closely related substances. The objective is to achieve final purity by removing any remaining trace impurities or closely related substances. The optimal selection and combination of purification techniques for Capture, Intermediate Purification and Polishing is crucial for an efficient purification.
Selection and combination of purification techniques Proteins are purified using purification techniques that separate according to differences in specific properties, as shown in Table 6. Table 6. Protein properties used during purification Protein property
Technique
Size
Gel filtration (GF)
Charge
Ion exchange (IEX)
Hydrophobicity
Hydrophobic interaction (HIC), Reversed phase (RPC)
Biorecognition (ligand specificity)
Affinity (AC)
Resolution
Recovery
Speed
Capacity
Every technique offers a balance between resolution, capacity, speed and recovery. Capacity, in the simple model shown, refers to the amount of target protein loaded during purification. In some cases the amount of sample that can be loaded will be limited by volume (as in gel filtration) or by large amounts of contaminants rather than the amount of the target protein. Speed is most important at the beginning of purification where contaminants, such as proteases, must be removed as quickly as possible. Recovery becomes increasingly important as the purification proceeds because of the increased value of the purified product. Recovery is influenced by destructive processes in the sample and by unfavourable conditions on the column.
88
Resolution is achieved by the selectivity of the technique and the efficiency of the chromatography matrix in producing narrow peaks. In general, resolution is most difficult to achieve in the final stages of purification when impurities and target protein are likely to have very similar properties. Select a technique to meet the objectives for the purification step. Choose logical combinations of purification techniques based on the main benefits of the technique and the condition of the sample at the beginning or end of each step. A guide to the suitability of each purification technique for the stages in CIPP is shown in Table 7. Table 7. Suitability of purification techniques for CIPP
Technique Main features
Sample start condition
Sample end condition
high resolution high capacity high speed
low ionic strength sample volume not limiting
high ionic strength or pH change
good resolution good capacity high speed
high ionic strength sample volume not limiting
high resolution high capacity high speed
specific binding conditions sample volume not limiting
GF
high resolution using Superdex media
limited sample volume (<5% total column volume) and flow rate range
buffer exchanged (if required) diluted sample
RPC
high resolution
sample volume usually not limiting additives may be required
in organic solvent, risk loss of biological activity
IEX
HIC
AC
Capture
Intermediate
Polishing
concentrated sample low ionic strength
concentrated sample specific elution conditions
concentrated sample
Minimize sample handling between purification steps by combining techniques to avoid the need for sample conditioning. The product should be eluted from the first column in conditions suitable for the start conditions of the next column (see Table 7). Ammonium sulfate, often used for sample clarification and concentration (see Appendix 3), leaves the sample in a high salt environment. Consequently HIC, which requires high salt to enhance binding to the media, becomes the ideal choice as the capture step. The salt concentration and the total sample volume will be significantly reduced after elution from the HIC column. Dilution of the fractionated sample or rapid buffer exchange using a desalting column will prepare it for the next IEX or AC step. Gel filtration is a non-binding technique unaffected by buffer conditions, but with limited volume capacity. GF is well suited for use after any of the concentrating techniques (IEX, HIC, AC) since the target protein will be eluted in a reduced volume and the components from the buffer will not affect the gel filtration process. Selection of the final strategy will always depend upon specific sample properties and the required level of purification. Logical combinations of techniques are shown in Figure 66.
89
Proteins with low solubility SDS extraction
GF (in non-ionic detergent)
SDS extraction
Solubilizing agents (urea, ethylene glycol non-ionic detergents)
HIC
HIC
GF
GF
Crude sample or sample in high salt concentration Sample clarification
Capture
GF GF desalt mode desalt mode
AC
IEX
Intermediate Purification Polishing
GF or RPC
GF or RPC
GF desalt mode
HIC IEX dilution may be needed
IEX
HIC
GF
GF
Clear or very dilute samples Capture
AC
IEX
Intermediate Purification Polishing
GF or RPC
GF or RPC
IEX
Precipitation (e.g. in high ionic strength)
HIC
Resolubilize
GF
Treat as for sample in high salt concentration
Fig. 66. Logical combinations of chromatography techniques.
For any capture step, select the technique showing the most effective binding to the target protein while binding as few of the contaminants as possible, i.e. the technique with the highest selectivity and/or capacity for the target protein. A sample is purified using a combination of techniques and alternative selectivities. For example, in an IEX-HIC-GF strategy, the capture step selects according to differences in charge (IEX), the intermediate purification step according to differences in hydrophobicity (HIC) and the final polishing step according to differences in size (GF). If nothing is known about the target protein use IEX-HIC-GF. This combination of techniques can be regarded as a standard protocol. IEX is a technique which offers different selectivities using either anion or cation exchangers. The pH of the purification can be modified to alter the charge characteristics of the sample components. It is therefore possible to use IEX more than once in a purification strategy, for capture, intermediate purification or polishing. IEX can be used effectively in the same purification scheme for rapid purification in low resolution mode during capture and in high resolution mode during polishing. Consider the use of both anion and cation exchange chromatography to give different selectivities within the same purification strategy.
90
Gel filtration as a polishing step Most commonly, separations by charge, hydrophobicity or affinity will have been used in earlier stages of a purification strategy so that high resolution gel filtration is ideal for the final polishing step. The product can be purified and transferred into the required buffer in one step and dimers and aggregates can be removed, as shown in Figure 67. Gel filtration is also the slowest of the chromatography techniques and the size of the column determines the volume of sample that can be applied. It is therefore most logical to use gel filtration after techniques that reduce sample volume so that smaller columns can be used.
A 280 nm
Column: Sample: Sample load: Buffer: Flow:
monomeric ZZ-Brain IGF
0.01
XK 16/60 packed with Superdex 75 prep grade Partly purified ZZ-brain IGF 1.0 ml 0.3 M ammonium acetate, pH 6.0 0.5 ml/min (15 cm/h)
0.005
Vo
Fraction 1
0
1
Vt
2
3
4
2
5
6
3
4 Time (h)
Fig. 67. Final polishing step: separation of dimers and multimers on Superdex 75 prep grade.
Media for polishing steps should offer highest possible resolution. Superdex is the first choice at laboratory scale and Superdex prep grade for large scale applications. RPC can also be considered for a polishing step, provided that the target protein can withstand the run conditions. Reversed phase chromatography (RPC) separates proteins and peptides on the basis of hydrophobicity. RPC is a high selectivity (high resolution) technique, usually requiring the use of organic solvents. The technique is widely used for purity check analyses when recovery of activity and tertiary structure are not essential. Since many proteins are denatured by organic solvents, RPC is not generally recommended for protein purification because recovery of activity and return to a correct tertiary structure may be compromised. However, in the polishing phase, when the majority of protein impurities have been removed, RPC can be an excellent technique, particularly for small target proteins that are not often denatured by organic solvents. CIPP does not mean that there must always be three purification steps. For example, capture and intermediate purification may be achievable in a single step, as may intermediate purification and polishing. Similarly, purity demands may be so low that a rapid capture step is sufficient to achieve the desired result. For purification of therapeutic proteins, a fourth or fifth purification step may be required to fulfill the highest purity and safety demands. The number of steps used will always depend upon the purity requirements and intended use for the protein.
91
Purification of humanised IgG4 monoclonal antibody A humanized IgG4 monoclonal antibody was expressed in a myeloma cell culture and purified by a combination of affinity chromatography and gel filtration. The antibody was captured by affinity chromatography using MabSelect™. Gel filtration on Superdex 200 prep grade was then used to separate the monomer from the dimer and larger polymers. Capture by affinity chromatography
Purification of the monomer (polishing step)
Sample:
Sample:
1282 ml myeloma cell culture containing humanized IgG4 (~0.33 mg/ml) Column: MabSelect (18 ml), XK 16/20 column Binding buffer: 20 mM sodium phosphate, 0.15 M NaCl, pH 7.4 Elution buffer: 100 mM sodium citrate, pH 3.0 Flow: 220 cm/h (7.4 ml/min) System: ÄKTAexplorer Operation: Equilibration: 5 column volumes (CV) binding buffer Sample application: 1 282 ml. Wash: 10 CV binding buffer. Elution: step gradient 100% 5 CV elution buffer
Column: Buffer: Flow: System: Operation:
7.5 ml of the pooled fractions from MabSelect column HiLoad 26/60 Superdex 200 prep grade 50 mM sodium phosphate, 0.15 M NaCl, pH 7.0 22.6 cm/h (2 ml/min) ÄKTAexplorer Equilibration 2 CV Sample application Isocratic elution 1 CV
CV = total column volume = Vt A 280nm (mAU)
A 280nm (mAU)
5000 600
Purified IgG 4 4000
500 Monomer 400
3000
300
2000 Dimer/multimer
200
1000
100
0
13579121620
0
400
800
1200
0
Waste
1600 ml
1 2 3 4 56 7 8 910 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60 62 64 66 68 70
0
100
200
Analysis: SDS-PAGE, silver staining Unreduced sample Lane 1: LMW Marker Lane 2: Crude sample from myeloma cell culture Lane 3: Pooled eluted sample from capture step Lane 4: Fraction 4–9 from polishing step Lane 5: Fraction 10–12 from polishing step Lane 6: Fraction 14–18 from polishing step 1
2
3
4
5
6
Reduced sample Lane 1: LMW Marker Lane 2: Pooled eluted sample from capture step Lane 3: Fraction 4–9 from polishing step Lane 4: Fraction 10–12 from polishing step Lane 5: Fraction 14–18 from polishing step 1
2
3
4
5
Mr 97 000 66 000
H - chain
45 000 30 000 20 100 14 400
Fig. 68. Two step purification of humanized IgG4.
92
L - chain
300
400 ml
Appendix 1 Column packing and preparation A well-packed column is essential for a high resolution fractionation on any gel filtration medium. Prepacked columns from Amersham Biosciences will ensure reproducible results and the highest performance. If the column volume or medium you require is not available as a prepacked column, contact you local Amersham Biosciences sales representative to inquire about our column packing services. Packing a column is a very critical stage in any gel filtration experiment. A poorly packed column will give rise to uneven flow, peak broadening, loss of resolution and can also affect achievable flow rates. If you decide to pack a gel filtration column yourself then the guidelines in this appendix will apply at any scale of operation. A Column Packing Video is available to demonstrate how to produce a well-packed column (see Ordering information). The video focuses particularly on the importance of column packing for gel filtration. Gel filtration is simple to perform once a well-packed column has been obtained. Providing that a column is used and maintained carefully it can be expected to give reproducible, high resolution results for a long time. Ensure that there is sufficient buffer for long, unattended runs or that the pump is programmed to stop the flow after a suitable time. Gel filtration columns that run dry must be repacked.
Columns for packing gel filtration media XK columns are fully compatible with the high flow rates achievable with modern media and a broad range of column dimensions is available. Ordering information for XK columns and main accessories can be found at the back of this handbook. For a complete listing of all spare parts refer to the Amersham Biosciences BioDirectory catalogue or web catalogue (www.chromatography.amershambiosciences.com).
93
Table 8. XK columns for packing gel filtration media Column
Column volume (ml) with one adaptor
XK 16/20
2 –34
XK 16/40
42 –74
XK 16/70
102 –135
XK 16/100
163 –195
XK 26/20
0 –80
XK 26/40
122 –196
XK 26/70
281 –356
XK 26/100
440 –315
XK 50/20
0 –275
XK 50/30
330 –510
XK 50/60
785 –1099
XK 50/100
1570 –1884
Adaptors are adjustable column end pieces that help to eliminate any disturbances to the surface of the packed medium as sample is applied and to prevent insoluble particles from entering and blocking the column. All XK columns are delivered with one AK adaptor, but a second adaptor can be used instead of a column end piece if a shorter bed height is required. TEFZEL tubing with M6 connectors, a thermostatic jacket, snap-on support net rings, dismantling tool and instructions are supplied with the XK column. Longer XK 50 columns can be difficult to pack under normal laboratory conditions. As alternatives, use our column packing services or connect two or more shorter XK columns (20 or 30 cm bed height) in series to achieve the required bed height.
94
Checking column efficiency Column performance should be checked at regular intervals by determining the theoretical plate number and peak symmetry. Prepacked columns are supplied with recommended values. Typical values for column performance: Superdex: Efficiency N>10 000, Peak symmetry As = 0.70–1.30 Sephacryl HR: Efficiency N>9 000, Peak symmetry As = 0.80–1.50 1. Equilibrate the packed column in distilled water at a linear flow of 60 cm/h. 2. Inject acetone (10 mg/ml in water) in a volume equivalent to 0.2% of the total packed column volume. 3. Monitor UV absorbance 280 nm from the time of injection until the acetone peak has eluted and the signal has returned to baseline. 4. Calculate column efficiency i.e. the number of theoretical plates (N): N = 5.54 (Ve / W1/2)2 × 1000/L
Absorbance
where Ve = peak elution (retention) volume W1/2 = peak width at half peak height L = bed height (mm) Ve and W1/2 are in same units Calculate the symmetry factor (As): w1/2
As = b/a
where a = first half peak width at 10% peak height b = second half peak width at 10% peak height
a
b
Ve
Volume
Column packing for high resolution fractionation using Superdex prep grade and Sephacryl High Resolution Superdex prep grade and Sephacryl High Resolution should be packed and equilibrated at a high flow rate using a column from the XK-series. XK columns are optimally designed for gel filtration with a bed design that ensures a uniform liquid flow and a dead space at the column outlet of less than 0.1% of the column volume in order to minimize dilution and to prevent remixing of separated peaks. XK columns are manufactured from materials which do not interfere with labile biological substances. They are easy to dismantle and reassemble for thorough cleaning, particularly important when handling biological samples. Ensure that the column and all components are clean and in good condition. It is particularly importance that the nets, net fasteners and glass tube are not damaged. Use well degassed buffers and equilibrate all materials to the temperature at which the separation will be performed. Avoid simple columns with large dead volumes as this will affect resolution.
95
For high resolution fractionation, use bed heights between 30–60 cm. Apply sample volumes equivalent to 1–2% of the column volume. The sample volume can be increased up to 4% if good resolution can be maintained. The settled medium should have a volume of 1.15 times that of the required packed column volume, see Table 8 for examples. 1. Sephacryl HR and Superdex prep grade are supplied swollen in a suspension containing 20% ethanol as a preservative. Suspend the medium by shaking gently and pour a sufficient quantity into a graduated glass cylinder or beaker. Avoid using magnetic stirrers, spatulas or glass rods since they may damage the matrix. 2. Wash the medium with 5–10 column volumes of distilled water on a glass filter and resuspend in distilled water to a final concentration of 50% settled medium. The medium must be thoroughly washed to remove the 20% ethanol storage solution. Residual ethanol may interfere with subsequent procedures.
To produce a more evenly dispersed slurry of Superdex prep grade, Tween™ 20 (250 ml per 500 ml washed slurry) can be added in order to reduce surface tension. 3. Wet the bottom filter by injecting distilled water through the effluent tubing. Close the end piece outlet. Mount filter and bottom end piece onto the column. 4. Attach the packing reservoir tightly to the column.
For XK 16 and XK 26 columns using a second column instead of a packing reservoir makes it easier to obtain a well-packed column. The second column is used with Packing Connector XK 16 or XK 26 as appropriate. 5. Mount the column and packing reservoir vertically on a laboratory stand. 6. Fill the column with distilled water to a height of 2 cm above the column end piece. Avoid air bubbles. 7. Degas the suspended medium under vacuum and carefully pour the suspended medium down the wall of the column using a glass rod. Avoid introducing air bubbles. Pour everything in a single operation and fill the reservoir to the top with distilled water. 8. Connect the pump outlet to the inlet on the packing reservoir. Open the column outlet and start the flow of buffer, see Table 9 for flow recommendations.
To achieve satisfactory column efficiency, Superdex prep grade must be packed in two steps: Step 1 for 2 hours or until the bed has reached a constant height and Step 2 for 60 minutes. Table 9 shows the flow rates for each step. Sephacryl HR can usually be packed satisfactorily using only the higher flow rate given in Step 2 of Table 9. Use the two step process if the column efficiency was unsatisfactory after the first attempt. 9. Stop the pump and remove the packing reservoir. Carefully fill the column with distilled water to form an upward meniscus at the top and insert the adaptor. Adjust the adapter to the surface of the packed bed. 10. Continue packing the column at the flow rate used in Step 2 for approximately 10 minutes. If the recommended flow rate cannot be obtained, use the maximum flow rate the pump can deliver.Mark the position of the top of the packed medium, stop the pump, close the column outlet, move the adaptor down onto to the surface of the medium and then push the adaptor a further 3 mm into the medium. The column is now ready to use. See Table 9 for maximum recommended flow rate and operating pressure for Sephacryl HR and Superdex prep grade media.
Maximum pressures (Sephacryl HR 0.3 MPa, 0.3 bar and Superdex prep grade 5 MPa, 5 bar) should not be exceeded during packing.
96
Always check the specific storage instructions supplied with the product. Table 9. Recommended flow rates for packing Sephacryl HR and Superdex prep grade Column
Bed height cm
Step 1 Sephacryl HR ml/min
Step 2 Sephacryl HR ml/min
Step 1 Superdex prep grade ml/min
Step 2 Superdex prep grade ml/min
XK 16/40
35
2
12–14
2
10–12
XK 16/70
65
2
12–14
2
10–12
XK16/100
95
2
12–14
2
10–12
XK 26/40
35
4
6–8
4
12
XK 26/70
65
4
6–8
4
12
XK 26/100
95
4
6–8
4
12
XK 50/20
10–15
9
12
10
20
XK 50/30
20–25
9
12
10
20
XK 50/60
55
9
12
10
20
XK 50/100
95
9
12
10
20
Controlling flow The safest and easiest way in which to control flow during column packing and chromatography separation is to use a pump controlled within an ÄKTAdesign chromatography system. Accurate and reproducible flow control is particularly important for efficient column packing and when repeating experiments or performing routine preparative work. The maximum flow rate achievable will depend on column diameter and buffer viscosity. Narrow columns allow a higher pressure and higher linear flow (cm/h) than wide columns. Always connect a pump so that buffer is pumped onto the column (rather than connecting the pump after the column and drawing buffer through the column). This reduces the risk of bubble formation due to suction effects. Do not exceed the maximum recommended values for pressure or linear flow for the medium (see Chapter 2). Exceeding these values may cause the medium to compress and reduce the flow rate and resolution during the separation. Do not exceed 75% of the packing flow rate during any separation. A peristaltic pump cannot achieve the highest flow rates or back pressures tolerated by Superdex and Sephacryl and so is not recommended for column packing or running high resolution fractionation on larger columns.
97
Column packing for group separations using Sephadex Sephadex is supplied as a dry powder and must be allowed to swell in excess buffer before use. After swelling adjust with buffer to form a thick slurry from which air bubbles are removed under vacuum. Approximately 75% settled medium is suitable. Fine particles can be decanted. Accelerate the swelling process by using a boiling water bath (Table 10). This also serves to degas the suspension. Allow the suspension to cool before use. Table 10. Bed volume and swelling times for Sephadex Medium
Approx. bed volume (ml/g)
Swelling time (h) +20 °C
Swelling time (h) +90 °C
Sephadex G-10
2–3
3
1
Sephadex G-25 (all grades)
4–6
3
1
Sephadex G-50 Fine
9–11
3
1
Ensure that the column and all components are clean and in good condition. It is particularly important that the nets, net fasteners and glass tube are not damaged. Use well degassed buffers and equilibrate all materials to the temperature at which the separation will be performed. Keep a packed column away from locations that are exposed to drafts or direct sunlight that can cause temperature changes and the formation of bubbles. For group separations, use up to 10 cm bed height. Sample volumes can be up to 30% of the column volume. Pack a quantity of medium up to 5 times the volume of the sample to be desalted. Note: These instructions assume that a column with two adaptors is used for packing. 1. Weigh out the correct amount of dry Sephadex and allow the medium to swell according to the instructions above. Avoid using magnetic stirrers, spatulas or glass rods since they may damage the matrix. 2. Wet the bottom filter by injecting distilled water through the effluent tubing. Close the end piece outlet. Mount filter and bottom end piece onto the column.
For XK 16 and XK 26 columns using a second column instead of a packing reservoir makes it easier to obtain a well-packed column. The second column is used with Packing Connector XK 16 or XK 26 as appropriate. 3. If the slurry volume is greater than the volume of the column, attach a packing reservoir to the column (Figure 69). 4. Mount the column and packing reservoir vertically on a laboratory stand. 5. Fill the column with distilled water or buffer to a height of approximately 2 cm above the column end piece. Avoid air bubbles. 6. Pour the well-mixed and well-degassed suspension in a single operation down the inside wall using a glass rod. Avoid introducing air bubbles. 7. Connect the pump outlet to the inlet of the packing reservoir. Open the column outlet and start the flow of buffer. Pass 2–3 column volumes of buffer through the column in order to stabilize the bed and equilibrate completely. Use a slightly higher flow rate than the flow rate to be used during separations. 8. Maintain the packing flow rate for at least 3 column volumes after a constant bed height is obtained. 9. Mark the bed height on the column and close the column outlet. Remove the packing reservoir. 10. Add buffer carefully to fill the column and form an upward meniscus (Figure 70).
98
11. Connect all tubings. Slacken the adaptor tightening mechanism and insert the adaptor at an angle into the column so that no air is trapped under the net. Slide the adaptor slowly down the column until the mark is reached. Note that the outlet of the adaptor should be open and the column outlet should be closed. 12. Adjust the tightening mechanism to give a sliding seal between the column wall and O-ring. Screw the adaptor onto the column. 13. Continue packing the column for approximately 10 minutes. Stop the pump, close the column outlet and move the top adaptor down onto the surface of the medium. Push the adaptor a further 3 mm into the medium. The column is now ready for equilibration.
1
2
3
2
3
Fig. 69. Using a packing reservoir. 1
Fig. 70. Adding the top adaptor.
Sephadex G-10, G-25 and G-50 obey Darcy's law, for example if the flow rate is doubled then the column pressure will double, hence maximum values for flow or operating pressures do not need to be considered (see Appendix 2 for an explanation of Darcy's law). Controlling flow The safest and easiest way in which to control flow during column packing and chromatography separation is to use a pump controlled within an ÄKTAdesign chromatography system. Accurate and reproducible flow control is particularly important for efficient column packing and when repeating experiments or performing routine preparative work. A peristaltic pump can be used with Sephadex packed in smaller columns. Always connect a pump so that buffer is pumped onto the column (rather than connecting the pump after the column and drawing buffer through the column). This reduces the risk of bubble formation due to suction effects. Always use a flow rate for column packing that is higher than the flow rate used for separation. 99
Packing under gravity Sephadex can be packed using a gravity feed system in which flow rates are controlled by differences in hydrostatic pressure, that is the operating pressure created by the difference between the free surface of the buffer in the buffer container and the column outlet. Use a safety loop as shown in Figure 71 to prevent air from entering the column. A
B AK 26 Operating pressure
XK 26/70
XK 26/70
Fig. 71a. Definition of operating pressure A and B. Pressure (cm water) is measured as the distance between the free surface in the column or reservoir and the end of the outlet tubing. A
B
Fig. 71b. Alternative safety loop arrangements: A. Place the safety loop after the column and place the end of the outlet tubing above the column. The flow stops when the buffer in the inlet tubing reaches the level of the outlet. B. Place the safety loop before the column with the column outlet tubing in any position above the lower loop on the inlet side. The flow stops when the buffer in the inlet tubing reaches the level of the outlet.
Temperature influences the viscosity of the buffer. For a given pressure head, lower flow rates will be reached in a cold room than at room temperature.
100
Custom Designed Products The Custom Products (CP) Group at Amersham Biosciences supplies prepacked columns, made according to the client's choice from our range of columns and media. Custom Designed Media (CDM) can be produced for specific industrial process separations when suitable media are not available from the standard range. The CDM group at Amersham Biosciences works in close collaboration with the user to design, manufacture, test and deliver media for specialized separation requirements. When a chromatography step is developed to be an integral part of a manufacturing process, the choice of column is important to ensure consistent performance and reliable operation. Amersham Biosciences provides a wide range of columns that ensures the highest performance from all our purification media and meets the demands of modern pharmaceutical manufacturing. Please ask your local representative for further details of CP and CDM products or services.
101
Appendix 2 Sephadex and Darcy's law Sephadex G-10, G-25 and G-50 may be assumed to behave as rigid spheres in gel filtration and therefore obey Darcy's Law: U = K DP L-1 (1) U = linear flow rate expressed in cm/h (see Appendix 5). DP = pressure drop over bed expressed in cm H2O L = bed height expressed in cm K = constant of proportionality depending on the properties of the bed material and the buffer. Assuming a buffer with viscosity of 1 cP: U = Ko DP L-1 (2) Ko = the "specific permeability" depending on the particle size of the medium and the water regain. Note that flow is proportional to the pressure drop over the bed and, assuming a constant pressure head, inversely proportional to the bed height. In practice this means that the pressure/flow considerations that must be made when using other gel filtration media do not apply to Sephadex and that a doubling of flow rate leads to a doubling in column pressure. To a good approximation, flow rate is independent of the column diameter. Flow at viscosities greater than 1 cP can be obtained by using the relationship: flow rate is inversely proportional to viscosity. High buffer viscosities can be compensated for by increasing the operating pressure and so maintaining high flow rate. Theoretical flow (not maximum) can be calculated from equation (2) by inserting values for DP and L. Specific permeabilities (K) are given in Table 11. Table 11. Specific permeabilities of Sephadex Sephadex type
Permeability K
Sephadex G-10
19
Sephadex G-25 Superfine Sephadex G-25 Fine Sephadex G-25 Medium Sephadex G-25 Coarse Sephadex G-50 Fine
102
9 30 80 290 36
Appendix 3 Sample preparation Samples for chromatographic purification should be clear and free from particulate matter. Simple steps to clarify a sample before beginning purification will avoid clogging the column, may reduce the need for stringent washing procedures and can extend the life of the chromatographic medium. Sample extraction procedures and the selection of buffers, additives and detergents are determined largely by the source of the material, the stability of the target molecule, the chromatographic techniques that will be employed and the intended use of the product. These subjects are dealt with in general terms in the Protein Purification Handbook and more specifically according to target molecule in the Recombinant Protein Handbook, Protein Amplification and Simple Purification and Antibody Purification Handbook, available from Amersham Biosciences.
Sample stability In the majority of cases, biological activity needs to be retained after purification. Retaining the activity of the target molecule is also an advantage when following the progress of the purification, since detection of the target molecule often relies on its biological activity. Denaturation of sample components often leads to precipitation or enhanced non-specific adsorption, both of which will impair column function. Hence there are many advantages to checking the stability limits of the sample and working within these limits during purification. Proteins generally contain a high degree of tertiary structure, kept together by van der Waals' forces, ionic and hydrophobic interactions and hydrogen bonding. Any conditions capable of destabilizing these forces may cause denaturation and/or precipitation. By contrast, peptides contain a low degree of tertiary structure. Their native state is dominated by secondary structures, stabilized mainly by hydrogen bonding. For this reason, peptides tolerate a much wider range of conditions than proteins. This basic difference in native structures is also reflected in that proteins are not easily renatured, while peptides often renature spontaneously. It is advisable to perform stability tests before beginning to develop a purification protocol. The list below may be used as a basis for such testing: • Test the stability and occurrence of proteolytic activity by leaving an aliquot of the sample at room temperature overnight. Centrifuge each sample and measure activity and UV absorbance at 280 nm in the supernatant. • Test pH stability in steps of one pH unit between pH 2 and pH 9. • Test salt stability with 0–2 M NaCl and 0–2 M (NH4)2SO4 in steps of 0.5 M. • Test the stability towards acetonitrile and methanol in 10% steps between 0 and 50%. • Test the temperature stability in +10 °C steps from +4 to +40 °C.
103
Sample clarification Centrifugation and filtration are standard laboratory techniques for sample clarification and are used routinely when handling small samples. It is highly recommended to centrifuge and filter any sample immediately before chromatographic purification. Centrifugation Centrifugation removes lipids and particulate matter, such as cell debris. If the sample is still not clear after centrifugation, use filter paper or a 5 µm filter as a first step and one of the filters below as a second step filter. • For small sample volumes or proteins that adsorb to filters, centrifuge at 10 000 g for 15 minutes. • For cell lysates, centrifuge at 40 000–50 000 g for 30 minutes. • Serum samples can be filtered through glass wool after centrifugation to remove any remaining lipids. Filtration Filtration removes particulate matter. Membrane filters that give the least amount of nonspecific binding of proteins are composed of cellulose acetate or PVDF. For sample preparation before chromatography, select a filter pore size in relation to the bead size of the chromatographic medium. Nominal pore size of filter 1 µm
Particle size of chromatographic medium 90 µm and upwards
0.45 µm
34 µm
0.22 µm
3, 10, 15 µm or when extra clean samples or sterile filtration is required
Check the recovery of the target protein in a test run. Some proteins may adsorb nonspecifically to filter surfaces. Desalting Desalting columns are suitable for any sample volume and will rapidly remove low molecular weight contaminants in a single step at the same time as transferring the sample into the correct buffer conditions. Centrifugation and/or filtration of the sample before desalting is still recommended. Detailed procedures for buffer exchange and desalting are given in Chapter 2, page 57. At laboratory scale, when samples are reasonably clean after filtration or centrifugation, the buffer exchange and desalting step can be avoided. For affinity chromatography or hydrophobic interaction chromatography, it may be sufficient to adjust the pH of the sample and, if necessary, dilute to reduce the ionic strength of the solution. Rapidly process small or large sample volumes. Use before and/or between purification steps, if needed (remember that each extra step can reduce yield and desalting also dilutes the sample).
104
Remove salts from proteins with molecular weight Mr >5 000. Use 100 mM ammonium acetate or 100 mM ammonium hydrogen carbonate if volatile buffers are required.
Specific sample preparation steps Specific sample preparation steps may be required if the crude sample is known to contain contamininants such as lipids, lipoproteins or phenol red that may build up on a column or if certain gross impurities, such as bulk protein, should be removed before any chromatographic step. Fractional precipitation Fractional precipitation is frequently used at laboratory scale to remove gross impurities from small sample volumes, and occasionally used in small-scale commercial production. Precipitation techniques separate fractions by the principle of differential solubility. Because protein species differ in their degree of hydrophobicity, increased salt concentrations can enhance hydrophobic interactions between the proteins and cause precipitation. Fractional precipitation can be applied to remove gross impurities in three different ways, as shown in Figure 72.
Clarification Bulk proteins and particulate matter precipitated
Supernatant
Extraction Clarification Concentration Target protein precipitated with proteins of similar solubility
Redissolve pellet*
Extraction Clarification Bulk proteins and particulate matter precipitated
Concentration Target protein precipitated
Chromatography
Redissolve pellet*
Remember: if precipitating agent is incompatible with next purification step, use Sephadex G-25 for desalting and buffer exchange e.g. HiTrap Desalting or PD-10 columns
*Remember: not all proteins are easy to redissolve, yield may be reduced
Fig. 72. Three ways to use precipitation.
105
Examples of precipitation agents are reviewed in Table 12. The most common precipitation method using ammonium sulfate is described in more detail. Table 12. Examples of precipitation techniques Precipitation agent
Typical conditions for use
Sample type
Comment
Ammonium sulfate
As described below.
>1 mg/ml proteins especially immunoglobulins.
Stabilizes proteins, no denaturation, supernatant can go directly to HIC.
Dextran sulfate
Add 0.04 ml 10% dextran sulfate and 1 ml 1 M CaCl2 per ml sample, mix 15 min, centrifuge 10 000 g, discard pellet.
Samples with high levels of lipoprotein e.g ascites.
Precipitates lipoprotein.
Polyvinylpyrrolidine
Add 3% (w/v), stir 4 hours, centrifuge 17 000 g, discard pellet.
Samples with high levels of lipoprotein e.g ascites.
Alternative to dextran sulfate.
Polyethylene glycol (PEG, Mr > 4 000)
Up to 20% w/v
Plasma proteins.
No denaturation, supernatant goes directly to IEX or AC, complete removal may be difficult.
Acetone (cold)
Up to 80% v/v at +0 °C. Collect pellet after centrifugation at full speed in an Eppendorf™ centrifuge.
May denature protein irreversibly. Useful for peptide precipitation or concentration of sample for electrophoresis.
Polyethyleneimine
0.1% w/v
Precipitates aggregated nucleoproteins.
Protamine sulfate
1% w/v
Precipitates aggregated nucleoproteins.
Streptomycin sulfate
1% w/v
Caprylic acid
(X/15) g where X = volume of sample.
Precipitation of nucleic acids. Antibody concentration should be >1 mg/ml.
Precipitates bulk of proteins from sera or ascites, leaving immunoglobulins in solution.
Details taken from: Scopes R.K., Protein Purification, Principles and Practice, Springer, (1994), J.C. Janson and L. Rydén, Protein Purification, Principles, High Resolution Methods and Applications, 2nd ed. Wiley Inc, (1998). Personal communications.
Ammonium sulfate precipitation Some proteins may be damaged by ammonium sulfate. Take care when adding crystalline ammonium sulfate: high local concentrations may cause contamination of the precipitate with unwanted proteins. For routine, reproducible purification, precipitation with ammonium sulfate should be avoided in favour of chromatography. In general, precipitation is rarely effective for protein concentrations below 1 mg/ml. Solutions needed for precipitation: Saturated ammonium sulfate solution (add 100 g ammonium sulfate to 100 ml distilled water, stir to dissolve). 1 M Tris-HCl, pH 8.0. Buffer for first purification step.
106
1. Filter (0.45 µm) or centrifuge the sample (10 000 g at +4 °C). 2. Add 1 part 1 M Tris-HCl, pH 8.0 to 10 parts sample volume to maintain pH. 3. Stir gently. Add ammonium sulfate solution, drop by drop. Add up to 50% saturation*. Stir for 1 hour. 4. Centrifuge 20 minutes at 10 000 g. 5. Remove supernatant. Wash the pellet twice by resuspension in an equal volume of ammonium sulfate solution of the same concentration (i.e. a solution that will not redissolve the precipitated protein or cause further precipitation). Centrifuge again. 6. Dissolve pellet in a small volume of the buffer to be used for the next step. 7. Ammonium sulfate is removed during clarification/buffer exchange steps with Sephadex G-25, using desalting columns (see Chapter 2, page 57). *The % saturation can be adjusted either to precipitate a target molecule or to precipitate contaminants.
The quantity of ammonium sulfate required to reach a given degree of saturation varies according to temperature. Table 13 shows the quantities required at +20 °C. Table 13. Quantities of ammonium sulfate required to reach given degrees of saturation at +20 °C Final percent saturation to be obtained 20
25
Starting percent saturation
30
35
40
45
50
55
60
65
70
75
80
85
90
95 100
Amount of ammonium sulfate to add (grams) per litre of solution at +20 °C
0
113 144 176 208 242 277 314 351 390 430 472 516 561 608 657 708 761
5
85 115 146 179 212 246 282 319 358 397 439 481 526 572 621 671 723
10
57
15
28
58
88 119 151 185 219 255 293 331 371 413 456 501 548 596 647
20
0
29
59
89 121 154 188 223 260 298 337 378 421 465 511 559 609
0
29
60
0
30
61
92 125 160 195 232 270 309 351 393 438 485 533
0
30
62
94 128 163 199 236 275 316 358 402 447 495
0
31
63
96 130 166 202 241 281 322 365 410 457
0
31
64
0
32
65
99 135 172 210 250 292 335 381
0
33
66 101 138 175 215 256 298 343
25 30 35 40
86 117 149 182 216 251 287 325 364 405 447 491 537 584 634 685
91 123 157 191 228 265 304 344 386 429 475 522 571
45 50 55 60 65 70 75 80 85 90 95
98 132 169 206 245 286 329 373 419
0
33 0
67 103 140 179 219 261 305 34 0
69 105 143 183 224 267 34 0
70 107 146 186 228 35 0
72 110 149 190 36 0
73 112 152 37 0
75 114 37
76
0
38
Resolubilization of protein precipitates Many proteins are easily resolubilized in a small amount of the buffer to be used in the next chromatographic step. However, a denaturing agent may be required for less soluble proteins. Specific conditions will depend upon the specific protein. These agents must always be removed to allow complete refolding of the protein and to maximize recovery of mass and activity. A chromatographic step often removes a denaturant during purification. Table 14 gives examples of common denaturing agents.
107
Table 14. Denaturing agent
Typical conditions for use
Removal/comment
Urea
2 M–8 M
Remove using Sephadex G-25.
Guanidine hydrochloride
3 M–6 M
Remove using Sephadex G-25 or during IEX.
2%
Remove using Sephadex G-25 or during IEX.
1.5%
Remove using Sephadex G-25 or during IEX.
Triton X-100 Sarcosyl N-octyl glucoside Sodium dodecyl sulfate Alkaline pH
2% 0.1%–0.5% >pH 9, NaOH
Remove using Sephadex G-25 or during IEX. Exchange for non-ionic detergent during first chromatographic step, avoid anion exchange chromatography. May need to adjust pH during chromatography to maintain solubility.
Details taken from: Scopes R.K., Protein Purification, Principles and Practice, Springer, (1994), J.C. Janson and L. Rydén, Protein Purification, Principles, High Resolution Methods and Applications, 2nd ed. Wiley Inc, (1998) and other sources.
See Chapter 2, page 57.
Removal of lipoproteins Lipoproteins and other lipid material can rapidly clog chromatography columns and it is advisable to remove them before beginning purification. Precipitation agents such as dextran sulfate and polyvinylpyrrolidine, described under Fractional precipitation, are recommended to remove high levels of lipoproteins from samples such as ascitic fluid. Centrifuge samples to avoid the risk of non-specific binding of the target molecule to a filter. Samples such as serum can be filtered through glass wool to remove remaining lipids.
Removal of phenol red Phenol red is frequently used at laboratory scale as a pH indicator in cell culture. Although not directly interfering with purification, phenol red may bind to certain purification media and should be removed as early as possible to avoid the risk of contamination. It is known to bind to anion exchange media at pH >7. Use a desalting column to simultaneously remove phenol red (a low molecular weight molecule) and transfer sample to the correct buffer conditions for further purification, as described in Chapter 2, page 57.
Removal of low molecular weight contaminants If samples contain a high level of low molecular weight contaminants, use a desalting column before the first chromatographic purification step, as described in Chapter 2, page 57.
108
Appendix 4 Selection of purification equipment Simple buffer exchange and desalting steps can be performed using a syringe or peristaltic together with prepacked HiTrap columns. A chromatography system is needed to deliver accurately controlled flow rates for high resolution separations. Standard ÄKTAdesign configurations Explorer 100
Purifier 10
FPLC
Prime
Syringe or peristaltic pump + HiTrap Desalting column
Gravity–fed columns
Simple, one step desalting, buffer exchange
ü
ü
ü
ü
ü
ü
Reproducible performance for routine separation
ü
ü
ü
ü
Optimization of one step separation to increase purity
ü
ü
ü
ü
System control and data handling for regulatory requirements, e.g. GLP
ü
ü
ü
Automatic method development and optimization
ü
ü
ü
Automatic buffer preparation
ü
ü
Automatic pH scouting
ü
ü
Automatic media or column scouting
ü
Automatic multi-step purification
ü
Scale up, process development and transfer to production
ü
Way of working
ÄKTAprime
ÄKTAFPLC™
ÄKTAexplorer ÄKTApurifier
109
Appendix 5 Converting from linear flow (cm/hour) to volumetric flow rates (ml/min) and vice versa It is convenient when comparing results for columns of different sizes to express flow as linear flow (cm/hour). However, flow is usually measured in volumetric flow rate (ml/min). To convert between linear flow and volumetric flow rate use one of the formulae below.
From linear flow (cm/hour) to volumetric flow rate (ml/min) Volumetric flow rate (ml/min) = =
Linear flow (cm/h) x column cross sectional area (cm2) 60 Y p x d2 x 60 4
where Y = linear flow in cm/h d = column inner diameter in cm
Example: What is the volumetric flow rate in an XK 16/70 column (i.d. 1.6 cm) when the linear flow is 150 cm/hour? Y = linear flow = 150 cm/h d = inner diameter of the column = 1.6 cm Volumetric flow rate =
150 x p x 1.6 x 1.6 ml/min 60 x 4
= 5.03 ml/min
From volumetric flow rate (ml/min) to linear flow (cm/hour) Linear flow (cm/h) =
Volumetric flow rate (ml/min) x 60 column cross sectional area (cm2)
= Z x 60 x
4 p x d2
where Z = volumetric flow rate in ml/min d = column inner diameter in cm
Example: What is the linear flow in an HR 5/5 column (i.d. 0.5 cm) when the volumetric flow rate is 1 ml/min? Z = Volumetric flow rate = 1 ml/min d = column inner diameter = 0.5 cm Linear flow = 1 x 60 x
4 p x 0.5 x 0.5
cm/h
= 305.6 cm/h
From ml/min to using a syringe 1 ml/min = approximately 30 drops/min on a HiTrap 1 ml column 5 ml/min = approximately 120 drops/min on a HiTrap 5 ml column
110
Appendix 6 Conversion data: proteins, column pressures Mass (g/mol)
1 µg
1 nmol
Protein
A280 for 1 mg/ml
10 000
100 pmol; 6 x 10
13
molecules
10 µg
IgG
50 000
20 pmol; 1.2 x 10
13
molecules
50 µg
IgM
1.20
100 000
10 pmol; 6.0 x 10
12
molecules
100 µg
IgA
1.30
150 000
6.7 pmol; 4.0 x 10
12
molecules
150 µg
Protein A
0.17
1 kb of DNA
= 333 amino acids of coding capacity
270 bp DNA
= 10 000 g/mol
1.35
Avidin
1.50
Streptavidin
3.40
Bovine Serum Albumin
0.70
= 37 000 g/mol 1.35 kb DNA
= 50 000 g/mol
2.70 kb DNA
= 100 000 g/mol
Average molecular weight of an amino acid = 120 g/mol.
Column pressures The maximum operating back pressure refers to the pressure above which the column contents may begin to compress. Pressure units may be expressed in megaPascals, bar or pounds per square inch and can be converted as follows: 1MPa = 10 bar = 145 psi
111
Appendix 7 Table of amino acids Three-letter code
Single-letter code
Alanine
Ala
A
Arginine
Arg
R
Amino acid
Structure HOOC CH3 H 2N NH2
HOOC CH2CH2CH2NHC H 2N
NH
HOOC
Asparagine
Asn
N
Aspartic Acid
Asp
D
CH2CONH2 H 2N HOOC CH2COOH H 2N HOOC
Cysteine
Cys
CH2SH
C H 2N HOOC
Glutamic Acid
Glu
CH2CH2COOH
E H 2N HOOC
Glutamine
Gln
Q
Glycine
Gly
G
Histidine
His
H
Isoleucine
Ile
I
CH2CH2CONH2 H 2N HOOC H H 2N HOOC
N CH2
NH
H 2N HOOC
CH(CH3)CH2CH3 H 2N HOOC
Leucine
Leu
L
CH3 CH2CH CH3
H 2N HOOC
Lysine
Lys
K
Methionine
Met
M
CH2CH2CH2CH2NH2 H 2N HOOC CH2CH2SCH3 H 2N HOOC
Phenylalanine
Phe
F
Proline
Pro
P
CH2 H 2N HOOC H 2N
NH
HOOC
Serine
Ser
S
Threonine
Thr
T
CH2OH H 2N HOOC CHCH3 H 2N
OH
HOOC
Tryptophan
Trp
W
CH2 H 2N
NH
HOOC
Tyrosine
Tyr
CH2
Y H 2N HOOC
Valine
Val
CH(CH3)2
V H 2N
112
OH
Formula
Mr
Middle unit residue (-H20) Formula Mr
C3H7NO2
89.1
C3H5NO
C6H14N4O2
174.2
C 4H 8N 2O 3
Charge at pH 6.0–7.0
Hydrophobic (non-polar)
Uncharged (polar)
71.1
Neutral
n
C6H12N4O
156.2
Basic (+ve)
132.1
C 4H 6N 2O 2
114.1
Neutral
C4H7NO4
133.1
C4H5NO3
115.1
Acidic(-ve)
C3H7NO2S
121.2
C3H5NOS
103.2
Neutral
C5H9NO4
147.1
C5H7NO3
129.1
Acidic (-ve)
C5H10N2O3
146.1
C 5H 8N 2O 2
128.1
Neutral
n
C2H5NO2
75.1
C2H3NO
57.1
Neutral
n
C 6H 9N 3O 2
155.2
C6H 7N3O
137.2
Basic (+ve)
C6H13NO2
131.2
C6H11NO
113.2
Neutral
n
C6H13NO2
131.2
C6H11NO
113.2
Neutral
n
C6H14N2O2
146.2
C6H12N2O
128.2
Basic(+ve)
C5H11NO2S
149.2
C5H9NOS
131.2
Neutral
n
C9H11NO2
165.2
C9H9NO
147.2
Neutral
n
C5H9NO2
115.1
C5H7NO
97.1
Neutral
n
C3H7NO3
105.1
C3H5NO2
87.1
Neutral
n
C4H9NO3
119.1
C4H7NO2
101.1
Neutral
n
C11H12N2O2
204.2
C11H10N2O
186.2
Neutral
C9H11NO3
181.2
C9H9NO2
163.2
Neutral
C5H11NO2
117.1
C5H9NO
99.1
Neutral
Hydrophilic (polar)
n n n n n
n
n
n n n
113
Appendix 8 Analytical assays during purification Analytical assays are essential to follow the progress of purification. They are used to assess the effectiveness of each step in terms of yield, biological activity, recovery and to help during optimization of experimental conditions. The importance of a reliable assay for the target molecule cannot be over-emphasized. When testing chromatographic fractions, ensure that the buffers used for purification do not interfere with the assay. Total protein determination Lowry or Bradford assays are used most frequently to determine the total protein content. The Bradford assay is particularly suited to samples where there is a high lipid content that may interfere with the Lowry assay. Purity determination Purity is most often estimated by SDS-PAGE. Alternatively, isoelectric focusing, capillary electrophoresis, reversed phase chromatography or mass spectrometry may be used. SDS-PAGE Analysis Reagents Required 6X SDS loading buffer: 0.35 M Tris-HCl (pH 6.8), 10.28% (w/v) SDS, 36% (v/v) glycerol, 0.6 M dithiothreitol (or 5% 2-mercaptoethanol), 0.012% (w/v) bromophenol blue. Store in 0.5 ml aliquots at -80 °C.
1. Add 2 µl of 6X SDS loading buffer to 5–10 µl of supernatant from crude extracts, cell lysates or purified fractions as appropriate. 2. Vortex briefly and heat for 5 minutes at +90 to +100 °C. 3. Load the samples onto an SDS-polyacrylamide gel. 4. Run the gel and stain with Coomassie™ Blue (Coomassie Blue R Tablets) or silver (PlusOne™ Silver Staining Kit, Protein).
The percentage of acrylamide in the SDS-gel should be selected according to the expected molecular weight of the protein of interest (see Table 15). Table 15. % Acrylamide in resolving gel
Separation size range
Single percentage: 5%
36 000–200 000
7.5%
24 000–200 000
10%
14 000–200 000
12.5%
14 000–100 000
15%
14 000–60 0001
5–15%
14 000–200 000
Gradient:
1
114
5–20%
10 000–200 000
10–20%
10 000–150 000
The larger proteins fail to move significantly into the gel.
Functional assays Immunospecific interactions have enabled the development of many alternative assay systems for the assessment of active concentration of target molecules. • Western blot analysis is used when the sensitivity of SDS-PAGE with Coomassie Blue or silver staining is insufficient. 1. Separate the protein samples by SDS-PAGE. 2. Transfer the separated proteins from the gel to an appropriate membrane, such as Hybond™ ECL™ (for subsequent ECL detection) or Hybond P (for subsequent ECL Plus™ detection). 3. Develop the membrane with the appropriate specified reagents.
Electrophoresis and protein transfer may be accomplished using a variety of equipment and reagents. For further details, refer to the Protein Electrophoresis Technical Manual and Hybond ECL instruction manual, both from Amersham Biosciences. • ELISAs are most commonly used as activity assays. • Functional assays using the phenomenon of surface plasmon resonance to detect immunospecific interactions (e.g. using BIACORE™ systems) enable the determination of active concentration, epitope mapping and studies of reaction kinetics. Detection and assay of tagged proteins SDS-PAGE, Western blotting and ELISAs can also be applied to the detection and assay of genetically engineered molecules to which a specific tag has been attached. In some cases, an assay based on the properties associated with the tag itself can be developed, e.g. the GST Detection Module for enzymatic detection and quantification of GST tagged proteins. Further details on the detection and quantification of GST and (His)6 tagged proteins are available in The Recombinant Protein Handbook: Protein Amplification and Simple Purification and GST Gene Fusion System Handbook from Amersham Biosciences.
115
Appendix 9 Storage of biological samples The advice given here is of a general nature and cannot be applied to every biological sample. Always consider the properties of the specific sample and its intended use before following any of these recommendations. General recommendations • Add stabilizing agents, if essential. Stabilizing agents are often required for storage of purified proteins. • Serum, culture supernatants and ascitic fluid should be kept frozen at -20 °C or -70 °C, in small aliquots. • Avoid repeated freeze/thawing or freeze drying/re-dissolving that may reduce biological activity. • Avoid conditions close to stability limits for example pH or salt concentrations, reducing or chelating agents. • Keep refrigerated at +4 °C in a closed vessel to minimize bacterial growth and protease activity. Above 24 hours at +4 °C, add a preserving agent if possible (e.g. merthiolate 0.01%). Sodium azide can interfere with many coupling methods and some biological assays and can be a health hazard. It can be removed by using a desalting column (see Chapter 2, page 57). General recommendations for purified proteins • Store as a precipitate in high concentration of ammonium sulfate, for example 4.0 M. • Freeze in 50% glycerol, especially suitable for enzymes. • Avoid the use of preserving agents if the product is to be used for a biological assay. Preserving agents should not be added if in vivo experiments are to be performed. Instead store samples in small aliquots and keep frozen. • Sterile filter to prolong storage time. • Add stabilizing agents, e.g. glycerol (5–20%), serum albumin (10 mg/ml), ligand (concentration is selected based on concentration of active protein) to help to maintain biological activity. Remember that any additive will reduce the purity of the protein and may need to be removed at a later stage. • Avoid repeated freeze/thawing or freeze drying/re-dissolving that may reduce biological activity. Sodium azide can interfere with many coupling methods and some biological assays. It can be removed by using a desalting column (see Chapter 2, page 57). Cryoproteins are a group of proteins, including some mouse antibodies of the IgG3 subclass, that should not be stored at +4 °C as they precipitate at this temperature. Keep at room temperature in the presence of a preserving agent.
116
Additional reading and reference material Code No.
Purification Antibody Purification Handbook
18-1037-46
Protein Purification Handbook
18-1132-29
Recombinant Protein Handbook: Protein Amplification and Simple Purification
18-1142-75
GST Gene Fusion System Handbook
18-1157-58
Affinity Chromatography Handbook: Principles and Methods
18-1022-29
Ion Exchange Chromatography Handbook: Principles and Methods
18-1114-21
Hydrophobic Interaction Chromatography Handbook: Principles and Methods
18-1020-90
Reversed Phase Chromatography Handbook: Principles and Methods
18-1112-93
Expanded Bed Adsorption Handbook: Principles and Methods
18-1124-26
Protein and Peptide Purification Technique Selection
18-1128-63
Fast Desalting and Buffer Exchange of Proteins and Peptides
18-1128-62
Gel Filtration Columns and Media Selection Guide
18-1124-19
Ion Exchange Columns and Media Selection Guide
18-1127-31
Chromatofocusing with Polybuffer and PBE, Handbook
18-1009-07
HIC Columns and Media Product Profile
18-1100-98
Affinity Columns and Media Product Profile
18-1121-86
Convenient Protein Purification, HiTrap Column Guide
18-1128-81
ÄKTAdesign Brochure
18-1158-77
ÄKTA 3D Kit Brochure
18-1160-45
GST Fusion System Brochure
18-1159-30
Protein Purifier Software
18-1155-49
Protein Purification: Principles, High Resolution Methods and Applications, J-C. Jansson and L.Rydén
18-1128-68
Sephadex LH-20: chromatography in organic solvents
18-1009-74
Preparative Gel chromatography on Sephadex-LH-20, H. Henke
18-1113-89
Column Packing Video (PAL)
17-0893-01
Column Packing Video (NTSC)
17-0894-01
Reference list HiTrap Desalting
18-1156-70*
Reference list HiPrep 26/10 Desalting
18-1156-89*
Reference list HiPrep Sephacryl S-100 HR
18-1156-86*
Reference list HiPrep Sephacryl S-200 HR
18-1156-87*
Reference list HiPrep Sephacryl S-300 HR
18-1156-88*
Reference list HiLoad Superdex 30 prep grade
18-1156-94*
Reference list HiLoad Superdex 75 prep grade
18-1156-95*
Reference list HiLoad Superdex 200 prep grade
18-1156-96*
Analysis Protein analysis–using the power of 2-D electrophoresis
18-1124-82
2D Electrophoresis Handbook
80-6429-60
Protein Electrophoresis Technical Manual
80-6013-88
ECL Western and ECL Plus Western Blotting Application Note
18-1139-13
The reference lists are only available at www.chromatography.amershambiosciences.com and many of the above items can also be downloaded.
117
Ordering information Product
Quantity
Code No.
Superdex Peptide PC 3.2/30
1 × 2.4 ml column
17-1458-01
Superdex 75 PC 3.2/30
1 × 2.4 ml column
17-0771-01
Superdex 200 PC 3.2/30
1 × 2.4 ml column
17-1089-01
Superdex Peptide HR 10/30
1 × 24 ml column
17-1453-01
Superdex 75 HR 10/30
1 × 24 ml column
17-1047-01
Superdex 200 HR 10/30
1 × 24 ml column
17-1088-01
HiLoad 16/60 Superdex 30 prep grade
1 × 120 ml column
17-1139-01
HiLoad 26/60 Superdex 30 prep grade
1 × 320 ml column
17-1140-01
HiLoad 16/60 Superdex 75 prep grade
1 × 120 ml column
17-1068-01
HiLoad 26/60 Superdex 75 prep grade
1 × 320 ml column
17-1070-01
HiLoad 16/60 Superdex 200 prep grade
1 × 120 ml column
17-1069-01
HiLoad 26/60 Superdex 200 prep grade
1 × 320 ml column
17-1071-01
Superdex 30 prep grade
25 ml
17-0905-10
Superdex 30 prep grade
150 ml
17-0905-01
Superdex 75 prep grade
25 ml
17-1044-10
Superdex 75 prep grade
150 ml
17-1044-01
High Resolution Fractionation Superdex
Superdex 200 prep grade
25 ml
17-1043-10
Superdex 200 prep grade
150 ml
17-1043-01
Superose 6 PC 3.2/30
1 × 2.4 ml column
17-0673-01
Superose 12 PC 3.2/30
1 × 2.4 ml column
17-0674-01
Superose 6 HR 10/30
1 × 24 ml column
17-0537-01
Superose 12 HR 10/30
1 × 24 ml column
17-0538-01
Superose 6 prep grade
125 ml
17-0489-01
Superose 12 prep grade
125 ml
17-0536-01
Superose
Sephacryl
118
HiPrep 16/60 Sephacryl S-100 HR
1 × 120 ml column
17-1165-01
HiPrep 26/60 Sephacryl S-100 HR
1 × 320 ml column
17-1194-01
HiPrep 16/60 Sephacryl S-200 HR
1 × 120 ml column
17-1166-01
HiPrep 26/60 Sephacryl S-200 HR
1 × 320 ml column
17-1195-01
HiPrep 16/60 Sephacryl S-300 HR
1 × 120 ml column
17-1167-01
HiPrep 26/60 Sephacryl S-300 HR
1 × 320 ml column
17-1196-01
Sephacryl S-100 HR
150 ml
17-0612-10
Sephacryl S-100 HR
750 ml
17-0612-01
Sephacryl S-200 HR
150 ml
17-0584-10
Sephacryl S-200 HR
750 ml
17-0584-01
Sephacryl S-300 HR
150 ml
17-0599-10
Sephacryl S-300 HR
750 ml
17-0599-01
Sephacryl S-400 HR
150 ml
17-0609-10
Sephacryl S-400 HR
750 ml
17-0609-01
Sephacryl S-500 HR
150 ml
17-0613-10
Sephacryl S-500 HR
750 ml
17-0613-01
Sephacryl S-1000 SF
750 ml
17-0476-01
Product
Quantity
Code No.
Desalting and Group Separations HiTrap Desalting
5 × 5 ml columns
17-1408-01
HiPrep 26/10 Desalting
1 × 53 ml column
17-5087-01
PD-10 Desalting Column
30 gravity-fed columns
17-0851-01
Empty PD-10 Desalting Column
50 gravity-fed empty columns
17-0435-01
NICK columns
20 gravity-fed columns
17-0855-01*
NICK columns
50 gravity-fed columns
17-0855-02*
NAP-5 columns
20 gravity-fed columns
17-0853-01*
NAP-5 columns
50 gravity-fed columns
17-0853-02*
NAP-10 columns
20 gravity-fed columns
17-0854-01*
NAP-10 columns
50 gravity-fed columns
17-0854-02*
NAP-25 columns
20 gravity-fed columns
17-0852-01*
NAP-25 columns
50 gravity-fed columns
17-0852-02*
Sephadex G-10
100 g
17-0010-01
Sephadex G-10
500 g
17-0010-02
Sephadex G-25 Coarse
100 g
17-0034-01
Sephadex G-25 Coarse
500 g
17-0034-02
Sephadex G-25 Fine
100 g
17-0032-01
Sephadex G-25 Fine
500 g
17-0032-02
Sephadex G-25 Medium
100 g
17-0033-01
Sephadex G-25 Medium
500 g
17-0033-02 17-0031-01
Sephadex G-25 Superfine
100 g
Sephadex G-25 Superfine
500 g
17-0031-02
Sephadex G-50 Fine
100 g
17-0042-01
Sephadex G-50 Fine
500 g
17-0042-02
Sephadex LH-20
25 g
17-0090-10
Sephadex LH-20
100 g
17-0090-01
Sephadex LH-20
500 g
17-0090-02
Gel Filtration LMW Calibration Kit Includes: Ribonuclease A (13 700), chymotrypsinogen A (25 000), ovalbumin (43 000), bovine serum albumin (67 000), Blue Dextran 2000
1 kit
17-0442-01
Gel Filtration HMW Calibration Kit Includes: Aldolase (158 000), catalase (232 000), ferritin (440 000), thyroglobulin (669 000), Blue Dextran 2000
1 kit
17-0441-01
Blue Dextran 2000
10 g
17-0360-01
Separation in organic solvents
Calibration Kits
*Prepacked columns suitable for desalting of oligonucleotides, DNA and proteins.
119
Product
Quantity
Code No.
XK 16/20 column
1
18-8773-01
XK 16/40 column
1
18-8774-01
XK 16/70 column
1
18-8775-01
XK 16/100 column
1
18-8776-01
XK 26/20 column
1
18-1000-72
XK 26/40 column
1
18-8768-01
XK 26/70 column
1
18-8769-01
XK 26/100 column
1
18-8770-01
XK 50/20 column
1
18-1000-71
XK 50/30 column
1
18-8751-01
XK 50/60 column
1
18-8752-01
XK 50/100 column
1
18-8753-01
Columns
All XK columns are delivered with one AK adaptor, TEFZEL tubing (0.8 mm i.d. for XK 16 and XK 26 columns, 1.2 mm i.d. for XK 50 columns, with M6 connectors, thermostatic jacket, support snap-on net rings, dismantling tool (XK 16 and XK 26 only), and instructions.
Accessories and spare parts Packing Connector XK 16
1
18-1153-44
Packing Connector XK 26
1
18-1153-45
SR 10/50 column
1
19-2638-01
SR 10/50J column*
1
19-1734-01
SR 25/45 column
1
19-0879-01
SR 25/100 column
1
19-0880-01
Solvent resistant columns
All SR columns are delivered complete with two SRA adaptors, PTFE tubing (2 × 50 cm), spare bed supports, tubing end fittings, flanging tool and instructions. *SR 10/50J includes a borosilicate glass jacket. Jackets are not available for other SR columns.
Accessories SRE 10 packing reservoir
1
For a complete lisiting refer to Amersham Biosciences BioDirectory or www.chromatography.amershambiosciences.com
120
19-2097-01
Handbooks from Amersham Biosciences
Antibody Purification ÄKTA, FPLC, PlusOne, HiLoad , HiTrap, HiPrep, Hybond, ECL, ECL Plus, BioProcess, MabSelect, Sephacryl, Sephadex, Superose, Sepharose, Superdex and Drop Design are trademarks of Amersham Biosciences Limited.
Handbook 18-1037-46
Amersham and Amersham Biosciences are trademarks of Amersham plc.
The Recombinant Protein Handbook
Gel Filtration
Coomassie is a trademark of ICI plc.
Protein Amplification and Simple Purification 18-1142-75
Principles and Methods 18-1022-18
MicroSpin is a trademark of Lida Manufacturing Corp. Triton is a registered trademark of Union Carbide Chemicals and Plastics Co.
Percoll
Tween is a registered trademark of ICI Americas, Inc.
Methodology and Applications 18-1115-69
Eppendorf and Multipipette are trademarks of Eppendorf-Netheler-Hinz GmbH.
Ficoll-Paque Plus
All goods and services are sold subject to the terms and conditions of sale of the company within the Amersham Biosciences group that supplies them. A copy of these terms and conditions is available on request. © Amersham Biosciences AB 2002 – All rights reserved.
Protein Purification
Reversed Phase Chromatography
Handbook 18-1132-29
Principles and Methods 18-1134-16
Ion Exchange Chromatography
Expanded Bed Adsorption
Principles and Methods 18-1114-21
Principles and Methods 18-1124-26
Affinity Chromatography
Chromatofocusing
Principles and Methods 18-1022-29
with Polybuffer and PBE 18-1009-07
Hydrophobic Interaction Chromatography
Microcarrier cell culture
using immobilized pH gradients
Principles and Methods 18-1020-90
Principles and Methods 18-1140-62
Principles and Methods 80-6429-60
For in vitro isolation of lymphocytes 18-1152-69
GST Gene Fusion System Handbook 18-1157-58
BIACORE is a trademark of Biacore AB.
Amersham Biosciences AB, Björkgatan 30, SE-751 84 Uppsala, Sweden Amersham Biosciences UK Limited, Amersham Place, Little Chalfont, Buckinghamshire HP7 9NA, England Biosciences Corp., 800 Centennial Avenue, PO Box 1327, Piscataway NJ 08855, USA Amersham Biosciences Europe GmbH, Munzinger Strasse 9, D-79111 Freiburg, Germany
2-D Electrophoresis
Amersham Biosciences K.K., Sanken Bldg. 3-25-1, Hyakunincho Amersham Shinjuku-ku, Tokyo 169-0073, Japan.
Production: RAK Design AB
Gel Filtration Handbook – Principles and Methods
Gel Filtration Principles and Methods
www.chromatography.amershambiosciences.com
18-1022-18 Edition AI
Affinity Chromatography Principles and Methods
Back to Collection 18-1022-29 Edition AC
Handbooks from Amersham Pharmacia Biotech
Antibody Purification Handbook 18-1037-46
The Recombinant Protein Handbook Protein Amplification and Simple Purification 18-1142-75
Protein Purification Handbook 18-1132-29
Ion Exchange Chromatography
Reversed Phase Chromatography
Principles and Methods 18-1114-21
Principles and Methods 18-1134-16
Affinity Chromatography
Expanded Bed Adsorption
Principles and Methods 18-1022-29
Principles and Methods 18-1124-26
Hydrophobic Interaction Chromatography
Chromatofocusing
Principles and Methods 18-1020-90
with Polybuffer and PBE 50-01-022PB
Gel Filtration
Microcarrier cell culture
Principles and Methods 18-1022-18
Principles and Methods 18-1140-62
Affinity Chromatography Principles and Methods
1
Content Introduction ............................................................................................................. 7 Symbols and abbreviations ......................................................................................................................... 8
Chapter 1 Affinity chromatography in brief ................................................................................ 9 BioProcess Media for large-scale production ................................................................. 12 Custom Designed Media and Columns ......................................................................... 12 Common terms in affinity chromatography ................................................................... 13
Chapter 2 Affinity chromatography in practice ......................................................................... 15 Purification steps ..................................................................................................................................... 15 Media selection ....................................................................................................................................... 16 Preparation of media and buffers ............................................................................................................... 16 Sample preparation and application ........................................................................................................... 17 Elution ................................................................................................................................................... 18 Flow rates ............................................................................................................................................... 21 Analysis of results and further steps ........................................................................................................... 21 Equipment selection ................................................................................................................................ 21 Troubleshooting ....................................................................................................................................... 22
Chapter 3 Purification of specific groups of molecules ............................................................ 25 Immunoglobulins ....................................................................................................... 25 IgG, IgG fragments and subclasses .............................................................................. 26 HiTrap Protein G HP, Protein G Sepharose 4 Fast Flow, MAbTrap Kit ............................................................. 28 HiTrap Protein A HP, Protein A Sepharose 4 Fast Flow, HiTrap rProtein A FF, rProtein A Sepharose 4 Fast Flow .............................................................................................................. 33
Monoclonal IgM from hybridoma cell culture ................................................................ 38 HiTrap IgM Purification HP ....................................................................................................................... 38
Avian IgY from egg yolk .............................................................................................. 40 HiTrap IgY Purification HP ........................................................................................................................ 40
Recombinant fusion proteins ...................................................................................... 42 GST fusion proteins ................................................................................................... 42 GST MicroSpin Purification Module, GSTrap FF, Glutathione Sepharose 4 Fast Flow, Glutathione Sepharose 4B ........................................................................................................................ 42
Poly (His) fusion proteins ........................................................................................... 46 His MicroSpin Purification Module, HisTrap Kit, HiTrap Chelating HP, Chelating Sepharose Fast Flow .................................................................................................................. 46
Protein A fusion proteins ............................................................................................ 51 IgG Sepharose 6 Fast Flow ........................................................................................................................ 51
Purification or removal of serine proteases, e.g. thrombin and trypsin, and zymogens ..................................................................... 53 HiTrap Benzamidine FF (high sub), Benzamidine Sepharose 4 Fast Flow (high sub) ........................................ 53
2
Serine proteases and zymogens with an affinity for arginine ........................................... 57 Arginine Sepharose 4B ............................................................................................................................. 57
DNA binding proteins ................................................................................................. 59 HiTrap Heparin HP, HiPrep 16/10 Heparin FF, Heparin Sepharose 6 Fast Flow ............................................... 59
Coagulation factors .................................................................................................... 64 HiTrap Heparin HP, HiPrep 16/10 Heparin FF, Heparin Sepharose 6 Fast Flow ............................................... 64
Biotin and biotinylated substances .............................................................................. 65 HiTrap Streptavidin HP, Streptavidin Sepharose High Performance ................................................................ 65
Purification or removal of fibronectin ........................................................................... 68 Gelatin Sepharose 4B ............................................................................................................................... 68
Purification or removal of albumin ............................................................................... 69 HiTrap Blue HP, Blue Sepharose 6 Fast Flow .............................................................................................. 69
NAD+-dependent dehydrogenases and ATP-dependent kinases ....................................... 72 5' AMP Sepharose 4B, HiTrap Blue HP, Blue Sepharose 6 Fast Flow ............................................................. 72
NADP+-dependent dehydrogenases and other enzymes with affinity for NADP+ ............... 74 2'5' ADP Sepharose 4B, Red Sepharose CL-6B ........................................................................................... 74
Glycoproteins or polysaccharides ................................................................................. 79 Con A Sepharose 4B, Lentil Lectin Sepharose 4B, Agarose Wheat Germ Lectin ............................................... 79 Con A for binding of branched mannoses, carbohydrates with terminal mannose or glucose (aMan > aGlc > GlcNAc) .......................................................................................................... 79 Lentil lectin for binding of branched mannoses with fucose linked a(1,6) to the N-acetyl-glucosamine, (aMan > aGlc > GlcNAc) N-acetylglucosamine binding lectins ......................................................................................................................................... 82 Wheat germ lectin for binding of chitobiose core of N-linked oligosaccharides, [GlcNAc(b1,4GlcNAc)1-2 > b GlcNAc] ......................................................................................................... 83
Calmodulin binding proteins: ATPases, adenylate cyclases, protein kinases, phosphodiesterases, neurotransmitters ................................................. 85 Calmodulin Sepharose 4B ......................................................................................................................... 85
Proteins and peptides with exposed amino acids: His, Cys, Trp, and/or with affinity for metal ions (also known as IMAC, immobilized metal chelate affinity chromatography) ...................................................... 87 HiTrap Chelating HP, Chelating Sepharose Fast Flow, His MicroSpin Purification Module, HisTrap Kit ........................................................................................... 87
Thiol-containing substances (purification by covalent chromatography) ........................... 91 Activated Thiol Sepharose 4B, Thiopropyl Sepharose 6B .............................................................................. 91
Chapter 4 Components of an affinity medium ........................................................................... 96 The matrix ............................................................................................................................................... 96 The ligand ............................................................................................................................................... 97 Spacer arms ............................................................................................................................................ 98 Ligand coupling ....................................................................................................................................... 99 Ligand specificity ..................................................................................................................................... 99
3
Chapter 5 Designing affinity media using pre-activated matrices ............................................ 100 Choosing the matrix ............................................................................................................................... 100 Choosing the ligand and spacer arm ......................................................................................................... 100 Choosing the coupling method ................................................................................................................. 100 Coupling the ligand ................................................................................................................................ 102 Binding capacity, ligand density and coupling efficiency ............................................................................ 103 Binding and elution conditions ................................................................................................................ 104
Coupling through the primary amine of a ligand .......................................................... 105 HiTrap NHS-activated HP, NHS-activated Sepharose 4 Fast Flow ................................................................ 105 CNBr-activated Sepharose ....................................................................................................................... 108 Immunoaffinity chromatography .............................................................................................................. 112
Coupling small ligands through amino or carboxyl groups via a spacer arm ...................................................................................................... 113 EAH Sepharose 4B and ECH Sepharose 4B .............................................................................................. 113
Coupling through hydroxy, amino or thiol groups via a 12-carbon spacer arm ...................................................................................... 116 Epoxy-activated Sepharose 6B ................................................................................................................. 116
Coupling through a thiol group .................................................................................. 120 Thiopropyl Sepharose 6B ........................................................................................................................ 120
Coupling other functional groups ............................................................................... 121
Chapter 6 Affinity chromatography and CIPP ........................................................................... 123 Applying CIPP .......................................................................................................... 124 Selection and combination of purification techniques .................................................. 124
Appendix 1 .......................................................................................................... 129 Sample preparation ................................................................................................. 129 Sample stability ..................................................................................................................................... 129 Sample clarification ............................................................................................................................... 130
Specific sample preparation steps ............................................................................. 131 Resolubilization of protein precipitates ..................................................................................................... 133
Buffer exchange and desalting .................................................................................. 134 Removal of lipoproteins ............................................................................................ 137 Removal of phenol red ............................................................................................. 137 Removal of low molecular weight contaminants .......................................................... 137
Appendix 2 .......................................................................................................... 139 Selection of purification equipment ........................................................................... 139
Appendix 3 .......................................................................................................... 140 Column packing and preparation ............................................................................... 140
Appendix 4 .......................................................................................................... 142 Converting from linear flow (cm/hour) to volumetric flow rates (ml/min) and vice versa ............................................................................. 142
Appendix 5 .......................................................................................................... 143 Conversion data: proteins, column pressures .............................................................. 143 Column pressures .................................................................................................................................. 143
Appendix 6 .......................................................................................................... 144 Table of amino acids ................................................................................................ 144
Appendix 7 .......................................................................................................... 146 Kinetics in affinity chromatography ........................................................................... 146
Appendix 8 .......................................................................................................... 151 Analytical assays during purification .......................................................................... 151
Appendix 9 .......................................................................................................... 153 Storage of biological samples .................................................................................... 153
Additional reading and reference material ............................................................. 154 Ordering information ............................................................................................ 155
Introduction Biomolecules are purified using purification techniques that separate according to differences in specific properties, as shown in Figure 1. Property
Technique*
Biorecognition (ligand specificity)
Affinity chromatography
Charge
Ion exchange chromatography
Size
Gel filtration (sometimes called size exclusion)
Hydrophobicity
Hydrophobic interaction chromatography Reversed phase chromatography
*Expanded bed adsorption is a technique used for large-scale purification. Proteins can be purified from crude sample without the need for separate clarification, concentration and initial purification to remove particulate matter. The STREAMLINE™ adsorbents, used for expanded bed adsorption, capture the target molecules using the same principles as affinity, ion exchange or hydrophobic interaction chromatography.
Gel filtration
Hydrophobic interaction
Ion exchange
Affinity
Reversed phase
Fig. 1. Separation principles in chromatographic purification.
Affinity chromatography separates proteins on the basis of a reversible interaction between a protein (or group of proteins) and a specific ligand coupled to a chromatographic matrix. The technique offers high selectivity, hence high resolution, and usually high capacity for the protein(s) of interest. Purification can be in the order of several thousand-fold and recoveries of active material are generally very high. Affinity chromatography is unique in purification technology since it is the only technique that enables the purification of a biomolecule on the basis of its biological function or individual chemical structure. Purification that would otherwise be time-consuming, difficult or even impossible using other techniques can often be easily achieved with affinity chromatography. The technique can be used to separate active biomolecules from denatured or functionally different forms, to isolate pure substances present at low concentration in large volumes of crude sample and also to remove specific contaminants. Amersham Pharmacia Biotech offers a wide variety of prepacked columns, ready to use media, and pre-activated media for ligand coupling.
7
This handbook describes the role of affinity chromatography in the purification of biomolecules, the principle of the technique, the media available and how to select them, application examples and detailed instructions for the most commonly performed procedures. Practical information is given as a guide towards obtaining the best results. The illustration on the inside cover shows the range of handbooks that have been produced by Amersham Pharmacia Biotech to ensure that purification with any chromatographic technique becomes a simple and efficient procedure at any scale and in any laboratory.
Symbols and abbreviations this symbol indicates general advice which can improve procedures or provide recommendations for action under specific situations. this symbol denotes advice which should be regarded as mandatory and gives a warning when special care should be taken. this symbol highlights troubleshooting advice to help analyse and resolve difficulties that may occur. chemicals, buffers and equipment. experimental protocol. PBS
8
phosphate buffered saline (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4).
Chapter 1 Affinity chromatography in brief Affinity chromatography separates proteins on the basis of a reversible interaction between a protein (or group of proteins) and a specific ligand coupled to a chromatographic matrix. The technique is ideal for a capture or intermediate step in a purification protocol and can be used whenever a suitable ligand is available for the protein(s) of interest. With high selectivity, hence high resolution, and high capacity for the protein(s) of interest, purification levels in the order of several thousand-fold with high recovery of active material are achievable. Target protein(s) is collected in a purified, concentrated form. Biological interactions between ligand and target molecule can be a result of electrostatic or hydrophobic interactions, van der Waals' forces and/or hydrogen bonding. To elute the target molecule from the affinity medium the interaction can be reversed, either specifically using a competitive ligand, or non-specifically, by changing the pH, ionic strength or polarity. In a single step, affinity purification can offer immense time-saving over less selective multistep procedures. The concentrating effect enables large volumes to be processed. Target molecules can be purified from complex biological mixtures, native forms can be separated from denatured forms of the same substance and small amounts of biological material can be purified from high levels of contaminating substances. For an even higher degree of purity, or when there is no suitable ligand for affinity purification, an efficient multi-step process must be developed using the purification strategy of Capture, Intermediate Purification and Polishing (CIPP). When applying this strategy affinity chromatography offers an ideal capture or intermediate step in any purification protocol and can be used whenever a suitable ligand is available for the protein of interest. Successful affinity purification requires a biospecific ligand that can be covalently attached to a chromatographic matrix. The coupled ligand must retain its specific binding affinity for the target molecules and, after washing away unbound material, the binding between the ligand and target molecule must be reversible to allow the target molecules to be removed in an active form. Any component can be used as a ligand to purify its respective binding partner. Some typical biological interactions, frequently used in affinity chromatography, are listed below: • Enzyme ! substrate analogue, inhibitor, cofactor. • Antibody ! antigen, virus, cell. • Lectin ! polysaccharide, glycoprotein, cell surface receptor, cell. • Nucleic acid ! complementary base sequence, histones, nucleic acid polymerase, nucleic acid binding protein. • Hormone, vitamin ! receptor, carrier protein. • Glutathione ! glutathione-S-transferase or GST fusion proteins. • Metal ions ! Poly (His) fusion proteins, native proteins with histidine, cysteine and/or tryptophan residues on their surfaces.
9
Affinity chromatography is also used to remove specific contaminants, for example Benzamidine Sepharose™ 6 Fast Flow can remove serine proteases, such as thrombin and Factor Xa. Figure 2 shows the key stages in an affinity purification.
1. Affinity medium is equilibrated in binding buffer.
2. Sample is applied under conditions that favour specific binding of the target molecule(s) to a complementary binding substance (the ligand). Target substances bind specifically, but reversibly, to the ligand and unbound material washes through the column.
3. Target protein is recovered by changing conditions to favour elution of the bound molecules. Elution is performed specifically, using a competitive ligand, or non-specifically, by changing the pH, ionic strength or polarity.Target protein is collected in a purified, concentrated form.
4. Affinity medium is re-equilibrated with binding buffer.
Absorbance
equilibration
adsorption of sample and elution of unbound material
begin sample application
1-2 cv
wash away unbound material
elute bound protein(s)
change to elution buffer
x cv
1-2 cv Column Volumes (cv)
Fig. 2. Typical affinity purification.
10
re-equilibration
>1 cv
1-2 cv
The high selectivity of affinity chromatography enables many separations to be achieved in one simple step, including, for example, common operations such as the purification of monoclonal antibodies or fusion proteins. A wide variety of prepacked columns, ready to use media, and pre-activated media for ligand coupling through different functional groups, makes affinity chromatography readily available for a broad range of applications. To save time, the HiTrap™ column range (Table 1) is excellent for routine laboratory scale applications in which the risk of cross-contamination between samples must be eliminated, for purification from crude samples or for fast method development before scaling up purification. HiTrap columns can be operated with a syringe, a peristaltic pump or any ÄKTA™design chromatography system. Several HiTrap columns can be connected in series to increase purification capacity and all columns are supplied with detailed protocols for use.
Table 1. HiTrap and HiPrep™ affinity columns for laboratory scale purification. Application
HiTrap and HiPrep columns
Isolation of human immunoglobulins IgG, fragments and subclasses
HiTrap rProtein A FF, 1 ml and 5 ml
IgG, fragments and subclasses
HiTrap Protein A HP, 1 ml and 5 ml
IgG, fragments and subclasses including human IgG3 strong affinity for monoclonal mouse IgG1 and rat IgG
HiTrap Protein G HP, 1 ml and 5 ml MAbTrap™ Kit
Avian IgY from egg yolk
HiTrap IgY Purification HP, 5 ml
Mouse and human IgM
HiTrap IgM Purification HP, 1 ml
Purification of fusion proteins (His)6 fusion proteins
HisTrap™ Kit HiTrap Chelating HP, 1 ml and 5 ml
GST fusion proteins
GSTrap™ FF, 1 ml and 5 ml
Other Group Specific Media Albumin and nucleotide-requiring enzymes
HiTrap Blue HP, 1 ml and 5 ml
Proteins and peptides with exposed His, Cys or Trp
HiTrap Chelating HP, 1 ml and 5 ml
Biotinylated substances
HiTrap Streptavidin HP, 1 ml
DNA binding proteins and coagulation factors
HiTrap Heparin HP, 1 ml and 5 ml HiPrep 16/10 Heparin FF, 20 ml
Trypsin-like serine proteases including Factor Xa, thrombin and trypsin
HiTrap Benzamidine FF (high sub), 1 ml and 5 ml
Matrix for preparation of affinity media. Coupling via primary amines
HiTrap NHS-activated HP, 1 ml and 5 ml
11
BioProcess Media for large-scale production Specific BioProcess™ Media have been designed for each chromatographic stage in a process from Capture to Polishing. Large capacity production integrated with clear ordering and delivery routines ensure that BioProcess Media are available in the right quantity, at the right place, at the right time. Amersham Pharmacia Biotech can assure future supplies of BioProcess Media, making them a safe investment for long-term production. The media are produced following validated methods and tested under strict control to fulfil high performance specifications. A certificate of analysis is available with each order. Regulatory Support Files contain details of performance, stability, extractable compounds and analytical methods. The essential information in these files gives an invaluable starting point for process validation, as well as providing support for submissions to regulatory authorities. Using BioProcess Media for every stage results in an easily validated process. High flow rate, high capacity and high recovery contribute to the overall economy of an industrial process. All BioProcess Media have chemical stability to allow efficient cleaning and sanitization procedures. Packing methods are established for a wide range of scales and compatible large-scale columns and equipment are available. Please refer to the latest BioProcess Products Catalogue from Amersham Pharmacia Biotech for further details of our products and services for large-scale production.
Custom Designed Media and Columns Prepacked columns, made according to the client's choice from the Amersham Pharmacia Biotech range of columns and media, can be supplied by the Custom Products Group. Custom Designed Media (CDM) can be produced for specific industrial process separations when suitable media are not available from the standard range. The CDM group at Amersham Pharmacia Biotech works in close collaboration with the user to design, manufacture, test and deliver media for specialized separation requirements. When a chromatographic step is developed to be an integral part of a manufacturing process, the choice of column is important to ensure consistent performance and reliable operation. Amersham Pharmacia Biotech provides a wide range of columns that ensures the highest performance from all our purification media and meets the demands of modern pharmaceutical manufacturing. Please ask your local representative for further details of CDM products and services.
12
Common terms in affinity chromatography
Matrix: for ligand attachment. Matrix should be chemically and physically inert.
Spacer arm: used to improve binding between ligand and target molecule by overcoming any effects of steric hindrance.
Ligand: molecule that binds reversibly to a specific target molecule or group of target molecules.
Binding: buffer conditions are optimized to ensure that the target molecules interact effectively with the ligand and are retained by the affinity medium as all other molecules wash through the column. Elution: buffer conditions are changed to reverse (weaken) the interaction between the target molecules and the ligand so that the target molecules can be eluted from the column. Wash: buffer conditions that wash unbound substances from the column without eluting the target molecules or that re-equilibrate the column back to the starting conditions (in most cases the binding buffer is used as a wash buffer). Ligand coupling: covalent attachment of a ligand to a suitable pre-activated matrix to create an affinity medium. Pre-activated matrices: matrices which have been chemically modified to facilitate the coupling of specific types of ligand.
13
14
Chapter 2 Affinity chromatography in practice This chapter provides guidance and advice that is generally applicable to any affinity purification. The first step towards a successful purification is to determine the availability of a suitable ligand that interacts reversibly with the target molecule or group of molecules. Ready to use affinity media, often supplied with complete separation protocols, already exist for many applications. The contents section of this handbook lists the full range of affinity media from Amersham Pharmacia Biotech according to the specific molecule or group of molecules to be purified. Application- and product-specific information and advice for these media are supplied in other sections of this handbook. Practical information specific to the use of pre-activated matrices for the preparation of affinity medium is covered in Chapter 5.
Purification steps 1. Affinity medium is equilibrated in binding buffer.
2. Sample is applied under conditions that favour specific binding of the target molecule(s) to a complementary binding substance (the ligand). Target substances bind specifically, but reversibly, to the ligand and unbound material washes through the column.
3. Target protein is recovered by changing conditions to favour elution of the bound molecules. Elution is performed specifically, using a competitive ligand, or non-specifically, by changing the pH, ionic strength or polarity. Target protein is collected in a purified, concentrated form.
4. Affinity medium is re-equilibrated with binding buffer.
Absorbance
equilibration
adsorption of sample and elution of unbound material
begin sample application
1-2 cv
wash away unbound material
elute bound protein(s)
re-equilibration
change to elution buffer
x cv
1-2 cv
>1 cv
1-2 cv
Column Volumes (cv)
Fig. 3. Typical affinity purification.
15
Figure 4 shows the simple procedure used to perform affinity purification on prepacked HiTrap columns. Equilibrate column with binding buffer
3 min
Apply sample Wash with binding buffer
5-15 min
Waste
Elute with elution buffer
2 min
Collect
Collect fractions
Fig. 4.
HiTrap columns may be used with a syringe, a peristaltic pump or a liquid chromatography system (see Selection of Purification Equipment, Appendix 2) and are supplied with a detailed protocol to ensure optimum results.
Media selection A ligand already coupled to a matrix is the simplest solution. Selecting prepacked columns such as HiTrap or HiPrep will not only be more convenient, but will also save time in method optimization as these columns are supplied with detailed instructions for optimum performance. If a ligand is available, but needs to be coupled to a pre-activated matrix, refer to Chapter 5. If no suitable ligand is available, decide whether it is worth the time and effort involved to obtain a ligand and to develop a specific affinity medium. In many cases, it may be more convenient to use alternative purification techniques such as ion exchange or hydrophobic interaction chromatography.
Preparation of media and buffers Storage solutions and preservatives should be washed away thoroughly before using any affinity medium. Re-swell affinity media supplied as freeze-dried powders in the correct buffer as recommended by the manufacturer. Use high quality water and chemicals. Solutions should be filtered through 0.45 µm or 0.22 µm filters. Reuse of affinity media depends on the nature of the sample and should only be performed with identical samples to prevent cross-contamination. If an affinity medium is to be used routinely, care must be taken to ensure that any contaminants from the crude sample can be removed by procedures that do not damage the ligand. Binding and elution buffers are specific for each affinity medium since it is their influence on the interaction between the target molecule and the ligand that facilitates the affinitybased separation. Some affinity media may also require a specific buffer in order to make the medium ready for use again.
16
Avoid using magnetic stirrers as they may damage the matrix. Use mild rotation or end-over-end stirring.
Sample preparation and application Samples should be clear and free from particulate matter. Simple steps to clarify a sample before beginning purification will avoid clogging the column, may reduce the need for stringent washing procedures and can extend the life of the chromatographic medium. Appendix 1 contains an overview of sample preparation techniques. If possible, test the affinity of the ligand: target molecule interaction. Too low affinity will result in poor yields since the target protein may wash through or leak from the column during sample application. Too high affinity will result in low yields since the target molecule may not dissociate from the ligand during elution. Binding of the target protein may be made more efficient by adjusting the sample to the composition and pH of the binding buffer: perform a buffer exchange using a desalting column or dilute in binding buffer (see page 134). Sample preparation techniques should ensure that components known to interfere with binding (the interaction between the target molecule and the ligand) are removed. Since affinity chromatography is a binding technique, the sample volume does not affect the separation as long as conditions are chosen to ensure that the target protein binds strongly to the ligand. It may be necessary to test for a flow rate that gives the most efficient binding during sample application since this parameter can vary according to the specific interaction between the target protein and the ligand and their concentrations. The column must be pre-equilibrated in binding buffer before beginning sample application. For interactions with strong affinity between the ligand and the target molecule that quickly reach equilibrium, samples can be applied at a high flow rate. However, for interactions with weak affinity and/or slow equilibrium, a lower flow rate should be used. The optimal flow rate to achieve efficient binding may vary according to the specific interaction and should be determined when necessary. Further details on the kinetics involved in binding and elution from affinity media are covered in Appendix 7. When working with very weak affinity interactions that are slow to reach equilibrium, it may be useful to stop the flow after applying the sample to allow more time for the interaction to take place before continuing to wash the column. In some cases, applying the sample in aliquots may be beneficial. Do not begin elution of target substances until all unbound material has been washed through the column by the binding buffer (determined by UV absorbance at 280 nm). This will improve the purity of the eluted target substance.
17
Elution There is no generally applicable elution scheme for all affinity media. Reference to manufacturer's instructions, the scientific literature and a few simple rules should result in an effective elution method that elutes the target protein in a concentrated form. Elution methods may be either selective or non-selective, as shown in Figure 5.
Method 1 The simplest case. A change of buffer composition elutes the bound substance without harming either it or the ligand. Method 2 Extremes of pH or high concentrations of chaotropic agents are required for elution, but these may cause permanent or temporary damage. Methods 3 and 4 Specific elution by addition of a substance that competes for binding. These methods can enhance the specificity of media that use group-specific ligands.
Fig. 5. Elution methods.
When substances are very tightly bound to the affinity medium, it may be useful to stop the flow for some time after applying eluent (10 min. to 2 h is commonly used) before continuing elution. This gives more time for dissociation to take place and thus helps to improve recoveries of bound substances. Selective elution methods are applied in combination with group-specific ligands whereas non-selective elution methods are used in combination with highly specific ligands. Forces that maintain the complex include electrostatic interactions, hydrophobic effects and hydrogen bonding. Agents that weaken these interactions may be expected to function as efficient eluting agents. The optimal flow rate to achieve efficient elution may vary according to the specific interaction and should be determined when necessary. Further details on the kinetics involved in binding and elution of target molecules from affinity media are covered in Appendix 7. A compromise may have to be made between the harshness of the eluent required for elution and the risk of denaturing the eluted material or damaging the ligand on the affinity medium. Ready to use affinity media from Amersham Pharmacia Biotech are supplied with recommendations for the most suitable elution buffer to reverse the interaction between the ligand and target protein of the specific interaction. Each of these recommendations will be based on one of the following elution methods:
18
pH elution A change in pH alters the degree of ionization of charged groups on the ligand and/or the bound protein. This change may affect the binding sites directly, reducing their affinity, or cause indirect changes in affinity by alterations in conformation. A step decrease in pH is the most common way to elute bound substances. The chemical stability of the matrix, ligand and target protein determines the limit of pH that may be used. If low pH must be used, collect fractions into neutralization buffer such as 1 M Tris-HCl, pH 9 (60–200 µl per ml eluted fraction) to return the fraction to a neutral pH. The column should also be re-equilibrated to neutral pH immediately. Ionic strength elution The exact mechanism for elution by changes in ionic strength will depend upon the specific interaction between the ligand and target protein. This is a mild elution using a buffer with increased ionic strength (usually NaCl), applied as a linear gradient or in steps. Enzymes usually elute at a concentration of 1 M NaCl or less. Competitive elution Selective eluents are often used to separate substances on a group specific medium or when the binding affinity of the ligand/target protein interaction is relatively high. The eluting agent competes either for binding to the target protein or for binding to the ligand. Substances may be eluted either by a concentration gradient of a single eluent or by pulse elution, see page 22. When working with competitive elution the concentration of competing compound should be similar to the concentration of the coupled ligand. However, if the free competing compound binds more weakly than the ligand to the target molecule, use a concentration ten-fold higher than that of the ligand. Reduced polarity of eluent Conditions are used to lower the polarity of the eluent promote elution without inactivating the eluted substances. Dioxane (up to 10%) or ethylene glycol (up to 50%) are typical of this type of eluent. Chaotropic eluents If other elution methods fail, deforming buffers, which alter the structure of proteins, can be used, e.g. chaotropic agents such as guanidine hydrochloride or urea. Chaotropes should be avoided whenever possible since they are likely to denature the eluted protein.
19
Gradient and step elution Figure 6 shows examples of step and gradient elution conditions. For prepacked affinity HiTrap columns, supplied with predefined elution conditions, a step elution using a simple syringe can be used. HiTrap columns can also be used with a chromatography system such as ÄKTAprime. The use of a chromatography system is essential when gradient elution is required.
A 280
A 280 Elution conditions
Linear change in elution conditions Binding conditions
Binding conditions
Time/vol.
Time/vol.
Fig. 6a. Step elution.
Fig. 6b. Gradient elution.
During development and optimization of affinity purification, use a gradient elution to scan for the optimal binding or elution conditions, as shown in Figure 7 and Figure 8.
A 280 nm
UV 280 nm Programmed elution buffer conc.
0.3
Imidazole (M) 0.5
Column: 0.4
0.2
Sample:
Binding buffer:
Elution buffer:
(His)6 fusion protein
0.3
0.2
Flow: System:
Clarified homogenate of E. coli expressing His fusion protein HiTrap Chelating HP 1 ml column charged with Ni2+ 20 mM sodium phosphate, 0.5 M sodium chloride, 10 mM imidazole, pH 7.4 20 mM sodium phosphate, 0.5 M sodium chloride, 0.5 M imidazole, pH 7.4 1 ml/min ÄKTAprime
0.1 0.1
1 2
0 0
45
0
65 min
Fig. 7. Gradient elution of a (His)6 fusion protein.
20
1: selected imidazole concentration for elution of impurities 2: selected imidazole concentration for elution of pure (His) 6 fusion protein
pH
A 280 nm
Sample:
0.6
7.0
pH selected for elution in a step gradient
6.0
0.4
Column: Binding buffer:
A 280
0.2
5.0
Elution buffer:
4.0
Flow: System:
pH
0
Cell culture supernatant containing monoclonal IgG1, 90 ml HiTrap rProtein A FF, 1 ml 100 mM sodium phosphate, 100 mM sodium citrate, 2.5 M sodium chloride, pH 7.4 100 mM sodium phosphate, 100 mM sodium citrate, pH-gradient from 7.4 to 3.0 1 ml/min ÄKTAFPLC™
3.0 150
200
250
ml
Fig. 8. Scouting for optimal elution pH of a monoclonal IgG1 from HiTrap rProtein A FF, using a pH gradient.
Flow rates It is not possible to specify a single optimal flow rate in affinity chromatography because dissociation rates of ligand/target molecule interactions vary widely. For ready to use affinity media follow the manufacturer's instructions and optimize further if required: -determine the optimal flow rate to achieve efficient binding -determine the optimal flow rate for elution to maximize recovery -determine the maximum flow rate for column re-equilibration to minimize total run times To obtain sharp elution curves and maximum recovery with minimum dilution of separated molecules, use the lowest acceptable flow rate.
Analysis of results and further steps The analysis of results from the first separation can indicate if the purification needs to be improved to increase the yield, achieve higher purity, speed up the separation or increase the amount of sample that can be processed in a single run. Commonly used assays are outlined in Appendix 8. It is generally recommended to follow any affinity step with a second technique, such as a high resolution gel filtration to remove any aggregates, or ligands that may have leached from the medium. For example, Superdex™ can be used to separate molecules, according to differences in size, and to transfer the sample into storage buffer, removing excess salt and other small molecules. The chromatogram will also give an indication of the homogeneity of the purified sample. Alternatively, a desalting column that gives low resolution, but high sample capacity, can be used to quickly transfer the sample into storage buffer and remove excess salt (see page 134).
Equipment selection Appendix 2 provides a guide to the selection of purification systems.
21
Troubleshooting This section focuses on practical problems that may occur when running a chromatography column. The diagrams below give an indication of how a chromatogram may deviate from the ideal during affinity purification and what measures can be taken to improve the results. Target elutes as a sharp peak. Satisfactory result A 280
Flow through (unbound material)
Eluted target
Elution buffer Binding buffer
ml
• If it is difficult or impossible to retain biological activity when achieving this result, either new elution conditions or a new ligand must be found. • If using low pH for elution, collect the fractions in neutralization buffer (60–200 µl 1 M Tris-HCl, pH 9.0 per ml eluted fraction).
Target is a broad, low peak that elutes while binding buffer is being applied A 280
Flow through (unbound material)
• Find better binding conditions.
Eluted target
Binding buffer
ml
Target elutes in a broad, low peak A 280
Flow through (unbound material) Elution buffer Eluted target
Binding buffer
ml
A 280
Flow through (unbound material) Elution buffer Binding buffer
Wait Eluted target
ml
• Try different elution conditions. • If using competitive elution, increase the concentration of the competitor in the elution buffer. • Stop flow intermittently during elution to allow time for the target molecule to elute and so collect the target protein in pulses (see second figure beneath). Note: This result may also be seen if the target protein has denatured and aggregated on the column or if there is non-specific binding.
Some of the target molecule elutes as a broad, low peak while still under binding conditions A 280
Flow through (unbound material)
Elution buffer Eluted target
Binding buffer
ml Flow through (unbound material)
A 280
Elution buffer Binding buffer
Eluted target
ml
22
• Allow time for the sample to bind and/or apply sample in aliquots, stopping the flow for a few minutes between each sample application (see second figure beneath).
Situation
Cause
Protein does not bind or elute as expected.
Sample has not been filtered properly. Clean the column, filter the sample and repeat.
Low recovery of activity, but normal recovery of protein.
Remedy
Sample has altered during storage.
Prepare fresh samples.
Sample has wrong pH or buffer conditions are incorrect.
Use a desalting column to transfer sample into the correct buffer (see page 134).
Solutions have wrong pH.
Calibrate pH meter, prepare new solutions and try again.
The column is not equilibrated sufficiently in the buffer.
Repeat or prolong the equilibration step.
Proteins or lipids have precipitated on the column.
Clean and regenerate the column or use a new column.
Column is overloaded with sample.
Decrease the sample load.
Microbial growth has occurred in the column.
Microbial growth rarely occurs in columns during use, but, to prevent infection of packed columns, store in 20% ethanol when possible.
Precipitation of protein in the column filter and/ or at the top of the bed.
Clean the column, exchange or clean the filter or use a new column.
Protein may be unstable or inactive in the elution buffer.
Determine the pH and salt stability of the protein. Collect fractions into neutralization buffer such as 1 M Tris-HCl, pH 9 (60–200 µl per fraction).
Lower yield than expected.
More activity is recovered than was applied to the column.
Enzyme separated from co-factor or similar.
Test by pooling aliquots from the fractions and repeating the assay.
Protein may have been degraded by proteases.
Add protease inhibitors to the sample and buffers to prevent proteolytic digestion. Run sample through a medium such as Benzamidine 4 Fast Flow (high sub) to remove serine proteases.
Adsorption to filter during sample preparation.
Use another type of filter.
Sample precipitates.
May be caused by removal of salts or unsuitable buffer conditions.
Hydrophobic proteins. Protein is still attached to ligand.
Use chaotropic agents, polarity reducing agents or detergents.
Different assay conditions have been used before and after the chromatographic step.
Use the same assay conditions for all the assays in the purification scheme.
Removal of inhibitors during separation. Reduced or poor flow through the column.
Presence of lipoproteins or protein aggregates.
Remove lipoproteins and aggregrates during sample preparation (see Appendix 1).
Protein precipitation in the column caused by removal of stabilizing agents during fractionation.
Modify the eluent to maintain stability.
Clogged column filter.
Replace the filter or use a new column. Always filter samples and buffer before use.
Clogged end-piece or adaptor or tubing.
Remove and clean or use a new column.
Precipitated proteins.
Clean the column using recommended methods or use a new column.
Bed compressed.
Repack the column, if possible, or use a new column.
Microbial growth.
Microbial growth rarely occurs in columns during use, but, to prevent infection of packed columns, store in 20% ethanol when possible.
23
Situation
Cause
Remedy
Back pressure increases during a run or during successive runs.
Turbid sample.
Improve sample preparation (see Appendix 1). Improve sample solubility by the addition of ethylene glycol, detergents or organic solvents.
Precipitation of protein in the column filter and/or at the top of the bed.
Clean using recommended methods. Exchange or clean filter or use a new column. Include any additives that were used for initial sample solubilization in the solutions used for chromatography.
Bubbles in the bed.
Column packed or stored at cool temperature and then warmed up.
Buffers not properly de-gassed.
De-gas buffers thoroughly.
Cracks in the bed.
Large air leak in column.
Check all connections for leaks. Repack the column if possible (see Appendix 3).
Distorted bands as sample runs into the bed.
Air bubble at the top of the column or in the inlet adaptor.
Re-install the adaptor taking care to avoid air bubbles.
Particles in buffer or sample.
Filter or centrifuge the sample. Protect buffers from dust.
Clogged or damaged net in upper adaptor.
Dismantle the adaptor, clean or replace the net. Keep particles out of samples and eluents.
Column poorly packed.
Suspension too thick or too thin. Bed packed at a temperature different from run. Bed insufficiently packed (too low packing pressure, too short equilibration). Column packed at too high pressure.
Distorted bands as sample passes down the bed.
24
Remove small bubbles by passing de-gassed buffer upwards through the column. Take special care if buffers are used after storage in a fridge or cold-room. Do not allow column to warm up due to sunshine or heating system. Repack column, if possible, (see Appendix 3).
Chapter 3 Purification of specific groups of molecules A group specific medium has an affinity for a group of related substances rather than for a single type of molecule. The same general ligand can be used to purify several substances (for example members of a class of enzymes) without the need to prepare a new medium for each different substance in the group. Within each group there is either structural or functional similarity. The specificity of the affinity medium derives from the selectivity of the ligand and the use of selective elution conditions.
Immunoglobulins The diversity of antibody-antigen interactions has created many uses for antibodies and antibody fragments. They are used for therapeutic and diagnostic applications as well as for immunochemical techniques within general research. The use of recombinant technology has greatly expanded our ability to manipulate the characteristics of these molecules to our advantage. The potential exists to create an infinite number of combinations between immunoglobulins and immunoglobulin fragments with tags and other selected proteins. A significant advantage for the purification of antibodies and their fragments is that a great deal of information is available about the properties of the target molecule and the major contaminants, no matter whether the molecule is in its a native state or has been genetically engineered and no matter what the source material. The Antibody Purification Handbook from Amersham Pharmacia Biotech presents the most effective and frequently used strategies for sample preparation and purification of the many different forms of antibodies and antibody fragments used in the laboratory. The handbook also includes more detailed information on antibody structure and classification, illustrated briefly here in Figures 9 and 10.
Fig. 9. H2L2 structure of a typical immunoglobulin.
25
Antibody classes Characteristic Heavy chain Light chain
IgG
IgM
IgA
IgE
IgD
g k or l
m k or l
a
e
k or l
k or l
d k or l
Y structure
Fig. 10. Antibody classes.
IgG, IgG fragments and subclasses The basis for purification of IgG, IgG fragments and subclasses is the high affinity of protein A and protein G for the Fc region of polyclonal and monoclonal IgG-type antibodies, see Figure 9. Protein A and protein G are bacterial proteins (from Staphylococcus aureus and Streptococcus, respectively) which, when coupled to Sepharose, create extremely useful, easy to use media for many routine applications. Examples include the purification of monoclonal IgG-type antibodies, purification of polyclonal IgG subclasses, and the adsorption and purification of immune complexes involving IgG. IgG subclasses can be isolated from ascites fluid, cell culture supernatants and serum. Table 2 shows a comparison of the relative binding strengths of protein A and protein G to different immunoglobulins compiled from various publications. A useful reference on this subject is also: Structure of the IgG-binding regions of streptococcal Protein G, EMBO J., 5, 1567–1575 (1986). Binding strengths are tested with free protein A or protein G and can be used as a guide to predict the binding behaviour to a protein A or protein G purification medium. However, when coupled to an affinity matrix, the interaction may be altered. For example, rat IgG1 does not bind to protein A, but does bind to Protein A Sepharose.
26
Table 2. Relative binding strengths of protein A and protein G to various immunoglobulins. No binding: -, relative strength of binding: +, ++, +++, ++++. Species
Subclass
Human
IgA IgD IgE IgG1 IgG2 IgG3 IgG4 A IgM IgY IgYB
Chicken Avian egg yolk Cow Dog Goat Guinea pig Hamster Horse Koala Llama Monkey (rhesus) Mouse
Pig Rabbit Rat
IgG1 IgG2
IgG1 IgG2a IgG2b IgG3 IgMA no distinction IgG1 IgG2a IgG2b IgG3
Sheep
Protein A binding
Protein G binding
variable -
-
++++ ++++ ++++ variable ++ ++ ++++ ++++ + ++ ++++ + ++++ +++ ++ variable +++ ++++ + +/-
++++ ++++ ++++ ++++ ++++ + ++ ++ ++ ++ ++++ + + ++++ ++++ ++++ +++ +++ +++ +++ + ++++ ++ ++ ++
A
Purify using HiTrap IgM Purification HP columns. B Purify using HiTrap IgY Purification HP columns.
Single step purification based on Fc region specificity will co-purify host IgG and may even bind trace amounts of serum proteins. For any preparation that must be free of even trace amounts of contaminating IgG, immunospecific affinity using anti-host IgG antibodies as the ligand to remove host IgG or using target specific antigen to avoid binding host IgG, ion exchange and/or hydrophobic interaction chromatography may be better alternatives (see Chapter 6). Both protein A and a recombinant protein A are available, with similar specificities for the Fc region of IgG. The recombinant protein A has been engineered to include a C-terminal cysteine that enables a single-point coupling to Sepharose. Single point coupling often results in an enhanced binding capacity. Genetically engineered antibodies and antibody fragments can have altered biological properties and also altered properties to facilitate their purification. For example, tags can be introduced into target molecules for which no affinity media were previously available thus creating a fusion protein that can be effectively purified by affinity chromatography. Details for the purification of tagged proteins are covered in the section Recombinant Fusion Proteins on page 42 of this handbook. For information on the purification of 27
recombinant proteins in general, refer to The Recombinant Protein Handbook: Protein Amplification and Simple Purification from Amersham Pharmacia Biotech.
HiTrap Protein G HP, Protein G Sepharose 4 Fast Flow, MAbTrap Kit Protein G, a cell surface protein from Group G streptococci, is a type III Fc-receptor. Protein G binds through a non-immune mechanism. Like protein A, it binds specifically to the Fc region of IgG, but it binds more strongly to several polyclonal IgGs (Table 2) and to human IgG3. Under standard buffer conditions, protein G binds to all human subclasses and all mouse IgG subclasses, including mouse IgG1. Protein G also binds rat IgG2a and IgG2b, which either do not bind or bind weakly to protein A. Amersham Pharmacia Biotech offers a recombinant form of protein G from which the albumin-binding region of the native molecule has been deleted genetically, thereby avoiding undesirable reactions with albumin. Recombinant protein G contains two Fc binding regions. Protein G Sepharose is a better choice for general purpose capture of antibodies since it binds a broader range of IgG from eukaryotic species and binds more classes of IgG. Usually protein G has a greater affinity than protein A for IgG and exhibits minimal binding to albumin, resulting in cleaner preparations and greater yields. The binding strength of protein G for IgG depends on the source species and subclass of the immunoglobulin. The dynamic binding capacity depends on the binding strength and also on several other factors, such as flow rate during sample application. Many antibodies also interact via the Fab region with a low affinity site on protein G. Protein G does not appear to bind human myeloma IgM, IgA or IgE, although some do bind weakly to protein A. Leakage of ligands from an affinity medium is always a possibility, especially if harsh elution conditions are used. The multi-point attachment of protein G to Sepharose results in very low leakage levels over a wide range of elution conditions. Purification options Binding capacity
Maximum operating flow
Comments
HiTrap Protein G HP
Human IgG, > 25 mg/column Human IgG, >125 mg/column
4 ml/min (1 ml column) 20 ml/min (5 ml column)
Purification of IgG, fragments and . subclasses, including human IgG3 Strong affinity for monoclonal mouse IgG1 and rat IgG. Prepacked columns.
MAbTrap Kit
Human IgG, > 25 mg/column
4 ml/min
Purification of IgG, fragments and subclasses, including human IgG3. Strong affinity for monoclonal mouse IgG1 and rat IgG. Complete kit contains HiTrap Protein G HP (1 x 1 ml), accessories, pre-made buffers for 10 purifications and detailed experimental protocols.
Protein G Sepharose 4 Fast Flow
Human IgG, > 20 mg/ml medium Cow IgG, 23 mg/ml medium Goat IgG, 19 mg/ml medium Guinea pig IgG, 17 mg/ml medium Mouse IgG, 10 mg/ml medium Rat IgG, 7 mg/ml medium
400 cm/h*
Supplied as a suspension ready for column packing.
*See Appendix 4 to convert linear flow (cm/h) to volumetric flow rate. Maximum operating flow is calculated from measurement in a packed column with a bed height of 10 cm and i.d. of 5 cm. 28
Purification examples Figure 11 shows the purification of mouse monoclonal IgG1 on HiTrap Protein G HP 1 ml. The monoclonal antibody was purified from a hybridoma cell culture supernatant. Immunodiffusion
Sample: Column: Flow: Binding buffer: Elution buffer: Electrophoresis:
12 ml mouse IgG1 hybridoma cell culture supernatant HiTrap Protein G HP, 1 ml 1.0 ml/min 20 mM sodium phosphate, pH 7.0 0.1 M glycine-HCI, pH 2.7 SDS-PAGE, PhastSystem™, PhastGel™ Gradient 10–15, 1 µl sample, silver stained Immunodiffusion: 1% Agarose A in 0.75 M Tris, 0.25 M 5,5-diethylbarbituric acid, 5 mM Ca-lactate, 0.02% sodium azide, pH 8.6 A 280 nm
Binding Elution Binding buffer buffer buffer
5.0
SDS PAGE Lane 1. Low Molecular Weight Calibration Kit, reduced Lane 2. Mouse hybridoma cell culture fluid, non-reduced, diluted 1:10 Lane 3. Pool I, unbound material, non-reduced, diluted 1:10 Lane 4. Pool II, purified mouse IgG1, non-reduced, diluted 1:10
Mr 97 000 66 000 45 000
2.5
30 000 20 100 14 000 pool I
0 5
10
Lane 1
pool II
15
20
25
30
2
3
4
ml
Fig. 11. Purification of monoclonal mouse IgG1 on HiTrap Protein G HP, 1 ml.
Figure 12 shows the purification of recombinant mouse Fab fragments, expressed in E. coli, using Protein G Sepharose 4 Fast Flow. Chimeric, non-immunogenic "humanized" mouse Fab, Fab' and F(ab')2 fragments are of great interest in tumour therapy since they penetrate tumours more rapidly and are also cleared from the circulation more rapidly than full size antibodies.
UV 280 nm Conductivity pH
A 280 nm 3.5
Sample:
Recombinant Fab fragment from E. coli. Medium: Protein G Sepharose 4 Fast Flow (1 ml) Flow: 0.2 ml/min (60 cm/h), or 0.3 ml/min (90 cm/h) Binding buffer: 0.15 M NaCl, 10 mM sodium phosphate, 10 mM EDTA, pH 7.0 Elution buffer: 0.5 M ammonium acetate, pH 3.0 Wash buffer: 1 M acetic acid, pH 2.5
Elution buffer 2.5
1.5
0.5 0 0.0
10.0
20.0
30.0
40.0
ml
Fig. 12. Purification of recombinant Fab fragments directed to the envelope protein gp120 of HIV-1 (anti-gp120 Fab), expressed in E. coli.
29
Performing a separation Column:
HiTrap Protein G HP, 1 ml or 5 ml
Recommended flow rates: 1 ml/min (1 ml column) or 5 ml/min (5 ml column) Binding buffer:
0.02 M sodium phosphate, pH 7.0
Elution buffer:
0.1 M glycine-HCl, pH 2.7
Neutralization buffer:
1 M Tris-HCl, pH 9.0
Centrifuge samples (10 000 g for 10 minutes) to remove cells and debris. Filter through a 0.45 µm filter. If required, adjust sample conditions to the pH and ionic strength of the binding buffer either by buffer exchange on a desalting column or by dilution and pH adjustment (see page 134). 1. Equilibrate column with 5 column volumes of binding buffer. 2. Apply sample. 3. Wash with 5–10 column volumes of the binding buffer to remove impurities and unbound material. Continue until no protein is detected in the eluent (determined by UV absorbance at 280 nm). 4. Elute with 5 column volumes of elution buffer*. 5. Immediately re-equilibrate with 5–10 column volumes of binding buffer. *Since elution conditions are quite harsh, it is recommended to collect fractions into neutralization buffer (60 µl – 200 µl 1 M Tris-HCl, pH 9.0 per ml fraction), so that the final pH of the fractions will be approximately neutral.
IgGs from most species and subclasses bind to protein G at near physiological pH and ionic strength. For the optimum binding conditions for IgG from a particular species, it is worth consulting the most recent literature. Avoid excessive washing if the interaction between the protein and the ligand is weak, since this may decrease the yield. Most immunoglobulin species do not elute from Protein G Sepharose until pH 2.7 or less. If biological activity of the antibody or antibody fragment is lost due to the low pH required for elution, try Protein A Sepharose: the elution pH may be less harsh. Desalt and/or transfer purified IgG fractions to a suitable buffer using a desalting column (see page 134). Reuse of Protein G Sepharose depends on the nature of the sample and should only be performed with identical samples to prevent cross-contamination. To increase capacity, connect several HiTrap Protein G HP columns (1 ml or 5 ml) in series. HiTrap columns can be used with a syringe, a peristaltic pump or connected to a liquid chromatography system, such as ÄKTAprime. For greater capacity pack a larger column with Protein G Sepharose 4 Fast Flow (see Appendix 3).
30
MAbTrap Kit
Fig. 13. MAbTrap Kit, ready for use.
MAbTrap Kit contains a HiTrap Protein G HP 1 ml column, stock solutions of binding, elution and neutralization buffers, a syringe with fittings and an optimized purification protocol, as shown in Figure 13. The kit contains sufficient material for up to 20 purifications of monoclonal or polyclonal IgG from serum, cell culture supernatant or ascitic fluid, using a syringe. The column can also be connected to a peristaltic pump, if preferred. Figure 14 shows the purification of mouse monoclonal IgG1 from cell culture supernatant with syringe operation and a similar purification with pump operation. Eluted fractions were analysed by SDS-PAGE as shown in Figure 15. Column: Sample:
HiTrap Protein G HP, 1 ml 10 ml mouse monoclonal cell supernatant, IgG1, anti-transferrin. Filtered through 0.45 µm filter Binding buffer: 20 mM sodium phosphate, pH 7.0 Elution buffer: 0.1 M glycine-HCl, pH 2.7
B) Pump operation, flow 2 ml/min A 280 nm
Elution
A) Syringe operation, approx. 60 drops/min 3.0
A 280 nm
3
2.0 2
1.0
1
0 1
4
7
10
13
16
19
22
25
28
31 ml
0
5
10
15
20
25
30
ml
Fig. 14. Purification of mouse monoclonal IgG1 from cell culture supernatant. A. with syringe operation. B. with pump operation.
Lanes 1 and 7. Lane 2. Lane 3. Lane 4. Lane 5. Lane 6.
Mr 97 000 66 45 30 20 14
000 000 000 100 000
Low Molecular Weight Calibration Kit, Amersham Pharmacia Biotech Crude cell culture supernatant, mouse IgG1, diluted 1:11 Flow through, using a peristaltic pump, diluted 1:10 Eluted mouse IgG1, using a peristaltic pump Flow through, using a syringe, diluted 1:10 Eluted mouse IgG1, using a syringe
1 2 3 4 5 6 7
Fig. 15. SDS-PAGE on PhastSystem using PhastGel 10–15, non-reduced, and silver staining.
31
Performing a separation Column:
HiTrap Protein G HP, 1 ml
Recommended flow rate: 1 ml/min Binding buffer:
Dilute buffer concentrate 10-fold
Elution buffer:
Dilute buffer concentrate 10-fold
Neutralization buffer:
Add 60–200 µl of neutralization buffer per ml fraction to the test tubes in which IgG will be collected
Centrifuge samples (10 000 g for 10 minutes) to remove cells and debris. Filter through a 0.45 µm filter. If required, adjust sample conditions to the pH and ionic strength of the binding buffer either by buffer exchange on a desalting column (see page 134) or by dilution and pH adjustment.
A
B
C
Fig. 16. Using HiTrap Protein G HP with a syringe. A: Dilute buffers and prepare sample. Remove the column’s top cap and twist off the end. B: Equilibrate the column, load the sample and begin collecting fractions. C: Wash and elute, continuing to collect fractions. 1. Allow the column and buffers to warm to room temperature. 2. Dilute the binding and elution buffers. 3. Connect the syringe to the column using the luer adapter supplied. 4. Equilibrate the column with 5 ml distilled water, followed by 3 ml diluted binding buffer. 5. Apply the sample. 6. Wash with 5–10 ml diluted binding buffer until no material appears in the eluent. 7. Elute with 3–5 ml diluted elution buffer. Collect fractions into tubes containing neutralization buffer. 8. Immediately re-equilibrate the column with 5 ml diluted binding buffer.
32
Media characteristics Ligand density
Composition
pH stability*
Mean particle size
HiTrap Protein G HP (MAbTrap Kit)
2 mg/ml
Ligand coupled to Sepharose HP by N-hydroxysuccinimide activation (gives stable attachment through alkylamine and ether links).
Long term 3–9 Short term 2–9
34 µm
Protein G Sepharose 4 Fast Flow
2 mg/ml
Ligand coupled to Sepharose 4 Fast Flow by cyanogen bromide activation.
Long term 3–9 Short term 2–9
90 µm
*Long term refers to the pH interval over which the medium is stable over a long period of time without adverse effects on its subsequent chromatographic performance. Short term refers to the pH interval for regeneration, cleaning-in-place and sanitization procedures.
Chemical stability Stable in all common aqueous buffers. Storage Wash media and columns with 20% ethanol (use approximately 5 column volumes for packed media) and store at +4 to +8 °C.
HiTrap Protein A HP, Protein A Sepharose 4 Fast Flow, HiTrap rProtein A FF, rProtein A Sepharose 4 Fast Flow Protein A is derived from a strain of Staphylococcus aureus and contains five regions that bind to the Fc region of IgG. As an affinity ligand, protein A is coupled to Sepharose so that these regions are free to bind IgG. One molecule of protein A can bind at least two molecules of IgG. Both protein A and a recombinant protein A are available from Amersham Pharmacia Biotech. These molecules share similar specificities for the Fc region of IgG, but the recombinant protein A has been engineered to include a C-terminal cysteine that enables a single-point coupling to Sepharose. Single point coupling often results in an enhanced binding capacity. The binding strength of protein A for IgG depends on the source species of the immunoglobulin as well as the subclass of IgG (see Table 2). The dynamic binding capacity depends on the binding strength and also on several other factors, such as flow rate during sample application. Although IgG is the major reactive human immunoglobulin, some other types have also been demonstrated to bind to protein A. Interaction takes place with human colostral IgA as well as human myeloma IgA2 but not IgA1. Some human monoclonal IgMs and some IgMs from normal and macroglobulinaemic sera can bind to protein A. Leakage of ligands from an affinity medium is always a possibility, especially if harsh elution conditions are used. The multi-point attachment of protein A to Sepharose results in very low leakage levels over a wide range of elution conditions. 33
Purification options Binding capacity
Maximum operating flow
Comments
HiTrap Protein A HP
Human IgG, > 20 mg/column Human IgG, > 100 mg/column
4 ml/min (1 ml column) 20 ml/min (5 ml column)
Purification of IgG, fragments and sub-classes. Prepacked columns.
Protein A Sepharose 4 Fast Flow*
Human IgG, > 35 mg/ml medium Mouse IgG, 3–10 mg/ml medium
400 cm/h**
Supplied as a suspension ready for column packing.
HiTrap rProtein A FF
Human IgG, > 50 mg/column Human IgG, > 250 mg/column
4 ml/min (1 ml column) 20 ml/min (5 ml column)
Purification of IgG, fragments and sub-classes. Enhanced binding capacity. Prepacked columns.
rProtein A Sepharose 4 Fast Flow*
Human IgG, > 50 mg/ml medium Mouse IgG, 8–20 mg/ml medium
300 cm/h**
Enhanced binding capacity. Supplied as a suspension ready for column packing.
*Protein A Sepharose 4 Fast Flow and rProtein A Sepharose Fast Flow have a higher binding capacity, a more rigid matrix and provide more convenient alternatives to Protein A Sepharose CL-4B, which must be rehydrated before column packing. **See Appendix 4 to convert linear flow (cm/h) to volumetric flow rate. Maximum operating flow is calculated from measurement in a packed column with a bed height of 10 cm and i.d. of 5 cm.
Purification examples Figure 17 shows the purification of mouse IgG2b from ascites fluid on HiTrap rProtein A FF 1 ml column using a syringe. The eluted pool contained 1 mg IgG2b and the silver stained SDS-PAGE gel confirmed a purity level of over 95%. Sample: Column: Binding buffer: Elution buffer: Flow:
1 ml of mouse ascites containing IgG2b, filtered through a 0.45 µm filter. The sample was a kind gift from Dr. N. Linde, EC Diagnostics, Sweden HiTrap rProtein A FF 1 ml 0.02 M sodium phosphate, pH 7.0 0.1 M sodium citrate, pH 3.0 Mr ~ 1 ml/min
IgG2b
97 000
A 280 nm 2.5
66 000 45 000
Elution buffer
2.0
30 000
1.5
20 100 14 000 1
1.0 0.5 0.0 0
2
4
Flow-through pool
6
8
10
12
14
Eluted IgG 2b pool
16 Volume (ml) Time (min)
2
3
4
SDS-PAGE on PhastSystem using PhastGel Gradient 10–15, silver staining Lane 1. Low Molecular Weight Calibration Kit, Amersham Pharmacia Biotech Lane 2. Starting material, diluted 10-fold Lane 3. Flow-through pool Lane 4. Eluted IgG2b pool
Fig. 17. Purification of mouse IgG2b from ascites on HiTrap rProtein A FF 1 ml column using a syringe.
Figure 18 shows a larger scale purification of monoclonal mouse IgG2a from a clarified hybridoma cell culture on rProtein A Sepharose Fast Flow. Sample loading was over 9 mg IgG/ml of medium, with a 95% recovery of highly purified antibody.
34
Column: A 280 nm 2.0
Sample: Binding buffer: Elution buffer: Flow:
rProtein A Sepharose Fast Flow, XK 16/20, bed height 4.8 cm (9.6 ml) 600 ml clarified cell culture containing 87.6 mg IgG2a 20 mM sodium phosphate, pH 7.0 20 mM sodium citrate, pH 4.0 5 ml/min (150 cm/h) Mr
1.5
97 000 66 000 45 000 1.0
30 000 20 100 14 400 0.5
1
0.0 0
200
400
600
2
3
4
Volume (ml)
Fig. 18a. Purification of a monoclonal IgG2a from clarified cell culture on rProtein A Sepharose 4 Fast Flow.
Fig. 18b. SDS-PAGE of starting material (lane 2) and eluate (lane 3). The samples were concentrated 10 times and reduced. Lane 1 and 4 are LMW markers. PhastSystem, PhastGel Gradient 10–15.
Performing a separation Column:
HiTrap Protein A HP, 1 ml or 5 ml, or HiTrap rProtein A FF, 1 ml or 5 ml
Recommended flow rates: 1 ml/min (1 ml columns) or 5 ml/min (5 ml columns) Binding buffer:
0.02 M sodium phosphate, pH 7.0
Elution buffer:
0.1 M citric acid, pH 3–6
Neutralization buffer:
1 M Tris-HCl, pH 9.0
Centrifuge samples (10 000 g for 10 minutes) to remove cells and debris. Filter through a 0.45 µm filter. If needed, adjust sample conditions to the pH and ionic strength of the binding buffer either by buffer exchange on a desalting column (see page 134) or by dilution and pH adjustment. A HiTrap column can be used with a syringe, a peristaltic pump or connected to a liquid chromatography system, such as ÄKTAprime. 1. Equilibrate the column with 5 column volumes of binding buffer. 2. Apply sample. 3. Wash with 5–10 column volumes of the binding buffer to remove impurities and unbound material. Continue until no protein is detected in the eluent (determined by UV absorbance at 280 nm). 4. Elute with 5 column volumes of elution buffer.* 5. Immediately re-equilibrate with 5–10 column volumes of binding buffer. *Since elution conditions are quite harsh, collect fractions into neutralization buffer (60 µl – 200 µl 1 M Tris-HCl, pH 9.0 per ml fraction), so that the final pH of the fractions will be approximately neutral.
35
Table 3 gives examples of some typical binding and elution conditions that have been used with Protein A Sepharose. Table 3. Species
Binding to free protein A
Protein A Sepharose binding pH
IgG1
++
6.0–7.0
3.5–4.5
IgG2
++
6.0–7.0
3.5–4.5
IgG3
–
8.0–9.0
< 7.0
IgG4
++
7.0–8.0
use step elution
Subclass
Protein A Sepharose elution pH Usually elutes by pH 3
Human
Cow
IgG2
++
2
Goat
IgG2
+
5.8
Guinea pig
IgG1
++
4.8
IgG2
++
IgG1
+
8.0–9.0
IgG2a
+
7.0–8.0
4.5–5.5
IgG2b
+
7
3.5–4.5
Mouse
Rat
4.3 5.5–7.5
IgG3
+
7
4.0–7.0
IgG1
+
> 9.0
7.0–8.0 < 8.0
IgG2a
–
> 9.0
IgG2b
–
> 9.0
< 8.0
IgG3
+
8.0–9.0
3–4 (using thiocyanate)
Binding strengths are tested with free protein A. They can be used as a guide to predict the binding behaviour to a protein A affinity medium. However, when coupled to an affinity matrix the interaction may be altered. For example, rat IgG1 does not bind to protein A, but does bind to Protein A Sepharose. IgGs from most species and subclasses bind protein A near to physiological pH and ionic strength. Avoid excessive washing if the interaction between the protein of interest and the ligand is weak, since this may decrease the yield. With some antibodies, such as mouse IgG1, it might be necessary to add sodium chloride up to 3 M in the binding buffer to achieve efficient binding when using protein A, for example 1.5 M glycine, 3 M NaCl, pH 8.9. Alternative elution buffers include: 1 M acetic acid, pH 3.0 or 0.1 M glycine-HCl, pH 3.0 or 3 M potassium isothiocyanate. Potassium isothiocyanate can severely affect structure and immunological activity. Use a mild elution method when labile antibodies are isolated. Reverse the flow of the wash buffer and elute with 0.1 M glycyltyrosine in 2 M NaCl, pH 7.0 at room temperature, applied in pulses. (Note: glycyltyrosine absorbs strongly at wavelengths used for detecting proteins). The specific elution is so mild that the purified IgG is unlikely to be denatured. To increase capacity, connect several HiTrap Protein A HP or HiTrap rProtein A FF columns (1 ml or 5 ml) in series or pack a larger column with Protein A Sepharose 4 Fast Flow or rProtein A Sepharose 4 Fast Flow (see Appendix 3).
36
Desalt and/or transfer purified IgG fractions into a suitable buffer using a desalting column (see page 134). Reuse of Protein A Sepharose and rProtein A Sepharose media depends on the nature of the sample and should only be performed with identical samples to prevent cross-contamination. Media characteristics Product
Ligand density
Composition
pH stability*
Mean particle size
HiTrap Protein A HP
3 mg/ml
Ligand coupled to Sepharose HP by N-hydroxysuccinimide activation (stable attachment through alkylamine and ether links).
Short term 2–10 Long term 3–9
34 µm
Protein A Sepharose 4 Fast Flow**
6 mg/m
Ligand coupled to Sepharose 4 Fast Flow by cyanogen bromide activation.
Short term 2–10 Long term 3–9
90 µm
HiTrap rProtein A FF
6 mg/ml
Ligand coupled to Sepharose 4 Fast Flow by epoxy activation, thioether coupling.
Short term 2–11 Long term 3–10
90 µm
rProtein A Sepharose 4 Fast Flow**
6 mg/ml
Ligand coupled to Sepharose 4 Fast Flow by epoxy activation, thioether coupling.
Short term 2–11 Long term 3–10
90 µm
*Long term refers to the pH interval over which the medium is stable over a long period of time without adverse effects on its subsequent chromatographic performance. Short term refers to the pH interval for regeneration, cleaning-in-place and sanitization procedures. **Protein A Sepharose 4 Fast Flow and rProtein A Sepharose 4 Fast Flow have a higher binding capacity, a more rigid matrix and provide more convenient alternatives to Protein A Sepharose CL-4B which must be rehydrated before column packing.
Chemical stability These media and columns tolerate high concentrations of urea, guanidine HCl and chaotropic agents. Storage Wash media and columns with 20% ethanol (use approximately 5 column volumes for packed media) and store at +4 to +8 °C.
37
Monoclonal IgM from hybridoma cell culture HiTrap IgM Purification HP The technique described here is optimized for purification of monoclonal IgM from hybridoma cell culture, but it can be used as a starting point to determine the binding and elution conditions required for other IgM preparations. Purification option
HiTrap IgM Purification HP
Binding capacity
Maximum operating flow
Comments
Human IgM, 5 mg/column
4 ml/min
Purification of monoclonal and human IgM. Prepacked 1 ml column.
HiTrap IgM Purification HP columns are packed with a thiophilic adsorption medium (2-mercaptopyridine coupled to Sepharose High Performance). The interaction between the protein and the ligand has been suggested to result from the combined electron donatingand accepting-action of the ligand in a mixed mode hydrophilic-hydrophobic interaction. Purification example Figure 19 shows results from the purification of monoclonal a-Shigella IgM from hybridoma cell culture supernatant. SDS-PAGE analysis demonstrates a purity level of over 80%. Results from an ELISA (not shown) indicated a high activity of the antibody in the purified fraction. A 280 nm
mS/cm
2.5
Sample:
75 ml of cell culture supernatent containing a-Shigella IgM, filtered through a 0.45 µm filter Column: HiTrap IgM Purification HP, 1 ml Binding buffer: 20 mM sodium phosphate buffer, 0.5 M potassium sulphate, pH 7.5 Elution buffer: 20 mM sodium phosphate buffer, pH 7.5 Cleaning buffer: 20 mM sodium phosphate buffer, pH 7.5, 30% isopropanol Flow: 1 ml/min
100 Flow through material
80
1.5 60
IgM
Elution buffer
Cleaning buffer
40
0.5
20
0
0 0
80
100
ml
Fig. 19a. Purification of a-Shigella IgM on HiTrap IgM Purification HP. Samples reduced with 2-mercaptoethanol
Lane 1. Low Molecular Weight Calibration Kit Lane 2. Cell culture supernatant, starting material, diluted 20-fold Lane 3. IgM, human Lane 4. IgG Lane 5. Flow-through pool, diluted 20-fold Lane 6. Eluted IgM, fraction 8, diluted 8-fold Lane 7. Eluted IgM, fraction 9, diluted 8-fold Lane 8. Washing out unbound material, pool diluted 3-fold
Mr
Mr 97 66 45 30 20 14
Non-reduced samples
97 66 45 30 20 14
000 000 000 000 100 400 1 2 3 4 5 6 7 8
000 000 000 000 100 400 1 2 3 4 5 6 7 8
Fig. 19b. SDS-PAGE on PhastSystem, using PhastGel 4–15 with silver staining.
38
Performing a separation Column:
HiTrap IgM Purification HP
Recommended flow rate: 1 ml/min Binding buffer:
20 mM sodium phosphate, 0.8 M (NH 4)2SO4, pH 7.5
Elution buffer:
20 mM sodium phosphate, pH 7.5
Wash buffer:
20 mM sodium phosphate, pH 7.5 with 30% isopropanol
The sample must have the same concentration of ammonium sulphate as the binding buffer. Slowly add small amounts of solid ammonium sulphate to the sample of hybridoma cell culture supernatant until the final concentration is 0.8 M. Stir slowly and continuously. Pass the sample through a 0.45 µm filter immediately before applying it to the column. To avoid precipitation of IgM, it is important to add the ammonium sulphate slowly. Purification 1. Wash column sequentially with at least 5 column volumes of binding, elution and wash buffer. 2. Equilibrate column with 5 column volumes of binding buffer. 3. Apply the sample. 4. Wash out unbound sample with 15 column volumes of binding buffer or until no material appears in the eluent (monitored at A280). 5. Elute the IgM with 12 column volumes of elution buffer. 6. Wash the column with 7 column volumes of wash buffer. 7. Immediately re-equilibrate the column with 5 column volumes of binding buffer.
Some monoclonal IgMs might not bind to the column at 0.8 M ammonium sulphate. Binding can be improved by increasing the ammonium sulphate concentration to 1.0 M. An increased concentration of ammonium sulphate will cause more IgG to bind, which might be a problem if serum has been added to the cell culture medium. If there is IgG contamination of the purified IgM, the IgG can be removed by using HiTrap Protein G HP, HiTrap Protein A HP or HiTrap rProtein A FF. Potassium sulphate (0.5 M) can be used instead of ammonium sulphate. Most monoclonal IgMs bind to the column in the presence of 0.5 M potassium sulphate and the purity of IgM is comparable to the purity achieved with 0.8 M ammonium sulphate. Some monoclonal IgMs may bind too tightly to the column for complete elution in binding buffer. The remaining IgM will be eluted with wash buffer, but the high content of isopropanol will cause precipitation of IgM. Perform an immediate buffer exchange (see page 134) or dilute the sample to preserve the IgM. Lower concentrations of isopropanol may elute the IgM and decrease the risk of precipitation. To increase capacity, connect several HiTrap IgM Purification HP columns in series. HiTrap columns can be used with a syringe, a peristaltic pump or connected to a liquid chromatography system, such as ÄKTAprime. Reuse of HiTrap lgM Purification HP depends on the nature of the sample and should only be performed with identical samples to prevent cross-contamination. 39
Media characteristics HiTrap IgM Purification HP
Ligand and density
pH stability*
2-mercaptopyridine 2 mg/ml
Long term 3–11 Short term 2–13
Mean particle size 34 µm
*Long term refers to the pH interval over which the medium is stable over a long period of time without adverse effects on its subsequent chromatographic performance. Short term refers to the pH interval for regeneration, cleaning-in-place and sanitization procedures.
Storage Wash the column with 5 column volumes 20% ethanol and store at +4 to +8 °C.
Avian IgY from egg yolk HiTrap IgY Purification HP Purification option
HiTrap IgY Purification HP
Binding capacity
Maximum operating flow
Comments
100 mg pure IgY/column (1/4 egg yolk)
20 ml/min
Purification of IgY from egg yolk. Prepacked 5 ml column.
HiTrap IgY Purification HP columns are packed with a thiophilic adsorption medium (2-mercaptopyridine coupled to Sepharose High Performance). The interaction between the protein and the ligand has been suggested to result from the combined electron donatingand accepting-action of the ligand in a mixed mode hydrophobic-hydrophilic interaction. Purification example Figure 20 shows the purification of a-Hb IgY from 45 ml of egg yolk extract (corresponding to one quarter of a yolk) and Figure 21 shows the SDS-PAGE analysis indicating a purity level of over 70%. A 280 nm
IgY
mS/cm
2.0
80
Sample:
Column: Binding buffer: Elution buffer: Cleaning buffer: Flow:
45 ml of egg yolk extract (corresponding to 1/4 of an egg yolk) containing a-Hb IgY, filtered through a 0.45 µm filter HiTrap IgY Purification HP, 5 ml 20 mM sodium phosphate buffer, 0.5 M potassium sulphate, pH 7.5 20 mM sodium phosphate buffer, pH 7.5 20 mM sodium phosphate buffer, pH 7.5, 30% isopropanol 5 ml/min
60
1.0
40 Elution buffer
Cleaning buffer
20
0
0
0
Fig. 20. Purification of avian IgY on HiTrap IgY Purification HP.
40
50
100
150
ml
Lane Lane Lane Lane Lane Lane Lane
Mr 97 66 45 30 20 14
000 000 000 000 100 000 1
2
3
4
5
6
1. 2. 3. 4. 5. 6. 7.
Low Molecular Weight Calibration Kit Egg yolk extract Flow-through pool Eluted IgY Egg yolk extract, diluted 4-fold Flow-through pool, diluted 4-fold Eluted IgY, diluted 4-fold
7
Fig. 21. SDS-PAGE of non-reduced samples on PhastSystem, using PhastGel 4–15%, Coomassie™ staining.
Performing a separation Column:
HiTrap IgY Purification HP
Recommended flow rate: 5 ml/min Binding buffer:
20 mM sodium phosphate, 0.5 M K2SO4, pH 7.5
Elution buffer:
20 mM sodium phosphate, pH 7.5
Wash buffer:
20 mM sodium phosphate, pH 7.5 with 30% isopropanol
As much as possible of the egg yolk lipid must be removed before purification. Water or polyethylene glycol can be used to precipitate the lipids. Precipitation with water is described below. Precipitation of the egg yolk lipid using water 1. Separate the egg yolk from the egg white. 2. Add nine parts of distilled water to one part egg yolk. 3. Mix and stir slowly for 6 hours at +4 °C. 4. Centrifuge at 10 000 g, at +4 °C for 25 minutes to precipitate the lipids. 5. Collect the supernatant containing the IgY. 6. Slowly add K2SO4 to the sample, stirring constantly, to reach a concentration of 0.5 M. 7. Adjust pH to 7.5. 8. Pass the sample through a 0.45 µm filter immediately before applying it to the column.
Purification 1. Wash the column with at least 5 column volumes of binding, elution and wash buffer. 2. Equilibrate with 5 column volumes of binding buffer. 3. Apply the sample. 4. Wash with at least 10 column volumes of binding buffer or until no material appears in the eluent, as monitored at A280. 5. Elute the IgY with 10 column volumes of elution buffer. 6. Wash the column with 8 column volumes of wash buffer. 7. Immediately re-equilibrate the column with 5 column volumes of binding buffer.
41
To improve recovery of total IgY or a specific IgY antibody, replace 0.5 M K2SO4 with 0.6–0.8 M Na2SO4. The sample should have the same concentration of Na2SO4 as the binding buffer. An increase in salt concentration will reduce the purity of the eluted IgY. The purity of the eluted IgY may be improved by using gradient elution with, for example, a linear gradient 0–100% elution buffer over 10 column volumes, followed by elution with 100% elution buffer for a few column volumes. To increase capacity, connect several HiTrap IgY Purification HP columns in series. A HiTrap column can be used with a syringe, a peristaltic pump or connected to a liquid chromatography system such as ÄKTAprime. Reuse of HiTrap IgY Purification HP depends on the nature of the sample. To prevent cross-contamination, columns should only be reused with identical samples. Media characteristics HiTrap IgY Purification HP
Ligand and density
pH stability*
2-mercaptopyridine 3 mg/ml
Long term 3–11 Short term 2–13
Mean particle size 34 µm
*Long term refers to the pH interval over which the medium is stable over a long period of time without adverse effects on its subsequent chromatographic performance. Short term refers to the pH interval for regeneration, cleaning-in-place and sanitization procedures.
Storage Wash the column with 5 column volumes 20% ethanol and store at +4 to +8 °C.
Recombinant fusion proteins The purification of recombinant proteins can often be simplified by incorporating a tag of known size into the protein. As well as providing a marker for expression and facilitating detection of the recombinant protein, an important role for the tag is to enable a simple purification by affinity chromatography. The two most commonly used tags are glutathioneS-transferase (GST) and 6 x histidine residues (His)6. Protein A fusion proteins have also been produced to take advantage of the affinity between IgG and protein A for affinity purification.
GST fusion proteins GST MicroSpin Purification Module, GSTrap FF, Glutathione Sepharose 4 Fast Flow, Glutathione Sepharose 4B Glutathione S-transferase (GST) is one of the most common tags used to facilitate the purification and detection of recombinant proteins and a range of products for simple, one step purification of GST fusion proteins are available (see Purification options). Purification and detection of GST-tagged proteins, together with information on how to handle fusion proteins when they are expressed as inclusion bodies, are dealt with in depth
42
in The Recombinant Protein Handbook: Protein Amplication and Simple Purification, available from Amersham Pharmacia Biotech. Purification options Binding capacity
Maximum operating flow
Comments
GST MicroSpin™ Purification Module
400 µg/column
n.a.
Ready to use, prepacked columns, buffers and chemicals. High throughput when used with MicroPlex™ 24 Vacuum (up to 48 samples simultaneously).
GSTrap FF 1 ml
10–12 mg recombinant GST/column
4 ml/min
Prepacked column, ready to use.
GSTrap FF 5 ml
50–60 mg recombinant GST/column
15 ml/min
Prepacked column, ready to use.
Glutathione Sepharose 4 Fast Flow
10–12 mg recombinant GST/ml medium
450 cm/h*
For packing high performance columns for use with purification systems and scaling up.
Glutathione Sepharose 4B
8 mg horse liver GST/ml medium
75 cm/h*
For packing small columns and other formats.
*See Appendix 4 to convert linear flow (cm/h) to volumetric flow rate. Maximum operating flow is calculated from measurement in a packed column with a bed height of 10 cm and i.d. of 5 cm.
Purification example Figure 22 shows a typical purification of GST fusion protein on GSTrap FF 1 ml with an SDS-PAGE analysis of the purified protein. Column: Sample: Binding buffer: Elution buffer:
GSTrap FF, 1 ml 8 ml cytoplasmic extract from E. coli expressing a GST fusion protein PBS, pH 7.3 50 mM Tris-HCl, pH 8.0 with 10 mM reduced glutathione 1 ml/min
Flow: Chromatographic procedure: 4 ml binding buffer, 8 ml sample, 10 ml binding buffer, 5 ml elution buffer, 5 ml binding buffer System: ÄKTAexplorer
Mr 97 000 66 000 45 000 30 000
A280 nm
% Elution buffer
Elution buffer
3.5 3.0
20 100 14 400
100
2.5
2.7 mg pure GST fusion protein
Wash
2.0
80
40
1.0
20
0.5
0
0 10.0 10.0
15.0 15.0
20.0 20.0
2
3
60
1.5
5.0 5.0
1
ml min
SDS-PAGE on ExcelGel™ SDS Gradient 8–18% using Multiphor™ II followed by silver staining. Lane 1. Low Molecular Weight (LMW) Calibration kit, reduced, Amersham Pharmacia Biotech Lane 2. Cytoplasmic extract of E. coli expressing GST fusion protein, 1 g cell paste/10 ml Lane 3. GST fusion protein eluted from GSTrap FF 1 ml
Fig. 22. Purification of GST fusion protein.
43
Performing a separation Binding buffer: 140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.3 Elution buffer: 50 mM Tris-HCl, 10 mM reduced glutathione, pH 8.0
A
B
C
Fig. 23. Using GSTrap FF with a syringe. A: Prepare buffers and sample. Remove the column’s top cap and twist off the end. B: Equilibrate column, load the sample and begin collecting fractions. C: Wash and elute, continuing to collect fractions. 1. Equilibrate the column with 5 column volumes of binding buffer. 2. Apply the sample. 3. Wash with 5–10 column volumes of binding buffer. 4. Elute with 5–10 column volumes of elution buffer. 5. Wash with 5–10 column volumes of binding buffer.
To improve yield try decreasing the flow rate or passing the sample through the column several times. For a single purification of a small quantity of product or for high throughput screening, GST MicroSpin columns are convenient and simple to use with either centrifugation or MicroPlex 24 Vacuum. To increase capacity, connect several GSTrap FF columns (1 ml or 5 ml) in series or, for even larger capacity, pack Glutathione Sepharose 4 Fast Flow into a suitable column (see Appendix 3). GSTrap FF columns can be used with a syringe, a peristaltic pump or a chromatography system. Enzyme-specific recognition sites are often included to allow the removal of the GST tag by enzymatic cleavage when required. Thrombin is commonly used for enzymatic cleavage, and must, subsequently, be removed from the recombinant product. HiTrap Benzamidine FF (high sub) 1 ml or 5 ml columns provide simple, ready-made solutions for this process (see page 53). Reuse of GSTrap FF depends on the nature of the sample. To prevent cross-contamination, columns should only be reused with identical samples.
44
Cleaning These procedures are applicable to Glutathione Sepharose 4 Fast Flow and Glutathione Sepharose 4B. 1. Wash with 2–3 column volumes of alternating high pH (0.1 M Tris-HCl, 0.5 M NaCl, pH 8.5) and low pH (0.1 M sodium acetate, 0.5 M NaCl, pH 4.5) buffers. 2. Repeat the cycle 3 times. 3. Re-equilibrate immediately with 3–5 column volumes of binding buffer. If the medium is losing binding capacity, this may be due to an accumulation of precipitated, denatured or non-specifically bound proteins. To remove precipitated or denatured substances: 1. Wash with 2 column volumes of 6 M guanidine hydrochloride. 2. Wash immediately with 5 column volumes of binding buffer. To remove hydrophobically bound substances: 1. Wash with 3–4 column volumes of 70% ethanol (or 2 column volumes of a non-ionic detergent (Triton™ X-100 1%)). 2. Wash immediately with 5 column volumes of binding buffer. Media characteristics Spacer arm
Ligand and density
pH stability*
Mean particle size
Glutathione Sepharose 4 Fast Flow (GSTrap)
10 carbon linker
Glutathione 120–320 µmoles/ml
Short term 6–9 Long term 6–9
90 µm
Glutathione Sepharose 4B
10 carbon linker
Glutathione 7–15 µmoles/ml
Short term 4–13 Long term 4–13
90 µm
*Long term refers to the pH interval over which the medium is stable over a long period of time without adverse effects on its subsequent chromatographic performance. Short term refers to the pH interval for regeneration, cleaning-in-place and sanitization procedures.
Chemical stability No significant loss of binding capacity when exposed to 0.1 M citrate (pH 4.0), 0.1 M NaOH, 70% ethanol or 6 M guanidine hydrochloride for 2 hours at room temperature. No significant loss of binding capacity after exposure to 1% SDS for 14 days. Storage Wash media and columns with 20% ethanol at neutral pH (use approximately 5 column volumes for packed media) and store at +4 to +8 °C.
45
Poly (His) fusion proteins His MicroSpin Purification Module, HisTrap Kit, HiTrap Chelating HP, Chelating Sepharose Fast Flow The (His)6 tag is one of the most common tags used to facilitate the purification and detection of recombinant proteins and a range of products for simple, one step purification of (His)6 fusion proteins are available (see Purification options). Polyhistidine tags such as (His)4 or (His)10 are also used. They may provide useful alternatives to (His)6 for improving purification results. For example, since (His)10 binds more strongly to the affinity medium, a higher concentration of eluent (imidazole) can be used during the washing step before elution. This can facilitate the removal of contaminants which may otherwise be co-purified with a (His)6 fusion protein. Chelating Sepharose, when charged with Ni2+ ions, selectively binds proteins if complexforming amino acid residues, in particular histidine, are exposed on the protein surface. (His)6 fusion proteins can be easily bound and then eluted with buffers containing imidazole. Purification and detection of His-tagged proteins, together with information on how to handle fusion proteins when they are expressed as inclusion bodies, are dealt with in depth in The Recombinant Protein Handbook: Protein Amplication and Simple Purification, available from Amersham Pharmacia Biotech. Purification options Binding capacity
Maximum operating flow
Comments
His MicroSpin Purification Module
100 µg/column
n.a.
Ready to use, prepacked columns, buffers and chemicals. High throughput when used with MicroPlex 24 Vacuum (up to 48 samples simultaneously).
HisTrap Kit
12 mg*/column
4 ml/min
As above, but includes buffers for up to 12 purifications using a syringe.
HiTrap Chelating HP 1 ml
12 mg*/column
4 ml/min
Prepacked column, ready to use.
HiTrap Chelating HP 5 ml
60 mg*/column
20 ml/min
Prepacked column, ready to use.
Chelating Sepharose Fast Flow
12 mg*/ml medium
400 cm/h**
Supplied as suspension for packing columns and scale up.
*Estimate for a (His)6 fusion protein of Mr 27 600, binding capacity varies according to specific protein. **See Appendix 4 to convert linear flow (cm/h) to volumetric flow rate. Maximum operating flow is calculated from measurement in a packed column with a bed height of 10 cm and i.d. of 5 cm.
46
Purification examples Figures 24 and 25 show the purification of recombinant proteins expressed in soluble form or as inclusion bodies and Figure 26 gives an example of simultaneous on-column purification and refolding of a recombinant protein expressed as an inclusion body. Soluble recombinant proteins Sample:
9 ml E. coli periplasm containing Protein A-(HisGly)4His. Diluted with 9 ml binding buffer. Column: HiTrap Chelating HP, 5 ml Metal ion: Zn2+ Flow: 1.0 ml/min Binding buffer: 50 mM sodium phosphate, 0.1 M NaCl, pH 8.0 Elution buffer: 50 mM sodium phosphate, 0.1 M NaCl, pH 4.0 Gradient: 20 ml elution buffer, step gradient Electrophoresis: SDS-PAGE, PhastSystem, PhastGel Gradient 8–25, 1 µl sample, Coomassie stained
A 280 nm 0.10
0.05
Mr 97 000 66 000 45 000
0 45
30 000 20 100 14 400
pool I
65
ml
Lane 1. Low Molecular Weight Calibration Kit (LMW), reduced Lane 2. Crude periplasmic fraction, reduced Lane 3. Pool I, purified Protein A-(HisGly)4His, reduced 1
2
3
Fig. 24. Purification of recombinant proteins on HiTrap Chelating HP, 5 ml, charged with Zn2+.
Recombinant protein expressed in inclusion bodies Sample:
8 ml cell extract containing (His)10-tagged protein. The clone was a kind gift from Dr. C. Fuller and S. Brasher, Department of Biochemistry, University of Cambridge, UK. Column: HiTrap Chelating HP, 1 ml Metal ion: Ni2+ Binding buffers: 20 mM sodium phosphate, 0.5 M NaCl, 100 mM imidazole, 8 M urea or 6 M guanidine hydrochloride, pH 7.4 Elution buffers: 20 mM sodium phosphate, 0.5 M NaCl, 500 mM imidazole, 8 M urea or 6 M guanidine hydrochloride, pH 7.4 Flow: Approx. 4 ml/min Equipment: Syringe Electrophoresis: SDS-PAGE, PhastSystem, PhastGel 10–15, 1 µl sample, silver staining
Purification in 8 M urea Lane 1. Low Molecular Weight Calibration Kit (LMW) Lane 2. Starting material, cell extract, diluted 20-fold Lane 3. Flow-through, diluted 10-fold Lane 4. Wash Lane 5. Elution (first two ml) Lane 6. Elution (last two ml) Lane 7. LMW
Mr 97 66 45 30 20 14
000 000 000 000 100 400 1 2 3 4 5 6 7
Purification in 6 M guanidine hydrochloride Lane 1. Low Molecular Weight Calibration Kit (LMW) Lane 2. Starting material, cell extract, diluted 10-fold Lane 3. Flow-through Lane 4. Wash Lane 5. Elution (first two ml) Lane 6. Elution (last two ml) Lane 7. LMW
Mr 97 66 45 30 20 14
000 000 000 000 100 400 1 2 3 4 5 6 7
Fig. 25. Purification of (His)10-tagged protein from inclusion bodies on HiTrap Chelating HP, 1 ml, charged with Ni2+.
47
One step, on-column, refolding and purification of recombinant proteins from inclusion bodies Ni2+-loaded HiTrap Chelating HP, 1 ml N-terminal (His)6 recombinant protein produced in E. coli Flow: 0.1–1 ml/min, sample loading and refolding 1 ml/min, wash and elution Binding buffer: 20 mM Tris-HCl, 0.5 M NaCl, 5 mM imidazole, 6 M guanidine hydrochloride, 1 mM 2-mercaptoethanol, pH 8.0 Washing buffer: 20 mM Tris-HCl, 0.5 M NaCl, 20 mM imidazole, 6 M urea, 1 mM 2-mercaptoethanol, pH 8.0 Refolding buffer: 20 mM Tris-HCl, 0.5 M NaCl, 20 mM imidazole, 1 mM 2-mercaptoethanol, pH 8.0 Refolding gradient: 30 ml Elution buffer: 20 mM Tris-HCl, 0.5 M NaCl, 500 mM imidazole, 1 mM 2-mercaptoethanol, pH 8.0 Elution gradient: 10 ml Fraction volumes: 3 ml sample loading, wash and refolding, 1 ml elution
A 280
Column: Sample:
Mr 97 000 66 000 45 000 30 000 20 100 14 400 1 2 3 4 5 6 7 8
1.0 Start refolding
0.75 fr. fr. fr. 38 40 42
fr. 49
Start elution
0.5
0.25
fr. 46
Manually using a syringe: • Sample loading • Gua-HCl wash • Urea wash
0 10 20
30 40
50
60
65
ml
Lane 1. Low Molecular Weight Calibration Kit (LMW) Lane 2. Starting material for Lane 1. LMW HiTrap Chelating HP, 1 ml Lane 2. Fraction 38 Mr Lane 3. Fraction 1 Gua-HCl Lane 3. Fraction 39 wash (manually) Lane 4. Fraction 40 97 000 Lane 4. Fraction 2 Gua-HCl 66 000 Lane 5. Fraction 41 wash (manually) 45 000 Lane 6. Fraction 42 Lane 5. Fraction 3 Gua-HCl 30 000 Lane 7. Fraction 46 wash (manually) 20 100 Lane 8. Fraction 49 Lane 6. Fraction 4 Gua-HCl 14 400 wash (manually) Lane 7. Fraction 1 Urea 1 2 3 4 5 6 7 8 wash (manually) Lane 8. Fraction 2 Urea Electrophoresis: SDS-PAGE. PhastSystem, PhastGel 10–15, wash (manually) reducing conditions, 1 µl sample, Coomassie Blue staining.
Fig. 26. One step refolding and purification of a (His)6-tagged recombinant protein on HiTrap Chelating HP, 1 ml, charged with Ni2+. The sample is bound to the column and all unbound material is washed through. Refolding of the bound protein is performed by running a linear 6–0 M urea gradient, starting with the wash buffer and finishing with the refolding buffer. A gradient volume of 30 ml or higher and a flow rate of 0.1–1 ml/min can be used. The optimal refolding rate should be determined experimentally for each protein. The refolded recombinant protein is eluted using a 10–20 ml linear gradient starting with refolding buffer and ending with the elution buffer.
Performing a separation Figure 27 shows the simplicity of a poly (His) fusion protein purification when using a prepacked HiTrap Chelating HP column. The protocol described has been optimized for a high yield purification of (His)6 fusion proteins and can be used as a base from which to scale up. An alternative optimization protocol designed to achieve high purity is supplied with the HisTrap Kit and is also described in The Recombinant Protein Handbook: Protein Amplification and Simple Purification from Amersham Pharmacia Biotech. Prepare column Wash with H2O Load with NiSO4 Wash with H2O
3 min
Equilibrate column with binding buffer
3 min
Waste
Apply sample Wash with binding buffer
5-15 min
Waste
Elute with elution buffer
2 min
Collect
Collect fractions
Fig. 27. HiTrap Chelating HP and a schematic overview of poly (His) fusion protein purification.
48
Nickel solution: 0.1 M NiSO4 Binding buffer: 20 mM sodium phosphate, 0.5 M NaCl, 10 mM imidazole, pH 7.4 Elution buffer: 20 mM sodium phosphate, 0.5 M NaCl, 500 mM imidazole, pH 7.4 1. Wash the column with 5 column volumes of distilled water.
Use water, not buffer, to wash away the column storage solution which contains 20% ethanol. This avoids the risk of nickel salt precipitation in the next step. If air is trapped in the column, wash the column with distilled water until the air disappears. 2. Load 0.5 column volumes of the 0.1 M nickel solution onto the column. 3. Wash with 5 column volumes of distilled water. 4. Equilibrate the column with 10 column volumes of binding buffer. 5. Apply sample at a flow rate 1–4 ml/min (1 ml column) or 5 ml/min (5 ml column). Collect the flow-through fraction. A pump is more suitable for application of sample volumes greater than 15 ml. 6. Wash with 10 column volumes of binding buffer. Collect wash fraction. 7. Elute with 5 column volumes of elution buffer. Collect eluted fractions in small fractions such as 1 ml to avoid dilution of the eluate. 8. Wash with 10 column volumes of binding buffer. The column is now ready for a new purification and there is rarely a need to reload with metal if the same (His)6 fusion protein is to be purified.
Imidazole absorbs at 280 nm. Use elution buffer as blank when monitoring absorbance. If imidazole needs to be removed, use a desalting column (see page 134). For a single purification of a small quantity of product or for high throughput screening His MicroSpin columns are convenient and simple to use with either centrifugation or MicroPlex 24 Vacuum. To increase capacity use several HiTrap Chelating HP columns (1 ml or 5 ml) in series. HiTrap Chelating HP columns (1 ml or 5 ml) can be used with a syringe, a peristaltic pump or a chromatography system. For even larger capacity, pack Chelating Sepharose Fast Flow into a suitable column (see Appendix 3). The loss of metal ions is more pronounced at lower pH. The column does not have to be stripped (i.e. all metal ions removed) between each purification if the same protein is going to be purified. In this case, strip and re-charge (i.e. replace metal ions) the column after 5–10 purifications. Reuse of purification columns depends on the nature of the sample and should only be performed with identical samples to prevent cross contamination.
49
Purification using HisTrap Kit HisTrap Kit includes everything needed for 12 purifications using a syringe. Three ready to use HiTrap Chelating HP 1 ml columns and ready-made buffer concentrates are supplied with easy-to-follow instructions.
Cleaning Removal of nickel ions before re-charging or storage: 1. Wash with 5 column volumes of 20 mM sodium phosphate, 0.5 M NaCl, 0.05 M EDTA, pH 7.4. 2. Wash with 10 column volumes of distilled water. 3. For storage, wash with 5 column volumes of 20% ethanol. Removal of precipitated proteins: 1. Fill column with 1 M NaOH and incubate for 2 hours. 2. Wash out dissolved proteins with 5 column volumes of water and a buffer at pH 7.0 until the pH of the flow-through reaches pH 7.0. Media characteristics Composition
Metal ion capacity
Chelating Sepharose High Performance (HiTrap Chelating HP)
Iminodiacetic acid coupled to Sepharose High Performance via an ether bond.
23 µmoles Cu /ml
Chelating Sepharose Fast Flow
Iminodiacetic acid coupled Sepharose Fast Flow via a spacer arm using epoxy coupling.
22–30 µmoles Zn /ml
2+
2+
pH stability*
Mean particle size
Short term 2–14 Long term 3–13
34 µm
Short term 2–14 Long term 3–13
90 µm
*Long term refers to the pH interval over which the medium is stable over a long period of time without adverse effects on its subsequent chromatographic performance. Short term refers to the pH interval for regeneration, cleaning-in-place and sanitization procedures.
Chemical stability Stable in all commonly used aqueous buffers and denaturants such as 6 M guanidine hydrochloride and 8 M urea. Storage Wash media and columns with 20% ethanol at neutral pH (use approximately 5 column volumes for packed media) and store at +4 to +8 °C. The column must be recharged with metal ions after long term storage to reactivate the medium.
50
Protein A fusion proteins IgG Sepharose 6 Fast Flow Recombinant fusion proteins containing a protein A tail and protein A can be purified on IgG Sepharose 6 Fast Flow. Purification option Product
Binding capacity/ml medium
IgG Sepharose Fast Flow
2 mg protein A at pH 7.5
Maximum operating flow 400 cm/h*
*See Appendix 4 to convert linear flow (cm/h) to volumetric flow rate. Maximum operating flow is calculated from measurement in a packed column with a bed height of 10 cm and i.d. of 5 cm.
Purification example Figure 28 shows automatic on-line monitoring of the production of a secreted fusion protein during fermentation. The fusion protein, ZZ-IGF-1 is insulin-like growth factor 1 fused with a derivative of protein A (designated ZZ), expressed in E. coli. A) Sample: Column: Binding buffer: Wash buffer: Elution buffer: A
Bacterial suspension containing ZZ-IGF-1 fusion protein, automatically sampled from fermentation broth, 500 µl IgG Sepharose 6 Fast Flow (0.5 x 2.5 cm) 0.05 M Tris-HCl, 0.05% Tween™ 20, pH 7.6 10 mM ammonium acetate, pH 4.6 0.2 M acetic acid, pH 3.2 B
A 280 nm
Binding Wash buffer buffer
Elution buffer
A 600 nm 120
Conc. (mg/ml) 600
Conc. A600
100
500
80
400
60
300
40
200
20
100
0.1
ZZ-IGF-1 0.05
0
0 10.0 Time (min)
10
20
30
40
0 50 Time (h)
Fig. 28. A) Chromatogram of a sample taken at one time point during fermentation. B) Results from automatic monitoring of the product concentration during fermentation. Concentration of ZZ-IGF-1 is determined by integration of the ZZ-IGF-1 peak obtained during each chromatographic analysis. Bacterial density is measured manually at A600 nm.
Performing a separation Binding buffer:
0.05 M Tris-HCl, 0.15 M NaCl, 0.05% Tween 20, pH 7.6
Wash buffer:
5 mM ammonium acetate, pH 5.0
Elution buffer:
0.5 M acetic acid, adjusted to pH 3.4 with ammonium acetate
Neutralization buffer: 1 M Tris-HCl, pH 9.0
51
1. Pack the column (see Appendix 3) and wash with at least 5 column volumes of binding buffer. 2. Equilibrate the column with approximately 5 column volumes of binding buffer. 3. Wash with 2–3 column volumes of acetic acid followed by 5 column volumes of binding buffer. 4. Apply the sample. 5. Wash with 10 column volumes binding buffer. 6. Wash with 2 column volumes of wash buffer or until no material appears in the eluent (determined by UV absorbance at A 280 nm). 7. Elute with 2–5 column volumes of elution buffer.* 8. Immediately re-equilibrate the column with binding buffer until the eluent reaches pH 7.0 (the IgG may denature if left at a lower pH). *Since elution conditions are quite harsh, it is recommended to collect fractions into neutralization buffer (60 µl – 200 µl 1 M Tris-HCl, pH 9.0 per ml fraction), so that the final pH of the fractions will be approximately neutral.
This method, while giving a concentrated eluate, can only be used if the fusion product is stable under the acid conditions. An alternative eluent is 0.1 M glycine-HCl, pH 3.0. Chaotropic agents may also be used for elution. Media characteristics IgG Sepharose 6 Fast Flow
Ligand
Composition
pH stability*
Particle size
Human polyclonal IgG
IgG coupled to Sepharose Fast Flow by the cyanogen bromide method.
Short term 3–10 Long term 3–10
90 µm
*Long term refers to the pH interval over which the medium is stable over a long period of time without adverse effects on its subsequent chromatographic performance. Short term refers to the pH interval for regeneration, cleaning-in-place and sanitization procedures.
Chemical stability Avoid reducing agents such as 2-mercaptoethanol or DTT since they may disrupt disulphide bonds within the IgG ligand. Storage Wash with 5 column volumes of 20% ethanol at neutral pH and store at +4 to +8 °C.
52
Purification or removal of serine proteases, e.g. thrombin and trypsin, and zymogens HiTrap Benzamidine FF (high sub), Benzamidine Sepharose 4 Fast Flow (high sub) Sample extraction procedures often release proteases into solution, requiring the addition of protease inhibitors to prevent unwanted proteolysis. An alternative to the addition of inhibitors is to use a group specific affinity medium to remove the proteases from the sample. The same procedure can be used to either specifically remove these proteases or purify them. The synthetic inhibitor para-aminobenzamidine is used as the affinity ligand for trypsin, trypsin-like serine proteases and zymogens. Benzamidine Sepharose 4 Fast Flow (high sub) is frequently used to remove molecules from cell culture supernatant, bacterial lysate or serum. During the production of recombinant proteins, tags such as GST are often used to facilitate purification and detection. Enzyme specific recognition sites are included in the recombinant protein to allow the removal of the tag by enzymatic cleavage when required. Thrombin is commonly used for enzymatic cleavage, and must often be removed from the recombinant product. HiTrap Benzamidine FF (high sub) provides a simple, ready to use solution for this process. Figure 29 shows the partial structure of Benzamidine Sepharose 4 Fast Flow (high sub) and Table 4 gives examples of different serine proteases.
S e p h a r o s e
OH
OH O
O
O
H N
NH N
NH 2
H
Fig. 29. Partial structure of Benzamidine Sepharose 4 Fast Flow (high sub). Table 4. Examples of different serine proteases. Source
Mr
pI
Thrombin
Bovine pancreas
23 345
Trypsin
Human plasma chain A Human plasma chain B
5 700 31 000
7.1
Urokinase
Human urine
54 000
8.9
Enterokinase
Porcine intestine heavy chain Porcine intestine light chain
134 000 62 000
4.2 6.4–8.5
Plasminogen
Human plasma
90 000
Prekallikrein
Human plasma
nd
Kallikrein
Human plasma Human saliva
86 000 nd
10.5
nd nd (plasma) 4.0 (saliva)
53
Purification options Binding capacity
Maximum operating flow
Comments
HiTrap Benzamidine FF (high sub)
Trypsin, > 35 mg/column Trypsin, > 175 mg/column
4 ml/min (1 ml column) 15 ml/min (5 ml column)
Prepacked columns**.
Benzamidine Sepharose 4 Fast Flow (high sub)
Trypsin, > 35 mg/ml medium
300 cm/h*
Supplied as a suspension ready for column packing**.
*See Appendix 4 to convert linear flow (cm/h) to volumetric flow rate. Maximum operating flow is calculated from measurement in a packed column with a bed height of 10 cm and i.d. of 5 cm. **Supplied in 0.05 M acetate, pH 4 containing 20% ethanol.
Purification examples Figure 30 shows an example of the removal of trypsin-like proteases from human plasma to prevent proteolysis of the plasma components, using a low pH elution. The activity test demonstrated that almost all trypsin-like protease activity is removed from the sample and bound to the column.
A 280
IU/litre A 405
Sample:
1 ml human plasma filtered through a 0.45 µm filter Column: HiTrap Benzamidine FF (high sub), 1 ml Binding buffer: 20 mM Tris-HCl, 0.5 M NaCl, pH 7.4 Elution buffer: 50 mM glycine, pH 3.0 0–100% elution buffer in one step Flow: 1.0 ml/min System: ÄKTAexplorer Protease activity: S-2288 from Chromogenix, Heamochrom Diagnostica AB A405 measurement. The activity is presented as the proteolytic activity/mg protein
3.0 1.00
A 280 2.5
0.80 2.0 0.60 1.5 0.40 1.0 0.20
0.5
0 0.0
5.0
10.0
15.0
ml
Fig. 30. Removal of trypin-like serine proteases from human plasma using HiTrap Benzamidine FF (high sub), 1 ml.
Figure 31 shows the effectiveness of using a GSTrap FF column with a HiTrap Benzamidine FF (high sub) for purification of a GST fusion protein, followed by cleavage of the GST tag via the thrombin cleavage site and subsequent removal of the thrombin enzyme. The GST fusion protein binds to the GSTrap FF column as other proteins wash through the column. Thrombin is applied to the column and incubated for 2 hours. A HiTrap Benzamidine FF (high sub) column, pre-equilibrated in binding buffer, is attached after the GSTrap FF column and both columns are washed in binding buffer followed by a high salt buffer. The cleaved protein and thrombin wash through from the GSTrap FF column, thrombin binds to the HiTrap Benzamidine FF (high sub) column, and the eluted fractions contain pure cleaved protein.
54
Sample:
2 ml clarified E. coli homogenate expressing a Mr 37 000 SH2-GST fusion protein with a thrombin cleavage site Columns: GSTrap FF, 1 ml and HiTrap Benzamidine FF (high sub), 1 ml Binding buffer: 20 mM sodium phosphate, 0.15 M NaCl, pH 7.5 High salt wash buffer: 20 mM sodium phosphate, 1.0 M NaCl, pH 7.5 Benzamidine elution buffer: 20 mM p-aminobenzamidine in binding buffer GST elution buffer: 20 mM reduced glutathione, 50 mM Tris, pH 8.0 Flow: 0.5 ml/min System: ÄKTAprime Protease treatment: 20 units/ml thrombin (Amersham Pharmacia Biotech) for 2 hours at room temperature Thrombin activity: S-2238 (Chromogenix, Haemochrom Diagnostica AB) was used as a substrate and its absorbance at 405 nm was measured High salt buffer wash
Elution of HiTrap Benzamidine FF (high sub)
Thrombin
Elution of GSTrap FF
A 280 nm
Thrombin activity A 405 nm
0.80 0.30
GST-tag
Thrombin 0.60
0.20 0.40
fr.21 fr.22
fr.14
fr.2
0.10 fr.6 fr.7 fr.8
Cleaved SH2 protein
0.20
0
0 0
10 A)
B)
15
20
A)
25
B)
50
ml
A)
B)
A) GSTrap FF, 1 ml B) HiTrap Benzamidine FF (high sub), 1 ml
Mr 97 000 66 000 45 000 30 000 20 100 14 400 1
2
3
4
5
6
7
8
9
Gel: Lane Lane Lane Lane
1. 2. 3. 4.
Lane Lane Lane Lane
5. 6. 7. 8.
Lane 9.
ExcelGel SDS Gradient 8–18%, Coomassie Blue staining Low Molecular Weight Calibration Kit (LMW) Clarified E. coli homogenate expressing SH2-GST fusion protein Flow-through from GSTrap FF (Fraction 2) SH2 GST-tag cleaved, washed off with binding buffer through both columns (Fraction 6) as above (Fraction 7) as above (Fraction 8) Elution of thrombin, HiTrap Benzamidine FF (high sub) Elution of GST-tag and some non-cleaved SH2-GST, GSTrap FF (Fraction 21) as above (Fraction 22)
Fig. 31. On-column cleavage of a GST fusion protein and removal of thrombin after on-column cleavage, using GSTrap FF and HiTrap Benzamidine FF (high sub).
Performing a separation Binding buffer: 0.05 M Tris-HCl, 0.5 M NaCl, pH 7.4 Elution buffer alternatives: - pH elution: 0.05 M glycine-HCl, pH 3.0 or 10 mM HCl, 0.05 M NaCl, pH 2.0 - competitive elution: 20 mM p-aminobenzamidine in binding buffer - denaturing eluents: 8 M urea or 6 M guanidine hydrochloride
55
1. Equilibrate the column with 5 column volumes of binding buffer. 2. Apply the sample. 3. Wash with 5–10 column volumes of binding buffer or until no material appears in the eluent (monitored by UV absorption at A280 nm). 4. Elute with 5–10 column volumes of elution buffer. Collect fractions in neutralization buffer if low pH elution is used*. The purified fractions can be buffer exchanged using desalting columns (see page 134). *Since elution conditions are quite harsh, collect fractions into neutralization buffer (60 µl – 200 µl 1 M Tris-HCl, pH 9.0 per ml fraction), so that the final pH of the fractions will be approximately neutral.
Since Benzamidine Sepharose 4 Fast Flow (high sub) has some ionic binding characteristics, the use of 0.5 M NaCl and pH elution between 7.4–8.0 is recommended. If lower salt concentrations are used, include a high salt wash step after sample application and before elution. The elution buffer used for competitive elution has a high absorbance at 280 nm. The eluted protein must be detected by other methods, such as an activity assay, total protein or SDS-PAGE analysis. The advantage with competitive elution is that the pH is kept constant throughout the purification. Cleaning Wash with 3–5 column volumes of 0.1 M Tris-HCl, 0.5 M NaCl, pH 8.5 followed with 3–5 column volumes of 0.1 M sodium acetate, 0.5 M NaCl, pH 4.5 and re-equilibrate immediately with 3–5 column volumes of binding buffer. Remove severe contamination by washing with non-ionic detergent such as 0.1% Triton X-100 at +37 °C for 1 minute. Media characteristics
Benzamidine Sepharose 4 Fast Flow (high sub)
Ligand density
Composition
pH stability*
Mean particle size
> 12 µmoles p-aminobenzamidine/ml
Amide coupling of ligand via a 14 atom spacer to highly cross-linked 4% agarose
Short term 1–9 Long term 2–8
90 µm
*Long term refers to the pH interval over which the medium is stable over a long period of time without adverse effects on its subsequent chromatographic performance. Short term refers to the pH interval for regeneration, cleaning-in-place and sanitization procedures.
Chemical stability All commonly used aqueous buffers. Storage Wash media and columns with 20% ethanol in 0.05 M sodium acetate, pH 4.0 (use approximately 5 column volumes for packed media) and store at +4 to +8 °C.
56
Serine proteases and zymogens with an affinity for arginine Arginine Sepharose 4B Arginine Sepharose 4B is an L-arginine derivative of Sepharose 4B that can be used for any biomolecule with a biospecific or charge dependent affinity for arginine, such as serine proteases and zymogens. Specific examples include prekallikrein, clostripain, prothrombin, plasminogen and plasminogen activator. The L-arginine is coupled via its a-amino group, leaving the guanidino and a-carboxyl groups free to interact with samples. Electrostatic and stereospecific effects may contribute to the binding and elution process depending upon the specific sample involved. Figure 32 shows the partial structure of Arginine Sepharose 4B. S e p h a r o s e
O CH2 CH CH2 O (CH2)4 O CH2 OH CHOH CH2 NH _
NH CH COO CH2 CH2 CH2 NH C NH2
Fig. 32. Partial structure of Arginine Sepharose 4B.
Purification option
Arginine Sepharose 4B
Binding capacity/ml medium
Maximum operating flow
Comments
No data available
75 cm/h*
Supplied as a suspension ready for column packing.
*See Appendix 4 to convert linear flow (cm/h) to volumetric flow rate. Maximum operating flow is calculated from measurement in a packed column with a bed height of 10 cm and i.d. of 5 cm.
Performing a separation Determine the capacity of the medium for the sample of interest over a range of different pH and flow rates. The sample must be at the same pH as the binding buffer for each experiment. 1. Pack the column (see Appendix 3) and wash with 5 column volumes of binding buffer. 2. Equilibrate the column with 10 column volumes of binding buffer. 3. Apply the sample. 4. Wash with at least 10 column volumes of binding buffer or until no material appears in the eluent (monitored by UV absorption at A280 nm). 5. Elute with 10–20 column volumes of elution buffer.
Biomolecules bound non-specifically can be eluted by: • step or gradient elution with increasing ionic strength (up to 1 M NaCl) • increasing concentration of urea or guanidine hydrochloride (up to 0.7 M)
57
Specifically bound biomolecules can be eluted by competitive elution with a buffer containing arginine or another competing agent for the target molecule. Cleaning Wash with 2–3 column volumes of alternate high pH (0.1 M Tris-HCl, 0.5 M NaCl, pH 8.5) and low pH (0.1 M sodium acetate, 0.5 M NaCl, pH 5.0). Repeat 3 times. Re-equilibrate immediately with 5 column volumes of binding buffer. Remove strongly bound proteins with 2–3 column volumns of 0.5 M NaOH or include 8 M urea or 6 M guanidine hydrochloride in the normal wash buffer to minimize adsorption. Remove severe contamination by washing with non-ionic detergent, e.g. Triton X-100 (0.1%) at +37 °C for 1 min. Re-equilibrate immediately with binding buffer. Media characteristics
Arginine Sepharose 4B
Ligand density
Composition
pH stability*
Mean particle size
14–20 µmoles/ml
Arginine is coupled by an epoxy coupling method through a long hydrophilic spacer and stable ether and alkylamine bonds.
Short term 2–13 Long term 2–13
90 µm
*Long term refers to the pH interval over which the medium is stable over a long period of time without adverse effects on its subsequent chromatographic performance. Short term refers to the pH interval for regeneration, cleaning-in-place and sanitization procedures.
Chemical stability Stable to all commonly used aqueous buffers. Storage Wash media and columns with 20% ethanol at neutral pH (use approximately 5 column volumes for packed media) and store at +4 to +8 °C.
58
DNA binding proteins HiTrap Heparin HP, HiPrep 16/10 Heparin FF, Heparin Sepharose 6 Fast Flow DNA binding proteins form an extremely diverse class of proteins sharing a single characteristic, their ability to bind to DNA. Functionally the group can be divided into those responsible for the replication and orientation of the DNA such as histones, nucleosomes and replicases and those involved in transcription such as RNA/DNA polymerases, transcriptional activators and repressors and restriction enzymes. They can be produced as fusion proteins to enable more specific purification (see page 42), but their ability to bind DNA also enables group specific affinity purification using heparin as a ligand. Heparin is a highly sulphated glycosaminoglycan with the ability to bind a very wide range of biomolecules including: • DNA binding proteins such as initiation factors, elongation factors, restriction endonucleases, DNA ligase, DNA and RNA polymerases. • Serine protease inhibitors such as antithrombin III, protease nexins. • Enzymes such as mast cell proteases, lipoprotein lipase, coagulation enzymes, superoxide dismutase. • Growth factors such as fibroblast growth factor, Schwann cell growth factor, endothelial cell growth factor. • Extracellular matrix proteins such as fibronectin, vitronectin, laminin, thrombospondin, collagens. • Hormone receptors such as oestrogen and androgen receptors. • Lipoproteins. The structure of heparin is shown in Figure 33. Heparin has two modes of interaction with proteins and, in both cases, the interaction can be weakened by increases in ionic strength. 1. In its interaction with DNA binding proteins heparin mimics the polyanionic structure of the nucleic acid. 2. In its interaction with coagulation factors such as antithrombin III, heparin acts as an affinity ligand. (A)
(B)
COO – O
O
OH
H2COR1
OH O
O
COO OH
OH
O HNR2
–
O OR1
Fig. 33. Structure of a heparin polysaccharide consisting of alternating hexuronic acid (A) and D-glucosamine residues (B). The hexuronic acid can either be D-glucuronic acid (top) or its C-5 epimer, L-iduronic acid (bottom). R1 = -H or -SO3–, R2 = -SO3– or -COCH3.
59
Purification options Binding capacity
Maximum operating flow
Comments
HiTrap Heparin HP
Bovine antithrombin III, 3 mg/column Bovine antithrombin III, 15 mg/column
4 ml/min (1 ml column) 20 ml/min (5 ml column)
Prepacked columns.
HiPrep 16/10 Heparin FF
Bovine antithrombin III, 40 mg/column
10 ml/min
Prepacked 20 ml column.
Heparin Sepharose 6 Fast Flow
Bovine antithrombin III, 2 mg/ml medium
400 cm/h*
Supplied as a suspension ready for column packing.
*See Appendix 4 to convert linear flow (cm/h) to volumetric flow rate. Maximum operating flow is calculated from measurement in a packed column with a bed height of 10 cm and i.d. of 5 cm.
Purification examples Figures 34, 35 and 36 show examples of conditions used for the purification of different DNA binding proteins.
Sample:
49 ml E. coli lysate (= 1 g cells) after passage through a 5 ml DEAE Sepharose Fast Flow column Column: HiTrap Heparin HP, 1 ml Flow: 1.0 ml/min Binding buffer: 20 mM Tris-HCl, 1 mM EDTA, 1 mM 2-mercaptoethanol, 2% glycerol, pH 8.0 Elution buffer: Binding buffer + 1.0 M NaCl Elution conditions: 25 ml elution buffer, linear gradient 0–100%
Mr 97 66 45 30 20 14
000 000 000 000 100 400
A 280 nm
1
2
3
4
5
0.4
% Elution buffer
0.3
100 0.2
50 0.1
SDS-PAGE, PhastSystem, PhastGel Gradient 8–25, 1 ml sample, silver stained. Lane 1. Weight (LMW) calibration kit, reduced Lane 2. Reverse transciptase, reduced Lane 3. Pool I from HiTrap Heparin HP, 1 ml reduced Lane 4. Unbound material from DEAE Sepharose FF, reduced Lane 5. Cell lysate, reduced
pool I 0 6
50
60
70
80 ml
Fig. 34. Partial purification of recombinant HIV-reverse transcriptase on HiTrap Heparin HP.
60
Sample:
Column: Binding buffer:
Elution buffer:
Flow:
30 ml extract containing Oct-1, filtered (0.45 µm) and transferred to binding buffer using a PD-10 desalting column HiTrap Heparin HP, 5 ml 20 mM Tris-HCl, 5% (v/v) glycerol, 0.1 mM EDTA, 10 mM 2-mercaptoethanol, 0.1 mM Pefabloc™, 0.5 M NaCl, pH 8 20 mM Tris-HCl, 5% (v/v) glycerol, 0.1 mM EDTA, 10 mM 2-mercaptoethanol, 0.1 mM Pefabloc, 2 M NaCl, pH 8 1 ml/min (30 cm/h)
A 280nm Flow through
Eluate
1.0
0.5
Pool 0.0 0.0
20.0
40.0
60.0
80.0 Volume (ml)
Mr 97 000
Mr
66 000 45 000
97 000 66 000 45 000
30 000 20 100
30 000 20 100 14 400
14 400
1
2
3
4
SDS-PAGE, PhastSystem, PhastGel 10–15, 1 µl sample, Coomassie Blue staining Lane 1. Starting material, E. coli extract, dil. 4-fold Lane 2. Flow-through Lane 3. Low Molecular Weight Calibration Kit (LMW) Lane 4. Eluate pool
4
3
2
1
Western blot of the electrophoresis gel using rabbit anti-Oct human-1 and alkaline phosphatase Lane 1. Starting material Lane 2. Flow-through Lane 3. Low Molecular Weight Calibration Kit (LMW) Lane 4. Eluate pool
Fig. 35. Partial purification of the recombinant DNA binding Oct-1 protein (courtesy of Dr Gunnar Westin, University Hospital, Uppsala, Sweden) using HiTrap Heparin HP, 5 ml.
61
Sample:
Column: Binding buffer: Elution buffer:
2000 ml partially purified sample from DEAE Sepharose CL-4B flow-through, pH 7.0 HiPrep 16/10 Heparin FF 50 mM sodium phosphate, pH 7.5 50 mM sodium phosphate, 1 M sodium chloride, pH 7.5 1.5 ml/min (45 cm/h)
Flow: Chromatographic procedure: Equilibration binding buffer: 80 ml Sample application: 2000 ml Wash with binding buffer: 100 ml Elution: 300 ml elution buffer as linear gradient 0–100% 1
Mr 97 000 66 000 45 000 30 000 20 100 14 400
1
1
2
3
4
5
6
7
8
9
10
fr. 13–14
(
A 280 (
)
) [NaCl] (M)
fr. 23–24
scCro8, fr. 54–55 fr. 69
0
Electrophoresis: SDS-PAGE, 12% gel, Coomassie Blue staining Lane 1. Pool from HiPrep 26/10 Desalting Lane 2. Flow-through pool from DEAE Sepharose CL-4B Lane 3. Low Molecular Weight Calibration Kit (LMW) Lane 4-10. Eluted fractions from HiPrep 16/10 Heparin FF Lane 4. Fraction 13 Lane 5. Fraction 14 Lane 6. Fraction 23 Lane 7. Fraction 24 Lane 8. Fraction 54 Lane 9. Fraction 55 Lane 10. Fraction 69
0
Fig. 36. scCro8 purification on HiPrep 16/10 Heparin FF.
Performing a separation Binding buffers: 20 mM Tris-HCl, pH 8.0 or 10 mM sodium phosphate, pH 7.0 Elution buffer:
20 mM Tris-HCl, 1–2 M NaCl, pH 8.0 or 10 mM sodium phosphate, 1–2 M NaCl, pH 7.0
1. Equilibrate the column with 10 column volumes of binding buffer. 2. Apply the sample. 3. Wash with 5–10 column volumes of binding buffer or until no material appears in the eluent (monitored by UV absorption at A280 nm). 4. Elute with 5–10 column volumes of elution buffer using a continuous or step gradient from 0–100% elution buffer.
Modify the selectivity of heparin by altering pH or ionic strength of the buffers. Elute using a continuous or step gradient with NaCl, KCl or (NH4)2SO4 up to 1.5–2 M. Cleaning Remove ionically bound proteins by washing with 0.5 column volume 2 M NaCl for 10–15 minutes. Remove precipitated or denatured proteins by washing with 4 column volumes 0.1 M NaOH for 1–2 hours or 2 column volumes 6 M guanidine hydrochloride for 30–60 minutes or 2 column volumes 8 M urea for 30–60 minutes. Remove hydrophobically bound proteins by washing with 4 column volumes 0.1% – 0.5% Triton X-100 for 1–2 hours. 62
Media characteristics Ligand density
Composition
pH stability*
Mean particle size
HiTrap Heparin HP
10 mg/ml
Heparin coupled to Sepharose High Performance using a N-hydroxysuccinimide coupling method to give stable attachment to the matrix through alkylamine and ether links.
Short term 5–10 Long term 5–10
34 µm
Heparin Sepharose 6 Fast Flow
5 mg/ml
Heparin coupled to Sepharose 6 Fast Flow by reductive amination to give a stable attachment even in alkaline conditions.
Short term 4–13 Long term 4–12
90 µm
HiPrep 16/10 Heparin FF
*Long term refers to the pH interval over which the medium is stable over a long period of time without adverse effects on its subsequent chromatographic performance. Short term refers to the pH interval for regeneration, cleaning-in-place and sanitization procedures.
Chemical stability 0.1 M NaOH (1 week at +20 °C), 0.05 M sodium acetate, pH 4.0, 4 M NaCl, 8 M urea, 6 M guanidine hydrochloride. Storage Wash media and columns with 0.05 M sodium acetate containing 20% ethanol (use approximately 5 column volumes for packed media) and store at +4 to +8 °C.
63
Coagulation factors HiTrap Heparin HP, HiPrep 16/10 Heparin FF, Heparin Sepharose 6 Fast Flow Blood coagulation factors form an extremely important group of proteins for research, medical and clinical applications. The information about the purification of DNA binding proteins (page 59) is applicable also to the purification of coagulation factors. Purification examples Sample: Column: Flow: Binding buffer: Elution buffer: Chromatographic procedure:
Electrophoresis:
30 ml bovine plasma diluted with 15 ml 0.1 M Tris, 0.01 M citrate, 0.225 M NaCl, pH 7.4 HiTrap Heparin HP, 1 ml 1.0 ml/min 0.1 M Tris, 0.01 M citric acid, 0.225 M NaCl, pH 7.4 0.1 M Tris, 0.01 M citric acid, 2 M NaCl, pH 7.4 8 ml 12.5% elution buffer, 45 ml sample, 27 ml 12.5% elution buffer, 26 ml 25% elution buffer, 26 ml 100% elution buffer. 2.9 mg antithrombin-III was eluted in peak II SDS-PAGE, PhastSystem, PhastGel Gradient 8–25, Mr 1 µl sample, silver stained 97 000
A 280 nm
66 000 A 280
45 000
0.8 % Elution buffer Flow through
0.6
peak I
peak II
30 000 20 100
100 0.4
14 400 1
50
0.2
0 10
50 60
70 80 90 100 110 120 130 ml
3
4
Lane 1. Low Molecular Weight Calibration Kit, reduced Lane 2. Peak II, reduced, diluted 2-fold Lane 3. Peak I, reduced, diluted 2-fold Lane 4. Unbound material, reduced, diluted 15-fold
Fig. 37. Purification of antithrombin III from bovine plasma on HiTrap Heparin HP, 1 ml.
64
2
Sample:
Pooled and frozen human plasma from 5 donors. 50 ml of thawed plasma filtered (0.45 µM). Plasma and binding buffer mixed in ratio 2:1 0.1 M Tris-HCl, 0.01 M citric acid, 0.225 M NaCl, pH 7.4 0.1 M Tris-HCl, 0.01 M citric acid, 0.330 M NaCl, pH 7.4 0.1 M Tris-HCl, 0.01 M citric acid, 2.0 M NaCl, pH 7.4 Heparin Sepharose 6 Fast Flow packed in HR 5/5 column
Binding buffer: Wash buffer: Elution buffer: Column: Chromatographic procedure: 5 ml binding buffer, 45 ml sample, 40 ml binding buffer, 15 ml wash buffer, 9 ml elution buffer A 280 nm 2
1
Wash Elution 1 C B 0 0
50
100
150
200
250 Time (min)
2
3 4 5
6 7
8
Isoelectric focusing-PAGE analysis of the peaks B and C from the affinity chromatography. Lanes 1 and 4. Peak C Lanes 2 and 7. IEF calibration kit Lanes 3 and 6. Antithrombin-III from Sigma (A7388) Lanes 5 and 8. Peak B The results show that pure antithrombin-III is present in the two peaks. NOR-PartigenAntithrombin-III test of peaks B and C shows a more active form of antithrombin-III concentrated in peak C.
Fig. 38. Purification of antithrombin-III from human plasma on Heparin Sepharose 6 Fast Flow. Peak B elutes with wash buffer. Peak C elutes with elution buffer and includes a more active form of antithrombin-III.
Performing a separation As for DNA binding proteins, see page 62. Since the heparin acts as an affinity ligand for coagulation factors, it may be advisable to include a minimum concentration of 0.15 M NaCl in the binding buffer. If an increasing salt gradient gives unsatisfactory results, use heparin (1–5 mg/ml) as a competing agent in the elution buffer.
Biotin and biotinylated substances HiTrap Streptavidin HP, Streptavidin Sepharose High Performance Biotin and biotinylated substances bind to streptavidin (a molecule isolated from Streptomyces avidinii) in a very strong interaction that requires denaturing conditions for elution. By coupling streptavidin to Sepharose a highly specific affinity medium is created and, using biotinylated antibodies, the strong interaction can be utilized for the purification of antigens. The biotinylated antibody-antigen complexes bind tightly to Streptavidin Sepharose and the antigen can then be eluted separately using milder elution conditions, leaving behind the biotinylated antibody. An alternative to labelling the antibody with biotin is to use 2-iminobiotin that binds to streptavidin above pH 9.5 and can be eluted at pH 4 (see Figure 39).
65
Purification options Binding capacity
Maximum operating flow
Comments
HiTrap Streptavidin HP
Biotin, > 300 nmol/column Biotinylated BSA, 6 mg/column
4 ml/min
Prepacked 1 ml column.
Streptavidin Sepharose High Performance
Biotin, > 300 nmol/medium Biotinylated BSA, 6 mg/medium
150 cm/h*
Supplied as a suspension ready for column packing.
*See Appendix 4 to convert linear flow (cm/h) to volumetric flow rate. Maximum operating flow is calculated from measurement in a packed column with a bed height of 10 cm and i.d. of 5 cm.
Purification example A 280 nm
% elution buffer
1.2 100
Sample: Column: Binding buffer: Elution buffer: Flow: System:
9.0 ml of a mixture of BSA and iminobiotinylated BSA, filtered through a 0.45 µm filter HiTrap Streptavidin HP, 1 ml 50 mM ammonium carbonate buffer, 0.5 M NaCl, pH 10.0 50 mM ammonium acetate buffer, 0.5 M NaCl, pH 4.0 1 ml/min (0.3 ml/min during sample application) ÄKTAexplorer
80 0.8 60
40 0.4
20
0
0 0
5
10
15
20
ml
Fig. 39. Purification of iminobiotinylated BSA on HiTrap Streptavidin HP, 1 ml.
Performing a separation: Biotinylated substances Binding buffer: 20 mM sodium phosphate, 0.15 M NaCl, pH 7.5 Elution buffer: 8 M guanidine-HCl, pH 1.5 Iminobiotinylated substances Binding buffer: 50 mM ammonium carbonate, 0.5 M NaCl, pH 10.0 Elution buffer: 50 mM ammonium acetate, 0.5 M NaCl, pH 4.0
1. Equilibrate the column with 10 column volumes of binding buffer. 2. Apply the sample. For the best results use a low flow rate, 0.1–0.5 ml/min, during sample application. 3. Wash with at least 10 column volumes of binding buffer or until no material appears in the eluent (monitored by UV absorption at A 280 nm). 4. Elute with 10–20 column volumes of elution buffer.* *Since elution conditions can be quite harsh, it is recommended to collect fractions into neutralization buffer (100 µl – 200 µl 1 M Tris-HCl, pH 9.0 per ml fraction), so that the final pH of the fractions will be approximately neutral or perform a rapid buffer exchange on a desalting column (see page 134).
66
The harsh conditions required to break the streptavidin-biotin bond may affect both the sample and the ligand. Streptavidin Sepharose columns cannot be re-used after elution under these conditions. Antigen purification Antigens can be purified from biotinylated antibody-antigen complexes bound to streptavidin. The following method was adapted for HiTrap Streptavidin HP from work published in Anal. Biochem. 163, 270–277 (1987), Gretch, D.R., Suter, M. and Stinski, M.F. Solubilization buffer: 20 mM sodium phosphate, 150 mM NaCl, pH 7.5 with 0.1% SDS, 1.0% Nonidet™-P-40, 0.5% sodium deoxycholate, 0.02% NaN3, 100 µg/ml PMSF Elution buffer:
0.1 M glycine-HCl, pH 2.2
1. Solubilize the antigen with an appropriate amount of solubilization buffer, clear the sample by centrifuging at 12 000 g for 15 min. 2. Add the biotinylated antibody and adjust the volume to 1 ml. 3. Incubate with end-over-end mixing, for at least 1 h or overnight. 4. Equilibrate the column with 10 column volumes of solubilization buffer. 6. Apply antibody-antigen solution to the column at a low flow rate such as 0.2 ml/min. If the sample volume is less than 1 ml, apply the sample, and leave for a few minutes to allow binding to take place. 7. Wash out unbound sample with 10 column volumes of solubilization buffer or until no material is found in eluent (monitored by UV absorption at A280 nm). 8. Elute with 5–10 column volumes of elution buffer.* *Since elution conditions are quite harsh, it is recommended to collect fractions into neutralization buffer (100 µl – 200 µl 1 M Tris-HCl, pH 9.0 per ml fraction), so that the final pH of the fractions will be approximately neutral or perform a rapid buffer exchange on a desalting column (see page 134).
Media characteristics Streptavidin Sepharose High Performance HiTrap Streptavidin HP
Composition
pH stability*
Mean particle size
Streptavidin is coupled to Sepharose High Performance using a N-hydroxysuccinimide coupling method.
Short term 2–10.5 Long term 4–9
34 µm
*Long term refers to the pH interval over which the medium is stable over a long period of time without adverse effects on its subsequent chromatographic performance. Short term refers to the pH interval for regeneration, cleaning-in-place and sanitization procedures.
Storage Wash media and columns with 20% ethanol (use approximately 5 column volumes for packed media) and store at +4 to +8 °C.
67
Purification or removal of fibronectin Gelatin Sepharose 4B Fibronectin is a high molecular weight glycoprotein found on the surfaces of many cell types and present in many extracellular fluids including plasma. Fibronectin binds specifically to gelatin at or around physiological pH and ionic strength. Purification option Gelatin Sepharose 4B
Binding capacity/ml medium
Maximum operating flow
Comments
1 mg human plasma fibronectin
75 cm/h*
Supplied as a suspension ready for column packing.
*See Appendix 4 to convert linear flow (cm/h) to volumetric flow rate. Maximum operating flow is calculated from measurement in a packed column with a bed height of 10 cm and i.d. of 5 cm.
Performing a separation Binding buffer: PBS: 140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4 Elution buffer alternatives: - 0.05 M sodium acetate, 1.0 M sodium bromide (or potassium bromide), pH 5.0 - Binding buffer + 8 M urea - Binding buffer + arginine
Fibronectin has a tendency to bind to glass. Use siliconized glass to prevent adsorption. Cleaning Wash 3 times with 2–3 column volumes of buffer, alternating between high pH (0.1 M TrisHCl, 0.5 M NaCl, pH 8.5) and low pH (0.1 M sodium acetate, 0.5 M NaCl, pH 4.5). Re-equilbrate immediately with 3–5 column volumes of binding buffer. Remove denatured proteins or lipids by washing the column with 0.1% Triton X-100 at +37 °C for one minute. Re-equilibrate immediately with 5 column volumes of binding buffer. Media characteristics Gelatin Sepharose 4B
Ligand density
Composition
pH stability*
Mean particle size
4.5–8 mg gelatin/ml
Gelatin linked to Sepharose using the CNBr method
Short term 3–10 Long term 3–10
90 µm
*Long term refers to the pH interval over which the medium is stable over a long period of time without adverse effects on its subsequent chromatographic performance. Short term refers to the pH interval for regeneration, cleaning-in-place and sanitization procedures.
Chemical stability Stable in all commonly used aqueous buffers. Storage Wash media and columns with 20% ethanol at neutral pH (use approximately 5 column volumes for packed media) and store at +4 to +8 °C.
68
Purification or removal of albumin HiTrap Blue HP, Blue Sepharose 6 Fast Flow The same procedure can be used either to purify albumin or to remove albumin as a specific contaminant before or after other purification steps. Albumin binds to Cibacron™ Blue F3G-A, a synthetic polycyclic dye that acts as an aromatic anionic ligand binding the albumin via electrostatic and/or hydrophobic interactions. Similar interactions are seen with coagulation factors, lipoproteins and interferon. Cibacron Blue F3G-A is linked to Sepharose to create Blue Sepharose affinity media. O
NH2 SO3Na
SO3Na NH N
O
NH
NH SO3Na
N N O
Sepharose
Fig. 40. Partial structure of Blue Sepharose Fast Flow and Blue Sepharose High Performance.
Use HiTrap Blue HP 1 ml or 5 ml columns to remove host albumin from mammalian expression systems, or when the sample is known to contain high levels of albumin that may mask the visualization of other protein peaks seen by UV absorption. Albumin can be a significant contaminant during the purification of immunoglobulins from ascites fluid, cell cultures or serum, chiefly because of its abundance in the original source material. Advice on the selection of techniques for the removal of albumin during antibody purification is given in The Antibody Purification Handbook from Amersham Pharmacia Biotech. Cibacron Blue F3G-A also shows certain structural similarities to naturally occurring molecules, such as the cofactor NAD+, that enable it to bind strongly and specifically to a wide range of proteins including kinases, dehydrogenases and most other enzymes requiring adenylyl-containing cofactors (see page 72). Purification options Binding capacity
Maximum operating flow
Comments
HiTrap Blue HP
Human serum albumin, 20 mg/column Human serum albumin, 100 mg/column
4 ml/min (1 ml column) 20 ml/min (5 ml column)
Prepacked columns.
Blue Sepharose 6 Fast Flow*
Human serum albumin, > 18 mg/ml medium
400 cm/h**
Supplied as a suspension ready for column packing.
*A convenient alternative to Blue Sepharose CL-6B, since rehydration is not required. **See Appendix 4 to convert linear flow (cm/h) to volumetric flow rate. Maximum operating flow is calculated from measurement in a packed column with a bed height of 10 cm and i.d. of 5 cm.
69
Purification examples Figure 41 shows the use of HiTrap Blue HP for purification of increasing amounts of human serum albumin. The process is easily scaled up by connecting several 1 ml or 5 ml HiTrap columns is series. In this example elution is achieved by increasing the ionic strength of the buffer. Changing the pH or the polarity of the buffer can also work. Figure 42 shows the use of Blue Sepharose 6 Fast Flow for the separation of human serum albumin from interferon b. Sample:
Human serum buffer exchanged on a PD-10 Desalting column to binding buffer. Filtered on a 0.45 µm filter Column: HiTrap Blue HP, 1 ml or 5 ml Flow: 2 ml/min (1 ml column), 4 ml/min (5 ml column) Binding buffer: 50 mM KH 2PO4, pH 7.0 Elution buffer: 50 mM KH 2PO4, 1.5 M KCl, pH 7.0
Configuration: 1×1 ml column Sample vol.: 0.7 ml human serum Yield: 16.7 mg HSA A 280 nm
Configuration: 2×1 ml column Sample vol.: 1.4 ml human serum Yield: 33.2 mg HSA
Configuration: 3×1 ml column Sample vol.: 2.1 ml human serum Yield: 52.0 mg HSA
A 280 nm
A 280 nm Binding Elution buffer
buffer
Binding Elution buffer buffer
Fractions 17–22
Binding Elution buffer buffer Fractions 16–21
Fractions 2–10
Fractions 2–10
1.0
1.0
Fractions 2–10
1.0
Fractions 15–20
10
20
10
ml
Configuration: 1×5 ml column Sample vol.: 3.5 ml human serum Yield: 98.5 mg HSA A 280 nm
20
ml
10
ml
Configuration: 3×5 ml columns connected in series Sample vol.: 10.5 ml human serum Yield: 286.9 mg HSA A 280 nm
Binding buffer
Elution buffer Binding buffer
Elution buffer
Fractions 22–30
1.0
20
1.0
Fractions 3–14
10
Fractions 67–83
20
30
ml
Fractions 11–46
10
20
30
40
50
60
70
80
90
100
Fig. 41. Scaling up on HiTrap Blue HP gives predictable separations and quantitatively reproducible yields.
70
110 ml
250
IFN-act. units
A 280 nm 0.12 IFNactivity
200
0.10
A 280nm 0.08 150
Sample:
Column: Flow: Binding buffer:
0.06 100 0.04 50
Elution buffer 1: Elution buffer 2:
0.5 ml interferon b (1 000 000 U/ml) in 0.1 M phosphate, pH 7.4, with 1 mg/ml of human serum albumin Blue Sepharose 6 Fast Flow (0.5 ml) Gravity feed 0.02 M phosphate, 0.15 M NaCl, pH 7.2 0.02 M phosphate, 2 M NaCl, pH 7.2 0.02 M phosphate, 2 M NaCl, 50% ethylene glycol, pH 7.2
0.02
0 0
5 Elution buffer 1
10
Volume (ml)
0
Elution buffer 2
Fig. 42. Purification of human serum albumin and interferon b on Blue Sepharose 6 Fast Flow.
In these examples elution is achieved by increasing the ionic strength of the buffer or changing the polarity of the buffer. Changing the pH of the buffer can also work, but the correct co-factor is preferable for the elution of specifically bound proteins. Performing a separation Binding buffer: 50 mM KH2PO4, pH 7.0 or 20 mM sodium phosphate, pH 7.0 Elution buffer: 50 mM KH2PO4, 1.5 M KCl, pH 7.0 or 20 mM sodium phosphate, 2 M NaCl, pH 7.0
1. Equilibrate the column with 5 column volumes of binding buffer. 2. Apply the sample, using a syringe or a pump. 3. Wash with 10 column volumes of binding buffer or until no material appears in the eluent (monitored by absorption at A280 nm). 4. Elute with 5 column volumes of elution buffer. More may be required if the interaction is difficult to reverse.
Cleaning Wash with 5 column volumes of high pH (0.1 M Tris-HCl, 0.5 M NaCl, pH 8.5) followed by low pH (0.1 M sodium acetate, 0.5 M NaCl, pH 4.5). Repeat 4–5 times. Re-equilibrate immediately with binding buffer. Remove precipitated proteins with 4 column volumes of 0.1 M NaOH at a low flow rate, followed by washing with 3–4 column volumes of 70% ethanol or 2 M potassium thiocyanate. Alternatively, wash with 2 column volumes of 6 M guanidine hydrochloride. Re-equilibrate immediately with binding buffer. Remove strongly hydrophobic proteins, lipoproteins and lipids by washing with 3–4 column volumes of up to 70% ethanol or 30% isopropanol. Alternatively, wash with 2 column volumes of detergent in a basic or acidic solution, e.g. 0.1% non-ionic detergent in 1 M acetic acid at a low flow rate, followed by 5 column volumes of 70% ethanol to remove residual detergent. Re-equilibrate immediately in binding buffer.
71
Media characteristics Ligand and density
Composition
pH stability*
Mean particl size
HiTrap Blue HP
Cibacron Blue F3G-A 4 mg/ml
Ligand coupled to Sepharose High Performance using the triazine method.
Short term 3–13 Long term 4–12
34 µm
Blue Sepharose 6 Fast Flow
Cibacron Blue F3G-A 6.7–7.9 µmoles/ml
Ligand coupled to Sepharose Fast Flow using the triazine method.
Short term 3–13 Long term 4–12
90 µm
*Long term refers to the pH interval over which the medium is stable over a long period of time without adverse effects on its subsequent chromatographic performance. Short term refers to the pH interval for regeneration, cleaning-in-place and sanitization procedures.
Chemical stability Stable in all commonly used aqueous buffers, 70% ethanol, 8 M urea and 6 M guanidine hydrochloride. Storage Wash media and columns with 20% ethanol (use approximately 5 column volumes for packed media) and store at +4 to +8 °C.
NAD+-dependent dehydrogenases and ATP-dependent kinases 5' AMP Sepharose 4B, HiTrap Blue HP, Blue Sepharose 6 Fast Flow NAD+-dependent dehydrogenases and ATP-dependent kinases interact strongly with 5' AMP so that selective elution with gradients of NAD+ or NADP+ enables the resolution of complex mixtures of dehydrogenase isoenzymes, using 5' AMP Sepharose 4B. Synthesis of 5' AMP Sepharose 4B takes place in several steps. Diaminohexane is linked to AMP via the N6 of the purine ring. The derivatized AMP is then coupled to Sepharose 4B via the aminohexane spacer. NAD+-dependent dehydrogenases and ATP-dependent kinases are also members of a larger group of proteins that will interact with Cibacron Blue F3G-A, a synthetic polycyclic dye that shows certain structural similarities to the cofactor NAD+. When used as an affinity ligand attached to Sepharose 6 Fast Flow or Sepharose HP, Cibacron Blue F3G-A will bind strongly and specifically to a wide range of proteins. Some proteins bind specifically due to their requirement for nucleotide cofactors, while others, such as albumin, lipoproteins, blood coagulation factors and interferon, bind in a less specific manner by electrostatic and/or hydrophobic interactions with the aromatic anionic ligand.
72
Purification options Binding capacity
Maximum operating flow
Comments
5' AMP Sepharose 4B
Lactate dehydrogenase, 10 mg/ml medium (0.1 M phosphate buffer, pH 7.0 at +20 °C)
75 cm/h*
High specificity for proteins with affinity for NAD +. Supplied as a freeze-dried powder, rehydration required.
HiTrap Blue HP
Human serum albumin, 20 mg/column Human serum albumin, 100 mg/column
4 ml/min (1 ml column)
General specificity for proteins with affinity for NADP+ and other proteins that react less specifically. Prepacked columns.
Blue Sepharose 6 Fast Flow
Human serum albumin, > 18 mg/ml medium
20 ml/min (5 ml column)
400 cm/h*
General specificity for proteins with affinity for NADP+ and other proteins that react less specifically. Supplied as a suspension ready for column packing.
*See Appendix 4 to convert linear flow (cm/h) to volumetric flow rate. Maximum operating flow is calculated from measurement in a packed column with a bed height of 10 cm and i.d. of 5 cm.
5' AMP Sepharose 4B Performing a separation Swell the required amount of powder for 15 min. in 0.1 M phosphate buffer, pH 7.0 (100 ml per gram dry powder) and wash on a sintered glass filter. Pack the column (see Appendix 3). Binding buffer: 10 mM phosphate, 0.15 M NaCl, pH 7.3
If the protein of interest binds to the medium via ionic forces, it may be necessary to reduce the concentration of NaCl in the binding buffer. Elution buffers: • use low concentrations of the free cofactor, NAD+ or NADP+ (up to 20 mM) with step or gradient elution.
If detergent or denaturing agents have been used during purification, these can also be used in the high and low pH wash buffers. Cleaning Wash 3 times with 2–3 column volumes of buffers, alternating between high pH (0.5 M NaCl, 0.1 M Tris-HCl, pH 8.5) and low pH (0.5 M NaCl, 0.1 M sodium acetate, pH 4.5). Re-equilibrate immediately with 3–5 column volumes of binding buffer. Remove denatured proteins or lipids by washing the column with 2 column volumes of detergent e.g. 0.1% Triton X-100 for 1 minute. Re-equilibrate immediately with 5 column volumes of binding buffer.
73
Media characteristics
5' AMP Sepharose 4B
Ligand density
Composition
2 µmoles/ml
N (6-aminohexyl-) 5' AMP coupled to Sepharose 4B using CNBr method**
6
pH stability*
Mean particle size
Short term 4–10 Long term 4–10
90 µm
*Long term refers to the pH interval over which the medium is stable over a long period of time without adverse effects on its subsequent chromatographic performance. Short term refers to the pH interval for regeneration, cleaning-in-place and sanitization procedures. **The attachment of the ligand via an alkyl linkage to the N6 amino group gives a stable product that is conformationally acceptable to most 5' AMP- or adenine nucleotide cofactor-requiring enzymes.
Chemical stability Stable to all commonly used aqueous buffers and additives such as detergents. Avoid high concentrations of EDTA, urea, guanidine hydrochloride, chaotropic salts and strong oxidizing agents. Exposure to pH >10 may cause loss of phosphate groups. Storage Store freeze-dried product below +8 °C under dry conditions. Wash media and columns with 20% ethanol at neutral pH (use approximately 5 column volumes for packed media) and store at +4 to +8 °C. HiTrap Blue HP, Blue Sepharose 6 Fast Flow The information supplied for the purification or removal of albumin (page 69) is applicable also to the purification of enzymes with an affinity for NAD+. Performing a separation As for albumin (see page 71), but note the following: For elution use low concentrations of the free cofactor, NAD+ or NADP+ (1–20 mM), or increase ionic strength (up to 2 M NaCl or KCl, 1 M is usually sufficient). For less specifically bound proteins: use higher concentrations of cofactor or salt or more severe eluents such as urea or potassium isothiocyanate. Polarity reducing agents such as dioxane (up to 10%) or ethylene glycol (up to 50%) may be used.
NADP+-dependent dehydrogenases and other enzymes with affinity for NADP+ 2'5' ADP Sepharose 4B, Red Sepharose CL-6B NADP+-dependent dehydrogenases interact strongly with 2'5' ADP. Selective elution with gradients of NAD+ or NADP+ has allowed the resolution of complex mixtures of dehydrogenase isoenzymes using 2'5' ADP Sepharose 4B. Synthesis of the medium takes place in several steps. Diaminohexane is linked to 2'5' ADP via the N6 of the purine ring. The derivatized ADP is then coupled to Sepharose 4B via the aminohexane spacer. Figure 43 shows the partial structure of 2'5' ADP Sepharose 4B.
74
NH
Sepharose
(CH2)n
NH
N
N N
N
O HO
P
O
CH2 O
O HO HO
O O
P O
Fig. 43. Partial structure of 2'5' ADP Sepharose 4B.
NADP+-dependent dehydrogenases are also members of a larger group of proteins that will interact with Procion™ Red, a synthetic polycyclic dye that shows certain structural similarities to naturally occurring NADP+. When used as an affinity ligand attached to Sepharose CL-6B, Procion Red HE-3B will bind strongly and specifically to a wide range of proteins. Some proteins bind specifically due to their requirement for nucleotide cofactors, while others, such as albumin, lipoproteins, blood coagulation factors and interferon, bind in a less specific manner by electrostatic and/or hydrophobic interactions with the aromatic anionic ligand. NaO3S
NaO3S
SO3Na N
N
N
N OH
OH
NH
NH N
SO3Na
SO3Na
N
SO3Na
N NH
N O
N
NH N
OH
Sepharose
Fig. 44. Partial structure of Red Sepharose CL-6B.
75
Purification options Binding capacity/ml medium
Maximum operating flow
Comments
2'5' ADP Sepharose 4B
Glucose-6-phosphate, dehydrogenase, 0.4 mg (0.1 M Tris-HCl, 5 mM EDTA, 1 mM 2-mercaptoethanol buffer, pH 7.6).
75 cm/h*
High specificity for proteins with affinity for NADP+. Supplied as a freeze-dried powder, rehydration required.
Red Sepharose CL-6B
Rabbit lactate dehydrogenase, 2 mg
150 cm/h*
General specificity for proteins with affinity for NADP+ and other proteins that react less specifically. Supplied as dry powder, rehydration required.
*See Appendix 4 to convert linear flow (cm/h) to volumetric flow rate. Maximum operating flow is calculated from measurement in a packed column with a bed height of 10 cm and i.d. of 5 cm.
2'5' ADP Sepharose 4B Purification example Figure 45 shows a linear gradient elution used for the initial separation of NADP+-dependent enzymes from a crude extract of Candida utilis.
Enzyme activity
B C
D
E
0.8 0.6 0.4 0.2
0
20
40
60
80
NADP + concentration (mM)
A
100 120 Fraction number
Fig. 45. Gradient elution with 0–0.6 mM NADP+. A: non-interacting protein, B: glucose-6-phosphate dehydrogenase, C: glutamate dehydrogenase, D: glutathione reductase, E: 6-phosphogluconate dehydrogenase. (Brodelius et al., Eur. J. Biochem. 47, 81–89 (1974)).
Performing a separation Swell the required amount of powder for 15 min. in 0.1 M phosphate buffer, pH 7.3 (100 ml per gram dry powder) and wash on a sintered glass filter (porosity G3). Pack the column (see Appendix 3). Binding buffer: 10 mM phosphate, 0.15 M NaCl, pH 7.3
If the protein of interest binds to the medium via ionic forces, it may be necessary to reduce the concentration of NaCl in the binding buffer. Elution buffers: • use low concentrations of the free cofactor, NAD+ or NADP+ (up to 20 mM) with step or gradient elution.
76
If detergent or denaturing agents have been used during purification, these can also be used in the low and high pH wash buffers. Cleaning Wash 3 times with 2–3 column volumes of buffers, alternating between high pH (0.1 M Tris-HCl, 0.5 M NaCl, pH 8.5) and low pH (0.1 M sodium acetate, 0.5 M NaCl, pH 4.5). Re-equilibrate immediately with 3–5 column volumes of binding buffer. Remove denatured proteins or lipids by washing the column with 2 column volumes of detergent e.g. 0.1% Triton X-100 for 1 minute. Re-equilibrate immediately with 5 column volumes of binding buffer. Media characteristics
2'5' ADP Sepharose 4B
Ligand density
Composition
2 µmoles/ml
N -(6-aminohexyl)adenosine 2'5' bisphosphate coupled to Sepharose 4B by CNBr method**
6
pH stability*
Mean particle size
Short term 4–10 Long term 4–10
90 µm
*Long term refers to the pH interval over which the medium is stable over a long period of time without adverse effects on its subsequent chromatographic performance. Short term refers to the pH interval for regeneration, cleaning-in-place and sanitization procedures. **Coupling via the N6 position of the NADP+ -analogue, adenosine 2'5' bisphosphate, gives a ligand that is stereochemically acceptable to most NADP+-dependent enzymes.
Chemical stability Stable to all commonly used aqueous buffers and additives such as detergents. Avoid high concentrations of EDTA, urea, guanidine hydrochloride, chaotropic salts and strong oxidizing agents. Exposure to pH > 10 may cause loss of phosphate groups. Storage Store freeze-dried product below +8 °C under dry conditions. Wash media and columns with 20% ethanol at neutral pH (use approximately 5 column volumes for packed media) and store at +4 to +8 °C. Red Sepharose CL-6B Performing a separation Swell the required amount of powder for 15 min. and wash with distilled water on a sintered glass filter (porosity G3). Use 200 ml water for each gram of dry powder, adding in several aliquots. One gram of freeze-dried material gives a final volume of approximately 4 ml. Pack a column (see Appendix 3). Binding buffer: Use a buffer at around neutral pH since proteins bind specifically to Red Sepharose CL-6B at this pH.
The binding capacity will depend upon parameters such as sample concentration, flow rate, pH, buffer composition and temperature. To obtain optimal purification with respect to capacity, determine the binding capacity over a range of different pH and flow rates.
77
Elution buffers: • use low concentrations of the free cofactor, NAD+ or NADP+ (up to 20 mM), or increase ionic strength up to 2 M NaCl or 1 M KCl.
If detergent or denaturing agents have been used during purification, these can also be used in the low and high pH wash buffers. Cleaning Wash 3 times with 2–3 column volumes of buffers, alternating between high pH (0.1 M Tris-HCl, 0.5 M NaCl, pH 8.5) and low pH (0.1 M sodium acetate, 0.5 M NaCl, pH 4.5). Re-equilibrate immediately with 3–5 column volumes of binding buffer. Remove denatured proteins or lipids by washing with 2 column volumes of 6 M guanidine hydrochloride or 8 M urea. Alternatively, wash the column with 2 column volumes of detergent in a basic or acidic solution, e.g. 0.1% Triton X-100 in 1 M acetic acid. Remove residual detergent by washing with 5 column volumes of 70% ethanol. In both cases wash immediately with 5 column volumes of binding buffer. Media characteristics
Red Sepharose CL-6B
Ligand and density
Composition
pH stability*
Mean particle size
Procion Red HE 3B 2 µmoles/ml
Ligand coupled to Sepharose CL-6B using the triazine method.
Short term 3–13 Long term 4–12
90 µm
*Long term refers to the pH interval over which the medium is stable over a long period of time without adverse effects on its subsequent chromatographic performance. Short term refers to the pH interval for regeneration, cleaning-in-place and sanitization procedures.
Chemical stability Stable with all commonly used aqueous buffers and additives such as 8 M urea and 6 M guanidine hydrochloride. Storage Store freeze-dried powders under dry conditions and below +8 °C. Wash media and columns with 20% ethanol in 0.1 M KH2PO4, pH 8.0 (use approximately 5 column volumes for packed media) and store at +4 to +8 °C.
78
Glycoproteins or polysaccharides Con A Sepharose 4B, Lentil Lectin Sepharose 4B, Agarose Wheat Germ Lectin Glycoproteins and polysaccharides react reversibly, via specific sugar residues, with a group of proteins known as lectins. As ligands for purification media, lectins are used to isolate and separate glycoproteins, glycolipids, polysaccharides, subcellular particles and cells, and to purify detergentsolubilized cell membrane components. Substances bound to the lectin are resolved by using a gradient of ionic strength or of a competitive binding substance. Media screening To select the optimum lectin for purification, it may be necessary to screen different media. The ligands, Concanavalin A (Con A), Lentil Lectin and Wheat Germ Lectin provide a spectrum of parameters for the separation of glycoproteins. Table 5 gives their specificity. Table 5. Specificity of lectins. Lectin
Specificity
Mannose/glucose binding lectins Con A, Canavalia ensiformis
Branched mannoses, carbohydrates with terminal mannose or glucose (aMan > aGlc > GlcNAc).
Lentil Lectin, Lens culinaris
Branched mannoses with fucose linked a(1,6) to N-acetyl-glucosamine, (aMan > aGlc > GlcNAc).
N-acetylglucosamine binding lectins Wheat Germ Lectin, Triticum vulgare
Chitobiose core of N-linked oligosaccharides, [GlcNAc(b1,4GlcNAc)1–2 > bGlcNac].
Con A for binding of branched mannoses, carbohydrates with terminal mannose or glucose (aMan > aGlc > GlcNAc) Concanavalin A (Con A) is a tetrameric metalloprotein isolated from Canavalia ensiformis (jack bean). Con A binds molecules containing a-D-mannopyranosyl, a-D-glucopyranosyl and sterically related residues. The binding sugar requires the presence of C-3, C-4 and C-5 hydroxyl groups for reaction with Con A. Con A can be used for applications such as: • Separation and purification of glycoproteins, polysaccharides and glycolipids. • Detection of changes in composition of carbohydrate-containing substances, e.g. during development. • Isolation of cell surface glycoproteins from detergent-solubilized membranes. • Separation of membrane vesicles into "inside out" and "right side out" fractions.
79
Purification options
Con A Sepharose 4B
Binding capacity/ml medium
Maximum operating flow
Comments
Porcine thyroglobulin, 20–45 mg
75 cm/h**
Supplied as a suspension ready for column packing*.
*Supplied in acetate buffer solution (0.1 M, pH 6) containing 1 M NaCl, 1 mM CaCl2, 1 mM MgCl2, 1 mM MnCl2, 20% ethanol. **See Appendix 4 to convert linear flow (cm/h) to volumetric flow rate. Maximum operating flow is calculated from measurement in a packed column with a bed height of 10 cm and i.d. of 5 cm.
Purification example Figure 46 shows the purification of a human cell surface alloantigen on Con A Sepharose 4B.
A 280 nm
Inhibition of antibody binding % 100 a-methyl-manoside
1.0
50
0.8 0.6 0.4 0.2
0 UB
1
2
3
4
5
6
7
8
9 10
Fig. 46. Purification of a cell surface antigen on Con A Sepharose 4B. Solid circles represent antigen activity and open circles represent protein profile. Reproduced courtesy of the authors and publishers. Reference: A novel heteromorphic human cell surface alloantigen, gp60, defined by a human monoclonal antibody. Schadendorf, D. et al., J. Immunol. 142, 1621 (1989).
Performing a separation Binding buffer: 20 mM Tris-HCl, 0.5 M NaCl, 1 mM MnCl2, 1 mM CaCl2, pH 7.4 Elution buffer: 0.1–0.5 M methyl-a-D-glucopyranoside (methyl-a-D-glucoside) or methyl-a-D-mannopyranoside (methyl-a-D-mannoside), 20 mM Tris-HCl, 0.5 M NaCl, pH 7.4
1. Pack the column (see Appendix 3) and wash with at least 10 column volumes of binding buffer to remove preservative. 2. Equilibrate the column with 10 column volumes of binding buffer. 3. Apply the sample, using a low flow from 15 cm/h, during sample application (flow rate is the most significant factor to obtain maximum binding). 4. Wash with 5–10 column volumes of binding buffer or until no material appears in the eluent (monitored by UV absorption at A280 nm). 5. Elute with 5 column volumes of elution buffer.
Recovery from Con A Sepharose 4B is decreased in the presence of detergents. If the glycoprotein of interest needs the presence of detergent and has affinity for either lentil lectin or wheat germ lectin, the media Lentil Lectin Sepharose 4B or Agarose Wheat Germ Lectin may provide a suitable alternative to improve recovery
80
For complex samples containing glycoproteins with different affinities for the lectin, a continuous gradient or step elution may improve resolution. Recovery can sometimes be improved by pausing the flow for some minutes during elution. Elute tightly bound substances by lowering the pH. Note that elution below pH 4.0 is not recommended and that below pH 5.0 Mn2+ will begin to dissociate from the Con A and the column will need to be reloaded with Mn2+ before reuse. Cleaning Wash with 10 column volumes of 0.5 M NaCl, 20 mM Tris-HCl, pH 8.5, followed by 0.5 M NaCl, 20 mM acetate, pH 4.5. Repeat 3 times before re-equilibrating with binding buffer. Remove strongly bound substances by: • washing with 0.1 M borate, pH 6.5 at a low flow rate • washing with 20% ethanol or up to 50% ethylene glycol • washing with 0.1% Triton X-100 at +37 °C for one minute Re-equilibrate immediately with 5 column volumes of binding buffer after any of these wash steps. Media characteristics
Con A Sepharose 4B
Ligand density
Composition
pH stability*
Mean particle size
10–16 mg/ml
Con A coupled to Sepharose 4B by CNBr method
Short term 4–9 Long term 4–9
90 µm
*Long term refers to the pH interval over which the medium is stable over a long period of time without adverse effects on its subsequent chromatographic performance. Short term refers to the pH interval for regeneration, cleaning-in-place and sanitization procedures.
Chemical stability Stable to all commonly used aqueous buffers. Avoid 8 M urea, high concentrations of guanidine hydrochloride, chelating agents such as EDTA, or solutions with pH < 4.0 as these remove the manganese from the lectin or dissociate Con A, resulting in loss of activity. Storage Wash media and columns with 20% ethanol in 0.1 M acetate, 1 M NaCl, 1 mM CaCl2, 1 mM MnCl2, 1 mM MgCl2, pH 6 (use approximately 5 column volumes for packed media) and store at +4 to +8 °C.
81
Lentil lectin for binding of branched mannoses with fucose linked a(1,6) to the N-acetyl-glucosamine, (aMan > aGlc > GlcNAc) N-acetylglucosamine binding lectins Lentil lectin binds a-D-glucose and a-D-mannose residues and is an affinity ligand used for the purification of glycoproteins including detergent-solubilized membrane glycoproteins, cell surface antigens and viral glycoproteins. Lentil lectin is the haemagglutinin from the common lentil, Lens culinaris. When compared to Con A, it distinguishes less sharply between glucosyl and mannosyl residues and binds simple sugars less strongly. It also retains its binding ability in the presence of 1% sodium deoxycholate. For these reasons Lentil Lectin Sepharose 4B is useful for the purification of detergent-solubilized membrane proteins, giving high capacities and extremely high recoveries. Purification options
Lentil Lectin Sepharose 4B
Binding capacity/ml medium
Maximum operating flow
Comments
Porcine thyroglobulin, 16–35 mg
75 cm/h*
Supplied as a suspension ready for column packing.
*See Appendix 4 to convert linear flow (cm/h) to volumetric flow rate. Maximum operating flow is calculated from measurement in a packed column with a bed height of 10 cm and i.d. of 5 cm.
Performing a separation Binding buffer: 20 mM Tris-HCl, 0.5 M NaCl, 1 mM MnCl2, 1 mM CaCl2, pH 7.4. Elution buffer:
0.1–0.5 M methyl-a-D-glucopyranoside (methyl-a-D-glucoside), 20 mM Tris-HCl, 0.5 M NaCl, pH 7.4
Buffers for soluble glycoproteins: Binding buffer: 20 mM Tris-HCl, 0.5 M NaCl, 1 mM MnCl2, 1 mM CaCl2, pH 7.4 Elution buffer: 0.3 M methyl-a-D-mannopyranoside, 20 mM Tris-HCl, 0.5 M NaCl, pH 7.4
Buffers for detergent-solubilized proteins: Equilibrate column with the buffer 20 mM Tris-HCl, 0.5 M NaCl, 1 mM MnCl2, 1 mM CaCl2, pH 7.4, to ensure saturation with Mn2+ and Ca2+ . Binding buffer: 20 mM Tris-HCl, 0.5 M NaCl, 0.5% sodium deoxycholate, pH 8.3 Elution buffer: 0.3 M methyl-a-D-mannopyranoside, 20 mM Tris-HCl, 0.5 M NaCl, 0.5% sodium deoxycholate, pH 8.3 1. Pack the column (see Appendix 3) and wash with at least 10 column volumes of binding buffer to remove preservative. 2. Equilibrate the column with 10 column volumes of binding buffer. 3. Apply the sample, using a low flow from 15 cm/h, during sample application (flow rate is the most significant factor to obtain maximum binding). 4. Wash with 5–10 column volumes of binding buffer or until no material appears in the eluent (monitored by UV absorption at A280 nm). 5. Elute with 5 column volumes of elution buffer using a step or gradient elution.
Below pH 5, excess Mn2+ and Ca2+ (1 mM) are essential to preserve binding activity. It is not necessary to include excess Ca2+ or Mn2+ in buffers if conditions that lead to their removal from the coupled lectin can be avoided. 82
For complex samples containing glycoproteins with different affinities for the lectin, a continuous gradient or step elution may improve resolution. Recovery can sometimes be improved by pausing the flow for some minutes during elution Elute tightly bound substances by lowering the pH, but not below pH 3. In some cases strongly bound substances can be eluted with detergent, for example 1.0% deoxycholate. Cleaning Wash with 10 column volumes of 0.5 M NaCl, 20 mM Tris-HCl, pH 8.5, followed by 0.5 M NaCl, 20 mM acetate, pH 4.5. Repeat 3 times before re-equilibrating with binding buffer. Remove strongly bound substances by: • washing with 0.1 M borate, pH 6.5 at a low flow rate • washing with 20% ethanol or up to 50% ethylene glycol • washing with 0.1% Triton X-100 at +37 °C for one minute Re-equilibrate immediately with 5 column volumes of binding buffer after any of these wash steps. Media characteristics
Lentil Lectin Sepharose 4B
Ligand density
Composition
pH stability*
Mean particle size
2.5 mg/ml
Lentil lectin coupled to Sepharose 4B by CNBr method.
Short term 3–10 Long term 3–10
90 µm
*Long term refers to the pH interval over which the medium is stable over a long period of time without adverse effects on its subsequent chromatographic performance. Short term refers to the pH interval for regeneration, cleaning-in-place and sanitization procedures.
Chemical stability To avoid loss of activity of the coupled lectin, avoid solutions having a pH below 3 or above 10, buffers that contain metal chelating agents such as EDTA, or high concentrations of guanidine hydrochloride or urea. Storage Wash media and columns with 20% ethanol (use approximately 5 column volumes for packed media) and store at +4 to +8 °C.
Wheat germ lectin for binding of chitobiose core of N-linked oligosaccharides, [GlcNAc(b1,4GlcNAc)1-2 > b GlcNAc] Wheat germ lectin can be used for group specific affinity purification of glycoproteins and polysaccharides. This lectin binds N-acetylglucosamine residues and reacts strongly with the chitobiose core of N-linked oligosaccharides. It also has affinity for N-acetylneuraminic acid. Wheat germ lectin is a dimeric, carbohydrate-free protein composed of two identical subunits, each with a molecular weight of approximately Mr 20 000.
83
Purification options
Agarose Wheat Germ Lectin
Binding capacity/ml medium
Maximum operating flow
Comments
No data available
75 cm/h*
Supplied as a suspension ready for column packing.
*See Appendix 4 to convert linear flow (cm/h) to volumetric flow rate. Maximum operating flow is calculated from measurement in a packed column with a bed height of 10 cm and i.d. of 5 cm.
Performing a separation Binding buffer: 20 mM Tris-HCl, 0.5 M NaCl, pH 7.4 Elution buffer: 0.5 M N-acetylglucosamine, 20 mM Tris-HCl, 0.5 M NaCl, pH 7.4
Agarose Wheat Germ Lectin can be used with detergents, such as 1% deoxycholate or 0.5% Triton X-100. 1. Pack the column (see Appendix 3) and wash with at least 10 column volumes of binding buffer to remove preservative. 2. Equilibrate the column with 10 column volumes of binding buffer. 3. Apply the sample, using a low flow from 15 cm/h, during sample application (flow rate is the most significant factor for maximum binding). 4. Wash with 5–10 column volumes of binding buffer or until no material appears in the eluent (monitored by UV absorption at A280 nm). 5. Elute with 5 column volumes of elution buffer.
Use 0–0.5 M N-acetylglucosamine, 20 mM Tris-HCl, 0.5 M NaCl, pH 7.4 with a continuous gradient or step elution to improve resolution of complex samples containing glycoproteins with different affinities for the lectin. Elute tightly bound substances with 20 mM acetate buffer, pH 4.5 or with an alternative sugar, for example triacetylchitotriose. Higher concentrations of eluting substances may be necessary and recovery may be improved by pausing the flow for some minutes during elution. Cleaning Wash with 5–10 column volumes of 20 mM Tris-HCl, 1 M NaCl, pH 8.5 and re-equilibrate immediately with binding buffer. Low concentrations of non-ionic detergents in the Tris-HCl buffer can be used if necessary, for example 0.1% Nonidet P-40. Media characteristics
Agarose Wheat Germ Lectin
Ligand density
Composition
pH stability*
Mean particle size
1–2 mg/ml
Wheat Germ Lectin coupled to Sepharose 4B by CNBr method
Short term 4–9 Long term 4–9
90 µm
*Long term refers to the pH interval over which the medium is stable over a long period of time without adverse effects on its subsequent chromatographic performance. Short term refers to the pH interval for regeneration, cleaning-in-place and sanitization procedures.
84
Chemical stability Avoid exposure to conditions below pH 4.0 as this causes dissociation of the wheat germ lectin dimer. Storage Wash media and columns with 20% ethanol (use approximately 5 column volumes for packed media) and store at +4 to +8 °C.
Calmodulin binding proteins: ATPases, adenylate cyclases, protein kinases, phosphodiesterases, neurotransmitters Calmodulin Sepharose 4B Calmodulin is a highly conserved regulatory protein found in all eukaryotic cells. This protein is involved in many cellular processes such as glycogen metabolism, cytoskeletal control, neurotransmission, phosphate activity and control of NAD+/NADP+ ratios. Calmodulin Sepharose 4B provides a convenient method for the isolation of many of the calmodulin binding proteins involved in these pathways. Calmodulin binds proteins principally through their interactions with hydrophobic sites on its surface. These sites are exposed after a conformational change induced by the action of Ca2+ on separate Ca2+-binding sites. The binding of enzymes may be enhanced if the enzyme substrate is present and enzyme-substrate-calmodulin-Ca2+ complexes are particularly stable. Purification options
Calmodulin Sepharose 4B
Binding capacity/ml medium
Maximum operating flow
Comments
No data available
75 cm/h*
Supplied as a suspension ready for column packing.
*See Appendix 4 to convert linear flow (cm/h) to volumetric flow rate. Maximum operating flow is calculated from measurement in a packed column with a bed height of 10 cm and i.d. of 5 cm.
Performing a separation Binding buffer: 50 mM Tris-HCl, 0.05–0.2 M NaCl, 2 mM CaCl2, pH 7.5 Elution buffer: 50 mM Tris-HCl, 0.05–0.2 M NaCl, 2 mM EGTA, pH 7.5
1. Pack the column (see Appendix 3) and wash with at least 10 column volumes of binding buffer to remove preservative. 2. Equilibrate the column with 10 column volumes of binding buffer. 3. Apply the sample, using a low flow from 15 cm/h, during sample application (flow rate is the most significant factor for maximum binding). 4. Wash with 5–10 column volumes of binding buffer or until no material appears in the eluent (monitored by UV absorption at A280 nm). 5. Elute with 5 column volumes of elution buffer.
85
Remove proteases as quickly as possible from the sample as the calmodulin-binding sites on proteins are frequently very susceptible to protease action (see page 53). Remove free calmodulin from the sample by hydrophobic interaction chromatography in the presence of Ca2+ on HiTrap Phenyl FF (high sub) or by ion exchange chromatography on HiTrap Q FF. Since some non-specific ionic interactions can occur, a low salt concentration (0.05–0.20 M NaCl) is recommended to promote binding to the ligand while eliminating any non-specific binding. Use chelating agents to elute the proteins. Chelating agents strip Ca2+ from the calmodulin, reversing the conformational change that exposed the protein binding sites. Calcium ions may also be displaced by a high salt concentration, 1 M NaCl. Cleaning Alternative 1 Wash with 3 column volumes of 0.05 M Tris-HCl, 1.0 M NaCl, 2 mM EGTA, pH 7.5 and re-equilibrate immediately with 5–10 column volumes of binding buffer. Alternative 2 Wash with 3 column volumes of 0.1 M ammonium carbonate buffer, 2 mM EGTA, pH 8.6 followed by 3 column volumes of 1 M NaCl, 2 mM CaCl2. Continue washing with 3 column volumes of 0.1 M sodium acetate buffer, 2 mM CaCl2, pH 4.4 followed by 3 column volumes of binding buffer. Remove severe contamination by washing with non-ionic detergent such as 0.1% Triton X-100 at +37 °C for 1 min. Media characteristics
Calmodulin Sepharose 4B
Ligand density
Composition
pH stability*
Mean particle size
0.9–1.3 mg/ml
Bovine testicular calmodulin coupled to Sepharose 4B by the CNBr method.
Short term 4–9 Long term 4–9
90 µm
*Long term refers to the pH interval over which the medium is stable over a long period of time without adverse effects on its subsequent chromatographic performance. Short term refers to the pH interval for regeneration, cleaning-in-place and sanitization procedures.
Chemical stability Stable in all commonly used aqueous solutions. Storage Wash media and columns with 20% ethanol (use approximately 5 column volumes for packed media) and store at +4 to +8 °C.
86
Proteins and peptides with exposed amino acids: His, Cys, Trp, and/or with affinity for metal ions (also known as IMAC, immobilized metal chelate affinity chromatography) HiTrap Chelating HP, Chelating Sepharose Fast Flow, His MicroSpin Purification Module, HisTrap Kit Proteins and peptides that have an affinity for metal ions can be separated using metal chelate affinity chromatography. The metals are immobilized onto a chromatographic medium by chelation. Certain amino acids, e.g. histidine and cysteine, form complexes with the chelated metals around neutral pH (pH 6–8) and it is primarily the histidine-content of a protein which is responsible for its binding to a chelated metal. Metal chelate affinity chromatography is excellent for purifying recombinant (His)6 fusion proteins (see page 46) as well as many natural proteins. Chelating Sepharose, the medium used for metal chelate affinity chromatography, is formed by coupling a metal chelate forming ligand (iminodiacetic acid) to Sepharose. Before use the medium is loaded with a solution of divalent metal ions such as Ni2+, Zn2+, Cu2+, Ca2+, Co2+ or Fe2+. The binding reaction with the target protein is pH dependent and bound sample is, most commonly, eluted by reducing the pH and increasing the ionic strength of the buffer or by including EDTA or imidazole in the buffer. The structure of the ligand, iminodiacetic acid, is shown in Figure 47. S e p h a r o s e
CH2COOH O CH2 CH CH2 O CH2 CH CH2 N CH2COOH OH OH
Fig. 47. Partial structure of Chelating Sepharose High Performance and Chelating Sepharose Fast Flow.
Metalloproteins are not usually suitable candidates for purification by chelating chromatography since they tend to scavenge the metal ions from the column. Purification options Binding capacity
Maximum operating flow
Comments
His MicroSpin Purification Module
100 µg/column
Not applicable
Ready to use, prepacked columns, buffers and chemicals for purification of (His) 6 fusion proteins.
HiTrap Chelating HP 1 ml
12 mg/column
4 ml/min
Prepacked column, ready to use.
HiTrap Chelating HP 5 ml
60 mg/column
20 ml/min
Prepacked column, ready to use.
HisTrap Kit
12 mg/column*
4 ml/min
Ready to use, prepacked columns, buffers and chemicals for purification of (His) 6 fusion proteins for up to 12 purifications using a syringe.
Chelating Sepharose Fast Flow
12 mg/ml medium
400 cm/h**
Supplied as suspension for packing columns and scale up.
*Estimate for a (His)6 fusion protein of Mr 27 600, binding capacity varies according to specific protein. **See Appendix 4 to convert linear flow (cm/h) to volumetric flow rate. Maximum operating flow is calculated from measurement in a packed column with a bed height of 10 cm and i.d. of 5 cm.
87
Purification example A 280 nm 1.0
Sample:
200 µl egg white (10% in binding buffer filtered through a glass filter) Column: HiTrap Chelating HP, 1 ml, Cu 2+-loaded according to the instructions Binding buffer: 0.02 M sodium phosphate, 1 M NaCl, pH 7.2 Elution buffer: 0.02 M sodium phosphate, 1 M NH4Cl, pH 7.2 Flow: 0.5 ml/min Elution: 8 ml linear gradient 0–100% elution buffer
0.5
0
5
10
15
20
25 ml
Fig. 48. Purification of egg white proteins on HiTrap Chelating HP 1 ml, using the metal ion Cu2+.
Development of a separation protocol Details of a specific purification protocol are given on page 49. This protocol can be used as a base from which to develop purification methods for other proteins and peptides with affinity for metal ions, as shown in Figure 48. Reuse of purification columns depends on the nature of the sample and should only be performed with identical samples to prevent cross contamination. Selecting the metal ion The following guidelines may be used for preliminary experiments to select the metal ion that is most useful for a given separation: • Cu2+ gives strong binding and some proteins will only bind to Cu2+. Load solution equivalent to 60% of the packed column volume to avoid leakage of metal ions during sample application. Alternatively, the medium can be saturated and a short secondary uncharged column of HiTrap Chelating HP or packed Chelating Sepharose Fast Flow should be connected in series after the main column to collect excess metal ions. • Zn2+ gives a weaker binding and this can, in many cases, be exploited to achieve selective elution of a protein mixture. Load solution equivalent to 85% of the packed column volume to charge the column. • Ni2+ is commonly used for poly (His) fusion proteins. Ni2+ solution equivalent to half the column volume is usually sufficient to charge the column. • Co2+ and Ca2+ are also alternatives. Charge the column with metal ions by passing through a solution of the appropriate salt through the column, e.g. 0.1 M ZnCl2, NiSO4 or CuSO4 in distilled water. Chloride salts can be used for other metals.
88
Several methods can be used to determine when the column is charged. If a solution of metal salt in distilled water is used during charging, the eluate initially has a low pH and returns to neutral pH as the medium becomes saturated with metal ions. The progress of charging with Cu2+ is easily followed by eye (the column contents become blue). When charging a column with zinc ions, sodium carbonate can be used to detect the presence of zinc in the eluate. Wash the medium thoroughly with binding buffer after charging the column. Choice of binding buffer A neutral or slightly alkaline pH will favour binding. Tris-acetate (0.05 M), sodium phosphate (0.02–0.05 M) and Tris-HCl (0.02–0.05 M) are suitable buffers. Tris-HCl tends to reduce binding and should only be used when metal-protein affinity is fairly high. High concentrations of salt or detergents in the buffer normally have no effect on the adsorption of protein and it is good practice to maintain a high ionic strength (e.g. 0.5–1 M NaCl) to avoid unwanted ion exchange effects. Chelating agents such as EDTA or citrate should not be included, as they will strip the metal ions from the medium. Choice of elution buffers Differential elution of bound substances may be obtained using a gradient of an agent that competes for either the ligand or the target molecules. An increased concentration of imidazole (0–0.5 M), ammonium chloride (0–0.15 M), or substances such as histamine or glycine with affinity for the chelated metal can be used. The gradient is best run in the binding buffer at constant pH. Since pH governs the degree of ionization of charged groups at the binding sites, a gradient or step-wise reduction in pH can be used for non-specific elution of bound material. A range of pH 7.0–4.0 is normal, most proteins eluting between pH 6.0 and 4.2. Deforming eluents such as 8 M urea or 6 M guanidine hydrochloride can be used. Elution with EDTA (0.05 M) or other strong chelating agents will strip away metal ions and other material bound. This method does not usually resolve different proteins. If harsh elution conditions are used, it is recommended to transfer eluted fractions immediately to milder conditions (either by collecting them in neutralization buffer or by passing directly onto a desalting column for buffer exchange (see page 134). The loss of metal ions is more pronounced at lower pH. The column does not have to be stripped between consecutive purifications if the same protein is going to be purified, as shown in Figure 49.
89
Samples: Binding buffer:
2.5 ml cell extract containing expressed GST-(His)6 20 mM sodium phosphate, 0.5 M NaCl, 20 mM imidazole, pH 7.4 Elution buffer: 20 mM sodium phosphate, 0.5 M NaCl, 500 mM imidazole, pH 7.4 Flow: 2 ml/min, 312 cm/h Note: No Ni2+re-loading of the column between the runs
mg eluted GST-(His) 6
3.0 2.5 2.0 1.5 1.0
Result: Run No. 1 2 3 4 5 6 7 8 9 10
Eluted GST-(His)6, total mg 2.76 2.82 2.83 2.72 2.71 2.65 2.64 2.63 2.54 2.59
0.5 0 1
2
3
4
5
7 6 Run No.
8
9
10
Fig. 49. 10 repetitive purifications of GST-(His)6 without reloading the column with Ni2+ between the runs.
Although metal leakage is very low, the presence of any free metal in the purified product can be avoided by connecting an uncharged HiTrap Chelating HP column in series after the first column and before the protein is eluted. This column will bind any metal ions removing them from the protein as it passes through the second column. Scale of operation To increase capacity use several HiTrap Chelating HP columns (1 ml or 5 ml) in series (note that back pressure will increase) or, for even larger capacity, pack Chelating Sepharose Fast Flow into a suitable column (see Appendix 3). Cleaning Remove metal ions by washing with 5 column volumes 20 mM sodium phosphate, 0.5 M NaCl, 0.05 M EDTA, pH 7.4. Remove precipitated proteins by filling the column with 1 M NaOH and incubate for 2 hours. Wash out dissolved proteins with 5 column volumes of water and a buffer at pH 7.0 until the pH of the flow-through reaches pH 7.0. Alternatively wash with a non-ionic detergent such as 0.1% Triton X-100 at +37 °C for 1 min. Remove lipid and very hydrophobic proteins by washing with 70% ethanol, or with a saw-tooth gradient 0%–30%–0% isopropanol/water.
90
Media characteristics Composition
Metal ion capacity
Chelating Sepharose High Performance
Iminodiacetic acid coupled to Sepharose High Performance via an ether bond.
23 µmoles Cu /ml
Chelating Sepharose Fast Flow
Iminodiacetic acid coupled Sepharose Fast Flow via a spacer arm using epoxy coupling.
22–30 µmoles Zn /ml
2+
2+
pH stability*
Mean particle size
Short term 2–14 Long term 3–13
34 µm
Short term 2–14 Long term 3–13
90 µm
*Long term refers to the pH interval over which the medium is stable over a long period of time without adverse effects on its subsequent chromatographic performance. Short term refers to the pH interval for regeneration, cleaning-in-place and sanitization procedures.
Chemical stability Stable in all commonly used aqueous buffers and denaturants such as 6 M guanidine hydrochloride, 8 M urea and other chaotropic agents. Storage Wash media and columns with 20% ethanol at neutral pH (use approximately 5 column volumes for packed media) and store at +4 to +8 °C. Before long term storage, remove metal ions by washing with five column volumes 20 mM sodium phosphate, 0.5 M NaCl, 0.05 M EDTA, pH 7.4. The column must be recharged with metal ions after long term storage.
Thiol-containing substances (purification by covalent chromatography) Activated Thiol Sepharose 4B, Thiopropyl Sepharose 6B Thiol-containing substances can be isolated selectively by covalent binding to an activated thiolated matrix via thiol-disulphide exchange to form a mixed disulphide bond. After washing away unbound material, the thiol-containing substance is eluted by reducing the disulphide bond. This technique is also known as covalent chromatography. The reaction scheme is shown in Figure 50.
Sepharose
S
S
+RSH
Sepharose
S
S
R+S N
N H reducing agent Sepharose
S
S R
Sepharose
SH+RSH+R’ S
S
R’
Fig. 50. Reaction scheme purification of a thiolated substance (RSH) on Activated Thiol Sepharose 4B or Thiopropyl Sepharose 6B. The reducing agent is a low molecular weight thiol such as dithiothreitol.
91
In Activated Thiol-Sepharose 4B the hydrophilic glutathione residue acts as a spacer group thereby decreasing steric interference with exchange reactions at the terminal thiol group. The partial structure is shown in Figure 52.
S e p h a r o s e
N CH (CH2)2 C NH CH CH2 S S COOH
O
CO
N
NHCH2 COOH
Fig. 52. Partial structure of Activated Thiol Sepharose 4B.
In Thiopropyl Sepharose 6B the 2-hydroxypropyl residue acts as a hydrophilic spacer group. The partial structure of Thiopropyl Sepharose 6B is shown in Figure 53.
S e p h a r o s e
O CH2 CH CH2 S S OH
N
Fig. 53. Partial structure of Thiopropyl Sepharose 6B.
Purification options Binding capacity/ml medium
Coupling conditions
Maximum operating flow
Comments
Activated Thiol Sepharose 4B
Mercaptalbumin, 2–3 mg
pH 4–8, 3–16 hours, +4 °C - room temp.
75 cm/h*
Low capacity derivative suitable for coupling of high molecular weight substances. Supplied as dry powder, rehydration required.
Thiopropyl Sepharose 6B
Ceruloplasmin, 14 mg
pH 4–8, 3–16 hours, +4 °C - room temp.
75 cm/h*
High capacity derivative suitable for coupling of low molecular weight substances. Supplied as dry powder, rehydration required.
*See Appendix 4 to convert linear flow (cm/h) to volumetric flow rate. Maximum operating flow is calculated from measurement in a packed column with a bed height of 10 cm and i.d. of 5 cm.
Both media react spontaneously and reversibly under mild reducing conditions or in the presence of denaturing agents with substances containing thiol groups.
92
Performing a separation Binding buffer: 20 mM Tris-HCl, 0.1–0.5 M NaCl, pH 7.0. If required, include 8 M urea or 6 M guanidine HCl to ensure that the protein is denatured and all thiol groups are accessible for the reaction. 1 mM EDTA can be added to remove trace amounts of catalytic heavy metals. Elution buffer alternatives: For covalently bound proteins: 0.025 M cysteine, 50 mM Tris-HCl, pH 7–8. To minimize reduction of intramolecular disulphide bridges: 5–20 mM L-cysteine, 50 mM Tris-HCl, 1 mM EDTA, pH 8.0 or 20–50 mM 2-mercaptoethanol, 50 mM Tris-HCl, 1 mM EDTA, pH 8.0. Note: When using Thiopropyl Sepharose, 2-thiopyridyl groups must be removed after the protein has bound. Wash the column with sodium acetate 0.1 M, 2-mercaptoethanol 5 mM, pH 4.0 before beginning elution.
N.B. Degas all buffers to avoid oxidation of free thiol groups.
If the proteins to be purified contain disulphide bonds, the disulphide bridges must be reduced, for example with 2-mercaptoethanol, (5 mM). Analyse the thiol content of the sample by thiol titration to ensure that the capacity of the medium will not be exceeded. Use preliminary titration studies with 2,2'-dipyridyl disulphide to provide a guide to optimal coupling conditions. A spectrophotometer can be used to determine the release of 2-thiopyridone (absorbance coefficient = 8.08 x 103 M-1 cm-1 at 343 nm) when the sample (1–5 mg in 1–3 ml binding buffer) reacts with 2, 2'-dipyridyl disulphide. Choose the conditions to suit the specific sample. Under standard conditions at pH 7.5, a few minutes is usually enough for a complete reaction. 1. Use a desalting column to transfer pre-dissolved sample into the binding buffer (see page 134) and to remove any low molecular weight thiol compounds and reducing agents that might interfere with the coupling reaction. 2. Weigh out the required amount of powder (1 g gives about 3 ml for Activated Thiol Sepharose 4B and 4 ml for Thiopropyl Sepharose 6B). 3. Wash and re-swell on a sintered glass filter (porosity G3), using degassed, distilled water or binding buffer (200 ml/g, 15 min at room temperature) to remove additives. 4. Prepare the slurry with binding buffer in a ratio of 75% settled medium to 25% buffer. 5. Pack the column (see Appendix 3) and equilibrate with binding buffer. 6. Load the sample at a low flow (5–10 cm/h) and leave in contact with the medium for at least one hour to ensure maximum binding. 7. Wash the column with binding buffer until no material appears in the eluent (monitored by UV absorption at A 280 nm). 8. Elute the target molecules with elution buffer using a low flow (5–10 cm/h).
The coupling reaction can be monitored and, in some cases, quantified by following the appearance of 2-thiopyridone in the eluent at 343 nm during the purification. Sodium phosphate or ammonium acetate can be used as an alternative to Tris-HCl. Resolve different thiol proteins by sequential elution: 5–25 mM L-cysteine < 0.05 M glutathione < 0.02–0.05 M 2-mercaptoethanol < and 0.02–0.05 M dithiothreitol in 50 mM TrisHCl, 1 mM EDTA, pH 7–8. 93
Reactivation Pass one to two column volumes of a saturated solution (approximately 1.5 mM) of 2,2'-dipyridyl disulphide, pH 8.0 through the medium. Prepare 2,2'-dipyridyl disulphide: 1. Make a stock solution by adding 40 mg 2,2'-dipyridyl disulphide to 50 ml buffer at room temperature and stirring the suspension for several hours. 2. Filter off insoluble material. 3. Adjust the pH. The solution will be approximately 1.5 mM with respect to 2,2'-dipyridyl disulphide.
Cleaning Wash with non-ionic detergent such as 0.1% Triton X-100 at +37 °C for 1 minute. Re-equilibrate immediately with a minimum of 5 column volumes of binding buffer. Media characteristics Density of thiol groups
Composition
pH stability*
Mean particle size
Thiopropyl Sepharose 6B
25 µmoles/ml
Mixed disulphide containing 2-thiopyridyl protecting groups attached to Sepharose 6B through a chemically stable ether linkage.
Short term 2–8 Long term 2–8
90 µm
Activated Thiol Sepharose 4B
1 µmole/ml
Mixed disulphide formed between 2,2'-dipyridyl disulphide and glutathione coupled to CNBr-activated Sepharose 4B.
Short term 2–8 Long term 2–8
90 µm
*Long term refers to the pH interval over which the matrix is stable over a long period of time without adverse effects on its subsequent chromatographic performance. Short term refers to the pH interval for regeneration, cleaning-in-place and sanitization procedures. When a molecule has been coupled to the thiolated matrix, the long term and short term pH stability of the medium will be dependent upon the nature of that molecule.
Chemical stability Stable to all commonly used aqueous buffers and additives such as detergents. Avoid azides. Storage Store freeze-dried powders below +8 °C. Wash media and columns with 20% ethanol at neutral pH (use approximately 5 column volumes for packed media) and store at +4 to +8 °C. Storage under nitrogen is recommended to prevent oxidation of thiol groups by atmospheric oxygen. Avoid using sodium azide, merthiolate or phenyl mercuric salts as bacteriostatic agents. Azide ions will react with the 2,2'-dipyridyl disulphide groups, although low concentrations (0.04%) have been used.
94
Do not store the suspension for long periods in the free thiol form. Thiol groups are susceptible to oxidation by atmospheric oxygen, especially at alkaline pH. Figure 53 shows the decrease in free thiol content of Thiopropyl Sepharose 6B on storage for moderate periods at three different pH values. The thiol content of partially oxidized medium is restored by treatment with reducing agent under conditions used for removing protecting groups (see below). 40
Free thiol content µmoles/ml
pH 4 pH 6
30
20
10
pH 8
0 0
10 Storage time days
20
Fig. 53. Loss of free thiol content of reduced Thiopropyl Sepharose 6B on storage at +4 °C. The reduced medium was stored in 0.1 M sodium acetate or phosphate, 0.3 M NaCl, 1 mM EDTA at the indicated pH values.
Removal of protecting groups Activated Thiol Sepharose 4B and Thiopropyl Sepharose 6B may easily be converted into the free thiol form (i.e. reduced) by removing the 2-thiopyridyl protecting groups with a reducing agent. 1. Prepare the medium as described earlier. Gently remove excess liquid on a glass filter (porosity G3). 2. Suspend the medium in a solution containing 1% (w/v) dithiothreitol or 0.5 M 2-mercaptoethanol, 0.3 M sodium bicarbonate, 1 mM EDTA, pH 8.4. 3. Use 4 ml of solution per gram of freeze-dried powder. 4. React for 40 minutes at room temperature, mixing gently. 5. Wash the medium thoroughly with 0.5 M NaCl, 1 mM EDTA in 0.1 M acetic acid. Use a total of 400 ml of solution per gram of original freeze-dried powder. Perform the washing in several steps.
Estimate the content of free thiol groups by measuring the absorption increase at 343 nm (see above) due to the 2-thiopyridone liberated in the wash solutions. The amount of thiol groups on the medium can be estimated by reacting an excess of 2,2'-dipyridyl disulphide with the medium and measuring the liberated 2-thiopyridone at 343 nm.
95
Chapter 4 Components of an affinity medium Matrix: for ligand attachment. Matrix should be chemically and physically inert.
Spacer arm: used to improve binding between ligand and target molecule by overcoming any effects of steric hindrance.
Ligand: molecule that binds reversibly to a specific target molecule or group of target molecules.
The matrix The matrix is an inert support to which a ligand can be directly or indirectly coupled. The list below highlights many of the properties required for an efficient and effective chromatographic matrix. • Extremely low non-specific adsorption, essential since the success of affinity chromatography relies on specific interactions. • Hydroxyl groups on the sugar residues are easily derivatized for covalent attachment of a ligand, providing an ideal platform for the development of affinity media. • An open pore structure ensures high capacity binding even for large biomolecules, since the interior of the matrix is available for ligand attachment. • Good flow properties for rapid separation. • Stability under a range of experimental conditions such as high and low pH, detergents and dissociating agents. Sepharose, a bead-form of agarose (Figure 54), provides many of the these properties. Agarose
HO
O CH 2 OH O O
O HO D-galactose
Fig. 54. Partial structure of agarose.
96
Structure of agarose gel
HO O 3–6 anhydro L-galactose
O
Sepharose has been modified and developed to further enhance these excellent properties, resulting in a selection of matrices chosen to suit the particular requirements for each application (see Table 6). In affinity chromatography the particle size and porosity are designed to maximize the surface area available for coupling a ligand and binding the target molecule. A small mean particle size with high porosity increases the surface area. Increasing the degree of crosslinking of the matrix improves the chemical stability, in order to tolerate potentially harsh elution and wash conditions, and creates a rigid matrix that can withstand high flow rates. These high flow rates, although not always used during a separation, save considerable time during column equilibration and cleaning procedures. Table 6. Sepharose matrices used with Amersham Pharmacia Biotech affinity media. Form
Mean particle size
Sepharose High Performance
6% highly cross-linked agarose
34 µm
Sepharose 6 Fast Flow
6% highly cross-linked agarose
90 µm
Sepharose 4 Fast Flow
4% highly cross-linked agarose
90 µm
Sepharose CL-6B
6% cross-linked agarose
90 µm
Sepharose CL-4B
4% cross-linked agarose
90 µm
Sepharose 6B
6% agarose
90 µm
Sepharose 4B
4% agarose
90 µm
The ligand The ligand is the molecule that binds reversibly to a specific molecule or group of molecules, enabling purification by affinity chromatography. The selection of the ligand for affinity chromatography is influenced by two factors: the ligand must exhibit specific and reversible binding affinity for the target substance(s) and it must have chemically modifiable groups that allow it to be attached to the matrix without destroying binding activity. The dissociation constant (kD) for the ligand - target complex should ideally be in the range 10-4 to 10-8 M in free solution. Interactions involving dissociation constants greater than 10-4 M, for example the binding reaction between an enzyme and a weak inhibitor, are likely to be too weak for successful affinity chromatography. Conversely, if the dissociation constant is lower than approximately 10-8 M, for example the affinity between a hormone and hormone receptor, elution of the bound substance without causing inactivation is likely to be difficult. If no information on the strength of the binding complex is available, a trial and error approach must be used. Refer to Appendix 7 for further details on the kinetics involved in affinity chromatography. Altering elution methods may help to promote successful affinity chromatography when the dissociation constant is outside the useful range (see Appendix 7). It is important to consider the region of the ligand that will be used for attachment to the matrix. For example, many proteins have several equivalent groups through which coupling can take place resulting in a random orientation of the ligand on the matrix. This may reduce the number of ligand molecules that are available in the correct orientation to bind during an affinity purification.
97
If several functional groups are available, couple the ligand via the group least likely to be involved in the specific affinity interaction. A range of pre-activated matrices for attachment of the ligand through different functional groups is available (see Table 7).
Spacer arms The binding site of a target protein is often located deep within the molecule and an affinity medium prepared by coupling small ligands, such as enzyme cofactors, directly to Sepharose may exhibit low binding capacity due to steric interference i.e. the ligand is unable to access the binding site of the target molecule, as shown in Figure 55a. In these circumstances a "spacer arm" is interposed between the matrix and the ligand to facilitate effective binding. Spacer arms must be designed to maximize binding, but to avoid non-specific binding effects. Figure 55 shows the improvement that can be seen in a purification as the spacer arm creates a more effective environment for binding.
b)
a)
A 280
A 280
Efficient binding target elutes in a single peak
Inefficient binding target elutes during binding and elution
0
5
10
15
20 25 Elution volume, ml
0
5
10
15
20 25 Elution volume, ml
Fig. 55. Using spacer arms. a) Ligand attached directly to the matrix. b) Ligand attached to the matrix via a spacer arm.
The length of the spacer arm is critical. If it is too short, the arm is ineffective and the ligand fails to bind substances in the sample. If it is too long, proteins may bind nonspecifically to the spacer arm and reduce the selectivity of the separation. As a general rule, use spacer arms when coupling molecules Mr < 1 000. Spacer arms are not generally needed for larger molecules. Table 7 shows the pre-activated media with different types of spacers arms that are available from Amersham Pharmacia Biotech.
98
Ligand coupling Several methods are available to couple a ligand to a pre-activated matrix. The correct choice of coupling method depends on the ligand characteristics. The use of commercially available, pre-activated media is recommended to save time and avoid the use of the potentially hazardous reagents that are required in some cases. Table 7. Examples of pre-activated media. NHS-activated Sepharose High Performance
12-atom hydrophilic spacer arm to couple via amino groups.
NHS-activated Sepharose 4 Fast Flow
As above.
CNBr-activated Sepharose 4 Fast Flow
Coupling via primary amino groups.
EAH Sepharose 4B
10-atom spacer arms to couple via amino groups.
ECH Sepharose 4B
9-atom spacer arms to couple via carboxyl groups.
Epoxy-activated Sepharose 6B
12-atom hydrophilic spacer arm to couple through hydroxyl, amino or thiol groups.
Activated Thiol Sepharose 4B
10-atom spacer arm for reversible coupling through free thiol groups.
Thiopropyl Sepharose 6B
4-atom hydrophilic spacer arm for reversible coupling of proteins and small thiolated ligands through thiol groups. Also reacts with heavy metal ions, alkyl and aryl halides and undergoes addition reactions with compounds containing C=O, C=C and N=N bonds.
Ligand specificity For purification of specific molecules or groups of molecules, many ligands are available coupled to an appropriate matrix (see Chapter 3). Ligands can also be isolated and purified to prepare a specific affinity medium for a specific target molecule. Coupling of ligands to pre-activated matrices is described in Chapter 5.
99
Chapter 5 Designing affinity media using pre-activated matrices Earlier chapters in this handbook have covered a wide range of ligands that have been coupled to Sepharose to provide ready to use affinity media for specific groups of molecules. However, it is also possible to design new media for special purposes. When a ready to use affinity medium is not available, a medium can be designed for the purification of one or more target molecules by coupling a specific ligand onto a pre-activated chromatographic matrix. For example, antibodies, antigens, enzymes, receptors, small nucleic acids or peptides can be used as affinity ligands to enable the purification of their corresponding binding partners. There are three key steps in the design of an affinity medium: • Choosing the matrix. • Choosing the ligand and spacer arm. • Choosing the coupling method.
Choosing the matrix Sepharose provides a macroporous matrix with high chemical and physical stability and low non-specific adsorption to facilitate a high binding capacity and sample recovery and to ensure resistance to potentially harsh elution and wash conditions. The choice of a preactivated Sepharose matrix depends on the functional groups available on the ligand and whether or not a spacer arm is required. Table 8 reviews the pre-activated matrices available.
Choosing the ligand and spacer arm The ligand must selectively and reversibly interact with the target molecule(s) and must be compatible with the anticipated binding and elution conditions. The ligand must carry chemically modifiable functional groups through which it can be attached to the matrix without loss of activity (see Table 8). If possible, test the affinity of the ligand: target molecule interaction. Too low affinity will result in poor yields since the target protein may wash through or leak from the column during sample application. too high affinity will result in low yields since the target molecule may not dissociate from the ligand during elution. Use a ligand with the highest possible purity since the final purity of the target substance depends on the biospecific interaction. As discussed in Chapter 4, when using small ligands (Mr < 5 000) there is a risk of steric hindrance between the ligand and the matrix that restricts the binding of target molecules. In this case, select a pre-activated matrix with a spacer arm. For ligands with Mr > 5 000 no spacer arm is necessary.
Choosing the coupling method Ligands are coupled via reactive functional groups such as amino, carboxyl, hydroxyl, thiol and aldehyde moieties. In the absence of information on the location of binding sites in the ligand, a systematic trial and error approach should be used.
100
Couple a ligand through the least critical region of the ligand to minimize interference with the normal binding reaction. For example, an enzyme inhibitor containing amino groups can be attached to a matrix through its amino groups, provided that the specific binding activity with the enzyme is retained. However, if the amino groups are involved in the binding reaction, an alternative, non-essential, functional group must be used. Avoid using a functional group that is close to a binding site or that plays a role in the interaction between the ligand and target molecule. If a suitable functional group does not exist, consider derivatizing the ligand to add a functional group. Table 8. Chemical group on ligand
Length of spacer arm
Structure of spacer arm
Product
Proteins, peptides, amino acids 10-atom
O
O
OH
amino
N
O
HiTrap NHS-activated HP NHS-activated Sepharose 4 Fast Flow
O N O
None
–
CNBr-activated Sepharose 4B CNBr-activated Sepharose 4 Fast Flow O
OH N
O
OH
10-atom
ECH Sepharose 4B
OH N
O
carboxyl
NH2
11-atom
EAH Sepharose 4B
OH S
O
thiol
4-atom N
N
10-atom
O
OH
12-atom
O
S S
Activated Thiol Sepharose 4B
N
N
HO
O
O
O
Sugars
O
O
Epoxy-activated Sepharose 6B
O
Epoxy-activated Sepharose 6B
OH O
O
O
12-atom
O
O
OH N
O
amino
Thiopropyl Sepharose 6B
N
OH
O
hydroxyl
S
O N
10-atom
HiTrap NHS-activated HP
O
O
OH
10-atom
N
O
OH
ECH Sepharose 4B
OH
12-atom
O
O
O
O
Epoxy-activated Sepharose 6B
OH
carboxyl
11-atom
N
O
NH2
EAH Sepharose 4B
Polynucleotides amino
None
mercurated base
4-atom
CNBr-activated Sepharose 4B CNBr-activated Sepharose 4 Fast Flow OH O
S
S N
Thiopropyl Sepharose 6B
Coenzymes, cofactors, antibiotics, steroids amino, carboxyl, thiol or hydroxyl
use matrix with spacer arm (see above)
101
Coupling the ligand 1. Prepare the ligand solution in coupling buffer, either by dissolving the ligand in coupling buffer or exchanging the solubilized ligand into the coupling buffer using a desalting column. 2. Prepare the pre-activated matrix according to the manufacturer's instructions. 3. Mix the ligand solution and the matrix in the coupling buffer until the coupling reaction is completed. 4. Block any remaining active groups. 5. Wash the coupled matrix alternately at high and low pH to remove excess ligand and reaction by-products. 6. Equilibrate in binding buffer or transfer to storage solution.
It is not usually necessary to couple a large amount of ligand to produce an efficient affinity medium. After coupling, wash the medium thoroughly using buffers of alternating low and high pH to remove non-covalently bound ligand. A high concentration of coupled ligand is likely to have adverse effects on affinity chromatography. The binding efficiency of the medium may be reduced due to steric hindrance between the active sites (particularly important when large molecules such as antibodies, antigens and enzymes interact with small ligands).Target substances may become more strongly bound to the ligand making elution difficult. The extent of nonspecific binding increases at very high ligand concentrations thus reducing the selectivity of the medium. Remember that the useful capacity of an affinity medium may be significantly affected by flow rate. For applications that require operating at high pH, the amide bond formed when using NHS-activated Sepharose is stable up to pH 13. Figure 56 shows the effect of ligand concentration on the final amount of ligand coupled to a matrix. Chymotrypsinogen
15
Chymotrypsin
Protein coupled mg
10
5
0 0
10
20 Protein added mg
30
40
Fig. 56. Effect of protein concentration on amount of protein coupled. Protein was coupled to 2 ml CNBr-activated Sepharose 4B in NaHCO3, NaCl solution, pH 8.
102
Table 9 summarizes recommended ligand concentrations according to the experimental conditions. Table 9. Experimental condition
Recommended concentration for coupling
Readily available ligands
10–100 fold molar excess of ligand over available groups
Small ligands
1–20 µmoles/ml medium (typically 2 µmoles/ml medium)
Protein ligands
5–10 mg protein/ml medium
Antibodies
5 mg protein/ml medium
Very low affinity systems
Maximum possible ligand concentration to increase the binding
For certain pre-activated matrices agents are used to block any activated groups that remain on the matrix after ligand coupling. These blocking agents such as ethanolamine and glycine may introduce a small number of charged groups into the matrix. The effect of these charges is overcome by the use of a relatively high salt concentration (0.5 M NaCl) in the binding buffer for affinity purification. A wash cycle of low and high pH is essential to ensure that no free ligand remains ionically bound to the coupled ligand. This wash cycle does not cause loss of covalently bound ligand.
Binding capacity, ligand density and coupling efficiency Testing the binding capacity of the medium after coupling will give an indication of the success of the coupling procedure and establish the usefulness of the new affinity medium. Several different methods can be used to determine the ligand density (µmoles/ml medium) and coupling efficiency. • The fastest and easiest, but least accurate, way to quantify the free ligand in solution is by spectrophotometry. Measure the ligand concentration before coupling and compare this with the concentration of the unbound ligand after coupling. The difference is the amount that is coupled to the matrix. • Spectroscopic methods or scintillation counting can also be used if the ligand has been suitably pre-labelled. The coupled ligand can be quantified by direct spectroscopy of the affinity medium suspended in a solution with the same refractive index, such as 50% glycerol or ethylene glycol. By-products of the coupling reaction, such as N-hydroxysuccinimide in the case of NHS-activated matrices, can be quantified by spectroscopy. • The medium can be titrated to determine ligand concentration. The titrant must be relevant to the ligand. • The most accurate method to determine ligand concentration is direct amino acid analysis or determination of characteristic elements. Note that these are destructive techniques. If the binding capacity for the target is insufficient there are several ways to try to increase the coupling efficiency: • Ensure that the ligand is of high purity. There may be contaminants present that are preferentially coupled.
103
• Increase the ligand concentration to increase the ligand density on the matrix, but avoid overloading the matrix as this may cause steric hindrance and so reduce the binding capacity again. • Modify reaction conditions such as pH, temperature, buffers or contact time. Most preactivated matrices are supplied with details of the preferred conditions for a coupling reaction that can be used as a basis for further optimization.
Binding and elution conditions Binding and elution conditions will depend on the nature of the interaction between the ligand and target. As for any affinity purification, the general guidelines outlined in Chapter 2 can be applied during development. For the first run, perform a blank run to ensure that any loosely bound ligand is removed. Immunospecific interactions can be strong and sometimes difficult to reverse. The specific nature of the interaction determines the elution conditions. Always check the reversibility of the interaction before attaching a ligand to an affinity matrix. If standard elution buffers do not reverse the interaction, try alternative elution buffers such as: • Low pH (below pH 2.5). • High pH (up to pH 11). • Substances that reduce the polarity of the buffer may facilitate elution without affecting protein activity such as dioxane (up to 10%), ethylene glycol (up to 50%). The following protocol can be used as a guideline for a preliminary separation: 1. Prepare the column (blank run) a. Wash with 2 column volumes binding buffer. b. Wash with 3 column volumes elution buffer. 2. Equilibrate with 10 column volumes of binding buffer. 3. Apply sample. The optimal flow rate is dependent on the binding constant of the ligand, but a recommended flow rate range is, for example, 0.5–1 ml/ min on a HiTrap NHS-activated HP 1 ml column. 4. Wash with 5–10 column volumes of binding buffer, or until no material appears in the eluent, as monitored by absorption at A280 nm. 5. Elute with 1–3 column volumes of elution buffer (larger volumes may be necessary). 6. If required purified fractions can be desalted and transferred into the buffer of choice using prepacked desalting columns (see page 134). 7. Re-equilibrate the column immediately by washing with 5–10 column volumes of binding buffer.
104
Coupling through the primary amine of a ligand HiTrap NHS-activated HP, NHS-activated Sepharose 4 Fast Flow NHS-activated Sepharose is designed for the covalent coupling of ligands (often antigens or antibodies) containing primary amino groups (the most common form of attachment) and is the first choice for the preparation of immunospecific media. The matrix is based on highly cross-linked agarose beads with 10-atom spacer arms (6-aminohexanoic acid) attached by epichlorohydrin and activated by N-hydroxysuccinimide (Figure 57). Non-specific adsorption of proteins (which can reduce binding capacity of the target protein) is negligible due to the excellent hydrophilic properties of the base matrix. The matrix is stable at high pH to allow stringent washing procedures (subject to the pH stability of the coupled ligand).
S e p h a r o s e
O O CH2 CH CH2 NH (CH2)5 CO O N OH O
Fig. 57. Partial structure of NHS-activated Sepharose bearing activated spacer arms.
Ligands containing amino groups couple rapidly and spontaneously by nucleophilic attack at the ester linkage to give a very stable amide linkage (Figure 58). The amide bond is stable up to pH 13 making NHS-activated Sepharose suitable for applications that require conditions at high pH. S e p h a r o s e
S e p h a r o s e
O O CH2 CH CH2 NH (CH2)5 CO O N
+ R NH2
OH O
O O CH2 CH CH2 NH (CH2)5 C NH R + HO N OH
O O
Fig. 58. Coupling a ligand to NHS-activated Sepharose.
105
Options Product
Spacer arm
Coupling conditions
Maximum operating flow
Comments
HiTrap NHS-activated HP
10-atom
pH 6.5–9, 15–30 min., +4 °C - room temp.
4 ml/min (1 ml column) 20 ml/min (5 ml column)
Pre-activated medium for coupling via primary amine group of a ligand. Prepacked 1 ml and 5 ml columns.
NHS-activated Sepharose 4 Fast Flow
10-atom
pH 6–9, 2–16 hours, +4 °C - room temp.
300 cm/h*
Supplied as a suspension ready for column packing.
*See Appendix 4 to convert linear flow (cm/h) to volumetric flow rate. Maximum operating flow is calculated from measurement in a packed column with a bed height of 10 cm and i.d. of 5 cm.
Figure 59 shows that over 30 mg IgG can be coupled to a 1 ml HiTrap NHS-activated HP column. The coupling process takes less than 15 minutes. The affinity medium is then ready to use for antigen purification. Protein coupled (mg) 40
30
20
10
20
60
40
80
100
Protein added (mg/1 ml column)
Fig. 59. Ligand coupling to HiTrap NHS-activated HP.
Preparation of HiTrap NHS-activated HP The protocol below describes the preparation of a prepacked HiTrap NHS-activated HP column and is generally applicable to NHS-activated Sepharose media. A general column packing procedure is described in Appendix 3. The activated matrix is supplied in 100% isopropanol to preserve the stability before coupling. Do not replace the isopropanol until it is time to couple the ligand. Buffer preparation Acidification solution: 1 mM HCl (kept on ice) Coupling buffer:
0.2 M NaHCO3, 0.5 M NaCl, pH 8.3
Blocking buffer:
0.5 M ethanolamine, 0.5 M NaCl, pH 8.3
Wash buffer:
0.1 M acetate, 0.5 M NaCl, pH 4.0
Coupling within pH range 6.5–9, maximum yield is achieved at around pH 8.
106
Ligand and column preparation 1. Dissolve the ligand in the coupling buffer to a final concentration of 0.5–10 mg/ml (for protein ligands) or perform a buffer exchange using a desalting column (see page 134). The optimal concentration depends on the ligand. Dissolve the ligand in one column volume of buffer. 2. Remove the top cap from the column and apply a drop of ice-cold 1 mM HCl to the top of the column to avoid air bubbles. 3. Connect the top of the column to the syringe or pump. 4. Remove the twist-off end.
Ligand coupling 1. Wash out the isopropanol with 3 x 2 column volumes of ice-cold 1 mM HCl. 2. Inject one column volume of ligand solution onto the column. 3. Seal the column. Leave for 15–30 minutes at +25 °C (or 4 hours at +4 °C).
Re-circulate the solution if larger volumes of ligand solution are used. For example, when using a syringe, connect a second syringe to the outlet of the column and gently pump the solution back and forth for 15–30 minutes or, if using a peristaltic pump, circulate the ligand solution through the column. Do not use excessive flow rates (maximum recommended flow rates are 1 ml/min (equivalent to approximately 30 drops/min when using a syringe) with HiTrap 1 ml and 5 ml/min (equivalent to approximately 120 drops/min when using a syringe) with HiTrap 5 ml). The column contents can be irreversibly compressed. Measure the efficiency of protein ligand by comparing the A280 values of the ligand solution before and after coupling. Note that the N-hydroxy-succinimide, released during the coupling procedure, absorbs strongly at 280 nm and should be removed from the used coupling solution before measuring the concentration of the remaining ligand. Use a small desalting column (see page 134) to remove N-hydroxy-succinimide from protein ligands. Alternative methods for the measurement of coupling efficiency are described on page 103 and in the HiTrap NHS-activated HP instructions. Washing and deactivation This procedure deactivates any excess active groups that have not coupled to the ligand and washes out non-specifically bound ligands. 1. Inject 3 x 2 column volumes of blocking buffer. 2. Inject 3 x 2 column volumes of wash buffer. 3. Inject 3 x 2 column volumes of blocking buffer. 4. Let the column stand for 15–30 min. 5. Inject 3 x 2 column volumes of wash buffer. 6. Inject 3 x 2 column volumes of blocking buffer. 7. Inject 3 x 2 column volumes of wash buffer. 8. Inject 2–5 column volumes of a buffer with neutral pH. The column is now ready for use.
107
Media characteristics Product
Ligand density
Composition
pH stability*
Mean particle size
HiTrap NHS-activated HP
10 µmoles/ml
6-aminohexanoic acid linked by epoxy coupling to highly cross linked agarose, terminal carboxyl group esterified with NHS.
Short term 3–12 Long term 3–12
34 µm
NHS-activated Sepharose 4 Fast Flow
16–23 µmoles/ml
As above
Short term 3–13 Long term 3–13
90 µm
*Long term refers to the pH interval over which the matrix is stable over a long period of time without adverse effects on its subsequent chromatographic performance. Short term refers to the pH interval for regeneration, cleaning-in-place and sanitization procedures. Stability data refers to the coupled medium provided that the ligand can withstand the pH.
Storage Store the column in a solution that maintains the stability of the ligand and contains a bacteriostatic agent, for example PBS, 0.05% NaN3, pH 7.2. pH stability of the media when coupled to the chosen ligand will depend upon the stability of the ligand itself. Sodium azide can interfere with many coupling methods and some biological assays. It can be removed by using a desalting column (see page 134).
CNBr-activated Sepharose CNBr-activated Sepharose offers a well-established option for the attachment of larger ligands and as an alternative to NHS-activated Sepharose. Cyanogen bromide reacts with hydroxyl groups on Sepharose to form reactive cyanate ester groups. Proteins, peptides, amino acids or nucleic acids can be coupled to CNBr-activated Sepharose, under mild conditions, via primary amino groups or similar nucleophilic groups. The activated groups react with primary amino groups on the ligand to form isourea linkages (Figure 60). The coupling reaction is spontaneous and requires no special chemicals or equipment. The resulting multi-point attachment ensures that the ligand does not hydrolyze from the matrix. The activation procedure also cross-links Sepharose and thus enhances its chemical stability, offering considerable flexibility in the choice of elution conditions. NH OH
HO
Sepharose
CNBr
O C NHR
RNH2
Sepharose
isourea
Fig. 60. Activation by cyanogen bromide and coupling to the activated matrix.
108
OH
Options Product
Spacer arm
Coupling conditions
Maximum operating flow
Comments
CNBr-activated Sepharose 4 Fast Flow
None
pH 7–9, 2–16 hours, +4 °C - room temp.
400 cm/h*
Supplied as a freezedried powder.
CNBr-activated Sepharose 4B
None
pH 8–10, 2–16 hours, +4 °C - room temp.
75 cm/h*
Supplied as a freezedried powder.
*See Appendix 4 to convert linear flow (cm/h) to volumetric flow rate. Maximum operating flow is calculated from measurement in a packed column with a bed height of 10 cm and i.d. of 5 cm.
There are many examples in the literature of the use of CNBr-activated Sepharose. Figure 61 shows the separation of a native outer envelope glycoprotein, gp120, from HIV-1 infected T-cells. Galanthus nivalis agglutinin (GNA), a lectin from the snowdrop bulb, was coupled to CNBr-activated Sepharose 4 Fast Flow to create a suitable affinity medium. A 280 nm 0.5
native gp120
0 24 hours
40 min
Time
Fig. 61. Separation of native gp120 protein on GNA coupled to CNBr-activated Sepharose 4 Fast Flow. From Gilljam, G. et al., Purification of native gp120 from HIV-1 infected T-cells. Poster presented at Recovery of Biological Products VII, Sept. 25-30, 1994, San Diego, CA, USA. Further details are available in the CNBr-activated Sepharose 4 Fast Flow datafile, from Amersham Pharmacia Biotech.
Buffer preparation Acidification solution: 1 mM HCl (kept on ice) Coupling buffer:
0.2 M NaHCO3, 0.5 M NaCl, pH 8.3
Blocking buffer:
1 M ethanolamine or 0.2 M glycine, pH 8.0
Wash buffer:
0.1 M acetate, 0.5 M NaCl, pH 4
Preparation of CNBr-activated Sepharose 4 Fast Flow and CNBr-activated Sepharose 4B 1. Suspend the required amount of freeze-dried powder in ice-cold 1 mM HCl (HCl preserves the activity of the reactive groups that hydrolyze at high pH). 2. Wash for 15 min. on a sintered glass filter (porosity G3), using a total of 200 ml 1 mM HCl per gram dry powder, added and sucked off in several aliquots. The final aliquot of 1 mM HCl is sucked off until cracks appear in the cake. 3. Transfer the matrix immediately to the ligand solution.
109
Preparation of the matrix should be completed without delay since reactive groups on the matrix hydrolyze at the coupling pH. Do not use buffers containing amino groups at this stage since they will couple to the matrix. Ligand preparation Dissolve the ligand in the coupling buffer to a final concentration of 0.5–10 mg/ml (for protein ligands) or perform a buffer exchange using a desalting column (see page 134). The optimal concentration depends on the ligand. Use a matrix:buffer ratio of 1:2.
Ligand coupling 1. Mix the ligand solution with suspension in an end-over-end or similar mixer for 2 hours at room temperature or overnight at +4 °C. A matrix: buffer ratio of 1:2 gives a suitable suspension for coupling. 2. Transfer the medium to blocking buffer for 16 hours at +4 °C or 2 hours at room temperature to block any remaining active groups. Alternatively, leave the medium for 2 hours in Tris-HCl buffer, pH 8. 3. Remove excess ligand and blocking agent by alternately washing with coupling buffer followed by wash buffer. Repeat four or five times. A general column packing procedure is described in Appendix 3.
Do not use magnetic stirrers as they may disrupt the Sepharose matrix. The coupling reaction proceeds most efficiently when the amino groups on the ligand are predominantly in the unprotonated form. A buffer at pH 8.3 is most frequently used for coupling proteins. The high salt content of the coupling buffer minimizes protein-protein adsorption caused by the polyelectrolyte nature of proteins. Coupling of a-chymotrypsinogen by the method described here typically yields about 90% coupled protein. It may be necessary to reduce the number of coupling groups on the matrix to preserve the structure of binding sites in a labile molecule, or to facilitate elution when steric effects reduce the binding efficiency of a large ligand. Reduced coupling activity may be achieved by controlled hydrolysis of the activated matrix before coupling, or by coupling at a lower pH. Pre-hydrolysis reduces the number of active groups available for coupling and reduces the number of points of attachment between the protein and matrix as well as the amount of protein coupled. In this way a higher binding activity of the product may be obtained. At pH 3, coupling activity is lost only slowly, whereas at pH 8.3 activity is lost fairly rapidly. A large molecule is coupled at about half as many points after 4 hours pre-hydrolysis at pH 8.3 (Figure 62).
110
100
Coupled substance, %
80
60
a-chymotrypsinogen A
40 glyleu B
20
A B
0 0
3 1 2 Time of pre-hydrolysis, hours
4
24
Fig. 62. Variation of coupling activity with time of pre-hydrolysis at pH 8.3. CNBr-activated Sepharose 4B was washed at pH 3 and transferred to 0.1 M NaHCO3, pH 8.3 for pre-hydrolysis. Samples were removed after different times and tested for coupling activity towards a-chymotrypsinogen (A) and glycyl-leucine (B).
Coupling at low pH is less efficient, but may be advantageous if the ligand loses biological activity when it is fixed too firmly, e.g. by multi-point attachment, or because of steric hindrance between binding sites which occurs when a large amount of high molecular weight ligand is coupled. Use a buffer of approximately pH 6. IgG is often coupled at a slightly higher pH, for example in 0.2–0.25 M NaHCO3, 0.5 M NaCl, pH 8.5–9.0. Media characteristics Product
Composition
Binding capacity per ml medium
pH stability*
Mean particle size
CNBr-activated Sepharose 4 Fast Flow
Cyanogen bromide reacts with hydroxyl groups on Sepharose to give a reactive product for coupling ligands via primary amino groups or similar nucleophilic groups.
a-chymotrypsinogen, 13–26 mg
Short term 3–11 Long term 3–11
90 µm
a-chymotrypsinogen, 25–60 mg
Short term 2–11 Long term 2–11
CNBr-activated Sepharose 4B
*Long term refers to the pH interval over which the matrix is stable over a long period of time without adverse effects on its subsequent chromatographic performance. Short term refers to the pH interval for regeneration, cleaning-in-place and sanitization procedures. Stability data refers to the coupled medium provided that the ligand can withstand the pH.
Storage Store the freeze-dried powder below +8 °C in dry conditions. Store the column in a solution that maintains the stability of the ligand and contains a bacteriostatic agent, for example, PBS, 0.05% NaN3, pH 7.2 or 20% ethanol in a suitable buffer. The pH stability of the medium when coupled to the chosen ligand will depend upon the stability of the ligand itself.
111
Immunoaffinity chromatography Immunoaffinity chromatography utilizes antigens or antibodies as ligands (sometimes referred to as adsorbents, immunoadsorbents or immunosorbents) to create highly selective media for affinity purification. Antibodies are extremely useful as ligands for antigen purification, especially when the substance to be purified has no other apparent complementary ligand. Similarly, highly purified antigens or anti-antibodies can provide highly specific ligands for antibody purification. The Antibody Handbook from Amersham Pharmacia Biotech covers the purification and application of antibodies in greater detail. Immunoaffinity media are created by coupling the ligand (a pure antigen, an antibody or an anti-antibody) to a suitable matrix. The simplest coupling is via the primary amine group of the ligand, using NHS-activated Sepharose or CNBr-activated Sepharose. Figure 63 illustrates a typical immunoaffinity purification.
Mr 97 000 66 000 45 000
A 280 nm Flow through material
Sample:
50 ml sheep anti-mouse Fc serum, filtered 0.45 µm Column: HiTrap NHS-activated HP 1 ml. Mouse IgG, (10 mg, 3.2 ml) was coupled in 0.2 M NaHCO 3, 0.5 M NaCl, pH 8.3, room temp., recycled with a peristaltic pump for 1 h. The coupling yield was 95% (9.5 mg). Flow: 1.0 ml/min Binding buffer: 75 mM Tris-HCl, pH 8.0 Elution buffer: 100 mM glycine-HCl, 0.5 M NaCl, pH 2.7 Electrophoresis: SDS-PAGE. PhastSystem. PhastGel Gradient 8–25 1 µl sample, Coomassie Blue stained
30 000 20 100
Binding Elution buffer buffer
2.0
14 000 1
2 Mr 97 000 66 000 45 000
1.0
30 000 20 100 14 000 1
20
40
60
80
100 ml
2
Lane 1. Eluted material, non-reduced Lane 2. Low Molecular Weight Calibration Kit, reduced
Fig. 63. Purification of anti-mouse Fc-IgG from sheep antiserum.
If there is no primary amine available (for example, this group may be required for the specific interaction), then pre-activated media for ligand attachment via carboxyl, thiol or hydroxyl groups can be considered. The guidelines given in Chapter 2, Affinity chromatography in practice, and Chapter 3, Purification of immunoglobulins, are applicable to immunoaffinity chromatography. Optimal binding and elution conditions will be different for each immunospecific reaction according to the strength of interaction and the stability of the target proteins.
112
Coupling small ligands through amino or carboxyl groups via a spacer arm EAH Sepharose 4B and ECH Sepharose 4B The partial structures of EAH Sepharose 4B and ECH Sepharose 4B are shown in Figure 64.
S e p h a r o s e
S e p h a r o s e
OH O CH2 CH CH2 NH(CH2)5 COOH
ECH Sepharose
OH O CH2 CH CH2 NH(CH2)6 NH2
EAH Sepharose
Fig. 64. Partial structures of ECH Sepharose 4B and EAH Sepharose 4B.
Ligands are coupled in a simple one-step procedure in the presence of a coupling reagent, carbodiimide. The carbodiimides may be regarded as anhydrides of urea. The N,N' disubstituted carbodiimides promote condensation between a free amino and a free carboxyl group to form a peptide link by acid-catalyzed removal of water. Thus EAH Sepharose 4B can be coupled with carboxyl-containing ligands and ECH Sepharose 4B can be coupled with ligands containing amino groups. The carbodiimide yields an isourea upon hydration. The coupling reaction is shown in Figure 65. O R COOH + R1N C NR2
NHR1
R C O C NHR2
Carbodiimide O
O
R C O C NHR1 R1NH C NHR2 + R3NH2
O R C NHR3+
NHR2 Active ester
Urea
Fig. 65. Carbodiimide coupling reaction.
Options Product
Spacer arm
Substitution per ml matrix
Coupling conditions
Maximum operating flow
Comments
EAH Sepharose 4B
10-atom
7–11 µmoles amino groups
pH 4.5, 1.5–24 hours, +4 °C - room temp.
75 cm/h*
Couple ligands containing free carboxyl groups. Supplied as a suspension ready for use.
ECH Sepharose 4B
9-atom
12–16 µmoles carboxyl groups
pH 4.5, 1.5–24 hours, +4 °C - room temp.
75 cm/h*
Couple ligands containing free amino groups. Supplied as a suspension ready for use.
*See Appendix 4 to convert linear flow (cm/h) to volumetric flow rate. Maximum operating flow is calculated from measurement in a packed column with a bed height of 10 cm and i.d. of 5 cm.
113
Preparation of coupling reagent Use a water-soluble carbodiimide such as N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) or N-cyclohexyl-N'-2-(4'-methyl-morpholinium) ethyl carbodiimide p-toluene sulphonate (CMC). These two carbodiimides have been used in a variety of experimental conditions and at a wide range of concentrations (Table 10). EDC often gives better coupling yields than CMC. Table 10. Examples of conditions used during coupling via carbodiimides. Coupled ligand
Carbodiimide
Conc. of carbodiimide mg/ml
pH
Reaction time
Methotrexate
EDC
18
6.4
1.5 h
UDP-glucuronic acid
EDC
32
4.8
24 h
p-amino-benzamidine
CMC
2
4.75
5h
Folic acid
EDC
5
6
2h
Mannosylamine
EDC
19
4.5–6.0
24 h
Use a concentration of carbodiimide greater than the stoichiometric concentration, usually 10–100 times greater than the concentration of spacer groups. The coupling reaction is normally performed in distilled water adjusted to pH 4.5–6.0 to promote the acid-catalyzed condensation reaction. Blocking agents are not usually required after the coupling reaction if excess ligand has been used. Always use freshly prepared carbodiimides. Coupling buffer: Dissolve the carbodiimide in water and adjust to pH 4.5 Wash buffer:
0.1 M acetate, 0.5 M NaCl, pH 4
Avoid the presence of amino, phosphate or carboxyl groups, as these will compete with the coupling reaction. Preparation of EAH and ECH Sepharose 4B Wash the required amount of matrix on a sintered glass filter (porosity G3) with distilled water adjusted to pH 4.5 with HCl, followed by 0.5 M NaCl (80 ml in aliquots/ml sedimented matrix). Ligand preparation Dissolve the ligand and adjust to pH 4.5. The optimal concentration depends on the ligand. Organic solvents can be used to dissolve the ligand, if necessary. If using a mixture of organic solvent and water, adjust the pH of the water to pH 4.5 before mixing it with the organic solvent. Solvents such as dioxane (up to 50%), ethylene glycol (up to 50%), ethanol, methanol and acetone have been used. If organic solvents have been used, use pH paper to measure pH since solvents may damage pH electrodes.
114
Ligand coupling 1. Add the ligand solution followed by the carbodiimide solution to the matrix suspension and leave on an endover-end or similar mixer. Use a matrix: ligand solution ratio of 1:2 to produce a suspension that is suitable for coupling. Typically the reaction takes place overnight either at +4 °C or room temperature. 2. Adjust the pH of the reaction mixture during the first hour (pH will decrease) by adding 0.1 M sodium hydroxide. 3. Wash at pH 8 and pH 4 to remove excess reagents and reaction by-products.
If a mixture of aqueous solution and organic solvent has been used, use this mixture to wash the final product as in Step 3. After Step 3 wash in distilled water, followed by the binding buffer to be used for the affinity purification. Do not use magnetic stirrers as they may disrupt the Sepharose matrix. Media characteristics Product
Composition
pH stability*
Mean particle size
EAH Sepharose 4B
Covalent linkage of 1,6-diamino-hexane by epoxy coupling creates a stable, uncharged ether link between a 10-atom spacer arm and Sepharose 4B.
Short term 3–14 Long term 3–14
90 µm
ECH Sepharose 4B
Covalent linkage of 6-aminohexanoic acid by epoxy coupling creates a stable, uncharged ether link between the 9-atom spacer arm and Sepharose 4B.
Short term 3–14 Long term 3–14
90 µm
*Long term refers to the pH interval over which the matrix is stable over a long period of time without adverse effects on its subsequent chromatographic performance. Short term refers to the pH interval for regeneration, cleaning-in-place and sanitization procedures. Stability data refers to the coupled medium provided that the ligand can withstand the pH.
Storage Store pre-activated matrices at +4 to +8 °C in 20% ethanol. Store the column in a solution that maintains the stability of the ligand and contains a bacteriostatic agent, for example, PBS, 0.05% NaN3, pH 7.2 or 20% ethanol in a suitable buffer. The pH stability of the media when coupled to a ligand will depend upon the stability of the ligand. Performing a separation See page 104 for a preliminary separation protocol and Chapter 2 for general guidelines.
115
Coupling through hydroxy, amino or thiol groups via a 12-carbon spacer arm Epoxy-activated Sepharose 6B Epoxy-activated Sepharose 6B is used for coupling ligands that contain hydroxyl, amino or thiol groups. Because of the long hydrophilic spacer arm, it is particularly useful for coupling small ligands such as choline, ethanolamine and sugars. The pre-activated matrix is formed by reacting Sepharose 6B with the bis oxirane, 1,4 bis-(2,3-epoxypropoxy-)butane. The partial structure is shown in Figure 66. S e p h a r o s e
O CH2 CH CH2 O (CH2)4 O CH2 CH CH2 O
OH
Fig. 66. Partial structure of Epoxy-activated Sepharose 6B.
A stable ether linkage is formed between the hydrophilic spacer and the matrix. Free oxirane groups couple via stable ether bonds with hydroxyl-containing molecules such as sugars, via alkylamine linkages with ligands containing amino groups, and via thioether linkages with ligands containing thiol groups. Options Product
Spacer arm
Substitution per ml matrix
Coupling conditions
Maximum operating flow
Comments
Epoxy-activated Sepharose 6B
12-atom
19–40 µmoles epoxy groups
pH 9–13, 16 hours several days, +20 - +40 °C
75 cm/h*
Supplied as a freeze-dried powder.
*See Appendix 4 to convert linear flow (cm/h) to volumetric flow rate. Maximum operating flow is calculated from measurement in a packed column with a bed height of 10 cm and i.d. of 5 cm.
Purification example A 280 nm
Fucose-specific lectin
Elution volume
Fig. 67. Chromatography of a crude extract of Ulex europaeus on fucose coupled to Epoxy-activated Sepharose 6B, column volume 11 ml. Extract was applied in 0.9% NaCl. Fucose-specific lectin was eluted with 5 ml fucose (50 mg/ml).
116
Alternative coupling solutions: Distilled water or aqueous buffers with sugars and carbohydrates are preferable. Carbonate, borate or phosphate buffers can be used. Sodium hydroxide may be used for solutions of high pH. Organic solvents such as dimethylformamide (up to 50%) and dioxane (up to 50%) may be used to dissolve the ligand. The same concentration of organic solvent should be included in the coupling solution.
Coupling procedure 1. Suspend the required amount of freeze-dried powder in distilled water (1 g freeze-dried powder gives about 3.0 ml final matrix volume). 2. Wash immediately for 1 hour on a sintered glass filter (porosity G3), using approximately 200 ml distilled water per gram freeze-dried powder, added in several aliquots. 3. Dissolve the ligand in the coupling buffer to a final concentration of 0.5–10 mg/ml (for protein ligands) or transfer solubilized ligands into the coupling buffer using a desalting column (see page 134). Adjust the pH of the aqueous phase. 4. Use a matrix:buffer ratio of 1:2, mix the matrix suspension with the ligand solution for 16 h at +25 to +40 °C in a shaking water bath. 5. Block remaining excess groups with 1 M ethanolamine for at least 4 h or overnight, at +40 to +50 °C. 6. Wash away excess ligand with coupling solution followed by distilled water, 0.1 M NaHCO 3, 0.5 M NaCl, pH 8.0 and 0.1 M NaCl, 0.1 M acetate, pH 4.0.
If organic solvents have been used, use pH paper to measure pH since solvents may damage pH electrodes. Using the higher temperatures can decrease coupling times. Do not use Tris, glycine or other nucleophilic compounds as these will couple to the oxirane groups. Do not use magnetic stirrers as they may disrupt the Sepharose matrix.
117
Coupled ligand µmoles/g conjugate
100
75
20 mg/ml 50
25
5 mg/ml
0 10
9
12
11
13
14 pH
Fig. 68. pH dependence of coupling N-acetyl-D-galactosamine to Epoxy-activated Sepharose 6B. Carbonate/bicarbonate buffers were used in the range pH 9–11, sodium hydroxide solution in the range pH 12–14. Ligand concentrations: 5 mg/ml and 20 mg/ml.
When a ligand contains more than one kind of group (thiol, amino and hydroxyl), the coupling pH will determine which of these groups is coupled preferentially. As a general rule, the order of coupling is e-amino > thiol > a-amino > hydroxyl although the exact result will depend on the detailed structure of the ligand. The time of reaction depends greatly on the pH of the coupling solution, properties of the ligand and the coupling temperature. The stability of the ligand and the carbohydrate chains of the matrix limit the maximum pH that can be used. Coupling is performed in the range pH 9–13 as shown in Figure 68 and the efficiency of coupling is pH and temperature dependent (Figure 69). pH 11.0, +45 °C
pH 10.5, +40 °C
Coupled glycylleucine %
100
75
50
25
0 0
8
16
24
32
40
Fig. 69. Efficiency of coupling glycyl-leucine to Epoxy-activated Sepharose 6B.
118
48 Time hours
Media characteristics Product
Composition
pH stability*
Mean particle size
Epoxy-activated Sepharose 6B
Sepharose 6B reacts with 1,4 bis-(2,3 epoxypropoxy-) butane to form a stable ether linkage.
Short term 2–14 Long term 2–14
90 µm
*Long term refers to the pH interval over which the matrix is stable over a long period of time without adverse effects on its subsequent chromatographic performance. Short term refers to the pH interval for regeneration, cleaning-in-place and sanitization procedures. Stability data refers to the coupled medium provided that the ligand can withstand the pH.
Storage Store the freeze-dried powder dry below +8 °C. Store the column in a solution that maintains the stability of the ligand and contains a bacteriostatic agent, for example, PBS, 0.05% NaN3, pH 7.2 or 20% ethanol in a suitable buffer. The pH stability of the media when coupled to a ligand will depend upon the stability of the ligand.
119
Coupling through a thiol group Thiopropyl Sepharose 6B The active thiol groups of Thiopropyl Sepharose 6B (see page 91 for product details) can be used to couple many types of small ligands to synthesize affinity media. • Heavy metal ions and derivatives can be used as ligands to react with thiol groups forming mercaptides. • Alkyl or aryl halide ligands give thioether derivatives. • Ligands containing C=O, N=N and, under certain conditions, C=C bonds undergo addition reactions. The medium is converted into the free thiol form, as described earlier, before ligands can be coupled. The hydroxypropyl group acts as a small spacer arm. Reactions of free thiol groups are shown in Figure 70.
S S N
3 Covalent Chromatography 2 DTT S
S
1 PDS
S Hg
COOH
R 4 NO2
S
1 5 2 5
SH
S
CH2
CONH 2
6 6
6
6 S
CH R OH
S
N
NHR
O
R S
S CH
O
CH2 CHO
Fig. 70. Reactions of thiol groups. Mixed disulphide formation (1), reversible by reducing agents such as dithiothreitol (DTT) (2). Mixed disulphide formation with 2, 2'-dipyridyl disulphide gives a 2-thiopyridyl derivative suitable for use in covalent chromatography (3). Reaction with heavy metals and their derivatives e.g. p-chloromercuribenzoate (4) leads to mercaptide formation. Treatment with alkyl or aryl halides gives thioether derivatives (5). Addition reactions (6) are possible with a wide variety of compounds containing C=O, C=C and N=N bonds.
Use Thiopropyl Sepharose 6B in the activated form to couple thiol-containing low molecular weight ligands, such as coenzyme A. If the ligand:protein interaction is so strong that elution requires denaturing conditions, the entire ligand-protein complex may be eluted by reduction with dithiothreitol or 2-mercaptoethanol. 120
Ligands containing amino groups can be attached to Thiopropyl Sepharose 6B or Activated Thiol-Sepharose 4B by multi-point attachment or coupling through a small number of groups using the heterobifunctional thiolating reagent, SPDP. The coupled molecules may be recovered by eluting with a reducing agent. This may be extremely useful when elution is difficult using other methods. The entire ligand-protein complex is eluted from the medium.
Coupling other functional groups EAH Sepharose 4B may be used as a starting material for coupling via alternative functional groups (Figure 71). Phenolic groups may be attached via diazonium derivatives (VII) or via the bromoacetamidoalkyl derivative (V) prepared by treating EAH Sepharose 4B with O-bromoacetyl-N-hydroxysuccinimide. This derivative also couples via primary amino groups. The spacer arm of EAH Sepharose 4B may be extended by reaction with succinic anhydride at pH 6 (VI) to form a derivative to which amino groups can be coupled by carbodiimide reaction. Carboxyl groups are coupled to EAH Sepharose 4B by the carbodiimide reaction (III). Thiol derivatives, prepared by reaction (IV), couple carboxyl groups in the presence of carbodiimide and the thiol ester bond may be cleaved specifically using hydroxylamine, thus providing a simple and gentle method for eluting the intact ligand-protein complex.
II carbodiimide
NH(CH2)5 COOH ECH Sepharose 4B
RNH2
I
RNH2
CNBr-Activated Sepharose
NH(CH2)5CONHR
NHR
III carbodiimide RCOOH
NH2
IV
NH(CH2)6NH2 EAH Sepharose 4B
NH(CH2)6NHCOR
CH2 S
NH(CH2)6NHCOCH(CH2)2SH
CH 2 V
VII
NH2 CH CO (homocysteine thiolactone) NH(CH2)6NHCOCH2Br
VI
O O BrCH2CON
=
O
(1)
O2N
C N3
O (O-bromoacetyl-N-hydroxysuccinimide)
(p-nitrobenzoylazide) (2)
Na2S2O4
O O O (succinic anhydride)
(sodium dithionite) (3)
HNO2 (nitrous acid)
NH(CH2)6NHCO
N+
N
NH(CH2)6NHCO(CH2)2COOH
Fig. 71. Reactions used to couple ligands to Sepharose.
121
122
Chapter 6 Affinity chromatography and CIPP Affinity chromatography separates proteins on the basis of a reversible interaction between a protein (or group of proteins) and a specific ligand coupled to a chromatographic matrix. With such high selectivity and hence high resolution for the protein(s) of interest, purification levels in the order of several thousand-fold with high recovery of active material are achievable. Samples are concentrated during binding and the target protein(s) is collected in a purified, concentrated form. Affinity purification can therefore offer immense time-saving over less selective multi-step procedures. Common operations such as the purification of antibodies or tagged fusion proteins can be performed in a single step. The concentrating effect enables large volumes to be processed. Target molecules can be purified from complex biological mixtures, native forms separated from denatured forms of the same substance, small amounts of biological material purified from high levels of contaminating substances. Affinity chromatography can also be used to remove specific contaminants, such as proteases. In many cases, the high level of purity achievable requires, at most, only a second step on a gel filtration column to remove unwanted small molecules, such as salts or aggregates. For an even higher degree of purity, or when there is no suitable ligand for affinity purification, an efficient multi-step process must be developed using the purification strategy of Capture, Intermediate Purification and Polishing (CIPP), shown in Figure 72. When applying this strategy affinity chromatography offers an ideal capture or intermediate step in any purification protocol and can be used whenever a suitable ligand is available for the protein(s) of interest. CIPP is used in both the pharmaceutical industry and in the research laboratory to ensure faster method development, a shorter time to pure product and good economy. Affinity chromatography can be used, in combination with other chromatography techniques, as an effective capture or intermediate step in a CIPP strategy.
Purity
This chapter gives a brief overview of the approach recommended for any multi-step protein purification. The Protein Purification Handbook from Amersham Pharmacia Biotech is highly recommended as a guide to planning efficient and effective protein purification strategies and for the selection of the correct medium for each step and scale of purification.
Polishing Achieve final high level purity
Intermediate purification Capture Preparation, extraction, clarification
Remove bulk impurities
Isolate, concentrate and stabilize
Step Fig. 72. Preparation and CIPP.
123
Applying CIPP Imagine the purification has three phases: Capture, Intermediate Purification and Polishing. Assign a specific objective to each step within the purification process. The purification problem associated with a particular step will depend greatly upon the properties of the starting material. Thus, the objective of a purification step will vary according to its position in the process. As shown in Figure 72, an important first step for any purification is correct sample preparation and this is covered in more detail in Appendix 1. In the capture phase the objectives are to isolate, concentrate and stabilize the target product. The product should be concentrated and transferred to an environment that will conserve potency/activity. During the intermediate purification phase the objectives are to remove most of the bulk impurities, such as other proteins and nucleic acids, endotoxins and viruses. In the polishing phase most impurities have already been removed except for trace amounts or closely related substances. The objective is to achieve final purity by removing any remaining trace impurities or closely related substances. The optimal selection and combination of purification techniques for Capture, Intermediate Purification and Polishing is crucial for an efficient purification.
Selection and combination of purification techniques Proteins are purified using purification techniques that separate according to differences in specific properties, as shown in Table 11. Table 11. Protein properties used during purification. Protein property
Technique*
Biorecognition (ligand specificity)
Affinity (AC)
Charge
Ion exchange (IEX)
Size
Gel filtration (GF)
Hydrophobicity
Hydrophobic interaction (HIC), Reversed phase (RPC)
*Expanded bed adsorption is a technique used for large-scale purification. Proteins can be purified from crude sample without the need for separate clarification, concentration and initial purification to remove particulate matter. The STREAMLINE adsorbents, used for expanded bed adsorption, capture the target molecules using the same principles as affinity, ion exchange or hydrophobic interaction chromatography. Resolution
Recovery
Speed Capacity
Every chromatographic technique offers a balance between resolution, capacity, speed and recovery. 124
Resolution is achieved by the selectivity of the technique and the efficiency of the chromatographic matrix in producing narrow peaks. In general, resolution is most difficult to achieve in the final stages of purification when impurities and target protein are likely to have very similar properties. The high selectivity of affinity chromatography typically gives a high resolution result. Capacity, in the simple model shown, refers to the amount of target protein that can be loaded during purification. In some cases the amount of sample that can be loaded will be limited by volume (as in gel filtration) or by large amounts of contaminants, rather than by the amount of the target protein. Since affinity chromatography is a binding technique the separation is unaffected by sample volume as long as the correct binding conditions are maintained during sample application and the total amount of target protein loaded onto the column does not exceed the binding capacity of the affinity medium. Speed is most important at the beginning of purification where contaminants such as proteases must be removed as quickly as possible. Modern affinity matrices enable high flow rates to be used for sample application as well as washing and reequilibration steps. For each application a flow rate can be selected to achieve an optimal balance between efficient binding and elution of the target protein and a fast separation. Recovery becomes increasingly important as the purification proceeds because of the increased value of the purified product. Recovery is influenced by destructive processes in the sample and by unfavourable conditions on the column. Affinity media provided with optimized separation protocols can give extremely high recoveries of target protein. Select the technique that meet the objectives for the purification step. Choose logical combinations of purification techniques based on the main benefits of the technique and the condition of the sample at the beginning or end of each step. A guide to the suitability of each purification technique for the stages in CIPP is shown in Table 12. Table 12. Suitability of purification techniques for CIPP. Technique Main features
Sample start condition
Sample end condition
high resolution high capacity high speed
low ionic strength sample volume not limiting
high ionic strength or pH change
good resolution good capacity high speed
high ionic strength sample volume not limiting
low ionic strength
high resolution high capacity high speed
specific binding conditions sample volume not limiting
specific elution conditions
GF
high resolution using Superdex
limited sample volume (<5% total column volume) and flow rate range
buffer exchanged (if required) diluted sample
RPC
high resolution
sample volume usually not limiting additives may be required
in organic solvent, risk loss of biological activity
IEX
HIC
AC
Capture
Intermediate
Polishing
concentrated sample concentrated sample
concentrated sample
125
Minimize sample handling between purification steps by combining techniques to avoid the need for sample conditioning. The product should be eluted from the first column in buffer conditions suitable for the start conditions of the next column (see Table 12). Ammonium sulphate, often used for sample clarification and concentration (see Appendix 1), leaves the sample in high salt. Consequently HIC, which requires high salt to enhance binding to the media, is ideal as the capture step. The salt concentration and the total sample volume will be significantly reduced after elution from the HIC column. Dilution of the fractionated sample or rapid buffer exchange using a desalting column will prepare it for the next IEX or AC step. Gel filtration is a non-binding technique unaffected by buffer conditions, but with limited volume capacity. GF is well suited for use after any of the concentrating techniques (IEX, HIC, AC, EBA) since the target protein will be eluted in a reduced volume and the components from the elution buffer will not affect the gel filtration process. Selection of the final strategy will always depend upon specific sample properties and the required level of purification. Logical combinations of techniques are shown in Figure 73. Proteins with low solubility SDS extraction
GF (in non-ionic detergent)
SDS extraction
Solubilizing agents (urea, ethylene glycol non-ionic detergents)
HIC
HIC
GF
GF
Crude sample or sample in high salt concentration Sample clarification
Capture
GF GF desalt mode desalt mode
AC
IEX
Intermediate Purification Polishing
GF or RPC
GF or RPC
GF desalt mode
HIC IEX dilution may be needed
IEX
HIC
GF
GF
Clear or very dilute samples Capture
AC
IEX
Intermediate Purification Polishing
GF or RPC
GF or RPC
IEX
Precipitation (e.g. in high ionic strength)
HIC
Resolubilize
GF
Treat as for sample in high salt concentration
Fig. 73. Logical combinations of chromatographic techniques. 126
For any capture step, select the technique showing the most effective binding to the target protein while binding as few of the contaminants as possible, i.e. the technique with the highest selectivity and/or capacity for the target protein. A sample is purified using a combination of techniques and alternative selectivities. For example, in an IEX-HIC-GF strategy, the capture step selects according to differences in charge (IEX), the intermediate purification step according to differences in hydrophobicity (HIC) and the final polishing step according to differences in size (GF). If nothing is known about the target protein use IEX-HIC-GF. This combination of techniques can be regarded as a standard protocol. Consider the use of both anion and cation exchange chromatography to give different selectivities within the same purification strategy. IEX is a technique which offers different selectivities using either anion or cation exchangers. The pH can be modified to alter the charge characteristics of the sample components. It is therefore possible to use IEX more than once in a purification strategy, for capture, intermediate purification or polishing. IEX can be used effectively in the same purification scheme for rapid purification in low resolution mode during capture and in high resolution mode during polishing. Consider reversed phase chromatography (RPC) for a polishing step, provided that the target protein can withstand the run conditions. RPC separates proteins and peptides on the basis of hydrophobicity. RPC is a high selectivity (high resolution) technique, requiring the use of organic solvents. The technique is widely used for purity check analyses when recovery of activity and tertiary structure are not essential. Since many proteins are denatured by organic solvents, RPC is not generally recommended for protein purification because recovery of activity and return to a native tertiary structure may be compromised. How ever, in the polishing phase, when the majority of protein impurities have been removed, RPC can be excellent, particularly for small target proteins that are not often denatured by organic solvents. CIPP does not mean that there must always be three purification steps. For example, capture and intermediate purification may be achievable in a single step, as may intermediate purification and polishing. Similarly, purity demands may be so low that a rapid capture step is sufficient to achieve the desired result. For purification of therapeutic proteins, a fourth or fifth purification step may be required to fulfil the highest purity and safety demands. The number of steps used will always depend upon the purity requirements and intended use for the protein.
127
128
Appendix 1 Sample preparation Samples for chromatographic purification should be clear and free from particulate matter. Simple steps to clarify a sample before beginning purification will avoid clogging the column, may reduce the need for stringent washing procedures and can extend the life of the chromatographic medium. Sample extraction procedures and the selection of buffers, additives and detergents are determined largely by the source of the material, the stability of the target molecule, the chromatographic techniques that will be employed and the intended use of the product. These subjects are dealt with in general terms in the Protein Purification Handbook and more specifically according to target molecule in the Recombinant Protein Handbook, Protein Amplification and Simple Purification and Antibody Purification Handbook, available from Amersham Pharmacia Biotech.
Sample stability In the majority of cases, biological activity needs to be retained after purification. Retaining the activity of the target molecule is also an advantage when following the progress of the purification, since detection of the target molecule often relies on its biological activity. Denaturation of sample components often leads to precipitation or enhanced non-specific adsorption, both of which will impair column function. Hence there are many advantages to checking the stability limits of the sample and working within these limits during purification. Proteins generally contain a high degree of tertiary structure, kept together by van der Waals' forces, ionic and hydrophobic interactions and hydrogen bonding. Any conditions capable of destabilizing these forces may cause denaturation and/or precipitation. By contrast, peptides contain a low degree of tertiary structure. Their native state is dominated by secondary structures, stabilized mainly by hydrogen bonding. For this reason, peptides tolerate a much wider range of conditions than proteins. This basic difference in native structures is also reflected in that proteins are not easily renatured, while peptides often renature spontaneously. It is advisable to perform stability tests before beginning to develop a purification protocol. The list below may be used as a basis for such testing: • Test pH stability in steps of one pH unit between pH 2 and pH 9. • Test salt stability with 0–2 M NaCl and 0–2 M (NH4)2SO4 in steps of 0.5 M. • Test the stability towards acetonitrile and methanol in 10% steps between 0 and 50%. • Test the temperature stability in +10 °C steps from +4 to +40 °C. • Test the stability and occurrence of proteolytic activity by leaving an aliquot of the sample at room temperature overnight. Centrifuge each sample and measure activity and UV absorbance at 280 nm in the supernatant.
