Biotechnology Annual Review, Volume 9 (Biotechnology Annual Review)

v

Preface It is indeed an exciting time to try to follow the new development in the field of biotechnology and its wider applications in the different areas. The whole genomes of over 1000 viruses and over 100 microbes can now be found in Entrez Genome. The genomes represent both completely sequenced organisms and those for which sequencing is still in progress. The three main domains of life – bacteria, archaea, and eukaryota – are represented, as well as many viruses and organelles. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db ¼ Genome The exponential increase of the sequence data lead to the development of the new ‘‘Bioinformatics’’ field in order to attempt making sense, at least biological sense, out of all the new and fast data. But it will take also other techniques such as ‘‘functional genomics’’ to link the gap between a specific phenotype or a treatment and a gene sequence. Functional genomics tools are therefore important for the accurate molecular diagnosis/ prognosis, target discovery and validation needed for drug development and novel targets for antibiotics development. Functional genomics are also important for the confirmation of therapy in pharmacogenomics studies. Since proteins commonly work with other proteins as part of cellular network(s), it is often essential to investigate these interacting protein partners by for example techniques such as TAP (Tandem Affinity Purification) (Gavin et al., 2002, Nature; 415(6868): 141–7), and Integrated genomic and proteomic analyses of metabolic network. Ideker et al., 2001, Science 292(5518): 929–34. Applications of functional genomics techniques are equally important for progress in biotechnology applications of plants, animals and other organisms. There are several tools that can be used to monitor gene expression, i.e. functional genomics: a – Proteomics, b – Differential display, c – DNA arrays (DNA) chips, d – Protein–DNA interaction (up- and down-regulation) and protein–protein interaction network. Biotechnology is in many respects shaping our life and affecting our means of production and the creation of jobs. Progress in the applications of biotechnology depends on a wide base of basic as well as applied sciences. The output of biotechnology has already proved itself in many diverse fields from health to biomining and from agriculture to enzyme ‘‘breeding.’’ It is therefore difficult to follow all of the current as well as the potential applications of biotechnology. The objective of the Biotechnology Annual Review series is to attempt to provide readers with the needed indepth knowledge of the unfolding field, by reviewing specific topics in biotechnology in each issue. The philosophy behind this series is to encourage good reviews to make it easier for readers to keep in touch with progress and applications of biotechnology. We also encourage reviewing topics that are related to regulatory affairs, social impact of biotechnology, biodiversity, biosafety, public acceptance and patent issues.

vi For suggestions and contributions to any relevant subject please contact a member of the editorial board as listed.

M. Raafat El-Gewely, Ph.D. Professor of Biotechnology Gene Function Group Institute of Medical Biology University of Tromsø, 9037 Tromsø, Norway Tel: þ 47-776-44654. Fax: þ 47-776-45350 E-mail: [email protected]

vii

EDITORIAL BOARD FOR VOLUME 9 CHIEF EDITOR Dr. M. Raafat El-Gewely Department of Molecular Biotechnology Institute of Medical Biology University of Tromsø 9037 Tromsø, Norway Phone: þ 47-77 64 46 54 Fax: þ 47-77 64 53 50 E-mail: [email protected] EDITORS Dr. MaryAnn Foote Associate Director Medical Writing Department Amgen, Thousand Oaks, CA 91320-1879, USA Phone: þ 1-805-447-4925 Fax: þ 1-805-498-5593 E-mail: [email protected] Dr. Guido Krupp Director & Founder artus GmbH Koenigstr. 4a D-22767 Hamburg, Germany Phone: þ 49-40-41 364 783 Fax: þ 49-40-41 364 720 E-mail: [email protected] website: http://www.artus-biotech.com ASSOCIATE EDITORS Dr. Marin Berovic Department of Chemical and Biochemical Engineering University of Ljubljana Hajdrihova 19, Ljubljana Slovenia E-mail: [email protected] Dr. Thomas M.S. Chang Artificial Cells & Organs Research Centre McGill University

3655 Drummond St., Room 1005 Montreal, Quebec, Canada H3G 1Y6 Phone: þ 1-514-398-3512 Fax: þ 1-514-398-4983 E-mail: [email protected] Dr. Thomas T. Chen Director & Professor Biotechnology Center University of Connecticut 184 Auditorium Road U-149 Storrs Connecticut 06269-3149, USA Phone: þ 1-860-486-5011/5012 Fax: þ 1-860-486-5005 E-mail: [email protected] Dr. Franco Felici Kenton Labs c/o Sigma-Tau via Pontina Km 30.400 00040 Pomezia, Roma (Italy) Phone: þ 39 06 51501520 Fax: þ 39 06 51962706 E-mail: [email protected] Dr. Leodevico L. Ilag Xerion Pharmaceuticals AG Fraunhoferstrasse 9 82152 Martinsried Germany Phone: þ 49 89 86307 201 Fax: þ 49 89 86307 222 E-mail: [email protected] Dr. Kuniyo Inouye Laboratory of Enzyme Chemistry Division of Applied Life Sciences Graduate School of Agriculture Kyoto University Sakyo-ku, Kyoto 606-8502, Japan Phone: þ 81-75-753 6266 Fax: þ 81-75-753 6265 E-mail: [email protected]

viii Dr. Alfons Lawen Senior Lecturer Department of Biochemistry and Molecular Biology Monash University, Clayton Campus Room 312, Building 13D Clayton, Victoria 3800 Phone: þ 61-3-9905 3711 Fax: þ 61-3-9905 4655 E-mail: [email protected] Dr. Jocelyn H. Ng Hirsch-Gereuth-strasse 56 81369 Munich Germany Phone: þ 49 89 78018945 E-mail: [email protected] Dr. Eric Olson Aurora Biosciences Corporation 11010 Torreyana Road San Diego, CA 92121, USA Phone: þ 1-858-404-5381 Fax: þ 1-858-404-6787 E-mail: [email protected] Dr. Steffen B. Petersen Biostructure and Protein Engineering Laboratory

Department of Biotechnology University of Aalborg Sohngaardsholmsvej 57 DK-9000 Aalborg Denmark Phone: þ 45-9-635 8469 Fax: þ 45-9-814 2555 E-mail: steff[email protected] Dr. Jack Preiss Professor of Biochemistry The Starch Bio-Engineering Group 201 Biochemistry Building Michigan State University East Lansing, MI 48824, USA Phone: þ 1-517-353-3137 Fax: þ 1-517-353-9334 E-mail: [email protected] Dr. Rene´ H. Wijffels Wageningen Agricultural University Department of Food Science Food and Bioprocess Engineering Group P.O. Box 8129 6700 EV Wageningen The Netherlands Phone: þ 31-317-484372 Fax: þ 31-317-482237 E-mail: rene.wijff[email protected]

ix

List of contributors Ana M. Azevedo Centro de Engenharia Biolo´gica e Quimica Instituto Superior Te´cnico Av. Rovisco Pais 1049-001 Lisboa, Portugal

Leodevico L. Ilag Xerion Pharmaceuticals AG Martinsried, Munich Germany E-mail: [email protected]

Joaquim M.S. Cabral Centro de Engenharia Biolo´gica e Quimica Instituto Superior Te´cnico Av. Rovisco Pais 1049-001 Lisboa, Portugal

Alfons Lawen Monash University Department of Biochemistry and Molecular Biology School of Biomedical Sciences, P.O. Box 13D Melbourne, Victoria 3800 Australia Phone: þ 61-3-9905 3711 Fax: þ 61-3-9905 3726/4655 E-mail: [email protected]

Toniann Derion Roche Molecular Systems Inc. 4300 Hacienda Drive Pleasanton, CA 94588, USA Phone: þ 1-925-730-8044 Fax: þ 1-925-225-0195 E-mail: [email protected] Luis P. Fonseca Centro de Engenharia Biolo´gica e Quimica Instituto Superior Te´cnico Av. Rovisco Pais 1049-001 Lisboa, Portugal MaryAnn Foote Amgen Inc. One Amgen Center Drive M/S 17-2-A Thousand Oaks, CA 91320-1789, USA Phone: þ 1-805-447-4925 Fax: þ 1-805-498-5593 E-mail: [email protected] Marian Giffin Amgen Inc. One Amgen Center Drive Thousand Oaks, CA 91320, USA Phone: þ 1-805-447-2149 Fax: þ 1-805-498-5593 E-mail: mgiffi[email protected] Sandra J. Hecker Hecker and Associates Arlington, VA, USA

Vero´nica C. Martins Centro de Engenharia Biolo´gica e Quimica Instituto Superior Te´cnico Av. Rovisco Pais 1049-001 Lisboa, Portugal Rosemary Mazanet Oracle Partners LP 200 Greenwich Avenue Greenwich, CT 06830, USA Phone: þ 1-203-862-7925 Fax: þ 1-203-862-7927 E-mail: [email protected] Sally McLeish Amgen Inc. One Amgen Center Drive Thousand Oaks, CA 91320, USA David T. Molowa Biotechnology Equity Research J P Morgan Chase New York, NY, USA Theresa K. Neumann Technical Director, Clinical Research Amgen Inc. One Amgen Center Drive Thousand Oaks, CA 91320-1699, USA

x Maria Teresa Neves-Petersen Department of Physics and Nanotechnology University of Aalborg Biostructure and Protein Engineering Group Sohngaardsholmsvej 49 DK-9000 Aalborg Denmark Jocelyn H. Ng IMI Consulting GmbH Auf dem Amtshof 3 30938 Burgwedel Germany E-mail: [email protected] Soonmyung Paik Division of Pathology National Surgical Adjuvant Breast and Bowel Project Four Allegheny Center Pittsburgh, PA 15212, USA Phone: þ 412-359-5013 Fax: þ 412-359-6878 E-mail: [email protected] Steffen B. Petersen Department of Physics and Nanotechnology University of Aalborg Biostructure and Protein Engineering Group Sohngaardsholmsvej 49

DK-9000 Aalborg, Denmark Phone: þ 45 9635 8469 Fax: þ 45 9635 9129 E-mail: [email protected] Duarte M.F. Prazeres Centro de Engenharia Biolo´gica e Quimica Instituto Superior Te´cnico Av. Rovisco Pais 1049-001 Lisboa, Portugal Phone: þ 351218419139 Fax: þ 351218419062 E-mail: [email protected] Christopher Preston F. Hoffmann-La Roche Basel Switzerland Tony Velkov Department of Biochemistry and Molecular Biology Monash University School of Biomedical Sciences, P.O. Box 13D Melbourne, Victoria 3800 Australia Vojislav Vojinovic´ Centro de Engenharia Biolo´gica e Quimica Instituto Superior Te´cnico Av. Rovisco Pais 1049-001 Lisboa, Portugal

xi

Contents Preface Editorial Board List of contributors

v vii ix

Biochips beyond DNA: technologies and applications Jocelyn H. Ng and Leodevico L. Ilag Non-ribosomal peptide synthetases as technological platforms for the synthesis of highly modified peptide bioeffectors – Cyclosporin synthetase as a complex example Tony Velkov and Alfons Lawen Horseradish peroxidase: a valuable tool in biotechnology Ana M. Azevedo, Vero´nica C. Martins, Duarte M.F. Prazeres, Vojislav Vojinovic´, Joaquim M.S. Cabral and Luis P. Fonseca Considerations for the planning and conduct of reproducibility studies of in vitro diagnostic tests for infectious agents Toniann Derion Clinical trial methods to discover and validate predictive markers for treatment response in cancer Soonmyung Paik Production of high-quality marketing applications: strategies for biotechnology companies working with contract research organizations Sandra J. Hecker, Christopher Preston and MaryAnn Foote Use of benchmarking in the development of biopharmaceutical products Marian Giffin and Sally McLeish The state of biopharmaceutical manufacturing David T. Molowa and Rosemary Mazanet Review of current authorship guidelines and the controversy regarding publication of clinical trial data MaryAnn Foote Protein electrostatics: a review of the equations and methods used to model electrostatic equations in biomolecules – Applications in biotechnology Maria Teresa Neves-Petersen and Steffen B. Petersen The development of supportive-care agents for patients with cancer Theresa K. Neumann and MaryAnn Foote

397

Index of authors

419

Keyword index

421

1

151 199

249

259

269 279 285

303

315

1

Biochips beyond DNA: technologies and applications Jocelyn H. Ng* and Leodevico L. Ilag IMI Consulting GmbH, Burgwedel, Germany Xerion Pharmaceuticals AG, Martinsried, Munich, Germany Abstract. Technological advances in miniaturization have found a niche in biology and signal the beginning of a new revolution. Most of the attention and advances have been made with DNA chips yet a lot of progress is being made in the use of other biomolecules and cells. A variety of reviews have covered only different aspects and technologies but leading to the shared terminology of ‘‘biochips.’’ This review provides a basic introduction and an in-depth survey of the different technologies and applications involving the use of non-DNA molecules such as proteins and cells. The review focuses on microarrays and microfluidics, but also describes some cellular systems (studies involving patterning and sensor chips) and nanotechnology. The principles of each technology including parameters involved in biochip design and operation are outlined. A discussion of the different biological and biomedical applications illustrates the significance of biochips in biotechnology. Keywords: protein, chips, arrays, microfluidics, lab-on-a-chip, micro total analysis systems, immunoassays, diagnostics, electrophoresis, drug screening, drug discovery, proteomics, expression profiling, photolithography, soft lithography, patterning, tissue array, nanotechnology, nanolithography, cell assays.

Introduction The completion of the sequence of the human genome commenced a new era in biology. For a long time, biology has been the poor cousin of the physical and chemical sciences due to the lack of quantitative rigor for characterizing biological phenomenon. This is mainly due to the inherent complexity of biological systems and the lack of tools to track these systems. Advances in technology and information sciences put humanity on the verge of conquering biological complexity. An effective way to address biological complexity is to isolate the different components of the system and to understand how the different components interact. Historically, biology has been focused on studying one component at a time. The advent of Systems Biology has accelerated the shift to understanding multiple components at a time, which is more appropriate to characterize biological phenomenon. One area that will have immediate impact toward a better understanding of biology is through the use of chip technology to capture the different components in a miniaturized, well-defined, and quantifiable environment. The applications of chip technology in biology dawned during the genomics *Corresponding author: IMI Consulting GmbH, Auf dem Amtshof 3, 30938 Burgwedel, Germany. Tel: þ 49 5139-99180. Fax: þ 49 5139-991877. E-mail: [email protected] BIOTECHNOLOGY ANNUAL REVIEW VOLUME 9 ISSN 1387-2656 DOI: 10.1016/S1387-2656(03)09001-X

ß 2003 ELSEVIER SCIENCE BV ALL RIGHTS RESERVED

2 revolution. The power of DNA chips became imminent with its applications in profiling the expression of thousands of genes arrayed in a silicon chip. From then on the concept of chips have been extended to a variety of biomolecules such as proteins and including cells leading to the coining of the word ‘‘biochips.’’ Although many reviews have been written on biochips (see Table 1), the aim of this particular review is to provide the users or potential users of non-DNA biochips with an understanding of a wide enough breadth to realize the different options that exist and, if pursued further, evaluate the effectiveness, Table 1. Literature references (books, review articles, or journal publications that review or provide an overview of the particular subject area). Category

Subject area

References

Book

Microsystem technology mTAS

Manz and Becker, editors [563] van den Berg et al., editors [564]

General

Protein arrays

Wagner and Kim [59] Mitchell [79] Templin et al. [125] Stoll et al. [565] Wilson and Nock [9] Cahill [348] Ekins and Chu [5] Zhu and Snyder [496] Schweitzer and Kingsmore [389] Kodadek [3] Mirzabekov and Kolchinsky [150] Frank [513] Blagoev and Pandey [367] Kallioniemi et al. [464] Freemantle [12] Mitchell [38] Reyes et al. [294] Auroux et al. [307] McDonald et al. [10]

Peptide arrays Cell-based microarrays Tissue arrays Microfluidics

Microfluidics on PDMS Technologies

Microfabrication Patterning proteins

Patterning cells

Soft lithography

Microfluidics microfabrication

(Continued.)

Qin et al. [104] Blawas and Reichert [116] Bernard et al. [218] Kane et al. [51] Kane et al. [51] Bhadriraju and Chen [566] Folch and Toner [238] Whitesides et al. [213] Xia and Whitesides [216] Zhao et al. [217] Reyes et al. [294] Becker and Ga¨rtner [61] Chova`n and Guttman [41]

3 Table 1. Continued. Category

Applications

Subject area

References

Microfluidics on PDMS

McDonald et al. [10] Anderson et al. [251] Jo et al. [252]

Microfluidics (mTAS)

Auroux et al. [307] McDonald et al. [10] Figeys and Pinto [568] Dolnı´ k et al. [75] Effenhauser et al. [411] Regnier et al. [532] Kutter [73] Regnier et al. [532] Kutter [73] Jenkins and Pennington [344] Hughes [296] Mousses et al. [569] Santini et al. [518] Wan [529] Ekins and Chu [524] Wang [529] Talary et al. [567] Kane et al. [51] Singhvi et al. [187] Bhadriraju and Chen [566]

Proteomics Capillary electrophoresis on chips

Chromatography on chips Protein expression profiling Dielectric separations Clinical and functional target validation Drug delivery Immunoassay and other ligand assays On-chip enzymatic assays Medical diagnostics Patterned cells

appropriateness, and ultimately the value of biochips (from those that may or will be commercially available to those that will be designed to suit particular needs). This review also provides a perspective for those interested in the biotechnological aspect of biochips: many technical issues are not trivial but that research leads to knowledge, discovery, and/or even achievement. Furthermore, a list of abbreviations and a glossary are provided as appendices to cope with the complex terminologies that have floated in the field.

Microfabrication of chips The development of a biochip involves several considerations: what type of experiments needs to be performed on the chip, what support is needed, how to apply the protein on the chip, and how to immobilize the protein without losing its functionality. The type of experiment will determine the chip format. In constructing a chip, an important consideration is what material works best or is compatible for a given type or range of required experiments. The chip in its final state goes through a number of steps from applying the proper surfaces that are compatible with the biomolecules of interest, in this case, proteins or cells, to the

4 techniques used to attach these to the surfaces in a functional form, i.e., keeping the proteins active or cells viable. Biochip formats Biochips have different configurations depending on the application. They include microfluidic chips, nanovials, nanoplates, three-dimensional pads, patterned arrays, and flat-surface spot arrays [1]. These can be classified mainly into two types: arrays and microfluidics. Arrays have sample or ligand molecules (e.g., antibodies) at fixed locations on the chip while microfluidics involves the transport of material, samples, and/or reagents, on the chip. A third type of chip is a biosensor where a chip can be composed of several components including electronic circuitry, electrodes, sample vials, or channels. When sufficiently miniaturized, in principle, a sensor system can also be arrayed. Examples of the different chip formats are shown in Fig. 1. Arrays An array encompasses a number of variations including nanovials, nanoplates, patterned arrays, and flat-surface spot arrays. So long as proteins or their ligand molecules are delivered in a regular fashion or present in certain groupings on the chip, an array is created, regardless of whether the same sample or different samples of the same type are deposited onto the chip. One important factor is spot size. An array, specifically a microarray, will have spot sizes in micrometer dimensions. Whether or not a filter membrane containing spots of a few millimeters in diameter, for example, should be considered an array is debatable. An array might contain variants of a bioactive peptide or protein (domains, mutants, or splice variants), components of a protein pathway, or the entire proteome of an organism [2]. The protein of interest can be in solution as in nanovials or held in place by some gel, matrix, or capture agent. Arrayed molecules referred to in this review include protein antigens, ligand molecules or binders (e.g., antibodies), cells, and tissues. In the case of proteins, the strategy is to have an array with only antigen molecules (antigen arrays), or its reverse, with only antibodies (antibody arrays). Other terms that have been used include protein function arrays (containing antigens) and protein detecting arrays (containing ligand molecules) [3]. Arrays have been a popular chip format in the biotechnology sector due to their adaptability to automation and high-throughput systems and because they are adapted from or a logical extension of the equivalent format containing DNA molecules. Although this may be a general notion, Roger Ekins (Department of Molecular Endocrinology, University College London Medical School) conceived, used, and patented ligand-binding assays in a microarray format in the 1980s [4,5]. Ekin’s patent covers ‘‘all microarray-based ligand assays’’ (including immunoassays) and DNA/RNA analysis [6]. The key concept regarding microarrays according to Ekins is that when a spot is small enough,

5

Fig. 1. (A) Microarray format. A glass-coated slide with 10,800 protein microspots. Array density is 1600 per cm2 and spot size is 150–200 mm in diameter printed via split-pin arraying. Protein G was printed 10,799 times and FRB was printed once in row 27, column 109 as indicated (visible in fluorescence analysis, see [118]). Reprinted with permission from [118]. Copyright 2000 American Association for the Advancement of Science. (B) Microfluidics format. A microfluidic device for electrophoretic separations. Copyright Agilent Technologies. (C) Sensor chip experimental setup. (a) Photograph of an experimental setup. The culture/sensor unit (b) is placed on the microscope stage. (b) Photograph of the two-channel version of the culture/sensor unit. The sensor chips from the bottoms of the two culture chambers. These chambers are connected to a sensor module on the right side, equipped with microelectrodes for pH and oxygen-concentration [48]. Copyright 1998 Elsevier Science Ltd. (D) Illustration of the sensor chip in (C). This design of the silicon sensor chip includes IDES-structures, ISFETs, oxygen sensors, temperature sensors, and a transparent aperture. Because each type of sensor element is at least present in pairs, a chip allows redundant data acquisition of each parameter [48]. Copyright 1998 Elsevier Science Ltd.

6

Fig. 1. Continued.

7 the signal obtained is independent of the sample volume and the amount of binder (e.g., antibody) within this spot, and dependent only on the concentration of target (antigen) molecules in solution that the binders in this spot are exposed to. When operating within this region, termed ambient analyte assay limits, greater sensitivity and faster results can be obtained [4,7,125]. (See Fig. 2; see also Biochip applications, Clinical Applications, Diagnostics, Immunoassays.) (However, microarrays can also be analyzed on the basis of the total mass of the analyte bound to capture antibodies rather than on concentration [8].) Nevertheless, such insights led to the idea of the microarray and that with a drop of sample, thousands of different substances can be analyzed in parallel [4]. The advantages of arrays lie in the minimal sample consumption and highthroughput capability inherent in its configuration. Compared to a 96-well microtiter plate, a single array spot may contain a million times less sample, which means less than a picogram or femtomole of protein [9]. And yet, the amount of information that can be generated is tremendous. Even chips that are biochemical sensors involving SPR-BIA (surface plasmon resonance-biomolecular interaction analysis) are amenable to an array format for high-throughput

Fig. 2. Signal and signal density in microspots. Signal density (signal/area, relative intensities, log scale) and signal (total intensity, log scale) or captured targets in microspots are shown for different concentrations of capture molecules. The capture molecules are immobilized with the same surface density on all spots. The signal (total signal) increases with increasing amount of capture molecules at growing spot size. When most of the targets are captured from the solution the signal reaches its maximum. By contrast, signal density (signal/area) increases with decreasing amount of capture molecules (decreasing spot size), reaching a constant level when the capture molecule concentration is <0.1/K (K is the association constant). Under these ambient analyte conditions, target concentration in solution is minimally altered by the amount of captured targets on the microspot. The figure was adapted from Ref. [328]. Reprinted with permission from Ref. [125]. Copyright 2002 Elsevier Science Ltd.

8 applications (for examples, see Table 2, Drug discovery and development, Proteomics and drug discovery tools) (see Biacore, HTS Biosystems, and Prolinx, Table 3; Nanoplex, Table 4). One of the relevant and widespread applications of arrays containing proteins or cells is in immunodiagnostics. The other is expression profiling along with the elucidation of protein–protein interactions, which the proteomics field has fostered. Other applications are enumerated in Table 2 (see also Biochip Applications). Nevertheless, the technology is not without its challenges. Difficulties arise in surface effects that lead to protein adsorption and denaturation and in detector sensitivity. A lot of research and development has gone into patterning and immobilization, exploring and using various surface chemistries to arrive at appropriate surfaces to anchor either sample or ligand molecules (see Technologies, Array technologies). The importance of detector sensitivity is obvious from the minute amounts of proteins that may be arrayed. In addition, some research has focused on the issue of tailor-made molecular tags that address specificity in detecting particular proteins in sandwich assays and in multiplexed analyses. Microfluidics Microfluidics involves manipulating fluids (gases and liquids) in a closed channel system with cross-sectional dimensions in the range of 5–200 mm [10–13] and total chip area is 2–3 cm2 [12,13]. A miniaturized gas chromatographic (GC) system on a silicon wafer, developed in the 1970s at Stanford University by S.C. Terry et al. is considered to be the first microfluidic device [14]. The technology, however, did not develop much further until molecular biology, particularly the area of genomics, made a major impact in academic, pharmaceutical, and biotechnological research. Because it was possible to carry out experiments such as separations and assays on a chip, a microfluidic chip was termed lab-on-a-chip. The type of experiments can range from biochemical or chemical synthetic reactions to analytical separations including electrophoresis. Extending this capability and incorporating different experimental steps so that a full analysis can be performed on a chip has led to the term m-TAS (micrototal analysis system), a concept discussed in a 1990 paper by Andreas Manz (Center for Analytical Sciences, Department of Chemistry, Imperial College of Science, Technology and Medicine, London, UK) [15]. Such a chip may incorporate functions such as fluid transport, mixing, incubation, and even particle sorting [16]. Microfluidics for aqueous systems, which are relevant for biochemical and biological applications, came out of the research labs of Andreas Manz [7,17–20], D. Jed Harrison (Department of Chemistry, University of Alberta, Edmonton, Canada) [21–26], J. Michael Ramsey (Oak Ridge National Laboratory, Oak Ridge, Tenn.) [27–32], and Richard A. Mathies (Department of Chemistry, University of California, Berkeley, CA, USA) [33–36] with technologies adapted from the microelectronics industry (photolithography and wet etching) [10,37].

Table 2. Summary of applications (a list of biochip applications including those cited in this review). Application area

Chip format

Proteomics and Drug discovery tools Electrophoresis, CE microfluidics Electrophoresis, CE microfluidics Electrophoresis, CE microfluidics Electrophoresis, CE microfluidics Electrophoresis, CE microfluidics Electrophoresis, CE Electrophoresis, CE

microfluidics microfluidics

Chromatography, EC Chromatography, EC Chromatography, EC Chromatography, EC Chromatography, EC

microfluidics microfluidics microfluidics microfluidics microfluidics

Description

Reference

Electrophoresis, early demonstration Electrophoresis, high-speed separations Electrophoresis, commercial chip Electrophoresis þ protein digestion and peptide separation Electrophoresis þ digestion, separation, and post-column labeling Analyze kinase A assay Immunoaffinity capillary electrophoresis

Manz et al. [15] Jacobson et al. [30,402] Owen [11], Kuschel [473] Gao et al. [474] Gottschlich et al. [570]

EC and ion-exchange chromatography; chips and capillaries have comparable resolution. Open-channel EC

Cohen et al. [475] Dodge et al. [80], Ensing and Paulus [471], Phillips and Chmielinska [472] Ericson et al. [423] Jacobson et al. [403]

Reverse-phase EC on peptides and amino acids; Throckmorton et al. [476] outperformed capillary separations in speed and efficiency Micellar electrokinetic chromatography; von Heeren et al. [418] cyclic channel system; theophylline analysis 2D EC/CE, 1st dimension: open channel EC, Gottschlich et al. [571] 2nd dimension: CE

Particle sorting Particle sorting Particle sorting Flow cytometry

microfluidics microfluidics microfluidics microfluidics

Trapping and separation of latex beads, TMV, below 1
m Green et al. [298] Dielectrophoresis of particles and cells Fiedler et al. [299] Separation, multicomponent fractionation Hughes [296] Flow cytometry of E. coli McClain et al. [477]

Mass spectrometry

sensor

Proteomics, chip-MS SPR-BIA applications

9

(Continued.)

Nelson et al. [44]

10

Table 2. Continued. Application area

Chip format

Description

Reference

Mass spectrometry Mass spectrometry

sensor array

MALDI/MS-BIA analysis SELDI-TOF MS

Mass Mass Mass Mass

array array microfluidics microarray

SELDI protein chips SELDI profiling, cancer research High-throughput CE/ESI-MS Sample chip for ESI-MS

Mass spectrometry

array

Multichannel device for ESI-MS

So¨nksen et al. [45] Merchant and Weinberger [54] Weinberger et al. [55] von Eggeling et al. [56] Zhang et al. [480] Prosser et al. [481], Schultz et al. [482] Liu et al. [483]

Expression profiling Expression profiling

array (antibody) array (cDNA expression)

Proteomic profiling in cancer Protein expression and antibody screening

Expression profiling

array (antigen)

Expression profiling Expression profiling Expression profiling

array (antigen) array (antigen) array(antibody)

Expression profiling

microarray (tissue)

multiplex protein profiling; immunoRCA on 75 cytokines, proteomic survey cytokine profiling chip, commercial SELDI profiling of tumor tissue lysates 60 antibodies against CD antigens on arrays; studied patterns from blood leukocytes of leukemic patients Biomarker profiling of tumor samples

Expression profiling Expression profiling

microarray (tissue) microarray (tissue)

Expression profiling

array (cell lysates)

Expression profiling ELISA

array (antibody)

spectrometry spectrometry spectrometry spectrometry

Analyzed prognostic markers in 553 breast carcinomas Verified sample quantity representative of whole tissue and use of archival tissue Molecular profiling of tumor samples by layered expression scanning Simultaneous detection of IgG as antigens (direct) and cytokines (sandwich) on PVDF-immobilized antibodies; ECL detection

Knezevic et al. [484] Lueking et al. [485], Bu¨ssow et al. [486] Schweitzer et al. [388] Wagner and Kim [59] von Eggeling et al. [56] Belov et al. [534] Kononen et al. [465], Nocito et al. [467] Torhorst et al. [466] Camp et al. [539] Englert et al. [468] Huang [490]

Expression profiling ELISA Expression profiling ELISA

microarray (antigen)

Molecular screening Molecular screening Molecular screening Molecular screening

arrays (antigen, antibody) array (cDNA expression) array (cDNA expression) array (antibody)

Molecular screening

array (target protein)

Molecular screening

microarray (yeast proteome)

Molecular screening

microarray

Molecular interactions Molecular interactions Molecular interactions

array

microarray

microarray microarray (yeast proteome)

Drug screening and testing Drug screening microarray Drug screening

sensor

Drug delivery

microfluidics

ELISA for autoimmune diagnostics, monitoring 18 autoantigens in human sera High-throughput ELISA with IgGs as antigens Arrays on membranes, binding interaction detected with HRP-conjugated antibodies; visualization by ECL Antibody specificity screening Screening antibody–antigen interactions High-throughput screening of antigen–antibody interactions; expression monitoring Identification of protein targets of small molecules High-throughput screening of biochemical activity (phosphoinositide-binding) Immunoassay and enzymatic reactions Screening protein interactions with p52, dsDNA, ssDNA, and simian virus 40 early pre-mRNA Tested 3 interactions: protein G with IgG, p50 with IkB, and FRB with FKBP12 Protein–protein interactions in yeast

Joos et al. [121] Mendoza et al. [74] Huang [572] Lueking et al. [485] Holt et al. [491] De Wildt et al. [340] MacBeath and Schreiber [118] Zhu et al. [128] Arenkov et al. [137] Ge [514] MacBeath and Schreiber [118] Zhu et al. [128]

Determining compound selectivity for subtypes of adrenergic receptors Multiparametric screening of the effects of cytochalasine B and D on the cytoskeleton of LS 174T cells.

Fang et al. [153]

Controlled release; silicon chip with electrochemical dissolution of chemicals

Santini et al. [517]

Ehret et al. [47]

(Continued.)

11

Application area

12

Table 2. Continued. Chip format

Description

Reference

array sensor (antibody)

Sapsford et al. [573]

microfluidics

Measurement of binding kinetics and the effect of spot size on the association rate IgG analysis with a post-separation CE chip

microfluidics

hCG in serum

Mangru and Harrison [405] Schneider et al. [71]

microfluidics

hCG in undiluted whole blood

Schneider et al. [72]

microfluidics

Analysis of serum cortisol with no extraction or sample preparation

Koutny et al. [454]

Immunoassay

microarray

Bernard et al. [249]

Immunoassay Immunoassay

array (antigen) sensor

Immunoassay

microarray

Immunoassay

microfluidics

Immunoassay

microfluidics

Immunoassay

microfluidics

Micromosaic immunoassay: patterning by microfluidics; rows of antigens perpendicular to sample solutions; antibody screening for immunodiagnostics AFM detection Multianalyte analysis with an integrated optical waveguide sensor Immobilized IgG in polyacrylamide gel pad; FITC-labeled antigen Automated heterogeneous assay with PA/rIgG affinity model in less than 5 min Micellar electrokinetic chromatography; cyclic channel system; theophylline analysis Simultaneous assays for ovalbumin and anti-estradiol

Competitive immunoassay Competitive immunoassay

sensor

Clinical applications Direct-binding immunoassay Direct-binding immunoassay Direct-binding immunoassay Direct-binding immunoassay Direct-binding immunoassay

microfluidics

Serum theophylline determination for point-of-care diagnostics Distinguish between IgG subclasses; immunoaffinity CE

Jones et al. [69] Plowman et al. [574] Arenkov et al. [137] Dodge et al. [80] von Heeren et al. [418] Cheng et al. [541] Schult et al. [575] Dodge et al. [80]

Competitive immunoassay

microfluidics

Electrophoresis; analysis of monoclonal antibodies and theophylline

Chiem and Harrison [25,525]

Sandwich immunoassay Sandwich immunoassay Sandwich immunoassay Sandwich immunoassay Sandwich immunoassay Sandwich immunoassay

array (antibody)

High-throughput screening of multiple cytokine expression in sera; ELISA with ECL detection High-throughput protein quantification; multiplexed ELISA; human cytokines Multiplexed ELISA; option for analyte quantification in complex biological samples Mass-sensing, multianalyte analysis of 4 human IgG subclasses Detecting hCG with FTTC-labeled secondary antibody Simultaneous multianalyte fluoroimmunoassays on a planar waveguide; antibody lanes patterned perpendicular to sample lanes Simultaneous multianalyte identification (bacterial, viral, and protein analytes) High-throughput ELISA with IgGs as antigens Multiplexed profiling of induced secretion of inflammatory cytokines with RCA detection

Huang et al. [526]

array (antibody) microarray (antibody) microarray (antibody) microarray array sensor

Sandwich immunoassay array sensor (antibody) Sandwich assay Fluorescent-sandwich immunoassay

microarray microarray

Enzymatic assay Enzymatic assay

microfluidics microarray

Enzymatic assay

microfluidics

Enzymatic assay

microarray (target proteins)

Diagnostics

array (antibody)

Diagnostics

microarray (antigen)

Reverse immunoassay

microarray

Wiese et al. [528] Silzel et al. [8] Arenkov et al. [137] Rowe et al. [119]

Rowe et al. [576] Mendoza et al. [74] Schweitzer et al. [388]

-galactosidase (120 pg), using RBG (7.5 ng) Assays on HRP, AP, and -D-glucuronidase; accelerated by microelectrophoresis Fluorogenic enzyme assay on -galactosidase after on-chip cell lysis and protein extraction Phosphorylation assay

Hadd et al. [32] Arenkov et al. [137]

Analyze blood leukocytes for diagnosing leukemia based on expessed CD antigens Screening 18 diagnostic markers for systemic rheumatic disease; ELISA ImmunoRCA on allergen-specific IgEs

Belov et al. [534]

Schilling et al. [542] MacBeath and Schreiber [118]

Joos et al. [121] Wiltshire et al. [535]

13

(Continued.)

Moody et al. [527]

14

Table 2. Continued. Application area

Chip format

Description

Reference

Reverse immunoassay

microarray (antigen)

ImmunoRCA for femtomolar antigen detection

Schweitzer et al. [387]

Biomarkers

microarray

Detection/quantitation of specific proteins and antibodies in complex solutions

Haab et al. [375]

Biomarkers

See also Proteomics and Drug Discovery tools, Expression Profiling on tissue microarrays microarray (antigen) Analysis of proteomic changes in cell lysates relevant to prostate cancer progression

Disease monitoring

Biological and medical research Biochemical assay microfluidics Enzymatic assays

See Enzymatic assays under Clinical applications

Sensor

microfluidics

Sensor

See also Drug screening and testing

Cellular manipulation Cell sorting

microfluidics microfluidics

Cell sorting Cell sorting

microfluidics microfluidics

Cell shape/behavior

patterning

Cell shape/behavior

patterning

Paweletz et al. [120]

-galactosidase (120 pg), using RBG (7.5 ng)

Hadd et al. [32]

Differences in electrical signals between 2 cell populations: normal and those exposed to an irritant or toxin

DeBusschere et al. [543]

Transported cells through a microchannel network With cell trapping algorithm, PDMS chip with pneumatic control Dielectrophoretic sorting of particles and cells Fluorescence-activated cell sorter

Li and Harrison [26] Fu et al. [16]

Regulating apoptotic behavior (vs. growth) by restricting extent of spreading Cell shape depends on surface characteristics

Fiedler et al. [299] Fu et al. [275] Chen et al. [52] Kleinfeld et al. [114]

Cell shape/behavior Cell shape/behavior

patterning patterning

Neurite outgrowth dependence on bioactive laminin Limiting cell extension controls growth and protein secretion Cell spreading changes hepatocyte behavior from differentiation to proliferation Development strategy for chip substrates to study chemical biology in cells Cell fusion

Hammarback et al. [546] Singhvi et al. [187]

Cell shape/behavior

patterning

(Cell behavior)

Patterning; microelectrode array

Cell behavior

microfluidics

Cell migration

Microfluidics (gradient formed)

Study of neutrophil chemotaxis in an interleukin-8 gradient

Joen et al. [240]

Toxicity

Microelectrode array

Brain slice cultures grown on chip to monitor electrophysiological signals

Kristensen et al. [70]

Mooney et al. [548] Yeo et al. [547] Chiu [540]

15

16

Table 3. Companies with biochip products and technologies. Chip company (References)

Chip products and services (Instrumentation/Software)

Technology

Agilent Technologies Palo Alto, CA www.chem.agilent.com [38], [11], [152]

LabChipÕ (see Caliper) (2100 BioAnalyzer with Cell Assay Extension cartridge available)

Electrophoretic DNA, RNA, protein analysis; cell assays (fluorescently labeled annexin-V detection for apoptosis; transfection efficiency monitoring by GFP detection or antibody staining); microfluidics on glass quartz

Aurora Biosciences Corp. San Diego, CA http://www.aurorabio.com (now PanVera LLC, a subsidiary of Vertex Pharmaceuticals) Biacore International AB Uppsala, Sweden http://www.biacore.com [79]; [146]

NanoWellTM assay plates (3456 wells)

UHTSS (ultra high-throughput screening system) with FRET based detection; cell-based assays; automated screening of 100,000 compounds and 2400 re-tests per day; automated plate replication for 96- and 384-well screening plates [57,58].

Sensor chips, 10 different SPR-based protein chips (gold-coated glass) Biacore 3000: automated SPR-based biosensor system

Technology involves (1) SPR which detects local concentration changes due to binding interactions at the surface, (2) the Sensor chips which provides for surface attachment of molecules of interest, and (3) a microfluidic system which delivers sample to the surface of the chip. Single spot format. LEAPS (Light-controlled Electrokinetic Assembly of Particles near Surfaces): optically programmable assembly of thousands of ‘‘bead’’ microparticles, computercontrolled illumination for biochemical analysis; sorting and light-directed transport of cells on chip Custom Bead Array Technology: high-volume production of customized bead arrays; monitoring molecular binding events; bead selection from encoded bead libraries, array assembly, and immobilization; decoding image with bead position and color tag provided; 4000 binding events recorded in one CCD camera snapshot.

BioArray Solutions Warren, NJ http://www.bioarrays.com [522], [580]

(offers complete assay solutions and on-demand high volume production of custom chips with proprietary assay designs and software analysis, chips in disposable fluidic cartridges which hold patient samples)

SMARTCHIP-Integrated Assay Solutions: bead array assay format with programmable fluidic sample/reagent delivery, temperature control, and image analysis Applications in protein profiling, quantitative assays, array cytometry, clinical diagnostics Biolog, Inc. Hayward, CA http://www.biolog.com

Phenotype MicroArraysTM(PM) Cell-based array that tests up to 2000 cellular properties simultaneously OmniLogTM instrument (reader/incubator that monitors cellular changes in 50 arrays simultaneously)

Integrated system of cellular assays, instrumentation, and software for HTS of cells; identify new drug targets by cellto-cell comparison, also for lead characterization and optimization. Cell-based assays available in PM format now number 760. Simultaneous measurement of effects on one gene in a cell under different growth conditions [456,580].

Biosite Diagnostics, Inc. San Diego, CA http://www.biosite.com [456], [522]

TriageÕ protein biochips 1. Cardiac system: point-of-care blood test to diagnose AMI (screens CK-MB, myoglobin, and troponin 1 markers) 2. BNP test (point-of-care blood test to diagnose CHF by measuring BNP marker) 3. DOA panel differential diagnosis of drugs of abuse, DOA, covers eight drug classes) 4. Micro Parasite Panel (for diagnosis of 3 pathogenic parasites) 5. C. difficile Panel (to identify Clostridium difficile, an infectious organism causing severe diarrhea especially in hospital settings)

TriageÕ protein biochip immunoassays: microfluidics in plastic; 100 assays in 15 min; picomolar sensitivities from several drops of whole blood or plasma; uses antibodies with a near-infrared fluorescent label which is read by a portable battery-powered fluorometer [522]. TriageÕ Cardiac system: microfluidics in immunodiagnostics, only a few ml of blood needed [456]; 10 antibodies arrayed in 6 zones [522]

(Continued.)

17

18

Table 3. Continued. Chip Company (References)

Chip products and services (Instrumentation/Software)

Technology

Caliper Technologies Mountain View, CA http://www.calipertech.com [38]

LabChipÕ system (for electrophoretic separation; for vacuum-driven cell assays; products and services from benchtop to HTS systems; see also Agilent) CaliperÕ 250 HTS system (uses LabChip, performs fluorogenic and mobility shift assays currently, other cell-based assays are under development) Library CardTM device (reagent array for HTS of chemical compound libraries)

Microfluidics lab-on-a-chip technology: quartz glass chips with photolithographically etched channels sealed by a second glass layer; fluid motion induced by electrokinetic pressure; demonstrated screening of 750,000 compounds in a drug library against a protein target, consuming 750 ng of protein (equivalent to 1 million compounds per
g of target) [38] Partnered with Agilent on LabChips

Ciphergen Biosystems, Inc. Fremont, CA http://www.ciphergen.com [146], [79]

ProteinChipÕ Biomarker System (tools for biomarker discovery from identifying potential markers to resolving multivariate patterns in 16-spot ProteinChip Latex Arrays, including the ProteinChip Reader and ProteinChip and Biomarker PatternsTM Software) ProteinChipÕ Products (8 spots/chip; 6 different surfaces including affinity capture applications; ProteinChip Reader) Biomarker Discovery CenterÕ Services (R and D facilities to help Ciphergen customers solve complex, biological research problems

SELDI ProteinChip technology for rapid separation, detection, analysis of proteins; protein capture chips for MS analysis Aluminum chip with 8 spots; 12 side by side is the size of a 96-well microtiter plate

Combimatrix Corp. Mukilteo, WA www.combimatrix.com [260]

(microelectrode array [260])

CMBX biological array processor system: semiconductor array chip coated with a 3D porous reaction layer in which DNA, RNA, peptides or small molecules can be synthesized or immobilized; virtual flask technology using chemistry to build virtual walls around each electrode beneath each microspot to avoid chemical contamination, each virtual flask has a diameter about that of human hair [260]; proprietary software to control each electrode at each microspot

Fluidigm Corp. South San Francisco, CA http://www.fluidigm.com (formerly Mycometrix) [11]; [38]; [67]

TopazTM Protein crystallization Starter Kit (chips with integral valves that meter and mix precise amounts of sample and reagents, to crystallize proteins from nanogram amounts)

MSL microfluidicsTM: using proprietary MSL (Multi-layer Soft Lithography) process to fabricate PDMS microfluidic chips with microscale pumps and valves, including a cell sorter and a rotary pump that accelerates reaction kinetics on a chip; enables precise fluid manipulation; chips have up to 600 valves and 12 control lines

Gyros AB Uppsala, Sweden http://www.gyros.com [11]

Gyrolab MALDI SP 1 (CD microlabs) (Gyrolab Workstation, Cutter and Adaptor for Bruker MALDI)

CDs that use centrifugal forces to move samples through microstructures (nanoliter scale versions to perform specific tasks, e.g., volume definition, chromatography columns and chambers for enzymatic reactions and cell growth). Microstructures have the appropriate surface chemistries. Monitoring results by LIF Concentrates, desalts, mixes with matrix and crystallizes protein samples for MALDI-MS; processes 96 samples in parallel per CD [11]

HTS Biosystems, Inc. Hopkinton, MA www.htsbiosystems.com [79]

FLEX CHIPTM: Kinetic Analysis System: BMT chip (for kinetic analysis of up to 400 binding events, 400 test sites/cm2) FLEX CHIPTM Reader (for data collection) CHEMI FLEXTM System (microarray biochip; chemiluminescence-based detection for quantitative multiplexed assays PHASE FLEXTM System (disposable HTS plates for cell-based assays)

ProteomatrixTM technology portfolio: 1. FLEX CHIPTM Kinetic Analysis System based on gratingcoupled SPR(GCSPR), parallel detection of binding events in microarray format without relying on a reporter molecule. Protein interactions; gold-coated plastic optical grating 2. CHEMI FLEXTM technology: chemiluminescence detection of quantitative multiplexed assays in microarray format with integrated fluidics and reagent reservoirs for expression proteomics, target screening 3. PHASE FLEXTM phase fluorescence system: Lifetime fluorescence system using standard dyes (GFP, FITC); fluorescence lifetime, characteristic of fluorescence emission and the excited state lifetime (or half-life), has advantages over intensity and ratiometric measurements (independent of the measurement platform, immune to photobleaching and fluorophore concentration, allows multilabel detection)

19

(Continued.)

20

Table 3. Continued. Chip Company (References)

Chip products and services (Instrumentation/Software)

Technology

Large Scale Biology Corp. Vacaville, CA http://www.lsbc.com [38]

(Plastic-based antibody arrays; protein chips developed for in-house clinical diagnostics [38])

ProGExTM proteomics technology for the separation, analysis, identification, and quantitation of proteins in biological samples Patented methods for recombinant animal viral nucleic acids to extend proteomics development capabilities in protein chips; study human proteins in animal cells Bioinformatics: eFLOW, a custom computing cluster used for high-throughput processing and analysis of genomic and proteomic sequence data and for computing applications involving protein chips and discovery of protein markers

LumiCyte, Inc. Fremont, CA http://www.lumicyte.com [79]

Uses Protein BioChips as a service for clinical diagnostics; selling SELDI-based services and information to the life science industry BioPhore KnowledgebaseTM

Seldiography (patented, proprietary SELDI process): detection and quantitative mapping of protein biomarkers from biological fluids; protein capture chips for MS analysis

Micronics,Inc.Redmond,WA http://www.micronics.net Ref: Chip description in Freemantle pp. 32–33 []

Provides lab card development and microplumbing solutions: 1. Liquid/Liquid Extraction System (LLES)(using H-FilterÕ technology) 2. Chemical Analysis System (using T-sensorÕ technology) 3.
FluidicTM Evaluation Kit (for testing different channel configurations, sold by Pierce Chemical Co.) 4. AccessTM Lab Card (for quantitative analtye detection, e.g. in immunoassays, in H-FilterÕ or TsensorÕ format)

Diffusion-based detection, filtration methods, and microcytometry Patented technology platforms: 1. H-FilterÕ technology: diffusion-based separation in aqueous laminar flow streams, preparation of complex samples, e.g. whole blood, without centrifugation or solvent extraction 2. T-sensorÕ technology: multiple input flows are simultaneously analyzed, differentiating large from small particles by diffusion 3. MicrocytometryTM: intergrated chemical diffuser and cytometer channel, allows uniform controlled lysing of blood cells prior to cytometry 4. ORCA MicrofluidicsTM: integrates sample preparation with point-of-care analysis, integrates H-FilterÕ and T-sensorÕ platforms in a disposable card

Nanostream, Inc. Pasadena, CA http://www.nanostream.com [38]

Customized microfluidic solutions

Snap-n-FlowTM: rapid prototyping modular approach to fabricating microfluidic components (e.g., 3 configurations for splitters, valves, filters, and mixers) Customized microfluidic solutions (e.g., a device for highthroughput separations; a multiplexed assay chip that performs serial dilutions and dose–response curves are generated; multiplexed cellular assays; optimization of cellular growth conditions)

Packard Bioscience Co. Meriden, CT http://www.packardbioscience.com [146], [152]

HydrogelTM coated slides (array substrates) SpotArrayTM 72 (for high-throughput contact printing, 1536 spots onto 72 slides in 1 h) SpotArray Enterprise (PiezoTipTM piezoelectric dispensing, 325-pL drops, 108 array slides per run, 20,000 spots per slide) ScanArra (Way scanners)

3-D Hydrogel technology to maintain protein activity in a porous polyacrylamide gel pad; proteins are immobilized via a coupling agent that covalently links protein amine groups to the gel. Piezo Tipnology (non-contact printing technology based on piezoelectric dispensing)

Prolinx, Inc. Bothell, WA http://www.prolinx.com [79], [456]

Versalinx Chemical Affinity Tools (for assay development) Versalinx Protein Microarray Technology (array substrate with universal high-capacity binding surface that retains protein activity and minimizes background)

VersalinxTM chemical affinity technology: synthetic chemical affinity technology involving the P(D)BA-SHA (phenyl(di)boronic acid-salicylhydoxamic acid) pair, complex formation enables solution-phase bioconjugation for immobilization, purification, and analysis with low NSB. Versalinx Protein Microarray Technology: developed for protein immobilization on array surfaces, based on the reversible complexation of P(D)BA-SHA; P(D)BA-conjugated proteins (and peptides) attach to SHA moieties displayed on chip surface; results in uniform spot morphology, high signal intensities and does not require blocking to prevent NSB. AcapellaTM Molecular Interaction Analysis System: instrumentation for label-free molecular interaction analysis based on SPR

(Continued.)

21

22

Table 3. Continued Chip Company (References)

Chip products and services (Instrumentation/Software)

Technology

Sense Proteomic Ltd. Babraham, Cambridge, UK www.senseprotomic.com

ExpressarrayTM p53 (for cancer research, 200 spots in 2.5 cm
2.5 cm chip) Sense Kv subunit protein array (for cardiovascular research, 96-well plate) Sense p450 protein array Sense human liver proteome array (both for drug metabolism and toxicology, available for compound screening services or for technology establishment in-house)

Functional protein arrays based on specific disease tissues COVETTM: high-throughput expression of proteins with a proprietary tag that serves both for affinity capture purification and a folding marker Protein Function Arrays: purification and printing of COVET proteins in arrays, preserving biological activity for protein activity profiling ScinticellTM: cell-based scintillation proximity assay, scintillant molecules inserted into the cell membrane and experiments are carried out with radio-ligands; to study receptor–ligand interactions in cells

SomaLogic, Inc. Boulder, CO http://www.somalogic.com [79]

Services: Research Proteomics: target identification, surrogate marker development Clinical Research: patient stratification and response monitoring in clinical trials; product development Commercial Diagnostics: screening, diagnosis, and patient management

Photoaptamers: as capture agents; 1015 library size PhotoSELEX: in vitro, combinatorial chemistry technique used to identify photoaptamers to specific target proteins, photochemically links them; evolutionary process that results in highly specific photoaptamers; automated process with current capacity of 200 new photoaptamers/week for development of high-density aptamer arrays

Umedik, Inc. Toronto, Ontario, Canada http://www.umedik.com [383]

DIA/PROTM BioChip (separates contaminating particles in a drop of fluid; contains assay(s) and pertinent biochemical analysis components; disposable plastic) DIA/PRO Electronic Reader (identifies and analyzes specific tests with bar coding)

ICEfloTM technology: on-chip particle separation, eliminating centrifugation. Works well with blood, saliva, urine, and feces samples. Applications in biomarker detection, diagnosis, food safety and testing, environmental testing

Zeptosens AG Witterswil, Switzerland http://www.zeptosens.com [565]

ZeptoMARK (protein microarray for proteomics) ZeptoREADER ZeptoVIEW software SensiChip (for low abundance gene expression analysis; planar waveguide substrate with 6 microarrays, each enclosed in a low-volume microfluidic structure) SensiChip Reader

PLL-PEG surface chemistry for immobilizing proteins on microarrays, minimizing NSB; cDNAs, 70mer oligonucleotides, or antibodies as recognition elements or capture probes; fluorescent planar waveguide detection by evanescent field excitation to minimize background and achieve detection limits in the zeptomole range; bioassays are multiplexed nucleic acid hybridization, immunoaffinity, and membrane receptor based ZeptoTM READER: capture molecules in a microarray are immobilized on a thin film (100–200 nm Ta2O5, a highrefractive index material) planar waveguide deposited on a transparent support [565]

Zyomyx, Inc. Hayward, CA http://www.zyomyx.com [59]

Protein Profiling BiochipTM System (for simultaneous multianalyte analysis from biological mixtures, e.g. serum, cell lysates; system includes fluorescent reader and assay components)

Possesses technologies in microsystem design, fluid mechanics, and non-contact printing (10 nL reservoirs and zero dead volume [59]); surface chemistry for protein immobilization; detection physics for optical parallel detection (also electronic biosensors and MS); and biochemistry for protein expression, purification, and characterization

This table is of companies with non-DNA biochip products and technologies. Although they may have DNA chip products and technologies, these are not mentioned. A number of chip companies have been excluded because either they have no products out yet or there is no clear description of the technology. Information was obtained from the companies’ respective websites, if no additional reference citations are indicated.

23

24 Microfluidics systems have several technical advantages: 1. Minimal/economical use of sample and reagents (nanoliter amounts) and limited evaporation in closed channels. This low reagent use minimizes costs and, with toxic reagents, minimizes or even totally eliminates, disposal and environmental concerns [11,38]. 2. High performance due to rapid mixing; reactions/processes are more accurate and faster, hence more efficient, when compared with normal systems (discussed below). This is an important factor in high-throughput applications [11,38]. 3. Smaller size, which means lower power consumption and more lab space, possibly even portability [11]. 4. Integration of mechanical, electronic, and optical capabilities, which not only leads to more reliable and reproducible results [11], but also enables m-TAS [38]. 5. Relatively inexpensive fabrication techniques, which translates to multiplexing and to mass production [11]. A summary of the advantages of microfluidics with respect to specific properties such as heat and mass transfer are summarized in Sanders and Manz [13]. In terms of applications, the advantages in biological and medical research stem from compatibility with biological systems, e.g., microfluidic channels and in vivo capillary systems have comparable sizes and fluid flow rates ( 10 mm, 0.1 cm/s) [39], and similar elasticities allow its use in devices for physiological research and diagnostics [10]. In microfluidic devices, fluid flow through the channels is characterized by what is referred to in separation science as a low Reynolds number [12,40–42]. This means the flow is laminar or turbulence-free, resulting in mixing that is controlled solely by diffusion (see also Glossary). When a system is 10-fold smaller, diffusion-controlled mass transport is 100 times faster. Hence, chemical separations, e.g., electrophoresis, can also be performed 100 times faster. The same type of relationship also holds with heat transport [12,13,41]. Because of more efficient power dissipation, higher voltage gradients can be used [13]. Synthetic reactions also result in higher yields and kinetic parameters are easily extracted from the data [41]. Reactions and separations done on microfluidic chips are hence described as high performance. Fluids are pumped through the channels by a number of techniques. The most common is electrokinetic or electroosmotic flow (EOF) where an applied electric field induces the bulk fluid in the channel to move [22,23]. (Other means of fluid transport are discussed under Technologies, Microfluidic technologies, Fluid Flow.) Hence a microfluidic chip includes other elements, which may comprise pumps, valves, mixing and reaction chambers, reagent and waste reservoirs, and detection cells [11]. Microfluidic systems are commonly used for electrophoretic separations, but other separation methods including electrochromatography and ion-exchange chromatography have also been done (see Table 2). Other applications include immunoassays, flow cytometry, sample injection of protein samples into mass

25 spectrometers, cell manipulation and sorting, including chemical gradient formation. Thus, the four major application areas are: miniaturized analytical systems, biomedical devices, chemistry and biochemistry tools, and systems for fundamental research [10]. A wide range of applications indicates a variety of measurements that can be performed (viscosity, pH, kinetics, molecular diffusion, and binding coefficients) and different sample types and reagents that have been used (whole blood, cellular suspensions, protein solutions, and various buffers) [11]. An interesting, recent development is the Lab CD, a compact disc system that does sample preparation by using centrifugal force to ‘‘spin out’’ the sample from the center of the disc outward through the channels (see Gyro in Table 3) [38]. Although microfluidics chips are gaining wide usage, it is not without problems and technical challenges. Because of the small dimensions and the higher surface to volume ratio, surface effects should be controlled or compensated for, if not prevented with proper surface treatment during chip fabrication or just prior to use. Protein adsorption has been a major issue but there are a variety of chemistries and patterning techniques to use on chip surfaces to render them adherent or nonadherent to proteins and cells (see Technologies, Array technologies). A second issue is detector sensitivity. Optical detection systems are path-length dependent. Hence, laser-induced fluorescence detection, an inherently sensitive method, has been used (see also other detection systems under Principles of Design and Operation, Microfluidic detection systems) [11]. A number of techniques address other issues including integrating required components on the chip (pumps, valves, reservoirs), pumping fluids efficiently, monitoring their flow, and avoiding bubble formation as well as evaporation in the microchannels (see Technologies, Microfluidic technologies). Nevertheless, research in microfluidics includes the development of novel microfabrication methods, the integration of novel components that increase chip complexity, and the fundamentals of fluid flow in narrow channels [43]. Specifically, the development of mTAS systems is focused on micro- and nanofluidic control, and the development of microreactors and high-throughput screening systems [41]. Sensors and other chips Chips that involve miniaturized experiments or sample spots and wells that are not within the specifications described earlier for microarrays or microfluidic systems are described here in this section. With further miniaturization or addition of certain procedures or components, they would fit the categories described above. Nevertheless, they have been used by the biotechnology community in various relevant applications. Several sensor chips perform miniaturized experiments; one is the biochemical sensor involving the principle of SPR-BIA [44–46]. Another is a sensor that can monitor cell signaling and metabolism where cells are cultured and their behavior recorded without any transport [47–49]. The whole field of patterning cells to

26 study their function also falls in this area [40,50–53] (see also Biochip Applications, Biological and Medical research). ProteinChip Arrays (see Ciphergen in Table 3) are chips used for surfaceenhanced laser desorption and ionization (SELDI) applications. Although formatted on a chip with 8 or 16 spots, they are not, by definition, microarrays. Each spot, which is about 2 mm in diameter, binds several nanomoles of protein [54]. They have been used especially in protein capture experiments and biomarker profiling with interesting and useful results [54–56] (see Table 2). Nanowells in multiwell plate format have samples that are not immobilized. They have been used in high-throughput cell-based arrays [57,58] (see Aurora Biosciences, Table 3) and the analysis of yeast kinases [136]. Materials Protein chips consist of a substrate (see Glossary) with or without a surface coating. Substrates can be made from glass, quartz, polymers, or even silicon. The coating or the appropriate surface chemistry that is applied to the chip surface (in arrays) or the channel walls (in microfluidic systems) is one of the most important considerations because the key issue is to preserve protein activity [10]. Other criteria for choosing the appropriate material include chemical inertness, nonspecific binding properties, uniform density of the molecular surface with accessible end groups for protein binding [59], and compatibility with other components of the system, often the detector. The characteristics and properties of chip materials are outlined in Table 5. Glass and oxidized silicon are very different materials yet share some similarities (see Table 5). They are the immediate choices for mechanical and electronic devices but their use in chips for biological or even chemical applications is not as straightforward. An exception for glass is that it has been used successfully for DNA separation and sequencing [33–36,60]. Their surfaces can be coated, however, with monomolecular thin films (e.g., SAMs) or functionalized polymers with appropriate protein-binding end groups [59]. One advantage in microfluidics is that the etching process used to fabricate the channels also cleans the surface [10]. Plastic has been used and come in a wide range of size and thickness. The attraction is that it is considerably cheaper than glass and can be disposable, convenient for single-use applications [38]. Chips fabricated from polymers have also been popular. There are three types of polymers classified according to their degree of pliability or softness, which is a function of the extent of molecular cross-linking within the material. These are thermoplastic, elastomeric, and duroplastic polymers [61]. To date, only the first two have been used for microfluidic devices. Duroplastics are more cross-linked, harder, and more brittle. As an important fabrication parameter, the glass transition temperature, Tg, of the polymer determines the temperatures at which it can be molded; some guidelines and precautions are given by Becker and

27 Ga¨rtner [61]. Polymers can have different mechanical and optical properties and resistance to various types of chemicals and reagents (organics, acids, and alkalines). In particular, a distinct mechanical property of elastomers is that its Young’s modulus can be varied over two orders of magnitude by controlling the amount of cross-linking between polymer chains [62]. Biodegradable polymers are also available. A list of polymers for microfabrication, along with their physical and chemical properties, is provided in Becker and Ga¨rtner [61]. For drug screening in particular but also for other analytical applications, suitable polymers are PDMS, PMMA, PC, and Teflon [41]. Advantages specific to polymers are the following: 1. Some polymers can seal effectively against silicon and glass, allowing hybrid devices to be designed and fabricated [62]. For example, microfluidic channels were constructed of SU-8, composite-quartz SU-8, and silicon to be able to perform UV/VIS spectroscopy [63]. 2. Fabrication conditions are not as stringent as with silicon [62,64], hence is favorable with academic researchers (see also Table 5). The additional advantages below benefit commercial applications: 3. Little capital equipment is needed to set up a fabrication facility [62]. 4. They are relatively inexpensive. For example, the elastomer PDMS is 50 times cheaper than an equivalent volume of silicon [62]. On the one hand, they can be easily mass-produced so that high-volume production of disposable units is possible [61,62]. On the other hand, it is affordable to produce small quantities [62]. 5. The rapid turnaround time for fabrication allows easier design modification and iteration [65]. 6. A low barrier of entry should speed up innovation and benefit both research and development in industry [62]. A large part of the literature on polymeric material for protein chips is focused on PDMS. It has several properties that make it well suited for patterning proteins and cells [66] as well as for microfluidic systems [10] (see Table 5). It is durable and has been used about 100 times over several months with ‘‘no noticeable degradation in its performance’’ [51]. It has a hydrophobic surface and adsorbs lipophilic compounds but the elastomer can be modified or its surface treated after manufacture to render it hydrophilic [10,67]. Its surface can be modified by plasma treatment (see Glossary) and subsequent formation of monomolecular films on the resulting oxidized surface [10,51,68]. The fact that it has poor thermal conductivity and is highly permeable to gases is a double-edged sword. For applications like electrophoresis, low thermal conductivity means a build-up in temperature due to resistive heating and results in peak broadening. However, for an incubator system, low thermal conductivity is desirable to maintain elevated temperatures. Gas permeability is not desirable when evaporation or oxidation can be a problem but is desirable for cell culture, for example, and in instances where liquid is to be exchanged [10]. Lastly, in microfluidic systems, several PDMS layers containing channels,

28

Table 4. Companies with biochip-related products and technologies. Chip-related Company (References)

Product/Services (Instrumentation/Software) TM

Technology

Advion BioSciences, Inc. Ithaca, NY http://www.advion.com

ESI Chip (100-nozzle chip for nanoelectrospray infusion of samples, automated) NanoMateTM (automated nanoelectrospray system compatible with several MS systems)

Electrospray nozzles on a chip: a sample aliquot (1–25
l) is transferred from a standard 96-well microtiter plate into a disposable conductive tip; the filled tip is aligned and sealed against a chip nozzle; spraying is initiated when head pressure and voltage are applied to the tip

Akceli, Inc. Cambridge, MA http://www.akceli.com [79]

(Seeks partnerships to apply reverse transfection technology in drug discovery)

Reverse transfection technology: enhances drug discovery and development (target identification and lead optimization, HTS); improves basic understanding of cellular pathways; enables high-throughput cell- and protein-based assays. Transfected cell microarrays: human cells are added to a slide with 10,000 or more different gene sequences in a hydrogel matrix. The cells are analyzed to study how the newly expressed proteins affect cellular networks and pathways

Affinity sensors Cambridge, UK http://www.affinity-sensors.co.uk [44]

IAsys (optical biosensor for direct analysis of unfiltered lysates, whole cells; measures kinetic parameters; choice of pre-derivatized surfaces)

Beecher Instruments, Inc. Sun Prairie, WI http://www.beecherinstruments.com [1]

ATA-27 automated arrayer (0.6–3 mm punch size; punch speed: 120-180 per h) MTA-1 manual arrayer (0.6 min punch size; 30–70 per h)

Tissue microarray instruments generate multiple specimen slides that contain hundreds of individual tissues

BioCrystal, Ltd. Westerville, OH http://www.biocrystal.com

BioPixelsTM (novel nanocrystalline fluorescent markers for tagging biomolecules) PhyloMTM (family of proprietary molecules with very specific binding properties for targeting, imaging, detection, etc.)

BioPixels are functionalized to enhance their stability and allow for coupling to biomolecules, provides high-resolution imaging due to their small size (<20 nm), resists photobleaching, results in high fluorescence intensity due to high quantum efficiency

Biomedical Photometrics, Inc. Waterloo, Ontario, Canada http://www.confocal.com [383]

Designs and manufactures laser scanning products to accommodate many types of biochips, materials, and even live specimen MACROscopeÕ (for tissue imaging and materials analysis scanning) DNAscopeTM (for microarray and biochip scanning) MACROviewTM analysis software (for image display and analysis)

Confocal laser imaging technology for biological and medical diagnostic applications MACROscopeÕ technology: enables design of high optical resolution and wide field-of-view simultaneously for fewer scanning steps

GeSiM mbH (Gesellschaft fu¨r Silizium-Mikrosysteme mbH) Grosserkmannsdorf, Germany http://www.gesim.de

Micro Pipettes (piezodispensers that deliver 30 pl up to 2 nl drops) Nano-Plotter NP 1.2TM (modular micropipeting system, 384 samples in 45 min) microfluidic components and services (silicon/ glass micromachining)

Microfluidic Manifold Systems: integrating fluidic structures (capillaries and reaction chambers) in a single chip; micromachining and rapid prototyping; customized lab-on-achip solutions

Phylos, Inc. Lexington, MA http://www.phylos.com [79], [146]

TRINECTINTM binding proteins as capture molecules for protein microarrays (seeking collaborations where partners will develop and commercialize protein arrays)

PROfusionTM technology: establishing a system to produce high quality binding proteins from TRINECTINTM scaffolds. TRINECTINTM binding proteins (8.5 kDa) are based on an IgG domain scaffold of fibronectin; these proteins are covalently linked to an mRNA tag; attachment to the chip is via the mRNA tag for consistent orientation; also being developed as biotherapeutics (E. coli expression; subnanomolar affinities)

29

(Continued.)

30

Table 4. Continued. Chip-related Company (References)

Product/Services (Instrumentation/Software)

Technology

Nanoplex Technologies, Inc. Mountain View, CA http://www.nanoplextech.com

(seeking partnerships to exploit technologies)

NanobarcodesTM Particles: self-encoded submicron metal stripes for multiplexed biological applications Nanoparticulate Raman Tags: SERS-based quantitation tags (a SERS band is 1/50 the width of a fluorescence band), for multiplexed analyte quantitation Self-assembled Metal Surfaces: nanoscale architecture by surface assembly from spherical colloidal gold nanoparticles in a planar arrangement on, e.g., glass Colloidal Gold Amplified SPR: instrumentation and methods for colloidal gold-amplified imaging SPR for high-throughput microarray applications with SPR sensitivity

Quantum Dot Corp. Hayward, CA http://www.gdot.com

QbeadTM microspheres (polymer latex beads with quantum dot bar coding for multiplexed assays) QcellTM encoded cells (as above, using cells instead of beads)

QdotTM bar codes: as bar codes in multiplexed assays. Quantum dot nanocrystals have 1) a semiconductor core whose composition and size determines the emission wavelength, 2) an inorganic coating to amplify optical properties, insulate the core, and provide chemical stability, and 3) an outer organic coating for chemical coupling to biomolecules

TeleChem International, Inc. Sunnyvale, CA http://www.arrayit.com Boguslavsky [146]

ArrayItTM Products: Microarray substrates (SuperAmine, SuperAldehyde, SuperEpoxy), Micro Spotting Pins, Printheads; SpotBotÕ personal microarrayer

Special protein microspotting pins and nickelplated substrates to immobilize histidine-tagged proteins

This table is a list of biochip-related companies. Although not a comprehensive list, these are companies that support chip technology, e.g. with detection systems or equipment and instrumentation. Companies that sell arrayers, in general, have been excluded unless they provide other chip-related products (see TeleChem). Companies like Affibody, Cambridge Antibody Technology, Dyax, and Protein Sciences have been excluded because although they may collaborate with chip companies, they do not produce chip-related products as their main line of business. Information was obtained from the companies’ respective websites, if no additional reference citations are indicated.

31

32

Table 5. Chip materials. Substrate

Advantages

Disadvantages

Silicon also silicon dioxide (SiO2) and silicon nitride (Si3N4) [70]

Surface negative charge supports EOF (oxidized Si) [10] Can be coated with monomolecular thin films or functionalized polymers displaying protein binding moieties [59] Can be coated onto glass substrate (140 nm silicon nitride waveguide layer deposited onto a BK-7 glass substrate) [71] Biocompatible [70]1 Compatible with a variety of microfabrication techniques already available [12,70]

Relatively expensive [10] Not UV/VIS transparent; cannot use optical detection [10] Sealing processes require a cleanroom, high voltages and temperatures [10]

Etching is expensive, time-consuming [10] Quite stiff; cannot make moving parts; surface chemistries require high temperatures [62] A semiconductor, cannot apply high voltages [12]

Quartz glass

Good electrical insulator hence suitable for electrophoresis [12] Transparent; UV transparent for absorption and fluorescence detection [10,12] Surface negative charge supports EOF [10] (see also very flat glass)

Amorphous, relatively difficult to etch vertically [10] Sealing processes require a cleanroom, high voltages and temperatures [10] Etching is expensive, time-consuming [10] Quite stiff, cannot make moving parts; Surface chemistries require high temperatures [62]

Very flat glass (Corning (GLW))

Chemically inert [74] analyses in organic solvents or low molecular weight organic materials [12] Has low background fluorescence, gives higher signal to noise ratio [152] Can be coated with monomolecular thin films or functionalized polymers displaying protein binding moieties [59] Compatible with a variety of microfabrication techniques already available [12]

(see quartz glass)

Polylysine-coated glass slide

Readily available [59] Direct protein immobilization [125] Positively charged [125] 2

Inconsistent spot morphologies due to non-specific, non-covalent bonds formed between surface and protein, resulting in poor reproducibility [59]

Nitrocellulose

Readily available [59] Direct protein immobilization [125]

Inconsistent spot morphologies due to non-specific, non-covalent bonds formed between surface and protein, resulting in poor reproducibility [59] Hydrophobic [125] 2

PVDF

Readily available [59] Direct protein immobilization [125]

Inconsistent spot morphologies due to non-specific, non-covalent bonds formed between surface and protein, resulting in poor reproducibility [59]

Plastic

Less expensive, more rugged, easy to work with, transparent [12] Inexpensive [62,67] Thermal sealing or use of adhesives possible [10] Good for biological applications; can be mass produced inexpensively [12]

Hydrophobic [59]

Organic polymers (general)

PDMS (an elastomer)

Should control surface chemistry [10] Incompatible with organic solvents or low molecular weight organics [10] Some geometries collapse because of elasticity [10] Poor thermal conductivity ( 0.2 Wm/K) [10]3 Most organics are soluble in the bulk polymer and swell the polymer [51] (see also Organic polymers)

33

(Continued.)