129
Sample clarification Centrifugation and filtration are standard laboratory techniques for sample clarification and are used routinely when handling small samples. It is highly recommended to centrifuge and filter any sample immediately before chromatographic purification. Centrifugation Centrifugation removes lipids and particulate matter, such as cell debris. If the sample is still not clear after centrifugation, use filter paper or a 5 µm filter as a first step and one of the filters below as a second step filter. • For small sample volumes or proteins that adsorb to filters, centrifuge at 10 000 g for 15 minutes. • For cell lysates, centrifuge at 40 000–50 000 g for 30 minutes. • Serum samples can be filtered through glass wool after centrifugation to remove any remaining lipids. Filtration Filtration removes particulate matter. Membrane filters that give the least amount of nonspecific binding of proteins are composed of cellulose acetate or PVDF. For sample preparation before chromatography, select a filter pore size in relation to the bead size of the chromatographic medium. Nominal pore size of filter 1 µm
Particle size of chromatographic medium 90 µm and upwards
0.45 µm
34 µm
0.22 µm
3, 10, 15 µm or when extra clean samples or sterile filtration is required
Check the recovery of the target protein in a test run. Some proteins may adsorb nonspecifically to filter surfaces. Desalting Desalting columns are suitable for any sample volume and will rapidly remove low molecular weight contaminants in a single step at the same time as transferring the sample into the correct buffer conditions. Centrifugation and/or filtration of the sample before desalting is still recommended. Detailed procedures for buffer exchange and desalting are given on page 134. At laboratory scale, when samples are reasonably clean after filtration or centrifugation, the buffer exchange and desalting step can be avoided. For affinity chromatography or hydrophobic interaction chromatography, it may be sufficient to adjust the pH of the sample and, if necessary, dilute to reduce the ionic strength of the solution. Rapidly process small or large sample volumes. Use before and/or between purification steps, if needed (remember that each extra step can reduce yield and desalting also dilutes the sample).
130
Remove salts from proteins with molecular weight Mr > 5 000. Use 100 mM ammonium acetate or 100 mM ammonium hydrogen carbonate if volatile buffers are required.
Specific sample preparation steps Specific sample preparation steps may be required if the crude sample is known to contain contamininants such as lipids, lipoproteins or phenol red that may build up on a column or if certain gross impurities, such as bulk protein, should be removed before any chromatographic step. Fractional precipitation Fractional precipitation is frequently used at laboratory scale to remove gross impurities from small sample volumes, and occasionally used in small-scale commercial production. Precipitation techniques separate fractions by the principle of differential solubility. Because protein species differ in their degree of hydrophobicity, increased salt concentrations can enhance hydrophobic interactions between the proteins and cause precipitation. Fractional precipitation can be applied to remove gross impurities in three different ways, as shown in Figure 74.
Clarification Bulk proteins and particulate matter precipitated Extraction Clarification Concentration Target protein precipitated with proteins of similar solubility Extraction Clarification Bulk proteins and particulate matter precipitated
Supernatant
Redissolve pellet*
Concentration Target protein precipitated with proteins of similar solubility
Chromatography Remember: if precipitating agent is incompatible with next purification step, use Sephadex TM G-25 for desalting and buffer exchange e.g. HiTrap Desalting or PD-10 columns
Redissolve pellet* *Remember: not all proteins are easy to redissolve, yield may be reduced
Fig. 74. Three ways to use precipitation.
131
Examples of precipitation agents are reviewed in Table 13. The most common precipitation method using ammonium sulphate is described in more detail. Table 13. Examples of precipitation techniques. Precipitation agent
Typical conditions for use
Sample type
Comment
Ammonium sulphate
As described below.
> 1 mg/ml proteins especially immunoglobulins.
Stabilizes proteins, no denaturation, supernatant can go directly to HIC.
Dextran sulphate
Add 0.04 ml 10% dextran sulphate and 1 ml 1 M CaCl2 per ml sample, mix 15 min, centrifuge 10 000 g, discard pellet.
Samples with high levels of lipoprotein e.g ascites.
Precipitates lipoprotein.
Polyvinylpyrrolidine
Add 3% (w/v), stir 4 hours, centrifuge 17 000 g, discard pellet.
Samples with high levels of lipoprotein e.g ascites.
Alternative to dextran sulphate.
Polyethylene glycol (PEG, Mr > 4000)
Up to 20% w/vol
Plasma proteins.
No denaturation, supernatant goes directly to IEX or AC, complete removal may be difficult.
Acetone (cold)
Up to 80% vol/vol at +0 °C. Collect pellet after centrifugation at full speed in an Eppendorf™ centrifuge.
May denature protein irreversibly. Useful for peptide precipitation or concentration of sample for electrophoresis.
Polyethyleneimine
0.1% w/v
Precipitates aggregated nucleoproteins.
Protamine sulphate
1% w/v
Precipitates aggregated nucleoproteins.
Streptomycin sulphate
1% w/v
Caprylic acid
(X/15) g where X = volume of sample.
Precipitation of nucleic acids. Antibody concentration should be > 1 mg/ml.
Precipitates bulk of proteins from sera or ascites, leaving immunoglobulins in solution.
Details taken from: Scopes R.K., Protein Purification, Principles and Practice, Springer, (1994), J.C. Janson and L. Rydén, Protein Purification, Principles, High Resolution Methods and Applications, 2nd ed. Wiley Inc, (1998). Personal communications.
Ammonium sulphate precipitation Some proteins may be damaged by ammonium sulphate. Take care when adding crystalline ammonium sulphate: high local concentrations may cause contamination of the precipitate with unwanted proteins. For routine, reproducible purification, precipitation with ammonium sulphate should be avoided in favour of chromatography. In general, precipitation is rarely effective for protein concentrations below 1 mg/ml. Solutions needed for precipitation: Saturated ammonium sulphate solution (add 100 g ammonium sulphate to 100 ml distilled water, stir to dissolve). 1 M Tris-HCl, pH 8.0. Buffer for first purification step.
132
1. Filter (0.45 µm) or centrifuge the sample (10 000 g at +4 °C). 2. Add 1 part 1 M Tris-HCl, pH 8.0 to 10 parts sample volume to maintain pH. 3. Stir gently. Add ammonium sulphate solution, drop by drop. Add up to 50% saturation*. Stir for 1 hour. 4. Centrifuge 20 minutes at 10 000 g. 5. Remove supernatant. Wash the pellet twice by resuspension in an equal volume of ammonium sulphate solution of the same concentration (i.e. a solution that will not redissolve the precipitated protein or cause further precipitation). Centrifuge again. 6. Dissolve pellet in a small volume of the buffer to be used for the next step. 7. Ammonium sulphate is removed during clarification/buffer exchange steps with Sephadex G-25, using desalting columns (see page 134). *The % saturation can be adjusted either to precipitate a target molecule or to precipitate contaminants.
The quantity of ammonium sulphate required to reach a given degree of saturation varies according to temperature. Table 14 shows the quantities required at +20 °C. Table 14. Quantities of ammonium sulphate required to reach given degrees of saturation at +20 °C. Final percent saturation to be obtained 20
25
Starting percent saturation
30
35
40
45
50
55
60
65
70
75
80
85
90
95 100
Amount of ammonium sulphate to add (grams) per litre of solution at +20 °C
0
113 144 176 208 242 277 314 351 390 430 472 516 561 608 657 708 761
5
85 115 146 179 212 246 282 319 358 397 439 481 526 572 621 671 723
10
57
86 117 149 182 216 251 287 325 364 405 447 491 537 584 634 685
15
28
58
20
0
29
59
89 121 154 188 223 260 298 337 378 421 465 511 559 609
0
29
60
91 123 157 191 228 265 304 344 386 429 475 522 571
0
30
61
0
30
62
94 128 163 199 236 275 316 358 402 447 495
0
31
63
96 130 166 202 241 281 322 365 410 457
0
31
64
0
32
65
99 135 172 210 250 292 335 381
0
33
66 101 138 175 215 256 298 343
25 30 35 40 45
88 119 151 185 219 255 293 331 371 413 456 501 548 596 647
92 125 160 195 232 270 309 351 393 438 485 533
50 55 60 65 70 75 80 85 90 95
98 132 169 206 245 286 329 373 419
0
33 0
67 103 140 179 219 261 305 34 0
69 105 143 183 224 267 34 0
70 107 146 186 228 35 0
72 110 149 190 36 0
73 112 152 37 0
75 114 37
76
0
38
Resolubilization of protein precipitates Many proteins are easily resolubilized in a small amount of the buffer to be used in the next chromatographic step. However, a denaturing agent may be required for less soluble proteins. Specific conditions will depend upon the specific protein. These agents must always be removed to allow complete refolding of the protein and to maximize recovery of mass and activity. A chromatographic step often removes a denaturant during purification. Table 15 gives examples of common denaturing agents.
133
Table 15. Denaturing agent
Typical conditions for use
Removal/comment
Urea
2 M–8 M
Guanidine hydrochloride
3 M–6 M
Remove using Sephadex G-25 or during IEX.
2%
Remove using Sephadex G-25 or during IEX.
Triton X-100 Sarcosyl N-octyl glucoside Sodium dodecyl sulphate
Remove using Sephadex G-25.
1.5%
Remove using Sephadex G-25 or during IEX.
2%
Remove using Sephadex G-25 or during IEX.
0.1%–0.5%
Alkaline pH
Exchange for non-ionic detergent during first chromatographic step, avoid anion exchange chromatography.
> pH 9, NaOH
May need to adjust pH during chromatography to maintain solubility.
Details taken from: Scopes R.K., Protein Purification, Principles and Practice, Springer, (1994), J.C. Janson and L. Rydén, Protein Purification, Principles, High Resolution Methods and Applications, 2nd ed. Wiley Inc, (1998) and other sources.
Buffer exchange and desalting Dialysis is frequently mentioned in the literature as a technique to remove salt or other small molecules and to exchange the buffer composition of a sample. However, dialysis is generally a very slow technique, requiring large volumes of buffer. During handling or as a result of proteolytic breakdown or non-specific binding to the dialysis membranes, there is a risk of losing material. A simpler and much faster technique is to use a desalting column, packed with Sephadex G-25, to perform a group separation between high and low molecular weight substances. Proteins are separated from salts and other small molecules. In a fast, single step, the sample is desalted, transferred into a new buffer and low molecular weight materials are removed. Desalting columns are used not only to remove low molecular weight contaminants, such as salt, but also for buffer exchange before or after different chromatographic steps and for the rapid removal of reagents to terminate a reaction. Sample volumes up to 30% of the total volume of the desalting column can be processed. Figure 75 shows a typical buffer exchange and desalting separation. The process can be monitored by following changes in UV absorption and conductivity. A 280 nm
(mS/cm)
0.25 0.20 0.15
10.0
protein
salt
0.10 5.0 0.05 0.00 0.0
1.0
2.0
Time (min)
Fig. 75. Buffer exchange of mouse plasma (10 ml) on HiPrep 26/10 Desalting.
134
For laboratory scale operations, Table 16 shows a selection guide for prepacked, ready to use desalting and buffer exchange columns. Table 16. Selection guide for desalting and buffer exchange. Column MicroSpin G-25 PD-10 (gravity feed column) HiTrap Desalting 5 ml HiPrep 26/10 Desalting
Sample volume
Sample elution volume
0.1–0.15 ml
0.1–0.15 ml
1.5–2.5 ml
2.5–3.5 ml
0.25–1.5 ml
1.0–2.0 ml
2.5–15 ml
7.5–20 ml
To desalt larger sample volumes - connect up to 5 HiTrap Desalting 5 ml columns in series to increase the sample volume capacity, e.g. 2 columns: sample volume 3 ml, 5 columns: sample volume 7.5 ml. - connect up to 4 HiPrep 26/10 Desalting columns in series to increase the sample volume capacity, e.g. 2 columns: sample volume 30 ml, 4 columns: sample volume 60 ml. Even with 4 columns in series, the sample can be processed in 20 to 30 minutes, at room temperature, in aqueous buffers. Instructions are supplied with each column. Desalting and buffer exchange can take less than 5 minutes per sample with greater than 95% recovery for most proteins. To prevent possible ionic interactions the presence of a low salt concentration (25 mM NaCl) is recommended during desalting and in the final sample buffer. Sample concentration does not influence the separation as long as the concentration of proteins does not exceed 70 mg/ml when using normal aqueous buffers. The sample should be fully dissolved. Centrifuge or filter to remove particulate material. For small sample volumes it may be possible to dilute the sample with the buffer that is to be used for chromatographic purification, but cell debris and particulate matter must still be removed. Volatile buffers such as 100 mM ammonium acetate or 100 mM ammonium hydrogen carbonate can be used if it is necessary to avoid the presence of NaCl. Alternative 1: Manual desalting with HiTrap Desalting 5 ml using a syringe 1. Fill the syringe with buffer. Remove the stop plug. To avoid introducing air into the column, connect the column "drop to drop" to the syringe (via the adapter provided). 2. Remove the twist-off end. 3. Wash the column with 25 ml buffer at 5 ml/min to remove completely the 20% ethanol (supplied as storage buffer). If air is trapped in the column, wash with degassed buffer until the air disappears. Air bubbles introduced onto the column by accident during sample application do not influence the separation. 4. Apply the sample using a 2–5 ml syringe at a flow rate between 1–10 ml/min. Discard the liquid eluted from the column. 5. If the sample volume is less than 1.5 ml, change to buffer and proceed with the injection until a total of 1.5 ml has been eluted. Discard the eluted liquid. 6. Elute the protein with the appropriate volume selected from Table 16. Collect the desalted protein in the volume indicated.
Note: 5 ml/min corresponds to approximately 120 drops/min when using a HiTrap 5 ml column.
135
The maximum recommended sample volume is 1.5 ml. See Table 17 for the effect of reducing the sample volume applied to the column. Table 17. Recommended sample and elution volumes using a syringe or Multipipette™. Sample load ml
Add bufferml
Elute and collect ml
Yield %
Remaining salt %
Dilution factor
0.25
1.25
1.0
> 95
0.0
4.0
0.50
1.0
1.5
> 95
< 0.1
3.0
1.00
0.5
2.0
> 95
< 0.2
2.0
1.50
0
2.0
> 95
< 0.2
1.3
A simple peristaltic pump can also be used to apply sample and buffers. Alternative 2: Simple desalting with ÄKTAprime ÄKTAprime contains pre-programmed templates for individual HiTrap Desalting 5 ml and HiPrep 26/10 Desalting columns.
Buffer Preparation Prepare at least 500 ml of the required buffer. 1. Follow the instructions supplied on the ÄKTAprime cue card to connect the column and load the system with buffer. 2. Select the Application Template. 3. Start the method. 4. Enter the sample volume and press OK.
Figure 76 shows a typical procedure using ÄKTAprime. The UV (protein) and conductivity (salt) traces enable pooling of the desalted fractions. A 280 nm
–– UV 280 nm –– Conductivity 0.15 (His)6 protein
Sample: 0.10
Salt
Column: Buffer:
0.05 Inject
0 0
1
2 min
Fig. 76. Desalting of a (His)6 fusion protein on ÄKTAprime.
136
(His)6 protein eluted from HiTrap Chelating HP with sodium phosphate 20 mM, sodium chloride 0.5 M, imidazole 0.5 M, pH 7.4 HiTrap Desalting 5 ml Sodium phosphate 20 mM, sodium chloride 0.15 M, pH 7.0
Removal of lipoproteins Lipoproteins and other lipid material can rapidly clog chromatography columns and it is advisable to remove them before beginning purification. Precipitation agents such as dextran sulphate and polyvinylpyrrolidine, described under Fractional precipitation, are recommended to remove high levels of lipoproteins from samples such as ascitic fluid. Centrifuge samples to avoid the risk of non-specific binding of the target molecule to a filter. Samples such as serum can be filtered through glass wool to remove remaining lipids.
Removal of phenol red Phenol red is frequently used at laboratory scale as a pH indicator in cell culture. Although not directly interfering with purification, phenol red may bind to certain purification media and should be removed as early as possible to avoid the risk of contamination. It is known to bind to anion exchange media at pH > 7. Use a desalting column to simultaneously remove phenol red (a low molecular weight molecule) and transfer sample to the correct buffer conditions for further purification, as described under Buffer exchange and desalting.
Removal of low molecular weight contaminants If samples contain a high level of low molecular weight contaminants, use a desalting column before the first chromatographic purification step, as described under Buffer exchange and desalting.
137
138
Appendix 2 Selection of purification equipment Many affinity chromatography experiments may be carried out using the simplest methods and equipment, for example step-gradient elution using a syringe together with prepacked HiTrap columns. When more complex elution methods are necessary, large sample volumes are being applied or the same column is to be used for many runs in series, it is wise to use a dedicated system. Standard ÄKTAdesign configurations Way of working
Explorer 100
Purifier 10
FPLC
Prime
Syringe+ HiTrap
Rapid, screening (GST or His tagged proteins)
Centrifugation+ MicroSpin "
Simple, one step purification
"
"
"
"
Reproducible performance for routine purification
"
"
"
"
Optimization of one step purification to increase purity
"
"
"
"
System control and data handling for regulatory requirements, e.g. GLP
"
"
"
Automatic method development and optimization
"
"
"
Automatic buffer preparation
"
"
Automatic pH scouting
"
"
Automatic media or column scouting
"
Automatic multi-step purification
"
Scale up, process development and transfer to production
"
"
ÄKTAprime
ÄKTAFPLC ÄKTAexplorer ÄKTApurifier
139
Appendix 3 Column packing and preparation Prepacked columns from Amersham Pharmacia Biotech will ensure reproducible results and the highest performance. However, if column packing is required, the following guidelines will apply at any scale of operation: With a high binding capacity medium, short, wide columns can be used for rapid purification, even with low linear flow rates. Ready to use affinity media are supplied with details of the binding capacity per ml of medium. Unless otherwise stated, estimate the amount of medium required to bind the target molecules and use two- to five times this amount to pack the column. Refer to the product instructions for more specific information regarding buffers, flow rates etc. For affinity media made from pre-activated matrices, determine the binding capacity of the medium. Estimate the amount of medium required to bind the target molecules and use two- to five times this amount to pack the column. A Column Packing Video is available to demonstrate how to produce a well-packed column (see Ordering information). The video focuses particularly on the importance of column packing for gel filtration, shown in the figure below.
1. Equilibrate all materials to the temperature at which the purification will be performed. 2. Eliminate air by flushing column end pieces with the recommended buffer. Ensure no air is trapped under the column net. Close column outlet leaving 1–2 cm of buffer in the column. 3. Gently resuspend the purification medium.
For media not supplied in suspension, use a medium: buffer ratio of approximately 1:2 to produce a suspension for mixing during rehydration. Avoid using magnetic stirrers since they may damage the matrix.
140
4. Estimate the amount of slurry (resuspended medium) required on the basis of the recommendations supplied. 5. Pour the required volume of slurry into the column. Pouring down a glass rod held against the wall of the column will minimize the introduction of air bubbles. 6. Immediately fill the column with buffer. 7. Mount the column top piece and connect to a pump. 8. Open the column outlet and set the pump to the desired flow rate.
If the recommended flow rate cannot be obtained, use the maximum flow rate the pump can deliver. Do not exceed the maximum operating pressure of the medium or column. 9. Maintain the packing flow rate for at least 3 column volumes after a constant bed height is obtained. Mark the bed height on the column.
Do not exceed 75% of the packing flow rate during any purification. 10. Stop the pump and close the column outlet. Remove the top piece and carefully fill the rest of the column with buffer to form an upward meniscus at the top. 11. Insert the adaptor into the column at an angle, ensuring that no air is trapped under the net. 12. Slide the adaptor slowly down the column (the outlet of the adaptor should be open) until the mark is reached. Lock the adaptor in position. 13. Connect the column to the pump and begin equilibration. Re-position the adaptor if necessary.
The medium must be thoroughly washed to remove the storage solution, usually 20% ethanol. Residual ethanol may interfere with subsequent procedures. Many media equilibrated with sterile PBS containing an antimicrobial agent may be stored at +4 °C for up to 1 month, but always follow the specific storage instructions supplied with the product. Columns for packing affinity media XK columns are fully compatible with the high flow rates achievable with modern media and a broad range of column dimensions is available. Columns XK 16/20 and XK 26/20 are recommended for affinity chromatography and their specifications are shown in Table 18. For a complete listing refer to the Amersham Pharmacia Biotech BioDirectory™ or web catalogue (www.apbiotech.com). In most cases, the column size required is governed by the capacity of the medium and the amount of substance to be purified. Table 18. Columns
Volume (ml)
Code no
XK 16/20
2–34
18-8773-01
XK 26/20
0–80
18-1000-72
XK 50/20
0–275
18-1000-71
141
Appendix 4 Converting from linear flow (cm/hour) to volumetric flow rates (ml/min) and vice versa It is convenient when comparing results for columns of different sizes to express flow as linear flow (cm/hour). However, flow is usually measured in volumetric flow rate (ml/min). To convert between linear flow and volumetric flow rate use one of the formulae below.
From linear flow (cm/hour) to volumetric flow rate (ml/min) Volumetric flow rate (ml/min) = =
Linear flow (cm/h) x column cross sectional area (cm2) 60 Y p x d2 x 60 4
where Y = linear flow in cm/h d = column inner diameter in cm
Example: What is the volumetric flow rate in an XK 16/70 column (i.d. 1.6 cm) when the linear flow is 150 cm/hour? Y = linear flow = 150 cm/h d = inner diameter of the column = 1.6 cm Volumetric flow rate =
150 x p x 1.6 x 1.6 ml/min 60 x 4
= 5.03 ml/min
From volumetric flow rate (ml/min) to linear flow (cm/hour) Linear flow (cm/h) =
Volumetric flow rate (ml/min) x 60 column cross sectional area (cm2)
= Z x 60 x
4 p x d2
where Z = volumetric flow rate in ml/min d = column inner diameter in cm
Example: What is the linear flow in an HR 5/5 column (i.d. 0.5 cm) when the volumetric flow rate is 1 ml/min? Z = Volumetric flow rate = 1 ml/min d = column inner diameter = 0.5 cm Linear flow = 1 x 60 x
4 p x 0.5 x 0.5
cm/h
= 305.6 cm/h
From ml/min to using a syringe 1 ml/min = approximately 30 drops/min on a HiTrap 1 ml column 5 ml/min = approximately 120 drops/min on a HiTrap 5 ml column
142
Appendix 5 Conversion data: proteins, column pressures Mass (g/mol)
1 µg
1 nmol
Protein
A280 for 1 mg/ml
10 000
100 pmol; 6 x 10
13
molecules
10 µg
IgG
50 000
20 pmol; 1.2 x 10
13
molecules
50 µg
IgM
1.20
100 000
10 pmol; 6.0 x 10
12
molecules
100 µg
IgA
1.30
150 000
6.7 pmol; 4.0 x 10
12
molecules
150 µg
Protein A
0.17
1 kb of DNA
= 333 amino acids of coding capacity
270 bp DNA
= 10 000 g/mol
1.35
Avidin
1.50
Streptavidin
3.40
Bovine Serum Albumin
0.70
= 37 000 g/mol 1.35 kb DNA
= 50 000 g/mol
2.70 kb DNA
= 100 000 g/mol
Average molecular weight of an amino acid = 120 g/mol.
Column pressures The maximum operating back pressure refers to the pressure above which the column contents may begin to compress. Pressure units may be expressed in megaPascals, bar or pounds per square inch and can be converted as follows: 1MPa = 10 bar = 145 psi
143
Appendix 6 Table of amino acids Three-letter code
Single-letter code
Alanine
Ala
A
Arginine
Arg
R
Amino acid
Structure HOOC CH3 H 2N NH2
HOOC CH2CH2CH2NHC H 2N
NH
HOOC
Asparagine
Asn
N
Aspartic Acid
Asp
D
CH2CONH2 H 2N HOOC CH2COOH H 2N HOOC
Cysteine
Cys
CH2SH
C H 2N HOOC
Glutamic Acid
Glu
CH2CH2COOH
E H 2N HOOC
Glutamine
Gln
Q
Glycine
Gly
G
Histidine
His
H
CH2CH2CONH2 H 2N HOOC H H 2N HOOC
N CH2
NH
H 2N HOOC
Isoleucine
Ile
CH(CH3)CH2CH3
I H 2N HOOC
Leucine
Leu
L
CH3 CH2CH CH3
H 2N HOOC
Lysine
Lys
K
Methionine
Met
M
CH2CH2CH2CH2NH2 H 2N HOOC CH2CH2SCH3 H 2N HOOC
Phenylalanine
Phe
F
Proline
Pro
P
CH2 H 2N HOOC H 2N
NH
HOOC
Serine
Ser
S
Threonine
Thr
T
CH2OH H 2N HOOC CHCH3 H 2N
OH
HOOC
Tryptophan
Trp
W
CH2 H 2N
NH
HOOC
Tyrosine
Tyr
CH2
Y H 2N HOOC
Valine
Val
CH(CH3)2
V H 2N
144
OH
Formula
Mr
Middle unit residue (-H20) Formula Mr
C3H7NO2
89.1
C3H5NO
C6H14N4O2
174.2
C 4H 8N 2O 3
Charge at pH 6.0–7.0
Hydrophobic (non-polar)
Uncharged (polar)
71.1
Neutral
#
C6H12N4O
156.2
Basic (+ve)
132.1
C 4H 6N 2O 2
114.1
Neutral
C4H7NO4
133.1
C4H5NO3
115.1
Acidic(-ve)
C3H7NO2S
121.2
C3H5NOS
103.2
Neutral
C5H9NO4
147.1
C5H7NO3
129.1
Acidic (-ve)
C5H10N2O3
146.1
C 5H 8N 2O 2
128.1
Neutral
#
C2H5NO2
75.1
C2H3NO
57.1
Neutral
#
C 6H 9N 3O 2
155.2
C6H 7N3O
137.2
Basic (+ve)
C6H13NO2
131.2
C6H11NO
113.2
Neutral
#
C6H13NO2
131.2
C6H11NO
113.2
Neutral
#
C6H14N2O2
146.2
C6H12N2O
128.2
Basic(+ve)
C5H11NO2S
149.2
C5H9NOS
131.2
Neutral
#
C9H11NO2
165.2
C9H9NO
147.2
Neutral
#
C5H9NO2
115.1
C5H7NO
97.1
Neutral
#
C3H7NO3
105.1
C3H5NO2
87.1
Neutral
#
C4H9NO3
119.1
C4H7NO2
101.1
Neutral
#
C11H12N2O2
204.2
C11H10N2O
186.2
Neutral
C9H11NO3
181.2
C9H9NO2
163.2
Neutral
C5H11NO2
117.1
C5H9NO
99.1
Neutral
Hydrophilic (polar)
# # # # #
#
#
# # #
145
Appendix 7 Kinetics in affinity chromatography The binding (adsorption) and elution (desorption) of a target protein (T) to and from an affinity ligand (L) can be considered in terms of the binding equilibria involved and the kinetics of adsorption and desorption. Binding equilibria: Non-selective elution by changing KD
L
+
T
LT
The standard definition of the equilibrium dissociation constant KD is shown below. Free ligand is the ligand that is not bound to a target protein and free target is the target which is not bound to a ligand. KD is the equilibrium dissociation constant
At equilibrium
KD =
[L] is the concentration of free ligand
[L][T] [LT]
[T] is the concentration of free target [LT] is the concentration of the ligand/target complex
Standard definition of the equilibrium constant.
Graves and Wu in Methods in Enzymology 34, 140–163 (1974) have shown that: Bound target Total target
~ ~
L0 KD + L0
KD is the equilibrium dissociation constant L0 is the concentration of ligand, usually 10-4 - 10-2 M
There are many assumptions and simplifications behind the derivation of this equation, but, although it is not an exact description, it does give a reasonable qualitative description. The ratio of bound to total target should be close to 1 during binding, i.e. almost all the target binds to the ligand. KD should be small compared to the ligand concentration, i.e. KD is 10-6 - 10-4 M when L0 is 10-4 - 10-2 M, to achieve efficient binding. Since KD can be changed by altering pH, temperature, ionic strength and other parameters, these parameters can be modified to cause elution in affinity chromatography. If the conditions are changed the binding equilibrium changes and to get a reasonable elution the dissociation constant must be increased by quite a large factor (Figure 77). During binding KD 10 -6 - 10-4 M
L+T
LT
During elution KD 10-1 - 10-2 M
LT
L+T
Fig. 77. Changes in binding and elution alter KD.
146
Expected results when changing conditions to alter KD KD changes when pH, ionic strength or temperature is changed. A 280
Flow through (unbound material) Elution buffer
Eluted target
Binding buffer
Target elutes as a sharp peak.
ml
KD low (10-6 M)
KD high (10-1 M)
Unexpected results when changing conditions to alter KD KD too high during binding. A 280
Flow through (unbound material)
Target binds as a broad peak and elutes as a broad, low peak while binding buffer is being applied.
Eluted target
Binding buffer
- Find better binding conditions to reduce KD. ml
KD too high (10-3 M)
KD too low during elution. A 280
Flow through (unbound material)
Target elutes in a long, low peak.
Elution buffer
- Try different elution conditions to increase KD.
Eluted target
Binding buffer
ml
KD low (10 -6 M)
KD still too low (10-3 M)
KD too low during binding. A 280
Flow through (unbound material)
Difficult or impossible to increase KD enough to elute the target without destroying it.
Elution buffer
Binding buffer Target not eluted
- Change ligand. ml
KD very low (10 -15 M)
147
Binding equilibria: Selective elution or competitive elution The examples shown have related to the changes in KD caused by non-selective elution techniques for affinity chromatography. However, competitive elution can also be interpreted in terms of changes in the binding equilibrium, as in the illustration below showing elution by adding a competing free ligand. A similar situation applies when adding a competing binding substance. L
T
+
C+T
LT
CT
Binding equilibrium for competing ligand. At equilibrium
KDComp =
KDComp is the equilibrium dissociation constant [C] is the concentration of free competing ligand
[C][T] [CT]
[T] is the concentration of free target [CT] is the concentration of the competing ligand/target complex
Graves and Wu in Methods in Enzymology 34, 140–163 (1974) have shown that: Eluted target Total bound target
~ ~
r r+1
rC0 KDCompL0 rC0 + KD r is the ratio between the volume of competitor added and the pore volume in the gel, assumed to be in the range 1–10 KD is the dissociation constant, coupled ligand KDComp is the dissociation constant, free competing ligand C0 is the concentration of competing ligand, usually 10-2 - 10-1 M L0 is the concentration of coupled ligand, usually 10-4 - 10-2 M
Again the derivation of the equation relies on some assumptions and simplifications and can only be expected to give a qualitative picture of what happens during binding. If KDComp and KD are similar then the concentrations of competing and coupled ligand should be similar to achieve effective elution. If KDComp is 10 x KD (i.e. the free competing ligand binds more weakly) then the concentration of competing ligand will need to be 10 x higher to achieve effective elution. If the competing ligand is not very effective in capturing the target protein at low concentrations so that the target is eluted from the column as a very broad peak, then a higher concentration of the competing ligand will be required to achieve elution. Since competing ligands are often expensive this is not a desirable situation. 148
Expected results with competitive elution KDComp too high or C0 too low. A 280
Flow through (unbound material) Elution buffer
Eluted target
Target elutes as a sharp peak.
Binding buffer
ml
KD for coupled form not too low (10-5 M)
KDComp for soluble competitor not too high (10-3 M)
Unexpected results with competitive elution A 280
Flow through (unbound material)
Target elutes in a long, low peak.
Elution buffer
- Increase competitor concentration or use a more effective competitor.
Eluted target
Binding buffer
ml
KD low (10-6 M)
KDComp too high or C0 too low
Kinetics of adsorption and desorption Overall kinetics are influenced by diffusion processes and slow kinetics during adsorption or desorption may create problems during an affinity separation. The effects of diffusion become noticeable for target molecules which are relatively large - they will diffuse more slowly than smaller target molecules thus taking longer to reach ligands in the interior of the gel and so slowing down the whole process.
Desorption is a first order reaction, i.e. the rate is not affected by ligand concentration. Expected results with fast on/off kinetics A 280
Flow through (unbound material) Elution buffer
Eluted target
Target elutes as a sharp peak.
Binding buffer
ml
Fast on
Fast off
149
Unexpected results - slow binding (adsorption) A 280
Flow through (unbound material)
Some of the target elutes under binding conditions as a broad, low peak.
Elution buffer
- Apply sample in aliquots to allow time for binding to take place.
Eluted target
Binding buffer
ml
Slow on
Fast off
Unexpected results - slow elution (desorption) A 280
Flow through (unbound material)
Target elutes as a long, low peak.
Elution buffer
- Change elution scheme.
Eluted target
Binding buffer
- Use pulsed elution (see page 22). ml
Fast on
150
Slow off
Appendix 8 Analytical assays during purification Analytical assays are essential to follow the progress of purification. They are used to assess the effectiveness of each step in terms of yield, biological activity, recovery and to help during optimization of experimental conditions. The importance of a reliable assay for the target molecule cannot be over-emphasized. When testing chromatographic fractions, ensure that the buffers used for purification do not interfere with the assay. Total protein determination Lowry or Bradford assays are used most frequently to determine the total protein content. The Bradford assay is particularly suited to samples where there is a high lipid content that may interfere with the Lowry assay. Purity determination Purity is most often estimated by SDS-PAGE. Alternatively, isoelectric focusing, capillary electrophoresis, reversed phase chromatography or mass spectrometry may be used. SDS-PAGE Analysis Reagents Required 6X SDS loading buffer: 0.35 M Tris-HCl (pH 6.8), 10.28% (w/v) SDS, 36% (v/v) glycerol, 0.6 M dithiothreitol (or 5% 2-mercaptoethanol), 0.012% (w/v) bromophenol blue. Store in 0.5 ml aliquots at -80 °C.
1. Add 2 µl of 6X SDS loading buffer to 5–10 µl of supernatant from crude extracts, cell lysates or purified fractions as appropriate. 2. Vortex briefly and heat for 5 minutes at +90 to +100 °C. 3. Load the samples onto an SDS-polyacrylamide gel. 4. Run the gel and stain with Coomassie Blue (Coomassie Blue R Tablets) or silver (PlusOne Silver Staining Kit, Protein).
The percentage of acrylamide in the SDS-gel should be selected according to the expected molecular weight of the protein of interest (see Table 19). Table 19. % Acrylamide in resolving gel
Separation size range
Single percentage: 5%
36 000–200 000
7.5%
24 000–200 000
10%
14 000–200 000
12.5%
14 000–100 000
15%
14 000–60 000
1
Gradient: 5–15%
1
14 000–200 000
5–20%
10 000–200 000
10–20%
10 000–150 000
The larger proteins fail to move significantly into the gel.
151
For information and advice on electrophoresis techniques, please refer to the section Additional reading and reference material. Functional assays Immunospecific interactions have enabled the development of many alternative assay systems for the assessment of active concentration of target molecules. • Western blot analysis is used when the sensitivity of SDS-PAGE with Coomassie Blue or silver staining is insufficient. 1. Separate the protein samples by SDS-PAGE. 2. Transfer the separated proteins from the gel to an appropriate membrane, such as Hybond™ ECL™ (for subsequent ECL detection) or Hybond P (for subsequent ECL Plus™ detection). 3. Develop the membrane with the appropriate specified reagents.
Electrophoresis and protein transfer may be accomplished using a variety of equipment and reagents. For further details, refer to the Protein Electrophoresis Technical Manual and Hybond ECL instruction manual, both from Amersham Pharmacia Biotech. • ELISAs are most commonly used as activity assays. • Functional assays using the phenomenon of surface plasmon resonance to detect immunospecific interactions (e.g. using BIACORE™ systems) enable the determination of active concentration, epitope mapping and studies of reaction kinetics. Detection and assay of tagged proteins SDS-PAGE, Western blotting and ELISAs can also be applied to the detection and assay of genetically engineered molecules to which a specific tag has been attached. In some cases, an assay based on the properties associated with the tag itself can be developed, e.g. the GST Detection Module for enzymatic detection and quantification of GST tagged proteins. Further details on the detection and quantification of GST and (His)6 tagged proteins are available in The Recombinant Protein Handbook: Protein Amplification and Simple Purification from Amersham Pharmacia Biotech.
152
Appendix 9 Storage of biological samples The advice given here is of a general nature and cannot be applied to every biological sample. Always consider the properties of the specific sample and its intended use before following any of these recommendations. General recommendations • Add stabilizing agents, if essential. Stabilizing agents are often required for storage of purified proteins. • Serum, culture supernatants and ascitic fluid should be kept frozen at -20 °C or -70 °C, in small aliquots. • Avoid repeated freeze/thawing or freeze drying/re-dissolving that may reduce biological activity. • Avoid conditions close to stability limits for example pH or salt concentrations, reducing or chelating agents. • Keep refrigerated at +4 °C in a closed vessel to minimize bacterial growth and protease activity. Above 24 hours at +4 °C, add a preserving agent if possible (e.g. merthiolate 0.01%). Sodium azide can interfere with many coupling methods and some biological assays and can be a health hazard. It can be removed by using a desalting column (see page 134). General recommendations for purified proteins • Store as a precipitate in high concentration of ammonium sulphate, for example 4.0 M. • Freeze in 50% glycerol, especially suitable for enzymes. • Avoid the use of preserving agents if the product is to be used for a biological assay. Preserving agents should not be added if in vivo experiments are to be performed. Instead store samples in small aliquots and keep frozen. • Sterile filter to prolong storage time. • Add stabilizing agents, e.g. glycerol (5–20%), serum albumin (10 mg/ml), ligand (concentration is selected based on concentration of active protein) to help to maintain biological activity. Remember that any additive will reduce the purity of the protein and may need to be removed at a later stage. • Avoid repeated freeze/thawing or freeze drying/re-dissolving that may reduce biological activity. Sodium azide can interfere with many coupling methods and some biological assays. It can be removed by using a desalting column (see page 134). Cryoproteins are a group of proteins, including some mouse antibodies of the IgG3 subclass, that should not be stored at +4 °C as they precipitate at this temperature. Keep at room temperature in the presence of a preserving agent. 153
Additional reading and reference material Code No.
Purification Antibody Purification Handbook
18-1037-46
Protein Purification Handbook
18-1132-29
Recombinant Protein Handbook: Protein Amplification and Simple Purification
18-1142-75
Gel Filtration Handbook: Principles and Methods
18-1022-18
Ion Exchange Chromatography Handbook: Principles and Methods
18-1114-21
Hydrophobic Interaction Chromatography Handbook: Principles and Methods
18-1020-90
Reversed Phase Chromatography Handbook: Principles and Methods
18-1112-93
Expanded Bed Adsorption Handbook: Principles and Methods
18-1124-26
Protein and Peptide Purification Technique Selection
18-1128-63
Fast Desalting and Buffer Exchange of Proteins and Peptides
18-1128-62
Gel Filtration Columns and Media Selection Guide
18-1124-19
Ion Exchange Columns and Media Selection Guide
18-1127-31
HIC Columns and Media Product Profile
18-1100-98
Affinity Columns and Media Product Profile
18-1121-86
Convenient Protein Purification, HiTrap Column Guide
18-1128-81
ÄKTAdesign Brochure
18-1129-05
Column Packing Video (PAL)
17-0893-01
Column Packing Video (NTSC)
17-0894-01
Analysis Gel Media Guide (electrophoresis)
18-1129-79
2D Electrophoresis Handbook
80-6429-60
Protein Electrophoresis Technical Manual
80-6013-88
ECL Western and ECL Plus Western Blotting Application Note
18-1139-13
Many of these items can be downloaded from www.apbiotech.com
154
Ordering information Product
Quantity
Code No.
HiTrap rProtein A FF
2 x 1 ml 5 x 1 ml 1 x 5 ml
17-5079-02 17-5079-01 17-5080-01
HiTrap Protein A HP
2 x 1 ml 5 x 1 ml 1 x 5 ml
17-0402-03 17-0402-01 17-0403-01
HiTrap Protein G HP
2 x 1 ml 5 x 1 ml 1 x 5 ml
17-0404-03 17-0404-01 17-0405-01
HiTrap Blue HP
5 x 1 ml 1 x 5 ml
17-0412-01 17-0413-01
HiTrap Heparin HP
5 x 1 ml 1 x 5 ml
17-0406-01 17-0407-01
HiTrap NHS-activated HP
5 x 1 ml 1 x 5 ml
17-0716-01 17-0717-01
HiTrap Chelating HP
5 x 1 ml 1 x 5 ml
17-0408-01 17-0409-01
Prepacked columns
HiTrap Streptavidin HP
5 x 1 ml
17-5112-01
HiTrap IgM Purification HP
5 x 1 ml
17-5110-01
HiTrap IgY Purification HP
1 x 5 ml
17-5111-01
GSTrap FF
2 x 1 ml 5 x 1 ml 1 x 5 ml
17-5130-02 17-5130-01 17-5131-01
HiTrap Benzamidine FF (high sub)
2 x 1 ml 5 x 1 ml 1 x 5 ml
17-5143-02 17-5143-01 17-5144-01
HiPrep 16/10 Heparin FF
1 x 20 ml
17-5189-01
MAbTrap Kit
HiTrap Protein G HP (1 x 1 ml), accessories, pre-made buffers for 10 purifications
17-1128-01
HisTrap Kit
3 x 1 ml HiTrap Chelating HP columns, pre-made buffers and accessories for uo to 12 purifications
17-1880-01
Protein A Sepharose CL-4B
1.5 g 25 ml
17-0780-01 17-0963-03
Protein A Sepharose 4 FF
5 ml 25 ml
17-0974-01 17-0974-04
rProtein A Sepharose FF
5 ml 25 ml
17-1279-01 17-1279-02
Protein G Sepharose 4 FF
5 ml 25 ml
17-0618-01 17-0618-02
Kits
Media
2´,5´ ADP Sepharose 4B
5g
17-0700-01
5´ AMP Sepharose 4B
5g
17-0620-01
Agarose Wheat Germ Lectin
5 ml
27-3608-02
Arginine Sepharose 4B
25 ml
17-0524-01
Benzamidine Sepharose 4 FF (high sub)
25 ml
17-5123-01
Blue Sepharose 6 FF
50 ml
17-0948-01
Calmodulin Sepharose 4B
10 ml
17-0529-01
Chelating Sepharose FF
50 ml
17-0575-01
155
Product
Quantity
Code No.
Con A Sepharose 4B
5 ml 100 ml
17-0440-03 17-0440-01
Gelatin Sepharose 4B
25 ml
17-0956-01
Glutathione Sepharose 4 FF
25 ml 100 ml 500 ml
17-5132-01 17-0532-02 17-0532-03
Glutathione Sepharose 4B
10 ml
17-0756-01
Heparin Sepharose 6 FF
50 ml 250 ml
17-0998-01 17-0998-25
IgG Sepharose 6 FF
10 ml
17-0969-01
Lentil Lectin Sepharose 4B
25 ml
17-0444-01
Red Sepharose CL-6B
10 g
17-0528-01
Streptavidin Sepharose HP
5 ml
17-5113-01
Pre-activated media and columns for ligand coupling HiTrap NHS-activated HP
5 x 1 ml 1 x 5 ml
17-0716-01 17-0717-01
NHS-activated Sepharose 4 FF
25 ml
17-0906-01
CNBr-activated Sepharose 4 FF
10 g
17-0981-01
CNBr-activated Sepharose 4B
15 g
17-0430-01
Activated CH Sepharose 4B
15 g
17-0490-01
ECH Sepharose 4B
50 ml
17-0571-01
Epoxy-activated Sepharose 6B
15 g
17-0840-01
EAH Sepharose 4B
50 ml
17-0569-01
Activated Thiol Sepharose 4B
15 g
17-0640-01
Thiopropyl Sepharose 6B
15 g
17-0420-01
2 x 2 ml
17-6002-35
Immunoprecipitation Immunoprecipitation Starter Pack Protein A Sepharose 4 Fast Flow Protein G Sepharose 4 Fast Flow
156
STREAMLINE, Sepharose, HiTrap, ÄKTA, MAbTrap, HisTrap, GSTrap, BioProcess, PhastSystem, PhastGel, FPLC, MicroSpin, Microplex, Multiphor, HiPrep, Sephadex, BioDirectory, Hybond, ECL, ECL Plus, ExcelGel and Superdex are trademarks of Amersham Pharmacia Biotech Limited or its subsidiaries. Amersham is a trademark of Nycomed Amersham plc Pharmacia and Drop Design are trademarks of Pharmacia Corporation BIACORE is a trademark of Biacore AB Multipipette and Eppendorf are trademarks of Eppendorf-Netheler-Hinz GmbH Tween is a trademark of ICI Americas Inc Cibacron is a registered trademark of Ciba-Geigy Corp Procion and Coomassie are trademarks of ICI plc. Triton is a trademark of Union Carbide Chemicals and Plastics Co. Nonidet is a trademark of Shell Co. Ltd. Pefabloc is a trademark of Pentafam AG.
All goods and services are sold subject to the terms and conditions of sale of the company within the Amersham Pharmacia Biotech group that supplies them. A copy of these terms and conditions is available on request. © Amersham Pharmacia Biotech AB 2001 – All rights reserved. Amersham Pharmacia Biotech AB Björkgatan 30, SE-751 84 Uppsala, Sweden Amersham Pharmacia Biotech UK Limited Amersham Place, Little Chalfont, Buckinghamshire HP7 9NA, England Amersham Pharmacia Biotech Inc 800 Centennial Avenue, PO Box 1327, Piscataway, NJ 08855, USA Amersham Pharmacia Biotech Europe GmbH Munzinger Strasse 9, D-79111 Freiburg, Germany Amersham Pharmacia Biotech KK, Sanken Bldg. 3-25-1, Hyakunincho Shinjuku-ku, Tokyo 169-0073, Japan
www.apbiotech.com Production: RAK Design AB
Antibody Purification –
Antibody Purification Handbook
Handbook
www.amershambiosciences.com
Back to Collection 18-1037-46 Edition AB
Handbooks from Amersham Pharmacia Biotech
Antibody Purification Handbook 18-1037-46
The Recombinant Protein Handbook Protein Amplification and Simple Purification 18-1142-75
Protein Purification Handbook 18-1132-29
Ion Exchange Chromatography
Reversed Phase Chromatography
Principles and Methods 18-1114-21
Principles and Methods 18-1134-16
Affinity Chromatography
Expanded Bed Adsorption
Principles and Methods 18-1022-29
Principles and Methods 18-1124-26
Hydrophobic Interaction Chromatography
Chromatofocusing
Principles and Methods 18-1020-90
with Polybuffer and PBE 18-1009-07
Gel Filtration
Microcarrier cell culture
Principles and Methods 18-1022-18
Principles and Methods 18-1140-62
Asia Pacific Tel: +852 2811 8693 Fax: +852 2811 5251 Australasia Tel: +61 2 9899 0999 Fax: +61 2 9899 7511 Austria Tel: 01 576 0616 20 Fax: 01 576 0616 27 Belgium Tel: 0800 73 888 Fax: 03 272 1637 Canada Tel: 1 800 463 5800 Fax: 1 800 567 1008 Central, East, South East Europe Tel: +43 1 982 3826 Fax: +43 1 985 8327 Denmark Tel: 45 16 2400 Fax: 45 16 2424 Finland & Baltics Tel: +358 (0)9 512 3940 Fax: +358 (0)9 512 1710 France Tel: 0169 35 67 00 Fax: 0169 41 9677 Germany Tel: 0761 4903 401 Fax: 0761 4903 405 Italy Tel: 02 27322 1 Fax: 02 27302 212 Japan Tel: 81 3 5331 9336 Fax: 81 3 5331 9370 Latin America Tel: +55 11 3667 5700 Fax: +55 11 3667 87 99 Middle East and Africa Tel: +30 (1) 96 00 687 Fax: +30 (1) 96 00 693 Netherlands Tel: 0165 580 410 Fax: 0165 580 401 Norway Tel: 2318 5800 Fax: 2318 6800 Portugal Tel: 21 417 7035 Fax: 21 417 3184 Russian & other C.I.S. & N.I.S. Tel: +7 (095) 232 0250,956 1137 Fax: +7 (095) 230 6377 South East Asia Tel: 60 3 8024 2080 Fax: 60 3 8024 2090 Spain Tel: 93 594 49 50 Fax: 93 594 49 55 Sweden Tel: 018 612 19 00 Fax: 018 612 19 10 Switzerland Tel: 01 802 81 50 Fax: 01 802 81 51 UK Tel: 0800 616 928 Fax: 0800 616 927 USA Tel: +1 800 526 3593 Fax: +1 877 295 8102 Hybond, HiTrap, GSTrap, Sephadex, Superdex, HisTrap, Sepharose, MAbTrap, ÄKTA, FPLC, HiPrep, PhastSystem, PhastGel, SOURCE, RESOURCE, HiLoad, STREAMLINE, ECL, ECL Plus, MicroSpin, Mono Q, Mono S, Monobeads, BioProcess and Drop Design are trademarks of Amersham Biosciences Limited. Amersham and Amersham Biosciences are trademarks of Amersham plc. BIACORE is a trademark of Biacore AB. Multipipette and Eppendorf are trademarks of Eppendorf-Netheler-Hinz GmbH. Tween is a trademark of ICI Americas Inc. Cibacron is a registered trademark of Ciba-Geigy Corp. Coomassie is a trademark of ICI plc. All goods and services are sold subject to the terms and conditions of sale of the company within the Amersham Biosciences group that supplies them. A copy of these terms and conditions is available on request. Amersham Biosciences UK Limited, Amersham Place, Little Chalfont, Buckinghamshire HP7 9NA, England. Amersham Biosciences AB, Björkgatan 30, SE-751 84 Uppsala, Sweden. Amersham Biosciences Corp., 800 Centennial Avenue, PO Box 1327, Piscataway NJ 08855, USA. Amersham Biosciences Europe GmbH, Munzinger Strasse 9, D-79111 Freiburg, Germany. Amersham Biosciences KK, Sanken Bldg. 3-25-1, Hyakunincho Shinjuku-ku, Tokyo 169-0073, Japan. © Amersham Biosciences AB 2002 – All rights reserved.
Antibody Purification Handbook
1
Content Introduction ............................................................................................................... 5 Chapter 1 Antibody structure, classification and production ....................................................... 7 Native sources .............................................................................................................. 7 Genetically engineered sources .................................................................................... 10
Chapter 2 Sample preparation ................................................................................................. 13 Sources and their associated contaminants ................................................................... 13 Extraction of recombinant antibodies and antibody fragments ......................................... 13 Clarification of serum, ascitic fluid, culture supernatant or cell lysates ............................ 15 Sample preparation before purification ......................................................................... 16
Chapter 3 Simple, rapid purification by affinity chromatography ............................................... 25 IgG classes, fragments and subclasses .......................................................................... 27 Using Protein G Sepharose media ................................................................................. 28 Using MAbTrap Kit ...................................................................................................... 31 Using Protein A Sepharose or rProtein A Sepharose media ............................................. 33 Fab, F(ab')2 fragments ................................................................................................. 36 IgA ............................................................................................................................. 38 IgD ............................................................................................................................. 38 IgE ............................................................................................................................. 38 IgM ............................................................................................................................ 38 Avian IgY from egg yolk ............................................................................................... 41 Making specific purification columns ............................................................................ 43
Chapter 4 Immunoprecipitation ................................................................................................ 49 Chapter 5 Multi-step purification strategies ............................................................................. 53 Selection and combination of purification techniques .................................................... 54 Selection of media for multi-step purification ................................................................ 58 Examples of multi-step purification .............................................................................. 60
Chapter 6 Removal of specific contaminants after initial purification ....................................... 69 Bovine immunoglobulins .............................................................................................. 69 Albumin and transferrin ............................................................................................... 70 a2-macroglobulin and haptoglobulin ............................................................................. 72 Dimers and aggregates ................................................................................................ 72 DNA and endotoxins .................................................................................................... 73 Affinity ligands ............................................................................................................ 73
2
Chapter 7 Large-scale purification ........................................................................................... 75 Considerations for monoclonal antibody purification ....................................................... 76 Combining sample preparation and capture for Fc-containing antibodies ......................... 77 BioProcess Media for production .................................................................................. 78
Appendix 1 .............................................................................................................. 79 Analytical assays during purification ............................................................................. 79
Appendix 2 .............................................................................................................. 81 Selection of purification equipment .............................................................................. 81
Appendix 3 .............................................................................................................. 82 General instructions for affinity purification with HiTrap columns .................................... 82
Appendix 4 .............................................................................................................. 84 Column packing and preparation .................................................................................. 84
Appendix 5 .............................................................................................................. 86 Use of sodium hydroxide for cleaning chromatographic media and systems ..................... 86
Appendix 6 .............................................................................................................. 88 Storage of biological samples ....................................................................................... 88
Appendix 7 .............................................................................................................. 90 Table of amino acids .................................................................................................... 90
Appendix 8 .............................................................................................................. 92 Converting flow rates from linear flow rates (cm/h) to volumetric flow rates (ml/min) and vice versa ............................................................................................................. 92
Appendix 9 .............................................................................................................. 93 Protein conversion data ............................................................................................... 93
Appendix 10 ............................................................................................................ 94 Principles and standard conditions for purification techniques ........................................ 94 Additional reading and reference material ................................................................... 101 Ordering information ................................................................................................. 102
3
4
Introduction The diversity of the antibody-antigen interaction and our ability to manipulate the characteristics of the interaction has created many uses for antibodies and antibody fragments, both for immunochemical techniques within general research and for therapeutic and diagnostic applications. The use of recombinant technology opens up the potential to create an infinite number of combinations between immunoglobulins, immunoglobulin fragments, tags and selected proteins, further manipulating these molecules to our advantage. The purpose of this handbook is to present the most effective and most frequently used strategies for sample preparation and purification of the many different forms of antibodies and antibody fragments used in the laboratory. Advice is given on how to plan a purification strategy, beginning with a consideration of the factors shown in Figure 1. Wherever possible, examples and practical protocols are included to provide a ready-to-use solution or at least a good starting point for further optimization of a specific purification. Purity required for final application It is hoped that this blend of general -Purity check and functional analysis -Importance and properties of guidance and specific examples will remaining impurities assist the reader in a successful approach to any purification. Physico-chemical characteristics -Size -Charge -pI -Stability
Scale of purification -µg -mg -g
Source -Sample preparation
Economy -Time and expense
Fig. 1. Factors to consider when planning purification.
Symbols this symbol indicates general advice which can improve procedures or provide recommendations for action under specific situations. this symbol denotes advice which should be regarded as mandatory and gives a warning when special care should be taken. this symbol highlights troubleshooting advice to help analyse and resolve difficulties that may occur.
5
6
Chapter 1 Antibody structure, classification and production Antibodies are members of a family of molecules, the immunoglobulins, that constitute the humoral branch of the immune system and form approximately 20% of the plasma proteins in humans. Different populations of immunoglobulins are found on the surface of lymphocytes, in exocrine secretions and in extravascular fluids. Antibodies are host proteins produced in response to foreign molecules or other agents in the body. This response is a key mechanism used by a host organism to protect itself against the action of foreign molecules or organisms. B-lymphocytes carrying specific receptors recognize and bind the antigenic determinants of the antigen and this stimulates a process of division and differentiation, transforming the B-lymphocytes into plasma cells. It is these lymphoid or plasma cells that predominantly synthesize antibodies.
Native sources Immunoglobulins All immunoglobulins, independent of their specificity, have a common structure with four polypeptide chains: two identical heavy (H) chains, each carrying covalently attached oligosaccharide groups; and two identical, non-glycosylated light (L) chains. A disulphide bond joins a heavy chain and a light chain together. The heavy chains are also joined to each other by disulphide bonds. These disulphide bonds are located in a flexible region of the heavy chain known as the hinge, a region of approximately 12 amino acids that is exposed to enzymatic or chemical cleavage. Each globular region formed by the folding of the polypeptide chains as a result of the disulphide bonding is termed a domain. All four polypeptide chains contain constant (C) and variable (V) regions, found at the carboxyl and amino terminal portions, respectively. Heavy and light chains have a single V region, while light chains possess a single C region. Heavy chains contain three C regions. The V regions of both heavy and light chains combine to form two identical antigen binding sites (the parts of the antibody which bind the antigen). Effector functions of antibodies, such as placental transport or antigen-dependent cellular toxicity, are mediated by structural determinants within the Fc region of the immunoglobulin. Figure 2 illustrates the basic H2L2 structure of a typical immunoglobulin.
Fig. 2.
7
Immunoglobulins are divided into five major classes according to their H chain components: IgG (g), IgA (a), IgM (µ), IgD (d) and IgE (e). There are two types of light chain, k and l. Individual molecules may contain k or l chains but never both. In man, the ratio of immunoglobulins containing k or l light chains is about 60:40, whereas in mouse the ratio is 95:5. Figure 3 and Table 1 give a summary of human and mouse antibody classes and their physicochemical characteristics. Antibody classes IgG
IgM
IgA
IgE
IgD
g k or l
m k or l
a
e
k or l
k or l
d k or l
Characteristic Heavy chain Light chain
Y structure
Fig. 3.
1. Antibodies of classes G, D and E are of monomeric type H2L2. 2. IgA in serum is mainly monomeric, but in secretions, such as saliva and tears, IgA is found as a dimer held together by the secretory piece and the J-polypeptide chain (H2L2)-SC-J-(H2L2). The dimer has four antigen binding sites. 3. IgM is composed of five monomeric units (H2L2)5 and has ten antigen binding sites. 4. IgG and IgA are further divided into subclasses that result from minor differences in the amino acid sequence within each class. In humans, four IgG subclasses IgG1, IgG2, IgG3 and IgG4 have g1, g2, g3 and g4 heavy chains, respectively. Mouse IgG has four IgG subclasses: IgG1, IgG2a, IgG2b and IgG3, with heavy chains g1, g2a, g2b and g3. These heavy chains have virtually the same size and similar electrophoretic properties, but their amino acid sequences differ considerably. Human IgA has two subclasses, IgA1 and IgA2, while mouse IgA has only one subclass. Immunoglobulin
Heavy chain
Light chain
Sedimentation coefficient
IgG1 IgG2 IgG3 IgG4 IgM IgA1 IgA2 IgA3 IgD IgE
l1 l1 l1 l1 µ a1 a2 a1 , a 2 d e
k, l k, l k, l k, l k, l k, l k, l k, l k, l k, l
7S 7S 7S 7S 19S 7S 7S 11S 7S 8S
Mol. Wt (Mr) 146 146 170 146 900 160 160 370 184 190
000 000 000 000 000 000 000 000 000 000
Mr heavy chain
Carbohydrate content (%)
50 000 50 000 60 000 50 000 68 000 56 000 52 000 52–56 000 68 000 72 000
2-3 2-3 2-3 2-3 12 7-11 7-11 11 12 12
Mr heavy chain
Carbohydrate content (%)
pI
2–3 2–3 2–3 2–3 12 7–11 12–14 12
7.0–8.5 6.5–7.5 5.5–7.0 – 4.5–7.0 4.0–7.0 – –
A280nm
pI
13.8
5.0–9.5 5.0–8.5 8.2–9.0 5.0–6.0 5.1–7.8 5.2–6.6 5.2–6.6 4.7–6.2 -
12.5 13.4
17.0 15.3
Table 1a. Physico-chemical properties of human immunoglobulins.
Immunoglobulin IgG1 IgG2a IgG2b IgG3 IgM IgA IgD IgE
Heavy chain
Light chain
Sedimentation coefficient
l1 l2a l2b l3 µ a d e
k, l k, l k, l k, l k, l k, l k, l k, l
7S 7S 7S 7S 19S 7S 7S 8S
Mol. Wt (Mr) 150 150 150 150 900 170 180 190
000 000 000 000 000 000 000 000
Table 1b. Physico-chemical properties of mouse immunoglobulins.
8
50 50 50 50 80 70 68 80
000 000 000 000 000 000 000 000
IgY immunoglobulin The use of avian antibodies, IgY, has several major advantages. Avian species produce an elevated antibody response to highly conserved, weakly immunogenic mammalian antigens. Because of the phylogenetic distance between birds and mammals, IgY can be used to provide a source of highly specific antibodies against mammalian antigens with minimum cross reactivity. The antibodies are most commonly produced in eggs. Eggs are more easily collected than blood samples and a few eggs per week can provide the same amount of immunoglobulin as repeated bleeding of an immunized rabbit.
Antibody fragments Partial enzymatic digestion of immunoglobulins generates biologically active antibody fragments that can be used to elucidate antibody structure or as specific reagents. These fragments can also be produced using recombinant technology. Fragmentation of immunoglobulins has created the potential for new applications. For example, chimeric, non-immunogenic 'humanized' mouse Fab, Fab' and F(ab')2 fragments are of great interest in tumour therapy since they penetrate tumours more rapidly and are also cleared from the circulation more rapidly than full size antibodies. The most common types of antibody fragments are listed below. Figure 4 shows the fragments created by enzymatic cleavage. Fab and Fc fragments: papain digestion creates two Fab (antigen binding) fragments and one Fc (crystallizable) fragment. F(ab')2 fragment: pepsin digestion creates a fragment containing two antigen binding sites and comprises two Fab units and the hinge. Fv fragment: an unstable fragment able to bind to an antigen. An Fv fragment has two V regions, VL and VH. Single chain Fv fragment (scFv): scFv is a stable variant of Fv, commonly produced by recombinant technology, in which a peptide linker connects the two V regions. Fd fragment: the N-terminal half of the H chain.
Fig. 4. Antibody fragments are created by enzymatic cleavage.
9
Polyclonal antibodies Most frequently, a host will produce a large number of antibodies that recognizes independent epitopes (the antibody binding site) on the antigen. Each specific antibody is produced by a different clone of plasma cells. Serum is a very good source of polyclonal antibodies. These antibodies are commonly used as reagents in immunochemical techniques, using crude serum as the source. Further purification may be required, either to isolate the group of polyclonal antibodies or to isolate a specific antibody from the group.
Monoclonal antibodies Hybridoma cells are created by isolating plasma cell precursors which are then fused with immortal cells. The hybridoma cells can be single cell cloned and expanded as individual clones that secrete only one antibody type, a monoclonal antibody. The high specificity of a monoclonal antibody is a significant advantage, particularly in therapeutic applications. Monoclonal antibodies are frequently used in the form of tissue culture supernatants harvested from the hybridoma culture, or as crude extracts produced from hybridoma cells grown as tumours in syngenic mice. Production of monoclonal antibodies using hybridoma technology has been successful for the production of mouse monoclonal antibodies, but this has meant that therapeutic applications have always been associated with the risk of immunogenic reactions (only human antibodies are non-immunogenic to humans).
Genetically engineered sources Recombinant technology is used increasingly for the manipulation and production of antibodies and their fragments. For antibodies to be most effective when used as a therapeutic agent they should have a long serum half-life, low immunogenicity, a high affinity for the antigen, and be able to neutralize the antigen's activity. These are all features that can be enhanced by genetic manipulation. To reduce immunogenicity, mouse-human chimeric antibodies have been produced, containing some human constant region sequences along with the mouse V regions. Another approach to reducing immunogenicity is to produce humanized monoclonal antibodies that contain human sequences. Antibody phage libraries and breeding mice that contain parts of the human immune system provide alternative sources of therapeutic antibodies with a fully human sequence. Figure 5 illustrates various modifications to monoclonal antibodies.
Fig. 5.
10
Antibody fragments The enzymatic mechanisms used to generate antibody fragments are shown in Figure 4. Antibody fragments are also produced using recombinant technology.
Antibody fusion proteins For research, diagnostic and therapeutic applications the potential uses for antibody fusion proteins are vast. Combining a fusion partner with all or part of an antibody can enable the antibody or fragment to access specific areas of the host (e.g. crossing the blood-brain barrier), carry an enzyme to a specific site (e.g. for therapy or to create a drug at site) or carry a toxin to a specific site for therapy.
Antibody fusion proteins are divided into two groups: 1. Fab and F(ab')2 fusions, in which the single or double antigen binding site(s) is retained and a fusion partner either replaces or is linked to the Fc domain. 2. Fc fusions, also known as immunoadhesions, in which the antigen recognition site is replaced by the fusion partner, but the Fc region is retained. Depending upon the type of immunoglobulin involved, an Fc fusion will retain effector functions and can confer a longer half life to the fusion protein.
Tagged fusion antibodies and fragments Amplification of a protein containing a tag of known size and biological function greatly simplifies subsequent isolation, purification and detection. For example, (His)6 or GST tags are now in common use to enable simple affinity purification at any scale. In some cases the protein yield can also be increased. Adding tags of this type is also extremely useful if the target molecule has no Fc region (an Fc region enables purification with Protein A Sepharose™ or Protein G Sepharose affinity media). Epitope tags (short peptide sequences to which strongly binding, highly specific antibodies have already been produced) are used for detection and purification in many immunological methods. Table 2 reviews some of the practical advantages and disadvantages of using tagged proteins. Advantages
Disadvantages
Cell compartments can be targeted
Tag may interfere with protein structure and affect folding and biological activity
Provide a marker for expression
Not always possible to remove the tag without modifying the sequence of interest
Simple purification using affinity chromatography under denaturing or non-denaturing conditions Easy detection Refolding achievable on a chromatography column Ideal for secreted proteins as the product is easily isolated from the growth medium
Table 2.
General guidelines for the amplification and purification of recombinant proteins are covered in detail in the Recombinant Protein Handbook: Protein Amplification and Simple Purification from Amersham Biosciences.
11
12
Chapter 2 Sample preparation Sources and their associated contaminants Antibodies and antibody fragments are produced from native and recombinant sources. Table 3 reviews some of the most common options. The choice of source material can affect the choice of techniques for sample preparation and purification due to the differences in specific contaminants and the quantity of target molecule required. However, in many cases, the high selectivity of an affinity purification medium for a specific molecule minimizes contamination and produces a sample of high purity in a single step. Molecular types
Significant contaminants
Quantity
Human serum
Polyclonal IgG, IgM, IgA, IgD, IgE
albumin, transferrin, a2-macroglobulin, other serum proteins
IgG 8–16 mg/ml IgM 0.5–2 mg/ml IgA 1–4 mg/ml IgE 10–400 ng/ml IgD up to 0.4 mg/ml
Hybridoma: cell culture supernatant with 10% foetal calf serum
Monoclonal
Phenol red, water, albumin, transferrin, bovine IgG, a2-macroglobulin, other serum proteins, viruses
Up to 1 mg/ml
Monoclonal
Albumin, transferrin (often added as supplements)
Up to 0.05 mg/ml
Ascites fluid
Monoclonal
Lipids, albumin, transferrin, lipoproteins, endogenous IgG, other host proteins
1–15 mg/ml
Egg yolk
IgY
Lipids, lipoproteins and vitellin
IgY 3–4 mg/ml
Tagged antibodies, antibody fusion proteins, Fab or F(ab')2 fragments
Proteins from the host, e.g. E. coli. General low level of contamination
Depends upon expression system
Proteins from the host, e.g. E. coli, phage
Depends upon expression system
Source: native
Hybridoma: cell culture supernatant serum free
Source: recombinant Extracellular protein expressed into supernatant Intracellular protein expression
Table 3.
An advantage of cell culture systems is the unlimited volume and quantity of material that can be produced. For ascites, there is a limited production and, in certain countries, significant legal restrictions on their production.
Extraction of recombinant antibodies and antibody fragments The source and location of the recombinant molecule, e.g. bacterial or mammalian, interor intra-cellularly expressed, soluble or in the form of inclusion bodies, will determine the extraction procedure. Selection of an extraction technique depends as much on the equipment available and scale of operation as on the type of sample. Examples of common extraction processes are shown in Table 5. Buffer components should be selected to stabilize the extraction conditions. Table 4 reviews some of the substances most commonly used in buffer systems.
13
Use procedures that are as gentle as possible since disruption of cells or tissues leads to the release of proteolytic enzymes and general acidification. Use additives (see Table 4) only if essential for stabilization of the product or to improve extraction. Select additives that are easily removed otherwise and additional purification step may be required. Additives such as 8 M urea or 6 M guanidine hydrochloride can be included if solubilization of the protein is needed (e.g. if the protein is expressed as an inclusion body). Typical conditions for use
Purpose
20 mM, pH 7.4
maintain pH minimize acidification caused by lysosomal disruption
NaCl
100 mM
maintain ionic strength of medium
EDTA
10 mM
reduce oxidation damage, chelate metal ions
Sucrose or glucose
25 mM
stabilize lysosomal membranes, reduce protease release
Buffer components Tris
Ionic or non-ionic detergent
DNAse and RNAse
solubilize poorly soluble proteins refer to The Recombinant Protein Handbook, Protein Amplification and Simple Purification for details on handling inclusion bodies 1 µg/ml
Protease inhibitors*
degradation of nucleic acids, reduce viscosity of sample solution Inhibits
PMSF
0.5–1 mM
APMSF
0.4–4 mM
serine proteases serine proteases
Benzamidine-HCl
0.2 mM
serine proteases
Pepstatin
1 µM
aspartic proteases
Leupeptin
10–100 µM
cysteine and serine proteases
Chymostatin
10–100 µM
chymotrypsin, papain, cysteine proteases
Antipain-HCl
1–100 µM
papain, cysteine and serine proteases
EDTA
2–10 mM
metal dependent proteases, zinc and iron
EGTA
2–10 mM
metal dependent proteases e.g. calcium
1, 4 dithiothreitol, DTT
1–10 mM
keep cysteine residues reduced
1, 4 dithioerythritol, DTE
1–10 mM
keep cysteine residues reduced
Mercaptoethanol
0.05%
keep cysteine residues reduced
5–10%
for stabilization, up to 50% can be used if required
Reducing agents
Others Glycerol
PMSF - Phenylmethylsulphonyl fluoride. APMSF - 4-Aminophenyl-methylsulphonyl fluoride. PMSF is a hazardous chemical. Half-life time in aqueous solution is 35 min. PMSF is usually stored as 10 mM or 100 mM stock solution (1.74 or 17.4 mg/ml in isopropanol) at -20°C. * Protease inhibitors are available in pre-made mixes from several suppliers. Details taken from Protein Purification, Principles and Practice, R.K. Scopes. 1994, Springer., Protein Purification, Principles, High Resolution Methods and Applications, J-C. Janson and L. Rydén, 1998, 2nd ed. Wiley Inc. and other sources.
Table 4. Common substances used in sample buffers.
14
Extraction process
Typical conditions
Protein source
Comment
Gentle Cell lysis (osmotic shock)
2 volumes water to 1 volume packed pre-washed cell
E. coli periplasm: intracellular proteins
lower product yield but reduced protease release
Enzymatic digestion
lysozyme 0.2 mg/ml, +37°C, 15 mins.
bacteria: intracellular proteins
lab scale only, often combined with mechanical disruption
Moderate Grinding with abrasive e.g. sand
follow equipment instructions
bacteria
Vigorous Ultrasonication or bead milling
"
cell suspensions: intracellular proteins in cytoplasm, periplasm, inclusion bodies
small-scale, release of nucleic acids may cause viscosity problems, inclusion bodies must be resolubilized
Manton-Gaulin homogenizer
"
cell suspensions
large-scale only
French press
"
bacteria
Table 5. Common sample extraction processes for recombinant antibodies and antibody fragments.
Extraction should be performed quickly, at sub-ambient temperatures, in the presence of a suitable buffer (see Table 4) to maintain pH and ionic strength and to stabilize the sample. If lysates are too viscous to handle (caused by a high concentration of host nucleic acid), continue to sonicate on ice for a longer period, or follow one of the following procedures: 1. Add DNase I to a final concentration of 10 µg/ml. 2. Add RNase A to a final concentration of 10 µg/ml and DNase I to 5 µg/ml, and incubate on ice for 10-15 min. 3. Draw the lysate through a syringe needle several times to avoid adding enzymes.
Clarification of serum, ascitic fluid, culture supernatant or cell lysates Centrifugation and filtration are standard laboratory techniques for sample clarification from any source and are used routinely when handling small samples. Lipids and lipoproteins can clog chromatographic columns and should be removed prior to purification. Ascitic fluid has a particularly high lipid content. See removal of specific impurities on page 16. Phenol red is often added to cell culture supernatants as a pH indicator. Since phenol red may bind to certain chromatographic media, it is advisable to remove it prior to purification. See removal of specific impurities on page 17.
Centrifugation Centrifugation removes lipids and particulate matter, such as cell debris. For small sample volumes or proteins that adsorb non-specifically to filters, centrifuge at 10 000 x g for 15 minutes. For cell lysates, centrifuge at 40 000-50 000 x g for 30 minutes. Serum samples can be filtered through glass wool after centrifugation to remove any remaining lipids.
15
Filtration Filtration removes particulate matter and is suitable for small sample volumes. For sample preparation before chromatography select a filter size in relation to the bead size of the chromatographic medium. Nominal pore size of filter
Particle size of chromatographic medium
1 µm
90 µm and upwards
0.45 µm
34 µm
0.22 µm
3, 10, 15 µm or when extra clean samples or sterile filtration is required
Check the recovery of the target protein in a test run. Some proteins may adsorb nonspecifically to filter surfaces.
Buffer exchange and desalting Desalting columns are suitable for buffer exchange of any sample volume and remove many smaller contaminants in a single step. Use before and/or between purification steps. Rapidly process small or large sample volumes. Remove salts from proteins with molecular weight Mr > 5 000. Detailed procedures for buffer exchange and desalting are given on page 20.
Sample preparation before purification The main tasks of the sample preparation stage prior to purification are: Removal of specific impurities, such as lipoproteins or phenol red, from the source material. Removal of gross impurities, such as bulk protein, from the source material. Buffer exchange and desalting to transfer sample to the correct buffer conditions (pH and salt concentration) and to remove unwanted small molecules.
At laboratory scale, when samples are reasonably clean after filtration and centrifugation, the buffer exchange and desalting step can be omitted, particularly if affinity chromatography is used for purification. It may be sufficient to adjust the pH of the sample and, if necessary, dilute to reduce the ionic strength of the solution.
Removal of specific impurities before purification Lipoproteins Lipoproteins and other lipid material can clog chromatography columns. It is advisable to remove them before beginning purification. Ascitic fluid often has a high content of lipid material. The alternatives described here are suitable for treatment of serum, ascites and cell culture supernatant. Centrifuge samples to avoid the risk of non-specific binding of the target molecule to a filter. Samples such as serum can be filtered through glass wool to remove remaining lipids. 16
Alternative 1: Dextran sulphate precipitates lipoproteins in the presence of divalent cations, such as Ca2+. The precipitate can be removed by centrifugation. 1. Add 0.04 ml 10% dextran sulphate solution and 1 ml 1 M CaCl2 per ml of sample. 2. Mix for 15 minutes. 3. Centrifuge (10 000 x g for 10 minutes). 4. Discard precipitate. 5. Exchange sample into a suitable buffer for purification using a desalting column (see page 20).
Alternative 2: Polyvinylpyrrolidine (PVP) produces a pH dependent precipitation effect. Note that 8% PVP precipitates b-lipoproteins and euglobulins at pH 7.0, but below pH 4.0 the lipoproteins do not precipitate. 1. Add solid PVP to the sample solution to a final concentration of 3% (w/v). 2. Stir for 4 hours at +4°C. 3. Centrifuge at 17 000 x g. 4. Discard precipitate. 5. Exchange sample into a suitable buffer for purification using a desalting column (see page 20).
Phenol red Phenol red is used at laboratory scale as a pH indicator in cell culture. Although not directly interfering with purification, phenol red may bind to certain purification media and should be removed as early as possible to avoid the risk of contamination. It is known to bind to anion exchange media at pH > 7. Use a desalting column to remove phenol red (a low molecular weight molecule) and transfer sample to the correct buffer conditions for further purification (see page 20).
Removal of gross impurities Low molecular weight contaminants If samples contain a high level of low molecular weight contaminants, use a desalting column, as already described, to prepare the sample for the first chromatographic purification
Fractional precipitation Fractional precipitation is frequently used at laboratory scale to remove gross impurities from small sample volumes, and occasionally used in small-scale commercial production. When using a HiTrap™ affinity purification column at laboratory scale, it is unlikely that fractional precipitation will be required. Precipitation techniques separate fractions by the principle of differential solubility. Because protein species differ in their degree of hydrophobicity, increased salt concentrations can enhance hydrophobic interactions between the proteins and cause precipitation. Fractional precipitation can be applied to remove gross impurities in three different ways, as shown in Figure 6. These techniques are reviewed in Table 6.
17
Clarification Bulk proteins and particulate matter precipitated
Supernatant
Extraction Clarification Concentration Target protein precipitated with proteins of similar solubility
Redissolve pellet*
Extraction Clarification Bulk proteins and particulate matter precipitated
Concentration Target protein precipitated
Chromatography
Redissolve pellet*
Remember: if precipitating agent is incompatible with next purification step, use Sephadex ™ G-25 for desalting and buffer exchange e.g. HiTrap Desalting or PD-10 columns
*Remember: not all proteins are easy to redissolve, yield may be reduced
Fig. 6. Three ways to use precipitation.
Most precipitation techniques are not suitable for large-scale preparation. Precipitation techniques are affected by temperature, pH and sample concentration. These parameters must be controlled to ensure reproducible results. Not all proteins are easy to redissolve, yield may be reduced. Precipitation agent
Comment
Ammonium sulphate
Stabilizes proteins. Helps reduce lipid content. Antibody concentration should be > 1 mg/ml. Sample can be concentrated into a pellet. Most samples retain native form. Excellent if HIC is subsequent purification step.
Caprylic acid
Sample remains in supernatant and is not concentrated. Antibody concentration should be > 1 mg/ml.
Polyethylene glycol
Stabilizes proteins. Frequently used for polyclonal antibodies and monoclonal IgM. IgMs precipitate more readily than IgGs. PEG-600 behaves as a Mr 50 000-100 000 globular protein in gel filtration and is easily separated from IgM, but difficult to remove from smaller molecules.
Ethacridine
Sample remains in supernatant and is not concentrated. Used mainly in commercial preparation. Precipitates lipids, DNA, viral particles and endotoxins. Toxic!
Table 6. Commonly used precipitation agents.
Ammonium sulphate precipitation Ammonium sulphate is used most frequently to precipitate, and thus concentrate, immunoglobulins from a crude source. Some proteins may be damaged by ammonium sulphate. Take care when adding crystalline ammonium sulphate: high local concentrations may cause contamination of the precipitate with unwanted proteins. For routine, reproducible purification, precipitation with ammonium sulphate should be avoided in favour of chromatography.
18
Adding an equal volume of saturated (or even 35-40% saturated) solution reduces contamination by transferrin and albumin. Discard any lipoproteins that may form a layer after centrifugation. Samples can be filtered through glass wool to remove any remaining lipids. Preparation of saturated ammonium sulphate solution Add 100 g ammonium sulphate to 100 ml distilled water, stir to dissolve.
1. Filter (0.45 µm) or centrifuge (refrigerated, 10 000 x g) the sample. 2. Add 1 part 1 M Tris-HCl, pH 8.0 to 10 parts sample volume to maintain pH. 3. Stir gently. Add saturated ammonium sulphate solution, drop by drop (solution becomes milky at about 20% saturation). Add up to 50% saturation*. Stir for 1 hour. 4. Centrifuge for 20 minutes at 10 000 x g. 5. Discard supernatant. Wash pellet twice by resuspension in an equal volume of ammonium sulphate solution of the same concentration (i.e. a solution that will not redissolve the precipitated protein or cause further precipitation). Centrifuge again, as in Step 4. 6. Dissolve pellet in a small volume of the start buffer. 7. Ammonium sulphate is removed during clarification/buffer exchange steps with Sephadex G-25, using desalting columns (see page 20), or during hydrophobic interaction purification. *The % saturation can be adjusted to either precipitate a target molecule or to precipitate contaminants.
The quantity of ammonium sulphate required to reach a given degree of saturation varies with temperature. Table 7 shows the quantities required at +20°C. Final percent saturation to be obtained 20
25
Starting percent saturation 0 5
30
35
40
45
50
55
60
65
70
75
80
85
90
95 100
Amount of ammonium sulphate to add (grams) per litre of solution at +20°C 113 144 176 208 242 277 314 351 390 430 472 516 561 608 657 708 761 85 115 146 179 212 246 282 319 358 397 439 481 526 572 621 671 723
10
57
86 117 149 182 216 251 287 325 364 405 447 491 537 584 634 685
15
28
58
20
0
29
59
89 121 154 188 223 260 298 337 378 421 465 511 559 609
0
29
60
91 123 157 191 228 265 304 344 386 429 475 522 571
0
30
61
92 125 160 195 232 270 309 351 393 438 485 533
0
30
62
0
31
63
96 130 166 202 241 281 322 365 410 457
0
31
64
98 132 169 206 245 286 329 373 419
0
32
65
99 135 172 210 250 292 335 381
0
33
66 101 138 175 215 256 298 343
25 30 35 40 45 50 55 60 65 70 75 80
88 119 151 185 219 255 293 331 371 413 456 501 548 596 647
94 128 163 199 236 275 316 358 402 447 495
0
33 0
67 103 140 179 219 261 305 34 0
69 105 143 183 224 267 34 0
70 107 146 186 228 35 0
72 110 149 190 36
85 90 95
0
73 112 152 37 0
75 114 37
76
0
38
Table 7. Quantities of ammonium sulphate required to reach given degrees of saturation at +20°C.
19
Caprylic acid precipitation Caprylic (octanoic) acid is as effective as ammonium sulphate and can be used to precipitate the bulk of proteins from sera and ascites. Using caprylic acid can help to avoid the formation of protein aggregates. Unlike ammonium sulphate, caprylic acid does not concentrate the immunoglobulins as these are left in solution. This technique is not recommended for cell culture supernatants because of low yields and sample dilution. Poorly soluble antibodies may precipitate with the contaminants. Check recovery. A protocol for caprylic acid precipitation of a monoclonal antibody from ascitic fluid is given here as a starting point from which other specific protocols can be developed. 1. Mix X ml ascitic fluid with 2X ml acetate buffer 50 mM, pH 4.0. 2. Adjust to pH 4.5 with 2 M HCl or NaOH. 3. Slowly add caprylic acid (X/15)g, stirring constantly. 4. Continue stirring for 30 minutes. 5. Centrifuge at 1 000 x g for 10 minutes. 6. Remove supernatant and adjust to pH 6.0 with 2 M NaOH. 7. Prepare sample for further purification as required, removing the caprylic acid using a desalting column.
Buffer exchange and desalting Dialysis is frequently mentioned in the literature as a technique to remove salt or other small molecules and to exchange buffer composition of a sample. However, dialysis is generally a slow technique, requiring large volumes of buffer. There is also a risk of losing material during handling, or because of proteolytic breakdown or non-specific binding to the dialysis membranes. A simpler and much faster technique is to use a desalting column packed with Sephadex G25 to performs a group separation between high and low molecular weight substances. Proteins are then separated from salts and other small molecules. In a fast, single step, the sample is desalted, exchanged into a new buffer and low molecular weight materials are removed. Desalting columns are used not only to remove low molecular weight contaminants, such as salt, but also for buffer exchange before or after different chromatographic steps and for the rapid removal of reagents to terminate a reaction. Sample volumes up to 30% of the total volume of the desalting column can be processed. Figure 7 shows a typical buffer exchange and desalting separation. The process can be monitored by following changes in UV absorption and conductivity.
20
A 280 nm
(mS/cm)
0.25 0.20 0.15
10.0
protein
salt
0.10 5.0 0.05 0.00 0.0
1.0
2.0
Time (min)
Fig 7. Buffer exchange of mouse plasma (10 ml) on HiPrep™ 26/10 Desalting.
For normal laboratory scale operations Table 8 shows a selection guide for prepacked, ready-to-use desalting and buffer exchange columns. Column
Sample volume
Sample elution volume
PD-10 (gravity feed column)
1.5–2.5 ml
2.5–3.5 ml
HiTrap Desalting 5 ml
0.25–1.5 ml
1.0–2.0 ml
HiPrep 26/10 Desalting
2.5–15 ml
7.5–20 ml
Table. 8. Selection guide for desalting and buffer exchange.
To desalt larger sample volumes -connect up to 5 HiTrap Desalting columns 5 ml in series to increase the sample volume capacity, e.g. 2 columns: sample volume 3 ml, 5 columns: sample volume 7.5 ml. -connect up to 4 HiPrep 26/10 Desalting columns in series to increase the sample volume capacity, e.g. 2 columns: sample volume 30 ml, 4 columns: sample volume 60 ml. Even with 4 columns in series, the sample can be processed in 20 to 30 minutes at room temperature in aqueous buffers. Instructions are supplied with each column. Desalting and buffer exchange can take less than 5 minutes per sample with greater than 95% recovery for most proteins. A salt concentration of at least 25 mM NaCl in the chosen buffer is recommended to prevent possible ionic interactions. Sample concentration does not influence the separation as long as the concentration of proteins does not exceed 70 mg/ml when using normal aqueous buffers. The sample should be fully dissolved. If necessary, centrifuge or filter to remove particulate material. For small sample volumes it may be possible to dilute the sample with the buffer that is to be used for chromatographic purification, but cell debris and particulate matter must still be removed.
21
Alternative 1. Manual desalting with HiTrap Desalting 5 ml using a syringe or pipette
1. Fill the syringe with buffer. Remove the stop plug. To avoid introducing air into the column, connect the column "drop to drop" to the syringe (via the adapter). 2. Remove the twist-off end. 3. Wash the column with 25 ml buffer at 5 ml/min to remove completely the 20% ethanol (supplied as storage buffer). If air is trapped in the column, wash with degassed buffer until the air disappears. Air bubbles introduced onto the column by accident during sample application do not influence the separation. 4. Connect the syringe to the column with the luer adapter supplied. 5. Apply the sample using a flow rate between 1-10 ml/min. Discard the liquid eluted from the column. 6. Change to the required buffer and elute the target protein with the volumes listed in Table 9. Collect the desalted protein in the volume indicated.
A multi-dispensing pipette (Eppendorf™ model 4780 Multipipette™) can also be used. To deliver more precise volumes for sample application and elution, use the M6 threaded stopper from the HiTrap column as an adapter by piercing a hole through the bottom of the stopper. Connect the modified "stopper" to the top of the column and, by using gentle force, drive the pipette tip (Combitip with a pipette tip mounted) into the stopper. When dispensing liquid with the Multipipette, do not exceed the maximum flow rate for the column. Take care that all liquid is dispensed for each stroke before a new stroke is delivered. The maximum recommended sample volume is 1.5 ml. If the sample volume is less than 1.5 ml, add buffer until a total of 1.5 ml buffer is eluted. Discard the eluted liquid (see Table 9). Sample load ml
Add buffer ml
Elute and collect ml
Yield %
Remaining salt %
0.25
1.25
1.0
> 95
0.0
4.0
0.50
1.0
1.5
> 95
< 0.1
3.0
1.00
0.5
2.0
> 95
< 0.2
2.0
1.50
0
2.0
> 95
< 0.2
1.3
Table 9. Recommended sample and elution volumes using a syringe or Multipipette.
A simple peristaltic pump can also be used to apply sample and buffers.
22
Dilution factor
Alternative 2. Simple desalting with ÄKTAprime
ÄKTA™prime contains pre-programmed templates for individual HiTrap Desalting 5 ml and HiPrep 26/10 Desalting columns.
Buffer Preparation Prepare at least 500 ml of the required buffer. 1. Follow the instructions supplied on the ÄKTAprime cue card to connect the column and load the system with buffer. 2. Select the Application Template. 3. Start the method. 4. Enter the sample volume and press OK.
Figure 8 shows a typical procedure using ÄKTAprime. The UV (protein) and conductivity (salt) traces enable pooling of the desalted fractions. AU 280nm
–– UV 280 nm –– Conductivity
0.15 (His)6 protein
Sample:
0.10
Salt
Column: Buffer: 0.05
(His)6 protein eluted from HisTrap™ with sodium phosphate 20 mM, sodium chloride 0.5 M, imidazole 0.5 M, pH 7.4 HiTrap Desalting 5 ml Sodium phosphate 20 mM, sodium chloride 0.15 M, pH 7.0
Inject
0 0
1
2 min
Fig. 8. Desalting of a (His)6 fusion protein on ÄKTAprime.
23
24
Chapter 3 Simple, rapid purification by affinity chromatography A significant advantage for the purification of antibodies and antibody fragments, from any source, is that a great deal of information is available about the properties of the target molecule and the major contaminants (see Table 3, page 13 and Table 15, page 53). When there is an immunospecific interaction affinity chromatography is often the first, and frequently the only, step required. Affinity purification offers high selectivity, hence high resolution, and, usually, high capacity for the target protein(s). The target molecule is concentrated into a smaller volume and purity levels as high as 99% are achievable in one step. This chapter focuses specifically on the solutions available for simple, rapid affinity purification in the laboratory. Recent advances in the production and purification of genetically engineered antibodies and antibody fragments have opened up many possibilities, not only to manipulate their biological properties, but also to facilitate their purification. For example, tags can be introduced into target molecules for which no affinity media were previously available thus providing an effective affinity purification. For additional information on the purification of recombinant proteins, including purification of GST and (His)6 tagged proteins, please refer to The Recombinant Protein Handbook: Protein Amplification and Simple Purification from Amersham Biosciences. Further details on the purification of protein A fusion proteins can be found in the handbook Affinity Chromatography: Principles and Methods from Amersham Biosciences. Affinity chromatography isolates a specific protein or a group of proteins with similar characteristics. The technique separates proteins on the basis of a reversible interaction between the protein(s) and a specific ligand coupled to a chromatographic matrix. The basic principles of affinity chromatography are outlined in Appendix 10. It is generally recommended to follow any affinity step with a second purification technique, such as a high resolution gel filtration. For example, Superdex™ can be used to separate any contaminant molecules on the basis of differences in size, and also to transfer the sample into storage buffer, and remove excess salt and other small molecules. In the case of antibodies, a gel filtration step is often used to separate dimeric and monomeric forms of the molecule, as shown in Figure 9. A 280
Monomer
0.70
Void volume
Total column volume
0.50
Sample: Column: Flow rate: Buffer:
0.30
Dimer
50 µl human IgG (9 mg/ml) Superdex 200 HR 10/30 0.25 ml/min 50 mM NaH2PO4, 0.15 M NaCl, pH 7.0
0.10 0 0
5.0
10.0
15.0
20.0
ml
Fig. 9. 25
Alternatively, a desalting column that gives a low resolution separation, but has high sample capacity can be used to transfer the sample into storage buffer and remove excess salt (see page 20). For laboratory scale affinity purification, a wide range of HiTrap columns is available. All columns are supplied with a detailed protocol that outlines the buffers and steps required for optimal results. Purification on a HiTrap column is used as a typical example for many of the applications described in this handbook and the buffers and procedures presented can be used for guidance when scaling up.
HiTrap Protein A HP, HiTrap rProtein A FF and HiTrap Protein G HP are designed for the isolation and purification of monoclonal and polyclonal IgG from serum, cell culture supernatants and ascites.
HiTrap columns can be used with a syringe, a peristaltic pump or a liquid chromatography system such as ÄKTAprime. General instructions for use are given in Appendix 3. For larger scale work, HiTrap columns can often be linked in series to increase the capacity. Most media are available for packing larger columns. Always check specific availability if the intention is to scale up. Custom-designed affinity media can be produced. Reuse of affinity media depends on the nature of the sample and should only be performed with identical samples to prevent cross-contamination. For sample preparation, follow procedures according to the source of the antibody, as recommended in Chapter 2. Use high quality water and chemicals. Filtration of buffers is recommended. Centrifuge or filter samples immediately before use. If the sample is too viscous, dilute with binding buffer. Sample binding properties can be improved by adjusting the sample to the composition of the binding buffer: perform a buffer exchange using a desalting column (see page 20) or dilute in binding buffer. Ready-to-use media are supplied in a range of prepacked column formats or as loose media, to suit the needs of a specific purification step (e.g. scale, resolution, speed). As an example, Table 10 presents recommendations for the most useful products for a laboratory scale purification of target molecules containing a protein A- or protein G binding region.
26
Step
Increasing scale
Capture
HiTrap Protein G HP, 1 ml or 5 ml HiTrap Protein A HP, 1 ml or 5 ml HiTrap rProtein A FF, 1 ml or 5 ml
Protein G Sepharose Fast Flow Protein A Sepharose Fast Flow rProtein A Sepharose Fast Flow
Polishing (select medium according to size of target molecule and contaminants)
Superdex 200 HR 10/30 Superdex 75 HR10/30
HiLoad™ 16/60 Superdex 200 pg HiLoad 26/60 Superdex 200 pg HiLoad 16/60 Superdex 75 pg HiLoad 26/60 Superdex 75 pg HiLoad 16/60 Superdex 30 pg HiLoad 26/60 Superdex 30 pg
Buffer exchange/desalting
HiTrap Desalting 5 ml
HiPrep 26/10 Desalting
Table 10.
The goal of a purification and the nature of the target molecules and contaminants may require the use of other purification techniques, such as ion exchange (IEX), hydrophobic interaction (HIC) and gel filtration (GF) chromatography. The strategy of Capture, Intermediate Purification and Polishing (CIPP) that is used to develop a multi-step purification protocol is explained more fully in Chapter 5.
IgG classes, fragments and subclasses The high affinity of protein A and protein G for the Fc region of polyclonal and monoclonal IgG-type antibodies forms the basis for purification IgG classes fragments and subclasses. Protein A and protein G are bacterial proteins from Staphylococcus aureus and Streptococcus, respectively. When immobilized to Sepharose, these proteins create extremely useful, easy-touse media for many routine applications. Examples include the purification of monoclonal IgG-type antibodies, purification of polyclonal IgG and its subclasses, and the adsorption and purification of immune complexes. IgG and IgG subclasses can be isolated from ascites fluid, cell culture supernatants, serum and other sources of recombinant protein. Table 11 shows a comparison of the relative binding strengths of protein A and protein G compiled from various publications. Binding strengths are tested with free protein A or protein G and can be used as guidelines to predict the binding behaviour to a protein A or protein G purification medium. However, when immobilized to a purification medium, the interaction may be altered. For example, rat IgG1 does not bind to protein A, but does bind to Protein A Sepharose.
27
Species
Subclass
Human
IgA IgD IgE IgG1 IgG2 IgG3 IgG4 IgM* IgY IgY**
Chicken Avian egg yolk Cow Dog Goat Guinea pig Hamster Horse Koala Llama Monkey (rhesus) Mouse
Pig Rabbit Rat
Protein A binding
Protein G binding
variable -
-
++++ ++++ ++++ variable ++ ++ ++++ ++++ + ++ ++++ + ++++ +++ ++ variable +++ ++++ + +/-
++++ ++++ ++++ ++++ ++++ + ++ ++ ++ ++ ++++ + + ++++ ++++ ++++ +++ +++ +++ +++ + ++++ ++ ++ ++
IgG1 IgG2
IgG1 IgG2a IgG2b IgG3 IgM1 no distinction IgG1 IgG2a IgG2b IgG3
Sheep * Purify using HiTrap IgM Purification HP columns. ** Purify using HiTrap IgY Purification HP columns.
Table 11. Relative binding strengths of protein A or protein G.
Single-step purification based on Fc region specificity will co-purify host IgG and may even bind trace amounts of serum proteins. To avoid even trace amounts of contaminating IgG, alternative techniques such as immunospecific affinity (using anti-host IgG antibodies as the ligand to remove host IgG or target specific antigen to avoid binding host IgG), ion exchange and hydrophobic interaction chromatography may be better choices (see Chapter 6). Both native protein A and a recombinant protein A are available from Amersham Biosciences. These molecules share similar specificity for the Fc region of IgG, but the recombinant protein A has been engineered to include a C-terminal cysteine that enables a single-point coupling when the protein is immobilized to Sepharose. Single-point coupling often results in an enhanced binding capacity.
Using Protein G Sepharose media Protein G, a cell surface protein from Group G streptococci, is a type III Fc-receptor. Protein G binds through a non-immune mechanism similar to that of protein A. Like protein A, it binds specifically to the Fc region of IgG, but it binds more strongly to several polyclonal IgGs (Table 11) and to human IgG3. Amersham Biosciences offers a recombinant form of protein G from which the albumin-binding region of the native molecule has been geneti28
cally deleted, thereby avoiding undesirable reactions with albumin. Recombinant protein G contains two Fc binding regions. Under standard buffer conditions, protein G binds equally well to all human subclasses and all mouse IgG subclasses, including mouse IgG1. Protein G also binds to rat IgG2a and IgG2b, which either do not bind or bind weakly to protein A. Many antibodies also interact via the Fab region with a low affinity site on protein G. Protein G Sepharose media are a better choice for general purpose capture of antibodies since they bind to a broader range of eukaryotic species and bind more classes of IgG. Protein G has a greater affinity than protein A for IgG and exhibits minimal binding to albumin, resulting in cleaner preparations and greater yields. The binding strength of protein G for IgG depends upon the source species of the immunoglobulin. The dynamic binding capacity depends upon the binding strength as well as other factors like flow rate during sample application. Leakage of ligands from an affinity matrix is always a possibility, especially if harsh elution conditions are used. The multi-point attachment of protein G to Sepharose media results in very low ligand leakage over a wide range of elution conditions.
Purification options Binding capacity/ml medium
Comments
HiTrap Protein G HP
Human IgG > 25 mg
Purification of IgG classes, fragments and subclasses, including human IgG3. Strong affinity to monoclonal mouse IgG1 and rat IgG. Prepacked 1 ml or 5 ml columns.
Protein G Sepharose 4 Fast Flow
Human IgG > 20 mg Cow IgG 23 mg Goat IgG 19 mg Guinea pig IgG 17 mg Mouse IgG 10 mg Rat IgG 7 mg
Supplied as a suspension ready for column packing.
MAbTrap™ Kit
Human IgG > 25 mg
Complete kit for the purification of monoclonal and polyclonal IgG from serum, cell supernatant and ascites fluid. Contains one HiTrap Protein G HP 1 ml column, binding, elution and neutralization buffers for at least 20 runs using a syringe, together with detailed experimental protocols.
29
Purification Figure 10 shows the purification of mouse monoclonal IgG1 on HiTrap Protein G HP 1 ml. The monoclonal was produced in a hybridoma cell culture. Sample: Column: Flow rate: Binding buffer: Elution buffer: Electrophoresis:
12 ml mouse IgG1 hybridoma cell culture fluid HiTrap Protein G HP, 1 ml 1.0 ml/min 20 mM sodium phosphate, pH 7.0 0.1 M glycine-HCI, pH 2.7 SDS-PAGE, PhastSystem™, PhastGel™ Gradient 10–15, 1 µl sample, silver stained Immunodiffusion: 1% Agarose A in 0.75 M Tris, 0.25 M 5,5-diethylbarbituric acid, 5 mM Ca-lactate, 0.02% sodium azide, pH 8.6
A 280 nm
Binding Elution Binding buffer buffer buffer
5.0
2.5
pool I
0 5
10
pool II
15
20
25
30
ml
SDS PAGE Immunodiffusion Lane 1. Mr Lane 2.
97 000 67 000 43 000
Lane 3. 30 000 Lane 4.
20 100 14 000 Lane 1
2
3
Low Molecular Weight Calibration Kit, reduced Mouse hybridoma cell culture fluid, non-reduced, diluted 1:10 Pool I, unbound material, non-reduced, diluted 1:10 Pool II, purified mouse IgG1, non-reduced, diluted 1:10
4
Fig. 10. Purification of monoclonal mouse IgG1 on HiTrap Protein G HP, 1 ml.
Performing a purification Column:
HiTrap Protein G HP, 1 ml or 5 ml.
Recommended flow rates: 1 ml/min (1 ml column) or 5 ml/min (5 ml column). Binding buffer:
0.02 M sodium phosphate, pH 7.0.
Elution buffer:
0.1 M glycine-HCl, pH 2.7.
Neutralization buffer:
1 M Tris-HCl, pH 9.0.
Centrifuge samples (10 000 x g for 10 minutes) to remove cells and debris. Filter through a 0.45 µm filter. If required, adjust sample conditions to the pH and ionic strength of the binding buffer by either buffer exchange on a desalting column (see page 20) or dilution and pH adjustment.
30
1. Equilibrate column with 2-3 column volumes of binding buffer. 2. Apply sample. 3. Wash with 5-10 column volumes of the binding buffer to remove impurities and unbound material. Continue until no protein is detected in the effluent (determined by UV absorbance at 280 nm). 4. Elute with 1-3 column volumes of elution buffer**. 5. Re-equilibrate with 5-10 column volumes of binding buffer. **Since elution conditions are quite harsh, it is recommended to collect fractions into a neutralization buffer (60-200 µl 1 M Tris-HCl, pH 9.0 per ml fraction), so that the final pH of the fractions will be approximately neutral.
Most immunoglobulin species do not elute from Protein G Sepharose until pH 2.7 or lower. If activity is lost due to the low pH required for elution, try Protein A Sepharose: the elution pH may be more gentle. IgGs from most species and subclasses bind to protein G at near physiological pH and ionic strength. Avoid excessive washing if the interaction between the protein of interest and the ligand is weak since this may decrease the yield. Desalt and/or transfer purified IgG fractions to a suitable buffer using a desalting column (see page 20). Reuse of Protein G Sepharose media depends on the nature of the sample and should only be performed with identical samples to prevent cross-contamination. For larger sample volumes, connect several HiTrap Protein G HP columns in series or pack a larger column with loose medium.
Storage Wash media and columns with 20% ethanol (5 column volumes for packed media) and store at +4° to +8°C.
Using MAbTrap Kit
MAbTrap Kit.
MAbTrap Kit contains one HiTrap Protein G HP 1 ml column, binding, elution and neutralization buffers, a syringe with fittings and an optimized purification protocol. The kit contains sufficient material for up to 20 purifications of monoclonal or polyclonal IgG from serum, cell culture supernatant or ascitic fluid. The column can be connected to a peristaltic pump or to a syringe, if preferred. Figure 11 shows the purification of mouse monoclonal IgG1 from cell culture supernatant with syringe operation and a similar purification with pump operation. Eluted fractions were analysed by SDS-PAGE as shown in Figure 12. 31
Column: Sample:
HiTrap Protein G HP, 1 ml 10 ml mouse monoclonal cell supernatant, IgG1, anti-transferrin. Filtered through 0.45 µm filter Binding buffer: 20 mM sodium phosphate, pH 7.0 Elution buffer: 0.1 M glycine-HCl, pH 2.7
B) Pump operation, flow rate 2 ml/min A 280 nm
A) Syringe operation
Elution 3.0
A 280nm
3
2.0 2
1.0
1
0 1
4
7
10
13
16
19
25
22
28
31 ml
0
5
10
15
20
25
30
Fig. 11. Purification of mouse monoclonal IgG1 from cell culture supernatant.
Lanes 1 and 7. Low Molecular Weight Calibration Kit, Amersham Biosciences Lane 2. Crude cell culture supernatant, mouse IgG1, diluted 1:11 Lane 3. Flow through, using a peristaltic pump, diluted 1:10 Lane 4. Eluted mouse IgG1, using a peristaltic pump Lane 5. Flow through, using a syringe, diluted 1:10 Lane 6. Eluted mouse IgG1, using a syringe
Mr 97 000
67 000 43 000 30 000 20 100 14 000 1
2
3
4
5
6
7
Fig. 12. SDS-PAGE on PhastSystem using PhastGel 10-15 and silver staining.
Performing a purification Contents of a MAbTrap Kit Column:
HiTrap Protein G HP, 1 ml.
Binding buffer:
50 ml, 10X concentrate, containing 20% ethanol as preservative.
Elution buffer:
15 ml, 10X concentrate.
Neutralization buffer: 25 ml, containing 20% ethanol as preservative. Connectors. Syringe 5 ml. Instructions for use.
32
ml
Centrifuge samples (10 000 x g for 10 minutes) to remove cells and debris. Filter through a 0.45 µm filter. If required, adjust sample conditions to the pH and ionic strength of the binding buffer by either buffer exchange on a desalting column (see page 20) or dilution and pH adjustment. 1. Allow the HiTrap Protein G HP column and buffers to warm to room temperature. 2. Dilute the buffers. 3. Connect the syringe to the column using the luer adapter supplied. 4. Equilibrate the column with 5 ml distilled water, followed by 3 ml diluted binding buffer. 5. Apply the sample. 6. Wash with 5-10 ml diluted binding buffer until no material appears in the eluent. 7. Elute with 3-5 ml diluted elution buffer. Collect fractions into tubes containing neutralization buffer. 8. Re-equilibrate the column with 5 ml diluted binding buffer.
Storage Wash the column with 10 ml 20% ethanol. Store the entire kit at +4° to +8°C.
Using Protein A Sepharose or rProtein A Sepharose media Protein A is derived from a strain of Staphylococcus aureus and contains five regions that bind to the Fc region of IgG. As an affinity ligand, protein A is immobilized to Sepharose so that these regions are free to bind. One molecule of immobilized protein A can bind at least two molecules of IgG. Both native protein A and a recombinant protein A are available from Amersham Biosciences. These molecules share similar specificity for the Fc region of IgG, but the recombinant protein A has been engineered to include a C-terminal cysteine that enables a single-point coupling when the protein is immobilized to Sepharose. Single-point coupling often results in an enhanced binding capacity. The binding strength of protein A for IgG depends upon the source species of the immunoglobulin. The dynamic binding capacity depends upon the binding strength as well as factors, like flow rate during sample application. Although IgG is the major human immunoglobulin, some other types have also been demonstrated to bind with protein A (see IgA and IgM page 38). Leakage of ligands from an affinity matrix is always a possibility, especially if harsh elution conditions are used. The multi-point attachment of protein A to Sepharose media results in very low ligand leakage over a wide range of elution conditions.
33
Purification options Binding capacity/ml medium
Comments
HiTrap Protein A HP
Human IgG > 20 mg
Purification of IgG classes, fragments and sub-classes. Prepacked 1 ml or 5 ml columns.
Protein A Sepharose 4 Fast Flow*
Human IgG > 35 mg Mouse IgG 3–10 mg
Supplied as a suspension ready for column packing.
HiTrap rProtein A FF
Human IgG > 50 mg
Purification of IgG classes, fragments and sub-classes. Enhanced binding capacity. Prepacked 1 ml or 5 ml columns.
rProtein A Sepharose 4 Fast Flow*
Human IgG > 50 mg Mouse IgG 8–20 mg
Enhanced binding capacity. Supplied as a suspension ready for column packing.
Protein A Sepharose 6MB
Human IgG > 5 mg
For purification of cells coated with antibodies.
*Protein A Sepharose 4 Fast Flow and rProtein A Sepharose Fast Flow have a higher binding capacity, a more rigid matrix and provide more convenient alternatives to Protein A Sepharose CL-4B which must be rehydrated prior to column packing.
Purification Figure 13 shows the purification of mouse IgG2b from ascites on a HiTrap rProtein A FF 1 ml column using a syringe. The eluted pool contained 1 mg IgG2b and the silver stained SDS-PAGE gel confirmed a purity level of over 95%. Sample: Column: Binding buffer: Elution buffer: Flow rate:
1 ml of mouse ascites containing IgG2b, filtered through a 0.45 µm filter. The sample was a kind gift from Dr. N. Linde, EC Diagnostics, Sweden HiTrap rProtein A FF 1 ml 0.02 M sodium phosphate, pH 7.0 0.1 M sodium citrate, pH 3.0 ~ 1 ml/min Mr 97 000
A 280nm
2.5
67 000 43 000
Elution buffer
2.0
30 000
1.5
20 100 14 000 1
1.0 0.5 0.0 0
2
4
6
Flow-through pool
8
10
12
14
16 Volume (ml) Time (min)
Eluted IgG 2b pool
2
3
4
SDS-PAGE on PhastSystem using PhastGel Gradient 10–15, silver staining Lane 1. Low Molecular Weight Calibration Kit, Amersham Biosciences Lane 2. Starting material, diluted 10-fold Lane 3. Flow-through pool Lane 4. Eluted IgG2b pool
Fig. 13. Purification of mouse IgG2b from ascites.
Performing a purification Column:
HiTrap Protein A HP, 1 ml or 5 ml, or HiTrap rProtein A FF, 1 ml or 5 ml.
Recommended flow rates: 1 ml/min (1 ml columns) or 5 ml/min (5 ml columns). Binding buffer:
34
0.02 M sodium phosphate, pH 7.0.
Elution buffer:
0.1 M citric acid, pH 3-6.
Neutralization buffer:
1 M Tris-HCl, pH 9.0.
Centrifuge samples (10 000 x g for 10 minutes) to remove cells and debris. Filter through 0.45 µm filter. If needed, adjust sample conditions to the pH and ionic strength of the binding buffer by either buffer exchange on a desalting column (see page 20) or dilution and pH adjustment. 1. Equilibrate the column with 2-3 column volumes of binding buffer. 2. Apply sample. 3. Wash with 5-10 column volumes of the binding buffer to remove impurities and unbound material. Continue until no protein is detected in the effluent (determined by UV absorbance at 280 nm). 4. Elute with 1-3 column volumes of elution buffer**. 5. Re-equilibrate with 5-10 column volumes of binding buffer. **Since elution conditions are quite harsh, collect fractions into a neutralization buffer (60-200 µl 1 M Tris-HCl, pH 9.0 per ml fraction), so that the final pH of the fractions will be approximately neutral.
Table 12 gives examples of some typical binding and elution conditions that have been used with Protein A Sepharose. Protein A binding
Protein A binding condition
IgG1
++
6.0–7.0
3.5–4.5
IgG2
++
6.0–7.0
3.5–4.5
IgG3
–
8.0–9.0
< 7.0
IgG4
++
7.0–8.0
use step elution
Cow
IgG2
++
2
Goat
IgG2
+
5.8
Guinea pig
IgG1
++
4.8
IgG2
++
IgG1
+
8.0–9.0
5.5–7.5
IgG2a
+
7.0–8.0
4.5–5.5
IgG2b
+
7
3.5–4.5
IgG3
+
7
4.0–7.0
IgG1
+
> 9.0
7.0–8.0 < 8.0
Species
Subclass
Protein A elution pH Usually elutes by pH 3
Human
Mouse
Rat
4.3
IgG2a
–
> 9.0
IgG2b
–
> 9.0
< 8.0
IgG3
+
8.0–9.0
3-4 (using thiocyanate)
Table 12.
Binding strengths are tested with free protein A or protein G and can be used as guidelines to predict the binding behaviour to a protein A or protein G purification medium. However, when immobilized to a purification medium the interaction may be altered. For example, rat IgG1 does not bind to protein A, but does bind to Protein A Sepharose. With some antibodies, such as mouse IgG1, it might be necessary to add sodium chloride up to a concentration of 3 M in the binding buffer to achieve efficient binding when using protein A. For example use 1.5 M glycine, 3 M NaCl, pH 8.9. Avoid excessive washing if the interaction between the protein of interest and the ligand is weak since this may decrease the yield.
35
IgGs from most species and subclasses bind protein A close to physiological pH and ionic strength. It is preferable to use a mild elution method, such as specific desorption, when labile antibodies are isolated. Reverse the flow of the wash buffer and elute with 0.1 M glycyltyrosine in 2 M NaCl, pH 7.0 at room temperature, applied in pulses. (Note: glycyltyrosine absorbs strongly at wavelengths used for detecting proteins). The specific elution is so mild that the purified IgG is unlikely to be denatured. Alternative elution buffers include: 1 M acetic acid, pH 3.0 or 0.1 M glycine-HCl, pH 3.0 or 3 M potassium isothiocyanate. Potassium isothiocyanate can severely affect structure and immunological activity. Desalt and/or transfer purified IgG fractions into a suitable buffer using a desalting column (see page 20). Reuse of Protein A Sepharose media depends on the nature of the sample and should only be performed with identical samples to prevent cross-contamination. For larger samples volumes, connect several HiTrap Protein A HP or HiTrap rProtein A FF columns in series, or pack a larger column with loose medium.
Storage Wash media and columns with 20% ethanol (5 column volumes for packed media) and store at +4° to +8°C.
Fab, F(ab')2 fragments
Protein G has a low affinity site for the Fab region (binding to C-H1 domains of heavy chains bound to Ck light chains). Consequently, Protein G affinity purification can sometimes be used for the purification of Fab and F(ab')2 fragments. Figure 14 shows the purification of recombinant mouse Fab fragments, expressed in E. coli, in a single affinity purification step using Protein G Sepharose 4 Fast Flow.
36
A 280 nm
0.6
Sample:
Column: Binding buffer: Elution buffer: Flow rate:
Recombinant mouse Fab, expressed in E. coli. Centrifuged medium from fermentor, 15 ml Protein G Sepharose 4 Fast Flow 50 mM Tris-HCl, 0.15 M NaCl, 0.05% Tween™, pH 7.4 0.2 M HAc, pH 2.8 0.8 ml/min
Fab fragments 0.4 0.2 M HAc
0.2
0
10
20
40
44 42 Volume (ml)
Fig. 14. Purification of recombinant mouse Fab fragments, expressed in E. coli using Protein G Sepharose 4 Fast Flow.
Performing a purification Column:
Protein G Sepharose Fast Flow.
Recommended flow rate: 0.8 ml/min. Binding buffer:
50 mM Tris-HCl, 0.15M NaCl, 0.05% Tween, pH 7.4.
Elution buffer:
0.2 M HAc, pH 2.8.
Centrifuge samples (10 000 x g for 10 minutes) to remove cells and debris. Filter through a 0.45 µm filter. If needed, adjust sample conditions to the pH and ionic strength of the binding buffer by either buffer exchange on a desalting column (see page 20) or dilution and pH adjustment. 1. Equilibrate column with 2-3 column volumes of binding buffer. 2. Apply sample. 3. Wash with 5-10 column volumes of the binding buffer to remove impurities and unbound material. Continue until no protein is detected in the eluent (determined by UV absorbance at 280 nm). 4. Elute with 1-3 column volumes of elution buffer**. 5. Re-equilibrate column with 5-10 column volumes of binding buffer. ** Since elution conditions are quite harsh, it is recommended to collect fractions into a neutralization solution (60-200 µl 1 M Tris-HCl, pH 9.0 per ml fraction) so that the final pH of the fractions will be approximately neutral.
Although protein A has no affinity for Fab regions, protein A affinity purification can sometimes be used to separate whole or partially digested IgG from F(ab')2 after proteolytic cleavage. Protein A does have an affinity for the variable region in the human heavy chain subgroup III so that F(ab')2 derived from this group can occasionally be purified with protein A affinity chromatography, although protein G is preferred.
37
Alternatively, produce the Fab or F(ab')2 fragment with a (His)6 or GST tag and purify using HisTrap Kit, HiTrap Chelating HP or GSTrap™ FF. For detailed protocols please refer to The Recombinant Protein Handbook: Protein Amplification and Simple Purification from Amersham Biosciences.
IgA Protein A can interact with human colostral IgA as well as human myeloma IgA2 but not IgA1. Polyclonal IgA from pig, dog and cat and monoclonal canine IgA have also exhibited binding affinity for protein A. For routine purification it may be worth developing an immunospecific purification with an anti-IgA monoclonal antibody coupled to a pre-activated affinity matrix to provide a high resolution, high selectivity affinity purification medium (see page 43). Alternatively, a multi-step purification strategy could be employed (see Chapter 5).
IgD Protein A and protein G do not bind to IgD. For routine purification it may be worth developing an immunospecific purification with an anti-IgD monoclonal antibody coupled to a pre-activated affinity matrix to provide a high resolution, high selectivity affinity purification medium (see page 43). Alternatively, a multi-step purification strategy could be employed (see Chapter 5).
IgE IgE is present at very low concentrations in both human and mouse serum and can make a simple purification more difficult to develop and perform. Protein A and protein G do not bind to IgE (see Table 11, page 28). For routine purification it may be worth developing an immunospecific purification with an anti-IgE monoclonal antibody coupled to a pre-activated affinity matrix to provide a high resolution, high selectivity affinity purification medium (see page 43). Alternatively, and for any initial purification, a multi-step purification strategy should be employed (see Chapter 5). Use ion exchange chromatography or hydrophobic interaction in the first step as this will purify and concentrate the IgE.
IgM The technique described in this section is optimized for purification of monoclonal IgM from hybridoma cell culture, but it can be used as a starting point to determine the binding and elution conditions required for other IgM preparations.
Purification options HiTrap IgM Purification HP
38
Binding capacity/ml medium
Comments
Human IgM – 5 mg
Purification of monoclonal and human IgM. Prepacked 1 ml column.
HiTrap IgM Purification HP columns are packed with a thiophilic adsorption medium (2-mercaptopyridine coupled to Sepharose High Performance). The interaction between the protein and the ligand has been suggested to result from the combined electron donatingand accepting-action of the ligand in a mixed mode hydrophilic-hydrophobic interaction.
Purification Figures 15 and 16 show results from the purification of monoclonal a-Shigella IgM from hybridoma cell culture supernatant. SDS-PAGE analysis demonstrated a purity level of over 80%. Results from an ELISA (not shown) indicated high activity in the purified fraction. mS/cm
mAU 2500
100
Sample:
Column: Binding buffer: Elution buffer: Cleaning buffer: Flow rate:
75 ml of cell culture supernatent containing a-Shigella IgM, filtered through a 0.45 µm filter HiTrap IgM Purification HP 20 mM sodium phosphate buffer, 0.5 M potassium sulphate, pH 7.5 20 mM sodium phosphate buffer, pH 7.5 20 mM sodium phosphate buffer, pH 7.5, 30% isopropanol 1 ml/min
Flow through material
2000
80
1500 60
IgM 1000 Elution buffer
Cleaning buffer
40
500 20
0
0 0
80
100
ml
Fig. 15. Purification of a-Shigella IgM on HiTrap IgM Purification HP.
Samples reduced with 2-mercaptoethanol
Non-reduced samples Lane 1. Low Molecular Weight Calibration Kit Lane 2. Cell culture supernatant, starting material, diluted 20-fold Lane 3. IgM, human Lane 4. IgG Lane 5. Flow-through pool, diluted 20-fold Lane 6. Eluted IgM, fraction 8, diluted 8-fold Lane 7. Eluted IgM, fraction 9, diluted 8-fold Lane 8. Washing out unbound material, pool diluted 3-fold
1 2 3 4 5 6 7 8
1 2 3 4 5 6 7 8
Fig. 16. SDS-PAGE on PhastSystem, using PhastGel 4-15, silver staining.
Performing a purification Column:
HiTrap IgM Purification HP.
Recommended flow rate: 1 ml/min. Binding buffer:
20 mM sodium phosphate, 0.8 M (NH4)2SO4, pH 7.5.
Elution buffer:
20 mM sodium phosphate, pH 7.5.
Wash buffer:
20 mM sodium phosphate, pH 7.5 with 30% isopropanol.
39
The sample must have the same concentration of ammonium sulphate as the binding buffer. Slowly add small amounts of solid ammonium sulphate to the sample from the hybridoma cell culture until the final concentration is 0.8 M. Stir slowly and continuously. Pass the sample through a 0.45 µm filter immediately before applying it to the column. Avoid precipitation of IgM. It is important to add the ammonium sulphate slowly.
Purification 1. Wash column with 5 column volumes of each buffer. 2. Equilibrate column with 5 column volumes of binding buffer. 3. Apply the sample. 4. Wash out unbound sample with 15 column volumes of binding buffer or until no material appears in the effluent (monitored at A280). 5. Elute the IgM with 12 column volumes of elution buffer. 6. Wash the column with 7 column volumes of cleaning buffer. 7. Re-equilibrate the column with 5 column volumes of binding buffer.
Some monoclonal IgM might not bind to the column at 0.8 M ammonium sulphate. Binding can be improved by increasing the ammonium sulphate concentration to 1.0 M. An increased concentration of ammonium sulphate will cause more IgG to bind, which might be a problem if serum has been added to the cell culture medium. If there is IgG contamination of the purified IgM, the IgG can be removed by using HiTrap Protein A HP, HiTrap rProtein A FF, or HiTrap Protein G HP. Ammonium sulphate can be exchanged for 0.5 M potassium sulphate. Most monoclonal IgM binds to the column in the presence of 0.5 M potassium sulphate and the purity of IgM is comparable to the purity achieved with 0.8 M ammonium sulphate. Some monoclonal IgM may bind too tightly to the column for complete elution. The remaining IgM will be eluted during cleaning, but the high content of isopropanol will cause precipitation of IgM. Perform an immediate buffer exchange (see page 20) or dilute the sample to preserve the IgM. Lower concentrations of isopropanol may elute the IgM and decrease the risk of precipitation. Reuse of HiTrap lgM Purification HP depends on the nature of the sample and should only be performed with identical samples to prevent cross-contamination. To increase capacity, connect several HiTrap IgM Purification HP columns in series. A HiTrap column can be used with a syringe, a peristaltic pump or connected to a liquid chromatography system, such as ÄKTAprime.
Storage Wash the column with 20% ethanol and store at +4° to +8°C. Protein A Sepharose media may offer an alternative solution to HiTrap IgM Purification HP since some human monoclonal IgM, some IgM from normal and macroglobulinaemic sera, and some monoclonal canine IgM and polyclonal IgA from pig, dog and cat can bind to protein A.
40
Avian IgY from egg yolk Purification option HiTrap IgY Purification HP
Binding capacity
Comments
100 mg pure IgY per 5 ml column
Purification of IgY from egg yolk. Prepacked 5 ml column.
HiTrap IgY Purification HP columns are packed with a thiophilic adsorption medium (2-mercaptopyridine coupled to Sepharose High Performance). The interaction between the protein and the ligand has been suggested to result from the combined electron donatingand accepting-action of the ligand in a mixed mode hydrophobic-hydrophilic interaction.
Purification Figures 17 and 18 show the purification of a-Hb IgY from 45 ml of egg yolk extract (corresponding to one quarter of a yolk) and the SDS-PAGE analysis showing a purity of over 70%. mAU
Sample:
Column: Binding buffer: Elution buffer: Cleaning buffer: Flow rate:
45 ml of egg yolk extract (corresponding to 1/4 of an egg yolk) containing a-Hb IgY, filtered through a 0.45 µm filter HiTrap IgY Purification HP 20 mM sodium phosphate buffer, 0.5 M potassium sulphate, pH 7.5 20 mM sodium phosphate buffer, pH 7.5 20 mM sodium phosphate buffer, pH 7.5, 30% isopropanol 5 ml/min
IgY
mS/cm
2000
80
1500
60
1000
40 Elution buffer
Cleaning buffer
20
500
0
0
0
50
100
150
ml
Fig. 17. Purification of IgY on HiTrap IgY Purification HP.
Lane Lane Lane Lane Lane Lane Lane
Mr 97 67 43 30 20 14
000 000 000 000 100 000 1
2
3
4
5
6
1. 2. 3. 4. 5. 6. 7.
Low Molecular Weight Calibration Kit Egg yolk extract Flow-through pool Eluted IgY Egg yolk extract, diluted 4-fold Flow-through pool, diluted 4-fold Eluted IgY, diluted 4-fold
7
Fig. 18. SDS-PAGE of non-reduced samples on PhastSystem, using PhastGel 4–15%, Coomassie™ staining.
41
Performing a purification Column:
HiTrap IgY Purification HP.
Recommended flow rate: 5 ml/min. Binding buffer:
20 mM sodium phosphate, 0.5 M K2SO4, pH 7.5.
Elution buffer:
20 mM sodium phosphate, pH 7.5.
Wash buffer:
20 mM sodium phosphate, pH 7.5 with 30% isopropanol.
As much as possible of the egg yolk lipid must be removed before purification. Water or polyethylene glycol can be used to precipitate the lipids. Precipitation with water is described here.
Precipitation of the egg yolk lipid using water 1. Separate the egg yolk from the egg white. 2. Add nine parts of distilled water to one part egg yolk. 3. Mix and stir slowly for 6 hours at +4°C. 4. Centrifuge at 10 000 x g, at +4°C for 25 minutes to precipitate the lipids. 5. Collect the supernatant containing the IgY. 6. Slowly add K2SO4 to the sample, stirring constantly, to reach a concentration of 0.5 M. 7. Adjust pH to 7.5. 8. Pass the sample through a 0.45 µm filter immediately before applying it to the column.
Purification 1. Wash the column with at least 5 column volumes of each buffer. 2. Equilibrate with 5 column volumes of binding buffer. 3. Apply the sample. 4. Wash with at least 10 column volumes of binding buffer or until no material appears in the effluent, as monitored at A280. 5. Elute the IgY with 10 column volumes of elution buffer. 6. Wash the column with 8 column volumes of cleaning buffer. 7. Re-equilibrate the column with 5 column volumes of binding buffer.
To improve recovery of total IgY or a specific IgY antibody, replace 0.5 M K2SO4 with 0.6-0.8 M Na2SO4 in the binding buffer. An increase in salt concentration will adversely affect the purity of the eluted IgY. The purity of the eluted IgY may be improved by using gradient elution with, for example, a linear gradient 0-100% elution buffer over 10 column volumes, followed by 100% elution buffer for a several column volumes. To increase binding capacity, connect several HiTrap IgY Purification HP columns in series. Reuse of HiTrap IgY Purification HP depends on the nature of the sample. To prevent cross-contamination, it should only be reused with identical samples. A HiTrap column can be used with a syringe, a peristaltic pump or connected to a liquid chromatography system, such as ÄKTAprime.
42
Storage Wash the column with 20% ethanol and store at +4° to +8°C.
Making specific purification columns If an affinity medium is not available, a ligand (such as a pure antigen or an anti-antibody) can be coupled to a suitable matrix to create an immunospecific affinity medium for purification. Although this process requires careful development and optimization, it is often worthwhile, for example when a specific protein needs to be prepared on a regular basis. The simplest coupling is via the primary amine group of the ligand, as described in this handbook. If there is no primary amine available (e.g. this group may be required for the specific interaction), then pre-activated media for ligand attachment via carboxyl, thiol or hydroxyl groups can be considered. These are described in the handbook, Affinity Chromatography: Principles and Methods from Amersham Biosciences. Media are supplied in different prepacked column formats, in suspension or as dry media. Table 13 presents recommendations for the products that would be most useful for laboratory scale purification. Step
Increasing scale
Capture
Specific ligand bound to HiTrap NHS-activated HP, 1 ml or 5 ml
Specific ligand bound to NHS-activated Sepharose Fast Flow
Polishing (select medium according to size of target molecule and contaminants)
Superdex 200 HR 10/30 Superdex 75 HR10/30
HiLoad HiLoad HiLoad HiLoad HiLoad HiLoad
Buffer exchange/desalting
HiTrap Desalting
HiPrep 26/10 Desalting
16/60 26/60 16/60 26/60 16/60 26/60
Superdex Superdex Superdex Superdex Superdex Superdex
200 pg 200 pg 75 pg 75 pg 30 pg 30 pg
Table 13.
Immunospecific purification is particularly useful if the target molecules bind weakly or not at all to protein A or protein G. A pure ligand is required that has a proven reversible high affinity for the target molecule. Using an antigen or an anti-antibody as a ligand will give a high degree of purification. Immunospecific purification can also be used to remove key contaminants. If possible, test the affinity of the interaction. Too low or too high affinity will result in poor yields after purification. The target protein may wash through or leak from the column, or the target molecule may not dissociate from the ligand during elution. Immunospecific interactions often require harsh elution conditions. It is recommended to collect fractions into a neutralization buffer, such as 60-200 µl 1 M Tris-HCl, pH 9.0 per ml fraction.
43
Purification examples Figure 19 shows the partial purification of an IgE-stimulating factor from a human T-cell line, using IgE as the specific affinity ligand coupled to HiTrap NHS-activated HP 1 ml column. A 280 nm
Sample:
Column: Binding buffer: Elution buffer: Flow rate:
0.016
2 ml of a 65-fold concentrated serum-free cell culture supernatant of the human T-cell line MO IgE coupled to HiTrap NHS-activated HP, 1 ml 20 mM sodium phosphate, 0.15 M NaCl, pH 7.4 100 mM glycine, 0.5 M NaCl, pH 3.0 0.25 ml/min
0.012 Binding Elution buffer buffer
0.008
0.004
0 Flow through
10
Eluent
20
30
40
50 ml
Fig. 19. Purification of an IgE-stimulating factor from a human T-cell line.
Figure 20 shows an example of the purification of anti-mouse Fc-IgG from sheep serum using mouse IgG1 coupled to HiTrap NHS-activated HP 1 ml column. Mr 97 000 67 000 43 000
A 280 nm
Sample: Column:
Flow rate: Binding buffer: Elution buffer: Electrophoresis:
50 ml sheep anti-mouse Fc serum filtered 0.45 µm HiTrap NHS-activated HP 1 ml. Mouse IgG, (10 mg, 3.2 ml) was coupled in 0.2 M NaHCO3, 0.5 M NaCl, pH 8.3, room temp., recycled with a peristaltic pump for 1 h. The coupling yield was 95% (9.5 mg). 1.0 ml/min 75 mM Tris-HCl, pH 8.0 100 mM glycine-HCl, 0.5 M NaCl, pH 2.7
Flow through material 2.0
14 000 1
2 Mr 97 000 67 000 43 000 30 000 20 100 14 000
1.0
1
SDS-PAGE. PhastSystem. PhastGel Gradient 8–25 1 µl sample, Coomassie stained
20
Fig. 20. Purification of anti-mouse Fc-IgG from sheep antiserum.
44
30 000 20 100
Binding Elution buffer buffer
40
60
80
100 ml
2
Lane 1: Desorbed material, non-reduced Lane 2: Low Molecular Weight Calibration Kit, reduced
Preparing NHS-activated media Product
Comments
HiTrap NHS-activated HP
Pre-activated medium for coupling via primary amine group of a ligand. Prepacked 1 ml and 5 ml columns.
NHS activated Sepharose 4 Fast Flow
Supplied as a suspension ready for column packing.
NHS-activated Sepharose media are chromatographic matrices specifically designed to allow the covalent coupling of ligands (often antigens or antibodies) containing primary amino groups (the most common form of attachment). The excellent hydrophilic properties of the base matrix minimize non-specific adsorption of proteins that can reduce the binding capacity of the target protein. Fifteen atoms spacer arms make the matrix suitable for the coupling of smaller molecules. The pH range for coupling is well suited to the stability characteristics of many immunoglobulins. The media are stable at high pH to allow stringent washing procedures (subject to the pH stability of the coupled ligand). The protocol below describes the preparation of a prepacked HiTrap NHS-activated HP column and a recommendation for a preliminary purification protocol. Many of these details are generally applicable to NHS-activated Sepharose media.
Buffer Preparation Acidification solution: 1 mM HCl (kept on ice). Coupling buffer:
0.2 M NaHCO3, 0.5 M NaCl, pH 8.3.
Use high quality water and chemicals. Filtration through 0.45 µm filters is recommended. Coupling within pH range 6-9, maximum yield is achieved at pH ~ 8. The activated product is supplied in 100% isopropanol to preserve the stability prior to coupling. Do not replace the isopropanol until it is time to couple the ligand.
HiTrap NHS-activated HP column preparation 1. Dissolve the ligand in the coupling buffer to a final concentration of 0.5-10 mg/ml (for protein ligands) or perform a buffer exchange using a desalting column (see page 20). The optimal concentration depends on the ligand. The optimal sample volume is one column volume. 2. Remove the top cap and apply a drop of ice cold 1 mM HCl to the top of the column to avoid air bubbles. 3. Connect the top of the column, via the connector supplied, to a syringe or a pump. 4. Remove the twist-off end.
Ligand coupling 1. Wash out the isopropanol with 3 x 2 column volumes of ice-cold 1 mM HCl. 2. Inject one column volume of ligand solution onto the column. 3. Seal the column and leave for 15-30 minutes at +25°C (or 4 hours at +4°C).
Do not use excessive flow rates. The medium can be irreversibly compressed. Re-circulate the solution if larger volumes of ligand solution are used. For example, when using a syringe, connect a second syringe to the outlet of the column and gently pump the solution back and forth for 15-30 minutes or, if using a peristaltic pump, simply re-circulate the sample through the column. If required, the coupling efficiency can be measured after this step. Procedures are supplied with each HiTrap NHS-activated HP column.
45
Washing and deactivation This procedure deactivates any excess active groups that have not coupled to the ligand and washes out non-specifically bound ligand. Buffer A: 0.5 M ethanolamine, 0.5 M NaCl, pH 8.3. Buffer B: 0.1 M acetate, 0.5 M NaCl, pH 4. 1. Inject 3 x 2 column volumes of Buffer A. 2. Inject 3 x 2 column volumes of Buffer B. 3. Inject 3 x 2 column volumes of Buffer A. 4. Let the column stand for 15-30 min. 5. Inject 3 x 2 column volumes of Buffer B. 6. Inject 3 x 2 column volumes of Buffer A. 7. Inject 3 x 2 column volumes of Buffer B. 8. Inject 2-5 column volumes of a buffer with neutral pH. The column is now ready for use.
A HiTrap column can be used with a syringe, a peristaltic pump or connected to a liquid chromatography system, such as ÄKTAprime.
Storage Store the column in a solution that maintains the stability of the ligand and contains a bacteriostatic agent.
Performing a purification on coupled NHS-activated media Optimal binding and elution conditions for purification of the target protein must be determined separately for each ligand (see below for elution buffer suggestions). Literature references and textbooks may give good guidelines. Below is a general protocol that can be used initially. For the first run, perform a blank run in the absence of sample to ensure the removal of loosely bound ligand. Use high quality water and chemicals. Filtration through 0.45 µm filters is recommended. Samples should be centrifuged immediately before use and/or filtered through a 0.45 µm filter. If the sample is too viscous, dilute with binding buffer. Sample binding properties can be improved by adjusting the sample to the composition of the binding buffer. Perform a buffer exchange using a desalting column (see page 20) or dilute the sample in binding buffer.
46
1. Prepare the column (blank run). a. Wash with 2 column volumes of start buffer. b. Wash with 3 column volumes of elution buffer. 2. Equilibrate with 5-10 column volumes of start buffer. 3. Apply sample. The optimal flow rate depends on the binding constant of the ligand, but a recommended flow rate range is 0.5-1 ml/min on HiTrap 1 ml column and 2-5 ml/min on HiTrap 5 ml column. 4. Wash with 5-10 column volumes of start buffer, or until no material appears in the eluent as monitored by absorption at A280. 5. Elute with 2-5 column volumes of elution buffer (larger volumes may be necessary). 6. If required, purified fractions can be desalted and exchanged into the buffer of choice using prepacked desalting columns (see Chapter 20). 7. Re-equilibrate the column by washing with 5-10 column volumes of start buffer.
Avoid excessive washing if the interaction between the protein of interest and the ligand is weak, since this may decrease the yield. If elution conditions are quite harsh, collect fractions into a neutralization buffer (60-200 µl 1 M Tris-HCl, pH 9.0 per ml fraction), so that the final pH of the fractions will be approximately neutral.
Elution buffers Immunospecific interactions can be very strong and sometimes difficult to reverse. The specific nature of the interaction determines the elution conditions and the reversibility of the interaction should always be checked before attaching a ligand to an affinity matrix. If standard elution buffers do not reverse the interaction, alternative elution buffers that may be useful are listed below: • Low pH (below pH 2.5). • High pH (up to pH 11). • Substances that reduce the polarity of a buffer may facilitate elution without affecting protein activity: dioxane (up to 10%), ethylene glycol (up to 50%). NHS-activated Sepharose is the first choice for the preparation of immunospecific media. CNBr-activated Sepharose media offer a well-established option for the attachment of larger ligands and can be an alternative to NHS-activated Sepharose.
47
48
Chapter 4 Immunoprecipitation Immunoprecipitation is a highly specific technique for the analytical separation of target antigens from crude cell lysates. By combining immunoprecipitation with other techniques, such as SDS-PAGE and immunoblotting, it can be used to detect and quantify antigens, determine relative molecular weights, monitor protein turnover and post-translational modifications, and check for enzyme activity. By using the high specificity of protein A and protein G for the Fc regions of IgG from a wide range of mammalian species, Protein A Sepharose 4 Fast Flow and Protein G Sepharose 4 Fast Flow media offer effective and rapid removal of immune complexes formed between an antigen and its specific antibody in the immunoprecipitation reaction. The Immunoprecipitation Starter Pack from Amersham Biosciences is ideal to begin working with immunoprecipitation. The pack includes Protein A Sepharose 4 Fast Flow (2 ml) and Protein G Sepharose 4 Fast Flow (2 ml) to enable work with a wide range of antibody species and selection of the optimal medium.
When using immunoprecipitation, procedures must often be optimized empirically to obtain satisfactory results. For example, determining cell lysis conditions is critical with regard to cell type and how the antigen is to be used and, whereas cells without cell walls (e.g. animal cells) are easily disrupted by treatment with mild detergent, other cells may need some type of mechanical shearing, such as sonication or Dounce homogenization. The protocol presented here describes a generic step-by-step method for immunoprecipitation by Protein A Sepharose Fast Flow and Protein G Sepharose Fast Flow. Refer to Table 11 on page 28 to see which medium is likely to be suitable for the antibody source and sub-type, or test using the Immunoprecipitation Starter Pack.
49
Immunoprecipitation protocol Preparation of Protein A Sepharose 4 Fast Flow or Protein G Sepharose 4 Fast Flow 1. Wash the medium three times with lysis buffer to remove 20% ethanol. 2. Between the washes centrifuge at 12 000 x g for 20 seconds and discard the supernatant. 3. Mix equal volumes of medium and lysis buffer to prepare a 50% slurry. 4. Store at +4ºC and mix well before use.
Cell lysis Adherent cells: Step 1. Remove all culture medium and wash twice with ice-cold PBS. Discard the supernatants and drain well. 6
7
Step 2. Place the tissue culture dish on ice. Add ice-cold lysis buffer to a concentration of 10 -10 cells/ml (1 ml to a cell culture plate, Ø 10 cm). Incubate on ice for 10-15 minutes with occasional agitation.
Cells in suspension: Step 1. Collect cells by centrifugation at 1 000 x g for 5 minutes and discard the culture medium supernatant. Resuspend the pellet in ice-cold PBS, centrifuge and discard the supernatant. Repeat the wash. 6
7
Step 2. Suspend the washed pellet in ice-cold lysis buffer at a concentration of 10 -10 cells/ml (approximately 10 cell volumes lysis buffer). Incubate on ice for 10-15 minutes, mixing gently. 3) Transfer the cells to a homogenization tube. 4) Disrupt the cells by sonication, Dounce homogenization or passage through a 21 Gauge needle. Keep the cells on ice to prevent an increase in temperature. 5) Centrifuge at 12 000 x g for 10 minutes at +4°C to remove particulate matter. 6) Transfer the lysate (the supernatant) to a fresh tube. Keep on ice.
Pre-clearing (optional) 1) Add 50-100 µl of prepared Protein A Sepharose 4 Fast Flow or Protein G Sepharose 4 Fast Flow suspension (50% slurry) to 1 ml cell lysate in an Eppendorf tube. 2) Gently mix for 1 hour at +4°C. 3) Centrifuge at 12 000 x g for 20 seconds. Save the supernatant.
Couple antigen to antibody 1) Aliquot samples (500 µl) into new Eppendorf tubes. 2) Add polyclonal serum (0.5-5 µl), hybridoma tissue culture supernatant (5-100 µl), ascites fluid (0.1-1 µl) or purified monoclonal or polyclonal antibodies (add the volume corresponding to 1-5 µg). For controls, use non-immune antibodies that are as close to the specific antibody as possible (for example, polyclonal serum should be compared to normal serum from the same species). 3) Mix gently for 1 hour at +4°C.
Precipitation of the immune complexes 1) Add 50 µl of prepared Protein A Sepharose 4 Fast Flow or Protein G Sepharose 4 Fast Flow suspension (50% slurry). 2) Mix gently for 1 hour at +4°C. 3) Centrifuge at 12 000 x g for 20 seconds and save the pellet. 4) Wash the pellet three times with 1 ml lysis buffer and once with wash buffer. Centrifuge at 12 000 x g for 20 seconds between each wash and discard the supernatants.
Be very careful when removing the supernatants to avoid loss of the immunocomplexes.
50
Dissociation and analysis 1) Suspend the final pellet in 30 µl SDS-PAGE sample buffer. 2) Heat to +95°C for 3 minutes. 3) Centrifuge at 12 000 x g for 20 seconds to remove the Sepharose. Carefully remove the supernatant. 4) Add 1 µl 0.1% bromophenol blue. 5) Analyse the supernatant by SDS-PAGE, followed by protein staining and/or immunoblotting for detection. Radiolabelled antigens are detected by autoradiography.
Buffers and solutions Cell lysis must be harsh enough to release the target antigen, but mild enough to maintain its immunoreactivity. Some commonly used lysis buffers are listed in Table 14. NP-40 (IGEPAL CA-630) and RIPA buffer release most soluble cytoplasmic or nuclear proteins without releasing chromosomal DNA and are a good choice for initial experiments. Parameters that affect the extraction of an antigen include salt concentration (0-1 M), nonionic detergents (0.1-2%), ionic detergents (0.01-0.5%) and pH (6-9). Buffers and solutions
Contents
Ability to disrupt cells
Lysis buffers Low salt
1% IGEPAL CA-630, 50 mM Tris, pH 8.0, 1 mM PMSF
NP-40 (IGEPAL CA-630)
150 mM NaCl, 1% IGEPAL CA-630, 50 mM Tris, pH 8.0, 1 mM PMSF
++
+
RIPA
150 mM NaCl, 1% IGEPAL CA-630, 0.5% sodium deoxycholate (DOC), 0.1% SDS, 50 mM Tris, pH 8.0, 1 mM PMSF
+++
High salt
500 mM NaCl, 1% IGEPAL CA-630, 50 mM Tris, pH 8.0, 1 mM PMSF
++++
Other buffers and solutions PBS
1 mM KH2PO4, 10 mM Na2HPO4, 137 mM NaCl, 2.7 mM KCl, pH 7.4
Wash buffer
50 mM Tris, pH 8
Sample buffer (reducing)
1% SDS, 100 mM DTT, 50 mM Tris, pH 7.5
Table 14.
Choice of antibody Polyclonal serum contains antibodies against multiple epitopes of an antigen. These antibodies help to stabilize the antigen-antibody- medium complexes, but can also create a problem of high background during analysis. Monoclonal antibodies are more specific, which reduces background, but may mean that less stable immune complexes are formed due to lower affinity. This can be overcome by using pools of different monoclonal antibodies.
51
Troubleshooting Target antigen cannot be detected due to incomplete release during lysis Try harsher lysis conditions.
High levels of background proteins on SDS-PAGE Specific cause: polyclonal serum may contain antibodies that recognize other antigens. Purify the antibody by affinity purification using a specific purification column. Try a different antibody. Non-specific cause: proteins binding to medium and/or to the plastic tubes or the presence of protein aggregates that co-precipitate with the immune complex. Pre-coat plastic tube with lysis buffer prior to addition of cell lysate. Add saturating amount of competitive protein (i.e. BSA, gelatine, acetone powders). Spin the lysate at 100 000 x g for 30 minutes to remove aggregated proteins before addition of the antibody. Spin the antibody at 100 000 x g for 30 minutes to remove particulate matter. Spin the antigen-antibody complex at 10 000 x g for 10 minutes prior to addition of medium to remove protein aggregates. Try a different antibody. Use more stringent washing conditions, for example: 1 M sodium chloride, 1 M potassium thiocyanate, 0.5 M lithium chloride, 0.2% SDS or 1% Tween 20. Alternate between high and low salt wash buffer, or wash the beads with distilled water. Prolong washing times and/or increase the number of washes. Titrate the optimal amounts of cell lysate, antibody and Protein A Sepharose 4 Fast Flow/ Protein G Sepharose 4 Fast Flow.
52
Chapter 5 Multi-step purification strategies As discussed in Chapter 3, a single, rapid purification step using affinity chromatography is often sufficient to achieve the level of purity and quantity of product required for research purposes. Antibodies or their fragments can be adequately purified for further use, and a polishing step (desalting/buffer exchange or high resolution gel filtration) is sufficient to remove unwanted small molecules, such as salts. If affinity chromatography cannot be used, or if a higher degree of purity is required, alternative techniques need to be combined effectively into a multi-step purification strategy. A significant advantage when working with native or recombinant antibodies or fragments is that there is often considerable information available about the product and contaminants, as shown in Table 15 below and in Table 3 on page 13. Molecular weight
Mr 150 000–160 000 (IgG) Mr 900 000 (IgM)
Isoelectric point (pI)
4–9, most > 6.0, often more basic than other serum proteins.
Hydrophobicity
IgG is more hydrophobic than many other proteins and so precipitates more readily in ammonium sulphate.
Solubility
IgG very soluble in aqueous buffers. Lowest solubility (specific to each antibody) near pI or in very low salt concentration.
Temperature stability
Relatively stable at room temperature (but specific to each antibody).
pH stability
Often stable over a wide pH interval, but unstable in very acidic buffers (specific to each antibody).
Carbohydrate content
2–3% for IgG, higher for IgM (12%), most carbohydrate is associated with Fc region of the heavy chains.
Table 15. Characteristics of native IgG and IgM.
Purity
With this information, and with detection assays and sample preparation and extraction procedures in place, a purification strategy of Capture, Intermediate Purification and Polishing (CIPP) can be applied (Figure 21). This strategy is used in both the pharmaceutical industry and in the research laboratory to ensure faster method development, a shorter time to pure product and good economy. This section gives a brief overview of the approach recommended for any multi-step protein purification together with typical examples. The Protein Purification Handbook from Amersham Biosciences is highly recommended as a guide to planning efficient and effective protein purification strategies and for the selection of the correct medium for each step and scale of purification.
Polishing Achieve final high level purity
Intermediate purification Capture Preparation, extraction, clarification
Remove bulk impurities
Isolate, concentrate and stabilize
Step Fig. 21. Preparation and CIPP.
53
Applying CIPP: Imagine the purification has three phases: Capture, Intermediate Purification and Polishing. Assign a specific objective to each step within the purification process. The purification problem associated with a particular step will depend greatly upon the properties of the starting material. Thus, the objective of a purification step will vary according to its position in the process. In the capture phase the objectives are to isolate, concentrate and stabilize the target product. The product should be concentrated and transferred to an environment that will conserve potency/activity. During the intermediate purification phase the objectives are to remove most of the bulk impurities, such as other proteins and nucleic acids, endotoxins and viruses. In the polishing phase most impurities have already been removed except for trace amounts or closely related substances. The objective is to achieve final purity by removing any remaining trace impurities or closely related substances. The optimal selection and combination of purification techniques for Capture, Intermediate Purification and Polishing is crucial for an efficient purification.
Selection and combination of purification techniques Proteins are purified using purification techniques that separate according to differences in specific properties, as shown in Table 16. Protein property
Technique
Charge
Ion exchange (IEX)
Size
Gel filtration (GF)
Hydrophobicity
Hydrophobic interaction (HIC), Reversed phase (RPC)
Biorecognition (ligand specificity)
Affinity (AC)
Charge, ligand specificity or hydrophobicity
Expanded bed adsorption (EBA) follows the principles of AC, IEX or HIC
Table 16. Protein properties used during purification.
Resolution
Recovery
Speed
Capacity
Every technique offers a balance between resolution, capacity, speed and recovery.
54
Capacity, in the simple model shown, refers to the amount of target protein loaded during purification. In some cases the amount of sample that can be loaded will be limited by volume (as in gel filtration) or by large amounts of contaminants rather than the amount of the target protein. Speed is most important at the beginning of purification where contaminants, such as proteases, must be removed as quickly as possible. Recovery becomes increasingly important as the purification proceeds because of the increased value of the purified product. Recovery is influenced by destructive processes in the sample and by unfavourable conditions on the column. Resolution is achieved by the selectivity of the technique and the efficiency of the chromatographic matrix in producing narrow peaks. In general, resolution is most difficult to achieve in the final stages of purification when impurities and target protein are likely to have very similar properties. Select a technique to meet the objectives for the purification step. Choose logical combinations of purification techniques based on the main benefits of the technique and the condition of the sample at the beginning or end of each step. A guide to the suitability of each purification technique for the stages in CIPP is shown in Table 17. Technique Main features
Sample start condition
Sample end condition
high resolution high capacity high speed
low ionic strength sample volume not limiting
high ionic strength or pH change
good resolution good capacity high speed
high ionic strength sample volume not limiting
low ionic strength
high resolution high capacity high speed
specific binding conditions sample volume not limiting
specific elution conditions
GF
high resolution using Superdex media
limited sample volume (<5% total column volume) and flow rate range
buffer exchanged (if required) diluted sample
RPC
high resolution
requires organic solvents
in organic solvent, risk loss of biological activity
IEX
HIC
AC
Capture
Intermediate
Polishing
concentrated sample concentrated sample
concentrated sample
Table 17. Suitability of purification techniques for the CIPP.
Minimize sample handling between purification steps by combining techniques to avoid the need for sample conditioning. The product should be eluted from the first column in conditions suitable for the start conditions of the next column (see Table 17). Ammonium sulphate, often used for clarification and concentration of antibodies (see page 18), leaves the sample in high salt consequently HIC, which requires high salt to enhance binding to the media, is ideal as the capture step. The salt concentration and the total sample volume will be significantly reduced after elution from the HIC column. Dilution of the fractionated sample or rapid buffer exchange using a desalting column will prepare it for the next IEX or AC step. 55
Gel filtration is a non-binding technique unaffected by buffer conditions, but with limited volume capacity. GF is well suited for use after any of the concentrating techniques (IEX, HIC, AC, EBA) since the target protein will be eluted in a reduced volume and the components from the elution buffer will not affect the gel filtration process. Selection of the final strategy will always depend upon specific sample properties and the required level of purification. Logical combinations of techniques are shown in Figure 22.
Proteins with low solubility SDS extraction
GF (in non-ionic detergent)
SDS extraction
Solubilizing agents (urea, ethylene glycol non-ionic detergents)
HIC
HIC
GF
GF
Crude sample or sample in high salt concentration Sample clarification
Capture
GF GF desalt mode desalt mode
AC
IEX
Intermediate Purification Polishing
GF or RPC
GF or RPC
GF desalt mode
HIC IEX dilution may be needed
IEX
HIC
GF
GF
Clear or very dilute samples Capture
AC
IEX
Intermediate Purification Polishing
GF or RPC
GF or RPC
IEX
Precipitation (e.g. in high ionic strength)
HIC
Resolubilize
GF
Treat as for sample in high salt concentration
Fig. 22. Logical combinations of chromatographic techniques.
For any capture step, select the technique showing the most effective binding to the target protein while binding as few of the contaminants as possible, i.e. the technique with the highest selectivity and/or capacity for the target protein. A sample is purified using a combination of techniques and alternative selectivities. For example, in an IEX-HIC-GF strategy, the capture step selects according to differences in charge (IEX), the intermediate purification step according to differences in hydrophobicity (HIC) and the final polishing step according to differences in size (GF).
56
If nothing is known about the target protein use IEX-HIC-GF. This combination of techniques can be regarded as a standard protocol. Consider the use of both anion and cation exchange chromatography to give different selectivities within the same purification strategy. IEX is a technique which offers different selectivities using either anion or cation exchangers. The pH of the purification can be modified to alter the charge characteristics of the sample components. It is therefore possible to use IEX more than once in a purification strategy, for capture, intermediate purification or polishing. IEX can be used effectively in the same purification scheme for rapid purification in low resolution mode during capture and in high resolution mode during polishing. Consider RPC for a polishing step, provided that the target protein can withstand the run conditions. Reversed phase chromatography (RPC) separates proteins and peptides on the basis of hydrophobicity. RPC is a high selectivity (high resolution) technique, requiring the use of organic solvents. The technique is widely used for purity check analyses when recovery of activity and tertiary structure are not essential. Since many proteins are denatured by organic solvents, the technique is not generally recommended for protein purification because recovery of activity and return to a correct tertiary structure may be compromised. However, in the polishing phase, when the majority of protein impurities have been removed, RPC can be excellent, particularly for small target proteins that are not often denatured by organic solvents. CIPP does not mean that there must always be three purification steps. For example, capture and intermediate purification may be achievable in a single step, as may intermediate purification and polishing. Similarly, purity demands may be so low that a rapid capture step is sufficient to achieve the desired result. For purification of therapeutic proteins, a fourth or fifth purification step may be required to fulfil the highest purity and safety demands. The number of steps used will always depend upon the purity requirements and intended use for the protein.
57
Selection of media for multi-step purification Having decided upon the most suitable purification techniques, the most suitable medium should be selected for each technique. Recommended prepacked columns to follow a standard purification protocol combining IEX, HIC and GF at laboratory scale are shown in Table 18. These recommendations are based on the assumption that the possibility of affinity purification has been excluded. Step
Starting scale
Increasing scale
Sample Preparation
HiTrap Desalting
HiPrep 26/10 Desalting
Capture IEX
HiTrap IEX Selection Kit (screen 7 different IEX media packed in 1 ml HiTrap columns to select optimal medium)
HiLoad HiLoad HiLoad HiLoad
16/10 26/10 16/10 26/10
Q Sepharose Fast Flow Q Sepharose Fast Flow SP Sepharose Fast Flow SP Sepharose Fast Flow
HiTrap HiTrap HiTrap HiTrap HiTrap HiTrap HiTrap
HiPrep HiPrep HiPrep HiPrep
16/10 16/10 16/10 16/10
Q XL SP XL DEAE FF CM FF
16/10 16/10 16/10 16/10
Phenyl FF (high sub) Phenyl FF (low sub) Butyl FF Octyl FF
Q FF, 1 ml or 5 ml SP FF, 1 ml or 5 ml DEAE FF, 1 ml or 5 ml CM FF, 1 ml or 5 ml Q XL, 1 ml or 5 ml SP XL, 1 ml or 5 ml ANX FF(high sub), 1 ml or 5 ml
Capture HIC
HiTrap HIC Selection Kit (screen 5 different HIC media packed in 1 ml HiTrap columns to select optimal medium) HiTrap Phenyl FF (high sub), 1 ml or 5 ml HiTrap Phenyl FF (low sub), 1 ml or 5 ml HiTrap Octyl FF, 1 ml or 5 ml HiTrap Butyl FF, 1 ml or 5 ml HiTrap Phenyl HP, 1 ml or 5 ml
HiPrep HiPrep HiPrep HiPrep
Intermediate Purification IEX
HiTrap Q HP, 1 ml or 5 ml HiTrap SP HP, 1 ml or 5 ml RESOURCE™ Q, 1 ml or 6 ml RESOURCE S, 1 ml or 6 ml Mono Q™ HR 5/5 Mono S™ HR 5/5
HiLoad 16/10 Q Sepharose HP HiLoad 26/10 Q Sepharose HP HiLoad 16/10 SP Sepharose HP HiLoad 26/10 SP Sepharose HP SOURCE 15Q (loose medium) SOURCE 15S (loose medium)
Intermediate Purification HIC
HiTrap Phenyl HP, 1 ml or 5 ml RESOURCE 15ISO RESOURCE 15PHE RESOURCE 15ETH
HiLoad 16/10 Phenyl Sepharose HP HiLoad 26/10 Phenyl Sepharose HP SOURCE™ 15ISO (loose medium) SOURCE 15PHE (loose medium) SOURCE 15ETH (loose medium)
Polishing GF grade
Superdex 200 HR 10/30
HiLoad 16/60 Superdex 200 prep
(listed in order of bead size: use smaller bead sizes as sample purity increases to maximize resolution)
HiLoad 26/60 Superdex 200 prep
(select medium according to
grade size of target molecules and contaminants)
Superdex 75 HR 10/30 Superdex 30 HR 10/30
HiLoad HiLoad HiLoad HiLoad
16/60 26/60 16/60 26/60
Superdex Superdex Superdex Superdex
75 75 30 30
prep prep prep prep
grade grade grade grade
Table 18. To increase
the binding capacity and for larger scale purification, HiTrap columns can easily be linked in series. Most media are available for packing in larger columns. Always check specific availability if the intention is to scale up. Custom-designed media and custom-packed columns can be produced. 58
If a purification is not intended for scale up (i.e. milligram quantities of product are needed), use high performance, prepacked media, such as Sepharose High Performance (IEX and HIC), SOURCE (IEX, HIC), Monobeads™ (IEX), or Superdex (GF) for all steps.
Selection of pH for purification by ion exchange Knowledge of the characteristics of antibodies and fragments helps considerably in the selection of the correct purification conditions, particularly with regard to the elimination of known contaminants. Table 19 gives examples of the isoelectric point values for antibodies and some common contaminants. Component
pI
Antibodies
4–9
Albumin
4.9
Transferrin
5.2–6.1
a2-macroglobulin
4.1–4.9
Table 19.
The selection of anion or cation exchange and the correct pH is crucial for a successful purification. The principles of ion exchange chromatography are outlined briefly in Appendix 10, together with practical advice on the development and optimization of a purification method. As shown below, the ion exchanger and buffer pH can be chosen according to the information available on the isoelectric point of the antibody.
Use buffer 0.5 pH units above pI on anion exchange e.g. HiTrap Q or Use buffer 0.5 pH units below pI on cation exchange e.g. HiTrap SP Yes
Is the isoelectric point of the antibody known? No
Use anion exchange, pH 7.0 or Use cation exchange, pH 4.0
Antibody does not bind – raise pH Antibody does not elute – lower pH Antibody does not bind – lower pH Antibody does not elute – raise pH
If the pI of the antibody is sufficiently different from the contaminants, it may be possible to minimize contamination by using a cation exchange medium (negatively charged) at a pH above the pI of the impurities and below that of the antibody. This will ensure that the antibody (positively charged) binds to the column and the impurities (which could include negatively charged nucleic acids and endotoxins) pass through.
59
Examples of multi-step purification The following examples demonstrate the successful application of the CIPP strategy to the purification of antibodies and antibody fragments. In many cases, with knowledge of the characteristics of the target protein and known contaminants techniques and elution conditions can be selected to yield a highly pure product in as few as two purification steps. The availability of a suitable affinity medium will often lead to a two-step purification process, combining affinity and gel filtration, as shown below, and as demonstrated by examples in Chapter 3.
Example 1: Two-step purification of mouse monoclonal IgG1 This example demonstrates the effectiveness of using a high selectivity, affinity purification for initial capture. In common with most antibody preparations, there is a possibility that IgG aggregates and/or dimers are present. To achieve highest purity it is therefore essential to include a gel filtration polishing step. A more detailed description of this purification can be found in Application Note 18-1128-93, available from Amersham Biosciences.
Target molecule Mouse monoclonal IgG1.
Source material Cell culture supernatant.
Extraction and clarification Cell culture supernatant is filtered through a 0.45 µm filter.
Capture The target protein is captured on a HiTrap rProtein A FF column. This step removes contaminating proteins, low molecular substances and significantly reduces the sample volume.
Scouting for optimal elution conditions In contrast to other IgG subclasses, most mouse monoclonal antibodies of the IgG1 subclass require a high salt concentration to bind to rProtein A. Figure 23 shows the results of a scouting experiment to define the optimal salt concentration for binding. Scouting is also used to select the optimal pH for elution of the monoclonal antibody (pH 4.5 was selected, results not shown). Using ÄKTAdesign systems for automatic scouting of optimal binding and elution conditions can improve the recovery of a specific antibody, and the optimized purification can be automated for routine use.
60
mAU
Sample:
Column: Binding buffer:
1200
Elution buffer: Flow: System:
900
Cell culture supernatant containing monoclonal IgG1, 90 ml HiTrap rProtein A FF, 1 ml 100 mM sodium phosphate, 0-3.5 M sodium chloride, pH 7.4 100 mM sodium citrate, pH 3 1 ml/min ÄKTAFPLC™
0.0 M NaCl 600 0.5 M NaCl 1.5 M NaCl
300
2.5 M NaCl 3.5 M NaCl
0 120
125
130
135
140
145
150
155
ml
Fig. 23. Automatic scouting of optimal salt concentration in the binding buffer on HiTrap rProtein A FF.
Optimization of binding and elution conditions gives a well-resolved peak containing IgG1, as shown in Figure 24. mAU
Sample:
2200 1800 Sample application
1400
Column: Binding buffer:
Wash with binding buffer
Elution
1000
IgG1 peak
Elution buffer: Flow: System:
Cell culture supernatant containing monoclonal IgG1, 100 ml HiTrap rProtein A FF, 1 ml 100 mM sodium phosphate, 2.5 M sodium chloride, pH 7.4 100 mM sodium citrate, pH 4.5 1 ml/min ÄKTAFPLC
600 200 90
120
ml
Fig. 24. Optimized capture step on HiTrap rProtein A FF.
Intermediate purification No intermediate step is required as the high selectivity of the capture step gives a sufficiently high level of purity so that only a final polishing step is necessary.
Polishing The polishing step shown in Figure 25 removes low or trace levels of contaminants, in this case IgG aggregates and/or dimers, by gel filtration on a HiLoad 16/60 Superdex 200 prep grade column.
61
Monomeric monoclonal IgG1
300
Sample:
A 280
250
Conductivity
Column: Buffer:
200 Total column volume
Flow: System:
150
Fraction from HiTrap rProtein A FF column containing monoclonal IgG1 (3 ml) HiLoad 16/60 Superdex 200 prep grade 50 mM sodium phosphate, 0.15 M sodium chloride, pH 7.4 1 ml/min ÄKTAFPLC
100 50 0 0
50
100
ml
150
Fig. 25. Polishing on HiLoad 16/60 Superdex 200 prep grade.
An affinity purification reduces sample volume and concentrates the sample. Gel filtration is the slowest of all the chromatographic techniques and the size of the column determines the volume of sample that can be applied. It is most logical to use gel filtration after techniques that reduce sample volume.
Yield and analysis Approximately 1.2 mg monoclonal antibody was recovered from 50 ml cell culture supernatant. The recovery from the capture and polishing steps was above 95%. Figure 26 shows the SDS PAGE analysis of selected fractions.
Lane 6.
Sample: Low Molecular Weight Calibration Kit Starting material (diluted 2-fold) Eluted IgG1 peak from HiTrap rProtein A FF column (diluted 10-fold) Flow through, HiTrap rProtein A FF Eluted IgG1 peak from HiLoad Superdex 200 prep grade (diluted 6-fold) Low Molecular Weight Calibration Kit
Gel: System:
10-15% SDS-PAGE PhastGel PhastSystem
Mr Lane 1. Lane 2. Lane 3.
97 000 67 000 43 000
Lane 4. Lane 5.
30 000 20 100 14 000 1
2
3
4
5
6
Fig. 26. Purity analysis using SDS-PAGE.
Example 2: Two-step purification of a mouse monoclonal IgG1 for diagnostic use The goal of this purification is production of a monoclonal antibody to achieve a level of purity sufficient for in vitro diagnostic use.
Target molecule Mouse monoclonal IgG1 anti-IgE.
Source material Hybridoma cell culture.
Clarification Sample is filtered and ammonium sulphate added to 0.05 M. This is to enhance binding to the HIC column, not to precipitate the monoclonal antibody.
62
Capture As shown in Figure 27 a HIC purification is chosen for the capture step because the antibody binds very strongly to the medium (Phenyl Sepharose High Performance) and most foetal calf serum proteins pass through the column. The sample is concentrated into a smaller volume for polishing. Screening of HIC media, using a HiTrap HIC Selection Kit or RESOURCE HIC Test Kit, is recommended to select the medium that gives the best results. Buffer conditions should be checked to select the concentration of ammonium sulphate that gives the highest binding selectivity for the antibody and avoids binding albumin. Start buffer:
20 mM potassium phosphate, 0.05 M ammonium sulphate, pH 7.0.
Elution buffer: 20 mM potassium phosphate, pH 7.0.
Performing the purification 1. Equilibrate column in start buffer. 2. Apply sample. 3. Wash the column with start buffer until the absorbance at 280 nm has returned to baseline. 4. Use the elution buffer to create a linear gradient (10 column volumes) from 0.5-0 M ammonium sulphate. 5. Wash with 2-3 column volumes of 100% elution buffer. 6. Re-equilibrate with 2-3 column volumes of start buffer.
A 280 nm
Sample: Column: Start buffer:
0.40 0.30
Elution buffer: Gradient:
0.20
Hybridoma cell culture supernatant, mouse IgG1, anti-IgE. Ammonium sulphate added to 0.5 M Phenyl Sepharose High Performance, 10 cm bed height 20 mM potassium phosphate, 0.5 M ammonium sulphate, pH 7.0 20 mM potassium phosphate, pH 7.0 0-100% elution buffer, 10 column volumes
0.10 0.00 50
50
100
150 Time (min)
Fig. 27. Capture on HiLoad 16/10 Phenyl Sepharose High Performance.
Intermediate purification No intermediate step is required as the capture step gives a purity level > 95%.
Polishing Gel filtration is a suitable polishing step (Figure 28). Using Superdex 200 prep grade a final purity of > 99% is achieved.
Performing the purification 1. Equilibrate column in phosphate buffered saline, pH 7.5 at 15 ml/min. 2. Apply sample (maximum sample volume 1-2% of total column volume). 3. Elute sample in one column volume of buffer. Collect fractions. 4. Wash with 2-3 column volumes of buffer.
63
A 280 nm
Sample: Column: Buffer: Flow rate:
0
500
1000
Fraction from HIC capture step Superdex 200 prep grade, 60 cm bed height Phosphate buffered saline, pH 7.5 15 ml/min
1500 Volume (ml)
Fig. 28. Polishing on Superdex 200 prep grade.
Example 3: Three-step purification of a recombinant Fab fragment This example demonstrates a three-step purification strategy in which the same purification principle is used in two different modes in the capture and polishing step: IEX for capture, HIC for intermediate purification and IEX for the polishing step. The objective of this work was to develop a strategy that could be scaled up for use as a routine procedure. A more detailed description of this work can be found in Application Note Code No. 18-1111-23 from Amersham Biosciences.
Target Molecule Recombinant antigen binding fragment (Fab) directed against HIV gp-120.
Source The anti-gp 120 Fab was expressed in the periplasm of the E. coli strain BM170 MCT61. E. coli pellets were stored frozen after being harvested and washed once.
Sample extraction, clarification and capture Thawed cells were lysed with sucrose. The lysate was treated with DNase I in the presence of 2 mM MgCl2 at pH 7.5 before the capture step. The Fab fragment was captured from non-clarified homogenate by expanded bed adsorption with STREAMLINE™ SP (cation exchanger). Expanded bed adsorption was chosen because the target protein could be captured directly from the crude homogenate in a single step, without the need for centrifugation or other preparatory clean-up steps. The technique is well suited for large-scale purification. At laboratory scale, a prepacked cation exchange column can be used together with suitable sample preparation before beginning purification. The result of the capture step is shown in Figure 29. The Fab fragment is concentrated and rapidly transferred into a stable environment by a step elution.
64
A 280 nm
Column: Adsorbent: Sample:
2.0
STREAMLINE 200 (i.d. 200 mm) STREAMLINE SP, 4.6 l 60 l high pressure homogenized E. coli suspension 50 mM sodium acetate, pH 5.0 50 mM sodium acetate, pH 5.0, 1 M NaCl 300 cm/h during sample application and wash 100 cm/h during elution
Start buffer: Elution buffer: 1.0
Flow:
Sample application
50
Washing, Start buffer
100
Elution, Elution buffer Pool
150
5 10 15 Volume (litres)
Fig. 29. Capture step using expanded bed adsorption.
Intermediate purification HIC was selected because the separation principle is complementary to IEX and, since the sample was already in a high salt buffer after elution from STREAMLINE SP, a minimum amount of sample conditioning was required. Hydrophobic properties are difficult to predict and it is always recommended to screen different media. A HiTrap HIC Selection Kit (containing five 1 ml columns prepacked with different HIC media suitable for use at production scale) was used to screen for the most appropriate medium. Buffer pH was kept at pH 5.0 to minimize the need for sample conditioning after capture. Results of the media screening are shown in Figure 30.
mAU 280nm
System: Sample: Columns:
Start buffer: Elution buffer: Gradient: Flow rate:
ÄKTAexplorer Fab fraction from STREAMLINE SP, 2 ml HiTrap HIC Selection Kit (1 ml columns): HiTrap Phenyl HP HiTrap Phenyl FF (low sub) HiTrap Phenyl FF (high sub) HiTrap Butyl FF HiTrap Octyl FF 1 ml (NH4)2SO4, 50 mM NaAc, pH 5.0 50 mM NaAc, pH 5.0 20 column volumes from 0-100% elution buffer 2 ml/min (300 cm/hr)
Conductivity (mS/cm)
400
150
300 100 HiTrap Phenyl HP 200 HiTrap Phenyl FF (low sub) Fab
HiTrap Phenyl FF (high sub)
100
50
HiTrap Butyl FF HiTrap Octyl FF
0
0 0.0
10.0
Time (min)
Fig. 30. HIC media scouting using HiTrap HIC Selection Kit.
Phenyl Sepharose 6 Fast Flow (high sub) was selected since the medium showed excellent selectivity for the target protein thereby removing the bulk contaminants. Optimization of elution conditions resulted in a step elution being used to maximize the throughput and the concentrating effect of the HIC technique. Figure 31 shows the optimized elution scheme and the subsequent scale up of the intermediate purification step.
65
System: Sample: Columns:
ÄKTAexplorer Fab fraction from STREAMLINE SP, 80 ml Phenyl Sepharose 6 Fast Flow (high sub) in XK 16/20 (10 cm bed height) Start buffer: 1 M (NH4)2SO4, 50 mM NaAc, pH 5.0 Elution buffer: 50 mM NaAc, pH 5.0 Gradient: Step gradient to 50% elution buffer Flow rate: 5 ml/min Column volume: 20 ml
Conductivity (mS/cm)
A 280 nm
System: Sample: Columns:
ÄKTAexplorer Fab fraction from STREAMLINE SP, 800 ml Phenyl Sepharose 6 Fast Flow (high sub) in XK 50/20 Start buffer: 1 M (NH4)2SO4, 50 mM NaAc, pH 5.0 Elution buffer: 50 mM NaAc, pH 5.0 Gradient: Step gradient to 50% elution buffer Flow rate: Equilibration: 100 ml/min Loading and elution: 50 ml/min Column volume: 200 ml Conductivity (mS/cm)
A 280 nm b) 2.00
a) 2.00 100
100
1.00 1.00 50
50
0
0 0
50
100
150
200
250 Volume (ml)
0
500
1000
1500
2000
2500 Volume (ml)
Fig. 31. Intermediate purification using HIC: optimization and scale-up.
Polishing Gel filtration was investigated as the natural first choice for a final polishing step to remove trace contaminants and transfer the sample to suitable storage conditions. However, in this example, gel filtration could not resolve a Mr 52 000 contaminant from the Mr 50 000 Fab fragment (results not shown). As an alternative another cation exchanger SOURCE 15S was used. In contrast to the cation exchange separation at the capture step, the polishing cation exchange separation was performed using a shallow gradient elution on a medium with a small, uniform bead size (SOURCE 15S) to give a high resolution result, as shown in Figure 32.
66
Conductivity (mS/cm)
A 280 nm 100
80.0
System: Sample:
ÄKTAexplorer Fab fraction from HIC purification 15 ml eluate diluted 7.5/100 Columns: RESOURCE S 6 ml Buffer A: 50 mM NaAc, pH 4.5 Buffer B: 50 mM NaAc, pH 4.5, 1 M NaCl Gradient: 50 column volumes Flow rate: 18.3 ml/min
80 60.0
60 40.0 Active Fab
40
20.0 20
0
100
200 Volume (ml)
Fig. 32. Optimized Fab polishing step.
Analytical assays Collected fractions were separated by SDS-PAGE and stained by Coomassie Blue using PhastSystem, following the protocols supplied with the instrument. Fab activity was measured by a goat-anti-human IgG Fab ELISA, an anti-gp120 ELISA and an in vitro assay which measured the inhibition of HIV-1 infection of T-cells. Nucleic acid was routinely monitored by measuring A260/A280. The correlation of a high A260/A280 ratio (> 1) with the presence of DNA was verified for selected samples by agarose gel electrophoresis and EtBr staining. Endotoxin determination employed a kinetic chromogenic Limulus assay (COAMATIC Chromogenics AB, Mölndal, Sweden).
67
68
Chapter 6 Removal of specific contaminants after initial purification For many applications at laboratory scale, contaminant molecules may not be a significant problem. Affinity chromatography will provide sufficient purity and, as long as the presence of any minor contaminants does not interfere with the intended application, the purified sample can be used directly. However, as outlined in Table 3 on page 13, source materials will be associated with major contaminants which may need to be removed either before purification begins (e.g. lipid material or phenol red) or after initial purification. Common contaminants are albumin, transferrin and host or bovine immunoglobulins that originate in ascites, cell culture and serum. These three contaminants pose three different purification problems: albumin because of its abundance, transferrin because of its similarity to the charge characteristics of many antibodies, and host or bovine immunoglobulins because of the similarity of their properties to those of the target molecule. For some cell culture preparations it is possible to decreased the level of serum during growth, thereby reducing or eliminating many of these impurities before purification. An alternative solution is to consider the use of a different host that does not require these supplements. The chromatographic technique best suited to separate specific contaminants from the target molecule will largely depend upon the physical characteristics of all the components. Select the technique according to the characteristics that are significantly different: ion exchange (for separation by differences in charge), hydrophobic interaction (for separation by differences in hydrophobicity), and gel filtration (for separation by size). See Appendix 10 for an overview of the principles of the chromatographic techniques. If the pI value of the antibody is sufficiently different from the contaminants it may be, possible to minimize contamination by using a cation exchange medium (negatively charged) at a pH above the pI of the impurities and below that of the antibody. This will ensure that the antibody (positively charged) binds to the column and the impurities (including negatively charged nucleic acids) pass through. When using ion exchange chromatography, apply the same principles for the removal of contaminants as those described on page 59.
Bovine immunoglobulins Co-purification of host or bovine immunoglobulins is a problem associated with any affinity purification when using a native source or a source to which supplements such as calf serum or bovine serum albumin are added. This contamination problem is particularly significant for monoclonal antibodies intended for in vivo human applications. Difficulties have also been encountered when murine monoclonal antibodies are the target molecule. The similarities between the physical characteristics of the contaminant and the target molecule require careful selection of the most suitable chromatographic technique for purification. Both hydrophobic interaction and ion exchange can be used.
69
Hydrophobicity of proteins is difficult to predict. It is recommended to screen several chromatographic media with different hydrophobicities (e.g. using the HiTrap HIC Selection Kit) to enable selection of the medium that gives the best results In this example, SOURCE 15ISO, 15PHE and 15ETH were tested. As shown in Figure 33, HiTrap HIC Selection Kit can be used to screen for the most suitable hydrophobic medium. A RESOURCE HIC Test Kit is also available. AU
% 100
1
0.50
Samples: Flow rate: Start buffer: Elution buffer: Columns:
0.8 mg pure mouse monoclonal IgG 1 ml/min 50 mM sodium phosphate, 1.0 M ammonium phosphate, pH 7.0 50 mM sodium phosphate, pH 7.0 1. HiTrap Phenyl HP 2. HiTrap Phenyl FF (low sub) 3. HiTrap Phenyl FF (high sub) 4. HiTrap Octyl FF 5. HiTrap Butyl FF
80 0.40 60
0.30
2 0.20
40
5 3
0.10
4
20
0.00
0 10.0
20.0
30.0
min
Fig. 33.
Albumin and transferrin If albumin is present at very high levels in the original sample, it may occasionally be seen as a contaminant even after affinity purification, but other impurities will wash through the column. IEX or HIC are usually the methods of choice for removing albumin and transferrin, separating the molecules on the basis of differences in their isoelectric points or differing hydrophobicities (see Appendix 10 for the principles of these techniques). After an IEX purification, albumin and transferrin may be present if their charge properties are similar to the target protein. In some cases, it may be possible to optimize the IEX step to improve the separation between the target protein and the contaminants by modifying the pH and elution conditions (see page 59). Since most monoclonal antibodies are more hydrophobic than albumin transferrin hydrophobic interaction chromatography can be used to bind the antibody and allow these contaminants to wash through the column.
Removal of albumin Blue Sepharose media may be a useful alternative for removing albumin. This affinity technique can be used to remove albumin either before or after other purification steps. The albumin binds in a non-specific manner by electrostatic and/or hydrophobic interactions with the aromatic anionic ligand, Cibacron™ Blue 3G-A, coupled to Sepharose.
70
Do not use Blue Sepharose media if the immunoglobulin or other target molecule has a hydrophobicity similar to that of albumin. Use HiTrap Blue HP 1 ml or 5 ml columns to remove host albumin from mammalian expression systems or when the sample is known to contain high levels of albumin that may mask the visualization of other protein peaks seen by UV absorption.
Purification options Capacity/ml medium*
Comments
HiTrap Blue HP
HSA 20 mg
Removal of albumin. Prepacked 1 ml and 5 ml columns.
Blue Sepharose 6 Fast Flow # packing.
HSA 16 mg
Supplied as a suspension ready for column
*Protein binding capacity will vary for different proteins. # Higher binding capacity than Blue Sepharose CL-6B.
Purification mS/cm
AU 280 nm UV 280 nm Conductivity
2.0
Sample: Column: Binding buffer: Elution buffer:
Human plasma, buffer exchanged to binding buffer with HiTrap Desalting HiTrap Blue HP 1 ml column 20 mM sodium phosphate, pH 7.0 20 mM sodium phosphate, 2.0 M sodium chloride, pH 7.0
Albumin
150
100
1.0
50
0
0 15
20
25
30 min
Fig. 34.
Performing a purification Binding buffer: 20 mM sodium phosphate, pH 7.0 Elution buffer: 20 mM sodium phosphate, 2.0 M NaCl, pH 7.0 1. Wash and equilibrate with 10 column volumes of binding buffer. 2. Apply the sample, collecting the flow-through (containing the target molecule) until no more protein is detected (determined by UV absorbance at 280 nm). 3. Elute the albumin from the column with 10 column volumes of elution buffer until no material appears in the flow eluent. 4. Wash with 6 column volumes of binding buffer.
Storage Store columns and media at +4° to +8°C with 20% ethanol.
71
a2-macroglobulin and haptoglobulin These and other minor proteins, such as ceruloplasmin, may be present in preparations made from native sources or in the presence of serum. Since a2-macroglobulin (Mr 820 000) is closely related in size to IgM it is easily separated from smaller molecules, such as IgG, by gel filtration. Similarly, haptoglobulin will separate from IgM on a suitable gel filtration column. In general, careful selection of the correct ion exchange medium and pH for purification can ensure that these contaminants are removed during an ion exchange purification. Blue Sepharose and Chelating Sepharose can also be considered for removing of a2-macroglobulin
Dimers and aggregates A frequent difficulty when purifying immunoglobulins is the appearance of dimers and other aggregates. Aggregates are often formed when working with proteins at higher concentrations. When high salt concentrations are involved, dimers or polymers can be formed during freezing and thawing. These aggregates can lower the biological activity of the sample. Gel filtration is the technique of choice for remving aggregates, and a medium such as Superdex 200 will give the best possible separation between monomer and dimer. It is used as the final polishing step in many purification strategies. The principles of gel filtration are outlined in Appendix 10. Gel filtration is highly recommended as a final polishing step after any affinity purification. The sample will be transferred into a final buffer at the correct pH and the low molecular weight molecules, such as salt, will be removed. Gel filtration is not a binding technique so the sample loading is limited to 1-3% of the total column volume in most cases. For purification with larger sample volumes, use HiLoad 16/60 Superdex 200 prep grade or HiLoad 26/60 Superdex 200 prep grade prepacked columns. Figure 35 shows an example of the purification of human IgG monomers and dimers on Superdex 200. A 280
Monomer
0.70
Void volume
Total column volume
0.50
Sample: Column: Flow rate: Buffer:
0.30
Dimer 0.10 0 0
Fig. 35.
72
5.0
10.0
15.0
20.0
ml
50 µl human IgG (9 mg/ml) Superdex 200 HR 10/30 0.25 ml/min 50 mM NaH2PO4, 0.15 M NaCl, pH 7.0
DNA and endotoxins For large-scale purification the need to assay for critical impurities is often essential as the products may be used for clinical or diagnostic applications. In practice, when a protein is purified for research purposes, it is often too time consuming to identify and set up specific assays for harmful contaminants, such as DNA and endotoxins. A practical approach is to purify the protein to a certain level and perform SDS-PAGE after storage to check for protease degradation. Suitable control experiments should be included within bio-assays to indicate if impurities are interfering with results. Nucleic acids often dissociate from proteins at high salt concentrations. This makes hydrophobic interaction chromatography a suitable technique for capturing the target protein and removing nucleic acids. Since DNA and endotoxins are negatively charged over a wide pH interval, a cation exchange chromatography step at a pH below the isoelectric point of the target molecule will bind the target and allow the negatively charged molecules to wash through the column. Consequently, if cation exchange is used as the initial capture step these contaminants will be removed at an early stage in purification. If endotoxins need to be removed from a purified product, anion exchange chromatography at a pH value slightly below the isoelectric point of the product will bind the endotoxins and the target molecule will wash through the column. Alternatively use a pH which binds both molecular species, but allows them to be clearly separated during gradient elution from the column.
Affinity ligands With any affinity chromatography medium there is a risk of ligand leakage from the matrix, particularly if harsh conditions are required to elute the target molecule. In many cases this leakage is negligible and a satisfactory purity is achieved. At laboratory scale, leakage of ligands is not a significant problem. However, in pharmaceutical production processes it must be shown that even trace amounts of ligand have been removed from the final product. Figure 36 shows an example of the removal of protein A from mouse IgG2b on a HiTrap SP HP 1 ml column. Levels of protein A leakage are usually extremely low so the sample has been spiked with protein A to visualize the protein A peak. AU
IgG
0.10
Column: Sample: Binding buffer: Elution buffer: Flow rate: Gradient:
HiTrap SP HP 1 ml Purified antibody (0.61 mg) spiked with recombinant protein A (1.8 mg) 20 mM sodium citrate, pH 5.2 20 mM sodium citrate, 1.0 M NaCl, pH 5.2 4 ml/min 0–45% elution buffer in 15 column volumes
0.08
0.06
Protein A
0.04
0.02
0.00 0.0
5.0
10.0
15.0
20.0 Volume (ml)
Fig. 36. Removal of protein A from mouse IgG2b by cation exchange chromatography on HiTrap SP HP. Recombinant protein A was spiked into mouse IgG2b previously purified on rProtein A Sepharose Fast Flow. 73
74
Chapter 7 Large-scale purification Large-scale purification requires careful planning and close collaboration with process specialists. This chapter gives only a brief review of some of the issues involved in largescale purification. A downstream production process must achieve the required purity and recovery with complete safety and reliability, at a given scale and within a given economic framework. Economy is a very complex issue. In commercial production, time to market can override extensive optimization in favour of recovery, capacity or speed. Robustness and reliability are always of great concern since a batch failure can have major consequences. Special safety issues may be involved in the purification of biopharmaceuticals, such as detection or removal of infectious agents, pyrogens, immunogenic contaminants and tumorigenic hazards. It may be necessary to use analytical techniques targeted towards specific contaminants in order to demonstrate that they have been removed to acceptable levels. High safety demands mean that considerable attention is paid not only to the purification strategy, but also to the source of the product. At production scale, considerable emphasis is placed on minimizing the contaminants that can enter the process from the very beginning, as well as focusing on the removal of impurities downstream. Currently, therapeutic antibodies are being developed in Chinese Hamster Ovary (CHO) cells and E. coli, as well as hybridomas. Expression levels are often higher than those used in research applications (up to several grams per litre of culture supernatant) and impurities are minimized by, for example, the use of protein-free or highly characterized culture media. With well established characteristics for both target molecule and contaminants and with detection assays and sample preparation procedures in place, the CIPP (Capture, Intermediate Purification, Polishing) strategy, as described in Chapter 5, is frequently used in industry. This strategy ensures faster method development and a short process to pure product and good economy. Each step has clearly defined goals in terms of concentration, stabilization, removal of critical contaminants and conditioning. Since a production process has goals and constraints that differ from those in the laboratory, CIPP can lead to a different combination of techniques as compared to traditional laboratory scale approaches. Process development is performed in a down-scaled model of the final production process. Scalability is therefore a key issue and media or conditions that are unproven or inappropriate at large-scale are not worth testing in the laboratory. In largescale capture, throughput will often be the focus during method development. It is important to consider all aspects: sample extraction and clarification, sample loading capacity, flow rate during equilibration, binding, washing, elution and cleaning. The need for cleaning-inplace procedures may even exclude media that were suitable at laboratory scale. In principle, a capture step is optimized to maximize capacity and/or speed at the expense of some resolution. However, there is usually considerable resolution and purification from molecules that have significant physicochemical differences compared to the target protein. Recovery will be of concern in any preparative situation, especially for production of a high value product, and it is important to assay for recovery during optimization of the capture step.
75
Considerations for monoclonal antibody purification CIPP can be applied to any protein purification, including any antibody class or fragment, whether from native or genetically engineered sources. In the case of native monoclonal antibodies, certain combinations of techniques can be prioritized for production of large quantities of material, since a great deal is known about their characteristics and the characteristics of their most common contaminants. These are illustrated in Table 20. Purification step
Technique/Products
Comments
AC
Protein A Sepharose 4 Fast Flow rProtein A Sepharose Fast Flow rmpProtein A Sepharose Fast Flow** Protein G Sepharose 4 Fast Flow
High purity.
EBA.AC
STREAMLINE rProtein A
As above, but offers simultaneous clarification and purification.
IEX
SP Sepharose Fast Flow
Cation exchange. Since pI of MAb is usually higher than that of main contaminants: major serum proteins (e.g. albumin, transferrin) are washed through the column. MAb often more stable in acidic conditions than many other proteins. High resolution and capacity.
EBA.IEX
STREAMLINE SP XL
As above, but offers simultaneous clarification and purification.
HIC
Phenyl Sepharose Fast Flow (high sub) Phenyl Sepharose Fast Flow (low sub)
Major serum contaminants (e.g. albumin, transferrin) washed through the column.
IEX
Q or SP Sepharose Fast Flow Q or SP Sepharose High Performance
Cation or anion exchange according to the charge properties of components remaining after capture step. Use cation exchange when pI of MAb is higher than that of main contaminants. Sepharose matrix selected according to the performance required (scale, speed etc.)
HIC
Phenyl Sepharose Fast Flow (high sub) Phenyl Sepharose Fast Flow (low sub) Phenyl Sepharose High Performance SOURCE 15ISO or 15PHE
Follows after EBA and/or IEX as samples are already in salt. Screen to determine the medium that gives the best purification.
IEX
Q Sepharose Fast Flow or SP Sepharose Fast Flow Q Sepharose High Performance or SP Sepharose High Performance
Cation or anion exchange, depending on previous step, remaining impurities and other purposes. Sepharose matrix selected according to the performance required (scale, speed etc.).
HIC
SOURCE 15ISO or 15PHE
Follows after AC or IEX. Separates contaminants such as bovine IgG.
GF
Superdex 200 prep grade
Follows after IEX, HIC or AC. Highest resolution for separation of dimers and aggregates.
Capture
Intermediate Purification
Polishing
** rmpProtein A Sepharose Fast Flow may help to minimize regulatory concerns at process scale since no mammalian components are involved in production or purification.
Table 20.
76
Combining sample preparation and capture for Fc-containing antibodies There are two approaches to the purification of Fc-containing antibodies. Conventionally, filtration is used to remove debris before the capture step on a packed bed of immobilized protein A. A modern alternative is expanded bed adsorption (EBA), a technique that clarifies, concentrates and captures the target molecules from cell culture supernatant in a single step. Apart from offering better yields, because of fewer steps and quicker removal of degrading agents, EBA has been found to be more gentle for the removal of cells, which consequently reduces the load of intracellular contaminants on subsequent steps. The principles of this technique are described in Appendix 10. EBA is particularly suited for large-scale recombinant protein and monoclonal antibody purification. Crude sample containing particles can be applied to the expanded bed without filtration or centrifugation. STREAMLINE adsorbents are specially designed for use in STREAMLINE columns. Together they enable the high flow rates needed for high productivity in industrial applications. The technique combines sample preparation and capture in a single step. STREAMLINE adsorbents are designed to handle feed directly from both fermentation homogenate and crude feedstock from cell culture/fermentation. In Figure 37 IgG1 is captured from a crude sample as it is applied to an expanded bed of STREAMLINE rProtein A, while cell debris, cells, particulate matter, whole cells, and contaminants pass through. Flow is reversed and the IgG1 is desorbed in the elution buffer. Column: Absorbent: Sample:
STREAMLINE 25 75 ml STREAMLINE rProtein A 1 l of unclarified fermentation broth after pH adjustment to 8.0 Binding buffer: 50 mM Tris-HCL, 150 mM NaCl, pH 8.0 Elution buffer: 50 mM sodium phosphate, 150 mM NaCl, pH 4.5 Flow rate: 300 cm/h during sample application and wash 100 cm/h during elution
Lane 1. Lane Lane Lane Lane Lane Lane
2. 3. 4. 5. 6. 7.
Low Molecualr Weight Calibration Kit (reduced) Purified IgG Cell culture broth Flow through Wash fraction Eluate Eluate (1:10 diluted)
A 280nm Equilibration
1.5
Sample application
Wash
Elution
Mr
MAb
97 000 1
67 43 30 20 14
0.5
Transferrin
000 000 000 100 000
Albumin
1 2 3 4 5
6 7 8
0 0
25
50
75
100 Time (min)
Fig. 37. Capture of IgG1 by EBA on STREAMLINE rProtein A and analysis by SDS PAGE using silver staining under non-reduced conditions.
77
BioProcess Media for production Specific BioProcess™ Media have been designed for each chromatographic stage in a process from Capture to Polishing. Large capacity production integrated with clear ordering and delivery routines ensure that these media are available in the right quantity, at the right place, at the right time. Amersham Biosciences can assure future supplies of BioProcess Media, making them a safe investment for long-term production. The media are produced following validated methods and tested under strict control to fulfil high performance specifications. A certificate of analysis is available with each order. Regulatory Support Files contain details of performance, stability, extractable compounds and analytical methods. The essential information in these files gives an invaluable starting point for process validation, as well as providing support for submissions to regulatory authorities. Using BioProcess Media for every stage results in an easily validated process. High flow rate, high capacity and high recovery contribute to the overall economy of an industrial process. All BioProcess Media have chemical stability to allow efficient cleaning and sanitization procedures. Packing methods are established for a wide range of scales and compatible large-scale columns and equipment are available.
78
Appendix 1 Analytical assays during purification Analytical assays are essential to follow the progress of purification. They are used to assess the effectiveness of each step in terms of yield, biological activity, recovery and to help during optimization of experimental conditions. The importance of a reliable assay for the target molecule cannot be over-emphasized. When testing chromatographic fractions, ensure that the buffers used for purification do not interfere with the assay.
Total protein determination Lowry or Bradford assays are used most frequently to determine the total protein content. The Bradford assay is particularly suited to samples where there is a high lipid content that may interfere with the Lowry assay.
Purity determination Purity is most often estimated by SDS-PAGE. Alternatively, isoelectric focusing, capillary electrophoresis, reversed phase chromatography or mass spectrometry may be used.
SDS-PAGE Analysis Reagents Required 6X SDS loading buffer: 0.35 M Tris-HCl (pH 6.8), 10.28% (w/v) SDS, 36% (v/v) glycerol, 0.6 M dithiothreitol (or 5% 2-mercaptoethanol), 0.012% (w/v) bromophenol blue. Store in 0.5 ml aliquots at -80°C. 1. Add 2 µl of 6X SDS loading buffer to 5-10 µl of supernatant from crude extracts, cell lysates or purified fractions as appropriate. 2. Vortex briefly and heat for 5 minutes at +90° to +100°C. 3. Load the samples onto an SDS-polyacrylamide gel. 4. Run the gel and stain with Coomassie Blue or silver stain.
The percentage of acrylamide in the SDS-gel should be selected according to the expected molecular weight of the protein of interest (see Table 21). % Acrylamide in resolving gel
Separation size range (Mr x 10-3)
Single percentage: 5%
36–200
7.5%
24–200
10%
14–200
12.5%
14–100
15%
14–60
1
Gradient: 5–15%
1
14–200
5–20%
10–200
10–20%
10–150
The larger proteins fail to move significantly into the gel.
Table 21.
79
If using horizontal SDS pre-cast gels, refer to the Gel Media Guide from Amersham Biosciences.
Functional assays Immunospecific interactions have enabled the development of many alternative assay systems for the assessment of active concentration of target molecules. Western blot analysis is used when the sensitivity of SDS-PAGE with Coomassie Blue or silver staining is insufficient. 1. Separate the protein samples by SDS-PAGE. 2. Transfer the separated proteins from the gel to an appropriate membrane, such as Hybond™ ECL™ (for subsequent ECL detection) or Hybond P (for subsequent ECL Plus™ detection). 3. Develop the membrane with the appropriate specified reagents.
Electrophoresis and protein transfer may be accomplished using a variety of equipment and reagents. For further details, refer to the Protein Electrophoresis Technical Manual and Hybond ECL instruction manual, both from Amersham Biosciences. ELISAs are most commonly used as activity assays. Functional assays using the phenomenon of surface plasmon resonance to detect immunospecific interactions (e.g. using BIACORE™ systems) enable the determination of active concentration, epitope mapping and studies of reaction kinetics.
Detection and assay of tagged proteins SDS-PAGE, Western blotting and ELISAs can also be applied to the detection and assay of genetically engineered molecules to which a specific tag has been attached. In some cases, an assay based on the properties associated with the tag itself can be developed, e.g. the GST Detection Module for enzymatic detection and quantification of GST tagged proteins. Further details on the detection and quantification of GST and (His)6 tagged proteins are available in The Recombinant Protein Handbook: Protein Amplification and Simple Purification from Amersham Biosciences.
80
Appendix 2 Selection of purification equipment Amersham Biosciences offers solutions from the simplest purification through to large-scale protein production. Using this guide will assist in the selection of the most appropriate solution to suit the immediate purification task and possible needs in the future. ÄKTAexplorer fast method and process development, scale up
ÄKTAFPLC high performance purification
ÄKTAprime simple purification
One-step purification
Optimization of one step to increase purity
Routine, reproducible purification
Protein folding after isolation
Automated multi-step purification
Method development and optimization
System control and data handling to follow regulatory requirements, e.g. GLP
Purification will be scaled up
Easy transfer of methods to production scale
Purification Needs
Syringe+ MicroSpin™ HiTrap Purification columns Modules manual purification
Rapid, high throughput screening (GST or His tagged proteins)
ÄKTAprime: simple purification of proteins
ÄKTAexplorer: fast method and process development and scale-up for proteins, peptides and nucleic acids
ÄKTAFPLC: high performance purification of proteins
81
Appendix 3 General instructions for affinity purification with HiTrap columns Alternative 1. Manual purification with a syringe
A
B
C
Using HiTrap with a syringe. A Prepare buffers and sample. Remove the column top cap and twist off the end. B Load the sample and begin collecting fractions. C Elute and continue collecting fractions. 1. Fill the syringe with binding buffer. 2. Connect the column to the syringe using the adapter supplied ('drop to drop' to avoid introducing air into the column). 3. Remove the twist-off end. 4. Equilibrate the column with 5 column volumes of binding buffer. 5. Apply the sample using the syringe. For best results, maintain a flow rate of 0.2-1 ml/min (1 ml column) and 1-5 ml/min (5 ml column) as the sample is applied. 6. Wash with 5-10 column volumes of binding buffer. Maintain flow rates of 1-2 ml/min (1 ml column) and 5-10 ml/min (5 ml column) during the wash. 7. Elute with 5-10 column volumes of elution buffer. Maintain flow rates of 1-2 ml/min (1 ml column) and 5-10 ml/min (5 ml column) during elution.
For large sample volumes, a simple peristaltic pump can be used to apply sample and buffers.
Alternative 2. Simple purification with ÄKTAprime ÄKTAprime contains pre-programmed templates for purification of IgG, IgM and IgY using the appropriate HiTrap columns.
82
Prepare at least 500 ml of each buffer. 1. Follow instructions supplied on the ÄKTAprime cue card to connect the column and load the system with binding buffer. 2. Select the Application Template. 3. Start the method. 4. Enter the sample volume and press OK to start.
Typical procedures using ÄKTAprime Preparing the buffers
Connecting the column
Preparing the fraction collector
Loading the sample
83
Appendix 4 Column packing and preparation A Column Packing Video is available to demonstrate how to produce a well-packed column (see ordering information).
1. Equilibrate all materials to the temperature at which the purification will be performed. 2. Eliminate air by flushing column end pieces with the recommended buffer. Ensure no air is trapped under the column net. Close column outlet leaving 1-2 cm of buffer in the column. 3. Gently resuspend the purification medium. 4. Estimate the amount of slurry (resuspended medium) required. 5. Pour the required volume of slurry into the column. Pouring down a glass rod held against the wall of the column will minimize the introduction of air bubbles. 6. Immediately fill the column with buffer. 7. Mount the column top piece and connect to a pump. 8. Open the column outlet and set the pump to the desired flow rate, for example, 15 ml/min in an XK 16/20 column.
If the recommended flow rate cannot be obtained, use the maximum flow rate the pump can deliver. Do not exceed the maximum operating pressure of the medium or column. 9. Maintain the packing flow rate for at least 3 bed volumes after a constant bed height is obtained. Mark the bed height on the column.
Do not exceed 75% of the packing flow rate during any purification. 10. Stop the pump and close the column outlet. Remove the top piece and carefully fill the rest of the column with buffer to form an upward meniscus at the top. 11. Insert the adapter into the column at an angle, ensuring that no air is trapped under the net. 12. Slide the adapter slowly down the column (the outlet of the adapter should be open) until the mark is reached. Lock the adapter in position. 13. Connect the column to the pump and begin equilibration. Re-position the adapter if necessary.
The medium must be thoroughly washed to remove the 20% ethanol storage solution. Residual ethanol may interfere with subsequent procedures. Most media when equilibrated with sterile PBS containing an anti-microbial agent may be stored at +4°C for up to 1 month, but always check the specific storage instructions supplied with the product.
84
Column selection XK columns are fully compatible with the high flow rates achievable with modern media and a broad range of column dimensions are available. For a complete listing refer to the Amersham Biosciences BioDirectory or web catalogue. Columns XK 16/20 column
Volume (ml)
Code no
2–34
18-8773-01
XK 26/20 column
0–80
18-1000-72
XK 16/70 column
102–135
18-8775-01
XK 26/70 column
281–356
18-8769-01
HR columns can be used for small scale chromatography applications. Columns
Volume (ml)
Code no
HR 5/2 column
0.2–0.59
18-0382-01
HR 5/5 column
0.8–1.2
18-0383-01
HR 10/2 column
0.08–2.43
18-1000-97
HR 10/10 column
6.4–8.7
19-7402-01
85
Appendix 5 Use of sodium hydroxide for cleaning chromatographic media and systems Sodium hydroxide is widely accepted for cleaning, sanitization and storage of chromatography media and systems. A detailed review of its use is given in Application Note 18-1124-57 available from Amersham Biosciences. The most suitable cleaning procedure for any affinity medium will depend on the nature of the contaminants to be removed and the stability of the affinity ligand. Many media are supplied in suspension in 20% ethanol. Use 20% ethanol as an alternative to sodium hydroxide for storage. Include 0.2 M sodium acetate to those products containing an SP ligand.
Description
Working pH
pH stability Short term cleaning
Gel filtration Sephadex G-25 Superdex prep grade
2–13 3–12
2–13 1–14
2–13 3–12
0.01 M NaOH 0.01 M NaOH
Ion exchange media DEAE Sepharose Fast Flow CM Sepharose Fast Flow SP Sepharose Fast Flow Q Sepharose Fast Flow SP Sepharose High Performance Q Sepharose High Performance SOURCE 15/30S SOURCE 15/30Q STREAMLINE SP XL
2–9 6–10(a) 4–13 2–12 4–13 2–12 2–13 2–12 4–13
1–14 2–14 3–14 1–14 3–14 1–14 1–14 1–14 3–14
2–13 4–13 4–13 2–12 4–13 2–12 2–13 2–12 4–13
0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01
M M M M M M M M M
NaOH NaOH NaOH NaOH NaOH NaOH NaOH NaOH NaOH
Hydrophobic interaction media Phenyl Sepharose Fast Flow (h/l sub) Phenyl Sepharose High Performance Butyl Sepharose Fast Flow Octyl Sepharose Fast Flow
3–13 3–13 3–13 3–13
2–14 2–14 2–14 2–14
3–13 3–13 3–13 3–13
0.01 0.01 0.01 0.01
M M M M
NaOH NaOH NaOH NaOH
Affinity Media Blue Sepharose 6 Fast Flow Chelating Sepharose Fast Flow Protein A Sepharose 4 Fast Flow rProtein A Sepharose Fast Flow STREAMLINE rProtein A Protein G Sepharose 4 Fast Flow
4–12 (b) 4–8.5 (c) 2*–9 2*–9(c) (c) 2*–9 (c) 2*–9
3–13 2–14 2*–10 2*–11 2*–11 2*–10
4–12 3–13 3–9 3–10 3–10 3–9
0.01 M NaOH 0.01 M NaOH 20% ethanol, pH 20% ethanol, pH 20% ethanol, pH 20% ethanol, pH
(a)
pH stability Long-term operational
Storage
6 6 6 6
* pH below 3 is sometimes required to elute strongly bound immunoglobulins. However, protein ligands may hydrolyze at very low pH.
Table 22. pH ranges for operation, cleaning and storage of media. Data underlined are estimates to the best of our knowledge and experience; complete studies on stability as a function of pH have not yet been performed. Working pH:
86
pH interval where the medium binds protein as intended or is needed for elution, without adverse long term effect. This differs from operational due to a) uncharged weak ion exchanger, b) broken complex with metal ions and c) no useful increase in binding.
Short-term pH: pH interval to which the medium can be subjected, for cleaning- or sanitization-in-place (accumulated 90–400 hours at room temperature) without significant change in function. Long-term:
pH interval where the medium can be operated without significant change in function.
Storage:
Recommended storage conditions to prevent microbial growth. The medium can be stored up to one year without significant change in function.
The data presented here give an overview over recommended ranges. For detailed information on chromatographic stability and leakage on Amersham Biosciences media, please contact our specialists in process chromatography or regulatory affairs. ‘Without significant change’ means that the media will pass our QC test again.
87
Appendix 6 Storage of biological samples The advice given here is of a general nature and cannot be applied to every biological sample. Always consider the properties of the specific sample and its intended use before following any of these recommendations.
General recommendations Serum, culture supernatants and ascitic fluid should be kept frozen at -20°C or -70°C, in small aliquots. Avoid repeated freeze/thawing or freeze drying/re-dissolving that may reduce biological activity. Avoid conditions close to stability limits (e.g. pH or salt concentrations, reducing or chelating agents). Keep refrigerated at +4°C in a closed vessel to minimize bacterial growth and protease activity. Above 24 hours at +4°C, add a preserving agent if possible (e.g. merthiolate 0.01%). Sodium azide can interfere with many coupling methods and some biological assays and can be a health hazard. It can be removed by using a desalting column (see page 20). Add stabilizing agents, if essential. Stabilizing agents are often required for storage of purified proteins.
General recommendations for purified proteins Store in high concentration of ammonium sulphate (e.g. 4.0 M). Freeze in 50% glycerol, especially suitable for enzymes. Avoid the use of preserving agents if the product is to be used for a biological assay. Preserving agents should not be added if in vivo experiments are to be performed. Instead store samples in small aliquots and keep frozen. Sodium azide can interfere with many coupling methods and some biological assays. It can be removed by using a desalting column (see page 20). Add stabilizing agents, e.g. glycerol (5-20%), serum albumin (10 mg/ml), ligand (concentration is selected based on concentration of active protein) to help to maintain biological activity, but remember that this is reducing the purity of the sample. Sterile filter. Avoid repeated freeze/thawing or freeze drying/re-dissolving that may reduce biological activity. Cryoproteins are a group of antibodies, including some mouse antibodies of the IgG3 subclass, that should not be stored at +4°C as they precipitate at this temperature. Keep at room temperature in the presence of a preserving agent.
88
89
Appendix 7 Table of amino acids Three-letter code
Single-letter code
Alanine
Ala
A
Arginine
Arg
R
Amino acid
Structure HOOC CH3 H2 N NH2
HOOC CH2CH2CH2NHC H2 N
NH
HOOC
Asparagine
Asn
N
Aspartic Acid
Asp
D
CH2CONH2 H2 N HOOC CH2COOH H2 N HOOC
Cysteine
Cys
CH2SH
C H2 N HOOC
Glutamic Acid
Glu
CH2CH2COOH
E H2 N HOOC
Glutamine
Gln
Q
Glycine
Gly
G
Histidine
His
H
Isoleucine
Ile
I
CH2CH2CONH2 H2 N HOOC H H2 N HOOC
N CH2
NH
H2 N HOOC
CH(CH3)CH2CH3 H2 N HOOC
Leucine
Leu
L
CH3 CH2CH CH3
H2 N HOOC
Lysine
Lys
K
Methionine
Met
M
CH2CH2CH2CH2NH2 H2 N HOOC CH2CH2SCH3 H2 N HOOC
Phenylalanine
Phe
F
Proline
Pro
P
CH2 H2 N HOOC H2 N
NH
HOOC
Serine
Ser
S
Threonine
Thr
T
CH2OH H2 N HOOC CHCH3 H2 N
OH
HOOC
Tryptophan
Trp
W
CH2 H2 N
NH
HOOC
Tyrosine
Tyr
CH2
Y H2 N HOOC
Valine
Val
CH(CH3)2
V H2 N
90
OH
Formula
Mr
Middle unit residue (-H20) Formula Mr
C3H7NO2
89.1
C3H5NO
C6H14N4O2
174.2
C4H8N 2O3
Charge at pH 6.0-7.0
Hydrophobic (non-polar)
Uncharged (polar)
71.1
Neutral
C6H12N4O
156.2
Basic (+ve)
132.1
C4H6N 2O2
114.1
Neutral
C4H7NO4
133.1
C4H5NO3
115.1
Acidic(-ve)
C3H7NO2S
121.2
C3H5NOS
103.2
Neutral
C5H9NO4
147.1
C5H7NO3
129.1
Acidic (-ve)
C5H10N2O3
146.1
C5H8N 2O2
128.1
Neutral
C2H5NO
75.1
C2H3NO
57.1
Neutral
C6H9N 3O2
155.2
C6H7N3O
137.2
Basic (+ve)
C6H13NO2
131.2
C6H11NO
113.2
Neutral
C6H13NO2
131.2
C6H11NO
113.2
Neutral
C6H14N2O2
146.2
C6H12N2O
128.2
Basic(+ve)
C5H11NO2S
149.2
C5H9NOS
131.2
Neutral
C9H11NO2
165.2
C9H9NO
147.2
Neutral
C5H9NO2
115.1
C5H7NO
97.1
Neutral
C3H7NO3
105.1
C3H5NO2
87.1
Neutral
C4H9NO3
119.1
C4H7NO2
101.1
Neutral
C11H12N2O2
204.2
C11H10N2O
186.2
Neutral
C9H11NO3
181.2
C9H9NO2
163.2
Neutral
C5H11NO2
117.1
C5H9NO
99.1
Neutral
Hydrophilic (polar)
91
Appendix 8 Converting flow rates from linear flow rates (cm/h) to volumetric flow rates (ml/min) and vice versa It is often convenient when comparing results for columns of different sizes to express flow rates in linear flow rate (cm/h). However, flow rates are usually measured in volumetric flow rate (ml/min). To convert between linear and volumetric flow rate use one of the formulae below.
From linear flow rate (cm/h) to volumetric flow rate (ml/min)
Volumetric flow rate (ml/min) = =
Linear flow rate (cm/h) x column cross sectional area (cm2) 60 Y p x d2 x 60 4
where Y = linear flow rate in cm/h d = column inner diameter in cm
Example: What is the volumetric flow rate in an XK 16/70 column (i.d. 1.6 cm) when the linear flow rate is 150 cm/h? Y = linear flow rate = 150 cm/h d = inner diameter of the column = 1.6 cm Volumetric flow rate =
150 x p x 1.6 x 1.6 ml/min 60 x 4
= 5.03 ml/min
From volumetric flow rate (ml/min) to linear flow rate (cm/h) Linear flow rate (cm/h) =
Volumetric flow rate (ml/min) x 60 column cross sectional area (cm2)
= Z x 60 x
4 p x d2
where Z = volumetric flow rate in ml/min d = column inner diameter in cm
Example: What is the linear flow rate in an HR 5/5 column (i.d. 0.5 cm) when the volumetric flow rate is 1 ml/min? Z = Volumetric flow rate = 1 ml/min d = column inner diameter = 0.5 cm Linear flow rate = 1 x 60 x = 305.6 cm/h
92
4 p x 1.6 x 1.6
cm/h
Appendix 9 Protein conversion data Mass (g/mol)
1 µg
1 nmol
Protein
A280 for 1 mg/ml
10 000
100 pmol; 6 x 10
13
molecules
10 µg
IgG
1.35
50 000
20 pmol; 1.2 x 10
13
molecules
50 µg
IgM
1.20
100 000
10 pmol; 6.0 x 1012 molecules
100 µg
IgA
1.30
150 000
6.7 pmol; 4.0 x 10
150 µg
Protein A
0.17
Avidin
1.50
1 kb of DNA
12
molecules
Streptavidin
3.40
Bovine Serum Albumin
0.70
= 333 amino acids of coding capacity = 37 000 g/mol
270 bp DNA
= 10 000 g/mol
1.35 kb DNA
= 50 000 g/mol
2.70 kb DNA
= 100 000 g/mol
Average molecular weight of an amino acid = 120 g/mol.
93
Appendix 10 Principles and standard conditions for purification techniques Affinity Chromatography (AC) AC separates proteins on the basis of a reversible interaction between a protein (or group of proteins) and a specific ligand coupled to a chromatographic matrix. The technique is ideal for a capture or intermediate step and can be used whenever a suitable ligand is available for the protein(s) of interest. AC offers high selectivity, hence high resolution, and usually high capacity for the protein(s) of interest. The target protein(s) is specifically and reversibly bound by a complementary binding substance (ligand). The sample is applied under conditions that favour specific binding to the ligand. Unbound material is washed away, and the bound target protein is recovered by changing conditions to those favouring desorption. Desorption is performed specifically using a competitive ligand, or non-specifically, by changing the pH, ionic strength or polarity. Samples are concentrated during binding and protein is collected in a purified, concentrated form. The key stages in a purification are shown in Figure 38. Affinity chromatography is also used to remove specific contaminants, for example Benzamidine Sepharose 6B can remove serine proteases.
Absorbance
equilibration
adsorption of sample and elution of unbound material
wash away unbound material
begin sample application
1-2 cv
elute bound protein(s)
re-equilibration
change to elution buffer
x cv
1-2 cv
>1 cv
1-2 cv
Column Volumes (cv)
Fig. 38. Typical affinity purification.
General troubleshooting Column has clogged Cell debris in the sample may clog the column. Clean the column and ensure that samples have been filtered or centrifuged.
No binding to the purification column Decrease the flow rate to improve binding. Check pH and buffer composition.
94
If re-using a prepacked column, check that the column has been regenerated correctly. Replace with fresh medium or a new column if binding capacity does not return after regeneration. Column capacity may have been exceeded. If using HiTrap columns (1 ml or 5 ml), link 2 or 3 columns in series to increase capacity or pack a larger column.
Poor elution from the column Decrease the flow rate to improve elution. Check pH and buffer composition.
Change to a different eluent. Further information Protein Purification Handbook Affinity Chromatography Handbook: Principles and Methods
Ion exchange (IEX) IEX separates proteins on the basis of differences in charge to give a very high resolution separation with high sample loading capacity. The separation is based on the reversible interaction between a charged protein and an oppositely charged chromatographic medium. Proteins bind when they are loaded onto a column. Conditions are then altered so that bound substances are eluted differentially. Elution is usually performed by increasing salt concentration or by changing pH. Changes are made stepwise or with a continuous gradient. Most commonly, samples are eluted with salt (NaCl), using gradient elution (Figure 39). Target proteins are concentrated during binding and collected in a purified, concentrated form. equilibration
sample application
gradient elution
wash
re-equilibration
high salt wash 1M
1-4 cv
[NaCl]
unbound molecules elute before gradient begins
tightly bound molecules elute in high salt wash
10-20 cv 2 cv
2 cv
0 Column volumes [cv]
Fig. 39. Typical IEX gradient elution.
The net surface charge of proteins varies according to the surrounding pH. When above its isoelectric point (pI) a protein will bind to an anion exchanger; when below its pI a protein will behind to a cation exchanger. Typically IEX is used to bind the target molecule, but it can also be used to bind impurities if required. IEX can be repeated at different pH values to separate several proteins that have distinctly different charge properties, as shown in Figure 40.
95
Selectivity pH of mobile phase Abs
Abs
Abs
Abs
V
V
V
V
Surface net charge
+
Cation
pH
0
Anion -
Abs
Abs
Abs
Abs
V
V
V
V
Fig. 40. Effect of pH on protein elution patterns.
Method development (in priority order) 1. Select the optimal ion exchanger using small columns as in the HiTrap IEX Selection Kit to save time and sample. 2 Scout for the optimal pH. Begin 0.5-1 pH unit away from the isoelectric point of the target protein if known. 3. Select the steepest gradient to give acceptable resolution at the selected pH. 4. Select the highest flow rate that maintains resolution and minimizes separation time. Check recommended flow rates for the specific medium.
To reduce separation times and buffer consumption, design and optimize a method for step elution as shown in Figure 41. It is often possible to increase sample loading when using step elution.
high salt wash 1M
2 cv elution of target molecule
[NaCl]
unbound molecules elute sample injection volume
elution of unwanted material
1-2 cv tightly bound molecules elute
1-2 cv equilibration
re-equilibration
2 cv 2 cv
0 Column volumes [cv]
Fig. 41. Step elution.
Further information Protein Purification Handbook Ion Exchange Chromatography Handbook: Principles and Methods
96
Hydrophobic interaction chromatography (HIC) HIC separates proteins on the basis of differences in hydrophobicity. The technique is ideal for the capture or intermediate steps in a purification. Separation is based on the reversible interaction between a protein and the hydrophobic surface of a chromatographic medium. This interaction is enhanced by high ionic strength buffer which makes HIC an ideal 'next step' after precipitation with ammonium sulphate or elution in high salt during IEX. Samples in high ionic strength solution (e.g. 1.5 M ammonium sulphate) bind as they are loaded onto a column. Conditions are then altered so that the bound substances are eluted differentially. Elution is usually performed by decreasing in salt concentration (Figure 42). Changes are made stepwise or with a continuous decreasing salt gradient. Most commonly, samples are eluted with a decreasing gradient of ammonium sulphate. Target proteins are concentrated during binding and collected in a purified, concentrated form. Other elution procedures include reducing eluent polarity (ethylene glycol gradient up to 50%), adding chaotropic species (urea, guanidine hydrochloride) or detergents, changing pH or temperature.
equilibration
sample application
gradient elution
salt free wash
re-equilibration
[ammonium sulphate]
1M
tightly bound molecules elute in salt free conditions
unbound molecules elute before gradient begins 10-15 cv
2 cv
2 cv 0 Column volumes [cv]
Fig. 42. Typical HIC gradient elution.
Method development (in priority order) 1. The hydrophobic behaviour of a protein is difficult to predict and binding conditions must be studied carefully. Use HiTrap HIC Selection Kit or RESOURCE HIC Test Kit to select the medium that gives the optimal binding and elution over the required range of salt concentration. For proteins with unknown hydrophobic properties begin with 0-100%B (0%B = 1 M ammonium sulphate). 2. Select the gradient that gives acceptable resolution. 3. Select the highest flow rate that maintains resolution and minimizes separation time. Check recommended flow rates for the specific medium. 4. If samples adsorb strongly to a medium, then conditions that cause conformational changes, such as pH, temperature, chaotropic ions or organic solvents can be altered. Conformational changes caused by these agents are specific to each protein. Use screening procedures to investigate the effects of these agents. Alternatively, change to a less hydrophobic medium.
To reduce separation times and buffer consumption, transfer to a step elution after method optimization, as shown in Figure 43. It is often possible to increase sample loading when using step elution, an additional benefit for larger scale purification.
97
equilibration
[ammonium sulphate]
1M
salt free wash elution of unwanted material
unbound molecules elute
1-2 cv
sample injection volume
elution of target molecule
re-equilibration
2 cv
tightly bound molecules elute
1-2 cv
2 cv
1-2 cv
0
Column volumes [cv]
Fig. 43. Step elution.
Further information Protein Purification Handbook Hydrophobic Interaction Chromatography Handbook: Principles and Methods
Gel filtration (GF) GF separates proteins on the basis of differences in molecular size. The technique is ideal for the final polishing steps in purification when sample volumes have been reduced (sample volume significantly influences speed and resolution in gel filtration). Samples are eluted isocratically (single buffer, no gradient Figure 44). Buffer conditions are varied to suit the sample type or the requirements for further purification, analysis or storage step, since buffer composition does not directly affect resolution. Proteins are collected in a purified form in the chosen buffer.
high molecular weight low molecular weight
UV
sample injection volume
intermediate molecular weight equilibration
1 cv Column Volumes (cv)
Fig. 44. Typical GF elution.
Further information Protein Purification Handbook Gel Filtration Handbook: Principles and Methods
98
Reversed phase chromatography (RPC) RPC separates proteins and peptides with differing hydrophobicity based on their reversible interaction with the hydrophobic surface of a chromatographic medium. Samples bind when they are loaded onto a column. Conditions are then altered so that the bound substances are eluted differentially. Due to the nature of the reversed phase matrices, the binding is usually very strong and requires the use of organic solvents and other additives (ion pairing agents). Elution is usually performed by increases in organic solvent concentration, most commonly acetonitrile. Samples, which are concentrated during the binding and separation process, are collected in a purified, concentrated form. The key stages in a separation are shown in Figure 45.
sample application
column equilibration
gradient elution
clean after gradient
re-equilibration
100%
unbound molecules elute before gradient begins
2-4 cv
5-40 cv
5 cv 0
2 cv Column Volumes [cv]
Fig. 45. Typical RPC gradient elution.
RPC is often used in the final polishing of oligonucleotides and peptides and is ideal for analytical separations, such as peptide mapping. RPC is not recommended for protein purification if recovery of activity and return to a correct tertiary structure are required, since many proteins are denatured in the presence of organic solvents.
Method Development 1. Select the medium from screening results. 2. Select the optimal gradient to give acceptable resolution. For unknown samples begin with 0-100% elution buffer. 3. Select the highest flow rate that maintains resolution and minimizes separation time. 4. For large-scale purification transfer to a step elution. 5. Samples that adsorb strongly to a medium are more easily eluted from a less hydrophobic medium.
Further information Protein Purification Handbook Reversed Phase Chromatography Handbook: Principles and Methods
99
Expanded bed adsorption (EBA) EBA is a single pass operation in which target proteins are purified from crude sample, without the need for separate clarification, concentration and initial purification to remove particulate matter. Crude sample is applied to an expanded bed of STREAMLINE adsorbent particles within a specifically designed STREAMLINE column. Target proteins are captured on the adsorbent. Cell debris, cells, particulate matter, whole cells, and contaminants pass through and target proteins are then eluted. Figure 46 shows a representation of the steps involved in an EBA purification and Figure 47 shows a typical EBA elution.
0.Sedimented adsorbent
1.Equilibration 2.Sample appl. (expanded) (expanded)
3.Washing (expanded)
4.Elution (packed bed)
5.Regeneration (packed bed)
Fig. 46. Steps in an EBA purification process.
equilibration
Begin sample application adorbance
adsorption of sample and elution of unbound material
Begin wash with start buffer
wash away unbound material
elute bound protein(s)
column wash
Change to elution buffer
Sample volumes
Volume
Fig. 47. Typical EBA elution.
Method development 1. Select suitable ligand to bind the target protein. 2. Scout for the optimal binding and elution conditions using clarified material in a packed column (0.02-0.15 litres bed volume of media). Gradient elution may be used during scouting, but the goal is to develop a step elution. 3. Optimize binding, elution, wash and cleaning-in-place procedures using unclarified sample in expanded mode at small-scale (0.02-0.15 litres bed volume of media). 4. Begin scale up process at pilot scale (0.2-0.9 litres bed volume of media). 5. Full scale production (up to several hundred litres bed volume of media).
Further information Protein Purification Handbook Expanded Bed Adsorption Handbook: Principles and Methods
100
Additional reading and reference material Code No.
Purification Protein Purification Handbook
18-1132-29
Recombinant Protein Handbook: Protein Amplification and Simple Purification
18-1142-75
Gel Filtration Handbook: Principles and Methods
18-1022-18
Ion Exchange Chromatography Handbook: Principles and Methods
18-1114-21
Hydrophobic Interaction Chromatography Handbook: Principles and Methods
18-1020-90
Affinity Chromatography Handbook: Principles and Methods
18-1022-29
Reversed Phase Chromatography Handbook: Principles and Methods
18-1112-93
Expanded Bed Adsorption Handbook: Principles and Methods
18-1124-26
Protein and Peptide Purification Technique Selection
18-1128-63
Fast Desalting and Buffer Exchange of Proteins and Peptides
18-1128-62
Gel Filtration Columns and Media Selection Guide
18-1124-19
Ion Exchange Columns and Media Selection Guide
18-1127-31
HIC Columns and Media Product Profile
18-1100-98
Affinity Columns and Media Product Profile
18-1121-86
Convenient Protein Purification, HiTrap Column Guide
18-1128-81
HiTrap Protein A and HiTrap Protein G Data File
18-1134-76
HiTrap IgM Purification Data File
18-1127-43
HiTrap IgY Purification Data File
18-1127-42
HiTrap NHS-activated Data File
18-1134-80
MAbTrap Kit Data File
18-1034-14
ÄKTAdesign Brochure
18-1129-05
Column Packing Video (PAL)
17-0893-01
Column Packing Video (NTSC)
17-0894-01
Rapid optimization and development of an automated two-step purification procedure for monoclonal IgG antibodies
18-1128-93
Rapid development of a purification process for a recombinant antigen binding fragment expressed in E. coli
18-1111-23
Use of sodium hydroxide for cleaning and sanitizing chromatography media and systems
18-1124-57
Analysis Gel Media Guide (electrophoresis)
18-1129-79
2D Electrophoresis Handbook
80-6429-60
Protein Electrophoresis Technical Manual
80-6013-88
ECL Western and ECL Plus Western blotting Application Note
18-1139-13
Many of these items can be downloaded from www.amershambiosciences.com
General reading Chamow, S. and Ashkenazi, A. (eds.) Antibody Fusion Proteins, John Wiley and Sons Inc. Publisher, New York (1999) Harlow, E. and Lane, D. (eds.) Using Antibodies: a laboratory manual, Cold Spring Harbor Laboratory Press, New York (1998) Gagnon, P., Purification Tools for Monoclonal Antibodies, Validated Biosystems, Inc.,Tucson (1996)
101
Ordering information Product Affinity: prepacked columns HiTrap rProtein A FF
HiTrap Protein A HP
HiTrap Protein G HP
MAbTrap Kit
HiTrap IgY Purification HP HiTrap IgM Purification HP HiTrap NHS-activated HP GSTrap FF
HisTrap Kit
HiTrap Chelating HP HiTrap Blue HP
Quantity
Code No.
5 x 1 ml 1 x 5 ml 2 x 1 ml 5 x 1 ml 2 x 1 ml 1 x 5 ml 5 x 1 ml 2 x 1 ml 1 x 5 ml HiTrap Protein G HP (1 x 1 ml), accessories, pre-made buffers for 10 purifications 1 x 5 ml 5 x 1 ml 5 x 1 ml 1 x 5 ml 2 x 1 ml 5 x 1 ml 1 x 5 ml 3 x 1 ml HiTrap Chelating HP columns, pre-made buffers and accessories for up to 12 purifications 5 x 1 ml 1 x 5 ml 5 x 1 ml 1 x 5 ml
17-5079-01 17-5080-01 17-5079-02 17-0402-01 17-0402-03 17-0403-01 17-0404-01 17-0404-03 17-0405-01 17-1128-01
Affinity: loose media (larger quantities available on request) Immunoprecipitation Starter Pack 2 x 2 ml Protein A Sepharose 4 Fast Flow Protein G Sepharose 4 Fast Flow Protein A Sepharose 4 Fast Flow 5 ml 25 ml rProtein A Sepharose 4 Fast Flow 5 ml 25 ml rmp Protein A Sepharose Fast Flow 5ml 25 ml STREAMLINE rProteinA 75 ml Protein A Sepharose 6MB 10 ml Protein G Sepharose 4 Fast Flow 5 ml 25 ml NHS-activated Sepharose Fast Flow 5 ml CNBr-activated Sepharose 4 Fast Flow 10 g Glutathione Sepharose 4 Fast Flow 25 ml 100 ml 500 ml Chelating Sepharose Fast Flow 50 ml IgG Sepharose 6 Fast Flow 10 ml Blue Sepharose 6 Fast Flow 50 ml IEX: Prepacked columns HiTrap IEX Selection Kit HiTrap Q XL 1 ml HiTrap SP XL 1 ml HiTrap ANX FF (high sub) 1 ml HiTrap DEAE FF 1 ml HiTrap CM FF 1 ml HiTrap Q FF 1 ml HiTrap SP FF 1 ml
102
7 x 1 ml
17-5111-01 17-5110-01 17-0716-01 17-0717-01 17-5130-02 17-5130-01 17-5131-01 17-1880-01
17-0408-01 17-0409-01 17-0412-01 17-0413-01 17-6002-35
17-0974-01 17-0974-04 17-1279-01 17-1279-02 17-5138-01 17-5138-02 17-1281-01 17-0469-01 17-0618-01 17-0618-02 17-0717-01 17-0981-01 17-5132-01 17-5132-02 17-5132-03 17-0575-01 17-0969-01 17-0948-01 17-6002-33
Product IEX: Prepacked columns HiTrap Q HP HiTrap SP HP HiTrap Q XL HiTrap SP XL HiTrap ANX FF (high sub) HiTrap DEAE FF HiTrap CM FF HiTrap Q FF HiTrap SP FF HiPrep 16/10 SP XL HiPrep 16/10 Q XL HiPrep 16/10 DEAE FF HiPrep 16/10 CM FF HiLoad 16/10 Q Sepharose Fast Flow HiLoad 26/10 Q Sepharose Fast Flow HiLoad 16/10 SP Sepharose Fast Flow HiLoad 26/10 SP Sepharose Fast Flow IEX: loose media Q Sepharose Fast Flow SP Sepharose Fast Flow DEAE Sepharose Fast Flow CM Sepharose Fast Flow ANX Sepharose 4 Fast Flow (high sub) SP Sepharose High Performance SOURCE 15Q SOURCE 15S STREAMLINE SP XL HIC: prepacked columns HiTrap HIC Selection Kit HiTrap Phenyl HP HiTrap Phenyl FF (low sub) HiTrap Phenyl FF (high sub) HiTrap Butyl FF HiTrap Octyl FF HiTrap Phenyl FF (high sub) HiTrap Phenyl FF (low sub) HiTrap Phenyl HP
Quantity
Code No.
5 x 1 ml 5 x 5 ml 5 x 1 ml 5 x 5 ml 5 x 1 ml 5 x 5 ml 5 x 1 ml 5 x 5 ml 5 x 1 ml 5 x 5 ml 5 x 1 ml 5 x 5 ml 5 x 1 ml 5 x 5 ml 5 x 1 ml 5 x 5 ml 5 x 1 ml 5 x 5 ml 1 x 20 ml 1 x 20 ml 1 x 20 ml 1 x 20 ml 1 x 20 ml 1 x 53 ml 1 x 20 ml 1 x 53 ml
17-1153-01 17-1154-01 17-1151-01 17-1152-01 17-5158-01 17-5159-01 17-5160-01 17-5161-01 17-5162-01 17-5163-01 17-5055-01 17-5154-01 17-5056-01 17-5155-01 17-5053-01 17-5156-01 17-5054-01 17-5157-01 17-5093-01 17-5092-01 17-5090-01 17-5091-01 17-1060-01 17-1062-01 17-1135-01 17-1136-01
25 ml 300 ml 25 ml 300 ml 25 ml 500 ml 25 ml 500 ml 25 ml 500 ml 75 ml 10 ml 50 ml 10 ml 50 ml 100 ml
17-0510-10 17-0510-01 17-0729-10 17-0729-01 17-0709-10 17-0709-01 17-0719-10 17-0719-01 17-1287-10 17-1287-01 17-1087-01 17-0947-20 17-0974-01 17-0944-10 17-0944-01 17-5076-05
5 x 1 ml
17-1349-01
5 x 1 ml 5 x 5 ml 5 x 1 ml 5 x 5 ml 5 x 1 ml 5 x 5 ml
17-1355-01 17-5193-01 17-1353-01 17-5194-01 17-1351-01 17-5195-01
103
Product
Quantity
Code No.
5 x 1 ml 5 x 5 ml 5 x 1 ml 5 x 5 ml 1 x 20 ml 1 x 20 ml 1 x 20 ml 1 x 20 ml 1 x 20 ml 1 x 53 ml
17-1357-01 17-5197-01 17-1359-01 17-5196-01 17-5095-01 17-5094-01 17-5096-01 17-5097-01 17-1085-01 17-1086-01
HIC: prepacked columns HiTrap Butyl FF HiTrap Octyl FF HiPrep 16/10 Phenyl FF (high sub) HiPrep 16/10 Phenyl FF (low sub) HiPrep 16/10 Butyl FF HiPrep 16/10 Octyl FF HiLoad 16/10 Phenyl Sepharose HP HiLoad 26/10 Phenyl Sepharose HP
HIC: loose media Phenyl Sepharose 6 Fast Flow (high sub) 25 ml 200 ml Phenyl Sepharose 6 Fast Flow (low sub) 25 ml 200 ml Butyl Sepharose 4 Fast Flow 25 ml 200 ml Octyl Sepharose 4 Fast Flow 25 ml 200 ml Phenyl Sepharose High Performance 75 ml SOURCE 15ETH 50 ml SOURCE 15ISO 50 ml SOURCE 15PHE 50 ml
17-0973-10 17-0973-05 17-0965-10 17-0965-05 17-0980-10 17-0980-01 17-0946-10 17-0946-02 17-1082-01 17-0146-01 17-0148-01 17-0147-01
Gel Filtration: prepacked columns (desalting and buffer exchange) PD-10 Desalting HiTrap Desalting HiPrep 26/10 Desalting
30 columns 5 x 5 ml 1 x 53 ml
17-0851-01 17-1408-01 17-5097-01
(high resolution) Superdex 200 HR 10/30 HiLoad 16/60 Superdex 200 pg HiLoad 26/60 Superdex 200 pg HiLoad 16/60 Superdex 75 pg HiLoad 26/60 Superdex 75 pg Superdex 200 prep grade Superdex 200 prep grade HiLoad 16/60 Superdex 30 pg HiLoad 26/60 Superdex 30 pg
1 x 24 ml 1 x 120 ml 1 x 320 ml 1 x 120 ml 1 x 320 ml 25 ml 150 ml 1 x 120 ml 1 x 320 ml
17-1088-01 17-1069-01 17-1071-01 17-1068-01 17-1070-01 17-1043-10 17-1043-01 17-1139-01 17-1140-01
25 ml 150 ml 25 ml 150 ml 25 ml 150 ml
17-1043-10 17-1043-01 17-1044-10 17-1044-01 17-0905-10 17-0905-01
10 sheets 10 sheets 2 for 1000 cm
RPN2020F RPN2020D RPN2109
Gel filtration: loose media Superdex 200 prep grade Superdex 75 prep grade Superdex 30 prep grade Western Blotting Hybond-P Hybond-ECL ECL Western Blotting Detection Reagents ECL Plus Western Blotting Detection System
104
for 1000 cm
2
RPN2132
Handbooks from Amersham Pharmacia Biotech
Antibody Purification Handbook 18-1037-46
The Recombinant Protein Handbook Protein Amplification and Simple Purification 18-1142-75
Protein Purification Handbook 18-1132-29
Ion Exchange Chromatography
Reversed Phase Chromatography
Principles and Methods 18-1114-21
Principles and Methods 18-1134-16
Affinity Chromatography
Expanded Bed Adsorption
Principles and Methods 18-1022-29
Principles and Methods 18-1124-26
Hydrophobic Interaction Chromatography
Chromatofocusing
Principles and Methods 18-1020-90
with Polybuffer and PBE 18-1009-07
Gel Filtration
Microcarrier cell culture
Principles and Methods 18-1022-18
Principles and Methods 18-1140-62
Asia Pacific Tel: +852 2811 8693 Fax: +852 2811 5251 Australasia Tel: +61 2 9899 0999 Fax: +61 2 9899 7511 Austria Tel: 01 576 0616 20 Fax: 01 576 0616 27 Belgium Tel: 0800 73 888 Fax: 03 272 1637 Canada Tel: 1 800 463 5800 Fax: 1 800 567 1008 Central, East, South East Europe Tel: +43 1 982 3826 Fax: +43 1 985 8327 Denmark Tel: 45 16 2400 Fax: 45 16 2424 Finland & Baltics Tel: +358 (0)9 512 3940 Fax: +358 (0)9 512 1710 France Tel: 0169 35 67 00 Fax: 0169 41 9677 Germany Tel: 0761 4903 401 Fax: 0761 4903 405 Italy Tel: 02 27322 1 Fax: 02 27302 212 Japan Tel: 81 3 5331 9336 Fax: 81 3 5331 9370 Latin America Tel: +55 11 3667 5700 Fax: +55 11 3667 87 99 Middle East and Africa Tel: +30 (1) 96 00 687 Fax: +30 (1) 96 00 693 Netherlands Tel: 0165 580 410 Fax: 0165 580 401 Norway Tel: 2318 5800 Fax: 2318 6800 Portugal Tel: 21 417 7035 Fax: 21 417 3184 Russian & other C.I.S. & N.I.S. Tel: +7 (095) 232 0250,956 1137 Fax: +7 (095) 230 6377 South East Asia Tel: 60 3 8024 2080 Fax: 60 3 8024 2090 Spain Tel: 93 594 49 50 Fax: 93 594 49 55 Sweden Tel: 018 612 19 00 Fax: 018 612 19 10 Switzerland Tel: 01 802 81 50 Fax: 01 802 81 51 UK Tel: 0800 616 928 Fax: 0800 616 927 USA Tel: +1 800 526 3593 Fax: +1 877 295 8102 Hybond, HiTrap, GSTrap, Sephadex, Superdex, HisTrap, Sepharose, MAbTrap, ÄKTA, FPLC, HiPrep, PhastSystem, PhastGel, SOURCE, RESOURCE, HiLoad, STREAMLINE, ECL, ECL Plus, MicroSpin, Mono Q, Mono S, Monobeads, BioProcess and Drop Design are trademarks of Amersham Biosciences Limited. Amersham and Amersham Biosciences are trademarks of Amersham plc. BIACORE is a trademark of Biacore AB. Multipipette and Eppendorf are trademarks of Eppendorf-Netheler-Hinz GmbH. Tween is a trademark of ICI Americas Inc. Cibacron is a registered trademark of Ciba-Geigy Corp. Coomassie is a trademark of ICI plc. All goods and services are sold subject to the terms and conditions of sale of the company within the Amersham Biosciences group that supplies them. A copy of these terms and conditions is available on request. Amersham Biosciences UK Limited, Amersham Place, Little Chalfont, Buckinghamshire HP7 9NA, England. Amersham Biosciences AB, Björkgatan 30, SE-751 84 Uppsala, Sweden. Amersham Biosciences Corp., 800 Centennial Avenue, PO Box 1327, Piscataway NJ 08855, USA. Amersham Biosciences Europe GmbH, Munzinger Strasse 9, D-79111 Freiburg, Germany. Amersham Biosciences KK, Sanken Bldg. 3-25-1, Hyakunincho Shinjuku-ku, Tokyo 169-0073, Japan. © Amersham Biosciences AB 2002 – All rights reserved.
Antibody Purification –
Antibody Purification Handbook
Handbook
www.amershambiosciences.com
18-1037-46 Edition AB
Ion Exchange Chromatography Principles and Methods
Back to Collection 18-1114-21 Edition AA
Ion Exchange Chromatography Principles and Methods
ISBN 91 970490-3-4
Contents 1.
Introduction ............................................................................................9
2.
Ion exchange chromatography .........................................................10 The theory of ion exchange .................................................................10 The matrix.....................................................................................11 Charged groups .............................................................................13 Resolution in ion exchange chromatography .................................13 Capacity factor ........................................................................15 Efficiency.................................................................................16 Selectivity.................................................................................17 Capacity ........................................................................................18
3.
Product Guide .......................................................................................20 MonoBeads ...................................................................................20 MiniBeads .....................................................................................20 SOURCE .......................................................................................20 Sepharose High Performance ion exchangers .................................21 Sepharose Fast Flow ion exchangers ..............................................21 Sepharose Big Beads ion exchangers...............................................21 STREAMLINE ion exchangers ......................................................21 DEAE Sepharose CL-6B and CM Sepharose CL-6B .......................22 DEAE Sephacel..............................................................................22 Sephadex ion exchangers ...............................................................22 Bulk quantities...............................................................................22 Equipment.....................................................................................22
4.
MonoBeads and MiniBeads ..............................................................23 MonoBeads.....................................................................................23 Properties ......................................................................................24 Chemical stability ....................................................................24 Physical stability ......................................................................25 Flow rate..................................................................................27 Capacity ..................................................................................27 Recovery..................................................................................28 Reproducibility ........................................................................29 Availability ....................................................................................30 MiniBeads .......................................................................................30 Properties ......................................................................................31 Chemical stability ....................................................................31 Physical stability ......................................................................32 Reproducibility ........................................................................33 Availability ....................................................................................33
2
5.
SOURCE ................................................................................................34 Properties ......................................................................................37 Chemical stability ....................................................................37 Flow rate..................................................................................38 Capacity ..................................................................................40 Recovery..................................................................................40 Reproducibility ........................................................................41 Availability ....................................................................................41
6.
Sepharose based ion exchangers .......................................................42 Chemical stability ..........................................................................42 Physical stability ............................................................................43 Sepharose High Performance ion exchangers.........................43 Properties ......................................................................................44 Physical stability ......................................................................44 Capacity ..................................................................................44 Flow rate..................................................................................46 Availability ....................................................................................46 Sepharose Fast Flow ion exchangers ........................................46 Properties ......................................................................................47 Physical stability ......................................................................47 Capacity ..................................................................................47 Flow rate..................................................................................49 Availability ....................................................................................50 Sepharose Big Beads ion exchangers .........................................50 Properties ......................................................................................51 Physical properties ...................................................................51 Capacity ..................................................................................51 Flow rate..................................................................................51 Availability ....................................................................................51 STREAMLINE SP and STREAMLINE DEAE ......................52 Properties ......................................................................................53 Physical stability ......................................................................53 Capacity ..................................................................................53 Availability ....................................................................................54 DEAE Sepharose CL-6B and CM Sepharose CL-6B .............54 Properties ......................................................................................54 Physical stability ......................................................................54 Capacity ..................................................................................55 Flow rate..................................................................................56 Availability ....................................................................................57
7.
DEAE Sephacel .....................................................................................58 Properties ......................................................................................58 Chemical stability ....................................................................58 Physical stability ......................................................................59
3
Capacity ..................................................................................59 Flow rate..................................................................................60 Availability ....................................................................................60
8.
Sephadex ion exchangers ...................................................................61 Properties ......................................................................................61 Chemical stability ....................................................................61 Physical stability ............................................................................62 Swelling ...................................................................................62 Ionic strength dependence ........................................................62 pH dependence ........................................................................62 Capacity ..................................................................................62 Availability ....................................................................................64
9.
Experimental design ............................................................................65 Choice of ion exchanger ..............................................................65 Specific requirements of the application .........................................65 Column separation, batch separation or...................................65 expanded bed adsorption The scale of the separation .......................................................65 The required resolution ............................................................65 The required throughput ..........................................................66 Scaleability...............................................................................66 Reproducibility ........................................................................67 Economy .................................................................................67 The molecular size of the sample components ................................67 Choice of exchanger group ............................................................68 Determination of starting conditions .......................................69 The isoelectric point .................................................................69 Test-tube method for selecting starting pH ...............................69 Electrophoretic titration curves (ETC) .....................................70 Chromatographic titration curves (retention maps) ..................74 Choice between strong and weak ion exchangers ...........................76 Choice of buffer.............................................................................76 Choice of buffer pH and ionic strength.....................................76 Choice of buffer substance .......................................................77 Test-tube method for selecting starting ionic strengths..............79
10. Experimental Technique.....................................................................80 Column chromatography ............................................................80 Choice of column...........................................................................80 Column design .........................................................................80 Column dimensions .................................................................81 Quantity of ion exchanger .............................................................81 Preparation of the ion exchanger ...................................................81 Pre-swollen ion exchangers ......................................................81
4
Pre-packed ion exchange media ...............................................81 Sephadex ion exchangers .........................................................82 Alternative counter-ions ...........................................................82 Decantation of fines .................................................................82 Packing the column........................................................................82 Column Packing Video Film.....................................................82 Checking the packing ...............................................................83 Equilibrating the bed ...............................................................84 Sample preparation........................................................................85 Sample concentration...............................................................85 Sample composition .................................................................85 Sample volume.........................................................................85 Sample viscosity .......................................................................85 Sample preparation ..................................................................86 Sample application ........................................................................87 Sample application with an adaptor .........................................87 Other methods of sample application.......................................89 Sample application onto a drained bed .....................................89 Sample application under the eluent .........................................89 Elution ..........................................................................................90 Change of pH ..........................................................................90 Change of ionic strength ..........................................................91 Gradient direction....................................................................91 Choice of gradient type ............................................................91 Resolution using a continuous gradient ....................................93 Choice of gradient shape ..........................................................94 Sample displacement ................................................................95 Gradient generation.......................................................................96 Gradient formation with two pumps or a single pump .............96 in combination with a switch valve Gradient Mixer ........................................................................96 Batch separation ............................................................................97 Expanded bed adsorption ...........................................................98 Expanded bed technology ..............................................................99 Basic principle of operation............................................................99 STREAMLINE adsorbents ..........................................................100 STREAMLINE columns ..............................................................100 Auxiliary equipment ....................................................................100 Regeneration ................................................................................101 Cleaning, sanitization and sterilization procedures ............101 Cleaning ......................................................................................101 Sanitization .................................................................................101 Sterilization .................................................................................101 Protocols for cleaning-in-place (CIP),...........................................102 sanitization and sterilization
5
SOURCE and Sepharose Based ion exchangers......................102 MonoBeads and MiniBeads columns......................................102 DEAE Sephacel and Sephadex based ion exchangers..............103 Storage of gels and columns......................................................103 Prevention of microbial growth..............................................103 Storage of unused media ........................................................104 Storage of used media ............................................................104 Storage of packed columns.....................................................104 Determination of the available and dynamic capacities .....104 Calculation ............................................................................106
11. Process considerations .....................................................................107 Defining the purpose ..................................................................108 The strategic focus ......................................................................109 Capture .......................................................................................109 Intermediate purification .............................................................111 Polishing......................................................................................111 Selection of chromatography media .......................................112 Base matrix properies and derivitization chemistry.................113 Bead size ................................................................................113 Documentation and technical support....................................113 Regulatory support ................................................................113 Vendor certification ...............................................................114 Delivery capacity ...................................................................114 Method design and optimization ............................................114 Binding conditions.......................................................................114 Elution ........................................................................................115 Sample load .................................................................................116 Flow rate .....................................................................................117 Selecting a column ......................................................................118 Aspects of column design .............................................................119 Flow distribution system ........................................................119 Material resistance and durability ..........................................119 Sanitary design.......................................................................119 Pressure vessel safety..............................................................120 Regulatory support ................................................................120 Ergonomics............................................................................120 Packing large scale columns......................................................120 Column configuration .................................................................120 Packing the column......................................................................121 Scale-up .........................................................................................121
6
12. Applications ........................................................................................124 The design of a biochemical separation ........................................124 Application examples ..................................................................127 Enzymes ................................................................................127 Isoenzymes ............................................................................128 Immunoglobulins...................................................................129 Nucleic acid separation ..........................................................129 Polypeptides and polynucleotides...........................................130 Antisense phosphorothioate oligonucleotides .........................132 Areas of application.....................................................................133 Purification of a recombinant Pseudomonas ...............................136 aeruginosa exotoxin A, PE553D Strategy..................................................................................141
13. Fault-finding chart .............................................................................143 14. Ordering information .......................................................................151 15. References ............................................................................................155
7
BioProcess
?@f?@?@?@?/K ?@e@@?@?@?@?V4 ? @ ? ? @@@@@@6XfO26 S,?W2@@Y@ @@@@@@@U?7UI'X@ I/?@)?V4@ ? ?@f?@?@?@?/K ? ?@e@@?@?@?@?V4 ? ?)X?'@?@?@@?W.? @ ?@)?V'e?@@?.Y? ? ? ? ?@f?@?@?@?/K ?@@?? @@@@@@6XeW2@@@ ?@e@@?@?@?@?V4 S,?W&@@X@ ? ? @@@@@@@U?7>(R@@ @ ? I/?@0Y?@@ ? O2@?? ? @@e@?@@@@@U@?? ? @@@@@@6Xe@?@?W ?@f?@?@?@?/K B@@?? ?@@?W.?@?@@??@? S,?J@@@?7 ?@e@@?@?@?@?V4 ?@@?.Y?@?@@?e? @@e@?@?@??@@?? @@@@@@@U?7@??J@ ? ? I/?@@??@@ @ ? ?@@?? ?@f?@?@?@?/K ? ? ? ? ?@e@@?@?@?@?V4 ?@?@e?@@ @@@@@@6Xe@6T26 ? ?@@??@e?@@?@?? O2@?? S,?J@@@R4 @Khf? @ ?@@?e?@g? @@@@@@@U?7@V'9? @@e@?@@@@@U@?? ? @@@@@@@?e?@e@ I/?@@?V4@ B@@?? ? ?@@?? ? ? @@e@?@?@??@@?? @@@@@@6Xe@@@6T ? ? S,?J@@@>@ ?@@?@??@?@@?e? ? @@@@@@@U?7@(R@> ? O2@?? ?@?@e?@@ I/?@0Y?@0 ? @@e@?@@@@@U@?? @Khf? ?@@?? ? B@@?? @@@@@@@?e@?@?@ ? @@e@?@?@??@@?? ? ?@@?e?@g? ? O2@?? ? ? @@e@?@@@@@U@?? ? B@@?? ?@?@e?@@ ?@@?? @@e@?@?@??@@?? @Khf? ? ? @@@@@@@?e@@?@? ? ? ?@?@e?@@ O2@?? @Khf? @@e@?@@@@@U@?? @@@@@@@??@f@ B@@?? ? @@e@?@?@??@@??
BioProcess Media are designed, manufactured and supported for industrial bioprocessing. This symbol is your guarantee of:
• • • • • •
Assured long term supply of large batches, on time and with the right quality. Full technical and regulatory support to assist in process validation. Scaleable performance from bench top to production hall. Compatible large scale columns and equipment. Well documented cleaning and sanitization methods. Media shown to perform well in real downstream applications – from Capture to Polishing, from synthetic oligonucleotides to recombinant proteins. • High and reliable productivity in the production hall. Media that fulfill the above described criteria are labelled with the BioProcess Media symbol in Chapter 3.
8
1. Introduction Adsorption chromatography depends upon interactions of different types between solute molecules and ligands immobilized on a chromatography matrix. The first type of interaction to be successfully employed for the separation of macromolecules was that between charged solute molecules and oppositely charged moieties covalently linked to a chromatography matrix. The technique of ion exchange chromatography is based on this interaction. Ion exchange is probably the most frequently used chromatographic technique for the separation and purification of proteins, polypeptides, nucleic acids, polynucleotides, and other charged biomolecules (1). The reasons for the success of ion exchange are its widespread applicability, its high resolving power, its high capacity, and the simplicity and controllability of the method. This handbook is designed as an introduction to the principles of ion exchange chromatography and as a practical guide to the use of the media available from Pharmacia Biotech. The handbook is illustrated with examples of different types of biological molecules which have been separated using ion exchange chromatography and different ways the technique can be used. For information on specific separations, the reader is recommended to consult the original literature.
9
2. Ion exchange chromatography The theory of ion exchange Separation in ion exchange chromatography depends upon the reversible adsorption of charged solute molecules to immobilized ion exchange groups of opposite charge. Most ion exchange experiments are performed in five main stages. These steps are illustrated schematically below. ?W&? ?*@? ?W26X? ?W26X? ?W&? ?@@@@? ?N@? ?.MB1? ?.MB1? W&@? ?@ @? ?J5? ?J5? ?W.Y@? ?@@6X? @? W.Y? ?*U? ?7Y?@? ?B1? @? ?W.Y ?N1? ?@@@@? @? @?e@? ?7Y? ?/KC5? @? ?/KC5? ?@@@@??@ ?V40Y??@ @??@hg ?V40Y??@ @? ?@ @? @? @? @? @? ?@ @? @? @? @? @? ?@ @? @? @? @? @? W2@6X?hf?@ ?@ @? @? @? @? @? 7
@@@@@@@@ @@@@@@@@ @@@@@@@@ @@@@@@@@ W2@@6Xe?W2@6X @?hf?@h?W-T.? ?@ @@@@@@@@f?W2@6Xg@? @? ?@ ?W.MeI/X??7
W2@@@6X? ?W&@@@@@)X ?7@@@@@@@1 ?@@@@@@@@@ ?@@@@@@@@@ ?@@@@@@@@@ ?3@@@@@@@5f?W2@6Xhf@?@? ?@ ?V'@@@@@(YfW.M?I/hf@?hf)X V4@@@0Y?f7Hf?)T-T26X?W2@@?@?W26X?)T-X?@)e?@?W26X?)T-X?W2@@?f @??@@@?@(R'UV1?7
Fig. 1. The principle of ion exchange chromatography (salt gradient elution).
The first stage is equilibration in which the ion exchanger is brought to a starting state, in terms of pH and ionic strength, which allows the binding of the desired solute molecules. The exchanger groups are associated at this time with exchangeable counter-ions (usually simple anions or cations, such as chloride or sodium). The second stage is sample application and adsorption, in which solute molecules carrying the appropriate charge displace counter-ions and bind reversibly to the gel. Unbound substances can be washed out from the exchanger bed using starting buffer. In the third stage, substances are removed from the column by changing to elution conditions unfavourable for ionic bonding of the solute molecules. This normally involves increasing the ionic strength of the eluting buffer or changing its pH. In Figure 1 desorption is achieved by the introduction of an increasing salt concentration gradient and solute molecules are released from the column in the order of their strengths of binding, the most weakly bound substances being eluted first.
10
The fourth and fifth stages are the removal from the column of substances not eluted under the previous experimental conditions and re-equilibration at the starting conditions for the next purification. Separation is obtained since different substances have different degrees of interaction with the ion exchanger due to differences in their charges, charge densities and distribution of charge on their surfaces. These interactions can be controlled by varying conditions such as ionic strength and pH. The differences in charge properties of biological compounds are often considerable, and since ion exchange chromatography is capable of separating species with very minor differences in properties, e.g. two proteins differing by only one charged amino acid, it is a very powerful separation technique. In ion exchange chromatography one can choose whether to bind the substances of interest and allow the contaminants to pass through the column, or to bind the contaminants and allow the substance of interest to pass through. Generally, the first method is more useful since it allows a greater degree of fractionation and concentrates the substances of interest. The conditions under which substances are bound (or free) are discussed in detail in the sections dealing with choice of experimental conditions, Chapter 9. In addition to the ion exchange effect, other types of binding may occur. These effects are small and are mainly due to van der Waals forces and non-polar interactions. Ion exchange separations may be carried out in a column, by a batch procedure or by expanded bed adsorption. All three methodologies are performed in the stages of equilibration, sample adsorption etc. described previously.
The matrix An ion exchanger consists of an insoluble matrix to which charged groups have been covalently bound. The charged groups are associated with mobile counterions. These counter-ions can be reversibly exchanged with other ions of the same charge without altering the matrix. It is possible to have both positively and negatively charged exchangers (Fig. 2). Positively charged exchangers have negatively charged counter-ions (anions) available for exchange and are called anion exchangers. Negatively charged exchangers have positively charged counter-ions (cations) and are termed cation exchangers. The matrix may be based on inorganic compounds, synthetic resins or polysaccharides. The characteristics of the matrix determine its chromatographic properties such as efficiency, capacity and recovery as well as its chemical stability, mechanical
11
O2@@@6K? W2(Me?I'6X? 7(Y?fV'1? ?J(Yg?V'L ?7H?hN1 ?@he?@ ?@he?@ ?@e@@@@@@@@@@ ?@he?@ ?3L?hJ5 ?N)Xg?W&H 3)X?fW&5? V4)Ke?O&0Y? I4@@@0M? ?O2@@@6K ?O2@@@@0M?eI4@@@@6K O2@0M? I4@6K? W20M ?I46X? ?O.M ?I/K ?W20Y?h@? V46X ?O2@@@6K O.M?he@? I/K? ?W2(M?eI'6XhfW20Yhf@? ?V46X? ?7(Yf?V'1he?W.M @? ?I/Xhf?W2@@@@6X? J(Y?gV'L?hW.Y?he@@@@@@@@@? V/X?heW&0MeI4)X 7Hh?N1?h7H @? ?N1?h?W.MgI/X? @?he@?g?J5? @? 3LhW&H?g?N)X @?he@?gW.Y? @? V/X?g75he31 @??@@@@@@@@@@?f?W.Y @? ?V/Xg@HheN@ @?he@?f?7H? @?fN1g@?he?@ 3Lh?J5?fJ5 @?f?3L?f@?he?@ N)X?gW&H?e?W.Y @?f?V/Xf@L?@@@@@@@@6X@ ?3)Xf?W&5f?7H?g@? @?gN1f31h?B@5 ?V4)K?eO&0Yf?@h@? @@@@@@@@@?e?@fV'L?g?J(Y ?I4@@@0Mg?@h@? @?g?3L?e?V/KgO.Y? J5h@? @?g?N1?fV'6KeO2(Y 7Hf@@@@@@@@@? @?h@?f?V4@@@@0Y? @?h@? ?O2@@@6Kg@?h@? @?h@? ?W2(M?eI'6X @? @?h@? ?7(Yf?V'1 @? @?h@?g?@heJ(Y?gV'L?hf3L ?J5? ?@he7Hh?N1?hfN1 ?7H? ?@he@?he@?hf?@ ?@ ?@he@?he@?hf?@ ?@ ?@@@@@@@@@g@?@@@@@@@@@?@?hf?@ ?@ ?@he@?he@?hf?@ ?3L? ?@he3Lh?J5?hfJ5 ?N1? ?@heN)X?gW&H?hf7H @? ?@he?3)Xf?W&5 @? @? ?V4)K?eO&0Y @? @? ?I4@@@0M @? @? @? 3L ?J5? N1 ?7H? ?@ ?@ ?3L?he@? @?heJ5 ?V/Xhe@? @?h?W.Y N1he@? @?h?7H? ?3L?h@? @?hJ5 ?V/Xf@@@@@@@@@? @@@@@@@@@?e?W.Y V/X?g@? @?gW.Y? ?N1?g@? @?g7H 3Lg@? @?f?J5? V/X?f@? @?fW.Y? ?V/K ?O.Y V46X ?W20Y? I/K? O.M? ?V46X? W20Y ?I/K ?O.M V46K ?O20Y?gW2@@@@6X ?W2@@@@6X?hI4@6K? O2@0M?g?W&0M??I4)X? W&0MeI4)Xhe?I4@@@@6K?eO2@@@@0MheW.M?f?I/X ?W.MgI/X? ?I4@@@0M ?W&HhN)X? W&H?g?N)X ?75?h?31? 75he31 ?@H?h?N@? @HheN@ ?@hf@? @?he?@ ?@?@@@@@@@@@e@? @??@@@@@@@@@?@ ?@L?h?J@? @LheJ@ ?31?h?75? 31he75 ?V'LhJ(Y? V'L?g?J(Y V/K?f?O.Y ?V/KgO.Y? ?V'6K??O2(Y? V'6KeO2(Y V4@@@@0Y ?V4@@@@0Y?
@?e?@@?e@?@??W2@@6X?@@e?@ ?@ @??@?@ ?J@Le?@@Le@?@??7
O2@@@6K? W2(Me?I'@ 7(Y?fV'@? ?J(Yf@?e 3L ?7H?f@?e N1 ?@g@?e?@ ?@g@?e J@ ?@e@@@@@@@@ @@ ?@g@?e ?3L?f@?eN@ J5 ?N)Xf@?e 7H 3)X?e@?W&@? V4)Ke?O&@ I4@@@0M? ?O2@@@6X ?O2@@@@0M?eI/@@@@6K O2@0M?he I4@6K? W20Mhg ?I46K? ?O.M ?I46X? ?W20Y? ?I/K ?O2@@@6K O.M? V46K ?W2(M?eI'6XhfW20Y I46X ?7(Yf?V'1he?W.Mh?@@@@@@@@@f I/X?he?W2@@@@6X? J(Y?e?@eV'L?h?7H? ?N1?heW&0MeI4)X 7Hf?@e?N1?hJ5 3Lh?W.MgI/X? @?f?@f@?g?W.Y V/X?gW&H?e?@e?N)X @?f?@f@?g?7H? ?N1?g75f?@f31 @??@@@@@@@@@@?gJ5 3Lg@Hf?@fN@ @?f?@f@?f?W.Y V/X?f@?f?@f?@ 3Lf?@e?J5?f?7H? ?N1?f@??@@@@@@@@@?@ N)X?e?@eW&H?fJ5 3Lf@Lf?@fJ@ ?3)Xe?@?W&5g7H ?@@@@@@@@@fN1f31f?@f75 ?V4)K?eO&0Yg@? ?@fV'L?e?@e?J(Y ?I4@@@0Mg?J5? ?3L?e?V/
[email protected]? ?7H?e?@@@@@@@@@ ?N1?fV'6KeO2(Y ?@ @?f?V4@@@@0Y? ?@ W2@@@6K? @? J5 W&.Me?I'6X? @? 7H 75 ?@eV'1? @? @? ?J(Y ?@e?V'L 3L @? ?7H? ?@f?@ ?@fN1 N1 ?J5? ?@e ?@ ?7H?hf@@@@@@@@@?f?@?@ @@@@@@@@?@ ?@ ?@ ?@e ?@f?@ ?@ ?3L? ?3L? ?@fJ5 ?@ ?N1? ?N)X ?@e?W&H J5 @? 31 ?@eW&5? 7H @? V' /Ke?O&0Y? @? 3L V4@@@0M? @? N1 @? ?@ @? ?@ ?J5? ?3L? ?7H? ?N1? ?@ 3L J5 N1 7H ?3L? ?J5? ?V/Xf?@@@@@@@@@hf W.Y? N1 @@@@@@@@@?f7H ?3L? ?J5? ?V/X W.Y? N1 7H ?3L? ?J5? ?V/K O.Y? V46X O20Y I/K? W20M ?V46X? ?O.M ?I/K ?O20Y? V46Khg ?O20M?gW2@@@@6X ?W2@@@@6X?hI4@6K?he@@@@0MheW.M?f?I/X O2@0M?g?W&0M??I4)X? W&0MeI4)Xhe?I4@@@@6K?eO. ?W.MgI/X? ?I4@@@0Y ?W&Hf@?eN)X? W&H?e?@e?N)X ?75?f@?e?31? 75f?@f31 ?@H?f@?e?N@? @Hf?@fN@ ?@g@?f@? @?f?@f?@ ?@e@@@@@@@@@?@? @??@@@@@@@@@?@ ?@L?f@?e?J@? @Lf?@fJ@ ?31?f@?e?75? 31f?@f75 ?V'Lf@?eJ(Y? V'L?e?@e?J(Y V/K?e@??O.Y ?V/
[email protected]? ?V'6K??O2(Y? V'6KeO2(Y V4@@@@0Y ?V4@@@@0Y?
W2@@6Xe?@e@@@@@??@eW2@@6Xe@@e?@ @? ?@e?@@?hg 7<eI/eJ@L?e@?e?@e7<eB1e@@L??@ @? J@@?hg @?g7R1?e@?e?@e@?e?@e@V1??@e?W26X?/X?W-T26X?@L @?f?J5?3Le@?e?@e@?e?@e@?3L?@e?7YV1?V/T.R@
Fig. 2. Ion exchanger types.
strength and flow properties. The nature of the matrix will also affect its behaviour towards biological substances and the maintenance of biological activity. The first ion exchangers were synthetic resins designed for applications such as demineralisation, water treatment, and recovery of ions from wastes. Such ion exchangers consist of hydrophobic polymer matrices highly substituted with ionic groups, and have very high capacities for small ions. Due to their low permeability these matrices have low capacities for proteins and other macromolecules. In addition, the extremely high charge density gives very strong binding and the hydrophobic matrix tends to denature labile biological materials. Thus despite their excellent flow properties and capacities for small ions, these types of ion exchanger are unsuitable for use with biological samples. The first ion exchangers designed for use with biological substances were the cellulose ion exchangers developed by Peterson and Sober (2). Because of the hydrophilic nature of cellulose, these exchangers had little tendency to denature proteins. Unfortunately, many cellulose ion exchangers had low capacities (otherwise the cellulose became soluble in water) and had poor flow properties due to their irregular shape. Ion exchangers based on dextran (Sephadex), followed by those based on agarose (Sepharose CL-6B) and cross-linked cellulose (DEAE Sephacel) were the first ion exchange matrices to combine a spherical form with high porosity, leading to improved flow properties and high capacities for macromolecules. Subsequently, developments in gel technology have enabled this macroporosity to be extended to the highly cross-linked agarose based media such as Sepharose High Performance, Sepharose Fast Flow and Sepharose Big Beads, and the synthetic polymer matrices, MonoBeads, and SOURCE. These modern media enable fast, high capacity, high resolution ion exchange chromatography to be carried out at both analytical and preparative scales. Non-porous polymer matrices, e.g. MiniBeads, are available for extremely high resolution micropreparative or analytical separations.
12
Charged groups The presence of charged groups is a fundamental property of an ion exchanger. The type of group determines the type and strength of the ion exchanger; their total number and availability determines the capacity. There is a variety of groups which have been chosen for use in ion exchangers (3); some of these are shown in Table 1. Table 1. Functional groups used on ion exchangers. Anion exchangers
Functional group
Diethylaminoethyl (DEAE)
-O-CH2-CH2-N+H(CH2CH3)2
Quaternary aminoethyl (QAE)
-O-CH2-CH2-N+(C2H5)2-CH2-CHOH-CH3
Quaternary ammonium (Q)
-O-CH2-CHOH-CH2-O-CH2-CHOH-CH2-N+(CH3)3
Cation exchangers
Functional group
Carboxymethyl (CM)
-O-CH2-COO-
Sulphopropyl (SP)
-O-CH2-CHOH-CH2-O-CH2-CH2-CH2SO3-
Methyl sulphonate (S)
-O-CH2-CHOH-CH2-O-CH2-CHOH-CH2SO3-
Sulphonic and quaternary amino groups are used to form strong ion exchangers; the other groups form weak ion exchangers. The terms strong and weak refer to the extent of variation of ionization with pH and not the strength of binding. Strong ion exchangers are completely ionized over a wide pH range (see titration curves on page 49) whereas with weak ion exchangers, the degree of dissociation and thus exchange capacity varies much more markedly with pH. Some properties of strong ion exchangers are: • Sample loading capacity does not decrease at high or low pH values due to loss of charge from the ion exchanger. • A very simple mechanism of interaction exists between the ion exchanger and the solute. • Ion exchange experiments are more controllable since the charge characteristics of the media do not change with changes in pH. This makes strong exchangers ideal for working with data derived from electrophoretic titration curves. (see Chapter 9)
Resolution in ion exchange chromatography This section discusses the main theoretical parameters which affect the separation in ion exchange chromatography. For more in-depth information the reader is referred to standard works on the subject (4, 5). The result of an ion exchange experiment, as with any other chromatographic separation, is often expressed as the resolution between the peaks of interest.
13
The resolution (Rs) is determined from the chromatogram as shown in Figure 3. ? ? @?e@? ?@ ? @?e@? ?@K? ? ?J@Le@@6X?W2@@?W26X?)T2@@6X?@@@?W26X?)T-X ? ?7R1e@?B1?*U?e7
Fig. 3. Determination of the resolution (Rs) between two peaks.
The resolution is defined as the distance between peak maxima compared with the average base width of the two peaks. Elution volumes and peak widths should be measured with the same units to give a dimensionless value to the resolution. Rs is a measure of the relative separation between two peaks and can be used to determine if further optimization of the chromatographic procedure is necessary. If Rs = 1.0 (Fig. 4) then 98% purity has been achieved at 98% of peak recovery, provided the peaks are Gaussian and approximately equal in size. Baseline resolution requires that Rs ³1.5. At this value purity of the peak is 100%. Note: A completely resolved peak is not equivalent to a pure substance. This peak may represent a series of components which are not resolvable using the selected separation parameter. The resolution achievable in a system is proportional to the product of the selectivity, the efficiency and the capacity of the system, the three most important parameters to control in column chromatography. The analytical expression for Rs is:
Rs = 1/4
(a - 1) a
k (1 + k)
( N)
? @? @ @? @ @? @ @? @ 3L ?J5 V/K? O.Y ?V46K? O20Y? ?I4@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@0Me? I'T(M? ? ?N@H ? @? ? ?
@? @? @? @? 3= @? V4@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@? I@M?
selectivity
capacity @? @? @? @? 3= @? V4@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@? I@M?
efficiency
14
W-T&K? ?W&?@? 7R@@@6X?W.eW26T2@?@@6T&@?@W26X?@@@? ?J@?@@?B1?*Ue7
)Ke@?O2@H ?N1? ?@ ?J5? V'?@6X@@@>5? @? ?@ ?7H? ?N@)Xe?7@@@1 @?hfV4@@@)Xe@@@Uhe?@?@@??@@?@?@??@?@?@?@@@@?he W@@=C5e?J@@?,e?@e?@ @? W@@=C5e?J@@?,e@?V1he?3T@@=C@@?@?@??@?@?@?3X?hf ?@0MI40Ye?.MI+Ye?@e?@ @?hf?@0MI40Ye?.MI+Ye@@@@he?V+MI40R'?@@@??@?@?@?V4@hf @? ?@K?f@? ?@@6K?e@??O2@ ?@@@@6X?@?@@@@ @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@)?@?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ I4@0M?e@?eI@M? W26Xhe@@6X @? ?W26X?he?W-X .MB)T-X?g@?B1 @? ?.MB)T-Xh?7R1 ?@@?)T-Xf@?C5 @? @@?)T-X?fJ@?@L? W.eI+R@>)X?e@@@U @? ?W.??I+R@>)Xf7@@@1? 7YfJ@@?,?e@?V1 @? ?7Y?e?J@@?,f@?e@? @@@@e.MI+Y?e@@@@ @? ?@@@@??.MI+Yf@?e@? @? @? @? @? @? @?
W-T&K? ?W&?@? 7R@@@6X?W.eW26T2@?@@6T&@?@W26X?@@@? ?J@?@@?B1?*Ue7)X?e7@@@1? @@6T.??@?@e@?@??@V+R@>)X??@@@U? @?40Y??@?3=C5?3=C5eJ@@?,?e@?e@? @?40Y??@?3=C5?3=C5eJ@@?,??@?V1? ?@?V40Y?V40Ye.MI+Y?e@?e@? ?@?V40Y?V40Ye.MI+Y??@@@@?
Fig. 4. Separation results with different resolutions.
Capacity factor The capacity or retention factor k is a measure of the retention of a component and should not be confused with loading capacity (mg sample/ml) or ionic capacity (mmol/ml). The capacity factor is calculated for each individual peak. For example k for peak 1 in Figure 5 is derived from the equation: VR1 - Vt capacity factor k = Vt In the equation for Rs, k is the average of k1 and k2. Adsorption techniques such as ion exchange chromatography can have high capacity factors since experimental conditions can be chosen which lead to peak retention volumes greatly in excess of Vt (Vt is also often denoted Vm). This can
15
be seen in contrast with the technique of gel filtration where capacity is limited since all peaks must elute within the volume (Vt - V0). ?@g@? @? ?@g@? @K J@L??W2@@?W2@@?W26X?)T2@@6X?@@@?W26X?)T-X? 7R1??7
Fig. 5. Hypothetical chromatogram. V0 = void volume, VR1 = elution volume for peak 1, VR2 = elution volume for peak 2, Vt = total volume, wb1 = peak width for peak 1, wb2 = peak width for peak 2.
Efficiency The column efficiency is related to the zone broadening which occurs on the column and can be calculated from the expression:
N = 5.54
VR1 2
where wh is the peak width
wh
at half peak height
( )
and is expressed as the number of theoretical plates (N) for the column under specified experimental conditions. Efficiency is frequently stated as the number of theoretical plates per metre chromatographic bed, or expressed as H (height equivalent to a theoretical plate, HETP), which is the bed length (L) divided by the plate number. H = L/N Since the observed value for N depends on experimental factors such as flow rate and sample loading, it is important that comparisons are done under identical conditions. In the case of ion exchange chromatography, efficiency is measured under isocratic conditions, using a substance which does not interact with the matrix, e.g. acetone.
16
One of the main causes of zone broadening in a chromatography bed is longitudinal diffusion of the solute molecules. The effect is minimized if the distances available for diffusion, in both the mobile phase and stationary phase, are minimized. In practice this is achieved by using small uniform bead sizes and important developments in ion exchange chromatography have been the introduction of 10 µm and 15 µm diameter particles such as MonoBeads and SOURCE, to give high efficiency preparative media. The highest efficiency is achieved with the non-porous, 3 µm diameter MiniBeads, designed for analytical and micropreparative applications. After bead size, the second major contributory factor to efficiency is good experimental technique. Badly, unevenly packed chromatography beds and air bubbles will lead to channelling, zone broadening and loss of resolution. Good separations require well packed columns and the importance of column packing increases in direct proportion to the performance required.
Selectivity The selectivity (a) defines the ability of the system to separate peaks i.e. the distance between two peaks. The selectivity factor can be calculated from the chromatogram (Fig. 5) using the expression a=
k2
=
k1
VR2 - V0
VR2
Å
VR1 - V0
VR1
Good selectivity is a more important factor than high efficiency in determining resolution (Fig. 6) since Rs is linearly related to selectivity but quadratically related to efficiency. This means that a four fold increase in efficiency is required to double the resolution under isocratic conditions. ?W2@6X @?hf@?hf@?f?@ W.M?I/ @?hf@?he@Kh?@K? 7Hf?W26X?W26X?W2@@?e?W2@6T26X?@?W26X?W26X?@@@?/X?W2@?@@@e?@ @??@@@?7
? ? ? ? ? ? ? ? ? ? ? ? ? @? ? @? ? ?J@L ? ?7R1 ? J@@@L? ? 7
@??@?V/?N@H? J5 .Y
Fig. 6. The effect of selectivity and efficiency on resolution.
17
Selectivity in ion exchange chromatography depends not only on the nature and number of the ionic groups on the matrix but also on the experimental conditions, such as pH and ionic strength. It is the ease and predictability with which these experimental conditions, and thus the selectivity, can be manipulated which gives ion exchange chromatography the potential of extremely high resolution.
Capacity The capacity of an ion exchanger is a quantitative measure of its ability to take up exchangeable counter-ions and is therefore of major importance. The capacity may be expressed as total ionic capacity, available capacity or dynamic capacity. The total ionic capacity is the number of charged substituent groups per gram dry ion exchanger or per ml swollen gel. Total capacity can be measured by titration with a strong acid or base. The actual amount of protein which can be bound to an ion exchanger, under defined experimental conditions, is referred to as the available capacity for the gel. If the defined conditions include the flow rate at which the gel was operated, the amount bound is referred to as the dynamic capacity for the ion exchanger. Available and dynamic capacities depend upon: The properties of the protein. The properties of the ion exchanger. The chosen experimental conditions. The properties of the protein which determine the available or dynamic capacity on a particular ion exchange matrix are its molecular size and its charge/pH relationship. The capacity of an ion exchanger is thus different for different proteins. On a porous matrix used for ion exchange chromatography, molecules which are small enough to enter the pores will exhibit a higher available capacity than those molecules which are restricted to the charged substituents on the surface of the gel. Similarly, since the interaction is ionic, the protein’s charge/pH relationship must be such that the protein carries the correct net charge, at a sufficiently high surface charge density, to be bound to a particular ion exchanger under the chosen buffer conditions. The properties of the ion exchange matrix which determine its available capacity for a particular protein are the exclusion limit of the matrix, and the type and number of the charged substituents. High available capacity is obtained by having a matrix which is macroporous and highly substituted with ionic groups which maintain their charge over a wide range of experimental conditions. Non-porous
18
matrices have considerably lower capacity than porous matrices, but higher efficiency due to shorter diffusion distances. The experimental conditions which affect the observed capacity are pH, the ionic strength of the buffer, the nature of the counter-ion, the flow rate and the temperature. The flow rate is of particular importance with respect to dynamic capacity, which decreases as the flow rate is increased. These conditions should always be taken into consideration when comparing available capacities for different ion exchangers. Methodologies for determining the available and dynamic capacities for an ion exchanger are given in Chapter 10.
19
3. Product Guide Pharmacia Biotech manufactures a wide range of ion exchange media suitable for analytical, micropreparative, small scale preparative, and process scale applications. The product range is summarized below.
MonoBeads (page 23) Mono Q and Mono S are strong ion exchangers based on MonoBeads, monodisperse 10 µm hydrophilic polymer particles. Mono Q and Mono S are the established standards for high performance ion exchange separations and are best suited for analytical and small scale preparative applications.
MiniBeads (page 30) MiniBeads, a non-porous matrix of monodisperse 3 µm hydrophilic polymer particles, is the base for two strong ion exchangers, Mini Q and Mini S. Both media are available pre-packed in Precision Columns 3.2/3, for micropreparative chromatography in SMART System. With a specially designed column holder, these columns can also be used in FPLC and HPLC systems. BioProcess
?@f?@?@?@?/K ?@e@@?@?@?@?V4 ? @ ? ? @@@@@@6XfO26 S,?W2@@Y@ @@@@@@@U?7UI'X@ I/?@)?V4@ ? ?@f?@?@?@?/K ? ?@e@@?@?@?@?V4 ? ?)X?'@?@?@@?W.? @ ?@)?V'e?@@?.Y? ? ? ? ?@f?@?@?@?/K ?@@?? @@@@@@6XeW2@@@ ?@e@@?@?@?@?V4 S,?W&@@X@ ? ? @@@@@@@U?7>(R@@ @ ? I/?@0Y?@@ ? O2@?? ? @@e@?@@@@@U@?? ? @@@@@@6Xe@?@?W ?@f?@?@?@?/K B@@?? ?@@?W.?@?@@??@? S,?J@@@?7 ?@e@@?@?@?@?V4 ?@@?.Y?@?@@?e? @@e@?@?@??@@?? @@@@@@@U?7@??J@ ? ? I/?@@??@@ @ ? ?@@?? ?@f?@?@?@?/K ? ? ? ? ?@e@@?@?@?@?V4 ?@?@e?@@ @@@@@@6Xe@6T26 ? ?@@??@e?@@?@?? O2@?? S,?J@@@R4 @Khf? @ ?@@?e?@g? @@@@@@@U?7@V'9? @@e@?@@@@@U@?? @@@@@@@?e?@e@ ? I/?@@?V4@ B@@?? ? ?@@?? ? ? @@e@?@?@??@@?? @@@@@@6Xe@@@6T ? ? S,?J@@@>@ ?@@?@??@?@@?e? ? @@@@@@@U?7@(R@> ? O2@?? ?@?@e?@@ I/?@0Y?@0 ? @@e@?@@@@@U@?? @Khf? ?@@?? ? B@@?? @@@@@@@?e@?@?@ ? @@e@?@?@??@@?? ? ?@@?e?@g? ? O2@?? ? ? @@e@?@@@@@U@?? ? B@@?? ?@?@e?@@ ?@@?? @@e@?@?@??@@?? @Khf? ? ? @@@@@@@?e@@?@? ? ? ?@?@e?@@ O2@?? @Khf? @@e@?@@@@@U@?? @@@@@@@??@f@ B@@?? ? @@e@?@?@??@@??
SOURCE (page 34)
SOURCE 15Q, SOURCE 15S, SOURCE 30Q and SOURCE 30S are strong ion exchangers based on the same type of rigid polymer matrix as MonoBeads, polystyrene/divinyl benzene beads. SOURCE 15Q and SOURCE 15S are based on 15 µm monodisperse particles while SOURCE 30Q and SOURCE 30S are based on 30 µm mondisperse particles. SOURCE ion exchange media are designed for high performance applications at both research and industrial scales. They provide high capacity at high flow rates and at a minimum of back-pressure, thus allowing short cycle times, high productivity and scaleability. SOURCE 15 matrices are ideal for purification when very high resolution (efficiency) is required. SOURCE 30 matrices gives, in comparison with SOURCE 15 matrices, slightly less resolution (lower efficiency) but at much lower back-pressure. This makes SOURCE 30 ideal for purification with more complex samples and larger volumes. Using SOURCE 30, a higher degree of purification can be obtained with high productivity. Typically working flow rate ranges for ion exchangers based on SOURCE 15 and SOURCE 30 are 30-600 cm/h and 300-1000 cm/h respectively.
20
Sepharose High Performance ion exchangers (page 43)
BioProcess
?@f?@?@?@?/K ?@e@@?@?@?@?V4 ? @ ? ? @@@@@@6XfO26 S,?W2@@Y@ @@@@@@@U?7UI'X@ I/?@)?V4@ ? ?@f?@?@?@?/K ? ?@e@@?@?@?@?V4 ? ?)X?'@?@?@@?W.? @ ?@)?V'e?@@?.Y? ? ? ? ?@f?@?@?@?/K ?@@?? @@@@@@6XeW2@@@ ?@e@@?@?@?@?V4 S,?W&@@X@ ? ? @@@@@@@U?7>(R@@ @ ? I/?@0Y?@@ ? O2@?? ? @@e@?@@@@@U@?? ? @@@@@@6Xe@?@?W ?@f?@?@?@?/K B@@?? ?@@?W.?@?@@??@? S,?J@@@?7 ?@e@@?@?@?@?V4 ?@@?.Y?@?@@?e? @@e@?@?@??@@?? @@@@@@@U?7@??J@ ? ? I/?@@??@@ @ ? ?@@?? ?@f?@?@?@?/K ? ? ? ? ?@e@@?@?@?@?V4 ?@?@e?@@ @@@@@@6Xe@6T26 ? ?@@??@e?@@?@?? O2@?? S,?J@@@R4 @Khf? @ ?@@?e?@g? @@e@?@@@@@U@?? @@@@@@@U?7@V'9? ? @@@@@@@?e?@e@ I/?@@?V4@ B@@?? ? ?@@?? ? ? ?
@@e@?@?@??@@?? @@@@@@6Xe@@@6T ? S,?J@@@>@ ?@@?@??@?@@?e? ? @@@@@@@U?7@(R@> ? O2@?? ?@?@e?@@ I/?@0Y?@0 ? @@e@?@@@@@U@?? @Khf? ?@@?? ? B@@?? @@@@@@@?e@?@?@ ? @@e@?@?@??@@?? ? ?@@?e?@g? ? O2@?? ? ? @@e@?@@@@@U@?? ? B@@?? ?@?@e?@@ ?@@?? @@e@?@?@??@@?? @Khf? ? ? @@@@@@@?e@@?@? ? ? ?@?@e?@@ O2@?? @Khf? @@e@?@@@@@U@?? @@@@@@@??@f@ B@@?? ? @@e@?@?@??@@??
Q and SP Sepharose High Performance are strong ion exchangers based on a 34 µm highly cross-linked agarose matrix, providing high physical and chemical stability. These media are ideal for intermediate and final purification. They should be used when resolution is the main objective. As resolution and efficiency are maintained with increasing column diameter and sample load, separations using these media are easy to scale up. Typical working flow rates are 50-150 cm/h. BioProcess
?@f?@?@?@?/K ?@e@@?@?@?@?V4 ? @ ? ? @@@@@@6XfO26 S,?W2@@Y@ @@@@@@@U?7UI'X@ I/?@)?V4@ ? ?@f?@?@?@?/K ? ?@e@@?@?@?@?V4 ? ?)X?'@?@?@@?W.? @ ?@)?V'e?@@?.Y? ? ? ? ?@f?@?@?@?/K ?@@?? @@@@@@6XeW2@@@ ?@e@@?@?@?@?V4 S,?W&@@X@ ? ? @@@@@@@U?7>(R@@ @ ? I/?@0Y?@@ ? O2@?? ? @@e@?@@@@@U@?? ? @@@@@@6Xe@?@?W ?@f?@?@?@?/K B@@?? ?@@?W.?@?@@??@? S,?J@@@?7 ?@e@@?@?@?@?V4 ?@@?.Y?@?@@?e? @@e@?@?@??@@?? @@@@@@@U?7@??J@ ? ? I/?@@??@@ @ ? ?@@?? ?@f?@?@?@?/K ? ? ? ? ?@e@@?@?@?@?V4 ?@?@e?@@ @@@@@@6Xe@6T26 ? ?@@??@e?@@?@?? O2@?? S,?J@@@R4 @Khf? @ ?@@?e?@g? @@e@?@@@@@U@?? @@@@@@@U?7@V'9? ? @@@@@@@?e?@e@ I/?@@?V4@ B@@?? ? ?@@?? ? ? ?
@@e@?@?@??@@?? @@@@@@6Xe@@@6T ? S,?J@@@>@ ?@@?@??@?@@?e? ? @@@@@@@U?7@(R@> ? O2@?? ?@?@e?@@ I/?@0Y?@0 ? @@e@?@@@@@U@?? @Khf? ?@@?? ? B@@?? @@@@@@@?e@?@?@ ? @@e@?@?@??@@?? ? ?@@?e?@g? ? O2@?? ? ? @@e@?@@@@@U@?? ? B@@?? ?@?@e?@@ ?@@?? @@e@?@?@??@@?? @Khf? ? ? @@@@@@@?e@@?@? ? ? ?@?@e?@@ O2@?? @Khf? @@e@?@@@@@U@?? @@@@@@@??@f@ B@@?? ? @@e@?@?@??@@??
Sepharose Fast Flow ion exchangers (page 46)
Sepharose Fast Flow ion exchangers are based on 90 µm highly cross-linked 6% agarose beads of high chemical and physical stability. The range consists of the weak exchangers DEAE Sepharose Fast Flow and CM Sepharose Fast Flow as well as the strong exchangers Q Sepharose Fast Flow and SP Sepharose Fast Flow. The exceptional flow characteristics make these ion exchangers the first choice for separating crude mixtures early in a purification scheme. Here, fast removal and a combination of good resolution and high flow rate are essential. Typical working flow rates for these media are 100-300 cm/h. Sepharose Fast Flow ion exchangers are ideal for purifications with high demands on productivity. BioProcess
?@f?@?@?@?/K ?@e@@?@?@?@?V4 ? @ ? ? @@@@@@6XfO26 S,?W2@@Y@ @@@@@@@U?7UI'X@ I/?@)?V4@ ? ?@f?@?@?@?/K ? ?@e@@?@?@?@?V4 ? ?)X?'@?@?@@?W.? @ ?@)?V'e?@@?.Y? ? ? ? ?@f?@?@?@?/K ?@@?? @@@@@@6XeW2@@@ ?@e@@?@?@?@?V4 S,?W&@@X@ ? ? @@@@@@@U?7>(R@@ @ ? I/?@0Y?@@ ? O2@?? ? @@e@?@@@@@U@?? ? @@@@@@6Xe@?@?W ?@f?@?@?@?/K B@@?? ?@@?W.?@?@@??@? S,?J@@@?7 ?@e@@?@?@?@?V4 ?@@?.Y?@?@@?e? @@e@?@?@??@@?? @@@@@@@U?7@??J@ ? ? I/?@@??@@ @ ? ?@@?? ?@f?@?@?@?/K ? ? ? ? ?@e@@?@?@?@?V4 ?@?@e?@@ @@@@@@6Xe@6T26 ? ?@@??@e?@@?@?? O2@?? S,?J@@@R4 @Khf? @ ?@@?e?@g? @@@@@@@U?7@V'9? @@e@?@@@@@U@?? ? @@@@@@@?e?@e@ I/?@@?V4@ B@@?? ? ?@@?? ? ? @@e@?@?@??@@?? @@@@@@6Xe@@@6T ? ? S,?J@@@>@ ?@@?@??@?@@?e? ? @@@@@@@U?7@(R@> ? O2@?? ?@?@e?@@ I/?@0Y?@0 ? @@e@?@@@@@U@?? @Khf? ?@@?? ? B@@?? @@@@@@@?e@?@?@ ? @@e@?@?@??@@?? ? ?@@?e?@g? ? O2@?? ? ? @@e@?@@@@@U@?? ? B@@?? ?@?@e?@@ ?@@?? @@e@?@?@??@@?? @Khf? ? ? @@@@@@@?e@@?@? ? ? ?@?@e?@@ O2@?? @Khf? @@e@?@@@@@U@?? @@@@@@@??@f@ B@@?? ? @@e@?@?@??@@??
Sepharose Big Beads ion exchangers (page 50)
Q and SP Sepharose Big Beads are strong ion exchangers designed for industrial applications. They are based on 100-300 µm highly cross-linked 6% agarose beads. The large particle size combined with high physical stability of the base matrix ensures rapid processing, even for viscous samples. Sepharose Big Beads is therefore the choice at the beginning of a purification scheme, where viscosity and back-pressure may limit the throughput attainable with ion exchangers based on smaller bead sizes, such as Sepharose Fast Flow ion exchangers. The medium should be chosen when large volumes are handled and fast adsorption is required and when resolution is of less importance. BioProcess
?@f?@?@?@?/K ?@e@@?@?@?@?V4 ? @ ? ? @@@@@@6XfO26 S,?W2@@Y@ @@@@@@@U?7UI'X@ I/?@)?V4@ ? ?@f?@?@?@?/K ? ?@e@@?@?@?@?V4 ? ?)X?'@?@?@@?W.? @ ?@)?V'e?@@?.Y? ? ? ? ?@f?@?@?@?/K ?@@?? @@@@@@6XeW2@@@ ?@e@@?@?@?@?V4 S,?W&@@X@ ? ? @@@@@@@U?7>(R@@ @ ? I/?@0Y?@@ ? O2@?? ? @@e@?@@@@@U@?? ? @@@@@@6Xe@?@?W ?@f?@?@?@?/K B@@?? ?@@?W.?@?@@??@? S,?J@@@?7 ?@e@@?@?@?@?V4 ?@@?.Y?@?@@?e? @@e@?@?@??@@?? @@@@@@@U?7@??J@ ? ? I/?@@??@@ @ ? ?@@?? ?@f?@?@?@?/K ? ? ? ? ?@e@@?@?@?@?V4 ?@?@e?@@ @@@@@@6Xe@6T26 ? ?@@??@e?@@?@?? O2@?? S,?J@@@R4 @Khf? @ ?@@?e?@g? @@@@@@@U?7@V'9? @@e@?@@@@@U@?? ? @@@@@@@?e?@e@ I/?@@?V4@ B@@?? ? ?@@?? ? ? @@e@?@?@??@@?? @@@@@@6Xe@@@6T ? ? S,?J@@@>@ ?@@?@??@?@@?e? ? @@@@@@@U?7@(R@> ? O2@?? ?@?@e?@@ I/?@0Y?@0 ? @@e@?@@@@@U@?? @Khf? ?@@?? ? B@@?? @@@@@@@?e@?@?@ ? @@e@?@?@??@@?? ? ?@@?e?@g? ? O2@?? ? ? @@e@?@@@@@U@?? ? B@@?? ?@?@e?@@ ?@@?? @@e@?@?@??@@?? @Khf? ? ? @@@@@@@?e@@?@? ? ? ?@?@e?@@ O2@?? @Khf? @@e@?@@@@@U@?? @@@@@@@??@f@ B@@?? ? @@e@?@?@??@@??
STREAMLINE ion exchangers (page 52)
STREAMLINE adsorbents, available as STREAMLINE DEAE and STREAMLINE SP, are specially designed for use in STREAMLINE columns for expanded bed adsorption. Together they enable the high flow rates needed for high productivity in industrial applications of fluidized beds. STREAMLINE adsorbents, based on cross-linked 6% agarose beads with a mean particle size of 200 µm, are designed to handle samples directly from both fermentation homogenates and crude samples from cell culture/fermentation at working flow rates of typically 200-400 cm/h.
21
DEAE Sepharose CL-6B and CM Sepharose CL-6B (page 54) DEAE and CM Sepharose CL-6B ion exchangers are based on 90 µm cross-linked 6% agarose beads. These two gels are the traditional agarose based ion exchangers from Pharmacia Biotech. Their performance has been demonstrated in several hundred applications for the separation of proteins, polysaccharides, nucleic acids, membrane components and other high molecular weight substances. Sepharose CL-6B based ion exchangers are typically used with working flow rates of up to 60 cm/h.
DEAE Sephacel (page 58) DEAE Sephacel is a beaded cellulose ion exchanger for separations over a wide molecular weight range (up to l x 106 for globular proteins). DEAE Sephacel is the medium of choice when a cellulose ion exchanger is needed for standard chromatography of proteins, nucleic acids or other biopolymers.
Sephadex ion exchangers (page 61) Sephadex ion exchangers are bead-formed media based on cross-linked dextran. They are available as strong and weak ion exchangers covering the pH range 2-10. Sephadex ion exchanger are suitable for batch-type applications.
Bulk quantities All Pharmacia Biotech ion exchangers are available in larger pack sizes or larger pre-packed columns. Contact your local Pharmacia Biotech supplier for further information.
Equipment Pharmacia Biotech also supply a full range of equipment for operating all of the ion exchangers covered in this handbook. Information regarding specific equipment is available upon request from Pharmacia Biotech.
22
4. MonoBeads and MiniBeads MonoBeads MonoBeads are unique, highly efficient, pH stable ion exchange media, specifically designed for high resolution separations of proteins, peptides, and oligonucleotides. An example of the type of separation which can be achieved is shown in Figure 7. ?W-X ?7R1 J@?@L? 7@@@)X @?e@)KO-X?W-X @?e(R4@?,?7R1 ?S@U?@?@ @@6X @KO&?,?3T5 ?W-Xf@?B1 @@0R+Y?V+Y ?*?)T-X?@?C5 ?W&?W26X?W26X? ?V+R@>)X@@@U ?*@?7@
Fig. 7. Characterization of venom from the White Faced Hornet by cation exchange chromatography (6). Conditions: Venom (7 mg) dissolved in 50 mM BICINE, pH 8.4 (buffer A); Column, Mono S HR 5/5; Buffer B, 0.35 M NaCl in Buffer A; Gradient, 0-100% B in 40 ml; flow rate, 1 ml/min; detection, 280 nm at 0.05 AUFS.
MonoBeads ion exchangers are based on a 10 µm beaded hydrophilic polystyrene/divinyl benzene resin which has been substituted with quaternary amine groups to yield the strong anion exchanger, Mono Q, or with methyl sulphonate groups to yield the strong cation exchanger, Mono S. Note: Substitution with the same ionic groups as Polybuffer Exchanger PBE 94 gives Mono P - the matrix used for high resolution chromatofocusing. For further information on the technique and media for chromatofocusing the reader should contact Pharmacia Biotech.
23
The name MonoBeads is derived from the unique monodisperse nature of the matrix. This monodispersity (Fig. 8) was accomplished through a process developed by Professor John Ugelstad of SINTEF, Trondheim, Norway.
Fig. 8. An electron micrograph of MonoBeads showing their distinct monodispersity.
The resolution which can be achieved on any chromatographic matrix is a result of a combination of the efficiency and selectivity of the system. Maximum efficiency is obtained through the use of small, perfectly spherical, monodisperse particles, optimally packed in a well designed column. All pre-packed MonoBeads columns have efficiencies at about 25 000 plates per metre. High efficiency, coupled with the excellent selectivity of the Q and S substituents, results in high resolution separations. Scale-up to SOURCE Q and S (see Chapter 5), Q and SP Sepharose High Performance and Q and SP Sepharose Fast Flow (see Chapter 6) is simple, since these gels have similar selectivities to MonoBeads based media.
Properties Chemical stability The gels are stable for continuous use in the pH range 2-12, although pH values as high as 14 can be used during cleaning and sanitizing procedures. MonoBeads can be used with solutions of most buffers used in biochemical separations of biomolecules and in water-alcohol (C1 - C4) and acetonitrile-water solutions. The resistance of the MonoBeads matrix to organic solvents allows complete cleaning and the use of conditions necessary for the solubilization of very hydrophobic samples. An example of the use of MonoBeads with organic solvents is given in Figure 9, which shows the analysis of the peptide bacitracin on Mono S using lithium chlorate as the eluting salt and 90% methanol as the liquid phase (7).
24
W-X? 7R1? ?J@?@L ?7@@@)X? ?@e?@)KO-X?W-K? ?@e?(R4@?,?7R@@@@@@@@@? S@U?@?@@?@@?@?@? ?@KO&?,?3T@@?@@?@?@? ?@@0R+Y?V+R'?@@?@?@? ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ @?he?@ ?@ @?he?@ ?@ @?he?@ ?@ @?he?@ ?@ @?he?@ ?@ @?he?@ ?@ @?he?@ ?@ @?he?@ ?@ @?he?@ ?@ @?he?@ ?@ @?he?@ ?@ @?he?@ ?@ @?he?@ ?@ @?he?@ ?@ @?he?@ ?@ @?he?@ ?@ @?heJ@ ?@ @?he7@ ?@ @?he@@ ?@ @?he@@ ?@ @?he@@ ?@ @?he@@ ?@ ?J@?he@@ O2@@@@@@@@@@@@@@e ?@ ?7@?he@@ O2@@@@0M ?@ ?@@?he@@ O20M ?@ ?@@?he@@ W20M ?@ ?@@?he@@ ?O.M ?@ ?@@?he@@ ?W20Y? ?@ ?@@?he@@ O.M? ?@ ?@@?he@@ W20Y ?@ ?@@?he@@ ?W.M ?@ ?@@?he@@ W.Y? ?@ ?@@?he@@ ?W.Y ?@ ?@@?he@@ W.Y? ?@ ?@@?he@@ ?W.Y ?@ ?@@?he@@ W.Y? ?@ ?@@?he@@ ?W.Y ?@ ?@@?he@@ W.Y? ?@ ?@@?he@@ ?W.Y ?@ ?@@?he@@L? W.Y? ?@ ?@@Lhe@V1?hf?W.Y ?@ ?@V1he@?@?hfW.Y? ?@ ?@?@he@?@?he?W.Y ?@ ?@?@he@?@?heW.Y? ?@ ?@?@he@?@?h?W.Y ?@ ?@?@he@?@?hW.Y? ?@ ?@?@he@?@?g?O.Y ?@ ?@?@he@?@?f?W20Y? ?@ ?@?@he@?@?fW.M? ?@ ?@?@he@?@?e?W.Y ?@ ?@?@he@?@?eW.Y? ?@ ?@?@he@?@??W.Y ?@ ?@?@he@?@?W.Y? @? ?@ ?@?@he@[email protected] @? ?@ ?@?@he@?@(Y? @L ?@ ?@?@he@?@H ?J@1 ?@ ?@?@he@@@? ?7Y@ ?@ ?@?@h?J@?@? ?@?@ ?@ ?@?@hW&5?@? ?@?@ ?@ ?@?@g?W&@H?@? ?@?@ ?@ ?@[email protected]@e@? ?@?3L? ?@ ?@?@f?W.Y?@e@? ?@?N1? ?@ ?@[email protected]??@e@? ?@e@? ?@ ?@?@f7He?@e@? ?@e@? ?@ ?@?@e?J5?e?@e@? ?@e@? ?@ ?@[email protected]?e?@e@? J5e@? ?@ J5?@?W.Yf?@e@? 7He3L ?@ [email protected]?f?@e@? @?eN1 ?@ @??@(Yg?@e@? @?e?@ ?@ @?J@H?g?@e@? @?e?@ ?@ @?7@h?@e@? @?e?@ ?@ @W@@h?@e@? @?e?@ ?@ @(Y@h?@e@? @?e?3L? ?@ ?J@H?@h?@e@? @?e?N1? ?@ W&@??@h?@e@? @?f@? ?@ ?W.Y@??@h?@e@? @?f@? ?@ W.Y?@??@h?@e@? @?f@? ?@ ?W.Ye@??@h?@e@? @?f3L ?@ ?7H?e@??@h?@e@? @?fN1 ?@ J5f@??@h?@e@? ?J5?f?@ ?@ ?W.Yf@??@h?@e@? ?7H?f?@ ?@ W.Y?f@??@h?@e@? ?@g?@ ?@ @?f?W.Yg@??@h?@e@? ?@g?@ ?@ @?fW.Y?g@??@h?@e@? ?@g?3L? ?@ @?e?W.Yh@??@h?@e@? ?@g?N1? ?@ @?e?7H?h@??@h?@e@? ?@h@? ?@ @?eJ5he@??@hJ5e@? ?@h@? ?@ ?J@L?W.Yhe@??@h7He@? ?@h@? ?@ ?7R)T.Y?he@??@h@?e3L ?@h@? ?@ ?@?@(Yhf@??@h@?eN1 ?@h3L ?@ ?@?@H?hf@??@h@?e?@ ?@hN1 ?@ ?@@@hf?J5??@h@?e?@ J5h?@ ?@ J@?@hf?7H??@h@?e?@ 7Hh?3L? ?@ 7@?@hf?@e?@h@?e?@ @?h?N1? ?@ ?J@5?@hf?@e?@h@?e?@ @?he3Le?@hg ?@ W&@H?3L?he?@e?3L?g@?e?3L??)X? @?heN1e?@hg ?@ ?W.Y@??N1?he?@e?N1?f?J5?e?N1?J@1? ?J5?he?3L??@L?hf ?@ W.Y?@?e@?he?@f3Lf?7H?f@?7R'L O.Y?he?N1??@1?hf ?@ ?W.Ye@?e@?heJ5fN1f?@g3T5?N1 ?W2@0Y 3L?@@?hf ?@ ?7U??J5?e3LfW-X?e7Hf?3=?eC5gV+Y??3L?hfO.M? N1?@@Lhf ?@ J@)X?7H?eN1?)X?7R1?e@?f?V46T20Yhe?V/XheO20Y ?3X@V1hf ?@ 7@V/X@f?3T@)T5?3T-X@?g?I+M V/K?fO2@@0M ?V4@?3L?he ?@ @5?V4@f?V+MI+Y?V+R4@? ?V4@@@@@0M ?N1?he ?@ ?O2@@@0Y 3Lhe ?@hO2@@@@@@@@@0M?@? N)X?h ?@@@@@@@@@0Mhe?J5? ?@)Kh ?@ ?7H? I4@@@@@?e ?@ J5 ?@ ?W.Y ?@ ?7H? ?@ J5 ?@ ?W.Y ?@ W.Y? ?@ 7H ?@ ?J5? ?@ O.Y? ?@@@@@@@@@@@@@@@0Y ?@ ?@ ?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@? ?@@@@@@?hg ?@e@?@@@@@?W26Xf ?@e@?@?@?@?7YV1f ?@e@?@?@?@?@@@@f ?@e@?@?@?@?3Xg ?@e@?@?@?@?V4@?f
Fig. 9. Separation of the peptide bacitracin on Mono S. (Work by Pharmacia Biotech, Uppsala, Sweden.)
Dimethylsulphoxide (DMSO) and similar solvents can be used, but will change the separation properties of the gels. Aqueous solutions of urea, ethylene glycol and similar compounds can be used but will increase the back-pressures due to their higher viscosities. Non-ionic detergents, zwitterionic detergents or detergents with the same charge as the ion exchange groups may be used. Oxidizing agents should be avoided.
Physical stability MonoBeads are based on highly rigid beads which means that they can be used at high flow rates. As a consequence of the monodisperse nature of the matrix these high flow rates do not result in high back-pressures. For example, an HR 5/5 column (5 mm inner diameter and 50 mm bed height) packed with a MonoBeads matrix normally generates a back-pressure of 1.0-1.5 MPa (10-15 bar) when operated at a flow rate of 1 ml/min (300 cm/h). Note: These back-pressures are beyond the operating limits of standard laboratory peristaltic pumps.
25
A summary of the characteristics of MonoBeads is shown in Table 2. Table 2. Characteristics of MonoBeads. Properties
Mono Q
Mono S
Type of gel
strong anion exchanger
strong cation exchanger
Charged group
-O-CH2-CHOH-CH2-O-CH2-CHOH-CH2-N+(CH3)3
-O-CH2-CHOH-CH2-O-CH -CHOH-CH SO -
Total ionic capacity (µmoles/ml gel)
270-370
140-180
Thyroglobulin (MW 669 000)
25
N.D.
HSA (MW 68 000)
65
N.D.
a-lactalbumin (MW 14 300)
80
N.D.
IgG (MW 150 000)
N.D.
75
Ribonuclease (MW 13 700)
N.D.
75
Typical protein recoveries (%)
90-100
90-100
Typical enzyme activity recoveries (%)
>80
>80
Average particle size (µm)
10 ±0.5
10 ±0.5
MW range (proteins)
up to 107
up to 107
working pH range*
3-11
3-11
pH stability** long term
2-12
2-12
short term
2-14
2-14
2
2
3
Total protein binding capacity (mg/ml gel)
N.D. = Not determined Solvent restrictions: The ion exchangers are stable in alcohol/water solutions (C1-C4). 100% dimethyl sulphoxide, dimethylformamide, and formic acid can change the separation properties of the gel. Avoid oxidizing and reactive reagents. Detergents can be used if they are non-ionic or have the same charge as the gel. * working pH range refers to the pH range over which the ion exchange groups remain charged and maintain consistently high capacity. ** pH stability, long term refers to the pH interval where the gel is stable over a long period of time without adverse effects on its subsequent chromatographic performance. pH stability, short term refers to the pH interval for regeneration and cleaning procedures.
26
Flow rate The rigid monodisperse nature of the media enables high flow rates to be used on MonoBeads columns. Normal recommended flow rates for high resolution separations are in the range 150 to 600 cm/h for HR 5/5 columns. Higher flow rates can be used during column washing and regeneration. In addition, the absence of buffering capacity means that buffer exchange and re-equilibration can be executed quickly and with small amounts of buffer. Details of the recommended flow rates to be used on the different columns are given in Table 3.
Capacity The high substitution levels coupled with the large pore size of the matrix, the exclusion limit for globular proteins is 107, give MonoBeads exchangers high capacities for large proteins as well as for smaller polypeptides and peptides. Typical saturation capacities are in the range of 60 mg protein per ml of gel and typical sample loading capacities are in the region of 25 mg of protein per ml of gel. Data on the saturation capacities for some specific proteins are given in Table 2. Table 3. Chromatographic properties of pre-packed columns of MonoBeads. Properties
PC 1.6/5
HR 5/5
HR 10/10
HR 16/10
BioPilot Column 35/100 60/100
Column volume (ml)
0.1
1
8
20
100
300
Column dimensions i.d. x bed height (mm)
1.6x50
5x50
10x100
16x100
35x100
60x100
0.01-0.40
0.5-2.0
up to 6
up to 10
up to 32
up to 94
5
5
4
3
2
2
Number of theoretical plates per meter (N/m)
25 000
25 000
25 000
25 000
25 000
25 000
Normal separation times (min)
5-20
5-20
40
40
60-90
60-90
Recommended working flow rate range (ml/min) Max operating pressure (MPa)
27
The titration curves for Mono Q and Mono S (Fig. 10) show no buffering capacity which means that the loading capacity does not vary with pH over the working range of the gel. ?@@?e@@ ?W2@@6X? ?@@?e@@W26T2@@??W26X??7
?W&?W26X ?*@?.MB1 ?N@?e?@ @?W.g@@@@@? @?7Yhe@? @?@@@@h@? @? @? @? @? @? @@6K @?I4@@@@6K @?gI4@@6K @?heI4@6K? W&eW&h@?hf?I4@6K *@e*@h@? I46X N@eN@h@? I/X? ?@e?@f@@@@@? ?V/K ?@e?@h@? V46X ?@e?@h@? I/X? @? ?V/X @? V/X? @? ?N1? @? 3L @? N1 @? ?3L? @? ?N1? @? 3L @? N1 ?W&?W26Xh@? ?3L? ?*@?7
? ? ? ? ? ? ? ? ? ? ? ? ?W&?e?W26X?h@?e@? ?@K?hg)T26X?@@e@?@@@?f*@f@@e?@@?@W&He7
@?e@? @@6X@?e@?heW&fW26Xh?@e?@ @K ?@h?W&?e?@@?e@@?@?W.??W2@6X?@ @?B@@@@@@?he*@f7
W26Xe?W26X? 7
W26Xf@6X? 7
W26XfW& 7
?@e?@e?@?W2@6T&? ?@@@@@e@@@@@?W26X?@e?@e?@?7
Fig. 10. Titration curves for Mono Q and Mono S. (Work by Pharmacia Biotech, Uppsala, Sweden.)
Recovery Non-specific interactions to the MonoBeads matrix are very low and consequently recoveries are high. Recoveries of protein mass are typically 90-100% and of protein activity greater than 80%. Examples of protein activity recoveries are shown in Table 4. Table 4. Protein activity recoveries (%) from MonoBeads columns. Protein
Mono Q
Mono S
b-Glucuronidase b-Glucosidase Phosphodiesterase Creatine Kinase Enolase Lactate Dehydrogenase Aldolase
106 N.D. 80 90 N.D. N.D. N.D.
N.D. 93 N.D. N.D. 95 102 94
N.D. = Not determined
28
Reproducibility The stability of the MonoBeads matrix together with controlled synthesis and column packing procedures ensure very reproducible separations both over time and from column to column. Figure 11 shows the reproducibility of a separation performed on three different Mono S columns. ? ?? ?? ? ?? ?? ?? ? ?? ?? ?? ? ?? ?? ? ?? ?W.? ?W2@@?)Xhe?7U??W&? W-X?eW-X??@f)X?W&?heW&?)X? @?he?W&?he@??@e?@@@e?@@@ ?7Y?e@1?@?W-X?@@@?@)??7@??@ 7R1?e7R1??@f@1?7@??W&??W-X?W&@?@1?@?@@@@@?e@@@@@?W-X?@W-X?@@@e)X?*@W)Xf?@6X@??@e?@X?fI@ ?? ?@@@@?@@?@?7R1?@?@?@H?J@@? @?@?e@?@??@f@@?@@??*@L?7R1?7Y@?@@?@?@?@?@?e@?@?@?7R1?@@R1?@?@?J@1?N@@@)f?@V@@@@@e?@)X ? ?@e?J@@?@?@@@T5?@?@?W.R'L 3T5?e3T5??@f@@T@@??N@,?3T5?3X@?@@?@?@?@?@?e@?@?@?3T5?@@T5?@?@?*U@e@@X?f?@W@@??@fS,f@? ?@@@?.R4@@?@0R+Y?@?@?.Y?V/?@ V+Y?@?V+Y??@f(R+R'?e(Y?V+Y?V4@?@@@@?@?@?@?e@?@?@?V+Y?(R+Y?@?@?V4@e(R/??@e?@(R'[email protected]?@e@? ?? ?@e?(Y? ?? ?W.? ?? ?W2@@?)Xhe?7U??W26K? W-X?eW-X??@f)X?W&?heW&?)X? @?he?W&?he@??@e?@@@e?@@@f?W-Xf@??@@@e?)X?W&e@?e@?W26X?@e? ?7Y?e@1?@?W-X?@@@?@)??7YS@@ 7R1?e7R1??@f@1?7@??W&??W-X?W&@?@1?@?@@@@@?e@@@@@?W-X?@W-X?@@@e)X?*@W)Xf?@6X@??@e?@X?fI@f?7R1f@??@X?e?@1?7@e@?e@?7
Fig. 11. Reproducible separations on three Mono S HR 5/5 columns. (Work by Pharmacia Biotech, Uppsala, Sweden.)
29
Availability Mono Q and Mono S are available pre-packed in columns HR 5/5, HR 10/10 and HR 16/10, containing 1, 8 and 20 ml of gel respectively. The media are also available in BioPilot Columns 35/100 and 60/100 containing respectively 100 and 300 ml bed volumes, for chromatography at BioPilot scale. MonoBeads ion exchangers are also available as Mono Q PC 1.6/5 and Mono S PC 1.6/5, prepacked columns specially designed for micropurification on SMART System. These columns can also be used in other high performance chromatography systems by using a column holder for Precision Columns. For further information about the column holder, please contact your Pharmacia Biotech representative. For ordering information, see Chapter 14.
MiniBeads MiniBeads is the base matrix for 3 µm high resolution ion exchange media. This non-porous matrix, consisting of monodisperse hydrophilic polymer beads is substituted with Q and S functional groups to give Mini Q and Mini S. Both media are packed in Precision Columns 3.2/3 (3.2 mm inner diameter and 30 mm bed height) for micropreparative chromatography on SMART System. With a specially designed column holder, these columns can also be used in FPLC and HPLC systems. MiniBeads are highly efficient pH-stable adsorbents designed for high performance micropurification of proteins, peptides and polynucleotides. The monodispersity permits the use of high flow rates at relatively low back-pressures. The main properties are listed in Table 5. Due to the smaller particle size, Mini Q and Mini S give faster separations with higher resolution of peaks than ion exchangers based on MonoBeads, see Figure 12. This resolution is crucial for success when separating complex samples in the pg to µg micropreparative scale.
30
Sample:
Chymotrypsinogen A, Ribonuclease A, Cytochrome C, Lysozyme (6:10:6:5), 25 µg/ml gel (6 µg Mini S, 2.5 µg Mono S ) Buffer A: 20 mM acetic acid, pH 5.0 Buffer B: Buffer A with 0.5 M lithium chloride Gradient: 0–100% B in 20 col. vols. (6 min Mini S, 10 min Mono S). Flow rate: 10 cm/min (800 µl/min Mini S, 200 µl/min Mono S) Instrument: SMART System with µPeak Monitor
?W-Xe@?e@? ?7R1e@?e@? J@?@L?@?e@? 7@@@1?@?e@? @?e@?3=?C5? @?e@?V4@0Y?
? ? ? ? ? ? ? ? ? ? ? ? @? ? @? ? @? ? @? ? @?he?@@@@6X? ? @?he?@e?B1? O2@@@@@@@@@@@@@?he? @?he?@f@? O20M ? @?he?@e?C5? W20M ? ?W26X?eW26KO2@(h@?he?@@@@@U? 7< ? ?7
@? @? @? @? @? ?W-X ?O2@@@@@@@@? @? ?7R1 ?W20M? @? J5?3L? W.M? @? 7H?N1? ?W.Y @? @?e@? ?@ W.Y? W26Xe?W26KO2@(?g@?hf?J@@@@@L ?@ ?W.Y 7
Fig. 12. Comparison of (A) Mini S PC 3.2/3 and (B) Mono S PC 1.6/5. Mini S PC 3.2/3 gives a faster separation and a better resolution of the peaks. Similar results have been found with Mini Q PC 3.2/3 and Mono Q PC 1.6/5. (Work by Pharmacia Biotech, Uppsala, Sweden.)
Properties Chemical stability MiniBeads may be used in aqueous solutions and nearly all organic solutions commonly used in chromatography of proteins, oligonucleotides and peptides. As examples, the matrix is stable to 100% acetonitrile, 75% acetic acid, 2 M NaOH and 1 M HCl. Dimethylsulphoxide (DMSO), dimethylformamide, and formic acid and similar solvents will change the separation properties of the gels. Aqueous solutions of urea, ethylene glycol and similar compounds can be used but will increase the back-pressure due to their higher viscosities. Non-ionic detergents, zwitterionic detergents or detergents with the same charge as the ion exchange groups may be used. Oxidizing agents should be avoided.
31
Physical stability The combination, non-porous and monodisperse beads gives MiniBeads very high physical stability. It has better flow kinetics and can withstand higher back-pressure (up to 10 MPa) than MonoBeads. Note: These back-pressures are beyond the operating limits of standard laboratory peristaltic pumps. Table. 5. Main properties of Mini Q PC 3.2/3 and Mini S PC 3.2/3. Properties
Mini Q PC 3.2/3
Mini S PC 3.2/3
Type of gel
strong anion exchanger
strong cation exchanger
Charged group
-O-CH2-CHOH-CH2-O-CH2-CHOH-CH2-N+(CH3)3
-O-CH2-CHOH-CH2-O-CH2-CHOH-CH2SO3-
Total ionic capacity (µmoles/ml gel)
60-90
16-30
Column dimensions i.d. x bed height (mm) 3.2 x 30
3.2 x 30
Column volume (ml)
0.24
0.24
Average particle size (µm)
3 µm
3 µm
a-amylas (MW 49 000)
Å 1.4
N.D.
Trypsin inhibitor (MW 20 100)
Å 1.4
N.D.
Ribonuclease (MW 13 700)
N.D.
Å 1.3
Lysozyme (MW 14 300)
N.D.
Å 1.3
Binding capacity (mg/column)
Max loading capacity (mg/column)
1-1.5
1-1.5
Practical loading capacity (µg/column)
²200
²200
Typical protein recoveries (%)
70-90
70-90
working pH range*
3-11
3-11
pH stability** long term
3-11
3-11
short term
1-14
1-14
Maximum flow rate (ml/min)
1.0
1.0
Operational pressure limit (MPa)
10
10
Normal separation times (min)
5-20
5-20
N.D. = Not determined * working pH range refers to the pH range over which the ion exchange groups remain charged and maintain consistently high capacity. ** pH stability, long term refers to the pH interval where the gel is stable over a long period of time without adverse effects on its subsequent chromatographic performance. pH stability, short term refers to the pH interval for regeneration and cleaning procedures.
32
Reproducibility Figure 13 shows a long life test and reproducibility test on Mini S PC 3.2/3. Long life and reproducibility are a result of the stabile nature of MiniBeads together with controlled synthesis and column packing procedures.
W-X?@?e@? 7R1?@?e@? ?J@?@?@?e@? ?7@@@@@?e@? ?@e?@@=?C5? ?@e?(R4@0Y?
Column: Sample:
Mini S PC 3.2/3 Chymotrypsinogen A, Ribonuclease A, Lysozyme 6 mg in proportions 1:3:1 20 mM acetic acid, pH 5.0 20 mM acetic acid, pH 5.0 with 0.4 M NaCl 400 ml/min 0–100% B in 12 min
?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@L? ?@ ?@1? ?@ ?@@? ?@ ?@@? ?@ ?@@? ?@ ?@@? ?@ ?@@? ?@ ?@@? ?@ ?@@? ?@ ?@@? ?@ ?@@? ?@ ?@@? ?@ J@@? ?@ ?@K?e?@h?W2@ ?@ 7Y@? ?@ ?@@@@??@W26X?W26T&@@?W26T2@@ ?@ @?@? ?@ ?@@?@??@@YV1?7
Buffer A: Buffer B: Flow rate: Gradient:
?@@@@@ ?W-Kf?@g/Xf ?@?@?@@@@@?W26X?e?7R@@@@@?@?@@@eN1f ?@?@?@?@?@?7YV1?e?@?@?@?@?@?@?@e?@f ?@?@?@?@?@?@@@@?e?@?@?@?@?@?@?@e?@f ?@?@?@?@?@?3X?f?@?@?@?@?@?@?@e?@f ?@?@?@?@?@?V4@f?@?@?@?@?@?@?@e?@f ?3L?hfJ5f ?V/?hf.Yf
Fig. 13. Long life test on Mini S PC 3.2/3. The chromatograms show the 1st, 5th and 201st separation of a series run on the same column. The same consistently good stability and reproducibility have been confirmed on Mini Q PC 3.2/3 (data not shown here). (Work by Pharmacia Biotech, Uppsala, Sweden.)
Availability Mini Q and Mini S are available pre-packed in Precision Columns 3.2/3. For ordering information, see Chapter 14.
33
5. SOURCE SOURCE ion exchangers are high performance media for fast and high resolution separations of biomolecules such as proteins, peptides and oligonucleotides. Examples are given in Figure 14, 18 and Figure 19. SOURCE media are available in two particle sizes, 15 and 30 µm, and as Column: RESOURCE S 1 ml 6.4 mm diam x 30 mm bed height anion and cation exchangers. The Sample: Snake venom 4 mg/ml, 0.1 ml Flow rate: 5 ml/min (180 cm/h strong anion exchangers SOURCE Buffer A: 20 mM sodium phosphate, pH 6.8 Buffer B: Buffer A + 0.4 M NaCl 15Q and 30Q are produced by substiGradient: 0 - 100% buffer B, 20 ml (20 col. vol.) System: FPLC System tution of the base matrices with quaternary amino groups. For the strong cation exchangers, SOURCE 15S and 30S, the matrices have been substituted with methyl sulphonate groups. Table 6 summarizes the general properties of SOURCE ion exchangers. ?? ??? ?? ?? ?? ?? ?@ ?? ?@e?@e@? ?@ ?@e?@e@? ?@ ?@e?@e@? ?@ ??? ?@e?@e@? ?@ ?@e?@e@? ?@ ?? ?@e?@e@? ?@ ?@e?@e@? ?@ ?? ?@e?@e@? ?@L? ?@e?@e@? ?@1? ?? ?@e?@e@? ?@@? ?@e?@e@? ?@@? ?? ?@e?@e@? ?@@? ?@e?@e@? ?@@? ?? ?@e?@e@? ?@@? ?@e?@e@? ?@@? ?? ?@e?@e@? ?@@? ?@e?@e@? ?@@? ?? ?@e?@e@? ?@@? ?@e?@e@? ?@@? ?? ?@e?@e@? ?@@? ?@e?@e@? ?@@? ?? ?@e?@e@? ?@@? ?@e?@e@? ?@@? ?? ?@e?@e@? ?@@? ?@e?@e@? ?@@? ?? ?@e?@e@? ?@@? ?@e?@e3L ?@@? ?? ?@e?@eN1 ?@@? ?@e?@e?@ ?@@? ?? ?@e?@e?@ ?@@? ?@e?@e?@ ?@@L ?? ?@e?@e?@ ?@V1 ?@e?@e?@ ?@?@ ?? ?@e?@e?@ ?@?@ ?@e?@e?@ ?@?@ ?? ?@e?@e?@ ?@?@ ?@e?@e?@ ?@?@ ?? ?@e?@e?@ ?@?@ ?@e?@e?@ ?@?@ ?? ?@e?@e?@ ?@?@ ?@e?@e?@ ?@?@ ?? ?@e?@e?@ ?@?@ ?@e?@e?@ ?@?@ ?@e?@e?@ ?@?@ W.e?W2@6XhW&?@hW&e@@6Xe?? ?@e?@e?@ ?@?@ ?W&He?7@@?e?@hf?J5??@e@?eN1 @? ?@ ? ?@e?@f@? @?e?@ ?@(R'?e?@hf?7H??@e@?e?@ @? ?@ ?@e?@f@? @?e?@ ?@H?f?@hf?@e?@e@?e?@ @? ?@ ??? ?@e?@f@? @?e?@ J@g?@hf?@e?@e@?e?@ @? ?@ ?@e?@f@? @?e?@hf?W&@g?3L?he?@e?@e@?e?@ @? ?@ ? ?@e?@f@? @[email protected]@g?N1?he?@e?@e@?e?@ @? ?@ ?@e?@f@? @?e?@hf7H?@h@?he?@e?@e@?e?@ @? ?@ ??? ?@e?@f3L @?e?@he?J5??@h@?he?@e?@e@?e?@ @? ?@ ?@e?@fN1 @[email protected]??@h@?he?@e?@e@?e?@ @? ?@ ? ?@e?@f?@ @?e?@he7He?@h@?he?@e?@e@?e?@ @? ?@ ?@e?@f?@ ?J5?e?3L?g?J5?e?@h@?he?@e?@e@?e?@ @? ?@ ??? ?@e?@f?@ ?7H?e?N1?gW.Y?e?@h@?he?@e?3L?@?e?@ @? ?@ ?@e?@f?@ ?@?@g@?f?W.Yf?@h@?he?@e?N1?@?e?@ @? ?@ ? ?@e?@f?@ ?@?@g@?f?7H?fJ5h@?he?@f@?@?e?3L? @? ?@ ?@e?@f?@ ?@@@g@?fJ5g7Hh@?he?@f@?@?e?N1? @? ?@ ??? ?@e?@f?@ ?@@@g@?e?W.Yg@?h@?he?@f@?@?f@? @? ?@ ?@e?@f?@ ?@@@g3Le?7H?g@?h@?he?@f@?@?f@? @? ?@ ? ?@e?@f?@ J@@@gN1eJ5h@?h@?he?@f@?@?f@? @? ?@ ?@e?@f?@ 75? ?3T@1?e7H @? @? ?@ ?@e?@h?@f?7@(Y? ?N@R'=?C5? @? @? ?@ ??? ?@e?@h?@f?@(Y @?N@@0Y? 3L @? ?@ ?@e?@h?@f?@H? @??@M? N1 @? ?@ ? ?@e?@h?3L?eJ@ ?3L? @? ?@ ?@e?@h?N1??W&5 ?N1? @? ?@ ??? ?@e?@he@L?7@H 3L @? ?@ ?@e?@he@)X@5? N1 @? ?@ ? ?@e?@he?S@0Y? ?3L? @? ?@ ?@?@[email protected]? ?V/X @? ?@ ??? ?@?@?@he7H V/K? @? ?@ ?@?@?@h?J5? ?V46K?h?W&?@? ?@ ? ?@[email protected]? ?I4@@6K?f?7@X@? ?@ ?@@R@Hh7H ?I4@@6K?C(R@@? ?@ ??? ?@@?@?g?J5? ?I4@0Y?3@? ?@ ?@@?@?gW.Y? ?N@@@@@@@?hf?@ ? ?@@?@?f?W.Y @?f@?hf?@ ?W-Xf?@5?@?f?7H? @?f3=hf?@ ??? ?7R1f?@Y?g?@ @?fV@@@@6K?g?@ ?@?@f?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ ? ?3T5hf?I@MfI@M?fI@M?fI@M?fI@M?e?I@Mf?I@Mf?I@Mf?I@MfI@M?fI@M?fI@M?e?I@Mf?I@M ?V+Y ??? ? ?W-Xe?W-X?W-X ?W-Xe?W-X?W-X ?@fW-X?W-X?he?@@@@@hfW.f?O@?f/X ?7R1e?7R1?7R1 ?.R/e?7R1?7R1 J@f7R1?7R1?hf?@?@?@@@@6?)X?e7H?@@@@@@?@@@?N1 ??? ?@?@e?@?@?@?@ ?@?@?@?@ 7@L?e@?@?@?@?hf?@?@?@?@?@@@)?e@??@?@?@@?@?@??@ ?3T5e?3T5?3T5 ?@K?e?3T5?3T5 3@(?e3T5?3T5?hf?@?@?@?@?@@Xf@??@?@?@@?@?@??@ ? ?V+Y?@?V+Y?V+Y ?@@@?@?V+Y?V+Y V+Y?@?V+Y?V+Y?hf?@?@?@?@?(R/f3L?@?@?@@?@?@?J5 V/hf.Y ??? ?? ?W-Xe@??@ ?7R1e@??@ ?@?@e@??@ J@@@L?3=C5 .M?I/?V40Y
Fig. 14. An example of a fast and high resolution separation on SOURCE. (Work by Pharmacia Biotech, Uppsala, Sweden.)
SOURCE is based on monodisperse, hydrophilized and rigid polystyrene/divinyl benzene beads with controlled pore structure. The beads have a uniform size distribution, see Figure 15, and allow perfect packing of stable chromatography beds. Monodispersity together with the absence of broken beads and fine particles give low operating back-pressures.
Fig. 15. Light microscope photograph of SOURCE. Note the uniform size distribution and absence of broken beads and bead fragments.
34
Table 6. Characteristics of SOURCE 15Q and 15S, and SOURCE 30Q and 30S. Properties
SOURCE 15Q
SOURCE 30Q
SOURCE 15S
Type of gel
strong anion exchangers
strong cation exchangers
Charged group
-O-CH2-CHOH-CH2-O-CH2-CHOH-CH2-N+(CH3)3
-O-CH2-CHOH-CH2-O-CH -CHOH-CH SO -
Matrix
Polystyrene/divinyl benzene
Bead form
Rigid, spherical, porous monodisperse
Mean particle size (µm)
15
2
SOURCE 30S
2
30
15
30
N.D.
80
80
3
Dynamic binding capacity* (mg/ml gel) Lysozyme (MW 14 500) N.D. 45
45
N.D.
N.D.
MW range (proteins)
BSA (MW 67 000)
up to 107
up to 107
up to 107
up to 107
working pH range**
2-12
2-12
2-12
2-12
pH stability*** long term
2-12
2-12
2-12
2-12
short term
1-14
1-14
1-14
1-14
Maximum flow rate (cm/h)
1800
2000
1800
20002
Recommended working flow rate range (cm/h)
30-600
300-1000
30-600
300-1000
4-40
4-40
4-40
1
Operating temperature (°C) 4-40
2
1
N.D. = Not determined Solvent restrictions: SOURCE is stable in alcohol/water solutions (C1-C4). 100% dimethyl sulphoxide, dimethylformamide, and formic acid can change the separation properties of the gel. Avoid oxidizing and reactive reagents. Detergents can be used if they are non-ionic or have the same charge as the gel. * Determined by frontal analysis at a flow rate of 300 cm/h, using a 5.0 mg/ml solution of protein in 20 mM sodium phosphate buffer, pH 6.8 (lysozyme) and 20 mM BIS TRIS PROPANE buffer, pH 7.0 (BSA). ** working pH range refers to the pH range over which the ion exchange groups remain charged and maintain consistently high capacity. *** pH stability, long term refers to the pH interval where the gel is stable over a long period of time without adverse effects on its subsequent chromatographic performance. pH stability, short term refers to the pH interval for regeneration and cleaning procedures. 1) Maximum flow rate to be applied, will depend on the pressure specification of the chromatographic system used. A linear flow rate of 1800 cm/h will give a pressure drop of approximately 10 MPa at a bed height of 3 cm. 2) Maximum flow rate to be applied, will depend on the pressure specification of the chromatographic system used. A linear flow rate of 2000 cm/h will give a pressure drop of approximately 10 MPa at a bed height of 10 cm.
A uniquely wide pore size distribution and a large specific surface area offer excellent resolution and capacity for a wide range of molecules; from small peptides and oligonucleotides up to large proteins. The performance is well maintained at high flow rates and high sample loads. This is illustrated in Figure 16 and 17, which show separations of model protein mixtures on SOURCE 30Q.
35
Column: Sample: Sample load: Eluent A: Eluent B: Flow rate: W-X?@?e@? 7R1?@?e@? ?J@?@?@?e@? ?7@@@@@?e@? ?@e?@@=?C5? ?@e?(R4@0Y?
Gradient:
SOURCE 30Q, 10 mm i.d. x 50 mm (4 ml) Mixture of lactoglobulin B and amyloglucosidase 1 mg/ml bed volume 20 mM BIS-TRIS PROPANE, pH 7.0 0.5 M sodium chloride, 20 mM BIS-TRIS PROPANE, pH 7.0 a) 4 ml/min (300 cm/h) b) 13 ml/min (1000 cm/h) 0-100% B, 20 column volumes W-X?@?e@? 7R1?@?e@? ?J@?@?@?e@? ?7@@@@@?e@? ?@e?@@=?C5? ?@e?(R4@0Y?
@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@? @? @? @? @? @? @? @? @? @? @? @L @? @? ?J@1 @? @? ?7Y@ @? @? ?@?@ @? @? ?@?@ @? @? ?@?@ @? @? ?@?@ @? @? ?@?@ @? @? ?@?@ @? @? ?@?@ @? @? ?@?@ @? @? ?@?@ @? @? ?@?@ @? @? @? ?@?@ @? @? @? ?@hf?@?@ ?J@L @? @? ?@hf?@?@ ?7R1 @? @? ?@hf?@?@ ?@?@ @? @? ?@hf?@?@ ?@?@ @? @? J@L?he?@?@ ?@?@ @? @? 7R1?he?@?@ ?@?@ @? @? @?@?he?@?@ J5?3L? @? @? @?@?he?@?3L? 7H?N1? @? @? @?@?he?@?N1? @?e@? @? @? @?@?he?@e@? @?e@? @? @? @?@?he?@e@? @?e@? @? @? @?@?he?@e@? @?e@? @? @? @?@?he?@e@? @?e@? @? @? @?@?he?@e@? @?e@? @? @? @?@?he?@e@? @?e@? @? @? @?@?he?@e@? @?e@? @? @? @?@?he?@e@? @?e@? @? @? @?@?he?@e@? @?e3L @? @? @?@?he?@e@? @?eN1 @? @? @?@?he?@e@? @?e?@ @? @? @?@?he?@e@? @?e?@ @? @? @?@?he?@e@? ?J5?e?@ @? @? @?@?he?@e@? ?7H?e?@ @? @? @?@?heJ5e@? ?@f?@ @? @? @?@?he7He@? ?@f?@ @? @? @?@?he@?e@? ?@f?@ @? @? @?@?he@?e@? ?@f?@ @? @? ?J5?@?he@?e@? ?@f?@ @? @? ?7H?@?he@?e@? ?@f?@ @? @? ?@e@?he@?e@? ?@f?@ @? @? ?@e@?he@?e@? ?@f?@ @? @? ?@e@?he@?e@? ?@f?@ @? @? ?@e@?he@?e@? ?@f?@ @? @? ?@e@?he@?e@? ?@f?@ @? @? ?@e@?he@?e@? ?@f?@ @? @? ?@e3Lhe@?e@? ?@f?3L? @? @? ?@eN1he@?e@? ?@f?N1? @? @? ?@e?@he@?e@? ?@g@? @? @? ?@e?@he@?e@? ?@g@? @? @? ?@e?@he@?e3L ?@g@? @? @? ?@e?@he@?eN1 ?@g@? @? @? ?@e?@he@?e?@ ?@g@? @? @? ?@e?@he@?e?@ ?@g@? @? @? ?@e?@he@?e?@ ?@g@? @? @? ?@e?@he@?e?@ ?@g@? @? @? ?@e?@he@?e?@ J5g@? @? @? ?@e?@he@?e?@ 7Hg@? @? @? ?@e?@he@?e?@ @?g@? @? @? J5e?@he@?e?@ @?g@? @? @? 7He?@he@?e?@ @?g@? @? @? @?e?@he@?e?@ @?g@? @? @? @?e?@he@?e?@ @?g@? @? @? @?e?@he@?e?@ @?g@? @? @? )Xe@?e?@he@?e?@ @?g3L @? @? @1?J5?e?@he@?e?@ @?gN1 @? @? @@T&H?e?@he@?e?@ @?g?@ @? @? ?J(R@@f?@he@?e?@ @?g?@ @? @? ?7H?3@f?@he@?e?@ @?g?@ @? @? ?@eV'f?@he@?e?@ @?g?@ @? @? ?@h?@he@?e?@ @?g?@ @? @? ?@h?@he@?e?3L? @?g?@ @? @? ?@h?@he@?e?N1? @?g?@ @? @? ?@h?@h?J5?f@? @?g?@ @? @? ?@h?@h?7H?f@? @?g?@ @? @? ?@h?@h?@g@?e?W2@he?J5?g?@ @? @? ?@h?@h?@g@?e?7R'L?h?7H?g?@ @? @? ?@h?3L?g?@g@@6KC5?N1?h?@h?@ @? @? ?@h?N1?g?@hI40Ye@?h?@h?3L? @? @? ?@he@?g?@ 3Lh?@h?N1? @? @? ?@he@?gJ5 N1h?@he@? @? @? ?@he@?g7H ?3L?g?@he@? @? @? J5he3Lf?J5? ?N1?gJ5he@? @? @? 7HheN1f?7H? 3Lg7Hhe@? @? @? @?he?3=?eJ5 N1f?J5?he@? @? @? ?J5?he?V46KO&H ?3=?eW.Y?he3= @? @? ?7H?hf?I4@@? ?V46KO.YhfV46K @? @? ?@ ?I40Y? I46K @? @? J5 I46K @? @??)X? ?O&H I46K @? @??@)K O2@@@? I4@6K? @? @??@V@@@@@@@@@@@@@@@@@@@@@@@@@@@@Y ?V@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@? @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@? ?@ ?@ ?@ ?@ ?@ ?@ W&?W26X? ?W26KO26X?hf?@@@@@@? W-K?f@?f?/X? *@?7
?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@L? ?@ ?@ J@L? J@1? ?@ ?@ 7R1? 7Y@? ?@ ?@ @?@? @?@? ?@ ?@ @?@? @?@? ?@ ?@ @?@? @?@? ?@ ?@ @?@? @?3L ?@ ?@ @?@? ?J5?N1 ?@ ?@ @?@? ?7H??@ ?@ ?@ @?@? ?@e?@ ?@ ?@ @?@? ?@e?@ ?@ ?@ ?J5?@? ?@e?@ ?@ ?@ ?7H?3L ?@e?@ ?@ ?@ ?@eN1 ?@e?@ ?@ ?@ ?@e?@ ?@e?@ ?@ ?@ ?@e?@ ?@e?3L? ?@ ?@ ?@e?@ ?@e?N1? ?@ ?@ ?@e?@ J5f@? ?@ ?@ ?@e?@ 7Hf@? ?@ ?@ ?@e?@ @?f@? ?@ ?@ @?he?@e?@ @?f@? ?@ ?@ @Lhe?@e?@ @?f@? ?@ ?@ ?J@1he?@e?@ @?f@? ?@ ?@ ?7Y@he?@e?@ @?f@? ?@ ?@ ?@?@heJ5e?@ @?f@? ?@ ?@ ?@?@he7He?@ @?f@? ?@ ?@ ?@?@he@?e?3L? @?f3L ?@ ?@ J5?@he@?e?N1? @?fN1 ?@ ?@ 7H?@he@?f@? @?f?@ ?@ ?@ @??@he@?f@? @?f?@ ?@ ?@ @??@he@?f@? @?f?@ ?@ ?@ @??@he@?f@? @?f?@ ?@ ?@ @??3L?h@?f@? ?J5?f?@ ?@ ?@ @??N1?h@?f@? ?7H?f?@ ?@ ?@ ?J5?e@?h@?f@? ?@g?@ ?@ ?@ ?@e?7H?e@?h@?f@? ?@g?@ ?@ ?@ ?@L??@f@?g?J5?f@? ?@g?@ ?@ ?@ ?@1??@f@?g?7H?f@? ?@g?3L? ?@ ?@ J@@L?@f@?g?@g@? ?@g?N1? ?@ ?@ 7
Fig. 16. The influence of increasing flow rate on resolution. (Work by Pharmacia Biotech, Uppsala, Sweden.)
Column: Sample: Sample load:
?W-Xe?@e?@ ?7R1e?@e?@ ?@?@e?@e?@ J@@@L??@e?@ 7
Eluent A: Eluent B: Flow rate: Gradient:
SOURCE 30S, 5 mm i.d. x 50 mm (1 ml) Mixture of chymotypsinogen, cytochrome C and lysozyme a)1 mg b) 10 mg 20 mM sodium phosphate, pH 6.8 0.5 M sodium chloride, 20 mM sodium phosphate, pH 6.8 1 ml/min (300 cm/h) 0-100% B, 20 column volumes
W26X 7
W&eW26X *@e7
? ? ? ? ? ? ? ? ? ? ? ? ? ? @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@g? @? ?@ ?@g? @? ?@ ?@g? @? ?@L? ?@g? @? ?@1? ?@g? @? ?@@? ?@g? @? ?@@? ?@g? @? ?@@? ?@g? @? J@@L ?@g? @? 7
?W-Xe?@e?@ ?7R1e?@e?@ ?@?@e?@e?@ J@@@L??@e?@ 7
?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ J@ ?@ ?@ 7@ ?@ ?@ @@ ?@ ?@ @@L? ?@ ?@ @V1? @? ?@ ?@ @?@? @? ?@ ?@ ?@ ?J5?@? ?J@? ?@ ?@ ?@ ?7H?@? ?7@? ?@ ?@ ?@L?hf?@e@? ?@@L ?@ ?@ ?@1?hf?@e@? ?@V1 ?@ ?@ ?@@?hf?@e@? J5?@ ?@ ?@ J@@?hf?@e@? 7H?@ ?@ ?@ 7Y@?hf?@e@? @??@ ?@ ?@ @?3Lhf?@e@? @??@ ?@ ?@ @?N1hf?@e@? @??@ ?@ ?@ @??@hf?@e@? @??@ ?@ ?@ @??@hf?@e@? @??@ ?@ ?@ @??@hf?@e@? @??@ ?@ ?@ @??@hf?@e@? ?J5??@ ?@ ?@ @??@hf?@e@? ?7H??@ ?@ ?@ @??@hf?@e@? ?@e?@ ?@ ?@ @??@hf?@e@? ?@e?@ ?@ ?@ ?J5??@hf?@e@? ?@e?@ ?@ ?@ ?7H??@hfJ5e3L ?@e?@ ?@ ?@ ?@e?@hf7HeN1 ?@e?3L? ?@ ?@ ?@e?@hf@?e?@ ?@e?N1? ?@ ?@ ?@e?@hf@?e?@ ?@f@? ?@ ?@ ?@e?3L?he@?e?@ ?@f@? ?@ ?@ ?@e?N1?he@?e?@ ?@f@? ?@ ?@ ?@f@?he@?e?@ ?@f@? ?@ ?@ ?@f@?he@?e?@ J5f@? ?@ ?@ ?@f@?he@?e?@ 7Hf@? ?@ ?@ J5f@?he@?e?@ @?f@? ?@ ?@ 7Hf@?he@?e?@ @?f@? ?@ ?@ @?f@?he@?e?@ @?f@? ?@ ?@ @?f@?he@?e?@ @?f@? ?@ ?@ @?f@?he@?e?@ @?f@? ?@ ?@ @?f@?he@?e?@hf?J5?f@? ?@ ?@ @?f@?he@?e?@hf?7H?f@? ?@ ?@ @?f@?he@?e?@hf?@g@? ?@ ?@ ?J5?f@?he@?e?@hf?@g@? ?@ ?@ ?7H?f@?he@?e?@hfJ5g@? ?@ ?@ ?@g@?h?J5?e?3L?h?O&Hg3L ?@ ?@ ?@g3Lh?7H?e?N1?g?@@@@?gN1 ?@ ?@ ?@gN1h?@g@?gJ(M?h?@ ?@ ?@ J5g?@h?@g@?g7Hhe?@ ?@ ?@ 7Hg?@h?@g@?f?J5?he?@ ?@ ?@ @?g?@hC5g@?f?7H?he?@ ?@ ?@ ?J5?g?@gW20Yg3LfJ5hf?@ ?@ ?@ ?7H?g?3L?e?W.MhN1f7Hhf?@ ?@ ?@ J5h?N1?eW.Y?h?3L??J5?hf?3L? ?@ ?@f@? ?W.Yhe3=?O.Yhe?N1?W&H?hf?N1? ?@ ?@e?J@L ?7H?heN@@0Y?hf3T&@ @? ?@ ?@e?7R1 J5hf?@M? V+M? @? ?@ ?@eJ5?@ ?O.Y 3L ?@ ?@e7H?@ ?O20Y? N)K? ?@ ?@e@??@@@6K ?O2@Y? ?@@6K? ?@ ?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ ?@ ?@ ?@ ?@ ?@ ?@ ?W2@6KO26Xg?@@@@@@? W.h@?f?/X? ?.M?B@@
Fig. 17. The influence of increasing sample load on resolution. (Work by Pharmacia Biotech, Uppsala, Sweden.)
SOURCE 15 and SOURCE 30 ion exchangers are designed for research and industrial applications, with emphasis on high performance, high capacity, high reproducibility, and easy scaleability. In comparison with media based on SOURCE 15 matrix, SOURCE 30 gives slightly less resolution (lower efficiency) but at much lower back-pressure. This makes SOURCE 30 ideal for purifications with more complex samples and larger volumes. Using SOURCE 30, a high degree of purification can be obtained with
36
high productivity. SOURCE 15 matrices are ideal for purification when very high resolution (efficiency) is required. SOURCE 15 media are available in pre-packed RESOURCE columns. These columns are convenient for lab research applications and method development.
Properties Chemical stability The hydrophilized polymeric matrix has high chemical stability and can be used over a wide pH range allowing flexibility in the choice of conditions for separation. The wide pH stability range, 1-14, allows cleaning and sanitization with harsh agents like 1 M NaOH. Additionally, in applications such as the purification of synthetic oligonucleotides, the wide pH stability also allows use of a high pH buffer to prevent aggregation, see Figure 18. Column:
Sample: Sequence: Eluent A: Eluent B: Flow rate: Gradient: System:
?W-X?@e?@ ?7R1?@e?@ J@?@?@e?@ 7@@@@@e?@ @?e@@=?C5 @?e(R4@0Y ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@g?@ /X @? ?@g?@ )X N1 ?@g?@ @1 W2@6X??3L?hf?W.? ?@g?@ ?J@@ .M?B1??N1?hf?.Y? ?@g?@ ?7R'L? @?e@? ?@g?@ ?@?N1? W2@@@?e@? ?@g?@ ?@e@L 7<e@?e@?hfW. ?@g?@ C5e31 3=?C@Le@?hf.Y ?@g?@ ?@(YeN@ V4@0R/?J5? ?@g?@ ?@H?e?@ ?7H? ?@g?@ ?@f?@ J5 @? ?@g?@ ?@f?@ .Y @? ?@g?@ J5f?3L? ?@g?@ 7Hf?N1? ?@gJ@ @?g@? ?W.? ?@g7@ @?g@? ?.Y? ?@g@@ @?g@? W26Xe?W26X?f?@g@@ @?g@? 7)X??@@@U? ?@f?@e@? @?hf@? J@@?,??@?V1? ?@f?@e@? @?hf@? .MI+Y??@@@@? ?@f?@e@? @?hf@? @? ?@f?@e@? @?hf3L @? ?@f?@e@? @?hfN1 ?@f?@e@? @?hf?@ ?@f?@e@? @?hf?@ ?W.? W2@6T26X ?@f?@e@? @?hf?@ ?.Y? 7@@>@
W26KO26Xe?W26X?hf@6KO26X?eW26Xf?W2@6Xf?@ @?f?W-Kf?@?/X? .MB@@
a) RESOURCE Q 1 ml (6.4 mm diam x 30 mm bed height) b) SOURCE 15Q in BioProcess Special, 240 ml (100 mm diam. x 30 mm bed height) a) 800 µmoles synthesis of 19 mer DNA oligo, load 5 mg b) As a), load 820 mg ATACCGATTAAGCAAGTTT 10 mM NaOH pH 12 A + 1.5 M NaCl a) 1.6 ml/min (300 cm/h), b) 385 ml/min (300 cm/h) 0.25-0.75 M NaCl in 30 column volumes a) FPLC, b) BioProcess 6 mm, controlled by UNICORN™ W-X?@?e@? 7R1?@?e@? ?J@?@?@?e@? ?7@@@@@?e@? ?@e?@@=?C5? ?@e?(R4@0Y? ?@ ?@ ?@ ?@ @?f/X ?@ @?fN1 ?@ @W26X??3L? ?@ @(MB1??N1? ?W26X? ?@ @He@?e@? ?7)X??@@@U? ?@e@?eJ@@?f?@ ?@e ?3L?hf?J5? J@@?,??@?V1? ?@e@?e7Y@Lf?@ J5e ?N1?hf?7H? .MI+Y??@@@@? ?3=C@Le@@@,e?@@@ 7He @?hf?@ ?V40R/e?I(Yf?@ @?e @?hf?@ ?@ @?e @?hf?@ ?@ ?J5?e @?hf?@ ?@ ?7H?e @?hf?@L? W2@6T26X ?@ ?@f @?hf?@1? 7@@>@@0Y @? @? ?@h?W.Ye?7H?he?I4@@@@@@@@@@@@@@@@@0M 3L @? [email protected]?eJ5 I40M N1 @?eW26KO2@( ?@h7He?W.Y ?3=? @?e.MB@@@@U ?@g?J5?eW&H? ?@@? ?V46K? @?f?@0MB1 ?@g?.Y?e75he@@e@@e@@e@@ ?I4@@?hf@@@?W.f?@ ?@he?J(Ye@@e@@ 7YfC5 ?@he?.Y? @@@@?@0Y ?@ ?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ ?@ @?e@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@? @? ?@ @?e @? ?@ @?e @? ?@ @?e @? ?W26X? W&?W26X?eW26XhfW26KO26Xe?W2@@? @6KO26X?eW26XgW2@6X?f@?hf?@gW-K?f@?/Xf ?7
Fig. 18. Purification of 19 mer DNA oligonucleotide on RESOURCE Q 1 ml scaled up to BioProcess Special column 100 mm diameter. Separation optimized for sample load, yield and purity of product. (Work by Pharmacia Biotech, Uppsala, Sweden.)
37
Flow rate SOURCE media are based on highly rigid beads which allows use at high flow rates. As a consequence of the monodisperse nature of the matrix, these high flow rates do not result in high back-pressures. The back-pressure from SOURCE media is much lower than for other media of the same particle size range. The low back-pressure offers the advantage of being able to run at very high flow rates on medium pressure equipment, as well as with acceptable flow rates on low pressure equipment. As an example, when a RESOURCE 1 ml column is used at the maximum flow rate of FPLC or HPLC systems (about 10 ml/min, 1800 cm/h) separations are completed in less then 3 minutes, see Figure 19. At flow rates of 1 ml/min (180 cm/h) the extremely low back-pressures (typically around 0.1 MPa, 1 bar, 15 psi) allow high resolution separations in about 20 minutes with systems based only on a peristaltic pump, see Figure 20. Column:
RESOURCE Q 1ml 6.40 mm diam x 30 mm bed height Pancreatin 5 mg/ml, 0.2 ml 9.6 ml/min (1800 cm/h) 20 mM BIS TRIS PROPANE, pH 7.5 Buffer A + 0.5 ml NaCl 0 - 80% buffer B, 20 ml (20 col. vol.) FPLC
? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? @?e?@@@@? ?@ ? @?e?@e@? ?@ ? @?e?@e@? ?@ ? @?e?@e@? ?@ ? @?e?@e@? ?@ ? @?e?@e@? ?@ ? @?e?@e@? ?@ ? @?e?@e@? ?@ ? @?e?@e@? ?@ ? @?e?@e@? ?@ ? @?e?@e@? ?@ ? @?e?@e@? ?@ ? @?e?@e@? ?@ ? @?e?@e@? ?@ ? @?e?@e@? ?@ ? @?e?@e@? ?@ ? @?e?@e@? ?@ ? @?e?@e@? ?@ ? @?e?@e@? ?@ ? @?e?@e@? ?@ ? @?e?@e@? ?@ ? @?e?@e@? ?@ ? @?e?@e@? ?@ ? @?e?@e@? ?@ ? @?e?@e@? J@ ? @?e?@e@? 7@ ? @?e?@e@? @@ ? @?e?@e@? @@ ? @?e?@e@? @@ ? @?e?@e@? @@ ? @?e?@e@? @@ ? @?e?@e@? @@ ? @?e?@e@? @@ ? @?e?@e@? @@ ? @?e?@e@? @@ ? @?e?@e@? @@ ? @?e?@e@? @@ ? @?e?@e@? @@ ? @?e?@e@? @@ ? @?e?@e@? @@ ? @?e?@e@? @@ ? @?e?@e@? @@ ? @?e?@e@? @@ ? @?eJ5e@? @@ ? @?e7He@? @@ ? @?e@?e@? @@ ? @?e@?e@? @@ W2@6X?heO2@?h?W&??@@6X?e? @?e@?e@? @@ ?W-Xg7)Xe@??@@??@?@(Y@?@??@@?@@@@@?@?e@??@@@U?e? @?e@?e@? @@ ?J@@?,e3=?V@??@?3U?@?3=?@@?3Xe@?@?e@??@?V1?e? @?e@?e@? @@ ?.MI+YeV4@@@??@?V4@@?V4@@@?V4@?@?@?e@??@@@@?e? @?e@?e@? @@ ? @?e@?e@? @@ ? @?e@?e@? @@ ? @?e@?e@? @@ W&?W26X?W26X ? W26XfW&g@?e@?e@? @@ *@?75f@?e?J5?f?@g@@ ?@ @? ? @??@g?@ @?e@@e?@e?W.R@Hf@?e?7H?f?@g@@ ?@ @? ? @??@g?@ @?e@@[email protected]?@?f@?e?@g?@g@@L? ?@ @? ? @??@g?@ ?J5?e@@e?@e7He@?f@?e?@g?@f?J(R1? ?@ @? ? @??@g?@ ?7H?e@@e?@?J5?e@?f@?e?@g?@f?7H?@? ?@ @? ? @??@g?@ ?@f@@[email protected]??J5?f@?e?@g?@f?@e@? ?@ @? ? @??@g?@ ?@f@@e?@(Ye?7H?f@?e?@g?3L?e?@e@? ?@ @? ? @??@g?3L? ?@f3@eJ@H?e?@g@?e?@g?N1?e?@e@? ?@ @? ? @??@g?N1? ?@fN@?W&@f?@g@?e?@h@?e?@e@? ?@ @? ? @??@h@? ?@[email protected]@fJ5g@?e?@h@?eJ5e3L ?@ @? ? @??@h@? ?@f?@(Y?@f7Hg@?e?@h3Le7HeN1 ?@ @? ? @??@h3L ?@fJ(Y??@f@?g@?eJ5hN1e@?e?@ ?@ @? ? @??@hN1 J5f7He?@e?J5?g@?e7Hh?@e@?e?@ ?@ @? ? @??@h?@ 7He?J5?e?@e?7H?g@?e@?h?3=C5?e?@ ?@ @? ? @??@h?@ @?eW.Y?e?@e?@h@?e@?h?V40Y?e?@ ?@ @? ? @??@h?3L? @??W.Yf?@e?@h@?e@? ?@ ?@ @? ? @?J@h?N1? @?W.Y?f?@e?@h@?e@? ?@ )Xhe?@ @? ? @?7@he@? @W.Yg?@eJ5h@?e@? ?3L? ?J@1he?@ @? ? @?@@he@? @(Y?g?@e7Hh@??J5? ?N1? ?7Y@he?@ @? ? @?@@he3Lhf?J@Hh?3L?@?h3L?7H? @? ?@?3L?h?@ @? ? @?@@heN1hf?75?h?N1?@?hV/T5 @? ?@?N1?h?@ @? ? @?@@he?@hf?@H?he@?@?h?V+Y 3@6X ?@e@?h?@ @? ? @?@@he?@he?@@@hf@?@? V+R1 J5e@?h?@ @? ? @?@@he?3L?hJ@@@hf@?@? ?3L? 7He3Lh?@ @? ? @@@@he?N1?g?W&R'@hf@W5? ?N1? @?eN1h?@ @? ? @@@@hf@?gW&5?V'hf@@H? @? @?e?@h?@ @? ? @@@@hf3Lf?W&@H? @@ @? @?e?@h?@ @? ? @@@@hfN1fW&>5 @@ 3L @?e?3L?g?@ @? ? @@?@hf?3L??W.R@H @@ N1 ?J5?e?N1?g?@ @? ? @@?@hf?N1??7HJ5? @5 ?3L? ?7H?f@?g?@ @? ? @@?@ 3=C5?7H? (Y ?V/X ?@g3Lg?@ @? ? @@?@ N@@HJ5 V/K?hf?@gN1g?@ @? ? @@?@ J@@T.Y ?V4@@6K?h?@g?3L?f?@ @? ? @@?@hf?W.MI+Y? ?I4@6KgJ5g?V/Kf?@ @? ? @@[email protected]? I4@@6K?O.YhV46Ke?@ @? ? @@?@he?W.Y I4@0Y?heI46K?@ @? ? @[email protected]? I4@@ @? ? @H?@h?W.Y ?@@@@6X? @? ? @??@h?7H? ?@e?B1? @? ? W26Xg@?heJ5 ?@f@? @? ? 7
Sample: Flow rate: Buffer A: Buffer B: Gradient: System:
Fig. 19. Separation of pancreatin (Sigma) on RESOURCE Q 1 ml at 9.6 ml/min (1800 cm/h). System: FPLC. (Work by Pharmacia Biotech, Uppsala, Sweden.)
38
? ? ? ?W-X ? ?7R1 ? J@?@ ? 7@@@@6KO-X?W-K ? @?e@V4@?,?7R@@@@@@@@@ ? @?e@??S@U?@?@@?@@?@?@ ? @?O&?,?3T@@?@@?@?@ ? @@0R+Y?V+R'?@@?@?@ ? ? ? ? ? @? ? W26Xe?W26KO2@(?g@? ? 7)Xe@??@@??@?@(Y@?@??@@?@@@@@?@?e@??@@@U?e? @?e?@f@? @??@ ?J@@?,e3=?V@??@?3U?@?3=?@@?3Xe@?@?e@??@?V1?e? @?e?@f@? @??@ ?.MI+YeV4@@@??@?V4@@?V4@@@?V4@?@?@?e@??@@@@?e? @?e?@f@? @??@ @? ? @?e?@f@? @??@ @? ? @?e?@f@? @??@ @? ? @?e?@f@? @??@ @? ? @?e?@f@? @??@ @? ? @?e?@f@? @??@ @? ? @?e?@f@? @??@ @? @? ?W&?W26X?W26X? ? @?e?@f@? @??@ @? @? ?@f?*@?75?heN)X?f@?h?@he@?e?@?7H?e?@ ?@ ? @?e@?g?@ @(Y?he?@1?f@?h?@he@?e?@?@f?@ ?@ ? @?e@?g?3L? @Hhf?3@?f@?h?3L?h@?e?@?@f?@ ?@ ? @?e@?g?N1? @?hf?N@?e?J5?h?N1?h@?e?@?@f?@he@?he?@ ? @?e@?h@? @? 3Le?7H?he@?h@?e?@?@f?@he@?he?@ ? @?e@?h@? @? N1eJ5hf@?h@?e?3X@f?3L?h@?he?@ ? @?e@?h@?heW.e@? ?@e7Hhf@?h@?e?N@5f?N1?h@?he?@ ? @?e@?h@?h?W.Ye@? ?@?J5?hf@?h@?f@Hg@? ?@ ? @?e@?h3Lh?.Y?e@? ?3T&H?hf3Lh@?f@?g@? ?@ ? @?e@?hN1hf?J5? ?N@5 N1h@?hf@? ?@ ? @?e@?h?@hf?7H? (Y ?@h@?hf@? ?@ ? @?e@?h?3L?eW.gJ5 ?@h@?hf3Lh@?he?@ ? @?e@?h?N1??W.Yg7H ?@g?J5?hfN1h@?he?@ ? @?e@?he@??.Y?g@? ?3L?f?7H?hf?@h@?he?@ ? @?e@?he@?he@? ?N1?fJ5 ?@h@?he?@ ? @?e@?he3Lh?J5? @?f7H ?@ ?@ ? @?e@?heS1h?7H? 3T-K?C5? ?@ ?@ ? @?e@?h?W&@=?gJ5 V+R4@0Y? ?3L? ?@ ? @?e@?hW.MI46K?e?O.Y ?N1? ?@ ? @?e@?h.Ye?I46KO20Y? @?g@?he?@ ? @?e@? ?I40M? @?g@?he?@ ? @?e@? 3Lg@?he?@ ? @?e@? N1g@?he?@ ? @?e@?e?W.? ?3L? ?@ ? @?e@?eW.Y? ?V/X ?@ ? @?e@?e.Y N1 ?@ ? ?W26X?g@?e@? ?3=? ?@ ? ?7
Column: Sample: Flow rate: Buffer A: Buffer B: Gradient: System:
RESOURCE S 1ml 6.40 mm diam x 30 mm bed height Snake venom 4 mg/ml, 0.1 ml 1 ml/min (180 cm/h) 20 mM sodium phosphate, pH 6.8 Buffer A + 0.4 ml NaCl 0 - 100% buffer B, 20 ml (20 col. vol.) GradiFrac with Peristaltic Pump P-1
Fig. 20. Separation of snake venom (Sigma) on RESOURCE S 1 ml at 1.0 ml/min (180 cm/h). System: GradiFrac equipped with Peristaltic Pump P-1. Although high performance separations with RESOURCE Q and S do not put special demands on the pump, resolution obtained on the column can be lost through mixing in dead spaces. Low dead volumes, accurate gradient generation, and a good detector and fraction collector are also essential for good results. (Work by Pharmacia Biotech, Uppsala, Sweden.) ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? @? ? @6KO@?@? ? @V@@@?@? ? @W(Y@@@? ?@he? @(Y?@?@? ?@6KO@?@he? (Y ?@V@@@?@he? ?@W(Y@@@he? ?@(Y?@?@he? @? ?W.?f?@ ?(Y?hg? @? W.Y?f?@ ? @?W-X??W-XeW.?@e?W-X?@f@? ?W.Yg?@f@??@eW-X? ? @?7R1??7R1e7H?@e?7R1?@f@? W.Y?g?@f@?J@e7R1? ?@?W-XeW.?@e?W-X?@hf ?@@@6KfO@e@KfO@e?O@K ? @?@?@??@?@e@??@e?@?@?@f@? ?W.Yh?@f@?7@L?@?@? J@?7R1e7HJ@e?7R1?@hf ?@?W@@@@?@@@e@@@?@@@@?@@@@@@??@@? ?@@6T.eW-X?? @?3T5?J@T5e@??@eJ@T5?@e@@@? W.Y?h?@@@e@?3@,?3T5? 7@?@?@e@?7@L??@?@?@hf ?@@@U@@@?@?@e@?@?@@@@?@?@@?@??@H? ?@X;@Ue7R1?? @[email protected]+Y?@f@? ?W.Yhe?@f@?V+Y?V+Y? 3@@@T5e@?3@(?J@T5?@hf ?@?V@@e?@?@e@?@?@??@?@?@@?@??@L? ?@)X@)X?@?@?? V/h?@f@? W.Y?he?@ V+MI+Ye3LV+Y?.R+Y?@e?@@?g ?@@@@@@@?@@@e@?@?@@@@?@@@5?@??@@? ?@@?eS@@>,?3T5?? @? ?W.Yhf?@ V/h?@e?N@?g ?@@0Y? ?@H?e.MI+Y?V+Y?? @? W.Y?hf?@ @?g ?@hg? @? ?W.Y ?@ @?g ?@hg? @? W.Y? ?@ @?e?@@@ @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@? ?@hg? @? ?W.Y ?@ @?e?@e @? ?@hg? @? W.Y? ?@ @?e?@e ?@?@@@hf@? ?@hg? @? ?W.Y ?@f@?W-X?@@@? @?e?@e ?@?@X?eW.?@@@@@e@? ?@hg? ?@@@e@@@??W-T-Xe@@@@@@?@f@? W.Y? ?@f@?7R1?@X @?e?@e@@@@@@@@@@@@@@@@@@@@@@f?@eS,e3L?@?@?@e@? ?@?@)Xe7H?@?@?@e@? ?@hg? I@e@Xe?7R@R1e?I'@X??@f@? ?W.Y ?@f@?@?@?@)X? @?e?@e J@hg? @)X??@?@?@fV4)X?@f@? W.Y? ?@f@?3T5??S,? @?e?@e [email protected]/?@?@?@e@? ?W&@hg? @?e?S,??@?3T@L??@eS,?@e@@@? ?O.Y ?@@@e@?V+Y??.Y? @?e?@e @? ?7Y@hg? @?@??.Y??3LV+R/[email protected]?@f@? ?W20Y? ?@ @?e?@e @? J5?@hg? ?V/?he?@f@? W.M? ?@ W-T2@@eW-T-X??@@@?@f@?e?@e @? 7H?@f@?W-X?W-X?? @? ?W.Y ?@ *?@@X?e7R'?,??@X??@f@?e?@e ?@?W-Xhf@?hf?J5??@e?J@?*>1?7R1?? @? W.Y? ?@ V'>@)Xe@?V'U??@)X?@e?J@?e?@e ?@?7R1eW.?@@@@@e@?hf?7H??@L??7@LV'@?@?@?? @? ?W.Y ?@ ?S(MS,e@??S,?eS,?@e?@@?e?@e@@@@@@@@f@@@@@@@@g?@?3T5e3L?@?@?@e@?he?W.Ye?@H??V+Y?.Y?V+Y?? ?@?@?@e7H?@?@?@e@?hfJ5e?@@??3@(?S5?3T5?? @? W.Y? ?@ ?.Y?.Ye3L?.Y?@?.Y?@e?N@?e?@e @? ?W.Y ?@ V/h?@f@?e?@e ?@?V+YeV/?@?@?@e@?he?7H?e?@hg? @? W.Y? ?@ @?e?@e @?heJ5f?@hg? @? ?W.Y ?@ @?e?@e @?he7Hf?@hg? ?@@@eW-X??W-T-Xe@@6T-X?@f@? W.Y? ?@e?@@6T-X? @?e?@e @?h?J5?f?@hg? ?@X?e7R1??7R@R1e@X;@R1?@f@? ?W.Y ?@fI'@R1? @?e?@e ?@@@hf@?h?7H?f?@hg? ?@)Xe@?@??@?@?@e@)X@?@?@f@? W.Y? ?@f?N@?@? @?e?@e ?@X?eW.?@@@@@e@?hJ5g?@hg? S)X?3T5??@?3T@L??S@@T5?@e@@@? ?W.Y ?@@@e@?3T5? @?e?@e @@e@@e@@e@@e@@e@?g?@)Xe7H?@?@?@e@?g?W.Yg?@hg? .R/?V+Y??3LV+R/??.MI+Y?@f@? W.Y? ?@f@?V+Y? @?e?@e S,e3L?@?@?@e@?g?7H?g?@hg? ?V/?he?@f@? ?W.Y ?@ @?e?@e .YeV/?@?@?@e@?gJ5h?@hg? @? W.Y? ?@ @?e?@e @?g7Hh?@hg? @? ?W.Y ?@ @?e?@e @?f?J5?h?@hg? @? W.Y? ?@ W-KO-XeW-T-X??W-X?@f@?e?@e @?fW.Y?h?@hg? @? ?W.Y ?@ *?@@R1e7R'?,??7R1?@f@?e?@e @?f7Hhe?@f@?W-X?W-X?? @? W.Y? ?@ V'U@?@e@?V'U??@?@?@e?J@?e?@e ?W-Xhf@?e?J5?he?@L??J@?.R/?7R1?? @? ?W.Y ?@ ?S@@T5e@??S,?J@T5?@e?@@?e?@e@@e@@g@@e@@he?*?,eW.?@@@@@e@?e?7H?he?@@??7@?f@?@?? ?W-Xe@@@??W-T-XeW-T2@@?@f@? W.Y? ?@e?W-T2@@? ?.MI+Ye3L?.Y?.R+Y?@e?N@?e?@e ?V'Ue7H?@?@?@e@?eJ5hf?@H??3@@6Ke3T5?? ?.R/e@[email protected]'@X??@f@?hf?W.Y ?@e?*?@@X V/h?@f@?e?@e S,e3L?@?@?@e@?e7Hhf?@e?V+?4@@?V+Y?? @)X??@?@?@fV4)X?@f@?hfW.Y? ?@e?V'>@)X? @?e?@e .YeV/?@?@?@e@??J5?hf?@hg? ?@K?e?S,??@?3T@L?@KeS,?@e@@@?hf7H ?@@@eS(MS,? @?e?@e @?W.Y?hf?@hg? ?@@@@??.Y??3LV+R/?@@@?.Y?@f@?he?J5? [email protected]?.Y? @?e?@@@ @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?7H ?@hg? ?V/?he?@f@?heW.Y? ?@ @?g ?J5? ?@hg? @?h?W.Y ?@ @?g ?7H? ?@hg? @?hW.Y? ?@ @?g J5 ?@hg? @?g?W.Y ?@ @?g 7H ?@hg? @?gW.Y? ?@ @?g ?J5? ?@hg? @?f?W.Y ?@ @?g W.Y? ?@hg? @?fW.Y? ?@ @?g 7H ?@hg? @?e?W.Y ?@ @?g ?J5? ?@hg? @?eW.Y? ?@ W-T2@@eW-T-X??@@@?@f@?g ?7H? ?@e?W-T2@@?W-X?? @??O.Y ?@ .R'@X?e7R+R/??@X??@f@?g J5 ?@e?*?@@Xe7R1?? @W20Y? ?@ V4)Xe@?f?@)X?@e?J@?g 7H ?@L??V'>@)X?@?@?? @@Y? ?@ @KeS,e@?@KfS,?@e?@@?g ?J5? ?@@?eS(MS,?3T5?? @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ @@@?.Ye3T@@@?@?.Y?@e?N@?g W.Y? W.?@H?e.Y?.Y?V+Y?? @?f?@f?@g@?f@?f?@f?@g@?f@?f?@ V+M?g?@f@?g 7H ?W.Y?@hg? @?fJ5f?@f?J5?e?J@LfJ5fJ@L?e?J5?f@?f?3L? @?g ?J5? W.Y??@hg? ?W-Xf@?e?W&UfJ@L?e?7H?e?7@@e?W.Yf@@1?eW&U?e?J@Le?@?S)X @?g ?7H? ?W.Ye?@hg? ?7R1f@?e?.R/f*?,?eJ@f?@X?e?7U?f?I@?e*?,?e?*>1e?@?7R1 @?g J5 W.Y?e?@hg? ?@?@f@?heV'U?e7@L?e?@)Xe?@)XheS@U?e?V'@e?@?@?@ @?g ?W.Y .Yf?@hg? ?3T5f@?e?@K?f?S,?e3@,?fS,e?3>,f?@f*?,?fS5e?@?3T5 @?g ?7H? ?@hg? ?V+Yf@?e?@@@f?.Y?eV+Y?f.Ye?V+Yf?@fV+Y?f.Ye?@?V+Y @?g J5 ?@hg? @?g 7H ?@hg? @?g ?J5? ?W.?g?@hg? /X ?@@@@? W-K?eO@e@KeO@e?O@? @?g ?7H? W.Y?g?@hg? N1 ?@e@W-T2@@@@?@@?)X?@?)Xe7R@@@@@@e@@@@@@?@@@@? @?g J5 ?W.Yh?@hg? ?3L? ?@@@@@R@@@@@@?@HJ@1?@@@)e@?@?@?@@e@?@?@@?@?@@? W-KO-XeW-T-X??W-X?@f@?g ?W.Y W.Y?h?@e?W-KO-X?W-X?? ?O2@6X?N1? ?@e@@T@@@@@@?@?*U@?@@X?e@?@?@?@@e@?@?@@?@?@@? .R'@R1e7R+R/??7R1?@f@?g ?7H? ?W.Yhe?@e?.R4@?,?7R1?? @(M?I/e@? ?@e(R+R40?4@?@?V4@?(R/?e3T5?@?@@e@?@?@@?@?3@? N@?@e@?f?@?@?@e?J@?g J5 ?.Y?he?@L?fS@U?@?@?? ?@6Xg?)X? ?@K?e?)X? @Hg@? V+Y? ?V'? @KC@T5e@?@KeJ@T5?@e?@@?g 7H ?@@??@KO&?,?3T5?? ?@S,e@KO.?@1?e@6T26T-X?W&?W&?@?@@6T-X??@@@@??@)Xe@?2@ @?g@? @@0R+Ye3T@@@?.R+Y?@e?N@?g ?J5? ?@H??@@0R+Y?V+Y?? ?@@U?J@@@H?@@?e@V@@V@@)?*@W&@?@?@@V@@)??@@?@??@V1?J@@@H 3=eO.e@? V+M?g?@f@?g ?7H? ?@hg? ?@V1?*U@@L?@@Le@W@@?3X??N@(Y@@@?@@?3Xe?@@?@??@W5?*U@@? V4@@0Y?J5? @?g J5 ?W.? ?@hg? ?@@@?V40R/?(R/e@(R'?V/?e(Y?@?4@@@?V/e?@@?@??@0Y?V4@@? ?7H? @?g ?W.Y W.Y? ?@hg? (Y J5 @?g ?7H?hf?W.Y ?@hg? .Y @?g J5 W.Y? ?@hg? @?g 7Hhf?W.Y ?@hg? @?g ?J5?hf?.Y? ?@hg? @?g ?7H? ?@hg? @?g J5 ?@hg? @?g ?W.Y ?@hg? ?W-T2@@? @?g ?7H?he?W.? ?@hg? ?.R'@X ?@?@@@eW.?@e?@@@?@f@?g J5hfW.Y? ?@hg? ?V4)X? ?@?@X?e7H?@e?@X??@f@?g 7Hhe?W.Y ?@e?W-X?@eW-X?? ?@K??S,?e@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@? ?@?@)Xe@??@e?@)X?@f@?g ?J5?heW.Y? ?@e?.R/?@e7R1?? ?@@@?.Y?eN@ ?@g@?f?@f?@g@?f?@g@?f@?f?@g@? ?@eS,e@??@fS,?@e?J@?g ?7H?h?W.Y ?@L?f?@e@?@?? ?@ ?@g@?f?@f?@g@?f?@g@?f@?f?@g@? [email protected]?@e@?.Y?@e?@@?g J5he?.Y? ?@@??@K??@e3T5?? ?@ ?@g@?f?@f?@g@?f?@g@?f@?f?@g@? V/h?@e?N@?g ?W.Y ?@H??@@@?@eV+Y?? ?@ ?@g@?f?@f?@g@?f?@g@?f@?f?@?@f@? @?g ?7H? ?@hg? ?@ ?@g@?f?@f?@g@?f?@g@?f@?fJ@g@? @?g J5 ?@hg? ?@ W.?W26T&?@?@@@?W.?@@@? ?@g@?f?@f?@g@?f?@g@?f@?f@@g@? @?g 7Hh?W.? ?@hg? J@ 7HW&(Y ?@@? ?@hg? ?@g@?f?@f?@g@?f?@g@?f@?f?@g@?f@?f?@eW.?N@?f?@f?@g@?f?@g@?f@?f?@g@? @?g ?@hg? ?@g@?f?@f?@g@?f?@g@?f@?f?@g@?f@?f?@L?.Ye@?f?@f?@g@?f?@g@?f@?f?@g@? @?f?@ ?@@? ?@hg? ?@g@?f?@f?@g@?f?@g@?f@?f?@g@?f@?f?@@?f@?f?@f?@g@?f?@g@?f@?f?@g@? @?fJ5 ?@ ?@hg? ?@g@?f?@f?@g@?f?@g@?f@?f?@g@?f@?f?@H?f@?f?@f?@g@?f?@g@?f@?f?@g@? @?e?W&H ?@@? ?@hg? J@g@?f?@f?@g@?f?@g@?f@?f?@g@?f@?f?@g@?f?@f?@g@?f?@g@?f@?f?@g@? @?e?75? ?@@? ?@hg? @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@? @?eJ(Y? @@ @? ?@hg? N@g@?f?@f?@g@?f?@g@?f@?f?@g@?f@?f?@g@?f?@f?@g@?f?@g@?f@?f?@g@? @??W&He ?@hg? ?@g@?f?@f?@g@?f?@g@?f@?f?@g@?f@?f?@g@?f?@f?@g@?f?@g@?f@?fJ@e@@e@? @?W&5?e ?@hg? ?@g@?f?@f?@g@?f?@g@?f@?f?@g@?f@?f?@g@?f?@f?@g@?f?@g@?f@?f@@g@? @W&@H?@@ @@ ?@hg? ?@g@?f?@f?@g@?f?@g@?f@?f?@g@?f@?f?@g@?f?@f?@g@?f?@g@?f@?fN@g@? ?W-Xf@@U@L?e@@ ?@hg? ?@g@?f?@f?@g@?f?@g@?f@?f?@g@?f@?f?@g@?f?@f?@g@?f?@g@?f@?@@e?@g@? ?7R1f@V@@@?e ?@hg? @?W-X?e?@g@?f?@f?@g@?f?@g@?f@?f?@f?J@?f@?f?@g@?f?@f?@g@?f?@g@?e@@@?f?@g@? ?@?@f@?@Mf ?@hg? @?7R1?e?@g@?f?@f?@g@?f?@g@?f@?f?@f?@@?f@?f?@g@?f?@f?@g@?f?@g@@@?e@?f?@g@? ?3T5f@?g ?@hg? @?@?@?eJ@g@?f?@f?@g@?f?@g@?f@?f?@eO@KV@?f@?f?@g@?f?@f?@g@?f?@g@?f@?f?@g@? ?V+Yf@@@@@@@@ @?3T5?e@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@? I@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@hg? I@M?eI@M?e?I@Mf?I@MfI@M?eI@M?e?I@MfI@M?fI@hg? @?V+Y?eN@g@?f?@f?@g@?f?@g@?f@?fW@(Mf@?f@?f?@g@?f?@f?@g@?f?@(Mf@?f@?f?@@@@0M?@? ? ?@g@?f?@f?@g@?f?@g@?f@?f@@H?f@?f@?f?@g@?f?@f?@g@??@@??@H?f@?f@?eO2@@g@? ? ?@g@?f?@f?@g@?f?@g@?f@??W.?N@g@?f@?f?@g@?f?@f?@g@?f?@g@?f@W2@0M?@g@? ?W-Xg?W-KO-X?W-X?f@?W-X?W-X?e?W.?W-X?W-X?e?W-KO-X?W-X?e?@?W-X?W-KO-X?h? ?@g@?f?@f?@g@?f?@g@?f@L?.Y??@g@?f@?f?@g@?f?@f?@g@?f?@g@??O2@@(M?e?@g@? ?7R1g ?.R'@R1?7R1?e?J@?7R1?7R1?e?7U?7R1?7R1?e?*?@@R1?7R1?e?@?7R1?7R@@R1?h? ?@g@?f?@f?@g@?f?@g@?e?J@@f?@g@?f@?f?@g@?f?@fJ@e@@e@?f?@g@@@0M?@Hf?@g@? ?@?@g ?N@?@?@?@?e?7@?@?@?@?@?e?@)X@?@?@?@?e?S@U@?@?@?@?e?@?@?@?@?@@?@?h? ?@g@?f?@f?@g@?f?@g@?eW&@Hf?@g@?f@?f?@g@?f?@f@@g@?f?@eO2@@@?f@?f?@g@? ?3T5g ?@KC@T5?3T5?e?3@@@T5?3T5?e?3>@@T5?3T5?e?*?@@T5?3T5?e?@?3T5?3T@@T5?h? J@g@?f?@f?@g@?f?@g@??O&Y@?f?@g@?f@?f?@g@?f?@fV@g@?f?@@@@Ye@?f@?f?@g@? ?V+Yg?@@0R+Y?V+Y?e?V+MI+Y?V+Y?e?V+MI+Y?V+Y?e?V+MI+Y?V+Y?e?@?V+Y?V+MI+Y?h? @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@? ? N@g@?f?@f?@g@?f?@g@?f@?f?@g@?f@?f?@g@??I40Y@f?@g@@@@0M?@g@?f@?f?@g@? ? ?@g@?f?@f?@g@?f?@g@?f@?f?@g@?f@?f?@g@@@?e?@f?@e?O2@@?f?@g@?f@?f?@g@?hfO2@@@?W26T2@6T26KO.?e@(f?@?)T.f@?g?@@@h?@(?fW&f@( ? ?@g@?f?@f?@g@?f?@g@?f@?f?@g@?f@?f?@e?@@?@?f?@f?@@@@0M?@?f?@g@?f@?f?@g@?he?@0Y@?@?7@=C@@T.eV/?@@??@@@@T.?e@L?@@?@??@@@@@f7U?V/?@@)?N@e@?V/X? ?@e@?@@6?)X?)X?@@?@?@W-X?@@@@@?@@@@eW.?@@@@@?@@@@?hf? ?@g@?f?@f?@g@?f?@g@?f@?f?@g@?f@?f?@@?f@?e?O&@@0M??@g@?f?@g@?f@?f?@g@? @MeV40R+R+R40MI+Yh?@?(R+Y?@?@@f@??@@0M??@e@)h?@f?N1? ?@e@?@?@@@)T@1?@H?@?@@R1?@@@@@?@@?@e7H?@?@?@?@@?@?hf? ?@@@e?@g@?f?@f?@g@?f?@g@?f@?f?@g@?e?J@??@@??@H?f@W2@@0Y@f?@g@?f?@g@?f@?f?@g@? @? @? ?@e@?@?@@XI'U@?@??@?@@T5?@@@@@?@@?@e3L?@?@?@?@@?@?hf? ?@X?e?@g@?f?@f?@g@?f?@g@?f@?f?@g@?e?@@?f?@e?O2@@(M?e?@f?@g@?f?@g@?f@?f?@g@? ?@@@@?@?(R/?V4@?@??@?(R+Y?@0?4@?@@?@eV/?@?@?@?@@?@?hf? ?@)XeJ@g@?f?@f?@g@?f?@g@?f@?f?@g@W@K?V@?f?@@@@@Y?@Yf?@f?@g@?f?@g@?f@?f?@g@?he?@@@hf?W-Xg@6T& ? S,e@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?he?@?@e?@g)X?7@1?@K?e@S@@he?@K??@@@@@ ? .YeN@g@?f?@f?@g@?f?@g@?f@?f?@@0M?e@?f@@@0M??@g@?f?@f?@g@?f?@g@?f@?f?@g@?he?@@@@@?@e?@@?@)?@@@?@@@e@@Y@@@h?@@@?@?@?@ ? ?@g@?f?@f?@g@?f?@g@?f@?e@@?@g@W2@@@@?f?@g@?f?@f?@g@?f?@g@?f@?f?@g@?he?@M?e?@g@H?@@@@0M?e@@@0M?f/T.??@M??@?@?@ ? ?@g@?f?@f?@g@?f?@g@?f@Lf?@e?O2@@(M?e@?f?@g@?f?@f?@g@?f?@g@?f@?f?@g@? @? V+Y? ? ?@g@?f?@f?@g@?f?@g@?f@@f?@@@@0M?@Hf@?f?@g@?f?@f?@g@?f?@g@?f@?f?@g@? ? ?@g@?f?@f?@g@?f?@g@?@@e@Y?O2@@@g@?f@?f?@g@?f?@f?@g@?f?@g@?f@?f?@g@?hfO)T-T.e?@?@hf@6X??@@?e@?g@@@?hW&?W&?f?@6X?@eW. ? ?@g@?f?@f?@g@?f?@f@@@?f@@@0M??@g@?f@?f?@g@?f?@f?@g@?f?@g@?f@?f?@g@?he?@@@0R@He?@?@e?)X?e?@e@V1??@f@?eO@e@@@?h7@?&@?W.e?@S,?@?O&Hf?@?@hg ? J@g@?f?@f?@g@?f?@W26Ke@@@@@@@?f?@g@?f@?f?@g@?f?@f?@g@?f?@g@?f@?f?@g@? 3T.??@@@e?@)?@@?@e@W5??@)Xe@L?@@@e@@@?@@f@?3@?N@?7Ye?@@U?@@@@T.?@@?@@@?'@?he ? @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@? V+Y??@?@?/X@H?e?@e@0Y??(R1e@@eI@e@@@?e?@fV'e@?@@@??@@)?@M?I+Y?e?@V@?V'?he ? N@g@?f?@f?@g@@@?e?@@@0Me@?f@?f?@g@?f@?f?@g@?f?@f?@g@?f?@g@?f@?f?@g@? ?V4@ ?@ ?I@? ?@ ? ?@g@?f?@f?@L??'6X@W@@@@@@g@?f@?f?@g@?f@?f?@g@?f?@f?@g@?f?@g@?f@?f?@g@? ? ?@g@?f?@f?@)K?S@@@(M?e?@g@?f@?f?@g@?f@?f?@g@?f?@f?@g@?f?@g@?f@?f?@g@?he?@6Xf?)X?hf?)X?g@?@?e?@ ?@?@K?he?W-X ? ?@g@?@?e?@?'6K?@@@@0M?@Hf?@g@?f@?f?@g@?f@?f?@g@?f?@f?@g@?f?@g@?f@?f?@g@?he?@S)K??@?@1??@@@@@?@f?@1??O2@?J@?@?eJ@hf?@X@?@@@?W&??@f?7@1 ? ?@g@?f?@?S@@@@g@?f?@g@?f@?f?@g@?f@?f?@g@?f?@f?@g@?f?@g@?f@?f?@g@?he?@@>@@?@?@@??@@@@@?@?@@??@@?'@@@?'@?@@@?'@e'@?@?@e@@?@@@?@@@?&@??@?@@??@@@?@@? ? ?W-Xe?@f@?@?eO2@@@0M??@g@?f?@g@?f@?f?@g@?f@?f?@g@?f?@f?@g@?f?@g@?f@?f?@g@?he?@@0M??@?@@??@@@@@?@f?@@?V4@@?V'?@MeV'eV'h?@?@?@@@?N@??@f?@@@ ? ?7R1e?@?W-Ke@@@@0M?@f?@g@?f?@g@?f@?f?@g@?f@?f?@g@?f?@f?@g@?f?@g@?f@?f?@g@? @? ? ?@?@eJ@W&R@@@@?f?@f?@g@?f?@g@?f@?f?@g@?f@?f?@g@?f?@f?@g@?f?@g@?f@?f?@g@? ? ?3T5e'@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@? @? ?@ ?@fW& ? ?V+YeV+M?e?I@MfI@M?eI@M?e?I@MfI@M?e?I@Me?I@MfI@M?e?I@Me?I@MfI@M?e?I@MfI@M?eI@M?e?I@MfI@M?e?I@Me?I@MfI@M?e?I@?he?@@@@6X?@@?@@?@?f@?@@@?e?@f?@K?f?O2@@@6KO2@?J@f7@e?@@? ? ?@?@?@1?@@?@@?@@@?@?@?@@@?@@?@f?@@@?@e@@@?@?@@@@@?'@?'@?@@@@?@@? ? ?@?@?@@?@@?@@?@?@?e@?@@@?e?@f?@M?f?I@?@?@MI4@?V'?V'?@0M??@@? ? ? W-X? ?@?W-X?W-X ?W-KO-X?W-X? ? 7R1? J@?7R1?7R1 ?*?@@R1?7R1? ?@?W-X?W-KO-X? @?W.eW-KO-X W-KO-X?W-KO-X? ? @?@? 7@?@?@?@?@ ?S@U@?@?@?@? ?@?.R/?7R@@R1? @?7Ue7R@@R1 .R'@R1?7R@@R1? ? 3T5? 3@@@T5?3T5 ?*?@@T5?3T5? ?@f?@?@@?@? @?@)X?@?@@?@ N@?@?@?@@?@? ? V+Y? V+MI+Y?V+Y ?V+MI+Y?V+Y? ?@?@K??3T@@T5? @?3>,?3T@@T5 @KC@T5?3T@@T5? ? ?@?@@@?V+MI+Y? @?V+Y?V+MI+Y @@0R+Y?V+MI+Y? ? ? ? ? ?@e@?hf?@?@he@Khf?@6K ? ?@e@?@@6?)X?)X?@@?@?@W-X?@@@@@?@@@@eW.?@@@@@?@@@@? ? ?@e@?@?@@@)T@1?@H?@?@@R1?@@@@@?@@?@e7H?@?@?@?@@?@? ? ?@e@?@?@@XI'U@?@??@?@@T5?@@@@@?@@?@e3L?@?@?@?@@?@? ? ?@@@@?@?(R/?V4@?@??@?(R+Y?@0?4@?@@?@eV/?@?@?@?@@?@? ? ? ? ? ?@@6K? ?W&K ?@?@@6T&KO@?W&?@?@?@6T-XeW&@@6T-T26X? ?@@@@V@@@@@W&@?@?@?@V@@)e7Y@@V@R@@V1? ?@e@?3Xe@(Y@@@?@?@?3X?e3X@@?3T@@W5? ?@e@?V/e(Y?@?4@@?@?V/?eV4@@?V+Y@(Y? (Y ?)X? ?W-T&?@?)X?@@?W.?W-X?@1?@@@@@@@@@? ?7R@@?@@@)?@H?7H?7R1?@@?@@?@?@@?@? ?3T@@T@@X??@e3L?3T5?@@?@@?@?@@?@? ?V+MI+MI/??@eV/?V+Y?@@@@@?@?@@?@? ?)X?g?W26X?W&?@@@f@? ?@)X?)X?@@?7Y@1?7@?@?@?)X?@? ?@V)T@1?@H?@?@@?@@?@@@T@1?@? ?@W@@U@?@??@?@@T@@?@?I'U@?@? ?@0MI4@?@??3T(R+R'?@eV4@?@? ?V+Yhf@?
?)X? ?31? ?W2@6X?N@? ?*U?V1e@? ?S@@@@e@? ?7
@@6K W&K?eW26X @?@@6T&KO@?W&?@?@?@6T-X??W&@@6T. @@@@V@@@@@W&@?@?@?@V@@)??7Y@@V@H 7@V1 @??@?3X??@(Y@@@?@?@?3Xe?3X@@?3LN@(Y 3@W5 @??@?V/??(Y?@?4@@?@?V/e?V4@@?V/ ?(Y? )Xg@@@@ W-T&?@?)X?@@?W.?W-X?@1?@@@@@ 7R@@?@@@)?@H?7H?7R1?@@?@@?@H @@?@ 3T@@T@@Xe@??3L?3T5?@@?@@?@?@@?@ @@?@ V+MI+MI/e@??V/?V+Y?@@@@@?@? )XheW26X?W&?@@@?@X@? @? @)X?)X?@@?e7Y@1?7@?@?@? @V)T@1?@H?e@?@@?@@?@@@L 3@@? @W@@U@?@f@?@@T@@?@?I/@@@? ?@@? @0MI4@?@f3T(R+R'?@?e V+Y?h?I@?
@?e?)X? @?e?31? @@@6XN@? @??B1?@? @?e@?@? @?e@?@? @??C5?@? @@@0Y?@? ?J5? ?.Y?
Fig. 21. Pressure versus flow rate curves that can be expected with SOURCE media. Curve in a) shows that RESOURCE 1 ml columns can be used with high pressure equipment or peristaltic pumps. The curves in b) are from a large scale column packed with SOURCE 15 media to 4 different bed heights. Curve c) shows the flow characteristics of the monosized SOURCE 30 matrix compared with polysized media with mean diameters of 35 and 50 µm respectively. The pressure flow data were determined in a 100 mm i.d. column with a bed height of 10 cm. (Work by Pharmacia Biotech, Uppsala, Sweden.) POROS is a registred trademark of Perseptive Biosystems. S HyperD F is a registred trademark of BioSepra.
39
Recommended flow rates for high resolution separations are in the range 30 to 600 cm/h for SOURCE 15Q and 15S and 300 to 1000 cm/h for SOURCE 30Q and 30S. Figure 21 shows pressure versus flow rate characteristics of SOURCE 15 and SOURCE 30.
Capacity SOURCE ion exchangers have high capacities for large proteins as well as for smaller polypeptides, peptides and oligonucleotides. This is a result of an optimized pore size distribution, a high substitution level and a large binding surface area. Figure 22 shows breakthrough curves at different flow rates on RESOURCE Q and RESOURCE S. SOURCE Q and S retain their charge and can therefore be used over a wide pH range, 2-12, without variation in loading capacity.
@@e?@@?he?@e?@@?e@? ?W-Xg@@e?@@?'6X?@?@?e?@e?@@L?J5? ?*?)T-X?e@@L?J@@?S@1?3T5?e?@e?@V1?7H? ?V+R@>)Xe@V1?7Y@@(Y@?S@U?e?@e?@?@?@ ?J@@?,e@?3T5?@@U?@?7R1?e?3=?C5?3T5e@@6T-KO-X?W-K? ?.MI+Ye@?V+Y?(R4@@?@?@?e?V4@0Y?V+Ye@?@(R4@?,?7R@@@@@@@@@? @@@?eS@U?@?@@?@@?@?@? ?J(?')KO&?,?3T@@?@@?@?@? ?.Y?V4@0R+Y?V+R'?@@?@?@?
?/X? ?N1? 3L W2@6X?N1 .M?V1??@ ?O2@@??@ ?@@0MW@??@ ?3X?O&@L?@ ?V4@0MI/T5 ?N@H ?J5? ?.Y?
?W&?W26X?W26X? ?*@?75fW20M?O.? @? 7R@He?W.M?O20Y? @? ?J5?@?eW.YW20M? @? ?7HJ5?e7HW.M? @?h?@e?@K? ?@@@?@hf@@@@fW& J5?7H??J5?7H @?h?@e?@@@@?W-X??)X?@@e?@e?@W-X?@@@@@?e@?W5?)X?*@W)X?he7H?@e?7HJ5? @?h?@e?@@?@?7@)?J@1?@He?@@@?@@R1?@@@@@?e@@@U:@1?N@@@)?he@??@eJ5?7H? @?h?@e?@@?@?3Xe*U@?@?e?@e?@@T5?@@@@@?e@?B@@U@??@@Xhf@??@e7HJ5 @?h?@@@?@@?@?V/eV4@?@?e?@e?(R+Y?@0?4@?e@??(R4@??(R/hf@??@e@?7H @? @??@?J@T5? @? @??@?7R@H? @? @K @??@W5?@ @?h?W.??@@@@@?J@@@? @?J@@HJ5 @?h?7H??@?@?@?7@?@? @?7Y@?7H @?h?3L??@?@?@?@@?@? @?@?@W5? @?h?V/??@?@?@?@@?@? @?@?@@H? @? ?@eW-X?W-X?he?J5?@?@@ @? ?@e7R1?7R1?he?7H?@?@@ @? ?@e@?@?@?@?he?@e@?@@ @? ?@e3T5?3T5?he?@e@?@5 @? ?@eV+Y?V+Y?heJ@L?@?@H @? ?O&@1?@@@? @? ?@@@@@@@@@@@@@@@@@@@@@@@@?@@@? @? ?I'@5?@@@? @? N@H?@W5? @? ?@e@@H? @? ?@?J@@ @? W-X?W-X?W-X?h?@?7@@ @? *?,?7R1?7R1?h?@?@@5 @? S@U?@?@?@?@?h?@?@@H @? *?,?3T5?3T5?h?@?@@? @? V+Y?V+Y?V+Y?h?@?@@? @? ?@?@5? @? ?@@@H? @? @@@@@@@@@@@@@@@@@@@@@@@@ @? ?@@@ @? ?@?@ @? ?@?@ @? J@X@ @? 7R@@ @? @?@5 @? @?@H @? @?@? @? @??W-X?W-X?W-Xhe@?@? @? @??7R1?7R1?7R1he@?@? @? @??@?@?@?@?@?@he@@@? @? @??3T5?3T5?3T5he@@@? @? @??V+Y?V+Y?V+Yhe@@@? @? @@@@@? @? @@@@@? @? ?@@@@@@@@@@@@@@@@@@@@@@@@@@? @? @?@? @? @@@? @? @@@? W2@6T26Xh@? @@5? 7@@>@@@0Y? @? O2@@?S@0M? @? ?O2@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@0M? @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@? @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ @? @? @? @? @? @? W&f?@K? W26KO26X ?W&?W26Xh?@@@@@eW2@@e@@6X?@6T26X?*@W26X?@@@@? .MB@@
? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?W&?W26X?W26X? ? ?*@?7@5g ? @? ?@?@@Hg ? @? ?@?@@?g ? @? J5?@@?g ? @? 7H?@@?g ? @? @?J@@?g ? @? @?7Y@?g ? @? ?J@?@W5?g ? @? ?7@?@@H?g ? @? ?@@?@@h ? @? ?@@T@@h ? @? ?@V@>5h ? @? ?@?@0Yh ? @? @HW2@?h ? @? ?J@?7>5?h ? @? ?7@T@@H?h ? @? J@V@>5he ? @? ?W&@T@@Hhe ? @? ?7@V@>5?he ? @? J@@T@@H?he ? @? ?W&@>@>5hf ? @? O&@>@?(Yhf ? @? W2@@>@?(Y?hf ? @? ?W&@@>@?(Yhg ? @? O&(MS@?(Y?hg ? @? O2@0YW&?(Y ? @? O2@(M?W&>(Y? ? @? O2@@@Y?O&@0Y ? @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@0M ? @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ ? @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ ? ?@ ?@ ?@ ?@f@@@@ ? ?@ ?@ ?@ ?@f ? ? ? W&?W26X?hW2@?W26X ?W&?W26X?W2@@?h@@@@@??W2@@??@@6X?@6T26X?*@W26X?@@@@f? ?W&?f@Kg? 7@?7
@@e?@@?he?@e?@@?e@? ?W-Xg@@e?@@?'6X?@?@?e?@e?@@L?J5? ?*?)T-X?e@@L?J@@?S@1?3T5?e?@e?@V1?7H? ?V+R@>)Xe@V1?7Y@@(Y@?S@U?e?@e?@?@?@ ?J@@?,e@?3T5?@@U?@?7R1?e?3=?C5?3T5e@@6T-KO-X?W-K? ?.MI+Ye@?V+Y?(R4@@?@?@?e?V4@0Y?V+Ye@?@(R4@?,?7R@@@@@@@@@? @@@?eS@U?@?@@?@@?@?@? ?J(?')KO&?,?3T@@?@@?@?@? ?.Y?V4@0R+Y?V+R'?@@?@?@?
@?e?/X? @?e?N1? @?f3L @@@6X?N1 @??B1??@ @?e@??@ @?e@??@ @??C5??@ @@@0Y?J5 7H ?J5? ?.Y?
Fig. 22. Breakthrough curves at different flow rates (superimposed). a) RESOURCE Q 1 ml. Sample: Bovine serum albumin (Sigma), 5 mg/ml. b) RESOURCE S 1 ml. Sample: Lysozyme, 5 mg/ml (Sigma). (Work by Pharmacia Biotech, Uppsala, Sweden.)
Recovery The recovery of protein mass is typically 90-100%. In the application described in Chapter 12, purification of exotoxin A, the recovery in the SOURCE 30Q step was 91%.
40
Reproducibility Emphasis during development has been on quality, reproducibility and scaleability, features which are particularly important for industrial applications under strict regulatory control. Through the combination of high quality assurance standards and a patented manufacturing process, the particle structure is consistent both bead-to-bead and batch-to-batch. Modal particle diameter varies between batches within ± 1 µm for SOURCE 15 and ±2 µm for SOURCE 30. The reproducibility of a separation of a standard protein mixture performed on four different production batches of SOURCE 30S, see Figure 23, is an example that reflects the reproducible qualities of SOURCE media. W-X?@?e@? 7R1?@?e@? ?J@?@?@?e@? ?7@@@@@?e@? ?@e?@@=?C5? ?@e?(R4@0Y? @? @? @? @? @? @? @? @? @? @? @? @? @? @? @? @? @? ?J@? @? ?7@? @? ?@@? @? ?@@? @? ?@@? @? ?@@? @? ?@@? @? ?@@? @? ?@@? @? ?@@? @? ?@@? @? ?@@? @? ?@@? @? ?@@? @? ?@@? @? ?@@? @? @? ?@@? @? @? ?@@? @? @? ?@@? @? @? ?@@? @L @? ?@@? ?J@1 @? ?@@? ?7Y@ @? ?@@? ?@?@ @? ?@@? ?@?@ @? ?@@? ?@?@ @? ?@@? ?@?@ @? J@@? ?@?@ @? 7Y@? ?@?@ @? @? @?@? ?@?@ @? @? @?@? ?@?@ @? @? @?@? ?@?@ @? @? @?3L ?@?@ @? @? @LN1 ?@?@ @L @? @1?@ ?@?@ @1 @? @@?@ ?@?@ @@ @? @@?@ ?@?@ @@ @? @@?@ ?@?@ @@ @? @@?@ ?@?@ @@ @? @@?@ ?@?@ @@ @? @@?@ ?@?@ @@ @? @@@@ ?@?@ @@ @? @?@@ ?@?@ @@ @? @?@@ ?@?@ @@ @? @?@@ ?@?@ @@ @? @?@@ ?@?@ @@ @? @?@@ ?@?3L? @@ @? @?@@ ?@?N1?hf?J@@ @? @?@@ ?@e@?hf?7Y@ @? @?@@ ?@e@?hf?@?@ @? @?@@ ?@e@?hf?@?@ @? @?@@ ?@e@?hf?@?@ @? @?@@ ?@e@?hf?@?@ @? @?@@ ?@e@?hf?@?@ @? @?@@ ?@e@?hf?@?@ @? @@@@ ?@e@?hf?@?@ @? @@@@ ?@e@?hf?@?3L? @? @@@@ ?@e@?hf?@?N1? @? @@@@ ?@e@?hf?@e@? @? @@@@ ?@e@?hf?@e@? @? @@@@ ?@e@?hf?@L?@? @? @@@@ ?@L?@?hf?@1?@? @? @@@@ ?@1?@?hf?@@?@? @? @?@@ ?@@?@?hfJ@@?@? @? @?@@ ?@@?@?hf7Y@?@? @? ?J@?@@ ?@@?@?hf@?@?@? @? ?7@?@@ ?@@@@?hf@?@?@? @? ?@@?@@ ?@@@@?hf@?@@@? @? ?@@?@@ J@@@@?hf@?@@@? @? ?@@?@@ 7@@@@?hf@?@@@? @? ?@@?@@ @@@@@?hf@?@@@? @? ?@@@@@ @@@@@?hf@?@@@? @? ?@@@@@ @@@@@?hf@?@@@? @? ?@@@@@ @@@@@?hf@?@@@? @? ?@@@@@ @@@@@?hf@?@@@? @? ?@@@@@ @@@@@?hf@?@@@? @? ?@@@@@ @@@@@?hf@?@@@? @? ?@@@@@ @@@@@?hf@?@@@? @? ?@@@@@ @@?@@?hf@?@@@? @? ?@@?@@ @@?@@?he?J@X@@@? @? ?@@?@@ @@?@@?he?7R@@@@? @? ?@@?@@ @@?@@?he?@?@@@@? @? ?@@?@@L? @@?@@?he?@?@@@@? @? ?@@?@@1? @@?@@?he?@?@@@@? @? ?@@?@@@? @5?@@?he?@?@@@@? @? ?@@?@@@? @H?@@?he?@?@@@@? @? ?@@?@@@? @??@@?he?@?@@@@? @? ?@@?@@@? @??@@Lhe?@?@@@@? @? ?@@?@@@? @??@@1he?@?@@@@? @? ?@@?@@@? @??@@@he?@?@@@@? @? ?@@?@@@? @??@@@he?@?@@@@L @?g@? ?@@?@@@? @??@@@he?@?@@@@1 @?g@? ?@@?@@@? @??@@@he?@?@@@@@ @?g@? ?@@?@@@? @??@@@he?@?@@@@@ @?g@? ?@@?@@@? @??@@@he?@@@@@@@ @?g@? ?@@?@@@? @??@@@heJ@@@@@@@ @?g@? ?@@?@@@? @?J@@@he7Y@@@@@@ @?g@? ?@@?@@@? @?7@@@he@?@@@@@@ @?g@? ?@@?@@@? @?@@@@he@?@@@@@@ @?g@? ?@@?@@@? @?@@@@he@?@@@@@@ @?g@? ?@@?@@@? @?@@@@he@?@@@@@@ @?g@? ?@@?@@@? @?@@@@he@?@@@@@@ @?g@? J@@?@@@? @?@@@@he@?@@@@@@ @?g@? 7@@?@@@? @?@@@@he@?@@V'@@ @?g@L @@@?@@@? @?@@@@he@?@@?N@@ @?g@1 @@@?3@@? @?@@@@he@?@@e@@ @?g@@ @@@?N@@? @?@?@@he@?@@e@@ @?g@@ @@@??@@? @?@?@@he@W@@e@@ @?g@@ @@@??@@? @?@?@@he@@Y@e@@ @?f?J@@ @@5??@@? @?@?@@he@@?@e@@ @?f?7Y@ @@H??@@? @?@?@@he@@?@e@@ @?f?@?@ @@e?@@? @?@?@@h?J@@?@e@@ @?f?@?@ @@e?@@? @?@?@@L?g?7Y@?@e@@ @?f?@?@ @@e?@@? @?@?@@1?gJ5?@@@e@@ @?f?@?@ @@e?@@L ?J@?@?@@@?g7H?@@@e@@ @?f?@?@ @@e?@@1 ?7@?@?@@@?f?J5??@@@e@@L? @?f?@?@ @@e?@@@ ?@@?@?@@@?f?7H??@@@e@@1? @?f?@@@ @@e?@@@ ?@@?@?@@@?f?@e?@@@e@@@? @?f?@@@ @@e?@@@ ?@5?@?@@@?f?@e?@@@e@@@? @?f?@@@ @@e?@@@ ?@H?@?@@@?fJ5e?@@@e@@@? @?f?@@@ @6X?@@e?@@@ J@e@?@@@?f7He?@@@e@@@? @?f?@@@ ?J(R1?@@e?@@@ 7@e@?3@@?f@?eJ@@@e@@@L @?f?@@@ ?7H?3X@@e?@@@ @@e@?N@@?e?J5?e7Y@@e@@V1 @?f?@@@ ?@eV'@@e?@@@hf?J@@e@??@@Le?7H?e@?@@e@@@@ @?f?@@@ J5e?N@@e?@@@hfO&Y@e@??@V1eC5f@?@@e@@@@ @?f?@@@L? 7Hf@@e?@@@h?O2@@@?@e@??@?3T20Yf@?@@e@@@@L? @?f?@@V1? ?J5?f@@e?@@@g?O20M?e?@e@??@?V+Mg@?@@e@@@V1? @?f?@@?3L W.Y?f@@e?@@@L?e@@@0M?f?@e@??@he@?@@e@@@?@? @?f?@@?N1 ?O.Yg@@e?@@V)K?C(Mh?@e@??@he@?@5e@@@?3L @?fJ@@??3L? O2@0Y?g@@e?@@?@@@0Y?h?@e@??@he@?@He3@@?N)K? @?e?O&@@L?V/K O2@@@@0Mhe@@e?@@? ?@e@??@he@?@?eN@@??@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@6K? @?O2@0M?@1eV4@@@@@@@@@@@@@@0M @@e?@@? ?@e@??@he@?@?e?@@? ?I4@@@@@@@@@@@@@@@@@@@@@@@@@@@@@hg @@0Mf@@ @@e?@@? ?@?J@??@he@?@?e?@@? @?g@@ @@e?@@? ?@?7@??@he@?@?e?@@? @?f?J@@ @@e?@@? ?@?@@??@L?h@?@?e?@@? @?f?7@@ @@e?@@? ?@?@@??@1?h@?@?e?@@? @?f?@@@ @@e?@@? ?@?@5??@@?h@?@?e?@@? @?f?@@@ @@e?@@? ?@?@H??@@?g?J@X@?e?@@? @?f?@@@ @@e?@@? ?@?@e?@@?g?7R@@?e?@@? @?f?@@@ @@e?@@? J5?@e?@@?gJ5?@@?e?@@? @?f?@@@ @@e?@@L 7H?@e?@@?g7H?@@?e?@@L @?f?@@@ @@e?@@1 @??@e?@@?f?J5??@@?e?@@1 @?f?@@@ @@e?@@@ @??@e?@@?f?7H??@@?e?@@@ @?f?@@@ @@e?@@@ @??@e?@@?f?@e?@@?e?@@@ @?f?@@@ @@e?@@@ @??@e?@@?f?@e?@@?e?@@@ @?f?@@@ W-X?@@e?@@@ @??@e?@@?fJ5e?@@?e?@@@ @?f?@@@ 7R1?@@e?@@@ @??@e?@@?f7He?@@?e?@@@ @?f?@@@ ?J5?@?@@e?3@@hf?J5??@e?@@?f@?e?@@?e?@@@ @?f?@@@ ?7H?@@@@e?N@@hf?7H??@e?@@?e?J5?e?@@?e?@@@ @?f?@@@ ?@f@@f@@hfJ5e?@e?@@Le?7H?e?@@?e?@@@ @?f?@@@ J5f@@f@@hf7He?@e?3@1eC5f?@@?e?@@@L? @?f?@@@L? 7Hf@@f@@he?C5?e?@e?N@@T20Yf?@@?e?@@V1? @?f?@@@1? ?J5?f@@f@@h?O20Y?e?@f@V+Mg?@@?e?@@?@? @?f?@?@@L W.Y?f@@f@@=??O-K?O20M?f?@f@?h?@5?e?@@?3L @?f?@@@V1 ?O.Yg@@f@V4@@0R4@0M?g?@f@?h?@H?e?@@?V/K? @?fJ@@@?3=? O2@6K?O2@0Y?g@@f@? ?@f@?h?@f?3@??V46K? @?e?O&@@@?V4@@@6Kf?O2@@@0M?I4@0Mhe@@f@? ?@f@?h?@f?N@?e?I4@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@hg @@@@@0Y@@@gI4@@@@@0M? @@f@? ?@f@?hJ@g@? @?f?@@@ @@f@? J@f@Lh7@g@? @?f?@@@ @5f@? 7@f@1g?J@@g@? @?f?@@@ @Hf@L @5f@@g?7Y@g@? @?f?@@@ @?f@1 @Hf@@gJ5?@g@L @?f?@@@ @?f@@ @?f@@g7H?@g@1 @?f?@@@ @?f@@ @?f@@g@??@g@@ @?f?@@@ @?f@@ @?f@@f?J5??@g@@ @?f?@@@ @?f@@ @?f@@f?7H??@g@@ @?f?@@@ ?)X?@?f@@ @?f@@f?@e?@g@@ @?f?@@@ J@)X@?f@@ ?J@?f@@fJ5e?@g@@ @?f?@@@ 7
Column: Sample:
Sample load: Eluent A: Eluent B: Flow rate: Gradient:
W26Xe?W26X? 7
W&?W26X?eW26X *@?7
SOURCE 30S, 4 separate batches, 7.5 mm i.d. x 50 mm (2.2 ml) Mixture of chymotrypsinogen, cytochrome C and lysozyme 0.32 mg/ml bed volume 20 mM sodium phosphate, pH 6.8 0.5 M sodium chloride, 20 mM sodium phosphate, pH 6.8 2.2 ml/min (300 cm/h) 0 - 100% B, 20 column volumes
?W26KO26X?eW26Xg?@@@@@ ?W-Kf?@g/Xf ?.MB@@
Fig. 23. Quality control evaluation of 4 production batches of SOURCE 30S. (Work by Pharmacia Biotech, Uppsala, Sweden.)
Availability SOURCE 15Q and 15S are available in pack sizes of 10 ml, 50 ml, 200 ml, 500 ml and 1 litre. SOURCE 30Q and 30S are available in pack sizes of 10 ml, 50 ml, 200 ml, 1 litre, and 5 litres. RESOURCE Q and S are pre-packed columns with SOURCE 15Q and 15S respectively. They are available in 1 and 6 ml sizes, packed to a bed height of 3 cm. For ordering information please refer to Chapter 14.
41
6. Sepharose based ion exchangers Pharmacia Biotech offers a range of ion exchange media based on agarose, which is cross-linked to produce Sepharose High Performance, Sepharose Fast Flow, Sepharose Big Beads, Sepharose CL-6B and STREAMLINE base matrices. Exchanger groups are attached to the gels by stable ether linkages to the monosaccharide units to give the final ion exchange gels. The polysaccharide chains of Sepharose based ion exchangers are arranged in bundles (Fig. 24). These bundles are further strengthened by different degrees of intra-chain cross-linking which provide a high matrix rigidity. The resulting structure is macroporous and the capacity of the gels very good for globular proteins up to 106 in molecular weight.
O@ ?W2@@5 ?@6X O&@@@Y ?3@)X? W2@@@@@@6K ?N@@)K?O2@@6X? ?O&@X@@@@@@@@? @@@@@@@@@@)X @@@V@@@@@@@@@? @@@@@@@??@@)K? @@@@@@@@@@@@@@@? @@@@@@@@@6K? ?O2@@@@@@?@@@@@@@@5? ?W2@@@@@@@@@@@@@@? ?O2@@@@@@@@@@@@@@@@(Y? O&@@@@@?@@@@@@@@@?hf?W2@@@@@@@@@@@@?@@@@(Y O2@@@@@@@@@@@@@@@@@@6KheO&@@@@@@@@@@@@@@@@@@H? ?'@@@@@@@@@@@@@@@@@@@@@@6KgO2@@@@@@@@@@@@@@@@@@@5 ?V4@@@@@@@@@@@@@@@@@@@@@@@6XeW2@@@@@@@@@@@@@@@@@@@@(Y @@@@@@@@@@@@@@@@@@@@@@@)KO&@@@@@@@@@@@@@@@@@@@@0Y? @@@@?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@(M? @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@0Y I4@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@0M ?I4@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@(M ?I'@@@@@@@@@@@@@@@?@@@@@@@@@@@@@@(Y? V4@@@@@@@@@@@?@@?@@@@@@@@@@@@@0Y ?I4@@@@@@@@?@@?@@@@@@@@@@@(M ?I'@@@@@@@@@@@@@@@@@@@@0Y? ?@@@@@@@@@@@@@@@@@@@ ?W&@?@@@@@@@@@@@@@@@@@L? O&@@@@@@@@@@@@@@@@@@@@)X W2@@@@@@@?@@@@@@@@@@@@@@@)K? ?W&@@@@@@@@@@@@@@@@@@@@@@@@@@@ O&@@@@@@@@@@@@@@@@@@@@@?@@@@@@ ?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@6X? ?@@@@@@@@@@@@@@@@@@@@?@@@@@@@@@@@@)X ?O2@@?W2@@@@@@@@@@@@@@@@@@V'@?@@@@@@@@@@@@@)X? O2@@@@@W&@@@@@@@@@@@@@@@@@@5?N@@@@@@@@@@@@@@@@)X ?W26?2@@@@@@@@@@@@@@@@@@@@@@@@@@0Ye@@@@@@@@@@@@@@@@@)X? ?7@@@@@@@@@@@@?@@@?@@@@@@@@@@@@?f3@@@@@@@@@@@@@@@@@)X ?@@@@@?@@@@@@@@@@@@@@@@@@@@@@@@?fV'@@@@@@@@@@@@@@@@@)X? ?@@?@@@@@@@@@@@@@@@@@@@@@@@@0Mg?V'@@@@@@@@@@@@@@@@@)?2@6K ?3@@@@@@@@@@@@@?@@@@@@@@@@@?heV4@@@@@@@@@@@@@@@@@@@@@@@@@? ?V4@@@@@@@@@@@@@@@@@@@@@@@@?hfI'@@@@@@@@@@@@@@@@@@@@@@@? I'@@@@@@@@@@@@@@@@@@(M ?V'@@@@@@@@@@@@@@@@@@@@@@@@? @@@@@@@@?@@@@@@@@0Y? V4@@@@@@@@@@@@@@?@@@@@@@@? W&@@@@@@@@@@@@@@@(M? I'@@@@@@@@@@@@@@@@@@?@@? ?W&@@@@?@@@@@@@??@@H ?V'@@@@@@@@@?@@@@@@@@@@? ?7@@@@@@@@@@@@@@@@@? V'@@@@@@@@@@?@@@@@@@@L ?@@@@@@@@@@@@@@@X? ?V'@@@@@@@@@@@@@@@?@@, ?3@@@@@@@@@@@@@@1? V4@@@@@@@@@@@@@@@@0Y ?V'@@@@@@@@@@@W@@? @@@@@@@@@@@@@X V4@@X@@@@X@@@Y@? @@@@@@@@@@@@@)X? ?B@@@@V@@@@@@L ?J@@@@@@@@@@@@@@1? @@@@@@@@@@@)X? ?7@@@@@@@@@@@@?@@? ?J@@@@@@@@@@@@1? J@@@@@@@@@@@@@@@5? ?7@@@@@@@@@@@@@L 7@@@@@@@@@@@@V40Y? ?3@?@@?@@@?@@@@1 ?J@@@@@@@@@@@@@? ?N@@@@@@@@@@@@@@L? W&@@@@@@@@@@@@5? @@@@@@@@@@@@@@1? 7@@@@@@@@@@@@@H? @@?@@@@@@@@@@@@L ?J@@@@@@@@@@@@@5 @@@@@@@@@@@@@@@1 ?7@@@@@@@@@@@@@H @@@@@@@@@@@@@@ J@@@@@@@@@@@@@5? 3@@@@@@@@@@@@@L? 7@@@@@@@@@@@@@H? N@@@@@@@@@@@@@1? @@@@@@@@@@@@@5 ?@@@@@@@@@@@@@@? ?J@@@@@@@@@@@@@H ?3@@@@@@@@@@@@@@@? W&@@@@@@@@@@@@5? ?N@@@@@@@@@@@@?@@?hfO26?&@@@@@@@@@@@@@H? 3@@@@@@@@@@@@@@?eO26K?O2@@@@@@@@@@@@@@@@@@@@5 N@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@Y W2@?f O26K ?@@@@@@@@@@@@@@@@?@@@@@@@@@@@@@@@@@@@@@@@@?@@@@@@@@@@@@@@@@@@6?2@@6?&@@?f @@6KO2@@@@@@@@6?@?@K?O2@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?@@@@@@@@@@@@@@@@@@@@@@5?f @@@@@@@?@@@@@@@@@@@@@@@@@@@@?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?@@@@0Y?f @@@@@@@@@@@@@@@@@@@??@@@@@@@@@@@@@@@?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?h ?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@e@@@@@@@@@@@@@@@@@@@@@@6Xf ?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@)X?e ?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@1?e ?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@X@@?@@@@@@@@@@@@@@@@@@@@@@@??@@@@@@@@@@@e@@@@@@@@@@@@@@@@@@@@@@@@@?e ?@@@@@@?@@@@@@@@@@@@@@@@@@e?@@V@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@??@@@@?e W@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@W@@@@@@@@@@@@@0Mhe@@@@@@@@@@@?e 7@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?@@@@@@@0?4@@@0Mg?@@Y@@@@@@@@ @@@@@@@@@@@?e 3@@@@@@@@@@@@@@@@@@@@@@?@@@@@@@@@?@@@@X? ?@@@@@@?@@@@ @@@@@@@@0Mf N@@@@@@@@@@@@@@@@@@@@@@@@@@@@?@@@X@@@@1? ?@@@@@@@@(M? @@@@0Mh ?@M? I4@@0M?@@@@@V@@@?@@? I4@@0Y ?@@@@@@@@@@@@? I4@0?4@@@?
The gels, particularly the most highly cross-linked forms, e.g. Sepharose Fast Flow and Sepharose High Performance ion exchangers, have good flow properties and stable bed volumes that are largely insensitive to changes in ionic strength and pH. They also show extremely low non-specific adsorption of macromolecules (8).
?@
Fig. 24. Structure of cross-linked agarose gels.
Chemical stability Sepharose ion exchangers tolerate extreme working conditions of temperature, pH and chemical agents. They are stable in water, salt solutions, and organic solvents. Details on the pH stability range for each gel is given in the appropriate section below. The ion exchangers can be used in solutions of non-ionic detergents such as Triton X-100® and with strongly dissociating solvents such as 8 M urea and 6 M guanidine hydrochloride (9). The Sepharose based ion exchangers also tolerate 1 M acetic acid, 30% isopropanol, 30% acetonitrile, 70% ethanol and 1 M NaOH. Under oxidizing conditions, limited hydrolysis of the polysaccharide chains may occur. The gel-forming fibres of agarose are stiff bundles of polysaccharide chains rather than flexible single chains (10). For this reason the water in the gel can be replaced by other solvents with relatively little effect on pore size. Sepharose ion exchangers Triton® is a registred trademark of Rohm and Haas Co.
42
can be used in polar organic solvents and in aqueous/organic mixtures. The gel matrix is stable in a wide variety of solvents including ethanol, dimethylformamide, tetrahydrofuran, acetone, dimethylsulphoxide, chloroform, dichloromethane, dichloroethane and dichloroethane/pyridine (50:50). Sepharose is very resistant to microbial attack due to the presence of the unusual sugar, 3,6-anhydro-L-galactose. However, most buffers can support bacterial growth and so a antimicrobial agent should be used in storage (see page 103).
Physical stability The highly cross-linked structure of the modern Sepharose based ion exchangers, e.g. Sepharose Fast Flow and Sepharose High Performance, not only gives them increased chemical stability but also results in improved physical stability. This improves flow properties enormously compared to Sepharose CL-6B gels. Crosslinking prevents fluctuations in bed volume under conditions of increasing ionic strength. Thus Sepharose ion exchangers can be regenerated and re-equilibrated repeatedly in the column. Repacking between experiments is thus eliminated, improving reproducibility. Sepharose ion exchangers can be used at temperatures up to 70 °C and can be sterilized repeatedly in the salt form by autoclaving at 121 °C, pH 7, for 30 minutes.
Sepharose High Performance ion exchangers Sepharose High Performance ion exchange media are based on 34 µm highly cross-linked agarose beads. The small bead size gives the media high efficiency, which in combination with high selectivity offers the possibility of high resolution standard chromatography separations. In addition, the use of identical functional groups to those used in Q and SP Sepharose Fast Flow, Mono Q and Mono S and SOURCE Q and SOURCE S media, at comparable substitution levels (i.e. same selectivity), simplifies scaling up from FPLC and up to BioPilot and BioProcess scales.
43
Properties Physical stability The high degree of cross-linking of Sepharose High Performance renders the media extremely stable physically. The high rigidity of the cross-linked agarose matrix eliminates volume variations due to changes in pH or ionic strength.
Capacity As is the case with all ion exchangers, the capacity depends upon the accessibility of the charged groups and their number. Sepharose High Performance gels have an exclusion limit of approximately 4 x 106 and are highly substituted with strong ion exchange groups. Thus they remain charged and maintain consistently high capacities for proteins over a broad working pH range (Table 7). This allows the selection of a pH value and buffer that best suit the properties of the sample. Titration curves for Q and SP Sepharose High Performance are similar to those for Q and SP Sepharose Fast Flow, which are shown in Figure 26. Capacity varies from case to case depending on protein properties and flow rate. As an example, the dynamic capacity for human serum albumin on Q Sepharose High Performance is approximately 70 mg per ml gel at 150 cm/h (start buffer 20 mM Tris, pH 8.2). The dynamic capacity for ribonuclease on SP Sepharose High Performance has been estimated to 55 mg/ml gel at 150 cm/h (start buffer 0.1 M sodium acetate, pH 6.0). The characteristics of the media are shown in Table 7. Characteristics specific for HiLoad, HiTrap and BioPilot columns pre-packed with Q and SP Sepharose High Performance are shown in Table 8.
44
Table 7. Characteristics of Q and SP Sepharose High Performance. Product
Q Sepharose High Performance
SP Sepharose High Performance
Type of gel
strong anion
strong cation
Total ionic capacity (µmol/ml gel)
140-200
140-200
BSA (MW 67 000)
70
N.D.
Ribonuclease (MW 13 700)
Dynamic binding capacity* (mg/ml gel) N.D.
55
Recommended working flow rate range (cm/h)
up to 150
up to 150
Approx. mean particle size (µm)
34
34
Particle size range (µm)
24-44
24-44
working pH range**
2-12
4-13
pH stability*** short term
1-14
3-14
long term
2-12
4-13
N.D. = Not determined * The dynamic binding capacity was determined at a linear flow rate of 150 cm/h using a 10.0 mg/ml solution of BSA in 20 mM Tris buffer, pH 8.2 and a 5.0 mg/ml solution of ribonuclease in 100 mM sodium acetate, pH 6.0. ** working pH range refers to the pH range over which the ion exchange groups remain charged and maintain consistently high capacity. *** pH stability, long term refers to the pH interval where the gel is stable over a long period of time without adverse effects on its subsequent chromatographic performance. pH stability, short term refers to the pH interval for regeneration and cleaning procedures.
Table 8. Characteristics of HiLoad, HiTrap and BioPilot columns pre-packed with Q and SP Sepharose High Performance. Product
HiLoad 16/10 26/10
HiTrap 1 ml 5 ml
BioPilot Column 35/100 60/100
Column dimensions (mm) 16x100 26x100 (inner diameter x bed height)
7x25
16x25 35x100
60x100
Bed volume (ml)
53-58
1
5
100
300
Maximum flow rate* (ml/min) 5
13
4
20
25 70
Recommended working flow rate range (ml/min)
up to 5
up to 13
1
5
up to 24 (for SP) up to 70 (for SP) up to 16 (for Q) up to 47 (for Q)
Max pressure* (MPa)
0.3
0.3
0.3
Number of theoretical plates per meter** (N/m)
>12 000 >12 000
20-22
N.D.
N.D.
0.31.1
1.1
>10 000
>10 000
* Max. pressures and flow rates should not be used routinely. ** Determined with acetone.
45
Flow rate The rigidity of Sepharose High Performance based ion exchangers together with the small particle size distribution of the 34 µm beads confers excellent flow properties. Flow rates achievable with Sepharose High Performance media and prepacked columns are given in Table 7 and 8.
Availability Q and SP Sepharose High Performance are available in packs of 75 ml, 1 and 5 litres. To ensure optimal performance and reproducibility Sepharose High Performance media are also available in pre-packed HiLoad columns with dimensions 16 and 26 mm in internal diameter and 10 cm in bed height. Q and SP Sepharose High Performance are also available in pre-packed, ready-touse 1 ml and 5 ml columns, HiTrap Q and HiTrap SP respectively. They are designed for method scouting, group separations, sample clean-up or sample concentration. For pilot scale separations, Q Sepharose High Performance is available in prepacked BioPilot columns of 100 ml (35/100) and 300 ml (60/100). SP Sepharose High Performance pre-packed in BioPilot columns 35/100 and 60/100 are available on request. For ordering information, please refer to Chapter 14.
Sepharose Fast Flow ion exchangers Sepharose Fast Flow ion exchangers are based on 90 µm agarose beads. A higher degree of cross-linking, compared to Sepharose CL-6B based ion exchangers, is used to give the media greatly improved physical and chemical stability. This high
?@@@@6X?@@@@@?e@@e@@@@@?hW2@6X?he?@@? @@@@@?he@@e?@@@@@?@@? ?@@??@1?@@f?J@@L?@@hf7@V4)?he?@@? @@ @@L??@@?e?@@? ?@@??@@?@@f?7@@1?@@hf3@e?O2@@6X?@@@6X@@@6X?@@6X?@@@@@@6X?W2@@@@@@6X?e@@f@@6X?W2@@@@@@??@@?e?@@W2@@@@6?2@?@@ ?@@??@@?@@@@@??@@@@?@@@@@?hV4@@@@@??@1?@@?@@@@?@1e?@1?@@@@e@1?*@??@@??@1?e@@@@@?e@1?*@?e@@H??@@@@@?@@@@??@@@@@@?@5 ?@@??@@?@@fJ@@@@?@@ @@@@@@@?@@?@@@@?@@W2@@@?@@@@e@@?V4@@@@@@@@?e@@e?W2@@@?V4@6X@@e?@@?e?@@@@??@@@@@@@@H ?@@??@5?@@e?O&@e@@@@he?'6Ke@@@?f@@?@@@@?@@@@@@@?@@@@e@5f@@@?g@@e?*@@@@f@@@@L??@@?e?@@@@??@@@@@@@@? ?@@@@0Y?@@@@@@@@e@@@@@@@@g?V4@@@0?4@@@@?@@@0?4@?@0?4@@@?@0?4@@0Y?@@@0?4@@@@?e@@e?V4@@@?@@@0MI4@??@@?e?@0?4@@0MI40?4@? @@ @@
?W2@@6X?@@@??@@@g?W2@6Xhf@@ ?@@@@@he?@@?e@@@@@?@@ ?7@?I4)?@@@??@@@g?7@V4)hf@@ ?@@?hf?@@Le@@f@@ ?@@?f@@@@@@@@g?3@?eO2@@6?2@@6X?@@@6X?@@6X?@@@@@@6X?W2@@@@@@6Xe?@@??O2@6X?W2@@@@@@@e@@f@@@@@6T26?2@?@@? ?@@?f@@@@@@@@g?V4@@@@@e@@@@?@1?@@?@1?e@1?@@@@??@1?*@e@@e@1e?@@@@@U?@1?*@?e?@@He@@@@@?@@@??@@Y@@@@?@5? ?@@?f@@@@@@@@he?@@@@@@@@@?@@?@@?@@W2@@@?@@@@??@@?V4@@@@@@@@e?@@??S@@@@?V4@6X?@@?e@@f@@@??@@?@@@@@@H? ?3@?O2(?@@?@@?@@g'6K??@@@f@@?@5?@@?@@@@@@@?@@@@??@5?e?@@@g?@@??*@@@@f@,?3@Le@@f@@@??@5?3@@@@@ ?V4@@0Y?@@?@@?@@gV4@@@0?4@@@@@@@0Y?@@?@0?4@@@?@0?4@@0Y?@@@0?4@@@@e?@@??V4@@@?@@@0Y?V4@e@@f@@@@@0Y?V40?4@ @@ @@
W2@@6Xf?@@??@@?e 7@?I4)f?@@??@@?e @@h?@@??@@?e @@h?@@@@@@?e @@h?@@??@@?e 3@?O26T26X?@@??@@@@@ ?O2@eV4@@0R+MS1?@@??3@?e ?O20M?h?W&@f?V4@6X ?O20M?heW&Y?hS, ?O20M?hf&@@@g@@0Y ?O20M? ?O20M? ?O20M? ?O20M? ?O2@
W2@6X?hW2@6X?he?@@? ?@@@@@he?@@?e?@@@@@?@@? ?W&(?')Xh7@V4)?he?@@? ?@@?hf?@@Le?@@?e?@@? ?7@H?N@1h3@e?O2@@6?2@@6X?@@@6T2@6X?@@@@@@@6X?W2@@@@@@6X??@@?e?@@6X?W2@@@@@@e?@@?e?@@@@@6T26?2@?@@ ?@@?e@@hV4@@@@@??@@@@?@1?@@?@@Y?@1?@@@@@e@1?*@??@@??@1??@@@@@e?@1?*@e?@@He?@@@@@?@@@e@@Y@@@@?@5 ?3@W2@@5hf@@@@@@@@@?@@?@@?@@@@@@?@@?@@e@@?V4@@@@@@@@??@@?eW2@@@?V4@6X@@?e?@@?e?@@@e@@?@@@@@@H ?V'@Y@@?g?'6Ke@@@?e?@@?@5?@@?@@@@@@?@@?3@e@5f@@@?f?@@?e*@@@@?e?@@@@Le?@@?e?@@@e@5?3@@@@@? V4@@@@g?V4@@@0?4@@@@@@@0Y?@@?@@@@@@?@@?V4@@0Y?@@@0?4@@@@??@@?eV4@@@?@@@0MI4@e?@@?e?@@@@@0Y?V40?4@? ?@@? ?@@?
W-X? *@)X W2@@6?2@e@@hf?@6X?@@?V'@1?W2@@6X?@@e@@?@@? 7@?I4@@@e@@hf?@@1?@@?e@5?7@?I4)?@@e@@?3@L @@f@@e@@hf?@@@?@@??7@H?@@?f@@e@@?N@1 @@f@@@@@@hf?@@@@@@??@@??@@?f@@@@@@e@@ ?@@@@?e@@f@@e@@g?@@@@??@@?@@@??@@??@@?f@@e@@e@@L? 3@?O2@@@e@@W26Xh?@@?3@@??@@??3@?O2(?@@e@@e@@)X V4@@0?4@e@@0MS1h?@@?V4@??3@L?V4@@0Y?@@e@@?J@@>1 ?W&@ ?N@1 ?7@V4@L? W&Y? @@ ?@@9?S,? &@@@ I4@0Y?
?W2@6X?@@@@6X?g?W2@6Xhf@@ @@@@@?he@@f@@@@@@@? ?7@V4)?@@??@1?g?7@V4)hf@@ @@ @@L?e@@e?@@? ?3@?e?@@??@@?g?3@?eO2@@6?2@@6X?@@@6T2@6X?@@@@@@@6X?W2@@@@@@6Xe@@f@@6X?W2@@@@@@?e@@e?@@W2@@6T26?2@?@@? ?V4@@6X@@??@5?g?V4@@@@@e@@@@?@1?@@?@@Y?@1?@@@@@??@1?*@e@@e@1e@@@@@?e@1?*@?e@@H?e@@@@@@@@@??@@Y@@@@?@5? ?@@@@@@0Y?he?@@@@@@@@@?@@?@@?@@@@@@?@@?@@??@@?V4@@@@@@@@e@@e?W2@@@?V4@6X@@f@@e?@@@@??@@?@@@@@@H? '6K??@@@@?he'6K??@@@f@@?@5?@@?@@@@@@?@@?3@??@5?e?@@@g@@e?*@@@@f@@@@L?e@@e?@@@@??@5?3@@@@@ V4@@@0?4@?heV4@@@0?4@@@@@@@0Y?@@?@@@@@@?@@?V4@@0Y?@@@0?4@@@@e@@e?V4@@@?@@@0MI4@?e@@e?@0?4@@0Y?V40?4@ @@ @@
W2@@6?2@e@@hf?W2@6XeW2@6X? 7@?I4@@@e@@hf?7@V4)?W&(?')T2@ @@f@@e@@hf?3@?e?7@H?N@@
Fig. 25. Partial structure of Sepharose Fast Flow ion exchange media.
46
? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?
stability allows the gels to be used at the higher flow rates required for modern laboratory separations as well as meeting the throughput and cleaning-in-place requirements of process scale chromatography. To give a complete range of ion exchange media Sepharose Fast Flow is available with the weak exchanger groups, DEAE and CM and the strong exchanger groups Q and SP. Figure 25 shows the partial structures of these media. Characteristics of the different ion exchangers based on Sepharose Fast Flow is listed in Table 9. Table 9. Characteristics of Q, SP, DEAE and CM Sepharose Fast Flow. Product
Q Sepharose SP Sepharose Fast Flow Fast Flow
DEAE Sepharose Fast Flow
CM Sepharose Fast Flow
Type of gel
strong anion
strong cation
weak anion
weak cation
Total ionic capacity (µmol/ml gel)
180-250
180-250
110-160
90-130
Recommended working flow rate range (cm/h)
100-300
100-300
100-300
100-300
Approx. mean particle size (µm)
90
90
90
90
Particle size range (µm)
45-165
45-165
45-165
45-165
working pH range*
2-12
4-13
2-9
6-10
pH stability** short term
1-14
3-14
1-14
2-14
2-12
4-13
2-13
4-13
long term
* working pH range refers to the pH range over which the ion exchange groups remain charged and maintain consistently high capacity. ** pH stability, long term refers to the pH interval where the gel is stable over a long period of time without adverse effects on its subsequent chromatographic performance. pH stability, short term refers to the pH interval for regeneration and cleaning procedures.
Properties Physical stability Sepharose Fast Flow ion exchangers are supplied pre-swollen and ready for packing or in pre-packed columns. The highly cross-linked nature of the matrix means that the bead size and bed volumes do not change with changes in ionic strength or pH.
Capacity As is the case with all ion exchangers the capacity is dependent upon the accessibility of the charged groups and their number. Sepharose Fast Flow ion exchangers are highly substituted and have an exclusion limit of approximately 4 x 106 giving
47
high capacity for proteins. Capacity data for Fast Flow ion exchange media are given in Table 10. Characteristics specific for pre-packed columns with Q and SP Sepharose Fast Flow, HiLoad columns, are shown in Table 11. Table 10. Capacity data for Sepharose Fast Flow ion exchangers. Ion Exchanger
Q Sepharose SP Sepharose DEAE Sepharose CM Sepharose Fast Flow Fast Flow Fast Flow Fast Flow
Total ionic capacity (µmol/ml gel)
180-250
180-250
110-160
90-130
Dynamic binding capacity* (mg/ml gel) Thyroglobulin (MW 669 000)
3
N.D.
3.1
N.D.
HSA (MW 68 000)
120
N.D.
110
N.D.
a-lactalbumin (MW 14 300)
110
N.D.
100
N.D.
IgG (MW 160 000)
N.D.
50
N.D.
15
Bovine COHb (MW 69 000)
N.D.
50
N.D.
30
Ribonuclease (MW 13 700)
N.D.
70
N.D.
50
N.D. = Not determined *Capacities were determined using the method described in Chapter 10 at a flow rate of 75 cm/h. For anion exchangers (DEAE and Q) the starting buffer was 0.05 M Tris, pH 8.3 and for cation exchangers (CM and S) 0.1 M acetate buffer, pH 5.0. Limit buffers were the respective start buffers containing 2.0 M NaCl.
Table 11. Characteristics of HiLoad columns pre-packed with Q and SP Sepharose Fast Flow. Product
HiLoad 16/10
HiLoad 26/10
Bed volume (ml)
20-22
53-58
Maximum flow rate* (ml/min)
10
26
Recommended working flow rate range (ml/min)
up to 7
up to 18
Max pressure* (MPa)
0.3
0.3
Number of theoretical plates per meter** (N/m)
>3000
>3000
* Max. pressures and flow rates should not be used routinely. ** Determined with acetone.
Q Sepharose Fast Flow and SP Sepharose Fast Flow are highly substituted with strong ion exchange groups. These groups remain charged and maintain consistently high capacities over broad working pH ranges of 2-12 and 4-13 respectively. This allows the selection of a pH value and buffer that best suit the properties of the sample. Titration curves for both gels are shown in Figure 26. The working pH ranges for DEAE Sepharose Fast Flow and CM Sepharose Fast Flow are 2-9 and 6-10 respectively.
48
?@e?@ ?@@6X@e?@ ?@?B@@@@@@ ?@e@@e?@ ?@?C@@e?@ ?@@0R'e?@ ?@
@?e@? @@6X@?e@? @?B@@@@@@? @??@@?e@? @?C@@?e@? @@0R'?e@? @?
?@
@?
?W&?W26X ?*@?.MB1 ?N@?e?@ @?W.f?@@@@@ @?7Yh?@ @?@@@@g?@ ?@ ?O2@@@@@@@@@@@ ?@ O2@@@@@@@@@@@@@@@@@@@@@0M? ?@ ?O2@@@@@@@@@@@@@@@@@@@@@0M ?@ O2@@@@@@@@@@@@@@@@@@@@@0M? ?@ ?O2@@@@@@@@@@@@@@@@@@@@@0M ?@ ?O2@@0M? ?@ ?O2@@0M? ?@@@ ?O20M? ?@ ?O20M? ?@ ?W20M? ?@ ?7
@@ ?@e@? @? ?W.? ?7Y? ?@@@@?
@@ ?W.? ?*U? ?N1? ?/KC5? ?V40Y?
?@ @@ ?J@@ ?7Y@L? ?@@@,? I(Y?
@@@? ?@@@U? ?@MB1? @? ?C5? @0Y?
?@@? @K @@6X @?B1 3=C5 V40Y
@@@@ @? ?J5? ?7H? ?@
@?eW26Xe?W&??@@?e@@e)Xe@?fW2@@6T&?e@? ?@@@@@e@?e7
?W&?W26X ?*@?.MB1 ?N@?e?@ @?W.f?@@@@@ @?7Yh?@ @?@@@@g?@ ?@ ?@ ?@ O2@@@@@@@@h ?@ O2@@@@@@@@@@@@@@@@0M ?@ ?O2@@@@@@@@@@@@@@@0M ?@ O2@@@@@@@@@@@@@@@0M? ?@ O2@@@@@@@@@@@@@@@@0M ?@@@ O2@@@@@@@@@@@@@@@@@@0Mg ?@ O2@@0M ?@ O2@@0M ?@ O20M ?@ O20M ?@ W20M ?@ ?W.M ?W&?W26Xg?@ W.Y? ?*@?7
@@ ?@e@? @? ?W.? ?7Y? ?@@@@?
?@@?f W.Y?e *Uf N1f /KC5f V40Yf
@? ?@@? J@@? 7Y@L @@@, ?I(Y
?@@6X? @@6X @MB1 ?@ C5 ?@0Y
?@6X @K @@6X @?B1 3=C5 V40Y
?@@@@?e ?@f J5f 7Hf @?f
?@e?W26X?eW&e@@e?@@??)X??@f?W2@@6T&e?@f @@@@@??@e?7
Fig. 26. Titration curves; approximately 5 ml Q and SP Sepharose Fast Flow in 50 ml 1 M KCl. (Work by Pharmacia Biotech, Uppsala, Sweden.)
Flow rate The optimal cross-linking of Sepharose Fast Flow confers excellent flow properties on the matrix. This is illustrated in Figure 27 which shows the relationship between flow rate and operating pressure for DEAE Sepharose Fast Flow. ?@@@@@ W& @K ?@e?@?W26X?@?@?@??@@??'6T&@W26Xe?W26T2@@@@f@@@? ?@e?@?7
?@@? J@@? 7@@L ?J@@@1 ?@@@@5 @H @? @? ?W&?W26X?W26X?h@? ?7@?7
Fig. 27. Flow rate as a function of pressure drop of DEAE Sepharose Fast Flow. Column: Pharmacia XK 50/30 fitted with 1/4 inch tubing. Gel bed height 15 cm. Gel volume 294 ml. Eluent 0.1 M NaCl. (Work by Pharmacia Biotech, Uppsala, Sweden.)
49
Flow rates achievable with Fast Flow media are above 300 cm/h at 0.1 MPa (1 bar) in an XK 50/30 column packed with a 15 cm high bed of gel. In laboratory separations where the best possible separation is frequently a major consideration this flow rate is frequently traded off against improved resolution. In industrial processing, the high throughput properties of Sepharose Fast Flow ion exchangers give significantly reduced cycle times and improved productivity.
Availability DEAE and CM Sepharose Fast Flow are available in packs of 500 ml, 10 and 60 litres. Q and SP Sepharose Fast Flow are available in packs of 25 ml, 300 ml, 5 and 60 litres and are also available pre-packed in HiLoad 16/10 and 26/10 columns. Q, DEAE and CM Sepharose Fast Flow are supplied in 20% ethanol and SP Sepharose Fast Flow in 20% ethanol with 0.2 M sodium acetate. For ordering information, please refer to Chapter 14.
Sepharose Big Beads ion exchangers Sepharose Big Beads ion exchangers are based on 100-300 µm agarose beads. A higher degree of cross-linking, compared to Sepharose CL-6B based ion exchangers, is used to give the media greatly improved physical and chemical stability. Due to its excellent physical stability and large bead size, Sepharose Big Beads can be run at high flow rates even with viscous samples. Table 12, gives the characteristics of Q and SP Sepharose Big Beads. Table 12. Characteristics of Q and SP Sepharose Big Beads. Product
Q Sepharose Big Beads
SP Sepharose Big Beads
Type of gel
strong anion
strong cation
Total ionic capacity (µmol/ml gel)
180-250
180-250
Recommended working flow rate range (cm/h)
1200-1800
1200-1800
Approx. mean particle size (µm)
200
200
Particle size range (µm)
100-300
100-300
working pH range*
2-12
4-13
pH stability** short term
2-14
3-14
2-12
4-13
long term
* working pH range refers to the pH range over which the ion exchange groups remain charged and maintain consistently high capacity. ** pH stability, long term refers to the pH interval where the gel is stable over a long period of time without adverse effects on its subsequent chromatographic performance. pH stability, short term refers to the pH interval for regeneration and cleaning procedures.
50
Properties Physical stability Sepharose Big Beads ion exchangers are supplied pre-swollen and ready for packing. The average bead diameter is 200 µm with a bead size distribution of 100300 µm. The highly cross-linked nature of the matrix means that the bead size and bed volumes do not change with changes in ionic strength or pH.
Capacity Q and SP Sepharose Big Beads are highly substituted with strong ion exchange groups. These groups remain charged and maintain consistently high capacities over broad working pH ranges of 2-12 and 4-13 respectively. This allows the selection of a pH value and buffer that best suit the properties of the sample. Figure 28 shows typical binding capacities of SP Sepharose Big Beads. W2@6X?e@@@6X?g?W.?g/X 7)X?@ 3=?O.??@@=?O.?3=C5e?@e?J@@?,?@ V4@0Y??(R4@0Y?V40Ye?@e?.MI+Y?@ ?3L?gJ5 ?V/?g.Y
@@6X?W2@6Xe?W-Xe@@6X?W26KO26X? @6K? @?B1?7U?I/e?7R1e@?S,?7
@? ?@@6X?W2@6X?eW-X??@?@eW26X ?@K? ?W&?W26X?W26X?f@? ?@@@@@ ?@?B1?7U?I/?e7R1??@@@e.MB1eW26T2@@@@?e?@@@ ?*@?7
Fig. 28. Typical binding capacities of SP Sepharose Big Beads media. The binding capacity was measured with frontal analysis in acetate pH 5 for bovine serum albumin and format pH 4.1 for b-lactoglobulin at linear flow rates of 12 and 300 cm/h. (Work by Pharmacia Biotech, Uppsala, Sweden.)
Flow rate Because of its flow characteristics, Q and SP Sepharose Big Beads is the choice for initial purification when high viscosity precludes the use of ion exchangers with smaller bead size, e.g. Sepharose Fast Flow ion exchangers. Even with viscosities as high as 2.5 times water a high flow rate (500 cm/h) is maintained in industrial column operation.
Availability Q and SP Sepharose Big Beads are available in packs of 1 and 10 litres. For ordering information, please refer to Chapter 14.
51
STREAMLINE SP and STREAMLINE DEAE STREAMLINE adsorbents are specifically developed for expanded bed adsorption, see page 98 for details, and are based on a modified Sepharose matrix, crosslinked 6% agarose. STREAMLINE adsorbents have been designed with a well-defined density distribution, which is required in expanded bed adsorption. This is achieved through inclusion of inert crystalline quartz material in the base matrix. Mean particle density is approximately 1.2 g/ml drained gel. The porosity is comparable to Sepharose Fast Flow ion exchangers, with an exclusion limit of 4 x 106 for globular proteins. Table 13. Characteristics of STREAMLINE ion exchange adsorbents. Product
STREAMLINE SP
STREAMLINE DEAE
Type of gel
strong cation
weak anion
Total ionic capacity (µmol/ml gel)
170-240
130-210
Dynamic binding capacity* (mg/ml gel) Lysozyme (MW 14 500)
70
N.D.
N.D.
55
Recommended working flow rate range (cm/h) - sample application
200-400
200-400
- elution
50-150
50-150
Approx. mean particle size (µm)
200
200
Particle size range (µm)
100-300
100-300
working pH range**
4-13
2-9
pH stability*** short term
3-14
1-14
long term
4-13
2-13
BSA (MW 67 000)
N.D. = Not determined *The binding capacity was determined in STREAMLINE 50 column at a linear flow rate of 300 cm/h using a 2.0 mg/ml solution of protein in phosphate buffer, pH 7.5 (lysozyme) and 50 mM Tris-HCl buffer, pH 7.5 (BSA). ** working pH range refers to the pH range over which the ion exchange groups remain charged and maintain consistently high capacity. *** pH stability, long term refers to the pH interval where the gel is stable over a long period of time without adverse effects on its subsequent chromatographic performance. pH stability, short term refers to the pH interval for regeneration and cleaning procedures.
52
Properties Physical stability STREAMLINE adsorbents are supplied pre-swollen and ready for use. The average bead diameter is 200 µm with a bead size distribution of 100-300 µm. The highly cross-linked nature of the matrix means that the bead size and bed volumes do not change with changes in ionic strength or pH.
Capacity As is the case with all ion exchangers the binding capacity of STREAMLINE adsorbents are dependent on each different molecule’s size and pI, the flow rate etc. In general, the adsorption characteristics of STREAMLINE adsorbents are similar to those of a packed bed in chromatography, a direct result of the stability of the expanded bed during feed application. This is illustrated in Figure 29 which compares the breakthrough curves for a model protein with STREAMLINE DEAE in packed mode and expanded mode at two scales of operation. W2@6X?W2@@6Xf 7
W26X 7
?@@?eW26X ?N@?e7
?@K?g@@ ?)X??@?@K? W&?@f)X @@@@eW26KO26T2@6KO26T26T2@6X@@@@?@@6X@@?W26T2@6X??@@6X@)??@@@@6KO26X??W26T&e7@@@W26X@) 7
W26XeW26X 7
W26XeW2@@ 7
W26Xe?W&? 7
W2@@he?@@?hf?@ O@h?O@Khe)X *UeO26T-X?@@6X@@?eW&KO26T2@6X@?W26X?e@@6KO2@@W)X?W26T2@@@6KO26T2@6X@) V4@@@@V@R1eW@@@@?e&@@@@YV@@
@?g@Khf)X ?@ O@gO@ O@f @?e@@6X@@6KO26T2@@@6X@)T26T2@?@?@eW-X?W2@@@6X@?W26X?e?W26T-T2@@6T2@6?2@6KO2@@W26KO2@@e@6T-X?W26KO2@@W26X @?e?W@@@?B@@
W26XeW26X 7
@@@6T2@?f)Xhf?@ O@gO@ O@f @??S@U@?W26X@)eW&KO26T2@6X@?W26X?e?W2@6T.?@@6T2@6?2@6KO2@@W26KO2@@e@6T-X?W26KO2@@W26X @@@0R@@?7
W26XeW26X 7
W26KO26X .MS@@
?W&?W26X W&@W&
W2@6T26X 7Y?;@
W26KO26X 7YS@@
?@@?W26KO26X ?N@?7
?W-Xh?@@?gO@e@@6X?W2@@?W-X? W-K?h?W-Xf@?he?O2@ ?)X?f?O@?g?O@?h?@K?h)T-X? ?7R1?@@6T2@6X@eW26KO2@@e@?V1?*U??O&R1? 7R@6T-X?W2@@?7@)T-X?@?eW&KO26KO2@@??@6T-X?W26T2@6X@)T26KO2@@??@@6KO2@@W@KO26T2@?@@6KO2@@@6X@(R1? ?@@@?@?B@@?B@@1?7YV@@
Fig. 29. Breakthrough capacity curve comparisons. Running conditions: BSA in 50 mM Tris-HCl, pH 7.5, linear flow rate 300 cm/h. (Work by Pharmacia Biotech, Uppsala, Sweden.)
STREAMLINE SP and STREAMLINE DEAE are highly substituted with ion exchange groups. These groups remain charged and maintain consistently high capacities over the working pH ranges of 4-13 and 2-9 respectively. This allows the selection of a pH value and buffer that best suit the properties of the sample.
53
Availability STREAMLINE SP and DEAE are available in packs of 300 ml and 7.7 litres. For ordering information, please refer to Chapter 14.
DEAE Sepharose CL-6B and CM Sepharose CL-6B DEAE Sepharose CL-6B and CM Sepharose CL-6B are macroporous bead-formed (mean particle size of 90 µm diameter) ion exchangers derived from the crosslinked agarose gel Sepharose CL-6B. DEAE or CM groups are then attached to the gel by ether linkages to the monosaccharide units to give the final ion exchange gel (Fig. 30). DEAE Sepharose CL-6B and CM Sepharose CL-6B have good chemical and physical stability and can be used to advantage in the ion exchange chromatography of proteins, polysaccharides, nucleic acids, membrane components and other high molecular weight substances. ? ? ?W2@@6X?f@@e@@g? ?7@?I4)?f@@e@@g? ?@@?h@@e@@g? ?@@?h@@@@@@g? ?@@?h@@e@@g? ?3@?O26T26X?@@e@@W2@@e? ?V4@@0R+MS1?@@e@@@Yf? O.hW&@?f?I4@6Xe? O20Yg?W&YheS,e? O20Mh?&@@@?g@@0Ye? W20M ? ?@@? ?O.M ? ?@@? ?O20Y? ? ?@@@@@@?he?O20M? ? ?@@?he?O20M? ? ?@@?h?W20M? ? O.M? ? O20Y ? O20M ? W20M ? .M ? ?@@@@6X?@@@@@?e@@e?@@@@@f@@?W2@@6X?he@@ W2@@6X?@@?fW2@@6X?@@@@6?2@?heW2@6X?h?W2@@6?2@??@@?f?W2@@6X?@@e@@f@6X?@@h?@@??@@?hf?W2@@6X?@@?@@@@?g? ?@@??@1?@@f?J@@L??@@?f?J@5?7@?I4)Khe@@ 7@?I4)?@@?f7@?I4)?@@??@@@@Lh?W&(?')Xh?7@?I4@@@??@@?f?7@?I4)?@@e@@f@@1?@@h?@@??@@?hf?7@?I4)?@@hf? ?@@??@@?@@f?7@@1??@@?f?7@H?3@?eV@@@6?2@@6X?@@@6X?@@@6?2@@@@@@6X?W2@@@@@@6X?e@@f?@@?f@@f?@@??@(Y@1h?7@H?N@1h?@@?e?@@??@@?f?@@?f@@e@@f@@@?@@h?@@??@@?hf?@@?f@@hf? ?@@??@@?@@@@@??@@@@??@@@@@e?@@??V4@@@@@e@@@@?@1?@@?@1?e?@@@@@@@e@1?*@??@@??@1?e@@f?@@?f@@@@6X?@@@@@e@@h?@@?e@@h?@@?e?@@@@@@?f?@@?f@@@@@@f@@@@@@h?@@@@@@?hf?@@?f@@hf? ?@@??@@?@@fJ@@@@L?@@?f?@@?f?@@@@@@@@@?@@?@@?@@W2@@@@@@?@@e@@?V4@@@@@@@@?e@@f?@@??@@@@@e@1?@@??@1?@@e@@@@e?3@L?J@5e@@@@e?@@?e?@@??@@?f?@@?f@@e@@f@@?@@@e@@@@e?@@??@@?hf?@@?f@@hf? ?@@??@5?@@e?O&@e@1?@@?f?@@?'6K??@@@f@@?@5?@@?@@@@?@@@@@?3@e@5f@@@?g3@?O2(?@@?e?I'@e@5?@@??@5?@@h?V')?&(Yh?3@?O2@@@??@@?W26X?3@?O2(?@@e@@@6X?@@?3@@h?@@??@@?hf?3@?O2(?@@hf? ?@@@@0Y?@@@@@@@@e@@?@@@@@@??3@LV4@@@0?4@@@@@@@0Y?@@?@0?4@@@@@@?V4@@0Y?@@@0?4@@@@?eV4@@0Y?@@@@@eV4@@0Y?@@@@0YJ@5heV4@0Y?h?V4@@0?4@??@@?.MS1?V4@@0Y?@@e@0MS1?@@?V4@h?@@??@@?hf?V4@@0Y?@@hf? ?N@1hf@@ ?7@H ?W&@ W&@? ? @@hf@@ ?@@? W&Y?hf?W&Y ? &@@@hf?&@@@?g/K ? V46X ? I/K? ? ?V46K? ? ?I46X? ? ?I/K ? V46X ? I/K? ? ?V46K? ? ?I46X? ? ?I/K ? V46X ? I/K? ? ?V46K? ? ?I46X??W2@@6X?f@@e@@g? ?I/??7@?I4)?f@@e@@g? ?@@?h@@e@@g? ?@@?h@@@@@@g? ?@@?e?O26X?@@e@@W2@@e? ?3@?O2@0MS1?@@e@@@Yf? ?V4@@0M?W&@?@@e@@@@6Xe? ?W&YheS,e? ?&@@@?g@@0Ye? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?@@@@? ?@@? ? ?@@? ? ?W2@@6X?@@@??@@@e@@?W2@@6X?he?@@? W2@@6X?@@?f?W2@@6?2@@@6X?@@heW2@6X?h?W2@@6X?@@e@@f?W2@@6X??W2@6X ?@6X?@@?e?@@@@@@? ? ?7@?I4)?@@@??@@@?J@5?7@?I4)?he?@@? 7@?I4)?@@?f?7@?I4@@@??@1?3@L?g?W&(?')Xh?7@?I4)?@@e@@f?7@?I4)?W&(?')X? ?@@1?@@?f?@@? ? ?@@?f@@@@@@@@?7@H?3@?e?W2@@6?2@@6X?@@@6T2@@6X?@@@@@@6X?W2@@@@@@6X?e@@f?@@?f?@@?e?@@??@5?N@1?g?7@H?N@1h?@@?f@@e@@f?@@?f7@H?N@1? ?@@@?@@?@@@6T@@? ? ?@@?f@@@@@@@@?@@??V4@@6?&@??@@@@?@1?@@?@@Y??@1?@@@@e@1?*@??@@??@1?e@@f?@@?f?@@@@6X@@@@@e?@@?g?@@?e@@h?@@?f@@@@@@f?@@?f@@e?@@? ?@@@@@@?e?@@< ? ?@@?f@@@@@@@@?@@?f?@@@@@@@@@@?@@?@@?@@@@@@@?@@@@e@@?V4@@@@@@@@?e@@f?@@?e@@@@@??@@@@??@1??@@??@@@@??3@L?J@5e@@@@e?@@?f@@e@@W26X?@@?f3@L?J@@@6X ?@@?@@@W2@@@@? ? ?3@?O2(?@@?@@?@@?@@?'6K??@@@@?e?@@?@5?@@?@@@?@@@?@@@@e@5f@@@?g3@?O2(?@@?fI'@??@@@@??@5??@@?g?V')?&(Yh?3@?O2(?@@e@@(MS1?3@?O2(?V')?&@0MS1 ?@@?3@@@@?@@@? ? ?V4@@0Y?@@?@@?@@?3@LV4@@@0MI4@@@@@@@0Y?@@?@@@@@@@?@0?4@@0Y?@@@0?4@@@@?eV4@@0Y?@@@@@e?V4@@0?4@@@0Y?J@5?hV4@0Y?h?V4@@0Y?@@e@0YW&@?V4@@0Y??V4@0M?W&@ ?@@?V40?4@@@@? ? ?N@1hf?@@? 7@H? W&Y? W&Y? ? @@hf?@@? @@ &@@@ &@@@ ? ? ? ? ? ?
Fig. 30. Partial structure of Sepharose CL-6B ion exchangers.
Properties Physical stability DEAE and CM Sepharose CL-6B are supplied pre-swollen and ready for packing. As stated earlier, the cross-linked nature of the matrix means that the bed volume changes very little with changes in ionic strength or pH (approximately 2% change when the pH is reduced from 10 to 4).
54
Capacity Since DEAE and CM Sepharose CL-6B are weak ion exchangers, the number of ligand groups which are charged and hence the capacity for macromolecules is dependent upon pH. This dependency is illustrated by the titration curves for DEAE and CM Sepharose CL-6B (Fig. 31). @?e@? @@6X@?e@? @?B@@@@@@? @??@@?e@? @?C@@?e@? @@0R'?e@? @? @?
@@@6X??@@@@??W-X?@@@@?eW2@6X?h@K ?W2@6X?@gW2@?@@6X @??B1??@f?7R1?@g7U?I/T26X?@@6X@@@?e'6?2@?W26KO.eW26Xe?7
@? @? @? @? @? @? @? @? @? @? @? @? ?O2@@? @? ?O2@@0M? @? ?O2@@0M? @? ?O2@@0M? ?W&?W26Xh@? ?O2@@0M? ?*@?7
W26X @6X?hf?W&? @?eW26Xe?W&??@@?e@@e)Xe@?fW2@@6T&?e@? .MB1 ?S,?hf?7@?hf?@@@@@e@?e7
@?e@? @@6X@?e@? @?B@@@@@@? @??@@?e@? @?C@@?e@? @@0R'?e@? @? @?
?W2@6X?@@?e@@eW2@6X?h@K ?W2@6T&?f?W2@e@@6X ?7
@? @? @? @? @? @? @? O2@@@?hf @? ?O2@@@@@0M @? O2@@@0M? @? ?O2@0M @? O2@0M? @? ?O2@0M @? ?O20M? @? ?O20M? @? ?W20M? ?W&?W26Xh@? O.M? ?*@?7
W26X @6X? W&f .MB1 ?S,? 7@f ?@ ?*U?hf?J@@f W. ?N1?hf?7Y@L?e 7Y ?/KC5?hf?@@@,?e @@@@hf?V40Y? I(Y?e
?@e?W26X?eW&e@@e?@@??)X??@f?W2@@6T&e?@f @@@@@??@e?7
Fig. 31. Titration curves; Approximately 5 ml DEAE and CM Sepharose CL-6B both in 50 ml 1 M KCl. (Work by Pharmacia Biotech, Uppsala, Sweden.)
The working pH ranges for the media are 2-9 for DEAE Sepharose CL-6B and 6-10 for CM Sepharose CL-6B. DEAE and CM Sepharose CL-6B have exclusion limits of approximately 4 x 106. Capacity data for Sepharose CL-6B ion exchangers are summarized in Table 14.
55
Table 14. Characteristics of Sepharose CL-6B ion exchangers.
Total ionic capacity (µmol/ml gel) Dynamic binding capacity* (mg/ml gel) Thyroglobulin (MW 669 000) IgG (160 000)
DEAE Sepharose CL-6B
CM Sepharose CL-6B
130-170
100-140
2.0
N.D.
N.D.
9.5
Bovine COHb (MW 69 000)
N.D.
75
HSA (MW 68 000)
170
N.D.
a-lactalbumin (MW 14 300)
150
N.D.
Ribonuclease (MW 13 700)
N.D.
120
Recommended working flow rate range (cm/h) up to 60
up to 60
Approx. mean particle size (µm)
90
90
Particle size range (µm)
45-165
45-165
working pH range**
2-9
6-10
pH stability*** short term
2-14
2-14
long term
3-12
4-13
N.D. = Not determined *Capacities were determined using the method described in Chapter 10 at a flow rate of 75 cm/h. For the anion exchanger (DEAE) the starting buffer was 0.05M Tris, pH 8.3 and for the cation exchangers (CM) 0.1 M acetate buffer, pH 5.0. Limit buffers were the respective start buffers containing 2.0 M NaCl. ** working pH range refers to the pH range over which the ion exchange groups remain charged and maintain consistently high capacity. *** pH stability, long term refers to the pH interval where the gel is stable over a long period of time without adverse effects on its subsequent chromatographic performance. pH stability, short term refers to the pH interval for regeneration and cleaning procedures.
Flow rate The cross-linked structure of Sepharose CL-6B ion exchangers allows flow rates of up to 100 cm/h to be used. Figure 32 illustrates the variation of flow rate with pressure drop for DEAE and CM Sepharose CL-6B.
56
?? ? ?? ? ??? ?? ?? ?? ?? ?? ?W&?W2@(?W26X? ?*@?7@@U?7@(Y? ?@gJ@(Y ? ?@f?W&(Y? ?@fW&(Y ??? ?@f7@H? ?@e?J@5 ? ?@eW&(Y ?@?W&(Y? ?? ?@?7(Y ?@W(Y? ?? ?@@Y ?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ ??? @? @? @? @? @? @? ? @? @? @? @? @? @? ??? ? ?W2@(? ?W&?W26X W&?W2@(? ?7@@U? ?*@?7
Fig. 32. Flow rate as a function of pressure drop in columns (5x10 cm bed volume) of DEAE and CM Sepharose CL-6B. pH 7.0; Ionic strength 0.02. (Work by Pharmacia Biotech, Uppsala, Sweden.)
As in all types of chromatography, resolution is dependent on flow rate (5). Therefore in applications where resolution is critically important high flow rates should be avoided or an exchanger based on Sepharose High Performance used. For applications where high flow rates and large throughput of material are required, ion exchangers based on Sepharose Fast Flow or Sepharose Big Beads should be used since these forms have been specially developed with these criteria in mind.
Availability DEAE and CM-Sepharose CL-6B are available in packs of 500 ml and 10 litres. The gels are supplied in 20% ethanol. For ordering information, please refer to Chapter 14.
57
7. DEAE Sephacel DEAE Sephacel is a bead-formed cellulose ion exchanger produced from high purity microcrystalline cellulose. Cellulose is a naturally occurring polymer consisting of b(1-4) linked glucose units. In the native state, adjacent polysaccharide chains are extensively hydrogen bonded, forming microcrystalline regions. These regions are interspersed with amorphous regions with less hydrogen bonding. Limited acid hydrolysis results in preferential loss of the amorphous regions and gives so-called microcrystalline cellulose. During the production of DEAE Sephacel the microcrystalline structure is broken down and the cellulose is regenerated to give a bead-formed (40-160 µm) gel. The gel is strengthened by cross-linking with epichlorohydrin, although the main structure-forming bonds are still hydrogen bonds. Functional groups are attached during the synthesis by ether linkages to glucose units of the polysaccharide chains to give the structure shown in Figure 33. ? ? ? W26Xe?@e?@f?W26X?e@?e@?g? ?W.MI/X??@[email protected]/Xe@?e@?g? ?7H??V/??@e?@f7HeV/e@?e@?g? ?@g?@@@@@f@?g@@@@@?g? ?3L??W.??@e?@[email protected]@?e@?W-X?e? ?V/KO.Y??@e?@?.R+R/KO.Ye@?e@?*?,?e? O.eV40Ye?@e?@f?V40Y?e@?e@?V'U?e? O20Y ?@K? ?S,?e? W20M ?@@@ ?.Y?e? ?O.M ? ?W20Y? ? O.M? ? O20Y ? W20M ? ?O.M ? ?W20Y? ? O.M? ? O20Y ? W20M ? ?O.M ? ?W20Y? ? O.M? ? O20Y ? W20M ? ?O.M ? ?@h?W20Y? ? [email protected]? ? ?@@@@@fO20Y ? ?@fW20M ? [email protected] ? ? ? ?@@@6X ?@h?@ @?@?f?@ W2@6X?heW26Xe@?e@?f?W26X?e@?e@?f)Xe@?h?@e?@ ?W26X??@?@@@ ? ?@eB1 ?@h?@ @?@Lf?@ ?W.M?I/Xh?W.MI/X?@?e@?fW.MI/Xe@?e@?f@1e@?h?@e?@ W.MI/X?@ ? ?@eC@T2@6T2@@6X?W2@@@W2@6KO2@@@e?W2@6T2@6Xe@?@1e?@?@W2@6KO2@@?W2@6X?g?7H?eN1h?7H??V/?@?e@?f7HeV/e@?e@?f@@L?@?h?@e?@ 7HeV/?@ ? ?@@@@>@Y?V@YeV1?7
Fig. 33. Partial structure of DEAE Sephacel.
Properties Chemical stability DEAE Sephacel is stable in aqueous suspension within the range pH 2-12. Hydrolysis may occur in strongly acidic solutions and the macromolecular structure is broken down in strongly alkaline solutions. The free base form of the DEAE group is inherently unstable at high pH values. Strong oxidizing agents should be avoided. DEAE Sephacel is susceptible to microbial attack, especially in the presence of phosphates, and should therefore be stored in the presence of antimicrobial agents when not in use (see page 103). Samples containing enzymes capable of hydrolysing b-glucosidic linkages should be purified on MonoBeads, MiniBeads, SOURCE, Sepharose or Sephadex based ion exchangers.
58
Physical stability The cross-linked bead form of DEAE Sephacel gives it increased physical stability compared to ordinary microgranular celluloses. It has a stable bed volume over a wide range of ionic strengths (approx. 5% change between I = 0.05 and I = 0.5) and pH values and can therefore be re-equilibrated in the column. DEAE Sephacel can be sterilized by autoclaving at pH 7 for 30 minutes at 121 °C. During autoclaving, minute quantities of carbohydrate are released; these can be washed away with sterile buffer solution.
Capacity DEAE Sephacel is macroporous and has an exclusion limit for proteins with molecular weights of approximately 1 x 106. The binding of substances with molecular weights substantially greater than 1 x 106 will be restricted to charged groups on the surface of the beads. Capacity data for DEAE Sephacel is summarized in Table 15. Table 15. Capacity data for DEAE Sephacel. Total ionic capacity
DEAE Sephacel
(µmol/mg gel)
(µmol/ml gel)
Dynamic binding capacity* (mg/ml gel) albumin thyroglobulin
130-150
100-140
160
10
*Capacity was determined using the method described in Chapter 10 at a flow rate of 75 cm/h. The starting buffer was 0.05 M Tris, pH 8.3 Limit buffer was start buffer containing 2.0 M NaCl.
The adsorption kinetics for bead-formed cellulose ion exchangers are substantially the same as for conventional cellulose ion exchangers (11). As with other weak ion exchangers the capacity varies with pH. The titration curve for DEAE Sephacel is shown in Figure 34. @?e@? @@6X@?e@? @?B@@@@@@? @??@@?e@? @?C@@?e@? @@0R'?e@? @? @? ?W&?W26X ?*@?.MB1 ?N@?e?@ @?W.g?@@@@@@6K? @?7Yhe@??I4@@@@@6K @?@@@@h@?hI4@@@@6K @? I4@@@@6K @? I4@@@6K? @? ?I4@6K @? I46K @? I4@6K? @? ?I46X? @? ?I/K @? V46X @? I/X? @? ?V/X @? V/X? @? ?V/X @? V/X? @? ?N1? @? 3L @? V/X? @? ?N1? @? 3L @? N1 ?W&?W26Xh@? ?3L? ?*@?7
Fig. 34. Titration of 1 g DEAE Sephacel in 1 M KCl. (Work by Pharmacia Biotech, Uppsala, Sweden.)
59
Flow rate As with other ion exchangers, resolution decreases with increasing flow rate. Flow rates of 10 cm/h are usually suitable for the resolution of protein mixtures on DEAE Sephacel. Figure 35 illustrates the variation of flow rate as a function of pressure drop for DEAE Sephacel. For applications requiring higher flow rates SOURCE or Sepharose based ion exchangers should be used. ?@@@@@ W& @K ?@e?@?W26X?@?@?@??@@??'6T&@W26Xe?W26T2@@@@f@@@?W26X?@?@?@@? ?@e?@?7
? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?@6KO26X?W26X?h@@6K W&he? S@@
W2@?W26X 7Ye75e?W.Y @??@@HeW.Y? @?J@5?e7H @?7@H??J5? @?@@eW.Y? @?@5?W.Y @@@HW.Y? @@@T.Y @@S(Y? W26Xh@@(Y 7
?W&?W26X?W26X? ?*@?7
W26KO26X?W26X? .MB@@
Fig. 35. Flow rate as a function of the pressure drop across beds of DEAE Sephacel. 0.1 M Tris-HCl buffer solution pH 7.6. (Work by Pharmacia Biotech, Uppsala, Sweden.)
Availability DEAE Sephacel is supplied in packs of 500 ml as a suspension in 20% ethanol. For ordering information, please refer to Chapter 14.
60
8. Sephadex ion exchangers Sephadex ion exchangers are produced by introducing functional groups onto Sephadex, a cross-linked dextran matrix. These groups are attached to glucose units in the matrix by stable ether linkages. Sephadex is suitable as a base for an ion exchanger matrix since it is hydrophilic and shows very low non-specific adsorption. Sephadex ion exchangers are derived from either Sephadex G-25 or Sephadex G-50 and swell readily in aqueous solutions. Ion exchangers based on Sephadex G-25 are more tightly cross-linked than those based on Sephadex G-50 and therefore swell less and have greater rigidity. Ion exchangers based on Sephadex G-50 are more porous than those based on Sephadex G-25 and therefore have a better capacity for molecules with molecular weights larger than 30 000. The full range of Sephadex ion exchangers is shown in Table 16. Anion and cation exchangers are designated as A-25 or A-50 and C-25 or C-50, respectively, depending on the matrix porosity. Table 16. Sephadex ion exchangers. Types
Description
Functional groups
Counter ion
DEAE Sephadex
A-25 A-50
Weakly basic anion exchanger
Diethylaminoethyl
Chloride
QAE Sephadex
A-25 A-50
Strongly basic anion exchanger
Diethyl-(2-hydroxypropyl)aminoethyl
Chloride
CM Sephadex
C-25 C-50
Weakly acidic cation exchanger
Carboxymethyl
Sodium
SP Sephadex
C-25 C-50
Strongly acidic cation exchanger
Sulphopropyl
Sodium
Properties Chemical stability Sephadex ion exchangers are insoluble in all solvents. They are stable in water, salt solutions, organic solvents, alkaline and weakly acidic solutions. In strongly acidic solutions, hydrolysis of the glycosidic linkages may occur and thus pH values below 2 should be avoided, particularly at elevated temperatures. Sephadex ion exchangers can also be used in the presence of denaturing solvents which can be important when substances are to be separated on the basis of their electrostatic properties alone (12, 13, 14). Exposure to strong oxidizing agents or dextranases should be avoided. During regeneration, the ion exchanger can be exposed to 0.2 M NaOH for a short time
61
without appreciable hydrolysis. Sephadex ion exchangers are susceptible to attack by dextranases and should be stored in the presence of an antimicrobial agent (see page 103).
Physical stability Swollen Sephadex ion exchangers can be sterilized by autoclaving for up to 30 min at 121 °C, at neutral pH in the salt form. During autoclaving, minute quantities of carbohydrate are released; these can be washed away with sterile buffer.
Swelling The swelling properties of Sephadex ion exchangers are related to those of the parent Sephadex G-types, those based on 50-types swelling more than those based on 25-types. Due to the presence of charged groups in the matrix, the swelling varies with ionic strength and pH.
Ionic strength dependence At low ionic strengths, repulsion between groups carrying the same charge on the matrix is maximal, and swelling of the gel is at its greatest. The degree of swelling decreases with increasing ionic strength. Note: Sephadex ion exchangers should not be swollen in distilled water since the bead structure may be broken down due to strong ionic interactions.
pH dependence The degree of dissociation and hence the extent to which an ion exchanger is charged is dependent on pH. Repulsion between charged groups is greatest at pH values where the ion exchanger is fully dissociated, and decreases at pH values close to the pK of the charged groups. Note: QAE Sephadex and SP Sephadex have swelling properties quite independent of pH since they are charged over a very wide pH range.
Capacity Due to differences in swelling characteristics, ion exchangers based on Sephadex G-25 have a much higher ionic capacity per ml gel than those based on Sephadex G-50. (Table 17).
62
Table 17. Total ionic capacity data for Sephadex based ion exchangers. Ion exchanger
DEAE
A-25
Sephadex
A-50
QAE
A-25
Sephadex
A-50
CM
C-25
Sephadex
C-50
SP
C-25
Sephadex
C-50
Total ionic capacity (µmol/mg dry gel)
(µmol/ml wet gel)
3.5 ±0.5
500 175
3.0 ±0.4
500 100
4.5 ±0.5
550 170
2.3 ±0.3
300 90
Thus for smaller biomolecules (MW < 30 000) the A-25 and C-25 types have a higher available capacity. In the molecular weight range 30 000 to 100 000 however, the A-50 and C-50 exchangers have higher available capacities due to their larger pore size. ? ? ? ?@e?@ @?e@? ? ?@@6X@e?@ @@6X@?e@? ? ?@?B@@@@@@ @?B@@@@@@? ? ?@e@@e?@ @??@@?e@? ? ?@?C@@e?@ @?C@@?e@? ? ?@@0R'e?@ @@0R'?e@? ? ?@ @? ? ?@ @? ? ? ? ?W&?W26X W&?W26X? ? ?*@?.MB1 *@?.MB1? ? ?N@?e?@f@@@@ N@f@?e?@@@@@ ? @?W.h?@ ?@?W.?h?@ ? @?7Yh?@ ?@?7Y?h?@ ? @?@@@@g?@ ?@?@@@@?g?@ ? ?@ O2@@@@@@@@@@@@@@@@@@@@@@@@@? ?@ O2@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@? ? ?@ ?O2@@@@@@@0M ?@ O2@@@@@@@@0M ? ?@ O2@@@@@@@0M? ?@ O2@@@@0M ? ?@ O2@@@@0M ?@ O2@@0M ? ?@ O20M ?@ ?O2@0M ? ?@ O20M ?@ ?O2@@0M? ? ?@ O20M ?@ ?O20M? ? ?@ O20M ?@ ?W20M? ? ?@ O20M ?@ ?7
Fig. 36. Titration of 0.1 gram of Sephadex ion exchangers in 50 ml 1 M KCl. (Work by Pharmacia Biotech, Uppsala, Sweden.)
63
If working with larger molecules (MW > 100 000), a higher available capacity is frequently observed with the A-25 and C-25 types since at these molecular weights binding is only occurring on the bead surface and the higher ionic capacity can be used to advantage. As capacity also depends upon the number of substituent groups which are charged under given buffer conditions, it will also vary with pH. The variation of the charge on Sephadex ion exchangers with pH is illustrated by their titration curves (Fig. 36). Dynamic capacity data for Sephadex ion exchangers are given in Table 18. Table 18. Dynamic capacity (mg/ml wet gel) data for Sephadex ion exchangers Protein (MW)
Thyroglobulin (669 000)
HSA (68 000)
a-lactalbumin (14 300)
IgG (160 000)
Bovine COHb (69 000)
Ion exchanger A-25
1.0
30.0
140.0
N.D.
N.D.
Sephadex A-50
DEAE
2.0
110.0
50.0
N.D.
N.D.
A-25
1.5
10.0
110.0
N.D.
N.D.
Sephadex A-50
QAE
1.2
80.0
30.0
N.D.
N.D.
C-25
N.D.
N.D.
N.D.
1.6
70.0
Sephadex C-50
CM
N.D.
N.D.
N.D.
7.0
140.0
SP
C-25
N.D.
N.D.
N.D.
1.1
70.
Sephadex C-50
N.D.
N.D.
N.D.
8.0
110.0
N.D. = Not determined Capacities were determined using the method described in Chapter 10 at a flow rate of 75 cm/h. For anion exchangers (DEAE and QAE) the starting buffer was 0.05M Tris, pH 8.3 and for cation exchangers (CM and SP) 0.1 M acetate buffer, pH 5.0. Limit buffers were the respective start buffers containing 2.0 M NaCl.
Availability Sephadex ion exchangers are supplied as dry powders in packs of 100 g and 500 g. Bulk quantities of 5 kg or more are available on request. For ordering information, please refer to Chapter 14.
64
9. Experimental design Choice of ion exchanger No single ion exchanger is best for every separation. The choice of matrix and ionic substituent depends on: 1. The specific requirements of the application 2. The molecular size of the sample components 3. The isoelectric points of the sample components
Specific requirements of the application Column separation, batch separation or expanded bed adsorption If the separation is to be carried out using a batch separation technique rather than column chromatography, the flow and packing characteristics of the matrix are of minor importance. The economy and high capacity of Sephadex based ion exchangers make them a natural choice. In large scale applications, capturing proteins from crude samples containing particulate matter, expanded bed adsorption using STREAMLINE has proven to be effective and cost efficient.
The scale of the separation The amount of sample to be processed is an important parameter when choosing an ion exchange medium. For laboratory scale separations, any of the Pharmacia Biotech range of ion exchangers can be used. However for large scale separations, which must satisfy the throughput and cleaning-in-place (CIP) requirements of industry, the choice of a BioProcess Media such as SOURCE, Sepharose High Performance, Sepharose Fast Flow, Sepharose Big Beads or STREAMLINE adsorbent is indicated. The same reasoning applies to experiments designed as method scouting for eventual scale-up since such procedures should be developed using the gel which will eventually be used at the larger scale. SOURCE media and Sepharose Fast Flow based exchangers are extremely well suited to this type of method optimization as well as routine laboratory separations.
The required resolution When choosing an ion exchanger it is important to decide the degree of resolution required from the separation. Normally analytical or semi-analytical separations
65
place high demands on resolution. In contrast, resolution is frequently traded off against capacity and speed in the case of preparative work. Resolution in ion exchange chromatography depends upon the selectivity and efficiency of the media. Maximum selectivity is often obtained by choosing one of the gels carrying the strong exchanger groups Q or S/SP, since strong ion exchangers can be used at any pH tolerated by the sample molecules. Maximum efficiency is obtained by choosing a gel based on a small particle size matrix. The media in order of their particle sizes and potential efficiencies are MiniBeads (3 µm) > MonoBeads (10 µm) > SOURCE 15 (15 µm) > SOURCE 30 (30 µm)> Sepharose High Performance (34 µm) > Sepharose Fast Flow/ Sepharose CL-6B/ Sephacel (90 µm) > Sephadex (40-125 µm in dry form) > STREAMLINE adsorbents/Sepharose Big Beads (200 µm). The media thus offering the highest degree of resolution are MiniBeads, MonoBeads and SOURCE 15 exchangers for high resolution in SMART, FPLC and HPLC systems and SOURCE 30 and Sepharose High Performance exchangers for high resolution standard chromatography.
The required throughput How much material which can be processed in a defined time is determined amongst other things by the capacity, the flow characteristics of the media and the size of the column. All of the ion exchangers available from Pharmacia Biotech have high capacities for macromolecules but differ considerably in their flow properties. The media which have optimal flow characteristics are MiniBeads for micropreparative chromotography in SMART System, MonoBeads and SOURCE 15 for high performance, FPLC separations, and SOURCE 30, Sepharose High Performance, Sepharose Fast Flow and Sepharose Big Beads media for laboratory and process scale preparative separations. The uniform size distribution of beads in SOURCE media provides comparatively low pressure drops over packed beds and thus makes SOURCE ion exchangers also ideal for scaling up in industrial applications such as the separation of closely related product variants.
Scaleability Frequently ion exchange separations are carried out initially on a small scale to optimize conditions before committing the sample to full scale separations. It is thus important to choose an ion exchanger which will allow simple and convenient scale up so that methods established on a small column can be applied more or less directly to the larger column. Detailed information on scaling up ion exchange separations is given in Chapter 11.
66
Reproducibility Reproducibility is obtained when the characteristics of the chromatography bed remain unchanged during the course of the separation and during regeneration of the column. The more rigid varieties of Pharmacia Biotech ion exchangers, such as MiniBeads, MonoBeads, SOURCE, Sepharose High Performance, Sepharose Fast Flow and Sepharose Big Beads, show no changes in bed size with changes in pH and ionic strength and can thus be washed and regenerated in the columns providing additional reproducibility. The use of media which are supplied pre-packed and tested, such as MiniBeads, MonoBeads, RESOURCE, HiTrap columns pre-packed with Sepharose High Performance and HiLoad columns pre-packed with Sepharose High Performance or Sepharose Fast Flow assures reproducibility since variability in column packing is eliminated.
Economy Column or batch procedures in which the ion exchanger is used once and thrown away, as well as applications requiring large amounts of gel, may make economy a major consideration. Sephadex A-50 and C-50 ion exchangers are the least expensive in terms of bed volume, followed by Sephadex A-25 and C-25 ion exchangers. Using expanded bed adsorption, STREAMLINE product line, reduces the number of operations in a process by fusing the function clarification, concentration and adsorption into one operation. It offers process developers the selectivity afforded by chromatography, the throughput of ultra-filtration and the convenience of small scale centrifugation.
The molecular size of the sample components The accessibility of the sample components to the charged groups will determine the available capacity of the ion exchanger for those particular substances. All of the ion exchange media supplied by Pharmacia Biotech, with the exception of Sephadex based media, have exclusion limits for globular proteins in excess of 1 x 106. Steric factors only affect the separation of charged solutes via their influence on the available capacity for each substance. When choosing ion exchangers it is unnecessary to consider the possibility of gel filtration effects on the sample. Sample molecules, although always larger than those of the eluent buffer, cannot migrate ahead of the eluting buffer since they then encounter conditions which favour their re-binding to the matrix. Only uncharged solutes will be fractionated according to size as in gel filtration. These uncharged molecules will normally be removed during the initial isocratic elution phase which proceeds the application of the gradient.
67
The exclusion limits for the different media and subsequent effects on available capacity are given in the relevant sections covering each gel type. When working with samples of unknown molecular weight the use of MonoBeads, SOURCE and Sepharose based ion exchangers is recommended since they are particularly easy to handle and have good capacities over a large molecular weight range.
Choice of exchanger group Substances are bound to ion exchangers when they carry a net charge opposite to that of the ion exchanger. This binding is electrostatic and reversible. In the case of substances which carry only one type of charged group the choice of ion exchanger is clear-cut. Substances which carry both positively and negatively charged groups, however, are termed amphoteric and the net charge which they carry depends on pH (Fig. 37). Consequently at a certain pH value an amphoteric substance will have zero net charge. This value is termed the isoelectric point (pI) and at this point substances will bind to neither anion or cation exchangers. The pH ranges in which the protein is bound to anion or cation exchangers and an arbitrary range of stability are shown in Figure 37. ? ? ? O@KO.? ? ?@@@@0Y? ? I@M? ? @? ? @? ?W2@@? ? @@@@@?e?@ ?7@(Y@@?@e@??@@@@@e?@e@?@? ? ?@hf?V/X ?@@@@0Y? @??B@@U?@@?@e3=?@@Xf?3=C5?@? ? ?@ V/X? I'X? @?e(R4@@@?@eV4@@V4@?e?V40Y?@? ? ?@ ?V/X ?S,?@? W5 ? ?@ V/X? ?7H?@? ?@0Y ? ?@ ?V/X ?3=C5? ? ?@ V/X? ?V40Y? ? ?@ ?V/X ?W&?f@Ke@?@?@?@? ? ?@ V/X? ?W&?@? ?W.??*@?'6X?@@6X@?@?@@@?/X?W.? ? ?@ ?V/X ?7@?@? ?*U??N@?S@1?@?B@@?@?@?@?N1?7H? ? ?@ V/X? ?3@W5? ?V/Xe@@(Y@?@??@@?@?@?@??@?@ ? ?@ ?V/X ?V@@U? S,e@@U?@?@?C@@?@?@?@??3T5 ? ?@ V/X? ?@@@@@)? .Ye(R4@@?@@0R'?@?@?@??N@H ? ?@ ?V/X ?J@? ? W2@@h?@ V/X? ?W&?@? ?@@? ? 7
Fig. 37. The net charge of protein as a function of pH.
The pH of the buffer thus determines the charge on amphoteric molecules during the experiment. In principle therefore, one could use either an anion or a cation exchanger to bind amphoteric samples by selecting the appropriate pH. In practice however, the choice is based on which exchanger type and pH give the best separation of the molecules of interest, within the constraints of their pH stability.
68
Methods for determining the optimum pH and corresponding ion exchanger type are discussed later in this chapter. Many biological macromolecules become denatured or lose activity outside a certain pH range and thus the choice of ion exchanger may be limited by the stability of the sample. This is illustrated in Figure 37. Below its isoelectric point a protein has a net positive charge and can therefore adsorb to cation exchangers. Above its pI the protein has a net negative charge and can be adsorbed to anion exchangers. However, it is only stable in the range pH 5-8 and so an anion exchanger has to be used. In summary: 1. If the sample components are most stable below their pI’s, a cation exchanger should be used. 2. If they are most stable above their pI’s, an anion exchanger should be used. 3. If stability is high over a wide pH range on both sides of pI, either type of ion exchanger can be used.
Determination of starting conditions The isoelectric point The starting buffer pH is chosen so that substances to be bound to the exchanger are charged. The starting pH should be at least 1 pH unit above the isoelectric point for anion exchangers or at least 1 pH unit below the isoelectric point for cation exchangers to facilitate adequate binding. Substances begin to dissociate from ion exchangers about 0.5 pH units from their isoelectric points at ionic strength 0.1 M (15). There are comprehensive lists of isoelectric points determined for proteins (16, 17) which can be useful in the design of ion exchange experiments. If the isoelectric point of the sample is unknown, a simple test can be performed to determine which starting pH can be used.
Test-tube method for selecting starting pH 1. Set up a series of 10 test-tubes (15 ml). 2. Add 0.1 g Sephadex ion exchanger or 1.5 ml Sepharose or Sephacel ion exchanger to each tube. 3. Equilibrate the gel in each tube to a different pH by washing 10 times with 10 ml of 0.5 M buffer (see page 78 for choice of buffers for ion exchange). Use a range of pH 5-9 for anion and pH 4-8 for cation exchangers, with 0.5 pH unit intervals between tubes.
69
4. Equilibrate the gel in each tube at a lower ionic strength (0.05 M for Sephadex or 0.01 M for Sepharose and Sephacel ion exchangers) by washing 5 times with 10 ml of buffer of the same pH but lower ionic strength. 5. Add a known constant amount of sample to each tube. 6. Mix the contents of the tubes for 5-10 minutes. 7. Allow the gel to settle. 8. Assay the supernatant for the substance of interest. The results may appear as shown in Figure 38 (a). The pH to be used in the experiment should allow the substance to be bound, but should be as close to the point of release as possible. If too low (or high) a pH is chosen, elution may become more difficult and high salt concentrations may have to be used. In Figure 38 the buffer chosen should be pH 7.0. @? @?e@? @?e@? @?e@? @?e@? @?e@? @?e@? @?e@? @?e@? @?e@? @? @? @?e@? @?e@? @?e@? @?e@? @?e@? @?e@? @?e@? @?e@? @?e@? @? @? @?e@? @?e@? @?e@? @?e@? @?e@? @?e@? @?e@? @?e@? @?e@? @? @? @?e@? @?e@? @?e@? @?e@? @?e@? @?e@? @?e@? @?e@? @?e@? @? @? @?e@? @?e@? @?e@? @?e@? @?e@? @?e@? @?e@? @?e@? @?e@? @? @? @?e@? @?e@? @?e@? @?e@? @?e@? @?e@? @?e@? @?e@? @?e@? @? @? @?e@? @?e@? @?e@? @?e@? @?e@? @?e@? @?e@? @?e@? @?e@? @? @? @?e@? @?e@? @?e@? @?e@? @?e@? @?e@? @?e@? @?e@? @?e@? @? @? @?e@? @?e@? @?e@? @?e@? @?e@? @?e@? @?e@? @?e@? @?e@? @? @? @?e@? @?e@? @?e@? @?e@? @?e@? @?e@? @?e@? @?e@? @?e@? @? @L ?J@?e@L ?J@?e@L ?J@?e@L ?J@?e@L ?J@?e@L ?J@?e@L ?J@?e@L ?J@?e@L ?J@?e@L ?J@?h?/X?fO@f?W&?hf?@ @?e@?he@)K? O&@?e@)K? O&@?e@)K? O&@?e@)K? O&@?e@)K? O&@?e@)K? O&@?e@)K? O&@?e@)K? O&@?e@)K? O&@?e@1 O&@?g'6XN1?eW2@@?W26T&@W26X?@@?@@@@@?@?@@@?W26X? @@6X@?e@?he@@@@@6K?hO2@@@?e@@@@@6K?hO2@@@?e@@@@@6K?hO2@@@?e@@@@@6K?hO2@@@?e@@@@@6K?hO2@@@?e@@@@@6K?hO2@@@?e@@@@@6K?hO2@@@?e@@@@@6K?hO2@@@?e@@@@@6K?hO2@@@?e@@@@@6K?hO2@@@?gS@1?@?e7
? ? ? ? ? ? ? ? ?
? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?W2@(?eW26X ?W2@(?eW2@(hfW2@?e?W26X?hfW2@?e?W2@(?hf@@@@e?W26X?hf@@@@e?W2@(?hf?W26X?eW26Xhf?W26X?eW2@(hf?W26X?eW26Xhe ?W26X?eW2@( ? ?7@@U?e7(MB1he?@e@?eJ@@?he?@e@?eJ@@?@MB1h ?@e@?e@MB1 ? @??@e?@e@He@?he?@e@?f@?he@??@f?@f@?he@??@e?W.? @??@e?W.?f@?he@??@f?N1?h?@e@?fN@H??@he?@e@?e7Y@Lhe?@e@?e7Y@Le?@h ?3=C@LfC5 ?@e@?f?@ ? 3=C@L??3=C5??C5?he?3=C@Lf@?he3=C@L?e?@e?C5?he3=C@L??7Y? 3=C@L??7Y?e?C5?he3=C@L??/KC5?h?3=C@Le/KC5eC5he?3=C@Le@@@,he?3=C@Le@@@(eC5h ? V40R/??V40Y?@0Y?he?V40R/f@?heV40R/?e?@e@0Y?heV40R/??@@@@?hfV40R/??@@@@?@0Y?heV40R/??V40Y?h?V40R/eV40Y?@0Yhe?V40R/e?I(Yhe?V40R/e?I(Y?@0Yh ?V40R/e?@0Y ? ? ? ? ? ? ? ? ? ? ? ? @? @?e@? @?e@? @?e@? @?e@? @?e@? @?e@? @?e@? @?e@? @?e@? @? ? @? @?e@? @?e@? @?e@? @?e@? @?e@? @?e@? @?e@? @?e@? @?e@? @? ? @? @?e@? @?e@? @?e@? @?e@? @?e@? @?e@? @?e@? @?e@? @?e@? @? ? @? @?e@? @?e@? @?e@? @?e@? @?e@? @?e@? @?e@? @?e@? @?e@? @? ? @? @?e@? @?e@? @?e@? @?e@? @?e@? @?e@? @?e@? @?e@? @?e@? @? ? @? @?e@? @?e@? @?e@? @?e@? @?e@? @?e@? @?e@? @?e@? @?e@? @?h?/X?fO@f?W&?hf?@ ? @? @?e@? @?e@? @?e@? @?e@? @?e@? @?e@? @?e@? @?e@? @?e@? @?f?W26XN1?eW2@@W26X?*@W26T2@?@@@@@??@?@@@?W26X? ? @? @?e@? @?e@? @?e@? @?e@? @?e@? @?e@? @?e@? @?e@? @?e@? @?f?7@
Fig. 38. Test-tube methods for selecting ion exchange conditions.
Electrophoretic titration curves (ETC) While information on the pI of the sample components gives valuable indications concerning the choice of starting conditions, it does not give a picture of how the charge on the molecules varies with pH (Fig. 37), nor indicate at what pH or on which exchanger type maximum resolution could be expected. Electrophoretic titration curves (Fig. 39) enable the determination of the charge pH relationship for the molecules present across a continuum of pH and are a particularly useful way of predicting suitable conditions for an ion exchange separation (18). An electrophoretic titration curve is obtained by electrophoresis of the sample at right angles to a pH gradient in a horizontal slab gel of agarose or polyacrylamide (19, 20). The pH gradient is established in the gel by isoelectric focusing prior to
70
sample application. A schematic description of the various steps is shown in Fig. 40. A detailed description of the method using PhastSystem electrophoresis systems is available upon request from Pharmacia Biotech.
?@g ?W2@@6X??@ ?@g W.M??I/X?@ ?@g 7H?@@?N1?@ ?@g @??@@??@?@ ?@g @@@@@@@@?@ ?@g 3X?@@?W5?@ ?@g V/X@@W.Y?@ ?@g ?V4@@0Y??@ ?@g ?@ ?@g ?@ ?@g ?@ ?@g ?@ ?@g @?f?@ ?@g ?J@Lf?@ ?@g ?7@1f?@ ?@g ?@@@L?e?@ ?@g J@@@1?e?@ ?@g @@@@@?e?@ ?@g @?f?@ ?@g @?f?@ ?@g @?f?@ ?@g @?f?@ ?@g @?f?@ ?@g @?f?@ ?@g @?f?@ ?@g @?f?@ ?@g @?f?@ ?@g @?f?@ ?@g @?f?@ ?@g @?f?@ ?@g @?f?@ ?@g @?f?@ ?@g @?f?@ ?@g @?f?@ ?@g @?f?@ ?@g @?f?@ ?@g @?f?@ ?@g @?f?@ ?@g ?@ ?@g ?@ ?@g ?@ ?@g ?@ ?@g ?@ ?@g W26Xf?@ ?@g 7Y@1f?@ ?@g 3X@5f?@ ?@g S@@Yf?@ ?@g 7@@@6Xe?@ ?@g @??@V1e?@ ?@g 3=C5?@e?@ ?@g S@0Y?@e?@ ?@g 7Yg?@ ?@g 3@@@f?@ ?@g ?@@@f?@ ?@g 7@X@f?@ ?@g @V@5f?@ ?@g 3X@?f?@ ?@g S@@@f?@ ?@g 7
Fig. 39. The electrophoretic titration curve of chicken breast muscle. (19)
?@@@@? @? ?@ ?@e?O2@6KO26X?@e@?W2@@@?@@6X?W2@@? ?@@@@@
W26Xg?@?@he?@ ?W.MI/gJ@?@he?@heW2@@6X ?7H?e?@@6X?@@@@@6X?W2@6T2@@?W2@6Xe?W.MeI/X? ?@g?V1?N@?@?B1?7@@@6K ?@fS@@@U? 3X@W5?f@? @??C5?@?e@? ?@g?3=?C5 @? 3L ?N@<eI46K ?@f7@@@@?e?@e@@@@@0Y? @? ?@f?3L?eJ5 ?@?@W5 @? ?@hf?V'Ug?@e?I@M @? ?@f?V/K?O.Y ?@?@@H @? ?3L?hfN1g?@ @? ?@gV4@0Y? J@@L @? ?V/Xhf?3L?f?@fW.?/X? @? ?@ ?W.?7
Prefocused ampholytes in a gel provides a stable pH gradient for electrophoresis
W2@6X?h ?W.M?I/Xh ?7H?@?N1h ?@e@??@h ?@@@@@@@h ?3X?@?W5e?@@?e ?V/[email protected]?@@?e V4@0Y?eJ@@Le 7@@1e ?J@@@@e ?@@@@5e ?W2@?@f@He ?7Y@?@f@?e ?@?@?@f@?e ?3X@W5f@?e ?V4@0Yh ?@@@@@6Xg ?@e?@V1g ?3=?C5?@g ?S@@0Y?@g ?7Y?he ?@@@@@h W5h ?W2@@Uh ?7Y@V1h ?@?@W5h ?3X@@Uh ?S@@@)h ?7
Electrophoresis is performed perpendicular to the pH gradient. Positively charged molecules migrate to the cathode and negatively charged to the anode.
? ? ? ? ? ? ? ? ? ? ? ? ? ? @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@? @?e?@e@? ?@? @?e?@e@? ?@? @?e?@@@@L ?@? @?e?@eB1e@? ?@? @?e?@eC5e3L ?@? @?e?@@@0YeN1 ?@? @?he?@ ?@? @?he?@ ?@? @?he?3L? ?@? @?he?N1? ?@? @?hf@? ?@? @?hf3L ?@? @?hfN1 ?@? @?hf?3L? ?@? @?hf?N1? ?@? @? 3L ?@? @? N1 ?@? @? ?3L? ?@? @? ?V/X ?@? @? V/X? ?@? @? ?N1? ?@? @? 3L ?@? @? V/X? ?@? @? ?V/X ?@? @? V/K? ?@? @? ?@e?V46X? ?@? @? ?@f?I/X ?@? @? ?@gV/K? ?@? @? ?@g?V46X? W2@6X?g?@? @? ?@h?I/K 7
Electrophoretic mobility of component B at pH values 5 and 9 correlates with its net charge
Fig. 40. The major steps in making electrophoretic titration curves.
Electrophoresis of the sample perpendicular to the pH gradient produces a series of curves, unique for each component, since the relative electrophoretic mobility of each component will be different depending on its net charge at given pH values. The pH value where each curve intersects the line of sample application represents the pH at which that particular component has a zero net charge, the pI for that component.
71
?@g@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@f?@ J@L?f@? ?@fJ@L?hg 7@1?f@? ?@f7@1?hg @@@Lf@? ?@f@@@Lhg ?J@@@1f@? ?@e?J@@@1hg ?@@@@@f@? ?@e?@@@@@hg ?@g@? ?@f?@ ?@g@? ?@f?@ ?@g@? ?@@?e@@ ?W2@6X ?@f?@ ?@g@? ?@@?e@@W26T2@@??W26X??7U?I/ ?@f?@ ?@g@? ?@@L?J@@@1 ?@h@?e?3=C5?@?e @@@@@(e@?'@@(f@? ?@hf?I46K?hf?V'@L? ?@g?C5?e?V40Y?@?e W@@UfS@@Uf@? ?I46X?hfV4)X ?@f?'@@U?hf 7)X? ?@f?7@?@?hf V40YfV40Yf@? ?I/Xg?V'?)X ?@f?3@W5?e?W2@@?f @? V/K?gN@R/X? ?@f?V@@U?e?7
?@g@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@f?@ J@L?f@? ?@fJ@L?hg 7@1?f@? ?@f7@1?hg @@@Lf@? ?@f@@@Lhg ?J@@@1f@? ?@e?J@@@1hg ?@@@@@f@? ?@e?@@@@@hg ?@g@? ?@f?@ ?@g@? ?@f?@ ?@g@? ?@f?@ ?@g@? ?@f?@ ?@g@? ?@f?@ ?@g@? ?@f?@ ?@g@? ?@f?@ ?@g@? ?@f?@ ?@g@? ?@f?@ ?@g@? ?@f?@ ?@g@? ?@f?@ ?@g@? ?@f?@ ?@g@? ?@f?@ ?@g@? ?@f?@ ?@g@? ?@f?@ ?@g@? ?@f?@ ?@g@? ?@f?@ ?@g@? ?@f?@ ?@g@? ?@f?@ ?@g@? ?@f?@ ?@g@? ?@f?@ ?@g@? ?@f?@ ?@g@? ?@f?@ ?@g@? ?@f?@ ?@g@? ?@f?@ ?@g@? ?@f?@ ?@g@? ?@f?@ W.?@f?@g@? ?@f?@ 7H?@f?@g@? ?@f?@ 3=C5hf@? ?@ V40Yhf@? ?@ @? ?@hfW2@6X?f @?2@@(hf@?he?/X? ?@he?W.M?@)Xf @@@@0Yhf@?he?V/K ?@f?W26X??7H??@V1f ?I'X @?hfV46X ?@f?7@V1??@?@@@@@f S1?@hf@?h?@6KeI/X? ?@f?3@@5??3L??@W5f 7@?@hf@?heI46K?S)K ?@e?@?V40Y??V/K?@(Yf 3@W5hf@?hfI4@@>@6K ?@e?@hV4@0Y?f V40Yhf@? ?I4@>@6K ?@ /Kg@? ?I4@@@6Kf?O2@@@@@@@@@@@@@@@@@@@@6K? ?@ @KgV46Kf@? ?@@@@@@@@@Y? ?I4@@@@@@6K? ?@f?W&?@?e?W&?@?f @@@@gS@6Xe@? O2@@@@@@@@@@@@@@@6K? ?I4@6K ?@f?7@?@?e?7@?@?f @@@UI/e@?h?@@@@@0MhfI4@@@@@@@@6K I46K ?@f?3@W5?e?3@W5?f W26Xf@?S,f@? I4@@@@@@@6K? I4@6K? ?@f?V40Y?e?V40Y?f 7@@@6KhV/X? ?@g?C5?e?V40Y?@?e @? @? ?I4@X?I46Xg?V/X ?@f?'@@U?hf @@@@@(e@?'@@(f@? ?I46X?I/K?gV/X? ?@f?S(R1?e?@K?g W@@UfS@@Uf@? ?I/X?V46X?f?N1? ?@f?7H?@?e?3@@@?f 7
@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ ?@g@? ?@f?@ J@L?f@? ?@fJ@L?hg 7@1?f@? ?@f7@1?hg @@@Lf@? ?@f@@@Lhg ?J@@@1f@? ?@e?J@@@1hg ?@@@@@f@? ?@e?@@@@@hg ?@g@? ?@f?@ ?@g@? ?@f?@ ?@g@? ?@f?@ ?@g@? ?@f?@ ?@g@? ?@f?@ ?@g@? ?@f?@ ?@g@? ?@f?@ ?@g@? ?@f?@ ?@g@? ?@f?@ ?@g@? ?@f?@ ?@g@? ?@f?@ ?@g@? ?@f?@ ?@g@? ?@f?@ ?@g@? ?@f?@ ?@g@? ?@f?@ ?@g@? ?@f?@ ?@g@? ?@f?@ ?@g@? ?@f?@ ?@g@?hf/K ?@f?@ ?@g@?hfV46X ?@f?@ ?@g@? I/K? ?@f?@ ?@g@? ?V46X? ?@f?@ ?@g@?h?@6Kf?I/K ?@f?@ ?@g@?heI4@@6KeS@6K ?@f?@ ?@g@? I4@@@US@6K ?@f?@ W.?@f?@g@? I4@UI46K ?@f?@ 7H?@f?@g@? I/K?I46X ?@f?@ 3=C5f?@g@? ?V46X?I/K? ?@f?@ V40Yf?@g@?h@@@6K?h?I/X?V46X? ?@f?@ @?he?I4@@@@6K?fV/X??I/X ?@hfW2@6X?f @?2@@(hf@? ?I4@@@6K?S)KeN1 ?@he?W.M?@)Xf @@@@0Yhf@? I4@@>@6X?3L? ?@f?W26X??7H??@V1f ?I'X @? ?I4@@)KV)X ?@f?7@V1??@?@@@@@f S1?@hf@? ?I'@@)K? ?@f?3@@5??3L??@W5f 7@?@hf@? V4@@@@@6K? ?@e?@?V40Y??V/K?@(Yf 3@W5hf@? I'@@XI4@@6K? ?@e?@hV4@0Y?f V40Yhf@? ?V'>)Ke?I4@6K ?@ /Kg@? V'?@6XfI46K ?@ @KgV46Kf@? ?N@
Fig. 41. Column selection based on electrophoretic titration curve analysis.
Maximum resolution can be expected at a pH where there is maximum separation between the titration curves for individual solutes, using the ion exchanger type indicated by the charge of the molecules at that particular pH. At this pH the difference in electrophoretic mobilities and hence net charges between the species is greatest. This principle is illustrated in Figure 41. The protein’s stability at the indicated pH must be taken into consideration before applying these conditions to the separation.
72
If maximum separation is observed at a pH where the sample molecules are positively charged, i.e. below their isoelectric points, maximum resolution will be obtained using a cation exchanger such as Mono S, SOURCE S or SP Sepharose Fast Flow. If the largest difference in electrophoretic mobility is found at a pH where the components of interest are negatively charged, i.e. above their isoelectric points, an anion exchanger such as Mono Q, SOURCE Q or Q Sepharose Fast Flow should be chosen. If maximum separation of the curves occurs at the position of sample application i.e. at the isoelectric points of the molecules, then maximum resolution may be achieved using the technique of chromatofocusing. Further information on techniques and media for chromatofocusing is available on request. Measurement of pH can be done using a surface electrode or by running pI calibration proteins as a narrow band at the top or bottom of the slab gel during the first dimension electrophoresis as the pH gradient is established in the gel. This section of the gel is removed and stained before the sample is applied for the second dimension electrophoresis and then afterwards replaced to estimate pH values. Staining the titration curve with a general protein stain such as Coomassie Blue does not give any information about the charge/pH relationship for specific proteins unless they can be clearly identified by their isoelectric points. To gain positive identification it is necessary to use a specific detection technique such as zymographic analysis or immunofixation as illustrated in Figure 42.
?? ?@f?? ?@ ?@f? W2@@6Xe?@ ?@f? ?W.Y@@V/X??@ ?@f? ?7Y?@@?V1??@ ?@f? ?@@@@@@@@??@ ?@f? ?3X?@@?W5??@ ?@f? ?V/X@@W.Y??@ ?@f? V4@@0Ye?@ ?@f? ?@ ?@f? ?@ ?@f? ?@ ?@f? ?@ ?@f? ?@ ?@f? ?@@?f?@ ?@f? J@@?f?@ ?@f? 7@@Lf?@ ?@f? ?J@@@1f?@ ?@f? ?@@@@5f?@ ?@f? @Hf?@ ?@f? @?f?@ ?@f? @?f?@ ?@f? @?f?@ ?@f? @?f?@ ?@f? @?f?@ ?@f? @?f?@ ?@f? @?f?@ ?@f? @?f?@ ?@f? @?f?@ ?@f? @?f?@ ?@f? @?f?@ ?@f? @?f?@ ?@f? @?f?@ ?@f? @?f?@ ?@f? @?f?@ ?@f? @?f?@ ?@f? @?f?@ ?@f? @?f?@ ?@f? @?f?@ ?@f? ?@ ?@f? ?@ ?@f? ?@ ?@f? ?@ ?@f? ?@ ?@f? ?@ ?@f? ?W26X?f?@ ?@f? ?7Y@1?f?@ ?@f? ?3X@5?f?@ ?@f? ?S@@Y?f?@ ?@f? ?7@@@6X?e?@ ?@f? ?@e@V1?e?@ ?@f? ?3=C5?@?e?@ ?@f? ?S@0Y?@?e?@ ?@f? ?7Y?g?@ ?@f? ?3@@@?f?@ ?@f? @@@?f?@ ?@f? ?7@X@?f?@ ?@f? ?@V@5?f?@ ?@f? ?3X@g?@ ?@f? ?S@@@?f?@ ?@f? ?7
?@ ?@ W2@( 7@@U @MB1 ?@ C5 ?@0Y
?@ ?@ W2@? 7Y @@6X @?B1 3=C5 V40Y
?@ ?@ @@@@ @? ?J5? ?7H? ?@
?@ ?@ ?W26X? ?*US,? ?S@@U? ?7
?@f? ?@f? ?W26X?e?? ?7
In addition to information regarding optimal starting conditions, the electrophoretic titration curves also reveal important information which will assist the interpretation of the chromatogram after the run.
Fig. 42. The electrophoretic titration curve of chicken breast muscle using zymogram detection for creatine kinase. (19)
73
Since the lines on the ETC reflect the degree of charge of the components at different pH’s, the curves may be used to predict the order in which the components will be eluted from the column. The molecular species with the lowest electrophoretic mobility at a certain pH has logically the lowest charge at that particular pH and should be the first substance eluted from the column in the gradient. Similarly the species showing highest electrophoretic mobility will be the most strongly retained on a column of opposite charge and should be eluted last. The order in which solutes are eluted cannot be predicted with 100% certainty from the titration curve since electrophoretic mobility depends on the total net charge on a molecule and ion exchange chromatography depends on the net charge on the solutes surface.
Chromatographic titration curves (retention maps) For those ion exchanger types which allow rapid separations, optimal starting pH and choice of anion or cation exchanger can be determined using chromatographic titration curves (18). In a specified salt gradient, the retention of particular molecular species is dependent upon the molecules net charge and charge density. These in turn depend upon the pH of the eluent. The chromatographic titration curve is based on this relationship between retention and buffer pH. The methodology is extremely simple. The sample is analysed in a series of rapid separations, carried out on a cation exchanger (Mono S) or an anion exchanger (Mono Q), using the same salt gradient but over a range of pH’s. A plot is then made, for each separated peak, of elution salt concentration, elution time or elution volume against pH. This will produce a series of curves as illustrated in Fig. 43. An analysis of this composite plot for the point of maximum separation will indicate at what pH and on which type of exchanger maximum resolution between any two or more components can be expected. The conditions used (column type, buffers, pH, etc.) during the rapid runs for the generation of the chromatographic titration curves can be directly applied when developing the final optimized procedure.
74
A 280nm
A 280nm
a) Mono S pH 3.0
C
A280nm
b) Mono S pH 5.0
f) Mono Q pH 11.0
B
B
B
A
A
C
C
A
Elution ionic strength
Data from seven chromatograms plotted as chromatographic titration curves - A, B, C. Elution conc.
a @@@@@@e W.e/Xe 7HeN1e @?e?@e 3=eC5e V4@@0Ye ?W2@@@e ?7@X?@e ?(R)T5e ?@@Ue @@@@@,e ?W(Ye ?W20Y?e W&Y?f &@@@@@e @@@@@@e @@@Xf I46Xe S,e O20Ye @@@Yf @@@@@@e
A
b
Mono S
B
?@@@@@e ?@g ?@@@@@e ?@M?f
c C 9 3
4
5
10
11
12
pH
6
@@@@@@e W.e/Xe 7HeN1e @?e?@e 3=eC5e V4@@0Ye ?W2@@@e ?7@X?@e ?(R)T5e ?@@Ue @@@@@,e ?W(Ye ?W20Y?e W&Y?f &@@@@@e @@@@@@e @@@Xf I46Xe S,e O20Ye @@@Yf @@@@@@e ?@@@@@e ?@g ?@@@@@e ?@M?f
d
e
Mono Q
f
Fig. 43. Chromatographic titration curves.
75
Choice between strong and weak ion exchangers Having selected a suitable starting pH to use on a cation or anion exchanger, it is necessary to choose between a strong and weak ion exchange group. In those cases where maximum resolution occurs at an extreme of pH and the molecules of interest are stable at that pH, the choice is clearly to use a strong exchanger. The majority of proteins however, have isoelectric points which lie within the range 5.5 to 7.5 and can thus be separated on both strong and weak ion exchangers. Some advantages in using a strong ion exchanger are discussed in Chapter 2.
Choice of buffer As with the choice of ion exchanger, there are a number of variables which have to be considered. These include: 1. The choice of buffer pH and ionic strength. 2. The choice of buffering substance. 3. The price of the buffer if it is to be used in production process.
Choice of buffer pH and ionic strength The choice of buffer pH has been discussed in the previous section. It should be pointed out, however, that in many applications the optimum separation may be achieved by choosing conditions so that major and troublesome contaminants are bound to the exchanger while the substance of interest is eluted during the wash phase (21). This procedure is sometimes referred to as “starting state elution”. Note: Concentration of sample does not occur with starting state elution. The highest ionic strength which permits binding of the selected substances and the lowest ionic strength that causes their elution should normally be used as the starting and final ionic strengths in subsequent column experiments (i.e. the starting and limiting buffers for gradient elution). A third and higher ionic strength buffer is frequently employed as a wash step before column regeneration and re-use. The required concentration of the start buffer will vary depending on the nature of the buffering substance. A list of some suitable buffers and suggested start concentrations is shown in Table 19. In the majority of cases a starting ionic strength of at least 10 mM is required to ensure adequate buffering capacity. Salts also play a role in stabilizing protein structures in solution and so it is important that the ionic strength should not be so low that protein denaturation or precipitation occurs. A major advantage of using Pharmacia Biotech ion exchangers is that they have excellent capacities and so the initial ionic strength of the buffer can be quite high without significantly affecting capacity for sample.
76
In the case of pre-packed ion exchangers and columns which can be run conveniently quickly, trial experiments using salt gradients will allow the determination of an optimal starting ionic strength. In the case of Sephadex based exchangers for batch applications or where column running times are prohibitively long, a simple test-tube technique is recommended as a test for a suitable ionic strength.
Choice of buffer substance If the buffering ions carry a charge opposite to that of the functional groups of the ion exchanger they will take part in the ion exchange process and cause local disturbances in pH. It is preferable, therefore, to use buffering ions with the same charge sign as the substituent groups on the ion exchanger. There are of course exceptions to this rule as illustrated by the frequency with which phosphate buffers are cited in the literature in connection with anion exchangers. In those instances when a buffering ion which interacts with the ionic groups on the matrix is used, extra care must be taken to ensure that the system has come to equilibrium before application of sample. In cases where substances purified by ion exchange chromatography have to be freeze dried it is advantageous to use volatile buffer systems. Examples of such systems are shown in Table 20.
77
Table 19. Buffer tables. Buffer substances for cation exchange chromatography pKa pH (25°C) interval
Substance
Conc. (mM)dT
2.00 2.88 3.13 3.81 *3.75 *4.21 *4.76 *5.68 *7.20
1.5-2.5 2.38-3.38 2.63-3.63 3.6-4.3 3.8-4.3 4.3-4.8 4.8-5.2 5.0-6.0 6.7-7.6
Maleic acid Malonic acid Citric acid Lactic acid Formic acid Butanedioic acid Acetic acid Malonic acid Phosphate
20 20 20 50 50 50 50 50 50
*7.55 *8.35
7.6-8.2 8.2-8.7
HEPES BICINE
50 50
dpKa/ (°C)
Counter-ion
Comments
Dicarboxylic acid Dicarboxylic acid Dicarboxylic acid
-0.0028
Na+ Na+/Li+ Na+ Na+ Na+/Li+ Na+ Na+/Li+ Na+/Li+ Na+
-0.0140 -0.0180
Na+/Li+ Na+
Dicarboxylic acid Often needs purification before use Zwitterionic Zwitterionic
Comments
-0.0024 +0.0002 -0.0018 +0.0002
Buffer substances for anion exchange chromatography pKa pH (25°C) interval
Substance
Conc. (mM)dT
dpKa/ (°C)
Counter-ion
*4.75
4.5-5.0
20
-0.015
Cl-
*5.68 *5.96 *6.46 *6.80 *7.76
5.0-6.0 5.5-6.0 5.8-6.4 6.4-7.3 7.3-7.7
N-methyl piperazine Piperazine L-histidine bis-Tris bis-Tris propane Triethanolamine
20 20 20 20 20
-0.015
*8.06
7.6-8.0
Tris
20
-0.028
Cl-/HCOOClClClCl-/ CH3COOCl-
*8.52
8.0-8.5
N-methyldiethanolamine
50
-0.028
SO2-/Cl-/ CH3COO-
*8.88
8.4-8.8
Diethanolamine
-0.025
Cl-
*8.64
8.5-9.0
20 at pH 8.4 50 at pH 8.8 20
-0.031
Cl-
20 20 20
-0.029 -0.026 -0.026
ClClCl-
20 20
-0.031 -0.026
ClCl-
*9.50 *9.73 *10.47 11.12 12.33
1,3-diaminopropane 9.0-9.5 Ethanolamine 9.5-9.8 Piperazine 9.8-10.3 1,3-diaminopropane 10.6-11.6 Piperadine 11.8-12.0 Phosphate
-0.017 -0.020
* Recommended on the basis of experiments performed in our laboratories.
78
Often needs purification before use and especially sensitive to temperature change.
Table 20. Volatile buffer systems. pH
Substance
Counter-ion
2.0
Formic acid
H+
2.3-3.5
Pyridine/formic acid
3.0-5.0
Trimethylamine/formic acid
HCOOHCOO-
3.0-6.0
Pyridine/acetic acid
CH3OO-
4.0-6.0
Trimethylamine/acetic acid
6.8-8.8
Trimethylamine/HCl
CH3COOCl-
7.0-8.5
Ammonia/formic acid
HCOO-
8.5-10.0
Ammonia/acid
7.0-12.0
Trimethylamine/CO2
CH3COOCO -
7.0-12.0
Triethylamine/CO2
CO3-
7.9
Ammonium bicarbonate
3
8.0-9.5
Ammonium carbonate/ammonia
HCO3CO -
8.5-10.5
Ethanolamine/HCl
Cl-
8.9
Ammonium carbonate
CO3-
3
Test-tube method for selecting starting ionic strengths 1. Set up a series of tubes with ion exchanger as detailed on page 69. 2. Equilibrate the gel in each tube with 0.5 M buffer at the selected starting pH (10 x 10 ml washes). 3. Equilibrate the gel in each tube to a different ionic strength, at constant pH, using a range from 0.05 M to 0.5 M NaCl for Sephadex ion exchangers and from 0.01 M to 0.3 M NaCl for Sephacel and Sepharose ion exchangers. This will require 5 x 10 ml washes. Intervals of 0.05 M NaCl are sufficient. 4. Add sample, mix and assay the supernatant to determine the maximum ionic strength which permits binding of the substance of interest and the minimum ionic strength required for complete desorption. In the hypothetical example shown in Figure 38 (b) the ionic strength for sample binding (start buffer) would be at most 0.15 M and for elution at least 0.3 M.
79
10. Experimental Technique There are three ways of performing an ion exchange separation: by column chromatography, by batch methods, and by expended bed adsorption. This section will mostly deal with column chromatography.
Column chromatography Choice of column Good results in column chromatography are not solely dependent on the correct choice of gel media. The design of the column and good packing technique are also important in realising the full separation potential of any gel. These factors are built into the pre-packed columns supplied by Pharmacia Biotech and should be considered before packing a chromatography column in the laboratory.
Column design The material used in the construction of the column should be chosen to prevent destruction of labile biological substances and minimize non-specific binding to exposed surfaces. The bed support should be designed so it is easily exchangeable to restore column performance whenever contamination and/or blockage in the column occurs. Bed supports made from coarse sintered glass or glass wool cannot be recommended because they soon become clogged, are difficult to clean and cause artifacts (22). Dead spaces must be kept to a minimum to prevent re-mixing of separated zones. The pressure specifications of the column have to match the back-pressure generated in the packed bed when run at optimal flow rate. This is particularly important when using high performance media with small bead size. Pharmacia Biotech has developed a series of standard columns suitable for ion exchange chromatography. All are easy to dismantle and reassemble to allow thorough cleaning, which is a particularly important aspect when handling biological samples. Further information on the full range of Pharmacia Biotech chromatography columns are available upon request. Larger chromatography columns, specially designed for pilot and process scale chromatography are also available. Some aspects regarding process scale columns are described in Chapter 11.
80
Column dimensions As for most adsorptive, high selectivity techniques, ion exchange chromatography is normally carried out in short columns. A typical ion exchange column is packed to a bed height of 5-15 cm. Once the separation parameters have been determined, scale-up is easily achieved by increasing the column diameter.
Quantity of ion exchanger The amount of ion exchanger required for a given experiment depends on the amount of sample to be chromatographed and on the available or dynamic capacity of the ion exchanger for the sample substances. For the best resolution in ion exchange chromatography, it is not usually advisable to use more than 10-20% of this capacity, although this value can be exceeded if resolution is adequate. The capacity data given for each specific ion exchanger in respective product Chapter serves as a guideline for calculating the required amount of ion exchanger needed for a given experiment.
Preparation of the ion exchanger Having chosen the appropriate ion exchanger and starting buffer it is essential that the exchanger is brought to equilibrium with start buffer before sample application. Preparation of Sephadex ion exchangers, which are supplied as powders, differs somewhat from the other ion exchangers available from Pharmacia Biotech, which are supplied pre-swollen and/or pre-packed.
Pre-swollen ion exchangers SOURCE, Sepharose based, and DEAE Sephacel ion exchange media are supplied ready to use. To prepare the gel, the supernatant is decanted and replaced with starting buffer to a ratio of approximately 75% settled gel to 25% buffer. If large amounts of ion exchangers are to be equilibrated with a weak buffer, the ion exchanger should first be equilibrated with a 10 times concentrated buffer solution at the correct pH, and then with a few volumes of starting buffer.
Pre-packed ion exchange media MiniBeads and MonoBeads are supplied pre-packed in PC and HR columns respectively. SOURCE 15 are available pre-packed and ready to use in RESOURCE columns, 1 or 6 ml. Pre-packed HiLoad columns are XK Columns pre-packed with Sepharose Fast Flow and Sepharose High Performance ion exchangers. Sepharose High Performance ion exchangers are also available prepacked in HiTrap columns, 1 and 5 ml. For pilot scale applications, Mono Q, Mono S and Q Sepharose High Performance are available in pre-packed BioPilot
81
Columns of 100 ml(35/100) and 300 ml (60/100). SP Sepharose High Performance pre-packed in BioPilot Columns are available on request. For the above mentioned pre-packed columns, the preparation consists of washing out the 20% ethanol packing solution with 5 column volumes of start buffer.
Sephadex ion exchangers Sephadex ion exchangers should be swollen at the pH to be used in the experiment. Complete swelling takes 1-2 days at room temperature or 2 hours (at pH 7) in a boiling water bath. Swelling at high temperature also serves to de-aerate the gel. Vigorous stirring (e.g. with a magnetic stirrer) and swelling in distilled water should be avoided due to the risk of damaging the beads. The required amount of ion exchanger should be stirred into an excess of starting buffer. Remove the supernatant and replace with fresh buffer several times during the swelling period. Instead of decantation, the ion exchanger can be washed extensively on a Büchner funnel after the initial swelling.
Alternative counter-ions If ion exchangers are to be used with counter-ions other than those supplied (i.e. other than sodium or chloride) then the following procedure should be used. Suspend the required amount of ion exchanger in and excess of 0.5-1.0 M solution of a salt of the new counter-ion. After sedimentation and decantation, re-suspend the ion exchanger in the buffer to be used in the experiment. Decant and re-suspend the ion exchanger in this buffer several times.
Decantation of fines Decantation of fines is not necessary with any Pharmacia Biotech ion exchangers.
Packing the column As with any other chromatographic technique, packing is a very critical stage in an ion exchange experiment. A poorly packed column gives rise to poor and uneven flow, zone broadening, and loss of resolution. Detailed packing instructions are to be found in the instructions supplied with respective media.
Column Packing Video Film A video film describing the correct methodologies for packing laboratory columns is available and can be ordered from your local distributor of Pharmacia Biotech products.
82
Checking the packing The bed should be inspected for irregularities or air bubbles using transmitted light from a lamp held behind the column. Be careful in the choice of any dye substances used for checking beds as many of them are strongly charged. For example, Blue Dextran 2000 binds strongly to anion exchangers. Testing the bed is easily done by injecting a test substance on the column and calculating the number of theoretical plates (N) or the height equivalent to a theoretical plate (H). Choose a test substance which shows no interaction with the media and which has a low molecular weight, to give full access to the interior of the beads. Acetone at a concentration of 1% (v/v) can be used with all kinds of chromatographic media and is easily detected by UV-absorption. Keep the sample volume small to ensure a narrow zone when the sample enters the top of the column. For optimal results, the sample volume should be ²0.5% of the column volume for a column packed with a medium of approximately 30 µm bead diameter and ² 2% of the column for a column packed with a medium of approximately 100 µm bead diameter. Keep the linear flow rate low to reduce zone spreading due to non-equilibrium at the front and rear of the zone. For 30 µm media the flow rate should be between 30-60 cm/h and for 100 µm media, 15-30 cm/h. Use the following equations to calculate the number of theoretical plates (N) and the hight equivalent to a theoretical plate (H). VR
2
()
N = 5.54 x
wh
H = L/N where VR is the volume eluted from the start of sample application to the peak maximum and wh is the peak width measured as the width of the recorded peak at half of the peak height, see Figure 44. L is the height of the packed bed. Measurements of VR and wh can be made in distance (mm) or volume (ml) but both parameters must be expressed in the same unit.
83
?@e?@ ?3L?J5 ?@ ?N1?7H ?@ @?@? J@L? 3T@T-X 7@1? V+R@@) 3@5? ?3X? N@H? ?V/? ?@ @? ?@ @? ?@?O@K ?O26K?@? ?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@? ?@?I@M ?I40M?@? ?@ @? ?@ @? ?@ ?J@L ?@ ?7@1 ?@ J@@@L? ?@ 7Y@V1? ?@ ?J5?@?3L ?@ ?7H?@?N1 ?@ ?@e@??@ ?@ ?@e@??@ ?@ ?@e@??@ ?@ J5e@??3L? ?@ 7He@??N1? ?@ @?e@?e@? ?@ @?e@?e@? ?@ @?e@?e@? ?@ @?e@?e@? ?@ @?e@?e@? ?@ @?e@?e3L ?@ ?J5?e@?eN1 ?@ ?7H?e@?e?@ ?@ ?@f@?e?@ ?@ ?@f@?e?@ ?@ ?@f@?e?@ ?@ ?@f@?e?3L? ?@ ?@f@?e?N1? ?@ J5f@?f@? ?@ 7Hf@?f@? ?@ @?f@?f@? ?@ @?f@?f@? ?@ @?f@?f@? ?@ @?f@?f3L ?@ @?f@?fN1 ?@ ?J5?f@?f?@ ?@ ?7H?f@?f?@ ?@ ?@g@?f?@ ?@ ?@g@?f?@ ?@ ?@g@?f?@ ?@ ?@g@?f?3L? ?@ ?@g@?f?N1? ?@ ?@g@?g@? ?@ J5g@?g@? ?@ 7Hg@?g@? ?@ @?g@?g@? ?@ @?g@?g3L ?@ @?g@?gN1 ?@ @?g@?g?@ ?@ @?g@?g?@ ?@ ?J5?g@?g?@ ?@ ?7YO@Kf@?eO2@6X@ ?@ ?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@? ?@ ?@?I@Mf@?eI40MI'X? ?@ @?e@?g?@h@?g?N1? @@ ?@ 3L?J5?g?@h@?h@? ?J@@ ?@ N1?7H?g?@h@?h@? ?@@@ ?@ ?@?@?@K?fJ5h@?h@? ?@ ?@ ?3T5?@@@f7Hh@?h@? ?@ ?@ ?V+Y?@?@f@?h@?h@? ?@ ?@ ?@?@f@?h@?h3L ?@ ?@ ?@?@f@?h@?hN1 ?@ ?@ @?h@?h?@ ?@ ?@ @?h@?h?@ ?@ ?@ @?h@?h?@ ?@ ?@ ?J5?h@?h?@ ?@ ?@ ?7H?h@?h?3L? ?@ ?@ ?@he@?h?N1? ?@ ?@ ?@he@?he@? ?@ ?@ ?@he@?he@? ?@ ?@ ?@he@?he@? ?@ ?@ ?@he@?he@? ?@ ?@ J5he@?he@? ?@eW2@6T26X ?@ 7Hhe@?he3L ?@e7@@>@)Xhe ?@ @?he@?he?@ ?@fC@@=C5e?J@@?,he ?@ @?he@?he?@ ?@e?@0MI40Ye?.MI+Yhe ?@ ?J5?he@?he?@ ?@ ?@ ?7H?he@?he?3L? ?@ ?@ ?@hf@?he?N1? ?@ ?@ ?@hf@?hf@? ?@ ?@ J5hf@?f@Kg@? ?@ ?@ 7He?'6Xg@?f@@6Xf@? ?@ ?@ @?e?S@1g@?f@?B1f@? ?@ ?@ ?J5?e@(Y@g@?f@??@f3L ?@ ?@ ?7H?e3U?@g@?f@?C5fN1 ?@ ?@ J5fV4@@g@?f@@0Yf?@ ?@ ?@ 7H @?hf?3L? ?@ ?@ ?J5? @?hf?N1? ?@ ?@ ?7H? @? 3L ?@ ?@ J5hf@Ke@?he@KeN1 ?@ ?@ 7H?@6Kh@@@?@?@6K?g@@@??3=? ?@ ?@ ?J5??@@@@@@@@@@@@@@?@?@@@@@@@@@@@@@??N@@@@@@@@@@@@@@@@@@@@@@@@ ?@ ?7H? @? 3X ?@f@@fW&?W26X?he ?@ J5 @? N1 ?@e?J@@f*@?7)X?e ?@ O.Y? @? V46XheJ@f7@f?@?3=C5?eJ@@?,?e ?@ O20Y @? I/K?h7@f@@f?@?V40Y?e.MI+Y?e ?@ O20M @? ?V46K?g@@ ?@ ?O2@0M @? ?I46K? ?@ ?O2@@@@@Y? @? ?V@@@@@@@6K? ?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ ?@ @? ?@ @? ?@ @? ?@ @? ?@ @K ?@ @@@? ?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@? @?e@Ke@? 3L?J@@6X@?@?@??@@@@@?W26X? N1?7@?B@@?@?@??@?@?@?7YV1? ?@?@@??@@?@?@??@?@?@?@@@@? ?3T@@=C@@?@?@??@?@?@?3X? ?V+MI40R'?@@@??@?@?@?V4@
Fig 44. A UV trace for acetone in a typical test chromatogram showing the HETP and As value calculations.
As a general rule of thumb, a good H value is about two to three times the mean bead diameter of the gel being packed. For a 90 µm particle packing, this means an H value of 0.018-0.027 cm. Another useful parameter for testing the packed bed is the symmetry factor (As) As =
b a
where a = 1st half peak width at 10% of peak height. (see Figure 44) b = 2nd half peak width at 10% of peak height. (see Figure 44) As should be as close as possible to 1. A reasonable As value for a short column such as an IEX column is 0.80-1.80. (For longer gel filtration columns it will probably fall within 0.70-1.30). An extensive leading edge is usually a sign that the gel has been packed too tightly and extensive tailing is usually a sign that the gel has been packed too loosely.
Equilibrating the bed Run at least two bed volumes of buffer through the ion exchange bed to allow the system to reach equilibrium. Counter-ion concentration, conductivity, and pH of the eluent should be checked against the ingoing solution. It is often sufficient just to measure the pH of the effluent.
84
Sample preparation Sample concentration The amount of sample which can be applied to a column depends on the dynamic capacity of the ion exchanger and the degree of resolution required. For the best resolution it is not usually advisable to use more than 10-20% of this capacity (23). Information on the available capacities for the different exchangers is given in the relevant product sections. Methods for determining available and dynamic capacities are given later in this chapter.
Sample composition The ionic composition should be the same as that of the starting buffer. If it is not, it can be changed by gel filtration on Sephadex G-25 using e.g. Pharmacia Biotech Disposable Column PD-10, Fast Desalting Column HR 10/10 or HiTrap Desalting Columns, dialysis, diafiltration or possibly by addition of concentrated start buffer.
Sample volume If the ion exchanger is to be developed with the starting buffer (isocratic elution), the sample volume is important and should be limited to between 1 and 5% of the bed volume. If however, the ion exchanger is to be developed with a gradient, starting conditions are normally chosen so that all important substances are adsorbed at the top of the bed. In this case, the sample mass applied is of far greater importance than the sample volume. This means that large volumes of dilute solutions, such as pooled fractions from a preceding gel filtration step or a cell culture supernatant can be applied directly to the ion exchanger without prior concentration. Ion exchange thus serves as a useful means of concentrating a sample in addition to fractionating it. If contaminants are to be adsorbed, and the component of interest is allowed to pass straight through, then the sample volume is less important than the amount of contaminant which is present. Under these conditions there will be no concentration of the purified component, rather some degree of dilution due to diffusion.
Sample viscosity The viscosity may limit the quantity of sample that can be applied to a column. A high sample viscosity causes instability of the zone and an irregular flow pattern. The critical variable is the viscosity of the sample relative to the eluent. A rule of thumb is to use 4 cP as the maximum sample viscosity. This corresponds to a protein concentration of approximately 5%. Approximate relative viscosities can be quickly estimated by comparing emptying times from a pipette.
85
If the sample is too viscous, due to high solute concentration, it can be diluted with start buffer. High viscosity due to nucleic acid contaminants can be alleviated by precipitation with a poly-cationic macromolecule such as polyethyleneimine or protamine sulphate. Nucleic acid viscosity can also be reduced by digestion with endonuclease. Such additives may however be less attractive in an industrial process since they will have to be proven absent from the final product.
Sample preparation In all forms of chromatography, good resolution and long column life time depend on the sample being free from particulate matter. It is important that “dirty” samples are cleaned by filtration or centrifugation before being applied to the column. This requirement is particularly crucial when working with small particle matrices, such as MiniBeads (3µm), MonoBeads (10 µm), SOURCE (15 and 30 µm) and Sepharose High Performance (34µm). The “grade” of filter required for sample preparation depends on the particle size of the ion exchange matrix which will be used. Samples which are to be separated on a 90 µm medium can be filtered using a 1 µm filter. For 3, 10, 15, 30 and 34 µm media, samples should be filtered through a 0.45 µm filter. When sterile filtration or extra clean samples are required, a 0.22 µm filter is appropriate. Samples should be clear after filtration and free from visible contamination by lipids. If turbid solutions are injected onto the column, the column lifetime, resolution and capacity can be reduced. Centrifugation at 10 000 g for 15 minutes can also be used to prepare samples. This is not the ideal method of sample preparation but may be appropriate if samples are of very small volume or adsorb nonspecifically to filters. Note: The latter may indicate that the substance in question may also adsorb strongly to chromatography matrices. Care should therefore be taken and perhaps a buffer additive such as glycerol or a detergent used. Crude samples containing lipids, salts, etc. can be passed through a suitably sized column of Sephadex G-25 e.g. Pharmacia Biotech Desalting Column PD-10, Fast Desalting Column HR 10/10 or HiTrap Desalting Column. Preliminary sample clean-up can be achieved simultaneously in this way. In expanded bed adsorption sample preparation is not as crucial as for column chromatography. Samples can be applied directly to the expanded bed without prior sample preparation, e.g. filtration, centrifugation etc. (Expanded bed adsorption is described in detail on page 98.)
86
Sample application There are a number of ways to apply the sample.
Sample application with an adaptor This is the recommended method for all ion exchange media with the exception of Sephadex based media and is always the method used with pre-packed columns or when upward elution is employed. The sample may be applied to the column via the adaptor in one of the following ways. Sample loops are a convenient way of applying small samples in a reproducible manner without interrupting the liquid flow on the column. Sample loops can be used in conjunction with LV-4 or SRV-4 valves (Fig. 45) or in conjunction with the manual valves V-7 and IV-7 or the motorized valves MV-7 and IMV-7 (Fig. 46).
?@g@? @? ?@g@? @?f@@@@@@@@@@@@g@? @? ?@g@? @? ?@g@? @? ?@g@? @? ?@g@? @? ?@g@? @?he@@@@@@g@? @? ?@g@? @? ?@g@? @? ?@g@? @? ?@g@? @?f@@@@@@@@@@@@g@? @? ?@g@? @? ?@g@? @? ?@g@? @? ?@g@? @? ?@g@? @?he@@@@@@g@? @? ?@g@? @? ?@g@? @? ?@g@? @? ?@g@? @? ?@g@? @@@@@@@@@@@@@@@@@@@@@@@@@? @? @? @@@@@@@@@@@@@@@@@@@@@@@@@? ?@he@? ?3L?g?J5? ?N1?gW.Y? 3Lg7H N1f?J5? ?@f?7H? ?@f?@ ?@f?@ ?@f?@ ?@@@@@@@ ?@f?@ ?@@@@@@@hfW26X ?@fW@hf75f?B@H V@6K?O@Kf?O2@YV@Yf?C5? @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@U? ?J(M @? ?I/X ?7H? @? N1 ?@ @? ?@ ?@ @? ?@ ?@ @? ?@ ?@ @? ?@ ?@ @? ?@ ?@ @? ?@ ?@ @? ?@ ?@ @? ?@ ?@ @? ?@ ?@ @? ?@ ?@ @? ?@ ?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ @?hI4@@@@@@@@@0M?h@? @@@@@@@@@@@@@@@? @? @? @?e?@@?g@? @? @? O2@@@@@@@@@@@@@@@?e?@@?g@? @? @? ?O2@@@@@@@@@@@@@@@@@@?e?@@?g@? @? @? ?W2@@@@0M?hf@?e?@@?g@? @? @? W&@@0M @?e?@@?g@? @? @? ?W&@(M @@@@@@@@@@@@@@@? @? @? W&@(Y? @? @? @? 7@(Y @? @? @? ?J@@H? @? @? @? ?7@5 @? @? @? ?@@H @? @? @? J@@? @? @? @? 7@5? @? @? @? @@H? 3L @?hf?J5? @@ N1 @?hf?7H? @@ ?3L?hf@?hfJ5 @@ ?V/Xhf@?he?W.Y @@ V/X?he@?heW.Y? @@ ?N1?he@?he7H @@ 3Lhe@?h?J5? @@ V/X?h@?hW.Y? @@ ?V/Xh@?g?W.Y @@@@@@@? N1h@?g?7H? @@@@@@@? ?3L?g@?gJ5 @@@@@@@? ?N1?g@?g7H @@@@@@@? @?g@?g@? @@@@@@@? @?g@?g@? @@@@@@@? @?g@?g@? @@@@@@@? @?g@?g@? @@@@@@@? @?g@?g@? @@@@@@@? @?g@?g@? @@@@@@@? @?g@?g@? @@@@@@@@@@@@@@@? @?g@?g@? @@@@@@@@@@@@@@@? @?g@?g@? @@@@@@@? @?g@?g@? @@@@@@@? @?g@?g@? @@@@@@@? @?g@?g@? @@@@@@@? @?g@?g@? @@@@@@@? 3Lg@?f?J5? @@@@@@@? N1g@?f?7H? @@@@@@@? ?3L?f@?fJ5 @@@@@@@? ?V/Xf@?f7H @@@@@@@? V/X?e@?e?J5? @@@@@@@? ?N1?e@?e?7H? @@@@@@@? 3Le@?eJ5 @@@@@@@? N1e@?e7H @@ ?@e@?e@? @@ ?@?J@?e@? @@ ?@W&@?e@? @@ ?@@@@@@@@? @@ ?O2@@0Y@@X?I4@6K @@ ?W20M?e?@@1fI46X @@L? W.M?f?@@@gI/X? 3@1? ?W.Yg?@@5g?V/X N@@L W.Y?gJ@@HhV/X? ?3@1 ?W.Yh7@5?h?V/X ?N@@L? ?7H?g?J@@H?@KhN1 3@1? J5hW&@5e3@@?g?3L? N@@L 7Hh7@@HeV'@?g?N1? ?3@)X? @?g?J@@5?e?V'?h@? ?V'@)X ?J5?gO&@(Y? 3L V'@)K? ?7H?fW2@@(Yh?W26K?eN1 ?V'@@@6K ?@f?O&@@0Y?h?&@@@@e?@ V4@@@@@6K? ?@?O2@@@@(M?hf?@@@W2@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@? ?I4@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@0Y J@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@? I4@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@0M ?W&@@@@0Y@ ?@gW&K?heW&@(M?e?@ ?3L?f*@@@h?O&@(YfJ5 ?N1?fV4@@g?W2@@(Y?f7H @? ?7@@0Yg@? 3L J@@?g?J5? N1hf?W&@5?g?7H? ?3L?h@KO&@@H?gJ5 ?V/Xh3@@@@5g?W.Y V/X?gN@@@@HgW.Y? ?V/Xg?3@@@?f?W.Y V/K?f?N@@@?fO.Y? ?V46K?f@@5?eO20Y ?I4@6Ke@@YO2@0M I4@@@@@0M? @@ @@ @@ @@ @@ @@ @@ @@ @@ @@ @@ @@ @@ ?J@@ W&@5 7@@H ?J@@5? W&@(Y? ?W&@(Y O&@(Y? O2@@@@(Y @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@0Y? @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@0M?
Syringe and needle
SA-5
SRV-1
To column SRV-4
Fig. 45. Sample application using an SA-5 in a sample loop system.
From pump
Sample applicators SA-5, SA-50. These are reservoirs which, used in combination with a suitable valve, e.g. SRV-4, allow the sample to be introduced via a closed sample loop system using a pump (Fig. 45). As well as their large capacity (up to 6 ml for the SA-5 and 45 ml for the SA-50) the sample applicator offers the advantage of serving as a pulse damper and bubble trap. W2@6X?f@? 7
?W2@6Xf?@ ?7
W2@6X?W26X@?@?@?@@@@@??@@@ @? 7
?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ J@L? J@L? ?@ ?@@6K? 7@1? @@6K 7@1? @@6K ?@ ?@?B@@?@e@@@@@?@@6X 3@5? @?B@@?@??@@@@@?@@6X? 3@5? @?B@@?@??@@@@@?@@6X? ?@ ?@?C@@?@e@?@?@?@?B1 N@H? @?C@@?@??@?@?@?@?B1? N@H? @?C@@?@??@?@?@?@?B1? ?@ ?@@0Y@?@e@?@?@?@??@ ?@ @@0Y@?@??@?@?@?@e@? ?@ @@0Y@?@??@?@?@?@e@? ?@ ?@e?@?@e@?@?@?@?C5 ?@ @?e@?@??@?@?@?@?C5? ?@ @?e@?@??@?@?@?@?C5? ?@ ?@e?@@@e@?@?@?@@0Y ?@ @?e@@@??@?@?@?@@0Y? ?@ @?e@@@??@?@?@?@@0Y? ?@ @? ?@ ?@ ?@ ?@ ?@ @? ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@e?W&? ?@eW& ?@eW& ?@e?*@? ?@e*@ ?@e*@ /K ?@e?N@? /K ?@eN@ ?/K? ?@eN@ V46K ?@f@? V46K ?@e?@ ?V46K? ?@e?@ I46K @@@@ J@f@? I46K @@@@ J@e?@ ?I46K? @@@@hg J@e?@ I'6K O2@@@@@@6K?O&@L?e@? I'6K O2@@@@@@6K?O&@L??@ ?I46K? O2@@@@@6K??O&@L??@ ?V4@6Khf@?hO20MgI4@@@@1? ?V4@6Khf@?hO20MgI4@@@@1? ?I46K?hf@?g?O2@0M?@ ?I4@@@@@1? ?I4@6KO)X?f?J5?gO20Mhe?@@@@? ?I4@6KO)X?f?J5?gO20MheI'@@@? ?I46K?O)X?f?J5?f?W20M?e?@ I'@@5? ?I4@@@)Xf?7H?fW2(MhfJ@@@@L ?I4@@@)Xf?7H?fW2@@@U?h@? @??I'@L? I4@@@@@@@Xhe@? ?@H??I46X?g ?C@@@@@@@Xhe@? @?e?I/? W&@@(MI+MB1?h@? @?eS@@? ?@(?@MB1he@? ?@f?B1?g ?O20Y@(?@MB1he@? ?W2@f@? 7@@@R1??7R1e@V1??7R1?7R1e7R1?7R1??7R1e@? @??7R1e7R1??7R1e7>@@@R1??7R1e@V1??7R1?7R1e7R1?7R1??7R1e@? @??7>,e7>,??7>,?@@@@?7R1??@ N1e@V1??7>,?7>,e7>,?7R1??7>,e@? @??@W5e@W5??@W5e@@Y@@T5??@W5e@W5??@W5?@W5e@W5?@W5??@W5e@? @??@W5e@W5??@W5e@@Y@@T5??3T5e@W5??@W5?@W5e@W5?3T5??@W5e@? @??@@He@@H??@@H?3T@@?@W5??@ J5e@W5??@@H?@@He@@H?@W5??@@He@? @?J@@He@@H??@@H?J@@?@V@H??@@He@@H??@@H?@@He@@H?@@H??@@He@? @?J@@He@@H??@@H?J@@?@V@H??S@He@@H??@@H?@@He@@H?S@H??@@He@? @?J@@??J@@eJ@@??S@Y@?@@H??@3=e@@=O&@@=C@@=O&@@=C@@=O&@@=e@? 7He@@H?J@@??@@??J@@e@@H?J@@?e@? @W&@@=?C@@=?C@@=O&@@X@@@=?C@@=e@@=?C@@=C@@=?C@@=C@@=?C@@=e@? @W&@@=?C@@=?C@@=O&@@X@@@=?O&@=e@@=?C@@=C@@=?C@@=O&@=?C@@=e@? @W&@@=O&@@=O&@@=O&@@@W@@=?C5 @0M?I4@0MI4@0MI40M?I'@?I4@0MI4@@@V4@0MI40MI4@0MI40MI4@0MI4@@@? @0M?I4@0MI4@0MI40M?I'@?I4@0MI4@@@V4@0MI40MI4@0MI40MI4@0MI4@@@? @0M?I40M?I40M?I40Me@(MI4@0Y V4@@@V40M?I40MI40M?I40MI40M?I4@@@? N@he@? N@he@? @Hg @? ?@he@? ?@he@? @?g @? ?@he@? ?@he@? @?g @? ?@he@? ?@he@? @?g @? ?@he@? ?@he@? @?g @? ?@he@??W2@6X @? ?@he@? @?g @? @@6X W&hf?@h?J@??7U?I/?'6X?@@@@@?@@6X?@?W26X @@6Xhf?W26X?h?@he@? ?@@6X?hf?@6Xg?J@Lg @? @?B)T26X?W.?g*@hf?@h?7@??@)?e?S@1?@?@?@?@?B1?@?7YV1 @?B)T26X?W.?f?.MB1?h?@he@? ?@?B)T26X?W.hS,g?7@@g @? @?C@@
@6X??@e?@e@@@6T2@6X?@@@@@? @@)X?@e?@e@??B@
?@e@??@eW-K?@@6X?@e?@ ?@?J@L?@e7R@@ I/?@e?@ ?@?.R/?@e@?@@@?e?@@@@@ ?3X@?@S,?@@@@@ '@?@e?@ ?N@5?3@H?@e?@ S5?@e?@ (Y?V'??@e?@@@0Y?@e?@
Fig. 46. Sample application using V-7, IV-7 or motorized MV-7 or IMV-7 valves.
87
Superloops can be used together with the manual valves V-7 and IV-7 or the motorised valves MV-7 and IMV-7 when larger volumes of sample have to be applied (Fig. 47). Superloops are available with capacities of 10, 50 and 150 ml. (The 150 ml Superloop is most often used in BioPilot System.) ? ? ?W2@ /K ?O@? ? ?7U? V'@?@??@@6KO26X?@@@?W26X?W26T2@6X? ? ?@)?'@@?@??@e@@@@@?@?@?@??@?@e@@e@?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@? ?V@?@??@?B@@YV1?@?@?7@@@@@?@@? ?@? ?@ @?hf@? @? @? O2@@@@@@@@@@@@@@@@@@@@@@@@@He?@ @@W@@0M?e?I4@@@@@@? ?@? ?@ @?eO2@@@6K?e@? @? @? W2@@@@@@@@@@@@@@@@@@@@@@@@@@@?f @@0Me?O2@6Ke?I4@@? ?@? ?@ @??@0Me?I4@@?@? @? @? ?O&@@@@@@@@@@@@@@@@@@@@@@@@@@@@?f 3Xf@(M?I'@?e?W5? ?@? @K ?@ @?hf@? @? @?hf?W2@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@6Xe V/K?e@He?N@?eW.Y? ?@? @@@6K? ?@ @??O2@@@@6K?e@? @? @?@@@@hO&@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@)K? ?V46K?@?f@??O.Y ?@? @@@@@@@@@@@@@@@@@@@@@@@? ?@ @@@0M?e?I4@@@@? @? @?heW2@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ ?I4@@?f@W20Y? ?@? @? ?@ ?J(Mhe?I'L @? @?e@?f?W&@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@1 )X @@@@@@@(M? ?@? @? ?@ ?7H?O2@@@@@@@6K?N1 @? @??J5?fW&@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ @@@@@@0Y ?@? @? ?@ ?@W2@@@@?@?@@@@6X@ @? @??7H?e?W&@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ @@eO2@? ?@ ?@? @? ?@e?W&? ?@@@@@@@?@?@@?@V@@ @? @??@f?7@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ @@@@@@@? ?@ ?@? @? ?@e?*@? ?@?@@@@@?@?@@?@?@5 @? 3LgJ@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@(M? @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ ?@? @? J@e?N@? ?3X@@@@@?@?@@?@?@H @? N1f?W&@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ ?@? @? ?O2@@@6Kg7@f@? ?N@@@@@@?@?@@?@?@? @? ?3=?eW&@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@H??.MB1? @@@@@@(Y?W26X? ?@? @? ?O2@@@@@@@@@@@6Ke@@L?e@? @@@@@@@@@@@?@?@? @? ?V'@6?&@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ ?@? @? O2@@@@@@@@@@@@@@@@@@@@1?e@? @@@@@@@@@@@@@@@? @? ?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@1??W.? @@@@@@L?f@? ?@? @?hfW2@@@@@@@@@@@@@@@@@@@@@@@? @(M??W@@X?eI'@? @? 7@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ ?@? @@e@@e/X @?@@@@g?W&@@@@@@@@@@@@@@@@@@@@@@@@L 3Ue?7@@L @? @?g@MB1eN@@@f?I4@@@@@@@@@@@(Mg?3@He ?@? ?@he?J(YW&@@@@>(M?@@@@@@@@@@@@@@@@@@@@@@@@@@@1 @?e?S@1?@H?7@Ye@@@@@@@@@@@@@@@@@@@@@@@@@@@@ @?e@(Y@?@e@??@@@@@ @? @?hC5e?@he?@e@@@@?@f7@f@?e ?@? ?@f?W.YW&@@@@>@@@@@@@@@@@@@@@@@@@@@@@@@@0MI'@ ?/X?g@??C@U?@?@e3=?@@? @? @?g?@0Ye?@he?@e@@@@?@e?J@@f@?e ?@? W.YW&@@@@>@@@@X??@(Me?I4@@@@@@@0MgN@ ?V/?g@@@0R4@@?@eV4@@@@@? @? @?hf?@he?@e@@@@?@e?7Y@L?e@?e ?@? /T.YW&@@@@>(R+MB1??@H? W&e?@ W(M? @? @?hf?@he?@e@@@@?@e?@@@,?e@?e ?@? S(YW&@@@@>(Yf@??@ 7@e?@ @??@hf?@X@ ?@(Y @? @?hf?@he?@e@@@@?@fI(Y?e@?e ?@? ?W.YW&@@@@>(Y?e?C5??@ ?J@@e?@ @? @?hf?@he?@e@@@@?@he@?e ?@? W.YW&@@@@>@Hf@0Y??@ ?7Y@L??@ @(e)X?W2@?@@@1??W2@6T26X?hf@? @?hf?@he?@e@@@@?@he@?e ?@? ?W.YW&@@@@>@@?h?@ ?@@@,??@ @@e@@e3Ue31?7@H?@@?@??7@<e?/X?f?@ I(Y??@ @@heV/X?N@?@@??@@?@??@e@@@@@?hf@? @?hf?@he?@e@@@@?@he@?e ?@? ?W.YW&@@@@>@@?e?V/??@eJ@ J@g@@e@@ ?S,??3T@@??@@?@??3=?@@X? @? @?hf?@he?@e@@@@?@he@?e ?@? W.YW&@@@@>(Y@?he@@e@@he@@e@@e@@e@@ ?.Y??N@R'??@@?@??V4@@V4@ @? @?hf?@he?@e@@@@?@he@?e ?@? ?W.YW&@@@@>(Y?@?heN@g@@e@@heN@ ?J@?he?W5? @? @?hf?@he?@e@@@@?@he@?e ?@? W.YW&@@@@>(Ye@?he?@ ?@ ?@@?he@0Y? @? @?hf?@he?@e@@@@?@he@?e ?@? O2@6KO&HW&@@@@>(Y?e@?he?@ ?@ @? @?hf?@he?@e@@@@?@he@?e ?@? ?W2@@@@@@@@W&@@@@>(Yf@?he?@ ?@ @? @?hf?@he?@e@@@@?@he@?e ?@? W&@@@@@@@@@@@@@@>(Y?f@?heJ@ ?@ @? @?hfJ@L?h?@e@@@@?@h?J@Le ?@? ?W&@@@@@@@@@@@@@@>(Yg@?h?W&@ J@L? @? @?hf7@1?h?@e@@@@?@h?7@)X? ?@? ?7@@@@@@@@@@@@@@S(Y?g@?hO.Y@ ?O&@)K @? @?he?C@@@=h?@e@@@@?@hC@@V/X ?@? J@@@@@@@@@@@@@@@@Hh@@@@@@@@@@0Y?@@@@@@@@@@@@@@@@@@@@@@0Y@V4@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@? @@@@@@@@@@@@@0Y@V4@@@@@@@@@@e@@@@?@@@@@@@@@0Y@?V/ 7@@@@@@@@@@@@@@@@? ?@ ?@ ?@he?@e@@@@?@he@?e@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@? ? ?J@@@@@@@@@@@@@@@@@L ?@ ?@ ?@he?@e@@@@?@he@?e ? ?7@@@@@@@@@@@@@@@@@1 ?@ ?@ ?@he?@e@@@@?@he@?e ? ?@@@@@@@@@@@@@@@@@@@ ?@ ?@ ?@he?@e@@@@?@he@?e ? ?3@@@@@@@@@@@@@@@@@@ ?@ ?@ ?@he?@e@@@@?@he@?e ? ?N@@@@@@@@@@@@@@@@@@ ?@ ?@ ?@he?@e@@@@?@he@?e ? 3@@@@@@@@@@@@@@@@@ ?@ ?@ ?@he?@e@@@@?@he@?e ? N@@@@@@@@@@@@@@@@5 ?@ ?@ ?@he?@e@@@@?@he@?e ? ?3@@@@@@@@@@@@@@@H ?@ ?@ ?@he?@e@@@@?@he@?e ? ?N@@@@@@@@@@@@@@5? ?@ ?@ ?@he?@e@@@@?@he@?e ? 3@@@@@@@@@@@@0Y? J@ J@L? ?@he?@e@@@@?@he@?e ? V4@@@@@@@@@0M? 7@ @@@? ?@he?@e@@@@?@he@?e ? I4@@@0M? @@ N@H? ?@he?@e@@@@?@he@?e @??@e@KgW& ? ?@ ?@e@??@K?f?W&?h?@he?@e@@@@?@h?J@Le ? ?@?J@L?@@6KO.??*@?W26XfJ@L?h?@e@@@@?@L?g?7@@e @?7R1?@W@@@UeN@?7YV1? @?J@L?@@6KO.e*@?W26X? ? ?@?7R1?@W@@@U??N@?7YV1f@@@?h?@@@@@@@@@)Xg?3@He ? ?3X@?@?@(Y@V/Xe@?@@@@fN@H?gW2@@@@@@@@@@V)K?f?N@?e N@5?3@@U?@?S,??@?3X? 3X@?@?@(Y@V/X??@?@@@@? ? ?N@5?3@@U?@?S,e@?3Xg?@g?W&@@@@@@@@@@@@@@6X?f@?e ? (Y?V+R4@@?.Ye@?V4@?hfW&@@@@@@@@@@@@@@@@)Xh ?(Y?V+R4@@?.Y??@?V4@ ? @??@e@KgW&hf?@e@??@hW& 7@@@@@@@@@@@@@@@@@@1h ? @?J@L?@@6KO.e*@?W26X?g?@?J@L?@?'6KO.e*@?W26X? ?J@@@@@@@@@@@@@@@@@@@@L?g ? @?7R1?@W@@@UeN@?7YV1?g?@?7R1?@?S@@@UeN@?7YV1? ?7@@@@@@@@@@@@@@@@@@@@1?g ? 3X@?@?@(Y@V/X??@?@@@@?g?3X@?@W@@(Y@V/X??@?@@@@? ?@?@@@@@@@@@@@@@@@@@@@@?g ? N@5?3@@U?@?S,??@?3X?h?N@5?3@R'U?@?S,??@?3X? J@@@@@@@@@@@@@@?@@@@@@@Lg ? ?(Y?V+R4@@?.Y??@?V4@he(Y?V'?V4@@?.Y??@?V4@ 7@@@@@@@@@@@@@@@@@@@@@@1g ? @@@@@@@@@@@@@@@@@@@@@@@@g ? @@@@@@@@@@@@@@@@@@@@@@@@g ? @@@@@@@@@@@@@@@@@@@@@@@5g ? 3@@@@@@@@@@@@@@@@@@@@@@Hg ? N@@@@@@@@@@@@@@@@@@@@@@?g ? ?@@@@@@@@@@@@@@@@@@@@@5?g ? ?3@@@@@?@@@@@@@@@@@@@@H?g ? ?N@@@@@@@@@@@@@@@@@@@5h ? 3@@@@@@@@@@@@@@@@@@Hh ? V4@@@@@@@@@@@@@@@@5?h ? I4@@@@@@?@@@@@@0Y?h ? I4@@@@@@@@@0M?he ? ?I4@@@0Mhg ?
Fig. 47. Sample application using a Superloop.
Syringe method (Fig. 48). The valves LV-3 and LV-4 can be used as syringe holders to give a very simple method for the application of small samples in standard chromatography. Using this method the sample is allowed to run onto the column under gravity. ?@@@@@@@@@@@@@ ?@@@@@@@@@@@@@ ?@f@@f?@ ?@f3@f?@ ?@fV'f?@ ?@he?@ ?@he?@ ?@he?@ ?@?O@KeO26X?@ W2@@@@@@@@@@?@@@@@@@@@@1?@ 7@@@@@@@@@@@?@V@?@?@?@?@?@ @?h?@?@he?@ @?h?@?@@@@@@@@@@@@@ @?h?@?@e?@e?@e?@ @?h?@?@e?@e?@e?@ @?h?@?@e?3L?J5e?@ @?h?@?@e?N1?7He?@ @?h?@?@f@?@?e?@ @?h?@?@f@?@?e?@ @?h?@?@f@?@?e?@ @@@@@@@@@@@@?@@@@@@@@@@@@@ @@@@@@@@@@@@?@@@@@@@@@@@@@ @@@@@@@@@@@@?@@@@@@@@@@@@@ @@@@@@@@@@@@?@@@@@@@@@@@@@ @@@@@@@@@@@@?@@@@@@@@@@@@@ @@@@@@@@@@@@?@@@@@@@@@@@@@ @@@@@@@@@@@@?@@@@@@@@@@@@@ @@@@@@@@@@@@?@@@@@@@@@@@@@ ?@@@@@@@ @@@@@@@@@@@@?@@@@@@@@@@@@@ ?3@@@@@5 @@@@@@@@@@@@?@@@@@@@@@@@@@ ?N@?e@H @@@@@@@@@@@@?@@@@@@@@@@@@@ @?e@? @@@@@@@@@@@@?@@@@@@@@@@@@@ @?e@? @@@@@@@@@@@@?@@@@@@@@@@@@@ @?e@? @@@@@@@@@@@@?@@@@@@@@@@@@@ @??J@? @@@@@@@@@@@@?@@@@@@@@@@@@@L? @??7@? @@@@@@@@@@@@@@@@@@@@@@@@@@@@1? @?J@@? @?heW@@Xhf@??O26K? @?@@@? @?he7
GM-1
Syringe barrel
LV-3
AK 16
XK 16/20
88
Fig. 48. Sample application using a syringe.
Sample reservoir (Fig. 49). In a similar way, a sample reservoir (e.g. R9, RK 16/26) can be connected via a 3-way valve to apply larger samples. ?W2@@@@@@@@@@@@6X? ?7@@@@@@@@@@@@@@1? ?@@@@@@@@@@@@@@@@? ?@ @? ?@f?@@@g@? ?@f?@@@f?J@? ?@?@fI@?@?@?@@?fW2@6X?@@e?@@?e?W&? ?@hf?N@?f7,e?@?B1? @@@@@@@@@@@@@@@@@??@@@@@@@@@@@@@@@@? ?@eB@@?N1f?@e3=C5?@@Ye?3=C5? @@@@@@@@@@@@@@@@@??@@@@@@@@@@@@@@@@? ?@e?@@??@f?@eV40Y?@@@@@?V40Y? @@@@@@@@@@@@@@@@@??@@@@@@@@@@@@@@@@? ?O2@6K @@@@@@@@@@@@@@@@@??@@@@@@@@@@@@@@@@? O2@0M?I4@@6K @@@@@@@@@@@@@@@@@??@@@@@@@@@@@@@@@@? W20MhI46X @@@@@@@@@@@@@@@@@??@@@@@@@@@@@@@@@@? ?W.MhfB1 @@@@@@@@@@@@@@@@@??@@@@@@@@@@@@@@@@? ?7H?hf?3L? @@@@@@@@@@@@@@@@@??@@@@@@@@@@@@@@@@? J5 ?V/X @@@@@@@@@@@@@@@@@??@@@@@@@@@@@@@@@@? ?W.Y N1 @@@@@@@@@@@@@@@@@??@@@@@@@@@@@@@@@@? ?7H? ?3L? @@@@@@@@@@@@@@@@@??@@@@@@@@@@@@@@@@? @@@? ?@ ?N1? @@@@@@@@@@@@@@@@@??@@@@@@@@@@@@@@@@? @?@? ?@ @? @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ @?@? ?@ @? @? W@@@@X ?@ ?J@?@? ?@ @? @?hf?W&@@@@)X? J@@@@@@@@@@? ?7@@@? ?@ @? @?hf?7@?e?@1? 7@@@@@@@@@@? ?@@@@? ?@ @? @?hf?@@?e?@@? @@@@@@@@@@@@@@@@@@@@@@@@@@6K ?@@@@L ?@ @? @?hf?3@?e?@5? 3@@@@@@@@@0M I4@6X? ?@@@@1 ?@ @? @?hf?V@@@@@@Y? V@e?I@M ?I/X ?@@@@@ ?@ @? @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ V/X? @@@@@@@@@@ ?@ @? @@@@ @@@@ ?V/X @@@@@@@@@@ ?@ @? @@@@ @@@@ N1 3@@@@@@@@@ ?@ @? ?3L? N@@@@@@@@5 ?@ @? ?N1? ?@?@@?@?@H ?@ @? 3L ?@@@@?@?@? J@ @? N1 ?@?@@?@?@? ?O&@@6K? @? ?@ ?@@@@?@?@? ?@@@@@@@@@ @? ?@ ?@?@@?@?@? ?@@@@@@@@@ @? ?@ ?@@@@?@?@? ?@@@@@@@@@ @? ?@ ?@?@@?@?@? ?@@@@@@@@5 @? ?@ ?@@@@?@?@? W@@@@@@? @? ?@ ?@@@@@@@@? ?W&@@@@@@1 @? ?@ ?@@@@@@@@? ?7@@@@@@@@ @? ?@ ?@@@@@@@@? ?@@@@@@@@@ @? ?@ ?@@@@@@@@? ?@@@@@@@@@ @? ?@ ?@@@@@@@@? ?@@@@@@@@@ @? ?@ ?@@@@@@@@? @@@@ @? ?@ ?@@@@@@@@? @@@@ @? ?@h?@f@?e@?e@6X?g?@@@@@@@@? @@@@ @? ?@h?@f3L?J5?e?S,?g?@@@@@@@@? @@@@ @? ?@h?@fN1?7Y?e?*U?g?@@@@@@@@? @@@@ @? ?@h?@f?@?@@@e?N1?gJ@@@@@@@@L @@@@ @? ?@h?@f?3T(M??/KC5?g7@@@@@@@@1 @@@@ @? ?@h?@@@@??V+Ye?V40Y?g@@@@@@@@@@ @@@@ @? ?@ @@@@@@@@@@ W2@@@@@@@@@@@6K?hf@? ?@ @@@@@@@@@@ 7@@@@@@@@@@@@@@@@@@?h@? ?@ ?@@@@@ @@@@@@@@@@@@@@@@@@@?h@? ?@ ?@@@@5 @@@@@@@@@@@@@@@@@@@?h@? ?@ ?@@@@H @@@@@@@@@@@@@@@?hf@? ?@ ?@@@@? @@@@@@@@@@@@@@@?hf@? ?@ @@@@@? ?@@@5? @@@@@@@@@@@@@@@?hf@? ?@ @@@@@? ?@H? @@@@@@@@@@@@@@@?hf@? ?@ @@@@@? ?@ ?O2@@@@@@@@@@@@@@@5?hf@? ?@ @@@@@? ?@hf@@@@@@@@@@@@@@@@@@@@@@H?hf@? ?@ 3@@@@? ?@hf@@@@@@@@@@@@@@@@@@@@@@L?hf@? ?@ ?@@@@L J5 I4@@@@@@@@@@@@@@@@1?hf@? ?@ 7@@@@1 7H ?I'@@@@@@@@@@@@@5?hf@? ?3L?he?J@@@@@@L? @? V'@@@@@@@@@@@@H?hf@? ?N1?heO&@@@@@@)Xhf?J5? ?N@?@?@??@@??@ @? 3LhO2@@@@@@@@@)K?heO.Y? @?@?@??@@??@ @? V/K??@@@@@@@@@@@@@@@@@@@@@@@@?eO20Y @?@?@??@@??@ @? ?V46X@@@@@@@@@@@@@@@@@@@@@@@@W2@0M @?@?@??@@??@ @? ?I'@@@@@@@@@@@@@@@@@@@@@@@@(M? @?@?@??@@??@ @? N@@@@@@@@@@@@@@@@@@@@@@@@H @?@?@??@@??@ @? ?@@@@@@@@@@@@@@@@@@@@@@@@? @?@?@??@@??@ @? I'@@@@@@@0M? @?@?@??@@??@ @? ?N@@@@@@ @?@?@??@@??@ 3@@@?@ @?@?@??@@??@ N@@@@@ @?@?@??@@??@ ?@@@@@ @?@?@??@@??@ ?@@@@@ @?@?@??@@??@ ?@@@@@ @?@?@??@@??@ ?@@@@@ @?@?@??@@??@ ?@@@@@ @?@?@??@@??@ ?@ @?@?@??@@??@ ?@ @?@@@@@@@??@ ?@ @?@@@@@@@??@ ?@ @?@@@@@@@??@ ?@ @?@@@@@@@??@ ?@ @?@@@@@@@??@ ?@ @?@@@@@@@??@ ?@ @?@@@@@@@??@ ?@ @?@@@@@@@??@ ?@ @?@@@@@@@??@ ?@ @?@@@@@@@??@ ?@ @?@@@@@@@??@ ?@ @?@@@@@@@??@ ?@ @?@@@@@@@??@ ?@ @?@@@@@@@??@ ?@ @?@@@@@@@??@ ?@ @?@@@@@@@??@ ?@ @?@@@@@@@??@g?@?@?@?W.?fW&?W2@f@?@?W26Xf ?@ @?@@@@@@@??@g?3T5?@W&H?f*@?7Y?f@@@?7
Fig. 49. Sample application using a reservoir. This is also an example of upward elution.
Other methods of sample application The following methods can be used with Sephadex based ion exchangers where it is not recommended to use an adaptor due to the variability of the bed height. They are not recommended for the more rigid ion exchanger types.
Sample application onto a drained bed This method requires the least equipment but is very difficult to do well. Allow eluent to drain to the bed surface, then pipette the sample onto the gel surface and allow it to drain into the gel. When all the sample has entered the bed, the top of the column is washed with aliquots of starting buffer and is connected up for elution. The drawback with this system is that disturbances to the bed surface result in uneven sample application and band skewing.
Sample application under the eluent Here excess eluent is left on top of the column. Some very thin capillary tubing is attached to a syringe and the free end is flared by gentle heating. The syringe is filled with sample which is then layered on top of the bed by positioning the end of
89
the tubing just above the surface and slowly pressing out the sample. Note that the sample must be denser than the eluent or made denser by the addition of a sugar, e.g. glucose (24). The column can then be connected for elution.
Elution If starting conditions are chosen such that only unwanted substances in the sample are adsorbed, then no change in elution conditions is required since the substance of interest passes straight through the column. Similarly no changes are required if sample components are differentially retarded and separated under starting conditions. This procedure is termed isocratic elution, and the column is said to be developed under starting conditions. Isocratic elution can be useful since no gradient apparatus is required for the run and, if all retarded substances elute, regeneration is not required. Normally, however, separation and elution are achieved by selectively decreasing the affinity of the solute molecules for the charged groups on the gel by continuously changing either buffer pH or ionic strength or possibly both. This procedure is termed gradient elution.
Change of pH As shown in Figure 37 on page 68, the net charge on a molecule depends on pH. Thus altering the pH towards the isoelectric point of a substance causes it to lose its net charge, desorb, and elute from the ion exchanger. Figure 50 shows use of a decreasing pH gradient in separation of haemocyanin fractions (25). ? ? W2@@6T2@@6X? ?@e?@ ? 7<eB@@??B1? ?@@6X@e?@ ? @?e?@@?e@? ?@?B@@@@@@ ? @?e?@@?e@?W-KO-X?W-X ?@e@@e?@ ? 3=eC@@??C5?.R4@?,?7R1 ?@?C@@e?@ ? V4@@0R4@@0Y?e?S@U?@?@ ?@@0R'e?@ ? @KO&?,?3T5 ?@ ? @@0R+Y?V+Y ?@ ? ? ? ?W&?e?W2@(? ?@@@@? ? ?*@?e?7@@U? ? ?N@?e?@MB1? ?@ ? @?g@?e?@@@@@ @@@@@?eJ5 ? @?f?C5?g?@ @?g7H ? @?@?e@0Y?g?@ @?g@? ? ?@ @? ? ?@ @? ? ?@ @? ? ?@g?@?@?@?@?@?@?@?@?@?@?@?@ @? ? ?@ @? ? ?@ ?@?@?@?@ @? ? ?@ @? ? ?@ ?/X? @? ? ?@ ?V/? @? ? ?@ /X @? ? ?@ V/ @? ? ?@ @? ? ?@ @? ? ?@ @? @? ? ?@ @? ? ?@ ?@?@ @? ? ?@ @? ? ?@ @? ? ?@ @? ?W&? @? ? ?@ ?)X? ?7@? @? ? ?@ ?@?@1? J@@? @? ? ?@ :@@L 7R'L @? ? ?@ ?@@@
Fig. 50. Elution pattern of whole stripped haemocyanin on DEAE Sepharose CL-6B. Sample applied in 0.1 M sodium phosphate buffer pH 6.8 and eluted with decreasing pH gradient (25). (Reproduced by kind permission of the authors and publisher.)
Since many proteins show minimum solubility in the vicinity of their isoelectric points, care and precautions must be exercised to avoid isoelectric precipitation on the column. The solubility of the sample components at the pH and salt concentrations to be used during separation should always be tested in advance.
90
Change of ionic strength At low ionic strengths, competition for charged groups on the ion exchanger is at a minimum and substances are bound strongly. Increasing the ionic strength increases competition and reduces the interaction between the ion exchanger and the sample substances, resulting in their elution. Figure 51 shows the elution of mouse IgM on SP Sepharose Fast Flow using a concentration gradient of NaCl.
Fig. 51. Isolation of mouse IgM on SP Sepharose Fast Flow. (Courtesy of Dr. H. F. J. Savelkoul, Erasmus University, Rotterdam, The Netherlands.)
?W-X ?7R1 W-X? J@?@ *?)T-X 7@@@@6KO-X?W-K V+R@>)X?hg @?e@V4@?,?7R@@@@@@@@@ J@@?,?hg @?e@??S@U?@?@@?@@?@?@ .MI+Y?hg @?O&?,?3T@@?@@?@?@ @@0R+Y?V+R'?@@?@?@ ?W&?W26X?W26X?e W26XfW& ?*@?7@
Gradient direction Guidelines for the choice of ascending or descending gradients are given in Table 21. Table 21. Choosing the direction of the gradient for elution.
Ion exchanger
Direction of pH gradient
Direction of ionic strength gradient
Anion exchanger
decreasing
increasing
Cation exchanger
increasing
increasing
Choice of gradient type The components in the sample usually have different affinities for the ion exchanger and so variations in the pH and ionic strength of the eluent can cause their elution at different times and thus their separation from each other. One can choose to use either continuous or stepwise gradients of pH or ionic strength. Stepwise pH gradients are easier to produce and are more reproducible than linear pH gradients. In the case of weak ion exchangers the buffer may have to titrate the ion exchanger and there will be a short period of re-equilibration before the new pH is reached. Gradients of pH can be also used in combination with ionic strength gradients.
91
Stepwise ionic strength gradients are produced by the sequential use of the same buffer at different ionic strengths. Stepwise elution is technically simple and offers the potential of high resolution in preparative applications. Care must be exercised in the design of the steps and the interpretation of results since substances eluted by a sharp change in pH or ionic strength elute close together. Peaks tend to have sharp fronts and pronounced tailing since they frequently contain more than one component. Tailing may lead to the appearance of false peaks if a buffer change is introduced too early. For these reasons a first separation using a continuous gradient is always recommended as a means of characterising the sample and an indication of suitable steps. The differences between continuous and stepwise gradient elution are shown in Figure 52. Continuous pH gradients are difficult to produce at constant ionic strength, since simultaneous changes in ionic strength, although small, also occur. Linear pH gradients cannot be obtained simply by mixing buffers of different pH in linear volume ratios since the buffering capacities of the systems produced are pH dependent. A relatively linear gradient can be produced over a narrow pH interval (Max. 2 pH units) by mixing two solutions of the same buffer salt adjusted, respectively, to 1 pH unit above and 1 pH unit below the pKa for the buffer. W2@6K?e)X ?W.R'>@@?J@)T2@6?2@@@@e@? ?7H?V@@@?7@V@@?@@@@?@H ?@W2@@@@X@@?@@T@X@@T5? ?@@0?40R4@@@0R+R40R+Y?@? @@@?eW&?@hW2@? ?J@@@T-T&@T@T26T2@?W&>5?@? ?7@@V@R@@V@R@@@@
@@@?eW&?@h@@@? ?J@@@T-T&@T@T26T2@?J@@5??@ ?7@@V@R@@V@R@@@@
? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? W-X? ? 7R1? ? @? ?J@?@? ? @? ?7@@@@6KO-X?W-K? ? @? ?@e?@V4@?,?7R@@@@@@@@@? ? @L ?@e?@eS@U?@?@@?@@?@?@? ? @@@1 ?@?O&?,?3T@@?@@?@?@? ? @@@5 ?@@0R+Y?V+R'?@@?@?@? ? 3@@H ?@ ? N@@? ?@ ? ?@@? ?@ ? ?@ ? ?@ ? ?@ @? ? ?@ @? ? W-X? ?@ @? ? 7R1? @? ?@ @? ? ?J@?@? @? ?@ @? ? ?7@@@@6KO-X?W-K? @? ?@ @? ? ?@e?@V4@?,?7R@@@@@@@@@? @? ?@ @? ? ?@e?@eS@U?@?@@?@@?@?@? @? ?@ @? ? ?@?O&?,?3T@@?@@?@?@? @? ?@ @? ? ?@@0R+Y?V+R'?@@?@?@? @? ?@ @? ? @L ?@ @? ? @? @1 ?@ @? ? @? @@ ?@ @? ? @? @@ ?@ @? ? @? @@ ?@ @? ? @? @@ ?@ @? ? @? ?@he@@ ?@ @? ? @? ?@he@@ ?@ @? ? @? ?@he@@ ?@ @? ? @? ?W.?g?/X? ?@he@@ [email protected]@?f/X @L ? @? ?7H?g?N1? ?@he@@ ?@hf7He@?fN1 @1 ? @? J5e?W2@6Xe3L ?@he@@ ?@he?J5?e@W26X??3L? @@ ? @? 7He?.M?B1eN1 ?@he@@ ?@he?7H?e@(MB1??N1? @@ ? @? @?g?@e?@ ?@he@@ ?@he?@f@He@?e@? @@ ? @? @?e?W2@@@e?@ ?@he@@ ?@he?@f@?e@?e@? @@ ? @? @?e?7,e@?@?@@f3T@LeS,e@@T@@??@@?@?e@?e@?@?@@@?e?@e?(R+R'?@?@?@eV+Y??@?@?@@@ ?@e?@@T@@?@?@?@e3>,??@?@?@@L ? ?@0Y@?eV+Y?@?V/?@(R40?4@@?@MI/?eN@@5?V4@?V4@0R/??@?@@??V+Y?@e@@@?V4@?V40R4@e?V+MeV+R/?@@@?.Y??(R+R'e@@?@e?.Y??@?@?@@?@??V+R/?@@@?.Ye(R+R'??@@?@??V+Ye@?@?@@?@eV+R/e.Ye(R+R'??@@?@?e@?e@?@?@@ ? @?he?(Y? ?@0Y @? ?@ @? ? ? ?@@@?@K?he?@ @?h@?g?@K?hf?)X?f?W&KhW&he?W&??@@?he?@@@ ?@e?W-XgO@?@K?eO@K?hg ? ?N@H?@@6?)X??W&??@X@?@eW.?W-T2@@?W.?W&?2@@@?@@?@?@?@W-X?@@@e@W@?eW&?@@6T-T2@@@@@@@??@)Xf?7@@@?W-X??W&@W-T&?@?W&KO&@??@@?@@6?)Xf?@?@?W-T2@@@@?@@?@?@?W-Xe?@e?7R1e@@@@@@?@@@@@@@@@@?hf ? @??@?@@@)??*@?C@@@?@e7H?7R@@?@?7H?7@@@?@@?@HJ@@@?@@R1?@?@e@@@Le*@?@?@@R@@@@@@@?@??@V1f?@@?@?7@)??7Y@@R@@?@?7@@@Y@??@@?@?@@@)f?@@@?7R@@@@@@?@HJ@@@?7@)e?@e?@?@e@?@?@@?@@?@?@@@?@?hf ? @??@?@@Xe?N@@@U@@?@e3L?3T@@?@?3L?3X?@?@@?@?*U@@?@@T5?@?@e@?@,eN@@@?@@T@@@@@@@?@??@W@L?e?@@?@?3Xe?3X@@T@@?@?3XI'X@??@@?@?@@X?f?@?@?3T@@@@@@?@?*U@@?3X?e?@e?3T5e@?@?@@?@@?@?@@@?@?hf ? @??@?(R/f@MI4@@?@eV/?V+R'?@?V/?V/?@?@@?@?V4@@?(R+Y?@?@e@?(Ye?@?@?(R+R40?4@@?@??@0R/T.??@@?@?V/e?V40R+R'?@?V/?V4@??@@?@?(R/?@?e?@?@?V+R40?4@?@?V4@@?V/?e?@?@?V+Ye@?@?@@?@@?@?@@@?@?@?he ? V+Y? ? ? ? ? )X?W&?he?W26X??@?@?@@@@??@@@e@@@? @1?7@W-T2@@?W-X??7
@@6T-XgO)X?W&e)X?W&?@@6T.?h@?@?eW.e?W-X @X;@R1e@@@@@@1?7@e@1?7@?@?V@Y?f@6X?@?@?e7Ue?7R1 @)X@?@e@?@?@@@?@@e@@?@@?@@@@@@f@V1?@@@?e@)X??@?@ ?S@@T5e@?@?@@@T@@e@@T@@?@?W@T5f@W5?@?@?e3>)X?3T@L? ?.MI+Ye@?@?@0R+R'e(R+R'?@@0R+Y?@e@(Y?@?@?eV+R/?V+R1? ?@e(Y @? ?@g?O)X?W&??)X?W&e?O2@?@g?@g?O)X?W&?@@6X?@@@@@@? ?@e?@@@@@@1?7@??@1?7@W2@@
Fig. 52. Continuous and stepwise gradient elution of b-galactosidase from Escherichia coli on Mono Q HR 5/5 (26). (Reproduced by kind permission of the authors and publisher.)
92
Continuous ionic strength gradients are the most frequently used type of elution in ion exchange chromatography. They are easy to prepare and very reproducible. Two buffers of differing ionic strength, the start and limit buffers, are mixed together and if the volume ratio is changed linearly, the ionic strength changes linearly. The limit buffer may be of the same buffer salt and pH as the start buffer, but at higher concentration, or the start buffer containing additional salt e.g. NaCl. Gradient elution generally leads to improved resolution since zone sharpening occurs during elution. In all forms of isocratic elution, a limiting factor with regards to achievable resolution is zone broadening as a result of longitudinal diffusion. In gradient elution, the leading edge of a peak is retarded if it advances ahead of the salt concentration or pH required to elute it. In contrast the trailing edge of the peak is exposed to continuously increasing eluting power. Thus the trailing edge of the peak has a relatively higher speed of migration, resulting in zone sharpening, narrower peaks and better resolution. Gradient elution also reduces zone broadening by diminishing peak tailing due to non-linear adsorption isotherms.
Resolution using a continuous gradient To optimize a separation it is important first to consider the objectives of the experiment, since the desired features of a separation i.e. speed, resolution and capacity, are often mutually exclusive. In the case of ion exchange separations the speed of separation is not solely related to the flow rate used in the experiment but also to the steepness or slope of the gradient applied. Novotny (27) has shown that the retention of charged molecules on an ion exchange column is related to the volume of the column and the molarity difference across it. This means that long shallow gradients will give maximum separation between peaks but that the separation time will be longer and peak broadening larger. In contrast short steep gradients will give faster separations and sharper peaks but the retention differences between peaks will be reduced. The effect of gradient slope on resolution is illustrated in Figure 53. It should be remembered that the sample loading also has a major influence on resolution since the width of the peaks is directly related to the amount of substance present. In practice it is recommended that trial experiments be carried out to allow the selection of optimal run parameters in terms of gradient shape and length. As a general rule a gradient of 0.05 to 0.5 M salt over a volume of 10 to 20 column volumes at the flow rate recommended for the medium (see individual media sections) can be used for initial investigative separations.
93
? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? W-X? ? 7R1? ? ?J@?@? ? ?7@@@@6KO-X?W-K? ? ?@e?@V4@?,?7R@@@@@@@@@? ? ?@e?@eS@U?@?@@?@@?@?@? ? ?@?O&?,?3T@@?@@?@?@? ? ?@@0R+Y?V+R'?@@?@?@? ? ? ? ?@ ? ?@ ? ?@ ?O2@@@@@@@@@@@@? ? ?@ ?O2@@@@@@@ @@@@@@@@@@@@@0M?he@? ? ?@ @? ?O2@@0M?g 3L ? ?@ @? O2@0M?he N1 ? ?@ @? O20Mhg ?@ ? ?@ @? O20M ?@ ? ?@ @? O20M ?@ ? ?@ @? O20M ?3L? ? ?@ @? O20M ?N1? ? ?@ @? O20M @? ? ?@ @? ?O2@0M @? ? ?@ @L ?O20M? @? ? ?@ @1 ?O20M? @? ? W26XfW&?W2@(?g?@ @@ ?O20M? @? ? 7[email protected]'@R1e@@@@@@1?@@e?@?@?@@@L??@V@@@@?g?@?@ 7@eN@V@@@W@e?@6X@?@?e?I@??7R1 ? 7@@V@R@@V@R@@@@)X??@@@U?e?@?@?@e?W.?e@??@e?@e?@e@@?@?@?@?@?@e@?@?f@?@@e@@?@?@?@?@?@e@@@0R/X? 7@@V@R@@V@R@@@@<e7@@Hh?@@@eW-X?e@?e @@?@@??@@@?J@@@? ? ?@?V1?f?3=?V@?@e3U?@@=?@?@@Xe?@?@e@?e3=C5g?@@@,?3=C5eJ@@?,??@?V1?e?@?@?@e?7Y?e3=C5e?3=O&@=C@@?@?@?@?@?@e@?@?f3T@@=C@@?@?@?@?@?@e3Xe?S,? ?J@W@X@?@@T5?@@W5??J@W5?hJ(?'L?*@,?e@?e(R+R'??@@@?V40R40Y @@T@@??@@@?*U@@=O. ? ?@@@@?@?e?V4@@@?@eV4@0R4@@?(R4@??@?@e@?eV40YhI(Y?V40Ye.MI+Y??@@@@?e?@?@?@e?@@@@?V40Ye?V40MI40R'?@@@?@?@?@e@?@?fV+MI40R'?@@@?@?@?@eV4@??.Y? ?@@0R4@@0R+Y?@@0Y??@@0Y?@?g.Y?V/?V+Y?e@?e ? ? ? @@@6K? ?@e?W-XgO@?@@@@@@@@@@?@? ?@K?eO@K?e@? ? ?J@??@@@?@@@@?W2@@@?W2@@e?@e?@e?7R1e@@@@@@ ? ?7@@@@@@?@@W5?78W@@?7@@5g?@e?@?@e@?@?@@ ?@@?@?@@@?@?@? ? J(MW@@@@@@@@HJ5?7>@T@@@Hg?@e?3T5e@?@?@@ ?@@?@?@@@?@?@? ? .Y?.R40?4@@@?.Y?@0R+R4@??@f?@?@?V+Ye@?@?@@?@@?@?@@@?@?@? ? ?W-X ? ?7R1 ? J@?@L? ? 7@@@)T-KO-X?W-K? ? @?e@(R4@?,?7R@@@@@@@@@? ? @?e3UeS@U?@?@@?@@?@?@? ? N)KO&?,?3T@@?@@?@?@? ? ?@@0R+Y?V+R'?@@?@?@? ? ? ? ? ? ?@ ? ?@ ? ?@ O2@@@@@@@@@@ ? ?@ O2@@@@@@@@@@@@0Mh?@ ? ?@ ?@ W20M ?@ ? ?@ ?@ O..M ?3L? ? ?@ ?@ ?@(Y ?N1? ? ?@ ?@ J(Y? @? ? ?@ ?@ 7He @? ? ?@ ?@ ?J5?e @? ? ?@ ?@ ?7H?e @? ? W26XfW&?W26X?g?@ ?@ J5f 3L ? 7)X??@@@U?e@?@?@?e?W.?N@e@?e@?e@??@?@@?@??@?@?@?@?@f?@?@@??@?@@?@??@?@?@?@@@@?V/X? ?@e?@f3=?V@?@??3U?@@=?@?@@X?e@?@??@e?3=C5?h@??3=C@@=C5?eJ@@?,??@?V1?e@?@?@?e?7Y?C@=C5?e3=O&@=C5?@@?@??@?@?@?@?@f?3T@@=C5?@@?@??@?@?@?3X?e?S,? ?@e?@fV4@@@?@??V4@0R4@@?(R4@e@?@??@e?V40Y?h@??V40MI40Y?e.MI+Y??@@@@?e@?@?@?e?@@@0R40Y?eV40MI40Y?@@@@??@?@?@?@?@f?V+MI40Y?@@@@??@?@?@?V4@e?.Y?
Fig. 53. Effect of gradient slope on resolution. (Work by Pharmacia Biotech, Uppsala, Sweden).
Choice of gradient shape Linear Gradients. It is strongly recommended that initial experiments with a new separation problem be carried out using linear gradient elution. The results obtained can then serve as a base from which optimization can be planned. If better resolution is required then the separation can be improved by altering the shape or slope of the gradient. Convex gradients can be used to improve resolution in the last part of the gradient or to speed up a separation when the first peaks are well separated and the last few are adequately separated.
94
Concave gradients can be used to improve resolution in the first part of the gradient or to shorten the separation time when peaks in the latter part of the gradient are more than adequately separated. Complex gradients can be generated to use the maximum resolution offered by isocratic resolution when required combined with steeper gradient portions where resolution is adequate or unnecessary (Fig. 54). Complex gradients offer the maximum flexibility in terms of combining resolution with speed during the same separation. A knowledge of the chromatographic behaviour of the sample obtained from previous separations using simpler gradients is essential. W-X? 7R1? ?J@?@? ?7@@@@6KO.eW-X? ?@e?@V'@Ue7R1? ?@e?@?N@)X?@?@? ?@?C@>,?3T5? ?@@0R+Y?V+Y? ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ )Xe@?eW-T2@@6Xe@@6T&?e@?e?@@@@@@@@@@@@@@@@@@@@? ?@ @)X?@?e7R@@eB1e@?B@@?e@?e?@ @? ?@ @V1?@??J@?@@e?@e@?C@@@@@@?eJ5 @? ?@ @?3X@??7@@@@e?@e@@0Y@?e@?e7H @? ?@ @?V'@??@e?@eC5e@?e@?e@?e@? @? ?@ @??V'??@e?@@@0Ye@?e@?e@?e@? @? ?@ ?J5? @? ?@ @? ?7H? 3L ?@ )Xe@?eW-T2@@6Xe@@6Ke@? ?@ N1 ?@ @)X?@?e7R@@eB1e@?B@@@@@@? ?@ ?@ ?@ @V1?@??J@?@@e?@e@?C(M?@? ?@fJ5 ?@ ?@ @?3X@??7@@@@e?@e@@0Ye@? ?@f7H ?@ ?@ @?V'@??@e?@eC5e@? ?@f@? ?@ ?@ @??V'??@e?@@@0Ye@? ?@f@? ?@ ?@ ?@e?J5? ?@ ?@ ?@e?7H? ?@ ?@ ?@e?@ ?@ ?@ ?@e?@ ?@ ?@ ?@ ?@eJ5 ?@ ?@ ?@ ?@e7H ?@ ?@ ?@ ?@e@? ?@ ?@ ?@ ?@e@? ?@ ?@ ?@ ?@e@? ?3L? ?@ ?@ ?@?J5? ?N1? ?@ ?@ ?@?7H? @? ?@ ?@ ?@?@ @? ?@ ?@ ?@?@ @? ?@ ?@ ?@W5 @? ?@ ?@ ?@@H @? ?@ ?@ ?@@? @? ?@ ?@ ?@@? @? ?@ ?@ ?@5? @? ?@ ?@ ?@H? @? ?@ ?@L? ?@L? @? ?@ ?@1? J@1? @? ?@ ?@@? 7@@? @? ?@ ?@@? @?@? @? ?@ ?@@? @?@? 3L ?@ ?@@? @?@? N1 ?@ ?@@? ?J@?@? ?@ ?@ ?@@? ?7@?@? ?@ ?@ ?@@? ?@@?@? ?@ ?@ ?@@? ?@@?@? ?@ ?@ ?@@? ?@@?@? ?@ ?@ ?@@? J@@?@? ?@ ?@ ?@@? 7Y@?@? ?@ ?@ ?@@? @?@?@? ?@ ?@ ?@@? @?@?@? ?@ ?@ ?@@? ?J5?@?@? ?@ ?@ ?@@? ?7H?@?@? ?@ ?@ ?@@? ?@e@?@? ?@ ?@ ?@@? ?@e@?@? ?@g?@@@@@@?e ?@ ?@@? J5e@?@? ?3L?gW@0Mf ?@ ?@@? 7He@?@? ?N1?f?@@Ug ?@ ?@@? @?e@?@? @?gV@6Kf ?@ ?@@? @?e@?@? @?f?@@@@@@?e ?@ ?@@? ?J5?e@?@? @?hg ?@ ?@@? ?7H?e@?@? @?hg ?@ ?@@? ?@f@?@? @?hg ?@ ?@@? ?@f@?@? @?hg ?@ ?@@? J5f@?@? @?f?@@@@@@?e ?@ ?@@? 7Hf@?@? @?g?W@Xf ?@ ?@@? @?f@?@? @?gW.R/X?e ?@ ?@@? @?f@?@? @?f?W.Y?N1?e ?@ ?@@L @?f@?@? @?f?.Y?e@?e ?@ ?@V1 ?J5?f@?@? @?gO26Kf ?@ ?@?@ ?7H?f@?@? 3Lf?@(MI'@?e ?@ ?@?@ ?@g@?@? N1f?@H??N@?e ?@ ?@ ?@?@ ?@g@?@? ?@f?3L??J5?e ?@hf?)X??@e?W-T2@@6K??@ ?@?@ J5g@?@? ?@f?V/??.Y?e ?@hf?@)X?@e?7R@@??B@@@@@@ ?@?@ 7Hg@?@? ?@hg ?@hf?@V1?@eJ@?@@?e@??@ ?@?@ @?g@?@? ?@f?@@@@@@?e ?@hf?@?3X@e7@@@@?e@??@ J5?@ @?g@?@? ?@hg ?@hf?@?V'@e@?e@??C5? 7H?@ ?J5?g@?@? ?@hg ?@hf?@eV'e@?e@@@0Y? @??@ ?7H?g@?@? ?@hg ?@ @??@ ?@h@?@? ?@fO2@@6Kf ?@ )Xe@?eW-T2@@6X?@e?@f@??@ ?@h@?@? ?@e?@0MeI4@?e ?@ @)X?@?e7R@@eB1?@e?@f@??@ J5h@?@? ?@hg ?@ @? @V1?@??J@?@@e?@?@@@@@f@??@ 7Hh@?@? ?@hg ?@ @? @?3X@??7@@@@e?@?@e?@f@??@ @?h@?@? ?@hg ?@ @L @?V'@??@e?@eC5?@e?@f@??@ @?h@?@? ?@hg ?@ ?J@1 @??V'??@e?@@@0Y?@e?@f@??@ ?J5?h@?3L ?3L?g@?f ?@ ?7Y@ @??@ ?7H?h@?N1 ?N1?g@?f ?@ ?@?@ @??@ ?@h?J5??@ @?g@?f ?@ ?@?@ @??@ ?@h?7H??@ @?g@?f ?@ ?@?@ @??@ ?@h?@e?@ @?g@?f ?@ ?@?@ @??@ J5h?@e?@ @?g@?f ?@ ?@?3L? ?@ @??@ 7Hh?@e?@ @?g@?f ?@ ?@?N1? ?@ @??@ @?h?@e?@ @?g@?f ?@ ?@e@? J@ @??@ @?h?@e?@ @?g@?f ?@ ?@e@? 7@ @??@ ?J5?h?@e?@ @?g@?f ?@ ?@e@? @@ @??@ ?7H?h?@e?@ @?g@?f ?@ ?@e@? @@ @??@ ?@he?@e?@ @?g@?f ?@ ?@e@? @@ @??3L? ?@he?@e?@ @?g@?f ?@ ?@e3L @@ @??N1? J5he?@e?@ @?hf ?@ ?@eN1 @@L?hf@?e@? 7Hhe?@e?@ @?hf ?@ ?@e?@ @V1?hf@?e@? @?he?@e?@ 3Lhf ?@ ?@e?@ @?@?hf@?e@? @?he?@e?@ N1?@6KeO2@?e ?@ ?@e?@ @?@?hf@?e@? ?J5?he?@e?@ ?@eI4@@0Mf ?@ ?@e?@ @?@?hf@?e@? ?7H?he?@e?@ ?@hf ?@ ?@e?@ @?@?he?J5?e@? ?@hf?@e?@ ?@hf ?@ ?@e?@ @?@?he?7H?e@? ?@hf?@e?@ ?@hf ?@ J5e?3L? @?@?he?@f@? J5hf?@e?@ ?@hf ?@ 7He?N1? @?@?he?@f@? 7Hhf?@e?@ ?@hf ?@ @?f@? @?@?he?@f@? ?J@?hf?@e?@ ?@hf ?@ @?f@? @?3Lhe?@f@? O&@?hf?@e?@ ?@hf ?@ @?f@? @?N1he?@f@?hfO2@@0M ?@e?@ ?@hf ?@ @?f@? @??@he?@f@?hO2@@0M ?@e?@ ?@hf ?@ @?f3L @??@he?@f@?fO2@@0M ?@e?@ ?@hf ?@ @?fN1 @??@he?@e?J@?O2@@0M ?@e?@ ?@hf ?@ @?f?@ @??@he?@eO&@@(M ?@e?3L? ?@hf ?@ @?f?@ @??@he?@@@0M?@H? ?@e?N1? ?3L?he ?@ @?f?3L? @??@gO2@@@@f?@ J5f@? ?N1?he ?@ @?f?N1? @??@eO2@@0Me?@f?@ 7Hf@? @?he ?@ @?g@? ?J@?C@@@0Mg?@f?@ @?f@? @?he ?@ @?g@? O&@@0Y@?h?@f?3L? @?f@? @?he ?@ @?g3L O2@@0Y@?e@?h?@f?N1? @?f3L @?he ?@f?@ @?gN1 O2@@0Mf@?e@?h?@g@? ?J5?fN1 @?he ?@f?@ @?g?@ ?O2@0Mh@?e@?hJ5g@? ?7H?f?@ @?he ?@f?@ @?g?@ ?O2@@0M?he@?e3Lh7Hg@? ?@g?@ @?he ?@f?@ @?g?3L? ?O2@@0M? @?eN1h@?g3L ?@g?3L? @?he ?@f?@L?hf@?g?N1? ?O2@@0M? ?J5?e?@h@?gN1 ?@g?N1? @?he ?@e?@@@1?hf@?h@? ?O2@@0M? ?7H?e?3L?g@?g?@ J5h@? @?he ?@e?3@@5?he?J5?h3Lhe?O2@@0M? ?@f?N1?f?J5?g?3L? 7Hh@? @?he ?@e?N@@H?he?7H?hN1g?O2@@0M? C5g3Lf?7H?g?N1? ?J@?h3L @?he ?@f3@hfJ@he?3=??O2@@0M? O2@@@@0YgN1fJ5he3L O&@?hN)X? @?he ?@fN@he?O&@he?S@@@@Y? O20Mhf?3=??W.YheN)K? O20Mhe?@)K 3Lhe ?@f?@@@@@@@@@@@@0M?g?O2@@0R4@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@0M ?V46T.Y?he?@@6K?hO2@@@@@@@@0M I4@6K? N1he ?@ ?O2@@0M? ?I+Y ?I4@@@@@@@@@0M ?I4@@@@@@@@@@@@@@@@@@@@@@??@he ?@ ?O2@@0M? ?@he ?@f?@@@@@@@@@@@@@@0M? ?@he ?@ ?@he ?@ ?@he ?@ ?@L?h ?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?h @?heI@M?he@?he?I@Mhe?@he?I@Mhe?@hfI@M?he@?heI@M?he@?heI@M?he@? @? @? ?@ ?@ @? @? @? @? @? ?@ ?@ @? @? @? ?W26X? W& W26X ?W&?W26X ?W&?W2@? ?W26KO26X? ?W26X?W& ?7
Fig. 54. Anion exchange chromatography of a mixture of pyridine nucleotides on Mono Q (28): Sample, 100 µl of 100 µM NAD, 100 µM NADH, 250 µM NADP and 250 µM NADPH; column, Mono Q HR 5/5; buffer A, 20 mM triethanolamine, pH 7.7; buffer B, buffer A with 1.0 M KCl; gradient, 0% B for 2 ml, 0-20% B in 15 ml, 20-100% B in 5 ml, 100% B for 3 ml; flow rate, 1 ml/min.
Sample displacement When a sample of solutes, such as proteins, is applied to the top of an ion exchange column the species with the highest charge density will bind at the top, displacing more weakly bound species or preventing such from binding. In effect a degree of separation occurs on the column during sample loading, with the solutes stacked on the column in order of their relative charges and strengths of binding. On application of a gradient the increasing salt concentration will cause the most weakly bound molecules to migrate and leave the column first. For this reason “reverse flow elution” should never be used in the ion exchange separation of complex mixtures. Under such conditions early desorbing substances would have to migrate through all other bound species, possibly displacing them, and lead to lost resolution.
95
Gradient generation Accurate and reproducible pH and ionic strength gradients are best formed using purpose designed equipment. The choice of gradient generating system will depend upon the type of ion exchange media and the required complexity of the gradient.
Gradient formation with two pumps or a single pump in combination with a switch valve Maximum flexibility in terms of gradient production is achieved by using two separate pumps for start and limit buffers or a single pump in combination with a switch valve. Using gradient programmers, e.g. GP-250 Plus or LCC-501 Plus, or using chromatography systems which are controlled via a controlling software, e.g. UNICORN and FPLCdirector, the proportions of the start and limit buffers which constitute the eluent being supplied to the column are programmed for specific times or volumes during the separation. The relative amounts of start and limit buffer then increase or decrease in a linear fashion between two such “breakpoints” to produce the gradient. The more breakpoints which have been programmed the more complex the gradient. Further information on controlling software, gradient programmers and related pump systems is available from Pharmacia Biotech.
Gradient Mixer The Pharmacia Gradient Mixer GM-1 can be used to make linear ionic strength or pH gradients of up to 500 ml in volume (Fig. 55). The mixing chamber should contain the starting buffer and the other chamber the limiting buffer. Although the Gradient Mixer GM-1 will not produce linear pH gradients it can be used to form reproducible continuous pH gradients from two solutions of different pH and similar ionic strength.
Fig. 55. Gradient elution system using the Gradient Mixer GM-1.
96
?W2@@@@@@@@@@@@6X? ?7@@@@@@@@@@@@@@1? ?@@@@@@@@@@@@@@@@? ?@ @? ?@g@@g@? ?@@@?@e@@?@?@e@? ?@ @? ?@ @? ?@@@?@f?@?@e@? ?@ @? ?@ @? ?@@6?@K?eO@?@K?@? ?@@@@@@@@@@@@@@@@? W2@@@@@@@@@@@@6X?@@T@T@T@T@T@T@X@? 7@@@@@@@@@@@@@@1?@V@R@R@R@R@R@R@@? @?hf?@?@@@@@@@@@@@@@@@@? @?hf?@?@f?@e?@f@? @?hf?@?@f?@e?@f@? @?hf?@?@f?@e?@f@? @?hf?@?@f?@eJ5f@? @?hf?@?@f?3L?7Hf@?g?W2@6X?@@?e@@fW& @?hf?@?@f?N1?@?f@?g?7,?N@??@hf@?@?f@?@? ?@ W&R)X@?N1?f?@?3=C5??@@Y?C@=C5hf@?@@@@@@@?@? ?@ &@?@@@e@?f?@?V40Y??@@@@0R40Yhf@?@@@@@@@?@? ?@ @?@?f@?@? ?@ @?@@@@@@@?@? ?@ @?@@@@@@@?@? ?@ @?@@@@@@@?@? ?@ @?@@@@@@@?@? ?@ @?@@@@@@@?@? ?@ @?@@@@@@@?@? ?@ @?@@@@@@@?@? ?@ @?@@@@@@@?@? ?@ @?@@@@@@@?@? ?@ @?@@@@@@@?@? J5 @?@@@@@@@?@? 7H @?@@@@@@@?@? @? @?@@@@@@@?@? @? @?@@@@@@@?@? @? @?@@@@@@@?@? @? @?@@@@@@@?@? @? @?@@@@@@@?@? @? @?@@@@@@@?@? @? @?@@@@@@@?@? @? @?@@@@@@@?@? @? @?@@@@@@@?@? @? @?@@@@@@@?@? @? @?@@@@@@@?@? @? @?@@@@@@@?@? @? @?@@@@@@@?@? @? @?@@@@@@@?@? @? @?@@@@@@@?@? @? @?@@@@@@@?@? @? @?@@@@@@@?@? @? ?J@?@@@@@@@?@L @? ?7@@@@@@@@@@@)X? @? J(M?h?B1? @? 7Hhf@? @? @?hf@@@6K? @? @@@@@@@@@@@@@@@@@@@@@@@?he@? @?hf@@@@@@@@@?he@? @?hf@@@@@@@@@?he@? @@@@@@@@@@@@@@@@@0M? @? @?hf@? @? @?hf@? @? @?hf@? @? @?hf@? @? @?hf@? @? @?hf@? @? @@@@@@@@@@@@@@@? @? @@@@@@@@@@@@@@@? @? @@@@@@@@@@@@@@@? @? 3@@@@@@@@@@@@@5? @? V4@@@@@@@@@@@0Y? @? @@@@@@@(M? @? @@@@@@@H @? @@@@@@@? @? @@@@@@@? @? 3@@@@@5? @? N@@@@@H? @? ?@@@@@ @? ?@@@@@ @? ?3@@@5 @? ?V4@@H @? @? @? @? ?J5? @? ?7H? 3L J5 V/X? 7H ?N1? ?J5? 3L W.Y? V/K? ?W.Y ?V46K?hfW.Y? ?I46K?h?O.Y ?I4@6Ke?O2@@0Y? I4@@@0M?
An example of a decreasing pH gradient produced by the Gradient Mixer GM-1 is shown in Figure 56. The gradient was produced from 0.1 M solutions of Tris (free base), pH 10.5 and Tris-HCl, pH 7.5. Usually changes in pH also produce small changes in ionic strength. These can be estimated by monitoring conductivity. ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?W&?W26Xe?W26X? ? ?*@?7
Fig. 56. Gradient from pH 10.5 to 7.5 in 400 ml produced using the Gradient Mixer GM-1. pH –, conductance - - -. (Work by Pharmacia Biotech, Uppsala, Sweden.)
Batch separation There is essentially no difference in separation procedure between a column developed by stepwise elution and a batch procedure. Either the substance of interest or contaminants may be attached to the ion exchanger. Although batch procedures are less efficient than column techniques they may offer advantages in particular cases. When very large sample volumes with low protein concentration have to be processed, the sample application time on a column can be very long and filtration of such a large sample can also be rather difficult to perform. Binding the sample in batch mode will be much quicker and there will be no need to remove particulate matter. A batch procedure can also be an attractive approach if high sample viscosity generates high back-pressure in a column procedure or if high back-pressure is generated by contaminants such as lipids, which may cause severe fouling and clogging of the column. Batch separation is a very rapid technique and no technical difficulties are caused by the swelling or shrinkage of Sephadex ion exchangers. The shrinkage may even be an advantage in some applications.
97
When working with batch ion exchange, the starting conditions are selected in the same way as in column chromatography, i.e. choose buffer pH an ionic strength to bind the substance of interest but to prevent as many contaminants as possible from binding. To maximize recovery, the starting conditions should be selected so that the protein of interest binds much stronger than is usual in column chromatography. Unless the proteins adsorbs to 100%, losses during subsequent washing will be inevitable, especially if the volume of liquid is large compared to the volume of adsorbent. To keep recovery high, the pH in a batch experiment may have to be several units away from the isoelectric point of the protein. Batch separation is carried out by stirring the ion exchanger previously equilibrated in the appropriate buffer with the solution to be treated until the mixture has reached equilibrium. This usually takes about one hour. The slurry is then filtered and washed with the buffer solution. In cases of incomplete adsorption this procedure should be repeated on the filtrate with a new batch of ion exchanger. Then elution buffer is added (1-2 times the volume of the sedimented gel) and stirred until desorption is complete, which can take up to 30 minutes or more. Finally, suction is used to filter the buffer containing the desorbed product of interest from the adsorbent. The gel can also be packed in a column after the washing step and be eluted stepwise in the same way as during normal column chromatography. Resolution will however be lower for such a combined batch and column procedure compared with a normal column procedure, since the sample is bound uniformly throughout the gel slurry and the subsequent chromatographic bed. Under these conditions stepwise elution is recommended since gradient elution will give broad bands and poor resolution. Batch chromatography is very useful for concentrating dilute solutions and separating the substances of interest from gross contaminants during the initial stages of a purification scheme. Note: Fines will be generated if the ion exchangers are stirred too vigorously. This will increase the time required for filtration.
Expanded bed adsorption Expanded bed adsorption is a unit operation that uses STREAMLINE adsorbents and columns for recovering proteins directly from crude samples. Proteins are recovered in a single pass without the need for prior clarification. STREAMLINE has proven effective in purification proteins from fermentation or cell culture in extracellular processes, and has demonstrated its suitability when used with broth from cell lysis and homogenization in intracellular processes with soluble proteins. STREAMLINE reduces the number of operations in a process by fusing the functions of clarification, concentration and capture (see page 109) in one operation.
98
It offers the selectivity afforded by chromatography, the throughput of ultra-filtration and the convenience of small scale centrifugation. Crude feed from the fermentor containing the desired product and undesired cells, cell debris and particulates is applied to the expanded bed. Target products are bound by the adsorbent while particulates and contaminants pass through unhindered. The desired molecule is then eluted as in packed bed chromatography.
Expanded bed technology Expanded bed adsorption is based on fluidization. The sedimented bed begins to expand as the adsorbent particles are raised by an upward liquid flow. The difference between a fluidized bed and expanded bed is that in an expanded bed the adsorbent particles display very little back-mixing. This is achieved through the unique design of the column and the adsorbents. The column has a special flow distributor at the bottom, the adsorbent particles have a well-defined size and density distribution. The particles are kept in suspension by the balance between upward flow rate and particle sedimentation velocity. As the bed expands with the upward liquid flow, the movement of any given particle is very small. This creates a stable, homogeneous expanded bed and a liquid flow which is characterized by a constant velocity profile, i.e. plug flow. The stability of the expanded bed give STREAMLINE characteristics that are similar to those of a packed bed in chromatography.
Basic principle of operation 1. STREAMLINE adsorbent is poured into STREAMLINE column and allowed to sediment (Fig. 57 a). 2. An upward liquid flow of equilibration buffer is applied to the column and STREAMLINE adsorbent particles are suspended in the flow, creating a stable fluidized bed (Fig. 57 b). 3. The sample, a mixture of soluble proteins, contaminants, cells, or cell debris, is passed upwards through the expanded bed. The target proteins are bound on STREAMLINE adsorbent while particulates and contaminants pass through the expanded bed unrestricted. Loosely bound material is washed out with the upward flow of the buffer (Fig. 57 c). 4. The liquid flow is reversed to downward flow. By using suitable buffer conditions, the bound proteins are eluted from STREAMLINE adsorbent in a sedimented bed mode. The eluate contains the target proteins, increased in con centration, free from particulates and ready for further purification (Fig. 57 d).
99
a
c
b
d
?@@@@? ?@@@ ?@@@ ?@e@? ?@?@ ?@?@ ?@e@? ?@?@ ?@?@ ?@e@? ?@?@ ?@?@ ?@e@? ?@?@ ?@?@ ?@e@? ?@?@ ?@?@ ?@e@? ?@?@ ?@?@ ?@e@? ?@?@ ?@?@ ?@e@? ?@?@ ?@?@ ?@e@? ?@?@ ?@?@ ?@e@? ?@?@ ?@?@ ?@e@? ?@?@ ?@?@ ?@e@? ?@?@ ?@?@ ?@e@? ?@?@ ?@?@ ?@e@? ?@?@ ?@?@ ?@e@? ?@?@ ?@?@ ?@e@? ?@?@ ?@?@ ?@e@? ?@?@ ?@?@ ?@e@? ?@?@ ?@?@ ?@e@? ?@?@ ?@?@ ?@e@? ?@?@ ?@?@ ?@e@? ?@?@ ?@?@ ?@e@? ?@?@ ?@?@ ?@@@@? ?@e@? ?@?@ ?@?@ ?@e@? ?@e@? ?@?@ ?@?@ ?@e@? ?@e@? ?@?@ ?@?@ ?@e@? ?@e@? ?@?@ ?@?@ ?@e@? ?@e@? ?@?@ ?@?@ ?@e@? ?@e@? ?@?@ ?@?@ ?@e@? O2@@e@@6K O2@@?@@@6K O2@@?@@6K? O2@@e@@6K ?O2@@@0M?@e@?I4@@@6K? O2@@@@0M?@?@eI4@@@6K? O2@@@@0M?@?@?I4@@@6K O2@@@@0M?@e@?I4@@@6K?hg O2@@@0M?f?@e@?f?I4@@@6K ?O2@@@0Mg?@?@g?I4@@@6K ?O2@@@0Mg?@?@gI4@@@@6K ?O2@@@0Mg?@e@?f?I4@@@6Khe O2@@0Mhe?@e@?heI4@@6K ?O2@@0M?he?@?@hfI4@@6K ?O2@@0M?he?@?@hfI4@6K? ?O2@@0M?he?@e@?heI4@@6Kg O20M ?@e@? I46Khf?W20M? ?@?@ I46Khf?O20M? ?@?@ ?I46K?he?W20M? ?@e@? I46Kf W20M ?@e@? I46XheW.M? ?@?@ I46Xhe@(M? ?@?@ ?I46X?hW.M? ?@e@? I46Xe ?W.M ?@e@? I/X?g?W.Y ?@?@ I/X?g?J(Y ?@?@ ?I/Xg?W.Y ?@e@? I/X? ?7U? ?@e@? ?S1?g?7U? ?@?@ ?S1?g?7H? ?@?@ S1g?7U? ?@e@? ?S1? ?@1? ?@e@? ?7@?g?@1? ?@?@ ?7@?g?@L? ?@?@ 7@g?@1? ?@e@? ?7@? ?@@= ?@e@? C@@?g?@@= ?@?@ C@@?g?@)X ?@?@ ?J@@g?@@= ?@e@? C@@? ?@V46K ?@e@? O20Y@?g?@V46K ?@?@ O20Y@?g?@@)K? ?@?@ O.Y@g?@V46K ?@e@? O20Y@? ?@eI46K ?@e@? O20Me@?g?@eI46K ?@?@ O20Me@?g?@?I46K? ?@?@ O20Y?@g?@eI46K ?@e@? O20Me@? ?@fI4@@6Khe?@e@?heO2@@0Mf@?g?@fI4@6K?he?@?@hfO2@@0Mf@?g?@e?I4@@6K?he?@?@hfO2@@0Me?@g?@fI4@6K?he?@e@?heO2@@0Mf@? ?@hI4@@@6K?f?@e@?f?O2@@@0Mh@?g?@g?I4@@@@6K?f?@?@g?O2@@@0Mh@?g?@g?I4@@@@6K?f?@?@g?O2@@@0Mg?@g?@g?I4@@@@6K?f?@e@?f?O2@@@0Mh@? ?@hf?I4@@@6K?@e@?O2@@@0M?hf@?g?@hf?I4@@@6K?@?@?O2@@@@0M?hf@?g?@hf?I4@@@6K?@?@?O2@@@@0M?he?@g?@hf?I4@@@6K?@?J@W2@@@@0M?hf@? ?@ S@@@e@@@U @?g?@ V@@@?@@@U? @?g?@ V@@@?@@@U? ?@g?@ I4@@?@@(M? @? ?@ ?O&@@@e@@@)K? @?g?@ O2@@@@?@@@)K @?g?@ O2@@@@?@@@)K ?@g?@ ?@?N@H @? ?@h?O2@@@@@@0M??@e@??I4@@@@@@6K?h@?g?@hO2@@@@@@0Me?@?@eI4@@@@@@6Khe@?g?@hO2@@@@@@0Me?@?@eI4@@@@@@6Kh?@g?@ ?@e@? @? ?@fO2@@@0M?h?@e@?h?I4@@@6Kf@?g?@e?O2@@@0Mhe?@?@heI4@@@@6Kf@?g?@e?O2@@@0Mhe?@?@heI4@@@6K?e?@g?@ ?@e@? @? ?@eO20M ?@e@? I46Ke@?g?@?W20M? ?@?@ I46Ke@?g?@?O20M? ?@?@ ?I46K??@g?@ ?@e@? @? ?@W20M ?@e@? I46X@[email protected]? ?@?@ I46X@?g?@@(M? ?@?@ ?I46X@g?@ ?@e@? @? ?@@< ?@e@? B@@?g?@@H ?@?@ B@@?g?@(Y ?@?@ ?B@@g?@ ?@e@? @? ?@5? ?@@@@? ?3@?g?@5? ?@@@ ?3@?g?@H? ?@@@ 3@g?@ ?@e@? @? ?@ @?g?@ @?g?@ ?@g?@ ?@e@? @? ?@1? ?7@?g?@1? ?7@?g?@L? 7@g?@ ?@e@? @? ?@@? ?@@?g?@@? ?@@?g?@)X @@g?@ ?@e@? @? ?@@@6K O2@@@?g?@@@6K O2@@@?g?@@)K? ?O2@@@g?@ ?@e@? @? ?@V4@@6K O2@@0Y@?g?@V4@@6K O2@@0Y@?g?@@@@6K? ?O2@@0Y@g?@ ?@e@? @? ?@eI4@@@@@@6K ?O2@@@@@0Me@?g?@eI4@@@@@@6K O2@@@@@@0Me@?g?@?I4@@@@@@6K? O2@@@@@0M??@g?@ ?@e@? @? ?@fI4@@@@@@@@@@@@6Kg?O2@@@@@@@@@@@0Mf@?g?@fI4@@@@@@@@@@@@6KgO2@@@@@@@@@@@@0Mf@?g?@e?I4@@@@@@@@@@@@6K?gO2@@@@@@@@@@@0M?e?@g?@ ?@e@? @? ?@heI4@@@@@@@@@@@@@@@@@@@@@@@0M?h@?g?@heI4@@@@@@@@@@@@@@@@@@@@@@0Mhe@?g?@h?I4@@@@@@@@@@@@@@@@@@@@@@@0Mh?@g?@ ?@e@? @? ?@ I4@@@@@@@0M? @?g?@ I4@@@@@@0M @?g?@ @@@@@@@@@? ?@g?@ ?@e@? @? ?@ @?g?@ @?g?@hfO2@@@@@@@@@@@@@@@@6Khf?@g?@ ?@e@? @? ?@ @?g?@ @?g?@g?O2@@@@@@@@@@@@@@@@@@@@@@@@@@6K?g?@g?@ ?@e@? @? ?@ @?g?@ @?g?@e?O2@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@6K?e?@g?@ ?@e@? @? ?@ @?g?@ @?g?@?O2@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@6K??@g?@ ?@e@? @? ?@ @?g?@ @?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@6X@g?@ ?@e@? @? ?@ @?g?@ @?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@g?@ ?@e@? @? ?@ @?g?@ @?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@g?@ ?@e@? @? ?@ @?g?@ @?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@g?@ ?@e@? @? ?@ @?g?@hf?O2@@@@@@@@@@@@@@@6K @?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@g?@ ?@e@? @? ?@ @?g?@g?O2@@@@@Y@@@@@@@@@@@@@@@V@@@@@6Kh@?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@g?@ ?@e@? @? ?@ @?g?@fO2@@Y?@@@@@@@@@@@@@@@@@@@@@@@@@@V@@6K?f@?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@g?@ ?@e@? @? ?@ @?g?@e?@@Y@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@6K?e@?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@g?@ ?@e@? @? ?@ @?g?@W@X@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@6X?@?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@g?@ ?@e@? @? ?@ @?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@)X@?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@g?@ ?@e@? @? ?@ @?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@g?@ ?@e@? @? ?@ @?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@g?@ ?@e@? @? ?@ @?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@g?@ ?@e@? @? ?@ @?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@g?@ ?@e@? @? ?@ @?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@g?@ ?@e@? @? ?@ @?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@g?@ ?@e@? @? ?@ @?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@g?@ ?@e@? @? ?@ @?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@g?@ ?@e@? @? ?@ @?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@g?@ ?@e@? @? ?@ @?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@g?@ ?@e@? @? ?@ @?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@g?@ ?@e@? @? ?@ @?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@g?@ ?@e@? @? ?@ @?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@g?@ ?@e@? @? ?@ @?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@g?@ ?@e@? @? ?@ @?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@g?@ ?@?J@? @? ?@ @?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@g?@ O2@@@@?@@@6K @? ?@ @?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@g?@hO2@@@@@@0Me?@?N@?I4@@@@@@6Khe@? ?@hf?O2@@@@@@@@@@@@@@@6K @?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@g?@fO2@@0Mhe?@e@?hI4@@@6K?f@? ?@g?O2@@@@@Y?@@@@@@@@@@@@@@@@@@@@6Kh@?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@g?@eW20M ?@e@?hf?I46K?e@? ?@fO2@@Y?@@@@@@@@@@@@@@@@@@@@@@@@@@V@@6K?f@?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@g?@?W.M ?@e@? ?I46X?@? ?@e?@@Y@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@6K?e@?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@[email protected]? ?@e@? ?I/X@? ?@W@X@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@6X?@?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@g?@(Y ?@e@? V'@? ?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@)X@?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@g?@ ?@@@@? @? ?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@g?@1? ?7@? ?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@g?@@? ?@@? ?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@g?@@@6K O2@@@? ?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@g?@@@V@6K O2@Y@@@? ?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@g?@@@@@V@@@@@6K O2@@@@@Y@@@@@? ?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@g?@@@@@@@fS@@@@@@@6KgO2@@@@@@@Uf@@@@@@@? ?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@g?@@@@@@@@@@@@YgS@@@@@@@@UgV@@@@@@@@@@@@? ?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@g?@@@@@@@@@@@@@@@@@@@@YgV@@@@@@@@@@@@@@@@@@@@? ?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@? ?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@? ?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@? ?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@? ?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@? ?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@? ?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@? ?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@? ?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@? ?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@? ?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@? ?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@? ?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@? ?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@? ?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@? ?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@g?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@? J@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?gJ@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?gJ@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@gJ@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@? 7@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?g7@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?g7@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@g7@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@? @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?g@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?g@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@g@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@? @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?g@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?g@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@g@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@? 3@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@5?g3@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@5?g3@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@5g3@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@5? V'@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@(Y?gV'@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@(Y?gV'@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@(YgV'@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@(Y? ?V4@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@0Yh?V4@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@0Yh?V4@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@0Y?g?V4@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@0Ye ?I4@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@0Mhf?I4@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@0Mhf?I4@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@0M?he?I4@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@0Mf I4@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@0M? I4@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@0M? I4@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@0M I4@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@0M?g ?I4@@@@@@@@@@@@@@@@@@@@@@@@@@@0M ?I4@@@@@@@@@@@@@@@@@@@@@@@@@@@0M ?I4@@@@@@@@@@@@@@@@@@@@@@@@@@0M? ?I4@@@@@@@@@@@@@@@@@@@@@@@@@@@0Mhe ?I4@@@@@@@@@@@@@@@0M ?I4@@@@@@@@@@@@@@@0M ?I4@@@@@@@@@@@@@@0M? ?I4@@@@@@@@@@@@@@@0M ?W&? ?7@L J@@)X? 7@@@1? ?J@@@@@L W&@@@@@1 7@@@@@@@L? ?J@@@@@@@@)X ?7@@@@@@@@@1 J@@@@@@@@@@@ @@@@@@@@ @@@@ @@@@ @@@@ @@@@ @@@@ @@@@ @@@@ @@@@ @@@@
Buffer
W& 7@L? ?J@@1? ?7@@@L J@@@@1 ?W&@@@@@L? ?7@@@@@@1? J@@@@@@@@L 7@@@@@@@@1 ?J@@@@@@@@@@ ?@@@@@@@ ?@@@ ?@@@ ?@@@ ?@@@ ?@@@ ?@@@ ?@@@ ?@@@ ?@@@
Feed
W& 7@L? ?J@@)X ?7@@@1 J@@@@@L? ?W&@@@@@1? ?7@@@@@@@L J@@@@@@@@)X? 7@@@@@@@@@1? ?J@@@@@@@@@@@? ?@@@@@@@@? ?@@@@? ?@@@@? ?@@@@? ?@@@@? ?@@@@? ?@@@@? ?@@@@? ?@@@@? ?@@@@?
Eluate
Fig. 57. The principle of operation of expanded bed adsorption.
STREAMLINE adsorbents STREAMLINE adsorbents are described in detail in Chapter 6.
STREAMLINE columns STREAMLINE columns are designed to suit the different stages of process development. STREAMLINE 50 column is designed for method optimization. A set-up with this column can handle 1-20 l from the fermentor at a throughput of 6 l/h. A set-up with STREAMLINE 200 column, for pilot scale and verification of the optimized methods, can handle 50-300 l of sample at a throughput of 100 l/h. STREAMLINE CD column, custom designed for industrial manufacturing, can handle production volumes of samples at a throughput of several thousands of litres per hour.
Auxiliary equipment To operate expanded bed adsorption in method optimization some auxiliary equipment is needed. For example, a peristaltic pump, manual valves, UV, pH and conductivity monitors and a recorder, all standard laboratory equipment. For production installations STREAMLINE is engineered to your specifications.
100
Regeneration After each cycle, bound substances must be washed out from the column to restore the original function of the media. Ion exchange adsorbents can normally be regenerated after each run by washing with a salt solution until an ionic strength of about 2 M has been reached. This should remove any substances bound by ionic forces. The salt should contain the counter-ion to the ion exchanger to facilitate equilibration. To prevent a slow build up of contaminants on the column over time, more rigorous cleaning protocols may have to be applied on a regular basis, see below.
Cleaning, sanitization and sterilization procedures Cleaning Cleaning-in-place (CIP) is the removal from the purification system of very tightly bound, precipitated or denatured substances generated in previous purification cycles. In some applications, substances such as lipids or denatured proteins may remain in the column bed instead of being eluted by the regeneration procedure. If contaminants accumulate on the column over a number of purification cycles, they may affect the chromatographic properties of the column. If fouling is severe, it may also block the column, increasing the back-pressure and reducing the flow rate. A specific CIP protocol should be designed according to the type of contaminants that are known to be present in the sample. NaOH is a very efficient cleaning agent that can be used for solubilizing irreversibly precipitated proteins and lipids. NaOH can effectively be combined with solvent or detergent based cleaning methods.
Sanitization Sanitization is the inactivation of microbial populations. When a packed column is washed with a sanitizing agent, the risk of contaminating the purified product with viable micro-organisms is reduced. The most commonly used sanitization method in chromatography today is to wash the column with NaOH. NaOH has a very good sanitizing effect and also has the addition advantage of cleaning the column.
Sterilization Sterilization, which is not synonymous with sanitization, is the destruction or elimination of all forms of microbial life in the system.
101
Protocols for cleaning-in-place (CIP), sanitization and sterilization. Suggested protocols for cleaning-in-place (CIP), sanitization and sterilization that can be applied to each specific ion exchanger from Pharmacia Biotech are summerized below.
SOURCE and Sepharose based ion exchangers CIP, sanitization and sterilization protocols for SOURCE and Sepharose based ion exchangers media are summarized in Table 22. Table 22. Suggested CIP, sanitization and sterilization protocols for SOURCE 15 and 30, and Sepharose based ion exchangers media from Pharmacia Biotech. Purpose
Procedure
Removal of precipitated proteins
4 bed volumes of 0.5-1.0 M NaOH at 40 cm/h followed by 2-3 bed volumes of water.
Removal of strongly bound hydrophobic proteins, lipoproteins and lipids
4-10 bed volumes of up to 70% ethanol or 30% isopropanol followed by 3-4 bed volumes of water. or 1-2 bed volumes of 0.5% non-ionic detergent (e.g. in 1 M acetic acid) followed by 5 bed volumes of 70% ethanol to remove the detergent, and 3-4 bed volumes of water.
Sanitization
0.5-1.0 M NaOH with a contact time of 30-60 min.
Sterilization
Autoclave the medium at 121 °C for 15 min.
MonoBeads and MiniBeads columns Due to the small particle size of MonoBeads and MiniBeads, they are more sensitive to particulate matter such as precipitated proteins from the sample or buffer solutions than the larger bead size matrices. Preventative measures to ensure cleanliness of the sample and buffers are essential to ensure long column life. Sample preparation procedures are described earlier in this Chapter. Should precipitated material be present, as indicated by a decrease in performance or an increase in back-pressure, the columns may be cleaned using the detailed instructions included with the column.