Inexpensive [67], 50 times cheaper than silicon on a per volume basis [62] Optically transparent to 280 nm [10], to 300 nm [51] Compatible with aqueous media and some alcohols [51] Non-toxic to cells, can be used for cell culture [10,51]; high permeability to gases [10]3 Cures at low temperatures, can be molded with delicate features, deforms and seals onto itself reversibly [10]; contacts nonplanar surfaces conformally [51]; material strength can be controlled by extent of crosslinking between polymer chains; can form a tight seal against silicon or glass so can construct hybrid devices; optical structures can be fabricated (optical gratings and waveguides) as well as difficult geometries (coils and basket weave structures) [62]

2

Substrate

34

Table 5. Continued. Advantages

Disadvantages

Excellent material for EOF and pressure pumping; its surface can be charged; elastomeric so easy to incorporate pumps and valves [10] Less stringent fabrication conditions than silicon; fabrication with non-lithographic techniques, i.e. soft lithography [62]; application of rapid prototyping and replica molding; replicates reproducibly [10], durable with repeated use [51] PDMS (Sylgard 184 by Dow Coming)

High transparency above 230 nm with little autofluorescence [61,105]; stiffer than RTV 615 (see below); chemically inert to acids and bases [16]

(see Organic polymers)

PDMS (RTV 615 by General Electric) COC

Less stiff compared to Sylgard 184 (see above) [16]

(see Organic polymers)

High chemical stability; optically transparent [61,90]

(see Organic polymers)

Polystyrene

(see Organic polymers)

Hydrophobic [9,125]2

Bacterial S-layers

Immobilization matrix for biomolecules [78]; immobilization matrices for binding of monolayers of functional molecules (e.g. enzymes, antibodies, and immunogens) in a geometrically well-defined way [76]; exhibit identical physicochemical properties on each molecular unit, down to the sub-nanometer scale; a broad spectrum of very precise chemical modifications can be applied [77]; can be coated or recrystallized on silicon wafers [78]

Not characterized enough

1

Biocompatible means that the material does not induce toxic reactions in tissues that the material is placed intimately in contact with. In particular, silicon, silicon dioxide, silicon nitride, and platinum were used [70]. 2 Hydophobicity and hydrophilicity of chip surfaces, as an advantage or disadvantage, ultimately depend on the particular proteins that are immobilized on the chip (in arrays). They also affect surface wettability in microfluidic systems. In many cases, the substrates are surface-treated or coated to provide a compatible system with a given application, and hence the inherent surface characteristics may not be a major consideration. 3 This property can also be an advantage or disadvantage depending on a particular application.

35 valves, and reservoirs, can be aligned and sealed for the fabrication of threedimensional devices [10]. For protein arrays, glass and silicon have been widely used. Some, however, prefer the use of polymers, because they are nonreactive and can be coated over other sturdier surfaces such as glass. Their fabrication also is relatively simpler. Silicon, silicon dioxide, silicon nitride, platinum, and gold are also suitable [69] and, in particular, considered biocompatible to tissues, i.e., they do not induce toxic reactions in tissue material that they are in contact with [51,70]. Although compatible with a variety of microfabrication techniques, silicon is a semiconductor, which means high voltages cannot be applied. This is taken advantage of on sensing chips where the entire chip is coated with a 500-nm thick silicon dioxide except the sensing regions, where a thickness of 40 nm was used [71,72]. For microfluidic systems, glass or quartz is inert to the chemicals (e.g., low molecular weight organics) and reagents used in the various processes that are carried out on chip [73–75]. Furthermore, transparency is necessary for detection systems involving absorbance and fluorescence. Glass slides and quartz glass continue to be used both academically and commercially. Quartz glass is used for commercially available chips for protein separation (see Caliper in Table 3). More recently, polymeric materials have also been used in chips that perform capillary electrophoresis [75]. Silicone rubber (PDMS) has properties that make it suitable for microfluidics, e.g., devices with moving parts such as pumps and valves are best made of soft materials [62]. In the area of nanotechnology, researchers have been studying the crystalline surface structures on archaea and bacteria. These surface layers, termed S-layers, are one of the most abundant cellular proteins on such organisms. They are composed of protein subunits that form self-assembled crystalline lattices of monomolecular thickness [76,77]. They have center spacings of less than 25 nm and therefore may find biomimetic and nanotechnological applications [77]. In particular, S-layers have the appropriate structures to be used as an immobilization matrix [78]. Although not in the mainstream, they have potential use in protein microchips. Technologies It would seem that, especially for arrays, the technology with which to fabricate protein chips would already be in place, given the wide usage of DNA microarrays and that commercially available DNA arrayers and contact printers could be adapted for use with proteins. This assumption, however, cannot be made. The making of a protein chip is more complicated; it involves more steps and more scientific know-how [79]. Proof of this is the many commercial entities, especially biotechnology companies, which have development projects on protein chips (see Table 3), not to mention the amount of literature covering this topic (see Table 1). Although some technologies are proprietary and therefore inaccessible, a number

36 of techniques have been published with success and will be discussed. The main issues in miniaturization are the more pronounced surface effects and detection sensitivity. When volumes are down to the picoliter, even nanoliter level, the surface to volume ratio increases dramatically [9]; proteins will denature especially due to dehydration and will adsorb on chip substrates such as glass, plastics, steal, etc. [9]. Counteracting these surface effects with various chemistries are an essential part of chip fabrication. The modes of detection are discussed in the subsequent section on Principles of Design and Operation. The technologies mentioned below are based on fabrication technologies used for glass, silicon, and polymers. They have been adapted from existing technologies used in other industries such as in the electronics and manufacturing sectors. Polymer technologies possess the flexibility with which different surface chemistries can be used depending on the application. For microfluidics, clearly, they have been used because of the various microstructures required on chip. Photolithography Photolithography is used to prepare the surface of a silicon or glass chip, similar to the etching of semiconductor microchips [37,79,80]. The first step involves silanization, which deactivates the surface, before a photoresist is applied or spun on and prebaked. After UV exposure, developing and postbaking, the unmasked regions are etched and then the mask is stripped off. The etched areas reveal structures that are to be specifically surface treated for protein binding (in arrays) [81,82] or that define the required channels (in microfluidics) [37,75,80]. Photolithography on gold has been described [69]. X-ray lithography on PMMA has been used for the fabrication of microfluidic devices [83]; the channels formed for microelectrophoresis were approximately 20 mm wide (depending on the optical mask used) and 50 mm deep (depending on X-ray exposure time). The literature on photolithography can be referred to for more details [84–88]. (See Fig. 3 for an illustration of the photolithographic process applied to microfluidics.) Photolithography is a well-established technology in the fabrication of microelectronic devices. Although it can result in features smaller than 1 mm, many chip applications today do not require features to be this small, especially in cell biology [51]. For academicians, disadvantages include the high cost of photolithographic equipment, the need to access cleanrooms, the relative inflexibility in the type of chemical functionalities that can be introduced onto surfaces, and the limitation to planar substrates [51]. Photolithography and wet etching have disadvantages for commercialization as well: 1. Cost of the substrate material: Compared to polymers, many types of glass (e.g., borofloat such as Corning Pyrex, borosilicate such as Schott B270, and photostructurable glass such as Schott Foturan) are at least 2–10 times more expensive and is a factor in mass production [61]. The situation is worse then for silicon, which is more expensive than glass. It is 50 times more expensive than PDMS [62].

37

Fig. 3. Photolithographic process for making chips. Reprinted with permission from Ref. [75]. Copyright 2000 Wiley-VCH Verlag GmbH & Co. KGaA.

2. Difficult to automate: With many steps in the photolithographic process, as these steps should be done sequentially, there is the increased fabrication time and the higher risk for errors. Since a number of reagents and chemicals (some even harmful) are required, there are the substantial costs and attention required in both their use and their proper disposal. 3. Design limitations: The etching process on glass or quartz allows only the fabrication of structures with shallow, semicircular cross sections. But there are applications that require different features, e.g., angled walls and different channel heights. These can be done on silicon by dry etching [89] but then the substrate cost becomes prohibitive. 4. Surface chemistry: With silicon and glass substrates, surface silicon bonds to biomolecules. Although silanization helps, this is an added step to an already long process [61,80]. Polymer fabrication methods Polymers are an attractive alternative for chip fabrication. Once a master is made, mass replication is possible. Nevertheless, single devices can also be fabricated with a number of techniques. Polymer fabrication methods can be categorized into two: replication techniques, amenable to mass production,

38 and single-device techniques, which are ideal for producing prototypes. These technologies have been used, in particular, for microfluidics, although arrays have been fabricated with these technologies. The most popular replication methods, hot embossing and injection molding, have used PMMA and PC polymers. COC is also promising for molecular biotechnology applications [90]. A table of polymer physical properties can be found in Becker and Ga¨rtner [61]. Master fabrication The master can be fabricated by a number of techniques depending on the precision required and the channel dimensions. Larger structures (>100 mm) can be made from computer numerical control (CNC) machining, as done on stainless steel [75]. The techniques below produce features <100 mm in size. Micromachining. Laser micromachining techniques (sawing, cutting, milling, turning) for miniaturized applications can produce structures down to tens of micrometers in size [41,61]. Advantages include a wide range of materials that can be machined, the short times required, and no additional steps of mask fabrication and lithography. A major drawback is that only simple structures can be made, i.e., straight walls; deep holes and structures with high aspect ratios cannot be fabricated well [61]. Micromachining techniques to produce 3D structures are discussed in Unger et al. [292]. Silicon micromachining. Silicon micromachining can be done by wet etching of silicon (the simplest technique) or dry etching, which includes the technique ASE (advanced silicon etch) [89]. Wet etching results in a trapezoidal channel whereas dry etching can result in deep vertical walls. The depth, however, depends on the etch rate: the faster the rate, the rougher the surface. Typical depths are 10–40 mm; with ASE, this is increased to 200 mm [61] (see also Glossary). Electroplating. Electroplating is a commonly used method for the fabrication of masters. This means the master produced is made out of nickel or an alloy like NiCo or NiFe. The process starts with photolithography up to the development of the photoresist. The resist-free regions are then electroplated to form a metal structure shim, which is further processed. Typical structures have heights of 10–40 mm. Heights of up to 1 mm can be achieved with the use of thick resists such as EPON SU-8 [61]. A variation of electroplating is the LIGA technique. A combination of lithography, electroplating and molding, very small channels with high aspect ratios can be made [75]. In the lithographic step, thick PMMA layers are ablated by either synchrotron radiation [91] or, in the case of laser-LIGA, pulsed UV irradiation [92]. With the electroplating step, the resulting nickel surface is quite smooth but there are some drawbacks, including a slow growth rate (10–100 mm/h) that has a radial dependency leading to an uneven thickness [61].

39 Replication technologies Polymer replication technologies are used in the commercialization of microfluidics systems because of the relatively low manufacturing cost [93]. The difficult and expensive step is the fabrication of the master or mold tool. Once this is made, it can be replicated into the desired polymer substrate by a number of techniques with a short turnaround time [61]. Beyond proper process control to minimize surface roughness, the main disadvantage is that undercuts or overhanging structures cannot be fabricated. Restrictions to replication that set constraints on how the master can be fabricated are summarized by Becker and Ga¨rtner [61]. Hot embossing. A widely used replication method for the fabrication of microfluidic systems is hot embossing [94–100]. The embossing tool and the polymer substrate are heated separately (helps extend the lifetime of the mold tool) in a vacuum chamber to just above the substrate’s Tg; this removes water vapor and prevents the formation of bubbles. Embossing is carried out at 0.5–2 kN/cm2 for several minutes until the mold–substrate sandwich cools to just below Tg. Turnaround times are short, e.g., 5–7 min for PMMA. Furthermore, high structural resolution can be achieved [61,75,101]. (See Fig. 4 for a diagram of a hot-embossing machine.) Injection molding. Adapted from macroworld polymer manufacturing, raw, granular polymer material is put into a cylinder with a heated screw (see Fig. 5). The cavity containing the polymer is heated to a temperature close to the melting point of the polymer. As the polymer melts, it flows into the mold cavity, which is subsequently cooled to allow the removal of the replica [61,93,102]. This process, termed variotherm, is necessary for microfabrication but is distinctly different from the macro process where the molten polymer is injected with 60–100 MPa (600–1000 bar) pressure into the evacuated mold cavity. Typical turnaround times are 1–3 min. The process requires optimization for proper replication; shrinkage may occur due to the large temperature drop from injection to ejection [61]. The fabrication of microfluidic devices using this technique has been described [93,103]. Casting. Because of material flexibility and low cost in producing planar channel structures [104], the casting of silicone rubber (PDMS) elastomers has been widely used by academic researchers. The elastomer is poured on top of the mold and cured or hardened at room temperature and atmospheric pressure [105], although heating can accelerate curing [75]. It is the simplest of the three replication techniques but requires long (minutes to hours) contact with the mold [75]. A miniaturized separation device fabricated using this technique was patented by Ekstro¨m et al. [106] in 1990. The elastomer is sandwiched between two glass plates to provide mechanical support and seal the channels [61]. An overview of these replication methods is given by Becker and Ga¨rtner [61]. Other polymer fabrication techniques that have not yet been used for microfluidics are also cited.

40

Fig. 4. Diagram of a hot embossing machine. Reprinted with permission from Ref. [61]. Copyright 2000 Wiley-VCH Verlag GmbH & Co. KGaA.

Fig. 5. Schematic diagram of an injection molding machine. Reprinted with permission from Ref. [61]. Copyright 2000 Wiley-VCH Verlag GmbH & Co. KgaA.

Single-device fabrication Laser ablation. Laser ablation with an excimer laser has been widely used for the fabrication of microfluidic devices [107,108]. Various geometries can be patterned with the use of a mask. The structures formed have little thermal damage, straight vertical walls, and well-defined depth [75,109,110]. Electroosmotic flow in laser-ablated channels has been demonstrated [108]. Accurate patterning of parallel arrays has also been done [82]. Polymers that have been used with this technique include PMMA, PS, PC, PET (or Melinex [75]), cellulose acetate, nitrocellulose, polyimide, poly(tetrafluoroethylene) (Teflon), and photoresists [82,107,108,111–113]. Photoresist technology. The use of photoresists is a well-established technology, similar to photolithography, and can be adapted for use with arrays or

41 microfluidics. The process involves spin casting a photoresist layer over a substrate, masking, and exposing to UV irradiation. Unmasked photoresist decomposes under UV exposure and this defines regions for patterning (in arrays). Additional layers of photoresist and other treatments are required to form channels and to seal them (in microfluidics). One of the early applications of photoresist technology was controlling cellular growth within amino-silanized regions [114]. In fabricating arrays, silanes are used for attaching proteins to silica (glass or quartz) or metal surfaces [115]. They do not interfere with the optical properties of quartz or the electrical properties of metal substrates. They form covalent bonds with proteins and can withstand the harsh solvents used to dissolve photoresists. Once the unmasked regions have been defined after UV exposure, the silane (usually amino-terminated for protein linkage) is bound to these exposed regions and residual photoresist is sonicated away with acetone. The chip is then incubated in a hydrophobic, ‘‘background’’ silane to define the nonadherent regions. The result is then a mixed silane monolayer with protein adherent and nonadherent regions [116]. The proper choice of background silane can minimize nonspecific protein binding. However, nonspecific binding can occur with incomplete surface coverage or gaps in the silane monolayer. An additional disadvantage is that residual photoresist as well as solvent can denature proteins [116]. In fabricating microfluidic systems the use of thick resists such as SU-8 results in channel heights of over 200 mm (normal photoresists usually have 0.5–3 mm thickness) [61]. Starting with a base layer of SU-8, which is exposed to UV light and postbaked, a second SU-8 layer is spin-coated over the first layer and then processed by one of the following three general processes: 1. Fill: A sacrificial layer of another polymer (e.g., Araldite) fills the gaps left on the second SU-8 layer after UV exposure. A third SU-8 layer is spun on top, exposed, and baked. The sacrificial layer is dissolved, thus defining closed microchannels. 2. Mask: The second SU-8 is not developed but instead a metal layer is laid on this as a mask. After the third SU-8 layer is spun on top and exposed to UV light, developing dissolves the resist directly underneath the metal, thus forming the channel. This process, however, is slow and would take several hours to produce a channel length of 1 cm [61,117]. 3. Lamination: This is similar to the fill process up until the channels are to be closed. In this case, a layer of dry SU-8 film is laminated on top to close the channel. Advantages are the short processing times, no dissolution steps, and the channels are completely sealed [61]. (See Fig. 6 for an illustration of these three processes done with SU-8.) Other polymer techniques (e.g., layering) specific to a particular format are discussed in their respective sections below. Others involving electrochemical or ultrasonic techniques are found in the literature [41,104]. Apart from the polymer replication technologies and photolithographically related methods described above, a set of microfabrication techniques has made

42

Fig. 6. Fabrication methods for microchannels using optical lithography with SU-8. Reprinted with permission from Ref. [61]. Copyright 2000 Wiley-VCH Verlag GmbH & Co. KGaA.

an impact in chip microfabrication. With much of the research on patterning proteins and cells done on ‘‘soft’’ PDMS elastomer, the term soft lithography came into use, stemming from research done in George M. Whitesides’ laboratory (Department of Chemistry and Chemical Biology, Harvard University) (see under Array technologies, B. Patterning, Soft lithography, and Microfluidic Technologies, A. Fabrication techniques, Soft lithographic methods for microfluidics). Array technologies The crux in producing a protein array is immobilizing proteins that remain functional. Despite all the technologies discussed above, arrays are still being produced on the simplest substrate, glass slides [71,72,118–121]. The key of course is application of the appropriate chemistry to treat or coat the surface for protein immobilization. A number of protein immobilization techniques are discussed below. An important part of protein immobilization is patterning. Although the use of both terms is at times interchangeable, patterning specifically pertains to immobilization at specific locations in 2D or 3D space [116]. Several patterning techniques are discussed below. The application of proteins or protein ligands on the chip is implied with immobilization or patterning. However, this should not always be assumed. At times, immobilization or patterning only refers to the preparation of the chip surface and how the proteins are to be bound but does not include how the proteins or ligands are applied or put on the chip. Hence, protein-printing methods are

43 discussed, some of which are patterning techniques themselves. Finally, an issue with protein immobilization is proper orientation of protein molecules, specifically antibodies, so that binding sites are effectively directed toward the bulk solution and away from the chip surface. Some of these orientation techniques are summarized at the end of this section. Protein immobilization The efficiency of protein immobilization depends on the chip surface, the method of applying the protein on the chip and the immobilization method itself. At optimum conditions, the resulting density may be as high as 105 protein molecules in a 50-mm diameter spot [59]. Immobilization can be achieved by one of three methods: physical (noncovalent) adsorption, the use of highaffinity ligands, and covalent bond formation [59,116]. These methods may require that either the chip surface or the protein undergo some modification or treatment. Noncovalent attachment. Physical adsorption is characterized by weak, noncovalent attractive forces such as ionic, hydrophobic, van der Waals, and hydrogen bonding [9,59,122]. Because of these nonspecific interactions, results lack reproducibility as evidenced by irregular spot morphologies. Furthermore, the immobilized protein can be denatured and sterically occluded [9,123,124]. This is the case when arrays are prepared from adhesive substrates, e.g., those that are hydrophobic such as nitrocellulose, PVDF, and PS, or positively charged such as polylysine (coated glass slides) and aminosilane (e.g., on glass or silicon) [9,59,125]. This type of immobilization is attractive because of its simplicity and is generally used in ELISAs [9]. High-affinity ligands. The use of high-affinity ligands result in strong noncovalent specific interactions between proteins and surface-bound ligands. The protein of interest then becomes attached to the surface only at a specific contact point, leaving it functional. Examples include biotinylated proteins on streptavidin-coated surfaces [126,127], His-tagged proteins on Ni2 þ -chelated surfaces [9,125,128], and the use of lectins, protein G and protein A [129–132]. Another technique called DNA-directed immobilization (DDI) uses a microarray of DNA–streptavidin conjugates as an immobilization matrix. Biotinylated proteins can then bind in a highly parallel yet chemically mild process [133] (see Fig. 7). Covalent attachment. In covalent attachment the chip surface is usually treated with an appropriate silane. The most common is an amine-reactive surface (brought about by reaction with an aminosilane reagent) that randomly conjugates to lysine residues on proteins [118,136,137]. An aldehyde-reactive surface reacts with oxidized glycoproteins [137] and with primary amines on proteins [9,118], forming a Schiff’s base linkage [118]. Thiol-terminated silane surfaces

44

Fig. 7. Schematic drawing of the ‘‘DNA-directed immobilization’’ (DDI). A set of covalent DNA– streptavidin (STV) conjugates (7a–c) are coupled with biotinylated antibodies to generate tagged conjugates (12a–c). A microarray of capture oligonucleotides is used as the immobilization matrix. Note that owing to the specificity of Watson–Crick base pairing, many different compounds can be site-specifically immobilized simultaneously in a single step. Adapted from Ref. [581]. Reprinted with permission from Ref. [133]. Copyright 2002 Elsevier Science Ltd.

have also been used [138–141] and also in combination with heterobifunctional crosslinkers [142]. Another technique involves a mixed monolayer of benzoquinone and oligo(ethylene glycol) groups. Benzoquinone provides the selectivity via ligand immobilization through a Diels–Alder cyclopentadiene conjugation while the ethylene glycol ‘‘repel’’ proteins and reduce nonspecific adsorption [143–145]. Industrialized approaches to protein immobilization have also used proprietary surface chemistries [146,147] (see Zyomyx in Table 3). Covalent immobilization chemistries have been shown to preserve protein functionality for up to 2 years [134,135]. Another set of chemistries involving covalent attachment is the use of photochemistry and heterobifunctional crosslinkers (see Patterning, Photochemical techniques). A method that combines covalent and strong noncovalent attachment is to derivatize the chip surface with poly(ethylene glycol) (PEG) [148,149]. PEG is effective for nonadherent regions of the chip surface. To render specific areas of the chip as protein-adherent or protein-binding, these regions are derivatized with biotin. Streptavidin binds to these surface biotin molecules and because it is tetrameric, it also binds biotinylated proteins to the chip surface. This method has been used to produce high-density protein microarrays [126,127].

45 Polyacrylamide gel pads. An immobilization technique that increases spot density as well as preserves biological activity is the use of gels. With the use of gel pads, the advantages are that evaporation is minimized and more proteins are immobilized because of a ‘‘third dimension’’ [150]. In fact, the immobilization capacity is said to be 100 times greater for proteins as well as oligonucleotides. After the polyacrylamide gel micromatrix is made and loaded with microliter amounts of protein solution, it is chemically activated. Chemical activation involves reducing the enamine bond formed between the aldehyde group on the glutaraldehyde-activated gel and the amino groups of the protein [151]. This step immobilizes the protein in the gel. In one account, microelectrophoresis accelerated diffusion in the gel [137]. Commercially available HydroGel-coated glass slides are made from ‘‘a modified polyacrylamide to optimize protein immobilization and spot morphology’’ [146] (see Packard Bioscience in Table 3). Immobilized proteins on these slides have shown retention of their activity in enzymatic and binding assays. Glass substrates are currently used although other materials may be used [152]. A reverse process applies proteins to the chip via a gel matrix (see Protein printing; Stamping). Immobilization of membrane proteins. An interesting and important development is the immobilization of membrane proteins. A majority of the current drug targets are membrane proteins. Hence, they should be addressed by biotechnological tools such as protein chips in multiplexed compound screening but also in more fundamental studies involving cell–cell communication. Chip surfaces, modified with -aminopropylsilane (GAPS), bind the membrane proteins via their associated lipids. This technique was tested on GPCRs (G-protein coupled receptors), which showed high specificity with no apparent artifacts. Incidentally, gold as a substrate exhibited less nonspecific binding than ultraflat glass [153]. Patterning Patterning is immobilizing proteins or cells in specific locations [116]. Protein patterning in dimensions of up to 100 mm is important for biochemical applications from binding assays to combinatorial screening [10,51] while patterning of cells is important for fundamental studies in cell biology [15,18,19] and for their use in cell-based sensors [17,39,65,154] and tissue engineering [42,51]. Patterning methods include: photoresist technology (discussed above under Polymer fabrication methods, Single-device fabrication), photochemical techniques, and techniques that involve the use of self-assembled monolayers (SAMs) [116], including soft lithography. Photochemical techniques. Photochemistry involves the use of labile chemical species that are activated, in this case, with UV irradiation [155–157]. There are three commonly used chemistries for photopatterning: arylazide chemistry, diazirine chemistry, and nitrobenzyl caging chemistry.

46 Arylazide photochemistry involves a reactive nitrene that can insert into C–H bonds [157]. One application used a photoactive biotin (nitroarylazide attached to the biotin tail) which binds to adsorbed avidin on the chip surface. Incubation in protein solution and UV exposure allows the nitrene formed to insert into the protein [158]. Another type of photochemistry involving diazirine has been used to immobilize proteins in various applications [155]. Upon absorbing 350 nm irradiation, diazirines form highly reactive carbenes, which insert into covalent bonds. The main disadvantage with arylazide and diazirine chemistries, including benzophenone, is that the protein has to be present during the UV exposure. This may inactivate the protein and with the short half-life of the nitrene, in the case of arylazide chemistry, protein conjugation is not optimal and results in low yields [159]. Arylazide ring substitution to bring the required UV activation to longer wavelengths is a possible solution [160]. Nitrobenzyl chemistry is also referred to as caging chemistry; labile groups are attached to a molecule to prevent its activity. Two caging groups that are used are NVOC (nitroveratryloxycarbonyl) and MeNPOC (-methyl-6-nitropiperonyloxycarbonyl) [161–166]. For a multianalyte sensor, antibodies were patterned onto waveguide surfaces where caged biotin (MeNPOC-biotin) was immobilized by conjugation to BSA, which was then adsorbed onto the glass substrate [165,167]. Upon UV exposure, the caging group breaks down into a ketone and carbon dioxide, harmless by-products, and the caged molecule, i.e., biotin, retains its activity [156]. The sensing elements were therefore formed from repeating a sequence of UV exposures in selected areas and incubation in streptavidin and then biotinylated antibody. Advantages with nitrobenzyl chemistry are the harmless by-products from the break up of the caging molecules, as mentioned, and that protein binding occurs after the UV irradiation. This provides some flexibility and specificity in the immobilization step. In the above example, different types of biotinylated antibodies can be applied to the chip [132]. In general these photopatterning chemistries described above can be used repeatedly on the same surface with different proteins or protein ligands. However, a concern is proper control of the background surface. Blocking with BSA is an option [168]. But a more important issue is UV light: the chip must be kept under subdued lighting until the photochemistry is to be done and proper care should be taken during photolithographic masking so that the derivatized surface is not disturbed (damaged or smeared) and UV scatter is avoided [116]. Other references on photoimmobilization include papers by Hensakul et al. [169], Morgan et al. [170], and Delamarche et al. [171]. Apart from the chemistries described, photopatterning can be performed with heterobifunctional crosslinkers [138,172–175]. Several are commercially available. Many have aryl azides as the photoactivatable group but benzophenone (BP) reagents have also been used because of higher crosslinking efficiencies [172,175–177]. They can be handled at ambient light and activated at longer wavelengths [172,178–180]. These rigid molecules have also been made more

47 flexible with hydrophilic spacers, e.g., the BP-oligo(ethylene glycol)-maleimides (BP-EGn-MI), for better accessibility of immobilized proteins or ligands to their binding partners in bulk solution. In photopatterning antibodies, BP-EG5-MI tested well on polystyrene. The BP group is attached to the PS surface and the MI end is available for protein binding via amino groups. Maximum sensitivity is achieved when the preparation is carried out stepwise on the chip rather than premixed [172]. Another heterobifunctional crosslinker is N-succinimidyl-4maleimidobutyrate [116,138]. Immobilization with heterobifunctional crosslinkers can be done on glass, silica, silicon, or platinum substrates [116,138,181]. Self-assembled monolayers (SAMs). On silica or metal surfaces, alkylsilanes or alkane thiols assemble into organized layers [182,276]. One of the earlier works involved patterning with n-octadecyltrimethoxysilane (OTMS) to produce hydrophobic SAMs on SiO2 [81]. The background regions were treated with UV irradiation and the use of a lithographic mask to strip off OTMS while the protein-binding regions were prepared by adsorption of BSA onto OTMS and layering biotin and streptavidin on top of this. With later developments came the use of mixed monolayers that defined the pattern on the chip. One end of the molecule binds to the chip surface and the other has an end group that can be varied to define adherent or protein-binding regions (hydrophilic) and nonadherent or background regions (hydrophobic). Examples of patterning mixed SAMs with UV irradiation are given in Blawas and Reichert [116]. The advantages of using substrates coated with SAMs are the variety of surface chemistries that can be achieved via the different end groups that can be attached [59] and the uniform surface characteristics that allows higher sensitivity and reproducibility, especially when compared to physical adsorption. Protein denaturation has therefore been minimized. The use of SAMs has tremendously improved the microfabrication of protein arrays. Binding protein molecules to alkanethiol SAMs by C–C bond formation using photoactivatable benzophenone has been demonstrated [183]. SAMs have been applied in different areas: polypeptide synthesis [166], microelectrode formation [184], cell adhesion [185–188], and protein adhesion [81,141,186,189–194]. Protein adsorption onto uncharged SAMs is dependent on protein size and the wettability of the surface. The general trend is that the less wettable the surface, the greater the adsorption. Exceptions, however, have been noted with both smaller and larger proteins [195]. SAMs that are protein-adherent, e.g., are those presenting Ni2 þ chelates for binding with His-tagged proteins from cell extracts [196], those presenting hexadecanethiol [116,197,198], those patterned with EDA ((N-(2-aminoethyl)(2-aminopropyl) trimethoxysilane on silica and platinum surfaces [188,199], and those ending with methyl groups [189]. Bovine capillary endothelial cells, e.g., attached to the fibronectin-coated methyl end groups (as opposed to ethylene-glycol end groups) and remained attached for at least 5 days [200]. SAMs that resist protein adsorption, and hence are useful as background SAMs, are those made from alkanethiols

48 !-substituted with oligomeric ethylene glycol [148,201], with tripropylene sulfoxide [202] or with sulfonate groups [203]. With UV irradiation, either strategy for patterning on SAMs is possible: start with an adherent surface (e.g., EDA) and render specific regions as nonadherent (UV exposure and subsequent binding to 13F (tridecafluoro-1,1,2,2-tetrahydrooctyl-1dimethylchlorosilane) to give a hydrophobic surface) [188,199,204] or start with a nonadherent surface (e.g., chemically bonded OTS) and render specific regions adherent (UV exposure and subsequent binding of silanol groups to EDA) [81,205]. Currently, SAMs of alkanethiols are the most common and are usually formed on gold. (Silver films can be used except in the case of live cells because Ag þ , which is released upon exposure to air or oxidants, is cytotoxic [51].) The procedure involves laying a titanium thin film (1–5 nm in thickness) on the substrate to promote the adhesion of gold, gold (10–200 nm in thickness) is then applied [206]. The gold film is subsequently exposed to alkanethiol vapor or solution [207,208], whereupon the sulfur atoms are coordinated to the gold surface and the close-packed alkyl chains are characteristically tilted 30 from the surface [51,203]. SAMs can be used over several days, e.g., in cell culture but they should be protected from temperatures above 70 C, UV light, and oxygen or ozone [51]. Patterning on SAMs can be done via a variety of techniques: UV microlithography [189], scanning probe lithography using AFM [209,210], microwriting [189], micromachining [104,189], stamping [189,211], microcontact printing [197,200], laser ablation [198], and photochemistry [171,211]. Some of these methods (stamping and microcontact printing) are discussed subsequently. Microwriting, micromachining, and stamping may have the advantage that UV exposure of the surface is not required. However, multiple protein patterning may not be possible because the protein solution is incubated in a single step whereas photochemical methods can pattern multiple proteins on a single surface [116]. But for high-throughput applications, these techniques are not suitable because of repeated processing. Protein printing methods by inkjet techniques or multiple-pin arrayers are more suitable (see Protein printing). Soft lithography. Soft lithographic methods have also been used on SAMs of alkanethiols on gold. Soft lithography can pattern biomolecules and cells as well as form microfluidic channels and both surface chemistry and the cellular environment can be controlled [51,213]. They are performed on elastomeric stamps with raised patterns (bas-relief) produced via replica molding. PDMS elastomer has been used extensively where the master is fabricated from photolithography [51]. (See Fig. 8 for an illustration of the fabrication of a PDMS stamp.) The following general protocols depend on feature sizes generated in the master. For features  20 mm, the photolithographic mask can be flexible transparencies made from high-resolution laser printing at 3387 dots/in. [64,154] with patterns drawn from Freehand or AutoCAD software. This is the

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Fig. 8. Schematic illustration of the procedure used to fabricate a PDMS stamp from a master having relief structures in photoresist on its surface. Reprinted with permission from Ref. [51]. Copyright 1999 Elsevier Science Ltd.

basis for the term ‘‘rapid prototyping’’ – costs little and is done in hours. For features >10 mm, microfiche patterns are used as photomasks [214]. Finally, for small features (2–20 mm), commercial laser writing is used for fabricating the PDMS stamp [215]. Minimizing the use of specialized equipment, Deng et al. [64] have described a fabrication procedure producing features as small as 15 mm starting with CAD files printed at a resolution of 600 dots/in. Access to a cleanroom, however, is still necessary. Soft lithographic patterning covers four techniques: microcontact printing (mCP) of alkanethiol SAMs on gold; patterning using microfluidic channels (or micromolding in capillaries, MIMIC); laminar flow printing [51,216–218] and patterning with 3D microfluidic systems [53]. 1. Microcontact printing (
CP): Microcontact printing involves the application of material (proteins, thiols, etc.) from an elastomeric stamp to specific regions on a substrate [51,200,219] (see Fig. 9 on mCP using soft lithography). The stamp is made of silicone rubber, PDMS, fabricated by replica molding on a micropatterned silicon wafer. As an elastomer, PDMS can provide conformal contact between stamp and substrate, ensuring a homogeneous transfer of ‘‘ink’’ to the substrate [197,219]. The ‘‘ink’’ is a thiol-terminated alkane [197] or, in a recent report, PTMP [219]. It provides the highest resolution

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Fig. 9. Microcontact printing (
CP) and fabrication of contoured substrates using soft lithography. (A) A stamp is inked with an alkanethiol and placed on a gold surface; the pattern on the stamp is transferred to the gold by the formation of an SAM on the regions that contacted the substrate. The bare areas of the gold are exposed to a different alkanethiol to generate a surface patterned with an SAM that presents different chemical functionalities in different regions. (B) The PDMS stamp can also be used as a master to mold harder polymers and generate contoured surfaces. After evaporation of a layer of gold, these surfaces can be functionalized by
CP of one alkanethiol with a flat stamp. The grooves of the substrate can then be exposed to an alkanethiol presenting a different functional group to produce a contoured surface with patterned chemical reactivity. Reprinted with permission from Ref. [51]. Copyright 1999 Elsevier Science Ltd.

among the soft lithographic methods with features smaller than 1 mm. When patterning on SAMs, controlled adsorption has been observed [51]. It also allows control of surface chemistry at the molecular level [51] to promote certain properties such as wettability [220–222], protein repellency [187,189], crystal growth [223–225], and adhesion [226,227]. The most widespread application of mCP is in the formation of patterned SAMs by the printing of alkanethiols onto Au [200,228], Ag [200,229,230], Cu [231], Pd [232], or of silanes onto Si/SiO2 [233–236].

51 Micropatterning of cells has been done with mCP on SAMs of alkanethiolates. The appropriate extracellular matrix is important to be able to pattern any adhesion-dependent cell line. In other patterning techniques, defining adherent and nonadherent regions require the adsorption, e.g., of albumin, which after several days starts to degrade and leak into the nonadherent regions. With mCP on SAMs, an advantage is that no leakage is observed either from albumin degradation or extracellular matrix. It should be noted, however, that the mCP process does affect cell shape and size [51]. Pattern resolution is typically 1–2 mm with island separations of 20–40 mm. Hence, this is one application where the resolution obtained with photolithography is not necessary. Patterning of cells is important fundamental studies in cell biology and the development of biosensors requiring precise cell control and positioning on the chip. Furthermore, because cells can be accurately arrayed, automated systems can be developed for their incorporation into drug screening with large compound libraries [52]. 2. Patterning using microfluidic channels: Patterning with microfluidic channels relies on restricting fluid flow to selected regions on the chip surface. The technique, micromolding in capillaries (MIMIC), developed by Kim et al. [237], involves allowing solutions to flow into channels formed by a PDMS mold in conformal contact with the substrate (see Fig. 10). PDMS seals reversibly and thus can be used to pattern proteins and cells by covalent or noncovalent attachment, or dissolution [10,238–240]. This technique allows the fabrication of 3D structures.

Fig. 10. Illustration of the procedure used to pattern proteins and cells using microfluidic channels. Reprinted with permission from Ref. [51]. Copyright 1999 Elsevier Science Ltd.

52 MIMIC has been used on several substrates such as glassy carbon or ceramics [241,242], sol-gel materials [243], inorganic salts [244], polymer beads [245], and colloidal particles [244]. It has also been used to pattern proteins using sol-gel microstructures on silicon [246]. MIMIC has been used to pattern immunoglobulins with submicron resolution on glass, gold, and PS for IgG detection in immunoassays [247,248]. In one application, capture agents were immobilized in stripes and samples were delivered in parallel channels perpendicular to the rows of capture agents. Binding reactions result in fluorescent signals [249]. Microfluidic channels were also used to pattern an immunosensor array to simultaneously detect clinical analytes [119]. To generate patterns of biotinylated ligand on a biodegradable polymer, Patel et al. [250] allowed a solution of avidin in elastomeric channels to flow over the biodegradable polymer PLA-PEG-biotin (biotin attached to the terminal PEG group of a polylactide-PEG copolymer). The avidin molecules bound to surface biotin moieties can then bind to biotinylated proteins or ligands in solution. Several cell patterning studies using MIMIC include the use of PLA-PEG-biotin to study the adhesion and spreading of bovine aortic endothelial cells and PC12 nerve cells [250] and the selective adherence of cells on various biomedical polymers and other substrates [238,239]. 3. Laminar flow patterning: This technique was developed by Takayama et al. and takes advantage of microfluidic laminar flow [40]. With laminar flow conditions, several different fluids can flow next to each other in a channel without mixing except at their interfaces where diffusion occurs. This provides a means to selectively pattern cells (or portions of cells) and culture medium in a highly controlled manner [10,40,51]. Escherichia coli and chick erythrocytes were patterned side by side via a hydrophilic PDMS channel mold reversibly sealed onto a silanized silicon chip; other fluids (nutrients, fluorescent labels, enzymes, drugs) were delivered to these immobilized cells by the same technique [40] (see Fig. 11). The technique is simple and allows multicomponent patterning in relatively few steps as well as patterning over delicate surfaces like mammalian cells [51]. It also should enable applications such as array screening of cells, cell-based sensors, and the use of microfluidic systems in metabolic and toxicological studies [40]. 4. Patterning using 3D microfluidic systems: The three-dimensional microfluidic system in PDMS is actually made from a 3D MIMIC technique [251] and is related to techniques for the 3D fabrication of microchannels [252]. It can be used to pattern multiple types of proteins and cells in a complex, discontinuous pattern on planar surfaces [53] (see Fig. 12). Protein printing Protein printing, spotting, or deposition encompasses a set of techniques that apply protein or protein ligand solutions to the chip. These techniques can be classified as either a contact or a noncontact printing method. Contact methods include several of the patterning techniques mentioned including mCP and the

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Fig. 11. Schematic representation of a laminar flow patterning experiment. (A) Top view of the capillary network. A poly(dimethylsiloxane) (PDMS) membrane containing micron-sized channels molded in its surface was placed on the flat surface of a Petri dish to form a network of capillaries. Micrographs were obtained for the area of the capillary system at which the inlet channels converge into a single main channel. (B) Two different cell types patterned next to each other. Chick erythrocytes and Escherichia coli were deposited selectively in their designated lanes by patterned flow of cell suspensions. Adherent cells were visualized with a fluorescent nucleic acid stain (Syto 9). (C) Pattern of selectively stained BCE cells. A suspension of BCE cells was introduced into capillary network (pretreated with fibronectin) and allowed to attach and spread. After removing nonadherent cells by washing with media, Syto 9 and media were allowed to flow from the designated inlets. (D) Patterned detachment of BCE cells by treatment with trypsin/EDTA. Cells were allowed to adhere and spread in a fibronectin-treated capillary network, and nonadherent cells removed by washing. Trypsin/EDTA and media were allowed to flow from the designated inlets. Pictures B and C are fluorescence micrographs taken from the top through PDMS. Picture D is a phase contrast image observed by an inverted microscope looking through the polystyrene Petri dish. White dotted lines identify channels not visible with fluorescence microscopy. Reprinted with permission from Ref. [51]. Copyright 1999 Elsevier Science Ltd.

other soft lithographic methods where a microfluidic system is used. Several more are discussed below. Noncontact printing comprises several techniques of which the inkjet is the most popular. Contact printing methods. 1. Multi-pin arrayers: Photolithographic methods have been the most common method employed in the fabrication of microarrays. Instruments used to print DNA arrays have been used with proteins. These arrayers are robotic systems that perform high-precision contact printing [118,121]. Contact printing arrayers with needle-like dispensing tips deliver sub-nanoliter to nanoliter sample volumes directly on the chip surface [125]. (This should be distinguished from mCP which uses a stamp.) In one account, with nanoliters of sample solution delivered, spot diameters of 150–200 mm are typical (1600 spots/cm2) [118]. A disadvantage is that with long printing times and small volumes of solution, the proteins dry out. One solution is to humidify the spotting chamber [118,253] and/or to add glycerol to the protein solutions [118]. A potential problem is that the chip

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Fig. 12. Patterning by using 3D microfluidic systems. Fluorescence (B) and phase contrast (C and D) pictures of two cell types, ECVs and BCEs, deposited on a tissue culture dish in a concentric square pattern by using the 3D stamp depicted in A. These cells were labeled fluorescently before being deposited. Before patterning cells, the PDMS stamp first was autoclaved for 20 min, then the channel walls were coated with BSA (30 mg/ml in pH 7.4 phosphate buffer for 1 h) for preventing cell attachment to the PDMS stamp. Suspensions of cells ( 5 million cells per ml) were introduced into the three sets of channels and were allowed to sediment and attach to the surface of the tissue culture dish. These cells were cultured with the stamp in place for  24 h to grow and spread into a confluent layer. The pictures were taken immediately after the PDMS stamp was removed; these cells were immersed under media and were alive. An expanded view of the lower right corner of C is shown in D. Reprinted with permission from Ref. [53]. Copyright 2000 National Academy of Sciences, USA.

surface may be damaged mechanically, which results in irregular spot shapes and other defects. This inhomogeneity that may result from contact pin printing may affect signal reproducibility [59]. 2. Scanning probe lithography: Scanning probe lithography is nanopatterning of SAMs using atomic force microscopy [209,210,254]. This is a form of contact printing because of local interactions between the chip surface and the tip of the AFM [254]. 3. Microcontact printing (
CP): Because an elastomeric stamp is used, it is possible to use different types of ‘‘inks.’’ Many studies involving mCP has been done on SAMs on gold [51]. A procedure is detailed in Kumar et al. [184]. Most of the literature describing this printing method was published in the 1990s

55 [184,197,216–218,228,255–257]. A novel approach is positive microcontact printing with the use of PTMP inked onto PDMS [219]. Since not all patterns on a stamp are stable or transfer properly, the use of protrusions (in þ mCP) rather than recessions (in mCP) on a stamp provides more stability. The use of PTMP also improves the wettability of the gold surface. The use of mCP to deliver hydrogel spots onto surfaces has been demonstrated [258]. 4. Stamping: Similar to putting a rubber stamp onto paper, a hydrogel stamper makes brief contact with an aminosilylated surface and deposits a protein film [212]. The stamper is a micromolded, elastic hydrogel loaded with the protein solution. The stamp prints a protein spot 10–80 mm in diameter as a submonolayer as observed from AFM (atomic force microscopy) studies and 125I labeling. Martin et al. [212] did a 4-element pattern in 2 min and complete patterning was done in 25 min. The stamping procedure can be configured for multiplex array fabrication, where stamping can be done by several stampers working in parallel. The protein loaded into the hydrogel is immobilized within the gel matrix via an amine-coupling agent. This is an advantage for the production of arrays since an immobilization step is not necessary. Noncontact printing methods. Noncontact printing comprises methods that use capillaries or inkjet technology to deposit nanoliter to picoliter drops onto the chip without touching the surface [125]. Although mechanical damage is minimized, the inside walls of the spotters should be made biocompatible to avoid protein inactivation [59]. 1. Inkjet printing: Of these ‘‘ink’’ techniques, inkjet technology is the most established and has been largely used for DNA arrays on glass slides [11,152] as well as ligand arrays [4,5]. Protein solutions are aspirated from a reservoir and less than 1 nl is spotted onto the chip to yield spot diameters of about 80 mm on polystyrene [4]. Some applications of inkjet printing in protein microdeposition include work by Roda et al. [259] and Silzel et al. [8]. 2. Piezoelectric printing: This is a variation of inkjet printing where, as opposed to back-filling, samples are aspirated from the front. The glass capillary dispensing tip has a piezo-electric collar that compresses the sample upon applying a voltage and dispenses 325-pl drops without surface contact [146,260]. An advantage with front-filling is less sample is required and there is minimal sample heating so that proteins remain active [152]. Piezoelectric printing has been used with in the production of hydrogel-coated slides [152] (see Packard Bioscience in Table 3). 3. Dip-pen nanolithography (DPN): This technique by Chad Mirkin (Department of Chemistry, Northwestern University, Evanston, Illinois) is a high-resolution patterning technique that can be used to fabricate protein arrays with spot diameters of approximately 100 nm [261–264] (see also Glossary). Molecules are transported from the AFM tip to the solid substrate via capillary action [264]. (See also Nanotechnology, Arrays: nano-lithography, Dip-pen nanolithography).

56 4. Electrospray deposition (ESD) is done on slightly conductive surfaces [265] through a dielectric grid ‘‘mask’’. The electrospray process stops as the distance between tip and chip substrate is increased about 100
m. Hence, the printing process can be automated using a computer-controlled x-y-z stage [265,266]. Spot sizes can be as small as 2–6
m in diameter but are usually  50
m under laboratory conditions [265]. Protein orientation For optimum performance and increased sensitivity, ligand molecules should have their binding sites directed away from the chip surface. This is especially important for immunodiagnostics. Immobilization of whole antibodies [267–269] and antibody fragments should be done with the proper orientation. The simplest way to achieve this is by the use of amino- or carboxy-terminal tags with recombinant proteins, e.g., with His-tags [128] or via an amino-terminal serine or threonine residue [270]. Depending on the application, it may be sufficient to orient whole antibodies by binding to immobilized protein A or G [271]. However, this is not very stable. Several methods have been developed specifically to orient antibodies and antibody fragments [272]. One method is by oxidizing and biotinylating the conserved glycosylation site on the Fc region of the antibody [9]. Another method involves obtaining the dimeric Fab0 fragments by cleaving with pepsin, reducing the disulfide bond that holds these fragments together in the hinge region, and conjugating the reduced disulfide to a thiol-reactive surface or biotin-maleimide. The resulting antibody or Fab0 fragments have their antigen-binding sites directed away from the chip surface [9]. Heterobifunctional crosslinkers have been used as well to immobilize antibodies. Densities of 74–220 ng/cm2 were achieved on glass cover slips [138]. (Assuming a 10 kDa protein in a 100 mm
100 mm spot, the spot would contain low to subfemtomole amounts, 0.74–2.20 fmol, or picogram amounts.) Carbohydrate-reactive crosslinkers bind to the carbohydrate moiety in the Fc region of the antibody. At least eight different crosslinkers were tested and no dependence or correlation could be found with length, reactive group present, or rigidity of the linkers. Compared with previous studies with thiolsilanes, a distinct difference is that these carbohydrate-reactive linkers showed higher activites. The immobilization procedure, however, is more complex and tends to cause some denaturation. Apart from ligand molecules, the protein molecules themselves can be oriented in a more random manner to enable different sides of the molecule to effectively interact with the components in bulk solution and obtain maximum detector sensitivity. In using an aldehyde silane reagent on glass slides, the aldehydes react with primary amines on the protein. Since a typical protein will have several surface lysines and have also the amino-terminal group free, the binding reactions with the aldehydes on the chip surface lead to bound protein molecules on the chip with varying orientations [118].

57 Microfluidic technologies Fabrication techniques Early microfluidic devices were used in DNA analysis [273,274] and cell sorting [275]. They were made from elastomers and, to this day, although glass and quartz are used even in commercially available microfluidics chips (see Table 3), the fabrication of polymer microfluidic devices is a growing field because of tremendous savings in time and cost compared to fabricating devices out of silicon and glass [10]. Currently, technological efforts are focused on the integration of more capabilities on chip, i.e., sample preparation and/or detection systems, high-volume production (millions of chips per year), and further developments in micro- and nanofabrication as well as in surface modifications [61]. In the fabrication of microfluidic devices, important considerations are the development time from design to working prototypes, the design and layout of the chip considering channel size and geometry, and the integration of the required components (e.g., for injection, separation, and/or detection) [10]. With polymers, microfluidic channels are formed by some of the technologies already discussed under arrays and several more to be discussed below. Two general categories are, as mentioned earlier, the replication technologies and the single-device technologies [61]. Replication technologies include molding, embossing, and casting (discussed above) as well as the use of SAMs and soft lithographic techniques (discussed below) [10,61]. As basic principles in patterning with soft lithography, rapid prototyping and replica molding are also used for microfluidic systems [10,65,154,216]. The single-device technologies include laser ablation and photoresist technology (discussed above), as well as stereolithography and layering techniques (discussed below). Techniques for fabricating and patterning 3D structures are discussed in Reyes et al. [294] and in Blawas and Reichert [116]. Soft lithographic methods for microfluidics. 1. Rapid prototyping: Rapid prototyping [65,154] combines high-resolution commercial printing, photolithography, and soft lithography to produce a master at a fraction of the time and cost that it takes to produce one by photolithography using a chrome mask (see also Glossary). Instead of a chrome mask, a high-resolution transparency is used as a photomask for the fabrication of the master by contact photolithography. The resulting master is a positive relief (protrusions) of SU-8 photoresist on a silicon wafer. For extended lifetimes, the master can be made of a single piece of hard polymer (structural polyurethane or epoxy) [10]. The major difference in resolution, >20 mm features against  500 nm for a chrome mask, can be improved with the use of image setters with >3386 dpi resolution. If features <20 mm are required, a chrome mask should be used instead. The channels formed have cross-sectional diameters in the 50–100-mm range. The resulting rough edges on two walls do not affect the performance

58 of a 50
50 mm channel, when compared to separations done with fused silica capillaries [10]. 2. Replica molding (casting): This method involves fabricating channels from bulk polymer. The technique involves using the positive master and creating a negative PDMS replica by casting the prepolymer. After an hour of curing, the PDMS replica is cooled and peeled off the master. Replica molding can be used with any master fabrication method [10]. Since the PDMS replica would have three walls after casting, a fourth wall then seals the channel. This fourth wall is a flat material that can be PDMS or some other material. Sealing can be done reversibly by conformal contact or irreversibly by sealing onto certain substrates upon exposure to air plasma [277] (see also Glossary). Reversible sealing is due to van der Waals interaction between PDMS and the material of the fourth wall. Although it is watertight, the seal will not hold greater than 5 psi pressure in the channels. Irreversible sealing is due to the covalent bonds formed upon exposure to the plasma. For example, when PDMS is irreversibly sealed against glass, Si–O–Si bonds are formed with the loss of water. The seal can withstand 30–50 psi. Duffy et al. [65] list the different materials that will work or not work with this plasma sealing technique. Plasma oxidation serves another purpose. PDMS is hydrophobic, difficult to wet, and to easily nucleate bubbles. Plasma oxidation transforms the PDMS surface to a hydrophilic one with exposed silanol groups. Although the adsorption of hydrophobic and negatively charged proteins is minimized, some still do adsorb [65]. An advantage is that these silanol groups support a cathodic EOF upon interacting with ionized or ionizable species in neutral or basic solutions. Hence, it is useful for capillary zone electrophoresis (CZE). This oxidized surface, however, is stable for only about 30 min and reverts back to its hydrophobic state [10]. Nevertheless, the oxidized PDMS surface can be modified by adsorption of charged and neutral polymers [65] or covalent attachment of trichlorosilanes [277,278]. Single-device techniques for microfluidics. 1. Stereolithography: Stereolithography is a method that requires no masks or steps aside from the laser time required to ‘‘draw’’ the structures on the chip (see also Glossary). The technique starts with the liquid polymer and as the focused laser hits the polymer surface, the polymer at the focal point cures and solidifies. The fabrication of a microfluidic device using this technique has been described [279]. The features of the chip are drawn on CAD software and this controls the movement of the laser and the chip container. Although the technique fits the label of rapid prototyping, a chip can take several hours to fabricate, depending on how many features need to be defined on the chip [61]. Stereolithography can be used to fabricate 3D structures. 2. Layering: The technique has been described by Carlos H. Mastrangelo and his group (Department of Electrical Engineering and Computer Science,

59 University of Michigan, Ann Arbor MI, USA) [280–282]. Thin layers of different polymers define the chip including a sacrificial layer in between that is etched away to define the channel [61] (see Fig. 13). Parylene can be used to define the channel walls and cover. Metal can be deposited onto the parylene by sputtering or evaporation to define electrode structures. The etchant, usually acetone, enters the chip via holes and carry with it dissolved material. The etching process can last for an extended period of time because the transport of etchant is done only by diffusion [61]. A similar technique uses multilayer structures on flexible rubber chips and is referred to as multilayer soft lithography. The interesting aspect of this proprietary technology is that miniature pumps and valves are built on the chip [11,38,67] (see Fluidigm in Table 3). Manufacturing technologies. Besides forming microchannels, a set of methods is employed to seal them and put in other structures such as through holes and electrodes. Bonding techniques such as lamination, gluing, applying heat

Fig. 13. Diagram of the fabrication process for layered structures. Reprinted with permission from Ref. [61]. Copyright 2000 Wiley-VCH Verlag GmbH & Co. KGaA.

60 and pressure, and welding, and other manufacturing steps including dicing and electrode fabrication are discussed in Becker and Ga¨rtner [61]. Microfluidic functions: fluid flow and control There are several methods of actuating flow based on principles such as electric field, pressure, and surface tension. A number of these techniques are discussed below. Apart from fluid transport, another important aspect in operating microfluidic systems is flow control. Various techniques exist from microvalves and the use of hydrogels and hydrodynamics to valveless flow switching and virtual walls. The integration of other components on the chip, especially optical components for detection, and interfacing to other instrumentation systems are discussed in the next section, Principles of Design and Operation. Fluid flow. Fluid flow in microchannels can be controlled via different principles. Some give only localized control, while others influence the entire fluidic system in the device. In a complex microfluidic system, the active control and switching of fluid flow is crucial. In other words, it should be possible to change flow rates at different stages of the experiment and at different points within the chip. This can be achieved with actuation principles that directly operate on fluids, such as electroosmotic force (EOF) and electrocapillary pressure, as opposed to local electrostatic generation of pressure or the use of centrifugal force [38]. However, more active control can be achieved if the fluids on the chip can be properly controlled by a system of pumps and valves as it is done in the macro world. These methods are discussed or cited below. For both EOF and pressure pumping, the use of PDMS as a chip material has distinct advantages. Its surface can be charged and, when sealed irreversibly, can withstand high pressure. It is also easy to incorporate membrane pumps and check valves because the material is elastomeric [10]. A number of actuation principles involving the induction of surface tension differences will also be discussed. 1. Electroosmotic flow: The early development of microfluidic chips in the 1990s can be attributed to the poor performance of microfabricated pumps. The early microfluidic devices used electrokinetic or EOF control. To this day, many microfluidic devices transport fluids in this manner [12]. A fluid transport method used for both soft and hard materials [283], electroosmotic pumping was first demonstrated by Harrison et al. [23]. Molecules pick up a charge from the channel walls and are driven through the channel via an applied voltage. The applied fields are high (over 2 kV/cm) [18]. Microfabricated electrodes made of platinum, chromium, or gold wires are located on the channel walls, e.g., at the interface of the polymeric substrate and the glass cover plate [38,284]. Gases generated electrolytically at the electrodes are removed by permeation through polymeric substrates. Furthermore, the flat flow profile, as opposed to a parabolic one in pressure systems, results in less band broadening. Therefore, besides simplicity in design and ease of operation, separation efficiencies are also

61 improved [73]. Variations to EOF have also been described. Electroosmotic hydraulic pumping has been demonstrated where fluid flow relies on the applied voltage and not the field strength. It is also independent of the entire channel length that is electroosmotically pumped [284]. Opposite flows can be generated with flowFET, EOF with field-effect flow control, analogous to a field-effect transistor (FET) [285]. This illustrates the potential of flow switching in microfluidic systems using EOF. The use of EOF in contrast to pressure-driven flow has several advantages: experimental simplicity, maintaining fluid flow without mechanical parts (pumps or valves), and minimal back pressure. The presence of an applied field already assists applications involving electrophoretic separation [41]. But it can also be a disadvantage in some cases. Electric fields are an important means of molecular separations; however, they pose some difficulties as a method for manipulating fluids. Pumping heterogeneous solutions causes ‘‘electrophoretic demixing’’ [283]. Changing solution conditions such as buffer composition or salt concentration requires some readjustment of the voltage applied. Thus, depending on the experiment, difficulties may arise from having to reset voltages to adjust for changes in ion concentration, unequal pressures, and evaporation [16]. Voltage control is adequate for a simple one-junction system but in more complicated systems with several switching junctions, voltage adjustments to compensate for different pressures and resistances throughout the device will be difficult [62]. 2. Manetohydrodynamics (MHD): A pumping method developed by Ingrid Fritsch (Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR, USA), magnetohydrodynamics relies on electric and magnetic fields to ‘‘push’’ fluids down the channel. Sample solutions are spiked with redox species such as ferric cyanide or nitrobenzene. When the fields are applied orthogonally to the direction of flow, the redox species move down the microchannel and drag along the rest of the fluid, effecting a flow. Typical flow velocities are up to a few millimeters a second, comparable to EOF. Voltage required is about one volt and the magnetic fields are ‘‘moderate.’’ Nonaqueous solutions (e.g., dichloromethane and ethylbromide) can be used which is useful for on-chip organic synthesis [38]. Lemoff and Lee [286] performed AC magnetohydrodynamic pumping where solution is driven along a circular silicon channel. 3. Electrocapillary pressure (ECP): A technique developed by Menno W.J. Prins (Philips Research Eindhoven, Eindhoven, Netherlands), ECP is a means of moving fluids through a channel by controlling the position of a fluid–fluid interface (see Fig. 14). Electrocapillary pressure refers to the voltage-dependent pressure on the meniscus of a fluid in a microchannel. The position of this meniscus is controlled by a localized force that is generated by the voltage on the electrode that is embedded in the channel walls, giving the method a high spatial resolution in terms of actuation [287]. Fluid transport is sustained by a proper sequence of voltages. It should be distinguished from electrowetting because it is

62

Fig. 14. Electrocapillary pressure. (A) Sketch of fluid in a microchannel. The fluid at the bottom (shown in gray) is electrically conducting (an aqueous salt solution). The top contains an insulating fluid (either air or a nonpolar oil). A voltage V is applied to the electrode; the aqueous solution is at ground potential. The pressures at the bottom and top openings of the microchannel are expressed as p1 and p2, respectively. The angle between the gravitational force and the long axis of the microchannel is given by . (B) Close-up of the meniscus edge. Inside the microchannel wall, a 20nm-thick aluminum electrode is present, separated from the aqueous solution by an insulating layer of thickness d. The insulating layer consists of an 11.5-
m thickness of parylene (deposited by chemical vapor polymerization of di-p-xylylene) with a 10-nm fluoropolymer coating (AF1600 of DuPont, deposited from solution). Because of the applied electric field, opposite charge densities are induced at the fluid/solid interface and in the subsurface electrode. The electrostatic attraction between the aqueous solution and the electrode enhances the coverage of the wall by the aqueous solution. Reprinted with permission from Ref. [287]. Copyright 2001 American Association for the Advancement of Science.

independent of the contact angle with the applied voltage [287]. It should also be distinguished from electrocapillarity in the classical mercury/electrode system where all fluids are electrically conducting [287]. ECP is electrostatic in nature with very low charge densities (104 C/m2) and displacement currents; electrochemical reactions and electrolytic bubbles are absent. Consequently, power consumption is very low. This pumping method does not rely on mechanical valves or pumps and is suitable for microfluidics applications that require ‘‘repeated low-power operation.’’ But as with electrokinetic flow, the surface chemistry in the channels has to be well controlled. The smaller the dimension of the channels, the greater is the influence of, and problems associated with, surface effects [38]. Because of precise control, flow switching is possible as well as guiding fluid flow through a 3D-channel network [287]. Fluid velocities can be several centimeters per second, almost 2 orders of magnitude higher than other methods for electrofluidic actuation [285,288]. 4. Thermocapillary pumping (TCP): Given a nanoliter- or picoliter-sized drop, TCP involves heating one end of this drop to create a surface tension difference with the opposite end of the same drop. This surface tension difference causes a

63 capillary pressure difference and induces motion. Typical TCP velocities run up to 20 mm/min (approximately 300 mm/s). References on this technique are Kataoka et al. [289] and Burns and Troian. [290]. 5. Electrochemistry: Fluids can be actively controlled electrochemically with the use of redox-active surfactants. Surfactant species are generated at one electrode and consumed at the other. Electrochemical control of the position of these species then controls fluid motion [288]. 6. Pneumatic actuation: Valves fabricated from elastomers can have precise fluid control over a wide range of conditions [62,292]. They can attain complete valve sealing, have fast response times, need low actuation forces, and have a small footprint. The first devices occupied an area of 100 mm
100 mm and now the valves’ working area has been reduced to 20 mm
20 mm. A peristaltic pump results from having three consecutive valves in a row. Flow rates are up to a few nanoliters per second, compatible with that which is required for assays, and dead volumes of both valves and pumps are negligible [62]. Mechanical pumping can transport and mix liquid samples [291,292]. Pressure-driven flow will allow the use of more types of solutions in microfluidic devices since it is less sensitive to solution properties than EOF [67]. 7. Light-driven fluid motion: Asymmetrical irradiation of a substrate surface with ‘‘photochromic azobenzene units’’ causes a gradient in surface free energy due to azobenzene photoisomerization. The direction and velocity of fluid motion is determined from the direction and steepness of the light intensity gradient [293].

Fluid control. A number of systems for flow control exist. A review by Reyes et al. [294] provides extensive literature coverage on this topic. Below, some techniques developed to control fluids, with or without the use of valves, are described. 1. Pneumatic actuation: The same principle discussed above in Fluid Flow is basically a fluid control device. A valve is created by introducing gas pressure (air or nitrogen) into a control line, which in turn deflects a thin membrane downward to seal off a channel. This downward sealing motion is made possible by having a two-layer system; all microchannels wherein sample flows through are in the bottom layer and the control lines for pneumatic actuation are in the top layer [16]. 2. Passive microvalves: Fluid control or control of the flow sequence is achieved by a network of passive microvalves (or restrictions). The valves can be modified according to the channel geometry and the sequence of fluid delivery is programmable [295]. 3. Valveless switches: One type of a valveless switch is the principle of dielectrophoresis. Dielectrophoresis is particle motion caused by polarization effects in nonuniform electric fields, i.e., AC rather than DC fields as in the case of electrophoresis [12,296–298]. In sorting mammalian cells, dielectrophoretic field cages and switches can trap and direct particles, respectively [299]. Another

64 method is pressure switching [300]. Both techniques were used as a valveless switch to separate cells and other particles in microchannels [16]. 4. Hydrodynamic flow control: Chien and Parce [301] designed a chip with hydrodynamic flow control. An algorithm and a strategy for calculating pressures were developed to control flow from multiple reservoirs via a multiport system on the chip. Its application in a study of enzyme kinetics was straightforward and reproducible and used only pressure-driven flow. The response was linear with a dynamic range of over 2 orders of magnitude. The key was an appropriate network structure to apply the proper pressure control [41]. 5. Self-regulating flow switch with hydrogels: Depending on the pH of the solution, hydrogels can either swell or shrink. The response time for this speeds up tremendously in micrometer dimensions to less than 10 s [302]. Stable hydrogel jackets were designed around prefabricated posts that provide support as well as volume, allowing a short diffusion path to speed up both sensing and actuating functions. This behavior is taken advantage of as self-regulating shut-off valves to stop fluid flow in particular microfluidic channels, without any external controls or circuitry. Response times, however, were much slower than mechanical valves [62]. Changing the hydrogel composition can modify the pH behavior. Furthermore, hydrogels have other characteristics besides pH sensitivity that may enable other functional components [302]. 6. Virtual walls with surface-directed flow: Fluid flow can be directed by patterned surfaces or polymerized coatings on a chip. At low pump pressures, fluid flow is confined to adherent surfaces and will avoid the hydrophobic nonadherent regions. Hence, it is possible to set a flow path without valves or channel walls and without the need of electrical power [38,291]. The chip surfaces or channel walls are treated with SAMs either by multistream laminar flow (laminar flow patterning) or photolithography. Multistream laminar flow required preformed channels where the depth used was  180 mm. Photolithography was performed with 2-nitrobenzyl photochemistry to have hydrophilic carboxylate groups in the exposed regions [303]. SAMs can be used to modify the surface wettability [291,304–306] and can be formed on substrates such as silica in minutes. Contact angle measurements showed complete coverage of a glass substrate with SAMs of trichlorosilanes (OTS and HFTS) in less than 2 min [291]. One function that surface-directed flow can perform is the concentration of samples on chip [291]. With a wide choice of materials and technologies, hybrid devices in microfluidics may likely be developed [38]. Different materials have different desirable properties, e.g., glass for optical windows and plastics for channels. Different technologies have different advantages in achieving a given end product with respect to fabrication time and features that can be built on the chip: microstructures with replica molding and 3D structures with stereolithography. For fluid manipulation and control, there are numerous techniques, some of which have been developed probably only within the last 3 years. It will be some time before the value of some of these technologies can be properly assessed. Furthermore although certain techniques may be robust enough, they are

65 proprietary technologies of commercial entities. And with their products still under development, it will be a while before one can pass judgment on winning combinations. Nevertheless, the enormous interest and research in the field will only benefit innovation and progress. The potential applications of microfluidic devices are outlined in McDonald et al. [10] and a two-part review on mTAS from Manz’s group provides more than 600 references on technologies and applications [294,307]. Nanotechnology Progress in the area of biochips has extended to nanotechnology. Both array and microfluidics chips are benefiting from technologies that can write nanometer features. Although producing these nanometer features may be more timeconsuming and expensive, this may apply only for fabricating the master or mold tool and, hence, should not deter their commercial or industrial use. The resolution of replication technologies is determined by the size of the features on the mold or master [62]; replica molding or casting has been used to replicate nanometer features. Mold replication would allow mass production of the device, which would offset the cost of fabricating the master. Nanoscale molecular tags, i.e., nanobarcodes and nanocrystals, are discussed under Principles of Design and Operation. Arrays: nanolithography Lithographic techniques that write nanometer features can collectively be called nanolithography. Several techniques used in fabricating nanoarrays are mentioned below. Zhao et al. [217] discusses soft lithographic methods for nanofabrication. Nanopatterning, along with three-dimensional patterning, has been a growing interest since the late 1990s [116]. Electron beam lithography (EBL). Although it is a time-consuming and expensive process, electron beam lithography can be used to make nanometer features on a master [62]. Certain chemicals change their properties when hit with an electron beam. With computer control of the beam position, structures can be written in the range of up to about a hundred nanometers [308,309]. Dip-pen nanolithography (DPN). Using DPN, arrays were fabricated with 100–350-nm features. Optimization should reduce this closer to 10 nm, the limit of conventional DPN [262–264]. Molecules transported via capillary action from the AFM tip to the substrate are chemisorbed [264] (see Fig. 15). An important feature is that there is almost no detectable background due to nonspecific binding (NSB) and the chemisorbed proteins remain biologically active [261]. An array was fabricated by patterning 16 mercaptohexa-decanoic acid (MHA) on a gold thin-film in the form of dots or grids. Protein non-adherent regions were treated with 11 mercaptoundecyl-tri(ethylene glycol) [261]. AFM recognised binding interactions when it detected a doubling in height indicating

66

Fig. 15. Schematic representation of DPN. A water meniscus forms between the AFM tip coated with ODT and the Au substrate. The size of the meniscus, which is controlled by relative humidity, affects the ODT transport rate, the effective tip-substrate contact area, and DPN resolution. Reprinted with permission from Ref. [264]. Copyright 1999 American Association for the Advancement of Science.

the presence of 2 proteins. It is interesting that under these nanoscale conditions, whatever the orientation of the IgG relative to the chip surface was, it reacted with the anti-IgG in the protein solution. (See also Array Technologies, Protein printing, noncontact printing methods.) Scanning probe lithography. Based on atomic force microscopy, scanning probe lithography is an alternative to photolithography in terms of fabricating structures less than 0.1 mm (100 nm). SAMs have been nanopatterned by scanning probe lithography [209,210,254] (see also Array Technologies, Protein Printing, Contact printing methods). Finely focused ion beam lithography (FFIB). FFIB is a short wavelength lithographic technique using 30-keV indium ions to write small features (defect pits) on a gallium arsenide surface having 60-nm diameters with 185-nm spacing between them [310]. Adsorption of human serum albumin was limited to the inside portion of these pits, as seen on TM-SFM (tapping mode scanning force microscopy) images. Hence, the spatially controlled adsorption of proteins was achieved on a nanometer scale. Since no such adsorption was observed with silica, the choice of material is important. It is speculated that even single molecules can be adsorbed. In wellordered arrays where well-defined lines or defects are created, the scanning probe tip can easily go or return to specific locations on the chip to within a few nanometers [310]. Potential applications therefore are the study of specific protein adsorption behavior as well as protein–protein interaction studies on single molecules [310]. Nano-imprint lithography (NIL). This embossing technique developed by Stephen Y. Chou and his group (Department of Electrical Engineering, Princeton University) can write nanometer features [62,311,312]. Relief patterns in a thermoplastic resist layer can be created by simply compressing it after it has been thermally softened [313]. However, problems occur in trying to get a clean release of the resist from the mold and the resulting imprinted structures become

67 distorted. With room-temperature NIL (RT-NIL), compression leads to the freevolume contraction and plastic deformation of the polymer [314]. Amorphous polymeric material has vacancies called free volume such that a polymeric film with a thickness of 100 nm can be compressed to a depth of 10–30 nm solely from compacting or collapsing the free volume. Beyond free volume compaction, further imprinting can be done through plastic deformation (see Glossary). S-layer proteins. Discussed under Materials, these cellular protein layers have a repeating pattern of nanometer dimensions. The S-layer proteins of Thermoproteus tenax, for example, have hexagonal channels that are 6 nm in diameter [315]. In one application, the S-layer was used as a template to create metal masks [316]. Microfluidics: integration of optical elements and nanostructures Integrating optical elements. Optical signals resulting from biochemical assays or other chip experiments need to be captured and recorded. The best solution would be to have light going in and out of the microfluidic devices and to have the necessary optics near the flow channels. Optical elements such as lenses and beam splitters can be fabricated within glass or elastomers [62]. Fine structures such as narrow line gratings and pillar arrays have been made with elastomers by embossing and replica molding [62,311,317]. Diffractive lenses can be defined on PMMA resist with subwavelength features by using electron beam lithography, rather than photolithography, followed by replication in quartz or silicon by using the PMMA mask in an ion etching process [62,318]. Silicon molds can be even etched at a faster rate with XeF2 and results in high-resolution features as small as 50 nm [62,319]. Some replication guidelines are given by Quake and Scherer [62]. Nanostructures. Some of the same technologies used for arrays such as electron beam lithography (EBL) can be applied to microfluidics to create nanostructures. EBL and highly anisotropic pattern transfer have been used to construct nanostructures with lateral dimensions as small as a few nanometers [62,320]. With this high-resolution lithography and anisotropic ion etching, nanopillars that were 6 nm in width and lines 10 nm in width were fabricated [62,321]. The limit in developing nanodevices is in constructing molecular nanostructures using single molecules as templates [62]. Examples include singlemolecule nanobridges in silicon and, out of single DNA molecules, nanowires less than 10 nm thick and nanoresonators with predicted gigahertz frequencies [62,322–324]. It would be interesting to see where all this will play a role in the biological and medical fields.