102
DEAE Sephacel and Sephadex based ion exchangers Due to the relatively large volume changes of Sephadex based gels in different solvents, we recommend cleaning and washing with organic solvents on a Büchner funnel, since the gel needs to be repacked after such treatment. Remove ionically bound proteins by washing the column with 0.5-1 bed volume of a 2 M NaCl solution. Remove precipitated proteins, hydrophobically bound proteins and lipoproteins by washing the column with 0.1 M NaOH solution, contact time 1-2 hours, followed by binding buffer until free from alkali. Alternatively, wash the column with 2 bed volumes of 6 M guanidine hydrochloride. Strongly hydrophobically bound proteins, lipoproteins and lipids can be removed by washing the gel with up to 70% ethanol or 30% isopropanol. Alternatively, wash the gel with 2 bed volumes of a non-ionic detergent in a basic or acidic solution. Use for example, 0.1-0.5% non-ionic detergent (e.g. Triton X-100) in 0.1 M acetic acid. After treatment with detergent always remove residual detergents by washing with 5 bed volumes of 70% ethanol. Re-equilibrate the ion exchanger with starting buffer.
Storage of gels and columns Prevention of microbial growth As well as endangering the sample, bacterial and microbial growth can seriously interfere with the chromatographic properties of ion exchange columns and may obstruct the flow through the bed. During storage an antimicrobial agent should always be added to the ion exchanger. Antimicrobial agents may be eluted from the columns during equilibration before starting a run. Recommended antimicrobial agents for anion exchangers: Equilibrate the column with 20% ethanol in 0.2 M acetate. Recommended antimicrobial agents for cation exchangers: Equilibrate the column with 20% ethanol or 0.01 M NaOH.
103
Storage of unused media Unused media should be stored in closed containers at +4 °C to +25 °C. Note that it is important that the media are not allowed to freeze as the structure of the beads may be disrupted by ice crystals. This disruption will generate fines.
Storage of used media Used media should be stored at a temperature of +4 °C to +8 °C in the presence of an antimicrobial agent, e.g. 0.01 M NaOH or 20% ethanol according to the recommendation given above. Note that it is important that the media are not allowed to freeze as the structure of the beads may be disrupted by ice crystals. This disruption will generate fines.
Storage of packed columns Packed columns should be stored at a temperature of +4 °C to +8 °C in the presence of an antimicrobial agent, e.g. 0.01 M NaOH or 20% ethanol according to the recommendation given above. For long-term storage, the packed column should be thoroughly cleaned before equilibration with the storage solution. Recycling the storage solution through the column or flushing the column once a week with fresh storage solution is recommended to prevent bacterial growth.
Determination of the available and dynamic capacities The available capacity of an ion exchanger can be determined by a batch test-tube method similar to that used for the determination of suitable buffer pH and binding and elution ionic strengths, see page 70 and Figure 38. In this case a series of solutions with different concentrations of the protein are added to a known quantity of ion exchanger, equilibrated at a suitable binding pH and ionic strength. Assaying the supernatants after mixing will show the maximum protein concentration which can be bound per ml of ion exchanger. For a more realistic and useful measurement of the available capacity of an ion exchanger, a dynamic method is recommended (see page 18 for definition of available and dynamic capacity). The type of equipment necessary for this determination is shown in Figure 58. FPLC System can also be used for this determination.
104
? ?? W2@6X? ?@ @? 7U?I/?'6X?@@@@@?@@6X?@?W26X??@@?W26KO.eW26X?@@?/X?W2@6X@?@@ ?? @)fS@1?@?@?@?@?B1?@?7YV1??@H?7YV@@Ue7YV1?@H?N1?7@?B@@?@H ?? ?'@@(Y@?@?@?@?@??@?@?@@@@??@e@@@0R/X?@@@@?@e?@?@@??@@?@? /K?S@@U?@?@?@?@?@?C5?@?3X?e?@e3Xe?S,?3Xe?@e?3T@@=C@@?@? ? V4@0MI4@@?@?@?@?@@0Y?@?V4@e?@eV4@??.Y?V4@??@e?V+MI40R'?@? @? ?? ?W.?hO@ @? /X ?? ?7HW.??/X?W2@@?@@@eW2@6T26XN1 ? ?@?*U??N1?7@?@?@?@e7@@@@@@@@h?@eS,e3T@@?@?@?@e3=?@@Xe?@ N@@@@@@@@@[email protected]@R'?@?@?@eV4@@V4@??@ ?? ?@@@@@@@@@@@@?f?3L?fJ@hfW5fJ5 ? ?@@@@@@@@@@@@?f?V/?f@@[email protected] ?@@@@@@@@@@@@? ?? ?@@@@@@@@@@@@? ?3@@@@@@@@@@@? ?? ?N@?g?@@@@@ @?g?@@@@@ ?? @?g?@ ? @?g?@ ?? ?W2@@@@@@@@@@@@6X? 3LgJ5 ?7@@@@@@@@@@@@@@1? N1f?W.Y ?@@@@@@@@@@@@@@@@? ?3L?eW.Y? ?? ?@ @? ?N1?e7H ? ?@g@@g@? @?e@? ?@?@f@@?@?@e@? @?e@? ?? ?@ @? @?e@? ?@ @? 3L?J5? ?? ?@?@g?@?@e@? S)?&U?f?@h@K W& @?e@Ke@?he?@f@?e@?e@6X? ?@ @? 7@@@1?f?@f'6X?@@6KO26X?@@?'6X?*@W26X?@@6X?W.e3L?J@@6X@?/X?W2@6Xe?@f3L?J5?e?S,? ?? ?@ @? @@@@@?f?@fS@1?@?B@@@@@@@@X@@@@@@@@@@@@?@@@@@@@6Ke?@@@@@@@@5 O&@@@@@V@@@?@@V@@??@e@@e@@?@@@@@?@@@6X?@@@@@@@(Y ?? @@@@X@@@@@@@5?@5?@@??@e@@e@5?3@?@@@@@@@)X@@@@@@(Y? ?? ?S@@@X@@@@H?@Y?@@??@e@@e@Y?V@@@@?@@@@@@@@@@@@H ?@@@@@Y?V@@??@e@@@@@??@L?3@e@@@@@??@@@@@X?e@?e@Lhe@@@6K?h?W2@hfW2@6X?f@?@?hW&he?@@@@?@@@@@??W-KO2@6X?e?W&??W26KO26X? ? ?@e@@@@@@@@@@@@@@@X@??@)KV@e@?e@??@@@@@)Xe@@@@@1he@??B@@e'6KO26T&@@?W26T2@@f7
Fig. 58. Experimental set-up for the determination of the dynamic capacity of an ion exchanger.
A defined amount of gel is introduced into the column (Normally a quantity of gel to give a bed volume of approximately 1 ml is sufficient). The gel is packed and equilibrated until the eluate is of the same pH as the starting buffer. The exact volume of gel is calculated from the known column diameter and the measured bed height. In the case of a pre-packed column the amount of gel is already predetermined. The protein solution (1 to 5 mg/ml in start buffer) is applied to the column by switching the sample application valve. To ensure that the column is fully loaded,
105
sample application is not interrupted until the recorder shows 50% full scale deflection, FSD, (0% = starting buffer; 100% = the protein solution in starting buffer). Sample application is stopped by re-setting the valve to allow passage of start buffer. Washing is continued until 0% full scale deflection is approached (Fig. 59). ?W26K? W.MI'@ ?W.YeN@ ?7H?e?@ J5f?@ 7Hf?@ @?f?@ @?f?@ ?J5?f?3L? ?7H?f?N1? ?@h@? ?@h@? ?@h@? ?@h@? ?@h@? ?@h@? ?@h@? ?@h@? J5h@? 7Hh@? @? @?h3L @?h?W26X?hf?@ @?hN1 @?h?7YS,??)X?@@@@@@6X?@?W-X @?h?@ @?h?@@@U?J@1?@?@?@@V1?@?7@) @?h?@ @?h?3XS,?*U@?@?@?@@W5?@?3X? @?h?@ @?h?V40Y?V4@?@?@?3@(Y?@?V/? @?h?@ @? V'Y? @?hf@?h?@ @?he @? ?@ @?h?@ @? ?@@?g?W2@ @?W-X?)Xf?@K??@ @?h?@g@@@@6Xe?W2@ @?he)X?@6X?@6X?@@?W.e)X?*@@?W-X?@@@ @?7R1?@)X?W&?@@@?@?@@@?W2@ @?hf@?h?@g@?e@1?@?*@@?W-X?@@@g@?he @?h?J@1?@V1?@V1?@@?7H?J@1?N@@?7R1?@?@h?@hf3X@?@?@@1?*@?@?@?@?@?@?7Y@ @?hf@?h?@g@@@@@@?@?N@@?7R1?@?@g@?he @?h?*U@?@W5?@W5?@@?3L?*U@e@@?3T5?@?@h?@hfN@5?3@@X@?N@@@?@?@?@?@?3X@ @?h?@g@?e@@?@e@@?3T5?@?@ @?h?V4@?@(Y?@(Y?@@?V/?V4@e@@?V+Y?@?@ ?(Y?V+R4@??@?@?@?@?@?@?N@@ @?h?3L?f@@@@@@@@e@@?V+Y?@?@ @?hf?(Y??(Y? @@ @?hf@?h?N1? @?he @? ?@ @?hf@?he@? @?he @L J@ @Lhe@L @@e@@e@@e@@e@@e@@e@@e@@e@@e@@e@@e@@e@@e@@e@@e@@e@@e@@e@@e@@e@@e@@e@@e@@e@@e@@e@6X?@@e@@e@@?J@@e@@e@@e@@e@@e@@e@@e@@e@@e@@e@@hg @H ?B1?he?7
W.f@@6Xe?@?@hf?@hW&fW2@?g?W2@ ?W&Hf@?V1?@?@?@eW&eW.e)X?@?W-Xe?W&@?W-T&@@?W-X?W.?*@@?W-X?@@@ ?75?f@@@@?@?@?@e*@L?7H?J@1?@?7@)e?7Y@?7@0Y@@?7@)?7H?N@@?7R1?@?@ J(Y?f@??@?@?@?@eN@,?3L?*U@?@?3X?e?3X@?3X??@@?3Xe3Le@@?3T5?@?@ .Yg@??@@@?@?@e?(Y?V/?V4@?@?V/?e?V4@?V/??@@?V/eV/e@@?V+Y?@?@
?@?@f?)X? W-T2@?he/Xe ?@?@?W-X?@1?@?@@@@@?W-X?e7R'@@?@@@@@?W-X?N1e ?3T5?7R1?@@?@?@?@?@?7@)?e@?N@@?@?@?@?7@)??@e ?N@H?3T5?@@?@?@?@?@?3Xf@??@@?@?@?@?3Xe?@e @??V+Y?@@@@?@?@?@?V/f3L?@@?@?@?@?V/eJ5e V/ .Ye
Fig. 59. Graph obtained with the setup in Figure 58 used in the determination of dynamic capacity.
After the wash phase, adsorbed protein is eluted with a stepwise change in ionic strength (e.g. 2 M NaCl) or pH. Fractions are collected as long as the UV-absorption is above 2% FSD. These fractions are pooled and the UV-absorption of the pool is measured.
Calculation The maximum amount of protein that can be bound to the column (A) at the chosen flow rate is: A=CxV C = The protein concentration in the pooled fractions (mg/ml). V = volume of pooled fractions. C = A280/ E A280 = absorbance of solution at 280 nm in 1 cm cell. E = absorbance of standard solution (1 mg/ml) at 280 nm in a 1 cm cell. The protein capacity is calculated as: A/gel volume This calculation assumes that the recovery of bound protein from the column is 100%. This can be checked by comparison with the quantity of protein applied to the column.
106
11. Process considerations When an ion exchange step is to be part of a purification sequence for a manufacturing process (in contrast to analytical chromatography or small scale preparative applications), method development work has to find conditions which give the highest throughput with the highest yield and the lowest possible cost. ”What kind of application will the product be used in?” What are the purity issues in relation to the source material and intended use of final product? What has to be removed? ”What kind of starting material do I have?” What are the major ”Headaches”? ”What final scale am I thinking of?” What consequences will this have for the technical approach? ”What is my purification strategy?” First, CAPTURE What will be the major purpose for the initial chromatographic step? Is my proposal rational? Then, INTERMEDIATE PURIFICATION What will be the major purpose with each subsequent chromatographic step? Finally, POLISHING What will be the major purpose of the polishing stage? Looking back upstream, does the overall balance and sequence of techniques appear logical? ”How do I get the most out of my process?” Will my process be more productive, safe, robust, economic and easier to use than one which our competitors could do? Will I arrive at the final process faster than our competitors?
Fig. 60. Questions that must be addressed to assure a rational process design
The design must ensure that the purity requirements of the final product are met, and also considering the special safety issues involved in production of biopharmaceuticals, such as infectious agents, pyrogens, immunogenic contaminants and tumorigenic hazards. In general, the purity issues must be addressed in relation to the nature of the source material and the intended use of the final product. It is important to define the impurities and contaminants which have to be removed from the source material during downstream processing.
107
Another important aspect of process development is to assure that scaleability, robustness and consistency are designed into the process from the very beginning. This is secured by careful selection of appropriate chromatography media, by the way a particular chromatographic step is optimized and by an early identification of different sources of variation and how they can be eliminated or controlled during processing. To assure a rational process design, a number of questions must be adressed. These questions are summerized in Figure 60 and will be discussed below.
Defining the purpose To reach the targets for yield and purity in as few steps as possible, and with the simplest possible design, it is not efficient to add one step to another until the purity requirements have been fulfilled. Instead a specific purpose is assigned to each step which is included in the complete scheme. The specific purification problem associated with a particular step, will depend greatly on the characteristics of the ingoing feed material to be processed in the step. Thus, the purpose of a particular step depends on if it is placed at the beginning, to handle a crude feed stock, in the middle, after partial purification or at the end when final purity is achieved. The different stages in downstream processing can be divided into capture, intermediate purification and polishing (Fig. 61). ? ? ? ? ? ? ? ? ? W2@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@6X? ? 7< ?B1? ? ?W&? @? @? ? ?7@? @? @? ? ?@@L @? @? ? J@@1 @? @? ? 7@@@ @?f@@@@ ?W&K ?@K?hf?O@?W2@? @? ? @@@@L? @?f@?W@T-X?@@@@@?W-X??@?@6Xe?*@@@?)X?W.??W-Xe?@@@@@@?@6X?@?@?@@@?&@@W)X?W&? @? ? ?J@@@@1? @?f@@@>@@)?@?@?@?7R1??@?@@)e?N@@HJ@1?7H??7@)e?@@?@?@?@V1?@?@?@?@?N@@@@)?*@L @? ? ?@@@@@@? @?f@?B@@Xe@?@?@?3T5??3T@X?f@@?*U@?3L??3X?e?@@?@?@?@W5?@?@?@?@??@@@X??N@)X? @? ? @? @?f@??(R/e@?@?@?V+Y??V+R/?f@@?V4@?V/??V/?e?@@?@?@?@(Y?@@@?@?@??@0R/?e(R1? @? ? @? @? (Y @? @? ? @? @? @? ? @? @?e?@K? ?@ ?W&? W&he@? ? @? @?e?@@@?W-X?@@@@@?W-X?@W-X?W2@?@?@?W-X?W&g)X?W2@?W2@?@6T-X?W2@e)X?*@W)X?W&?fW-X?*@W.h@? ? @? @?e?@?@?7R1?@?@?@?7R1?@@R1?7Y@?@?@?7@)?*@L?e?J@1?7Y@?7Y@?@V@@)?7Y@?J@1?N@@@)?*@Lf7@)?N@@Hh@? ? @? @?e?@?@?3T5?@?@?@?3T5?@@T5?3X@?@?@?3XeN@)Xe?*U@?3X@?3X@?@?3X??3X@?*U@e@@X??N@)X?e3Xe?@@Lh@? ? @? @?e?@?@?V+Y?@?@?@?V+Y?(R+Y?N@@?@@@?V/e?(R1e?V4@?N@@?N@@?@?V/??N@@?V4@e(R/?e(R1?eV/e?(R/?@g@? ? @? @? ?@@?he?@h@@e@@h@@ @? @? ? @? @? @? ? @? @?fW-X?e?@K??@ W26Kg?@hf?@?)X?hfW&he?W&?e?W&?g@? ? @? @?f7R1?W.?@@@?@?W-Xe@?@6X?e*@@@@@e)X?@e?@6X?@?@@@?@@@)X?W.??)X?@@6T&@e?W&?e)X?*@W)X?*@L?W.?e@? ? @? @?f@?@?7H?@?@?@?7@)e@?@@)?eN@@@?@?J@1?@e?@V1?@?@@H?@?@V1?7H?J@1?@?@@Y@e?*@L?J@1?N@@@)?N@1?7H?e@? ? @? @?e?J@@@T@L?@?@?@?3X?e3T@Xf?@@@?@?*U@?@e?@W5?@?@@??@?@?@?@e*U@?@?@@X@e?N@,?*U@e@@X?e@@?@f@? ? @? @?e?.M?I+R/?@?@?@?V/?eV+R/f?@@@?@?V4@?@e?@(Y?@@@@??@?@?@W5eV4@?@?(R4@f(Y?V4@e(R/?e3@W5f@? O2@@@@6K ? @? @? ?(Y?he?@0Y V40Yf@? O2@@@@@@0MfI46K ? @? @? @? ?O2@@@@@@@(M I46K ? @? @? @?hf?@@@@@@@@@@@@(Y? V@6X ? @? @? @? ?W(Yhe?W2@@@@@@@@) ? @? @? @? W.Y?heW.M? ? @? @? @? ?O.Yhe?O.Y ? @? 3= ?C5? ?W20Y?h?W20Y? ? @? V4@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@0Y? W.M?heW.M? ? @? ?W.Yhe?W.Y ? @? W&H?he?.Y? ? @? @@@@6Xg?@@?@@g7@f@@ ? @? @@e@1g?@@?h@@ ? @? @@e@@?W2@6X?@@?@6?2@6X?@@@6X?@@?)T26X?W26T& ? @? @@e@5?7@?@1?@@?@@@@?@)?@@?@1?@@?@@Y@1?7(R@@ ? @? @@@@0Y?@@?@@?@@?@@V4@@e@@?@@?@@?@@?@@?@H?@@ ? @? @@f?@@?@@?@@?@@e?@1?@@?@@?@@?@@?@@?@L?@@ ? @? @@f?3@?@5?@@?@@@6X@5?@@?@@?@@?@@?@@?3)X@@ ? @? @@f?V4@0Y?@@?@0?4@0Y?@@?@@?@@?@@?@@?V4@@@ ? @? @5 ? @? @@0Y ? @? ? @? ? @? W.hf?W.? ? @? ?O.YhfO.Y? ? @? ?W20Y?heW20Y ? @? W.M?he?W.M ? @? ?O.YhfO.Y? ? @? ?W20Y?heW20Y ? @? W.M?he?W.M ? @? ?O.YhfO.Y? ? @? ?W20Y?heW20Y ? @? W.M?he?W.M ? @? ?O.YhfO.Y? ? @? ?W20Y?heW20Y ? @? W.M?he?W.M ? @? .YhfO.Y? ? @? ?@@@@6X?he@6T2@@@@hf@@?@0Y ? @? ?@@??@1?he?V@@X?hf?J@@ ? @? ?@@??@@?@@?@@?@?2@@@@@@?@@?W2@@?W2@6T&@@@@@?W2@6X?)T26X? ? @? ?@@??@5?@@?@@?@@(Y@@@@H?@@?7@V@?*U?@@Y@@?@@?7@?@1?@@Y@1? ? @? ?@@@@0Y?@@?@@?@@H?@@@@e@@?@@?eS@@@@?@@?@@?@@?@@?@@?@@? ? @? ?@@?f@@?@@?@@e@@@@e@@?@@?e7@?@@?@@?@@?@@?@@?@@?@@? ? @? ?@@?f3@W@@?@@e@@@@e@@?3@W@?3@W@@W@@?@@?3@?@5?@@?@@? ? @? ?@@?fV40R'?@@e@@@@e@@?V4@@?V40R40R4@@@?V4@0Y?@@?@@? ? @? ? @? ? @? ? @? W.hf?W.? ? @? ?W.YhfW.Y? ? @? O.Y?he?O.Y ?W2@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@6Xg? @? W20Yhe?W20Y? ?7@@)?@?@?@?7R1e@?@@)?e@V1?@?@?@@@?e?@@?@?@?@V1?@?@?@?@?N@@@@)?*@Lf?@g? @? W.M?he?W.Y ?@e?@?B@@X??@?@?@?3T5e3T@Xf@W5?@?@?@@@Le?@@?@?@?@W5?@?@?@?@??@@@X??N@(f?@g? @? ?W.YhfW.Y? ?@e?@e(R/??@?@?@?V+YeV+R/f@0Y?@@@?@0R/e?@@?@?@?@(Y?@@@?@?@??@0R/?e(Y?@e?@g? @? W.Y?he?W.Y ?@ (Y ?@g? @? ?O.YhfO.Y? ?@ ?@g? @? ?W20Y?heW20Y ?@e?W-Xf@Ke@? W&hW&heW-X??W-Xh?@g? @? W.M?he?W.M ?@e?7R1?W.?@@@?@?W-X??@?@6Xe?@6X?@6T-X?W&@?@?@?W.?*@eW&e?)X?*@)X?*@)X?W.f?@g? @? ?W.YhfW.Y? ?@e?@?@?7H?@?@?@?7@)??@?@@)e?@V1?@V@R1?7Y@?@?@?7H?N@e*@L?J@1?N@@)?N@V1?7Hf?@g? @? ?7U?hf.Y ?@eJ@@@T@L?@?@?@?3Xe?3T@X?e?@W5?@?3T5?3X@?@?@?3L??@eN@,?*U@??@X?e@?@?@?f?@g? @? ?W2@@6X?he?@1? [email protected]?I+R/?@?@?@?V/e?V+R/?e?@(Y?@?V+Y?V4@?@@@?V/??@e?(Y?V4@??@@?e@?@W5?f?@g? @? W&(MI'1?heJ@@? ?@ ?(Y? @0Y?f?@g? @? 7@H??V'T2@6X?)T26T&@@@@@?@@?@?26T2@6X? ?@ ?@g? @? @@fV'U?@1?@@R'@Y@@?@@?@@?@@(R@@?@1? ?@ ?@g? @? @@f?V@@@@?@@?N@?@@?@@?@@?@@H?@@@@@? ?@ ?@g? @? 3@L??W2@@?@@?@@?J@?@@?@@?@@?@@e@@ ?@ ?@g? @? V')KO&@@@W@@?@@W&5?3@W@@W@@?@@e3@W2(? ?3=? C5g? @? ?V4@@0MI40R4@@@@0Y?V40R40R'?@@eV4@0Y? ?V4@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@0Yg? @? ?@@? ? @? ?@@? ? @? ? @? ?W.?hf?W.? ? @? W.Y?hfO.Y?e?W2@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@6X ? @? ?O.YhfW20Yf?7
@@@6X?e?O@?)X @??@1?@?@@@@@)X?W. @@@@@?@?@?@?@V1?7H @?e@?@?@?@?@?@?@? @?e@@@?@?@?@?@W5? @0Y?
Fig. 61. Different stages in downstream processing
When ion exchange chromatography is applied in capture, the purpose will be to adsorb the protein of interest quickly from the crude feed stock and isolate it from critical contaminants such as proteases and glycosidases. The product should be concentrated and transferred to an environment which will conserve potency/acti-
108
vity. At best, significant removal of other critical contaminants can also be achieved. In polishing, on the other hand, most impurities have already been removed except for trace amounts or closely related substances such as microheterogeneous structural variants of the product. When ion exchange chromatography is applied in such a step the purpose will be to reduce these variants and trace contaminants to a level that will be acceptable for final product quality by applying scaleable high resolution ion exchange chromatography techniques. When ion exchange chromatography is applied in intermediate purification, i.e. steps performed on clarified feed between capture and polishing, the purpose is to remove most of the significant impurities such as proteins, nucleic acids, endotoxins and viruses down to safe levels.
The strategic focus In any chromatographic step there are four main performance properties to adjust to reach a fully optimized procedure (Fig. 62), resolution, speed, capacity and recovery. )X ?@@@6X @?f?J@1 ?@eS)T2@6KO2@6T2@6X@?@??@?@@@?W2@6T2@6X ?@@@@>@Y?V@@U?;@)X? 7Hh?3@@@R1? ?J5?h?N@@@W@L ?7H?he@@@@>)X? J5hf3@@@@R1? 7HhfN@@@@W@L ?J5?hf?@@@@@>)X? ?7H?hf?@@@@@@R1? J5 ?3@@@@@W@L 7H ?N@@@@@@>)X? ?J5? @@@@@@@R1? W.Y? @@@@@@@W@L 7H 3@@@@@@@>)X? ?J5? N@@@@@@@@R1? ?7H? ?@@@@@@@@W@L J5 ?3@@@@@@@@>)X? 7H ?N@@@@@@@@@R1? ?J5? @@@@@@@@@W@L ?7H? @@@@@@@@@@R1 J5 3@@@@@@@@@@@L? 7H N@@@@@@@@@@S)X ?J5? ?@@@@@@@@@@@R1 W.Y? ?3@@@@@@@@@@@@L? 7H ?N@@@@@@@@@@@S)X ?J5? @@@@@@@@@@@@R1 ?7H? @@@@@@@@@@@@@@L? J5 3@@@@@@@@@@@@S)X 7H N@@@@@@@@@@@@@R1 ?J5? ?@@@@@@@@@@@@@@@L? ?7H? ?3@@@@@@@@@@@@@S)X J5 ?N@@@@@@@@@@@@@@R1 7H @@@@@@@@@@@@@@@@L? ?J5? @@@@@@@@@@@@@@@S)X W.Y? 3@@@@@@@@@@@@@@@R1 7H N@@@@@@@@@@@@@@@@@L? ?J5? ?@@@@@@@@@@@@@@@@S)X ?7H? ?3@@@@@@@@@@@@@@@@R1 J5 ?N@@@@@@@@@@@@@@@@@@L? 7H @@@@@@@@@@@@@@@@@V1? ?J5? @@@@@@@@@@@@@@@@@@@L ?7H? 3@@@@@@@@@@@@@@@@@@)X? J5 N@@@@@@@@@@@@@@@@@@V1? ?W.Y ?@@@@@@@@@@@@@@@@@@@@L ?7H? ?3@@@@@@@@@@@@@@@@@@@)X? J5 ?N@@@@@@@@@@@@@@@@@@@V1? 7H @@@@@@@@@@@@@@@@@@@@@L ?J5? @@@@@@@@@@@@@@@@@@@@@)X? ?7H? 3@@@@@@@@@@@@@@@@@@@@V1? J5 N@@@@@@@@@@@@@@@@@@@@@@L 7H ?@@@@@@@@@@@@@@@@@@@@@@)X? ?J5? ?@@@@@@@@@@@@@@@@@@@@@@V1? ?7H? ?3@@@@@@@@@@@@@@@@@@@@@@@L J5 ?N@@@@@@@@@@@@@@@@@@@@@@@)X? ?W.Y @@@@@@@@@@@@@@@@@@@@@@@V1? ?7H? 3@@@@@@@@@@@@@@@@@@@@@@@@L J5 N@@@@@@@@@@@@@@@@@@@@@@@@)X? 7H ?@@@@@@@@@@@@@@@@@@@@@@@@V1? ?J5? ?@@@@@@@@@@@@@@@@@@@@@@@@@@L ?7H? ?3@@@@@@@@@@@@@@@@@@@@@@@@@1 J5 ?N@@@@@@@@@@@@@@@@@@@@@@@@@@L? 7H @@@@@@@@@@@@@@@@@@@@@@@@@@)X ?J5? 3@@@@@@@@@@@@@@@@@@@@@@@@@@1 ?7H? N@@@@@@@@@@@@@@@@@@@@@@@@@@@L? J5 ?@@@@@@@@@@@@@@@@@@@@@@@@@@@)X ?W.Y ?@@@@@@@@@@@@@@@@@@@@@@@@@@@@1 ?7H? ?3X@@@@@@@@@@@@@@@@@@@@@@@@@@@L? J5 ?N@@@@@@@@@@@@@@@@@@@@@@@@@@@@)X 7H @@@@@@@@@@@@@@@@@@@@@@@@@@@@@1 ?J5? 3@@@@@@@@@@@@@@@@@@@@@@@@@@@@@L? ?7H? N@@@@@@@@@@@@@@@@@@@@@@@@@@@@@)X J5 ?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@1 7H ?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@L? ?J5? ?3X@@@@@@@@@@@@@@@@@@@@@@@@@@@@@)X ?7H? ?N@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@1 J5 @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@L? ?W.Y 3X@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@)X ?7H? N@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@1 J5 ?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@L? 7H ?@?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@1? ?J5? ?3X@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@L ?7H? ?N@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@)X? J5 @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@1? ?W2@@? O@he7H @?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@L ?@h ?7Y?e)T26KO2@6T2@6KO2@@h?J5? 3X@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@)X?hW26X @?J@h ?3@6X?@(MB@@Y?V@Y?V@@@Y?V@@
Fig. 62. The optimal process must be defined within the context of competing goals.
In general, optimization of any of these can only be realized at the expense of the others. The relative priority of these parameters will vary depending on whether a particular chromatographic step is for capture, intermediate purification or polishing. This will steer the optimization of the critical processing parameters in any particular step, as well as the selection of the most suitable chromatography matrix to be used in the step.
Capture In a typical capture situation, throughput (i.e. capacity and speed) will be very important for processing of large sample volumes, keeping the scale of equipment
109
as small as possible and giving the shortest possible cycle time. Binding capacity for the product in the presence of the impurities will be one of the most critical parameters to reduce the scale of work as much as possible. High speed may be required to reduce sample application time, particularly if proteolysis or other destructive effects occur. The characteristics of the feed and the anticipated final scale of work will form the basis for the balance between capacity and speed in a capture situation (Fig. 63).
@@@6X? @?f?@?@ @??B1? @LfJ@ @??C@T2@6KO2@@?W2@6X@1e@?@@@@?W2@6T2@6X @@@@>@Y?V@@Ue?7)X? 7Hf?@@R1? ?J5?f?3@W@L W.Y?f?N@@>)X? 7Hh@@@R1? ?J5?h@@@W@L ?7H?h3@@@R1 J5heN@@@@@L? 7Hhe?@@@@S)X ?J5?he?3@@@@R1 ?7H?he?N@@@@@@L? J5 @@@@@S)X 7H @@@@@@R1 ?J5? 3@@@@@@@L? W.Y? N@@@@@@S)X 7H ?@@@@@@@R1 ?J5? ?3@@@@@@@@L? ?7H? ?N@@@@@@@@)X J5 @@@@@@@@V1 7H @@@@@@@@@@L? ?J5? 3@@@@@@@@@)X ?7H? N@@@@@@@@@V1 J5 ?@@@@@@@@@@@L? ?W.Y ?3@@@@@@@@@@)X ?7H? ?N@@@@@@@@@@V1 J5 @@@@@@@@@@@@L? 7H 3@@@@@@@@@@@)X ?J5? N@@@@@@@@@@@V1 ?7H? ?@@@@@@@@@@@@@L? J5 ?@@@@@@@@@@@@@)X 7H ?3@@@@@@@@@@@@V1 ?J5? ?N@@@@@@@@@@@@@@L? ?7H? @@@@@@@@@@@@@@1? J5 3@@@@@@@@@@@@@@L ?W.Y N@@@@@@@@@@@@@@)X? ?7H? ?@@@@@@@@@@@@@@@1? J5 ?@@@@@@@@@@@@@@@@L 7H ?3@@@@@@@@@@@@@@@)X? ?J5? ?N@@@@@@@@@@@@@@@@1? ?7H? @@@@@@@@@@@@@@@@@L J5 3@@@@@@@@@@@@@@@@)X? 7H N@@@@@@@@@@@@@@@@@1? ?J5? ?@@@@@@@@@@@@@@@@@@L W.Y? ?@@@@@@@@@@@@@@@@@@)X? 7H ?3@@@@@@@@@@@@@@@@@@1? ?J5? ?N@@@@@@@@@@@@@@@@@@@L ?7H? @@@@@@@@@@@@@@@@@@@)X? J5 3@@@@@@@@@@@@@@@@@@@1? 7H N@@@@@@@@@@@@@@@@@@@@L ?J5? ?@@@@@@@@@@@@@@@@@@@@)X? ?7H? ?@@@@@@@@@@@@@@@@@@@@@1? J5 ?3@@@@@@@@@@@@@@@@@@@@@L ?W.Y ?N@@@@@@@@@@@@@@@@@@@@@)X? ?7H? @@@@@@@@@@@@@@@@@@@@@@1? J5 3@@@@@@@@@@@@@@@@@@@@@@L 7H N@@@@@@@@@@@@@@@@@@@@@@1 ?J5? ?@@@@@@@@@@@@@@@@@@@@@@@L? ?7H? ?@@@@@@@@@@@@@@@@@@@@@@@)X J5 ?3@@@@@@@@@@@@@@@@@@@@@@@1 7H ?N@@@@@@@@@@@@@@@@@@@@@@@@L? ?J5? @@@@@@@@@@@@@@@@@@@@@@@@)X ?7H? 3@@@@@@@@@@@@@@@@@@@@@@@@1 J5 N@@@@@@@@@@@@@@@@@@@@@@@@@L? ?W.Y ?@@@@@@@@@@@@@@@@@@@@@@@@@)X ?7H? ?@@@@@@@@@@@@@@@@@@@@@@@@@@1 J5 ?3@@@@@@@@@@@@@@@@@@@@@@@@@@L? 7H ?N@@@@@@@@@@@@@@@@@@@@@@@@@@)X ?J5? @@@@@@@@@@@@@@@@@@@@@@@@@@@1 ?7H? @@@@@@@@@@@@@@@@@@@@@@@@@@@@L? J5 3@@@@@@@@@@@@@@@@@@@@@@@@@@@)X 7H N@@@@@@@@@@@@@@@@@@@@@@@@@@@@1 ?J5? ?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@L? W.Y? ?3@@@@@@@@@@@@@@@@@@@@@@@@@@@@)X 7H ?N@@@@@@@@@@@@@@@@@@@@@@@@@@@@@1 ?J5? @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@L? ?7H? @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@)X J5 3@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@1 7H N@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@L? ?J5? ?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@)X ?7H? ?3@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@1 J5 ?N@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@L? 7H @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@1? ?J5? @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@L W.Y? 3@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@)X? 7H N@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@1? ?J5? ?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@L ?7H? ?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@)X? ?W26X? @?hJ5 ?3@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@1? ?7@Y?V@@
Fig. 63. In a typical capture situation the strategic planning will focus on capacity and speed. If scale of work is the main problem, binding capacity will have higher priority than speed, i.e. it will be more important to utilize the total binding capacity of the bed than to apply very high flow rates during sample application. If time is the main problem, speed will have higher priority than binding capacity, i.e. it will be more important to apply high flow rates, even if this means that a somewhat larger bed volume is needed for processing of a specific amount of feed.
In capture, an important factor impacting capacity is the selectivity during sample adsorption. Typically binding conditions are selected to avoid binding contaminating substances so that more binding sites are available for the protein of interest. This also allows stepwise elution of the product in concentrated form. Recovery is another parameter that will be of great concern in any preparative situation, especially for production of a high value product. Recovery generally becomes more important further downstream because of the increased value of the purified product. Recovery is influenced by destructive processes in the sample and unfavourable conditions on the column. Resolution of similar components is not of greatest concern in a typical capture situation. However, there is usually significant resolution and purification from molecules with gross physicochemical difference from the product. In principle, a capture step is designed to maximize capacity and/or speed at the expense of some resolution. The separation from impurities is usually achieved during binding of the product which can simply be eluted, in concentrated form, by a step.
110
Intermediate purification In intermediate purification steps, achieving resolution of similar components will be more and more important further downstream. Capacity will still be important to maintain productivity, i.e. amount of product produced per volume of chromatography media and time unit. As in a capture step, selectivity during sample adsorption will be important, not only to achieve high binding capacity, but also to contribute to the purification by achieving a degree of separation already during sample application. However, in contrast to a capture step, selectivity during sample desorption from the column becomes important as we go downstream. This is usually achieved by applying a more selective desorption principle, such as a continuos gradient or a multi-step elution procedure. Hence, the delicate problem in an intermediate purification step will be to decide on the optional balance between capacity and resolution (see Fig. 64). Speed will usually be less critical in a typical intermediate purification step due to the fact that impurities causing proteolysis and other destructive effects should have been removed in the capture step and also due to the fact that sample volume has been reduced at this stage. ? ? ? ? ? ? ? ? ? ? ? ? ?)X? ? ?@)K ? ?@@@6K ? J@@@@@6X ? 7Y@@@@@)K? ? ?J5?@@@@@@@6X? ? ?7H?3@@@@@@@,? ? J5eN@@@@@@(Y? ? 7He?@@@@@@? ? ?J5?e?@@@@@@1 ? W.Y?e?@@@@?@@L? ? 7Hg?@@W@@)X ? ?J5?g?@@@>@@1 ? ?7H?g?3@@@Y@@L? ? J5h?N@@@W@@1? ? 7Hhe@@@@Y@@L ? ?J5?he3@@@@@@)X? ? ?7H?heN@@@@W@@1? ? J5hf?@@@@@Y@@L ? 7Hhf?@@@@@@@@)X? ? ?J5?hf?3@@@@@W@@1? ? W.Y?hf?N@@@@@@Y@@L ? 7H @@@@@@@@@)X? ? ?J5? 3@@@@@@W@@1? ? ?7H? N@@@@@@@Y@@L ? J5 ?@@@@@@@@@@)X? ? 7H ?@@@@@@@@W@@1? ? ?J5? ?3@@@@@@@@Y@@L ? ?7H? ?N@@@@@@@@@@@1 ? J5 @@@@@@@@@?@@L? ? ?W.Y 3@@@@@@@@@@@)X ? ?7H? N@@@@@@@@@@@@1 ? J5 ?@@@@@@@@@@?@@L? ? 7H ?3@@@@@@@@@@@@)X ? ?J5? ?N@@@@@@@@@@@@@1 ? ?7H? @@@@@@@@@@@?@@L? ? J5 @@@@@@@@@@@@@@)X ? 7H 3@@@@@@@@@@@@@@1 ? ?J5? N@@@@@@@@@@@@?@@L? ? ?7H? ?@@@@@@@@@@@@@@@)X ? J5 ?3@@@@@@@@@@@@@@@1 ? ?W.Y ?N@@@@@@@@@@@@@?@@L? ? ?7H? @@@@@@@@@@@@@@@@1? ? J5 @@@@@@@@@@@@@@@@@L ? 7H 3@@@@@@@@@@@@@@@@)X? ? ?J5? N@@@@@@@@@@@@@@@@@1? ? ?7H? ?@@@@@@@@@@@@@@@@@@L ? J5 ?3@@@@@@@@@@@@@@@@@)X? ? 7H ?N@@@@@@@@@@@@@@@@@@1? ? ?J5? @@@@@@@@@@@@@@@@@@@L ? W.Y? @@@@@@@@@@@@@@@@@@@)X? ? 7H 3@@@@@@@@@@@@@@@@@@@1? ? ?J5? N@@@@@@@@@@@@@@@@@?@@L ? ?7H? ?@@@@@@@@@@@@@@@@@@@@)X? ? J5 ?3@@@@@@@@@@@@@@@@@@@@1? ? 7H ?N@@@@@@@@@@@@@@@@@@?@@L ? ?J5? @@@@@@@@@@@@@@@@@@@@@1 ? ?7H? @@@@@@@@@@@@@@@@@@@@@@L? ? J5 3@@@@@@@@@@@@@@@@@@@@@)X ? ?W.Y N@@@@@@@@@@@@@@@@@@@@@@1 ? ?7H? ?@@@@@@@@@@@@@@@@@@@@@@@L? ? J5 ?3@@@@@@@@@@@@@@@@@@@@@@)X ? 7H ?N@@@@@@@@@@@@@@@@@@@@@@@1 ? ?J5? @@@@@@@@@@@@@@@@@@@@@@@@L? ? ?7H? @@@@@@@@@@@@@@@@@@@@@@@@)X ? J5 3@@@@@@@@@@@@@@@@@@@@@@@@1 ? 7H N@@@@@@@@@@@@@@@@@@@@@@@@@L? ? ?J5? ?@@@@@@@@@@@@@@@@@@@@@@@@@)X ? ?7H? ?3@@@@@@@@@@@@@@@@@@@@@@@@@1 ? J5 ?N@@@@@@@@@@@@@@@@@@@@@@@@@@L? ? ?W.Y @@@@@@@@@@@@@@@@@@@@@@@@@@1? ? ?7H? @@@@@@@@@@@@@@@@@@@@@@@@@@@L ? J5 3@@@@@@@@@@@@@@@@@@@@@@@@@@)X? ? 7H N@@@@@@@@@@@@@@@@@@@@@@@@@@@1? ? ?J5? ?@@@@@@@@@@@@@@@@@@@@@@@@@@@@L ? ?7H? ?3@@@@@@@@@@@@@@@@@@@@@@@@@@@)X? ? J5 ?N@@@@@@@@@@@@@@@@@@@@@@@@@@@@1? ? 7H @@@@@@@@@@@@@@@@@@@@@@@@@@@@@L ? ?J5? @@@@@@@@@@@@@@@@@@@@@@@@@@@@@)X? ? W.Y? 3@@@@@@@@@@@@@@@@@@@@@@@@@@@@@1? ? 7H N@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@L ? ?J5? ?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@)X? ? ?7H? ?3@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@1? ? J5 ?N@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@L ? 7H @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@1 ? ?J5? 3@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@L? ? ?7H? N@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@)X ? J5 ?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@1 ? 7H ?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@L? ? ?J5? ?3@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@)X ? W.Y? ?N@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@)?)X? ? 7H @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@1? ? ?J5? 3@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@? ? ?7H? N@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@? ? ?W26X? @?hJ5 ?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?hW26X @??@h? ?7@0M? ? I4@@@0M? ? ? ? ? ? ? @@@6X? ? @??B1? ? @??C@T2@6KO26X?W2@6T2@?W2@@6T2@??@?W.? ? @@@@>@Y?V@@@Y?V@@Ue?7
Fig. 64. In a typical intermediate purification situation the strategic planning will focus on resolution and capacity. The requirements for resolution must be defined in context with the nature of the feed and the purity requirements in the final product. Capacity must also be considered to ensure a high productivity. The optimal balance must be defined which then will decide how selectivity parameters should be optimized during sample application to achieve the requirements for resolution (purification) and capacity in the system.
Polishing In a polishing step the prime issue will be resolution, since this is the last chance to reach the required quality of the product by removal of trace contaminants such as host proteins, structural variants of product, reagents, leachables, endotoxins, nucleic acids and viruses.
111
O2@@@@@@@@@6K? ? ?O2@@@@@@@@@@@@@@@6K ? W2@@@@@@@@@@@@@@@@@@@@@6X? ? ?O&@@@@@@@@@@@@@@@@@@@@@@@)K ? ?W2@@@@@@@@@@@@@@@@@@@@@@@@@@@6X ? O&@@?@@@@@@@@@@@@@@@@@@@@@@@@@@)K? ? W2@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@6X? ? ?W&@@?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@)X ? ?7@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@1 ? J@@X@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@L? ? ?W&@V@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@)X ? ?7@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@1 ? J@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@L? ? ?W&@?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@)X ? ?7@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@1 ? ?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ ? J@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@L? ? 7@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@1? ? @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@? ? ?J@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@L ? ?7@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@1?W2@@@@6X? ? ?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@W&@@@@@@)K O2@@@@@@@@@@@@@@@@@@@@6X ? ?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@YgV@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@)X? ? ?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@1? ? ?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@? ? ?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@? ? ?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?g@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@5? ? ?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@(Y? ? ?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@0Y ? ?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@V'@@@@@@(?4@@@@@@@@@@@@@@@@@@@@0M? ? ?3@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@5?V4@@@@0Y? ? ?N@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@H ? @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@? ? 3@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@5? ? N@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@H? ? ?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ ? ?3@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@5 ? ?V'@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@(Y ? N@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@H? ? ?3@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@5 ? ?V'@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@(Y ? N@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@H? ? ?3@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@5 ? ?V'@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@(Y ? V4@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@0Y? ? I'@@@@@@@@@@@@@@@@@@@@@@@@@@@@@(M? ? ?V4@@@@@@@@@@@@@@@@@@@@@@@@@@@0Y ? ?I'@@@@@@@@@@@@@@@@@@@@@@@(M ? V4@@@@@@@@@@@@@@@@@@@@@@H? ? @@@@@@@@@@@@@@@@@@@@L? ? @?I4@@@@@@@@@@@@@@@@1? ? ?J5?hf@@@@@@@L ? W.Y?hf3@@@@@@)X? ? 7H N@@@@@@@1? ? ?J5? ?@@@@@@@@L ? ?7H? ?@@@@@@@@)X? ? J5 ?3@@@@@@@@1? ? 7H ?N@@@@@@@@@L ? ?J5? @@@@@@@@@)X? ? ?7H? 3@@@@@@@@@1? ? J5 N@@@@@@@@@@L ? ?W.Y ?@@@@@@@@@@)X? ? ?7H? ?3@@@@@@@@@@1? ? J5 ?N@@@@@@@@@@@L ? 7H @@@@@@@@@@@)X? ? ?J5? @@@@@@@@@@@@1? ? ?7H? 3@@@@@@@@@@@@L ? J5 N@@@@@@@@@@@@)X? ? 7H ?@@@@@@@@@@@@@1? ? ?J5? ?3@@@@@@@@@@@@@L ? W.Y? ?N@@@@@@@@@@@@@)X? ? 7H @@@@@@@@@@@@@@1? ? ?J5? 3@@@@@@@@@@@@@@L ? ?7H? N@@@@@@@@@@@@@@)X? ? J5 ?@@@@@@@@@@@@@@@1? ? 7H ?@@@@@@@@@@@@@@@@L ? ?J5? ?3@@@@@@@@@@@@@@@)X? ? ?7H? ?N@@@@@@@@@@@@@@@@1? ? J5 @@@@@@@@@@@@@@@@@L ? 7H 3@@@@@@@@@@@@@@@@)X? ? ?J5? N@@@@@@@@@@@@@@@@@1? ? W.Y? ?@@@@@@@@@@@@@@@@@@L ? 7H ?@@@@@@@@@@@@@@@@@@1 ? ?J5? ?3@@@@@@@@@@@@@@@@@@L? ? ?7H? ?N@@@@@@@@@@@@@@@@@@)X ? J5 @@@@@@@@@@@@@@@@@@@1 ? 7H @@@@@@@@@@@@@@@@@@@@L? ? ?J5? 3@@@@@@@@@@@@@@@@@@@)X ? ?7H? N@@@@@@@@@@@@@@@@@@@@1 ? J5 ?@@@@@@@@@@@@@@@@@@@@@L? ? ?W.Y ?3@@@@@@@@@@@@@@@@@@@@)X ? ?7H? ?N@@@@@@@@@@@@@@@@@@@@@1 ? J5 @@@@@@@@@@@@@@@@@@@@@@L? ? 7H 3@@@@@@@@@@@@@@@@@@@@@)X ? ?J5? N@@@@@@@@@@@@@@@@@@@@@@1 ? ?7H? ?@@@@@@@@@@@@@@@@@@@@@@@L? ? J5 ?@@@@@@@@@@@@@@@@@@@@@@@)X ? 7H ?3@@@@@@@@@@@@@@@@@@@@@@@1 ? ?J5? ?N@@@@@@@@@@@@@@@@@@@@@@@@L? ? W.Y? @@@@@@@@@@@@@@@@@@@@@@@@)X ? 7H 3@@@@@@@@@@@@@@@@@@@@@@@@1 ? ?J5? N@@@@@@@@@@@@@@@@@@@@@@@@@L? ? ?7H? ?@@@@@@@@@@@@@@@@@@@@@@@@@)X ? J5 ?@@@@@@@@@@@@@@@@@@@@@@@@@@1 ? 7H ?3@@@@@@@@@@@@@@@@@@@@@@@@@@L? ? ?J5? ?N@@@@@@@@@@@@@@@@@@@@@@@@@@)X ? ?7H? @@@@@@@@@@@@@@@@@@@@@@@@@@@1 ? J5 3@@@@@@@@@@@@@@@@@@@@@@@@@@@L? ? ?W.Y N@@@@@@@@@@@@@@@@@@@@@@@@@@@)X ? ?7H? ?@@@@@@@@@@@@@@@@@@@@@@@@@@@@1 ? J5 ?3@@@@@@@@@@@@@@@@@@@@@@@@@@@@L? ? 7H ?N@@@@@@@@@@@@@@@@@@@@@@@@@@@@)X ? ?J5? @@@@@@@@@@@@@@@@@@@@@@@@@@@@@1 ? ?7H? @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@L? ? J5 3@@@@@@@@@@@@@@@@@@@@@@@@@@@@@)X ? 7H N@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@1 ? ?J5? ?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@L? ? W.Y? ?3@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@)X ? 7H ?N@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@1 ? ?J5? @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@L? ? ?7H? @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@)X ? J5 3@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@1 ? W2@6X? ?O@?h7H N@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@L? ? 7U?I/?@@6KO26X?W26KO2@@?g?J5? ?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@)Xg?W2@6X ?@?W-Xg? @)K?e@?B@@YV1?7YV@@
Fig. 65. In a typical polishing situation the strategic planning will focus almost entirely on resolution. However, since more expensive, high resolution media will be used, it will also be important to verify that the high resolution achieved during the scouting experiments at laboratory scale can be maintained also when preparative loadings are applied in the final production scale.
Selectivity during sample desorption from the column will be very important and can be maximized by working on the shape and slope of a continuos gradient elution technique. The resolution required may not be achieved by working on the selectivity alone, but high efficiency media with small bead size usually have to be used in a typical polishing situation (see Fig. 65).
Selection of chromatography media When chromatography media are to be selected for use in an industrial process there are a number of important selection criteria to take into consideration to assure a safe and smooth transfer from research phase to routine production. ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@he? ?J(M I'L?h? ?7H?eO2@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@6K? ?N1?h? J5eW20M ?I46X? @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@6X? W2@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ 7He7< ?B1? ?J@X ?I/X 7Y@?e?W@? W@?@e@@@@@@@@6Kf3Lh? W@fI'@?eN1h? ?J5??J5? 3L ?7R@@6X? ?W&?e?W2@hN1 @?@@@@?@@W)X?@@@@@@@?W-X?W&@?@?@ @@W)X??N@?e?@h? ?7H??7H?he@@6X W&KO@? ?O2@ N1gW2@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@6Xf?@?@?B1?W-X?W.?@?@?@@@@@?W-X?@@@?*@?)X?*@@W-X?@@@??@ @?@@?@?N@@@)?@?@?@?@?7@)?7Y@?@W5 @?N@@@)?e@?e?3L?g? J5e?@hf@?S,e)X?W&?W-X?e@@@@@??)X?*@@@@?@@e?@6X?@6T-X?@6X?W-X?@@@@@?W-X?W&?h?@f?W.M B1f?@?@e@?7R1?7H?@?@?@?@?@?7@)?@?@?N@W@1?N@@@R1?@?@??@ @?@@?@e@@X??@?@?@?@?3X??3X@?@@U@??@@Xf@?e?N1?g? 7He?@hf@@@U?J@1?*@W&@)?e@?@?@?J@1?N@@?@?@@e?@V1?@V@R1?@V1?7@)?@e@@?7@)?*@Lh?@f?7H? ?@f?@?@?C5?3T5?3L?@?@?@?@?@?3X??@?@e@@U@e@@@T5?@?@??@ @?@@?@e(R/??@?@?@?@?V/??V4@?(R/ @??(R/f@?f3Lg? ?J5?e?@hf@?V1?*U@?N@@@Xf@?@?@?*U@??@@?@?@@e?@W5?@?3T5?@W5?3X??@e@@?3X??N@,h?@f?@e@@@@hf@?e?W&?he?@f?@?@@0Y?V+Y?V/?@@@?@?@?@?V/??@?@e(R4@e@0R+Y?@?@??@ @? @?fN1g? ?7H?e?@hf@@@@?V4@e@MI/f@?@?@?V4@??@@?@?@@e?@(Y?@?V+Y?@(Y?V/??@e@@?V/?e(Yh?@f?@e@?W@T-X?W2@?@?@?@?)X?*@W-X?@@6X?W.?@f?@ ?@ @? @?f?@g? J5f?@ ?(Y?g?(Y? ?@f?@e@@@>@@)?7Y@?@?@?@W@1?N@@R1?@?B1?7H?@f?@ ?@ @?he?O@?W2@?g?W2@e @@@?f@?f?N1?f? @?f?3L?f? 7Hf?@ ?@f?@e@?B@@Xe3X@?@?@?@@U@e@@T5?@e@?@??@f?@hfW&e?W&?g@Kg@?g?@f?@ @?e@6X?@?@?@@@?&@@W.?e)X?*@@W. ?J5?f?@ ?@f?@e@??(R/eN@@?@@@?(R4@e(R+Y?@e@W5??@f?@f)X?@@@?W&@e?*@[email protected]@@@?@@@?@W.?e)X?@f?@ @?e@V1?@?@?@?@?N@@@H??J@1?N@@@H @?@?@?f@?g3Lf? ?7H?f?@ ?@f?@he?@@? @0Y??@f?@e?J@1?@?@?7Y@e?N@@@@@He@?@?@?@?@@H??J@1?@f?@ @?e@W5?@?@?@?@??@@@L??*U@e@@@L @?@?@?f@?gN1f? J5g?@ W2@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@6X?h?@f?@ ?@f?@e?*U@?@?@?3X@f@@XI'Le@?@?@?@?@@L??*U@?@f?@ @?e@(Y?@@@?@?@??@0R/??V4@e@0R/ @?@?f@?g?3L?e? 7Hg?@ ?W.M ?I/Xh?@f?@ O@g?@f?@e?V4@?@?@?V4@f(R/?V/e@?@?@?@?(R/??V4@?@f?@ @?e(Y @?g?N1?e? ?J5?g?@ W2@@@@@@@@@@@6K?g?7H? N1h?@f?@g?W&??@?@@6X?@6X?W-X?@@@@g?@f?@ ?@ 3L ?J5?h@?e? ?7H?g?@ 78he?I'@g?@?W26X?gO@?W2@hW2@?he?@h?@f?@g?*@L?@?@@V1?@V1?7R1?@??@g?@f?@ ?@ V/K? O.Y?h3Le? J5h?@ @W26X??@?@@@@?N@g?@?7YS,??)X?@@@@?&@@?@@@?)X?*@@W-X?@@@f?@h?@f?@g?N@(?@?@@W5?@W5?3T5?@??@g?@f?@ ?O@?hf?@ ?V4@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@0YheN1e? 7Hh?@ @@@6X?hW-X?hN1e?3=? C5g ?O2@@@@@@@@@@@@@@@@@@ @@@@@@@@@@@@@@@@@@@6K? W2@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@6K?hf@@@5he?@he?@?@?@?@g ?@L? ?7H?he?@ N1 7(Y?h@?e@?hV'1?h?V'@6K ?O2@R@(Yhe?@L?h?@?3T5?@g J@1? ?@hf?@ ?@L? ?J(Yhe@?e@?h?V'LheV'@@@@6Kh?O2@@@@@@(Y?he?@)Xh?@?V+Y?@g ?W&Y@? ?O2@@@@@@@@@6K ?@hf?@@@@?he?@1? ?7H?he@?e@?heN1he?V4@@@@@@@@@@@@@@@@@@@@@0Yhf?3@)X? W&@@5? ?O2@@0M?hI4@@6K ?@L?he?@ J@@? ?@hf@@@@@?he?@L?hfI4@@@@@@@@@@@@@@@0M? ?N@@)K ?O&@?@H? ?W20M? I46X ?3)Xhe?@hf?W&@5? ?@L?he@?e@?heJ@1? I4@@@0M? 3@@@6K @@Y@@5 W.M?g?@@@6XhI/X? ?N@)X?h?@hfW&U@H? ?@)Xhe@?e@?h?W&Y@? V'@@@@@@6Kh?O2@@@ @@@@(Y ?W.Yh?@eB1h?V/X 3@)K ?O&>@5 ?3@)X?h@?e@?hW&@@5? ?V'@@@@@@@@@@@@@@@@@@@@@ @@@0Y? W.Y?h?@eC5heV/X? O2@@@@@@@@@6K? V4@@6K ?O2@R@(Y ?N@@)K ?O&@?@H? V4@@@@@@@@@?e@@@@@@@5 @0M? 7Hhe?@@@@Uhe?N1?heO2@@0Mh?I4@@6K?hfI'@@@@6Kh?O2@@@@@@(Y? @@@@6K ?O2@Y@@5 ?I4@@@@@@@@@@@@@@@0Y @?he?@eB1hf@LhW20M ?I46X?he?V4@@@@@@@@@@@@@@@@@@@@@0Y ?I4@@@@@6Kh?O2@@@Y@@@0Y ?I4@@@0Mg @?he?@eC5hf@1g?W.M ?I/X I4@@@@@@@@@@@@@@@0M? ?I4@@@@@@@@@@@@@@@Y@@@@@0M @Lhe?@@@0Yhe?J@@gW.Y?h@@@6X?hV/X? I4@@@0M? ?I4@@@@@@@e?@@@@@@@0M 3)X? W&@5f?W.Yhe@??I/Xh?V/X ?I4@@@@@@@@@0M N@)X ?W&U@Hf?7H?he@?eN1heN1 ?I4@0M ?3@)K? O&R@5?f?@hf@?e?@he?@ O2@@@@@@@@@6K? ?V4@@6K? O2@@@(Y?f?@hf@?eJ5he?@L? O2@@0Mh?I4@@6K? ?I'@@@@6K?hO2@@@@@@0Yg?@L?he@??O.YheJ@1? O2@@@@@@@@@6K? W20M ?I46X? V4@@@@@@@@@@@@@@@@@@@@0Mh?3)Xhe@@@0Y?h?W&U@?hfO2@@0Mh?I4@@6K? ?W.Mh@??W2@g?I/X ?I4@@@@@@@@@@@@@@0M?he?N@)X? W.R@5?heW20M ?I46X? W.Y?h@?W.M?hV/X? I4@@@0M? 3@)K ?O&U?@H?h?W.M ?I/X ?W.Yhe@W&Hhe?V/X V'@@6K ?O2@R@@5heW.Y? V/X? ?7H?he@@@LhfN1 ?V4@@@@@6Kh?O2@@@Y@@@0Yh?W.Yhe@@@@@?h?V/X ?@hf@?I/X?he?@L? ?@@@@6X? ?I4@@@@@@@@@@@@@@@Y@@@@@0Mhe?7H?he@? N1hf@@@@@? ?@L?he@??N)Xhe?@1? W2@@@@he J(M?W@1? ?I4@@@@@@@e?@@@@@@@0Mhf?@hf@? ?@L?he@?e@? O2@@@@@@@@@6K? ?31?he@?e@)heJ@@? 7@X?I'L?h 7He7@5? ?I4@@@@@@@@@0M ?@hf@@@@@?he?@1?he@?e@? O2@@0Mh?I4@@6K?he?N@L ?W&@5? 3@1??N1?h ?J5?e@@H? ?I4@0M ?@L?he@? J@@?he@?e@?hfO20M ?I46K?he3)X? W&U@H? N@@?e3Lh ?7H?e@@ ?@)Xhe@?hf?W&@5?he@?e@?heW2(Mhe@?h?I'6X?hV')K ?O&>@5 ?@@?eN1h J5e?J@@ ?3@)X?h@@@@@?hW&U@H?he@?e@?he7(Y?he@?heV'1?h?V'@6K ?O2@R@(Y ?@@Le?3L?g 7He?7@5 ?N@@)K ?O&>@@hf@?e3Lh?J(Yhf@?he?V'LheV'@@@@6Kh?O2@@@@@@(Y? ?3@1e?N1?g ?J5?e?@@H @@@@6K ?O2@R@@5he?J5?eN1h?7H?hf@?hfN1he?V4@@@@@@@@@@@@@@@@@@@@@0Y ?N@@f3Lg ?7H?eJ@@? ?I4@@@@@6Kh?O2@@@@@@@0Yhe?7H?e?@h?@ @?hf?@L?hfI4@@@@@@@@@@@@@@@0M? @@L?eN1g J5f7@5? ?I4@@@@@@@@@@@@@@@@@@@@@0Mhf?@f?@h?@L?hf@?hfJ@1? I4@@@0M? 3@1?e?3L?f 7Hf@@H? I4@@@@@@@@@@@@@@@0M? ?@f?@h?@)Xhf@?he?W&Y@? N@@?e?N1?f ?J5?f@@ I4@@@0M? ?@f?@h?3@)X? W&@@5? ?@@?f3Lf ?7H?e?J@@ ?@f?@h?N@@)K ?O&@?@H? ?@@LfN1f C5f?7@@ ?@f?@he@@@@6K ?O2@Y@@5 ?@@1f?3=?e @@(Yf?3@@@@ ?@@@@@f?3@@@?g?I4@@@@@6Kh?O2@@@Y@@@0Y ?@@@@5f?V'@@? 3@Y?g@@@5 ?3@@h@@5?h?I4@@@@@@@@@@@@@@@Y@@@@@0M ?3@@@?gV@5? V'@6X?eO&@@0Y ?V4@@6K?eO&@0Y?he?I4@@@@@@@e?@@@@@@@0M ?V4@@)K?eW2@(Y? ?V4@)KO2@@0M ?I4@@6?2@(M? ?I4@@@@@@@@@0M ?I4@@6KO&@0Ye ?I'@@@0M ?I4@@@0Y ?I4@0M ?I4@@@(Mf V40M ?I@M ?I40Y?f
Fig. 66. Pre-selection of separation media.
112
The number of separation media on the market is quite enormous and it will not be possible to include all different alternatives in the initial media screening. The number of media have to be reduced to cut down time and effort spent on the method scouting phase of process development. Only those media supporting the issues of scaleability and suitability for the different stages in a process, listed inside the filter in Figure 66, should be considered for testing.
Base matrix properties and derivitization chemistry Base matrix properties and derivitization chemistry govern the chemical and physical stability of the chromatography media and are very closely related to the scaleability of the complete process. The design in of scaleability by selection of suitable media will assure that the media can be packed in large scale columns without change in performance and flow/pressure characteristics and that efficient maintenance procedures can be applied to secure a long media life time. The possibility of any toxic substances leaking from the media into the product stream is also closely related to the properties of the base matrix and the chemistry used for coupling spacers and ligands to the matrix.
Bead size The particle size and the range of particle size distribution may also have an impact on scaleability in the sense that particle size is closely related to the backpressure generated in the column during a chromatographic run. The optimal bead size in any particular chromatographic step will depend on the characteristics of the feed and the degree of purification required in this step. In a final polishing step, for instance, there will be a need for smaller beads, i.e. high efficiency, to accomplish separation of closely related compounds. In such a step, media with a narrow particle size distribution will help to give a lower back-pressure at a given bed height and flow rate compared to media with a wider particle size distribution.
Documentation and technical support Comprehensive documentation is required for chromatography media to be used in industrial processes, to facilitate the work with setting up and validating the complete process. Chromatography media used in production processes are treated as raw materials. As with any type of raw material, acceptance criteria have to be established and every new batch of media has to be subjected to tests before it can be brought into production.
Regulatory support Information on possible extractable compounds and what kind of methods to use to quantify these compounds should be provided by the vendor. Leakage data on the most relevant extractable compounds should be available.
113
Vendor certification Vendor certification programmes should be initiated for all vendors of critical chemicals or materials and should certainly include the chromatography media supplier. Such programmes should be implemented to assure a long term, reliable supply of chromatography media of high quality and consistency.
Delivery capacity The delivery capacity of the vendor is an important issue during the vendor certification phase to secure timely deliveries of large quantities of media when the purification process is scaled up. The largest batch size that the vendor can provide should be discussed and put in relation to the column size that will be used in the final production scale. The stock situation and lead times should be discussed to estimate the consequences for continuity in production in case of an urgent need for a new batch of media. Long-term contracts, based on forecasts provided by the user, should be discussed to secure future timely deliveries. Long-term delivery guarantees should also be discussed to assure that the same quality of chromatography media can be delivered during the entire life cycle of the product to be purified.
Method design and optimization The main purpose of optimising a chromatographic step is to reach the pre-defined purity level with highest possible product recovery by choosing the most suitable combination of the critical chromatographic parameters. In process chromatography, in contrast to analytical or small scale preparative chromatography, this also has to be accomplished as quickly, cheaply and easily as possible. The method must be designed carefully to be robust despite variations in feed stock and other conditions in the production hall. The following sections will give some guidelines for optimising the critical operational parameters which affect the maximum utilization of an ion exchange chromatography step to be used in a production process.
Binding conditions Selectivity during adsorption to an ion exchanger is optimized by careful selection of pH and ionic strength of the start buffer. A pH far away from the isoelectric point of the molecule of interest will give stronger binding and increased capacity but may also have a negative impact on selectivity due to increased binding of contaminating molecules. If retention of the molecule of interest is low at selected start conditions, due to the pH being very close to the isoelectric point, it will start to elute from the column during sample application (isocratic elution) when the sample volume is increased in a preparative situation. The choice of optimal pH
114
will always be a balance between selectivity and capacity which in turn depends on the purpose and strategic focus of any particular chromatographic step. The buffer system should be selected to give maximum buffering power at the least possible ionic strength to ensure high binding capacity of the molecule of interest. To achieve this, the pKa of the buffer should ideally not be more than 0.5 pH units away from the pH being used. Generally, 10 mM of a buffer is the minimum desirable level. Ideally, one of the buffering species should also be uncharged, and so not contribute to the ionic strength. In large scale applications, economic considerations often limit the choice to acetate, citrate, phosphate, or other inexpensive components. The pH and conductivity in the binding buffer can sometimes cause aggregation/ precipitation in the sample when it has been equilibrated to start conditions. If aggregates are formed they may be excluded by the beads and lost in the flow through fraction with loss of recovery as a consequence. The extent of aggregates/precipitate formation depends on the pre-column residence time, after sample has been transferred to start conditions. This problem is often recognized as a scale-up problem since the pre-column residence time may increase considerably upon scale up.
Elution Elution from ion exchangers is usually accomplished by applying a continuos or stepwise increase of the ionic strength of the eluting buffer, thereby weakening the electrostatic interaction between the bound molecule and the adsorbent. Depending on the purpose and strategic focus, as previously outlined, different desorption principles can be applied to achieve the objectives of any particular chromatographic step in the most optimal way. • Stepwise elution • Gradient elution • Isocratic elution Stepwise elution is often preferred in large scale applications since it is technically more simple than elution with continuos gradients. Stepwise elution will also decrease buffer consumption, shorten cycle times and allow the molecule of interest to be eluted in a more concentrated form. Single-step elution and two-step elution can be characterized as being a ’’group separation” technique. This type of elution is usually applied in initial chromatographic steps (capture) where the purpose is to remove bulk impurities and substances differing greatly from the product. In a large scale initial chromatographic step, using a crude feed material, media with a large bead size are favoured to avoid problems with high back-pressure and reduced media life time due to high viscosity and severe fouling during sample application. In such an application it will be very difficult, unless the selectivity is extremely high, to resolve closely rela-
115
ted contaminants from the molecule of interest, even when applying very shallow gradients. The strategy will be to resolve the ”group” of substances that the molecule of interest belongs to from ”group(s)” containing the contaminating substances. This can most conveniently be achieved by eluting one ”group” at a time by applying one or several steps with increasing eluting strength. In later purification steps however, applying feed material that has been partly purified and using chromatography media with higher resolving power, it will be easier to resolve closely related substances by applying multi-step or gradient elution techniques. Resolution is maximized by working on the shape or slope of a gradient or the eluting strength of different steps in a multi-step procedure. Such eluting techniques can be characterized as being ”fine separation” techniques as opposed to the ”group separation” refered to above. In final purification steps (polishing), where the main focus will be to reach the predefined purity of the molecule of interest, resolution is maximized by applying shallow gradients or even isocratic elution using high resolution media with small bead size. When stepwise elution is applied, one has to keep in mind the danger of getting artefact peaks when a subsequent step is administered too early after a tailing peak. For this reason it is recommended to use continuos gradients in the initial experiments to characterize the sample and its chromatographic behaviour. Elution by pH gradients is not generally applied. This is because, changing the pH by applying a pH gradient is frustrated by the buffering power of the molecules adsorbed on the column and, in case of weak ion exchangers, the buffering of the adsorbent groups themselves. For stepwise applications, pH elution can be quite successful. The pH change will be delayed compared with the new buffer front because of these titrations, but eventually the bound molecule is desorbed, coincident with a rapid pH change.
Sample load When the selectivity parameters have been defined to achieve the most optimal balance between resolution, capacity, speed and recovery, in ion exchange chromatography, as for most other adsorption techniques, there are then basically two alternative routes to follow for optimization of sample load and flow rate to achieve highest possible productivity in the system. I. In a typical capture situation the sample will be applied to the column, nonbound substances will be washed out from the column and the compound of interest will be eluted from the column with a simple step elution procedure. The difference in eluting strength, between the different steps will usually be large, i.e. it will be possible to elute one group of compounds while the others are still retained on the column. In this mode, the entire bed volume can be utilized for sample bin-
116
ding and the prime consideration when optimising for highest possible productivity is to define the highest possible sample load over the shortest possible sample application time with acceptable loss in yield. The dynamic binding capacity for the protein of interest should be determined by frontal analysis, i.e. by continuously applying sample on the column up to the point where the compound of interest starts leaking off at the column outlet. PAGE, ELISA or other appropriate techniques are used for the determination of the break-through profile of the compound of interest. II. In many intermediate purification steps, and always in a polishing step, the requirements for resolution will set the limit for the amount of sample that can be applied to the column. Sample is mainly bound in the upper part of the bed since there will be a need for a certain bed height to achieve separation between closely related substances moving down the column with different velocities in a shallow gradient of elution buffer. Maximum sample loading is defined by running a series of experiments with gradually increased sample load. Optimal conditions will be the maximum sample load that provides a resolution still high enough to meet the pre-defined purity requirements.
Flow rate The maximum flow rate that can be applied in any particular ion exchange chromatography step will differ between different parts of the chromatographic cycle. Since low molecular weight substances show high diffusion rates, i.e. are transported rapidly between the mobile phase and stationary phase, the flow rate during equilibration, washing and regeneration procedures is limited primarily by the rigidity of the chromatography media and by system constraints regarding pressure specification. Larger molecules, i.e. the substances to be separated during the chromatographic run, show a lower diffusion rate which will limit the flow rate that can be applied during sample adsorption and desorption. In a typical capture situation, the flow rate during sample application has to be controlled so that the residence time in the column allows for a complete binding without leakage in the flow through fraction. Maximum flow rate is defined by running the frontal analysis test (break-through) refered to above at a number of different flow rates. Optimal conditions will depend on the requirements for speed and capacity in the system. If speed, i.e. sample application time, is critical due to proteolysis or other detrimental effects in the feed material, a higher flow rate may have to be used on the expense of the binding capacity in terms of amount of sample that can be applied per volume of media. If speed is not a big issue, binding capacity can be increased on the expense of flow rate which will reduce the scale of work in the final production process. Occasionally, high back-pressure, due to
117
the viscosity and crude nature of the feed, may set the limit for maximum flow rate during sample application. During elution, the flow rate will affect resolution between compounds to be separated and also the concentration of these compounds in the product pool. When there is a need for high concentration in the product pool, a lower flow rate may have to be applied to minimise the volume (dilution) of the eluted product/fractions. This is particularly important in a step preceding a gel filtration step, where the sample volume is limiting for loading capacity.
Selecting a column When a chromatographic step is being developed to be a part of a manufacturing process and the time has come for scaling up, the next crucial step in ensuring a reliable product quality and maximum production economy is the decision about which column to use. Information on large scale columns from Pharmacia Biotech is available upon request. Different demands are put on a column for production compared with one used for the initial laboratory scale and scale-up experiments. Flexibility, which is needed in laboratory scale and scale-up is achieved by using a column with a movable adaptor. In production, consistency in performance and safety of the end product are the main concerns. Here the column packing has to be reproducible, materials of construction have to be well characterized for leakage and the design mechanically stable. A number of criteria have to be considered. These criteria are more dependent on the scale of operation than on the media and are thus very similar in their importance for ion exchange, hydrophobic interaction, gel filtration and affinity chromatography. Their ranking and importance change when moving through a chromatographic process is shown in Figure 67. ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? @? ? @? ? @? ? @? ? @? ? @? ? @? ? @? @@@6X? ?@@? ? @@@@@? @@?@1? ?@@? ? N@@@H? @@?@@W2@6?2@@@6T2@@6X?@@6KO2@@@W2@@@hg? ?@@@ @@?@@@@@@@@@@@@@Y@@@1?@@@@@@?@@@@@@?hg? ?3@5 @@?@@@@?e@@@@@@@@@@@?@@@@@@?@@?@@@,hg? ?N@H @@@0MI4@@@@@@@@0?4@@@?@@@0?4@@@@@@0Yhg? @? ? ? @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ ? @? ?@ ? @? ?@ ? @? ?@he@??@?@f?@K?eW2@? ?W&?hg? @? ?@he@??@?@?W2@?@@@e*@@?W-X?@@@@@??@@?)X?*@W)Xhf? @? ?@he@@@@?@?7Y@?@?@eN@@?7R1?@@@@@??@HJ@1?N@@@)hf? @? ?@he@??@?@?3X@?@?@e?@@?3T5?@@@@@??@?*U@e@@X?hf? @? ?@he@??@?@?N@@?@?@e?@@?V+Y?@0?4@??@?V4@e(R/??@he? @? ?@ @@ ?@he? @? ?@ ? @? ?@he?@ @? ? @? ?@he?@f)X?@6T2@?W-Xf@?@6X?@?@?@?@@@@@?W-X?he? @? ?@he?@e?J@1?@V@Y@?7@)f@?@V1?@?@?@?@?@?@?7@)?he? @? ?@he?@e?*U@?@?3X@?3X?f3T@T5?@?@?@?@?@?@?3Xhf? @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@he?@@@?V4@?@?N@@?V/?fV+R+Y?@?@@@?@?@?@?V/hf? @? @@ ? @? ? @? ?W26X??@?@@@@? ? @? ?7@@)?*@W&R1?@?@?@?N@@@R1?@?@f?@f?@?@@@@??@@@U??@?@@@@?? @@@@@@@@@@@@@@@@@@@? ?@?B@@X??N@@@T5?@?@?@??@@@T5?@?@f?3=O.??@?@f?3XS,??@?@f? ?@e(R/?e@MI+Y?@?@@@??@0R+Y?@?@?@e?V40Y??@?@?@e?V40Y??@?@f? ?@hf?@ ? ? @? ? @? ? @? ? @? ? @? ? @? ? @? ? @? ? @? ? @? ? @@@@@? ? N@@@H? ? ?@@5 ? ?3@H ? ?V'? ? ? ? ? ? ?@@@6X ?@@@6Xhe@@he?@@? ? ?@@?@)K?hf?@@?@)K?h@@he?@@L ? ?@@?@@@@@@?@@@@@6Xe?@@?@@@@@@@6KO2@@@?@@@6?2@6X?@@@ ? ?@@@0Y@@@@?@@@@@@)e?@@@0Y@@@@?@@@@?@@?@@@@@@V4)?@@H ? ?@@?e3@@@?@@@@?f?@@?e@@@@?@@@@?@@?3@@@@@?e?@@L ? ?@@?eV4@@?@0?4@@@e?@@?e@0?4@0MI4@@@?V4@0?4@@@?@@@ ? ? ? ? ? ? ?W2@@6?2@?h@@ @@h@@f?@@? ?7@?I4@@@?h@@L?hf@@ ?@@? ?3@?e?@@@@@6?2@@@@@@?@@@@6X?@@@6X@@@@@6?2@@@@@@6X?@@? ?V4@@6X@@?@@@@@@(Y@@H?@@@@@1e@@@@@@@@@@@@@@@@@@@1?@@? ?@@@@@@@@@@@H?@@L?@@@@@@?'@@@@@@@@e?@@?@@@@@@?@@? ?@@@@0?4@@@@@@@@e@@@?@@@@@@?V4@@@@@@@@@@@@?@@@@@@?@@?
?@@@6Xh@@?W2@@?h@@?@@? @@ ?@@?@)K?hO&@?he@@ @@ ?@@?@@@@@@?@@@@@@@@@@@@6T2@@6X@@@@@@@6X?@@6XeW2@@@?@@@@6KO2@@6?2@6X ?@@@0Y@@@@?@@@@@?@@@@@V4@U@@@@@@?@@@?@1?@@@1e*@@@e@@@@@@@@?@@@@@@) ?@@?e3@@@?@@?@@?@@@@@eS@@@@@@@?@@@?@5?@@@@e?@@@(?@@@@@@@@?@@@@? ?@@?eV4@@?@@?@@?@@@@@@@0R4@@@@@@@@@@0Y?@@@@e@@@0Y?@@@@@0?4@@@V4@@@ ?@5? @@@0Y? W26Xh?W-XfO@ 7
@Ke?W&? W&?@fW& @@@@?*@W)X?@@@@@@@?W-X?W&@?@?)X?*@W)X? @@?@?N@@@)?@?@?@?@?7@)?7Y@?@W@1?N@@@)? @@?@e@@X??@?@?@?@?3X??3X@?@@U@??@@X @@?@e(R/??@?@?@?@?V/??V4@?(R4@??(R/ O@?W2@hW2@? ?@6X?@?@?@@@?&@@W.e?)X?*@@W-X?@@@ ?@V1?@?@?@?@?N@@@HeJ@1?N@@@R1?@?@ ?@W5?@?@?@?@e@@@Le*U@??@@@T5?@?@ ?@(Y?@@@?@?@e@0R/eV4@??@0R+Y?@?@ ?(Y?
?@@@@?e?@@?e?@K??@ ?@e@W-X?@@?W&?@@@?@?@@@?W2@ ?@@@@@R1?@@?&@?@?@?@?@?@?7Y@ ?@eI'T5?@@?N@@@?@?@?@?@?3X@ ?@e?V+Y?@@??@?@?@?@?@?@?N@@ @@
118
Fig. 67. In the initial capturing, handling large volumes at high flow rates is important. When moving towards the final steps, the demand for low dead volume columns and systems to achieve high resolution becomes more and more important.
Aspects of column design Flow distribution system The most important factor in process column construction is to design the flow distribution system to give as even a flow distribution as possible at the column inlet and outlet. The aim is to retain HETP values of the same order as in the laboratory column. This is usually achieved by a construction where the radial backpressure is negligible compared to the axial back-pressure at the column inlet, see Figure 68. The simplest method is to place a coarse mesh net between the column end piece and the finer mesh net retaining the bed, to create channels for radial distribution. This may be combined with multiple inlet/outlet ports depending on column diameter. Depth filters are more easily clogged due to the relatively large filter surface. This may be a severe disadvantage in continuous operation. Column inlet
?@@@@@@@@@@@@@@@@@@@@@@@ ?@ ?@ ?@ @??@ @??@ @?J@ @?7@ @?@@ @?@@ @?@@ @?@@ @?@@ @?@@ @?@@ @?@@ ?W.? @?@@ W.Y? @?@@hf?W.Y @@@@hfW.Y? @@@@hf7H @??@he?J5? @??@heO&Y? @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@??@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?h @? @??@hW(M? @?h @? @[email protected] @?h @? @??@ @?h @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@??@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?h @? @?h @? @?h @? @?h @? ?O@K @?h @? ?@@@@@@@@@@@@@@@@@ @?h @? ?I@Mhe?@ @?h @? ?@ @?h @? ?@ @?h @? ?@ @?h @? ?@ @?h @? ?@ @?h @? ?@ @?h @? ?@ @?h @? ?@ @?h @? ?@ @?h @? ?@ @?h @? ?@ @?h @? ?@ @?h @? ?@ @?h @? ?@ @?h @? ?@ @?h @? ?@ @?h @? ?@ @?h @? J@L? @?h @? 7@@? @?h @? 3@H? @?h @? N@ @?h @? ?@ @?h @? @?h @? @?h @? @?h @? @?h @? @?h @? @?h @? @?h @? @?h @? @?h @? @?h @? @?h @? @?h @? @?f?O2@ @? @?e?O20M? @? @??O20M?e @? @W20M?f @? @(M?g @? ?O2@@Hh @? ?O20M?@?h @? ?O20M?e@?h @? ?O20M?f@?h @? ?O20M?g@?h @? ?W20M?h@?h @? ?.M?he@?h @? @?h @? @?h @? @?h @? @?h @? @?h @? @?h @? @?h @? @?h @? @?h @? @?h @? @?h @? @?h @? @?h @? @?h @? @?h @? @?h @? @?h @? @?h @? @?h @? @?h @? @?h @? @?h @? @?h @? @?h @? @?h @? @?h @? @?h @? @?h @? @?h @? @?h @? @?h @? @?h @? @?h @? @?h @? @?h @? @?h @? @?h @? @?h @? @?h @? @?h @? @?h @? O26Khf?O2@6K O26K @?h @?eO2@@@@@@6Khe?O2@0MI46Kh?O20M?I46KheO20MI46Khe?O2@@6K?g?J@?h @?O20MgI4@6K?fO2@0M?fI4@6K?e?O20M?fI46Kf?O2@0MfI4@6K?e?O2@@0M??I46K?fO&@?h @@0Mhe?I46K?O20Mhe?I46KO20M?hI46K?O20M?h?I46KO20M?g?I4@6KO20Mhe @M ?I4@0M ?I40M?hfI4@0M?hf?I40M?hfI40Mhf
Column lid
dP dr
dP dL
Packed bed
Fig. 68. Radial and axial backpressure in a column distribution system.
Material resistance and durability Wetted components must be constructed from materials with high chemical resistance against the harsh chemicals which are frequently used for cleaning-in-place (CIP) and sanitization procedures such as 1 M NaOH, salts, acids, etc. Stainless steel of high grades is most commonly used for very large columns. However, stainless steel does not always have sufficient corrosion resistance when high salt concentrations in acidic solution are used. In this case fluoroplastic coated stainless steel is recommended. Materials should be chosen to minimize leakage and be tested for toxicity.
Sanitary design Effective cleaning and sanitizing of packed columns depends on the total column design, including the absence of threaded fittings and the smoothness of wetted surfaces. It is important to minimize dead volumes in the column to hinder bacterial attachment and facilitate cleaning. Columns constructed from calibrated borosilicate glass allow the use of thin O-rings in the adaptor and end piece and
119
give minimum dead volumes. Borosilicate also has a smooth and durable surface which facilitates cleaning. Plastic columns are usually less expensive but most plastics do not meet pharmaceutical industry demands for chemical resistance, hygienic design and in-line cleaning. They may however be well suited for scale-up experiments.
Pressure vessel safety Large scale columns should be regarded as pressure vessels even if the actual working pressures usually are kept low. Large volumes of organic solvents may be handled during cleaning which calls for explosion proof equipment. The column design has to meet local regulations to be approved.
Regulatory support Regulatory support for process scale columns should be available from the column supplier to provide information on materials necessary for registration of the process including chemical stability, toxicological tests, physical data and column construction.
Ergonomics For easy handling of process columns it is important that they are constructed in a stable way and are easy to pack and clean. Large columns should preferably have lockable wheels. In laboratory columns, the bed height can easily be adjusted using moveable adaptors. In large columns, adaptors may be impractical and too heavy to handle. Therefore, columns with fixed end pieces are selected in many applications. Valves should be easy to reach and remove when the column is taken apart.
Packing large scale columns Column configuration Process columns with a moveable adaptor are essentially packed in the same way as laboratory columns with adaptors. In essence, this means that the gel slurry is compressed by a flowing liquid until the bed height has stabilized, at which point the flow is stopped and the adaptor is lowered onto the gel surface and secured in place. Large scale columns are, however, frequently supplied with fixed end pieces. This calls for a different packing technique. An extension tube is fitted on top of the column as a reservoir for the gel slurry. When the bed has been packed and settled at the join between the extension tube and the column, the extension tube is removed and the top column lid is secured in place. With this method it is important to
120
calculate the exact amount of media that is required to get the appropriate bed height.
Packing the column Detailed packing instructions for ion exchange media in process columns will not be given here. Please refer to the instructions supplied with respective media and respective column.
Scale-up When the IEX step has been optimized at laboratory scale, the method can be scaled-up. Provided that scaleability has been ”designed-in” during the development phase, scale-up to final production scale should be straightforward. ”Design-in” of scaleability has to do with how the chromatographic step has been designed and optimized (robustness, simplicity, costs, capacity etc.) and the choice of appropriate chromatography media (chemical stability, physical stability, bead size, cost etc.). Some general guidelines for scaling up are outlined in Table 23. Table 23. Scale-up guidelines. Maintain
Increase
Check system factors
Bed height
Column diameter
Distribution system
Linear flow rate
Volumetric flow rate
Wall effects
Sample concentration Gradient volume/bed volume
Sample load
Extra column zone spreading
Increasing the bed volume by increasing the column diameter and increasing volumetric flow and sample load accordingly, will ensure the same cycle time as in the laboratory scale method development. The column bed height, linear flow rate, sample concentration and ratio of sample to gel, all optimized at laboratory scale, will be kept the same. If a gradient is used for elution, the ratio of gradient volume to bed volume will remain constant and, therefore, the time required for the gradient to develop and the effect on resolution, will remain the same on the larger column. The same principle is applied for the volume of each step in a step elution procedure. Different system factors may affect performance after scale-up. If the large scale column has a less efficient flow distribution system, or the large scale system introduces large dead volumes, peak broadening may occur. This will cause extra dilution of the product fraction or even loss of resolution if the application is sensitive to variations in efficiency (plate number) in the system used.
121
scaling up to a larger diameter column means that most of the bed support generated by the friction against the column wall is lost. This can give increased bed compression and poorer flow/pressure characteristics. If all the above aspects are taken into consideration, chromatographic variability is normally not a major issue when scaling up. Non-chromatographic factors may have a more significant effect on performance during scaling up. These factors include: changes in sample composition and concentration that often occur as the fermentation scale increases, precipitation in the feed stock due to longer holding times when large volumes are handled, nonreproducibility of the buffer quality due to inadequate equipment for consistently preparing large quantities of buffer solutions, and microbial growth in feed-stock or buffers due to increased handling and longer holding times. Figure 69 shows a 700-fold scale up of a model protein separation on SOURCE 30S going from a 2.2 ml column to a 1.57 liter column in one step.
122
Column: Sample: Sample load: Eluent A: Eluent B: Flow rate: Gradient: System:
SOURCE 30S, a) 7.5 mm i.d. x 50 mm (2.2 ml) b) 200 mm i.d. x 50 mm (1.57 l) Mixture of chymotrypsinogen, cytochrome C and lysozyme 0.32 mg/ml bed volume 20 mM sodium phosphate, pH 6.8 20 mM sodium phosphate + 0.5 M NaCl, pH 6.8 300 cm/h ; a) 2.2 ml/min b) 1.57 l/min 0-100% B; 20 column volumes a) FPLC System b) BioProcess Engineering System
?? ?? ? ?? ?? ? ?? ? ?? ?@ ?? ?@ ?@ ? ?@ ?@ ?@ W.?? ?@ ?@ ?O.Y? ?@ ?@ ?W20Y?? ?@ J@L? W.M?e? ?@ 7R1? ?W.Yf? ?@ @?@? O.Y?f? ?@ @?@? W20Yg? ?@ @?@? ?W.Mh? ?@ @?@? O.Y?h? ?@ @?@? W20Yhe? ?@ @?@? ?W.Mhf? ?@ ?@ @?@? W.Y?hf? ?@ ?@ @?@? ?O.Yhg? ?@ ?@ @?@? ?W20Y?hg? ?@ J@L? @?@? W.M? ? ?@ 7R1? ?J5?@? ?W.Y ?? W26Xe?W26X?W&eW26Xg?@ @?@? ?7H?@? O.Y? 7@@(MB1?e@??@he?J@@?@e@?e@??@ J@@?@MB1e?@e@?hf?@MB@@e@?e@??@ ?? ?W.?f@?e@??@ N@@??@e?@e@? ?N@He@?e@??@he?7Y@?@e@?e@??@ 7Y@Le?@e?@e@? @@e@?e@??@ ?7Y?e?C5?e3=C5hf/KC@@=C5e?3=C5? ?/KC5??C5?e3=C5he?@@@@@=C5?e3=C5 @@@(eC5e?3=C5? ?C@@=C5?e3=C5 ? ?@@@@?@0Y?@?V40YhfV40MI40Y?@?V40Y? ?V40Y?@0Y?@?V40YhfI@MI40Y?@?V40Y ?I(Y?@0Y?@?V40Y? @0MI40Y?@?V40Y ?? ? @?e@Ke@? ?W2@@?W&K?he/Xe?? 3L?J@@6X@?@?@??@@@@@?W26X?e?7Y@@?&@@@?W26X?W.eN1e? N1?7@?B@@?@?@??@?@?@?7YV1?e?@?@@?N@@H?7YV1?*Ue?@e? ?@?@@??@@?@?@??@?@?@?@@@@?e?@?@@??@@??@@@@?V/X??@e? ?3T@@=C@@?@?@??@?@?@?3X?f?@?@@??@@??3X?e?S,??@e? ?V+MI40R'?@@@??@?@?@?V4@f?@?@@??@@??V4@e?.Y??@e? ?3L? J5e? ?V/? .Ye? ? ?? W-X? 7R1? ?J@?@L ?7@@@)T-KO-X?W-K ?@e?@(R4@?,?7R@@@@@@@@@ ?@e?3U??S@U?@?@@?@@?@?@ ?N)KO&?,?3T@@?@@?@?@ @@0R+Y?V+R'?@@?@?@
/X N1 W2@6X??3L? .M?B1??N1? @?e@? W2@@@?e@? 7<e@?e@? 3=?C@Le@? V4@0R/?J5? ?7H? J5 .Y
Fig 69. 700-fold scale up from a 2.2 ml lab-scale column to a 1.57 litre production scale FineLINE 200 column. (Work by Pharmacia Biotech, Uppsala, Sweden.)
123
12. Applications Ion exchange has proven to be one of the major methods of fractionation of labile biological substances. From the introduction of the technique in the 1960s´ to the development of modern high performance media, ion exchange chromatography has played a major role in the separation and purification of biomolecules and contributed significantly to our understanding of biological processes. The examples given in the following section have been drawn from the published literature as well as from work in our own laboratories. For detailed information on specific subjects the reader is referred to the original work.
The design of a biochemical separation Ion exchange chromatography, in common with other separation techniques in the life sciences, is rarely sufficient as the sole purification stage in the separation or analysis of complex biological samples. Ion exchange is frequently combined with other techniques which separate according to other parameters such as size (gel filtration), hydrophobicity (hydrophobic interaction chromatography or RPC) or biological activity (affinity chromatography). Popularity of fractionation techniques 90 80
Homogenization
70 60 50 Precipitation
40
GF IEX AC
30 20 10
1
2
3
4
5 6 7 Stage in purification scheme
Fig. 70. Frequency of use of fractionation techniques (1). (Reproduced by kind permission of the authors and publisher.)
Not only is the choice of techniques important. The order in which they are employed will also play a role in determining the speed, the convenience and the overall yield for the purification.
124
Sample loading, sample dilution and impurity contamination are usually maximal at the beginning of a separation scheme. At this stage the high capacity, high selectivity and concentrating effect of ion exchange makes the technique ideal. This suitability is reflected in Figure 70 which shows the frequency of use of different fractionation techniques in published protein purification schemes (1). The use of multi-dimensional chromatography with ion exchange as a first step is well illustrated by the separation of monoclonal IgG2b from cell culture medium (Fig. 71). An initial purification and concentration of the antibody from 500 ml cell culture medium by cation exchange chromatography on SP Sepharose High Performance was followed by a second fractionation by gel filtration using Superdex 200 prep grade. ?@?@?@?@heW&eW-KO.??@?@?W-XeW.e?@@@eW.h@K @?@??@eO@K?e?@@@f?O@? ?W26K?e?)X? ?@?@?@?@eW-X?)[email protected]'@U??@?@?7R1e7Ye?@?@e7YeW&?26X@@@??@?26T-KO@?W-Xe@?@??@W2@@@@e?@?@W)X?@@@W-X?@@@@@@@?@?2@6T.?W-X W.MS@@6K?@)T2@@@6T2@e@?he?@@@?@?@e7R)T@1?7Y@fN@)X?@?@?@?@e@@@??@@@e@@@?7@@@V@@?@?J@@@V@R@@@W&@)e@@@??@@Y@@?@e?@@@@@)?@?@@R1?@?@?@?@W@@@?@@H?7@) *UO&@XS@@@>@@?@?@@Y@ ?@?@?@?@?C@T@@U@?3X@e@KC@>,?@?@?3T5e3T5??@f3T5?3X?@W@@?@?*U@@?3T(Y@@@X?e@?@??@@X@@?@e?@?I'Xe@?@@T5?@?@?@?@@U@@?@@L?3X? V40MI40R+R4@@?@?@@?@e@?he?@?@?@?@@0R+MI4@?V4@e@@0R+Y?@?@?V+YeV+Y??@fV+Y?V/?@(R'?@?V4@@?V+Y?@MI/?e@?@??(R@@5?@e?@eV/e@?(R+Y?@?@?@?(R4@@?(R/?V/? ?(Y? ?@0Y ?)X?W&hf?@he@?e@?eO2@?g?W-Kg@?e?@@6T-X?W-X?f?O@? ?@ ?@?@ ?W26K?he?@ ?@1?7@W-T2@@?W-KO.?@W-X?@@@?@X@?e@W2@@8h?7R@@?@?@?@?e?@X;@R1?7R1??@@@@@@??W&?W&?2@@?@@@@@@??@?@6T&KO)X??W.?W&X@?@ W&@>@@6T2@@6T26KC@T&?@hf?@@?@@@R@@?@?7R@@H?@@R1?@?@W@@@?e@@Y@@?@@g?@?@HJ@@@?@?e?@)X@?@?@?@??@?@?@@??*@W&@@@?@?@@?@?@??@?@V@@@@@)??7H?7@@@?@ *@S@@@@@Y@?@@YS@@R@5 ?@@T@@@T@@?@?3T@@L?@@T5?@?@@U@@?e@@X@@9V@g?@?@?*U@@?@?fS@@T5?3T5??@?@?@@??N@@@X?@?@?@@?@?@??@?@?3XI'Xe?3L?3X?@?@ V40MI4@@?@?3@@0R'?(Y?@hf?(R+R+R+R'?@?V+MI/?(R+Y?@?(R4@@?e(R@@V4@@e?@e?3T5?V4@@?@?@?e.MI+Y?V+Y??@?@?@@?e@MI/?@?@@@@?@?@??@?@?V/?V/e?V/?V/?@?@ ?N@? @@g?@e?V+Yg@?@? @? ?@@5 W& W2@@heW&f?V@HW&K?e@@@?W-KO-XgO@e@?@?eW26X ?W.?@?7@?@@?@?@6T-X??W&@@@?@?@@?W&KO&@e@@@@@?7@@@e@Xe7R@@R1e@@@@@@e@?@?e7,?@he?@)X@?@??@?@?@@@?@@??@@?@@?@@@@@@?g?@?@e@?@?@@@?@@?@@@eJ@@@eJ@f@V@@@@e?@)Xe@)X? J@S@@>@>@T5?7@@<eJ@@U S@@T5??@?@?@@@T@@??@@T@@?@?W@T5?e?@K??3T5e@?@?@@@T@@?@@@e*U@@=O&@f@W@@?@fS,e?S,? @@0MI4@0R+Y?@?@?e.MI/[email protected]+Y??@?@?@0R+R'??(R+R'?@@0R+Y?@??@@@?V+Ye@?@?@0R+R'?@@@eV40R40R'?@e@(R'[email protected]?@?.Y? @? ?@e(Y ?@@6T-X?f?O)X?W&??)X?W&?@@6T.g@?e)X?W&??@@@f?W2@?@g?@?@e?@@@e@@@? ?W2(e?W&?@?g?W2(hf?@X;@R1??@@@@@@1?7@??@1?7@?@?V@Yg@?e@1?7@??@@@e)X?7@>@T5?7@@<eJ@S, S@@T5??@?@?@@@T@@??@@T@@?@?W@T5?f@?e@@T@@??@@@?*U@?3=O&@L?e@W@@?@fS)X??S,? @@0MI4@0R+Y?@?@?e@@[email protected]+Y??@?@?@0R+R'??(R+R'?@@0R+Y?@?e@?e(R+R'??@@@?V4@?V40R'1?e@(R'[email protected]/??.Y? @? ?V'?e(Y ?W2(?@K?hf?@ ?@ ?@@?h@?hf?W-KO-X?he@6K?eW2@@@?f?O@?@Ke?O@Ke?@ ?7(YJ@@@6?)T.?W2@@@??@W@e@?gW&e@?2@@@@@6X?@W)X?e@?26X?@6X@@?W.e@?@?@W-T2@@??@f?*?@@R1??W.?@@@@@?@@@@e7@@R1??W.?@@@@@?@@@@e7R'?,??@@@@@@?@@@@@@@@@@?@ 7@@@?@?@@?@@R@@?@?g?V'@@?@??7H?@?@?@?@@?@e@?S@U??@?@?@@?@@?@?@@@?@?@ 3X?@?@?@@?@@T@@?@?hS@@T5??3L?@?@?@?@@?@e@?*?,??@?@?@@?@@?@?@@@?@?@ V/?@?@@@@?(R+R'?@[email protected]+Y??V/?@?@?@?@@?@e3LV+Y??@?@?@@?@@?@?@@@?@?@ V/ ?@
W2@6X?fW2@? @K ?W&? ?@ @?h?W&?e?@f?W2@6X 7
?@@?f@?@?g?@@? B)T(R@Y@@?@?S)?2@??)T. ?@@U?@?@@?@?7@@@H??@@U ?@V1?@?@@?@?3X?@e?@V1 ?@@@?@@@@?@?V/?@e?@@@ W.fW& 7He?W&@ @?eW.Y@ @??W.Y?@ 3L?.Y??@ V/f?@ ?@@@g?@fW&he@? @?W-X?W-X? ?W.?W. ?@?@W-KO-X?@W@KO&@f?@KO.?@?@@?@?)X? @?7R1?7R1? O&H?7Y ?@@@@R@@R1?@@@@@Y@fJ@@@H?@?@@?@@@)? @?@?@?@?@? ?@?@@@@@@5?J@@@@@@@@ ?@?I'T@@T5?@@XI'X@f*U@@L?@?@@T@@Xh?@?@?@@@?@?@?@@@e@?3T5?3T5? W-X??W.?f?@?@f@H?7,f?@?@ ?@ @@@@@?@?@?W&?2@@e@X@? ?@f@?fJ@@@ V+R/?V+Yf?@?@ ?@ @?@?@W@@@?7@@@?@?J@@@? @?e?O&0M? ?@?@ ?@ @?@?@@U@@?3X?@?@?*U@@? ?@f@?e@0M? ?@?@ ?@ @?@?(R4@@?V/?@?@?V4@@? @?fO2@? ?@?@ ?@ ?@f@?eW2@< ?@?@ ?@ @?e.Y@? ?@?@ ?@ @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?f?@f@?f@? ?@?@ ?@ @? @?he@?f@? ?@?@ ?@ @? @?f?@f@?f@? ?@?@ ?@ @?f@? W-X?e@?f?@?@ ?@ )X ?@f@?f@?h@?W-X? 7R1??J@?f?@?@ ?@ )Xf?J@1 @?f@?g?J@?7R1? @?@??7@Lf?@?@ ?@ ?J@1f?7Y@ ?@f@?f@?g?7@?@?@? 3T@L?3@,e?@@@?@ ?@ ?7R'L?e?@?@ @?f@@@@f?3@@@T5? V+R/?V+Yf?@?@ ?@ ?@?V/Xe?@?@ ?@f@Lf@?g?V+MI+Y? ?@?@ ?@ J5eN1e?@?@ ?J@1f@? ?@?@ ?@ 7He?@e?@?@ ?@e?7Y@f@? ?@?3L?hf?@ @?e?@eJ5?@ ?@?@f@? ?@?N1?hf?@ @?e?@e7H?@ ?@e?@?@f@? ?@e@?hf?@ @?e?3L?@??3L? ?@?@f@? ?@e@?hf?@ ?J5?e?N1?@??N1? ?@e?@?@f@? ?@e@?hf?@ ?7H?f@?@?e@? ?@?@f@? ?@e@?hf?@ ?@g@?@?e@? ?@e?@?@f@? ?@e@?hf?@ ?@g@?@?e@? ?@?@f@? W-X??W-Xf?@e@?hf?@ J5g@?@?e@? ?@e?@?@f@? 7R1??.R/f?@e@?hf?@ 7Hg3X@?e@? ?@?@f@?g?W-KO-X? @?@?h?@e@?hf?@ @?gN@5?e@? @?e?@?@f@?g?.R'@R1? 3T@L?@K?e?@@@e@?hf?@ @?g?(Y?e@?hf@?@?f?@?@f@@@@g?N@?@? V+R/?@@@f?@e@?hf?@ ?J5?hf3Lg@?@?@?h?@?@f@?g?@KC@T5? ?@e@?hf?@ ?W26T&H?hfS)T2@?@? ?@?@f@?g?@@0R+Y? ?@e@?hf?@ ?7
@@?@@@@@ @@f?@@@@@
Lane 1 and 5: Lane 2: Lane 3: Lane 4:
? ? ?@?@?@?@heW&eW-KO.??@?@?W-XeW.e@?@?@6KO@?26T&@?W-X?@@e.R'@R@@R1??@6X?@6T&?26X??W2@@@?)X?W&@W)Xhg? ?W&?hW-KO-KO-X? ?W&? ? ?W26K?e?)X? ?@?@?@?@eW-X?)[email protected]'@U??@?@?7R1e7Ye W.MS@@6K?@)T2@@@6T2@e@?he?@@@?@?@e7R)T@1?7Y@fN@)X?@?@?@?@e@@@? *UO&@XS@@@>@@?@?@@Y@ ?@?@?@?@?C@T@@U@?3X@e@KC@>,?@?@?3T5e3T5?@?@?@V@@@@@V@Y@?7@)?@@fN@?@@?@??@V1?@V@@@@V1??7Y@@HJ@1?7Y@@@)hg? @?@?@W@@X?@?3X@?3Xe@@e@KC@T@@T5??@W5?@?3X?@W5??3X@@?*U@?3X@@X?hg? V40MI40R+R4@@?@?@@?@e@?he?@?@?@?@@0R+MI4@?V4@e@@0R+Y?@?@?V+YeV+Y? @@@?@(MI/?@?V4@?V/e@@e@@0R+MI+Y??@(Y?@?V/?@(Y??N@@5?V4@?V40R/?hg? (Y ?(Y?g(Yf@0Y? ? ? ?W-Xe@@@?f?O@?he?@f?W&?f@@?@?@?W&?2@@?)X?@e@@6T-X?@@@@@?W.??)X?7@?W-T2@@??W-X?@6T.?@@@??@?2@6T2@?W-X?? ?O@?e?@e@K W& @K ? ?W26K?he?@ ?*?,e@Xe?@@@@@@??@6KO-X?W-X@W)X?W&@??@@@ W&@>@@6T2@@6T26KC@T&?@hf?S@Ue@)X??@?@?@@??@V@@R1?7R@@@@)?7Y@??@?@ N@W@@@?7@@@?@W@1?@e@@V@R1?@?@?@?7H?J@1?@@?7R@@?@??7@)?@@@H?@?@?J@@@?@@Y@?7@)?? *@S@@@@@Y@?@@YS@@R@5 ?*?)X??S,??@?@?@@??@W@@T5?3T@@@Xe3X@??@?@ ?@@U@@?3X?@?@@U@?@e@@?3T5?@?@?@?3L?*U@?@@?3T@@?@??3X??@@@L?@?@?*U@@?@@X@?3Xe? V40MI4@@?@?3@@0R'?(Y?@hf?V+R/??.Y??@?@?@@??@(MI+Y?V+R+R/eV4@??@?@ ?(R4@@?V/?@?(R4@?@e@@?V+Y?@?@?@?V/?V4@?@@?V+R'?@??V/??@0R/?@?@?V4@@?(R@@?V/e? ?N@? ?(Y? ?@@?f? @? ? ? @K @Ke W. ?@?@@6T-X?hf@?eO2@??@ ? ?W.?@@@@6T-X?@@@@@?@?@?W-KO2@@@?)X?@6X@@@? 7H?@?26X?@6T26T-X?@@f?@?@X;@R1??@@@@6T2@?e@W2@@8e?@ ? ?7H?@?@@V@R1?@?@?@W@@@?7R@@Y@@HJ@1?@V@@?@? @?J@@@V1?@V@@V@R1?@@f?@?@)X@?@??@?@?@@Y@?e@@Y@@?@@?@ ? ?3L?@?@@?3T5?@?@?@@U@@?3T@@X@@?*U@?@W@@?@L @?*U@@W5?@W@@?3T5?@@L?e?@eS@@T5??@?@?@@X@?e@@X@@9V@?@ ? ?V/?@?@@?V+Y?@?@?(R4@@?V+MB@@5?V4@?@(R'?@@ W.e3LV4@@(Y?@(R'?V+Y?@@@[email protected]+Y??@?@?(R@@?e(R@@V4@@?@ ? ?@0Yf?(Y?f.YeV/eI(Y??(Y? ?@@?f@@f?@ ? ? ?W.?g?O2@g@@6T-XgO)X?W&f ?W2@ @Khe?@K? ? ?W2(e?W&?@? ?7Y??@?W&?2@@@?W-Xe@X;@R1e@@@@@@1?7@eW&W&R@@Y@@?@?@@?@?@??@V@@?@@R1?*@?@V@@?@W@1?@@@) ?W-KO&@@?@?@@@@@@??@6X@@6T-X?W&?@6X@@@?)X?@?)X ? ?7@UO-T&@?@?W-T2@@ ?@@@?@?7@@@?@@?7@)e@)X@?@e@?@?@@@?@@e*@ ? J@S@@>@>@T5?7@@< ?3T5?@?3X?@?@@?3X?e?S@@T5e@?@?@@@T@@eN@ &@T@@X@@?@?@@?@?@??@W@@?@@T5?N@@@W@@?@@U@?@@X? ? @@0MI4@0R+Y?@?@??@ ?V+Y?@?V/?@?@@?V/?e?.MI+Ye@?@?@0R+R'e?@ ?I+MI4@@?@@@@?@?@??@(R'?(R+Ye@Y@(R'?(R4@?(R/??@ ? ?(Y?he(Yhf?@ ? ? ?W-Xe?@?@@@e)X?W&??@@@f?W2@?@g?@J@[email protected]/? ?@e?@@@eW-X? ? ?7R1e?@?@X?e@1?7@??@@@e)X?7@YgV@@@@@@@@@Y? V@@Y ? @?@?f@@@@@@?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ ? 3T5?g?W@>(Mhe@? ?@hg ?@ @? ? V+Y?g?.R+Y?h?C@L ?@L?hf ?@?W&U?W-Xhf?@?W-XN@@@fW-K?eO@K?hf?@ J5 3= ? ?@@@?)X? ?@@@@@@?hf ? ?@X;@R1? I'@Xhg ?@?@?@?@?@hf?@f?@)Xf@?@?@?@@@?@?@?@@?7@@@@?@ ?@?7R1?7R1hf?@?.R/?@X?f7R@@@@@@@@@?@?@@?W&KO@?@ ? ?@)X@?@? ?V4)X?hf ? S@@T5? @??S,?hf ?@?3T5?3T5hf?@?@K?eS,f@?@?@?@@@?@?@?@@?3X??@@@ ? .MI+Y? @??.Y?hf ?@?V+Y?V+Yhf?@?@@@e.Yf3T5?@?@@@?@?@@@@?V/??@Y@ ? V+Y? ?@ ? ? @? ?@hg ?@ @? ? @? ?@hg ?@ @? ? @? C5hg J5 @? ? ?@?J@L?W-Xhf@?@@0YW-X?he W-KO&U?W-Xhf?W-T2@@?W-X?eW-K?eO2@? ? ?@?7R1?7R1hf@?@Xe7R1?he .R'@R1?7R1hf?.R'@Xe7R1?e7R@@@@@@@? ? ?@?@?@?@?@hf@?@)X?@?@?he @KC@T5?3T5hf?@K??S,?3T5?e@?@?@?@@@? N@?@?@?@ ?V4)X?@?@?e@?@?@?@@@? ? ?@?3T5?3T5hf@??S,?3T5?he ? ?@?V+Y?V+Yhf@??.Y?V+Y?he @@0R+Y?V+Yhf?@@@?.Y?V+Y?e3T5?@?@@@? ? V+Y?e?I@? ? ? ? ?
Marker proteins HMW kit Starting material (crude cell culture medium) Pooled material after cation exchange chromatography Pooled material after gel filtration
Native electrophoresis using PhastSystem with PhastGel 8-25 and silver staining
1
2
3
4
5
Fig. 71. Purification of rat monoclonal IgG2b from cell culture supernatant. (Work by Pharmacia Biotech, Uppsala, Sweden.
The sample composition with regards to ionic strength and pH should be taken into consideration when designing the separation scheme. In ion exchange chro125
matography solutes bind to the gel at low ionic strength and are eluted from the column at a higher ionic strength. The converse situation occurs in hydrophobic interaction chromatography. Thus if these two techniques are to be used in a separation scheme it is logical to have them adjacent to each other. This principle is illustrated in Figure 72 which shows the purification of human a2-macroglobulin from Cohn Fraction III. After initial purification by affinity chromatography on Blue Sepharose CL-6B to remove albumin, the sample was applied to a Q Sepharose High Performance column and eluted with an increasing salt concentration gradient. Relevant fractions were then pooled and a2-macroglobulin was purified to homogeneity by hydrophobic interaction chromatography on a Phenyl Sepharose High Performance column. ? ? ? @? ?@K? W&eW26KheW2@?hfW&?@K? O@K?fW&K?h?W2@hfW&K?hg? @W26X?@@@?eW26Xe@?)T26X@@@e'6X?@@@?W2@@?W26X??W26X?*@e*@@@@??W26X?e @@@@?@?@@Y@he@?@?@?@?C@T@@U@?3X@??@KC@>,?@?@?3T5??3T&@?e3T5?3X?@W@@?@?*U@@?3T(Y@@@X?e@?@??@@X@@?@e?@?I'Xe@?@@T5?@?@?@?@@U@@?@@L?3X? ? V40MI40R+R4@@?@?@@?@e@?g@?@?@?@@0R+MI4@?V4@??@@0R+Y?@?@?V+Y??V4@@?eV+Y?V/?@(R'?@?V4@@?V+Y?@MI/?e@?@??(R@@5?@e?@eV/e@?(R+Y?@?@?@?(R4@@?(R/?V/? @?eW-X? ? ?(Y? ?@0Y @?e.R/? ?W-Xe@@@??)X?W&hf? @? ?7R1e@Xe?@1?7@hf? ?@?@@@?W-XgO@f?@f@Kg@?@?he?@K?he?O@Ke?@he?@hW.?@e@?W-X? ?@K?e@? @?e@Kf@@@? ?@@??@@??@@@@@e?@?@e@)X??@@?@@hf? ?W26K?he?@hf?@eI@?7R1e@@@@@@eW-X@e@?@@6?)Xe@?@W-X?@@@@@?@?@@@@6T-T&?@?W2@@@@??@?@@?@KO.?@?@W-T2@@e7H?@e@?7R1??@@@@6T2@??@6X?@6T-T&?W&X@@@@?@? @?@?@@@?f@? ?@f?3T@L??S,??@@T@@hf? W&@>@@6T2@@6T26KC@T&?@h?@f?@?@e@?@?@@e7R@@e@?@?@@@)e@?@@R1?@@@@@?@?@?@@V@R@@?@?7Y@@?@??@?@HJ@@@H?@?@@R@@?@e@??@e@?@?@??@?@?@@Y@??@V1?@V@R@@?7@@@@?@?@? @? ?@h?@f?V+R/??.Y??(R+R'hf? *@S@@@@@Y@?@@YS@@R@5he?@e@??3T5e@?@?@@e3T@@e@?@?@@X?e@?@@T5?@@@@@?@?@?@@?3T@@?@?3X@@?@??@?@?*U@@L?@?@@T@@?@e@??@e@?3T5??@?@?@@X@??@W5?@?3T@@?3X?@@?@?@? @? ?@e@?f?@ ? V40MI4@@?@?3@@0R'?(Y?@h?@e@??V+Ye@?@?@@eV+R'e@?@?(R/?e@?(R+Y?@0?4@?@?@?@@?V+R4@@?N@@5?@??@?@?V40R/?@?(R+R'?@e3L?@e@?V+Y??@?@?(R@@??@(Y?@?V+R'?V/?@@?@?@? @? @?f?@ ? ?N@? @0Y? V/ ?@@??(Y? @? @? @?f?@ ? @? @? ?@e@?f?@ ? @? ?W&?he?@6X?)X?g?W.?g?@K? ?W2@e@?e?W.?@6X? @? ?@e@?f?@ ? @?@6T-T2@@@@e?)X??@6T26T&?@?7@?W-T&?@?@e?@S,?@1?@?W-X??7Y??W&?26X@@@e@?26T-KO@?W-X??7@@V1?eN@@@?3X?@W5? @? ?@e@?f?@ ? @?@?V+R'?@?@eV4@??@(R'?V/?V+R'?V+R4@@?(Y??@@@?@@@@?V/e?V+Y?V/?@(R'?@?V4@@?V+Y?@MI/e?V40Y?@@0Y?V+R4@@?e?@?@?V/?@(Y? @? ?@e@?f?@ ? ?(Y? (Y (Y @? @?f?@ ? @? @Lf?@ ? @? ?@e@1f?@ ? ?W2(e?W&?@?g?W-XhW-KO-XgO)X?W&e)X?@g@@6KO@ ?W-T2@@?f?O)X?W&??@@@g?W.??W26X?h?@?@e?@@@eW-X? @? ?@e@@f?@ ? ?7@UO-T&@?@?W-T2@??7>,[email protected]'@R1e@@@@@@1?7@e@)X@?W&?eN@V@@@W@e?@6T26T-X?@6X?@?2@@?W-X??*?@@Xe?@@@@@@1?7@??@@@e)Xe?7Y??7@>@T5?7@@<eJ@@UheN@?@e@?@?@@@?@@e@V@@?&@?e?@?@?@@@L??@V@@V@R1?@V)T@@@?@?7@)??V'>@)X??@?@?@@@?@@??@@@?J@1e?@@@?@e@?f?@V1?@@@ @? @@f?@ ? @@0MI4@0R+Y?@?@?e.MI/?@g@KC@T5e@?@?@@@T@@e@W@@?N@@@??@?@?@?@,??@W@@?3T5?@W@@U@@?@?3XfS(MS,??@?@?@@@T@@??@@@?*U@e?3T5?3=C5?f?@W5?@?@f@?e@K @? ?@e@@f?@ ? @@0R+Ye@?@?@0R+R'e@0R'e@Me?@?@?@?(Y??@(R'?V+Y?@(MI4@@?@?V/f.Y?.Y??@?@?@0R+R'??@@@?V4@e?V+Y?V40YW&?@??@(Y?@?@f@?@?@@@? @? O2@?hg ?@e@@f?@ ? ?(Y?g(Y ?&@?@??(Y? @? O20Y@?hg @@f?@ ? ?@@?g?V'? @?he?O2@0Me@?hg @@f?@ ? W-KO-XgO)X?W&e)X?@g@@6KO@ ?@@6T-KO-XgO)X?W&e@@@?fW2@?@?g@?@?e@@@??W-X @?h?W20M?f@?hg ?@?J@@f?@ ? ?W2(e?W&?@?g?W2(h.R'@R1e@@@@@@1?7@e@)X@?W&?eN@V@@@W@e?@6T26T-X?@6X?@?2@@?W-X??@X;@R@@R1e@@@@@@1?7@e@@@??)X?7<e@?e?@6X@?@?e?I@??.R/ @?hW.M?g3Lhg ?@?7Y@f?@ ? ?7@UO-T&@?@?W-T2@??7@U?@hN@?@e@?@?@@@?@@e@V@@?&@?e?@?@?@@@L??@V@@V@R1?@V)T@@@?@?7@)??@)X@?@@?@e@?@?@@@?@@e@@@?J@1?@??J@?e?@V@@@@? ?W-XeW.g@?g?W.YhN1hg ?@?@f?@ ? J@S@@>@>@T5?7@@<eJ@S,h@KC@T5e@?@?@@@T@@e@W@@?N@@@??@?@?@?@,??@W@@?3T5?@W@@U@@?@?3XfS@@T@@T5e@?@?@@@T@@e@@@?*U@?3=O&@Le?@W@@?@?e?@e?@K? ?7R1e7Ug@?gW.Y?h?@hg ?@?@f?@ ? @@0MI4@0R+Y?@?@?e@@0Y?@g@@0R+Ye@?@?@0R+R'e@0R'e@Me?@?@?@?(Y??@(R'?V+Y?@(MI4@@?@?V/f.MI+MI+Ye@?@?@0R+R'e@@@?V4@?V40R'1e?@(R'?@?e?@?@?@@@ ?@?@e@)X?f@?f?W.Yhe?@hg @?he?@?@?@f?@ ? ?(Y?g(Y V'e?(Y? ?3T@L?3>,?e@@@?f?7H?he?@hg ?J@?hf?@?@f?@ ? ?V+R/?V+Y?f@?f?@hf?@hg ?7@Lhf?@?@f?@ ? ?@ ?@@?h@? ?@@6T-X?he@6K?eW-T.f@?gO@?@K?eO@K?e@? @?fJ5hf?@hg ?@V1h?@e?@?@f?@ ? ?W2(?@K?hf?@hW&e@?2@@@@@6X?@W)X?e@?26X?@6X@@?W.e@?@?@W-T2@@??@ I4@?,??W.?@@@@@?@@@@e7R@Ue?J@?e@@@@@@?@@@@@@@@@@?@? @?f7Hhf?@hg J5?@e)Xh?@?@f?@ ? ?7(YJ@@@6?)T.?W2@@@??@W@e@?e*@?C@@@?@?@@V1?@@@)??J@@@V1?@V@@@?7H?J@@@?@@R@@?@? I'U??7H?@?@?@?@@?@e@?@)X??7@Le@?@?@@?@@?@?@@@?@?@? @?f@?hf?@hg ?W&H?@e@1?@g?@?@f?@ ? J(Y?7@XW@@@@H?7R'@@??@@5gN@@@U@@?@?@@W5?@@Xe?*U@@W5?@W@@@?3L?*U@@?@@T@@?@? @??S,??3L?@?@?@?@@?@e@?3>)X?3@,e@?@?@@?@@?@?@@@?@?@? @?f@?hf?@hg W&@??3L?@@L?g?@?@f?@ ? .Ye(R40R+?@e@?V4@??@0Ye@?e?@MI4@@?@?3@(Y?(R/e?V4@@(Y?@(R4@?V/?V4@@?(R+R'?@??@ @??.Y??V/?@?@?@?@@?@e3LV+R/?V+Ye@?@?@@?@@?@?@@@?@?@? @?f@?hf?3L?hf 7<e?V/T(R/Xg?@?@f?@ ? V+Y?h?I(Ye(Y V/ @? @?f@?hf?N1?hf @?fV+Y?N1?W-Xe?@?@f?@ ? @?f@? @?hf @[email protected]/X??@?3L?e?@ ? ?@K?hf@?hfW&K?g@@hf@KhfO@K?e@?he@?he?W-KO-X?W-X?he@Ke?W-T-Xe@?e@@@?f?O@?@Ke?O@K?O@? @?f@? @?hf ?J5?e?@f?@@H?N1??@?N1?e?@ ? @@@@@6X?W&?@@@eW-T&?@?@e@?@6T-T2@@@@e7@@@?W-Xe@@?W-T2@@@@?@?@@@@6T-X?@?)T2@@@@e@?@@?@KO.?@?@W-X?@@@?@e?.R4@?,?.R/??W.?@@@@@?@@@??7R+R/?J@?e?I@??@@@@@@?@@@@@@@@@@@? @?e?J5? @?hf W&f?7H?h?@@?e3L?@e@?e?@ ? @@@@@@1?*@?@?@L?7R@@?@?@e@?@V@R@@?@?@e@@?@?7@)e@@?7R@@@@@@?@?@?@@V@R1?@?@@Y@@?@e@?@HJ@@@H?@?@@R1?@?@hS@U?f?7H?@?@?@?@?@??@f?7@Lg?@?@?@@?@@?@?@@@?@@? @?e?7H? @?hf ?W&@L?e?@?W.?hfV/X@e@?e?@ ? @@@@@?@?N@@@?@,?3T@@?@?@e@?@?3T@@?@?@e@@?@?3X?e@@?3T@@@@@@?@?@?@@?3T5?@?@@X@@?@e@?@?*U@@L?@?@@T5?@?@f?@KO&?,?@Ke?3L?@?@?@?@?@??@?@K??3@)X??@e?@?@?@@?@@?@?@@@?@@? @?e?@ 3Lhf ?7@@1?e?@?.Y?hf?N@@e@?e?@ ? @0?4@@@??@?@?(Y?V+R4@@?@e@?@?V+R'?@?@e@@?@?V/?e@@?V+R40?4@?@?@?@@?V+Y?@@0R@@5?@e@?@?V40R/?@?(R+Y?@?@?@e?@@0R+Y?@@@??V/?@?@?@?@?@??3T@@@?V+R/??@e?@?@?@@?@@?@?@@@?3@? @?e?@ N1hf ?@@@@Le?@ @@e@?e?@ ? ?@0Y ?V+M ?V'? @?e?@ ?3L?he J@@@@)T2@@ 3@e@?e?@ ? @?e?@ ?N1?he 7@@@@@@< N@e@?e?@ ? W2@?hf?)X?e?@ ?W-KO-X?W.hf@Ke?W.?@??W-XeW-X?f?O@?@Ke?O@K?O@? @?e?@ 3Lhe @@@@@@@? ?@e@?e?@ ? W2@@@?)X?W&@@W)X?@@@@??W-X?@1?@@?@W-X?@@@?@? ?.R40R/?7Ue?W.?@@@@@?@@@??7H?@??*>1e*?,??@@@@@@?@@@@@@@@@@@? @?e?@ V/X?h ?J@@@@@@@? @?e?@ ? 7Y@@HJ@1?7Y@@@@)?@?@@??7@)?@@?@@?@@R1?@?@? @)X??7H?@?@?@?@?@??@e@??V'@eS@U??@?@?@@?@@?@?@@@?@@? @?e?@ ?V/Kh ?7@@@@@@@? @?e?@ ? 3X@@?*U@?3X@@@X??@?@@??3X??@@?@@?@@T5?@?@? ?@KO@Ke3>,??3L?@?@?@?@?@??@e@?eS@L?*?,??@?@?@@?@@?@?@@@?@@? @?e?@ V4@6K?f ?@@@@@@@@? @?e?@ ? N@@5?V4@?V4@0R/??@?@@??V/??@@@@@?(R+Y?@?@?@? ?@@@@@@?V+Y??V/?@?@?@?@?@??3L?@?e.R/?V+Y??@?@?@@?@@?@?@@@?3@? @?eJ5 ?I4@@6K? J@@@@@@@@? @?e?@ ? ?@0Y ?V/? ?V'? @?e7H ?I4@V4@@6K /K 7@@@@@@@@? 3Le?@ ? @?e@? @@@@@@@@@? N1e?@ ? @?e@? I4@6K?he@@@@@@@@@? ?@e?@ ? @?e@? ?I4@6Kf?O2@@@@@@@@@@? ?3L??@f?W-XeW-X? ? ?W-XeW-X?f@?e@? I4@@@@@@@@@@@@@@@@@? ?V/X?@f?7R1e7R1? ? ?7R1e7R1?f@?e@? ?@e?@@@@@@@@@@? V)X@f?@?@e@?@? ? ?@?@e@?@?f@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@f?3T@L?3T5? ? ?3T@L?3T5? ?@g @? @? @?g?V+R/?V+Y? ? ?V+R/?V+Y? ?@g @? @L @? ? @?J@L?f ?@?J@Lhe@@@@@1 @@6T@LgW-K?eO@K?hf?@f? ?J@?7R1?f J@?*?,he@XeI@ ?I40R/g7R@@@@@@@@@?@?@@?W&KO@?@f? ?7@?@?@?f 7@?S@Uhe@)X? @?@?@?@@@?@?@?@@?7@@@@?@f? ?3@@@T5?f 3@@@?,he?S,?@? ?@?@K?g@?@?@?@@@?@?@?@@?3X??@@@f? ?V+MI+Y?f V+MI+Yhe?.Y?@? ?@?@@@g3T5?@?@@@?@?@@@@?V/??@Y@f? V+Y? ?@f? ?@g @? @? @? ? ?@g @? @? @? ? ?@g @?hf?J5? @? ? ?W-KO-X?W-X?e @@6T-X?W-Xg?W.?W&U?W-X? W-KO-X?W-XfW-K?eO2@? ? ?*?@@R1?7R1?e @X;@R1?7R1g?7U?*?,?7R1? *>@@?,?7R1f7R@@@@@@@? ? ?V'U@?@?@?@?e @)X@?@?@?@g?@)KS@U?@?@? V'@S@U?@?@f@?@?@?@@@? ? S@@T5?3T5?e ?S@@T5?3T5g?3>@@?,?3T5? ?S@@?,?3T5f@?@?@?@@@? ? .MI+Y?V+Y?e ?.MI+Y?V+Yg?V+MI+Y?V+Y? ?.MI+Y?V+Yf3T5?@?@@@? ? V+Y?e?I@? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?@e?@g?O@Khe?@K?g@Ke@?g@?fW& W&@?W26X?@@@e?W26X@@@e@6T26X?@@@@@e'6X?*@W26X?W2@@@@??'6?2@6X?@@@?/X?W.?W26X?*@hf? @? ?@K? W& ?@K?hfW&hf? ?@e?@?/X?W2@@@@@?W26T2@6X?@@@?W26X?@@6X@?W26Xe@?@@@?*@?W26T2@??'6KO26T&@ ?@@@@@?N1?7@e@@H?7@@?@?@@Y@hf@?@?@?@?C@T@@U@?3X@??@KC@>,?@?@?3T5?e@??@?@?3X?@?@L?J@??3T5?3X??@W@@?@@U@?@?3T(Y@@@X?e@?@?@?3X@@?@e?@?I'X?@?@?3T@@?@?@?@@U@?@?@@L?3X? @@f?@@@@@ @@h?V+M ? V40MI40R+R4@@?@?@@?@e@?h@?@?@?@@0R+MI4@?V4@??@@0R+Y?@?@?V+Y?e@??@?@?V/?@?@)T&@??V+Y?V/??@(R'?(R4@?@?V+Y?@MI/?e@?@?@?N@@5?@e?@eV/?@?@?V+R'?@?@?(R4@?@?(R/?V/? ? ?I+Mhe?(Y? ?@0Y ? ?W-Xe?@ ?W-Xe@@6T-Xe)X?W&?g? ?7R1eJ@ ?7R1e?I'@R1e@1?7@?g? ?W26K?he?@ ?@?W-X?W-XgO@hf@?e?W&??@he?@hf@Khe?W26X?eW.h@K @?@??@eO@K? ?@?@e7@L? ?@?@fN@?@e@@?@@?g? W&@>@@6T2@@6T26KC@T&?@he?@?7R1?7R1e@@@@@@e@6KO-KO-X?@W@KO&@??@?@@?@KO.?@?@W-X?@@6?@?e@@6T-X?@@@@@?7,?@?heN@?@e@?@?@@@?@@e*@W&R@@Y@?@@?@@?@?@??@V@@?@?7R@@@?@V@@?@?J@@@?7@)?e@?@?g@@?@@?e?*@W&R@@Y@@?@?@@?@?@e*@?@?@@@V1?@?@W@@@?7@)f@V@@@@ @?g?@g@@@? @? ? J@S@@>@>@T5?7@@@>@T5?7@@<eJ@S,he3T@L?@K??S,??@?@?@@@T@@??N@@@T@@X@?@@?@@?@?@e@W@@?@?3T(Y@@@W@@?@?*U@@?3X?e?@W5?@?@f@?e@K @?h@?e?@e?@ ?@ @? ? @@0MI4@0R+Y?@?@?e@@0Y?@hV+R/?@@@?.Y??@?@?@0R+R'?e@MI+MI4@?@@@@@?@?@e@(R'?@?V+Y?@Y@(R'?@?V4@@?V/?@??@(Y?@?@f@?@?@@@? @?h@?e?@e?@ ?@ @? ? (Yhe?(Y?hf@??(Y? @?hf?@e?@ ?@ @? ? @?hf?@e?3L?hg ?@ @? ? W. @? @@h?@he?@?W-X?@@@he?@K?eW.?@e?W-XgO@e@KeO@e?O@? @?h@?e?@e?N1?hg ?@ @? ? ?W2(?@K?hf?@he7Ye?@?2@@@@@6X?@W)Xe?@?26X?@6X@@?W.??@?@?@W-T2@@e@??@?.R/?@X?eW.?@@@@@?@@@e7HJ@e?.R/e@@@@@@e@@@@@@?@@@@? @?h@?e?@f@?hg ?@ @? ? ?7(YJ@@@6?)T.?W2@@@??@W@e@?f@@@?J@@@?@?@@V1?@@@)eJ@@@V1?@V@@@?7H?J@@@?@@R@@?@f?@f?@)Xe7H?@?@?@?@?@e@?7@L?g@?@?@@e@?@?@@?@?@@? @?hf?@f@?hg ?@L? @? ? J(Y?7@XW@@@@H?7R'@@??@@5h3T5?*U@@?@?@@W5?@@X?e*U@@W5?@W@@@?3L?*U@@?@@T@@?@f?@?@K?eS,e3L?@?@?@?@?@e@?3@(?W&K?e@?@?@@e@?@?@@?@?@@? @?hf?@f@?hg ?@1? @? ? .Ye(R40R+?@e@?V4@??@0Ye@?fV+Y?V4@@?@?3@(Y?(R/?eV4@@(Y?@(R4@?V/?V4@@?(R+R'?@e@??@?@@@e.YeV/?@?@?@?@?@e3LV+Y?&@@@e@?@?@@e@?@?@@?@?3@? @?h@?e?@f@?hg ?@@? @? ? ?V+YheI(Y??(Y? V/ ?V'? @?h@?e?@f@?hg ?@@? @? ? @?hf?@f@?hg ?@@? @? ? @?hf?@f@?hg J@@? @? ? ?)T-T-X?e?@K? @?e?@K?f?@?@hf@KhfO@K?e@?he@? @?h?@e?@f@?hg 7Y@? @? ? ?@@R@@)KO@?@@@e?W-T&?@@e?W-X@??@?@@6?)X??@?@W-X?@@@@@?@?@@@@6T-T&?@?W2@@@@e@?@@?@KO.?@?@W-T2@@? @?h?@e?@f@?hg @?@? @? ? ?@@?@@@@@@?@?@e?7R@@?@@e?7R@@??@?@?@@@)??@?@@R1?@@@@@?@?@?@@V@R@@?@?7Y@@?@e@?@HJ@@@H?@?@@R@@?@? @?hf?@f3Lhg @?@? @? ? ?@5?3@?@?@@@?@?@?3T@@?@@e?3T@@??@?@?@@Xe?@?@@T5?@@@@@?@?@?@@?3T@@?@?3X@@?@e@?@?*U@@L?@?@@T@@?@? @?hf?@fN1hg @?3L @?f?W-XeW-KO-Xhg? ?(Y?V4@@?@?@?@e?V+R4@@@e?V+R'??@?@?(R/e?@?(R+Y?@0?4@?@?@?@@?V+R4@@?N@@5?@e@?@?V40R/?@?(R+R'?@? @?h?@e?@f?@hg @?N1 @?f?7R1e.R4@?,hg? ?@0Y @?h?3L?J@f?@hg @??@ @?f?@?@f?S@Uhg? @?h?V/?@@e@@?@@@e@@e@@e@@ @@e@@e@@e@6X?f@??@ @@@?e?3T@L?@KO&?,hg? ?W-KO-KO-XgO)X?W&e@@@?gW.eW26XgW-X?W-KO-Xhe?@6Ke?W-T.?e@@@?f?O@?@Ke?O@K?O@? @?hfN@f?@hg ?B1?f@??@ @?f?V+R/?@@0R+Yhg? ?@?@e?.R4@?@@R1e@@@@@@1?7@e@@@??)X?e7Ye7)X??@e?@?@?@@?@@?@?@@@?@@? @?hf@?f?@hg @??3L? @? ? V4@@e?@@0R+MI+Ye@?@?@0R+R'e@@@?V4@?eV+Y?V40YW&e@?@@@?V+MI+YeV/?@?@?@?@@?@??3LV+R/??@e?@?@?@@?@@?@?@@@?3@? @?hf@?f?@hg ?@f@??N1? @? ? &@ ?V/? ?V'? @?hf@?f?3L?hf ?@f@?e@? @? ? ?@@?hS@L? @?hf@?f?N1?hf @?e@? @?he@? ? W2@?g?W2@ )Xe?W&?he@@@?)Xhe?@6Ke?W.?@?e@@@?f?O@?@Ke?O@K?O@? @?hf@?g3Lhf @?e@? ?J@Lhe@? ? 78e?@@?@KO&@@?W&?2@@@e?W&X@1?@?7@?W-T2@@?@e@X;@R1eW.?@@@@@?@@@@??7H?@?e?I@??@@@@@@?@@@@@@@@@@@? @?hf@?gN1hf @?e@?e3L ?7@1he@? ? @?@@?@HJ@@@Y@@?7@@@?@@e?7@@@@?@?@@?7R@@?@f@)X@?@e7H?@?@?@?@@?@??@e@?g?@?@?@@?@@?@?@@@?@@? @?hf@?g?3L?he @?e@?eN1 ?@@@he@? ? 39V@?@?*U@@X@@?3X?@?@@e?3X?@@?@?@@?3T@@?@f?S@@T5e3L?@?@?@?@@?@??@e@?e?@e?@?@?@@?@@?@?@@@?@@? @?hf@?g?N1?he @?e?3=? ?@@@he@? ? V4@@?@?V40R4@@?V/?@?@@e?V/?@@@@?@@?V+R'?@?@e?.MI+YeV/?@?@?@?@@?@??3L?@?@??@e?@?@?@@?@@?@?@@@?3@? @?hf@?h@?he @?e?V'@ J@@@L?h@? ? ?V/? ?V'? @?hf@?h3Lhe @?e@?fV'L? 7@@@1?h@? ? @?hf@?hN1he @?e@?f?N1? @@@@@?h@? ? ?@f@? @Kf@Kf?@eW-X?f?O@? @?hf@?h?@he ?J5?g@? @@@@@?h@? ? ?W2@g?O@?g@?he?@e?J@?e@@@@6T2@e@6T26T-X?@?)X?@@@@e@@@@e?@e*?,??@@@@@@? @?hf@?h?3L?h ?7H?g3L @@@@@?h@? ? ?7@@T2@@6T2@@W-T2@@?@?@?h?@e?7@Le@?@?@@Y@e@V@@V@R1?@@@)?@@?@e@@?@e?@eS@U??@?@?@@? @?hf@?h?V/Xh ?@?@hN1 @@@@@Lh@? ? J(MB@R'XS@UW@@>@@Xe@?he?@e?3@,e@?@?@@X@e@W@@?3T5?@@Xe@@?@e@@?@e?@e*?,??@?@?@@? @?hf@?heV/X?g ?@h?3L?hf@@@@@1h@? ? .Ye@?V40R40MI40R4@?@?@?h?@?@?V+Ye@?@?(R@@e@(R'?V+Y?(R/e@@?@e@@?@e?@eV+Y??@?@?@@? @?hf@?he?V/Xg ?@L?g?N1?he?J@@@@@@h@? ? @@e(Y @?hf@?hfV/K?f ?@@?h@?he?7@@@@@@h@? ? @?hf@?hf?V46K?e ?@H?h3Lhe?@@@@@@@L?g@? ? @?hf@? ?I46K? ?@e?@gN)X?h?@@@@@@@1?g@? ? @?hf@? ?I4@ ?@f?@?@@??@)KO2@?fJ@@@@@@@@Lg@? ? ?W-XeW-X?f@?hf@? @@@6K? ?@hfI4@Ug7@@@@@@@V1g@?f?W-XeW-KO-Xhg? ?7R1e7R1?f@?hf@? ?I4@@6K?he?@ I46T&K?C@@@@@@@@?3=?f@?f?7R1e7R@@R1hg? ?@?@e@?@?f@?e?O-T&Kf@? ?V@@@@@@@@@@@@@@ V@@@@@Y@@@@@@@?V@@6Ke@?f?@?@e@?@@?@hg? ?3T@L?3T5?f@@@@@@R@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?f?3T@L?3T@@T5hg? ?V+R/?V+Y?hW@T@@? @? @? ?V+R/?V+MI+Yhg? ?W&?(Y@? @? @? ? ?.R'U?3L @? ?J5? ? ?N1?S)X? W.?J@L @?W&U?W-X??W-Ke?O@K @?he? ?J@?*?,? 7U?*?, @?*?,?*?,??7R@@@@@@@@@?@?@@?W&KO@?@?he? ?7@LV'U? @)KS@U @?V'U?V'U??@?@?@?@@@?@?@?@@?7@@@@?@?he? ?3@(?S,? 3>@@?, @??S,??S,??@?@?@?@@@?@?@?@@?3Xe@@@?he? ?V+Y?.Y? V+MI+Y @??.Y??.Y??3T5?@?@@@?@?@@@@?V/e@Y@?he? ?V+Y @?he? @? @? @? ? @? @? @? ? ?J5? ?C5? ?J5? ? @?W&U?W-X? W-T2@0YW-X @?W.Y?W-X??W-Ke?O2@ ? @?*?,?7R1? *?@@X??7R1 ?J@?7Ue7R1??7R@@@@@@@ ? @?S@U?@?@? V'>@)X?@?@ ?7@?@)X?@?@??@?@?@?@@@ ? @?*?,?3T5? ?S(MS,?3T5 ?3@@@>,?3T5??@?@?@?@@@ ? @?V+Y?V+Y? ?.Y?.Y?V+Y ?V+MI+Y?V+Y??3T5?@?@@@ ? ?V+YfI@ ? ? ? ?
M 1 2 3 4 5
Lane M: Marker proteins HMW-kit Lane 1: Cohn fraction III, 5% in 20 mM bis-Tris propane and 35 mM sodium sulphate Lane 2: Flow-through fraction from Blue Sepharose CL-6B. 6.5 mg protein. Lane 3: Pooled fractions from Q Sepharose High Performance. 2.4 mg protein. Lane 4: Purified a2-M from Phenyl Sepharose High Performance. 80 µg protein. Lane 5: 3 incubated 2 hours with 200 mM methylamine, pH 8.0 Native PAGE using PhastSystem with PhastGel 4-15 and silver staining.
Fig. 72. Purification of human a2-macroglobulin. (Work by Pharmacia Biotech, Uppsala, Sweden.)
126
Towards the end of a separation scheme the complexity and the volume of sample to be handled is smaller, but in most cases the need for higher resolution is increased. Ion exchange chromatography, particularly with MonoBeads, SOURCE, or Sepharose High Performance media can also be used at this stage (Fig. 73). ?@@@@@@? ?@@? ?@6T2@@@@?he?@@?@@ W2@@hf?@@?@@g?@@? ?@@? J@@L V@@X J@@? ?W&@X?hf?@@?@@g?@@? ?@@?@@?@?@@?W2@6X?e?W2@6T&@@)T2@6X?)T26X?e?)T26X?@@?@@?@?2@@@@@@?@@?W2@@?W2@6T&@@@@@?W2@6X?)T26XfW2@6T&@@@?eW2@@eW2@6X@@?@@?@@?@@?@@?W2@6KO2@6X?W2@6X ?@@?@@?@?@@?7@?@1?e?*@?@(Y@@V@@?@1?@@R'1?e?@@R'1?@@?@@?@@(Y@@@@H?@@?7@V@?*U?@@Y@@?@@?7@?@1?@@Y@1f7@?@@Y@@H?e7@V@e7@?@@@@?@@?@@?@@?@@?*U?@@@@?@)?7@?@1 ?@@?3@W@X@5?@@?@@?@@?V4@@??@@?@@@@@?@@?N@?e?@@?N@?@@?@@?@@H?@@@@??@@?@@e?S@@@@?@@?@@?@@?@@?@@?@@f@@?@@?@@f@@f@@@@@@@?@@?@@?@@?@@?S@@@@V4@@??@@@@@ ?@@?N@@R@@H?@@?@@?g@1?@@?@@f@@?J@?e?@@?J@?@@?@@?@@??@@@@??@@?@@e?7@?@@?@@?@@?@@?@@?@@?@@f@@?@@?@@f@@f@@e?@@?@@?@@?@@?@@?7@?@@?e@1?@@? ?@@??@@?@@e3@?@5?e?'6X@5?3@W@@W2(?@@W&5?e?@@W&5?3@W@@?@@??@@@@??@@?3@W@?3@W@@W@@?@@?3@?@5?@@?@@f3@?@5?@@f3@W@e3@W2@@@?@@?3@W@@?@@?3@W@@@6X@5?3@W2( ?@@??@@?@@eV4@0Y?e?V4@0Y?V40R4@0Y?@@@0Y?e?@@@0Y?V40R'?@@??@@@@??@@?V4@@?V40R40R4@@@?V4@0Y?@@?@@fV4@0Y?@@fV4@@eV4@0?4@?@@?V40R'?@@?V40R4@@@0Y?V4@0Y @@g?@@? @@g?@@?
@? @K @K @K @@@?f?)X?f@K @W-T2@@??W-X?@6T.?@@@??@?2@6T2@?W-X??W.?@@@@6T-T2@@@@?)X?)T-X?W2@@@?@?26X?@@@? N@HW-T26T@1?W26X@@@6T-T-T&?@he@@R@@?@??7@)?@@@H?@?@?J@@@?@@Y@?7@)??7H?@?@@V@R@@?@?@W@1?@@R1?7Y@@HJ@@@V1?@?@? J5?7@@UV@Y@?7Y@@@XW@@>@R@5hf@@T@@?@??3X??@@@L?@?@?*U@@?@@X@?3Xe?3L?@?@@?3T@@?@?@@U@?@@T5?3X@@?*U@@W5?@?@L .Y?@MI4@@?@?@?@0R'@@@6T2@@6T26KC@T&?@?g?@)X@?@?@?@??@@?@??7R@@?eN@H??W26T&eW&?2@?W-T5e7H?@?@?@@Y@?7@)??7H?7@@@@?@?@@W@1?*@W&@@@@L? ?*@S@@@@@Y@?@@YS@@R@5?heS@@T5?3T5??@@?@??3T@@?eJ@L??7@@?@?@@Y@he?@@T@@@T@@?@?3T5e3T&@e@?@?@?B1e?S,??@S, V40MI40R+R4@@?@?@@?@e@?g?(R+R+R+R'?@?V+YeV4@@e@?@?@??@e?.Y??@0Y @?eW-X?f?O@?@Ke?O@K ?W2(?@K?hf?@ @?e7R1??@@@@@@?@@@@@@@@@@ ?7(YJ@@@6?)T.?W2@@@??@W@e@?h@?e@?@??@?@?@@?@@?@?@@@?@ J(Y?7@XW@@@@H?7R'@@??@@5hf@?e3T5??@?@?@@?@@?@?@@@?@ .Ye(R40R+?@e@?V4@??@0Ye@?h@?@?V+Y??@?@?@@?@@?@?@@@?@ ?W-KO-X?f?O)X?W&??@@6KO@?f@?@??W2@?@g?@?@e?@@@eW. W2(?eW&?@hW-X?h?.R'@R1??@@@@@@1?7@??N@V@@@W@?e@?@??7,?@?h?N@?@??@?@?@@@?@@?e@?@?@@@?e@@@??@eJ@f@V@@@@h@)X? ?J@S@@>@>@T5?7@@,? ?@@0MI4@0R+Y?@?@e?.MI/?@?g?@@0R+Y??@?@?@0R+R'?e@?@?@?@Me@?@??V40R'1?e@(R'?@f@?@?V+Y? ?V'?e(Y @@@?f?W-Xe@@@??)X?W&e@@@?e?W2@e@? W2(?eW&?@hW2(?he@?@?f?7R1e@Xe?@1?7@e@@@??@?&@>@T5?7@@@@@@@?e3=g?W&HfI46X @?fV46K @?@?fV4@6K?eO2@0M?B@Y@@@@?eV46KfO&@?gI/T2@@6K?O26K?hf?J@?gI4@@ @?@?g?S@@@@@YfJ@?@@@@?fI4@@@@0Mh?V+MeI4@0MI46K?heO&@? @?@?gO&@0MI4@6K?O&@?@@@@? ?I4@@@@@@@@@@@0M @@@@@@@@@@@YgV@@@Y??@@@@? @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ @? @? @? @? @? @? ?W-X ?@?W-X W-KO-X W-KO-X ?7R1 ?@?7R1 .R'@R1 *?@@R1 ?@?@ ?@?@?@ N@?@ V'U@?@ ?3T5 ?@?3T5 @KC@T5 ?S@@T5 ?V+Y ?@?V+Y @@0R+Y ?.MI+Y ?W-T-T&? ?7R'?@@? ?@?V'U@? ?@eS@@? ?3L?.Y@? ?V/?e@?
?@@@6Kh?W-Ke?O@?eO@ ?N@?@@@@@6?)X??7R@@@@@@?@@@@ @?@@?@?@@@)??@?@?@?@@?@?@@ @?@@?@?@@Xe?@?@?@?@@?@?@@ @?@@?@?(R/e?3T5?@?@@?@?3@ ?V+YhV'
? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? @? @K @Ke/T-T2@@@@?)X?)T-X?W2@@@?@?26X?@@@?f? @Kg? ?@@@g)Xf?@K? @W-T2@@??W-X?@6T.?@@@??@?2@6T2@?W-X??W.?@@@@ ?N@HW-T26T@1?W26X@@@6T-T-T&?@?h@@R@@?@??7@)?@@@H?@?@?J@@@?@@Y@?7@)??7H?@?@@ N@R@@?@?@W@1?@@R1?7Y@@HJ@@@V1?@?@?f? ?J5?7@@UV@Y@?7Y@@@XW@@>@R@5?he@@T@@?@??3X??@@@L?@?@?*U@@?@@X@?3Xe?3L?@?@@ ?3T@@?@?@@U@?@@T5?3X@@?*U@@W5?@?@Lf? ?.Y?@MI4@@?@?@?@0R'@@@6T2@@6T26KC@T&?@?h@@@@@)T@1?@@eV'U??@?@V@R@@?@?@??*@?@?7@@@V1 ? ?*@S@@@@@Y@?@@YS@@R@5?he@?I'XI'U@?@@L??S,??@?@?3T@@?@?@??N@@@?3X?@W5 ?@ ? ?V40MI4@@?@?3@@0R'?(Y?@?h@??V/?V4@?(R/??.Y??@?@?V+R'?@?@?e@?@?V/?@(Y ?@ ? N@ ?(Y? ? ?@ ? ?)X?W&hfW.e?@?@e@@@@e@@@?@@@@f ? W26Kf)X ?@1?7@W-T2@@?W-Xe7Ye?@?@e@?W5e@Xe@@X?f ? ?W.MS@@6K?@)T2@@@6T2@??@g?@@?@@@R@@?@?7R1e@@@??@@@e@@@Ue@)X?@@)Xf ? ?*UO&@XS@@@>@@?@?@@Y@?h?@@T@@@T@@?@?3T5e3T5??@?@e@?B1e?S,?@?S,f ? ?V40MI40R+R4@@?@?@@?@??@g?(R+R+R+R'?@?V+YeV+Y??@?@e@??@e?.Y?@?.Yf ? ? ? @?eW-X?f?O@?@Ke?O@K ? W2(?@K @?hf@?e7R1??@@@@@@?@@@@@@@@@@ ? 7(YJ@@@6?)T.?W2@@@e@W@??@h@?e@?@??@?@?@@?@@?@?@@@?@ ? ?J(Y?7@XW@@@@H?7R'@@e@@5?he@?e3T5??@?@?@@?@@?@?@@@?@ ? ?.Y??(R40R+?@??@?V4@e@0Y??@h@?@?V+Y??@?@?@@?@@?@?@@@?@ ? ? ? ?W-KO-X?f?O)X?W&? ?@?@e?W-XeW. ? ?W2(e?W&?@?gW-X?h?.R'@R1??@@@@@@1?7@?e@KO.?W-X?@?)X?@?)Xf@@ J@?@e?*?,e7U ? ?7@UO-T&@?@?W-T2@?7>,?@?h?N@?@??@?@?@@@?@@??J@@@H?7@)?@W@1?@@@)f@H 7@@@e?V'Ue@)X? ? J@S@@>@>@T5?7@@,? ? @@0MI4@0R+Y?@?@??.MI/?@?g?@@0R+Y??@?@?@0R+R'??V40R/?V/??(R4@?(R/??@e@, ? ?@e(YV'[email protected]?.R+Y? ? ? ? @@@?gW-X??W-Xe)X?W&??@@@fW2@??@f ? ?W2(e?W&?@?g?W2(he@?@?g7R1??.R/e@1?7@??@@@e@?&<e?@f ? ?7@UO-T&@?@?W-T2@??7@Uhe@@@??W-Xe@?@?g@@?@@??@@@?J@@@?e?@f ? J@S@@>@>@T5?7@@<eJ@S)X?g?J(?'L?*@,e3T@L?@K?e@@T@@??@@@?*U@@=O.?@f ? @@0MI4@0R+Y?@?@?e@@0R/?g?.Y?V/?V+YeV+R/?@@@e(R+R'??@@@?V40R40Y?@f ? ? ? ?W-Xe?@eW-KO-Xg?@6Xe?@K?e?W-X?W.?f O@K? ? ?W26X?gO2@?g@?h?7R1e?@e7R@@R1e?W.??@S,e?@@@@??.R/?7U?e@@@?@@@?@? @@@@@@@? ? W.R')T2@@6T2@@5?W-T2@?@?@?g?@?@L??@e@?@@[email protected]??@@Ue?@@?@?f?@)Xe@H ? *U?V@@R'@@@U?@H?7@@Y@?@?h?3T@@??@e3T@@T5?W.Ye?@V1e?@@?@??@K??3>,e@?@?@@@?@? @?@@@?@? ? V4@@@@?V40R4@@e@?@?@?@?@?g?V+Me?@eV+MI+Y?.Y?e?@@@e?@@?@??@@@?V+Ye@? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? @@@? ? @?@? ? @@@? ? ?J(?'L ? ?.Y?V/ ?@@?@@@@@? ? ? ?@@?f@@@@@? ? ? ?W-Xe@@@? ? ?7R1e@X ? ?@?@e@)X? ? ?3T@L??S,??@@@@? W.g ? ?V+R/??.Y?f@? 7Hg ? @? ?J5?g ? @? W.Y?g ? @? ?W.Yh ? @? ?7H?h ? @? @? J5he ? @? @? ?W.Yhe ? @? @? ?7H?he ? @? @? J5hf ? @? @? ?W.Yhf ? @? @? ?7H?hf ? @? @? J5hg ? @? @? ?W.Yhg ? @? @? W.Y?hg ? @? @? 7H ? @? @? ?J5? ? @? @? W.Y? ? @? @? 7H ? @? @? ?J5? ? @? @? W.Y? ? @? @? 7H ? @? @? ?J5? ? @? @? W.Y? ? @? @L ?W.Y ? @? @1 ?7H? ? @? @@ J5 ? @? @@hf?W.Y ? @? @@hf?7H? ? @? @@hfJ5 ? @? @@he?W.Y ? @? @@he?7H? ? @? @@heJ5 ? @? @@he.Y ? @? @@H?gW. ? @? @@h7H ? @? @@g?J5? ? @? @@gW.Y? ? @? @@g7H ? @? @@f?J5? ? @? @@fW.Y? ? @? @@f7H ? @? @@e?J5? ? @? @@eW.Y? ? @? @@?W.Y ? @? @@?7H? ? @? @@W5 ? @? @@(Y ? @? @@H? ? @? @@ ? @? ?J@@ ? @? ?7@@ ? @? J@@@ ? @? ?W.Y@@ ? @? W.Y?@@ ? @? 7He@@ ? @? ?J5?e@@ ? @? W.Y?e@@ ? @? 7Hf@@ ? @? ?J5?f@@ ? @? W.Y?f@@ ? @? 7Hg@@L? ? @? ?J5?g@@1? ? @? W.Y?g@@@? ? @?hf?W.Yh@@@? ? @?hf?7H?h@@@? ? @?hfJ5he@@@? ? @?he?W.Yhe@@@? ? @?he?7H?he@@@? ? @?heJ5hf@@@? ? @?h?W.Yhf@@@? ? @?h?7H?hf@@@? ? @?hJ5 @@@? ? @?g?W.Y @@@? ? @?gW.Y? @@@? ? @?g7H @@@? ? @?f?J5? @@@? ? @?fW.Y? @@@? ? @?f7H @@@? ? @?e?J5? @@@? ? @?eW.Y? @@@? ? @?e7H @@@L ? @??J5? @@@1 ? @?W&U? @@@@L? ? @W.R1? @@@V)XeW-X? ? @@HJ@?e?@K?eO26Kh@@@?@)KO.R/K ? @5?7@LeC@@6T20MI46Kg@@@??I40Y?V46K ? @HJ(R/T20M?I+MfI4@6K?e@@@?hI46K?O-KeO-X? ? @W.Y?V+Mhf?I46K?@@@?heI4@0R4@@0R/T2@@@@@@@@@@@f ? @@Y? ?V@@@@@? V@Y?hg ? @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@f ? @? @? ? @? @? ? @? @? ? ?W-X ?@?J@L W-KC@L ? ?7R1 ?@?7R1 .R'@R1 ? ?@?@ ?@?@?@ N@?@ ? ?3T5 ?@?3T5 @KC@T5 ? ?V+Y ?@?V+Y @@0R+Y ? ? ? ? @@@?@Kh?W-Ke?O@?eO@f ? N@H?@@@@@6?)X??7R@@@@@@?@@@@f ? ?@e@@?@?@@@)??@?@?@?@@?@?@@f ? W-T-T& ?@e@@?@?@@Xe?@?@?@?@@?@?@@f ? 7R'?@@ ?@e@@?@?(R/e?3T5?@?@@?@?3@f ? @?V'U@ ?V+YhV'f ? @??S@@ ? 3L?.Y@ ? V/e?@ ? ? ? ?
Fig. 73. Use of ion exchange in the final purification of cellulase. (Work by Pharmacia Biotech, Uppsala, Sweden.)
Application examples Ion exchange chromatography has been used successfully to separate all classes of charged biological molecules. The following are some representative examples.
Enzymes In the purification of biologically active proteins such as enzymes the recovery of biological activity is usually as important as the recovery of protein mass or degree of homogeneity. Ion exchange chromatography has played a role in the purification of thousands of enzymes, and using modern matrices with optimized separation conditions gives extremely high recoveries. This is exemplified by the separation of enzymes from chicken breast muscle on Mono Q (Fig. 74). The recovery of creatine kinase in this separation was 89%.
127
? W. @? ?@@@g?@f@@hf?O2@?@fW& @Kg?)X?@? ? 7Ye?@?2@@@@@6X?@W)X?@hf?@?@?@?2@@?@?)X?@@f?@6T&?@?@@@@?@W@KO&@eW.?@6T-Xe@?@?@@@6?)X??@1?@?@@@?@KO@?W-Xg? @@@?J@@@?@?@@V1?@@@) ?@@@T@@@?@?@W@1?@@f?@V@@?@?@?@@?@@@@@Y@e7H?@V@@)?J@@@?@@?@@@)??@@?@?@?@W@@@@W&@)g? 3T5?*U@@?@?@@W5?@@X? ?@?I'U@@?@?@@U@?@@L?e?@W@@?@?@?@@?@@XI'X@e3L?@?3X??*U@@?@@?@@Xe?@@X@?@?@@U@?@@@X?g? V+Y?V4@@?@?3@(Y?(R/??@hf?@eV4@@?@?(R4@?@@)T.??@(R4@@?@?@@?(R/?V4@eV/?@?V/??V4@@?@@?(R/e?(R4@?@?(R4@?@MI/?g? ?V+Y I+Y??(Y? ? ? W2@?f)X )X?W&?he?W26X??@?@?@@@@??@@@e@@@? ? 7<eW-X?@1?@@@@@@@@@e@?he@1?7@W-T2@@?W-X??71?e?W.?e? ?@ @@@@@@ @[email protected]? @??J@V@@)?J@@@?@@?@@@)e@@1?@?@?@W@@@@W&@)e@V@@)?7H?7R@@?@@@@@Hf?S@UI'@?eW.Y?e? ?@ ?J@@@@@@ @??@eW20Y 3=O&@?3Xe*U@@?@@?@@X?e@V'X@?@?@@U@?@@@X?e@?3Xe3L?3T@@T@@X?@?f?*?,?S5??W.Yf? ?@ ?7@@@@@@ @??3=O.M V40R'?V/eV4@@?@@?(R/?e@?V4@?@?(R4@?@MI/?e@?V/eV/?V+MI+MI/?@?/T.??V+Y?.Y??.Y?f? ?@ ?@@@@@@@ @??V@(Y? V+Y? ? ?@ ?@@@@@@@ @W2@@H ? ?@ ?@@@@@@@L? @(M?@? ? ?@ ?@@@@@@@1? ?W2@@He@? ? ?@ ?@@@@@@@@? W.M?@?e@? ? ?@ ?@@@@@@@@? 7He@?e@? ? ?@ ?@@@@@@@@? ?J5?e@?e@? ? ?@ ?@@@@@@@@? W.Y?e@?e@? ? ?@ ?@@@@@@@@? ?O.Yf@?e@? ? ?@ ?@@@@@@@@? ?W20Y?f@?e@? ? ?@ ?@@@@@@@@? O.M?g@?e@? ? ?@ ?@@@@@@@@? W20Yh@?e@? ? ?@ ?@@@@@@@@? ?O.Mhe@?e@? ? ?@ ?@@@@@@@@? ?W20Y?he@?e@? ? ?@ ?@@@@@@@@? W.M?he?J5?e@? ? ?@ ?@@@@@@@@? ?W.Yhf?7H?e3L ? ?@ ?@@@@@@@@? O.Y?hf?@fN1 ? ?@ ?@@@@@@@@?hfW20Y ?@f?@ ? ?@ ?@@@@@@@@Lhe?W.M ?@f?@ ? ?@ ?@@@@@@@@1heW.Y? ?@f?@ ? ?@ ?@@@@@@@@@h?O.Y ?@f?3L? ? ?@ ?@@@@@@@@@g?O20Y? ?@f?N1? ? ?@ @?e?@@@@@@@@@f)T20M? ?@g@? ? ?@ @?e?@@@@@@@@@f@@
Fig. 74. Separation of creatine kinase from a partially purified preparation of chicken breast muscle on Mono Q. (Work by Pharmacia Biotech, Uppsala, Sweden.)
Isoenzymes Normally the isoforms of an enzyme have approximately the same molecular weight. This makes their separation impossible by gel filtration. However, the small differences in charge properties resulting from altered amino acid composition enable the separation of isoenzymes using ion exchange chromatography. ?? ?)X??@hfW2@6X??)X?hf?W&?)Xe?@?W-Xh@@@@?@hf@?@?g@@6Xe?W26KO2@(?h@?eW&hfW2@?he? ?@)X?@e'6X?g7@
128
N-Acetyl b-D-glucosaminidases have been widely investigated in the diagnosis of haematological malignancies. In the case of common acute lymphoblastic leukaemia, an isoenzyme, referred to as “Intermediate 1 Form” has been reported (29). Using high resolution ion exchange chromatography (Fig. 75) this previously “single” peak has been resolved into a number of component isoenzymes which had previously only been detectable using isoelectric focusing.
Fig. 75. Separation of Leukaemic cell N-Acetyl b-D-glucosaminidase isoenzymes by anion exchange chromatography on Mono Q. NA-Glu activities associated with distinct peaks (Ia-IXa) are indicated in relation to the NaCl gradients (29). (Reproduced by kind permission of the authors and publisher.)
Immunoglobulins Ion exchange is frequently used for the purification of immunoglobulins. Figure 71 shows the purification of rat monoclonal IgG2b from cell culture supernatant. As illustrated in Figure 76 the technique can also be applied to the purification of monoclonal immunoglobulin from ascites fluid. ? ?)X?W&@?@@@@?@@6?)X?e@?@W@?W.?@1?W&KO@?e@?@1?7@?W&@? O@he@@@?f?)X?h@?)X?W&??W&? ? ?W26K?he?@ ?@1?7@ ? W&@>@@6T2@@6T26KC@T&?@ ?@@?@@ @?@@?@?@?@@@)?e@@@@@W&H?@@?7@@@@Le@?@@?@@?7Y@? ? *@S@@@@@Y@?@@YS@@R@5 ?@@T@@ ? V40MI4@@?@?3@@0R'?(Y?@ ?(R+R'@?@@?@?@?@@Xe?J(?'X@@@L?@@?3Xe@,e@?@@?@@?3X@? @@@@?@?@?(R/e?.Y?V40MI/?@@?V/e(Ye@?@@@@@?V4@? ? ?N@? ? @? ? @?@@ ? ?W26K?he?@he?@ @?@?@?f?O@? ?@@@@@@? ? W&@>@@6T2@@6T26KC@T&e?@@@@6T@T-T2@@@@?W&?@?g@?@@ ?@?@?@@? ? *@S@@@@@Y@?@@YS@@R@5e?@@@XV@R'>@@?@?@?75?h@?e@??@?@?@@? ? V40MI4@@?@?3@@0R'?(Ye?@MI4@@?V4@@?@?@?(Y?@?g@??@ ?@?@?@@? ? ?N@? ? @? ? ?W26X??W.?h@K @?@?@?eO@K?e?@@@fO@ ? ?W26X?e?)X? ?7,?@?@?3T5? 31f@?@)X?@?@?@?@? ? *UO&@XS@@@>@@?@?@@Y@ ?J@@X@ ? V40MI40R+R4@@?@?@@?@e@? ?.MI4@ ?V/?e@?V+Y?@?@?V+Y? ? ? ? ?W-Xe@@@@@@?@@@@@@@@@@? O@?@K?eO@K? ? ?W2(?@K?hf?@ ?.R/e ? ?7(YJ@@@6?)T.?W2@@@??@W@e@? @?@?@@?@@?@?@@@?@? ? J(Y?7@XW@@@@H?7R'@@??@@5 ?@K?e@?@?@@?@@?@?@@@?@? ? .Ye(R40R+?@e@?V4@??@0Ye@? ?@@@e@?@?@@?@@?@?@@@?@? ? ? ? ?@@6T.@??@@@@@@@@@@??N@He@?e@?@?e@6X@?@e?*?,e7R1? ?@@@e@@@?@@@?f?@?@e?W-XeW-X? ? ?W2@?)X?h@?eW-X? ?@X;@H ? ?7@@X@)T.?W-T2@?@?e7>,?@? ?@)X@? @??@?@?@@?@?@?e@?e@@@?@@@?e@V@@@@e?S@Ue@?@? ? ?@?V@@>@H?7@@Y@?@??J@@U? S@@L @??@?@?@@?@?@?e@?e@??:(?'Le@W@@?@e?*?,?J@T5? ? ?@@@0R4@e@?@?@?@??.MI/?@? .MI/ ?@?@?@@?@?@?e@?e@@@0Y?N1e@(R'?@e?V+Y?.R+Y? ? ?@e(Y ? ? @@@? W-X??W-T2@@??)X?W&e@@@?fW2@?@? ? ?W2@?)X?h@?eW2(? @?@? 7R1??*?@@Xe?@1?7@e@@@??)X?7<e@? ? ?7@@X@)T.?W-T2@?@?e7@U?@? @@@??*@,e3T@LeS(MS,??@@T@@e@@@?*U@?3=O&@? ?W-Xe@?@??V'>@)X??@@?@@e@@@?J@1?@??J@? ? ?@?V@@>@H?7@@Y@?@??J@S,? ?J(?'L ? ?@@@0R4@e@?@?@?@??@@0Y?@? ?.Y?V/ ?V+YeV+R/e.Y?.Y??(R+R'e@@@?V4@?V40R'? ? ? ? ?W-Xe ?@6X W-X?f?O@? ? ?W26X?gO2@?g@? ?7R1e ?W.??@S,e?W-T&?@?)X?@@?.R/??@@@@@@?@@6?@? ? W.R')T2@@6T2@@5?W-T2@?@?@? ?@[email protected]?@V1e?3T@@T@@X??@e@Ke?@?@?@@?@?@?@, W.Y??@@Ue?7R@@?@@@)?@H?f?@?@?@@?@?@@@L ? *U?V@@R'@@@U?@H?7@@Y@?@? ?3T5e ? V4@@@@?V40R4@@e@?@?@?@?@? ?V+Y?@ ?@@@e?V+MI+MI/??@e@@@??@?@?@@?@?@?(Y ? ? ? ? ?W-Xe?@e7R1?eW.e@S,?eW-T&?@?@?2@?e@?J@e?@@@@@@?@@6?@? ?@eW-X?g@6X? @??@g?O@? ? ?7R1e ? ?@?@L??@e3T5?W.Y?e@V1?e3T@@T@@X?@f@?3@,??@?@?@@?@?@?@, ?@e@?@??W.Ye@@U?e7R@@?@@@@@H?e@?7@L??@?@?@@?@?@@@L ? ?3T@@? ? ?V+Me ?@eV+Y?.Yf@@@?eV+MI+MI/?@f@?V+Y??@?@?@@?@?@?(Y ? ? ? @?W.@?7R1?eW.e@S,?eW-T&?@?@?2@??J@?e@@@@@@@@@?W& W-X?g@6X? @?gO@K? ? @?7H ? ?W-X W2@6X?f@?hfW& ?@?W2@hf?W-Xg@@6X?/X?he@?@? @?@?@??W.Ye@@U?e7R@@?@@@@@H??7@Le@?@?@@@?@?&@L? ? ?7R1 7U?I/T26X?@?/X?W2@6T2@@?*@e?W26KO26T2@@@@e@@6KO26X?W.??@?&@@?W26T2@@f?7>)X?f@?B1?N1?he@?3L@?3T5?W.Y?e@V1?e3T@@T@@X?@e?3@,e@?@?@@@?@?N@,? ? J@?@ @)eV@)X?@@@Ue@?hg V+Y?.Yf@@@?eV+MI+MI/?@e?V+Ye@?@?@@@?@??(Y? ? @?e@V4@?,?7R@@@@@@@@@ /K?S@@=C5?@??3T@@Xe@?@??@e?3=O&@=C@@?@?@e@?C@@=C5eS,?@e@@?3=C@@?@f?@eJ@@?,?@?V1e@?hg ? @?e@??S@U?@?@@?@@?@?@ V4@0MI40Y?@??V+MI4@?@?@??@e?V40MI40R'?@?@e@@0MI40Ye.Y?@e@@?V40R'?@[email protected]+Y?@@@@e@?hg ? @?O&?,?3T@@?@@?@?@ @? ?3L?he?J5?hg ? @@0R+Y?V+R'?@@?@?@ @? ?V/?he?.Y?hg ? ? ? ?W&?W26X?W26X?hg ? ?*@?7@
Fig. 76. Ion exchange purification of mouse monoclonal IgG1 from ascites fluid. (Reproduced by kind permission of Dr. LeRoy Baker, Eli Research Laboratories, Eli Lilly and Company, Indianapolis, USA.)
Nucleic acid separation Nucleic acids, being charged molecules, can also be fractionated and purified using ion exchange chromatography. A recent application of the technique in this area is the purification of plasmids from bacterial cultures. This process is traditionally done by centrifugation using CsCl gradients. Figure 77 shows the separation of plasmid HB101 (pBR322) by anion exchange chromatography on Q Sepharose High Performance. Subsequent analysis showed the electrophoretic purity of the plasmid to be equivalent to that obtained by centrifugation, as was its behaviour in ligation and transformation assays. The time, however, required for the preparation was 1 hour using the chromatographic method and approximately 8 hours using centrifugation.
129
? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?@ @K @K ?W&? @Khf?W&?h@Khe?W&?g@?f@?g?@?)X?f?@?@hf)XhfW&g? ?@W26X?@@@e?W26X??@?)T26X@@@??'6X?@@@?W2@@?W26XeW26X@@@??@6T26X?@@@@@??'6X?*@W26X?W2@@@@e'6?2@6X?@@@?/X?W.?W26X?*@?e'6X?e@@6X?'6X?W26T&@W26T2@?@??'6X@?e?'6X?@?@1?e'6X@?@?@@@?W26X?e@)X?W2(?e'6X?*@W26Xe? ?@@@)X?@?@?e?@@@?@?@?@?@e?@?@?@?@@@ ? S@@T5?g?@?3T5?3T5e?@?@eS,?3T5f?@KC@T5?3T5?f@KeS,?3T5gS@@T5?3T5?fS(MS,?3T5?e?@@@@@T5?3T5e?@?@?@?@@@ ? .MI+Y?g?@?V+Y?V+Ye?@[email protected]?V+Yf?@@0R+Y?V+Y?f@@@?.Y?V+Yg.MI+Y?V+Y?f.Y?.Y?V+Y?e?@V@MI+Y?V+Ye?3T5?@?@@@ ? ?@ ?@hf?V+YfI@ ? ?@ ?@ ?@ ? ?@ ?@ ?@ ? ?@ ?@ ?@ ? ?@ ?@ ?@ ? ? ?W-T2@@? ?@@6T-X? ?@@@@@@?e?W-Ke?O@K @? ? ?.R'@X ?@X;@R1? I'@Xf?7R@@@@@@@@@?@?@@?W&KO@?@? ? ?V4)X? ?@)X@?@? ?V4)X?e?@?@?@?@@@?@?@?@@?7@@@@?@? ? ?@K??S,? S@@T5? @??S,?e?@?@?@?@@@?@?@?@@?3Xe@@@? ? ?@@@?.Y? .MI+Y? @??.Y?e?3T5?@?@@@?@?@@@@?V/e@Y@? ? ?V+Y @? ? ? ? ? ?@?@?@?@heW&e?@?W.??@?@?W-XeW26Xe?W.?g?@K? ?@?@e@??O@Kf@@@?fO@ W26Kf)X ?@?@?@?@eW-X?)X?W&@e?@?7U??@?@?7R1e7@@?@?@@Y@?he?@?@?@?@?C@T@@U@?3X@e?@?3>,?@?@?3T5e3T&@e?3T5?3X?@W@@?@?*U@@?3T(Y@@@Xe?@?@e@@X@@?@?e@?I'X??@?@@T5?@?@?@?@@U@@?@@L?3X ?V40MI40R+R4@@?@?@@?@??@h?@?@?@?@@0R+MI4@?V4@e?@?V+Y?@?@?V+YeV4@@e?V+Y?V/?@(R'?@?V4@@?V+Y?@MI/e?@?@e(R@@5?@?e@??V/??@?(R+Y?@?@?@?(R4@@?(R/?V/ (Y @0Y? ?W-XeW-X?f?O@?f@?hf?@?)X?e?@@? ?@e@?@??@6Xe?@eW-X?@?eW-K??@6X?@@@@?W-KO-KO-X?@? W26Khf@?hf?.R/e7R1??@@@@@@??W-X@?e?@?2@@e?@X@?@1??@X@@?@@6?)XeW-X?@@@?@@?@KO.?@?eW-X@e@?@??@S,e?@e7R1?@?e7R@6X@S,?@?W5?*?@0R40R/?@? ?W&@>@@6T2@@6T26KC@T&?@? @?@??@?@?@@??7R@@?eJ@@@?@eJ@@@?@@?J@@@@?@?@@@)e7@)?@@@?@HJ@@@H?@?e7R@@e@@@??@@Ue?@e@?@?@?e@?@V@@@U?@@@U?V'U?g@? ?*@S@@@@@Y@?@@YS@@R@5?he?@K?e3T5??@?@?@@??3T@@?e*U@@?@e*U@@?@@T&U@@@?@?@@X?e3Xe@@@?@?*U@@L?@?e3T@@e@?@??@V1e?@e3T5?@?e@?@W@@V1?@?B1??S@6KO@Ke@? ?V40MI4@@?@?3@@0R'?(Y?@?h?@@@@?V+Y??@?@?@@??V+R'?eV4@@?@eV4@@?(R+R4@@@?@?(R/?eV/e@@@?@?V40R/?@?eV+R'e@?@??@@@e?@eV+Y?@?e3X@(R4@@?@e@??.R4@@@@@?@? N@ V40Y @? ?@ ?W26K? @??W-KO-X?f?O@?hf?@@@?@f?W&??)X?hfO@f@?gW& ?W.?W-T2@@6T-X?W&?@6KO-T2@@?W&@@@@@?W2@??)T-X??J@??7R@@R1??@@@@@@?e@?2@@@@@6X?@@@?@W)X?W&@??@)X?@KO.?@?W&?2@@e@X@??W.?@?7@?@@?@?@6T-X? ?7H?7R@@?@V@@)?*@?@V@@R@@?@?7Y@@@?@?7Y@??@@R1??7@L?@?@@?@??@?@?@@??J@@@?@?@@V1?@@@?@@@)?7Y@??@V)T@@@H?@?7@@@?@?J@@@??7H?@?@@?@@?@?@V@@)? ?3L?3T@@?@?3X??N@@@W@@T@@?@?3X@@@?@?3X@??@@T5??3@,?3T@@T5??@?@?@@??*U@@?@?@@W5?@@@?@@Xe3X@??@W@@U@@L?@?3X?@?@?*U@@??3L?@?@@?@@?@?@?3X ?V/?V+R'?@?V/?e@Y@(MI+R'?@?V4@@@?@?N@@??(R+Y??V+Y?V+MI+Y??@?@?@@??V4@@?@?3@(Y?@@@?(R/eV4@??@0MI40R/?@?V/?@?@?V4@@??V/?@@@@?@@@@?@?V/ (Y ?@@? V+Y? @?W-X?f?O)X?W&??@@6KO@?f@?@??W2@?@f?@g?O)X?W&??@@@@@6X?@@@?@@@e?W-Xe@@@??)X?W&e@@@?fW2@?@?g@?@?eW-X??W-X W2(?eW&?@g?W-Xhf@?7R1??@@@@@@1?7@??N@V@@@W@?e@?@??7,?@he@?@?@??@?@?@@@?@@?e@?@?@@@?e@@@??@eJ@f?@e?@?@?@@@?@@??@@@@??@e@??@@@e?@?@g?@@?@@e@@@?J@1?@??J@?e?@V@@@@?eS@U??@?@ ?J@S@@>@>@T5?7@@@>@T5?7@@@@R1??W.?@@@@@?@@@@e7Y@Xe?@@@@@@?@@@@@@@@@@?@ 7(YJ@@@6?)T.?W2@@@e@W@??@f?V'@@?@??7H?@?@?@?@@?@e@?@)X??@?@?@@?@@?@?@@@?@?@ ?J(Y?7@XW@@@@H?7R'@@e@@5?hS@@T5??3L?@?@?@?@@?@e@??S,??@?@?@@?@@?@?@@@?@?@ ?.Y??(R40R+?@??@?V4@e@[email protected]+Y??V/?@?@?@?@@?@e3L?.Y??@?@?@@?@@?@?@@@?@?@ V/ ?@
1 2 3 4 5
Lane 1: Crude alkaline lysate of HB 101 (pBR322) Lane 2: Fraction 1-20 (hydrolyzed RNA) Lane 3: OC-peak (chromosomal DNA) Lane 4: CCC-peak (supercoiled DNA) Lane 5: pBR 322 purified by CsCl gradients Electrophoresis in horizontal 0.8 % agarose gels (GNA-100). DNA was vizualized by ethidiumbromide staining and illumination with UV light.
Fig. 77. Ion exchange purification of plasmid DNA. (Work by Pharmacia Biotech, Uppsala, Sweden.)
Polypeptides and polynucleotides Ion exchange chromatography is not limited in its application to macromolecules such as proteins and nucleic acids. The technique can be used in the separation ofpeptides as illustrated by the separation of cyanogen bromide fragments of collagen (Fig. 78). W-X? 7R1? @?@? ?J@@@Le?W&?W&K? ?.M?I/T.?*@?7@@@?@@@@@ ?N@U?N@?3@@@?@?@?@ @)e@?V4@@?@?@?@
?W.?e@@@??W.?g?@K? ?@?@e@??O@Kf@@@?fO@g@@@@@?@?2@6T.?W-X??N@@Y@@Uf@?7Ue?@@??7R1e ?/KO26T.f@?W.e?@@??W-Xe ?W26K?e?)X? ?7Y?e@?@??7Y??W&?26X@@@e@?26T-KO@?W-X??@?@e@W2@@@@?e@?@W)X?@@@W-X?@@ W.MS@@6K?@)T2@@@6T2@e@?h?@@@e@@@??@@@?7@@@V@@?@?J@@@V@R@@@W&@)??@@@e@@Y@@?@?e@@@@@)?@?@@R1?@H @?@?@W@@@?@@H?7@)?e@@?@@1f@?@)X??@@??@?@e *UO&@XS@@@>@@?@?@@Y@hf?3T5e@?e?3T5?3X?@W@@?@?*U@@?3T(Y@@@Xe?@?@e@@X@@?@?e@?I'X??@?@@T5?@? @?@?@@U@@?@@L?3Xe?J@@X@V'L?e@?3>,??@@??3T5e V40MI40R+R4@@?@?@@?@e@?h?V+Ye@?e?V+Y?V/?@(R'?@?V4@@?V+Y?@MI/e?@?@e(R@@5?@?e@??V/??@?(R+Y?@? @?@?(R4@@?(R/?V/e?.MI4@?V/?e@?V+Y??@@??V+Ye (Y @0Y? ?W-KO. W2@?h?)X?gW&e@KfW-X?f?O@?f@? )Xe?W&?@?g?@@@ ?*?@@Ue?@@@@6T2@?e?W&@@?W&?W&?W-X@1?@?)X?W&@e@@@@e.R/??@@@@@@??W-X@? ?W26K?he?@ ?V'U@)X??@?@?@@Y@?e?7Y@@?&@W&@W&R@@@?@@@)?7Y@e@@?@g?@?@?@@??7R@@? @)T&?7@?@?W&?2@??@?@ @V@@?@@?@?7@@@H??@@@ W&@>@@6T2@@6T26KC@T&?@hfS@@>,??@?@?@@X@?e?3X@@?N@(Y@@@T@@@T@@X??3X@e@@?@e@Ke?@?@?@@??3T@@? @W@@?@@?@?3X?@eJ(?'L? *@S@@@@@Y@?@@YS@@R@5 .MI+Y??@?@?(R@@?@??V4@@??(Y?@MI+R+R+MI/??V4@e@@?@e@@@??@?@?@@??V+R'? @0R4@@@?@?V/[email protected]?V/? V40MI4@@?@?3@@0R'?(Y?@ ?@@?@? ?N@? @? ?W-KO-X?f?O)X?W&??@?@?W2@eW26KO26X?@?@f?W-Xe)X?W&?hg ?@?@e?W-XeW-X? ?W2(e?W&?@?gW-X?he?.R'@R1??@@@@@@1?7@??@?@?7,?@?he?N@?@??@?@?@@@?@@??@@@?@f@??@@??@?@@@he@@?@@?@?@?@V@@)T@1e @6X@?@e?*?,e*?,? @V@@@@e?V'UeS@U? J@S@@>@>@T5?7@@@>@T5?7@@<eJ@S,he?J(?'T&@,?e@?e@@T@@??@@@?*U@@=O.?@ @@0MI4@0R+Y?@?@?e@@0Y?@h?.Y?V+R+Y?e@?e(R+R'??@@@?V40R40Y?@
W-X?eW-X? 7R1?e*?,? @?@?eS@U?f?O@? 3T5?e*?,?e@@@@@? V+Y?@?V+Y?g@? @?e?@ @?e?@ W26X?@@6X?W-X? @?e?@ 7,?e?@g@1 @?e@?@? ?@ V40Y?@@@@?V+Y?e?@f?J@@L? @?e@?3L J@ ?7,?e@@@@@?e@??@ @?@? @?@?he@?e@? ?@ V+Y?@?V+Y?g@?e@??@ @?@? @?@?he@?e3L ?@ @?e@??@ @?@? @?@?h?J5?eN1 ?@ @?e@??@ @?@? @?@?h?7H?e?@ ?@ @?e@??@ @?@? ?J5?@?h?@f?@ ?@ @?e@??@ @?@? ?7H?@?hJ5f?@ ?@ @?e@??3L? @?@? ?@e@?h7Hf?@ ?@ @?e@??N1? @?@? ?@e@?h@?f?@ ?@ @?e@?e@? @?@? ?@e@?h@?f?@ ?@ @?e@?e@? @?@? ?@e@?h@?f?@ ?@ @?e@?e@? @?@? ?@e@?h@?f?@ ?@ @?e@?e@? @?@? ?@e@?h@?f?@ ?@ @?e@?e@? ?J5?@? ?@e@?h@?f?3L?g?W-Xe?@ ?@ @?e@?e@? ?7H?@? J5e3Lh@?f?N1?g?7R/X?J@ ?@f?@eW-X?W-X? @?e@?e@? ?@e@? 7HeN1h@?g@?gJ5?N1?7@ ?@f?@e7R1?7R1? @?e@?e@? ?@e@? @?e?@h@?g@?g7He3X@@ ?@f?@e@?@?@?@? @?e@?e@? ?@e@? @?e?@h@?g@?g@?eN@@@L? ?@@@@@@@@@@@@@@@@??@e3T5?3T5? @?e@?e@? ?@e@? @?e?@h@?g@?f?J5?e?@?B1? ?@he?@f?@eV+Y?V+Y? @?e@?e@? ?@e@? @?e?@h@?g@?f?7H?e?@e@? ?@he?@ @?e@?e@? ?@e@? @?e?@h@?g3=O@e?@h@? ?@he?@ @?e@?e@? ?@e@? @?e?@h@?gN@@@e?@h@? ?@he?@ @?e@?e@? ?@e@? @?e?@h@?g?@@@L?J5h@? ?@he?@ @?e@?e@? ?@e@? ?J5?e?@h@?g?@MB1?7Hh@? ?@he?@ @?e@?e@? ?@e@? ?7H?e?@h@?he@?@?h@? ?@he?@ @?e@?e@? ?@e3L ?@f?@h@?he@?@?h3L ?@he?@ @?e@?e@? ?@eN1 ?@f?@h@?he3X@?hN1 ?@he?@ @?e@?e@? ?@e?@ ?@f?@h@?heN@5?h?@ ?@he?@ @?e@?e@? ?@e?@ ?@f?@h@?he?@H?h?@ ?@he?@ @?e@?e@? ?@e?@ ?@f?@h@?he?@he?@ ?@he?@ @?e@?e@? ?@e?@ ?@f?3L?g@? ?@@@ ?@he?@ @?e@?e3L ?@e?@ ?@f?N1?g@? I'L? ?@he?@ @?e@?eN1 ?@e?@ ?@g@?g@? ?N1? ?@he?@ @?e@?e?@ ?@e?@ J5g@?g@? @? ?@he?@ @?e@?e?@ ?@e?@ 7Hg@?g@? @? ?@he?@ @?e@?e?@ ?@e?@ ?@e@?g@?g@? 3= ?@he?@ W-X?e?@h@?e@?e?@ ?@e?@ J@e@?g@?g@? N@@@ ?@he?@ 7R1?eJ@h@?e@?e?@ ?@e?@ 7@L?@?g@?g@? ?@Y@ ?@he?@ @?@?e7@L?e@@@@@?e@?e?@ J5e?@ @V1?@?g@?f?J5? ?3L? ?@he?@ 3T5?e3@,?g@?e@?e?@ 7He?@ @?@?@?g@?f?7H? ?N1? ?@he?@ V+Y?@?V+Y?g@?e@?e?@ @?e?@ ?J5?@?@?g@?f?@ @? ?@he?@ @?e@?e?@ @?e?@ ?7H?@?@?g@?f?@ @? ?@he?@ @?e@?e?@ @?e?@ ?@e@W5?g@?f?@ @? ?@he?@ @?e@?e?@ @?e?@ ?@e3@H?g@?f?@ 3L ?@he?@ @?e@?e?@ @?e?@ ?@eN@h@?f?@ N1 ?@he?@ @?e@?e?@ @?e?@ ?@e?@h@?f?@ ?@ ?@he?@ @?e@?e?@ @?e?@ ?@e?@h3Lf?@ ?@ ?@he?@ @?e@?e?@ @?e?@ ?@e?@hN1f?@ ?@ ?@he?@ @?e@?e?@ @?e?@ ?@hf?@f?@ ?@ ?@he?@ @?e@?e?@ @?e?@ ?@hf?@f?@ ?3L? ?@he?@ @?e@?e?@ @?e?@ ?@hf?@f?@ ?N1? ?@he?@ @?e@?e?@ @?e?@ ?@hf?@f?@ @? ?@he?@ @?e@?e?@ @?e?@ ?@hf?@f?@ @? ?@he?@ @?e@?e?@ @?e?@ ?@hf?@f?@ @? ?@he?@ @?e@?e?@ @?e?@ ?@hf?3L?e?@ 3L ?@he?@ @?e@?e?@ @?e?@ ?@hf?V/Xe?@ N1 ?@he?@ @?e@?e?3L? @?e?@ J5 N1e?@ ?@ ?@he?@ @?e@?e?N1? @?e?@ 7H ?@e?@ ?@ ?@he?@ @?e@?f@? @?e?@ @? ?@e?@ ?@ ?@he?@ @?e@?f@? @?e?3L? @? ?@e?@ ?3L? ?@he?@ @?e@?f@? @?e?N1? @? ?@eJ5 ?N1? ?@he?@ @?e@?f@? @?f@? @? ?@e7H @? ?@he?@ @?e@?f@? @? @?f@? @? ?@e@? @? ?@he?@ @?e@?f@? @? @?f@? @? ?3L?@? @? ?@he?@ @?e@?f@? @?hf?J5?f@? @? ?N1?@? @? ?@he?@ @?e@?f@? ?J@?hf?7H?f@?hf?)X?e@? 3T5? 3L ?@he?@ @?e@?f@? ?7@?hf?@g@?hf?@)Xe@? N@H? N)?@ ?@he?@ @?e@?f@? ?@@?hf?@g@?hf?@V1?J@? ?@ ?@@@L? ?@he?@ @?e@?f@? ?@@Lhf?@g@?hfJ5?3T&@? ?@MB1? ?@he?@ @?e@?f@? ?@V1hf?@g@?hf7H?V+M @? ?@he?@ @?e@?f@? ?@?@hf?@g@?hf@? @? ?@he?@f@@@?W-X? @?e@?f@? ?@?@hf?@g@?hf@? 3L ?@he?@f@Xe7R1? @?e@?f@? ?@?@hf?@g@?hf@? N1 ?@he?@f@)X?@?@? ?@f?@h@?e@?f@? ?@?@hf?@g@?hf@? ?@ ?@he?@@@@??I/?@?@? @?@?e@?@?g@?e@?f@? @??@hf?@g@?hf@? N@ ?@he?@f?@e?@ @?@?hf@?e@?f@? @??@hf?@g@?hf@? ?3L? ?@he?@ 3T5?e@Kf@@@@@?e@?f@? @??@hf?@g@?he?J5? ?N1? ?@he?@ V+Y?@?@@@?g@?e@?f@? @??@hf?@g@?he?7H? @? ?@he?@ @?e@?f3L @??@hf?@g3Lhe?@ @? ?@he?@ @?e@?fN1 @??@hfJ5gN1e?W&?f?@ 3L ?@he?@ @?e@?f?@ @??@hf7Hg?@e?7@Lf?@ N)T2@@ ?@he?@ @?e@?f?@ @??@hf@?g?3=?C(R1fJ5 ?@0MI'L? ?@he?@ @?e@?f?@ @??@hf@?g?N@@0Y?@f7H ?N1? ?@he?@ @?e@?f?@ @??3L?h?J5?h@Me?3L??J5? @?he?O2@@@he?@ @?e@?f?@ @??N1?h?7H?hf?N1??7H? @?f?O2@@@@0M?hf?@ @?e@?f?@ @?e@?h?@ 3=C5 @@@@@@@0M? ?@ @?e@?f?@ @?e@?h?@ N@@H O2@@@@@@ ?@ @?e@?f?@ ?J5?e@?h?@ ?@5? O2@@@@0MfI'L? ?@ @?e@?f?@ ?7H?e@?h?@ ?(Y? ?O2@@@@@0Mhe?N1? ?@ @?e@?f?@ ?@f@?hJ5 ?O2@@@@0M? 3L ?@ @?e@?f?@ ?@f@?h7H O2@@@@@0M? N1 ?@ @?e@?f?3L? ?@f@?h@? O2@@@@0M ?3L? ?@ @?e@?f?N1? ?@f@?h@? ?O2@@@@@0M ?V/K ?@ @?e@?g@? ?@f@?f?@@@@? ?O2@@@@0M? V46X ?@ @?e@?g@? ?@f@?f?@ O2@@@@@0M? B1 ?@ @?e@?g@? ?@f@?fC5 O2@@@@0M ?3L? ?@ @?e@?g3L ?@f3T2@@@@H O2@@@@0M ?N1? ?@ @?e@?gN1 ?@fN@(M?I@? ?O2@@@@@0M 3L ?@ @?e@?g?@ ?@f?(Y? ?O2@@@@0M? N)K? ?@ @?e@?g?@@6X?he?@ O2@@@@@0M? ?@@@@@@?hf?@ @?e@?h?I/Xhe?@ O2@@@@0M ?@ @?e@?heV/X?fW-K??@he?O2@@@@@0M ?@ @?e@?he?V/[email protected]@f?O2@@@@0M? ?@ @?e@?hfV+M?B@Y??V@@@@@@@0M? ?@ @?e@? C@@@@@0M?W@X ?@@6X? W26Xe?@@6X?f@@@@he?@@@h?W2@6X ?@ @?e@?heO2@@@@0Mg?7R1 ?@?S,? 7,?3T5?3T5 *?,?3T5?3T5? ?@@@?V+Y?V+Y ?V+Y?V+Y?V+Y ?V+Y?V+Y?V+Y V+Y?V+Y?V+Y? @@@@6Xe?W2@ )X ?W-Kf?@?/X? @?e@1?@?*@@?W-X?@@@e?@?)T-X?@1?@?@@@@@?W-Xe?7R@@@@@?@?N1? @@@@@@?@?N@@?7R1?@?@e?@?@@R1?@@?@?@?@?@?7@)e?@?@?@?@?@e@? @?e@@?@e@@?3T5?@?@e?3T@@T5?@@?@?@?@?@?3X?e?@?@?@?@?@e@? @@@@@@@@e@@?V+Y?@?@e?V+MI+Y?@@@@?@?@?@?V/?e?3T5?@?@?@?J5? ?V+Yg?.Y?
Fig. 78. Separation of CNBr-peptides from a-1 chains of collagen type 1. (Work by Pharmacia Biotech, Uppsala, Sweden.)
130
In peptide mapping applications ion exchange chromatography can be used advantageously as a complement to reverse phase chromatography since both offer high resolution but separate according to different parameters (30). W-X? 7R1? ?W-X ?J@?@? ?*?)T-X? ?7@@@@6T2@@??@h?V+R@>)X ?@e?@V'@XeJ@?@@@@@@@@??J@@?, ?@e?@?V4)X?7@?@?@@?@?@??.MI+Y ?@e?S,?3@@@?@@?@?@? ?@@@?.Y?V+?@?@@?@?@?
?)X?W&hfW26Xe@?@?@@@@e@@@??@@@ ?W&?W26X?W26X? ?W26K?e?)X? ?@1?7@W-T2@@?W-Xe7@@?@?@@Y@he?@@T@@@T@@?@?3T5e3T&@e@?@?@?B1e?S,??@S, ?@ @?@??@?@e@??@@@@@ V40MI40R+R4@@?@?@@?@e@?g?(R+R+R+R'?@?V+YeV4@@e@?@?@??@e?.Y??@0Y ?@ @?3=C5?3=C5?f?@ ?@ @?V40Y?V40Y?f?@ ?@ ?@ ?@@6KO@?h?@?@eW2@?@??W-KO-X?f?O)X?W&?g@?@?eW-X??W-X ?@ ?@ ?W2(e?W&?@?gW-X?h?N@V@@@W@?g?@?@e7<e@??.R'@R1??@@@@@@1?7@?e?@6X@?@?e*?,??.R/ ?@ ?@ ?7@UO-T&@?@?W-T2@?7>,?@?h@?@?@@@LeW-X??@@@e@??J@?e?N@?@??@?@?@@@?@@?e?@V@@@@?eS@U? ?@ ?@ J@S@@>@>@T5?7@@1??@1?7@e@@@??@?&@>@T5?7@@<eJ@S,h?J(?'T&@,??3T@L??S5??@@T@@e@@@?*U@@=O.?@? ?@e?@@? ?@ @@0MI4@0R+Y?@?@?e@@0Y?@g?.Y?V+R+Y??V+R/??.Y??(R+R'e@@@?V40R40Y?@? ?@e?@@? ?@ ?@e?@@? ?@ ?@e?@@? ?@ ?@e?@@? ?@ ?@e?@@? ?@ ?@e?@@? ?@ ?@e?@@? ?@ ?@e?@@? ?@ ?@L??@@? ?@ ?@1??@@? ?@ ?@@??@@? ?@ ?@@??@@? ?@ ?@@??@@L ?@ ?@ ?@@??@V1 ?@ ?@ ?@@??@?@ ?@ ?@ ?@@??@?@ ?@ ?@ ?@@??@?@ ?@ W26X?W&?e@6X?e@Kg ?@ ?@@??@?@ ?@ .MB1?*@?e?S,?e@@6T2@6X ?@ ?@@??@?@ ?@ ?@?N@?e?*U?e@?B@@?B1 ?@ ?@@??@?@ ?@ [email protected]@?e?N1?e@??@@??@ ?@ @?he?@@??@?@ ?@ ?@e7Yf@??/KC5?e@?C@@?C5 ?@ @?he?@@??@?@ ?@ ?@e@@@@e@??V40Y?e@@0Y@@0Y ?@ @?he?@@??@?@ ?@ ?@ @?e ?@L?hf@?he?@@?J5?@ ?@ ?@ @?e ?@1?hf@?he?@@?7H?@ ?@ ?@ ?@@?hf@?heJ@@?@??@ ?@ ?@ ?@@?hf@?he7Y@?@??@ ?@ ?@hf@?hf ?@@?hf@@@?h@?@?@??@ ?@ ?@hf@?hf ?@@?hf@@@?h@?@?@??@ ?@ ?@g@?f@?hf ?@@?hf@@@?h@?3X@??@ ?@ ?@g@?f@?hf ?@@?he?J@@@?h@?N@@??@ ?@ ?@L?f@?f@?hf ?@@?he?7Y@@?h@??@@??3L? ?@ ?@1?f@?f@?hf ?@@?he?@?@@?h@??@@??N1? ?@ @? ?@@?f@?f@?hf ?@@?he?@?3@?h@??@@?e@? ?@ @? ?@@?f@Lf@?hf ?@@?he?@?V'Lh@??@@?e@? ?@ @? ?@@?f@1f@?hf ?@@?he?@eN1h@??@@?e@? ?@ @? ?@@?f@@f@?hf ?@@?he?@e?@h@??@@?e@? ?@ @? ?@@?f@@f@?hf ?@@?he?@e?@h@??@@?e@? ?@ @? @? ?@@?f@@e?J@?hf ?@@?he?@e?@h@??@@?e@? ?@ @? @? ?@@?f@@e?7@Lhf ?@@?he?@e?@h@??@@?e@? ?@ @? @? ?@@?f@@e?@V1hf ?@@?he?@e?@h@??@@?e@? ?@ @? @? ?@@?f@@e?@?@hf ?@@?he?@e?@h@??@@?e@? ?@ @? @? J@@?f@@e?@?@hf ?@@?he?@e?@h@??@@?e@? ?@ @L @? 7Y@?f@@e?@?@hf ?@@Lhe?@e?@h@??@@?e@? ?@ @1 @? @?@?f@@e?@?@hf ?@V1he?@e?@h@??@@?e@? ?@ @@ @L @?@?f@@L??@?@hf ?@?@he?@e?@h@??@@?e@? ?@ @@ @1 @?@?f@V1??@?@hf ?@?@he?@e?@h@??@@?e@? ?@ @@ @@ @?@?f@?@??@?@hf ?@?@he?@e?@h@??@@?e@? ?@ @@ @@ @?@?f@?@??@?@hf ?@?@he?@e?@h@??@@?e@? ?@ @@ @@ @?@?f@?@??@?@e@?h ?@?@he?@e?@h@??@@?e3L ?@ @@ @@ @?@?f@?@??@?@e@?h ?@?@he?@e?3L?g@??3@?eN1 ?@ @@ @@ @?@?f@?@??@?@e@?h ?@?@heJ5e?N1?g@??N@?e?@ ?@ @@ @@ @?@?f@?@??@?@e@?h ?@?@he7Hf@?g@?e@?e?@ ?@ @@ @@ @?@?f@?@??@?@e@?h ?@?@he@?f@?g@?e@?e?@ ?@ @@ @@ @?3Lf@?@??@?@e@?h ?@?@he@?f@?g@?e@?e?@ ?@ @@ @@ @?N1f@?@??@?@e@Lh ?@?@he@?f@?g@?e@?e?@ W2@6T26Xg?@ @@ @@ @??@f@?3L?@?@e@1h ?@?@he@?f@?g@?e@?e?@ 7@@>@@
Fig. 79. Ion exchange separation of DNA restriction fragments form pBR322 cleaved by HaeIII (31). (Reproduced by kind permission of the authors and publishers.)
Analogously, ion exchange can be used to separate oligonucleotides such as restriction fragments of DNA (31) (Fig. 79) and even individual nucleotides (32, 33, 34) (Fig. 80). ?@@@ ?@?@ ?@@@ J(?'L? .Y?V/?@6T& ?:(R'@?@@@@@ @0Y?V'?@@@@@ ?W-Xe?@ ?7R1e?@ ?@?@e?@fO@ ?3T@L??@e?@@@ ?V+R/??@f?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ @? ?@ ?J@? ?W.? ?@ ?7@L ?7H? ?@ ?3@, J5 ?@ ?V+Y ?W.Y ?@ ?7H? ?@ J5 ?@ ?@ ?W.Y ?@ ?@ ?7H? ?@ ?@ J5 ?@ ?@ ?W.Y @?g?W2@?)X?W&?@@@ ?@ ?@ ?7H? @?g?7,?@fJ5?@h?@g@? ?@ ?W-Xh?@@?f?V+Yf?W.Y?@h?@g@? ?@ ?*?,h?@@?hf?7H??@h?@g@? ?W.?h@?@@@6X??@@@ ?@ ?V'Uh?@@?hfJ5e?@h?@g@? ?7U?h@?@@?B1??@?@ ?@ S,h?@@?he)T.Ye?@h?@g@? ?@)Xh@?@@e@??@@@ ?@ .Yh?@@?e@@@?f@@H?e?@h?@g@? ?3>)X?g@?@@?C5??@ ?@ ?@@?e@Xe?@e@5f?@h?@L?f@? ?V+R/?g@@@@@0Y??@ ?@ ?@@?e@)X??@?J@Hf?@eW-X?e?@1?f@? ?@ ?@@?e?S,??@?7@?f?@e*>1?e?@@?f@? ?@ ?@h?@@?e?.Y??@W@@?f?@eV'@?e?@@?f@? ?@@@h@@@@@6X?@@@? ?@ ?@h?@@?g?@(Y@?f?@L??S5?e?@@?f@? I@h@?@@?B1?@?@? ?@heW-X?e?@h?@@?g?@H?@?f?@1??.Y?e?@@?f@L @@@@e@?@@@? [email protected]/?e?@h?@@?f@?J@e@?f?@@?g?@@?f@1 @?g?J(?'@?C5?@? ?@ ?@h?@@?f@W&@e@?f?@@?g?@@?f@@ @?@?f?.Y?V4@0Y?@? ?@he@Kf?@L?g?@@?f@@Y@e@Lf?@@?gJ@@?f@@ ?@h@?@@@?e?@1?g?@@?f@5?@L?@1f?@@?eW&?W&Ug@@ ?@h@?g?@@?g?@@?e?J@H?@1?@@f?@@?e7@?7R1g@@ ?W-Xg?W2@e@@6X?@@@ ?@h@?g?@@?g?@@?eW&@??@@?@@f?@@?e@@?@?@g@@ ?*?,g?78?e@?B1?@?@ ?@h@?e@?e?@@?g?@@?e7Y@??@@?@@f?@@?e@@?3T5g@@ ?S@Ug?@?@@?@??@?@@@ ?@h@?e@?e?@@?g?@@??J5?@??@@?@@f?@@?e@5?V+Yg@@ ?*?)X?f?39V@?@?C5?@ ?@hf@?e?@@?g?@@?W.Y?@??@@?@@f?@@?e@Hhe@@ ?V+R/?f?V4@@?@@0Y?@ ?@hf@?e?@@?g?@@?7He@??@@?@@f?@@?e@?@??@@?e?J@@ ?@h?@e@?e?@@?g?@@W5?e@??@@?@@f?@@?e@?@??@@?e?7Y@ ?@h?@e@?e?@@?g?@@(Y?e@??@@?@@f?@@?e@?@??@@?e?@?@ ?W-Xg?W2@?@@@e@@@? ?@h?@e@?e?@@?g?@@?f@??@@?@@f?@@?e@?@??@@?e?@?@ ?*>1g?7
@@@6K?hW-K?eO@e?O@? N@?@@@@@6?)Xe7R@@@@@@?@@@@? ?@?@@?@?@@@)e@?@?@?@@?@?@@? ?@?@@?@?@@X?e@?@?@?@@?@?@@? ?@?@@?@?(R/?e3T5?@?@@?@?3@? V+Y?g?V'?
? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? @? @K @K @K ? @@@?f?)X?g?@ @W-T2@@??W-X?@6T.?@@@??@?2@6T2@?W-X??W.?@@@@6T-T2@@@@?)X?)T-X?W2@@@?@?2@ @@@? ? N@HW-T26T@1?W26T26T@T2@@6T-T&??@h@@R@@?@??7@)?@@@H?@?@?J@@@?@@Y@?7@)??7H?@?@@V@R@@?@?@W@1?@@R1?7Y@@HJ@@@H@?@?@? ? J5?7@@UV@Y@?7Y@@Y@@R'UW@>@R@5?he@@T@@?@??3X??@@@L?@?@?*U@@?@@X@?3Xe?3L?@?@@?3T@@?@?@@U@?@@T5?3X@@?*U@@L@?@?@L ? .Y?@MI4@@?@?@?@@?@@?V'@R4@?(Y??@h(R+R'?@??V/??@0R/?@?@?V4@@?(R@@?V/e?V/?@?@@?V+R'?@?(R4@?(R+Y?N@@5?V4@@, ? ?V'? ?@@? ?@0Ye?I(Y @?@)T. ?I+Y ? ? @@@?f?O@? ?@f@?W-X?eW2@?@@ @?gW&eW&hf? W26Khf@? @?@??@@@@@@?@@@?@?@@6T-XeW-X@f@?.R/??W&@@?@@?W&?26T&?2@@?@e@@@@?@e @?@W@KO-X?7@?W&@W@KO@?g? ?W&@>@@6T2@@6T26KC@T&?@? @@@??@?@?@@?@@@?@?@@V@@)e7R@@f@?f?7Y@@?@@?7@@@V@@@@?@?@e@?@@?@?@ @@@@@R1?@@?7Y@@@@@@Lg? ?*@S@@@@@Y@?@@YS@@R@5? ?J(?'L?@?@?@@?@@@?@?@@?3X?e3T@@f@?@Ke?3X@@?@@?3X?@?3X?@?@?@e@?@@?@?@@?(R/?V+Y?@@?V40R/e(Yg? @@XI'T5?@@?3X@@Xe@,g? ?V40MI4@@?@?3@@0R'?(Y?@? ?.Y?V/?@?@?@@?@@@?@@@@?V/?eV+R'f@?@@@??V4@@?@@?V/?@?V/?@?@?@e@?@@@@e N@ ? ?@ ? ?)X?W&hfW26Xe@?@?@@@@e@@@??@@@ ? W26Kf)X ?@1?7@W-T2@@?W-Xe7@@?@?@@Y@? ?@@T@@@T@@?@?3T5e3T&@e@?@?@?B1e?S,??@S, ? ?V40MI40R+R4@@?@?@@?@??@ ?(R+R+R+R'?@?V+YeV4@@e@?@?@??@e?.Y??@0Y ? ? ? ? @?e@@@?f?O@?@Ke?O@K ? W2(?@K @? @?e@Xe?@@@@@@?@@@@@@@@@@ ? 7(YJ@@@6?)T.?W2@@@e@W@??@ @?e@)X??@?@?@@?@@?@?@@@?@ ? ?J(Y?7@XW@@@@H?7R'@@e@@5? @?e?S,??@?@?@@?@@?@?@@@?@ ? ?.Y??(R40R+?@??@?V4@e@0Y??@ @?@??.Y??@?@?@@?@@?@?@@@?@ ? ? ? @?W-X?f?O)X?W&??)T.e?@?@e@@6T26X?h?@?@e?W-XeW-X?he ? W2(?eW&?@g?W-X @?7R1??@@@@@@1?7@??@@Ue?@?@e@?@@,?@ @?@?@??@?@?@@@?@@??@@1e?@@@e@@@@e@?f?@V1?@@@e?S@Ue@?@?he ? ?J@S@@>@>@T5?7@@@>@T5?7@@,??@?@?@@@?@he? ?V4@@@@?V40R4@@??@?@?@?@?@ ?V+Y?.Y?e?@@@e?@?V+R'?eV+Y??@?@?@@@?31?eV+M?e@??.Y??.Y?e?@@@e@?@?@? ?@@0R+Y??@?@?@@@?@he? ?V'? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? W2@6X? W2@? ?W&? ?@h?W&?@??O@? 7U?I/T26X?@@6X?'6X?@@?'6X?*@@?W26T2@@?e?W26T&@?e@@@?@?@??W26X@W26X?W26T&@?@W2@@?W26KO. @)eV@YV1?@?B)KS@1?@H?S@1?N@@?7
Fig. 80. Ion exchange separation of nucleotides. (Work by Pharmacia Biotech, Uppsala, Sweden.)
131
Antisense phosphorothioate oligonucleotides Phosphorothioate analogs of DNA have been identified as promising candidates for oligonucleotide therapy, a major advantage being their in-vivo resistance to degradation by nucleases. Technology for automated gram-scale synthesis of phosphorothioate DNA oligomers has developed rapidly in recent years. In contrast, the large scale purification technology for therapeutic or diagnostic oligonucleotides has received little attention, and has largely been based on scaling up methods suitable for analysis or research. In this application example, a novel method* for purifying synthetic phosphothioate oligonucleotides in large scale using SOURCE 30Q is shown. The method includes adsorption of trityl-on oligonucleotide on SOURCE 30Q, washing with 10 mM NaOH and 2 M sodium chloride to remove non-tritylated failure sequences, on-column cleavage of the trityl groups using 0.4% trifluoroacetic acid, washing with 10 mM NaOH and eluting the oligonucleotide with a sodium chloride gradient to further purify it from shorter sequences. After elution, SOURCE 30Q is regenerated with 30% isopropanol in 2 M sodium chloride to wash away the adsorbed trityl-groups. A 25-mer phosphorothioate oligonucleotide produced on OligoPilot II DNA/RNA Synthesizer was purified with this method. A 25% ammonia solution containing the crude oligonucleotide mixture obtained after synthesis was applied directly onto a 0.8 liter SOURCE 30Q column. The chromatogram from the gradient elution is shown in Figure 81. Analysis of the pool revealed a yield of 1.56 g product with a purity of 97% as determined by capillary electrophoresis, see Fig 82. The overall recovery was approximately 70%. The complete process (cleavage and purification) took less then three hours. ?W-X ?7R1 J@?@L? 7@@@)T-KO-X?W-K? @?e@(R4@?,?7R@@@@@@@@@? @?e3UeS@U?@?@@?@@?@?@? N)KO&?,?3T@@?@@?@?@? ?@@0R+Y?V+R'?@@?@?@? @? @? @? @? @? @? @? @? ?@@6T-X? ?O2@@6K?gO2@@@@@@@?g @? J(MI+R1? ?O20M??I4@@@@@@@0Mhg @? 7Hf@? ?W20M? @? @?f3L W.M? @? @?fN1 ?W.Y @? ?J5?f?@ O.Y? @? ?7H?f?3L? W20Y @? ?@g?N1? ?O.M @? ?@h@? ?W20Y? @? ?@h@? W.M? @? ?@h3L ?W.Y @? J5hN1 W.Y? @? 7Hh?@ ?W.Y @? @?h?@ W.Y? @? @?h?@ ?W.Y @? @?h?@ ?7H? @? @?h?3L? J5 ?W&?e?W2@(?g@? @?h?N1? ?W.Y ?*@?e?7@@U?g@? @?he@? W.Y? ?N@?e?@MB1?g@? @?he@? ?W.Y @?g@??@@@@@@? @?he@? W.Y? @?f?C5?g@? @?he@? ?W.Y @?@?e@0Y?g@? @?he@? W.Y? @? @?he@? ?W.Y @? ?J5?he3L W.Y? @? ?7H?heN1 7H @? ?@hf?@ ?J5? @? ?@hf?@ W.Y? @? ?@hf?@ ?W.Y @? ?@hf?@ ?7H? @? ?@hf?@ J5 @? ?@hf?@ ?W.Y @? ?@hf?3L?hfW.Y? @? ?@hf?N1?hf7H @? ?@ @?he?J5? @? ?@ @?heW.Y? @? ?@ @?h?W.Y @? ?@ @?h?7H? @? ?@ @?hJ5 @? ?@ @?h7H @? ?@ @?g?J5? @? ?@ 3Lg?7H? @? ?@ N1gJ5 @? ?@ ?@f?W.Y @? ?@ ?@f?7H? @? ?@ ?@fJ5 @? ?@ ?@f7H @? ?@ ?@e?J5? @? ?@ ?@e?7H? @? ?@ ?@eJ5 @? ?@ ?@?W.Y @? J5 ?@?7H? @? 7H ?@W5 @? @? ?3@H ?W&?e?W26X?g@? @? @? ?*@?e?7(Y? @? @? ?@@H @? @? ?@5? @? @? ?@H? 3L @? J@ N1 @? ?W&@ ?@ W26Xe?W2@(?g@? W.Y@ ?@ 7
?W2@(?eW26X ?7@@U?e7
?W&?W26Xe?W26X? ?*@?7
W&?W2@(?eW26X *@?7@@U?e7
W26KO26Xe?W26X? .MB@@
?W26KO2@(?eW26X ?.MB@@@@U?e7
?@e?@K??@ W2@@?W&Khe?/X?g ?3L?J@@6X@?@?@e@@@@@?W26Xf7Y@@?&@@@?W26X?W.??N1?g ?N1?7@?B@@?@?@e@?@?@?7YV1f@?@@?N@@H?7YV1?*U?e@?g @?@@e@@?@?@e@?@?@?@@@@f@?@@e@@e@@@@?V/Xe@?g 3T@@=C@@?@?@e@?@?@?3Xg@?@@e@@e3XfS,e@?g V+MI40R'?@@@e@?@?@?V4@?f@?@@e@@eV4@?e.Ye@?g 3L ?J5?g V/ ?.Y?g
*patent pending
132
Fig. 81. Preparative purification of 25-mer phosphorothioate oligonucleotide on SOURCE 30Q. (Work by Pharmacia Biotech, Uppsala, Sweden.)
Samples:
Capillary: Buffer: Running conditions: Data collection:
All samples were desalted on NAP 10 Columns a) Pool b) Fraction 4 c) Fraction 10 µPAGE (5% T, 5% C), capillary length: 40 cm (J&W Scientific, FISON) Tri-borate and urea buffer (J&W Scientific, FISON) 8 kV, 10 s (sample) 16 kV, 30 min (run) FPLCdirector
?@@@?@?@ ?@?@?@?@ ?@@@?@?@ J(?'X@?@ .Y?V4@@@ @? @? @? @? @? @? @? @? @? @? @? @? @? @? @? @? @? @? @? @? @? @? @? @? @? @? @? @? @? @? ?W-XeW-KO-X?W-Xf@? @? ?7R1e7R@@?,?7R1f@? @? ?@?@e@?@V'U?@?@f@? @? ?3T@L?3T5?S,?3T5e@@@? @? ?V+R/?V+Y?.Y?V+Yf@? @? @? @? @? @? @? @? @? @? @? @? @? @? @? @? @? @? @? @? @? @? @? @? @? @? @? @? @? @? @? @? ?W-XeW-KO-X?@@@f@? @? ?7R1e7R@(R/?@X?f@? @? ?@?@e@?@?e?@)Xf@? @? ?3T@L?3T@)K?eS,e@@@? @? ?V+R/?V+R4@@e.Yf@? @? @? @? @? @? @? @? @? @? @? @? @? @? @? @? @? @? @? @? @? @? @? @? @? @? @? @? @? @? @? ?J@L @? ?7R1 ?W-XeW-KO-X?W-Xf@? ?@?@ ?7R1e7R@(R/?7R1f@? ?@?@ ?@?@e@?@?e?@?@e@@@? ?@?@ ?3T@L?3T@)K??3T5f@? ?@?@ ?V+R/?V+R4@@?V+Yf@? ?@?@ @? ?@?@ @? ?@?@ @? ?@?@ @? ?@?@ @? ?@?@ @? ?@?@ @? ?@?@ @? ?@?@ @? ?@?@ @? ?@?@ @? ?@?@ @? ?@?@ @? ?@?@ @? ?@?@ @? ?@?@ ?W-XeW-X?@??@@@f@? ?@?@ ?7R1e7R1?@??@X?f@? ?@?@ ?@?@e@?@?@??@)Xe@@@? ?@?@ ?3T@L?3T5?@?eS,f@? ?@?@ ?V+R/?V+Y?@?e.Yf@? ?@?@ @? ?@?@ @? ?@?@ @? ?@?@ @? ?@?@ @? ?@?@ @? ?@?@ @? ?@?@ @? ?@?@ @? ?@?@ @? ?@?@ @? ?@?@ @? ?@?@ @? ?@?@ @? ?@?@ @? ?@?@ ?W-XeW-X?@??W-Xf@? ?@?@ ?7R1e7R1?@??7R1f@? ?@?@ ?@?@e@?@?@??@?@e@@@? ?@?@ ?3T@L?3T5?@??3T5f@? ?@?@ ?V+R/?V+Y?@??V+Yf@? ?@?@ @? ?@?@ @? ?@?@ @? ?@?@ @? ?@?@ @? ?@?@ @? ?@?@ @? ?@?@ @? ?@?@ @? ?@?@ @? J5?@ @? 7H?@ @?e?@?@ @??@ @?eJ@T5 ?O-T5??3=? @??O&R@Yhf?O2@@@@@@@@@@@@@@@@0R+Y??V'6K? @@@@@?@@@@@@@@@@@@@@@0M? V4@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ @M?I@?@M ?N)K?S)T&K @@@@R@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ ?@?@ ?@ @? @? J@T5 ?@ @L ?J@L 7R@H ?@?@@@ ?W-X@)X? W-T&@@ @?@? ?@?@X? ?.R'@V1? .R'@X? ?@?@)X ?N@?@? V4)X ?@eS, ?@KC@T5? @KeS, [email protected] ?@@0R+Y? @@@?.Y @@@?@Kh?W-Ke?O@?eO@ N@H?@@@@@6?)X??7R@@@@@@?@@@@ ?@e@@?@?@@@)??@?@?@?@@?@?@@ ?@e@@?@?@@Xe?@?@?@?@@?@?@@ ?@e@@?@?(R/e?3T5?@?@@?@?3@ ?V+YhV'
a)
?@@@?@?@ ?@?@?@?@ ?@@@?@?@ J(?'X@?@ .Y?V4@@@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?W-XeW-X?@??W-Xf?@ ?@ ?7R1e7R1?@??.R/f?@ ?@ ?@?@e@?@?@?h?@ ?@ ?3T@L?3T5?@??@K?f?@ ?@ ?V+R/?V+Y?@??@@@e?@@@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?W-XeW-X?@??W-Xf?@ ?@ ?7R1e7R1?@??7R1f?@ ?@ ?@?@e@?@?@??@?@f?@ ?@ ?3T@L?3T5?@??3T5f?@ ?@ ?V+R/?V+Y?@??V+Ye?@@@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?W-XeW-KO-X?W-Xf?@ ?@L? ?7R1e7R@@R1?*?,f?@ ?@1? ?@?@e@?@@?@?S@Uf?@ ?@@? ?3T@L?3T@@T5?*?,e?@@@ ?@@? ?V+R/?V+MI+Y?V+Yf?@ ?@@? ?@ ?@@? ?@ ?@@? ?@ ?@@? ?@ ?@@? ?@ ?@@? ?@ ?@@? ?@ ?@@? ?@ ?@@? ?@ ?@@? ?@ ?@@? ?@ ?@@? ?@ ?@@? ?@ ?@@? ?W-XeW-KO-X?W.?f?@ ?@@? ?7R1e7R@@R1?7U?f?@ ?@@? ?@?@e@?@@?@?@)Xf?@ ?@@? ?3T@L?3T@@T5?3>,e?@@@ ?@@? ?V+R/?V+MI+Y?V+Yf?@ ?@@? ?@ ?@@? ?@ ?@@? ?@ ?@@? ?@ ?@@? ?@ ?@@? ?@ ?@@? ?@ ?@@? ?@ ?@@? ?@ ?@@? ?@ ?@@? ?@ @??@@? ?@ @??@@? ?@ @??@@? ?@ @??@@? ?W-XeW-KO-Xe@?f?@ @??@@? ?7R1e7R@@R1?J@?f?@ @??@@? ?@?@e@?@@?@?7@Le?@@@ @??@@? ?3T@L?3T@@T5?3@,f?@ ?J@??@@? ?V+R/?V+MI+Y?V+Yf?@ ?7@??@@? ?@ ?@@??@@? ?@ ?@@??@@? ?@ ?@@??@@? ?@ ?@@??@@? ?@ J@@?C@@? ?@ 7@@@@Y@? ?@ ?J@(Y@@?@? ?@ W&@H?@@?@? ?@ ?W&R'??@@?3L ?@ ?75?e?3@?N1 ?@ J@H?e?V'??@ ?@ 7@h?3L? ?@e?W-T.? ?J@@h?N)K ?@eO&R@Y? O.M?he@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ ?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@0Y ?@KO-T&K ?@@@R@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ W@T(M? ?@ ?@ ?@ .R+Y ?@ ?3L? ?@ ?@?@@@ W-KS)X W-X@@@ ?@?@X? .R'@R1 .R'@X? ?@?@)X N@?@ V4)X ?@eS, @KC@T5 @KeS, [email protected] @@0R+Y @@@?.Y ?@@@6Kh?W-Ke?O@?eO@ ?N@?@@@@@6?)X??7R@@@@@@?@@@@ @?@@?@?@@@)??@?@?@?@@?@?@@ @?@@?@?@@Xe?@?@?@?@@?@?@@ @?@@?@?(R/e?3T5?@?@@?@?3@ ?V+YhV'
b)
? ? ? ? ?@@@?@?@e ? ?@?@?@?@e ? ?@@@?@?@e ? J(?'X@?@e ? .Y?V4@@@e ? ? W-X??W-KO-X?W-X?he ? 7R1??7R@(R/?7R1?he ? @?@??@?@f@?@?he ? 3T@L?3T@)Ke3T5??@@@g @? ? V+R/?V+R4@@?V+Y?e?@g @? ? ?@g @? ? ?@g @? ? ?@g @? ? ?@g @? ? ?@g @? ? ?@g @? ? ?@g @? ? ?@g @? ? ?@g @? ? ?@g @? ? ?@g @? ? ?@g @? ? ?@g @? ? ?@g @? ? ?@g @? ? ?@g @? ? ?@g @? ? ?@g @? ? ?@g @? ? ?@g @? ? ?@g @? ? ?@g @? ? ?@g @? ? ?@g @? ? ?@g @? ? ?@g @? ? ?@g @? ? W-X??W-X?@e@@@?e?@g @? ? 7R1??7R1?@e@Xf?@g @? ? @?@??@?@?@e@)X?e?@g @? ? 3T@L?3T5?@e?S,??@@@g @? ? V+R/?V+Y?@e?.Y?e?@g @? ? ?@g @? ? ?@g @? ? ?@g @? ? ?@g @? ? ?@g @? ? ?@g @? ? ?@g @? ? ?@g @? ? ?@g @? ? ?@g @? ? ?@g @? ? ?@g @? ? ?@g @? ? ?@g @? ? ?@g @? ? ?@g @? ? ?@g @? ? ?@g @? ? ?@g ?J@L ? ?@g ?7R1 ? ?@g ?@?@ ? ?@g ?@?@ ? ?@g ?@?@ ? ?@g ?@?@ ? ?@g ?@?@ ? ?@g ?@?@ ? ?@g ?@?@ ? ?@g ?@?@ ? W-X??W-X?@eW-X?e?@g ?@?@ ? 7R1??7R1?@e7R1?e?@g ?@?@ ? @?@??@?@?@e@?@?e?@g ?@?@ ? 3T@L?3T5?@e3T5??@@@g ?@?@ ? V+R/?V+Y?@eV+Y?e?@g ?@?@ ? ?@g ?@?@ ? ?@g ?@?@ ? ?@g ?@?@ ? ?@g ?@?@ ? ?@g ?@?@ ? ?@g ?@?@ ? ?@g ?@?@ ? ?@g ?@?@ ? ?@g ?@?@ ? ?@g ?@?@ ? ?@g ?@?@ ? ?@g ?@?@ ? ?@g ?@?@ ? ?@g ?@?@ ? ?@g ?@?@ ? ?@g ?@?@ ? ?@g ?@?@ ? ?@g ?@?@ ? ?@g ?@?@ ? ?@g ?@?@ ? ?@g ?@?@ ? ?@g ?@?@ ? ?@g ?@?@ ? ?@g ?@?@ ? ?@g ?@?@ ? ?@g ?@?@ ? ?@g ?@?@ ? W-X??W-KO-X?@@@?e?@g ?@?@ ? 7R1??7R@@R1?@Xf?@g ?@?@ ? @?@??@?@@?@?@)X?e?@g ?@?@ ? 3T@L?3T@@T5??S,?e?@g ?@?@ ? V+R/?V+MI+Y??.Y??@@@g ?@?@ ? ?@g ?@?@ ? ?@g ?@?@ ? ?@g ?@?@f?@ ? ?@g ?@?@f?@ ? ?@g ?@?@f?@f@? ? ?@g ?@?@f?@L?e@? ? ?@g ?@?@f?@1??J@? ? ?@g ?@?@f?@@??7@? ? ?@g J5?@f?@@??@@@6K ? ?@g 7H?@f?@@??@?@@@@? ? ?@g @??@fJ@@@@@?@?4@= ? ?@g @X@??3=O)T&@@0M?fI'@6X? ? ?@?O@?)X @@@??V4@@0M?h?V4@)Kh?O2@@@@@@@@?e? ?@@@@T@)@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@0M I4@@@@@@@@@0M?he? ?@?@V@
c)
Fig. 82. Capillary electrophoresis of the pool and side fractions from the preparative purification of 25-mer phosphorothioate oligonucleotide shown in Fig. 81. (Work by Pharmacia Biotech, Uppsala, Sweden.)
Areas of application In the preceding chapters the examples which have been used to illustrate the principles and practice of ion exchange chromatography have mostly been based on analytical and preparative applications from the research laboratory. Ion exchange chromatography also has many important applications in the field of industrial and pilot scale preparations. Many blood products such as albumin and IgG (35) as well as the products of recombinant DNA technology, such as growth factors and pharmaceutically important enzymes (Fig. 83), are purified using this technique. An example of a pilot scale purification of a recombinant protein using ion exchange chromatography is given at the end of this chapter. For further information on the application of ion exchange chromatography at pilot and process scales the reader is advised to contact Pharmacia Biotech. Analytical applications of ion exchange chromatography are to be found in diverse areas such as quality control of purified products or process monitoring in biotechnology. Figure 84 shows the use of cation exchange in monitoring a fermentation process for the production of ß-galactosidase.
133
?@ ?@ ?@ ?@ ?@ ?@e@? )X @? ?J@)X? @? ?7
?@?@ ?@?@ ?@?@ ?@?@ ?@?@
W26Xf)X ?W.R'1?W-T@) ?*U?V@?7@@8? ?V4@@@?@?@?@
W26XeW.h?@K? @@@?h?@@@@? 7@@W26T2@@?@@6T2@@?W2@@6?2@??@W@?W2@@@6T5?@?h?@e@@1?W&?26T-X?W&@eW.?W&X@@e@@@?W-T2@@@6T-KO2@?W&?2@@?)X?@?)X??W-X?@e@6T&KO.?W-X?@@@@@@)X@1 ?*@S(Y@@@@@@@??@?@?@?4@??@0Y?@?@?4@@e@?h?@e@@@?3X?@?3X??3X@e3L?3X?@@e@?@?3T@@?@?@@T@@X@?3X?@?@@U@?@@Xe?3T5?@e@?3XI'L?3T5?@?@?@@W@@@ ?@?*U@@?@@hg ?V'>5? ?@e@@@?V/?@?V/??V4@eV/?V/?@@e@?@?V+R'?@?(R+MB@@?V/?@?(R4@?(R/e?V+Y?@e@?V/?V/?V+Y?@?@?@@0R4@ ?@?V4@@?@@hg V+Y? @@=? @K O@eW&e?S@6K? @@@@?@?@@@@@?@?2@@eW&?@?@@6X?W&?26T-X?@@@?W&@eW&@@@@@@@@?@?@?)X?W&?W-X @?@@?@?@?@?@W@@@?@e*@?@?@@V1?7@@@V@R1?@@@?7Y@e7Y@@@?@?@@?@?@W@1?*@W&@) @?@@?@?@?@?@@U@@?@eN@@@?@@W5?3X?@?3T5?@@@?3X@e3X@@@?@?@@?@?@@U@?N@@@X? @?@@@@?@?@?(R4@@?@e?@?4@@@(Y?V/?@?V+Y?@@@?V4@eV4@@@?@?@@@@?(R4@??@MI/? ?I(Y W26Khf@? W-X??W-Xe@??@ ?W&?hfW&he)X?@K? ?W&@>@@6T2@@6T26KC@T&?@? *?,??*?,e@??@?@6T-T2@@@@??@6T26T&?@?7@?W-T&?@?@e?W&@W)X?W&e@X@1?@@@@?W2@? ?*@S@@@@@Y@?@@YS@@R@5? V'U??S@Ue@??@?@V@R@@?@?@??@V@@V@@@@?@@?7R@@?@@@L??7Y@@@)?*@?C@@@@?@@?@?7Y@? ?V40MI4@@?@?3@@0R'?(Y?@? ?S)X?*?,e@??@?@?3T@@?@?@??@W@@?3XI'T@@?3T@@?@?@,??3X@@XeN@@@U@@@?@@?@?3X@? N@ ?.R/?V+Ye@??@?@?V+R'?@?@??@(R'?V/?V+R'?V+R4@@?(Y??V40R/e?@MI4@@@?@@?@?N@@? ?@ ?@H? ?@@? ?W.?h@?J@L?he?@K??O@?h@@ W&?@?W&?26X??7HW.??)X?e@?.R/??@@@@6T2@??@@@@@@??W2@?W&X@@ *@?@?7@@@V1??@?7H?J@1?e@?f?@?@?@@Y@??@?@?@@??7Y@?7@@@@ N@@@?3X?@W5??@?3L?*U@?e@?@Ke?@?@?@@X@??@?@?@@??3X@?3X?@@ ?@?@?V/?@(Y??3LV/?V4@?e@?@@@??@?@?(R@@??@?@?@@??N@@?V/?3@ (Ye?V/? ?@@?he@@fV' W26Kf)X @6X?@?f@@6K W2@?f)Xhf@6X??@@@e?@?@?W-Xe@@e@@@?hg ?W.MS@@6K?@)T2@@@6T2@??@ @S,?@W-Xe@?@@6T-KO.?W&KO@?W&?e7<eW-X?@1?@@@@@@@@@e@S,??@?@e?@?@?*?,e@@e@X ?*UO&@XS@@@>@@?@?@@Y@? @@U?@@R1e@@@@V@R@@H?7@@@@W&@Le@??W&R1?@@?@@?@?@@?@e@@U??@@@e?@?@?V'Ue@@e@)X?hg ?V40MI40R+R4@@?@?@@?@??@ @V1?@@T5e@??@?3T@@L?3X??@(Y@,e3=O&@T5?@@?@@?@?@@?@e@V1??@f?@?@eS,e@@e?S)Xhg @@@?(R+Ye@??@?V+MI/?V/??(Y?(YeV40MI+Y?@@@@@?@?@@?@e@@@??@f?@[email protected]@@e?.R/hg W2@6X?fW&e?W&?@K @?he?@e@?eW-X??O2@@?hO@ 7Y@S,?W&KO&@eW&@?@@@@@6?@?2@@?W&?@W-T2@@?W&e?@e@?e*?,?@@@Xe?W.?@@@@@@ @?@@U?7@@@Y@e7Y@?@@?@?@@@@@?@?&@?@@R@@?@?&@L??@e@?eV'U?@@@)X??7H?@?@?@@ @?@V1?3XI'X@e3X@?@@?@?@@X?@?@?N@@@@T@@?@?N@,??@e@?e?S,?@@?S,??3L?@?@?@@ 3T@@@?V/?V4@eV4@?@@?@?(R/?@?@e@?(R+R'?@??(Y??@e@?@??.Y?@@?.Y??V/?@?@?3@ N@@>@T5?7@@
@?@@g@?@? @?@@g@?@? @?@@g3T5? @?@@gN@H? @?@@g?@?@ ?@ ?@
Fig. 83. Process scale purification of recombinant superoxide dismutase by ion exchange chromatography on Q Sepharose Fast Flow. (Work by Pharmacia Biotech, Uppsala, Sweden.
@? @K @K @K ?@@@g)Xf?@K? @W-T2@@??W-X?@6T.?@@@??@?2@6T2@?W-X??W.?@@@@6T-T2@@@@?)X?)T-X?W2@@@?@?26X?@@@? ?N@HW-T26T@1?W26X@@@6T-T-T&?@?h@@R@@?@??7@)?@@@H?@?@?J@@@?@@Y@?7@)??7H?@?@@V@R@@?@?@W@1?@@R1?7Y@@HJ@@@V1?@?@? ?J5?7@@UV@Y@?7Y@@@XW@@>@R@5?he@@T@@?@??3X??@@@L?@?@?*U@@?@@X@?3Xe?3L?@?@@?3T@@?@?@@U@?@@T5?3X@@?*U@@W5?@?@L ?.Y?@MI4@@?@?@?@0R'@@@6T2@@6T26KC@T&?@ ?@)X@?@?@?@??@@?@??7H?@W@1?@?@@?@@@@@Y@??@V1?J@@@H?@?7@@@?@W@1?@e@@@)?@?@?@?@@@@@?@@?J@1?@@?7R@@?@??*@?C@@@?@?@@V1?@@@)g *@S@@@@@Y@?@@YS@@R@5 S@@T5?3T5??@@?@??3L?@@U@?@?@@?@@XI'X@??@W5?*U@@L?@?3X?@?@@U@?@e@@X??@?@?@?@@X?@?@@?*U@?@@?3T@@?@??N@@@U@@?@?@@W5?@@X?g V40MI4@@?@?3@@0R'?(Y?@ .MI+Y?V+Y??@5?@??V/?(R4@?@?@@?(R/?V4@??@0Y?V40R/?@?V/?@?(R4@?@e(R/??@?@?@?(R/?@?@@?V4@?@@?V+R'?@?e@MI4@@?@?3@(Y?(R/?g ?N@? ?(Y? ?V+Yhf @? ?)X?W&hfW26Xe@?@?@@@@e@@@??@@@ ?W26K?e?)X? ?@1?7@W-T2@@?W-Xe7@@?@?@@Y@ ?@@T@@@T@@?@?3T5e3T&@e@?@?@?B1e?S,??@S, V40MI40R+R4@@?@?@@?@e@?hf?(R+R+R+R'?@?V+YeV4@@e@?@?@??@e?.Y??@0Y @?eW-X?f?O@?@Ke?O@K ?W2(?@K?hf?@ @?e7R1??@@@@@@?@@@@@@@@@@ ?7(YJ@@@6?)T.?W2@@@??@W@e@?he@?e@?@??@?@?@@?@@?@?@@@?@ J(Y?7@XW@@@@H?7R'@@??@@5 @?e3T5??@?@?@@?@@?@?@@@?@ .Ye(R40R+?@e@?V4@??@0Ye@?he@?@?V+Y??@?@?@@?@@?@?@@@?@ ?@@@gO)X?W&e@6X?@?f@@6KO@he@?@?e@@@??W-X ?W2(e?W&?@?gW-X? ?@X?e@@@@@@1?7@e@S,?@W@?eN@V@@@W@f?@6X@?@?e@Xe?*?, ?7@UO-T&@?@?W-T2@?7>,?@?hf?@)Xe@?@?@@@?@@e@@U?@@@?e?@?@?@@@L?e?@V@@@@?e@)X??S@U J@S@@>@>@T5?7@@@>@T5?7@@<eJ@S, ?J(?'L?*@,e3T@L?3>,e@@T@@??@@@?*U@@=O.?@ @@0MI4@0R+Y?@?@?e@@0Y?@hf?.Y?V/?V'YeV+R/?V+Ye(R+R'??@@@?V40R40Y?@ ?W-Xe?@eW-KO-Xg?@6Xe?@K?e?W-X?W-XgO@K? ?W26X?gO2@?g@?hf?7R1e?@e7R@@R1e?W.??@S,e?@@@@??.R/?7R1e@@@@@@@@@? W.R')T2@@6T2@@5?W-T2@?@?@?he?@?@L??@e@?@@[email protected]??@@Ue?@@?@?f?@?@e@?@?@@@?@? *U?V@@R'@@@U?@H?7@@Y@?@?hf?3T@@??@e3T@@T5?W.Ye?@V1e?@@?@??@K??3T5e@?@?@@@?@? V4@@@@?V40R4@@e@?@?@?@?@?he?V+Me?@eV+MI+Y?.Y?e?@@@e?@@?@??@@@?V+Ye@?@?@@@?@?
@@@? @?@? @@@? ?J(?'L ?.Y?V/ ?@@?@@@@@? ?@@?f@@@@@? @?eW-X? @?e7R1? @?e@?@? @?e3T5?@@@@@? ?W.? @?@?V+Y?f@?f@? ?@?7H? @?f@? ?@W5 @?f@? ?@@H @?f@? ?@5? @?f@? ?@H? @?he@?W& @?f@? J@g?@@(?W2@?)X?@W.?@?W-KO@?@W&@e@KO@?W-X @?f@? 7@g?@@U?7Y@W@1?@@H?@?7R@@@?@@Y@?J@@@@W&@) @?f@? ?J@@g?@V1?3X@@U@?@@L?@?3T(Y@@@@X@?*U@?@@@X? @?f@? ?7Y@g?@@@?N@@V4@?(R/?@?V+Y?@?(R4@?V4@?@MI/? @?f@? J5?@g?@M?e@@ @?f@? 7H?@ @?f@? ?J5??@ @?f@? ?7H??@ @?f@? J5e?@ @?f@? 7He?@ @?f@? ?J5?e?@ @?f@L ?7H?eJ@L? @?f@1 J5f7R1? @?f@@ 7Hf@?@? @?f@@ ?J5?f@?@? @?f@@ ?7H?f@?@? @?f@@ J5g@?@? @?f@@ 7Hg@?@? @?f@@ ?J5?g@?@? @?f@@g@? ?7H?g@?@? @?f@@g@? J5h@?@? @?f@@g@? ?W.Yh@?@? @? @?f@@g@? ?7H?h@?@? @? @?f@@g@? J5he@?@? @? @?f@@e@?e@? 7Hhe@?@? @? @?f@@e@?e@? ?J5?he@?@? @L @?f@@e@?e@? ?7H?he@?@? @)X? @?f@@e@?e@? J5hf@?@? @@1? @?f@@?J@?e@? 7Hhf@?@? @@@? @?f@@?7@?e@? ?J@?hf@?@? @@@? @?e?J@@?@@?e@? ?7@?hf@?@? @@@? @?e?7Y@?@@?e@? J@@?hf@?@? @@@? @?e?@?@?@@??J@? 7Y@?hf@?@? @?@? @?e?@?@?@@??7@? ?J5?@?hf@?@? @?@? @?e?@?3X@@L?@@? ?7H?@?hf@?@? @?@? @?e?@?N@@V1?@@? J5e@?hf@?@? @?@? @?e?@e@5?@?@@? 7He@?hf@?@? @?@??@ @?e?@e@H?@?@@? ?J5?e@?hf@?@? @?@??@ @?e?@e@??@?@@L ?7H?e@?hf@?@? @?@??@ @?e?@e@??@?@V1 J5f@Lhf@?@? @?@??@L? @?e?@e@??@?@?@ 7Hf@1hf@?@? @?@??@1? @?e?@f?@?@?@ ?J5?e?J@@hf@?@? @?@??@@? @?e?@f?@?@?@ ?7H?e?7Y@hf@?@? @?@??@@? @?e?@f?@?@?@ J5f?@?@he?J@?3L @?3=C@@?e?)X? @?e?@f?@?@?@ 7Hf?@?@he?7@?N1 @?N@(Y@?eJ@)K @?e?@f?@?@?@ ?J5?f?@?@he?@@??@ @??(Y?@?e7@@U? I40Y @?O&@?hf?J@0MS,? @@0M ?7
134
Fig. 84. Monitoring the production of b-galactosidase (36).
Other areas of application include food research where FPLC ion exchange can be used in the study of wheat varietals (Fig. 85) and in clinical research where ion exchange chromatography has been used in studies such as the relationship between post-partum depression and b-endorphin secretion (Fig. 86) and the correlation of proteinuria with different renal conditions (Fig. 87). A chromatogram of the urine from patients exhibiting tubular proteinuria, due to acute pyelonephritis, severe burns or renal transplants, shows distinct peaks corresponding to b2-microglobulin, retinol binding protein and a1-acid glycoprotein. The disappearance of these peaks could be correlated with the reversal of their causal lesion (39). @? @K @K @K @@@?g)Xf?@K? @W-T2@@??W-X?@6T.?@@@??@?2@6T2@?W-X??W.?@@@@6T-T2@@@@?)X?)T-X?W2@@@?@?26X?@@@? N@H?W-T26T@1?W26X@@@6T-T-T&?@?h@@R@@?@??7@)?@@@H?@?@?J@@@?@@Y@?7@)??7H?@?@@V@R@@?@?@W@1?@@R1?7Y@@HJ@@@V1?@?@? J5e7@@UV@Y@?7Y@@@XW@@>@R@5?he@@T@@?@??3X??@@@L?@?@?*U@@?@@X@?3Xe?3L?@?@@?3T@@?@?@@U@?@@T5?3X@@?*U@@W5?@?@L .Ye@MI4@@?@?@?@0R'@@@6T2@@6T26KC@T&?@? ?@?@@?@@W@1?7Y@@@?@??7@)e@@@@HJ@1?7H?@??@?@V@R@@?@?@?eN@f7R@@?@@Y@?7R@@H? ?*@S@@@@@Y@?@@YS@@R@5? ?39V@?@@@U@?3X@@@?@??3X?e@@@@?*U@?3L?@??@?@?3T@@?@?@?e?3=O.?3T@@?@@X@?3T@@ ?V40MI4@@?@?3@@0R'?(Y?@? ?V4@@?@0R4@?V4@@@?@??V/?e@@@@?V4@?V/?@??@?@?V+R'?@?@?e?V40Y?V+R'?(R4@?V+R' N@ ?)X?W&hfW26Xe@?@?@@@@e@@@??@@@ W26Kf)X?@ ?@1?7@W-T2@@?W-Xe7@@?@?@@Y@? ?@@T@@@T@@?@?3T5e3T&@e@?@?@?B1e?S,??@S, ?V40MI40R+R4@@?@?@@?@??@ ?(R+R+R+R'?@?V+YeV4@@e@?@?@??@e?.Y??@0Y @?eW-X?f?O@?@Ke?O@K W2(?@K @? @?e7R1??@@@@@@?@@@@@@@@@@ 7(YJ@@@6?)T.?W2@@@e@W@??@hf@?e@?@??@?@?@@?@@?@?@@@?@ ?J(Y?7@XW@@@@H?7R'@@e@@5? @?e3T5??@?@?@@?@@?@?@@@?@ ?.Y??(R40R+?@??@?V4@e@0Y??@hf@?@?V+Y??@?@?@@?@@?@?@@@?@ @?W-X?f?O)X?W&??W2@e@@@?@@6T.?h@?@?e?@?W-Xe?@ W2(?eW&?@hW-X? @?7R1??@@@@@@1?7@??7,?@? @?@?@??@?@?@@@?@@??@f@@@?@@@@@@f?@V@@@@?e?@?@?@e7@L? ?J@S@@>@>@T5?7@@@>@T5?7@@
?@ ?@K? ?@K? ?@K? @@@?f?)X?f@K ?@W-T2@@eW-X?@6T.?@@@e@?2@6T2@?W-XeW.?@@@@6T-T2@@@@?)X?)T-X?W2@@@?@?26X?@@@ N@HW-T26T@1?W26X@@@6T-T-T&?@g?@@R@@?@e7@)?@@@H?@?@?J@@@?@@Y@?7@)e7H?@?@@V@R@@?@?@W@1?@@R1?7Y@ @HJ@@@V1?@?@ J5?7@@UV@Y@?7Y@@@XW@@>@R@5h?@@T@@?@e3Xe@@@L?@?@?*U@@?@@X@?3X?e3L?@?@@?3T@@?@?@@U@?@@T5?3X@@?*U@@W5?@?@L? .Y?@MI4@@?@?@?@0R'@
W26Khf@? 78e?@@?)X?W&@@@@@eW-X??@@@@@?)X?W.?@e@?@6T-T2@@@@e?@@@e?)X?W.7@W)X??@@? ?W&@>@@6T2@@6T26KC@T&?@?hf@?@@?@@W@1?7Y@@@?@e7@)??@@@@HJ@1?7H?@e@?@V@R@@?@?@f?@@6T@1?7H @@@@)? ?*@S@@@@@Y@?@@YS@@R@5? 39V@?@@@U@?3X@@@?@e3Xe?@@@@?*U@?3L?@e@?@?3T@@?@?@f?@?S@U@?3L@(R/ @@@X ?V40MI4@@?@?3@@0R'?(Y?@?hfV4@@?@0R4@?V4@@@?@eV/e?@@@@?V4@?V/?@e@?@?V+R'?@?@f?@@0R4@?N1 N@ ?@(Y )X?W&?he?W26X??@?@?@@@@??@@@e@@@? W26Kf)X?@ @1?7@W-T2@@?W-X??7@@?@?@@Y@? @@T@@@T@@?@?3T5??3T&@??@?@?@?B1?eS,e@S,? ?V40MI40R+R4@@?@?@@?@??@hf(R+R+R+R'?@?V+Y??V4@@??@?@?@e@?e.Ye@0Y? ?@e?W-XgO@?@K?eO@K? W2(?@K @?hf?@e?7R1e@@@@@@?@@@@@@@@@@? 7(YJ@@@6?)T.?W2@@@e@W@??@h?@e?@?@e@?@?@@?@@?@?@@@?@? ?J(Y?7@XW@@@@H?7R'@@e@@5?he?@e?3T5e@?@?@@?@@?@?@@@?@? ?.Y??(R40R+?@??@?V4@e@0Y??@h?@?@?V+Ye@?@?@@?@@?@?@@@?@? ?@?W-XgO)X?W&eW2@??@@@?@@6T.h?@?@f@?W-X?e@?g W2(?eW&?@hW-X?hf?@?7R1e@@@@@@1?7@e7<e?@?@?@?@@Yg@6X@?@f@?7R1??J@?g 7@UO-T&@?@?W-T2@e7>,?@?he?@?@?@e@?@?@@@?@@e@?e?@@@?@@@@@@?f@V@@@@f@?@?@??7@Lg ?J@S@@>@>@T5?7@@@>@T5?7@@
?W-Xe?@eW-KO-Xg?@6Xe?@K?e?W-X?W-XgO@K? W26Xg?O2@g?@ ?7R1e?@e7R@@R1e?W.??@S,e?@@@@??.R/?7R1e@@@@@@@@@? ?W.R')T2@@6T2@@5?W-T2@?@?@hf?@?@L??@e@?@@[email protected]??@@Ue?@@?@?f?@?@e@?@?@@@?@? ?*U?V@@R'@@@U?@H?7@@Y@?@ ?3T@@??@e3T@@T5?W.Ye?@V1e?@@?@??@K??3T5e@?@?@@@?@? ?V4@@@@?V40R4@@??@?@?@?@?@hf?V+Me?@eV+MI+Y?.Y?e?@@@e?@@?@??@@@?V+Ye@?@?@@@?@?
?W-X ?7R1 J@?@L? 7@@@1? @?e@L @?e@@f@@@@?@@@@@ @??@@?@@@@?@?@?@ ?J@@f?I4@?@?@?@ O&5hf?@h?J@?f?@e?@ ?@ ?W.R@Hhf?@h?7@Lf?@e?@ ?@ W.Y?@?hf?@f)Xe?@V1f?@e?@ ?@ ?W.Ye@?hf?@f@1e?@?@f?@e?@ ?@ W.Y?e@?hf?@f@@eJ5?@fJ5e?@ ?@ ?W.Yf@?hf?@e?J@@e7H?@f7He?@ ?@ W.Y?f@?hf?3L??7R'L?@??3L?e@?e?3L? ?@ ?W.Yg@?hf?N1?J5?N1?@??N1?e@?e?N1? ?@ ?7H?g@? @?7He@W5?e@??J5?f@? ?@h?@hJ5h@? 3X@?e@@H?e3L?7H?f@? ?@h?@L?f?W.Yg?J5? V'@?e@@fV/T5g3L ?@h?@1?fW.Y?g?7H? ?V'?e@@f?V+YgN1 ?@h?@@?e?W.Yh?@ ?3=? ?@h?@@?eW.Y?h?@ ?V46K? ?@h?@@??W.Yhe?@ ?I46X? ?@hJ@@?W.Y?he?@ ?I/X ?@[email protected]?@ V/X? ?@h@?@(Y?hfJ5 ?V/X ?@h@?(Y .Y V/@6X? ?@g?W2(?@ @? ?@g?7(Y?@hf?J5? ?I/? ?@gJ@[email protected]? ?@f?W&@e?@hW2@@0Y ?@fW&U@e?@g?O.M ?@e?O&>@@e?3=O2@@@@0Y? ?@?'@@@0M?e?V40M? ?@?S(M [email protected]? ?@@Y ?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ ?@ ?@ ?@ ?@ ?@ ?@ W26X 7
?W&?W26X ?*@?7
W-X? 7R1? ?J@?@L ?7@@@1 ?@e?@L? ?@e?@@?e?@@@@?@@@@@? ?@e@@?@@@@?@?@?@? J@@?fI4@?@?@?@? ?O&< @@@@@? W.hf @? ?W.Yhf @? ?@ W.Y?hf @? ?@ ?W.Yhg @? ?@ W.Y?hg @? ?@ ?W.Y @? J@ W.Y? @? 7@ ?W.Y @? @@ W.Y? @? ?J@@ ?W.Y @? ?7@@ W.Y? @? ?@V'L? ?W.Y @? ?@?N1? W.Y? @? ?@e@? 7H @? J5e@? ?J5? @? 7He@? W.Y? @? @?e@? ?W.Y @? @?e@? W.Y? @? @?e@? ?W.Y @? @?e@? W.Y? @? @?e@? ?W.Y @? @?e@? W.Y? @? ?J5?e@?hf?W.Y @? ?7H?e@?hfW.Y? @? ?@f@?he?W.Y @? ?@f@?heW.Y? @? ?@f@?h?W.Y @? ?@f@?hW.Y? @? ?@f@?g?W.Y @? ?@f@?gW.Y? @? ?@f@?f?W.Y @? J5f3LfW.Y? @? 7HfN1e?W.Y @? @[email protected]? @? @?f?@?W.Y @? @?f?@W&U? @? @?f?@@>1? @? @?fJ@V@@? @? @?e?W&@W@@? @? @?eW.Y@@Y@? @? ?J5?e7H?@5?@? @? ?7H??J5??(Y?@? @? [email protected]?f@? @? ?@?W.Yg@? @? [email protected]?g@? @? @? ?@(Yh@? @? @? J@H?h@?hf?J@? @? ?W&@he@?hf?7@L @? W&>5he3Lhf?@V1 @? ?W.R@HheN1hf?@?@ @? W.Y?@?he?@hf?@?@ @? ?W.Ye@?he?@h@?eJ5?@ @? W.Y?e@?he?@h@?e7H?@ @? ?W.Yf@?he?@g?J@?e@??3L? @? W.Y?f@?he?@g?7@Le@??N1? @? ?W.Yg@?he?@g?@V1e@?e@? @? W.Y?f?J5?he?@g?@?@?J5?e@? @? ?W.Yg?7H?he?@g?@?@?7H?e@? @? W.Y?g?@hf?@gJ5?@?@f@? @? ?W.Yh?@hf?@f@?&H?@?@f@? @? W.Y?h?@hf?@f@@@??3X@f3L @? ?W.Yhe?@hf?@f@V@??N@5fN1 @? W.Y?he?@hf?3L??J5?f@Hf?@ @?he@?e?W.YhfJ5hf?N1??7H?f@?f?@ @?he@?eW.Y?hf7H @??@g@?f?@ @?he@L?W.Y @? 3L?@hf?3L? @?h?J@)T.Y? @? N)T5hf?N1? @?h?7Y@(Y @? ?3@H 3L @?h?@?@H? @? ?V'? V/X? @?h?@?@ @? ?V/X @?h?@@@ ?J5? N1 @?h?@?@ ?.Y? ?@ @?g?W2@?@ ?@ ?N1? @?gW&@H?@ ?@ 3L @?f?W.Y@??@ ?@ N1 @?fW.Y?@??3L? ?@ ?3=? @?e?W.Y?J5??V/X J5 ?V46K? @?eW.Y?W&H?eV/K?hf7H ?I4@6Khg @??O&Y?O&@f?V46K?he@? I4@?hf @W2@@@@0M?g?I4@@@@@@@@@@@@? @@(M @@Y? @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@f @? ?@hf @? ?@hf @? ?@hf
W26KO26Xf@@@@@@ ?W-Kf?@g/X .MB@@
?W26X? ?7
W&?W26X? *@?7
W26KO26Xe?@@@@@@?@@@@@?W26Xf7R@@@@@?@?@@@??N1?e W-K?f@?f?/X?e .MB@@
Fig. 85. Protein profiles of wheat varietal gliadins (37). Absorbance, ) 280 nm MM (
Proportion of buf fer B ( 1 23 A
1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3
b-endorphin immunoreactivity , ) pg/tube 1 23 Sample. B 10 100 Column: 90 9 Flow rate: Buffer A. 80 8 70 7 Buffer B. 60 6 Gradient: 50 5 40 4 Detection: Fraction size: 30 3
0.2
2
20
0.1
1
10
1
10 20
30 40 50 Fraction No.
1
10
20 30
2 ml plasma from pregnant woman in labour Mono S HR 5/5 1.2 ml/min 0.05 M NH4 Ac, pH 5.5, 20% acetonitrile 0.05 M NH4 Ac, pH 5.5, 20% acetonitrile 2 min 0% B, 0-100% in 17 min 280 nm and RIA 0.6 ml
40 50 Fraction No.
Cation exchange FPLC of a 2-ml plasma sample collected from a pregnant woman in labour . A. The elution pattern ( ) of UV-absorbing material and the concentration of buf fer B (---). B. Elution ofb-endorphin immunoreactivity: the position of elution of referenceb-endorphin in (1). 125 [ ] b-endorphin (2) andb-lipotropin are shown by the vertical lines.
Fig. 86. Cation exchange of chromatography by plasma b-endorphin (38).
135
A280nm
b2 m
ALB
RBP + TSF
Sample: Column: Flow rate: Buffer A: Buffer B: Gradient: Detection:
0.5 ml desalted urine Mono Q HR 5/5 2 ml/min 6.25 mM Bis-Tris propane, pH 7.5 BufferA + 0.35 M NaCl, pH 9.5 0-100% in 25 ml 280 nm, 0.05AUFS
AGP + a1m
0
10
20
30 Vol (ml)
Urine protein chromatogram from a renal-transplant patient, illustrating the distinct peaks of the low MW proteins. The retinal-binding proteins (RBP) is contaminated with a small amount of transferrin (TSF). b2m = b2 -microglobulin,AGP =a1-acid glycoprotein,a1m = a1 -microglobulin, ALB = albumin. Diagonal line illustrates gradient from ferA buf to buffer B.
Fig. 87. Anion exchange of urine in renal proteinuria (39).
Purification of a recombinant Pseudomonas aeruginosa exotoxin A, PE553D This application shows a purification process for a genetically modified recombinant Pseudomonas aeruginosa exotoxin A (MW 55 000) expressed in the periplasm of E. coli. The process was developed for large scale production of modified toxin, conjugation to a polysaccharide and use as a vaccine. The purification strategy used chromatography media differentiated to tackle the problems of capture, intermediate purification and polishing (for further information please refer to Chapter 11.) The result was a highly purified exotoxin A from crude cell homogenate using only four chromatography steps, and taking less than half the time of a more conventional approach. Exotoxin A was captured directly from unclarified E. coli homogenate by expanded bed adsorption using STREAMLINE DEAE adsorbent in a STREAMLINE 200 column (Fig. 88). The following intermediate purification step was hydrophobic interaction chromatography (HIC) on Phenyl Sepharose 6 Fast Flow (high sub) packed in a BPG 200 column (Fig. 89). This step removed a substantial part of the UV absorbing material (including nucleic acids) that could interfere with the following steps. The second intermediate purification step on SOURCE 30Q, packed in FineLINE 100 column, removed the majority of the remaining contaminants (Fig. 90). The polishing step was HIC on SOURCE 15PHE (Fig 91). The process resulted in a pure protein, according to PAGE and RPC analysis (Fig 92 and 93), and the overall recovery was 51 % (Table 24).
136
Column: Medium: Sample:
Buffer A: Buffer B: Flow rate: System:
STREAMLINE 200 (i.d. 200 mm) STREAMLINE DEAE, 4.7 l 4.7 kg of cells were subjected to osmotic shock and suspended in a final volume of 180 litres 50 mM Tris buffer, pH 7.4 before application onto the expanded bed. 50 mM Tris buffer, pH 7.4 50 mM Tris, 0.5 M sodium chloride, pH 7.4 400 cm/h during sample application and wash 100 cm/during elution BioProcess Modular
?@@@ ?@?@ ?@@@ J(?'L? .Y?V/T.?e?@@@@?@@@@@? N@U?@@?@@@@?@?@?@? ?@)?fI4@?@?@?@? ?W2@@? ?7Y? ?W-Xe?W-X ?@@@@? ?'@@@@f ?.R/e?7R1 ?@@X ?@ ?V'XW5f ?@?@ S@@? O26X ?@ N@@Hf ?@K?e?3T5e?@@@ ?@@U ?@(MI/X? ?@ J@@Lf ?@@@?@?V+Yf?@ ?O2@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@6K S@@?hf?@H??N1? ?@ 7Y@1f ?@heW2@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@0M? I4@6X? ?'@U ?@f@? ?@ 3X@5f ?@h?W.M ?B1? ?S@)X?hf?@f@? ?@ S@@Uf ?@h?7H? 3L ?7@@?hf?@f@? ?@ 3=C5f ?@h@? ?@ ?V@Y ?@f@? ?@ V40Yf ?@h@? ?@ @@@@@?hf?@f@? ?@ ?@h@? ?@ ?@f@? ?@ O.f ?@h@? ?@ ?W.?@?hf?@f@? ?@ @@@@0Yf ?@h@? ?@ ?*U:5?hf?@f@? ?@ ?@h@? ?@ ?N@@U?hf?@f@? ?@ W2@@f ?@h@? ?@ ?J(R1?hf?@f@? ?@ ?W&Yg ?@h@? ?@ ?*U:5?hf?@f@? ?@ ?&@@@@f ?@h@? ?@ ?V40Y?hf?@f@? ?@ ?@h@? ?@ ?@f@? ?@ @@@@6Xe ?@h@? ?@ ?W2@@?hf?@f@? ?@ 3XW(R1e ?@h@? ?@ ?7@W5?hf?@f@? ?@ N@@U?@e ?@h@? ?@ ?@@@U?hf?@f@? ?@g@??W-KO-X?e?@?@@)f ?@h@? ?@ ?@?B1?hf?@f3L ?@g@??7R@@R1?fJ@@?f ?@h@? ?@ ?@e@?hf?@fN1 ?@g@??@?@@?@?f7Y@1f ?@h@? ?@ ?@@@@@@?he?@f?@ ?@@@@?e@??3T@@T5?f3X@5f ?@h@? ?@ ?@f?@ ?@g@??V+MI+Y?fV40Yf ?@h@? ?@ ?@f?@ ?@ ?@h@? ?@ ?@f?@ ?@ ?@@@@@f ?@h@? ?3L? '@@@@?hf?@f?@ ?@ ?@g ?@h@? ?N1? V'XW5?hf?@f?@ ?@ ?@g ?@h@? @? ?N@@H?hf?@f?@ ?@ ?@@@@@f @?e?W-Xf?@h@? @? ?J@@L?hf?@f?@ ?@ @?e?7R1f?@h@? @? ?7Y@1?hf?@f?@ ?@ @?e?@?@f?@h@? @? ?3X@5?hf?@f?@ ?@ @?e?3T5e?@@@h@? @? ?S@@U?hf?@f?@ ?@ @??@?V+Yf?@h@? @? ?7,?3T5 ?@h@?e7H ?@ ?3X@5?hf?@f?@ ?@f?V+Y?V+Y ?@h@?e@? ?@ ?V40Y?hf?@f?@ ?@ ?@h@? ?@ ?@f?@ ?@ ?@h@? ?@ ?@@6X?hf?@f?@ ?@ ?@h@? ?@ ?S,?hf?@f?@ ?@ ?@h@? ?@ ?@@@U?hf?@f?@ ?@ ?@h@? ?@ ?@?@1?hf?@f?@ ?@ ?@g?J@? ?@ ?3X@5?hf?@f?@ ?@ ?@g?@@? ?@ ?V40Y?hf?@f?@ ?@ ?@g?N@? ?@ ?@f?@ ?@ ?@h@? ?@ ?@K? ?@f?@ ?@ ?@h@? ?@ ?@@@@?hf?@f?@ ?@ ?@L??W.?e@? ?@ ?@f?@ ?@g@??W-X ?@@?W.Y?e@? ?@ ?@6K ?@f?@ ?@f?J@??7R1 ?@H?.Yf@? ?@ S@@?hf?@f?@ ?@f?7@L?@?@ ?@h@? ?@ ?@@U ?@f?@ ?@@@@??3@,?3T5 ?@h@? ?@ S@@?hf?@f?@ ?@f?V+Y?V+Y ?@h@? ?3L? ?'@U ?@f?@ ?@ ?@h@? ?N1? ?S@)X?hf?@f?@ ?@ ?@h@? @? ?7
Column: Medium: Sample: A: B: C: Flow rate:
Fig. 88. Capture by expanded bed adsorption on STREAMLINE DEAE.
BPG 200/500 (i.d. 200 mm) Phenyl Sepharose 6 Fast Flow (high sub), 4.7 L (150 mm bed height) 4.5 L of the previous pool were adjusted to 0.6 M ammonium sulphate and applied onto the column 50 mM phosphate, 0.7 M ammonium sulphate, pH 7.4 20 mM phosphate, pH 7.4 Distilled water 120 cm/h
? ? ? ? ? ? ? ? ?W-X ? W.R/X? ? 7H?N1? ? ?O2@@@@6K? ?J5?e3L ? ?W20M?e?I46K? ?7H?eN1 ? W.M?g?I46X? ?@f?@ ? @? ?O.Yhe?I/X ?@f?@ @? ? @? ?W20Y?hfN1 ?@f?@ @? ? @? W.M? ?3L? ?@f?@ @L ? @? ?W.Y ?N1? ?@f?@ ?J@1 ? @? ?7H? 3L ?@f?@ ?7Y@ ? @? J5 N1 ?@f?3L? ?@?@ ? @? 7H ?@ ?@f?N1? ?@?@ ? @? @? ?@ ?@g@? ?@?@ ? @? ?J5? ?3L? ?@g@? ?@?@ ? @? ?7H? ?N1? ?@g@? ?@?@ ? @? J5 @? ?@g@? ?@?3L? ? @? 7H @? ?@g@? ?@?N1? ? @? @? 3L ?@g@? ?@e@? ? @? @? N1 ?@g3L ?@e@? ? @? ?J5? ?@ ?@gN1 ?@e@? ? @? ?7H? ?@ ?@g?@ ?@e@? ? @? ?@ ?@ ?@g?@ ?@e@? ? @? ?@ ?3L? ?@g?@ ?@e@? ? @? ?@ ?N1? ?@g?@ ?@e@? ? @? J5 @? ?@g?@ ?@e@? ? @? 7H @? ?@g?3L? ?@e@? ? @? @? @? ?@g?N1? ?@e@? ? @? @? @? ?@h@? ?@e@? ? @? @? 3L ?@h@? ?@e@? ? @? @? N1 ?@h@? ?@e3L ? @? @? ?@ ?@h@? ?@eN1 ? @? @? ?@ ?@h@? ?@e?@ ? @? @? ?@ ?@h3L ?@e?@ ? @? @? ?@ ?@hN1 ?@e?@ ? @? @? ?@ ?@h?@ ?@e?@ ? @? ?J5? ?3L? ?@h?@ ?@e?@ ? @? ?7H? ?N1? ?@h?@ ?@e?@ ? @? ?@ @? J5h?@ ?@e?@ ? @? ?@ @? 7Hh?@ ?@e?@ ? @? ?@ @? @?h?@ ?@e?@ ? @? ?@ @? @?h?3L? ?@e?@ ? @? ?@ 3L @?h?N1? ?@e?@ ? @? ?@ N1 @?he@? ?@e?@ ? @? ?@ ?@ @?he@? ?@e?@ ? @? ?@ ?@ @?he@? J5e?@ ? @? ?@ ?@ @?he@? 7He?@ ? @? ?@ ?@ @?he@? @?e?3L? ? @? ?@ ?@ @?he3L @?e?N1? ? @? ?@ ?3L? @?heN1 @?f@? ? @? ?@ ?N1? @?he?@ @?f@? ? @? ?@ @? @?he?@ @?f@? ? @? ?@ @? @?he?@ @?f@? ? @? ?@ @? @?he?@ @?f@? ? @? J5 @? @?he?@ @?f@? ? @? 7H 3L @?he?3L? @?f@? ? @? @? N1 @?he?N1? @?f@? ? @? @? ?@ @?hf@? @?f@? ? @? @? ?@ @?hf@? @?f@? ? @? @? ?@ @?hf@? @?f@? ? @? @? ?@ @?hf@? @?f@? ? @? @? ?@ @?hf@? @?f@? ? @? @? ?3L? @?hf@? @?f@? ? @? @? ?N1? @?hf3L @?f3L ? @? @? @? @?hfN1 @?fN1 ? @? @? @? @?hf?@ @?f?@ ? @? @? @? @?hf?@ @?f?@ ? @? @? @? @?hf?@ @?f?@ ? @? @? 3L @?hf?@ @?f?@ ? @? @? N1 @?hf?@ @?f?@ ? @? @? ?@ @?hf?3L? @?f?@ ? @? @? ?@ @?hf?N1? @?f?@ ? @? ?J5? ?@ @? @? @?f?@ ? @? ?7H? ?@ @? @? @?f?@ ? @? ?@ ?@ @? @? @?f?@ ? @? ?@ ?3L? @? @? @?f?@ ? @? ?@ ?N1? @? @? @?f?@ ? @? ?@ @? @? 3L @?f?@ ? @? ?@ @? @? N1 @?f?@ ? @? ?@ @? @? ?@ @?f?@ ? @? ?@ @? @? ?@ @?f?3L? ? @? ?@ 3L @? ?@ @?f?N1? ? @? ?@ N1 @? ?@ @?g@? ? @? ?@ ?@ @? ?@ @?g@? ? @? ?@ ?@ @? ?@ @?g@? ? @? ?@ ?@ @? ?3L? @?g@? ? @? ?@ ?@ @? ?N1? @?g@? ? @? ?@ ?@ @? @? @?g@? ? @? ?@ ?3L? @? @? @?g@? ? @? J5 ?N1? @? @? @?g@? ? @? 7H @? @? @? @?g@? ? @? @? @? @? 3L ?J5?g@? ? @? @? @? @? N1 ?7H?g@? ? @? @? @? @? ?@ ?@h@? ? @? @? 3L @? ?@ ?@h@? ? @? @? N1 @? ?@ ?@h@? ? @? @? ?@ ?J5? ?@ ?@h@? ? @? @? ?@ ?7H? ?3L? ?@h3L ? @? @? ?@ ?@ ?N1? ?@hN1 ? @? @? ?@ ?@ @? ?@h?@ ? @? @? ?3L? ?@ @? ?@h?@ ? @? @? ?N1? ?@ @? ?@h?@ ? @? @? @? ?@ @? ?@h?@ ? @? @? @? ?@ 3L ?@h?@ ? @? @? @? ?@ N1 ?@h?@ ? @? @? 3L ?@ ?@ ?@h?@ ? @? @? N1 ?@ ?@ ?@h?@ ? @? ?J5? ?@ ?@ ?3L? ?@h?@ ? @? ?7H? ?@ ?@ ?N1? ?@h?@ ? @? ?@ ?@ ?@ 3L ?@h?3L? ? @? ?@ ?@ ?@ N1 ?@h?N1? ? @? ?@ ?3L? ?@ ?3L? ?@he@? ? @? ?@ ?N1? ?@ ?N1? ?@he@? ? @? ?@ @? ?@ 3L ?@he@? ? @? ?@ 3L ?@ V/X? ?@he3L ? @? ?@ N1 ?@ ?N1? ?@heN1 ? @? ?@ ?3L? ?@ 3L ?@he?3L? ? @? ?@ ?N1? ?@ V/X? ?@he?N1? ? @? ?@ 3L ?@ ?V/X ?@hf3L ? @? ?@ N1 ?@ N1 ?@hfN1 ? @? ?@ ?3L? ?@ ?3L? J5hf?3L? ? @? ?@ ?N1? ?@ ?V/K 7Hhf?V/X ? @? ?@ 3L ?@ V46K @? V/K?hg? @? J5 N1 ?@ I46X @? ?V46X?hf? @? 7H ?3L? ?@ I/K? @? ?I/Xhf? @? @? ?V/X ?@ ?V46X? @? V/K?he? @? ?J5? V/X? ?@f?@@@g?@ ?I/K @? ?V46K?h? @? ?7H? ?V/X ?@f?@?@W-KO-X?@ V46K ?J5? ?I4@h? @? J5 V/X? J5f?@@@@R@@R1?@ I46K ?7H? ? @? 7H ?V/K 7Hf?@?I'T@@T5?@ I46KhfJ5 ? @? @? V46K @?f?@eV+MI+Y?@ I4@6K?g?O.Y ? @?hf?J5? I46K @? ?I4@@@@@@@@0Y? ? @?hf?7H? I46K ?J@L ? @?hfC5 I4@6K? O.R)K? O@ ? @?heW20Y ?I4@@@@@@@@@@@@6K?fO20Y?@@@@@@@@@@@@@@@@@@@@@@@ ? @?h?W.Mf?W.?hf?@ ?@@?h@? ?I4@@@@@0Me?@ ?@ ? @?hO.Y?f?7Y?e@?2@@@@@6X?@W)X?e@?26X?@6X@@?W.e@?@?@W-T2@@? ?@ ?@ ? @?gO20Yg?@@@?J@@@?@?@@V1?@@@)??J@@@V1?@V@@@?7H?J@@@?@@R@@?@? ? @?fO20Mh?3T5?*U@@?@?@@W5?@@Xe?*U@@W5?@W@@@?3L?*U@@?@@T@@?@? ? @?eO2(Mhe?V+Y?V4@@?@?3@(Y?(R/e?V4@@(Y?@(R4@?V/?V4@@?(R+R'?@?he@K @K @Khe? @W2@@@H? V'Y?h?I(Ye(Y @@6Ke@KO2@? @@6Ke@KO2@? @@6Ke@?f? @@@@@@ @@@@6X@@@@@?hfO26Ke?O@?@KgO26K @@@@6X@@@@@? @@@@6X@?f? @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?f? @V4@@@ ?@W(Y@?@@?@?W@?@(M?@?@ ?I4@0Me@V4@@?hf?@@X?@?@@?@?@@@@e?@@X @@@0M?@V4@@? ?@ @@@0M?@?f? @?eI@ ?@@U?@?@@?@?7@@@H??@@@ @??I@?hf?@V1?@?@@?@?3X?@e?@V1 @0M?e@??I@? ?@ @0M?e@?f? @? ?@V1?@?@@?@?3X?@eJ(?'L? ?@@@?@@@@?@?V/?@e?@@@ ?3=O.? ? @? ?@@@?@@@@?@?V/[email protected]?V/? ?V40Y? ? @? ? @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?? @? @? @? @? ? @? @? @? @? ? @? @? @? @? ? ? ? ?W2@ W&?W26X? W&?W26X? ?W26X?W& ? ?7Y? *@?.MB1? *@?*US,? ?.MB1?7@ ? ?@@6X? N@f@? N@?S@@U? 3T@@ ? ?@?B1? ?@?W.? ?@?7
W-X? 7R1? ?J@?@? ?7@@@@6KO-X?W-K? ?@e?@V4@?,?7R@@@@@@@@@? ?@e?@eS@U?@?@@?@@?@?@? ?@?O&?,?3T@@?@@?@?@? ?@@0R+Y?V+R'?@@?@?@?
Fig. 89. Intermediate purification by hydrophobic interaction chromatography on Phenyl Sepharose 6 Fast Flow (high sub).
137
Column: Medium: Sample: Buffer A: Buffer B: Gradient: Flow rate:
FineLINE 100 (i.d. 100 mm) SOURCE 30Q, 375 ml (50 mm bed height) from the previous pool, diluted 1 to 3 with distilled water 1.5 l/cycle were applied 20 mM phosphate, pH 7.4 Buffer A + 1.0 sodium chloride 0 to 50% B, 20 column volumes 600 cm/h
?W-X? ?7R1? ?@@@? J(?'L W-KO-KO-K?g .Y?V/?S@>@@@@T@@V@@V@H *?@@R@@R@@6T26T. ?&@0R+MI+R'?@@?@?
W26Xe?@@6T26X 7
W26Xe?W&?W26X 7
W26XeW26KO26X 7
?@@6K?? ?S@6X ?@@@UI/ J@?S,?? @@@0Y??? /T2@(?? V4@@U?? ?S,?? ?@@0Y?? ? /T2@@?? S@Y?@?? *@@@@?? N@e@?? ?3=C5?? ?S@@U?? ?&@@1?? @?? ?C5?? W2@@U?? *@@@1?? N@e@?? ?3=C5?? ?N@@U?? ?J@@)?? ?7
W26XeW26KO26X 7
W26Xe?@@?W26X 7
@@@6X?g?@ @??S)T26KO26X@ @@@0R@
W26XeW26X 7
W26XeW26X .MS,e7
?W&?eW26X W&@Le7
W2@@eW26X 7Yf7
W26XeW26X 7YS,e7
?@@?W26XeW26X ?N@?7
?@@?W26XeW26X ?N@?.MS,e7
/X?W. @? ?W.?@@?@K?h?/X N1?7H W26X@?@??@@6T-KO26X??7H?@??@@@6T26KO)X?N1 ?@?@? 7
Fig. 90. Intermediate purification by ion exchange chromatography on SOURCE 30Q.
?@@??@@@h/Ke? ?N@?J@X?e@6T-X?S@@6X @?@@)Xe@V@R1?7@?B1 @?eS,e@?@?@?@@e@ @?@@0Ye@?@?@?@@e@? ?
Column: Medium: Sample: Buffer A: Buffer B: Gradient: Flow rate:
35 mm i.d. SOURCE 15 HPE from the previous step, adjusted to 1.0 M ammonium sulphate, 0.5 l/cycle was applied 1.0 M ammonium sulphate, 50 mM phosphate, pH 7.4 50 mM phosphate 0-45%B, 15 column volumes 200 cm/h
?W-X ?7R1 ?@?@ J@@@ .Me@6KO)T-Kg ?V@@@@?@@@@@@? @@0?(R+R+?4@@?
W26X?@@?e@@@ 7
W26X?@@??W26X 7
?/K?? ?S@@@ W.MW5 ?W&YO.Y ?7@@@U? ?@?4@)?? ?@6X? S,? ?@@U? ?/T@@1? ?S@YW5? ?&@@@U? W(R1? *U:5? V40Y?? ?@@@? W5? O2@U? ?'@@@1? ?V'XW5? V40Y?? @@@@? 3Xe? V4@@?? W2@@? *UW5? V40Y? @@?@@? @H?N@? 3=?C5? V4@0Y?
W26X?W26X?@@@ 7
@@6Xg?@ @?S,g?@ @@0YW-X?W-X@ @?e*?,?*?@@ @?eV+Y?V+R'
W26X?W26KO26X 7
138
W26KO2@ .MS@@
?W&?W2@ W&@?7
W26KO2@ 7YS@@
@@@? @?heW.g@?e?/X N@H? 7Hhe?N1 ?@e @@6T-X?W26X?@?@6T-X?@@6Xe@ ?@e@@?@?@?V40Y?3T5?@?@?@@?@?J5 @@V@R1?*@@(?@?@V@R1?@@V1e@ ?@e V+Y?h?.Y
Fig. 91. Polishing by hydrophobic interaction chromatography on SOURCE 15PHE.
a Lane 1 LMW markers Lane 2 pool from SOURCE 15 PHE Lane 3 pool from SOURCE 30Q Lane 4 pool from Phenyl Sepharose 6 Fast Flow (high sub) Lane 5 LMW markers
1
2
3
4
5
b
1
2
3
Lane 1 pool from Phenyl Sepharose 6 Fast Flow (high sub) Lane 2 pool from SOURCE 30Q Lane 3 pool from SOURCE 15PHE
Fig. 92. a) SDS-PAGE on PhastGel Gradient 8-25 b) Native PAGE on PhastGel Gradient 8-25 Table 24 Purification step
Volume, litre
Total protein, gram
Exotoxin A, gram*
Step recovery*
Bacterial extract STREAMLINE DEAE Phenyl Sepharose 6 Fast Flow (high sub)
180
351
10.8
13.5
140
8.54
79
11.4
41
6.60
77
SOURCE 30Q SOURCE 15PHE
30.2 12.2
12.6 n.d.
6.04 5.5
91 91
*Activity was determined with a radial immunodiffusuin assay using Goat anti-exotoxin A antibodies (List, USA).
139
Column: Sample:
?W-X? ?7R1? ?@?@? J@@@L 7@T5?3T@@V@@V@R1 ??&@0R+Y?V+R'?@@?@?@
W26Xe?W2@6T26X? 7
a)
Sample load: A: B: Gradient: Flow rate: System:
W26XfW&?W26X? 7
µRPC C2/C18, SC 2.1/10 a) pool from Phenyl Sepharose 6 Fast Flow (high sub) b) Pool from SOURCE 30Q c) Pool from SOURCE 15 PHE 50µL 0.1% trifluoroacetic acid (TFA) in water 0.1% TFA in acetonitrile 25-75% B over 47 minutes 150 µL/min SMART System
W26Xe?W26KO26X? 7
W26Xe?W26KO26X? 7
W26XfW&?W26X? 7
W26Xe?W26KO26X? 7
W26Xe?W26X .MB1e?7
?W&?e?W26X W&@?e?7
W2@@e?W26X 7Yf?7
W26Xe?W26X @?e@ @? ?@h@?e@? 7
?W-X? ?7R1? ?@?@? J@@@L 7@T5?3T@@V@@V@R1 ??&@0R+Y?V+R'?@@?@?@
W26Xe?W26KO26X? 7
b)
W26Xe?W26KO26X? 7
W26XfW&?W26X? 7
W26Xe?W26KO26X? 7
W26Xe?W26X .MB1e?7
?W&?e?W26X W&@?e?7
W2@@e?W26X 7Yf?7
W26Xe?W26X 7
@?e@ @? ?@h@?e@? 3L?J5 @? J5h@?e3L N1?7H W26X@?@??@?@6T-X?W26X?e7H?@6T-Xe@?eN1 ?@?@? 7
?W-X? ?7R1? ?@?@? J@@@L 7@T5?3T@@V@@V@R1 &@0R+Y?V+R'?@@?@?@
W26XfW&?W26X? 7
W26Xe?W26KO26X? 7
c)
W26Xe?W26KO26X? 7
W26XfW&?W26X? 7
W26Xe?W26KO26X? 7
W26Xe?W26X 7
W26Xe?W26X .MB1e?7
?W&?e?W26X W&@?e?7
W2@@e?W26X 7Yf?7
W26Xe?W26X 7
@?e@ @? ?@h@?e@? 3L?J5 @? J5h@?e3L N1?7H W26X@?@??@?@6T-X?W26X?e7H?@6T-Xe@?eN1 ?@?@? ?3T5?7
Fig. 93. Reversed phase chromatography analysis of pools obtained after the purification steps.
140
Strategy The starting point for developing the purification strategy was experience from a successful downstream process for purification of another modified Pseudomonas aeruginosa exotoxin, LysPE38 (Fig. 94). Refinements included reducing the number of steps by the introduction of STREAMLINE DEAE for capture, and achieving high flow rates through the use of high performance SOURCE media for late intermediate purification and polishing.
Fig. 94. Comparison of the two purification schemes.
141
This approach of using media designed for different stages in a downstream process speeded up process development and shortened the production schedule. Comparison with the earlier process for purification of a similar exotoxin demonstrates the dramatic savings in time achieved. Interestingly, the use of ion exchange at more than one stage is a common characteristic of large-scale processes. Note that the role and mode of use of the ion exchanger is very different in the different stages. In the example shown, the anion exchanger, STREAMLINE DEAE, is used for expanded bed adsorption of the product from the crude, unfiltered E. coli lysate. STREAMLINE is optimised to handle such crude feedstocks. Particles, the bulk of the impurities and much of the water are removed in a group separation. Sample application conditions are optimised to achieve maximum selectivity during capture of the product and the stepwise elution conditions achieve maximum concentration during elution. Later in the process, anion exchange is again used, but this time to remove proteins which have very similar characteristics to the product. This time SOURCE 30Q is used with gradient elution. The uniform, small diameter particles help to achieve good resolution with excellent flow rates and low back-pressures. The linear salt gradient further improves resolution. Note also that a different pH has been chosen to increases the binding strength of the product and further improve the resolution during elution. In other examples ion exchange can be used repeatedly in the same process under even more divergent conditions, in one step to bind only impurities and allow the product to pass through and later to bind the product. Furthermore, anion and cation exchange can be combined in the same scheme or pH and salt gradient elution can be alternated to achieve removal of different impurities. All of this is an indication of why ion exchange is such a useful technique in industrial purification.
142
13. Fault finding chart Problem
Cause
Column is clogged. Presence of lipoproteins or protein aggregates.
No flow through the column
Reduced or poor flow through the column.
Remedy Prior to chromatography, precipitate with 10% Dextran Sulphate or 3% polyvinylpyrrolidone.
Precipitation of proteins in the column caused by removal of stabilizing agents during fractionation.
Modify the eluent to maintain stability.
Filter is clogged.
Replace the filter. Always filter samples and buffer before use.
Microbial growth has occurred in the column.
Microbial growth rarely occurs in columns during use, but steps should always be taken to prevent infection of packed columns, buffers and gel suspensions. Store gel in the presence of 20% ethanol or an antimicrobial agent, see page 103.
Outlet closed.
Open outlet.
No flow from pump.
With peristaltic pumps check the condition of the tubings. Check for leaks at all connections.
Clogged end-piece or adaptor or tubing.
Remove and clean, if possible.
Bed surface blocked by precipitated proteins.
Clean using recommended methods.
143
Problem
Cause
Remedy
Reduced or poor flow through the column.
Bed compressed.
Repacking the column may be necessary.*
Microbial growth.
Microbial growth rarely occurs in columns during use, but steps should always be taken to prevent infection of packed columns, buffers and gel suspensions. Store gel in the presence of 20% ethanol or an antimicrobial agent, see page 103.
Fines.
Do not use a magnetic stirrer; it can break the beads.
Back pressure Turbid sample has been increases during a applied to the column. run or during successive runs.
Improve sample solubility by the addition of monoethylene glycol, detergents or organic solvents.
Clogged column filter.
Prefilter buffers and samples. Change filter.
Precipitation of protein in the column filter and/ or at the top of the gel bed.
Clean the column and exchange or clean the filter. Change pH and/or add urea. Develop a procedure with detergents. Additives which were used for initial sample solubilization should be included in the solutions used for chromatography.
* Does not apply for pre-packed MonoBeads, MiniBeads, RESOURCE, HiTrap, HiLoad and BioPilot columns.
144
Problem
Cause
Remedy
Back-pressure Precipitation of increases during a lipoproteins at increased run or during ionic strength. successive runs.
Lipoproteins may be precipitated prior to chromatography by the addition of 10% dextran sulphate and1 M calcium chloride to final concentrations of 0.2% and 0.5 M respectively.
The protein does not elute in the salt gradient.
Incorrect buffer pH.
Use a buffer pH closer to the pI of the protein.
Ionic strength too low.
Use a more concentrated limit buffer.
The protein does not elute.
Solutions have wrong pH.
Calibrate your pH meter, prepare new solutions and try again.
Protein elutes in the wash phase.
Ionic strength of start buffer is too high.
Decrease ionic strength of start buffer.
The ionic strength of the sample is too high or the pH is wrong.
Buffer exchange on sample.
The column is not properly equilibrated.
Repeat or prolong the equilibration step.
Ionic detergents or other additives are adsorbed to the column.
Clean the column.
The gradient slope is too steep.
Use a shallower gradient or a plateau in the gradient.
Microbial growth has occurred in the column.
See above.
Flow rate is too high.
Run the separation at a lower flow rate.
The resolution obtained is less than expected.
145
Problem
Cause
Remedy
The resolution obtained is less than expected.
Proteins or lipids have precipitated on the column.
Clean and regenerate the column.
Improper filtration of the sample before application to the column.
Regenerate the column, filter the sample and repeat the chromatography step.
Aggregate formation of proteins in sample and strong binding to gel.
Use urea or zwitterions, betaine up to 10% or taurine up to 4%.
Column is poorly packed.
Check the packing by running a coloured compound and observing the band. Repack the column if necessary.*
Too much sample mass has been loaded onto the column.
Decrease the sample load.
The column is dirty.
Clean and regenerate the column.
Detector cell volume is too big.
Change the flow cell.
Large mixing spaces in or after column.
Reduce all post column volumes.
Overloaded column.
Decrease the sample load and repeat the run.
Leading or very rounded peaks observed in the chromatogram.
* Does not apply for pre-packed MonoBeads, MiniBeads, RESOURCE, HiTrap, HiLoad and BioPilot columns.
146
Problem
Cause
Remedy
Leading or very rounded peaks observed in the chromatogram.
Column is poorly packed.
Check the packing by running a coloured compound and observing the band. Repack the column if necessary.*
Column needs regeneration.
Clean and regenerate the column. If this does not help replace with a new one.
Tailing of the peak Sample too viscous. is observed in the chromatogram.
Previous elution profile cannot be reproduced.
Previous elution profile cannot be reproduced.
Reduce the amount of protein.
Precipitation of protein in the column filter and/ or at the top of the gel bed.
Remove nucleic acids. Clean the column and exchange or clean the filter.
Incorrect buffer pH and ionic strength.
Prepare new solutions.
The sample has altered during storage.
Prepare fresh sample.
Proteins or lipids have precipitated on the column.
Clean and regenerate the column.
Sample has not been filtered properly.
Regenerate the column, filter the sample carefully and repeat this step.
Incomplete equilibration.
Equlibrate until conductivity is constant.
Aggregate formation of proteins in sample and strong binding to gel.
Use urea or zwitterions, betaine up to 10% or taurine up to 4%.
* Does not apply for pre-packed MonoBeads, MiniBeads, RESOURCE, HiTrap, HiLoad and BioPilot columns.
147
Problem
Cause
Remedy
Low recovery of activity while normal recovery of protein.
Sample substance may not be stable in the elution buffers and is therefore inactivated.
Determine the pH and salt stability of the protein.
Enzyme separated from co-factor or similar.
Test by pooling fractions and repeating the assay.
Microbial growth.
Microbial growth rarely occurs in columns during use, but steps should always be taken to prevent infection of packed columns, buffers and gel suspensions. Store gel in the presence of 20% ethanol or an antimicrobial agent, see page 103.
Protein amount in The protein may have the eluted fractions been degraded by is much less than proteases. expected. Adsorbtion to filter during sample preparation.
Use another type of filter or use detergents.
Microbial growth has occurred in the column.
Microbial growth rarely occurs in columns during use, but steps should always be taken to prevent infection of packed columns, buffers and gel suspensions. Store gel in the presence of 20% ethanol or an antimicrobial agent, see page 103.
Protein amount in Non-specific adsorption. the eluted fractions is much less than expected.
148
Add protease inhibitors to the buffers to prevent proteolytic digestion.
Try adding ethylene glycol (e.g. 10%) to the buffers to prevent any hydrophobic interactions.
Sample precipitates.
May be caused by removal of salts or sample dilution.
Hydrophobic proteins.
Chaotropic salts may be used for elution.
Problem
Cause
Remedy
More activity is recovered than was applied to the column
Different assay conditions have been used before and after the chromatographic step.
Use the same assay conditions for all the assays in your purification scheme.
Removal of inhibitors during separation.
Replace if necessary.
Wrong sensitivity range on detector.
Adjust.
Sample absorbs poorly at the chosen wavelength.
Use a different wavelength.
Recorder range incorrectly set.
Adjust.
Excessive zone broadening
Check the column packing and re-pack if necessary.
Peaks too small.
Bubbles in the bed. Column packed or stored at cool temperature and then warmed up.
Eluent not properly de-gassed.
Small bubbles can often be removed by passing well de-gassed buffer upwards through the column. Column may need to be re-packed. Take special care if buffers are used after storage in a fridge or coldroom. Do not allow column to warm up due to sunshine or heating system. A waterjacket is a good safeguard. Use de-gassed buffers. De-gas the eluent thoroughly.
Cracks in the bed. Large air leak in column.
Check all connections for leaks. Repack the column*.
Distorted bands as Air bubble at the top sample runs into of the column or the bed. in the inlet adaptor.
Re-install the adaptor taking care to avoid air bubbles.
* Does not apply for pre-packed MonoBeads, MiniBeads, RESOURCE, HiTrap, HiLoad and BioPilot columns.
149
Problem
Cause
Remedy
Distorted bands as sample runs into the bed.
Particles in eluent or sample.
Filter or centrifuge the sample. Protect eluents from dust.
Clogged or damaged net in upper adaptor.
Dismantle the adaptor, clean or replace the net. Keep particles out of samples and eluents.
Distorted bands as Column poorly packed. sample passes down the bed.
Gel suspension too thick or too thin. Bed packed at a temperature different from run. Bed insufficiently packed (too low packing pressure, too short equilibration). Column packed at too high pressure.
Negative peaks at Refractive index effects. solvent front.
Buffer exchange the sample to start buffer.
Strange peaks in chromatogram.
Buffer impurities.
Clean the buffer by running it through precolumn. Use high quality reagents.
Peaks on blank gradients.
Incomplete elution.
Wash the column according to recommended method.
Spikes in chromatogram.
Air bubble trapped in UV cell.
Use de-gassed solutions.
UV baseline rises with gradient.
Salt concentration, micelle formation.
Work well below or above the CMC or change the gradient so that the increase in UV absorption does not occur while the samples are eluting.
Impurities in buffers.
Use high quality reagents.
150
14. Ordering information Product MonoBeads Mono Q, PC 1.6/5 Mono Q HR 5/5 Mono Q HR 10/10 Mono Q HR 16/10 Mono Q 35/100 Mono Q 60/100 Mono S, PC 1.6/5 Mono S HR 5/5 Mono S HR 10/10 Mono S HR 16/10 Mono S 35/100 Mono S 60/100
Quantity/Pack Size 1 1 1 1 1 1 1 1 1 1
Code No.
1
17-0671-01 17-0546-01 17-0556-01 17-0506-01 17-1001-01 17-1002-01 17-0672-01 17-0547-01 17-0557-01 17-0507-01 17-1021-01 17-1022-01
MiniBeads Mini Q, PC 1.6/5 Mini S, PC 1.6/5 Precision Column Holder
1 1 1
17-0671-01 17-0671-01 17-1455-01
SOURCE Q RESOURCE Q 1 ml RESOURCE Q 6 ml SOURCE 15Q SOURCE 15Q SOURCE 15Q SOURCE 15Q SOURCE 15Q SOURCE 30Q SOURCE 30Q SOURCE 30Q SOURCE 30Q SOURCE 30Q
1 1 10 ml 50 ml 200 ml 500 ml 1l 10 ml 50 ml 200 ml 500 ml 1l
17-1177-01 17-1179-01 17-0947-20 17-0947-01 17-0947-05 17-0947-02 17-0947-03 17-1275-10 17-1275-01 17-1275-02 17-1275-03 17-1275-04
SOURCE S RESOURCE S 1 ml RESOURCE S 6 ml SOURCE 15S SOURCE 15S SOURCE 15S SOURCE 15S SOURCE 15S SOURCE 30S SOURCE 30S SOURCE 30S SOURCE 30S SOURCE 30S
1 1 10 ml 50 ml 200 ml 500 ml 1l 10 ml 50 ml 200 ml 500 ml 1l
17-1178-01 17-1180-01 17-0944-10 17-0944-01 17-0944-05 17-0944-02 17-0944-03 17-1273-20 17-1273-01 17-1273-02 17-1273-03 17-1273-04
151
Product
Quantity/Pack Size
Code No.
5 x 1 ml 5 x 5 ml 1 1 1 1 75 ml 1l 5l
17-1153-01 17-1154-01 17-1064-01 17-1066-01 17-1011-21 17-1012-21 17-1014-01 17-1014-03 17-1014-05
5 x 1 ml 5 x 5 ml 1 1 1 1 75 ml 1l 5l
17-1151-01 17-1152-01 17-1137-01 17-1138-01 17-1031-21 17-1032-21 17-1087-01 17-1087-03 17-1087-04
1 1 25 ml 300 ml 5l
17-1060-01 17-1062-01 17-0510-10 17-0510-01 17-0510-04
1 1 25 ml 300 ml 5l
17-1135-01 17-1136-01 17-0729-10 17-0729-01 17-0729-04
25 ml 500 ml 10 l 60 l
17-0709-10 17-0709-01 17-0709-05 17-0709-60
25 ml 500 ml 10 l 60 l
17-0719-10 17-0719-01 17-0719-05 17-0719-60
Q Sepharose High Performance HiTrap Q HiTrap Q HiLoad 16/10 Q Sepharose HP HiLoad 26/10 Q Sepharose HP BioPilot Column Q Sepharose HP 35/100 BioPilot Column Q Sepharose HP 60/100 Q Sepharose High Performance Q Sepharose High Performance Q Sepharose High Performance SP Sepharose High Performance HiTrap SP HiTrap SP HiLoad 16/10 SP Sepharose HP HiLoad 26/10 SP Sepharose HP BioPilot Column SP Sepharose HP 35/100 BioPilot Column SP Sepharose HP 60/100 SP Sepharose High Performance SP Sepharose High Performance SP Sepharose High Performance
Q Sepharose Fast Flow HiLoad 16/10 Q Sepharose Fast Flow HiLoad 26/10 Q Sepharose Fast Flow Q Sepharose Fast Flow Q Sepharose Fast Flow Q Sepharose Fast Flow SP Sepharose Fast Flow HiLoad 16/10 SP Sepharose Fast Flow HiLoad 26/10 SP Sepharose Fast Flow SP Sepharose Fast Flow SP Sepharose Fast Flow SP Sepharose Fast Flow DEAE Sepharose Fast Flow DEAE Sepharose Fast Flow DEAE Sepharose Fast Flow DEAE Sepharose Fast Flow DEAE Sepharose Fast Flow CM Sepharose Fast Flow CM Sepharose Fast Flow CM Sepharose Fast Flow CM Sepharose Fast Flow CM Sepharose Fast Flow
152
Product
Quantity/Pack Size
Code No.
Q Sepharose Big Beads Q Sepharose Big Beads
1l 10 l
17-0989-03 17-0989-05
SP Sepharose Big Beads SP Sepharose Big Beads
1l 10 l
17-0657-03 17-0657-05
STREAMLINE DEAE STREAMLINE DEAE
300 ml 7.5 l
17-0994-01 17-0994-02
STREAMLINE SP STREAMLINE SP
300 ml 7.5 l
17-0993-01 17-0993-02
500 ml 10 l 500 ml 10 l
17-0710-01 17-0710-05 17-0720-01 17-0720-05
500 ml 10 l
17-0500-01 17-0500-05
DEAE Sephadex A-25 DEAE Sephadex A-25 DEAE Sephadex A-25 DEAE Sephadex A-25
100 g 500 g 5 kg 40 kg
17-0170-01 17-0170-02 17-0170-03 17-0170-07
DEAE Sephadex A-50 DEAE Sephadex A-50 DEAE Sephadex A-50 DEAE Sephadex A-50
100 g 500 g 5 kg 40 kg
17-0180-01 17-0180-02 17-0180-03 17-0180-07
QAE Sephadex A-25 QAE Sephadex A-25 QAE Sephadex A-25
100 g 500 g 5 kg
17-0190-01 17-0190-02 17-0190-03
QAE Sephadex A-50 QAE Sephadex A-50 QAE Sephadex A-50
100 g 500 g 5 kg
17-0200-01 17-0200-02 17-0200-03
CM Sephadex C-25 CM Sephadex C-25 CM Sephadex C-25 CM Sephadex C-25
100 g 500 g 5 kg 40 kg
17-0210-01 17-0210-02 17-0210-03 on request
Sepharose Big Beads
STREAMLINE
Sepharose CL-6B DEAE Sepharose CL-6B DEAE Sepharose CL-6B CM Sepharose CL-6B CM Sepharose CL-6B
Sephacel DEAE Sephacel DEAE Sephacel Sephadex
153
Product
Quantity/Pack Size
Code No.
CM Sephadex C-50 CM Sephadex C-50 CM Sephadex C-50
100 g 500 g 5 kg
17-0220-01 17-0220-02 17-0220-03
SP Sephadex C-25 SP Sephadex C-25 SP Sephadex C-25 SP Sephadex C-25
100 g 500 g 5 kg 40 kg
17-0230-01 17-0230-02 17-0230-03 on request
SP Sephadex C-50 SP Sephadex C-50 SP Sephadex C-50
100 g 500 g 5 kg
17-0240-01 17-0240-02 17-0240-03
154
15. References 1.
The right step at the right time. Bio/Technology, 4, 954-958 (1986), Bonnerjera, J., Oh, S., Hoare, M., Dunhill, P.
2.
Chromatography of Proteins. I. Cellulose ion exchange adsorbents. J. Amer. Chem. Soc. 78 (1956) 751 755, Peterson, E.A., Sober, H.A.
3.
Chromatography of proteins on ion-exchange adsorbents. Meth. Enzymol. 22 (1971) 273—286, Himmelhoch, S.R.
4.
Chromatography: a laboratory handbook of chromatographic and electrophoretic techniques. Heftman, E. (Ed.), Van Noostrand Rheinhold Co., New York (1975).
5.
Dynamics of chromatography, Part 1, Principles and theory. Giddings, J.C., Keller, R.A. (Eds.), Marcel Dekker Inc., New York (1965).
6.
Ion exchange chromatographic characterization of stinging insect vespid venoms. Toxicon (Pergamon Press), 22,1 (1984) 154-160, Einarson, R., Renck, B.
7.
Physicochemical considerations in the use of MonoBeads for the separation of Biological Molecules. Protides of the Biological Fluids, 30 (1982) 629-634, Söderberg, L. et al.
8.
Gel Filtration in Theory and Practice, Pharmacia Biotech, S-75182 Uppsala, Sweden.
9.
The separation of human globin chains by ion-exchange chromatography on CM Sepharose CL-6B. Hemoglobin 3 (1979)13—20, Sparham, S.J., Huehns, E.R.
10. Agar derivatives for chromatography, electrophoresis and gel-bound enzymes. I. Desulphated and reduced cross-linked agar and agarose in spherical bead form. J Chromatogr. 60(1971)161—177, Porath, J., Janson, J.-C., Laas, T. 11. Ion exchanger from pearl-shaped cellulose gel. Nature 223 (1969) 499—500, Determann, H., Meyer, N., Wieland, T. 12. Chromatography of mixed oligonucleotides on DEAE-Sephadex. Biochemistry 3 (1964) 626—629, Rushizky, G.W., Bartos, E.M., Sober, H.A. 13. DEAE-Sephadex chromatography of guanylate oligomers using guanidinium chloride. Biochim. Biophys. Acta 277 (1972) 290-300, Olson, A.C., Volkin, E. 14. The synthesis of triaminoacyl-insulins and the use of the t-butyloxy-carbonyl group for the reversible blocking of the amino groups of insulin. Biochemistry 6 (1967) 3559—3568, Levy, D., Carpenter, F.H. 15. A simple method for estimating isoelectric points. Anal. Biochem. 11(1965) 374—377, Lampson, G.P., Tytell, A.A. 16. Isoelectric points and molecular weights of proteins: a table. J. Chromatogr.127 (1976)1—28, Righetti, P.G., Caravaggio, T. 17. Isoelectric points of proteins: a table. AnaL Biochem. 86 (1978) 620—647, Malamud, D., Drysdale, J.W. 18. Basic principles used in the selection of MonoBeads ion exchangers for the separation of biopolymers. Protides of the Biological Fluids, 30 (1982) 621-628, Fägerstam, L.G. et al. 19. Use of electrophoretic titration curves for predicting optimal conditions for fast ion exchange chromatography of proteins. J. Chromatogr. 266 (1983) 409-425, Haff, L.A., Fägerstam, L.G., Barry, A.R.
155
20. ”Isoelectric Focusing: Principles and Methodes”, Technical Booklet Series (1982), Pharmacia Biotech, Uppsala, Sweden. 21. Interrelationships of human-interferon gamma with lymphotoxin and monocyte cytotoxin. J. Exp. Med. 159 (1984) 824-843, Stone-Wolff, D.S., Yip, Y.K., Kelker, H.C. et al. 22. Glass wool as a potential source of artifacts in chromatography. J. Chromatogr. 152 (1978) 514—516, Schwartz, D.P. 23. Ion Exchange Chromatography. Protein Purification, Principles, High resolution methods and Applications, Janson, J.C., Ryden, L. (Eds) VCH, Publishers Inc. New York. (1989) 107-148, Karlsson, E., Ryden, L., Brewer, J. 24. Gel Filtration Chromatography. L. Fischer. Elsevier, Amsterdam (1980) 25. Arthropod hemocyanin structure: isolation of eight subunits in the scorpion. Arch. Biochem. Biophys. 193 (1979)140—149, Lamy, J., Lamy, J., Weill, J. 26. Rapid isolation of Escherichia Coli b-galactosidase by fast protein liquid chromatography. J. Chromatogr. 393 (1987) 462-465, Motorin, Y.A. et al. 27. Chromatography of proteins and peptides on Sephadex ion-exchangers: dependence of the resolution on the elution schedule. FEBS Lett. 14 (1971) 7—10, Novotny, J. 28. High Performance ion-exchange separation of oxidised and reduced nicotinamide adenine dinucleotides. Anal. Biochem. 142 (1984) 232-234, Orr, G.A., Blanchard, J.S. 29. FPLC of leukaemia cell N-Acetyl ß-D-Hexosaminidases. Leukaemia Res.11 (1987) 437-444, Scott, C.S., Patel, M., Stark, A.N., Roberts, B.E. 30. Presented at Sixth International Congress on Methods in Protein Sequence Analysis, Seattle, Washington, USA. (1986) Bhikhabhai, R., Lindblom, H., Källman, I., Fägarstam, L. 31. Fractionation of DNA restriction fragments with ion exchangers for high performance liquid chromatography. European Journal of Biochemistry 155 (1986) 203-212, Müller, W. 32. Inositol triphosphates in carbochal-stimulated rat parotid glands. Biochem. J., 223 (1984) 237-243, Irvine, R.F., Letcher, A.J., Lander, D.J., Downes, C.P.; 33. Inositol bis-, tris-, and tetrakis- phosphate(s): Analysis in tissue by HPLC. Proc. Natl. Acad. Sci. USA., 83 (1986) 4162-4166, Meek, J.L. 34. Release of intra-cellular Ca2+ and elevation of inositol triphosphates by secretagogues in parietal and chief cells isolated from rabbit gastric mucosa. Biochim. Biophys. Acta., 88 (1986) 116-125, Chew, C.S., Brown, M.R. 35. Albumin from human plasma: preparation and in vitro properties. in Separation of Plasma proteins. J.M. Curling, ed., Pharmacia Fine Chemicals AB, Uppsala, Sweden. (1983) 51-58. Berglöf, J.H., Eriksson, S., Suomela, H., Curling, J.M. 36. FPLC for monitoring microbial and mammalian cell cultures. Bio/Tecchnology 2 (1984) 777-781, Frej, A.K. et al. 37. Varietal identification by rapid chromatography (FPLC) of wheat gliadins. 3rd Conference, Royal Australian Institute, Brisbane, Australia. (1983). Batey, I. 38. Rapid extraction and separation of plasma b-endorphin by cation exchange chromatograpy. J. Chromatogr., 297 (1984) 399-403, Stenman, U-H., et al. 39. Applications of Fast Protein Liquid Chromatography in the separation of plasma proteins in urine and cerebrospinal fluid. Clin. Chem., 29 (1983) 1635-1640, Cooper, E.H. et al.
156
Before any part of this handbook is reproduced, please request permission of Pharmacia Biotech. The following designations are trademarks owned by Pharmacia AB: Sephadex, Sephacel, Sepharose, STREAMLINE, HiLoad, HiTrap, MonoBeads, MiniBeads, SOURCE, RESOURCE, FPLC, FPLCdirector, UNICORN, SMART, OligoPilot II, FineLine, BPG, BioPilot, BioProcess, PhastSystem, PhastGel.
157
158
159
Percoll Methodology and Applications
Back to Collection 18-1115-69 Edition AC 1
Handbooks from Amersham Biosciences
Antibody Purification Handbook 18-1037-46
The Recombinant Protein Handbook Protein Amplification and Simple Purification 18-1142-75
Protein Purification Handbook 18-1132-29
2
Ion Exchange Chromatography
Reversed Phase Chromatography
Principles and Methods 18-1114-21
Principles and Methods 18-1134-16
Affinity Chromatography
Expanded Bed Adsorption
Principles and Methods 18-1022-29
Principles and Methods 18-1124-26
Hydrophobic Interaction Chromatography
Chromatofocusing
Principles and Methods 18-1020-90
with Polybuffer and PBE 50-01-022PB
Gel Filtration
Microcarrier cell culture
Principles and Methods 18-1022-18
Principles and Methods 18-1140-62
Percoll Methodology and Applications
3
Content Introduction ................................................................................................. 7 Principles of density gradient centrifugation .................................................... 8 Separation by density (Isopycnic centrifugation) ............................................................. 9 Separation by size (Rate zonal centrifugation) ................................................................ 9
Percoll - physical properties ......................................................................... 10 Particle size composition ............................................................................................ 10 Viscosity ................................................................................................................... 10 Density ..................................................................................................................... 10 pH and osmolality ..................................................................................................... 11 Behavior of the colloid ............................................................................................... 11
How to make and use gradients of Percoll ..................................................... 12 Making and diluting a stock solution of Percoll ............................................................. 12 Diluting stock solutions to lower densities .................................................................... 12 The one-step procedure for diluting Percoll .................................................................. 14 Diluting Percoll to a desired osmolality ........................................................................ 15 Effects of osmolality on apparent buoyant density of cells and subcellular particles .......... 17 Factors affecting gradient formation and shape ............................................................. 18 Discontinuous gradients ............................................................................................. 20 Continuous linear and non-linear gradients ................................................................... 20 Preformed self-generated gradients ............................................................................. 21 Gradients formed in situ ............................................................................................. 21 Maximum sample loading ........................................................................................... 22 A model experiment to standardize conditions .............................................................. 22
How to fractionate and analyze gradients of Percoll ........................................ 23 Density determination using Density Marker Beads ....................................................... 23 Density Marker Beads - properties .................................................................................................. 23 Effects of ionic strength and sucrose concentration on density of Density Marker Beads ....................... 24 Using Density Marker Beads .......................................................................................................... 25
Other methods for measuring density ........................................................................... 25 Fractionation of gradients ........................................................................................... 26 Cell sorting and counting ............................................................................................ 26 Protein determination and enzyme assay ...................................................................... 26
Removal of Percoll after centrifugation ......................................................... 27 Washing ................................................................................................................... 27 High speed centrifugation .......................................................................................... 27 Other methods .......................................................................................................... 28
4
Practical notes ........................................................................................... 29 Care and cleaning of equipment ..................................................................................................... 29 Storage of Percoll ......................................................................................................................... 29 Sterilization of Percoll solutions ..................................................................................................... 29 Aggregates of silica particles .......................................................................................................... 29
Applications ............................................................................................... 30 Blood cells ............................................................................................................... 30
Applications - Blood Cells ............................................................................ 31 Applications - Other Cell Types .................................................................... 38 Applications - Microorganisms ..................................................................... 46 Applications - Subcellular Particles .............................................................. 48 Appendix 1 - Summary methodology charts ................................................... 54 Scheme 1. Separation of cells on gradients of Percoll ...................................................................... 54 Scheme 2. Separation of subcellular particles on gradients of Percoll ................................................ 55
References ................................................................................................. 56 Ordering Information ................................................................................... 80 Products for Cell Separation and Culture ......................................................................................... 80 Products for Purification of RNA .................................................................................................... 81 Products for Purification of DNA .................................................................................................... 81 Kits for cDNA Synthesis ................................................................................................................ 81
5
6
Introduction Since its introduction in 1977, the silica colloid Percoll™ has become the density gradient medium of choice for thousands of researchers worldwide. Its nearly ideal physical characteristics facilitate its use in separating cells, organelles, viruses, and other subcellular particles. Percoll is especially useful as a first step to enrich for cell populations before attempting finer resolution or extraction of nucleic acids. A considerable savings of time and resources may be realized using Percoll as a first step before employing these methods. For Biological particles, the ideal gradient medium has been described as one having the following characteristics (79): • covers a sufficient density range for isopycnic (see p. 9 Figure 1) banding of all biological particles of interest • possesses physiological ionic strength and pH • is iso-osmotic throughout the gradient • has low viscosity • is non-toxic • will not penetrate biological membranes • is supplied sterile and is resterilizable • will form self-generated gradients by centrifugation at moderate g-forces • is compatible with biological materials • is easily removed from purified materials • does not affect assay procedures • will not quench radioactive assays Percoll is exceptional among the available media in that it fulfills the above criteria, and also provides these additional advantages: • It can form both continuous and discontinuous gradients. • Stability of gradients means that gradients can be premade to give reproducible results. • Analysis of gradients is simple with colored Density Marker Beads (available from Amersham Biosciences). • Further experiments with isolated materials are not affected by Percoll. • The success of thousands of researchers has been documented in the Percoll Reference List. This manual provides the basic methodology for making and using gradients of Percoll. In addition, the Application Tables in the latter part of this manual provide numerous references for using Percoll to isolate various cells, microorganisms, organelles and subcellular particles.
7
Principles of density gradient centrifugation When a suspension of particles is centrifuged, the sedimentation rate of the particles is proportional to the force applied. The physical properties of the solution will also affect the sedimentation rate. At a fixed centrifugal force and liquid viscosity, the sedimentation rate is proportional to the size of the particle and the difference between its density and the density of the surrounding medium. The equation for the sedimentation of a sphere in a centrifugal field is:
v=
d2 (rp - rl) 18h
xg
where v = sedimentation rate d = diameter of the particle (hydrodynamically equivalent sphere) rp = particle density rl = liquid density h = viscosity of the medium g = centrifugal force
From this equation, the following relationships can be observed: • The sedimentation rate of a particle is proportional to its size. • The sedimentation rate is proportional to the difference between the density of the particle and that of the surrounding medium. • The sedimentation rate is zero when the density of the particle is equal to the density of the surrounding medium. • The sedimentation rate decreases as the viscosity of the medium increases. • The sedimentation rate increases as the centrifugal force increases.
8
Separation by density (Isopycnic centrifugation) In this technique, the density range of the gradient medium encompasses all densities of the sample particles. Each particle will sediment to an equilibrium position in the gradient where the gradient density is equal to the density of the particle (isopycnic position). Thus, in this type of separation, the particles are separated solely on the basis of differences in density, irrespective of size.
Note: When considering biological particles, it is important to remember that the osmolality of the medium can significantly alter the size and apparent buoyant density of membrane-bound particles. A high external osmolality will cause membrane-bound particles to shrink while a low osmolality in the medium will cause the particles to swell.
r1 r2 Rate zonal cent.
Isopycnic cent.
Time
Figure 1. Diagrammatic representation of rate zonal and isopycnic centrifugation. r1 = buoyant density of the less dense (blue) particles r2 = buoyant density of the more dense (red) particles (Courtesy of H. Pertoft, reproduced by kind permission.)
Percoll
Buoyant density g/ml
Figure 1 illustrates the two types of centrifugal separation (see below for rate zonal centrifugation). When using Percoll, it is common to separate particles isopycnically rather than on the basis of size differences (but see Figure 19, page 30, where both techniques are used.).
r
Plasma membranes Platelets
Ribosomes
Lymphocytes
Herpes virus Granulocytes
Lysosomes
1.1 Erythrocytes
Mitochondria
Nuclei
Sucrose
Figure 2 shows that particles centrifuged in gradients of Percoll under physiological conditions (280-320 mOs/kg H2O) have much lower apparent buoyant densities than in sucrose or Metrizamide (see also Table 1, page 18).
1.2
Lysosomes Mitochondria
Plasma membranes
Microbodies Herpes virus
Ribosomes
Nuclei
1.3 10
0
10
1
10
2
10
3
10
4
10
5
10
6
10
7
8
9
10 10 Svedberg units S
Figure 2. Approximate sedimentation rates and isopycnic banding densities of particles in a rat liver homogenate, herpes virus and human blood cells in gradients of Percoll (green) compared with sucrose gradients (blue). Svedberg units = sedimentation coefficient, 1S = 10-13 sec. (27, reproduced by kind permission of the authors and publisher).