Principles of design and operation This section on principles of design and operation provides the logic behind the tools, the rationale behind the choice of which materials and methods

68 make sense in any one particular chip application as well as discusses the other aspects involved in operating a biochip, e.g., the detection system. This section is divided into a discussion on arrays and microfluidics. A separate section is dedicated to cell and tissue biochips because there are inherently different issues to be addressed as well as a different set of required conditions for operation.

Arrays: capture agents, molecular tags, detection Generally, a key element in microarray is the occurrence of a binding event, e.g., antigen–antibody interaction. As such, there are many situations involving all ligand-binding events where a microarray format is applicable [125]. How many samples constitute a microarray? This number can vary greatly from less than 1000/cm2 [324] to a range of 1600–1800 protein spot cm2 [118,128]. Another example is a dipeptide array with over 250,000 elements/cm2 [166]. But in comparison, an array of cDNA sequences can be over 400,000/cm2 [325]. In the final analysis, the relevant issues still are preserving protein activity and detector sensitivity. The protein should be active in order to take part in a binding interaction. And when this interaction occurs, it should be detectable. Protein concentrations should be within detection range. For proteins to be immobilized on arrays MacBeath and Schreiber [118] observed saturated fluorescence signals above 1 mg/ml and kept the concentration at 100 mg/ml. Hence, for high-throughput applications, a microliter of sample may be enough to spot a whole array. Proteins in free solution, however, need to be at much lower concentrations. Sample concentration at the lower limits of detection is a more critical issue. Specific binding of Cy5-FKBP12 (Human immunophilin) could be detected at 150 pg/ml ( 12.5 pM) [118]. Ekins et al. [125,326–329] quantified proteins such as thyroid-stimulating hormone (TSH) and Hepatitis B surface antigen (HbsAG) down to femtomolar levels or the equivalent of 106 molecules/ml. For high-throughput applications, a microliter of sample may be enough to spot a whole array [118]. But for detection systems, although there may be inherent sensitivity, background interference should be minimized (see Array detection systems, Nonspecific binding). Assuming one already has a large library or repertoire of binders to target proteins in the sample and the goal is to simultaneously identify and quantify different types of molecules and interreactions, the choice in strategy is between immobilization via a capture agent or molecular tagging [330]. Immobilization involves keeping one set of molecules bound at specific sites on an array and the other free in solution to react with it. Although techniques to orient protein molecules exist (see Microfabrication of chips, Technologies, Array Technologies, Protein orientation), there is still the possibility and limitation that an interaction site on a bound molecule may be blocked and inaccessible. Molecular tagging, on the other hand, allows molecules to be unconfined. However, methods of identification and detection of the tag and the interacting

69 partner become more complicated. Both strategies are discussed in the following sections. Capture agents Capture agents need to be stably positioned on the chip. They must also be able to bind their target proteins present in nanomolar or even picomolar solutions [8,59,331]. Capture molecules include antibodies, antibody mimics, and aptamers [79,125,146,331]. Antibodies. Antibodies are the most prominent type of capture molecules. However, because monoclonal antibody production is labor-intensive, the development and use of alternative methods become imperative [125]. Antibody arrays have since been made [332–339], some using an scFv antibody library [335,340–347] isolated by phage display [332,333]. Monoclonal antibodies are known to have high binding specificity and be stable enough to last several weeks [348]. Nevertheless, recombinant scFv are relatively stable. Immobilization of the antibody on the chip surface can be done via reductive amination, direct avidin– biotin attachment, via epoxide linkers or by immobilization to the GOPS (3glycidoxypropyldimethyl ethoxysilane) film [72,136,349]. The last method, which involves direct, covalent coupling, minimizes losses and is straightforward when compared with avidin–biotin coupling [72]. Antibody mimics. A disadvantage with antibodies is that they tend to denature with heat or pH changes. Another limitation is that sandwich assays, which require a second antibody, may give unreliable results when the antibodies bind to nontarget proteins. Alternative capture molecules are other protein scaffolds or antibody mimics [125,146,331]. They are more stable than antibodies, are smaller, and can be synthesized chemically. A 100-amino-acid domain of human fibronectin has been used as a pseudo-antibody. The variants are called trinectins and a proprietary mRNA display is performed on a library size of 1013 [350]. Another scaffold, an affibody, uses a 58-amino acid domain of a staphylococcus surface protein where the binding surface is 13 amino acids with IgG molecules [351]. The library size is claimed to be as large as 1016 in theory whereas phage display can only accommodate about 1011 members [334,335]. Affibodies, from heterologous expression of microorganisms [125,352,353], are labeled with 2 fluorophores such that when they bind their target protein, the labeled segments move away from each other. This results in a change in fluorescence thereby signaling a binding event [354]. This phenomenon has been termed fluorescence resonance energy transfer (FRET) [57,354,355] (see also Array detection systems). Aptamers. Aptamers are single-stranded nucleic acid structural units that have been used as capture molecules [125,334,356–359]. There are also peptide aptamers [125,361]. A key advantage with their use is that they are synthesized in vitro and hence can be chemically modified efficiently [356]. Single strands

70 of DNA can mimic antibodies as they can assume three-dimensional shapes. Selection is done via SELEX (systematic evolution of ligands by exponential enrichment) [356,360] (Brody et al.; Brody & Gold 2000) or mRNA display [125,362]. It should be noted that RNA aptamers are susceptible to nucleases in complex protein mixtures [344,363]. A photoaptamer system has been developed where thymidine has been replaced with bromodioxyuracil in a 1015 DNA library [360]. In this system, aptamers are covalently attached to the chip and target proteins in the sample bind to their respective aptamers on the chip. Upon exposure to UV light, the captured target proteins are cross-linked via the bromodioxyuracil. After washing unbound proteins, detection can be done via a general protein stain. This is a particular advantage since aptamers are not protein molecules; hence, the readout can be reliable and rapid. In contrast to DNA microarrays, fluorescent labeling will not work in this case because different proteins take up the label to different extents [331,360,364]. Templin et al.[125] presents a table of capture molecules including their source, the techniques used to produce them, and corresponding references. Other capture molecules not discussed here include synthetic receptor ligands generated by combinatorial chemistry [365,366], enzyme substrates [118,136,137], and the potential use of molecularly imprinted polymers (MIPs) for affinity capture [344]. Molecular tags Radioactive or fluorescent labels are most commonly used in detecting binding interactions [80,344,367] (see also Array detection systems). But with fluorescence measurements, specificity can be a problem. Organic dye molecules (e.g., fluorescein, rhodamine, and cyanine dyes) and light scattering particles such as gold or silver have broad spectra making it impossible to generate unique tags for different types of molecules within a useful wavelength range [330]. Alternatives include semiconductor crystals called quantum dots and metallic bar-coded rods called nanobarcodes. Quantum dots. Quantum dots are nanocrystals of semiconductors (e.g., calcium selenide) that have much narrower spectral bandwidths [368–371]. They emit light at a number of precise wavelengths and the exact color depends on dot size. One advantage is stability; the dots can withstand hours of repeated cycles of excitation and fluorescence, compared to conventional dyes that last only minutes. The other advantage is activation of many colored dots using a single laser; several single wavelength activations of conventional dyes would imply the use of multiple lasers, where the cumulative energy would damage biological material [368,370–372]. They are an important innovation in cellular imaging and biosensing. Their applications are in screening drug leads and in providing a rapid readout for proteins present in a cell or tissue. These dots have been linked to antibodies to label target proteins in human cancer cells [370]. Currently under study are the extension of their excitation wavelength range beyond the visible

71 (trying indium arsenide), the mechanism by which cells take up the dots, and their surface chemistry (increase or change their binding specificity; make them stable, resistant to oxidation and high salt concentrations) [368] (see also BioCrystal and Quantum Dot in Table 4). Nanobarcodes. Nanobarcodes are metallic rods 0.5 mm in diameter and about 10 mm long with electrochemically deposited thin metal stripes. Rods have been made with as many as 13 distinguishable stripes using seven different metals (Pt, Pd, Ni, Co, Ag, Cu, Au) in 10 nm to mm segments. With two metals, 4160 unique barcodes are possible; with three, the number increases to 8.0
105 distinct stripes [373]. Since derivatization chemistry for metal surfaces is well developed, the rods are attached to a variety of sample molecules via a number of techniques [373]. The barcodes are read by reflectance microscopy and the analytes are detected by fluorescence or mass spectrometry [373]. Hence, theoretically, many types of bioanalytical assays can be performed in a multiplex fashion. There are, however, technical challenges to be overcome: the making of these nanobarcodes and the attachment to analyte molecules can be automated but thus far is not a multiplexed or highly parallel process. In addition, the readout of a system with multiple barcodes has not yet been demonstrated. To be able to read thousands of different bar-coded samples is a complex image acquisition and pattern recognition problem. After an assay, the rods are placed on a flat surface at a low enough density and should overlap only to the extent that the coding information is not obscured. Decoding requires imaging resolution approaching the wavelength of visible light [330,373]. In a sandwich immunoassay, Nicewarner-Pena et al. [373] showed that barcoded human IgG and rabbit IgG were analyzed simultaneously with secondary antibodies given specific fluorescence labels. The reflectivity image was enough to distinguish the two different IgG molecules so the experiment could have been performed even with only one fluorophore (see Nanoplex in Table 4). DNA labels. Co-tagging beads with short pieces of DNA is another labeling strategy termed ‘‘encoded synthetic libraries.’’ Since DNA sequences in large numbers can be made quite easily, this method does not have the limitations associated with spectral coding [374]. A similar approach is being used commercially [330].

Array detection systems In array systems, the detection of binding interactions or of specific molecules themselves depends highly on their abundance and on the binding affinities [375]. Since the range of concentrations of different proteins in a biological sample can be several orders of magnitude, dynamic range is an important factor in the choice of a detection system. The other factor to consider is quantitation. Firstly, the amount of deposited protein in a microsport may often be only femtograms, thus extremely sensitive detection will be required to detect them and their interactions [59]. Secondly, for the quantitation itself, some important issues are

72 the chip surface itself that has to be structurally well-defined and inert to nonspecific protein adsorption and the method of protein immobilization that should be generally applicable to most polypeptides but with adequate selectivity [143]. Signal intensities can be increased with proper site-specific orientation of molecules on the chip, especially with antibodies (see also Array Technologies, Protein orientation). This should give a higher binding probability than with conventional ELISA [59]. Nevertheless, signal intensities from binding interactions should be relatively quite high because the reactions are confined to a small area where there is a high local concentration. Spot size and spot density are responsible for the optimal signal level in miniaturized systems [125]. In a two-color labeling procedure, Haab et al. [375] detected different concentrations of captured antigens in the picomolar range. A few nanoliters of a picomolar solution correspond to femtogram amounts on the chip. In a peptide example, high local densities reduced nonspecific binding and permitted the detection of low-affinity interactions [376]. Nonspecific binding. Nonspecific binding (NSB) effects should be addressed especially in complex biological samples such as serum or whole blood [72]. Approaches to reduce NBS effects involve the following: signal amplification, sample dilution, surface-blocking treatments, and signal referencing. All except signal amplification involve reducing background noise. Sample dilution is not an option. Signal referencing aims to cancel out NSB signal either by direct optical interferometric referencing or indirect electronic background substraction, which may lead to signal degradation due to the loss of phase information [72]. In practice, there are several ways to reduce nonspecific binding. Jones et al. [69] used a monolayer of octadecanethiol (ODT); its hydrophobicity minimizes the NSB of protein material to the grid regions. Many have reduced nonspecific binding by introducing a monolayer of bovine serum albumin (BSA) on unspotted surfaces of the array chip [81,116,118]. In order not to obscure peptides and small proteins, BSA-NHS (BSA-N-hydroxysuccinimide) can be used instead [118], detailed protocols are given in Science Online (http:// www.sciencemag.org/feature/data/1053284.shl). Mendoza et al. [74] blocked nonspecific binding sites by preincubating with Blocker Casein in PBS. In peptide arrays, the SPOT synthesis method, which involves the derivatization of Fmoc--alanine groups by hydroxyl groups on cellulose, reduces nonspecific interactions [377,378]. Several have resorted to a monolayer presenting poly(ethylene glycol) (PEG) groups in the background regions of the chip to make them nonadherent to proteins [51,71,72,116,143]. Background signals are nearly zero due to the absence of nonspecific adsorption [143]. In experiments that involve binding with scFv antibodies, detection sensitivity can be improved via scFv multimerization [379,380] and signals can be amplified with the use of biotin tyramine [381,382]. Detection with labels. In line with the foregoing sections on capture agents and molecular tags, detection systems should address the fact that the molecules have

73 labels or are label-free. For systems involving molecular tags, the most widely used detection method is the measurement of fluorescence. The analytes can be fluorescently labeled directly or indirectly via labeled secondary antibodies in a sandwich format [59]. Alternatively, the incorporation of fluorescently labeled nucleotides during replication of template DNA provides increased signal intensity in a technique called immunorolling circle amplification. Other options for label detection methods include luminescence, chemiluminescence, and radioactivity [59,125]. Fluorescence detection is done using charge-coupled device (CCD) cameras or laser scanners with confocal detection optics [125]. Confocal laser imaging with full quantitation is available commercially [383] (see Umedik and BioMedical Photometrics in Tables 3 and 4, respectively). Software solutions that do image analysis and classify results are also available [1]. A variation in fluorescence detection is fluorescence resonance energy transfer (FRET) with affibody capture molecules that are engineered with two tags, a donor and an acceptor fluorophore. When the acceptor fluorophore is in close proximity, as when binding occurs, the fluorescence emission of the donor disappears as it transfers energy to the acceptor and the resulting emission is at a longer wavelength. The appearance/disappearance of fluorescence emission at the two different wavelengths can then be monitored [57,354,355,384]. An advantage of this technique is that because the readings from two wavelengths are ratioed, signal artifacts, noise, and random errors are minimized. Such a detection scheme is useful for cell-based or biochemical assays and was the basis for a commercial high-throughput screening system [57] (see Aurora Bioscience in Table 3). With fluorescent labels an improvement in sensitivity was observed with waveguide technology [119,385] and an even greater improvement with planar waveguide technology [385,386] (see Fig. 16). Sandwich assays, such as enzyme-linked immunosorbent assays (ELISAs) are an alternative to the detection of molecular labels. Since different samples can take up molecular labels to different extents, this variability is not a factor in sandwich assays where no labels are required [9]. Joos et al. [121] had a microarray ELISA for immune diagnostics and detected 40 fg of a protein standard. Because two different reagents are needed to recognize the protein, the specificity of the method is higher [9]. The use of ELISAs for microarrays, however, is met with several criticisms [9]. One is that the method may not be robust enough because each protein spot on the chip requires two binders. It is very difficult to make different ELISAs work under the same conditions on one chip [79]. Mendoza et al. [74] performed high-throughput ELISA. The chip had a 96-well format on optically flat glass plate with a black Teflon mask; each well had 144 elements made up of four identical 6
6 arrays, each 6
6 array having eight antigens at least in duplicate along with markers and blanks. Each antigen’s monoclonal antibody was biotinylated while detection was done with alkaline phosphatase-streptavidin conjugate and ELFÕ (Fluorescent alkaline phosphatase substrate). In such a multianalyte array analysis, it is important to carefully

74

Fig. 16. Planar waveguide technology. Capture antibodies are immobilized on the planar waveguide in a microarray format. A preincubated cocktail mix of sample and fluorescently labeled detection antibodies is added onto the planar waveguide microarray. The formed sandwich immuno-complex can be detected without washing away unbound fluorescently labeled detection antibodies. Laser light of the desired wavelength is coupled into planar waveguide and generates an evanescent field, which extends only a few hundred nanometers into the solution. Therefore, only the surface-bound fluorophores are selectively excited. Individual signals correlating to the amount of captured analytes on each microspot are monitored by means of imaging detectors, such as CCD cameras. Reprinted with permission from Ref. [385]. Copyright 2001 Elsevier Science Ltd.

screen each monoclonal antibody clone against all antigens in the array to avoid cross-reactivities. An interesting detection method is immunorolling circle amplification (immunoRCA). In this method, an oligonucleotide is coupled to a binder and upon binding and washing, a circular template DNA is added. In the presence of DNA polymerase and fluorescently labeled nucleotides, many copies of the template DNA are produced if the binder is present and the signal is amplified from the incorporation of the labeled nucleotides. Because of signal amplification, this technique has high sensitivity and a very wide dynamic range making it suitable for single molecule detection. Antigens at femtomolar levels can be detected where the antibodies are each labeled with a unique oligonucleotide primer. Multiplexed immunoassays can therefore be carried out and multiple antigens can be quantitated simultaneously [385,387–389]. Label-free detection. When capture molecules are used and the sample molecules are label-free, detection is based on the physicochemical changes that occur upon binding of the target protein to the capture molecule. Label-free detection schemes overcome the limitations of a sandwich assay in terms of cross-reactivity and the need for reagents [59]. Examples of label-free detection include surface plasmon resonance (SPR), mass spectrometry, and atomic force microscopy [59,125]. SPR involves surface refractive index changes due to changes in mass upon protein binding; an optical sensor measures the change in the angle of incident

75 light [72]. Some commercial systems are available [1,146,344] (see Biacore, HTS Biosystems, and Prolinx in Table 3). Mass spectrometry (MS) can be used to identify sample molecules and covalently bound interacting partners, such as in the photoaptamer system. An advantage is that the measurement does not require that the protein is still active or in its folded state. Surface-enhanced laser desorption/ionization (SELDI) chips with different treated surfaces enable the capture of specific protein molecules and analyzes them in a time-of-flight (TOF) MS and, more recently, in a quadrupole-TOF instrument to achieve higher resolution for more precise mass measurements [1]. It has been used to analyze differential protein expression and profiling to identify disease marker proteins [55]. Proper identification requires enriching the sample first by affinity separation and then identifying via Edman sequencing, western blot, or MS partial sequencing or peptide fingerprinting [56]. SPR-biomolecular interaction analysis (SPR-BIA) has been done in combination with a matrix-assisted laser desorption ionizationtime of flight (MALDI-TOF) MS instrument [44,45]. In addition, MS can be used for quality control of proteins [390]. A drawback, however, is that MS is not quantitative [344]. Atomic force microscopy (AFM) provides topographic imaging of the chip surface [2,69,261,391]. In particular, it has sufficient sensitivity to detect a height increase upon antibody–antigen binding through cantilever deflections. It takes 1–5 min to scan an area more than 100 mm2 [69]. This may be a problem for highthroughput applications. However, there are current efforts to adapt this to a high-throughput format [392]. Other detection systems include the use of piezoelectric devices in quartz crystal microbalances which measure changes in refractive index upon binding (has low sensitivity for smaller proteins below 10 kDa) [344,393–395]; the use of ion channels with subpicomolar sensitivity (is relatively complex) [396,397]; ellipsometry, an optical technique [59]; and electrochemistry applied to enzymatic systems [125,344,398–401]. A detection scheme involving both imaging and quantitation was in a kinase chip. A high-resolution phosphorimager was used to quantify phosphoryl transfer [136]. Microfluidics: channel dimensions, separations, integration, simulation, detection Channel dimensions Design parameters for microfluidic systems include the channel size and geometry [10]. Channel dimensions have to be carefully considered: length should be short enough for a minimum transport distance to mixing points or reaction chambers but long enough to prevent hydrostatic induced flow [80]. Furthermore the depth should be enough to allow a detectable volume while avoiding the effects of hydrostatic pressure. Supply channels for reagent and sample should be separate to avoid cross-contamination and facilitate

76 automation. The layout of the channels, the reagent reservoir, and the points where mixing will occur have to be optimized (see Simulation). For capillary electrophoresis, channel depths are typically 15–40 mm and widths are 60–200 mm [75,402–405]. Typical chip sizes are 2–3 cm2 [12] and are made of silicon, glass, quartz, plastic, or polymer a few millimeters thick. The chips are also covered with a glass plate, the fourth wall of the channels. In a microfluidic system for heterogeneous immunoassays, the chip dimensions were 6.7
2.8 cm, the channel dimensions were 50 mm
20 mm, and the reaction chamber was 165 pl [80]. In achieving the separation of two dye compounds in under a millisecond, Jacobson et al. [30] had a separation length of 200 mm. But channel lengths for separation also go up to a few mm [405]. Using multilayer soft lithography and elastomeric material, it is possible to build active systems with valves and pumps. Furthermore, the resulting devices are reduced in size by more than two orders of magnitude compared with devices using silicon [292].

Separations In electrophoretic separations, the length determines the separation efficiency and resolution. Two means of increasing the channel length have problems with peak broadening: a meandering path that avoids sharp bends [406] and a square channel configuration for synchronous cyclic capillary electrophoresis (SCCE) [407,408]. Effenhauser et al. [409] had optimized parameters including short injection plugs, a high electric field strength, and a short effective separation length to obtain rapid separation in seconds with extremely high efficiencies. There is a limit to how much field strength can be applied in order to prevent excessive Joule heating [406], arcing between electrodes, and to avoid dielectric breakdown of the chip substrate itself [73]. One solution is having wide channels that narrow down over a short distance (the potential drop being limited to the narrow section of the channel) (see Fig. 17). The separation length was 200 mm and the field strength was 53 kV/cm. Separation for two dye compounds was achieved in less than 1 ms [30]. Another solution is increasing the field strength by having a microelectrode array a few millimeters apart inside a channel. By synchronizing the applied voltage with the moving sample bands in the channel, high field strengths are obtained at relatively low voltages and thereby achieving faster separations [410]. There were, however, some practical problems with compatibility between buffer composition and electrode materials, not to mention electrolytic effects [73]. CE chips have been made from the micromachining of glass substrates [30,37,405]. A CE chip using a glass/quartz substrate is commercially available (see Agilent and Caliper in Table 3). The use of cross-channels and double T injectors allowed the electrophoretic analysis of well-defined sample quantities on microchips [411]. Depending on the mode of operation (injection or separation), the electric fields are applied across a channel but to a lesser extent, at the orthogonal channel or reservoirs to prevent sample bleeding,

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Fig. 17. Schematic of microchip used for high-speed electrophoretic separations. (Inset) Enlargement of the injection valve and separation channel. Reprinted with permission from Ref. [30]. Copyright 1998 American Chemical Society.

a procedure termed ‘‘pinching’’ [30,80,412]. (See Fig. 17 for the reservoir configuration.) The first miniaturized system for capillary electrophoresis (CE) on PDMS was developed by Effenhauser et al. [10,105]. Their device sealed reversibly and hence could be re-used after cleaning. Without any surface modification, the channel walls were uncharged and hydrophobic, thus supporting only a weak EOF. A later development was a CE device also on PDMS but irreversibly sealed by plasma oxidation [413,414]. Since this process also modifies the surface, separation of amino acids and negatively charged proteins were achieved. But for positively charged proteins, the channels were coated with PolybreneÕ , a polymer containing quaternary amines, to make the charge ladder (successive acylation of lysines on a protein) positively charged. This reduced adsorption and reversed the EOF toward the anode. In several instances, the addition of zwitterions reduced adsorption of proteins and their derivatives [415]. Electrophoretic separations on chip is far advanced compared to on-chip chromatography. Difficulties arise from the use of stationary phases that in the macroworld has relied on pressure pumping. Because of the incompatibility of packed microchannels and EOF, most stationary phases that have been implemented in microfluidics use micellar phases [416–419] or are coated on

78 channels for an ‘‘open column’’ configuration [29,403,420,421]. Both methods proved to be cumbersome. New types of stationary phases are being developed and are compatible with chromatography using EOF, hence termed electrochromatography [73]. The criteria are that they pack or fill a column, they have compound selectivity, and they sustain EOF [73]. The type of stationary phase that fit this description are those that involve in situ polymerization. Usually, the polymerization is thermally induced as with polyacrylamide or methacrylates, styrene-divinylbenzene polymers, and those based on silicates, the sol-gel approaches [73]. But in one case, the polymerization was photo-induced and this is interesting in terms of being able to pattern microchannel manifolds [73,422]. With polymer-filled microchannels, the same resolution and van Deemter plots were obtained from separations done in fused silica capillaries and in microchips, of both low molecular weight (alkyl phenones, antidepressants) and high molecular weight analytes (proteins) [423]. In a different application, reagent-carrying beads were packed in a microchip for on-chip solid phase extraction; packing was done by applying both pressure and electrokinetic flow [424]. Integration A key concept in integration and of mTAS is being able to put together different techniques and technologies so that the whole is more than the sum of its parts [73]. A high level of integration would accommodate different components that are required in a device from sample preparation to detection. The design of the chip must then consider how these components will be fabricated and how should the actual experiment be carried out (where to position these components and how will the sample be transported to and from different components). In general, this is more feasible when the components have as few moving parts as possible to avoid complicated chip fabrication and risk of breakdown during operation [10]. Miniaturization of sample preparation has several benefits such as reduced sample requirements, faster analysis times, and minimized exposure to contaminants [425]. There are a few examples where sample preparation has been integrated on chip. Sample derivatization and enrichment, with or without separation has been done [27,412,426–429], so with incorporation of a dialysis sampling unit [430] and interfacing to an FIA unit [431]. Though not often applicable, postcolumn derivatization has also been done on-chip after separation [432]. The incorporation of electrodes has always been part of microfluidic microfabrication [61,287]. The integration of electrodes for electrochemical detection has also been done [73]. A more critical aspect is the integration of optical components such as light sources, waveguides, optical filters, refractive elements, diffraction grating, and even light-sensitive diodes (see also Nanotechnology) [24,433–437]. Especially for highly sensitive fluorescence detection, the integration of micro-optical elements or, at least, the proper interfacing to optical detection systems is essential.

79 Interfacing to detection systems may not be straightforward mainly because different detection schemes have different requirements. Mass spectrometry (MS) is one detection system where there has been quite a lot of effort put into interfacing with chip devices. The impetus has been both from chromatographers and mass spectrometrists because of the advantages and capabilities in hyphenating the technique. Most of the interfacing has been done with electrospray ionization. (See Fig. 18 for two types of chip–MS interfaces.) In general, better and consistent electrospraying is achieved if capillary tubing is used after the separation channel [73,425,438,439] (see Fig. 19). Oleschuk and Harrison [425] have a review on microfluidic devices interfaced to mass spectrometry including a section on sample preparation. Reyes et al. [294] has a section in their review on interfacing chips to mass spectrometers. Although there have been attempts to miniaturize a mass spectrometer, doing so does not enhance sensitivity or improve the speed of analysis. It would be more convenient to improve on interfacing rather than reconstructing a new system [73,425]. Simulation Simulation is an efficient way to design microfluidic devices from CAD software and to virtually test fluid and mass transport in both two- and three-dimensional

Fig. 18. Schematic drawings of two different chip-mass spectrometer interfaces: a disposable nanoelectrospray emitter (a) and a sheath flow electrospray interface (b). Reprinted with permission from Ref. [438]. Copyright 1999 American Chemical Society.

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Fig. 19. Schematic diagram of different electrospray interfaces that have been developed for chip-ESI-MS: (A) spraying directly from an exposed channel at the edge of a chip; (B) liquid junction capillary interface; (C) gold-coated capillary interface; and (D) coaxial sheath flow configuration. HV denotes points to which the electrospray voltage is applied. Reprinted with permission from Ref. [425]. Copyright 2000 Elsevier Science Ltd.

flow systems. Computational fluid dynamics simulation can provide the information required from fluid mixing and transport to biomolecule separation and cell sorting [440]. Some specific methods include: 1. Galerkin fine-element method: a general, steady-state approach to simulate 2D and 3D fluid flow, thermal fields, and chemical concentrations in microfabricated devices [441]. 2. FlumeCAD (Coventor, Inc.): a fine-element method for the general design and simulation of electrophoretic, electroosmotic, and pressure systems. It can be used to analyze electrokinetic injections [442] and to predict the electrophoretic motion in microchannels [443].

81 3. Multicomponent continuous media mechanics: describes electrokinetic motion in microchannel networks [444]. Ermakov et al. [445] developed a comprehensive computer program to simulate mass transport in any 2D channel structure, which considers electroosmosis and electrophoresis as well as diffusion. Besides fluid flow, their simulation of microfluidic systems also assisted in optimizing operational parameters for gated or pinched injection (cross-channel type) [446,447], where each technique has its own set of parameters and operational sequence. It has been demonstrated that T-structures are suited for sample mixing and cross channels, for electrokinetic focusing [448]. A microfluidic separation chamber for cell sorting was modeled on a three-step process [41]: (1) electrode geometry was optimized with the Ansoft Maxwell 2D-field simulator modeling the different electric fields in the device, (2) effective cell separation from various mixtures was modeled with MATLAB software based on dielectric properties [449], and (3) flow and thermal conditions were predicted using lattice-Boltzmann simulation [450]. Additional information regarding surface adsorption properties and surface interfacial forces can also be entered. Microfluidic detection systems For microfluidic systems, laser-induced fluorescence (LIF) in a confocal system is highly sensitive and is the most popular for microchip applications involving separation [41,451]. As channel dimensions are reduced, so too are detection volumes. The ultimate limit is single molecule detection, which can be done with LIF [73,105,452,453]. A detection cell with improved sensitivity can detect 6 mM fluorescein [24,75]. However, the use of LIF in a multichannel protein microchip remains a challenge [75]. Chemiluminescence detection limits, e.g., 7–35 nM HRP-F1 (fluorescein conjugate of horseradish peroxidase) [405], are 50–100 times better than on-chip absorption detection [24]. These limits, however, are higher than the mid-picomolar detection limits for on-chip LIF [25,454]. An alternative to detecting fluorescence is detecting changes in index of refraction. Two components manipulate the light beams: liquid-core waveguides and diffraction gratings [10]. Advantages of the method include universality (i.e., analytes do not need labeling), concentration-dependence and optical simplicity (i.e., less expensive than fluorescence) [433]. However, disadvantages include its temperature-dependence and poor detection limits [436]. Interfacing microfluidic devices to a mass spectrometer can be viewed in two ways: the MS as the detector or the chip as the sample introduction method. Depending on the type of instrument chosen, Mass Spectrometry gives high resolution, is sensitive enough for trace analysis, and can provide structural information and identify analytes. Since efficient means for interfacing the chips to electrospray ionization sources are in place (see Integration), the developments have been in increasing the sample handling capabilities of the chips [425].

82 Other detection schemes include electrochemical detection, Raman spectroscopy, and holographic refractive index detection [75].

Cell and tissue biochips Cells on chips Some applications involving cells on chips are not conveniently categorized into established formats. Patterned cells on a chip to study cellular behavior is a typical example. One that is clearly in an array format, that of single live cells, is being developed (see below). Several examples integrate different functions on the chip, and as it is necessary to transport material (sample and reagents), they fall under microfluidics. In some cases, cells on chips are used to sense microenvironmental changes, and hence are sensor chips. The different examples cited below exemplify a wide range of applications. Cell patterning has been done by a number of researchers to study various cellular responses including the influence of cell shape and size on cell differentiation and function (see also Biochip Applications, Biological and medical research). Folch et al. [238,239] used deep (20–30 mm) channels on PDMS to transport material more slowly (for comparison, see Delamarche et al. [247,248]). As a base layer, collagen or fibronectin was patterned over different substrates before applying cells on them. A second cell type was applied to regions on the chip that did not require this protein base layer for adhesion [238]. Microfluidic channels were also used to pattern the cells directly onto the substrate such that two different cell types were patterned simultaneously [239]. Other examples of cell patterning have been described. Growth of bacterial cultures on a PDMS device has also been demonstrated [41]. The use of live cells and maintaining their viability is the crux of cell biochips. One type of cell array, single live cell arrays, is being developed by David Walt (Department of Chemistry, Tufts University) using optical imaging fiber (OIF) technology at a density of 10 million wells per cm2. MH 3T3 mouse fibroblast cells are used in this sensor. With one cell per well, cell positions are encoded and physiological and genetic responses are simultaneously recorded (e.g., pH and O2 measurements, promotor responses via fluorescence produced by the lacZ and phoA reporter genes, and protein–protein interactions using the yeast two-hybrid system) [455,456]. Assuming the cells remain viable and enough cells are monitored for statistical significance, the use of such a system for screening the physiological effects of a combinatorial drug library can be speculated [456]. With live cell microarrays, signal transduction pathways, protein–protein interactions, expression cloning, and cell localization for encoded proteins can be determined or performed. A current limitation is the availability of cDNAs for such experiments. One method that uses live cell microarrays for cDNA expression is the living protein chip for expression monitoring where proteins

83 were expressed in human embryonic kidney (HEK) cells to examine their effects on the cells themselves, e.g., the induction of apoptosis [457]. The HEK293 cells are added to DNA–lipid complexes (reverse transfection) arrayed on a glass slide. These cultured cells can be made to express excess amounts of a protein whose cDNA has been introduced into these cells; the cDNAs need only to be spotted on to the array and the cells take them up. The microspots are 120–150 mm in diameter consisting of a cluster of 30–80 fluorescent cells. With these living arrays, the proteins do not need purification because their concentrations are so high relative to the HEK cell’s endogenous proteins. The proteins expressed are 10–15% of the transfected plasmid [458]. These cells can even express membrane proteins like GPCRs. The expressed proteins can then be used for functional studies, e.g., its interaction with small molecules. After the protein of interest is expressed within the cells, the array is removed from the culture and the cells are fixed. The cells are then visualized or subjected to other detection schemes [367,455]. In live cell microarrays, visualization is done via fluorescently labeled antibodies or by direct fluorescence microscopy with GFP-tagged proteins. Other methods of detection for cell microarrays include western blots and radiolabeled ligands [367]. The transport, cultivation, and manipulation of living cells have been performed on microfluidic devices [26,459]. One interesting device is a cell sorter. This integrated cell sorter incorporated several coordinated functions on a microfluidic device that included sorting, sample dispensing, flushing, recovery, and absorption of fluid perturbations [16]. Sorting required the use of peristaltic pumps, dampers, and switch valves. An actuated valve on this sorter can have an active volume as low as 1 pl while the volume allotted for optical detection is about 100 fl [16]. This pressure-driven device has a T-cross for sorting [16,275,292] as it was in a previous electrokinetic sorter [275]. Electrokinetic flow, however, presented some difficulties so this improved sorter with microvalves and micropumps comes even closer to mTAS. Using multilayer soft lithography for fabrication, this device has a top layer where all the valves and pumps are located and a bottom layer that has all the fluidic channels. For sorting, the channels start with a width of 30 mm which tapers down to 20 mm and finally to 6 mm at the sorting T. By detecting fluorescent signals, certain valves are opened or closed so that the cells are pushed into one or the other branches of the T-junction [292]. The literature presents more details on the valve dimensions, the use of dampers, and their locations [16]. The fabrication of switching valves and peristaltic pumps for sample dispensing and switching [292] and rotary pumps for mixing and incubation [460,461] have been described previously. Another cell sorter was used to separate two types of white blood cells, T-lymphocytes and granulocytes. This device was also fabricated in PDMS, since glass and silicon dioxide proved to be too adhesive to effectively carry out the experiments with these cells. This chip had a network of small channels

84 with different lengths mimicking capillaries in vivo where cells are subjected to size restrictions. As predicted, the cells’ passage through the microchannel network depended on their size and nuclear morphology [462]. For the separation of particles and cells in microfluidic systems, besides the components and methods described above which concentrated mainly on fluid flow and control, i.e., pumps and valves, some additional components have been cited in the literature. For incubation purposes, spatially fixed temperature zones are situated along various sections of the microchannel [20]. For dispensing reagents to use in enzymatic reactions and dilution, multiple ports and plugs can be incorporated [26,463]. Another type of biochip involving live cells was a sensor chip used in multiparametric cellular monitoring for drug screening [47] (see also Biochip Applications, Drug Screening). Functional studies usually comprise measuring the effects of drugs on intracellular Ca þ concentration, pH, and membrane potential in cells using fluorescent dyes. Hence, different microelectronic sensors were used: pH-ISFET transducers and pH glass electrodes to measure extracellular acidification, a Clark-type oxygen electrode to measure cellular oxygen uptake, interdigitized electrode structures (IDES) to detect changes in cell adhesion and morphology, and temperature sensors. Furthermore, maintaining a physiological environment involved thermostated chambers and a fluid exchange system. Two test versions were made; one on glass and the other on silicon. Details on their operation were presented [47].

Tissue microarrays Tissue microarrays (TMAs) were developed for molecular pathological studies on a genomic scale [464,537]. Molecular alterations in thousands of tissue specimens can be analyzed in parallel. They can be complementary to DNA arrays because they would help in validating and understanding the physiological relevance of the gene changes taken from DNA array data. However, TMAs can also be used to test the toxicity of drug leads by evaluating the drug target distribution in normal tissue, hence, accelerating the drug discovery process in being able to predict clinical failure [456]. Tissue microarrays (TMAs) have been applied in the high-throughput molecular profiling of tumor specimens [465,537]. A robot was designed to punch out cylinders (0.6 mm wide, 3–4 mm high) from 1000 individual tumor biopsies embedded in paraffin and to array them in a 45
20 mm paraffin block. The robot is now commercially available (see Beecher Instruments, Table 4). The tumor sections are analyzed in parallel by immunohistochemistry and in situ hybridization [1,465]. An important advantage is that archival tissue (paraffin blocks dated 20–40 years that are fixed in 4% buffered formalin) can be used since it may be difficult to gather fresh samples in a high-throughput setting [464]. Most of the profiling done has been in cancer; the TMA studies done on various cancers including tissue types, sample size, methods used, molecular

85 targets, and the corresponding references are outlined in Kallioniemi et al. [464]. The types of studies performed include the frequency of molecular alterations in large tumors (as part of a genomic scale study in molecular pathology), tumor progression, identification of prognostic markers, and validation of novel genes as diagnostic and therapeutic targets [464,466]. The information on the cellular origin of the molecular targets is also inherently available. TMAs are meant to be research tools to study population-related trends and not to diagnose individual cases. Different types of TMAs are available for cancer research such as multi-tumor arrays (containing different tumors), tumor progression arrays (tumors at different stages), and prognostic arrays (tumors with clinical endpoints) [467]. Molecular profiling of tumor samples can also be done using the technique referred to as layered expression scanning [468]. Cells or tissue samples are made to pass through a layered array of capture membranes (nitrocellulose or agarose gel layers) where each layer is linked to a specific antibody or cDNA sequence. Target proteins are then confined to specific layers so that a molecular profile for each cell type is generated.

Biochip applications Drug discovery and drug development Proteomics and drug discovery tools The term proteomics can mean any of several different strategies aimed at unraveling the human proteome. The proteomics field can be categorized so as to provide a means of better understanding the complex field which leads to several approaches at solving the problem, each complementary to the others [469,470]. Profiling and cataloging proteins in normal and diseased tissue constitute expression proteomics, an area where protein arrays have been applied. Another important part of current proteomics research is in cell-map or interaction proteomics involving the investigation of molecular interactions and the determination of proteins that are involved in signaling pathways. All this is to be able to gain insight into biological processes, which is an end in and of its own. However, pharmaceutical research aims to identify or validate protein function in order to discover novel diagnostic and therapeutic drug targets, the realm of functional proteomics. To them, protein arrays help to identify diseaserelated proteins, or better still, determine their function. Last but not least, protein arrays can also help identify novel, therapeutic compounds. Hence, in achieving any or all of the above, biochips are becoming an indispensable tool in proteomics and drug discovery. Separations. One of the first demonstrations of the usefulness of doing electrophoresis on microchips was in 1990 [15]. With cross-channels and double

86 T injector setups, well-defined sample quantities could be separated by on-chip electrophoresis [411]. An electrophoretic separation completed in less than a millisecond has been carried out [30]. Such a separation may be useful for ultrahigh throughput drug discovery, in monitoring millisecond timescale kinetics, or as a final cleanup or separation step, e.g., in multidimensional separations. In a technique called immunoaffinity capillary electrophoresis, antibodies were immobilized directly onto fused-silica capillaries [80,471,472]. A commercially sold lab-on-a-chip can perform electrophoresis on 10 protein samples (cell lysates, column fractions, or purified proteins) in less than 30 min. The chip is put into a ‘‘reader’’ with software that is linked to allow the separations to be viewed in real time. After the analysis is done and data is stored, the chip can be disposed of without any toxic waste chemicals to worry about [11,473] (see also Agilent in Table 3). On a per chip basis, it is less costly than standard SDS-PAGE and has been used for recombinant protein expression analysis, optimization of protein purification procedures and for QA/QC of protein-based drugs [11]. An integrated microfluidic system that incorporates protein digestion and peptide separation for protein identification has also been developed [474]. The chip contains a trypsin membrane reactor for rapid protein digestion. The resulting peptides are concentrated and separated by capillary isotachophoresis (CITP)/capillary zone electrophoresis (CZE) prior to MS analysis. Cohen et al. [475] used on-chip electrophoretic separation to assess the result of an enzyme assay for kinase A. On-chip dilution of the peptide substrate was also performed to determine kinetic constants. A reverse-phase chromatographic separation of peptides and amino acids on a microchip ‘‘outperformed capillary-based separations in both speed and efficiency’’; six peptides were separated in 45 s with up to 600,000 plates/m [476]. For other examples of on-chip separations, see Table 2. The separation of cells is covered under Bioparticle manipulation and sorting (see below). Bioparticle manipulation and sorting. Bioparticle separation and manipulation is based on the principle of dielectrophoresis. It has been used in biotechnological applications including the purification, fractionation, and concentration of cells and microorganisms. The size limit of the method has been shown to be below 1 mm with the trapping and separation of latex beads and tobacco mosaic virus (TMV) [298]. Strategies from a binary separation to multicomponent fractionation in microfluidic devices are discussed by Hughes [296]. As a means of separation, negative dielectrophoresis is implemented via electrode arrays in the microchannel and are driven with high-frequency AC. Laminar flow carries the particles past the electrodes while the AC strength determines particle trajectory. A particle sorter has been described to concentrate the sample down to a narrow stream, break up aggregates, trap and finally sort [299]. Flow velocities are at approximately 10 mm/s. When combined with sensitive optical detection, the technique may be applied to perform miniaturized flow cytometry [299].

87 Flow cytometry. Flow cytometry of E. coli has been carried out in a microfluidic device. Labeling efficiencies for the dyes and the antibody were greater than 94% [477]. Such an application is promising for drug discovery. (See also Bioparticle manipulation and sorting, above, and Biological and medical research, Cell manipulation and sorting.) Mass spectrometry. Besides being a mode of detection, mass spectrometry in itself has been an indispensable tool in proteomics, especially in protein identification through partial sequencing and peptide fingerprinting as well as in quality control of reagents and in drug screening. At low array density, SELDI mass spectrometry and SPR-BIA, although they operate on very different principles, can monitor intermolecular interactions and can be linked to some means of identifying the interacting partners [44,45,54,55,344,478,479]. SELDI has also been used quite extensively for profiling and biomarker discovery. (See also Clinical Applications, Biomarkers.) Whole tissue extracts or microdissected material can be analyzed [56] (see also Table 2). In a high-throughput CE/MS analysis, automated sampling from a 96-well microtiter plate held vertically in front of an electrospray mass spectrometer, was followed by CE separation through a microfabricated device fitted for electrokinetic and pressure-induced fluid flow [480]. Other high-throughput options include electrospray nozzles on a microchip [481,482] (See Advion, Table 4) as well as a multichannel device for high-throughput electrospray infusion equipped with individual electrospray exit ports via a microchannel/ electrode system [483]. MS can also be used for screening applications as well as for quality control of small molecules, reagents, and proteins [390]. Expression profiling. Proteomic profiling involves measuring and recording the expression patterns of proteins in a given tissue, cell, or subcellular component. Comparing expression patterns of normal versus diseased states, provides some insight on possible disease-related proteins, as diagnostic markers or as drug targets. Confirmation of disease-relatedness, however, needs to be validated [470] (see also Identification of protein function). In the case of cancer, the aim of proteomic profiling is to understand how protein expression patterns influence the tissue microenvironment as well as the initiation and progression of tumor growth. Because most of the proteins identified in connection with oral cavity cancer, both in normal cells and tumor cells, are involved in signal transduction pathways, complex cellular signaling must play a key role in cancer progression [484]. Such a hypothesis may be answered with chip technology. The combination of 2D-electrophoresis and mass spectrometry can study only a limited number of proteins. The use of protein profiling arrays can complement or replace 2D-gel electrophoresis and would have a significant impact on proteomics [344]. It can be a general resource for expression or interaction (cell map) proteomics, i.e., high-throughput screening of gene expression and receptor–ligand interactions [1,485,486]. In a cytokine profiling chip, Fab antibody fragments are deposited on array posts and oriented for increased

88 detector sensitivity. Lower limits of detection achieved so far is 0.1 pM and the quantitation limit is 0.5 pM. Apart from performing multianalyte cytokine profiling, these chips can be used for the identification of possible drug targets or diagnostic markers using expression pattern data [59] (see Zyomyx, Table 3). 1. cDNA arrays. Initial screening of gene expression profiles via cDNA arrays is rapid but other methods are required to validate whether differences in hybridization signals actually correlate to physiologically relevant differences in gene expression [487]. From running DNA arrays to profiling proteins encoded by differentially expressed cDNA clones requires a high-throughput approach for parallel protein expression analysis. (See Fig. 20 on how cDNA libraries on chips are processed.) A large number of cDNA clones have to be expressed simultaneously with the appropriate vector system [1,128]. Gene expression data from DNA arrays and/or protein expression profiling data from 2D electrophoresis and MS can be correlated with data from protein chips [488,489]. DNA sequence information and protein expression levels can also be linked. Expressing cDNAs of interest can also be done via live cell microarrays [367,457] (see also Principles of Design and Operation, Cell and tissue biochips). 2. Enzyme-linked immunosorbent assay (ELISA). In principle, expression profiling can also be done using high-throughput enzyme-linked immunosorbent assay (ELISA) [74] (see also sandwich assays in Principles of Design and Operation; Arrays: capture agents, molecular tags, detection, Array detection systems; Detection with labels). The profiling of cytokine levels in biological samples with high specificity and sensitivity has been done by microarray

Fig. 20. High-density arrays of cDNA libraries. Using a picking/spotting robot, cDNA clones are picked from agar plates into 384-well microtiter plates, and identical patterns are spotted at high density onto nylon or polyvinylidene difluoride filter membranes that are subsequently processed for DNA hybridization (DNA filters) or protein detection (Protein filters), respectively. For microarrays, cDNA inserts are first amplified by high-throughput PCR, or proteins are expressed in microtiter plates before spotting. Reprinted with permission from Ref. [1]. Copyright 2000 Elsevier Science Ltd.

89 sandwich immunoassays [490]. To avoid cross-reactivity, the antibodies have to be screened against all antigens on the array. (See also Table 2.) 3. Tissue microarrays (TMAs). In cancer, molecular profiling of tumor samples with TMAs generated data that correlated with tumor progression and frequency of molecular alterations in tumors, as well as the identification of prognostic and diagnostic markers, and drug targets [464–468]. At the National Human Genome Research Institute, a multi-tumor array ( 5000 specimens, and sections from 36 normal and 800 metastatic tissues) and a normal tissue array (76 tissue and 332 cell types) have been built [465]. Kallioniemi et al. [464] outlines the different studies that have been done with TMAs on a variety of cancers. With the technique called layered expression scanning, a type of tissue array, it is possible to produce a molecular profile for each cell type present in the tissue [468]. Molecular screening 1. Specificity screening. An offshoot of molecular profiling is antibody specificity screening against complex mixtures of proteins. Since antibodies may cross-react with unrelated proteins, protein arrays would help in determining antibody specificity. However, determination of cross-reactivity using protein chips is limited by the number and type of proteins arrayed on the chip. High-throughput specificity screening was done with 92 expression clones from the human fetal brain cDNA expression library, hEx1, using selected monoclonal antibodies and scFv fragments from phage display [485]. This was done as a quality control step to check for cross-reactivity as well as to detect common epitopes. This reverse antibody array would be particularly important for reagents used in immunohistochemistry and physiological studies on whole cells or tissues. Walter et al. [1] described the first use of a high-density protein filter. They screened the hEx1 cDNA library array with monoclonal antibodies against mouse GAPDH (glycerol aldehyde phosphate dehydrogenase), the heat shock protein 90-alpha (HSP90), and tubulin-alpha. Holt et al. [491] screened protein array filters of hEx1 with antibody fragments to generate specific antibodies without the need to immunize or resort to display methods. 2. Screening for modification, expression, activity. Biochips can be used for the simultaneous determination of chemical modifications of many proteins on a given chip [59,128]. For example, molecular screening with antibody arrays can be used for detecting posttranslational modifications or smaller molecules, such as peptide hormones [492,493] and carbohydrates [494,495]. Furthermore, biochips can be used to monitor the expression of one or more proteins in a mixture such as a cell extract just as 2D gel electrophoresis has been widely used to profile protein expression and detect protein modifications [340]. Profiling biochemical activities in parallel for a large number of proteins would be a task that biochips should fulfill [59]. In a microarray format, the proteins should be properly immobilized on the chip surface and should maintain their

90 activity (not be denatured) [496]. Quantitating inhibitor–enzyme interactions [137] and screening a yeast proteomic array for phosphoinositide-binding activity [128] have been done. Similar studies that have analyzed protein biochemical activity in microwell plates [136,497] have the potential to be carried out in miniaturized chip or nanowell format. 3. Protein availability for screening. More often than not, the amount of protein isolated from 2D gels is not sufficient for screening, much less for monoclonal antibody production [344,498–500]. Even with advances in antibody production by phage display [501–506], significant amounts of protein are still required. De Wildt et al. [340] compared mass array screening with several rounds of phage display in screening antigen–antibody interactions. Some antibodies were selected with only 0.005% or 0.0005% recombinant protein in the lysate. Furthermore, parallel screening should be able to identify antibodies that recognize differentially expressed proteins [340]. Nevertheless, an important consideration is an adequate supply or source of proteins. With this comes the improvement of protein expression systems. A dual expression system in E. coli and Pichia pastoris has been used to produce soluble proteins in the native state [507]. Soluble expressed proteins find uses in antibody generation, crystallization, NMR studies, therapeutics, and in vitro interaction studies. A parallel, miniaturized expression of recombinant proteins in E. coli and in the baculovirus system has been developed [488,508]. An arrayed expression library can be a source of recombinant proteins [390]. The Resource Center of the German Human Genome Project (RZPD, http://rzpd.de) has been an important resource in this regard. For example, the human fetal brain cDNA expression library hEx1 (RZPD library no. 800) was used in experiments by Leuking et al. [1,485]. Although the availability of full-length human cDNAs is limited, there are on-going projects that address this [457,509–512]. 4. SPOT synthesis peptide arrays. Using peptide arrays, the SPOT synthesis method can detect low-affinity antibody interactions or receptor contact sites [376]. This is due to reduced nonspecific binding and the increased binding affinity because of high local peptide density on the cellulose substrate [377,378]. Although not sufficiently miniaturized, SPOT synthesis involves straightforward and reliable experimental protocols, inexpensive equipment, a highly parallel process, and adaptability to different assays and screening methods. These peptide arrays prepared by the SPOT technique have been used in molecular recognition studies in molecular immunology and can be used in in situ screening of chemical libraries [513]. Molecular interactions and signaling pathways. Studies on molecular interactions include peptide–protein, protein–protein, and protein–drug interactions and can also extend to epitope mapping [348]. Early research in this area involved the use of a low-density universal protein array (UPA) system to screen the interactions

91 of p52 against 48 purified proteins spotted on nitrocellulose [514]. This system can also be used to study protein interactions with DNA, RNA, and other ligands as well as small chemical entities. Hence, it can be used to screen drug targets. MacBeath and Schreiber [118] studied the limit of their system (FKBP12rapamycin-binding domain) and found specific binding was detected using concentrations as low as 150 pg/ml (approximately 12.5 pM). This implies being able to detect such interactions in cell lysates when using fluorescently labeled proteins. Their microarray also works in detecting transient signals, e.g., enzyme–substrate interactions as with kinases. Zhu et al. [128] studied interacting proteins using a yeast proteome microarray. Since it was possible to observe almost the entire yeast proteome on one array, this strategy helps to map out pathways of protein–protein interactions, which is currently done by two-hybrid assays. One difference is that because much less material is needed on the chip (10–950 fg per protein) [9,128], the method is 10–100 times faster [79]. The study of multiprotein complexes is an extension of interaction studies. It is important for understanding cellular processes [348] and a basis for studying signal transduction. Signaling involves posttranslational modifications, e.g., phosphorylations, which cannot be accessed by DNA methods. Since protein complexes are formed in stimulated cells, they cannot be studied in in vitro cell-free systems. Hence, live cell microarrays can be used to study signal tranduction. Signaling can be induced by a number of stimuli and assays are available in different formats. Activation is detected in microarrays expressing hundreds of cDNAs (e.g., phosphorylation of a downstream kinase) or with antibodies specifically recognizing the relevant protein form [367]. A kinase chip specifically for evaluating the function of protein kinases in cell signaling is discussed below (see Identification of protein function). There have been studies done, albeit not chip applications, on protein interactions and epitope mapping with synthetic peptides [377,378,515]. Geysen et al. [515] used a peptide array to identify an immunogenic epitope preset on the VP1 coat protein of the foot-and-mouth-disease virus. Such studies can be done today more efficiently in microarrays.

Identification of protein function. The goal of molecular profiling is the identification of disease-related proteins that may be novel drug targets and prognostic and diagnostic markers. One possible strategy is to have organ and disease-specific protein arrays [348]. However, a disease-associated protein does not necessarily imply an appropriate drug target or biomarker. Molecular profiling catalogs the up- and down-regulation of proteins, the functional relevance however, for any one of these over- or under-expressed proteins should be determined by another means [470]. The probability of functional relevance increases if this protein’s participation in a signaling cascade is established. The definitive criterion for function should then be the causative role of the protein in a given disease or physiological state.

92 Many types of functional studies can be carried out on protein arrays. Live cell arrays are appropriate for large-scale characterization of protein function [367]. Information on protein function can be derived from the analysis of biochemical activities [118,128,136,496,497,516]. Kinase chips are specifically used to determine function in the context of cell signaling, to evaluate substrate selectivity, to identify physiologically relevant enzyme–substrate pairs, and to discover novel inhibitors [128]. MacBeath and Schreiber [118] described their preliminary results in developing microarrays to study function in human proteins. Their major efforts involved immobilizing functionally active, folded proteins and detecting the interactions. They plan to assign function at a broader level, running miniaturized assays in parallel. Whether it is necessary to express and purify thousands of proteins or cellular systems or cell lysates sufficient to use to carry out functional arrays remains to be seen. Identification of novel therapeutic compounds. The use of protein arrays to identify and validate novel therapeutics necessitates the development of assays. For example, identifying relevant enzyme–substrate pairs is a means to discovering new kinase inhibitors with the use of protein chips [349]. Some commercial entities intend to sell arrays containing whole protein families for use in drug lead optimization [260]. Before actual optimization studies begin, the viability of a compound as a drug lead is determined from its binding kinetics to a specific protein drug target. SPR-BIA technology can be used for this purpose. A commercial collaboration intends to carry out SPR-BIA technology in an array format for the next step, characterizing lead compounds via ADMET (Adsorption, Distribution, Metabolism, Excretion and Toxicity) assays [383]. Drug screening and testing Protein chips can screen potential lead compounds at high throughput and can be used for toxicological testing as well. Arrays are well suited for this application when there is only a limited supply of cells or amount of drugs available for testing [47], especially in the high-throughput screening of lead compounds. Microfluidics has been used to screen 750,000 compounds from a drug library against a protein target, consuming only 750 ng of the protein (see also Caliper in Table 3). Although most protein arrays are based on soluble proteins, progress has been made in arraying of functional membrane proteins, which represent the majority of drug targets [153]. In experiments designed to detect protein–lipid interactions, proteins that were bound to low molecular weight compounds were identified. With specificity toward other proteins, lipids, and even small molecules, these functional protein arrays will enable the direct screening of entire proteomes for protein–drug interactions [79,128]. They can aid in determining the selectivity and specificity of drug leads in further downstream testing. For example, genetic variants of HIV reverse transcriptase and protease enzymes can be screened for efficacy against drug-resistant enzymes. In a clinical setting, pharmacoproteomic screening of

93 known population genetic variants of HMG-CoA reductase should help define patient types so that the most efficacious statin can be prescribed to a particular patient [59]. Increased demand for high-throughput cell-based formats to screen for drug leads will require engineered cell lines with diverse yet well-controlled characteristics for more critical applications. To identify and validate lead compounds, in vitro cellular screening is necessary before further animal testing. Furthermore, it may be helpful in cellular pharmacokinetics, i.e., analyzing drug action from the onset to reversibility of effects [47]. There is also demand for using primary tissue arrays from patients as a better predictor of disease. Arrays of functional cells or tissues, ideally primary diseased tissue, can provide better understanding of disease physiology and may serve as surrogate for the disease eventually replacing the use of animal disease models. Considering the argument of pharmacogenomics, if drug response is influenced by genetic factors, diseasespecific protein chips or primary tissue arrays from patients can be used to determine whether individuals will respond favorably or adversely to a given drug. Some may not even show a response due to a defect in the drug target. Such information is relevant and should be exploited for the development of new therapeutics, as well as the interpretation of clinical trial results and even treatment [348]. Cell biosensors. Multiparametric cell monitoring with the Cell-MonitoringSystem CMSÕ [48,49], can detect relative changes in cellular behavior in situ. Different sensors on the chip monitored different parameters (see also Cells on chips). Cellular oxygen exchanges would indicate mitochondrial and photosynthetic activity, changes in cell adhesion or morphology would correspond to physiological responses, and extracellular acidification rates were monitored to determine metabolic activity. Fast pH changes (minutes) would correspond to cellular activation events such as receptor-mediated signaling; slow changes (hours or longer) meant cell growth or proliferation or cell death, respectively. The signals from the different sensors were correlated to give a proper interpretation of the cellular response to a particular drug. Cell-based assays would be simpler since metabolic activity does not play a role but nevertheless, there is the restriction that only adherently growing cells can be used [47]. After the successful testing of this sensor chip, an array format is a logical extension for high-throughput screening. The major hurdles would be in managing a more complex fluidic system and having appropriate data acquisition and processing [47]. Screening tissue arrays. Tissue microarrays can be used to predict the probable toxicity of lead compounds by evaluating the distribution of drug targets in normal tissue [465]. Monitoring drug metabolism and toxicity. Protein biochips may be utilized in monitoring drug metabolism and toxicity especially during preclinical

94 pharmacokinetic studies, to supplement in vitro and in vivo model systems. Furthermore, biochips used in drug metabolism and toxicology studies could be tools to optimally design clinical trials and exclude individuals with potentially deleterious drug-metabolizing enzyme (DME) profiles [59]. Xenobiotics or their metabolites can tightly bind to their corresponding DMEs and inhibit enzymatic activity. This is the major cause of adverse drug–drug interactions observed in patients on an incompatible drug regimen [59]. DMEs can be classified according to phases of metabolism: 1. Biotransformation: includes oxygenases, e.g., cytochrome P450s and flavin monooxygenases (FMOs), that catalyze the hydroxylation and demethylation of hydrophobic xenobiotics. 2. Bioconjugation: includes transferases that take biotransformed intermediates as substrates and conjugate polar adducts, to make xenobiotics more hydrophilic enabling renal excretion. Potential toxicity can be determined from information on xenobiotic–DME interaction. For example, xenobiotic toxicity involves a measurable increase in DME expression levels, particularly the P450s. Since many of the preclinical pharmacokinetic and pharmacodynamic analyses are performed in rats, protein biochips containing the human-equivalent P450s should lead to more medically relevant results [59] and may eventually circumvent the use of animals for toxicology studies. Drug metabolism biochips would have two assay components: (1) DME expression profiling and (2) activity profiling of DMEs in the presence of selected xenobiotics. An ELISA-type analysis can be used for profiling the DME, which can be derived from cell or tissue extracts. Protein microchips may therefore have a significant impact on the development of safer drugs through the comprehensive profiling of drugs or leads for effects on DMEs. However, challenges remain especially toward the expression of membrane-bound DMEs, which have complicated folding pathways due to the presence of the prosthetic heme group [59]. Drug delivery. Drug efficacy is affected by the means in which drugs are delivered in the human system. Silicon microchips enable drug release rates to be varied over time. The chips can store and release multiple chemicals on demand. In the future, integration of microprocessor control or biosensors may enable smart implants or tablets for controlled drug delivery [517,518] (see also Table 2).

Clinical applications Immunoassays To reiterate the principle in carrying out assays in microarray format, as one goes down with the amount of binder on the chip, the detected signal is a function of the concentration of target or analyte molecules in solution and is

95 independent of the solution or sample volume and the amount of binder in the microspot [4,326]. This is because the fraction of analyte captured reflects the solution concentration [121]. There is a limit of course to the amount of binder as statistics starts to be a problem if the number goes too low or as detection systems are challenged in sensitivity. Microarrays that operate in this realm, termed ambient analyte assay limits, yield faster results and obtain greater sensitivity due to optimal signal to noise ratios [4,121]. These conditions are represented in spot sizes less than 100 mm2 containing 106–108 binders [121]. On the other hand, mass-sensing arrays can yield overall stronger signals because practically all the analyte molecules in solution are captured. However, incubation times can take 1–3 h. In this case, spots are about 200 mm in diameter with 1010 binders [8,121]. The most common use of microarrays is in immunoassays [9]. In particular, antibody-based immunoassays are the most common type of diagnostic assay because of antibody specificity; technologies that involve the use of these molecules develop at a rapid pace [519]. The assay is usually run in a multiplexed mode where the antibodies or other capture agents are immobilized and then exposed to a biological sample [9]. There are four immunoassay formats: directbinding, sandwich (ELISA), competitive, and displacement [520]. Direct-binding and sandwich assays are the most common. There are some reports on the use of competitive assays and displacement assays, which are usually associated with high surface area/volume systems. Direct-binding immunoassays. Immunoassays can be homogeneous or heterogeneous. Homogeneous assays refer to a binding reaction where both components are in the same phase, i.e., in solution, while heterogeneous assays have one component in solution and the other fixed to a solid support, analogous to the use of capture agents in a microarray. In homogeneous assays, it is not always easy to detect a binding event. A solution to this is to do sample separation on chip, e.g., capillary electrophoresis. However, with such a format, only a limited number of assays are available [80]. Furthermore, a problem with precipitation is often observed when the antigens are large proteins and more so when the antibodies are polyclonal. This implies that the homogeneous format is more applicable for small antigens or haptens, such as drugs and some hormones [405]. In heterogeneous assays, one of the binding partners, usually the antibody, is attached to the chip surface. A key advantage of solid-phase immunoassays is that dilute solutions can be concentrated on the chip surface, making the binding event more easily detectable. Mangru and Harrison [405] have performed an IgG immunoassay using chemiluminescence detection in a postseparation reactor capillary electrophoresis chip. In another case, an optical immunoassay microfluidic chip was developed to analyze human chronic gonadotrophin (hCG) in human serum [71] and in undiluted, whole blood [72]. Sensitivities were in the nanomolar

96 range: 0.1 ng/ml (human serum) and 0.5–5 ng/ml (whole blood), whereas the clinically relevant range is 0.3–1.5 ng/ml [72]. With rather high detection limits at 200–300 picograms, their conclusions were that better control of NSB effects is expected with improved surface blocking and this should in turn lower the detection limits. A microchip electrophoretic immunoassay analysis on serum cortisol was done in 30 s and within range of clinical interest (1–60 mg/dl); no extraction or sample preparation was done [454]. A fluorescence-based immunosensor was developed for simultaneous multianalyte analysis [119,521]. Recognition elements on the chip capture sample analytes and the detection system involves a small diode laser, CCD camera, and image analysis software to correlate the positions of fluorescent signal and analyte identity. Although only spiked samples were tested, physiologically relevant concentrations of staphylococcal enterotoxin B (SEB) (a common cause of food poisoning), F1 antigen from Yersinia pestis (the etiologic agent of plague), and D-dimer (a marker for sepsis and thrombosis) were detected. Rapid detection of all three was performed by sandwich fluoroimmunoassays performed on a planar waveguide [119]. For other immunoassay applications, see also Table 2. For point-of-care diagnostics, biochips are promising, especially since immunosensors for clinical diagnostics would require one-time-use, disposable chips [172] (Liu et al., p. 760). In a microtiter well plate where molecules have to travel a distance in the order of millimeters, incubation times take several hours because it will take most analytes that long to diffuse throughout the entire well volume. Therefore, a much-reduced size is necessary and would be compatible with a chip format. Another complicating factor is the fact that automation equipment (e.g., fluid handling/dispensing systems or robots) is bulky [80] (Dodge p. 3400–3401). The solution lies in microfluidics [62,522]. Most diagnostic assays have reagents bound to a solid support as in ELISA. When probing for multiple targets, the assay sensitivity becomes limited by the distribution of sample over a bigger area and by how much analyte can be pulled down from solution. Fluidic flow that transports material to different possible binding sites is a more efficient means of performing the assay and increasing detector signals, rather than passive diffusion. Complete recycling of sample can be achieved by active pumping, allowing optimal sensitivity [62]. Since assays are done in a flowing system, i.e., nonequilibrium conditions, analysis times can be short, e.g., less than 5 min [80]. This is a major factor in considering a microfluidic platform for automating heterogeneous immunoassays. In simulating this time advantage with biotin pixels and neutravidin-coated beads, passive diffusion for 4 h resulted in only a fraction of bound pixels whereas active pumping for 4 min resulted in greater than 80% of the beads binding to the pixels [62]. A low-cost diagnostic chip could be one based on dielectrophoresis technology. This technique is gentler on cells than DC techniques and can separate particles including various cells and bacteria [12]. It is capable of single

97 particle detection but at least 10 are preferred for better statistics. Organisms can also be tagged with antibodies coated on polystyrene beads. Current prototypes are on glass prepared by laser ablation and photolithography; commercial versions are expected to be on plastic [12]. Competitive assays. Competitive assays, by definition, involve measuring the labeled antibodies that are unbound [4,523]. (It is distinguished from noncompetitive or direct-binding assays where only occupied binding sites are measured. Direct-binding, however, represents a better strategy because it generally gives higher sensitivity [4].) Microfluidic channel layouts and the operational sequence for a two-step competitive assay are illustrated in Dodge et al. [80]. Chromatography-type competitive assays in both single and sequential addition modes are possible [80]. The competitive immunoassay of the drug theophylline in human serum involved an immunoreactor with subsequent capillary electrophoretic separation on the chip and fluorescence detection [25,525]. Sandwich assays. Parallel detection of 25 cytokines was performed in a simple, low-density filter array in a sandwich format where detection limits were at physiological concentrations [526]. In contrast, sandwich ELISAs performed on high-density microarrays have also been done [74,527,528]. They can be used for detection of infectious agents or in cancer diagnostics [74,490]. Examples of other sandwich assays on microarrays include the simultaneous detection of multiple IgG subclasses [8], antigen–antibody reactions on a polyacrylamide gel substrate with immobilized capture molecules [137], and a fluorescence-based immunosensor (as mentioned above) [119] (see also Table 2). Enzymatic assays. Apart from immunoassays, enzyme assays can also be used to screen for certain substrates in a clinical diagnostic setting. The benefits of performing enzymatic assays on microchips are the analytical power and minimal reagent use in microfluidic systems combined with the selectivity and amplification factors that come with biocatalysis [529]. Hence, for clinical testing of substrates, on-chip enzymatic assays have advantages over conventional systems in terms of speed and performance, not to mention sample volume since reagents and the enzymes themselves can be costly. At nanoliter volumes, these chips reduce their enzyme consumption by about four orders of magnitude over standard assays. Enzymes can convert 102–105 substrate molecules to product within 1 s and thus has been used in FIA [529,530] and CE. An enzymatic assay procedurally involves a derivatization reaction where a nondetectable species is transformed into a detectable one [529,533] and hence has steps including mixing, reactions, and separations. Fluid control was via electrokinetic pumping with voltages applied at the enzyme, sample, and buffer reservoirs. The application of this type of on-chip assay to complex samples such as biological fluids would benefit from additional sample processing functions on chip such

98 as cleanup and preconcentration [529]. Wang [529] summarized applications of on-chip enzymatic assays and provided references. Diagnostics Protein chips benefit clinical diagnostics and the diagnosis of complex ailments (e.g., allergies, cancer, and autoimmunity) [1]. Diagnosis of leukemias and lymphomas is currently done using a combination of morphology, immunophenotype, cytochemistry, and karyotype. The different leukemias are differentiated by particular subsets of the 247 cluster of differentiation (CD) antigens on the plasma membrane. Belov et al. [534] constructed arrays with 60 different antibodies against CD antigens. These arrays can capture whole cells because leukocytes express subsets of CD antigens (dependent on leukemia type) on their plasma membranes. The binding pattern of leukocytes from leukemic blood samples on these arrays can be used for diagnosis, as well as drug discovery [9]. Conventional flow cytometry can analyze only up to three CDs in any one assay. Since results from this microarray method compare well with those from flow cytometry, faster and more extensive immunophenotyping should be now possible. Autoimmune diagnostics profile the presence or absence as well as specificity of antibodies involved in autoimmune diseases [348]. The presence of certain serum antibodies then would enable the diagnosis of various autoimmune diseases. An example is the unambiguous diagnosis of systemic rheumatic disease based on simultaneous screening 18 autoantigens (diagnostic markers) by microarray ELISA [121]. Antibody titers were determined with high accuracy with less than 1 ml of patient serum [121]. It is also possible to diagnose allergies by using reverse immunoassays (i.e., immobilized antigens or tissue extracts) in microarray format [390,535]. Biomarkers The discovery of biomarkers, both for prognostics and diagnostics, is a very active research field. Protein expression profiling of cancer-specific biomarkers is a relatively recent development in diagnostics [59]. Sera and other body fluids, both in normal and diseased states, are profiled to identify novel biomarker candidates. Sometimes large antibody arrays are used [348]. Since screening single biomarkers cannot properly diagnose the disease and their clinical value is questionable (e.g., in the case of CA125, an ovarian cancer protein biomarker) [59], protein microarrays are used to measure the abundance of specific proteins and characterize patterns of up to thousands of proteins. Haab et al. [375] tested the specificity and sensitivity of their assay with 115 antibody/antigen pairs in complex solutions using quantitative comparative fluorescence. Of all the arrayed samples, 50% of the antigens and 20% of the antibodies gave specific and accurate results on their binding partner at or below 0.34 and 1.6 mg/ml, respectively. This means the sensitivities are sufficient for many clinically relevant proteins in blood samples, even more so since antibodies used in clinical

99 diagnostics have much higher affinities than those used in research. For example, threshold prognostic concentration values of various breast cancer markers are as follows: c-erbB-2, 15 mg/ml; CEA, 5 mg/ml; and CA 15.3, 35 mg/ml [375,536], all within the limit detected in this study. Other concentration ranges for other clinically relevant proteins were cited [375]. Such an approach, combinatorial biomarker profiling, covers multiple cellular pathways thus increasing the probability of correct diagnosis, or even prognosis. It is promising in early cancer detection and in selecting therapeutic strategies for specific patient groups. In therapeutic strategies that fail, a compensatory mechanism may lead to the discovery of secondary drug targets. Wagner and Kim [59] listed differentially expressed proteins relevant in oral cavity and prostate cancers. Prognostic and diagnostic markers can also be analyzed via tissue microarrays (TMA). Three TMA studies on breast cancer corroborate the estrogen and progesterone receptors and the HER-2 oncoprotein as biomarkers [464,466,538,539]. Disease monitoring Organ and disease-specific protein arrays can be used in expression profiling to identify disease-related proteins. These arrays can also quantify specific sample proteins in terms of abundance, localization, and modification [146] as the disease progresses. On a large scale, arrays can provide the means to characterize the pattern for clinical or research purposes [375]. Hence, there is overlap with applications in diagnostics and drug discovery. By studying protein regulation and expression, clinicians and researchers alike can predict predisposition to disease. Once the disease is manifest, data gathered can be used for monitoring disease progression, determining response to treatment, and providing overall prognosis [348]. It is also possible to screen for molecular markers and diagnostic and/or therapeutic targets in patient-matched tissue during disease progression [120]. Kononen et al. [465] used tissue arrays to do molecular profiling of tumor specimens obtained from 1000 tissue biopsies. This involved the simultaneous analysis of samples from many different patients at different stages of the disease. Englert et al. [468] also used tissue arrays in rapid molecular profiling of tumor samples (see also Principles of Design and Operation, Cell and tissue biochips, Tissue microarrays). Paweletz et al. [120] used protein arrays to support a hypothetical model of prostate cancer progression. The researchers needed to gain insight into certain molecular events in how the disease progressed in individual patients. Whole protein lysates obtained from laser capture microdissection of histopathologically relevant cells were immobilized in reverse-phase arrays. They had a 20 mm
30 mm slide with 1000 individual cellular lysates from 1 ml of lysate per spot. They expect that protein arrays will be used in the highthroughput analysis of the proteomic changes in tissue cells before and after treatment of the disease [120].

100 Biological and medical research The use of biochips in basic biological and medical research has provided information that was heretofore inaccessible. Molecular profiles of cell types provided by tissue microarrays and the layered expression scanning technique [464,468] are relevant in understanding biological processes. In addition, many other types of experiments performed on microchips from assays to mimicking cellular migration in tissues and neuronal behavior in vivo give new insight into processes relevant in cellular biology and medicine. Biochemical assays Technology has led to advances in the understanding of genes and proteins particularly in how these biomolecules function in a cell. Comparing functional properties as well as screening for novel substrates can be performed simultaneously in an array format. Both biochemical [32,540] and enzymatic [118,125,137,542] assays on chips have been performed (see also Table 2). Sensors Cellular responses have been studied with the use of biosensor chips that incorporate several components to detect different types of response. The multiparametric microsensor chip is one example [47–49] (see Drug screening and testing). Another is a cell-based biosensor that detects a differential response in cell populations when exposed to irritants [10,543]. Cell manipulation and sorting The ability to manipulate and separate cells facilitates the further study of cells and has practical applications. Microchip cell sorters have advantages over conventional FACS machines: reduced background fluorescence from precise control of the interrogation volume, increased sensitivity through the use of optics with high aperature numbers, and chip disposability [16,275,544]. Although the throughput is higher with a FACS machine, a chip format allows more flexibility in the types of experiments (including kinetic studies) that can be performed as well as the incorporation of other chip functions [16]. Electrokinetic manipulation of cells was done by Li and Harrison [26]. Flow cytometry on a microchip was used to sort and isolate fused cells and their viability in cell culture can be checked on chip as well [540,545]. (See also Principles of design and operation, Cell and tissue biochips, Cells on chips; and Biochip applications, Drug discovery and drug development, Proteomics and drug discovery tools, Bioparticle manipulation and separation.) Cell shape and cellular behavior A fundamental question in human biology that also has a disease-relevant aspect is the central role of cell shape in cell function. Cells micropatterned by SAMs

101 of alkanethiolates on gold were used to address these questions. As a matter of fact, the control of cell position is central to the developments in cellular biosensors and tissue engineering. It has been observed that nerve cells attach to and extend neurites only within patterned adhesive areas on chips [114,175,185,546]. The size and shape of these patterns affect cellular behavior [175,187]. By progressively reducing the micropatterned islands of extracellular matrix where endothelial cells were confined to, the transition from cell growth to apoptosis was observed. Therefore, apoptosis increases with ‘‘cell rounding’’ [52]. Furthermore, understanding the mechanism involved in apoptotic signaling implies the possible development of new anticancer therapies, e.g., through the inhibition of angiogenesis (capillary formation) to block solid tumor growth. Cell growth and differentiation are responses to changes in ligand composition within the ECM [547–549], so is tumor metastasis [547,550]. Cell shape also seems to regulate differentiation [187,548]. Studies of cellular processes were performed using a dynamic substrate where immobilized ligands were electrochemically released [547]. Micropatterning technology is critical then for controlling cellular responses in vitro and for future work in tissue engineering where cell–cell and cell–ECM interactions can be studied [52]. Cell fusion has also been studied by microfluidics [540]. Cell migration and protein gradients Generally, by immobilization techniques, cell migration can be controlled by manipulating substrate adhesiveness [175]. However, gradients of various molecules may be the determining factor in cellular migration within specific locations in a particular tissue. Hence, studies on cellular migration have benefited from biochips with gradients of biological molecules. However, gradient profile (the degree of steepness) may be the key in effecting this migratory behavior. Concentration differences are detected by receptors on the cell membrane; thus, only when these receptors detect a change will the cell elicit a response. For example, only when a sharp change is sensed do neutrophils exhibit chemotactic behavior in an interleukin-8 gradient [240]. Protein gradients in microfluidic systems can be used to study chemotaxis (directed cellular migration in chemoattractant gradients). Cell receptors bound to certain soluble molecules are potential targets in cancer metastasis, wound healing, and inflammation [240,551–553]. Other examples of gradients of physiological relevance include the morphogen gradient in Drosophila embryos [554,555] and angiogenic activity as a function of distance to the tumor site [556]. Microgradients have been made in two ways: by the use of photocrosslinkers [175] and by multistream laminar flow [540]. With the use of photolinkers, a laser rasters through the chip surface at progressively faster speeds where the amount of gradient molecules immobilized at a particular chip location is a function of the exposure time to the laser. Multistream laminar flow, on the other hand, involves protein and chemical gradients formed within microchannels [557–560]. Gradients can consist of ECM proteins, growth factors, and peptides [175].

102 Tissue engineering Different types of basic biological research contribute to the understanding of tissue structures [52,187,546,561]. Certain cells may have to be situated at specific locations to achieve organized structures relevant to tissue formation [51]. Tissues formed in vitro can study how cells native to these tissues migrate. Cell migration enables the investigation of matrices and cellular paths that aid the formation of ‘‘innervated, vascularized tissue’’ [175]. The use of 3D microfluidic systems can study functionality in tissue architecture by addressing the molecular interactions between cells and cell types that are the bases in processes such as embryonic morphogenesis, formation of the blood–brain barrier, and tumor angiogenesis [53]. Toxicity An example of arrays used in medical research was the study of electrophysiological parameters that are influenced by neurotoxicity in the brain. In the future, this research can help in understanding how electrophysiological activity is developed or organized in the brain. It can also lead to screening methods that detect toxic effects that lead to neurodenegeneration. Rat hippocampal and corticostriatal brain slice cultures were grown on perforated silicon in microelectrode arrays [70,562]. The neurons in culture developed normally and showed the susceptibility to the neurotoxin trimethyltin (TMT) and the excitotoxin NMDA (N-methyl-D-aspartate), as would neurons in vivo. Conclusion Recent advances in miniaturization, incorporation of complex components and adaptation of chips for non-DNA molecules signals a new era in biology. The shift towards studying biology as a complex interacting system (systems biology) requires the use of robust and reliable experimental systems that are amenable to analysis and quantitation of complex behavior. The use of biochips represents a platform to accelerate studies in systems biology and its applications in medicine and other fields are only limited by one’s imagination. Further advances in this field and nanotechnology will lead to convergence of the physical, chemical, and biological sciences with great impact on biotechnology and improving quality of life. The economic potential is enormous. Protein arrays were a $45 million business in 2000 and expected to be $500 million by 2006 [79] whereas microfluidics alone has been estimated to be a $1.5 billion business by 2005 [38]. The biochip business as a whole was already a $300 million business in 2000 and is growing annually at 40% [38]. However, for the nascent technologies to reach its full potential, several critical factors need to be addressed. Currently, the main limiting factor is the availability of appropriate reagents that can be incorporated on chips and not the technology for fabricating the chip itself. Stability of the capture reagents or

103 proteins needs to be addressed in order to preserve their functionality and to facilitate uniform coupling of these reagents on the chip for reliable detection. Continuous innovation is rapidly developing in search of alternative ligands to antibodies and proteins. Advances in detection technology will help in addressing specificity and increasing signal-to-noise ratio. Another critical factor will be to determine which specific capture agents should be included in a chip. Arraying thousands of proteins or capture reagents is meaningless if the results cannot be interpreted, thus it would be necessary to array the proper molecules. Regardless of these limitations, this field is still in its infancy yet making giant strides. It will be clear in time which technologies are more robust. Nevertheless, exciting developments in the technology and applications are expected in the coming years. 1. Acknowledgments We are grateful to Larry Gold, Olli-P. Kallioniemi, and Thomas Joos for providing reprints of their articles; Holger Becker, Konrad Bu¨ssow and Gerald Walter, Jed Harrison, Stephen Jacobson, Chad Mirkin and David Ginger, Christof Niemeyer, Menno Prins, Thomas Joos, Meike Kuschel, Gavin MacBeath, George M. Whitesides, Ravi Kane, and Shuichi Takayama, for granting permission and/or providing figures from their publications; Bernard Westerop who furnished us with all the figures we needed from Elsevier publications; Agilent Technologies, the American Chemical Society, the American Association for the Advancement of Science, the National Academy of Sciences (USA), and Wiley-VCH Verlag GmbH & Co. KGaA, who gave copyright permission for the figures used; Leopold L. Ilag for help with the references; and to editor Raafat El-Gewely and daughter Andrea N. Ilag for their patience since this review was much longer than expected. Appendix 1: Abbreviations AP AFM AMI ATP BCE BNP CAD CCD CE CHF CK-MB COC

alkaline phosphatase atomic force microscopy acute myocardial infarction (heart attack) adenosine triphosphate bovine adrenal capillary endothelial (cells) B-type natriuretic peptide (cardiac hormone) computer-aided design charge-coupled device capillary electrophoresis congestive heart failure creatinine kinase-MB cycloolefin copolymer

104 CNC CZE DOA DME DPN EBL ECL ECM ECP ECV EDA ELISA EOF EPON SU-8 ESD ESI FFIB FIA FKBP12 FRET GFP GOPS GPCR HEK HFTS HMG-CoA HTS IDES ISFET LIF LIGA MALDI MEMS MHA MHD MIMIC MIP MS NIL NSB ODT OTCS OTMS OTS

computer numerical control (machining) capillary zone electrophoresis drugs of abuse drug metabolizing enzymes dip-pen nanolithography electron beam lithography enhanced chemiluminescence (detection) extracellular matrix electrocapillary pressure human bladder cancer cells [53] N-(2-aminoethyl)(2-aminopropyl) trimethoxysilane enzyme-linked immunosorbent assay (see Glossary) electroosmotic flow (see Glossary) electrospray deposition electrospray ionization finely focused ion beam (lithography) flow injection analysis human immunophilin fluorescence resonance energy transfer green fluorescent protein 3-glycidoxypropyldimethyl ethoxysilane G-protein coupled receptor Human embryonic kidney (cells) heptadeca-fluoro-1,1,2,2-tetrahydrodecyltrichlorosilane 3-hydroxy-3-methylglutaryl coenzyme A high throughput screening interdigitized electrode structures Ion-sensitive field effect transistor laser-induced fluorescence (see Glossary) matrix-assisted laser desorption ionization microelectromechanical systems (American usage) 16-mercaptohexadecanoic acid magnetohydrodynamics micromolding in capillaries molecularly imprinted polymer mass spectrometer; mass spectrometry nano-imprint lithography nonspecific binding 1-octadecanethiol n-octadecyltrichlorosilane, more reactive than OTMS n-octadecyltrimethylsilane N-octadecyltrichlorosilane

105 PBS PC PDMS PEEK PET PLL-PEG PMMA PS PTMP PVDF Re RCA RMS SAMs SBIR SDS-PAGE SELDI SELEX SERS SPL SPR SU-8 Tg TM-SFM TMA TMV mCP mTAS

phosphate buffered saline polycarbonate poly(dimethylsiloxane) polyetheretherketone poly(ethylene terephthalate) poly(L-lysine) grafted with poly(ethylene glycol), polycationic polymethylmethacrylate polystyrene pentaerythritol-tetrakis(3-mercaptopropionate) polyvinylidene fluoride Reynolds number (see Glossary) rolling circle amplification root mean square self-assembled monolayers small business innovation research sodium dodecyl sulfate–polyacrylamide gel electrophoresis surface-enhanced laser desorption ionization systematic evolution of ligands by exponential enrichment) surface-enhanced Raman Spectroscopy scanning probe lithography surface plasmon resonance (see Glossary) glass transition temperature (see Glossary) tapping mode scanning force microscopy tissue microarray tobacco mosaic virus microcontact printing micro Total Analysis Systems

Appendix 2: Glossary Bold-faced words indicate terms that are defined in this Glossary. Actuator. (1) A peripheral device which translates electrical signals into mechanical actions; e.g., a stepper motor which acts on an electrical signal received from a computer instructing it to turn its shaft a certain number of degrees or a certain number of rotations. [FDA, US Glossary of Computerized System and Software Development Terminology 1998, http://www.fda.gov/ora/ inspect_ref/igs/gloss.html] (2) An actuator, the reverse of a sensor, is a device that converts an electrical signal to an action. Actuators are further divided into three categories: simple actuators that move valves or beams using one simple physical law, micromotors, more complex in the design and the possibilities, and microrobots which are the latest release in microtechnology. [National Textile Center, ‘‘MEMS: An Overview’’ 1998,

106 http://www2.ncsu.edu/unity/lockers/project/ntcprojects/projects/F98-S12/memsoverview.html, also in http://www.genomicglossaries.com/content/labels_sig_and_detection_gloss.asp] Atomic force microscopy (AFM). Scanning probe microscopy (SPM) involves methods of imaging surfaces with atomic or near-atomic resolution wherein a small tip is scanned across the surface of a sample in order to construct a 3D image of the surface. AFM is a type of SPM where, in particular, any type of surface can be probed by the molecular forces exerted by the surface against the tip. The sample must be less than 25 mm thick, up to 200 mm in diameter, and have an area of interest less than 5 mm total relief. Advantages of the technique include high lateral and vertical resolution, little or no sample preparation or alteration, digital data acquisition, manipulation, and storage of surface images [http://www.cea.com/tech.htm#afm1]. The newer proximal probe can be used in a number of modes of operation. Included are a contact mode, in which the tip touches the specimen surface and senses internuclear repulsive forces between nuclei in the tip and sample, and a noncontact mode that exploits electrostatic or van der Waals forces [http://www.sunyx.de/sunyx_engl/htm/tech/ glossary.html] Air plasma. See Plasma Anisotropic. Not exhibiting the same physical properties in all directions [http://www.mdacomposites.org/Glossary.htm] Anisotropic etching. (1) Etching which involves different etch rates in different directions in the material [http://www.csa.com/hottopics/mems/gloss.html]. (2) Etching that results from reactive ion etching (RIE) which can etch surfaces normal to the direction of bombarding ions at a faster rate than surfaces parallel to the ion motion [adapted from http://www.icknowledge.com/glossary/r.html]. See also Reactive Ion Etch Aspect ratio. (1) Ratio of length to diameter [http://www.mdacomposites.org/Glossary.htm]. (2) The ratio of width-to-height or height-to-width in a feature formed by ablation or lithography [http://www.tamsci.com/library/library.html] Bas-relief. Patterns or structures that are raised or protrudes from a flat background [Webster’s New World Dictionary ß 1990 Simon & Schuster, Inc.] Capillary pressure. (1) Difference in pressure across the interface between two fluids [http://water.usgs.gov/pubs/wri/wri974097/text/glossary.html]. (2) The difference in the pressure for the nonwetting phase and the pressure for the wetting phase [http://www.ces.clemson.edu/ees/lee/napl/interfacial_tension.htm]. See website for a more in-depth explanation Casting. A microfabrication method that can be used for replication. The elastomer precursor and its curing agent are poured over the mold. After curing, which may take several hours, the soft elastomer copy or replica is peeled off the mold. The formed microstructures can simply be placed against a planar surface, e.g., a glass slide, a plastic sheet, or a printed circuit/ board containing electrodes to form closed channels (adapted from Ref. [61]).

107 Charge-coupled device (CCD) and CCD camera. A CCD is a charge-coupled device – a silicon chip whose surface is divided into light-sensitive pixels. When a photon (light particle) hits a pixel, it registers a tiny electric charge that can be counted. With large pixel arrays and high sensitivity, CCDs can create highresolution images under a variety of light conditions. A CCD camera incorporates a CCD to take such pictures. [Xenogen website, Glossary, http:// www.xenogen.com/glossary.html; also in http://www.genomicglossaries.com/ content/imaging_glossary.asp and http://www.itl.arizona.edu/Library/glossary. htm (with animation on a CCD device).] Chemisorption. (1) Adsorption involving very strong bonding forces [http://www.swbic.org/education/env-engr/tools/glossary.html#C]. (2) A process, related to adsorption, in which atoms or molecules of reacting substances are held to the surface atoms of a catalyst by electrostatic forces having about the same strength as chemical bonds. Chemisorption differs from adsorption chiefly in the strength of the bonding, which is much stronger in chemisorption [http://www.msu.edu/ svobodar/glossary.htm]. (3) The assimilation of gas, vapor, or dissolved matter by the surface of another substance resulting from electron transfer and a bond-forming chemical reaction between the surface and the gas, vapor, or dissolved matter [http://www.lungusa.org/pub/cleaners/air_clean_app4.html] Chemoattractant. A substance exhibiting chemical properties that attract biological cells during chemotaxis [http://www.neuroprobe.com/global/glossary.html] Chemotaxis. (1) Directed migration of cells in gradients of soluble molecules termed chemoattractants [240]. (2) Directional movement (migration) of biological cells or organisms in response to concentration gradients of chemicals, whereby the cells are attracted or repelled by substances exhibiting chemical properties [http://www.neuroprobe.com/global/glossary.html] Confocal laser imaging. See Confocal Scanning Laser Microscopy Confocal Scanning Laser Microscopy (CSLM). Also referred to as Laser Scanning Confocal Microscopy (LSCM), is used to obtain high-resolution images and 3-D reconstructions of a variety of biological specimens. In CSLM, a laser light beam is expanded to make optimal use of the optics in the objective. Through an x–y deflection mechanism, this beam is turned into a scanning beam, focused to a small spot by an objective lens onto a fluorescent specimen. The mixture of reflected light and emitted fluorescent light is captured by the same objective and (after conversion into a static beam by the x–y scanner device) is focused onto a photodetector (photomultiplier) via a dichroic mirror (beam splitter). The reflected light is deviated by the dichroic mirror while the emitted fluorescent light passes through in the direction of the photomultiplier. A confocal aperture (pinhole) is placed in front of the photodetector, such that the fluorescent light (not the reflected light) from points on the specimen that are not within the focal plane (the so-called out-of-focus light) where the laser beam was focused will be largely obstructed by the pinhole. In this way, out-of-focus

108 information (both above and below the focal plane) is greatly reduced. This becomes especially important when dealing with thick specimens. The spot that is focused on the center of the pinhole is often referred to as the ‘‘confocal spot.’’ (A simple arrangement of a LSCM illustrating the confocal principle can be viewed in the website.) A 2-D image of a small partial volume of the specimen centered on the focal plane (referred to as an optical section) is generated by performing a raster sweep of the specimen at that focal plane. As the laser scans across the specimen, the analog light signal, detected by the photomultiplier, is converted into a digital signal, contributing to a pixel-based image displayed on the attached computer monitor. The relative intensity of the fluorescent light, emitted from the spot that the laser hits, corresponds to the intensity of the resulting pixel in the image (typically 8-bit grayscale). The plane of focus (Z-plane) is selected by a computercontrolled fine-stepping motor, which moves the microscope stage up and down. Typical focus motors can adjust the focal plane in as little as 0.1 micron increments. A 3-D reconstruction of a specimen can be generated by stacking 2-D optical sections collected in series. It should be noted that most laser scanning confocal microscopes consist of a confocal unit attached to a conventional fluorescence microscope. (The general setup of an entire LSCM system is illustrated in the website.) [Adapted from http://www.cs.ubc.ca/spider/ladic/intro.html.] Compared to another form of laser scanning microscopy, multiphoton laser scanning microscopy (MPLSM), CLSM is less sensitive. MPLSM gives a sharper image and the photomultiplier is situated very close to the specimen without all the intervening optics and pinhole as in the case with CLSM [Bruce Jenks, Department of Cellular Animal Physiology, University of Nijmegen, Netherlands, http://www.sci.kun.nl/celanphy/Bruce%20web/scanning%20microscopy.htm, also in http://www.genomicglossaries.com/content/Microscopy.asp]. Conformal contact. (1) The condition where a mold is in contact with a master or a substrate with a mold such that there are no gaps and all the features are represented in the other material in reverse. (2) To conform to the configuration of the object in contact with [adapted from the definition of conformal coating in http://developer.intel.ru/download/design/packtach/gloss.pdf] Contact angle. (1) The angle of intersection of the interface between two fluids at a solid surface. It is measured from the solid surface through the water phase, or in an oil and gas test through the oil phase [http://www.glossary. oilfield.slb.com/Display.cfm?Term ¼ contact%20angle]. (2) Depending on the surface tension of different materials, a well-defined angle is obtained for the contact of a liquid on a solid. The contact angle is defined as the angle between the tangent to solid–vapor interface and the line of the solid–liquid interface. The angle is chosen to be that containing the liquid. The angle can be zero if the liquid fully wets the solid surface; 180 for immiscible liquids; and 90 for meniscus maintaining fluids. The advancing and receding contact angles of a liquid on a surface are often different [http://www.sunyx.de/sunyx_engl/htm/

109 tech/glossary. html, with additional information from http://simscience.org/ membranes/advanced/glossary/c.html] Copolymer. Polymers that are formed from one or more monomers so that the molecule consists of chemically heterogeneous components [http://www.ch.kcl.ac.uk/kclchem/staff/arr/glossold.HTM#Colloid] Cure. (1) A change in the physical properties of a material via chemical reaction or by reaction to temperature–time profile [http://www.tkb-4u.com/ glossarylist/glossary_ac.php]. (2) To irreversibly change the properties of a thermosetting resin by chemical reaction, that is, condensation, ring closure, or addition. Cure may be accomplished by addition of curing (cross-linking) agents, with or without heat and pressure [http://www.mdacomposites.org/ Glossary.htm]. (3) The irreversible process of polymerizing a thermosetting epoxy in a temperature-time profile [http://e-teknet.com/pcb_glossary.htm] Curing agent. A catalytic or reactive agent that, when added to a resin, causes polymerization. Also called hardener [http://www.mdacomposites.org/ Glossary.htm] Dicing. The action of slicing a silicon wafer into its respective parts is called die. Due to the precision needed for such a feat, a special saw, similar to a circular saw only with a blade approximately 2 inches in diameter, is used for dicing silicon [adapted from http://www.itl.arizona.edu/Library/ glossary.htm] Dielectrophoresis. The induced motion of polarizable particles in nonuniform alternating electric fields. The effect is governed by the relative magnitudes of the dielectric properties of the medium and the particles [296,298] Dip pen nanolithography (DPN). An AFM-based soft-lithographic technique where stable nanostructures are formed from the chemisorption of molecules that are transferred to the substrate surface. When T-substituted alkanethiols are patterned on a gold substrate, a monolayer is formed in which the thiol headgroups form relatively strong bonds to the gold and the alkane chains extend roughly perpendicular to surface. The thiol lattice formed is identical to that of a monolayer obtained via solution deposition of alkanethiols on gold. Creating nanostructures using DPN is a single-step process which does not require the use of resists. One of the most important attributes of DPN is that because the same device is used to image and write a pattern, patterns of multiple molecular inks can be formed on the same substrate in very high alignment [Chad Mirkin group, Department of Chemistry, Northwestern University, US ‘‘Surface Science and Dip Pen Nanolithography’’ 2001, adapted from http://www.chem.northwestern.edu/  mkngrp/dippen.html, also in http://www.genomicglossaries.com/content/ miniaturization_glossary.asp]. Dry etch. See Etch Electrocapillarity. (1) The change in surface tension due to electric potential gradients in the presence of a surface charge [Ken Casson, Department of Chemical Engineering, University of Alabama,

110 http://www.eng.ua.edu/ checlass/Seminar/spring2001/0426.html]. (2) An actuation principle that uses surface tension gradient caused by electric potential difference [Jung hoon Lee and Chang-Jin ‘‘CJ’’ Kim, Mechanical and Aerospace Engineering Department, University of California, Los Angeles, CA, http://cjmems.seas.ucla.edu/papers/Jung_imece98.PDF]. (3) The dependence of the interfacial tension on the electrical state of the interphase [IUPAC Compendium of Chemical Terminology, 1986, 58, 446. 2nd ed. 1997, http:// www.iupac.org/goldbook/E01941.pdf] Electron beam lithography (EBL). A method of fabricating sub-micron and nanoscale features by exposing electrically sensitive surfaces to an electron beam. The method is similar to photolithography, but uses electrons rather than photons. Since the wavelength of an electron is far smaller than that of a photon, diffraction is not a limit to the resolution. While EBL is more expensive and less parallel than photolithography, its resolution is higher and it is frequently used to create photolithographic masks [http://www.allaboutmems.com/glossary.html#E] Electroosmotic flow (EOF). Most surfaces have an electric charge and a double layer of counter ions forms at the walls of the microchannels. When an electric field is applied across the channel, the ions in the double layer move toward the electrode of opposite polarity. This movement is transferred by viscous forces into convective motion of the bulk fluid and a uniform velocity profile is created [11]. Enzyme-linked immunosorbent assay (ELISA). An immunoassay for detecting antigens involving the coupling of an enzyme (e.g., alkaline phosphatase or horseradish peroxidase) to the detection reagent such as antibody that leads to a colorimetric readout upon reaction in the presence of a substrate. The direct ELISA assay format starts with an antigen where an antibody selectively binds to the antigen and a secondary antibody covalently linked to an enzyme recognizes the first antibody leading to a colorimetric readout upon addition of the appropriate substrate. The ELISA ‘‘sandwich assay’’, utilizes two antibodies that simultaneously bind the same antigen: one antibody is immobilized onto the surface, and the other one is fluorescently labeled or conjugated to an enzyme that can produce a fluorescent, luminescent or colored product when supplied with the appropriate substrate. The antigen is between two antibodies hence it has been termed a sandwich assay (sandwich assay definition adapted from Ref. [9]) EPON SU-8. See SU-8 Etch. (1) A process using a chemical bath (wet etch) or a plasma (dry etch) that removes unwanted substances from the wafer surface [http://www.logicorp.net/glossary_terms.htm, http://www.forums.pctechguide.com/glossary/wordfind.php?wordInput ¼ Etch]. (2) Removal of portions of a layer of conductive material from a usually insulating base through chemical or electrolytic means. In wet etching, the material is dissolved when immersed in a chemical solution. In dry etching, the material is sputtered or dissolved using reactive ions or a vapor phase etchant

111 [http://www.csa.com/hottopics/mems/gloss.html]. Hydrofluoric acid (HF) or potassium hydroxide (KOH) are used as etchants [61] Evanescent field. In a waveguide, a time-varying field having an amplitude that decreases monotonically as a function of transverse radial distance from the waveguide, but without an accompanying phase shift [http://www.atis.org/tg2k/_evanescent_field.html, http://www.bandwidthmarket.com/resources/glossary/E6.html, http://www.its.bldrdoc.gov/projects/devglossary/_evanescent_field.html] Expression cloning. A molecular cloning approach in which expression of the gene to produce an identifiable gene-product is the basis of the selection of the desired clone. One such strategy uses antibodies to recognize epitopes expressed as part of a fusion protein in E. coli. Another variation involves the production of protein molecules by in vitro transcription of cloned cDNAs, followed by microinjection into Xenopus oocytes to allow expression of ion channels for patch-clamp methods [adapted from http://www.pain.med.umn.edu/csn/e66/66-E.html]. Finite element. A discrete entity used to subdivide the geometry of a structure in order to create an idealized structure for finite element analysis. Each finite element is a simple shape (such as a rectangle or a triangle) for which the software has information to write the governing equations in the form of a stiffness matrix. The unknowns for each element are the displacements at the points where the elements are connected. The behavior of the structure is approximated by the aggregate behavior of all of the elements [http://www.ces.clemson.edu/ lonny/courses/me818/glossary.html]. Finite element analysis. (1) An approximate method for calculating the behavior of a real structure. An idealized model structure is created by subdividing the geometry of a structure into elements which are connected at nodes. The method is based on the generalized method of weighted residuals using locally based approximations [http://www.ces.clemson.edu/ lonny/ courses/me818/glossary.html]. (2) A computer-based analysis method which calculates the response of the model by solving a set of simultaneous equations that represent the behavior of the structure under external loading [http:// www.cosmosm.com/support/glossary.htm]. (3) A technique for predicting the response of structures and materials to environmental factors such as forces, heat and vibration. The process starts with the creation of a geometric model. Then, the model is subdivided (meshed) into small pieces (elements) of simple shapes connected at specific node points. In this manner, the stress–strain relationships are more easily approximated. Finally, the material behavior and the boundary conditions are applied to each element [http://www.cosmosm.com/support/ fea1.htm] Glass transition temperature (Tg). (1) The temperature at which many polymers/high molecular weight substances begin to solidify. Cooling below this temperature results in a solid that is as hard as glass and brittle [61]. (2) The temperature at which a polymer changes from hard and brittle to soft and pliable

112 [http://www.psrc.usm.edu/macrog/glossary.htm, http://www.epolybags.com/GLOSSARY.htm]. Hydrogel. (1) A water-based gel; a gel whose liquid constituent is water [http://www.solgel.com/educational/glossary.htm]. (2) The hydrogel polymer network is a cross-linked poly-(6-acryloyl--O-methyl-galactopyranoside, a highly hydrophilic polymer with ordered pendant-type sugar repeat units. When permitted to swell in protein solution, an initially dry polymer will reach an equilibrium solution content of about 99.1 wt% [212,577,578] Injection molding. One of the widespread standard processes to fabricate polymer parts. The process starts with the raw polymer material in granular form. These granules are fed into the cylinder (Fig. 5), a heated screw, where the pellets start to melt. This melt is then transported forward toward the mole cavity. Typical temperatures in this region range from 200 C (for polymers such as PMMA and PS), over  280 C (for PC), up to  350 C for materials like PEEK. The molten material is then injected under a high pressure (typically 60–100 MPa ¼ 60–1000 bar) into the evacuated cavity, which contains the mold insert/as the master structure. With microfabrication, therefore less material injected into the cavity and the surface-to-volume ratio increases, the cavity has to be heated closer to the melting point of the polymer material to allow the polymer to flow into the small structure of the mold insert. The cavity will then be cooled to allow the ejection of the microstructured part. This process, called variotherm, allows the fabrication of smaller structures than the equivalent cold-cavity process for macroscopic systems, but increases the cycle time due to the heating and cooling (adapted from Ref. [61]) Joule heating. The production of heat in a conductor as a result of the passage of an electric current through the conductor. The quantity of heat produced is given by Joule’s law (Q ¼ I2Rt where Q is the heat produced when an electric current I flows through a resistance R for a given time t.) [http:// physics.miningco.com/library/dict/bldefjouleheating.htm, http://physics.about. com/library/dict/bldefjouleslaw.htm?terms ¼ Joule%27s þ law] Laminar flow. Streamlined flow with no turbulence [http://www.allaboutmems.com/glossary.html#E]. See also Reynolds number Laser ablation. The photoablation process involves absorption of a shortwavelength pulse to break covalent bonds in long-chain polymer molecules with production of a shock wave that ejects decomposed polymer fragments [75,109]. A typical laser ablation setup consists of an excimer laser (ArF: 193 nm, 10–100 Hz; or KrF: 248 nm, several kHz), a mask or aperture, and an xy-table on which the substrate is mounted. The mask defines the ablated region, while moving the substrate on the x–y stage underneath the mask completes the pattern. Depending on the substrate material and on the energy available per laser pulse, typical ablation rates per laser pulse range between some hundred nanometers and 5 mm. The accuracy of the process in the x–y direction is largely determined by the energy distribution in the beam (which is normally constant to about 5% across the mask or aperture) and the quality of the x–y stage.

113 Typically, they are of the order of a few microns. Depth control, which ultimately also determines the wall roughness, is of the order of 0.1 mm. Due to the interaction of the laser light and the polymer material, however, certain surface modifications are induced in comparison to the untreated material [61,108]. See also Pulsed lasers. Layering. (1) Providing layers of various materials to complete the fabrication of components on the silicon surface [http://www.cabotcmp.com/glossary_semiconductor.htm]. (2) It is compatible with integrated circuit (IC) technologies and involves the growth of thin layers of polymers on planar substances and the use of sacrificial layers to create open volumes between these layers. A thin layer of parylene is deposited onto a substrate, either silicon or polycarbonate. Onto this layer, metal can be deposited using evaporation or sputtering methods to form electrode structures. This structure is then covered with a thin chromium layer and a layer of a sacrificial polymer. The thickness of this sacrificial polymer defines the channel height. The next step consists of depositing an additional layer of parylene on top of this structure to form the channel walls and cover. This represents a big advantage because the closing of the channel structure is naturally included in this fabrication method. Through-holes are defined in the parylene which allow fluid connections in the completed system as well as access of the etchant (usually acetone) for the subsequent sacrificial etch. Due to the comparatively small channel cross-section, this etch can take several hours as the dissolved material can only be transported by diffusion. Cast silicone structures on top of the parylene layer then define fluid reservoirs (see Fig. 13) [61,280–282] LIGA. A German acronym for a microfabrication technique that combines lithography (Lithographie), electroplating (Galvanoformung, Galvanoplastie), and molding (Abformung) [41,61]. Originating from the Nuclear Research Center in Karlsruhe, Germany, the technique involves deep-etch X-ray lithography, electroplating, and injection molding. [http://ranier.hq.nasa.gov/Sensors_page/ Terms.html]. It is suitable for making parts with depths significantly greater than lateral dimensions from metals, metal alloys, plastics or ceramics. Synchrotron radiation is used to create a pattern in an X-ray resist, usually PMMA. The use of synchrotron radiation allows lateral feature sizes that are as small as a few microns in dimension, and straight, smooth vertical sidewalls that can be as tall as several millimeters. Once the PMMA is exposed, it is chemically developed and used as a mold for electroforming metal or metal alloys [http://www.eetimes.com/reshaping/microstructures/OEG20020912S0029]. Lithography. The transfer of a pattern or image from one medium to another, as from a mask to a wafer. Photolithography involves the use of light to effect this pattern transfer. Microlithography refers to the process as applied to images with features in the micrometer range [http://www.cabotcmp.com/glossary_semiconductor.htm], also in [61]. See also Microlithography, Photolithography. Magnetohydrodynamics. Better known in the astrophysics of plasmas and liquid metal pumping, MHD is the study of plasma motion and dynamics in the presence of a magnetic field [http://www.spacescience.org/ExploringSpace/

114 Glossary/1.html]. It involves three physical fields that are at right angles to each other: electric, magnetic, and flow. Application of an electric and magnetic field in small channels or microscopic reactor or sensor vessels containing aqueous or nonaqueous solution results in controlled flow or stirring [adapted from http://www.uark.edu/depts/cheminfo/uarkchem/facultystaff/faculty/fritsch/]. Mask. A device that acts as a barrier to the passage of a reagent (often light, see Photolithography). A pattern of holes in the mask allows selective passage of reagent and results in a corresponding pattern of reagent deposition or photodeprotection on a surface placed behind the mask [IUPAC Combinatorial Chemistry, also in http://www.genomicglossaries.com/content/microarrays.asp]. See also Photomask Master. A microfabricated mold tool representing the negative (inverse) structure of the desired polymer structure [61]; a ‘‘negative’’ from which microdevices are replicated. The composition of the master used in production may depend on the production run. Masters made from metal or other hard materials may be used in manufacturing when the production run is large because of their durability; the expense of the master becomes negligible after many uses. For prototyping new devices and for limited run productions, however, metal or silicon molds are time-consuming and expensive to make, especially in a program of research and development where several iterations are necessary for the development of a final design. A master in SU-8 photoresist on a silicon wafer is durable and can be used indefinitely, although proper precaution should be taken by the user against breaking the fragile silicon wafer or when releasing the photoresist from the wafer. Replication of the master as one piece in a hard polymer, e.g., structural polyurethane or epoxy, can further extend its lifetime [10] Micromachining. (1) The application of integrated circuit fabrication processes to the manufacture of miniature machines, sensors and switches [http://www. ultratech.com/products/glossary.shtml]. (2) Machining (i.e., performing various cutting or grinding operations on a piece of work) for the purpose of fabricating memsmechanical parts [http://www.csa.com/hottopics/mems/gloss.html] Microcontact printing (lCP). (1) For patterning, a technique that uses the relief pattern on the surface of an elastomeric PDMS stamp to form patterns of SAMs on the surfaces of substrates. The stamp is ‘inked’ with a solution of an alkanethiol in ethanol, dried, and brought into conformal contact with the gold surface for 10–20 s. The alkanethiol is transferred to the gold substrate only in the regions where the PDMS stamp contacts the substrate. Subsequent exposure of the remaining bare gold substrate to a second alkanethiol generates a surface patterned into regions presenting different terminal groups (see Fig. 9) [51,228,579]. (2) A planar or rolling stamp is used to transfer molecules of the ‘‘ink’’ to the surface of the substrate by contact, only really suited to single layer fabrication [http://www.npl.co.uk/npl/cmmt/functional/func_process.html] Molecularly imprinted polymers (MIPs). A new class of materials that have artificially created receptor structures. Since their discovery in 1972, MIPs have attracted considerable interest from scientists and engineers involved with the

115 development of chromatographic absorbents, membranes, sensors and enzyme and receptor mimics. [Piletsky S et al. Molecular imprinting: at the edge of the third millennium. Trends in Biotechnology 2001 Jan:19(1):9–12, Jan. 2001, also in http://www.genomicglossaries.com/content/biomaterials.asp]. Monolithic. (1) Formed from a single crystal, such as a monolithic silicon chip. (2) Produced in or on a monolithic chip, as a monolithic circuit. A monolithic integrated circuit (MIC) is an IC having elements formed in place on or within a semiconductor substrate, with at least one element being formed within the substrate [http://www.csa.com/hottopics/mems/gloss.html] Microlithography. (1) The science and art of building a mask pattern on top of a substrate (e.g., quartz glass) and then transferring this pattern into another substrate. This process is repeated many times to form an integrated circuit, MEMS, etc. [http://www.tamsci.com/library/glossary.html]. (2) A manufacturing process for producing highly accurate, microscopic, two-dimensional patterns in a photosensitive resist material. These patterns are replicas of a master pattern on a durable photomask, typically made out of a thin patterned layer of chromium on a transparent glass plate [http://www.tamsci.com/library/ glossary.html]. See also Lithography. Multilayer soft lithography. See Soft lithography Multilayer structures. See Soft lithography, Multilayer soft lithography Nanopatterning. Creating patterns on the order of nanometers, the same size as a protein, by placing the proteins in specific locations [116] Nanotechnology. (1) The application of science to developing new materials and processes by manipulating molecular and atomic particles [http:// www.nanoelectronicsplanet.com/glossary/article]. (2) The science of building microscopic devices out of individual molecules or small numbers of molecules. The devices are measured in nanometers. A nanometer is one millionth of a millimeter, or about 10 carbon atoms long. The products of nanotechnology are likely to include tiny mechanical devices and computer circuitry far smaller than is possible with today’s semiconductor technology [http://www.trnmag.com/ Glossary/NglossaryN.html]. Phage. A collective term for viruses that infect bacterial hosts. Phage display. A technique in which phage is engineered to display proteins on the virus particle’s surface by fusing a recombinant peptide or protein (e.g., antibodies) with the virus capsid or coat protein genes. The phage may then be used to screen for specific ligands that bind to an immobilized antigen. Photolithography. (1) A type of lithographic process used in the manufacturing of semiconductor devices, integrated circuits, and photomasks. It comprises applying a layer of material known as photoresist or simply resist, exposing the resist to UV light, developing the exposed resist [http://www.tamsci.com/library/ glossary.html]. (2) Process by which selective masking generates light patterns which direct chemical transformations to certain areas of a photosensitive surface. Coupling of different building blocks to discrete sites may give rise to

116 spatially addressable arrays of compounds. [IUPAC Combinatorial Chemistry, also in http://www.genomicglossaries.com/content/microarrays.asp] See also Lithography, Photoresist Photomask or Photolithographic mask. (1) A template used in photolithography that allows selective exposure of a photosensitive surface [http:// www.allaboutmems.com/glossary.html#E] (2) A plate of material that is transparent at a certain wavelength (e.g., SiO2 at UV wavelengths) coated according to a certain pattern with material that does not transmit (mostly reflects) at the same wavelength (e.g., chrome, aluminum, dielectric, etc.) [http://www.tamsci.com/library/glossary.html] Photoresist. (1) A layer of material that will react when exposed to actinic energy, UV light (or X-ray, e-beam etc.). The resist layer exposed to UV light, in certain portions, undergoes a change in its solubility. It is then developed by washing it with a basic developer solution, thereby removing the nonirradiated (in a negative-tone resist) or irradiated (in a positive tone resist) portions [http://www.tamsci.com/library/glossary.html]. (2) A class of polymers used in microsystem technology and for microfluidic systems. Irradiation, with electrons, ions, X-rays, UV or visible light, leads to a photochemical reaction of the resist material, which is coated onto a carrier substrate (typically either silicon or another polymer). In the case of the so-called positive tone resists (or singly positive resists), the solubility of the irradiated areas increases; in negative tone resists, it decreases. The irradiated or nonirradiated areas, respectively, will be removed by a developer. For deep X-ray lithography, the standard resist material is PMMA which acts as a positive tone resist. Polyactide or copolymers of lactide and glycolide can be used as resist material for X-ray lithography. In the longer wavelength range, SU-8 is a resist material developed by IBM for UV-lithography which allows the fabrication of structures with heights of more than 1000 mm [61]. Piezoelectric. (1) A substance that becomes electrically charged by pressure [http://www.geology.wisc.edu/ jill/glossary.html, http://www.genomicglossaries.com/content/microarrays.asp]. (2) Having the ability to generate a voltage when mechanical force is applied, or to produce a mechanical force when a voltage is applied, as in a piezoelectric crystal [http://www.csa.com/hottopics/mems/gloss.html]. Plasma. A conductive assembly of charged particles, neutrals and fields that exhibit collective effects. Plasmas carry electrical currents and generate magnetic fields. Because plasmas are conductive, respond to electric and magnetic fields, and can be efficient sources of radiation, they can be used in innumerable applications when special sources of energy or radiation are required (adapted from http://www.plasma.org/basics.htm]. Plasma oxidation. (1) Plasma oxidation involves the growth rather than the deposition of high quality thin layers of silicon oxide, at room temperature. The technique employs O produced in an oxygen plasma [http://www.liv.ac.uk/ EEE/research/sse/project4.htm]. (2) The process involved when a silicon

117 substrate is in contact with a plasma at floating potential during oxidation [http://researchwed.watson.ibm.com/journal/rd/431/hess.html]. (3) PDMS comprises repeating units of –O–Si(CH3)2–. Exposing a PDMS replica to air plasma introduces polar groups on the surface. One view is that the methyl groups (Si–CH3) are replaced with silanol groups (Si–OH). These silanol groups then condense with appropriate groups (OH, COOH, ketone) on another surface when the two layers are brought into conformal contact. For PDMS and glass, this reaction yields Si–O–Si bonds after loss of water. These covalent bonds form the basis of a tight, irreversible seal: attempting to break the seal results in failure in the bulk PDMS. The seal withstands pressures of 30–50 psi. It is possible to seal PDMS irreversible to the surfaces of a number of materials: PDMS, glass, Si, SiO2, quartz, silicon nitride, polyethylene, polystyrene, and glassy carbon. This method, however, does not work with all polymers, e.g., Saran, polyimide, polycarbonate, and poly(methylmethacrylate) [10,65]. Plastic deformation. (1) Atoms in a material are permanently rearranged due to a stress-induced change. Removal of stress does not result in a restoration of the original shape [http://www.phy.davidson.edu/StuHome/BeKinneman/metal/ glossary.htm]. (2) The permanent change in shape or size of a body without fracture, produced by a sustained stress beyond the elastic limit of the material [http://www.vishay.com/brands/measurements_group/guide/glossary/plas_def.htm]. Polymers. Macromolecular substances with a relative molecular mass between 10 and 100 kDa with more than 1000 monomeric units. The polymerization process requires an initiator substance or a change in physical parameters (light, pressure, temperature). Due to the great length of the polymer chain, polymers are bulk materials. In most cases, polymers are amorphous but in some cases, microcrystalline, where the polymer chain is longer than the size of the crystallites. The chain length of the polymer molecules varies in bulk material; therefore, a polymeric material does not have a precisely defined melting temperature. Instead, a melt interval exists where the material becomes a highly viscous mass. The decomposition temperature is another characteristic point above which the thermal cracking of the material starts and the material ceases to function (adapted from Ref. [61]). Prototyping. See Rapid prototyping Pulsed lasers. Lasers can be continuous wave (cw) or pulsed. Pulsed lasers with very short pulse duration (on the order of nanoseconds) are critical for achieving ablation since the laser energy has to be delivered in a very short amount of time. Hence, the laser energy will be confined to a very thin layer of material inducing intense photochemical or photothermal reaction, which results in ablation [http://www.tamsci.com/library/glossary.html]. Rapid prototyping. (1) Any of a variety of processes that avoids tooling time in producing prototypes or prototype parts and therefore allows (generally nonfunctioning) prototypes to be produced within hours or days rather than weeks. These prototypes are frequently used to quickly test the product’s

118 technical feasibility or consumer interest [http://www.shapetomorrow.com/ resources/r.html#rapid]. (2) The combination of high-resolution commercial printing, photolithography, and soft lithography, allowing microsystems to be designed and fabricated rapidly and inexpensively [10,65,154]. For microfluidic systems, it begins with creating a design for a device in a computer-aided design (CAD) program. A high-resolution commercial image setter then prints this design on a transparency. This transparency serves as the photomask in contact photolithography to produce a positive relief as a master; it is used for the replication (via casting) of PDMS devices [10]. Reactive ion etch (RIE). A technique whereby radio frequency radiation is coupled with a low-pressure gas to ionize the gas producing disassociation of the gas molecules into more reactive specie, and the substrate being etched is biased to induce ion bombardment. Compounds containing carbon (C) and halogens such as, fluorine (F), chlorine (Cl) or Bromine (Br) are typically used as gases. When the compounds dissociate in the plasma, both highly reactive halogen atoms or halogen compounds, and polymers that may deposit on the substrate blocking the highly reactive species are generated. Ions accelerated towards the substrate being etched by the applied or induced bias remove polymers on substrate surfaces oriented normal to the direction of ion motion, polymers coat substrate surfaces that are oriented parallel to the ion motion and block etching of those surfaces. Ion bombardment may also activate or accelerate chemical etching reactions. RIE therefore has the capability to etch surfaces normal to the direction of ion motion at a higher relative rate and surfaces parallel to the ion motion at a lower relative rate resulting in anisotropic etching. Typical RIE conditions are low pressure, low ionization levels and high ion energies relative to other dry etch techniques [http://www.icknowledge.com/glossary/r.htm]. Refractive index. (1) The ratio of the velocity of the radiation (e.g., light) in vacuum to the velocity of radiation (e.g., light) in the material [http:// www.tamsci.com/library/glossary.html]. (2) The refractive index indicates the extent to which a light beam is deflected when passing from vacuum into a given substance [http://www.photometer.com/en/abc/abc_015.htm]. Registration. Proper positioning and exact alignment, term usually used in printing and image assembly [adapted from http://www.uniprint.com/terms/ r.htm and http://www.ideaexchange.sappi.com/home.asp?pid ¼ 89]. Example sentence from Chiu et al. [53]: ‘‘Patterning of multiple ‘‘colors’’ . . . requires multiple steps of registration.’’ Replica molding. The process involves the casting of prepolymer against a master and generating a negative replica of the master in PDMS with micronscale relief patterns, i.e., ridges on the master appear as valleys in the replica. The PDMS is cured in an oven at 60 C for 1 h, and the replica is then peeled from the master. Access holes for channels and reservoirs for buffer can be added in the replication step by appropriate placement of posts on the master or punched out of the cured layer by using a borer [10,51].

119 Reverse transfection. A microarray-based system for the functional analysis in mammalian cells of many genes in parallel. Mammalian cells are cultured on a glass slide printed in defined locations with solutions containing different DNAs. Cells growing on the printed areas take up the DNA, creating spots of localized transfection within a lawn of nontransfected cells. By printing sets of complementary DNAs (cDNAs) cloned in expression vectors, microarrays can be made whose features are groups of live cells that express a defined cDNA at each location. These ‘‘transfected cell microarrays’’ should be of broad utility for the high-throughput expression cloning of genes, particularly in areas such as signal transduction and drug discovery. For many applications, these arrays can serve as substitutes for protein microarrays, particularly for proteins that are difficult to purify, such as membrane proteins. [David Sabatini ‘‘Reverse transfection’’ Whitehead Institute, MIT, Cambridge, MA, USA, http://staffa.wi.mit.edu/sabatini_public/reverse_transfection/frame.htm, also in http://www.genomicglossaries.com/content/microarrays.asp]. Reynolds number (Re). (1) A dimensionless parameter relating the ratio of inertial to viscous forces in a specific fluid flow configuration, and is a measure of the tendency to the liquid to develop turbulence [51]. 2. The nature of a flow of fluid is characterized by the Reynolds number, Re ¼ l/, where  is the density of the fluid,  is the velocity, l characterizes the shape and dimensions of the channel (the cylinder diameter), and  is the viscosity of the fluid. Flows that have Re<2000 are laminar. When two or more streams with low Re are combined into a single stream, the combined streams flow side by side without turbulent mixing; mixing occurs only by diffusion [10,41,440]. Self-assembled monolayer (SAM). (1) A layer of material that is grafted at a surface or interface formed from a reagent that forms a single uniform layer. (A grafted layer is chemically attached by bonds to an interface rather than adsorbed. This provides distinction between chemisorbed and physisorbed species.) Typical reagents will use the reaction of a thiol with gold or a trichlorosilane with SiOH groups. A wide variety of groups can be attached in this way to modify the stability of colloidal particles or to change surface properties [http://www.ch.kcl.ac.uk/kclchem/staff/arr/glossold.HTM#SAM]. (2) The resist used in the Harvard/NIST method is a self-assembled monolayer of organic molecules called alkanethiolates which is adsorbed on the surface of the gold. An alkanethiolate molecule can be thought of as a ball and chain in a weightless environment. The thiol ball of the molecule bonds strongly to the gold, while the hydrocarbon chain floats away from the surface. On gold and a number of other metallic and oxide surfaces, these and other appropriate compounds will self assemble into a single layer of tightly packed molecules a few nanometers thick, much thinner than a typical photoresist [http://physics.nist.gov/News/Releases/n95-28.html]. Silicone rubber. PDMS [116] Silane (SiH4). (1) A gas that readily decomposes into silicon and hydrogen, silane is often used to deposit silicon-containing compounds. It also reacts with

120 ammonia to form silicon nitride, or with oxygen to form silicon dioxide [http://www.appliedmaterials.com/products/glossary.html]. (2) A pyrophoric, toxic gas with a TLV (threshold limit value) of 5 ppm, and is combustible in the range of  1–96%. Silane is widely used to deposit silicon-containing films in a variety of CVD (chemical vapor deposition) reactions [http://www.icknowledge.com/glossary/s.html]. Silanization. Silanization of items prevents compounds from adhering to the inside walls of the glassware. To silanize glass, the product is totally immersed in a silane solution for up to 30 min, followed by appropriate rinses chemically binding a nonreactive silicone layer to the glass surface, thus deactivating the surface and allowing for more accurate quantitative analysis [http://www.sun-intl.com/html/glossary.cfm]. Sintering. The process in which fine particles of a material become chemically bonded at a temperature that is sufficient for atomic diffusion [http://www.solgel.com/educational/glossary.htm]. Soft lithography. (1) A set of nonphotolithographic, microfabrication techniques for patterning surfaces used in biochemistry and biology. An elastomeric material with the patterns embedded as a bas-relief on the surface acts as the pattern transfer agent. These methods have the characteristic that routine access to a cleanroom is not necessary when producing most structures relevant to microfluidics (20–100 mm). They are also able to pattern transfer to curved materials. This method can be used to fabricate channels in bulk polymer [10,51]. (2) A technique where patterns are incorporated by curing the elastomer on a micromachined mould. Features that can be made include channels, diffraction gratings, and three-dimensional structures, such as coils and rings. These can be as small as 80 nm in diameter but typical channel widths are 50–100 mm [62,67]. Multilayer soft lithography is a micromachining technique that exploits the elasticity and the surface chemistry of silicone elastomers in order to create monolithic microvalves within microfabricated devices [292]. This technique is based on rapid prototyping and replica molding methods of soft lithography. A monolithic chip can be made of multiple layers of elastomeric channels [16]. Each layer is cast separately and bonded irreversibly to the next using the elastomer’s chemical curing process, eradicating the problems of adhesion failure and thermal stress that occur when layering hard materials. This technique was developed by Stephen R. Quake and his group (Department of Applied Physics, California Institute of Technology, Pasadena, CA, USA) and is proprietary technology of Fluidigm Corp. (see Table 3) [67] Sol-gel. Transition of a system from a liquid sol (colloidal suspension of minute solid particles in a liquid) to a viscous gel in which the suspended particles are organized in a loose, but definite three-dimensional arrangement. The thin film gel is dried (this process can be repeated several times to achieve the required film thickness) and finally sintered [http://www.npl.co.uk/npl/cmmt/functional/func_process.html].

121 Sol-gel coating. A coating produced by the sol–gel process of glassmaking, in which glass is formed at low temperatures from suitable compounds by chemical polymerization in a liquid phase; a gel is formed from which glass may be derived by the successive elimination of interstitial liquid and the collapse of the resulting solid residue by sintering [http://www.solgel.com/educational/glossary.htm]. Spin-coating. Solution is poured onto the substrate surface, which is then spun to expel excess fluid and create a uniform thickness [http://www.npl.co.uk/npl/cmmt/functional/func_process.html]. Scanning probe lithography (SPL). A high resolution patterning technique that uses a sharp tip in close proximity to a sample to pattern nanometer-scale features even below 30 nm with nanometer-scale alignment registration. It is a relatively simple, inexpensive, reliable method and can be used on various substrates. Some applications include nanometer-scale research, maskless semiconductor lithography, and photomask patterning. With better control of the tip, scanning speed has been increased to millimeters per second. Both noncontact and in-contact writing have been demonstrated as controlled writing of sub-100 nm lines over large steps on the substrate surface. (Adapted from: Soh HT, Guarini KW, Quate CF. Scanning Probe Lithography. (Microsystems, Volume 7) Kluwer Academic Publishers, 224 pp., July 2001, ISBN: 0-7923-73618; http://www.neutrino.co.jp/abi_mict/0-7923-7361-8.PDF] SU-8. A photocurable, epoxy-based, negative photoresist that is used to make high aspect ratio features in electroplating, polymer casting and UV-LIGA. LIGA yields better results but due to cost considerations, it is well suited as a mold for electroplating because of its relatively high thermal stability (Tg>200 C for the cross-linked, i.e., exposed, resist). SU-8 is based on EPON SU-8 epoxy resin (from Shell Chemical) that has been originally developed, and patented by IBM (US Patent No. 4882245 (1989) and others). This photoresist typically is 200 mm but can be as thick as 2 mm and aspect ratio >20 have been demonstrated with standard contact lithography equipment. These astounding results are due to the low optical absorption in the UV range which only limits the thickness to 2 mm for the 365-nm wavelength where the photoresist is the most sensitive (i.e., for this thickness 100% absorption occurs). Normal resists range from 0.5 to 3 mm in thickness. Microchannels with depths of some tens of micrometers can be fabricated. Although it is durable and can be used indefinitely, it not easy to process, has a large internal stress, and once developed, is difficult to remove. [Adapted from http://aveclafaux.freeservers.com/SU-8.html and http://www.allaboutmems.com/glossary.html#E, with additional information from McDonald et al. [10], Becker and Ga¨rtner [61], and http://www.mems-exchange.org/catalog/UMich-B-0120/]. Substrate. (1) The material of which something is made and from which it derives its special qualities. In electronics, it is the physical material on which a MEMS circuit is fabricated; used primarily for mechanical support and insulating purposes, as with ceramic, plastic, and glass substrates;

122 semiconductor and ferrite substrates may also provide useful electrical functions [http://www.csa.com/hottopics/mems/gloss.html]. (2) A substance which an enzyme acts on. Systems biology. (1) The simultaneous study of complex interactions of multiple levels of biological information including DNA, RNA, proteins and biochemicals. [http://www.paradigmgenetics.com/content/aboutus/glossary.asp] (2) A powerful approach to studying genes and proteins, made possible through technological advances. Unlike traditional biology that has examined single genes or proteins in isolation, systems biology simultaneously studies the complex interaction of many levels of biological information-genomic DNA, mRNA, proteins, functional proteins, informational pathways, and informational networks, to understand how they work together. [Institute for Systems Biology, Seattle WA, US ‘‘What is systems biology?,’’ http://www.systemsbiology.org/overview.html, also in http://www.geocities.com/pribond/bioinfo/glossary/cell-biology.htm]. Teflon. Poly(tetrafluoroethylene), can be used for laser ablation [75]. Titer. (1) The term refers to antibody titer, which is a measure of the concentration of specific antibodies to selected microbes that are circulating in an individual’s bloodstream [http://www.healingwithnutrition.com/cdisease/chronicfatigue/glossary.html]. (2) A titer is the amount of a substance found in certain tests [http://www.mtio.com/lupus/lfanp2.htm]. Waveguide. (1) Structure that guides electromagnetic waves along its length. An optical fiber is an optical waveguide [http://www.csp.com/html/tech_library/ fiber_glossary/fbgl_t-z.htm]. (2) A material medium that confines and guides a propagating electromagnetic wave. In the optical regime, a waveguide used as a long transmission line consists of a solid dielectric filament (optical fiber), usually circular in cross section. In integrated optical circuits, an optical waveguide may consist of a thin dielectric film. [http://www.atis.org/tg2k/_waveguide.html, also in http://www.fiber-optics.info/glossary-wxyz.htm#Waveguide] Wet etch. See Etch Young’s modulus. A measure of the strength of a material expressed as the stress divided by the strain [http://www.youngsmodulus.com]. References 1. 2. 3. 4. 5. 6.

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Non-ribosomal peptide synthetases as technological platforms for the synthesis of highly modified peptide bioeffectors – Cyclosporin synthetase as a complex example Tony Velkov and Alfons Lawen* Monash University, Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, P.O. Box 13D, Melbourne, Victoria 3800, Australia Abstract. Many microbial peptide secondary metabolites possess important medicinal properties, of which the immunosuppressant cyclosporin A is an example. The enormous structural and functional diversity of these low-molecular weight peptides is attributable to their mode of biosynthesis. Peptide secondary metabolites are assembled non-ribosomally by multi-functional enzymes, termed non-ribosomal peptide synthetases. These systems consist of a multi-modular arrangement of the functional domains responsible for the catalysis of the partial reactions of peptide assembly. The extensive homology shared among NRPS systems allows for the generalisation of the knowledge garnered from studies of systems of diverse origins. In this review we shall focus the contemporary knowledge of non-ribosomal peptide biosynthesis on the structure and function of the cyclosporin biosynthetic system, with some emphasis on the re-direction of the biosynthetic potential of this system by combinatorial approaches. Keywords: peptide antibiotics, peptolides, cyclosporin A, SDZ 214-103, cyclosporin synthetase, immunosuppresant, modular enzymes, multi-functional enzymes, non-ribosomal peptide synthetase, thiotemplate mechanism, secondary metabolites, polyketide synthases, N-methylation, peptide assembly, precursor-directed biosynthesis, in vitro biosynthesis, combinatorial biosynthesis, a-amino acids, b-alanine, a-hydroxy acids, non-ribosomal code, 40 -phosphopantetheine.

Introduction, products of the enzymes Biologically active, low molecular weight peptide secondary metabolites of microbial origin have found a niche in medicine, agriculture and biological research, by virtue of their enormous structural and functional diversity [1]. Pharmacologically important properties of some of these compounds include immunosuppressive, antibiotic, anti-viral, anti-tumour and cytostatic effects [2]. The biosynthesis of these bioactive peptide metabolites proceeds non-ribosomally and is catalysed by complex multi-functional enzymes, termed non-ribosomal peptide synthetases (NRPS). The phenomenon of non-ribosomal synthesis represents nucleic acid-free information transfer, which is a direct violation of Crick’s central dogma that states information is lost, once it has passed from nucleic acid to polypeptide [3]. Most non-ribosomal peptides are products of the secondary metabolism of soil microbes, primarily the Actinomycetes, Bacilli and filamentous fungi [4]. Marine microorganisms have also emerged as a source of biologically active peptide secondary metabolites [5]. The fungal species *Corresponding author: Tel: þ 61 3 9905 3711. Fax: þ 61 3 9905 3726 E-mail: [email protected] BIOTECHNOLOGY ANNUAL REVIEW VOLUME 9 ISSN 1387-2656 DOI: 10.1016/S1387-2656(03)09002-1

ß 2003 ELSEVIER SCIENCE BV ALL RIGHTS RESERVED

152 Tolypocladium inflatum Syn. Beauveria nivea [6,7] has been of intense scientific interest due to the immunogenic pharmacological importance of its secondary metabolite, cyclosporin A (CsA; Fig. 1a). Cyclosporins are low molecular weight neutral hydrophobic cyclic undecapeptide products of a NRPS system, cyclosporin synthetase (CySyn) [8]. Other examples of clinically important NRPS products include the b-lactam antibiotic (penicillins, cephalosporins and cephamycins) precusor d-(a-aminoadipyl)-cysteinyl-D-valine (ACV) [9], the vancomycin antibiotics [10] and the anti-tumour drug bleomycin [11]. Furthermore, many pathogenic bacteria employ NRPSs in the biosynthesis of siderophore-dependent virulence factors, notable examples include yersiniabactin from the plague-causing Yersinia sp [12], vibriobactin from Vibrio cholerae [13,14], enterobactin from Escherichia coli [15], myxochelin from Stigmatella

Fig. 1. (a) The chemical structure of cyclosporin. The three non-proteogenic amino acids, are D-alanine, (4R)-4-[(E)-2-butyl]-4-methyl-L-threonine (Bmt), L-2-aminobutyric acid. (b) The crystal structure of cyclosporin A. (c) The crystal structure of cyclosporin A bound to human cyclophilin A. M1 to M11, incorporation catalysed by modules 1 to 11.

153 aurantiaca [16] and mycobactin of Mycobacterium tuberculosis [17]. The development of new pharmacological agents for the treatment of the ever increasing number of emerging multi-drug resistant bacterial strains and the siderophore-dependent bacteria is therefore of the utmost importance. To this end, a complete understanding of the biosynthetic activities of NRPS systems is pre-requisite to the exploitation and re-direction of their biosynthetic functions for the production of novel compounds and variations of therapeutic non-ribosomal peptides. The history of CsA Tolypocladium inflatum was first isolated from soil samples appropriated by Sandoz Ltd. (Basel, Switzerland) employees from Wisconsin (U.S.A) and from Hardanger Vidda (Norway). T. inflatum was initially mis-identified as the fungus imperfectus Trichoderma polysporum [18]. Recently, the sexual stage Cordyceps subsessilis, was described [19]. Initially CsA was developed as a fungicide, however, due to its narrow therapeutic spectrum investigations into this clinical application were abandoned. The subsequent discovery of its immunosuppressive activity [20,21] lead to investigations which eventuated in its approval for clinical implementation to prevent graft rejection in transplantation surgery [22–24]. Binding of CsA to cyclophilin [25,26] produces an inhibition of calcium signalling in T-cells following antigen recognition [27,28]. This selective suppression of T-cell immunity afforded by CsA therapy [20] has enabled the routine transplantation of organs previously untenable and significantly reduced patient morbidity [29–31]. CsA is also of considerable utility in the treatment of certain auto-immune conditions [32,33]. Structure-function investigations together with the resolution of the NMR and X-ray structures of the cyclophilin-CsA complex revealed the amino acids at positions 1, 2, 9, 10 and 11 are involved in cyclophilin binding [25,34–37]. In addition to its immunosuppressive action, CsA exhibits several other biological activities, including anti-fungal [38], anti-inflammatory [39,40], anti-parasitic [41–43], anti-human immuno-deficiency viral [44,45] and anti-alopecia [46,47] properties. Owing to the spectrum of bioactivity of these metabolites, considerable effort has been invested to identify new cyclosporins and for the increased production of important congeners. Cyclosporins, fungal secondary metabolites The cyclosporins exhibit noticeable differences in their amino acid composition and bioactivity. In addition to the main product CsA, over 25 cyclosporins have been isolated from submerged cultures of T. inflatum [48–52]. The natural cyclosporins differ from the CsA sequence by one to two amino acids, displaying positional variations predominantly at position 2 (Abu) and by the presence or absence of one to two N-methyl groups at certain amino acid positions [52]. Biosynthesis can be directed towards specific congeners by supplementation of

154 the culture with the corresponding precursors [53,54]. Cyclosporins are produced by fungi imperfecti. CsA and its naturally occurring congeners have been isolated from 17 other fungal taxa [52,55]. Moreover, two novel cyclosporins [Thr2, Leu5, Ala10] CsA and [Thr2, Ile5] CsA have been isolated from strain F/88-3089/11 of Acremonium luzulae (Fuckel) W Gams and strain F/93-4641/04 of the Leptostroma anamorph of Hypoderma eucalypti Cooke and Harkin [56]. Despite the fact that cyclosporins are produced by many fungal taxa, the functional roles they play within the producer organism remains enigmatic. In the case of Cryphanectrica parasitica cydosporum, CsA has been found to induce gene expression via a cyclophilin mediated pathway [57]. Considering cyclosporins are highly complex with respect to structure, function and their biosynthesis, it is difficult to imagine that the complex biosynthetic machinery required for cyclosporin assembly would be conserved if it would not give the organism any advantage [58]. Structure of cyclosporin A The cyclic structure of CsA (Fig. 1a), in addition to the three non-proteogenic composite amino acids, D-alanine, (4R)-4-[(E)-2-butyl]-4-methyl-L-threonine (Bmt), L-2-aminobutyric acid and seven N-methylated peptide bonds, are indicative of its non-ribosomal origins. The numbering of the amino acids in the CsA molecule corresponds to the sequence of identification of each residue by sequential Edman degradation (Fig. 1a) [59]. The structural analysis of CsA crystals by X-ray diffraction studies reveals a rigid conformation (Fig. 1b) [60]. The backbone of the molecule between residues 11 and 7 forms a b-fragment consisting of an anti-parallel b-sheet with a type II b-turn between residues 2 and 5. Residues 7 to 11 form an open loop structure. The rigidity of the structure can be attributed to a number of unique structural properties. Predominantly, the four intra-molecular hydrogen bonds maintain the rigidity of the backbone structure. This is evident from the increase in the number of backbone conformations observed in polar solvents due to formation of inter-molecular hydrogen bonds with the solvent molecules [61]. In addition to its four intra-molecular hydrogen bonds, the molecule exhibits a cis-amide bond between the N-methyl leucine residues atpositions 9 and10. Moreover,the N-methyl moiety of MeVal11 in the loop makes backbone contacts, which further contributes to the rigidity of the structure. N-demethylation of specific residue positions has been shown to influence hydrogen bonding and the backbone conformation of the molecule [62]. Interestingly, this is not the structure involved in complex formation with its target protein, cyclophilin (Fig. 1c). In this complex, CsA adopts an all trans conformation, which maintains hydrogen bonds only between the hydroxyl of the MeBmt1 side chain and the carbonyl oxygen of the MeLeu4 position [36,37]. Biosynthesis of cyclosporins The biosynthesis of non-ribosomal peptides in addition to the NRPS system responsible for the condensation of the monomeric units and in certain cases

155 modification of the peptide backbone, often involves several independent enzyme systems that elaborate the production of substrates or ancillary enzymes that function to modify the peptide product, during or subsequent to assembly. Cyclosporin production involves a number of independent enzyme systems that cooperate in trans to generate and assemble the monomeric units of the cyclosporin molecule (Fig. 2). The undecapeptide backbone is assembled on the multi-functional protein thiotemplate, CySyn, a very complex high molecular

Fig. 2. The biochemical pathway of cyclosporin biosynthesis. Cyclosporin assembly is facilitated by a number of enzyme systems. In addition to the classical amino acid biosynthetic pathways, the production and channelling of precursor amino acids to cyclosporin synthetase is elaborated by specialised Bmt polyketide and alanine racemase enzyme systems dedicated to cyclosporin production. The activation of cyclosporin synthetase to the catalytically active form occurs posttranslationally by the covalent attachment of 11 40 -phosphopantetheine groups, catalysed by a specific 40 -phosphopantetheine transferase.

156 mass NRPS [8,63]. CySyn is one of the most elaborate and intensive systems known, capable of catalysing a total of 40 partial reaction steps, in the synthesis of the cyclo-undecapeptide product: 11 amino acyladenylation reactions, 10 transpeptidations, 7 N-methylations, 10 chain elongation reactions and a final cyclisation reaction. The latter of course, is another peptide bond formation, so all in all, it the biosynthetic process entails 11 amide bond formation reactions [64]. Non-ribosomal peptides range in size between 2 and 15 residues in length [65], it is believed that chain lengths greater than 15 residues are not observed due to the enormous size of the NRPS that would be required for assembly [66]. The massive CySyn polypeptide is representative of the upper limits of molecular sizes of these enzymes. A molecular mass of 1.69 MDa (15,281 amino acids), was delineated from the sequence of the CySyn gene, simA, which constitutes an intron-less genomic open reading frame of 45.8 kb [67]. Until recently, the largest system described, CySyn has been overshadowed by the discovery of a 62.8 kb continuous open reading frame encoding an 18 modules NRPS peptaibol synthetase from Trichoderma virens [68]. Initially the hydrodynamic shape of CySyn was studied by sedimentation velocity ultracentrifugation. The data garnered from this study suggested the native enzyme is an oblate ellipsoid structure with a diameter of about 300 A˚, a thickness of around 46 A˚ and a central opening of 50–60 A˚, potentially to allow for peptide elongation [63]. Recently published transmission electron micrographs of negatively stained CySyn macromolecules revealed two distinct structures, one appeared to be a large globular structure of 25 nm whereas the second appeared to be a long chain of globular elements [69]. Another family of multi-functional enzymes involved in the production of cyclosporins are the type 1 polyketide synthases (PKS). In analogy to NRPS systems, PKSs function to assemble the carbon backbone of a polyketide product from the decarboxylative condensation of acyl coenzyme A substrates by successive formation of C–C or in certain cases C–O bonds on a multimodular protein thiotemplate [70–72]. Similarly to NRPSs, PKS systems utilise 40 -phosphopantetheine (Ppant) prosthetic groups attached to an acyl carrier protein of each module for the internal channelling of intermediates between reactive centres [70–72]. Furthermore, not unlike-NRPS systems, PKS introduce structural diversity into the ketide backbone through various modifying functions [72]. The biosynthesis of the non-proteogenic C9 amino acid Bmt is elaborated by a PKS that catalyses the formation of the polyketide backbone by the head-to-tail condensation of four acetate units, forming the 3(R)-hydroxy-4(R)-methyl-6-(E)-octenoic acid thioester, the C-methyl in the carbon chain is derived from AdoMet [73,74]. The polyketide 3(R)-hydroxy-4-(R)-methyl-6-(E)octenoyl-CoA is then transformed into the b-amino acid form which is utilised by CySyn as a substrate for cyclosporin biosynthesis. The remaining amino acid constituents of the CsA molecule are synthesised by the classical biosynthetic pathways [75].