Separation by size (Rate zonal centrifugation) In this type of separation, the size difference between particles affects the separation along with the density of the particles. As can be seen from the above equation, large particles move faster through the gradient than small particles, and the density range is chosen so that the density of the particles is greater than the density of the medium at all points during the separation (see Figure 1). The run is terminated before the separated zones reach the bottom of the tube (or their equilibrium positions). 9
Percoll - physical properties Percoll is available from Amersham Biosciences. Composition Density
silica sol with nondialyzable polyvinylpyrrolidone (PVP) coating 1.130 + 0.005 g/ml
Conductivity
1.0 mS/cm
Osmolality
<25 mOs/kg H2O 10 + 5 cP at 20 ºC
Viscosity pH Refractive Index
9.0 + 0.5 at 20 ºC 1.3540 + 0.005 at 20 ºC
Percoll is non-toxic
Particle size composition The physical properties of Percoll have been extensively studied by Laurent et al. (45, 46, 47). Electron microscopic examination (Figure 3) shows the silica to be in the form of a polydisperse colloid composed of particles from 15 to 30 nm in size, with a mean particle diameter of 21-22 nm. Hydrodynamic measurements (viscometry and sedimentation) give values of 2930 nm and 35 nm in 0.15 M NaCl and water, respectively, for the mean particle diameter, indicating a layer of hydration on the particles which is more pronounced at low ionic strength.
Figure 3. Electron microscopy of Percoll particles. Negative contrast with 1% uranyl acetate at pH 4.6 (21, reproduced by kind permission of the authors and publisher).
Chromatography of Percoll on Sepharose™ 4B (22) has demonstrated the presence of only 1-2% free PVP. Inclusion of PEG in the eluant did not result in any loss of PVP from the silica, indicating that the PVP is firmly bound. Calculations based on the nitrogen content of the colloid indicate that the PVP coating is a monomolecular layer.
Viscosity The viscosity of Percoll is a function of the ionic strength, and is lower in saline solutions at physiological ionic strength (e.g. 0.15 M NaCl) than in water or in 0.25 M sucrose (22). This has the effect of making gradient formation in 0.15 M NaCl much faster than in 0.25 M sucrose when solutions are centrifuged under identical conditions (page 18). Under working conditions, the viscosity of Percoll solutions is 1-15 cP, facilitating extremely rapid banding of particles in gradients of Percoll.
Density Percoll is supplied as a 23% (w/w) colloidal solution in water having a density of 1.130 + 0.005 g/ml. Gradients ranging from 1.0-1.3 g/ml are achievable by centrifugation as described elsewhere in this booklet. All biological particles having sedimentation coefficient values of >60S can be successfully banded on gradients of Percoll, and most have buouyant densities of <1.13 g/ml in Percoll (see Figure 2). 10
pH and osmolality Percoll has a pH of about 9.0, adjustable to pH 5.5-10.0 without any change in properties. If the pH is dropped below 5.5, gelling may occur. Gelling can also be caused by the presence of divalent cations, an effect which is exacerbated by elevated temperatures. Percoll has a very low osmolality (<25 mOs/kg H2O) and can therefore form a density gradient without producing any significant osmolality gradient itself. This makes it possible to work with density gradients which are iso-osmotic and adjusted to physiological conditions throughout. This is very important for obtaining preparations of cells having extremely high viabilities (23), and intact morphology (31). Due to this fact, gradients of Percoll also provide an opportunity to observe the effect of osmolality on the apparent buoyant density of cells and subcellular particles (see page 17 and ref. 27).
Behavior of the colloid Density g/ml
Percoll particles have an inner core of silica which 4 3 2 1 1.14 is very dense (r = 2.2 g/ml) and an average hydrated particle size of 29–30 nm in 0.15 M NaCl 1.12 and 35 nm in water (46). Thus, when a solution of Percoll (in 0.15 M saline or 0.25 M sucrose) is 1.10 centrifuged at >10,000 x g in an angle-head rotor, Starting density 1.08 the coated silica particles will begin to sediment. in This results in an uneven distribution of particles, 15 m in 1.06 30 m and thus forms a density gradient. Since Percoll is n min mi 60 90 a polydisperse colloid, its component particles 1.04 1 will sediment at different rates, creating a very 1.02 smooth gradient. Electron microscopic analysis of 2 3 4 gradients by high speed centrifugation in an 0 10 20 30 40 50 60 anglehead rotor shows that the material at the Distance from meniscus mm bottom of the tube is considerably enriched in Figure 4. Isometric gradient formation by Percoll in an anglelarger particles (Pertoft, personal communication). head rotor, 8 x 14 ml (MSE Superspeed centrifuge) starting The gradient forms isometrically (i.e. less dense density 1.07 g/ml in 0.15 M NaCl. Running conditions: on top and more dense on the bottom) around the 20,000 x g for 15, 30, 60 and 90 minutes. Gradient density was monitored by means of colored Density Marker Beads. starting density and becomes on average See Figure 12, page 23 (Work from Amersham Biosciences, progressively steeper with time (Figure 4). Uppsala, Sweden). Prolonged centrifugation of Percoll at high gforces results in all the colloid sedimenting to form a hard pellet (see "Removal of Percoll", page 27). It is important to note that if a gradient of Percoll is spun at >10,000 x g in a swinging-bucket type rotor, the colloid will rapidly sediment into a pellet and not form a suitable gradient. The colloid does not perceptibly diffuse over time, resulting in the formation of very stable gradients. Therefore, both discontinuous and continuous gradients can be prepared weeks in advance, giving great reproducibility over the course of an experiment.
11
How to make and use gradients of Percoll Making and diluting a stock solution of Percoll In order to use Percoll to prepare a gradient, the osmolality of Percoll (undiluted) must first be adjusted with saline or cell culture medium to make Percoll isotonic with physiological salt solutions. Adding 9 parts (v/v) of Percoll to 1 part (v/v) of 1.5 M NaCl or 10x concentrated cell culture medium is a simple way of preparing a Stock Isotonic Percoll (SIP) solution. Final adjustment to the required osmolality can be carried out by adding salts or distilled water. Cell density depends on osmolality (see e.g. Figure 6); because of this, the osmolality of the stock solution should be checked routinely with an osmometer to ensure reproducibility between experiments. For subcellular particles which aggregate in the presence of salts, the Stock Isotonic Percoll (SIP) can be made by adding 9 parts (v/v) of Percoll to 1 part (v/v) of 2.5 M sucrose. The density of the SIP solution can be calculated from the following formula:
Vx = Vo
where Vx Vo ro
= = =
r10
=
ri
=
(ro- ri) (ri - r10)
thus ri =
Voro + Vxr10 V x + Vo
volume of diluting medium (ml) volume of undiluted Percoll (ml) density of Percoll (1.130 + 0.005 g/ml; see Certificate of Analysis for exact density) density of 1.5 M NaCl = 1.058 g/ml (minor differences for other salts) density of 2.5 M sucrose = 1.316 g/ml (minor differences for other additives) density of SIP solution produced (g/ml)
Thus, for SIP in saline, ri = 1.123 g/ml and for SIP in sucrose, ri = 1.149 g/ml, assuming ro = 1.130 g/ml.
Diluting stock solutions to lower densities Solutions of stock isotonic Percoll (SIP) are diluted to lower densities simply by adding 0.15 M NaCl (or normal strength cell culture medium) for cell work, or with 0.25 M sucrose when working with subcellular particles or viruses. The following formula can be used to calculate the volumes required to obtain a solution of the desired density.
12
Vy = Vi where Vy Vi ri ry
r Example:
= = = =
=
(ri-r) (r-ry) volume of diluting medium in ml volume of SIP in ml density of SIP in g/ml density of diluting medium in g/ml (density of 0.15 M NaCl is ~1.0046 g/ml) * (density of 0.25 M sucrose is ~1.032 g/ml)* density of diluted solution produced in g/ml
To dilute 55 ml of SIP to a final density of 1.07 g/ml, determine the amount of 0.15 M NaCl required.
Volume of 0.15 M NaCl required = 55 x
1.123 - 1.07 1.07 - 1.0046
= 44.6 ml
ro =1.135 ro =1.130 ro =1.125 1.14 ro =1.135 ro =1.130 ro =1.125
1.12
M
Su cr os e
1.10
1.04
Na Cl M
wi th
0. 15
1.06
Di lu tio n
wi th
0. 25
1.08
Di lu tio n
Note: The graph shown in Figure 5 can also be used as an empirical guide to the density of solutions produced by diluting SIP with 0.15 M saline or 0.25 M sucrose. This graph refers to the dilution of SIP where SIP is 90% (v/v) undiluted Percoll osmotically adjusted by addition of 10% (v/v) saline or sucrose. To avoid confusion, it is therefore preferable to refer to the actual density of the working solution (or to state % SIP) rather than to refer to the solution as a percentage of Percoll in iso-osmotic saline or sucrose. This is particularly important when using the one-step dilution procedure described below, where a working solution of known density is obtained by diluting Percoll (undiluted) plus concentrated salts or sucrose to a final volume with distilled water.
Density g/ml
The above formula is useful for achieving densities that will be very close to the actual density desired. However, slight variations in volumes and densities of diluting media will affect final density. For determining actual densities, we recommend measuring the final density of Percoll solutions using a densitometer or refractometer (see page 25).
1.02
10 20 30 40 50 60 70 80 90 100 % of Stock Isotonic Percoll (SIP) in final solution
* from CRC Handbook of Chemistry and Physics, 67th edition (1986-1987), CRC Press, D253 and D262.
Figure 5. Dilution of Stock Isotonic Percoll (SIP) with iso-osmotic saline or sucrose solution. Po is the density of the Percoll (undiluted). SIP is prepared as described on page 12. The calibration lines shown are for guidance only. For accurate density measurements, refer to the formula given in the text. (Work from Amersham Biosciences, Uppsala, Sweden.)
13
The one-step procedure for diluting Percoll Percoll (undiluted) may de diluted directly to make a final working solution of known density by the following procedure. In a measuring cylinder, add 1.5 M NaCl or 2.5 M sucrose to 1/10 of the final desired volume (e.g. 10 ml for 100 ml of working solution). To this, add the required volume of Percoll (undiluted), calculated using the formula shown below. Make up to the final volume with distilled water.
Vo = V where Vo V r ro
= = = =
r10
=
Example:
r - 0.1r10 - 0.9 ro - 1 volume of Percoll (undiluted) (ml) volume of the final working solution (ml) desired density of the final solution (g/ml) density of Percoll (undiluted) (g/ml) (see Certificate of Analysis for exact density) density of 1.5 M NaCl = 1.058 (g/ml) (minor differences for other salts) density of 2.5 M sucrose = 1.316 (g/ml) (minor differences for other additions)
To prepare 100 ml of working solution of Percoll of density 1.07 g/ml in 0.15 M NaCl. To 10 ml of 1.5 M NaCl, add 1.07 - 0.1058 - 0.9
Volume of Percoll required = 100 x 0.13 = 49.4 ml (if Percoll density is 1.130 g/ml) and make up to 100 ml with distilled water. The above formula is useful for achieving densities that will be very close to the actual density desired. However, slight variations in volumes and densities of diluting media will affect final density. For determining highly accurate densities, we recommend measuring the final density of Percoll solutions using a densitometer or refractometer (see page 25). Graphs similar to the one shown in Figure 5 can be drawn to relate the volume of Percoll (undiluted) to the final density.
14
Diluting Percoll to a desired osmolality To make isotonic Percoll for most mammalian cells, it is common to dilute 9 parts of Percoll (undiluted) with 1 part of 1.5 M NaCl or 2.5 M sucrose solution. This Stock Isotonic Percoll (SIP) is then further diluted with physiological buffers according to needs. However, while this procedure has proved successful, it is rather simplistic and does not take into account the effect of having solid silica particles present (i.e. that 100 ml of Percoll stock contains a certain volume of solid silica, making the total aqueous volume less than 100 ml). Due to the volume occupied by silica, the electrolytes in the stock solution have a higher effective concentration than in physiological salt solution, and SIP made in this way will be hyperosmolal. Thus, determining the actual osmolality of the SIP has always been recommended. Vincent and Nadeau (555) discuss the problem elegantly and described an equation which can be used to calculate the number of parts of Percoll which should be added to one part of 10x concentrated physiological salt buffer to obtain a SIP of any desired osmolality. The authors determined the fraction of the total volume of a Percoll stock solution which is occupied by silica and thus determined the ratio of volume of aqueous solution to that of total Percoll stock solution.
Vp = Vc where Vp Vc Oc Of R
= = = = =
Op
=
O c - Of R(Of - Op) number of parts of Percoll to be added number of parts of solute concentrate (e.g. 1.5 M NaCl) to be added osmolality of solute concentrate (e.g. 1.5 M NaCl = 2880 mOsm) desired osmolality ratio of aqueous volume to total volume of Percoll (typically = 0.85 for NaCl and 0.80 for sucrose) osmolality of Percoll undiluted (see Certificate of Analysis)
The key variable in this equation is R, which is a measure of the real aqueous volume of a Percoll solution. The value of R is a function of the hydrodynamic volume occupied by the Percoll particles. This, in turn is a function of the ionic strength of the medium - that is, as ionic strength increases, hydrodynamic volume decreases. Thus, there is a difference in the R value of 1.5 M NaCl and 2.5 M sucrose. To obtain a SIP of osmolality = 320 mOs/kg H2O adjusted with 1.5 M NaCl (i.e. 10x concentrated physiological saline): 2880 - 320 Vp = 1
= 10.04 0.85 (320 - 20)
assuming:
2880 = 20
=
osmolality of 1.5 M NaCl (10x concentrated physiological saline) osmolality of Percoll undiluted
Therefore to obtain a SIP of 320 mOs/kg H2O, one would add 10 parts Percoll to 1 part 1.5 M NaCl.
15
The ratio of concentrated solute solution (i.e. 1.5 M NaCl, etc.) to SIP is called Rx where:
Rx =
Vc V p + Vc
Using this formula, one can calculate the amount of Percoll (undiluted) required to make a final working solution of known density and osmolality.
Vo = V where Vo V r Rx
= = = =
ro
=
r10
=
r-Rxr10-(1-Rx) ro -1 Volume of Percoll undiluted (ml) Volume of final working solution (ml) desired density of final working solution (g/ml) fraction of total volume which is solute concentrate (i.e. 1.5 M NaCl, etc.) density of Percoll undiluted (g/ml) (see Certificate of Analysis) density of 1.5 M NaCl (1.058 g/ml), 2.5 M sucrose (1.316 g/ml), etc.
Thus, for 100 ml of SIP of osmolality = 320 mOs/kg H2O adjusted with NaCl and density = 1.07 g/ml:
1.07 - 1/11 x 1.058 - (1-1/11) Vo = 100
= 49.8 ml 1.13 - 1
The final solution contains 9.1 ml of 1.5 M NaCl (1/11 x 100 = 9.1), 49.8 ml Percoll undiluted and 34 ml (i.e. 100 - 58.9 = 41.1) of distilled water.
16
30
200 300 mOsm 400 1.10
20
ient grad sity Den
1.05
Number of cells x 106
Hepatocytes (viable)
1.15
10
Non-viable cells
1
2
5 7 3 4 6 Distance from meniscus cm
8
1.15 70 60 50
Density g/ml
Figure 6. Fractionation of rat liver hepatocytes cells (35 x 106 cells in a volume of 2 ml) on a self-generated Percoll gradient (8 ml solution with a density of 1.065 g/ml). The osmolality of the Percoll solution was varied by adding NaCl to 200 mOsm, 300 mOsm and 400 mOsm. Centrifugation was performed in a Beckman rotor 30.2 for 15 min at 35,000 x g at a temperature of 4 ºC. Density gradient determined using DMB (see p. 23). (27, reproduced by kind permission of the authors and publisher).
Enzyme activity % of total
The very low osmolality of Percoll has facilitated the study of the interrelation of the separation medium osmolality with the apparent buoyant density of particles. Figure 6 shows the effects of banding rat liver hepatocytes in Percoll gradients having osmolalities of 200, 300 and 400 mOsmol/kg H2O (mOsm). The apparent buoyant density of the cells increases with increasing osmolality, due to removal of water from the cells. The same effect has been observed with mitochondria (Figure 7) and with lysosomes (Table 1). Even small changes in osmolality cause a large change in the apparent buoyant densities of these organelles. The actual recorded buoyant densities of particles banded in Percoll gradients at physiological osmolality are therefore much more likely to correspond to those existing in vivo, than when the particles are banded in sucrose or other centrifugation media.
Density g/ml
Effects of osmolality on apparent buoyant density of cells and subcellular particles
1.10
40 30 1.05 20 10 0
Dens ity gradie
0
5
nt
10 Fraction number
15
20
Figure 7. The density distribution of mitochondria from rat liver cells after incubation in iso-osmotic buffer (red) and buffer containing 17.5% albumin (green). Centrifugations were performed in a Beckman 65 rotor (23º angle) for 30 minutes at 40,000 x g. (59, reproduced by kind permission of the authors and publisher).
17
Table 1. Changes in buoyant density of lysosomes after incubation in serum albumin. Incubation medium Albumin % Sucrose %
Osmolality of medium (mOm/l)
Average density of lysosomes (g/ml)
-
8.5
284
1.045
2.5
8.5
288
1.058
5
8.5
292
1.074
7.5
8.5
300
1.078
10
8.5
310
1.091
20
8.5
374
1.110
30
8.5
503
1.148
40
8.5
800
1.177
A lysosomal fraction from rat hepatocytes was recovered from a Percoll/0.25 M sucrose gradient at a density of 1.0 - 1.05 g/ml and incubated in the media described in the table for 1 hour at 37 ºC. The buoyant density was then redetermined in a gradient of Percoll/0.25 M sucrose (27, reproduced by kind permission of the authors and publisher).
Although the hydrated volume of Percoll particles is smaller in the presence of 0.15 M NaCl than in Percoll/0.25 M sucrose, the sedimentation rate of the particles is faster due to the lower viscosity of Percoll in saline. Thus, when Percoll is made isoosmotic with a final concentration of 0.15 M saline or a tissue culture medium of equivalent ionic strength, it will form a self-generated gradient about 2-3 times faster than the equivalent Percoll solution made iso-osmotic with a final concentration of 0.25 M sucrose. Centrifugation and time are interrelated in that it is the total (g-force) x (time) which determines the shape of the gradient. A minimum of approximately 10,000 x g should be used for Percoll in 0.15 M saline and about 25,000 x g for Percoll in 0.25 M sucrose in order to self-generate gradients in anglehead rotors. Rotor geometry has a marked effect on gradient shape under given conditions as shown in Figure 8. As the angle approaches vertical, the path-length for formation of the gradient becomes shorter and the gradient forms more rapidly. Figures 9 and 10 demonstrate that the initial concentration of Percoll also has some effect on the shape of the gradient formed. Centrifugation in vertical rotors will form gradients of Percoll very rapidly. Care must be taken, however, to ensure that the compacted pellet of Percoll which may be formed under high speed centrifugation conditions does nor contaminate the gradient during fractionation. 18
Distance from the meniscus cm
Factors affecting gradient formation and shape 2 40º 4 6
2
23.5º
4 6
2
14º
4 6 8 1.00
1.10
1.20
Density g/ml
Figure 8. The effect of rotor angle on gradient development using Percoll. Starting density was 1.065 g/ml in 0.15 M NaCl. Running conditions: 30,000 x g for 14 minutes. Colored lines refer to the positions of the colored density marker beads. (45, reproduced by kind permission of the authors and publisher.)
Density g/ml
The use of swinging bucket rotors for self-generation of gradients is not recommended, due to the long path length and unequal g-force along the tube. However Jenkins et al. (personal communication and ref. 87) report some advantages in using these types of rotors for subcellular fractionation of liver organelles.
1.14
1.12 90% 1.10
80% 70%
1.08
60%
When starting work with self-generated gradients, it is advisable to conduct a model experiment with colored Density Marker Beads (see page 22) to produce a series of standard curves under known conditions which are characteristic of the anglehead rotor to be used for subsequent experiments.
1.06
50% 40%
1.04
30% 20%
1.02 60
50 40 30 20 10 Band position from bottom of tube mm
Figure 9. Use of colored Density Marker Beads to show gradient shape. Gradients formed from solutions of Percoll varying from 90% to 20% of stock isotonic Percoll in 0.15 M NaCl. Running conditions 23º angle-head rotor 30,000 x g, 15 minutes. (Work from Amersham Biosciences, Uppsala, Sweden.)
1.14
Density g/ml
Zonal rotors can be used to form gradients of Percoll in situ. Gradients formed in zonal rotors have the same characteristics as those generated in angle-head rotors. Because of their large sample volumes, it is recommended that the separation conditions in a nonzonal rotor be empirically determined prior to scale-up in a zonal rotor. Zonal rotors have been used in the large scale purification of viruses (21) and for subfractionation of lysosomes (24).
1.12
1.10
1.08
90% 80% 70%
1.06
60% 50%
1.04
40% 30% 20%
1.02 60 50 40 30 20 10 Band position from bottom of tube mm
Figure 10. Use of colored Density Marker Beads to show gradient shapes. Dilutions of Percoll as in Figure 9, running conditions: 23º angle-head rotor, 60,000 x g, 15 minutes. Steeper gradients were formed by the greater g-force. (Work from Amersham Biosciences, Uppsala, Sweden.)
19
Discontinuous (step) gradients Banded cells 1.004 1.062 1.064 1.066 1.068
7 390 x g
6 5
30 min
4 3 2
1.070 1.080
Fraction number
To form a discontinuous gradient, SIP is diluted to a series of different densities as described on page 12. The solutions of different density are then carefully layered in order of density one on top of another, starting with the most dense at the bottom of the tube. This is most conveniently done using a pipette or a syringe fitted with a wide-bore needle. It is important to keep the tip of the instrument against the wall of the tube just above the surface of the liquid to avoid a "splash" and mixing at the interface. Formation of a sharp band of cells at a interface will occur only if there is a sharp change in density.
Density of Percoll solution g/ml
Discontinuous gradients offer great flexibility and ease of use. Often, only a cushion of Percoll or a single step is all that's required to achieve excellent enrichment or resolution of a target cell type. For example, most blood cells can be enriched using discontinuous gradients (66,69) (see also Figure 11).
1 PBMC in Percoll solution
Figure 11. Separation of lymphocytes and monocytes by discontinuous density centrifugation in Percoll. 1.5-2.0 x 107 PBMC (peripheral blood mononuclear cells) isolated on Ficoll Paque were mixed in 11.25 ml of Percoll in Hanks BSS containing 1% HEPES buffer (density = 1.080 g/ml) and underlayered below the steps shown in the figure (69, reproduced by kind permission of the authors and publisher).
Centrifugation is performed using relatively gentle condition, such as 400 x g for 15-20 minutes in a bench-top centrifuge. These gentle conditions result in the isopycnic banding of cells at the relevant interfaces. The low-g conditions and short run time will not cause sedimentation of the Percoll and will not affect the gradient in any way.
Continuous linear and non-linear gradients Continuous gradients are characterized by a smooth change in density from the top to the bottom of the tube. Instead of the obvious interfaces present in the discontinuous gradient, a continuous gradient can be thought of as having an infinitive number of interfaces. Therefore, isopycnic banding of cells occurs at the precise density of the cell. To form such a gradient, SIP is first diluted to produce two solutions of known density at the limits of the range required, and then mixed using a dual-chamber gradient maker (e.g. Amersham Biosciences Gradient Mixer GM-1). A linear gradient spanning the range between the limits of the two starting solutions is formed. A single-channel peristaltic pump (e.g. Amersham Biosciences Peristaltic Pump P-1) in combination with a gradient mixer can be used to generate linear, convex, and concave gradients, depending upon the relative diameters of the tubing used. A very narrow range of densities from top to bottom of the gradient can be formed to effect a maximum resolution of viable cells. Heavier cells usually pellet, while non-viable cells are found at the top of the gradient. For example, erythrocytes will pellet if the density at the bottom of the gradient does not exceed 1.08 g/ml. Density Marker Beads can be used as an external marker in a tube containing an identical gradient to that in the sample tube.
20
The centrifugation conditions necessary to achieve a separation are the same as those for the discontinuous gradients. Examples of separations performed on continuous gradients include the purification of Leydig cells (31), lactotrophs (19), bone marrow cells (52), intestinal epithelial cells (18), marine microalgae (28, 60) and chloroplasts (49, 58, 76, 88, 109).
Preformed self-generated gradients Preforming a gradient by centrifugation can be a convenient alternative to using a gradient maker or pump. As described earlier, Percoll will sediment when subjected to significant g-forces (i.e. >10,000 x g). When preforming a gradient, SIP is diluted to a density that lies in the middle of the range in which maximum resolution is required. Two centrifuge tubes are filled with gradient material - one for the experiment and one containing Density Marker Beads. This second tube serves both as a counterbalance and as an external method for monitoring the gradient. The tubes are centrifuged in an angle-head rotor (e.g. 30,000 x g for 15 minutes), and the gradient forms isometrically around the starting density (Figure 4). The relatively "flat" region of the gradient should encompass the range required for maximum resolution of the target cells. This can be confirmed by observing the shape of the gradient in the tube containing the Density Marker Beads. The gradient becomes progressively steeper with time. It has been shown that the shape of the gradient is approximately linear related to the total g-force and time of the centrifugation (22). After forming the gradient, isopycnic banding of cells can be accomplished by low-speed centrifugation for 15-20 minutes at 400 x g. If an estimate of cell density is required, a volume equal to that of the cell suspension is layered on top of the tube containing the Density Marker Beads,. This serves as both a way to estimate cell density and as a counter-balance.
Gradients formed in situ The sedimentation coefficients of subcellular particles and viruses are usually too low to allow banding on preformed gradients at low g-forces. Therefore, it is often convenient to mix the suspension of biological particles with Percoll and to band the particles on a gradient formed in situ. Gradients of Percoll formed by centrifugation are metastable - i.e. they will change continuously during high speed centrifugation. The rate of sedimentation of the colloid is slow enough to allow the banding of small viruses and cell organelles with "S" values >60S as the gradient is formed in situ. A common method for forming gradients in situ is to prepare a SIP, using 9 parts of Percoll to 1 part of 2.5 M sucrose. The SIP is then diluted to the desired density using 0.25 M sucrose. (Although sucrose is typically used to make in situ gradients, cell culture media can also be used). When mixing the sample directly with gradient material, the effect on the overall density of the Percoll solution can be calculated from the formula on page 13. Premixing of the sample with the gradient material is convenient when it is desirable to accurately measure the buoyant density of the particles. However, it may be better to layer the experimental sample on top of the gradient material, particularly in cases where it is desirable to separate subcellular particles from soluble proteins. The soluble proteins will remain in the buffer layer above the gradient and subcellular particles will separate in the Percoll gradient in situ. Centrifugation must be carried out in an angle-head rotor. A balance tube containing Density Marker Beads in place of experimental sample is used to monitor the gradient. An appropriate model experiment similar to the one described on page 22, should be carried out first to establish the gradient formation characteristics of the rotor to be used.
21
Maximum sample loading There are no standard rules governing the maximum quantity of cells or subcellular material which can be separated on gradients of Percoll. For subcellular fractionation, successful purification can be achieved with a total loading of 1-5 mg of protein in a samlpe volume of 0.5 ml on 10 ml of gradient material (Pertoft, personal communication).
A model experiment to standardize conditions The exact shape and range of gradients formed during centrifugation is influenced by the model and angle of the rotor used, and by the size of the centrifuge tubes. The following experiment is designed to enable you to establish a series of gradient curves for a particular rotor and tubes, and can be used as a reference for all future experiments. The example chosen is for 10 ml gradients, but this may be scaled up for larger tube sizes. 1. Mix 49.5 ml of Percoll with 5.5 ml of 1.5 M NaCl to make a SIP. 2. Mix SIP from step 1 with 0.15 M NaCl to make a series of 10 ml experimental samples (total centrifuge tube size = 13.5 ml) as shown in the following table: Tube No.
1
2
3
4
5
6
7
8
9
10
Percoll (SIP) (ml)
10
9
8
7
6
5
4
3
2
1
0.15 M NaCl (ml)
-
1
2
3
4
5
6
7
8
9
3. Add 10 µl of a suspension of each type of Density Marker Beads to each tube according to the instructions supplied in the pack. 4. Balance and cap the tubes, and mix them by inverting several times. 5. Place the tubes in the angle-head rotor (if there are only 8 spaces, omit tubes 1 and 10). 6. Centrifuge at 30,000 x g for 15 minutes. 7. Carefully remove the tubes and using millimeter graph paper, measure to the nearest 0.5 mm the distance of each band from the bottom of the tube. 8. Plot the gradient shape for each tube by calibrating each band with the exact boyant density for each Marker Bead. 9. Re-mix the contents of each tube by inversion and repeat the centrifugation, this time using 60,000 x g for 15 minutes. 10. Measure the gradients and plot the results as before. Calculate the exact density of the dilution using the formula (see page 13). Figures 9 and 10 show typical examples of a series of curves generated using Percoll in 0.15 M NaCl. The experiment can be repeated using Percoll in 0.25 M sucrose; in this case, running conditions should be 50,000 x g for 25 minutes followed by 100,000 x g for 25 minutes.
22
How to fractionate and analyze gradients of Percoll Density determination using Density Marker Beads Density Marker Beads are dyed derivatives of Sephadex™. There are ten color-coded bead types, each with a specific density. They have been specifically formulated for use in Percoll gradients and will not work with other media. Using Density Marker Beads as an external marker facilitates monitoring of the gradient shape and range. The position of cells or organelles within the gradient may be accurately located before fractionation using preformed gradients (73,83). The densities of the Density Marker Beads cover the buoyant densities of the vast majority of cells and organelles to be separated in Percoll. In addition to providing a very rapid and simple method for density measurement, using Density Marker Beads provides more accurate data than other methods, since distortion of gradients by fractionation before analysis is completely avoided. Density Marker Beads are also very useful for standardizing running conditions before carrying out an actual experiment, using the model experiment described previously to generate a series of gradient curves specific for a particular rotor and tube type.
Density Marker Bead - properties Each vial contains freeze-dried cross-linked dextran beads having an accurately determined density in Percoll. Nine of the ten bead types can be used for gradients of Percoll containing 0.15 M NaCl or 0.25 M sucrose. Vial 5 is used exclusively for Percoll with 0.15 M NaCl and vial 10 contains beads to be used only for Percoll with 0.25 M sucrose. Volume of beads swollen in water:
0.7 ml/vial
Density of each bead type:
calibrated to ± 0.0005 g/ml
Total density range covered: 1.017 - 1.142 g/ml for Percoll in 0.15 M NaCl 1.037 - 1.136 g/ml for Percoll in 0.25 M sucrose The exact density of each type of bead is specific for each manufactured lot, and is printed on the label of each box.
90 min 60 min 15 min
30 min
Figure 12. Banding of Density Marker Beads in gradients of Percoll as described in Figure 4. (Work from Amersham Biosciences, Uppsala, Sweden.)
23
Effects of ionic strength and sucrose concentration on density of Density Marker Beads 9
8
1.12
7 6 5 4
1.08
3 1.04
2 1
1.00
0.05
0.15
0.25
Concentration of NaCl (M)
Figure 13. Effects of salt concentration on the recorded densities of Density Marker Beads in gradients of Percoll. Numbers refer to different bead types; the exact density of specific lots is printed on the box label. (Work from Amersham Biosciences, Uppsala, Sweden.)
9 8 1.12
7 6 10
1.08
4 3 2 1
1.04
1.00
1.15
Density Marker Beads Digital densitometer
1.10
1.05
0.10
0.20
0.30
Concentration of sucrose (M)
Figure 14. Effects of sucrose concentration on the recorded densities of Density Marker Beads in gradients of Percoll. Numbers refer to different bead types; the exact density of specific lots is printed on the box label. (Work from Amersham Biosciences, Uppsala, Sweden.)
24
1.16
1.16
Density g/ml
Buoyant density g/ml
Figure 15 shows the correlation of densities calibrated with Density Marker Beads and by a digital densitometer. This latter method may be used as a crosscheck when working with Percoll in systems outside normal physiological conditions.
Buoyant density g/ml
The densities of Density Marker Beads printed on the label of each box are those recorded under the most widely used conditions - i.e. when Percoll is made iso-osmotic with physiological saline or 0.25 M sucrose. The actual buoyant density of the beads will vary slightly with ionic strength or sucrose concentration (osmolality). Figure 13 shows variations of density with ionic strength and Figure 14 shows variations with sucrose concentration. When working with systems outside the normal range of ionic strength or osmolality, these figures may be used as a guideline for calibration of bead densities.
20
30
40
50
60
70
Volume (ml)
Figure 15. Correlation of recorded densities of a Percoll gradient in 0.15 M NaCl using Density Marker Beads and a digital densitometer (DMA 46, Anton Paar A.G.). Fraction size 2.64 ml, centrifuge MSE Superspeed 75, rotor 10 x 100 ml, angle 18º, 40,000 x g for 60 minutes. (Work from Amersham Biosciences, Uppsala, Sweden.)
Using Density Marker Beads The beads must be swollen with water prior to use; 1.0 ml of sterile water is added to each vial and the beads are allowed to swell overnight. For long term storage of beads in water, it is advisable to add a preservative such as Merthiolate® (0.01% w/v). The quantity of beads required for each experiment will depend on the size of the centrifuge tube, but 10-15 µl of suspension is usually sufficient for 10 ml of Percoll. When dispensing the beads with a micropipette, it is useful to snip off the end of the disposable plastic tip to avoid clogging by the beads. The size of the Density Marker Beads is sufficiently small for them to pass through tubing, monitoring equipment, etc., without problems. Density Marker Beads have been used to monitor gradients of Percoll in zonal centrifuge rotors. Density Marker Beads are used as external markers, in a centrifuge tube containing identical gradient material to the one used for the experiment. They should not be mixed with the cell sample. Density Marker Beads are added to the control tube, which is then used as a counter-balance in the rotor during the centrifugation. The shape of the gradient is measured as described in the model experiment on page 22. Detailed instructions for use are included in each box of Density Marker Beads. Note: Density Marker Beads can only be used to calibrate gradients of Percoll. The densities printed on the label do not apply to gradients of other media.
Other methods for measuring density
Refractive Index
Several techniques can be used to monitor the density of Percoll solutions after fractionation. Weighing of empty and filled glass micropipettes is accurate but tedious. It is also possible to measure the isopycnic equilibrium point of samples in a precalibrated gradient made from nonaqueous organic liquids (12). Refractive index has a linear correlation with the density of a Percoll solution as shown in Figure 16. Direct measurement using a densitometer (e.g. DMA 3, Anton Paas A.G.) is an accurate alternative to using Density Marker Beads (see Figure 15).
1.36 se
cro
5M
1.35
ll
rco
h wit
Pe
n
ll i
rco
Su
0.2
5 0.1
Cl
Na
Pe
1.34
1.33 1.00
1.05
1.10
1.15
1.20 Density g/ml
Figure 16. Refractive index as a function of density of a Percoll gradient. (Work from Amersham Biosciences, Uppsala, Sweden.)
25
Fractionation of gradients After centrifugation, the gradient can be fractionated by puncturing the bottom of the tube and collecting the outflow into fractions, or by a number of other techniques (1, 28). A simple and convenient method is to collect the fractions from the top of the tube by displacement with a dense medium such as undiluted Percoll, or a 60-65% sucrose solution. Upon pumping this dense material to the bottom of the tube, fractions can be drawn off the top. Zonal rotors may be emptied by pumping a denser solution to the distal part of the rotor and collecting fractions from the center.
Cell sorting and counting Percoll does not interfere with fluorescent activated cell sorting (FACS) (911, 1042), or with electronic counting instruments (12). The DNA content of gradient fractions can also be used as a measurement of cell number (12).
Protein determination and enzyme assay Percoll causes a background color with Folin-Ciocalteau and Lowry reagents, and all measurements should use Percoll solutions for the preparation of the blank. Higher protein concentrations can be determined using the biuret reaction (85). Terland et al. (89) recommend the Coomassie blue method of Bradford (90), since Percoll does not interfere with color development. Vincent and Nadeau (518) have reported a modification of Bradford´s method which involves precipitation of Percoll in a NaOH Triton® X-100 mixture. Cell organelles are often identified primarily by the presence of specific enzymes. Many enzyme assays can be carried out in the presence of Percoll without interference. Pertoft and Laurent (21) described an experiment in which the enzymes 5'-nucleotidase (plasma membranes), glucose-6-phosphatase (microsomes), b-glucuronidase (lysosomes) and succinic dehydrogenase (mitochondria) from rat liver homogenates were analyzed in the presence of Percoll. In all cases, the activities were at least as high in Percoll as in the controls indicating that the determinations were not influenced by the medium. Labile succinic dehydrogenase activity was stabilized by Percoll. Aryl sulphatase, alkaline phosphatase, acid phosphatase, b-galactosidase, N-acetyl-a-D-glucosaminidase and b-glucosaminidase have also been analyzed in the presence of Percoll without interference from the medium (21). Due to light scattering by Percoll, it is preferable to use enzyme assays which utilize fluorescence rather than absorbance for detection of activity. For further details of enzyme measurements in Percoll, see references 13, 43, 53, 54, 78 and 89.
26
Removal of Percoll after centrifugation Since Percoll is non-toxic to biological materials and does not adhere to membranes, it is usually unnecessary to remove Percoll from the purified preparation. Cells can be transferred directly to cell culture systems (23, 57), virus infectivity is unimpaired (21), and organelles can be used for metabolic studies (21) without any effect caused by the gradient material. The following methods can be used to eliminate the gradient material if desirable.
Washing (Low speed centrifugation) Living cells can be separated from Percoll medium by washing with physiological saline (5 volumes saline to 1 volume of Percoll cell suspension). The washing may be repeated two or three times and the cell collected between each washing step by centrifugation at 200 x g for 2-10 minutes. Studies with radioactively labelled Percoll (Table 2) have shown that no detectable residual Percoll is left adhering to cells washed in this way. Electron micrographs by Enerbäck et al. (9) (Figure 17) and Schumacher et al. (31) show cell preparations with no visible contaminating particles from the gradient material.
Figure 17. Electron micrograph of mast cells isolated by gradient centrifugation on Percoll (9, reproduced by kind permission of the authors and publisher).
Washing (High speed centrifugation) For viruses and subcellular particles which are too small to be pelleted by low speed centrifugation as described above, the biological material can be separated from coated silica particles by high speed centrifugation in a swinging bucket rotor or angle-head rotor. The undiluted fraction obtained from the first centrifugation run is placed in a centrifuge tube and spun in a swinging bucket rotor at 100,000 x g for 2 hours, or 90 minutes in an angle-head rotor (100,000 x g) to pellet the Percoll. The biological material remains above the hard pellet of Percoll (12, 39). Table 2. Removal of Percoll from rat liver hepatocytes
[125I]-Iabelled Percoll was used to isolate hepatocytes in Eagle´s MEM at a density of 1.07-1.09 g/ml. [125I] (cpm) 5 ml of the original cell suspension in Percoll
35,680
Cell pellet (from 5 ml of the original cell suspension in Percoll) washed with 80 ml of Eagle´s MEM and centrifuged at 200 x g for 10 minutes
71
Washing repeated once
0
Cells from 2 ml of the cell suspension were seeded on a 6 cm Petri dish and 80% of the cells attached to the dish. After four washings with 5 ml portions of Eagle´s MEM, the cells were detached with 0.01% trypsin plus 0.25% EDTA.
0
(Original work by Pertoft et al. reproduced by kind permission)
27
Other methods Chromatography by gel filtration on Sephacryl™ S-1000 Superfine will separate Percoll from larger particles (e.g. subcellular particles), which are eluted in the void volume. Removal of Percoll from microsomal vesicles by gel filtration on Sephacryl S-1000 Superfine has been reported (275). The authors followed the elution pattern by assaying for the microsomal marker enzyme NADPHcytochrome c reductase (Figure 18). The resulting microsomal fraction was examined by electron microscopy and found to be almost free from Percoll (less than 0.5% compared with the initial sample).
125 l(cpm
x 10 -3)
Preliminary experiments using electrophoresis to separate lysosomes and viruses from Percoll have been reported (21), but the methodology is difficult and results are often unpredictable (Pertoft, personal communication). 10 A 280 [
8 6
A 280 5
4
3
125
l ]-labelled Percoll VO
Vt
2 1 0 0
25
50 1h
75 ml
Figure 18. Gel filtration of microsomes obtained from gradients of Percoll containing 125I-labelled Percoll on Sephacryl S-1000 Superfine. Vo = void volume, Vt = total volume. (275, reproduced by kind permission of the authors and publisher).
28
Practical notes Care and cleaning of equipment Polycarbonate tubes should be used with Percoll as the particles do not adhere to the walls of these tubes. Solutions of Percoll usually produce a small pellet of compacted silica at the bottom of the tube after centrifugation and deposits on the wall of tubing used for fractionation etc. These deposits may be difficult to remove when dry. Therefore, it is recommended that all equipment is washed immediately after use. Spillages of Percoll can be removed by washing with water.
Storage of Percoll Percoll is supplied sterile and can be stored at room temperature for at least two years. At -20 ºC, it can only be stored for six months. If stored at -20 ºC, gradients form upon thawing, necessitating a mixing of the contents of the bottle before use. Preformed gradients can be stored for weeks without a change in gradient shape, provided that the gradient is sterile and is not physically disturbed.
Sterilization of Percoll solutions Percoll is supplied sterile and can be resterilized by autoclaving at 120 ºC for 30 minutes without any change in properties. Autoclaving of Percoll solutions must be carried out in the absence of salts or sucrose (i.e. do not autoclave SIP). When autoclaving undiluted Percoll, it is recommended that minimum contact with air be maintained to avoid particle aggregation at the Percoll/air interface. This can be accomplished by using a narrow-necked bottle when autoclaving. If these particles form, they may be removed by low speed centrifugation. If any significant evaporation occurs during autoclaving, the volume should be replenished with sterile water so that the density is not affected.
Aggregates of silica particles It is an inherent tendency of all silica colloids to form aggregates, either during autoclaving as described above, or upon prolonged storage. These aggregates may be observed in some batches of Percoll either as a slight precipitated sediment or as a faint white band which has a density of 1.041.05 g/ml. This band may form during gradient formation in the centrifuge or during low speed centrifugation of a preformed gradient. The aggregated silica does not interfere with the separation of biological particles as almost all cells and organelles have buoyant densities in Percoll of greater than 1.05 g/ml.
29
Applications Blood cells The entire spectrum of cell types present in blood can be resolved on preformed gradients of Percoll. The method described by Pertoft et al. (55) (Figure 19) utilizes both rate zonal (separation by size) and isopycnic (separation by density) techniques. Diluted blood was layered on top of a preformed self-generated gradient and centrifuged for 5 minutes at 400 x g, during which time the thrombocytes or platelets (which are appreciably smaller than the other cells present) did not penetrate into the gradient. 15 min at 800 x g 1010 9
10 1.10
sity
Den
Platelets
ient
grad
108
Number of cells
Density g/ml
5 min at 400 x g
107 106
1.05
0.5•106 MNC 1.00
1 1
PMNC
RBC
2
3
4
5 6 Volume ml
7
8
0
8
5 6 3 7 4 2 Distance from the meniscus cm 9
10
Figure 19. Separation of human blood cells in a gradient of Percoll. The tubes were filled with 10 ml of 70% (v/v) Percoll in 0.15 M NaCl (p=1.086 g/ml), and the gradient performed by spinning in a 14º angle rotor at 20,000 x g for 15 minutes. Two ml of gradient material was removed from the bottom of the tube using a syringe, and 2 ml of 50% heparinized blood on 0.15 M NaCl was layered on top of the gradient. Centrifugation was carried out as indicated. Densities were monitored using Density Marker Beads. MNC = Mononuclear cells, PMNC = Polymorphonuclear cells, RBC = Red Blood cells (55, reproduced by kind permission of the authors and publisher.)
The plasma layer containing the thrombocytes was removed and replaced by saline, and centrifugation was continued at 800 x g for 15 minutes, resulting in isopycnic banding of mononuclear cells (lymphocytes and monocytes), polymorphonuclear cells and erythrocytes. The position and densities of the banded cells were monitored using Density Marker Beads in an identical gradient contained in a second centrifuge tube. Although the above method demonstrates the utility of Percoll for fractionating whole blood, most blood cells can be appreciably enriched using a simple step gradient. A simple step gradient often gives acceptable yields and purity for downstream processing. The Application Tables below contain a number of examples of purification of blood cells and other cell types using different types of Percoll gradients.
30
The following tables were complied to assist the researcher in selecting references most likely to contain relevant information regarding use of Percoll for a particular cell or tissue type.
Applications - Blood Cells Lymphocytes Species
Gradient type
Tissue Type
Comments
Downstream application
Ref. #
human
continuous
blood
Percoll density centrifugation resulted in significant down-regulation of L-selectin surface reactivity.
Immunoflourescence
891
human
continuous
tonsil
A Percoll density gradient was used for separation of large (low density) in vivo activated cells from small (high density) resting cells.
cell culture, FACS, granulocytemacrophage colony stimulating factor (GM-CSF) assays, and Northern blots
892
human
continuous
spleen, tonsil
Large B lymphocytes from tonsils (in vivo activated cells) obtained by Percoll gradient centrifugation displayed higher IL-4R levels than resting cells.
cell culture, Northern blots, FACS
893
human
continuous
peripheral blood
Percoll was used to separate proliferating form nonproliferating cells.
tritiated thymidine incorporation
11
human
continuous
tonsil, peripheral blood
This procedure yielded >90% viable cells and has proved quite helpful in renewing overgrown cultures.
proliferation and cytotoxicity assays
16
human
continuous
blood
Percoll was used to separate monocytes from lymphocytes.
cell culture, coagulation activity, immunoradiometric assays
40
human
discontinuous (3-layer)
intestine
Lymphocytes were enriched in the interface between 66.7 and 44% Percoll. Further purification was performed using magnetic beads.
flow cytometric analysis, immunoperoxidase procedure, cell culture
894
human
discontinuous (6-layer)
peripheral blood
Percoll was used to separate large granular lymphocytes (LGL) from peripheral mononuclear cells.
detection of CD5LOW+ in the LGL population
895
human
discontinuous
tonsil
Percoll gradient was used for the separation of small (high density) and large (low density) cells.
cell culture, apoptosis assays, immunoassay for G-CSF, bioassay for GM-CSF, northern blot analysis
896
human
discontinuous
intestine
proliferation assays, measurement of cytotoxicity, H1 receptor binding studies
897
human
discontinuous
peripheral blood
Percoll was used for the isolation of low density cells.
FACS, immunoflourescence, nonspecific esterase staining
898
human
discontinuous
peripheral blood
Lymphoctes were recovered from low density Percoll fractions.
suppression of NK-cell proliferation by freshly isolated monocytes
899
human
discontinuous
tonsil
Percoll was used to isolate follicular dendrite cells (FDCs).
cell sorting, B cell proliferation by FDCs
900
human
discontinuous
bone marrow
Percoll was used to isolate leukemic cells from bone marrow.
establishment of a leukemic cell line
901
human
discontinuous
peripheral blood
After separation on Percoll, a virtually pure population of activated cells was obtained, as estimated by the presence of the 4F2 marker and of the transferrin receptor.
immunoflourescence and assay of phospholipid metabolism
902
human
discontinuous (1-step)
blood
Lymphocyte purity was >99% and the population of monocytes was enriched 82-90%.
induction and assay of lymphokine (IL-2)-activated killer (LAK) cell activity
903
human
discontinuous (4-layer)
blood
Percoll was used for separation of large granular lymphocytic (LGL) cells from T cells.
Giemsa staining, cell activation with interleukin-2 (IL-2)
904
human
discontinuous (4-layer)
tonsil
Percoll was used for B cell enrichment.
flow cytometry
905
31
Applications - Blood Cells (cont.) Lymphocytes (cont.) Species
Gradient type
Tissue Type
human
discontinuous (5-layer)
blood
Large granular lymphocytes (LGL) were collected from the low density fractions, whereas T cells were located in the higher density bottom fraction.
FACS, cell culture, cytotoxicity assays
906
human
discontinuous (5-layer)
blood
Monocytes were purified up to 90% and lymphocytes to >99%.
cell counting (hemocytometer) and cell culture assays
69
human
discontinuous (7-layer)
peripheral blood
cytotoxicity assay, flow cytometry analysis, and complementdependent lysis
907
human
selfgenerating
peripheral blood
Percoll was used to separate viable and nonviable cells. Yields were slightly higher and erythrocyte contamination was slightly lower with Percoll than with Ficoll-Isopaque.
cytotoxicity assays
83
canine
continuous
blood
Percoll was used for enrichment and depletion of antibody-positive cells.
reverse hemolytic plaque assay and cell-mediated lympholysis
908
canine
discontinuous (4-step and 2-layer)
whole blood
A final sedimentation of purified measurement of NK activity lymphocytes through a 45/50% Percoll gradient concentrated natural killer (NK) activity into a single band of lymphocytes.
909
mouse
continuous (3-layer)
intestine
Enrichment increased from 44.1% (single filtration) to 52.4% (multiple filtration) after nylon wool filtration, and from 70.3% (single filtration) to 82.8% (multiple filtration) after Percoll fraction.
flow cytometry
910
mouse
continuous (5-layer)
spleen
Percoll was used for separation of virgin and memory T cells.
cell proliferation assays, FACS
911
mouse
discontinuous (3-layer)
spleen
Percoll was used for separation of B cells.
protein phosphorylation assay
912
mouse
discontinuous (3-layer)
intestine
Percoll was used for isolation of intestinal intraepithelial lymphocytes (IEL).
DNA analysis by flow cytometry, mRNA-cDNA dot blots, PCR
913
mouse
discontinuous (4-layer)
spleen
Percoll was used for isolation of small, resting B cells.
cell cycle analysis by flow cytometry
914
bovine
discontinuous
mammary
Purified cells were >80% pure.
Wright´s Giemsa staining, cell culture
915
32
Comments
Downstream application
Ref. #
Monocytes Species
Gradient Type
Tissue Type
human
discontinuous (minigradient)
peripheral blood
With the Percoll minigradient, cells could be obtained in 90-100% from the patients at all time points after bone marrow transplant (BMT).
Comments
cytogenic analysis
916
human
continuous
blood
The isolated mononuclear leukocyte (MNL) fraction contained >80% cells giving a positive reaction for a-naphthyl acetate esterase ( a-NAE).
cell culture
917
human
continuous
peripheral blood
Percoll was used to isolate monocytes with >85% purity and >95% viability.
cell culture with cytokines
918
human
continuous
blood
Percoll has proved very practical for the separation of monocytes from blood and of macrophages from ascites and synovial fluids.
cell culture
34
human
continuous
blood
Percoll gradients were used for the separation of monocytes from lymphocytes.
cell culture
40
human
continuous
blood
A one-step procedure was used for obtaining a high-yield suspension of monocytes of 20% purity, which does not require washing before cultivation. A two-step method gave better than 90% pure monocytes at a lower yield.
cell counts, Fc-receptor presence and phagocytosis assays
57
human
continuous
peripheral blood
MNL were separated into two fractions with Percoll: one consisting mostly of monocytes and the other lymphocytes.
fungal (Coccidioides immits) killing assay
919
human
discontinuous
blood
Monocyte purity was 95%.
cell culture
920
human
discontinuous
whole blood
Percoll gradient was used for enrichment of hematopoietic progenitor cells.
assay for colony formation
921
human
discontinuous
blood
RNA isolation, northern blot analysis and RT-PCR
922
human
discontinuous
bone marrow
DNA hybridization studies
923
human
discontinuous (1-layer)
blood
Lymphocyte purity was >99% and the population of monocytes was enriched 82-90%.
induction and assay of lymphokine (IL-2)-activated killer (LAK) activity
903
human
discontinuous (1-layer)
blood
PMN recovery was >90% and RBC contamination <5%.
northern blot analysis
924
human
discontinuous (1-layer)
peripheral blood
Monocytes were ≥95% pure.
northern blot analysis, nuclear runoff experiments, S1 protection assay
925
human
discontinuous (4-layer)
peripheral blood
Cells obtained from the 65% to 75% interface were 99% granulocytes.
analysis and western blot analysis genomic DNA isolation and PCR
926
human
discontinuous (1-layer)
peripheral blood
With the 1-step gradient, the purity of the monocytes was 93-96%.
Giemsa staining and cell culture
927
human
discontinuous (5-layer)
peripheral blood
Percoll-isolated monocyte/ macrophages as identified by Wright-Giemsa stain.
interactions between monocyte/ macrophage and vascular smooth muscle cells
928
human
discontinuous (5-layer)
blood
Monocytes were purified up to 90% and lymphocytes to 99% purity.
cell recovery counting and cell culture assays
69
human
discontinuous
peripheral blood
cell enumeration with Coulter counter, RNA isolation, and northern blot analysis
929
equine
discontinuous (1-layer)
peripheral blood
cell recovery assays
930
All MNCs were recovered on Percoll gradients without any neutrophil contamination.
Downstream Application
Ref. #
33
Applications - Blood Cells (cont.) Erythrocytes Species
Gradient Type
Tissue Type
Comments
Downstream Application
Ref. #
human
continuous
whole blood
Percoll was used for separating young and old erythrocytes.
immunoflourescence analysis of complement receptor type 1 (CR1) and CD59, proteolytic cleavage of CR1 in vivo.
931
human
continuous
blood
Percoll was used to separate Plasmodium falciparum-parasitized erythrocytes from nonparasitized erythrocytes.
isolation of erythrocyte membranes lipid peroxidation, vitamin E and transmembrane reducing system analysis
932
human
continuous
blood
A rapid method for the age fractionation of human erythrocytes by Percoll density gradient centrifugation was described.
flame photometry, enzyme assays
77
human
discontinuous (4-layer and 8-layer)
blood
A rapid method using Percoll to fractionate erythrocytes according to age was described.
analysis of the decline of enzymatic activity in aging erythrocytes
933
human
discontinuous (4-layer)
blood
ELISAs, proteolytic digestion of membranes
934
human
discontinuous (4-layer)
blood
The position of Density Marker Beads (Amersham Biosciences) was used to collect cells with densities 3 3 < 1.00 g/cm or > 1.119 g/cm .
yield stress experiment: a sensitive index of cell: cell adhesion of deoxygenated suspensions of sickle cells
935
human
discontinuous
blood
Percoll gradient was used to separate erythrocytes into 4 density fractions.
platelet-activating factor (PAF) acetylhydrolase activity and membrane fluidity
936
human
discontinuous
blood
Erythrocytes loaded with L-asparaginase using a hypotonic dialysis process were separated into eight fractions.
L-asparaginase activity
937
human
discontinuous
blood
Discontinuous gradient of the range 1.080-1.115 g/cm3 with each layer differing in density by 0.005 g/ml produced nine cell fractions.
enzyme assays
66
human
discontinuous (5-layer)
blood
study of RBC deformability and cell age
938
human
discontinuous (8-layer)
blood
Percoll was used for density separation of RBC loaded with inositol hexaphosphate (IHP) by reverse osmotic lysis.
haemoglobin distribution, distribution of IHP concentrations
939
human
discontinuous (9-layer)
blood
A detailed comparison between two cell-loading techniques for inositol hexaphosphate was performed by monitoring the RBC distribution patterns on Percoll density gradients.
oxygen affinity, hematological parameters and organic phosphate content measurements
940
Mastomys natalensis
continuous
blood
Percoll was used to separate Plasmodium berghei-paraitized erythrocytes from non parasitized cells.
cAMP level in RBCs
941
mouse
continuous (self-forming)
blood
Fractionation of RBC yielded five distinct populations that maintained their densities upon recentrifugation in a second gradient.
transbilateral movement and equilibrium distribution of lipid
942
mouse
continuous
peripheral blood
Percoll was used for density gradient separation of chemically-induced erythrocytes.
fixing, staining and flow cytometric analysis of micronucleated polychromatic (MPCE) and micronucleated nonchromatic (MNCE) erythrocytes
943
mouse
discontinuous
peripheral blood
Erythrocytes were contaminated with only 0.001% nucleated cells.
glucose phosphate isomerase (GPI) assay
944
34
Erythrocytes (cont.) Species
Gradient Type
Tissue Type
rat
discontinuous
whole blood
Percoll was used to separate Plasmodium berghei-infected RBCs.
Comments
oxygen dissociation analysis
Downstream Application
Ref. # 945
rabbit
discontinuous (7-layer)
blood
Rabbit red blood cells were reproducibly fractionated into populations of various stages of maturation.
measurement of cytosolic protease activities
946
trout
discontinuous
blood
The gradient in the region of 45-65% Percoll produced three red cell fractions which is due to multiplicity of haemoglobin components.
antioxidant enzyme activities and membrane fluidity analysis
947
Downstream Application
Ref. #
Natural killer cells Species
Gradient Type
Tissue Type
Comments
human
discontinuous
peripheral blood
The Percoll (preculture) step facilitated the density separation of resting cells from larger lymphocytes.
NK- and T-cell activation, immunoflourescence
948
human
discontinuous
blood
K562 cells which adhere to NK cells were separated together. Enrichment of NK cells was 71.3%.
cytotoxicity studies, morphological characterization
61
human
discontinuous (2-layer)
peripheral blood
The low density fraction (42.5-47.5% Percoll) which showed a 4-fold enrichment in NK activity was used.
NK activity and kinetic constant determinations, measurement of the effect of divalent cations on NK activity, and effect of ATP on NK cell-surface markers
949
human
discontinuous (6-layer)
blood
Further purification using magnetic beads resulted in a pure preparation.
cytotoxic assay
950
human
discontinuous (8-layer)
peripheral blood
Recovery was >80% while viability, as judged by trypan blue exclusion, was >95%.
natural-killer cell stimulatory effect, phenotype evalution by immunoflourescence
951
mouse
discontinuous (3-layer)
lung
The cells at the 50/55% interface were the richest in NK cell activity.
adoptive transfer to reconstitute NK activity in NK-depleted mice
952
mouse
discontinuous (6-layer)
spleen
NK cells were enriched in the lower density Percoll fraction, while natural cytotoxic T-cells (NCT) were distributed between both higher and lower density fractions.
cytotoxicity of NK cells was measured
953
mouse
discontinuous (6-layer)
liver
All NK activity was above 1.08 g/ml density. Interfaces at 1.04 and 1.06 gave a 2X enrichment of NK progenitors.
PCR, western blot analysis, and cytotoxicity assays
954
35
Applications - Blood Cells (cont.) Neutrophils Species
Gradient Type
Tissue Type
human
discontinuous (1-layer)
whole blood
human
discontinuous (2-layer)
whole blood
human
discontinuous (4-layer)
peripheral blood
human
discontinuous (4-layer)
human
Comments Neutrophils pelleted in the 1.077 g/ml cushion.
Downstream Application FACS analysis, intracellular Ca and superoxide anion measurements
Ref. # ++
955
polymorphonuclear neutrophil (PMN) labelling by immunoflourescence, adherance assay and superoxide assay
956
Percoll was used to separate monocytes and lymphocytes.
immunoflourescence and flow cytometry
957
whole blood
Eosinophils and neutrophils were isolated following dextran sedimentation.
flow cytometry and measurement of lactoferrin release
958
discontinuous
blood
Cell preparation was layered onto a Percoll cushion to remove monocytes. After lysis of the erythrocytes, primarly neutrophils, with the remaining cells being predominantly eosinophils.
immunoflourescence studies
959
human
discontinuous
blood
The neutrophils were >95% pure.
indirect immunoflourescence, immunoelectron microscopy and FACS analysis, O2 consumption
960
human
continuous, nonlinear (2-layer)
blood
Percoll was used for subcellular fractionation of azurophil granules, specific granules, gelatinase granules, plasma membranes, and secretory vesicles.
ELISAs for NGAL, gelatinase, lactoferrin and myeloperoxidase
961
mouse
continuous
peritoneum
An ~97% pure polymorphonuclear neutrophilic leukocyte (PMN) preparation was obtained using Percoll.
electrophoretic analysis, GM-CSF assay, and cell morphology and counts
70
Species
Gradient Type
Tissue Type
Comments
Downstream Application
Ref. #
human
discontinuous
peripheral blood
Eosinophils were purified using Percoll gradients followed by immunomagnetic beads. Using this procedure, the eosinophil purity was always >95% and the viability was >98%.
FACS analysis, eosinophil migration assays, Ca++ measurements
962
human
discontinuous (2-layer)
blood
The recovery of eosinophils was 40-60%, the viability >98% as tested by trypan blue exclusion, and the purity >85%.
chemotaxis and intracellular Ca++ measurements
963
human
discontinuous (2-layer)
blood
Eosinophil purity was >95%, and the method did not induce priming of the eosinophils.
serum-treated Zymosan (STZ) binding and placenta-activating factor (PAF) measurments
964
human
discontinuous (2-layer)
blood
Eosinophil purity was always >85% and the recovery ranged from 40-60%. Viability was >98%.
chemotaxis assay
965
human
discontinuous (3-layer)
peripheral blood
Eosinophil purity was 95-99%, viability using trypan blue was >98%, and recovery was 40-60%.
density distribution analysis, cell culture
966
human
discontinuous (4-layer)
whole blood
The effect of dextran sedimentation on the density of neutrophils and eosinophils was analyzed.
flow cytometry and measurement of lactroferrin release
958
Eosinophils
36
Basophils Species
Gradient Type
Tissue Type
Comments
human
continuous
peripheral blood
Basophils were purified by Percoll density gradient separation and cell sorting. The procedure yielded 95% purity with a total yield estimated to range from 5-28%.
flow cytometry, histamine release, electron microscopy
Downstream Application
Ref. # 967
human
continuous
bone marrow
The purity of basophils in the low density fraction (<1.063 g/ml) was generally >75% of the cells.
histamine content and release
968
human
discontinuous
peripheral blood
Highly purified basophils were obtained by Percoll gradient followed by negative selection using flow cytometry.
effects of cytokines on human basophil chemotaxis
969
human
discontinuous (2-layer)
blood
The majority of the basophils were located at the 1.070-1.080 interface. The purity in this fraction was 36-63%.
further purification by negative selection using immunomagnetic beads
970
human
discontinuous (2-layer)
blood
Highly purified basophils were obtained by Percoll gradient followed by negative selection using flow cytometry.
histamine release assay, chemotactic assay
971
human
discontinuous (3-layer)
whole blood
Basophils were purified to >80% using Percoll gradient followed by treatment with monoclonal antibodies to remove contaminants.
flow cytometry and leukotriene C4 generation following calcium ionophore stimulation
972
human
discontinuous (3-layer)
peripheral blood
Basophil purity was 85-96% using Percoll.
cell stimuli and mediator release assay
973
rat
discontinuous
blood
further purification by immunomagnetic beads, immunoflourescence, electron microscopy
974
37
Applications - Other Cell Types Liver cells Species
Gradient Type
Tissue Type
Comments
Downstream Application
Ref. #
human
continuous
liver
Purification of cryo-preserved hepatocytes on Percoll density gradients increased the percentage of viable cells from 55 to 87%.
primary cell culture, electron microscopy, viability assay radiolabelled protein synthesis, secretion assay, metabolic studies, toxicological studies
975
rat
continuous
liver
Percoll offered a good way to obtain an enriched population of Kupffer cells. Recovery was 82%, viability 87% and purity 67%.
peroxidatic reaction
20
rat
continuous
liver
Percoll gradients were used to isolate hepatocyte plasma membranes and mitochondrial membranes.
phase contrast microscopy, cell binding experiments
33
rat
continuous
liver
Rat liver cells furnished subpopulations of parenchymal cells (hepatocytes) having buoyant densities of 1.07-1.09 g/ml, and non-parenchymal cells (mostly phagocytosing Kupffer cells) at a density of 1.04-1.06 g/ml.
cell culture
55
rat
NA
liver
Final preparations contained less than 5% nonviable cells as judged by trypan blue exclusion.
cell culture
71
rat
continuous
liver
Percoll gradients were used to franctionate nonparenchymal cells into Kupffer cells, stellate and endothelial cells.
light and flourescence microscopy, carboxyesterase and Glutathione-Stransferase (GST) activities
976
rat
discontinuous (2-layer)
liver
Percoll provided a simple, low cost, and rapid method for the isolation, purification and cultivation of rat liver sinusoidal endothelial cells (LEC).
electron microscopy, cell culture, trypan blue exclusion
977
rat
discontinuous (2-step)
liver
Percoll gradients were used to separate fat storing cells (FSC) from liver endothelial cells (LEC) and Kupffer cells (KC).
cell culture
978
rat
continuous
liver
Following the removal of damaged cells by centrifugation in Percoll, the mean viability of cryo-preserved hepatocytes, tested by trypan blue exclusion, was 88.6% (±1.3%).
cell viability and study of xenobiotic metabolism
979
rat
continuous
liver
Percoll was used to remove dead cells from cryopreserved cells. Cell viability was 88 ±1% after the Percoll step.
cell viability and study of xenobiotic metabolism
980
rat
continuous
liver
If cryo-preserved cells were purified by a Percoll centrifugation after thawing, the enzyme activities were not significantly different from those of freshly isolated parenchymal cells, and the viability was 86%.
Lowry protein assay, cytochrome assay, enzyme assays
981
rat
continuous
liver
Percoll separation yielded cryopreserved cells with a viability and metabolic capacity not measurably different from freshly isolated cells.
protein determination, enzyme assays and metabolism of testosterone and benzo(a) pyrene (BaP)
982
rat
discontinuous (2-layer)
liver
Percoll two-step gradients were used to separate Kupffer cells (KC) and liver endothelial cells (LEC). Preparations of KC were 85-92% homogenous while the LEC preparation was at least 95% pure.
light microscopy, electron microscopy and peroxidase staining
983
38
Liver cells (cont.) Species
Gradient Type
Tissue Type
rat
discontinuous (5-layer)
liver, spleen
Percoll gradients were used to separate both spleen and liver cells. Spleen and liver cell viability was over 95%.
Comments
trypan blue viability assay, cell culture
Downstream Application
Ref. # 984
rat
continuous
liver biopsy
Percoll was used for separation of hepatocytes and non-parenchymal cells, as well as subfractionation.
cell enumeration using Coulter counter, immunocytochemistry, DNA extraction, Southern blot analysis, assay of marker enzymes and protein in subcellular fractions, electron microscopy
985
Downstream Application
Ref. #
Leydig cells Species
Gradient Type
Tissue Type
human
continuous
testis
Percoll-purified Leydig cells were 70-80% pure based on staining for 3 betahydroxysteroid dehydrogenase.
Comments
cell culture, stimulation of testosterone production
986
human
continuous
testis
Percoll-purified Leydig cells were 80-90% pure as determined by 3 betahydroxysteroid dehydrogenase staining.
immunocytochemical localization of apolipoprotein E (apoE)
987
human
discontinuous (4-layer)
testis
Percoll gradients were used to isolate human Leydig cell mesenchymal precursors.
cell culture
988
human
discontinuous (5-layer)
testis
Percoll gradient centrifugation permitted isolation of two Leydig cell fractions.
cell culture
989
mouse
continuous (linear)
testis
Two groups were obtained: group 1 had densities of 1.0667-1.0515 g/ml; group 2 had densities of 1.0514-1.0366 g/ml.
in vitro testosterone production electron microscope stereology
990
porcine
discontinuous
testis
Purity of Leydig cells was >85%.
effect of hydrocortisone (HS) and adrenocorticotropic hormone (ACTH) on testosterone production
991
rat
continuous
testis
Rat Leydig cells were purified from testis using elutriation followed by Percoll gradient centrifugation.
cell culture, the effect of human chorionic gonadotropin (hcG) on its gene regulation and protein secretion
992
rat
continuous
testis
cell culture, the effect of GHreleasing hormone (GHRH) on Leydig cell steroidogensis
993
rat
continuous
testis
Rat Leydig cells were purified from testis using elutriation followed by Percoll gradient centrifugation. Band 2(of 3) contained >95% Leydig cells (average density was 1.075 g/ml).
cell culture in presence of 125Ilabelled hcG, testosterone and cAMP production
994
rat
continuous
testis
Comparison of Leydig cells of different densities were made.
viability staining, cell culture
995
rat
continuous
testis
viability staining, in vitro testosterone production, SDS-PAGE electrophoresis
996
rat
continuous
testis
cell culture in presence of human chorionic gonadotropin (hcG), phase contrast microscopy, light microscopy and electron microscopy
31
rat
discontinuous (2-step)
testis
cell culture in the presence of interleukin-1 (IL-1)
997
rat
continuous testis (self-generating)
Leydig cell precursors and pure (96%) Leydig cells were isolated on Percoll gradients.
cell culture in presence of human chorionic gonadotropin (hcG)
998
rat
discontinuous
testis
The purity of Leydig cells ranged from 90–95%.
cell culture in presence of human chorionic gonadotropin (hcG)
999
rat
discontinuous and continuous
testis
In the discontinuous gradient, the densest fraction contained a high proportion of Leydig cells whereas the lighter fraction contained mostly non-Leydig cells.
125 I-labelled iododeoxyuridine incorporation
Isolation by Percoll gradient resulted in complete retention of morphological and biological integrity and a purity of 90-95%.
1000
39
Applications - Other Cell Types (cont.) Spermatozoa Species
Gradient Type
human
discontinuous
Percoll density centrifugation was a more efficient technique than swim-up for the selection of spermatozoa from fresh semen samples and worked equally well for cryo-preserved samples.
flow cytometry
1001
human
discontinuous (2-layer)
For oligozoospermic samples, the Percoll pre-layered motile count was significantly higher than the two other sperm washing methods evaluated.
concentration and mobility assays
1002
human
discontinuous (2-layer)
The percentage of sperm recovery was higher with Percoll than with the swim-up method.
recovery and mobility assays, acrosome reaction, oocyte penetration assay
1003
human
discontinuous (2-layer)
Percoll gradients resulted in better recovery and motility compared to SpermPrep filtration and swim-up techniques. Percoll also separated the spermatozoa from the seminal plasma, thereby removing bacteria and free oxygen radicals.
recovery and motility assays
1004
human
discontinuous (2-layer)
The use of Percoll resulted in a marked improvement in motility, but the ATP concentration remained low.
ATP and motility characteristics of sperm
1005
human
discontinuous (2-layer)
The Percoll and centrifuged-wash treatments showed a correlation between the sperm’s decondensation and penetration abilities.
cryo-preservation, sperm penetration assay, sperm nuclear decondensation assay
1006
human
discontinuous (2-layer)
Both swim-up and Percoll techniques significantly improve the percentage motility, curvilinear velocity, mean amplitude of lateral head displacement (ALH) and percentage normal morphology compared with the original semen samples.
motility assays, zona-free hamster egg penetration test
1007
human
discontinuous (2-layer)
The effects of swim-up, Percoll and Sephadex™ techniques on sperm membrane integrity were compared.
hypo-osmotic swelling (HOS) test
1008
human
discontinuous (2-layer)
The characteristics of spermatozoa selected using multiple-tube swim-up and discontinuous Percoll centrifugation were compared.
motility assays, hypo-osmotic swelling (HOS) test, acrosome assessment hamster egg penetration assay (HEPA)
1009
human
discontinuous (2-layer)
Percoll-purified sperm were used to assay the effect of trypsin inhibition on the acrosome reaction and spermatozoa penetration.
acrosome reaction, sperm penetration assay
1010
human
discontinuous (2-layer)
Percoll gradients gave excellent yields when the lower layer contained 81% (v/v) Percoll.
spermatozoa recovery and motility assays
1011
human
discontinuous (2-layer)
Recovery of motile sperm was 70.4-95% in the denser fraction and 5.2-30.0% in the other fractions.
lectin binding assays
1012
human
discontinuous (2-layer)
Discontinuous Percoll gradients were used to separate sperm into high and low density populations.
hamster oocyte penetration test, motility assay, lipid peroxidation analysis
1013
human
discontinuous (2-layer)
The 80% fractions generally exhibited high rates of sperm-oocyte fusion without leukocyte contamination.
hamster oocyte penetration test, acrosome reaction
1014
human
discontinuous (2-layer)
In the 80% sperm fractions (oligospermic), ATP content, total lactate dehydrogenase (LDH) and LDH-X activity were not statistically different from the corresponding normospermic fractions.
simultaneous measurement of LDH, LDH-X, sperm creatine phosphokinase (CPK) activities, and ATP content
1015
human
discontinuous
Highly motile, functionally competent sperm were (2-step) isolated in the high-density region of the gradient, which is characterized by a low capacity for generation of reactive oxygen species (ROS).
ROS generation in oligozoospermic and fertile donor ejaculates
1016
human
discontinuous (2-layer)
hamster oocyte penetration test, acrosome reaction and motility characteristics
1017
40
Comments
Downstream Application
Ref. #
Spermatozoa (cont.) Species
Gradient Type
human
discontinuous (3-layer)
Autoantibody-carrying spermatozoa from infertile men were processed using a discontinuous Percoll gradient.
Comments
direct immunobead test
Downstream Application
Ref. # 1018
human
discontinuous (5-layer)
Sperm recovered from different Percoll layers and incubated in a capacitating medium (B2 Menezo) had an excellent ability to maintain motility, regardless of their Percoll fraction.
motility assay
1019
human
discontinuous (6-layer)
Percoll centrifugation selected a germinal population that is denser and with less stainable chromatin.
flow cytometry
1020
human
discontinuous (hyperosmotic and iso-osmotic)
When comparing hyperosmotic versus conventional Percoll gradients, significantly higher total and motile sperm recovery rates were found with the hyperosmotic in both the normal and abnormal group.
motility and morphology analysis
1021
human
discontinuous (6-layer)
Percoll centrifugation appeared to be quite efficient, as 32% of the initial concentration was recovered.
motility and morphology assays, transmission electron microscopy
1022
human
discontinuous (2-layer)
The purity and efficiency of sorting were high (98% for both Percoll and the swim-up method).
flow cytometry
1023
bovine
discontinuous
Percoll was thought to improve semen and preserve acrosome integrity.
acrosome microscopy evaluation
1024
hamster
continuous
Caput epididymal spermatoazoa, with a specific gravity of 1.10-1.12 g/ml, were isolated without contamination by other cells.
lipid extraction and fractionation electron microscopy
1025
macaque continuous
Percoll separation resulted in increased sperm-zona binding and did not affect the percentage of acrosomereacted sperm bound to the zona or the percent motility and percentage of acrosome-reacted sperm in suspension.
zona binding experiments, acrosome reaction, motility assays
1026
Bone marrow cells Species
Gradient Type
Tissue Type
normal human
discontinuous (2-layer)
bone marrow
Megakaryocytes were at the interface between 1.020 g/ml and 1.050 g/ml.
magnetic beads for further purification, flow cytometry
1027
normal human
discontinuous
blood
B-cells were recovered at least 95% pure. Gradients removed B-cell blasts very effectively.
flow cytometry
1028
HIV discontinuous infected, (2-layer) normal and immune thrombocytopenic purpura human
bone marrow
Cells at the 1.020/1.050 interface were enriched 10-fold in megakaryocytes, while those at the 1.050/1.070 interface were immature cells.
megakaryocyte cultures prepared from immature cells for in situ hybridization
1029
normal human
discontinuous (2-layer)
bone marrow
Percoll density fractionation resulted in the depletion of greater than 95% of total marrow cells and an increase in megakaryocyte frequency from about 0.05% to 3-7%.
preparation of RNA and subsequent PCR, flow cytometry
1030
normal and arthritic human
discontinuous (3-layer)
bone marrow
Cells prepared were suitable for cell culture.
colony plaque assay, immunoflourscence, flow cytometry, protein colony blotting, RNA-colony blotting
1031
peripheral blood
Low density cells post- and pre-transplant were prepared for analysis.
magnetic beads for further purification, PCR
1032
normal discontinuous and (4-layer) leukemic human
Comments
Downstream Application
Ref. #
41
Applications - Other Cell Types (cont.) Bone marrow cells (cont.) Species
Gradient Type
Tissue Type
normal human
discontinuous (7-layer)
bone marrow
T-cells obtained using Percoll were enriched about two-fold in the highdensity fractions of marrow cells and depleted by about four- to five-fold in the lowest-density fraction as compared with Ficoll-purified cells.
flow cytometry, mixed lymphocyte reaction assay, natural killer cell assay, cell culture
1033
normal human
discontinuous (1-layer)
bone marrow
Bone marrow cells were prepared using Percoll to remove RBC.
isolation of CD34+ cells using soybean agglutinin-coated flasks, progenitor cell assays, and flow cytometry
1034
marmoset
discontinuous (1-layer)
bone marrow
Bone marrow megakaryocytes from both interleukin-6 (IL-6) treated and untreated animals could be separated in Percoll.
flow cytometry
1035
primate
discontinuous (1-layer)
bone marrow
Bone Marrow was isolated from both normal monkeys and interleukin-6 (IL-6) treated monkeys.
cell enumeration, FACS, digital imaging microscopy and electron microscopy
1036
monkey
discontinuous (1-layer)
bone marrow peripheral and blood
Light density cells were prepared from aspirates over a 60% cushion.
cell culture and identification of various colony types
1037
mouse
discontinuous (1-layer)
bone marrow
Red blood cells were removed from bone-marrow preparations with a single 70% Percoll cushion.
culture of hematopoietic precursers, effects of interleukin-10 (IL-10) on proliferation, alkaline phosphatase activity, collagen synthesis assay, osteocalcin, preparation of RNA, and electron microscopy
1038
mouse
discontinuous (3-layer)
bone marrow
Bone marrow progenitor cells were suitable for culture.
effects of interleukin-3 (IL-3) and lipoplysaccharide (LPS) on cultured cells
1039
mouse
discontinuous (3-layerlayer)
bone marrow
Cells prepared were depleted of lymphoid and macrophage-lineage cells by addition of monoclonal antibody plus complement.
FACS analysis, hematopoietic progenitor cell culture, reconstitution of lethally irradiated mice
1040
mouse
discontinuous (3-layer)
bone marrow
Percoll was used to separate bone marrow fractions containing mostly blasts and lymphoid cells from those containing a high level of colonyforming units-spleen (CFU-S) counts.
FACS analysis, chemotaxis assay, assay of colonyforming units-spleen (CFU-S)
1041
mouse
discontinuous (3-layer)
proteasetreated calvarial bone sections
Percoll gradients gave distinct subpopulations of cells based upon the results of various assays.
primary cell culture, flow 1042 cytometry, insulin-like growth factor I (IGF-I) assay, binding of epidermal growth factor, alkaline phosphatase determination
mouse
discontinuous (4-layer)
bone marrow
Normal suppressor cell activity was maintained after separation.
suppressor cell activity assay
1043
mouse
discontinuous (4-layer)
bone marrow
Cells at a 1.06/1.07 g/ml density were used in subsequent studies.
reconstitution of lethally irradiated animals
1044
mouse
discontinuous (5-layer)
bone marrow, spleen
flow cytometry, reconstitution of lethally irradiated mice
1045
rat
discontinuous (3-layer)
bone marrow
culture of hematopoietic progenitor cells
1046
42
Comments
About 75% of the input CFUmegakaryocytes (CFU-MK) were recovered in the fraction between 1.063 and 1.982 g/ml Percoll. CFU-MK were enriched only in this density fraction.
Downstream Application
Ref. #
Bone marrow cells (cont.) Species
Gradient Type
Tissue Type
rabbit
continuous
bone marrow
feline
discontinuous (1-layer)
bone marrow
Comments
Downstream Application implantation into in vivo placed diffusion chamber, cytochemical staining, and electron microscopy
Marrow mononuclear cells from both feline immunodeficiency virus-infected cats and normal cats were isolated.
culture of hematopoietic progenitor cells
Ref. # 38
1047
Macrophages Species
Gradient Type
Tissue Type
human
discontinuous
lung
Alveolar macrophages were purified from contaminating granulocytes using a discontinuous Percoll gradient.
Comments
superoxide (SO) release
Downstream Application
Ref. # 1048
human
discontinuous (4-layer)
brochoalveolar lavage
Percoll gradients gave >95% alveolar macrophage (AM) purity.
cell viability assay, light microscopy
1049
human
discontinuous (4-layer)
lung
Use of Percoll resulted in near total purification of alveolar macrophages (AM) from other cells.
superoxide (SO) anion release
1050
human
discontinuous (4-layer)
decidual tissue
When cells were purified further with Percoll, the percentage of CD-14positive cells increased by 52%.
secretion of platelet-activating factor (PAF) acetylhydrolase
1051
human
discontinuous
pulmonary
>97% of the cells of fractions 1-4 were (4-layer) shown to be alveolar macrophages (AM) in a previous study.
nonspecific esterase staining, flow cytometric DNA analysis
1052
human
discontinuous (4-layer)
lung
This method was used to study alveolar macrophage (AM) heterogeneity. The increased numbers of hypodense AM found in the asthmatic patients were unlikely to be due to the procedure.
cell viability, esterase and peroxidase activity assays, electron microscopy, generation of superoxide anion and thromboxane B2
1053
human
discontinuous (5-layer)
peripheral blood
Percoll-isolated monocyte/macrophages were harvested from the top layer and routinely contained 75/90% monocytes/macrophages as identified by Wright-Giemsa stain.
interactions between monocyte/ macrophage and vascular smooth muscle cells
928
mouse
continuous and discontinuous
peritoneum
The total cell yield was 100.0% ±0.8%, and as measured by the trypan blue exlusion test, the cell viability was completely preserved.
light microscopy, trypan blue exclusion, esterase activity assay, peroxidase activity assay, cell immunophenotyping, bacterial phagocytic assays
1054
mouse
discontinuous (4-layer)
cultured cells
Percoll did not have a detectable effect on the cytolytic activity of cultured macrophages or on their viability.
phagocytic and cytolytic assays
30
mouse, rat
continuous and discontinuous
peritoneum
A continuous gradient followed by a discontinuous gradient was used to isolate all cell populations according to their actual density. This procedure yielded cells of high viability with preservation of critical cell function.
trypan blue exclusion
1055
rat
discontinuous (5-layer)
lung
The Percoll fractions were designated I to IV in order of increasing density with a percent distribution of cells of about 5, 15, 50 and 30%, respectively. Cell viability was >95%.
fluorescence microscopy
1056
rat
discontinuous (5-layer)
lung
Cell viability was >95% by trypan blue exclusion and >95% were identified as alveolar macrophages (AM) in unfractionated and fractionated cells by Giemsa and nonspecific esterase stains.
effects of pulmonary surfactant and protein A on phagocytosis, light microscopy
1057
rat
continuous
bronchoalveolar lavage
The various fractions comprised approximately 90-99% macrophages in virtually all instances.
esterase activity, surface expansion of Ia antigen by an immunoperoxidase technique
1058
43
Applications - Other Cell Types (cont.) Mast cells Species
Gradient Type
Tissue Type
Comments
Downstream Application
Ref. #
mouse
NA
peritoneum
Purity of the mast cells was nearly 100%, as checked by Memacolor fast staining.
qualitative and quantitative PCR analysis
1059
mouse
continuous
peritoneum
Starting from a peritoneal cell population containing 4% mast cells, a mast cell purification of up to 95% was obtained.
electron microscopy and ultrastructural cytochemistry
rat
discontinuous
peritoneum
Mast cell purity with Percoll was >95%.
direct interaction between mast and non-mast cells, histamine release assay
1060
rat
continuous
peritoneum
Mast cells purified on Percoll gradients were more than 90% pure by toluidine blue staining, and the viability was >98% by the trypan blue exclusion test.
flourometric assay to measure histamine release
1061
rat
continuous
peritoneum
Mast cells can be isolated with high yields and purity by centrifugation on gradients of Percoll.
light and electron microscopy, cytofluorometry
9
rat
continuous (sequential)
peritoneum
The purity of mast cells purified over sequential Percoll gradients was evaluated by measurement of the contribution of eosinophil peroxidase to mast cell peroxidase activity.
histamine release and peroxidase activity
8
1062
Thymocytes Species
Gradient Type
Tissue Type
Downstream Application
Ref. #
mouse
discontinuous (5-layer)
thymus
Percoll was used for separation of immature thymocytes.
Comments
in vitro stimulation by mitogens, isolation of nuclei, isolation and gel electrophoresis DNA, enzyme assays
1063
rat
discontinuous
thymus
Percoll was used for separation of normal and apoptotic thymocytes.
flow cytometry
1064
rat
discontinuous (4-layer)
thymus
Percoll was used for separation of cells possessing the characteristically condensed nuclear chromatin associated with apoptosis from apparently normal thymocytes.
electron microscopy, Coulter counter analysis, flow cytometry, DNA analysis
1065
rat
discontinuous (4-layer)
thymus
Percoll was used for isolation of a transitional population of preapoptotic thymocytes.
DNA analysis, isolation of nuclei and DNA autodigestion, light and electron microscopy
1066
rat, mouse
discontinuous (3-layer)
thymus
Percoll was used to separate large and small thymocytes. An extremely high level of viability was maintained.
phase contrast microscopy and autoradiography
62
Miscellaneous cells Cell type
Species
Gradient Type
Tissue Type
pancreatic islets
human, mouse
continuous
pancreas
The use of Percoll eliminated the problems of high viscosity, undesired osmotic properties and, in some cases, also toxic effects.
density determination and insulin secretion
endothelial
human
continuous linear gradient
whole blood
Final recovery of endothelial cells was 91.6%.
immunofluorescence
44
Comments
Downstream Application
Ref. # 5
1067
Miscellaneous cells (cont.) Cell type
Species
Gradient Type
Tissue Type
trophoblasts
rat
continuous
placenta
various
NA
NA
viable vs. nonviable
human, rat
apoptotic
Comments
Downstream Application
Ref. #
Percoll gradient centrifugation yielded efficient separation of rat placental lactogen-II (rPL-II) producing cells from digested tissue from labyrinth and junctional zones of the chorioallantoic placenta.
development of in vitro rat placental trophoblast cell culture system
1068
NA
This paper compared different approaches to cell separation. According to the authors, Percoll is generally the most useful media for isopycnic centrifugation of most kinds of cells.
none
1069
discontinuous (2-layer)
various tumor tissue
Interface showed a viability of >90%, but the yield of viable cells decreased dramatically if the tissue resection was not immediately processed.
trypan blue viability assay, 2-D PAGE
1070
human
discontinuous (7-layer)
promyelocytic leukemic cell line
The step gradient used generated three main cell bands and a cell pellet, the pellet was very enriched for apoptotic cells (85-90%).
DNA isolation
1071
lymphoblast
human
continuous
whole blood
Lymphoblasts were enucleated using a Percoll gradient containing cytochalasin B.
electrofusion
1072
brain capillary endothelial
rat
continuous pre-made
brain
Subsequent Percoll gradient centrifugation resulted in a homogenous population of capillary endothelial cells capable of attachment to collagen and incorporation of tritiated thymidine.
cell culture, light microscopy electron microscopy
170
neurons
rabbit
discontinuous and rate zonal
dorsalroot ganglia
Neurons were isolated with a viability of 80% and a purity of >90%.
cell culture, light and electron microscopy
480
nonmyogenic separated from myogenic
chicken
discontinuous
breast muscles
Separation of cells from embryonic muscle allowed direct analysis of cell-specific proteins without the need for cell culturing.
cell culture, microscopy, DNA/protein analysis
680
megakaryocytes
human
discontinuous
bone marrow
Isolation of megakaryocytes was reproducibly better in Percoll than in BSA.
Ficoll 400 centrifugation to further purify, complement receptor assay
155
chondrocytes
rat
discontinuous
bone marrow
Cell viability was >95% while yield varied depending on aggregation of cells.
cell culture, quantitation of proteoglycans and collagen
629
spermiophages
turkey
discontinuous
sperm
Spermiophages fixed immediately after Percoll isolation resembled those in freshly ejaculated semen except for an apparent increase in the number of mitochondria.
light and electron microscopy, cell culture
1073
NA
human
continuous
parathyroid gland
Densities of parathyroid glands were measured using various density gradient media. For densities >1.0 g/ml, Percoll proved superior to any of the other gradient liquids investigated.
glandular density determination
2
45
Applications - Microorganisms Microorganisms Species
Type
Gradient Type
Bacter -oides sp.
bacteria
discontinuous (4-layer)
NA
Percoll was used to assess the degree of capsulation of the twelve Bacteroides strains grown in 3 different media.
Ehrlichia ristcii
bacteria
continuous
cultured cells
Percoll was used to purify CO2 production assay, Ehrlichia risticii from an infected Coomassie brilliant blue murine macrophage dye binding assay cell line (P388D).
1075
Ehrlichia risticii
bacteria
continuous
cultured cells
Ehlichia risticii was purified from an infected murine macrophage cell line (P388D).
CO2 production assay, Coomassie brilliant blue dye binding assay
1076
Porphyromonas gingivalis
bacteria
continuous
NA
Percoll was used to separate unbound cells from salivacoated bead (SHAP)-bound cells.
binding and binding inhibition assays
1077
Treponema pallidum
bacteria
continuous
NA
Percoll-purified treponemes from 5-day infections were immobilized significantly more slowly than the purified treponemes from 7- and 8-day infections.
influence of different sera on in vitro immobilization of Percoll-purified Treponema pallidum
1078
Theileria sp.
bacteria
discontinuous (2-layer)
bovine erythrocytes
A purification method for viels from Theileria-infected bovine erythrocytes was developed.
light and electron microscopy and 1- and 2-D polyacrylamide gel electrophoresis
1079
Babesia bigemina
protozoa
continuous and discontinuous (4-layer)
bovine erythrocytes
Babesis bigemina-infected erythrocytes were successfully concentrated at least 20 times by Percoll and PercollRenografin density gradients.
enzymatic studies and starch gel electrophoresis
1080
Babesia equi
protozoa
continuous
horse erythrocytes
The piroplasms of Babesia equi were purified by lysis of infected horse erythrocytes and Percoll density-gradient centrifugation.
protein characterization of B. equi piroplasms
1081
Plasmodium berghei and P. chabundi
protozoa
continuous
mouse blood
Percoll was used for the separation of host erythrocyte membrane from malarial parasites. The recovery of the erythrocyte membranes was ~65-70%, whereas parasite recovery was 80-90%, and the relative purity was ~85-90%.
electron microscopy, electrophoresis, immuno-blotting, marker enzyme analysis and pulse chase analysis
1082
Babesia bovis
protozoa
continuous
bovine erythrocytes
A 65% Percoll concentration was found to be optimal for Babesia bovis merozoite (i.e. mature exoerythrocytic stage) separation. A 100% Percoll stock solution was optimal for enrichment of infected erythrocytes.
parasite viability assay
1083
Entamoeba histolytica
protozoa
discontinuous (2-layer)
faecal cyst
Percoll purification provided a good yield even from a moderate faecal cyst load in a single stool sample.
E. histolytica for use as antigen
1084
Vairimorpha necatrix
protozoa
continuous
caterpillar
Percoll was used to purify spores. 40% of the original spores were recovered with nearly all refractile (90% or more). Contaminating bacteria were not seen.
infection of cultured cells
1085
46
Host Tissue
Comments
Downstream Application light microscopy
Ref. # 1074
Microorganisms (cont.) Species
Type
Gradient Type
Host Tissue
Comments
Downstream Application
Ref. #
rice transitory yellowing virus (RTYV)
virus
continuous
rice plant leaf
Typical purification runs gave about 140-850 mg of purified virus per 100 g of infected material.
Lowry protein assay, electron microscopy, SDS-PAGE, ELISAs, western blots
1086
Rubivirus (rubella virus)
virus
continuous
cultured cells
Comparison of Percoll and sucrose gradients for purifying Rubella gave a yield of 72% with Percoll compared to 8.6% with a sucrose gradient.
hemagglutinating titer assays
1087
Herpes simplex virus
virus
continuous
NA
Percoll was used to purify herpes simplex virus.
none
56
dinoflagellates, diatoms, bluegreen bacteria
marine microalgae
continuous
NA
Most of the marine species recovered were in a condition that would permit direct physiological measurements of photosynthesis, respiration, ion adsorption and specific growth rates.
light microscopy, motility assay, photosynthesis assay
60
mycoplasmalike organism (MLO)
NA
discontinuous
lettuce (Lactuca sativa )
Electron microscopy showed a high concentration of MLOs with well-preserved cellular structures.
electron microscopy, ELISA
1088
47
Applications - Subcellular Particles Plasma membranes Species
Gradient Type
human
continuous (selfgenerating)
platelets
A method for rapid isolation of platelet plasma membrane was described, based on the use of [3H]-concanavalin A as a membrane marker and self-generating gradients of Percoll.
radioactive tracer studies, enzyme and protein assays
rat, human
continuous
liver biopsy
Plasma membrane enzymatic marker and membrane transport assays indicated that isolated membranes retained their functional integrity.
membrane enzyme assays and measurement of amino acid transport by membrane vesicles
1089
rat
continuous
uterus
The plasma membrane markers, 5'-nucleotidase and cholesterol, were enriched in the fractions near the top of the gradient, while the sarcoplasmic reticulum marker enzyme, rotenoneinsensitive NADH-cytochrome-c reductase, was in the lower part.
Ca++ uptake and release assays enzyme assays, cholesterol and progesterone assays, and western blot
1090
rat
continuous (3-layer)
brain
Synaptic plasma membranes were prepared by Ficoll and Percoll density gradients.
phospolipase C assay, marker enzyme assays
1091
rat
discontinuous (2-layer)
cultured cells
Two subcellular fractions, one enriched in plasma membranes and the other enriched in endoplasmic reticulum membranes, were obtained by Percoll gradient fractionation.
electron microscopy, determination of enzymatic markers, enzyme activity, calcium uptake and release
1092
rat
continuous
liver
The plasma membrane marker, 5'-nucleotidase, was enriched, whereas the cytosolic (endoplasmic reticulum) enzyme, glucose-6-phosphatase, was impoverished, indicating vesicle purity.
vesicle amino acid transport assay
1093
rat
continuous
liver
Percoll gradients were used to isolate hepatocytes, plasma membranes and mitochondrial membranes.
phase-contrast microscopy, cell binding experiments
33
rat
continuous
liver
Use of Percoll for the low speed nuclear pellet resulted in plasma membrane markers and Ins (1,4,5)P3 binding activity being purified together.
marker enzyme determinations, Ins(1,4,5)P3 binding, Bradford protein assay, SDS-PAGE
1094
rat
continuous
liver
Percoll purified hepatic plasma membranes were used to examine the transport of amino acids.
arginine transport activity, enzyme marker assays
1095
bovine
continuous
cultured aortic endothelial cells
Plasma membranes were labelled with trace amounts of [3H]-cholesterol and cell homogenates were fractionated on sucrose and Percoll gradients.
enzyme assays and SDS-PAGE/ligand blots
1096
bovine
discontinuous (3-layer)
adrenal gland
The procedure provided a fraction rich in plasma membranes.
solubilization of plasma membranes, affinity chromatography, radiolabelling of plasma membrane, enzyme assays
sheep
continuous (selfgenerating)
perirenal fat adipocytes
The fatty acid content of plasma membranes was analyzerd.
fatty acid analysis using gaschromatography
1097
Chinese hamster
continuous (selfgenerating)
cultured chinese hamster ovary (CHO) cells
A procedure yielded plasma membrane fractions that were enriched 3-fold and practically free of lysosomes; pure endoplasmic reticulum (ER) and mitochondrial fractions were obtained as well.
lipid analysis, enzyme assays
1098
48
Tissue/ Cell Type
Comments
Downstream Application
Ref. # 54
78
Plasma membranes (cont.) Species
Gradient Type
Tissue Type
skate (Raja erinacea)
continuous (selfgenerating)
liver
Marker enzyme studies indicated that plasma membranes isolated with Percoll gradients were highly enriched in the basolateral domain of the liver plasma membrane and largely free of contamination by intracellular organelles or canalicular membranes.
Comments
enzyme assays, fluorescence anisotropy measurements, alanine transport, protein and lipid determination
Downstream Application
Ref. # 1099
Fungi (Penicillium chrysogenum)
continuous
NA
The majority of contaminating membranes were removed by Percoll step gradients.
enzyme assays, electron microscopy, membrane fusion, transport studies, Lowry protein assay
1100
Fungi (Penicillium cyclopium)
continuous
NA
Right-side-out plasma membrane vesicles were prepared using two-phase partitioning and Percoll gradients.
ATPase activities, electron microscopy
1101
Lysosomes Species
Gradient Type
Tissue Type
Downstream Application
Ref. #
human
continuous
cultured fibroblasts
Only lysosomes ,sedimented in the bottom third of 30-40% Percoll density gradients.
Comments
adenosine deaminase and N-acetyl-b-hexosaminidase assays
1102
human
continuous
cultured fibroblasts
A crude mitochondrial lysosomal preparation of fibroblasts was separated into high-density fractions (lysosomal markers) and low-density fractions (mitochondrial markers).
enzyme assays, SDS-PAGE electrophoresis, immunoblotting
1103
mouse
continuous
liver
After homogenization, lysosomes equilibrated in the dense regions of Percoll gradients.
electron microscopy, Bradford protein assay, enzymatic assays
1104
rat
continuous
cultured hepatocytes
Lysosomal fractions were used to assay for endocytic transport of lysosomal membrane glycoprotein from cell surface to lysosomes.
purification of lysosomal membrane glycoprotein, Lowry protein assay, proteinhorseradish peroxidase assay
1105
rat
continuous (selfgenerating)
liver
Analysis of relevant marker enzymes showed considerably purified lysosomal particles in the density range of 1.04-1.11 g/ml.
Lowry protein assay, enzyme assays, free isoelectron focusing
24
rat, buffalo
continuous (differential and isopycnic)
kidney
The method gave a 25-40 fold enrichment in lysosomal marker enzymes with <0.5% contamination from mitochondrial and peroxisomal markers.
preparation of membrane vesicles, electron microscopy, protein assay
1106
porcine
continuous
cultured kidney epithelial cells
The method allowed for the relatively easy preparation of enriched fractions of endosomes and lysosomes.
distribution and structure of vacuolar H+ ATPase, radiolabelling detection, hexosaminidase activity and alkaline phosphatase activity
1107
49
Applications - Subcellular Particles (cont.) Mitochondria Species
Gradient Type
Tissue Type
Comments
plant
discontinuous
etiolated tissue and green leaf tissue
For etiolated tissue mitochondria, about 90% of catalase contamination was removed. For green leaf mitochondria, about 95% of chlorophyll, 80% of catalase and 65% of glycollate oxidase were removed.
cytochrome c oxidase (CCO) activity, membrane activity, respiratory control and substrate oxidation measurements
Downstream Application
Ref. # 12
plant
discontinuous (3-layer)
etiolated tissue and green leaf tissue
Separation of mitochondria from chloroplast material was possible under isoosmotic conditions, and in a relatively short time.
chlorophyll, cytochrome c oxidase and glycollate oxidase activities
43
rabbit, porcine
discontinuous
heart
Percoll was especially suitable for in vitro studies on mitochondria from both normal and diseased hearts.
electron microscopy, enzyme activities
1108
rat
discontinuous
liver
Isolated rat liver mitochondria were split into three density fractions when applied to a Percoll gradient.
staining of mitochondrial populations, flow cytometry
1109
Plasmodium berghei (protozoa)
continuous
NA
The purified mitochondria were obtained at the interface with a density of 1.05 g/ml.
mitochondrial marker enzyme assays, phase-contrast and electron microscopy
1110
turkey
discontinuous (3-layer)
sperm
Mechanical disruption, sonication and centrifugation over Percoll was an effective procedure to isolate the mitochondria.
fluorescence and electron microscopy, cytochrome oxidase assay, oxygen consumption, mitochondrial DNA isolation
1111
Downstream Application
Ref. #
Granules Species
Gradient Type
human
discontinuous (3-layer)
whole blood
neutrophils
Specific and gelatinase granules were separated on a three-layer Percoll gradient.
myeloperoxidase, alkaline phosphatase, lactoferrin, gelatinase, B12 binding protein, b2 microglobulin, cytochrome b558, and CD116 assays
1112
human
discontinuous (2-layer)
whole blood
neutrophils
Subcellular fractionation resulted in a band containing gelatinase and specific granules and a band containing plasma membrane and secretory vessels.
receptor localization, enzyme marker assays
1113
human
discontinuous (2-layer)
whole blood
neutrophils
Percoll gradient centrifugation resulted in a bottom band containing azurophil granules, a top band of plasma membrane and secretory vesicles, and a clear supernatant containing cytosol.
marker enzyme assays, ELISA
1114
human
discontinuous (2-layer)
whole blood
neutrophils
Percoll was used for subcellular fractionation of plasma membranes, specific granules and azurophilic granules.
subcellular localization of myeloperoxidase alkaline phosphatase, and vitamin B12 binding protein
1115
human
continuous
whole blood
primary cultured lymphocytes
Percoll gradients were used for the isolation of large granular lymphocyte (LGL) cytoplasmic granules.
macrophage tumorcidal assay
1116
50
Tissue Type
Cell Type
Comments
Granules (cont.) Species
Gradient Type
Tissue Type
Cell Type
mouse
continuous
mastocytoma
mast cell
rat
continuous
parotid gland
bovine
discontinuous (3-layer)
Paracentrotus lividus (sea urchin)
discontinuous (2-layer)
Comments
Downstream Application
Ref. #
Density gradient centrifugation was carried out in Percoll/0.25 M sucrose.
uptake and degradation of mast cell granules by mouse peritoneal macrophages
17
NA
A secretory granular fraction (SG) and a plasma membranerich fraction (PM) were isolated using differential and Percoll gradient centrifugation.
enzyme assays, interactions of SG with PM
1117
adrenal gland
NA
Using Percoll to isolate chromaffin granules did not increase the yield, but it did eliminate the need for exposure of the granules to extreme hypertonic conditions during isolation.
electron microscopy, glutaraldelyde fixation for preparation of affinity column
NA
NA
Lytic molecules were contained within small (0.1-0.25 mm) granules (cytolytic granules) which could be isolated by Percoll gradients.
hemolytic and enzymatic activities
78
1118
Plant organelles Organelle
Species
Gradient Type
Tissue Type
Comments
Downstream Application
Ref. #
mitochondria
castor bean
continuous
seed (endosperm)
Highly purified mitochondria were obtained with the Percoll gradient.
mitochondrial cytidyltransferase assay
1119
mitochondria
sunflower
continuous
seed
No organellar contamination was seen in the pellet sections.
Lowry protein assay, characterization of NADPdependent isocitrate dehydrogenase (NADP-IDH), SDS-PAGE and native gel electrophoresis, gel filtration, electron microscopy
1120
mitochondria
maize, faba bean, wheat, tobacco, sugar beet
discontinuous
leaf
The purified intact mitochondria exhibited high respiratory controls and P/O ratios and were cleared of most of the chlorophyll.
in vitro radioactive labelling of the products of mitochondrial protein synthesis and their analysis by SDS-PAGE
1121
chloroplast
tobacco (Nicotiana tabacum)
discontinuous
leaf
The yield from the Percoll gradient was 4.63 x 107 chloroplasts/g of chlorophyll/ chloroplast.
Extraction of chloroplast proteins, Bradford protein assay, SDS-PAGE, protein blotting and immunological reactions
1122
chloroplast
Pea continuous (Pisum sativum) and spinach (Spinacea oleracea)
leaf
The purified chloroplasts were capable of light-dependent protein synthesis at rates comparable to those previously reported.
in vitro reconstitution of protein transport and fractionation of chloroplast stromal protein
76
chloroplast
spinach continuous (Spinacea linear oleracea) gradient
leaf
A clear separation of intact chloroplasts sustaining high photosynthetic activities occured.
enzyme assays, photosynthetic CO2 fixation, and O2 evolution
88
51
Applications - Subcellular Particles (cont.) Plant organelles (cont.) Organelle
Species
Gradient Type
Tissue Type
cytoplasts
various
discontinuous
leaf
protoplasts
barley
discontinuous (4-layer)
nuclei
carrot
plastids
barley, pea, maize
Comments
Downstream Application
Ref. #
Cytoplasts were obtained by centrifugation of leaf protoplasts on Percoll gradients.
cytoplast staining, laser microscopy, cytoplast: protoplast fusion
1123
seed (aleurone layer)
After the Percoll gradient, the protoplasts were obtained in relatively high yield and showed good viability.
transient expression of CAT activity by transfected barley protoplasts
1124
discontinuous (3-layer)
suspension cells
This method yielded an average of 2 x 105 nuclei from 2 g of suspension cultured cells 6 (approximately 2 x 10 cels). Greater than 80% of the nuclei appeared fully intact following the Percoll gradient.
cytochrome c oxidase and reductase assays, and in vitro RNA synthesis
1125
continuous
leaf and seed endosperm
Plastids obtained using Percoll exhibited high degrees of intactness (89.1% and greater) and purity.
starch synthesis, enzyme assays
1126
Miscellaneous organelles Organelle
Species
Gradient Type
Tissue Type
Comments
nuclei
chicken
continuous (selfgenerated)
skeletal muscle
Percoll density gradient centrifugation provided a convenient method for the isolation of transcriptionally active nuclei applicable to a variety of tissues.
in vitro transcription
832
nuclei
Neurospora crassa (fungi)
continuous
whole organism
Percoll was a very effective alternative to LUDOX for the purification of Neurospora nuclei from crude nuclear preparations.
electron microscopy, DNA, RNA and protein purification
1127
nuclei and subcellular fractionation
NA
continuous (self -generated)
cultured NIH and KNIH cells
Percoll centrifugation allowed efficient fractionation and preservation of enzymatic activity.
b-galactosidase and galactosyltransferase activity
597
flagella
human
discontinuous
sperm
The interface between 80% Percoll and 0.9% NaCl contained the flagella and their fragments.
phase-contrast and electron microscopy, SDS-PAGE
1128
flagella
human
discontinuous
sperm
Separation of flagella from sperm heads at 25 kHz and subsequent Percoll centrifugation resulted in a high yield of flagella in the interface.
SDS-PAGE
1129
endosomes
human
continuous
cultured hepatoma cells
Percoll gradients were used to separate endosomes from lysosomes. The conditions of centrifugation were chosen specifically to permit resolution of early, intermediate and late endosomes.
b-hexosaminidase activity, Bradford protein assay
1130
52
Downstream Application
Ref. #
Miscellaneous organelles (cont.) Organelle
Species
Gradient Type
Tissue Type
Comments
Downstream Application
endosomes
human
continuous
cultured B cells
Percoll was used to isolate intracellular major histocompatability complex (MHC) molecules in a preparative scale from endosomal compartments.
plasma membrane, endoplasmic reticulum, lysosomes and mitochondria
human
continuous
liver biopsy
Percoll permited rapid analytical marker enzyme assays subcellular fractionation. Resolution of organelles was good, and recoveries were high (86-105%).
melanosomes, lysosomes, peroxisomes
human
continuous
cultured melanocytes
Subcellular fractionation was used to determine the relationship between melanosomes, lysosomes and peroxisomes.
enzyme activity assays, immunoflourescence and immuno-electron microscopy
1132
azygospores
Condiobolas obscuros (fungi)
discontinuous
whole organism isolated from soil
Recovery was 64% on average for a variety of soil types.
microscopy
246
chromosomal and mitotic clusters
human
continuous and discontinuous (selfgenerated)
cultured HeLa 53 and CHO cells
Chromosomes were isolated free of cytoplasmic contamination.
microscopy, western blotting
677
peroxisomes
rat
continuous and discontinuous (selfgenerated)
liver
enzyme assay, fatty acid oxidation studies
53
various
human
continuous (selfgenerated)
blood
Percoll-sucrose gradients were used to purify subcellular fractions to assay for catalase.
indirect immunocytofluorescence microscopy, ultrastructural immunogold, enzyme activity assays
1133
cytosol, lysosomes, Golgi elements
NA
continuous (selfgenerated)
cultured monoblastic cell line
Percoll gradients were used to separate subcellular organelles into various fractions.
marker enzyme assays
1134
microbodies
Cladosporium resinae (fungi)
discontinuous
whole organism
Best results were obtained with a discontinuous Percoll gradient which yielded a fraction enriched in microbodies and one enriched in mitochondria.
catalase and cytochrome oxidase assays
1135
subcellular fractionation
human
continuous (selfgenerated)
cultured HL-60 cells
Percoll centrifugation allowed efficient fractionation and preservation of enzymatic activity.
peroxidase, b-glucuronidase and acid phosphatase assays
727
lipid vesicles
Torpedo californica
continuous (selfgenerated)
electroplax Intact vesicles were isolated. tissue
phosphate determination
392
sequence analysis of pooled and single peptides, fluorescence labelling and binding assay
Ref. # 1131
13
53
Appendix 1 - Summary methodology charts Scheme 1. Separation of cells on gradients of Percoll. Prepare an osmotically-adjusted Stock Isotonic Percoll (SIP) which is isotonic with cell preparation
Dilute to desired starting densities with physiological saline solution (page 13)
Either
Or
Preform the gradient by layering, gradient former or by centrifugation (e.g. 30,000 x g for 15-45 min. in anglehead rotor) (page 21)
Mix cell preparation with diluted iso-osmotic Percoll to form a homogenous suspension
Use Density Marker Beads to monitor the gradient (page 23)
Layer the cell suspension on preformed gradient and centrifuge (e.g. 400800 x g for 10-20 min. in swinging bucket or angle-head rotor)
Centrifuge (e.g. 30,000 x g for 15-45 min. in an anglehead rotor)
Fractionate gradient by upward displacement (page 26)
Use cell fraction directly Remove Percoll from cells by washing (page 27)
Downstream applications: Cell culture - Cytodex 1 and 3 RNA extraction - RNA Extraction Kit (27-9270-01), QuickPrep Total RNA Extraction Kit (28-9271-01), QuickPrep mRNA Purification Kit (27-9254-01), QuickPrep Micro mRNA Purification Kit (27-9255-01) Genomic DNA extraction - RapidPrep Micro and Macro Genomic DNA Isolation Kit for Cells and Tissue (27-5225-01 and 27-5230-01 respectively) 54
Scheme 2. Separation of subcellular particles and some viruses on gradients of Percoll. Make Stock Isotonic Percoll (SIP) by adding 1 volume of 2.5 M sucrose to 9 volumes of Percoll (page 12)
Dilute stock Percoll to lower density by addition of 0.25 M sucrose (page 13)
Mix preparation of particles with Percoll solution in 0.25 M sucrose
Centrifuge (e.g. 60,000 x g for 15-45 min. in an angle-head rotor) - Particles band on the self-generated gradients as it is formed (page 21)
Use Density Marker Beads to monitor the gradient (page 23)
Fractionate gradient by upward displacement (page 26)
Analyze fractions for enzyme activities etc. (page 26)
Either
Or
Use fraction directly for further experiments
Remove Percoll by one of two techniques
High speed centrifugation Centrifuge at 1000,00 x g in a swinging bucket or angle-head rotor for 1.5-2 hours. Biological material stays on top of the hard pellet of Percoll formed (page 27)
Gel filtration chromatography Sephacryl S-1000 Superfine will retard Percoll; collect biological particles in the void volume (page 27)
55
References Note: In portions of the list of references below, the numbering is not sequential. This is due to the way in which the list was constructed. All references with numbers lower than 891 have been extracted from the original Percoll Reference List (1992), and the numbering used in that List was maintained in this Manual. All references higher than 891 are new and are sequential. 1. Parenchymal cell mass determinations in human parathyroid glands. Åkerström, G., Johansson, H. et al., Presented at Societé International de Chirurgie, San Francisco (August 1979). 2. Density determinations of human parathyroid glands by density gradients. Åkerström, G., Pertoft, H., Grimelius, L. et al., Acta Path. Microbiol. Scand. Sect. A. 87, 91–96 (1979). 3. Estimation of parathyroid parenchymal cell mass using density gradients. Åkerström, G., Grimelius, L., Johansson, H. et al., Am. J. Pathol. 99, 685–694 (1980). 4. Washing and concentration of human semen by Percoll density gradients and its application to AIH. Iizuka, R. et al., Arch. Androl. 20, 117–124 (1988). 5. Rapid isolation of pancreatic islets from collagenase digested pancreas by sedimentation through Percoll at unit gravity. Buitrago, A., Gylfe, E., Henriksson, C. et al., Biochem. Biophys. Res. Commun. 79, 823–828 (1977). 6. Isolation and characterization of cells from rat adipose tissue developing into adipocytes. Björntorp, P., Karlsson, M., Pertoft, H. et al., J. Lipid Res. 19, 316–324 (1978). 7. Presence of alloreactive Ia antigens on murine intestine epithelial cells. Curman, B., Kämpe, O., Rask, L. et al., Scand. J. Immunol. 10, 11–15 (1979). 8. Separation of lymphoid cells, mast cells and macrophages on Percoll density gradient. Courtoy, R., Simar, L.J. and Delrez, M. Presented at a Meeting of the Belgian Society of Immunology. (December 1978). 9. Isolation of rat peritoneal mast cells by centrifugation on density gradients of Percoll. Enerbäck, L. and Svensson, I. J. Immunol. Methods. 39, 135–145 (1980). 10. Distribution of Ia-antigen-like molecules on non-lymphoid tissues. Forsum, U., Klareskog, L. and Peterson, P.A. Scand. J. Immunol. 9, 343–349 (1979). 11. Separation of human lymphocytes on the basis of volume and density. Hutchins, D. and Steel, C.M. In Separation of Cells and Subcellular Elements (Peeters, H., Ed.) Pergamon Press, Oxford and New York (1979). 12. Separation of chloroplasts from mitochondria utilising silica sol gradient centrifugation. Jackson, C., Dench, J.E., Halliwell, B. et al., Presented at The Wolffson Conference, University of Surrey (July 1978). 13. Simple analytical subcellular fractionation of liver biopsies with Percoll. Jenkins, W.J., Clarkson, E., Milson, J. et al., Clin. Sci. 57, 29P (1979). 14. Coloured beads as density markers in isopycnic centrifugation. Kågedal, L. and Pertoft, H. Abstracts, 12th FEBS Meeting, Dresden (1978). 15. Separation of blood cells from healthy and leukemic donors in Percoll gradients. Rogge, H., Christ, H., Grosshans, E. et al., Abstracts, 12th FEBS Meeting, Dresden (1978).
56
16. Long term growth in vitro of human T cell blasts with maintenance of specificity and function. Kurnick, J.T., Grönvik, K.-O., Kimura, A.K. et al., J. Immunol 122, 1255–1260 (1979). 17. Uptake and degradation of mast cell granules by mouse peritoneal macrophages. Lindahl, U., Pertoft, H. and Seljelid, R. Biochem. J. 182, 189–193(1979). 18. Purification and steroidogenic responses of isolated rat luteal cells. McNamara, B.C., Booth, R. and Stansfield, D.A. Presented at International Union of Biochemistry Meeting Toronto (1979). 19. Separation of lactotrophs from hyperplastic rat adenohypophysis using Percoll density gradients. Milligan, J.V. Abstracts, Canad. Fed. Biol. Sci. Meeting, Vancouver (1979). 20. Isolation and characterization of hepatocytes and Küpffer cells. Page, D.T. and Garvey, J.S. J. Immunol. Methods 27, 159–173 (1979). 21. Isopycnic separation of cells and cell organelles by centrifugation in modified colloidal silica gradients. Pertoft, H. and Laurent, T.C. In Methods of Cell Separation Vol. 1. (Catsimpoolas, N., Ed.) Plenum Press, New York 25–65 (1977). 22. Density gradients prepared from colloidal silica particles coated polyvinylpyrrolidone (Percoll). Pertoft, H., Laurent, T.C., Låås, T. et al., Anal. Biochem. 88, 271–282 (1978). 23. The viability of cells grown or centrifuged in a new density gradient medium, Percoll. Pertoft, H., Rubin, K., Kjellén, L. et al., Exp. Cell Res. 110, 449–457 (1977). 24. Heterogeneity of lysosomes originating from rat liver parenchymal cells. Metabolic relationship of subpopulations separated by density gradient centrifugation. Pertoft, H., Wärmegård, B. and Höök, M. Biochem. J. 174, 309–317 (1978). 25. Characterization of rabbit sperm by equilibrium sedimentation in Percoll during frequent ejaculation. Oshio, S., Kaneko, S. and Mohri, H. Arch. Androl. 17, 189–194 (1986). 26. A two-step procedure for the purification of hepatitis B surface antigen (HbsAg). Einarsson, M., Kaplan, L. and Pertoft, H. Vox Sanguinis 41, 91–97 (1981). 27. The use of density gradients of Percoll for the separation of biological particles. Pertoft, H., Laurent, T.C., Seljelid, R. et al., In Separation of Cells and Subcellular Elements, (Peeters, H., Ed.) Pergamon Press Oxford and New York 67–72 (1979). 28. Collection of dinoflagellates and other marine microalgae by centrifugation in density gradients of a modified silica sol. Price, C.A., Reardon, E.M. and Guillard, R.R.L. Limnol. Oceangr. 123, 548–553 (1978). 29. Adhesion of rat hepatocytes to collagen. Rubin, K., Oldberg, Å., Höök, M. et al., Exp. Cell Res. 117, 165–177 (1978). 30. Macrophage functional heterogeneity: evidence for different antibody-dependent effector cell activities and expression of Fc-receptors among macrophage subpopulations. Serio, C., Gandour, D.M and Walker, W.S. J. Reticuloendothelial Soc. 25, 197–216 (1979). 31. Rapid isolation of mouse Leydig cells by centrifugation in Percoll density gradients with complete retention of morphological and biochemical integrity. Schumacher, M., Schäfer, G., Holstein, A.F. et al., FEBS LETT. 91, 333–338 (1978).
57
32. Epithelial rat liver cells have cell surface receptors recognizing a phosphorylated carbohydrate on lysosomal enzymes. Ullrich, K., Mersmann, G., Fleischer, M. et al., Hoppe-Seyler's Z. Physiol. Chem. 359, 1591–1598 (1978). 33. Specific binding of rat liver plasma membranes by rat liver cells. Öbrink, B., Wärmegård, B. and Pertoft, H. Biochem. Biophys. Res. Commun. 77, 665–670 (1977). 34. Liberation of a fibrogenic factor from human blood monocytes, ascites cells, cultured histiocytes and transformed mouse macrophages by treatment with SiO2. Aalto, M., Kulonen, E., Rönnemaa, T. et al., Scand. J. Clin. Lab. Invest. 39, 205–214 (1979). 35. Parenchymal cell mass determinations in human parathyroid glands and its application in a material of hyperparathyroidism. Åkerström, G., Grimelius, L., Johansson, H. et al., World. J. Surg. 5, 555–563 (1981). 36. In vitro induction of self reactive T lymphocyte memory in cultures of syngeneic peanut agglutinin-negative mouse thymocytes and spleen cells. Born, W. and Wekerle, H. Immunobiol. 156, 243–244 (1979). 37. Characteristics of cultured brain capillaires. Bowman, P.D., Betz, A.L. and Goldstein, G.W. J. Cell Biol. 83, 95a (1979). 38. Characterization of bone marrow osteoprogenitor cell lines. Budenz, R.W. and Bernard, G.W. J. Cell Biol. 83, 32a (1979). 39. Isolation, characterization and cultivation of human trophoblastic cells. Calaminus, J.M., Brüggen, J. and Sorg, C. Immunobiol. 156, 287 (1979). 40. Characterization of procoagulant activity produced by cultures of human monocytes and lymphocytes separated in colloidal silica-polyvinylpyrrolidone gradients. Giddings, J.C., Piovella, F., Ricetti, M. et al., Clin. Lab. Haematol. 2, 121–128 (1980). 41. Purification of human T and B cells by a discontinuous density gradient of Percoll. Gutierrez, C., Bernabe, R.R., Vega, J. et al., J. Immunol. Methods 29, 57–63 (1979). 42. Production of Ficoll, Percoll and albumin gradients by the freeze-thaw method. Haff, L.A. Prep. Biochem. 9, 149–156 (1979). 43. Isolation of intact higher-plant mitochondria. Jackson, C. and Moore, A.L. Plant Organelles, Methodological Surveys (B) Biochemistr, Vol. 9, (Reid, E., Ed.) Ellis Horwood Ltd, Chichester, West Sussex, UK 1–12 (1979). 44. Human MLC activated suppressor cells - enrichment on discontinuous density gradients. Kabelitz, D., Fink, U. and Reichert, A. Immunobiol. 156, 218 (1979). 45. Physical chemical characterization of Percoll. I. Particle weight of the colloid. Laurent, T.C., Pertoft, H. and Nordli, O. J. Colloid Interface Sci. 76, 124–132 (1980). 46. Physical chemical characterization of Percoll. II. Size and interaction of colloidal particles. Laurent, T.C., Ogston, AJ.G., Pertoft, H. et al., J. Colloid Interface Sci. 76, 133–141 (1980). 47. Physical chemical characterization of Percoll. III. Sodium binding. Laurent, T.C. and Pertoft, H. J. Colloidal Interface Sci. 76, 142–145 (1980). 48. Peanut agglutinin. IV. A tool for studying human mononuclear cell differentiation. London, J., Perrot, J.Y., Berrih, S. et al., Scand. J. Immunol. 9, 451–459 (1979). 58
49. Conversion of 4-hydroxyphenylpyruvic acid into homogentisic acid at the thylakoid membrane of Lemna gibba. Löffelhardt, W. and Kindl, H. FEBS LETT. 104, 332–334 (1979). 50. Further purification of rat spermatogenic cells by density centrifugation. Meistrich, M.L., Longtin, J.L. and Brock, W.A. J. Cell Biol. 83, 226a (1979). 51. Protozoan parasite-induced proliferative response of primed T lymphocytes. Moedder, E., Engers, H. and Louis, J. Immunobiol. 156, 205 (1979). 52. In vitro evidence suggests a direct action of adjuvants on myeloid precursor cells in the bone marrow. Monner, D.A.L. and Mühlradt, P.F. Immunobiol. 156, 189–190 (1979). 53. Regulation of peroxisomal fatty acid oxidation. Osmundsen, H. and Neat, C.E. FEBS LETT. 107, 81–85 (1979). 54. Asymmetric distribution of arachidonic acid in the plasma membrane of human platelets. A determination using purified phospholipases and a rapid method for membrane isolation. Perret, B., Chap, H.J. and Douste-Blazy, L. Biochim. Biophys. Acta 556, 434–446 (1979). 55. Cell separations in a new density medium, Percoll. Pertoft, H., Hirtenstein, M. and Kågedal, L. Cell Populations, Methodological Surveys (B) Biochemistry Vol. 9. (Reid, E., Ed.) Ellis Horwood Ltd, Chichester, West Sussex, UK 67–80 (1979). 56. Purification of herpes simplex virus using Pecoll. Pharmacia Fine Chemicals, Pertoft, H. Separation News 3 (1980). 57. Separation of human monocytes on density gradients of Percoll. Pertoft, H., Johnsson, A., Wärmegård, B. et al., J. Immunol. Methods 33, 221–230 (1980). 58. Isolation of chloroplasts in silica-sol gradients. Price, C.A., Bartolf, M., Ortiz, W. et al., Plant Organelles, Methodological Surveys (B) Biochemistry Vol. 9. (Reid, E., Ed.) Ellis Horwood Ltd, Chichester, West Sussex, UK 25–33 (1979). 59. Fractionation of subcellular components by centrifugation in Percoll density gradients. Pertoft, H. and Hirtenstein, M. Presented at The Wolfson Conference, University of Surrey, Guildford, UK (July 1978). 60. Harvesting of marine microalgae by centrifugation in density gradients of Percoll. Reardon, E.M., Price, C.A. and Guillard, R.R.L. Cell Populations, Methodological Surveys (B) Biochemistry Vol. 9. (Reid, E., Ed.) Ellis Horwood Ltd, Chichester, West Sussex, UK (1979). 61. Morphological and functional characterization of isolated effector cells responsible for human natural killer activity to fetal fibroblasts and to cultured cell line targets. Saksela, E., Timonen, T., Ranki, A. et al., Immunological Rev. 44, 71–123 (1979). 62. A rapid method for the separation of large and small thymocytes from rats and mice. Salisbury, J.G., Graham, J.M. and Pasternak, C.A. J. Biochem. Biophys. Methods 1, 341–347 (1979). 63. Separation of lymphocytes, polymorphonuclear leucocytes and lysosomes by density gradient centrifugation. Seale, T.W. In Manual of Procedures for the Seminar on Biochemical Hematology, (Sunderman, F.W., Ed.) The Institute for Clinical Science Inc., Philadelphia, PA 355–367 (1979).
59
64. Disaggregation and separation of rat liver cells. Seglen, P.O. Cell Populations, Methodological Surveys (B) Biochemistry Vol. 9. (Reid, E., Ed.) Ellis Horwood Ltd, Chichester, West Sussex, UK 25–46 (1979). 65. Separation of cell populations from embryonic chick neural retina. Sheffield, J.B., Lynch, M. and Pressman, D. J. Cell Biol. 83, 34a (1979). 66. The effect of erythrocyte aging on some vitamin and mineral dependent enzymes. Spooner, R.J., Percy, R.A. and Rumley, A.G. Clin. Biochem. 12, 289–290 (1979). 67. Fractionation, morphological and functional characterization of effector cells responsible for human natural killer activity against cell-line targets. Timonen, T., Saksela., E. Ranki, A. et al., Cell. Immunol. 48, 133–148 (1979). 68. Recognition of human urine a-N-acetylglucosaminidase by rat hepatocytes. Involvement of receptors specific for galactose, mannose-6-phosphate and mannose. Ullrich, K., Basner, R., Gieselmann, V. et al., Biochem. J. 180, 413–419 (1979). 69. Discontinuous density gradient separation of human mononuclear leucocytes using Percoll as gradient medium. Ullmer, A.J. and Flad, H.-D. J. Immunol. Methods 30, 1–10 (1979). 70. Isolation and surface labeling of murine polymorphonuclear neutrophils. Watt, S.M., Burgess, A.W. and Metcalf, D. J. Cell Physiol. 100, 1–22 (1979). 71. Primary cultures of rat hepatocytes synthesize fibronectin. Voss, B., Allam, S., Rauterberg, J. et al., Biochem. Biophys. Res. Commun. 90, 1348–1354 (1979). 72. In vitro characterization of anti-tumor effector mechanisms in rats bearing spontaneous tumors. Zöller, M. and Matzku, S. Immunobiol. 156, 276 (1979). 73. Percoll methodology. Pharmacia Biotech. Separation News 1 (1979). 74. Fractionation of blood cells. In Centrifugation: A Practical Approach. Jurd, R.D. and Rickwood, D. (Rickwood, D., Ed.) Information Retrival Ltd, 1 Falconberg Court, London W1V 5FG, UK 143–152 (1978). 75. Choice of media for centrifugal separations. In Centrifugation: A Practical Approach. Jurd, R.D. and Rickwood, D. (Rickwood, D., Ed.) Information Retrieval Ltd, 1 Falconberg Court, London W1V 5FG, UK 15–31 (1978). 76. Post-translational transport into intact chloroplasts of a precursor to the small subunit of ribulose-1,5-bisphosphate carboxylase. Chua, N.-H. and Schmidt, G.W. Proc. Nat. Acad. Sci. USA 75, 6110–6114 (1978). 77. Human erythrocyte fractionation in Percoll density gradients. Rennie, C., Thompson, S., Parker, A. et al., Clin. Chim. Acta 98, 119–125 (1979). 78. Isolation of a protein from the plasma membrane of adrenal medulla which binds to secretory vesicles. Meyer, D. and Burger, M. J. Biol. Chem. 254, 9854–9859 (1979). 79. Techniques of preparative, zonal, and continuous flow ultracentrifugation. Beckman® Instruments Inc. Spinco division, USA, Griffith, O.M. 80. Cytophysical studies on living normal and neoplastic cells. Mateyko, G.M. and Kopac, M.J. Ann. N.Y. Acad. Sci. 105, 185–218 (1963).
60
81. The use of gradients of colloidal silica for the separation of cells and subcellular particles. Pertoft, H. and Laurent, T.C. Modern Separation Methods of Macromolecules and Particles Vol. 2. (Gerritsen, T., Ed.) Wiley Interscience, John Wiley & Sons, New York and London 71–90 (1968). 82. The separation of cells and subcellular particles by colloidal-silica density centrifugation. Wolff, D.A. In Methods in Cell Biology Vol. 10. (Prescott D.M., Ed.) Academic Press New York and London 85–104 (1975). 83. A rapid method for the separation of functional lymphoid cell populations of human and animal origin on PVP-silica (Percoll) density gradients. Kurnick, J.T., Österberg, L., Stegagno, M. et al., Scand. J. Immunol. 10, 563–573 (1979). 84. A novel reagent for the fluorometric assay of primary amines. Weigele, M., DeBarnardo, S.L., Tengi, J.D. et al., J. Am. Chem. Soc. 94, 5927–5928 (1972). 85. Application of the Weichselbaum biuret reagent to the determinations of spinal fluid protein. Ditterbrandt, M. Am. J. Clin. Pathol. 18, 439–441 (1948). 86. Isolation of highly purified rat liver mitochondria for the study of the biotransformation of drugs. Blume, H. (Article in German). Archiv der Pharmazie 312, 561–572 (1979). 87. Subcellular distribution of liver copper in normal subjects, patients with primary biliary cirrhosis and Wilson's disease. Jenkins, W.J., Evans, S. and Epstein, O. Clin. Sci. 58, 14 (1979). 88. Isolation of intact chloroplasts from spinach leaf by centrifugation in gradients of modified silica Percoll. Tahabe, T., Nishimuru, M. and Akazawa, T. Agric. Biol. Chem. 43, 2137–2142 (1979). 89. Isolation and characterization of noradrenalin storage granules of bovine adrenal medulla. Terland, O., Flatmark T. and Kryvi, T. Biochim. Biophys. Acta 553, 460–468 (1979). 90. A rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principle of protein-dye bindning. Braford, M. Anal. Biochem. 72, 248–254 (1976). 91. A parapoxvirus isolated from nasal secretion of a calf with respiratory disease. Moreno-Lopéz, J. and Lif, I. Vet. Microbiol. 4, 85–88 (1979). 92. Isolation of rat mast cell granules with intact membranes. Krüger, P.G., Lagunoff, D. and Wan, H. Exp. Cell Res. 129, 82–93 (1980). 93. Long term maintenance of HLA-D restricted T cells specific for soluble antigens. Kurnick, J., Altevogt, P., Lindblom, J. et al., Scand. J. Immunol. 11, 131–136 (1980). 94. A rapid centrifugation step method for the separation of erythrocytes, granulocytes and mononuclear cells on continuous density gradients of Percoll. Segal, A., Fortuno, A. and Herd, T. J. Immunol. Methods 32, 209–214 (1980). 95. Isolation of blood monocytes by use of Percoll. Seljelid, R. and Pertoft, H. In Methods for Studying Mononuclear Phagocytes. (Edelson, P., Koren, H. and Adams, D.O., Eds.) Academic Press, London and New York (1980). 96. Isolation of human NK cells by density gradient centrifugation. Timonen, T. and Saksela, E. J. Immunol. Methods 36, 285–291 (1980).
61
97. Separation of human eosinophils in density gradients of polyvinylpyrrolidone-coated silica gel (Percoll). Gärtner, I. Immunology 40, 133–136 (1980). 98. Separation of human bone marrow cells in density gradients of polyvinylpyrrolidone-coated silica gel (Percoll). Olofsson, T., Gärtner, I. and Olsson, I. Scand. J. Haematol. 24, 254–262 (1980). 99. Improved separation of human peripheral T-cells using PVP-coated colloidal silica particles (Percoll). Feucht, H.E., Hadam, M.R., Frank, F. et al., In Separation of Cells and Subcellular Elements. (Peeters, H., Ed.) Pergamon Press, Oxford and New York 73–76 (1979). 100. Surface markers of a purified peritoneal eosinophil population from Mesocestoides cordiinfected BALB/c male mice. Hogarth, P.M., Cruise, K.M., McKenzie, I.F.C. et al., J. Immunol. 124, 406–411 (1980). 101. Separation of human blood monocytes and lymphocytes on a continuous Percoll gradient. Gmelig-Meyling, F. and Waldmann, T.A. J. Immunol. Methods, 33, 1–9 (1980). 102. Collagenase - Percoll isolation and intraportal transplantation of pancreatic islets in the mouse. Henriksson, C. and Soome, A. Presented at Societé Internationale de Chirurgie, San Fransisco (August 1979). 103. Calcium uptake and release by skeletal-muscle mitochondria. Mickelson, J.R. and Marsh, B.B. Cell Calcium 1, 119–128 (1980). 104. Rapid separation of rat peritoneal mast cells with Percoll. Németh, A. and Rölich, P. Eur. J. Cell Biol. 20, 272–275 (1980). 105. Maximal steroidogenic capacity of mouse Leydig cells. Kinetic analysis and dependence on protein kinase activation and cAMP accumulation. Schumacher, M., Schäfer, G., Lichtenberg, V. et al., FEBS LETT. 107, 398–402 (1979). 106. Separation of mitochondria from contaminating subcellular structures utilizing silica sol gradient centrifugation. Jackson C., Dench, J.E.., Hall, D.O. et al., Plant Physiol. 64, 150–153 (1979). 107. Human parathyroid cells in vitro - the occurrence of an autonomous cell population in adenomas and uremic hyperplasias. Ljunghall, S., Åkerström, G. And Rudberg, C. In 15th European Symposium on Calcified Tissues, Helsinki (1980), (Kaitila, I. and Penttinen, O., Eds.) Springer International, supplement to Calcified Tissue International 31, 88 (1980). 108. A Percoll gradient for the separation of malaria infected eryhrocytes from low parasitized blood. Biochemical studies of malaria parasites in a Plasmodium vinckei/ NMRI-mouse system. Kientsch, R., Engel, W.D., Ziegler, A. et al., Submitted to Mol. Biochem. Parasitol (1980). 109. A rapid method for isolation of purified, physiologically active chloroplasts, used to study the intracellular distribution of amino acids in pea leaves. Mills, W.R. and Joy, K.W. Planta 148, 75–83 (1980). 110. Use of a novel rapid preparation of fat-cell plasma membranes employing Percoll to investigate the effects of insulin and adrenaline on membrane protein phosphorylation within intact fat cells. Belsham, G. J., Denton, R.M. and Tanner, M.J.A. Biochem. J. 192, 457–467 (1980). 111. A simple and fast method for the isolation of basolateral plasma membranes from rat small intestinal epithelial cells. Scalera, V., Storelli, C., Storelli-Joss, C. et al., Biochem. J. 186, 177–181 (1980). 62
112. Induction of peroxisomal b-oxidation in rat liver by high fat diets. Neat, C.E., Thomassen, M.S. and Osmundsen, H. Biochem. J. 186, 369–371 (1980). 113. Isolation of bacteria from yogurt on gradients of Percoll generated in situ. Standard course experiment. Pharmacia Fine Chemicals. 155. Human megakarayocytes. I. Characterization of the membrane and cytoplasmic components of isolated marrow megakaryocytes. Rabellino, E.M., Nachmann, R.L., Williams, N. et al., J. Exp. Med. 149, 1273–1287 (1979). 170. Primary culture of capillary endothelium from rat brain.Bowman, P.D., Betz, A.L., Ar, D et al., In Vitro 17, 353–362 (1981). 246. Conidiobolus obscurus in arable soil: a method for extracting and counting azygospores. MacDonald, R.M. and Spokes, J.R. Soil. Biol. Biochem. 13, 551–554 (1981). 275. Removal of Percoll from microsomal vesicles by gel filtration on Sephacryl S-1000 Superfine. Hjorth, R. and Pertoft, H. Biochim. Biophys. Acta 688, 1–4 (1982). 392. Investigation of liposomes and vesicles reconstituted with acetylcholine receptor employing Percoll density gradient centrifugation. Spillecke, F. and Neumann, E. Symposium on Neuroreceptors, Berlin, Neuroreceptors 243–252 (1981). 480. Pure population of viable neurons from rabbit dorsal root ganglia, using gradients of Percoll. Goldenberg, S.S.S. and DeBoni, U. J. Neurobiol. 14, 195–206 (1983). 518. A micromethod for the quantitation of cellular proteins in Percoll with the Coomassie brilliant blue dye-binding assay. Vincent, R. and Nadeau, D. Anal. Biochem. 135, 355–362 (1983). 547. Lymphocyte transformation test in diagnosis of nickel allergy. Nordlind, K. Int. Archs. Allergy Appl. Immunol. 73, 151–154 (1984). 555. Adjustment of the osmolality of Percoll for the isopycnic separation of cells and cell organelles. Vincent, R. and Nadeau, D. Anal. Biochem. 141, 322–328 (1984). 597. Processing and lysosomal localization of a glycoprotein whose secretion is transformation stimulated. Gal, S., Willingham, M.C. et al., J. Cell. Biol. 100, 535–544 (1985). 677. Improved methods for the isolation of individual and clustered mitotic chromosomes. Gasser, S.M. and Laemmli, U.K. Exp. Cell Res. 173, 85–98 (1987). 680. Skeletal muscle cell populations. Yablonka-Reuveni, Z. and Nameroff, M. Histochem. 87, 27–38 (1987). 727. Biochemical and ultrastructural effects of monensin on the processing, intracellular transport, and packaging of myeloperoxidase into low and high density compartments of human leukemia (HL-60) cells. Akin, D.T. et al., Arch. Biochem. Biophys. 257, 451–463 (1987). 832. Isolation of transcriptionally active nuclei from striated muscle using Percoll density gradients. Hahn, C, C. and Covault, J. Anal. Biochem. 190, 193–197 (1990). 891. Down-regulation of L-selectin surface expression by various leukocyte isolation procedures. Stibenz, D. and Buhrer, C. Scand. J. Immunol. 39, 59–63 (1994).
63
892. Production of hematopoietic growth factors by human B lymphocytes: mechanisms and possible implications. Pistoia, V., Corcione, A., Baldi, L. et al., Stem Cells 11, (suppl.2) 150–155 (1993). 893. Differential expression of IL-4 receptors in human T and B lymphocytes. Mozo, L., Rivas, D., Zamorano, J. et al., J. Immunol. 150, 4261–4269 (1993). 894. Isolation of functionally active intraepithelial lymphocytes and enterocytes from human small and large intestine. Lundqvist, C., Hammarstrom, M.-L., Athlin, L. et al., J. Immunol. Methods 152, 253–263 (1992). 895. The presence of CD5LOW+NK cells in normal controls and patients with pulmonary tuberculosis. Ishiyama, T., Watanabe, K., Fukuchi, K. et al., Immunol. Lett. 37, 139–144 (1993). 896. Spontaneous production of granulocyte colony-stimulating factor in vitro by human B-lineage lymphocytes is a distinctive marker of germinal center cells. Corcione, A., Baldi, L., Zupo, S. et al., J. Immunol. 153, 2868–2877 (1994). 897. Intestinal mucosal lymphocytes have H1 receptors: H1 antagonists reduce their proliferation and cytotoxicity. Roberts, A.I., Leone, V.M. and Ebert, E.C. Cell. Immunol. 156, 212–219 (1994). 898. The number of CD1a+ large low-density cells with dendritic cell features is increased in the peripheral blood of HIV+-patients. Ree, H.J., Liau, S., Yancovitz, S.R. et al., Clin. Immunol. Immunopathol. 70, 190–197 (1994). 899. Cell-to-cell mediated inhibition of natural killer cell proliferation by monocytes and its regulation by histamine H2-receptors. Hellstrand, K. and Hermodsson, S. Scand. J. Immunol. 34, 741–752 (1991). 900. Follicular dendritic cells inhibit human B lymphocyte proliferation. Freedman, A.S., Munro, J.M., Rhynhart, K. et al., Blood 80, 1284–1288 (1992). 901. Constitutive expression and role in growth regulation of interleukin-1 and multiple cytokine receptors in a biphenotypic leukemic cell line. Cohen, A., Grunberger, T., Vanek, W. et al., Blood 78, 94–102 (1991). 902. Monoclonal anti-CD23 antibodies induce a rise in [Ca2+]i and polyphosphoinositide hydrolysis in human activated B cells. Kolb, J.-P., Renard, D., Dugas, B. et al., J. Immunol. 145, 429–437 (1990). 903. Interleukin-2-inducible killer activity and its regulation by blood monocytes from autologous lymphocytes of lung cancer patients. Sone, S., Kunishige, E., Fawzy, F. et al., Jpn. J. Cancer Res. 82, 716–723 (1991). 904. Lymphokine-activated killer cell regulation of T-cell mediated immunity to Candida albicans. Wei, S., Blanchard, D.K., McMillen, S. et al., Infect. Immun. 60, 3586–3595 (1992). 905. Coregulation of the APO-1 antigen with intercellular adhesion molecule-1 (CD54) in tonsillar B cells and coordinate expression in follicular center B cells and in follicle center and mediastinal B-cell lymphomas. Moller, P., Henne, C., Leithauser, F. et al., Blood 81, 2067–2075 (1993). 906. Interleukin-2 activated T cells (T-LAK) express CD16 antigen and are triggered to target cell lysis by bispecific antibody. Nitta, T., Nakata, M., Yagita, H. et al., Immunol. Lett. 28, 31–38 (1991).
64
907. Antitumor activity, growth, and phenotype of longterm IL-2 cultures of human NK and T lymphocytes. Fuchshuber, P.R., Lotzová, E., Pollock, R.E. Lymphokine and Cytokine Res. 10, 51–59 (1991). 908. Functional characterization of canine lymphocyte subsets. Hotzl, C., Kolb, H.J., Holler, E. et al., Ann. Hematol. 63, 49–53 (1991). 909. Measurement of NK activity in effector cells purified from canine peripheral lymphocytes. Knapp, D.W., Leibnitz, R.R., DeNicola, D.B. et al., Vet. Immunol. Immunopathol. 35, 239–251 (1993). 910. A rapid method for isolating murine intestine intraepithelial lymphocytes with high yield and purity. Mosley, R.L. and Klein, J.R. J. Immunol. Meth. 156, 19–26 (1992). 911. Differential sensitivity of virgin and memory T lymphocytes to calcium ionophores suggests a buoyant density separation method and a model for memory cell hyporesponsiveness to Con A. Miller, R.A., Flurkey, K., Molloy, M. et al., J. Immunol. 147, 3080–3086 (1991). 912. Characterization of protein phosphorylation by 2,3,7,8,-tetrachlorodibenzo-para-dioxin in murine lymphocytes: indirect evidence for a role in the suppression of humoral immunity. Snyder, N.K., Kramer, C.M., Dooley, R.K. et al., Drug and Chem. Toxicol. 16, 135–163 (1993). 913. Cytokine synthesis by intestinal intraepithelial lymphocytes. Yamamoto, M., Fujihashi, K., Beagley, K.W. et al., J. Immunol. 150, 106–114 (1993). 914. The heavy metal lead exhibits B cell-stimulatory factor activity by enhancing B cell Ia expression and differentiation. McCabe, Jr., M.J. and Lawrence, D.A. J. Immunol. 145, 671–677 (1990). 915. Antibacterial activity of bovine mammary gland lymphocytes following treatment with interleukin-2- J. Sordillo, L.M., Campos, M. and Babiuk, L.A. Dairy Sci. 74, 3370–3375 (1991). 916. Use of probe to repeat sequence of the Y chromosome for detection of host cells in peripheral blood of bone marrow transplant recipients. Przepiorka, D., Thomas, E.D., Durnamn, D.M. et al., Am. J. Clin. Pathol. 95, 201–206 (1991). 917. Differences in "antioncogene" p53 expression in human monocytes and lymphocytes in vitro. Osipovich, O.A., Sudarikov, A.B., Kolesnikova, T.S. et al., Biull. Eksp. Biol. Med. 113, 638–640 (1992). 918. Regulation of tumor necrosis factor-alpha production and gene expression in monocytes. Kohn, F.R., Phillips, G.L. and Klingemann, H.-G. Bone Marrow Transplantation 9, 369–376 (1992). 919. Killing of Coccidioides immitis by human peripheral blood mononuclear cells. Ampel, N.M., Bejarano, G.C. and Galgiani, J.N. Infect. Immun. 60, 4200–4204 (1992). 920. Monocyte dysfunction in patients with Gaucher disease: evidence for interference of glucocerebroside with superoxide generation. Liel, Y., Rudich, A., Nagauker-Shriker, O. et al., Blood 83, 2646–2653 (1994). 921. Effect of human immunodeficiency virus-1 envelope glycoprotein on in vitro hematopoiesis of umbilical cord blood. Sugiura, K., Oyaizu, N., Pahwa, R. et al., Blood 80, 1463–1469 (1992).
65
922. Point mutations in the beta-subunit of cytochrome b558 leading to X-linked chronic granulomatous disease. Bolscher, B.G.J.M., de Boer, M., de Klein, A. et al., Blood 77, 2482–2487 (1991). 923. Acceleration of chronic myeloid leukemia correlates with calcitonin gene hypermethylation. Malinen, T., Palotie, A., Pakkala, S. et al., Blood 77, 2435–2440 (1991). 924. Expression of c-jun protooncogene in human myelomonocytic cells. Bertani, A., Polentarutti, N., Sica, A. et al., Blood 74, 1811–1816 (1989). 925. Expression of a heat-inducible gene of the HSP70 family in human myelomonocytic cells: regulation by bacterial products and cytokines. Fincato, G., Polentarutti, N., Sica, A. et al., Blood 77, 579–586 (1991). 926. Minimal residual disease is more common in patients who have mixed T-cell chimerism after bone marrow transplantation for chronic myelogenous leukemia. MacKinnon, S., Barnett, L., Heller, G. et al., Blood 83, 3409–3416 (1994). 927. Expression of C-myb and B-myb, but not A-myb, correlates with proliferation in human hematopoietic cells. Golay, J., Capucci, A., Castellano, M. et al., Blood 77, 149–158 (1991). 928. Interactions between the monocyte/macrophage and the vascular smooth muscle cell. Zhang, H., Downs, E.C., Lindsey, J.A. et al., Arteriousclerosis and Thrombosis 13, 220–230 (1993). 929. Splice site mutations are common cause of X-linked chronic granulomatous disease. de Boer, M., Bolscher, B.G.J.M., Dinauer, M.C. et al., Blood 80, 1553–1558 (1992). 930. Isolation of equine peripheral blood mononuclear cells using Percoll. May, S.A., Hooke, R.E. and Lees, P. Res. Vet. Sci. 50, 116–117 (1991). 931. Proteolytic cleavage of CR1 on human erythrocytes in vivo: evidence for enhanced cleavage in AIDS. Pascual, M., Danielsson, C., Steiger, G. et al., Eur. J. Immunol. 24, 702–708 (1994). 932. Lipid peroxidation in Plasmodium falciparum-parasitized human erythrocytes. Simoes, A.P.C.F., van den Berg, J.J.M., Roelofsen, B. et al., Arch. Biochem. Biophys. 298, 651–657 (1992). 933. Clinical utility of fractionating erythrocytes into Percoll density gradients. Mosca, A., Paleari, R., Modenese, A. et al., Red Blood Cell Aging 227–238 (1991). 934. A monoclonal antibody monitoring band 3 modifications in human red blood cells. Giuliani, A., Marini, S., Ferroni, L. et al., Molec. Cell. Biochem. 117, 43–51 (1992). 935. Deoxygenation-induced changes in sickle cell-sickle cell adhesion. Morris, C.L., Rucknagel, D.L. and Joiner, C.H. Blood 81, 3138–3145 (1993). 936. Density-associated changes in platelet-activating factor acetylhydrolase activity and membrane fluidity of human of human erythrocytes. Yoshida, H., Satoh, K., Ishida, H. et al., Ann. Hematol. 69, 139–146 (1994). 937. Density gradients separation of L-asparaginase-loaded human erythrocytes. Garin, M.I., Kravtzoff, R., Chestier, N. et al., Biochem. Mol. Biol. International 33, 807–814 (1994). 938. Determinations of red blood cell deformability in relation to cell age. Bosch, F.H., Werre, J.M., Schipper, L. et al., Eur. J. Haematol. 52, 35–41 (1994).
66
939. Density gradient separation of inositol hexaphosphate loaded in red blood cells in various preparation conditions. Bourget, G., Boucher, L. and Ropars, C, In The Use of Resealed Erythrocytes as Carriers and Bioreactors. (Magnani, M. and DeLoach, J.R., Eds.) Plenum Press, NY 27–33 (1992). 940. IHP entrapment into human erythrocytes: comparison between hypotonic dialysis and DMSO osmotic pulse. Mosca, A., Paleari, R., Russo, V. et al., In The Use of Resealed Erythrocytes as Carriers and Bioreactors, (Magnani, M. and DeLoach, J.R., Eds.) Plenum Press, NY 19–26 (1992). 941. Cyclic, AMP level in red blood cells of Plasmodium berghei-infected Mastomys natalensis. Khare, S. and Ghatak, S. Experientia 47, 236-238 (1991). 942. Exposure of phosphatidylserine in the outer leaflet of human red blood cells. Connor, J., Pak, C.C. and Schroit, A.J. J. Biol. Chem. 269, 2399–2404 (1994). 943. Flow cytometric detection of micronuclei induced by chemicals in poly- and normochromatic erythrocytes of mouse peripheral blood. Cao, J., Beisker, W., Nusse, M. et al., Mutagenesis 8, 533–541 (1993). 944. Temporal replacement of donor erythrocytes and leukocytes in nonanemic W44J/W44J and severely anemic W/W+ mice. Barker, J.E., Greer, J., Bacon, S. et al., Blood. 78, 1432–1437 (1991). 945. Oxygen transport properties in malaria-infected rodents - a comparison between infected and noninfected erythrocytes. Schmidt, W., Correa, R., Boning, D. et al., Blood 83, 3746–3752 (1994). 946. Multicatalytic and 26S ubiquitin/ATP-stimulated proteases in maturing rabbit red blood cells. Di Cola, D., Pratt, G. and Rechsteiner, M. FEBS 280, 137–140 (1991). 947. Biochemical characterization of density-separated trout erythrocytes. Falcioni, G., Grelloni, F., Bonfigli, A.R. et al., Biochem. International 28, 379–384 (1992). 948. Loss of activation-induced CD45RO with maintenance of CD45RA expression during prolonged culture of T cells and NK cells. Warren, H.S. and Skipsey, L.J. Immunol. 74, 78–85 (1991). 949. Negative modulation of human NK cell activity by purinoreceptors: Effect on exogenous adenosine triphosphate. Krishnaraj, R. Cell. Immunol. 141, 306–322 (1992). 950. Lysis of neuroblastoma cell lines by human natural killer cells activated by interleukin-2 and interleukin-12. Rossi, A.R., Pericle, F., Rashleigh, S. et al., Blood 83, 1323–1328 (1994). 951. Natural killer-stimulatory effect of combined low-dose interleukin-2 and interferon beta in hairy-cell leukemia patients. Liberati, A.M., De Angelis, V., Fizzotti, M. et al., Cancer Immunol. Immunother. 38, 323–331 (1994). 952. Mouse hypersensitivity pneumonitis: depletion of NK cells abrogates the spontaneous regression phase and leads to massive fibrosis. Denis, M. Exp. Lung Res. 18, 761–773 (1992). 953. Natural cytotoxic T cells responsible for anti-CD3-induced cytotoxicity in mice. Yanagita, Y., Nishimura, T., Goa, X. et al., Immunol. Lett. 31, 137–142 (1992). 954. Generation of natural killer cells from both FcgRII/III+ and FcgRII/III- murine fetal liver progenitors. Moingeon, P., Rodewald, H.-R., McConkey, D. et al., Blood 82, 1453–1462 (1993). 955. Hypoxic human umbilical vein endothelial cells induce activation of adherent polymorphonuclear leukocytes. Arnould, T., Michiels, C. and Remacle, J. Blood 83, 3705–3716 (1994). 67
956. Platelet-activating factor-induced polymorphonuclear neutrophil priming independent of CD11B adhesion. Read, R.A., Moore, E.E., Moore, F.A. et al., Surgery 114, 308–313 (1993). 957. Human neutrophils synthesize thrombomodulin that does not promote thrombin-dependent protein C activation. Conway, E.M., Nowakowski, B. and Steiner-Mosonyi, M. Blood 80, 1254–1263 (1992). 958. Induction of low density and up-regulation of CD11b expression of neutrophils and eosinophils by dextran sedimentation and centrifugation. Berends, C., Dijkhuizen, B., de Monchy, J.G.R. et al., J. Immunol. Meth. 167, 183–193 (1994). 959. Intracellular localization of glycosyl-phosphatidyl-inositol-anchored CD67 and FcRIII (CD16) in affected neutrophil granulocytes of patients with paroxysmal nocturnal hemoglobinuria. Jost, C.R., Gaillard, M.L., Fransen, J.A.M. et al., Blood 78, 3030–3036 (1991). 960. Membrane surface antigen expression on neutrophils: a reappraisal of the use of surface markers for neutrophil activation. Kuijpers, T.W., Tool, A.T.J., van der Schoot, C.E. et al., Blood 78, 1105–1111 (1991). 961. Identification of neutrophil gelatinase-associated lipocalin as a novel matrix protein of specific granules in human neutrophils. Kjeldsen, L., Bainton, D.F., Sengelov, H. et al., Blood 83, 799–807 (1994). 962. RANTES- and interleukin-8-induced responses in normal human eosinophils: effects of priming with interleukin-5. Schweizer, R.C., Welmers, B.A.C., Raaijmakers, J.A.M. et al., Blood 83, 3697–3704 (1994). 963. Human platelets secrete chemotactic activity for eosinophils.Burgers, J.A., Schweizer, R.C., Koenderman, L. et al., Blood. 81, 49–55 (1993). 964. Granulocyte-macrophage colony-stimulating factor, interleukin-3 (IL-3), and IL-5 greatly enhance the interaction of human eosinophils with opsonized particles by changing the affinity of complement receptor type-3. Blom, M., Tool, A.T.J., Kok, P.T.M. et al., Blood 83, 2978–2984 (1994). 965. Modulation and induction of eosinophil chemotaxis by granulocyte-macrophage colonystimulating factor and interleukin-3. Warrings, R.A.J., Koenderman, L., Kok, P.T.M. et al., Blood 77, 2694–2700 (1991). 966. Migration of primed human eosinophils across cytokine-activated endothelial cell monolayers. Moser, R., Fehr, J., Olgiati, L. et al., Blood 79, 2937–2945 (1992). 967. Purification of human basophils by density and size alone. Kepley, C., Craig, S. and Schwartz, L. J. Immunol. Meth. 175, 1–9 (1994). 968. Purification of normal human bone-marrow-derived basophils. Arock, M., Mossalayi, M.D., Le Goff, L. et al., Int. Arch. Allergy Immunol. 102, 107–111 (1993). 969. Effects of cytokines on human basophils chemotaxis. Tanimoto, Y., Takahashi, K. and Kimura, I. Clin. Exper. Allergy 22,1020–1025 (1992). 970. Purification of human blood basophils by negative selection using immunomagnetic beads. Bjerke, T., Nielsen, S., Helgestad, J. et al., J. Immunol. Meth. 157, 49–56 (1993).
68
971. Purification of human blood basophils using negative selection by flow cytometry. Tanimoto, Y., Takahashi, K., Takata, M. et al., Clin. Exper. Allergy 22, 1015–1019 (1992). 972. Purification of human blood basophils and leukotriene C4 generation following calcium ionophore stimulation. Mita, K., Akiyama, K., Hayakawa, T. et al., Prostaglandins, Leukotrienes and Essential Fatty Acids 49, 783–788 (1993). 973. Effect of nerve growth factor on the release of inflammatory mediators by mature human basophils. Bischoff, S. and Dahinden, C.A. Blood 79, 2662–2669 (1992). 974. Infection of Nippostrongylus brasiliensis induces normal increase of basophils in mast cell-deficient Ws/Ws rats with a small deletion of the kinase domain of c-kit. Kasugai, T., Okada, M., Morimoto, M. et al., Blood 81, 2521–2529 (1993). 975. Thawed human hepatocytes in primary culture. Dou, M., de Sousa, G., Lacarelle, B. et al., Cryobiol. 29, 454–469 (1992). 976. The distribution of non-specific carboxylesterases and glutathione S-transferases in different rat liver cells. Gad, M.Z. Biochem. Pharmacol. 48, 139–144 (1994). 977. Methods in laboratory investigation: assessment of a method of isolation, purification, and cultivation of rat liver sinusoidal endothelial cells. Braet, F., De Zanger, R., Sasaoki, T. et al., Lab. Invest. 70, 944–952 (1994). 978. Differential expression of platelet-derived growth factor a- and b-receptors on fat-storing cells and endothelial cells of rat liver. Heldin, P., Pertoft, H., Nordlinger, H. et al., Exper. Cell Res. 193, 364–369 (1991). 979. Preservation of the rate and profile of xenobiotic metabolism in rat hepatocytes stored in liquid nitrogen. Zaleski, J., Richburg, J. and Kauffman, F.C. Biochem. Pharmacol. 46, 111-116 (1993). 980. Inhibition of biotransformation of xenobiotic p-nitroanisole after cryopreservation of isolated rat hepatocytes. Petrenko, A.Y. and Mazur, S.P. Cryobiol. 30, 158-163 (1993). 981. A method for the cryopreserved rat liver parenchymal cells for studies of xenobiotics. Diener, B., Utesch, D., Beer, N. et al., Cryobiol. 30, 116–127 (1993). 982. Characterization of cryopreserved rat liver parenchymal cells by metabolism of diagnostic substrates and activities of related enzymes. Utesch, D., Diener, B., Molitor, E. et al., Biochem. Pharmacol. 44, 309–315 (1992). 983. Salmonella choleraesuis and Salmonella typhimurium associated with liver cells after intravenous inoculation of rats are localized mainly in Küpffer cells and multiply intracellularly. Nnalue, N.A., Shnyra, A., Hultenby, K. et al., Infect. Immun. 60, 2758–2768 (1992). 984. Erythroid colony formation by fetal rat liver and spleen cells in vitro: inhibition by a low relative molecular mass component of fetal spleen. Nagel, M.-D. and Nagel, J. Development 114, 213–219 (1992). 985. Fate of DNA targeted to the liver by asialoglycoproein receptor-mediated endocytosis in vivo. Chowdhury, N.R., Wu, C.H., Wu, G.Y. et al., J. Biol. Chem. 268, 11265–11271 (1993). 986. Epidermal growth factor stimulates testosterone production of human Leydig cells in vitro. Syed, V., Khan, S.A. and Nieschlag, E. J. Endocrinol. Invest. 14, 93–97 (1991).
69
987. Immunocytochemical localization and endogenous synthesis of apolipoprotein E in testicular Leydig cells. Schleicher, R.L., Zheng, M. and Zhang, M. Biol. Reprod. 48, 313–324 (1993). 988. Isolation of human Leydig cell mesenchymal precursors from patients with the androgen insensitivity syndrome: testosterone production and response to human chorionic gonadotropin stimulation in culture. Chemes, H., Cigorraga, S., Bergada, C. et al., Biol. Reprod. 46, 793–801 (1992). 989. Enhancement of testosterone secretion by normal adult human Leydig cells by co-culture with enriched preparations of normal adult human Sertoli cells. Lejeune, H., Skalli, M., Sanchez, P. et al., International. J. Androl. vol 4, 27–34 (1993). 990. Heterogeneity of adult mouse Leydig cells with different buoyant densities. Chamindrani Mendis-Handagama, S.M.L. and De Kretser, D.M. J. Androl. 13, 274–282 (1992). 991. Effect of cortisol on testosterone production by immature pig Leydig cells. Li, P.S. J. Steroid Biochem. Molec. Biol. 38, 205–212 (1991). 992. Insulin-like growth factor-binding protein-2: the effect of human chorionic gonadotropin on its gene regulation and protein secretion and its biological effect in rat Leydig cells. Wang, D., Nagpal, M.L., Lin, T. et al., Mol. Endocrinol. 8, 69–76 (1994). 993. Germ cell localization of a testicular growth hormone-releasing hormone-like factor. Srivastava, C.H., Collard, M.W., Rothrock, J.K. et al., Endocrinol. 133, 83–89 (1993). 994. Separation and characterization of Leydig cells and macrophages from rat testes. Dirami, G., Poulter, L.W. and Cooke, B.A. J. Endocrinol. 130, 357–365 (1991). 995. D5-3b-hydroxysteroid dehydrogenase-isomerse activity in two distinct density Leydig cells from immature rats. Differences in responsiveness to human chorionic gonadotropin or 8-bromoadensoine 3',5'-monophosphate. Murono, E.P. and Washburn, A.L. Biochem. Biophys. Acta 1091, 55–62 (1991). 996. A possible role for a low molecular weight peptide in regulation of testosterone production by rat Leydig cells. Ramaraj, P., Subbarayan, V.S.R. and Jagannadha, R.A. Indian J. Biochem. Biophys. 28, 536–540 (1991). 997. Interleukin-1a-induced changes in androgen and cyclic adenosine 3',5'- monophosphate release in adult rat Leydig cells in culture. Moore, C. and Moger, W.H. J. Endocrinol. 129, 381–390 (1991). 998. Enhanced stimulation of 5a-reductase activity in cultured Leydig cell precursors by human chorionic gonadotropin. Murono, E.P., Washburn, A.L. and Goforth, D.P. J. Steroid Biochem. Molec. Biol. 48, 377–384 (1994). 999. Rat seminiferous tubular culture medium contains a biological factor that inhibits Leydig cell steroidogenesis; its purification and mechanism of action. Zwain, I.H. and Cheng, C.Y. Mol. Cell. Endocrinol. 104, 213–227 (1994). 1000. In vitro DNA synthesis in Leydig and other interstitial cells of the rat testis. Moore, A., Findlay, K. and Morris, I.D. J. Endocrinol. 124, 247–255 (1992). 1001. Flow cytometric comparison between swim-up and Percoll gradient techniques for the separation of frozen-thawed human spermatozoa. Chen, Y., Obhrai, M.S., Chapman, J. et al., Int. J. Fertil. 37, 315–319 (1992). 70
1002. Comparative evaluation of three sperm-washing methods to improve sperm concentration and motility in frozen-thawed oligozoospermic and normozoospermic samples. Bongso, A., Jarina, A.K., Ho, J. et al., Archiv. Androl. 31, 223–230 (1993). 1003. Human spermatozoa selected by Percoll gradient or swim-up are equally capable of binding to the human zona pellucida and undergoing the acrosome reaction. Morales, P., Vantman, D., Barros, C. et al., Human Reprod. 6, 401–404 (1991). 1004. Isolation of motile spermatozoa: comparison of Percoll centrifugation, SpermPrep filtration, and swim-up techniques. Ziebe, S. and Andersen, C.Y. J. Assist. Repro. Genet. 10, 485–587 (1993). 1005. Adenosine triphosphate and motility characteristics of fresh and cryopreserved human spermatozoa. McLaughlin, E.A., Ford, W.C.L. and Hull, M.G.R. International. J. Androl. 17, 19–23 (1994). 1006. Association of human sperm nuclear condensation and in vitro penetration ability. Chan, P.J. and Tredway, D.R. Andrologia. 24, 77–81 (1992). 1007. Differential sperm performance as judged by the zona-free hamster egg penetration test relative to differing sperm penetration techniques. Chan, S.Y.W. and Tucker, M.J. Human Reprod. 7, 255–260 (1992). 1008. Effect on swim-up, Percoll and Sephadex sperm separation methods on the hypo-osomotic swelling test. Check, J.H., Katsoff, D., Kozak, J. et al., Human Reprod. 7,109–111 (1992). 1009. Comparison of characteristics of human spermatozoa selected by the multiple-tube swim-up and simple discontinuous Percoll gradient centrifugation. Chan, S.Y.W, Chan. Y.M. and Tucker, M.J. Andrologia 23, 213–218 (1991). 1010. Inhibition of the acrosome reaction by trypsin inhibitors and prevention of penetration of spermatozoa through the human zona pellucida. Llanos, M., Vigil, P., Salgado, A.M. et al., J. Reprod. Fertil. 97, 173–178 (1993). 1011. Sperm recovery techniques to maximize fertilizing capacity. Mortimer, D. Reprod. Fertil. Dev. 6, 25–31 (1994). 1012. Human zona pellucida recognition associated with removal of sialic acid human sperm surface. Lassalle, B. and Testart, J. J. Reprod. Fertil. 101, 703–711 (1994). 1013. Relationship between iron-catalysed lipid peroxidation potential and human sperm function. Aitken, R.J., Harkiss, D. and Buckingham, D. J. Reprod. Fertil. 98, 257–265 (1993). 1014. Leukocytic infiltration into the human ejaculate and its association with semen quality, oxidative stress, and sperm function. Aitken, R.J., West, K. and Buckingham, D. J. Androl. 15, 343–352 (1994). 1015. Simultaneous measurement of sperm LDH, LDH-X, CPK activities and ATP content in normospermic and oligozoospermic men. Orlando, C., Krausz, C., Forti, G. et al., International. J. Androl. 17, 13–18 (1994). 1016. Differential contribution of leucocytes and spermatozoa to the generation of reactive oxygen species in the ejaculate of oligozoospermic patients and fertile donors. Aitken, R.J., Buckingham, D., West, K. et al., J. Reprod. Fert. 94, 451–462 (1992).
71
1017. Analysis of the extent to which sperm movement can predict the results of ionophore-enhanced functional assays of the acrosome reaction and sperm-oocyte fusion. Aitken, J., Buckingham, D. and Harkiss, D. Human Reprod. 9, 1867–1874 (1994). 1018. Density differences between spermatozoa with antisperm autoantibodies and spermatozoa covered with antisperm antibodies from serum. Almagor, M., Margalioth, E.J. and Yaffe, H. Human Reprod. 7, 959–961 (1992). 1019. Movement characteristics of human spermatozoa collected from different layers of a discontinuous Percoll gradient. Saad, A. and Guerin, J.E. Andrologia 24, 149–153 (1992). 1020. Comparison of the chromatin stainability of human spermatozoa separated by discontinuous Percoll gradient centrifugation: a flow cytometric contribution. Pasteur, X., Maubon, I., Sabido, O. et al., Anal. Quant. Cytol. Histol. 14, 96–104 (1992). 1021. Selection of human spermatozoa by a hyperosmotic two-layer Percoll gradient. Chan, Y.-M. Abuzeid, M.I., Malcomnson, J.H. et al., Fertil. Steril. 61, 1097–1102 (1994). 1022. Ultrastructural comparison of human spermatozoa along a Percoll density gradient. Barthelemy, C., Fricot, G., Hamamah, S. et al., Int. J. Fertil. 37, 362–367 (1992). 1023. Flow cytometric sorting of living, highly motile human spermatozoa based on evaluation of their mitochondria. Auger, J., Leonce, S., Jouannet, P. et al., J. Histochem. Cytochem. 41, 1247–1251 (1993). 1024. Evaluating acrosome reaction steps with brightfield and differential interference contrast microscopy techniques. Steinholt, H.C., Chandler, J.E. and Tirado, V. J. Dairy Sci. 74, 3822–3826 (1991). 1025. Lipid composition of hamster epididymal spermatozoa. Awano, M., Kawaguchi, A. and Mohri, H. J. Reprod. Fertil. 99, 375–383 (1993). 1026. Separate effects of caffeine and dbcAMP on macaque sperm motility and interaction with the zona pellucida. Vandevoort, C.A., Tollner, T.L. and Overstreet, J.W. Mol. Reprod. Dev. 37, 299–304 (1994). 1027. Effects of the stem cell factor, c-kit ligand, on human megakaryocytic cells. Avraham, H., Vannier, E., Cowley, S. et al., Blood 79, 365–371 (1992). 1028. Interferon-a downregulates the abnormal intracytoplasmic free calcium concentration of tumor cells in hairy cell leukemia. Genot, E., Bismuth, G., Degos, L. et al., Blood 80, 2060–2065 (1992). 1029. Infection of megakaryocytes by human immunodeficiency virus in seropositive patients with immune thrombocytopenic purpura. Louache, F., Bettaieb, A., Henri, A. et al., Blood. 78, 1697–1705 (1991). 1030. Fibrinogen g-chain mRNA is not detected in human megakaryocytes. Lange, W., Luig, A., Dolken, G. et al., Blood 78, 20–25 (1991). 1031. Growth and detection of human bone marrow B lineage colonies. McGinnes, K., Keystone, E., Bogoch, E. et al., Blood 76, 896–905 (1990).
72
1032. Myeloid and lymphoid chimerism after T cell-depleted bone marrow transplantation: evaluation of conditioning regiments using the polymerase chain reaction to amplify human minisatellite regions of genomic DNA. MacKinnon, S., Barnett, L., Bourhis, J.H. et al., Blood 80, 3235–3241 (1992). 1033. T-cell subsets and suppressor cells in human bone marrow. Schmidt-Wolf, I.G.H., Dejbakhsh-Jones, S., Ginzton, N. et al., Blood 80, 3242–3250 (1992). 1034. The heparin binding PECAM-1 adhesion molecule is expressed by CD34+ hematopoietic precursor cells with early myeloid and B-lymphoid cell phenotypes.Watt, S.M., Williamson, J., Genevier, H. et al., Blood 82, 2649–2663 (1993). 1035. Long-term interleukin-6 administration stimulates sustained thrombopoiesis and acute-phase protein synthesis in a small primate - the marmoset. Ryffel, B., Car, B.D., Woerly, G. et al., Blood 83, 2093–2102 (1994). 1036. Effect of human interleukin-6 on megakaryocyte development and thrombocytopoiesis in primates. Stahl, C.P., Zucker-Franklin, D., Evatt, B.L. et al., Blood 78, 1467–1475 (1991). 1037. Studies of an improved Rhesus hematopoietic progenitor cell assay. Winton, E.F., Jacobs, P.C., Rozmiarek, S.K. et al., Exp. Hematol. 20, 401–404 (1992). 1038. Interleukin-10 inhibits the osteogenic activity of mouse bone marrow. Van Vlasselaer, P., Borremans, B., Van Den Heuvel, R. et al., Blood 82, 2361–2370 (1993). 1039. Interleukin-3 and lipopolysaccharide interact to inhibit proliferation of mouse bone marrow cells. Silva, Z.Z.A., Furlanetto, M.P., Marques, E.K. et al., Immunol. Lett. 40, 55–58 (1993). 1040. Separation of hematopoietic stem cells into two populations and their characterization. Ogata, H., Taniguchi, S., Inaba, M. et al., Blood 80, 91–95 (1992). 1041. Production of hematopoietic stem cell-chemotactic factor by bone marrow stromal cells. Cherry, B., Yasumizu, R., Toki, J. et al., Blood 83, 964–971 (1994). 1042. Characterization of bone cells isolated on discontinuous Percoll gradients: distribution in sequentially derived populations. Wong, G.L., Ng, M.C., Calabrese, D.W. et al., J. Bone and Mineral Res. 6, 969–976 (1991). 1043. LPS activation of bone marrow natural suppressor cells. Holda, J.H. Cell. Immunol. 141, 518–527 (1992). 1044. Enrichment and functional characterization of Sca-1+WGA+, Lin-WGA+, Lin-Sca-1+, and Lin-Sca-1+WGA+ bone marrow cells from mice with an Ly-6a haplotype. Jurecic, R., Van, N.T. and Belmont, J.W. Blood 82, 2673–2683 (1993). 1045. Different subsets of T cells in the adult mouse bone marrow and spleen induce or suppress acute graft-versus-host disease. Palathumpat, V., Dejbakhsh-Jones, S., Holm, B. et al., J. Immunol. 149, 808–817 (1992). 1046. Purification of rat megakaryocyte colony-forming cells using a monoclonal antibody against rat platelet glycoprotein IIb/IIIa. Miyazaki, H., Inoue, H., Yanagida, M. et al., Exp. Hematol. 20, 855–861 (1992). 1047. Hematopoiesis in asymptomatic cats infected with feline immunodeficiency virus. Linenberger, M.L., Shelton, G.H., Persik, M.T. et al., Blood 78, 1963–1968 (1991).
73
1048. Enhanced reactive oxygen species metabolism of air space cells in hypersensitivity pneumonitis. Calhoun, W.J. J. Lab. Clin. Med. 117, 443–452 (1991). 1049. Alveolar macrophage subpopulations in patients with active pulmonary tuberculosis. Kuo, H.-P. and Yu, C.-T. Chest 103, 1773–1778 (1993). 1050. Enhanced superoxide production by alveolar macrophages and air-space cells, airway inflammation, and alveolar macrophage density changes after segmental antigen bronchoprovocation in allergic subjects. Calhoun, W.J., Reed, H.E., Moest, D.R. et al., Am. Rev. Respir. Dis. 145, 317–325 (1992). 1051. Secretion of platelet-activating factor acetylhydrolase by human decidual macrophages. Narahara, H., Nishioka, Y. and Johnston, J.M. J. Clin. Endocrinol. Metab. 77, 1258–1262 (1993). 1052. Airway macrophages from patients with asthma do not proliferate. Chanez, P., Vago, P., Demoly, P. et al., J. Allergy Clin. Immunol. 92, 869–877 (1993). 1053. Increased numbers of hypodense alveolar macrophages in patients with bronchial asthma. Chanez, P., Bousquet, J., Couret, I. et al., Am. Rev. Respir. Dis. 144, 923–930 (1991). 1054. Mouse peritoneal macrophages: characterization of functional subsets following Percoll density gradients. Plasman, N. and Vray, B. Res. Immunol. 14, 151–163 (1993). 1055. Separation of murine peritoneal macrophages using Percoll density gradients. Vray, B. and Plasman, N. J. Immunol. Meth. 174, 53–59 (1994). 1056. Fluorescence demonstration of cathepsin B activity in fractionated alveolar macrophages. Sakai, K., Nii, Y., Ueyama, A. et al., Cell. Molec. Biol. 37, 353–358 (1991). 1057. Effects of pulmonary surfactant and surfactant protein A on phagocytosis of fractionated alveolar macrophages: relationship to starvation. Sakai, K., Kweon, M.N., Kohri, T. et al., Cell. Molec. Biol. 38, 123–130 (1992). 1058. The effects of asbestos inhalation on the distribution and enhancement of immunoassociated antigen expression of alveolar macrophage subpopulation. Inamoto, T., Georgian, M.M., Kagan, E. et al., J. Vet. Med. Sci. 54, 829–836 (1992). 1059. Immunoglobulin E plus antigen challenge induces a novel intercrine/chemokine in mouse mast cells. Kulmburg, P.A., Huber, N.E., Scheer, B.J. et al., J. Exp. Med. 176, 1773–1778 (1992). 1060. Potentiation of antigen-induced histamine release from rat peritoneal mast cells through a direct interaction between mast cells and non-mast cells. Inagaki, N., Kawasaki, H., Ueno, M. et al., Life Sci. 54, 1403–1409 (1994). 1061. Effect of phospholipase A2 inhibitor ONA-RS-082, on substance P-induced histamine release from rat peritoneal mast cells. Kurosawa, M., Hisada, T. and Ishizuka, T. Int. Arch. Allergy Immunol. 97, 226–228 (1992). 1062. Eosinophil peroxidase accounts for most if not all of the peroxidase activity associated with isolated rat peritoneal mast cells. Rickard, A. and Lagunoff, D. Int. Arch. Allergy Immunol. 103, 365–369 (1994).
74
1063. Sensitivity in vitro of mature and immature mouse thymocytes to dexamethasone cytotoxicity and its correlation to poly ADP-ribosylation. Hoshino, J., Beckmann, G. and Kroger, H. Biochem. International 27, 105–116 (1992). 1064. Dexamethasone and etoposide induce apoptosis in rat thymocytes from different phases of the cell cycle. Fearnhead, H.O., Chwalinski, M., Snowden, R.T. et al., Biochem. Pharmacol. 48, 1073–1079 (1994). 1065. Characterization of apoptosis in thymocytes isolated from dexamethasone-treated rats. Sun, X.-M., Dinsdale, D., Snowden, R.T. et al., Biochem. Pharmacol. 44, 2131–2137 (1992). 1066. Formation of large molecular weight fragments of DNA is a key committed step to apoptosis in thymocytes. Cohen, G.M., Sun, X.-M, Fearnhead, H. et al., J. Immunol. 153, 507–516 (1994). 1067. Immunologic detection of endothelial cells in human whole blood. Sbarbati, R., de Boer, M., Marzilli, M. et al., Blood 77, 764–769 (1991). 1068. Stimulation of rat placental lactogen-II (rPL-II) secretion by cultured trophoblasts by insulin: development of a rat placental cell culture system and effects of peptide hormones on rPL-TT secretion in vitro. Kishi, K., Itoh, M., Kanemori, S. et al., J. Reprod. Fertil. 99, 519–527 (1993). 1069. Sedimentation for the separation of cells. Pretlow, T.G. and Pretlow, T.P. Methods: A Companion to Methods in Enzymology 2, 183–191 (1991). 1070. Nonenzymatic extraction of cells from clinical tumor material for analysis of gene expression by two dimensional polyacrylamide gel electrophoresis. Franzen, B., Linder, S., Okuzawa, K. et al., Electrophoresis 14, 1045–1053 (1993). 1071. HL-60 cells induced to differentiate towards neutrophils subsequently die via apoptosis. Martin, S.J., Bradley, J.G. and Cotter, T.G. Clin. Exp. Immunol. 79, 448–453 (1990). 1072. Cytoplasmic transfer of the mtDNA nt 8993 T-G(ATP6) point mutation associated with the Leigh syndrome into mtDNA-less cells demonstrates cosegregation with a decrease in state III respiration and ADP/O ratio. Trounce, I., Neill, S: and Wallace, D.C. Proc. Natl. Acad. Sci. USA 91, 8334–8338 (1994). 1073. Morphological obervations of turkey (Meleagris galopavo) spermiophages maintained in tissue culture. Perez, B.S., Derrick, Jr, F.C., Korn, N. and Thurston, R.J. Poultry Sci. 73, 1597–1606 (1994). 1074. The influence of growth medium on serum sensitivity of Bacteroids species. Allan, E. and Poxton, I.R. J. Med. Microbiol. 41, 45–50 (1994). 1075. Inhibition of binding, entry, or intracellular proliferation of Ehrlichia risticii in P388D1 cells by anti-E. risticii serum, immunoglobulin G, or Fab fragment. Messick, J.B. and Rikihisa, Y. Infect. Immun. 62, 3156–3161 (1994). 1076. Inhibition of infection of macrophages with Ehrlichia risticii by cytochalasins, monodansylcadaverine, and taxol. Rikihisa, Y., Zhang, Y. and Park. J. Infect. Immun. 62, 5126–5132 (1994). 1077. Synthetic peptides analogous to the fimbrillin sequence inhibit adherence of Porphyromonas gingivalis. Lee, J.-Y., Sojar, H.T., Bedi, G.S. et al., Infect. Immun. 60, 1662–1670 (1992).
75
1078. The influence of different sera on the in vitro immobilisation of Percoll purified Treponema pallidum, Nichols strain. Engelkens, H.J.H., Kant, M., Onvlee, P.C. et al., Genitourin. Med. 68, 20–25 (1992). 1079. Preliminary biochemical characterization of veil structure purified from Theileria sergenti-, T. buffeli and T. orientalis-infected bovine erythrocytes. Sugimoto, C., Kawazu, S., Sato, M. et al., Parasitol 104, 207–213 (1992). 1080. Concentration and enzyme content of in vitro-cultured Babesia bigemina-infected erythrocytes. Vega, C.A., Buening, G.M.., Rodriguez, S.D. et al., J. Protozool. 33, 514–518 (1986). 1081. Protein characterization of Babesia equia piroplasms isolated from infected horse erythrocytes. Ali, S., Sugimoto, C., Matsuda, M. et al., Parasitol. Res. 79, 639–643 (1993). 1082. Rapid transport of the acidic phosphoproteins of Plasmodium berghei and P. chabaudi from the intra-erythrocytic parasite to the host membrane using a miniaturized fractionation procedure. Wiser, M.F. and Lanners, H.N. Parasitol. Res. 78, 193–200 (1992). 1083. Babesia bovis: purification and concentration of merozoites and infected bovine erythrocytes. Rodriquez, S.D., Buening, G.M., Vega, C.A. et al., Exp. Parasitol. 61, 236–243 (1986). 1084. Improved method for the concentration and purification of faecal cysts of Entamoeba histolytica for use as antigen. Jyothi, Foerster, B., Hamelmann, C. et al., J. Trop. Med. and Hygiene 96, 249–250 (1993). 1085. Vairimorpha necatrix: infectivity for and development in a lepidopteran cell line. Kurtti, T.J., Munderloh, U.G. and Noda, H. J. Inv. Pathol. 55, 61–68 (1990). 1086. Purification and partial characterization of rice transitory yellowing virus. Chiu, R.-J., Yau-Heiu, H., Chen, M.-J. et al., Am: Phytopathol. Soc. 80, 777–783 (1990). 1087. Purification of rubella virus by isopycnic gradients: continuous Percoll versus discontinuous sucrose. Bustos, J., Zamora, P., Mejia, E. et al., Arch. Virol. 118, 285–288 (1991). 1088. Purification of mycoplasma-like organisms from lettuce with aster yellows disease. Jiang, Y.P. and Chen, D.A. Am. Phytopathol. Soc. 77, 949–953 (1987). 1089. Growth hormone regulates amino acid transport in human and rat liver. Pacitti, A.J., Inoue, Y. and Plumley, D.A. Ann. Surg. 216, 353–362 (1992). 1090. Oxytocin pretreatment of pregnant rat uterus inhibits Ca2+ uptake in plasma membrane and sarcoplasmic reticulum. Magocsi, M. and Penniston, J.T. Biochem. Biophys. Acta 1063, 7–14 (1991). 1091. Lysophosphoinostide-specific phospholipase C in rat brain synaptic plasma membranes. Tsutsumi, T., Kobayashi, T., Ueda, H. et al., Neurochem. Res. 19, 399–406 (1994). 1092. Pharmacological characterization of inositol 1,4,5-triphosphate binding sites: relation to Ca2+ release. Mouillace, B., Devilliers, G. and Jard. S. Eur. J. Pharmacol. 225, 179–193 (1992). 1093. Influence of fasting on glutamine transport in rat liver. Espat, N.J., Copeland, E.M. and Souba, W.W. J. Parenter. Enter. Nutr. 17, 493–500 (1993). 1094. Inositol 1,4,5-triphosphate binding sites copurify with the putative Ca-storage protein calreticulin in rat liver. Enyed, P., Szabadkai, G., Krause, K.-H. et al., Cell. Calcium 14, 485–492 (1993).
76
1095. Stimulation of hepatocyte system y+-mediated L-arginine transport by an inflammatory agent. Pacitti, A.J., Copeland, E.M. and Souba, W.W. Surgery 11, 403–411 (1992). 1096. Cellular localization and characterization of proteins that bind high density lipoprotein. Hokland, B., Mendez, A.J. and Oram, J.F. J. Lipid Res. 33, 1335–1342 (1992). 1097. Dietary soybean oil changes lipolytic rate and composition of fatty acids in plasma membranes of ovine adipocytes. Jenkins, T.C, Thies, E.J. and Fotouhi, N. J. Nutr. 124, 566–570 (1994). 1098. Isolation of the plasma membrane and organelles from Chinese hamster ovary cells. Cezanne, L., Navarro, L. and Tocanne, J.-F. Biochim. Biophys. Acta 1112, 205–214 (1992). 1099. Isolation of Raja erinacea basolateral liver plasma membranes: characterization of lipid composition and fluidity. Smith, D.J. and Ploch, S.A. J. Exp. Zool. 258, 189–195 (1991). 1100. Structural and functional properties of plasma membranes from the filamentous fungus Penicillium chrysogenum. Hillenga, D.J., Versantvoort, H.J.M., Driessen, A.J.M. et al., Eur. J. Biochem. 224, 581–587 (1994). 1101. Preparation of right-side-out plasma membrane vesicles from Penicillium cyclopium: a critical assessment of markers. Ugalde, U.O., Hernandez, A., Galindo I. et al., J. Gen. Microbiol. 138, 2205–2212 (1992). 1102. Demonstration of adenosine deaminase activity in human fibroblast lysosomes. Lindley, E.R. and Pisoni, R.L. Biochem. J. 290, 457–462 (1993). 1103. Specific storage of subunit c of mitochondrial ATP synthase in lysosomes of neuronal ceroid lipofuscinosis (Batten's disease). Kominami, E., Ezaki, J., Muno, D. et al., J. Biochem. 111, 278–282 (1992). 1104. Effect of pH and ATP on the equilibrium density of lysosomes. Mayorga, L.S., De Veca, M.G., Colombo, M.I. et al., J. Cell. Physiol. 156, 303–310 (1993). 1105. Cycling of an 85-kDa lysosomal membrane glycoprotein between the cell surface and lysosomes in cultured rat hepatocytes. Akasaki, K., Michihara, A., Fukuzawa, M. et al., J. Biochem. 116, 670–676 (1994). 1106. Preparation of membrane vesicles from kidney cortex lysosomes using amino acid methyl ester. Harikumar, P. Biochem. Molecul. Biol. International 30, 1005–1011 (1993). 1107. Distribution and structure of the vacuolar H+ ATPase in endosomes and lysosomes from LLC-PK1 cells. Somsel Rodman, J., Stahl, P.D. and Gluck, S. Exp. Cell Res. 192, 445–452 (1991). 1108. Rapid preparation of subsarcolemmal and interfibrillar mitochondrial subpopulation from cardiac muscle. Chemnitius, J,-M., Manglitz, T., Kloeppel, M. et al., Int. J. Biochem. 4, 589–596 (1993). 1109. Developmental changes in rat liver mitochondrial populations analyzed by flow cytometry. Lopez-Mediavilla, C., Orfao, A., San Miguel, J. et al., Exp. Cell Res. 203, 134–140 (1992). 1110. Plasmodium berghei: partial purification and characterization of the mitochondrial cytochrome c oxidase. Krungkrai, J., Krungkrai, S.R. and Bhumiratana, A. Exp. Parasitol. 77, 136–146 (1993). 1111. Isolation and characterization of mitochondria from turkey spermatozoa. McLean, D.J., Korn, N., Perez, B.S. et al., J. Androl. 14, 433–438 (1993).
77
1112. Isolation and characterization of gelatinase granules from human neutrophils. Kjeldsen, L., Sengelov, H., Lollike, K. et al., Blood 83, 1640–1649 (1994). 1113. Subcellular localization and translocation of the receptor for N-formylmethionyl-leucylphenylalanine in human neutrophils. Sengelov, H., Boulay, F., Kjeldsen, L. et al., Biochem. J. 299, 473–479 (1994). 1114. Subcellular localization and dynamics of Mac-1 (a[m]b[2]) in human neutrophils. Sengelov, H., Kjeldsen, L., Diamond, M.S. et al., J. Clin. Invest. 92, 1467–1476 (1993). 1115. Subcellular localization of heparanase in human neutrophils. Matzner, Y., Vlodavsky, L., Bar-Ner, M. et al., J. Leuk. Biol. 51, 519–524 (1992). 1116. Granules of human CD3+ large granular lymphocytes contain a macrophage regulation factor(s) that induces macrophage H2O2 production and tumoricidal activity but decreases cell surface Ia antigen expression. Roussel, E. and Greenberg, A.H. Cell. Immunol. 134, 31–41 (1991). 1117. Properties of plasma membrane-induced amylase release from rat parotid secretory granules: effects of Ca2+ and Mg-ATP. Mizuno, M., Kameyama, Y., Yashiro, K. et al., Biochem. Biophys. Acta 1116, 104–111 (1992). 1118. Isolation of cytolytic granules from sea urchin amoebocytes. Pagliara, P. and Canicatti, C. Eur. J. Cell. Biol. 60, 179–184 (1993). 1119. Phosphatidylethanolamine synthesis by castor bean endosperm. Wang, X. and Moore, Jr, T.S. J. Biol. Chem. 266, 19981-19987 (1991). 1120. Characterization of a mitochondrial NADP-dependent isocitrate dehydrogenase in axes of germinating sunflower seeds. Attucci, S., Rivoal, J., Brouquisse, R. et al., Plant Science 102, 49–59 (1994). 1121. Microanalysis of plant mitochondrial protein synthesis products: detection of variant polypeptides associated with cytoplasmic male sterility. Boutry, M., Faber, A.-M., Charbonnier, M. et al., Plant Molec. Biol. 3, 445–452 (1984). 1122. Association of TMV coat protein with chloroplast membranes in virus-infected leaves. Reinero, A. and Beachy, R.N. Plant Molec. Biol. 6, 291–301 (1986). 1123. The methods for isolation of cytoplasts in several crop plants. Watanabe, M. and Yamaguchi, H. Japan. J. Breed. 38, 43–52 (1988). 1124. Barley aleurone layer cell protoplasts as a transient expression system. Gopalakrishnan, B., Sonthayanon, B., Rahmatullah, R. et al., Plant Molec. Biol. 16, 463–467 (1991). 1125. Rapid isolation of nuclei from carrot suspension culture cells using a BioNebulizer. Okpodu, C.M., Robertson, D., Boss, W.F. et al., BioTechniques 16, 154–158 (1994). 1126. Purification of highly intact plastids from various heterotrophic plant tissues: analysis of enzymatic equipment and precursor dependency for starch biosynthesis. Neuhaus, H.E., Batz, O., Thom, E. et al., Biochem. J. 296, 395–401 (1993). 1127. Nuclear buoyant density determination and the purification and characterization of wild-type Neurospora nuclei using Percoll density gradients. Talbot, K.J. and Russell, P.J. Plant Physiol. 70, 704–708 (1982).
78
1128. Outer dense fibres of human spermatozoa: partial characterization and possible physiological functions. Henkel, R., Stale, T., Mertens, N. et al., International J. Androl. 17, 68–73 (1994). 1129. Isolation and partial characterization of the outer dense fiber proteins from human spermatozoa. Henkel, R., Stalf, T. and Miska, W. Biol. Chem. Hoppe-Seyler 373, 685–689 (1992). 1130. Endocytosis and lysosomal delivery of tissue plasminogen activator-inhibitor 1 complexes in Hep G2 cells. Underhill, D.M., Owensby, D.A., Morton, P.A. et al., Blood 80, 2746–2754 (1992). 1131. Characterization of peptides bound to extracellular and intracellular HLA-DR1 molecules. Max, H., Halder, T., Kropshofer, H. et al., Human Immunol. 38, 193–200 (1993). 1132. Subcellular fractionation of cultured normal human melanocytes: new insights into the relationship of melanosomes with lysosomes and peroxisomes. Smit, N.P.M., van Roermund, C.W.T., Aerts, H.M.F.G. et al., Biochim. Biophys. Acta 1181, 1–6 (1993). 1133. Changes in the localization of catalase during differentiation of neutrophilic granulocytes. Ballinger, C.A., Chamindrani Mendis-Handagama, S.M.L., Kalmar, J.R. et al., Blood 83, 2654–2668 (1994). 1134. Biosynthesis and processing of cathepsin G and neutrophil elastase in the leukemic myeloid cell line U-937. Lindmark, A., Persson, A.-M. and Olsson, I. Blood 76, 2374–2380 (1990). 1135. Spheroplast formation and partial purification of microbodies from hydrocarbon-grown cells of Cladosporium resinae.J. Carson, D.B. and Cooney, J.J. Ind. Microbiol. 3, 111–117 (1988).
79
Ordering Information Products for Cell Separation and Culture Pr oduct Product ™
Percoll
Density Marker Beads Ficoll-Paque™ PLUS Ficoll™ PM 70
Quantity
Code No.
1 l, sterile
17-0891-01
250 ml, sterile
17-0891-02
10 vials
17-0459-01
6 x 100 ml
17-1440-02
6 x 500 ml
17-1440-03
100 g
17-0310-10
500 g
17-0310-50
1 kg
17-0310-01
5 kg
17-0310-05
100 g
17-0300-10
500 g
17-0300-50
1 kg
17-0300-01
5 kg
17-0300-05
40 kg
17-0300-08
Cytodex™ 1
25 g
17-0448-01
Cytodex™ 3
10 g
17-0485-01
Ficoll™ PM 400
Panel A. Separation of human blood cells in a gradient of Percoll. Bottom layer contains red blood cells, the middle band is polymorphonuclear cells (e.g. granulocytes) and the top band is mononuclear cells. Panel B: Percoll, our exceptional density gradient medium, is available in easy to open, resealable bottles.
80
Products for Purification of RNA Pr oduction Production
Quantity
Code No.
†
27-9255-01
1 kit†
27-9254-01
QuickPrep ™ Total RNA Extraction Kit
1 kit†
27-9271-01
RNA Extraction Kit
1 kit†
27-9270-01
(2 purifications)
1 kit†
27-9258-01
(4 purifications)
1 kit†
27-9258-02
CsTFA (Solution)
100 ml
17-0847-02
Oligo(dT)-Cellulose
500 mg
27-5543-01
1g
27-5543-02
5g
27-5543-03
QuickPrep
™
Micro mRNA Purification Kit
1 kit
(24 purifications) QuickPrep ™ mRNA Purification Kit (4 purifications)
(extracts RNA from up to 4 g of tissue) mRNA Purification Kit
Type 7
Products for Purification of DNA Pr oduct Product
Quantity
RapidPrep
™
Micro Genomic
Code No.
†
27-5225-01
1 kit†
27-5230-01
1 kit†
27-9281-01
1 kit
(DNA Isolation Kit for Cells and Tissue) RapidPrep™ Macro Genomic (DNA Isolation Kit for Cells and Tissue) FlexiPrep™ Kit
Kits for cDNA Synthesis Pr oduct Product
Quantity
Code No.
†
27-9262-01
cDNA Synthesis Kit (5 reactions)
1 kit†
27-9260-01
First-Strand cDNA Synthesis Kit (55 reactions)
1 kit†
27-9261-01
Ready-To-Go™ T-Primed First-Strand Kit
1 kit†
27-9263-01
TimeSaver
™
cDNA Synthesis Kit (5 reactions)
1 kit
(50 reactions) †
Product must be shipped cold. There is an extra charge for insulated container and refrigerant.
81
82
Percoll, Sephadex, Sephacyl, Ficoll, Ficoll-Paque, Cytodex, QuickPrep, TimeSaver, RapidPrep and Ready-To-Go are trademarks of Amersham Biosciences Limited. Merthiolate is a trademark of Eli Lilly & Co. Amersham is a trademark of Amersham plc. Pharmacia and Drop Design are trademarks of Pharmacia Corporation. The data presented herein have been carefully complied from our records, which we believe to be accurate and reliable. We make, however, no warranties or representations with respect hereto, nor is freedom from any patent to be inferred. Before any part of this manual is reproduced, please request permission from Amersham Biosciences. The products described in this literature are intended for in vitro use only. Nothing in this literature should be construed as either a recommendation or an authorization to use these products for in vivo applications. All goods and services are sold subject to the terms and conditions of sale of the company within the Amersham Biosciences group that supplies them. A copy of these terms and conditions is available on request. © Amersham Biosciences AB 2001 – All rights reserved. Amersham Biosciences AB Björkgatan 30, SE-751 84 Uppsala, Sweden Amersham Biosciences Amersham Place, Little Chalfont, Buckinghamshire HP7 9NA, England Amersham Biosciences Inc 800 Centennial Avenue, PO Box 1327, Piscataway, NJ 08855 USA Amersham Biosciences Europe GmbH Munzinger Strasse 9, D-79111 Freiburg, Germany Amersham Biosciences KK, Sanken Bldg. 3-25-1, Hyakunincho Shinjuku-ku, Tokyo 169-0073 Japan
83
www.amershambiosciences.com
84
Protein Purification
Handbook Back to Collection 18-1132-29 Edition AB
HiTrap, Sepharose, STREAMLINE, Sephadex, MonoBeads, Mono Q, Mono S, MiniBeads, RESOURCE, SOURCE, Superdex, Superose, HisTrap, HiLoad, HiPrep, INdEX, BPG, BioProcess, FineLINE, MabTrap, MAbAssistant, Multiphor, FPLC, PhastSystem and ÄKTA are trademarks of Amersham Pharmacia Biotech Limited or its subsidiaries. Amersham is a trademark of Nycomed Amersham plc Pharmacia and Drop Design are trademarks of Pharmacia & Upjohn Inc Coomassie is a trademark of ICI plc All goods and services are sold subject to the terms and conditions of sale of the company within the Amersham Pharmacia Biotech group which supplies them. A copy of these terms and conditions of sale is available on request. © Amersham Pharmacia Biotech AB 1999 All rights reserved. Amersham Pharmacia Biotech AB SE-751 84 Uppsala Sweden Amersham Pharmacia Biotech UK Limited Amersham Place Little Chalfont Buckinghamshire England HP7 9NA Amersham Pharmacia Biotech Inc 800 Centennial Avenue PO Box 1327 Piscataway NJ 08855 USA
Protein Purification Handbook
Contents Introduction........................................................................................................7 Chapter 1 Purification Strategies - A Simple Approach ......................................................9 Preparation ............................................................................................10 Three Phase Purification Strategy ..........................................................10 General Guidelines for Protein Purification ............................................12 Chapter 2 Preparation ......................................................................................................13 Before You Start .................................................................................... 13 Sample Extraction and Clarification ......................................................16 Chapter 3 Three Phase Purification Strategy ....................................................................19 Principles ................................................................................................19 Selection and Combination of Purification Techniques ..........................20 Sample Conditioning ..............................................................................26 Chapter 4 Capture ..................................................................................................29 Chapter 5 Intermediate Purification ........................................................................37 Chapter 6 Polishing ................................................................................................40 Chapter 7 Examples of Protein Purification Strategies ............................................45 Three step purification of a recombinant enzyme ..................................45 Three step purification of a recombinant antigen binding fragment ......49 Two step purification of a monoclonal antibody ....................................54 One step purification of an integral membrane protein ..........................57 Chapter 8 Storage Conditions ................................................................................61 Extraction and Clarification Procedures ................................................ 62 Chapter 9 Principles and Standard Conditions for Purification Techniques ............73 Ion exchange (IEX) ................................................................................73 Hydrophobic interaction (HIC) ..............................................................79 Affinity (AC) ..........................................................................................85 Gel filtration (GF) ..................................................................................88 Reversed phase (RPC) ............................................................................92 Expanded bed adsorption (EBA) ............................................................95
Introduction The development of techniques and methods for protein purification has been an essential pre-requisite for many of the advancements made in biotechnology. This booklet provides advice and examples for a smooth path to protein purification. Protein purification varies from simple one-step precipitation procedures to large scale validated production processes. Often more than one purification step is necessary to reach the desired purity. The key to successful and efficient protein purification is to select the most appropriate techniques, optimise their performance to suit the requirements and combine them in a logical way to maximise yield and minimise the number of steps required. Most purification schemes involve some form of chromatography. As a result chromatography has become an essential tool in every laboratory where protein purification is needed. The availability of different chromatography techniques with different selectivities provides a powerful combination for the purification of any biomolecule. Recombinant DNA developments over the past decade have revolutionised the production of proteins in large quantities. Proteins can even be produced in forms which facilitate their subsequent chromatographic purification. However, this has not removed all challenges. Host contaminants are still present and problems related to solubility, structural integrity and biological activity can still exist. Although there may appear to be a great number of parameters to consider, with a few simple guidelines and application of the Three Phase Purification Strategy the process can be planned and performed simply and easily, with only a basic knowledge of the details of chromatography techniques. Advice codes: general advice for any purification
advice for large scale purification
advice for micro scale purification
shortcuts
advice on media selection
7
8
Chapter 1
Purification Strategies - a simple approach Apply a systematic approach to development of a purification strategy. The first step is to describe the basic scenario for the purification. General considerations answer questions such as: What is the intended use of the product? What kind of starting material is available and how should it be handled? What are the purity issues in relation to the source material and intended use of the final product? What has to be removed? What must be removed completely? What will be the final scale of purification? If there is a need for scale-up, what consequences will this have on the chosen purification techniques? What are the economical constraints and what resources and equipment are available? Most purification protocols require more than one step to achieve the desired level of product purity. This includes any conditioning steps necessary to transfer the product from one technique into conditions suitable to perform the next technique. Each step in the process will cause some loss of product. For example, if a yield of 80% in each step is assumed, this will be reduced to only 20% overall yield after 8 processing steps as shown in Figure 1. Consequently, to reach the targets for yield and purity with the minimum number of steps and the simplest possible design, it is not efficient to add one step to another until purity requirements have been fulfilled. Occasionally when a sample is readily available purity can be achieved by simply adding or repeating steps. However, experience shows that, even for the most challenging applications, high purity and yield can be achieved efficiently in fewer than four well-chosen and optimised purification steps. Techniques should be organised in a logical sequence to avoid the need for conditioning steps and the chromatographic techniques selected appropriately to use as few purification steps as possible. Limit the number of steps in a purification procedure
9
Yield (%) 10 80 95% / step 60 90% / step
40
85% / step 20
80% / step 75% / step
0 1
2
3
4
5
6
7
8 Number of steps
Fig.1. Yields from multi-step purifications.
Preparation The need to obtain a protein, efficiently, economically and in sufficient purity and quantity, applies to every purification. It is important to set objectives for purity, quantity and maintenance of biological activity and to define the economical and time framework for the work. All information concerning properties of the target protein and contaminants will help during purification development. Some simple experiments to characterise the sample and target molecule are an excellent investment. Development of fast and reliable analytical assays is essential to follow the progress of the purification and assess its effectiveness. Sample preparation and extraction procedures should be developed prior to the first chromatographic purification step. With background information, assays and sample preparation procedures in place the Three Phase Purification Strategy can be considered.
Three Phase Purification Strategy Imagine the purification has three phases Capture, Intermediate Purification and Polishing. In the Three Phase Strategy specific objectives are assigned to each step within the process: In the capture phase the objectives are to isolate, concentrate and stabilise the target product. During the intermediate purification phase the objective is to remove most of the bulk impurities such as other proteins and nucleic acids, endotoxins and viruses. In the polishing phase the objective is to achieve high purity by removing any remaining trace impurities or closely related substances. The selection and optimum combination of purification techniques for Capture, Intermediate Purification and Polishing is crucial to ensure fast method development, a shorter time to pure product and good economy. 10
Purity
The final purification process should ideally consist of sample preparation, including extraction and clarification when required, followed by three major purification steps, as shown in Figure 2. The number of steps used will always depend upon the purity requirements and intended use for the protein.
Polishing Achieve final high level purity
Intermediate purification Capture Preparation, extraction, clarification
Remove bulk impurities
Isolate, concentrate and stabilise
Step Fig. 2. Preparation and the Three Phase Purification Strategy
11
Guidelines for Protein Purification The guidelines for protein purification shown here can be applied to any purification process and are a suggestion as to how a systematic approach can be applied to the development of an effective purification strategy. As a reminder these guidelines will be highlighted where appropriate throughout the following chapters. Define objectives for purity, activity and quantity required of final product to avoid over or under developing a method Define properties of target protein and critical impurities to simplify technique selection and optimisation Develop analytical assays for fast detection of protein activity/recovery and to work efficiently Minimise sample handling at every stage to avoid lengthy procedures which risk losing activity/reducing recovery Minimise use of additives additives may need to be removed in an extra purification step or may interfere with activity assays Remove damaging contaminants early for example, proteases Use a different technique at each step to take advantage of sample characteristics which can be used for separation (size, charge, hydrophobicity, ligand specificity) Minimise number of steps extra steps reduce yield and increase time, combine steps logically
KEEP IT SIMPLE!
12
Chapter 2
Preparation Before You Start The need to obtain a protein, efficiently, economically and in sufficient purity and quantity, applies to any purification, from preparation of an enriched protein extract for biochemical characterisation to large scale production of a therapeutic recombinant protein. It is important to set objectives for purity and quantity, maintenance of biological activity and economy in terms of money and time. Purity requirements must take into consideration the nature of the source material, the intended use of the final product and any special safety issues. For example, it is important to differentiate between contaminants which must be removed and those which can be tolerated. Other factors can also influence the prioritisation of objectives. High yields are usually a key objective, but may be less crucial in cases where a sample is readily available or product is required only in small quantities. Extensive method development may be impossible without resources such as an ÄKTA™design chromatography system. Similarly, time pressure combined with a slow assay turnaround will steer towards less extensive scouting and optimisation. All information concerning properties of the target protein and contaminants will help during purification development, allowing faster and easier technique selection and optimisation, and avoiding conditions which may inactivate the target protein. Development of fast and reliable analytical assays is essential to follow the progress of the purification and assess effectiveness (yield, biological activity, recovery).
Define objectives Goal: To set minimum objectives for purity and quantity, maintenance of biological activity and economy in terms of money and time. Define purity requirements according to the final use of the product. Purity requirement examples are shown below. Extremely high > 99%
Therapeutic use, in vivo studies
High 95- 99 %
X-ray crystallography and most physico-chemical characterisation methods
Moderate < 95 %
Antigen for antibody production N-terminal sequencing
13
Identify 'key' contaminants Identify the nature of possible remaining contaminants as soon as possible. The statement that a protein is >95% pure (i.e. target protein constitutes 95% of total protein) is far from a guarantee that the purity is sufficient for an intended application. The same is true for the common statement "the protein was homogenous by Coomassie™ stained SDS-PAGE". Purity of 95% may be acceptable if the remaining 5% consists of harmless impurities. However, even minor impurities which may be biologically active could cause significant problems in both research and therapeutic applications. It is therefore important to differentiate between contaminants which must be removed completely and those which can be reduced to acceptable levels. Since different types of starting material will contain different contaminant profiles they will present different contamination problems. It is better to over-purify than to under-purify. Although the number of purification steps should be minimised, the quality of the end product should not be compromised. Subsequent results might be questioned if sample purity is low and contaminants are unknown. Contaminants which degrade or inactivate the protein or interfere with analyses should be removed as early as possible. The need to maintain biological activity must be considered at every stage during purification development. It is especially beneficial if proteases are removed and target protein transferred into a friendly environment during the first step. A downstream production process must achieve the required purity and recovery with complete safety and reliability, and within a given economic framework. Economy is a very complex issue. In commercial production the time to market can override issues such as optimisation for recovery, capacity or speed. Robustness and reliability are also of great concern since a batch failure can have major consequences. Special safety issues may be involved in purification of biopharmaceuticals, such as detection or removal of infectious agents, pyrogens, immunogenic contaminants and tumorigenic hazards. It may be necessary to use analytical techniques targetted towards specific contaminants in order to demonstrate that they have been removed to acceptable levels. 14
Define properties of target protein and critical impurities Goal: To determine a 'stability window' for the target protein for easier selection and optimisation of techniques and to avoid protein inactivation during purification. Check target protein stability window for at least pH and ionic strength. All information concerning the target protein and contaminant properties will help to guide the choice of separation techniques and experimental conditions for purification. Database information for the target, or related proteins, may give size, isoelectric point (pI) and hydrophobicity or solubility data. Native one and two dimensional PAGE can indicate sample complexity and the properties of the target protein and major contaminants. Particularly important is a knowledge of the stability window of the protein so that irreversible inactivation is avoided. It is advisable to check the target protein stability window for at least pH and ionic strength. Table 1 shows how different target protein properties can affect a purification strategy. Table 1. Protein properties and their effect on development of purification strategies. Sample and target protein properties
Influence on purification strategy
Temperature stability
Need to work rapidly at lowered temperature
pH stability
Selection of buffers for extraction and purification Selection of conditions for ion exchange, affinity or reversed phase chromatography
Organic solvents stability
Selection of conditions for reversed phase chromatography
Detergent requirement
Consider effects on chromatographic steps and the need for detergent removal. Consider choice of detergent.
Salt (ionic strength)
Selection of conditions for precipitation techniques and hydrophobic interaction chromatography
Co-factors for stability or activity
Selection of additives, pH, salts, buffers
Protease sensitivity
Need for fast removal of proteases or addition of inhibitors
Sensitivity to metal ions
Need to add EDTA or EGTA in buffers
Redox sensitivity
Need to add reducing agents
Molecular weight
Selection of gel filtration media
Charge properties
Selection of ion exchange conditions
Biospecific affinity
Selection of ligand for affinity medium
Post translational modifications
Selection of group specific affinity medium
Hydrophobicity
Selection of medium for hydrophobic interaction chromatography
15
Develop analytical assays Goal: To follow the progress of a purification, to assess effectiveness (yield, biological activity, recovery) and to help during optimisation. Select assays which are fast and reliable. To progress efficiently during method development the effectiveness of each step should be assessed. The laboratory should have access to the following assays: • • • •
A rapid, reliable assay for the target protein Purity determination Total protein determination Assays for impurities which must be removed
The importance of a reliable assay for the target protein cannot be overemphasised. When testing chromatographic fractions ensure that the buffers used for separation do not interfere with the assay. Purity of the target protein is most often estimated by SDS-PAGE, capillary electrophoresis, reversed phase chromatography or mass spectrometry. Lowry or Bradford assays are used most frequently to determine the total protein. The Bradford assay is particularly suited to samples where there is a high lipid content which may interfere with the Lowry assay. For large scale protein purification the need to assay for target proteins and critical impurities is often essential. In practice, when a protein is purified for research purposes, it is too time consuming to identify and set up specific assays for harmful contaminants. A practical approach is to purify the protein to a certain level, and then perform SDS-PAGE after a storage period to check for protease cleavage. Suitable control experiments, included within assays for bio-activity, will help to indicate if impurities are interfering with results.
Sample Extraction and Clarification Minimise sample handling Minimise use of additives Remove damaging contaminants early Definition: Primary isolation of target protein from source material. Goal: Preparation of a clarified sample for further purification. Removal of particulate matter or other contaminants which are not compatible with chromatography.
16
The need for sample preparation prior to the first chromatographic step is dependent upon sample type. In some situations samples may be taken directly to the first capture step. For example cell culture supernatant can be applied directly to a suitable chromatographic matrix such as Sepharose™ Fast Flow and may require only a minor adjustment of the pH or ionic strength. However, it is most often essential to perform some form of sample extraction and clarification procedure. If sample extraction is required the chosen technique must be robust and suitable for all scales of purification likely to be used. It should be noted that a technique such as ammonium sulphate precipitation, commonly used in small scale, may be unsuitable for very large scale preparation. Choice of buffers and additives must be carefully considered if a purification is to be scaled up. In these cases inexpensive buffers, such as acetate or citrate, are preferable to the more complex compositions used in the laboratory. It should also be noted that dialysis and other common methods used for adjustment of sample conditions are unsuitable for very large or very small samples. For repeated purification, use an extraction and clarification technique that is robust and able to handle sample variability. This ensures a reproducible product for the next purification step despite variability in starting material. Use additives only if essential for stabilisation of product or improved extraction. Select those which are easily removed. Additives may need to be removed in an extra purification step. Use pre-packed columns of Sephadex™ G-25 gel filtration media, for rapid sample clean-up at laboratory scale, as shown in Table 2. Table 2. Pre-packed columns for sample clean-up. Pre-packed column
Sample volume loading per run
Sample volume recovery per run
Code No.
HiPrep™ Desalting 26/10 HiTrap Desalting Fast Desalting PC 3.2/10 PD-10 Desalting
2.5 -15 ml 0.25 - 1.5 ml 0.05 - 0.2 ml 1.5 - 2.5 ml
7.5 1.0 0.2 2.5
17-5087-01 17-1408-01 17-0774-01 17-0851-01
-
20 ml 2.0 ml 0.3 ml 3.5 ml
Sephadex G-25 gel filtration media are used at laboratory and production scale for sample preparation and clarification of proteins >5000. Sample volumes of up to 30%, or in some cases, 40% of the total column volume are loaded. In a single step, the sample is desalted, exchanged into a new buffer, and low molecular weight materials are removed. The high volume capacity, relative insensitivity to sample concentration, and speed of this step enable very large sample volumes to be processed rapidly and efficiently. Using a high sample volume load results in a separation with minimal sample dilution (approximately 1:1.4). Chapter 8 contains further details on sample storage, extraction and clarification procedures. 17
Sephadex G-25 is also used for sample conditioning i.e. rapid adjustment of pH, buffer exchange and desalting between purification steps. Media for consideration: Sephadex G-25 gel filtration For fast group separations between high and low molecular weight substances Typical flow velocity 60 cm/h (Sephadex G-25 SuperFine, Sephadex G-25 Fine), 150 cm/h (Sephadex G-25 Medium).
Combine Sample Clean-up and Capture in a single step If large sample volumes will be handled or the method scaled-up in the future, consider using STREAMLINE™ expanded bed adsorption. This technique is particularly suited for large scale recombinant protein and monoclonal antibody purification. The crude sample containing particles can be applied to the expanded bed without filtration or centrifugation. STREAMLINE adsorbents are specially designed for use in STREAMLINE columns. Together they enable the high flow rates needed for high productivity in industrial applications of fluidised beds. The technique requires no sample clean up and so combines sample preparation and capture in a single step. Crude sample is applied to an expanded bed STREAMLINE media. Target proteins are captured whilst cell debris, cells, particulate matter, whole cells, and contaminants pass through. Flow is reversed and the target proteins are desorbed in the elution buffer. Media for consideration: STREAMLINE (IEX, AC, HIC) For sample clean-up and capture direct from crude sample. STREAMLINE adsorbents are designed to handle feed directly from both fermentation homogenate and crude feedstock from cell culture/fermentation at flow velocities of 200 - 500 cm/h, according to type and application. Particle size: 200 µm Note:
18
cm/h: flow velocity (linear flow rate) = volumetric flow rate/cross sectional area of column.
Chapter 3
Three Phase Purification Strategy Principles
Purity
With background information, assays, and sample preparation and extraction procedures in place the Three Phase Purification Strategy can be applied (Figure 3). This strategy is used as an aid to the development of purification processes for therapeutic proteins in the pharmaceutical industry and is equally efficient as an aid when developing purification schemes in the research laboratory.
Polishing Achieve final high level purity
Intermediate purification Capture Preparation, extraction, clarification
Remove bulk impurities
Isolate, concentrate and stabilise
Step Fig. 3. Preparation and the Three Phase Purification Strategy.
Assign a specific objective to each step within the purification process. In the Three Phase Strategy a specific objective is assigned to each step. The purification problem associated with a particular step will depend greatly upon the properties of the starting material. Thus, the objective of a purification step will vary according to its position in the process i.e. at the beginning for isolation of product from crude sample, in the middle for further purification of partially purified sample, or at the end for final clean up of an almost pure product. The Three Phase Strategy ensures faster method development, a shorter time to pure product and good economy. In the capture phase the objectives are to isolate, concentrate and stabilise the target product. The product should be concentrated and transferred to an environment which will conserve potency/activity. At best, significant removal of other critical contaminants can also be achieved. 19
During the intermediate purification phase the objectives are to remove most of the bulk impurities, such as other proteins and nucleic acids, endotoxins and viruses. In the polishing phase most impurities have already been removed except for trace amounts or closely related substances. The objective is to achieve final purity. It should be noted that this Three Phase Strategy does not mean that all strategies must have three purification steps. For example, capture and intermediate purification may be achievable in a single step, as may intermediate purification and polishing. Similarly, purity demands may be so low that a rapid capture step is sufficient to achieve the desired result, or the purity of the starting material may be so high that only a polishing step is needed. For purification of therapeutic proteins a fourth or fifth purification step may be required to fulfil the highest purity and safety demands. The optimum selection and combination of purification techniques for Capture, Intermediate Purification and Polishing is crucial for an efficient purification process.
Selection and Combination of Purification Techniques Resolution
Speed
Capacity
Recovery
Every technique offers a balance between resolution, capacity, speed and recovery.
Minimise sample handling Minimise number of steps Use different techniques at each step Goal: Fastest route to a product of required purity. For any chromatographic separation each different technique will offer different performance with respect to recovery, resolution, speed and capacity. A technique can be optimised to focus on one of these parameters, for example resolution, or to achieve the best balance between two parameters, such as speed and capacity. A separation optimised for one of these parameters will produce results quite different in appearance from those produced using the same technique, but focussed on an alternative parameter. See, for example, the results shown on page 49 where ion exchange is used for a capture and for a polishing step. 20
Select a technique to meet the objectives for the purification step. Capacity, in the simple model shown, refers to the amount of target protein loaded during purification. In some cases the amount of sample which can be loaded may be limited by volume (as in gel filtration) or by large amounts of contaminants rather than the amount of the target protein. Speed is of the highest importance at the beginning of a purification where contaminants such as proteases must be removed as quickly as possible. Recovery becomes increasingly important as the purification proceeds because of the increased value of the purified product. Recovery is influenced by destructive processes in the sample and unfavourable conditions on the column. Resolution is achieved by the selectivity of the technique and the efficiency of the chromatographic matrix to produce narrow peaks. In general, resolution is most difficult to achieve in the final stages of purification when impurities and target protein are likely to have very similar properties. Every technique offers a balance between resolution, speed, capacity and recovery and should be selected to meet the objectives for each purification step. In general, optimisation of any one of these four parameters can only be achieved at the expense of the others and a purification step will be a compromise. The importance of each parameter will vary depending on whether a purification step is used for capture, intermediate purification or polishing. This will steer the optimisation of the critical parameters, as well as the selection of the most suitable media for the step. Proteins are purified using chromatographic purification techniques which separate according to differences in specific properties, as shown in Table 3. Table 3. Protein properties used during purification. Protein property
Technique
Charge
Ion exchange (IEX)
Size
Gel filtration (GF)
Hydrophobicity
Hydrophobic interaction (HIC), reversed phase (RPC)
Biorecognition (ligand specificity)
Affinity (AC)
Charge, ligand specificity or hydrophobicity
Expanded bed adsorption (EBA) follows the principles of AC, IEX or HIC
21
Choose logical combinations of purification techniques based on the main benefits of the technique and the condition of the sample at the beginning or end of each step. Minimise sample handling between purification steps by combining techniques to avoid the need for sample conditioning. A guide to the suitability of each purification technique for the stages in the Three Phase Purification Strategy is shown in Table 4. Technique
Main features
Capture
Intermediate
Polish
Sample Start condition
Sample End condition
IEX
high resolution high capacity high speed
★★★
★★★
★★★
low ionic strength sample volume not limiting
High ionic strength or pH change concentrated sample Low ionic strength concentrated sample specific elution conditions concentrated sample buffer exchanged (if required) diluted sample
HIC
good resolution good capacity high speed
★★
★★★
★
AC
high resolution high capacity high speed
★★★
★★★
★★
GF
High resolution using Superdex™ media
★
★★★
limited sample volume (<5% total column volume) and flow rate range
RPC
high resolution
★
★★★
requires organic solvents
high ionic strength sample volume not limiting specific binding conditions sample volume not limiting
in organic solvent, risk loss of biological activity
Table 4. Suitability of purification techniques for the Three Phase Purification Strategy
Avoid additional sample conditioning steps. The product should be eluted from the first column in conditions suitable for the start conditions of the next column. The start conditions and end conditions for the techniques are shown in Table 4. For example, if the sample has a low ionic strength it can be applied to an IEX column. After elution from IEX the sample will usually be in a high ionic strength buffer and can be applied to a HIC column (if necessary the pH can be adjusted and further salt can be added). In contrast, if sample is eluted from a HIC column, it is likely to be in high salt and will require dilution or a buffer exchange step in order to further decrease the ionic strength to a level suitable for IEX. Thus it is more straightforward to go from IEX to HIC than vice-versa. Ammonium sulphate precipitation is a common sample clarification and concentration step at laboratory scale and in this situation HIC (which requires high salt to enhance binding to the media) is ideal as the capture step. The salt concentration and the total sample volume will be significantly reduced after elution from the HIC column. Dilution of the fractionated sample or rapid buffer exchange using a Sephadex G-25 desalting column will prepare it for the next IEX or AC step. 22
GF is well suited for use after any of the concentrating techniques (IEX, HIC, AC, EBA) since the target protein will be eluted in a reduced volume and the components from the elution buffer will not affect the gel filtration separation (gel filtration is a non-binding technique with limited volume capacity and unaffected by buffer conditions). Selection of the final strategy will always depend upon specific sample properties and the required level of purification. Logical combinations of techniques are shown in Figure 4.
Crude sample or sample in high salt concentration Sample clarification*
Capture
GF desalt mode
GF desalt mode
AC
IEX
HIC IEX dilution may be needed
Intermediate
Polish
GF
GF desalt mode
GF
IEX
HIC
GF
GF
* Alternatively samples can be filtered and, if required, their ionic strength can be reduced by dilution.
Clear or very dilute samples
Capture
AC
IEX
Intermediate Purification Polishing
GF or RPC
GF or RPC
IEX
Precipitation (e.g. in high ionic strength)
HIC
Resolubilise
GF
Treat as for sample in high salt concentration
Fig. 4. Logical combinations of chromatographic steps.
For any capture step, select the technique showing the strongest binding to the target protein while binding as few of the contaminants as possible i.e. the technique with the highest selectivity and/or capacity for the protein of interest.
23
A sample is purified using a combination of techniques and alternative selectivities. For example, in an IEX-HIC-GF Three Phase Strategy the capture step selects according to differences in charge (IEX), the intermediate purification step according to differences in hydrophobicity (HIC) and the final polishing step according to differences in size (GF). Figure 5 shows a standard Three Phase strategy purification.
Capture by IEX Basic proteins STREAMLINE SP or SP Sepharose XL Suggested binding buffer: 20 mM sodium phosphate, pH 7 Suggested elution buffer: Binding buffer + 0.5 M NaCl Acidic proteins STREAMLINE DEAE or Q Sepharose XL Suggested binding buffer: 50 mM Tris.HCl, pH 8 Suggested elution buffer: Binding buffer + 0.5 M NaCl
Intermediate purification by HIC Phenyl Sepharose 6 Fast Flow (high sub) Suggested binding buffer: 50 mM sodium phosphate, pH 7 + 1.5 M ammonium sulphate Suggested elution buffer: 50 mM sodium phosphate, pH 7
Polishing by GF Superdex 75 prep grade or Superdex 200 prep grade Suggested buffer: as required by subsequent use
Fig. 5. A standard purification protocol.
If nothing is known about the target protein use IEX-HIC-GF. This combination of techniques can be regarded as a standard protocol.
Consider the use of both anion and cation exchange chromatography to give different selectivities within the same purification strategy. IEX is a technique which offers different selectivities using either anion or cation exchangers. The pH of the separation can be modified to alter the charge characteristics of the sample components. It is therefore possible to use IEX more than once in a purification strategy, for capture, intermediate purification or polishing. IEX can be used effectively both for rapid separation in low resolution mode during capture, and in high resolution mode during polishing in the same purification scheme. Figure 6 shows an example for the purification of cellulase in which advantage is taken of the alternative selectivities of anion and cation exchange to create a simple two step process.
24
Sample: Column: Flow: Buffer A: Buffer B. Gradient:
500 ml of T. reesei crude cellulases in buffer A, 2.5 mg Mono Q™ HR 5/5 1.0 ml/min 20 mM Tris-HCl, pH 7.6 A + 0.5 M NaCl 0% B for 4 min, 0-40% in 21 min, 40-100% B in 15 min
Peak 3 from step 1 Mono S™ HR 5/5 1.0 ml/min 20 mM acetate, pH 3.6 A + 0.2 M NaCl 0-100% B in 26 min
A280nm 0.5
A280nm 0.5
0
Sample: Column: Flow rate: Buffer A: Buffer B. Gradient:
10
(3)
20
0
30 Time (min)
(3)
10
20 Time (min)
Fig. 6. Two step purification of a cellulase.
Consider RPC for a polishing step provided that the target protein can withstand the run conditions. Reversed phase chromatography (RPC) separates proteins and peptides on the basis of hydrophobicity. RPC is a high selectivity (high resolution) technique, requiring the use of organic solvents. The technique is widely used for purity check analyses when recovery of activity and tertiary structure are not essential. Since many proteins are denatured by organic solvents, the technique is not generally recommended for protein purification where recovery of activity and return to a correct tertiary structure may be compromised. However, in the polishing phase, when the majority of protein impurities have been removed, RPC can be excellent, particularly for small target proteins which are not often denatured by organic solvents. If a purification is not intended for scale up (i.e. only milligram quantities of product are needed), use high performance, pre-packed media such as Sepharose High Performance (IEX, HIC), SOURCE™ (IEX, HIC), MonoBeads™ (IEX), or Superdex (GF) for all steps. Recommended media for a standard protocol Purification step
Media
Quantity
Code No.
Capture Capture Capture Capture Intermediate purification Polishing Polishing Sample clarification/conditioning Sample clarification/conditioning Sample clarification/conditioning
STREAMLINE SP STREAMLINE DEAE HiPrep™ 16/10 SP XL HiPrep 16/10 Q XL HiPrep Phenyl (high sub) HiLoad™ 16/60 Superdex 75 prep grade HiLoad 16/60 Superdex 200 prep grade Pre-packed PD-10 Column HiTrap Desalting HiPrep 26/10 Desalting
300 ml 300 ml 1 column 1 column 1 column 1 column 1 column 30 columns 5 columns 1 column
17-0993-01 17-0994-01 17-5093-01 17-5092-01 17-5095-01 17-1068-01 17-1069-01 17-0851-01 17-1408-01 17-5087-01
25
Sample Conditioning Although additional sample handling between purification steps should be avoided, it may be necessary to adjust the buffer conditions of an eluted product (pH, ionic strength and/or buffering ions) to ensure compatibility with the following purification technique. Sephadex G-25 is an ideal media for rapid desalting and pH adjustment by buffer exchange between purification steps. Sample volumes of up to 30%, or in some cases 40%, of the total column volume are loaded. In a single step, the sample is desalted, exchanged into a new buffer, and low molecular weight materials are removed. Figure 7 shows a typical desalt/buffer exchange separation. The high volume capacity and speed of this step enable very large sample volumes to be processed rapidly and efficiently. The high sample volume load results in a separation with minimal sample dilution. Sephadex G-25 is also used for rapid sample clean-up at laboratory scale.
A280 nm (mS/cm)
0.25
0.20 10.0
0.15
protein
salts
0.10 5.0 0.05
0.00 0.0
1.0
2.0
(min) Time
Fig 7. Buffer exchange of mouse plasma on HiPrep 26/10 Desalting.
Use pre-packed columns of Sephadex G-25 for rapid sample conditioning at laboratory scale, as shown in Table 5. Table 5. Pre-packed columns for rapid desalting and buffer exchange. Pre-packed column
Sample volume loading per run
Sample volume recovery per run
Code No.
HiPrep Desalting 26/10 HiTrap Desalting Fast Desalting PC 3.2/10 PD-10 Desalting
2.5 -15 ml 0.25 - 1.5 ml 0.05 - 0.2 ml 1.5 - 2.5 ml
7.5 1.0 0.2 2.5
17-5087-01 17-1408-01 17-0774-01 17-0851-01
26
-
20 ml 2.0 ml 0.3 ml 3.5 ml
Dilution can be used as an alternative to desalting before application to an ion exchange column.
Media for consideration:
Sephadex G-25 Gel filtration For fast group separations between high and low molecular weight substances. Typical flow velocities 60 cm/h (Sephadex G-25 SuperFine, Sephadex G-25 Fine), 150 cm/h (Sephadex G-25 Medium). In the following chapters Capture, Intermediate Purification and Polishing are discussed in more detail. Note:
cm/h: flow velocity (linear flow rate) = volumetric flow rate/cross sectional area of column.
27
Chapter 4
Capture Resolution
Speed
Capacity
Recovery
Remove damaging contaminants early Definition: Initial purification of the target molecule from crude or clarified source material. Goals: Rapid isolation, stabilisation and concentration. Use a high capacity, concentrating technique to reduce sample volume, to enable faster purification and to allow the use of smaller columns. Focus on robustness and simplicity in the first purification step. Do not try to solve all problems in one step when handling crude material. In the capture phase, the objective is to isolate, concentrate and stabilise the target product efficiently by optimising speed and capacity. The product is concentrated and transferred to an environment which will conserve activity. Capture is often a group separation using a step elution on ion exchange or affinity chromatography. Ideally, removal of critical contaminants is also achieved. It is sometimes possible to achieve a high level of purification if a highly selective affinity media is used. Binding capacity for the protein in the presence of the impurities will be one of the most critical parameters to optimise and reduce the scale of work. For example, when ion exchange chromatography is used as a capture step, the goal is to adsorb the target protein quickly from the crude sample and isolate it from critical contaminants such as proteases and glycosidases. Conditions are selected to avoid binding of contaminants so that the capacity for the target protein is maximised. High speed may be required to reduce sample application time, particularly if proteolysis or other destructive effects threaten the integrity of the target protein. Transfer to a step elution during method development to increase speed and capacity of the capture step. 29
The most common technique for a capture step is ion exchange chromatography (IEX) which has high binding capacity. IEX media are resistant to harsh cleaning conditions which may be needed after purification of crude samples. Typically proteins are eluted from an IEX column using a salt gradient. However, during method development, a transfer to a step elution will give a simple, robust separation with a shorter run time and decreased buffer consumption. It is often possible to use high sample loadings since the focus is not on resolution (high sample loadings will decrease resolution). High speed and capacity and low buffer consumption are particularly advantageous for large scale purification, as shown in Figure 8. A280 nm
%B
a 100
3.0 EGF 2.0 150
Column:
BPG™ 300/500 packed with Phenyl Sepharose 6 Fast Flow (high sub) EGF in yeast supernatant ammonium sulphate added to 0.5 M Sample load: 80 L containing 2.56 g EGF Starting buffer: 20 mM sodium phosphate, pH 7.0 + 0.5 M ammonium sulphate Elution buffer: 20 mM sodium phosphate, pH 7.0 Flow loading: 210 L/h, 300 cm/h Flow elution: 42 L/h, 60 cm/h
Sample:
1.0
15
0 0
150 Volume (I)
100
50
a) Purification of recombinant epidemal growth factor (EGF) - capture step. A280 nm
Conductivity
b
Column: Adsorbent: Sample:
Buffer A: Buffer B: Flow: Gradient: Eluate: Spec. act.
0
5.0
10.0
15.0
20.0
INdEX™ 70 (70 mm i.d.) Q Sepharose XL, 385 mL bed volume Recombinant a-amylase produced in E. coli, homogenized, 2.2 L diluted in distilled water to 15.4 L, 7.2 mS/cm, 10 mM CaCl2, centrifuged 20 mM Tris-HCl, pH 8, 10 mM CaCl2 20 mM Tris-HCl, pH 8, 1 M NaCl, 10 mM CaCl2 300 cm/h, 12 L/h 20 bed volumes 0-1 M NaCl 1.48 L, 3.8 bed volumes a-amylase: 6420 U/L
Volume (l)
b) Pilot scale purification of recombinant a-amylase from E. coli -capture step. A 280 nm
Column:
c
Sample: Sample volume: Starting buffer: Elution buffer: Flow:
2.0
1.5
1.0
0.5
0.0 0
200
400
600
Volume (m)
c) Purification of IgG2a from clarified cell culture - capture step.
Fig. 8. Examples of capture steps. 30
rProtein A Sepharose Fast Flow, XK 16/20, bed height 4.8 cm (9.6 mL) clarified cell culture containing IgG2a 600 mL containing 87.6 mg IgG2a 20 mM sodium phosphate, pH 7.0 20 mM sodium citrate, pH 4.0 5 mL/min (150 cm/h)
For large scale capture, throughput will often be the focus during method development. It is important to consider all aspects: sample extraction and clarification, sample loading capacity, flow rate during equilibration, binding, washing, elution and cleaning, and the need for cleaning-in-place procedures. In principle, a capture step is designed to maximise capacity and/or speed at the expense of some resolution. However, there is usually significant resolution and purification from molecules which have significant physicochemical differences compared to the target protein. Recovery will be of concern in any preparative situation, especially for production of a high value product, and it is important to assay for recovery during optimisation of the capture step. Examples of capture steps are shown on page 30. Media for capture steps should offer high speed and high capacity.
Sepharose XL (IEX) For capture steps handling crude mixtures at laboratory and process scale. Fast removal and a combination of high capacity and good resolution at high flow rates are the main characteristics. Recommended flow velocity is 100-500 cm/h. Particle size: 90 µM. Available in pre-packed columns and as bulk media.
Sepharose Big Beads (IEX) For capture steps handling viscous samples or very large sample volumes. Sepharose Big Beads are for the capture step in processes where high sample viscosity precludes the use of ion exchange media with smaller bead sizes. Recommended flow velocity is up to 300 cm/h. This medium should be chosen when fast adsorption is required and resolution is of less importance. The flow characteristics of Big Beads may also be useful when processing very large volumes under conditions requiring an extremely high volumetric throughput. Flow velocities in these situations can exceed 1000 cm/h. Particle size: 200 µM. Available as bulk media.
STREAMLINE (IEX, AC, HIC) For sample clean-up and capture direct from crude sample. STREAMLINE adsorbents are designed to handle feed directly from both fermentation homogenate and crude feedstock from cell culture/fermentation at flow velocities of 200 - 500 cm/h, according to type and application. Particle size: 200 µM. Available as bulk media.
31
Other media for consideration:
Sepharose Fast Flow (IEX, HIC) These media offer the widest range of selectivities and an excellent alternative for purification of crude samples at any scale. They offer a fast separation combined with good resolution. Recommended flow velocity is 100-300 cm/h. Particle size: 90 µM. Available in pre-packed columns and as bulk media. Note:
cm/h: flow velocity (linear flow rate) = volumetric flow rate/cross sectional area of column.
If a purification is not intended for scale up (i.e. milligram quantities of product are needed), use high performance media such as Sepharose High Performance (IEX, HIC) or MonoBeads (IEX), or SOURCE (IEX, HIC). All these media are available in pre-packed columns. For microscale purification use MonoBeads or MiniBeads™ (IEX), Phenyl Superose™ (HIC) or NHS-activated Superose (AC) columns. For 'one time' purification or with a readily available sample, sacrifice yield for purity by taking a narrow cut from a chromatographic peak during the first purification step. Use HiTrap Ion Exchange and HiTrap HIC Test Kits for media screening and simple method optimisation. If the starting material is reasonably clean, a single step purification on highest resolution MonoBeads (IEX) may be sufficient to achieve required purity at laboratory scale. If a biospecific ligand is available, consider using affinity chromatography as the capture step. If the media is to be used routinely, ensure that any contaminants from the crude sample can be removed by column regeneration procedures which do not damage the affinity ligand. AC will give a highly selective capture step to improve resolution from contaminants, but speed may need to be reduced to maintain a high binding capacity. If the starting material is reasonably clean a single step purification on a prepacked HiTrap affinity column may be sufficient to achieve required purity at the milligram scale, as shown in Figure 9. HiTrap affinity columns are available in a wide range of selectivities (see Table 6, page 34). If the starting material is concentrated, has a low volume and there is no intention to scale up, Superdex gel filtration media can offer a mild first step, requiring little or no optimisation. Conversely, gel filtration is not suitable in a typical capture step where the sample volume is large or dilute or will be scaled up. 32
A 280 nm
A 405 nm
Column: Sample:
HiTrap Chelating, 1 ml 5 ml cytoplasmic extract containing (His)-tagged glutathione-S-transferase Binding buffer: 20 mM phosphate buffer, 0.5 M NaCl, 20 mM imidazole, pH 7.4 Elution buffer: 20 mM phosphate buffer, 0.5 M NaCl, 500 mM imidazole, pH 7.4 Flow: 2 mL/min (312 cm/h)
Fig. 9. HiTrap chelating column used to purify histidine tagged glutathione-S-transferase from cytoplasmic extract.
33
Table 6. Recommended HiTrap affinity columns for laboratory scale separation. Application
HiTrap column
Code No.
Quantity/ components
Approximate binding capacity per ml gel
Isolation of immunoglobulins IgG classes, fragments and subclasses
HiTrap rProtein A
17-5079-01 17-5080-01 17-5029-02
5 x 1 ml 1 x 5 ml 2 x 1 ml
human IgG 50 mg/ml
IgG classes, fragments and subclasses
HiTrap Protein A
17-0402-01 17-0402-03 17-0403-01
5 x 1 ml 2 x 1 ml 1 x 5 ml
human IgG 20 mg/ml
IgG classes, fragments and subclasses including human IgG3 strong affinity to monoclonal mouse IgG1 and rat IgG
HiTrap Protein G
17-0404-01 17-0404-03 17-0405-01
5 x 1 ml 2 x 1 ml 1 x 5 ml
human IgG 25 mg/ml
Monoclonal and polyclonal IgG from ascites fluid, serum and cell culture supernatant
MAbTrap™ GII
17-1128-01
HiTrap Protein G column (1 ml), accessories, pre-made buffers for 10 purifications
as above
Mouse recombinant Single chain antibody Fragment variable (ScFv) produced in E.Coli
RPAS Purification Module
17-1362-01
HiTrap Anti-E column, accessories, pre-made buffers for 20 purifications
0.17 mg ScFv/5 ml
IgY antibodies from egg yolk
HiTrap IgY Purification
17-5111-01
1 x 5 ml
IgY 20 mg/ml
IgM
HiTrap IgM Purification
17-5110-01
5 x 1 ml
IgM 5 mg/ml
Group Specific Media: Glycoproteins or polysaccharides Specificity: branched mannoses, carbohydrates withterminal mannose or glucose(a Man> a Glc> GlcNAc)
HiTrap Con A
17-5105-01
5 x 1 ml
transferrin 4 mg/ml
Specificity: branched mannoses with fucose linked a(1,6) to the N- acetylglucosamine, (a Man> a Glc> GlcNAc) N- acetylglucosamine binding lectins
HiTrap Lentil Lectin
17-5106-01
5 x 1 ml
thyroglobulin 4 mg/ml
34
Application
HiTrap column
Code No.
Quantity/ components
Approximate binding capacity per ml gel
Specificity: Terminal ß -galactose, (Gal ß 1,3 GalNAc > a and ß Gal)
HiTrap Peanut Lectin
17-5108-01
5 x 1 ml
asialofetuin 3 mg/ml
Specificity: chitobiose core of N-linked oligosaccharides, [GlcNAc (ß 1,4GlcNAc) 1- 2 > ß GlcNAc]
HiTrap Wheat Germ Lectin
17-5107-01
5 x 1 ml
ovomuroid 4 mg /ml
Specificity: as listed for each column
HiTrap Lectin Test Kit
17-5109-01
4 x 1 ml columns of HiTrap Con A HiTrap Lentil Lectin HiTrap Peanut Lectin HiTrap Wheat Germ Lectin
as listed above
Group Specific Media Various Nucleotiderequiring enzymes, coagulation factors, DNA binding proteins, a2-macro-globulin
HiTrap Blue
17-0412-01 17-0413-01
5 x 1 ml 1 x 5 ml
HSA 20 mg/ml
Proteins and peptides with exposed amino acids: His (Cys, Trp) e.g. a-2-macro-globulin and interferon
HiTrap Chelating
17-0408-01 17-0409-01
5 x 1 ml 1 x 5 ml
(His)6-tagged protein (27.6 kD) 12 mg /ml
Histidine-tagged fusion proteins
HisTrap™
17-1880-01
HiTrap Chelating column (1 ml), accessories, pre-made buffers
as above
Biotin and biotinylated substances
HiTrap Streptavidin
17-5112-01
5 x 1 ml
biotinylated BSA 6 mg/ml
Coagulation factors, lipoprotein lipases, steroid receptors, hormones, DNA binding proteins, interferon, protein syntheses factors
HiTrap Heparin
17-0406-01 17-0407-01
5 x 1 ml 1 x 5 ml
ATIII (bovine) 3 mg/ml
Matrix for preparation of affinity media Coupling of primary amines
HiTrap NHS-activated
17-0716-01 17-0717-01
5 x 1 ml 1 x 5 ml
ligand specific
Recommended separation conditions All HiTrap columns are supplied with a detailed protocol to ensure optimum results Maximum flow rates: HiTrap 1 ml column: up to 4 ml/min HiTrap 5 ml column: up to 20 ml/min
35
For crude, large volume samples containing particles, consider using STREAMLINE expanded bed adsorption STREAMLINE expanded bed adsorption is particularly suited for large scale recombinant protein and monoclonal antibody purification. STREAMLINE adsorbents are specially designed for use in STREAMLINE columns. The technique requires no sample clean up and so combines sample preparation and capture in a single step. As shown in Figure 10, crude sample is applied to an expanded bed of STREAMLINE media, target proteins are captured whilst cell debris, particulate matter, whole cells, and contaminants pass through. Flow is reversed and the target protein is desorbed in the elution buffer.
System: Column: Medium: Sample:
Buffer A: Buffer B: Flow:
BioProcess™ Modular STREAMLINE 200 (i.d. 200 mm) STREAMLINE DEAE, 4.7 L 4.7 kg of cells were subjected to osmotic shock and suspended in a final volume of 180 L 50 mM Tris buffer, pH 7.4, before application onto the expanded bed. 50 mM Tris buffer, pH 7.4 50 mM Tris, 0.5 M sodium chloride, pH 7.4 400 cm/h during sample application and wash 100 cm/h during elution
A 280 nm
100 1.0 80
Height of expanded bed (cm)
2.0
60 40 20 Sample application
100
Washing Buffer A
200
Elution Buffer B
260 Volume (L)
Fig. 10. Purification of a recombinant protein Pseudomonas aeruginosa exotoxin A capture step.
See Chapter 3, page 23 for suggested logical combinations of techniques for Capture, Intermediate Purification and Polishing.
36
Chapter 5
Intermediate Purification Resolution
Speed
Capacity
Recovery
Use different techniques at each step Minimise number of steps Definition: Further removal of bulk contaminants. Goal: Purification and concentration. In the intermediate purification phase the focus is to separate the target protein from most of the bulk impurities such as other proteins, nucleic acids, endotoxins and viruses. An ability to resolve similar components is of increased importance. The requirements for resolution will depend upon the status of the sample produced from the capture step and the purity requirements for the final product. Capacity will still be important to maintain productivity. Speed is less critical in intermediate purification since the impurities causing proteolysis or other destructive effects should have been removed, and sample volume should have been reduced, in the capture step. The optimal balance between capacity and resolution must be defined for each specific application. This then decides how the separation conditions should be optimised during method development. The technique must give a high resolution separation. Elution by a continuous gradient will usually be required. As in a capture step, selectivity during sample adsorption will be important, not only to achieve high binding capacity, but also to contribute to the purification by achieving a further separation during sample application. However, in contrast to a capture step, selectivity during sample desorption from the column is also important and is usually achieved by applying a more selective desorption principle, such as a continuous gradient or a multi-step elution procedure, as shown in Figure 11. Examples of Intermediate Purification steps are shown on page 38. Use a technique with a complementary selectivity to that which was used for the capture step. 37
a) Purification of recombinant protein Pseudomonas aeruginosa exotoxin A -intermediate purification step.
Column: Medium: Sample:
A280 nm 0.50
Buffer A: Buffer B: Gradient: Flow:
0.40 0.30
FineLINE™ 100 (i.d. 100 mm) SOURCE 30Q, 375 mL (50 mm bed height) from the previous pool, diluted 1 to 3 with distilled water 1.5 L/cycle were applied 20 mM phosphate, pH 7.4 Buffer A + 1.0 M sodium chloride 0 to 50% B, 20 column volumes 600 cm/h
0.20 0.10 Pool 0.00 0.0
2.0
4.0
6.0
8.0
10.0 12.0 Volume (L)
b) Purification of recombinant Annexin V-intermediate purification step. A280 nm
Column: Buffer A: Buffer B: Sample: Gradient: Flow:
XK 16/20 Butyl Sepharose 4 Fast Flow 20 mM Sodium phosphate, pH 7.0, 1 M (NH4)2SO4 20 mM Sodium phosphate, pH 7.0, Partically expressed Annexin V expressed in E. coli, 5 ml 0 to 50% B, 20 column volumes 100 cm/h
Annexin V
0
60
Time (min)
Fig. 11. Intermediate purification steps.
Media for intermediate purification should offer high capacity and high resolution with a range of complementary selectivities.
SOURCE (IEX) For fast, high resolution and high capacity intermediate purification. SOURCE media are for high throughput, high capacity and high resolution purification. They are the natural choice at laboratory scale. Frequently, if filtered samples are used, the intermediate purification step can be combined with the capture step. A flow velocity up to 2000 cm/h is possible. SOURCE 30 is also a good choice at large scale for intermediate purification. Particle size: 15 µM. Available in pre-packed columns and as bulk media. Particle size: 30 µM. Available as bulk media.
38
Sepharose High Performance (IEX, HIC, AC) For high resolution and high capacity intermediate purification. These media are ideal for intermediate purification at large scale and should be used when resolution and capacity are a priority. Recommended flow velocity is up to 150 cm/h. Particle size: 34 µM. Available in pre-packed columns and as bulk media.
Sepharose Fast Flow (IEX, HIC, AC) Proven in large scale production of pharmaceuticals during intermediate purification steps. These media are the accepted standard for general applications in the laboratory and at large scale. They are available in the widest range of techniques and selectivities and are able to withstand harsh cleaning-in-place conditions. They offer a fast separation combined with good resolution. Recommended flow velocity is 100-300 cm/h. Particle size: 90 µM. Available in pre-packed columns and as bulk media. Note: cm/h: flow velocity (linear flow rate) = volumetric flow rate/cross sectional area of column. Use HiTrap IEX, HiTrap HIC and RESOURCE HIC Test Kits for media screening and simple method optimisation, as shown in the table below. Kit
Code No.
HiTrap IEX Test Kit HiTrap HIC Test Kit RESOURCE HIC Test Kit
17-6001-01 17-1349-01 17-1187-01
If a purification is not intended for scale up (i.e. only milligram quantities of product are needed), use high performance media such as Sepharose High Performance (IEX, HIC) MonoBeads (IEX) or SOURCE 15 (IEX, HIC). For microscale purification use Monobeads, MiniBeads (IEX) or Phenyl Superose PC (HIC) columns.
See Chapter 3, page 23 for suggested logical combinations of techniques for Capture, Intermediate Purification and Polishing.
39
Chapter 6
Polishing Resolution
Speed
Capacity
Recovery
Use different techniques at each stage Definition: Final removal of trace contaminants. Adjustment of pH, salts or additives for storage. Goal: End product of required high level purity. In the polishing phase the focus is almost entirely on high resolution to achieve final purity. Most contaminants and impurities have already been removed except for trace impurities such as leachables, endotoxins, nucleic acids or viruses, closely related substances such as microheterogeneous structural variants of the product, and reagents or aggregates. To achieve resolution it may be necessary to sacrifice sample load or even recovery (by peak cutting). Recovery of the final product is also a high priority and a technique must be selected which ensures the highest possible recovery. Product losses at this stage are more costly than in earlier stages. Ideally the product should be recovered in buffer conditions ready for the next procedure. The technique chosen must discriminate between the target protein and any remaining contaminants The high resolution required to achieve this discrimination is not always reached by using a high selectivity technique alone, but usually requires selection of a high efficiency media with small, uniform bead sizes.
40
A280 nm monomeric ZZ-Brain IGF
Column:
I
a 0.01
Sample: Sample load: Buffer:
I
Flow: 0.005
VO
I Fraction 1
0
1
XK 16/60 packed with Superdex 75 prep grade partly purified ZZ-brain IGF 1.0 ml 0.3 M ammonium acetate pH 6.0 0.5 ml/min (15 cm/h)
Vt
2
3 I4
5
I
6
2
I
3
4 Time (h)
Fig 12. Separation of dimers and multimers -polishing step.
Typically, separations by charge, hydrophobicity or affinity will have already been used so that a high resolution gel filtration is ideal for polishing. The product is purified and transferred into the required buffer in one step and dimers or aggregates can often be removed, as shown in Figure 12. To remove contaminants of similar size, an alternative high resolution technique using elution with shallow gradients is usually required, as shown in Figure 13.
A280 nm
%B 100
0.10
Sample: Column: Buffer A: Buffer B: Gradient: Flow:
62.5 ml EGF pool after IEX purification SOURCE 15 RPC, 35 x 100 mm 0.05% TFA, 5% acetonitrile in water 0.05% TFA, 80% acetonetrile in water 0–100% B in 40 column volumes 50 ml/min, (300 cm/h)
80 0.08 60
0.06
0.04
40
0.02
20
0 0
20.0
40.0
0 60.0 Time (min)
Fig 13. Final polishing step of recombinant epidermal growth factor, using reversed phase chromatography. Method developed on pre-packed RESOURCE™ RPC and scaled up on SOURCE 15 RPC.
Gel filtration is also the slowest of all chromatography techniques and the size of the column determines the volume of sample that can be applied. It is therefore most logical to use gel filtration after techniques which reduce sample volume so that smaller columns can be used. 41
When scaling up a purification it is important to verify that the high resolution achieved from the laboratory scale polishing step is maintained when applying preparative sample volumes to large scale columns. Media for polishing steps should offer highest possible resolution.
Superdex (GF) High productivity gel filtration media for polishing. Superdex media are high resolving gel filtration media for short run times and good recovery. Superdex is the first choice at laboratory scale and Superdex prep grade for large scale applications. Typical flow velocity is up to 75 cm/h. Particle size Superdex: 13 µM. Available in pre-packed columns. Particle size Superdex prep grade: 34 µM. Available in pre-packed columns and as bulk media.
MonoBeads (IEX) Media for polishing at laboratory scale when highest resolution is essential. These media offer high capacity and highest resolution separations at laboratory scale. Typical flow velocity is 150-600 cm/h. Particle size: 10 µM. Available in pre-packed columns.
SOURCE 15 (IEX, HIC, RPC) Media for rapid high resolution polishing. SOURCE 15 are for rapid high capacity, high resolution separations for laboratory and large scale applications. The pore structure of these media enables maintained resolution at high loading and high flow rates. Recommended flow velocity is 150-1800 cm/h. Particle size: 15 µM. Available in pre-packed columns and as bulk media.
Sephasil Protein/Sephasil Peptide (RPC) Media for high resolution polishing and analysis. Sephasil media are silica-based RPC media with three different selectivities, C4, C8 or C18. Recommended flow velocity is 180-1450 cm/h (particle size dependent). Particle size: 5 or 12 µm. Available in pre-packed columns and as bulk media.
42
Other media for consideration.
SOURCE 30 (IEX) SOURCE 30 media are for high throughput, high capacity and high resolution purification. However, these media can be an alternative choice for polishing offering a flow velocity of up to 2000 cm/h at large scale. Particle size: 30 µM. Available as bulk media. Note:
cm/h: flow velocity (linear flow rate) = volumetric flow rate/cross sectional area of column. For microscale purification use Superdex PC (GF), MiniBeads (IEX) or Phenyl Superose PC (HIC) columns.
See Chapter 3, page 23 for suggested logical combinations of techniques for Capture, Intermediate Purification and Polishing.
43
Chapter 7
Examples of Protein Purification Strategies The Three Phase Purification Strategy has been successfully applied to many purification schemes from simple laboratory scale purification to large, industrial scale production. Examples highlighted in this chapter demonstrate applications in which a standard protocol was applied i.e. sample extraction and clarification, capture, intermediate purification and polishing. There are also examples where strategies were developed requiring even fewer steps, by following the general guidelines for protein purification given in this handbook and selecting the most appropriate technique and media to fulfil the purification objectives. In most of these examples methods were developed using ÄKTAdesign chromatography systems.
Example 1.
Three step purification of a recombinant enzyme This example demonstrates one of the most common purification strategies: IEX for capture, HIC for intermediate purification and GF for the polishing step. The objective of this purification was to obtain highly purified protein for crystallisation and structural determination. A more detailed description of this work can be found in Application Note 18-1128-91.
Target Molecule Deacetoxycephalosporin C synthase (DAOCS), an oxygen-sensitive enzyme.
Source Material Recombinant protein over-expressed in soluble form in the cytoplasm E. coli bacteria
45
Sample Extraction and Clarification Cells were suspended in lysis buffer, 50 mM Tris-HCl, 1 mM EDTA, 2 mM DTT, 0.2 M benzamidine-HCl, 0.2 mM PMS, pH 7.5 and lysed using ultrasonication. Streptomycin sulphate (1%) and polyethyleneimine (0.1%) were added to precipitate DNA. The extract was clarified by centrifugation. EDTA, DTT, Benzamidine-HCl and PMSF were used in the lysis buffer to inhibit proteases and minimise damage to the oxygen sensitive-enzyme. Keeping the sample on ice also reduced protease activity.
Capture The capture step focused on the rapid removal of the most harmful contaminants from the relatively unstable target protein. This, together with the calculated isoelectric point of DAOCS (pI = 4.8), led to the selection of an anion exchange purification. A selection of anion exchange columns, including those from HiTrap IEX Test Kit, were screened to select the optimum medium (results not shown) before using a larger column for the optimisation of the capture step. Q Sepharose XL, a high capacity medium, well suited for capture, was chosen. As shown in Figure 14, optimisation of the capture step allowed the use of a step elution at high flow rate to speed up the purification. This was particularly advantageous when working with this potentially unstable sample. System: Column: Sample: Sample volume: Buffer A: Buffer B: Flow: A 280 nm
ÄKTAFPLC™ HiPrep 16/10 Q XL Clarified E. coli extract 40 ml 50 mM Tris-HCl, 1 mM EDTA, pH 7.5; 2 mM DTT, 0.2 M benzamidine-HCl, 0.2 mM PMSF A + 1.0 M NaCl 10 ml/min (300 cm/h)
mS/cm A 280 nm
400
mS/cm
A 280 nm
mS/cm
80 3000
300
80
60 2000
200
1000
1000
0
100
200
300
ml
20
20
0
0
40
40
20 0
60
2000
40 100
80
3000
60
0 0
100
200 ml
0
0 0
50
100
150
200 ml
Fig. 14. Capture using IEX and optimisation of purification conditions. The elution position of DAOCS is shaded.
46
Intermediate Purification Hydrophobic interaction chromatography (HIC) was selected because the separation principle is complementary to ion exchange and because a minimum amount of sample conditioning was required. Hydrophobic properties are difficult to predict and it is always recommended to screen different media. The intermediate purification step was developed by screening pre-packed hydrophobic interaction media (RESOURCE HIC Test Kit) to select the optimum medium for the separation (results not shown). RESOURCE ISO was selected on the basis of the resolution achieved. In this intermediatestep, shown in Figure 15, the maximum possible speed for separation was sacrificed in order to achieve higher resolution and allow significant reduction of remaining impurities. System: Column: Sample: Sample volume: Buffer A:
Buffer B: Gradient: Flow:
ÄKTAFPLC SOURCE 15ISO, packed in HR 16/10 column DAOCS pool from HiPrep 16/10 Q XL 40 ml 1.6 M ammonium sulphate, 10% glycerol, 50 mM Tris-HCl, 1 mM EDTA, 2 mM DTT, 0.2 mM benzamidine-HCl, 0.2 mM PMSF, pH 7.5 50 mM Tris-HCl, 10% glycerol, 1 mM EDTA, 2 mM DTT, 0.2 mM benzamidine-HCl, 0.2 mM PMSF, pH 7.5 0–16% B in 4 CV, 16–24% B in 8 CV, 24–35% B in 4 CV, 100% B in 4 CV 5 ml/min (150 cm/h)
A 280 nm
400 300 200 100 0 0
100
200
ml
Fig. 15. Intermediate purification using HIC.
Polishing The main goal of the polishing step was to remove aggregates and minor contaminants and transfer the purified sample into a buffer suitable for use in further structural studies. Superdex 75 prep grade, a gel filtration media giving high resolution at relatively short separation times, was selected since the molecular weight of DAOCS (34500 kDa) is within the optimal separation range for this medium. Figure 16 shows the final purification step.
47
System: Column: Sample: Sample volume: Buffer: Flow:
ÄKTAFPLC HiLoad 16/60 Superdex 75 prep grade Concentrated DAOCS pool from SOURCE 15ISO 3 ml 100 mM Tris-HCl, 1 mM EDTA, 2 mM DTT, 0.2 mM benzamidine-HCl, 0.2 mM PMSF, pH 7.5 1 ml/min (30 cm/h)
A 280 nm
1000 800 600 400 200 0 0
20
40
60
80
100
ml
Fig. 16. Polishing using gel filtration.
Analytical assays Figure 17 shows the analysis of collected fractions by SDS-PAGE and silver staining using Multiphor™ II or PhastSystem™, following the separation and staining protocols supplied with the instruments.
1
2
3
4
5
Lane 1, 6:
LMW Marker Kit.
Lane 2:
Cell homogenate
Lane 3:
DAOCS pool from Q Sepharose XL
Lane 4:
DAOCS pool from SOURCE 15ISO
Lane 5:
DAOCS pool from Superdex 75 pg
6
Fig. 17. Analysis of purification steps using SDS PAGE.
The final product was used successfully in X-ray diffraction studies as shown in Figure 18.
Fig. 18. Crystals (a), diffraction pattern (b) and high resolution electron density map (c) of purified DAOCS. 48
Example 2.
Three step purification of a recombinant antigen binding fragment This example demonstrates a three stage purification strategy in which the same purification principle is used in two different modes in the capture and polishing step : IEX for capture, HIC for intermediate purification and IEX for the polishing step. The objective of this purification was to scale up the purification for use as a routine procedure. A more detailed description of this work can be found in Application Note 18-1111-23.
Target Molecule Recombinant antigen binding fragment (Fab) directed against HIV gp-120.
Source Material The anti-gp 120 Fab was expressed in the periplasm of the E. coli strain BM170 MCT61. E. coli pellets were stored frozen after being harvested and washed once.
Sample Extraction, Clarification and Capture Thawed cells were lysed with sucrose. The lysate was treated with DNase in the presence of 2 mM MgCl2 at pH 7.5, before the capture step. The Fab fragment was captured from non-clarified homogenate by using expanded bed adsorption with STREAMLINE SP (cation exchanger). Expanded bed adsorption was chosen because the target protein was captured directly from the crude homogenate in a single step, without the need for centrifugation or other preparatory clean-up steps. The technique is well suited for large scale purification. The result of the capture step is shown in Figure 19. The Fab fragment is concentrated and transferred rapidly into a stable environment, using a step elution.
49
Column: Adsorbent: Sample: Buffer A: Buffer B: Flow:
STREAMLINE 200 (i.d. 200 mm) STREAMLINE SP, 4.6 L 60 L high pressure homogenized E. coli suspension 50 mM sodium acetate, pH 5.0 50 mM sodium acetate, pH 5.0, 1 M NaCl 300 cm/h during sample application and wash 100 cm/h during elution
A280 nm 2.0
1.0
Sample application
50
Washing, Buffer A
100
Elution, Buffer B Pool
150
5 10 15 Volume (litres)
Fig. 19. Capture step using expanded bed adsorption.
Intermediate Purification Hydrophobic interaction chromatography (HIC) was selected because the separation principle is complementary to ion exchange and because a minimum amount of sample conditioning was required since the sample was already in a high salt buffer after elution from STREAMLINE SP. Hydrophobic properties are difficult to predict and it is always recommended to screen different media. A HiTrap HIC Test Kit (containing five 1 ml columns pre-packed with different media suitable for production scale) was used to screen for the most appropriate medium. Buffer pH was kept at pH 5.0 to further minimise the need for sample conditioning after capture. Results of the media screening are shown in Figure 20.
50
System: Sample: Columns:
Buffer A: Buffer B: Gradient: Flow:
ÄKTAexplorer Fab fraction from STREAMLINE SP, 2 ml HiTrap HIC Test Kit (1 ml columns), Phenyl Sepharose High Performance, Phenyl Sepharose 6 Fast Flow (low sub), Phenyl Sepharose 6 (high sub), Butyl Sepharose 4 Fast Flow, Octyl Sepharose 4 Fast Flow 1 ml (NH4)2SO4, 50 mM NaAc, pH 5.0 50 mM NaAc, pH 5.0 20 column volumes 2 ml/min (300 cm/hr)
A280nm
Conductivity (mS/cm)
400
150
Conductivity
300 100 200
Fab
50
100
0 0 0.0
10.0
Time (min)
Fig. 20. HIC media scouting using HiTrap HIC Test Kit.
Phenyl Sepharose 6 Fast Flow (high sub) was selected since the medium showed excellent selectivity for the target protein thereby removing the bulk contaminants. Optimisation of elution conditions resulted in a step elution being used to maximise the throughput and the concentrating effect of the HIC purification technique. Figure 21 shows the optimised elution and the subsequent scale up of the intermediate purification step.
51
System: Sample: Columns: Buffer A: Buffer B: Gradient: Flow:
ÄKTAexplorer Fab fraction from STREAMLINE SP, 80 ml Phenyl Sepharose 6 Fast Flow (high sub) in XK 16/20 (10 cm bed height) 1 M (NH4)2SO4, 50 mM NaAc, pH 5.0 50 mM NaAc, pH 5.0 Step gradient to 50% B 5 ml/min
System: Sample: Columns: Buffer A: Buffer B: Gradient: Flow:
Conductivity (mS/cm)
A280 nm
Conductivity (mS/cm)
A280 nm
ÄKTAexplorer Fab fraction from STREAMLINE SP, 800 ml Phenyl Sepharose 6 Fast Flow (high sub) in XK 50/20 1 M (NH4)2SO4, 50 mM NaAc, pH 5.0 50 mM NaAc, pH 5.0 Step gradient to 50% B Equilibration: 100 ml/min Loading and elution: 50 ml/min
b) a)
2.00
2.00 100
100
1.00
1.00 50
50
0 0
50
100
150
200
0
250 Volume (ml)
0
500
1000
1500
2000
2500 Volume (ml)
Fig. 21. Intermediate purification using HIC: optimisation and scale-up.
Polishing Gel filtration was investigated as the natural first choice for a final polishing step to remove trace contaminants and transfer the sample to a suitable storage conditions. However, in this example, gel filtration could not resolve a 52 KDa contaminant from the 50 KDa Fab fragment (results not shown). As an alternative another cation exchanger SOURCE 15S was used. In contrast to the cation exchange step at the capture step, the polishing cation exchange step was performed using a shallow gradient elution on a media with a small, uniform size (SOURCE 15 S) to give a high resolution result, as shown in Figure 22.
52
System: Sample: Columns: Buffer A: Buffer B: Gradient: Flow:
ÄKTAexplorer Fab fraction from HIC separation 15 ml eluate diluted 7.5/100 RESOURCE S 6 ml 50 mM NaAc, pH 4.5 50 mM NaAc, pH 4.5, 1M NaCl 50 column volumes 18.3 ml/min
Conductivity (mS/cm)
A280 nm 100
80.0
80 60.0
60 40.0 Active Fab
40
20.0 20
0
100
200 Volume (ml)
Fig. 22. Optimised Fab polishing step.
Analytical assays Collected fractions were separated by SDS-PAGE and stained by Coomassie using PhastSystem, following the separation and staining protocols supplied with the instrument. Fab was measured by a goat-anti-human IgG Fab ELISA, an anti-gp120 ELISA and an in vitro assay which measured the inhibition of HIV-1 infection of T-cells. Nucleic acid was routinely monitored by measuring A260/A280. The correlation of a high A260/A280 ratio (>1) with the presence of DNA was verified for selected samples by agarose gel electrophoresis and EtBr staining. Endotoxin determination employed a kinetic chromogenic Limulus assay (COAMATIC Chromogenics AB, Mölndal, Sweden).
53
Example 3.
Two step purification of a monoclonal antibody This example demonstrates the effectiveness of using a high selectivity affinity chromatography technique as a capture step, since only a second gel filtration polishing step was needed to achieve the required level of purity. The objective of this work was to produce an efficient, routine procedure for monoclonal antibody purification. A more detailed description of this work can be found in Application Note 18-1128-93.
Target Molecule Mouse monoclonal IgG1 antibodies.
Source Material Cell culture supernatant.
Sample Extraction and Clarification Salt concentration and pH were adjusted to those of the binding buffer in the capture step. Samples were filtered through a 0.45 µm filter before chromatography.
Capture Affinity or ion exchange chromatography are particularly suitable for samples such as cell culture supernatants as they are binding techniques which concentrate the target protein and significantly reduce sample volume. For monoclonal antibody purification capture of the target protein can be achieved by using a highly selective affinity chromatography medium. In this example a HiTrap rProtein A column was used. Although general standard protocols were supplied with this pre-packed columns, it was decided to further optimise the binding and elution conditions for the specific target molecule. Most mouse monoclonal antibodies of the IgG 1 sub-class require high salt concentrations to bind to immobilised Protein A, therefore a salt concentration was selected which gave the largest elution peak area and absence of antibodies in the flow-through. Results from the scouting for optimal binding conditions are shown in Figure 23. Scouting for the optimum elution pH also helped to improve antibody recovery. Optimisation of binding and elution conditions gave a well resolved peak containing IgG1, as shown in Figure 24. 54
System: Sample: Column: Binding buffer: Elution buffer: Flow:
ÄKTAFPLC Cell culture supernatant containing monoclonal IgG1, 90 ml HiTrap rProtein A, 1 ml 100 mM sodium phosphate, 0-3.5 M sodium chloride pH 7.4 100 mM sodium citrate pH 3 1 ml/min
A 280 nm
1200
900 0.0 M NaCl 600 0.5 M NaCl 1.5 M NaCl 300 2.5 M NaCl 3.5 M NaCl
0 120
125
130
135
140
145
150
155
ml
Fig. 23. Automatic scouting for optimal binding conditions.
System: ÄKTAFPLC Sample: Cell culture supernatant containing monoclonal IgG1, 100 ml Column: HiTrap rProtein A, 1 ml Binding buffer: 100 mM sodium phosphate, 2.5 M sodium chloride pH, 7.4 Elution buffer: 100 mM sodium citrate, pH 4.5 Flow: 1 ml/min
A 280 nm 2200 1800 Sample application
1400
Wash with binding buffer
Elution IgG1 peak collected in Superloop
1000 600 200 90
120
ml
Fig. 24. Optimised capture step on HiTrap rProtein A.
55
Intermediate Purification No intermediate purification was required as the high selectivity of the capture step also removed contaminating proteins and low-molecular substances giving a highly efficient purification.
Polishing In most antibody preparations there is a possibility that IgG aggregates and/or dimers are present. It was therefore essential to include a gel filtration polishing step, despite the high degree of purity achieved during capture. The polishing step removes low or trace levels of contaminants. Superdex 200 prep grade gel filtration media was selected as it has the most suitable molecular weight separation range for IgG antibodies. Figure 25 shows the final purification step.
System: Sample:
ÄKTAFPLC Fraction from HiTrap rProtein A column containing monoclonal IgG1 (3 ml) HiLoad 16/60 Superdex 200 prep grade 50 mM sodium phosphate, 0.15 M sodium chloride, pH 7.4 1 ml/min
Column: Buffer: Flow:
A 280 nm
300 250
A280
200 150
Conductivity
100 50 0 0
50
100
150
ml
Fig. 25. Gel filtration on HiLoad 16/60 Superdex 200 prep grade.
Analytical assay Collected fractions were separated by SDS-PAGE and silver stained using PhastSystem, following the separation and staining protocols supplied with the instrument. Lane 1. LMW-standard Lane 2. Starting material (diluted 2 x) Lane 3. Eluted IgG1 peak from HiTrap rProtein A column (diluted 10 x) Lane 4. Flow through, HiTrap rProtein A Lane 5. Eluted IgG1 peak from HiLoad Superdex 200 prep grade (diluted 6 x) Lane 6. LMW-standard Gel: 10–15 % SDS-PAGE PhastGel System: PhastSystem Lane 1
2
3
4
5
6
Analysis of purification steps using SDS PAGE. 56
Example 4.
One step purification of an integral membrane protein This example demonstrates that, with the use of a suitably tagged recombinant protein, selected detergents and an appropriate chromatographic medium, a successful purification can be achieved in a single chromatographic step. The objective was to purify a recombinant histidine-tagged integral membrane protein sufficiently to allow characterisation under non-denaturing conditions. A more detailed description of this work, including results for size and charge homogeneity, can be found in Application Note 18-1128-92.
Target Molecule Histidine-tagged cytochrome bo 3 ubiquinol oxidase from E. coli.
Source Material The histidine-tagged cytochrome bo 3 ubiquinol oxidase was expressed in the membrane of E. coli.
Sample Extraction and Clarification Membrane Preparation Integral membrane proteins require the use of detergents for extraction. The concentration and type of detergent that is suitable for a particular extraction must be tested for each situation. Cells were harvested by centrifugation and frozen at –80 °C. Frozen cells were mixed with 200 mM Tris-HCl, pH 8.8, 20 mM Na2 -EDTA, 500 mM sucrose and brought to room temperature, stirring gently. 10 mg/ml lysozyme in buffer was added and the solution stirred for 30 min. Cells were sedimented by centrifugation and supernatant removed. Pellets were resuspended in 5 mM Na2 -EDTA, pH 8.0, with PMSF, and stirred for 10 min. MgCl2 (final concentration 10 mM) and a few crystals of Dnase I were added and stirred for 5 min. The solution was sonicated for 3 x 1 min. Unbroken cells were removed by centrifugation Membrane particles were isolated by high speed centrifugation, resuspended in 50 mM Tris-HCl (pH 8.0), 250 mM NaCl and sedimented again at high speed. Membrane pellets were stored frozen.
57
Membrane Solubilisation Membrane pellets were thawed, ice cold 1% dodecyl-b-D-maltoside (a non-ionic detergent) in 20 mM Tris-HCl, pH 7.5, 300 mM NaCl, 5 mM imidazole was added. The solution was stirred on ice for 30 min. Non-solubilised material was removed by centrifugation. The presence of non-ionic detergent avoided denaturing conditions and interference with purification steps whilst maintaining membrane protein solubility.
Capture Due to the instability of membrane proteins and their tendency to associate it is often essential to use fast purification protocols at low temperatures. Attachment of a histidine tag allowed the use of a HiTrap Chelating column giving a highly selective affinity chromatography capture step, shown in Figure 26. This technique also removed contaminating proteins, DNA, lipids and low-molecular substances and allowed equilibration of detergent-protein complexes with the detergent solution. The technique was unaffected by the presence of the non-ionic detergent. Buffers and separation procedure followed the recommendations provided with the HiTrap Chelating column.
System: Column: Sample: Binding buffer: Elution buffer: Gradient: Temperature: Flow:
ÄKTAFPLC HiTrap Chelating, 1 ml Detergent extracts of E. coli membranes 20 mM Tris-HCl, pH 7.5, 5 mM imidazole, 0.03% dodecyl-b-D-maltoside, 300 mM NaCl 20 mM Tris-HCl, pH 7.5, 500 mM imidazole, 0.03% dodecyl-b-D-maltoside, 300 mM NaCl 0–60%, 20 column volumes +5 °C 1 ml/min
A 280 nm
%B 100
1500
80 Fraction 2
1000
60
Fraction 1 40 500
20 0
0 0
10
20
30 ml
Fig. 26. One Step Purification on a HiTrap Chelating column.
58
Intermediate Purification and Polishing No intermediate or polishing steps were needed as the high selectivity of the capture step produced a membrane protein of sufficient purity to allow further characterisation i.e. a single step purification was achieved (as shown by electrophoresis).
Analytical assay Lane 1: Low Molecular Weight Calibration kit (LMW) 14 400, 20 100, 30 000, 43 000, 67 000, 94 000 Lane 2: Detergent extract of Escherichia coli membranes Lane 3: Flow-through material Lane 4: Fraction 1 from HiTrap Chelating 1 ml Lane 5: Fraction 2 from HiTrap Chelating 1 ml
Lane 1
2
3
4
5
Fig. 27. SDS electrophoresis on PhastSystem using PhastGel 8–25%, silver staining.
To confirm final purity, collected fractions were separated by SDS-PAGE and silver stained by PhastSystem, following the separation and staining protocols supplied with the instrument. Figure 27. Shows that four subunits of cytochrome bo 3 were present in both fractions. Fraction 2 was essentially pure, whereas contaminants were seen in Fraction 1.
59
Chapter 8
Sample Storage Conditions Recommendations for biological samples Keep refrigerated in a closed vessel to minimise bacterial growth and protease activity. Avoid conditions close to stability limits (for example, extreme pH, pH values close to the isoelectric point of the target protein or salt concentrations, reducing or chelating agents). For storage times longer than 24 hours a bacteriostatic agent may be added, but this should be selected with care to ensure compatability with subsequent procedures. For long term storage keep proteins frozen or freeze dried in small aliquots (to avoid repeated freeze/thawing or freeze drying/re-dissolving which may reduce biological activity). Samples which will be freeze dried should be dissolved in volatile buffers, examples shown in Table 7. It should also be noted that concentration gradients can develop during freezing and thawing which may create extreme conditions causing protein denaturation. If essential add stabilising agents. These are more often required for storage of purified proteins. Table 7. Volatile buffer systems. pH range
Volatile buffer systems used in ion exchange chromatography Buffer system Counter-ion pK-values for buffering ions
2.0 2.3-3.5 3.0-5.0 3.0-6.0 4.0-6.0 6.8-8.8 7.0-8.5 8.5-10.0 7.0-12.0 7.9 8.0-9.5 8.5-10.5 8.5
Formic acid Pyridine/formic acid Trimethylamine/formic acid Pyridine/acetic acid Trimethylamine/acetic acid Trimethylamine/HCl Ammonia/formic acid Ammonia/acetic acid Trimethylamine/carbonate Ammonium bicarbonate Ammonium carbonate/ammonia Ethanolamine/HCl Ammonium carbonate
H+ HCOOHCOOCH3COOCH3COOClHCOOCH3COOCO32HCO3CO32ClCO32-
3.75 3.75; 3.75; 4.76; 4.76; 9.25 3.75; 4.76; 6.50; 6.50; 6.50; 10.0 6.50;
5.25 9.25 5.25 9.25 9.25 9.25 9.25 9.25 9.25 9.25
Recommendations for purified proteins Store in high concentration of ammonium sulphate (e.g. 4M). Freeze in 50% glycerol, especially suitable for enzymes. Add stabilising agents e.g. glycerol (5-20%), serum albumin (10 mg/ml), ligand (concentration is selected based on the concentration of the active protein). Sterile filter to avoid bacterial growth. 61
Sample Extraction and Clarification Procedures Sample extraction Extraction procedures should be selected according to the source of the protein, such as bacterial, plant or mammalian, intracellular or extracellular. Use procedures which are as gentle as possible since disruption of cells or tissues leads to the release of proteolytic enzymes and general acidification. Selection of an extraction technique is dependent as much upon the equipment available and scale of operation as on the type of sample. Examples of common extraction processes are shown in Table 8. Extraction should be performed quickly, at sub-ambient temperatures, in the presence of a suitable buffer to maintain pH and ionic strength and stabilise the sample. Samples should be clear and free from particles before beginning a chromatographic separation.
62
Table 8. Common sample extraction processes. Extraction process
Typical conditions
Protein source
Comment
Gentle
2 volumes water to 1 volume packed pre-washed cells
erythrocytes E.coli periplasm: intracellular proteins
lower product yield but reduced protease release
Enzymatic digestion
lysozyme 0.2 mg/ml, 37 °C, 15 mins.
bacteria: intracellular proteins
lab scale only, often combined with mechanical disruption
Hand homogenisation
follow equipment instructions
liver tissue
Mincing (grinding)
"
muscle
Moderate
follow equipment instructions
muscle tissue, most animal tissues, plant tissues
Grinding with abrasive e.g. sand
"
bacteria, plant tissues
Vigorous Ultrasonication or bead milling
follow equipment instructions
cell suspensions: intracellular proteins in cytoplasm, periplasm, inclusion bodies
small scale, release of nucleic acids may cause viscosity problems inclusion bodies must be resolubilised
Manton-Gaulin homogeniser
follow equipment instructions
cell suspensions
large scale only
French press
follow equipment instructions
bacteria, plant cells
Fractional precipitation
see section on fractional precipitation
extracellular: secreted recombinants, monoclonal antibodies, cell lysates
Cell lysis (osmotic shock)
Blade homogeniser
precipitates must be resolubilised
Details from Protein Purification, Principles and Practice, R.K. Scopes and other sources.
Buffers and additives With knowledge of the target protein stability window and other properties, additives can be kept to a minimum. This can help to avoid problems of interference with assays or other procedures and will avoid the need for an extra purification step to remove additives at a later stage in purification. Examples of buffers and additives, together with their use, are shown in Table 9.
63
Table 9. Common substances used in sample preparation. Typical conditions for use
Purpose
Buffer components Tris
20 mM, pH7.4
maintain pH minimise acidification caused by lysosomal disruption
NaCl
100 mM
maintain ionic strength of medium
EDTA
10 mM
reduce oxidation damage, chelate metal ions
Sucrose or glucose
25 mM
stabilise lysosomal membranes, reduce protease release
Detergents lonic or non-ionic detergents
See Table 10
extraction and purification of integral membrane proteins solubilisation of poorly soluble proteins
DNAse and RNAse
1 µg/ml
degradation of nucleic acids, reduce viscosity of sample solution
Protease inhibitors* PMSF
0.5 - 1 mM
Inhibits serine proteases
APMSF
0.4 - 4 mM
serine proteases
Benzamidine-HCl
0.2 mM
serine proteases
Pepstatin
1 µM
aspartic proteases
Leupeptin
10 - 100 µM
cysteine and serine proteases
Chymostatin
10 - 100 µM
chymotrypsin, papain, cysteine proteases
Antipain-HCl
1 - 100 µM
papain, cysteine and serine proteases
EDTA
2 - 10 mM
metal dependent proteases, zinc and iron
EGTA
2 - 10 mM
metal dependent proteases e.g. calcium
Reducing agents 1, 4 dithiothreitol, DTT
1 - 10 mM
keep cysteine residues reduced
1, 4 dithioerythritol, DTE
1 - 10 mM
Mercaptoethanol
0.05%
Others Glycerol
5 - 10%
" " for stabilisation, up to 50% can be used if required
PMSF - Phenylmethylsulfonyl fluoride APMSF - 4-Aminophenyl-methylsulfonyl fluoride PMSF is a hazardous chemical. Half-life time in aqueous solution is 35 min. PMSF is usually stored as 10 mM or 100 mM stock solution (1.74 or 17.4 mg/ml in isopropanol) at - 20° C. * Protease inhibitors are available in pre-made mixes from several suppliers. Details taken from Protein Purification, Principles and Practice, R.K. Scopes. 1994, Springer., Protein Purification, Principles, High Resolution Methods and Applications, J-C. Janson and L. Rydén, 1998, 2nd ed. Wiley Inc. and other sources.
64
Detergents Non-ionic detergents are used most commonly for extraction and purification of integral membrane proteins. Selection of the most suitable detergent is often a case of trial and error. The detergent should be used at concentrations near or above its critical micellar concentration i.e. the concentration at which detergent monomers begin to associate with each other. This concentration is dependent upon the type of detergent and the experimental conditions. Examples of ionic and non-ionic detergents are shown in Table 10. Adjustment of the detergent concentration necessary for optimum results is often a balance between the activity and yield of the protein. During purification procedures it may be possible to reduce the concentration of detergent compared to that used for extraction. However, some level of detergent is usually essential throughout purification procedures to maintain solubility. Detergents can be exchanged by adsorption techniques (Ref: Phenyl Sepharose mediated detergent exchange chromatography: its application to exchange of detergents bound to membrane protein. Biochemistry 23, 1984, 6121-6126, Robinson N.C., Wiginton D., Talbert L.) Table 10. Examples of ionic and non-ionic detergents. Sodium dodecyl sulphate
0.1 - 0.5%
denatures proteins, used for SDS-PAGE use non-ionic detergents to avoid denaturation
Triton-X-100
0.1 %
non-ionic detergent for membrane solubilisation. Note: may absorb strongly at 280 nm!
Nonidet-P-40
0.05 - 2%
"
Dodecyl b D maltoside
1%
"
Octyl b D glucoside
1 - 1.5%
"
For further information on detergents: Protein Purification, Principles, High Resolution Methods and Applications, J-C. Janson and L. Rydén, 1998, 2nd ed. Wiley Inc.
Sample Clarification Centrifugation Use before first chromatographic step Removes lipids and particulate matter For small sample volumes and those which adsorb non-specifically to filters: Centrifuge at 10000g for 15 minutes For cell homogenates: Centrifuge at 40 000-50 000g for 30 minutes
65
Ultrafiltration Use before first chromatographic step Removes salts, concentrates sample
Ultrafiltration membranes are available with different cut off limits for separation of molecules from 1000 up to 300000 daltons. The process is slower than gel filtration and membranes may clog. Check the recovery of the target protein in a test run. Some proteins may adsorb non-specifically to filter surfaces.
Filtration Use before first chromatographic step Removes particulate matter Suitable for small sample volumes. For sample preparation before chromatography select filter size according to the bead size of the chromatographic medium. Filter size 1 µm 0.45 µm 0.22 µm
Bead size of chromatographic medium 90 µm and upwards 3, 10, 15, 34 µm sterile filtration or extra clean samples
Check the recovery of the target protein in a test run. Some proteins may adsorb non-specifically to filter surfaces.
Gel filtration (for sample clarification or conditioning) Use before or between chromatographic purification steps. For rapid processing of small or large sample volumes. Removes salts from samples >5000 daltons. Sephadex G-25 is used at laboratory and production scale for sample preparation and clarification. Typically sample volumes of up to 30% of the total column volume are loaded. In a single step, the sample is desalted, exchanged into a new buffer, and low molecular weight materials are removed. The high volume capacity and speed of this step enable very large sample volumes to be processed rapidly and efficiently. The high sample volume load results in a separation with minimal sample dilution. A typical elution is shown in Figure 28.
66
Fig 28. Typical elution profile for sample desalting and buffer exchange.
Methodology Select a pre-packed desalting column from the table below or pack a column. Pre-packed column
Sample volume loading per run
Sample volume recovery per run
Code No.
HiPrep Desalting 26/10 HiTrap Desalting Fast Desalting PC 3.2/10 PD-10 Desalting
2.5 -15 ml 0.25 - 1.5 ml 0.05 - 0.2 ml 1.5 - 2.5 ml
7.5 1.0 0.2 2.5
17-5087-01 17-1408-01 17-0774-01 17-0851-01
-
20 ml 2.0 ml 0.3 ml 3.5 ml
Column packing The following guidelines apply at all scales of operation: Column dimensions = typically 10 - 20 cm bed height. Quantity of gel = five times volume of sample. For column packing Sephadex G-25 is available in a range of bead sizes (Superfine, Fine, Medium and Coarse). Changes in bead size alter flow rates and sample volumes which can be applied (see Figure 29). For laboratory scale separations use Sephadex G-25 Fine with an average bed height of 15 cm. Individual product packing instructions contain more detailed information on packing Sephadex G-25. 200
maximum flow rate cm/h flow velocity (linear flow rate)
% of column volume
100
maximum sample volume
Superfine
Fine
Medium
Coarse
increasing bead size
Fig. 29. Sephadex G-25: sample volume and flow rate varies with bead size. 67
Fractional precipitation For extraction and clarification at laboratory scale Partially purifies sample, may also concentrate Use before the first chromatographic step Most precipitation techniques are not suitable for large scale preparations. Precipitation techniques are affected by temperature, pH and sample concentration. These parameters must be controlled to ensure reproducible results. Precipitation can be used in three different ways, as shown in Figure 30. Clarification Bulk proteins and particulate matter precipitated
Supernatant
Extraction Clarification Concentration Target protein precipitated with proteins of similar solubility Extraction Clarification Bulk proteins and particulate matter precipitated
Resolubilise pellet*
Concentration Target protein precipitated
Chromatography Remember: if precipitating agent is incompatible with next purification step, use Sephadex G25 for desalt and buffer exchange
Resolubilise pellet* *Remember: not all proteins are easy to resolubilise, yield may be reduced
Fig. 30. Three ways to use precipitation.
Precipitation techniques are reviewed in Table 11 and the two common methods are described in more detail.
68
Table 11. Examples of precipitation techniques. Precipitation agent
Typical conditions for use
Sample type
Comment
Ammonium sulphate
as described
>1mg/ml proteins especially immunoglobulins
stabilizes proteins, no denaturation, supernatant can go directly to HIC
Dextran sulphate
as described
samples with high levels of lipoprotein e.g ascites
precipitates lipoprotein
Polyvinylpyrrolidine
Add 3% (w/v), stir 4 hours, centrifuge, discard pellet
"
alternative to dextran sulphate
Polyethylene glycol (PEG, M.W. >4000)
up to 20% wt/vol
plasma proteins
no denaturation supernatant goes direct to IEX or AC. Complete removal may be difficult
Acetone
up to 80% vol/vol at 0 °C
useful for peptide precipitation or concentration of sample for electrophoresis
may denature protein irreversibly
Polyethyleneimine
0.1% w/v
Protamine sulphate
1%
Streptomycin sulphate
1%
precipitates aggregated nucleoproteins " precipitation of nucleic acids
Details taken from Protein Purification, Principles and Practice, R.K. Scopes. 1994, Springer., Protein Purification, Principles, High Resolution Methods and Applications, J-C. Janson and L. Rydén, 1998, 2nd ed. Wiley Inc.and other sources
Ammonium sulphate precipitation Materials Saturated ammonium sulphate solution Add 100g ammonium sulphate to 100 ml distilled water, stir to dissolve 1 M Tris-HCl pH 8.0 Buffer for first chromatographic purification step Procedure 1. Filter (0.45mm) or centrifuge (refrigerated, 10000g) sample. 2. Add 1 part 1M Tris-HCl pH 8.0 to 10 parts sample volume to maintain pH. 3. Stir gently. Add ammonium sulphate solution, drop by drop (solution becomes milky at about 20% saturation). Add up to 50% saturation*. Stir for 1 hour. 4. Centrifuge 20 minutes at 10000g. 5. Discard supernatant. Wash pellet twice by resuspension in an equal volume of ammonium sulphate solution of the same concentration (i.e. a solution that will not redissolve the precipitated protein or cause further precipitation). Centrifuge again. 6. Dissolve pellet in a small volume of the chromatographic buffer.
69
7. Ammonium sulphate is removed during clarification/buffer exchange steps with Sephadex G25 or during hydrophobic interaction separations. *The % saturation can be adjusted to either precipitate a target molecule or to precipitate contaminants. The quantity of ammonium sulphate required to reach given degrees of saturation varies according to temperature. Table 12 shows the quantities required at 20 °C. Table 12. Quantities of ammonium sulphate required to reach given degrees of saturation at 20 °C. Values calculated according to Protein Purification, R. K. Scopes (Springer-Verlag, New York),Third Edition, p. 346, 1993. Final percent saturation to be obtained 20
25
30
35
Starting percent saturation
40
45
50
55
60
65
70
75
80
85
90
95
100
Amount of ammonium sulphate to add (grams) per liter of solution at 20 °C
0
113
144
176
208
242
277
314
351
390
430
472
516
561
608
657
708
761
5
85
115
146
179
212
246
282
319
358
397
439
481
526
572
621
671
723
10
57
86
117
149
182
216
251
287
325
364
405
447
491
537
584
634
685
15
28
58
88
119
151
185
219
255
293
331
371
413
456
501
548
596
647
20
0
29
59
89
121
154
188
223
260
298
337
378
421
465
511
559
609
0
29
60
91
123
157
191
228
265
304
344
386
429
475
522
571
0
30
61
92
125
160
195
232
270
309
351
393
438
485
533
0
30
62
94
128
163
199
236
275
316
358
402
447
495
0
31
63
96
130
166
202
241
281
322
365
410
457
0
31
64
98
132
169
206
245
286
329
373
419
0
32
65
99
135
172
210
250
292
335
381
0
33
66
101
138
175
215
256
298
343
0
33
67
103
140
179
219
261
305
0
34
69
105
143
183
224
267
0
34
70
107
146
186
228
0
35
72
110
149
190
0
36
73
112
152
0
37
75
114
0
37
76
0
38
25 30 35 40 45 50 55 60 65 70 75 80 85 90 95
For further details showing variation of % saturation versus temperature and a review of precipitation tachniques see Guide to Protein Purification, Methods in Enzymology, Vol. 182, p. 291 Acad. Press 1990.
70
Dextran sulphate precipitation Materials 10% dextran sulphate 1M calcium chloride Buffer for first chromatographic purification step. Procedure 1.Add 0.04ml dextran sulphate solution and 1 ml calcium chloride solution to every 1 ml of sample. Mix 15 minutes. 2.Centrifuge (10000g, 10 minutes), discard precipitate. Dextran sulphate is removed during a clarification/buffer exchange Sephadex G25 step.
Resolubilisation of protein precipitates Many proteins are easily resolubilised in a small amount of the buffer to be used in the next chromatographic step. However, an agent, selected from Table 13, may be required for less soluble proteins. Specific conditions will depend upon the specific protein. These agents must always be removed to allow complete re-folding of the protein and maximise recovery of mass and activity. A chromatographic step often removes a denaturant during purification. Table 13. Examples of denaturing agents. Denaturing agent
Typical conditions for use
Removal/comment
Urea
2 - 8M
remove using Sephadex G25
Guanidine hydrochloride
3 - 8M
remove using Sephadex G25 or during IEX
Triton X-100 with
2%
”
1.5%
”
N-octyl glucoside
2%
”
Sodium dodecyl sulphate
0.1 - 0.5%
exchange for non-ionic detergent during first chromatographic step, avoid anion exchange chromatography
alkaline pH
> pH 9, NaOH
may need to adjust pH during chromatography to maintain solubility
Sarcosyl
Details taken from Protein Purification, Principles and Practice, R.K. Scopes. 1994, Springer., Protein Purification, Principles, High Resolution Methods and Applications, J-C. Janson and L. Rydén, 1998, 2nd ed. Wiley Inc.and other sources
71
Expanded bed adsorption (STREAMLINE) For large scale recombinant protein and monoclonal antibody purification. The technique requires no sample clean up and enables clarification, concentration and capture in a single step. EBA can be regarded as a technique in which sample preparation and capture are combined in a single step. Crude sample is applied to an expanded bed of STREAMLINE media, target proteins are captured whilst cell debris, cells, particulate matter, whole cells, and contaminants pass through. Flow is reversed and the target proteins are desorbed in the elution buffer.
72
Chapter 9
Ion Exchange (IEX) Chromatography, Principles and Standard Conditions IEX separates proteins with differences in charge to give a very high resolution separation with high sample loading capacity. The separation is based on the reversible interaction between a charged protein and an oppositely charged chromatographic medium. Proteins bind as they are loaded onto a column. Conditions are then altered so that bound substances are eluted differentially. This elution is usually performed by increases in salt concentration or changes in pH. Changes are made stepwise or with a continuous gradient. Most commonly, samples are eluted with salt (NaCl), using a gradient elution (Figure 31). Target proteins are concentrated during binding and collected in a purified, concentrated form. equilibration
sample application
gradient elution
wash
re-equilibration
high salt wash 1M
1-4 cv
tightly bound molecules elute in high salt wash
[NaCl]
unbound molecules elute before gradient begins
10-20 cv 2 cv
2 cv
0 Column volumes [cv]
Fig. 31. Typical IEX gradient elution.
The net surface charge of proteins varies according to the surrounding pH. When above its isoelectric point (pI) a protein will bind to an anion exchanger, when below its pI a protein will behind to a cation exchanger. Typically IEX is used to bind the target molecule, but it can also be used to bind impurities if required. IEX can be repeated at different pH values to separate several proteins which have distinctly different charge properties, as shown in Figure 32. This can be used to advantage during a multi-step purification, as shown in the example on page 24.
73
Selectivity pH of mobile phase Abs
Abs
V
Abs
V
Abs
V
V
+
Surface net charge
cation
pH
0
anion
–
Abs
Abs
V
Abs
V
Abs
V
V
Fig 32. Effect of pH on protein elution patterns.
Choice of ion exchanger For most purification steps it is recommended to begin with a strong exchanger, allowing work over a broad pH range during method development. Use a strong anion exchanger (Q) to bind the target if the isoelectric point is below pH 7.0 or unknown.
Strong ion exchangers Q (anion exchange), S and SP (cation exchange) are fully charged over a broad pH range (pH 2 - 12).
Weak ion exchangers DEAE (anion exchange) and CM (cation exchange) are fully charged over a narrower pH range (pH 2 - 9 and pH 6 - 10, respectively), but give alternative selectivities for separations.
Sample volume and capacity Ion exchange chromatography is a binding technique, independent of sample volume, provided that the ionic strength of the sample is low and the target molecule highly charged. The total amount of protein which is loaded and binds to the column should not exceed the total binding capacity of the column. For optimal separations when performing gradient elution, use approximately one fifth of the total binding capacity of the column
74
trace enrichment µg and less
Mini Beads
Q, S
• extreme resolution • micropurification and analysis • upper-medium pressure systems
Mono Beads
Q, S
• purification and analysis • medium pressure systems
Q, S
• high speed • high capacity • ideal for scale up • low-medium pressure systems
Sepharose High Performance
Q, SP
• lab / pilot scale separation of samples • low-medium pressure systems • clean up of small samples, use HiTrap columns
Sepharose Fast Flow
Q, SP, DEAE, CM
Sepharose XL
Q, SP
• very high capacity to reduce manufacturing costs • low-medium pressure systems
Sepharose Big Beads
Q, SP
• initial capture of viscous samples • low-medium pressure systems • industrial scale
STREAMLINE
DEAE, SP
mg and less
mg and more SOURCE 15
Preparative separation mg and more
SOURCE 30 mg - kg Partially purified or lab scale Crude material or production scale g - kg
• fast separations of crude samples • ideal for scale up • low-medium pressure systems • method scouting, use HiTrap columns
high capacity and flow Preparative separation Group separations
highest capacity, industrial production high viscosity samples crude feedstream samples
Fig. 33. Ion exchange media selection guide. (Code no. 18-1127-31)
• clarification, filtration, capture in one step • low-medium pressure systems • industrial scale
Resolution
Analytical or semi-preparative
75
Media selection Parameters such as scale of purification, resolution required, speed of separation, sample stability and media binding capacity, should be considered when selecting a chromatographic medium. Figure 33 on page 75 shows a guide to selection of ion exchange media.
Sample Preparation Correct sample preparation ensures good resolution and extends the life of the column. To ensure efficient binding during sample application samples should be at the same pH and ionic strength as the starting buffer. Samples must be free from particulate matter, particularly when working with bead sizes of 34 µm or less (see Chapter 8 for details of sample clarification procedures).
Column Preparation Pre-packed columns To increase speed and efficiency in method development, use small pre-packed columns for media scouting and method optimisation. HiTrap IEX Test Kit is ideal for this type of work, as shown in Figure 34. Sample: nm
Columns: Buffer A: Buffer B: Flow:
Ribonuclease A, human apo-transferrin, a-lactalbumin HiTrap IEX Test Kit 20 mM piperazine, pH 9.7 20 mM piperazine, 1 M NaCl pH 9.7 2 ml/min (310 cm/h)
Fig. 34. Media selection using 2 columns from HiTrap IEX Test Kit.
Using pre-packed columns at any scale will ensure reproducible results and high performance.
Column packing
The following guidelines apply at all scales of operation: Column dimensions = typically 5 - 15 cm bed height. Quantity of gel = estimate amount of gel required to bind the sample, use five times this amount to pack a column. See individual product packing instructions for more detailed information on a specific medium. 76
Buffer Preparation Buffering ions should have the same charge as the selected medium, with a pKa within 0.6 pH units of the working pH. Buffer concentration should be sufficient to maintain buffering capacity and constant pH during sample application and while an increase in salt concentration is applied. When working with a sample of unknown charge characteristics, try these conditions first:
Anion Exchange Gradient: 0-100% elution buffer in 10 - 20 column volumes Start buffer A: 20 mM Tris-HCl, pH 8.0 Elution buffer B: 20 mM Tris-HCl + 1M NaCl, pH 8.0
Cation Exchange Gradient: 0-100% B in 10 - 20 column volumes Start buffer A: 20 mM Na2HPO4.2H2O, pH 6.8 Elution buffer B: 20 mM Na2HPO4.2H2O + 1M NaCl, pH 6.8
Method Development (in priority order) 1. Select optimum ion exchanger using small columns such as pre-packed HiTrap columns to save time and sample. 2. Scout for optimum pH. Begin 0.5-1 pH unit away from the isoelectric point of the target protein if known (see Figure 32 showing changes in elution versus pH). 3. Select the steepest gradient to give acceptable resolution at the selected pH. 4. Select the highest flow rate which maintains resolution and minimises separation time. Check recommended flow rates for the specific medium. 5. For large scale purification and to reduce separation times and buffer consumption transfer to a step elution after method optimisation as shown in Figure 35. It is often possible to increase sample loading when using step elution.
Fig. 35. Step elution. 77
Cleaning, sanitisation and sterilisation Procedures vary according to type of sample and medium. Guidelines are supplied with the medium or pre-packed column.
Storage of media and columns Recommended conditions for storage are supplied with the medium or pre-packed column.
Further information Ion Exchange Chromatography: Principles and Methods Code no. 18-1022-19.
BioProcess Mediafor large scale production Specific BioProcess Media have been designed for each chromatographic stage in a process from Capture to Polishing. Large capacity production integrated with clear ordering and delivery routines ensure that BioProcess media are available in the right quantity, at the right place, at the right time. Amersham Pharmacia Biotech can assure future supplies of BioProcess Media, making them a safe investment for long term production. The media are produced following validated methods and tested under strict control to fulfil high performance specifications. A certificate of analysis is available with each order. Media
BioProcess
Regulatory support files contain details of performance, stability, extractable compounds and analytical methods. The essential information in these files gives an invaluable starting point for process validation, as well as providing support for submissions to regulatory authorities. Using BioProcess Media for every stage results in an easily validated process. High flow rates, high capacity and high recovery contribute to the overall economy of an industrial process. All BioProcess Media have chemical stability to allow efficient cleaning and sanitisation procedures. Packing methods are established for a wide range of scales and compatible large scale columns and equipment are available.
78
Hydrophobic Interaction Chromatography (HIC), Principles and Standard Conditions HIC separates proteins with differences in hydrophobicity. The technique is ideal for the capture or intermediate steps in a purification. The separation is based on the reversible interaction between a protein and the hydrophobic surface of a chromatographic medium. This interaction is enhanced by high ionic strength buffer which makes HIC an ideal 'next step' after precipitation with ammonium sulphate or elution in high salt during IEX. Samples in high ionic strength solution (e.g. 1.5 M ammonium sulphate) bind as they are loaded onto a column. Conditions are then altered so that the bound substances are eluted differentially. Elution is usually performed by decreases in salt concentration (Figure 36). Changes are made stepwise or with a continuous decreasing salt gradient. Most commonly, samples are eluted with a decreasing gradient of ammonium sulphate. Target proteins are concentrated during binding and collected in a purified, concentrated form. Other elution procedures include reducing eluent polarity (ethylene glycol gradient up to 50%), adding chaotropic species (urea, guanidine hydrochloride) or detergents, changing pH or temperature.
equilibration
sample application
gradient elution
salt free wash
re-equilibration
[ammonium sulphate]
1M
tightly bound molecules elute in salt free conditions
unbound molecules elute before gradient begins 10-15 cv
2 cv
2 cv 0 Column volumes [cv]
Fig. 36. Typical HIC gradient elution
79
Choice of hydrophobic ligand Very hydrophobic proteins bind tightly to very hydrophobic ligands and may require extreme elution conditions, e.g. chaotropic agents or detergents, for the target protein or other contaminants. To avoid this problem it is recommended to screen several hydrophobic media, using HiTrap HIC Test Kit or RESOURCE HIC Test Kit. Begin with a medium of low hydrophobicity if the sample has very hydrophobic components. Select the medium which gives the best resolution and loading capacity at a reasonably low salt concentration. Typically the strength of binding of a ligand to a protein increases in the order: ether, isopropyl, butyl, octyl, phenyl. However, the nature of the binding (both the selectivity and the binding strength)- can vary and must be tested in individual cases.
Sample volume and capacity HIC is a binding technique and therefore rather independent of sample volume, provided that conditions are chosen to strongly bind the target protein. The total amount of protein which is loaded and binds to the column should not exceed the total binding capacity of the column. For optimal separations when performing gradient elution, use approximately one fifth of the total binding capacity of the column
Media selection In HIC the characteristics of the chromatographic matrix as well as the hydrophobic ligand affect the selectivity of the medium. This should be considered, together with parameters such as sample solubility, required resolution, scale of purification and availability of the medium at the scale intended. Figure 37 on page 81 shows a guide to selection of HIC media.
Sample Preparation Correct sample preparation ensures good resolution and extends the life of the column. To ensure efficient binding during sample application samples should be at the same pH as the starting buffer and in high ionic strength solution (e.g. 1.5 M ammonium sulphate or 4M NaCl). Samples must be free from particulate matter, particularly when working with bead sizes of 34 µm or less (see Chapter 8 for details of sample clarification procedures).
80
Partially purified or lab scale Crude material or production scale g–kg
Superose
alkyl phenyl
• purification and analysis
SOURCE 15
ethyl isopropyl phenyl
• high speed • high capacity • ideal for scale up
Sepharose HP
phenyl
• small/pilot scale of pretreated samples
Sepharose FF
butyl oktyl phenyl
• fast separations of crude samples • ideal for scale up • method scouting, use HiTrap columns
mg and more g and more, high speed and capacity mg and kg
Preparative separation Group separations
high capacity and flow
Fig. 37. Hydrophobic interaction media selection guide.
Increasing resolution
Preparative separations
mg and less
Increasing bead size
Analytical or semi-preparative
81
Column Preparation Pre-packed columns To increase speed and efficiency in method development use small pre-packed columns for media scouting and method optimisation. HiTrap HIC Test Kit and RESOURCE HIC Test Kit are ideal for this work. Using pre-packed columns at any scale will ensure reproducible results and high performance. Figure 38 shows an example of media screening with HiTrap HIC Test Kit A280nm
Conductivity (mS/cm)
400
System: Sample: Columns:
150
Conductivity
300 100 200
Fab
Buffer A: Buffer B: Gradient: Flow:
ÄKTAexplorer Fab fraction from STREAMLINE SP, 2 ml HiTrap HIC Test Kit (1 ml columns), PhenylSepharose High Performance, Phenyl Sepharose 6 Fast Flow (low sub), Phenyl Sepharose 6 (high sub), Butyl Sepharose 4 Fast Flow, Octyl Sepharose 4 Fast Flow 1 ml (NH4)2SO4, 50 mM NaAc, pH 5.0 50 mM NaAc, pH 5.0 20 column volumes 2 ml/min (300 cm/hr)
50
100
0 0 0.0
10.0
Time (min)
Fig. 38. Media screening with HiTrap HIC Test Kit.
Column packing The following guidelines apply at all scales of operation: Column dimensions = typically 5 - 15 cm bed height. Quantity of gel = estimate amount of gel required to bind the sample, use five times this amount to pack a column. See individual product packing instructions for more detailed information on a specific medium.
Buffer Preparation Buffering ion selection is not critical for hydrophobic interaction. Select a pH compatible with protein stability and activity. Buffer concentration must be sufficient to maintain pH during sample application and changes in salt concentration. When working with a sample of unknown hydrophobic characteristics, try these conditions first: Gradient: 0-100% B in 10 - 20 column volumes Start buffer A: 50 mM sodium phosphate pH 7.0 + 1 - 1.5 M ammonium sulphate Elution buffer B: 50 mM sodium phosphate pH 7.0 82
Method Development (in priority order) 1. The hydrophobic behaviour of a protein is difficult to predict and binding conditions mst be studied carefully. Use a HiTrap HIC Text Kit or a RESOURCE HIC Test Kit to select the medium which gives optimum binding and elution over the required range of salt concentration. For proteins with unknown hydrophobic properties begin with 0-100%B (0%B=1M ammonium sulphate). 2. Select the gradient which gives acceptable resolution. 3. Select the highest flow rate which maintains resolution and minimises separation time. Check recommended flow rates for the specific medium. 4. For large scale purification and to reduce separation times and buffer consumption, transfer to a step elution after method optimisation, as shown in Figure 39. It is often possible to increase sample loading when using step elution, an additional benefit for large scale purification.
Fig. 39. Step elution.
5. If samples adsorb strongly to a gel then conditions which cause conformational changes, such as pH, temperature, chaotropic ions or organic solvents can be altered. Conformational changes caused by these agents are specific to each protein. Use screening procedures to investigate the effects of these agents. Alternatively, change to a less hydrophobic medium.
Cleaning, sanitisation and sterilisation Procedures vary according to type of sample and medium. Guidelines are supplied with the medium or pre-packed column.
83
Storage of media and columns Recommended conditions for storage are supplied with the medium or pre-packed column.
Further information Hydrophobic Interaction Chromatography: Principles and Methods Code no. 18-1020-90
BioProcess Media- for large scale production Specific BioProcess Media have been designed for each chromatographic stage in a process from Capture to Polishing. Large capacity production integrated with clear ordering and delivery routines ensure that BioProcess media are available in the right quantity, at the right place, at the right time. Amersham Pharmacia Biotech can assure future supplies of BioProcess Media, making them a safe investment for long term production. The media are produced following validated methods and tested under strict control to fulfil high performance specifications. A certificate of analysis is available with each order. Media
BioProcess
Regulatory support files contain details of performance, stability, extractable compounds and analytical methods. The essential information in these files gives an invaluable starting point for process validation, as well as support providing for submissions to regulatory authorities. Using BioProcess Media for every stage results in an easily validated process. High flow rates, high capacity and high recovery contribute to the overall economy of an industrial process. All BioProcess Media have chemical stability to allow efficient cleaning and sanitisation procedures. Packing methods are established for a wide range of scales and compatible large scale columns and equipment are available.
84
Affinity Chromatography (AC), Principles and Standard Conditions AC separates proteins on the basis of a reversible interaction between a protein (or group of proteins) and a specific ligand attached to a chromatographic matrix. The technique is ideal for a capture or intermediate step and can be used whenever a suitable ligand is available for the protein(s) of interest. AC offers high selectivity, hence high resolution, and usually high capacity (for the protein(s) of interest). The target protein(s) is specifically and reversibly bound by a complementary binding substance (ligand). The sample is applied under conditions that favour specific binding to the ligand. Unbound material is washed away, and the bound target protein is recovered by changing conditions to those favouring desorption. Desorption is performed specifically, using a competitive ligand, or non specifically, by changing the pH, ionic strength or polarity. Samples are concentrated during binding and protein is collected in purified, concentrated form. The key stages in a separation are shown in Figure 40. Affinity chromatography is also used to remove specific contaminants, for example Benzamidine Sepharose 6B removes serine proteases.
UV
equilib ration
adsorption of sample and elution of unbound material
begin sample application
1-2 cv
wash away unbound material
elute bound protein (s)
gel regeneration
change to elution buffer
x cv
1-2 cv
>1 cv
1-2 cv
Column Volumes [cv]
Fig. 40. Typical affinity separation.
85
Sample volume and capacity AC is a binding technique, independent of sample volume provided that conditions are chosen to strongly bind the target protein. Total binding capacity (target protein(s) bound per ml gel) is defined for all commercially available affinity media.
Media selection Parameters such as scale of purification and commercial availability of affinity matrices should be considered when selecting affinity media. To save time and ensure reproducibility use prepacked columns for method development or small scale purification. HiTrap affinity columns are ideal for this work. Table 6 on page 34 shows examples of prepacked affinity columns. Specific affinity media are prepared by coupling a ligand to a selected gel matrix, following recommended coupling procedures. Further details on other affinity media are available in the Affinity Chromatography Product Profile (Code No. 18-1121-86).
Sample Preparation Correct sample preparation ensures efficient binding and extends the life of a column. Removal of contaminants which may bind non-specifically to the column, such as lipids, is crucial. Stringent washing procedures may damage the ligand of an affinity medium, destroying the binding capacity of the column. Samples must be free from particulate matter (see Chapter 8 for details of sample clarification procedures).
Column Preparation Pre-packed columns Pre-packed columns ensure reproducible results and highest performance.
Column packing The following guidelines apply at all scales of operation: Column dimensions = short wide dimensions Quantity of gel = calculate according to known binding capacity of medium, use 2-5 times excess capacity. See individual product packing instructions for more detailed information on a specific medium.
86
Buffer Preparation Binding, elution and regeneration buffers are specific to each affinity medium. Follow instructions supplied with the medium or column. 1. Select the correct specificty for the target protein. Follow manufacturer's instructions for binding or elution conditions and check recommended flow rates for the specific medium. 2. Select optimum flow rate to achieve efficient binding 3. Select optimum flow rate for elution to maximise recovery. 4. Select maximum flow rate for column regeneration to minimise run times.
Cleaning, sanitisation and sterilisation Procedures vary according to type of sample and medium. Guidelines are supplied with the medium or pre-packed column.
Storage of media and columns Follow manufacturer's instructions.
Further information Affinity Chromatography: Principles and Methods Code no. 18-1022-29.
BioProcess Media- for large scale production Specific BioProcess Media have been designed for each chromatographic stage in a process from Capture to Polishing. Large capacity production integrated with clear ordering and delivery routines ensure that BioProcess media are available in the right quantity, at the right place, at the right time. Amersham Pharmacia Biotech can assure future supplies of BioProcess Media, making them a safe investment for long term production. The media are produced following validated methods and tested under strict control to fulfil high performance specifications. A certificate of analysis is available with each order. Media
BioProcess
Regulatory support files contain details of performance, stability, extractable compounds and analytical methods. The essential information in these files gives an invaluable starting point for process validation, as well as providing support for submissions to regulatory authorities. Using BioProcess Media for every stage results in an easily validated process. High flow rates, high capacity and high recovery contribute to the overall economy of an industrial process. All BioProcess Media have chemical stability to allow efficient cleaning and sanitisation procedures. Packing methods are established for a wide range of scales and compatible large scale columns and equipment are available.
87
Gel Filtration (GF) Chromatography, Principles and Standard Conditions GF separates proteins with differences in molecular size. The technique is ideal for the final polishing steps in a purification when sample volumes have been reduced (sample volume significantly influences speed and resolution in gel filtration). Samples are eluted isocratically (single buffer, no gradient Figure 41). Buffer conditions are varied to suit the sample type or the requirements for further purification, analysis or storage step, since buffer composition does not directly affect resolution. Proteins are collected in purified form in the chosen buffer.
high molecular weight low molecular weight
UV
sample injection volume intermediate molecular weight equilibration
1 cv Column Volumes [cv]
Fig. 41. Typical GF elution.
Sample volume and capacity To achieve highest resolution the sample volume must not exceed 5% of the total column volume. Gel filtration is independent of sample concentration, although above 50 mg/ml protein viscosity effects may cause 'fingering'. Extremely viscous samples should be diluted.
88
Fractionation range (globular proteins) Peptides
Superdex High recovery High stability High selectivity
High selectivity (0.1-600 kD) Wide Mw range (1-5,000 kD)
Preparative & analytical (0.1-5,000 kD) Preparative /Macro fractionation (1-500,000 kD)
Small proteins
Analytical separation
Polynucleotides Proteins DNA-fragment
Preparative separation
10
3
10
4
10
5
Superdex 75 Superdex 200 Superdex 30 prep grade
Small proteins Polynucleotides
Superdex 75 prep grade
Proteins DNA-fragment
Superdex 200 prep grade
High recovery Wide Mw fractionation range High selectivity (0.5-600 kD) Wide Mw range ( 1-5,000 kD)
Semi-preparative Analytical separation Preparative separation
Intermediate fractionation range Wide fractionation range Intermediate fractionation range Small proteins
Sephacryl Macromolecule separation Product line covering wide fractionation range
Wide fractionation range
Proteins
Proteins Large proteins
Macro molecules
Sephadex
e er
Sephacryl S-200 HR Sephacryl S-300 HR
Sephacryl S-500 HR Sephacryl S-1000 SF
Proteins
th
Superose 12 prep grade
Sephacryl S-100 HR
Small particles Virus
Peptides/small proteins
ar
Superose 6 prep grade
Sephacryl S-400 HR
Group separation
St
Superose 12
Purification of macromolecules
Desalting
Fig. 42. Gel filtration media selection guide Code: 18-1124-19.
Superose 6
Fractionation of macromolecules
Small peptides
10
6
10
7
10
8
High resolution
Peptides
Fractionation Group separation Desalting
2
Superose
Analytical (0.1-5,000 kD) Preparative (0.5-5,000 kD)
Semi-preparative
10 Superdex Peptide
Sephadex G-10 Sephadex G-25 SF Sephadex G-25 F Sephadex G-25 M Sephadex G-50 F
Exclusion limit Exclusion limit Exclusion limit
89
Media selection Parameters such as molecular weight of target proteins and contaminants, resolution required, scale of purification should be considered when selecting gel filtration media. Figure 42 on page 89 shows a guide to selection of GF media.
Sample Preparation. Correct sample preparation ensures good resolution and extends the life of the column. Sample buffer composition does not directly affect resolution. During separation the sample buffer is exchanged with buffer in the column. Viscous samples, which could cause an increase in back pressure and affect column packing, should be diluted. Samples must be free from particulate matter, particularly when working with bead sizes of 34µm or less (see Chapter 8 for details of sample clarification procedures)
Column Preparation Pre-packed columns Pre-packed columns ensure reproducible results and highest performance.