157 In addition to CsA, many non-ribosomal peptides display D-amino acids [66]. Apart from the structural diversity endowed by D-amino acids, the side chain chirality of these residues provides resistance to proteolysis and also presents stereochemical constraints on the biosynthetic machinery. The occurrence of D-amino acids can eventuate via one of the two mechanisms. One possibility is the direct incorporation of the D-isomer by a gate-keeping initiation module of the NRPS, such as in the case of CySyn. Alternatively, epimerisation of the L-amino acid to the corresponding D-isomer can take place during peptide elongation, as is the case in gramicidin S synthetase-I [76], tyrocidine synthetase [77] and ACV synthetase [9]. These systems possess integral epimerisation (E)-domains of about 45 kDa, which catalyse the racemisation of the thioesterified L-amino acid of the amino acyl-S-Ppant or peptidyl-S-Ppant intermediate to the D-isomer. Recent evidence suggests epimerisation preferentially occurs on the peptidyl-S-intermediate [77]. This strategy appears to be the general mechanism employed by NRPSs. CySyn which is unable to catalyse the isomerisation of L-alanine to the D-isomer relies on an external racemase enzyme for the provision of the D-amino acid substrate for incorporation into the cyclosporin molecule. Hoffmann et al. [78] have reported the purification of a pyridoxyl phosphatedependent oligomeric alanine racemase from T. inflatum strain 7939/45 that appears to be responsible for the supply of D-alanine for CsA biosynthesis. Subcellular fractionation, together with immuno-electron microscopy, indicates CsA is localised within the fungal vacuole, with the CySyn enzyme and the cognate alanine racemase associated with the vacuolar membrane [69]. These findings are consistent with the possible vacuolar targeting sequence we detected in the deduced sequence of CySyn (Fig. 3). Due to the low concentration of D-alanine in the fungal cell milieu, it is possible that the alanine racemase channels the D-alanine substrate to the CySyn loading module via a direct protein–protein interaction. The cyclosporin biosynthetic machinery may operate as a metabolon comprised of the CySyn NRPS, Bmt PKS and alanine racemase. In addition, these findings suggest the product CsA is synthesised into the vacuolar lumen and released via vacuolar and cytoplasmic membrane trafficking. Given that the cyclophilin of T. inflatum is sensitive to CsA [Bang and Lawen, unpublished data; [79], the vacuolar compartmentalisation of CsA storage may represent a self-protective mechanism whereby the producer isolates its cyclophilins from cyclosporin production. As mentioned earlier, the cyclosporins are products of secondary metabolism. Presently, there is a poor understanding of the regulatory and metabolic events controlling the production of secondary metabolites. The production of cyclosporins appears to be regulated by several factors. High producer strains of T. inflatum produce 60 g/L of CsA, in comparison medium producer strains produce 1.5 g/L. This 40-fold difference in CsA production is not accounted for by the two-fold difference in CySyn levels detected between the two strains, nor due to a kinetic difference between the CySyn systems from each strain as the rate of product formation was the same [80]. Therefore the levels of alanine

158

Fig. 3. Multiple sequence alignment of the 11 modules of cyclosporin synthetase. The domain boundaries are indicated by arrows. The putative vacuolar targeting motif is indicated as VAC. The N-methyltransferase domain regions are abbreviated as N-MET. The putative cyclisation domain at the C-terminus of the 11th module is labelled as M11 Cy-domain. The conserved motif regions of each NRPS domain class are labelled and colour-coded according to the table, which lists the core sequences of the motif regions in single-letter amino acid code; x indicates any amino acid; alternative amino acids for a given position are in parentheses. The amino acid positions responsible for discrimination of amino acid substrates are boxed in purple and labelled S1-3.

159

Fig. 3. Continued.

racemase and Bmt PKS together with the availability of substrates from the primary amino acid pool may also be contributing factors. Since D-alanine serves as the starting unit for cyclosporin assembly [64], together with the high affinity of CySyn for D-alanine [78] and as indicated by the absence of free D-alanine in

160

Fig. 3. Continued.

extracts of T. inflatum [52], it appears the alanine racemase activity acts as the dominant rate limiting factor in the cyclosporin biosynthetic process. An in depth knowledge of the factors that regulate the CsA biosynthetic process would facilitate the overproduction of desired congeners.

161

Fig. 3. Continued.

Modular structure of NRPS The foundation of the complex biosynthetic activities of NRPSs arises from the ordered architecture of their functional components. NRPSs are composed of a conserved multi-modular arrangement of functional domains that catalyse

162

Fig. 3. Continued.

the partial reactions of peptide synthesis (Fig. 4). These domains represent the catalytic units of each module such that the sequential polymerisation of the constituent amino acids of the product takes place by virtue of their gross organisation in the peptide synthetase. In the process of peptide assembly these modules operate in a concerted vectorial fashion to elaborate the N- to C-terminal assembly of the peptide product. In many cases, including CySyn, the order of the modules from the N- to C-terminus of the NRPS polypeptide is co-linear with the sequence of the peptide product such that the repeating series of modules forms an ordered macromolecular assembly line that defines the

163

Fig. 4. (a) Schematic diagram of the domain architecture of the type I and II modules of cyclosporin synthetase. Type I modules consist of a C-A-T domain arrangement; in addition type II modules possess a N-methyltransferase domain between the A- and T-domains. (b) The linear modular architecture of cyclosporin synthetase. Peptide assembly proceeds in a N- to C-terminal fashion, with the successive addition of an amino acid by each module. The mature linear peptide is release by the function of a putative cyclisation domain situated at the end of the 11th module.

amino acid sequence of the peptide product. The repeating series of modules, each 1000–1400 amino acids in length, are highly conserved across NRPSs and are believed to function in a semi-autonomous manner [66]. The semiautonomous catalytic nature of modules and their composite domains, is

164 supported by the finding that these units retain their catalytic activities when proteolytically excised from the peptide synthetase [81,82] or heterologously expressed [83,84]. The fundamental module unit consists of catalytic domains responsible for substrate amino acid activation (A-domain), thiolation (T-domain, synonymous with peptidyl carrier protein), and condensation (C-domain) [66]. Together these domains form the C-A-T catalytic triad of the type I NRPS domain, the functional repeating unit that represents the minimal domain set necessary for a single cycle of peptide elongation [85,86]. In addition to these core domains, peptide synthetases that modify their product during elongation possess modules with specific tailoring domains [87]. One problem imposed by this form of organisation is that the sequence and directionality of reactions has to be strictly controlled in order to prevent the aberrant initiation of a peptide at any one of the several active sites involved. Initially it was well accepted that there was a strict co-linearity between NRPS modules and the sequence of amino acids in the product. However, with the increasing number of NRPS gene sequences it has come to light that co-linearity is not an absolute rule in these systems. It seems nature has evolved far more complicated domain organisations to expand the biosynthetic potential of these systems to generate more structurally complex products. Marahiel and colleagues [88] have proposed a classification of NRPS systems into three categories based on domain architecture and the different assembly mechanisms. Type A: Linear NRPSs wherein the sequence and length of the peptide are dictated by the linear order and number of modules, respectively. CySyn falls under this category. Type B: Iterative NRPSs, not unlike-type A NRPSs, these systems assemble their products in a linear fashion. However, iterative NRPS differ from linear NRPSs, by the fact that they consist of a minimal repertoire of modules for the assembly of one set of the repeated sequence element in the peptide product. Iterative NRPS systems re-use their modules to assemble a multimeric product consisting of repeating units which are then oligmerised on a terminal TE domain or T-C didomain. Thus, iterative NRPS systems are essentially linear NRPS systems, which utilise a terminal thioesterase (TE)-domain or T-C didomain for oligmerisation of the repeating units to yield the final product. They are exemplified by enniatin synthetase [89]. Type C: Non-linear NRPS. A number of interesting examples have been reported wherein permutations of the conventional C-A-T domain order of type A and B NRPSs is observed. Examples of biosynthetic systems that can be classified under the type C category include those responsible for the synthesis of bleomycin [11], syringomycin [90,91], ferrichrome [92], mycosubtilin [93] and the virulence conferring siderophores yersiniabactin [12], mycobactin [17] and vibriobactin [13,14]. A notable exception to the co-linearity of domain organisation with the peptide product is seen in the biosynthesis of the phytotoxic lipodepsinonapeptide, syringomycin. In the syringomycin biosynthetic gene cluster two peptide synthetases have been identified, syringomycin B (SyrB) and syringomycin E (SyrE). The SyrE polypeptide consists of eight conventional C-A-T modules

165 with a C-T didomain insert after the eighth module. The SyrB polypeptide simply consists of an A-T didomain that functions to activate and chaperone the incorporation of L-threonine, the ninth amino acid of the syringomycin sequence. In the assembly of the complete cyclic lipononapeptide, firstly, SyrE functions to incorporate the fatty acid moiety and assemble the first eight amino acids in the syringomycin sequence in a linear fashion, generating the lipooctapeptidyl intermediate. Then SyrB introduces the ninth amino acid L-threonine at the last (ninth) module of SyrE, to yield the lipononapeptidyl chain which is in turn translocated to the C-terminal TE-domain for cyclisation and release. Due to the lack of the integral A-domain in the ninth module, it is believed that the T-domain of this module undergoes aminoacylation in trans via the function of the A-domain on the separate SyrB polypetide [90,91]. In eukaryotes, NRPS systems are invariably single polypeptides which harbour all the functional domains necessary for complete product formation from their amino or hydroxy acid precursors [66]. In prokaryotic systems, however, the amino acid incorporating modules are often spread over a number of polypeptides, which assemble in order to form a functional oligomer capable of complete peptide synthesis. This difference in the structural and functional organisation of peptide synthetase systems between prokaryotes and eukaryotes parallels the differences between the structural organisation of their respective fatty acid synthase systems [94]. Furthermore, in eukaryotes, NRPS genes are encoded by large intron-less genes, whereas bacterial NRPS genes usually are composed of several exons [66]. The modular architecture of CySyn is typical of linear (type A) NRPS. The polypeptide is composed of 11 conserved modules, which are composed of homologous NRPS functional domains responsible for the activation, modification and polymerisation of the constituent amino acids of the undecapeptide product [67]. CySyn contains four type I C-A-T modules (modules 1, 6, 9, 11) and seven type II modules that display an additional 430 amino acid N-methyltransferase (N-MTase) domain insert between the A- and T-domains (modules 2, 3, 4, 5, 7, 8, 10) (Fig. 4) [67]. Unlike many initiation modules which solely consists of an A-T didomain which functions to initiate chain elongation, the first module of CySyn displays a C-domain. This region may be responsible for catalysing ring closure of the full-length peptide chain. In line with the co-linear relationship between the protein template and amino acid order in the product, modules 1, 6, 9 and 11 are responsible for the specific recognition and binding of unmethylated amino acids 8, 2, 5 and 7 of cyclosporin, respectively and modules, 2, 3, 4, 5, 7, 8 and 10 function to incorporate and N-methylated amino acids 1, 3, 4, 6, 8, 9, 10 and 11, respectively (Fig. 1a). The co-linearity between the CySyn protein template and the CsA sequence has also been substantiated by biochemical analysis of isolated proteolytic fragments [67]. The eleventh, N-terminal, domain of CySyn can be assigned as the L-alanine activating module based on N-terminal sequence analysis of an isolated 130 kDa proteolytic fragment that was capable of activating L-alanine. This finding is

166 corroborated by the observation that L-alanine is the last amino acid to be incorporated into the cyclosporin chain [64]. In addition to the presence of N-MTase domains in modules purported to incorporate N-methylated amino acids, primary level analysis of the substrate specificity of the A-domain of each module in accordance to the defined non-ribosomal code reveals that the substrate selectivity of A-domains of each module of CySyn from the N- to C-terminus mirrors the amino acid sequence of the product [95]. Taken together these findings support a co-linearity between the product sequence and the protein template. The modular architecture of CySyn may represent an evolutionary mechanism whereby lower eukaryotes have economised the molecular machinery to conserve energy, such that transcription/translation only has to be initiated once, and by lack of introns eliminating the need to splice/ligate the transcript. This level of organisation is certainly more economical than the oligomeric organisation observed in prokaryotic NRPS systems. It is likely that peptide synthetases evolved from a common ancestor via gene duplication and fusion events. The structural differences across the peptide products of this enzyme family possibly result from module swapping through recombinatorial or horizontal transfer events. It is plausible to postulate that eukaryotic NRPS systems represent an advantageous evolutionary convergence of genes of individual polypeptides of an arcane complex that have fused under evolutionary pressure to form a single operon with an ORF encoding a single functional polypeptide, capable of catalysing all of the functions of the ancestral enzyme complex, however, being much more efficient with respect to its stability under normal cellular conditions such as low concentrations of the complex proteins. Such unison relieves the cell of the arduous task of maintaining the stringent conditions required for complex assembly. Some NRPS biosynthetic operons are connected to other operons, together constituting complex networks that serve to organise and regulate developmental processes such as shift down processes in the event of nutritional limitations or sporulation [96,97]. Non-ribosomal peptide assembly In analogy to the activation of amino acid substrates catalysed by aminoacyltRNA synthetases in ribosomal peptide synthesis [98], in the reaction sequence of an elongation cycle catalysed by a simple C-A-T elongation module, activation of the substrate amino acid occurs at the adenylation domain (A-domain) through the generation of a transient aminoacyl adenylate [64,66]. The activated amino acid is then covalently tethered to the cysteamine thiol of the Ppant prosthetic cofactor of the cognate modular T-domain, to yield aminoacyl-SPpant [99–101]. Similarly to fatty acid synthase and PKS systems, the Ppant acts as a swinging arm that translocates the nascent peptidyl (or aminoacyl) intermediate between modular active sites. In a given C-A-T elongation module, the T-domain moves the invariably monomeric aminoacyl-S-Ppant nucleophile

167 to the acceptor site of the cognate upstream C-domain of the module. Peptide bond formation then ensues with the peptidyl-S-Ppant or aminoacyl-S-Ppant electrophile (the latter monomeric intermediate only applies if the upstream module is a A-T didomain initiation module) from the upstream T-domain (belonging to the preceding module), which is positioned at the donor site of the C-domain [88]. The condensation event translocates the extended peptidyl chain from the Ppant arm of the upstream T-domain to the Ppant of the downstream cognate modular T-domain, thereby allowing the nascent product to move one step forward in the assembly line. The consummation of an elongation cycle occurs when the Ppant swings the peptidyl chain to the donor (electrophile) site of the downstream C-domain of the next module. Once again peptide bond formation with the aminoacyl-S-Ppant nucleophile at the acceptor site of the C-domain translocates the now extended peptidyl chain to the T-domain of the next module. Once Ppant dissociates from the acyl constituent, the regenerated cysteamine thiol of the arm is re-loaded with another amino acid by the A-domain, the arm then swings back to the acceptor site of its cognate modular C-domain (upstream of the T-domain) awaiting for the next incoming peptidyl-S-Ppant electrophile. The nascent enzyme bound peptide is elongated with successive rounds of trans-thiolations and trans-peptidations by the interplay of Ppant internal carriers until the peptide is extended to its full length. Although this mechanism of peptide assembly appears to be rather laborious, this idiosyncrasy allows NRPSs to supersede the limitations observed with the conventional ribosomal mode of peptide assembly and thereby greatly expands the structural diversity of their products. The predicted amino acid sequence of CySyn displays 11 Ppant attachment sites, one for each amino acid activating module (Fig. 3). Thus, at a given time a single synthetase molecule can carry multiple chain intermediates of varying lengths (Fig. 4b). This stepwise assembly of the tethered amino acids in a N- to C-terminal fashion is well accepted and is termed the multiple carrier thiotemplate model of non-ribosomal peptide synthesis [101]. For some time this appeared to be the central dogma of non-ribosomal peptide synthesis, however, as eluded earlier, more recently exceptions to this rule have become apparent. In addition to the established co-linearity between the cyclosporin amino acid sequence and the conserved NRPSs domain elements identified by sequence homology analysis of the CySyn gene [67], evidence that the multiple carrier thiotemplate mechanism is employed by CySyn arises from the observation that analysis of intermediates released via performic acid hydrolysis identified all stages of CsA intermediates [64]. The complex biosynthetic reactions catalysed by these systems require the coordination of inter-modular/domain substrate transfers into a vectorial motion allowing for chain growth. It is believed the ordered channelling of intermediates between active sites on the protein template is, to some extent, achieved via inter-domain and inter-modular communication mediated by flexible intra-polypeptide or inter-polypeptide linkers in the case of separate NRPS protein units [102–104]. Although, the precise role of these linker regions

168 remains enigmatic, it is presumed they facilitate the vectorial channelling of intermediates by directing inter-domain/modular communication between reactive centres. The fact that the distance between inter-modular active sites is likely to be greater than the 20 A˚ distance, which is within the reach of the Ppant arm, highlights the necessity for flexible inter-domain linkers. To a great extent the crucial role of intra/inter-polypeptide linkers towards the proper functioning of the inter modular transfer of substrates has been elucidated for PKS [104]. Similarly to the scenario observed with PKS systems, it is expected that NRPS linker pairs exhibit selectivity for protein–protein interactions of naturally consecutive modules. Nevertheless, the precise nature of these interactions and the selectivity of naturally matched linker pairs will have to be investigated in order to make the construction of hybrid NRPS fully tenable. In most NRPS systems a specialised domain situated at the C-terminus of the last module, catalyses the release of the mature peptidyl chain from the enzyme. Many prokaryotic NRPS systems utilise a C-terminal TE-domain (
250 amino acids in length) for the release of the mature peptide product via hydrolysis, cyclisation or oligomerisation [105]. In this process, the mature peptidyl chain is transferred from the T-domain of the last elongation module onto an active site serine or, in certain cases, cysteine of the TE-domain, thereby regenerating the Ppant prosthetic group of the terminal module in preparation for the next peptidyl intermediate [106]. Trauger et al. [84] showed the TE-domain of tyrocidine synthetase is capable of catalysing the macrocyclisation reaction when expressed heterologously as an individual unit. However, the cyclisation reaction was dependent upon the chiral recognition of the side chains of residues at the N-termini of the mature peptide. It has been suggested that this selectivity towards the N-terminal nucleophile group serves as a mechanism to prevent cyclisation of immature chain lengths. The recently published X-ray crystallographic structure of the surfactin synthetase Srf-C subunit TE domain provides conclusive evidence for the macrocyclisation mechanism employed by this system [107]. It was suggested that TE-mediated macrocycle formation is achieved by the juxtapositioning of the thioesterfied N-termini of the mature peptidyl chain with the free C-termini via directed folding within a structural bowel cavity formed by the TE scaffold. Evidence also suggests the mature peptidyl chain is pre-organised to allow for the proper presentation of the termini to the cyclisation domain cavity for ring closure, this is accomplished, to some extent by intra-molecular backbone hydrogen-bonding [108]. In the biosynthesis of the cyclic antibiotic tyrocidine A, key residues near the N- and C-termini of the decapeptide are involved in the formation of intra-molecular hydrogen bonds, to allow for the pre-organisation of the linear peptide backbone, such that the N- and C-termini are presented in the correct orientation for macrocyclisation by the TE-domain [108]. It is expected the extent of N-methylation is likely to influence the degree of internal H-bonding of the cyclosporin molecule whilst it is covalently tethered to the synthetase. Potentially, this may influence the

169 efficiency of pre-organisation of the linear undecapeptide backbone for presentation of the N- and C-termini for ring closure. Recent studies have demonstrated the portability of TE domains from heterogeneous NRPS for the construction of hybrid systems with desired termination reaction [109,110]. The portability and generality of the TE-mediated cyclisation reaction will provide an avenue for the synthesis of a plethora of potential therapeutic macrocyclic peptide molecules. In addition to carboxyterminal TE-domains, certain systems utilise external TE enzymes [111,112]. These discrete TE enzymes are believed to function to deacylate the Ppant cysteamine thiol of T-domains domains mis-charged by non-specific thioesterifications. In some systems the release of the completed product does not involve a TE function or specialised cyclisation domain; they release the mature peptidyl-S-Ppant intermediate via a NAD(P)H-dependent reduction to the corresponding amino aldehyde [16,113] or an amino alcohol [114]. In the biosynthesis of alkaloid cyclopeptides in the ergot fungi, the linear peptide chain is released via diketopiperazine formation [115]. With the exception of ACV synthetase, which utilises the aforementioned TE-domain for release of a linear peptide product with a free terminal carboxyl group [9], other fungal NRPS systems employ a unique C-terminal domain for cyclisation and release of the mature peptide [105]. In addition to the specialised C-terminal C-domain, a 500 amino acid stretch at the N-terminus of CySyn in the 11th module (Fig. 3), that is non-homologous to the other NRPS domains, may also facilitate the ring closure reaction [67]. In this respect it is conceivable that the N- and C-terminus of CySyn interact to elaborate the cyclisation and release of the mature peptide. The underlying mechanisms controlling the timing and co-ordination of reaction steps that occur at the aminoacyl- or peptidyl-S-Ppant stage of each elongation cycle on each module and the co-ordination of these events in a vectorial fashion to elaborate successive chain elongation events, remain enigmatic. Elongation must proceed in a co-ordinated motion to avoid random mis-initiation at different modules, stalling of the assembly line due to the formation of terminal chains that cannot be further elongated or aberrant hydrolysis of peptidyl-S-Ppant chain intermediates. It is tempting to speculate that these orchestrations involve an intricate global conformational transition transmitted across the protein template via the aforementioned inter-modular/ domain linker regions. In order for the construction of hybrid NRPSs to become more tangible, all these unknowns will have to be resolved. Domains of cyclosporin synthetase By virtue of the variation in the organisation of the various domain types, the biosynthetic potential of NRPSs in terms of the structural diversity of their products by far exceeds that of conventional ribosomal peptide assembly. In contrast to the ribosomal system, which is limited to 20 proteogenic amino acids, non-ribosomal pathways utilise a veritable plethora a-hydroxy, carboxylic and

170 unusual non-proteogenic amino acid substrates (>300) [116]. These unusual substrates can be derived from primary metabolism (such as, 2-aminobutyrate, the position 2 amino acid of CsA) or from specialised biosynthetic pathways (such as the C9 amino acid Bmt, the position 1 amino acid of CsA). In addition to the expanded structural spectrum of monomeric units, NRPS introduce further structural diversity into their products via auxiliary modifying functions that reside in either externally associated or integral enzymatic species [87,112]. Integral tailoring domains act in cis to modify the peptide chain during elongation while it is covalently tethered to the Ppant. External enzyme species act in trans via protein–protein interactions with the synthetase whilst intermediates are still covalently tethered, or post-assembly, following release. In addition to the N-methyltransferase (N-MTase) domains discussed herein in more detail, several other forms of modifying domains have been observed in NRPS systems [87,112]. Whereas the mechanistic analysis of domains forming the C-A-T catalytic triad has progressed during recent years, characterisation of the modification domains is still in its infancy. Further diversity is produced by variations in the backbone structure of the assembled peptide product, linear, cyclic, branched-cyclic and heterocycles are common themes [85]. This vast structural diversity endows these compounds with ability to bind important cellular macromolecules, which often eventuates in useful biological effects, which is of great evolutionary value to the producing organism. In this section we shall delve into the structural and functional knowledge of the domains that are the molecular machines of NRPS assembly lines, with an impetus on the domains of the CySyn system. A-domain Similarly to aminoacyl-tRNA synthetase, the binding specificity, recognition and activation of the substrate amino acids as aminoacyl adenylates is mediated by the A-domain of each module [95,117]. Despite this common function, the two systems are non-homologous on the structural and sequence level [98,118]. The A-domain functions to catalyse the reaction of the a-carboxyl group of the amino acid substrate with the a-phosphate of ATP yielding the aminoadenylate which in turn is covalently tethered to the terminal sulfhydryl of the Ppant prosthetic group of the cognate modular T-domain [66]. In general, the amino acid substrate recognition pockets of the A-domains are fairly unspecific at the level of primary selection of precursors. It therefore comes as no surprise that non-ribosomal peptides often display permutations in their sequence. Non-ribosomal peptides are often produced as a homologous series of related peptides as opposed to single products. The preference for certain substrates at the primary selecting surfaces of the A-domains is very obvious in the case of CySyn, of the possible 72,000 analogues of the cyclo-undecapeptide [119], only 32 have been isolated to date [52]. The A-domain regions of CySyn appear to be fairly non-specific at certain amino acid recognition centres

171 [120,121]. The naturally occurring cyclosporins differ from the CsA sequence in positions 1, 2, 4, 5, 7 and 11 [122]. The trend with respect to the variability evident across the naturally occurring cyclosporins is predominantly in the exchangeability of amino acids in positions 2 and 8 [123]. However, the substrate specificities of A-domains with respect to the stereochemistry of the amino acid substrate appears to be quite strict, the N-terminal initiation module of CySyn displays a 10,000-fold preference for the D-epimer of alanine over the L-form [78]. The resolution of the three-dimensional structure of the phenylalanineactivating A-domain of gramicidin synthetase A (PheA) together with its substrates phenylalanine and ATP has provided a valuable insight into the molecular basis of substrate recognition and catalysis [124]. The A-domain catalytic unit belongs to the acyladenylate forming superfamily, which includes the luciferases and acyl-CoA ligases [121,125–128]. All members of this superfamily catalyse the ATP-dependent activation of the carboxyl group of their respective substrates as adenylates. On the primary level, firefly luciferase and the phenylalanine-A domain of GrsA share only 16% sequence identity, despite this discrepancy the topology of their respective crystal structure models is well conserved [124,128]. The PheA structure consists of a large N-terminal subdomain which is responsible for providing most of the substrate recognition determinants and a smaller C-terminal subdomain. The A-domains of NRPS display several highly conserved motif elements (A1 to A8) that are also common to other members of the acyladenylate forming enzyme superfamily (Fig. 3) [112,124]. Many of the invariant or highly conserved amino acid positions within the core sequences (A1 to A8) provide key interactions with the co-factor AMP and with the a-amino or a-carboxyl groups of the substrate amino acid. A sequence analysis of CySyn revealed the presence a synthetase-specific domain (SSD) of approximately 54 amino acids situated downstream of the A-domain in type I modules and downstream of the N-methyltransferase (N-MTase) domain in type II modules [121]. This region is only common to NRPSs and shows a high degree of sequence conservation (approximately 50%) [121]. Previously, the motif elements A9 and A10 have been assigned to the A-domain, however, the sequence alignment of the 11 modules of CySyn indicates these motifs are found within the SSDs of each module, therefore we have re-assigned them as SSD1 and SSD2, respectively (Fig. 3). The PheA crystal structure indicates that discrimination between the side chains of potential amino acid substrates is mediated by 10 residues that form the amino acid binding pocket within the N-terminal subdomain. Excluding two conserved lysine and aspartate residues that stabilise the a-carboxyl and a-amino groups of the amino acid substrate, respectively, this sequence region is poorly conserved. Taken together with a sequence analysis of the corresponding positions in heterogeneous A-domains it was possible to define a selectivity-conferring ‘‘non-ribosomal’’ code for A-domains [95]. Not unlike codon usage in its ribosomal counterpart, the non-ribosomal code appears to be redundant, as a

172 number of different 10 residue sequences could be defined for the activation of certain amino acids. The high degree of sequence identity between NRPS A-domains of diverse origins (
30–60%) allows for the PheA structure to be employed as a template for molecular modelling of NRPS A-domains to facilitate the prediction of substrate selectivity and its alteration by site-directed mutagenesis [95,121,129,130]. Moreover, knowledge of the selectivity-conferring non-ribosomal code has allowed for the prediction of the sequence of the peptide products of biochemically uncharacterised NRPS systems deduced from genome sequencing of model organisms [130,131]. In contrast to amino acid activating A-domains, aryl-acid activating A-domains of siderophore NRPS systems display an altered substrate conferring code. The recent resolution of the crystal structure of the stand-alone aryl-acid activating A-domain DhbE involved in bacillibactin biosynthesis has shed light on the specificity conferring code for these regions [132]. The definition of the substrate specificity conferring determinants of aryl-acid activating A-domains, will allow for the elucidation of the specificity of aryl acid activating A-domains of NRPSs genes of unknown function and potentially for the genetic alteration of the specificity of these regions. C-domain The C-domain (  450 residues in length) catalyses the nucleophilic substitution reaction between the upstream donor acyl- or peptidyl-S-Ppant electrophile, with the a-amino of the downstream acceptor aminoacyl-S-Ppant nucleophile. The catalytic mechanism of the C-domain remains an open question. However, the highly conserved double histidine motif HHxxxDGWS is essential for catalysis of the condensation reaction. Mutational analysis indicates the second histidine and the aspartate are functionally indispensable [133,134]. The His motif is also common to other members of the acyl transferase superfamily such as chloramphenicol acetyltransferase (CAT) and dihydrolipoamide acetyltransferase [134,135]. In the case of CAT the downstream histidine acts as a general base in the catalysis of the activation of a hydroxyl moiety of chloramphenicol [136]. Recent evidence has provided the providence for a model based on the proposed functional analogy to chloramphenicol acetyl-transferase, which purports a catalytic triad of residues is involved in C-domain catalysis [137]. It is believed that one of the histidines facilitates peptide bond formation by augmenting the nucleophilic nature of the amine nitrogen [135,137]. Based on the fact that amide bond formation is favoured proximal to a thioester, it has also been speculated that condensation may take place as a result of the proximal orientation of the donor peptidyl-S- Ppant and acceptor aminoacyl-S- Ppant substrates [138]. However, exceptions to this conventional elongation strategy have been observed. In such systems specialised cyclisation domains mediate heterocyclisation of cysteine to thiazoline or heterocyclisation of serine and threonine to oxazoline [138,139]. A further variation of the common integral C-domain is

173 observed in the vibriobactin biosynthetic system [13,14]. This system utilises a stand-alone C-domain protein which catalyses the transfer of a free soluble amine molecule to the peptidyl-S-Ppant electrophile donor. In the conventional C-A-T module, the multiple-carrier thiotemplate model dictates all of the aminoacyl and peptidyl reaction intermediates are covalently tethered to the enzyme and channelled from active site to active site in an ordered succession. To circumvent the confines of this system these C-domains possess a specialised acceptor nucleophile site that allows them to sequester free small molecules from the cellular milieu [13,14,140,141]. The recent resolution of the X-ray crystal structure of the stand-alone C-domain enzyme, VibH of the vibriobactin biosynthetic biosynthetic system has shed some light on the mechanism of the condensation reaction [138]. The VibH momomer is composed of two aba domain regions joined by a 56-residue linker helical region representative of a pseudo-dimer structure. It serves as a paradigm for both cyclisation and E-domains domains, both of which exhibit the conserved His motif [135]. If the specialised C-terminal C-domain of CySyn is indeed homologous to the VibH protein, as indicated by the presence of the functional His motif, it is tenable to envisage that cyclisation of the mature cyclosporin undecapeptide takes place via the pre-organisation of the backbone by internal hydrogen bonding, which optimally positions the N- and C-terminal ends on opposing sides of the solvent channel allowing for condensation of the ends. Recent studies have shown the C-domain displays an inherent editing function [86,142,143]. In an ingenious study the chemical barrier imposed by the amino acid substrate specificity of the A-domain was by-passed by artificially mis-priming the T-domain with chemically synthesised amino acyl-CoAs via the action of the surfactin phosphopantetheinyl transferase [86]. These chemically synthesised aminoacylated-coenzyme analogues served as probe for the selectivity of the acceptor and donor sites of the C-domain. The employment of various acyl-CoAs revealed the C-domain displays specificity towards the size and stereochemistry of the side chain of the aminoacyl-S-Ppant nucleophile at the downstream ‘‘acceptor’’ site whereas the upstream ‘‘donor’’ site, which accommodates the electrophilic amino acyl- or peptidyl from the downstream modular T-domain was found to be relatively non-selective [86,142]. It appears that the C-domain is, to some extent, responsible for controlling the directionality of assembly by the suppression of internal mis-initiation of chain elongation. It is likely that the downstream C-domain nucleophile acceptor site selectively binds the aminoacyl-S-Ppant intermediate delaying its passage until condensation has occurred, thereby preventing further reactions such as aberrant initiation of elongation at the downstream elongation module [142]. Further, evidence in support of an editing function at the C-domain acceptor site comes from the observation that internal C-A-T elongation modules can be converted to initiation modules by deletion of the C-domain [143]. In this study, the recombinant C-A-T modular form of the tyrocidine synthetase elongation

174 module 3 (TycB3) was incapable of initiating elongation and assembly of the dipeptidyl-S-Ppant product, whereas the construct with the C-domain deletion (A-T-E) was, indicating a gate-keeping role for the C-domain. Moreover, the modular positioning of the C-domain seems to dictate its functionality; C-domain inserts at the C-terminal side of E-domains are functionally distinct from those found in the usual C-A-T arrangement [77]. A sequence alignment of the C-domain regions of different NRPS systems indicated groupings that correlate with function, it was found that C-domains that catalyse the condensation of L-L amino acids form distinct groups from those that catalyse D-L condensations [144]. T-domain The T-domain is situated downstream of the A-domain and is 80–100 amino acids in length. The Ppant prosthetic cofactor is covalently associated through its phosphate group with the hydroxyl moiety of the side chain of an invariant serine residue of the conserved core sequence LGG(H/D)-S-(L/I) in the T-domain of each module (Fig. 3) [100,101,145–147]. This motif represents the consensus phosphopantetheine binding sequence element and is also found in the related ACPs of fatty acid synthases and PKS [94,148]. The sequence of the CySyn gene displays 11 T-domain regions, one for each of the 11 amino acid incorporating modules [67]. Biochemical evidence that CySyn utilises the cysteamine thiol of covalently associated Ppant prosthetic groups to form thioester bonds with substrate amino acids stems from two observations. Firstly, pantetheine has been found to be present in the CySyn enzyme [8]. Secondly, we have demonstrated that the substrate amino acids are activated as thioesters, evident from the instability of covalent enzyme–substrate complexes at alkaline pH and release of CsA intermediates with performic acid hydrolysis [64]. As eluded in the preceding discussion the cysteamine thiol of the Ppant prosthetic group serves as the thioester attachment site and in analogy to tRNAs, serve as an inter-modular taxi for the aminoacyl and peptidyl intermediates [100,101]. The elucidation of the solution structure of the T-domain of B. brevis tyrocidine synthetase revealed a homologous fold to the functionally related fatty acid synthetase acyl carrier protein (ACP) and PKS-ACP, despite the marginal degree of sequence identity between them [147]. Nevertheless, the conservation of structure>function>sequence in decreasing order is commonplace among enzyme families. The structure consists of a distorted anti-parallel four a-helix bundle with a long connecting loop between a-helix 1 and 2. Unlike the acidic surface charge of its ACP counterparts, the surface of the T-domain is relatively non-polar, which most likely provides the surface for productive domain– domain interactions with adjacent domains. The invariant serine attachment site for the Ppant cofactor is situated within the long flexible loop region directly at the interface between the loop and a-helix 2. This a-helix 2 element appears to be important for the interaction of the T-domain with a downstream E domain in

175 this modular context. It has been recently shown T-domains found downstream of E-domains possess specific determinants responsible for productive interactions with the upstream E-domain [149]. Furthermore, an alignment of the T-domains of various NRPSs revealed that T-domains that interact with tailoring domains are distinct from T-domains found in the type I C-A-T modular context [149,150]. However, there is no evidence for a binding pocket for the amino acid substrate or the Ppant cofactor in the T-domain structure, suggesting there is no substrate selectivity imposed by the T-domain. This is also exemplified by functional studies wherein functional domain fusions between T-domains and non-cognate modular A- and C-domains have been produced [151,152]. However, in the case of siderophore NRPS systems it seems the T-domain contributes to substrate discrimination. In the course of peptide assembly, specific domain–domain interactions take place between the T-domain with C- and A-domain regions through multiple determinants unique to that particular system [153]. In these systems the cognate T-domain and A-domain regions interact in trans, it will be interesting to see if the same phenomenon applies to T- and A-domains that interact in cis. These data exemplify the correct pairing of cognate domains for the specificity and maintenance of proper peptide assembly. Furthermore, these findings suggest cognate domain pairs co-evolved in the evolution of the NRPS system. This postulate is further supported by the aforementioned T-E domain interactions [149] The conversion of the T-domain from the inactive apo form to the catalytically competent holo form (Ppant- T-domain) occurs post-translationally [154]. The covalent attachment of Ppant group to the invariant serine in the T-domain of each module is catalysed by a specialised Ppant transferase (Fig. 2) [155,156]. The recent resolution of the three dimensional structure of the surfactin Ppant transferase has provided valuable information on the mechanism of these systems and the basis of their ability to mis-load NRPS T-domains with artificial acyl-S-Ppant derivatives [157]. In the latter respect, the model structure indicates in the bound state, the terminal cysteamine of the Ppant does not contact the protein and instead protrudes away from the binding site into the solution environment. N-Methyltransferase domain One unusual property of many peptides of non-ribosomal origin is that the amide nitrogens in these molecules are often methylated. Examples of N-methylated non-ribosomal peptides include the cyclosporins, SDZ 214-103 [158], SDZ 90-215 [159], enniatin [160], actinomycin D [161] and pristinamycin [162]. The NRPS systems responsible for the assembly of these peptides often possess integral N-MTase domains that catalyse the transfer of the S-methyl group of AdoMet to the a-nitrogen of the thioiesterfied amino acid, releasing S-adenosylL-homocysteine (AdoHcy) as a reaction product (Fig. 4). C- and O-methylations are also common, these functions have been detected in certain hybrid

176 PKS/NRPS systems [87,163]. In addition to the seven N-methylated peptide bonds in the CsA molecule, analysis of the cDNA sequence of the CySyn gene, indicates seven of the eleven modules display an additional domain insert between the A and T-domains, which display the GxGxG signature sequence for AdoMet-dependent methyltransferases (Fig. 3) [67,164]. The presence of a functional N-MTase activity in the CySyn polypeptide was first demonstrated by photoaffinity labelling with [14C-methyl]AdoMet and by the ability of the purified enzyme to transfer the sulfonium methyl group from [14C-methyl] AdoMet to the CsA product [8]. Moreover, it was found that the enzyme does not accept N-methylated precursor amino acids as substrates [64]. Our recent stochiometric photoaffinity labelling studies have shown the enzyme displays seven AdoMet binding sites [164a,b], which conform with the seven N-MTase regions identified in the CySyn cDNA sequence. These integral modifying domains represent additional inserts of
430 amino acids between the A and T domains (C-A-(N-MTase)-T) (Fig. 4a) [67]. Considering the N-MTase domain separates the active centres of the upstream C-domain from the T-domain by a distance of
430 amino acids, the distance between reactive centres is significantly greater than the reach of the 20 A˚ Ppant arm. It is tenable to imagine that the N-MTase domain is organised into a globular fold with its cognate modular domains. This scenario would allow the N-MTase active site to N-methylate the thioester-bound amino acid without affecting the spacing between the reactive centres. It has also been suggested that the N-MTase domain is located peripherally to the site of the condensation reaction [161]. The portability of the N-MTase domain was demonstrated with the actinomycin NRPS system [161], where a basic C-A-T elongation module could be replaced with another that possesses an N-MTase domain (C-A-(N-MTase)-T). The cognate C-domain of the fusion protein did not show any preference towards non-methylated amino acids and was capable of catalysing peptide bond formation with the unnatural N-methylated aminoacyl substrate. However, peptide bond formation with the N-methylated substrate was dependent upon the stereochemistry of the a-carbon of the donor aminoacyl-S-Ppant of the preceding module. Condensation of the N-methyl amino acid was only compatible with the naturally paired substrate. This cannot be attributed to the editing function of the C-domain as the upstream aminoacyl electrophile interacts with the donor site of the C-domain, which has been shown to be relatively non-selective towards the donor side chain [86,142]. It is believed that only the natural intermediate is positioned properly for reaction with the unnatural N-methyl amino acid derivative, which presents a more complex bonding partner due to the steric hindrance presented by the methyl group. Furthermore, the ability of the acceptor site of the upstream C-domain to accept the unnatural N-methyl amino acid substrate indicates the C-domain does not show a pronounced selectivity towards this modification. C domains upstream and downstream of E- possess stereospecific acceptor and donor sites, respectively, for the N-terminal side chain of the donor peptidyl-S-

177 Ppant intermediate and the side chain of the acceptor aminoacyl-S-Ppant intermediate [77]. This specialised property of C-domains that border E-domains can also be construed in terms of C-domains, that handle N-methyl amino acids. It is purported that E-domains must possess higher catalytic turnover rates than the downstream C-domain in order to avoid stalling of the peptidyl chain due to the stereoselective barrier imposed by the donor site donor site of the downstream C-domain [77]. It is tempting to speculate that a similar chemical selectivity towards the donor and acceptor aminoacyl side chains exists at the acceptor and donor sites of the proximal C-domains upstream and downstream of N-MTase domains, respectively. In analogy to the E-domain scenario, the N-MTase domain must possess a greater catalytic capacity than the downstream C-domain such that the side chain of the Ppant anchored residue is N-methylated faster than the impending peptide bond formation event at the downstream C-domain. It is also likely that the timing of these events is under some form of conformational control. Unlike the strict chiral barrier imposed by the C-domain immediately downstream of E-domains, C-domains downstream of N-MTase domains appear to show less selectivity for N-methylated-aminoacyl intermediates, as indicated by the aforementioned study [161]. Moreover, this postulate is substantiated by the observation that the assembly of the peptide chain can proceed in the absence of AdoMet, albeit at markedly reduced rates in comparison to the methylated product [160,165]. The production of the demethyl congener of the partial product of CsA, cyclo-(D-alanyl-L-leucyl) diketopiperazine (demethyl-D-DKP) by CySyn proceeds at a slower rate than the corresponding methylated congener; similarly, the rate of biosynthesis of demethyl-enniatin by enniatin synthetase was found to be 90% slower in the absence of AdoMet. This would account for the fact that of the seven N-methylated residues in the cyclosporin structure, a maximum of two demethyl positions are observed across the natural cyclosporins [119]. However, the influence of the pattern of backbone N-methylation on the preorganisation of the peptide backbone for cyclisation may also be a contributing factor. This may impose restrictions for the insertion or deletion of N-MTase domains when constructing recombinant forms of this system. Across the naturally occurring cyclosporins, demethyl residues occur at positions 1,6, 9, 10 and 11 [52,122]. In particular demethylations are most common at positions 1,6 and 10 suggesting the corresponding modular N-MTase centre possess a marginally reduced catalytic efficiency. Precursor-directed synthesis of peptide analogues CySyn is perhaps the NRPS, which has been best explored for its suitability for the precursor-directed synthesis of CsA analogues. As there was an immediate need for high yield fermentation of CsA, early research into enhancing its yield by feeding of suitable precursor amino acids gave some insight into the biosynthetic system responsible for CsA formation. The first study published on

178 that issue [54] reported an up to 7.5-fold increase of cyclosporin production by adding the amino acids DL-Abu, L-Thr, L-Val, L-Nva and L-Ala, the highest increase observed after the addition of valine. While addition of 2-aminobutyric acid led to the almost exclusive formation of CsA, addition of the latter four amino acids also triggered the formation of CsC, CsD, CsG and CsB, analogues, which have L-Abu at position 2 of CsA replaced by the respective amino acid. The stimulation of CsA production by addition of L-Val to the fermentation broth has since been reproduced by several laboratories [166–168] and is commercially exploited in CsA fermentation. Addition of amino acids not normally incorporated into CsA can yield novel analogues as exemplified by the formation of [MeIle4]CsA (Cs29) upon addition of D-Thr to the fermentation [169]. As D-Thr is not a direct precursor for the biosynthesis of [MeIle4]CsA, it is believed to be metabolised by the fungus to isoleucine. Direct supply to the fermentation, however, does not yield increased amounts of [MeIle4]CsA, most likely again due to other metabolic use of isoleucine by the fungus. The problems of fungal metabolism can be easily overcome by using an in vitro biosynthetic system. However, this approach is limited by the need to supply all precursor amino acids needed for CsA biosynthesis and by the amount of enzyme available. Especially the former is problematic, as Bmt has to be either fermented using a strain of T. inflatum, which is blocked in CsA synthesis [170] or chemically synthesised. Already the first claim of complete in vitro biosynthesis of CsA included a claim of in vitro biosynthesis of 5 analogues in addition to CsA [171]. However, the enzyme preparation used in these experiments did not appear to contain any intact CySyn and in the light of later results the data in this publication are most likely reflecting artefacts as discussed earlier [172]. After the preparation of intact enzyme had been established [8], sufficient enzyme was available to systematically explore the specificity of the individual modules for the incorporation of a range of amino acids into the various positions of CsA [120,123]. In order to upscale in vitro cyclosporin production and to overcome the problems with the instability of the enzyme at standard incubation temperatures, preparative cyclosporin formation was routinely performed at 4 C for 7 days. With this procedure it was possible to yield g amounts of cyclosporins, sufficient for Fast Atom Bombardment (FAB) mass spectrometry analysis and a cell culture based semi-quantitative evaluation of their immunosuppressive activity. Table 1 summarises the various CsA analogues obtained by this procedure and their immunosuppressive activities, if tested. The structures of all these analogues have been established by co-chromatography with authentic references and/or FAB mass spectrometry. The substrate specificities of the various modules are discussed elsewhere [52,120,172]. Various fungi produce cyclosporins, some have an apparently different spectrum of analogues produced than Tolypocladium inflatum. Whether these differences are the result of different precursor availability or of variations in the substrate specificities of the individual enzymes has yet to be analysed. Examples include the formation of [Leu4]CsA and MeLeu1]CsA by

179 Table 1. Verified cyclosporin analogues synthesised in vitro. Cyclopeptide R

Cyclosporin A (Sandimmun ) Cyclosporin A [Me-L-2-amino-3-hydroxy-4methyloctanoic acid1]CsA [Me-L-3- cyclohexylalanine1]CsA [MeLeu1]CsA [MeLeu1,Nva2]CsA ( ¼ CsO) [Me-L-2-aminooctanoic acid1]CsA [Me-3-hydroxy-3-cyclohexyl-L-alanine1]CsA [Me-L-2-aminooct-6-enoic acid1]CsA [Me-L-2-amino-3-hydroxy-4,8dimethylnonanoic acid1]CsA [Me-cyclo-(dihydro-Bmt)1]CsA [Me- L-Ser1]CsA [Ala2]CsA ( ¼ CsB) [Thr2]CsA ( ¼ CsC) [Val2]CsA ( ¼ CsD) [Nva2]CsA ( ¼ CsG) [L-allylglycine2]CsA [L-aThr2]CsA [L-Cys2]CsA [Nva2,5]CsA ( ¼ CsM) [Nva2,5, MeNva11]CsA [Val4]CsA ( ¼ CsQ) [MeIle4]CsA ( ¼ Cs29) [Nva5, MeNva11]CsA [aIle5, aMeIle11]CsA [Ile5, MeIle11]CsA [Cyclopropylglycine5, MeCyclopropylglycine11] [Leu6]CsA ( ¼ CsU) [Gly7]CsA [Abu7]CsA ( ¼ CsV) [bAla7]CsA [Gly7,8]CsA [Abu7, D-Abu8]CsA [b-chloro-D-Ala8]CsA [b-fluoro-D-Ala8]CsA [D-Abu8]CsA [D-Ser8]CsA [D-Phe8]CsA [D-vinylglycine8]CsA [D-Cys8]CsA [D-Lys8]CsA [Gly8]CsA [bAla8]CsA

Biosynthesis

Analysis

Activity

natural enzymatic enzymatic

FAB FAB HPTLC

þ þ þ þ þ þ nd

enzymatic enzymatic enzymatic enzymatic enzymatic enzymatic enzymatic

HPTLC HPTLC HPTLC HPTLC FAB HPTLC HPTLC

nd nd nd nd þ þ nd nd

enzymatic enzymatic enzymatic enzymatic enzymatic enzymatic enzymatic enzymatic enzymatic enzymatic enzymatic enzymatic enzymatic enzymatic enzymatic enzymatic enzymatic enzymatic enzymatic enzymatic enzymatic enzymatic enzymatic enzymatic enzymatic enzymatic enzymatic enzymatic enzymatic enzymatic enzymatic enzymatic enzymatic

HPTLC HPTLC HPTLC HPTLC HPTLC HPTLC HPTLC HPTLC HPTLC HPTLC FAB HPTLC FAB FAB FAB HPTLC FAB HPTLC FAB FAB FAB FAB FAB HPTLC HPTLC FAB HPTLC HPTLC FAB HPTLC HPTLC FAB FAB

nd nd nd nd nd nd nd nd nd nd þ nd  þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ nd nd þ þ

þ

þ(þ) þ þ(þ) þ þ þ þ þ þ þ þ þ þ

þ(þ) þ

The identity of the various cyclosporins was either established by co-chromatography with authentic reference in three different solvents or by FAB mass spectroscopy [120,123,205]. The immunosuppressive activity is given in a seminquantitative form ( þ þ þ , high, þ þ , moderate, þ , week and , no activity).

180 Table 2. Verified SDZ-214-103 analogues synthesised in vitro. Cyclopeptide

Biosynthesis

Analysis

Activity

SDZ 214-103 SDZ 214-103 [Me-L-2-amino-3-hydroxy-4methyloctanoic acid1]-SDZ 214-103 [3-Hydroxynva2]-SDZ-214-103 [Leu4]-SDZ 214-103 [Ile4]-SDZ 214-103 [Abu7]-SDZ-214-103 [D-lactic acid8]-SDZ-214-103 [D-2-hydroxybutyric acid8]-SDZ-214-103 [D-2-hydroxy-n-valeric acid8]-SDZ-214-103 [D-2-hydroxy-3-methylvaleric acid8]-SDZ-214-103 [D-2-hydroxyisocaproic acid8]-SDZ-214-103 [MeAbu11]-SDZ 214-103 [MeaIle11]-SDZ 214-103

natural enzymatic enzymatic

FAB FAB HPTLC

þ þ þ þ þ þ nd

enzymatic enzymatic enzymatic enzymatic enzymatic enzymatic enzymatic enzymatic enzymatic enzymatic enzymatic

FAB HPTLC HPTLC FAB FAB FAB FAB FAB FAB FAB FAB

þ nd nd þ þ þ þ þ þ þ þ

þ þ

þ(þ) þ þ þ þ þ þ þ (þ) þ þ(þ) þ(þ)

The identity of the various cyclosporins was either established by co-chromatography with authentic reference in three different solvents or by FAB mass spectroscopy [120,158,172]. The immunosuppressive activity is given in a seminquantitative form ( þ þ þ , high, þ þ , moderate and þ , week activity).

Tolypocladium terricola [173], [Thr2, Leu5, Leu10]CsA by Stachybotrys chartarum [174], [Thr2, Leu5, Ala10]CsA by Acremonium luzulae (Fuckel) W Gams [56] and [Thr2, Ile5]CsA by Leptostroma anamorph of Hypoderma eucalytii Cooke & Harkn [56]. One example where the biosynthesis of the cyclosporin analogue is clearly the result of a different enzyme, is the peptolide SDZ 214-103 ( ¼ [Thr2, Leu5, 8 10 D-Hiv , Leu ]CsA). This peptolide is produced by the fungus Cylindrotrichum oligospermum (Corda) Bonorden and is distinguished from CsA in three positions; the major difference being the exchange of D-Ala by D-hydroxy isovaleric acid (D-Hiv) [158]. SDZ 214-103 synthetase is not capable of incorporating D-amino acids into position 8 and CySyn is incapable of incorporating D-hydroxy acids into position 8 [120], most likely indicating that the module for this position is the main one that is changed between the two enzymes. Taking a similar approach as described for the in vitro biosynthesis of novel cyclosporins, a range of novel SDZ 214-103 analogues has been produced and analysed (Table 2) [120,172]. Alteration or inactivation of pathways responsible for the supply of amino acid precursors to the NRPS is another attractive approach to metabolic engineering of novel products [175]. The relatively low specificity of most NRPS A-domains would allow for the recognition and incorporation of altered precursors derived from the modified pathway. Alternatively, the natural pathway can be inactivated and the culture broth supplemented with an analogue. Thus, the PKS responsible for supplying CySyn with Bmt could be engineered, such that it produces Bmt-variants with functional groups that can

181 be chemically modified. As in vitro biosynthetic approaches are limited to small scale biosynthesis, there is a need for direct manipulation of the selectivity of the protein template at the genetic level. Combinatorial biosynthesis of non-ribosomal peptides One of the most exciting facets of research into these multi-functional biocatalysts is the production of chimeras that are capable of synthesising novel peptides. Over the years a variety of molecular biology methodologies have been established for the cloning of peptide synthetase genes [85,176–178]. Together with the recent advances in automated sequencing technology and bioinformatics, we are well equipped to implement combinatorial design projects to produce altered non-ribosomal products. Furthermore, due to the use of high throughput automated screening techniques, large numbers of peptide secondary metabolites are routinely discovered. The high degree of sequence identity conserved across functionally homologous domains of NRPSs often allows for the prediction of the architecture of the synthesising NRPS from the structure of the peptide product. Conversely, the same principles permit the prediction of the product of putative biosynthetic gene clusters detected in genome sequences. Their modular architecture allows for combinatorial approaches for the construction of hybrids for the biosynthesis of novel products. To this end, knowledge of the structural/functional properties and the size limits of each domain type is paramount for the construction of productive domain fusions. At present many of these factors remain enigmatic, particularly with respect to the nature of the interactions between specific modules. One potential problem with the construction of recombinant peptide synthetases is the preservation of the functional conformation. Knowledge of the three-dimensional arrangement of the domains within an elongation module and the integrated modular arrangement of a native NRPS system is of great value for the design of recombinant systems. Unfortunately, to date efforts to obtain crystals of NRPS modules have been unsuccessful. Another important aspect, which remains to be fully explored, is the portability of various domain types, both within the maternal system and into heterologous systems. Indeed, there is a need for consideration into the matching of compatible domain interfaces when designing hybrid systems as was highlighted by the apparent editing function of C-domains at the acceptor site [86,142]. Genetic level combinatorial approaches for the rational design of hybrid NRPSs include module/domain exchange, insertion or deletion and the alteration of the substrate specificity of the A-domain via site-directed mutagenesis [85,88]. Most of these combinatorial techniques have been successfully applied to PKS systems that also synthesise therapeutically important compounds, such as the erythromycin antibiotics [179–184]. By virtue of the mechanistic and architectural similarities between these systems, this knowledge will serve as a paradigm for parallel studies on NRPS systems.

182 Several examples of functional hybrid NRPS proteins, genetically engineered by module and domain exchanges, were recently reported [109,151,152,161,185–187]. These studies have been paramount in defining the inter-domain/modular boundaries suitable for artificial fusions. A notable example is the engineering of a hybrid surfactin synthetase complex via in vivo recombination. The replacement of a leucine-activating module with modules of different specificities from the gramicidin S and ACV synthetase systems, generated hybrid NRPS species capable of assembling the predicted surfactin variants [185]. However, the levels of surfactin variants produced by the recombinant strains were very low compared to surfactin production, indicating the alterations to the native molecular architecture possibly affect the optimal spatial configuration of the catalytic domains and the ability of downstream modules to process the un-natural intermediates. The exchange of CySyn modules that display a stringent substrate selectivity (such as module 7, which displays a high specificity for Gly) with modules, which possess a broader substrate specificity (such as module 6 (Abu)), should allow for the production of a greater spectrum of cyclosporins via precursor-directed synthesis, than is accessible with the native enzyme. The work from Biochimie Kundl has demonstrated that the combinatorial biosynthesis of cyclosporins is tenable. A plasmid transformation system has been developed for T. inflatum, consisting of the promoter element derived from genomic cyclophilin fused to a bacterial hygromycin phosphotransferase gene [188]. Using this system, T. inflatum protoplasts were transformed at high frequency with plasmid constructs containing internal fragments of the CySyn gene. A successful homologous cross-over event between the cloned fragment and the genomic CySyn DNA was evident by the detection of cyclosporin non-producing transformants. The insertions were also verified by southern hybridisations. The high frequency of CySyn knockout transformants that were obtained, suggests that T. inflatum possesses a single genomic copy of the CySyn gene. These CySyn knockout mutants can also serve as hosts for CySyn genes mutated in vitro or for the fermentative production of D-alanine and Bmt precursors [189,190]. More recently, Leitner et al. [190] have described the generation of a recombinant vector containing the entire CySyn DNA sequence and the transformation of T. inflatum cells with this construct. The implementation of this transformation system for T. inflatum will also allow for the selection of transformants with multiple inserts of the CySyn gene. In this manner, strains with enhanced cyclosporin production capabilities can be generated expediently compared to conventional mutagenesis and strain selection methodology [190]. Furthermore, the use of the isolated CySyn gene potentially allows for the generation of cyclosporin variants via site-directed mutagenesis of the A-domain specificity determinants and/or the deletion/insertion or exchange of individual modules and domains. The authors have also employed the CySyn DNA for screening of microbes for CySyn genes that may have been overlooked in product screening tests due to the inactive state of their CySyn gene. If the

183 isolated gene encodes a CySyn enzyme that produces pharmacologically important cyclosporin analogues, it may be desirable to reactivate and transform the gene into T. inflatum for the production of this cyclosporin variant. Alternatively, the CySyn DNA of T. inflatum can be recombined with the heterologous CySyn DNA isolated from producers of different cyclosporins to construct a hybrid synthetase with the desired product profile. This procedure can also be employed for the re-programming of the protein template to direct biosynthesis towards cyclosporin congeners, which are normally minor products in CsA fermentation cultures. Another enigmatic aspect of these systems is the nature of the protein–protein interactions, which govern substrate channelling between modules of different subunits of NRPS complexes. In PKS systems it has been elucidated that these interactions are to some extent governed by short linkers at the N- and C-termini of the protein subunits [104,191,192]. The fusion of heterologous PKS modules has been facilitated by the engineering of the inter-polypeptide modular linkers [191], a similar approach may prove useful in the combinatorial engineering of NRPS systems. In the case of PKSs the channelling of intermediates between modules could be re-directed by the matching of compatible linkers. Analogous inter-polypeptide linker regions have been identified in NRPS and hybrid NRPS/ PKS systems [103]. The functional significance of these poorly conserved regions remains unknown, in particular this issue must be fully clarified for rational combinatorial engineering of these systems to become tenable. Fusions at the linker regions appear to be the most attractive approach, hybrid NRPSs systems constructed via this method exhibit good product yields and processivity [151,152,193]. Although fusions at conserved sequence elements within the domains and at arbitrary restriction sites within the NRPS genes have proven successful in certain instances [194], the engineering of fusion points at the linker regions is a more attractive option as it is less likely to disrupt the native conformational continuity of the synthetase. The compatibility between specific domains types must also be taken into consideration when engineering heterologous inter-domain fusions. The strategy of inter-modular fusions also circumvents the editing function imposed by the acceptor site of the C-domain. Modifications to the backbone length and structure by the insertion/deletion of modules [193] or by the translocation of TE or cyclase domains represent another manipulation strategy of great practical potential [90]. In the case of biosynthetic systems that utilise integral domains or external enzymes to modify the peptide scaffold, modifications to the product can be introduced by the deletion/insertion or substitution of the genes encoding these tailoring functions. This may require the proper partnering of compatible upstream and downstream domains to permit the proper processing of the modified product. In the case of E-domain insertions this will require correct matching with the bordering C-domains because, as alluded to earlier, in this modular context the proximal upstream and downstream C-domains are stereoselective towards the donor and acceptor sites [77]. Moreover, recent evidence indicates there is some form of

184 productive communication between cognate modular T-domains and E-domains [149]. All these factors will have to be considered and clarified to fully allow combinatorial design projects for specific systems. In addition to the example noted earlier [185] there have been many reports wherein hybrid NRPS systems have exhibited a reduced rate of product formation [152,161,186,187,195]. This consistency suggests the domain–domain interactions in these engineered systems may not be entirely optimal. Alternatively, the reduced product turnover maybe due to an incompatibility between the foreign substrate and the donor electrophile and acceptor nucleophile sites of the C-domains. Another interesting aberration in function was observed in a hybrid PKS wherein skipping of interpolated modules occurs via the ACP-to-ACP transfer of the growing polyketide chain [196]. A more subtle approach, which is likely not to perturb the architecture of the synthetase, is the site-directed mutagenesis of the substrate specificity determinants of the A-domain regions. The elucidation of the structural basis of amino acid specificity of the A-domain has paved the way for the rational alteration of A-domain selectivity [97,137]. This powerful strategy has been successfully implemented to alter the substrate specificity of the A-domain regions of tyrocidine synthetase and surfactin synthetase NRPS systems [97,136]. One limitation of this procedure is, that so far, successful alterations of A-domain specificity have been achieved only with a limited number of mutations within the substrate binding pocket; thereby allowing for the acceptance of only minor changes in the shape and polarity of the substrate side chain [129]. Moreover, knowledge of the ‘‘non-ribosomal’’ code allows for the implementation of combinatorial engineering strategies that involve the exchange of A-domain regions to give the desired product [129]. Given the increasing pool of A-domain specificities obtained from the mapping of NRPS gene clusters and genome sequencing projects of model organisms, this strategy is a promising approach for the alteration of substrate specificity. Molecular modelling and sequence comparisons of the 11 A-domains of CySyn indicated the recognition of amino acid substrates is dictated by three amino acid positions (Fig. 3) [121]. It was possible to define an amino acid recognition code for CySyn, this knowledge will allow for the rational design of mutants for the production of combinatorial libraries of cyclosporins. The vast domain combinations and permutations in the order of modules observed across native NRPSs shows that nature has utilised the molecular toolbox of NRPS domains to perform some metabolic engineering on its own. Interesting examples are the mixed NRPS/PKS systems that incorporate both NRPS and PKS modules. Accordingly, the peptide-polyketide products of hybrid NRPS/PKS systems are constituted from amino acids and short carboxylic acids [179,180]. The natural cross-over between these two multi-functional enzyme families presents further prospects for the combinatorial biosynthesis of novel compounds [103]. Notable examples of the co-operative activity of cognate PKS and NRPS modules include the rapamycin [197], yersiniabactin [12],

185 epothilone [119], bleomycin [11] and mycosubtilin [93] biosynthetic systems. In such systems NRPS and PKS modules function either as singular enzymes in trans, or cis as integrated domains within a single polypeptide [103]. The cis mode of operation involves a functional hybridisation between NRPS and PKS units to mediate the direct vectorial elongation of the product. Trans systems do not involve a direct functional hybridisation between NRPS and PKS proteins, in this case, there is no direct transfer of intermediates between the two enzyme systems. Cyclosporin biosynthesis falls under the latter class, in this instance a polyketide synthase mediates the biosynthesis of the unusual C9 amino acid Bmt [73,74]. Another fascinating convergence between mechanistically related biosynthetic systems is seen in the biosynthesis of the lipopeptide mycosubtilin, which is synthesised by a hybrid NRPS/fatty acid synthase complex [93]. The added diversity presented by these hybrid systems significantly increases the potential for the engineering of artificial constructs from combinations of these systems. To date most of the research has been focused on the engineering of recombinant linear NRPSs, partly due to the predictability and amenability of the linear modular architecture of this class of NRPSs to genetic engineering strategies. However, non-linear NRPSs such as mixed NRPS/PKS systems are also an attractive target for rational design experiments. To date, the determinants of the domain–domain interactions in these systems are poorly understood and will require further investigation to allow for combinatorial exploitation. The engineering of heterologous strains for the production of the desired novel metabolite is another ideal of combinatorial biosynthesis. In addition to serving as a platform for the production of peptide libraries, the heterologous expression of NRPS genes can also be effected to beneficially modify the phenotype of the heterologous host, for example the production of an insecticidal compound in crop plants [198]. One of the main obstacles for in vivo recombination methodology is the lack of natural competence of producer strains, which highlights the need for vector design and the development of an efficient transformation system for the surrogate host strain. Given the antibiotic properties of many of non-ribosomal peptides, it is also necessary to endow the heterologous host with self-resistance genes. The selected heterologous host should possess the necessary cellular processes for the production and maintenance of the biosynthetic machinery for product formation. The production of non-ribosomal peptides in a heterologous host requires the co-expression of a Ppant transferase that post-translationally activates the NRPS. The heterologous expression of NRPSs is also complicated by improper post-translational processing, instability and degradation of these enzyme systems due to their large size [187,198–201]. Owing to the fact that some of these systems utilise unusual non-proteogenic amino acids, the intrinsic amino acid pool of the heterologous host will often be inadequate for peptide production. Thus, in certain cases there will be a need to co-transfect the heterologous host with the respective substrate biosynthetic genes. Moreover, the complete biosynthesis of non-ribosomal

186 peptides often requires tailoring enzymes that modify the peptide scaffold, hence, the heterologous production of the biologically active form of the desired product may require the co-expression of the cognate tailoring enzymes. Homologous recombination methodology has been successfully employed to engineer a B. subtilis strain for the production of foreign NRPS peptides [202]. In this example the integration and heterologous expression of the bacitracin biosynthetic gene cluster from B. licheniforms together with the affiliated selfresistance genes was achieved by deletion and insertion into the chromosomal region encoding the innate surfactin biosynthetic gene cluster. The resultant B. subtilis strain exhibited bacitracin resistance comparable to the native producer and produced bacitracin at elevated levels compared to B.licheniforms indicating functional expression of the foreign biosynthetic and resistance genes. Another notable example of the production of a foreign metabolite in a heterologous host is the production of 6-deoxyerythronolide B, the macrocyclic core of the antibiotic erythromycin in a genetically engineered E. coli strain [201]. In addition to combinatorial strategies based on modification of the protein templates, a novel approach was recently implemented that exploits the versatility of isolated NRPS domains. The isolated NRPS TE domain has been utilised to cyclise and release biomimetic linear peptides bound to a solid-phase resin support [203]. This combination of solid-phase peptide synthesis together with natural product biosynthesis greatly expands the possibilities for combinatorial biosynthesis of novel peptide libraries. The authors also express the potential of the biomimetic solid-phase resin as an artificial T-domain support for a variety of natural products, which in analogy to the TE domain, will allow for the investigation of other isolated NRPS domains. Apart from the genetic level combinatorial approaches described herein, a number of alternative methods for the metabolic engineering of natural products has been described [175]. Furthermore, possibilities to modify products of NRPS systems biologically have been explored to some extent [204]. However, with the advances and versatility of the genetic technologies of the modern laboratory, together with the large pool of NRPS genes available for manipulation, metabolic engineering at the genetic level is by far the most direct and attractive approach.

Conclusion Irrespective of the extensive knowledge collected so far, the discovery and characterisation of additional NRPS systems remains of utmost importance considering that the products of such systems are often of great therapeutic benefit. Moreover, the complexity and the multitude of the possible combinations of modules remains to be explored. All in all, there still remains a number of mechanistic facets of the NRPS pathway in general and with respect to the individual systems that remain enigmatic.

187 Abbreviations ACV Bmt C-domain Cs CySyn D-Hiv E-domain FAB N-MTase NRPS Ppant PheA PKS SSD T-domain TE-domain

d-(a-amioadipyl)-cysteinyl-D-valine; A-domain; adenylation domain (4R)-4-[(E)-2-butyl]-4-methyl-L-threonine condensation domain cyclosporin cyclosporin synthetase D-hydroxy isovaleric acid epimerisation domain fast atom bombardment N-methyltransferase non-ribosomal peptide synthetase 40 -phosphopantetheine phenylalanine activating domain of gramicidine synthetase A polyketide synthetase synthetase-specific domain thiolation domain thioesterase domain

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199

Horseradish peroxidase: a valuable tool in biotechnology Ana M. Azevedo, Vero´nica C. Martins, Duarte M.F. Prazeres*, Vojislav Vojinovic´, Joaquim M.S. Cabral, and Luı´ s P. Fonseca Centro de Engenharia Biolo´gica e Quı´mica, Instituto Superior Te´cnico, Av. Rovisco Pais, 1049-001 Lisboa, Portugal Abstract. Peroxidases have conquered a prominent position in biotechnology and associated research areas (enzymology, biochemistry, medicine, genetics, physiology, histo- and cytochemistry). They are one of the most extensively studied groups of enzymes and the literature is rich in research papers dating back from the 19th century. Nevertheless, peroxidases continue to be widely studied, with more than 2000 articles already published in 2002 (according to the Institute for Scientific Information). The importance of peroxidases is emphasised by their wide distribution among living organisms and by their multiple physiological roles. They have been divided into three superfamilies according to their source and mode of action: plant peroxidases, animal peroxidases and catalases. Among all peroxidases, horseradish peroxidase (HRP) has received a special attention and will be the focus of this review. A brief description of the three super-families is included in the first section of this review. In the second section, a comprehensive description of the present state of knowledge of the structure and catalytic action of HRP is presented. The physiological role of peroxidases in higher plants is described in the third section. And finally, the fourth section addresses the applications of peroxidases, especially HRP, in the environmental and health care sectors, and in the pharmaceutical, chemical and biotechnological industries. Keywords: horseradish peroxidase, hydrogen peroxide, compound I, compound II, benzhydroxamic acid, ferulic acid, heme proteins, plant peroxidases, applications, physiological role, indole acetic acid, structure, calcium, glycans, biosensors, reporter systems.

Introduction Peroxidases are widely distributed in nature and can be easily extracted from most plant cells and from some animal organs and tissues. They are among the first enzymes to have been discovered, with references dating back to the 19th century, already describing a peroxidatic activity in biological systems. Scho¨nbein observed the oxidation of certain organic compounds (such as guaiacol) by hydrogen peroxide (H2O2), as early as 1855. The name peroxidase was first used by Linossier, who isolated it from pus in 1898. In 1917, Willsta¨tter and Stoll introduced the Purpurogallinzahl number (PZ-number), which was probably the first attempt to define units for a non-hydrolytic enzyme [1,2]. Horseradish peroxidase (HRP) and yeast cytochrome c peroxidase (CcP) are some of the most intensively studied peroxidases. In 1976, Welinder determined the first complete primary structure of a peroxidase, HRP [3]. Following this, *Corresponding author: Tel: þ 351218419139. Fax: þ 351218419062. E-mail: [email protected] BIOTECHNOLOGY ANNUAL REVIEW VOLUME 9 ISSN 1387-2656 DOI: 10.1016/S1387-2656(03)09003-3

ß 2003 ELSEVIER SCIENCE BV ALL RIGHTS RESERVED

200 in 1980, Yonetani published the primary sequence of CcP [4], while the first X-ray crystal peroxidase resolved structure, that of CcP, was obtained in the laboratory of Joseph Kraut [5, 6]. Peroxidase superfamilies Peroxidases have been divided into different superfamilies: plant peroxidases, animal peroxidases and catalases. Plant peroxidase superfamily The plant peroxidases superfamily, which contains peroxidases from both prokaryotic and eukaryotic origin, can be divided into three classes, based on structural similarities and certainly in a suspected common evolutionary origin [7]. Class I: Peroxidases of prokaryotic origin Members of this class of peroxidases can be found in organelles of prokaryotic origin, namely in plastids and mitochondria and include yeast cytochrome c peroxidase (CcP) [5, 6], chloroplast and cytosolic ascorbate peroxidase (AsP) [8], and the gene-duplicated catalase-peroxidase (Cat-Per) [9]. A common feature of these peroxidases is the lack of bound carbohydrates, disulphide bonds, calcium ions and signal peptides for secretion. Class II: Secreted fungal peroxidases Contrary to class I peroxidases, class II peroxidases have a signal peptide sequence responsible for their secretion through the endoplasmatic reticulum. They possess about 5% carbohydrates, two calcium ions and four conserved, disulphide bonds. Class II peroxidases include lignin peroxidase (LiP) [10], manganese peroxidase (MnP) [11], both from white-rot fungi such as Phanerchaete chrysosporium, Phlebia radiata and Lentinula edodes, the peroxidase from the hyphomycete Arthromyces ramosus (ARP) [12] and the black inkcap mushroom peroxidase from Coprinus cinereus (CiP) [13]. Class III: Classical secretory plant peroxidases Horseradish peroxidase (HRP), peanut peroxidase (PNP) [14], soybean peroxidase (SoP) [15], turnip peroxidase (TuP) [16], tobacco peroxidase (TobP) [17], tomato peroxidase (TomP) [18] and barley peroxidase (BaP) [19] are examples of class III peroxidases. They contain an N-terminal signal peptide for secretion, two conserved calcium ions, four conserved disulphide bridges in different locations from those in class II, an extra helical region that plays a role in access to the heme edge and a carbohydrate content between 0 and 25%. Some authors have suggested the definition of a fourth group of plant peroxidases, grouping more unusual peroxidases that do not fit into this threefamily classification, such as chloroperoxidases and diheme peroxidases [20].

201 Animal peroxidase superfamily While plant peroxidases are monomeric proteins with a non-covalently bound heme, animal peroxidases are usually dimeric with a covalently attached prosthetic group.

Class I – Halide peroxidases Peroxidases in this class can catalyse the oxidation of halides by H2O2 into their corresponding hypohalous acids, through a halogenation cycle. They have two distinct reaction cycles, the normal peroxidase cycle and the halogenation cycle in which the enzyme is reduced back to the native form directly through oxidation of the halide. Myeloperoxidase, eosinophil peroxidase, lactoperoxidase and thyroid peroxidase are examples of class I animal peroxidases that differ from each other in their halide specificity. Myeloperoxidase (MPO), originally named verdoperoxidase due to its green colour, can be found in the granules of myelocytes, the precursors of neutrophils (a type of leukocytes). MPO is a homodimeric protein where each monomer contains a light and a heavy chain [21]. The heme is a derivative of protoporphyrin IX in which three of its side chains substitutes form covalent bonds with the amino acid residues of the protein, namely Glu242, Asp94 and Met243 [22]. Most of the H2O2 produced by neutrophils is consumed by myeloperoxidase. Many species of bacteria are killed by the combined action of MPO/H2O2/Cl
[23]. Eosinophil peroxidase (EPO) can be found inside eosinophils (another type of leukocytes) and is capable of oxidising bromide to hypobromous acid. EPO also oxidises chloride, but with a lower affinity [24]. Thyroid peroxidase (TPO) can be found inside the mammalian thyroid gland where it plays a major role in the biosynthesis of thyroid hormones. In fact, TPO is responsible for the iodination of tyrosine residues of the thyroglobulin (Tg) protein and for the consecutive coupling of mono or di-iodotyrosines with di-iodotryrosines for the formation of the thyroid hormones tri-iodothyronine (T3) and thyroxine (tetra-iodothyronine, T4), respectively (Fig. 1) [21]. Thyroglobulin is a large glycoprotein, synthesised by thyroid epithelial cells that contains 134 tyrosines [25].

Fig. 1. Schematic representation of the thyroid peroxidase-catalysed formation of thyroxine.

202 Lactoperoxidase (LPO) is an essential component of the defence mechanism of mammalian secretory fluids, such as milk, tears, sweat and saliva. LPO is able to catalyse the H2O2 oxidation of iodide, thiocyanate and tyrosine [26, 27]. Class II – Prostaglandin synthases Prostaglandin H2 synthase is a dimeric, membrane-bound, glycoprotein that can be found primarily inside in the endoplasmatic reticulum. Each monomer contains three independent folding units: an N-terminal epidermal growth factor domain, a membrane binding domain and a C-terminal catalytic domain, containing two distinct active sites that account for both peroxidatic and cyclooxygenatic activities. The cyclooxygenase active site catalyses the formation of prostaglandin G2 (a hydroperoxide) from arachidonic acid, an essential fatty acid, and the peroxidase active site catalyses the reduction of prostaglandin G2 into prostaglandin H2 [28]. Prostaglandin H2 synthase occurs as two isoenzymes: PH2S-1 is constitutive in most mammalian tissues and is responsible for keeping the stomach lining intact and maintaining functional kidneys. In contrast, PH2S-2 is produced only under certain conditions and catalyses the biosynthesis of prostaglandins, which trigger pain and inflammatory responses [29]. Prostaglandins are potent mediators of inflammatory reactions, and are therefore common targets for drugs like aspirin. Aspirin, acetaminophen and ibuprofen belong to a class of drugs called nonsteroidal anti-inflammatory drugs (NSAIDs), which act on the cyclooxygenase portion of the enzyme prostaglandin synthase, inhibiting the production of prostaglandins. The NSAID aspirin has the ability to covalently modify the enzyme. It acetylates a serine residue in the active site of cyclooxygenase, permanently disabling the enzyme (Fig. 2). At that point, prostaglandin synthesis will not resume until more prostaglandin synthase is made [30, 31].

Catalase superfamily Catalases are heme tetrameric enzymes that catalyse the dismutation of H2O2: 2 H2 O2 ! 2 H2 O þ O2 With a unique tyrosyl proximal ligand and four heme groups, one for each subunit, they are structurally different from the other peroxidases. Nevertheless

Fig. 2. Acetylation of the serine residue of PH2S by aspirin.

203 they have several features in common, namely a similar heme prostethic group and the ability to catalyse some of the peroxidases reactions and vice versa [25]. These heme proteins can be found in high concentrations in cell compartments called peroxisomes. H2O2 is a powerful oxidising agent with potential damage effect to cells. Thus, catalase prevents an excessive accumulation of peroxide, allowing important cellular processes, which produce H2O2, to take place safely. They have one of the highest turnover numbers of all known enzymes a characteristic that reinforces the importance of these enzymes as peroxide detoxifiers [32]. Horseradish peroxidase When, in 1810, Planche observed that soaking a piece of horseradish roots into a tincture of guaiacum resin led to the development of a strong colour, he was not aware that the compound responsible for that colour change, HRP, would become the most widely studied of all peroxidases [2]. Since then, HRP has emerged as a valuable tool in biotechnology, finding wide applications in a range of areas. Isoenzymes It is now accepted that peroxidases occur as a large family of isoenzymes. Isoenzymes (or isozymes) are different molecular forms of the same enzyme, which catalyse the same biochemical reaction but have distinct physical, chemical and kinetic properties arising from small differences in their amino acid sequence [33]. Before the development of chromatographic techniques, it was already known that multiple forms of HRP existed throughout the whole plant. Theorell was one of the first authors to isolate two forms of peroxidases from horseradish roots, which presented different absorption spectra and physico-chemical properties [34]. HRP I was basic and contained a low carbohydrate content, while HRP II was neutral and highly glycosylated. These two HRP forms were later on recognised as isoenzymes, and not as modifications of a single form. In 1958, Paul [35] isolated five different forms of peroxidase from horseradish roots using ion-exchange chromatography on carboxymethyl cellulose (CMC). These were labelled with the capital letters A, B, C, D and E. In 1966, Shannon and co-workers [36] confirmed these results and further reported that using DEAE-cellulose chromatography, fraction A could be resolved into three additional fractions, designated as A1, A2 and A3. These authors performed an extensive study, reported in a series of papers, on the characterisation of HRP isoenzymes in terms of their physical, catalytic and structural properties [37–39]. The research group of Morita isolated and characterised five neutral (B1, B2, B3, C1 and C2) [40] and six basic isoenzymes (E1, E2, E3, E4, E5 and E6) [41]. Neutral isoenzymes contain a high carbohydrate content and have similar

204 physico-chemical and kinetic properties. Their main difference appears to be their behaviour in isoelectric focussing gels. Nevertheless, the isoelectric point values vary significantly with the focussing method and conditions used. Basic isoenzymes contain a lower content of carbohydrates than the neutral isoenzymes. Isoenzymes E1 and E2 contain a carbohydrate content between 12.8 and 14.1%, and a pI around 10.6, while isoenzymes E3–E6 have an appreciably lower carbohydrate content (0.8–4.2%), and extremely high pI values of over 12. Reaction cycle Peroxidases catalyse the oxidation of a wide variety of electron donor substrates, such as phenols, aromatic amines, thioanisoles and iodide, by H2O2. The reaction is a three-step cyclic process, in which the enzyme is first oxidised by H2O2 and then reduced back to the native form in two sequential steps involving the formation of two enzyme intermediates, Compounds I and II (Fig. 3). The first step consists in the cleavage of the H2O2 molecule, with the concomitant production of water and incorporation of one of the oxygen atoms of H2O2 into Compound I. Compound I was first identified by Theorell in 1941, four years after the identification of Compound II by Keilin and Hartree [25]. When George correctly proposed the above reaction cycle, in which Compound I contained two oxidising equivalents compared to the native enzyme and Compound II one equivalent, he postulated an iron(V) state for Compound I [42]. It is now known that Compound I contains an oxoferryl group (FeIV¼O), in which the iron is in a þ 4 oxidation state, and a porphyrin p-cation radical.

Fig. 3. Reaction cycle of HRP, showing the enzyme intermediates, Compounds I and II.

205 Compound I is then capable of oxidising a wide range of reducing substrate molecules (AH) by a mechanism involving a single-electron transfer, in which the p-cation radical is first discharged, leading to the formation of the second enzyme intermediate called Compound II. Compound II, which also contains an oxoferryl group (FeIV¼O), is then reduced by a second substrate molecule (AH) to the native ferric enzyme (FeIII). During this one-electron reduction, the ferryl iron returns to its ferric state, whereas the oxygen accepts two protons to form a water molecule and is released from the heme. Everse has proposed that the bond between the iron and oxygen in both Compounds I and II is not a conventional double bond. In fact, except for its length, all experimental observations indicate that the oxoferryl bond may be similar to the heme-O2 bonds in oxy-hemoglobin and oxy-myoglobin and also to the bonds between the oxygen atoms in ozone, i.e., a bi-radical, three-centre four electron p-bond [43]. HRP activity assays The detection of HRP activity is widely used in labelling systems and a large number of procedures have been developed for that purpose. Although H2O2 is the natural substrate, numerous reducing molecules, which are used to monitor HRP activity, are often referred to as peroxidase substrates. Many chromogenic substrates can be used in colorimetric and fluorimetric assays. These substrates are hydrogen donors that upon oxidation form a coloured product that can be monitored spectrophotometrically. Table 1 lists some of the most commonly used substrates of HRP. Almost all phenol and aniline derivatives (e.g. alkyl, halo) are able to reduce HRP Compound I to the native enzyme. Other reactions catalysed by HRP involve chemiluminescence, in which light emission occurs. The most common chemiluminescent substrates are luminol and other related hydrazides. Enhancers like p-iodophenol and luciferin are often used to improve light emission. Amino acid sequence Horseradish Peroxidase is a heme protein with 308 amino acid residues (Fig. 4). The N-terminal residue is blocked by a pyrrolidenecarboxyl residue ( ) that appears to be buried inside the polypeptide chain. The C-terminal peptides were sequenced with and without a serine residue, indicating a rather labile Asn-Ser peptide bond [3, 44]. HRP isoenzyme C (HRPC) polypeptide chain is N-glycosylated in eight specific asparagines and contains four disulphide bridges between Cys11–Cys91, Cys44–Cys49, Cys97–Cys301 and Cys177–Cys209. Two calcium ions (Ca2 þ ) are bound per molecule, one in the distal and the other in the proximal side [3, 44]. Distal indicates location far from the heme and on the contrary, proximal is a distinction in place that indicates location close to the heme.

206 Table 1. Common substrates for HRP. Common name

Synonym

Detection method

ABTS Benzidine TMB DAB Guaiacol Pyrogallol Phenol p-Cresol o-Dianisidine p-Toluidine Tolidine Hydroquinone Resorcinol Catechol 4-Aminoantipyrine p-Anisidine o-Phenylenediamine Luminol Ferulic acid Caffeic acid

2,20 -Azino-di(3-ethylbenzothiazolin-6-sulfonate) 4,40 -Diaminobiphenyl 3,30 ,5,50 -Tetramethylbenzidine 3,30 -Diaminobenzidine 2-Methoxyphenol 1,2,3-Trihidroxybenzene Hydroxybenzene 4-Methylphenol 3,30 -Dimethoxybenzidine 1-Amino-4-methylbenzene 3,30 -Dimethylbenzidine 1,4-Dihydroxybenzene 1,3-Dihydroxybenzene 1,2- Dihydroxybenzene 4-Amino-2,3-dimethyl-1-phenyl-3-pyrazolinone 1-Amino-4-methoxyphenol 1,2-Diaminophenol 3-Aminophthalhydrazide 4-Hydroxy-3-methoxycinnamic acid 3,4-Dihydroxycinnamic acid

Spectrophotometric Spectrophotometric Spectrophotometric Spectrophotometric Spectrophotometric Spectrophotometric Spectrophotometric Spectrophotometric Spectrophotometric Spectrophotometric Spectrophotometric Spectrophotometric Spectrophotometric Spectrophotometric Spectrophotometric Spectrophotometric Spectrophotometric Fluorimetric Fluorimetric Fluorimetric

Fig. 4. The amino acid sequence of HRP isoenzyme C, determined by Welinder [3].

The polypeptide chain has a molecular weight of 33,890 Da determined from its amino acid sequence [3], the heme group weighs 572 Da and the two calcium ions 80 Da, adding up to a molecular weight of 34,542 Da. If the carbohydrate moiety constitutes 20%, then the total molecular weight is around 42 kDa. Nevertheless this calculation does not account for bound water and counter ions.

207 Heme prosthetic group The heme prosthetic group is ferriprotoporphyrin IX (Fig. 5), which is made of four pyrrole rings joined by methene bridges with iron(III) centring the molecule. Three different substitutes are found in the pyrrole rings: four methyl, two vinyl and two propionate side chains. In nature, iron is usually six-coordinated, but in native enzymes, the iron is five-coordinated. In HRP, positions 1–4 are occupied by the four pyrrole nitrogen atoms. Position 5 is located on the proximal side of the heme and is occupied by the imidazole side chain of a histidine residue, the proximal histidine (His170). Position 6 in the native resting enzyme is vacant and located on the distal side of the heme. The linkage from position 5 to the proximal histidine is probably best described as being covalent, and can be broken in acid solution. The propionate side chains form hydrogen bonds with neighbouring residues. Finally, weak hydrophobic forces, in which the organic porphyrin is in Van der Waals contact with hydrophobic amino acids, complete the binding of ferriprotoporphyrin IX to the protein [25].

Crystal structure The determination of the crystal structures of peroxidases is greatly hampered by the presence of attached carbohydrates, which do not facilitate crystal growth. This is the main reason why the first crystal structures published were from yeast CcP [6] and from two catalases [45, 46], enzymes that are not glycosylated. The problem of carbohydrate attachment has now been overcome by the use of recombinant DNA technology. Peroxidases can thus be expressed in Escherichia coli, a host that does not contain the necessary machinery for glycosylation, secretion or proper folding. In this way the overexpressed protein

Fig. 5. Structure of ferriprotoporphyrin IX (2,7,12,18-tetramethyl-3,8-divinylporphine-13,17dipropionic acid).

208 accumulates in the form of inclusion bodies and has to be subsequently extracted, refolded and activated by the addition of calcium, heme, urea and oxidised glutathione in proper concentrations [47]. HRPC was expressed in E. coli from a synthetic gene comprising the 308 amino acid residues determined by Welinder and an additional N-terminal methionine, required to initiate translation. Smith and co-workers observed that the binding of calcium ions was an obligatory step in the folding pathway and that it had to take place before correct disulfide bridge formation and heme incorporation were completed [47]. When the original crystal structure of CcP was determined at 2.5 A˚ resolution, only partial amino acid sequence information was available and consequently there were some errors in assigning some residues to their proper location. The most prominent feature of this structure was the existence of 10 helical segments, helices A to J, which accounted for half of the residues. The structure also contained three anti-parallel
sheets. In 1984, the CcP crystal structure was refined to 1.7 A˚ resolution and an additional helix was found between helices B and C, which was named B0 [5]. All peroxidases from class I and II contain these 11 helices. The first complete crystal structure of a class III plant peroxidase (peanut peroxidase) was only published in 1996 [14]. Schuller compared the structure of four representative members of the three classes in the plant peroxidase superfamily, namely CcP and ascorbate peroxidase (APX) from class I, lignin peroxidase (LIP) from class II and the peanut peroxidase (PNP) from class III [14]. All of them contain the 10 prominent helices of CcP (helices A to J) and 13 structurally conserved regions (SCR) which have a deviation of less than 2.5 A˚. The crystal structure of the recombinant HRP (Fig. 6) was obtained by Gajehede and co-workers, in 1997 [48], at a 2.15 A˚ resolution and only 306 amino acid residues, form the total 308, were visible. Apparently the C-terminal peptide Asn307–Ser308 was missing. HRPC is folded into two clearly defined domains. The heme occupies the crevice between both domains, sandwiched between helix B and helix F. The -helices A to J are found in similar positions to the ones in other peroxidases. In addition, HRP contains three extra -helices (D0 , F0 and F00 ), which are not found in other classes (Table 2). The small helix, D0 , is inserted between helices D and E and is common to class III peroxidases (HRP, PNP and BaP). But the most striking feature of class III peroxidases is a long insertion (of 34 amino acid residues in HRPC) between helices F and G, the so-called FG insertion. This insertion contains the helices F0 and F00 and is maintained by a disulphide bridge (between Cys177 and Cys209). A general sequence alignment of class III peroxidases shows that this region is highly variable within this class. In particular, the loop connecting F0 and F00 helices varies in length and amino acid composition. It has been suggested that these differences may affect the character and the accessibility of the substrate access channel in this region [48].

209

Fig. 6. Ribbon representation of the 3-D structure of HRP. Blue ribbons –  helices; yellow arrows –
sheets; red spheres – calcium ions. The heme is located in the centre of the molecule (C, white; O, red; N, blue; Fe, magenta).

HRP and other class III peroxidases lack the short helix B’, which is found in all known class I and II peroxidases. Two anti-parallel
-strands flank the helical insertion FG and restrain its structural flexibility (Table 2). Other features of HRP structure are reviewed in the following sections. The understanding of any redox enzyme requires detailed structures of the different redox intermediates. This is a rather difficult task because electrons liberated during the X-ray crystallographic analysis can alter the redox state of the active site. Very recently, Berglund and co-workers were able to obtain a three-dimensional movie of the X-ray-driven catalytic pathway of HRP [49]. Their data collection strategy consisted in applying different X-ray doses on individual crystals, at various rotation angles. A mechanism for the reduction of bonded dioxygen species to two molecules of water was proposed. This mechanism involved four successive, one-electron, reductions with the concomitant uptake of a proton, from Compound III (FeIII–O –O
H þ ) to the ferrous HRP (FeII þ 2H2O þ H þ ), via the intermediate forms of Compound I (Porph . , FeIV¼O þ H2O), Compound II (FeIV¼O, H þ þ H2O) and ferric HRP (FeIII þ 2H2O). .

Heme active site The key features found in the heme pocket of HRP (Fig. 7) are common to all other peroxidases. The proximal heme ligand is His170, which is covalently

210 Table 2. The secondary structure of the recombinant HRP isoenzyme C, determined by Gajhede [48]. Secondary structure

a-Helix a-Helix a-Helix a-Helix a-Helix a-Helix a-Helix b-Strand a-Helix a-Helix b-Strand a-Helix a-Helix a-Helix a-Helix

Fig. 7. HRP heme active site.

Common name

A B C D D0 E F 1 F0 F00 2 G H I J

Amino acid residues Beginning

Ending

Val14 Ile32 Phe77 Cys97 Leu131 Leu145 Ser160 Lys174 Met181 Thr199 Leu218 Lys232 Gln245 Ile260 Thr270

Ser28 Cys44 Ala90 Leu111 Asn137 Asn154 Leu166 Gln176 Leu184 Leu208 Gln220 Glu238 Ser252 Asn268 Met284

211 bonded to the iron atom. There is a hydrogen-bonding network, between the heme propionates and the residues Gln176, Ser73, Ser35 and Arg31, strengthening the binding of the heme to the protein [48]. The binding of H2O2 occurs in the distal heme pocket, which is formed by Arg38, Phe41 and His42. Two of these residues (Arg38 and His42) are conserved in all plant peroxidases and have been implicated in the acid–base catalytic cleavage of the O–O bond. The essential role of both these residues as proton donor/acceptors has been confirmed by site-directed mutagenesis. The replacement of His42 by non-polar residues such as Ala (H42A), Leu (H42L) and Val (H42V) leads to a drastic decrease in the rate of Compound I formation of more than five orders of magnitude [50, 51]. The catalytic activity can be partially rescued by the introduction of another histidine residue (e.g. the double mutant H42A:F41H or H42V:R38H) [52], by replacement with a charged residue (H42E) [53] or by the addition of an exogenous imidazol (e.g. 2-substituted imidazoles) [54]. Arg38 replacement also leads to a decrease in the rate of Compound I formation but not as drastically as with His42 [55]. The double mutant H42E:R38S shows one of the slowest rates of Compound I formation, confirming the role of these two residues in the catalytic reduction of H2O2 [55]. These site-directed mutagenesis studies also suggest that His42 has a critical role as an acid–base catalyst and that Arg38 is not directly involved in the acid–base catalysis but enhances the efficiency of the reaction and the binding of ligands. A complex hydrogen bond network seems to ensure the correct positioning of the catalytic residues. A hydrogen bond between His42 N1 hydrogen and Asn70 O1 oxygen increases the basicity of the catalytic His42 and fixes it at the correct position for participation in the acid–basic catalytic reduction of H2O2. It has been observed that the breakage of this hydrogen bond induces severe damage in the enzyme activity [56, 57]. Asn70 N2 is also hydrogen bonded to the oxygen backbone of Glu64, which is in turn hydrogen bonded to a calcium ligand water (Wat12). Arg38 is also hydrogen bonded to His42 N"2, via two water molecules (Wat123 and Wat92) [48]. A strong hydrogen bond is also present between the proximal histidine His170 N1 and Asp247 O1, which is also believed to increase the basicity of the proximal histidine, stabilising high oxidation intermediates and maintaining the resting heme in its five-coordinated state. A strong hydrophobic contact between His170 with Phe221 also promotes the stacking of these two residues [48]. Substrate binding pocket The aromatic substrates binding region in plant peroxidases is a flexible, solventexposed region that allows for fast exchange of solvent molecules and small aromatic compounds. It cannot be considered a substrate binding site in the classical sense [58]. It is generally accepted that aromatic substrates interact with

212 peroxidases via the exposed heme edge rather than with the ferryl oxygen of Compound I or II. Chemical modification with suicide substrate inhibitors has shown that these substrate-like molecules covalently bind to the carbon-20 and carbon-18-methyl, both of which are oriented towards the access channel of the heme pocket [59]. Gajhede and co-workers showed that HRP contains a hydrophobic patch of three peripheral phenylalanine residues (Phe68, Phe142 and Phe179), which constrains the entrance to the exposed heme edge, and suggested that this aromatic region was responsible for HRP ability to bind aromatic substrates [48]. Other hydrophobic residues, such as Leu138, Pro139, Ala140, Pro141 and Gly69 surround the substrate access channel. Phe68 and Gly69 are involved in hydrophobic contacts with the distal heme pocket at the top of the entrance. The amino acid residues between Leu138 and Phe142 form a hydrophobic plateau at the bottom of the substrate access channel. Leu138 and Pro179 form a hydrophobic wall to the left and right of the substrate, respectively [58]. Benzhydroxamic acid binding site Benzhydroxamic acid, BHA, substrate molecule is a weak phenolic acid with no established physiological role (Fig. 8). In 1998, Henriksen and co-workers [60] published the first three-dimensional structure of a peroxidase–substrate complex, the structure of recombinant HRP complexed with BHA. The crystallographic analysis of HRP–BHA complex showed that BHA binds to the distal pocket and demonstrated the existence of an aromatic binding pocket.

Fig. 8. Schematic representation of the hydrogen bond network of HRP–BHA complex [60].

213 The hydrophilic part of BHA forms hydrogen bonds to the distal catalytic His42 and Arg38, to the backbone oxygen of Pro139 and with a water molecule placed above the heme iron. The hydrophobic pocket is provided by residues Phe68, Gly69, Ala14, Pro141, Phe179 and by heme C18, C18-methyl and C20. A large reorientation of Phe68 near the access channel upon BHA binding is observed: the aromatic ring of this residue becomes almost perpendicular to the face of the aromatic ring of BHA, forming a lid on the hydrophobic pocket [60]. Ferulic acid binding site Ferulic acid (FA) is a phenolic cinnamic acid derivative found in the plant cell wall, which acts as an in vivo substrate of plant peroxidases (Fig. 9). Henriksen and co-workers have resolved the X-ray structure of the binary HRP–ferulic acid complex and the ternary HRP–cyanide–ferulic acid complex to 2.0 and 1.45 A˚ [61]. They observed that the binary complex showed a high disorder in the electron density of the substrate and three binding modes were reported. One of them, however, was not physiologically possible, since it contained the carboxylate facing the interior, which in vivo is esterified to the hydroxyl groups of polysaccharides. In the ternary complex, a cyanide molecule was found co-ordinated in the sixth position of the heme iron and there was only a single FA binding mode. This single FA binding site was identical to the most populated conformation of the binary complex. The binding of FA is mediated by hydrogen bonds and

Fig. 9. Schematic representation of the hydrogen bond network of HRP–CN–FA complex [61].

214 hydrophobic contacts. The aromatic hydroxyl is hydrogen bonded to Arg38 N2 and also to the active site water molecule. This water molecule is hydrogen bonded both to the backbone oxygen of Pro139 and to the cyanide nitrogen. The hydrogen bond network is completed with the hydrogen bond between the methoxy oxygen of FA and Arg38 N2. The hydrophobic contact is made through Ph68, Gly69, Pro139, Ala140, Phe142, Phe179 and heme C20 and C18-methyl. In both complexes (HRP–FA and HRP–CN–FA), a slight side chain rearrangement near the active site is observed. Phe68 is also repositioned, as in the HRP–BHA complex, but in an open lid orientation. This probably explains the higher dissociation constant of the ternary complex (3.8 mM) when compared with the HRP–BHA complex (2.4 M in the resting state and 0.15 mM in cyanide-ligated state) [62].

Calcium binding pocket Calcium ions are the most abundant metal ions found in protein structures deposited in the Brookhaven Protein Data Bank (PDB). Calcium ions have the ability to bind to oxygen atoms with a coordination number that usually varies between 6 and 7. These oxygen atoms belong either to the backbone carbonyl, to the side chain carbonyl (from Gln and Asn), to the side chain carboxyl (from Glu and Asp), to the side chain hydroxyl (from Thr, Ser and Tyr) or to water oxygens. All plant peroxidases in classes II and III contain two calcium binding sites, one proximal and another distal to the heme. These sites are thought to play an important role in defining the heme pocket architecture [63–65]. The number of coordination of both calcium ions is seven, implying a pentagonal bi-pyramidal geometric conformation, and hence the existence of a bidentate residue (formed by the combination of a backbone (bb) and a sidechain (sc) oxygen) (Table 3). Table 3. The O-donor ligands of distal and proximal calcium ions in HRP [48]. Distal calcium

Proximal calcium

Ligand

Oxygen atom

Ligand

Oxygen atom

Asp 43 Asp 43 Val 46 Gly 48 Asp 50 Ser 52 Water

–C¼O, bb –COOH, sc –C¼O, bb –C¼O, bb –COOH, sc –OH, sc H–O–H

Thr 171 Thr 171 Asp 222 Thr 225 Thr 225 Ile 228 Asp230

–C¼O, bb –OH, sc –COOH, sc –C¼O, bb –OH, sc –C¼O, bb –COOH, sc

bb, backbone; sc, sidechain.

215

Fig. 10. Systematic representation of the major glycan structure attached to HRP. Asn, asparagine; Fuc, fucose; GlcNac, N-acetylglucosamine; Man, mannose and Xyl, xylose.

Glycan content N-linked glycans of all plant glycoproteins are covalently bonded through an amide bond to asparagine residues (Asn) belonging to a common consensus sequence, Asn–Xxx–Ser/Thr, where Xxx is any amino acid residue other than proline and aspartic acid [66, 67]. The determined amino acid sequence of HRP showed that there were nine potential N-glycosylation sites, only eight of which were effectively glycosylated (Asn13, Asn57, Asn158, Asn186, Asn198, Asn214, Asn255 and Asn268) [3]. HRP glycans are composed of mannose (Man), xylose (Xyl), fucose (Fuc) and N-acetylglucosamine (GlcNAc) and account for ca. 20% of the total enzyme molecular weight. In 1988, McMannus and co-workers [68] identified the major HRP glycan that comprises 80% of the total carbohydrate content (Fig. 10). These authors released the oligosaccharides from the protein by hydrazinolysis, purified them by gel filtration, analysed the composition of the major fraction and determined its structure by high resolution 1H NMR spectroscopy [68]. Other authors confirmed the structure of the major glycan. In 1991, Kurosaka and co-workers [69] raised an antiserum against HRP that recognised the nervous system of Drosophila and other insects, namely the carbohydrate moieties expressed on some neural proteins. The structure of the major HRP carbohydrate unit, which they thought to be responsible for the antigenic response of the neural cells, was then determined, confirming the structure proposed by McMannus. Further studies were performed in order to elucidate the nature and distribution of HRP minor carbohydrates. Montgomery and colleagues [70,71] analysed the oligosaccharides released by digestion with glycanase A using MALDITOF1 mass spectroscopy. They confirmed once more the structure of the major glycan and identified some minor species as members of the (Xyl)xManm(Fuc)f GlcNAc2 family with x ¼ 0 or 1; m ¼ 2, 4, 5, 6; f ¼ 0 or 1. Traces of high-mannose oligosaccharides were also detected. These studies revealed a high heterogeneity of the glycans at each N-glycosylation site [70,71]. Takahashi and co-workers also found heterogeneity in HRP glycans but in a different way [72]. These authors did not detect the presence of high-mannose 1

Matrix-assisted laser desorption/ionisation time-of-flight (MALDITOF).

216 type glycans and identified different minor species. They also reported that different batches of HRP isoenzyme c from the same commercial source had considerable differences in the oligosaccharide composition. All glycosylation sites point away from the molecule and are evenly distributed throughout the protein surface, suggesting that one of the main function of the glycans is to maintain the protein conformational structure and to increase the protein solubility in water [48, 73]. So far few studies have directly addressed the role of the glycans attached to peroxidases. Deglycosylated HRP It has been shown that HRP glycans are resistant to the action of several endoglycosydases (such as N-glycanase, Endo H2 and Endo F3), normally used to deglycosylate many glycoproteins. Consequently several chemical deglycosylation procedures have been developed [74, 75]. In 1995, Tams and Welinder developed a protocol using anhydrous trifluoromethane sulfonic acid in the presence of phenol, which yielded a fully active enzyme and removed all carbohydrates, except the asparagine-linked (GlcNAc)2 glycans [76]. They showed that the glycans had no effect on HRP specific activity and reaction kinetics. However, the deglycosylated HRP (d-HRP) showed a greatly reduced solubility in salt solution. Smith and co-workers had already obtained an active and non-glycosylated HRP by heterologous expression in E. coli [47]. It is believed that glycosylation decreases dynamic fluctuations throughout the molecule, i.e., the glycans restrain movements within the protein domain. Consequently, the higher transition state energy needed for unfolding, will be attained less frequently. In fact, the unfolding of d-HRP in guanidinum chloride was 2–3 times faster than the unfolding of native HRP at pH 7, 23 C [77]. Glycosylation may also stabilise the polypeptide chain against uncontrolled proteolysis [78] and free radical induced protein crosslinking [79]. It also plays an important role in intracellular sorting, externalisation of glycoproteins and embryonic development and differentiation [78]. Catalytic mechanisms Formation of Compound I Different mechanisms for the formation of Compound I have been proposed throughout the years. Nevertheless, the most widely accepted mechanism is based on the model proposed by Poulos and Kraut in 1980 for CcP (Fig. 11) [80]. According to these authors the reaction starts when H2O2 enters the heme crevice and binds to the heme iron. This initial interaction between the peroxide and the iron consists in the formation of a ligand bond between FeIII and one of the peroxide oxygens (the -oxygen-O), and in the subsequent abstraction of a 2 3

Endo-
-acetylglucosaminidase H (Endo H). Endo-
-acetylglucosaminidase F (Endo F).

217

Fig. 11. Mechanism of Compound I formation.

proton by the distal His42. The intermediate enzyme complex formed is referred in the literature as Compound 0. This compound is very unstable and has only been detected in cryosolvents at negative temperatures using peroxidase mutants with low activity [81, 82]. The next step consists in the heterolytic cleavage of the O–O bond. His42 transfers the abstracted proton to the
-oxygen (O
) and the positively charged guanidinium side chain of Arg38 stabilises the negative charge that is generated at the O
, promoting the cleavage of the O–O bond. A water molecule is then produced, while the O remains bonded to the heme iron. A fast intramolecular electron transfer then occurs in the heme active site and the oxygen atom acquires two electrons, one withdrawn from the iron and the other from the porphyrin ring [80]. In the mechanism described above, the distal histidine plays the main role as both proton acceptor from O and proton donor to O
. The distal arginine facilitates the cleavage of the O–O bond. Some authors have proposed the involvement of different residues. If histidine residues are modified with a specific reagent (diethyl pyrocarbonate), the enzyme is still able to reduce H2O2 and produce Compound I, while Compound II formation is blocked [83]. It was concluded that His42 was not directly involved in the formation of Compound I and that the carboxylate side chain of Asp43 could participate in the reaction [83]. Recent work suggests that there is no reason to invoke His42 as both proton donor and acceptor [84]. Instead, Arg38 could be the only residue able to donate a proton to the peroxide O
, a mechanism that was supported with molecular dynamics simulation studies. Formation of Compound I at acid pH The formation of Compound I requires the abstraction of a proton from the oxygen bound to the heme iron by a neighbouring residue, a role that has been attributed to the distal histidine. However, at low pH, His42 is protonated and thus cannot accept a proton from the peroxide. Filizola and Loew [84] proposed that the crystallographic water molecule Wat400 could serve as proton acceptor from the peroxide O and that both Arg38 and His42 could play the role of proton donor. They supported the role of Wat400 with the calculated formation

218 of a stable hydrogen bond between the oxygen of this water molecule and the hydrogen in the peroxide O. An alternative mechanism to the one described above, also proposed by Filizola and Loew [84], is based on a dynamic interchange of the peroxide oxygen atoms as ligands to the heme iron. Loew and colleagues had previously reported that CcP formed a stable complex with HOOH in which the oxygen atoms systematically exchange places as ligands to the iron [85, 86]. The first step in this mechanism is proton donation by Arg38 to the oxygen atom bonded to the iron. Subsequently, the iron ligand changes, allowing the formation of a hydroxywater complex. The water molecule Wat400 then accepts the proton of the ligand oxygen, leading to the formation of the oxywater complex. Rodrı´ guez-Lo´pez and co-workers presented data that was not consistent with the role of Arg38 as the proton donor at any pH. They suggested a mechanism in which the protonated His42 donates a proton to the peroxide O
and further abstracts the proton from the O [87]. Mechanism of Compounds I and II reduction The mechanism of Compounds I and II reduction has not been as intensively studied as the mechanism of Compound I formation. It is generally accepted that when the substrate molecule binds to Compound I, an electron is transferred to the porphyrin ring, via the exposed heme edge, and the p-cation radical disappears. It is also known, from NMR and resonance Raman spectroscopy studies, that this reduction is accompanied by the uptake of a proton, and that this proton does not bind to the oxy-ferryl group [88–90]. For the case of a phenolic substrate, the hydrogen bond between Arg38 N2 and the phenolic oxygen is thought to assist the proton transfer from the phenol substrate to the protein (Fig. 12). The final destination of this proton has been suggested to be the imidazole side chain of the distal histidine [91]. As Compound II is formed

Fig. 12. Reduction of Compound I by a phenol substrate molecule. B: represents a protein group that mediates the abstraction of the phenolic proton to His42.

219

Fig. 13. Mechanism of reduction of Compound II.

the radical phenolic substrate leaves the protein and is substituted by a fresh substrate molecule. The reduction of Compound II occurs by a similar mechanism but this time the final destination of both proton and electron is the ferryl oxygen (Fig. 13). As the ferryl heme iron (FeIV) is reduced to the ferric state (FeIII), the ferryl oxygen accepts two protons (one from the substrate molecule and the other from the distal His) to form a water molecule that is released from the heme iron. Henriksen and co-workers have suggested that both proton transfer from the substrate molecules could be mediated by a water molecule situated in the active site that is hydrogen bonded to Pro139 [61]. Gajhede proposed a similar mechanism in which the water molecule originated from the peroxide reduction was not removed from the active site and provided the highway for proton transfer [58]. Physiological role of plant peroxidases Plant peroxidases are constitutive enzymes mainly found in the cell wall, vacuoles and transport organelles and also on rough endoplasmatic reticulum. They play an important role in plant physiological responses including auxin catabolism [92], modification of the cell wall [93], lignification [17, 94], pathogen defence [95] and wound healing [96]. Auxin catabolism Auxins are plant hormones with an important role in plant growth and development, including the control of elongation, division and differentiation of cells. One of the most important auxins produced by plants is indole-3-acetic acid (IAA). The oxidation of the IAA by HRP has been shown to occur in the absence of added H2O2. Product analysis and kinetic studies strongly support a complex, radical mechanism, in which many intermediates and end products of IAA oxidation are formed (e.g. indole-3-methanol, indole-3-aldehyde, indole-3-methylhydroperoxide).

220 Extensive studies on IAA oxidation have been carried out for almost half a century, leading to an accumulation of contradictory experimental results and interpretations. Two main reaction pathways have been proposed to describe the oxidation of IAA, the classical peroxidase cycle and an oxygenase (or oxidase) cycle. The specific pathway followed seems to depend upon the experimental conditions used. At neutral and basic pH, the most likely mechanism involves the normal peroxidase cycle, comprising Compounds I and II, associated with a highly efficient branched chain reaction. This reaction is initiated by a slow production of a trace of the IAA cation radical, that in turn leads to the formation of indole-3-methylhydroperoxide. Each hydroperoxide molecule formed can initiate a new HRP cycle. The dominant mechanism of IAA oxidation at acidic pH occurs via an oxygenase-like pathway involving Compound III and the ferrous enzyme (FeII–HRP) [97]. Peroxidases may also contribute to the synthesis of ethylene, another important phytohormone, responsible for the promotion of maturation and abscission of fruits and for the regulation of senescence and fading of flowers and abscission of petals and leaves. Modification of cell walls The cell wall is sometimes considered the site of primary action of plant peroxidases, where they play an integral role in the cell wall biosynthesis. Peroxidases are involved in the synthesis of minor cell wall components, such as lignin, which adds rigidity and protects the plant against pathogens, and suberin, a wax that reduces water loss from cells [1, 98]. Lignin is a complex phenolic structure resulting from the free-radical polymerisation of hydroxycinnamyl alcohols (namely p-coumaryl, coniferyl and sinapyl alcohol) within the plant cell wall. Plant peroxidases catalyse the last enzymatic step in the biosynthesis of lignin, the conversion of the monolignols into their free-radical forms by H2O2. The H2O2 required for this reaction is supplied by the oxidation of NADH, catalysed by a cell wall bound peroxidase. Plant peroxidases are not only responsible for the lignification and suberisation of the cell wall, but are also involved in cross-linking processes between phenoxy free radicals, produced during monolignols oxidation, with other cell wall constituents, such as proteins and polysaccharides, rendering cell walls resistant to mechanical and enzymatic disruption. Pathogen defence Resistance of plants to a wide variety of pathogens (including bacteria, fungi, virus and nematodes) is frequently a result of the rapid establishment of a localised response by the plant at the regions of attempted infection. There are multiple biochemical components of response which act together to halt the spread of an invading organism. One of these responses consists in the cell wall

221 strengthening near the infection site, for example by deposition of extracellular molecular barriers such as lignin, which is polymerised by peroxidases as described above [99]. Applications of plant peroxidases Peroxidases have a considerable potential for application in many different areas. Nevertheless, their commercial application is especially well established in analytical diagnostics, especially, in biosensors and immunodetection. Biosensors Horseradish peroxidase is one of the most widely used enzymes in analytical applications. Due to its characteristics, HRP meets all the requirements for a successful use in analytical systems (e.g. specificity in reaction, flexibility in assay, stability, sensitivity in range of analyte detection, as well as availability in pure form at reasonable cost). Besides that, the ability of HRP to catalyse the oxidation of numerous chromogenic substrates enables the use of spectrophotometric detection systems, including fluorescence and luminescence, opening way to a wide range of procedures. Moreover, the nature of the catalysedreaction (reduction/oxidation reactions) also allows the use of electrochemical detection procedures, and thus the development of electronic biosensors. Biosensors are becoming more and more important tools in medicine, quality control, food and environmental monitoring and research [100]. They can be defined as analytical devices that combine a biological component with a suitable transducer, which converts the biological signal into an electrical signal. The biocomponent can comprise tissues, cells, organelles or molecules (e.g. antibody, cellular receptor, protein and enzyme). According to the measuring principle of the transducer, biosensores have been divided into electrochemical, optical, calorimetric/thermometric, acoustic, evanescent wave, surface plasma resonance and piezoelectric biosensors. The electrochemical biosensors are the most widely used type of biosensors and are based on the generation of an electrochemical signal during the interaction of the biological component with the analyte. Depending upon the electrochemical property measured they can be further divided into amperometric (current flow at constant potential, in redox processes), potentiometric (potential changes at constant current, using generally ion-selective electrodes) and conductimetric biosensors (conductance changes in the ionic environment) [100,101]. Since HRP is capable of reducing H2O2 and also some organic peroxides, HRP-based biosensors can be used to control and monitor these peroxides, in pharmaceutical, environmental and dairy industries [102], in bleaching operations in the textile and paper industries [103], in air and water ozonisation processes and in food products [104]. The principle of detection is rather simple (Fig. 14). If an HRP-modified electrode is placed in a solution containing a

222

Fig. 14. Mechanism of direct electroenzymatic reduction of a peroxide molecule at HRP-modified electrode.

peroxide (ROOH) and set at a sufficiently negative potential, then a proportionality between the reduction current and the peroxide concentration is observed (the peroxide oxidises the enzyme and the electrode reduces it back to its native form). Mediators, small redox molecules (e.g. ferrocene-Fc), can be used to enhance the intensity of the current generated [100]. The on-line monitoring and control of bioprocesses led to the development of real-time measuring systems, in which biosensors are integrated with flow injection analysis (FIA) systems [105]. FIA is an analytical set-up that allows a rapid and continuous on-line monitoring ex situ, automation and sample manipulation (dilution, mixing with reagents, filtration). This reduces the problems related to dependence of biosensors on a great number of parameters that can change in situ during the process [105, 106]. Besides H2O2 and some organic peroxides, HRP sensors can also be used to monitor and control the concentration of the reducing substrates, specially phenolic derivatives (Table 4). The detection principle used in these sensors is generally spectrophotomeric and involves the use of enzyme reactor columns with the enzyme immobilised in solid supports (such as controlled pore glass, CPG). These sensors are usually used in combination with FIA systems. If HRP is coupled with a hydrogen peroxide producing oxidase (Fig. 15), then the system becomes sensitive to the oxidase substrate, enabling the control and monitoring of a wide range of analyts such as glucose, ethanol, cholesterol, lactate, uric acid, pyruvate, amino acids, and many others (Table 5). Reporter systems HRP is widely used as an enzyme label in medical diagnostics and research applications. Universal covalent conjugates of proteins, antibodies and other molecules with HRP, offer a wide range of amplifying possibilities. They are useful and versatile tools for ultra-sensitive detection in immunoassays, nucleic acid detection, histo- and cytochemical applications.

223 Table 4. Examples of analytes that can be monitored using HRP as sensing probe. System Monoenzymatic H2O2

In marine waters

Detection

Description

Reference

Amperometric Amperometric

HRP/sol-gel/Eastman AQ HRP/osmium/polypyrrole film/glassy carbon HRP þ 4-hydroxyphenylacetic acid HRP þ scopoletin Luminol/liquid core waveguide (LCW)-based instrument; FIA Luminol/HRP/sol-gel/optical fibre

[107] [108]

HRP/polyvinylferrocenium film HRP/Fc-phenylenediamine film/ glassy carbon electrode Retinoic acid/HRP–retinol binding protein–CPG

[103] [113]

[115]

Amperometric

HRP/Si–Ti/DNA/carbon paste electrode Luminol/HRP/sol–gel/optical fibers HRP/SAM/Glod electrode

[117]

Spectrophotomeric

Inhibition of HRP/ABTS

[118]

Fluorescent Fluorescent Chemiluminescent Chemiluminescent

Organic peroxides

Amperometric Amperometric Thermometric

Phenols

Amperometric Chemiluminescent

L-Ascorbic

acid

[109] [110] [111] [112]

[114]

[116]

SAM, self-assembled monolayers.

Fig. 15. Schematic representation of a bi-enzymatic biosensor incorporating HRP and an

oxidase enzyme.

224 Table 5. Examples of bi-, tri- and tetra-enzymatic sensors based on several oxidases and HRP for the detection, monitoring and control of a wide range of substrates. System Bi-enzymatic Amino acid Alcohols Higher alcohols Cholesterol Choline Ethanol

Galactose Glucose In rabbits blood In human serum

L-Glutamate

In nerve cells culture Lactate In silage material In animal cell culture Lysine Free fatty acids Hipoxanthine

Detection

Description

Reference

Amperometric Amperometric Spectrophotomeric Amperometric Amperometric Chemiluminescent Amperometric Fluorescent Semi-conductor capacitance Amperometric Spectrophotomeric Amperometric Chemiluminescent Fluorescent Semi-conductor capacitance Amperometric Amperometric Amperometric Spectrophotometric Amperomeric Amperomeric Spectrophotometric Amperometric

Amino acid oxidase/HRP/graphite–Teflon electrode Alcohol oxidase/HRP/osmium hydrogel/carbon paste electrode Alcohol oxidase/HRP/ABTS Cholesterol oxidase/HRP/polymeric film/graphite electrode Cholesterol oxidase/HRP/Fe(CN)6/graphite–Teflon electrode; reversed micelles Luminol/choline oxidase/HRP/sepharose PVA–SbQ photopolymer Alcohol oxidase/HRP/carbon paste electrode HRP-alcohol oxidase co-immobilised on-chitosan beads Alcohol oxidase/HRP/fluoride-sensitive EIS chip

[119] [120] [121] [122] [123] [124] [120] [125] [126]

Galactose oxidase/HRP/Fc/electrode Glucose oxidase/HRP/Sol–gel/variamine blue Glucose oxidase/polyion membrane/HRP–Fc/glassy carbon electrode Luminol/glucose oxidase/HRP Glucose oxidase/HRP/tetra-substituted amino aluminum phthalocyanine Glucose oxidase/HRP/fluoride-sensitive EIS chip

[127] [128] [129] [130] [131] [126]

Glutamate oxidase/HRP/osmium–redox polymer/graphite electrode Glutamate oxidase/HRP/osmium–gel/carbon electrode Lactate oxidase/HRP/Fc/graphite–Teflon electrode Lactate oxidase/HRP/4-chlorophenol þ 4-aminoantipyrine Lactate oxidase/HRP/UV-polymerisable screen printable paste L-lysine-a-oxidase/rHRP/gold electrode Acyl-CoA oxidase/HRP/p-clorophenol þ 4-aminoantipyrine Xanthine oxidase/HRP/Fc/graphite–Teflon composite electrode

[132] [133] [134] [135] [136] [137] [138] [139]

Oxalate Pyruvate

Amperometric Amperometric

Putrescine Sulfite

Chemiluminescent Semi-conductor capacitance Amperometric Amperometric Chemiluminescent Semi-conductor capacitance

Uric acid

Xanthine Tri-enzymatic Cholesterol

Amperometric

Lactose

Amperometric Amperometric

Pectin Phosphate

Spectrophotometric Semi-conductor capacitance

Tetra-enzymatic Maltose Sucrose Citric Acid

Amperometric

Oxalate oxidase/HRP/silica gel–TiO2–toluidine blue/carbon paste electrode Pyruvate oxidase/HRP/threalose (or lactitol)-cationic poly-L-amino/carbon paste electrode Diamine oxidase/HRP/luminol Sulfite oxidase/HRP/fluoride-sensitive EIS chip

[140] [141] [142] [126]

Urease oxidase/HRP/Fc/glassy carbon Uricase/HRP/carbon paste electrode Luminol/Uricase/HRP Xanthine oxidase/HRP/fluoride-sensitive EIS chip

[143] [144] [145] [126]

Cholesterolesterase/cholesterol oxidase/HRP/Fc–CH3OH/carbon paste electrode
-galactosidase/glucose oxidase/HRP/glassy carbon electrode
-galactosidase/glucose oxidase/HRP/Nafion-N-methyl phenazine methosulfate modified electrode Pectinesterase/alcohol oxidase/HRP/ABTS Nucleoside–phosphorylase/xanthine oxidase/HRP/fluoride-sensitive EIS chips

[146]

[149] [126]

Amyloglucosidase/mutarotase/glucose oxidase/HRP/fluoride-sensitive EIS chip Invertase/mutarotase/glucose oxidase/HRP/fluoride-sensitive EIS chip Citrate lyase/oxaloacetate decarboxylase/pyruvate oxidase/HRP/Pt electrode

[126] [126] [150]

[147] [148]

EIS, electrolyte isolator semiconductor.

225

226 Immunoassays The term immunoassay describes a wide range of assays used to detect and quantify antigens and antibodies. A typical immunoassay involves: the immobilisation of an antigen/antibody onto a solid support (e.g. polystyrene plate, bead, membrane); a primary antibody specifically raised against the antigen; a secondary antibody, which is labelled with an enzyme; and the addition of a substrate, which gives a detectable (e.g. coloured) end-product (Fig. 16). HRP conjugates have been extensively used in immunoassays, such as enzyme-linked immunosorbent assays (ELISA), Western-Blotting and immuno-histochemistry (IHC) techniques. HRP is well suited for the preparation of enzyme-conjugated antibodies [151] and antigens [152] due to its relatively good stability and relatively small molecular size, but especially due to its ability to yield chromogenic products with high turnover numbers [153]. Moreover, the availability of substrates for colorimetric, fluorimetric and chemiluminescent assays provide numerous detection options [154–159]. For the past 20 years, Western blotting has been used as a simple and effective procedure for detecting proteins. Antigens are first separated according to molecular weight by gel electrophoresis, then blotted onto a membrane (e.g., nitrocellulose and nylon-based). Afterwards, the procedure resembles an ELISA test. The binding of specific antibodies to the immobilised proteins (antigens) can be readily visualised by indirect HRP labelling immunoassay techniques, usually using a chromogenic substrate which produces an insoluble product [160]. Histo- and cytochemistry The application of HRP in histo- and cytochemistry is well established and documented [161], either alone as protein tracer [162] or conjugated with antibodies for immunoperoxidase labelling [163,164].

Fig. 16. Schematic representation of a typical immunoassay.

227 Immuno-cyto(-histo)chemistry is an essential element and major tool both in diagnostic and research. HRP is the most frequently used label for a large number of molecules used in immuno-cyto(-histo)logic techniques. In the presence of H2O2, HRP catalyses the oxidation of a large number of substrates (e.g. phenols, naphthols, diamines, aminophenols, indophenols), forming detectable products visible by light and electron microscopy, permitting precise cell/tissue localisation. The ultrastructural examination of cells, organelles and tissues can be performed using detectable molecules (tracers). These tracer molecules should present long term retention in the target structure and should be biologically inert and non-toxic, especially when used within live cells and tissues. HRP meets all these demands and has been used to investigate numerous processes, including the physiological role of several substances [165], the flow in capillaries [166], the connectivity in neuronal cell [167,168], the translocation of dyes through gap junctions [169], cell division [170] and drug delivery through liposomes [171]. Furthermore, HRP can be used to track the movements of labelled cells in culture, tissues or intact organisms [172]. HRP–antibody conjugates are often used in the diagnosis of tumours, especially to distinguish among tumours that appear similar on standard histologic stains. These conjugates can also be used in the classification of lymphomas and leukaemias, detection of micrometastases in tissue, identification and qualitative estimation of hormone receptors, as prognostic markers in tumour evaluation and in the diagnosis of viral and other infectious agents (e.g. CMV, herpes, legionella). They also enable the phenotype of cells to be established with direct visualisation of morphology [173]. DNA detection The detection of specific nucleic acid sequences using complementary DNA probes is of utmost importance in diagnostics and research. In order to detect the hybridisation between the target DNA sequence and the DNA probe, a label needs to be incorporated in the probe. A decade ago radioactivity was the most common label, but in recent years non-radioactive DNA probes in association with enzymes, such as HRP have become more and more used. The most straightforward approach is to directly cross-link HRP to the DNA probe. Alternatively, the DNA probe can be linked to a molecule, such as biotin or digoxigenin, which is then detected with complementary molecules (streptavidine and anti-digoxigenin, respectively) conjugated to HRP [174]. Chromogenic or chemiluminescent substrates are then used to generate a coloured product or light emission, signalling the hybridisation event. In situ hybridisation (ISH) techniques are used to localise specific nucleic acid sequences within tissues or cytological preparations, in chromosomes, or in whole mounts. ISH has proved to be an invaluable molecular tool in research and diagnostics. However, its applicability can be limited by its restricted detection sensitivity. In recent years, therefore, several strategies have been

228 developed to amplify either the nucleic acid targets (target amplification), or the immuno-cytochemical detection signals (signal amplification) in situ. In general, target amplification techniques combine the polymerase chain reaction (PCR) and ISH to visualise specific amplified DNA and RNA sequences in cell and tissue preparations, originating very often complex results. As a consequence, other amplification approaches have been developed in order to intensify the signal. One of the most promising techniques is tyramide signal amplification (TSA) [175]. TSA is an enzyme-mediated detection method that specifically uses the catalytic activity of HRP to generate high-density labelling of a target protein or nucleic acid sequence in situ. Microarrays Immobilisation of biomolecules on solid matrices has been of great interest, in particularly in micro- and nano-matrices because of their technological promise [176]. Microarray platforms will change immunochemical and nucleic acid-based analysis, by using immobilised probes and labelled targets, rather than fixed targets and labelled probes. Microarrays can be divided in two types of biosensors, depending on the nature of the recognition event. Bioaffinity devices rely on the selective affinity between a ligand and a receptor (e.g. antigen– antibody and complementary oligonucleotides) and biocatalytic devices, using an immobilised enzyme to recognise a target substrate [177]. HRP appears suitable for such nano-electronic devices because it catalyses a large number of electron-transfer reactions with natural and synthetic substrates. It can be used either directly immobilised on the microarray [178], or as a labelling agent for nucleic acids, antibodies and other proteins [179,180]. These HRP-based or associated microarrays (or biochips) may be used in numerous applications such as expression analysis, recombination and gene mapping, mutation analysis, etc [181,182]. Bioremediation and wastewater treatment The ability of HRP to catalyse the free-radical formation of a variety of aromatic pollutants followed by spontaneous polymerisation can be potentially used in bioremediation and wastewater treatment. Phenol, substituted-phenols (chlorophenols, methylphenols, naphthol) and azo dyes constitute examples of such hazardous compounds which can be found in a variety of wastewaters from different industrial origins (textile, petrochemical, paper, chemical) or in sediments and soils contaminated by accidental spills or uncontrolled discharges. A considerable number of publications describe the use of HRP together with H2O2 to remove phenolic compounds from synthetic model effluents [183–186], and also from real industrial effluents [187–190]. Table 6 shows some representative studies. Although real wastewaters could be thought of as more deleterious to HRP stability, Wagner and Nicell have reported that in the case of

229 Table 6. Representative studies of wastewater detoxification with HRP. Wastewater

Major pollutants

Coal derived Foundry

Phenols Phenol

Petroleum refinery Kraft pulping

Phenol Phenol, catechols, etc.

Model Model

Chlorophenols Pentachlorophenol

Model

Azo dyes (Remazol, Cibacron) Phenol and chlorophenols

Model

Observations

PEG stabilises HRP, 97–99% removal PEG, chitosan stabilise HRP lignin derivatives in the effluent stabilise HRP HRP immobilised on magnetite PEG and chitosan ineffective as stabilisers, residual toxicity higher

Reference [190] [189] [188] [187] [186] [185]

[183] Lower toxicity with chitosan Higher toxicity with PEG

[184]

a Kraft pulping effluent, the lignin derivatives present in the wastewater matrix can protect the enzyme from inactivation by reaction products [187]. The majority of the degradation products formed are insoluble polymers, which are less harmful and can be removed by coagulation and precipitation followed by filtration or sedimentation. The addition of natural coagulants such as chitosan or mineral coagulants such as aluminium sulphate can aid in the precipitation of polymerisation products. In many instances, chitosan is also proved to be an efficient stabiliser of HRP [185,188]. In spite of the promises of the HRP-based wastewater treatment technology, significant problems prevent its widespread use and large-scale application. The inactivation of HRP by reaction products such as free radicals or by H2O2 increases enzyme costs and thus constitutes one of the major barriers. Additionally, in certain circumstances, trace amounts of soluble, low molecular weight products can be produced which are more toxic than the original compound [184]. This toxicity however, declines with the treatment time in most cases, apparently indicating that toxic species further react to non-toxic compounds. It has also been observed that the presence of chitosan reduces the final toxicity [184]. Organic synthesis The advantages of enzymes as catalysts of chemical transformations are widely recognised. Not only do they make it possible to carry out reactions under mild, environmentally friendly conditions (e.g. aqueous media, at room temperature, under normal pressure) by cleaner catalytic transformations, using H2O2 or oxygen as the oxidant, but they also often show remarkable

230 chemo-, regio- and stereospecificity. Peroxidases, and in particular HRP, are able to catalyse numerous selective oxidations of reducing substrates and to resolve chiral hydroperoxides by enantioselectively reducing them to alcohols [191,192]. The broad scope of peroxidases as catalysts of potentially useful transformations in organic synthesis is due to their ability to catalyse different types of reactions. The reactions catalysed by peroxidases can be divided into four main categories [193,194]: 1. Oxidative dehydrogenations .

2RH þ H2 O2 ! 2 R þ 2H2 O 2. Oxygen transfer reactions R þ H2 O2 ! RO þ H2 O 3. Oxidative halogenations RH þ H2 O2 þ HX ! RX þ 2 H2 O 4. H2O2 dismutation 2H2 O2 ! 2H2 O þ O2 Unlike other heme peroxidases, HRP is only able to catalyse oxidative dehydrogenations that consist in the classical peroxidase reaction cycle (involving Compounds I and II), and some oxygen transfer reactions. Oxidative dehydrogenations Polymer synthesis One of the major applications of HRP in preparative organic synthesis is as a mild polymerisation catalyst [195]. The polymerisation of phenols and anilines (aromatic amines) has been extensively studied. In fact a number of homo-polymers and co-polymers have been synthesised from substituted phenolic and aromatic compounds using HRP and H2O2 as the oxidising agent [196]. Recently a water-soluble polyaniline has been synthesised by HRP-catalysed, polyelectrolyte-assisted polymerisation. The presence of sulfonated polystyrene promotes a more linear, para-directed polymerisation, resulting in a conducting emeraldine salt form of polyaniline [197]. The polymerisation of derivatives of tyrosine has also been achieved with HRP. Recently, HRP was used to catalyse the oligomerisation of ferulic acid to

231 a tyrosine-containing tripeptide originating a wide range of new cross-linked products [198]. HRP has also been used to catalyse the free-radical polymerisation of vinyl monomers, such as acrylamide, acrylic acid and methacrylates, such as methyl, phenylethyl, 2-hydroxyethyl methacrylate [199, 200]. N- and O-dealkylations Other particularly interesting HRP-catalysed reactions, via the classical oxidative pathway, are N- and O-dealkylations of aromatic compounds, a transformation that in preparative organic chemistry usually requires rather drastic reaction conditions. The O-demethylation of a methoxyellipticine is shown in Fig. 17. Other targeted aromatic substrates include alkylamines such as para-substituted N,N-dimethylanilines.

Oxygen transfer reactions From a synthetic point of view, selective oxygen transfer reactions are the most interesting oxidative transformations catalysed by peroxidases. In fact, the enantioselective introduction of an oxygen atom into an organic substrate is an area of great industrial and scientific interest since there are few reliable chemical processes capable of performing this specific task. Moreover, peroxidases are able to carry these reactions under mild controlled conditions using low-cost and environment compatible oxidants and solvents. These oxygenase-type reactions can be divided into three groups: (1) heteroatom oxidation, including S-oxidations and N-oxidations; (2) epoxidation and (3) CH bond oxidation, e.g. benzyllic/allylic alcohol and indole oxidations. Sulfoxidations Colonna and co-workers were the first to report that HRP was capable of catalysing asymmetric sulfoxidations of several aryl–alkyl sulfides, under the appropriated conditions (Fig. 18). They observed that this asymmetric synthesis was only observed with phenyl methyl sulfides and para-substituted derivatives. Nevertheless, by site-directed mutagenesis, the enantioselectivity of HRP oxidation of sulphides can be considerably increased. Indeed, the replacement of Phe41 by a smaller amino acid such as leucine (F41L) improves the access of the substrate to the oxy-ferryl group, enabling

Fig. 17. O-Demethylation of 9-methoxyellipticine catalysed by HRP.

232

Fig. 18. Asymmetric oxidation of the phenyl methyl sulphide (thioanisole). Enantiomeric excess (ee) of the S enantiomer is 77% (native HRP) and 97% (F41L HRP) [201].

Table 7. Peroxygenase activity of HRP and HRP mutants for the thioanisole oxidation and respective enantiomer excess. Enzyme

Peroxygenase activity (nmol s
1 mmol
1)

e.e. (% S enantiomer)

Reference

Native HRP

58 6 8 6 56 167 317 15 131 135 111 15 11 2600 16

77 83 52 72 77 97 97 97 65 10 10 92 93 45 36

[201] [50] [53] [202] [193] [193] [201] [202] [50] [201] [193] [50] [50] [53] [53]

Phe41 ! Leu

Phe41 ! Ala Phe41 ! Tyr His42 ! Ala His42 ! Val His42 ! Glu His42 ! Gln

the transfer of the ferryl oxygen directly to the substrate [201]. Many other mutations have been performed to increase both the oxygenase activity and enantioselectivity of HRP (Table 7). The discrepancy observed in the values obtained by different authors is probably due to the experimental conditions used, namely temperature, pH and substrate concentrations. N-oxidations Like S-oxidations, N-oxidations occur via direct transfer of the ferryl oxygen from Compound I to the nitrogen atom of the substrate. Few examples of HRP-catalysed N-oxidations have been described in the literature. Kalliney and Zaks have reported that HRP catalyses the oxidation of the nitroso- and hydroxylamino- derivatives of the antibiotic everninomicin (EVN) into the active nitro-EVN (Fig. 19). The everninomicins are a class of orthosomycin oligosaccharide antibiotics produced by fermentation of Micromonospora carbonaceae as a mixture of products that vary only in the oxidation state of the nitrogen atom (NHOH-EVN, NO-EVD and NO2-EVD) [203].

233

Fig. 19. N-oxidation of hydroxylamino- and nitroso- to nitro-EVN catalysed by HRP.

Fig. 20. N-oxidation of arylamidoximes. (X ¼ p-CH3; p-Cl; p-NO2; m-NO2; p-CH3O).

Another N-oxidation catalysed by HRP is the oxidation of arylamidoximes by H2O2, under mild conditions (Fig. 20). The non-enzymatic oxidation of arylamidoximes generally affords a mixture of compounds, including the corresponding amide and nitrile, as well as dimeric products, while their oxidation in the presence of HRP yields the corresponding O-(arylimidoyl)arylamidoximes. Epoxidation The search for practical methods for the enantioselective epoxidation of organic compounds (such as olefins) is of great importance not only from the synthetic point of view but also in biological systems. An important application of chiral epoxides is the production of the so-called
-blockers like the HIV protease inhibitor Crixivan. In general, native HRP does not perform epoxidations. Nevertheless, Savenkova and co-workers [52] observed styrene oxidation by native HRP and obtained three end products, styrene oxide (42%), phenylacetaldehyde (36%) and benzaldehyde (22%) (Fig. 21). On the other hand, Ozaki and Ortiz de Montellano [201] just detected traces of products of styrene oxidation, but observed that HRP was able to epoxidise trans-
-methylstyrene. Although native HRP catalyses epoxidation reactions at extremely low rate, various mutants (F41L, F41T, H42E, H42A, H42V and F41H/H42A) can form optically active styrene oxide and styrene oxide derivatives (cis- and trans
-methyl) with high reaction rates [50, 52, 53, 201]. However, the synthetic importance of these reactions is limited by the co-formation of aldehydes and ketones as by-products. HRP also catalyses an indirect oxidation of alkenes to epoxides: the glutathione or 4-methylphenol dependent co-oxidation of styrene to styrene oxide. CH-bond oxidation The selective hydroxylation of hydrocarbons by chemical methods is a demanding task in preparative chemistry. HRP has been found capable of

234

Fig. 21. Epoxidation of styrene catalysed by HRP.

Fig. 22. Production of L-DOPA from the HRP-catalysed hydroxylation of L-tyrosine.

catalysing the hydroxylation of some aromatic compounds by molecular oxygen in the presence of dihydroxyfumaric acid as a hydrogen donor. In 1981, Klibanov and co-workers optimised the hydroxylation reaction conditions and were able to increase the yield of these reactions up to 70%. L-DOPA (L-3,4-dihydroxyphenylalanine) is a widely used drug for the treatment of Parkinson’s disease, that also possesses an anti-tumour activity. It has been produced from L-tyrosine using HRP-catalysed hydroxylation (Fig. 22). Other two important drugs that have been produced using this enzymatic hydroxylation are D-(
)-3,4-dihydroxyphenylglycine, which has potential applications on the synthesis of semi-synthetic antibiotics like cephalosporins, from D-(
)-p-hydroxyphenylglycine, and adrenaline (L-epinephrine) from L-(
)-phenylephrine. Biomedical applications Cancer gene therapy Gene therapy for cancer treatment represents a promising approach that has shown potential to selectively eradicate tumour cells, while avoiding damage to healthy tissues [204]. Approaches based on the delivery of genes encoding non-toxic enzymes to confer sensitivity to specific prodrugs (gene-directed enzyme/prodrug therapy, GDEPT) have been extensively and successfully used in experimental systems as well as in clinical trials [205]. GDEPT is a two-step targeting strategy designed to improve the selectivity of anti-tumour agents. The first step consists in the introduction of a vector containing a therapeutic gene, which encodes a foreign enzyme, in tumour cells. Then, a specific prodrug is administered, and consequently converted into a cytotoxic drug by the enzyme expressed in the target tumour. Greco and colleagues have proposed a new GDEPT system based on the association of HRP with the plant hormone indole-3-acetic acid. In vitro studies

235 have demonstrated that, in HRP-transfected human cells, cytotoxic prodrug activation was prompt and efficient [206]. IAA is well tolerated by humans and its non-specific activation in normal tissue is unlikely to take place. No toxicity has been detected in cells incubated alone with either IAA or HRP. The oxidation of IAA by HRP leads to the formation of a radical-cation, which upon decarboxylation originates cytotoxins. These cytotoxins can then form conjugates with thiols and most probably with DNA and other biological nucleophiles [207]. This novel enzyme–prodrug system has been found to be effective against hypoxic and anoxic tumour regions in addition to normoxic tumour cells. This enzyme–prodrug combination exhibits a significant bystander effect independent of cell contact and induces a substantial enhancement of radiation-mediated toxicity. The HRP/IAA system has also the potential to be used in other anti-cancer strategies. Besides GDEPT, specific HRP targeting to the tumour could be achieved with HRP-conjugated antibodies (ADEPT – antibody-directed enzyme/ prodrug therapy) [207, 208] or polymers (PDEPT – polymer-directed enzyme/ prodrug therapy) [207, 209].

Diagnostic test kits Many biotechnology companies have engaged in the development and distribution of medical diagnostics and vaccines for world health care and diseases/ disorders management. The current trend is to encourage self-testing for a variety of ailments and diseases. Many of these companies offer a wide range of diagnostic test kits that provide instant and accurate results, requiring only one or two drops of blood from a fingertip. HRP is one of the many biological components used in these test kits. Two of the most widely used test kits are the glucose and cholesterol blood sensors.

Glucose sensor Diabetes mellitus is a metabolic disorder, whose earliest manifestation is the loss of control of the blood glucose level. Diabetes conditions can be controlled, if glucose levels are regulated to be within the normal physiological range. This need to maintain normal physiological levels of glucose led to the development of a series of sensing devices capable of measuring glucose levels in physiological fluids both in vivo and in vitro. Most of these glucose sensors are based on electrochemical measurements, using enzymes as recognition tools. Glucose sensors are generally based on the enzyme glucose oxidase, that catalyses the oxidation of glucose to gluconolactone with the concomitant production of H2O2. In many glucose sensors, glucose oxidase is coupled to HRP that further reduces H2O2 to water and allows the determination of glucose.

236 Cholesterol sensor The determination of serum cholesterol concentration is one of the most widely performed assays in biochemistry. Elevated serum cholesterol is supposed to be a risk factor in the development of heart diseases (e.g. arteriosclerosis and myocardial infarction). Cholesterol in blood is predominantly esterified with fatty acids and associated with lipoproteins. Total cholesterol, i.e. the sum of free and esterified cholesterol, can be accurately measured enzymatically using cholesterol oxidase and cholesterol esterase coupled to peroxidase. Cholesterol esterase, which has a broad specificity towards the various fatty acid residues, is used to cleave cholesterol esters to free cholesterol. Subsequently, cholesterol oxidase transforms the steroid alcohol into cholest-4-ene-3-one and H2O2, which can be determined with HRP. Cholesterol travels through the bloodstream by special carriers of fats and proteins called lipoproteins. The two major lipoproteins are high-density lipoprotein (HDL) and low-density lipoprotein (LDL) and some of the available test kits can even differentiate these two types of cholesterol.

Future trends Peroxidases such as HRP are involved in many fundamental physiological aspects of animal and plant life. The usefullness of HRP as a biotechnology tool has helped stear intensive research in all aspects of its structure and action. Nevertheless, many questions still remain unanswered (e.g. the exact physiological role of HRP, the mechanism of IAA oxidation, the exact role of the active site residues in catalysis, the role of glycans, etc), which will continue to drive future research. HRP has a high commercial value, due to its versatile and wide applicability, from organic synthesis to biomedicine. The future will certainly bring a consolidation of the more mature applications (e.g. immunodetection, diagnostic kits) and the growth of the incipient ones (e.g. wastewater treatment). Organic synthesis will certainly profit from the generation of HRP variants with improved selectivity and stability properties, by site-directed mutagenesis and directed molecular evolution techniques. One of the newest, most promising and exciting applications involve the in vivo synergistic action of HRP (expressed by a DNA vaccine) with prodrugs in the treatment of cancer.

Acknowledgements The authors would like to acknowledge the precious help of Dr. Gabriel Monteiro and Dr. Gonc¸alo Cabrita. A.M. Azevedo and V. Vojinovic´ also acknowledge Fundac¸a˜o para a Cieˆncia e Tecnologia for financial support (BD/ 18216/98 and SFRH/BD/5495/01).