Column packing In gel filtration good column packing is essential. The resolution between two separated zones increases as the square root of column length. The following guidelines apply: Column dimensions: =minimum 50 cm bed height (Sephacryl media) minimum 30 cm bed height (Superdex, Superose media) Bed volume = depending on sample volume per run (up to 5% of bed volume) See individual product packing instructions for more detailed information on a specific medium.
Buffer Preparation Selection of buffering ion does not directly affect resolution. Select a buffer in which the purified product should be collected and which is compatible with protein stability and activity. Buffer concentration must be sufficient to maintain buffering capacity and constant pH. Ionic strength can be up to 150 mM NaCl in the buffer, to avoid non-specific ionic interactions with the matrix (shown by delays in peak elution). When working with a new sample try these conditions first Buffer: 0.5 M sodium phosphate, pH 7.0 + 0.15 M NaCl or select the buffer in which the sample should be eluted for the next step
90
Method Development (in priority order) 1. Select the medium which gives the best separation of target proteins from contaminants. 2. Select the highest flow rate which maintains resolution and minimises separation time. Check recommended flow rates for the specific medium. Lower flow rates improve resolution of high molecular weight components, faster flow rates may improve resolution of low molecular weight components 3. Determine the maximum sample volume which can be loaded without reducing resolution (sample volume should be 0.5-5% of total column volume). 4. To further improve resolution increase column length by connecting two columns in series
Cleaning, sanitisation and sterilisation Procedures vary according to type of sample and medium. Guidelines are supplied with the medium or pre-packed column.
Storage of media and columns Recommended conditions for storage are supplied with the medium or pre-packed column.
Further information Gel Filtration: Principles and Methods Code no. 18-1022-18.
BioProcess Media- for large scale production Specific BioProcess Media have been designed for each chromatographic stage in a process from Capture to Polishing. Large capacity production integrated with clear ordering and delivery routines ensure that BioProcess media are available in the right quantity, at the right place, at the right time. Amersham Pharmacia Biotech can assure future supplies of BioProcess Media, making them a safe investment for long term production. The media are produced following validated methods and tested under strict control to fulfil high performance specifications. A certificate of analysis is available with each order. Media
BioProcess
Regulatory support files contain details of performance, stability, extractable compounds and analytical methods. The essential information in these files gives an invaluable starting point for process validation, as well as providing support for submissions to regulatory authorities. Using BioProcess Media for every stage results in an easily validated process. High flow rates, high capacity and high recovery contribute to the overall economy of an industrial process. All BioProcess Media have chemical stability to allow efficient cleaning and sanitisation procedures. Packing methods are established for a wide range of scales and compatible large scale columns and equipment are available. 91
Reversed Phase Chromatography (RPC), Principles and Standard Conditions RPC separates proteins and peptides with differing hydrophobicity based on their reversible interaction with the hydrophobic surface of a chromatographic medium. Samples bind as they are loaded onto a column. Conditions are then altered so that the bound substances are eluted differentially. Due to the nature of the reversed phase matrices, the binding is usually very strong and requires the use of organic solvents and other additives (ion pairing agents) for elution. Elution is usually performed by increases in organic solvent concentration, most commonly acetonitrile. Samples, which are concentrated during the binding and separation process, are collected in a purified, concentrated form. The key stages in a separation are shown in Figure. 43. sample application
column equilibration
gradient elution
clean after gradient
re-equilibration
100% B
unbound molecules elute before gradient begins
2-4 cv
5-40 cv
5 cv 0
2 cv Column Volumes [cv]
Fig. 43. Typical RPC gradient elution.
RPC is often used in the final polishing of oligonucleotides and peptides and is ideal for analytical separations, such as peptide mapping. RPC is not recommended for protein purification if recovery of activity and return to a correct tertiary structure are required, since many proteins are denatured in the presence of organic solvents.
92
Choice of hydrophobic ligand Select C4, C8 or C18 n-alkyl hydrocarbon ligands according to the degree of hydrophobicity required. Highly hydrophobic molecules bind tightly to highly hydrophobic ligands, e.g. C18. Screen several RPC media. Begin with a medium of low hydrophobicity, e.g. C8, if the sample has very hydrophobic components (more likely with larger biomolecules, such as proteins). Select the medium which gives the best resolution and loading capacity. A polymer based medium such as SOURCE RPC can offer significant advantages over silica based media as it can be used across the pH range 1-14 providing not only an alternative selectivity to silica but also a wider working pH range for method optimisation.
Sample volume and capacity RPC is a binding technique, often independent of sample volume. Total capacity is strongly dependent upon experimental conditions and the properties of the gel and sample. For optimal conditions during gradient elution screen for a sample loading which does not reduce resolution.
Media selection In RPC the chromatographic medium as well as the hydrophobic ligand affect selectivity. Screening of different RPC media is recommended.
Sample Preparation Samples should be free from particulate matter and, when possible, dissolved in the start buffer.
Column Preparation Reversed phase columns should be ‘conditioned’ for first time use, after long term storage or when changing buffer systems.
Buffer Preparation Try these conditions first when sample characteristics are unknown: Start buffer A: 0.065% TFA (trifluoroacetic acid) in water Elution buffer B: 0.05% TFA in acetonitrile Gradient: 2-80% elution buffer in 20 column volumes
93
Method Development 1. Select medium from screening results. 2. Select optimum gradient to give acceptable resolution. For unknown samples begin 0-100%B. 3. Select highest flow rate which maintains resolution and minimises separation time. 4. For large scale purification transfer to a step elution. 5. Samples which adsorb strongly to a gel are more easily eluted by changing to a less hydrophobic medium.
Cleaning, sanitisation and sterilisation Procedures vary according to type of sample and medium. Guidelines are supplied with the medium or pre-packed column.
Storage of media and columns Recommended conditions for storage are supplied with the medium or pre-packed column.
Further information Reversed Phase: Principles and Methods Code No. 18-1112-93.
BioProcess Media- for large scale production Specific BioProcess Media have been designed for each chromatographic stage in a process from Capture to Polishing. Large capacity production integrated with clear ordering and delivery routines ensure that BioProcess media are available in the right quantity, at the right place, at the right time. Amersham Pharmacia Biotech can assure future supplies of BioProcess Media, making them a safe investment for long term production. The media are produced following validated methods and tested under strict control to fulfil high performance specifications. A certificate of analysis is available with each order. Regulatory support files contain details of performance, stability, extractable compounds and analytical methods. The essential information in these files gives an invaluable starting point for process validation, as well as providing support for submissions to regulatory authorities. Using BioProcess Media for every stage results in an easily validated process. High flow rates, high capacity and high recovery contribute to the overall economy of an industrial process. All BioProcess Media have chemical stability to allow efficient cleaning and sanitisation procedures. Packing methods are established for a wide range of scales and compatible large scale columns and equipment are available. Media
BioProcess
94
Expanded Bed Adsorption (EBA), Principles and Standard Conditions EBA is a single pass operation in which target proteins are purified from crude sample, without the need for separate clarification, concentration and initial purification to remove particulate matter. Crude sample is applied to an expanded bed of STREAMLINE adsorbent particles within a specifically designed STREAMLINE column. Target proteins are captured on the adsorbent. Cell debris, cells, particulate matter, whole cells, and contaminants pass through and target proteins are then eluted. Figure 44a shows a representation of the steps involved in an EBA purification and Figure 44b shows a typical EBA elution.
0.Sedimented adsorbent
1.Equilibration (expanded)
2.Sample appl. (expanded)
3.Washing (expanded)
4.Elution 5.Regeneration (packed bed) (packed bed)
Fig. 44a. Steps in an EBA purification process.
equilibration
Begin sample application adorbance
adsorption of sample and elution of unbound material
Begin wash with start buffer
Sample volumes
wash away unbound material
elute bound protein (s)
column wash
Change to elution buffer
Volume
Fig. 44b. Typical EBA elution.
95
Selection of STREAMLINE adsorbent Selection of adsorbent is based on the same principles that are used for chromatography. Select the medium showing the strongest binding to the target protein which binds as few of the contaminants as possible i.e. the medium with the highest selectivity and/or capacity for the protein of interest.
Sample volume and capacity All STREAMLINE media are binding techniques, independent of sample volume. The total amount of protein which is loaded should not exceed the total binding capacity of the column.
Sample Preparation STREAMLINE is able to handle crude, particulate feedstock, eliminating the need for significant sample preparation steps. Adjustment of pH or ionic strength may be required according to the separation principle being used (IEX, AC, HIC)
Column Preparation For preliminary method scouting STREAMLINE media is used in packed bed mode in an XK 16 or XK 26 chromatography column. When used in expanded bed mode the media must be packed in specially designed STREAMLINE columns, following the manufacturer's instructions.
Buffer Preparation Buffer preparation will depend upon the chosen separation principle.
Method Development 1. Select suitable ligand to bind the target protein. 2. Scout for optimal binding and elution conditions using clarified material in a packed column (0.02 - 0.15 litres bed volume of media). Gradient elutions may be used during scouting, but the goal is to develop a step elution. 3. Optimise binding, elution, wash and cleaning-in-place procedures using unclarified sample in expanded mode at small scale (0.02 - 0.15 litres bed volume of media) 4. Begin scale up process at pilot scale (0.2 - 0.9 litres bed volume of media) 5. Full scale production (up to several hundred litres bed volume of media)
96
Cleaning, sanitisation and sterilisation Guidelines are supplied with each STREAMLINE adsorbent.
Storage of STREAMLINE adsorbents and columns Recommended conditions for storage are supplied with STREAMLINE adsorbents and columns.
Further Information Expanded Bed Adsorption: Principles and Methods Code No. 18-1124-26
97
Additional reading and reference material Monoclonal Antibody Purification including MAbAssistant™ Recombinant Protein Handbook Gel Filtration Principles and Methods Ion Exchange Chromatography Principles and Methods Hydrophobic Interaction Chromatography Principles and Methods Affinity Chromatography Principles and Methods Reversed Phase Chromatography Principles and Methods Expanded Bed Adsorption Principles and Methods
Code Code Code Code Code Code Code Code
No. No. No. No. No. No. No. No.
18-1037-46 18-1105-02 18-1022-18 18-1114-21 18-1020-90 18-1022-29 18-1112-93 18-1124-26
Gel Filtration Columns and Media Selection Guide Ion Exchange Columns and Media Selection Guide HIC Columns and Media Product Profile Affinity Chromatography Columns and Media Product Profile
Code Code Code Code
No. No. No. No.
18-1124-19 18-1127-31 18-1100-98 18-1121-86
Sample Clean-up, Proteins and Peptides Convenient Protein Purification - HiTrap™ Column Guide Protein and Peptide Purification Technique Selection Protein Purification - major techniques poster Protein Purification - strategies poster
Code Code Code Code Code
No. No. No. No. No.
18-1128-62 18-1129-81 18-1128-63 18-1123-93 18-1129-75
Protein Purification, Principles, High Resolution Methods and Applications, J-C. Janson and L. Rydén, 1998, 2nd ed. Wiley VCH Code No. 18-1128-68 Handbook of Process Chromatography, G.Sofer and L.Hagel, 1997, Academic Press Code No. 18-1121-56 Protein Purification, Principles and Practice, R.K. Scopes. 1994, Springer Advanced Texts in Chemistry Ed. Springer Verlag New York Inc.
Handbook
Printed in Sweden by Snits & design AB / Graphium Västra Aros 9904
Protein Purification
Amersham Pharmacia Biotech AB, SE-751 84 Uppsala, Sweden
Reversed Phase Chromatography Principles and Methods
Back to Collection 18-1134-16 Edition AB
Reversed Phase Chromatography
Contents 1. Introduction .............................................................................................. 5 Theory of reversed phase chromatography ............................................... 6 The matrix ................................................................................................ 9 The ligands ............................................................................................. 11 Resolution in reversed phase chromatography ........................................ 13 Resolution .............................................................................................. 13 Capacity factor ....................................................................................... 14 Efficiency ................................................................................................ 15 Selectivity ................................................................................................ 17 Binding capacity ..................................................................................... 18 Critical parameters in reversed phase chromatography ........................... 19 Column length ................................................................................... 19 Flow rate ............................................................................................ 19 Temperature ....................................................................................... 20 Mobile phase ..................................................................................... 20 Organic solvent .................................................................................. 20 Ion suppression .................................................................................. 21 Ion pairing agents .............................................................................. 22 Gradient elution ................................................................................. 23 Mode of use ............................................................................................ 24 Desalting ............................................................................................ 24 High resolution separations ............................................................... 25 Large scale preparative purification ................................................... 25 Stages in a purification scheme .......................................................... 26 Capture .............................................................................................. 26 Intermediate stages ............................................................................. 27 Polishing ............................................................................................ 27 2. Product Guide ......................................................................................... 29 SOURCE™ RPC ..................................................................................... 30 Product description ............................................................................ 30 High chemical stability ...................................................................... 32 Excellent flow/pressure characteristics ................................................ 34 High capacity ..................................................................................... 35 Availability ........................................................................................ 36 µRPC C2/C18 ......................................................................................... 37 Product description ............................................................................ 37 Chemical and physical stability .......................................................... 38 Flow/pressure characteristics .............................................................. 38 Capacity ............................................................................................ 38 Availability ........................................................................................ 39 Sephasil™ Protein/Sephasil Peptide ......................................................... 39 Product description ............................................................................ 39 Chemical and physical stability .......................................................... 40 Flow/pressure characteristics .............................................................. 40 Availability ........................................................................................ 40
3. Methods ................................................................................................. 41 Choice of separation medium ................................................................. 41 Unique requirements of the application .............................................. 41 Resolution .......................................................................................... 41 Scale of the purification ..................................................................... 42 Mobile phase conditions .................................................................... 42 Throughput and scaleability .............................................................. 42 Molecular weight of the sample components ...................................... 42 Hydrophobicity of the sample components ........................................ 43 Class of sample components .............................................................. 43 Choice of mobile phase ........................................................................... 44 The organic solvent ............................................................................ 44 pH ..................................................................................................... 46 Ion pairing agents .............................................................................. 47 Sample preparation ................................................................................. 49 Mobile phase preparation ....................................................................... 50 Storage of mobile phase ..................................................................... 50 Solvent disposal ................................................................................. 50 Detection ................................................................................................ 51 Ghosting ............................................................................................ 51 Mobile phase balancing ..................................................................... 51 Column conditioning .............................................................................. 52 Elution conditions ................................................................................... 53 Column re-equilibration ......................................................................... 55 Column cleaning ..................................................................................... 55 Column storage ...................................................................................... 56 4. Applications ............................................................................................ 57 Designing a biochemical purification ...................................................... 57 Naturally occurring peptides and proteins .............................................. 58 Purification of platelet-derived growth factor (PDGF) ........................ 59 Trace enrichment ............................................................................... 59 Purification of cholecystokinin-58 (CCK-58) from pig intestine ......... 60 Recombinant peptides and proteins ........................................................ 62 Process purification of inclusion bodies .............................................. 63 Purification of recombinant human epidermal growth factor ............. 63 Chemically synthesised peptides .............................................................. 65 Purification of a phosphorylated PDGF α-receptor derived peptide .... 65 Structural characterisation of a 165 kDa protein ............................... 66 Protein fragments from enzyme digests ................................................... 66 Protein characterisation at the micro-scale ......................................... 66 Protein identification by LC-MS ......................................................... 69 Chemically synthesised oligonucleotides ................................................. 70 5. Fault finding chart .................................................................................. 72 6. References ............................................................................................... 81 7. Ordering information ............................................................................. 84
Chapter 1
Introduction Adsorption chromatography depends on the chemical interactions between solute molecules and specifically designed ligands chemically grafted to a chromatography matrix. Over the years, many different types of ligands have been immobilised to chromatography supports for biomolecule purification, exploiting a variety of biochemical properties ranging from electronic charge to biological affinity. An important addition to the range of adsorption techniques for preparative chromatography of biomolecules has been reversed phase chromatography in which the binding of mobile phase solute to an immobilised n-alkyl hydrocarbon or aromatic ligand occurs via hydrophobic interaction. Reversed phase chromatography has found both analytical and preparative applications in the area of biochemical separation and purification. Molecules that possess some degree of hydrophobic character, such as proteins, peptides and nucleic acids, can be separated by reversed phase chromatography with excellent recovery and resolution. In addition, the use of ion pairing modifiers in the mobile phase allows reversed phase chromatography of charged solutes such as fully deprotected oligonucleotides and hydrophilic peptides. Preparative reversed phase chromatography has found applications ranging from micropurification of protein fragments for sequencing (1) to process scale purification of recombinant protein products (2). This handbook is intended to serve as an introduction to the principles and applications of reversed phase chromatography of biomolecules and as a practical guide to the reversed phase chromatographic media available from Amersham Pharmacia Biotech. Among the topics included are an introductory chapter on the mechanism of reversed phase chromatography followed by chapters on product descriptions, applications, media handling techniques and ordering information. The scope of the information contained in this handbook will be limited to preparative reversed phase chromatography dealing specifically with the purification of proteins, peptides and nucleic acids.
5
Theory of reversed phase chromatography The separation mechanism in reversed phase chromatography depends on the hydrophobic binding interaction between the solute molecule in the mobile phase and the immobilised hydrophobic ligand, i.e. the stationary phase. The actual nature of the hydrophobic binding interaction itself is a matter of heated debate (3) but the conventional wisdom assumes the binding interaction to be the result of a favourable entropy effect. The initial mobile phase binding conditions used in reversed phase chromatography are primarily aqueous which indicates a high degree of organised water structure surrounding both the solute molecule and the immobilised ligand. As solute binds to the immobilised hydrophobic ligand, the hydrophobic area exposed to the solvent is minimised. Therefore, the degree of organised water structure is diminished with a corresponding favourable increase in system entropy. In this way, it is advantageous from an energy point of view for the hydrophobic moieties, i.e. solute and ligand, to associate (4).
b
a
Protein
Protein
c
+
Protein
Structured water Matrix
Fig. 1. Interaction of a solute with a typical reversed phase medium. Water adjacent to hydrophobic regions is postulated to be more highly ordered than the bulk water. Part of this ‘structured’ water is displaced when the hydrophobic regions interact leading to an increase in the overall entropy of the system.
Reversed phase chromatography is an adsorptive process by experimental design, which relies on a partitioning mechanism to effect separation. The solute molecules partition (i.e. an equilibrium is established) between the mobile phase and the stationary phase. The distribution of the solute between the two phases depends on the binding properties of the medium, the hydrophobicity of the solute and the composition of the mobile phase. Initially, experimental conditions are designed to favour adsorption of the solute from the mobile phase to the stationary phase. Subsequently, the mobile phase composition is modified to favour desorption of the solute from the stationary phase back into the mobile phase. In this case, adsorption is considered the extreme equilibrium state where the distribution of solute molecules is essentially 100% in the stationary phase. Conversely, desorption is an extreme equilibrium state where the solute is essentially 100% distributed in the mobile phase.
6
1 Starting conditions
2 Adsorption of sample substances
3 Start of desorption
4 End of desorption
5 Regeneration
Fig. 2. Principle of reversed phase chromatography with gradient elution.
Reversed phase chromatography of biomolecules generally uses gradient elution instead of isocratic elution. While biomolecules strongly adsorb to the surface of a reversed phase matrix under aqueous conditions, they desorb from the matrix within a very narrow window of organic modifier concentration. Along with these high molecular weight biomolecules with their unique adsorption properties, the typical biological sample usually contains a broad mixture of biomolecules with a correspondingly diverse range of adsorption affinities. The only practical method for reversed phase separation of complex biological samples, therefore, is gradient elution (5). In summary, separations in reversed phase chromatography depend on the reversible adsorption/desorption of solute molecules with varying degrees of hydrophobicity to a hydrophobic stationary phase. The majority of reversed phase separation experiments are performed in several fundamental steps as illustrated in Figure 2. The first step in the chromatographic process is to equilibrate the column packed with the reversed phase medium under suitable initial mobile phase conditions of pH, ionic strength and polarity (mobile phase hydrophobicity). The polarity of the mobile phase is controlled by adding organic modifiers such as acetonitrile. Ion-pairing agents, such as trifluoroacetic acid, may also be appropriate. The polarity of the initial mobile phase (usually referred to as mobile phase A) must be low enough to dissolve the partially hydrophobic solute yet high enough to ensure binding of the solute to the reversed phase chromatographic matrix. In the second step, the sample containing the solutes to be separated is applied. Ideally, the sample is dissolved in the same mobile phase used to equilibrate the chromatographic bed. The sample is applied to the column at a flow rate where optimum binding will occur. Once the sample is applied, the chromatographic bed is washed further with mobile phase A in order to remove any unbound and unwanted solute molecules. 7
Bound solutes are next desorbed from the reversed phase medium by adjusting the polarity of the mobile phase so that the bound solute molecules will sequentially desorb and elute from the column. In reversed phase chromatography this usually involves decreasing the polarity of the mobile phase by increasing the percentage of organic modifier in the mobile phase. This is accomplished by maintaining a high concentration of organic modifier in the final mobile phase (mobile phase B). Generally, the pH of the initial and final mobile phase solutions remains the same. The gradual decrease in mobile phase polarity (increasing mobile phase hydrophobicity) is achieved by an increasing linear gradient from 100% initial mobile phase A containing little or no organic modifier to 100% (or less) mobile phase B containing a higher concentration of organic modifier. The bound solutes desorb from the reversed phase medium according to their individual hydrophobicities. The fourth step in the process involves removing substances not previously desorbed. This is generally accomplished by changing mobile phase B to near 100% organic modifier in order to ensure complete removal of all bound substances prior to re-using the column. The fifth step is re-equilibration of the chromatographic medium from 100% mobile phase B back to the initial mobile phase conditions. Separation in reversed phase chromatography is due to the different binding properties of the solutes present in the sample as a result of the differences in their hydrophobic properties. The degree of solute molecule binding to the reversed phase medium can be controlled by manipulating the hydrophobic properties of the initial mobile phase. Although the hydrophobicity of a solute molecule is difficult to quantitate, the separation of solutes that vary only slightly in their hydrophobic properties is readily achieved. Because of its excellent resolving power, reversed phase chromatography is an indispensable technique for the high performance separation of complex biomolecules. Typically, a reversed phase separation is initially achieved using a broad range gradient from 100% mobile phase A to 100% mobile phase B. The amount of organic modifier in both the initial and final mobile phases can also vary greatly. However, routine percentages of organic modifier are 5% or less in mobile phase A and 95% or more in mobile phase B. The technique of reversed phase chromatography allows great flexibility in separation conditions so that the researcher can choose to bind the solute of interest, allowing the contaminants to pass unretarded through the column, or to bind the contaminants, allowing the desired solute to pass freely. Generally, it is more appropriate to bind the solute of interest because the desorbed solute elutes from the chromatographic medium in a concentrated state. Additionally, since binding under the initial mobile phase conditions is complete, the starting concentration of desired solute in the sample solution is not critical allowing dilute samples to be applied to the column. 8
The specific conditions under which solutes bind to the reversed phase medium will be discussed in the appropriate sections in greater detail. Ionic binding may sometimes occur due to ionically charged impurities immobilised on the reversed phase chromatographic medium. The combination of hydrophobic and ionic binding effects is referred to as mixed-mode retention behaviour. Ionic interactions can be minimised by judiciously selecting mobile phase conditions and by choosing reversed phase media which are commercially produced with high batch-to-batch reproducibility and stringent quality control methods.
The matrix Critical parameters that describe reversed phase media are the chemical composition of the base matrix, particle size of the bead, the type of immobilised ligand, the ligand density on the surface of the bead, the capping chemistry used (if any) and the pore size of the bead. A reversed phase chromatography medium consists of hydrophobic ligands chemically grafted to a porous, insoluble beaded matrix. The matrix must be both chemically and mechanically stable. The base matrix for the commercially available reversed phase media is generally composed of silica or a synthetic organic polymer such as polystyrene. Figure 3 shows a silica surface with hydrophobic ligands. Fig. 3. Some typical structures on the surface of a silica-based reversed phase medium. The hydrophobic octadecyl group is one of the most common ligands.
—Si—OH
—
—Si
—
—Si
Residual silanol group
O
Ether; source of silanols — —
CH3
—Si—O—Si—(CH2)17—CH3
Octadecyl group
CH3 — —
CH3
—Si—O—Si—CH2—CH3
C2 capping group
CH3
9
Silica was the first polymer used as the base matrix for reversed phase chromatography media. Reversed phase media were originally developed for the purification of small organic molecules and then later for the purification of low molecular weight, chemically synthesised peptides. Silica is produced as porous beads which are chemically stable at low pH and in the organic solvents typically used for reversed phase chromatography. The combination of porosity and physical stability is important since it allows media to be prepared which have useful loading capacities and high efficiencies. It is worth noting that, although the selectivity of silica-based media is largely controlled by the properties of the ligand and the mobile phase composition, different processes for producing silica-based matrices will also give media with different patterns of separation. The chemistry of the silica gel allows simple derivatisation with ligands of various carbon chain lengths. The carbon content, and the surface density and distribution of the immobilised ligands can be controlled during the synthesis. The primary disadvantage of silica as a base matrix for reversed phase media is its chemical instability in aqueous solutions at high pH. The silica gel matrix can actually dissolve at high pH, and most silica gels are not recommended for prolonged exposure above pH 7.5. Synthetic organic polymers, e.g. beaded polystyrene, are also available as reversed phase media. Polystyrene has traditionally found uses as a solid support in peptide synthesis and as a base matrix for cation exchange media used for separation of amino acids in automated analysers. The greatest advantage of polystyrene media is their excellent chemical stability, particularly under strongly acidic or basic conditions. Unlike silica gels, polystyrene is stable at all pH values in the range of 1 to 12. Reversed phase separations using polystyrene-based media can be performed above pH 7.5 and, therefore, greater retention selectivity can be achieved as there is more control over the degree of solute ionisation. —CH2—CH—CH2—CH—CH2—CH—CH2—CH—CH2—CH
—CH2—CH—CH2—CH—CH2—CH—CH2—CH—CH2—CH
—CH2—CH—CH2—CH—CH2—CH—CH2—CH—CH2—CH
—CH2—CH—CH2—CH—CH2—CH—CH2—CH—CH2—CH
—CH2—CH—CH2—CH—CH2—CH—CH2—CH—CH2—CH
Fig. 4. Partial structure of a polystyrene-based reversed phase medium.
10
50–100 Å Narrow pore
300 Å Wide pore
Restricted mass transfer
More efficient mass transfer
Fig. 5. Reverse phase media with wide pores allow the most efficient transfer of large solute molecules between the mobile and the stationary phases.
The surface of the polystyrene bead is itself strongly hydrophobic and, therefore, when left underivatised unlike silica gels that have hydrophobic ligands grafted to a hydrophilic surface. The porosity of the reversed phase beads is a crucial factor in determining the available capacity for solute binding by the medium. Note that this is not the capacity factor (k´) but the actual binding capacity of the medium itself. Media with pore sizes of approximately 100 Å are used predominately for small organic molecules and peptides. Media with pore sizes of 300 Å or greater can be used in the purification of recombinant peptides and proteins that can withstand the stringent conditions of reversed phase chromatography.
The ligands The selectivity of the reversed phase medium is predominantly a function of the type of ligand grafted to the surface of the medium. Generally speaking, linear hydrocarbon chains (n-alkyl groups) are the most popular ligands used in reversed phase applications. Some typical hydrocarbon ligands are shown in Figure 6. Fig. 6. Typical n-alkyl hydrocarbon ligands. (A) Two-carbon capping group, (B) Octyl ligand, (C) Octadecyl ligand. CH3 (A) —O—Si—CH2—CH3 CH3 CH3 (B) —O—Si—CH2—CH2—CH2—CH2—CH2—CH2—CH2—CH3 CH3 CH3 (C) —O—Si—CH2—CH2—CH2—CH2—CH2—CH2—CH2—CH2—CH2—CH2—CH2—CH2—CH2—CH2—CH2—CH2—CH2—CH CH3
11
Although it is not possible to predict theoretically which ligand will be best for a particular application, a good rule of thumb is: the more hydrophobic the molecule to be purified, the less hydrophobic the ligand needs to be. The more hydrophilic molecules tend to require strongly hydrophobic immobilised ligands in order to achieve sufficient binding for separation. Typically, chemically synthesised peptides and oligonucleotides are efficiently purified on the more hydrophobic C18 ligands. Proteins and recombinant peptides, because of their size, behave as hydrophobic molecules and most often bind very strongly to C18 ligands. They are usually better separated on C8 ligands. The less hydrophobic eight carbon alkyl chain is less disruptive to the protein and peptide structures since lower concentrations of organic solvent are required for elution. In addition to ligand structure, the density of the immobilised hydrocarbon ligands on the silica surface also influences the selectivity shown by reversed phase media. Therefore, reproducible chemical derivatisation of the silica surface is critical for efficient reversed phase chromatography with consistent batch-to-batch selectivity. The hydrocarbon ligands are generally coupled to the silica gel via silanol groups on the silica surface using chlorotrialkylsilane reagents. Not all of the silanol groups will be substituted in this coupling reaction. The C18 and C8 reagents are large and bulky so that steric hindrance often prevents complete derivatisation of all the available silanol groups. The residual silanol groups are believed to be responsible for the deleterious mixed mode ion exchange effects often present during reversed phase separation of biomolecules. In order to reduce these damaging side effects, the residual silanol groups are reacted with smaller alkylsilane reagents (chlorotrimethyl- and chlorotriethylsilanes) where steric effects do not interfere with complete coverage of the silanol groups remaining on the surface of the silica gel. This process is referred to as “end-capping”. The extent of end-capping also affects selectivity, so reproducibility in the capping step is critical for a well behaved reversed phase medium. The derivatisation reaction is shown in Figure 7. Fig. 7. Substitution of silica with octadecyl chains by reaction with monochlorodimethyloctadecylsilane.
CH3 —Si—OH + Cl—Si—(CH2)17—CH3 CH3
CH3 —Si—O—Si—(CH2)17—CH3 + HCl CH3
The particle size of the bead, as measured by its diameter, has important consequences for the size of the chromatographic bed which can be usefully packed, and for the efficiency of the separation. The larger particle size media are generally used for large scale preparative and process applications due to their increased capacity and lower pressure requirements at high flow rates. Small scale preparative and analytical separations use smaller particles since separation efficiency, i.e. peak width, is directly related to particle size (see section on 12
Fig. 8. Determination of the resolution between two peaks. v2 v1
R s=
w1
v2–v1 (w2 + w1)/2
w2
efficiency and selectivity). Analytical and small scale preparative applications are usually performed with 3 and 5 µm beads while larger scale preparative applications (pilot and process scale) are usually performed with particle sizes of 15 µm and greater. Micropreparative and small scale preparative work can be accomplished using particle sizes of 3 µm since the limited capacity of the small columns packed with these media is not a severe problem when only small quantities of material are purified.
Resolution in reversed phase chromatography Resolution Adequate resolution and recovery of purified biological material is the ultimate goal for reversed phase preparative chromatography. Resolution, Rs, is generally defined as the distance between the centres of two eluting peaks as measured by retention time or volume divided by the average width of the respective peaks (Fig. 8). For example, an Rs value of 1.0 indicates 98% purity has been achieved (assuming 98% peak recovery). Baseline resolution between two well formed peaks indicates 100% purity and requires an Rs value greater than 1.5 (Fig. 9). Calculating Rs is the simplest method for quantitating the actual separation achieved between two solute molecules. This simple relationship can be expanded to demonstrate the connection between resolution and three fundamental parameters of a chromatographic separation. The parameters have been derived from chromatographic models based on isocratic elution but are still appropriate when used to describe their effects on resolution when discussing gradient elution (consider a continuous linear gradient elution to be a series of small isocratic elution steps). The parameters that contribute to peak resolution are column selectivity, column efficiency and the column retention factor. 13
B
A
98% A
A
98% B
Rs=1
100% A
B
100% B
Rs=1.5
Fig. 9. Separation results with different resolution.
Rs=
1 (α-1) 4
(
α
N
)
k´ 1 + k
Resolution Rs is a function of selectivity α, efficiency (number of theoretical plates N) and the average retention factor, k´, for peaks 1 and 2.
Capacity factor The capacity factor, k´, is related to the retention time and is a reflection of the proportion of time a particular solute resides in the stationary phase as opposed to the mobile phase. Long retention times result in large values of k´. The capacity factor is not the same as the available binding capacity which refers to the mass of the solute that a specified amount of medium is capable of binding under defined conditions. The capacity factor, k´, can be calculated for every peak defined in a chromatogram, using the following equations.
Capacity factor=k´=
moles of solute in stationary phase moles of solute in mobile phase
k´=
TR – TO TO
VR – VO VO
where TR and VR are the retention time and retention volume, respectively, of the solute, and To and Vo the retention time and retention volume, respectively, of an unretarded solute.
14
Fig. 10. Hypothetical chromatogram. VO=void volume, VC=total volume, V1, V2, and V3 are the elution (retention) volumes of peaks 1, 2 and 3, respectively.
1
2
3
vc v0 v1 v2
v3
In the resolution equation previously described, the k´ value is the average of the capacity factors of the two peaks being resolved. Unlike non-adsorptive chromatographic methods (e.g. gel filtration), reversed phase chromatography can have very high capacity factors. This is because the experimental conditions can be chosen to result in peak retention times greatly in excess of the total column volume.
Efficiency (N) The efficiency of a packed column is expressed by the number of theoretical plates, N. N is a dimensionless number and reflects the kinetics of the chromatographic retention mechanism. Efficiency depends primarily on the physical properties of the chromatographic medium together with the chromatography column and system dimensions. The efficiency can be altered by changing the particle size, the column length, or the flow rate. The expression “number of theoretical plates” is an archaic term carried over from the theoretical comparison of a chromatography column to a distillation apparatus. The greater the number of theoretical plates a column has, the greater its efficiency and correspondingly, the higher the resolution which can be achieved. The column efficiency (N) can be determined empirically using the equation below based upon the zone broadening that occurs when a solute molecule is eluted from the column (Fig. 11). The number of theoretical plates, N, is given by N = 5.54 (V1/W1/2)2 where V1 is the retention volume of the peak and W1/2 is the peak width (volume) at half peak height.
15
Abs
Fig. 11. Measurements for determining column efficiency. V1 is the retention volume of the peak and W1/2 is the peak width (volume) at half peak height.
v1 Peak height
w1/2
Volume
The number of theoretical plates is sometimes reported as plates per metre of column length (N/L). The height equivalent to a theoretical plate, H, is given by H = L/N where L is the column length and N is the number of theoretical plates. Any parameter change that increases N will also increase Rs. The relationship between the two is defined by the square root of N. For example, an increase in N from 100 to 625 will improve the resolution by a factor of 2.5, rather than 6.25. The main contribution to column efficiency (N) is particle size and the efficacy of the column packing procedure. It should be noted that the smaller the particle size, the more difficult it is to pack an efficient column. This is the reason why reversed phase media with particle sizes less than 10 µm are commercially available only in pre-packed formats.
16
Selectivity Selectivity (α) is equivalent to the relative retention of the solute peaks and, unlike efficiency, depends strongly on the chemical properties of the chromatography medium. The selectivity, α, for two peaks is given by α = k2´ /k1´ = V2 - V0/V1 – V0 = V2/V1 where V1 and V2 are the retention volumes, and k2´ /k1´ are the capacity factors, for peaks 1 and 2 respectively, and V0 is the void volume of the column. Selectivity is affected by the surface chemistry of the reversed phase medium, the nature and composition of the mobile phase, and the gradient shape. Fig. 12. Selectivity comparison between different silica based media at pH 2.0 and pH 6.5. A mixture of closely related angiotensin peptides was used as sample. (Work by Amersham Pharmacia Biotech AB, Uppsala, Sweden.) 1. 2. 3. 4. 5. 6. 7. 8.
Columns:
Val4-lle7-AT III (RVYVHPI) Ile7-AT III (RVYIHPI) Val4-AT III (RVYVHPF) Sar1-Leuß-AT II (Sar-RVYIHPL) (Sar=sarcosine, N-methylglycine) AT III (RVYIHPF) AT II (DRVYIHPF) des-Asp1-AT I (RVYIHPLFHL) AT I (DRVYIHPFHL)
Sephasil Protein C4
c)
b)
7+8 5+6
2+3
pH 2
7+8 5+6
pH 2
pH 2
4 34 1
7+8
d)
7+8
pH 2
1
µRPC C2/C18
Sephasil Peptide C18
Sephasil Peptide C8
5+6
a)
Eluent A (pH 2): Eluent B (pH 2): Eluent A (pH 6.5): Eluent B (pH 6.5): Flow: System: Gradient:
a) and e) Sephasil Protein C4 5 µm 4.6/100 b) and f) Sephasil Peptide C8 5 µm 4.6/100 c) and g) Sephasil Peptide C18 5 µm 4.6/100 d) and h) µRPC C2/C18 ST 4.6/100 0.065% TFA in distilled water 0.05% TFA, 75% acetonitrile 10 mM phosphate 10 mM phosphate, 75% acetonitrile 1 ml/min ÄKTApurifier 5–95% B in 20 column volumes
3
2 1
4 1
2
6 4 5 2 3
0.0
5.0 10.0 15.0 20.0 25.0 min
e)
0.0
3+4 1
5.0 10.0 15.0 20.0 25.0 min
2
6 5 8
43 2
pH 6.5
7
5.0 10.0 15.0 20.0 25.0 min
0.0
g)
f)
6 5
pH 6.5
0.0
2
h)
8
pH 6.5
5.0 10.0 15.0 20.0 25.0 min
1
6 43 8 2 5
1
3
4 7+8
7
pH 6.5
5+6
7
1
0.0
5.0 10.0 15.0 20.0 25.0 min
0.0
5.0 10.0 15.0 20.0 25.0 min
0.0
5.0 10.0 15.0 20.0 25.0 min
0.0
5.0 10.0 15.0 20.0 25.0 min
17
Good selectivity
Poor selectivity
High efficiency
Low efficiency
High efficiency
Low efficiency
Fig. 13. The effect of selectivity and efficiency on resolution.
Both high column efficiency and good selectivity are important to overall resolution. However, changing the selectivity in a chromatographic experiment is easier than changing the efficiency. Selectivity can be changed by changing easily modified conditions like mobile phase composition or gradient shape.
Binding capacity The available binding capacity of a reversed phase medium is a quantitative measure of its ability to adsorb solute molecules under static conditions. The dynamic binding capacity is a measure of the available binding capacity at a specific flow rate. Both values are extremely important for preparative work. The amount of solute which will bind to a medium is proportional to the concentration of immobilised ligand on the medium and also depends on the type of solute molecule being adsorbed to the medium. The available and dynamic binding capacities depend on the specific chemical and physical properties of the solute molecule, the properties of the reversed phase medium (porosity, etc.) and the experimental conditions during binding. The porosity of the bead is an important factor which influences binding capacity. The entire hydrophobic surface of macroporous media is available for binding solute. Large solute molecules (i.e. high molecular weight) may be excluded from media of smaller pore size and only a small fraction of the whole hydrophobic surface will be used. When maximum binding capacity is required, a medium with pores large enough to allow all the molecules of interest to enter freely must be used.
18
Critical parameters in reversed phase chromatography Column length The resolution of high molecular weight biomolecules in reversed phase separations is less sensitive to column length than is the resolution of small organic molecules. Proteins, large peptides and nucleic acids may be purified effectively on short columns and increasing column length does not improve resolution significantly. The resolution of small peptides (including some peptide digests) may sometimes be improved by increasing column length. For example, the number of peaks detected when a tryptic digest of carboxamidomethylated transferrin was fractionated by RPC increased from 87 on a 5 cm long column to 115 on a 15 cm long column and 121 on a 25 cm long column (6). The partition coefficients of high molecular weight solutes are very sensitive to small changes in mobile phase composition and hence large molecules desorb in a very narrow range of organic modifier concentration. The retention behaviour of large molecules may be considered to be governed by an on/off mechanism (i.e. a large change in partition coefficient) which is insensitive to column length. When small changes in organic modifier concentration result in small changes in the partition coefficient, longer column lengths increase resolution. The use of gradient elution further reduces the significance of column length for the resolution of large biomolecules by reversed phase chromatography. Gradients are required since most biological samples are complex mixtures of molecules that vary greatly in their adsorption to the reversed phase medium. Due to this variety of adsorption affinities, the mobile phase must have a broad range of eluting power to ensure elution of all the bound solute molecules. Under these conditions, especially with moderate to steep gradient slopes, column length is not a critical factor with regard to resolution.
Flow rate Flow rate is expected to be an important factor for resolution of small molecules, including small peptides and protein digests, in reversed phase separations. However, reversed phase chromatography of larger biomolecules, such as proteins and recombinantly produced peptides, appears to be insensitive to flow rate. In fact, low flow rates, typically used with long columns, may actually decrease resolution due to increased longitudinal diffusion of the solute molecules as they traverse the length of the column. The flow rate used during the loading of the sample solution is especially significant in large scale preparative reversed phase chromatography, although not critical during analytical experiments. Dynamic binding capacity will vary depending on the flow rate used during sample loading. When scaling up a purification, the dynamic binding capacity should be determined in order to 19
assess the optimum flow rate for loading the sample. Dynamic binding capacity is a property of the gel that reflects the kinetics of the solute binding process. The efficiency of this step can have enormous consequences for the results of a large scale preparative purification.
Temperature Temperature can have a profound effect on reversed phase chromatography, especially for low molecular weight solutes such as short peptides and oligonucleotides. The viscosity of the mobile phase used in reversed phase chromatography decreases with increasing column temperature. Since mass transport of solute between the mobile phase and the stationary phase is a diffusion-controlled process, decreasing solvent viscosity generally leads to more efficient mass transfer and, therefore, higher resolution. Increasing the temperature of a reversed phase column is particularly effective for low molecular weight solutes since they are suitably stable at the elevated temperatures.
Mobile phase In many cases, the colloquial term used for the mobile phases in reversed phase chromatography is “buffer”. However, there is little buffering capacity in the mobile phase solutions since they usually contain strong acids at low pH with large concentrations of organic solvents. Adequate buffering capacity should be maintained when working closer to physiological conditions.
Organic solvent The organic solvent (modifier) is added to lower the polarity of the aqueous mobile phase. The lower the polarity of the mobile phase, the greater its eluting strength in reversed phase chromatography. Although a large variety of organic solvents can be used in reversed phase chromatography, in practice only a few are routinely employed. The two most widely used organic modifiers are acetonitrile and methanol, although acetonitrile is the more popular choice. Isopropanol (2propanol) can be employed because of its strong eluting properties, but is limited by its high viscosity which results in lower column efficiencies and higher backpressures. Both acetonitrile and methanol are less viscous than isopropanol. All three solvents are essentially UV transparent. This is a crucial property for reversed phase chromatography since column elution is typically monitored using UV detectors. Acetonitrile is used almost exclusively when separating peptides. Most peptides only absorb at low wavelengths in the ultra-violet spectrum (typically less than 225 nm) and acetonitrile provides much lower background absorbance than other common solvents at low wavelengths.
20
The retention, or capacity factor (k´), for a given solute is a function of the mobile phase polarity. The elution order can be affected by changing the type of organic modifier or by the addition of ion pairing agents. Changes in elution order are most pronounced for proteins that are denatured in organic solvents. Denaturation of the protein can result in a change in its hydrophobicity. Ion suppression The retention of peptides and proteins in reversed phase chromatography can be modified by mobile phase pH since these particular solutes contain ionisable groups. The degree of ionisation will depend on the pH of the mobile phase. The stability of silica-based reversed phase media dictates that the operating pH of the mobile phase should be below pH 7.5. The amino groups contained in peptides and proteins are charged below pH 7.5. The carboxylic acid groups, however, are neutralised as the pH is decreased. The mobile phase used in reversed phase chromatography is generally prepared with strong acids such as trifluoroacetic acid (TFA) or ortho-phosphoric acid. These acids maintain a low pH environment and suppress the ionisation of the acidic groups in the solute molecules. Varying the concentration of strong acid components in the mobile phase can change the ionisation of the solutes and, therefore, their retention behaviour. The major benefit of ion suppression in reversed phase chromatography is the elimination of mixed mode retention effects due to ionisable silanol groups remaining on the silica gel surface. The effect of mixed mode retention is increased retention times with significant peak broadening.
Fig. 14. Typical effects of mixed-mode retention. Peaks are broader and skewed, and retention time increases.
(A) Reversed phase chromatography
(B) Mixed-mode
21
Mixed mode retention results from an ion exchange interaction between negatively charged silanol groups exposed on the surface of the silica and the positively charged amino groups on the solute molecules. Silanol groups on the surface of silica-based media can arise from two primary sources. The first is due to inadequate end-capping procedures during the manufacture of the gel. It is critical to choose a manufacturer that produces a gel with reproducibly low mixed mode retention effects, since these artefact can affect resolution. The other source of surface silanol groups is column ageing. The silica gel surface is continually eroded during the life of the column, resulting in exposed silanol groups and progressive deterioration in column performance. Prolonged exposure to aqueous solutions can accelerate column ageing. The low pH environment (usually less than pH 3.0) of typical reversed phase mobile phases suppresses the ionisation of these surface silanol groups so that the mixed mode retention effect is diminished. Ion suppression is not necessary when dealing with reversed phase media based on polystyrene or other synthetic organic polymers. Polystyrene media are stable between pH 1-12 and do not exhibit the mixed mode retention effects that silica gels do with mobile phases at high pH. Ion pairing agents The retention times of solutes such as proteins, peptides and oligonucleotides can be modified by adding ion pairing agents to the mobile phase. Ion pairing agents bind to the solute by ionic interactions, which results in the modification of the solute hydrophobicity. Examples of ion pairing agents are shown in chapter 3.
Fig. 15. Ion pair formation with (A) anionic or (B) cationic ion pairing agents.
Positively charged peptide
+
+ +
– + –
– + Negatively charged ion pairing agent with hydrophobic surface
+ –
+
+ –
– +
Negatively charged oligonucliotide
–
– –
22
+ –
+ –
+
Positively charged ion pairing agent with hydrophobic surface
– +
– + –
++
Both anionic and cationic ion pairing agents are used depending on the ionic character of the solute molecule and the pH of the mobile phase. For example, a typical ion pairing agent for peptides at pH less than 3.5 is trifluoroacetic acid. The ion pairing agent used with oligonucleotides, which contain a negative charge at neutral to high pH, is typically triethylamine. In some cases the addition of ion pairing agents to the mobile phase is an absolute requirement for binding of the solute to the reversed phase medium. For example, retention of deprotected synthetic oligonucleotides, i.e. without the trityl protecting group attached, requires triethylamine in the mobile phase. The same is true for hydrophilic peptides where binding is negligible in the absence of a suitable ion pairing agent such as trifluoroacetic acid. The concentration of ion pairing agents in the mobile phases is generally in the range 0.1 - 0.3%. Potential problems include possible absorbance of UV light by the ion pairing agent and changes in extinction coefficient with concentration of organic modifier. This can result in either ascending or descending baselines during gradient elution.
Gradient elution Gradient elution is the method of choice when performing preparative reversed phase chromatography of biomolecules. The typical gradients for preparative reversed phase chromatography of proteins and peptides are linear and binary, i.e. involving two mobile phases. Convex and concave gradients are used occasionally for analytical purposes particularly when dealing with multicomponent samples requiring extra resolution either at the beginning or at the end of the gradient. The concentration of organic solvent is lower in the initial mobile phase (mobile phase A) than it is in the final mobile phase (mobile phase B). The gradient then, regardless of the absolute change in percent organic modifier, always proceeds from a condition of high polarity (high aqueous content, low concentration of organic modifier) to low polarity (lower aqueous content, higher concentration of organic modifier). Gradient shape (combinations of linear gradient and isocratic conditions), gradient slope and gradient volume are all important considerations in reversed phase chromatography. Typically, when first performing a reversed phase separation of a complex sample, a broad gradient is used for initial screening in order to determine the optimum gradient shape. After the initial screening is completed, the gradient shape may adjusted to optimise the separation of the desired components. This is usually accomplished by decreasing the gradient slope where the desired component elutes and increasing it before and after. The choice of gradient slope will depend on how 23
closely the contaminants elute to the target molecule. Generally, decreasing gradient slope increases resolution. However, peak volume and retention time increase with decreasing gradient slope. Shallow gradients with short columns are generally optimal for high molecular weight biomolecules. Gradient slopes are generally reported as change in percent B per unit time (%B/ min.) or per unit volume (%B/ml). When programming a chromatography system in time mode, it is important to remember that changes in flow rate will affect gradient slope and, therefore, resolution. Resolution is also affected by the total gradient volume (gradient time x flow rate). Although the optimum value must be determined empirically, a good rule of thumb is to begin with a gradient volume that is approximately ten to twenty times the column volume. The slope can then be increased or decreased in order to optimise resolution.
Mode of use Desalting Desalting is a routine laboratory procedure in which low molecular weight contaminants are separated from the desired higher molecular weight biomolecules. The procedure is sometimes simply referred to as buffer exchange. Non-chromatographic techniques for buffer exchange include ultra-filtration and dialysis. Desalting is used in the laboratory primarily for sample preparation, e.g. desalting fractions obtained by other methods such as ion exchange chromatography. Size exclusion chromatography (gel filtration) with Sephadex™ G-25 is commonly used for desalting proteins and nucleic acids, and Sephadex G-10 is used for desalting small peptides. Size exclusion chromatography is a valuable method for desalting due to its simplicity and gentleness, although it suffers from the unwanted side effect of sample dilution. Proteins, peptides and oligonucleotide samples can be conveniently desalted using reversed phase chromatography. When desalting samples using reversed phase techniques, the samples can be recovered and reconstituted into small volumes thereby avoiding the sample dilution effects of gel filtration. The sample is passed through a small reversed phase column where it binds and concentrates on the reversed phase medium. Unlike gel filtration, reversed phase is an adsorption technique and sample volume is not limited. Reversed phase chromatography columns can concentrate large volumes of dilute samples at the same time as desalting them. After the entire sample has been processed, the bound solute is eluted using a small volume of low polarity mobile phase, typically acetonitrile. If the solvent is volatile, as acetonitrile is, it can then be removed by evaporation and the sample residue re-suspended in the desired volume of new buffer. 24
(A)
Gel filtration Protein
(B) Salt
Protein etc
Reversed phase chromatography Salt
Non-polar eluent
vo
vc
vo
vc
Fig. 16. Desalting by (A) gel filtration and (B) reversed phase chromatography. The large molecules elute first in gel filtration; the salt elutes without changing the eluent. The salt elutes first in reversed phase chromatography; a less-polar eluent is needed to elute proteins and other molecules which are retained on the column.
High resolution separations Reversed phase chromatography is most typically used as a high resolution technique, where its inherent robustness is especially advantageous. However, certain applications push the resolving power of the reversed phase technique to its limit. These tend to be in the intermediate stages of preparative applications or when isolating structurally similar components from a complex mixture. Examples include isolation of specific peptides from enzymatic digests or purification of oligonucleotides from a complicated mixture of oligonucleotide contaminants. In these cases, a great many peaks must be resolved from each other and recovered in sufficient amounts for further analysis. Reversed phase media of very small particle size, typically 3 and 5 µm beads, are usually required together with painstaking attention given to details such as column temperature, gradient slope and mobile phase composition. When dealing with smaller solutes, such as short oligonucleotides, digested protein fragments and short peptides, the optimisation of other factors such as flow rate and column length may also be necessary in order to maximise resolution.
Large scale preparative purification The large scale purification of biomolecules such as synthetic oligonucleotides and peptides, and recombinant peptides and proteins by reversed phase chromatography requires both high resolution separation together with the ability to scale up the purification. In these cases, the purification is optimised using a small particle reversed phase medium and then scaled up accordingly using a medium with similar selectivity but with a larger particle size. The techniques of scale up used with reversed phase chromatography are similar to those used with other chromatographic techniques such as ion exchange. Specific examples of preparative, large scale reversed phase purification of biomolecules are shown in chapter 4.
25
Start material Purification stage
Demands Throughput tolerate crude feed cleaning in place
Capture
Intermediate purification
Resolution reproducibility cleaning in place
Polishing
Pure product
Fig. 17. Stages in a purification scheme.
Stages in a purification scheme Once the source for a biomolecule has been determined, whether microbial, chemical, natural or other, and the starting material has been produced in sufficiently large quantities, the desired substance must then be purified from contaminants present in the crude sample. There are essentially three functional stages in the purification of a biomolecule from a crude preparation or extract. These are referred to as Capture, Intermediate Purification and Polishing. The suitability of any separation technique, including reversed phase chromatography, at any stage of purification will always depend on the specific sample, the specific separation problem at hand and the intended use of the purified material. Capture Capture is the first step in the purification procedure. At this stage, the sample volume is usually at its largest and the sample may contain particulates or viscous materials. The purpose of the capture step is to isolate, concentrate and stabilise the target molecule from the crude preparation rapidly, and with good recovery. The capture step is not expected to be highly resolving but is required to isolate the molecule of interest from contaminating substances that are dissimilar to the desired molecule. The capture step may be considered as a group separation rather than as a high resolution purification. Consequently, time, capacity and recovery are more important than resolution in a successful capture step. Reversed phase chromatography is a suitable method for the capture of synthetic peptides and synthetic oligonucleotides. However, it is usually less suitable for capture of peptides and proteins from biological sources. This is because of the presence of lipids and other highly hydrophobic solutes which bind strongly, reduce the dynamic capacity for the molecule of interest, and can be difficult to remove from the column. Additionally, the small particle size of most reversed phase media requires particulates to be removed from the sample to prevent the column from clogging. Ion exchange chromatography and hydrophobic interaction chromatography using bead diameters greater than 90 µm are better suited for capture in these instances. 26
Fig. 18. Steps during capture.
Apply sample
Elute unbound sugars, salts etc
Elute peptides, proteins etc
Intermediate stages In the intermediate purification phase the focus is to separate the target molecule from most of the bulk impurities such as other proteins, peptides, nucleic acids, endotoxins and viruses. An ability to resolve similar components is of increased importance since contaminants at this stage are often similar to the target molecule in terms of functional or structural properties. The critical requirements are recovery and resolution. Reversed phase chromatography is a suitable technique for this stage of the purification because of the high resolution that can be achieved. Polishing Polishing is the final step in the preparation of a pure product. The polishing step is used to remove trace contaminants and impurities. The purified biomolecule should be in a form suitable for its intended use. Contaminants at the polishing stage are often very similar to the target molecule. Typical contaminants may include “conformers” and structural variants of the target molecule. Structural variants can include dimers, oligomers, aggregates, oxidised amino acids, protease-clipped molecules, desamidated amino acids etc. Other microheterogeneities may also occur. In process related applications, polishing also removes final traces of leachables, endotoxins, viruses etc. The goals of the polishing stage might be product purity of 100% in less than two steps with a recovery of greater than 99%. Polishing can be performed using size exclusion, especially when dimers and aggregates must be removed. However, when dealing with slight structural variants and micro-heterogeneities, reversed phase chromatography with its excellent resolving power is the method of choice. 27
28
Chapter 2
Product Guide Reversed phase media from Amersham Pharmacia Biotech provide a broad range of selectivity for different applications for use at analytical, laboratory and production scale. Table 1 below reviews briefly the main characteristics of the media together with their application suitability. Media
Medium
Particle size (approx)
SOURCE™ 5RPC
Polystyrene/ divinylbenzene
5 µm monosized
SOURCE 15RPC
Polystyrene/ divinylbenzene
15 µm monosized
Preparative purification of proteins, peptides and oligonucleotides. Alternative selectivity to silica, especially for separations performed at high pH. Excellent pressure/ flow characteristics.
SOURCE 30RPC
Polystyrene/ divinylbenzene
30 µm monosized
Large scale purification of proteins, peptides and oligonucleotides. Alternative selectivity to silica, especially for separations performed at high pH. Excellent pressure/ flow characteristics.
µRPC C2/C18
Silica
3 µm
High efficiency media, for peptide mapping, analysis and micropurification.
Sephasil™ Protein C4 Silica Sephasil Peptide C8 Sephasil Peptide C18
5 µm
High resolution analysis and purification. Suitable for recombinant and synthetic peptides.
Sephasil Protein C4 Silica Sephasil Peptide C8 Sephasil Peptide C18
12 µm
Preparative purification of peptides, proteins and oligonucleotides.
Sephasil C8 Sephasil C18
5 µm
SMART™ system pre-packed columns. Micropurification and analysis.
Table 1
Silica
Applications High resolution analysis and small scale purification. Alternative selectivity to silica, especially for separations performed at high pH. Ideal for recombinant and synthetic peptides and oligonucleotides.
29
SOURCE RPC Product Description SOURCE RPC media are designed for analytical and preparative chromatography of synthetic peptides, oligonucleotides and proteins. SOURCE RPC is based on rigid, monosized, polystyrene/divinyl benzene beads (Fig. 19) that give rapid, reproducible, high capacity separations with excellent resolution at high flow rates. SOURCE RPC is a useful alternative to RPC matrices based on silica, especially for separations which must be performed at high pH or when different selectivity or higher capacity are required. Fig. 19. Scanning electron micrograph of SOURCE 15RPC. Note the uniform size distribution.
5 µm
The pore size distribution, batch-to-batch reproducibility (Fig. 20) and excellent scalability (Fig 21) of SOURCE RPC ensure outstanding chromatographic properties at any scale of operation.
30
Fig. 20. Reproducibility of three production batches of SOURCE 15RPC. (Work by Amersham Pharmacia Biotech AB, Lillestrøm, Norway.)
Sample:
(Ile7) angiotensin III (0.5 mg/ml) (Val4) angiotensin III (0.5 mg/ml) Angiotensin III (0.5 mg/ml) Angiotensin I (0.5 mg/ml) 25 µl applied. RESOURCE™ RPC, 1 ml (i.d. 6.4, length 30 mm) 0.1% TFA in water 0.1% TFA, 60% acetonitrile in water 15-65% B in 20 min 1 ml/min
Column: Eluent A: Eluent B: Gradient: Flow:
Batch 1 Batch 2
Retention time (min) 13
Batch 3 12 11 10 9 8 IIe7 Angio III
Fig. 21. Excellent scalability of SOURCE 30RPC.
VaI4 Angio III
Angio III
Angio I
Column:
SOURCE 30RPC, 10 mm i.d. x 300 mm column (24 ml) 200 mm i.d. x 300 mm column (10 l) Sample: Mixture of Angiotensin II, Ribonuclease A and Insulin Sample load: 0.064 mg/ml medium, total load Solution A: 0.1% TFA/0.05 M NaCl Solution B: 0.1% TFA/60% n-propanol Flow: 150 cm/h Gradient: 20–70% B, 5 column volumes (cv)
A280 2.0
HR 10/30 column
nm
1.0
0 0
A280 2.0
1
2
3
4
5
6
7
8
9
6
7
8
9
(cv)
FineLINE 200L FineLINE™ 200L column column
nm
1.0
0 0
1
2
3
4
5
(cv)
31
Characteristics of SOURCE RPC are shown in Table 2. SOURCE 5RPC
SOURCE 15RPC
SOURCE 30RPC
Base matrix and stationary phase
Polystyrene/divinyl Polystyrene/divinyl benzene benzene
Polystyrene/divinyl benzene
Particle size
5 µm
15 µm
30 µm
Particle size distribution
monosized
monosized
monosized
Typical separation flow velocity (cm/hr)
100 -480
200 - 900
100 - 1 000
pH stability (operational)
1 - 12
1 - 12
1 - 12
pH stability (cleaning range)
1 - 14
1 - 14
1 - 14
Dynamic binding capacity (per ml medium at 300 cm/h)
~ 80 mg bacitracin/ml
~ 10 mg BSA/ml ~ 50 mg insulin/ml
~ 14 mg BSA/ml ~ 72 mg insulin/ml
Table 2.
High Chemical Stability SOURCE RPC has an operating range between pH 1 - 12 allowing a free choice for running conditions. Since peptide solubility is frequently pH dependent, successful separations of some peptides may require conditions at high pH. SOURCE RPC therefore offers much greater pH stability and flexibility than silica based reversed phase matrices. Figure 22 demonstrates the chemical stability of SOURCE 30RPC, showing the separation of angiotensins before and after incubation of the medium for one week at 40°C in 1M HCl and, similarly, in 1M NaOH. The extremely high pH tolerance (1 - 14) gives full flexibility for frequent cleaning procedures improving both media lifetime and overall economy at every scale of application. Figure 23 shows results from a SOURCE 5RPC 4.6/150 column on which a thousand runs were performed, including four cleaning-inplace steps with 1 M NaOH and 1.0 M HCl, during a 21 days cycle. The resolution and retention times remained unchanged.
32
Column: Sample:
SOURCE 30RPC, 5 mm i.d. x 50 mm column (1 ml) Mixture of (Iie7) Angiotensin III, (Val4) Angiotensin III, Angiotensin III and Angiotensin II Sample load: 0.13 mg/ml media of each peptide Solution A: 0.1% TFA Solution B: 0.1% TFA/60% Acetonitrile Flow: 300 cm/h Gradient: 15–65% B, 20 column volumes (cv)
A214 nm = before incubation = after incubation with 1 M HCl
A214 nm
= before incubation = after incubation with 1 M NaOH
0.10 0.10
0.05
0.05
0.00
0.00
0.0
5.0
10.0
15.0
20.0
0.0
(min)
5.0
10.0
15.0
20.0
(min)
Fig. 22. Separation of model protein mixture on SOURCE 30RPC before and after incubation for one week at 40 °C in 1 M HCl, and similarly, in 1 M NaOH. Column: System: Sample mixture:
Sample concentration: Sample volume: Eluent A: Eluent B: Flow: Gradient:
SOURCE 5RPC ST 4.6/150 ÄKTA™explorer 10S system 1. (lle7) Angiotensin III 2. (Val4)Angiotensin III 3. Angiotensin III 4. Angiotensin I 0.1 mg/ml of each peptide 20 µl 20 mM Boric acid/NaOH, pH 10.0 Acetonitrile 1.0 ml/min 5–35% B over 15 minutes (6 CV)
A214 nm
A214 nm 3
4
2
a)
1
4
200
b)
3
200
2 1
100
100
0 8.0
0 10.0
12.0
14.0
min
8.0
10.0
12.0
14.0
min
Fig. 23a and 23b. Separation of peptides on SOURCE 5RPC ST 4.6/150. Figure a shows the first injection of the peptide mixture and Figure b the 1000th injection. CIP with 1 M HCl and 1 M NaOH was performed after 275, 400, 600 and 800 runs.
33
SOURCE RPC media and FineLINE™, HR, RESOURCE RPC and ST columns are resistant to all solvents commonly used in reversed phase chromatography, such as 0.1% TFA in water and 0.1% TFA in acetonitrile. SOURCE RPC is resistant to other organic solvents such as methanol, isopropanol, ethanol, acetic acid, and tetrahydrofuran. Due to the inert aromatic/hydrocarbon structure of the polystyrene/divinylbenzene matrix, SOURCE RPC is stable to more disruptive chemical reagents, such as 6 M guanidine hydrochloride and 0.1% SDS.
Excellent Flow/ Pressure Characteristics The uniform bead size and spherical shape of SOURCE RPC beads give stable densely packed beds with excellent flow properties unlike media with a wide range of particle sizes. The low operating back-pressure generated by SOURCE RPC allows higher flow rates to be used during separations and cleaning procedures while still giving excellent resolution, as demonstrated in Figure 24 which shows the performance maintained by SOURCE 30RPC even at high flow rates.
Column: Sample: Sample load: Solution A: Solution B: Flow: Gradient:
A280 nm 1.0
SOURCE 30RPC, 10 mm i.d. x 100 mm column (8 ml) Mixture of Ribonuclease A, Insulin and Albumin 1 mg/ml medium, total load 0.1% TFA 0.1% TFA/60% Acetonitrile 150 and 600 cm/h 20–80% B, 20 column volumes (cv)
Flow of 150 cm/h
A280 nm 1.0
0.5
0.5
0
0
0
50
100
(min)
0
Flow of 600 cm/h
10
20
(min)
Fig. 24. The influence of increasing flow velocity on resolution.
Actual pressure values generated during a run will depend upon the solvent used and the operating temperature. Figure 25 shows pressure versus flow curves with several solvents for RESOURCE RPC columns, packed with SOURCE 15RPC, and FineLINE columns, packed with SOURCE 30RPC.
34
a Pressure (MPa)
b Pressure (MPa)
3.0
Isopropanol
3.0
Isopropanol
Water Ethanol
Ethanol
2.0
2.0 Acetonitrile
Water 1.0
1.0 Acetonitrile
3.0
3.0 0
180
360
540
720
900 1080 1260 1440
0
180
360
540
720
Linear flow (cm/h)
900 1080 1260 1440 Linear flow (cm/h)
Fig. 25a. Pressure:flow curves for (a) RESOURCE RPC, 1 ml and (b) RESOURCE RPC, 3 ml with various organic solvents and water. (Work by Amersham Pharmacia Biotech AB, Lillestrøm, Norway.) a) 15 cm bed height b) 30 cm bed height 2-Propanol Ethanol
Acetonitrile Water
2-Propanol
10
8
Pressure (bar)
Pressure (bar)
10
6 4 2
Acetonitrile Water
Ethanol 8 6 4 2
0
0
0
200
400
600
800
1000
0
1200
200
400
600
Flow velocity (cm/h)
Flow velocity (cm/h)
Fig. 25b. Pressure/flow characteristics of SOURCE 30RPC in various organic solvents and water at room temperature. The pressure/flow velocity data were determined in a FineLINE column with a) 15 cm and b) 30 cm bed height.
High Capacity The controlled uniform pore size distribution in SOURCE RPC is responsible for the high capacities obtained for peptides, proteins and oligonucleotides. The dynamic binding capacity of SOURCE 15RPC is illustrated in Figure 26 while Figure 27 shows an example of the performance maintained by SOURCE 30RPC even with high sample loads. Fig. 26. Dynamic binding capacity of SOURCE 15RPC. (Work by Amersham Pharmacia Biotech AB, Lillestrøm, Norway.)
%
Insulin
60 50 40 1800 cm/h 30
360 cm/h
20
900 cm/h
10
180 cm/h
00 0.0
10.0
20.0
Volume (ml)
35
Column:
SOURCE 30RPC, 10 mm i.d. x 100 mm column (8 ml) Sample: Mixture of Ribonuclease A, Insulin and Albumin Sample load: 1 and 10 mg/ml media, total load Solution A: 0.1% TFA Solution B: 0.1% TFA/60% Acetonitrile Flow: 150 cm/h Gradient: 20–80% B, 20 column volumes (cv)
a)
A280 nm 1.0
b)
A280
Sample load of 10 mg nm
2.0
Sample load of 1 mg
1.0
0.5
0
0
0
50
100
(min)
0
50
100
(min)
Fig 27. The influence of increasing sample load on resolution.
Availability SOURCE 15RPC and SOURCE 30RPC are available in 10 ml, 200 ml, 500 ml, 1 litre and 5 litre pack sizes and should be packed in HR or FineLINE columns according to the scale of the separation. SOURCE 15RPC is also supplied pre-packed in PEEK or stainless steel ST columns: RESOURCE RPC 1 ml is recommended for rapid screening experiments where as RESOURCE RPC 3 ml and SOURCE 15RPC 4.6/100 are better suited for applications where higher resolution is necessary. SOURCE 5RPC is supplied in stainless steel 4.6/150 columns and is recommended for small scale or analytical separations which require the higher resolution which can be achieved using the smaller 5 µm bead size. All pre-packed columns are fully compatible with ÄKTAdesign and other high performance liquid chromatography systems. Ordering information is shown in Section 7. Table 3. Pre-packed SOURCE RPC columns Column
Dimensions Recom(i.d. x bed mended height) mm flow (ml/min)
Efficiency Maximum Maximum N/m flow operating (ml/min) pressure (MPa, bar, psi)
RESOURCE 1 ml RESOURCE 3 ml SOURCE 15RPC ST 4.6/100 SOURCE 5RPC ST4.6/150
6.4/30 6.4/ 100 4.6/100 4.6/100
> 12 000 > 12 000 > 20 000 > 60 000
36
1.0 - 5.0 1.0 - 5.0 0.5 - 2.5 1.0
10 10 5.0 1.5
3, 30, 435 3, 30, 435 4, 40, 580 40, 400, 5800
µRPC C2/C18 Product Description µRPC C2/C18 is a porous microparticulate silica (3 µm) to which C2 and C18 alkyl chains have been covalently bonded. µRPC C2/C18 is ideally suited for peptide mapping, analysis and micropurification. The extremely small particle size ensures high efficiency and excellent resolution of complex samples, as shown in Figure 28. Fig. 28. Separation of a tryptic digest of equine and bovine cytochrome c on µRPC C2/C18 SC 2.1/10.
System: Column: Sample: Buffers: Flow: Gradient:
SMART™ System µRPC C2/C18 SC 2.1/10 Equine and bovine cytochrome c digested with trypsin A. 0.15% trifluoroacetic acid (TFA) in water B. 0.14% TFA in acetonitrile/water, 60/40 250 µl/min 0% B for 2 min 0–33% B for 20 min 33–55% B for 42 min 55–100% B for 5 min
A %B
80
0.08 equine cyt c 0.06
60
0.04
40 bovine cyt c
0.02
20
conc. %B 0
0 10.0
20.0
30.0
40.0
50.0
min
37
µRPC C2/C18 is supplied in 2 pre-packed column formats. µRPC C2/C18 PC 3.2/3 can be used at relatively high flow rates in many micro-preparative applications. The longer bed height of µRPC C2/C18 SC 2.1/10 produces extremely high resolution of complex mixtures where long, shallow elution gradients are often used. Characteristics of these columns are shown in Table 4.
Dimensions i.d. x bed height (mm)
µRPC C2/C18 PC 3.2/3 (glass column)
µRPC C2/C18 SC 2.1/10 (stainless steel column)
3.2 x 30
2.1 x 100
Efficiency (N/m)
>90 000
>100 000
Operational pressure limit (MPa, bar,psi)
15, 150, 2250
25, 250,3625
Pore size
120 Å
120 Å
Particle size
3 µm
3 µm
Recommended flow (ml/min)
0.01 - 1.2
0.01 - 0.25
Practical loading capacity (µg protein/peptide/column)
0.2 - 500
0.01 - 500
Table 4.
Chemical and Physical Stability µRPC C2/C18 can be used with aqueous and organic solvents miscible in water in the pH range 2 - 8. As with all silica based media extended exposure to pH extremes should be avoided as the matrix begins to degrade at pH values greater than 7 - 8 and less than 2 - 3. SOURCE 5RPC media should be chosen whenever separation conditions demand pH conditions to be above pH 8.0. Additives such as guanidine hydrochloride, urea, formic acid (< 60%) and detergents may be used with µRPC C2/C18. Both columns may be operated over the temperature range of 4 - 40 °C up to the maximum pressures shown in Table 4.
Flow/Pressure Characteristics Higher flow rates than those specified in Table 4 are possible with low viscosity solvents, but the integrity of the packing may be compromised if the pressure limit is exceeded.
Capacity The maximum capacity of peptides for the µRPC C2/C18 PC 3.2/3 column is approximately 1 - 3 mg and for µRPC C2/C18 SC 2.1/10 approximately 1 - 2 mg. However, to minimise the risk of losing unbound material in the flow-through fractions or losing resolution, lower and more practical loading ranges are recommended. The detection limit for one peak may be below 1 ng under optimal conditions. 38
Availability Both columns are designed specifically for use with SMART System. They can be connected to ÄKTApurifier or other high performance chromatography systems via a Precision Column Holder (Code No. 17-1455-01). Ordering information is shown in Section 7.
Sephasil Protein/Sephasil Peptide Product Description Sephasil are porous silica-based media giving excellent resolution and offer alternative selectivities compared to SOURCE RPC media. Carefully controlled production conditions ensure batch-to-batch reproducibility for consistent performance in both analytical and process scale applications. Sephasil Protein and Sephasil Peptide are available with three different selectivities C4, C8 and C18. Sephasil Protein C4 is based on a wide-pore 300Å silica that makes it particularly suitable for proteins, whereas Sephasil Peptide is based on a 100Å silica which is more suitable for smaller biomolecules. Sephasil 5 µm media are recommended for high resolution analysis and purification and Sephasil 12 µm media for preparative purification of peptides, proteins or oligonucleotides. Characteristics of Sephasil pre-packed columns are shown in Table 5. Column (bonded phase particle size dimensions i.d. mm/bed height mm)
Pore size (Å)
Specific pore volume (ml/g)
Maximum Efficiency Recomoperating (N/m) mended pressure* flow (MPa, bar, psi) (ml/min)
Sephasil Protein C4 5 µm ST 4.6/100 Sephasil Peptide C8 5 µm ST 4.6/100 Sephasil Peptide C18 5 µm ST 4.6/100
300 100 100
0.6 0.7 0.7
25, 250,3625 “ “
>70 000 “ “
0.5 - 2.0 “ “
Sephasil Protein C4 5 µm ST 4.6/250 Sephasil Peptide C8 5 µm ST 4.6/250 Sephasil Peptide C18 5 µm ST 4.6/250
300 100 100
0.6 0.7 0.7
25, 250,3625 “ “
>70 000 “ “
“ “ “
Sephasil Protein C4 12 µm ST 4.6/250 300 Sephasil Peptide C8 12 µm ST 4.6/250 100 Sephasil Peptide C18 12 µm ST 4.6/250 100
0.6 0.7 0.7
25, 250,3625 “ “
>40 000 “ “
0.5 - 2.0 “ “
Sephasil Protein C4 12 µm ST 10/250 300 Sephasil Peptide C8 12 µm ST 10/250 100 Sephasil Peptide C18 12 µm ST 10/250 100
0.6 0.7 0.7
25, 250,3625 “ “
>40 000 “ “
2-8 “ “
Sephasil Protein C4 12 µm ST 20/250 300 Sephasil Peptide C8 12 µm ST 20/250 100 Sephasil Peptide C18 12 µm ST 20/250 100
0.6 0.7 0.7
25, 250,3625 “ “
>40 000 “ “
5 - 20 “ “
Sephasil Protein C4 12 µm ST 50/250 300 Sephasil Peptide C8 12 µm ST 50/250 100 Sephasil Peptide C18 12 µm ST 50/250 100
0.6 0.7 0.7
14, 140, 2000 “ “
>40 000 “ “
20 - 60 “ “
*refers to pressure above which bed compression may begin
Table 5.
39
Chemical and Physical Stability The chemical and physical stability of Sephasil media ensure consistent performance. Sephasil is resistant to all solvents commonly used in reverse phased chromatography with an operational pH range of pH 2 - 8. As with all silica based media, extended exposure to pH extremes should be avoided as the matrix begins to degrade at pH values greater than 7 - 8 and less than 2 - 3. SOURCE RPC media should be considered as an alternative if the separation conditions demand pH conditions to be above pH 7.5. Additives such as guanidine hydrochloride, urea, formic acid (< 60%) and detergents may be used. Columns may be used at pressures up to 25 MPa over a temperature range of 4 - 70 °C.
Flow /Pressure Characteristics The flow/pressure characteristics of Sephasil Protein and Sephasil Peptide columns are shown in Table 5. The final pressure values generated during a run will depend upon the solvent used, the operating temperature as well as the bed height of the column. Similarly, higher flow rates may be possible with low viscosity solvents, but the integrity of the medium may be compromised if the pressure limit is exceeded.
Availability Sephasil Protein and Sephasil Peptide are available in the pre-packed columns shown in Table 5 and in 100g and 1kg pack sizes. Sephasil columns are fully compatible with ÄKTAdesign and other high performance chromatography systems. Ordering information is shown in Section 7.
40
Chapter 3
3. Methods The following sections will discuss the practical steps involved in planning and implementing a reversed phase separation. Critical aspects of selecting the appropriate stationary phase, mobile phase and gradient conditions are discussed. Practical considerations of sample preparation, solvent handling, and potential pitfalls which may be encountered will also be presented.
Choice of separation medium The proper choice of reversed phase medium is critical for the success of a particular application. This choice should be based on the following criteria: 1) 2) 3) 4)
The unique requirements of the application, including scale and mobile phase conditions. The molecular weight, or size of the sample components. The hydrophobicities of the sample components. The class of sample components.
Unique requirements of the application Resolution It is essential to choose a medium which will yield the required resolution for a given purification. Applications involving fractionation of multi-component samples, such as peptide mapping, require extremely high resolution. Preparative reversed phase applications, such as the purification of synthetic peptides, are more concerned with throughput, and resolution is routinely traded off against both speed and capacity. Resolution in reversed phase chromatography depends on both the selectivity and the efficiency of the column. Selectivity for a specific separation depends on the nature of the immobilised hydrophobic ligand, the chemistry used to derivatise the silica matrix and chromatographic conditions used, such as the composition of the mobile phase. The maximum efficency of a reversed phase column depends on the process used to pack the column and the particle size of the medium. The smallest particles, e.g. µRPC C2/C18, give the highest efficiency followed by the larger 5 µm media, such as SOURCE 5RPC, Sephasil Protein 5 µm and Sephasil Peptide 5 µm. Lower efficiency is seen with larger 12 µm, 15µm and 30 µm particles such as Sephasil Protein 12 µm, Sephasil Peptide 12 µm, SOURCE 15RPC and SOURCE 30 RPC.
41
Scale of the purification For micro-purification narrow bore columns packed with 3 or 5 µm particles should be used e.g. µRPC C2/C18 or SMART columns Sephasil C8 and Sephasil C18. The ligands C2/C18, C8 and C18 will offer significantly different selectivities from which to choose. For typical laboratory scale purification 5 µm media can be selected e.g. SOURCE 5RPC, Sephasil Protein 5 µm and Sephasil Peptide 5 µm. It may be more suitable to use 12 µm media for particularly crude samples or SOURCE 15RPC media, if a high pH or alternative selectivity to silica is required for a successful separation. Media with larger particle sizes should always be chosen for pilot and process scale purification, using their equivalent smaller particle size media for method scouting prior to scale up e.g. RESOURCE RPC pre-packed columns exhibit similar selectivity to SOURCE 30RPC. Mobile phase conditions Choice of reversed phase matrix may also be influenced by the composition of the mobile phase. If the stability or solubility of the sample components dictates the use of a specific solvent system, then the stability of the base matrix in that solvent needs to be considered. For example, when the reversed phase chromatography will involve mobile phases with pH above 7.5, a polystyrenebased matrix, such as SOURCE RPC, should be used. Throughput and scaleability The amount of sample material that can be processed within a defined time (i.e. throughput) is determined by such properties as capacity, flow characteristics and the size of the column the medium is packed into. SOURCE 15RPC, SOURCE 30RPC, Sephasil Protein 12 µm and Sephasil Peptide 12 µm have been optimised for throughput in large scale preparative chromatography with easy scale up under high performance conditions.
Molecular weight of the sample components The accessibility of the sample components to the immobilised hydrophobic ligands will determine the available capacity of the reversed phase medium for a specific biomolecule. Accessibility depends greatly on the pore size and the pore volume of the bead. Reversed phase media with large pores will typically give better capacity and resolution with larger biomolecules than media with small pores. Generally, pore sizes of 300 Å or larger are recommended for the separation of proteins. Pore sizes less than 300 Å are recommended for the separation of peptides and oligonucleotides. The pore sizes and capacities for the reversed phase media supplied by Amersham Pharmacia Biotech are given in Chapter 2.
42
Hydrophobicity of the sample components Unlike other chromatographic techniques, such as ion exchange and size exclusion chromatography, it is difficult, if not impossible, to predict the retention of biomolecules in reversed phase chromatography. Attempts to predict retention on the basis of hydrophobicity factors derived from studies of standard peptides have been the subject of several studies. One which provides an algorithm which has been shown to mimic quite closely the retention behaviour of protein samples subjected to proteolytic digestion has been described by Sakamoto (7). The general benefit from such studies has been an improved understanding of the binding process. The important parameters affecting the retention of a peptide appear to be a combination of the amino acid sequence of the peptide together with any secondary structure, such as a-helices and b-pleated sheets, that the peptide may possess. The situation for proteins is further complicated by their tertiary structure. The selection of a reversed phase medium must, therefore, be made empirically, depending on the nature of the biomolecule components in the sample. Some prediction of resolution can be made based on the hydrophobicity of the sample relative to the immobilised ligand, i.e. the more hydrophobic the sample, the less hydrophobic the immobilised ligand should be. Consequently, a medium with C8 ligands, is generally recommended for preparative purification of more hydrophobic biomolecules than those purified by the corresponding C18 medium. Choice of reversed phase matrix may also be influenced by the composition of the mobile phase. When the reversed phase chromatography will involve mobile phases with pH above 7.5, a polystyrene-based matrix, such as SOURCE RPC, should be used. When the stability of the sample components dictates the use of a specific solvent system, then the stability of the base matrix in that solvent needs to be considered.
Class of sample components Reversed phase chromatography is used for purification of many classes of biomolecules. Whilst the conditions of reversed phase chromatography usually have no harmful effects on the chemical integrity of oligonucleotides and peptides, the question of the stability and biological activity of proteins must be considered carefully. Polypeptide interactions with a hydrophobic surface and organic solvents generally leads to some loss of tertiary structure. The loss in tertiary structure may then give rise to different conformational states for a given biomolecule (8) and each of these states may interact differently with the reversed phase medium. The widespread use of reversed phase chromatography for the large-scale purification of recombinant and synthetic proteins and peptides, such as insulin, growth hormone, growth factors and many others, indicates, however, that problems caused by denaturation can often be overcome. Loss of structure and, consequently, loss of activity can be minimised by proper treatment. The kinetics
43
be reversed by transferring the protein to conditions under which its native structure is favoured. Note that complex enzymes and multi-component proteins are more likely to lose activity than small peptides or highly stabilised and crosslinked proteins. However, when a protein or peptide is purified for primary structure determination, denaturation is not a problem unless precipitation occurs and reversed phase chromatography has found widespread use in preparing pure biomolecules for subsequent sequencing.
Choice of mobile phase The mobile phase in reversed phase chromatography of biomolecules generally contains a “buffer” component, an organic modifier and, often, an ion pairing agent added to the mobile phase to affect selectivity. All solvents, buffering salts, ion pairing agents, as well as the water used to prepare the mobile phases, must be of high chemical purity and should be free of any metal ions. Solvents, salts and water should be labelled as “HPLC grade” to ensure sufficient purity. Chemical purity is important in preparative reversed phase chromatography, since any contaminants in the mobile phase may affect the chromatography by producing unwanted extra peaks, ghost peaks, and may contaminate the purified biomolecule.
The organic solvent The organic solvent is added to the mobile phase to lower its polarity. Two solvents with different polarity, such as an aqueous low pH solution and acetonitrile, may be mixed together resulting in a solvent with polarity intermediate between those of the original components. The lower the polarity of the solvent mixture, the higher its eluting power in reversed phase chromatography. There is a large number of water miscible organic solvents that can be used in reversed phase chromatography but few of them are used in practice. The properties of some typical organic solvents used in reversed phase chromatography are shown in Table 6. Table 6. Solvents used in reversed phase chromatography.
Solvent
Boiling point (°C)
UV cut-off* (nm)
Viscosity (cP at 20 °C)
Comments More powerful denaturant than alcohols. Toxic.
Acetonitrile
82
190
0.36
Ethanol Methanol 1-propanol (n-propanol) 2-propanol (iso-propanol) Water
78 65 98
210 205 210
1.20 0.60 2.26
Viscous
82
210
2.30
Viscous
<190
1.00
100
* Absorbance is approx. 1 for HPLC-grade solvent at this wavelength.
44
Acetonitrile and methanol are the most commonly used since both have low viscosity (even when mixed in aqueous solution) and are UV transparent. 2-Propanol has the advantage of lower polarity and therefore higher eluting strength. It is UV transparent and is excellent for cleaning the reversed phase column. However, use of 2-propanol as the organic modifier results in high viscosity mobile phases. High viscosity mobile phases are undesirable since they result in poor mass transport of solute between the mobile and stationary phases and high back-pressure even at moderate to low flow rates. Figure 29 shows the change in pressure during gradient formation due to the viscosity effects of various solvents. Fig. 29. Variation of pressure during elution with a gradient from 100% water to 100% organic solvent at constant flow. Note that the pressure increases by ca. 50% from the starting value (pure water) during elution with methanol. The increase in the case of acetonitrile is much smaller, ca. 10%. (Work by Amersham Pharmacia Biotech AB, Uppsala, Sweden.) Column: C18 silica, 10 mm (i.d. 4.6 mm, length 250 mm). Eluent A: 100% water Eluent B: (A) 100% acetonitrile (B) 100% methanol Gradient: 0-100% B in 10 min 100% B for 2 min 100-0% B in 3 min 0% B for 2.5 min Flow: 1.75 ml/min
100
80
60 acetonitrile
40
20
0
2
4
6
8
10
12
14
16 Time (min)
100
80
60
methanol
40
20
0
2
4
6
8
10
12
14
16 Time (min)
45
Only high quality organic solvents should be used in reversed phase chromatography. HPLC grade solvents are desirable since small particulates that can clog reversed phase columns have been removed. They provide adequate chemical purity with good transparency to low wavelength UV light. Organic solvent in the eluting buffer is generally removed from the recovered biomolecule by evaporation and residual organic contaminants, such as acrylic acid, can be damaging to the biological integrity of the recovered sample. UV transparency is especially important since many reversed phase separations are monitored below 220 nm for optimum detection sensitivity (e.g. peptides, proteins lacking significant amounts of Trp or Tyr, etc.). The UV cut-off value for an organic solvent used in reversed phase chromatography should ideally be below 210 nm. This will provide better baseline stability when running gradients in which the content of organic modifier is varied.
pH Reversed phase separations are most often performed at low pH values, generally between pH 2 - 4. The low pH results in good solubility of the sample components and ion suppression, not only of acidic groups on the sample molecules, but also of residual silanol groups on the silica matrix. Acids such as trifluoroacetic acid, heptafluorobutyric acid and ortho-phosphoric acid in the concentration range of 0.05 - 0.1% or 50 - 100 mM are commonly used. Mobile phases containing ammonium acetate or phosphate salts are suitable for use at pH’s closer to neutrality. Note that phosphate buffers are not volatile. Polystyrene-based reversed phase matrices, such as SOURCE RPC, allow reversed phase chromatography to be performed routinely at pHs well above neutral. The advantage of performing chromatography at these elevated pH values include increased control of selectivity and, in some cases, improved solubility and yield of active sample components. Basic peptides often tail during elution from reversed phase columns at low pH. Better resolution of basic peptides is often achieved above pH 8. The acids, bases or buffering salts used to control pH must also be transparent to UV below 220 nm and must be soluble under the low polarity conditions of the mobile phases used in reversed phase chromatography. It is convenient, but not essential, that the buffer salts and acids used to prepare reversed phase mobile phases are volatile. Volatile buffer components can be removed from the eluted sample by evaporation along with the organic component. If non-volatile salts are used in the mobile phase, they must be separated from the recovered sample by an additional desalting step.
46
Sample: Angiotensin II (0.25 mg/ml), Angiotensin III (0.25 mg/ml) Column: RESOURCE RPC 3 ml (i.d. 6.4 mm, length 100 mm). Eluent A: (A) 0.1% TFA in water, pH 2 (B) NaOH 10 mM in water, pH 12 Eluent B: A) 0.1% TFA, 60% acetonitrile in water (B) NaOH 10 mM, 60% acetonitrile in water Gradient: 10-65% B in 10 min Flow: 2 ml/min
b)
A214nm
a)
0.14
pH 2
0.12
Angiotensin II Angiotensin III
A214nm
pH 12 0.40
0.10 0.30 Angiotensin II
0.08
Angiotensin III
0.20
0.06
0.04 0.10 0.02
0.00
0
5.00
10.00
15.00 Time (min)
5.00
10.00
15.00 Time (min)
Fig. 30. Separation of angiotensin II and angiotensin III at a) pH 2 and b) pH 12. The selectivity is changed significantly by changing the pH. (Work by Amersham Pharmacia Biotech AB, Uppsala, Sweden.) Table 7. Examples of mobile phases for use at different pH.
Buffer
Approximate pH
Hydrochloric acid Phosphoric acid Trifluoroacetic acid (TFA) Triethylammonium phosphate (TEAP) Ammonium acetate Sodium hydroxide
2-3 2-3 2-3 6 6-7 12
Comments Non-volatile. Non-volatile. Non-volatile. Only with stationary phases based on organic polymers.
Ion pairing agents The third component usually added to the mobile phase is an ion pairing agent. As described in Chapter 1, ion pairing agents are thought to bind by ionic interaction to the solute molecules to increase the hydrophobicity of the solute molecule and change selectivity. Aside from the fact that some reversed phase separations (such as purification of synthetic oligonucleotides) absolutely require the use of ion pairing agents, their greatest advantage is in affecting selectivity
47
Retention time (min)
20
Sample: Sample volume: Column:
Peptide 1 Peptide 2 Peptide 3
(A) Neutral peptides
Eluent A:
15
Eluent B: Gradient: Flow:
10
Standard peptides. See Table 8. 20 ml Organic polymer-based matrix (i.d. 5 mm, length 200 mm). TFA in water. TFA concentrations as in diagrams. TFA, 95% acetonitrile in water. TFA concentrations as in diagrams. 15-100% B at 1% B/min 1 ml/min
5 0.1
0
0.2
20
Retention time (min)
Retention time (min)
% TFA in mobile phase A (B) Basic peptides
15
10
20
(C) Acidic peptides Peptide 9 Peptide 10
15
10 Peptide 6 Peptide 7 Peptide 8
5
5 0.1
0
0.1
0
0.2 % TFA in mobile phase A
0.2 % TFA in mobile phase A
Fig. 31. Variation of retention time with concentration of the anionic ion pairing agent (TFA) for (A) neutral, (B) basic and (C) acidic peptides. (Work by Amersham Pharmacia Biotech Inc., Piscataway, NJ, USA.)
thereby improving the chances for complete resolution of sample components. The retention behaviour of the sample components may be affected by both the type and concentration of ion pairing agent used. The effects of varying the concentration of trifluoroacetic acid on peptide retention time depend on the type of peptide (neutral, acidic or basic) (Fig. 31).
Table 8. Standard neutral, basic and acidic peptides used for Fig. 31.
Peptide Type
Amino acid sequence
1 2 3 6 7 8 9 10
Trp Glu Glu Tyr Asn Arg Lys Trp
Neutral Neutral Neutral Basic Basic Basic Acidic Acidic
Val His Ala Arg Arg Pro Gly Ala
Pro Trp Asp Pro Val Lys Asp Gly
Thr Ala Pro Pro Tyr Pro Glu Gly
Asn Tyr Asn Gly Val Gln Glu Asp
Val Gly Lys Phe His Gln Ser Ala
Gly Leu Phe Ser Pro Phe Leu Ser
Ser Gln Tyr Pro Phe Gly Leu Gly
Glu Pro Gly Phe Asn Leu Ala Glu
Ala Phe Gly Leu Met Arg Leu Met
Some ion pairing agents, such as trifluoroacetic acid, also act to maintain the pH of the mobile phase. Typical concentration ranges for ion pairing agents in the mobile phase are 0.01% to 0.1% or between 10 - 100 mM. Some common anionic and cationic ion pairing agents are shown in Table 9.
48
Table 9. Ion pairing agents.
Ion pairing agent
Formula of pairing ion
Comments
Anionic Trifluoroacetic acid (TFA)
CF3COO–
Low UV-absorbance. Volatile, low pH, More hydrophobic than TFA. Volatile, low pH More hydrophobic than TFA. Volatile, low pH
Pentafluoroproprionic acid (PFPA) CF3CF2COO– Heptafluorobutyric acid (HFBA)
CF3CF2CF2COO–
Ammonium acetate Phosphoric acid
CH3COO– H2PO4–, HPO4 2–, PO4 3–
Cationic Tetramethylammonium chloride Tetrabutylammonium chloride Triethylamine
+N(CH3)4 +N(C4H9)4 NH+(C2H5)3
Less hydrophobic than TFA.
Again, the requirements for the ion pairing agent are that it is sufficiently pure, it is UV transparent below 220 nm and it is soluble under the low polarity conditions of the mobile phase. Additionally, volatile ion pairing agents ensure that sample recovery will not require a separate desalting step.
Sample preparation The sample should ideally be dissolved in the initial mobile phase. If this is not possible due to stability or solubility problems, formic acid, acetic acid or salt can be added to the sample to increase solubility. These additives do not usually effect the separation so long as the volume of the sample loaded is small compared to the column volume. The only effect when large sample volumes are applied may be an extra peak or two eluting in the void volume after sample injection. In situations where the sample is soluble in mobile phase A (or a reasonable facsimile thereof) and the sample component of interest will bind to the column, the volume of the sample usually does not have any effect on the subsequent chromatography. Since reversed phase chromatography is an adsorption technique, a large volume of dilute sample can be concentrated and purified in a single step. It is recommended that the sample is used directly after solubilization to minimise any undesirable side reactions such as oxidation of the sample components. All samples should be centrifuged at 10,000 g for 10 min prior to injection of sample or, alternatively, filtered through a 0.22 µm (or 0.45 µm) sterile filter. Be sure to choose a solvent resistant filter if the sample solution contains organic modifier. For particularly dirty samples, desalting through a large particle version of a reversed phase or a size exclusion medium can be done to remove gross contaminants that may foul a more valuable reversed phase column. Some columns have a replaceable, in line ”guard” column that prevents dirty samples from fouling the main body of the reversed phase column. 49
It is important to maintain sample solubility throughout the loading process in order to avoid precipitation of the sample on the column. Sample precipitation can be avoided by not overloading the column and by ensuring that the sample is sufficiently soluble in the sample solvent prior to injection on the column.
Mobile phase preparation As stated before, all solvents and additives used to prepare the mobile phases should be of HPLC grade or, when not available, of the highest purity grade. The solvents used to prepare mobile phases should also be de-gassed in a sonic bath for 10 - 15 minutes to prevent gas formation under elution conditions. Alternatively, they may be de-gassed under vacuum with magnetic stirring or by sparging with helium. All mobile phases to which solids have been added should be filtered through 0.22 µm filters before use to prevent particles from clogging the reversed phase column. When adding volatile ion pairing agents to the mobile phases (e.g. trifluoroacetic acid), the ion pairing agent should be added after de-gassing is complete. In this way, the concentration of ion pairing agent is not altered by the de-gassing process.
Storage of mobile phases A general rule is to use freshly prepared mobile phases in reversed phase chromatography. If the solvents have to be stored, it is important that the reservoirs are closed to avoid changes in the solvent composition caused by evaporation. Aqueous solutions at neutral pH should not be stored for more than 2 - 3 days because of the risk of microbial growth. For long term storage, store all solvents covered at 4 °C. Cold solvents must be allowed to come to running temperature and then be de-gassed before use. This will reduce the risk of bubble formation in the system.
Solvent disposal Unlike the aqueous salt solutions used as mobile phases for other types of biomolecule chromatography, reversed phase chromatography employs organic solvents in addition to aqueous buffers. The mobile phases must be handled as toxic chemical waste and disposed of properly. This includes methanol, which itself is not a significant chemical pollutant but will probably contain additives such as TFA, HFBA, etc. Under process conditions where large volumes of organic solvents are used to prepare mobile phases, it is generally more economical to recycle waste mobile phase. Skin exposure to organic solvents should be avoided, especially with acetonitrile, as should breathing of any fumes. It is advisable to work with these solvents under a fume hood when preparing mobile phases. The same precautions should be exercised when dealing with organic amines such as triethylamine (TEA) which is used as an ion pairing agent at neutral and basic pH. 50
Extreme care must be followed when dealing with mineral acids, such as hydrochloric acid, ortho-phosphoric acid, etc. and perfluorinated organic acids such as TFA, PFPA and HFBA. These acids can cause severe skin burns and inhalation of fumes should be avoided. These substances should be handled with the utmost caution and always within the secure confines of a well ventilated fume hood. Be sure to wear adequate protection such as gloves, goggles and a lab coat.
Detection It is essential to use reagents and solvents of high purity to ensure minimum detection limits for optimum sensitivity. All organic solvents and many additives, such as ion pairing agents, absorb in the UV range and the detection limit is related to the wavelength (9). Detection below 220 nm is necessary when separating short peptides that lack aromatic amino acid residues such as Trp and Tyr. Synthetic oligonucleotides absorb in the region 250 - 260 nm and proteins absorb at 280 nm. Detection difficulties are generally manifest when separating short peptides since they require the shortest wavelengths for detection. The solvents and additives recommended in this manual have been chosen on the basis of providing optimal separation in combination with low background absorbance (see Tables 6, 7, 9).
Ghosting A common problem caused by poor quality mobile phase components is a phenomenon referred to as “ghosting”. Trace levels of organic impurities in the mobile phases can bind to the reversed phase medium and concentrate during the equilibration and sample loading steps. Upon elution of the column with organic modifier, the bound organic contaminants desorb and appear in the chromatogram as unknown, or “ghost” peaks. The size of a ghost peak will usually depend on the equilibration time and the level of organic impurities in the mobile phase. Ghosting may also be caused by incomplete elution of sample components in a previous run. A blank gradient, with no sample, should be run as a check, especially if subsequent runs are to be performed with high sensitivity detection.
Mobile phase balancing When reversed phase columns are eluted with a gradient in the mobile phase, it is frequently noticed that the baseline drifts. During a typical run, where the proportion of mobile phase B in the gradient increases, the baseline can progressively increase or decrease in an approximately linear fashion (see, for example Fig. 30). The drifting baseline may originate from an ion pairing agent (or strong acid component) or an organic modifier that absorbs significantly at the wavelength used to monitor the chromatography (10).
51
The background absorbance of the initial mobile phase A is corrected for during the equilibration of the column. As the run is executed, the chemical environment of the mobile phase changes dramatically as the proportion of organic component is steadily increased. The absorption properties of the buffer components can change as the solvent characteristics of the eluting mobile phase change during progress of the gradient. If this happens there will be a gradual increase (or decrease) in the UV-absorption of the mobile phase as the gradient forms and a drifting baseline. It is interesting to note that the progress of the gradient is usually represented by the electronic signal from the two mixing pumps, but the baseline drift is a better reflection of the actual “chemical” gradient being produced. A drifting baseline can be compensated by using different concentrations of UVabsorbing ion pairing agents (or buffer acids) in mobile phases A and B. In this way, the “concentrations” with respect to UV-absorption properties are balanced, and the baseline should approximate a straight line. Because of batch-to-batch variations in the absorption properties of the components of the mobile phases and other differences between the conditions in different runs, it is not practical to give hard and fast recommendations for particular solvent systems. For gradients from TFA in water to TFA in acetonitrile it will usually be found that the concentration of TFA in acetonitrile will be need to be 10-30% lower than in water. However, the balanced concentrations of UVabsorbing components should be determined empirically. The difference in concentration of ion pairing agent between the two mobile phases is generally not large enough to affect the chromatography adversely.
Column conditioning Reversed phase columns should be “conditioned” for first time use, after long term storage or when mobile phase conditions are changed significantly. The mobile phases used in the conditioning should be the same as those used in the subsequent chromatography. The general procedure for conditioning most reversed phase columns is as follows: 1)
Wash the column with approximately 3 column volumes of mobile phase B at a low to moderate flow rate appropriate for the particular column.
2)
Run a 2 - 3 column volume linear gradient from 100% mobile phase B to 100% mobile phase A at same flow rate as above.
3)
Equilibrate the column with at least 5 column volumes of mobile phase A. Continue equilibration until all monitor signals are stable.
4)
Every time the mobile phase system is changed, a blank run should be performed to check for any artifacts that might appear due to UVabsorbing impurities in the mobile phase. Again, return to 100% A and equilibrate to a stable baseline prior to sample injection.
52
Elution conditions Reversed phase separations can be achieved using either a stepwise or a continuous gradient to elute sample components. Step gradients (i.e. a series of isocratic elutions at different % B) are useful for applications such as desalting, but for separations requiring high resolution, a linear, continuous gradient is required. Step gradients are also ideal when performing process scale applications providing the desired resolution can be obtained, as less complex instrumentation is required to generate step gradients. Additionally, step gradients can be generated more reproducibly than linear gradients. The ideal gradient shape and volume must be empirically determined for a particular separation. Generally, the sample to be purified is chromatographed using a broad range linear gradient to determine where the molecule of interest will elute. The initial conditions usually consist of mobile phase A containing 10% or less organic modifier and mobile phase B containing 90% or more organic modifier. The initial gradient runs from 0% B to 100% B over 10 - 30 column volumes. At a flow of 1 ml/min with a 1 ml column, this corresponds to a gradient slope between 3 - 10% B/min. A blank gradient is usually run prior to injecting sample in order to detect any baseline disturbances resulting from the column or impurities originating in the mobile phase. Gradients can be measured either in volume mode or in time mode. In reversed phase chromatography, changes in flow (at constant gradient slope) appear to have little effect on resolution (Fig. 24). At constant flow, however, gradient slope has a significant effect on column resolution. After the initial gradient has been run, the resolution of the peaks is evaluated to see whether improvement is needed or not. As shown previously, resolution can be improved by adjusting the components of the mobile phase but resolution can also be improved by using a shallower gradient (Fig. 32). This can be achieved either by increasing the gradient time or volume or by using a segmented gradient (Fig. 33). When only one of the sample components is of interest, as in a large scale peptide or protein purification, the segmented technique is more economical with respect to both time and mobile phase consumption. Shallow gradients are especially useful in increasing the resolution of protein separations since the retention times for large protein molecules are particularly sensitive to subtle changes in mobile phase polarity.
53
Sample: Synthetic peptide: NVILTKPEVSEGTEVTVK Column: Sephasil Peptide C18 5 µm ST 4.6/100 Eluent A: 0.06% trifluoroacetic acid (TFA), pH 2.5 Eluent B: 84% acetonitrile in 0.055% TFA Gradient: (A) 0 - 60% (B) 20 - 35% Flow: 1 ml/min
A280 nm
80
%B 100
0–60%
80
60
60
40
40
20
20 0
0 0.0
10.0
20.0
30.0
A280 nm
ml
%B 100
40.0 20–35%
80
30.0 60 20.0
40
10.0
0.0 0.0
20 0 10.0
20.0
30.0
ml
Fig. 32. Effect of gradient slope on resolution of a synthetic peptide at constant flow. (Work by Amersham Pharmacia Biotech AB, Uppsala, Sweden)
54
Fig. 33. Schematic chromatograms showing effects expected in a segmented gradient compared with a linear gradient. Where the gradient is steeper (segments A and C), resolution is lost. Where the gradient is shallower (segment B), resolution is improved.
A
B
C
Column re-equilibration Irrespective of the final concentration of the gradient used for the separation, the column should always be equilibrated with several column volumes of 100% mobile phase B to remove any remaining sample components. If the separation gradient is completed at lower level than 100% B, a gradient up to 100% B should be run, followed by equilibration at 100% B. The column is then reequilibrated with mobile phase A, preferably in a gradient to avoid any risk of damaging the column packing by a too abrupt change in the composition of the mobile phase.
Column cleaning Periodic column cleaning is recommended, especially when dealing with samples containing particulate matter and any column fouling contaminants. Increased back-pressure, loss of resolution or discoloration at the top of the bed (for glass columns) are all signs that the column may need to be cleaned. It should be noted that with silica-based reversed phase media, loss of resolution due to peak broadening may also indicate the presence of silanol groups on the silica gel surface or a head space in the column caused by dissolution of the matrix during frequent use above pH 7. Silanol groups can also form as a natural process of gel ageing during prolonged exposure to aqueous solvents during the normal course of the column lifetime. Poor resolution from the effect of exposed silanol groups can be minimised by ion suppression using low pH mobile phases. A common procedure for cleaning a typical reversed phase column is as follows: 1) 2) 3)
Equilibrate the column at a low flow rate with several column volumes of mobile phase A containing 0.1% TFA in water. Run a gradient (approximately 20 - 30 column volumes) from 100% A to 100% mobile phase B where mobile phase B is 0.1% TFA in 2-propanol. Equilibrate the column at 100% B for several column volumes and then bring the column back to 100% A using another gradient.
55
4)
Equilibrate the column with several column volumes of mobile phase A before equilibrating the column with the initial mobile phase to be used in the subsequent chromatography. If the new mobile phase A is significantly different from 0.1% TFA in water then the column should be introduced to the new mobile phase A using a linear gradient.
Custom packed columns, such as those used in large scale and process applications, can also be cleaned with the medium in place since the reversed phase matrix (whether silica- or polystyrene-based) does not significantly shrink or swell with solvent changes. SOURCE RPC can be cleaned using more aggressive chemical agents due to the extreme stability of the polystyrene-based matrix. A very effective cleaning procedure is equilibration with several column volumes of sodium hydroxide solution up to 0.5 M. Sodium hydroxide is an extremely effective cleaning agent and the ability to use it for cleaning is a major advantage of using SOURCE 15RPC or SOURCE 30RPC for large scale work.
Column storage Reversed phase media based on silica gel should not be stored in aqueous solution. This is due to the inherent instability of silica under aqueous conditions. Silica-based reversed phase columns and media are usually stored in a pure organic solvent such as methanol (acetonitrile is not recommended) free from TFA or any other additives. Polystyrene-based reversed phase media can be store in either pure methanol or 20% ethanol.
56
Chapter 4
Applications Reversed phase chromatography has proven itself to be an indispensable technique in the purification of biomolecules. The technique was originally developed in the 1960s for the separation of small organic molecules. In recent years, with the advent of high performance media and instrumentation, reversed phase chromatography has been applied to the purification of biomolecules such as peptides, proteins and oligonucleotides. Reversed phase chromatography has proven so successful for biomolecule purification in the research laboratory that it is now routinely applied for process scale purification of synthetic peptides, and recombinant peptides and proteins. The examples illustrated in the following section have been obtained from the published literature and from work in the laboratories at Amersham Pharmacia Biotech.
Designing a biochemical purification Except for a few specific applications, reversed phase chromatography is rarely used by itself in a biochemical purification. Reversed phase is usually combined with other chromatography techniques such as gel filtration and ion exchange chromatography. The choice of the additional techniques along with the sequence in which they are employed is critical to a successful purification. At the beginning of a purification protocol sample loading, sample dilution and impurity contamination are usually at their highest. At this stage, high capacity, low resolution techniques that also result in sample concentration are usually employed. At later stages in the protocol, higher and higher resolution techniques are utilised. The choice of a general protocol depends on the type of biomolecule being purified as well as its source. From the point of view of reversed phase chromatography, the biomolecules that we have so far focused on in this booklet may be grouped as follows: • Naturally occurring peptides and proteins • Recombinant peptides and proteins • Chemically synthesised peptides • Protein fragments from enzyme digests • Chemically synthesised oligonucleotides All these groups of biomolecules differ in several ways which prove important in their purification. Examples of the use of reversed phase chromatography in solving some typical purification problems are presented in the following pages, together with some general considerations for designing successful purification strategies. 57
Naturally occurring peptides and proteins Peptides and proteins are purified from their natural sources for many reasons. Due to the complexity of the starting material, their purification usually requires a series of steps using several complementary chromatography techniques (see Protein Purification Handbook Code no. 18-1132-29 for guidelines on purification strategies). Peptides and proteins often occur naturally in very small quantities and large amounts of starting material are usually required in order to get a reasonable amount of pure material. Several different separation techniques are applied in sequence to achieve a very high degree of purity. During the capture phase the main points are to reduce volume and eliminate the majority of gross contaminants while the later steps remove the components with similar characteristics. Early chromatographic steps could include ion exchange and gel filtration, using low to medium performance media. The final purification step would then employ high performance reversed phase chromatography, combined if necessary with high performance gel filtration. The purity throughout the various stages of the purification can be monitored using electrophoresis on PhastSystem™ . There are several points to consider when using reversed phase chromatography to purify molecules from complex biological sources. Firstly, if a protein or peptide is to be used in studies of its function or, for example, structure-activity relationships, then it will be important to ensure that its biological activity is maintained throughout the purification procedure. This is not a critical problem with peptides and small proteins, but larger proteins tend to denature under reversed phase conditions. The proper choice of reversed phase medium and elution conditions, including time of exposure to potentially denaturing conditions, must be optimised in order to maintain biological integrity. The presence of proteases may also present difficulties when dealing with polypeptides from natural sources. It is advisable to work rapidly at the initial stages of purification in order to minimise the contact time the desired peptide or protein will have with the protease. If proteases prove to be an intractable problem, specific chemical protease inhibitors may be added to the buffers. A third problem common with materials from natural sources is aggregation. Fortunately, the low polarity mobile phases used in reversed phase chromatography are often good solvents for just those polypeptides which are poorly soluble in aqueous buffer solutions at physiological pH. Soluble aggregates which do cause problems can be separated from the corresponding monomers by gel filtration.
58
Purification of platelet-derived growth factor (PDGF) Reversed phase chromatography was used for the rapid purification of dimeric Platelet-Derived Growth Factor (PDGF, Mr 30 000). In the initial method, cation exchange chromatography was combined with a second purification step using reversed phase chromatography. Further studies showed that the ion exchange step could be omitted to give a simple and rapid one-step method. A freeze/thaw extract of pig blood platelets was clarified by centrifugation and the supernatant was dialysed overnight against 30 volumes of 10 mM sodium phosphate, pH 7.4. After further centrifugation, the clear extract was applied to a RESOURCE RPC 3 ml column. The column was eluted with a gradient from TFA (0.1%) in water to TFA (0.1%) in acetonitrile (Fig. 34). PDGF was purified to apparent homogeneity in a single run. Fig. 34. Rapid purification of Plateletderived Growth Factor (PDGF) from freeze/thaw extract of porcine blood platelets by reversed phase chromatography. (Work by Amersham Pharmacia Biotech AB, Lillestrøm, Norway.)
Sample: Column: Eluent A: Eluent B: Gradient: Flow:
Platelet derived growth factor (Porcine blood platelet extract containing 10 mg/ml protein). 100 µl applied. RESOURCE RPC 3 ml (6.4 x 100 mm) 0.1% TFA in water 0.1% TFA in acetonitrile 4 ml 0% B, 4-48 ml, 0-60% B 2 ml/min (370 cm/h)
A280 nm
%B
100 PDGF
50
0 0
5.0
10.0
Time (min)
Trace enrichment Since naturally occurring peptides are typically present in very low concentrations in biological sources, the use of reversed phase techniques for trace enrichment from large volumes of dilute solution is of special interest. For example, a narrowbore column with a total bed volume of 240 µl (µRPC C2/C18 PC 3.2/3) can easily concentrate proteins from over 40 times as much (10 ml) of a dilute sample (Fig. 35). The solutes concentrated on the column can then be separated in the usual way, in this case by a linear gradient of acetonitrile concentration. Note that sample volumes differing 100-fold gave very similar results (Fig. 35 a and b).
59
Sample: Sample volume: Column: Eluent A: Eluent B: Gradient: Flow:
Ribonuclease A (10 µg), Cytochrome C (2.5 µg), Albumin (10 µg), Catalase (10 µg) (A) 100 µl, (B) 10 ml µRPC C2/C18 PC 3.2/3 (i.d. 3.2 mm, length 30 mm) 0.1% TFA in water 0.1% TFA in acetonitrile 0-60% B in 10 min 300 µl/min
Fig. 35. Trace enrichment of standard proteins followed by gradient elution on a narrow-bore reversed phase column, (i.d. 3.2 mm, length 30 mm). Sample volume (A) 100 µl, (B) 10 ml. (Work by Amersham Pharmacia Biotech AB, Uppsala, Sweden.)
A
280 nm
Purification of cholecystokinin-58 (CCK-58) from pig intestine This purification shows that, by selection of appropriate techniques and manipulation of elution conditions, a pure product can be obtained from very crude material after only a few chromatographic steps. In this example cation exchange chromatography (HiLoad™ 26/10 SP Sepharose™) was used for the initial capture of an ethanol-precipitated extract of intestinal peptides. Fractions which showed bio-activity were collected and purified further using an HR 16/10 column packed with SOURCE 15RPC. Figure 36 a shows the result of the reversed phase purification on one of the fractions. CCK-58, the molecule of interest, was present in region 11. The run was repeated several times and the bioactive material corresponding to region 11 was combined and lyophilised. The next purification step was carried out on a Sephasil Peptide 5 µm ST 4.6/100 column which gave an alternative selectivity to SOURCE RPC. As can be seen in Figure 36 b the bioactivity was found under the major peak of the chromatogram. This elution used an aqueous methanol gradient elution. For further purification the same column was used again but with an aqueous acetonitrile gradient. The peptides eluted from the final purification step (Figure 36 c) were shown to be pure by capillary zone electrophoresis, MALDI TOF mass spectrometry and amino acid composition analysis (11).
60
System: Column: Sample: Eluent A: Eluent B: Gradient:
Flow: Bioassay:
ÄKTA purifier HR 16/10 column packed with SOURCE 15RPC 50 ml of fraction 7 from a cation exchange step 0.1% TFA in water 0.1% TFA in acetonitrile/water 0% B for 2 column volumes (CV), 0–20% B for 1 CV, 20–55% B for 11CV, 55–100% B for 1 CV, 100% B for 2 CV 6 ml/min Guinea pig gall bladder concentration in vivo
a) CCK activity mU/µl
A II
200 1500 %B
150
1000 100
500
0
System: Column: Sample:
0
100
200
ÄKTA purifier Sephasil Peptide C18 5 µm ST 4.6/100 450 µg of dry peptide material from bioactivity region II 0.1% TFA in water 0.1% TFA in methanol 60% B for 2 column volumes (CV), 60–81% B for 20 CV, 83–100% B for 2 CV, 100% B for 2 CV 6 ml/min Guinea pig gall bladder concentration in vivo
Eluent A: Eluent B: Gradient:
Flow: Bioassay:
50
I
214 nm 230 nm 280 nm
0
300
System: Column: Sample: Eluent A: Eluent B: Gradient: Flow: Bioassay:
ml
ÄKTA purifier Sephasil Peptide C18 5 µm ST 4.6/100 Fraction 13 from the chromatography step b 0.1% TFA in water 0.1% TFA in 90% acetonitrile/water 30% B for 2 column volumes (CV), 30–48% B for 16 CV, 48–100% B for 2 CV, 100% B for 2 CV 0.6 ml/min Guinea pig gall bladder concentration in vivo
c)
b) CCK activity mU/µl
A 13
400
%B 300
60 30.0
200
200
100
214 nm
0
0 10.0
40
20.0
20.0
30.0
40.0
ml
280 nm
0.0 16.0
20
214 nm
10.0
280 nm
230 nm
0.0
%B
40.0
14
300
100
CCK activity mU/µl
A
230 nm
0 18.0
20.0
22.0
24.0
ml
Fig. 36. RPC steps from the purification of cholecystokinin.
61
Recombinant peptides and proteins Some of the problems associated with isolating peptides and proteins from their native sources can be overcome by producing them through recombinant techniques. Peptides and proteins produced in this way are used for basic research, diagnostic and therapeutic purposes. Recombinant peptides and proteins can be expressed by vectors in different hosts, e.g. E. coli, yeast, mammalian cells or viruses. A vector (plasmid or chromosome) is the vehicle used to transport the gene coding for the polypeptide into the host cell. The cell reproduction mechanism then produces many copies of the desired recombinant DNA molecule or clone. The cells containing the correct clone are grown and the gene which has been introduced is expressed to provide a continuous supply of the desired peptide or protein. The expressed peptide or protein may be found either in the culture medium or inside the cells. Peptides or proteins which are secreted into the culture medium are relatively simple to purify (12) and are generally not as susceptible to proteases. Polypeptides which are retained inside the cells are more difficult to purify since they must first be released by lysis (13). If the concentration of polypeptide within the cell is too high, precipitation can occur resulting in insoluble inclusion bodies. These inclusion bodies must be dissolved using a chaotropic agent such as guanidine hydrochloride (6 M) and the polypeptide carefully renatured (14). When refolding methods exist, it can be advantageous to produce proteins via inclusion bodies as only centrifugation, washing and solubilisation are necessary to obtain a relatively pure starting material (15). High purity can then be achieved through the addition of a single polishing step. The purification of extracellular recombinant peptides and polypeptides is usually complicated by large starting volumes requiring an initial capture step to concentrate the sample. This inconvenience can be overcome by introducing a specific terminal sequence, such as protein A (16), glutathione transferase (17) or polyamino acids, at the plasmid level which allows the polypeptide to be purified by affinity chromatography. After initial purification and concentration, followed if necessary by chemical or enzymatic removal of the affinity handle, the recombinant polypeptide may then be finally purified by reversed phase chromatography. Intracellular polypeptides must be released by cell lysis (18, 19) before the initial purification can begin. After removal of cellular debris, the initial capture steps aim at removing endotoxins, nucleic acids and any remaining cell debris or potentially coagulating substances such as lipids. As in the case of secreted polypeptides, initial purification is facilitated by use of an affinity handle. If an affinity handle cannot be used then other concentrating chromatographic techniques, such as ion exchange, must be used in the initial capture steps. Reversed phase chromatography, sometimes in combination with high resolution gel filtration, is used in the final polishing steps to produce a homogeneous polypeptide.
62
The purification of recombinant polypeptides can be monitored by techniques like analytical reversed phase chromatography, amino acid analysis, peptide mapping, bioassay or polyacrylamide electrophoresis.
Process purification of inclusion bodies As previously discussed, relatively pure starting material can be produced by expressing a recombinant protein as insoluble inclusion bodies, as long as a satisfactory solubilisation and refolding protocol is available. High purity can then be achieved by a single polishing step. Since reversed phase chromatography is known to be a particularly efficient means of removing endotoxins it would appear to be especially suitable as a polishing step for proteins which are to be used in biological systems. However, until recently, only silica based media were available for large scale purification of inclusion bodies. These media have low binding capacity, are easily fouled and almost impossible to regenerate. High back pressures are generated which lead to longer separation times and, consequently, lower recoveries. These limitations have now been overcome by the use of SOURCE 30RPC, a media specifically designed for large scale purification. The 30 µm monosized beads of SOURCE 30RPC not only generate very low back pressures, but give high recoveries and are easily sanitised. An example of the use of SOURCE 30RPC for large scale purification of inclusion bodies can be found in Downstream Vol. 28, p.14 -17, 1998 Code no: 18-1132-72, Amersham Pharmacia Biotech.
Purification of recombinant human epidermal growth factor A protocol was developed for purifying recombinant human Epidermal Growth Factor (rhEGF), Mr ca. 6 000, expressed as an extracellular product by Saccharomyces cerevisiae. The purification procedure involved a capture step followed by hydrophobic interaction chromatography on Phenyl Sepharose 6 Fast Flow (high sub) and ion exchange chromatography on Q Sepharose High Performance. The first two steps were efficient for purification and gave good recovery. However, the product pool after ion exchange chromatography still contained small amounts of impurities that were unresolved from the main product. These stubborn product variants were removed in a polishing step using reversed phase chromatography on SOURCE 15RPC (Fig. 37). A small scale experiment (Fig. 39 a) showed that elution with a gradient of acetonitrile concentration (1.3% B/min) resulted in a product which gave a single peak in analytical reversed phase chromatography on C2/C18 silica.
63
a Sample: Column: Eluent A: Eluent B: Gradient: Flow:
b Sample: Column: Eluent A: Eluent B: Gradient: Flow:
2.14 ml (0.34 mg EGF) partially purified rhEGF. RESOURCE RPC, 3 ml (i.d. 6.4 mm, length 100 mm) 0.05% TFA, 5% acetonitrile in water 0.05% TFA, 80% acetonitrile in water 1.3% B/min 1.6 ml/min; 300 cm/h
AU
62.5 ml (10 mg EGF) partially purified rhEGF. SOURCE 15RPC (i.d. 35 mm, length 100 mm) 0.05% TFA, 5% acetonitrile in water 0.05% TFA, 80% acetonitrile in water 1.3% B/min 50 ml/min; 300 cm/h
%B 100 AU
%B 100
80 0.10
0.060
80 0.08 60 0.040
60
0.06 40 0.04
40
0.02
20
0.020 20
0 0 0
20.0
40.0
60.0
80.0 Time (min)
0 0
20.0
40.0
0 60.0 Time (min)
Fig. 37. Preparative purification of rhEGF on SOURCE 15RPC at different scales. Sample load (a) 0.34 mg EGF, (b) 10 mg EGF. (Work by Amersham Pharmacia Biotech AB, Uppsala, Sweden.)
The result obtained on the 3 ml column (RESOURCE RPC, 3 ml) could be scaled up directly by a factor of 30 (Fig. 37 b). A column with a larger diameter was used to accommodate the larger volume of medium required for the increased mass of sample. Increasing column length to increase the column volume is inefficient since both the separation time and the operating pressure are increased with little or no increase in resolution. The volumetric flow (ml/min) was increased in proportional to the increase in column cross-sectional area to compensate for the increased column volume. In this way the flow velocity (cm/h) and the separation time were held constant. The differences between the results obtained at the two scales were negligible. The recovery was 92% and the recovered material was shown to be homogeneous using analytical reversed phase chromatography.
64
Chemically synthesised peptides Synthesis of peptides containing fewer than 20 amino acids is now a routine laboratory procedure. Peptides up to 100 amino acid residues can be synthesised, but with more difficulty. Essentially two different chemistries are used for peptide synthesis, but both procedures result in similar major contaminants in the final product. These contaminants include peptides with amino acid deletions, peptides with truncated amino acid sequences and peptides with modified amino acid residues. Small organic molecules, such as phenol and thiols, are also present, resulting from the removal of the synthesised peptide from its solid support. Synthetic peptides of less than 20 amino acid residues can often be purified to the required level by a single reversed phase separation. To monitor purity an analytical reversed phase medium is used (with a different selectivity compared to the medium used for purification), usually in combination with mass spectrometry. Reversed phase media should be selected according to the selectivity for the target peptide and also according to the scale of purification. For microgram quantities a 5µm medium will give good separation (see Figure 38) whereas larger quantities may require 15µm or 30 µm media to achieve sufficient capacity and speed for an efficient purification. Other purification techniques, such as ion exchange, may be used in combination with reversed phase for the purification of larger synthetic peptides.
Purification of a phosphorylated PDGF α-receptor derived peptide Peptide pY574α is an 18 amino acid residue phosphorylated peptide constituting part of the intracellular domain of the PDGF α-receptor. The peptide was designed and synthesised for use as a ligand for subsequent affinity purification of potential signal transduction proteins present in cell lysates.
Figure 40 shows the single reversed phase separation used to achieve required purity. PY574α is readily soluble under alkaline conditions, but poorly soluble under acidic conditions. This eliminates the possibility of using silica gels because of their incompatability with alkaline conditions. SOURCE RPC was therefore selected to allow a high yield purification under alkaline conditions. The 5 µm medium SOURCE 5RPC gave sufficient capacity, speed and resolution (20).
65
A %B 100
pY574α
80
200
60 100
System: Sample: Column: Eluent A: Eluent B: Gradient: Flow:
ÄKTA purifier Peptide pY574 α 200 µg of crude material SOURCE 5RPC ST 4.6/150 NH4HCO3, 0.1 mol/l, pH 7.9 60% acetonitrile 10–40% B over 75 ml 0.67 ml/min
40 230 nm
0
20
268 nm
–50
278 nm
0 0
50
100
min
Fig. 38. Purification of the PDGF α -receptor derived peptide using SOURCE 5RPC ST4.6/150 at pH 7.9.
Structural characterisation of a 165 kDa protein In order to analyse its structure, a native 165 kDa protein was subjected to tryptic digestion. If cleavage according to the specificity of trypsin was complete, the digest was expected to contain more than 150 fragments. As shown in Figure 39, this very large number of peptides was separated on a µRPC C2/C18 ST 4.6/100 column using a shallow gradient (21). The separation was monitored at 215 nm, 254 nm and 280 nm. The 215 nm is specific for the peptide bond, thus revealing all eluted peptides. The other two wavelengths are useful for monitoring the aromatic amino acid residues. By use of peak absorbance ratio calculations fragments containing phenylalanine, tyrosine and tryptophan can be specifically detected which is a significant help during subsequent structural analysis of the collected fragments (22).
A215 nm
System: Column: Sample:
%B conductivity gradient
80 40.0
Eluent A: Eluent B: Gradient:
60 215 nm
20.0
40 0.0
20
254 nm 280 nm
0
Flow:
ÄKTA purifier µRPC C2/C18 ST 4.6/100 Tryptic digest of a native 165 kDa protein; 1 nmol 0.060% TFA in water 0.055% TFA in 84% acetonitrile 0% B for 2 column volumes (CV), 0–50% B for 392 CV (650 min) 50–100% B for 55 CV (91 min) 100% B for 10 CV (17 min) 1 ml/min
0 200
400
ml
Fig.39. Separation of a tryptic digest of a native 165 kDa protein using ÄKTApurifier and µRPC C2/C18 3 µm ST 4.6/100
66
Protein fragments from enzyme digests Protein characterisation at the micro-scale Reversed phase chromatography plays a central role in a cluster of techniques (Fig. 42), including polyacrylamide gel electrophoresis (PAGE), mass spectrometry and micro-scale high performance LC, of increasing importance in high sensitivity characterisation of proteins (23, 24, 25, 26, 27)
Protein separation: SDS-PAGE or 2-D PAGE
Blotting
Edman sequencing
In-gel digestion
Peptide separation: reversed phase chromatography SMART System
micro-scale RPC
UV-detection
Flow splitter
Fraction collection
ESI-MS
MALDI-TOF MS
Data base
Protein identification
Fig. 40. Techniques for high sensitivity characterisation of proteins.
Many gene products are first isolated in very small amounts as bands or spots in SDS-PAGE or 2D-PAGE. Micro-scale reversed phase chromatography allows partial amino acid sequences, either terminal or internal, to be obtained for these proteins, thereby permitting their subsequent identification by matching against very large protein structure data-bases (28, 29). Although partial N-terminal sequences can sometimes be obtained from blotted proteins, in-gel digestion of the protein to produce characteristic peptide fragments may be more generally applicable. The combination of in-gel protein digestion (30, 31, 32, 33) with micro-scale high performance reversed phase purification of the fragments, followed by Edman sequence analysis and sequence matching against protein sequence data bases is highly efficient. Hellman and Gonez (34) were able to identify human glial fibrillary protein (hGFAP) after electrophoretic separation of glioma cell proteins even when the polyacrylamide gel had been dried onto paper for storage. The peptides from the in-gel digest were extracted and subsequently fractionated by micro-scale high performance reversed phase chromatography using SMART System (Amersham Pharmacia Biotech) (Fig. 41). The peptide in fraction 22 (Fig. 42) was sequenced. 67
Sample:
Protein digested with trypsin in situ in an SDS-PAGE gel, extracted with acetonitrile (60%) and redissolved in eluent A Column: µRPC C2/C18 SC 2.1/10 (i.d. 2.1 mm, length 100 mm) Eluent A: 0.065% TFA in water Eluent B: 0.05% TFA in acetonitrile Gradient: 1% B for 6 min 1-41% B in 80 min 41% B for 10 min 41-81% B in 10 min Flow: 0.1 ml/min
Fig. 41. Fractionation of peptides obtained by in-gel digestion of a single band from an electrophoretic separation of glioma cell proteins. (SMART Bulletin hGFAP (1993). Hellman, U., Gonez, J.)
Three matches were obtained in a search of a protein sequence data base and, since only one of the matches corresponded to a protein of human origin, the protein could be identified as hGFAP. A214 nm
22
Time (min)
Fig. 42. Enlargement of the indicated part of Figure 43. The peptide in fraction 22 was sequenced. (SMART Bulletin hGFAP (1993). Hellman, U., Gonez, J.)
68
Protein identification by LC-MS Developments in mass spectrometry now allow highly accurate estimates of the masses of peptides to be made after separation by reversed phase chromatography. The column eluent may be split so that part is delivered to an ElectrosprayIonisation Mass Spectrometer (ESI-MS) for on-line analysis (35), while the major part of the eluent is collected in a fraction collector for off-line analysis, including amino acid sequencing and Matrix-Assisted Laser-Desorption-Ionisation Time-OfFlight Mass Spectrometry (MALDI-TOF MS). Reversed phase chromatography coupled to ESI-MS provides high precision data for protein identification by matching against a protein-fragment data-base. In studies of two forms of apolipoprotein A, Apo A1 and Apo A1-M, Renlund et al. (36) have given an elegant demonstration of how structural modifications of a protein can be elucidated by the high resolving power of reversed phase chromatography in combination with ESI-MS. Apo A1 is the major protein component of plasma high density lipoproteins (HDL) and plays an important role in the reverse transport of cholesterol from tissues (37). High plasma levels of Apo A1 are associated with a low incidence of coronary artery disease. Apolipoprotein A1-Milano (Apo A1-M) is a genetic variant of normal Apo A1 (38), characterised by the replacement of Arg at position 173 by Cys. In vitro studies have indicated a threefold increase in the cholesterol transporting ability of Apo A1-M. The carriers of Apo A1-M have quite low levels of HDL, but they do not show any signs of arteriosclerosis. Apolipoprotein A1 was purified from normal human plasma. Apo A1-M was produced in an excreting E. coli system and isolated using conventional LC techniques followed by reversed phase HPLC. Both proteins were digested with Lys-C specific endopeptidase, and the peptides were separated on a µRPC C2/C18 SC 2.1/10 column connected to SMART System (Amersham Pharmacia Biotech) equipped with a Flow Splitter. The flow split ratio (MS flow/Total flow) was regulated to give a flow to the electrospray mass spectrometer (VG Platform, Fisons, Manchester, UK) in the range 5-10 µl/min. Figure 43 shows a separation of 400 pmol each of Apo A1-M and Apo A1. The most significant difference between these chromatograms is in the retention of the last peak, indicated by K15A1-M and K15A1 respectively. The ESI mass spectra of the peptides eluted in these peaks are shown in Figures 44 and 45. The mass difference between the two fragments is 52.92 Da, which deviates by 0.18 Da from the mass difference, 53.10, between Arg and Cys. This would indicate, with an error in the mass estimation less than 4 x 10-3%, that an Arg should be substituted with a Cys in Apo A1-M.
69
Samples:
Column: Eluent A: Eluent B: Gradient: Flow:
Upper: Lys-C endopeptidase digest of Apo A1-M (400 pmol) Lower: Lys-C endopeptidase digest of Apo A1 (400 pmol) µRPC C2/C18 SC 2.1/10 (i.d. 2.1 mm, length 100 mm) 0.25% pentafluoroproprionic acid (PFPA) in water 0.25% PFPA in acetonitrile 10-60% B in 200 min 25 µl/min
A215 nm
Fig. 44. ESI-MS mass spectrum obtained from peptide material from peak K15A1-M in Fig. 45. Mm for K15A1-M was 4773.54. See the text for details. Renlund, S., Wadensten, H., Persson, P. et al. (36).
Fig. 43. Reversed projection of separations of Lys-C endopeptidase digests of ApoA1-M (upper) and ApoA1 (bottom) by reversed phase chromatography. Renlund, S., Wadensten, H., Persson, P. et al. (36).
Fig. 45. ESI-MS mass spectrum obtained from peptide material from peak K15A1 in Fig. 45. Mm for K15A1 was 4826.46. See the text for details. Renlund, S., Wadensten, H., Persson, P. et al. (36).
Chemically synthesised oligonucleotides Automated solid phase synthesis is a commonly used procedure for preparing oligonucleotides 20 to 30 bases long for use as DNA probes and templates for PCR (39) reactions. Much longer oligonucleotides, 100 bases or more, are also synthesised. The main contaminants are truncated sequences together with smaller amounts of oligonucleotides which contain modified bases. After gel filtration to remove small organic contaminants which are produced during chemical cleavage of the oligonucleotide from its solid support, the desired oligonucleotide can generally be purified to sufficient purity by a single pass through a reversed phase column. Separation of the complete sequence from incomplete sequences is simplified if the purification is performed with the 5´ trityl protecting group still attached as none of the truncated sequences produced as side products during the synthesis contain the protecting group. The large hydrophobic trityl group contains three benzene ring structures and causes the complete sequence to elute significantly later than the truncated sequences (Fig. 46). 70
5' O
HO
Base 3
O Free 5' hydroxyl group
O
O=P
Base 2
O– O O
O=P
Base 1
O– 3'
OH
5' C—O
O
Base 3
O O
O=P Trityl protecting group
O
Base 2
–
O O=P O
O
Base 1
–
3'
OH
Fig. 46. Partial structure of a synthetic oligonucleotide with the trityl protecting group (A) off and (B) on.
The separation is carried out close to neutral pH with triethylammonium acetate as the ion pairing agent and a linear gradient of increasing acetonitrile concentration. Purification of a typical synthetic oligonucleotide (5´ DMTr-T TCT AGC TCA ACC GGT CAA) at different scales is shown in Figure 47.
71
Sample: Column: Eluent A: Eluent B: Gradient: Flow:
Purification of crude synthetic 19-base oligonucleotide, trityl on, by reversed phase chromatography at different sample loads. (A) 65 µg, (B) 650 µg, (C) 6500 µg RESOURCE RPC 1 ml Triethylamine acetate (0.1 M, pH 7.0) in water 100% acetonitrile 5% B for 5 min 5-40% B in 20 min 1 ml/min
A254 nm
%B
A 65 µg 100
50
0 A254 nm
10.0
Time (min) %B
B 650 µg 100
50
0
10.0
A254 nm
Time (min) %B
C 6500 µg 100
50
0
10.0
Time (min)
Fig. 47. Reversed phase purification of a synthetic 19-base oligonucleotide with the trityl protecting group on. (Work by Amersham Pharmacia Biotech AB, Uppsala, Sweden.)
Due to the excellent resolving power of the reversed phase technique, additional purification steps are generally not required.
72
Chapter 5
Fault finding chart Symptom
Cause
Remedy
Column is clogged
Column filter is clogged.
Replace the filter. Always filter samples and mobile phase before use. Clean or replace the column. Prior to chromatography, precipitate with 10% Dextran Sulphate or 3% PVP. Clean and regenerate the column. Modify the eluent to maintain stability.
Presence of particulates, lipoproteins or protein aggregates.
No flow through the column
Reduced or poor flow through the column
Precipitation of proteins in the column caused by removal of stabilising agents during fractionation. No flow from pump.
End-piece or adapter or tubing is blocked. Bed surface blocked by precipitated proteins.
Bed is compressed. Back pressure increases during a run or during successive runs
Precipitation of sample in the column and/or at the top of the bed.
Check the pump and system for leaks. Make sure the injector valve is in the correct position. Remove and clean, if possible. Clean the column by recommended procedures or change the precolumn. Replacing the column may be necessary. Clean the column and exchange or clean the filter or clean/replace the precolumn. Alternatively, replace any additives initially used to solubilize the sample so long as they are compatible with the reversed phase medium.
73
Symptom
Cause
Remedy
Back pressure increases during a run or during successive runs
Turbid sample has been applied to the column.
Improve sample solubility by increasing the concentration of organic modifier in the initial mobile phase. Alternatively, adjust the pH of the initial mobile phase to increase sample solubility. Replace the filter. Always filter samples and mobile phase before use. Adjust the pH so that the sample is not denatured. Increase the concentration of organic modifier in mobile phase B. Switch to a reversed phase column with a less hydrophobic immobilised ligand. Alternatively, change to an organic modifier with more efficient elution properties. Add or increase the concentration of ion pairing agent. Alternatively, switch to a column with a more hydrophobic immobilised ligand or change to an organic modifier with less efficient elution properties. Adjust the pH so that the sample binds. Clean the column by recommended procedures.
Column filter is clogged.
Sample does not elute in the organic solvent gradient
Unsuitable pH. Final concentration of organic modifier in the gradient is too low. Eluting power of the organic modifier is too low.
Sample components elute in the equilibrium phase
Sample not hydrophobic enough to adsorb to column.
Unsuitable pH. Impurities adsorbed to the column.
74
Symptom
Cause
Remedy
Resolution is less than expected
Concentration of organic modifier in the initial mobile phase is too high. Column is not properly equilibrated. Gradient slope is too steep.
Decrease the concentration of organic modifier. Repeat or prolong the equilibration step. Use a shallower gradient or a plateau in the gradient. Add or adjust the concentration of ion pairing agent. Change the flow cell.
Poor selectivity.
Detector cell volume is too big. Column is poorly packed.
Proteins or lipids have precipitated on the column. Column was overloaded.
Sample has not been filtered. Protein in the sample has aggregated and bound strongly to the medium. Mixed mode retention behaviour due to surface silanols on the silica gel. Large mixing spaces in or after column. Flow rate is too high.
Column ageing.
Check packing by doing a plate count and re-pack or replace the column if necessary. Clean and regenerate the column. Change the eluent to maintain stability. Clean and regenerate the column. Decrease the sample load. Filter the sample before applying it to the column. Increase organic content of initial mobile phase. Lower the pH to suppress ionisation of silanol groups or replace the column. Reduce all post column volumes. Run the separation at a lower flow rate (not a problem with proteins). Adjust mobile phase pH to suppress ionisation of surface silanols or replace the pre-column. If necessary, replace the column.
75
Symptom
Cause
Remedy
Leading or very rounded peaks
Column is poorly packed.
Check packing by doing a plate count and re-pack or replace the column if necessary. Clean and regenerate the column. Replace the precolumn or column if necessary. Clean and regenerate the column. Decrease the sample load. Clean the column by recommended procedures and replace the top filter. Alternatively, replace the pre-column. Reduce sample concentration. Clean and regenerate the column. Change the eluent to maintain stability.
Column needs regeneration.
Column was overloaded.
Tailing peaks
Precipitation of sample in the column and/or at the top of the bed.
Sample too viscous. Previous elution profile cannot be reproduced
Proteins or lipids have precipitated on the column. Incomplete column equilibration.
Low recovery of activity while normal recovery of sample mass
76
Sample has not been filtered. Incorrect mobile phase pH or loss of organic modifier by evaporation. Sample has altered during storage. Sample components denatured or inactivated in the mobile phase.
Equilibrate until the baseline is stable (5 - 10 column volumes). Filter the sample before applying it to the column. Prepare fresh mobile phase. Prepare fresh sample. Determine the pH and organic solvent stability of the sample. Decrease the separation time in order to limit exposure of sample components to mobile phase. Alternatively, use a reversed phase medium with a less hydrophobic ligand requiring milder elution
Symptom
Sample amount in the eluted fractions is much less than expected
Cause
Enzyme sample separated from cofactor or similar. Sample has been degraded by proteases or nucleases. Sample precipitates.
Basic proteins adsorbed to column by ionic retention.
More activity is recovered than was applied to the column
Peaks too small
Sample adsorbed to filter during preparation. Removal of inhibitors during separation. Different assay conditions have been used before and after the chromatographic step. Sensitivity range incorrectly set on the detector or recorder. Excessive zone broadening.
Remedy conditions. Alternatively, change the organic modifier. Test by pooling fractions and repeating the assay. Add inhibitors or minimise separation time. Decrease the sample load or change mobile phase conditions to maintain stability. Increase pH of mobile phase or add/adjust ion pairing agent concentration in the mobile phase. Use a different type of filter. Replace if necessary. Use the same assay conditions for all the assays in your purification scheme. Adjust.
Check the column packing by doing a plate count. Repack if necessary. Broad peaks may be due to column ageing. Adjust mobile phase pH to suppress ionisation of surface silanols or replace the pre-column. If necessary, replace the column.
77
Symptom
Cause
Remedy
Peaks to small
Sample absorbs poorly at the chosen wavelength. Column packed or stored at cool temperature and then warmed up.
Monitor at a different wavelength. Small bubbles can often be removed by passing well de-gassed mobile phase upwards through the column. The column may need to be re-packed. Take special care if mobile phases are used after storage in a refrigerator or cold room. Do not allow column to warm up
Cracks in the bed
Large air leak in the column.
Check all connections for leaks. Re-pack the column.
Distorted bands as the sample runs into the bed
Net in upper adapter is clogged or damaged.
Dismantle the adapter, clean or replace the net.
Bubbles in the bed
Filter or centrifuge the sample. Protect eluents from dust. Re-install the adapter (if Air bubble trapped at the top of the column or in the present) taking care to avoid air bubbles. inlet adapter. Column is poorly packed. Re-pack the column. Be careful not to pack at excessively high pressures. Particles in eluent or sample.
Distorted bands as the sample passes down the bed Negative peaks at solvent front Strange peaks in the chromatogram Peaks on blank gradients
78
Make sure sample is dissolved in initial mobile phase. Impurities in mobile phase. Use high quality reagents. Refractive index effects.
Organic impurities in water used to prepare mobile phase A.
Use high quality reagents. Pre-filter water through large particle reversed phase medium before preparing the mobile phase if necessary.
Symptom
Spikes in the chromatogram UV baseline rises with gradient
Retention time of same sample component increases over time
Cause
Remedy
Incomplete elution from previous run.
Wash the column according to the recommended method. Use de-gassed solutions.
Air bubble trapped in the UV cell. Eluents A and B absorb differently at the chosen wavelength.
Impurities in mobile phase. Mixed mode retention behaviour due to surface silanols on the silica gel.
Peak width increases over time
Mixed mode retention behaviour due to surface silanols on the silica gel.
Excessive baseline noise
UV absorption by component of mobile phase.
Impurities in mobile phase.
Adjust the concentrations of mobile phase components which absorb at the chosen wavelength or monitor at a different wavelength. Use high quality reagents. Lower the pH to suppress ionisation of silanol groups or replace the column. Lower the pH to suppress ionisation of silanol groups or replace the column. Monitor at different UV wavelength or reduce concentration of UV absorbing component (usually the ion pairing agent). If organic modifier is absorbing, change to one with a lower UV cut-off. Use high quality reagents.
79
80
Chapter 6
References 1. Renlund, S., Erlandsson, I., Hellman, U., Silberring, J., Wernstedt, C., Lindström, L., Nyberg, F. Micropurification and amino acid sequence of β-casomorphin-8 in milk from a woman with postpartum psychosis. Peptides 14, 1125-1132, 1993. 2. Olson, C.V., Reifsnyder, D.H., Canonva-Davis, E., Ling, V.T., Builder, S.E, Preparative isolation of recombinant human insulin-like growth factor 1 by reversed-phase high-performance liquid chromatography. J. Chromatogr. A 675, 101-112, 1994. 3. Dorsey, J.G., Cooper, W.T. Retention mechanisms of bonded-phase liquid chromatography. Anal. Chem. 66, 857A-867A, 1994. 4. Physical chemistry of macromolecules. Tanford, C. Wiley, London. 1961. 5. Standard chromatographic conditions for size-exclusion, ion-exchange, reversed-phase and hydrophobic interaction chromatography. Hodges, R.S., Mant, C.T. in High Performance Liquid Chromatography of Peptides and Proteins: Separation, Analysis and Conformation. (ed. Mant, C.T., Hodges, R.S.) CRC Press, Boca Raton, 1991, pp. 11-22. 6. Reversed-phase HPLC separation of sub-nanomole amounts of peptides obtained from enzymatic digests. Stone, K.L., LoPresti, M.B., Crawford, J.M., DeAngelis, R., Williams, K.R. in High Performance Liquid Chromatography of Peptides and Proteins: Separation, Analysis and Conformation. (ed. Mant, C.T., Hodges, R.S.) CRC Press, Boca Raton. 1991. pp 669-677. 7. Sakamoto, Y., Kawakami, N. and Sasagawa, T. Prediction of peptide retention times. J. Chromatogr. 442, 69-79, 1988. 8. Reversed-phase chromatography of proteins. Corran, P.H. in HPLC of macromolecules, a practical approach. (ed. Oliver, R.W.A.) IRL Press, Oxford. 1989, pp 127-156. 9. McCown, S.M., Southern, D., Morrison, B.E. Solvent properties and their effects on gradient elution high performance liquid chromatography. III Experimental findings for water and acetonitrile. J. Chromatogr. 352, 493509, 1986. 10. Winkler, G., Wolschann, P., Briza, P., Heinz, F.X., Kunz, C. Spectral properties of trifluoroacetic acid-acetonitrile gradient systems for separation of picomole quantities of peptides by reversed-phase high performance liquid chromatography. J. Chromatogr. 347, 83-88, 1985. 11. Purification of peptides from natural sources: isolation and characterisation of cholecystokinin-58 (CCK-58) from pig intestine. Application Note ÄKTApurifier, 18-1119-52, 1997. 81
12. Liao, Y.-F., Lal, A., and Moremen, K.W. Cloning, expression, purification and characterisation of the human broad specificity lysosomal acid alphamannosidase. J. Biol. Chem. 271 (8), 28348-28358, 1966. 13. Frangioni, J.V. and Neel, B.G. Solubilisation and purification of enzymatically active glutathione-S-transferase (pGEX) fusion proteins. Anal. Biochem. 210, 179-187, 1993. 14. Belew, M., Zhou, Y., Wang, S., Nyström, L-E, Janson, J-C. Purification of recombinant human granulocyte-macrophage colony stimulating factor from the inclusion bodies produced by transformed E.coli cells. J.Chrom. A. 679, 67-83, 1990. 15. Werner, M.H. et al., FEBS Lett 345, 125-130, 1994. 16. Affinity Chromatography. Principles and Methods, Amersham Pharmacia Biotech, 18-1022-29, 1997. 17. GST Gene Fusion System 3rd ed., rev.1, Amersham Pharmacia Biotech, 18-1123-20, 1997. 18. Johansson, H.J., Jägersten, C., and Slorach, J. Large scale recovery and purification of periplasmic recombinant protein from E.coli using expanded bed adsorption chromatography followed by a new ion exchange medium. J. Biotechnology 48, 9-14, 1996. 19. Purification of a recombinant Pseudomonas aeruginosa exotoxin expressed in E.coli. Application Note 212, BioProcess Media, Amersham Pharmacia Biotech, 18-1020-94. 20. Purification of a phosphorylated PDGF α-receptor derived peptide at high pH using a polymer stationary phase. Amersham Pharmacia Biotech, Application Note 18-1132-63. 21. Efficient isolation of protein fragments for structure analysis, Application Note 18-1119-53, Amersham Pharmacia Biotech. 22. Micropurification by RPC for protein/peptide sequence analysis, Technical Note, SMART System, 18-1022-96, Amersham Pharmacia Biotech. 23. Aebersold, R., Hess, D., Morrison, H.D., Yungwirth, T., Chow, D.T., Affolter, M., Amankwa, L. Recent advances and new targets in high-sensitivity protein characterization. J. Protein Chem. 13, 465-466, 1994. 24. Jensen, O. N., Podtelejnkov, A. V. and Mann, M. Identification of the components of simple protein mixtures by high-accuracy peptide mass mapping and data base searching. Anal. Chem., 69, 4741-4750, 1997. 25. Haynes, P. A., Fripp, N. and Aebersold, R. Identification of gel-separated proteins by liquid chromatography-electrospray tandem mass spectrometry: Comparison of methods and their limitations. Electrophoresis, 19, 939-945, 1998. 26. Yates, J.R. III. Mass spectrometry in the age of the proteome. J. Mass Spectrom. 33, 1-19, 1998. 82
27. Wilkins, M.R., Williams, K.L., Appel, R.D. and Hochstrasser, D.F. (Eds) Proteome Research: New frontiers in Functional genomics. Springer-Verlag Berlin Heidelberg 1997. 28. Bleasby, A.J., Wootton, J.C. Construction of validated, non-redundant composite protein sequence databases. Protein Eng. 3, 153-159, 1990. 29. Pappin, D.J.C., Hojrup, P., Bleasby, A.J. Rapid identification of proteins by peptide mass fingerprinting. Current Biol. 3, 327-332, 1993. 30. Eckerskorn, C., Lottspeich, F. Combination of two-dimensional gel electrophoresis with microsequencing and amino acid composition analysis: improvement of speed and sensitivity in protein characterization. Electrophoresis 11, 554-561, 1990. 31. Rosenfeld, J., Capdevielle, J., Guillemot, J.C., Ferrara, P In-gel digestion of proteins for internal sequence analysis after one- or two-dimensional gel electrophoresis. Anal. Biochem. 203, 173-179, 1992. 32. Hellman, U., Wernstedt, C., Góñez, J., Heldin, C.-H. Improvement of an “in-gel“ digestion procedure for the micropreparation of internal protein fragments for amino acid sequencing. Anal. Biochem. 224, 451-455, 1995. 33. Shevenchenko, A., Wilm, M., Vorm, O. and Mann, M. Mass spectrometric sequencing of proteins from silver-stained polyacrylamide gels. Anal. Chem. 68, 850-858, 1996. 34. Sequence analysis and identification of hGFAP after in situ enzymatic digestion in an electrophoretic gel. SMART Bulletin hGFAP. Hellman, U., Gonez, J. Amersham Pharmacia Biotech. 35. Electrospray LC-MS using SMART System Application Note, SMART System, Amersham Pharmacia Biotech, 18-1104-38. 36. Renlund, S., Wadensten, H., and Persson, P. Studies of different forms of apolipoprotein A1 and insulin-like growth factor I by electrospray LC-MS. Nishi, N. (Ed): Peptide Chemistry, Protein Research Foundation, Osaka, 6972, 1996. 37. Brewer, H. B., Fairwell, T., LaRue, A., Ronan, R., Houser, A., Bronzert, T. J. The amino acid sequence of human ApoA-I, an apolipoprotein isolated from high density lipoprotein. Biochem. Biophys. Res. Commun. 80, 623-630,1978. 38. Molecular characterization of native and recombinant apolipoprotein A IMilano dimer. The introduction of an interchain disulfide bridge remarkably alters the physicochemical properties of apolipoprotein A-I. J. Biol. Chem. 269 (1994) 32168-32174. Calabresi, L., Vecchio, G., Longhi, R., Gianazza, E., Palm, G., Wadensten, H., Hammarstršm, A., Olsson, A., Karlstršm, A., Sejlitz, T., Ageland, H., Sirtori, C. R., Franceschini, G. 39. The PCR process is covered by U.S. Patents 4,683,195 and 4,683,202 owned by Hoffmann-La Roche Inc. Use of the PCR process requires a license. Nothing here should be construed as an authorisation or implicit license to practice PCR under any patents held by Hoffmann-La Roche Inc. 83
Chapter 7
Ordering Information Product
Column material
Quantity/Pack size
Code no.
SOURCE 5 RPC SOURCE 5RPC ST 4.6/150
Steel
1
17-5116-01
SOURCE 15 RPC RESOURCE RPC,1 ml RESOURCE RPC, 3 ml SOURCE 15RPC ST 4.6/100 SOURCE 15RPC SOURCE 15RPC SOURCE 15RPC SOURCE 15RPC SOURCE 15RPC
PEEK PEEK Steel -
1 1 1 10 ml 200 ml 500 ml 1 litre 5 litre
17-1181-01 17-1182-01 17-5068-01 17-0727-20 17-0727-02 17-0727-03 17-0727-04 17-0727-05
SOURCE 30 RPC SOURCE 30RPC SOURCE 30RPC SOURCE 30RPC SOURCE 30RPC SOURCE 30RPC
-
10 ml 200 ml 500 ml 1 litre 5 litre
17-5120-20 17-5120-02 17-5120-03 17-5120-04 17-5120-05
Sephasil Protein Sephasil Protein C4 5 µm ST 4.6/100 Sephasil Protein C4 5 µm ST 4.6/250 Sephasil Protein C4 12 µm ST 4.6/250 Sephasil Protein C4 12 µm ST 10/250 Sephasil Protein C4 12 µm ST 20/250 Sephasil Protein C4 12 µm ST 50/250 Sephasil Protein C4 12 µm Sephasil Protein C4 12 µm
Steel Steel Steel Steel Steel Steel -
1 1 1 1 1 1 100 g 1 kg
17-6000-24 17-6000-21 17-6000-27 17-6000-57* 17-6000-58* 17-6000-59* 17-6000-51* 17-6000-52*
Sephasil Peptide Sephasil Peptide C8 5 µm ST 4.6/100 Sephasil Peptide C8 5 µm ST 4.6/250 Sephasil Peptide C18 5 µm ST 4.6/100 Sephasil Peptide C18 5 µm ST 4.6/250 Sephasil Peptide C8 12 µm ST 4.6/250 Sephasil Peptide C8 12 µm ST 10/250 Sephasil Peptide C8 12 µm ST 20/250 Sephasil Peptide C8 12 µm ST 50/250 Sephasil Peptide C8 12 µm Sephasil Peptide C8 12 µm Sephasil Peptide C18 12 µm ST 4.6/250 Sephasil Peptide C18 12 µm ST 10/250 Sephasil Peptide C18 12 µm ST 20/250 Sephasil Peptide C18 12 µm ST 50/250 Sephasil Peptide C18 12 µm Sephasil Peptide C18 12 µm
Steel Steel Steel Steel Steel Steel Steel Steel Steel Steel Steel Steel -
1 1 1 1 1 1 1 1 100 g 1 kg 1 1 1 1 100 g 1 kg
17-6000-25 17-6000-22 17-6000-26 17-6000-23 17-6000-28 17-6000-60* 17-6000-61* 17-6000-62* 17-6000-53* 17-6000-54* 17-6000-29 17-6000-63* 17-6000-64* 17-6000-65* 17-6000-55* 17-6000-56*
µRPC µRPC C2/C18 PC 3.2/3# µRPC C2/C18 SC 2.1/10# µRPC C2/C18 ST 4.6/100
Glass Steel Steel
1 1 1
17-0703-01 17-0704-01 17-5057-01
Sephasil Sephasil C8 SC 2.1/10## Sephasil C18 SC 2.1/10##
Steel Steel
1 1
17-0769-01 17-0904-01
*available on request #PC and SC columns are designed for SMART System. Precision Column Holder (17-1455-01) is required for attachment to ÄKTAdesign systems or other HPLC systems. ## Sephasil C8 and Sephasil C18 media available only in pre-packed columns for SMART System
84
Trademarks RESOURCE, SOURCE, Sephasil, Sephadex, Sepharose, HiLoad, SMART, ÄKTA, FineLINE, and Phast System are trademarks of Amersham Pharmacia Biotech Limited or its subsidiaries Amersham is a trademark of Nycomed Amersham plc Pharmacia and Drop Design are trademarks of Pharmacia & Upjohn Inc Terms and Conditions of Sale All goods and services are sold subject to the terms and conditions of sale of the company within the Amersham Pharmacia Biotech group which supplies them. A copy of these terms and conditions is available on request. © Amersham Pharmacia Biotech UK Limited 1999 - All rights reserved Amersham Pharmacia Biotech UK Limited Amersham Place Little Chalfont Buckinghamshire England HP7 9NA Amersham Pharmacia Biotech AB SE-751 84 Uppsala Sweden Amersham Pharmacia Biotech Inc 800 Centennial Avenue PO Box 1327 Piscataway NJ 08855 USA
85
Gel Filtration Handbook – Principles and Methods
Gel Filtration Principles and Methods
www.chromatography.amershambiosciences.com
18-1022-18 Edition AI
Microcarrier cell culture principles & methods
Back to Collection 18-1140-62 Edition AA
to order: Tel: +852 2811 8693 Fax: +852 2811 5251 Australasia Tel: +61 2 9894 5152 Fax: +61 2 9899 7511 Austria Tel: 01 576 0616 20 Fax: 01 576 0616 27 Belgium Tel: 0800 73 888 Fax: 03 272 1637 Canada Tel: 1 800 463 5800 Fax: 1 800 567 1008 Central, East, South East Europe Tel: +43 1 982 3826 Fax: +43 1 985 8327 Denmark Tel: 45 16 2400 Fax: 45 16 2424 Finland Tel: 09 512 3940 Fax: 09 512 1710 France Tel: 0169 35 67 00 Fax: 0169 41 9677 Germany Tel: 0761 4903 401 Fax: 0761 4903 405 Italy Tel: 02 27322 1 Fax: 02 27302 212 Japan Tel: 81 3 5331 9336 Fax: 81 3 5331 9370 Latin America Tel: +55 11 3667 5700 Fax: +55 11 3667 87 99 Middle East and Africa Tel: +30 (1) 96 00 687 Fax: +30 (1) 96 00 693 Netherlands Tel: 0165 580 410 Fax: 0165 580 401 Norway Tel: 2318 5800 Fax: 2318 6800 Portugal Tel: 01 417 7035 Fax: 01 417 3184 Russian Federation Tel: +7 (095) 232 0250,956 1137 Fax: +7 (095) 230 6377 South East Asia Tel: 60 3 724 2080 Fax: 60 3 724 2090 Spain Tel: 93 594 49 50 Fax: 93 594 49 55 Sweden Tel: 018 612 19 00 Fax: 018 612 19 10 Switzerland Tel: 01 802 81 50 Fax: 01 802 81 51 UK Tel: 0800 616 928 Fax: 0800 616 927 USA Tel: +1 800 526 3593 Fax: +1 800 329 3593
Asia Pacific
Cytodex, Sephadex, Sepharose and Ficoll are trademarks of Amersham Pharmacia Biotech Limited or its subsidiaries. Amersham is a trademark of Nycomed Amersham plc. Pharmacia and Drop Design are trademarks of Pharmacia & Upjohn. Amersham Pharmacia Biotech AB Björkgatan 30, SE-751 84 Uppsala, Sweden. Amersham Pharmacia Biotech UK Limited Amersham Place, Little Chalfont, Buckinghamshire HP7 9NA, England. Amersham Pharmacia Biotech Inc 800 Centennial Avenue, PO Box 1327, Piscataway, NJ 08855 USA. Amersham Pharmacia Biotech Europe GmbH Munzinger Strasse 9, D-79111 Freiburg, Germany. All goods and services are sold subject to the terms and conditions of sale of the company within the Amersham Pharmacia Biotech group that supplies them. A copy of these terms and conditions is available on request. © Amersham Pharmacia Biotech AB 1999 – All rights reserved.
Microcarrier cell culture principles & methods
Contents
1. Background .............................................................................................. 7 1.1 Introduction ....................................................................................... 7 1.2 . Adhesion of cells to culture surfaces ................................................. 9 1.3 . The development of microcarriers for animal cell culture ............... 11 1.4 . Applications of microcarrier culture ............................................... 14 1.4.1 . Cell types cultured on Cytodex microcarriers ........................ 15 1.4.2 . Production of large numbers of cells ...................................... 17 1.4.3 . Production of viruses and cell products ................................. 18 1.4.4 . Studies on cell function, metabolism and differentiation ........ 22 1.4.5 . Proteolytic enzyme-free subcultivation and cell transfer ......... 25 1.4.6 . Microscopy ............................................................................ 26 1.4.7 . Harvesting mitotic cells ......................................................... 26 1.4.8 . Transportation and storage of cells ........................................ 27 2. Cytodex microcarriers ............................................................................ 29 2.1 . Requirements for an optimum microcarrier ................................... 29 2.2 . Cytodex 1 ...................................................................................... 30 2.3 . Cytodex 3 ...................................................................................... 30 2.4 . Which Cytodex microcarrier to use? .............................................. 34 2.5 . Availability and storage ................................................................. 35 3. Microcarrier cell culture methods ........................................................... 37 3.1 . General outline of procedure .......................................................... 37 3.2 . Microcarrier culture vessels ........................................................... 38 3.2.1 . Requirements ......................................................................... 38 3.2.2 . Laboratory scale microcarrier culture vessels ......................... 39 3.2.3 . Large scale microcarrier culture vessels ................................. 45 3.2.4 . Siliconizing culture vessels ..................................................... 45 3.3 . Preparing Cytodex microcarriers for culture .................................. 46 3.4 . Initiating a microcarrier culture ..................................................... 47 3.4.1 . Equilibration before inoculation ............................................ 47 3.4.2 . Initial stirring ......................................................................... 48 3.4.3 . Concentration of microcarriers .............................................. 51 3.4.4 . Inoculation density ................................................................ 52 3.4.5 . Inoculum condition ................................................................ 53 3.4.6 . Culture media during the initial culture phase ....................... 54 3.4.7 . Relationship between plating efficiency and culture procedure56 3.5 . Maintaining a microcarrier culture ................................................ 58 3.5.1 . Stirring speed ......................................................................... 58 3.5.2 . Replenishment of culture medium .......................................... 61 3.5.3 . Maintaining cultures at confluence ........................................ 65
3.6 . Monitoring the growth of cells and microscopy ............................. 66 3.6.1 . Direct observation by microscopy .......................................... 66 3.6.2 . Counting cells released after trypsinization ........................... 67 3.6.3 . Counting released nuclei ........................................................ 67 3.6.4 . Fixing cells ............................................................................. 67 3.6.5 . Staining cells .......................................................................... 76 3.7 . Harvesting cells and subculturing .................................................. 77 3.7.1 . Chelating agents .................................................................... 78 3.7.2 . Proteolytic enzymes ............................................................... 78 3.7.3 . Hypotonic treatment .............................................................. 80 3.7.4 . Cold treatment ....................................................................... 81 3.7.5 . Sonication .............................................................................. 81 3.7.6 . Lignocaine for harvesting macrophages ................................. 81 3.7.7 . Modifications to harvesting procedures for large scale cultures 81 3.7.8 . Separating detached cells from microcarriers ......................... 82 3.7.9 . Measurement of cell viability ................................................. 82 3.7.10 . Subculturing techniques ....................................................... 83 3.7.11 . Re-use of Cytodex ............................................................... 84 4. General considerations ............................................................................ 85 4.1 . Culture media ................................................................................ 85 4.1.1 . Choice of culture medium ...................................................... 85 4.1.2 . General comments on components of culture media .............. 88 4.1.3 . Practical aspects of culture media .......................................... 91 4.2 . Serum supplements ........................................................................ 91 4.2.1 . The purpose of serum in culture media .................................. 91 4.2.2 . Choice and concentration of serum supplement ..................... 92 4.2.3 . Variability of sera .................................................................. 96 4.2.4 . Serum free media ................................................................... 97 4.3 . Gas supply ..................................................................................... 97 4.3.1 Gas supply and exchange in microcarrier cultures ................... 98 4.3.2 . Oxygen .................................................................................. 99 4.3.3 . Carbon dioxide ...................................................................... 99 4.3.4 Purity of the gas supply ......................................................... 100 4.4 Culture pH ..................................................................................... 100 4.4.1 . pH optima for cell culture .................................................... 100 4.4.2 . Buffers and the control of pH .............................................. 102 4.4.3 . Minimizing accumulation of lactate ..................................... 104 4.5 . Osmolarity ................................................................................... 105 4.6 Frezing cells for storage ................................................................. 106 4.6.1 Procedure for freezing and thawing ....................................... 106 4.6.2 Storage medium ..................................................................... 107 4.7 Contamination ......................................................................... 107
5. Optimizing culture conditions and trouble shooting ............................. 111 6. Appendix .............................................................................................. 115 6.1 Cells cultured on Cytodex microcarriers ........................................ 115 6.2 Examples of microcarrier culture protocols .................................... 121 6.2.1 Diploid human fibroblast and the production of interferon ... 121 6.2.2 African Green monkey kidney cells (Vero) and the production of Simian Virus 40 .......................................................................... 122 6.2.3 Primary monkey or dog kidney cells ...................................... 123 6.2.4 Primary chicken embryo fibroblasts ...................................... 123 6.2.5 Baby hamster kidney cells (BHK) ........................................... 125 6.3 Methods for determining the protein and DNA content of cells grown on microcarriers ........................................................................ 125 6.4 Abbreviations ................................................................................ 126 7. References ............................................................................................. 127
Background 1.1 Introduction Cell culture techniques have become vital to the study of animal cell structure, function and differentiation an for the production of many important biological materials such as vaccines, enzymes, hormones, antibodies, interferon’s and nucleic acids. Microcarrier culture introduces new possibilities and for the first time makes possible the practical high yield culture of anchorage-dependent cells. In microcarrier culture cells grow as monolayers on the surface of small spheres (fig. 1) which are usually suspended in culture medium by gentle stirring. By using microcarriers in simple suspension culture systems it is possible to achieve yields of several million cells per milliliter. Cytodex® microcarriers have been specifically developed by Pharmacia Biotech for the high yield culture of a wide range of animal cells (section 6.1) in culture volumes ranging from a few milliliters to several hundred liters. The special requirements of the microcarrier system (section 2.1) are best fulfilled by the dextran-based beads which are subsequently derivitized to form the three types of Cytodex microcarriers. • The surface characteristics of the microcarriers have been optimized for efficient attachment and spreading of cells. • The size and density are optimized to facilitate even suspension and give good growth and high yields for a wide variety of cells. • The matrix is biologically inert and provides a strong but non-rigid substrate for stirred microcarrier cultures. • The microcarriers are transparent and allow easy microscopic examination of the attached cells. Experience with Cytodex for a wide variety of applications has confirmed the importance and benefits of the microcarrier technique. Fig. 1. Scanning electron micrograph of pig kidney cells (IBR-S2) growing on Cytodex 72 h after inculation. (Original photograph by G. Charlier, INVR, Brussels, Belgium, reproduced by kind permission.)
7
New opportunities and applications for animal cell culture Cytodex provides convenient surfaces for the growth of animal cells and can be used in suspension culture systems or to increase the yield of cells from standard monolayer culture vessels and perfusion chambers. Applications include production of large quantities of cells, viruses and cell products, studies on differentiation and cell function, perfusion culture systems, microscopy studies, harvesting mitotic cells, isolation of cells, membrane studies, storage and transportation of cells, assays involving cell transfer and studies on uptake of labelled compounds (see section 1.4. for a description of these applications). Increased production capacity The very large culture surface area to volume ratio offered by the microcarrier system (e.g. 30 cm2 in 1 ml using 5 mg Cytodex 1) provides high cell yields without having to resort to bulky equipment and tedious methodology. For a given quantity of cells or their products microcarrier cultures demand much less space than other types of monolayer cultures. The possibility to culture cells in small compact culture systems is especially important when working with pathogenic organisms. Improved control Suspension culture systems provide excellent opportunities for the control of culture parameters (e.g. pH, gas tensions etc). The microcarrier technique provides a method for growing anchorage-dependent cells in a system having all the advantages of suspension culture. The improved control possibilities with microcarrier culture allow for a homogenous culture system having a wide variety of process designs (1). Monitoring and sampling microcarrier cultures is simpler than with any other technique for producing large numbers of anchorage-dependent cells. Reduced requirements for culture medium When compared with other monolayer culture techniques stirred microcarrier cultures yield 2-4 times as many cells for a given volume of medium. The superior yields with microcarrier culture have been reported for a wide variety of systems including chicken fibroblasts (2,3), pig kidney cells (4), fish cells (5), Chinese hamster ovary cells (6), human fibroblasts (7), primary monkey kidney cells (8) and transformed mouse fibroblasts (9). This reduction in requirement for medium means considerable savings in cell culture costs (6,9), particulary when expensive serum supplements such as foetal calf serum are used. Reduced requirements for labour Because large numbers of cells can be cultured in small volumes (more than 109 cells/litre) fewer culture vessels are required when working with microcarrier cultures. For example, with microcarrier culture one technician can handle a vaccine production equivalent to 900 roller bottles per week (10). One litre of microcarrier culture can yield as many cells as up to 50 roller bottles (490 cm2 bottles, 2). The simplified procedures required with microcarriers reduce the labour necessary for routine production and save on cleaning and preparation of glassware. Separation of cells from the culture medium is simple; when the stirring is stopped the microcarriers with cells attached settle under the influence of gravity and the supernatant can be removed. Unlike true suspension cell culture systems, no centrifugation steps are necessary. 8
Lower risk of contamination In cell culture the risk of contamination is related to the number of handling steps (opening and closing of culture vessels) required to produce a given quantity of cells or their products. Microcarrier culture provides a method for reducing the number of handling steps. There is a much reduced risk of contamination when the production of large quantity of cells is from a single microcarrier culture rather than several hundred roller bottles (6). The principles and methods necessary to achieve the best results with microcarrier culture are described in this book. Although this technique is one of the most advanced in animal cell culture it need not be restricted to experienced cell culturist. Since cell culture is being used by a wide variety of scientists this book is written for both beginners and those experienced in cell culture and only a basic knowledge of cell culture is assumed. This book aims at describing the principles and techniques of cell culture with Cytodex so that the reader is able to deduce optimum procedures with a minimum of effort. The principles aim at a flexible and systematic approach. They are essential to making the most off microcarrier culture and to achieving consistent results with high yields. All methods described her have been developed for use with Cytodex and are not necessary suitable for use with other surfaces for cell culture.
1.2 Adhesion of cells to culture surfaces The adhesion of cells to culture surfaces is fundamental to both traditional monolayer culture techniques and microcarrier culture. Since the proliferation of anchorage-dependent cells can only occur after adhesion to a suitable culture surface (11), it is important to use surfaces and culture procedures which enhance all of the steps involved in adhesion. Adhesion of cells in culture is a multistep process and involves a) adsorption of attachment factors to the culture surface, b) contact between the cells and the surface, c) attachment of the cells to the coated surface and finally d) spreading of the attached cells (11, fig. 2).
Adsorption
Attachment
Contact
Spreading
Cell Cell
Cell
Cell
MHS
MHS-
-CIG Substrate
Substrate
-CIG Substrate
-CIG Substrate
Fig. 2. Simplified outline of steps involved in adhesion of animal cells to culture sufaces. The whole process involves divalent cations and glycoproteins adsorbed to the culture surface. Under usual culture conditions the attachment proteins vitronectin and fibronectin originates from the serum supplement in the medium. MHS is synthesized by the cells. CIG - fibronectin or vitronectin. MHS – multivalent heparan sulphate. (Adapted from refs. 11, 17, 30).
9
The culture surface must be hydrophilic and correctly charged before adhesion of cells can occur (11). All vertebrate cells possess unevenly distributed negative surface charged (12) and can be cultured on surfaces which are either negatively or positively charged (11,13-16). Examples of suitable culture surfaces bearing charges of different polarities are glass and plastic (negatively charged) and polylysine coated surfaces or Cytodex 1 microcarriers (positively charged). Since cells can adhere and grow on all of these surfaces, the basic factor governing adhesion and growth of cells is the density of the charges on the culture surface tather than the polarity of the charges (15,17). Two factors in culture medium are essential for adhesion of cells to culture surfaces divalent cations and protein(s) in the medium or adsorbed to the culture surface (11). In the absence of protein and divalent cations cells attach to a culture surface only by non-specific adsorption (11,18). The protein molecule essential for full adhesion of cells to a culture surface is now known to be a glycoprotein (19-21). The “critical charge densities” noted for microcarriers (16,22-24, fig 4) and other culture surfaces (14) are more likely to be related to interactions between attachment glycoprotein(s) and the charged surface rather than direct electrostatic interaction between the cells and the culture surface (17). Attachment glycoproteins found in the serum in culture medium are fibronectin and vitronectin, secreted from certain cells (19,25-27). Vitronectin and/or fibronectin must be adsorbed on the culture surface before they can promote cell attachment and spreading (28) and they are subsequently incorporated into the extracellular matrix of the spread cells (27). Under normal culture conditions multivalent heparan sulphate proteoglycans mediate adhesion of cells to culture surfaces by co-ordinate binding to glycoproteins on the cell surfaces and the CIG adsorbed on the culture surface (20). In order to achieve good adhesion of the cells to culture surfaces it is necessary that the requirement for an attachment glycoprotein is satisfied. Many established and transformed cell types secrete only very small amounts of fibronectin and require a fibronectin or serum supplement in the culture medium before adhesion occurs (18,25). Certain types of cells such as diploid fibroblasts can secrete significant quantities of fibronectin and do not require an exogenous source of this glycoprotein for attachment (19,29). When initiating a culture it is usual practice to let the culture surface come into contact with medium containing serum before cells are added to the culture. Culture medium supplemented with 10% (v/v) foetal calf serum contains approximately 2-3 µg fibronectin/ml (27) and a large proportion of the fibronectin adsorbs to culture surfaces within a few minutes (18). Serum-free media often require addition of fibronectin (1-50 µg/ml) before many cells can attach to culture surfaces. A minimum of 15 ng of adsorbed fibronectin/cm2 is required for spreading of an established type of cell, BHK (25). Therefore, standard culture procedures usually ensure that the culture surface (plastic, glass or Cytodex microcarrier) is coated with adequate amounts of glycoproteins involved in cell attachment.
10
Fig. 3. Transmission electron micrograph of pig kidney cells growing on a Cytodex microcarrier. (Original photograph by B. Meigneir and J. Tektoff, IFFA-Mérieux. Lyon, France, reproduced by kind permission.)
Culture procedures affect the rate at which cells attach to surfaces. In the case of microcarrier culture, microcarriers and cells are often in a stirred suspension. Under such conditions attachment of cells to Cytodex usually occurs to the same extent as with static culture systems, however with some cell types an initial static culture period is required so that all the steps of adhesion (fig. 2) are fully completed. The way in which microcarrier culture procedures are designed for each type of cell is closely related to the adhesion properties of the cell and the rate at which all steps of adhesion are completed. Ways of determining optimal procedures for individual cell types are discussed in sections 3 and 5. Figure 3 illustrates the close attachment of cells to Cytodex.
1.3 The development of microcarriers for animal cell culture The idea of culturing anchorage-dependent animal cells on small spheres (microcarriers) kept in suspension by stirring was first conceived by van Wezel (31). In the first experiments van Wezel (31) used the beaded ion-exchange gel, DEAE-Sephadex® A-50 as a microcarrier. This type of microcarrier proved useful in initial experiments since it provided a charged culture surface with a large surface area/volume ratio, a beaded form, good optical properties and a suitable density. 11
Glass spheres were not suitable because their high density required stirring speeds for suspension which were not compatible with cell growth (17,31). Several workers have suggested that the ideal microcarrier should have properties similar to those of DEAE-Sephadex A-50 (16,22,32-35). Other ion-exchange beads all proved to be inferior to DEAE-Sephadex A-50 (13,14,16). Using DEAE-Sephadex A-50 at a concentration of 1 mg/ml van Wezel demonstrated that a homogeneous microcarriersystem could be used for the large scale culture of anchorage-dependent cells, including diploid human fibroblasts (31). This early work illustrated the potential of the microcarrier technique for producing virus and latter experiments established that this technique could be scaled-up for a variety of large-scale production processes (1,33). Since the yield of anchorage-dependent cells depends on the surface area available for growth, it was believed that the maximum cell density (yield) in microcarrier cultures would depend on the microcarrier surface area (1). However, when the quantity of DEAE-Sephadex A-50 exceeded 1-2 mg/ml toxicity was encountered and there was not a proportional increase in cell yield (1,32). This toxicity was manifested by the failure of many types of cell to survive the early stages of culture, long lag periods and limited cell yields at the plateau stage of culture.
Cell growth
The explanations for this phenomen have been varied but it is now known that the degree of substitution of DEAE-Sephadex A-50 was not optimal for cell growth (13,14,22,23). The toxicity was probably due to excessive ion-exchange capacity in the micro-environment of the cell rather than too large a total exchange capacity in the culture (22). Although early experiments on microcarrier culture were not
1
2
3 Substitution (meq/g)
Fig. 4. The relationship between the total degree of DEAE substitution of Sephadex G-50 and growth of cells on the resulting microcarriers. The data were pooled from several studies (16, 22, 23 and unpublished work from Pharmacia Fine Chemicals) and concern growth of several strains of human fibroblasts, primary monkey cells and established monkey kidney cell lines in cultures containing 3–5 mg microcarriers/ml. The degree of substitution of DEAE-Sephadex A-50 is 3.5 meq/g.
12
Cells/ml x 10 5
16.0
Fig. 5. The growth of primary monkey kidney cells on Cytodex 1 microcarriers and in glass bottles. (Data from van Wezel, A. L., reprodused by kind permission).
Cytodex
Monolayer 4.0
1.0
0.25
100
200
300 Hours
controlled or optimized for various culture parameters such as plating efficiency, inoculation density, serum and culture medium, the work of Kuchler et al (36), Inooka (37) and Horng and McLimans (13,14) suggested that alterations in the ion-exchange capacity of DEAE-Sephadex A-50 could lead to improvements in cell attachment and growth. The ion-exchange capacity of DEAE-Sephadex A-50 could be altered by changing the culture environment (e.g. ionic strength, pH) but such changes were very limited since cells require physiological conditions for growth. This problem was overcome by the development of microcarriers with a much lower degree of substitution than DEAE-Sephadex A-50 and which also fulfilled the requirements for an optimal microcarrier (16,22,23,33,35). Figure 4 shows the effect that different degrees of substitution of Sephadex with DEAE-chloride has on cell growth. Using Sephadex as the starting material and substituting the matrix with DEAE groups to 1.5 meq/g dry product, it was possible to achieve a microcarrier, Cytodex 1, which was suitable for the growth of a wide variety of cells (17,23,38,39). By using Cytodex 1 the toxic effects associated with DEAE-Sephadex A-50 are avoided and microcarrier concentrations well in excess of 1 mg/ml can be used with concomitant increases in cell yield. Hence this reduced charge microcarrier was the first product to allow the full potential of microcarrier culture to be exploited at culture volumes up to several hundred litres (10,40,41). Cytodex 1is specifically designed for animal cell culture and satisfies the general requirements for an optimal microcarrier (16,17, section 2.1). When correct culture conditions are used the growth rate of most cells on Cytodex 1 microcarrier is comparable to that achieved on plastic or glass culture surfaces (fig. 5).
13
The development of Cytodex 1 microcarriers has taken into account the requirements for attachment of cells (section 1.2) and the procedures necessary for maximum growth of a wide variety of cells in microcarrier culture (section 3). The possibilities for microcarrier culture of animal cells have been increased further by the development of Cytodex 2 and Cytodex 3 microcarriers (section 2.3 and 2.4). Since charged groups are necessary only for cell attachment they need only be confined to the surface of the microcarriers. Cytodex 3 microcarriers represent a new concept in microcarrier culture. Instead of using syntethic charged groups to promote cell attachment, these microcarriers have a surface layer of denatured collagen. Thus the surface upon which cells attach is similar to that found in vivo. Such a surface is important for maximum plating efficiency, growth and function of certain cell types and lends itself to unique possibilities for harvesting cells from microcarrier cultures (section 3.7.2). Nilsson and Mosbach (42) have also examined this approach to microcarrier culture. The relative properties and uses of the various types of Cytodex microcarriers are outlined in section 2.
1.4 Applications of microcarrier culture Microcarrier culture techniques offer many new possibilities for animal cell culture and the applications fall into three categories, a) high-yield production of cells, viruses or cell products, b) studies on cells in vitro and c) routine cell culture techniques. With Cytodex these applications can be realized for a very wide variety of different cells. The applications of microcarrier culture are advancing rapidly and additional information can be obtained from Pharmacia Fine Chemicals.
Fig. 6. Human-mouse hybrid cells growing on Cytodex microcarriers. The parental cells for the hybrid were human lymphocytes and HGRT mouse cells. (Original photograph by B. Winchester, Queen Elizabeth Collage, London, UK, reproduced by kind permission).
14
Fig. 7. Hybrid of mouse spleen cells and human bladder tumour cells growing on Cytodex microcarriers. (Original photograph by A. O’Toole, London Hospital Res. Lab., London, UK, reproduced by kind permission).
1.4.1 Cell types cultured on Cytodex microcarriers The cell types successfully cultured on microcarriers are listed in Section 6.1. and examples of virtually all classes of cultured animal cells are represented. Cytodex microcarriers are cell culture surfaces of general applicability and provided a cell is capable of attachment in vitro it will be able to attach to the microcarriers. Cytodex 3 also provides an improved culture surface for many types of cells which attach or function poorly on glass or plastic culture surfaces (section 2.4). Mammalian, avian, fish and insect cells have been cultured on Cytodex. These cells are of wide histotypic origin and include primary cells, diploid cell strains and established or transformed cell lines. Hybrid cell lines and cells of tumour origin can be cultured on Cytodex microcarriers (figs. 6,7,8). Selection of the most suitable microcarrier for these cells depends on the cell type and the application (section 2.5). Examples of cells growing on Cytodex microcarriers are shown in Plates 1-9 and other figures throughout this book. Fig. 8. Human osteosarcoma cells cultured on Cytodex microcarriers. (Original photograph by B. Westermark and J. Pontén. Wallenberg Lab., Uppsala, Sweden, reproduced by kind permission).
15
The only types of cells which have proveded to be difficult to grow on microcarriers in stirred cultures have been some of lymphoid origin. Such cells attach only weakly to cell culture surfaces and can be dislodged from the microcarriers if the culture is stirred too vigorously. Lymphocytes and lymphoblastoid cells have been successfully cultured on Cytodex microcarriers (43, plate 4, G Alm, pers. comm., 184) and good attachment can be achieved when correct procedures are used (section 3.4,3.5). Cytodex is not mitogenic in cultures of lymphocytes (43). Anchorage-independent cells can be grown on Cytodex. Although these cells can be grown in free suspension culture there are often distinct advantages to using microcarriers while still retaining the benefits of suspension culture techniques.
Cells/ml
• Higher culture densities can often be achieved with microcarriers and productivity of the culture can be increased. • Separation of the cells from the liquid phase of the culture is more simple when using microcarriers - long sedimentation times, complicated filters or centrifuges are not required when harvesting the cells. • Cultures containing microcarriers are more homogeneous. By allowing the microcarriers to settle, dead cells and debris can be removed in the supernatant fluid. The culture can be therefore be enriched for living cells. 7
10
a
b
6
10
10
5
4
10
2
4
6
8
2
4
6
8 Days
Fig. 9. The growth of various types of cells on Cytodex microcarriers in stirred cultures. a. Human fibroblasts (MRC-5, —■—), chicken fibroplasts (—▲—), human nasophararangeal carcinoma cells (KB, —●—; data reproduced by kind permission of S.Toyama, Inst. Virus Res., Kyoto University, Kyoto, Japan). a. Mouse fibroblasts (J-129, —■—), normal rat kidney cells (NRK, —▲—), rhesus monkey kidnney cells (LLC-MK2, —●—; data reproduced by kind permission of S.Toyama, Inst. Virus Res., Kyoto University, Kyoto, Japan).
16
• Cells growing on culture surfaces often use medium more efficiently than the same cells growing in free suspension (44). Microcarriers provide a method for reducing the medium requirements of suspension cultures. • The yield of many strains of virus is greater when the cell substrate is grown attached to a culture surface. Some viruses (e.g. Herpes) grow poorly in free suspension culture systems. In general cells have the same growth kinetics on Cytodex microcarriers as they do on standard glass or plastic culture surfaces (figs. 5,25). Provide culture conditions are optimal (sections 3.4,3.5) most cells will retain their characteristic morphology, population doubling time and saturation density when growing on Cytodex microcarriers (figs. 5,9). Cells which grow with a pronounced fibroblast like morphology may have reduced saturation density on microcarriers since the spherical growth surface cannot be completely covered by the parallel array of cells.
1.4.2 Production of large numbers of cells A major area of application for microcarrier culture is the production o large numbers of cells. The advantages of the microcarrier system (section 1.1) can be used to obtain high yields of cells from small culture volumes. Cultures can often be initiated with 105 cells/ml or less and at the plateau stage the yield is usually more than 106 cells/ml (fig.9). This high yield of cells per unit culture volume and the large increase in cell number during the culture cycle (10-fold or more) make microcarrier culture an attractive technique for production of cells from a wide range of culture volumes. Applications for small culture volumes include situations when only few cells are available to initiate a culture (e.g. clinical diagnosis, cloned material). Microcarriers can be used to increase the culture surface area in small volumes and at the same time keep the density of cells/ml as high as possible. Maintaining high densities of cells leads to conditioning of the culture medium and stimulation of cell growth. With traditional monolayer techniques for small cultures it is not possible to achieve
Fig.10. Chinese hamster fibroblasts growing on Cytodex microcarriers contained as a static culture in a Petri dish. (Original photograph by T. Utakoji, Cancer Inst., Japanese Fondation for Cancer Res., Tokyo, Japan, reproduced by kind permission).
17
Stage Production volume:
Primary
Secondary
Tertiary
10 L
80 L
650 L
Quartenary
Perfusion Control tests:
Sterility
Sterility Virus Mycoplasma
Sterility Oncogenicity
Mycoplasma Virus
Fig.11. Scaling up microcarriers cultures for the production of large numbers of cells. Example illustrates the subcultivation system for production of polio vaccine from monkey (Cynomolgus) kidney cells growing on Cytodex microcarriers. (Adapted from van Wezel, A.L., van der Velden-de Groot, C.A.M., van Herwaarden, J.A.M., Develop. Biol. Standard. 46 (1981) 151).
a high culture surface area/volume ratio (approx. 4 cm2/ml in Petri dishes). Microcarrier cultures provide a surface area/volume ratio of approx. 20 cm2/ml. The increase in culture surface area means that a greater yield of cells is achieved before subculturing is necessary. In the area of clinical diagnosis or production of cloned material this technique leads to a reduction in the time required to grow cells for biochemical analyses (45). Figure 10 illustrates the high yield of cells which can be obtained from microcarrier cultures contained in traditional monolayer culture vessels. Microcarrier culture also provides a method for rapid scaling-up with a minimum number subculture steps (fig. 11). Scaling-up can be through the entire range of culture volumes and can also be achieved in the absence of subculturing steps by using a continuos propagation technique (6). This technique provides sustained periods of exponential growth. Large culture volumes of several litres or more are mainly used for production of viruses or cell products (section 1.4.3). The yield of cells from large-scale cultures using Cydotex is usually 109 cells/litre or more.
1.4.3 Production of viruses and cell products Cells cultured on microcarriers are often used as substrates for the production of viruses or cell products and the microcarrier method is compatible with standard production procedures. Cytodex can be used for the production of all substances which can be produced in animal cell culture. A wide variety of viruses can be produced using Cytodex, including viruses sensitive to growth in suspension cultures, e.g. Herpes (Table 1). The microcarrier system allows cultivation of large quantities of virus in compact culture units and provides an improved system for the production of many vaccines (1,40,46,48). Vaccines produced in the microcarrier system include polio, rubella, rabies, influenza, and foot-and-mouth disease (FMD) vaccines (1,10,40,41,46,48-50). Figures 12 and 13 illustrate the growth of Vero cells and the production of Herpes simplex virus on microcarriers. 18
37˚
9.0
35˚
10
8.8 9
8.6 8.4
8
HSV-2
log10 TCID50 (- -)
Fig.13. The growth of Vero cells and Herpes simplex virus (HSV-2 in stirred cultures containing Cytodex microcarriers. Fig. 12. shows photomicrographs of this culture. (Griffiths, B., Thornton, B., McEntee, I., Eur. J. Cell Biol. 2 (1980) 606, reproduced by kind permission).
log10 total cells (- -)
Fig.12. Culture of Vero cells on Cytodex microcarriers used for the production of Herpes simplex virus (HSV-2). The culture was infected with HSV-2 after approx. 50 h. CPE-cytopathic effect. (Original photograph by B. Griffiths, CAMR, Porton Down, UK, reproduced by kind permission).
8.2 0
20
40
60
80
100
120 Hours
Table 1. Some viruses which have been grown in cultures using Cytodex microcarriers. Polio Rabies Rubella Influenza Sindbis Sendai Marek’s Measles
Rous sarcoma Herpes Simian virus 40 Polyoma Pseudorabies Vaccinia Adenovirus Parvovirus
Foot-and-mouth Vesicular stomatitis Group B arboviruses Equine rhinopneumonitis Bovine rhinotrachteitis Endogeneous C-type Papova virus Respiratory syncytial virus
The advantages of using microcarrier culture for vaccine production include increased productivity, reduced costs and reduced contamination when compared with other cell culture methods (section 1.1). Sinskey et al (51) observed that the volumetric productivity of Sindbis virus in microcarrier cultures is in excess of 50-fold greater than that of roller bottles. Van Wezel et al (49,59) have developed a “Unit Process” for the production of polio and rabies vaccines using Cytodex microcarriers and the productivity and efficiency of such a system is illustrated in Table 2. An example of a cell culture scheme for the production of inactivated polio vaccine is shown in Figure 11. Serial cultivation on Cytodex reduces the requirement for a source of primary cells and provides a production culture of 650 litres. 19
Table 2. Processing of polio virus type I from microcarrier cultures using Cytodex. Step
Vol L
D-antigen DU/ml
Recovery %
Albumin µg/ml
IgG µg/ml
Virus suspension Clarification Concentration Gel filtration Ion-exchange Sterile filtration Monovalent vaccine
240 248 1 4,5 4,5 7,5 7,4
76 64,2 17,530 3,465 3,465 1,964 1,753
100 87 96 85 85 81 71
n.d. 1,000 >30,000 0,23 0,03 <0,03 <0,03
n.d. 300 >30,000 2,0 <0,23 <0,23 <0,23
PN content: after gel filtration, 40mg/ml; after ion exchange, 8 µg/ml. Gel filtration was with Sepharose® 6B in a Pharmacia K 215/100 column and ion-exchange chromatography was performed with DEAE-Sephadex® A-50. (van Wezel, A.L., van Herwaarden, J.A.M., van de Heuvel-de Rijk, E.W., Devel. Biol. Stand. 42 (1979) 65, by kind permission of the authors and the publisher.). Table 3. The average yields of polio virus from large scale cultures using Cytodex. Vero cellsa: 1.2x106 cells/ml with 1 g Cytodex 1/litre. Polio virus:
Type I
Type II
Type III
D-Antigen (DU/ml) Infectivity (log10 TCID50)
85 8,1
20 8,2
56 7,5
Monkey kidney cellsb: 106 cells/ml with 1-2 g Cytodex 1/litre. Polio virus
Type I
Type II
Type III
D-Antigen (DU/ml) Infectivity (log10 TCID50)
80 >8
30 >8
40 >8
a b
Montagnon, B., Fanget, B.. Nicolas, A.J., Devel. Biol. Standard. 47 (1981) 55. van Wezel, A.L., van Steenis, G., Hannik, Ch.A. et al, Devel. Biol. Standard. 44 (1978) 159.
Von Seefried and Chun (46) reported high yields of polio virus having high infectivity (8.84 log 10 TCID50/ml or more) when using human fibroblasts (MRC-5) growing on Cytodex. Vero cells growing on Cytodex have been used for the production of a stable polio vaccine from culture volumes of 140 litres (41). Polio virus production can also be taken as an example illustrating yields of virus from microcarrier cultures. The yield of polio virus from cultures using Cytodex is summarized in Table 3. Giard et al (2) reported that the yield of polio type III virus from microcarrier cultures (6.5 pfu/cell) was greater than the yield from roller bottles (4.0 pfu/cell). Similarly, Mered et al (52) observed that the yield of polio virus/cell was greater from microcarrier cultures than from culture flasks. Cytodex has been used for the production of rabies vaccine by multiple harvests from primary dog kidney cell cultures (48,50). The infective titre of the harvests from these cultures was 6.0±1.0 log10 LD50/ml in mice.
20
FMD vaccines have been produced from pig kidney cells growing on Cytodex and the vaccines were of good quality with long storage life (10). The FMD vaccines gave good protection of animals with no abnormal local reactions and it was not necessary to concentrate the antigen (10). Spier and Whiteside (53) have compared the production of FMD virus from BHK cells grown on microcarriers and in suspension. Microcarrier culture of FMD virus Type 0 gives a virus suspension with higher infectivity and complement-fixing activity than suspension culture. The complement-fixing activity of FMD virus Type A from microcarrier cultures was at least 5 times that obtained from suspension cultures (53). A large-scale controlled fermenter and Cytodex have been used for the prolonged culture of cells persistently infected with papova virus (54). Manousos et al (55) studied the production of oncornavirus in long-term microcarrier cultures and noted that an advantage of this technique was that addition of new microcarriers to confluent cultures caused a new wave of cell growth an virus production. The production of other types of viruses in microcarrier culture is described in references 2,51,56. Microcarrier culture provides a potential method for the mass-production of fish virus vaccines. Nicholson observed that the production of infectious pancreatic necrosis virus from microcarrier cultures (44,5 TCID50/cell) was nearly 3-fold greater than production of virus from culture flasks (16.0 TCID50/cell). Interferon has been produced in high yield from microcarrier cultures. The first report (57) described yields of 4x103 IU HuIFNß/106 human fibroblasts. A more detailed study examined various parameters and yield were increased to levels comparable to those obtained from traditional monolayer systems (58). Clark and Hirtenstein (59) optimized culture procedures for cell growth and modified the induction procedure to result in yields of 3x104 IU HuIFNß/106 human fibroblasts. This yield correspondend to 2x104 IU HuIFNß/mg of Cytodex and the technique could be used to produce 3x108 IU HuIFNß/5 litre culture. A procedure for producing HuIFNß is included in section 6.2.1. By using Cytodex microcarriers in roller bottles Kronenberg obtained improved yields (approx. 8-fold) of mouse fibroblast interferon (L. Kronenberg, pers. comm., 185) The cultures used for these experiments are illustrated in Figure 22. Cytodex has also been used for the production of immune interferon, HuIFNg (G. Alm, pers. comm., 184) Microcarrier culture has enabled the growth of large numbers of human colon carcinoma cells for the production of carcinoembryonic antigen (60) and the production of plasminogen activator from transformed mouse fibroblasts (K. Danø, pers. comm., 186). Further information on the production and purification of specific viruses and cell products from microcarrier cultures can be obtained from Pharmacia Biotech.
21
1.4.4 Studies on cell function, metabolism and differentiation Microcarriers can be used as convenient culture surfaces in many cell biology studies. The ability to culture cells at high densities in a homogeneous culture system provides unique opportunities for studies of cell function, metabolism and differentiation. In addition microcarriers make it easier to manipulate and observe the cells. When compared with traditional monolayer techniques which only provide for two-dimensional cultures, the microcarrier system allows for very high culture densities and when confluent microcarriers are packed together, a three-dimensional culture can be achieved. Cytodex microcarriers are compatible with cell function and differentiation in vitro and a wide variety of different studies have been reported. The choice of the most suitable microcarrier is described in section 2.5 and for most studies with differentiating systems Cytodex 3 is the microcarrier of choice (table 5). Several examples serve to illustrate the use of microcarriers in cell biology studies. Pawlowski et al (61) used Cytodex to study the differentiation of chick embryo skeletal muscle cells. Normal myogenesis occured on the microcarriers which were also used for microscopy studies (plate 1). After 4 days of culture 62% of the microcarriers had myotubes with extensive myofibril formation (61). With an even more sensitive cell system Moser and Stoffels (62) studied the differentiation of newborn rat heart muscle cells. The microcarrier method provided homogeneous and easily manipulated cultures. The heart cells spread and proliferated on the microcarriers and expressed pacemaker membrane properties. Between 20-30% of confluent monolayers on the microcarriers exhibited spontaneous beating activity (62). The release of insulin from foetal rat pancreas islet cells growing on Cytodex has been studied by Bone et al (63,64). These studies demonstrated that Cytodex is suitable for maintaining highly specialized endocrine cells in culture. The
Dissociation
Hypothalamic releasing factors Chromatographic column superperfusion system
Monitor hormone secretion
22
Micro carrier culture (3-5 days)
95% of cells attach
Fig. 14. The superperfusion culture of primary rat pituitary cells growing on Cydodex 1 microcarriers. The scheme is based on studies by Smith and Vale (65, 66).
Fig. 15. Primary rat interior pituitary cells attached to Cydodex 1 microcarriers and incubated for 5 days after dissociation. (Smith, M.A. and Vale, W.W. Endocrinol. 107 (1980) 1425, by kind permission of the authors and publisher.)
microcarriers provided a method for the uniform suspension culture of functioning pancreas cells and allowed for easy manipulation of the cells (64). The pancreas cells sustained synthesis and release of insulin during a 7 day growth period on the microcarriers (plate 3) and the release could be modulated by glucose and stimulated with theophylline (64). Microcarriers have been used in novel culture systems to study the function of differentiated cells. Smith and Vale (65,66) have developed a superperfusion column technique for the study of rat anterior pituitary cells and the modulation of pituitary secretions by gonadotrophins and cocarcinogens (fig. 14). The system provided responsive and well-defined high density cultures which maintained the ability to secrete hormones fore long periods of time. The dissociated pituitary cells attached to the Cytodex microcarriers (fig. 15) and remained responsive to hypothalamic releasing factors (65,66, fig. 16). Approximately 95% of the cells attached and the culture system could be used to study transient phenomena and desensitization (65). A variety of other differentiated cells have been studied using Cytodex microcarriers. Ryan et al (67) developed a microcarrier culture system for studying the role of bovine pulmonary endothelial cells and C. Busch (pers. comm., 187) has used Cytodex 3 in studies of endothelial cells from brain capillary and pulmonary artery (plate 2). Porcine thyroid cells cultured on Cytodex exhibited an epithelial morphology and were capable of releasing thyroglobulin (68).
23
FSH secretion (ng/min)
LH secretion (ng/min)
a 400 300 200
b 100 75 50 25
100
0
3
6
9
12
15
18
21
0
24
3
6
9
12
15
18
21
24
Hours Fig. 16. Response of rat anterior pituitary cells growing on Cydodex 1 microcarriers to pulses of gonadotropin-releasing hormone (GnRH). The cells were cultured in the system illustrated in fig. 14 and were exposed to 15 min pulses of 30 nM GnRH every 2 h. Flow rate was 0.2 ml/min and fractions were collected every 20 min. a. Secretion of lutenizing hormone. b. Secretion of follicle stimulating hormone. (Smith, M.A. and Vale, W.W., Endocrinol. 108 (1981) 752, by kind permission of the authors and publisher.)
Vosbeck and Roth (69) used microcarrier culture to study the effects of different treatments on intercellular adhesion. Confluent monolayers of cells were cultured on microcarriers and intercellular adhesion was examined by studying the binding of 32 P-labelled cells to the monolayers (69). Lymphocytes have been grown on microcarriers for studies of stimulation (43). Cytodex 1 alone was not mitogenic for lymphocytes but potentiated stimulation by Con A (fig. 17). The microcarriers have been used to study the relationship between anchorage, cell density and stimulation of lymphocytes (43). Microcarriers are also used in studies of animal cell plasma membranes. Lai et al (70) used Cytodex to study the influence of adhesion on the fluidity on Chinese hamster ovary cell plasma membranes. By using electron spin resonance technique it was possible to compare cells growing in free suspension culture and attached to microcarriers in suspension culture. Cytodex microcarriers are compatible with spinlabelling and provided a technique whereby cells could be easily transferred and assayed without removal from the culture surface (70). Microcarrier culture can also be used for the isolation of plasma membranes with less than 1% contamination from internal membrane markers (71). The procedure is suitable for cells capable of attachment. Cells are first allowed to attach and spread on the microcarriers. Hypotonic lysis is followed by brief sonication to disrupt the cells. The cell debris is then removed and membranes attached to the microcarriers can be used directly for assays of membrane-associated enzymes (71).
24
-3
cpm x 10
Fig. 17. The stimulation of human lymphocytes by Con A in the presence (❍) or absence (●) of Cydodex microcarriers. The microcarriers alone were not mitogenic. (Sundqvist, K. and Wagner, L., Immunology 43 (1981) 573, by kind permission of the authors and the publisher.)
80 70 60 50 40 30 20 10
0
5
10
15
20
25
30
50
µg Con A/ml
1.4.5 Proteolytic enzyme-free subcultivation and cell transfer In many studies it is important to be able to harvest or transfer cultured cells without using proteollytic enzymes or chelating agents. Such agents often alter cell viability and the integrity of the plasma membranes. By using microcarriers it is possible to subculture cells or scale-up cultures without using proteolytic enzymes or chelating agents. Microcarriers also provide convenient surfaces for cell growth and cells can be transferred from culture vessel to culture vessel or used directly for experiments without having to be removed from the microcarriers. Horst et al (72) and Ryan et al (67) observed that cells can migrate between microcarriers and the surfaces of cell culture flasks or Petri dishes. Cytodex microcarriers were allowed to settle onto monolayers of mouse fibroblasts and cells migrated onto the microcarriers which could then be transformed to another culture vessel (72). This method of transfering cells has also been used for bovine pulmonary artery endothelial cells; such cells are normally sensitive to treatment with proteolytic enzymes (67). A variant of this technique is to allow cells to migrate from confluent microcarriers onto new microcarriers. Crepsi andt Thilly (6) could maintain prolonged periods of exponential growth of CHO cells by diluting confluent microcarrier cultures and adding new microcarriers. The transfer of cells between microcarriers was enhanced by using a medium with low Ca2+ concentration (6). New microcarriers can also be added to confluent microcarrier cultures during periods of virus production and Manousos et al (55) used this technique to cause a new wave of cell proliferation and production of oncornavirus. It is also possible to scale up microcarrier cultures of human fibroblasts by allowing confluent microcarriers to settle with new microcarriers and after a few hours, migration of cells results in inoculation of the new microcarriers (P. Talbot, pers. comm., 188) 25
The suitability of this technique is limited by the mobility of cells. Some types of cells, e.g. hepatocytes, exhibit only limited mobility and do not migrate between microcarriers and other culture surfaces. The chance of cell transfer between microcarriers is increased by allowing the culture to remain static for several hours. Occasional stirring should eliminate any tendency for the microcarriers to aggregate. Microcarriers also have other applications in the transfer of cells. For example, Cytodex can be incubated with peritoneal fluid and after 10 min macrophages adhere to the microcarriers and can be separated from the other peritoneal cells by simple differential sedimentation (H. Slater, pers. comm., 189). The macrophages attached to the microcarriers can then be transfered to other culture vessels for study. Microcarriers can also be used for cloning cells. Cultures can be inoculated with approximately one cell/microcarrier and after allowing time for cell attachment those microcarriers bearing only one cell can be transfered by Pasteur pipette into cloning wells. In this way the microcarriers provide an easily manipulated culture surface. Similarly confluent microcarriers can be embedded in semi-solid medium to form feeder layers.
1.4.6 Microscopy Cytodex microcarriers can be used as cell culture substrates for a variety of microscopy studies using standard techniques such as scanning electron microscopy (fig. 1, plates 1,4,8), transmission electron microscopy (fig. 3) and different types of light microscopy illumination and cytochemistry (plates 2,3,5-7,9). The advantages of using microcarriers for microscope is that such culture substrates are easy to manipulate and cells do not need to be harvested when embedding techniques are used. The dextran-based matrix of Cytodex microcarriers (section 2.1) can be penetrated by the usual embedding media before sectioning. By using confluent microcarriers transverse sections through cells adhering to the culture substrate can be readily obtained (fig. 3) Routine samples from microcarrier cultures can be processed for detailed microscopical examination and cultures containing many coverslips can be avoided. Similarly samples of experimental cultures can be processed for microscopy without requiring large numbers of cells . Details of microscopy with Cytodex microcarriers can be found in section 3.6.
1.4.7 Harvesting mitotic cells Microcarrier culture provides an efficient method for harvesting mitotic cells (73,74). The technique is based on the observation that mitotic cells are attached only weakly to cell culture surfaces and can be detached by shaking (75). The use of monolayer culture vessels for this technique is limited by the small surface area for cell growth and microcarriers provide the large surface area necessary for recovering high yields of mitotic cells.
26
Exponential cultures of cells growing on microcarriers in suspension can be treated with mitotic inhibitors (e.g. Colcemid) and by selecting the appropriate stirring speed, mitotic cells can be dislodged and collected in the medium. Mitchell and Wray (73) reported that CHO cells harvested from Cytodex microcarriers by this method had a mitotic index of up to 88%, a considerable improvement over the mitotic index of 41% obtained when harvesting mitotic cells by shaking from a culture flask. Ng et al (74) treated exponential cultures of CHO cells on microcarriers with Colcemid (100 mg/ml) for 2.5 h and then harvested mitotic cells by increasing stirring speed. The increased stirring speed dislodged the mitotic cells and harvests of more than 4x104 mitotic cells/ml of microcarrier culture could be obtained. These cells had a mitotic index of 85-95% (74).
1.4.8 Transportation and storage of cells The large surface area/volume ratio of microcarriers is advantageous when transporting and storing culturing cells. Large numbers of cells (up to approx. 107 cells/ ml) can be transported or stored whilst still attached to the culture substrate. This technique avoids transportation of large numbers of monolayer culture vessels (e.g. flasks) and also eliminates the need to store or transport suspensions of anchorage-dependent cells. The advantage of transporting or storing cells attached to a culture surface rather than a suspension is that loss of cells associated with harvesting and replating is avoided. After transportation or thawing from storage the cells are already attached to the culture surface and can continue to function and proliferate. Procedures for storing most anchorage-dependent cells whilst still attached to Cytodex microcarriers are described in section 4.6. and even cultured insect cells can be frozen and stored in liquid nitrogen when attached to microcarriers (E. Duda, pers. comm., 190)
27
28
2. Cytodex microcarriers 2.1 Requirements for an optimum microcarrier In order for a microcarrier to be suitable for animal cell culture at all scales it must fulfill certain basic criteria (16). • Surface properties must be such that cells can adhere with a degree of spreading which permits proliferation. For homogeneous growth of cells the surface of the microcarrier must have an even, continuos contour. The surfaces of all microcarriers in the culture should have consistent properties. • Density of the microcarriers should be slightly greater than that of the surrounding medium, thus facilitating easy separation of cells and medium. The density should also be sufficiently low to allow complete suspension of the microcarriers with only gentle stirring. Under standard culture conditions the optimum density for microcarriers is 1.030-1.045 g/ml. • Size distribution should be narrow so that even suspension of all microcarriers is achieved and that confluence is reached at approximately the same stage on each microcarrier. Best growth of cells occurs when microcarriers have a size distribution which lies within limits of diameter in culture of 100-230 µm. • Optical properties should be such that routine observation of cells on microcarriers can be achieved using standard microscopy techniques. The microcarriers should also permit use of routine cytology procedures. • Non-toxic microcarriers are required not only for survival and good growth of the cells but also when cell culture products are used for veterinary or clinical purposes. • Non-rigid microcarrier matrices are required for good cell growth when the culture is stirred. Collisions between microcarriers occur in stirred cultures and a compressible matrix reduces the possibility of damage to the microcarriers and the cells. All Cytodex microcarriers are designed to meet the above requirements and are based on a spherical matrix of cross-linked dextran. This matrix was chosen because it provides a microcarrier with suitable physical properties and also because dextran in non-toxic, having widespread use in clinical applications and for the preparation of pharmaceutically important materials (see ref. 76) Cytodex microcarriers are non-toxic and provide surfaces which can be used for the cultivation of a wide variety of cell types (section 6.1). These microcarriers have a size distribution and density compatible with the culture procedures required for optimal cell growth. Furthermore Cytodex microcarriers are sufficiently strong to withstand normal culture procedures and conditions but are non-rigid and have excellent optical properties.
29
Cytodex 1 charges throughout matrix
CH2CH3 cross-linked dextran - O - CH2CH2 - N
.HCL CH2CH3
Cytodex 3 collagen layer coupled to surface
OH cross-linked dextran - O - CH2 - CH - CH2 - NH - ( LYS.COLLAGEN)
Fig. 18. Schematic representation of the three alternative types of Cydodex microcarriers.
2.2 Cytodex 1 Cytodex 1 microcarriers are based on a cross-linked dextran matrix which is substituted with positively charged N,N-diethylaminoethyl (DEAE) groups to a degree which is optimal for cell growth (fig. 4). The charged groups are found throughout the entire matrix of the microcarrier (fig. 18). Published procedures for substituting cross-linked dextran with DEAE groups to form microcarriers for cell culture (3,22,35,55) can lead to the formation of a high proportion (up to 35%) of tandem charged groups (see 24). The chemical reaction conditions used to produce Cytodex 1 microcarriers are controlled so that formation of such tandem groups is minimized (only approx. 15% of groups); thus stability and homogeneity of the charged groups is enhanced and possible leakage of charged groups from the microcarriers is minimized. Windig et al (77) used pyrolysis mass spectroscopy to examine the possible presence of leaked DEAE-dextran in concentrated polio vaccines prepared from microcarrier cultures using Cytodex 1. If leakage of such groups occurred it was found to be below the limits of detection, i.e. less than 20 ppm (77). The physical characteristics of Cytodex 1 are summarized in Table 4.
2.3 Cytodex 3 Cytodex 3* microcarriers are based on an entirely different principle for microcarrier culture. While most surfaces used in cell culture posses a specific density of small charged molecules to promote attachment and growth of cells (e.g. glass, plastic, Cytodex 1,), certain proteins can also provide a surface for the growth
*patents pending
30
of cells in culture. The connective tissue protein, collagen, has proved to be a valuable cell culture substrate. Cytodex 3 microcarriers consist of a surface layer of denatured collagen covalently bound to a matrix of cross-linked dextran (fig. 18). The amount of denatured collagen bound to the microcarrier matrix is approx. 60 µg/cm2 and results in maximum cell yields (24). The denatured collagen (MW 60,000-200,000) is derived from pig skin type I collagen which has been extracted and denatured by acid treatment, concentrated and purified by an ion exchange step and steam sterilized before being coupled to the microcarrier matrix. These microcarriers combine the advantages of collagen coated culture surfaces (see below) with the advantages and possibilities of microcarrier culture (section 1.1, 1.4). Cytodex 3 microcarriers can also be used as a general purpose collagen-coated cell culture substrate. Most normal epithelial cells will attach more efficiently to collagen than to other cell culture surfaces. Consequently, collagen-coated culture surfaces are used frequently for establishing primary cultures and for growing cells which are normally difficult to grow in culture (78, 79). Collagen-coated surfaces are valuable because they permit differentiation of cells in vitro even at very sparse or colonel culture densities (79,80). Such surfaces are also advantageous when culturing for extended periods since they delay the detachment of the cell sheet that eventually occurs in long-term mass culture on uncoated surfaces (81). A variety of different types of cells are routinely cultured on collagen-coated surfaces and include hepatocytes, fibroblasts, chondrocytes, epidermal cells, myoblasts and mammary epithelial cells (82). Differentiation of myoblasts at sparse densities in vitro depends on the presence of collagen bound to the culture surface (80, 81). Myoblasts attach and spread more satisfactorily on collagen than on standard cell culture surfaces (81) and growth is also stimulated (83). Hepatocytes can be cultured more successfully on collagen surfaces. The collagen permits freshly isolated hepatocytes to attach with maximum efficiency and spreading is more rapid than on any other cell culture surface (84). Since exogenous fibronectin is not required for attachment of hepatocytes to collagen (85) this culture surface is the most suitable surface for culture of hepatocytes in protein-free media (84). A collagen culture substrate also allows for more extended studies of hepatocytes in vitro with improved retention of differentiated function (86,87). Folkman et al (88) have described the culture of capillary endothelial cells from a variety of sources on collagen and excellent growth of endothelial cells on Cytodex 3 has been demonstrated (plate 2). Geppart et al (89) reported that there was better maintenance of differentiated alveolar type II cell function when using collagen as the culture substrate. Surface-bound collagen can also be used for the culture of fibroblasts (90, 91) and Ceriejido et al (92) described the use of cross-linked collagen for the culture of the epithelial kidney cell line, MDCK. Secondary bovine embryo kidney cells have a higher plating efficiency when grown on Cytodex 3 than when grown on Cytodex 1. This improvement in plating efficiency leads to higher cell yields (fig. 19).
31
Cells/ml
10
10
Fig. 19. The growth of secondary bovine embryo kidney cells on Cytodex 1 (—●—) and Cytodex 3 (—❍—) microcarriers. The cells were a mixed population with predominantly epithelial morphology. (Gebb, Ch., Clark, J.M., Hirtenstein, M.D. et al, Develop. Biol. Standard. (1981), in press, by kind permission of the authors and the publisher).
6
5
100
200 Hours
Cytodex 3 microcarriers are coated with denatured Type I collagen because this is the most generally useful form of collagen for cell culture . Although certain types of cells may show a specificity for attachment to a particular form o native collagen, this specificity is much less apparent when denatured collagen is used. Adsorption of the attachment glycoprotein, fibronectin, to the culture surface is known to be important in the adhesion of many cells (section 1.2) and fibronectin is believed to be also involved in the attachment of many cells to both native and denatured forms of collagen (93). Fibronectin binds equally well to all types of collagen (94) but shows a preference for denatured forms (82,95,96) and binds more rapidly to Sephadex® beads coated with denatured collagen than to beads coated with native collagen (97).
Table 4. Some physical characteristics of Cytodex microcarriers.
Density* (g/ml) Size* d50 (µm) d5-95 (µm) Approx. area* (cm2/g dry weight) Approx. no microcarriers /g dry weight Swelling* (ml/g dry weight)
Cytodex 1
Cytodex 3
1.03 180 131-220 4,400 6.8x106 18
1.04 175 133-215 2,700 4.0x106 14
Size is based on diameter at 50 % of the volume of a sample of microcarriers (d50), or the range between the diameter at 5 % and 95 % of the volume of a sample of microcarriers (d5-95). Thus size is calculated from cumulative volume distributions. *In 0,9% NaCl.
32
Table 5. Selection of Cytodex microcarrier based on cell type and application area. The choice is also modified under certain conditions (section 2.5).
a
b c d e
f
1d
1 3
1,3 3
1,3 3
1
1,3
1,3
Cytodex 3 1,3 1 3 3 Cytodex 3 1,3 1
eqithelial
1,3
fibroplast
1,3
Established
Normal diploid
1,3
cell lines
tumour
1
cell strains
mixed
Cultures for other cell biology studies Differentiated culture systemsf Microscopy Uptake studies Isolating cells with membranes intact Harvesting mitotic cells
epithelial
Cultures for production General purpose cell production Production of virus Production of cell products
fibroplast
Application areab
Primaryc
Cell typea
1
1,3e
1 3
1,3 3
1
1,3
Definition of cell type is based on morphology in culture. Cells of epithelial, endothelial and mesothelial origin usually have an epithelial morphology in culture. More details on applications can be found in section 1.4. Also includes secondary, tertiary etc. cultures which have not become established. Cytodex 3 can be used for high yields of certain cell strains, e.g. FS-4. Cytodex 3 is more suitable than Cytodex 1 for cell lines with pronounced epithelial morphology e.g. BSC-1. For example hepatocytes, hepatomas, muscle cells, endocrine cells etc.
In the past, a major difficulty with using collagen coated surfaces has been the rapid loss of collagen from the surface. Kleinman et al (93) observed that up to 40% of the collagen is lost from coated Petri dishes within 90 minutes of adding culture medium. Floating collagen gels (79) have greater retention of collagen but are difficult to prepare and often shrink during the culture period (87). Because the denatured collagen is cross-linked to the microcarrier matrix, the problems of shrinkage, cracking and leakage of collagen which are found with standard techniques for coating cell culture surfaces are avoided with Cytodex 3 microcarriers. A further advantage of using Cytodex 3 microcarriers as a general purpose collagen-coated culture surface is that beause the microcarriers have a porous matrix, nutrients also have access to the basal cell surfaces. Such access is not possible with collagen-coated plastic or glass surfaces. The denatured collagen layer on Cytodex 3 microcarriers is susceptible to digestion by a variety of proteases, including trypsin and collagenase (section 3.7.2) and provides unique opportunities for removing cells from the microcarriers. The physical characteristiques of Cytodex 3 are summarized in table 4.
33
2.4 Which Cytodex microcarrier to use? Choosing the correct Cytodex microcarrier will depend on the type of cell being cultured and the purpose of the culture (table 5). Cytodex 3 should be used for situations not included in table 5. Certain conditions or culture requirements may modify the choice of microcarrier indicated in table 5. In addition to being a microcarrier Cytodex 3 can be used as a general purpose collagen-coated cell culture substrate. • Cells having a low plating efficiency When a particular type of cell tends to have a low plating efficiency (i.e. less than approx. 15%) it is important to use the microcarrier which enables attachment of the maximum number of cells. In table 5 there are listed certain situations where more than one type of microcarrier can be used. In some cultures it may be necessary to use the microcarrier which results in the highest plating efficiency. When culturing cells with fibroblast-like morphology, Cytodex 1 is a suitable alternative. For cells having an epithelial-like morphology, plating efficiency is greater with Cytodex 3 than Cytodex 1. Selecting the microcarrier in this way can also result in higher cell yields, simply because the plating efficiency is improved (fig. 19). • Small numbers of cells available for inoculation. It is not always possible to use large quantities of cells to inoculate a microcarrier culture and it may be necessary to start with a suboptimal number of cells. This situation often arises when working with primary cultures derived from small quantities of tissue. Under such circumstances it is important to use the microcarrier which results in the highest plating efficiency (see above). For primary cultures inoculated with less than approx. 5 viable cells/microcarrier Cytodex 3 is recommended instead of Cytodex 1. If only small numbers of cells from established cell lines having a low plating efficiency (i.e. less than approx. 10%) are available for inoculation, then Cytodex 3 i preferred. • Requirement for improved harvesting and scaling-up When it is necessary to remove cells from the microcarriers with the maximum possible recovery, viability and preservation of membrane integrity, it is advisable to use Cytodex 3 in combination with a proteolytic enzyme for harvesting. Cytodex 3 should also be considered for scaling-up cultures when it is important to harvest the maximum number of cells in the best possible condition for inoculating the next microcarrier culture. Under such conditions Cytodex 3 should also be considered for cells with fibroblasts morphology. The sometimes slower growth of fibroblasts on Cytodex 3 is usually compensated by the improved harvesting and viability of the harvested cells. Hence it is advantageous to use Cytodex 3 for scaling up cultures and for fibroblasts the final production culture can then use Cytodex 1.
34
The improved harvesting techniques possible with Cytodex 3 are described in section 3.7. • Requirement for removal of medium components or maximum recovery of cell products In many microcarrier cultures and as part of production procedures it is important to be able to wash medium components or cell products from the culture. The amount of protein which binds to Cytodex microcarriers is extremely small. For example, using Cytodex in chromatography experiments only 4,3% of the total protein present in 100% newborn calf serum adsorbs to Cytodex 1 (1 ml serum/900 mg microcarrier in column); and less than 1.1% adsorbs to Cytodex 3. The amount of protein adsorbed to the microcarriers from culture medium supplemented with the usual 10% serum is therefore very small. Table 6 illustrates that it is possible to wash out serum proteins to a much greater extent from cultures with Cytodex 3 than from cultures using Cytodex 1. When it is important to remove medium components easily or to achieve maximum recovery of cell products Cytodex 3 are preferred. Table 6. Removal of serum proteins from cultures using Cytodex microcarriers. (Results kindly supplied by Dr. A.L. ven Wezel). Sample* Culture fluid First wash Second wash Third wash
Cytodex 1 Albumin
IgG
Cytodex 3 Albumin
IgG
>1/2,000 >1/2,000 >1/2,000 1/8,000
>1/125 1/500 1/4,000 1/16,000
>1/2,000 >1/2,000 1/32,000 1/128,000
>1/125 1/500 1/16,000 1/32,000
* Procedure: Secondary monkey kidney cells were grown for 9 days and cultures were washed with Medium 199 without serum (First wash). The cultures were resupended in Medium 199 without serum in incubated overnight. The wash fluid was removed (Second wash). The culture was resuspended in Medium 199 (without serum) and infected with polio virus. After 3 days culture fluids were harvested (Third wash). The third wash corresponds to the harvest of virus-containing culture fluids when producing vaccines from microcarrier cultures using Cytodex. Proteins were determined by countercurrent electrophoresis and a dilution of 1/32,000 corresponded to 1 mg albumin/ml or 1 mg IgG/ml.
2.5 Availability and storage All Cytodex microcarriers are supplied as a dry powder and must be hydrated and sterilized before use (section 3.3). The following pack sizes are available: Cytodex 1 Cytodex 3
25 g 10 g
100 g 100 g
500 g 500 g
2.5 kg 2.5 kg
5 kg 5 kg
Packs of Cytodex should be opened and stored under dry conditions. Stored unopened at room temperature Cytodex microcarriers are stable for more than 8 years.
35
36
3. Microcarrier cell culture methods 3.1 General outline of procedure Microcarrier culture is a versatile technique for growing animal cells and can be used in a variety of different ways for a wide range of applications (section 1.4). Although microcarrier culture is an advanced technique it is based on standard animal cell culture procedures and does not require complicated or sophisticated methods. Information on cell culture methods can be found in references 165,166,175,177. Microcarrier culture procedures are based on what is already known about the cell type to be cultured. Information about morphology, plating efficiency and growth properties of a cell type in traditional monolayer culture is invaluable when deducing the most suitable microcarrier culture procedure. The best procedure is the one which ensures maximum attachment of the inoculum to the microcarriers and results in rapid, homogeneous growth of cells to the highest possible culture density. The aim of section 3 is to outline the basic principles of microcarrier culture. These priciples provide the background necessary for deducing the best culture procedures for a wide variety of cells without needing to spend time on extensive preliminary experiments. Examples of specific culture procedures are given in section 6.2. A general outline of the microcarrier culture procedure can be defined by several simple steps: Step 1. Choose the most suitable Cytodex microcarrier based on cell type and application (section 2.5). 2. Select the most suitable culture vessel for the application. The best results and highest yields are obtained from microcarriers maintained in suspension (section 3.2), but a static culture is often use in an initial experiment (step 4). 3. Hydrate and sterilize the microcarriers (section 3.3). 4. Conduct an initial experiment with the microcarriers in a bacteriological Petri dish. The time required for attachment of cells to the microcarriers will subsequently influence the culture procedure used during the initial stages of the culture cycle (section 3.4). A rounded morphology and any tendency of the cells to clump will also define what stirringspeeds should be used (section 3.5). A rapid fall in pH of the culture (the medium turns yellow within 3 days) will indicate that modification to the medium may be necessary for cultures having a high density (section 3.5.2,4.1). 5. Carry out the microcarrier culture in the chosen vessel. The most suitable inoculation density, concentration of microcarriers and stirring speed (when applicable) can be deduced from what is known about the cell type and from the results of step 4. Section 3.4 and 3.5 describe the principles of choosing the most suitable conditions.
37
6. Optimize the culture procedure and conditions if necessary. Occasionally the results of step 5 indicate that further experiments are needed to increase the efficiency of the culture. The above steps are usually performed with the culture medium normally used for a given type of cell. It may be necessary to change or modify the medium (section 3.4.6,3.5.2,4.1) and to alter the supply of gases (section 4.3). In addition, greater economy of inoculum and serum may be achieved by altering the culture conditions (section 3.4,4.2). More information on optimizing culture conditions and trouble shooting is found in section 5.
3.2 Microcarrier culture vessels 3.2.1 Requirements In principle microcarrier culture is a very flexible technique and cultures can be contained in virtually any type of cell culture vessel. Microcarriers can be used simply to increase the surface area of static cultures in traditional vessels or can be used in genuine stirred suspension cultures where the full benefits of the microcarrier technique can be realized. In all situations the culture vessel must be non-toxic and sterile. The best results from microcarrier cultures are obtained when using equipment which gives even suspension of the microcarriers with gentle stirring and possibilities for adequate exchange of gases with the culture medium. Erratic stirring motions should be avoided since these lead to detachment of rounded mitotic cells from the microcarriers. The shape of the culture vessel and stirring mechanism should be chosen to prevent sedimentation of microcarriers in any part of the culture vessel. For this reason vessels with slightly rounded bases are preferred. It is important to avoid exposing the culture to vibration. Stirring mechanisms should be checked for vibration. Magnetic stirring units are often a source of vibration and a simple method for reducing transmission of vibration to the culture vessel is to place a thin piece of plastic foam between the culture vessel and the surface of the base unit. Note: If magnetic stirring units are placed in humidified incubators the electrical circuits should be isolated. The choice of vessel for microcarrier culture depends on the purpose of the study and the desired culture volume. Laboratory scale microcarrier cultures are generally less than approximately 5 liters and can be contained in a wide variety of vessels. Large scale microcarrier cultures range from approximately 5 to several hundred liters and must be maintained in specially designed vessels (fermenters) allowing monitoring and control of parameters such as gas tensions and pH.
38
A
B
C
D
Fig. 20. Various stirred culture systems for laboratory scale microcarriers culture. All systems are stirred by a magnetic base stirring unit. A. Traditional spinner vessel of type supplied by Bellco Glass Inc. or Wheaton Scientific. Stirring speed 50–60 rpm. B. Spinner vessel modefied for microcarrier culture: Paddle impeller (Bellco Glass Inc.). Stirring speed 20–40 rpm. C. Rod-stirred microcarrier culture contained in a vessel with idented base (Techne (Cambridge) Ltd.) A magnet is present in the bulb of the stirring rod. Stirring speed 20–30 rpm. D. Spinner vessel modefied for microcarrier culture: Plough impeller. Vessel with round bases were supplied by Wheaton Scientific and the plough-shaped impeller was fashioned from Teflon® . Stirring speed 15–30 rpm. (Hirtenstein, M., Clark, J.M., Gebb, Ch., Develop. Biol. Standard. (1981), in press, by kind permission of the authors and the publisher).
3.2.2 Laboratory scale microcarrier culture vessels Spinner and rod-stirred vessels The most suitable vessels for general purpose laboratory scale microcarrier culture are those having a stirring rod or impeller driven by a magnetic base unit (fig. 20). Cultures stirred by a bulb-shaped rod (fig. 20) produce higher yields of cells than cultures stirred by the spinner principle (fig. 21) and are more suitable for cells with a low plating efficiency. The spinner vessel has been used for many years for the suspension culture of anchorage-independent cells. The culture is stirred by a suspended teflon-coated bar magnet which is driven by a magnetic stirring base unit. The stirrer bar should never come into contact with the inside surface of the vessel during culture since this may damage the microcarriers. Similarly, spinner vessels having a bearing which is immersed in the culture medium are not suitable, since the microcarriers can circulate through the bearing and become crushed. When using
39
spinner vessels the position of the impeller should be adjusted so as to minimize sedimentation of microcarriers under the axis of rotation. This is usually accomplished by positioning the end of the impeller a few millimeters (approx. 5 mm) from the bottom of the spinner vessel. Spinner vessels used under closed culture conditions are suitable for cultures ranging in volume from 2 ml to 1-5 liters. If greater culture volumes are required, then an open, monitored culture system is advisable (section 3.2.3). The maximum culture volume that can be conveniently used in closed spinner vessels depends on cell type, how rapidly culture conditions change throughout the culture cycle and how often the culture medium is replaced. For example, the rapid accumulation of acid in cultures of some established cell lines requires either frequent changes of medium or other methods of controlling pH and gas tensions (section 4.3,4.4). The capacity of the closed culture system is limited by the gas exchange possibilities (i.e. the volume of the culture, the surface area of the gas/medium interface and the volume of the gas headspace, (fig. 33, section 3.5.2). While good results with many cell types can be obtained from traditional spinner vessels, recently developed vessels and magnetic stirrers can be obtained from Bellco Glass Inc. (Vineland, NJ, USA) who have modified the traditional spinner vessel for use with microcarriers (fig. 20). Wheaton Scientific (Milville, NJ, USA) and Wilbur Scientific Inc. (Boston, MA,USA) also supply spinner vessels and magnetic stirring bse units which are suitable for microcarrier culture (22). Culture vessels modified with rounded bases can be supplied on request (fig. 20). An inexpensive spinner vessel for culture volumes of between 2 and 20 ml can be made from scintillation vials (98) Such a vessel can be used with magnetic stirring base units where the stirring speed ranges from approximately 10 rpm to 40 rpm. An improved principle for keeping microcarriers in suspension is the asymmetric stirring action provided by a suspended stirring rod. Techne (Cambrighe) Ltd. (Duxford, Cambridge, UK) have developed a technique for stirring microcarrier cultures where a bulb-shaped rod with one end fixed above the culture moves with a circular motion in a culture vessel having a rounded and indented base (fig. 20). This system provides a more gentle and even circulation of microcarriers and eliminates the sedimentation of microcarriers often observed when using spinner vessels (99). The rod-stirred microcarrier vessel is used in combination with a low speed magnetic base stirring unit and results in yields of cells significantly greater than those achieved with spinner vessels. The increased yield is particularly apparent for cells which have a low plating efficiency (fig. 21). Such a system can be used for cultures with volumes ranging from approximately 100 ml to 3 liters. Further information on equipment for rod-stirred microcarrier cultures can be obtained from Pharmacia Biotech.
40
Cells/ml
7
Fig. 21. A comparison of the growth of human fibroblasts (MRC-5) in microcarrier cultures contained in either standard magnetic spinner vessels (—●—) or bulb-shaped rod stirred culture vessel (—❍—). Culture volume was 600 ml and microcarriers were stirred in both systems at a speed just sufficient to achieve even suspension (50–60 rpm, spinner vessel; 20–30 rpm, rod stirred culture). Cultures contained 3 mg Cytodex 1/ml. (From Pharmacia Fine Chemicals AB, Uppsala Sweden).
10
6
10
5
10
4
10
1
3
5
7 Days
Roller bottles Although spinner and rod-stirred cultures provide maximum yields from Cytodex, the microcarriers can be used to increase the yield of cells, virus or interferon from roller bottles. Under roller bottle conditions the yield of cells per unit weight or unit area of Cytodex is approximately 50% of that from the microcarriers when used under optimal conditions in rod-stirred cultures. Nevertheless by using Cytodex microcarriers it is possible to obtain a 5-10 fold increase in growth area in each roller bottle. This increase in growth area is paralleled by a corresponding increase in cell number and yield of virus or interferon. The yield will be dependent on availability of nutrients from the medium, control of pH and an adequate supply of oxygen (see below). There are three alternative methods for using Cytodex with roller bottles. Method 1: Cytodex can be used to increase the yield from roller bottles by using standard roller techniques. The microcarriers adhere to the surface of the roller bottle and provide a fixed surface for cell growth. The amount of microcarrier added is not critical but approximately 1.5 mg/cm2 of roller bottle surface is adequate. The microcarriers can be added with the initial supply of culture medium. It is not necessary to remove unbound microcarriers and the rolling speed should be that normally used for at given type of cell. Method 2: The microcarriers can be used for ”suspension” culture in roller bottles. Siliconized roller bottles (section 3.2.4) should be filled 1/2-2/3 full with medium 41
containing Cytodex at a concentration of not more than 3 mg/ml. The bottles are inoculated with the usual number of cells used for suspension microcarrier cultures (section 3.4.4), flushed with a mixture of 95% air: 5% CO2 and sealed. The most suitable speed of rotation is greater than that normally associated with roller bottle culture. A speed of 5-15 rpm should be sufficient to ensure good but gentle stirring of the microcarriers. Stringent aseptic techniques are required since the medium comes into contact with the inside of the bottle neck and cap. Modified culture procedures (section 3.4.2) can be used with this method. Method 3: This method was developed by L. Kronenberg (185, patent pending). Cells are grown to confluence in rotating roller bottles using standard techniques. Once confluence is achieved, the culture medium is replaced by fresh medium containing microcarriers. Approximately 1.5 mg Cytodex in 0.5 ml medium/cm2 of roller bottle surface is adequate. The bottles are rotated at 2-4 rpm for approximately 15-30 mins during which time an even layer of microcarriers adheres to the confluent layer of cells. The rotation speed is then reduced to that normally used for roller bottle culture of the given type of cell (0.25-1 rpm). Cells migrate onto the microcarriers and form additional confluent layers while the original monolayer on the surface of the bottle remains stable. This technique effectively accomplishes two subcultures in the one vessel and allows a reduction of medium consumption per cm2 or per 106 cells by 50-75% (185). During the microcarrier phase of the culture it is not usually necessary to replenish the medium.
Fig. 22. Roller bottle cultures of mouse L-cells using Cytodex to increase the yield. Confluent cultures were drained and replenished with 400 ml medium containing 1.5 g Cytodex 1 (roller bottles approx. 1400 cm2). The monolayers were photographed at a) 8 h, b) 24 h, c) 72 h and d) 96 h. (Original photographs by L. Kronenberg, Lee Bio Molecular Res. Labs. Inc., San Diego, USA, reproduced by kind permission).
42
The choice of method depends on the circumstances and cell type. Method 2 requires rolling machinery capable of higher speeds of rotation and yields using this method for certain types of cells may be limited by ability to control pH in the closed roller bottle. The success of Method 3 depends on the ability of cells to migrate from the confluent monolayer onto the microcarriers. Method 3 is the procedure of choice since the techniques are more simple and reliable than the other two methods. The microcarriers are also fixed or immobilized (fig. 22) and this means that such cultures are compatible with the usual virus/interferon harvesting schemes used with roller bottles and Method 3 has been used to increase the yield of interferon from roller bottle culture (L. Krononenberg, pers. comm., 185) Rocking bottles The yield of cells from culture bottles (e.g. Roux bottles) can be increased severalfold by using Cytodex. The microcarriers can be easily kept in suspension by using a rocking platform to keep the culture in motion and by supplementing the medium with 5-10% (w/v) Ficollâ 400 (S. Smit, pers. comm., 191) The Ficoll 400 increases the density of the culture medium and thus allows the microcarriers to float more easily. The procedures outlined in section 3.4 are applicable to using this culture method. The rate of rocking should be just sufficient to ensure slow movement of the microcarriers. Air-lift and fluid-lift culture systems An alternative method for keeping microcarriers in suspension is to use an upward flow of air or culture medium though the culture . The success of air-lift microcarrier culture depends on using gas of high purity an on defining the gas requirements of
Microcarrier culture
Gas
Filter
Heating jacket
Medium reservoir Heating jacket
Filter Peristaltic pump Fig. 23. Schematic diagram of fluid lift microcarrier culture system. The filters should have a mesh sufficient to exclude Cytodex microcarriers (approx. 100 µm). An example of this type of system is described in reference 39. The culture can be contained in a chromatographic column (Pharmacia K 50/ 60) with medium circulated at 17 ml/min with a Pharmacia P-3 pump.
43
each cell type (section 4.3). Only those cell types which remain relatively strongly attached to culture surfaces during mitosis can be grown with this method. The erratic movement caused by the gas bubbles causes greater shear-forces than when using circulating culture medium to achieve suspension. A simple fluid lift microcarrier culture system is illustrated in figure 23. After inoculation of cells into such a system it is necessary to have a static culture period. Once the cells have attached, circulating medium can be used to keep the microcarriers in suspension. This system produces somewhat better cell yields than spinner vessels (39) but is more difficult to use for general culture under aseptic conditions. Perfusion culture Cytodex can be used to greatly increase the culture surface area of perfusion chambers. Alternatively an efficient perfusion culture can be constructed by filling a chromatographic column with microcarriers covered with cells and then perfusing medium through the column. Such a system is illustrated in figure 14. A suitable column for perfusion culture is a Pharmacia K 9/15 column (code no. 19-0870-01) fitted with an 80mm mesh nylon net (Pharmacia code no. 19-2268-01). Dishes, tubes and multi-well trays The surface area of cell culture Petri dishes and wells can be greatly increased by using microcarriers. The yield of cells from Petri dishes can be improved at least two-fold by adding microcarriers (5 mg Cytodex /ml) and the yield from wells can be increased five-fold using the same concentration of microcarriers. The microcarriers can be added as a suspension in the culture medium and the dish or well can be inoculated with the usual number of cells. With this procedure the microcarriers do not attach firmly to the surface of the culture vessel and the culture should be aspirated gently with a pipette every few days so that extensive aggregation of microcarriers is avoided. Alternatively, the microcarriers can be added in PBS to the cell culture dish or well. After a few minutes the microcarriers attach to the culture surface, the PBS is removed and the dish or well can be rinsed carefully with culture medium. The culture is then inoculated in the usual manner. Cytodex can also be added to confluent dishes for subculturing without enzymes or chelating agents (section 1.4.4). In this method, cells migrate from the confluent monolayer onto the microcarriers. The microcarriers can then be removed and used to inoculate other cultures or for biochemical studies. Bacteriological Petri dishes can be used when the growth of cells is to be restricted only to the microcarriers. Such a culture is a useful first step when working for the first time with the microcarrier culture of a particular type of cell (section 3.1). For such a preliminary experiment the dish should contain 3-5 mg Cytodex/ml and is inoculated with approx. 1-2 x 105 cells/ml. Culture tubes can also be used for microcarrier culture. However to ensure even growth of cells on the microcarriers the tubes must be kept in smooth motion. Nilsson and Mosbach (42) successfully used an end-over-end rotator for growing a variety of cells in tube microcarrier cultures.
44
3.2.3 Large scale microcarrier culture vessels The requirements for large scale microcarrier culture equipment are similar to those for small scale cultures and because of the large culture volumes involved, equipment for monitoring and controlling several culture parameters is required. A variety of different configurations have been used successfully for large scale microcarrier culture (1, 4, 7, 32, 33, 40, 41, 47, 54, 55). However, to date the most suitable commercially available system has been a modified ”Bilthoven Unit” (100) supplied by Contact Holland (Ridderkerk, Holland). Such units have been used for culture volumes up to several hundred liters (40, 41) for production of cell products such as interferon and virus vaccines. Large scale equipment used for culturing microorganisms and suspension animal cell cultures is generally not suitable for microcarrier culture. Such equipment must be modified to account for the suspension properties of the microcarriers, the slow stirring speeds required and the culture procedures discussed in section 3.4. Optimal function of the large scale microcarrier culture process usually requires that specific design features are adopted for each application and situation. Further information on process design and equipment for large scale microcarrier culture can be obtained from Pharmacia Biotech.
3.2.4 Siliconizing culture vessels Whenever glass surfaces are used with microcarriers the inside surface of the vessel should be siliconized to prevent the microcarriers sticking to the glass. It is also useful to siliconize other glassware (e.g. pipettes, bottles etc.) which may be used for transferring or storing hydrated microcarriers. The best siliconizing fluids are those based on dimenthyldichlorosilane dissolved in an organic solvent. A small volume of siliconizing fluid is added to the clean culture vessel and is used to wet all surfaces which may come into contact with the microcarriers. Excess fluid is drained from the vessel which is then allowed to dry. The vessel is washed thoroughly with distilled water (at least twice) and sterilized by autoclaving. One coating with siliconizing fluid is sufficient for many experiments. Examples of suitable fluids are: Sigmacote Repelcote Dimethyldichlorosilane Prosil-28 Silicone Oil Siliclad
Sigma Chemical Co., Cat. no SL-2 Hopkins and Williams, Cat. no. 9962-70 BDH, Cat. no 33164 PCR Research Chemicals Midland Silicones Ltd., Cat. no MS 1107, use as 2-5% (v/v) solution in ethyl acetate. Clay-Adams, Cat. no 1950.
It is not necessary to siliconize polished stainless steel culture vessels.
45
3.3 Preparing Cytodex microcarriers for culture The dry Cytodex microcarriers are added to a suitably siliconized glass bottle (section 3.2.) and are swollen in Ca2+, Mg2+-free PBS (50-100 ml/g Cytodex) for at least 3 h at room temperature with occasional gentle agitation. The hydration process can be accelerated by using a higher temperature e.g. 37°C. The supernatant is decanted and the microcarriers are washed once with gentle agitation for a few minutes in fresh Ca2+, Mg2+-free PBS (30-50 ml/g Cytodex). The PBS is discarded and replaced with fresh Ca2+,Mg2+-free PBS (30-50 ml/g Cytodex) and the microcarriers are sterilized by autoclaving with steam from purified water (115°C, 15 min., 15 psi). It is not recommended that conditions for autoclaving exceed 120°C, 20 min., 20 psi. When hydrating Cytodex 3 initial surface tension may occasionally prevent wetting and sedimentation of the microcarriers. Should this occur, Tween 80 can be added to the PBS used for the first hydration rinse (2-3 drops, Tween 80/100 ml PBS). Note. Cytodex 3 do not swell to the same extent as Cytodex 1 (table 4). Prior to use the sterilized microcarriers are allowed to settle, the supernatant is decanted and the microcarriers are rinsed in warm culture medium (20-50 ml/g Cytodex). This rinse reduces dilution of the culture medium by PBS trapped between and within the microcarriers (a step of particular importance when using small culture volumes or cells with low plating efficiencies). Then the microcarriers are allowed to settle, the supernatant is removed and the microcarriers are resuspended in a small volume of culture medium and transferred to the culture vessel. It is not necessary to treat the microcarriers with serum or to have serum in the rinsing medium. Other sterilization methods It is also possible to sterilize the microcarriers by other methods. After swelling the microcarriers in Ca2+, Mg2+-free PBS they are allowed to settle, the supernatant is decanted and replaced by 70% (v/v) ethanol in distilled water. The microcarriers are washed twice with this ethanol solution and then incubated overnight in 70% (v/v) ethanol (50-100 ml/g Cytodex). The ethanol solution is removed and the microcarriers are rinsed three times in sterile Ca2+, Mg2+-free PBS (50 ml/g Cytodex) and once in culture medium (20-50 ml/g Cytodex) before use. Cytodex 1 can also be sterilized with gamma irradiation (2.5 megarads). The sterilizing step is performed with the dry microcarriers before swelling in sterile PBS with the procedure described above. For large scale cultures the microcarriers can be swollen and sterilized in situ in fermenter vessels possessing an in-line steam sterilization system. This makes dispensing the microcarriers into the fermenter a more simple step and reduces the risk of contamination.
46
The above ranges of solute volume to weight of microcarrier allow for different types of cell. For those cells with a low plating efficiency the larger solute volume/ microcarrier weight should be used when swelling and sterilizing. Conversely, microcarriers to be used with cells having a high plating efficiency can be prepared in the minimal quantities of solute. A reduction in pH of culture medium upon addition of the microcarriers indicates that hydration and equilibration are not complete. If this decrease in pH is observed, the microcarriers should be rinsed once more in medium before use.
3.4 Initiating a microcarrier culture The initial phase of a microcarrier culture is usually the most critical stage in the culture cycle (38, 101). The success of the culture depends on correct procedures being followed when starting the culture and during the early phase of growth. Furthermore, the exact procedure is different for each type of cell and will depend on its growth properties in culture. Growth properties such as the rate and strength of attachment to culture surfaces and plating efficiency must be taken into account when selecting conditions of inoculation and stirring speed. When initiating a microcarrier culture the following points should always be considered. In most cases the conditions optimal for a given type of cell can be deduced from what is already known about its growth properties in culture (e.g. in Petri dishes or roller bottles) and also from preliminary experimens with microcarriers in Petri dishes (section 3.1.).
3.4.1 Equilibration before inoculation Conditions for attachment should be optimal from the moment the cells are inoculated in the culture. A long period of equilibration after the culture has been inoculated should be avoided. Ensuring that the culture is equilibrated before inoculation assists in obtaining the maximum possible plating efficiency of cells in the inoculum. The PBS in the sterile microcarriers should be removed by a rinse in warm culture medium (section 3.3). The culture temperature should be adjusted to a level optimal for cell attachment. In practice this temperature is usually the same as that use for the growth stage of culture (normally 35-37°C). Every attempt should be made to ensure that the culture pH is within the limits optimal for cell attachment (usually pH 7.1-7.4, section 4.4). Gas mixtures to be used during the initial stage of culture (section 4.3) should be allowed to exchange with the culture medium before inoculation. These factors are particularly important at large culture volumes (more than 500 ml) when it takes a longer time for equilibration. Small culture volumes (500 ml or less) can be equilibrated by incubating the culture vessel containing medium at 37°C
47
% cells attached to micro carriers
100
80
60
40
20
4
8
12 Hours
Fig. 24. The rate of attachment of various types of cells to Cytodex 1 microcarriers. All cultures were inocluated with 105 cells/ml and contained 5 mg Cytodex 1/ml. Stirring was continuous (50 rpm). — — chicken fibroblasts, —▲— monkey cells (Vero), —❑— human fibroblast (MRC-5), —■— mouse fibroblasts (L-929). (From Pharmacia Fine Chemicals AB, Uppsala, Sweden).
and in an atmosphere of 95% : 5% Co2 (section 4.3). After a few minutes the culture will be ready for inoculation. Stirring can be used to hasten the process of gas exchange. Equilibration of cultures with very large volumes may take 2-3 hours. The exact procedure used for equilibration should always be noted if reproducible results are to be obtained.
3.4.2 Initial stirring The key to achieving maximum yields from microcarrier cultures is to ensure that all microcarriers are inoculated with cells from the very beginning of the culture. Transfer of cells from one microcarrier to another occurs only infrequently during the culture cycle and it is therefore important to ensure that the maximum possible number of cells from the inoculum attach to the microcarriers. One way of initiating a microcarrier culture is to inoculate the cells into the final volume of medium containing microcarriers and immediately begin stirring. Figure 24 illustrates that under such conditions different types of cell attach to the microcarriers at different rates. The rate and proportion of cells attaching to the microcarriers can be increased if the culture remains static with gentle intermittent stirring during the early attachment stage. If at the same time the cell-microcarrier mixture is contained in a reduced volume (e.g. in 1/3 of the final volume) then the cells have a greater chance of coming into contact with a microcarrier ant the conditioning effects on the medium are also much greater. The ability of an anchorage-dependent cell to attach to a culture surface is reduced if the cell is kept in free suspension for increasing lengths of time. For cell types which have an intrinsically slow rate of attachment it is important that culture conditions allow the cells to attach as rapidly as possible.
48
% cells attached to micro carriers
100
Monkey kidney cells (Vero)
Human fibroblasts (MRC-5)
80
60
40
20
4
8
12
4
8
12 Hours
Fig. 25. The effect of initial culture procedure on the attachment efficiency of cells. Cultures containing 5 mg Cytodex 1/ml final volume were inoculated with 105 cells/ml final volume and were either stirred immediately (—❑—, 60 rpm) or cultured in a reduced culture volume with intermittent stirring(—■—). The modefied initial culture procedure (—■—) involved stirring for 1 min (30 rpm) every 45 min in 1/3 of the final culture volume. After 3 h the culture was diluted to the final volume and stirred (60 rpm). Attachmment of cells to plastic Petri dished is indicated (—●—). (From Pharmacia Biotech AB, Uppsala, Sweden.)
Figure 25 shows the effect of using an attachment period with intermittent stirring and reduced initial culture volume on the rate of attachment on human fibroblasts and monkey kidney cells to microcarriers. For each type of cell both culture procedures use the same total number of cells and microcarriers but the modified procedure leads to a more efficient utilization on the inoculum. This procedure results in attachment efficiencies comparable to those observed in plastic Petri dishes. The increased efficiency of attachment which results from initiating the culture in a reduced volume and with intermittent stirring results in an increase in cell yield at the plateau stage of culture (fig. 26). The improvements in cell yield when using this technique are most apparent where cultures are started with low inoculation densities. This modified procedure at the initial stage of culture can be used for all types of cells and is particularly recommended when working with cells which have a low plating efficiency, (e.g. primary cell cultures and normal diploid cell strains) and if sufficient cells are not available to start the culture with optimum inoculation densities (section 3.4.4). 49
Cells/ml
10
10
10
10
7
Monkey kidney cells (Vero)
Human fibroblasts (MRC-5)
6
5
4
1
3
5
7
1
3
5
7
9 Days
Fig. 26. The effect of inoculation density and initial culture procedure on the growth of monkey kidney cells and human fibroblasts on Cytodex 1 microcarriers. Cultures were inoculated with cells and stirred immediately in the final volume (—●—, 60 rpm) or were cultured in a reduced volume during the attachment stage of culture (—❍—). The modified initial culture procedure (—❍—) involved stirring for 1 min (30 rpm) every 60 min in 1/3 of the final culture volume. After 4 h (Vero) or 6 h (MRC-5) the culture was diluted to the final volume and stirred (60 rpm). All cultures contained 3 mg Cytodex 1/ml final volume. (From Pharmacia Biotech AB, Uppsala, Sweden and Clark, J., Hirtenstein, M. Annals N.Y. Acad. Sci. 369 (1981) 33, by kind permission of the authors and publisher.)
The details of a modified initial culture procedure depend on the type of cell being cultured. A preliminary experiment with a stationary microcarrier culture in a bacteriological Petri dish will enable estimation of the time required for attachment of cells to the microcarriers and will also reveal any tendency for aggregation of cells and microcarriers under static culture conditions (section 3.1). When starting most cultures the cells and microcarriers are incubated in 1/3 of the final volume. The culture is stirred for 2 min. every 30 min. at the speed used during the growth phase of the culture (section 3.4.1). After 3-6 hrs continuous stirring is commenced at a speed just sufficient to keep the microcarriers in suspension (section 3.5.1) and the volume of the culture is increased with pre warmed (37°C) culture medium. The longer attachment time is usually used for primary cells with an epithelial morphology. For cells with a low plating efficiency (less than 10%) or for cultures started with suboptimal numbers of cells (section 3.4.7) the culture volume can be maintained at 50% of the final volume for the first three days of culture and then fresh medium is added to reach the final volume. In this way the population of cells can be cultured at greater densities during the period the culture is most susceptible to dilution. Slow
50
continuous stirring during the attachment stage of culture is necessary for cell types which tend to clump when allowed to settle (e.g. primary chicken embryo fibroblasts). In such cases the initial stirring speed need only be approximately 25% of that normally used for the growth phase of the culture (section 3.5.1). Griffiths et al. (7) and Moser et al. (62) have observed that the modified initial culture procedure was essential for good results when growing human fibroblasts and heart muscle cells, respectively.
3.4.3 Concentration of microcarriers The yield of cells from microcarrier culture is directly related to the surface area for growth and hence to the concentration of microcarriers. In most situations Cytodex microcarriers are used in stirred cultures at a concentration of 0.5-5.0 mg/ml final volume. With some types of cells (e.g. certain established cell lines) it is possible to achieve good growth at lower concentrations of microcarriers but these are cells which can grow at low culture densities. Provided an adequate supply of medium is ensured (section 3.5.2) and gas tensions and pH can be controlled (section 4.3., 4.4) it is possible to work with cultures containing more than 5 mg Cytodex/ml and in some situations it is possible to achieve 5-10 x 106 cells/ml. If consistent difficulties in maintaining culture conditions (pH and gas tensions) or in providing a sufficient supply of nutrients are encountered during the later stages of the culture cycle then decreasing the concentration of microcarriers should be considered.
% micro carriers bearing cells
Within the range 0.5-5 mg Cytodex/ml final volume the proportion of microcarriers bearing cells at the plateau stage of culture (and hence the yield) depends on the
100
Monkey kidney cells (Vero)
Human fibroblasts (MRC-5)
80
60
40
20
1
2
3
4
5
1
2
3
5 4 mg Cytodex/ml
Fig. 27. The effect of microcarrier concentration on the proportion of microcarriers bearing cells at the plateau stage of culture. Cultures were inoculated with 5 viable Vero cells/microcarrier or 10 viable MRC-5 cells/microcarrier and stirred immediately at 60 rpm. The proportion of microcarriers bearing cells was determined after 7 days (Vero) or 9 days (MRC-5). Cultures were maintained under conditions where supply of medium and control of pH wre not limiting cell growth. (From Pharmacia Biotech AB, Uppsala, Sweden.
51
concentration of microcarriers (fig. 27). Under conditions where the absolute concentration of cells and microcarriers is low, the chance of a cell coming in contact with a microcarrier is small and therefore a greater proportion of microcarriers remain devoid of cells at the plateau stage of culture. If low concentrations of microcarriers must be used than the proportion of microcarriers bearing cells at the plateau stage of culture and the yield can be increased by using the modified initial culture procedure (section 3.4.2). Provided a correct inoculation density is used (section 3.4.4) a concentration of 3 mg Cytodex/ml final volume is usually the optimal concentration for general microcarrier culture and results in the greatest proportion of microcarriers bearing cells. The yield of cells/cm2 from cultures containing lower concentrations of microcarriers (less than 2 mg/ml) depends on the ability of the cells to grow under less dense conditions. In order to obtain the maximum yield of cells/cm2 from cultures containing higher concentrations of microcarriers (more than 4 mg Cytodex/ml) the culture medium may need to be replenished more often than when growing cells at a low concentration (section 3.5.2). This is simply because a given volume o medium can support the growth of only a finite number of cells. As an approximate guide to expected cell yields it can be assumed that the culture has a density of 105 cells/cm2 at confluence. This corresponds to 6 x 105 cells/mg Cytodex 1, 5.5 x 105 cells/mg Cytodex 2 and 4.6 x 105 cells/mg Cytodex 3. The exact yield will depend on the characteristic saturation density of the cell type and on the supply of medium.
3.4.4 Inoculation density It is a general cell culture phenomenon that the survival and growth of cells depends to a large extent on the inoculation density and conditioning effects. These conditioning effects are dependent on the density of the culture and a low density leads to relatively poor growth. With respect to anchorage dependent cells one of the most critical parameters at inoculation is the number of cells/cm2 of culture surface area, cells with low plating efficiencies being particularly sensitive to culture under conditions of low density. It is therefore important to take into account the large surface area provided by Cytodex microcarriers. Since the efficiency of attachment of cells to Cytodex is similar to that observed in Petri dishes (fig. 25) the microcarrier cultures should be inoculation with approximately the same number of cells/cm2 as used when starting other types of monolayer cultures. The number of cells/cm2 used to inoculate the culture will depend on the plating efficiency of the cells (section 3.4.5) When inoculating a culture it is generally necessary to use more primary or normal diploid cells than established cells. Inoculation density effects both the proportion of microcarriers bearing cells at the plateau stage of culture (fig. 28) and the yield from the culture (fig. 26). Figure 28 shows that approximately 10 human fibroblasts/microcarrier are required for maximum utilization of the microcarriers, whereas only 5 monkey kidney cells/ microcarrier are required. Horng and McLimans (14) reported that approximately
52
% micro carriers bearing cells
100
Monkey kidney cells (Vero)
Human fibroblasts (MRC-5)
80 60 40 20
5
10
15
5 10 15 Inoculation density (cells/micro carrier)
Fig. 28. The effect of inoculation density on the proportion of microcarriers bearing cells at the plateau stage of culture. Culture contained 3 mg Cytodex 1/ml final volume and were either stirred continuously at 60 rpm (—●—) or were started with a reduced volume and intermittent stirring before the culture was diluted to the final volume and stirred at 60 (—❍—). Details of the modified culture procedure are given with fig. 26. The proportion of microcarriers bearing cells was determined after 7 days (Vero) or 9 days (MRC-5). (From Pharmacia Biotech AB, Uppsala, Sweden.)
5 cells/microcarrier were required when inoculating cultures with anterior calf pituitary cells. If sufficient cells are not available to inoculate the culture the modified initial culture procedure discussed in section 3.4.2 can be used. Since this procedure results in a more efficient utilization of the inoculum, more microcarriers bear cells at the plateau stage of culture (fig. 28) and cell yields are increased (fig. 26). Further details on inoculation density can be found in section 3.4.7.
3.4.5 Inoculum condition The plating efficiency of cells depends on the stage of the culture cycle from which the inoculum is taken. Cells in exponential growth have a higher plating efficiency than cells which come from a resting population. The yield from microcarrier cultures can be increased 2-3 fold by inoculating with cells taken from exponentially growing cultures rather than confluent cultures (101). When possible, microcarrier cultures should always be inoculated with cells taken from actively dividing cultures at approximately 70-80% confluence. In this way a greater percentage of cells in the inoculum can attach to the microcarriers and contribute to the growth of the culture. The inoculum should be evenly dispersed and preferably a single cell suspension. Excessive centrifugation during concentration of the inoculum should be avoided since this leads to aggregation of cells and reduced viability after attempts at resuspension. Centrifugation for 5 min. at 200-300 g is normally sufficient. Suspension of the inoculum in the medium to be used in the culture avoids dilution of medium components. Exposing the inoculum to sudden changes in temperature, pH or osmolarity should also be avoided. 53
3.4.6 Culture media during the initial culture phase A general discussion on culture media can be found in section 4.1 and replenishment is considered in section 3.5.2 The nutritional requirements of cells are not necessarily the same throughout the culture cycle and for optimal result it may be desirable to alter the formulation of the culture medium at some stage in the culture cycle. This is usually necessary if the culture is to span a very wide range of densities e.g. from 5x104 cells/ml at inoculation to 3x106 cells/ml at confluence. In this section alterations to the medium during the initial stage of culture will be considered. The main difference between culturing cells as monolayers on microcarriers or on other culture surfaces is that microcarrier cultures span a wider range of cell densities for any single culture. Microcarrier cultures must often be started with a low number of cells/cm2 . Nutritional requirements of cells growing under conditions of low density are usually more stringent than for cells growing under high densities (44) and the plating efficiency of cells can be improved by ensuring that the culture medium contains certain components in sufficient quantities. The necessity for supplementing the culture medium during the initial culture period depends on the culture medium, the inoculation density and the type of cell. For cells which tend to have high plating efficiencies (e.g. most established and transformed cell lines), additional supplementation of common culture media is usually not necessary. In contrast, many primary cells and normal diploid cell strains require additional supplementation of common culture media if maximum yields are to be obtained. Such supplementation need only be included during the initial growth phase and is no longer required when the medium is replenished later in the culture cycle. Table 7 illustrates that the growth of cells in cultures inoculated with small numbers of cells is better when a more ”complete” medium with a large number of components (Medium 199) is used rather than when a more sparse medium (DME, BME) is used. At high inoculation densities the plating efficiency of cells is improved and culture growth is greater in the medium with the highest concentration of amino acids and vitamins (DME). Table 7. The effect of various culture media on the initial growth of monkey kidney cells (Vero) in microcarrier cultures. All media contained 10% (v/v) foetal calf serum and 5 mg Cytodex 1/ml. Cultures were stirred at 60 rpm from the moment of inoculation. Inoculation density
Cells attached to microcarriers (%) of inoculum) DME BME Medium 199 24 h 48 h 24 h 48 h 24 h 48 h
High 1.5x105 cells/ml
210
460
180
350
150
280
Low 1.1x104 cells/ml
60
70
45
55
95
145
Examples of media which are useful when culturing cells at low densities or cells having a low plating efficiency are Medium 199, McCoy’s 5A, Ham’s F10 or Ham’s F12. The plating efficiency of cells inoculated at low density into Ham’s F10 or
54
Ham’s F12 media can be increased by doubling the concentration of amino acids and vitamins. More recent cloning media are described by Ham and McKeehan (44). The difficulty with using cloning media is that they often lack sufficient reserves of nutrients to support growth of cells at high culture densities. This is particularly important if maximum yields are to be obtained at the plateau stage of culture when more than 106 cells/ml are present. A medium such as Medium 199 is relatively poor at supporting the growth of high densities of cells (table 7, fig. 34). An alternative approach is to use a culture medium which can support the growth of high densities of cells and to supplement this medium to improve growth of cells at low culture densities. In general, components which are beneficial during the initial stage of microcarrier culture and which are not found in several common media include pyruvate, non-essential amino acids (182), adenine, hypoxanthine and thymidine. Table 12 present a useful general purpose medium based on DME which is supplemented for the initial stage of culture (59). The advantage of such a medium is that it has a higher concentration of amino acids and vitamins than most other media and can therefore support the growth of greater densities of cells. In addition the medium can be used for a wide variety of cells and when necessary, stock solutions of the above components can be added for the initial stage of culture. Griffiths et al.(38) described a medium (MEM/MC) which was superior to others in its ability to promote attachment of human fibroblasts (MRC-5) to Cytodex 1 in large scale cultures. The modified medium was MEM with added glucose (1 mg/ml), glutamine (0.3 mg/ml), pyruvate (0.1 mg/ml), Eagle’s non-essential amino acids (182), adenine (10 µg/ml), thymidine (10 µg/ml), hypoxanthine (3 µg/ml), inositol (2 µg/ml), choline chloride (2 µg/ml), tryptose phosphate broth (30 ml/liter, or approximately 1 mg/ml) and HEPES (5 mM). This medium was then supplemented with 5% (v/v) calf and 5% (v/v) foetal calf serum. MEM/MC resulted in attachment of cells to microcarriers nearly 2-fold greater than in CMRL 1066 and 4-fold greater than in standard MEM supplemented with serum. A shorter lag period was observed and after 24 h cell yield in MEM/MC was 5-fold greater than in MEM and nearly 2-fold greater than in CMRL 1066. Increasing the concentration of the serum supplement can also improve the attachment and growth of cells during the initial stages of culture (section 4.2.2). Different types of serum supplement may also be used to promote cell attachment (section 4.2.2). In cases of cells which are very difficult to grow in culture, or when cultures must be started at very low densities, conditioned medium may be used. The medium can be removed from actively growing or confluent cultures of, for example, fibroblasts, thoroughly clarified by centrifugation and then mixed (often 50:50) with a medium suitable for growing cells at clonal densities.
55
3.4.7 Relationship between plating efficiency and culture procedure Knowledge of the growth properties of a particular type of cell in general monolayer culture can be used to deduce microcarrier culture procedures which are near optimal for that type of cell. This information often means that extensive preliminary experiments can be avoided and only minor adjustments to the culture procedure may subsequently be required to achieve the best results from the microcarrier culture. Comparative studies of the conditions required to culture a wide variety of cells in microcarrier culture have revealed that plating efficiency is one of the most useful growth parameters to consider when developing a culture procedure (38,39,101). Plating efficiencies vary considerably between different types of cells and are a measure of the cell’s ability to survive a susbculture step and contribute to the proliferation of the next culture. The plating efficiency of any particular type of cell is not a fixed value and can be influenced to a large extent by changing the culture procedure and/or conditions. If a cell possesses as intrinsically low plating effiency or if only small numbers of cells are available for inoculation then it is important to use culture procedures and conditions which enhance the plating efficiency. Table 8 illustrates the relationships between plating efficiency and the initial culture variables. These relationships have been observed for a wide variety of cell types (38, 39, 101). For any type of cell the essential elements of the initial culture procedure can be deduced from the plating efficiency. Primary cell suspensions normally have plating efficiencies of less than 10%, and the plating efficiency of normal diploid cell strains is usually between 10 and 30%. Most established cell lines have plating efficiencies greater than 30%. In some cases the culture procedure may need to be modified because of specific growth properties of the cell type. For example, weak or slow attachment to culture surfaces and a rounded morphology suggest that stirring during the attachment phase should be very gentle and less frequent. In contrast, a tendency towards aggregation upon inoculation indicates that continuous but slow stirring will be required thoughout the entire initial culture period. Such peculiarities of growth can be checked by first performing a growth test on microcarriers in bacteriological Petri dish cultures every time a new type of cell is to be cultured on microcarriers (section 3.1). Table 8. The relationship between the plating efficiency of a cell and those parameters known to be critical during the initial phase of a microcarrier culture. Parameter <10%
Plating efficiency* 10-30%
<30%
Cells/microcarrier at inoculation (Section 3.4.4)
high (>10)
intermediate (5-10)
low (<5)
Initial culture volume (Section 3.4.2)
small (20-30% of final volume)
intermediate (30-60% of final volume)
large (100% of final volume)
Initial stirring speed** (Section 3.4.2)
static/intermittent
continuous (approx 10 rpm) (approx 40-60 rpm)
Additional medium supplements (section 3.4.6)
required
advantageous
* See text. ** Actual speed depends on design of stirrer and culture vessel.
56
not required
The definition of plating efficiency used when developing a culture procedure need not be rigid. In the most accurate sense plating efficiency is the proportion of cells (%) which can form colonies when plated at low density into a Petri dish (e.g. 200500 cells/6 cm dish). In this case the plating efficiency is measured as ”cloning efficiency”. Plating efficiency is also indicated by the routine ”split” or subculturing ratio. A cell type which is subcultured routinely with a low split ratio (1:2) will usually require the initial microcarrier culture procedures necessary for a cell with a low plating efficiency (table 8). Cells which can be subcultured with a high split ratio (e.g. 1:20) have a high plating efficiency and modified initial culture procedures are not usually necessary. Table 9 presents suitable inoculation densities for the microcarrier culture of some common established cell lines. The differences in inoculation densities reflect differences in plating efficiency. The inoculation densities refer to cultures stirred continuously from the moment of inoculation and if fewer cells are available, intermittent stirring in a reduced initial culture volume should be considered (section 3.4.2) Table 9. A guide to comparative inoculation densities for some common established cell lines. Inoculation density
Cell line
2x105/ml
Don, Detroit 532, NCTC 2544, RPMI 2650, SIRC
5
10 /ml
Chan conjunctiva, BGM, BSC-1, CV-1, Y-1, Morris hepatoma, tumor virus transformed hamster cells (most), GL-V3, Pt-K-1
8-9x104/ml
Chang liver, HeLa, MDCK, MDBK, HT 1080, LLC-MK2, LLC-RK, J111, L-132, Vero, Neuro-2a, RK 13
5-8x104/ml
CHO, HaK, Detroit 6, Detroit 98, Girardi heart, HEp2, KB, WISH, Chimp liver, 3T3, PK-15, C6, BHK 21, HTC, PyY, McCOY, L 929, A9, 3T6
These inoculation densities refer to cultures containing 3 mg Cytodex/ml and stirred from the moment of inoculation in the final culture volume. Lower inoculation densities can be used in combination with modified initial culture procedures (section 3.4.2).
If the plating efficiency or routine subculturing split ratio of a particular type of cell is not known when the first cultures should contain 3 mg Cytodex/ml final volume and be inoculated with at least 105 cells/ml final culture volume. If necessary the size of the inoculum can be modified for subsequent cultures.
57
3.5 Maintaining a microcarrier culture Once a microcarrier culture has been initiated certain procedures and precautions are required in order to maintain proliferation of cells and to achieve the maximum yield from the culture. If the culture is essentially non-proliferating as in the case of some primary cultures, (e.g. hepatocytes) conditions must enhance function and survival of the cells for as long as possible. Although the following comments will be restricted to proliferating cultures exactly the same principles should be considered when maintaining non-proliferating cultures. During the microcarrier culture cycle the changes in the density of the cell population are usually greater than 10-fold. Such growth leads to conditioning of the medium and thereby encourages the growth of cells in the culture until a saturation density is reached and there is density-dependent inhibition of proliferation (many established and transformed cell lines do not show suck inhibition). At the same time, oxygen and medium components are utilized and toxic products of metabolism accumulate. When maintaining a microcarrier culture these changes must be taken into account. In addition it should be kept in mind when optimizing the culture procedure that conditions optimal for growth of cells under low density (e.g. gas tensions, pH etc.) are not necessarily optimal at later stages of culture (section 4.3, 4.4, 5). Note: The most important aspect of maintaining a microcarrier culture and obtaining best results is to anticipate changes in the culture. For example, from the first few cultures with a particular type of cell it is possible to observe if or when pH changes occur, when oxygen supply or medium components are depleted or if aggregation of the microcarriers occurs. Once the stage at which these changes occur is known corrective measures should be taken before such changes occur. It is often difficult to return to optimal conditions and obtain good results once large deviations have occurred and in such cases irreparable damage to the culture usually ensues.
3.5.1 Stirring speed Although other culture systems can be used (section 3.2.2) the most suitable method for maintaining microcarrier cultures is in stirred suspensions. The purpose of stirring the culture is a) to ensure that the entire surface of the microcarriers is available for cell growth, b) to create a homogeneous culture environment, c) to avoid aggregation of microcarriers by cell overgrowth and d) to facilitate exchange of gases between the culture headspace and the medium. In principle the stirring speed should be just sufficient to keep all the microcarriers in suspension. After the initial culture period, during which there is normally intermittent stirring or a static attachment period (section 3.4.2), the culture should be stirred continuously. The rate at which the culture is stirred influences greatly the growth an final yield of cells (fig. 29) and this effect is related to an integrated shear factor (fig. 30). Slower stirring speeds reduce shearing forces on cells attached to the microcarriers but if the stirring rate is too slow, growth is reduced (fig. 29).
58
Cells/ml
10
Fig. 29. Effects of stirring speed on the growth of Vero cells on Cytodex 1 microcarriers (30 rpm ▲, 40 rpm ❑, 60 rpm ●, 90 rpm , 120 rpm ■). All cultures were 250 ml and contained 3 mg Cytodex 1/ml. Culture vessels were traditional magnetic spinner vessels and the cultures were stirred from the moment of inoculation in the final culture volume. (Hirtenstein, M and Clark, J. In “Tissue culture in medical research” eds Richards, R and Rajan, K., Pergamon Press, Oxford, 1980, pp 97, by kind permission of the authors and publisher.)
7
6
10
5
10
4
10
1
3
5
7 Days
This effect is due mainly to inadequate gas diffusion and sedimentation and aggregation of microcarriers. If the stirring speed is too fast the less strongly attached cells (mainly mitotic cells) are dislodged from the microcarriers and there is no net increase in cell number. Such a phenomenon can be used to advantage for harvesting mitotic cells (section 1.4.7). Excessive stirring speed cause a general loss of cells from the microcarriers and poor cell yields (fig. 29, 30). Figure 30 illustrates that a population of cells growing on microcarriers is less sensitive to shear forces if initial attachment phase is included in the culture procedure (section 3.4.2). It is during the attachment phase of a stirred culture that the adverse effects of excessive shear forces are most noticeable. The stirring speed used during the growth and plateau phase of the culture depends on the type of cell being cultured and on the design of the stirrer. Most primary cells and normal diploid strains attach firmly to culture surfaces and can withstand higher shear forces than more weakly attaching cells such as many established or transformed cell lines. However, with respect to attachment the most critical stage of the cell cycle is during mitosis and most types of cells do not differ greatly with respect to strength of attachment during this stage. Therefore similar stirring speeds tend to be used for all cell types during the exponential phase of growth. When cultures are to be maintained at high densities a slight increase in stirring speed will improve gas
59
Immediate agitation
24h attachment
4
m
-6
Max cell density (cells/ml x 10 )
Fig. 30. The effect of shear force on the productivity of microcarrier cultures of chicken embryo fibroblasts. (Sinskey, A.J., Fleischaker, R.J., Tyo, M.A. et al. Annals N.Y. Acad. Sci. 369 (1981) 47, by kind permission of the authors and publisher.)
6
2
40
80
120 -1
Integrated shear factor (sec )
exchange and can be used to improve the supply of oxygen to the cells. However, if the culture is still dividing an increase in stirring speed must be limited and should not result in detachment of mitotic cells. Some cells (mainly transformed cells and some fibroblast strains, e.g. chicken fibroblasts) tend to form aggregates of microcarriers during the later stages of the culture cycle (plate 5) and in these cases a slight increase in stirring speed will reduce the chance of aggregation. Increases in stirring speed must be considered carefully if there is any tendency for the cell monolayers to detach from the microcarriers. This detachment can be related to culture conditions and is discussed below. The other aspect of selecting a correct stirring speed is the design of the stirrer and culture vessel and the volume of the culture. The optimal stirring speed when using traditional magnetic spinner vessels is usually 50-70 rpm (fig. 29) and speeds of 15-30 rpm are used with the modified spinner vessels or cultures stirred with bulb-shaped rods (section 3.2.2). Higher speeds are often required when using the same design of stirrer with culture volumes larger than 500 ml. A gradual increase in stirring speed to these levels over a few days is a desirable procedure. The stirring speed when working with large scale culture volumes also depends in the design of the stirrer. Progressive increases in stirring speed from 50 to 100-150 rpm during the culture cycle are frequently used for cultures of 100 liters or more (4, 10, A. van Wezel, pers. comm., 192). With all types of stirring equipment it is important to avoid sedimentation of the microcarriers, particularly during later stages of the culture cycle. The accumulation of microcarriers which often occurs under the stirring axis in magnetic spinner
60
pfu/cell.h
1200 60 rpm 800
100 rpm 200 rpm
400 100 rpm
150 rpm 200 rpm
40 80 -1 Integrated shear factor (sec ) Fig. 31. The effect of shear force on the production of Sindbis virus from microcarrier cultures of chicken embryo fibroblasts. Virus productivity was averaged for the final 8 h of infection. Cultures were contained in magnnetic spinner vessels and stirring was with either a 4.4. cm (■) or 7.5 cm (●) impeller. (Sinskey, A.J., Fleischaker, R.J., Tyo, M.A. et al. Annals N.Y. Acad. Sci. 369 (1981) 47, by kind permission of the authors and publisher.)
vessels can be reduced by positioning the stirrer as close to the base as possible whilst still leaving clearance for circulation of microcarriers. Such sedimentation does not occur with cultures stirred by a bulb-shaped rod, especially if contained in culture vessels with convex bases (section 3.2.2, fig. 20) When microcarrier cultures are used for production of viruses or cell products the stirring speed should usually be reduced during the production phase. In most cases a reduction to a speed one half that used during the growth phase of the culture is optimal. Excessive stirring or shear forces result in decreased yields of viruses or cell products (fig 31).
3.5.2 Replenishment of culture medium Careful replenishment of medium during the culture cycle is an important aspect of maintaining microcarrier cultures. There are three reasons for replenishing the medium: a. Replacing essential nutrients which are depleted by cell growth. b. Removing products of metabolism which inhibit growth or survival. c. Assisting in control of pH. Through careful planning of medium replenishment it is possible to achieve maximum yields of cells for a given volume of medium. The frequency and extent of medium replenishment depends on cell type, culture density, culture medium and gas tensions. Rapidly dividing cells and cultures at high densities require more frequent replenishment than low density cultures or slowly dividing cultures. Rapid cell division and high cell densities lead to depletion of medium components and a decrease in culture pH. At the same time meatabolites such as lactate, ammonia and even specific growth inhibitors accumulate (102, 183).
61
The ideal replenishment scheme is the one which results in the smallest fluctuation of nutrient concentrations and pH during the culture cycle. For this reason a continuous flow of medium is a preferred method for culture maintenance (103, 104). However, for small scale cultures or experiments with cell densities up to 3-5x106 cells/ml batch replenishment of medium is more convenient. The usual procedure is to start with replenishment of 50% of the medium volume every 3 days, If necessary, a modified scheme can then be developed in order to get the best yield form the culture. For example, it is common practice to observe the culture every day and to determine culture density (section 3.6). When samples are taken for these observations 10-20% of the medium volume can be replaced with fresh medium. In order to take advantage of conditioning effects the replenishment should not take place within the first two days of culture, This replenishment scheme requires very little extra effort and usually results in higher yields. In this way it is possible to avoid sudden changes in culture conditions, reduce fluctuations in nutrient concentration, reduce accumulation of toxic metabolites and to assist in the regulation of gas tensions and pH (sections 4.3, 4.4). When the cultures contain several million cells/ml and are in the exponential or plateau phase more frequent replenishment will be required or a modified culture medium can be used (see below). Note: Whenever fresh medium is added it should have the same temperature as the culture. The fresh medium should have a pH and osmolality optimal for cell growth (section 4.4, 4.5) Another approach to medium replenishment, especially during the later stages of exponential growth, is to feed the culture with a modified medium. During this stage of culture, nutrient and growth factor requirements are not the same as at the beginning of the culture. Many medium components used when initiating the culture, e.g. non-essential amino acids, nucleosides, etc. (section 3.4.6, 6.4) can be omitted and greater economy can be achieved by reducing the concentration of the serum supplement (section 4.2.2). The type of culture medium can also be changed during the culture cycle. For example, if cultures are initiated with Medium 199, this medium can be replenished during the culture by addition of DME. This change is beneficial because although Medium 199 is good for growth at low culture densities (table 7), DME is superior for high culture densities (fig. 34). Persistent difficulties with controlling pH at high cell densities can be overcome by modifying the carbon source in the medium, slightly increasing the oxygen tension or by using daily additions of glutamine in the presence of reduced concentrations of glucose (section 4.4.3). If excessive aggregation of the culture occurs and cannot be controlled by adjusting the stirring speed (section 3.5.1), then the calcium and magnesium concentration in the medium can be reduced. A simple method for reducing the concentration of these ions is to use mixtures of media involving suspension culture versions of culture media (e.g. Spinner MEM). A 50:50 mixture of this medium with the usual culture medium normally overcomes difficulties with aggregation, without affecting cell growth.
62
When nutrient depletion is the factor limiting growth in a culture rather than accumulation of toxic metabolites or a decrease in pH, a different approach can be adopted.Growth limitation is caused by depletion of only certain medium components and table 10 lists the components in MEM depleted by the growth of human fibroblasts. Provided other factors are not limiting, replenishment of these components leads to continued cell growth. Figure 32 illustrates that a stock solution containing these components can be added to microcarrier cultures of chicken fibroblasts and cell yields can be as good as those achieved when complete medium is used for replenishment. This method provides for greater economy bur depends on good control of pH and definition of limiting nutrients. Table 10. Depletion of nutrients from MEM by human diploid fibroblasts (MRC-5). The data show the expected sequence of depletion in the presence of 10% (v/v) foetal calf serum and are taken from Lambert, K. and Pirt, S.J. J. Cell. Sci. 17 (1975) 397-411. Concentration in MEM
Expected yield (cells/ml)
Glutamine Cystine Choline HCI Glucose Inositol Pyridoxine
0.292 mg/ml 0.094 mg/ml 1 mg/ml 2 mg/ml 2 mg/ml 1 mg/ml
2.05x105 2.15x105 2.53x105 4.04x105 5.44x105 7.90x105
Cells/ml
Nutrient
7
10
6
10
5
10
1
3
5
7 Days
Fig. 32. The depletion of medium components during growth of secondary chicken fibroblasts in microcarrier culture. Cells were cultured in DME supplemented with 15 mM HEPES. 5% (v/v) calf serum, 1% (v/v) chicken serum and 1% (w/v) tryptose phosphate broth and containing Cytodex 1 microcarriers (5 mg/ml). After 3 days the culture medium was replaced by fresh medium (—❍—) or was removed and supplemented with cystine (30 µg/ml), glutamine (0.3 mg/ml), inositol (2 µg/ml), glucose (2 mg/ml), choline HCl (1 µg/ml) and 1% (v/v) calf serum, 1% (v/v) chicken serum. The medium was well mixed and returned to the culture (—▲—). Control cultures were not refed (—●—). Replenishment of all the medium was necessary after 7 days if the microcarriers were to be kept confluent. (Clark, J., Hirtenstein, M. Annals N.Y. Acad. Sci. 369 (1981) 33, by kind permission of the authors and publisher.)
63
3
m
-6
Max cell yield (cells/ml x 10 )
4
2
1
0.2
0.4
0.6
0.8
1.0 VM/VT
Fig. 33. The effect of culture volume and headspace volume on cell yields from a closed microcarrier culture system. Cultures were contained in traditional magnetic spinner vessels (Bellco) with a total internal volume (VT) of 500 mls. Cultures of various volumes (VM) were inoculated with Vero cells (8x104 cells/ml) and stirred at 50–60 rpm and cell yield was determined at the plateau stage of the culture cycle (day 8). The culture vessels were sealed and briefly gassed with 95% air: 5% CO2 every day. 50% of the culture medium was changed on day 3 and day 6. (Hirtenstein, M., Clark, J.m., Gebb, Ch., Develop. Biol. Standard (1981), in press, by kind permission of the authors and publisher.)
All these aspects of medium replenishment are applicable to large scale microcarrier culture. An advantage with large scale cultures is that culture parameters such as pH and gas tensions are better controlled than with small scale closed culture systems and it is thus easier to optimize replenishment schemes. Closed culture systems are frequently used for microcarrier culture at laboratory scale. With such systems the vessels are sealed and the supply of gas is only renewed when the culture is opened for sampling or replenishment of the medium. One important aspect to consider when working with such culture systems in the ratio of the culture volume to the total internal volume of the culture vessel. Fig. 33 illustrates that the extent to which a spinner vessel is filled with culture influences greatly the maximum yield of cells/ml from the culture. Therefore, for reproducible results closed culture vessels should always be filled to the same extent and not more than half full. The reduction in cell yield in closed culture vessels which are more than half full is probably due to a decreased supply of oxygen and reduced headspace volume for buffering the usual CO2-bicarbonate system. This phenomenon is not encountered with open culture systems having a continuous gas supply. More information on culture media, gas and pH control can be found in sections 4.1, 4.3 and 4.4 respectively.
64
3.5.3 Maintaining cultures at confluence Production from microcarrier cultures sometimes requires that monolayers of cells are maintained at confluence on the microcarriers for several days or even weeks. Fibroblast interferon, certain viruses and urokinase are examples of culture products where viable and functional monolayers of cells must be kept on the microcarriers for extended periods. Mered et al (3) describe the maintenance of monolayers of chicken embryo fibroblasts and Vero cells on microcarriers for periods of one month or more without detachment or loss of viability. Dog kidney cells can be maintained on the microcarriers for periods of up to 38 days during rabies vaccine production (48). Similar procedures are used to maintain all types of cells at confluence on Cytodex and these procedures are the same as those used to maintain confluence in other monolayer culture systems. Cells which are contact-inhibited for proliferation (e.g. primary cells, diploid cell strains and several established cell lines) require stable culture conditions which promote viability and function of the quiescent population of cells. Such cells often form monolayers which become only weakly attached to culture surfaces when the saturation density is achieved. A typical example is the tendency for highly confluent monolayers of chicken embryo fibroblasts to detach from Petri dishes, roller bottles or microcarriers. This phenomenon can be prevented by careful control of pH, a reduced supply of serum supplements and by a consistent supply of fresh medium. For cell types which do not show contact-inhibition of proliferation. A reduction in the formation of multilayers of cells on the microcarriers and must also be accompanied by careful control of pH. Multilayers of cells growing on the microcarriers (plate 5) are very sensitive to changes in culture conditions and may detach after only small fluctuations in pH or nutrient supply. The following are general points which should always be considered when maintaining microcarrier cultures at confluence. • pH. It is most important to maintain optimum culture conditions, especially with respect to pH. Once a drift in pH occurs (usually a decrease) cells will tend to detach even after the pH has been returned to the optimal level. The most common cause for a decrease in pH at the later stage of the culture cycle is an accumulation of lactate (section 4.4.3). • Osmolarity. When pH is being controlled by addition of acid/base or buffers it is important to avoid changes in osmolarity (section 4.5). • Serum concentration. The most usual way of maintaining cultures at confluence is to reduce the concentration of the serum supplement (section 4.2.2). A reduction from the usual 5-10% (v/v) supplement to 2-5% (v/v) is required for cells which are contact inhibited for proliferation. For cells which continue to divide after confluence has been achieved lower concentrations of serum should be considered (down to 0.5% v/v). • Medium replenishment. The concentration of nutrients should be kept as constant as possible and toxic products of metabolism should not be allowed to accumulate. Thus a consistent supply of medium should be ensured and daily replacement of 10-20% of the medium usually gives the best results.
65
For many cell types depletion of medium components is not as rapid at confluence as during earlier stages of the culture cycle and the main function of the medium replenishment is to control pH. Temperature shocks should always be avoided when replenishing the medium and all solutions should be prewarmed to the culture temperature. Daily addition of low concentrations of glutamine instead of occasional addition of medium with high concentrations of glutamine avoids unnecessary accumulation of ammonia, a toxic product of glutamine decomposition (120,194). Hence glutamine-free medium and daily addition of approximately 0.1-0.2 mM glutamine assists in maintenance of viable monolayers by providing a more constant level of this essential amino acid without excessive accumulation of ammonia. If cultures are not contact inhibited for proliferation and continue to divide after confluence, higher levels of glutamine in the presence of low concentrations of glucose will assist in maintaining pH (section 4.6.3). • Antibiotics. When possible the concentration of antibiotics in the culture medium should be decreased for long-term maintenance of confluent cultures (section 4.7). Details on the control of stirring speed and gas tensions can be found in sections 3.5.1 and 4.4 respectively.
3.6 Monitoring the growth of cells and microscopy 3.6.1 Direct observation by microscopy Examining cells by microscope is a vital part of microcarrier culture technique. For routine observation the growth and condition of the cells can be assessed simply with phase contrast optics. A small sample of evenly suspended culture is placed on a microscope slide and a coverslip is gently lowered over the sample. To avoid crushing the microcarriers the coverslip should come to rest slightly above the slide and this can be accomplished by placing small pieces of broken coverslip between the slide and the coverslip. The formation of haloes occasionally occurs with phase optics and can be avoided by increasing the refractive index of the medium, for example by the addition of serum or Ficoll® 400 (as an isotonic 30% (W/v) stock). If permanent preparations are required the sample must be fixed (section 3.6.4) and can then be stained (section 3.6.5). Quantitation of cell growth can be achieved by counting cells attached to individual microcarriers but this is generally too time-consuming to be useful for routine purposes. More efficient and rapid methods for determining cell number are described below.
66
3.6.2 Counting cells released after trypsinization A 1 ml sample of evenly suspended culture is placed in a test tube and after the microcarriers have settled the supernatant is removed and the microcarriers are briefly washed in 2 ml of Ca2+, Mg2+-free PBS containing 0.02% (w/v) EDTA, pH 7.6. When the microcarriers have settled this solution is decanted and replaced by 1 ml of a1:1 mixture of 0.25% (w/v) trypsin in Ca2, Mg2+-free PBS and EDTA (0.02%, w/v) in Ca2, Mg2+-free PBS. The pH of this mixture should be 7.6. The tube is incubated at 37°C for 15 min. with occasional agitation. The microcarriers are allowed to settle and the supernatant is transferred to another test tube. The microcarriers are washed with 2 ml culture medium containing serum (5-10%, v/v) and the supernatant is pooled with the first supernatant. The cell suspension is centrifuged (300 gav, 5 min., 4°C), the supernatant is discarded and the pellet is resuspended in 2 ml Ca2+, Mg2+-free PBS containing 0.05% (w/v) trypan blue. The concentration of cells in the suspension can be counted in a haemocytometer or electronic counter and the concentration of the cells in the culture can be expressed per ml or per cm2 of microcarrier surface area (see table 4). Including trypan blue in the re-suspending solution allows estimates of cell viability to be made at the same time (section 3.7.9). A similar method can be used when using collagenase in combination with Cytodex 3 microcarriers (section 3.7.2).
3.6.3 Counting released nuclei A simpler way of monitoring cell growth is to count released nuclei as described by Sanford et al (105). In this method, modified by van Wezel (32), cells growing on the microcarriers are incubated in a hypotonic solution and nuclei released by lysis are stained by a dye in this solution. A 1 ml sample of evenly suspended culture is centrifuged (22 gav, 5 min.) and the supernatant is discarded. The pelted microcarriers are resuspended in 1 ml 0.1 M citric acid containing 0.1% (w/v) crystal violet. The contents of the tube are mixed well (e.g. with a “Whirlimixer” or by several traverses over a corrugated surface) and then incubated for 1 hr at 37°C. Evaporation of the contents of the tube must be avoided by using either a humidified incubator or by sealing the tube with plastic film. After incubation the contents of the tube are mixed as above and the released stained nuclei are counted with a haemocytometer. The microcarriers in the sample do not interfere with the counting. The samples can be stored for up to one week at 4°C. This method of determining the number of cells in the culture is most accurate when cultures are evenly suspended and when culture conditions have avoided aggregation of microcarriers and cells (section 5).
3.6.4 Fixing cells When fixation and staining are necessary, e.g. for preservation of samples, cytochemistry, electron microscopy etc., any of the usual cell culture fixation and staining procedures can be used with Cytodex microcarriers.
67
Plate 1: Top: Chicken embryo skeletal muscle cells (myoblasts) 2 days after inoculation onto Cytodex.
Bottom: Chicken embryo skeletal muscle cells (myoblasts) 7 days after inoculation onto Cytodex. At this stage the microcarrier is confluent and the myoblasts have fused to form myotubes. (Original photographs by Przybylski, R., Pawlowski, R., Loyd, R., Department of Anatomy, Scool of Medicine, Case Western Reserve University, Clevland, OH 44106, USA; work supported by the Muscular Dystrophy Assosiation and National Institutes of Health. Reproduced by kind permission.)
68
Plate 2: Top: A confluent monolayer of mouse brain capillary endothelial cells on Cytodex 3. Bottom: Bovine pulmonary artery endothelial cell growing on Cytodex 3. (Original photographs by Busch, C., Department of Pathology, University of Uppsala, Uppsala, Sweden. Reproduced by kind permission.)
69
Plate 3: Top: Demonstration of insulin synthesis by immunostaining of foetal rat pancreas cell growing on Cytodex 3. (Original photographs by Bone, A., Swenne, I., Department of Medical Cell Biology, Biomedical Centre, Uppsala, Sweden. Reproduced by kind permission.) Plate 4: Bottom: Scanning electron micrograph of human lymphoblastoid cells proliferating on Cytodex. (Original photographs by Christie, W., Gallacher, A., MRC Clinical and Population Cytogenetics Unit, Western General Hospital, Edinburgh EH4 2XU, Scotland. Reproduced by kind permission.)
70
Plate 5: Top: Human cervical carcinoma cells (HeLa) growing on Cytodex microcarriers 3 days after inoculation. Note pronounced epithelial morphology. Bottom: Confluent microcarrier culture of human glioma cells. The cells do not exhibit contact inhibition of proliferation and hence multilayers form at confluence. (Original photographs by Pharmacia Fine Chemicals, Uppsala, Sweden. The glioma cells were kindly supplied by K. Nilsson, Wallenberg Laboratory, Uppsala, Sweden.)
71
Plate 6: Top: Monolayers of diploid human embryo fibroblasts (MRC-5) on Cytodex microcarriers 7 days after inoculation. Bottom: A confluent culture of human kidney cells (Flow 4000/Clone 2) on Cytodex 3. (Original photographs by Pharmacia Fine Chemicals, Uppsala, Sweden.)
72
Plate 7: Examples of three common types of cells in microcarrier culture. Top left: Chicken embryo fibroblasts in motion 24 hours after inoculation. Top righ: Confluent monolayers of chicken embryo fibroblasts. Bottom left: Chinese hamster ovary cells (CHO) 4 days after inoculation, culture density 7x106 cells/ml. Bottom righ: Diploid human foreskin fibroblasts (FS-4). These cells are often used for interferon production in microcarrier cultures (see refs 51, 58). (Original photographs by Tyo, M., Southern Biotech Inc., 3500 E. Fletcher Ave., Suite 321, Tampa, FL 33612, USA. Reproduced by kind permission.)
73
Plate 8: Scanning electron micrographs of pig kidney cells growing on Cytodex in cultures used for the production of foot-and-mouth disease vaccine. (Original photographs by Megnier, B., Tektoff, J., IFFA-Mérieux, 254 rue Marcel Mérieux, Lyon, F-69342 France. Reproduced by kind permission.)
74
Plate 9: A predominantly epithelial monolayer of primary dog kidney cells growing on Cytodex. (Original photographs by Pharmacia Biotech Ab.)
75
The most common fixatives are methanol and ethanol. When using such alcohol’s maximum preservation of cell morphology can be achieved by first rinsing the cells and microcarriers with warm PBS and then pre-fixing in 50% (v/v) alcohol in PBS. After 10 min. the 50% alcohol is replaced by two 10 min. changes of cool 70% v/v) alcohol in PBS. Finally this fixative can be replaced by 70% (v/v) alcohol in water Alternatively, a modified Carnoy’s fixative (3 parts methanol, 1 part glacial acetic acid and containing 2% (v/v) chloroform) can be used after the cells and microcarriers have been rinsed in PBS. Better preservation of morphology will be achieved when aldehyde fixatives are used. Either 10% (v/v) formalehyde in PBS or 2-5% (v/v) glutaraldehyde in PBS can be used and the material should be fixed overnight at 4°C. Glutaraldehyde fixation results in the best preservation of morphology and the fixed material can be used for electron microscopy studies (plate 1). Further processing of the fixed cells attached to the microcarriers depends on the purpose of the study. For example, using standard procedures the material can be dehydrated in a graded series of alcohol solutions, cleared in xylene and embedded in paraffin (14). For electron microscopy cells growing on the microcarriers can be fixed, embedded, sectioned and stained by the usual procedures. Dehydration of the microcarriers in acetone instead of alcohol avoids the use propylene oxide which has been reported to alter the surface of microcarriers (55).When sections through cells attached to a solid surface are required, cells growing on Cytodex microcarriers are easier and more convenient to process than cells growing on the surface of Petri dishes or coverslips. When processing the microcarriers with cells attached for microscopy it must be remembered that the times taken for each step should allow for penetration of the microcarrier matrix by the solute or embedding agentdoubling the usual process times for embedding ensures good penetration of the matrix. Examples of transmission and scanning electron microscopy of cells attached to Cytodex can be seen in figures 1 and 3 and plates 1, 4, 8. Pawlowski et al (61) used the following procedures for preparation of the cells in plate 1. Cytodex with cells attached was allowed to settle onto coverslips coated with gelatin (1%) and fixed in half-strength Karnovsky’s fixative (4% glutaraldehyde, 1% paraformaldehyde, 0.1 M sodium cacodylate buffer, pH 7.8 and 12 mg CaCl2/ 100ml) for 30 min. The microcarriers were then washed briefly in 0.1 M cacodylate buffer (pH 7.8) and post-fixed in 1% osmium tetroxide for 1 hour. The samples were dehydrated in a graded ethanol series and critical point dried with carbon dioxide. The coverslip were “sputter-coated” with gold for 1.5 mins at 25 mA and 1.5 kV (61; R. Przybylski, pers. comm., 193)
3.6.5 Staining cells The most suitable routine procedures for staining cells growing on Cytodex microcarriers use either Geimsa stain or Harris’ haematoxylin. The latter stain can be used when better nuclear detail is required.
76
Staining with Giemsa stain Microcarriers with cells attached are rinsed in a small volume of warm PBS. Fixation in 50% (v/v) methanol in PBS for 10 min. is then followed by dehydratration in a cool graded methanol series (70, 90, 95% v/v solutions in PBS) to absolute methanol, with about 5 min. at each concentration. The material is stained for 5 min. in May-Grünwald’s stain (alternatively Jenner’s or Wright’s stain) and for a further 10 min. in dilute Giemsa (1:10 volumes in distilled water). A brief rinse in water will educe staining to the required intensity. Staining with Haematoxylin The Cytodex with cells attached is rinsed in a small volume of warm PBS. After fixation in 50% (v/v) methanol PBS for 10 min the microcarriers are fixed for a further 10 min in cool 70% (v/v) methanol in PBS. The fixative is removed and 10 ml distilled water containing 2-3 drops of haematoxylin are added. The material is left overnight at room temperature and then rinsed in tap water for 20 min. If desired, the cells can be counter-stained in an aqueous solution of eosin-Y for 30 sec and for permanent storage the material can be dehydrated in a series of alcohol solutions (50, 70, 90, 95% v/v solutions in distilled water) and two changes of absolute alcohol. The material can then be cleared in xylene and mounted. Many other more specific staining procedures are possible. However, it should be noted that because of the nature of the carbohydrate matrix of the microcarriers, it may not be possible to use some carbohydrate-specific stains. Some protein-specific stains will also stain the collagen layer on Cytodex 3. When staining dense monolayers of cells it may be necessary to use slightly longer times for rinsing. This will ensure that free stain is washed from the microcarrier matrix. When mounting the microcarriers for examination it is important that they are not crushed by the glass coverslip. This can be avoided by raising the coverslip above the surface of the slide with small fragments of broken coveslips placed on the slide. Plate 3 shows an example of immunostained cells attached to Cytodex microcarriers.
3.7 Harvesting cells and subculturing Removal of cells from microcarriers is usually required when subculturing and scaling-up, and also when large numbers of cells are required for biochemical analyses. However, it is important to note that for many biochemical studies, e.g. isotope incorporation studies, it may not be necessary to remove the cells from Cytodex. The only precaution required is that the microcarriers are well washed with buffer and then precipitation agent (usually 5-10% trichloroacetic acid) so that all un-incorporated isotope is washed from the microcarrier matrix.
77
Various methods can be used to remove cells from Cytodex and it is important to choose procedures which minimize damage to cells during harvesting. In most cases the usual cell culture methods are used with only minor modifications to standard techniques. The type of harvesting procedure can usually be deduced from that normally used to harvest a particular type of cell from other types of monolayer culture. Enzymatic methods for removing cells from the microcarriers (section 3.7.2) are most commonly employed. Since cells are less strongly attached at alkaline pH it is useful to use solutions with pH 7.6 to ensure that all acidic culture medium is removed prior to harvesting. Unless otherwise stated all solutions should be pre-warmed to 37°C.
3.7.1 Chelating agents Chelating agents such as EDTA can be used to remove certain epithelial and transformed cells from Cytodex. After removing and discarding the medium, the microcarriers are washed twice in Ca2+, Mg2+-free PBS containing 0.05% (w/v) EDTA (100 ml/g Cytodex). The microcarriers are then incubated at 37°C with fresh Ca2+, Mg2+-free PBS containing 0.05% (w/v) EDTA (approx. 50 ml/g Cytodex). The mixture should be stirred continuously in the culture vessel (fig. 20) at approximately 60 rpm for at least 10 min. The stirring speed may need to be increased or aspiration with a pipette may be required for some types of cell. When the cells have detached from the microcarriers the EDTA is neutralized by adding culture medium (100 ml/g Cytodex). The detached cells can be separated from the microcarriers as described in section 3.7.8. In general, chelating agents alone are not sufficient for removal of most cell types and are therefore usually used in combination with protoelytic enzymes. Long periods of exposure to EDTA may be harmful to some fibroblast strains and this type of cell is rarely removed by EDTA alone.
3.7.2 Proteolytic enzymes Enzymes are normally used for routine harvesting of a wide variety of cells from Cytodex microcarriers. In general trypsin, VMF Trypsin (Worthington), Pronaseâ (Sigma) or Dispaseâ (Boehringer) are used with Cytodex 1, Cytodex 2 and Cytodex 3 and in addition collagenase is used in combination with Cytodex 3. Trypsin is the most commonly used general protease, although Pronase has advantages for harvesting cells from primary cultures and Dispase can be used for cells which are sensitive to trypsin. Trypsin The stirring is stopped and the microcarriers are allowed to settle. The medium is drained from the culture and the microcarriers are washed for 5 min. in Ca2+, Mg2+free PBS containing 0.02% (w/v) EDTA, pH 7.6. The amount of EDTA-PBS solution should be 50-100 ml/g Cytodex. The EDTA-PBS is removed and replaced by trypsin-EDTA and incubated at 37°C with occasional agitation. After 15 min. the action of the trypsin is stopped by addition of culture medium containing 10% (v/v)
78
serum (20-30 ml medium/g Cytodex). An alternative method for inactivating the trypsin is to add soybean trypsin inhibitor (0.5 mg/ml). Chicken serum does not contain trypsin inhibitors. Any cells remaining on the microcarriers at this stage can be removed by gentle agitation. The detached cells can be separated from the microcarriers as described in section 3.7.8. The success of harvesting with trypsin depends on complete removal of medium and serum from the culture and the microcarriers before the trypsin is added (serum contains trypsin inhibitors). The pH is critical when harvesting with trypsin and care must be taken to ensure that harvesting is done between pH 7.4 and 8.0. It is important to expose cells to trypsin for as short a period as possible. For sensitive types of cells trypsinization at 4°C may be preferable (156) but the advantages of such a procedure must be weighed against the increased time for detachment and the occasional tendency for aggregation. With some types of cell, attachment to Cytodex is very strong, e.g. FS-4 human fibroblasts and an additional wash with EDTA-PBS is required before the EDTAtrypsin is added. Detachment of cells from the microcarriers can be enhanced by continuous stirring in the enzyme solution at a speed slightly greater than that used for normal culture (section 3.5.1). Trypsin-EDTA solution: The solution can be prepared in Ca2+, Mg2+-free PBS but the following solution is preferred for retention of maximum cell viability. NaCl KCI Na2HPO4 Glucose
122 mM 3.0 mM 1mM 4.0 mM
Phenol red 3.3 mM EDTA 0.02% (w/v) Tris (hydroxymethyl) aminomethane 2% (w/v) pH 7.8-8.0
To this solution is added trypsin at the usual concentration used for a given type of cell and for most cells 100 mg trypsin/ml is sufficient. Strongly adhering cells such as FS-4, may require 500 mg trypsin/ml. Trypsin solutions can be sterilized by filtration though a 0.2 mm sterile filter. Since trypsin solutions are subject to self-digestion, it is important to divide freshly prepared solutions into small aliquots and store frozen until required. Crude trypsin solutions have high content of DNA and RNA (106) and therefore pure recrystallized enzyme is preferred for many biochemical and somatic cell genetics studies. Crude trypsin also shows large batch-batch variation in toxicity and difficulties with cell growth can often be traced to a specific batch of trypsin. When possible, new batches of trypsin should be tested for toxicity. The procedures for harvesting cells with Dispase or Pronase are similar to those used for trypsin. The activity of Dispase is not inhibited by serum and thus harvesting must be accompanied by thorough washing of the cells. Collagenase Standard cell culture harvesting procedures using trypsin and chelating agents alter cell viability and remove large amounts of surface-associated molecules from the 79
cells (107-110). If subsequently studies require intact cell membranes or if rapid harvesting with maximum yields is required without impairment of cell viability then an alternative method of harvesting the cells must be used. The use of collagenase to harvest the cells growing on Cytodex 3 provides such a method and with this method the enzyme digests the culture surface rather than the surface of the cell. Thus cells harvested with collagenase are generally more viable and have greater membrane integrity than those harvested with trypsin. Harvesting cells from Cytodex 3 with collagenase is the method of choice when using the cells to start cultures at low densities. Collagenase is a proteinase with a high degree of specificity for collagen (111) and can be used for the rapid harvesting of cells from collagen-coated surfaces. For example, Michalopoulos and Pitot (86) reported easy and rapid harvesting of hepatocytes from collagen-coated surfaces and Sirica et al (87) obtained 100% recovery of rat hepatocytes from a collagen surface within 10 minutes. In addition to simplifying harvesting of cells from cultures, a combination of collagenase and collagen-coated surfaces can be used for the selective removal of different cell types (78,112). The rate of release of cells from the collagen in the presence of collagenase depends on cell type, with fibroblasts generally being released more rapidly than epithelial cells. The procedure for harvesting cells with collagenase is as follows. The stirring is stopped and the microcarriers are allowed to settle. The medium is drained form the culture and the microcarriers are washed for 5 min. in two changes of Ca2+, Mg2+-free PBS containing 0.02% (w/v) EDTA, pH 7.6 (50 ml PBS/g Cytodex 3). Standard PBS can be used instead for this step if chelating agents are to be avoided. The PBS is removed and replaced by collagenase solution (see below). Approximately 30-50 ml of this solution should be used per g Cytodex 3. The microcarriers are then mixed well in the collagenase solution and incubated with occasional agitation at 37°C. After approximately 15 min. the collagenase solution is diluted with fresh culture medium (50 ml medium/g Cytodex 3) and any cells remaining on the microcarriers are dislodged by aspiration with a pipette or by gentle agitation. The detached cells can be separated from the microcarriers as described in section 3.7.8. Collagenase requires Ca2+ and Mg2+ and therefore chelating agents should not be used during the harvesting step. When using the procedures described in section 3.7.8, steps directed at inactivating the collagenase are not usually required. the dilution factor and cysteine in the medium are sufficient to reduce the enzyme activity. If collagenase must be removed completely, washing cells by centrifugation is the most convenient method. Collagen solution: The solution should be prepared in PBS or Krebs II buffer (47)and sterilized by filtration through a 0.2 mm sterile filter. Collagenase is usually used at a concentration of 100-500 mg/ml.
3.7.3 Hypotonic treatment Incubation in hypotonic solution can be used for harvesting cells which do not have strong adhesion properties, e.g. some established and transformed cell lines. The osmotic shock associated with the hypotonic solution causes the cells to adopt 80
rounded morphology and they can then be shaken from the microcarriers. The Cytodex microcarriers with cells attached are washed twice in hypotonic saline (8 g NaCl, 0.4 g KCI, 1 g glucose in 1 liter distilled water) and incubated in fresh hypotonic saline (50 ml/g Cytodex) at 37°C for 15 min. with gentle agitation. Lai et al (70) used hypotonic treatment to harvest CHO cells from Cytodex 1 microcarriers. Cell recoveries are usually less with this method than when enzymes are used. An advantage however of harvesting with hypotonic saline is that the harvesting does not involve exogenous protein.
3.7.4 Cold treatment Incubation at low temperatures causes many types of cells to detach form culture surfaces. Cytodex microcarriers with cells attached can be incubated in culture medium without serum at 4°C for 8 h and a significant proportion of cells will detach from the microcarriers. The sudden fall in temperature associated with a change from warm to cold culture medium often leads to a more rounded cell morphology and the cells can then be gently shaken from the microcarriers. In general the use of temperature shifts for harvesting cells is associated with low viability and this method is best reserved for established cell lines when other methods are not desirable.
3.7.5 Sonication Sonication alone cannot be used for harvesting intact cells from Cytodex microcarriers. In combination with the methods above low intensity sonication can be used to increase cell yields. Sonication can be used to rupture cells and leave membrane fragments attached to intact microcarriers (71;S. Smit, pers. comm., 191).
3.7.6 Lignocaine for harvesting macrophages Some cells are extremely difficult to remove from culture surfaces e.g. macrophages. Although the methods described above can be used to harvest macrophages they are usually associated with poor recovery and low viability. An alternative is to use 30 mM lignocaine in PBS ( pH 6.7) for 15 min. at 22°C (113).
3.7.7 Modifications to harvesting procedures for large scale cultures The above procedures have been described for small scale cultures. In principle exactly the same procedures are used for large scale cultures although in certain situations modifications may be necessary, Modifications are usually associated with attempts to obtain maximum recovery of cells when processing large volumes of concentrated suspensions of microcarriers. Van Wezel et al (40) describe the use of a trypsinization apparatus for harvesting primary monkey kidney cells from Cytodex 1. This apparatus is based on a Vibromixer (Model El, Chempec. Inc.). Spier et al (114) describe the use of a narrow-bore tube for stripping cells from microcarriers. Using a 3.5 cm long capillary tube with a bore of 1.2 mm it was possible to obtain greater than 90% recovery of cells from the microcarriers (114). Further information on procedures for harvesting cells in specific large scale culture situations can be obtained from Pharmacia Biotech.
81
3.7.8 Separating detached cells from microcarriers There are several methods for separating detached cells from Cytodex. • Differential sedimentation. Recovery of cells by differential sedimentation takes advantage of the fact that cells and microcarriers sediment at different rates. For routine harvesting and subculturing when maximum recovery is not required differential sedimentation is the most simple way of obtaining a preparation of cells essentially free from microcarriers. After completion of the harvesting steps described above culture medium is added (50-100 ml/g Cytodex) and the microcarriers are allowed to settle. After approximately 5 min. the culture vessel is tilted to 45°C and the cells can be collected into the supernatant. Better recovery can be achieved if the microcarriers are washed one more time with medium and the supernatant collected. The pooled supernatants can be used directly to inoculate the next culture. Alternatively, the products of the harvesting steps can be transferred into a narrow container with a high head, e.g. test-tube or measuring cylinder. After 5 min. the microcarriers settle to the bottom of the container and the cells can be collected in the supernatant. Using these techniques it is possible to recover more than 80% of cells in the harvest suspension. A short period of centrifugation (200 g av, 2 min.) can be used to hasten sedimentation of microcarriers. If greater recoveries are required then filtration should be used. • Filtration. Filtration can be used when it is important to obtain very high recoveries of harvested cells without contamination from microcarriers. Any sterilizable filter with a mesh of approximately 100 mm which is non-toxic for animal cells is suitable (e.g. nylon or stainless steel filters). Sintered glass filters may also be used, however full recovery of cells may not be possible with such a filter. A filter which is convenient to use for small scale work is the “Cellector” supplied by Bellco Glass inc. (Vineland, NJ, USA) or similar filter and holder supplied by Cell-Rad (Lebanon, PA, USA). Alternatively, suitable nylon net can be obtained from Zurich Bolting (Rüschlikon, Switzerland) or Small Parts inc. (Miami, FA, USA). • Density gradient centrifugation. Provided there exists a difference in density between the cells and the microcarriers density gradient centrifugation can be used to obtain a preparation of cells free from microcarriers. Manousos et al (55) used discontinuous density gradient centrifugation in Ficoll/Hypaque (density 1.077 g/ml) to achieve an efficient separation with no contamination of the cells by microcarriers. Ficoll-Paqueâ from Pharmacia Biotech is supplied sterile ready for use and can be used for this separation.
3.7.9 Measurement of cell viability Exclusion of dyes provides a convenient measure of cell viability (115). Trypan blue is the dye most commonly used since it can be used with both living material and also material fixed with glutaraldehyde. Trypan blue is the only dye to give reproducible results both before and after fixation.
82
Trypan blue solution is prepared in PBS (4 mg/ml). Approx. 0.9 ml of diluted cell suspension is mixed with 0.1 ml trypan blue solution. After 5 min. at room temperature the viable (unstained) and non-viable (stained) cells are counted in a haemocytometer. This counting can be done in connection with determination of cell concentration. Staining tests should be performed at pH 7.3-7.6.
3.7.10 Subculturing techniques Subculturing cells from one microcarrier culture to another usually involves the steps discussed in section 3.4, 3.5 and 3.7. Cells harvested from one microcarrier culture can be used directly to inoculate the next culture containing fresh microcarriers. Transfer of a few microcarriers in the inoculum from the previous culture has no effect on subsequent culture development. For one subculture cycle it is possible when scaling-up to harvest cells from the microcarriers with trypsin, inactive the trypsin with medium containing serum and then to add fresh microcarriers. In this way the culture contains old and new microcarriers and procedures outlined in section 3.4 are used with corresponding increases in culture volume to maintain a constant concentration of microcarriers. The yield from such cultures is less that obtained when only new microcarriers are used. It is not possible to use this method when using enzymes to harvest cells from Cytodex 3. An alternative potential method for scaling up is to simply add fresh microcarriers when the culture approaches confluence. Manaus et al (55) demonstrated that addition of a further 1 mg of microcarriers/ml of culture could be used to lengthen the life of RD cell cultures and to improve production of oncornavirus. The success of this method of scaling-up depends on the ability of cells to move from the confluent microcarriers and to inoculate the microcarriers. Culture conditions need to be adjusted such that the chance of such a transfer is maximized. In the case of MRC-5 human fibroblasts, static periods of culture with intermittent stirring to avoid aggregation, are required before signigncant inoculation of the new microcarriers can occur (P. Talbot, pers. comm., 188). A reduction in the calcium concentration of the culture medium can be used to facilitate the transfer of cells between microcarriers (6). Horst et al (72) described the use of Cytodex 1 for subcultivation of cells without the use of harvesting procedures. In these experiments mouse fibroblasts migrated in static cultures from monolayer surfaces onto the microcarriers. More information on proteolytic enzyme-free subcultivation can be found in section 1.4.4.
83
3.7.11 Re-use of Cytodex Using microcarriers for more than one culture/harvest cycle is not recommended. The re-use of surfaces for cell culture requires alternate washing in strongly acidic and basic solutions. These washing steps are required in order to remove the debris remaining after the harvesting steps. The use of such procedures for washing Cytodex 1 is not recommended since the extreme of pH may alter both the microcarrier matrix and the degree of substitution. Used microcarriers can be washed in sterile PBS directly after harvesting and used for a further culture step, but attachment of cells is poor and yields are less than 70% of those obtained with fresh microcarriers. Re-use of the microcarriers for a third culture step has not been feasible with all cell types tested. For some cell strains re-use of the microcarriers is impossible. Cytodex 3 microcarriers cannot be re-used when the cells have been harvested by enzymatic methods.
84
4. General considerations 4.1 Culture media 4.1.1 Choice of culture medium A wide variety of different media can be used for the culture of any given type of cell. If culture medium support the growth of a particular type of cell in other culture systems then it will usually support the growth of cells on Cytodex microcarriers. Therefore, when selecting a medium for microcarrier culture the most suitable starting point is to use the medium which has previously been reported to support the growth of the particular type of cell. Once the basic procedures for growing a particular type of cell in microcarrier culture have been resolved the medium may need to be modified if maximum yields are to be obtained. Such modifications may be necessary simply because the microcarrier culture cycle usually spans a wide range of culture densities and the supply of nutrients must take into account the different requirements for growth at different culture densities. A more rich medium is often needed for the initial stages of a microcarrier culture and especially if low cell densities are used. In such cases the cells must survive under almost cloning conditions with only a few cells/cm2 and stirring eliminates the formation of “micro environments” which occurs in static monolayer cultures. Since the cells have very little conditioning effect on the medium at this stage of culture one way of improving growth is to use a medium which contains components important for growth at low density. These components are particularly important for cells with low plating efficiencies (section 3.4.6). The culture medium must have a large reserve of essential nutrients in order to support the growth of cells towards the end of the culture cycle when more than 106 cell/ml are usually present (section 3.5.2). Figure 34 illustrates that different media support the growth of cells in microcarrier culture to different extents. When cultures are initiated at a low density, Medium 199 often results in higher plating efficiencies and gives better yields than either DME or BME. In contrast, DME supports the growth of cells to much higher densities than BME or Medium 199; presumably because the concentrations of certain essential amino acids and vitamins are several-fold greater in DME than in the other media (44). Table 11 present a list of media which can be considered for culturing cells of different species. Although this list is far from complete and can be influenced by the tissue of origin of the cells, it is usually the case that a medium based on DME is suitable for the microcarrier culture of most types of cells. Section 6.1 lists the types of cells cultured on Cytodex microcarriers and a major proportion were cultured using DME as the base medium. In order to improve the growth of cells during the early stages of the culture cycle the DME can be supplemented with components which improve plating efficiency and growth at low densities (section 3.4.6). A medium which is recommended for general microcarrier culture is shown in table 12. This medium is based on DME and should be used if other formulations are not known to be more appropriate. 85
Cells/ml
Fig. 34. The effect of various culture media on the growth of monkey kidney cells (Vero) on Cytodex microcarriers. (DME, ●; BME, ❍, Medium 199, ). All media were supplemented with 10% (v/v) foteal calf serum. Cultures contained 5 mg Cytodex 1/ml and were stirred at 60 rpm for the culture period. In all experiments 50% of the medium was changed on day 3. (Clark, J.M., Hirtenstein, M.D., Annals N.Y. Acad. Sci. 369 (1981) 33, by kind permission of the authors and publisher).
7
10
6
10
5
10
10
4
3
1
5
7 Days
Table 11. Examples of culture media which can be used for cells of different species. The list is not exhaustive but covers the most commonly used media. The media are supplemented with serum and often with non-essential amino acids if not already present in the formulation. See Ham and McKeehan (44) for more information on formulations and section 6.1 for information on culture media used for microcarrier culture of specific types of cells. General
Monkey Human
Rat Rabbit
Mouse
Chicken
Hamster
MEM DME 199 F12K MCDB 202
MEM DME 199 BME L15 5a RPMI 1640 CMRL 1969 MCDB 104 MCDB 202 IMEM-Z0
MEM 5a F12 F12K MCDB 104
DME CMRL 1415 MCDB 202 MCDB401
DME 199 F12K MCDB 202 F10 5a
DME F10 F12
86
Table 12. A general purpose medium for high yield microcarrier culture.
Inoculation medium (section 3.4.6) DME* 10-4M alanine 10-4M asparagine 10-4M aspartic acid 10-4M glutamic acid 10-4M proline 10-4M thymidine 3x10-7M adenine 10-5M hypoxanthine 3x10-6M pyruvate 10-3M HEPES 10-2M Serum (see section 4.2) Replenishment medium (section 3.5.2) DME HEPES 10-2M Serum (usually lower concentration than in inoculation medium, see section 4.2). A supplement of inositol 10-4M and choline 10-4 is advantageous when maintaining high culture densities at low serum concentrations. In certain cases the following components may be used to replenish the culture (fig. 32). Cystine 30 mg/ml Glutamine 0.3 mg/ml Inositol 2 mg/ml Glucose 2 mg/ml Choline HCI 1 mg/ml These additions are used only in the presence of low serum concentrations (5%, v/v, or less) and only when the pH is at a level suitable for cell survival and growth (section 4.4.1).
Maintenance medium Based on replenishment medium, occasionally with modifications (see section 3.5.3, 4.1.2, 4.3.2). * DME contains the non-essential amino acid serine.
When culturing cells with low or intermediate plating efficiency (i.e. less than approximately 30%) the basic DME medium is supplemented for the initial stages of culture until the medium is replenished. Addition of non-essential amino acids is the single most important supplement for improving plating efficiency and growth in cultures with low densities (fig. 35). Whenever cultures are initiated at densities of less than approximately 7x104 cells/ml in cultures containing 3 mg Cytodex/ml, a medium such as that described in table 12 should be used for the initial stages of culture. Modifications to the initial culture procedure will also improve the yields from the culture (section 3.4.2). Microcarrier cultures are usually replenished with the basic formulation of DME. Under conditions when very high cell densities are being cultured (more than approximately 3x106 cells/ml) or when the cultures are rapidly dividing during the later stages of the culture cycle e.g. many established cell lines) the replenishment
87
medium is supplemented with additional inositol and choline. These increased concentrations of inositol and choline are important if the replenishment medium contains only low concentrations of serum (section 3.5.2). Increasing the concentration of these components can reduce the frequency or extent of medium replenishment, provided other factors do not become limiting (section 3.5.2 and 4.4). The medium described in table 12 should be supplemented with the usual concentration of serum (section 4.2.2). Buffer systems for the control of pH are discussed in section 4.4.2.
4.1.2 General comments on components of culture media While the requirements for growth of cells in microcarrier culture are similar to those for other monolayer methods it is important to consider several components of media if the culture conditions are to be optimized. Amino acids The requirement for essential amino acids becomes larger when non-essential amino acids are not provided. The beneficial effect of non-essential amino acids is illustrated in figure 35. There is an extremely rapid utilization of amino acids during the lag phase of growth and a long lag phase will cause a reduction in the maximum cell population when amino acids are growth-limiting (180). Long lag phases are often encountered with primary cells and diploid human fibroblasts and amino acid depletion can occur at low cell densities and in the absence of exponential growth. In the case of diploid human fibroblasts growing in MEM the concentration of amino acids becomes division restricting within 72-96 h after the plating (116, table 10). Although no single amino acid may ever reach total depletion, medium replenishment is required. Cystine, glutamine, isoleucine and serine are the amino acids utilized most rapidly, even in microcarrier cultures of diploid human fibroblasts (46). It is usually these amino acids which are depleted first by a variety of types of cells. Deficiencies in supply of any one of the essential amino acids stresses cultured cells and may inhibit cell division, induce chromosome damage, and increase lysosomal activity and cell size (117-118). There is a long recovery period after such restriction (see 118). Restriction of amino acid supply is a frequent occurrence with many culture procedures and it is important to avoid limiting concentrations or imbalances in amino acid levels if high cell yields are to be achieved. One way of avoiding imbalances or wide fluctuations in the levels of amino acids (or other medium components) is to follow a strict scheme for replenishment of the medium (section 3.5.2). It is important to note that additions such as lactalbumin hydrolysate often provide unphysiological mixtures of amino acids and may even result in changes in karyotype (181). These complex mixtures should be used with caution when working with many primary cells and cells with low plating efficiencies which have not been adapted to growth in medium containing these supplements.
88
Cells/ml
10
10
10
10
Fig. 35. The effect on nonessential amino acids on the growth of monkey kidney cells (Vero) on Cytodex microcarriers. The culture media were DME supplemented with 10% (v/v) foetal calf serum (●) or DME supplemented with 10% (v/v) foetal calf serum, alaine, aspargine, asparcit acid, glutamic acid and proline; all 10–4M (❍). Cultures contained 3 mg Cytodex 1/ml and were stirred at 60 rpm for the entire culture period. In all experiments 50% of the culture medium was changed on day 3. All cultures contained 20 mM HEPES. (From Pharmacia Biotech AB, Uppsala, Sweden).
7
6
5
4
1
3
5
7 Days
Since large imbalances or excessive concentrations reduce growth, amino acids should not be supplied at levels which differ widely from the original formulation of the medium. The only exceptions are glutamine and cystine which may need to be supplied at concentrations different from those in the original formulation. Glutamine plays a vital role in metabolism and is a precursor for nucleic acid synthesis and also an important carbon source. Glutamine is the most unstable of amino acids and decomposes in culture medium to form pyrrolidone carboxylic acid and ammonia (120). Regular addition of glutamine can be used to replenish the culture medium and compensate for the decomposition. Increasing the concentration of glutamine to 2.5 mM during the initial stages of culture usually results in better cell growth, especially at low cell densities (121). Consistent supply of glutamine is also important because of the likely role of this amino acid in the formation of molecules involved in cell-substrate adhesion (122).
89
The optimum concentration of cystine depends to a large extent on the serum concentration and batch (118) and if low concentrations of serum are used the cystine concentration may need to be reduced. For general microcarrier culture the cystine levels described in the original formulations of the various culture media are adequate but for optimization of a particular process it may be valuable to examine the effect of different concentrations of cystine. When replenishing the culture medium without complete replacement new cystine can be added approximately every 3-4 days at the concentration described in the formulation (section 3.5.2, figure 32). Nucleic acid precursors A supply of components such as adenosine, guanosine, cytidine, uridine (each 105 M) and thymidine (3x10-7M) is often beneficial, particularly in cases where folic acid is in short supply and when cells are cultured at low densities. Most media contain 1-4 mg/ml folic acid but Medium 199 contains only 1/100 of this amount. Therefore in some cases, e.g. primary cells or when culturing normal diploid cells, it may be necessary when using Medium 199 to add extra folic acid or thymidine (123). A simplified supplement of nucleic acid precursors for a general purpose medium is included in the medium in table 12. Carbon sources and lactate The growth of cells in culture depends on a source of carbon. In most of the commonly used culture media this source of carbon is provided by glucose (5-20 mM) and glutamine (0.7-5 mM). Glucose is also essential for continued attachment of cells to the microcarriers and if the concentration of glucose falls below approximately 20mM, detachment of cells occurs (124). Media containing glucose should be supplemented with pyruvate (1mM) for the growth of cells under conditions of low density (table 12). The type of carbon source in culture medium influences the formation of lactate. The use of different carbon sources in controlling pH is discussed in section 4.4.3. Vitamins and choline Addition of retinoids can promote adhesion of cells which adhere weakly to substrates (125). Retinol or retinoic acid can be added at up to 1 mg/ml for the improvement of cell adhesion and their effect may be due to influences on synthesis of specific glycoconnjugates of the cell surface (125). A sufficient supply of choline is vital to successful microcarrier culture. The main fate of choline is incorporation into membrane phospolipids and when choline levels decrease, the resulting pertubation of membrane composition causes rounding of cells and decreased adhesion (126). It is for this reason that the medium in table 12 has increased levels of choline for later stages of the culture cycle. Additional choline is important when using low concentrations of serum (less than 5%). Culture media should not contain ascorbic acid when producing RNA tumour viruses (127). In contrast, ascorbic acid enhances the yield of interferon from microcarrier cultures (59).
90
Polymers A high molecular weight component may be a necessary supplement for the culture of some types of cells at low cell densities (128). When working with cells possessing very low plating efficiencies (less than 1%) and low inoculation densities (less than 8x104 cells/ml) it is beneficial during the early stages after inoculation of the culture to include a polymer in the medium. The polymers improve cell survival but have no effect on growth. Suitable polymers are Ficollâ 400, Dextran T-70, Dextran T-500 (all available from Pharmacia Biotech) or methylcellulose and a concentration of 1 mg/ml is sufficient. If urea cycle components and other products of metabolism accumulate to toxic levels at the later stages of the culture cycle and replacement of medium is not possible, carboxymethyl cellulose (0.1 mg/ml) can be added to reduce the effects of these toxic components. Ficollâ 400 can also be used to reduce turbulence when culturing cell types which attach very weakly to culture surfaces, e.g. some established cell lines such as lymphoblastoid cells.
4.1.3 Practical aspects of culture media The water and reagents used for preparing culture media should be of the highest possible purity. All glass bottles used for storage of medium should be of high quality glass with a low content of heavy metals and should be well washed (129). In order to approve reproducibility of microcarrier cultures all procedures for preparing and supplementing culture media should be standardized and serum supplements should be added as a stock solution just prior to culture. Additional information on use and storage can be obtained from the many suppliers of culture media.
4.2 Serum supplements 4.2.1 The purpose of serum in culture media A serum supplement in usually an essential component of culture media for animal cell culture and in the absence of serum most cells fail to proliferate. Sera used to supplement culture media come form a variety of sources and are used at concentrations ranging from 0.5% to 30% (v/v). In microcarrier culture it is usual to use a serum supplement of 5-10% (v/v) for general purpose cultures. While culture media are chemically defined the serum supplement is undefined, especially with respect to those components responsible for promoting growth of cell cultures. The serum serves two vital functions. Firstly in assists attachment of cells to the culture surface, probably by supplying exogenous glycoproteins involved in the attachment process. Secondly, growth factors and hormones in the serum promote proliferation of cells. The serum also has a protective effect on cells in culture and enhances viability. A further function of the serum is to provide protease inhibitors which inactivate the trypsin used in routine subculturing procedures (section 3.7.2).
91
4.2.2 Choice and concentration of serum supplement The choice of serum for the growth of a particular type of cell is often based on tradition or convenience. Sera from different species, and even from different batches from the one species differ widely in their ability to promote attachment and proliferation of cells in culture. Foetal calf serum has been a common supplement because of a high fetuin content and a low content of gamma-globulin and fat (130). This serum often has the ability to promote the growth of more fastidious types of cells. Foetal calf serum is also unique in having high levels of biotin (131) and therefore provides a source of this growth promoting component which is not present in some media formulations (e.g. MEM, DME, L-15). The main disadvantage of foetal calf serum is that it is much more expensive than many other sera and supply is often limited. Alternative sera are required for microcarrier cultures of several hundred liters where foetal calf serum could be at a concentration of 5-10% (v/v). A further disadvantage of foetal calf serum is that it is one of the most variable sera with respect to hormone levels (132, table 13), and also contains significant levels of arginase, an enzyme which can deplete the medium of the essential amino acid arginine (130). Figure 36 shows how different sera influence the culture of mammary epithelial cells. Certain sera are good at assisting attachment but poor in promoting cell division. Although the pattern in figure 36 may not be the same for all types of cells it illustratese the principle that for the culture of any particular type of cell a variety of different sera should be tested. Table 13. Cell growth and variation of components in batches of foetal calf serum from commercial suppliers. The data are complied from several sources (132, 138-141). A measure of variations is provided by CoV (standard deviation/mean x 100).
Plating efficiency* Cell growth** Protein Haemoglobin Lactate dehydrogenase Gamma globulin Total lipids Cholesterol Free fatty acids Uric acid Free-cortisol Growth hormone Insulin Estrone
Unit
Range
CoV (%)
% 10-4 cells/cm2 g% mg % IU mg % mg % mh % mEq/liter mg/dl ng/ml ng/ml mU/ml pg/ml
0.6-19.6 0.57-19.5 1.68-5.30 10-110 300-3320 0-470 140-440 20-90 0.1-0.6 2.71-11.8 4-34 4,1-167 0,5-13,7 11-71
52 66 10 49 36 111 17 24 43 67 79 68 30 48
* Based on colony formation of primary hamster embryo cells ** Based on the number of human foetal lung cells after 72 h.
92
Attachment efficiency (%)
Fig. 36. The effect of various sera on the attachment efficiency and thymidine incorporation in cultures of mouse mammary epithelial cells. Cells were cultured in medium supplemented with 20% serum. Abbreviations; B-bovine, L-lamb, H-horse, G-goat, FB-foetal bovine, P-pig, M-mouse, Rt-rat, Hu-human, Rb-rabbit. (Feldman, M., Wong, D., In Vitro 13 (1977) 275, by kind permission of the authors and the publisher.)
15 H
B L
G
10
FB
P 5 M
Rt
Hu
Rb
NS 5
10 3
15
20
25 3
H-TdR incorporation (com x 10 /well)
A given type or concentration of serum may not necessarily be optimal for all cell types or all stages of the culture cycle. For example, a high concentration of horse serum (20% or more) favors attachment of mouse glial precursor cells but low concentration (5-10%) favor proliferation and differentiation (133). Certain sera may inhibit differentiation of cells in culture. The inhibition of chondrogenesis in chicken limb-bud cells by mouse serum (134) illustrates that some sera have very specific effects. Harrington and Godman (135) describe a factor in the alphaglobulin fraction of some sera which inhibits the proliferation of certain cell lines. The serum supplement is often the most expensive component of cell culture. Therefore efficient microcarrier culture (particularly for production purposes requires a flexible approach towards selection of serum supplements. Several different strategies can be adopted and by using one or more of these it is possible to reduce considerably the cost of serum supplements whilst still maintaining high cell yields Reducing the serum concentration The concentration of the serum supplement can often be reduced below the levels traditionally used for a given type of cell. Maintaining optimal culture conditions (e.g. pH and gas tensions) is particularly important if high cell yields are to be obtained in the presence of lower serum concentrations. Giard and Fleischaker (58) reported that 5% (v/v) foetal calf serum was more suitable for microcarrier culture of human fibroblasts than the more usual 10% (v/v) supplement. A period of adaptation or ”training” using successive decreases in serum concentrations may improve the growth of cells in reduced serum concentrations.
93
Unless special media are used (44), the plating efficiency of cells at low culture densities is proportional to the concentration of the serum and maximum plating efficiency usually occurs with 10-20% serum. At the beginning of the culture the role of serum in attachment and protection of cells is important and higher concentrations are often required than at later stages of the culture. At higher cell densities the medium becomes conditioned and cell proliferation depends to a lesser extent on the serum concentration. Hence the requirement for a serum supplement depends on the stage of the culture cycle and is related to the functions of the serum. The concentration of the serum need not be constant throughout the culture cycle. A typical procedure in microcarrier culture is to use a 10% supplement for the first three days of culture (or until the culture contains approximately 1-3 x 105 cells/ml) and then to use a medium with only 5% serum for replenishment (section 3.5.2). Once the culture has reached confluence the concentration of serum is reduced further (section 3.5.3), often as a low as 0.5%. Horng and McLimans (13, 14) noted that shedding of confluent monolayers could be avoided by decreasing the serum concentration. When cultures are used for production of viruses, interferon or other cell products, it is common to omit serum entirely during the production stage. Protein hydrolysates such as lactalbumin hydrolysate or tryptose phosphate broth can replace to a large extent the growth promoting properties of serum, particularly when growing established cell lines and some primary cells. These undefined mixtures of amino acids and polypeptides are often used at a concentration of 0.25-0.5% (w/v). Changing to another type of serum In many cases newborn or donor calf serum can replace the more expensive, less plentiful foetal calf serum supplement. Only a few types of cells require foetal calf serum and include amniotic cells, biopsy material and other primary cultures where the density of cells is very low. Other types of cells or cultures may show better growth in medium supplemented with foetal calf serum but after a period of adaptation acceptable cell yields can often be obtained in other sera. The choice of alternative sera will depend on availability and the scale of the culture, but for larger scale microcarrier cultures good quality calf, adult bovine, horse and lamb sera should all be considered as possible alternatives. Blending different sera By blending different sera it is possible to reduce the cost of the serum supplement and still maintain high yields form microcarrier cultures. A mixture of foetal calf serum and newborn calf serum (50:50) will often result in cell yields identical to those obtained in the same concentration of foetal calf serum (fig. 37). Various sera differ in their ability to assist attachment and promote cell division (fig. 36). Therefore mixing of sera known to support these individual functions can result in improved growth of the culture (136).
94
Cells/ml
107
106
105
10
4
1
3
5
7 Days
Fig. 37. The effect of various types of serum supplement on the growth of monkey kidney cells (Vero) in microcarrier cultures. Cells were cultured in modefied DME medium (table 12) containing 3 mg Cytodex 1/ml and various serum supplements. (—●—) 10% foetal calf serum, (—▲—) 5% foetal calf serum and 5% newborn calf serum, (—❍—) 5% foetal calf serum changed to 5% newborn calf serum on day 3, (—■—) 10% newborn calf serum, (—❑—) 10% horse serum. (Clark, J.M., Hirtenstein, M.D., Annals N.Y. Acad. Sci. 369 (1981) 33, by kind permission of the authors and publisher).
Different sera for various stages of the culture cycle Since sera differ in their ability to assist attachment of cells and to promote proliferation (fig. 36) the best serum supplement will depend on the stage of the culture cycle. Sera active in promoting attachment of cells and growth under conditions of low cell density can be used during the initial stages of culture. Once the culture enters exponential growth, the growth promoting function of serum is then important and sera providing this function can be used at the lowest effective concentration. For example, maximum yields of mouse mammary epithelial cells can be obtained by plating in medium containing 20 % horse, bovine or lamb serum and them changing to medium containing 5 % rabbit serum after 48-72 h (136). Foetal calf serum can be used to stimulate the growth of chicken embryo fibroblasts at low cell densities, whereas horse serum supplemented with 10 mM haemoglobin is more efficient for cultures at high cell densities (137). Figure 37 illustrates that the use of foetal calf serum for the first three days and then changing to medium containing newborn calf serum results in yields of human 95
fibroblasts equal to cultures maintained for the entire period in foetal calf serum. This is a common procedure in microcarrier culture and can be combined with a reduction in serum concentration as the culture proceeds. If the culture is to be maintained for longer periods this approach to providing a serum supplement is more economical than blending sera.
4.2.3 Variability of sera One of the most important factors controlling the success and reproducibility of cell cultures is the variation between batches of a given type of serum (132, 138-141). This effect is particularly noticeable with the microcarrier technique where there is often a wide range of culture densities during the growth of a particular culture. The early stages of the microcarrier culture cycle are the most sensitive to variations in the quality of the serum and when culturing cells with a low plating efficiency (less than approx. 25%) or when starting cultures at a low density (less than 8 x 104 cells/ml with 3 mg Cytodex/ml) it is important to use a serum supplement of the highest possible quality. Batches of a particular type of serum show large variations with respect to a number of components and this variation results in widely different plating efficiencies and yields, even if other culture conditions are optimal for growth (table 13). The batch of serum can also determine which components from the medium will become limiting. For example, the batch of serum is critical in determining the divisionlimiting concentrations of cystine (118) and variable levels of arginase can rapidly deplete the medium of arginine (130). Successful cell culture depends to a large extent on using the best batches of serum. Many cases of failure or heterogeneity in microcarrier cultures are associated with poor batches of serum. Since microcarrier culture is a method which is directed towards achieving the highest possible yields form a given volume of medium it is important to screen batches of serum whenever possible. Selection of the best batches of serum should be on the basis of plating tests in microcarrier culture. Batches of serum giving maximum cell attachment and growth in Petri dishes are not necessarily the most suitable batches for microcarrier culture (table 14). This effect may reflect different affinities the toxic components in the serum have for various culture surfaces. Table 14. Effect of various batches of foetal calf serum on attachment efficiency of human fibroblasts (MRC-5) in Petri dish and microcarrier cultures.
Batch 1 Petri dish Microcarriers Yield in microcarrier cultures after 8 days (106 cells/ml)
69.2, 49.4,
64.7 53.6
0.5,
0.7
Attachment (%) Batch 2
Batch 3
61.6, 58.3 66.1, 64.3
52.8, 50.1 55.1, 58.6
1.5,
1.0
0.7,
1.0
All cultures were inoculated with 5.6 x 103 cells/cm2. Microcarrier cultures contained 3 mg Cytodex 1/ ml and were static in bacteriological Petri dishes. Attachment was determined after 24 h. Results are from independent duplicate experiments. (Clark, J. Hirstenstein, M.D., Gebb, Ch., Develop. Biol. Standard. 1981, in press, by kind permission of the authors and the publisher.)
96
Batches of sera can be screened by simple plating and growth tests on microcarriers contained in bacteriological Petri dishes. A simple test, modified from that described by Federoff an Hall (133), is to plate 104-105 cells/ml in HBSS containing 3 mg Cytodex/ml and 50% serum of the batch to be tested. If a large number of cells are still unattached or granular after 24 hrs the batch is rejected. A further test when screening batches of serum is to determine the attachment efficiency and growth of cells in static cultures containing Cytodex (table 14). In this test, medium containing 10% serum should be used. These tests can be performed in parallel with standard plating efficiency tests in monolayer culture (141). Serum is a potential source of contamination and only batches free from microorganisms, including viruses, bacteriophage and mycoplasma, should be used.
4.2.4 Serum free media The undefined nature of serum supplements and their variation in quality makes the use of serum-free media one of the goals of cell culture. Recent developments in formulating media (142) show that a wide variety of cells can be cultured in the absence of a serum supplement provided certain components are added to the medium. Certain formulations can support the growth of cells in microcarrier culture (143). The components which can be used to replace serum in microcarrier cutures include fibronectin, transferring, insulin and epidermal growth factor. Most serum-free media formulations are probably suitable for microcarrier culture but the stirred nature of this culture system means that additional, high molecular weight components such as serum albumin or Ficollâ are required in order to protect the cells (144). Soybean trypsin inhibitor (0.5 mg/ml) should be used when harvesting cells to be cultured in serum-free media.
4.3 Gas supply Supply of correct amounts of O2 and CO2 is important to achieving high yields with microcarrier culture. Both O2 and CO2 have metabolic functions and CO2 is also usually involved in the control of culture pH (section 4.4). The gas requirements of individual cells are the same whether grown in microcarrier culture or other systems. Unlike static culture systems, stirred microcarrier cultures have even gas tensions throughout the culture volume and the possibility of monitoring in the culture gives the opportunity for accurate control. Balin et al (76) observed the beneficial effects of medium movement on reducing microenvironments having different gas tensions. It is important to note that the gas tensions currently in use are often traditional values and may not be optimal for the growth of a particular type of culture. Therefore examination alternative gas tensions can be valuable area when optimizing the microcarrier culture conditions.
97
4.3.1Gas supply and exchange in microcarrier cultures The supply of gas to static microcarrier cultures is the same as that for other monolayer techniques. Towards the end of the culture cycle when culture density is high it may be necessary to direct a stream of gas (usually 95% air: 5% CO2) over the surface of the culture for a few seconds when taking samples or changing the medium. Stirred microcarrier cultures having volumes up to approximately 500 ml-1 liter are usually kept as closed systems. Provided the vessel is not more than 50% full high yields can be obtained without a continuous supply of new gas (section 3.5, fig. 33). The gas in the headspace is renewed when taking samples or replenishing the medium (section 3.5.2). The headspace is briefly flushed with gas (15-20 sec of 95% air: 5 % CO2 forced through a Pasteur pipette) before sealing the vessel. This procedure usually supplies sufficient O2 and CO2 to satisfy the metabolic requirements of the cells. An alternative is to place the culture vessel with unsealed caps in a incubator having a humanized atmosphere (95% r.h.) with a constant supply of 95% air: 5 % CO2 . The exchange of gas by diffusion between the headspace and the culture is a relatively slow process and an important function of stirring microcarrier cultures is to improve this exchange. Because the recently modified vessels for stirred microcarrier cultures (section 3.2.2) operate at lower stirring speeds than the traditional spinner vesssels, the rate of gas exchange is reduced. It may be necessary to improve the supply of gas during the final stages of the culture cycle when using stirring speeds of less than 30 rpm or when using culture volumes exceeding 250 ml. The following steps can be taken to improve the supply of gas and they may also need to be considered when culturing primary cells or established cell lines, especially when culture densities exceed 2-3 x 106 cells/ml. These additional steps are not usually necessary for ormal diploid cell strains. • A continuous supply of 95% air: CO2 can be provided. • Gas tensions in the culture headspace can be increased, e.g. by using gas mixtures with a higher concentration of O2. • Stirring speed can be increased. The increase can be between 25-50% but will depend on the cell type and the degree of confluence (section 3.5.1). • The replenishment medium (section 3.5.2) can be gassed with the appropriate mixture before adding to the culture. This method is a simple way of achieving higher tensions of O2. in the culture medium. • Medium can be recirculated through a gas exchange vessel outside the culture (39). These steps can also be used for improving the supply of gas in large scale microcarrier cultures. The purpose of such steps is usually to improve the supply of O2 to cultures during the later stages of the culture cycle. These steps should be considered when cell yields are lower tha expected, when there is a sudden decline in growth rate or in combination with control of pH (section 4.4) Note: Sparging of microcarrier cultures should be avoided. The erratic movement of gas bubbles can damage the cells and dislodge them from the microcarriers.
98
4.3.2 Oxygen Oxygen is a key element for metabolism and the exact requirement for this gas in cell culture depends on the cell type, medium and stage of the culture cycle (145). Static monolayer cultures are relatively anaerobic and many established cell lines in common use are adapted to such conditions. In these cases it may not be necessary to have high tensions of O2 to satisfy the metabolic requirements of the cells. Primary cultures usually require more aerobic conditions and the O2 tension should be similar to that found in the tissue of origin. Oxygen tensions optimal for growth of normal diploid cell strains tend to be intermediate between those required for primary cells and established cell lines. The partial pressure of O2 in body fluids (pO2 approx. 95 mm Hg in human arterial plasma) is less than that of air (pO2 approx. 150 mm Hg at sea level) and although most culture media have been developed for use in approximately 20% O2 the tension optimal for cell growth is often substantially lower (145-151). In general, cultures should not be overgassed with O2 since high tensions (above ambient) can be toxic and reduce growth rate. Higher tensions O2 can be more toxic at alkaline culture pH (147). Oxygen tensions affect proliferation rather than cell attachment. Under conditions of low culture densities, low tensions of O2 (1-6%) are optimal for the growth of both normal diploid cell strains and established cell lines (147, 148). During the exponential growth phase the optimal tension of O2 is usually slightly greater (148152). For example, a pO2 of 9% is optimal for L-cells and at this tension accumulation of ammonia is at a minimum (152). The optimal pO2 for growth of diploid human fibroblasts is less than 5% (149). At the laboratory scale (up to 1 liter cultures) it is usually sufficient to use 95% air: 5% CO2 as a source of O2 throughout the culture cycle. The actual tension of O2 in the medium is lower than in the gas mixture (often 10-12%) and provided cultures are not inoculated with very low numbers of cells a satisfactory plating efficiency will be achieved. If lower tensions of O2 are required, e.g. when working with low culture densities (less than 5 x 104 cells/ml with 3 mg Cytodex/ml), the medium can be degassed by vacuum or by flushing with nitrogen. The low tension of O2 is maintained until the culture density increases to approximately 5 x 104 cells/ml and then 95% air: 5 % CO2 can be used. Under conditions of low culture densities the medium should contain HEPES buffer to control pH (section 4.4.2). When gas tensions can be monitored and controlled the most appropriate procedure is to start the culture with a low pO2 (2-5%) and increase the tension during the culture cycle to about 15-20% at the end of the exponential phase of growth. This increase in pO2 will also assist in controlling pH (section 4.4.2). The tension of pO2 should be measured by immersing a suitable electrode in the medium.
4.3.3 Carbon dioxide Solution of CO2 in medium results in the formation of HCO 3 an essential ion for the growth of cells. The requirement for HCO 3 is independent of its buffering 99
action, but since CO2, HCO3 and pH are intimately related it has been difficult to define the tension of CO2, optimal for cell growth (153). In the mixture 95% air: 5% CO2, the concentration of CO2 was selected originally on the basis of being the concentration in the alveolar spaces of the lung (153). This concentration was intended for studies on lung fibroblasts but has now become routine for general cell culture. The tension of CO2, optimal for cell growth may be in the range of 0.5-2.0% with the exact value depending on cell type (153). To date most work with microcarrier cultures has involved CO2, tensions of 5-10% and high cell yields have been obtained. Improved cell yields as a result of lower CO2 tensions remain to be demonstrated. While it may be difficult to work with lower CO2 tensions in routine small scale cultures the opportunity for control of gas tensions and pH with most large scale systems could be used to define the CO2 tension which is optimal for growth. Note: Leibovitz L-15 medium does not rely on CO2 for buffering and control of pH and can be used when low tensions of CO2 are required. The role of CO2 in control of pH is the most important aspect to consider when optimizing conditions for high cell yields and is discussed in section 4.4.
4.3.4 Purity of the gas supply All gases used for cell culture should be of the highest possible quality. It is important that the gas supply is essentially free of CO, nitrous oxide and hydrocarbons (153). A membrane and/or cotton wool filter should be used to remove any particulate matter in gases which are introduced directly into cultures. Wide variations may exist in the actual pCO2, levels of commercial gas mixtures (153) and by using certified sources this variable can be minimized.
4.4 Culture pH Since pH influence cell survival, attachment, growth and function, maintaining the correct pH is central to obtaining optimal cell growth and high yields. Controlling pH is particularly important when using microcarrier culture because cultures can rapidly become acidic at high culture densities. This decrease in pH is one of the most common causes of poor results in microcarrier culture and is due to accumulation of lactate. Methods for controlling pH include buffering to minimize the effects of lactate on culture pH (section 4.4.2) or altering culture conditions such that the cells produce less lactate (section 4.4.3). Note: The effect of temperature on pH should always be taken into account and if possible pH should be measured at the culture temperature. Unless stated otherwise all values refer to pH at 37 °C.
4.4.1. pH optima for cell culture In cell culture it is common to use a pH of 7.2-7.4 and the wide fluctuations in pH which often occur during the culture cycle and after medium replenishment (pH 7-8) have an adverse effect on cell yields (154). Föhring et al (54) concluded that a 100
Cells/ml
Fig. 38. Effect of pH on the plating effiency of cells. Plating was determined at 12–16 hr by 3 H- thymidine incorporation. (—●—) human embryo lung fibroblasts, (—❑—) SV40-transformed W138, (—❍—) HeLa. (Ceccarini, C. In Vitro 1 (1975) 78, by kind permission of the authors and the publisher.)
7
10
6
10
5
10
10
4
1
3
5
7 Days
constant pH was the most important parameter in determining growth rates and yields of cells and virus in microcarrier culture. The attachment and plating efficiency of cells depends on the pH of the medium (fig. 38). One of the most critical stages of culture with respect to pH is just after inoculation (155) and in order to achieve the highest possible plating efficiency the culture should have a pH of less than 7.6. When initiating a culture the medium is often exposed to the atmosphere for some minutes and at the time of inoculation the pH can be as high as 8.0. Therefore it is important to ensure that the medium is exposed to the atmosphere for as short a period as possible. In routine microcarrier culture it is advisable to equilibrate the medium for a few minutes with 95% air: 5% CO2 before inoculation (section 3.4.1). HEPES buffer can also be used to ensure that the medium is not too alkaline during the early stages of the culture cycle (section 4.4.2). Diploid human fibroblasts are particularly sensitive to alkaline conditions (pH grater than 7.6) during the attachment stages of culture (fig. 38). When the culture pH can be controlled diploid human fibroblasts should be cultured at pH 7.4-7.5 for the first 1-2 days of culture and then the pH can be increased to 7.6-7.8 for the exponential phase of growth. The pH should be decreased to 7.4-7.5 during the plateau stage of culture to ensure continued adhesion of the resting monolayers (section 3.5.3). 101
Table 15: The effect on pH on cell growth. Cell type Human Normal
SV 40-transformed Tumour Rabbit lens Mouse fibroblasts
a b
c
Strain
pH for optimal growtha
Increased growth at optimal pHb
KL2 MS2A Penny WI38 WI26VA HeLa
7.5-7.7c 7.6 7.5-7.8c 7.8 7.3-7.5c 7.0 6.9 7.5-7.8c 7.0-7.5c
3.2 1.9 2.1 2.6 1.2 1.3 3.7 1.8 1.0
3T3 L929
pH measured at 25°C Relative to growth in bicarbonate-buffered medium, pH fluctuates from pH 8 to 7 after each refeeding. Essentially equal over indicated range.
(Data from Ceccarini, C., and Eagle, H., Proc. Nat. Acad, Sci. US 68 (1971) 229-233, by kind permission of the authors and publisher)
The pH which is optimal for cell growth may not be the same as that which is optimal for cell attachment. Table 15 shows that by maintaining cultures at a pH optimal for cell growth substantial increases in yields can be achieved. Different types of cells may have different pH optima for growth (183, table 15). By maintaining the pH at a level optimal for growth it is possible to reduce the serum requirement by at least one half (155). In general, human fibroblasts are grown at a higher pH (7.6-7.8) than established cells (pH 7.0-7.4) and it is usual to culture primary cells at a pH of 7.2-7.4. The optimum poH for growth of human foreskin fibroblasts (e.g. FS-4) at low culture densities is more alkaline than the optimum pH for human lung fibroblasts (e.g. MRC-5, 156). When culturing these cells during the growth phase at a density of 105 cells/ml or less the pH should be 7.7-7.8 for FS-4 cells and 7.5-7.6 for MRC-5 cells.
4.4.2 Buffers and the control of pH The method used to monitor pH depends on the precision which is required. Good results with small scale culture (up to 1 liter) can be obtained simply by acting on changes in the colour of the phenol red indicator in the medium. In larger scale cultures it is normal to use electrodes to monitor pH. McLimans (104) describes how to control pH when preparing medium. Most media utilize a CO2/HCO 3 buffer system but the capacity of this system is often not sufficient to be able to prevent a decreasing pH towards the end of the culture cycle. If a cell type produces large amounts of CO2 then media based on Hank’s salt solution (0.33 NaHCO3/liter) are more suitable than media based on Earle’s solution (2.20 g NaHCO3/liter). Alternatively, a if cell type tends to produce large quantities of lactate then formulations with a higher concentration of HCO 3 should be used. It is normal to use media containing Earle’s salt solution in
102
Plating efficiency (%)
80
60
40
20
6.8
7.2
7.6
8.0
8.4 pH
Fig. 39. Effect of control of pH on the growth of monkey kidney cells (Vero) in microcarrier cultures. Cultures (400 ml) were contained in sealed spinner vessels and stirred continuously at 50 rpm. Culture medium (DME, based on Earle’s salt solution) was with (—●—) or without (—❍—) 10 mM HEPES. The pH of the medium without HEPES was initially 7.4 but varied between 7.0 and 8.0 during the culture cycle. The pH of the medium containing HEPES was maintained at 7.3–7.4. The culture medium was changed on day 4. The difference in cell yields was largely due to a higher proportion of empty microcarriers in the culture without pH control. (From Pharmacia Biotech AB, Uppsala, Sweden.)
combination with 95% air: 5% CO2 and media containing Hank’s salt solution with lower concentrations of CO2 in the gas phase. Therefore microcarrier cultures contained in sealed spinner vessels usually require media based on Earle’s salt solution. Addition of HEPES delays the onset of pH drift and usually increases cell yield (fig. 39). The HEPES assists in maintaining pH during the attachment period of culture and plating efficiency is enhanced. Routine addition of 10-25 mM HEPES is advisable when the best results are required from laboratory scale microcarrier cultures. Whenever the tension of CO2 is low (less than 5%) there is a lower stability of the HCO 3 system and HEPES should be used. The exact amount of HEPES should be no more than is required to maintain the pH and it is advisable to start with 10 mM. Formulations of buffers designed to give good control of pH at specific values can be found in references 157, 158.
103
In large scale microcarrier cultures small changes in pH can be controlled by addition of HCO 3 or increasing the tension of CO2 and addition of NaOH or HCI can be used to control larger changes. The opportunity for constant monitoring and control with large scale systems means that HEPES is no longer essential for high cell yields. Culture pH can also be controlled when replenishing with fresh medium (section 3.5.2). Care should be taken not to change the osmolarity of the culture medium when adding buffers for pH control (section 4.5).
4.4.3 Minimizing accumulation of lactate One of the most effective ways of avoiding difficulties with controlling a decline in pH is to use culture conditions which limit the formation of lactate. Some established cell lines produce large quantities of lactate and towards the end of the culture cycle when densities are high a rapid decrease in pH can occur even int the presence of addiotional buffer systems. This decrease in pH can result in reduced cell growth, viability and also detachment of cells from the microcarriers. Various methods can be used to limit production of lactate so that changes in pH are within the buffering capacity of the medium. Cultured cells degrade glucose to either CO2 or lactic acid and, depending on the redox state of the culture, high concentrations of glucose result in the formation of correspondingly high levels of lactate. When optimizing culture conditions an effective method for supplying glucose for obtaining maximum cell growth is to use 80 µM glucose for the initial stages of culture and to use daily refeeding with glucose to maintain a concentration of 25-40 µM in the presence of greater than 50 µM glutamine (124). This procedure reduce the production of lactate and encourages the use of glutamine as a source of carbon. Glutamine is a major source of energy and the degree of convention to lactate is less than when glucose is used as the energy source (124, 159, 160). A maintained or even increased supply of glutamine can also be used to reduce the formation of lactate (150). The amount of lactate secreted by transformed cells can be reduced by biotin (161) and replenishment media (e.g. table 12) can be supplemented with this component. Zielke et al (124) described a medium which can be used for the high yield cultivation of human fibroblasts with only minimal production of lactate. This medium is based on MEM supplemented with hypoxantine (100 µM), glycine (100 µM), thymidine (40 µM) and uridine (100 µM). An increased oxygen tension can be used to reduce the formation of lactate in the presence of glucose (146, 148). The oxygen tension can be increased during the culture cycle and can be used to encourage a more aerobic metabolism when culture density is high. Oxygen tensions of up to 20% can be used (section 4.3.2). Increasing the oxygen tension to approximately 15-20% of particularly useful for reducing lactate accumulation in stationary cultures of human fibroblasts (149). Another method of reducing formation of lactate is to use a carbohydrate other than glucose in the culture medium. Fructose or galactose (2-10 mM) can be used instead of glucose and result in greatly reduced levels of lactate (159, 162). When using carbohydrates other than glucose it is important to maintain a good supply of glutamine since this component becomes a major source of energy (159). Glutamine 104
levels should be increased to 4 mM during the exponential stage of growth when using these alternative carbohydrates. Changing the carbon source to fructose or galactose is a convenient method for maintaining very dense populations of cells (more than 3-5 x 104 cells/ml, with the minimum amount of medium. Imamura et al (162) reported that fructose (5-20 mM) was as effective for maintaining cell growth as glucose (20 mM) in high yield microcarrier cultures but resulted in virtually no decrease in culture pH. Under identical conditions diploid human fibroblasts produce only approximately ¼ as muck lactate when using fructose or galactose instead of glucose as the carbon source (163). Note: Leibovitz L-15 medium contains galactose instead of glucose and can be considered when experimenting with alternative carbon sources. The use of carbon sources and controlling accumulation of lactate should always be considered when optimizing medium replenishment schemes (section 3.5.2). To summarize, useful procedures for avoiding a sudden fall in pH at the later stages of the microcarrier culture include improving the supply of glutamine, increasing the oxygen tension and if necessary supplementing the medium with biotin. If lactate accumulation is still excessive then alternative carbon sources should be used. These measures need only be taken if changes in pH prove to be too great for the capacity of the buffer systems in the medium.
4.5 Osmolarity The growth and function of cells in culture depends on maintaining an appropriate osmolarity in the culture medium (164). Some cells (e.g. HeLa and other established cell lines) can tolerate wide fluctuations in osmolarity. In contrast primary cells and normal diploid strains are very sensitive to changes in osmolarity and high yields can only be obtained if the osmolarity of the culture medium is kept within a narrow range. In the absence of evidence to the contrary the osmolarity of the medium used for the culture of any particular type of cell should be kept constant at a value in the range 280-320 mOsm/litre, normally 290-300 mOsm/litre. By controlling osmolarity it is possible to achieve more reproducible cultures. Whenever the source of a particular culture medium is changed the osmolarity should be checked. Osmolarity of media produced by commercial suppliers may differ, probably because of differences in interpretation of original formulations (164). Microcarrier culture is no different from any other culture technique in its requirement for a controlled osmolarity. However, high yield cultures often require various additions to the culture medium during the culture cycle. These additions can include buffers (HEPES), acid (HCI), base (NaOH) and nutrients addition of NaCl and the correct amount required to achieve a particular osmolarity is calculated as follows (104).
105
The osmolarity of the medium is measured and the amount of stock NaCl (1 mg/ml) which must be added to achieve the desired osmolarity is calculated. 1 mg NaCl/ml = 1 ml stock (mOsm) = 32 mOsm increases. D–O Hence ——— = X, 32 where D (mOsm) = desired mOsm O (mOsm) = observed mOsm X = ml of stock NaCl (mOsm) to be added per milliliter of medium. Measurement of osmolarity by freezing point depression is the most practical method (164). Dilution of nutrients in the medium by addition of large volumes of buffers or saline solutions should be avoided as much as possible.
4.6 Frezing cells for storage 4.6.1Procedure for freezing and thawing The following procedures can be used for a wide variety of primary, normal and established cell cultures. For the storage of cells as a suspension, exponentially growing cells are harvested from the microcarriers by the usual EDTA-trypsin or collagenase procedure (section 3.7.2) and resuspended (107 cells/ml) in storage medium (section 4.6.2). The cell suspension is centrifuged (400 g, 5 min, 4OC), the supernatant is discarded and the pellet is gently resuspended in cool storage medium at a concentration of 3-5 x 106 cells/ml. One ml aliquots of the cell suspension are transferred to chilled sterile ampoules which are the cooled to -70OC at -1OC/min. This rate of cooling can be achieved by using a 5 cm-thick expanded-polystyrene box. The ampoules can then be transferred to the vapour or liquid phase of liquid nitrogen for long therm storage. Cells may also be stored whilst still attached to the surface of Cytodex microcarriers. The cells to be stored are grown on the microcarriers until approximately 75 % confluent. The microcarriers are then rinsed in cool (4oC) storage medium (section 4.6.2) containing one-half the final concetration of cryoprotectant e.g. glycerol or dimethylsulphoxide. After 5 min the supernatant is removed by gentle centrifugation and replaced by cool storage medium containing the final concentration of cryoprotectant. After a further 5 min the supernatant is removed and the microcarriers are resuspended in storage medium at a concentration of 5-10 x 106 cells/ml. Aliquots of this suspension are then stored as described above. During the washing steps at least 50 ml storage medium are required per g of microcarriers.
106
The additional washing steps for storage of cells attached to Cytodex microcarriers make sure that the microcarrier matrix is well penetrated with storage medium and thus breakage of the microcarriers during the freezing is prevented. Storage of cells on the Cytodex microcarriers has the advantage that harvesting is not required and cell viability is greater when the cells are frozen. Since the cells are already attached to the microcarriers, recovery after thawing is also improved. This can be particularly important for cells with a low plating efficiency where often less than 10 % of the cells in a stored suspension can attach to a cell culture surface and proliferate after thawing. Recovery of frozen cells is achieved by rapidly thawing the ampoule in a 37O C water bath and transferring the contents to a centrifuge tube. The cells and microcarriers are washed once in five volumes of growth medium, allowed to sediment and the supernatant is discarded. The cells and microcarriers are resuspended in fresh growth medium containing the usual serum supplement and transferred to the culture vessel for cultivation under standard conditions for 24 hr. After this incubation the microcarriers will be confluent and the cells can then be harvested in the usual way and used for further cultures (section 3.7). An advantage of using cells attached to microcarriers during the thawing step is that the storage medium can be easily removed from the cell preparation without the need for centrifugation.
4.6.2Storage medium The most suitable storage medium is either 5 % (v/v) dimethylsulphoxide in growth medium or 10 % (v/v) glycerol in growth medium. The growth medium should contain a 10 % (v/v) heat-inactivated serum supplement. If glycerol is to be used in combination with freezing cells attached to cytodex microcarriers a higher concentration (20 %, v/v) should be used. If this concentration of glycerol cannot be used with a particular type of cell then the material should be rinsed at least one more time in storage medium containing 10 % (v/v) glycerol. The cryoprotective agents should be of reagent grade and accumulation of oxidative products in the stock can be avoided by freezing ampoules of sterilized material. Dimethylsulphoxide is often preferred because it penetrates the cells more rapidly than glycerol: however, the time of exposure of cells to this agent at above freezing temperatures should be as brief as possible. For very sensitive types of cells it can be desirable to use a mixture of 5 % (v/v) dimethylsuflphoxide in foetal calf serum. When preparing the storage medium the cryoprotectant should be mixed well with the growth medium. The storage medium should be prepared immediately before use and should have a pH of 7.2-7.4.
4.7 Contamination Prevention of contamination by microorganisms is an essential part of all animal cell culture. The risk of contamination can be eliminated by efficient sterilization methods, effective aseptic techniques and antibiotics. Such measures are described in detail elsewhere (165-167).
107
The routine use of antibiotics in cell culture media is not recommended because a) they lead to a relaxation of aseptic techniques, b) resistant microorganisms develop, c) microbial growth may be controlled but biochemical alterations may still be produced and d) antibiotics often have adverse effects on cell growth and function. Therefore all routine cell culture in our laboratories at Pharmacia Fine Chemicals is performed in the absence of antibiotics. Antibiotics can depress the growth rate and reduce longivity of animal cells in culture (168, 169). Routine concentrations of penicillin and streptomycin cause at least a 20 % reduction in yield from cultures of human fibroblasts compared with the same cells grown in the absence of antibiotics for several passages (169). If microcarrier cultures are to be maintained at confluence for long periods of time the omission of antibiotics reduces any tendency for the cell layer to detach from established cell lines. Established cell lines may have been selected for growth in antibiotics and may be less sensitive than primary cells (170). Antibiotics can inhibit protein synthesis in primary cultures (170) and accumulate to high concentrations in fibroblast lysosomes (171). In certain situations it may be necessary to use antibiotics. Antibiotics are required when working with primary cell cultures or cell lines suspected of being contaminated. The antibiotics can be withdrawn as soon as tests on the cells show them to be free from contamination. Antibiotics may also be necessary for culture systems were there may be a large risk of contaminating during the period of culture. In large scale cultures the adverse effects of antibiotics are counteracted by the economical consequences of contamination. Therefore antibiotics are usually necessary for at least the initial stages of the culture production cycle. During the early stages of culture samples of medium can be taken for sterilitui tests and if the medium is sterile when the culture approaches confluence, antibiotics can be withdrawn from the replenishment medium or at least the concentration can be reduced. The most suitable antibiotics to use are penicillin (100 U/ml), streptomycin (100 µg/ml) or gentamycin (50 µg/ml) as antibacterial agents and nystatin ( 50 µg/ml) to eliminate growth of fungi and yeasts. These antibiotics can be used individually or in combination. The cytotoxicity of antibiotics increases in low-serum and serumfree media and the quantity of antibiotics should also be reduced in proportion to the serum concentration. Cell cultures must be frequently checked for mycoplasma. Mycoplasma have a wide range of effects on cultured cell growth and function (172) and most importantly for microcarrier culture, mycoplasma compete with cells for nutrients in the culture medium. Mycoplasma contamination can result in rapid depletion of the essential amino acid arginine from the culture medum and an increased accumalation of ammonia (172). It is this depletion and imbalace in amino acid composition of the medium which has serious consequences for cell cultures. Extensive mycoplasma
108
infection usually leads to failure of the microcarrier culture. High density cultures of animal cells require all the nutrients available in the medium and can only suffer if they must compete with mycoplasma. The most simple and effective method for routine screening for mycoplasma is to use the following fluorescent staining method (173, 174) The cells to be tested are grown on glass coverslips in 5 cm Petri dishes until approximately 70 % confluent. Without removing all the culture medium, 2 ml of modified Carnoy´s fixative (3:1 absolute methanol: glacial acetic acid) are added gently to the dish containing the coverslips. After 2 min at room temperature the fixative is replaced by fresh fixative for 5 min. The coverslips are then rinsed briefly in fresh fixative and air-dried. A stock solution (0.05 mg/ml) of the benzimidazole fluorochrome Hoexhst 33258 (American Hoechst, Somerville, NJ, USA or Riedel-De Haen AG, Seelze-Hannover, FDR) is prepared in HBSS without phenol red, pH 7.0. This solution is diluted to 0.05 µg/ml with HBSS and coverslip is washed 3 times in distilled water and mounted in 0.1 M acetate buffer, pH 5.5. A fluorescence microscope is used to check the presence of fluorescent particles at the periphery of the cells. Suitable filters are Zeiss 53/44 barrier filter and a BG-excitation filter. Infected cultures should be discarded. In exceptional cases measures may be taken to try and eliminate the mycoplasma (167, 172, 175).
109
110
5. Optimizing culture conditions and trouble shooting Optimizing culture conditions When microcarrier culture is to be used regularly for a particular type of cell it is advisable to optimize the culture procedures and conditions. Optimization is particularly important when microcarrier cultures are used for routine production of cell, viruses or cell products. The aim of optimization is to provide conditions which are suited to each type of cell and to each stage of the culture cycle. By using the most suitable conditions and procedures it is possible to maximize utilization of the microcarrier surface area and increase yields, to reduce the amount of cells needed for inoculation, to reduce the culture lag period, to improve reproducibility and to improve economy of serum and medium. Optimization should take place in several stages and only after the basic culture procedure has been established. The steps can be summarized as follows. 1. Control of inoculum condition Routine work requires that the inoculum should be of consistent quality and have the highest possible plating efficiency (section 3.4.5). The cells should be checked routinely for the presence of mycoplasma (section 4.7). 2. Modification of initial culture procedure The static attachment period, initial culture volume and concentration of microcarriers should be adjusted so as to permit the highest possible plating efficiency (section 3.4). 3. Adjusting stirring speeds The stirring speed may need to be adjusted for each stage of the culture cycle so that there is maximum cell growth with no aggregation of the microcarriers (section 3.4.2, 3.5.1). Most optimal procedures involve a slight but progressive increase in stirring speed during the culture cycle. 4. Defining a schedule for replenishment of medium Replenishment medium can be used to slowly dilute the culture to the final volume. Such a procedure can be used to assist in control of pH and to prevent wide fluctuations in the concentration of nutrients (section 3.5.2). A continuous perfusion system can also be considered. The most important aspect of successful replenishment and obtaining high cell yields is to anticipate changes in pH and nutrient concentrations. 5. Modifying the culture medium The culture medium may need to be modified for certain stages of the culture cucle (section 3.4.6, 3.5.2, 4.1). The components of the medium should be altered if there are persistent difficulties in controlling pH (section 4.4.2, 4.4.3). 111
6. Reducing the requirement for serum supplements Reducing the serum concentration and using different sera for different stages of the culture cycle should be investigated (section 4.2.2). Expensive supplements, e.g. foetal calf serum, usually only need to be present in the initial reduced culture volume; replenishment medium used for increasing the culture volume can often contain a reduced concentration of a cheaper type of serum (sections 3.5.2, 4.2.2) Batches of sera should be routinely screened with growth tests using microcarriers (section 4.2.3). 7. Modifying the gas supply The gas tensions optimal for cell attachment and growth should be examined. A progressive increase in oxygen tenson during the culture cycle usually leads to improved control of pH and higher cell yields (sections 4.3.2, 4.4.3). The sueply of gas (95 % air: 5 % CO2 ) should be used when possible to control pH. 8. Control of pH The modification above (medium, gas supply) should also be considered with respect to control of pH. If possible the pH of the culture should be optimized for cell attachment, growth and maintenance of confluent monolayers (section 3.5.3, 4.4.1). When working regularly with cultures having a volume of one litre or more it is convenient to use a sterile pH electrode for monitoring the culture instead of acting on the basis of phenol red indicator.
Trouble shooting When working with stirred microcarrier cultures for the first time some difficulties may be encountered. The following points list the occasional areas of difficulty and the most likely solutions. These points can also serve as a checklist when culturing each new type of cell. 1. Medium turns acid upon addition of microcarriers. • Check that the microcarriers have been propberly prepared and hydrated (section 3.3) 2. Medium is alkaline at inoculation stage. • Gas the culture vessel and equilibrate with 95 % air: 5 % CO2 (section 3.4.1). 3. Loss of microcarriers on surface of culture vessel. • Check that the culture vessel has been properly siliconized (section 3.2.4). 4. Poor attachment of cells and slow initial growth • Ensure that the culture vessel is non-toxic and well washed after siliconization. • Dilution of culture by PBS remaining after sterilization, rinse microcarriers in growth medium (section 3.3). • Modify initial culture conditions, increase length of static attachment period, reduce initial culture volume or increase the size of inoculum (section 3.4). • Control condition of inoculum (section 3.4.5).
112
• Eliminate vibration transmitted from stirring unit (section 3.2.1). • Change to a more enriched medium for the initial culture phase (section 3.4.6). • Check quality of serum supplement. • Check for contamination by mycoplasma. 5. Microcarriers with no cells attached • Modify initial culture conditions, increase length of static attachment period, reduce initial culture volume (section 3.4). • Check that inoculation density is correct (3.4.4). • Improve circulation of the microcarriers. 6. Aggregation of cells and microcarriers • Modify initial culture conditions, reduce time that the culture is allowed to remain static(section 3.4.2) • Increase stirring speed during growth phase (section 3.5.2), improve circulation of microcarriers. • Reduce concentration of serum supplement as culture approaches confluence (section 4.2.2). • Reduce concentration of Ca2+ and Mg 2+in the medium (section 3.5.2). 7. Rounded morphology of cells and poor flattering during growth phase • Replenish medium (section 3.5.2). • Check osmolarity (section 4.5) and pH (section 4.4) of culture medium. • Reduce concentration of antibiotics if low concentrations of serum are beeing used (5 % or less). • Check for contamiation by mycoplasma (section 4.7). 8. Rounding of cells when medium is changed • Check temperature, pH and osmolarity of replenishment medium. • Reduce serum concentration. 9. Cessation of growth during culture cycle • Replenish medium (section 3.5.3) or change to a different medium (section 4.1.1). • Check that pH is optimal for growth (section 4.4). • Re-gas culture vessel or improve supply of gas (section 4.3). • Reduce stirring speed (section 3.5.1). • Check form contamination by mycoplasma (section 4.7). 10. Difficulties controlling pH • Check that buffer system is appropriate (section 4.4.2). • Improve supply of gas to culture vessel, lower concentration of CO2 in headspace or increase supply of oxygen (section 4.3). • Improve the supply of glutamine, supplement the medium with biotin or use an alternative carbon source, e.g galactose (section 4.4.3).
113
11. Difficulties maintaining confluent monolayers • Check that pH (section 4.3) and osmolarity (section 4.5) are optimal. • Reduce the concentration of the serum supplement (section 4.2.2). • Improve schedule for medium replenishment (section 3.5.3). • Reduce the concentrations of antibiotics (section 4.7). 12. Broken microcarriers • Ensure that dry microcarriers are handled carefully. • Check design of culture vessel/impeller and ensure that bearing is not immersed in culture (section 3.2.2).
114
6. Appendix Cells cultured on Cytodex microcarriers The purpose of these two lists is to allow rapid identification of cell types and also to recommend suitable inoculation densities and culture media. Table 17 provides a cross-referenc list of cell types, by tissue origin, which can be used to identify the specific cell lines in table 16. the lists do not include somatic cell hybrids which have been cultured on Cytodex microcarriers (e.g. figs. 6,7). Table 16. Cell line specific list of cell types cultured on Cytodex microcarriers. Cell
Species
Origin
Typea
Mediumb
Inoc Commentsd Densityc
A9 Amniotic BGM BHK BSC-1 Carcinoma Carcimona Carcimona Carcinoma Chang “D“ Chang CHO CR-1 CV-1 C6 Detroit 6 Detroit 98 Detroit 532 Don Endothelium Endothelium Endothelium Endothelium EPC FHM Fibroblast Fibroblast Fibroblast Fibroblast Fibroblast Fibroblast
Mouse Human Af. Green monkey Syrian hamster Af. Green monkey Human Human Human Rat (Lewis) Human Human Chinese hamster Chimpanzee Af. Green monkey Rat Human Human Human Chinese hamster Bovine Rabbit Mouse Human Carp Fat head minnow Mouse Chicken Rat Rabbit Human Human
E P E E E E E E E E E E E E E E E ND E P P P P E E P, ND P, ND P, ND ND P, ND P, ND
1 2 3, 4 5 1, 2 1, 4 4 1, 4 8 21 1, 4, 23 1, 4, 8, 24 1, 12 1, 2, 4 21 1, 4 1, 4 1, 8 1, 2, 3, 11 2 2 2 2 5, 13 5, 13 1, 2, 8, 12 17, 18, 19 1, 2, 8, 12 1, 2, 8, 12 1, 2, 8, 9 2, 11
L H H L H M H M H M L M H L L L H H H H H H H H H, M H H, M H H, M H
Fibroblast Fibrosarcoma Flow 2002 Flow 4000 FS-4 Girardi heart Glial Glioma G1-V3 HaK
Muntjac Mouse Human Human Human Human Rat Human Af. Green monkey Syrian hamster
Areolar fibroblast Amniotic fluid Kidney Kidney Kidney Colon Squamous cell Thyroid Lung Conjunctiva Liver Ovary Embryo lung Kidney Glial tumour Bone marrow Bone marrow Down’s foreskin Lung Pulmonary artery Coronary artery Brain capillary Coronary artery Epithelioma Whole fish Embryo Embryo Embryo Embryo Embryo Xeroderma pigmentosum Adult skin Fibrosarcoma Embryo lung Embryo kidney Foreskin fibroblast Atrial appendage Brain Brain tumour Kidney Kidney
ND E ND ND ND E ND E E E
21 4, 22 1, 2, 8 1, 2, 8 1, 2, 8 1, 4 1, 2, 8 1, 2, 8, 11 1, 12, 15 1, 8, 23
M M M H H M H M H L
1 2 3 20
4 4 5
4 4 6
7 7 24 24 24 24 24 8
25 9, 24 10,24 4 11 11
115
Typea
Cell
Species
Origin
HEL 299 HeLa Hep2 HTC HT 1080 IBR IMR-90 Insect Insect Insect J 111 KB
Human Human Human Rat Human Pig Human Drosophila Trichoplusia Spodoptera Human Human
Kidney Kidney Kidney Kidney Kidney Liver Liver Liver Lung LLC-RK1 LLC-RK2 L-cells L-132 L-929 Lymphoblastoid Lymphocytes Macrophage Macrophage Macrophage Macrophage McCoy MDBK MDCK Melanoma Melanoma Melanoma Morris HM1CM1 MRC-5, MRC-9 Muscle Muscle Mv 1 Lu NCTC-2544 Neuro-2a NRK NZ-white Osteosarcoma Pancreas Pituitary Pituiary Pituitary PK-15 Pt-K-1
Dog Rabbit Monkey Human Bovine Chimpanzee Human Rat Cat Rabbit Rhesus monkey Mouse Human Mouse Human Human Human Mouse Mouse Rat Human Bovine Dog Dog Human Mouse Rat Human Chicken Rat Mink Human Mouse Rat Rabbit Human Rat Bovine Human Rat Pig Potoroo
Embryo lung ND Cervical carcinoma E Larynx carcinoma E Morris hepatoma E Fibrosarcoma E Kidney E Lung fibroblast ND Embryo E Ovaries E Ovaries E Monocytic leukemiaE Nasopharangeal E carcinoma Kidney P, ND Kidney P, ND Kidney P, ND Kidney ND Kidney ND Liver E Hepatocytes P Hepatocytes E Embryo lung ND Kidney E Kidney E Areolar fibroblast E Embryo lung E Areolar fibroblast E Lymphoblastoid E peripheral blood P Peripheral blood P Peritoneal P Peripheral blood P Peritoneal P Sunovial fluid E Kidney E Kidney E Melanoma E Melanoma E Melanotic tumour E Hepatoma E Embryo lung ND Myoblasts P Myscle-fibroblasts E Lung E Skin epithelium E Neuroblastoma E Kidney E Kidney E Bone tumour E Pancreas P Pituitary P, ND Pituitary P Pituitary P Kidney E Kidney E
116
Mediumb
Inoc Commentsd Densityc
1, 2 H 1, 4 M 1, 8, 11, 16 L 1, 4, 11, 13 L 1, 8 M 1, 8, 13 M 1, 2 H 10, 25 H 10, 25 H 10, 25 H 1, 4, 11 M 13, 14 L 1, 2 H 1, 2 H 1, 2 H 1, 2, 20 H 1, 2 H 1, 4, 12 L 2, 20 H 2, 20 H 1, 2 H 1, 12, 15 M 13, 15 M 1, 8 L 1, 20 M 1, 8 L 2, 6, 15, 20 M 1, 6, 15, 20 H 1, 6, 15, 20 H 1, 6, 15, 20 H 1, 6, 15, 20 H 1, 6, 15, 20 H 1, 4 L 1, 8 M 1, 3, 15 M 6 L 13 L 2, 11, 22 H 1, 8, 14 H 1, 2, 7 H 1, 2, 17, 25 H 4 L 30 H 27 H 1, 4 M 1, 8, 12 M 1, 2 M 1, 20 M 1, 2, 6 H 1,28 H, M 2, 8, 16 H 2, 8, 16 H 1, 12 M 1, 2 H
4, 12 13 24 14, 17 14, 17 14 4 15
16
17 17 17 18 18 18 18 19 25 24
4, 20
21
Typea
Cell
Species
Origin
PyY P38801 RD RK-13 RPMI 2650 RTG SC-1 SIRC Tb 1 lu Thyroid Vero WISH WI-38 Y-1
Syrian hamster Mouse Human Rabbit Human Rainbow trout Mouse Rabbit Bat Pig Af. Green monkey Human Human Mouse
3T3 3T6
Mouse Mouse
Polyoma-BHK E Macrophage E Rhabdomyosarcoma E Kidney E Nasal carcinoma E Gonad E Embryo E Cornea E Lung ND,E? Thyroid P, ND Kidney E Amnion E Embryo lung ND Adrenal cortex E tumour Embryo fibroblast E Embryo fibroblast E
Mediumb
Inoc Commentsd Densityc
5 L 6 M 2 H 1, 2 8 M 1 H 5, 13 H 1, 8 H 1, 2, 8 H 30 H 29 H 1, 3, 7, 8, 9 M 1, 2 M 1, 2, 7 H 22 H 8 8
23 7
4 24
M H
Additional transformed cells
Typea
Mediumb
Inoc. Density
SV40 - Mouse fibroblast (3T3) RSV - rat kidney(NRK) RSV - rat subcutaneous tumour (XC) MSV - mouse fibroblast (3T3) SV40 - human fibroblast (WI-38) MSV - dog epithelial (doC11, MDCK)
E E E E E E
8, 12 8 1, 8 1, 8, 12 1, 8, 12 1, 8, 12
L L M L M M
a
22
Commentsa
type of cell line
P - Primary culture. Cells which have been dissociate from the tissue, usually by enzymes, and inoculated directly onto the microcarriers. ND - Normal diploid cell line. These cells are normal in-so-far as they have the expected genetic complement, do not form tumours and have a finite lifespan in culture. E - Established or transformed cell lines. Cell which can be cultured indefinitely. Such cells are often transformed in the sense that they can form tumours when injected into a suitable host animal. Many cell lines of this type are of tumour origin. Cell lines in this category are usually easier to grow than those in the two categories above. b
Culture media
For any particular type of cell several different media will prove satisfactory. The following list of media refers to successful culture of the above cell types on cytodex microcarriers. Media 1 and 2 are the most universally applicable media and unless there are indications to the contrary these two media are to be recommended for microcarrier culture. Media used for large scale microcarrier culture are often different from those listed below. In such cases, the expensive serum supplements are partially replaced by the cheaper embryo extracts or hydrolysates. Details of media can be found in sections 3, 4, 6, 3.5.2 and 4.1. Abbreviations are listed in section 6.4.
117
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. c
DME, NEAA, 5 %FCS, 5 % CS. DME, NEAA, NUCS, 10 % FCS. DME, 5 % FCS, 5 % CS. MEEM, 10 % FCS. Glasgow modification of Eagle’s medium, 10 % CS, 10 % TPB. RPMI 1640, 10 % FCS. MEM, 7 % FCS 5 % CS, 3 % TPB, NEAA. DME, 10 % FCS. CMRL 1969, 10 % FCS. TC100, 10 % FCS. MEM, 20 % FCS. DME, 5 % FCS. MEM, 10 % CS. MEM, 5 % CS, NEAA. Medium 199, 5 % FCS. DME, 20 % FCS. Medium 199, 10 % FCS, 10 % TPB. DME, 1 % CHS, 5 % CS, 10 % TPB. F10, 2.5 % CHS, 20 % CS, 0,5 % BEE. Medium 199, 10 % FCS. F10, 10 % FCS or up to 20 % FCS. F10, 15% HS, 2.5 % FCS. BME, 10 % FCS. F12, 10 % FCS. Grace’s medium, 10 % FCS, 0.5 % LH. L-15, 10 % HS. NCTC 135, 10 % HuS. McCoy 5 A, 10 % FCS. NCTC 109, 20 % CS. MEM, NEAA, reduced bicarbonate (0.85 g/l), 10 % FCS.
Inoculation density
Includes only approximate classification of cells according to their optimal inoculation densities. These densities refer to cultures containing 3 mg Cytodex per millilitre and which are stirred continuosly in the final culture volume. If the cells are not available in sufficient quantities to be able to use these inoculation densities, the procedures outlined in section 3.4 can be used to obtain good results with fewer cells. L - Low inoculation density, 20-70x103 cells/ml. M - Medium inoculation density, 70-100x103 cells/ml. H - High inoculation density,100-300x103 cells/ml. It is recommended that newcomers to this technique begin with inoculation densities at the upper end of each range d
Comments
1. 2. 3. 4. 5. 6. 7. 8.
118
Negative for hypoxanthine-guanine phosphoribosyl transferase. Resistant to thioguani and often used as a parent in somatic cellhybridization. Hybrids using this cell have also been cultured on Cytodex microcarriers. Cultures must be started in a small volume, preferably 2-5 ml in wells or small dishes. Morphology may become rounded during later stages of culture. Stirring speed should be reduced and steps taken to control pH (section 4.4). Contaminated with HeLa. Produces large quantity of lactate, pH shift can be delayed by 10-20 ml mM HEPES in medium. From individual with Down’s syndrome and therefore does not have normal diploid karyotype. These cells should be grown at lower temperatures (25-30oC). Mutant cells of this type are extremly difficult to grow in all culture systems. Care should be taken when harvesting and the period of exposure to trypsin should be as short as possible.
9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.
Very large cells and less than 100 cells/microcarrier are found at confluence. This cell is of interest for urokinase production. These cells are often used for the production of HuIFNß. Clumping of these cells can occur during later stages of microcarrier culture. This can be avoided by increasing the stirring speed slightly at the stage when the culture reaches confluence. A very hardy cell line which can resist wide fluctuations in temperature, nutrition and environment without loss of viability. Frequent renewal of medium (3 times/week) is essential for high cell yields. Very careful techniques are required for these sensitive cells. Optimal culture conditions are different from those for mammalian cells. Cultures can be started with as few as 104 cells/ml. Cells should not be exposed to high levels of CO2. Serum quality is a particularly important variable (section 4.2.3). Some lines can grow in suspension without microcarriers. Such lines should be subcultured in static monolayer culture before microcarrier culture. Stirring speed may need to be reduced in order to make sure that such cells remain on the microcarriers. Removal of these cells from culture surfaces is difficult (section 3.7.6). Experiments can often be performed with the cells remaining on the microcarriers. May require high concentrations of trypsin for efficient removal from culture surfaces. Frequent renewal of medium and control of pH to 7.3 is essential. pH shift at later stages of culture should be controlled, otherwise cells detach. These cells produce large quantities of fibroblast interferon. Stirring speed should be decreased during later stages of culture. High cell yields require HEPES as a buffer against lactate accumulation (section 4.4.2). These cells grow as dense clusters. Faster stirring speeds may be required to avoid clumping and production of mucoid layers. Good condition of the inoculum is essential (section 3.4.5) Cells should be taken from a growing rather than a stationary culture. Rinsing culture with EDTA solution before trypsin is essential for rapid harvesting of cells (section 3.7.2).
119
Table 17. Tissue Specific list of cell types cultured on Cytodex microcarriers Tissue
Cell line
Adrenal Amnion Amniotic cells Bone Marrow
Mouse cortex tumour — Y-1 Human — WISH Human amniotic fluid Human — Detroit 6 Human — Detroit 38 Human nasal — RPMI 2650 Human Larynx — HEp 2 Human oral — KB Human cervical — HeLa Human colon Human thuroid Human — Chang “D“ Rabbit — SIRC Rabbit coronary endothelium Human coronary endothelium Mouse brain capillary endothelium Bovine pulmonary artery endothelium Human — NITC 2544 Human foreskin — FS-4 Human foreskin Detroit 532 Human — SV40 — transformed WI-38 Mouse — SC-1, 3T3, 3T6, L-cells, L-929, A9 Mouse — transformed Mouse — embryo Chicken — embryo Human — embryo Rat — embryo Rabbit — embryo Human — Xeroderma pigmentosum Muntjac — adult skin Human — HT 1080 Mouse Rainbow trout gonad — RTG Fat head minnow — FHM Carp eptihelioma — EPC Rat Rat — C6 Human Human atrial appendage — Girardi heart Rat — HTC, Morris MH1C1 Drosophila Spodoptera Trichoplusia Human embryo Human embryo — Flow 4000, L-132 Bovine embryo — MDBK Monkey — primary Dog — primary, MDCK, transformed Rabbit — primary, NZ white, LLC-RK RK-13 Rat — NRK, transformed Pig — PK-15, IBR Syrian hamster — HaK, BHK, transformed Potroo — Pt-k-1 Rhesus monkey — LLC-MK2 African Green monkey — Vero, CV-1 BSC-1, BGM, GL-V3 Human monocytic — J111
Carcinoma
Conjunctiva Cornea Endothelium
Epithelium Fibroblast
Fibrosarcoma Fish Glial Glial tumour Glioma Heart Hepatoma Insect Kidney
Leukemia
120
Tissue
Cell line
Liver
Human primary hepatocytes Rat primary hepatocytes Chimpanzee Human — Chang liver Chinese hamster — Don chimpanzee embryo — CR-1 Human embryo — L-132, MRC-5, MRC-9, WI-38, IMR-90, Flow 2002, HEL 299 Cat embryo Bat — Tb 1 Lu Mink — Mv 1 Lu Human — lymphoblastoid Human — lymphocytes Mouse — peritoneal, peripheral blood Rat — peritoneal Human — peripheral blood Mouse — P388D1 Human Mouse Chicken myoblasts Rat muscle-derived fibroblasts Mouse — Neuro-2a Human Chinese hamster — CHO Rat Rat Bovine Human — RD Human — McCoy Pig
Lung
Lymphoid Macrophage
Melanoma Muscle Neuroblastoma Osteosarcoma Ovary Pancreas Pituitary Rhabdomyosarcoma Synovial fluid Thyroid
6.2 Examples of microcarrier culture protocols The protocols are signed for use with rod stirred cultures or recently modified spinner vessels (section 3.2.2). Cell numbers and volumes can be altered proportionately if smaller or larger culture volumes are required. The procedures should be followed after reading the information in sections 3 and 4.
6.2.1 Diploid human fibroblast and the production of interferon The cells (MRC-5, WI-38, FS-4 etc.) should be used at as low a passage number as possible. Optimal results will be obtained when the cells are used between passages 10 and 25. The culture medium is described in table 12. For a culture with a final volume of 500 ml, 2 g sterile Cytodex 1 and 100 ml of medium are added to the culture vessel. This concentrated mixture of microcarriers in inoculated with 6x107 cells and incubated at 37oC. The cells are given sufficient time to attach and to begin to flatten on the microcarriers. This may take 6 h and during this period the culture should be stirred at 20 rpm for 2 min every 60 min. for those strains with a tendency to aggregate, slow (10 rpm) continuous stirring is required. Once the cells have flattened, the culture volume is increased to 250 ml with fresh medium and the culture is stirred at a speed just sufficient to keep all the microcarriers in suspension (20-30 rpm). After 2-3 days, 100 ml of medium is 121
discarded and the culture is diluted with fresh medium to 500 ml. Provided medium components do not become limiting, it is possible to achieve culture densities of 3x106 cells/ml in 9-10 days. It is usually necessary to change some of the medium on day 5. Interferon production When the culture has reached confluence (8-10 d) the serum concentration is reduced to 2.5 % and the cells are cultured for a further 3 days. The culture medium is removed and the culture washed twice with 100 ml PBS and once with 100 ml DME. DME containing 5 mg/ml human plasma protein (or albumin) and 50 IU of fibroblast interferon/ml (optional) is added to 200 ml. The culture is gently stirred at 10-20 rpm. After 16 h the medium is removed and the culture is briefly washed in 50 ml DME containing DEAE-Dextran (100 µg/ml) 200 ml DME containing cycliheximide (10 µg/ml ) and poly(I).poly(C) (20 g/ml) complexed with DEAEDextran ((100 µg/ml) are added and the culture is stirred gently at 10-20 rpm. After 5 h actinomycin D (0.75 µg/ml) is added and gentle stirring is continued for 3 h. The medium is removed and the culture is washed three times in 50 ml DME with 10 min for each wash. 200 ml DME containing human or bovine albumin (Cohn Fract. V, 0.5 mg/ml), ascorbic acid (10—5 M) and pyruvate (10—3 M) are added and the culture is stirred at 10-20 rpm for 16-20 h and the culture fluid is collected as a source of crude interferon. This procedure is based on those described in references 58 and 59. Note: Better results can be obtained if antibiotics are not used and all solutions are prewarmed. The interferon production phase is carried out at 34oC.
6.2.2 African Green monkey kidney cells (Vero) and the production of Simian Virus 40 A culture medium based on DME supplemented with 10 % foetal calf serum is most suitable (table 12). For a 1 litre culture 1-5x107 cells are added to 3 g of Cytodex 1 contained in 250 ml of medium. The culture is allowed to settle for 5-6 h with occasional stirring (20 rpm for 2 min every 60 min). If clumping of cells and microcarriers occurs, it may be necessary to stir continuously at 10 rpm. Once the cells have attached, the culture is diluted to 500 ml with fresh medium and stirring is commenced at a speed just sufficient to keep the microcarriers in suspension (approx. 20 rpm). After 1-2 days the culture is diluted to the final volume with fresh medium. At least 50 % of the culture medium should be replaced on about day 3-4. After 6-7 days the culture should contain 2-4x106 cells/ml. Virus production The confluent microcarriers are washed twice in 100 ml medium without serum and are resuspended in medium containing 1 % (v/v) foetal calf serum. Serum levels above 5 % (v/v) often inhibit infection. The volume of medium should be just sufficient to cover the microcarriers. Virus stock is added at a MOI of approximately 10 PFU/cell. Cell number is determined prior to infection by the usual procedures (section 3.6.3). After adsorption at 37 o for 2 h with occasional stirring,
122
the culture volume is increased to 500 ml with fresh medium containing 10 % (v/v) foetal calf serum. From this point on, the medium should contain a supplement of glucose (2 g/litre), non-essential amino acids and nucleosides (table 12 or section 6.4). Stirring should be just sufficient to keep the microcarriers in suspension (20-30 rpm). After 3-4 days the volume is increased to 1 litre with fresh medium and after 6-7 days a cytopathic effect is evident. At this point the medium can be collected or changed and collected 4 days later. During these later stages of culture, the volume can be decreased so as to increase the virus titre/ml. It is important that the pH should remain as close as possible to 7.2-7.4.
6.2.3 Primary monkey or dog kidney cells This type of cell is often cultured on Cytodex 3 microcarriers for the production of large quantities of virus for vaccines. The kidneys from a single animal are briefly perfused via the renal vein with a solution of 0.25 % (W/v) trypsin and 0.1 % (W/v) collagenase in PBS at 37oC, pH 7.4. The tissue is then cut into small pieces with scalpels and after washing in cold PBS incubated at 4oC in Hanks’ salt solution containing 0.025 M sodium citrate and 0.25 % (w/v) trypsin. After incubation for 20 h in 20-50 ml of this solution with gentle agitation the mixture is then warmed to 37oC and the tissues pieces are rinsed in medium containing serum (see below), resuspended in fresh medium and dispersed by a mixer. Debris is removed by passing the cell preparation through a 100 µm filter. Approximately 10 8 viable cells/ g wet weight of kidney can be obtained. For a 1 litre culture approximately 4-6x107 cells are added to 4 g of Cytodex 1 suspended in 500 ml of culture medium. The culture is placed at 37 oC and immediately stirred at a speed just sufficient to keep all the microcarriers in suspension (20-30 rpm). After about 12 h the culture is diluted to 1 litre with fresh medium. Provided medium componets do not become limiting during the later stages of culture, it is possible to achieve culture densities of greater than 2x106 cells/ml in about 7-8 days. It is usually necessary to change some of the medium on day 4; replacing 50 % of the volume with fresh medium is sufficient. The final cell yield can be influenced by the age and source of the kidneys. For small scale cultures a suitable medium is MEM supplemented with 10 % (v/v) newborn calf serum. A more economical alternative for large scale cultures is MEM supplemented with 5-8 % (v/v) newborn calf serum and 0.25 % (w/v) lactalbumin hydrolysate.
6.2.4 Primary chicken embryo fibroblasts A suitable culture medium for chicken embryo fibroblasts is DME supplemented with 1 % chicken serum, 5 % calf serum and 10 % tryptose phosphate broth. Preparation of primary chick embryo fibroblasts Embryos are removed aseptically from 9-11 day eggs and placed in a sterile Petri dish. The embryo is decapitated and transferred to a 50 ml centrifuge tube (plastic, conical). The embryo is minced briefly with a spatula, rinsed twice with 10 ml warm Ca2+-free, Mg2+-free PBS to remove erythrocytes and minced further with a fine spatula to 1 mm3 pieces. To this minced tissue is added 10 ml of warm 0.25 % (w/v)
123
trypsin in Tris saline and the suspension is pipetted up and down 10 times in a wide mouth pipette. This dissociation should be as gentle as possible and is repeated after the mixture has been incubated for 5-10 min at 37oC. The mixture is allowed to settle, the supernatant is collected and trypsin activity is neutralized by adding an equal volume of cold medium containing serum. The remaining pieces of tissue are dissociated as above and the cell suspensions are combined. The debris is washed several times by pipetting up and down in warm Tris salin and collecting the supernatant suspension. The final, pooled cell suspension is centrifuged (600 rpm, 10 min) and the pellet is gently resuspended in 20 ml culture medium containing serum. The yield from such an embryo is approximately 1-2x108viable cells. The above procedure gives a suspension of cells from the whole embryo. Under usual culture conditions fibroblast cells predominate, especially in the secondary culture. If a less heterogeneous primary inoculum is required, the skin can be dissected from the entire embryo. This is easily done with forceps and scalpels and the skin can be minced into small pieces in a Petri dish with scalpels. The skin pieces are washed briefly in Ca2+ -, Mg2+-free PBS and then dissociated as above. Material isolate from this source contains a greater proportion of fibroblasts. A difficulty which is sometimes encountered with material from certain strains of chickens, is excessive aggregation. Isolate chicken embryo cells tend to be rather sticky and this can be reduced by using collagenase instead of trypsin. In many situations a mixture of 0.1 collagenase in Tris saline results in cell suspensions with a reduced tendency to aggregate. EDTA should be omitted when using collagenase for tissue dissociation. When dissociation is complete a small amount of EDTA solution is added to inhibit the collagenase. Initiating the culture For a 1 litre culture 3 g of sterilized Cytodex microcarriers are added to the culture vessel in approximately 300 ml of culture medium. The culture is inoculated with 2x108 cells (approximately equivalent to the number of cells isolated from one egg) and stirred at about 10 rpm for 3-6 hours. This gentle stirring allows rapid attachment of a high proportion of cells (greater than 85 %) without excessive aggregation of cells and microcarriers. After this period of slow stirring the speed is increased to a rate just sufficient to keep all the microcarriers in suspension. After 24 hours the culture volume is increased to 500 ml with fresh medium. The above figure is representative for both primary and secondary cultures. Since these cells have finite lifespan the size of the inoculum must usually be increased when working with later subcultures. After 2 days the culture volume is increased to 700 ml with fresh medium and the final volume (1 litre) is achieved with fresh medium on day 3. It is usually necessary to change some of the medium during the period of rapid cell growth. Thus for best results 50 % of the medium should be changed on day 4-5. Confluence is achieved after 7-8 days and at least 90 % of the microcarriers should be covered with cells. The yield is usually 2-3x106 cells/ml using this procedure. 124
6.2.5Baby hamster kidney cells (BHK) A Suitable culture medium is Glasgow modification of Eagle’s medium supplemented with 10 % tryptose phosphate broth and 5-10 % calf serum. For a one litre culture approximately 6-8x107 cells are inoculated into 250 ml of medium containing 3 g of Cytodex 1 or Cytodex 3. The culture is stirred gently at 20 rpm for 2 min every 60 min. After 3-4 h the culture is diluted to 500 ml with fresh medium and stirred continuously at a speed just sufficient to keep the microcarriers in suspension (20-30 rpm). The culture is diluted to one litre after 2 days and it is usual to replace at least 50 % of the medium with fresh medium on day 4. Confluence is achieved after 5-6 days and corresponds to a culture density of 4-5x106 cells/ml.
6.3 Methods for determining the protein and DNA content of cells grown on microcarriers Protein content A few millilitres of microcarrier culture suspension are removed and added to a suitable tube. The microcarriers are allowed to settle, the medium is discarded and the microcarriers are washed three times in PBS. 1 ml of fresh PBS is added and the mixture is sonicated until all the cells have been thoroughly disrupted. The microcarriers are sedimented by centrifugation and a 100µl sample of the supernatant is removed and protein concentration is determined by the method of Lowry et al (see ref. 178). Fresh microcarriers in PBS and treated as above can be used to provide a reference sample. If necessary an additional sample from the culture can be used to determine cell number (section 3.6.2). Note: The denatured-collagen layer on Cytodex 3 microcarriers will contribute to the protein concentration when vigorous sonication is used. DNA content A few millilitres of microcarrier culture suspension are removed and added to a suitable tube. The microcarriers are allowed to settle, the medium is discarded and the microcarriers are washed with PBS. The supernatant is discarded and 1 ml of PBS containing Mithramycin (10µl/ml) and Mg C12 (15 mM) added. The mixture is sonicated until all the cells have been thourghly disrupted. The microcarriers are removed by centrifugation and the supernatant is measured in a fluorimeter (Ex. 440 nm; Em. 540 nm) with reference to standards containing 0.2-16.0 µg DNA/ml. This method is based on the technique described by Hill and Whatley (179). An additional sample from the culture can be used to determine cell number (section 3.6.3).
125
6.4 Abbreviations BEE BSA cAMP CIG CHS CS DEAE DME EDTA FCS HBSS HEPES HS HuS LH MEM MOI NEAA NUCS PBS PFU TPB
126
Beef embryo extract Bovine serum albumin Adenosine cyclic monophosphate Cold insoluble globulin Chicken serum Calf serum (newborn or donor) Diethylaminoethyl Dulbecco’s modification of Eagle’s medium Ethylenediamine tetraacetic acid Foetal calf serum Hank’s Balanced Salt Solution Hydroxyethylpiperazine ethane sulphonic acid Horse serum Human serum Lactalbumin hydrolysate Minimal Essential Medium Multiplicity of infection Non-essential amino acids (glycine, alanine, aspartic acid, asparagine, glutamic acid, each 0.1 mM; proline, serine, both 0.2 mM) Nucleosides (adenosine, guanosine, cytidine, uridine, each 30 µM; thymidine, 10 µM) Phosphate buffered saline Plaque forming units Tryptose phosphate broth.
7. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
Homogeneous cultivation of animal cells for the production of virus and virus products .Biotechnol. Bioeng. 11 (1969) 875-885, van Hemert,P., Kilburn, D. G., van Wezel, A.L. Virus production with a newly developed microcarrier system. Appl. Environ.Microbiol.34 (1977) 668-672, Giard, D.J.,Thilly, W.G., Wang, D.I.C. et al. Cell growth optimization in microcarrier culture. In Vitro 16 (1980) 859-865, Mered, B., Albrecht,P., Hopps, H.E. Cell culture on beads used for the industrial production of foot-and-mouth disease virus. Develop. Biol. Standard. 42 (1979) 141-145, Meignier, B. Growth of fish cell lines on microcarriers. Appl. Environ. Microbiol. 39 (1980) 394-397 Nicholson, B.L. Continuous cell propagation using low-charge microcarriers. Biotechnol. Bioeng. 23 (1981) 983-993, Crespi, C.L., Thilly, W.G. Production of Herpes viruses in microcarrier cultures of human diploid and primary chick fibroblast cells. Eur.J.Cell Biol. 22 (1980) 606, Griffiths, B., Thornton, B., McEntee, I. New trends in the preparation of cell substrates for the production of virus vaccines. Progr. Immunobiol. Standard.5 (1972) 187-192, van Wezel, A.L. Save with Cytodex TM 1. Separation News 4 (1979) 5, Pharmacia Fine Chemicals, Uppsala, Sweden. Foot and mouth disease virus production on microcarrier-grown cells. Develop. Biol. Standard 46 (1980) 249-256, Meignier, B., Mougeot, H., Favre, H. Cellular adhesiveness and extra-cellular substrata. Int.Rev. Cytol. 53 (1978) 65-144, Grinell,F. Density, distribution and mobility of surface anions on a normal/transformed cell pair. Exp.Cell Res. 118 (1979) 215-227, Borysenko, J.Z.,Woods, W. Primary culture of mammalian cells on microcarrier surface. Horng, C-B.C., Ph.D. Thesis, State University of New York, Buffalo, N.Y., USA, 1975. Primary suspension culture of calf anterior pituiary cells on a microcarrier surface. Biotechnol. Bioeng.17 (1975) 713-732, Horng, C-B., McLimans, W. Adhesion and spreading of cells on charged surfaces. J. Theor. Biol.49(1975)417-424, Maroudas, N.G. The large scale cultivation of diploid cell strains in microcarrier culture. Improvement of microcarriers. Develop. Biol. Standard.37 (1977) 143-147, van Wezel, A.L. Microcarriers for animal cell culture: a brief review of theory and practice. Develop. Biol. Standard. 46 (1980) 109-116, Hirtenstein, M., Clark, J., Lindgren, G. et al. Cell adhesion and spreading factor. Partial purification and properties. Exp. Cell Res. 110 (1977) 175-190, Grinnell, F., Hays. D.G., Minter, D.
127
19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34.
35. 36. 37.
128
Initial adhesion of human fibroblasts in serum-free medium: possible role secreted fibronectin. Cell 17 (1979) 117-129, Grinnell, F., Feld, M.K. Glycosaminoglycans that bind cold-insoluble globulin in cell-substratum adhesion sites of murine fibroblasts. Proc. Nat. Acad. Sci. USA 77 (1980) 6662-6666, Laterra,J., Ansbacher, R.,Culp, L.A. Fibronectins-adhesive glycoproteins of cell surface and blood. Nature 275 (1978) 179-184, Yamada, K.M., Olden, K. Optimization of growth surface parameters in microcarrier cell culture. Biotechnol. Bioeng. 21 (1979) 821-845, Levine, D.W., D.I.C.,Thilly, W.G. Cytodex TM 1:Beaded microcarriers for cell culture, Pharmacia Fine Chemicals, Technical Booklet Series, Uppsala, Sweden 1978. Alternative surfaces for microcarrier culture of animal cells. Develop. Biol. Standard. (1981, in press) Gebb, Ch., Clark, J.M., Hirtenstein, M.D. et al Molecular requirements for the adhesion and spreading of hamster fibroblasts. Exp.Cell Res. 121 (1979) 307-314, Hughes, R.C., Pena, S.D., Clark, J. Cold-insoluble globulin mediates the adhesion of rat liver cells to plastic petri dishes. Biochem. Biophys.Res.Commun. 79(1977) 726-733, Höök,M.,Rubin, K., Oldberg, A.et al. Distribution of fetal bovine serum fibronectin and endogenous rat cell fibronectin in extra-cellular matrix. J. Cell Biol. 84 (1979) 255-259, Hayman, E.G., Rouslahti, E. Studies of cell adhesion and recognition II. J. Cell Biol. 88 (1981) 138-148, Carter,W.G., Rauvla, H., Hakomori, S. Spreading of human fibroblasts in serum-free medium: Inhibition by dithiothreitol and the effect of cold insoluble globulin (plasma fibronectin). J. Cell Physiol. 104 (1980) 321-334, Grinell, F.,Feld, M.K. Fibronectin and prottttttteoglycans as determinants of cell-substratum adhesion. J. Supramol. Struct. 11 (1979) 401-427, Culp, L.A.,Murray, B.A.,Rollins,B.J. Growth of cell strains and primary cells on microcarriers in homogeneous culture. Nature 216 (1967) 64-65, van Wezel, A.L. Microcarrier cultures of animal cells. In “Tissue Culture:Methods and Applications” (Kruse,P.F.Pattersson, M.K.,eds) Academic Press, New York, 1973, pp. 372-377, van Wezel, A.L. Large scale cultivation of animal cells in microcarrier culture. Process Biochem. 13 (1978) 6-8, van Wezel, A.L., van der Velden-de Groot,C.A.M. Perspectives of mass culture and the mammalian cell. Presented at the W. Alton Jones Cell Science Center Workshop entitled “Large scale culture of plant and animal cells”. May 1976, Lake Placid, New York, Mc Limans, W.F., Gailani, S., Horng, C-B. Microcarrier cell culture: New methods for research-scale application. Somatic Cell Genet. 3 (1977) 149-155, Levine, D.W., Wong, J.S., Wang, D.I.C. et al. The mechanism of cell binding and cell-sheet formation in L strain fibroblasts. Exp. Cell Res. 20 (1960)428-437,Kuchler, R.J., Marlowe, M.L., Merchant, D.J. The adsorption of suspended MH129F cells to DEAE-Sephadex particles. Tohoku J. Agric. Res. 20 (1969) 19-26. Inooka,S.
38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49.
50. 51. 52. 53. 54. 55.
Attachment and proliferation of animal cells on microcarriers (Cytodex 1). In “Tissue culture in medical research” (Richards, R.J., Rajan, K.T., eds), Pergamon Press, Oxford, pp. 97-104, 1980, Hirtenstein. M., Claark, J. Optimizing culture conditions for the production of animal cells in microcarrier culture. Annals N.Y. Acad. Sci 369(1981) 33-46, Clark,J.M.,Hirtenstien, M.D. The production of inactivated polio vaccine on serially cultivated kidney cells from captive-bred monkeys. Develop. Biol. Standard. 46 (1980) 151-158, van Wezel, A.L., van der Velden-de Groot, C.A.M., van Herwaarden, J.A.M. The large-scale cultivation of Vero cells in microcarrier culture for virus vaccine production: preliminary results for killed poliovirus vaccine. Develop. Biol. Standard. 47 (1981) 55-64, Montagnon, B.J., Fanget, B.,Nicolas, A.J. Preparation of immobilized animal cells. FEBS Lett. 118 (1980) 145-150,, Nilsson, K., Anchorage and lymphocyte function II. Contact with non-cellular surfaces, cell density and T-cell activation. Immunology 43 (1981) 573-580, Sundqvist, K.G., Wanger, L. Media and growth requirements. Methods in Enzymology 58 (1979) 44-93, Ham, R.G., McKeehan, W.L. Prenatal diagnosis of X-linked diseases. Clin. Genet. 18 (1980) 147-150, Bernstein, R.E. Serially subcultivated cells as substrates for poliovirus production for vaccine. Develop. Biol. Standard. 47 (1981) 25-33, von Seefried, A., Chun, J.H. Synthesis and extrusion of collagen by freshly isolated cells from chick embryo tendon. Biochim. Biophys. Acta. 250 (1971) 358-369, Dehm, P.,Prockop, D.I. Production of an inactivated rabies vaccine in primary dog kidney cells. Develop. Biol.Standard. 40 (1978) 69-75, van Wezel, A.L., van Steenis, G. Large-scale concentration and purification of virus suspension from microcarrier culture for the preparation of inactivated virus vaccines. Develop. Biol. Standard. 42 (1979) 65-69, van Wezel, A.L., van Herwaarden, J.A.M., van de Heuvel-de-Rijk. New approach to the production of concentrated and purified inactivated polio and rabies tissue culture vaccinces. Develop. Biol. Standard.41 (1978) 159-168, van Wezel, A.L., van Steenis, G., Hannik, C.A. et al. Production of cell derived products: virus and interferon. Annals N.Y. Acad. Sci. 369 (1981) 47-64, Sinskey, A.J., Fleischaker, R.J., Tyo, M.A,et el. Propagation of poliovirus in microcarrier cultures of three monkey kidney cell lines. J.Biol. Standard.9 (1981) 137-145, Mered,B., Albrecht, P.,Hoppa,H.E. et al. Production of foot-and-mouth disease virus from BHK 21 C13 cells grown on the surface of DEAE Sephadex A50 beads, Biotechnol. Bioeng. 18 (1976) 659667, Spier, R.E., Whiteside, J.P. Propagation of mammalian cells and of virus in a self-regulating fermentor. Proc. Soc. Exp. Biol. Med. 164 (1980) 222-228, Föhring, B., Tjia, S.T., V.M. et al. Feasibility studies of oncornavirus production in microcarrier cultures. In Vitro 15 (1980) 507-515, Manousos, M., Ahmed, M., Torchio, C, et al.
129
56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 74.
130
Multiplication of normal and virus-infected diploid cells cultivated on microcarriers. Develop. Biol. Standard. 42 (1978) 153-158, Frappa, J., Beaudry, Y., Quillon, J.P. et al. Human interferon production with diploid fibroblast cells grown on microcarriers. Biotechnol. Bioeng. 21 (1979) 433-442, Giard, D.J., Loeb, D.H., Thilly, W.G. Examination of parmeters affecting human interferon production with microcarrier-grown fibroblast cells. Antimicrob. Agents Chemother. 18 (1980) 130-136, Giard, D.J., Fleischaker, R.J. High yield culture of human fibroblasts on microcarriers: a first step in production of fibroblastderived interferon (human beta interferon). J Interferon Res. 1 (1981) 391-400, Clark, J.M., Hirtenstein, M.D. Growth of human colon carcinoma cells on beaded microcarriers. J.Cell Biol. 83 (1979) 302 A, Pagé, M., Dufour, C. Skeletal muscle development in culture on beaded microcarriers, Cytodex 1. J. Cell Biol. 83 (1979) 115 A, Pawlowski, Krajic R., Loyd R., Przybylski, R. Differentiation of heart mucle cells from newborn rats on Cytodex 1 microcarriers. Eur. J. Appl. Microbiol. Biotechnol. In press, Moser, J.G., Stoffelsl, U. Culture and growth of free pancreatic islet cells on microcarrier beads. Diabetalogia 19 (1980) 259A, Bone, A.J., Hellerström, C. Microcarrier beads: a new approach to pancreatic islet cell culture. In Vitro (1981) In Press, Bone, A.J., Swenne, I. Superfusion of rat anterior pituitary cells attached to Cytodex beads; validation of a technique Endocrinology 107 (1980) 1425-1431. Smith, M.A., Vale, W.W. Desentization to gonadotropin-releasing hormone observed in superfused pituitary cells on Cytodex beads. Endocrinology 108 (1981) 752-759, Smith, M.A., Vale, W.W. Methods for microcarrier culture of bovine pulmonary artery endothelial cells avoiding the use of enzymes. Tissue and Cell 12 (1980) 619-635, Ryan, U.S., Mortara, M., Whitaker,C. Demonstration of growth in porcine thyroid cell culture. Biochimie 61 (1979) 923-930, Fayet. G., Hovsepian, S. Assay of intercellular adhesiveness using cell-coated Sephadex beads as collecting particles. J. Cell Sci, 22 (1976) 657-670, Vosbeck, K., Roth, S. ESR studies on membrane fluidity of Chinese hamster ovary cells groown on microcarriers and in suspension. Exp. Cell Res. 130 (1980) 437-442. Lai, CS., Hopwood, L.E., Swartz, H.M. Plasmamembrane isolation on DEAE-Sephadex beads. Biochim. Biophys. Acta 602 (1980) 207-212, Gottlib, L.J., Searls, D.B. Subcultivation of mammalian cells with a Sephadex derivative instead of proteinases. Eur. J. Cell Biol. 22 (1980) 599, Horst, J., Kern, M., Ulmer, E. Miotic cell populations obtained from a microcarrier culturing system. Exp. Cell Res. 123 (1979) 452-455, Mitchell, K.J., Wray, W. Selection of miotic Chinese Hamster Ovary cells from microcarriers. Anal. Biochem. 109 (1980) 231-238, Ng, J.J.Y., Crepsi, C.L., Thilly. W.G.
75. 76. 77. 78. 79. 80. 81. 82. 83. 84. 85. 86. 87. 88. 89. 90. 91. 92. 93.
Growth and nucleic acid synthesis in synchronously dividing populations of HeLa cells. Exp. Cell Res. 30 (1963) 344-362, Terasima, T., Tolmach, T.J. Dextran bibliography. Science and Education Administration, United States Department of Agriculture, Washington D.C., Miscellaneous Publication No. 1355, 1978. Control on the absence of DEAE-polysaccharides in DEAE-Sephadex purified poliovirus suspensions by pyrolysis mass spectrometry. Develop. Biol. Standard.47 (1981) 169-177, Windig, W., Haverkamp, J., van Wezel, A.L. Collagen substrata for studies on cell behavior. J. Cell Biol. 54 (1972) 626637, Elsdale, T., Bard, J. New techniques for culturing differentiated cells: reconstituted basement membrane rafts. Methods in Enzymology 58 (1979) 263-278, Reid, L.M., Rojkind, M. Rat skeletal muscle cells, In “Tissue Culture: Methods and Applications “ (Kruse, P.F., Patterson, M.K., eds) Academic Press, New York, 1973, pp 106114, Yaffe, D. Skeletal myoblasts in culture. Methods in Enzymology 58 (1979) 511-527, Koningsberg, I.R. Structurally distinct collagen types, Ann. Rev. Biochem. 49 (1980) 957-1003, Bornstein, P., Sage, H. The effect of different collagen types used as substrata on myogenesis in tissue culture. Cell Biol. Int. Reports 4 (1980) 841-850, John, H.A., Lawson, H. Adsorptopm, simple binding and complex binding of rat hepatocytes to various in vitro substrata. Exp. Cell Res. 129 (1980) 239-249, Gjessing, R., Seglen, P.O. Adhesion of rat hepatocytes to collagen. Exp. Cell Res. 117 (1978) 165-177, Rubin, K., Oldberg, Å., Höök, M. et al. Primary culture of parenchymal liver cells on collagen membranes. Exp. Cell Res. 94 (1975) 70-78, Michalopoulos, G., Pitot, H.C. Fetal phenotypic expression by adult rat hepatocytes on collagen gel/nylon meshes. Proc. Nat. Acad. Sci. USA 76 (1979) 283-287, Sirica, A.E., Richards, W., Tsukada, Y. et al. Long-term culture of capillary endothelial cells. Proc. Nat. Acad. Sci. USA 76 (1979) 5217-5221, Folkman, J., Haudenschild, C., Zetter, B. Primary culture of rat alveolar type II cells on floating collagen membranes. Exp. Cell Res. 128 (1980) 363-374, Geppert, E.F., Williams, M.C., Mason, R.J. Use of collagen-hydroxyethylmethacrylate hydrogels for cell growth. Proc. Nat. Acad. Sci. USA 77 (1980) 2064-2068, Civerchia-Perez, L., Faris, B., La Pointe, G. et al. Cellular adhesion fo collagen. Exp. Cell Res. 116 (1978) 470-474, Linsenmayer, T.F., Gibney, E., Toole, B.P. et al. Polarized monolayers formed by epithelial cells on a permeable and translucent support. J. Cell Biol. 77 (1978) 853-880, Cereijido, M., Robbins, E.S., Dolan, W.J. et al. Preparation to collagen substrates for cell attachment: Effect of collagen concentration and phosphate buffer. Anal. Biochem, 94 (1979) 308-312, Kleinman, H.K., McGoodwin, E.B., Rennard, S.I. et al.
131
94. 95. 96. 97. 98. 99. 100. 101. 102. 103. 104. 105. 106. 107. 108. 109. 110. 111. 112. 113.
132
Fibronectin-collagen binding and requirement during cellular adhesion. Biochem. J. 186 (1980) 551-559, Gold, L.I., Pearlstein, E. Affinity of fibronectin to collagens of different genetic types and to fibrinogen. J. Exp. Med. 147 (1978) 1584-1595, Engvall, E., Rouslahti, E., Miller, E.J. Role of collagenous matrices in the adhesion and growth of cells. J. Cell Biol. 88 (1981) 473-485, Kleinman, H.K., Klebe, R.J., Martin, G.R. Heparin enhances the rate of binding of fibronectin to collagen. Biochem. J. 187 (1980) 521-524, Johansson, S., Höök, M. An inexpensive, disposable minispinner for the cultivation of mammalian cells in suspension. Anal. Biochem, 102 (1980) 203-205, Zechel, K. A comparison of various labboratory scale culture configurations for microcarrier culture of animal cells. Develop. Biol. Standard. (1981) In press, Hirtenstein, M.D., Clark, J.M. The “Bilthoven Unit” for submerged cultivation of microorganisms, Biotechnol. Bioeng. 6 (1964) 381-401, Van Hemert, P. Critical parameters in the microcarrier culture of animal cells. Develop. Biol. Standard. 46 (1980) 117-124, Clark, J.,Hirtenstein, M., Gebb, C. Conrol of growth of kidney epithelial cells in culture. Hormones and Cell Culture, Cold Spring Harbor Conferences on Cell Profileration 6 (1979) 455459, Holley, R.W. The bulk growth of animal cells in continous suspension culture. J. Gen. Microbiol, 21 (1959) 702-720, Cooper, P.D., Wilson, J.N., Burt, A.M. Mass culture of mammalian cells. Methods in Enzymology 58 (1979) 194211, McLimans, W.F. The measurement of proliferation in tissue cultures by enumeration of cell nuclei. J. Nat. Cancer Inst. 11 (1951) 773-795, Sanford, K.K., Earle, W.R., Evans, V.J. et al. The DNA and RNA content of crude and crystalline trypsin used to trypsinize animal cell cultures: kinetics of trypsinization. Eur. J. Cell Biol. 22 (1980) 388, Kasche, V., Probst, K., Maass, J. The release of radioactive nucleic acids and mucoproteins by trypsin and EDTA treatment of baby hamster cells in tissue culture. Biochem J. 119 (1970) 707-714, Snow, C., Allen, A. Glycoprotein coat of the TA3 cell 1. Removal of carbohydrate and protein material from viable cells. J.Nat. Cancer Inst. 45 (1970) 637-647, Codington, J.F., Sanford, B.H., Jeanloz, R.W. Cell coat in tumour cells - effects of trypsin and EDTA; a biochemical and morphological study. Oncology 33 (1976) 11-23, Anghileri, L.J., Dermietzel, P. On the nature of the external surface of cultured human embryo fibroblasts. Differentiation 3 (1975) 91-97, Kelly, R.O., Lauer, R.B. Collagenases, Ann. Rev. Biochem. 49 (1980) 1063-1078, Harper, E. Isolation and identification of epithelial-like cells in culture by a collagenaseseparation technique. In Vitro 14 (1978) 746-753, Kanoza, R.J., Brunette, D.M., Pardon, A.D. et al. A modified “low pH” lignocaine mehtod to isolate human monocytes: a comparison with other separation procedures. J. Immunol.Methods 41 (1981) 279-288, Zanella, A., Mantovaniaa, A., Mariani, M. el al.
114. Trypsinization of BHK 21 monolayer cells grown in two large scale unit process systems. Biotechnol. Bioeng. 19 (1977) 1735-1738, Spier, R.E., Whiteside, J.P., Bolt, K. 115. Measurement of growth and viability of cells in culture. Methods in Enzymology 58( (1979) 141-152, Patterson, M.K. 116. The quantitative utilization of amino acids and glucose and contact inhibition of growth in cultures of human diploid cell WI-38. J. Cell Sci.6 (1970) 739749, Griffiths, J.B. 117. Chromosome aberrations in cultured cells deprived of single amino acids. Exp. Cell Res. 55 (1969) 393-409, Freed, J.J., Schatz, S.A. 118. Effects of cystine or glutamine restriction on human diploid fibroblasts in culture. In Vitro 14 (1978) 787-794, Corfield, V.A., Hay, R.J. 119. Effects of arginine deprivation upon chromosome aberrations, SCE’s and survival of CHO cells treated with mutagenic agents. Mutation Res. 62 (1979) 495-504, MacRae, W.D., MacKinnon, E.A., Stich, H.F. 120. Spontaneous decompotion of glutamine in cell culture media. Exp. Cell Res. 28 (1962) 360-364, Tritsch, G.L., Moore, G.E. 121. Improved medium for clonal growth of human diploid fibroblasts at low concentrations of serum protin. In Vitro 13 (1977) 399-416, McKeehan, W.L, Mc Keehan K.A., Hammond, S.L. et al. 122. Relationship of adhesiveness of cells in culture with specific enzyme activity. Exp. Cell Res. 122 (1979) 149-157, Grodin, M., Nyström., R. Scordato, J. et al. 123. The role of thymidine uptake in the control of cell proliferation. Exp. Cell Res. 119 (1979) 393-396, Taylor-Papadimitriou, J., Rozengurt, E. 124. Reciprocal regulation of glucose and glutamine utilization by cultured human diploid fibroblasts. J. Cell Physiol. 95 (1978) 41-48, Zielke, H.R., Ozand, P.T., Tildon, J.T.et al. 125. Retinoid-induced adhesion in cultured transformed mouse fibroblasts. J. Nat. Cancer Inst. 62 (1979) 1473-1477, Adamo, S., DeLuca, L.M., Akalovsky, I. et al. 126. Alterations in L fribroblast lipid metabolism and morpology during choline deprivation. Exp. Cell Res. 122 (1979) 103-113, Lynch, R.D., Schneeberger, E.E., Geyer, R.P. 127. Ascorbic acid inhibits replication and infectivity of avian RNA tumor virus. Proc. Natl. Acad. Sci. USA 77 (1980) 2711-2715, Bissell, M.J., Hatie, C., Farson, D.A, et al. 128. Cultivation of mammalian cells in defined media with protein and nonprotein supplements.. J.. Cell Biol. 30 (1966) 539-553, Healy, G.M., Parker, R.C. 129. Trace metal levels in commercially prepared tissue culture media. Tissue and Cell 12 (1980) 613-617, Glassman, A.B., Rydzewski, R.S., Bennett, C.E. 130. Human diploid cell cultures: their usefulness in the study of genetic variations in metabolism. In “Growth, Nutrion, and Metabolism of Cells in Culture” (Rothblat, G.H., Cristofalo, V.J. eds) Academic Press, New York 1972, pp 327-369, Mellman, W.J., Cristofalo, V.J. 131. Growth stimulation of BHK cells in culture by free biotin in serum. J Nat. Cancer Inst. 64 (1980) 639-644, Moskowitz, M., Cheng, D.K.S., Moscatello, D.K. et al.
133
132. Variability of hormone concentrations and ratios in commercial sera used for tissue culture. J. Nat. Cancer Inst. 50 (1973) 559-562, Esber, H.J., Payne, I.J., Bogden, A.E. 133. Effect of horse serum on neural cell differentiation in tissue culture. In Vitro 15 (1979) 641-648, Federoff, S.,Hall, C. 134. Inhibition of chondrogenesis by normal mouse serum in cultured chick limb cells. Exp. Cell Res. 130 (1980) 21-30, Parker, C.L., Paulsen, D.F., Rosebrook, J.A. et al. 135. A selective inhibitor of cell proliferation from normal serum. Proc. Nat. Acad. Sci. USA 77 (1980) 423-427, Harrington, W.N., Godman, G.C. 136. The effects of various mammalian sera on attachment efficiency and thymidine incorporation in primary cultures of mouse mammary epithelial cells. In Vitro 13 (1977) 275-279, Feldman, M.K., Wong, D.L. 137. Proliferation and morphology of chick embryo cells cultured in the precence of horse serum and hemoglobin. In Vitro 15 (1979) 587-592, Verger, C. 138. Fetal bovine serum: a multivariate standard. Proc. Soc. Exp. Biol. Med. 149 (1975) 344-347, Honn, K.V., Singley, J.A., Chavin, W. 139. Quality control studies on fetal bovine serum used in tissue culture. In Vitro 7 (1972) 174-189, Boone, C.W., Mantel, N., Caruso, T.D. et al. 140. Effects of steroid hormones in fetal bovine serum on plating and cloning of human cells in vitro. In Vitro 12 (1976) 23-30, Milo, G.E., Malarkey, W.B., Powell, J.E. et al. 141. Plating efficiency for primary hamster embryo cells as an index of efficacy of fetal bovine serum for cell culture. Appl. Microbiol. 26 (1973) 525-528, Goodheart, C.R., Casto, B.B., Zwiers, A. 142. Serum-free culture: a unifying approach. Cell 22 (1980) 649-655, Barnes., D., Sato, G 143. Serum supplements and serum-free media: Applicability for microcarrier culture of animal cells. Develop. Biol Standard. (1981, in press), Clark, J.M., Gebb, Ch., Hirtenstein, M.D. 144 High yield microcarrier culture of animal cells in low-serum and serum-free media. Eur.J.Cell Biol. 22 (1980) 606, Clark, J.M.,Gebb, Ch., Hirtenstein, M.D. 145. Effects of oxygen on growth in several established cell lines. J.Nat.Cancer Inst. 28 (1962) 897-909, Pace, D.M., Thompson, J.R, van Camp, W.A. 146. The effects of oxygen tension on the growth and metabolism of a mammalian cell. Exp. Cell Res. 25 (1961) 101-113, Brosemer, R.W., Rutter, W.J. 147. Influence of oxygen and pH on plating efficiency and colony development of WI-38 and Vero cells. Exp Cell Res. 86 (1974) 152-156, Taylor, W.G., Richter, A., Evans, V.J. et al. 148. Density-dependent effects of oxygen on the growth of mammalian fibroblasts in culture.J. Cell Physiol. 95 (1978) 33-40, Taylor, W.G., Camalier, R.F., Sanford, K.K. 149. The effect of oxygen tension on the growth and metabolism of WI-38 cells. J.Cell Physiol, 89 (1976) 235-250, Balin, A.K., Goodman, D.B.P., Rasmussen, H. et al. 150. The effect of dissolved oxygen partial pressure on the growth and caarbohydrate metabolism of mouse LS cells. J. Cell Sci. 4 (1969) 25-37, Kilburn, D.G., Lilly, M.D., Self, D.A. et al. 134
151. The cultivation of animal cells at controlled dissolued oxygen partial pressure. Biotechnol. Bioeng. 10 (1968) 801-814, Kilburn, D.G., Webb, F. C. 152. Growth and metabolism of L cells in a chemically defined medium in a controlled environment culture system. Appl. Microbiol. 21 (1971) 928-933, Taylor, G.W., Kondig, J.P., Nagle, S.C. et al. 153. The gaseous environment of the mammalian cell in culture. In “Growth, Nutrition, and Metabolism of Cells in Culture” (Rothblat, G.H., Christofalo, V.J. eds) Academic Press, New York, 1972, pp 137-170, McLimans, W.F. 154. pH as a determinant of cellular growth and contact inhibition. Proc. Nat. Acad. Sci. US 68 (1971) 229-233, Ceccarini, C., Eagle, H. 155. Effect of pH on plating efficiency, serum requirement, and incorporation of radioactive precursors into human cells. In Vitro 11 (1975) 78-86, Ceccarini, C. 156. Dermal fibroblasts, Methods Cell Biol. 21A (1980) 255-276, Ham, R.G. 157. Induction and reversal of contact inhibition of growth by pH modification. Nature 233 (1971) 271-273, Ceccarini, C., Eagle, H. 158. Buffer combinations for mammalian cell culture. Science 174 (1971) 500-503, Eagle H. 159. Evidence that glutamine, not sugar, is the major energy source for cultured HeLa cells. J.Biol.Chem. 254 (1979) 2669-2676, Reitzer, L.J., Wice, B.M., Kennell, F. 160. Lactate: a major product of glutamine metabolism by human diploid fibroblasts. J. Cell Physiol. 104 (1980) 433-441, Zielke, H.R., Sumbilla, C.M., Sevdalian, D.A. et al. 161. Reduction of lactic acid secreted from SV3T3 cells by a serum factor and biotin and its relationship to cell growth. Biochem.Biophys. Res. Commun. 93 (1980) 1036-1043, Young, D.V., Nakano, E.T. 162. Utilization of carbohydrate by Madin-Darby canine kidney (MDCK) cells in high density suspension of dextran microcarriers. Fed. Proc. 39 (1980) 2145, Imamura, T., Crespi, C.L., Bruengraber, H. 163. Growth and glycolysis in the human diploid cell strain WI-38. Proc.Soc. Exp. Biol. Med. 118 (1965) 1109-1113, Cristofalo, V.J., Kritchersky, D. 164. Determination and survey of osmolality in culture media, In “Tissue Culture: Methods and Applications” (Kruse, P.F., Patterson, M.K. eds) Academic Press, New York, 1973, pp 703-709, Waymouth, C. 165. Cell and tissue culture. Paul, J., E & S. Livingstone, Edinburgh, 4th edition, 1970. 166. Tissue culture: methods and applications, Kruse, P.F., Patterson, M.K. eds., Academic Press, New York, 1973. 167. Contamination in tissue culture, Fogh, J. ed. Academic Press, New York, 1973. 168. The effect of antibiotics on the growth and longivity of human dipoid fibroblasts in vitro. Acta Path. Microbiol. Scand. 81B (1973) 593-605, Litwin.J. 169. Effects of some antibiotics on the growth of human diploid skin fibroblasts in cell culture. In vitro 15 (1979) 114-119, Goetz, I.E., Moklebust, R., Warren, C.J.
135
170. Effects of some antibiotics on protein synthesis and degradation in primary cultures of rat hepatocytes. In vitro 17 (1981) 71-76, Schwarze, P.E., Seglen, P.O. 171. Gentamicin-induced lysosomal phospholipidosis in cultured rat fibroblasts. Lab. Invest. 40 (1979) 481-491, Aubert-Tulkens, G., van Hoof, F., Tulkens, M.D. 172. Mycoplasmas and cell cultures. Bacteriol. Rev. 35 (1971) 206-227, Stanbridge, E. 173. In situ detection of mycoplasma contamination in cell cultures by fluorescent Hoechst 33258 stain. Exp. Cell Res. 104 (1977) 255-262, Chen T.R. 174. A direct comparison of procedures for the detection of mycoplasma in tissue culture. J. Immunol. Methods 38 (1980) 315-324, Hessling, J.J., Miller, S.E., Levy, N.L. 175. Cell culture for biochemists. Adams, R.L.P., Elsevier/North Holland Biomedical Press, Amsterdam 1980. 176. Cell culture. Methods in Enzymology (Jacoby, W.B., Pastan I.H., eds), Academic Press, New York, vol. 58, 1979. 177. Journal of Tissue Culture Methods (formerly TCA Manual), Tissue Culture Association, 1 Bank St., Suite 210, Gatihersburg, MD 20760, USA. 178. Review of the Folin phenol protein quantitation method of Lowry, Rosebrough, Farr and Randall. Anal, Biochem. 100 (1979) 201-220, Peterson, G.L. 179. A rapid microassay for DNA. FEBS Lett. 56 (1975) 20-23, Hill, B.T., Whatley, S. 180. The uptake of amino acids by mouse cells (strain LS) During growt in batch culture and chemostat culture: the influence of growth rate. Proc. Roy. Soc. B 168 (1967) 421-438, Griffiths, J.B., Pirt, S.J. 181. Growth characteristics, cytogenetic stability and virus susceptibility of cell lines from New Zealand rabbits. Develop. Biol. Standard. 37 (1977) 35-39, Early, E.M. 182. Amino acid metabolism in mammalian cell culture. Science 130 (1959) 432437, Eagle, H. 183. The effect of environmental pH on the growth of normal and malignan cells. J. Cell Physiol. 82 (1973) 1-8, Eagle, H. 184. Alm, G., Department of Medical Virology, Biomedical Centre, Uppsala, Sweden. 185. Kronenberg, L., LEE BioMolecular Research Laboratories, Inc., 11211 Sorrento Valley Road, Suite M, San Diego, Ca 92121,USA. 186. Danö, K., Laboratory of Tumour Biology, Institute of Medical Microbiology, University of Copenhagen, Juliane Maries Vej 28, 2100 Copenhagen, Denmark. 187. Busch, K., Department of Pathology, University of Uppsala, Uppsala, Sweden. 188. Talbot, P., Wellcome Research Laboratories, Langly Court, Beckenham, Kent BR3 3BS, Great Britain. 189. Slater, H., Department of Biochemistry, Royal Infirmary, Glasgow, Great Britain. 190. Duda, E., Mta Sszegedi Biologiai Koezpont, Biokemiai Intezete, Odesszai Krt 42, H-6726 Szeged, Hungary.
136
191. Smit, S., Department of Chemical Pathology, Erasmus University, Rotterdam, The Netherlands. 192. van Wezel, A., Rijksinstituut voor de Volksgezondheid, P.O.B. 1, NL-3720 BA Bilthoven, The Netherlands. 193. Przybylski R.J., Department of Anatomy, School of Medicine, Case Western Reserve University, 2119 Abington Road, Cleveland OH 44106, USA. 194. Ammonia loading in cell culture systems. Cell Biol. Int. Reprots 5 (1981) 653660, Mc Limans, W.F., Blumenson, L.E., Repasky, E. et al.
137
138
The Recombinant Protein Handbook Protein Amplification and Simple Purification
Back to Collection 18-1142-75 Edition AB
Handbooks from Amersham Pharmacia Biotech
Antibody Purification Handbook 18-1037-46
The Recombinant Protein Handbook Protein Amplification and Simple Purification 18-1142-75
Protein Purification Handbook 18-1132-29
Ion Exchange Chromatography
Reversed Phase Chromatography
Principles and Methods 18-1114-21
Principles and Methods 18-1134-16
Affinity Chromatography
Expanded Bed Adsorption
Principles and Methods 18-1022-29
Principles and Methods 18-1124-26
Hydrophobic Interaction Chromatography
Chromatofocusing
Principles and Methods 18-1020-90
with Polybuffer and PBE 50-01-022PB
Gel Filtration
Microcarrier cell culture
Principles and Methods 18-1022-18
Principles and Methods 18-1140-62
The Recombinant Protein Handbook Protein Amplification and Simple Purification
1
Contents Introduction ............................................................................................................. 5 Symbols and abbreviations .............................................................................................................. 5
CHAPTER 1 .............................................................................................................. 6 Choice of host for protein amplification ......................................................................... 6 Choice of vectors ......................................................................................................... 7 Vectors for non-fusion proteins ......................................................................................................... 7 Vectors for fusion proteins ............................................................................................................... 8 Choice of fusion tag ........................................................................................................................ 8
CHAPTER 2 .............................................................................................................. 9 Protein amplification ................................................................................................... 9 Sample extraction .......................................................................................................................... 9 Troubleshooting protein amplification ............................................................................................... 9
CHAPTER 3 ............................................................................................................ 13 GST fusion proteins ................................................................................................... 13 Amplification ............................................................................................................................... 13 Purification .................................................................................................................................. 14 Detection of GST fusion proteins .................................................................................................... 21 Purification and detection troubleshooting ...................................................................................... 28 Tag removal by enzymatic cleavage ................................................................................................. 30 PreScission Protease cleavage and purification ................................................................................ 31 Thrombin cleavage and purification ................................................................................................ 35 Factor Xa cleavage and purification ................................................................................................ 37 Removal of thrombin, Factor Xa or other serine proteases ................................................................. 39
CHAPTER 4 ............................................................................................................ 41 (His)6 fusion proteins ................................................................................................. 41 Amplification ............................................................................................................................... 41 Purification .................................................................................................................................. 41 Detection of (His)6 fusion proteins .................................................................................................. 53 Purification and detection troubleshooting ...................................................................................... 56 Tag removal by enzymatic cleavage ................................................................................................. 58
CHAPTER 5 ............................................................................................................ 59 Handling inclusion bodies .......................................................................................... 59 Solubilization of inclusion bodies ................................................................................................... 59 Refolding of solubilized recombinant proteins ................................................................................. 60
CHAPTER 6 ............................................................................................................ 63 Harvesting and extraction of recombinant proteins ........................................................ 63
CHAPTER 7 ............................................................................................................ 67 Buffer exchange and desalting of recombinant proteins ................................................. 67
CHAPTER 8 ............................................................................................................ 71 Simple purification of other recombinant proteins ......................................................... 71 Ready to use affinity purification columns ....................................................................................... 71 Making a specific purification column ............................................................................................ 73 Purification .................................................................................................................................. 75
2
CHAPTER 9 ............................................................................................................ 77 Multi-step purification of recombinant proteins (fusion and non-fusion) .......................... 77 Selection and combination of purification techniques ....................................................................... 78
Appendix 1 ............................................................................................................ 86 Map of the GST fusion vectors showing reading frames and main features ....................... 86 Glutathione S-transferase (GST) .................................................................................. 87
Appendix 2 ............................................................................................................ 88 Amino acids table ...................................................................................................... 88
Appendix 3 ............................................................................................................ 90 Protein conversion data .............................................................................................. 90
Appendix 4. ........................................................................................................... 90 Centrifuges, rotors and carriers for use with MicroPlex 24 .............................................. 90
Appendix 5 ............................................................................................................ 91 Characteristics, cleaning and storage of Glutathione Sepharose ...................................... 91 Characteristics, cleaning and storage of Chelating Sepharose ......................................... 92
Appendix 6 ............................................................................................................ 93 Column packing and preparation ................................................................................. 93
Appendix 7 ............................................................................................................ 95 Converting from linear flow (cm/hour) to volumetric flow rates (ml/min) and vice versa ...... 95
Appendix 8 ............................................................................................................ 96 Selection of purification equipment ............................................................................. 96
Appendix 9 ............................................................................................................ 97 Principles and standard conditions for purification techniques ....................................... 97 Affinity Chromatography (AC) ......................................................................................................... 97 Ion Exchange (IEX) ....................................................................................................................... 97 Hydrophobic Interaction Chromatography (HIC) ............................................................................... 99 Gel Filtration (GF) Chromatography .............................................................................................. 100 Reversed Phase Chromatography (RPC) ........................................................................................ 101
Additional reading and reference material .................................................................. 104 Ordering information ................................................................................................ 105
3
General Purification of fusion proteins Native conditions
Binding buffer
Denaturing conditions
Binding buffer including 8 M Urea or 6 M Gua-HCl
Cell lysis
Binding to affinity media
Binding buffer
Elution buffer: Binding buffer with increased amount of imidazole ((His)6 fusion proteins) or glutathione (GST fusion proteins)
Binding buffer (as above) added 10-50 mM imidazole for (His) 6 fusion protein
Wash
Elute
Elution buffer: Binding buffer (as above) with increased amount of imidazole ((His)6 fusion proteins)
Pure denatured fusion protein
Pure fusion protein
Refolding
Fusion protein Cell protein
4
Introduction This handbook is intended for the general reader interested in the amplification and purification of recombinant proteins and for everyday use at the laboratory bench. The use of recombinant proteins has increased greatly in recent years, as has the wealth of techniques and products used for their amplification and purification. The advantages of using a fusion protein to facilitate purification and detection of the recombinant proteins are now widely recognised. This handbook introduces the reader to the initial considerations to be made when deciding upon host, vector and use of a fusion or non-fusion protein and covers general guidelines for successful protein amplification. General advice is also given on harvesting and extraction, handling of inclusion bodies, tag removal and removal of unwanted salts and small molecules. The more that is known about the characteristics of a protein, the more easily it can be isolated and purified. Consequently, fusion proteins are simple and convenient to work with and, for many applications, a single purification step, using a commercially available affinity chromatography column, is sufficient. This is clearly demonstrated in the specific chapters on the amplification, purification and detection of the two most common fusion proteins (GST and (His)6 tagged proteins) which include simple practical protocols for use in the laboratory. The handbook also gives suggestions for the successful purification of other fusion proteins by a single affinity chromatography step. In situations where no fusion system is available, or when a higher degree of purity is required, a multi-step purification will be necessary. This can also become a straightforward task by following a Three Phase Purification Strategy reviewed in the final chapter.
Symbols and abbreviations this symbol gives general advice that can improve procedures and provides recommendations for action under specific situations. this symbol denotes advice that should be regarded as mandatory and gives a warning when special care should be taken in a procedure. this symbol gives troubleshooting advice to help analyse and resolve any difficulties which may occur. reagents and equipment required. experimental protocol. PBS
phosphate buffered saline.
5
CHAPTER 1 Choice of host for protein amplification Several host systems are available including bacteria, yeast, plants, filamentous fungi, insect or mammalian cells grown in culture and transgenic animals. The final choice of host will depend upon the specific requirements and applications for the recombinant protein. Table 1 reviews commonly used host systems with their advantages and disadvantages. The choice of host affects not only the amplification of the protein, but also the way in which the product can be subsequently purified. In order to decide which host is most suitable the amount and the degree of purity of the product as well as its biological integrity and potential toxicity should be considered. For example, bacterial expression systems are not suitable if post-translational modification is required to produce a fully functional recombinant product. The location of product within the host will affect the choice of methods for isolation and purification of the product. For example, a bacterial host may secrete the protein into the growth media, transport it to the periplasmic space or store it as insoluble inclusion bodies within the cytoplasm. Host Bacteria e.g.Escherichia coli
Bacteria e.g. Staphylococcus aureus
Easy to grow with high yields (product can form up to 50% of total cell protein) Product can be designed for secretion into the growth media Secretes fusion proteins into the growth media
Mammalian cells
Same biological activity as native proteins Mammalian expression vectors available Can be grown in large scale cultures
Yeasts
Lacks detectable endotoxins Generally Regarded As Safe (GRAS) Fermentation relatively inexpensive Facilitates glycosylation and formation of disulphide bonds Only 0.5% native proteins are secreted so isolation of secreted product is simplified Well established large scale production and downstream processing Facilitates glycosylation and formation of disulphide bonds Safe, since few arthropods are adequate hosts for baculovirus Baculovirus vector received FDA approval for a clinical trial Virus stops host protein amplification. High level expression of product
Cultured insect cells Baculovirus vector
Table 1 (continued).
6
Advantages Many references and much experience available Wide choice of cloning vectors Gene expression easily controlled
Disadvantages No post-translational modification
Biological activity and immunogenicity may differ from natural protein High endotoxin content in gram negative bacteria
Does not express such high levels as E. coli
Pathogenic Cells can be difficult and expensive to grow Cells grow slowly Manipulated cells can be genetically unstable Low productivity as compared to micro-organisms Gene expression less easily controlled Glycosylation not identical to mammalian systems
Lack of information on glycosylation mechanisms Product not always fully functional Few differences in functional and antigenic properties between product and native protein
Host Fungi e.g.Aspergillus sp.
Advantages Well established systems for fermentation of filamentous fungi Growth inexpensive A.niger is GRAS Can secrete large quantities of product into growth media, source of many industrial enzymes
Plants
Disadvantages High level of expression not yet achieved Genetics not well characterized No cloning vectors available
Low transformation efficiency Long generation time
Table 1.
Choice of vectors In order to clone the gene of interest all engineered vectors have a selection of unique restriction sites downstream of a transcription promotor sequence. The choice of vector family is governed by the host. Once the host has been selected, many different vectors are available for consideration, from simple expression vectors to those that secrete fusion proteins. However, as for the selection of a suitable host system, the final choice of vector should take into consideration the specific requirements of the application and will, of course, be influenced by the behaviour of the target protein. One key factor that has led to the increased use of fusion protein vectors is that amplification of a fusion protein containing a tag of known size and biological function can greatly simplify subsequent isolation, purification and detection. In some cases the protein yield can also be increased. Table 2 reviews some of the features of fusion protein amplification that may influence the final choice of vector. Maintenance and cloning protocols are highly specific for each vector and the instructions provided by the supplier should be followed carefully. Advantages Fusion proteins Targetting information can be incorporated into a tag Provide a marker for expression Simple purification using affinity chromatography under denaturing or non-denaturing conditions Easy detection Refolding achievable on a chromatography column Ideal for secreted proteins as the product is easily isolated from the growth media Non- fusion proteins No cleavage steps necessary
Disadvantages Tag may interfere with protein structure and affect folding and biological activity Cleavage site is not always 100% specific if tag needs to be removed
Purification and detection not as simple Problems with solubility may be difficult to overcome, reducing potential yield
Table 2.
Vectors for non-fusion proteins Table 3 shows examples of non-fusion vectors. Vector family pTrc 99 A pKK223-3 pSVK 3 PSVL SV40 pMSG
Comments E. coli vector for expression of proteins encoded by inserts lacking a start codon, inducible by IPTG For over-expression of proteins under the control of the strong tac promotor in E. coli For in vivo expression in mammalian cell lines For high level transient expression in mammalian cells For inducible expression in mammalian cells
Table 3.
7
Vectors for fusion proteins Table 4 shows examples of vectors for fusion proteins together with the required purification product. Vector family pGEX
Tag Glutathione S-transferase
PQE
6 x Histidine
pET
6 x Histidine
pEZZ 18 (non-inducible expression) pRIT2T(expression inducible by temperature change)
IgG binding domain of protein A IgG binding domain of protein A
Purification Products GST MicroSpin™ Purification Module GSTrap™ FF Glutathione Sepharose™ Fast Flow His MicroSpin Purification Module HisTrap™ Kit HiTrap™ Chelating HP Chelating Sepharose Fast Flow His MicroSpin Purification Module HisTrap Kit HiTrap Chelating HP Chelating Sepharose Fast Flow IgG Sepharose 6 Fast Flow IgG Sepharose 6 Fast Flow
Table 4. Please refer to Chapter 3 and 4 for further details of purification products for GST and (His)6 fusion proteins.
Choice of fusion tag The two most commonly used tags are glutathione S-transferase (GST tag) and 6 x histidine residues (His)6 tag. As for the selection of host and vectors, the decision to use either a GST or a (His)6 tag must be made according to the needs of the specific application. Table 5 highlights some key features of these tags that should be considered. GST tag Can be used in any expression system Purification procedure gives high yields of pure product Selection of purification products available for any scale pGEX6P PreScission™ protease vectors enable cleavage and purification in a single step Site-specific proteases enable cleavage of tag if required
GST tag easily detected using an enzyme assay or an immunoassay Simple purification. Very mild elution conditions minimize risk of damage to functionality and antigenicity of target protein
GST tag can help stabilize folding of recombinant proteins Fusion proteins form dimers
(His)6 tag Can be used in any expression system Purification procedure gives high yields of pure product Selection of purification products available for any scale Small tag may not need to be removed e.g. tag is poorly immunogenic so fusion partner can be used directly as an antigen in antibody production Site-specific proteases enable cleavage of tag if required. N.B. Enterokinase sites that enable tag cleavage without leaving behind extra amino acids are preferable (His)6 tag easily detected using an immunoassay Simple purification, but elution conditions are not as mild as for GST fusion proteins. Purification can be performed under denaturing conditions if required. N.B. Neutral pH but imidazole may cause precipitation Desalting to remove imidazole may be necessary (His)6 - dihydrofolate reductase tag stabilizes small peptides during expression Small tag is less likely to interfere with structure and function of fusion partner Mass determination by mass spectrometry not always accurate for some (His)6 fusion proteins*
Table 5. *Geoghegan, K.F., et al., Anal Biochem 267 (1), 169–184 (1999).
Polyhistidine tags such as (His)4 or (His)10 are also used. They may provide useful alternatives to (His)6 if there are specific requirements for purification, as discussed on page 42.
8
CHAPTER 2 Protein amplification Cell culture conditions are dependent upon the host system. Follow the instructions of the supplier. Before performing a large scale purification, check protein amplification in the culture or do a small pilot experiment to establish optimum conditions for expression. Monitor expression during growth and induction by one or more of the detection methods referred to in this handbook. Retain small samples at key steps in all procedures for analysis of the purification method. Yield of fusion proteins is highly variable and is affected by the nature of the fusion protein, the host cell, and the culture conditions. Fusion protein yields can range from 0–10 mg/ml. Table 6 can be used to approximate culture volumes based on an average yield of 2.5 mg/ml. Protein Culture Volume Volume of sonicate
12.5 µg
50 µg
1 mg
10 mg
5 ml
20 ml
400 ml
4l
50 mg 20 l
0.5 ml
1 ml
20 ml
200 ml
1000 ml
Table 6.
Sample extraction The various methods for sample extraction are reviewed in Chapter 6.
Troubleshooting protein amplification (for specific details on GST or (His)6 fusion proteins, see page 21 for detection of GST fusion proteins or page 53 for detection of (His)6 fusion proteins).
High basal level of expression • Add 2% glucose to the growth medium. This will decrease the basal expression level associated with the upstream lac promoter but will not affect basal level expression from the tac promoter. The presence of glucose should not significantly affect overall expression following induction with IPTG. • Basal level expression (i.e. expression in the absence of an inducer, such as IPTG), present with most inducible promoters, can affect the outcome of cloning experiments for toxic inserts; it can select against inserts cloned in the proper orientation. Basal level expression can be minimized by catabolite repression (e.g. growth in the presence of glucose). The tac promoter is not subject to catabolite repression. However, with the pGEX vector system there is a lac promoter located upstream between the 3´-end of the lacIq gene and the tac promoter. This lac promoter may contribute to the basal level of expression of inserts cloned into the pGEX multiple cloning site, and it is subject to catabolite repression.
9
No protein is detected in bacterial sonicate • Check DNA sequences. It is essential that protein-coding DNA sequences are cloned in the proper translation frame in the vectors. Cloning junctions should be sequenced to verify that inserts are in-frame. • Optimize culture conditions to improve yield. Investigate the effect of cell strain, medium composition, incubation temperature and induction conditions. Exact conditions will vary for each fusion protein expressed. • Analyse a small aliquot of an overnight culture by SDS-PAGE. Generally, a highly expressed protein will be visible by Coomassie™ blue staining when 5–10 µl of an induced culture whose A600 is ~1.0 is loaded on the gel. Non-transformed host E. coli cells and cells transformed with the parental vector should be run in parallel as negative and positive controls, respectively. The presence of the fusion protein in this total cell preparation and its absence from a clarified sonicate may indicate the presence of inclusion bodies. • Check for expression by immunoblotting. Some fusion proteins may be masked on an SDS-polyacrylamide gel by a bacterial protein of approximately the same molecular weight. Immunoblotting can be used to identify fusion proteins in these cases. Run an SDS-polyacrylamide gel of induced cells and transfer the proteins to a nitrocellulose or PVDF membrane (such as Hybond™-C or Hybond-P). Detect fusion protein using anti-GST or anti-His antibody.
Most of fusion protein is in the post-sonicate pellet • Check cell disruption procedure. Cell disruption is seen by partial clearing of the suspension or by microscopic examination. Addition of lysozyme (0.1 volume of a 10 mg/ml lysozyme solution in 25 mM Tris-HCl, pH 8.0) prior to sonication may improve results. Avoid frothing as this may denature the fusion protein. • Reduce sonication since over-sonication can lead to co-purification of host proteins with the fusion protein. • Fusion protein may be produced as insoluble inclusion bodies. Try altering the growth conditions to slow the rate of translation, as suggested below. It may be necessary to combine these approaches. Exact conditions must be determined empirically for each fusion protein. - Lower the growth temperature (within the range of +20 to +30 °C) to improve solubility. - Decrease IPTG concentration to < 0.1 mM to alter induction level. - Alter time of induction. - Induce for a shorter period of time. - Induce at a higher cell density for a short period of time. - Increase aeration. High oxygen transport can help prevent the formation of inclusion bodies.
10
It may be necessary to combine the above approaches. Exact conditions must be determined empirically for each fusion protein. • Alter extraction conditions to improve solubilization of inclusion bodies (see Chapter 5).
Quantification of fusion proteins Fusion proteins must be purified to homogeneity and quantified using a standard protein assay. • The relative yield of fusion protein can often be determined by measuring the absorbance at 280 nm (suitable for both GST and (His)6 fusion proteins). • The yield of protein may also be determined by standard chromogenic methods (e.g. Lowry, BCA, Bradford, etc.). • Immunoassays can be used for quantification if a suitable standard curve can be produced. In this case, the fusion protein does not have to be purified for quantification as long as a purified standard is available. The immunoassay technique is also particularly suitable for screening large numbers of samples when a simple yes/no answer is required, as, for example, when testing fractions from a purification.
11
12
CHAPTER 3 GST fusion proteins Amplification Glutathione S-transferase (GST) Gene Fusion System is an integrated range of products for the amplification, purification and detection of GST fusion proteins in E. coli. The characteristics of GST are shown in Table 7 and Figure 1 shows the structure of Glutathione Sepharose used in the purification steps. Glutathione S-transferase
Naturally occurring Mr 26 000 protein Can be expressed in E. coli with full enzymatic activity
Properties as determined in pGEX-1N Dimer Molecular Weight Km (glutathione) Km (CDNB) pI (chromatofocusing) GST class
Mr 58 500 0.43 ± 0.07 mM 2.68 ± 0.77 mM 5.0 hybrid of Alpha and Mu characteristics
Table 7.
O
O C
H
N C
CH 2
CH 2 O
N
C H
O
S
OH
O
Fig. 1. Glutathione is attached to Sepharose by coupling to the oxirane group using epoxy-activation. The structure of glutathione is complementary to the binding site of the glutathione S-transferase binding site.
NH 3 +
C O
H
C
O
General considerations for the amplification of fusion proteins are discussed in Chapter 2. In the GST gene fusion system expression is under control of the tac promoter, which is induced using the lactose analogue isopropyl b-D-thiogalactoside (IPTG). Induced cultures should be left to express GST fusion proteins for several hours before the cells are harvested.
The host E. coli BL21 is a protease-deficient strain specifically selected to give a high level of expression of GST fusion proteins. Genotype Growth conditions Long term storage
-
-
-
F , ompT, hsdS (rB , mB ), gal (52, 53) Resuspend lyophilized cultures in 1 ml of L-broth. Grow overnight before plating onto L-broth media plates Mix equal volumes of stationary phase culture (grown in L-broth) and glycerol. Store at -70 °C. Revive frozen glycerol stocks by streaking onto L-broth media plates
Table 8.
Use an alternative strain for cloning and maintenance of the vector (e.g. JM105) as BL21 does not transform well. Using E. coli strains that are not protease-deficient may result in proteolysis of the fusion protein, seen as multiple bands on SDS-PAGE or Western blots. 13
The vectors pGEX vectors (pGEX-T, pGEX-P, pGEX-X, pGEX-2TK) are available in all three reading frames with a range of cleavage recognition sites as shown in Table 9. The same multiple cloning sites in each vector ensure easy transfer of inserts. The vectors carry the lacIq gene, so there are no specific host requirements for expression of fusion proteins. Vector control regions and the reading frame of the multiple cloning site for each pGEX vector are shown in Appendix 1. pGEX-6P-1, pGEX-6P-2, pGEX-6P-3 pGEX-4T-1, pGEX-4T-2, pGEX-4T-3 pGEX-5X-1, pGEX-5X-2, pGEX-5X-3 PGEX-2TK Allows detection of expressed proteins by direct labelling in vitro
PreScission Protease Thrombin Factor Xa Thrombin, c-AMP dependent protein kinase
Table 9.
pGEX6P PreScission Protease vectors offer the most efficient method for cleavage and purification of GST fusion proteins. Site specific cleavage is performed with simultaneous immobilization of the protease on the column. The protease has a high activity at a low temperature so that all steps can be performed in the cold room to protect protein integrity. Cleavage enzyme and GST tag are removed in a single step.
Purification For simple, one step purification of GST fusion proteins, several products have been designed to meet specific purification needs, as shown in Table 10. Column (prepacked) or Media**
Amount of GST fusion protein for a single purification
Comment
GST MicroSpin Purification Module
Up to 400 µg
Ready to use, prepacked columns, buffers and chemicals High throughput when used with MicroPlex™ 24 Vacuum (up to 48 samples simultaneously)
GSTrap FF 1 ml
10–12 mg
Prepacked column, ready to use
GSTrap FF 5 ml
50–60 mg
Prepacked column, ready to use
Glutathione Sepharose 4B
8 mg per ml
For packing small columns and other formats
Glutathione Sepharose 4 Fast Flow
10–12 mg per ml
For packing high performance columns for use with purification systems and scaling up
Table 10. Summary of purification options for GST fusion proteins. **Characteristics of GSTrap FF and Glutathione Sepharose are given in Appendix 5.
Re-use of purification columns depends upon the nature of the sample and should only be performed with identical samples to prevent cross contamination. Batch preparation procedures are frequently mentioned in the literature. However the availability of prepacked columns and easily packed high flow rate Glutathione Sepharose provide faster, more convenient alternatives. Batch preparations are occasionally used if it appears that the tag is not fully accessible or when the protein in the lysate is at very low concentrations (both could appear to give a low yield from the first purification step). A more convenient alternative to improve yield is to decrease the flow rate or pass the sample through the column several times.
14
Monitor purification steps by using one or more of the detection methods referred to in this handbook. The choice of purification equipment should also be made according to the needs of the purification. Appendix 8 provides a guide to aid in the selection of the correct purification solution and key points to consider are highlighted here. • For a single purification of a small quantity of product or for high throughput screening MicroSpin columns using centrifugation or MicroPlex 24 Vacuum are convenient and simple to use. • For purification of larger quantities of fusion proteins GSTrap FF columns provide the ideal solution and can be used with a syringe, a peristaltic pump or a chromatography system. • To increase capacity use several GSTrap FF columns (1 ml or 5 ml) in series or, for even larger capacity requirements, pack Glutathione Sepharose 4 Fast Flow into a suitable column (details of column packing procedures are outlined in Appendix 6). • For simple and reproducible purification a chromatography system such as ÄKTA™prime is a significant advantage, recording the purification process and eliminating manual errors. • For laboratory environments in which all experimental data must be recorded and traceable, where method development, optimization and scale up are needed, a computer controlled ÄKTAdesign chromatography system is recommended. • Experiments such as protein refolding or method optimization require linear gradient elution steps that can only be performed by a chromatography system.
GST MicroSpin Purification Module The GST MicroSpin Purification Module is useful for screening small or large numbers of lysates and for checking samples during the optimization of amplification or purification conditions. Each module contains reagents sufficient for 50 purifications.
•
10X PBS:
•
Reduced glutathione: 0.154 g
1.4 M NaCl, 27 mM KCl, 101 mM Na2HPO4, 18 mM KH2PO4, pH 7.3
•
Dilution buffer:
50 mM Tris-HCl, pH 8.0
•
IPTG:
500 mg
•
MicroSpin columns:
50 units
Reagents are prepared as follows: 1X PBS:
Dilute 10X PBS with sterile water. Store at +4 °C.
Glutathione elution buffer: Pour the entire contents of dilution buffer into the bottle containing the reduced glutathione. Shake until completely dissolved. Store as 1–20 ml aliquots at -20 °C. IPTG 100 mM:
Dissolve contents of the IPTG vial in 20 ml sterile water. Store as 1 ml aliquots at -20 °C.
15
Alternative 1. High throughput purification using MicroPlex Vacuum Do not apply more than 600 µl of sample at a time to a MicroSpin column. This procedure will accommodate lysates from 2 to 12 ml of culture.
Also required: • Vacuum source capable of providing 220 mm Hg (e.g. a water vacuum). •
Side arm flask, 500 ml or 1 litre.
•
Single or double hole rubber stop.
•
Vacuum tubing.
•
MicroPlex 24 Vacuum apparatus (one or two).
1. Assemble the MicroPlex 24 Vacuum following the instructions supplied. 2. Resuspend the Glutathione Sepharose in each MicroSpin column by vortexing gently. 3. Remove the caps and snap off the bottom closures from the MicroSpin columns. Place the columns in the manifold, filling any unused holes with the plugs provided with MicroPlex 24 Vacuum. 4. Ensure the stopcock is in the closed position (i.e. perpendicular to the vacuum tubing) and that the manifold is placed squarely on the gasket. 5. Turn on vacuum supply at source. Open the stopcock (i.e. parallel to the vacuum tubing). After the column storage buffer has been drawn through all the columns into the collection tray, close the stopcock. 6. Allow 10–15 seconds for the vacuum pressure to dissipate. Remove the manifold and place it on a paper towel. 7. Apply up to 600 µl of lysate to the column and incubate at room temperature for 5–10 minutes. 8. Open the stopcock. After the lysates have been drawn through all the columns into the collection tray, close the stopcock. 9. Add 600 µl of 1X PBS wash buffer to each column. Open the stopcock. After buffer has been drawn through all the columns into the collection tray, close the stopcock. 10. Allow 10–15 seconds for the vacuum pressure to dissipate. Remove the manifold and reassemble the apparatus with a clean collection tray. Additional 600 µl washes may be performed if desired. 11. Add 200 µl of Glutathione elution buffer to each column. Incubate at room temperature for 5–10 minutes. 12. Open the stopcock. After elution buffer has been drawn through all the columns into the collection tray, close the stopcock. 13. Allow 10–15 seconds for the vacuum pressure to dissipate. Remove the manifold. Cover eluates with sealing tape until required for analysis.
Note: Yields of fusion protein may be increased by repeating the elution step two or three times and pooling the eluates.
Troubleshooting See Purification and Detection Troubleshooting page 28.
Alternative 2. Purification of multiple samples using a microcentrifuge Do not apply more than 600 µl of sample at a time to a MicroSpin column. This procedure will accommodate lysates from 2 to 12 ml of culture.
16
1. Resuspend the Glutathione Sepharose in each column by vortexing gently. 2. Loosen the column caps one-fourth turn. Remove (and save) bottom closures. 3. Place each column into a clean 1.5 or 2 ml microcentrifuge tube. Spin for 1 minute at 735 g. 4. Discard the buffer from each centrifuge tube and replace the bottom closures. 5. Apply up to 600 µl of lysate to the column. 6. Recap each column securely and mix by gentle, repeated inversion. Incubate at room temperature for 5–10 minutes. 7. Remove (and save) the top caps and bottom closures. Place each column into a clean, pre-labelled 1.5 or 2 ml microcentrifuge tube. 8. Spin for 1 minute at 735 g to collect flow through. 9. Place each column into a clean, pre-labelled 1.5 or 2 ml microcentrifuge tube. 10. Apply 600 µl of 1X PBS wash buffer to each column and repeat spin procedure. Additional 600 µl washes with 1X PBS may be performed if desired. 11. Add 100–200 µl of Glutathione elution buffer to each column. Replace top caps and bottom closures. Incubate at room temperature for 5–10 minutes. 12. Remove and discard top caps and bottom closures and place the column into a clean 1.5 or 2 ml microcentrifuge tube. 13. Spin all columns again to collect eluate. Save for analysis.
Note: Yields of fusion protein may be increased by repeating the elution step two or three times and pooling the eluates.
Troubleshooting See Purification and Detection Troubleshooting page 28.
Alternative 3. Purification using MicroPlex Centrifugation Do not apply more than 600 µl of sample at a time to a GST MicroSpin column. This procedure will accommodate lysates from 2 to 12 ml of culture. See Appendix 4 for recommended centrifugation systems. 1. Assemble the MicroPlex 24 unit following the instructions supplied. Two units can be processed simultaneously to handle 48 samples. 2. Resuspend the Glutathione Sepharose in each column by vortexing gently. 3. Remove the caps from the MicroSpin columns and snap off the bottom closures. Place the columns in the manifold. 4. Centrifuge the unit for 2 minutes following the instructions supplied. 5. Add up to 600 µl of lysate to each column. Incubate at room temperature for 5–10 minutes. 6. Centrifuge the unit for 2 minutes following the instructions supplied. If desired remove the manifold from each collection try and place on a clean paper towel. Reassemble each unit with a fresh collection tray. 7. Apply 600 µl of 1X PBS wash buffer to each column and repeat spin procedure. Additional 600 µl washes with 1X PBS may be performed if desired. Remove the manifold from each collection tray and place it on clean paper. 8. Add 100–200 µl of glutathione elution buffer to each column. Incubate at room temperature for 5–10 minutes. 9. Centrifuge the unit for 2 minutes following the instructions supplied. Cover the eluted samples with sealing tape until required for analysis.
Note: Yields of fusion protein may be increased by repeating the elution step two or three times and pooling the eluates.
Troubleshooting See Purification and Detection Troubleshooting page 28. 17
Purification using GSTrap FF 1 ml or 5 ml columns GSTrap FF columns can be operated with a syringe, a peristaltic pump or a liquid chromatography system such as ÄKTAprime. Figure 2 shows a schematic of the simple steps needed for successful purification using a 1 ml GSTrap FF column.
Equilibrate column with binding buffer
3 min
Apply sample wash with binding buffer
5-15 min
Waste
Elute with elution buffer
2 min
Collect
Collect fractions
Fig. 2. Simple purification of GST fusion proteins using GSTrap FF.
Re-use of any purification column depends on the nature of the sample and should only be performed with identical fusion proteins to prevent cross-contamination. GSTrap FF columns (1 ml or 5 ml) can be connected in series to increase binding capacity and hence scale of purification. Larger columns can be packed with Glutathione Sepharose 4 Fast Flow (see Appendix 6 for column packing).
Sample and buffer preparation Use high quality water and chemicals. Filtration through 0.45 µm filters is recommended. Samples should be centrifuged immediately before use and/or filtered through a 0.45 µm filter. If the sample is too viscous, dilute with binding buffer. Sample binding properties can be improved by adjusting the sample to the composition of the binding buffer: dilute in binding buffer or perform a buffer exchange using a desalting column (see Chapter 7).
18
Alternative 1. Manual purification with a syringe Binding buffer: 1X PBS, pH 7.3 (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.3). Elution buffer: 50 mM Tris-HCl, 10 mM reduced glutathione, pH 8.0.
A
B
C
Fig. 3. Using GSTrap FF with a syringe. A Prepare buffers and sample. Remove the column’s top cap and twist off the end. B Load the sample and begin collecting fractions. C Wash and elute and continue collecting fractions. 1. Fill the syringe with binding buffer. 2. Connect the column to the syringe using the adapter supplied ("drop to drop" to avoid introducing air into the column). 3. Remove the twist-off end. 4. Equilibrate the column with 5 column volumes of binding buffer. 5. Apply the sample using the syringe. For best results, maintain a flow rate of 0.2–1 ml/min (1 ml column) and 1–5 ml/min (5 ml column) as the sample is applied.* 6. Wash with 5–10 column volumes of binding buffer. Maintain flow rates of 1–2 ml/min (1 ml column) and 5–10 ml/min (5 ml column) during the wash.* 7. Elute with 5–10 column volumes of elution buffer. Maintain flow rates of 1–2 ml/min (1 ml column) and 5–10 ml/min (5 ml column) during elution.*
* One ml/min corresponds to approximately 30 drops/min when using a syringe with a HiTrap 1 ml column and five ml/min corresponds to approximately 120 drops/min when using a HiTrap 5 ml column.
For large sample volumes a simple peristaltic pump can be used to apply sample and buffers.
Alternative 2. Simple purification with ÄKTAprime ÄKTAprime contains a pre-programmed template for purification of GST fusion proteins using a single GSTrap FF column, as shown below. This provides a standard purification protocol which can be followed exactly or optimized as required. % Elution buffer
Elution
100 System preparation & column equilibration
50
Sample Wash
11 10 11 Total separation time = 37 min + sample application time
Reequilibration
6
Min
19
Binding buffer: 20 mM sodium phosphate, 0.15 M NaCl, pH 7.3 (or the buffer used in Alternative 1). Elution buffer: 50 mM Tris-HCl, 10 mM reduced glutathione, pH 8.0. Prepare at least 500 ml of each eluent. 1. Follow instructions supplied on the ÄKTAprime cue card (Code No. 18-1138-06). 2. Select the Application Template. 3. Start the method. 4. Enter the sample volume and press OK to start.
Connecting the column.
Preparing the fraction collector.
Fig. 4. Typical procedures when using ÄKTAprime.
Figure 5 shows a typical purification of GST fusion protein on GSTrap FF 1 ml, using a chromatography system, and an SDS-PAGE analysis of the purified protein. Column: Sample:
GSTrap FF 1 ml 8 ml cytoplasmic extract from E. coli expressing a GST fusion protein PBS, pH 7.3 50 mM Tris-HCl, pH 8.0 with 10 mM reduced glutathione 1 ml/min
Binding buffer: Elution buffer: Flow: Chromatographic procedure: 4 CV binding buffer, 8 ml sample, 10 CV binding buffer, 5 CV elution buffer, 5 CV binding buffer (CV = column volume) System: ÄKTAexplorer
A280 Elution buffer
3.5 3.0 2.5
2.7 mg pure GST fusion protein
Wash
2.0
Lane 1: Low Molecular Weight (LMW) Calibration kit, reduced, Amersham Pharmacia Biotech (10 µl prepared for silver stain) Lane 2: Cytoplasmic extract of E. coli expressing GST fusion protein, 1 g cell paste/10 ml (5 µl sample from collect. fraction + 35 µl sample cocktail -> 10 µl applied) Lane 3: GST fusion protein eluted from GSTrap FF 1 ml (5 µl sample from collect. fraction + 35 µl sample coctail -> 10 µl applied )
% Elution buffer
Mr
100
45 000
80
30 000
60
20 100 14 400
97 000 66 000
1.5 40
1.0
20
0.5
1
0
0 5.0 5.0
10.0 10.0
15.0 15.0
20.0 20.0
ml min
Fig. 5a. Purification of GST fusion protein on GSTrap FF 1 ml.
3
Fig. 5b. SDS-PAGE on ExcelGel™ SDS Gradient 8–18% using Multiphor™ II (Amersham Pharmacia Biotech) followed by silver staining.
Troubleshooting See Purification and Detection Troubleshooting page 28. 20
2
Optimization of GST fusion protein purification Following the instructions supplied for each prepacked GSTrap FF column will generally provide very good results. Dimer formation is inevitable with GST fusion proteins since GST itself is a homodimer when folded. Use gel filtration to remove the dimers. A column prepacked with Superdex™ will give the highest possible resolution between two molecules of similar molecular weight. One of the most important parameters affecting the binding of GST fusion proteins to Glutathione Sepharose is the flow rate. Since the binding kinetics between glutathione and GST are relatively slow, it is important to keep the flow rate low during sample application to achieve maximum binding capacity. Volumes and times used for elution may vary among fusion proteins. Further elution with higher concentrations of glutathione (20–50 mM) may improve yield. At concentrations above 15 mM glutathione the buffer concentration should also be increased to maintain the pH within the range 8–9.
Detection of GST fusion proteins Table 11 reviews the methods available for detection of GST fusion proteins. These methods can be selected according to the experimental situation, for example, SDS-PAGE analysis, performed frequently during amplification and purification to monitor results, may not be the method of choice for routine monitoring of samples from high throughput screening. Functional assays based on the properties of the protein of interest (and not the GST tag) are useful, but need to be developed for each specific protein. Detection method GST 96 Well Detection module for ELISA assay
GST Detection Module for enzymatic assay Western blot analysis using anti-GST antibody and ECL™ detection systems
SDS-PAGE with Coomassie or silver staining Functional assays
Comments Ideal for screening expression systems and chromatographic fractions. Useful when amount of expressed protein is unknown or when increased sensitivity is required. Rapid assay, ideal for screening. Highly specific, detects only GST fusion protein. Little or no background detectable when used with optimized concentrations of secondary HRP conjugated antibody. ECL detection systems enhance detection in Western blot. ECL provides adequate sensitivity for most recombinant expression applications. For higher sensitivity use ECL Plus™. Provides information on size and % purity. Detects fusion protein and contaminants. Useful to assess activity of the purified GST fusion protein, but may require development and optimization.
Table 11. Detection methods for GST fusion proteins.
21
Alternative 1. SDS-PAGE Analysis 6X SDS loading buffer: 0.35 M Tris-HCl, 10.28% (w/v) SDS, 36% (v/v) glycerol, 0.6 M dithiothreitol (or 5% 2-mercaptoethanol), 0.012% (w/v) bromophenol blue, pH 6.8. Store in 0.5 ml aliquots at -80 °C. 1. Add 2 µl of 6X SDS loading buffer to 5–10 µl of supernatant from crude extracts, cell lysates or purified fractions as appropriate. 2. Vortex briefly and heat for 5 minutes at +90 to +100 °C. 3. Load the samples onto an SDS-polyacrylamide gel. 4. Run the gel for the appropriate length of time and stain with Coomassie Blue (Coomassie Blue R Tablets) or silver (PlusOne Silver Staining Kit, Protein).
The percentage of acrylamide in the SDS-gel should be selected according to the expected molecular weight of the protein of interest (see Table 12). % Acrylamide in resolving gel
Separation size range (M r x 10-3)
Single percentage: 5%
36–200
7.5%
24–200
10%
14–200
12.5%
14–100
15%
14–601
5–15%
14–200
Gradient:
1
5–20%
10–200
10–20%
10–150
The larger proteins fail to move significantly into the gel.
Table 12.
For information and advice on electrophoresis techniques, please refer to the section Additional reading and reference material.
Troubleshooting • If the fusion protein is absent, it may be insoluble or expressed at very low levels: refer to Troubleshooting protein amplification (page 9). • If no fusion protein is detected by Coomassie Blue, try silver staining or Western blotting to enhance sensitivity. Transformants expressing the fusion protein will be identified by the absence from total cellular proteins of the parental Mr 29 000 GST and by the presence of a novel, larger fusion protein. Parental pGEX vectors produce a 29 kDa GST fusion protein containing amino acids that are coded for by the pGEX multiple cloning site. In some cases both the Mr 29 000 GST and fusion protein may be present. This can be caused by translational pausing at the junction between GST and the fusion partner or a mixed culture between cells with parental plasmid and cells with fusion plasmid. Interpretation is sometimes complicated when fusion proteins break down and release the GST moiety Mr 26 000. Such cases are usually recognized by the appearance of the Mr 26 000 species, and a series of larger, partial proteolytic fragments above it. 22
Alternative 2. GST Detection Module The GST Detection Module is designed for the rapid enzymatic detection of GST fusion proteins produced using the pGEX vectors using the GST substrate 1-chloro-2,4 dinitrobenzene (CDNB). The GST-mediated reaction of CDNB with glutathione produces a conjugate that is measured by absorbance at 340 nm using a UV/vis spectrophotometer, such as an Ultrospec™ 1000, or a plate reader. The CDNB assay is performed in less than 10 minutes on crude bacterial sonicates, column eluates, or purified GST fusion protein. Figure 6 shows typical results from a CDNB assay. Each detection module contains reagents sufficient for 50 detections. A 340 Eluate (0.8 µg)
0.6
0.4
0.2
Sonicate (53 µg)
1
2 Time (minutes)
3
4
Fig. 6. Typical results of a CDNB assay for GST fusion proteins. 53 µg of total protein from an E. coli TG1/pGEX-4T-Luc sonicate and 0.8 µg of total protein eluted from Glutathione Sepharose were assayed according to the instructions for the GST Detection Module.
•
10X Reaction buffer: 1 M potassium phosphate buffer, pH 6.5.
•
CDNB:
•
Reduced glutathione powder for glutathione solution. Dissolve 100 mM reduced glutathione in sterile distilled water. Aliquot into microcentrifuge tubes. Store at -20 °C. Avoid more than five freeze/thaw cycles.
•
Goat/anti-GST antiserum is also supplied for use in Western blots.
100 mM 1-chloro-2,4-dinitrobenzene (CDNB) in ethanol.
Measurement of GST activity by CDNB assay CDNB is toxic. Avoid contact with eyes, skin and clothing. In case of accidental contact, flush affected area with water. In case of ingestion, seek immediate medical attention. pGEX-bearing cells must be lysed before performing a CDNB assay. 1. In a microcentrifuge tube, combine the following: - Distilled water 880 µl - 10X Reaction buffer 100 µl - CDNB 10 µl - Glutathione solution 10 µl - Total Volume 1000 µl 2. Cap and mix by inverting the tube several times.
CDNB may cause the solution to become slightly cloudy. The solution should clear upon mixing.
23
3. Transfer 500 µl volumes of the above solution into two UV-transparent cuvettes. Add sample (5–50 µl) to the "sample cuvette". To the "blank cuvette", add a volume of 1X reaction buffer equal to the sample volume in the sample cuvette. 4. Cover each cuvette with wax film and invert to mix. 5. Place the blank cuvette in the spectrophotometer and blank at 340 nm. Measure the absorbance of the sample cuvette at 340 nm and simultaneously start a stopwatch or other timer. 6. Record absorbance readings at 340 nm at one-minute intervals for 5 minutes by first blanking the spectrophotometer with the blank cuvette and then measuring the absorbance of the sample cuvette. 7. Calculate the A340 /min/ml sample Calculations DA340 /min/ml =
A340 (t2) - A340 (t1) (t2 - t1)(ml sample added)
Where: A340 (t2) = absorbance at 340 nm at time t2 in minutes A340 (t1) = absorbance at 340 nm at time t1 in minutes DA340 /min/ml values can be used as a relative comparison of GST fusion protein content between samples of a given fusion protein.
Adapt the assay to give absolute fusion protein concentrations by constructing a standard curve of DA340/min versus fusion protein amount. The activity of the GST moiety can be affected by the folding of the fusion partner. Absorbance readings obtained for a given fusion protein may not reflect the actual amount of fusion protein present.
Troubleshooting • The reaction rate is linear provided that an A340 of approximately 0.8 is not exceeded during the five-minute time course. Plot initial results to verify that the reaction rate is linear over the time course. Adjust the amount of sample containing the GST fusion protein to maintain a linear reaction rate. • If a low absorbance is obtained using the CDNB assay, a Western blot using the Anti-GST Antibody may reveal high levels of protein expression. • Under standard assay conditions at +22 °C and in the absence of GST, glutathione and CDNB react spontaneously to form a chemical moiety that produces a baseline drift at DA340/min of approximately 0.003 (or 0.015 in 5 minutes). Correct for baseline drift by blanking the spectrophotometer with the blank cuvette before each reading of the sample cuvette.
Alternative 3: GST 96 Well Detection Module The GST 96 Well Detection Module provides a highly sensitive ELISA assay for testing clarified lysates and intermediate purification fractions. Each detection module contains reagents sufficient for 96 detections:
24
•
GST 96 Well detection plates in which each well is coated with anti-GST antibody, blocked and dried.
•
Horse-radish peroxidase conjugated to goat polyclonal anti-GST antibody.
•
Purified recombinant glutathione S-transferase test protein.
Additional reagents to be prepared: PBS:
140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4.
Wash buffer:
0.05% Tween™ 20 in PBS (500 ml/96 well plate). Store at room temperature until needed.
Blocking buffer: 1 x conc. 3% non-fat dry milk in PBS with 0.05% Tween 20 (10 ml/96 well plate). 2 x conc. 6% non-fat dry milk in PBS with 0.1% Tween 20 (5 ml/96 well plate).
Prepare fresh buffers daily. As each fusion protein is captured uniquely, prepare standards of rGST protein and the target fusion protein using a dilution series from 100 ng/100 µl to 10 pg/µl in 1X blocking buffer if quantification is required. Run recombinant GST (rGST) protein as a standard control in every assay.
Screening of GST expression clones or chromatographic fractions 1. Bring each test sample to a final volume of 50 µl with 1X PBS. 2. Mix with 50 µl of 2X blocking buffer. 3. For screening: dilute rGST protein standard to 1 ng/100 µl in 1X blocking buffer. 4. For quantification: use dilution series from 100 ng/100 µl to 10 pg/µl in 1X blocking buffer for rGST protein and for the target fusion protein. 5. Remove one 96-well plate from the foil pouch. If using less than 96 wells, carefully remove the well strips from the holder by pushing up on the wells from below. Store unused swell strips in the pouch with the dessicant provided. 6. Pipette 100 µl of sample into each well. 7. Incubate for 1 hour at room temperature in a humidified container or incubator. 8. Empty contents of the well by flicking the inverted plate. (Biohazardous material should be pipetted or aspirated into a suitable container.) 9. Blot the inverted well or strips on to a paper towel to remove excess liquid. 10. Wash each well 5 times with wash buffer (inverting and flicking out the contents each time). 11. Blot the inverted well or strips on to a paper towel to remove excess wash buffer. 12. Dilute HRP/anti-GST conjugate 1:10 000 (1 µl:10 ml) in 1X blocking buffer. One 96 well plate will require 10 ml of the diluted solution. 13. Add 100 µl of diluted HRP/anti-GST conjugate to each well and incubate for 1 hour at room temperature in a humidified container or incubator. 14. Empty well contents and wash twice with wash buffer as previously described. 15. Add soluble horseradish peroxidase substrate* to each well and incubate according to supplier's instructions.
*3,3',5,5'-tetramethyl benzidine (A450) or 2',2'-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid) diammonium salt (ABTS™) (A410) have been used successfully. 16. Read plate absorbance in a microplate reader or spectrophotometer.
Troubleshooting See also Purification and Detection Troubleshooting page 28.
25
Low absorbance detected in samples • Check that samples were sufficiently induced and lysed (see Troubleshooting protein amplification page 9). • If clarified lysate is being tested, mix initial GST sample with 2X blocking buffer to give a final concentration of 1X blocking buffer.
Poor day to day reproducibility between identical samples • Ensure that all incubation times are consistent. Reduction in GST capture incubation time can be reduced to > 30 minutes with slightly reduced signal, but HRP/anti-GST conjugate incubation time can significantly reduce signal with every 15 minute decrease.
Alternative 4. Western blot analysis Amplification and purification can also be monitored by Western blot analysis, using ECL or ECL Plus detection systems to enhance sensitivity. Anti-GST Antibody Blocking/Incubation buffer: 5% (w/v) non-fat dry milk and 0.1% (v/v) Tween 20 in PBS (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.3) Wash buffer: 0.1% v/v Tween 20 in PBS (as above) Secondary Antibody to detect the anti-GST antibody (such as anti-goat IgG HRP conjugate). 1. Separate the protein samples by SDS-PAGE.
Although anti-GST antibody from Amersham Pharmacia Biotech has been cross-adsorbed with E. coli proteins, low levels of cross-reacting antibodies may remain. It is recommended always to run samples of E. coli sonicates that do not contain a recombinant pGEX plasmid and samples that contain the parental pGEX plasmid as controls. 2. Transfer the separated proteins from the electrophoresis gel to an appropriate membrane, such as Hybond ECL (for subsequent ECL detection) or Hybond P (for subsequent ECL or ECL Plus detection).
Electrophoresis and protein transfer may be accomplished using a variety of equipment and reagents. For further details, refer to the Protein Electrophoresis Technical Manual and Hybond ECL Instruction Manual from Amersham Pharmacia Biotech.
Blocking of membrane 1. Transfer the membrane onto which the proteins have been blotted to a container such as a Petri dish. 2. Add 50–200 ml of blocking/incubation buffer. 3. Incubate for 1–16 hours at ambient temperature with gentle shaking.
Longer incubation times with blocking buffer may reduce background signal. 4. Decant and discard the buffer.
26
Anti-GST antibody 1. Prepare an appropriate dilution of anti-GST antibody with blocking/incubation buffer, e.g. 5–10 µl of antibody to 50 ml of buffer. Refer to Amersham Pharmacia Biotech Application Note 18-1139-13 for further information on optimization. 2. Pour the antibody-buffer mixture into the container with the membrane. 3. Incubate for 1 hour at ambient temperature with gentle shaking. 4. Decant and discard the antibody-buffer. 5. Rinse twice with 20–30 ml of blocking or wash buffer to remove most of the unbound antibody. 6. Decant and discard the rinses. 7. Wash the membrane with 20–30 ml of blocking or wash buffer for 10–60 minutes at ambient temperature with gentle shaking. 8. Discard the wash and repeat.
Secondary antibody 1. Dilute an appropriate anti-goat secondary antibody with blocking/incubation buffer according to the manufacturer's recommendation. Refer to Amersham Pharmacia Biotech Application Note 18-1139-13 for further information on optimization. 2. Pour the antibody-buffer mixture into the container with the membrane. 3. Incubate for 1 hour at ambient temperature with gentle shaking. 4. Decant and discard the antibody-buffer. 5. Rinse twice with 20–30 ml of blocking or wash buffer to remove most of the unbound antibody. 6. Decant and discard the rinses. 7. Wash the membrane with 20–30 ml of blocking or wash buffer for 10–60 minutes at ambient temperature with gentle shaking. 8. Discard the wash and repeat. 9. Develop the blot with the appropriate substrate for the conjugated secondary antibody.
ECL and ECL Plus detection systems require very little antibody to achieve a sufficient sensitivity so the amount of antibody (primary and secondary) used in the protocols can be minimized. Smaller quantities of antibody-buffer mixtures can be used by scaling down the protocol and performing the incubations in sealable plastic bags.
Troubleshooting See also Purification and Detection Troubleshooting page 28.
Multiple bands seen on Western blot analysis • Anti-GST antibody from Amersham Pharmacia Biotech has been cross-absorbed against E. coli proteins and tested for its lack of non-specific background binding in a Western blot. Some sources of the anti-GST antibody may contain antibodies that react with various E. coli proteins present in the fusion protein sample. Cross-adsorb the antibody with an E. coli sonicate to remove anti-E. coli antibodies. This E. coli must not contain the pGEX plasmid.
27
Purification and detection troubleshooting Column has clogged • Cell debris in the sample may clog the column. Clean the column according to Appendix 5 and ensure that samples have been filtered or centrifuged.
Fusion protein does not bind to purification column • Over-sonication may have denatured the fusion protein. Check by using a microscope to monitor cell breakage. Use mild sonication conditions during cell lysis. • Sonication may be insufficient: Check using a microscope or monitor by measuring the release of nucleic acids at A260. Addition of lysozyme (0.1 volume of a 10 mg/ml lysozyme solution in 25 mM Tris-HCl, pH 8.0) prior to sonication may improve results. • Add 5 mM DTT prior to cell lysis. This can significantly increase binding of some GST fusion proteins to Glutathione Sepharose. • Check that the column has been equilibrated with a buffer 6.5 < pH < 8.0 (e.g. PBS) before application of the fusion protein. The correct pH range is critical for efficient binding. • Decrease the flow rate to improve binding. • If re-using a column, check that the column has been regenerated correctly (see Appendix 5). Replace with fresh Glutathione Sepharose or a new column if binding capacity does not return after regeneration. • Check the binding of a cell sonicate prepared from the parental pGEX plasmid. If GST produced from the parental plasmid binds with high affinity, then the fusion partner may have altered the conformation of GST, thereby reducing its affinity. Try reducing the binding temperature to +4 °C and limit the number of washes. • Column capacity may have been exceeded. If using GSTrap FF columns (1 ml or 5 ml) link 2 or 3 columns in series to increase capacity or pack a larger column. • Fusion protein may be in the inclusion bodies, although using a GST-tag reduces the chance of this problem occurring.
Fusion protein is poorly eluted • Increase concentration of glutathione in the elution buffer. Above 15 mM glutathione the buffer concentration should be increased to maintain pH. • Increase pH of the elution buffer. Values up to pH 9 may improve elution without requiring an increase in the concentration of glutathione. • Increase ionic strength of the elution buffer by addition of 0.1–0.2 M NaCl. Note that very hydrophobic proteins may precipitate under high salt conditions. If this is the case, addition of a non-ionic detergent may improve results (see below). • Decrease the flow rate to improve elution.
28
• Add a non-ionic detergent (0.1% Triton™ X-100 or 2% N-octyl glucoside) to the elution buffer to reduce non-specific hydrophobic interactions that may prevent solubilization and elution of fusion proteins • Try over-night elution at room temperature or +4 °C.
Multiple bands seen on SDS-PAGE or Western blot analysis Multiple bands result from partial degradation of fusion proteins by proteases, or denaturation and co-purification of host proteins with the GST fusion protein due to over-sonication. • Check that a protease-deficient host such as E. coli B21 has been used. • Add protease inhibitors such as 1 mM PMSF to the lysis solution. A non-toxic, water soluble alternative to PMSF is 4-(2-amino-ethyl)- benzenesulfonyl fluoride hydrochloride (AEBSF), commercially available as Pefabloc™ SC from Boehringer Mannheim. • Use prepacked GSTrap FF columns or Glutathione Sepharose 4 Fast Flow. These can be used at higher flow rates to process samples more quickly and so avoid degradation. • Decrease sonication. Addition of lysozyme (0.1 volume of a 10 mg/ml lysozyme solution in 25 mM Tris-HCl, pH 8.0) prior to sonication may improve results. Avoid frothing as this may denature the fusion protein. • Include an additional purification step (see Chapter 9). A variety of proteins known as chaperonins that are involved in the correct folding of nascent proteins in E. coli may co-purify with GST fusion proteins, including a Mr 70 000 protein (see below). Serine protease inhibitors must be removed prior to cleavage by thrombin or Factor Xa. Use HiTrap Benzamidine FF (high sub) (see page 39).
Mr 70 000 protein co-purifies with the GST-fusion protein Pre-incubate the protein solution with 2 mM ATP, 10 mM MgSO4, 50 mM Tris-HCl (pH 7.4) for 10 minutes at +37 °C prior to purification in order to dissociate the complex. This Mr 70 000 protein is probably a protein product of the E. coli gene dnaK and involved in the degradation of "abnormal" proteins in E. coli. Reports suggest that this protein can be removed by ion exchange chromatography (Analects and Separations p24, 1996, Amersham Pharmacia Biotech and (http://bionet.hgmp.mrc.ac.uk/hypermail/methods/ methods.199406/0813.html)) or by passage of the sample over ATP agarose (Myers, M., BIOSCI posting, 7 July 1993). Thain, A., et al. Trends Genet. 12, 209–210 (1996) and Sherman, M. and Goldberg, A.L., J. Biol. Chem, 269, 31479–31483, (1994) suggest washing the column with ATP or GroES rather than using a subsequent IEX step.
29
Tag removal by enzymatic cleavage In most cases, functional tests can be performed using the intact fusion with GST. If removal of the GST tag is necessary, it is highly recommended to produce the fusion proteins with a PreScission Protease cleavage site. The GST tag then can be removed and the protein purified in a single step on the column (see Figure 7). This protease also has the useful property of being maximally active at +4 °C thus allowing cleavage to be performed at low temperatures and so improving the stability of the target protein. Thrombin or Factor Xa recognition sites may be cleaved either while bound on the column or in solution after elution from the column (see Figure 8). The protease used for cleavage can be removed using Benzamidine Sepharose, a purification medium with a high specificity for serine proteases (see page 39). On-column cleavage is generally recommended as the method of choice since many potential contaminants can be washed through the column and the target protein eluted with a higher level of purity. For the removal of thrombin and Factor Xa, a GSTrap FF and a HiTrap Benzamidine FF (high sub) column can be connected in series so that cleaved product passes directly from the GSTrap FF into the HiTrap Benzamidine FF (high sub). Samples are cleaved and proteases removed in a single step (see page 39). The amount of enzyme, temperature and length of incubation required for complete digestion varies according to the specific GST fusion protein produced. Determine optimal conditions in pilot experiments. Remove samples at various time points and analyse by SDS-PAGE to estimate the yield, purity and extent of digestion. Approximate molecular weights for SDS-PAGE analysis: PreScission Protease
Mr 46 000
Bovine thrombin
Mr 37 000
Bovine Factor Xa
Mr 48 000
If protease inhibitors have been used in the lysis solution, they must be removed prior to cleavage by PreScission Protease, thrombin or Factor Xa (the inhibitors will usually be eluted in the flow-through when sample is loaded onto a GSTrap FF column). Enzyme
Inhibitor
PreScission protease
100 mM ZnCl2 (> 50% inhibition) 100 µM chymostatin 4 mM Pefabloc
Factor Xa and thrombin
AEBSF, APMSF, antithrombin III, Antipain, a1-antitrypsin, aprotinin, chymostatin, hirudin, leupeptin, PMSF
Factor Xa only
Pefabloc FXa
Thrombin only
Pefabloc TH Benzamidine
Cleavage of fusion proteins is most commonly performed on milligram quantities of fusion protein suitable for purification on GSTrap FF. The following protocols describe a manual cleavage and purification using a syringe and a 1 ml GSTrap FF column. The protocols can be adapted for use with GST MicroSpin columns to work at smaller scales or scaled up onto larger columns to run on ÄKTAdesign systems.
30
PreScission Protease cleavage and purification PreScission Protease is a fusion protein of GST and human rhinovirus 3C protease. The protease specifically recognizes the amino acid sequence Leu-Glu-Val-Leu-Phe-Gln¡Gly-Pro cleaving between the Gln and Gly residues. Since the protease is fused to GST, it is easily removed from cleavage reactions using GSTrap FF or Glutathione Sepharose. This protease also has the useful property of being maximally active at +4 °C thus allowing cleavage to be performed at low temperatures and so improving the stability of the target protein.
Enzymatic cleavage PreScission cleavage buffer: 50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, pH 7.0. PreScission Protease.
Cleavage should be complete following a 4 hour treatment at +5 ºC with at least 10 units/mg of fusion protein. Incubation times may be reduced by adding a greater amount of PreScission Protease. (continued on page 34)
31
Cleavage of GST tag using PreScission Protease 1
Add cell lysate to prepacked Glutathione Sepharose (GST MicroSpin or GSTrap FF columns)
3
Elute with reduced Glutathione
4
Cleave eluted fusion protein with PreScission Protease
Off column cleavage
2 Wash On column cleavage
3
Cleave fusion protein with PreScission Protease
Fig. 7. Flow chart of the affinity purification procedure and PreScission Protease cleavage of glutathione S-transferase fusion proteins.
Cleavage of GST tag using Thrombin or Factor Xa 1
Add cell lysate to prepacked GST MicroSpin or GSTrap FF columns
3
Elute with reduced Glutathione
4
Cleave eluted fusion protein with site-specific protease (Thrombin or Factor Xa)
Off column cleavage
2 Wash On column cleavage
3
Cleave fusion protein with site-specific protease (Thrombin or Factor Xa)
If using GSTrap FF, connect the column directly to a HiTrap Benzamidine FF (high sub) before elution. Cleaved product passes directly from the GSTrap FF into the HiTrap Benzamidine FF (high sub). Samples are cleaved and the protease removed in a single step (see page 39).
GSTrap FF
HiTrap Benzamidine FF (high sub)
Fig. 8. Flow chart of the affinity purification procedure and Thrombin or Factor Xa cleavage of glutathione S-transferase fusion proteins.
32
5
HiTrap Desalting column
6
Add sample to GST MicroSpin or GSTrap FF columns
7
4
Collect eluate
8
Analyse protein e.g. on SDS-PAGE or Mass Spec
5
Analyse protein e.g. on SDS-PAGE or Mass Spec
Collect flow through
Ettan™ MALDI-ToF mass spectrometer
5
HiTrap Desalting column
6
Add sample to GST MicroSpin or GSTrap FF columns
7
Collect eluate
9 8
Remove protease if necessary. Use HiTrap Benzamidine FF (high sub) Analyse protein e.g. on SDS-PAGE or Mass Spec
Sepharose
Glutathione
5 4 4
Collect flow through
5
Analyse protein e.g. on SDS-PAGE or Mass Spec
Collect flow through
6
Analyse protein e.g. on SDS-PAGE or Mass Spec Remove protease if necessary. Use HiTrap Benzamidine FF (high sub)
Glutathione S-transferase Cloned protein GST fusion protein Thrombin or Factor Xa PreScission Protease
33
Alternative 1. On-column cleavage and purification Assume: 8 mg GST fusion protein bound/ml gel, using GSTrap FF 1 ml column (adjust volumes for larger columns) 1. Fill the syringe with binding buffer. 2. Connect the column to the syringe using the adapter supplied ("drop to drop" to avoid introducing air into the column). 3. Remove the twist-off end. 4. Equilibrate the column with 5 column volumes of binding buffer. 5. Apply the sample using the syringe. For best results, maintain a flow rate of 0.2–1 ml/min as the sample is applied. 6. Wash with 5–10 column volumes of binding buffer. Maintain flow rates of 1–2 ml/min during the wash. 7. Wash the column with 10 column volumes of PreScission cleavage buffer. 8. Prepare the PreScission Protease mix: mix 80 µl (160 units) of PreScission Protease with 920 µl of PreScission cleavage buffer at +5 °C. 9. Load the PreScission Protease mix onto the column using a syringe and the adapter supplied. Seal the column with the top cap and the domed nut supplied. 10. Incubate the column at +5 °C for 4 hours. 11. Fill a syringe with 3 ml of PreScission cleavage buffer. Remove the top cap and domed nut. Avoid introducing air into the column. Begin elution and collect the eluate (0.5 ml–1 ml/tube).
N.B. The eluate will contain the protein of interest, while the GST moiety of the fusion protein and the PreScission Protease will remain bound to GSTrap FF.
Alternative 2. Off-column cleavage and purification Assume: 8 mg GST fusion protein bound/ml gel, using GSTrap FF 1 ml column (adjust volumes for larger columns) 1. Follow steps 1–6 above. 2. Elute with 5–10 column volumes of elution buffer. Maintain flow rates of 1–2 ml/min during elution. 3. Remove the reduced glutathione from the eluate using a quick buffer exchange on HiTrap Desalting or HiPrep™ 26/10 Desalting depending on the sample volume. 4. Add 1 µl (2 units) of PreScission Protease for each 100 µg of fusion protein in the eluate. If the amount of fusion protein in the eluate has not been determined, add 80 µl (160 units) of PreScission Protease. 5. Incubate at +5 °C for 4 hours. 6. Once digestion is complete, apply the sample to an equilibrated GSTrap FF column to remove the GST moiety of the fusion protein and the PreScission Protease.
NB. The protein of interest will be found in the flow-through, while the GST moiety of the fusion protein and the PreScission Protease will remain bound to the column.
Troubleshooting Incomplete PreScission Protease cleavage • Check that the PreScission Protease to fusion protein ratio is correct (although saturation of the purification column is rarely a problem). • Increase the incubation time to 20 hours or longer at +5 °C and increase the amount of PreScission Protease used in the reaction. • Verify presence of the PreScission Protease cleavage site. Compare the DNA sequence of the construct with the known PreScission Protease cleavage sequence. Verify that the optimal PreScission Protease recognition site, Leu-Glu-Val-Leu-Phe-Gln¡Gly-Pro, has not been altered. 34
• Remove possible PreScission Protease inhibitors by extensive washing of the purification column before cleaving with PreScission Protease. The presence of Zn2+ as well as Pefabloc SC or chymostatin may interfere with PreScission Protease activity.
Multiple bands seen on SDS gel after cleavage • Determine when the bands appear. Additional bands seen prior to PreScission Protease cleavage may be the result of proteolysis in the host bacteria. E. coli BL21 is a recom mended protease-deficient strain. • Check the sequence of the fusion partner for the presence of additional PreScission Protease recognition sites. PreScission Protease optimally recognizes the sequence Leu-Glu-Val-Leu-Phe-Gln¡Gly-Pro and cleaves between the Gln and Gly residues but similar secondary sites may exhibit some propensity for cleavage. Adjusting reaction conditions (e.g. time, temperature, salt concentration) may result in selective cleavage at the desired site. If adjustment of the conditions does not correct the problem, reclone the insert into a pGEX T (thrombin) or pGEX X (Factor Xa) expression vector.
Fusion partner is contaminated with PreScission Protease after purification • Pass the sample over fresh Glutathione Sepharose to remove residual PreScission Protease (the Glutathione Sepharose may have been saturated with GST fusion protein in the first purification). Alternatively, a conventional ion exchange chromatography separation can be developed to remove the PreScission Protease and other minor contaminants (see Appendix 9).
Thrombin cleavage and purification Enzymatic cleavage PBS:
140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO 4, pH 7.3.
Thrombin Solution: Dissolve 500 units in 0.5 ml of PBS pre-chilled to +4 °C. Swirl gently. Store solution in small aliquots at -80 °C to preserve activity.
With a specific activity > 7 500 units/mg protein one unit of thrombin will digest > 90% of 100 µg of a test fusion protein in 16 hours at +22 °C in elution buffer. A unit is approximately equal to 0.2 NIH units. Cleavage should be complete following overnight treatment with < 10 units/mg of fusion protein. Thrombin can be removed using Benzamidine Sepharose, a purification medium with a high specificity for serine proteases (see page 39). A GSTrap FF and a HiTrap Benzamidine FF (high sub) column can be connected in series so that cleaved product passes directly from the GSTrap FF into the HiTrap Benzamidine FF (high sub). Samples are cleaved and the thrombin removed in a single step (see page 39).
35
Alternative 1. On-column cleavage and purification Assume: 8 mg GST fusion protein bound/ml gel, using GSTrap FF 1 ml column (adjust volumes for larger columns) 1. Follow steps 1–6 under on-column Prescission Protease cleavage. 2. Prepare the thrombin mix: mix 80 µl thrombin solution (1 unit/µl ) with 920 µl PBS. 3. Load the thrombin mix onto the column using a syringe and the adapter supplied. Seal the column with the top cap and the domed nut supplied. 4. Incubate the column at room temperature (+22 to +25 °C) for 2–16 hours. 5. Fill a syringe with 3 ml PBS. Remove the top cap and domed nut. Avoid introducing air into the column. Begin elution and collect the eluate (0.5 ml–1 ml/tube).
N.B. The eluate will contain the protein of interest and thrombin, while the GST moiety of the fusion protein will remain bound to GSTrap FF.
Alternative 2. Off-column cleavage and purification Assume: 8 mg GST fusion protein bound/ml gel, using GSTrap FF 1 ml column (adjust volumes for larger columns) 1. Follow steps 1–6 under on-column Prescission Protease cleavage. 2. Elute with 5–10 column volumes of elution buffer. Maintain flow rates of 1–2 ml/min during elution. 3. Add 10 µl (10 units) of thrombin solution for each mg of fusion protein in the eluate. If the amount of fusion protein in the eluate has not been determined, add 80 µl (80 units) thrombin solution. 4. Incubate at room temperature (+22 to +25 °C) for 2–16 hours. 5. Once digestion is complete, remove the reduced glutathione using a quick buffer exchange on HiTrap Desalting or HiPrep 26/10 Desalting depending on the sample volume. 6. Apply the sample to an equilibrated GSTrap FF column to remove the GST moiety of the fusion protein.
N.B.The purified protein of interest and thrombin will be found in the flow-through.
Troubleshooting Incomplete thrombin cleavage • Check that the thrombin to fusion protein ratio is correct. • Increase the reaction time to 20 hours at +22 to +25 °C and increase the amount of thrombin used in the reaction. • Check the DNA sequence of the construct to verify the presence of the thrombin site. Verify that the thrombin site has not been altered. • Check that protease inhibitors have been removed.
Multiple bands seen on SDS-PAGE analysis after cleavage • Determine when the bands appear. Additional bands seen prior to cleavage may be the result of proteolysis in the host bacteria. E. coli BL21 is a protease-deficient strain that is recommended.
36
• Check the sequence of the fusion partner for the presence of additional thrombin recognition sites. Optimum cleavage sites for thrombin are given in 1) and 2) below. Ref: Chang, J-Y., Eur. J. Biochem. 151, 217 (1985). 1) P4-P3-Pro-Arg/Lys¡P1´-P2´ where P3 and P4 are hydrophobic amino acids and P1´ and P2´ are non-acidic amino acids. The Arg/Lys¡P1´ bond is cleaved. Examples: P4
P3
Pro
R/K¡P1´
A)
Met
Tyr
Pro
Arg¡Gly
P2´ Asn
B)
Ile
Arg
Pro
Lys¡Leu
Lys
C)
Leu
Val
Pro
Arg¡Gly
Ser
In A, the Arg¡Gly bond is cleaved very quickly by thrombin. In B, the Lys¡Leu bond is cleaved. C is the recognition sequence found on the thrombin series of pGEX plasmids, the Arg¡Gly bond is cleaved. 2) P2-Arg/Lys¡P1´, where either P2 or P1' is Gly. The Arg/Lys¡P1´ bond is cleaved. Examples: P2
R/K¡P1´
A)
Ala
Arg¡Gly
B)
Gly
Lys¡Ala
In A, the Arg¡Gly bond is cleaved efficiently. In B, the Lys¡Ala bond is cleaved. Adjusting time and temperature of digestion can result in selective cleavage at the desired thrombin site. If adjustment of conditions does not correct the problem, reclone the insert into a pGEX-6P (PreScission) or pGEX (Factor Xa) expression vector.
Factor Xa cleavage and purification Enzymatic cleavage Factor Xa cleavage buffer: 50 mM Tris-HCl, 150 mM NaCl, 1 mM CaCl2, pH 7.5. Factor Xa solution:
Dissolve 400 units of Factor Xa in +4 °C water to give a final solution of 1 unit/µl. Swirl gently.
Store solution in small aliquots at -80 °C to preserve activity.
With a specific activity of > 800 units/mg protein, one unit of Factor Xa will digest > 90% of 100 µg of a test fusion protein in 16 hours at +22 °C in Factor Xa cleavage buffer. Cleavage should be complete following overnight treatment at +22 °C with a Factor Xa to substrate ratio of at least 1% (w/w). Factor Xa can be removed using Benzamidine Sepharose, a purification medium with a high specificity for serine proteases (see page 39). A GSTrap FF and a HiTrap Benzamidine FF (high sub) column can be connected in series so that cleaved product passes directly from the GSTrap FF into the HiTrap Benzamidine FF (high sub). Samples are cleaved and the Factor Xa removed in a single step (see page 39). Heparin Sepharose has been used for this application but, since benzamidine has a higher specificity for Factor Xa, the protease will be removed more effectively.
37
Factor Xa consists of two subunits linked by disulphide bridges. Since glutathione can disrupt disulphide bridges, it should be removed from the sample prior to the cleavage reaction. Glutathione can be easily and rapidly removed from the fusion protein using a desalting column (Chapter 7) with Factor Xa cleavage buffer as eluent.
Alternative 1. On-column cleavage and purification Assume: 8 mg GST fusion protein bound/ml gel, using GSTrap FF 1 ml column (adjust volumes for larger columns) 1. Follow steps 1–6 under on-column Prescission Protease cleavage. 2. Wash GSTrap FF with 10 column volumes of Factor Xa cleavage buffer. 3. Prepare the Factor Xa mix: Mix 80 µl Factor Xa solution with 920 µl Factor Xa cleavage buffer. 4. Load the Factor Xa mix onto the column using a syringe and the adapter supplied. Seal the column with the top cap and the domed nut. 5. Incubate the column at room temperature (+22 to +25 °C) for 2–16 hours. 6. Fill a syringe with 3 column volmes of Factor Xa cleavage buffer. Remove the top cap and domed nut. Avoid introducing air into the column. Begin elution and collect the eluate (0.5 ml–1 ml/tube).
N.B. The eluate will contain the protein of interest and Factor Xa, while the GST moiety of the fusion protein will remain bound to GSTrap FF.
Alternative 2. Off-column cleavage and purification Assume: 8 mg GST fusion protein bound/ml gel, using GSTrap FF 1 ml column (adjust volumes for larger columns) 1. Follow steps 1–6 under on-column Prescission Protease cleavage. 2. Elute with 5–10 column volumes of elution buffer. Maintain flow rates of 1–2 ml/min during elution. 3. Remove the reduced glutathione from the eluate using a quick buffer exchange on HiTrap Desalting or HiPrep 26/10 Desalting depending on sample volume. 4. Add 10 units of Factor Xa solution for each mg fusion protein in the eluate. If the amount of fusion protein in the eluate has not been determined, add 80 µl (80 units) of Factor Xa solution. 5. Incubate the column at room temperature (+22 to +25 °C) for 2–16 hours. 6. Once digestion is complete, apply the sample to an equilibrated GSTrap FF column to remove the GST moiety of the fusion.
N.B. The protein of interest will be found in the flow-through together with Factor Xa.
Troubleshooting Incomplete Factor Xa cleavage • Factor Xa requires activation of Factor X with Russell's viper venom. Factor Xa from Amersham Pharmacia Biotech has been preactivated, but other sources may not be activated. Activation conditions: Incubate 1% (w/w) of Russell's viper venom to Factor X in 8 mM Tris-HCl (pH 8.0), 70 mM NaCl, 8 mM CaCl2 at +37 °C for 5 minutes. • Check the DNA sequence of the construct to verify the presence of the Factor Xa site. Verify that the Factor Xa site has not been altered. The recognition sequence for Factor Xa is Ile-Glu-Gly-Arg¡X, where X can be any amino acid except Arg or Pro. • Check that the Factor Xa to fusion protein ratio is correct. • Check that glutathione has been removed as recommended.
38
• In some cases increasing substrate concentration up to 1 mg/ml may improve results. • Add < 0.5% w/v SDS to the reaction buffer. This can significantly improve Factor Xa cleavage with some fusion proteins. Various concentrations of SDS should be tested to find the optimum concentration. • Increase incubation time to 20 hours or longer at +22 °C and increase the amount of Factor Xa (for some fusion proteins, Factor Xa can be increased up to 5%). • Check that protease inhibitors have been removed.
Multiple bands on SDS-PAGE gel after cleavage • Determine when the bands appear. Additional bands seen prior to cleavage may be the result of proteolysis in the host bacteria. E. coli BL21 is a protease-deficient strain that is recommended. • Check the sequence of the fusion partner for the presence of Factor Xa recognition sites. Factor Xa is highly specific for the recognition sequence Ile-Glu-Gly-Arg¡. The bond following the Arg residue is cleaved. Adjusting time and temperature of digestion can result in selective cleavage at the desired Factor Xa site. If adjustment of conditions does not correct the problem, reclone the insert into a pGEX-6P (PreScission) or pGEX T (thrombin) expression vector.
Removal of thrombin, Factor Xa or other serine proteases Before enzymatic cleavage it may be necessary to remove proteases from the sample and after enzymatic cleavage it may be necessary to remove thrombin or Factor Xa. Benzamidine 4 Fast Flow (high sub) provides a convenient and highly specific medium for the removal of serine proteases not only from enzymatic digests, but also from cell culture supernatant, bacterial lysate or serum.
Purification Options Column (prepacked) or Medium
Binding capacity for serine proteases
Comments
HiTrap Benzamidine FF (high sub), 1 ml
> 35 mg trypsin
Prepacked 1 ml column
HiTrap Benzamidine FF (high sub), 5 ml
> 175 mg trypsin
Prepacked 5 ml column
Benzamidine Sepharose 4 Fast Flow (high sub)
> 35 mg trypsin/ml medium
For column packing and scale-up
Binding buffer: 0.05 M Tris-HCl, 0.5 M NaCl, pH 7.4 Elution buffer alternatives: - 0.05 M glycine-HCl, pH 3.0 - 10 mM HCl, 0.5 M NaCl, pH 2.0 - 20 mM para-aminobenzamidine in binding buffer (competitive elution) - 8 M guanidine hydrochloride or 6 M urea (denaturing solutions)
39
1. Equilibrate the column with 5 column volumes of binding buffer. 2. Apply the sample. 3. Wash with 5–10 column volumes of binding buffer or until no material appears in the effluent (monitored by UV absorption at A280 nm). 4. Elute with 5–10 column volumes of elution buffer. Collect fractions in neutralization buffer*. The purified fractions can be buffer exchanged using desalting columns (see Chapter 7).
*Since elution conditions are quite harsh, collect fractions into neutralization buffer (60 µl–200 µl 1 M Tris-HCl, pH 9.0 per ml fraction), so that the final pH of the fractions will be approximately neutral.
Figure 9 shows the isolation of a GST fusion protein on a GSTrap FF column and oncolumn cleavage with thrombin (as described previously). After the thrombin incubation a HiTrap Benzamidine FF (high sub) column is placed in series below the GSTrap FF column. As binding buffers are passed through the two columns the cleaved fusion protein and thrombin are washed from the GSTrap FF column onto the HiTrap Benzamidine FF (high sub) column. This column binds the thrombin enabling the collection of pure thrombin-free protein in the eluent. Sample:
2 ml clarified E.coli homogenate expressing a Mr 37 000 SH2-GST fusion protein with a thrombin protease site GSTrap FF, 1 ml and HiTrap Benzamidine FF (high sub), 1 ml 20 mM sodium phosphate, 0.15 M NaCl, pH 7.5 20 mM sodium phosphate, 1.0 M NaCl, pH 7.5 20 mM p-aminobenzamidine in binding buffer 20 mM reduced glutathione, 50 mM Tris, pH 8.0 0.5 ml/min ÄKTAprime 20 units/ml thrombin protease (Amersham Pharmacia Biotech) for 2 hours at room temperature S-2238 (Chromogenix, Haemochrom Diagnostica AB) was used as a substrate and its absorbance at 405 nm was measured
Columns: Binding buffer: High salt wash buffer: Benzamidine elution buffer: GST elution buffer: Flow: System: Protease treatment: Thrombin activity:
High salt buffer wash
Elution of HiTrap Benzamidine FF (high sub)
Thrombin
Mr
Elution of GSTrap FF
A 280 nm
A 405 nm
97 000 66 000 45 000
0.80
30 000 0.30
GST-tag
Thrombin
20 100 14 400
0.60
1
2
3
4
5
6
7
8
9
0.20
Gel:
0.40 Cleaved SH2 protein
Lane 1. 0.10 fr.14
fr.6 fr.7 fr.8
fr.2
fr.21 fr.22
0.20
Lane 2. Lane 3. Lane 4.
0
0 0
10 A)
B)
15 A)
20 B)
B)
A) GSTrap FF, 1 ml B) HiTrap Benzamidine FF (high sub), 1 ml
25
50 ml A)
Lane 5. Lane 6. Lane 7. Lane 8. Lane 9.
ExcelGel SDS Gradient 8-18%, Coomassie staining Low Molecular Weight Calibration Kit (LMW) Clarified E.coli homogenate expressing SH2-GST fusion protein Flow-through from GSTrap FF (Fraction 2) SH2 GST-tag cleaved, washed out with binding buffer through both columns (Fraction 6) as above (Fraction 7) as above (Fraction 8) Elution of thrombin from HiTrap Benzamidine FF (high sub) Elution of GST-tag and some non-cleaved SH2-GST from GSTrap FF (Fraction 21) as above (Fraction 22)
Fig. 9. On-column cleavage of a GST fusion protein and removal of thrombin after on-column cleavage, using GSTrap FF and HiTrap Benzamidine FF (high sub).
40
CHAPTER 4 (His)6 fusion proteins Amplification General considerations for the amplification of fusion proteins are discussed in Chapter 2.
The vectors and hosts There is a wide variety of hosts and vectors for the amplification of (His)6 fusion proteins. The factors that should be considered when selecting the host and vector are discussed in Chapter 1.
Purification Figure 10 gives an overview of a typical purification flow scheme for (His)6 fusion proteins, including purification under denaturing conditions. On-column purification and refolding of (His)6 fusion proteins is described in Chapter 5.
Native conditions
Denaturing conditions
Binding buffer including 8 M Urea or 6 M Gua-HCl
Cell lysis
Binding buffer
Binding to affinity media
Binding buffer
Binding buffer (as above) including 10-50 mM imidazole
Wash
Elution buffer: Binding buffer with increased amount of imidazole
Elute
Elution buffer: Binding buffer (as above) with increased amount of imidazole
Pure denatured fusion protein
Pure fusion protein
Fusion protein
Refolding
Cell protein
Fig. 10.
41
For simple, one step purification of (His)6 fusion proteins, there is a range of products designed to meet specific purification needs, as shown in Table 13. These products can also be used for the purification of fusion proteins containing shorter or longer polyhistidine tags, such as (His)4 or (His)10. Under the standard binding and elution conditions described in this handbook, the shorter (His)4 will bind more weakly and the longer (His)10 will bind more strongly as compared to (His)6. This difference in binding strength can be used to advantage during purification. For example, since (His)10 binds more strongly, a higher concentration of imidazole can be used during the washing step before elution. This can facilitate the removal of contaminants which may otherwise be co-purified with a (His)6 fusion protein. Chelating Sepharose, when charged with Ni2+ ions, selectively retains proteins if complexforming amino acid residues, in particular histidine, are exposed on the protein surface. (His)6 fusion proteins can be easily desorbed with buffers containing imidazole. If the (His)6 fusion proteins are expressed as inclusion bodies, see Chapter 5 for solubilization, refolding and purification information. Column (prepacked) or Media**
Amount of protein for a single purification
Comment
His MicroSpin Purification Module
Up to 100 µg
Ready to use, prepacked columns, buffers and chemicals High throughput when used with MicroPlex 24 Vacuum (up to 48 samples simultaneously)
HiTrap Chelating HP 1 ml
Up to 12 mg*
Prepacked column, ready to use
HisTrap Kit
Up to 12 mg/column*
As above, but includes buffers for up to 12 purifications using a syringe
HiTrap Chelating HP 5 ml
Up to 60 mg*
Prepacked column, ready to use
Chelating Sepharose Fast Flow
12 mg (His)6 fusion protein* per ml
For packing columns and scale up
*estimate for a protein of Mr 27 600, binding capacity varies according to specific protein
Table 13. Summary of purification options for (His)6 fusion proteins. **Characteristics of HiTrap Chelating HP and Chelating Sepharose Fast Flow are given in Appendix 5.
Re-use of purification columns depends upon the nature of the sample and should only be performed with identical samples to prevent cross contamination. Batch preparation procedures are frequently mentioned in the literature. However the availability of prepacked columns and easily packed high flow rate Chelating Sepharose provide faster, more convenient alternatives. Batch preparations are occasionally used if it appears that the tag is not fully accessible or when the protein in the lysate is at very low concentrations (both could appear to give a low yield from the first purification step). A more convenient alternative to improve yield is to decrease the flow rate or pass the sample through the column several times.
42
Monitor purification steps by one or more of the detection methods referred to in this book. The choice of purification equipment should also be made according to the needs of the purification. Appendix 8 provides a guide to aid in the selection of the correct purification solution and key points to consider are highlighted below. • For a single purification of a small quantity of product or for high throughput screening, MicroSpin columns, using centrifugation or MicroPlex 24 Vacuum respectively, are convenient and simple to use. • For purification of larger quantities of fusion proteins HisTrap Kit or HiTrap Chelating HP columns are ready to use with a syringe, a peristaltic pump or a chromatography system. • To increase capacity use several HiTrap Chelating HP columns (1 ml or 5 ml) in series or, for even larger capacity requirements, pack Chelating Sepharose Fast Flow into a suitable column (details of column packing procedures are outlined in Appendix 6). • For simple and reproducible purification a chromatography system such as ÄKTAprime is a significant advantage, recording the purification process and eliminating manual errors. • For laboratory environments in which all experimental data must be recorded and traceable or where method development, optimization and scale up are required, a computer controlled ÄKTAdesign chromatography system is recommended. • Experiments such as protein refolding or method optimization require linear gradient elution steps that can only be performed by a chromatography system.
His MicroSpin Purification Module The His MicroSpin Purification Module is useful for screening small or large numbers of lysates and for checking samples during the optimization of amplification or purification conditions. Each module contains reagents sufficient for 50 purifications.
•
10X PBS:
•
8X Phosphate/NaCl buffer: 160 mM phosphate, 4 M NaCl, pH 7.4
1.4 M NaCl, 27 mM KCl, 101 mM Na2HPO4, 18 mM KH2PO4, pH 7.3
•
4 M Imidazole elution buffer (avoid contact with skin or eyes)
•
IPTG:
500 mg
•
His MicroSpin columns:
50 units
Reagents are prepared as follows: 1X PBS:
Dilute 10X PBS with sterile water. Store at +4 °C.
PNI20 wash buffer:
Mix 6.25 ml of 8X phosphate/NaCl buffer with 0.25 ml imidazole elution buffer. Add distilled water to a final volume of 50 ml.
PNI400 elution buffer: Mix 6.25 ml of 8X phosphate/NaCl buffer with 5 ml imidazole elution buffer. Add distilled water to a final volume of 50 ml. IPTG 100 mM:
Dissolve contents of the IPTG vial in 20 ml sterile water. Store as 1 ml aliquots at -20 °C.
43
Alternative 1. High throughput purification using MicroPlex Vacuum Do not apply more than 400 µl of sample at a time to a His MicroSpin column. This procedure will accommodate lysates from 2 to 8 ml of culture.
Also required: •
Vacuum source capable of providing 220 mm Hg (e.g. a water vacuum).
•
Side arm flask, 500 ml or 1 litre.
•
Single or double hole rubber stop.
•
Vacuum tubing.
•
MicroPlex 24 Vacuum apparatus (one or two).
1. Assemble the MicroPlex 24 Vacuum following the instructions supplied. 2. Remove the caps from the MicroSpin columns. Place the columns in the manifold, filling any unused holes with the plugs provided with MicroPlex 24 Vacuum. 3. Apply 400 µl of lysate to the column.
If using < 400 µl adjust volume to 400 µl with 1X PBS beforehand. 4. Recap each column securely and mix by gentle, repeated inversion. Incubate at room temperature for 5–10 minutes. 5. Remove columns from the manifold and remove top caps and bottom closures. Return columns to the manifold. 6. Ensure the stopcock is in the closed position (i.e. perpendicular to the vacuum tubing) and that the manifold is placed squarely on the gasket. 7. Turn on vacuum supply at source. Open the stopcock (i.e. parallel to the vacuum tubing). After lysates have been drawn through all the columns into the collection tray, close the stopcock. 8.
Allow 10–15 seconds for the vacuum pressure to dissipate. Remove the manifold and place it on a paper towel.
9. Discard eluate and re-use collection plate or save eluate in the collection plate for later analysis and use a new collection plate for the next step. 10. Add 600 µl of PNI20 wash buffer to each column. Open the stopcock. After buffer has been drawn through all the columns into the collection tray, close the stopcock. 11. Allow 10–15 seconds for the vacuum pressure to dissipate. Remove the manifold and reassemble the apparatus with a clean collection tray. Additonal 600 µl washes with PI20 may be performed if desired. Washes with higher concentrations of imidazole may remove impurities, but decrease yield of the fusion protein. 12. Add 200 µl of PNI400 elution buffer to each column. Incubate at room temperature for 5–10 minutes. 13. Open the stopcock. After elution buffer has been drawn through all the columns into the collection tray, close the stopcock. 14. Allow 10–15 seconds for the vacuum pressure to dissipate. Remove the manifold. Cover eluates with sealing tape until required for analysis.
Note: Yields of fusion protein may be increased by repeating the elution step two or three times and pooling the eluates.
Troubleshooting See Purification and Detection Troubleshooting page 56. 44
Alternative 2. Purification of multiple samples using a microcentrifuge Do not apply more than 400 µl of sample at a time to a His MicroSpin column. This procedure will accommodate lysates from 2 to 8 ml of culture. 1. Remove (and save) each column cap. 2. Apply 400 µl of lysate to the column.
If using < 400 µl adjust volume to 400 µl with 1X PBS beforehand. 3. Recap each column securely and mix by gentle, repeated inversion. Incubate at room temperature for 5–10 minutes. 4. Remove (and save) the top cap and bottom closure from each column. Place column into a clean 1.5 or 2 ml microcentrifuge tube. Spin for 1 minute at 735 g. 5. Remove the MicroSpin columns and place into a clean 1.5 or 2 ml microcentrifuge tube. 6. Apply 600 µl of PNI20 wash buffer to each column. Repeat spin procedure. Additonal 600 µl washes with PI20 may be performed if desired. Washes with higher concentrations of imidazole may remove impurities, but decrease yield of the fusion protein. 7. Replace the bottom closure on each column. Add 100–200 µl of PNI400 elution buffer to each column. Replace top cap and incubate at room temperature for 5–10 minutes. 8. Remove and discard top cap and bottom closure from each column and place the column into a clean 1.5 or 2 ml microcentrifuge tube. 9. Spin all columns again to collect eluate. Save for analysis.
Note: Yields of fusion protein may be increased by repeating the elution step two or three times and pooling the eluates.
Troubleshooting See Purification and Detection Troubleshooting page 56.
45
Alternative 3. Purification using MicroPlex Centrifugation Do not apply more than 400 µl at a time to a His MicroSpin column. This procedure will accommodate lysates from 2 to 8 ml of culture. See Appendix 4 for recommended centrifugation systems. 1. Assemble the MicroPlex 24 unit following the instructions supplied. Two units can be processed simultaneously to handle 48 samples. 2. Remove the caps from the MicroSpin columns and place the columns in the manifold. 3. Apply 400 µl of lysate to each column.
If using < 400 µl adjust volume to 400 µl with 1X PBS beforehand. 4. Recap each column securely and mix by gentle, repeated inversion. Incubate at room temperature for 5–10 minutes. 5. Remove columns from the manifold and remove top caps and bottom closures. Return columns to the manifold. 6. Centrifuge the unit for 2 minutes following the instructions supplied. 7. Add 600 µl of PNI20 wash buffer to each column. 8. Centrifuge the unit for 2 minutes following the instructions supplied. Remove the manifold from each collection tray and place on a clean paper towel. Reassemble each unit with a fresh collection tray. Additonal 600 µl washes with PI20 may be performed if desired. Washes with higher concentrations of imidazole may remove impurities, but decrease yield of the fusion protein. 9. Add 100–200 µl of PNI400 elution buffer to each column. Incubate at room temperature for 5–10 minutes. 10. Centrifuge the unit for 2 minutes following the instructions supplied. Cover the eluted samples with sealing tape until required for analysis.
Note: Yields of fusion protein may be increased by repeating the elution step two or three times and pooling the eluates.
Troubleshooting See Purification and Detection Troubleshooting page 56.
46
Purification using HiTrap Chelating HP 1 ml or 5 ml columns HiTrap Chelating HP columns are prepacked and ready for use. They can be operated with a syringe, a peristaltic pump or a liquid chromatography system such as ÄKTAprime. Figure 11 illustrates the simple steps required for purification of a (His)6 fusion protein.
Prepare column wash with H2O load with NiSO4 wash with H2O
Equilibrate column with binding buffer
3 min
3 min
Waste
Apply sample wash with binding buffer
5-15 min
Waste
Elute with elution buffer
2 min
Collect
Collect fractions
Fig. 11. HiTrap Chelating HP (1 ml) and a schematic overview of (His)6 fusion protein purification.
Re-use of any purification column depends on the nature of the sample and should only be performed with identical fusion proteins to prevent cross-contamination. HiTrap Chelating HP columns (1 ml and 5 ml) can be connected in series to increase the binding capacity. At larger scale, columns can be packed with Chelating Sepharose Fast Flow (for column packing see Appendix 6).
Sample and buffer preparation Use high quality water and chemicals. Filtration through 0.45 µm filters is recommended. Samples should be centrifuged immediately before use and/or filtered through a 0.45 µm filter. If the sample is too viscous, dilute with binding buffer. Sample binding properties can be improved by adjusting the sample to the composition of the binding buffer: dilute in binding buffer or perform a buffer exchange using a desalting column (see Chapter 7). If the fusion protein is expressed as an inclusion body, add 6 M guanidine hydrochloride or 8 M urea to all buffers. During purification there is a balance between the amount of imidazole needed to prevent non-specific binding of contaminants and the amount of imidazole needed to elute the (His)6 fusion protein, conditions may need to be optimized. 47
Alternative 1. Purification using HisTrap Kit HisTrap Kit includes everything needed for 12 preparations using a syringe. Three ready-to-use HiTrap Chelating HP 1 ml columns and ready-made buffer concentrates are supplied with easy-to-follow instructions.
•
HiTrap Chelating HP columns
•
Phosphate buffer, 8X stock solution, pH 7.4 2 x 50 ml
3 x 1 ml
•
2 M imidazole, pH 7.4
50 ml
•
0.1 M NiSO4
10 ml
•
Syringe 5 ml
•
All necessary connectors
Purification protocols Column preparation: loading with nickel ions 1. Fill syringe with distilled water. Remove stopper and connect the column to the syringe with the adapter provided ("drop to drop" to avoid introducing air into the column). 2. Remove the twist-off end. 3. Wash the column with 5 ml distilled water, using the syringe.
Use water, not buffer, to wash away the storage solution (precipitation of nickel salts may occur if the nickel solution comes into contact with the 20% ethanol in the storage solution). If air is trapped in the column, wash the column with distilled water until the air disappears. 4. Disconnect syringe from the column, fill syringe with 0.5 ml of the 0.1 M nickel solution supplied and load onto the column. 5. Wash column with 5 ml distilled water, using the syringe. The column is now ready for use. When high yield is more important than highest purity, follow Protocol A on page 49. To achieve the highest purity, follow Protocol B on page 50.
48
A. Basic protocol for high yield In all steps use the syringe supplied. Note that 1 ml/min corresponds to approximately 30 drops/min when using a syringe with a HiTrap 1 ml column and 5 ml/min corresponds to approximately 120 drops/min when using a HiTrap 5 ml column. 1. Prepare 24 ml binding buffer. Mix 3 ml phosphate buffer 8X stock solution with 0.12 ml 2 M imidazole and add water up to 24 ml. Check pH and adjust to pH 7.4–7.6 if necessary. This buffer contains 20 mM phosphate, 0.5 M NaCl and 10 mM imidazole. 2. Prepare 8 ml elution buffer. Mix 1 ml phosphate buffer 8X stock solution with 2 ml 2 M imidazole and add distilled water up to 8 ml. Check pH and adjust to pH 7.4–7.6 if necessary. This buffer contains 20 mM phosphate, 0.5 M NaCl and 500 mM imidazole. 3. Equilibrate the column with 10 ml binding buffer. Ensure that the column has been loaded with nickel ions before this step. 4. Apply sample at a flow rate 1–3 ml/min. Collect the flow-through fraction. A pump is more suitable for application of sample volumes greater than 15 ml. 5. Wash with 10 ml binding buffer. Collect wash fraction. 6. Elute with 5 ml elution buffer. Avoid dilution of the purified product by collecting the eluate in 1 ml fractions. 7. Check the purification by analysing aliquots of starting material, flow through and eluent, for example, by SDS-PAGE. The purified protein is most likely to be found in the 2nd + 3rd ml of the elution step.
Imidazole absorbs at 280 nm. Use elution buffer as the blank when monitoring absorbance. If imidazole needs to be removed use a desalting column (see Chapter 7). 8. After the protein has been eluted, regenerate the column by washing with 10 ml of binding buffer. The column is now ready for a new purification if the same (His)6 protein is to be purified and there is rarely a need to reload with metal (see Figure 12).
Samples: 2.5 ml GST-(His)6 cell extract Binding buffer: 1X binding buffer, 20 mM imidazole, pH 7.4 Elution buffer: 1X binding buffer, 500 mM imidazole, pH 7.4 Flow: 2 ml/min, 312 cm/h Note: No Ni2+ re-loading of the column between the runs
mg eluted GST-(His) 6
3.0 2.5 2.0 1.5 1.0 0.5 0 1
2
3
4
5
7 6 Run No.
8
9
Result: Run No. 1 2 3 4 5 6 7 8 9 10
Eluted GST-(His)6, total mg 2.76 2.82 2.83 2.72 2.71 2.65 2.64 2.63 2.54 2.59
10
Fig. 12. 10 repetitive purifications of GST-(His)6 without reloading the HiTrap Chelating HP 1 ml column with Ni2+ between the runs.
Troubleshooting See Purification and Detection Troubleshooting page 56.
49
B. Optimization protocol for high purity In all steps use the syringe supplied. Note that 1 ml/min corresponds to approximately 30 drops/min when using a syringe with a HiTrap 1 ml column and 5 ml/min corresponds to approximately120 drops/min when using a HiTrap 5 ml column. 1. Prepare buffers according to Table 14. Use 1X phosphate buffer including 10 mM imidazole as binding buffer and 6 steps ranging up to 500 mM imidazole as elution buffers. Check pH after mixing and adjust to pH 7.4–7.6 if necessary. Imidazole concentration in buffer (mM)
Phosphate buffer 8X stock solution pH 7.4 (ml)
2 M Imidazole pH 7.4 (ml)
Final volume (ml)*
10
3.0
0.12
24
20
1.0
0.08
8
40
1.0
0.16
8
60
1.0
0.24
8
100
1.0
0.40
8
300
1.0
1.20
8
500
1.0
2.00
8
*Use distilled water to adjust to final volume
Table 14. General mixing table using HisTrap Kit buffers for one purification. 2. Load the column with nickel ions according to "Column preparation" (page 48). 3. Equilibrate the column with 10 ml binding buffer (1X phosphate buffer, 10 mM imidazole, pH 7.4). 4. Apply the sample. Collect the flow-through fraction. 5. Wash with 10 ml binding buffer. Collect the wash fraction. 6. Begin elution with 5 ml 1X phosphate buffer containing 20 mM imidazole. Avoid dilution by collecting the eluate in 1 ml fractions. 7. Proceed with the next imidazole concentration, i.e. elute with 5 ml 1X phosphate buffer containing 40 mM imidazole. Collect the eluate in 1 ml fractions as above. 8. Proceed with the buffers of increasing imidazole concentration, as described in steps 6 and 7. The purified protein is most likely to be found in the 2nd + 3rd ml of one of the elution steps. 9. Check the collected fractions, for example, by SDS-PAGE. 10. Regenerate column by washing with 10 ml binding buffer.
From the results select the optimal binding and elution buffers. The optimum elution buffer is the one which eluted the (His)6 fusion protein. The optimum binding buffer is the elution buffer used in the step before, with a lower concentration of imidazole. Using the highest possible concentration of imidazole in the binding buffer will give the highest purity. These buffers can be used for any purification of an identical protein. Imidazole absorbs at 280 nm. Use elution buffer as the blank when monitoring absorbance. If imidazole needs to be removed use a desalting column (see Chapter 7). Perform a blank run to elute non-specifically bound metal ions. Add 5 column volumes binding buffer followed by 5 column volumes elution buffer. Re-equilibrate the column with 5–10 column volumes of binding buffer before sample application. For large sample volumes a simple peristaltic pump or an ÄKTAdesign chromatography system can be used to apply sample and buffers.
Troubleshooting See Purification and Detection Troubleshooting page 56. 50
Alternative 2. Simple purification with ÄKTAprime ÄKTAprime contains a pre-programmed template for automatic column preparation and purification of (His)6 fusion proteins on a single HiTrap Chelating HP column, as shown below. This provides a standard purification protocol which can be followed exactly or optimized as required. Wash
% Elution buffer 100
System preparation & water wash
Elution
Ni-loading 50
Water wash Equilibration
Buffer wash
Sample Reequilibration
625
11
11
20
17
5 Min
Total separation time = 77 min + sample application time
Binding buffer:
20 mM sodium phosphate, 0.5 M NaCl, pH 7.4
Elution buffer:
20 mM sodium phosphate, 0.5 M NaCl, 0.5 M imidazole, pH 7.4
Eluent:
Distilled water
Metal loading solution: 0.1 M NiSO4 Prepare at least 500 ml of each buffer. 1. Follow the instructions supplied on ÄKTAprime cue card Code No: 18-1138-02. 2. Select the Application Template. 3. Start the method. 4. Enter the sample volume and press OK.
Connecting the column. Preparing the fraction collector. Fig. 13. Typical procedures when using ÄKTAprime.
51
Sample:
Column: Binding buffer: Elution buffer: Flow: Result:
5 ml cytoplasmic extract containing (His)-tagged Glutathione S-transferase, GST-(His)6 The clone was a kind gift from Dr. J. Lidholm, Pharmacia Corporation, Sweden HiTrap Chelating HP 1 ml, Ni 2+-loaded according to the instructions 1X phosphate buffer, 20 mM imidazole, pH 7.4 1X phosphate buffer, 500 mM imidazole, pH 7.4 2 ml/min, 312 cm/h Eluted GST-(His)6, 4 ml, A280: 1.65 Total amount: 4.46 mg
A 280 nm
A 405 nm
Lane Lane Lane Lane Lane Lane Lane Lane
1.0
A 280
1: 2: 3: 4: 5: 6: 7: 8:
Low Molecular Weight Calibration Kit (LMW) Starting material, cytoplasmic extract, dil. 1:20 Flow-through, dil. 1:10 Wash Eluted GST-(His)6, dil 1:20 Eluted GST-(His)6, dil 1:10 GST standard, 0.5 mg/ml LMW
0.70
0.5
0.60 0.50 0.40 A 405
0.0
Flow through
0.0
5.0
Sample Binding application buffer
W ash
10.0
Eluted pool
15.0
Elution buffer
Mr
0.30
97 000
0.20
66 000 45 000
0.10
30 000 20 100 14 400
20.0
Volume (ml)
Binding buffer
Fig. 14a. Purification of histidine-tagged Glutathione S-transferase from a cytoplasmic extract.
1
2
3
4
5
6
7
8
Fig. 14b. SDS electrophoresis on PhastSystem™ using PhastGel™ 10–15, silver staining.
Fig. 14.
Figure 14 shows an example of the purification of a (His)6 fusion protein using a HiTrap Chelating HP 1 ml column.
Troubleshooting See Purification and Detection Troubleshooting page 56. An example of the purification and refolding of an insoluble (His)6 fusion protein is shown on page 61 and clearly demonstrates the advantage of using a chromatography system for this type of work.
52
Detection of (His)6 fusion proteins Table 15 reviews the methods available for detection of (His)6 fusion proteins. These methods can be selected according to the experimental situation, for example, SDS-PAGE analysis, performed frequently during amplification and purification to monitor results, may not be the method of choice for routine monitoring of samples from high throughput screening. Functional assays specific for the protein of interest are useful but not often available. Detection method ELISA assay using anti-His antibody Western blot analysis using anti-His antibody and ECL detection systems
SDS-PAGE with Coomassie or silver staining Functional assays
Comments Highly specific, detects only (His)6 fusion protein. Highly specific, detects only (His)6 fusion protein. Little or no background when used at optimized concentrations with secondary HRP conjugated antibody. ECL detection systems enhance detection in Western blot. ECL provides adequate sensitivity for most recombinant expression applications. For higher sensitivity use ECL Plus. Provides information on size and % purity. Detects fusion protein and contaminants. Useful to assess if the purified (His)6 fusion protein is active. Not always available. May require development and optimization.
Table 15. Detection methods for (His)6 fusion proteins.
Alternative 1: SDS-PAGE analysis 6X SDS loading buffer: 0.35 M Tris-HCl, 10.28% (w/v) SDS, 36% (v/v) glycerol, 0.6 M dithiothreitol (or 5% 2-mercaptoethanol), 0.012% (w/v) bromophenol blue, pH 6.8. Store in 0.5 ml aliquots at -80 °C. 1. Add 2 µl of 6X SDS loading buffer to 5–10 µl of supernatant from crude extracts, cell lysates or purified fractions as appropriate. 2. Vortex briefly and heat for 5 minutes at +90 to +100 °C. 3. Load the samples onto an SDS-polyacrylamide gel. 4. Run the gel for the appropriate length of time and stain with Coomassie Blue (Coomassie Blue R Tablets) or silver (PlusOne Silver Staining Kit, Protein).
The percentage of acrylamide in the SDS-gel should be selected according to the expected molecular weight of the protein of interest (see Table 16). % Acrylamide in resolving gel
Separation size range (Mr x 10-3)
Single percentage: 5%
36–200
7.5%
24–200
10%
14–200
12.5%
14–100
15%
14–60
1
Gradient: 5–15%
1
14–200
5–20%
10–200
10–20%
10–150
The larger proteins fail to move significantly into the gel.
Table 16.
53
If there appears to be aggregation of protein in the gel, use at least 7.5 mM 2-mercaptoethanol in the SDS-PAGE sample buffer. This may be caused by aggregation between N terminal histidine helices. For information and advice on electrophoresis techniques, please refer to the section Additional reading and reference material.
Troubleshooting • If the fusion protein is absent, it may be insoluble or expressed at very low levels; refer to protein amplification troubleshooting (page 9). • If no fusion protein is detected by Coomassie Blue, try silver staining or Western blotting to enhance sensitivity.
Alternative 2. Western blot analysis Amplification and purification can be monitored by Western blot analysis using ECL or ECL Plus detection systems to enhance sensitivity, if required. Anti-His Antibody Blocking/Incubation buffer: 5% (w/v) non-fat dry milk and 0.1% (v/v) Tween 20 in PBS (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.3) Wash buffer:
0.1% v/v Tween 20 in PBS (as above)
Secondary Antibody to detect the anti-His antibody (such as antibody to mouse Ig, HRP-linked Whole Ab, NA931). 1. Separate the protein samples by SDS-PAGE.
Anti-His antibody from Amersham Pharmacia Biotech is a monoclonal preparation avoiding the presence of low levels of cross-reacting antibodies. However, it is recommended to always run a sample of an E. coli sonicate that does not contain a recombinant (His)6 plasmid as a control. 2. Transfer the separated proteins from the electrophoresis gel to an appropriate membrane, such as Hybond ECL (for subsequent ECL detection) or Hybond P (for subsequent ECL or ECL Plus detection).
Electrophoresis and protein transfer may be accomplished using a variety of equipment and reagents. For further details, refer to the Protein Electrophoresis Technical Manual and the Hybond ECL instruction manual from Amersham Pharmacia Biotech.
Blocking of membrane 1. Transfer the membrane onto which the proteins have been blotted to a container such as a Petri dish. 2. Add 50–200 ml of blocking/incubation buffer. 3. Incubate for 1–16 hours at ambient temperature with gentle shaking.
Longer incubation times (up to 16 hours) with blocking buffer may reduce background signal. 4. Decant and discard the buffer.
54
Anti-His antibody 1. Prepare an appropriate dilution of anti-His antibody with blocking/incubation buffer e.g. 5–10 µl of antibody to 50 ml of buffer. Refer to Amersham Pharmacia Biotech Application Note 18-1139-13 for further information on optimization. 2. Pour the antibody-buffer mixture into the container with the membrane. 3. Incubate for 1 hour at ambient temperature with gentle shaking. 4. Decant and discard the antibody-buffer. 5. Rinse twice with 20–30 ml of blocking or wash buffer to remove most of the unbound antibody. 6. Decant and discard the rinses. 7. Wash the membrane with 20–30 ml of blocking or wash buffer for 10–60 minutes at ambient temperature with gentle shaking. 8. Discard the wash and repeat.
Secondary antibody 1. Dilute an appropriate anti-mouse secondary antibody with blocking/incubation buffer according to the manufacturer's recommendation. Refer to Amersham Pharmacia Biotech Application Note 18-1139-13 for further information on optimization. 2. Pour the antibody-buffer mixture into the container with the membrane. 3. Incubate for 1 hour at ambient temperature with gentle shaking. 4. Decant and discard the antibody-buffer. 5. Rinse twice with 20–30 ml of blocking or wash buffer to remove most of the unbound antibody. 6. Decant and discard the rinses. 7. Wash the membrane with 20–30 ml of blocking or wash buffer for 10–60 minutes at ambient temperature with gentle shaking. 8. Discard the wash and repeat. 9. Develop the blot with the appropriate substrate for the conjugated secondary antibody.
ECL and ECL Plus detection systems require very little antibody to achieve a sufficient sensitivity so the amount of antibody (primary and secondary) used in the protocols can be minimized. Smaller quantities of antibody-buffer mixtures can be used by scaling down the protocol and performing the incubations in sealable plastic bags.
Troubleshooting See also Purification and Detection Troubleshooting page 56.
Multiple bands seen on Western blot analysis Anti-His antibody from Amersham Pharmacia Biotech is a monoclonal preparation and has been tested for its lack of non-specific background binding in a Western blot. Some sources of the anti-His antibody may contain antibodies that react with various E. coli proteins present in the fusion protein sample. Cross-adsorb the antibody with an E. coli sonicate to remove anti-E. coli antibodies. This E. coli should not contain a His-tag encoding plasmid.
55
Purification and detection troubleshooting Column has clogged • Cell debris in the sample may clog the column. Clean the column according to Appendix 5 and ensure that samples have been filtered or centrifuged.
Protein is precipitating in solution or on the column • Solubilizing agents include: 2 M NaCl, 50 mM CHAPS, 50% glycerol, 8 M urea, 6 M guanidine hydrochloride, 0.1–2% Triton X-100, 0.1–2% Tween 20. Mix gently for 30 minutes. Reducing agents such as 2-mercaptoethanol may also help the solubilization process. Triton X-100 has a high absorbance at A280 and cannot be removed by buffer exchange procedures.
Fusion protein does not bind to the column • Over-sonication may have denatured the fusion protein. Check by using a microscope to monitor cell breakage. Use mild sonication conditions during cell lysis. • Sonication may be insufficient: Check using a microscope or monitor by measuring the release of nucleic acids at A260. Addition of lysozyme (0.1 volume of a 10 mg/ml lysozyme solution in 25 mM Tris-HCl, pH 8.0) prior to sonication may improve results. • Check that the correct buffers and pH have been used. Ensure that no chelating or reducing agents are present in the sample. • If re-using a column check that it has been regenerated correctly (see Appendix 5). Replace with fresh Chelating Sepharose or a new column if the binding capacity does not return after regeneration. • Decrease the concentration of imidazole in the binding buffer (MicroSpin columns are prepacked with 20 mM imidazole, wash the column once with phosphate/NaCl buffer and re-equilibrate with buffer containing the desired concentration of imidazole). • Tag may be inaccessible. Purify under denaturing conditions or move tag to opposite end of the protein. For denaturing conditions use 20 mM sodium phosphate, 8 M urea or 6 M guanidine hydrochloride and imidazole concentrations in the range 10–500 mM, pH 7.4. • Tag may be degraded. Check that tag is not associated with any part of the protein of interest. • Column capacity is exceeded. Join 2 or 3 HiTrap Chelating HP 1 ml or 5 ml columns in series or use an even larger column. • Fusion protein may be in inclusion bodies. Add solubilization agents. • Column may not have been correctly charged with nickel ions. Repeat column preparation steps.
56
Fusion protein is poorly eluted • The elution conditions are too mild: increase concentration of imidazole in the elution buffer. Concentrations > 400 mM may be more effective. Elute with an increasing imidazole gradient or decreasing pH to determine the optimal elution conditions. • Fusion protein may be precipitating (see Protein is precipitating). • Try over-night elution at room temperature or +4 °C. Do not go below pH 3.5, as metal ions will be stripped off the column.
Purification needs to be optimized • Following the instructions supplied with HiTrap Chelating HP columns will generally give good results. Further optimization for a specific fusion protein may be possible by adjusting the imidazole concentration in the start and elution buffers. • Optimize the imidazole concentration in the binding and elution buffers: Follow instructions on page 50 (HisTrap Kit protocols). Higher concentrations of imidazole in the binding buffer will remove more impurities from the column resulting in a higher purity of fusion protein in the elution buffer. • Yields of (His)6 fusion proteins may sometimes be increased by repeating the elution step two or three times and pooling the eluents or by reducing the flow rate of elution. Increasing the purity by increasing the imidazole concentration may decrease the yield of protein as some of the (His)6 fusion proteins may be washed away. Always determine the optimum combination of purity and yield for the specific application.
Multiple bands seen on SDS-PAGE or Western blot analysis • Multiple bands result from partial degradation of fusion proteins by proteases, or denaturation and co-purification of host proteins with the GST fusion protein due to over-sonication. • Over-sonication can lead to co-purification of host proteins with the fusion protein. Check conditions as described earlier. Avoid frothing. Decrease sonication times. Add lysozyme. • Follow optimization instructions on page 50 (HisTrap Kit protocols). • Increase detergent levels (e.g. up to 2% Triton X-100 or 2% Tween 20) or add glycerol (up to 50%) to the binding buffer to disrupt non-specific interactions. • Add 2-mercaptoethanol (up to 20 mM) to disrupt disulphide bonds. N.B. this can affect binding properties. • If contaminants are truncated forms of fusion protein, check for premature termination sites (N-terminal tag) or internal translation starts (C-terminal tag). Work at +4 °C or add protease inhibitors to prevent proteolysis.
57
• If contaminants have a high affinity for nickel ions add imidazole to the sample at the same concentration as in the binding buffer. • Use further chromatographic purification steps (see Chapter 9). • Use a protease deficient host or add protease inhibitors.
Tag removal by enzymatic cleavage In most cases, functional tests can be performed using the intact (His)6 fusion protein. If removal of the tag is necessary, then procedures similar to GST tag removal can be followed i.e. specific recognition sites are incorporated to allow subsequent enzymatic cleavage. The precise protocols required for cleavage and purification will depend upon the original vectors and the properties of the specific enzymes used for cleavage. There is no PreScission Protease recognition site available for use with (His)6 fusion proteins. rTEV protease (Life Technologies Cat. No. 10127-017) has a (His)6 tag and recognizes the amino acid sequence Glu-Asn-Leu-Tyr-Phe-Gln¡Gly. Glu, Tyr, Gln and Gly are needed for cleavage between the Gln and Gly residues(¡). N-terminal (His)6 tags can be removed. The advantage of this enzymatic cleavage is that the protein of interest can be repurified using the same Chelating Sepharose. The (His)6 tag and the (His)6 tag rTEV protease will both bind to the column and the protein of interest can be collected in the flow through. The amount of enzyme, temperature and length of incubation required for complete digestion varies according to the specific fusion protein produced. Determine optimal conditions in preliminary experiments. Remove samples at various time points and analyse by SDS-PAGE to estimate the yield, purity and extent of digestion. Approximate molecular weights for SDS-PAGE analysis: rTEV protease
Mr 29 000
Carboxypeptidase A*
Mr 94 000
* for the removal of C-terminal (His)6 tags.
Some cleavage procedures will require a second purification step to be performed to remove the protease or other contaminants. Conventional chromatographic separation techniques such as ion exchange or hydrophobic interaction chromatography will need to be developed (see Appendix 9).
58
CHAPTER 5 Handling inclusion bodies Amplification can often be controlled so that recombinant protein accumulates in the intracellular space or is secreted into the periplasmic space or out into the culture medium. While secretion is advantageous in terms of protein folding, solubility and disulphide bonding, the yield is generally much higher when using intracellular expression. However, recombinant protein accumulated intracellularly is frequently laid down in the form of inclusion bodies, insoluble aggregates of mis-folded protein lacking biological activity. So, whilst the presence of inclusion bodies can make preliminary isolation steps very simple, the isolation of proteins from inclusion bodies often leads to difficulties with re-folding and full recovery of biological activity. Table 17 summarizes the advantages and disadvantages of working with recombinant products expressed as inclusion bodies. Advantages High expression levels can reduce fermentation costs Easily monitored by SDS-PAGE or immunoblotting Cytoplasmic proteins are easily washed away
Disadvantages Re-folding shifts difficulties and costs downstream Amplification cannot be monitored directly by functional assays Minor contaminants are often hydrophobic, poorly soluble membrane proteins and cell wall fragments Major contaminants are oligomers and misfolded or proteolyzed forms of the protein which can be difficult to separate If the protein does not fold well, another expression system will be needed
Table 17.
Solubilization of inclusion bodies The solubility of a recombinant protein can be made more favourable by modification of culture conditions (see Chapter 1 and 2). If culture modifications do not significantly improve the yield of soluble fusion proteins, then common denaturants such as 4–6 M guanidine hydrochloride, 4–8 M urea, detergents, alkaline pH (> 9), organic solvents or N-lauroyl-sarcosine can be used to solubilize inclusion bodies. For each denaturant the success of solubilization will be affected by time, temperature, ionic strength, the ratio of denaturant to protein and the presence of reducing reagents. Solubilized proteins can often be purified at this stage by using a suitable purification technique that will also remove the denaturant and allow refolding of the protein (see below). Success of affinity purification in the presence of denaturing agents will depend on the nature of the fusion protein. For example: GST fusion proteins from inclusion bodies have been solubilized and purified in the presence of 2–3 M guanidine hydrochloride or urea. Other denaturants (up to 2% Tween 20; 1% CTAB; or 0.03% SDS) have also been used.
59
Refolding of solubilized recombinant proteins Following solubilization, proteins must be properly refolded to regain function. Denaturing agents must always be removed to allow refolding of the protein and formation of the correct intramolecular associations. Critical parameters during refolding include pH, presence of reducing reagents, the speed of denaturant removal, and the relative concentrations of host proteins and recombinant protein. Table 17 compares conventional methods for refolding of insoluble recombinant proteins with on-column affinity purification and refolding. Refolding techniques Step dialysis Dilution into near neutral pH Gel filtration
On-column refolding
Advantages/Disadvantages Takes several days Uses large volumes of buffer Dilutes the protein of interest Slow Requires a second column to be run Only small volumes can be processed per column Fast and simple No sample volume limitations
Table 17. Comparison of methods for protein refolding.
On-column refolding Using a (His)6 fusion protein enables the use of a simple, but efficient, purification and oncolumn refolding procedure that produces soluble protein exhibiting the desired biological activity. The protocol shown in Figure 15 has been used successfully for several different (His)6 fusion proteins. E. coli culture Cell paste
Cell disruption
Centrifugation (5–10 000 g)
Pellet Wash & Centrifugation
Isolated Inclusion Bodies
Solubilization Purification & Refolding on Ni2+ -loaded HiTrap Chelating HP
Fig. 15. General scheme for the extraction, solubilization and refolding of (His)6 fusion proteins produced as inclusion bodies in E. coli cells.
High concentrations of chaotropic agents (such as urea or guanidine hydrochloride) enhance the binding of the histidine tract to immobilized divalent metal ions. Consequently, (His)6 fusion proteins can be solubilized by chaotropic extraction and bound to Chelating Sepharose. Removal of contaminating proteins and refolding by exchange to non-denaturing buffer conditions can be performed before elution of the protein from the column (Colangeli et al., J. Chrom. B, 714, 223–235 (1998)). 60
Once refolded, the protein may be purified further by other techniques (see Chapter 9) if a higher degree of purity is required.
Purification and on-column refolding of an insoluble (His)6 fusion protein from a 100 ml E. coli culture Resuspension buffer:
20 mM Tris-HCl, pH 8.0
Isolation buffer:
2 M urea, 20 mM Tris-HCl, 0.5 M NaCl, 2% Triton X-100, pH 8.0
Binding buffer:
6 M guanidine hydrochloride, 20 mM Tris-HCl, 0.5 M NaCl, 5 mM imidazole, 1 mM 2-mercaptoethanol, pH 8.0
Wash buffer:
6 M urea, 20 mM Tris-HCl, 0.5 M NaCl, 20 mM imidazole, 1 mM 2-mercaptoethanol, pH 8.0
Refolding buffer:
20 mM Tris-HCl, 0.5 M NaCl, 20 mM imidazole, 1 mM 2-mercaptoethanol, pH 8.0
Elution buffer:
20 mM Tris-HCl, 0.5 M NaCl, 0.5 M imidazole, 1 mM 2-mercaptoethanol, pH 8.0
For column preparation: Distilled water 100 mM NiSO4
Phosphate buffers can also be used instead of Tris-HCl (see earlier purification protocols for (His)6 fusion proteins). Use high purity water and chemicals. Filter buffers through a 0.45 µm filter before use. Prepare at least 500 ml of each buffer when using ÄKTAprime. After preliminary investigations it may be possible to increase the concentration of imidazole and narrow the elution gradient. The optimum concentration and gradient elution will be dependent on the fusion protein. See the optimization protocol for high purity on page 50.
Disruption, wash and isolation of inclusion bodies 1. Resuspend the cell paste from 100 ml culture in 4 ml resuspension buffer. 2. Disrupt cells with sonication on ice (e.g. 4 × 10 sec.). 3. Centrifuge at high speed for 10 min. at +4 °C. 4. Resuspend pellet in 3 ml cold isolation buffer and sonicate as above. 5. Centrifuge at high speed for 10 min. at +4 °C. 6. Repeat steps 4 and 5.
At this stage the pellet material can be washed once in buffer lacking urea and stored frozen for later processing.
Solubilization and sample preparation 1. Resuspend pellet in 5 ml binding buffer. 2. Stir for 30–60 min. at room temperature. 3. Centrifuge for 15 min. at high speed, +4 °C. 4. Remove remaining particles by passing sample through a 0.45 µm filter.
Optimal concentration of reducing 2-mercaptoethanol (0–5 mM) must be determined experimentally for each individual protein.
61
Refolding and purification The requirement for linear gradient formation for refolding and elution makes the use of a chromatography system essential. When using ÄKTAprime, select the Application Template for automatic refolding and purification of His-tagged proteins and follow the instructions on the cue card (code no: 18-1139-43). The following steps will be performed automatically.
Preparation of the column 1. Wash HiTrap Chelating HP 1 ml column with 5 ml distilled water. 2. Load 0.5 ml 0.1 M NiSO4 solution and continue to wash with 5 ml distilled water. 3. Equilibrate column with 5–10 ml binding buffer.
Loading and washing 1. Load sample and wash column with 10 ml binding buffer. 2. Wash with 10 ml wash buffer.
Refolding 1. Refolding of the bound protein is performed by the use of a linear 6–0 M urea gradient, starting with the wash buffer above and finishing with the refolding buffer. A gradient volume of 30 ml or higher and a flow rate of 0.1–1 ml/min can be used. The optimal refolding rate should be determined experimentally for each protein. 2. Continue to wash with 5 ml of refolding buffer after the gradient has come to its endpoint.
Elution and Purification 1. Elute refolded recombinant protein using a 10–20 ml linear gradient starting with refolding buffer and ending with the elution buffer. 2. Check collected fractions with, for example, SDS-PAGE and pool as suitable. Figure 16 illustrates an example of this on-column refolding and purification procedure.
A 280
Ni2+-loaded HiTrap Chelating HP 1 ml N-terminal (His)6 recombinant protein 1.0 produced in E. coli 0.1–1 ml/min, sample loading and refolding Flow: 1 ml/min, wash and elution 0.75 Binding buffer: 20 mM Tris-HCl, 0.5 M NaCl, 5 mM imidazole, 6 M guanidine hydrochloride, 1 mM 2-mercaptoethanol, pH 8.0 Washing buffer: 20 mM Tris-HCl, 0.5 M NaCl, 20 mM imidazole, 0.5 6 M urea, 1 mM 2-mercaptoethanol, pH 8.0 Refolding buffer: 20 mM Tris-HCl, 0.5 M NaCl, 20 mM imidazole, 1 mM 2-mercaptoethanol, pH 8.0 Refolding gradient: 30 ml Elution buffer: 20 mM Tris-HCl, 0.5 M NaCl, 500 mM imidazole, 0.25 1 mM 2-mercaptoethanol, pH 8.0 Elution gradient: 10 ml Fraction volumes: 3 ml sample loading, wash and refolding 0 1 ml elution
Column: Sample:
Start refolding fr. fr. fr. 38 40 42
fr. 49
Start elution Manually using a syringe: • Sample loading • Gua-HCl wash • Urea wash
10 20 30 40
50
Fig. 16.
Imidazole is easily removed using a desalting column (see Chapter 7). 62
fr. 46
60
65
ml
CHAPTER 6 Harvesting and extraction of recombinant proteins This section reviews the most common harvesting and extraction procedures for recombinant proteins. Samples should be clear and free from particles before beginning purification. Extraction procedures should be selected according to the source of the protein, such as bacterial, plant or mammalian, intracellular or extracellular. Selection of an extraction technique is dependent as much upon the equipment available and scale of operation as on the type of sample. Examples of common extraction processes are shown in Table 18. Use procedures which are as gentle as possible since disruption of cells or tissues leads to the release of proteolytic enzymes and general acidification. Extraction should be performed quickly, at sub-ambient temperatures, in the presence of a suitable buffer to maintain pH and ionic strength and stabilize the sample. Extraction process Gentle Cell lysis (osmotic shock)
Typical conditions
Protein source
Comment
2 volumes water to 1 volume packed pre-washed cells lysozyme 0.2 mg/ml, +37 °C, 15 mins.
E. coli periplasm: intracellular proteins bacteria: intracellular proteins and periplasm
lower product yield but reduced protease release lab scale only, often combined with mechanical disruption
Moderate Grinding with abrasive e.g. sand Vigorous Ultrasonication or bead milling
follow equipment instructions
bacteria, cells/tissues
“
Manton-Gaulin homogeniser French press Fractional precipitation
“ “ see section on fractional precipitation
cell suspensions: intracellular proteins in cytoplasm, periplasm, inclusion bodies cell suspensions bacteria, cells/tissues extracellular: secreted recombinant proteins, cell lysates
Enzymatic digestion
small scale, release of nucleic acids may cause viscosity problems, inclusion bodies must be resolubilized large scale only precipitates must be resolubilized
Table 18. Common sample extraction processes.
If lysates are too viscous to handle (caused by the presence of a high concentration of host nucleic acid) continue to sonicate for a longer period or follow one of the following procedures: •
Add DNase I to a final concentration of 10 µg/ml and incubate for 10–15 min.
•
Add RNase A to a final concentration of 10 µg/ml and DNase I to a final concentration of 5 µg/ml, and incubate on ice for 10–15 min.
•
To avoid the use of enzymes, draw the lysate through a syringe needle several times.
To achieve optimal conditions for cell growth, induction of your recombinant fusion protein and cell lysis, refer to recommended protocols. The following is a general protocol for sample preparation.
63
Extraction buffer: PBS, pH 7.4 or other recommended buffer. Additives such as 8 M urea or 6 M guanidine hydrochloride can be included if solubilization of the protein is needed (e.g. if the protein is expressed as an inclusion body).
To prevent non-specific binding of host cell proteins, 5–50 mM imidazole can be included in the extraction buffer when working with (His)6 fusion proteins. 1. Harvest cells by centrifugation (e.g. at 7 000–8 000 g for 10 minutes or 1 000–1 500 g for 30 minutes at +4 °C). 2. Discard supernatant. Place bacterial pellet on ice. 3. Using a pipette, resuspend the cell pellet by adding 50 µl of ice-cold extraction buffer pH 7–8.5 per ml of cell culture. 4. Disrupt suspended cells e.g. sonicate on ice in short 10 seconds bursts. Save an aliquot of the sonicate for analysis by SDS-PAGE.
Sonicate for the minimum time necessary to disrupt the cells. Prolonged sonication may destroy protein functionality. Avoid frothing as this may denature the fusion protein and can lead to co-purification of host proteins with the fusion protein. 5. Sediment cell debris by centrifugation (e.g. at 12 000 g for 10 minutes at +4 °C). 6. Carefully transfer the supernatant, without disturbing the pellet, to a fresh container. Save aliquots of the supernatant and the cell debris pellet for analysis by SDS-PAGE.
Samples containing 8 M urea can be analysed directly, but, if 6 M guanidine hydrochloride is present, this must be exchanged for 8 M urea by using a prepacked desalting column (see Chapter 7) before loading onto an electrophoresis gel. 7. The sample should be fully dissolved prior to loading the column and adjusted to pH 7–8 by dilution or buffer exchange on a desalting column. 8. Store sample at -20 °C when not in use.
Fractional precipitation Fractional precipitation is frequently used at laboratory scale to remove gross impurities from small sample volumes, and occasionally used in small-scale commercial production. When using a HiTrap affinity purification column (such as GSTrap FF or HiTrap Chelating HP) at laboratory scale, it is unlikely that fractional precipitation will be required. Precipitation techniques separate fractions by the principle of differential solubility. Because protein species differ in their degree of hydrophobicity, increased salt concentrations can enhance hydrophobic interactions between the proteins and cause precipitation. Fractional precipitation can be applied to remove gross impurities in three different ways, as shown in Figure 17. Precipitation techniques are affected by temperature, pH and sample concentration. These parameters must be controlled to ensure reproducible results. Not all proteins are easy to redissolve, yield may be reduced. Most precipitation techniques are not suitable for large-scale preparation.
64
Clarification Bulk proteins and particulate matter precipitated
Supernatant
Extraction Clarification Concentration Target protein precipitated with proteins of similar solubility Extraction Clarification Bulk proteins and particulate matter precipitated
Redissolve pellet*
Concentration Target protein precipitated with proteins of similar solubility
Chromatography
Redissolve pellet*
Remember: if precipitating agent is incompatible with next purification step, use Sephadex™ G-25 for desalting and buffer exchange e.g. HiTrap Desalting or PD-10 columns
*Remember: not all proteins are easy to redissolve, yield may be reduced
Fig. 17. Three ways to use precipitation.
Precipitation agents are reviewed in Table 19 and the most common method is described in more detail. Precipitation agent
Typical conditions for use
Sample type
Comment
Ammonium sulphate
as described on page 66
Dextran sulphate
Add 0.04 ml 10% dextran sulphate and 1 ml 1 M CaCl per ml sample, mix 15 min., centrifuge 10 000 g, discard pellet Add 3% (w/vol), stir 4 hours, centrifuge, discard pellet up to 20% w/vol
> 1 mg/ml proteins especially immunoglobulins samples with high levels of lipoprotein e.g ascites
stabilizes proteins, no denaturation, supernatant can go directly to HIC precipitates lipoprotein
as above
alternative to dextran sulphate no denaturation, supernatant goes direct to IEX or AC, complete removal may be difficult may denature protein irreversibly
Polyvinylpyrrolidine Polyethylene glycol (PEG, Mr > 4 000)
Acetone (cold)
Polyethyleneimine
up to 80% vol/vol at +0 °C collect pellet after centrifugation at full speed in an Eppendorf centrifuge 0.1% w/vol
Protamine sulphate Streptomycin sulphate
1% w/vol 1% w/vol
plasma proteins
useful for peptide precipitation or concentration of sample for electrophoresis
precipitates aggregated nucleoproteins as above precipitation of nucleic acids
Table 19. Examples of precipitation techniques. Details taken from: Protein Purification, Principles and Practice. Scopes, R.K., Springer (1994). Protein Purification, Principles, High Resolution Methods and Applications. Janson, J.C. and Rydén, L., 2nd ed. Wiley Inc. (1998). Other personal communications.
For general advice on buffer selection and the use of detergents please refer to the Sample Preparation chapter in the Affinity Chromatography, Principles and Methods Handbook from Amersham Pharmacia Biotech.
65
Ammonium sulphate precipitation Solutions required Saturated ammonium sulphate: add 100 g ammonium sulphate to 100 ml distilled water, stir to dissolve. 1 M Tris-HCl, pH 8.0. Buffer for first purification step. 1. Filter (0.45 µm) or centrifuge (10 000 g, refrigerated at +4 °C) sample. 2. Add 1 part 1 M Tris-HCl, pH 8.0 to 10 parts sample volume to maintain pH. 3. Stir gently. Add saturated ammonium sulphate solution, drop by drop (solution becomes milky at about 20% saturation). Add up to 50% saturation*. Stir for 1 hour. 4. Centrifuge 20 minutes at 10 000 g. 5. Discard supernatant. Wash pellet twice by resuspension in an equal volume of ammonium sulphate solution of the same concentration (i.e. a solution that will not redissolve the precipitated protein or cause further precipitation). Centrifuge again, as in step 4. 6. Dissolve pellet in a small volume of the purification buffer. 7.
Ammonium sulphate is removed during clarification/buffer exchange steps with Sephadex G-25 (see Chapter 7) or during hydrophobic interaction separations.
*The % saturation can be adjusted either to precipitate a target molecule or to precipitate contaminants.
The quantity of ammonium sulphate required to reach a given degree of saturation varies according to temperature. Table 20 shows the quantities required at +20 °C. Final percent saturation to be obtained 20
25
Starting percent saturation
30
35
40
45
50
55
60
65
70
75
80
85
95 100
0
113 144 176 208 242 277 314 351 390 430 472 516 561 608 657 708 761
5
85 115 146 179 212 246 282 319 358 397 439 481 526 572 621 671 723
10
57
86 117 149 182 216 251 287 325 364 405 447 491 537 584 634 685
15
28
58
20
0
29
59
89 121 154 188 223 260 298 337 378 421 465 511 559 609
0
29
60
91 123 157 191 228 265 304 344 386 429 475 522 571
0
30
61
92 125 160 195 232 270 309 351 393 438 485 533
0
30
62
0
31
63
96 130 166 202 241 281 322 365 410 457
0
31
64
98 132 169 206 245 286 329 373 419
0
32
65
0
33
25 30 35 40 45 50 55 60 65 70 75 80 85
88 119 151 185 219 255 293 331 371 413 456 501 548 596 647
94 128 163 199 236 275 316 358 402 447 495
0
99 135 172 210 250 292 335 381 66 101 138 175 215 256 298 343 33 0
67 103 140 179 219 261 305 34 0
69 105 143 183 224 267 34 0
70 107 146 186 228 35 0
72 110 149 190 36 0
90 95
Table 20. Quantities of ammonium sulphate required to reach given degrees of saturation at +20 °C.
66
90
Amount of ammonium sulphate to add (grams) per litre of solution at +20 °C
73 112 152 37 0
75 114 37
76
0
38
CHAPTER 7 Buffer exchange and desalting of recombinant proteins Dialysis is frequently mentioned in the literature as a technique used to remove salt or other small molecules and exchange the buffer composition of a sample. However dialysis is generally a slow technique, requiring large volumes of buffer and with a risk of losing material during handling or because of proteolytic breakdown or non-specific binding of samples to the dialysis membranes. A simpler and much faster technique is to use desalting columns that perform a group separation between high and low molecular weight substances. The columns are packed with Sephadex G-25, a gel filtration product that separates molecules on the basis of size. In a single step, the sample is desalted, transferred into a new buffer, and low molecular weight materials are removed. Desalting columns are used not only for the removal of low molecular weight contaminants such as salt, but also to transfer the sample into a different buffer before or after purification. They are also used for the rapid removal of reagents to terminate a reaction. Sample volumes up to 30% of the total volume of the desalting column can be processed. The high speed and high capacity of the separation allows even large sample volumes to be processed rapidly and efficiently. Sample concentration does not influence the separation as long as the concentration of proteins does not exceed 70 mg/ml when using normal aqueous buffers. Figure 18 shows a typical desalting and buffer exchange separation, monitored by following changes in UV absorption and conductivity.
A 280 nm
(mS/cm)
0.25 0.20 0.15
10.0 protein
salt
0.10 5.0 0.05 0.00 0.0
1.0
2.0
Time (min)
Fig. 18. Desalting and buffer exchange of mouse plasma (10 ml), using a HiPrep 26/10 Desalting column.
Table 21 shows a selection guide for prepacked, ready to use desalting and buffer exchange columns. Column MicroSpin G-25 HiTrap Desalting HiPrep 26/10 Desalting
Column volumn 0.5 ml 5 ml 53 ml
Sample volume 0.1–0.15 ml 0.25–1.5 ml 2.5–15 ml
Volume after elution 0.1–0.15 ml 1.0–2.0 ml 7.5–20 ml
Table 21. Selection guide for desalting columns.
67
To desalt larger sample volumes - connect up to 5 HiTrap Desalting columns in series to increase the sample volume capacity e.g. 2 columns: sample volume 3 ml, 5 columns: sample volume 7.5 ml. - connect up to 4 HiPrep 26/10 Desalting columns in series to increase the sample volume capacity, e.g. 2 columns: sample volume 30 ml, 4 columns: sample volume 60 ml. Even with 4 columns in series the sample can be processed in 20 to 30 minutes. Instructions are supplied with each column. Desalting and buffer exchange can take less than 5 minutes per sample with greater than 95% recovery for most proteins. To prevent possible ionic interactions the presence of a low salt concentration (25 mM NaCl) is recommended during desalting and in the final sample buffer. The sample should be fully dissolved. Centrifuge or filter (0.45 µm filter) to remove particulate material if necessary. The protocols below describe desalting and buffer exchange using HiTrap Desalting. These procedures can be adapted to process smaller sample volumes (on MicroSpin G-25 columns) or larger sample volumes (on HiPrep 26/10 Desalting columns using a chromatography system).
Alternative 1. Desalting with HiTrap Desalting using a syringe or pipette
1. Fill the syringe with buffer. Remove the stop-plug. To avoid introducing air into the column, connect the column "drop to drop" to the syringe (via the adapter provided). 2. Remove the twist-off end. 3. Wash the column with 25 ml buffer at 5 ml/min to completely remove the storage buffer which contains 20% ethanol. If air is trapped in the column, wash with degassed buffer until the air disappears. Air introduced onto the column by accident during sample application does not influence the separation.
Note: 5 ml/min corresponds to approximately 120 drops/min when using a HiTrap 5 ml column.
To deliver more precise volumes, a multi-dispensing pipette (Eppendorf model 4780 Multipipette™) can also be used for sample application and elution. Use the M6 threaded stopper from the HiTrap column as an adapter by piercing a hole through the bottom-end of the stopper. Connect the modified "stopper" to the top of the column and, by using gentle force, drive the pipette tip (Combitip with a pipette tip mounted) into the stopper. When dispensing liquid with the Multipipette, do not exceed the maximum flow rate for the column. Take care that all liquid is dispensed for each stroke before a new stroke is delivered.
68
4. Apply the sample using a 2–5 ml syringe or a Multipipette at a flow rate between 1–10 ml/min. Discard the liquid eluted from the column. If the sample volume is less than 1.5 ml, change to buffer and proceed with the injection until a total of 1.5 ml has been eluted. Discard the eluted liquid. 5. Elute the protein with the appropriate volume selected from Table 22. Collect the desalted protein in the volume indicated.
The maximum recommended sample volume is 1.5 ml. See Table 22 for the effect of reducing the sample volume applied to the column. Sample load ml 0.25 0.50 1.00 1.50
Add buffer ml 1.25 1.0 0.5 0.0
Elute and collect ml 1.0 1.5 2.0 2.0
Yield % >95 >95 >95 >95
Remaining salt % 0.0 <0.1 <0.2 <0.2
Dilution factor 4.0 3.0 2.0 1.3
Table 22. Recommended sample and elution volumes using a syringe or Multipipette.
A simple peristaltic pump can also be used to apply sample and buffers.
Alternative 2. Simple desalting with ÄKTAprime ÄKTAprime contains pre-programmed templates for individual HiTrap Desalting and HiPrep 26/10 Desalting columns. Prepare at least 500 ml of the required buffer. 1. Follow the instructions supplied on ÄKTAprime cue cards, Desalting on HiTrap Desalting (code no: 18-1138-03) or Desalting on HiPrep 26/10 Desalting (code no: 18-1138-04). 2. Select the Application Template. 3. Start the method. 4. Enter the sample volume (up to 1.5 ml) and press OK.
Figure 19 shows a typical desalting procedure using ÄKTAprime. The UV and conductivity traces enable the appropriate desalted fractions to be pooled. AU 280 nm
–– UV 280 nm –– Conductivity
0.15
Sample: His tagged protein eluted with sodium phosphate 20 mM, sodium chloride 0.5 M, imidazole 0.5 M, pH 7.4 Column: HiTrap Desalting 5 ml Buffer: Sodium phosphate 20 mM, sodium chloride 0.15 M, pH 7.0
0.10
0.05 Inject
0 0
1
min
Fig. 19. Desalting of a (His)6 fusion protein on ÄKTAprime.
69
70
CHAPTER 8 Simple purification of other recombinant proteins GST MicroSpin and His MicroSpin Purification Modules, GSTrap FF, HiTrap Chelating HP and HisTrap Kit are products that use affinity chromatography to isolate and purify a specific fusion protein. However, many other fusion and non-fusion proteins can also be isolated to a satisfactory degree of purity by a single step purification using affinity chromatography.
Single step purification using specific affinity chromatography.
Affinity chromatography isolates a specific protein or a group of proteins with similar characteristics. The technique separates proteins on the basis of a reversible interaction between the protein(s) and a specific ligand attached to a chromatographic matrix. Whenever a suitable ligand is available for the protein(s) of interest, a single affinity purification step offers high selectivity, hence high resolution, and usually high capacity for the target protein(s). The basic principles of affinity chromatography are outlined in Appendix 9.
Ready to use affinity purification columns Table 23 shows the applications for which affinity purification with HiTrap columns are already available. All columns are supplied with a detailed protocol that outlines the buffers and steps required for optimum results. For larger scale work HiTrap columns can often be linked together in series to increase the capacity of a single purification step. Media are also available for packing larger columns.
71
Application
Column
Capacity (mg/ml affinity medium)
IgG, fragments and subclasses from all sources
HiTrap rProtein A FF
Human IgG, 50 mg/ml
IgG, fragments and subclasses from all sources
HiTrap Protein A HP
Human IgG, 20 mg/ml
IgG, fragments and subclasses including human IgG3. Strong affinity for monoclonal mouse IgG1 and rat IgG
HiTrap Protein G HP
Human IgG, 25 mg/ml
IgG, fragments and subclasses including human IgG3. Strong affinity for monoclonal mouse IgG1 and rat IgG
MAbTrap™ Kit (HiTrap Protein G HP column (1 ml), accessories, pre-made buffers)
Human IgG, 25 mg/ml
Isolation of immunoglobulins
IgY from egg yolk
HiTrap IgY Purification HP
IgY, 20 mg/ml
Monoclonal and human IgM
HiTrap IgM Purification HP
Mouse IgM, 5 mg/ml
Albumin, various nucleotide-requiring enzymes
HiTrap Blue HP
HSA, 20 mg/ml
Proteins and peptides with exposed amino acids: His (Cys, Trp) His-tagged proteins
HiTrap Chelating HP
(His)6 fusion protein (Mr 27 600), 12 mg/ml
Biotin and biotinylated substances
HiTrap Streptavidin HP
Biotinylated BSA, 6 mg/ml
Coagulation factors, lipoprotein lipases, steroid receptors, hormones, DNA binding proteins, interferon, protein synthesis factors
HiTrap Heparin HP
Antithrombin III (bovine), 3 mg/ml
Removal of proteolytic activity or purification of trypsin or trypsin-like serine proteases
HiTrap Benzamidine FF (high sub)
Trypsin, 35 mg/ml
Proteins and peptides with exposed amino acids: His (Cys, Trp). His-tagged proteins
HisTrap Kit (HiTrap Chelating HP 1 ml, (His)6 fusion protein (Mr 27 600), buffers for up to 12 purifications 12 mg/ml using a syringe)
GST-tagged proteins
GSTrap FF
Glutathione-S-transferase, 10–12 mg/ml
HiTrap NHS-activated HP
ligand specific
Group Specific Media
Matrix for preparation of affinity columns Coupling of primary amines
Table 23. Ready to use HiTrap columns for affinity purification.
72
Making a specific purification column In cases when a ready made affinity medium is unavailable, it may be considered worthwhile to develop a “home-made” affinity purification column, for example, when a specific recombinant protein needs to be prepared efficiently on a regular basis. The ligand must be prepared (following procedures that are specific for the type of ligand e.g. by raising antibodies), tested for affinity to the target protein and purified before linking to a chromatographic matrix. For further details on general purification strategies for proteins see Chapter 9 and the Protein Purification Handbook from Amersham Pharmacia Biotech. A detailed account of the principles of affinity chromatography can be found in the Affinity Chromatography, Principles and Methods Handbook also available from Amersham Pharmacia Biotech.
Preparation of HiTrap NHS-activated HP to create a simple affinity purification column NHS-activated Sepharose High Performance is a chromatographic matrix specifically designed for the covalent coupling of ligands containing primary amino groups (the most common form of attachment). The matrix is based on highly cross-linked agarose beads with 10-atom spacer arms attached to the matrix by epichlorohydrine and activated by N-hydroxysuccinimide. The substitution level is ~10 µmol NHS-groups/ml gel. Non-specific adsorption of proteins (which can reduce binding capacity of the target protein) is negligible due to the excellent hydrophilic properties of the base matrix. The protocol below describes the preparation of a prepacked HiTrap NHS-activated HP column and is generally applicable to all NHS-activated Sepharose products. Optimum binding and elution conditions for purification of the target protein must be determined separately for each ligand. This procedure can be performed using a HiTrap column with a syringe, a peristaltic pump or a liquid chromatography system such as ÄKTAprime. The activated matrix is supplied in 100% isopropanol to preserve stability prior to coupling. Do not replace the isopropanol until it is time to couple the ligand.
Buffer preparation Acidification solution: 1 mM HCl (ice-cold) Coupling buffer:
0.2 M NaHCO3, 0.5 M NaCl, pH 8.3
Use high quality water and chemicals. Filtration through 0.45 µm filters is recommended. Coupling within pH range 6.5–9, maximum yield is achieved at pH ~8.
73
Ligand and column preparation 1. Dissolve desired ligand in the coupling buffer to a concentration of 0.5–10 mg/ml (for protein ligands) or perform a buffer exchange using a desalting column (see Chapter 7). The optimum concentration depends on the ligand. Optimum sample volume is equivalent to one column volume. 2. Remove top-cap and apply a drop of ice-cold 1 mM HCl to the top of the column to avoid air bubbles. 3. Connect the top of the column to the syringe or system. 4. Remove the twist-off end.
Ligand coupling 1. Wash out the isopropanol with 3 x 2 column volumes of ice-cold 1 mM HCl.
Do not use excessive flow rates (maximum recommended flow rates are 1 ml/min (equivalent to approximately 30 drops/min when using a syringe) with HiTrap 1 ml and 5 ml/min (equivalent to approximately 120 drops/min when using a syringe) with HiTrap 5 ml). The column contents can be irreversibly compressed. 2. Immediately inject one column volume of ligand solution onto the column. 3. Seal the column. Leave to stand for 15–30 minutes at +25 °C (or 4 hours at +4 °C).
If larger volumes of ligand solution are used, re-circulate the solution. For example, when using a syringe connect a second syringe to the outlet of the column and gently pump the solution back and forth for 15–30 minutes or, if using a peristaltic pump, simply re-circulate the sample through the column. If required, the coupling efficiency can be measured at this stage. These procedures are supplied with each HiTrap NHS-activated HP column.
Washing and deactivation This procedure deactivates any excess active groups that have not coupled to the ligand and washes out non-specifically bound ligands. Buffer A:
0.5 M ethanolamine, 0.5 M NaCl, pH 8.3
Buffer B:
0.1 M acetate, 0.5 M NaCl, pH 4
1. Inject 3 x 2 column volumes of buffer A. 2. Inject 3 x 2 column volumes of buffer B. 3. Inject 3 x 2 column volumes of buffer A. 4. Let the column stand for 15–30 min. 5. Inject 3 x 2 column volumes of buffer B. 6. Inject 3 x 2 column volumes of buffer A. 7. Inject 3 x 2 column volumes of buffer B. 8. Inject 2–5 column volumes of a buffer with neutral pH.
The column is now ready for use. Store the column in storage solution, e.g. 0.05 M Na2HPO4, 0.1% NaN3, pH 7. Sodium azide can interfere with many coupling methods and some biological assays. It can be removed by using a desalting column (see Chapter 7).
74
Purification Optimum binding and elution conditions for purification of the target protein must be determined separately for each ligand. Literature references and textbooks may give good guidelines. Below is a general protocol that can be used initially. Use high quality water and chemicals. Filtration through 0.45 µm filters is recommended. Perform a blank run to ensure that loosely bound ligand is removed. Samples should be centrifuged immediately before use and/or filtered through a 0.45 µm filter. If the sample is too viscous, dilute with binding buffer. Sample binding properties can be improved by adjusting the sample to the composition of the binding buffer. Dilute in binding buffer or perform a buffer exchange using a desalting column (see Chapter 7).
Prepare the column (blank run) 1. Wash with 3 column volumes of binding buffer. 2. Wash with 3 column volumes of elution buffer. 3. Equilibrate with 10 column volumes of binding buffer.
Purification 1. Apply sample. Optimal flow rate is dependent on the binding constant of the ligand, but a recommended flow rate range is, for example, 0.2–1 ml/min on a HiTrap 1 ml column. 2. Wash with 5–10 column volumes of binding buffer, or until no material appears in the eluent.
Avoid excessive washing if the interaction between the protein of interest and the ligand is weak, since this may decrease the yield. 3. Elute with 1–3 column volumes of elution buffer (larger volumes may be necessary). 4. If required purified fractions can be desalted and exchanged into the buffer of choice using prepacked desalting columns (see Chapter 7).
Re-equilibrate the column Re-equilibrate the column by washing with 5–10 column volumes of binding buffer.
75
76
CHAPTER 9 Multi-step purification of recombinant proteins (fusion and non-fusion) Fusion systems are simple and convenient and, for many applications, a single purification step using affinity chromatography is sufficient to achieve the desired level of purity. However, if there is no suitable fusion system so that affinity chromatography cannot be used, or if a higher degree of purity is required, a multi-step purification will be necessary. A significant advantage when working with recombinant products is that there is often considerable information available about the product and contaminants. With this information, detection assays and sample preparation and extraction procedures in place, a purification strategy of Capture, Intermediate Purification and Polishing (CIPP) can be applied (Figure 20). This strategy is used in both the pharmaceutical industry and in the research laboratory to ensure faster method development, a shorter time to pure product and good economy.
Purity
This section gives a brief overview of the approach recommended for any multi-step protein purification. The Protein Purification Handbook (from Amersham Pharmacia Biotech) is highly recommended as a guide to planning efficient and effective protein purification strategies.
Polishing Intermediate purification Capture Preparation, extraction, clarification
Achieve final high level purity
Remove bulk impurities
Isolate, concentrate and stabilize
Fig. 20. Preparation and CIPP.
Step
CIPP is applied as follows: Imagine the purification has three phases Capture, Intermediate Purification and Polishing. Assign a specific objective to each step within the purification process. The purification problem associated with a particular step will depend greatly upon the properties of the starting material. Thus, the objective of a purification step will vary according to its position in the process i.e. at the beginning for isolation of product from crude sample, in the middle for further purification of partially purified sample, or at the end for final clean up of an almost pure product. In the capture phase the objectives are to isolate, concentrate and stabilize the target product. The product should be concentrated and transferred to an environment that will conserve potency/activity.
77
During the intermediate purification phase the objectives are to remove most of the bulk impurities, such as other proteins and nucleic acids, endotoxins and viruses. In the polishing phase most impurities have already been removed except for trace amounts or closely related substances. The objective is to achieve final purity by removing any remaining trace impurities or closely related substances. The optimum selection and combination of purification techniques for Capture, Intermediate Purification and Polishing is crucial for an efficient purification.
Selection and combination of purification techniques Proteins are purified using techniques that separate according to differences in specific properties, as shown in Table 24. Protein property Charge Size Hydrophobicity Biorecognition (ligand specificity)
Technique* Ion exchange (IEX) Gel filtration (GF) Hydrophobic interaction (HIC), Reversed phase (RPC) Affinity (AC)
Table 24. *Expanded bed adsorption is a technique used for large-scale purification. Proteins can be purified from crude sample without the need for separate clarification, concentration and initial purification to remove particulate matter. The STREAMLINE adsorbents, used for expanded bed adsorption, capture the target molecules using the same principles as affinity, ion exchange or hydrophobic interaction chromatography.
Resolution
Speed
Recovery
Capacity
Every chromatographic technique offers a balance between resolution, capacity, speed and recovery. Resolution is achieved by the selectivity of the technique and the ability of the chromatographic matrix to produce narrow peaks. In general, resolution is most difficult to achieve in the final stages of purification when impurities and target protein are likely to have very similar properties. Capacity, in the simple model shown, refers to the amount of target protein that can be loaded during purification. In some cases the amount of sample which can be loaded may be limited by volume (as in gel filtration) or by large amounts of contaminants, rather than by the amount of the target protein.
78
Speed is of the highest importance at the beginning of purification where contaminants such as proteases must be removed as quickly as possible. Recovery becomes increasingly important as the purification proceeds because of the increased value of the purified product. Recovery is reduced by destructive processes in the sample and unfavourable conditions on the column. Select a chromatographic technique to meet the objectives for the purification step. Choose logical combinations of purification techniques based on the main benefits of the technique and the condition of the sample at the beginning or end of each step. A guide to the suitability of each purification technique for the stages in CIPP is shown in Table 25. Technique Main features
Sample start condition
Sample end condition
high resolution high capacity high speed
low ionic strength sample volume not limiting
high ionic strength or pH change
good resolution good capacity high speed
high ionic strength sample volume not limiting
low ionic strength
high resolution high capacity high speed
specific binding conditions sample volume not limiting
specific elution conditions
GF
high resolution using Superdex
limited sample volume (<5% total column volume) and flow rate range
buffer exchanged (if required) diluted sample
RPC
high resolution
sample volume usually not limiting additives may be required
in organic solvent, risk loss of biological activity
IEX
HIC
AC
Capture
Intermediate
Polishing
concentrated sample concentrated sample
concentrated sample
Table 25. Suitability of purification techniques for the CIPP.
Minimize sample handling between purification steps by combining techniques to avoid the need for sample conditioning. The product should be eluted from the first column in a buffer suitable for the start conditions required for the next technique (see Table 25). Ammonium sulphate precipitation is a common sample clarification and concentration step and so HIC (which requires high salt to enhance binding to the media) is ideal as the capture step. The salt concentration and the total sample volume will be significantly reduced after elution from the HIC column. Dilution of the fractionated sample or rapid buffer exchange using a desalting column will prepare it for the next IEX or AC step. GF is well suited for use after any of the concentrating techniques (IEX, HIC, AC) since the target protein will be eluted in a reduced volume and the components from the elution buffer will not affect the gel filtration separation. Gel filtration is a non-binding technique with limited volume capacity and is unaffected by buffer conditions. Selection of the final strategy will always depend upon specific sample properties and the required level of purification. Logical combinations of techniques are shown in Figure 21. 79
Proteins with low solubility SDS extraction
GF (in non-ionic detergent)
SDS extraction
Solubilizing agents (urea, ethylene glycol non-ionic detergents)
HIC
HIC
GF
GF
Crude sample or sample in high salt concentration Sample clarification
Capture
GF GF desalt mode desalt mode
AC
IEX
Intermediate Purification Polishing
GF or RPC
GF or RPC
GF desalt mode
HIC IEX dilution may be needed
IEX
HIC
GF
GF
Clear or very dilute samples Capture
AC
IEX
Intermediate Purification Polishing
GF or RPC
GF or RPC
IEX
Precipitation (e.g. in high ionic strength)
HIC
Resolubilize
GF
Treat as for sample in high salt concentration
Fig. 21. Logical combinations of chromatographic steps.
For any capture step, select the technique showing the strongest binding to the target protein while binding as few of the contaminants as possible, i.e. the technique with the highest selectivity and/or capacity for the protein of interest. A sample is purified using a combination of techniques and alternative selectivities. For example, in an IEX-HIC-GF strategy the capture step selects according to differences in charge (IEX), the intermediate purification step according to differences in hydrophobicity (HIC) and the final polishing step according to differences in size (GF). If nothing is known about the target protein use IEX-HIC-GF. This combination of techniques can be regarded as a standard protocol. Consider the use of both anion and cation exchange chromatography to give different selectivities within the same purification strategy.
80
IEX is a technique which offers different selectivities using either anion or cation exchangers. The pH can be modified to alter the charge characteristics of the sample components. It is therefore possible to use IEX more than once in a purification strategy, for capture, intermediate purification or polishing. IEX can be used effectively both for rapid separation in low resolution mode during capture, and in high resolution mode during polishing in the same purification scheme. Consider RPC for a polishing step provided that the target protein can withstand the run conditions. Reversed phase chromatography (RPC) separates proteins and peptides on the basis of hydrophobicity. RPC is a high selectivity (high resolution) technique, requiring the use of organic solvents. The technique is widely used for purity check analyses when recovery of activity and tertiary structure are not essential. Since many proteins are denatured by organic solvents, the technique is not generally recommended for protein purification where recovery of activity and return to a native tertiary structure may be compromised. However, in the polishing phase, when the majority of protein impurities have been removed, RPC can be excellent, particularly for small target proteins that are not often denatured by organic solvents. CIPP does not mean that all strategies must have three purification steps. For example, capture and intermediate purification may be achievable in a single step, as may intermediate purification and polishing. Similarly, purity demands may be so low that a rapid capture step is sufficient to achieve the desired result. For purification of therapeutic proteins a fourth or fifth purification step may be required to fulfil the highest purity and safety demands. The number of steps used will always depend upon the purity requirements and intended use for the protein. The following example demonstrates the successful application of CIPP in the purification of a recombinant protein.
Three step purification of a recombinant enzyme using ÄKTAFPLC This example demonstrates one of the most common purification strategies used when high purity levels are required: IEX for capture, HIC for intermediate purification and GF for the polishing step. The objective was to obtain highly purified deacetoxycephalosporin C synthase (DAOCS), an oxygen-sensitive enzyme that had been produced by over-expression in soluble form in the cytoplasm of E. coli bacteria. A more detailed description of this work can be found in Application Note 18-1128-91.
Sample extraction and clarification Cells were suspended in Tris-based lysis buffer, pH 7.5 and lysed using ultrasonication. Streptomycin sulphate and polyethyleneimine were added to precipitate DNA. The extract was clarified by centrifugation. EDTA, DTT, benzamidine-HCl and PMSF were used in the lysis buffer to inhibit proteases and minimize damage to the oxygen sensitive-enzyme. Keeping the sample on ice also reduced protease activity.
81
Capture The capture step focused on the rapid removal of the most harmful contaminants from the relatively unstable target protein. This, together with the calculated isoelectric point of DAOCS (pI = 4.8), led to the selection of an anion exchange purification. A selection of anion exchange columns, including those from the HiTrap IEX Selection Kit, was screened to find the optimum medium (results not shown). Optimization of the capture step (in Figure 22) allowed the use of a step elution at high flow rate to speed up the purification. A 280 nm
mS/cm
80 3000 60 2000 40 1000
System: Column: Sample: Sample volume: Binding buffer: Elution buffer: Flow:
ÄKTAFPLC HiPrep 16/10 Q XL Clarified E. coli extract 40 ml 50 mM Tris-HCl, 1 mM EDTA, 2 mM DTT, 0.2 M benzamidine-HCl, 0.2 mM PMSF, pH 7.5 Binding buffer + 1.0 M NaCl 10 ml/min (300 cm/h)
20
0
0
200 ml
100
0
Fig. 22. Capture using IEX. The elution position of DAOCS is shaded.
Intermediate Purification Hydrophobic interaction chromatography (HIC) was selected because the separation principle is complementary to ion exchange and because a minimum amount of sample conditioning was required. Hydrophobic properties are difficult to predict and it is always recommended to screen different media. After screening, RESOURCE™ ISO was selected on the basis of the resolution achieved. In this intermediate step, shown in Figure 23, the maximum possible speed for separation was sacrificed in order to achieve higher resolution and allow significant reduction of impurities. System: Column: Sample: Sample volume: Binding buffer:
A 280 nm
400 300 200 100 0
0
100
200
ml
ÄKTAFPLC SOURCE™ 15ISO, packed in HR 16/10 column DAOCS pool from HiPrep 16/10 Q XL 40 ml 1.6 M ammonium sulphate, 10% glycerol, 50 mM Tris-HCl, 1 mM EDTA, 2 mM DTT, 0.2 mM benzamidine-HCl, 0.2 mM PMSF, pH 7.5 Elution buffer (B): 50 mM Tris-HCl, 10% glycerol, 1 mM EDTA, 2 mM DTT, 0.2 mM benzamidine-HCl, 0.2 mM PMSF, pH 7.5 Gradient: 0–16%B in 4 CV, 16-24%B in 8 CV, 24–35%B in 4 CV, 100%B in 4 CV Flow: 5 ml/min (150 cm/h) CV = column volume
Fig. 23. Intermediate purification using HIC. The elution position of DAOCS is shaded.
82
Polishing The main goal of the polishing step, shown in Figure 24, was to remove aggregates and minor contaminants and transfer the purified sample into a buffer suitable for use in structural studies. The final product was used successfully in X-ray diffraction studies. Ref: Structure of a cephalosporin synthase. Valegard, K., Terwisscha van Scheltinga, A.C., Lloyd, M., Hara, T., Ramaswamy, S., Perrakis, A., Thompson, A., Lee, H.J., Baldwin, J.E., Schofield, C.J., Hajdu, J. and Andersson, I. Nature 394, 805–809 (1998). A 280 nm System: Column: Sample: Sample volume: Buffer:
1000 800 600 400
Flow:
200 0
0
20
40
60
80
100
ÄKTAFPLC HiLoad™ 16/60 Superdex 75 prep grade Concentrated DAOCS pool from SOURCE 15ISO 3 ml 100 mM Tris-HCl, 1 mM EDTA, 2 mM DTT, 0.2 mM benzamidine-HCl, 0.2 mM PMSF, pH 7.5 1 ml/min (30 cm/h)
ml
Fig. 24. Polishing step using gel filtration. The elution position of DAOCS is shaded.
Three step purification of a recombinant phosphatase using ÄKTAprime The objective of this application was to produce a pure phosphatase (rPhosphatase) with retained biological activity. The phosphatase gene was overexpressed and the protein was produced in soluble form in the cytoplasm of Escherichia coli. Using the pre-programmed method templates of ÄKTAprime with prepacked HiPrep and HiLoad columns ensured quick and easy method development. The purification strategy consisted of a capture step by ion exchange chromatography, intermediate purification by hydrophobic interaction chromatography and polishing by gel filtration. Active rPhosphatase (35 mg) was purified within 8 hours. A more detailed description of this work can be found in Application Note 18-1142-32.
Sample preparation and extraction The E. coli cells were suspended in lysis buffer, 1 g cells to every 10 ml lysis buffer (50 mM Tris-HCl, 1 mM EDTA, 2 mM DTT, pH 7.4). The suspended cells were lysed by ultra-sonication, 6 x 20 bursts with 60 seconds cooling between each burst. DNA was removed by precipitation with 1% w/v streptomycin sulphate. The sample was clarified by centrifugation, 15 minutes at 22 000 g, before it was applied to the first chromatography column.
83
Capture The main purpose of the capture step was to concentrate the rPhosphatase and remove most of the contaminants. The ÄKTAprime system pump was used to apply 200 ml of the clarified E. coli supernatant, diluted 1:2 with water, to a HiPrep 16/10 DEAE FF column. A pre-programmed method template for ion exchange chromatography was used for the separation. Fractions of the eluate were collected and analysed with an enzyme immunoassay detecting alkaline phosphatase activity at an absorbance of 405 nm. The purity of the fractions containing rPhosphatase was determined by SDS-PAGE. A 280 nm 2.0
A 405 nm 4.0
UV absorbance conductivity
3.0
1.0
2.0
1.0
0 300
500
700
ml
Sample:
200 ml clarified E. coli supernatant, diluted 1:2 with water, pH 6.6 Column: HiPrep 16/10 DEAE FF, Vt = 20 ml Start buffer: 25 mM Tris-HCl, pH 7.4, 10% glycerol,1 mM EDTA, 2 mM DTT Elution buffer (B): 1 M NaCl in start buffer Flow: 5 ml/min (150 cm/h) Run parameters: Equilibration 0% B 2 CV Sample application Wash 1 0% B 4 CV Elution 0–50% B in 20 CV 50% B for 1 CV Wash 2 100% B 2 CV Vt = column volumn (CV)
Fig. 25. Capture step using ion exchange. The phosphatase activity is represented by the green bars (absorbance at 405 nm).
Intermediate purification Hydrophobic interaction chromatography was used for intermediate purification because of its compatibility with samples containing a high salt concentration. The pooled fractions from the ion exchange column were purified on HiLoad 16/10 Phenyl Sepharose High Performance, using a pre-programmed method template in ÄKTAprime. The fractions containing rPhosphatase were pooled and concentrated to 10 ml on an Amicon™ 50 ml stirred-cell using a Diaflow™ PM10 filter. Reducing the sample volume enables a smaller gel filtration column to be used for the final polishing step. A 280 nm
A 405 nm
2.0
UV absorbance conductivity
3.0
2.0
1.0 1.0
0 0
200
400
600
ml
Sample:
170 ml rPhosphatase containing pool from HiPrep 16/10 DEAE FF in 1.6 M ammonium sulphate, pH 7.0 Column: HiLoad 16/10 Phenyl Sepharose HP, Vt = 20 ml Start buffer: 25 mM Tris-HCl, pH 7.4 in 1.4 M ammonium sulphate, 1 mM EDTA, 2 mM DTT Elution buffer (B): 25 mM Tris-HCl in 10% glycerol, 1 mM EDTA, 2 mM DTT, pH 7.4 Flow: 5 ml/min (150 cm/h) Run parameters: Equilibration 0% B 2 CV Sample application Wash 1 0% B 3 CV Elution 0–100% B in 20 CV Wash 2 100% B 2 CV Vt = column volumn (CV)
Fig. 26. Intermediate purification step using hydrophobic interaction. The phosphatase activity is represented by the green bars (absorbance at 405 nm).
84
Polishing The final polishing step used a pre-programmed method template to run a gel filtration separation on a HiLoad 16/60 Superdex 75 prep grade column. The purity of the fractions containing rPhosphatase was checked with SDS-PAGE (Figure 28) and by mass spectrometry (results not shown). A 280 nm 2.0
A 405 nm
4.0
UV absorbance
Sample: 3.0
Column: 1.0
4 ml concentrated eluate, containing rPhosphatase from the HiLoad 16/10 Phenyl Sepharose HP HiLoad 16/60 Superdex 75 pg, Vt = 120 ml 25 mM Tris-HCl, 300 mM NaCl, 1 mM EDTA, 2 mM DTT, pH 7.4 0.5 ml/min (15 cm/h)
2.0
Buffer:
1.0
Vt = column volumn (CV)
Flow:
Vt
Vo
0 0
50
100
ml
Fig. 27. Polishing step using gel filtration. The phosphatase activity is represented by the green bars (absorbance at 405 nm).
Mr 97 000 66 000
Lane Lane Lane Lane Lane
45 000 30 000
1. 2. 3. 4. 5.
E. coli Supernatant Eluate from the capture step (ion exchange) Eluate from the intermediate step (hydrophobic interaction) Eluate from the polishing step (gel filtration) LMW marker
20 100 14 400 1
2
3
4
5
Fig. 28. Purity check by SDS-PAGE using Multiphor II and ExcelGel SDS Gradient 8–18%. The proteins were stained with Coomassie Brilliant Blue.
85
Appendix 1 Map of the GST fusion vectors showing reading frames and main features pGEX-2TK (27-4587-01) Thrombin
Kinase
Leu Val Pro Arg Gly Ser Arg Arg Ala Ser Val CTG GTT CCG CGT GGA TCT CGT CGT GCA TCT GTT GGA TCC CCG GGA ATT CAT CGT GAC TGA Stop codons BamH I Sma I EcoR I
pGEX-4T-1 (27-4580-01) Thrombin Leu Val Pro Arg Gly Ser Pro Glu Phe Pro Gly Arg Leu Glu Arg Pro His Arg Asp CTG GTT CCG CGT GGA TCC CCG GAA TTC CCG GGT CGA CTC GAG CGG CCG CAT CGT GAC TGA Stop codons EcoR I Sma I Sal I Xho I Not I BamH I
pGEX-4T-2 (27-4581-01) Thrombin Leu Val Pro Arg Gly Ser Pro Gly Ile Pro Gly Ser Thr Arg Ala Ala Ala Ser CTG GTT CCG CGT GGA TCC CCA GGA ATT CCC GGG TCG ACT CGA GCG GCC GCA TCG TGA Stop codon BamH I EcoR I Sma I Sal I Xho I Not I
pGEX-4T-3 (27-4583-01) Thrombin Leu Val Pro Arg Gly Ser Pro Asn Ser Arg Val Asp Ser Ser Gly Arg Ile Val Thr Asp CTG GTT CCG CGT GGA TCC CCG AAT TCC CGG GTC GAC TCG AGC GGC CGC ATC GTG ACT GAC TGA Stop codons BamH I EcoR I Sma I Sal I Xho I Not I
pGEX-5X-1 (27-4584-01) Factor Xa Ile Glu Gly Arg Gly Ile Pro Glu Phe Pro Gly Arg Leu Glu Arg Pro His Arg Asp ATC GAA GGT CGT GGG ATC CCC GAA TTC CCG GGT CGA CTC GAG CGG CCG CAT CGT GAC TGA Stop codons BamH I EcoR I Sma I Sal I Xho I Not I
pGEX-5X-2 (27-4585-01) Factor Xa Ile Glu Gly Arg Gly Ile Pro Gly Ile Pro Gly Ser Thr Arg Ala Ala Ala Ser ATC GAA GGT CGT GGG ATC CCC GGA ATT CCC GGG TCG ACT CGA GCG GCC GCA TCG TGA Stop codon BamH I Not I EcoR I Sma I Sal I Xho I
pGEX-5X-3 (27-4586-01) Factor Xa Ile Glu Gly Arg Gly Ile Pro Arg Asn Ser Arg Val Asp Ser Ser Gly Arg Ile Val Thr Asp ATC GAA GGT CGT GGG ATC CCC AGG AAT TCC CGG GTC GAC TCG AGC GGC CGC ATC GTG ACT GAC TGA Stop codons EcoR I Sma I Sal I Xho I BamH I Not I
pGEX-6P-1 (27-4597-01) PreScission Protease Leu Glu Val Leu Phe Gln Gly Pro Leu Gly Ser Pro Glu Phe Pro Gly Arg Leu Glu Arg Pro His CTG GAA GTT CTG TTC CAG GGG CCC CTG GGA TCC CCG GAA TTC CCG GGT CGA CTC GAG CGG CCG CAT BamH I EcoR I Sma I Sal I Xho I Not I
pGEX-6P-2 (27-4598-01) PreScission Protease Leu Glu Val Leu Phe Gln Gly Pro Leu Gly Ser Pro Gly Ile Pro Gly Ser Thr Arg Ala Ala Ala Ser CTG GAA GTT CTG TTC CAG GGG CCC CTG GGA TCC CCA GGA ATT CCC GGG TCG ACT CGA GCG GCC GCA TCG Not I EcoR I Sma I Sal I Xho I BamH I
pGEX-6P-3 (27-4599-01) PreScission Protease Leu Glu Val Leu Phe Gln Gly Pro Leu Gly Ser Pro Asn Ser Arg Val Asp Ser Ser Gly Arg CTG GAA GTT CTG TTC CAG GGG CCC CTG GGA TCC CCG AAT TCC CGG GTC GAC TCG AGC GGC CGC EcoR I Sma I Sal I Xho I Not I BamH I Bal I ut
gl
Ptac
S ne h io at
ase nsfer
Tth111 I Aat II
-tra
p Am
BspM I
r
pSj10 Bam7Stop7
Pst I
pGEX ~4900 bp
Nar I EcoR V
la
AlwN I
p4.5
c q I
BssH II Apa I BstE II Mlu I
86
pBR322 ori
pGEX- 4T- 1 27- 4580- 01
pGEX- 4T- 2 27- 4581- 01
pGEX- 4T- 3 27- 4583- 01
pGEX- 5X- 1 27- 4584- 01
pGEX- 5X- 2 27- 4585- 01
pGEX- 5X- 3 27- 4586- 01
pGEX- 6P- 1 27- 4597- 01
pGEX- 6P- 2 27- 4598- 01
pGEX- 6P- 3 27- 4599- 01 205- 211
Glutathione S- Transferase Region
tac promoter- 10
205- 211
205- 211
205- 211
205- 211
205- 211
205- 211
205- 211
205- 211
205- 211
tac promoter- 35
183- 188
183- 188
183- 188
183- 188
183- 188
183- 188
183- 188
183- 188
183- 188
183- 188
lac operator
217- 237
217- 237
217- 237
217- 237
217- 237
217- 237
217- 237
217- 237
217- 237
217- 237
Ribosome binding site for GST
244
244
244
244
244
244
244
244
244
244
Start codon (ATG) for GST
258
258
258
258
258
258
258
258
258
258
Coding region for thrombin cleavage
918- 935
918- 935
918- 935
918- 935
NA
NA
NA
NA
NA
NA
Coding region for factor Xa cleavage
NA
NA
NA
NA
921- 932
921- 932
921- 932
NA
NA
NA
Coding region for PreScission Protease cleavage
NA
NA
NA
NA
NA
NA
NA
918- 938
918- 938
918- 938
Coding for kinase recognition site
936- 950
NA
NA
NA
NA
NA
NA
NA
NA
NA
Multiple Cloning Site
951- 966
930- 966
930- 967
930- 965
934- 969
934- 970
934- 971
945- 981
945- 982
945- 980
Promotor- 10
1330- 1335
1330- 1335
1331- 1336
1329- 1334
1333- 1338
1334- 1339
1335- 1340
1345- 1350
1346- 1351
1344- 1349
Promoter- 35
1307- 1312
1307- 1312
1308- 1313
1306- 1311
1310- 1315
1311- 1316
1312- 1317
1322- 1327
1323- 1328
1321- 1326
Start codon (ATG)
1377
1377
1378
1376
1380
1381
1382
1392
1393
1391
Stop codon (TAA)
2235
2235
2236
2234
2238
2239
2240
2250
2251
2249
r
b -lactamase (Amp ) Gene Region
Lacq Gene Region Start codon (GTG)
3318
3318
3319
3317
3321
3322
3323
3333
3334
3332
Stop codon (TGA)
4398
4398
4399
4397
4401
4402
4403
4413
4414
4412
2995
2995
2996
2994
2998
2999
3000
3010
3011
3009
2302- 2998
2302- 2998
2303- 2999
2301- 2997
2305- 3001
2306- 3002
2307- 3003
2317- 3013
2318- 3014
3216- 3012
5' pGEX Sequencing Primer binding
869- 891
869- 891
869- 891
869- 891
869- 891
869- 891
869- 891
869- 891
869- 891
869- 891
3' pGEX Sequencing Primer binding
1041- 1019
1041- 1019
1042- 1020
1040- 1018
1044- 1022
1045- 1023
1046- 1024
1056- 1034
1057- 1035
1055- 1033
U13851
U13853
U13854
U13855
U13856
U13857
U13858
U78872
U78873
U78874
Plasmid Replication Region Site of replication initiation Region necessary for replication Sequencing Primers
GenBank Accession Number
Complete DNA sequences and rrestriction estriction site data ar e available at the Amersham Phar macia Biotech W eb Site (http:// www are Pharmacia Web www.. apbiotech. com).
Glutathione S-transferase (GST)
SELECTION GUIDE – pGEX Vector Control Regions pGEX- 2TK 27- 4587- 01
87
Appendix 2 Amino acids table Three-letter code
Single-letter code
Alanine
Ala
A
Arginine
Arg
R
Amino acid
Structure HOOC CH3 H2N NH2
HOOC CH2CH2CH2NHC H2N
NH
HOOC
Asparagine
Asn
N
Aspartic Acid
Asp
D
CH2CONH2 H2N HOOC CH2COOH H2N HOOC
Cysteine
Cys
CH2SH
C H2N HOOC
Glutamic Acid
Glu
CH2CH2COOH
E H2N HOOC
Glutamine
Gln
Q
Glycine
Gly
G
Histidine
His
H
CH2CH2CONH2 H2N HOOC H H2N HOOC
N CH2
NH
H2N HOOC
Isoleucine
Ile
I
Leucine
Leu
L
CH(CH3)CH2CH3 H2N HOOC
CH3 CH2CH CH3
H2N HOOC
Lysine
Lys
K
Methionine
Met
M
CH2CH2CH2CH2NH2 H2N HOOC CH2CH2SCH3 H2N HOOC
Phenylalanine
Phe
F
Proline
Pro
P
CH2 H2N HOOC H2N
NH
HOOC
Serine
Ser
S
Threonine
Thr
T
CH2OH H2N HOOC CHCH3 H2N
OH
HOOC
Tryptophan
Trp
W
CH2 H2N
NH
HOOC
Tyrosine
Tyr
CH2
Y H2N HOOC
Valine
Val
CH(CH3)2
V H2N
88
OH
Formula
Mr
Middle unit residue (-H 20) Formula Mr
C3H7NO2
89.1
C3H5NO
C6H14N4O2
174.2
C 4H 8N 2O 3
Charge at pH 6.0–7.0
Hydrophobic (non-polar)
Uncharged (polar)
71.1
Neutral
!
C6H12N4O
156.2
Basic (+ve)
132.1
C 4H 6N 2O 2
114.1
Neutral
C4H7NO4
133.1
C4H5NO3
115.1
Acidic(-ve)
C3H7NO2S
121.2
C3H5NOS
103.2
Neutral
C5H9NO4
147.1
C5H7NO3
129.1
Acidic (-ve)
C5H10N2O3
146.1
C 5H 8N 2O 2
128.1
Neutral
!
C2H5NO2
75.1
C2H3NO
57.1
Neutral
!
C 6H 9N 3O 2
155.2
C6H7N3O
137.2
Basic (+ve)
C6H13NO2
131.2
C6H11NO
113.2
Neutral
!
C6H13NO2
131.2
C6H11NO
113.2
Neutral
!
C6H14N2O2
146.2
C6H12N2O
128.2
Basic(+ve)
C5H11NO2S
149.2
C5H9NOS
131.2
Neutral
!
C9H11NO2
165.2
C9H9NO
147.2
Neutral
!
C5H9NO2
115.1
C5H7NO
97.1
Neutral
!
C3H7NO3
105.1
C3H5NO2
87.1
Neutral
!
C4H9NO3
119.1
C4H7NO2
101.1
Neutral
!
C11H12N2O2
204.2
C11H10N2O
186.2
Neutral
C9H11NO3
181.2
C9H9NO2
163.2
Neutral
C5H11NO2
117.1
C5H9NO
99.1
Neutral
Hydrophilic (polar)
! ! ! ! !
!
!
! ! !
89
Appendix 3 Protein conversion data Mass (g/mol)
1 µg
1 nmol
Protein
A280 for 1 mg/ml
10 000
100 pmol; 6 x 10
13
molecules
10 µg
IgG
50 000
20 pmol; 1.2 x 10
13
molecules
50 µg
IgM
1.20
100 000
10 pmol; 6.0 x 10
12
molecules
100 µg
IgA
1.30
150 000
6.7 pmol; 4.0 x 10
12
molecules
150 µg
Protein A
0.17
1 kb of DNA
= 333 amino acids of coding capacity
270 bp DNA
= 10 000 g/mol
1.35
Avidin
1.50
Streptavidin
3.40
Bovine Serum Albumin
0.70
= 37 000 g/mol 1.35 kb DNA
= 50 000 g/mol
2.70 kb DNA
= 100 000 g/mol
Average molecular weight of an amino acid = 120 g/mol.
Appendix 4 Centrifuges, rotors and carriers for use with MicroPlex 24
90
Separate carriers needed?
Number of places
Maximum g-force and rpm
S2096 (#361111)
no
2
1 100 g, 3 000 rpm
DuPont
H1000B (#11706)
yes #11093
2
1 900 g, 3 000 rpm
#83794
Fisher
#04-976-420
yes #04-975-410 MT
2
1 506 g, 3 500 rpm
#C1725-3 (Baxter #)
Heraeus
#C1725-10 #C1725-35
yes
4
1 280 g, 2 800 rpm
Centrifuge model
Manufacturer’s number Manufacturer
Rotor
GS-15 GS-15R
#360908 #360902
Beckman
Sorvall RT-6000B
#07983
6K Megafuge 1.0 Z-320
#C-0320
Hermle
#C-0320-50
no
2
1 506 g, 3 500 rpm
GP8 GP8R
#3121 #3122
IEC
#216 or #228
yes #5785
2 or 4
2 340 g, 3 500 rpm
HN-SII
#2355
IEC
#244
no
2
1 450 g, 3 575 rpm
MP4 MP4R
#2437 #2438
IEC
#244
no
2
1 450 g, 3 575 rpm
C312 CR312
#11175087 #11175090
Jouan
E4 (#11174153)
yes #11174168
4
2 290 g, 3 300 rpm
1130
Not required
Kubota
S11222
no
2
1 107 g, 3 000 rpm
1140
Not required
Kubota
S11222
no
2
1 107 g, 3 000 rpm
Appendix 5 Characteristics, cleaning and storage of Glutathione Sepharose Glutathione Sepharose 4B is recommended for packing small columns and other formats. Glutathione Sepharose Fast Flow is excellent for packing high performance columns for use with purification systems and for scaling up.
Characteristics Ligand density
7–15 µmole glutathione per ml Glutathione Sepharose 4B 120–320 µmole glutathione per ml Glutathione Sepharose 4 Fast Flow
Capacity
up to 8 mg recombinant GST per ml Glutathione Sepharose 4B up to 10 mg recombinant GST per ml Glutathione Sepharose 4 Fast Flow
Ligand and spacer arm
Glutathione and 10 carbon linker arm
Molecular weight exclusion limit
~2 x 10
Particle size
45–165 µm (90 µm)
7
Chemical stability No significant loss of binding capacity when exposed to 0.1 M citrate (pH 4.0), 0.1 M NaOH, 70% ethanol or 6 M guanidine hydrochloride for 2 hours at room temperature. No significant loss of binding capacity after exposure to 1% SDS for 14 days.
Cleaning Re-use of purification columns and media depends upon the nature of the sample and should only be performed with identical samples to prevent cross contamination. If required, Glutathione Sepharose 4B and Glutathione Sepharose 4 Fast Flow can be regenerated for re-use as follows: 1. Wash with 2–3 column volumes of alternating high pH (0.1 M Tris-HCl, 0.5 M NaCl, pH 8.5) and low pH (0.1 M sodium acetate, 0.5 M NaCl, pH 4.5) buffers. 2. Repeat the cycle 3 times. 3. Re-equilibrate with 3–5 column volumes of PBS, pH 7.4.
If Glutathione Sepharose appears to be losing binding capacity, it may be due to an accumulation of precipitated, denatured or non-specifically bound proteins.
To remove precipitated or denatured substances: 1. Wash with 2 column volumes of 6 M guanidine hydrochloride. 2. Immediately wash with 5 column volumes of PBS, pH 7.4.
To remove hydrophobically bound substances: 1. Wash with 3–4 column volumes of 70% ethanol (or 2 column volumes of 1% Triton X-100). 2. Immediately wash with 5 column volumes of PBS, pH 7.4.
91
For long-term storage (>1 month): 1. Wash the column twice with 10 column volumes of PBS, pH 7.4. 2. Repeat washes using 20% ethanol. 3. Store at +4 °C. 4. Re-equilibrate the column with 5–10 column volumes of PBS, pH 7.4 before re-use.
Characteristics, cleaning and storage of Chelating Sepharose Characteristics Capacity
up to 12 mg (His)6 fusion protein per ml Chelating Sepharose 4 Fast Flow 12 mg (His)6 fusion protein (Mr 27 600) per ml Chelating Sepharose High Performance (in HiTrap columns)
Ligand
Iminodiacetic acid
Molecular weight exclusion limit
~2 x 10
Particle size
45–165 µm (90 µm) Chelating Sepharose Fast Flow
7
24–44 µm (34 µm) Chelating Sepharose High Performance
Chemical stability Stable in all commonly used aqueous buffers and denaturants such as 6 M guanidine hydrochloride, 8 M urea and chaotropic agents.
Cleaning Re-use of purification columns depends upon the nature of the sample and should only be performed with identical samples to prevent cross contamination.
To remove precipitated proteins 1. Fill column with 1 M NaOH and incubate for 2 hours. 2. Wash out dissolved proteins with 5 column volumes of water and a buffer at pH ~7 until the pH of the flow-through reaches pH ~7.0.
To remove nickel ions prior before recharging or storage 1. Wash column with 5 column volumes 20 mM sodium phosphate, 0.5 M NaCl, 0.05 M EDTA, pH 7.4. 2. Wash with 10 column volumes of distilled water. 3. For storage wash with 5 column volumes of 20% ethanol. 4. Seal columns with the fittings provided and store at +4 to +8 °C.
The loss of metal ions is more pronounced at lower pH. The column does not have to be stripped between each purification if the same protein is going to be purified. In this case, perform stripping and re-charging of the column after 5–10 purifications. The column must be recharged with nickel ions after regeneration.
92
Appendix 6 Column packing and preparation A Column Packing Video is also available to demonstrate how to produce a well-packed column (see ordering information).
1. Equilibrate all materials to the temperature at which the purification will be performed. 2. Eliminate air by flushing column end pieces with recommended buffer. Ensure no air is trapped under the column net. Close column outlet leaving 1–2 cm of buffer in the column. 3. Gently resuspend the purification medium. 4. Estimate the amount of slurry (resuspended medium) required. 5. Pour the required volume of slurry into the column. Pouring down a glass rod held against the wall of the column will minimize the introduction of air bubbles. 6. Immediately fill the column with buffer. 7. Mount the column top piece and connect to a pump. 8. Open the column outlet and set the pump to the desired flow rate. For example: 15 ml/min in an XK 16/20 column
If the recommended flow rate cannot be obtained use the maximum flow rate the pump can deliver. 9. Maintain the packing flow rate for at least 3 column volumes after a constant bed height is obtained. Mark the bed height on the column.
Do not exceed 75% of the packing flow rate during any purification. Do not exceed the maximum operating pressure of the medium or column. 10. Stop the pump and close the column outlet. Remove the top piece and carefully fill the rest of the column with recommended buffer to form an upward meniscus at the top. 11. Insert the adapter into the column at an angle, ensuring that no air is trapped under the net. 12. Slide the adapter slowly down the column (the outlet of the adapter should be open) until the mark is reached. Lock the adapter in position. 13. Connect the column to the pump and begin equilibration. Re-position the adapter if necessary.
The medium must be thoroughly washed especially if 20% ethanol has been used as the storage solution. Residual ethanol may interfere with subsequent procedures. Many media, when equilibrated with PBS containing an anti-microbial agent, may be stored at +4° C for up to 1 month, but always follow the specific storage instructions supplied with the product. 93
Column selection XK columns are fully compatible with the high flow rates achievable with modern media and a broad range of column dimensions are available. For a complete listing refer to the Amersham Pharmacia Biotech BioDirectory or web catalogue. Columns XK 16/20 column XK 26/20 column XK 16/70 column XK 26/70 column
Volume (ml) 2–34 0–80 102–135 281–356
Code no 18-8773-01 18-1000-72 18-8775-01 18-8769-01
HR columns can be used for small scale chromatography applications. Columns HR 5/2 column HR 5/5 column HR 10/2 column HR 10/10 column
94
Volume (ml) 0.2–0.59 0.8–1.2 0.08–2.43 6.4–8.7
Code no 18-0382-01 18-0383-01 18-1000-97 19-7402-01
Appendix 7 Converting from linear flow (cm/hour) to volumetric flow rates (ml/min) and vice versa It is often convenient when comparing results for columns of different sizes to express flow as linear flow (cm/hour). However, flow is usually measured in volumetric flow rate (ml/min). To convert between linear flow and volumetric flow rate use one of the formulae below.
From linear flow (cm/hour) to volumetric flow rate (ml/min) Volumetric flow rate (ml/min) = =
Linear flow (cm/h) x column cross sectional area (cm2) 60 Y p x d2 x 60 4
where Y = linear flow in cm/h d = column inner diameter in cm
Example: What is the volumetric flow rate in an XK 16/70 column (i.d. 1.6 cm) when the linear flow is 150 cm/hour? Y = linear flow = 150 cm/h d = inner diameter of the column = 1.6 cm Volumetric flow rate =
150 x p x 1.6 x 1.6 ml/min 60 x 4
= 5.03 ml/min
From volumetric flow rate (ml/min) to linear flow (cm/hour) Linear flow (cm/h) =
Volumetric flow rate (ml/min) x 60 column cross sectional area (cm2)
= Z x 60 x
4 p x d2
where Z = volumetric flow rate in ml/min d = column inner diameter in cm
Example: What is the linear flow in an HR 5/5 column (i.d. 0.5 cm) when the volumetric flow rate is 1 ml/min? Z = Volumetric flow rate = 1 ml/min d = column inner diameter = 0.5 cm Linear flow = 1 x 60 x
4 p x 0.5 x 0.5
cm/h
= 305.6 cm/h
From ml/min to using a syringe 1 ml/min = approximately 30 drops/min on a HiTrap 1 ml column 5 ml/min = approximately 120 drops/min on a HiTrap 5 ml column
95
Appendix 8 Selection of purification equipment Many simple purification steps may be carried out using the simplest methods and equipment, for example step-gradient elution using a syringe together with prepacked HiTrap columns. When more complex elution methods are necessary or the same column is to be used for many runs in series, it is wise to use a dedicated system. Standard ÄKTAdesign configurations Way of working
Explorer 100
Purifier 10
FPLC
Prime
Syringe+ HiTrap
Rapid, screening (GST or His tagged proteins)
"
Simple, one step purification
"
"
"
"
Reproducible performance for routine purification
"
"
"
"
Optimization of one step purification to increase purity
"
"
"
"
System control and data handling for regulatory requirements e.g. GLP
"
"
"
Automatic method development and optimization
"
"
"
Automatic buffer preparation
"
"
Automatic pH scouting
"
"
Automatic media or column scouting
"
Automatic multi-step purification
"
Scale up, process development and transfer to production
"
"
ÄKTAprime
ÄKTAFPLC
ÄKTAexplorer ÄKTApurifier 96
Centrifugation+ MicroSpin
Appendix 9 Principles and standard conditions for purification techniques Affinity Chromatography (AC) AC separates proteins on the basis of a reversible interaction between a protein (or group of proteins) and a specific ligand attached to a chromatographic matrix. The technique is ideal for a capture or intermediate step and can be used whenever a suitable ligand is available for the protein(s) of interest. AC offers high selectivity, hence high resolution, and usually high capacity (for the protein(s) of interest). The target protein(s) is specifically and reversibly bound by a complementary binding substance (ligand). The sample is applied under conditions that favour specific binding to the ligand. Unbound material is washed away, and the bound target protein is recovered by changing conditions to those favouring desorption. Desorption is performed specifically, using a competitive ligand, or non-specifically, by changing the pH, ionic strength or polarity. Samples are concentrated during binding and protein is collected in purified, concentrated form. The key stages in a purification are shown in Figure 29. Affinity chromatography is also used to remove specific contaminants, for example Benzamidine Sepharose 4 Fast Flow can remove serine proteases.
Absorbance
equilibration
wash adsorption of away sample and unbound elution of unbound material material
begin sample application
1-2 cv
elute bound protein(s)
gel regeneration
change to elution buffer
x cv
1-2 cv
>1 cv
1-2 cv
Column Volumes [cv]
Fig. 29. Typical affinity purification.
Further information Protein Purification Handbook Affinity Chromatography Handbook: Principles and Methods Chapters 3 and 4 in this handbook for the purification of GST and His tagged proteins, respectively.