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Considerations for the planning and conduct of reproducibility studies of in vitro diagnostic tests for infectious agents Toniann Derion* Roche Molecular Systems, Inc., 4300 Hacienda Drive, Pleasanton, CA 94588, USA Abstract. The US Food and Drug Administration (FDA) requires reproducibility studies for premarket approval of in vitro diagnostic (IVD) tests. Results of reproducibility studies provide an estimate of the variability of the IVD test among study sites, reagent lots, site operators, within a single test run, and over multiple test days. In planning the study, discuss the product registration strategy, including the intended use of the product and desired label claims, and define the study team. Design the sample panel according to the limit of detection or quantitation of the test, dynamic range of the test, FDA guidelines, sample matrix, and genotype. Consider legal and ethical issues for obtaining the panel parent specimen, such as minimizing the privacy risk and keeping promises to donors. During the study, review data promptly to determine invalid runs, discover trends in the data that may require additional operator training, ensure correct completion of case report forms, and resolve queries quickly. At the end of the study, gather the study team to review and improve processes. Use the outcome to set expectations of other functional areas and to provide product feedback. Keywords: in vitro diagnostic, IVD, reproducibility study, premarket approval, precision, unlinked specimens, panel parent, variability, monitoring, data management, clinical research – PMA, regulatory agency (ies) – FDA, informed consent, medical device.

Introduction Premarket approval by the US Food and Drug Administration (FDA) of in vitro diagnostic (IVD) tests that detect infectious agents requires the conduct of studies to assess reproducibility (precision) of the test. The purpose of a reproducibility study is to estimate the variability of the test among study sites, reagent lots, site operators (laboratory personnel performing the test), within a single test run, and over multiple days (run to run). In designing the reproducibility study, one must consider the intended use of the product and the desired product label claims, such as limit of detection or quantitation of the test, dynamic range, and genotype. In a reproducibility study, site operators test a sample panel, i.e., a series of samples with known concentrations of the analyte the test is intended to detect (and perhaps one or more negative samples) with multiple reagent lots. Each set of samples representing the panel has a unique panel identification (ID) number, and each sample within the set has a unique sample ID. The panel is tested in a series of runs, so that when testing is finished, each operator will have completed a predetermined number of valid runs for each kit lot (and specimen matrix [e.g., EDTA plasma], if applicable). *Tel: þ 1 925 730 8044. Fax: þ 1 925 225 0195. E-mail: [email protected] BIOTECHNOLOGY ANNUAL REVIEW VOLUME 9 ISSN 1387-2656 DOI: 10.1016/S1387-2656(03)09004-5

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250 This chapter will focus on considerations for the successful planning and conduct of reproducibility studies of IVD tests for infectious agents, with particular attention paid to ethical and practical considerations for the design and production of the panel, and salient monitoring and data management activities. Planning the reproducibility study Planning should begin with a meeting among representatives from key functional areas, such as regulatory affairs, clinical research, business development or marketing, and research and development to discuss the product registration strategy. The specific purpose of the meeting is to discuss product performance, guidelines for the study design [1–3], timing of reagent availability for use in the study; and to agree on intended product label claims, scope and design of the reproducibility study, and the submission date of the marketing application. Use the information gathered in this meeting to draft a brief, high-level study protocol outline that can be submitted to FDA to obtain consensus on the study design (if warranted) and from which to write the protocol. When all parties agree on the approach, identify the team that will be responsible for managing all aspects of the study. The team should include representatives from clinical research, biostatistics, and data management and have a leader or project manager. Define roles and responsibilities of all team members, develop a timeline for the study, and review relevant standard operating procedures or work processes. One of the more time-consuming prestudy tasks is to design and produce the panel to be tested in the study. Many issues should be considered for the design and production of the panel, including legal and ethical considerations for obtaining the parent specimen (i.e., the donor or source specimen from which the panel members will be made). Panel parent specimen: ethical considerations Before the panel is constructed, one must identify the source of the parent specimen and the legal and ethical issues surrounding how this material is obtained. A key Institutional Review Board (IRB) concern is minimization of the risk to privacy, which is best accomplished by the absence of any link between the specimen donor and the sponsor (and, therefore, the research site). This means that source identifiers are either not recorded at the time the specimen is drawn (i.e., the source is anonymous), or severed so completely (unlinked) after specimen collection that test results could not possibly be traced to the donor (Table 1) [4]. According to Erica Heath, MBA (President, Independent Review Consulting, Inc., personal communication), important ethical issues for researchers include keeping any promises made to patients and being respectful in the use of the

251 Table 1. National Bioethics Advisory Commission [3] categories of human biologic materials. Specimen category

Alternate term

Definition

Unidentified Unlinked

Anonymous Anonymized

Coded

Linked/identifiable

Specimens to which identifiers were never attached Lack of identifiers or codes that can link a sample to an identified specimen or person Specimens supplied by repositories from identified specimens with a code rather than personally identifying information Samples supplied by repositories from identified specimens with a personal identifier (e.g., name or patient number)

Identified

specimen (e.g., some ethnic groups prohibit the use of body parts). If informed consent was not obtained for use of the specimen, and the specimen was unlinked from the identity of the source, the specimen can (generally speaking) be used for any ethical purpose. This is because the source (donor) is protected from ‘‘intrusion,’’ is not a ‘‘human subject,’’ and no specific ‘‘promise’’ was made to the source regarding or restricting use of the specimen (promises are often stated in consent forms). If, however, the donor gave consent (whether or not the consent form was IRB-approved and whether or not the specimen was unlinked), the specimen can be used only for purposes congruent with, or at least not inconsistent with, the purpose(s) stated in the consent form. Use of the specimen for any other purpose is disrespectful to the donor – if not unethical – as it breaks the promise made, even if the donor’s privacy remains intact through the unlinking process. If the specimen is unlinked the sample may be used without proof of informed consent, because receiving proof of consent would effectively nullify the donor’s privacy protection. For prospective collection of the panel parent specimen(s), use a consent form with an inclusive purpose section. If the specimen might be used for a secondary or tertiary purpose in the future, the consent form should reflect this potential wider application, as the sponsor’s future ability to use the specimen will depend upon the scope of the original consent. In general, this consent form should be IRB-reviewed [5]. Panel design and production To begin the panel design process, prepare a written request suitable for an internal panel production group or an external vendor that includes the study protocol number and title (as appropriate) and that describes in detail the panel design requirements. Such a document should minimally include the design requirements suggested in Table 2. Design the sample panel according to the limit of detection or quantitation of the test, dynamic range (if applicable), relevant guidelines [1–3], sample matrix, and genotype (if applicable).

252 Table 2. Suggested panel design requirements and considerations. Design requirement

Considerations

Purpose Panel matrix

For what will the panel be used and by whom?
e.g., serum, EDTA plasma
Source (e.g., vendor)
Screening for antigen and/or antibody negativity)

Parent specimen(s)




Source (cultured organism or donor specimen) Genotype(s)/subtype(s) if applicable
Required nominal concentration (and methodology)
Matrix
Informed consent


Panel design

Serial or parallel dilutions of parent specimen Numbers and approximate concentrations of panel members
Number of analyte-negative panel members



Number of panels Fill volume

Number of tubes needed at each concentration (including extras)
Volume in each tube
Enough to allow duplicate or triplicate testing per panel member (if applicable)

Contact information





Approval signatures

Sponsor contact (for external vendor) Department contact (for internal panel production group)

Clinical Research Regulatory Affairs
Biostatistics
Project team leader for the product



In determining the total number of panels needed, consider: Number of tests required for qualification of each panel member; the 95% confidence interval about the mean should be sufficiently narrow (e.g.,  10%; consult the project biostatistician)
Number of panels the site operators will need to train and to practice the test, as well as to qualify (i.e., pass a proficiency test before starting study testing)
Number of panels needed for study testing and extra test runs if invalid runs occur during the study
Number of additional panels needed (e.g., 30% more) in the event panel tubes are lost or mishandled during shipping As was done for the panel design requirements, prepare a document that details procedures for panel production. Suggested panel production procedures are shown in Table 3 and a suggested overall process is illustrated in Fig. 1. Test the parent specimen before production of the panel to ensure the parent meets predetermined specifications (set forth in collaboration with the biostatistician) for nominal concentration of the analyte. Test the matrix material to confirm antigen and/or antibody negativity. After the panel parent value has been assigned, it may be necessary to prepare ‘‘seed stocks,’’ or ‘‘secondary parents,’’ to minimize error introduced by large dilutions of parent stock. It may be necessary to prepare the secondary parents at more than one concentration,


253 Table 3. Suggested panel production procedures and considerations Production procedure

Considerations

Obtain panel matrix




Obtain panel parent




Matrix definition, source (vendor), qualification procedure

Source (e.g., cultured virus or donor specimen) Genotype(s)/subtype(s)
Nominal concentration (and methodology)
Qualification procedure


Assign parent value

Methodology employed (e.g., electron microscopy) Number of replicates tested and dilution factor
Estimated concentrations with each method and 95% confidence intervals
Criteria for parent value assignment



Secondary, tertiary, and quaternary parents from serial dilutions of original (primary) parent (as applicable)
Minimizes error introduced by large dilutions of parent stock

Prepare ‘‘secondary’’ parents




QC parent stock




Prepare panel




Confirm accurate dilutions of secondary, tertiary, and quaternary stocks (as applicable)
Biostatistician determines number of replicates to be tested and acceptable coefficient of variation


Label and QC panels

Who will make the panel (internal group or external vendor)? Procedure (e.g., parallel dilutions, fill-volume of tubes)

Biostatistician provides randomization schedule for labels Review filled tubes against specifications and panel batch record
Procedure for testing finished panel



Acceptance criteria




Approval signatures




Mean concentration and width of confidence interval about the mean

Clinical research Regulatory affairs
Biostatistics
Research leader


depending upon the range of concentrations the panel will need to cover. Results from tests of the secondary parents should satisfy predetermined specifications (e.g., mean analyte concentration). When the parent specimen meets the acceptance criteria, and the value (analyte concentration) has been assigned, production of the panel may begin. Before filling the panel tubes, label the tubes according to packaging specifications. These specifications should describe for each panel member the tube type and size, fill volume of the tubes, tube label type (e.g., one that withstands cryogenic conditions) and the total number of tubes needed at each analyte concentration. The packaging specifications should also include an example of the tube label, including panel lot number, panel ID number, and sample ID number. The biostatistician should randomize the sample IDs before the labels are printed. Specifications for the storage of the panel should include package type and label and required storage temperature.

254

Fig. 1. Panel design and production process.

Individuals from clinical research, biostatistics (and other functional areas as appropriate) should provide written approval of all panel design and production documents. After approval, these documents should become part of the sponsor’s study file.

255 When panel production is complete, test the panel to determine if it meets the acceptance criteria described in the production procedure. These criteria might include, for example, evaluation of extreme values. The panel is ready for use in the study when this process has been completed successfully. Protocol, case report forms, and edit checks A number of monitoring and data management activities can be underway while the panel is under production. From the protocol outline developed previously, write a study protocol that describes at minimum the test procedures, evaluation of test results, procedure for data handling, and statistical methods. If the panels will be provided to the sites in blinded and randomized fashion, do not disclose the analyte concentrations of the panel members in the protocol; however, consider specifying that the operators process the samples in consecutive order as indicated by the last digit of the sample ID. This process will facilitate later determination of sample aliquots that may have been accidentally switched during sample processing. Design the case report forms (CRFs) to capture identifiers such as site, operator initials, run number (each run should have a unique number), consecutive test day, and reagent lot number. In general, the CRFs should capture processing order of sample aliquots and workflow information (e.g., important time points in the sample preparation process). In addition, errors that occur during the run, such as instrument errors or failures or human errors, should be captured, which will allow the biostatistician and clinical representative to later determine whether or not a run should be excluded from analysis by using pre-established criteria for run exclusions. Generally, CRFs should not be developed until the protocol is approved. When draft CRFs become available, the site operators should perform a practice run with them to ensure that CRF layout and chronology are logical from the operators’ perspective. The data management and monitoring groups should work closely together to develop edit checks that the data manager will use to generate data queries. If reproducibility studies will be conducted on other assays in the future, and the sponsor anticipates that CRFs will be very similar across studies, the team may want to build a library of edit checks for use in all such studies. Operator qualification criteria The purpose of operator qualification is to ensure that each operator can perform the assay according to study requirements. Operator qualification ensures uniformity of process and increases the likelihood of high data quality. To that end, the study team must develop a set of criteria by which to evaluate the operators for proficiency. These criteria should include, for example, the numbers of allowable false positive and false negative results, the number of

256 allowable invalid sample results, and the allowable difference in a result from the expected concentration. In addition, qualification of the operators should include the study monitor’s evaluation of the process used to perform the assay. Process criteria may include adherence to the study protocol and standard laboratory practices, proper operation and maintenance of instruments, proper workflow, correct CRF completion, and proper use of personal protective equipment. Appropriate study team members should approve the qualification criteria document, which should be filed in the sponsor’s study file. Operator qualification should occur just before study testing begins (see ‘‘Site initiation visit’’). After approval of the study protocol, CRFs, and operator qualification criteria, provide these documents to the data management group. The database programmer will then create both the operator qualification database and the study database.

Study start-up Reagent kits and instruments Adhere to FDA requirements, if applicable, for unique critical raw materials in each reagent lot to be evaluated in the study. Before shipment of kits to the study sites, affix on each kit a color-coded sticker that indicates the study lot number as this will enable the operators to distinguish one study kit lot from another at a glance, which may prevent inadvertent use of the wrong kit lot. At the study site, perform installation/operational qualification (IOQ), according to the manufacturer’s IOQ protocol, on any instruments to be used in the study. Provide instructions for instrument maintenance to the operators before study start, including guidance on routine maintenance, maintenance intervals, and provide a log for recording all maintenance and service performed during the study. This log should be kept in the site study file.

Investigators’ meeting Conduct an investigators’ meeting when the study database is almost ready to be released into production, e.g., about 2 weeks before study testing will begin. Attendees should include the principal investigators (PIs), site operators, and the sponsor’s study team. Present and discuss the protocol, CRFs, query resolution process, and operator qualification process, and reserve time for questions and answers. An investigators’ meeting ensures that all parties understand the test procedures and know whom to contact during the study if questions or problems arise. Train the operators to perform the assay; discuss any results generated. At the conclusion of training, test the operators’ understanding of the material covered.

257 Site initiation visit After the investigators’ meeting, the monitor should perform a site initiation visit (SIV) at each site participating in the study. At the SIV, the monitor should review the protocol and CRFs, and the operators should undergo qualification. During qualification, the operators perform the assay in their laboratory environment while the study monitor observes. The study team then evaluates the results according to the approved criteria. When the operator has qualified to begin study testing, the sponsor should provide written notification of this to the operator; this notification should be filed in the site study file. The monitor may re-evaluate the operator’s proficiency at an interim monitoring visit if warranted. At the SIV, the monitor also should confirm proper receipt, storage, and accountability of the study panels and reagent kits, and verify that sufficient consumables (e.g., pipette tips) are available to the operators for the study. The monitor should also review the study binder for all required regulatory documents and confirm that any equipment to be used is functioning properly. After the visit, a follow-up letter should be sent to the PI that details any action items needing resolution. Conducting the reproducibility study Data review After the site operators have successfully qualified, all required regulatory documents have been obtained, all necessary study supplies have been provided to the sites, and the database has been released into production, study testing may begin. During the testing phase, the operators should send the completed CRFs (if paper CRFs are used) and test results (i.e., instrument printouts) from each run to the study monitor daily. This process allows the monitor to review data promptly, which is important (especially at the beginning of the study) for several reasons: (1) to discover patterns or trends in the data that may require the operator to undergo additional training; (2) to assess whether an operator needs to perform an additional run to replace one that is invalid; and (3) to ensure that operators are completing the CRFs correctly. It is much more desirable to discover errors or problems within the first few days of study testing than it is to discover them several weeks into testing when intervention will have less of an effect on overall data quality. In addition, prompt, ongoing data review allows timely query resolution. Other considerations If a run needs to be repeated, the same operator should perform the run with the same study kit lot, preferably before he or she completes the required number of

258 runs on that lot. This process will help keep runs performed with the same kit lot clustered together in time, which may be desirable statistically and may also reduce the potential for site operators to use the wrong lot. During the study, hold regular study team meetings to discuss study status, adherence to the timeline, problems or issues that arise, and to review data if warranted. Detailed minutes should be taken to capture topics discussed, decisions made, and issues needing resolution. When testing is complete, all data have been entered and verified, all queries have been resolved, and the database has been locked, the monitor should perform a close-out visit at each study site. At this visit, the monitor should verify the accuracy and completeness of the study files, including the study data, panel and reagent accountability, and communication records. The monitor should also review with the PI any matters requiring follow up and any contractual agreements regarding publication of study data. The study is considered finished when the final report for the study has been approved. At that time, assemble the team (including study site personnel) to review the trial process. Use information and insight gathered at this meeting to improve the trial process, help set expectations of personnel in other functional areas, and provide feedback on the product to other parts of the organization. Acknowledgments The author gratefully acknowledges the assistance and support of the Clinical Affairs Department at Roche Molecular Systems, Inc., Pleasanton, California. References 1.

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U.S. Department of Health and Human Services, Food and Drug Administration, Center for Devices and Radiological Health. Premarket approval applications for in vitro diagnostic devices pertaining to Hepatitis C viruses (HCV): assays intended for diagnosis, prognosis, or monitoring of HCV infection, Hepatitis C, or other HCV-associated disease; draft guidance for industry and FDA. April 27, 2001. U.S. Department of Health and Human Services, Food and Drug Administration, Center for Biologics Evaluation and Research (CBER). Guidance for industry in the manufacture and clinical evaluation of in vitro tests to detect nucleic acid sequences of human immunodeficiency viruses types 1 and 2. December 1999. NCCLS. Evaluation of precision performance of clinical chemistry devices; approved guidance, EP5-A. February 1999;19(2):1–43. National Bioethics Advisory Commission (NABC). Research involving human biological materials: ethical issues and policy guidance. Rockville MD: NABC, 1999. Gibbs J. Regulations and standards: human tissue acquisition: new regulatory and ethical issues. IVD Technol 2000;6(2):22–25.

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Clinical trial methods to discover and validate predictive markers for treatment response in cancer Soonmyung Paik* Division of Pathology, National Surgical Adjuvant Breast and Bowel Project, Four Allegheny Center, Pittsburgh, PA 15212, USA Abstract. Although postoperative chemotherapy in the treatment of cancer appears to have reached the limit of cytoreduction, this may be due to chemotherapeutic agents that are administered nonselectively rather than attainment of the true limit of cytoreduction. Molecular profiles of tumor cells may determine tumor response to chemotherapy, and therefore the selective use of chemotherapy based on prediction will ultimately provide a cure for breast cancer. In this paper, design strategies for clinical trials aimed at disclosing predictive markers are discussed. Keywords: biotechnology – medical biotechnology, clinical research – phase 3, predictive markers, genomic markers, preoperative chemotherapy trial, clinical trial, cancer clinical trial, microarray.

The limit of cytoreduction and the need for predictive markers Although it is logical to expect that the addition of a more potent or noncross-resistant chemotherapy to an existing cancer treatment regimen will provide clinical benefit in the treatment of solid tumors, the results of recent clinical studies challenge this concept and support the idea of a ‘‘limit of cytoreduction’’ [1]. Trials that have compared high-dose chemotherapy with stem-cell support versus standard-dose chemotherapy have produced largely negative results [2], and two large studies conducted by the Cancer and Leukemia Group B (CALGB)/Intergroup [3] and the National Surgical Adjuvant Breast and Bowel Project (NSABP) showed very small or no overall benefit from the addition of paclitaxel to four cycles of adriamycin and cyclophosphamide (AC). While these results seem to indicate that there is no reason to pursue more chemotherapy-based strategies in the future, it is possible that the limit of cytoreduction shown in these studies occurred because only subsets of patients benefit from specific chemotherapy regimens, resulting in a dampened overall effect. At least three lines of clinical evidence support this argument. First, in the NSABP’s B-18 study, complete pathological response of the index tumor to four cycles of AC preoperative chemotherapy was seen in only 9% of the cases, which was shown to be a strong predictor of clinical outcome [4], demonstrating that only a very small subset of breast cancers seems to respond to standard chemotherapy. Preliminary analyses of response data from the NSABP’s B-27 trial [5] indicate that an additional 10% of the patients responded to taxotere (unpublished result). Secondly, in both the CALGB and NSABP studies of *Tel: þ 412 359 5013. Fax: þ 412 359 6878. E-mail: [email protected] BIOTECHNOLOGY ANNUAL REVIEW VOLUME 9 ISSN 1387-2656 DOI: 10.1016/S1387-2656(03)09005-7

ß 2003 ELSEVIER SCIENCE BV ALL RIGHTS RESERVED

260 paclitaxel, there was a trend toward clinical benefit in the subset of patients who did not receive tamoxifen (unpublished results). Finally, several studies suggest that benefit from a doxorubicin-based regimen is largely restricted to those patients who are diagnosed with tumors overexpressing the HER-2 protein [6,7]. These results underscore the importance of identifying the predictors of response to specific adjuvant systemic therapy (referred to as ‘‘predictive markers’’ in this chapter). The power of predictive factors is three-fold: they assist in selecting patients most likely to benefit from treatment; they spare patients from the toxicity of therapy known a priori to be ineffective; and they provide mechanistic insights that can further our basic understanding of tumor biology.

Problems of the current approach taken in clinical trials to disclosing predictors of response Although there have been many attempts to find ideal prognosticators and predictors of response to therapy during the past two decades, the only accepted marker in clinical use currently is estrogen receptor status. This limitation is the result of the inherent limitations imposed by study design issues in the current clinical trials system. In studies that address marker-by-treatment interaction, approximately four times as many events are required to have the same statistical power as when treatment effect is addressed [8]. For example, in a hypothetical situation in which 1300 patients are randomized equally to two adjuvant treatment arms with the expected total number of events being 425, the power to detect a hazard ratio of 1.5 between the two treatments would be 99%. With a hypothetical marker prevalence of 23%, and provided that all tumor blocks are available for assay, the power to detect a hazard ratio of 1.5 between markernegative versus marker-positive cohorts (this marker being a prognostic factor) would be reduced to 73%; however, to detect marker-by-treatment interaction, the power to detect a hazard ratio of 1.5 would decrease to only 47%. Therefore, studies using the specimens procured through phase 3 adjuvant treatment trials usually are seriously underpowered to address marker-by-treatment interactions. Another reason for the lack of success in identifying predictive markers in conventional clinical trials is that molecular targets for chemotherapeutic agents are not clearly defined. Experiments using model systems comprising mutant strains of yeast have shown that chemotherapeutic agents once thought to have defined molecular targets actually are rather promiscuous about their target specificity [9]. This specificity is most likely because of the redundancy in DNA repair and the cell-cycle control mechanism in higher organisms renders assaying for a single or a handful of predefined molecular targets unable to provide clear prediction of response to specific drugs. An unbiased look at the entire genome and its expression pattern may be necessary to identify true predictors of response. Unfortunately, within the current multicenter adjuvant trial process, it is difficult to set up tissue banking in such a way as to collect the ideal materials

261 required for genomic scale assays, in part because a patient’s decision to enroll in a given clinical trial is usually made after diagnostic procedures are performed. A new paradigm: the preoperative chemotherapy trial as a platform for the discovery-oriented approach Is it possible to devise a systematic trial process that contains a built-in tool for the identification and confirmation of predictive markers? Preoperative chemotherapy trials may be ideal for this purpose. Results from NSABP Protocol B-18 suggest that pathologic response of the index tumor is an independent surrogate marker for eventual clinical outcome in patients receiving preoperative chemotherapy with four cycles of AC [4]. In trial B-18, 1532 women were randomly assigned to preoperative or postoperative AC therapy. Clinical tumor response to preoperative therapy was graded as complete (cCR), partial (cPR), or no response (cNR). Tumors with a cCR were further categorized as either pathologic complete response (pCR) or invasive cells (pINV). Outcome was better in women whose tumors showed a pCR than in women with a pINV, cPR, or cNR (relapse-free survival [RFS] rates, 85.7, 76.9, 68.1, and 63.9%, respectively; P<0.0001), even when baseline prognostic variables were controlled. This result demonstrates that response of the index tumor can be used as a surrogate endpoint for clinical outcome in preoperative chemotherapy trials. Two major advantages exist for preoperative trials compared with postoperative adjuvant trials. First, marker-by-treatment interaction study is more feasible in preoperative trials, since it does not lack statistical power. Second, and more importantly, in preoperative studies, the procurement of fresh tissue is possible. Therefore, discovery-oriented approaches such as cDNA microarray or array-based comparative genomic hybridization (CGH) can be used. During the past few years, methods that permit an unbiased look at the entire human genome and its transcriptome have been developed as side products of the Human Genome Project. Array-based CGH hybridization is a technique that uses an array of bacterial artificial chromosome (BAC) clones of the fragmented human genome so that the entire human genome is represented; this allows the evaluation of gene copy number changes such as deletion or amplification at high resolution. Since there are about 3 billion base pairs in the human genome, the use of 3000 BACs provides a survey of gene copy number changes with 1 megabase resolution [10]. Array-based CGH hybridization is a very powerful discovery tool since once amplified or deleted chromosome areas are identified, BAC clones can be used immediately for fluorescence in situ hybridization (FISH) for clinical application. cDNA microarray is a technique similar to arraybased CGH and allows examination of the expression level of more than 10,000 genes in a single assay [11]. Some laboratories in the private sector already claim to have arrays that cover the entire human transcriptome or all expressed genes (for an example, see http://www.eosbiotech.com). Combination of these newer

262 technologies with preoperative therapy trials will provide an ideal platform through which to discover predictive markers of response to systemic therapy. Feasibility of discovery-oriented preoperative trials Several important issues need to be addressed before array technology can be used effectively in preoperative therapy trials. The application of microarray technology, in particular to clinical specimens, has been limited by the fact that significant amounts of template RNA (50–200 mg total RNA and 1–5 mg mRNA) are required. The typical yield of total RNA from core needle biopsy or fine-needle aspiration specimens is only 1–3 mg. Thus, some method of amplifying the amount of starting RNA without bias or amplifying the hybridization signal is necessary to render the cDNA microarray useful in the clinical research setting. Linear amplification of RNA has been used to generate sufficient RNA to construct cDNA libraries and to generate probes in microarray analyses. The basis of this approach is the incorporation of T7 promoter sequences in the oligo-dT primer during reverse transcriptase and the subsequent addition of T7 polymerase to generate complementary RNA in a linear fashion. Despite the common use of linear amplification of RNA, a comprehensive evaluation of amplification bias that may distort the expression profile has not been done. To address this problem, Wang et al. systematically assessed the fidelity of RNA amplification against total RNA on a 2008-gene cDNA microarray [12]. They found that this method permitted a 105-fold amplification of antisense RNA (aRNA) from limiting amounts of starting total RNA. Using RNA amplification, the ability to detect differentially expressed genes (‘‘outliers’’) degenerated at 125 ng of input total RNA. At lower levels of input RNA (10–31 ng total RNA), false-positive outliers emerged due to the dynamics of the optical system. The lost information could be recovered and the false information corrected by a second round of RNA amplification. Using these combined approaches, aRNA, derived from as low as 10 ng of input RNA, maintained linearity and reproducibility equal to 100 mg total RNA or 2 mg poly(A)-RNA. Moreover, the performance of the aRNA was excellent: aRNA arrays preserved expression profiles and identify outlier genes in the same manner as did standard total RNA. This amplification method is now available in kit form from two companies (Arcturus Engineering [www.arctrur.com] and Ambion [www.ambion.com]). Another problem that has prevented implementation of microarray techniques in multicenter trial settings is the logistics of frozen tissue collection. This problem has been resolved through the introduction of RNAlater (Ambion). It has been shown that RNA is stable without degradation for a week, even at room temperature when the tissue is stored in RNAlater [13,14]. When laboratories use core-biopsy specimens rather than an entire index tumor, the question of representation should be addressed because of possible tumor heterogeneity. According to Perou et al. [15], gene expression patterns

263 obtained from multiple samples from the same patients (index tumor versus metastasis, or prechemotherapy versus postchemotherapy) always cluster together when compared with those obtained from different patients. Therefore, despite concerns about tumor cell heterogeneity, regional variation of an overall gene expression pattern may not be as great as suspected. Since there are approximately 30,000–50,000 genes in human cells, the data that will be generated from the trials will be extremely complex and will require an unusual statistical analysis of the tumor response – gene expression correlation. One example of the kind of analytical tool that will be needed has been developed by a group at the Massachusetts Institute of Technology based on a ‘‘neighborhood algorithm’’ that was used to autoclassify subtypes of leukemia [16]. In that study, the investigators used a microarray of more than 8000 genes to examine the mRNA expression pattern in tumor cells obtained from patients diagnosed with acute myelocytic leukemia and acute lymphoblastic leukemia. In the end, the use of a set of 10–100 genes identified from the original 8000-plus genes permitted the investigators to accurately classify the two types of leukemia. In another study [17], Alizadeh et al. using a cDNA microarray of 17,856 genes enriched for genes expressed in germinal center B cells and hierarchical cluster analysis, identified two molecularly distinct forms of diffuse large B-cell lymphoma (DLBCL) that had gene expression patterns indicative of different stages of B-cell differentiation [17]. One type expressed genes characteristic of germinal center B cells (‘‘germinal center B-like DLBCL’’); the second type expressed genes normally induced during in vitro activation of peripheral blood B cells (‘‘activated B-like DLBCL’’). Patients with germinal center B-like DLBCL had significantly better overall survival than did those with activated B-like DLBCL. More recently, a Stanford University group examined a small cohort of patients with breast cancer receiving clinical follow-up and found three distinct subgroup of breast cancer based purely on gene expression pattern – estrogen receptor positive, HER2 positive, and basal epithelial-like [15]. Further analyses of the same cohort demonstrated that these subgroups do have different clinical outcome [18]. Without a doubt, these studies demonstrate that genomic science is rapidly evolving, and more sophisticated analysis tools will be developed in the next few years. Conceptual protocol Since 1997, the NSABP has been exploring ways to incorporate discovery aims into the preoperative chemotherapy protocols it conducts. NSABP believes the ideal design would be to multiplex both drugs and markers in a two-phase trial [19]. With this sort of protocol, in the first discovery phase of the trial, multiple drugs are randomly assigned in a preoperative therapy setting, and pathologic response rate is obtained for each drug. Then gene expression or marker panel data are correlated with response to each drug to identify potential predictors of response. The most important aspect of this concept is that identified predictors

264 are immediately validated in the second validation phase of the trial, in which drugs are assigned based on predictive markers, with the expectation that response rate will be substantially increased; however, with the relative paucity of available drugs and in the absence of any preliminary data demonstrating the feasibility of identifying predictive markers using microarray in the preoperative setting, it is difficult to implement this concept. Incorporation of discovery–validation into current trials While it is easy to build in a discovery phase as part of a treatment comparison trial such as NSABP B-27 (in which preoperaptive AC was compared with preoperative AC-T), it will be impossible to build a validation phase into a conventional trial design. Since only a few drug regimens are used in the discovery phase in our studies, most patients will not belong to a ‘‘predictable’’ group (i.e., have a gene expression pattern predictive of sensitivity to a drug) during the validation phase. For example, if the discovery phase occurs in B-27, then in the subsequent validation phase in which treatments are assigned based on gene expression pattern, 80% of the cases would belong to the group predicted to be resistant to ACT. At that point, those 80% can be assigned to new drugs (or randomized to a newer drug or to ACT), making the validation phase for the initial drugs serve also as the discovery phase for the newer drugs. Since this process can go on until 100% of patients with breast cancer enrolled in NSABP trials are members of the ‘‘predictable’’ group, theoretically breast cancer can be cured, eventually. One potential pitfall is that chemotherapy response might not be specific to each drug but rather more dependent on duration of therapy. For example, the reason that ACT is better than AC in NSABP’s B-27 study could be due to the fact that ACT is longer in duration than AC (an argument that European colleagues used to reject approval of adjuvant paclitaxel in Europe). Obviously, the validation phase of the trial must be done to be able to answer such a question. Even if duration of therapy is the primary issue, the data generated from such study would still be extremely valuable since genes selectively expressed in resistant tumors then can become the ideal target for new drug development. The NSABP is currently pursuing a concept for the B-27 replacement trial, in which patients will be randomized to ACT or ACT plus capecitabine. A discovery–validation concept was incorporated into the design of this trial. The initiation of a validation phase will depend on the success of our identifying potential predictors using core biopsy specimens collected from the B-27 ‘‘replacement trial’’ (i.e., the trial that follows once B-27 is completed). At the same time, the NSABP has initiated preoperative therapy protocols for patients with locally advanced breast cancer, with a typical sample size of fewer than 100 cases. Therefore, by the time the B-27 replacement trial is completed, NSABP will have candidate predictive markers for multiple drugs that can be fed directly into the validation phase of that protocol.

265 The NSABP is encouraged by the fact that the pharmaceutical industry is beginning to show very strong interest in this trial approach and desires access to gene expression profiles of those patients who belong to the ‘‘non-predictable’’ group, since their genes might be an ideal target for the development of novel therapeutics. One concern in incorporating the discovery concept into the NSABP B-27 replacement trial was sample size – as it is large enough. The B-27 replacement trial will have a sample size of 2700 in a two-arm design. Because the trial will examine the entire genome of approximately 30,000–50,000 expressed genes, many false-positive observations are to be expected; therefore, it will be important to have a validation set within the discovery phase. If the first 300 cases in each arm are used as a discovery set and the remainder as a validation set, this approach will yield a better confirmation of the potential predictive markers than any statistical adjustment for multiple comparisons. Conservatively assuming that all predictive markers behave independently, the number of cases can be calculated that will be required to detect significant (at the  ¼ 0.001 level) associations between markers and rates of response to therapy for various scenarios. If marker prevalence is 20% (that is, 20% of the tumors are positive for a marker), only 80 patients will be needed to achieve more than 99.9% power to detect the difference in response rates of 90% in the marker-positive cohort, versus 10% in the marker-negative cohort. Even with a 50% versus 20% response rate difference, 300 patients would be needed in both arms to achieve more than 87% power. Consequently, the proposal is to use 300 patients in each arm, and because the NSABP is looking for markers with the discriminating power of almost 100% (i.e., accuracy of classification of near 100%), there will be enough power not to miss such markers even in the discovery phase of the study. Thus, the complete sample size of roughly 1100 per arm (assuming that 90% of patient specimens are evaluable) in this study should allow NSABP to perform confirmatory analyses to a great degree of precision. Conclusion Individualizing systemic therapy based on predictive markers has been the Holy Grail for medical oncologists during the past decade. Even with the advent of techniques that allow profiling of gene expression or copy number changes, only carefully designed clinical trials will eventually lead to the discovery of a set of predictive markers to achieve that goal. 1. Acknowledgments The author thanks Barbara C. Good, PhD, for editorial assistance with this manuscript.

266 References 1. Norton L. Evolving concepts in the systemic drug therapy of breast cancer. Semin Oncol 1997;24:S10-3–S10-10. 2. Hortobagyi GN. High-dose chemotherapy for primary breast cancer: facts and anecdotes. J Clin Oncol 1999;17:25–29. 3. Henderson IC, Berry DA, Demetri GD, Cirrincione CT, Goldstein LJ, Martino S, Ingle JN, Cooper MR, Hayes DF, Tkaczuk KH, Fleming G, Holland JF, Duggan DB, Carpenter JT, Frei E 3rd, Schilsky RL, Wood WC, Muss HB, Norton L. Improved outcomes from adding sequential Paclitaxel but not from escalating Doxorubicin dose in an adjuvant chemotherapy regimen for patients with node-positive primary breast cancer. J Clin Oncol. 2003 Mar 15;21(6):976–83. 4. Fisher B, Bryant J, Wolmark N, Mamounas E, Brown A, Fisher ER, Wickerham DL, Begovic M, DeCillis A, Robidoux A, Margolese RG, Cruz AB Jr, Hoehn JL, Lees AW, Dimitrov NV, Bear HD. Effect of preoperative chemotherapy on the outcome of women with operable breast cancer. J Clin Oncol 1998 Aug;16(8):2672–85. 5. Mamounas EP. NSABP protocol B-27. Preoperative doxorubicin plus cyclophosphamide followed by preoperative or postoperative docetaxel. Oncology (Huntingt) 1997;11:37–40. 6. Paik S, Bryant J, Tan-Chiu E, Yothers G, Park C, Wickerham DL, Wolmark N. HER2 and choice of adjuvant chemotherapy for invasive breast cancer: National Surgical Adjuvant Breast and Bowel Project Protocol B-15. J Natl Cancer Inst 2000 Dec 20;92(24):1991–8. 7. Paik S, Bryant J, Park C, Fisher B, Tan-Chiu E, Hyams D, Fisher ER, Lippman ME, Wickerham DL, Wolmark N. erbB-2 and response to doxorubicin in patients with axillary lymph node-positive, hormone receptor-negative breast cancer. J Natl Cancer Inst 1998 Sep 16;90(18):1361–1370. 8. Peterson B and Geroge SL. Sample size requirements and length of study for testing interaction in a 2
k factorial design when time-to-failure is the outcome [corrected]. Control Clin Trials 1993;14:511–522. 9. Simon JA, Szankasi P and Nguyen DK. Differential toxicities of anticancer agents among DNA repair and checkpoint mutants of Saccharomyces cerevisiae. Cancer Res 2000;60:328–333. 10. Pollack JR, Perou CM, Alizadeh AA, Eisen MB, Pergamenschikov A, Williams CF, Jeffrey SS, Botstein D, Brown PO. Genome-wide analysis of DNA copy-number changes using cDNA microarrays. Nat Genet 1999 Sept;23(1):41–46. 11. DeRisi J, Penland L, Brown PO, Bittner ML, Meltzer PS, Ray M, Chen Y, Su YA, Trent JM. Use of a cDNA microarray to analyse gene expression patterns in human cancer [see comments]. Nat Genet 1996 Dec;14(4):457–460. 12. Wang E, Miller LD, Ohnmacht GA, Liu ET, Marincola FM. High-fidelity mRNA amplification for gene profiling. Nat Biotechnol 2000 April;18(4):457–459. 13. Grotzer MA, Patti R, Geoerger B, Eggert A, Chou TT, Phillips PC. Biological stability of RNA isolated from RNATlater-treated brain tumor and neuroblastoma xenografts. Med Pediatr Oncol 2000 Jun;34(6):438–442. 14. Florell SR, Coffin CM, Holden JA, Zimmerman JW, Gerwells, Summers BK, Jones DA, Leachman SA. Preservation of RNA for functional genomic studies: a multidisciplinary tumor bank protocol. Mod Pathol 2001 Feb;14(2):116–128. 15. Perou CM, Sorlie T, Eisen MB, van de Rijn M, Jeffrey SS, Rees CA, Pollack JR, Ross DT, Johnsen H, Akslen LA, Fluge O, Pergamenschikov A, Williams C, Zhu SX, Lonning PE, Borresen-Dale AL, Brown PO, Botstein D. Molecular portraits of human breast tumours. Nature 2000 Aug 17;406(6797):747–752. 16. Golub TR, Slonim DK, Tamayo P, Huard C, Gaasenbeek M, Mesirov JP, Coller H, Loh ML, Downing J, Caligiuri MA, Bloomfield CD, Lander ES. Molecular classification of cancer: class

267 discovery and class prediction by gene expression monitoring. Science 1999 Oct 15;286(5439):531–537. 17. Alizadeh AA, Eisen MB, Davis RE, Ma C, Lossos IS, Rosenwald A, Boldrick JC, Sabet H, Tran T, Yu X, Powell JI, Yang L, Marti GE, Moore T, Hudson J Jr, Lu L, Lewis DB, Tibshirani R, Sherlock G, Chan WC, Greiner TC, Weisenburger DD, Armitage JO`, Warnke R, Levy R, Wilson W, Grever MR, Burd JC, Botstein D, Brown PO, Staudt LM. Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling. Nature 2000 Feb 3;403(6769):503–511. 18. Sorlie T, Perou CM, Tibshirani R, Aas T, Geisler S, Johnsen H, Hastie T, Eisen MB, van de Rijn M, Jeffrey SS, Thorsen T, Quist H, Matese JC, Brown PO, Botstein D, Eystein Lonning P, Borresen-Dale AL. Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. Proc Natl Acad Sci USA 2001 Sep 11;98(19): 10869–10874. 19. Paik S. Incorporating genomics into the cancer clinical trial process. Semin Oncol 2001;28:305–309.

269

Production of high-quality marketing applications: strategies for biotechnology companies working with contract research organizations Sandra J. Hecker1, Christopher Preston2, and MaryAnn Foote3,* 1

Hecker and Associates, Arlington, VA, USA F. Hoffmann-La Roche, Basel, Switzerland 3 Amgen Inc., One Amgen Center Drive, M/S 17-2-A, Thousand Oaks, CA 91320-1789, USA 2

Abstract. Many biotechnology and pharmaceutical companies use clinical research organizations (CROs) to assist in the writing and preparation of clinical documents intended for submission to health authorities. Start-up companies often require the expertise of a CRO to prepare their first regulatory documents. Larger or more experienced companies often require CRO staff to assist at times of multiple simultaneous submissions. The timely production of high-quality new drug marketing applications requires close collaborations between the drug company and the CRO. The views of both CRO and industry in ensuring best practices are discussed. Keywords: biotechnology – medical biotechnology, clinical research – phase 1, clinical research – phase 2, clinical research – phase 3, clinical research – IND, clinical research – BLA, clinical research – use of contract research organization (CRO), regulatory agency (ies) – FDA, regulatory agency (ies) – EMEA, regulatory agency (ies) – CPMP.

Introduction In the 1990s, drug development typically required 12 years to bring a drug from its initial discovery in the laboratory to marketing approval and patient availability [1]. For many patients critically in need of innovative therapies, the time required to bring a drug to market is unacceptably long. Drug development time needs to be shortened without sacrificing the quality of the research, and the pharmaceutical and biotechnology industries are evaluating many new technologies to reduce research time. One area that has not received much attention is the preparation of regulatory documents required to obtain marketing approval. In 2000, contract research organizations (CROs) were estimated to be playing an increasingly more prominent role in drug development, including the preparation of regulatory documents [2]. As Vincent-Gattis et al. suggested, drug sponsors (i.e., pharmaceutical and biotechnology companies) need to change from being transactors to becoming partners with CROs for the development of drugs. Start-up companies, because they often are too small and too inexperienced to take their good product candidates through the regulatory process alone, frequently partner with a CRO. *Corresponding author: Tel: þ 1 805 447 4925. Fax: þ 1 805 498 5593; E-mail: [email protected] BIOTECHNOLOGY ANNUAL REVIEW VOLUME 9 ISSN 1387-2656 DOI: 10.1016/S1387-2656(03)09006-9

ß 2003 ELSEVIER SCIENCE BV ALL RIGHTS RESERVED

270 Large companies, because they have many ongoing research projects competing for staff, often partner with a CRO to extend their workforce. This chapter will discuss the successful writing of regulatory documents from both the drug sponsor and the CRO perspectives. Regulatory documents Numerous documents are required for submission to regulatory authorities to gain marketing approval for drugs, biologics, or medical devices (Table 1). It is beyond the scope of the chapter to provide detailed outlines of these documents. Guidance is available in various websites [3–7]. The common denominator of all these documents, however, is clarity in writing and in presentation. Not only is the content critical; the presentation of the data in a ‘‘user-friendly’’ manner also is essential. Drug sponsors must realize that external reviewers (i.e., regulatory authorities) need to be educated on all aspects of their proposed drug candidates. In addition to accurate and complete but concise content, all editorial priorities (e.g., clear copies, lack of typographic errors, easy-to-read format) should be addressed. Thus, it is imperative that all writers of these documents, whether the sponsor’s or the CRO’s, be well trained in logical presentation of data and clarity of writing. Drug company perspective Many, if not all, large pharmaceutical and biotechnology companies have medical writing departments. Although the organization and the reporting structure may differ among companies, some basic tenets appear to apply to all. Most companies, large and small, staff medical writing departments only to ‘‘trough’’ work levels. Such staffing ensures that a critical number of writers who are intimately familiar with the company’s products are available, while avoiding the possible need for downsizing or dismissal that could occur with a larger number of in-house writers during times of fewer staff needs. Companies that staff this way often routinely contract with freelance writers and CROs to write clinical study reports (CSRs), especially those based on terminated drug candidates. If efficacy is no longer an issue because a company no longer intends to seek marketing approval for the product, a writer not familiar with all the nuances required for marketing efforts can write a CSR suitable for fulfilling regulatory needs. Sometimes, however, a company with limited regulatory experience will contract with a CRO for a full New Drug Application (NDA) in the United States, Marketing Authorisation Application (MAA) in Europe, or new global Common Technical Document (CTD). Most companies do not contract out the writing of investigator’s brochures, expert reports, or the integrated summary of safety and integrated summary of efficacy because of the depth of drug expertise and strategy required by these documents, unless the contract writer’s capabilities are known by the company.

Table 1. Documents required for submission to regulatory authorities to gain marketing approval. Document

Regional area applicable

Purpose

US

Allows for testing of drugs/biologics in humans, healthy volunteers or patients, to obtain clinical data for the subsequent marketing application Original submission to gain a license for marketing of a biologic Additional submission(s) to gain marketing approval for other indications, formulations, etc Summarizes the efficacy results Summarizes the safety results Original submission to gain marketing approval for a drug

US only Investigational New Drug Application (IND) Biologic License Application (BLA) Biologic License Supplement (BLS)

US US

Integrated Summary of Efficacy (ISE) Integrated Summary of Safety (ISS) New Drug Application (NDA)

US US US

Europe Only Clinical Trial Exemption (CTX)

EU (UK)

Clinical Trial Application (CTA)

EU

Marketing Authorisation Application (MAA)

EU

Worldwide Common Technical Document Clinical Study Reports Investigator’s Brochure

EU, US, Japan EU, US, Japan, Australia, ROW EU, US, Japan, Australia, ROW

Allows for testing of drugs/biologics in patients to obtain clinical data for the subsequent marketing application. A CTX is usually not needed to test a drug in healthy volunteers Allows for testing of drugs/biologics in healthy volunteers and/or patients to obtain clinical data for the subsequent marketing application. A CTA may or may not be required to be submitted before testing a product in humans, depending on the specific EU country. Original submission to gain marketing approval for a drug.

New global marketing application that takes the place of the regional MAA, NDA, and JNDA Summarizes the clinical and statistical findings of a given protocol Provides the investigational drug ‘‘label’’ (i.e., gives an investigator enough information to decide to participate in trials)

271

EU, European Union; JNDA, Japanese New Drug Application; MAA, Marketing Authorization Application; ROW, rest of world; UK, United Kingdom; US, United States.

272 When a company decides to contract with a CRO, it expects that the CRO is staffed with qualified writers who are dedicated to the task and who are capable of working on a given document. Contracts with CROs can be established in various ways: by specific jobs (i.e., writing of a set number of CSRs), by product (i.e., preparing a filing for a new drug candidate), or by fixed term (e.g., writing for a year). When a drug company engages a CRO to write regulatory documents, the company expects that the documents produced by those writers will be indistinguishable from documents produced by ‘‘in-house’’ writers. To ensure quality and consistency, a drug company usually gives the CRO specific information (Table 2). Typically a drug sponsor will provide technical background and scientific details about the drug, which the writer will transform into the necessary regulatory documents. Not all drug companies supply the same amount and type of information, however. Importantly too, the drug sponsor must provide contact information with a liaison within the company to facilitate project efficiency. The liaison should be someone who can answer technical and logistical questions from the CRO writer, facilitate data transfer, and maintain timelines. Table 2. Materials supplied by drug sponsors vary and may be quite inclusive. Data

Purpose/content

Case report forms

Contain the original data on the patient, drug, device, etc. Very confidential. Often summarized and sent that way Not always feasible because of security issues, but invaluable to CRO if available. Eliminates the need for the CRO to contact sponsor staff to answer simple queries Allows CRO to prepare documents according to sponsor’s requirements and to allow all documents in a submission to look the same Information provided by regulatory agencies to assist sponsors in understanding requirements and the reason for them. Useful if CRO has not worked extensively in the area and may include the sponsor’s interpretation of the guidance Provides the investigational drug ‘‘label’’ (i.e., gives an investigator enough information to decide to participate in trials) Samples of previously submitted documents or ‘‘mock-ups’’ of sample documents Given to allow CRO staff to learn the details of the project or the drug quickly. Often includes sponsor’s reasons for decisions on formulation, dosage, endpoints, etc. Sets the priorities for the CRO. Adherence to timelines often determines payment schedule to CRO by the sponsor Guide to conduct of the study. Tells writer how trial was to be conducted. Amendments to the protocol should also be supplied to the CRO May be a standard guide (i.e., AMA Style Guide) or may be one prepared by the sponsor; ensures consistency in usage, formatting, abbreviations, etc., for all documents in a submission

Direct links to data Electronic templates Guidelines

Investigator’s brochure Model documents Project/drug training Proposed plan timelines Protocols Style guide

AMA, American Medical Association; CRO, contract research organization.

273 The drug sponsor expects that the CRO’s writers will have had experience in writing documents for the United States Food and Drug Administration and European Union agencies, as relevant, and that they have the scientific background to understand and interpret the data about which they are writing. A drug sponsor almost always expects early involvement of the CRO’s writer, especially when that writer will be the principal or only writer of most – if not all – parts of the marketing application. Face-to-face meetings that allow CRO input and thorough discussion are necessary. When a drug sponsor gives a project to a CRO, it is understood that the CRO has the resources to complete the work on time and that it has no conflicting projects. Adherence to company standards and use of the same software versions as the drug sponsor are expected of the CRO writer. Drug sponsors cite a number of areas where problems can occur. Of great concern is the lack of continuity. Many CROs have a high rate of turnover in personnel, requiring the drug sponsor staff to train several waves of new CRO staff. Drug sponsors also are wary of CROs that subcontract parts of the project, fearing for lack of consistency or quality. Most drug sponsors believe that it is not easy to find a good, qualified, and responsible CRO. Once such a CRO – with a successful track record – is found, drug sponsors usually are willing to pay a premium for that company’s services. Sometimes a given writer at a CRO is the cohesive force in a writing project. In these situations, the drug sponsor can move projects as a given writer moves from CRO to CRO. Contract research organization perspective A CRO writing group has the same goals as the drug sponsor: timely production of high-quality document(s) and a successful working relationship. Experienced CRO personnel believe that an effective working relationship between a drug sponsor and its writing group is a necessary framework for success. Three additional elements that dramatically affect success are information, process, and personnel. Information The CRO cannot start the writing process without background information, so it is to the drug sponsor’s advantage to provide current and complete background information quickly (e.g., relevant protocol and all protocol amendments for a CSR, investigator’s brochure, internal research reports, completed animal study reports, and manufacturing information for a clinical trial or marketing application). Most CROs prefer to receive this background information electronically for ease of incorporation of the information into the new document(s). Immediately upon receipt, the CRO is responsible for checking that the documents – both the electronic files and paper copies provided – are complete, legible, and appropriate for their intended use. Occasionally, CRO

274 review of materials provided by a naı¨ ve drug sponsor reveals the need for other project materials that were not provided (e.g., early exploratory clinical study data that may seem irrelevant to a sponsor unaware that FDA wants to see all clinical study data for a product). As more drug sponsors form research partnerships, a CRO may be faced with working with more than one drug sponsor per drug candidate. Information may be lacking, and the CRO must determine whether the drug sponsor developed the product from the beginning (i.e., from the laboratory), has purchased a drug presently in clinical trials, or is developing a marketed drug for new indications. Each case will provide a unique challenge for the CRO. A CRO writer often needs to reconcile information from investigator’s brochures from two drug sponsors because of takeover, alliances, or collaborations. Other materials required by a CRO include drug sponsor corporate style guides, table formatting guidelines, document templates, and current versions of software, which are necessary for the CRO-written components to be indistinguishable from sponsor-written documents. Process Success requires a smooth process to ensure that each party understands and meets the needs and expectations of the other. Miscommunication or mistaken assumptions invariably cause delay or rework, often when time is at a premium to meet drug-sponsor goals. Drug sponsors often have process expectations that they may not express because they are unaware that not all companies operate as they do. For this reason, experienced CRO staff will query the sponsor on process details to clarify sponsor expectations. (Table 3 lists some potential problems.) Agreement and understanding must be reached on details of the document review process. Experienced CROs strongly advocate limiting review to prespecified sponsor staff in each relevant discipline (e.g., writing, clinical, safety, regulatory, preclinical, manufacturing, any other relevant expertise for the specific document), and to a prespecified number of reviews, usually two to prevent time-intensive, wasteful extra review cycles. A precise timeline, highly recommended, ensures that each step of the writing and review process is delineated to the team, each task is clearly assigned to specific staff, and the time for each step in the process is noted. Table 3. Drug sponsors should disclose all potential delays to a filing. Several items have the potential to delay filings. Preclinical toxicology or other studies not available until very close to filing dates Very large populations
Very ill populations
Number of narratives anticipated, their format, and time of delivery



275 To facilitate creation of documents that will not need major revision, some CROs submit to the sponsor for review an ‘‘annotated outline’’ showing intended information in a brief, sometimes bulleted form, and providing a suggested structure of the document and sequence of its points (major reorganization is possible). An annotated outline can be changed considerably, if the sponsor wishes, without much loss of time. An early draft, the next version in the process, accommodates fine-tuning of information in each section, while the final draft, with few changes expected, lays out the proposed final document and its precise wording. The final draft must include all attachments and be a complete document. The final draft may also be the signature draft (i.e., the final version). To maximize efficiency of review, an experienced CRO writer will not submit a document for sponsor review until it is complete. Likewise, during review, sponsor reviewers must review the draft completely and provide all comments to the CRO within the specified timeframe. Deciding at the beginning of the project how to handle late sponsor reviews (agreed to by both sponsor and CRO) is also useful; the CRO cannot always make up time when there is a final immovable deadline and the sponsor is late with reviews, unless the sponsor accepts that project quality may suffer or cost may increase. A good CRO writer will not change sections of a document that the sponsor has already reviewed, unless the sponsor asks for changes. Sponsor reviewers usually handle many projects and deadlines; having to reread an entire large document (e.g., pivotal study report) each time it comes back for review is extremely inefficient. After the first review, sponsors should request that changed information in subsequent review versions be brought to their attention, through track changes or version compare software, thus saving reviewer time. Personnel Experienced CROs know that cultural differences between, and even within, drug sponsors and CROs can greatly affect the timely production of a quality document. Experienced CROs, benefiting from experience working with a broad range of sponsors and projects, must assess what factors will be most relevant for the given project. Considerations include assessment of global teams and being sensitive to specific needs a given team may have. Cultural caveats include assuring that all team members’ questions and needs have been met to their satisfaction. Experienced CRO writers advocate repeating back to a team member for whom English is not the first language what the CRO staff thinks was agreed upon. Lines of authority must be known – which sponsor team members have what authority – to ensure that project decisions are handled appropriately and that the sponsor’s needs are met. Important too, is consideration of the timing of video or conference calls for global teams writing major documents (e.g., marketing applications). Team spirit improves when meeting times vary over the course of the project, so inconvenience is distributed across the globe. Often a creative approach to

276 meetings is needed to ensure that no one group always meets in the middle of the night. It is important to remember national holidays and to not place important deadlines at this time. Complications may arise in sponsor partnership situations in which two or more pharmaceutical or biotech companies have allied themselves to develop a product jointly. During the marketing application process, good writers will make a point to assess differences in corporate philosophy that can affect whether joint team meetings effectively handle critical issues and thus meet corporate goals. ‘‘Standard’’ document or table format and structure can vary widely between companies. For ease of review of the marketing application by the health authorities, jointly worked out corporate standards must be adopted and implemented before the start of writing or much time will be spent later sorting out these issues. Rapport and goodwill between sponsor and CRO staff must be carefully built and maintained from the beginning of the project. This communication and trust facilitates handling of the unexpected and sometimes sensitive project problems that can arise when mountains of data are reviewed and arranged in a regulatory application on the inevitably very short timeline. Seasoned CRO staff will stay calm and effectively problem-solve during times of unexpected project crisis. Effective CRO staff will understand drug sponsor pressures and work tirelessly to make life easier for the drug sponsor. Conclusions Drug development is a highly regulated industry with specific written documentation required for gaining marketing approval. Small companies often do not have dedicated staff to write critical regulatory documents, and established companies may require assistance because of staffing issues or corporate priorities. Often the collaboration between a drug sponsor and a CRO is critical for regulatory filings. A successful collaboration is based on teamwork and the smooth integration of both companies to reach a mutual goal. A knowledgeable CRO with experienced writers can facilitate projects to increase efficiency. In the situation of a new, small drug sponsor, the CRO staff may have more experience: many CRO staff are quite accustomed to accelerated project timelines, inexperienced sponsor staff, and working under difficult circumstances. A good drug sponsor/CRO relationship can be beneficial for both parties: a CRO can coach a naı¨ ve sponsor on regulatory documentation needs, and a busy large drug sponsor can benefit from having a CRO handle projects with minimal distraction for the sponsor. Acknowledgments The authors heartily thank Jim Yuen for his critical editing of the manuscript.

277 References 1.

2.

3. 4. 5. 6. 7.

Workman P. Emerging molecular therapies, small-molecule drugs. In: Principles of Molecular Oncology. Bronchud MH, Foote MA, Peters WP, Robinson MO, (eds), Totowa, NJ: Humana Press, pp. 421–437. 2000. Vincent-Gattis M, Webb C and Foote MA. Clinical research strategies in biotechnology. In: Biotechnology Annual Review. El-Gewely MR, (ed), Vol. 5, pp. 259–267. Elsevier Press, 2000. United States Food and Drug Administration. http://www.fda.gov (accessed 29 January 2002). European Society of Regulatory Affairs. http://www.esra.org (accessed 29 January 2002). Drug Information Association. http://www.diahome.org (accessed 30 January 2002). International Conference on Harmonisation. http://www.pharmweb.net (accessed 30 January 2002). Regulatory Affairs Professional Society. http://www.raps.org (accessed 30 January 2002).

279

Use of benchmarking in the development of biopharmaceutical products Marian Giffin* and Sally McLeish Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA Abstract. As the pharmaceutical and biotechnology industry enters the 21st century, the pressure on companies to maintain the level of productivity required for consistent year-on-year growth is increasing. Benchmarking has become a tool for obtaining the information needed to support continuous improvement and gain a competitive advantage. During the process of benchmarking, best practices can be identified while giving management the ability to improve on existing performance in an objective, well-informed manner. When used appropriately, benchmarking provides a new perspective on traditional methods while enabling companies to monitor their performance. Keywords: Deming Prize, quality control, total quality management, industry trends.

Introduction Benchmarking has its roots in the manufacturing environment, beginning in the 1950s with W. Edwards Deming [1]. Deming gave a series of lectures and seminars in Japan in which he taught the basic principle of statistical quality control to executives, managers, and engineers. The results of his teachings led to the establishment of quality control in companies. The Deming Prize was established in Japan in honor of Deming’s innovative way of thinking and is an award given to Japanese companies or divisions of companies that have achieved distinctive performance improvement through the application of Total Quality Management (TQM). In the early and mid-1980s, many industry and government leaders realized that a renewed emphasis on quality was no longer an option for companies in the United States but a necessity for doing business in an ever expanding, and more demanding, competitive world market. The Malcolm Baldridge National Quality Award (MBNQA) was established by the Department of Commerce, National Institute of Standards and Technology in 1987 to promote total quality management as an increasingly important approach for improving the competitiveness of United States companies. The award was envisioned as a standard of excellence that would help United States companies achieve worldclass quality. The MBNQA has since evolved from a quality focus to an award for performance excellence [2]. No business concept was more important to economic revival in the United States in the 1990s than reengineering. The concept of reengineering was *Corresponding author: Tel: þ 1 805 447 2149. Fax: þ 1 805 498 5593. E-mail: mgiffi[email protected] BIOTECHNOLOGY ANNUAL REVIEW VOLUME 9 ISSN 1387-2656 DOI: 10.1016/S1387-2656(03)09007-0

ß 2003 ELSEVIER SCIENCE BV ALL RIGHTS RESERVED

280 introduced by Hammer and Champy [3]. Hammer and Champy described how the radical redesign of a company’s processes, organization, and culture could achieve greatly improved performance. Hammer and Champy discovered that not only was quality important, but processes, systems, and technology were also of equal importance. Biopharmaceutical interest in benchmarking As the pharmaceutical and biotechnology industry grew, competitive pressure in the research, development, manufacturing operations, and sales areas increased. In the early to mid-1990s, management of biopharmaceutical companies relied largely on internal qualitative assessments to establish company objectives and to monitor performance. It became apparent, however, that companies could not rely exclusively on these assessments to support long-term process improvements. As a result, many companies began to use benchmarking, a comparison of existing company practices compared with external business practices, to support process improvements. External benchmarking organizations were established to provide anonymous pharmaceutical and biopharmaceutical industry data. Some organizations that collect and distribute this information are CMR International (Surrey, United Kingdom) and Tufts Center for the Study of Drug Development (Boston, MA). Because of the significant expenses associated with clinical development, the biopharmaceutical industry was particularly interested in the reduction of clinical development cycle times, reduction of costs, improvement of efficiency, and the reduction of review and approval times. The pharmaceutical and biopharmaceutical industry found that once comparisons were made to companies with like processes, as well as comparisons across the world’s regulatory agencies, improvement opportunities could be identified and achieved. In the late 1990s, the biopharmaceutical industry began to look outside their own industry, to industries with similar practices, to incorporate data into their benchmarking programs. Regulatory departments looked at the publishing industry for best practices relating to the publication of large, complex documents. Biopharmaceutical research and discovery found similarities with exploration and development in the oil and gas industry in terms of probabilities of success. Biopharmaceutical companies who benchmarked their processes against nonpharmaceutical industries were rewarded with new and innovative ideas that contributed to significant process improvements [4]. Benchmarking comparison of biopharmaceutical and pharmaceutical industry Before 1997, the clinical development times for biopharmaceutical products (any therapeutic biologic compound) were significantly faster than the clinical development times for traditional pharmaceutical products or new chemical entities (NCEs).

281

Fig. 1

While clinical development times declined by 22% for NCEs in the early to late 1990s, the clinical times for biopharmaceuticals increased 37% over the same 10-year period (Fig. 1). The increased length of time for biopharmaceutical development, particularly recombinant proteins, could be caused by several factors. The technology currently used to manufacture and characterize biopharmaceutical products is more sophisticated than that used in the 1980s and early 1990s. In addition, biopharmaceuticals are being developed for increasingly complex indications. The additional complexity of the disease interventions and the technology used to make biopharmaceuticals might have contributed to the increase in clinical development cycle times [5]. As evidenced by these trends, current mean clinical development time for biopharmaceuticals are nearly identical to that for NCEs [6]. Benchmarking of regulatory approvals Benchmarking allows trend analyses to be done and future performance to be forecast. Currently, industry trends are emerging that show that pipelines (potential new products) are static, the number of regulatory submissions is decreasing (Fig. 2), and the output of new active substances (NAS) is decreasing. Biopharmaceutical companies are beginning to focus on-line extensions, in lieu of NAS, possibly due to higher success rates of line extensions (Fig. 3). Clinical development strategies are being reengineered to ensure fewer, more successful trials, thus reducing costs [7]. Benchmarking of cycle time and research and development expenditure Two trends that receive a great deal of attention are cycle time and Research and Development (R&D) expenditure. Tufts Center Director, Dr Kenneth I Kaitin, reports ‘‘The biotechnology revolution is picking up speed and slowing down at the same time.’’ The number of products entering clinical development has

282

Fig. 2

Fig. 3

increased, but the process of development and approval is taking longer. Since 1982, clinical development times for biopharmaceutical products have more than doubled, from an average of 33 months to 68 months (Fig. 4). During this same period, the time required for approval by the Food and Drug Administration has decreased from an average of 24 months to an average of 15 months, but recently increased to just under 20 months [8].

283

Fig. 4

Longer clinical development time is related to a number of factors, including the growing number of newer and more sophisticated technologies, the focus on increasingly complex disease indications, the demand for higher standards for characterization of product safety and efficacy, and a growing tendency to develop new products for global markets. The average cost to develop a new prescription drug in 2001 is US$802 million compared with an estimated US$231 million 1987 [9]. The largest challenge facing the biopharmaceutical industry is to contain R&D costs and reduce development times without compromising clinical test design. Joseph DiMasi, director of economic analysis at the Tufts Center, attributes much of the increase in the total cost of new drug development beyond inflation to increasing clinical trial costs. Clinical trial costs are increased by difficulties in recruiting patients into clinical trials and the increased focus on developing drugs to treat chronic and degenerative diseases. Included in the drug cost analysis are expenses of project failures and the impact that long development times have on investment costs. The estimate accounts for out-of-pocket clinical costs, out-of-pocket discovery, and preclinical development costs, clinical success and phase attrition rates, as well as the cost of capital. Discussion Benchmarking allows a biopharmaceutical company to identify opportunities for improvement and to proactively direct efforts to become better than its competitors. Benchmarking is based in the philosophy of continuous improvement and is a change management tool. Benchmarking identifies gaps in performance and opportunities for improvement [10]. Although benchmarking is a valuable endeavor, it may be years before companies reap the benefits of

284 their process improvements since changes made today will not manifest for several years. Currently, no one company has been able to achieve best in class in every area, perhaps because companies have decided to target particular processes or functions where they believe they can have the most impact to their overall development effectiveness. It is preferable for a biopharmaceutical company to be better than its competitors in core processes and functions, as these processes have the highest strategic importance. References 1. The W. Edwards Deming Institute. http://www.deming.org (accessed 5 August 2002). 2. The Malcolm Baldridge National Quality Award. http://www.isixsigma.com (accessed 5 August 2002). 3. Michael Hammer, James Champy. Reengineering the Corporation. New York, NY: HarperBusiness, 1993;240 pp. 4. Cooke-Davies T. Benchmarking Project Management Practices: Creating a culture for projects to succeed. Paper presented at: CMR Project Management Meeting, June 2000, Philadelphia. 5. Reichert JM. New biopharmaceuticals in the USA: trends in development and marketing approvals 1995–1999. Trends in Biotechnology 2000;18:9. 6. Tufts Center for the Study of Drug Development, Drug Impact Report, vol. 2; October, 2000; http://csdd.tufts.edu; accessed 26 September 2002. 7. Ogg MS, van de Haak MA and Halliday RG. Activities of the international pharmaceutical industry 2000: pharmaceutical investment and output. CMR International. http:// www.cmr.org; accessed 26 September 2002. 8. Tufts Center for the Study of Drug Development, News Release November 13, 2001. 9. Tufts Center for the Study of Drug Development, News Release November 30, 2001. 10. McNair CJ and Leibfried KHJ. Benchmarking: a tool for continuous improvement. Essex Junction, VT: Oliver Wight Publications, Inc., 1992;344 pp.

285

The state of biopharmaceutical manufacturing David T. Molowa1 and Rosemary Mazanet2,* 1

Biotechnology Equity Research, J P Morgan Chase, New York, NY, USA Oracle Partners LP, 200 Greenwich Avenue, Greenwich, CT 06830, USA

2

Abstract. The manufacturing of protein-based biopharmaceuticals is done in bacterial or mammalian cell cultures. While bacterial cultures are inexpensive, dependable, and approved by regulatory authorities, many complex proteins cannot be manufactured this way. Complex proteins must be manufactured in mammalian cell cultures to produce active products. Mammalian cell culture capacity is limited and has slowed the delivery of necessary biopharmaceutical products to patients. The nature of the production capacity problem and future outlook are critically examined. Keywords: bacterial cell culture, biotechnology, E. coli, mammalian cell culture, protein manufacturing, transgenics.

Introduction Manufacturing capacity for certain protein-based therapeutics is in short supply. Enbrel (product of American Home Products and Immunex, recently acquired by Amgen Inc.) is the best-known example of the supply problem. Enbrel had the potential to achieve sales of greater than US$ 1 billion in 2002, but sales were below this level because of a lack of manufacturing capacity. The implications of capacity constraints are far reaching for the industry. Capacity constraint has an impact on companies’ balance sheets as the companies are forced to make significant investments in manufacturing capacity, process improvements, and alternative production technologies. Most companies would rather outsource their manufacturing and use their capital for investment in Research and Development (R&D) and acquisitions; however, they have little choice but to take on the financial burden and risk. Delays in the development and commercialization of some products may occur because of the lack of adequate clinical and commercial product supply. In the extreme situation, development of some biopharmaceutical products could be halted because the investment required to produce commercial quantities would make the total return on investment no longer attractive. Companies with excess capacity will have leverage over those companies that do not. To our knowledge, there are few companies that will have excess capacity before 2004. Going forward, third-party manufacturers will have more leverage in negotiations. In addition, they will be more selective in choosing projects and less willing to take on risk. *Corresponding author: Tel: þ 1 203 862 7925. Fax: þ 1 203 862 7927. E-mail: [email protected] BIOTECHNOLOGY ANNUAL REVIEW VOLUME 9 ISSN 1387-2656 DOI: 10.1016/S1387-2656(03)09008-2

ß 2003 ELSEVIER SCIENCE BV ALL RIGHTS RESERVED

286 Considering current manufacturing economics, products that must be chronically administered at amounts >5 mg/kg/week may not be commercially viable using current technology. Based on conversations with individuals involved in biopharmaceutical and contract manufacturing, consultants, and regulatory personnel, we estimate that capacity will triple by year 2006. Unfortunately, demand for capacity will quadruple during the same time period. In this chapter, we report research concerning the state of biopharmaceutical manufacturing. Biopharmaceutical manufacturing Biopharmaceutical manufacturing refers to the production of protein-based therapeutics such as Epogen (Amgen Inc), Enbrel (Amgen/American Home Products [AHP]), and Rituxan (Genentech/IDEC Pharmaceuticals). In contrast to small-molecule therapeutics, such as Lipitor (Pfizer) and Celebrex (Pharmacia/Pfizer), which are produced by chemical synthesis for less than US$5/g, proteins are produced in living cells at a cost of US$100–1000/g. First-generation protein-based therapeutics, such as insulin, growth hormone, and Neupogen (Amgen) are produced by inserting the gene encoding the desired protein into a simple bacterial host known as Escherichia coli, using classic recombinant DNA technology developed in the 1970s and 1980s. Large quantities of E. coli are grown using traditional fermentation methods, and the protein of interest is separated from the bacterial proteins using several purification steps. E. coli production, sometimes referred to as microbial production, is advantageous in that it has an established regulatory track record and is well characterized. E. coli is inexpensive to culture and replicates quickly. In 2000, adequate microbial manufacturing capability existed; however, in 2002, the capacity is lacking. We estimate that capacity utilization for microbial production is currently 90%. Not all therapeutic proteins can be produced in E. coli, however, because the bacteria are unable to modify proteins after they are produced, which can cause misfolded and inactive proteins. For these reasons, monoclonal antibodies, fusion proteins, and other proteins that must be modified cannot be produced in E. coli and must be produced in mammalian-derived cells (e.g., Chinese hamster ovary [CHO] cells). Mammalian-derived cell production of biopharmaceuticals also has a good regulatory track record, and allows for the modification of proteins with potentially reduced immunogeneity. Compared with E. coli production, however, the construction of the genetically engineered cells is more time consuming, production is less efficient and more expensive, and purification is more complicated. By most estimates, capacity utilization rates for mammalian cell culture production is approaching 100%.

287 Protein manufacturing in mammalian cell cultures The manufacturing process begins with the construction of the master cell bank (Fig. 1). The construction of the master cell bank involves the genetic engineering of a host mammalian cell, typically CHO cells, to produce the

Fig. 1. Biopharmaceutical process chain.