Ion Exchange (IEX) IEX separates proteins with differences in charge to give a very high resolution separation with high sample loading capacity. The separation is based on the reversible interaction between a charged protein and an oppositely charged chromatographic medium. Proteins bind as they are loaded onto a column. Conditions are then altered so that bound substances are eluted differentially. Elution is usually performed by increasing salt concentration or changing pH. Changes are made stepwise or with a continuous gradient. Most commonly, samples are eluted with salt (NaCl), using a gradient elution (Figure 30). Target proteins are concentrated during binding and collected in a purified, concentrated form. 97
equilibration
sample application
gradient elution
wash
re-equilibration
high salt wash 1M
1-4 cv
tightly bound molecules elute in high salt wash
[NaCl]
unbound molecules elute before gradient begins
10-20 cv 2 cv
2 cv
0 Column volumes [cv]
Fig. 30. Typical IEX gradient elution.
The net surface charge of proteins varies according to the surrounding pH. When above its isoelectric point (pI) a protein will bind to an anion exchanger, when below its pI a protein will behind to a cation exchanger. Typically IEX is used to bind the target molecule, but it can also be used to bind impurities if required. IEX can be repeated at different pH values to separate several proteins that have distinctly different charge properties, as shown in Figure 31. Selectivity pH of mobile phase Abs
Abs
V
Abs
V
Abs
V
V
+
Surface net charge
Cation
pH
0
Anion -
Abs
Abs
V
Abs
V
Abs
V
V
Fig. 31. Effect of pH on protein elution patterns.
Method development (in priority order) 1. Select optimum ion exchanger using small columns as in the HiTrap IEX Selection Kit to save time and sample. 2.
Scout for optimum pH. Begin 0.5–1 pH unit away from the isoelectric point of the target protein if known.
3. Select the steepest gradient to give acceptable resolution at the selected pH. 4. Select the highest flow rate that maintains resolution and minimizes separation time. Check recommended flow rates for the specific medium.
To reduce separation times and buffer consumption, transfer to a step elution after method optimization as shown in Figure 32. It is often possible to increase sample loading when using step elution. 98
high salt wash
[NaCl]
2 cv
sample injection volume
elution of target molecule
unbound molecules elute
1-2 cv
elution of unwanted material 1-2 cv
tightly bound molecules elute
equilibration
re-equilibration
2 cv
2 cv Column volumes [cv]
Fig. 32. Step elution.
Further information Protein Purification Handbook Ion Exchange Chromatography Handbook: Principles and Methods
Hydrophobic Interaction Chromatography (HIC) HIC separates proteins with differences in hydrophobicity. The technique is ideal for the capture or intermediate steps in a purification. Separation is based on the reversible interaction between a protein and the hydrophobic surface of a chromatographic medium. This interaction is enhanced by high ionic strength buffer which makes HIC an ideal “next step” after precipitation with ammonium sulphate or elution in high salt during IEX. Samples in high ionic strength solution (e.g. 1.5 M ammonium sulphate) bind as they are loaded onto a column. Conditions are then altered so that the bound substances are eluted differentially. Elution is usually performed by decreases in salt concentration (Figure 33). Changes are made stepwise or with a continuous decreasing salt gradient. Most commonly, samples are eluted with a decreasing gradient of ammonium sulphate. Target proteins are concentrated during binding and collected in a purified, concentrated form. Other elution procedures include reducing eluent polarity (ethylene glycol gradient up to 50%), adding chaotropic species (urea, guanidine hydrochloride) or detergents, changing pH or temperature. equilibration
sample application
gradient elution
salt free wash
re-equilibration
[ammonium sulphate]
1M
unbound molecules elute before gradient begins
tightly bound molecules elute in salt free conditions
10-15 cv 2 cv
2 cv 0 Column volumes [cv]
Fig. 33. Typical HIC gradient elution.
99
Method development (in priority order) 1. The hydrophobic behaviour of a protein is difficult to predict and binding conditions must be studied carefully. Use HiTrap HIC Selection Kit or RESOURCE HIC Test Kit to select the medium that gives optimum binding and elution over the required range of salt concentration. For proteins with unknown hydrophobic properties begin with 0–100%B (0%B=1M ammonium sulphate). 2. Select the gradient that gives acceptable resolution. 3. Select the highest flow rate that maintains resolution and minimizes separation time. Check recommended flow rates for the specific medium. 4. If samples adsorb strongly to a medium then conditions that cause conformational changes, such as pH, temperature, chaotropic ions or organic solvents can be altered. Conformational changes caused by these agents are specific to each protein. Use screening procedures to investigate the effects of these agents. Alternatively, change to a less hydrophobic medium.
To reduce separation times and buffer consumption, transfer to a step elution after method optimization, as shown in Figure 34. It is often possible to increase sample loading when using step elution.
[ammonium sulphate]
equilibration
unbound molecules elute
salt free wash elution of unwanted material
sample injection volume
1-2 cv
elution of target molecule
1-2 cv
re-equilibration
2 cv
tightly bound molecules elute
2 cv Column volumes [cv]
Fig. 34. Step elution.
Further information Protein Purification Handbook Hydrophobic Interaction Chromatography Handbook: Principles and Methods
Gel Filtration (GF) Chromatography GF separates proteins with differences in molecular size. The technique is ideal for the final polishing steps in purification when sample volumes have been reduced (sample volume significantly influences speed and resolution in gel filtration). Samples are eluted isocratically (single buffer, no gradient, Figure 35). Buffer conditions are varied to suit the sample type or the requirements for further purification, analysis or storage, since buffer composition does not directly affect resolution. Proteins are collected in purified form in the chosen buffer.
100
UV absorbance
high molecular weight low molecular weight
sample injection volume
intermediate molecular weight equilibration
1 cv Column Volumes (cv)
Fig. 35. Typical GF elution.
Further information Protein Purification Handbook Gel Filtration Handbook: Principles and Methods
Reversed Phase Chromatography (RPC) RPC separates proteins and peptides with differing hydrophobicity based on their reversible interaction with the hydrophobic surface of a chromatographic medium. Samples bind as they are loaded onto a column. Conditions are then altered so that the bound substances are eluted differentially. Due to the nature of the reversed phase matrices, the binding is usually very strong and requires the use of organic solvents and other additives (ion pairing agents). Elution is usually performed by increases in organic solvent concentration, most commonly acetonitrile. Samples, which are concentrated during the binding and separation process, are collected in a purified, concentrated form. The key stages in a separation are shown in Figure 36. column equilibration
sample application
gradient elution
100%
clean after gradient
re-equilibration
2-4 cv
[CH3CN/0.1%TFA]
wash out unbound molecules before elution begins
10-15 cv
2-4 cv 0
2 cv Column Volumes [cv]
Fig. 36. Typical RPC gradient elution.
RPC is often used in the final polishing of oligonucleotides and peptides and is ideal for analytical separations, such as peptide mapping. RPC is not recommended for protein purification if recovery of activity and return to a correct tertiary structure are required, since many proteins are denatured in the presence of organic solvents.
101
Method development 1. Select medium from screening results. 2. Select optimum gradient to give acceptable resolution. For unknown samples begin with 0–100% elution buffer. 3. Select highest flow rate which maintains resolution and minimizes separation time. 4. For large scale purification transfer to a step elution. 5. Samples that adsorb strongly to a medium are more easily eluted by changing to a less hydrophobic medium.
Further information Protein Purification Handbook Reversed Phase Chromatography Handbook: Principles and Methods
Expanded Bed Adsorption (EBA) EBA is a single pass operation in which target proteins are purified from crude sample, without the need for separate clarification, concentration and initial purification to remove particulate matter. Crude sample is applied to an expanded bed of STREAMLINE™ adsorbent particles within a specifically designed STREAMLINE column. Target proteins are captured on the adsorbent. Cell debris, cells, particulate matter, whole cells, and contaminants pass through and target proteins are then eluted. Figure 37 shows a representation of the steps involved in an EBA purification and Figure 38 shows a typical EBA elution.
0.Sedimented adsorbent
1.Equilibration 2.Sample appl. (expanded) (expanded)
3.Washing (expanded)
4.Elution (packed bed)
Fig. 37. Steps in an EBA purification process.
UV absorbance
equilibration
Begin sample application adorbance
Sample volumes
Fig. 38. Typical EBA elution.
102
adsorption of sample and elution of unbound material
Begin wash with start buffer
wash away unbound material
elute bound protein(s)
column wash
Change to elution buffer
Volume
5.Regeneration (packed bed)
Method development 1. Select suitable ligand to bind the target protein. 2. Scout for optimal binding and elution conditions using clarified material in a packed column (0.02–0.15 litres bed volume of media). Gradient elution may be used during scouting, but the goal is to develop a step elution. 3. Optimize binding, elution, wash and cleaning-in-place procedures using unclarified sample in expanded mode at small scale (0.02–0.15 litres bed volume of media). 4. Begin scale up process at pilot scale (0.2–0.9 litres bed volume of media). 5. Full scale production (up to several hundred litres bed volume of media).
Further information Protein Purification Handbook Expanded Bed Adsorption Handbook: Principles and Methods
103
Additional reading and reference material Code No. Gel Media Guide (electrophoresis) Protein Electrophoresis Technical Manual Protein Purification Handbook Protein and Peptide Purification Technique Selection Fast Desalting and Buffer Exchange of Proteins and Peptides Gel Filtration Handbook Principles and Methods Ion Exchange Chromatography Handbook Principles and Methods Chromatofocusing with Polybuffer and PBE Hydrophobic Interaction Chromatography Handbook Principles and Methods Affinity Chromatography Handbook Principles and Methods Reversed Phase Chromatography Handbook Principles and Methods Expanded Bed Adsorption Handbook Principles and Methods Antibody Purification Handbook Convenient Purification, HiTrap Column Guide ÄKTAdesign Brochure Protein Purification, Principles, High Resolution Methods and Applications, J.C. Janson and L. Rydén, 1998, 2nd ed. Wiley VCH
18-1129-79 80-6013-88 18-1132-29 18-1128-63 18-1128-62 18-1022-18 18-1114-21 50-01-022PB 18-1020-90 18-1022-29 18-1134-16 18-1124-26 18-1037-46 18-1129-81 18-1129-05 18-1128-68
GST fusion proteins GST Gene Fusion Manual Data File, GSTrap FF and Glutathione Sepharose 4 Fast Flow Miniposter, "Rapid Purification of GST-fusion proteins from large sample volumes"
18-1123-20 18-1136-89 18-1139-51
Purification and on-column cleavage of GST-tagged proteins Application Note, "Efficient, rapid protein purification and on-column cleavage using GSTrap FF columns"
18-1146-70
(His)6 fusion proteins Data File, HisTrap Kit Data File, HiTrap Chelating HP 1 ml and 5 ml Miniposter, "Purification of Poly(His)-tagged Recombinant Proteins using HisTrap Kit"
18-1212-00 18-1134-78 18-1116-26
On-column refolding of (His)6 fusion proteins Application Note, “Rapid and efficient purification and refolding of a (His)6-tagged recombinant protein produced in E. coli as inclusion bodies”
18-1134-37
Multi-step purification Application Note, “Purification of a labile, oxygen sensitive enzyme for crystallization and 3D structural determination” Application Note, “Purification of a recombinant phosphatase using preprogrammed generic templates for different chromatographic techniques”
18-1128-91 18-1142-32
Detection Application Note, “ECL Western and ECL Plus Western blotting”
18-1139-13
Column Packing Column Packing Video PAL Column Packing Video NTSC
104
17-0893-01 17-0894-01
Ordering information Product
Quantity
Code No.
25 25 25 25 25 25 25 25 25
27-4580-01 27-4581-01 27-4583-01 27-4584-01 27-4585-01 27-4586-01 27-4597-01 27-4598-01 27-4599-01
GST fusion proteins Protein Amplification PGEX- 4T-1 PGEX- 4T-2 PGEX- 4T-3 PGEX- 5X-1 PGEX- 5X-2 PGEX- 5X-3 PGEX- 6P-1 PGEX- 6P-2 PGEX- 6P-3 All vectors include E. coli B21 Purification GST MicroSpin Purification Module MicroPlex 24 Vacuum GSTrap FF
Glutathione Sepharose 4 Fast Flow
Glutathione Sepharose 4B
Detection GST Detection Module GST 96 Well Detection Module Anti-GST antibody
µg µg µg µg µg µg µg µg µg
50 purifications 1 system 2 x 1 ml 5 x 1 ml 1 x 5 ml 25 ml 100 ml 500 ml 10 ml 100 ml
27-4570-03 27-3567-01 17-5130-02 17-5130-01 17-5131-01 17-5132-01 17-5132-02 17-5132-03 17-0756-01 27-4574-01
50 reactions 96 reactions 0.5 ml
27-4590-01 27-4592-01 27-4577-01
50 purifications 1 system HiTrap Chelating HP columns (3 x 1 ml), accessories, pre-made buffers for up to 12 purifications 5 x 1 ml 1 x 5 ml 50 ml 500 ml 5l
27-4770-01 27-3567-01 17-1880-01
170 µl 1 ml
27-4710-01 NA931
500 units 400 units 500 units
27-0846-01 27-0849-01 27-0843-01
2 x 1 ml 5 x 1 ml 1 x 5 ml
17-5143-02 17-5143-01 17-5144-01
25 ml
17-5123-10
(His)6 fusion proteins Purification His MicroSpin Purification Module MicroPlex 24 Vacuum HisTrap Kit
HiTrap Chelating HP Chelating Sepharose Fast Flow
Detection Anti-His antibody HRP conjugated (anti-mouse) IgG
17-0408-01 17-0409-01 17-0575-01 17-0575-02 17-0575-04
Cleavage Enzymes Thrombin Factor Xa PreScission Protease Removal of thrombin and Factor Xa HiTrap Benzamidine FF (high sub)
Benzamidine Sepharose 4 Fast Flow (high sub)
105
Product
Quantity
Code No.
1 vial 1g 5g 10 g
27-1542-01 27-3054-03 27-3054-04 27-3054-05
10 sheets 10 sheets
RPN2020F RPN2020D
Companion Products E. coli B21 Isopropyl b-D-thiogalactoside (IPTG)
Western Blotting Hybond-P Hybond-ECL ECL Western Blotting Detection Reagents ECL Plus Western Blotting Detection System HiTrap Columns: Affinity HiTrap rProtein A FF
HiTrap Protein A HP
HiTrap Protein G HP
MAbTrap Kit
HiTrap IgY Purification HP HiTrap Blue HP GSTrap FF
HiTrap Chelating HP HisTrap Kit
HiTrap Streptavidin HP HiTrap Heparin HP HiTrap Benzamidine FF (high sub)
HiTrap NHS-activated HP
HiTrap Columns: IEX HiTrap IEX Selection Kit 7 x 1 ml
HiTrap Q HP
106
for 1000 cm
2
RPN2109
for 1000 cm
2
RPN2132
2 x 1 ml 5 x 1 ml 1 x 5 ml 2 x 1 ml 5 x 1 ml 1 x 5 ml 2 x 1 ml 5 x 1 ml 1 x 5 ml HiTrap Protein G HP (1 x 1 ml), accessories, pre-made buffers for 10 purifications 1 x 5 ml 5 x 1 ml 5 x 1 ml 1 x 5 ml 2 x 1 ml 5 x 1 ml 1 x 5 ml 5 x 1 ml 1 x 5 ml HiTrap Chelating HP (3 x 1 ml), accessories, pre-made buffers for up to 12 purifications 5 x 1 ml 5 x 1 ml 1 x 5 ml 2 x 1 ml 5 x 1 ml 1 x 5 ml 5 x 1 ml 1 x 5 ml
17-5079-02 17-5079-01 17-5080-01 17-0402-03 17-0402-01 17-0403-01 17-0404-03 17-0404-01 17-0405-01 17-1128-01
HiTrap Q XL, 1 ml HiTrap SP XL, 1 ml HiTrap ANX FF (high sub), 1 ml HiTrap DEAE FF, 1 ml HiTrap CM FF, 1 ml HiTrap Q FF, 1 ml HiTrap SP FF, 1 ml 5 x 1 ml 5 x 5 ml
17-6002-33
17-5111-01 17-5110-01 17-0412-01 17-0413-01 17-5130-02 17-5130-01 17-5131-01 17-0408-01 17-0409-01 17-1880-01
17-5112-01 17-0406-01 17-0407-01 17-5143-02 17-5143-01 17-5144-01 17-0716-01 17-0717-01
17-1153-01 17-1154-01
Product HiTrap Columns: IEX HiTrap SP HP HiTrap Q XL HiTrap SP XL HiTrap ANX FF (high sub) HiTrap DEAE FF HiTrap CM FF HiTrap Q FF HiTrap SP FF
HiTrap Columns: HIC HiTrap HIC Selection Kit 5 x 1 ml
HiTrap Phenyl FF (high sub) HiTrap Phenyl FF (low sub) HiTrap Phenyl HP HiTrap Butyl FF HiTrap Octyl FF
Desalting and Buffer Exchange MicroSpin G-25 Columns HiTrap Desalting HiPrep 26/10 Desalting
Quantity
Code No.
5 x 1 ml 5 x 5 ml 5 x 1 ml 5 x 5 ml 5 x 1 ml 5 x 5 ml 5 x 1 ml 5 x 5 ml 5 x 1 ml 5 x 5 ml 5 x 1 ml 5 x 5 ml 5 x 1 ml 5 x 5 ml 5 x 1 ml 5 x 5 ml
17-1151-01 17-1152-01 17-5158-01 17-5159-01 17-5160-01 17-5161-01 17-5162-01 17-5163-01 17-5055-01 17-5154-01 17-5056-01 17-5155-01 17-5053-01 17-5156-01 17-5054-01 17-5157-01
HiTrap Butyl FF, 1 ml HiTrap Octyl FF, 1 ml HiTrap Phenyl FF (low sub), 1 ml HiTrap Phenyl FF (high sub), 1 ml HiTrap Phenyl HP, 1 ml 5 x 1 ml 5 x 5 ml 5 x 1 ml 5 x 5 ml 5 x 1 ml 5 x 5 ml 5 x 1 ml 5 x 5 ml 5 x 1 ml 5 x 5 ml
17-1349-01
17-1355-01 17-5193-01 17-1353-01 17-5194-01 17-1351-01 17-5195-01 17-1357-01 17-5197-01 17-1359-01 17-5196-01
50 columns 5 x 5 ml 1 x 53 ml
27-5325-01 17-1408-01 17-5087-01
107
108
Ettan, Hybond, HiTrap, GSTrap, Sephadex, Superdex, HisTrap, Sepharose, MAbTrap, ÄKTA, FPLC, HiPrep, PhastSystem, PhastGel, SOURCE, RESOURCE, HiLoad, PreScission, ExcelGel, Multiphor, STREAMLINE, ECL, MicroPlex and Ultrospec are trademarks of Amersham Pharmacia Biotech Limited or its subsidiaries. Amersham is a trademark of Nycomed Amersham plc Pharmacia and Drop Design are trademarks of Pharmacia & Upjohn Multipipette is a trademark of Eppendorf-Netheler-Hinz GmbH Tween is a trademark of ICI Americas Inc MicroSpin is a trademark of Lida Manufacturing Corp Pefabloc is a registered trademark of Pentafam AG Triton is a registered trademark of Union Carbide Chemicals and Plastics Co ABTS is a registered trademark of Boehringer Mannheim GmbH Coomassie is a trademark of ICI plc Amicon is a trademark of Millipore Inc. Diaflow is a trademark of Cannon-Rubber Limited Licensing Information All materials for research use only. Not for diagnostic or therapeutic purposes. A license for commercial use of pGEX vectors must be obtained from AMRAD Corporation Ltd., 17-27 Cotham Road, Kew, Victoria 3101, Australia. PreScission Protease is licensed from the University of Singapore, 10 Kent Ridge Crescent, Singapore 0511. PreScission Protease is produced under commercial license from AMRAD Corporation Ltd., 17-27 Cotham Road, Kew, Victoria 3101, Australia. All goods and services are sold subject to the terms and conditions of sale of the company within the Amersham Pharmacia Biotech group that supplies them. A copy of these terms and conditions is available on request. © Amersham Pharmacia Biotech AB 2001 – All rights reserved. Amersham Pharmacia Biotech AB Björkgatan 30, SE-751 84 Uppsala, Sweden Amersham Pharmacia Biotech UK Limited Amersham Place, Little Chalfont, Buckinghamshire HP7 9NA, England Amersham Pharmacia Biotech Inc 800 Centennial Avenue, PO Box 1327, Piscataway, NJ 08855, USA Amersham Pharmacia Biotech Europe GmbH Munzinger Strasse 9, D-79111 Freiburg, Germany Amersham Pharmacia Biotech K.K. Sanken Building, 3-25-1, Hyakunincho, Shinjuku-ku, Tokyo 169-0073, Japan
www.apbiotech.com Production: RAK Design AB
Ficoll-Paque PLUS For in vitro isolation of lymphocytes
Back to Collection 18-1152-69 Edition AB
Handbooks from Amersham Biosciences
Antibody Purification Handbook 18-1037-46
The Recombinant Protein Handbook Protein Amplification and Simple Purification 18-1142-75
Protein Purification Handbook 18-1132-29
Ion Exchange Chromatography
Reversed Phase Chromatography
Principles and Methods 18-1114-21
Principles and Methods 18-1134-16
Affinity Chromatography
Expanded Bed Adsorption
Principles and Methods 18-1022-29
Principles and Methods 18-1124-26
Hydrophobic Interaction Chromatography
Chromatofocusing
Principles and Methods 18-1020-90
with Polybuffer and PBE 50-01-022PB
Gel Filtration
Microcarrier cell culture
Principles and Methods 18-1022-18
Principles and Methods 18-1140-62
Ficoll-Paque PLUS For in vitro isolation of lymphocytes
Content Introduction ................................................................................................. 5 Ficoll-Paque PLUS ....................................................................................... 6 The separation principle ............................................................................................... 6 A recommended standard method ................................................................................. 7 Equipment and solutions required .................................................................................................... 8 Preparation of the sample ............................................................................................................... 8 Procedure for isolation of lymphocytes .............................................................................................. 8 Washing lymphocytes free of platelets ............................................................................................. 10 Typical results from our laboratories ............................................................................................... 10 Notes .......................................................................................................................................... 10 Troubleshooting inadequate performance ........................................................................................ 12
Properties of lymphocytes isolated by the Ficoll-Paque PLUS method ............................. 13 Further applications of Ficoll-Paque PLUS ................................................................... 13 Availability and storage ................................................................................................................. 14 Precautionary note ........................................................................................................................ 14
References ................................................................................................. 15 Ordering Information ................................................................................... 18 Selected products for cell science from Amersham Biosciences ......................................................... 18
4
Introduction Isolation of lymphocytes from whole human blood is often required in such clinical investigations as histocompatibility testing and the assay of cell-mediated immune responses, as well as in many areas of immunological research. Ficoll-Paque™ PLUS is a sterile, ready to use density gradient medium for purifying lymphocytes in high yield and purity from small or large volumes of human peripheral blood, using a simple and rapid centrifugation procedure based on the method developed by Bøyum (1). Ficoll-Paque PLUS can also be used to prepare purified lymphocytes from sources other than human peripheral blood. Ficoll-Paque PLUS from Amersham Biosciences is: • A sterile endotoxin tested (<0.12 EU/ml) solution of Ficoll™ 400 and sodium diatrizoate with a density of 1.077 + 0.001 g/ml. • Recommended for small- or large-scale isolation of viable lymphocytes in high yield from whole human peripheral blood. • Subjected to rigorous quality control function testing, which guarantees reproducible performance from batch to batch. • Supplied in bottles sealed with a rubber septum closure, which facilitates aseptic withdrawal of solution. • Available in convenient pack sizes: 6 x 100 ml for research requirements and 6 x 500 ml for daily routine lymphocyte isolation. Detailed instructions for use are included with each pack. • Stable for at least 3 yr when stored at 4–25 °C and protected from light. Separation of normal whole human peripheral blood by the procedure recommended in this booklet typically yields a lymphocyte preparation with: • 60 + 20% recovery of the lymphocytes present in the original blood sample, • 95 + 5% monocular cells, • >90% viability of the separated cells, • 3 + 2% granulocytes, • 5 + 2% erythrocytes and • <0.5% of the total platelet content of the original blood sample.
5
Ficoll-Paque PLUS Ficoll-Paque PLUS is an aqueous solution of density 1.077 + 0.001 g/ml containing 5.7 g Ficoll 400 and 9 g sodium diatrizoate with 0.0231 g calcium disodium ethylenediamintetraacetic acid in every 100 ml. Ficoll 400 is a synthetic high molecular weight (Mw 400 000) polymer of sucrose and epichlorohydrin which is readily soluble in water. The molecules of Ficoll 400 are highly branched, approximately spherical and compactly coiled with a Stokes’ radius of a about 10 nm. Ficoll 400 has a low intrinsic viscosity (17 ml/g) compared with linear polysaccharides of the same molecular weight (cf. dextran Mw 400 000: /h/ 49 ml/g) and solutions of Ficoll 400 have low osmotic pressures. Sodium diatrizoate is a convenient compound to use with Ficoll 400 since it forms solutions of low viscosity with high density. Sodium diatrizoate (Mr 635.92) is the sodium salt of 3,5-diacetamido2,4,6-triiodobenzoic acid.
COONa I
I N C CH3
H3C C N O H
I
H O
Since sodium diatrizoate is light-sensitive, Ficoll-Paque PLUS must be stored protected from light. The function of sodium diatrizoate in Ficoll-Paque PLUS is to provide the optimal density and osmolarity necessary for the efficient removal of other cells from the lymphocytes. Ficoll-Paque PLUS is supplied as a sterile solution in a bottle with a rubber septum closure. To maintain sterility, aseptic techniques should be used when withdrawing solution and the rubber septum should not be removed. Ficoll-Paque PLUS should be stored between 4 °C and 25 °C and protected from direct light. Storage of unopened bottles in the dark will increase their shelf-life. Deterioration of Ficoll-Paque PLUS is indicated by the appearance of a distinct yellow colour or particulate material in the clear solution. Ficoll-Paque PLUS showing such deterioration should be discarded.
The separation principle Lymphocyte isolation using Ficoll-Paque PLUS is based on methodology established through the extensive studies of Bøyum (1,2,3) and investigations carried out in our own laboratories. Separation media consisting of a mixture of Ficoll 400 and an iodinated density gradient medium such as sodium diatrizoate have been very widely used for purifying human lymphocytes following the publication of Bøyum’s pioneering work in 1968. For lymphocyte separation, defibrinated or anticoagulant-treated blood is diluted with an equal volume of balanced salt solution and layered carefully over Ficoll-Paque PLUS (without intermixing) in a centrifuge tube. After a short centrifugation at room temperature (typically at 400 gav for 30–40 min) lymphocytes, together with monocytes and platelets, are harvested from the interface between the Ficoll-Paque PLUS and sample layers. This material is then centrifuged twice in balanced salt solution to wash the lymphocytes and to remove the platelets. 6
Several factors contribute to the success of this separation. On centrifugation, cells in the blood sample sediment towards the blood/Ficoll-Paque PLUS interface, where they come in contact with the Ficoll 400 present in Ficoll-Paque PLUS. Red blood cells are efficiently aggregated by this agent at room temperature. Aggregation increases the rate of sedimentation of the red cells, which rapidly collect as a pellet at the bottom of the tube, where they are well separated from lymphocytes. Granulocytes also sediment to the bottom of the Ficoll-Paque PLUS layer. This process is facilitated by an increase in their densities caused by contact with the slightly hypertonic Ficoll-Paque PLUS medium. Thus, on completion of centrifugation, both granulocytes and red blood cells are found at the bottom of the tube, beneath the Ficoll-Paque PLUS. Lymphocytes, monocytes, and platelets are not dense enough to penetrate into the Ficoll-Paque PLUS layer. These cells therefore collect as a concentrated band at the interface between the original blood sample and the Ficoll-Paque PLUS. This banding enables the lymphocytes to be recovered with high yield in a small volume with little mixing with the Ficoll-Paque PLUS medium. Washing and centrifugation the harvested cells subsequently removes platelets, any contaminating Ficoll-Paque PLUS and plasma. The resulting cell suspension then contains highly purified, viable lymphocytes and monocytes and is suitable for further studies.
A recommended standard method Lymphocyte purification using Ficoll-Paque PLUS can be carried out over a wide range of blood sample volumes. With its high yield, this method can be adapted to the processing of very small amounts of blood, such as may be obtained from children. Because of its rapidity and simplicity it is also the method of choice for emergency tissue typing procedures (4). For maximum reproducibility of separation it is recommended that a standardized procedure be used. The following procedure has been evaluated in our laboratories and is recommended for separation of normal blood samples on Ficoll-Paque PLUS. Simple changes can easily be made to suit a particular centrifugation system. To standardize the technique, blood volume and diameter of the centrifuge tube should be chosen first. These factors determine the height of the blood sample in the tube and consequently the centrifugation time. Increasing the height of the blood sample in the tube increases red cell contamination. The separation is, however, not appreciably affected by changing the diameter of the tube. Hence a larger volume can be separated with the same degree of purification in a tube of larger diameter if the height of the blood sample in the tube and the separation time are kept constant. The yield and degree of purity of the lymphocytes depend to a considerable extent on the efficiency of red cell removal. When erythrocytes in whole blood are aggregated, some lymphocytes are trapped in the clumps and therefore sediment with the erythrocytes. This tendency to trap lymphocytes is reduced by diluting the blood. Dilution gives a better lymphocyte yield and reduces the size of the red cell clumps. Aggregation of erythrocytes is enhanced at higher temperatures (37 °C), which consequently decreases the yield of lymphocytes. At lower temperatures (4 °C), however, the rate of aggregation is decreased but the time of separation is increased, which also decreases the yield of lymphocytes. A compromise temperature of 18–20 °C gives optimal results.
7
Equipment and solutions required 1. Two 10 ml glass test-tubes for each blood sample to be processed. The test-tubes should be siliconized (see “Notes”, page 10). 2. Balanced salt solution. At least 20 ml for each sample to be processed. The balanced salt solution may be prepared from two stock solutions, A and B. Solution A Conc. g/l Anhydrous D-glucose CaCl2 . 2H2O MgCl2 . 6H20 KCl TRIS
5.5 5.0 9.8 5.4
x 10-3 M (0.1%) x 10-3 M x 10-4 M x 10-3 M 0.145 M
1.0 0.0074 0.1992 0.4026 17.565
Dissolve in approximately 950 ml distilled water and add conc. HCl until pH is 7.6 before adjusting the volume to 1 l. Solution B Conc. g/l NaCl
0.14 M
8.19
To prepare the balanced salt solution, mix 1 volume of solution A with 9 volumes of solution B. Prepare the solution freshly each week. Other standard salt solutions may be used. 3. Pasteur pipettes (3 ml). One for each sample to be processed. These pipettes should be siliconized (see “Notes”, page 10). 4. A low speed centrifuge. 5. Glass centrifuge tubes. Two centrifuge tubes for each blood sample to be processed. Internal diameter approximately 1.3 cm, volume 15 ml. The centrifuge tubes should be siliconizied (see “Notes”, page 10). For larger or smaller samples see “Notes”, page 10. 6. Ficoll-Paque PLUS. 3 ml for each sample being processed. For larger or smaller samples see “Notes”, p 10. 7. Syringe with needle. Needed for withdrawing Ficoll-Paque PLUS from the bottle under aseptic conditions.
Preparation of the sample Fresh blood should be used to ensure high viability of isolated lymphocytes. Prepare the sample at +18 to +20 °C. 1. To a 10 ml test-tube add 2 ml of defibrinated- or anticoagulant-treated blood and an equal volume of balanced salt solution (final volume 4 ml). 2. Mix by drawing the blood and the buffer in and out of a Pasteur pipette.
Procedure for isolation of lymphocytes 1. Remove the blue cap on the bottle of Ficoll Paque PLUS (Fig. 1). 2. Invert the bottle of Ficoll-Paque PLUS several times to ensure mixing. Using the syringe with needle attached, pierce the septum and withdraw the required volume of Ficoll-Paque PLUS (3 ml for each centrifuge tube) from the inverted bottle (Fig 2). If this method is employed, each bottle 8
will deliver at least 100 ml Ficoll-Paque PLUS. 3. Add Ficoll-Paque PLUS (3 ml) to the centrifuge tube. 4. Carefully layer the diluted blood sample (4 ml) onto the Ficoll-Paque PLUS (Fig 3). Important. When layering the sample do not mix the Ficoll-Paque PLUS and the diluted blood sample. 5. Centrifuge at 400gav for 30–40 min at 18–20 °C. 6. Draw off the upper layer using a clean Pasteur pipette, leaving the lymphocyte layer undisturbed at the interface (Fig 4 and Fig 5). Care should be taken not to disturb the lymphocyte layer. The upper layer, which contains the plasma, may be saved for later use.
Fig 1.
Fig 3.
Blood sample
Fig 2. Ficoll-Paque PLUS
Fig 4.
Plasma Platelets Lymphocytes Ficoll-Paque PLUS Granulocytes Erythrocytes Fig 5.
Upper layer removed Lymphocytes Ficoll-Paque PLUS Granulocytes Erythrocytes
9
Washing lymphocytes free of platelets 1. Using a clean Pasteur pipette transfer the lymphocyte layer to a clean centrifuge tube. It is critical to remove all the material at the interface but in a minimum volume. Removing excess Ficoll-Paque PLUS causes granulocyte contamination; removing excess supernatant results in platelet contamination. 2. Add at least 3 volumes (6 ml) of balanced salt solution to the lymphocytes in the test-tube. 3. Suspended the cells by gently drawing them in and out of a Pasteur pipette. 4. Centrifuge at 60–100 gav for 10 min at 18–20 °C. 5. Discard the supernatant. 6. Suspend the lymphocytes in 6–8 ml balanced salt solution by gently drawing them in and out of a Pasteur pipette. 7. Centrifuge at 60–100 gav for 10 min at 18–20 °C. 8. Discard the supernatant. The lymphocytes should now be suspended in the medium appropriate to the application.
Typical results from our laboratories Lymphocytes: 60 + 20% recovery of lymphocytes from the original blood sample 95 + 5% of cells present in the lymphocyte fraction are mononuclear leukocytes >90% viability (measured by trypan blue exclusion) Other cells:
3 + 2% granulocytes 5 + 2% erythrocytes <0.5% of the total platelet content of the original blood sample
Notes Preparation of glassware. All glassware that comes in contact with the sample should be siliconized before use. Glassware should be immersed in a 1% silicone solution for 10 seconds (where no specific coating procedure is recommended by the manufacturer), washed thoroughly with distilled water and dried in an oven. The best siliconizing fluids are those based on dimethyldichlorosilane dissolved in an organic solvent. Examples of suitable fluids are: Sigmacote Repelcote Dimethyldichlorosilane Prosil-28 Silicone Oil Siliclad
Sigma Chemicals Co., Cat. No. SL-2. Hopkins and Williams, Cat. No. 9962-70 BDH, Cat. No. 33164 PCR Research Chemicals. Midland Silicones Ltd., Cat. No. MS 1107, use as 2—5% (v/v) solution in ethyl acetate. Clay-Adams, Cat. No. 1950.
Alternatively, tissue culture plasticware may be used. Anticoagulants, Heparin, EDTA, citrate, acid citrate dextrose (ACD), and citrate phosphate dextrose (CPD) may be used as anticoagulants for the blood sample. Defibrinated blood requires no anticoagulant. Defibrination, however, results in a lower lymphocyte yield and may cause increased contamination by red cells (3). It also causes selective loss of monocytes. Bøyum has found that a slightly purer 10
lymphocyte preparation is obtained using EDTA instead of heparin as anticoagulant (3). It has also been noted in the purification of lymphocytes from sources other than peripheral blood that addition of heparin may cause gelling of cell suspensions (5). Larger blood samples. Larger volumes of blood may be processed with the same efficiency of separation by using centrifuge tubes of increased diameter while maintaining approximately the same heights of Ficoll-Paque PLUS (2.4 cm) and blood sample (3.0 cm) as in standard method described above. Increasing the tube diameter does not affect the separation time required. Smaller blood samples. Smaller volumes of blood can be processed rapidly by a modification of the method of Bøyum (6). A micromethod suitable for use in tissue typing of blood cells from cadavers has also been described (4). Sample storage. Blood samples should be processed as soon as possible after collection to ensure optimal results. Storage for 24 h at room temperature has been reported to result in reduced lymphocyte yield, altered expression of surface markers and reduced response to mitogenic stimulation (7). Pathological blood samples. The standard method described above has been developed for the purification of lymphocytes from peripheral blood of normal, healthy, human donors. Different results may be obtained with samples taken from donors with infections or other pathological conditions, e.g. cancer (see “Further Applications”, page 13). Platelet removal. The washing procedure described in this standard method will give efficient removal of platelets from the lymphocytes in the majority of cases. If difficulty is experienced, centrifugation through a 4–20% sucrose gradient layered over Ficoll-Paque PLUS may be used to remove platelets (8). Alternatively, the platelets may be removed by aggregation with adenosine-5-diphoshate (ADP) before separating the lymphocytes (9).
11
Troubleshooting inadequate performance If used according to the recommended standard procedure, Ficoll-Paque PLUS may be expected to give trouble-free isolation of human peripheral blood lymphocytes with results as shown under “Typical results from our laboratories”, page 10. Deviations in certain experimental parameters may lead to poor results and the troubleshooting chart given here is intended to assist in the rapid identification and correction of the problem causing reduced performance. Deviation in Performance
Likely Source of Problem
Comments
Increased red blood cell and contamination of the lymphocytes.
A. Temperature too low
The density of Ficoll-Paque PLUS is greater at low temperature and red blood cells are aggregated less well, so granulocytes and red blood cells are prevented from entering the Ficoll-Paque PLUS layer. Raise the temperature to 18–20 °C.
B. Centrifugation speed too slow and/or centrifugation time too short.
Adequate time and g-force must be used to ensure complete sedimentation of non-lymphoid cells.
Low yield and viability of lymphocytes.
Temperature too high.
Ficoll-Paque PLUS is less dense at high temperature and some lymphocytes may penetrate into the Ficoll-Paque PLUS layer. Cell viability may also be affected. Reduce the temperature to 18–20 °C.
Low yield of lymphocytes with normal viability.
Blood not diluted 1:1 with balanced salt solution; unusually high hematocrit.
The high cell density results in a large numbers of lymphocytes being trapped by red blood cell aggregates. Dilute the blood sample further.
Low yield of lymphocytes with increased granulocyte contamination.
Vibration of the centrifuge rotor, leading to stirring of the gradient.
Vibration may cause broadening of the lymphocyte band and mixing with the underlying cells. Check that the rotor is properly balanced. Choose rotor speed to avoid natural resonant frequencies.
Low yield of lymphocytes, low viability, and contamination by other cell types.
Sample contains cells with abnormal densities; densities different from those in normal human blood.
May be encountered with pathological blood samples, non-human blood samples, or samples from sources other than peripheral blood. Percoll ™, a medium for density gradient centrifugation, may be more suitable than Ficoll-Paque PLUS for such separations.
12
Properties of lymphocytes isolated by the Ficoll-Paque PLUS method Since its introduction in 1968, the lymphocyte separation method of Bøyum (1,2) has been used in numerous immunological investigations as well as in routine diagnostic studies. This widespread adoption indicates the superior results obtained with this technique and its freedom from impairment of lymphocyte function. Nevertheless, certain effects of the separation procedure have been seen and these are noted below, since research situations may arise in which they are of significance. Separation with Ficoll-Paque PLUS has been reported to lead to adsorption of cytophilic IgG to the mononuclear leukocytes (10), resulting in erroneously high estimates of the number of Ig-bearing lymphocytes and too low estimates of the number of cells bearing Fc receptors. This interference can be avoided by washing the blood cells with balanced salt solution before isolation, thus removing the IgG present in the plasma that gives rise to these artifacts. Selective loss of a population of lymphocytes that form rosettes with autologous red blood cells has been reported to occur using the standard procedure (11,12) and evidence was found that this is the result of a specific lymphocyte-red blood cell interaction, not a non-specific trapping (12). This population was found to account for ca. 6% of the lymphocytes initially present in the blood sample and could be recovered almost quantitatively by resuspending the red cell pellet in medium and recentrifuging over a gradient of slightly higher density than normal, i.e. 1.083 g/ml (12). Lymphocytes separated by the Bøyum procedure have been reported (13) to show enhanced stimulation in mixed lymphocyte cultures as compared with lymphocytes in “leukocyte-rich plasma” (not exposed to Ficoll-Paque PLUS). This enhanced reaction was postulated to depend at least partially on the removal in the Ficoll-Paque PLUS method of neutrophils that appear otherwise to have a suppressive effect on the mixed lymphocyte reaction (13). Ficoll-Paque PLUS-separated lymphocytes have also been reported to show increased levels of “spontaneous” blastogenesis, as measured by 3H-thymidine incorporation in cultures not stimulated by mitogens, but the cause of this enhancement was not established (14). Diminished response of Ficoll-Paque PLUS-separated lymphocytes to mitogenic stimulation by phytohaemagglutinin (PHA) as compared to lymphocytes prepared by centrifugal elutriation has been reported in one instance (15).
Further applications of Ficoll-Paque PLUS A great many modifications and extensions of the method have come into use follwing the introduction of the technique by Bøyum in 1968 and its subsequent widespread adoption. For example, monocytes (which are recovered in the lymphocyte fraction, using the standard procedure decribed in this booklet) can be removed, if desired, by incubating the blood sample with iron (or iron carbonyl) before separation on Ficoll-Paque PLUS. The monocytes phagocytose the iron particles and become denser, with the result that they sediment through the Ficoll-Paque PLUS layer on centrifugation and collect in the red blood cell pellet at the bottom of the tube (3). An important and widely used extension of the original technique is its application, in combination with selective “rosetting” (clustering), to the isolation of lymphocyte subclasses. In the most often used case, the purified lymphocytes obtained by the standard procedure (with or without monocyte removal) are incubated with an excess of sheep red blood cells (ratio of red blood cells to lymphocytes at least 50:1), whereupon the T lymphocytes spontaneously form “rosettes” (clusters) with the sheep red blood cells. On centrifugation for a second time over Ficoll-Paque PLUS, the T lymphocyte rosettes sediment to the bottom of the tube together with the excess red blood cells, leaving the other (non-rosetting) lymphocytes at the interface (3).
13
Such techniques for the separation of lymphocyte subclasses, as well as the standard method for isolating the entire lymphocyte population, have been widely applied to studies of lymphocyte functions and surface markers in disease states as compared to normal controls. For such comparative studies it is important that the lymphocyte purification method should not lead to preferential enrichment or loss of any particular lymphocyte subclass. Evidence that the standard Ficoll-Paque PLUS procedure is free from this kind of distortion has been presented by Häyry et al. (16), although the existence of the minor subset of autologous rosette-forming lymphocytes described by Hokland and Heron (11) was not recognized in the earlier work. Caution is, however, necessary in applying the Ficoll-Paque PLUS technique to pathological blood specimens, since it has been found that the resulting lymphocyte layer may be contaminated with immature granulocytes in patients with certain infections (17), and particularly cancer (18,19). In the latter case, elevated numbers of monocytes may also be present (20). However, in a study of immunocompromised patients with aplastic anemia or acute leukemia, lymphocyte isolation proceeded normally using Ficoll-Paque PLUS and resulted, in combination with purification of granulocytes from the red blood cell pellet by dextran sedimentation, in substantially increased rates of virus recovery from the blood samples (21). Ficoll-Paque PLUS has been used with success to separate cells from a variety of sources other than peripheral blood, even though its properties have been optimized specifically for blood lymphocyte isolation. Thus, separation over Ficoll-Paque PLUS facilitated detection and identification of malignant cells in abdominal and pleural fluids (22) and similar conclusions have been drawn using FicollPaque PLUS mixtures of densities other than 1.077 g/ml (23,24). Separation on Ficoll-Paque PLUS has also been reported to assist in establishing cultures of amniotic fluid cells and to facilitate their subsequent cytogenic analysis (25). Ficoll-Paque PLUS can also be used to isolate lymphocytes from species other than man. In some cases, e.g. cow, goat, and rabbit, it may be necessary to alter the standard procedure to achieve good results (3) and it should be remembered that the density of Ficoll-Paque PLUS (1.077 g/ml), although optimized for the isolation of human lymphocytes, may not give optimal yield and purity of lymphocytes from other species. However, isolation methods using Ficoll-Paque PLUS of standard density have been described for mouse (26), dog (27), monkey (28), cow (29,30), rabbit (31), horse (32), pig (32,33), and even fish (34) lymphocytes. Where it is desired to work with solutions of densities other than 1.077 g/ml it may be convenient to use the alternative centrifugation medium Percoll™ (a descriptive handbook is available free on request), since iso-osmotic solutions of different densities are very easily prepared with this medium, facilitating the optimization of a particular separation. Separation with Percoll has also been reported to give improved lymphocyte yields and purities in some cases (35–37).
Availability and storage Ficoll-Paque PLUS is available in packs of 6 x 100 ml (Code No. 17-1440-02) and 6 x 500 ml (Code No. 17-1440-03) as a sterile, ready to use liquid in glass bottles with rubber septum caps. Full instructions for use are included with each pack. Ficoll-Paque PLUS should be stored between 4 °C and 25 °C protected from light, under which conditions it will maintain sterility and stability for 3 yr. Storage in the cold will prolong shelf-life. Freezing of this product is not recommended, but if frozen accidentally, the bottle should be inverted several times after thawing to ensure a homogeneous solution.
Precautionary note This material is intended for in vitro diagnostic use for the isolation of lymphocytes and other research applications. 14
References 1. Isolation of mononuclear cells and granulocytes from human blood. (Paper IV). Bøyum, A. Scand J Clin Lab Invest 21 Suppl, 97, 77–89 (1968). 2. Isolation of leucocytes from human blood - further observations. (Paper II). Bøyum, A. Scand J Clin Lab Invest 21 Suppl, 97, 31–50 (1968). 3. Isolation of lymphocytes, granulocytes and macrophages. Bøyum, A. Scand J Immunol 5 Suppl, 5, 9–15 (1976). 4. Micromethod for rapid separation of lymhocytes from peripheral blood. Fotino, M., Merson, E.J., Allen, F.H. Ann Clin Lab Sci, 1, 131–133 (1971). 5. Gel formation with leucocytes and heparin. Almeida, A.P., Beaven, M.A. Life Sci, 26, 549–555 (1980). 6. Tissue typing using a routine one-step lymphocyte separation procedure. Harris, R., Ukaejiofo, E.O. Brit J Haematol, 18, 229–235 (1970). 7. Altered lymphocyte markers and blastogenic responses associated with 24 hour delay in processing of blood samples. Kaplan, J., Nolan, D., Ree, A. J Immunol Methods, 50, 187–191 (1982). 8. Purification of lymphocytes and platelets by gradient centrifugation. Perper, R.J., Zee, T.W., Mickelson, M.M. J Lab Clin Med, 72, 842–848 (1968). 9. Platelet aggregation technique used in the preparation of lymphocyte suspensions. Vives, J., Parra, M., Castillo, R. Tissue Antigens 1, 276–278 (1971). 10. Quantitation of Fc receptors and surface immunoglobulin is affected by cell isolation procedures using Plasmagel and Ficoll-Hypaque. Alexander, E.L., Titus, J.A., Sega, D.M. J Immunol Methods, 22, 263–272 (1978). 11. Analysis of the lymphocyte distribution during Isopaque-Ficoll isolation of mononuclear cells from human peripheral blood. Hokland, P., Heron, I. J Immunol Methods, 32, 31–39 (1980). 12. The Isopaque-Ficoll method re-evaluated: Selective loss of autologous rosette-forming lymphocytes during isolation of mononuclear cells from human peripheral blood. Hokland, P., Heron, I. Scand J Immunol, 11, 353–356 (1980). 13. Reactivity in mixed cultures of mononuclear leucocytes separated on Ficoll-Hypaque. Bain, B., Pshyk, K. Proceedings 7th Leucocyte Culture Conference, (Ed. Daguillard, F.) Academic Press, New York, 29–37 (1973). 14. Effect of Ficoll-Hypaque separation on activation and DNA synthesis of human blood lymphocytes. Farnes, P., Barker, B.E. In: Regulatory Mechanisms in Lymphocyte Activation, ( Ed. Lucas, D.O.) Academic Press, New York, 368–370 (1977). 15. Comparison of lymphocyte function after isolation by Ficoll-Hypaque flotation or elutriaton. Berger, C.L.,Edelson, R.L. J Invest Dermatol, 73, 231–235 (1979). 16. Lack of subclass selection during density purification of human lymphocytes. Häyry, P., Tötterman, T.H., Ranki, A. Clin Exp Immunol, 28, 341–346 (1977).
15
17. Special requirements for isolation of purified lymphocyte populations in infected patients. Dougherty, P.A., Balch, C.M. Fed Proc, 40, 1120 (1981). 18. Changes in Ficoll-Hypaque gradients with advancing stage of lung cancer. Check, I.J., Hunter, R.L. Fed Proc, 38, 1224 (1979). 19. Contamination of mononuclear cell suspensions obtained from cancer patients by the Bøyum method. Currie, G.A., Hedley, D.W., Nyholm, R.E., et al. Brit J Cancer, 38, 555–556 (1978). 20. Non-lymphoid cells obtained by the Bøyum technique and their significance in cancer patients. Kluin-Nelemans, J.C., van Helden, H.P.T. J Clin Lab Immunol, 4, 99–102 (1980). 21. Comparison of rates of virus isolation from leukocyte populations separated from blood by conventional and Ficoll-Paque/Macrodex methods. Howell, C.L., Miller, M.J., Martin, W.J. J Clin Microbiol, 10, 533–537 (1979). 22. Gradient separation of normal and malignant cells. II. Application to in vivo tumour diagnosis. Minami, R., Yokota, S., Teplitz, R.L. Acta Cytol, 22, 584–588 (1978). 23. A quick method for concentration and processing cancer cells from serous fluids and fineneedle nodule aspirates. Elequin, F.T., Muggia, F.M., Ghossein, N.A., et al. Acta Cytol, 21, 596–599 (1977). 24. The comparative diagnostic accuracy of cancer-cell detection obtained with Ficoll-Hypaque gradient separation and standard centrifugation technics on body-cavity fluids. Katz, R.L., Lukeman, J.M. Amer J Clin Pathol, 74, 18–24 (1980). 25. Enhancement of human amniotic cell growth by Ficoll-Paque gradient fractionation. Chang, H-C., Jones, O.W., Bradshaw, C., et al. In Vitro. 17, 81–90 (1981). 26. A simple method for the isolation of murine peripheral blood lymphocytes. Chi, D.S., Harris, N.S. J Immunol Methods, 19, 169–172 (1978). 27. Isolation of various canine leucocytes and their characterization by surface marker analysis. Ho, C.K., Babiuk, L.A. Immunol, 35, 733–740 (1978). 28. Lymphocyte isolation, rosette formation, and mitogen stimulation in rhesus monkeys. Taylor, D.W., Marchette, N.J., Siddiqui, W.A. Develop Comp Immunol, 2, 539–546 (1978). 29. The bovine lymphoid system: Binding and stimulation of peripheral blood lymphocytes by lectins. Pearson, T.W., Roelants, G.E., Lundin, L.B., et al. J Immunol Methods, 26, 271–282 (1979). 30. Acid a-naphthyl acetate asterase: presence of activity in bovine and human T and B lymphocytes. Yang, T.J., Jantzen, P.A., Williams, L.F. Immunol, 38, 85–93 (1979). 31. Humoral and formed elements of blood modulate the response of peripheral blood monocytes. I. Plasma and serum inhibit and platelets enhance monocyte adherence. Musson, R.A., Henson, P.M. J Immunol, 122, 2026–2031 (1979). 32. Comparative study of six methods for lymphocyte isolation from several mammalian sources and determination of their carbohydrate composition. Hueso, P., Rocha, M. (Article in Spanish) Rev Esp Fisiol, 34, 339–344 (1978). 33. Separation of porcine blood cells by means of Ficoll-Paque. Wittman, G. (Article in German) Zbl Vet Med B, 27, 253–256 (1980). 16
34. A comparison of the methods used for the separation of fish lymphocytes. Blaxhall, P.C. J Fish Biol, 18, 177–181 (1981). 35. Separation of human peripheral blood monocytes on continuous density gradients of Polyvinylpyrrolidone-coated silica gel (Percoll). Brandslund, I., Møller-Rasmussen, J., Fisker, D., et al. J Immunol Methods, 48, 199–211 (1982). 36. Efficient separation of human T lymphocytes from venous blood using PVP-coated colloidal silica particles (Percoll). Feucht, H.E., Hadam, M.R., Frank, F., et al. J Immunol Methods, 38, 43–51 (1980). 37. An improved technique for the isolation of lymphocytes from small volumes of peripheral mouse blood. Mizobe, F., Martial, E., Colby-Germinario, S., et al. J Immunol Methods, 48, 269–279 (1982).
17
Ordering Information Selected products for cell science from Amersham Biosciences Product
Quantity
Code No.
Ficoll PM 400
A hydrophilic polymer of high moleculer weigt for density gradient centrifugation.
100 g
17-0300-10
Percoll
An unique density gradient centrifugation medium for fraction of cells, subcellular particles and viruses.
250 ml
17-0891-02
Density Marker Beads
Small coloured beads of accurately known densities, for calibration of density gradients of Percoll.
10 vials
17-0459-01
Protein A
The purified IgG-binding protein from Staphylococcus aureus with many applications in the localization and quantitation of antigens and antibodies.
5 mg
17-0872-05
Protein A-Sepharose™ CL-4B
For preparation and immobilization of IgG.
25 ml
17-0963-03
Phytohaemagglutinin
A purified PHA preparation for chromosome analysis and investigation of lymphocytes.
50 mg
27-3707-01
Protein A-Sepharose 6MB
For separation of cells by affinity chromatography.
10 ml
17-0469-01
Wheat germ Lectin
For separation of cells by affinity chromatography.
1g
17-0750-09
Concanavalin A
For separation of cells by affinity chromatography.
500 mg
17-0450-01
CNBr Activated Sepharose 6MB
For separation of cells by affinity chromatography.
15 g
17-0820-01
Microcarriers for cell culture.
25 g
17-0448-01
Microcarriers for cell culture.
10 g
17-0485-01
Cytodex
™
Cytodex 3
1
These and other products are available in the catalog “BioDirectory 2002” and in individual technical booklets which are avilable free on request.
18
Percoll, Ficoll, Ficoll-Paque, Sepharose and Cytodex are trademarks of Amersham Biosciences Limited. Amersham is a trademark of Amersham plc. Pharmacia and Drop Design are trademarks of Pharmacia Corporation. The data presented herein have been carefully compiled from our records, which we believe to be accurate and reliable. We make, however, no warranties or representations with respect hereto, nor is freedom from any patent to be inferred. Before any part of this manual is reproduced, please request permission from Amersham Biosciences. The products described in this literature are intended for in vitro use only. Nothing in this literature should be construed as either a recommendation or an authorization to use these products for in vivo applications. All goods and services are sold subject to the terms and conditions of sale of the company within the Amersham Biosciences group that supplies them. A copy of these terms and conditions is available on request. © Amersham Biosciences AB 2001 – All rights reserved. Amersham Biosciences AB Björkgatan 30, SE-751 84 Uppsala, Sweden Amersham Biosciences Amersham Place, Little Chalfont, Buckinghamshire HP7 9NA, England Amersham Biosciences Inc 800 Centennial Avenue, PO Box 1327, Piscataway, NJ 08855 USA Amersham Biosciences Europe GmbH Munzinger Strasse 9, D-79111 Freiburg, Germany Amersham Biosciences KK, Sanken Bldg. 3-25-1, Hyakunincho Shinjuku-ku, Tokyo 169-0073 Japan
www.amershambiosciences.com
Production: RAK Design AB
GST Gene Fusion System – Handbook
GST Gene Fusion System Handbook
www.amershambiosciences.com Back to Collection 18-1157-58 Edition AA
Handbooks from Amersham Biosciences
ÄKTA, ECL, Ettan, ExcelGel, FlexiPrep, FPLC, GFX, GSTPrep, GSTrap, HiLoad , HiTrap, HiPrep, Hybond, MicroPlex, MicroSpin, Multiphor, PhastGel, PreScission, Ready-To-Go, Sephaglas, Sepharose, and Superdex are trademarks of the Amersham Biosciences group. Amersham and Amersham Biosciences are trademarks of Amersham plc. ABTS is a registered trademark of Roche Molecular Biochemicals. Coomassie is a trademark of ICI plc. GenBank is a registered trademark of the National Institutes of Health. Gene Pulser is a registered trademark of Bio-Rad Laboratories. MicroSpin is a trademark of Lida Manufacturing Corp. Pefabloc is a registered trademark of Pentafam AG. Triton is a registered trademark of Union Carbide Chemicals and Plastics Co. Tween is a registered trademark of ICI Americas, Inc. Ultrospec is a registered trademark of Biochrom Ltd. Whatman is a registered trademark of Whatman Paper Ltd. Licensing information
Antibody Purification
All materials for research use only. Not for diagnostic or therapeutic purposes.
Handbook 18-1037-46
A license for commercial use of pGEX vectors must be obtained from AMRAD Corporation Ltd., 17-27 Cotham Road, Kew, Victoria 3101, Australia. PreScission Protease is licensed from the University of Singapore, 10 Kent Ridge Crescent, Singapore 0511. PreScission Protease is produced under commercial license from AMRAD Corporation Ltd., 17-27 Cotham Road, Kew, Victoria 3101, Australia.
The Recombinant Protein Handbook Protein Amplification and Simple Purification 18-1142-75
The Polymerase Chain Reaction (PCR) is covered by patents owned by Roche Molecular Systems and F Hoffmann-La Roche Ltd. A license to use the PCR process for certain research and development activities accompanies the purchase of certain reagents from licensed suppliers such as Amersham Biosciences and Affiliates when used in conjunction with an authorized thermal cycler.
Protein Purification
Reversed Phase Chromatography
Handbook 18-1132-29
Principles and Methods 18-1134-16
Ion Exchange Chromatography
Expanded Bed Adsorption
Principles and Methods 18-1114-21
Principles and Methods 18-1124-26
Affinity Chromatography
Chromatofocusing
Principles and Methods 18-1022-29
with Polybuffer and PBE 50-01-022PB
2-D Electrophoresis
Hydrophobic Interaction Chromatography
Microcarrier cell culture
Principles and Methods 80-6429-60
Principles and Methods 18-1020-90
Principles and Methods 18-1140-62
Gel Filtration
Percoll
IEF, SDS-PAGE and 2-D Electrophoresis
Principles and Methods 18-1022-18
Methodology and Applications 18-1115-69
Principles and Methods 80-6484-89
Ficoll-Paque Plus For in vitro isolation of lymphocytes 18-1152-69
GST Gene Fusion System Handbook 18-1157-58
using immobilized pH gradients
Purchase of Ready-To-Go PCR Beads is accompanied by a limited license to use it in the Polymerase Chain Reaction (PCR) process solely for the research and development activities of the purchaser in conjunction with a thermal cycler whose use in the automated performance of the PCR process is covered by the up-front license fee, either by payment to Perkin-Elmer or as purchased, i.e. an authorized thermal cycler. Taq DNA polymerase is sold under licensing arrangements with Roche Molecular Systems, F Hoffman-La Roche Ltd and the Perkin-Elmer Corporation. Purchase of this product is accompanied by a limited license to use it in the Polymerase Chain Reaction (PCR) process for research in conjunction with a thermal cycler whose use in the automated performance of the PCR process is covered by the up-front license fee, either by payment to Perkin-Elmer or as purchased, i.e. an authorized thermal cycler. All goods and services are sold subject to the terms and conditions of sale of the company within the Amersham group that supplies them. A copy of these terms and conditions is available on request. © Amersham Biosciences AB 2001– All rights reserved. Amersham Biosciences AB Björkgatan 30, SE-751 84 Uppsala, Sweden Amersham Biosciences Amersham Place, Little Chalfont, Buckinghamshire HP7 9NA, England Amersham Biosciences Corp 800 Centennial Avenue, PO Box 1327, Piscataway, NJ 08855, USA
Sample Preparation for Electrophoresis:
Amersham Biosciences Europe GmbH Munzinger Strasse 9, D-79111 Freiburg, Germany Amersham Biosciences Sanken Building, 3-25-1, Shinjuku-ku, Tokyo 169-0073, Japan
GST Gene Fusion System Handbook
Front cover shows the structure of glutathione S-transferase (human, class mu) (GSTM2-2) form A (E.C. 2.5.1.18) mutant with Trp 214 replaced by Phe (W214F). The protein was expressed in HeLa cells, as reported in Raghunathan, S. et al. Crystal structure of human class mu glutathione transferase GSTM2-2. Effects of lattice packing on conformational heterogeneity. J. Mol. Biol. 238, 815–832 (1994).
1
Contents Chapter 1 Overview ................................................................................................................. 5 Selecting an expression strategy .................................................................................... 5 Symbols used in this handbook .................................................................................................................. 7
Chapter 2 Cloning the gene or gene fragment into a pGEX expression vector .............................. 9 pGEX vectors .............................................................................................................. 9 The host ................................................................................................................... 11 Insert DNA ............................................................................................................... 11 Summary of procedures ............................................................................................. 11 1. Restriction digestion of pGEX vectors ...................................................................... 12 2. Dephosphorylation of linearized pGEX vector ............................................................ 13 3. Ligation of insert to pGEX DNA ............................................................................... 14 4. Preparation of competent cells and transformation with pGEX DNA ............................ 14 5. Screening ............................................................................................................. 16 6. Small-scale isolation of pGEX DNA .......................................................................... 17 7. Large-scale isolation of pGEX DNA .......................................................................... 18
Chapter 3 Monitoring expression, optimizing growth, and preparing large-scale cultures .......... 19 Summary of procedures ............................................................................................. 20 Optimizing for soluble expression versus working with inclusion bodies ........................... 20 8. Screening pGEX recombinants for fusion protein expression ....................................... 21 9. Preparation of large-scale bacterial sonicates ........................................................... 23
Chapter 4 Purification of GST fusion proteins .......................................................................... 25 Selecting an affinity chromatography product ............................................................... 25 General considerations for purification of GST fusion proteins ........................................ 27 Selecting equipment for purification ............................................................................ 28 Summary of procedures ............................................................................................. 30 Purification using the GST MicroSpin Purification Module ............................................. 30 10.1. Purification of multiple samples using GST MicroSpin columns with a microcentrifuge .......................... 31 10.2. High-throughput purification using GST MicroSpin columns with MicroPlex Vacuum .............................. 32
Purification using GSTrap FF 1 ml or 5 ml columns ...................................................... 33 11.1. Manual purification using GSTrap FF column with a syringe ................................................................ 34 11.2. Simple purification using a GSTrap FF column with ÄKTAprime ........................................................... 35
Preparative purification using GSTPrep FF 16/10 column .............................................. 37 12. Preparative purification using GSTPrep FF 16/10 column ........................................ 38 Purification using Glutathione Sepharose 4B medium ................................................... 38 13.1. Batch purification using Glutathione Sepharose 4B ............................................................................ 38 13.2. Batch/column purification using Glutathione Sepharose 4B ................................................................. 40
Purification using Glutathione Sepharose 4 Fast Flow ................................................... 41 14. Column purification using Glutathione Sepharose 4 Fast Flow .................................. 41
2
Chapter 5 Detection of GST fusion proteins ............................................................................. 43 Summary of procedures ............................................................................................. 43 15. GST 96-Well Detection Module for ELISA .............................................................. 43 16. GST Detection Module with CDNB enzymatic assay ................................................ 45 17. Western blot using anti-GST antibody .................................................................... 47 18. SDS-PAGE with Coomassie blue or silver staining ................................................... 49
Chapter 6 Removal of GST tag by enzymatic cleavage ............................................................. 51 Summary of procedures ............................................................................................. 54 PreScission Protease cleavage and purification ............................................................. 55 19.1. PreScission Protease cleavage and purification of GST fusion protein bound to GSTrap FF ...................... 55 19.2. PreScission Protease cleavage and purification of GST fusion protein eluted from GSTrap FF .................. 56 19.3. PreScission Protease cleavage and purification of GST fusion protein bound to Glutathione Sepharose 4 Fast Flow or Glutathione Sepharose 4B ......................................................... 57 19.4. PreScission Protease cleavage and purification of GST fusion protein eluted from Glutathione Sepharose 4 Fast Flow or Glutathione Sepharose 4B ......................................................... 57
Thrombin cleavage and purification ............................................................................. 58 20.1. Thrombin cleavage and purification of GST fusion protein bound to GSTrap FF ...................................... 58 20.2. Thrombin cleavage and purification of GST fusion protein eluted from GSTrap FF .................................. 59 20.3. Thrombin cleavage and purification of GST fusion protein bound to Glutathione Sepharose 4 Fast Flow or Glutathione Sepharose 4B ......................................................... 60 20.4. Thrombin cleavage and purification of GST fusion protein eluted from Glutathione Sepharose 4 Fast Flow or Glutathione Sepharose 4B ......................................................... 60
Factor Xa cleavage and purification ............................................................................. 61 21.1. Factor Xa cleavage and purification of GST fusion protein bound to GSTrap FF ...................................... 62 21.2. Factor Xa cleavage and purification of GST fusion protein eluted from GSTrap FF .................................. 63 21.3. Factor Xa cleavage and purification of GST fusion protein bound to Glutathione Sepharose 4 Fast Flow or Glutathione Sepharose 4B ......................................................... 63 21.4. Factor Xa cleavage and purification of GST fusion protein eluted from Glutathione Sepharose 4 Fast Flow or Glutathione Sepharose 4B ......................................................... 64
Removal of proteases using Benzamidine Sepharose 4 Fast Flow (high sub) .................... 65 22. Removal of thrombin and Factor Xa using HiTrap Benzamidine FF (high sub) ............ 67
Chapter 7 Applications .......................................................................................................... 69 Purification ............................................................................................................... 69 Rapid purification of GST fusion proteins using GSTrap FF 1 ml and 5 ml columns ......................................... 69 Rapid purification using Glutathione Sepharose 4 Fast Flow packed in XK 16/20 column ................................ 72 High-throughput purification of GST fusion proteins using the MicroSpin GST Purification Module ................... 73
Purification and cleavage ........................................................................................... 74 On-column cleavage and sample clean-up .................................................................................................. 74
Detection of GST fusion proteins ................................................................................. 80
Troubleshooting guide ............................................................................................ 83 Protein expression ..................................................................................................... 83 Purification and detection .......................................................................................... 85
3
Detection .................................................................................................................. 87 Cleavage ................................................................................................................... 87 PreScission Protease ................................................................................................................................ 87 Thrombin ................................................................................................................................................ 88 Factor Xa ................................................................................................................................................ 89
Appendix 1 ............................................................................................................ 91 Characteristics of GST and of host bacterial strain ........................................................ 91
Appendix 2 ............................................................................................................ 92 Control regions for pGEX vectors ................................................................................. 92
Appendix 3 ............................................................................................................ 93 Electroporation .......................................................................................................... 93
Appendix 4 ............................................................................................................ 95 Sequencing of pGEX fusions ....................................................................................... 95
Appendix 5 ............................................................................................................ 96 Cleaning, storage, and handling of media/columns ........................................................ 96
Appendix 6 ............................................................................................................ 99 Cross-adsorption of anti-GST antiserum with E. coli proteins .......................................... 99
Appendix 7 .......................................................................................................... 101 Converting from linear flow (cm/h) to volumetric flow rates (ml/min) and vice versa ........ 101
Appendix 8 .......................................................................................................... 102 Amino acids table .................................................................................................... 102
Appendix 9 .......................................................................................................... 104 Protein conversion data ............................................................................................ 104
References .......................................................................................................... 105 Additional reading ................................................................................................ 107 Ordering information ............................................................................................ 108
4
Chapter 1 Overview The Glutathione S-transferase (GST) Gene Fusion System is a versatile system for the expression, purification, and detection of fusion proteins produced in Eschericia coli. The system is based on inducible, high-level expression of genes or gene fragments as fusions with Schistosoma japonicum GST (1). Expression in E. coli yields fusion proteins with the GST moiety at the amino terminus and the protein of interest at the carboxyl terminus. The protein accumulates within the cell’s cytoplasm. GST occurs naturally as a Mr 26 000 protein that can be expressed in E. coli with full enzymatic activity. Fusion proteins that possess the complete amino acid sequence of GST also demonstrate GST enzymatic activity and can undergo dimerization similar to that observed in nature (2, 3, 4). The crystal structure of recombinant S. japonicum GST from pGEX vectors has been determined (5) and matches that of the native protein. Appendix 1 shows the characteristics of GST, as determined in pGEX-1N (6). GST fusion proteins are purified from bacterial lysates by affinity chromatography using immobilized glutathione. GST fusion proteins are captured by the affinity medium, and impurities are removed by washing. Fusion proteins are eluted under mild, non-denaturing conditions using reduced glutathione. The purification process preserves protein antigenicity and function. If desired, cleavage of the protein from GST can be achieved using a site-specific protease whose recognition sequence is located immediately upstream from the multiple cloning site on the pGEX plasmids. Fusion proteins can be detected using colorimetric or immunological methods. The GST Gene Fusion System has been used successfully in many applications including molecular immunology (7), the production of vaccines (8, 9), and studies involving proteinprotein (10) and DNA-protein (11) interactions.
Selecting an expression strategy Table 1 summarizes the choices to consider when devising a strategy for fusion protein expression and purification. Table 1. Criteria for devising expression and purification strategy Choice of
Criteria
Comments
Vector
Reading frame
Fusion protein must be in the same frame as the GST reading frame.
Cloning sites
Must be compatible with the ends of the insert DNA.
Protease cleavage site
Choose among PreScission™ Protease, Thrombin, and Factor Xa. PreScission Protease vectors offer the most efficient method for cleavage and purification. Cleavage site must be absent in protein to be expressed.
Insert
Reading frame and orientation Must have an open reading frame in the correct orientation. Size
Must be less than 2 kb long, preferably much less.
Fragment ends
Must be compatible with the vector’s cloning sites such that the junctions are maintained.
5
Table 1. Criteria for devising expression and purification strategy (continued) Choice of
Criteria
Comments
Host cells
Cloning and maintenance
Choose a strain that transforms well, such as JM105, but not one carrying the recA1 allele.
Expression
Use BL21, which is protease-deficient and designed to maximize expression of full-length fusion protein.
Growth conditions
Medium, temperature, induction conditions, aeration, positive selection, handling of inclusion bodies
Evaluate different parameters to optimize expression of fusion protein. Lowering the growth temperature, increasing aeration, and altering induction conditions should be investigated first.
Purification method
For initial screening
Batch method with Glutathione Sepharose™ 4B, for 2–3 ml culture. MicroSpin™ GST Purification Module, with prepacked columns containing Glutathione Sepharose 4B, for cultures up to 12 ml, using either microcentrifuge or MicroPlex™ 24 Vacuum.
For large-scale cultures
MicroSpin GST Purification Module provides high throughput when used with MicroPlex 24 Vacuum (up to 48 samples simultaneously). Yields up to 400 µg of fusion protein.
Preparative purification
GSTrap™ FF 1 ml and 5 ml prepacked columns containing Glutathione Sepharose 4 Fast Flow. May be run in series to increase yields. Five ml columns yield 50–60 mg of fusion protein*. GSTPrep™ FF 16/10 prepacked column containing Glutathione Sepharose 4 Fast Flow. May be run in series to increase yields. Twenty ml column for convenient preparative purification of GST fusion proteins, yielding 200–240 mg of fusion protein*. Glutathione Sepharose 4B, for batch purification and for packing small columns. Yields 8 mg/ml*. Glutathione Sepharose 4 Fast Flow, for packing high-performance columns for use with purification systems and scaling up. Yields 10–12 mg/ml*.
Detection method
Type of detection method
GST 96-Well Detection Module for ELISA. Uses 100 µl of sample/well. Ideal for screening expression systems and chromatographic fractions. Useful when amount of expressed protein is unknown or when increased sensitivity is required. Gives estimate of relative level of expression. GST Detection Module with CDNB enzymatic assay. Uses 5–50 µl of sample. Rapid assay; ideal for screening. Gives estimate of relative level of expression. Western blot using anti-GST antibody. Uses 5–10 µl of sample. Highly specific; detects only GST fusion protein. Little or no background detectable when using detection systems with optimized concentrations of secondary HRP-conjugated antibody. SDS-PAGE with Coomassie™ or silver staining. Uses 5–10 µl of sample. Provides information on size and percent purity. Detects fusion protein and contaminants.
Cleavage option
On-column or off-column
On-column cleavage is generally recommended since many potential contaminants can be washed out and the target protein eluted with a higher level of purity. Off-column cleavage is suggested if optimization of cleavage conditions is necessary.
Choice of protease
PreScission Protease: The GST tag can be removed and the protein purified in a single step on the column. Because the protease is maximally active at 4 °C, cleavage can be performed at low temperatures, thus improving stability of the target protein. Thrombin or Factor Xa sites can be cleaved either while the fusion protein is bound to the column or in solution after elution from the column. Either protease can be removed using Benzamidine Sepharose Fast Flow (high sub).
* Yield is protein dependent.
6
These topics are discussed in detail in the following chapters. The handbook includes procedures (Fig 1) and examples showing use of the GST system, as well as a troubleshooting guide and extensive appendices.
Clone gene or gene fragment into pGEX vector
Monitor and optimize expression of fusion protein
Culture recombinant cells (large-scale)
Harvest cells and lyse
Purify GST fusion protein
Detect and analyze GST fusion protein in the purified fraction
Fig 1. A typical protocol for expression and purification of GST fusion proteins. On- or off-column cleavage of the GST tag is an option.
Symbols used in this handbook This symbol indicates general advice that can improve procedures or provide recommendations for action under specific situations. This symbol denotes advice that should be regarded as mandatory and gives a warning when special care should be taken. chemicals, buffers, and equipment experimental protocol
7
8
Chapter 2 Cloning the gene or gene fragment into a pGEX expression vector pGEX vectors GST fusion proteins are constructed by inserting a gene or gene fragment into the multiple cloning site of one of the ten pGEX vectors. Expression is under the control of the tac promoter, which is induced by the lactose analog isopropyl b-D thiogalactoside (IPTG). All pGEX vectors are also engineered with an internal lacIq gene. The lacIq gene product is a repressor protein that binds to the operator region of the tac promoter, preventing expression until induction by IPTG, thus maintaining tight control over expression of the insert. In addition to offering chemically inducible, high-level expression, the vectors allow mild elution conditions for release of fusion proteins from the affinity medium. Thus, effects on antigenicity and functional activity of the protein are minimized. The vectors have a range of protease cleavage recognition sites as shown in Table 2. Table 2. Protease cleavage sites of pGEX vectors Vector
Cleaved by
pGEX-6P-1, pGEX-6P-2, pGEX-6P-3
PreScission Protease
pGEX-4T-1, pGEX-4T-2, pGEX-4T-3
Thrombin
pGEX-5X-1, pGEX-5X-2, pGEX-5X-3
Factor Xa
pGEX-2TK Allows detection of expressed proteins by direct labelling in vitro (12)
Thrombin
Collectively, the pGEX-P, pGEX-T, and pGEX-X series vectors provide all three translational reading frames beginning with the EcoR I restriction site (Fig 2). The same multiple cloning sites (MCS) in each vector ensure easy transfer of inserts. pGEX-6P-1, pGEX-4T-1, and pGEX-5X-1 can directly accept and express cDNA inserts isolated from lgt11 libraries. pGEX-2TK has a different MCS from that of the other vectors. pGEX-2TK is uniquely designed to allow the detection of expressed proteins by directly labelling the fusion products in vitro (12). This vector contains the recognition sequence for the catalytic subunit of cAMP-dependent protein kinase obtained from heart muscle. The protein kinase site is located between the thrombin recognition site and the MCS. Expressed proteins can be directly labelled using protein kinase and [g–32P]ATP and readily detected using standard radiometric or autoradiographic techniques. Refer to Appendix 2 for a listing of the control regions of the pGEX vectors. Complete DNA sequences and restriction site data are available at the Amersham Biosciences web site (http://www.amershambiosciences.com) and from GenBank™. GenBank accession numbers are listed in Appendix 2.
9
pGEX-2TK (27-4587-01) Thrombin
Kinase
Leu Val Pro Arg Gly Ser Arg Arg Ala Ser Val CTG GTT CCG CGT GGA TCT CGT CGT GCA TCT GTT GGA TCC CCG GGA ATT CAT CGT GAC TGA Stop codons BamH I Sma I EcoR I
pGEX-4T-1 (27-4580-01) Thrombin Leu Val Pro Arg Gly Ser Pro Glu Phe Pro Gly Arg Leu Glu Arg Pro His Arg Asp CTG GTT CCG CGT GGA TCC CCG GAA TTC CCG GGT CGA CTC GAG CGG CCG CAT CGT GAC TGA Stop codons EcoR I Sma I Sal I Xho I Not I BamH I
pGEX-4T-2 (27-4581-01) Thrombin Leu Val Pro Arg Gly Ser Pro Gly Ile Pro Gly Ser Thr Arg Ala Ala Ala Ser CTG GTT CCG CGT GGA TCC CCA GGA ATT CCC GGG TCG ACT CGA GCG GCC GCA TCG TGA Stop codon BamH I EcoR I Sma I Sal I Xho I Not I
pGEX-4T-3 (27-4583-01) Thrombin Leu Val Pro Arg Gly Ser Pro Asn Ser Arg Val Asp Ser Ser Gly Arg Ile Val Thr Asp CTG GTT CCG CGT GGA TCC CCG AAT TCC CGG GTC GAC TCG AGC GGC CGC ATC GTG ACT GAC TGA Stop codons BamH I EcoR I Sma I Sal I Xho I Not I
pGEX-5X-1 (27-4584-01) Factor Xa Ile Glu Gly Arg Gly Ile Pro Glu Phe Pro Gly Arg Leu Glu Arg Pro His Arg Asp ATC GAA GGT CGT GGG ATC CCC GAA TTC CCG GGT CGA CTC GAG CGG CCG CAT CGT GAC TGA Stop codons BamH I EcoR I Sma I Sal I Xho I Not I
pGEX-5X-2 (27-4585-01) Factor Xa Ile Glu Gly Arg Gly Ile Pro Gly Ile Pro Gly Ser Thr Arg Ala Ala Ala Ser ATC GAA GGT CGT GGG ATC CCC GGA ATT CCC GGG TCG ACT CGA GCG GCC GCA TCG TGA Stop codon BamH I Not I EcoR I Sma I Sal I Xho I
pGEX-5X-3 (27-4586-01) Factor Xa Ile Glu Gly Arg Gly Ile Pro Arg Asn Ser Arg Val Asp Ser Ser Gly Arg Ile Val Thr Asp ATC GAA GGT CGT GGG ATC CCC AGG AAT TCC CGG GTC GAC TCG AGC GGC CGC ATC GTG ACT GAC TGA Stop codons EcoR I Sma I Sal I Xho I BamH I Not I pGEX-6P-1 (27-4597-01) PreScission Protease Leu Glu Val Leu Phe Gln Gly Pro Leu Gly Ser Pro Glu Phe Pro Gly Arg Leu Glu Arg Pro His CTG GAA GTT CTG TTC CAG GGG CCC CTG GGA TCC CCG GAA TTC CCG GGT CGA CTC GAG CGG CCG CAT BamH I EcoR I Sma I Sal I Xho I Not I
pGEX-6P-2 (27-4598-01) PreScission Protease Leu Glu Val Leu Phe Gln Gly Pro Leu Gly Ser Pro Gly Ile Pro Gly Ser Thr Arg Ala Ala Ala Ser CTG GAA GTT CTG TTC CAG GGG CCC CTG GGA TCC CCA GGA ATT CCC GGG TCG ACT CGA GCG GCC GCA TCG Not I EcoR I Sma I Sal I Xho I BamH I
pGEX-6P-3 (27-4599-01) PreScission Protease Leu Glu Val Leu Phe Gln Gly Pro Leu Gly Ser Pro Asn Ser Arg Val Asp Ser Ser Gly Arg CTG GAA GTT CTG TTC CAG GGG CCC CTG GGA TCC CCG AAT TCC CGG GTC GAC TCG AGC GGC CGC EcoR I Sma I Sal I Xho I Not I BamH I Bal I ut
gl
Ptac
ase nsfer
Tth111 I Aat II
-tra
o
hi
at
S ne
Am
BspM I
p
r
pSj10 Bam7Stop7
Pst I
pGEX ~4900 bp
Nar I EcoR V
la
AlwN I
p4.5
c q I
BssH II Apa I BstE II Mlu I
pBR322 ori
Fig 2. Map of the glutathione S-transferase fusion vectors showing the reading frames and main features. See Appendix 2 for the control regions of the ten vectors.
10
Select the proper vector to match the reading frame of the cloned insert. pGEX-6P PreScission Protease vectors offer the most efficient method for cleavage and purification of GST fusion proteins. Site-specific cleavage is performed with simultaneous immobilization of the protease on the column. The protease has high activity at low temperature so that all steps can be performed in the cold room to protect the integrity of the target protein. Cleavage enzyme and GST tag are removed in a single step, as described in Chapter 6.
The host Although a wide variety of E. coli host strains can be used for cloning and expression with the pGEX vectors, there are specially engineered strains that are more suitable and that may maximize expression of full-length fusion proteins. Strains deficient in known cytoplasmic protease gene products, such as Lon, OmpT, DegP or HtpR, may aid in the expression of fusion proteins by minimizing the effects of proteolytic degradation by the host (13–16). Using E. coli strains that are not protease-deficient may result in proteolysis of the fusion protein, seen as multiple bands on polyacrylamide gels (SDS-PAGE) or Western blots. E. coli BL21, a strain defective in OmpT and Lon protease production, has been specifically selected to give high levels of expression of GST fusion proteins. It is the host of choice for GST fusion expression studies. Details on the genotype and handling of E. coli BL21 are found in Appendix 1. A lyophilized (noncompetent) culture of E. coli BL21 is supplied with all pGEX vectors and is also available separately. Use an alternative strain for cloning and maintenance of the vector (e.g. JM105) as BL21 does not transform well. However, do not use an E. coli strain carrying the recA1 allele for propagation of pGEX plasmids. There have been reports that these strains can cause rearrangements or deletions within plasmid DNA.
Insert DNA Insert DNA must possess an open reading frame and should be less than 2 kb long. Whether subcloned from another vector or amplified by PCR, the insert must have ends that are compatible with the linearized vector ends. Using two different restriction enzymes will allow for directional cloning of the insert into the vector. Directional cloning will optimize for inserts in the correct orientation.
Summary of procedures In the procedures below, the gene or gene fragment is cloned into the appropriate pGEX vector, and the host cells used for the cloning steps are transformed. The presence of the insert is verified, then a stock of DNA is prepared that can be used repeatedly in various procedures such as sequencing, mutagenesis, and cloning. Table 3 lists the procedures described in this chapter.
11
Table 3. Procedures for cloning the gene or gene fragment into a pGEX expression vector Procedure
Description
Comments
1
Restriction digestion of pGEX vectors
If digesting with two enzymes, consider gelpurifying the DNA before proceeding.
2
Dephosphorylation of linearized pGEX vector
Use recommended amount of enzyme so heat inactivation will be complete.
3
Ligation of insert to pGEX DNA
Using Ready-To-Go™ T4 DNA Ligase will reduce incubation time substantially.
4
Preparation of competent cells and transformation with pGEX DNA
Transform uncut pGEX DNA in parallel with recombinant DNA prepared above. Carry out all steps aseptically.
Screening using Ready-To-Go PCR Beads
Protocol uses the pGEX 5' and 3' Sequencing Primers. Ready-To-Go PCR Beads minimize pipetting steps.
Screening using standard PCR
Also uses the pGEX Sequencing Primers.
5
5.1
5.2 6
Small-scale isolation of pGEX DNA
Standard miniprep.
7
Large-scale isolation of pGEX DNA
Kit-based, but standard procedures also work well.
1. Restriction digestion of pGEX vectors Reagents required pGEX DNA 10× One-Phor-All Buffer PLUS (OPA+): 100 mM Tris acetate, 100 mM magnesium acetate, 500 mM potassium acetate, pH 7.5 (optional) Restriction enzyme
Many restriction enzymes are compatible with OPA+ (see the Amersham Biosciences catalog for details), and its recipe is provided here as a convenience. The buffer is also recommended for use in the dephosphorylation and ligation procedures that follow. Steps 1. Prepare the following reaction mixture. Volumes may vary depending on the amount of pGEX DNA to be digested. We recommend a final DNA concentration in the reaction mixture of 0.1 µg/µl. 5 µg of pGEX DNA 5–10 µl of 10× One-Phor-All Buffer PLUS (OPA+) or buffer supplied with enzyme 5–10 µl of optional components (e.g. BSA, Triton™ X-100, NaCl, etc.) 10–25 units of restriction enzyme H2O to 50 µl 2. Incubate at the appropriate temperature for 2–16 h. 3. Examine a small aliquot of the reaction by agarose gel electrophoresis to verify that the pGEX DNA has been digested to completion. 4. If digestion with a second enzyme is required, adjust the concentration of OPA+ and/or additional components, and the reaction volume as appropriate, add new enzyme, and continue incubation. 5. Monitor the progress of the digestion as in step 3.
Be alert for incomplete or failed double digestion. Continue digestion if necessary. 6. Dephosphorylate the pGEX DNA with an alkaline phosphatase if it is to be used following digestion with a single restriction enzyme (Procedure 2). If using OPA+, dephosphorylation can be performed in the same tube immediately following digestion.
12
If the pGEX DNA was digested with two restriction enzymes, consider agarose-gelpurifying the linearized vector prior to dephosphorylation. This can be conveniently accomplished with Sephaglas™ BandPrep Kit.
2. Dephosphorylation of linearized pGEX vector Reagents required Calf intestinal alkaline phosphatase 10× One-Phor-All Buffer PLUS (OPA+): 100 mM Tris acetate, 100 mM magnesium acetate, 500 mM potassium acetate, pH 7.5 Phenol: Redistilled phenol saturated with TE buffer containing 8-hydroxy quinoline (17) Chloroform/isoamyl alcohol: Reagent-grade chloroform and isoamyl alcohol, mixed 24:1 3 M sodium acetate, pH 5.4, aqueous solution Ethanol (70%, 95%) TE buffer: 10 mM Tris-HCl, pH 8.0, 1 mM EDTA
Steps 1. Dilute sufficient calf intestinal alkaline phosphatase for all dephosphorylations to be performed. When diluted, 1–2 µl should provide 0.1 unit to the reaction. For dilution, use 10× OPA+ and H2O to give a final buffer concentration of 1× OPA+. 2. Add 0.1 unit (1–2 µl of diluted enzyme) of alkaline phosphatase to the digested pGEX DNA and incubate for 30 min at 37 °C.
In radiolabel and transformation studies, dephosphorylation appears complete within 5 min when using 0.5× or 1× OPA+. When 2× OPA+ is used, an incubation period of 15–30 min is required for complete dephosphorylation. 3. Heat inactivate the alkaline phosphatase at 85 °C for 15 min.
Heat inactivation is complete for concentrations of alkaline phosphatase of 0.1 unit or less, but is not effective for concentrations greater than 1 unit. 4. Add an equal volume of phenol to the aqueous sample. Vortex for 1 min and centrifuge for 5 min at full speed to separate the phases. 5. Transfer the upper aqueous phase to a fresh tube and add an equal volume of chloroform/isoamyl alcohol. Vortex for 1 min, then centrifuge for 5 min at full speed to separate the phases. 6. Transfer the upper aqueous phase to a fresh tube and add 0.1 volume of 3 M sodium acetate, pH 5.4 and 2.5 volumes of 95% ethanol. Mix and place at -20 °C for 15 min. 7. Centrifuge at 4 °C for 15 min, remove the supernatant, and wash the pellet with 1 ml of 70% ethanol. 8. Recentrifuge for 2 min, drain thoroughly, and either air-dry the DNA pellet or dry it under vacuum. 9. Dissolve the DNA pellet in 10–20 µl of TE buffer.
pGEX DNA can be stored at -20 °C for later use. Avoid repeated freezing and thawing.
13
3. Ligation of insert to pGEX DNA Ready-To-Go T4 DNA Ligase can be used to achieve ligations in 30–45 min. An alternate procedure is described below. Reagents required Insert DNA ATP, 100 mM 10× One-Phor-All Buffer PLUS (OPA+): 100 mM Tris acetate, 100 mM magnesium acetate, 500 mM potassium acetate, pH 7.5 T4 DNA ligase
Steps
• The linearized pGEX DNA and insert DNA should be present at a vector to insert ratio of 1:5 moles of ends. The moles of ends of linear DNA can be calculated with the following formula: moles of ends = 2 × (g of DNA)/[(# of bp) × (649 Daltons/bp)] Example: 100 ng of pGEX DNA (0.06 pmol of ends) would require 100 ng of a 1 kb insert (0.3 pmol of ends).
• For ligation of cohesive ends, the final reaction mix should contain 1 mM ATP (diluted) and 0.5–5 units of T4 DNA ligase, and should be incubated for 1–4 h at 10 °C. • For ligation of blunt ends, the final reaction mix should contain 0.1–1 mM ATP (diluted) and 10–15 units of T4 DNA ligase, and should be incubated for 2–16 h at 4–16 °C. 1. Based upon the above considerations, prepare the following reaction mixture specific for your application: 1–5 µl of linearized pGEX DNA 1–5 µl of insert DNA 2 µl of 10× One-Phor-All Buffer PLUS (OPA+) 0.2 µl of 100 mM ATP 0.5–15 units of T4 DNA ligase H2O to 20 µl 2. Incubate for either 1–4 h at 10 °C (cohesive ends) or 2–16 h at 4–16 °C (blunt ends). 3. Terminate the reaction by heating at 65 °C for 10 min.
The ligation reaction can be used directly to transform competent cells. Otherwise, it can be stored at -20 °C until needed.
4. Preparation of competent cells and transformation with pGEX DNA In these procedures, E. coli host cells are made competent and then transformed with either uncut pGEX DNA or recombinant pGEX DNA. If electroporation is used to transform the cells, see Appendix 3. Otherwise, proceed as described below. Transform 1 ng of uncut (supercoiled) vector DNA in parallel with recombinant pGEX ligations to determine the efficiency of each competent cell preparation. This protocol is based on the procedure of Chung et al. (18).
14
All steps in this procedure should be carried out aseptically. Reagents required Use double-distilled H2O for preparation of all solutions. Glycerol stock of E. coli host strain LB medium and LB medium plates (prepared fresh): Combine 10 g tryptone, 5 g yeast extract, and 10 g NaCl in 900 ml H2O. Stir to dissolve, and adjust volume to 1 l. Sterilize by autoclaving. To prepare as a solid medium, add 1.2–1.5% agar. TSS (transformation and storage solution) (ice-cold): For 100 ml: combine 1.0 g tryptone, 0.5 g yeast extract, 0.5 g NaCl, 10.0 g polyethylene glycol (Mr 3350), 5.0 ml dimethylsulfoxide (DMSO), and 5.0 ml MgCl2 (1 M) in 70 ml of sterile distilled H2O. Stir until dissolved. Adjust the pH to 6.5 with HCl or NaOH. Adjust to 100 ml with sterile distilled H2O. Sterilize by filtering through a 0.2 µm filter. Store at 4 °C. Stable for up to 6 months. LBG medium (LB + 20 mM glucose): Dissolve 10 g tryptone, 5 g yeast extract, and 5 g NaCl in 900 ml of distilled H2O. Sterilize by autoclaving. After the medium has cooled to 50–60 °C, add 10 ml of sterile 2 M glucose. Adjust to 1 l with sterile distilled H 2O. To prepare as a solid medium, add 1.2–1.5% agar. LBAG medium and plates (LBG + 100 µg/ml ampicillin): See recipe for LBG medium, above. After autoclaving, cool the medium to 50 °C, then aseptically add 1 ml of a 100 mg/ml ampicillin stock solution (final concentration 100 µg/ml). To prepare as a solid medium, add 1.2–1.5 % agar. Ampicillin stock solution: Dissolve 400 mg of the sodium salt of ampicillin in 4 ml of H 2O. Sterilize by filtration and store in small aliquots at -20 °C. Glycerol: 80% in sterile distilled H2O
Steps Preparation of competent cells 1. Using sterile technique, streak an E. coli host strain (e.g. JM105, BL21, etc.) from a glycerol stock onto an LB medium plate. Incubate overnight at 37 °C. 2. Isolate a single colony and inoculate 50–100 ml of LB broth. Incubate at 37 °C with shaking at 250 rpm. Grow cells to an A600 of 0.4–0.5.
It is critical that the absorbance is not more than 0.5. This will take approximately 3–6 h. Pre-warming the broth to 37 °C will shorten the growth time. 3.
Sediment the cells at approximately 2500 × g for 15 min at 4 °C, then gently resuspend in 1/10 volume (5–10 ml) of ice-cold TSS and place on ice.
Cells must be used for transformations within 2–3 h. Transformation of competent cells 1. For each ligation reaction, as well as for the uncut vector control and the negative control (untransformed competent E. coli host cells), add 1 ml of freshly prepared competent E. coli host cells to separate pre-chilled 50 ml sterile disposable centrifuge tubes. Store on ice. 2. Add 20 µl of each ligation reaction or 1 ng of uncut vector to the competent cells, swirl gently to mix, and place on ice for 45 min. Do not add any DNA to the negative control but instead add 20 µl of sterile distilled H2O. 3. Incubate the tubes in a 42 °C water bath for 2 min, then chill briefly on ice. 4. For each sample, immediately transfer 100 µl of the transformed cells to a 17 × 100 mm tube (Falcon) containing 900 µl of LBG medium (pre-warmed to 37 °C) and incubate for 1 h at 37 °C with shaking (250 rpm).
15
5. Plate 100 µl of the diluted, transformed cells from the ligated samples and 10 µl of the diluted, transformed cells from the uncut vector sample onto separate LBAG plates. Also plate 100 µl of the untransformed, competent E. coli host cells. Incubate the plates at 37 °C overnight, then proceed to screening using Procedure 5. 6. To prepare a frozen stock culture, add 100 µl of the diluted, transformed cells containing the pGEX DNA to 1 ml of LBAG medium and incubate for 30 min at 37 °C with shaking at 250 rpm. After incubation, add 200 µl of sterile 80% glycerol and mix with a pipet tip. Store at -70 °C.
5. Screening The pGEX 5' and 3' Sequencing Primers can be used in the rapid screening of transformants by PCR, in conjunction with Ready-To-Go PCR Beads (Procedure 5.1) or in standard PCR (Procedure 5.2). Screening is needed to verify that the insert is in the proper orientation and the correct junctions are present such that the reading frame is maintained. 5.1. Screening using Ready-To-Go PCR Beads Reagents required Ready-To-Go PCR Beads pGEX 5' Sequencing Primer (5 pmol/µl) pGEX 3' Sequencing Primer (5 pmol/µl)
Steps 1. Resuspend a bead in 25 µl of H2O as per standard instructions. 2. Add 10 pmol each of pGEX 5' and 3' Sequencing Primers to the resuspended bead. 3. Gently touch a sterile micropipet tip to the bacterial colony to be screened and then transfer to the resuspended PCR bead. Pipet gently to disperse bacterial cells.
Avoid transferring too much of the bacterial colony. Results are better when cell numbers are low. Streak some of the bacteria remaining on the micropipet tip onto an LB medium grid plate as a source for Procedures 6 and 7. 4. Overlay the reaction mixture with 50 µl of mineral oil. 5. Amplify in a thermal cycler with the following cycle parameters: 35 cycles: 95 °C for 1 min 58 °C for 1 min 72 °C for 2 min 6. Transfer the aqueous phase from under the oil layer to a clean tube. Analyze 10–20 µl by agarose gel electrophoresis.
5.2. Screening using standard PCR Reagents required Taq DNA polymerase at 5 U/µl 10× Taq buffer as recommended by supplier
16
dNTP mix: For each reaction, add 0.2 µl each of 100 mM dATP, 100 mM dCTP, 100 mM dGTP, and 100 mM dTTP to 15.2 µl of H2O for a final concentration of 0.2 mM in a 100 µl reaction. pGEX 5' Sequencing Primer (5 pmol/µl) pGEX 3' Sequencing Primer (5 pmol/µl)
Steps 1. Mix the following components in a 0.65 ml tube: 10 µl of 10× Taq polymerase buffer 16 µl of dNTP mix 5 µl of pGEX 5' Sequencing Primer 5 µl of pGEX 3' Sequencing Primer H2O to 99.5 µl 2. Gently touch a sterile micropipet tip to the bacterial colony to be screened and transfer to the above PCR mixture. Pipet gently to disperse bacterial cells.
Avoid transferring too much of the bacterial colony. Results are better when cell numbers are low. Streak some of the bacteria remaining on the micropipet tip onto an LB medium grid plate as a source for Procedures 6 and 7. 3. Add 0.5 µl of 5 U/µl Taq DNA polymerase. 4. Overlay the reaction mixture with 50 µl of mineral oil. 5. Amplify in a thermal cycler with the following cycle parameters: 25–35 cycles: 94 °C for 1 min 55 °C for 1 min 72 °C for 2 min 6. Transfer the aqueous phase from under the oil layer to a clean tube. Analyze 20–40 µl by agarose gel electrophoresis.
6. Small-scale isolation of pGEX DNA Rapid and phenol-free isolation of plasmid DNA is greatly simplified by the use of FlexiPrep™ Kit or GFX™ Micro Plasmid Prep Kit. An alternate procedure is described below. Reagents required Solution I: 100 mM Tris-HCl, pH 7.5, 10 mM EDTA, 400 µg of heat-treated RNase I per ml of Solution I Solution II: 0.2 M NaOH, 1% (w/v) SDS Solution III: 3 M potassium, 5 M acetate. To prepare 100 ml, mix 60 ml of 5 M potassium acetate, 11.5 ml of glacial acetic acid, and 28.5 ml of distilled H2O. Isopropanol Phenol: Redistilled phenol saturated with TE buffer containing 8-hydroxy quinoline (17) Chloroform/isoamyl alcohol: Reagent-grade chloroform and isoamyl alcohol, mixed 24:1 Phenol/chloroform: Equal parts of redistilled phenol and chloroform/isoamyl alcohol (24:1), each prepared as described above 3 M sodium acetate, pH 5.4, aqueous solution Ethanol (70%, 95%) TE buffer: 10 mM Tris-HCl, pH 8.0, 1 mM EDTA 17
Steps 1. Transfer 1.5 ml of an overnight culture of E. coli to a microcentrifuge tube and centrifuge at full speed for 30 s to pellet the cells. 2. Remove the supernatant by aspiration without disturbing the cell pellet, leaving the pellet as dry as possible. 3. Resuspend the pellet in 200 µl of solution I by vigorously vortexing. 4. Add 200 µl of solution II and mix by inverting the tube several times. Incubate at room temperature for 5 min. 5. Add 200 µl of solution III and mix by inverting the tube several times. Place on ice for 5 min. 6. Centrifuge at full speed for 5 min at room temperature. 7. Carefully decant the supernatant into a clean centrifuge tube. 8. Add 420 µl (0.7 volume) of ambient-temperature isopropanol to the supernatant and vortex to mix. Incubate for 5 min at room temperature. 9. Centrifuge at full speed for 10 min. Decant the supernatant and invert the tube to drain. 10. Resuspend the DNA pellet in 200 µl of TE buffer by vortexing. 11. Add 200 µl of phenol to the aqueous sample. Vortex for 1 min and centrifuge for 5 min at full speed to separate the phases. 12. Transfer the upper aqueous phase to a fresh tube and add 200 µl of chloroform/isoamyl alcohol. Vortex for 1 min, then centrifuge for 5 min at full speed to separate the phases. 13. Transfer the upper aqueous phase to a fresh tube and add 20 µl of 3 M sodium acetate and 500 µl of 95% ethanol. Mix and place at -20 °C for 15 min. 14. Centrifuge at 4 °C for 15 min, remove the supernatant, and wash the pellet with 1 ml of 70% ethanol. 15. Recentrifuge for 2 min, drain thoroughly, and air-dry the DNA pellet or dry it under vacuum. 16. Dissolve the DNA pellet in 20 µl of TE buffer.
pGEX DNA can be stored at -20 °C for later use. Avoid repeated freezing and thawing.
7. Large-scale isolation of pGEX DNA Rapid, large-scale isolation of plasmid DNA from cultures up to 500 ml is greatly simplified by the use of FlexiPrep Kit. Reagents required 2× YTA medium (2× YT + 100 µg/ml ampicillin): Prepare 2× YT medium by dissolving 16 g tryptone, 10 g yeast extract, and 5 g NaCl in 900 ml of distilled H2O. Adjust the pH to 7.0 with NaOH. Adjust the volume to 1 l with distilled H2O. Sterilize by autoclaving for 20 min. After autoclaving, cool the medium to 50 °C, then aseptically add 1 ml of a 100 mg/ml ampicillin stock solution (final concentration 100 µg/ml).
Steps 1. Grow an appropriate volume of pGEX-containing E. coli in 2× YTA medium overnight. 2. Dilute an inoculum of the overnight culture at least 1:100 into the desired volume of the same medium prewarmed to the growth temperature. 3. Grow with aeration to an A 600 of 1–2. 4. Isolate plasmid DNA using FlexiPrep Kit, or protocols from reference 17.
18
Chapter 3 Monitoring expression, optimizing growth, and preparing large-scale cultures pGEX vectors carry the lacIq gene, so there are no specific host requirements for propagation of the plasmids or for expression of fusion proteins. As previously noted, E. coli BL21 does not transform well, and an alternate strain (e.g. JM105) is recommended for maintenance of the plasmid. For all expression studies, however, BL21 is the strain of choice. Once it has been established that the insert is in the proper orientation and the correct junctions are present (Chapter 2), the next step is to optimize fusion protein expression. Key to this step is the capability to screen crude lysates from many clones so that optimal expression levels and growth conditions can be readily determined. Once conditions are established, the researcher is ready to prepare large-scale bacterial sonicates of the desired clones. Various methods for the purification of fusion proteins are available. In this chapter, the focus is on obtaining relatively small samples quickly, to permit the screening of many putative clones simultaneously. To this end, we recommend two purification methods for initial screening. In the first method, a crude lysate suitable for screening from 2–3 ml of culture is prepared, using a batch purification method with Glutathione Sepharose 4B. The second method uses the GST MicroSpin Purification Module, which can isolate protein from up to 12 ml of culture. Procedures using the MicroSpin Purification Module can be performed using a standard microcentrifuge or in conjunction with MicroPlex 24 Vacuum. With this latter method, fusion protein can be purified from up to 48 samples in less than 1 h. The batch method with Glutathione Sepharose 4B is presented in this chapter. Procedures for use of the GST MicroSpin Purification Module are presented in Chapter 4. Various detection methods are also available for screening lysates for expression of GST fusion proteins. SDS-PAGE is described below. More information and detailed procedures for several other methods can be found in Chapter 5. After clones expressing the fusion protein have been selected, growth conditions should be evaluated for optimal expression. Media, growth temperature, culture density, induction conditions, and other variables should be evaluated. It is important to assure sufficient aeration and to minimize the time spent in each stage of growth, as well as to use positive selection for the plasmid (antibiotic resistance). The presence of inclusion bodies may affect optimization of expression, and is discussed in detail below. Monitor both cell density (A600) and protein expression at each step. Yield of fusion protein is highly variable, depending on the nature of the fusion protein, the host cell, and the culture conditions. Fusion protein yields can range up to 10 mg/l (19). Table 4 can be used to approximate culture volumes based on an average yield of 2.5 mg/l. Table 4. Estimate of culture volume based on average yield Fusion protein yield Culture volume Volume of sonicate
12.5 µg
50 µg
1 mg
10 mg
5 ml
20 ml
400 ml
4l
50 mg 20 l
0.5 ml
1 ml
20 ml
200 ml
1000 ml
19
Summary of procedures This chapter includes a simple procedure for preparing crude lysates for initial screening and a procedure for preparing a large-scale bacterial sonicate (see Table 5). Refer to Chapter 4 for additional purification options and to Chapter 5 for additional detection options. Table 5. Procedures for screening and preparing cultures of fusion proteins Procedure
Description
Comments
8
Screening pGEX recombinants for fusion protein expression
Prepare sonicate from 2 to 3 ml of culture; use SDSPAGE for detection of fusion protein.
9
Preparation of large-scale bacterial sonicates
Prepare sonicate from 0.2 to 10 l of culture, then proceed to a purification method in Chapter 4.
Optimizing for soluble expression versus working with inclusion bodies High-level expression of foreign fusion proteins in E. coli often results in formation of inclusion bodies. Inclusion bodies comprise dense, insoluble aggregates that are failed folding intermediates (20, 21). Formation of inclusion bodies can be advantageous in purifying an active form of an expressed fusion protein that otherwise may be unstable in the soluble fraction. However, the steps needed to solubilize and refold the fusion protein can be highly variable and may not always result in high yields of active protein. The advantages and disadvantages of inclusion bodies are summarized in Table 6. Table 6. Advantages and disadvantages of inclusion bodies Advantages
Disadvantages
High expression levels can reduce fermentation costs.
Steps to refold the protein shift difficulties and costs downstream.
Expression is easily monitored by SDS-PAGE or immunoblotting.
Expression cannot be monitored directly by functional assays.
Inclusion bodies can be isolated to high purity and used directly as antigen.
Minor contaminants are often hydrophobic, poorly soluble membrane proteins and cell wall fragments.
Fusion proteins are generally protected from proteolytic breakdown.
Major contaminants are oligomers and misfolded or proteolyzed forms of the protein that can be difficult to separate.
Small fusions present in inclusion bodies refold with good efficiency.
If the protein does not refold well, another expression system will be needed.
If the presence of inclusion bodies is not deemed a deterrent at this point, the researcher can proceed to optimizing growth conditions and preparing large-scale bacterial sonicates of the fusion-producing cells (Procedure 9 below). If, on the other hand, the presence of inclusion bodies is deemed a deterrent, there are two options to consider: • Optimize as much as possible for soluble expression. • Accept the formation of inclusion bodies but develop strategies to solubilize and refold the protein. A variety of growth parameters can be investigated, either solely or in combination, that may provide a good yield of non-degraded fusion protein in the soluble fraction. Steps to investigate include: 20
• Lowering the growth temperature to between 20 °C and 30 °C. • Increasing aeration. • Altering induction conditions. In general, induction at lower cell densities (A600 = 0.5) usually results in greater yields of the fusion protein in a soluble form. However, in some cases it may be beneficial to grow the cells to a higher cell density (> 1 A600 unit) for a shorter period of time, or simply to induce for a shorter period of time. Growing the cells to a higher cell density and either omitting induction by IPTG or reducing the IPTG concentration to 0.1 mM leads to lower yields, but more of the fusion protein is likely to be obtained in an intact form. Although not limited to discussion of the GST protein fusion system, the chapter on prokaryotic expression in reference 22 provides an excellent discussion of many aspects of working with expression systems. If the plasmid has been propagated in a host other than E. coli strain BL21, it should be transferred into BL21 for the expression of fusion protein, using Procedure 4 from Chapter 2. Retain small samples at key steps, including before induction, at various times after induction, and during the purification steps, to analyze growth and purification methods. Once analysis is complete, prepare glycerol stocks of the positive clones and store at -80 °C.
8. Screening pGEX recombinants for fusion protein expression Sections of this procedure have been adapted with permission from Current Protocols in Molecular Biology, Vol. 2, Supplement 10, Unit 16.7. Copyright © 1990 by Current Protocols. The following steps may be used prior to large-scale purification to check clones for expression of the desired fusion protein. Due to the small scale of the screening process (~ 5 µg of fusion protein), affinity purification should only be performed in a batch method using a Glutathione Sepharose 4B bed volume of 10 µl. Reagents required Preparation of the medium: Bulk Glutathione Sepharose 4B prepared to 50% slurry as described below in the procedural steps. 1× PBS (ice-cold): Dilute 10× PBS with sterile H2O. Store at 4 °C. 10× PBS is 1.4 M NaCl, 27 mM KCl, 100 mM Na2HPO4, 18 mM KH 2PO4, pH 7.3. Preparation of lysate: 2× YTA medium (2× YT + 100 µg/ml ampicillin): Prepare 2× YT medium by dissolving 16 g tryptone, 10 g yeast extract, and 5 g NaCl in 900 ml of distilled H2O. Adjust the pH to 7.0 with NaOH. Adjust the volume to 1 l with distilled H2O. Sterilize by autoclaving for 20 min. After autoclaving, cool the medium to 50 °C, then aseptically add 1 ml of a 100 mg/ml ampicillin stock solution (final concentration 100 µg/ml). 100 mM IPTG: Dissolve 500 mg of isopropyl-b-D-thiogalactoside (IPTG) in 20 ml of distilled H2O. Filter-sterilize and store in small aliquots at -20 °C. Elution buffer: 50 mM Tris-HCl, 10 mM reduced glutathione, pH 8.0. Dispense in 1–10 ml aliquots and store at -20 °C until needed. Avoid more than five freeze/thaw cycles. SDS-PAGE analysis: 6× loading buffer: 0.35 M Tris-HCl (pH 6.8), 10.28% (w/v) SDS, 36% (v/v) glycerol, 0.6 M dithiothreitol (or 5% b-mercaptoethanol), 0.012% (w/v) bromophenol blue. Store in 0.5 ml aliquots at -70 °C.
21
Steps Preparation of the medium
Glutathione Sepharose 4B as supplied is approximately a 75% slurry. The following procedure results in a 50% slurry. Based on the bed volume requirements, dispense 1.33 ml of the original Glutathione Sepharose 4B slurry per ml of bed volume required. 1. Determine the amount of Glutathione Sepharose 4B required.
Although only 10 µl of prepared slurry is needed for each screening analysis, additional slurry should be prepared if it will also be used for larger-scale purification procedures (see Procedures 13.1 and 13.2 in Chapter 4). 2. Gently shake the bottle of Glutathione Sepharose 4B to resuspend the medium. 3. Use a pipet with a wide-bore tip to remove sufficient slurry for use and transfer the slurry to an appropriate container/tube. 4. Sediment the medium by centrifuging at 500 × g for 5 min. Carefully decant the supernatant. 5. Wash the Glutathione Sepharose 4B by adding 10 ml of cold (4 °C) 1× PBS per 1.33 ml of the original slurry of Glutathione Sepharose 4B dispensed. Invert to mix.
Glutathione Sepharose 4B must be thoroughly washed with 1× PBS to remove the 20% ethanol storage solution. Residual ethanol may interfere with subsequent procedures. 6. Sediment the medium by centrifuging at 500 × g for 5 min. Decant the supernatant. 7. For each 1.33 ml of the original slurry of Glutathione Sepharose 4B dispensed, add 1 ml of 1× PBS. This results in a 50% slurry. Mix well prior to subsequent pipetting steps.
Preparation of lysate 1. Pick and transfer several colonies of E. coli transformed with the pGEX recombinants into separate tubes containing 2 ml of 2× YTA medium.
For comparison, it is advisable to inoculate a control tube with bacteria transformed with the parental pGEX plasmid. 2. Grow liquid cultures to an A600 of 0.6–0.8 (3–5 h) with vigorous agitation at 30–37 °C.
Lower temperatures, even as low as 20 °C, may be used if inclusion bodies are problematic (see Troubleshooting, page 84). 3. Induce fusion protein expression by adding 2 µl of 100 mM IPTG (final concentration 0.1 mM).
A higher concentration (up to 1 mM IPTG) may be used at this screening stage. 4. Continue incubation for an additional 1–2 h. 5. Transfer 1.5 ml of the liquid cultures to labelled microcentrifuge tubes. 6. Centrifuge in a microcentrifuge for 5 s and discard the supernatants. 7. Resuspend each pellet in 300 µl of ice-cold 1× PBS. Transfer 10 µl of each cell suspension into separate labelled tubes (for later use in SDS-PAGE analysis).
Except where noted, keep all samples and tubes on ice.
22
8. Lyse the cells using a sonicator equipped with an appropriate probe.
Lysis is complete when the cloudy cell suspension becomes translucent. The frequency and intensity of sonication should be adjusted such that complete lysis occurs in 10 s, without frothing (frothing may denature proteins). Keep on ice. Crude sonicates can be screened for the relative level of expression of GST fusion proteins using the GST substrate CDNB (1-chloro-2,4-dinitrobenzene). See Procedure 16, Chapter 5. 9. Centrifuge the lysate in a microcentrifuge for 5 min to remove insoluble material. Save a 10 µl aliquot of the insoluble material for analysis by SDS-PAGE. Transfer the supernatants to fresh tubes. 10. Add 20 µl of a 50% slurry of Glutathione Sepharose 4B (prepared as described above) to each supernatant and mix gently for 5 min at room temperature. 11. Add 100 µl of 1× PBS, vortex briefly, and centrifuge for 5 s to sediment the Glutathione Sepharose 4B beads. 12. Discard the supernatants. Repeat this 1× PBS wash twice for a total of three washes. 13. Elute the fusion protein by adding 10 µl of elution buffer. Suspend the Glutathione Sepharose 4B beads and incubate at room temperature for 5 min. 14. Centrifuge in a microcentrifuge for 5 min to sediment the Glutathione Sepharose 4B beads, then transfer the supernatants to fresh tubes.
SDS-PAGE analysis 1. Transfer 10 µl of each supernatant from step 14 (above) to fresh tubes. 2. To these aliquots, and to the 10 µl samples retained following steps 7 and 9 (above), add 2 µl of 6× SDS loading buffer. 3. Vortex briefly and heat for 5 min at 90–100 °C. 4. Load the samples onto a 10% or 12.5% SDS-polyacrylamide gel. 5. Run the gel for the appropriate length of time and stain with Coomassie blue or silver stain to visualize the parental GST (made in control cells carrying the parental pGEX vector) and the fusion protein.
Transformants expressing the desired fusion protein will be identified by the absence from total cellular proteins of the parental GST and by the presence of a novel, larger fusion protein. Parental pGEX vectors produce a Mr 29 000 GST fusion protein containing amino acids coded for by the pGEX multiple cloning site.
9. Preparation of large-scale bacterial sonicates Reagents required 2× YTA medium (2× YT + 100 µg/ml ampicillin): Prepare 2× YT medium by dissolving 16 g tryptone, 10 g yeast extract, and 5 g NaCl in 900 ml of distilled H2O. Adjust the pH to 7.0 with NaOH. Adjust the volume to 1 l with distilled H2O. Sterilize by autoclaving for 20 min. After autoclaving, cool the medium to 50 °C, then aseptically add 1 ml of a 100 mg/ml ampicillin stock solution (final concentration 100 µg/ml). 100 mM IPTG: Dissolve 500 mg of isopropyl-b-D-thiogalactoside (IPTG) in 20 ml of distilled H2O. Filter-sterilize and store in small aliquots at -20 °C. 1× PBS (ice-cold): Dilute 10× PBS with sterile H2O. Store at 4 °C. 10× PBS is 1.4 M NaCl, 27 mM KCl, 100 mM Na2HPO4, 18 mM KH 2PO4, pH 7.3. 20% Triton X-100
23
Steps 1. Use a single colony of E. coli cells containing a recombinant pGEX plasmid to inoculate 2–100 ml of 2× YTA medium. 2. Incubate for 12–15 h at 37 °C with vigorous shaking. 3. Dilute the culture 1:100 into fresh pre-warmed 2× YTA medium, and grow at 30–37 °C with shaking until the A600 reaches 0.5–2.
Lower temperatures, even as low as 20 °C, may be used if inclusion bodies are problematic (see Troubleshooting, page 84). To ensure adequate aeration, fill flasks to only 20–25% capacity (e.g. 20 ml in a 100 ml flask). Optimize the growth temperature and A600 for induction as these will vary with each fusion protein. 4. Add 100 mM IPTG to a final concentration of 0.1–1.0 mM and continue incubation for an additional 2–6 h. The optimal concentration can only be determined empirically. 5. Transfer the culture to appropriate centrifuge containers and centrifuge at 7700 × g (e.g. 8000 rpm in a Beckman JA20 rotor) for 10 min at 4 °C to sediment the cells. 6. Discard the supernatant and drain the pellet. Place on ice. 7. Using a pipet, completely suspend the cell pellet by adding 50 µl of ice-cold 1× PBS per ml of culture. 8. Disrupt the suspended cells using an appropriately equipped sonicator for the suspended volume. Sonicate on ice in short bursts.
Save an aliquot of the sonicate for analysis by SDS-PAGE as described in Procedure 8 above. Cell disruption is evidenced by partial clearing of the suspension or may be checked by microscopic examination. Avoid frothing as this may denature the fusion protein. Oversonication can also lead to co-purification of host proteins with the GST fusion protein. Detection of GST activity can be performed at this stage using one of the methods described in Chapter 5. 9. Add 20% Triton X-100 to a final concentration of 1%. Mix gently for 30 min to aid in solubilization of the fusion protein. 10. Centrifuge at 12 000 × g (e.g. 10 000 rpm in a Beckman JA20 rotor) for 10 min at 4 °C. Transfer the supernatant to a fresh container. Save aliquots of the supernatant and the cell debris pellet for analysis by SDS-PAGE as described in Procedure 8. These samples can be used to identify the fraction in which the fusion protein is located.
Analyze the aliquots as soon as possible; the longer they remain at 4 °C, the greater the risk of proteolysis. 11. Proceed with one of the purification procedures detailed in Chapter 4.
24
Chapter 4 Purification of GST fusion proteins GST fusion proteins are easily purified from bacterial lysates by affinity chromatography using glutathione immobilized to a matrix such as Sepharose (Fig 3). When applied to the affinity medium, fusion proteins bind to the ligand, and impurities are removed by washing with binding buffer. Fusion proteins are then eluted from the Glutathione Sepharose under mild, non-denaturing conditions that preserve both protein antigenicity and function. If separation of the cloned protein from the GST affinity tag is desired, the fusion protein can be digested with an appropriate site-specific protease while the fusion protein is bound to Glutathione Sepharose. Alternatively, the fusion protein can be digested following elution from the medium (see Chapter 6 for both of these alternatives). Cleavage of the bound fusion protein eliminates the extra step of separating the released protein from GST because the GST moiety remains bound to the medium while the cloned protein is eluted using wash buffer (23).
O
O C
H
N C
CH 2
CH 2 O
N
C H
O
S
OH
O
H
C NH 3 +
C O
O
Fig 3. Terminal structure of Glutathione Sepharose. Glutathione is attached to Sepharose by coupling to the oxirane group using epoxy-activation. The structure of glutathione is complementary to the glutathione S-transferase binding site.
Selecting an affinity chromatography product Products designed to meet specific purification needs are available for purification of GST fusion proteins, as shown in the selection guide in Table 7. All of these products rely on affinity chromatography using gravity flow, centrifugation, vacuum, syringe, or pump action to purify the protein. A comparison of the physical characteristics of Glutathione Sepharose 4B and Glutathione Sepharose 4 Fast Flow is given in Table 8.
25
Table 7. Selection guide summarizing purification options for GST fusion proteins Prepacked column or bulk media
Amount of fusion protein for a single purification
Comments
MicroSpin GST Purification Module
Up to 400 µg
Prepacked columns, buffers, and chemicals that are ready to use. High throughput when used with MicroPlex 24 Vacuum (up to 48 samples simultaneously).
GSTrap FF 1 ml and 5 ml columns
1 ml columns: 10–12 mg 5 ml columns: 50–60 mg
Prepacked columns ready to use with either a syringe, a pump, or a chromatographic system. May be run in series to increase yields.
GSTPrep FF 16/10 column
200–240 mg
Prepacked column, ready to use. For convenient preparative purifications.
Glutathione Sepharose 4B
8 mg/ml
For batch purification and for packing small columns.
Glutathione Sepharose 4 Fast Flow
10–12 mg/ml
For packing high-performance columns for use with purification systems and scaling up.
Packed with Glutathione Sepharose 4 Fast Flow.
Packed with Glutathione Sepharose 4 Fast Flow.
Table 8. Comparison of Glutathione Sepharose 4B and Glutathione Sepharose 4 Fast Flow Physical characteristics
Glutathione Sepharose 4B
Ligand
Glutathione and 10-carbon linker arm
Glutathione Sepharose 4 Fast Flow Glutathione and 10-carbon linker arm
Ligand concentration
200–400 µmol glutathione/g washed and dried resin
120–320 µmol glutathione/ml medium
Binding capacity
> 8 mg recombinant glutathione S-transferase/ml medium
10–12 mg recombinant glutathione S-transferase/ml medium
Dynamic binding
NA
11 mg GST fusion protein/ml medium Mr: 43 kDa (GSTrap FF 1 ml at 1 ml/min)
Mean particle size
90 µm
90 µm
Bead structure
4% agarose
Highly cross-linked 4% agarose
Chemical stability
Stable to all commonly used aqueous buffers. Exposure to 0.1 M NaOH, 70% ethanol, or 6 M guanidine hydrochloride for 2 h at room temperature or to 1% (w/v) SDS for 14 d causes no significant loss of activity.
Stable to all commonly used aqueous buffers and 6 M guanidine hydrochloride for 1 h at room temperature
pH stability
4–13
6–9
Storage temperature
4–8 °C
4–8 °C
Storage buffer
20% ethanol
20% ethanol
Note: Binding capacity of GST fusion proteins to Glutathione Sepharose 4B and Glutathione Sepharose 4 Fast Flow is protein dependent and may therefore vary between different proteins. Decreasing the flow rate during purification may increase yield.
26
General considerations for purification of GST fusion proteins Yield of fusion protein is highly variable and is affected by the nature of the fusion protein, the host cell, and the culture conditions used. Fusion protein yields can range from 1 mg/l up to 10 mg/l (19). Table 9 can be used to approximate culture volumes based on an average yield of 2.5 mg/l. Table 9. Reagent volume requirements for different protein yields Fusion protein yield Culture volume Volume sonicate Glutathione Sepharose bed volume* 1× PBS † Glutathione elution buffer
50 mg
10 mg
1 mg
50 µg
20 l
4l
400 ml
20 ml
1l
200 ml
20 ml
1 ml
10 ml
2 ml
200 µl
10 µl
100 ml
20 ml
2 ml
100 µl
10 ml
2 ml
200 µl
10 µl
*To obtain the desired bed volume, use twice the volume of 50% Glutathione Sepharose slurry prepared in the procedures that follow (i.e. 1 ml of 50% Glutathione Sepharose slurry will give a bed volume of 0.5 ml). The bed volume is always 1/100 of the sonicate volume. † This volume is per wash. Three washes are required per sample in the following procedures.
Use high-quality water and chemicals for sample and buffer preparation. Samples should be centrifuged immediately before use and/or filtered through a 0.45 µm filter. If the sample is too viscous, dilute with binding buffer to prevent column clogging. One of the most important parameters affecting the binding of GST fusion proteins to Glutathione Sepharose is the flow rate. Since the binding kinetics between glutathione and GST are relatively slow, it is important to keep the flow rate low during sample application to achieve maximum binding capacity. The binding properties of the target protein can be improved by adjusting the sample to the composition of the binding buffer. Dilute in binding buffer or perform a buffer exchange using a desalting column such as HiTrap Desalting 5 ml or HiPrep 26/10 Desalting. Volumes and times used for elution may vary among fusion proteins. Further elution with higher concentrations of glutathione (20–50 mM) may improve yield. At concentrations above 15 mM glutathione, the buffer concentration should also be increased to maintain the pH within the range 8–9. Flow-through, wash, and eluted material from the column should be monitored for GST fusion proteins using SDS-PAGE in combination with Western blot if necessary. Following the elution steps, a significant amount of fusion protein may remain bound to the medium. Volumes and times used for elution may vary among fusion proteins. Additional elutions may be required. Eluates should be monitored for GST fusion protein by SDS-PAGE or by CDNB assay (see Chapter 5). Dimer formation is inevitable with GST fusion proteins since GST itself is a homodimer when folded. The presence of dimers should not interfere with purification, but if desired, a prepacked column with the gel filtration medium Superdex™ can be used to purify monomers from dimers and other aggregates (see Table 10).
27
Table 10. Prepacked columns for purifying monomeric proteins from dimers and other aggregates Sample volume
Separation range Mr 3000–70 000
Mr 10 000–600 000
< 250 µl
Superdex 75 HR 10/30
Superdex 200 HR 10/30
250 µl–4.5 ml
HiLoad™ 16/60 Superdex 75 prep grade
HiLoad 16/60 Superdex 200 prep grade
2.5 ml–12.5 ml*
HiLoad 26/60 Superdex 75 prep grade
HiLoad 26/60 Superdex 200 prep grade
* If the sample has a volume greater than 12.5 ml, it should be concentrated or fractionated in several runs. Note: The sample volume is a critical factor in the separation. Try to use a small sample volume for increased separation performance.
Batch preparation procedures are frequently mentioned in the literature. However, the availability of prepacked columns and easily packed Glutathione Sepharose 4 Fast Flow provides faster, more convenient alternatives. Batch preparations are occasionally used if it appears that the GST tag is not fully accessible or when the concentration of protein in the bacterial lysate is very low (both could appear to give a low yield from the affinity purification step). A more convenient alternative to improve yield is to decrease the flow rate or pass the sample through the column several times (recirculation). Purification steps should be monitored using one or more of the detection methods described in Chapter 5. The GST Detection Module, for example, is designed to identify GST fusion proteins using a biochemical or immunological assay, and is convenient to use for optimizing expression and monitoring steps in the purification of a GST fusion protein. The yield of fusion protein in purified samples can be estimated by measuring the absorbance at 280 nm. The concentration of the GST affinity tag can be approximated by 1 A280 ≈ 0.5 mg/ml. (This is based on the extinction coefficient of the GST monomer using a Bradford protein assay. Other protein determination methods may result in different extinction coefficients.) The yield of protein in purified samples can also be determined by standard chromogenic methods (e.g. Lowry, BCA, Bradford, etc.). If a Lowry or BCA type method is to be used, the sample must first be dialyzed against 2000 volumes of 1× PBS to remove glutathione, which can interfere with protein measurement. The Bradford method can be performed in the presence of glutathione. Re-use of purification columns and affinity media depends upon the nature of the sample and should only be performed with identical samples to prevent cross-contamination.
Selecting equipment for purification The choice of equipment will depend on the specific purification. Many purification steps can be carried out using simple methods and equipment as, for example, step-gradient elution using a syringe in combination with prepacked HiTrap™ columns. When more complex elution methods are necessary, such as linear gradients, or the same column is to be used for many runs in series, it is wise to use a dedicated system. Table 11 provides a guide to aid in selecting the correct purification format.
28
Table 11. Selection guide for purification equipment Way of working
MicroSpin + Centrifugation
HiTrap + Syringe
Standard ÄKTA™ design configurations Explorer Purifier FPLC™ Prime 100 10
!
Rapid, high-throughput screening
!
!
!
!
Reproducible performance for routine purification
!
!
!
!
!
Optimization of one-step purification to increase purity
!
!
!
!
System control and data handling for regulatory requirements, e.g. GLP
!
!
!
Automatic method development and optimization
!
!
!
Automatic buffer preparation
!
!
Automatic pH scouting
!
!
Automatic media or column scouting
!
Automatic multi-step purification
!
Scale-up, process development, and transfer to production
!
Simple, one-step purification
For a single purification of a small quantity of product or for high-throughput screening, MicroSpin columns using centrifugation or MicroPlex 24 Vacuum are convenient and simple to use. For purification of larger quantities of fusion proteins, GSTrap FF columns and GSTPrep FF 16/10 columns provide ideal formats. To increase capacity, use several GSTrap FF columns (1 ml or 5 ml) or two GSTPrep FF 16/10 columns (20 ml) in series or, for even larger capacity requirements, pack Glutathione Sepharose 4 Fast Flow into a suitable column. For simple and reproducible purification, use of a chromatography system such as ÄKTAprime is advantageous since it can monitor and record the purification process, thus eliminating manual errors. For laboratory environments in which all experimental data must be recorded and traceable and where method development, optimization, and scale-up are needed, a computer-controlled ÄKTAdesign chromatography system is recommended. Experiments such as protein refolding or method optimization that require linear gradient elution steps can be performed only by a chromatography system.
ÄKTAFPLC
ÄKTAexplorer ÄKTApurifier
ÄKTAprime
29
Summary of procedures Procedures are included for use of both prepacked columns and media available from Amersham Biosciences for GST fusion protein expression (Table 12). In several instances, alternative methods are provided. Table 12. Procedures for purification of GST fusion proteins Procedure
Description
Comments
10
10.1
Purification of multiple samples using GST MicroSpin columns with a microcentrifuge
Accommodates lysates from 2 ml to 12 ml of culture. Yield may be increased by repeating the elution step and pooling the eluates.
10.2
High-throughput purification using GST MicroSpin columns with MicroPlex Vacuum
Rather than centrifugation, a vacuum source draws the liquid through the affinity medium.
11.1
Manual purification using GSTrap FF column with a syringe
One-step purification, 12 mg/1 ml column and 60 mg/5 ml column. Columns may be used in series to increase yield.
11.2
Simple purification using a GSTrap FF column with ÄKTAprime
Automatic, preprogrammed application template for GST fusion proteins.
Preparative purification using GSTPrep FF 16/10 column
One-step preparative purification of up to 240 mg of GST fusion protein.
13.1
Batch purification using Glutathione Sepharose 4B
Flexible method able to accommodate 50 µl to 10 ml of Glutathione Sepharose 4B.
13.2
Batch/column purification using Glutathione Sepharose 4B
Can be scaled to purify 50 µg to 50 mg of GST fusion protein. Is a hybrid procedure that binds the protein in the batch method and elutes in a column.
Column purification using Glutathione Sepharose 4 Fast Flow
Packing own columns and scaling-up.
11
12 13
14
Purification using the GST MicroSpin Purification Module The GST MicroSpin Purification Module is useful for screening small or large numbers of bacterial lysates and for checking samples during the optimization of expression or purification conditions. Each module contains reagents sufficient for 50 purifications using MicroSpin columns prepacked with Glutathione Sepharose 4B. Sample application, washing, and elution can be performed using a standard microcentrifuge (Procedure 10.1) or in conjunction with MicroPlex 24 Vacuum (Procedure 10.2). GST fusion proteins can be purified from up to 48 samples in less than 1 h with the dual manifold system of MicroPlex 24 Vacuum. Each MicroSpin column contains a 50 µl bed volume of Glutathione Sepharose 4B, sufficient for purifying up to 400 µg of recombinant GST.
30
10.1. Purification of multiple samples using GST MicroSpin columns with a microcentrifuge Refer to page 27, General considerations for purification of GST fusion proteins, before beginning this procedure. Do not apply more than 600 µl of sample at a time to a GST MicroSpin column. This procedure will accommodate lysates produced from 2 to 12 ml of culture. Components in GST MicroSpin Purification Module 10× PBS: 1.4 M NaCl, 27 mM KCl, 100 mM Na 2HPO4, 18 mM KH2PO4, pH 7.3. To prepare 1× PBS for use, dilute 10× PBS with sterile H2O. Store at 4 °C. Reduced glutathione: 0.154 g. To prepare elution buffer, pour the entire 50 ml volume of dilution buffer supplied with the module into the bottle containing the reduced glutathione. Shake until completely dissolved. Store as 1–20 ml aliquots at -20 °C. Dilution buffer: 50 mM Tris-HCl, pH 8.0 IPTG: 500 mg. To prepare 100 mM IPTG, dissolve contents of the IPTG vial in 20 ml of sterile H2O. Store as 1 ml aliquots at -20 °C. MicroSpin columns: 50 units Equipment required: Microcentrifuge
Steps 1. Resuspend the Glutathione Sepharose 4B in each column by vortexing gently. 2. Loosen the column caps one-fourth turn. Remove (and save) bottom closures. 3. Place each column into a clean 1.5 or 2 ml microcentrifuge tube. Spin for 1 min at 735 × g. 4. Discard the buffer from each centrifuge tube and replace the bottom closures. 5. Apply up to 600 µl of lysate to a column. 6.
Recap each column securely and mix by gentle, repeated inversion. Incubate at room temperature for 5–10 min.
7. Remove (and save) the top caps and bottom closures. Place each column into a clean, pre-labelled 1.5 or 2 ml microcentrifuge tube. 8. Spin for 1 min at 735 × g to collect the flow-through. 9. Place each column into a clean, pre-labelled 1.5 or 2 ml microcentrifuge tube. 10. Apply 600 µl of 1× PBS wash buffer to each column and repeat the spin procedure. Additional 600 µl washes with 1× PBS can be performed if desired. 11. Add 100–200 µl of elution buffer to each column. Replace top caps and bottom closures. Incubate at room temperature for 5–10 min. 12. Remove and discard top caps and bottom closures and place each column into a clean 1.5 or 2 ml microcentrifuge tube. 13. Spin all columns again to collect the eluates. Save for analysis.
Yields of fusion protein can be increased by repeating the elution step two or three times and pooling the eluates.
31
10.2. High-throughput purification using GST MicroSpin columns with MicroPlex Vacuum Refer to page 27, General considerations for purification of GST fusion proteins, before beginning this procedure. Do not apply more than 600 µl of sample at a time to a GST MicroSpin column. This procedure will accommodate lysates produced from 2 to 12 ml of culture. Equipment required Vacuum source capable of providing 220 mm Hg (e.g. a water vacuum) Side-arm flask, 500 ml or 1 l Single- or double-hole rubber stopper Vacuum tubing MicroPlex 24 Vacuum apparatus (one or two) GST MicroSpin Purification Module (see Procedure 10.1 for listing of components)
Steps 1. Assemble the MicroPlex 24 Vacuum according to the instructions supplied with the instrument. 2. Resuspend the Glutathione Sepharose 4B in each MicroSpin column by vortexing gently. 3. Remove the caps and snap off the bottom closures from the MicroSpin columns. Place the columns in the manifold, filling any unused holes with the plugs provided with MicroPlex 24 Vacuum. 4. Check the stopcock to make sure that it is in the closed position (i.e. perpendicular to the vacuum tubing) and that the manifold is placed squarely on the gasket. 5. Turn on the vacuum supply at the source. Open the stopcock (i.e. parallel to the vacuum tubing). After the column storage buffer has been drawn through all the columns into the collection tray, close the stopcock. 6. Allow 10–15 s for the vacuum pressure to dissipate. Remove the manifold and place it on a paper towel. 7. Apply up to 600 µl of lysate to the column and incubate at room temperature for 5–10 min. 8. Open the stopcock. After the lysates have been drawn through all the columns into the collection tray, close the stopcock. 9. Add 600 µl of 1× PBS wash buffer to each column. Open the stopcock. After the buffer has been drawn through all the columns into the collection tray, close the stopcock. 10. Allow 10–15 s for the vacuum pressure to dissipate. Remove the manifold and reassemble the apparatus with a clean collection tray.
Additional 600 µl 1× PBS washes can be performed if desired. 11. Add 200 µl of elution buffer to each column. Incubate at room temperature for 5–10 min. 12. Open the stopcock. After the elution buffer has been drawn through all the columns into the collection tray, close the stopcock. 13. Allow 10–15 s for the vacuum pressure to dissipate. Remove the manifold. Cover the eluates with sealing tape until required for analysis.
Yields of fusion protein can be increased by repeating the elution step two or three times and pooling the eluates.
32
Purification using GSTrap FF 1 ml or 5 ml columns GSTrap FF affinity columns provide a convenient format for fast and easy one-step purification of GST fusion proteins produced using the pGEX series of expression vectors, as well as other glutathione S-transferases, and glutathione binding proteins. The columns are specially designed 1 ml and 5 ml HiTrap columns prepacked with Glutathione Sepharose 4 Fast Flow, a high-capacity affinity medium with excellent flow properties. See Table 13. Table 13. Column parameters of GSTrap FF columns Column type
GSTrap FF 1 ml and 5 ml prepacked columns
Prepacked medium
Glutathione Sepharose 4 Fast Flow
Column dimensions (internal diameter × height)
0.7 cm × 2.5 cm (GSTrap FF 1 ml) 1.6 cm × 2.5 cm (GSTrap FF 5 ml)
Column volume
1 ml and 5 ml
Maximum back pressure
0.3 MPa, 3 bar
Maximum flow rate
4 ml/min (GSTrap FF 1 ml) 15 ml/min (GSTrap FF 5 ml)
Recommended flow rates
Sample loading: 0.2–1 ml/min (GSTrap FF 1 ml) 1–5 ml/min (GSTrap FF 5 ml) Wash and elution: 1–2 ml/min (GSTrap FF 1 ml) 5–10 ml/min (GSTrap FF 5 ml)
References 24–30 provide examples of use of GSTrap FF columns for purification of GST fusion proteins. Sample application, washing, and elution can be performed using a syringe with a supplied adapter, a peristaltic pump, or a liquid chromatography system such as ÄKTAdesign (see Table 11 for equipment choices). For easy scale-up, two to three columns can be connected together in series simply by screwing the end of one column into the top of the next. Figure 4 shows a schematic of the simple steps needed for successful purification using a 1 ml GSTrap FF column.
Equilibrate column with binding buffer
3 min
Apply sample wash with binding buffer
5– 15 min
Waste
Elute with elution buffer
2 min
Collect
Collect fractions
Fig 4. Simple purification of GST fusion proteins using a GSTrap FF column.
33
The column is made of polypropylene, which is biocompatible and non-interactive with biomolecules. The top and bottom frits are manufactured from porous polyethylene. Columns are delivered with a stopper on the inlet and a twist-off end on the outlet. Both ends have M6 connections (6 mm metric threads). Every package includes all necessary components for connection of the columns to different types of equipment. GSTrap FF columns are directly compatible with existing purification protocols for GST fusion proteins, including on-column proteolytic cleavage methods. If removal of the GST moiety is required, the fusion protein can be digested with an appropriate site-specific protease while bound to the medium or, alternatively, after elution (see Chapter 6). Oncolumn cleavage eliminates the extra step of separating the released protein from GST since the GST moiety remains bound (23). GSTrap FF columns cannot be opened or refilled. For quick scale-up of purifications, two or three GSTrap FF columns (1 ml or 5 ml) can be connected in series (back pressure will be higher). Glutathione Sepharose 4 Fast Flow is also available in prepacked 20 ml GSTPrep FF 16/10 columns (Procedure 12) and as a loose medium for packing high-performance columns (Procedure 14). Re-use of any purification column depends on the nature of the sample and should only be performed with identical fusion proteins to prevent cross-contamination.
11.1. Manual purification using GSTrap FF column with a syringe One of the simplest methods for GST fusion protein purification is the use of GSTrap FF columns in combination with a syringe, as shown in Figure 5.
A
B
C
Fig 5. Using a GSTrap FF column with a syringe. A) Prepare buffers and sample. Remove the column’s top cap and twist off the end. B) Load the sample and begin collecting fractions. C) Wash and elute and continue collecting fractions.
Refer to page 27, General considerations for purification of GST fusion proteins, before beginning this procedure. Reagents and equipment required Binding buffer: 1× PBS (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.3) Elution buffer: 50 mM Tris-HCl, 10 mM reduced glutathione, pH 8.0 Syringe
34
Steps 1. Fill a syringe with binding buffer. 2. Connect the column to the syringe using the adapter supplied. Avoid introducing air into the column. 3. Remove the twist-off end. 4. Equilibrate the column with five column volumes of binding buffer. 5. Apply the sample using the syringe. For best results, maintain a flow rate of 0.2–1 ml/min (1 ml column) and 1–5 ml/min (5 ml column) as the sample is applied.* 6. Wash with 5–10 column volumes of binding buffer. Maintain flow rates of 1–2 ml/min (1 ml column) and 5–10 ml/min (5 ml column) during the wash.*
Optional: Collect the flow-through (in 1 ml fractions for the 1 ml column and 2 ml fractions for the 5 ml column) and reserve until the procedure has been successfully completed. Retain a sample for analysis by SDS-PAGE or by CDNB assay to measure the efficiency of protein binding to the medium. 7. Elute with 5–10 column volumes of elution buffer and collect fractions. Maintain flow rates of 1–2 ml/min (1 ml column) and 5–10 ml/min (5 ml column) during elution.* *One ml/min corresponds to approximately 30 drops/min when using a syringe with a HiTrap 1 ml column, and 5 ml/min corresponds to approximately 120 drops/min when using a HiTrap 5 ml column. Note: The flow rate is critical for the yield. A lower flow rate may increase the binding capacity and yield.
For large sample volumes, a simple peristaltic pump can be used to apply sample and buffers.
11.2. Simple purification using a GSTrap FF column with ÄKTAprime ÄKTAprime, in combination with pre-installed templates for purifications and prepacked columns, is designed to perform the most common protein purification steps at the touch of a button. It provides significant advantages in speed, capacity, and fraction selection over manual methods. A set of cue cards includes detailed information on each procedure. Almost any sample volume can be loaded when using ÄKTAprime. High flow rates allow fast separations, and with on-line monitoring, UV, conductivity, or pH can be measured during a purification. The pre-programmed application template in ÄKTAprime for purification of GST fusion proteins using a single GSTrap FF column is shown in Figure 6. This provides a standard purification protocol that can be followed exactly or optimized as required. Typical procedures and results are shown in Figures 7 and 8, respectively. % Elution buffer
Elution, 11 min
100 System preparation & column equilibration, 11 min
50
Sample Wash, 10 min
Re-equilibration, 6 min
11 11 10 Total separation time = 37 min + sample application time
6
min
Fig 6. Purification of GST fusion proteins using a GSTrap FF column and ÄKTAprime.
35
Refer to page 27, General considerations for purification of GST fusion proteins, before beginning this procedure. Reagents required Binding buffer: 20 mM sodium phosphate, 0.15 M NaCl, pH 7.3 (or the buffer used in Procedure 11.1) Elution buffer: 50 mM Tris-HCl, 10 mM reduced glutathione, pH 8.0 Prepare at least 500 ml of each buffer.
Steps 1. Follow the instructions supplied on the ÄKTAprime cue card. 2. Select the Application Template. 3. Start the method. 4. Enter the sample volume and press OK to start.
Connecting the column.
Preparing the fraction collector.
Fig 7. Typical procedures when using ÄKTAprime.
A) Column: Sample:
Binding buffer: Elution buffer:
GSTrap FF 1 ml 8 ml cytoplasmic extract from E. coli expressing a GST fusion protein PBS, pH 7.3 50 mM Tris-HCl, pH 8.0 with 10 mM reduced glutathione 1 ml/min
Flow: Chromatographic procedure: 4 CV binding buffer, 8 ml sample, 10 CV binding buffer, 5 CV elution buffer, 5 CV binding buffer (CV = column volume)
A280 Elution buffer
3.5 3.0 2.5
2.7 mg pure GST fusion protein
Wash
2.0
% Elution buffer
Mr
100
97 000 66 000
80 60
1.5 40
1.0
B) Lane 1: Low Molecular Weight (LMW) Calibration kit, reduced, Amersham Biosciences (10 µl prepared for silver stain) Lane 2: Cytoplasmic extract of E. coli expressing GST fusion protein, 1 g cell paste/10 ml (5 µl sample from collect. fraction + 35 µl sample cocktail -> 10 µl applied) Lane 3: GST fusion protein eluted from GSTrap FF 1 ml (5 µl sample from collect. fraction + 35 µl sample cocktail -> 10 µl applied)
45 000 30 000 20 100 14 400
20
0.5
0
0 5.0 5.0
10.0 10.0
15.0 15.0
20.0 20.0
ml min
1
2
3
Fig 8. Purification of GST fusion protein on a GSTrap FF 1 ml column A) Chromatogram. B) SDS-PAGE on ExcelGel™ SDS Gradient 8–18% using Multiphor™ II (Amersham Biosciences) followed by silver staining.
36
Preparative purification using GSTPrep FF 16/10 column GSTPrep FF 16/10 columns are specially designed 20 ml HiPrep™ columns, ready to use for easy, one-step preparative purification of GST fusion proteins, other glutathione S-transferases, and glutathione binding proteins. Prepacked with Glutathione Sepharose 4 Fast Flow, the columns exhibit high binding capacity and excellent flow properties. For easy scale-up, columns can be connected in series.
The column is made of polypropylene, which is biocompatible and non-interactive with biomolecules. Both ends have M6 connections (6 mm metric threads), together with the included connectors. Separations can be easily achieved using a chromatography system such as ÄKTAdesign. Refer to Table 11 for a selection guide to purification equipment and to Table 14 for a list of GSTPrep FF 16/10 column parameters. GSTPrep FF 16/10 columns cannot be opened or refilled. For quick scale-up of purifications, two GSTPrep FF 16/10 columns can be connected in series (back pressure will be higher). Glutathione Sepharose 4 Fast Flow is also available as prepacked 1 ml and 5 ml GSTrap FF columns (Procedures 11.1 and 11.2) and as a loose medium for packing high-performance columns (Procedure 14). Re-use of any purification column depends on the nature of the sample and should only be performed with identical fusion proteins to prevent cross-contamination. Table 14. Column parameters of GSTPrep FF 16/10 columns Column type
GSTPrep FF 16/10 prepacked columns
Prepacked medium
Glutathione Sepharose 4 Fast Flow
Column dimensions (internal diameter × height)
16 × 100 mm
Column volume
20 ml
Maximum pressure over the packed bed during operation
0.15 MPa, 1.5 bar
HiPrep column hardware pressure limit
0.5 MPa, 5 bar
Maximum flow rate
10 ml/min (300 cm/h)
Recommended flow rates
1–10 ml/min (30–300 cm/h) (protein dependent)
37
12. Preparative purification using GSTPrep FF 16/10 column Refer to page 27, General considerations for purification of GST fusion proteins, before beginning this procedure. Reagents required Binding buffer: 1× PBS (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.3) Elution buffer: 50 mM Tris-HCl, 10 mM reduced glutathione, pH 8.0
Initial purification/optimization 1. Apply the centrifuged and/or filtered sample (in binding buffer) to the column at a flow rate of 1–5 ml/min (30–150 cm/h). 2. Wash the column with 5–10 column volumes of binding buffer at 2–10 ml/min (60–300 cm/h). 3. Elute the bound protein with 5–10 column volumes of elution buffer at a flow rate of 2–10 ml/min (60–300 cm/h).
Due to the relatively slow binding kinetics between GST and glutathione, it is important to keep the flow rate low during sample loading/elution. The binding capacity is protein dependent and therefore the yield will vary from protein to protein. Optional: Collect the flow-through and reserve until the procedure has been successfully completed. Retain a sample for analysis by SDS-PAGE or by CDNB assay to measure the efficiency of protein binding to the medium For cleaning, storage, and handling information, refer to Appendix 5.
Purification using Glutathione Sepharose 4B medium Glutathione Sepharose 4B is available in 10 ml, 300 ml, and function-tested 100 ml lab packs for affinity purification of GST fusion proteins in batch- or column-based methods. The batch method is very flexible, as purification can be performed with 10 µl to 10 ml of Glutathione Sepharose 4B. Both batch and batch/column purification schemes are presented below. For larger-scale column purifications, the high flow properties and binding capacity of Glutathione Sepharose 4 Fast Flow make it an excellent choice for scale-up.
13.1. Batch purification using Glutathione Sepharose 4B The following batch protocol can be conveniently scaled to purify as little as 50 µg or as much as 50 mg of GST fusion protein using Glutathione Sepharose 4B. Refer to page 27, General considerations for purification of GST fusion proteins, before beginning this procedure. Reagents required Glutathione Sepharose 4B Binding buffer: 1× PBS (ice-cold) (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.3) Elution buffer: 50 mM Tris-HCl, 10 mM reduced glutathione, pH 8.0. It is possible to dispense in 1–10 ml aliquots and store at -20 °C until needed. Avoid more than five freeze/thaw cycles.
38
Steps Preparation of the medium
Glutathione Sepharose 4B, as supplied, is approximately a 75% slurry. The following procedure results in a 50% slurry. 1. Referring to Table 9, determine the bed volume of Glutathione Sepharose 4B required for your application. 2. Gently shake the bottle of Glutathione Sepharose 4B to resuspend the medium. 3. Use a pipet with a wide-bore tip to remove sufficient slurry, and transfer the slurry to an appropriate container/tube. 4. Sediment the medium by centrifuging at 500 × g for 5 min. Carefully decant the supernatant. 5. Wash the Glutathione Sepharose 4B by adding 10 ml of cold (4 °C) 1× PBS per 1.33 ml of the original slurry of Glutathione Sepharose 4B dispensed. Invert to mix.
Glutathione Sepharose 4B must be thoroughly washed with 1× PBS to remove the 20% ethanol storage solution. Residual ethanol may interfere with subsequent procedures. 6. Sediment the medium by centrifuging at 500 × g for 5 min. Decant the supernatant. 7. For each 1.33 ml of the original slurry of Glutathione Sepharose 4B dispensed, add 1 ml of 1× PBS. This results in a 50% slurry. Mix well prior to subsequent pipetting steps.
Batch purification
Bed volume is equal to 0.5× the volume of the 50% slurry used. 1. Add 2 ml of the 50% slurry of Glutathione Sepharose 4B equilibrated with 1× PBS to each 100 ml of bacterial sonicate (i.e. use a 1 ml bed volume per 100 ml of sonicate). 2. Incubate for 30 min at room temperature. Use gentle agitation such as end-over-end rotation.
At this stage, the medium with adsorbed fusion protein can be packed into a suitable column to facilitate washing and elution steps. If a column is used, refer to the column purification procedure in the following section for instructions on washing and elution. 3. Sediment the medium by centrifuging at 500 × g for 5 min. Carefully decant the supernatant (= flow-through). 4. Wash the medium with 10 bed volumes of 1× PBS. Invert to mix. 5. Sediment the medium by centrifuging at 500 × g for 5 min. Carefully decant the supernatant (= wash). 6. Repeat steps 4 and 5 twice for a total of three washes.
Optional: Reserve the flow-through and wash until the procedure has been successfully completed. Retain a sample for analysis by SDS-PAGE or by CDNB assay to measure the efficiency of protein binding to the medium. Bound fusion protein can be eluted directly at this stage using elution buffer, or the protein can be cleaved on the medium to liberate the protein of interest from the GST moiety (see Chapter 6 for various options). 7. Elute the bound protein from the sedimented medium by adding 1.0 ml of elution buffer per 1 ml bed volume of the original slurry. 8. Mix gently to resuspend the medium. Incubate at room temperature for 10 min to elute the fusion protein from the medium. Use gentle agitation such as end-over-end rotation.
39
9. Sediment the medium by centrifuging at 500 × g for 5 min. Carefully decant the supernatant (= eluted protein) into a fresh centrifuge tube. 10. Repeat steps 7–9 twice for a total of three elutions. Check the three eluates separately for purified protein and pool those eluates containing protein.
13.2. Batch/column purification using Glutathione Sepharose 4B Refer to page 27, General considerations for purification of GST fusion proteins, before beginning this procedure. Reagents and equipment required Glutathione Sepharose 4B Binding buffer: 1× PBS (ice-cold) (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.3) Elution buffer: 50 mM Tris-HCl, 10 mM reduced glutathione, pH 8.0. It is possible to dispense in 1–10 ml aliquots and store at -20 °C until needed. Avoid more than five freeze/thaw cycles. Suitable disposable columns, for example Empty Disposable PD-10 Columns. These columns have a total volume capacity (including medium and sample) of ~ 13 ml, but they can be used with smaller volumes.
Steps Preparation of the medium
Steps 1–7 below are identical to those at the start of the batch procedure above. Glutathione Sepharose 4B, as supplied, is approximately a 75% slurry. The following procedure results in a 50% slurry. 1. Referring to Table 9, determine the bed volume of Glutathione Sepharose 4B required for your application. 2. Gently shake the bottle of Glutathione Sepharose 4B to resuspend the medium. 3. Use a pipet to remove sufficient slurry for use and transfer the slurry to an appropriate container/tube. 4. Sediment the medium by centrifuging at 500 × g for 5 min. Carefully decant the supernatant. 5. Wash the Glutathione Sepharose 4B by adding 10 ml of cold (4 °C) 1× PBS per 1.33 ml of the original slurry of Glutathione Sepharose 4B dispensed. Invert to mix.
Glutathione Sepharose 4B must be thoroughly washed with 1× PBS to remove the 20% ethanol storage solution. Residual ethanol may interfere with subsequent procedures. 6. Sediment the medium by centrifuging at 500 × g for 5 min. Decant the supernatant. 7. For each 1.33 ml of the original slurry of Glutathione Sepharose 4B dispensed, add 1 ml of 1× PBS. This results in a 50% slurry. Mix well prior to subsequent pipetting steps.
Batch/column purification
Bed volume is equal to 0.5× the volume of the 50% slurry used. 1. Add 2 ml of the 50% slurry of Glutathione Sepharose 4B equilibrated with 1× PBS to each 100 ml of bacterial sonicate (i.e. use a 1 ml bed volume per 100 ml of sonicate). 2. Incubate for 30 min at room temperature. Use gentle agitation such as end-over-end rotation. 3. Use a pipet to transfer the medium to a disposable column (e.g. Empty Disposable PD-10 Column mounted in its rack folded from the package). 4. Tap the column to dislodge any trapped air bubbles in the medium bed. Allow the medium to settle. 5. Open the column outlet and allow the column to drain. 40
Gentle downward pressure provided with a gloved thumb over the top of the column may be required to start the flow of liquid. 6. Wash the medium by adding 10 bed volumes of 1× PBS. Open the column outlet and allow the column to drain. Repeat twice more for a total of three washes. 7. After the column with bound protein has been washed and drained, close the column outlet. 8. Elute the fusion protein by adding 1 ml of elution buffer per 1 ml bed volume. Incubate the column at room temperature for 10 min to elute the fusion protein. 9. Open the column outlet and collect the eluate. This contains the fusion protein. 10. Repeat the elution and collection steps twice more. Pool the three eluates.
Optional: Collect the flow-through in fractions and reserve until the procedure has been successfully completed. Retain a sample for analysis by SDS-PAGE or by CDNB assay to measure the efficiency of protein binding to the medium. Because of its superior flow rates, we recommend Glutathione Sepharose 4 Fast Flow (Procedure 14) for use when column purification is desired.
Purification using Glutathione Sepharose 4 Fast Flow Physical characteristics of Glutathione Sepharose 4 Fast Flow are listed in Table 8. For information on cleaning and storage of columns packed with Glutathione Sepharose 4 Fast Flow, refer to Appendix 5. Suggested columns for this application include: • HR 10/10 (10 mm i.d.) for bed volumes of 6.4–8.7 ml and bed heights of 8–11 cm. • XK 16/20 (16 mm i.d.) for bed volumes of 2–34 ml and bed heights of 1–17 cm. • XK 26/20 (26 mm i.d.) for bed volumes of 0–80 ml and bed heights of 0–15 cm. Use the Packing Connector HR or XK to produce a well-packed column. A Column Packing Video is available to demonstrate appropriate packing techniques.
14. Column purification using Glutathione Sepharose 4 Fast Flow Refer to page 27, General considerations for purification of GST fusion proteins, before beginning this procedure. Glutathione Sepharose 4 Fast Flow is supplied in 20% ethanol as a bacteriostat. Decant the solution and replace it with binding buffer before use. In general, we recommend a bed height of 5–15 cm to allow the use of high flow rates. Prepacked GSTrap FF 1 ml and GSTrap FF 5 ml columns (Procedures 11.1 and 11.2) and GSTPrep 16/10 columns (20 ml; Procedure 12) are also available. Reagents required Glutathione Sepharose 4 Fast Flow Binding buffer: 1× PBS (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.3) Elution buffer: 50 mM Tris-HCl, 10 mM reduced glutathione, pH 8.0
41
Steps Column packing 1. Assemble the column (and packing reservoir if necessary). 2. Remove air from the column dead spaces by flushing the end-piece and adapter with binding buffer. Ensure that no air has been trapped under the column bed support. Close the column outlet, leaving the bed support covered with binding buffer. 3. Resuspend the medium stored in its container by shaking (avoid stirring the sedimented medium). Mix the binding buffer with the medium to form a 50–70% slurry (sedimented bed volume/slurry volume = 0.5–0.7). 4. Pour the slurry into the column in a single continuous motion. Pouring the slurry down a glass rod held against the column wall will minimize air bubble formation. 5. If using a packing reservoir, immediately fill the remainder of the column and reservoir with binding buffer. Mount the adapter or lid of the packing reservoir and connect the column to a pump. Avoid trapping air bubbles under the adapter or in the inlet tubing. 6. Open the bottom outlet of the column and set the pump to run at the desired flow rate. Ideally, Glutathione Sepharose 4 Fast Flow is packed at a constant pressure of approximately 0.1 MPa (1 bar).
If the packing equipment does not include a pressure gauge, use a packing flow velocity of approximately 450 cm/h (10 cm bed height, 25 °C, low-viscosity buffer). 450 cm/h corresponds to 6 ml/min in a HR 10/10 column or 15 ml/min in a XK 16/20 column. If the recommended pressure or flow rate cannot be obtained, use the maximum flow rate allowed by the pump. This should also give a sufficiently well-packed bed. Do not exceed 75% of the packing flow velocity in subsequent chromatographic procedures using the same pump. 7. When the bed has stabilized, close the bottom outlet, and stop the pump. 8. If using a packing reservoir, disconnect the reservoir, and fit the adapter to the column. If using a HR column, carefully place the top filter on top of the bed before fitting the adapter. 9. With the adapter inlet disconnected, push down the adapter approximately 2 mm into the bed, allowing the solution to flush the adapter inlet. The bottom outlet of the column should be closed. 10. Connect the pump, open the bottom outlet, and continue packing. The bed will be further compressed at this point and a space will be formed between the bed surface and the adapter. 11. Close the bottom outlet. Disconnect the column inlet, and lower the adapter approximately 2 mm into the bed. Connect the pump. The column is now ready to use.
Column purification 1. Equilibrate the column with approximately five column volumes of binding buffer (1× PBS). 2. Apply the centrifuged and/or filtered sample. 3. Wash the column with 5–10 column volumes of binding buffer or until no material appears in the flow-through. 4. Elute the bound protein with 5–10 column volumes of elution buffer.
Optional: Collect the flow-through and reserve until the procedure has been successfully completed. Retain a sample for analysis by SDS-PAGE or by CDNB assay. The reuse of Glutathione Sepharose 4 Fast Flow depends on the nature of the sample and should only be performed with identical samples to prevent cross-contamination.
42
Chapter 5 Detection of GST fusion proteins Several methods are available for detection of GST fusion proteins, with method selection largely depending on the experimental situation. For example, SDS-PAGE analysis, although frequently used for monitoring results during expression and purification (see Chapter 3), may not be the method of choice for routine monitoring of samples from high-throughput screening. Functional assays based on the properties of the protein of interest (and not the GST tag) are useful, but must be developed for each specific protein. These latter assays are not covered in this handbook. See Table 15 for a description of the procedures that follow.
Summary of procedures Table 15. Procedures for detection of GST fusion proteins Procedure
Description
Comments
15
GST 96-Well Detection Module for ELISA
Uses 100 µl of sample/well. Ideal for screening expression systems and chromatographic fractions. Useful when amount of expressed protein is unknown or when increased sensitivity is required. Gives estimate of relative level of expression.
16
GST Detection Module with CDNB enzymatic assay
Uses 5–50 µl of sample. Rapid assay; ideal for screening. Gives estimate of relative level of expression.
17
Western blot using anti-GST antibody
Uses 5–10 µl of sample. Highly specific, detects only GST fusion protein. Little or no background detectable when using detection systems with optimized concentrations of secondary HRP-conjugated antibody. ECL™ detection systems enhance detection in Western blots. ECL provides adequate sensitivity for most recombinant expression applications. For higher sensitivity, use ECL Plus. Provides information on size.
18
SDS-PAGE with Coomassie or silver staining
Uses 5–10 µl of sample. Provides information on size and percent purity. Detects fusion protein and contaminants.
15. GST 96-Well Detection Module for ELISA The GST 96-Well Detection Module provides a highly sensitive ELISA for testing clarified lysates and intermediate purification fractions for the presence of GST fusion proteins (see Figs 9 and 10). Samples are applied directly into the wells of the plates, and GST fusion proteins are captured by specific binding to anti-GST antibody that is immobilized on the walls of each well. Captured GST fusion proteins are then detected with HRP/Anti-GST conjugate provided in the module. Standard curves for quantitation of fusion proteins can be constructed using purified recombinant GST, which is included as a control. Each detection module contains reagents sufficient for 96 detections. Each plate is an array of 12 strips with eight wells per strip, such that as few as eight samples (one strip) can be assayed at a time. The GST 96-Well Detection Module can also be used with antibody directed against a GST fusion partner to screen and identify clones expressing the desired GST fusion protein.
43
A 450 1.50
0.75
0 0.01
0.1
1
10 100 ng rGST/well
1000
10 000
Fig 9. Sensitive detection of recombinant GST using the GST 96-Well Detection Module. Recombinant GST protein was prepared in 1× blocking buffer, and 100 µl volumes were applied directly to the wells of a GST 96-well capture plate. After 1 h binding at room temperature, the wells were washed in wash buffer and incubated with a 1:1000 dilution of HRP/Anti-GST conjugate for 1 h. Detection was performed using 3, 3',5,5'-tetramethyl benzidine (TMB) substrate, and the absorbance of each well was measured at 450 nm.
Fig 10. Screening of bacterial lysates for GST fusion protein expression using the GST 96-Well Detection Module.
Each fusion protein is captured uniquely; therefore, if quantitation is required, prepare standards of recombinant GST protein and the target fusion protein (if available) using a dilution series from 1 ng/µl to 10 pg/µl in 1× blocking buffer. Include recombinant GST protein as a standard control in every assay. Prepare fresh buffers daily. Components of GST 96-Well Detection Module GST 96-Well Detection Plates (each well is coated with goat polyclonal anti-GST antibody, blocked, and dried) Horseradish peroxidase conjugated to goat polyclonal anti-GST antibody (HRP/Anti-GST) Purified recombinant glutathione S-transferase (GST) standard protein
44
Additional reagents required for ELISA PBS (1×): 140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO 4, pH 7.3 Wash buffer: 0.05% Tween™ 20 in PBS (500 ml/96-well plate). Store at room temperature until needed. Blocking buffer (1×): 3% non-fat dry milk in PBS with 0.05% Tween 20 (10 ml/96-well plate) Blocking buffer (2×): 6% non-fat dry milk in PBS with 0.1% Tween 20 (5 ml/96-well plate) Substrate
Steps 1. Bring each test sample to a final volume of 50 µl with 1× PBS. 2. Add 50 µl of 2× blocking buffer to each sample. 3. For screening, dilute the recombinant GST protein standard to 1 ng/100 µl in 1× blocking buffer. 4. For quantitation, prepare a dilution series from 1 ng/µl to 10 pg/µl in 1× blocking buffer for both the recombinant GST protein and the target fusion protein (when available). 5. Remove one 96-well plate from its foil pouch.
If using fewer than 96 wells, carefully remove the well strips from the holder by pushing up on the wells from below. Store unused well strips in the pouch with the desiccant provided. 6. Pipette 100 µl of sample into each well. 7. Incubate for 1 h at room temperature in a humidified container or incubator. 8. Invert the plate and flick sharply to empty the contents of the wells.
Biohazardous material should be pipetted or aspirated into a suitable container. 9. Blot the inverted plate or well strips onto a paper towel to remove excess liquid. 10. Wash each well five times with wash buffer by inverting and flicking out the contents each time. 11. Blot the inverted plate or well strips onto a paper towel to remove excess wash buffer. 12. Dilute the HRP/anti-GST conjugate 1:10 000 (1 µl:10 ml) in 1× blocking buffer.
One 96-well plate will require 10 ml of the diluted conjugate. 13. Add 100 µl of diluted HRP/anti-GST conjugate to each well and incubate for 1 h at room temperature in a humidified container or incubator. 14. Empty the well contents and wash twice with wash buffer as previously described. 15. Add soluble horseradish peroxidase substrate* to each well and incubate according to the supplier’s instructions. *3,3',5,5'-tetramethyl benzidine (A450) and 2',2'-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid) diammonium salt (ABTS™) (A410) have been used successfully.
16. Read plate absorbance in a microplate reader or spectrophotometer.
16. GST Detection Module with CDNB enzymatic assay GST fusion proteins produced using pGEX vectors can be detected enzymatically using the GST substrate 1-chloro-2,4 dinitrobenzene (CDNB) (31, 32), included in the GST Detection Module. The GST-mediated reaction of CDNB with glutathione produces a conjugate that is measured by absorbance at 340 nm using either a plate reader or a UV/vis spectrophotometer, such as an Ultrospec™ 1100 pro. Assay results are available in less than 10 min for
45
crude bacterial sonicates, column eluates, or purified GST fusion protein. Figure 11 shows typical results from a CDNB assay. Each GST Detection Module contains reagents sufficient for 50 assays. A 340 Eluate (0.8 µg)
0.6
0.4
0.2
Sonicate (53 µg)
1
2 Time (min)
3
4
Fig 11. Typical results of a CDNB assay for GST fusion proteins. 53 µg of total protein from an E. coli TG1/pGEX-4TLuc sonicate and 0.8 µg of total protein eluted from Glutathione Sepharose were assayed according to instructions included with the GST Detection Module.
Components of GST Detection Module used with the CDNB enzymatic assay 10× reaction buffer: 1 M KH2PO4 buffer, pH 6.5 CDNB: 100 mM 1-chloro-2,4-dinitrobenzene (CDNB) in ethanol Reduced glutathione powder: Prepare a 100 mM solution by dissolving the reduced glutathione powder in sterile distilled H2O. Aliquot into microcentrifuge tubes. Store at -20 °C. Avoid more than five freeze/thaw cycles.
CDNB is toxic. Avoid contact with eyes, skin and clothing. In case of accidental contact, flush affected area with water. In case of ingestion, seek immediate medical attention. pGEX-bearing cells must be lysed prior to performing a CDNB assay. Steps 1. In a microcentrifuge tube, combine the following: Distilled H2O 10× reaction buffer CDNB Glutathione solution Total volume
880 100 10 10
µl µl µl µl
1000 µl
2. Cap the tube and mix the contents by inverting several times.
CDNB may cause the solution to become slightly cloudy. However, the solution should clear upon mixing. 3. Transfer 500 µl volumes of the above CDNB solution into two UV-transparent cuvettes labelled sample and blank. Add sample (5–50 µl) to the sample cuvette. To the blank cuvette, add 1× reaction buffer equal in volume to that of the sample in the sample cuvette. 4. Cover each cuvette with wax film and invert to mix. 5. Place the blank cuvette into the spectrophotometer and blank at 340 nm. Measure the absorbance of the sample cuvette at 340 nm and simultaneously start a stopwatch or other timer. 6. Record absorbance readings at 340 nm at 1 min intervals for 5 min by first blanking the spectrophotometer with the blank cuvette and then measuring the absorbance of the sample cuvette.
46
7. Calculate the A340/min/ml sample as follows:
Calculations DA340/min/ml =
A340(t2) – A340(t1) (t2 – t1)(ml sample added)
Where: A340 (t2) = absorbance at 340 nm at time t2 in min A340 (t1) = absorbance at 340 nm at time t1 in min
DA340/min/ml values can be used as a relative comparison of GST fusion protein content between samples of a given fusion protein. Adapt the assay to give absolute fusion protein concentrations by constructing a standard curve of DA340/min versus fusion protein amount. Purified sample of the fusion protein is required to construct the curve. Activity of the GST moiety can be affected by folding of the fusion partner. Absorbance readings obtained for a given fusion protein may not reflect the actual amount of fusion protein present.
17. Western blot using anti-GST antibody Expression and purification of GST fusion proteins can also be monitored by Western blot analysis, using ECL or ECL Plus detection systems to enhance sensitivity. Reagents required Anti-GST antibody (goat polyclonal) Blocking/incubation buffer: 5% (w/v) non-fat dry milk and 0.1% (v/v) Tween 20 in 1× PBS (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO 4, pH 7.3) Wash buffer: 0.1% (v/v) Tween 20 in 1× PBS (as above) Secondary antibody to detect the anti-GST antibody (such as anti-goat IgG HRP conjugate) Appropriate membrane, such as Hybond ECL (for subsequent ECL detection) or Hybond P (for subsequent ECL or ECL Plus detection)
Steps Electrophoretic separation of proteins 1. Separate the protein samples by SDS-PAGE.
Although anti-GST antibody from Amersham Biosciences has been cross-adsorbed with E. coli proteins, low levels of cross-reacting antibodies may remain. Samples of E. coli sonicates that do not contain a recombinant pGEX plasmid and samples that contain the parental pGEX plasmid should always be run as controls. 2. Transfer the separated proteins from the electrophoresis gel to membrane.
Electrophoresis and protein transfer can be accomplished using a variety of equipment and reagents. For further details, refer to the Protein Electrophoresis Technical Manual and Hybond ECL Instruction Manual from Amersham Biosciences.
47
Blocking of membrane 1. Transfer the membrane onto which the proteins have been blotted into an appropriately sized container, such as a Petri dish. 2. Add 50–200 ml of blocking/incubation buffer to the container. 3. Incubate for 1–16 h at ambient temperature with gentle shaking. 4. Decant and discard the buffer.
Longer incubation times with blocking/incubation buffer may reduce background signal. Incubation of membrane blot with primary antibody 1. Prepare an appropriate dilution of anti-GST antibody with blocking/incubation buffer (e.g. 5–10 µl of antibody in 50 ml of buffer).
Refer to Amersham Biosciences Application Note 18-1139-13 for further information on optimization of antibody concentration for Western blotting. 2. Pour the antibody-buffer mixture into the container with the membrane. 3. Incubate for 1 h at ambient temperature with gentle shaking. 4. Decant and discard the antibody-buffer. 5. Rinse the membrane twice with 20–30 ml of blocking/incubation or wash buffer to remove most of the unbound antibody. 6. Decant and discard the rinse buffers. 7. Wash the membrane with 20–30 ml of blocking/incubation or wash buffer for 10–60 min at ambient temperature with gentle shaking. 8. Discard the buffer and repeat the wash from step 7.
Incubation of membrane blot with secondary antibody 1. Dilute an appropriate anti-goat secondary antibody with blocking/incubation buffer according to the manufacturer’s recommendation.
Refer to Amersham Biosciences Application Note 18-1139-13 for further information on optimization of antibody concentration for Western blotting. 2. Pour the antibody-buffer mixture into the container with the membrane. 3. Incubate for 1 h at ambient temperature with gentle shaking. 4. Decant and discard the antibody-buffer. 5. Rinse twice with 20–30 ml of blocking/incubation or wash buffer to remove most of the unbound antibody. 6. Decant and discard the rinse buffers. 7. Wash the membrane with 20–30 ml of blocking/incubation or wash buffer for 10–60 min at ambient temperature with gentle shaking. 8. Discard the buffer and repeat the wash step using wash buffer.
Use wash buffer not blocking/incubation buffer for step 9. The protein in blocking/incubation buffer would cause problems in the development step. 9. Develop the blot with a substrate that is appropriate for the conjugated secondary antibody.
48
ECL and ECL Plus detection systems require very little antibody to achieve a sufficient sensitivity; therefore, the amount of antibody (primary and secondary) used in the protocols can be minimized. Smaller quantities of antibody-buffer mixtures can be used by scaling down the protocol and performing the incubations in sealable plastic bags.
18. SDS-PAGE with Coomassie blue or silver staining SDS-PAGE is useful for monitoring fusion protein levels during expression and purification. Transformants expressing the desired fusion protein are identified by the absence from total cellular proteins of the parental GST and by the presence of a novel, larger fusion protein. Parental pGEX vectors produce a Mr 29 000 GST fusion protein containing amino acids coded for by the pGEX multiple cloning site. Reagents required 6× SDS loading buffer: 0.35 M Tris-HCl, 10.28% (w/v) SDS, 36% (v/v) glycerol, 0.6 M dithiothreitol (or 5% 2-mercaptoethanol), 0.012% (w/v) bromophenol blue, pH 6.8. Store in 0.5 ml aliquots at -80 °C.
Steps 1. Add 1–2 µl of 6× SDS loading buffer to 5–10 µl of supernatant from crude extracts, cell lysates, or purified fractions, as appropriate. 2. Vortex briefly and heat for 5 min at 90–100 °C. 3. Centrifuge briefly, then load the samples onto an SDS-polyacrylamide gel. 4. Run the gel for the appropriate length of time and stain with Coomassie blue (Coomassie Blue R Tablets) or silver (PlusOne Silver Staining Kit, Protein).
The percentage of acrylamide in the SDS-gel should be selected based on the expected molecular weight of the protein of interest (see Table 16). Table 16. Selecting the appropriate gel composition for protein separation Percent acrylamide in resolving gel
Separation size range (Mr ×103)
Single percentage 5%
36–200
7.5%
24–200
10%
14–200
12.5%
14–100
15%
14–60*
Gradient 5–15%
14–200
5–20%
10–200
10–20%
10–150
*The larger proteins fail to move significantly into the gel.
For information and advice on electrophoresis techniques, please refer to the section Additional reading on page 107.
49
50
Chapter 6 Removal of GST tag by enzymatic cleavage In most cases, functional tests can be performed using the intact fusion with GST. However, if removal of the GST tag is necessary, fusion proteins containing a PreScission Protease, thrombin, or Factor Xa recognition site can be cleaved either while bound to Glutathione Sepharose or in solution after elution. On-column cleavage is generally recommended as the method of choice since many potential contaminants can be washed out and the target protein eluted with a higher level of purity. Cleavage after elution is suggested if optimization of cleavage conditions is necessary. It is highly recommended that fusion proteins be produced with a PreScission Protease cleavage site. The GST tag then can be removed and the protein purified in a single step on the column (see Fig 12 on next page). Because this protease is maximally active at 4 °C, cleavage can be performed at low temperatures, thus improving the stability of the target protein. Thrombin or Factor Xa recognition sites can be cleaved either while the fusion protein is bound to the column or in solution after elution from the column (see Fig 13 on next page). For removal of thrombin and Factor Xa, GSTrap FF and HiTrap Benzamidine FF (high sub) columns can be connected in series so that cleaved product passes directly from the GSTrap FF into the HiTrap Benzamidine FF (high sub). Thus, samples are cleaved and proteases removed in a single step (see Fig 13 and application example 6 in Chapter 7) (see Procedure 22 for additional information). Samples should be removed from the protease digest mixture at various time points and analyzed by SDS-PAGE to estimate the yield, purity, and extent of digestion. Table 17. Approximate molecular weights for SDS-PAGE analysis Protease
Molecular weight
PreScission Protease*
46 000
Bovine thrombin
37 000
Bovine Factor Xa
48 000
* PreScission Protease is a fusion protein of glutathione S-transferase and human rhinovirus type 14 3C protease (31).
The amount of enzyme, temperature, and length of incubation required for complete digestion varies according to the specific GST fusion protein produced. Optimal conditions should always be determined in pilot experiments. If protease inhibitors (see Table 18) have been used in the lysis solution, they must be removed prior to cleavage with PreScission Protease, thrombin, or Factor Xa. (The inhibitors will usually be eluted in the flow-through when sample is loaded onto a GSTrap FF column.)
51
Cleavage of GST tag using PreScission Protease 1
Add cell lysate to prepacked GST MicroSpin or GSTrap FF column
3
Elute with reduced glutathione
4
Cleave eluted fusion protein with PreScission Protease
Off-column cleavage
2 Wash On-column cleavage
3
Cleave fusion protein with PreScission Protease
Fig 12. Flow chart of the affinity purification procedure and PreScission Protease cleavage of glutathione S-transferase fusion proteins.
Cleavage of GST tag using thrombin or Factor Xa 1
Add cell lysate to prepacked GST MicroSpin or GSTrap FF column
3
Elute with reduced glutathione
4
Cleave eluted fusion protein with site-specific protease (thrombin or Factor Xa)
Off-column cleavage
2 Wash On-column cleavage
3
Cleave fusion protein with site-specific protease (thrombin or Factor Xa)
If using GSTrap FF, connect the column directly to a HiTrap Benzamidine FF (high sub) before elution. Cleaved product passes directly from the GSTrap FF into the HiTrap Benzamidine FF (high sub). Samples are cleaved and the protease removed in a single step.
GSTrap FF
HiTrap Benzamidine FF (high sub) Fig 13. Flow chart of the affinity purification procedure and thrombin or Factor Xa cleavage of glutathione S-transferase fusion proteins.
52
5
HiTrap Desalting column
6
Add sample to GST MicroSpin or GSTrap FF column
7
4
5
HiTrap Desalting column
6
Add sample to GST MicroSpin or GSTrap FF column
Collect eluate
8
Analyze protein e.g. on SDS-PAGE or by mass spectrometry
5
Analyze protein e.g. on SDS-PAGE or by mass spectrometry
Collect flow-through
7
Collect eluate
9
Remove protease if necessary, using HiTrap Benzamidine FF (high sub)
Ettan™ MALDI-ToF mass spectrometer
Sepharose
Glutathione
8
Analyze protein e.g. on SDS-PAGE or by mass spectrometry
Glutathione S-transferase Cloned protein
4 4
Collect flow-through
5
Analyze protein e.g. on SDS-PAGE or by mass spectrometry
5
Analyze protein e.g. on SDS-PAGE or by mass spectrometry
6
Remove protease if necessary, using HiTrap Benzamidine FF (high sub)
Collect flow-through
GST fusion protein Thrombin or Factor Xa PreScission Protease
53
Table 18. Inhibitors of the various proteases Enzyme
Inhibitor
PreScission Protease
100 mM ZnCl2 (> 50% inhibition) 100 µM chymostatin 4 mM Pefabloc™
Factor Xa and thrombin
AEBSF, APMSF, antithrombin III, Antipain, a1-antitrypsin, aprotinin, chymostatin, hirudin, leupeptin, PMSF
Factor Xa only
Pefabloc FXa
Thrombin only
Pefabloc TH Benzamidine
Summary of procedures Cleavage of fusion proteins is most commonly performed on milligram quantities of fusion protein suitable for purification on GSTrap FF columns. Protocols that follow describe manual cleavage and purification using a syringe and a 1 ml or 5 ml GSTrap FF column. The protocols can be adapted for use with GST MicroSpin columns to work at smaller scales. For quick scale-up of purifications, two or three GSTrap FF columns can be connected in series (back pressure will be higher). Further scaling-up is possible using GSTPrep FF 16/10 columns or columns packed by the user with Glutathione Sepharose 4 Fast Flow. Protocols are included for column or batch format using this medium. Table 19 lists the cleavage procedures found in this chapter. Table 19. Procedures for removal of GST by enzymatic cleavage Procedure
Description
PreScission Protease cleavage and purification 19.1
PreScission Protease cleavage and purification of GST fusion protein bound to GSTrap FF
19.2
PreScission Protease cleavage and purification of GST fusion protein eluted from GSTrap FF
19.3
PreScission Protease cleavage and purification of GST fusion protein bound to Glutathione Sepharose 4 Fast Flow or Glutathione Sepharose 4B
19.4
PreScission Protease cleavage and purification of GST fusion protein eluted from Glutathione Sepharose 4 Fast Flow or Glutathione Sepharose 4B
Thrombin cleavage and purification 20.1
Thrombin cleavage and purification of GST fusion protein bound to GSTrap FF
20.2
Thrombin cleavage and purification of GST fusion protein eluted from GSTrap FF
20.3
Thrombin cleavage and purification of GST fusion protein bound to Glutathione Sepharose 4 Fast Flow or Glutathione Sepharose 4B
20.4
Thrombin cleavage and purification of GST fusion protein eluted from Glutathione Sepharose 4 Fast Flow or Glutathione Sepharose 4B
Factor Xa cleavage and purification 21.1
Factor Xa cleavage and purification of GST fusion protein bound to GSTrap FF
21.2
Factor Xa cleavage and purification of GST fusion protein eluted from GSTrap FF
21.3
Factor Xa cleavage and purification of GST fusion protein bound to Glutathione Sepharose 4 Fast Flow or Glutathione Sepharose 4B
21.4
Factor Xa cleavage and purification of GST fusion protein eluted from Glutathione Sepharose 4 Fast Flow or Glutathione Sepharose 4B
Removal of proteases 22
Removal of proteases using HiTrap Benzamidine FF (high sub)
One ml/min corresponds to approximately 30 drops/min when using a syringe with HiTrap 1 ml column, and 5 ml/min corresponds to approximately 120 drops/min when using HiTrap 5 ml column.
54
PreScission Protease cleavage and purification PreScission Protease is a fusion protein of GST and human rhinovirus 3C protease (33). The protease specifically recognizes the amino acid sequence Leu-Glu-Val-Leu-PheGln¡Gly-Pro, cleaving between the Gln and Gly residues (34). Since the protease is fused to GST, it is easily removed from cleavage reactions using GSTrap FF or Glutathione Sepharose. Because this protease is maximally active at 4 °C, cleavage can be performed at low temperatures, thus improving the stability of the target protein.
19.1. PreScission Protease cleavage and purification of GST fusion protein bound to GSTrap FF Reagents required PreScission cleavage buffer: 50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, pH 7.0 PreScission Protease Binding buffer: 1× PBS (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.3)
Cleavage should be complete following a 4 h treatment at 5 °C with at least 10 units of enzyme/mg of fusion protein. Incubation times can be reduced by adding more PreScission Protease. Steps
Assume: 8 mg GST fusion protein bound/ml medium. 1. Fill the syringe with binding buffer. 2. Connect the column to the syringe using the adapter supplied. Avoid introducing air into the column. 3. Remove the twist-off end. 4. Equilibrate the column with five column volumes of binding buffer. 5.
Apply the sample using the syringe. For best results, maintain a flow rate of 0.2–1 ml/min (for both 1 ml and 5 ml columns) as the sample is applied.
6. Wash the column with 5–10 column volumes of binding buffer. Maintain flow rates of 1–2 ml/min (1 ml column) or 1–5 ml/min (5 ml column). 7. Wash the column with 10 column volumes of PreScission cleavage buffer. 8. Prepare the PreScission Protease mix: - For GSTrap FF 1 ml columns, mix 80 µl (160 units) of PreScission Protease with 920 µl of PreScission cleavage buffer at 5 °C. - For GSTrap FF 5 ml columns, mix 400 µl (800 units) of PreScission Protease with 4.6 ml of PreScission cleavage buffer at 5 °C. 9. Load the PreScission Protease mix onto the column using a syringe and the adapter supplied. Seal the column with the top cap and the domed nut supplied. 10. Incubate the column at 5 °C for 4 h. 11. Fill a syringe with 3 ml (1 ml column) or 15 ml (5 ml column) of PreScission cleavage buffer. Remove the top cap and domed nut from the column and attach the syringe. Avoid introducing air into the column. 12. Begin elution. Maintain flow rates of 1–2 ml/min (1 ml column) or 1–5 ml/min (5 ml column), and collect the eluate (0.5–1 ml/tube for 1 ml column, 1–2 ml/tube for 5 ml column).
Note: The eluate will contain the protein of interest, while the GST moiety of the fusion protein and the PreScission Protease will remain bound to GSTrap FF.
55
19.2. PreScission Protease cleavage and purification of GST fusion protein eluted from GSTrap FF Reagents required PreScission cleavage buffer: 50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, pH 7.0 PreScission Protease Binding buffer: 1× PBS (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.3) Elution buffer: 50 mM Tris-HCl, 10 mM reduced glutathione, pH 8.0
Steps
Assume: 8 mg GST fusion protein bound/ml medium. 1. Fill the syringe with binding buffer. 2. Connect the column to the syringe using the adapter supplied. Avoid introducing air into the column. 3. Remove the twist-off end. 4. Equilibrate the column with five column volumes of binding buffer. 5. Apply the sample using the syringe. For best results, maintain a flow rate of 0.2–1 ml/min (for both 1 ml and 5 ml columns) as the sample is applied. 6. Wash the column with 5–10 column volumes of binding buffer. Maintain flow rates of 1–2 ml/min (1 ml column) or 1–5 ml/min (5 ml column). 7. Elute with 5–10 column volumes of elution buffer. Maintain flow rates of 1–2 ml/min (1 ml column) or 1–5 ml/min (5 ml column). Collect the eluate (0.5–1 ml/tube for 1 ml column, 1–2 ml/tube for 5 ml column). Pool fractions containing the GST fusion protein (monitored by UV absorption at A280). 8. Remove the free reduced glutathione from the eluate using a quick buffer exchange on HiTrap Desalting or HiPrep 26/10 Desalting, depending on the sample volume.
Desalting will remove the free glutathione. Glutathione bound to the GST protein can be removed by dialyzing the eluate against PreScission cleavage buffer. 9. Add 1 µl (2 units) of PreScission Protease for each 100 µg of fusion protein in the eluate. 10. Incubate at 5 °C for 4 h. 11. Once digestion is complete, apply the sample to an equilibrated GSTrap FF column as described above (steps 1–7) to remove the GST moiety of the fusion protein and the PreScission Protease.
Note: The protein of interest will be found in the flow-through, while the GST moiety of the fusion protein and the PreScission Protease will remain bound to the column.
If the amount of fusion protein in the eluate has not been determined, add 80 µl (160 units) of PreScission Protease for fusion protein eluted from a GSTrap FF 1 ml column or add 400 µl (800 units) of PreScission Protease for fusion protein eluted from a GSTrap FF 5 ml column.
56
19.3. PreScission Protease cleavage and purification of GST fusion protein bound to Glutathione Sepharose 4 Fast Flow or Glutathione Sepharose 4B This procedure assumes that the GST fusion protein has been bound and washed as described in a purification procedure from Chapter 4. Reagents required PreScission Protease PreScission cleavage buffer: 50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol (DTT), pH 7.5
Steps
Assume: 8 mg GST fusion protein bound/ml medium. 1. Wash the fusion-protein-bound Glutathione Sepharose medium with 10 bed volumes of PreScission cleavage buffer. Bed volume is equal to 0.5× the volume of the 50% Glutathione Sepharose slurry used. 2. Prepare the PreScission Protease mix: For each ml of Glutathione Sepharose bed volume, prepare a mixture of 80 µl (160 units) of PreScission Protease and 920 µl of PreScission cleavage buffer at 5 °C. 3. Load the PreScission Protease mixture onto the column. Seal the column. If a batch format is used, centrifuge the suspension at 500 × g for 5 min, decant the supernatant, and add the PreScission Protease mixture to the Glutathione Sepharose pellet. Gently shake or rotate the suspension. 4. Incubate at 5 °C for 4 h. 5. Following incubation, elute the column with approximately three bed volumes of PreScission cleavage buffer. Collect the eluate in different tubes to avoid dilution of the fusion protein. Analyze the contents of each tube. If a batch format is used, centrifuge the suspension at 500 × g for 5 min to pellet the Glutathione Sepharose. Carefully transfer the eluate to a tube.
Note: The eluate will contain the protein of interest, while the GST portion of the fusion protein and the PreScission Protease will remain bound to the Glutathione Sepharose medium.
19.4. PreScission Protease cleavage and purification of GST fusion protein eluted from Glutathione Sepharose 4 Fast Flow or Glutathione Sepharose 4B Reagents required PreScission Protease PreScission cleavage buffer: 50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol (DTT), pH 7.5
Steps
Assume: 8 mg GST fusion protein bound/ml medium. 1. Following elution of the GST fusion protein from either a batch or column purification format as described in Chapter 4, remove the reduced glutathione from the eluate using a quick buffer exchange on HiTrap Desalting, a PD-10 disposable column, or HiPrep 26/10 Desalting, depending on sample volume.
Desalting will remove the free glutathione. Glutathione bound to the GST protein can be removed by dialyzing the eluate against PreScission cleavage buffer. 2. Add 1 µl (2 units) of PreScission Protease for each 100 µg of fusion protein in the eluate.
If the amount of fusion protein in the eluate has not been determined, add 80 µl (160 units) of PreScission Protease for each ml of Glutathione Sepharose bed volume from which the fusion protein was eluted. 57
3. Incubate at 5 °C for 4 h. 4. Once digestion is complete, apply the sample to washed and equilibrated Glutathione Sepharose to remove the GST moiety of the fusion protein and the PreScission Protease. 5. Incubate for 20–30 min at room temperature (22–25 °C). 6. Sediment the medium by centrifuging at 500 × g for 5 min and carefully transfer the supernatant to a new tube.
Note: The protein of interest will be found in the supernatant, while the GST moiety and the PreScission Protease will remain bound to the Glutathione Sepharose medium.
Thrombin cleavage and purification With a specific activity > 7500 units/mg protein, one unit of thrombin will digest > 90% of 100 µg of a test fusion protein in 16 h at 22 °C in elution buffer. One unit is approximately equal to 0.2 NIH units. Cleavage should be complete following overnight treatment with < 10 units of enzyme/mg of fusion protein. Thrombin can be removed using Benzamidine Sepharose FF (high sub), a purification medium with a high specificity for serine proteases (see Procedure 22). A GSTrap FF column and a HiTrap Benzamidine FF (high sub) column can be connected in series so that cleaved product passes directly from the GSTrap FF into the HiTrap Benzamidine FF (high sub). Samples are cleaved and the thrombin removed in a single step.
20.1. Thrombin cleavage and purification of GST fusion protein bound to GSTrap FF Reagents required 1× PBS (binding buffer): 140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.3 Thrombin solution: Dissolve 500 units in 0.5 ml of 1× PBS pre-chilled to 4 °C. Swirl gently. Store solution in small aliquots at -80 °C to preserve activity.
Steps
Assume: 8 mg GST fusion protein bound/ml medium. 1. Fill the syringe with binding buffer. 2. Connect the column to the syringe using the adapter supplied. Avoid introducing air into the column. 3. Remove the twist-off end. 4. Equilibrate the column with five column volumes of binding buffer. 5. Apply the sample using the syringe. For best results, maintain a flow rate of 0.2–1 ml/min (for both 1 ml and 5 ml columns) as the sample is applied. 6. Wash the column with 5–10 column volumes of binding buffer. Maintain flow rates of 1–2 ml/min (1 ml column) or 1–5 ml/min (5 ml column). 7. Prepare the thrombin mix: - For GSTrap FF 1 ml columns, mix 80 µl (80 units) of thrombin solution with 920 µl of 1× PBS. - For GSTrap FF 5 ml columns, mix 400 µl (400 units) of thrombin solution with 4.6 ml of 1× PBS. 8. Load the thrombin mix onto the column using a syringe and the adapter supplied. Seal the column with the top cap and the domed nut supplied. 9. Incubate the column at room temperature (22–25 °C) for 2–16 h.
58
10. Fill a syringe with 3 ml (1 ml column) or 15 ml (5 ml column) of 1× PBS. Remove the top cap and domed nut from the column. Avoid introducing air into the column. Begin elution. Maintain flow rates of 1–2 ml/min (1 ml column) or 1–5 ml/min (5 ml column). 11. Collect the eluate (0.5 ml–1 ml/tube for 1 ml column, 1–2 ml/tube for 5 ml column).
Note: The eluate will contain the protein of interest and thrombin, while the GST moiety of the fusion protein will remain bound to GSTrap FF.
After cleavage using thrombin, the enzyme can be removed from the eluted protein using HiTrap Benzamidine FF (high sub) (see Procedure 22).
20.2. Thrombin cleavage and purification of GST fusion protein eluted from GSTrap FF Reagents required 1× PBS (binding buffer): 140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.3 Elution buffer: 50 mM Tris-HCl, 10 mM reduced glutathione, pH 8.0 Thrombin solution: Dissolve 500 units in 0.5 ml of 1× PBS pre-chilled to 4 °C. Swirl gently. Store solution in small aliquots at -80 °C to preserve activity.
Steps
Assume: 8 mg GST fusion protein bound/ml medium. 1. Fill the syringe with binding buffer. 2. Connect the column to the syringe using the adapter supplied. Avoid introducing air into the column. 3. Remove the twist-off end. 4. Equilibrate the column with five column volumes of binding buffer. 5.
Apply the sample using the syringe. For best results, maintain a flow rate of 0.2–1 ml/min (for both 1 ml and 5 ml columns) as the sample is applied.
6. Wash the column with 5–10 column volumes of binding buffer. Maintain flow rates of 1–2 ml/min (1 ml column) or 1–5 ml/min (5 ml column). 7. Elute with 5–10 column volumes of elution buffer. Maintain flow rates of 1–2 ml/min (1 ml column) or 1–5 ml/min (5 ml column). Collect the eluate (0.5–1 ml/tube for 1 ml column, 1–2 ml/tube for 5 ml column). Pool fractions containing the fusion protein (monitored by UV absorption at A280). 8. Add 10 µl (10 units) of thrombin solution for each mg of fusion protein in the eluate.
If the amount of fusion protein in the eluate has not been determined, add 80 µl (80 units) of thrombin solution for fusion protein eluted from a GSTrap FF 1 ml column or add 400 µl (400 units) of thrombin solution for fusion protein eluted from a GSTrap FF 5 ml column. 9. Incubate at room temperature (22–25 °C) for 2–16 h. 10. Once digestion is complete, remove the reduced glutathione using a quick buffer exchange on HiTrap Desalting or HiPrep 26/10 Desalting, depending on the sample volume. Exchange with 1× PBS (binding buffer).
Desalting will remove the free glutathione. Glutathione bound to the GST protein can be removed by dialyzing the eluate against Thrombin cleavage buffer (1× PBS).
59
11. Apply the sample to an equilibrated GSTrap FF column to remove the GST moiety of the fusion protein.
Note: The purified protein of interest and thrombin will be found in the flow-through.
After cleavage using thrombin, the enzyme can be removed from the eluted protein using HiTrap Benzamidine FF (high sub) (see Procedure 22).
20.3. Thrombin cleavage and purification of GST fusion protein bound to Glutathione Sepharose 4 Fast Flow or Glutathione Sepharose 4B This procedure assumes that the GST fusion protein has been bound and washed as described in a purification procedure from Chapter 4. Reagents required 1× PBS: 140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.3 Thrombin solution: Dissolve 500 units in 0.5 ml of 1× PBS pre-chilled to 4 °C. Swirl gently. Store solution in small aliquots at -80 °C to preserve activity.
Steps
Assume: 8 mg GST fusion protein bound/ml medium. 1. Wash the fusion-protein-bound Glutathione Sepharose with 10 bed volumes of 1× PBS. Bed volume is equal to 0.5× the volume of the 50% Glutathione Sepharose slurry used. 2. Prepare the thrombin mix: For each ml of Glutathione Sepharose bed volume, prepare a mixture of 80 µl (80 units) of thrombin and 920 µl of 1× PBS. 3. Load the thrombin mixture onto the column. Seal the column. If a batch format is used, centrifuge the suspension at 500 × g for 5 min, decant the supernatant, and add the thrombin mixture to the Glutathione Sepharose pellet. Gently shake or rotate the suspension. 4. Incubate at room temperature (22–25 °C) for 2–16 h. 5. Following incubation, elute the column with approximately three bed volumes of 1× PBS. Collect the eluate in different tubes to avoid dilution of the fusion protein. Analyze the contents of each tube. If a batch format is used, centrifuge the suspension at 500 × g for 5 min to pellet the Glutathione Sepharose. Carefully transfer the eluate to a tube.
Note: The eluate will contain the protein of interest and thrombin, while the GST portion of the fusion protein will remain bound to the Glutathione Sepharose medium.
After cleavage using thrombin, the enzyme can be removed from the eluted protein using HiTrap Benzamidine FF (high sub) (see Procedure 22).
20.4. Thrombin cleavage and purification of GST fusion protein eluted from Glutathione Sepharose 4 Fast Flow or Glutathione Sepharose 4B Reagents required 1× PBS: 140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.3 Thrombin solution: Dissolve 500 units in 0.5 ml of 1× PBS pre-chilled to 4 °C. Swirl gently. Store solution in small aliquots at -80 °C to preserve activity.
60
Steps
Assume: 8 mg GST fusion protein bound/ml medium. 1. Following elution of the GST fusion protein from either a batch or column purification format as described in Chapter 4, remove the reduced glutathione from the eluate using a quick buffer exchange on HiTrap Desalting, a PD-10 column, or HiPrep 26/10 Desalting, depending on the sample volume. 2. Add 10 µl (10 units) of thrombin solution for each mg of fusion protein in the eluate.
If the amount of fusion protein in the eluate has not been determined, add 80 µl (80 units) of thrombin solution for each ml of Glutathione Sepharose bed volume from which the fusion protein was eluted. 3. Incubate at room temperature (22–25 °C) for 2–16 h. 4. Once digestion is complete, remove the reduced glutathione using a quick buffer exchange on HiTrap Desalting, a PD-10 column, or HiPrep 26/10 Desalting, depending on the sample volume.
Desalting will remove the free glutathione. Glutathione bound to the GST protein can be removed by dialyzing the eluate against thrombin cleavage buffer (1× PBS). 5. Apply the sample to washed and equilibrated Glutathione Sepharose to remove the GST moiety of the fusion protein. 6. Incubate for 20–30 min at room temperature (22–25 °C). 7. Sediment the medium by centrifuging at 500 × g for 5 min and carefully transfer the supernatant to a new tube.
Note: The supernatant will contain the protein of interest and thrombin, while the GST portion of the fusion protein will remain bound to the Glutathione Sepharose medium.
After cleavage using thrombin, the enzyme can be removed from the eluted protein using HiTrap Benzamidine FF (high sub) (see Procedure 22).
Factor Xa cleavage and purification With a specific activity of > 800 units/mg protein, one unit of Factor Xa will digest > 90% of 100 µg of a test fusion protein in 16 h at 22 °C in Factor Xa cleavage buffer. Cleavage should be complete following overnight treatment at 22 °C with a Factor Xa to substrate ratio of at least 1% (w/w). Factor Xa can be removed using Benzamidine Sepharose FF (high sub), a purification medium with a high specificity for serine proteases (see Procedure 22). A GSTrap FF column and a HiTrap Benzamidine FF (high sub) column can be connected in series so that cleaved product passes directly from the GSTrap FF into the HiTrap Benzamidine FF (high sub). Samples are cleaved and the Factor Xa removed in a single step. Heparin Sepharose has also been used for this application; however, since benzamidine has a higher specificity for Factor Xa, the protease will be removed more efficiently. Factor Xa consists of two subunits linked by disulfide bridges. Since glutathione can disrupt disulfide bridges, it should be removed from the sample prior to the cleavage reaction. Free glutathione can be easily and rapidly removed from the sample using a desalting column with Factor Xa cleavage buffer as eluent, followed by dialysis to remove any glutathione bound to the GST protein.
61
21.1. Factor Xa cleavage and purification of GST fusion protein bound to GSTrap FF Reagents required 1× PBS (binding buffer): 140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.3 Factor Xa cleavage buffer: 50 mM Tris-HCl, 150 mM NaCl, 1 mM CaCl2, pH 7.5 Factor Xa solution: Dissolve 400 units of Factor Xa in 4 °C H2O to give a final solution of 1 unit/µl. Swirl gently. Store solution in small aliquots at -80 °C to preserve activity.
Steps
Assume: 8 mg GST fusion protein bound/ml medium. 1. Fill the syringe with binding buffer. 2. Connect the column to the syringe using the adapter supplied. Avoid introducing air into the column. 3. Remove the twist-off end. 4. Equilibrate the column with five column volumes of binding buffer. 5. Apply the sample using the syringe. For best results, maintain a flow rate of 0.2–1 ml/min (for both 1 ml and 5 ml columns) as the sample is applied. 6. Wash the column with 5–10 column volumes of binding buffer. Maintain flow rates of 1–2 ml/min (1 ml column) or 1–5 ml/min (5 ml column). 7. Wash the column with 10 column volumes of Factor Xa cleavage buffer. 8. Prepare the Factor Xa mix: - For GSTrap FF 1 ml columns, mix 80 µl (80 units) of Factor Xa solution with 920 µl of Factor Xa cleavage buffer. - For GSTrap FF 5 ml columns, mix 400 µl (400 units) of Factor Xa solution with 4.6 ml of Factor Xa cleavage buffer. 9. Load the Factor Xa mix onto the column using a syringe and the adapter supplied. Seal the column with the top cap and the domed nut. 10. Incubate the column at room temperature (22–25 °C) for 2–16 h. 11. Fill a syringe with 3 ml (1 ml column) or 15 ml (5 ml column) of Factor Xa cleavage buffer. Remove the top cap and domed nut from the column. Avoid introducing air into the column. Begin the elution. Maintain flow rates of 1–2 ml/min (1 ml column) or 1–5 ml/min (5 ml column). 12. Collect the eluate (0.5–1 ml/tube for 1 ml column, 1–2 ml/tube for 5 ml column).
Note: The eluate will contain the protein of interest and Factor Xa, while the GST moiety of the fusion protein will remain bound to GSTrap FF.
After cleavage using Factor Xa, the enzyme can be removed from the eluted protein using HiTrap Benzamidine FF (high sub) (see Procedure 22).
62
21.2. Factor Xa cleavage and purification of GST fusion protein eluted from GSTrap FF Reagents required 1× PBS (binding buffer): 140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.3 Elution buffer: 50 mM Tris-HCl, 10 mM reduced glutathione, pH 8.0 Factor Xa cleavage buffer: 50 mM Tris-HCl, 150 mM NaCl, 1 mM CaCl2, pH 7.5 Factor Xa solution: Dissolve 400 units of Factor Xa in 4 °C H2O to give a final solution of 1 unit/µl. Swirl gently. Store solution in small aliquots at -80 °C to preserve activity.
Steps
Assume: 8 mg GST fusion protein bound/ml medium. 1. Fill the syringe with binding buffer. 2. Connect the column to the syringe using the adapter supplied. Avoid introducing air into the column. 3. Remove the twist-off end. 4. Equilibrate the column with five column volumes of binding buffer. 5.
Apply the sample using the syringe. For best results, maintain a flow rate of 0.2–1 ml/min (for both 1 ml and 5 ml columns) as the sample is applied.
6. Wash the column with 5–10 column volumes of binding buffer. Maintain flow rates of 1–2 ml/min (1 ml column) or 1–5 ml/min (5 ml column). 7.
Elute with 5–10 column volumes of elution buffer. Maintain flow rates of 1–2 ml/min (1 ml column) or 1–5 ml/min (5 ml column). Collect the eluate (0.5 ml/tube for 1 ml column, 1–2 ml/tube for 5 ml column). Pool fractions containing the GST fusion protein (monitored by UV absorption at A280).
8. Remove the reduced glutathione from the eluate using a quick buffer exchange on HiTrap Desalting or HiPrep 26/10 Desalting, depending on sample volume. Exchange with 1× PBS (binding buffer).
Desalting will remove the free glutathione. Glutathione bound to the GST protein can be removed by dialyzing the eluate against cleavage buffer. 9. Add 10 units of Factor Xa solution for each mg of fusion protein in the eluate.
If the amount of fusion protein in the eluate has not been determined, add 80 µl (80 units) of Factor Xa solution for fusion protein eluted from a GSTrap FF 1 ml column or add 400 µl (400 units) of Factor Xa solution for fusion protein eluted from a GSTrap FF 5 ml column. 10. Incubate at room temperature (22–25 °C) for 2–16 h. 11. Once digestion is complete, apply the sample to an equilibrated GSTrap FF column as described above (steps 1–7) to remove the GST moiety of the fusion.
Note: The protein of interest will be found in the flow-through together with Factor Xa.
After cleavage using Factor Xa, the enzyme can be removed from the eluted protein using HiTrap Benzamidine FF (high sub) (see Procedure 22).
21.3. Factor Xa cleavage and purification of GST fusion protein bound to Glutathione Sepharose 4 Fast Flow or Glutathione Sepharose 4B This procedure assumes that the GST fusion protein has been bound and washed as described in a purification procedure from Chapter 4. 63
Reagents required Factor Xa cleavage buffer: 50 mM Tris-HCl, 150 mM NaCl, 1 mM CaCl2, pH 7.5 Factor Xa solution: Dissolve 400 units of Factor Xa in 4 °C H2O to give a final solution of 1 unit/µl. Swirl gently. Store solution in small aliquots at -80 °C to preserve activity.
Steps
Assume: 8 mg GST fusion protein bound/ml medium. 1. Wash the fusion-protein-bound Glutathione Sepharose medium with 10 bed volumes of Factor Xa cleavage buffer. Bed volume is equal to 0.5× the volume of the 50% Glutathione Sepharose slurry used. 2. Prepare the Factor Xa mix: For each ml of Glutathione Sepharose bed volume, prepare a mixture of 80 µl (80 units) of Factor Xa and 920 µl of Factor Xa cleavage buffer. 3. Load the Factor Xa mix onto the column. Seal the column. If a batch format is used, centrifuge the suspension at 500 × g for 5 min, decant the supernatant, and add the Factor Xa mix to the Glutathione Sepharose pellet. Gently shake or rotate the suspension. 4. Incubate at room temperature (22–25 °C) for 2–16 h. 5. Following incubation, elute the column with approximately three bed volumes of Factor Xa cleavage buffer. Collect the eluate in different tubes to avoid dilution of the fusion protein and analyze it. If a batch format is used, centrifuge the suspension at 500 × g for 5 min to pellet the Glutathione Sepharose medium. Carefully transfer the eluate to a tube.
Note: The eluate will contain the protein of interest and Factor Xa, while the GST portion of the fusion protein will remain bound to the Glutathione Sepharose medium.
After cleavage using Factor Xa, the enzyme can be removed from the eluted protein using HiTrap Benzamidine FF (high sub) (see Procedure 22).
21.4. Factor Xa cleavage and purification of GST fusion protein eluted from Glutathione Sepharose 4 Fast Flow or Glutathione Sepharose 4B Reagents required Factor Xa cleavage buffer: 50 mM Tris-HCl, 150 mM NaCl, 1 mM CaCl2, pH 7.5 Factor Xa solution: Dissolve 400 units of Factor Xa in 4 °C H2O to give a final solution of 1 unit/µl. Swirl gently. Store solution in small aliquots at -80 °C to preserve activity.
Steps
Assume: 8 mg GST fusion protein bound/ml medium. 1. Following elution of the GST fusion protein from either a batch or column purification format as described in Chapter 4, remove reduced glutathione from the eluate using a quick buffer exchange on HiTrap Desalting, a PD-10 column, or HiPrep 26/10 Desalting, depending on sample volume.
Desalting will remove the free glutathione. Glutathione bound to the GST protein can be removed by dialyzing the eluate against Factor Xa cleavage buffer. 2. Add 10 µl (10 units) of Factor Xa solution for each mg of fusion protein in the eluate.
If the amount of fusion protein in the eluate has not been determined, add 80 µl (80 units) of Factor Xa solution for each ml of Glutathione Sepharose bed volume from which the fusion protein was eluted.
64
3. Incubate at room temperature (22–25 °C) for 2–16 h. 4. Once digestion is complete, apply the sample to washed and equilibrated Glutathione Sepharose to remove the GST moiety of the fusion protein. 5. Incubate for 20–30 min at room temperature. 6. Sediment the medium by centrifuging at 500 × g for 5 min and carefully transfer the supernatant to a new tube.
Note: The protein of interest will be found in the supernatant together with Factor Xa.
After cleavage using Factor Xa, the enzyme can be removed from the eluted protein using HiTrap Benzamidine FF (high sub) (see Procedure 22).
Removal of proteases using Benzamidine Sepharose 4 Fast Flow (high sub) To protect the fusion protein from proteolytic degradation prior to enzymatic cleavage with PreScission Protease, thrombin, or Factor Xa, it may be necessary to remove proteases from the sample. Additionally, following enzymatic cleavage, it may be necessary to remove thrombin or Factor Xa from the sample. Benzamidine Sepharose 4 Fast Flow (high sub) provides a convenient and highly specific medium for the removal of trypsin and trypsin-like serine proteases, not only from enzymatic digests but also from cell culture supernatants, bacterial lysates, or serum. In the procedure that follows, the GST fusion protein is present in the flow-through and wash, whereas the protease remains bound to the medium until eluted. Elution buffers are listed in the procedure so that after the GST fusion protein has been collected, the bound protease can be eluted. This step is necessary if the medium is to be reused. See Appendix 5 for more information on eluting the protease from the column. Benzamidine Sepharose 4 Fast Flow is available in either prepacked 1 ml or 5 ml HiTrap columns or in packages for scaling up purifications. HiTrap columns can be operated with a syringe together with the supplied adapters, a pump, or a liquid chromatography system, e.g. ÄKTAdesign. See Table 20 for a selection guide of purification options. Characteristics of HiTrap Benzamidine FF (high sub) are summarized in Table 21. Table 20. Selection guide for purification options to remove thrombin and Factor Xa Column (prepacked) or medium
Binding capacity for trypsin
Comments
HiTrap Benzamidine FF (high sub), 1 ml
> 35 mg trypsin
Prepacked 1 ml column
HiTrap Benzamidine FF (high sub), 5 ml
> 175 mg trypsin
Prepacked 5 ml column
Benzamidine Sepharose 4 Fast Flow (high sub)
> 35 mg trypsin/ml medium
For column packing and scale-up
65
Table 21. Characteristics of HiTrap Benzamidine FF (high sub) Column dimensions (i.d. × h)
0.7 × 2.5 cm (1 ml) and 1.6 × 2.5 cm (5 ml)
Column volumes
1 ml and 5 ml
Ligand
p-Aminobenzamidine (pABA)
Spacer
14-atom
Ligand concentration
> 12 µmol p-Aminobenzamidine/ml medium
Binding capacity
> 35 mg trypsin/ml medium
Mean particle size
90 µm
Bead structure
Highly cross-linked agarose, 4%
Maximum back pressure
0.3 MPa, 3 bar
Recommended flow rates
1 ml/min (1 ml column) and 5 ml/min (5 ml column)
Maximum flow rates
4 ml/min (1 ml column) and 20 ml/min (5 ml column)
Chemical stability
All commonly used aqueous buffers
pH stability short term*
pH 1–9
pH stability long term*
pH 2–8
Storage temperature
4–8 °C
Storage buffer
20% ethanol in 0.05 M acetate buffer, pH 4
*The ranges given are estimates based on our knowledge and experience. Please note the following: pH stability, short term refers to the pH interval for regeneration, cleaning-in-place, and sanitization procedures. pH stability, long term refers to the pH interval where the medium is stable over a long period of time without adverse effects on its chromatographic performance.
The column is made of polypropylene, which is biocompatible and non-interactive with biomolecules. The top and bottom frits are manufactured from porous polyethylene. Columns are delivered with a stopper on the inlet and a twist-off end on the outlet. Both ends have M6 connections (6 mm metric threads). The column cannot be opened or refilled. Buffer and sample preparation Water and chemicals used for buffer preparation should be of high purity. Buffers should be filtered through a 0.45 µm filter and de-gassed before use. Samples should be centrifuged and/or filtered through a 0.45 µm filter immediately before applying to the column. If the sample is too viscous, dilute it with binding buffer to prevent column clogging. Recommended binding and wash buffers HiTrap Benzamidine FF (high sub) has some ionic binding characteristics; therefore, binding and wash buffers should contain at least 0.5 M salt and have a pH of 7.4–8. An appropriate buffer would be 0.05 M Tris-HCl, 0.5 M NaCl, pH 7.4. If a lower salt concentration is used, include a high-salt wash step prior to elution of the bound protease. Recommended elution buffers See Appendix 5 for a discussion of elution options for removing bound protease from the medium.
66
22. Removal of thrombin and Factor Xa using HiTrap Benzamidine FF (high sub) Reagents required Binding buffer: 0.05 M Tris-HCl, 0.5 M NaCl, pH 7.4 Elution buffer alternatives (for removing the protease after the GST fusion protein has come through in the flowthrough and wash; see Appendix 5): - 0.05 M glycine-HCl, pH 3.0 - 10 mM HCl, 0.5 M NaCl, pH 2.0 - 20 mM p-Aminobenzamidine in binding buffer (competitive elution) - 8 M urea or 6 M guanidine hydrochloride (denaturing solutions)
Steps
Recommended flow rates are 1 ml/min (1 ml column) or 5 ml/min (5 ml column). 1. Fill the pump tubing or syringe with distilled H2O. Connect the column to the syringe, using the adapter supplied, or to the pump tubing. Avoid introducing air into the column. 2. Remove the twist-off end. 3. Wash the column with five column volumes of distilled H 2O to remove the storage buffer (0.05 M acetate buffer, pH 4, containing 20% ethanol). 4. Equilibrate the column with five column volumes of binding buffer. 5. Apply the sample using a syringe fitted to the luer adapter or by pumping it onto the column. Recommended flow rates for sample application are 1 ml/min for 1 ml column and 5 ml/min for 5 ml column. Collect any flow-through and reserve. 6. Wash the column with 5–10 column volumes of binding buffer, collecting fractions (0.5–1 ml fractions for 1 ml column and 1–3 ml fractions for 5 ml column) until no material appears in the effluent (monitored by UV absorption at A280). 7. Pool fractions from flow-through and wash that contain the GST fusion protein (monitored by UV absorption at A280). 8. For re-use of column, elute the bound protease with 5–10 column volumes of the elution buffer of choice (see above and Appendix 5). If the eluted thrombin or Factor Xa is to be retained, buffer-exchange the fractions containing the protease using HiTrap Desalting or PD-10 Desalting column. If a low pH elution buffer has been used, collect fractions in neutralization buffer. 9.
After all protease has been eluted, wash the column with binding buffer so it is ready for re-use. See Appendix 5 for additional cleaning and storage information. *Since elution conditions are quite harsh, collect fractions into neutralization buffer (60–200 µl of 1 M Tris-HCl, pH 9.0 per ml fraction collected), so that the final pH of the fractions will be approximately neutral.
An example showing isolation of a GST fusion protein on a GSTrap FF column and on-column cleavage with thrombin, followed by purification on HiTrap Benzamidine FF (high sub), can be found in Chapter 7.
67
68
Chapter 7 Applications Examples of the use of the GST gene fusion system to purify, cleave, and detect GST fusion proteins include the following:
Purification • Rapid purification of GST fusion proteins using GSTrap FF 1 ml and 5 ml columns • Rapid purification using Glutathione Sepharose 4 Fast Flow in XK 16/20 column • High-throughput purification of GST fusion proteins using the MicroSpin GST Purification Module
Purification and cleavage • On-column cleavage and sample clean-up
Detection of GST fusion proteins • Detection of GST fusion proteins in bacterial lysates using a 96-well Detection Plate For more details, refer to the source material listed at the end of each example.
Purification GSTrap FF columns prepacked with Glutathione Sepharose 4 Fast Flow provide a convenient format that is easy to use for one-step purification of GST fusion proteins. In the applications described below, examples are presented showing typical purifications using these columns. Applications are also included for large sample volume purification using Glutathione Sepharose 4 Fast Flow packed in a XK 16/20 column and for rapid, high-throughput sample processing using MicroSpin GST Purification Module in conjunction with MicroPlex Vacuum.
Rapid purification of GST fusion proteins using GSTrap FF 1 ml and 5 ml columns Example 1. Purification of a phosphatase SH2 domain GST fusion protein GSTrap FF 1 ml and 5 ml prepacked columns were used in conjunction with ÄKTAexplorer 10 to purify a fusion protein containing the SH2 domain of a phosphatase fused with GST (SH2-GST) (Mr 37 000). Approximately 2 mg of fusion protein was recovered from 2 ml of clarified E. coli homogenate using GSTrap FF 1 ml for estimation of expression level (Fig 14A). The eluted material contained mostly SH2-GST, with only a small amount of GST detected (Fig 14B). No detectable contaminants were observed using SDS-PAGE and silver staining.
69
A)
Sample: Column: Flow rate:
Sample application Wash
A 280 4
3
2 ml clarified lysate GSTrap FF 1 ml 2 ml/min (sample application and washing); 0.5 ml/min (elution) Binding buffer: 20 mM phosphate buffer, 150 mM NaCl, pH 7.3 Elution buffer: 20 mM reduced glutathione, 50 mM Tris-HCl, pH 8.0 Instrument: ÄKTAexplorer 10
B) Mr 94 000 67 000 43 000 30 000 20 100 14 400
2
1 Elution
1
0 0.0
5.0
10.0
15.0
20.0 Volume (ml)
Lane Lane Lane Lane Lane Lane
1: 2: 3: 4: 5: 6:
2
3
4
5
6
Low Molecular Weight markers (LMW) Sample Flow-through fraction Last wash fraction Eluate LMW
Fig 14. Purification of SH2 phosphatase domain-GST fusion protein using GSTrap FF 1 ml and ÄKTAexplorer 10. A) Two ml of clarified E. coli homogenate containing a Mr 37 000 SH2-GST fusion protein was applied to the column and the resulting chromatogram recorded. B) Fractions were analyzed by SDS-PAGE on an 8–25% PhastGel™ with silver staining for detection. Source: Haneskog, L. et al., Scientific poster: Rapid purification of GST-fusion proteins from large sample volumes, Amersham Biosciences, code number 18-1139-51.
Example 2. Scaling-up purification of a GST fusion protein A GST fusion protein was purified from 8 ml and 40 ml of a clarified cell lysate using GSTrap FF 1 ml and 5 ml columns, respectively. Samples were applied to columns preequilibrated with 1× PBS, pH 7.3. After washing the columns with 10 column volumes (CV) of 1× PBS, GST fusion protein was eluted using reduced glutathione (Fig 15). Each run was completed in 25 min using ÄKTAexplorer 10. Analysis by SDS-PAGE indicated the isolation of highly pure GST fusion protein (Fig 27, lanes 3–4). Fusion protein yields were 2.7 mg from GSTrap FF 1 ml and 13.4 mg from GSTrap FF 5 ml. Example 3. GSTrap FF column purification of open reading frames cloned as GST fusions As part of a structural genomics project where the aim was to determine three-dimensional structures of proteins at high speed, four open reading frames (ORFs) were cloned as GST fusions and expressed in E. coli. The proteins were purified on a small scale using GSTrap FF 1 ml columns and ÄKTAprime. These conditions allowed quick purification of the proteins from cell extracts, making it possible to easily evaluate the expression, purity, and stability of each protein. Although protein expression levels were good, under these non-optimized conditions a degradation pattern was observed for all the purified proteins (Fig 16).
70
A)
A 280 % Elution buffer
A280 Elution buffer
3.5
% Elution buffer
B) Elution buffer
3.5 3.0
100
Wash
3.0
100
2.5
2.7 mg pure GST fusion protein
Wash
2.0
80 60
1.5
2.5
13.4 mg pure GST fusion protein
2.0
40
1.0
20
0.5 20
0.5
0
0
0
0 5.0 5.0
10.0 10.0
15.0 15.0
20 4
ml min
20.0 20.0
Sample: 8 ml clarified lysate Column: GSTrap FF 1ml Binding buffer: 1× PBS (150 mM NaCl, 20 mM phosphate buffer, pH 7.3) Elution buffer: 10 mM reduced glutathione, 50 mM Tris-HCl, pH 8.0 Flow rate: 1 ml/min Chromatographic procedure: 4 column volumes (CV) binding buffer, 8 ml sample 10 CV binding buffer, 5 CV elution buffer 5 CV binding buffer Instrument: ÄKTAexplorer 10
60
1.5
40
1.0
80
60 12
40 8
100 20
80 16
ml min
Sample: 40 ml clarified lysate Column: GSTrap FF 5 ml Binding buffer: 1× PBS (150 mM NaCl, 20 mM phosphate buffer, pH 7.3) Elution buffer: 10 mM reduced glutathione, 50 mM Tris-HCl, pH 8.0 Flow rate: 5 ml/min Chromatographic procedure: 4 column volumes (CV) binding buffer, 40 ml sample 10 CV binding buffer, 5 CV elution buffer 5 CV binding buffer Instrument: ÄKTAexplorer 10
Fig 15. Purification of a GST fusion protein on GSTrap FF 1 ml and 5 ml columns in combination with ÄKTAexplorer 10. Eight and 40 ml of cytoplasmic extract from E. coli expressing a GST fusion protein were applied to GSTrap FF 1 ml (A) and GSTrap FF 5 ml (B), respectively. Sources: Data File: GSTrap FF 1 ml and 5 ml, Glutathione Sepharose 4 Fast Flow, Amersham Biosciences, code number 18-1136-89. Haneskog, L. et al., Fast and simple purification of GST fusion proteins using prepacked GSTrap affinity columns, Life Science News 4, 16 (2000). See also online Life Science News archive.
Refer to the SDS-PAGE analysis of the above GST fusion protein in Figure 27, lanes 1–4 (page 81).
Clone A, Mr 37 000 S
FT
E
Clone B, Mr 36 000
Mr
S
E
Clone C, Mr 39 000
Mr
S
FT
E
Clone D, M r 36 000
Mr
S
FT
E
Mr
Mr
Mr
Mr
Mr
97 000 66 000
97 000
97 000
97 000
66 000
66 000
45 000
45 000
45 000
45 000
30 000 20 100 14 400
30 000
30 000
20 100 14 400
20 100 14 400
66 000
30 000 20 100 14 400
A)
FT
B)
C)
D)
Fig 16. SDS-PAGE analysis of four different human open reading frames (A-D) expressed as GST fusions. Proteins were loaded onto ExcelGel SDS Gradient, 8–18% for electrophoresis and stained with Coomassie blue. S = sample loaded onto the GSTrap FF column; FT = flow-through during sample load and wash; E = eluted fusion protein; Mr = low molecular weight marker proteins. The arrow indicates the location of the GST fusion protein. Source: Sigrell, J. A., Scientific poster: Purification of GST fusion proteins, on-column cleavage and sample clean-up. Initial purification screen within a structural genomics program, Amersham Biosciences, code number 18-1150-20.
71
Rapid purification using Glutathione Sepharose 4 Fast Flow packed in XK 16/20 column Example 4. Purification of eukaryotic GST fusion protein present at low levels in large sample volumes For this application, 34 ml of Glutathione Sepharose 4 Fast Flow medium was packed in a XK 16/20 column and used for purification of a eukaryotic GST fusion protein expressed at low levels. This method provides a realistic alternative to optimizing a fermentation process or preparing a new gene construct to obtain higher expression levels. A large volume (1.5 l) of clarified cell culture medium from human embryo kidney cells (HEK293 cells) expressing small amounts of a Mr 120 000 glycosylated and secreted protein was applied to the Glutathione Sepharose 4 Fast Flow column. Preliminary data had shown expression levels of 0.5–1.5 µg of the GST fusion protein per ml of culture medium. The column was washed with binding buffer, and 1 mg of pure protein was eluted using a stepgradient of glutathione (Fig 17), all within a period of 5 h. The protein was concentrated by ultrafiltration (cut-off Mr 10 000) and used for successful crystallization trials (Fig 18). A)
B)
A 280 0.35
Mr 200 000
Sample application Wash
0.30
116 000 97 000
0.25
67 000 55 000
0.20
36 000 31 000
0.15
21 000 Elution
0.10
0.05
1
0.0 0
Sample:
500
1000
2
3
4
1500 Volume (ml)
1500 ml clarified cell culture medium from HEK293 expressing a Mr 120 000 glycosylated and secreted protein Column: Glutathione Sepharose 4 Fast Flow (34 ml, 1.6 × 17 cm) packed in XK 16/20 column Flow rate: 10 ml/min (sample application and washing); 1 ml/min (elution) Binding buffer: 140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.3 Elution buffer: 20 mM reduced glutathione, 50 mM NaCl, 50 mM Tris-HCl, pH 8.0 Instrument: ÄKTAexplorer 100
Lane Lane Lane Lane
1: 2: 3: 4:
Molecular weight markers Sample Flow-through Eluate
Fig 17. Purification (A) and SDS-PAGE analysis using silver staining (B) of a eukaryotic protein present at low levels in large sample volumes. This application is reproduced with kind permission of Johan Öhman and Eva Rupp-Thuresson, Pharmacia Corp., Stockholm, Sweden.
72
Fig 18. Crystals of the eukaryotic GST fusion protein obtained in the initial crystallization trial. Source: Haneskog, L. et al., Scientific poster: Rapid purification of GST-fusion proteins from large sample volumes, Amersham Biosciences, code number 18-1139-51.
High-throughput purification of GST fusion proteins using the MicroSpin GST Purification Module Example 5. Purification of human myoglobin GST fusion protein The MicroSpin GST Purification Module provides an ideal format for the simple and rapid purification of GST fusion proteins from large numbers of small-scale bacterial lysates, especially when it is used in conjunction with MicroPlex 24 Vacuum. By connecting two MicroPlex 24 Vacuum units in parallel, up to 48 Glutathione Sepharose 4B MicroSpin Columns can be processed simultaneously. In this application, GST fusion proteins from 24 randomly selected transformants were purified according to the instructions provided with the module and analyzed by SDS-PAGE (Fig 19). Seven of the 24 colonies tested expressed a GST fusion protein corresponding in molecular weight to that of GST-myoglobin. Each remaining colony expressed a GST-sized protein consistent with that encoded by the parent vector without an insert. Mr
Mr
97 000 66 000
97 000 66 000
45 000
GST/ myoglobin
45 000
GST/ myoglobin
30 000
GST
30 000
GST
20 100
20 100
14 400
14 400 M
G
1
2 3
4 5
6
7 8
9 10 11 12
M
G 13 14 15 16 17 18 19 20 21 22 23 24
Fig 19. Rapid screening of randomly selected E. coli transformants for expression of human myoglobin GST fusion protein. Aliquots (15 µl) of each eluted product were loaded into the lanes of a 12% polyacrylamide Tris-glycine-SDS gel and run for 2 h at 90 V. M = LMW Marker Kit, G = purified recombinant GST. Lanes 1–24 contain products eluted from the MicroSpin Columns using reduced glutathione. Source: See Figure 20.
When purified GST-myoglobin fusion protein obtained from the rapid screening procedure was treated with Factor Xa, two protein products were observed, a Mr 29 000 product and a Mr 14 000 product corresponding in size to that of the expected GST domain and human myoglobin, respectively (Fig 20). These results demonstrate not only expression of the desired GST-myoglobin fusion protein by the selected clone, but also that fusion proteins purified during rapid screening with the Glutathione Sepharose 4B MicroSpin Columns can be cleaved with site-specific proteases.
73
factor Xa-cleaved GST-myoglobin
purified GST-myoglobin
factor Xa-cleaved pGEX-5X GST
pGEX-5X GST
control GST
M
Mr 97 000 66 000
45 000 — GST-myoglobin
30 000
— pGEX-5X GST — factor Xa-released GST
20 100 — factor Xa-released myoglobin
Fig 20. Cleavage of putative GST-myoglobin fusion protein with factor Xa. After purification using the MicroSpin GST Purification Module, protein from a representative parent clone and GST-myoglobin fusion protein clone were dialyzed with two changes of 1× PBS overnight, then treated with factor Xa for 20 h at room temperature. M = LMW Marker Kit. Sources: Bell, P. A. et al., Rapid screening of multiple clones for GST fusion protein expression, Life Science News 1, 14 (1998). See also online Life Science News archive. Data File: MicroSpin GST Purification Module, Amersham Biosciences, code number 18-1128-13.
Purification and cleavage On-column cleavage and sample clean-up Example 6. Purification and on-column cleavage of GST fusion SH2 domain using thrombin and GSTrap FF. Sample clean-up using HiTrap Benzamidine FF (high sub) column in series with GSTrap FF The following application describes the purification of a Mr 37 000 SH2-GST fusion protein on a GSTrap FF 1 ml column, followed by on-column cleavage with thrombin (Fig 21). After the thrombin incubation step, a HiTrap Benzamidine FF (high sub) 1 ml column was placed in series below the GSTrap FF column. As the columns were washed with binding buffer and later with high salt buffer, the cleaved SH2 fusion protein and thrombin were washed from the GSTrap FF column onto the HiTrap Benzamidine FF (high sub) column. Thrombin was captured by this second column, thus enabling the collection of pure thrombin-free protein in the eluent (Fig 21A). Complete removal of thrombin was verified using the chromogenic substrate S-2238 (Chromogenix, Haemochrom Diagnostica AB; supplier in US is DiaPharma) for detection of thrombin activity (Fig 21B). This whole procedure could be completed in less than one day.
74
Gel: ExcelGel SDS Gradient 8–18%, Coomassie blue staining Lane 1: Low Molecular Weight Marker Kit (LMW) Lane 2: Clarified E. coli homogenate containing SH2-GST fusion protein with a thrombin cleavage site Lane 3: Flow-through from GSTrap FF (fraction 2) Lane 4: SH2 domain (GST-tag cleaved off), washed out with binding buffer through both columns (fraction 6) Lane 5: Same as lane 4 (fraction 7) Lane 6: Same as lane 4 (fraction 8) Lane 7: Elution of thrombin from HiTrap Benzamidine FF (high sub) Lane 8: Elution of GST-tag and some non-cleaved SH2-GST from GSTrap FF (fraction 21) Lane 9: Same as lane 8 (fraction 22)
A) Mr 97 000 66 000 45 000
— SH2-GST
30 000
— GST
20 100 — SH2
14 400 1
2
3
4
5
6
7
8
9
High-salt buffer wash
B)
Elution of HiTrap Benzamidine FF (high sub)
Thrombin
Elution of GSTrap FF
A 280 nm
Thrombin activity A 405 nm
0.80 0.30
GST-tag 0.60 Column wash
Thrombin 0.40
fr.21 fr.22
fr.14
fr.2
0.10 fr.6 fr.7 fr.8
Cleaved SH2 protein
0.20
0.20
0
0 0
10 A)
A)
15 A)
20 B)
25
50
ml
A)
B)
A) GSTrap FF, 1 ml B) HiTrap Benzamidine FF (high sub), 1 ml
Sample:
2 ml clarified E. coli homogenate containing a Mr 37 000 SH2-GST fusion protein with a thrombin cleavage site Columns: GSTrap FF 1 ml and HiTrap Benzamidine FF (high sub) 1 ml Binding buffer: 20 mM sodium phosphate, 0.15 M NaCl, pH 7.5 High salt wash buffer: 20 mM sodium phosphate, 1.0 M NaCl, pH 7.5 Benzamidine elution buffer: 20 mM p-Aminobenzamidine in binding buffer GST elution buffer: 20 mM reduced glutathione, 50 mM Tris, pH 8.0 Flow rate: 0.5 ml/min System: ÄKTAprime Protease treatment: 20 U/ml thrombin protease (Amersham Biosciences) for 2 h at room temperature Thrombin activity: Measured at 405 nm using S-2238 (Chromogenix, Haemochrom Diagnostica AB; supplier in US is DiaPharma) as substrate
Fig 21. Purification of SH2 domain-GST fusion protein with on-column cleavage and post-cleavage removal of thrombin using GSTrap FF and HiTrap Benzamidine FF (high sub) columns. A) SDS-PAGE analysis of various sample processing steps. B) Chromatogram and thrombin activity curve demonstrating all steps in the purification of the SH2 domain. Sources: Sigrell, J. A., Scientific poster: Purification of GST fusion proteins, on-column cleavage and sample clean-up, Amersham Biosciences, code number 18-1150-20. Data File: HiTrap Benzamidine FF (high sub) and Benzamidine Sepharose 4 Fast Flow (high sub), Amersham Biosciences, code number 18-1139-38.
75
Example 7. Purification of human hippocalcin using GSTrap FF columns in series with on-column cleavage by PreScission Protease The gene for human hippocalcin, a member of the neurone-specific calcium-binding protein family, was cloned into a pGEX vector containing a PreScission Protease site adjacent to the GST-tag. The expressed fusion protein was captured on a GSTrap FF 1 ml column. The column was then incubated overnight at 4 °C and for an additional 2 h at room temperature with PreScission Protease (which is GST-tagged itself). Following on-column cleavage, a second GSTrap FF 1 ml column was placed in series after the first to remove any PreScission Protease, uncleaved GST-fusion, or free GST-tag that could co-elute with the sample during the additional wash with binding buffer (Fig 22). For every gram of wet E. coli cells, 10 mg of pure, non-tagged hippocalcin was obtained. A)
Elution of GSTrap FF GST-tag and PreScission Protease
fr.12
A 280 nm 0.80 PreScission Continued Protease column wash 0.60
Column wash
0.20
fr.5 fr.6
fr.2
0.40
Hippocalcin
0 0
10
20
30
40
ml
GSTrap FF 2× GSTrap FF
Sample: Columns: Binding and wash buffer: GST elution buffer: Flow rate: Instrument: Protease treatment:
2 ml clarified E. coli homogenate containing expressed GST-hippocalcin, Mr 43 000 GSTrap FF 1 ml 50 mM Tris-HCl, pH 8.0, 0.15 M NaCl, 1 mM CaCl2, 1 mM DTT, 10% glycerol 20 mM reduced glutathione, 50 mM Tris-HCl, pH 8.0 0.5 ml/min ÄKTAprime 80 U/ml PreScission Protease overnight at 4 °C and then 2 h at room temperature
B) 1
2
3
4
5
6
Gel:
Mr Lane 1:
97 000 66 000 45 000 30 000 20 100 14 400
GST-hippocalcin PreScission Protease GST hippocalcin
Lane 2: Lane 3: Lane 4: Lane 5: Lane 6:
ExcelGel SDS Gradient, 8–18%, Coomassie blue staining Clarified E. coli homogenate containing expressed GST-hippocalcin Flow-through (fraction 2) GST-hippocalcin Pure hippocalcin after on-column cleavage (fraction 5) Same as lane 4, but fraction 6 Eluted fraction from GSTrap FF containing PreScission Protease and GST-tag released by cleavage (fraction 12)
Fig 22. Purification of human hippocalcin-GST fusion protein with on-column cleavage and post-cleavage removal of PreScission Protease using GSTrap FF columns. A) Chromatogram showing purification of hippocalcin. B) SDS-PAGE analysis of various sample processing steps. Source: Sigrell, J. A., Scientific poster: Purification of GST fusion proteins, on-column cleavage and sample clean-up, Amersham Biosciences, code number 18-1150-20.
76
Example 8. Purification and on-column cleavage of TLP40-GST fusion protein using GSTrap FF columns and PreScission Protease The gene coding for TLP40 protein was subcloned into pGEX-6P-1 and transformed into E. coli BL21. GST fusion proteins were purified from clarified lysates using two GSTrap FF 5 ml columns connected in series and ÄKTAexplorer 10. 1× PBS was used as binding buffer. The flow rate for loading was 1 ml/min, a rate found optimal for fusion protein binding. Loaded columns were washed with 1× PBS until the absorbance baseline stabilized, after which the buffer was changed to PreScission buffer (50 mM Tris-HCl, 100 mM NaCl, 1 mM EDTA, 1 mM DTT, pH 8.0). Columns were equilibrated with PreScission buffer until both the UV absorbance and conductivity baselines stabilized, after which buffer flow was stopped. For PreScission Protease digestion, 2 units of enzyme/100 µg of bound GST-fusion protein was used. PreScission Protease was diluted in PreScission buffer (9 ml total volume for two columns in series) and manually injected into the columns at a flow rate of 5–7 ml/min. This increased flow allows for uniform distribution of enzyme through the GSTrap FF 5 ml columns. Following injection, the columns were closed, sealed and incubated for 12–16 h at 4 °C. Prior to elution, a 1 ml GSTrap FF column (pre-equilibrated with PreScission buffer) was connected downstream to the GSTrap FF proteolytic cleavage columns. This configuration minimized loss of cleaved product and allowed for rapid baseline recalibration before peak elution. The 1 ml column also acted as a filter to capture any released cleaved GST protein, uncleaved GST-fusion protein and unbound PreScission Protease. Elution of cleaved protein occurred immediately upon flow start-up (Fig 23A). The eluted material contained TLP40 cleavage product with no contaminating proteins detected (Fig 23B). After target protein elution, GST, unbound GST-fusion protein, and PreScission Protease were eluted with reduced glutathione [as applied in a full one-step gradient (100%)]. SDSPAGE analysis of various fractions showed isolation of highly pure TLP40 after on-column cleavage (Fig 23B). This application is reproduced with kind permission of Dr. Darcy Birse, University of Stockholm, Sweden.
77
A)
B) % Elution buffer 100
A280
Mr 97 000
Buffer C eluted GST-tag
4
66 000 45 000
cleaved TLP40 fraction
3
80
60
30 000 2
20 100
Buffer B
Buffer A
1
40
PreScission Protease
20
F2 23456789 12 14 16 18 20 22 24 26
0 0
Sample:
50
100
150
0
200 ml
50 ml clarified lysate from E. coli containing TLP40-GST fusion protein 2× GSTrap FF 5 ml columns connected in series Binding buffer (A): 1× PBS, pH 7.4 Wash buffer (B): PreScission buffer, pH 8.9 Elution buffer (C): Reduced glutathione buffer, pH 8.0 Instrument: ÄKTAexplorer 10
Column:
1 Lane Lane Lane Lane Lane Lane Lane
1: 2: 3: 4: 5: 6: 7:
2
3
4
5
6
7
Low molecular weight markers (LMW) Total protein extract of non-induced culture Total protein extract of induced culture Supernatant after 70 000 × g centrifugation step Supernatant after 300 000 × g centrifugation step Flow-through from GSTrap FF column Native TLP40 cleavage product eluted after on-column digestion with PreScission Protease
Fig 23. Purification and SDS-PAGE analysis of TLP40-GST fusion protein. A) Purification and on-column cleavage of fusion protein using GSTrap FF 5 ml and PreScission Protease in combination with ÄKTAexplorer 10. The flow rate for sample loading and injecting the protease were 1 ml/min and 5–7 ml/min, respectively. B) Fractions from the purification steps were analyzed by SDS-PAGE using a 3.5–12% polyacrylamide gel. The gel was stained with Coomassie blue. Sources: Application Note: Efficient, rapid protein purification and on-column cleavage using GSTrap FF columns, Amersham Biosciences, code number 18-1146-70. Knaust, R. et al., An efficient and rapid protein purification and on-column cleavage strategy using GSTrap FF columns, Life Science News 6, 12–13 (2000). See also online Life Science News archive. Dian, C. et al., in Abstracts of the 4th Annual CHI Protein Expression Meeting, McLean, VA (April 4–6, 2001).
Example 9. Scaling-up purification of a phosphatase SH2 domain GST fusion protein and on-column cleavage using thrombin To obtain the pure SH2 domain without the GST tag, chromatography was scaled-up using 100 ml of clarified E. coli homogenate and GSTrap FF 5 ml, and on-column cleavage was carried out overnight with thrombin prior to elution of released SH2 domain with binding buffer (Fig 24). The eluted SH2 domain fraction contained 2 mg of protein, while the GST fraction that was subsequently eluted with reduced glutathione contained 4 mg. SDS-PAGE analysis and silver staining indicated that the SH2 domain was pure and that protease cleavage was complete (Fig 24). Mass spectrometry revealed essentially two peaks corresponding to the single-charged (m/z 12 472) and double-charged (m/z 6 241) protein (Fig 25); this agrees with the expected Mr of the SH2 domain (Fig 24). The spectra contained no other peak in the m/z window used (inset in Fig 25).
78
A) Sample application
Wash A280
A280 4
0.4 Elution with binding buffer
3
0.3
Start incubation with protease
2
0.2
Wash
Elution with elution buffer
0.1
1
0
0 0
50
Sample: Column: Flow rate: Binding buffer: Elution buffer: Protease treatment: Instrument:
100
150
Volume (ml)
0
20
100
120 Volume (ml)
100 ml clarified lysate GSTrap FF 5 ml 10 ml/min (sample application and washing); 2.5 ml/min (elution) 20 mM phosphate buffer, 150 mM NaCl, pH 7.3 10 mM reduced glutathione, 50 mM Tris-HCl, pH 8.0 20 U/ml thrombin (Amersham Biosciences) for 14 h at room temperature ÄKTAexplorer 10
B) Lane Lane Lane Lane Lane
Low Molecular Weight markers (LMW) Sample Flow-through fraction Last wash fraction Eluate containing cleaved material (the SH2 domain) eluted with binding buffer, first part of peak Lane 6: As in lane 5, middle part of peak Lane 7: As in lane 5, last part of peak Lane 8: Elution of GST tag from GSTrap FF
Mr 97 000 66 000 45 000 30 000 20 100 14 400 1
2
3
4
5
6
7
1: 2: 3: 4: 5:
8
Fig 24. Purification of SH2 domain using GSTrap FF 5 ml and ÄKTAexplorer 10 with on-column cleavage of the GST tag using thrombin. A) One hundred ml of clarified E. coli homogenate containing a Mr 37 000 SH2-GST fusion protein was applied to the column. The GST tag was removed with thrombin prior to elution of released SH2 domain with binding buffer and the resulting chromatogram recorded. B) Fractions were analyzed by SDS-PAGE on 8–25% PhastGel with silver staining for detection. 12472.0
6240.82 6240.82
Fig 25. MALDI-ToF MS analysis of the SH2 domain. Source: Haneskog, L. et al., Scientific poster: Rapid purification of GST-fusion proteins from large sample volumes, Amersham Biosciences, code number 18-1139-51. 79
Example 10. On-column cleavage of a GST fusion protein using thrombin To demonstrate the efficiency of on-column cleavage in conjunction with purification, a GST fusion protein containing the recognition sequence for thrombin was applied to GSTrap FF 1 ml. After washing, the column was filled by syringe with 1 ml of thrombin solution (20 U/ml in 1× PBS) and sealed using the supplied connectors. After incubation for 16 h at room temperature, the target protein minus the GST moiety was eluted using 1× PBS, and the bound GST was subsequently eluted using elution buffer (Fig 26). The cleavage reaction yield was 100%. Intact GST fusion protein was not detected in the eluate by SDS-PAGE (Fig 27, lane 5). A)
B)
A 280
A 280
3.5
3.5
3.0
% Elution buffer
3.0
Wash
2.5
100
2.5 Incubation 16 h room temp.
2.0 1.5
1.5
1.0
1.0
0.5
0.5
0
0 5.0
10.0
15.0
10 ml clarified cytoplasmic extract from E. coli expressing a GST fusion protein Column: GSTrap FF 1 ml Binding buffer: 1× PBS (150 mM NaCl, 20 mM phosphate buffer, pH 7.3) Elution buffer: 10 mM reduced glutathione, 50 mM Tris-HCl, pH 8.0 Flow rate: 1 ml/min Chromatographic procedure: 4 column volumes (CV) binding buffer, 10 ml sample 10 CV binding buffer, fill column with 1 ml thrombin solution using a syringe Instrument: ÄKTAexplorer 10
60
Target protein
40 20 0
2.0
min
Sample:
80 Free GST
2.0
4.0
6.0
8.0
10.0
12.0 min
Column:
GSTrap FF 1 ml column after 16 h incubation with thrombin Binding buffer: 1× PBS (150 mM NaCl, 20 mM phosphate buffer, pH 7.3) Elution buffer: 10 mM reduced glutathione, 50 mM Tris-HCl, pH 8.0 Flow rate: 1 ml/min Chromatographic procedure: 8 column volumes (CV) binding buffer (elution of cleaved target protein), 5 CV elution buffer (elution of free GST and non-cleaved GST fusion protein) 5 CV binding buffer Instrument: ÄKTAexplorer 10
Fig 26. On-column thrombin cleavage of a GST fusion protein. A) Equilibration, sample application, and washing of a GST fusion protein on GSTrap FF 1 ml were performed using ÄKTAexplorer 10. After washing, the column was filled by syringe with 1 ml of thrombin (20 U/ml 1× PBS) and incubated for 16 h at room temperature. B) GST-free target protein was eluted using 1× PBS. GST was eluted using 10 mM reduced glutathione. The GST-free target protein fraction also contained a small amount of thrombin (not detectable by SDS-PAGE; see Fig 27, lane 6). The thrombin can be removed using a HiTrap Benzamidine FF (high sub) column. Source: See Figure 27.
Detection of GST fusion proteins Example 11. Detection of GST fusion proteins in bacterial lysates using the GST 96-Well Detection Module The GST 96-Well Detection Module provides a convenient format for rapidly screening as many as 96 samples per plate for the presence of GST fusion proteins. The module uses immobilized goat polyclonal anti-GST antibody to capture GST fusion proteins from complex mixtures and exhibits very low, non-specific background binding. Using a chromogenic substrate, the system can detect as little as 1 ng of recombinant GST (Fig 28), providing a level of sensitivity that is 10 to 100 times greater than capture plates using immobilized glutathione. 80
Mr 97 000 GST fusion protein
66 000
GST-free protein
45 000
GST tag 30 000 20 100 14 400
1
2
3
4
5
6
7
Lane 1: Low Molecular Weight (LMW) Calibration kit, reduced Lane 2: Cytoplasmic extract of E. coli containing GST fusion protein, 1 g cell paste/10 ml Lane 3: GST fusion protein eluted from GSTrap FF 1 ml (from Fig 15A) Lane 4: GST fusion protein eluted from GSTrap FF 5 ml (from Fig 15B) Lane 5: GST-free target protein eluted from GSTrap FF 1 ml after 16 h thrombin cleavage (from Fig 26) Lane 6: Free GST eluted from GSTrap FF 1 ml after thrombin cleavage (from Fig 26) Lane 7: Thrombin solution (20 U/ml) Lane 8: LMW, reduced
8
Fig 27. Analysis of fractions from Figs 15 and 26 by SDS-PAGE on ExcelGel SDS Gradient 8–18% using Multiphor II followed by silver staining. Fusion protein was eluted using 10 mM reduced glutathione (lanes 3 and 4). On-column cleavage with thrombin (20 U/ml) was performed with GSTrap FF 1 ml loaded with fusion protein. The GST-free target protein was eluted with 1× PBS (lane 5), and the GST moiety was eluted with 10 mM glutathione (lane 6). Sources: Data File: GSTrap FF 1 ml and 5 ml Glutathione Sepharose 4 Fast Flow, Amersham Biosciences, code number 18-1136-89. Haneskog, L. et al., Fast and simple purification of GST fusion proteins using prepacked GSTrap affinity columns, Life Science News 4, 16 (2000). See also online Life Science News archive.
A 450 1.50
0.75
0 0.01
0.1
1
10 100 ng rGST/well
1000
10 000
Fig 28. Detection of recombinant GST using the GST 96-Well Detection Module. The indicated amounts of recombinant GST Protein were prepared in 1× blocking buffer, and 100 µl volumes of each were transferred to the wells of a GST 96-Well Detection Plate. After 1 h binding at room temperature, the wells were washed and then incubated with a 1:1000 dilution of HRP/Anti-GST Conjugate for 1 h. Using the substrate 3, 3', 5, 5'-tetramethylbenzidine (TMB) for detection, the absorbance of each well was measured at 450 nm. Source: See Figure 29.
81
The following application demonstrates that fusion proteins immobilized through their GST domains can be detected with antibodies directed against the fusion partner. A luciferase gene fragment was inserted into the multiple cloning site of the GST gene fusion vector pGEX-6P-1. Lysates from 64 randomly selected transformant colonies of E. coli BL21 were screened for GST fusion expression. Aliquots (50 µl) of each lysate were mixed 1:1 (v/v) with blocking buffer and applied to the wells of a GST 96-Well Detection Plate. Serial dilutions of recombinant GST were used as controls. After a 1 h incubation at room temperature, wells containing culture lysates were washed and incubated with rabbit antiluciferase, followed by HRP/anti-rabbit IgG conjugate for detection. Control rGST wells were incubated with HRP/Anti-GST Conjugate. All wells were developed using TMB, and the A450 of each well was measured in a 96-well plate reader. Wells containing luciferase GST fusion protein are evident by their strong positive signal (Fig 29). rGST control 1
2
3
Anti-luciferase detection 4
5
6
7
8
9
10
11
12
Fig 29. Screening of bacterial lysates for luciferase GST fusion protein expression using the GST 96-Well Detection Module. Cultures of randomly selected E. coli colonies transformed with a pGEX-6P-1/luciferase construct were grown, induced and lysed in a 96-well plate. Aliquots (50 µl) of each cleared lysate were transferred to the wells in columns 4–12 of a GST 96-Well Detection Plate to capture expressed GST-luciferase fusion proteins. Captured fusion proteins were detected using rabbit anti-luciferase, anti-rabbit IgG/peroxidase conjugate and TMB substrate. Columns 1–3 contain serial dilutions of recombinant GST. Recombinant GST was detected as described in Fig 28. Sources: Bell, P. A. et al., Rapid screening of multiple clones for GST fusion protein expression, Life Science News 1, 14 (1998). See also online Life Science News archive. Data File: GST 96-Well Detection Module, Amersham Biosciences, code number 18-1128-14.
82
Troubleshooting guide Protein expression A high basal level of expression is observed Basal level expression (i.e. expression in the absence of an inducer, such as IPTG) is associated with most inducible promoters. It can affect the outcome of cloning experiments for toxic inserts by selecting against inserts cloned in the proper orientation. Basal level expression can be minimized by catabolite repression (e.g. growth in the presence of glucose). The tac promoter responsible for the expression of the fusion protein in pGEX vectors is not subject to catabolite repression. However, with the pGEX vector system there is a lac promoter located upstream between the 3'-end of the lacIq gene and the tac promoter. This lac promoter might contribute to the basal level of expression of inserts cloned into the pGEX multiple cloning site, and it is subject to catabolite repression. • Add 2% glucose to the growth medium. This will decrease the basal level expression associated with the upstream lac promoter but will not affect basal level expression from the tac promoter. The presence of glucose should not significantly affect overall expression following induction with IPTG. No protein is detected in the bacterial sonicate • Check DNA sequences. It is essential that protein-coding DNA sequences are cloned in the proper translation frame in the vectors. Cloning junctions should be sequenced to verify that inserts are in-frame. For convenience, use the pGEX 5' and 3' Sequencing Primers (see Appendix 4 for more information on the primers). The reading frame of the multiple cloning site for each pGEX vector is shown in Figure 2, Chapter 2. • Optimize culture conditions to improve yield. Cell strain, medium composition, incubation temperature, and induction conditions can all affect yield. Exact conditions will vary for each fusion protein expressed. • Analyze a small aliquot of an overnight culture by SDS-PAGE. Generally, a highly expressed protein will be visible by Coomassie blue staining when 5–10 µl of an induced culture with an A600 of ~ 1.0 is loaded onto the gel. Non-transformed host E. coli cells and cells transformed with the parental vector should be run in parallel as negative and positive controls, respectively. If fusion protein is present in this total cell preparation and absent from a clarified sonicate, this may indicate the presence of inclusion bodies (see next page). • Check for expression by immunoblotting, which is generally more sensitive than stained gels. Some fusion proteins may be masked on an SDS-polyacrylamide gel by a bacterial protein of approximately the same molecular weight. Immunoblotting can be used to identify fusion proteins in most of these cases. Run an SDS-polyacrylamide gel of induced cells and transfer the proteins to a nitrocellulose or PVDF membrane (such as Hybond-C or Hybond-P). Detect fusion protein using anti-GST antibody. Alternatively, purify the extract using GSTrap FF, GSTPrep FF 16/10, or Glutathione Sepharose 4 Fast Flow prior to SDS-PAGE analysis. • Select a new, independently transformed isolate and check for expression.
83
Most of the fusion protein is in the post-sonicate pellet • Check the cell disruption procedure. Cell disruption is indicated by partial clearing of the suspension or by microscopic examination. Addition of lysozyme (0.1 volume of a 10 mg/ml lysozyme solution in 25 mM Tris-HCl, pH 8.0) prior to sonication might improve results. Avoid frothing as this might denature the fusion protein. • Reduce sonication since over-sonication can lead to co-purification of host proteins with the fusion protein. • Fusion protein may be produced as insoluble inclusion bodies. Try altering the growth conditions to slow the rate of translation, as suggested below. It may be necessary to combine these approaches. Exact conditions must be determined empirically for each fusion protein. – Lower the growth temperature (within the range of 20–30 °C) to improve solubility (35, 36). – Decrease the IPTG concentration to < 0.1 mM to alter induction level. – Alter the time of induction. – Induce for a shorter period of time. – Induce at a higher cell density for a short period of time. – Increase aeration. High oxygen transport can help prevent the formation of inclusion bodies. • Alter extraction conditions to improve solubilization of inclusion bodies. Protein can sometimes be solubilized from inclusion bodies using common denaturants such as 4–6 M guanidine hydrochloride, 4–8 M urea, alkaline pH > 9, organic solvents (20, 37), 0.5–2% Triton X-100, 0.5–2% N-lauroylsarcosine (Sarcosyl) (38, 39), or other detergents. Other variables that affect solubilization include time, temperature, ionic strength, the ratio of denaturants to protein, and the presence of reducing reagents (20, 37). For reviews, see references 20, 35, 37, and 40. • Following solubilization with denaturants, proteins must be correctly refolded to regain function. Denaturants can be removed by desalting or by dilution or dialysis to allow refolding of the protein and formation of the correct intramolecular associations. Detergent-solubilized proteins may have retained their native structure. Critical parameters during refolding include pH, the presence of reducing reagents, and the speed of denaturant removal (20, 37, 41). Once refolded, protein can be purified by ion exchange, gel filtration, or affinity chromatography. • Fusion proteins can be purified to some extent while denatured. In some instances when GST fusion proteins form inclusion bodies, solubilization and binding to GSTrap FF columns can be achieved in the presence of 2–3 M guanidine hydrochloride or by using up to 2% Tween 20. Binding to Glutathione Sepharose can also be achieved in the presence of 1% CTAB, 10 mM DTT or 0.03% SDS (19). Successful purification in the presence of these agents may depend on the nature of the fusion protein.
84
Purification and detection The column has clogged • Cell debris in the sample may clog the column. Clean the column using the protocol outlined in Appendix 5 and make sure that samples have been filtered using a 0.45 µm filter and centrifuged at > 30 000 × g. Dilute samples if they are too viscous. The fusion protein does not bind to the purification medium • Decrease the flow rate to improve binding. • Over-sonication may have denatured the fusion protein. Check the lysate microscopically to monitor cell breakage. Use mild sonication conditions during cell lysis. • Sonication may be insufficient. Check microscopically or monitor nucleic acid release by measuring the A260. Addition of lysozyme (0.1 volume of a 10 mg/ml lysozyme solution in 25 mM Tris-HCl, pH 8.0) prior to sonication may improve results. Avoid frothing as this may denature the fusion protein. • Add 1–10 mM DTT prior to cell lysis. This can significantly increase binding of some GST fusion proteins to Glutathione Sepharose. The optimal concentration must be determined empirically for each fusion protein. • Check that the column has been equilibrated with a buffer 6.5 < pH < 8.0 (e.g. 1× PBS) before applying fusion protein. The correct pH range is critical for efficient binding. • If re-using a column, check that the column has been cleaned and regenerated correctly (see Appendix 5). Replace with fresh Glutathione Sepharose or a new prepacked column if binding capacity does not return after regeneration. • Check the binding of a cell sonicate prepared from the parental pGEX plasmid. If GST produced from the parental plasmid binds with high affinity, then the fusion partner may have altered the conformation of GST, thereby reducing its affinity. Try reducing the binding temperature to 4 °C and limit the number of washes. • Column capacity may have been exceeded. If using GSTrap FF columns (1 ml or 5 ml), either link two or three columns in series to increase capacity, use a GSTPrep FF 16/10 column, or pack a larger column. • Fusion protein may be in inclusion bodies. See discussion on page 84. The fusion protein is poorly eluted • Decrease the flow rate to improve elution. • Increase the concentration of glutathione in the elution buffer. Above 15 mM glutathione, the buffer concentration should be increased to maintain pH (42). As an example, try 50 mM Tris-HCl, 20–40 mM reduced glutathione, pH 8 as elution buffer. • Increase the pH of the elution buffer. Values up to pH 9 may improve elution without requiring an increase in the concentration of glutathione. • Increase the ionic strength of the elution buffer by adding 0.1–0.2 M NaCl. Note that very hydrophobic proteins may precipitate under high salt conditions. If this is the case, addition of a non-ionic detergent may improve results (see below).
85
• Increase the volume of elution buffer. In some cases, especially after on-column cleavage of fusion protein, a larger volume of buffer may be necessary to elute the fusion protein. • Add a non-ionic detergent (e.g. 0.1% Triton X-100 or 2% N-octyl glucoside) to the elution buffer to reduce non-specific hydrophobic interactions that may prevent solubilization and elution of fusion proteins (39). • Try overnight elution at room temperature or 4 °C. Note: The longer the duration of purification, the greater the risk of protein degradation. Multiple bands are seen on SDS-PAGE or Western blot analysis Multiple bands result from partial degradation of fusion proteins by proteases, or denaturation and co-purification of host proteins with the GST fusion protein due to over-sonication. • Check that a protease-deficient host such as E. coli B21 has been used. • Add protease inhibitors such as 1 mM PMSF to the lysis solution. A non-toxic, water soluble alternative to PMSF is 4-(2-amino-ethyl)-benzenesulfonyl fluoride hydrochloride (AEBSF) (43), commercially available as Pefabloc SC from Roche Molecular Biochemicals. Note: Serine protease inhibitors must be removed prior to cleavage by thrombin or Factor Xa. Use HiTrap Benzamidine FF (high sub) (see Procedure 22). • Use prepacked GSTrap FF columns or Glutathione Sepharose 4 Fast Flow. These can be used at higher flow rates (compared with gravity columns) to process samples more quickly and thus avoid degradation. • Decrease sonication. Addition of lysozyme (0.1 volume of a 10 mg/ml lysozyme solution in 25 mM Tris-HCl, pH 8.0) prior to sonication may improve results. Avoid frothing as this may denature the fusion protein. • Include an additional purification step. A variety of proteins known as chaperonins are involved in the correct folding of nascent proteins in E. coli (44) and may co-purify with GST fusion proteins. These include, but are not limited to: DnaK (Mr ~ 70 000, reference 45), DnaJ (Mr ~ 37 000, reference 46), GrpE (Mr ~ 40 000, reference 47), GroEL (Mr ~ 57 000, reference 48), and GroES (Mr ~ 10 000, reference 49). Several methods for purifying GST fusion proteins from these co-purifying proteins have been described (see below). • Anti-GST antibody from Amersham Biosciences has been cross-absorbed against E. coli proteins and tested for its lack of non-specific background binding in a Western blot. However, other sources of the anti-GST antibody may contain antibodies that react with various E. coli proteins present in the fusion protein sample. Cross-adsorb the antibody with an E. coli sonicate to remove anti-E. coli antibodies. The E. coli used for crossabsorption must not contain the pGEX plasmid. See Appendix 6 for procedural details. Mr 70 000 protein co-purifies with the GST fusion protein Pre-incubate the protein solution with 2 mM ATP, 10 mM MgSO4, 50 mM Tris-HCl, pH 7.4 for 10 min at 37 °C prior to purification to dissociate the complex. This Mr 70 000 protein is probably a product of the E. coli gene dnaK and involved in the degradation of “abnormal” proteins in E. coli (50). Reports suggest that this protein can be removed by ion exchange chromatography (50) or by passage of the sample over ATP agarose (51).
86
Various reports (50, 52) suggest washing the column with ATP or GroES rather than using a subsequent ion exchange step.
Detection Results with the GST Detection Module are problematic • The reaction rate of the CDNB assay is linear provided that an A340 of ~ 0.8 is not exceeded during the 5-min time course. Plot initial results to verify that the reaction rate is linear over the time course. Adjust the amount of sample containing the GST fusion protein to maintain a linear reaction rate. • Depending on how the fusion protein is folded, some GST fusions will show very low activity with the CDNB assay. Whether for this or for any other reason, if a low absorbance is obtained using the CDNB assay, a Western blot using anti-GST antibody may reveal high levels of fusion protein expression. • Under standard assay conditions at 22 °C and in the absence of GST, glutathione and CDNB react spontaneously to form a chemical moiety that produces a baseline drift at DA340 /min of ~ 0.003 (or 0.015 in 5 min). Correct for baseline drift by blanking the spectrophotometer with the blank cuvette before each reading of the sample cuvette. Results with the GST 96-Well Detection Module are problematic • If low absorbance is detected in the samples, check that host cells were sufficiently induced and the samples were sufficiently lysed (see Troubleshooting protein expression). • If clarified lysate is being tested, mix the initial GST sample with 2× blocking buffer to give a final concentration of 1× blocking buffer. • If poor day-to-day reproducibility between identical samples is experienced, verify that all incubation times are consistent. GST capture incubation time can be decreased with slightly reduced signal, but do not incubate for less than 30 min. Every 15-min decrease in HRP/anti-GST conjugate incubation time can significantly reduce signal.
Cleavage PreScission Protease PreScission Protease cleavage is incomplete • Check that the ratio of PreScission Protease to fusion protein is correct. • Increase the incubation time to 20 h or longer at 5 °C, and increase the amount of PreScission Protease used in the reaction. • Verify that a PreScission Protease cleavage site is present in the fusion protein. Compare the DNA sequence of the construct with the known PreScission Protease cleavage sequence. Verify that the optimal PreScission Protease recognition site, Leu-Glu-Val-Leu-PheGln¡Gly-Pro, has not been altered. • Remove possible PreScission Protease inhibitors by extensive washing of the purification column before cleaving with PreScission Protease. The presence of Zn2+, as well as Pefabloc SC or chymostatin, may interfere with PreScission Protease activity. 87
Following cleavage and SDS gel analysis, multiple bands are seen on the gel • Determine when the bands appear. Additional bands seen prior to PreScission Protease cleavage may be the result of proteolysis in the host bacteria or in the purification steps leading up to the cleavage steps. E. coli BL21 is a protease-deficient strain that is recommended for use. • Check the sequence of the fusion partner for the presence of additional PreScission Protease recognition sites. PreScission Protease optimally recognizes the sequence Leu-Glu-Val-Leu-Phe-Gln¡Gly-Pro and cleaves between the Gln and Gly residues; however, similar secondary sites may exhibit some propensity for cleavage. Adjusting reaction conditions (e.g. time, temperature, salt concentration) may provide selective cleavage at the desired site. If adjustment of the conditions does not correct the problem, reclone the insert into a pGEX-T (thrombin) or pGEX-X (Factor Xa) expression vector. The fusion partner is contaminated with PreScission Protease after purification • Pass the sample over a new GSTrap FF column or fresh Glutathione Sepharose to remove residual PreScission Protease. It is possible that the Glutathione Sepharose may have been saturated with GST fusion protein in the first purification, although saturation of the purification column is rarely a problem.
Thrombin Cleavage with thrombin is incomplete • Check that the ratio of thrombin to fusion protein is correct. • Increase the reaction time to 20 h at 22–25 °C, and increase the amount of thrombin used in the reaction. • Check the DNA sequence of the construct to verify the presence of the thrombin site. Verify that the thrombin site has not been altered. • Check that protease inhibitors have been removed. Following cleavage and SDS gel analysis, multiple bands are seen on the gel • Determine when the bands appear. Additional bands seen prior to thrombin cleavage may be the result of proteolysis in the host bacteria or in the purification steps leading up to the cleavage step. E. coli BL21 is a protease-deficient strain that is recommended for use. • Check the sequence of the fusion partner for the presence of additional thrombin recognition sites. Optimum cleavage sites for thrombin are given in 1) and 2) below. 1) P4-P3-Pro-Arg/Lys¡P1'-P2' where P3 and P4 are hydrophobic amino acids and P1' and P2' are non-acidic amino acids. The Arg/Lys¡P1' bond is cleaved. Examples: P4
P3
Pro
R/K¡P1'
A)
Met
Tyr
Pro
Arg¡Gly
P2' Asn
B)
Ile
Arg
Pro
Lys¡Leu
Lys
C)
Leu
Val
Pro
Arg¡Gly
Ser
In A, the Arg¡Gly bond is cleaved very quickly by thrombin. In B, the Lys¡Leu bond is 88
cleaved. C is the recognition sequence found on the thrombin series of pGEX plasmids; the Arg¡Gly bond is cleaved. 2) P2-Arg/Lys¡P1', where either P2 or P1' is Gly. The Arg/Lys¡P1' bond is cleaved. Examples: P2
R/K¡P1'
A)
Ala
Arg¡Gly
B)
Gly
Lys¡Ala
In A, the Arg¡Gly bond is cleaved efficiently. In B, the Lys¡Ala bond is cleaved. Adjusting time and temperature of digestion can result in selective cleavage at the desired thrombin site. If adjustment of conditions does not correct the problem, reclone the insert into a pGEX-6P (PreScission) or pGEX-X (Factor Xa) expression vector.
Factor Xa Cleavage with Factor Xa is incomplete • Factor Xa requires activation of Factor X with Russell’s viper venom. Factor Xa from Amersham Biosciences has been preactivated, but other sources may not be activated. To activate, incubate Factor Xa with Russell’s viper venom at a ratio of 1% (w/w) in 70 mM NaCl, 8 mM CaCl2, 8 mM Tris-HCl, pH 8.0 at 37 °C for 5 min. • Check the DNA sequence of the construct to verify the presence of the Factor Xa site. Verify that the Factor Xa site has not been altered. The recognition sequence for Factor Xa is Ile-Glu-Gly-Arg¡X, where X can be any amino acid except Arg or Pro. • Check that the Factor Xa to fusion protein ratio is correct. • Check that glutathione has been removed as recommended. • In some cases, increasing the substrate concentration up to 1 mg/ml may improve results. • Add < 0.5% w/v SDS to the reaction buffer. This can significantly improve Factor Xa cleavage with some fusion proteins. Various concentrations of SDS should be tested to find the optimum concentration. • Increase incubation time to 20 h or longer at 22 °C and increase the amount of Factor Xa (for some fusion proteins, Factor Xa can be increased up to 5%). • Check that protease inhibitors have been removed. Following cleavage and SDS gel analysis, multiple bands are seen on the gel • Determine when the bands appear. Additional bands seen prior to cleavage may be the result of proteolysis in the host bacteria or in the purification steps leading up to the cleavage step. E. coli BL21 is a protease-deficient strain that is recommended for use. • Check the sequence of the fusion partner for the presence of additional Factor Xa recognition sites. Factor Xa is highly specific for the recognition sequence Ile-Glu-Gly-Arg¡. The bond following the Arg residue is cleaved. Adjusting the time and temperature of digestion can result in selective cleavage at the desired Factor Xa site. If adjustment of conditions does not correct the problem, reclone the insert into a pGEX-6P (PreScission) or pGEX-T (thrombin) expression vector. 89
90
Appendix 1 Characteristics of GST and of host bacterial strain Properties of Glutathione S-transferase Glutathione S-transferase is a naturally occurring Mr 26 000 protein that can be expressed in E. coli with full enzymatic activity. The properties below were determined in pGEX-1N (6). Dimer molecular weight
Mr 58 500
Km (glutathione)
0.43 ± 0.07 mM
Km (CDNB)
2.68 ± 0.77 mM
pI (chromatofocusing)
5.0
GST class
hybrid of Alpha and Mu characteristics
Properties and handling of E. coli BL21 Genotype
F-, ompT, hsdS (rB-, mB-), gal (53, 54).
Growth conditions
Resuspend lyophilized cultures in 1 ml of LB medium*. Grow overnight at 37 °C before plating onto LB agar plates.
Long-term storage
Mix equal volumes of stationary phase culture (grown in LB medium) and glycerol. Store at -70 °C. Revive frozen glycerol stocks of BL21 by streaking onto LB agar plates.
Recommended usage
The protease-minus nature of BL21 makes it useful as an expression host. Since BL21 does not transform well, use an alternate strain for cloning and maintenance of the vector.
* LB medium (prepared fresh): 10 g tryptone, 5 g yeast extract, 10 g NaCl. Combine tryptone, yeast extract, and NaCl in 900 ml H2O. Stir to dissolve, and adjust volume to 1 l. Sterilize by autoclaving. To prepare as a solid medium, add 1.2–1.5% agar.
91
92
Stop codon (TGA)
Plasmid Replication Region
U13853
1041–1019
869–891
2302–2998
2995
4398
3318
2235
1377
1330–1335 1307–1312
930–966
NA
NA
NA
918–935
258
U13854
1042–1020
869–891
2303–2999
2996
4399
3319
2236
1378
1331–1336 1308–1313
930–967
NA
NA
NA
918–935
258
244
217–237
205–211 183–188
pGEX-4T-2 27-4581-01
U13855
1040–1018
869–891
2301–2997
2994
4397
3317
2234
1376
1329–1334 1306–1311
930–965
NA
NA
NA
918–935
258
244
217–237
205–211 183–188
pGEX-4T-3 27-4583-01
U13856
1044–1022
869–891
2305–3001
2998
4401
3321
2238
1380
1333–1338 1310–1315
934–969
NA
NA
921–932
NA
258
244
217–237
205–211 183–188
pGEX-5X-1 27-4584-01
U13857
1045–1023
869–891
2306–3002
2999
4402
3322
2239
1381
1334–1339 1311–1316
934–970
NA
NA
921–932
NA
258
244
217–237
205–211 183–188
pGEX-5X-2 27-4585-01
U13858
1046–1024
869–891
2307–3003
3000
4403
3323
2240
1382
1335–1340 1312–1317
934–971
NA
NA
921–932
NA
258
244
217–237
205–211 183–188
pGEX-5X-3 27-4586-01
U78872
1056–1034
869–891
2317–3013
3010
4413
3333
2250
1392
1345–1350 1322–1327
945–981
NA
918–938
NA
NA
258
244
217–237
205–211 183–188
pGEX-6P-1 27-4597-01
U78873
1057–1035
869–891
2318–3014
3011
4414
3334
2251
1393
1346–1351 1323–1328
945–982
NA
918–938
NA
NA
258
244
217–237
205–211 183–188
pGEX-6P-2 27-4598-01
U78874
1055–1033
869–891
3216–3012
3009
4412
3332
2249
1391
1344–1349 1321–1326
945–980
NA
918–938
NA
NA
258
244
217–237
205–211 183–188
pGEX-6P-3 27-4599-01
Complete DNA sequences and restriction site data are available at the Amersham Biosciences web site (http:// www.amershambiosciences.com).
U13851
1041–1019
GenBank Accession Number
869–891
3' pGEX Sequencing Primer binding
2302–2998
5' pGEX Sequencing Primer binding
Sequencing Primers
Region necessary for replication
Site of replication initiation
2995
3318
4398
Start codon (GTG)
LacI q Gene Region
1377
2235
Start codon (ATG)
1330–1335 1307–1312
Stop codon (TAA)
Promoter –10 –35
b-lactamase (Ampr) Gene Region
936–950
951–966
NA
Coding region for PreScission Protease cleavage
Multiple Cloning Site
NA
Coding region for factor Xa cleavage
Coding for kinase recognition site
918–935
Coding region for thrombin cleavage
258
Start codon (ATG) for GST
244
217–237
217–237
244
205–211 183–188
pGEX-4T-1 27-4580-01
205–211 183–188
Ribosome binding site for GST
lac operator
tac promoter –10 –35
Glutathione S- Transferase Region
SELECTION GUIDE – pGEX Vector Control Regions pGEX-2TK 27-4587-01
Appendix 2
Control regions for pGEX vectors
Appendix 3 Electroporation Preparation of cells Reagents required 2× YT medium: Dissolve 16 g tryptone, 10 g yeast extract, and 5 g NaCl in 900 ml of distilled H2O. Adjust the pH to 7.0 with NaOH. Adjust the volume to 1 l with distilled H2O. Sterilize by autoclaving for 20 min. To prepare as a solid medium, add 1.2–1.5% agar. 1 mM HEPES: 0.26 g HEPES, sodium salt. Dissolve in 900 ml distilled, deionized H2O. Adjust the pH to 7.0. Adjust the volume to 1 l with distilled H2O. Sterilize by autoclaving. 10% glycerol in 1 mM HEPES, pH 7.0: Aseptically add 10 ml sterile glycerol to 90 ml sterile 1 mM HEPES, pH 7.0. 10% glycerol in distilled, deionized H2O: Add 10 ml glycerol to 90 ml distilled, deionized H2O. Sterilize by autoclaving. Isopropanol TE buffer: 10 mM Tris-HCl (pH 8.0), 1 mM EDTA Phenol: Redistilled phenol saturated with TE buffer containing 8-hydroxy quinoline (17) Chloroform/isoamyl alcohol: Reagent-grade chloroform and isoamyl alcohol, mixed 24:1 Phenol/chloroform: Equal parts of redistilled phenol and chloroform/isoamyl alcohol (24:1), each prepared as described above 3 M sodium acetate, pH 5.4: Aqueous solution Ethanol, 70%, 95%
Steps 1. Inoculate 10 ml of 2× YT medium with an E. coli host strain from an LB or 2× YT medium plate. Incubate at 37 °C overnight with shaking. 2. Inoculate 1 l of 2× YT medium with the 10 ml overnight culture of host cells. Incubate for 2–2.5 h at 37 °C with shaking at 250 rpm until an A600 of 0.5–0.7 is achieved. 3. Place the flask on ice for 15–30 min. 4. Spin at 4000 × g for 20 min at 4 °C. 5. Decant the supernatant and resuspend the cells in 1 l of ice-cold sterile 1 mM HEPES, pH 7.0. 6. Spin as described above. Decant the supernatant and resuspend the cells in 500 ml of ice-cold sterile 1 mM HEPES, pH 7.0. 7. Spin as described above. Decant the supernatant. Wash the cells in 20 ml of sterile 1 mM HEPES, pH 7.0, containing 10% glycerol. 8. Spin as described above. Decant the supernatant. Resuspend the cells in a total volume of 2–3 ml of sterile 10% glycerol in distilled, deionized H2O. 9. Dispense in 50–100 µl aliquots and proceed to the Electroporation protocol or freeze on dry ice and store at -70 °C. 10. Extract the ligated pGEX vector (as well as the uncut vector) once with an equal volume of phenol/chloroform and once with an equal volume of chloroform/isoamyl alcohol. 11. Remove the aqueous phase and add 1/10 volume of 3 M sodium acetate, pH 5.4 and 2.5 volumes of 95% ethanol. 12. Place on dry ice for 15 min and then spin in a microcentrifuge for 5 min to pellet the DNA.
93
13. Remove the supernatant and wash the pellet with 1 ml of 70% ethanol. Spin for 5 min, discard the supernatant, and dry the pellet. 14. Resuspend each DNA pellet in 20 µl of sterile distilled H2O. Alternatively, the DNA can be gel band-purified.
The DNA must be completely free of salt prior to electroporation. Electroporation We recommend that 1 ng of uncut (supercoiled) vector DNA be transformed in parallel with insert/pGEX ligations to determine the efficiency of each competent cell preparation. Reagents required
The following protocol was developed using a Bio-Rad Gene Pulser™. Salt-free DNA SOC medium: To 20 g of tryptone, 5 g of yeast extract and 0.5 g of NaCl, add distilled H2O to 1 l. Dispense into bottles at 100 ml per bottle and autoclave for 20 min. When cool, add 1 ml of sterile 1 M MgCl2, 1 ml of sterile 250 mM KCl and 2.78 ml of sterile 2 M (36%) glucose (see below) for each 100 ml of medium. LBAG plates: Prepare LBG medium by dissolving 10 g tryptone, 5 g yeast extract, and 5 g NaCl in 900 ml of distilled H2O. Sterilize by autoclaving. After the medium has cooled to 50 °C, add 10 ml of sterile 2 M glucose (see below), then aseptically add 1 ml of a 100 mg/ml ampicillin stock solution (final concentration 100 µg/ml), see below. Adjust to 1 l with sterile distilled H2O. To prepare as a solid medium, add 1.2–1.5% agar. 2 M glucose: Dissolve 36 g in 70 ml of H2O. Add H2O to 100 ml and filter-sterilize using a 0.2 µm filter. Ampicillin stock solution: Dissolve 400 mg of the sodium salt of ampicillin in 4 ml of H2O. Sterilize by filtration and store in small aliquots at -20 °C.
Steps 1. If electroporation-competent cells have been frozen, thaw vials on ice. Otherwise, proceed directly to the following step using freshly prepared cells. 2. Transfer 50 µl of cells to a pre-chilled 0.2 cm cuvette. 3. Add 2 µl of salt-free DNA from the insert/pGEX ligations (from step 14 above) or 1 ng of supercoiled control DNA (e.g. pUC 18) and disperse. Place on ice for 1 min. 4. Program the electroporator to give 25 µF, 2.5 kV at 200 ohms. Dry the cuvette with a tissue and place it into the electroporation chamber. Pulse once (should yield a pulse with a time constant of 4.5–5 msec). 5. Immediately add 1 ml of fresh SOC medium to the cuvette and invert to resuspend the cells. Transfer the contents of the cuvette to a 15 ml disposable culture tube.
Note: For a negative control, repeat steps 2–5 without adding DNA. 6. Incubate all tubes for 1 h at 37 °C with shaking (250 rpm). 7. Plate 100 µl of the transformed cells from the ligated samples and 100 µl of the negative control onto separate LBAG plates. Dilute the sample of the transformed cells from the uncut vector 10-fold and plate 10 µl onto a LBAG plate. Incubate the plates at 37 °C overnight.
94
Appendix 4 Sequencing of pGEX fusions Sequencing pGEX vectors can be sequenced using the pGEX 5' and 3' Sequencing Primers. The sequences and the binding regions of these primers are given below: pGEX 5' Sequencing Primer 5'-d[GGGCTGGCAAGCCACGTTTGGTG]-3' The pGEX 5' Sequencing Primer binds at nucleotides 869–891 on all ten pGEX vectors. pGEX 3' Sequencing Primer 5'-d[CCGGGAGCTGCATGTGTCAGAGG]-3' The pGEX 3' Sequencing Primer binds at the following locations on the pGEX vectors: Vector
Binding site
pGEX-2TK
1041–1019
pGEX-4T-1
1041–1019
pGEX-4T-2
1042–1020
pGEX-4T-3
1040–1018
pGEX-5X-1
1044–1022
pGEX-5X-2
1045–1023
pGEX-5X-3
1046–1024
pGEX-6P-1
1056–1034
pGEX-6P-2
1057–1035
pGEX-6P-3
1055–1033
For information concerning control regions in the pGEX vectors, see Appendix 2.
95
Appendix 5 Cleaning, storage, and handling of media/columns Glutathione Sepharose 4B and Glutathione Sepharose 4 FF media • Glutathione Sepharose 4B is recommended for packing small gravity-flow columns and for batch purifications. • Glutathione Sepharose Fast Flow is excellent for packing high-performance columns for use with purification systems and for scaling-up. See Chapter 4, Table 8 for the physical characteristics of these media. Chemical stability Glutathione Sepharose shows no significant loss of binding capacity when exposed to 70% ethanol or 6 M guanidine hydrochloride for 2 h at room temperature or to 1% SDS for 14 d. Cleaning Re-use of purification columns and media depends upon the nature of the sample and should only be performed with identical samples to prevent cross-contamination. If Glutathione Sepharose appears to be losing binding capacity, it may be due to an accumulation of precipitated, denatured or non-specifically bound proteins. To remove precipitated or denatured substances: 1. Wash with two column volumes of 6 M guanidine hydrochloride. 2. Immediately wash with five column volumes of 1× PBS, pH 7.4. To remove hydrophobically bound substances: 1. Wash with 3–4 column volumes of 70% ethanol or two column volumes of 1% Triton X-100. 2. Immediately wash with five column volumes of 1× PBS, pH 7.4. For storage: Wash medium and column twice with 20% ethanol at neutral pH (use approximately five column volumes for packed medium) and store at 4–8 °C. GSTPrep FF 16/10 columns The column is supplied in 20% ethanol. If the column is to be stored for more than 2 d after use, clean and store as described below. See Chapter 4, Table 14 for the physical characteristics of GSTPrep FF 16/10 columns. Re-use of GSTPrep FF 16/10 columns depends upon the nature of the sample and should only be performed with identical samples to prevent cross-contamination.
96
Do not open the column. Buffer and solvent resistance • De-gas and filter all solutions through a 0.45 µm filter to increase column lifetime. • Daily use: All commonly used aqueous buffers, pH 6–9. • Cleaning: Guanidine hydrochloride, up to 6 M for 1 h at room temperature. Ethanol up to 70%. Non-ionic detergents, e.g. Triton X-100. • Avoid: Solutions < pH 6. Cleaning in place If the column appears to be losing binding capacity, it may be due to an accumulation of precipitated, denatured or non-specifically bound proteins. To remove precipitated or denatured substances: 1. Wash with 40 ml of 6 M guanidine hydrochloride. 2. Immediately wash with 100 ml of 1× PBS, pH 7.4, at a flow rate of 5 ml/min. To remove hydrophobically bound substances: 1. Wash with 60–80 ml of 70% ethanol or 40 ml of 1% Triton X-100. 2. Immediately wash with 100 ml of 1× PBS, pH 7.4, at a flow rate of 5 ml/min. For storage: Wash column with at least 100 ml of 20% ethanol at neutral pH at a flow rate of 5 ml/min and store at 4–8 °C. Handling Many chromatography systems are equipped with pressure gauges to measure the pressure at a particular point in the system, usually just after the pumps. The pressure measured here is the sum of the pre-column pressure, the pressure drop over the gel bed, and the post-column pressure. It is always higher than the pressure drop over the bed alone. We recommend keeping the pressure drop over the bed below 1.5 bar. Setting the upper limit of your pressure gauge to 1.5 bar will ensure the pump shuts down before the gel is overpressured. If necessary, post-column pressure of up to 3.5 bar can be added to the limit without exceeding the column hardware limit. To determine post-column pressure, proceed as follows: To avoid breaking the column, the post-column pressure must never exceed 3.5 bar. 1. Connect a piece of tubing in place of the column. 2. Run the pump at the maximum flow you intend to use for chromatography. Use a buffer with the same viscosity as you intend to use for chromatography. Note this back pressure as total pressure. 3. Disconnect the tubing and run the same flow rate used in step 2. Note this back pressure as pre-column pressure.
97
4. Calculate the post-column pressure as total pressure minus pre-column pressure. If the post-column pressure is higher than 3.5 bar, take steps to reduce it (shorten tubing, clear clogged tubing, or change flow restrictors) and perform steps 1–4 again until the post-column pressure is below 3.5 bar. When the post-column pressure is satisfactory, add the post-column pressure to 1.5 bar and set this as the upper pressure limit on the chromatography system. Benzamidine Sepharose 4 Fast Flow With Benzamidine Sepharose 4 Fast Flow (high sub) columns, the GST fusion protein is present in the flow-through and wash. The protease (thrombin or Factor Xa) remains bound to the column until eluted using one of the buffers described below. Recommended elution buffers Elution of the bound protease can be performed using either a step or a continuous gradient. 1. Substances bound through ionic interactions can be eluted by increasing the salt concentration to 1.0 M. 2. Affinity-bound substances can be eluted in different ways: • Low pH elution buffers: 0.05 M glycine, pH 3.0 or 10 mM HCl, 0.5 M NaCl, pH 2.0. For each ml of fraction to be collected, add 60–200 µl of 1 M Tris-HCl, pH 9 to the collection tube. This will prevent denaturation of the eluted protein at low pH. • Competitive elution buffer: 20 mM p-Aminobenzamidine in binding buffer. Note: This elution buffer has a very high A280. Therefore, the eluted protein must be detected by methods other than absorbance, such as activity measurement (if possible), total protein, or SDS-PAGE analysis. The advantage of competitive elution is that pH can be kept constant during the run. 3. Other possible elution buffers: Denaturing agents such as 8 M urea or 6 M guanidine hydrochloride. After elution of the protease, wash the column with five column volumes of binding buffer (0.05 M Tris-HCl, 0.5 M NaCl, pH 7.4). For longer-term storage, store in a buffer containing 20% ethanol in 0.05 M acetate buffer, pH 4 at 4–8 °C.
98
Appendix 6 Cross-adsorption of anti-GST antiserum with E. coli proteins Some sources of anti-GST antibody may contain anti-E. coli antibodies that will react with E. coli proteins contaminating a fusion protein sample. Use the following protocols to prepare an immobilized E. coli sonicate that can be used to cross-adsorb anti-E. coli antibodies. The antibody available from Amersham Biosciences has been cross-adsorbed with E. coli BL21 proteins and therefore requires no additional cross-adsorption. The following protocols will treat 240 ml of anti-GST antiserum. Preparation of sonicate Reagents required 2× YT medium: Dissolve 16 g tryptone, 10 g yeast extract, and 5 g NaCl in 900 ml of distilled H2O. Adjust the pH to 7.0 with NaOH. Adjust the volume to 1 l with distilled H2O. Sterilize by autoclaving for 20 min. To prepare as a solid medium, add 1.2–1.5% agar. Coupling buffer: Dissolve 29 g NaCl and 8.4 g NaHCO3 in 800 ml distilled, deionized H2O. Adjust the pH to 8.3 with HCl. Bring final volume to 1 l with distilled, deionized H2O. Store at room temperature for no longer than 1 week.
Steps 1. Use a single colony of non-transformed E. coli cells to inoculate 30 ml of 2× YT medium. 2. Incubate for 12–15 h at 37 °C with vigorous shaking. 3. Transfer 25 ml of the culture into 2 l of pre-warmed 2× YT medium contained in a 4-l flask. Incubate at 37 °C with vigorous shaking until the A600 reaches 2.5. 4. Transfer the culture to 42 °C and continue incubating for an additional hour. 5. Transfer the culture to appropriate centrifuge bottles and centrifuge at 7700 × g for 10 min (or 5000 × g for 30 min) at 4 °C to sediment the cells. 6. Discard the supernatant and resuspend the cells in coupling buffer to an A600 of 80. The final volume should be approximately 50–75 ml. 7. Transfer the cell suspension to a container appropriate for sonication. 8. Place the container on ice and disrupt the cells using an appropriately equipped sonicator.
Sonication should achieve > 90% cell lysis as determined by microscopic examination. 9. Store the sonicate at -70 °C until needed.
Preparation of immobilized sonicate Reagents required CNBr-activated Sepharose 4B Coupling buffer: Dissolve 29 g NaCl and 8.4 g NaHCO3 in 800 ml distilled, deionized H2O. Adjust the pH to 8.3 with HCl. Bring the final volume to 1 l with distilled, deionized H2O. Store at room temperature for no longer than 1 week. Acetate buffer: Dissolve 8.2 g sodium acetate, 29 g NaCl in 800 ml distilled, deionized H2O. Adjust the pH to 4.0 with acetic acid. Bring the final volume to 1 l with distilled, deionized H2O. Store at room temperature. Tris buffer: Dissolve 12.1 g Tris-base, 29 g NaCl in 800 ml distilled, deionized H2O. Adjust the pH to 8.0 with HCl. Bring the final volume to 1 l with distilled, deionized H2O. Store at room temperature. 1× PBS: 140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.3
99
Steps 1. Prepare 15 g of CNBr-activated Sepharose 4B according to the manufacturer’s instructions. 2. To the 15 g of prepared CNBr-activated Sepharose 4B, add 45 ml of E. coli sonicate (thawed if necessary). Close the container securely and incubate at 4 °C for 12–16 h with gentle shaking (do not use magnetic stirring). 3. Transfer the suspension to an appropriately sized sintered filter funnel attached to a vacuum source. Apply a light vacuum to remove the solution. 4. Wash the medium with 500 ml of coupling buffer by slowly pouring the buffer into the funnel while stirring the medium with a glass stir rod. Apply a light vacuum to remove the solution. 5. Turn off the vacuum. Add 40 ml of acetate buffer. Stir for 5 min with a glass stir rod. Apply a light vacuum to remove the solution. 6. Turn off the vacuum. Add 40 ml of Tris buffer. Stir for 5 min with a glass stir rod. Apply a light vacuum to remove the solution. 7. Repeat steps 5 and 6 for a total of three times. 8. Add 100 ml of 1× PBS to the medium. Stir to suspend. 9. Split the suspension equally into four 100 ml centrifuge bottles. Add 50 ml of 1× PBS to each of the four bottles. Centrifuge at 500 × g for 10 min at 4 °C. Aspirate the supernatant, taking care not to disturb the medium.
Cross-adsorption of anti-GST antiserum with immobilized E. coli sonicate 1. Add 60 ml of crude anti-GST antiserum to each of the four bottles containing the immobilized E. coli sonicate. 2. Incubate at room temperature for 1 h with gentle shaking (do not use magnetic stirring). 3. Remove the medium by filtering the serum-medium suspension over Whatman™ 40 ashless filter paper. 4. The supernatant contains the cross-adsorbed anti-GST antiserum, which should be stored at 4 °C.
100
Appendix 7 Converting from linear flow (cm/h) to volumetric flow rates (ml/min) and vice versa When comparing results for columns of different sizes, it is often convenient to express flow as linear flow (cm/h). However, flow is usually measured in volumetric flow rate (ml/min). To convert between linear flow and volumetric flow rate, use one of the following formulas.
From linear flow (cm/h) to volumetric flow rate (ml/min) Volumetric flow rate (ml/min) = Linear flow (cm/h) × column cross-sectional area (cm2) 60 =
Y p × d2 × 60 4
where Y = linear flow in cm/h d = column inner diameter in cm
Example: What is the volumetric flow rate in an XK 16/70 column (i.d. 1.6 cm) when the linear flow is 150 cm/h? Y = linear flow = 150 cm/h d = inner diameter of the column = 1.6 cm Volumetric flow rate =
150 × p × 1.6 × 1.6 ml/min 60 × 4
= 5.03 ml/min
From volumetric flow rate (ml/min) to linear flow (cm/h) Linear flow (cm/h) =
Volumetric flow rate (ml/min) × 60 column cross-sectional area (cm2)
= Z × 60 ×
4 p × d2
where Z = volumetric flow rate in ml/min d = column inner diameter in cm
Example: What is the linear flow in an HR 5/5 column (i.d. 0.5 cm) when the volumetric flow rate is 1 ml/min? Z = Volumetric flow rate = 1 ml/min d = column inner diameter = 0.5 cm Linear flow = 1 × 60 ×
4 p × 0.5 × 0.5
cm/h
= 305.6 cm/h
From ml/min to using a syringe 1 ml/min = approximately 30 drops/min on a HiTrap 1 ml column 5 ml/min = approximately 120 drops/min on a HiTrap 5 ml column
101
Appendix 8 Amino acids table Three-letter code
Single-letter code
Alanine
Ala
A
Arginine
Arg
R
Amino acid
Structure HOOC CH3 H2N NH2
HOOC CH2CH2CH2NHC H2N
NH
HOOC
Asparagine
Asn
N
Aspartic Acid
Asp
D
CH2CONH2 H2N HOOC CH2COOH H2N HOOC
Cysteine
Cys
CH2SH
C H2N HOOC
Glutamic Acid
Glu
CH2CH2COOH
E H2N HOOC
Glutamine
Gln
Q
Glycine
Gly
G
Histidine
His
H
Isoleucine
Ile
I
CH2CH2CONH2 H2N HOOC H H2N HOOC
N CH2
NH
H2N HOOC
CH(CH3)CH2CH3 H2N HOOC
Leucine
Leu
L
CH3 CH2CH CH3
H2N HOOC
Lysine
Lys
K
Methionine
Met
M
CH2CH2CH2CH2NH2 H2N HOOC CH2CH2SCH3 H2N HOOC
Phenylalanine
Phe
F
Proline
Pro
P
CH2 H2N HOOC H2N
NH
HOOC
Serine
Ser
S
Threonine
Thr
T
CH2OH H2N HOOC CHCH3 H2N
OH
HOOC
Tryptophan
Trp
W
CH2 H2N
NH
HOOC
Tyrosine
Tyr
CH2
Y H2N HOOC
Valine
Val
CH(CH3)2
V H2N
102
OH
Formula
Mr
Middle unit residue (-H20) Formula Mr
C3H7NO2
89.1
C3H5NO
C6H14N4O2
174.2
C 4H 8N 2O 3
Charge at pH 6.0–7.0
Hydrophobic (non-polar)
Uncharged (polar)
71.1
Neutral
"
C6H12N4O
156.2
Basic (+ve)
132.1
C 4H 6N 2O 2
114.1
Neutral
C4H7NO4
133.1
C4H5NO3
115.1
Acidic(-ve)
C3H7NO2S
121.2
C3H5NOS
103.2
Neutral
C5H9NO4
147.1
C5H7NO3
129.1
Acidic (-ve)
C5H10N2O3
146.1
C 5H 8N 2O 2
128.1
Neutral
"
C2H5NO2
75.1
C2H3NO
57.1
Neutral
"
C 6H 9N 3O 2
155.2
C 6H 7N 3O
137.2
Basic (+ve)
C6H13NO2
131.2
C6H11NO
113.2
Neutral
"
C6H13NO2
131.2
C6H11NO
113.2
Neutral
"
C6H14N2O2
146.2
C6H12N2O
128.2
Basic(+ve)
C5H11NO2S
149.2
C5H9NOS
131.2
Neutral
"
C9H11NO2
165.2
C9H9NO
147.2
Neutral
"
C5H9NO2
115.1
C5H7NO
97.1
Neutral
"
C3H7NO3
105.1
C3H5NO2
87.1
Neutral
"
C4H9NO3
119.1
C4H7NO2
101.1
Neutral
"
C11H12N2O2
204.2
C11H10N2O
186.2
Neutral
C9H11NO3
181.2
C9H9NO2
163.2
Neutral
C5H11NO2
117.1
C5H9NO
99.1
Neutral
Hydrophilic (polar)
" " " " "
"
"
" " "
103
Appendix 9 Protein conversion data Mass (g/mol)
1 µg
1 nmol
A280 for 1 mg/ml
10 000
100 pmol; 6 × 10
13
molecules
10 µg
IgG
50 000
20 pmol; 1.2 × 10
13
molecules
50 µg
IgM
1.20
100 000
10 pmol; 6.0 × 10
12
molecules
100 µg
IgA
1.30
150 000
6.7 pmol; 4.0 × 10
12
molecules
150 µg
Protein A
0.17
1 kb of DNA
= 333 amino acids of coding capacity
270 bp DNA
= 10 000 g/mol
= 37 000 g/mol 1.35 kb DNA
= 50 000 g/mol
2.70 kb DNA
= 100 000 g/mol
Average molecular weight of an amino acid = 120 g/mol.
104
Protein
1.35
Avidin
1.50
Streptavidin
3.40
Bovine Serum Albumin
0.70
References 1. Smith, D. B. and Johnson, K. S., Gene 67, 31–40 (1988). 2. Parker, M. W. et al., J. Mol. Biol. 213, 221–222 (1990). 3. Ji, X. et al., Biochemistry 31, 10169–10184 (1992). 4. Maru, Y. et al., J. Biol. Chem. 271, 15353–15357 (1996). 5. McTigue, M. A. et al., J. Mol. Biol. 246, 21–27 (1995). 6. Walker, J. et al., Mol. Biochem. Parasitol. 61, 255–264 (1993). 7. Toye, B. et al., Infect. Immun. 58, 3909–3913 (1990). 8. Fikrig, E. et al., Science 250, 553–556 (1990). 9. Johnson, K. S. et al., Nature 338, 585–587 (1989). 10. Kaelin, W. G. et al., Cell 64, 521–532 (1991). 11. Chittenden, T. et al., Cell 65, 1073–1082 (1991). 12. Kaelin, W. G. et al., Cell 70, 351–364 (1992). 13. Baker, T. A. et al., Proc. Natl. Acad. Sci. USA 81, 6779–6783 (1984). 14. Strauch, K. L. and Beckwith, J., Proc. Natl. Acad. Sci. USA 85, 1576–1580 (1988). 15. Grodberg, J. and Dunn, J. J., J. Bacteriol. 170, 1245–1253 (1988). 16. Sugimura, K. and Higashi, N., J. Bacteriol. 170, 3650–3654 (1988). 17. Sambrook, J., Fritsch, E. F. and Maniatis, T., Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989). 18. Chung, C. T. et al., Proc. Natl. Acad. Sci. USA 86, 2172–2175 (1989). 19. Smith, D. B. and Corcoran, L. M., in Current Protocols in Molecular Biology Vol. 2 (Ausubel, F. M. et al., eds.), John Wiley and Sons, Inc., New York, p. 16.7.1 (1990). 20. Marston, F. A. O., Biochem. J. 240, 1–12 (1986). 21. Hober, S. and Uhlen, M., in Protein Engineering in Industrial Biotechnology (Alberghina, L., ed.), Harwood Academic Publishers, Amsterdam, Netherlands, pp. 24–39 (1999). 22. Bell, P., in Molecular Biology Problem Solver: A Laboratory Guide (Gerstein, A. S., ed.), John Wiley and Sons, Inc., New York, pp. 461–490 (2001). 23. Gearing, D. P. et al., Biotechnology 7, 1157–1161 (1989). 24. Young, B. P. et al., EMBO J. 20, 262–271 (2001). 25. Singh, S. P. et al., Eur. J. Biochem. 268, 2912–2923 (2001). 26. Sato, K. et al., J. Cell Biol. 152, 935–944 (2001). 27. Kneidinger, B. et al., J. Biol. Chem. 276, 5577–5583 (2001). 28. Yu, M. et al., J. Biol. Chem. 275, 24984–24992 (2000). 29. Tamaru, T. et al., J. Neurosci. 20, 7525–7530 (2000). 30. Crombie, R. and Silverstein, R., J. Biol. Chem. 273, 4855–4863 (1998).
105
31. Habig, W. H. et al., J. Biol. Chem. 249, 7130–7139 (1974). 32. Mannervik, B. and Danielson, U. H., CRC Crit. Rev. Biochem. 23, 283–337 (1988). 33. Walker, P. A. et al., Biotechnology 12, 601–605 (1994). 34. Cordingley, M. G. et al., J. Biol. Chem. 265, 9062–9065 (1990). 35. Schein, C. H., Biotechnology 7, 1141–1149 (1989). 36. Schein, C. H. and Noteborn, M. H. M., Biotechnology 6, 291–294 (1988). 37. Schein, C. H., Biotechnology 8, 308–317 (1990). 38. Gentry, D. R. and Burgess, R. R., Protein Expr. Purif. 1, 81–86 (1990). 39. Frangioni, J. V. and Neel, B. G., Anal. Biochem. 210, 179–187 (1993). 40. Kelley, R. F. and Winkler, M. E., in Genetic Engineering Vol. 12 (Setlow, J. K., ed.), Plenum Press, New York, pp. 1–19 (1990). 41. Pigiet, V. P. and Schuster, B. J., Proc. Natl. Acad. Sci. USA 83, 7643–7647 (1986). 42. Kaelin, W., personal communication. 43. Mintz, G. R., BioPharm 6, 34–38 (1993). 44. Buchberger, A. et al., J. Mol. Biol. 261, 328–333 (1996). 45. Buchberger, A. et al., EMBO J. 13, 1687–1695 (1994). 46. Zylicz, M. et al., J. Biol. Chem. 260, 7591–7598 (1985). 47. Schönfeld, H.-J. et al., J. Biol. Chem. 270, 2183–2189 (1995). 48. Ellis, R. J. and van der Vies, S. M., Ann. Rev. Biochem. 60, 321–347 (1991). 49. Jackson, G. S. et al., Biochemistry 32, 2554–2563 (1993). 50. Yu-Sherman, M. and Goldberg, A. L., EMBO J. 11, 71–77 (1992). 51. Myers, M., BIOSCI posting (7 July 1993). 52. Thain, A. et al., Trends in Genet. 12, 209–210 (1996). 53. Studier, F. W. and Moffatt, B. A., J. Mol. Biol. 189, 113–130 (1986). 54. Grodberg, J. and Dunn, J. J., J. Bact. 170, 1245–1253 (1988).
106
Additional reading Code No.
GST fusion system Data File, “ GSTrap FF and Glutathione Sepharose 4 Fast Flow”
18-1136-89
Data File, “ MicroSpin GST Purification Module”
18-1128-13
Scientific poster, “ Rapid purification of GST-fusion proteins from large sample volumes”
18-1139-51
Application Note, “ Efficient, rapid protein purification and on-column cleavage using GSTrap FF columns”
18-1146-70
Scientific poster, “ Purification of GST fusion proteins, on-column cleavage, and sample clean-up”
18-1150-20
Application Note, “ ECL Western and ECL Plus Western blotting”
18-1139-13
Data File, “ GST 96-Well Detection Module”
18-1128-14
Note: See also the Life Science News articles listed in Chapter 7.
Handbooks Recombinant Protein Handbook: Protein Amplification and Simple Purification*
18-1142-75
Antibody Purification Handbook
18-1037-46
Affinity Chromatography Handbook, Principles and Methods
18-1022-29
Expanded Bed Adsorption Handbook, Principles and Methods
18-1124-26
Gel Filtration Handbook, Principles and Methods
18-1022-18
Hydrophobic Interaction Chromatography Handbook, Principles and Methods
18-1020-90
Ion Exchange Chromatography Handbook, Principles and Methods
18-1114-21
Protein Purification Handbook
18-1132-29
Reversed Phase Chromatography Handbook, Principles and Methods
18-1134-16
Protein Electrophoresis Technical Manual
80-6013-88
* The Recombinant Protein Handbook includes information on recombinant protein expression and purification, including GST fusion proteins. It has less information specifically on the GST Gene Fusion System than this handbook, but includes more general advice and information for working with His-tagged protein purification.
Column packing Column Packing Video PAL
17-0893-01
Column Packing Video NTSC
17-0894-01
Additional reading Data File, “ HiTrap Benzamidine FF (high sub) and Benzamidine Sepharose 4 Fast Flow”
18-1139-38
Application Note, “ Purification of a labile, oxygen sensitive enzyme for crystallization and 3-D structural determination”
18-1128-91
Application Note, “ Purification of a recombinant phosphatase using preprogrammed generic templates for different chromatographic techniques”
18-1142-32
ÄKTAdesign Brochure
18-1129-05
Convenient Purification, HiTrap Column Guide
18-1129-81
Affinity Chromatography Columns and Media, Product Profile
18-1121-86
Protein and Peptide Purification, Technique Selection Guide
18-1128-63
Fast Desalting and Buffer Exchange of Proteins and Peptides
18-1128-62
Gel Media Guide (electrophoresis)
18-1129-79
Protein and Peptide Purification Technique Selection
18-1128-63
Protein Purification, Principles, High Resolution Methods and Applications, J.C. Janson and L. Rydén, 1998, 2nd ed. Wiley VCH
18-1128-68
107
Ordering information Product
Quantity
Code No.
pGEX- 6P-1
25 µg
27-4597-01
pGEX- 6P-2
25 µg
27-4598-01
pGEX- 6P-3
25 µg
27-4599-01
pGEX- 5X-1
25 µg
27-4584-01
pGEX- 5X-2
25 µg
27-4585-01
pGEX- 5X-3
25 µg
27-4586-01
pGEX- 4T-1
25 µg
27-4580-01
pGEX- 4T-2
25 µg
27-4581-01
pGEX- 4T-3
25 µg
27-4583-01
pGEX 2TK
25 µg
27-4587-01
GST MicroSpin Purification Module
50 purifications
27-4570-03
GSTrap FF
2 × 1 ml 5 × 1 ml 1 × 5 ml
17-5130-02 17-5130-01 17-5131-01
Cloning and expression
All vectors include E. coli B21 (E. coli BL21 also available separately; see below under Companion products.)
Purification Prepacked columns and media
GSTPrep FF 16/10
1 × 20 ml
17-5234-01
Glutathione Sepharose 4 Fast Flow
25 ml 100 ml 500 ml
17-5132-01 17-5132-02 17-5132-03
Glutathione Sepharose 4B
10 ml 100 ml 300 ml
17-0756-01 27-4574-01 17-0756-04
Detection GST 96-Well Detection Module
96 reactions
27-4592-01
GST Detection Module
50 reactions
27-4590-01
Anti-GST Antibody
0.5 ml
27-4577-01
Cleavage enzymes and media for removal of thrombin and Factor Xa PreScission Protease
500 units
27-0843-01
Factor Xa
400 units
27-0849-01
Thrombin
500 units
27-0846-01
HiTrap Benzamidine FF (high sub)
2 × 1 ml 5 × 1 ml 1 × 5 ml
17-5143-02 17-5143-01 17-5144-01
Benzamidine Sepharose 4 Fast Flow (high sub)
25 ml
17-5123-10
Companion products Reagents E. coli BL21 (All pGEX vectors include E. coli B21) Isopropyl b-D-thiogalactoside (IPTG)
108
1 vial
27-1542-01
1g 5g 10 g
27-3054-03 27-3054-04 27-3054-05
Sephaglas BandPrep Kit
100 average purifications
27-9285-01
T4 DNA Ligase, FPLCpure, Cloned
200 units 1000 units
27-0870-03 27-0870-04
Product
Quantity
Code No.
Companion products (continued) Reagents Ready-To-Go T4 DNA Ligase
50 reactions
27-0361-01
Adenosine 5'-Triphosphate, 100 mM Solution (ATP)
25 µmol
27-2056-01
Ready-To-Go PCR Beads (0.2 ml tubes/plate)
96 reactions 5 × 96 reactions
27-9553-01 27-9553-02
Ready-To-Go PCR Beads (0.5 ml tubes)
100 reactions
27-9555-01
Taq DNA Polymerase (cloned)
50 units† † 250 units 5000 units† 10 × 50 units† 5 × 250 units† 250 units‡ 4 × 250 units‡ ‡ 10 × 250 units 25 000 units‡
T0303Y T0303V T0303X RPN0303Y RPN0303Z 27-0798-04 27-0798-05 27-0798-06 27-0798-64
Supplied with: 10× PCR buffer containing 100 mM Tris-HCl (pH 8.6), 500 mM KCl, 15 mM MgCl2, 1% Triton X-100. Separate MgCl2 solution (25 mM) also provided.
†
Supplied with: 10× PCR buffer containing 100 mM Tris-HCl (pH 9.0), 500 mM KCl, and 15 mM MgCl2. Separate MgCl2 solution (25 mM) also provided.
‡
Taq DNA Polymerase (T. aquaticus)
250 units 4 × 250 units 10 × 250 units 1000 units 5000 units 10 000 units 25 000 units
27-0799-04 27-0799-05 27-0799-06 27-0799-61 27-0799-62 27-0799-63 27-0799-64
dNTP Set, 100 mM Solutions (dATP, dCTP, dGTP, dTTP)
4 × 25 µmol 4 × 100 µmol 4 × 500 µmol
27-2035-01 27-2035-02 27-2035-03
pGEX 5' Sequencing Primer
0.05 A260 unit
27-1410-01
pGEX 3' Sequencing Primer
0.05 A260 unit
27-1411-01
FlexiPrep Kit
1 kit
27-9281-01
GFX Micro Plasmid Prep Kit
1 kit
27-9601-01
MicroPlex 24 Vacuum
1 system
27-3567-01
Empty Disposable PD-10 Columns
50
17-0435-01
MicroSpin G-25 Columns
50 columns
27-5325-01
HiTrap Desalting
5 × 5 ml
17-1408-01
HiPrep 26/10 Desalting
1 × 53 ml
17-5087-01
Disposable PD-10 Desalting Columns
30 columns
17-0851-01
CNBr-activated Sepharose 4B
15 g
17-0430-01
Superdex Peptide HR 10/30
1 × 24 ml
17-1453-01
Superdex 75 HR 10/30
1 × 24 ml
17-1047-01
Columns, media, and equipment
HR and XK columns (see catalog or web site for range) ÄKTAprime and other ÄKTAdesign chromatography systems (see catalog or web site for range)
Superdex 200 HR 10/30
1 × 24 ml
17-1088-01
HiLoad 16/60 Superdex 30 prep grade
1 × 120 ml
17-1139-01
HiLoad 16/60 Superdex 75 prep grade
1 × 120 ml
17-1068-01
HiLoad 16/60 Superdex 200 prep grade
1 × 320 ml
17-1069-01
109
Product
Quantity
Code No.
HiLoad 26/60 Superdex 30 prep grade
1 × 320 ml
17-1140-01
HiLoad 26/60 Superdex 75 prep grade
1 × 120 ml
17-1070-01
HiLoad 26/60 Superdex 200 prep grade
1 × 320 ml
17-1071-01
Ultrospec 1100 pro Spectrophotometer
1
Inquire
Hybond-P
10 sheets
RPN2020F
Hybond ECL
10 sheets
RPN2020D
ECL Western Blotting Detection Reagents
for 1000 cm2
RPN2109
ECL Plus Western Blotting Detection System
for 1000 cm2
RPN2132
Columns, media, and equipment (continued)
Western blotting
110
111
112
Handbooks from Amersham Biosciences
ÄKTA, ECL, Ettan, ExcelGel, FlexiPrep, FPLC, GFX, GSTPrep, GSTrap, HiLoad , HiTrap, HiPrep, Hybond, MicroPlex, MicroSpin, Multiphor, PhastGel, PreScission, Ready-To-Go, Sephaglas, Sepharose, and Superdex are trademarks of the Amersham Biosciences group. Amersham and Amersham Biosciences are trademarks of Amersham plc. ABTS is a registered trademark of Roche Molecular Biochemicals. Coomassie is a trademark of ICI plc. GenBank is a registered trademark of the National Institutes of Health. Gene Pulser is a registered trademark of Bio-Rad Laboratories. MicroSpin is a trademark of Lida Manufacturing Corp. Pefabloc is a registered trademark of Pentafam AG. Triton is a registered trademark of Union Carbide Chemicals and Plastics Co. Tween is a registered trademark of ICI Americas, Inc. Ultrospec is a registered trademark of Biochrom Ltd. Whatman is a registered trademark of Whatman Paper Ltd. Licensing information
Antibody Purification
All materials for research use only. Not for diagnostic or therapeutic purposes.
Handbook 18-1037-46
A license for commercial use of pGEX vectors must be obtained from AMRAD Corporation Ltd., 17-27 Cotham Road, Kew, Victoria 3101, Australia. PreScission Protease is licensed from the University of Singapore, 10 Kent Ridge Crescent, Singapore 0511. PreScission Protease is produced under commercial license from AMRAD Corporation Ltd., 17-27 Cotham Road, Kew, Victoria 3101, Australia.
The Recombinant Protein Handbook Protein Amplification and Simple Purification 18-1142-75
The Polymerase Chain Reaction (PCR) is covered by patents owned by Roche Molecular Systems and F Hoffmann-La Roche Ltd. A license to use the PCR process for certain research and development activities accompanies the purchase of certain reagents from licensed suppliers such as Amersham Biosciences and Affiliates when used in conjunction with an authorized thermal cycler.
Protein Purification
Reversed Phase Chromatography
Handbook 18-1132-29
Principles and Methods 18-1134-16
Ion Exchange Chromatography
Expanded Bed Adsorption
Principles and Methods 18-1114-21
Principles and Methods 18-1124-26
Affinity Chromatography
Chromatofocusing
Principles and Methods 18-1022-29
with Polybuffer and PBE 50-01-022PB
2-D Electrophoresis
Hydrophobic Interaction Chromatography
Microcarrier cell culture
Principles and Methods 80-6429-60
Principles and Methods 18-1020-90
Principles and Methods 18-1140-62
Gel Filtration
Percoll
IEF, SDS-PAGE and 2-D Electrophoresis
Principles and Methods 18-1022-18
Methodology and Applications 18-1115-69
Principles and Methods 80-6484-89
Ficoll-Paque Plus For in vitro isolation of lymphocytes 18-1152-69
GST Gene Fusion System Handbook 18-1157-58
using immobilized pH gradients
Purchase of Ready-To-Go PCR Beads is accompanied by a limited license to use it in the Polymerase Chain Reaction (PCR) process solely for the research and development activities of the purchaser in conjunction with a thermal cycler whose use in the automated performance of the PCR process is covered by the up-front license fee, either by payment to Perkin-Elmer or as purchased, i.e. an authorized thermal cycler. Taq DNA polymerase is sold under licensing arrangements with Roche Molecular Systems, F Hoffman-La Roche Ltd and the Perkin-Elmer Corporation. Purchase of this product is accompanied by a limited license to use it in the Polymerase Chain Reaction (PCR) process for research in conjunction with a thermal cycler whose use in the automated performance of the PCR process is covered by the up-front license fee, either by payment to Perkin-Elmer or as purchased, i.e. an authorized thermal cycler. All goods and services are sold subject to the terms and conditions of sale of the company within the Amersham group that supplies them. A copy of these terms and conditions is available on request. © Amersham Biosciences AB 2002– All rights reserved. Amersham Biosciences AB Björkgatan 30, SE-751 84 Uppsala, Sweden Amersham Biosciences Amersham Place, Little Chalfont, Buckinghamshire HP7 9NA, England Amersham Biosciences Corp 800 Centennial Avenue, PO Box 1327, Piscataway, NJ 08855, USA
Sample Preparation for Electrophoresis:
Amersham Biosciences Europe GmbH Munzinger Strasse 9, D-79111 Freiburg, Germany Amersham Biosciences Sanken Building, 3-25-1, Shinjuku-ku, Tokyo 169-0073, Japan
Production: RAK Design AB
GST Gene Fusion System – Handbook
GST Gene Fusion System Handbook
www.amershambiosciences.com
18-1157-58 Edition AA