288 protein of interest. CHO cells are often used for mammalian cell cultures because they multiply quickly, are relatively hardy, grow well in culture, and are well known by regulators. Some more specialized cells may be required when unusual modifications are needed. For example, Xigris (Eli Lilly) is produced in African green monkey kidney cells because of the need for three modifications. When a cell line is created that produces high levels of the desired protein and can be grown as inexpensively as possible, a master cell bank is created. The master cell bank serves as the source of all cells used in the production of clinical and commercial quantities of the biopharmaceutical protein. The creation and characterization of the master cell bank typically takes between four weeks and four months. Once a master cell line has been created, the next step is bioprocessing, which includes two phases of activity: the growth of the cells and the recovery and purification of the protein product. Currently, two methods are used to grow cells: batch fermentation and continuous perfusion fermentation. Since >90% of current manufacturing processes use batch fermentation, we will focus on this production process. In batch fermentation, cells derived from the master cell bank are progressively grown in larger and larger volumes over a period of 3–4 weeks to provide a seed culture for large fermentation tanks. This gradual step-up in volume allows for the most rapid growth of a large volume of cells. The large stainless steel tanks typically hold 10,000–20,000 liters of culture medium. Once the seed culture is added to the large tank, the cells are grown to optimal density over 10–14 days. The determination of the optimal growth conditions takes many months and ties up valuable production capacity. Variables that must be taken into consideration include components of the culture media in which the cells are grown, cell density, protein stability, and others. Once optimal density has been reached, the cells are collected by filtration and are broken apart to release all the proteins inside the cell. The protein of interest (i.e., the biopharmaceutical protein) is purified through a series of two to four chromatography (separation) steps. Processes to inactivate and remove viral contaminants must be included in the next step of production. The final purified product is formulated and filled into vials. The purification process takes about 30 days while the entire manufacturing process takes approximately 10 weeks per batch. On average, CHO cells produce 0.25 g of product/liter cell culture medium. For monoclonal antibodies, purified bulk yield is usually 50%, translating into 5 kg finished product/10,000 l fermentor. If we assume that each fermentor can be run 20 times/year, the theoretical total purified bulk yield is 100 kg/10,000-l reactor per year. The largest mammalian cell culture manufacturing facility is in Vacaville, California (owned by Genentech) and has a capacity of 100,000 liters. This facility has a theoretical maximal capacity of >1000 kg/year. Due to the need for process development, scale-up, maintenance, and manufacturing of clinical trial material, however, the actual capacity is much lower than the theoretical maximum.

289 The regulatory environment The manufacturing of biopharmaceuticals is highly regulated. The United States Food and Drug Administration (FDA) has specific guidelines for the manufacturing of biopharmaceuticals. Much information is required by the FDA before it can grant approval of a new biopharmaceutical. First, the manufacturer must describe the cell line used and demonstrate that it is free of bacteria, fungi, mycoplasms, adventitious viruses, and retroviruses. Next, each lot of unprocessed bulk material must be tested for viral, bacterial, and mycoplasm contamination. After purification, the bulk material must be shown to be pathogen free, pure, sterile, and contaminant free. The molecular integrity of the product must be demonstrated. For the final filled product, the quantity, potency, purity, and sterility of the product must be documented. The stability of the final product must also be documented. All tests conducted to establish this information must be validated and the reproducibility and variability of each assay must be documented using defined procedures. While the cost of production of proteins is well in excess of that of small molecule pharmaceuticals, that is not to say that traditional pharmaceutical gross margins cannot be achieved with biopharmaceuticals. Due to the severity of the diseases that are often targeted by biopharmaceutical, premium pricing is common. We estimate that the average cost of goods for a monoclonal antibody is US$200–700/g, excluding royalties and the contract manufacturer’s profit. We further estimate that on average, biopharmaceutical companies receive US$9000/g, translating into gross margins of 85–90%. The average price per gram of several protein-based biopharmaceuticals is given in Table 1.

Table 1. Average price per gram for selected protein-based biopharmaceuticals. 2001 demand is a JP Morgan estimate. Product

Company

Actual price in 2000 (US$/g)

Estimated demand in 2001 (kg)

Rituxan Enbrel Remicade Herceptin Synagis Activase ReoPro Zenapax Simulect Mylotarg

Genentech Immunex/Amgen Centocor Genentech MedImmune Genentech Eli Lilly F. Hoffman-LaRoche Novartis Celltech

5000 5214 7227 5811 14,301 27,225 56,430 17,480 79,078 424,532

220 200 110 100 20 8 7 5 1 0.1

290 Why is there a shortage of manufacturing capacity? The question often asked is how did the current manufacturing capacity shortage happen. The answer has a number of components, the first of which is the unfulfilled promise of first-generation monoclonal antibodies and other biopharmaceutical proteins. More than 10 years ago, there was great expectation for the ‘‘magic bullets,’’ but they have not delivered them on their promise. As a result of these failures, the industry has been reluctant to pursue monoclonal antibodies. The successes since 1996, namely Rituxan, Enbrel, Remicade, Synagis, and Herceptin, caught many manufacturers unprepared for success (Table 2). Second, the dose of monoclonal antibodies and fusion protein is 10–100 times greater than that of first-generation therapeutic proteins. We estimate that Genentech will need to produce 255 kg of Rituxan to meet the demand in 2002. This amount compares with the 2 kg of Epogen we estimate that Amgen will need to produce to meet the demand in 2002. Consequently, new products consume more capacity than the earlier approved products. Third, there is a long lead-time in the design, construction, and validation of new manufacturing facilities. On average, it takes approximately four years from the decision to build a new facility to regulatory approval for its use. Table 2. FDA-approved drugs produced in mammalian cells. Year approved

Product

Company

Class/type

1986 1987 1989 1992 1992 1993 1993 1993 1996 1997 1998 1998 1998 1998 1998 1998 1999 2000 2000 2001 2001 2001

Murononab-CD3 Activase Epogen Cerezyme Recombinate Pulmozyme ReoPro Kogenate Avonex Rituxan Zenapax Remicade Simulect Synagis Enbrel Herceptin BeneFix TNKase Mylotarg Campath Aranesp Xigris

Ortho Genentech Amgen Genzyme AHP/Baxter Genentech JNJ/Centocor Bayer Biogen IDEC/Genentech/Roche PDL/Roche Centocor/JNJ Novartis MedImmune Amgen/AHP Genentech AHP Genentech Celltech/AHP Millennium/Berlex Amgen Eli Lilly

MMAb Protein Protein Protein Protein Protein cMAb Protein Protein cMAb hzMAb cMAb cMAb cMAb Fusion protein hzMAb Protein Protein hzMAb hzMAb Protein Protein

AHP, American Home Products; JNJ, Johnson & Johnson; PDL, Protein Design Labs; cMAB, chimeric monoclonal antibody; hzMAb, humanized monoclonal antibody; mMAb, murine monoclonal antibody.

291 Current mammalian cell culture manufacturing capacity Use of commercial biopharmaceutical therapeutics is increasing (Fig. 2). We have updated our current estimate of mammalian cell culture production capacity. We estimate that currently there are 475,000 l of capacity. Of this, the biopharmaceutical industry controls 360,000 l (Table 3), and contract manufacturers control 115,000 l (Table 4). Genentech has the largest capacity with an estimated 200,000 l; Boehringer Ingelheim has the second largest (75,000 l), most of which is used for the production of Enbrel. Other companies with significant capacity include Novartis, Lonza, Diosynth, Baxter, and F. Hoffman-LaRoche. Third-party contract manufacturers With the exception of the more-established biotechnology companies (i.e., Amgen, Biogen, Genentech, Genzyme General), the biotechnology industry has relied on third-party contract manufacturers for the production of clinical and commercial-scale quantities of protein-based biopharmaceuticals. The two primary contract manufacturers have been at full capacity for two years. Typically, contract manufacturers enter into two types of contracts with biotechnology companies. One type of contract is a development and service agreement, which deals with the production of clinical material for phase 1–2

Fig. 2. Approval of mammalian cell culture produced products over time.

292 Table 3. Current estimated mammalian cell culture capacity for industry. Product

Company

Volume (l)

No. of fermentors

D2E7 Epogen/Aranesp Factor VIII Avonex pipeline products

Abbott Amgen Baxter Biogen

2

CTLA4-Ig ReoPro/Remicade ReoPro/Remicade TPA/TNK/Pulmozyme Rituxin/Herceptin Cerezyme/Fabrazyme Pipeline products Rituxin/Zevalin Pipeline products Pipeline products Pipeline products Unknown Synagis Pipeline products Zenapax/Herceptin Diagnostic products Unknown Total

Bristol Myers Squibb Centocor/JNJ (facility 1) Centocor/JNJ (facility 2) Genentech

6000 Roller bottles 2500 2000 2000 2000 2000 10,000 12,500 12,500 2000 2000 3000 5000 2000 350 12,500 2500 750 10,000 1000 2000 360,000

Genzyme Human Genome Sciences IDEC Pharmaceuticals ICOS Immunex Medarex Novartis MedImmune Protein Design Labs F. Hoffman-LaRoche

9 3 3 5 2 8 8 4 2

4 2 2 2 3

JNJ, Johnson & Johnson.

Table 4. Current estimated mammalian cell culture manufacturing capacity for contract manufacturers. Product

Company

Volume (l)

No. of fermentors

Enbrel/Campath Unknown Bexxar/Synagis Xigris/Erbitux/ABX-IL8 5g 1.1

Boehringer Ingelheim Diosyth DSM Lonza

6

Unknown Total

Rentschler

12,500 20,000 2000 2000 5000 1000 115,000

1 3 2 2

clinical trials and process development. The development work is typically on a fixed fee-for-service basis. Productivity-related incentive payments may be included. The other type of contract is a long-term production contract, in which the customer contracts for a given number of runs per year for a set number of years. The customer is committed to pay a certain minimum amount based on

293 a negotiated price per gram of finished product or per run or a combination of both. Payments vary from contract to contract or with time through the life of the contract. These payments typically contain penalties for the termination of a contract and can be quite onerous. Due to the lack of capacity, contract manufacturers are currently charging a reservation fee for access to future capacity. Apparently the guaranteed minimum is great and some companies must reconsider continuing certain programs at this point. They risk agreeing to pay for production capacity for a product that fails in the clinic. Contract manufacturers consider a variety of factors when deciding which projects they will accept. These factors include the customer’s long-term manufacturing strategy and anticipated final scale of operations. Building a new biopharmaceutical manufacturing facility As a result of a lack of capacity at third-party contract manufacturers, many biotechnology companies are building their own manufacturing facilities. Once the decision has been made to build a new manufacturing facility, the company must decide what type of process (batch or continuous perfusion fermentation) will be used and how much capacity it will need. Simultaneously, the company must find and purchase or lease the land on which the new facility will be constructed. Negotiations with state and local authorities include appropriate zoning, financial aid, and potential tax benefits. The company must also secure financing at this point. The next steps are the conceptual and preliminary engineering by a specialized firm, and can require a year to complete. Detailed engineering plans follow and last approximately one year. The construction of the plant can move along at a slight lag to the ongoing engineering process. Actual construction takes approximately two years and is handled by construction managers. Toward the end of the construction process, start-up and validation of the facility begins. Three to five lots of commercial-grade material must be produced and analyzed before filing of an Establishment License Application (ELA) with regulatory authorities. This filing is followed by regulatory review, facilities inspection, a response to questions from the inspecting regulatory agency, and final approval. On average, the entire process from decision to approved facility may take 4–5 years and cost US$250–400 million (Fig. 3). One issue with which many biotechnology companies are beginning to grapple is the hiring of experienced personnel to manage and run these facilities. Not only does a lack exist for managers, but also for staff experienced in the daily operations of a tightly regulated biopharmaceutical manufacturing facility. Estimating demand for manufacturing capacity We have tried to estimate the future demand for production capacity with the caveat that the estimates are based on numerous assumptions (Table 5).

294

Fig. 3. Large-scale mammalian cell culture manufacturing plant timeline.

Table 5. Assumptions for average demand per product. Average dose

3 mg/kg

Average number of doses Average demand/patient/year Average market size Average penetration Peak no. of patients treated Average peak demand

10 2g 100,000 patients 30% 30,000 60 kg/year

We began by estimating the average peak demand per product. To arrive at this number, we assume that the average dose is 3 mg/kg body weight and that the patient is given an average of 10 doses. These numbers are the average of products on the market or in the late stages of development. Using these assumptions, a patient would receive an average of 2 g of product per year. We further estimate that the average market size is 100,000 patients and that peak penetration is 30% or 30,000 patients. Multiplying this number by the average annual dose calculated earlier, gives an average peak demand of 60 kg/year. The next step is to determine how many liters of capacity are required to produce 60 kg of finished product. First we assume an average yield of

295 50%, which means that we must have enough capacity to produce 120 kg of crude product. If we assume that the industry average yield is 0.25 g/l (range: 0.1–1.5 g/l), this translates into 480,000 l of capacity to produce 120 kg crude product (Fig. 4). For the average product we have described, two 12,000 l reactors running 20 batches per year would be required to produce that amount of material. We estimate that demand for finished product in 2002 will be 863 kg (Table 6). This amount represents a 36% increase compared with the previous year’s estimate. The success of Rituxan and the relatively high dose required by each patient makes it the product with the highest demand at 255 kg. Following closely behind is Enbrel, with annual demand of 250 kg. The recent strong growth of Remicade, due in part to Enbrel’s capacity issues, brings Remicade to third place with 180 kg. It is difficult to estimate the demand for Erbitux (ImClone) because the FDA refused to accept its file. Demand for 863 kg of finished product is the equivalent of 14.4 products, assuming an average production requirement of 60 kg/year. Using our calculations and estimate of 475,000 l of current capacity, we estimate there is enough capacity to produce 19.5 products. This suggests that the industry is currently at 74% capacity. It must be remembered, however, that commercial product cannot be produced all at one time. Some capacity is always tied up in process

Fig. 4. Illustration of product demand.

296 Table 6. Estimated demand for mammalian cell culture capacity in 2002. Product

Company

Dose/year (g)

No. of patients

Demand (kg)

Rituxin Enbrel Remicade Herceptin Erbitux Synagis Pulmozyme ReoPro Zygris Zevalin Cerzyme Epogen/Procrit Campath Total demand

Genentech/IDEC Immunex/AHP JNJ Genentech ImClone/BMS MedImmune Genentech JNJ/Eli Lilly Eli Lilly IDEC Genzyme Amgen/JNJ Ilex

4.3 2.5 1.8 3.5 6.0 0.14 1.0 0.023 0.2 0.5 1.0 0.08 0.75

60,000 100,000 100,000 26,000 5000 138,000 12,000 300,000 25,000 5000 3100 500,000 21,000

255 250 180 91 30 20 12 7 5 3 3 3 1 863

AHP, American Home Products; BMS, Bristol Meyers Squibb; JNJ, Johnson & Johnson.

development, scale-up, validation runs, and the production of clinical trial material. We would argue that the industry is currently running at 100% capacity; thus the problem that Immunex/AHP have regarding Enbrel production. To take our demand estimates one step further, we have made certain assumptions regarding the approval of new products through 2006. We based these assumptions on historical success rates for biopharmaceuticals at various stages of development. These success rates were 25% for products in phase 1, 33% for products in phase 2, 65% for products in phase 3, and 90% for products under regulatory review. We also used industry average time lines when assuming what year pipeline products would be approved for marketing. Scanning the current industry pipeline, we identified six products that were currently under review, eight products in phase 3, 27 products in phase 2, and 30 products in phase 1. As many companies do not disclose products in phase 1, our estimate of 30 candidates in phase 1 is probably low. We estimate that 41 products that must use mammalian cell culture techniques will be marketed in 2006 compared with the 20 that are marketed today. While this number represents only a doubling of products, it also represents a more than quadruple increase in demand for capacity using our estimates (Table 7). Future mammalian cell culture manufacturing capacity We estimate that by the end of 2006, 1,054,000 l of additional capacity will come on line. Approximately 17% (169,000 l) will come from contract manufacturers (Table 8) and 83% (885,000 l) will come from the biopharmaceutical industry (Table 9). Among the contract manufacturers, Boehringer Ingelheim and Lonza

297 Table 7. Mammalian cell culture capacity demand. Data for years 2002–2006 are estimates. Year

No. of biologic approvals

No. of cumulative approvals

Demand (kg)

1986 1987 1988 1989 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006

1 1 0 1 0 0 1 1 2 0 1 1 6 1 2 2 4 4 3 5 5

1 2 2 3 3 3 4 5 7 7 8 9 15 16 18 20 24 28 31 36 41

0.1 1.0 1.0 1.5 1.8 2.1 2.5 2.7 4.0 5.0 5.5 35.0 100.0 250.0 425.0 635.0 865.0 1180.0 1575.0 2070.0 2700.0

Table 8. Future additional estimated mammalian cell culture capacity for contract manufacturing. Company

Volume (l)

No. of fermentors

Estimated completion date

Boehringer Ingelheim DSM

12,500 2000 10,000 20,000 5000 2000 164,000

6

2004 2003 2005 2004 2004 2004

Lonza Rentschler Total

2 3 2

will be adding the most capacity. Boehringer Ingelheim is doubling its current capacity in Germany by adding 75,000 l, which should come on line in 2004. With regard to the biopharmaceutical industry, Amgen/American Home Products are adding the most capacity by far. The Rhode Island facility should add 64,000 l of capacity toward the end of 2002, while other facilities in Rhode Island and Ireland should contribute an additional 345,000 l in 2005 and 2006. Biogen has started plans to add 90,000 l of capacity in 2002 and another 90,000 l in 2005. IDEC has stated that it plans to add 120,000 l of capacity in 2005.

298 Table 9. Future additional estimated mammalian cell culture capacity for industry. Company

Volume (l)

No. of fermentors

Estimated completion date

Abgenix

12,000 2000 6000 20,000 15,000 15,000 12,500 10,000 15,000 8000 20,000 12,500 15,000 10,000 10,000 885,000

2 4 1

2002/2003 2002/2003 2002 2003/2004 2002 2002 2002 2004 2005 2002 2005 2005 2006 2005 2002

Abbott Baxter Biogen Genentech Human Genome Sciences IDEC Pharmaceuticals Amgen/AHP

Protein Design Labs ImClone Total

6 6 4 2 8 8 9 6 6 2 3

AHP, American Home Products.

Table 10. Total estimated mammalian cell culture manufacturing capacity.

Industry capacity Contract capacity Total capacity Fold increase

Year 2001

Year 2006

360,000 l 115,000 l 475,000 l 1.0

1,245,000 l 284,000 l 1,529,000 l 3.2

In summary, we estimate that total commercial-scale mammalian cell manufacturing capacity will exceed 1,500,000 l by the end of 2006. This amount represents more than three-fold increase over current levels of capacity (Table 10). Clearly our estimate of future demand is based on numerous assumptions and does not take into account the potential for process improvements. We believe that there could be a growing gap between supply and demand, although a large amount of capacity is currently in the process of being constructed (Fig. 5). The industry’s response to the growing problem While slow to react initially, the industry has begun to address the issue of biopharmaceutical manufacturing capacity. Three methods are available to companies to resolve the capacity problem. The first and most obvious way is to build additional manufacturing capacity. Contract manufacturers and well-financed biopharmaceutical companies are building facilities that we estimate should be more than triple the capacity in five years.

299

Fig. 5. Growing gap between supply and demand.

A second approach is to improve yields in existing facilities. MedImmune and Boehringer Ingelheim have developed processes to improve the yield of Synagis and Enbrel, respectively. The easiest way to improve yields is to optimize the growth conditions for the engineered cells in the bioreactor, which can be done by either changing the nutrients in the growth media, adding additional nutrients during the growth phase, altering the density to which cells are allowed to grow, or altering the oxygen content of the bioreactor. Boehringer Ingelheim has been able to enhance the yield of Enbrel by 10–30% by altering growth conditions. Another more difficult means of enhancing yields is to create new more productive cell lines. This approach is more time consuming than the traditional approach due to the requirements of the regulatory authorities. Any new cell line and its product must be fully characterized. Human ‘‘bridging studies’’ may need to be done to demonstrate that the protein produced in the new cell line behaves the same way as the protein produced in the older, less efficient cell line. One last approach to enhancing yield is to try to improve the downstream purification process. Alternative production technologies While fermentation and cell culture are the two primary methods of producing protein-based therapeutics, there are other ways to make these products.

300 Table 11. Alternate production technologies. The number of reactors and their volumes are not known for any company. Company

Product

Biological system

Genzyme Transgenics Genzyme Transgenics Crop Tech Corp Meristem Therapeutics TransXenoGen Large Scale Biology

Remicade CTLA4-16 Unknown Unknown Unknown Cancer vaccines

Goats Goats Tobacco Corn, tobacco Chicken eggs Tobacco

Transgenic mammals can produce human proteins in their milk while transgenic chickens can produce recombinant proteins in their eggs. Transgenic corn and tobacco offer another potential means of producing large quantities of protein economically. The potential advantage of transgenic animals is that they are capable of producing very large complex proteins at very high expression levels. Scale-up is easy and production is low relative to cell culture (Table 11). The primary disadvantage to using transgenic plants or animals is the lack of regulatory experience with transgenics. In addition, the public perception of transgenic technology in general is not favorable. Genzyme Transgenics has developed transgenic goats that can produce a variety of pharmaceutical products in their milk, such as Remicade. Feedback from industry sources suggests that these goats will have a role in large-scale commercial manufacturing of protein therapeutics in the future. Other potential bottlenecks In addition to the lack of adequate capacity for the bulk production of biopharmaceuticals, we have heard of two other steps in the production process that are beginning to become bottlenecks. These include lyophilization and regulatory inspections. Lyophilization is the process of freeze-drying the finished purified product. Many biopharmaceuticals are supplied as sterile, lyophilized powders in glass vials. Lyophilization, therefore, is required before the filling and finishing steps. It is our understanding that lyophilization capacity is currently very limited, but that new capacity is being added. We understand that the shortage of experienced FDA inspectors is increasing and that resources are limited to add more inspectors. All new facilities must be inspected by the FDA before licensure, and all existing facilities are subject to surprise inspections approximately every two years. Industry experts anticipate that the surge in the number of new products and new facilities may result in regulatory delays in the future.

301 Who gains from the capacity constraint? While the biopharmaceutical industry will likely continue to suffer as a consequence of inadequate manufacturing capacity, some companies should benefit from the building of new capacity. Most notably, Invitrogen, the primary producer of cell culture media, will experience an increased demand for media as new products are approved and new facilities come on line. Cell culture media is the fluid in which cells are grown and it contains all the essential nutrients needed for optimal cell growth. Importantly, once a specific media is incorporated into an FDA-approved process, it is extremely rare that it changes. Consequently, it becomes an annuity for the provider of the media. Other raw material providers may also gain from the expansion of the manufacturing capacity. Biopharmaceutical industry to shoulder economic burden The implications of these findings are troubling for the biopharmaceutical industry. The lack of adequate third-party manufacturing capacity will force biopharmaceutical companies to invest in manufacturing capacity at a time when their product development risk remains high. Investments in manufacturing facilities deplete capital resources that would otherwise be used to enhance shareholder value through investments in research and development. The industry will have no choice but to make these high-risk investments. If not, biotechnology companies run the risk of significant delays in the development and commercialization of new products. We argue that companies should consider entering into manufacturing consortia to reduce risk and capital investments. While a lack of adequate manufacturing capacity is a problem for the biopharmaceutical industry, the building of capacity is an advantage for companies that supply raw materials used during the manufacturing process. In our opinion, one of the primary beneficiaries will be commercial-scale cell culture media market. Implications for the biopharmaceutial industry The implications of the lack of mammalian cell culture capacity for the biopharmaceutical industry are far reaching. Initially, it will have an impact on companies’ balance sheets. Many companies are already being forced to use their own capital to fund the construction of new facilities, with the average facility costing between US$250 and 400 million. Most companies would rather not take on the financial risk and burden of building a new facility. They would rather invest their money in R&D or use it to acquire products, technology, or companies. Considering the pricing flexibility of the industry, it often makes greater economic sense to outsource manufacturing. With no third-party capacity, however, companies must now take on the risk of building facilities for

302 products that may fail in clinical development. The industry is extremely fortunate that many companies in need of capacity were able to raise significant amounts of capital in 2001. If this had not taken place, the industry would be in even greater trouble than it currently finds itself. Not all companies were so fortunate. We would not be surprised to see manufacturing consortiums develop where several companies share the cost and risk of building new facilities. In addition to investments in new facilities, we anticipate significant investment in process improvements and alternative production technologies such as transgenics. Due to the less-certain regulatory path of these new technologies, these investments must be made in conjunction with investments in traditional manufacturing technology. More important than these investment issues is the potential for the delay or discontinuation of the development of products in the clinic. Not only is commercial-scale capacity in short supply, but clinical material is also in tight supply. Availability of clinical material may soon be on the critical path of biopharmaceuticals in development. Clinical trials may be delayed or facilities that usually produce commercial product will be required to produce clinical trial material. Bridging studies would need to be performed before commercialization and would add to development time and risk. Some products that passed initial return-on-investment checkpoints in go/no go decisions may need to be discontinued when the added expense of the construction of a manufacturing plant is factored into the equation. We believe anyone with additional manufacturing capacity will be in command when negotiating with those in need of capacity. To our knowledge, Biogen is the only company that will have significant excess capacity within the next two years. We believe that Biogen management has no intention of using this capacity for simple contract manufacturing. Instead, we believe the company will use it to help bolster its product pipeline. Biogen is one of the few companies that can offer both manufacturing capability as well as a commercial infrastructure to a potential partner. The current capacity of Boehringer Ingelheim will be doubled in early 2004. While part of this capacity is already committed, the company can be highly selective in taking on new projects. Boehringer Ingelheim may use this asset strategically to gain marketing rights to certain products. Boehringer Ingelheim would be less likely to take on risky projects unless it was financially induced to do so. In addition, it would be unlikely that it would take on projects that would consume a large percentage of its capacity. For this reason, as well as cost issues, we believe that a product that must be chronically administered at amounts greater than 5 mg/kg/week may not be commercially viable using today’s technology.

303

Review of current authorship guidelines and the controversy regarding publication of clinical trial data MaryAnn Foote* Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA Abstract. Publication of clinical trial data is the final step in the scientific method and an important method by which pharmaceutical and biotechnology companies, i.e., drug sponsors, disseminate information about their products. Because of the nature of large, multicenter trials, multiple investigators from many institutions may be considered as authors of these papers. Controversy concerning the rights of academic institutions and the rights of drug sponsors has been widely debated. This chapter summarizes the controversy and the current policies. Keywords: biotechnology – medical biotechnology, clinical research – publication of trials, good publication practices, uniform requirements for biomedical journals, Vancouver conventions.

Introduction The scientific method consists of observation, questioning, hypothesis formation and testing, and publication of results of hypothesis testing. The last step, publication or some form of dissemination of the results, is a critical and inherent step of the process. Recently, publication of clinical trial results has become the topic of numerous essays, editorials, and news items. In September 2001, 11 journals simultaneously published a paper by Davidoff et al. (Table 1) calling for more involvement of physicians and academic centers in the research required for gaining marketing approval for drugs. At issue is the ability of drug sponsors to satisfactorily collect and honestly report on the efficacy and, particularly, the safety of their product candidates. The drug industry, both pharmaceutical and biotechnology, has always acknowledged that drug development would not be possible without the cooperation, insight, and assistance of physician investigators and their patients. Most physician investigators are likewise quick to acknowledge that drug development requires the money, personnel, and expertise of the drug industry. The drug industry invests much money in the development of drugs, techniques, and molecules. A recent report by Tufts Center for the Study of Drug Development calculates the cost at more than $800,000,000 per drug [1]. The cost of drug development is one reason drug sponsors consider all data collected during the process to be proprietary. Editorials and news wire reports are quick to ‘‘blame’’ the industry for unsavory practices, including suppression of publications, but research suggests that improprieties are known on both sides. *Tel: þ 1 805 447 4925. Fax: þ 1 805 498 5593. E-mail: [email protected] BIOTECHNOLOGY ANNUAL REVIEW VOLUME 9 ISSN 1387-2656 DOI: 10.1016/S1387-2656(03)09009-4

ß 2003 ELSEVIER SCIENCE BV ALL RIGHTS RESERVED

304 Table 1. Journals that published in 2001 the article by Davidoff F, DeAngelis CD, Drazen JM, et al., Sponsorship, authorship, and accountability. Journal

Volume

Page(s)

Ann Intern Med Arch Otolaryngol Head Neck Surg CMAJ JAMA Lakartidningen Lancet Med J Aust N Engl J Med Obstet Gynecol Rev Esp Cardiol Tidsskr Nor Laegeforen

135 127 165 286 98 358 175 345 98 54 121

463–466 1178–1180 786–788 1232–1234 4694–4696 854–856 294–296 825–827 1143–1146 1247–1250 2531–2532

This chapter examines the issue of protecting drug sponsors’ intellectual property and ensuring the author’s right to publish. Guidelines for publishing Several publishing guidelines exist, including the Uniform Requirements for Submissions to Biomedical Journals [2]. Nearly every journal has instructions to authors, and some of these discuss authorship criteria and responsibilities. Potential authors should always read these instructions and note immediately any authorship criteria that must be addressed. While many of the journals do not provide specific guidance on determining authorship, some do reference the Uniform Requirements. Many journals, however, acknowledge that authors are legally responsible for their work and that all authors must be able to describe their contributions that afford them authorship. Other journals are interested in knowing that all authors have read the manuscript and have agreed to be authors. Table 2 lists some journals and their definition of author, if any. Beyond specific instructions to the author for a given journal, the Uniform Requirements, and other guidelines have been written to help authors accurately, clearly, and fairly report the results of clinical trials and to identify criteria for authorship on manuscripts. Uniform requirements The Uniform Requirements for manuscripts submitted to Biomedical Journals addresses the issue of giving credit (authorship) where credit is due (i.e., to the researcher who did the work). The Uniform Requirements states: ‘‘Each author should have participated sufficiently in the work to take public responsibility for the content. Authorship credit should be based only on substantial contributions to (a) conception and design, or acquisition of data, or analysis and

Table 2. Definition of authorship for some biomedical journals. The journals are representative of ones that routinely publish clinical trial data. All website were accessed in May 2002 and were accurate as of that date. Journal

Definition of Authorship

Web site

Ann Intern Med

Authors must contribute directly to the intellectual content of the paper, and the corresponding author must list the specific contributions of all authors. Authors should meet all criteria (i.e., conceived and planned work or interpreted the results, wrote the paper or made substantive suggestions, and approved final manuscript) to allow them to take public responsibility for the content of the paper. The journal states that positions of administrative leadership, contributing patients to a study, and collecting and preparing the data for analysis are not, by themselves, criteria for authorship. The journal encourages financial disclosure and acknowledgment of substantial contributions by nonauthors. No definition of author; refers to Uniform Requirements. Journal requires that all named authors have agreed that the manuscript should be submitted to the journal. All authors are expected to disclose in a cover letter any commercial affiliations as well as consultancies, stock or equity interests, and patent-licensing arrangements that could be considered to pose a conflict of interest regarding the submitted article. Journal requires each author to have participated sufficiently in the work to take public responsibility for the content. It states that authorship should be given only to those substantially contributing to concept and design, analysis and interpretation, and drafting or revising article for important intellectual content. Journal requires the corresponding author to ensure that all authors have agreed to its content and an article is in submission. The journal accepts no responsibility for matters of authorship.

http://www.annals.org

Blood Bone Marrow Transplant Br J Haematol

Cancer

Cancer Res

http://www.blackwell-synergy.com

http://www.interscience.wiley.com

http://www.cancerres.aacrjournals.org

305

(Continued.)

http://www.bloodjournal.org http://www.naturesj.com/bmt/instructions.html

306

Table 2. Continued. Journal

Definition of Authorship

Web site

Exp Hematol

All authors must sign a statement at the time of submission stating that they have contributed significantly to the research described in the paper and have read and approved the final manuscript. Journal asks that significant contributors to the manuscript be acknowledged (after granting permission). No definition of author, but all authors must concur that they have seen and approved the manuscript. No definition of author; suggests that Uniform Requirements may be useful to authors. No definition of author. Adheres to Uniform Requirements as criteria for authorship. Lead author must ensure that all authors agree on the content of the manuscript before it is published, and authors are encouraged to acknowledge contributions of nonauthors in the acknowledgment section. Authors of research articles, reviews, or editorials are expected to disclose at the time of submission any financial arrangement they may have with a company whose product figures prominently in the submitted manuscript or with a company making a competing product. Journal follows the Uniform Requirements. Authorship is limited to those who have contributed substantially to the work, and the corresponding author must obtain permission from all authors for the submission of each version of the paper and for any change in authorship.

http://www.elsevier.com

J Biol Chem J Clin Oncol J Immunol Lancet Nature

N Engl J Med

Proc Natl Acad Sci USA

http://www.jbc.org http://www.jco.org http://www.jimmunol.org http://www.thelancet.com http://www.nature.com

http://www.nejm.org

http://www.pnas.org

307 interpretation of data; and to (b) drafting the article or revising it critically for important intellectual content; and on (c) final approval of the version to be published. Conditions (a), (b), and (c) must all be met.’’ Many peer-reviewed journals cite the Uniform Requirements, sometimes referred to as the ‘‘Vancouver Conventions,’’ in their instructions to authors. Before the establishment of the Uniform Requirements, it was a common practice to routinely add the name of the laboratory or chief physician to all papers written by anyone in the group. Good Publication Practices The Good Publication Practices (GPP) Guidelines for Pharmaceutical Companies (Liz Wager, personal communication) states: ‘‘Pharmaceutical companies’ relations with clinicians, academics, medical journals, and the public have often been characterized by conflicting interests and tensions and these negative aspects have received considerable attention. Companies should endeavor to publish the results from all of their clinical trials. These publications should present the results of the research accurately, objectively, and in a balanced fashion.’’ The GPP guidelines are written from an industry perspective, unlike the Uniform Requirements, which are written from the journal editors’ viewpoint. As such, the GPP guidelines acknowledge the use of drug sponsors’ writers as liaisons with the investigators and confirms the right of drug sponsors to review all publications before submission. A unique aspect of the GPP guidelines is that the criteria are applied equally to drug sponsor-employed authors and nonindustry authors. At the time of the preparation of this chapter, the GPP guidelines have not been published but have been widely discussed within a number of drug and biotech companies and are awaiting a publication decision from a major medical journal. What is the problem? In an editorial in the journal Blood published soon after the publication of the Davidoff et al. (2001) paper, the journal editor stated, ‘‘It has been said that laws are imposed on 99% of the population to help prevent the improprieties of the remaining 1%,’’ [3] suggesting that this journal believed that it published manuscripts that fairly and accurately reported clinical trial data. Another editorial stated, ‘‘Almost all new drugs are developed by the industry, and many companies have high ethical standards. . .’’ [4]. Given the existence of the Uniform Requirements, individual journal requirements, and the GPP guidelines, one could ask: What is the problem? Are physician-investigator rights to publish clinical trial data tightly controlled by drug sponsors? Why shouldn’t drug sponsors have the right to protect their patent positions? Are further checks necessary? As background for discussion, two extreme examples of publishing practices are presented.

308 The Synthroid Controversy In 1987, Flint Laboratories (subsequently acquired by Boots Pharmaceuticals and later Knoll Pharmaceutical Company) engaged researchers at the University of California, San Francisco, to study four preparations of thyroid medication. A preliminary investigation had suggested that a drug preparation manufactured by Flint Laboratories was bioequivalent to other thyroid preparations [5] and Flint Laboratories thought that another publication about the bioequivalence of its thyroid drug, Synthroid, would benefit them. Reprints of positive studies published in a peer-reviewed journal are routinely used in the drug industry for promotional purposes. The FDA monitors the use of such reprints in the United States. The study and its planned publication encountered difficulties. Several editorials and letters to the editor detail the controversy [6–8]; the data will be summarized. The interested reader is encouraged to review the original publications on this topic. The contract signed by the researchers included the statements ‘‘...all data obtained by the investigator. . .are also considered confidential and are not to be published or otherwise released without written permission of Flint Laboratories.’’ Such wording is consistent with other drug sponsor contracts. The results of the in vivo study completed in 1990, showed that Synthroid was bioequivalent to three other thyroid products, some of which were less costly. Dong et al. have reported that when they attempted to publish the results of their study, Flint Laboratories prohibited publication based on their original signed contract [7]. In subsequent legal proceedings, documents surfaced that suggested that in 1989 Flint Laboratories stated they had ‘‘concerns about the study execution’’ and could not reach agreement with the lead investigator. Boots Pharmaceuticals, who now owned the product, involved the chancellor of the University where the investigators were employed, asserting that there had been study deficiencies and statistical analysis problems, as well as ethics infractions. The controversy escalated when the researchers sent their paper to the Journal of American Medical Association in 1994, with an accompanying letter critical of Boots Pharmaceuticals. Expert reviewers selected by the journal read the paper and recommended that it be published. Twelve days before the paper was to be published, however, the authors asked for its withdrawal, setting off an inquiry by the journal. The authors stated that Knoll Pharmaceutical Company (who now owned the product) refused to allow the paper to be published and threatened legal action against the investigators and the institution. More intricate legal battles ensued, but finally an agreement was reached between Knoll Pharmaceutical Company and the investigator, and the paper was published [9]. The journal published an accompanying editorial [7] and commentary from Knoll Pharmaceutical Company [8]. From this case, is it fair to conclude that industry involvement in research and publications is controlling and unethical? Did the drug sponsor act irresponsibly

309 in its attempts to stop publication of a paper that could weaken its marketing position? While this example may be an extreme one, it must be noted that not all investigators maintain integrity in their private, nonindustry-sponsored publications. The Bezwoda Scandal In 1995, a researcher in South Africa published the results of a trial comparing high-dose chemotherapy with forms of less-intensive chemotherapy [10,11]. These reports were heralded as proof of the efficacy of high-dose chemotherapy for the treatment of patients with breast cancer, and the results were subsequently published or presented many times (Table 3). Bezwoda was an independent investigator who did not receive industry funding for his original study. Initially, no one questioned that only 90 patients were needed to prove a theory that larger trials had not been able to prove or that Bezwoda had done such an important study without industry funding or knowledge of the study. Because the results were so positive, the number of patients treated with highdose chemotherapy increased [12]. By February 2001, the 1995 paper had been cited 354 times and served to intensify the ongoing debate over high-dose chemotherapy [13]; however, an audit of another study by Bezwoda revealed major irregularities in that study [14], leading to heightened concern about the validity of the original 1995 study. When confronted with the data, the South African Medical Research Council invited a full audit of the data, and the results of this audit were published by Weiss et al. [13]. The audit and subsequent articles and editorials are well known and will be summarized. The interested reader is encouraged to review the original Table 3. Bezwoda’s results were published or presented many times.














Bezwoda WR, Seymour L, Vorobiof DA. High dose cyclophosphamide, mitroxantrone and VP16 (HD-CNV) as first line treatment for metastatic breast cancer. Proc Am Soc Clin Oncol. 1992; 11:64 (abstract). Bezwoda WR, Seymour L, Dansey RD. High dose chemotherapy with hematopoietic rescue as primary treatment for metastatic breast cancer: A randomised trial. Eur J Cancer. 1995; 31A:S76 (abstract). Bezwoda WR, Seymour L, Dansey RD. High-dose chemotherapy with hematopoietic rescue as primary treatment for metastatic breast cancer: A randomized trial. J Clin Oncol. 1995; 13:2483–289. Bezwoda WR. High-dose chemotherapy with haematopoetic rescue: application to primary management of metastatic breast cancer. Eur J Cancer Care. 1997; 6:10–15. Bezwoda WR. High-dose chemotherapy with haematopoietic rescue in breast cancer: from theory to practice. Cancer Chemother Pharmacol. 1997; 40:S79–S87. Bezwoda WR. Primary high dose chemotherapy for metastatic breast cancer: update and analysis of prognostic factors. Proc Am Soc Clin Oncol. 1998; 17:115a (abstract). Bezwoda WR. High dose chemotherapy with haematopoietic rescue in breast cancer. Hematol Cell Ther. 1999; 41:58–65.

310 publications on this topic. Briefly, the audit found records for only 61 of the 90 patients reported in the trial. When these 61 patients were reviewed for compliance to entry criteria, only 27 patients had sufficient records to verify eligibility criteria, and 18 of the 27 patients did not meet one or more eligibility requirements. Twenty-five of the reported 90 patients received therapy temporally associated with enrollment dates, and only 22 of the patients actually received high-dose chemotherapy. The auditors reported that the details of treatment from individual patients were at ‘‘great variance’’ from the published data. The final coupe de grace: the protocol was written nine years after the study started. Nine other trials published by Bezwoda were not reviewed or approved by the appropriate institutional review committees, despite statements to the contrary in the publications. When confronted with the mounting negative audit findings, Bezwoda became less cooperative and finally uncooperative. Eventually, he admitted that he had falsified data, and he was dismissed from the University. His 1995 paper is marked with a retraction in Medline. Given all these data, is it fair to extrapolate that investigators cannot be trusted to publish accurate, precise, and valid data? Had the study been done by a drug sponsor, certain documents such as a protocol, investigators brochure, institutional review board approval, and written informed consent forms, would have been required for patients to be enrolled into the trial. A monitoring plan, statistical analysis plan, and study-stopping rules would have been defined. Is it fair to state that the only groups capable of conducting a controlled randomized trial are drug sponsors? Discussion The Synthroid Controversy and the Bezwoda Scandal are two examples of publication practices gone awry that are frequently cited as proof that improvement is needed. Fortunately, these two examples reflect extremes in the realm of publications. Most drug sponsors have statements in their contracts and/or clinical trial protocols specifying that they have the right to review a paper before it is submitted to a journal. The purpose of the right to review an intended publication is to protect proprietary positions, which can be compromised inadvertently by subtle wording changes. Most drug sponsors have statements prohibiting one site of a multicenter study from publishing its own data to the detriment of the primary publication of data from all centers. Data from one site are not as powerful as pooled data from a large multicenter study and may not be clinically meaningful or have sufficient statistical power. If data from a single center are published independently, the more powerful primary publication will probably not be published in a top-tier, peer-reviewed journal because of prior publication. Lest it be thought that publication policies are the domain of drug sponsors, the International Committee on Harmonisation guidelines states that a publication policy should be included in the study protocol or as a separate agreement to ensure that clinical trial results are disseminated [15].

311 Publication of clinical trial results is the final step of the scientific process and allows other scientists to build on the work of others. Publication of valid clinical trial data is important in the marketing of drugs too. Given the costs involved in drug research, it is unreasonable to assume or to insist that drug sponsors allow outside investigators to publish as they wish without some review of intended publications. In recent years, more journals have encouraged the publication of negative studies, i.e., clinical trials that did not meet their intended endpoints. While some studies do not reach predefined endpoints and the results do not have statistically significant results because of simple bad luck, other trials fail because of poor design or insufficient knowledge of a drug’s characteristics. Using knowledge of these latter failed studies, investigators may be able to move the field forward by designing new studies that address the issues and provide answers for the study failures. Journals that require description of contributions to justify authorship allow employees of drug sponsors who meet the criteria for authorship to be so named. As stated in the introductory section of this chapter, drug companies are known to have the money, staff, and expertise to design and run a study, analyze the data, and write the study report and publication. By allowing drug sponsor staff who does critical work, not solely monitoring, enrolling, or entering data, to be authors, the publication process becomes transparent and the role of drug sponsor and investigator better understood. Drug sponsors have the staff and resources to ensure proper compliance with the protocol, to resolve data errors in the transcription process, and to select the proper statistical methodology and ensure the proper performance of computations. Some independent investigators are unaware that the FDA and other agencies regularly review and check statistical calculations. Drug sponsor employees who do essential work in monitoring, data checking, manuscript editing, etc., should be named in the Acknowledgment section of a paper. It should be noted, however, that some drug sponsors surveyed for this paper do not have publication policies and some who do, allow all employees who had anything to do with the clinical trial to be authors. These companies typically are smaller start-up enterprises. Some policy points are given in Table 4. One area of contention is the writing of manuscripts by drug sponsors or their vendors, which then seek a physician, particularly an opinion leader, who will agree to be an author. Manuscripts so written and authored are generally Table 4. Company policies vary concerning the publication of results from clinical trials. Company statistician requested/required to be an author Company has clause in contract permitting review by the company
Anyone at company who has worked on paper (including data manager who prepared graphs, clinical research assistants, clinical managers) is an author/can be an author
Only company staff actively involved in design of trial, not those involved in collection or analysis of data, are authors



312 derivative manuscripts, i.e., reviews, and are usually published in drug companysponsored supplements. Journals frequently number supplements in such a way that it is evident that these publications are not truly peer reviewed. Some physicians believe that this practice should be avoided and that those who allow the use of their name as such do so at the risk of their own professional reputation. Interestingly, most of the journal requirements and publishing guidelines do not question the ethics of a clinical study design or execution, only the ethics of publishing the results. This bias suggests that medical journals may not be concerned whether the regulatory authorities or the internal review or ethics boards allow a trial that excludes women or the elderly, or a trial that uses the sick babies of indigent women. Clearly, a policy that ensures the rights and safety of research participants, enforces the applicable laws and regulations regarding the conduct of clinical trials, and encourages the publication of all clinical trial results is warranted. However, as written and enforced now, the Uniform Requirements, GPP guidelines, and company-specific policies can be followed, should be followed, and are compatible. Drug sponsors are fully aware of the value of publication of the results of properly designed, conducted, and analyzed randomized clinical trials. Many drug sponsors routinely follow the Uniform Requirements, and encourage publication of randomized clinical trial data. By continuing to follow these processes, drug sponsors set the standards for other drug companies. The role of the drug industry in the publication of randomized clinical trial results could be strengthened if, as a group, they agreed on issues such as use of internal medical writers, identification of internal drug sponsor staff who meet authorship criteria, financial disclosure for all authors and contributors in the Acknowledgment section, and commitment to publication of all randomized clinical trials. Acknowledgements I thank the following people for sharing how proprietary information and publications are handled by their company or clients, for sending references, or for reading and critically editing this paper: Donna Causey, MD; Lyn Frumkin, MD, PhD; Cindy Hamilton, PharmD, Hamilton House, Virginia Beach, Virginia; Douglas Haneline, PhD, Ferris State University, Big Rapids, Michigan; Sandra J Hecker, Hecker and Associates, Arlington, Virginia; Frankie Ann Holmes, MD, Texas Oncology, Houston, Texas; Bertrand Liang, MD, Amgen Inc., Thousand Oaks, California; Marianne Mallia, Texas Heart Institute, Houston, Texas; Christopher Preston, PhD, F Hoffmann-La Roche, Basel, Switzerland; JoAnn Schuh, DVM, PhD, Applied Veterinary Pathobiology, Bainbridge Island, Washington; Susan Siefert, Cyberonics; Friendswood, Texas; Liz Wager, GPP; Mark Winnett, Stressgen Biotechnologies, Collegeville, Pennsylvania; and James Yuen, Amgen Inc, Thousand Oaks, California.

313 References 1. Tufts Center for the Study of Drug Development. http://www.tufts.edu/med/csdd/ Nov30CostStudyPressRelease.html (accessed 6 June 2002). 2. International Committee of Medical Journal Editors. Uniform requirements for manuscripts submitted to biomedical journals. N Engl J Med 1997;336:309–315. 3. Kaushansky K. Removing the cloud from industry-sponsored, multicentered clinical trials. Blood 2001;98:2001 (editorial). 4. Smith R. Maintaining the integrity of the scientific record. Br Med J 2001;323:588 (editorial). 5. Dong BJ, Young R and Rapaport B. The nonequivalence of thyroid products. Drug Intell Clin Pharmacol 1986;20:77–78. 6. Eckert CH. Bioequivalence of levothyroxine preparations: industry sponsorship and academic freedom. JAMA 1997;277:1200–1201 (editorial). 7. Rennie D. Thyroid storm. JAMA 1997;277:1238–1243 (editorial). 8. Spigelman MK. Bioequivalence of levothyroxine preparations for treatment of hypothyroidism. JAMA 1997;277:1199–1200 (letter). 9. Dong BJ, Hauck WW, Gambertoglio JG, et al. Bioequivalence of generic and brand-name levothyroxine products in the treatment of hypothyroidism. JAMA 1997;277:1205–1213. 10. Bezwoda WR, Seymour L and Dansey RD. High dose chemotherapy with hematopoietic rescue as primary treatment for metastatic breast cancer: a randomised trial. Eur J Cancer 1995a;31A:S76. 11. Bezwoda WR, Seymour L and Dansey RD. High-dose chemotherapy with hematopoietic rescue as primary treatment for metastatic breast cancer: a randomized trial. J Clin Oncol 1995b; 13:2483–289. 12. Antman KH. High-dose chemotherapy in breast cancer: the end of the beginning? Biol Blood Marrow Transplant 2000;6:49–475. 13. Weiss RB, Rifkin RM, Stewart FM, et al. High-dose chemotherapy for high-risk primary breast cancer: an on-site review of the Bezwoda study. Lancet 2000;18:355:999–1003. 14. Weiss RB, Gill CG and Hudis CA. An on-site audit of the South African trial of high-dose chemotherapy for metastatic breast cancer and associated publications. J Clin Oncol 2001;19:2771–2777. 15. International Conference on Harmonisation, Guidance for Industry, E6 Good Clinical Practice: Consolidated Guidance, April 1996, http://www.ifpma.org/pdfifpma/e6.pdf, accessed 4 March 2002.

Note Added in Proof The GPP have been published (Wager E, Field EA and Grossman L. Good publication practice for pharmaceutical companies. Curr Med Res Opinion 2003;19:149–154.)

315

Protein electrostatics: A review of the equations and methods used to model electrostatic equations in biomolecules – Applications in biotechnology Maria Teresa Neves-Petersen and Steffen B. Petersen* Department of Physics and Nanotechnology, University of Aalborg, Biostructure and Protein Engineering Group, Sohngaardsholmsvej 49, DK-9000 Aalborg, Denmark Abstract. The molecular understanding of the initial interaction between a protein and, e.g., its substrate, a surface or an inhibitor is essentially an understanding of the role of electrostatics in intermolecular interactions. When studying biomolecules it is becoming increasingly evident that electrostatic interactions play a role in folding, conformational stability, enzyme activity and binding energies as well as in protein–protein interactions. In this chapter we present the key basic equations of electrostatics necessary to derive the equations used to model electrostatic interactions in biomolecules. We will also address how to solve such equations. This chapter is divided into two major sections. In the first part we will review the basic Maxwell equations of electrostatics equations called the Laws of Electrostatics that combined will result in the Poisson equation. This equation is the starting point of the Poisson–Boltzmann (PB) equation used to model electrostatic interactions in biomolecules. Concepts as electric field lines, equipotential surfaces, electrostatic energy and when can electrostatics be applied to study interactions between charges will be addressed. In the second part we will arrive at the electrostatic equations for dielectric media such as a protein. We will address the theory of dielectrics and arrive at the Poisson equation for dielectric media and at the PB equation, the main equation used to model electrostatic interactions in biomolecules (e.g., proteins, DNA). It will be shown how to compute forces and potentials in a dielectric medium. In order to solve the PB equation we will present the continuum electrostatic models, namely the Tanford–Kirkwood and the modified Tandord–Kirkwood methods. Priority will be given to finding the protonation state of proteins prior to solving the PB equation. We also present some methods that can be used to map and study the electrostatic potential distribution on the molecular surface of proteins. The combination of graphical visualisation of the electrostatic fields combined with knowledge about the location of key residues on the protein surface allows us to envision atomic models for enzyme function. Finally, we exemplify the use of some of these methods on the enzymes of the lipase family. Keywords: protein electrostatics, laws of electrostatics, Maxwell equations of electrostatics, Poisson equation, Poisson–Boltzmann equation, Tanford–Kirkwood model, electrostatic potential distribution, molecular surface, pKa, dielectric constant, titratable residues, Debye–Hu¨ckel.

INTRODUCTION 1. Understanding the basic equations of electrostatics in order to model electrostatic interactions in proteins ‘‘Nature has simplicity and therefore a great beauty’’ Richard P. Feynman

Physics, the old Greek name for Nature, is the starting point of any field in science allowing us to describe how Nature works, even if we do not understand *Corresponding author: Tel: þ 45 9635 8469. Fax: þ 45 9635 9129. E-mail: [email protected] BIOTECHNOLOGY ANNUAL REVIEW VOLUME 9 ISSN 1387-2656 DOI: 10.1016/S1387-2656(03)09010-0

ß 2003 ELSEVIER SCIENCE BV ALL RIGHTS RESERVED

316 why Nature works that way. We cannot explain why Nature behaves in this peculiar way. Most theories will continue to evolve with time, others will not stand the test of time, and only a few pass this test. Some of the very basic questions we can ask within the scope of this chapter, dedicated to modelling electrostatic interactions in proteins, are: how does a charge perturb the space around it as it does? Why is the space surrounding a charge perturbed by it? What is the nature of this space? What are the fundamental equations of electrostatics? How were the equations used to model electrostatic interactions in proteins derived? In some sense we are very fortunate that some of these questions do not have immediate and perfectly understandable answers. This allows us to have the pleasure of speculation throughout our lives. In order to guide the reader this chapter has been divided in two main sections: (1) the basics of electrostatics; and (2) modelling protein electrostatics in proteins. The first section is dedicated to fundamental equations and laws of electrostatics that will hopefully fulfil the curiosity of the mind interested in the physics of electrostatics. We will go through the theory needed to derive the two laws (two of the Maxwell’s equations) of electrostatics. And why? Well, these two laws combined into a single equation will allow us to arrive at the Poisson equation of the electrostatic potential that in the second part of this chapter will be used as the starting point for the study of electrostatic interactions in proteins. On the other hand, if we know how these equations came about we will know under which conditions such equations are valid, i.e., the limitations of the laws of electrostatics. Knowing these limitations we will be better prepared to criticise the advantages and limitations of the methodologies used to model electrostatic interactions in proteins. The fulfilment that we feel when we understand how a particular formula came about and under which circumstances it can be used brings us the depth and the necessary knowledge needed in order to apply it properly in, e.g., the applied science domain. In Part 1 of this chapter we will consider electrostatic interactions between charges in vacuum, not including the effect that the dielectric constant of the media might have on such interactions. But much of electrostatic interactions have to do with charges and fields in media whose respective electric responses must be taken into account. In order to model electrostatic interactions in proteins we will consider the solute – protein molecules and the solvent molecules as dielectric media characterised by a particular dielectric constant. Therefore, we call them dielectrics. In Part 2 we shall then derive the equations of electrostatics when there are dielectrics. We will see that the Poisson equation derived in Part 1 from the fundamental equations of classical electrostatics is the starting point for modelling electrostatic interactions in proteins since it will allow us to arrive at the Poisson equation for a dielectric medium. In addition, it is reasonable to assume that the protein surrounds itself with an atmosphere of counterions, as described by the Debye–Hu¨ckel theory of electrolytes [1]. In this case the Poisson–Boltzmann (PB) equation, usually in its linear form, is solved. Solving such equation correctly parameterised will allow us to know the

317 electrostatic potential distribution at the location of each atom belonging to the protein. Summarising, in Part 2 we will cover the equations used to model electrostatic interactions in proteins, the different models that have been used to describe the electrostatic interactions in such macromolecules, as well as one method used to find the electrostatic potential distribution in proteins, i.e., methods used to solve the PB equation. We will start with simple delightful concepts of physics. Slowly the complexity will increase and we will see ourselves travelling from the world depicting the interaction between a pair of point charges to the beginning of the universe of complexity of an arbitrary distribution of charges. The beginning of this universe is still simple and understandable through simple concepts of physics. Slowly we will get closer to the protein and realise that the way of computing the electrostatic potential is different. The concepts of electrical force, electric field and electric potential will be introduced since they will many times be mentioned. In the following sections, a vector will be written in bold throughout the text and with an arrow above its symbol if in an equation. Both vectors and scalars will be written in italic. PART 1 – THE BASICS OF ELECTROSTATICS 2. Electromagnetic forces: electrostatics and electrodynamics 2.1. Introduction to electrical forces ‘‘Let us consider a force like gravitation which varies inversely as the square of the distance, but which is about a billion–billion–billion–billion times stronger. And with another difference. There are two kinds of ‘‘matter’’, which we can call positive and negative. Like kinds repel and unlike kinds attract – unlike gravity where there is presumably only attraction. What would happen? A bunch of positives would repel with an enormous force and spread out in all directions. A bunch of negatives would do the same. But an evenly mixture of positives and negatives would do something totally different. The net result would be that the terrific forces would balance themselves out almost perfectly, by forming tight, fine mixtures of the positive and the negative, and between two separate gathering of such mixtures there would be practically no attraction or repulsion at all. The electrical force is such a force. All the matter is a mixture of positive protons and negative electrons which are attracting and repelling with this great force. So perfect is the balance, however, that when we stand near someone else we do not feel any force at all. If there were even a very small unbalance we would know it. If a person was standing at an arm’s length from someone else and each of them had one percent more electrons than protons, the repelling force would be incredible. The repulsion would be enough to lift a ‘‘weight’’ equal to that of the entire earth! The force that holds the atoms together, and the chemical forces that hold molecules together, are really electrical forces acting in regions where the balance of charge is not perfect, or where the distances are very small. We would like to give another example that illustrates the magnitude and relevance of electrical forces. Let us think about a nucleus. In a nucleus there are several protons, all of which are positive. Why don’t they push themselves apart? And what would happen to the nucleus if they did? It turns out that in nuclei there are, in addition to electrical forces, non-electrical forces, called nuclear forces, which are greater that electrical forces and which are able to hold

318 the protons together in spite of the electrical repulsion. The nuclear forces, however, have a short range – their force falls off much more rapidly than 1/r2. And this has an important consequence. If a nucleus has too many protons in it, it gets too large, and it will not stay together. An example is uranium, with 92 protons. The nuclear forces act mainly between each proton (or neutron) and its nearest neighbour, while the electrical forces act over larger distances, giving a repulsion between each proton and all of the others in the nucleus. The more protons in a nucleus, the stronger is the electrical repulsion, until, as in the case of uranium, the balance is so delicate that the nucleus is almost ready to fly apart from the repulsive electrical forces. If such a nucleus is just ‘‘tapped’’ lightly (as can be done by sending in a slow neutron), it breaks into two pieces, each with positive charge, and these pieces fly apart by electrical repulsion. The energy which is liberated is the energy utilised to create the atomic bomb. This energy is usually called ‘‘nuclear’’ energy, but it is really ‘‘electrical’’ energy released when electrical forces have overcome the attractive nuclear forces.’’ Richard P. Feynman

Like a gravitational force, electrical forces decrease as the square of the distance between charges. This relationship is called Coulomb’s law, and will be addressed in this chapter. But this law is not precisely true when charges are moving – the electrical forces depend also on the motions of the charges in a complex way. One part of the force between moving charges is called the magnetic force. It is really one aspect of an electrical effect. This is why the subject is called ‘‘electromagnetism’’. The above-mentioned forces all depend on the distance between the bodies, but other things being equal they can be ranked as follows in order of magnitude: Strong nuclear > Electromagnetic > Weak nuclear > Gravitational It is rather peculiar that both gravitational and electrostatic forces follow the same fundamental equation. 2.2. Electromagnetism It has been found from experiment that the force that acts on a particular charge – no matter how many charges there are or how they are moving – depends only on the position of that particular charge, on the velocity of the charge and on the amount of charge. We can write the force F on a charge q moving with a velocity v as (non-relativistic force) Lorentz force
 F~ ¼ q E~ þ v~
B~

ð1Þ

E is the electric field and B the magnetic field at the location of the charge. The important thing is that the electrical forces from all the other charges in the universe can be summarised by giving just these two vectors. Their values will depend on where the charge is, and may change with time. Furthermore, if we replace that charge with another charge, the force on the new charge will be just in proportion to the amount of charge so long as all the rest of the charges in the world do not change their positions or motions. In reality, each charge produces

319 forces on all other charges in the neighbourhood and may cause these other charges to move, and so in some cases the fields can change if we replace our particular charge by another. 2.3. Principle of superposition One of the most important simplifying principles about the way the fields are produced is this: suppose a number of charges moving in some manner would produce a field E1, and another set of charges would produce E2. If both sets of charges are in place at the same time (keeping the same locations and motions they had when considered separately), then the field produced is the sum E~ ¼ E~1 þ E~2

ð2Þ

This fact is called the principle of superposition of fields. It holds also for magnetic fields. This principle means that if we know the law for the electric and magnetic fields produced by a single charge moving in an arbitrary way, then all the laws of electrodynamics are complete. If we want to know the force on charge A we need only to calculate the E and B produced by each of the charges B, C, D, etc., and then add the Es and the Bs from all the charges to find the fields, and from them the forces acting on charge A. However, it is not simple to give a formula for the force that one charge produces on another. It is true that when charges are standing still the Coulomb’s force law is simple, but when charges are moving about the relations are complicated by delays in time and by the effects of acceleration, among others. 3. Maxwell’s equations The complete classical theory of the electromagnetic field is contained in the following four equations, the Maxwell’s equations [2]. Maxwell’s equation: ~  E~ ¼
r "0

ð3Þ

~ ~
E~ ¼  @B r @t

ð4Þ

~ ~ ~
B~ ¼ @E þ j c2 r @T "0

ð5Þ

~  B~ ¼ 0 r

ð6Þ

320 where
(rho), the ‘‘electric charge density’’, is the amount of charge per unit volume, and j, the ‘‘electrical current density’’, is the rate at which charge flows through a unit area per second, and the gradient operator is defined as   @ @ @ ~ r¼ , , @x @y @z The situations that are described by these equations can be very complicated. The easiest circumstance to treat is one in which nothing depends on time – called the static case, electrostatics or magnetostatics. 4. Electrostatics Electrostatics is the branch of electromagnetism dealing with static electric fields and will be further developed in the present chapter. Its application to the study of the interaction between charged atoms in the proteins and solvent is largely dependent on the following approximations: – All charges are permanently fixed in space, or if they move, they move as a steady flow in a closed circuit. In these circumstances, all of the terms in the Maxwell’s equations which are time derivatives of the field are zero. This implies that we assume that the behaviour of a molecule in solution can be described in terms of a spatial and temporal average static structure. Protein structures determined by X-ray diffraction or NMR are normally used as models for the average structure. The electrostatic field and interactions between charged groups in the average structure can be taken as an average of the instantaneous charges in the real, or dynamic, structure. – A charged particle is instantaneously aware of a change in position of any other charge [3], i.e., relativistic or retardation effects do not play a role. – The electric field lines can originate or terminate only on electric charges. A given line of electric field in space is continuous and unbroken from its origin. – The electric field of a point charge at rest as having an isotropic radial pattern centred on the charge. – As long as the principle of superposition is valid (section 2.3 and section 9.1 in Part 2). – The coupling between electric and magnetic fields can be neglected. Electricity and magnetism are distinct phenomena so long as charges and currents are static, allowing electrostatics to be studied independently of magnetism. Under these circumstances, all of the terms in the Maxwell equations which are time derivatives of the field are zero. In this case, the Maxwell equations for electrostatics become [2]: ~  E~ ¼
r "0

ð7Þ

~
E~ ¼ 0 r

ð8Þ

321 We are saying that what is true for electrostatics is false for electrodynamics, because all terms with time derivatives are left off. Thus, the Coulomb’s law is in general false (true only for statics) whereas Lorentz’ law is always true. These two equations are the laws of electrostatics and in this chapter we will work through a number of calculations which will help us to deduct the first equation also called the Gauss’ law, and the second equation. It will be shown that Gauss’ law is equivalent to the Coulomb’s law mentioned in the next section. 4.1. Coulomb’s law The Coulomb’s law states that between two charges at rest there is a force directly proportional to the product of the charges and inversely proportional to the square of the distance between them. The force is along the straight line from one charge to the other. Coulomb’s law: 1 q1 q2 F~1 ¼ e~12 ¼ F~2 4p"0 r212

ð9Þ

F1 is the force on charge q1, e12 is the unit vector in the direction to q1 from q2, and r12 is the distance between q1 and q2. The force F2 on q2 is equal and opposite to F1. The constant of proportionality, for historical reasons, is written as 1/4p"0. In the mks unit system it is defined as exactly 107 times the speed of light squared. Since the speed of light is approximately 3
108 m/s, the constant is approximately 9
109, and the unit turns out to be newton times meter2 per coulomb2 or volt times meter per coulomb. When there are more than two charges present we must supplement Coulomb’s law with another fact of nature: the force on any charge is the vector sum of the Coulomb forces from each of the other charges. This is called ‘‘the principle of superposition’’. However, this principle cannot be applied in dealing with non-linear phenomena, since higher-order terms have to be included in order (see Part 2, section 9.1). 4.2. Electric field When applying Coulomb’s law, it is convenient to introduce the idea of an electric field. We say that the field E(1) is the force per unit of charge on q1 (due to all other charges). Dividing Eq. (9) by q1, we have, for one other charge besides q1, 1 q2 E~ð1Þ ¼ e~12 4p"0 r212

ð10Þ

322 E(1) also describes something about point (1) even if q1 was not there – assuming that all other charges keep their same positions. E(1) is the electric field at point (1). If there are many charges present, the field E at any point (1) is a sum of the contributions from each of the other charges. Each term of the sum will look like Eq. (10). Let qj be the magnitude of the jth charge, and r1j the displacement from qj to the point (1), we write E~ð1Þ ¼

X j

1 qj e~1j 4p"0 r21j

ð11Þ

Electric fields are vector functions of x, y and z (static conditions). It is precisely because E can be specified at every point in space that it is called a ‘‘field’’. A ‘‘field’’ is any physical quantity which takes on different values at different points in space (see Fig. 1). Often it is convenient to ignore the fact that charges come in packages like electrons and protons, and think of them as being spread out in a continuous smear – or in a ‘‘distribution’’, as it is called. This is acceptable as long as we are not interested in what is happening on too small scale. We describe a charge distribution by the ‘‘charge density’’,
(x, y, z). If the amount of charge in a small volume
V2 located at the point (2) in
q2, then
is defined by
q2 ¼
ð2Þ
V2

ð12Þ

To use Coulomb’s law with such a description, we replace the sums of Eq. (11) by integrals over all volumes containing charges. Then we have 1 E~ð1Þ ¼ 4p"0

Z all space


ð2Þ~ e12 dV2 r212

ð13Þ

Fig. 1. A vector field can be represented by drawing lines which are tangent to the direction of the field vector at each point, and by drawing the density of lines proportional to the magnitude of the field vector. The magnitudes and directions of the arrows indicate the values of the vector field at the points from which the arrows are drawn.

323 With the integrals we can find the fields produced by a sheet of charge, from a line of charge, from a spherical shell of charge or from any specified distribution. We shall go on to discuss the electric potential. 4.3. Electric potential The idea of electric potential is related to the work done in carrying a charge from a point to another. There is some distribution of charge, which produces an electric field. We ask about how much work it would take to carry a small charge from one place to another. The work done against the electrical forces in carrying a charge along some path is the negative of the component of the electrical force in the direction of the motion, integrated along the path. If we carry a charge from one point a to point b, Z

b

W ¼

F~  d l~ ¼ 

Z

a

b

ðFx dx þ Fy dy þ Fz dzÞ

ð14Þ

a

where F is the electrical force vector on the charge at each point, and dl is the differential vector displacement along the path (see Fig. 2). It is more interesting for our purposes to consider the work that would be done in carrying one unit of charge. Then the force on the charge is numerically the same as the electric field. Calling the work done against electrical forces in this case Wunit, we write Z

b

Wunit ¼ 

E~  d l~ ¼ 

a

Z

b

ðEx dx þ Ey dy þ Ez dzÞ

ð15Þ

a

We consider first what happens in the field due to a single charge q. Let point a be at the distance r1 from q, and point b at r2. Now we carry a different charge, which we call the ‘‘test’’ charge, and whose magnitude we choose to be one unit, from a to b. Let us start with the easiest possible path to calculate. We carry our test charge first along the arc of a circle, then along the radius, as shown in Fig. 3(a). To calculate the work done we think the following way [2]: first, there is no work done at all on the path from a to a0 . The field is radial (from Coulomb’s law), so it is at right angles to the direction of motion. Next, on the path from a0 to b, the field is in the direction of motion and varies with 1/r2. Thus, work done on the test charge in carrying it from a to b would be [2] Z

b

 a

q E~  d l~ ¼  4p"0

Z

b a0

  dr q 1 1 ¼  r2 4p"0 ra rb

ð16Þ

324

Fig. 2. The work done in carrying a charge from a to b is the negative of the integral of F dl along the path taken.

Fig. 3. The work when carrying this charge from a to b is the same along any chosen path. From Ref. [2].

Let us imagine a second possible path from a to b (Fig. 3b). It goes for a while along an arc of a circle, then radially for a while, then along an arc again, then radially, and so on. Every time we go along the circular parts, we do no work. Every time we go along the radial parts, we must just integrate 1/r2. Along the first radial stretch, we integrate from ra to ra0 , then along the next radial stretch from ra0 to ra00 , and so on. The sum of all these integrals is the same as a single integral directly from ra to rb. We get the same answer for this path that we did for the first path we tried. It is clear that we would get the same answer for any path, smooth or not. Z W unit ¼  a!b

b

E~  d l~

ð17Þ

a

Since the work done depends only on the endpoints, it can be represented as the difference between two numbers. Let  (a) stand for the work done against the field in going from a reference point P0 to a, and let  (b) be the work done in

325

Fig. 4. The work done in going along any path from a to b is the negative of the work from some point P0 to a plus the work from P0 to b.

going from P0 to b (Fig. 4). The work in going from a to b can be written as Z

b



E~  d l~ ¼ ðbÞ  ðaÞ

ð18Þ

a

Once we have chosen some arbitrary reference point, a number  is determined for any point in space:  is then a scalar field. It is a function of x, y, z. We call this scalar function the electrostatic potential at any point: Electrostatic potential: Z

P

ðPÞ ¼ 

E~  d l~

ð19Þ

P0

For convenience, we will often take the reference point at infinity, where the potential is considered zero. Then, for a single charge at the origin, the potential  is given for any point (x, y, z) using the following equation: ðx, y, zÞ ¼

q 1 4p"0 r

ð20Þ

The electric field from several charges can be written as the sum of the electric field from the first, from the second, from the third, etc. When we integrate the sum to find the potential we get a sum of integrals. Each integral is the potential from one of the charges. We conclude that the potential  from several charges is the sum of the potentials from all the individual charges. There is a superposition principle also for potentials. Using the same kind of arguments by which we found the electric field from a group of charges and for a distribution of charges, we can get the complete formulas for the potential  at a point we call (1): ð1Þ ¼

X j

1 qj 4p"0 r1j

ð21Þ

326 The potential  has physical significance: it is the potential energy which a unit charge would have if brought to the specified point in space from some reference point. ð1Þ ¼

1 4p"0

Z


ð2Þ dV2 r12

ð22Þ

4.4. Electric field and electrostatic potential The electric field vector E can be obtained easily from the electrostatic potential  by taking its derivative. Consider two points, one at x and one at (x þ dx), but both at the same y and z, and ask how much work is done in carrying a unit charge from one point to the other. The path is along the horizontal line from x to x þ dx. The work done is the difference in the potential at the points:
W ¼ ðx þ
x, y, zÞ  ðx, y, zÞ ¼

@
x @x

ð23Þ

But the work done against the field for the same path is Z
W ¼ 

E~  d l~ ¼ Ex
x

ð24Þ

We see that Ex ¼ 

@ @x

ð25Þ

Similarly, Ey ¼ 

@ @y

Ez ¼ 

@ @z

ð26a,26bÞ

or, summarising ~ E~ ¼ r

ð27Þ

This equation is the differential for Eq. (19). Any problem with specified charges can be solved by computing the potential from Eq. (21) or Eq. (22) and using Eq. (27) to get the field. Equation (27) also agrees with what was found from vector calculus, that for any scalar field  Z

b a

~   d l~ ¼ ðbÞ  ðaÞ r

ð28Þ

327 The advantage of computing  rather than E is that there is only one integral for  while there are three for E (because E is a vector). It turns out that in many practical cases it is easier to calculate  and then take the gradient to find the electrical field, than it is to evaluate the three integrals for E. 4.5. First law of electrostatics: Gauss’ law Let us consider a surface, for example a sphere with radius r, centred on a point charge q, as shown in Fig. 7. What is the flux of E out of the closed surface that contains the point charge q? If the radius of the little sphere is r, the value of E everywhere on its surface is 1 q 4p"0 r2 and is directed always normal to the surface. We find the total flux through S0 if we multiply this normal component of E by the surface area: Flux through the surface S0 1 q q ð4pr2 Þ ¼ 4p"0 r2 "0

ð29Þ

Flux ¼ (average normal component)  (surface area) a number independent of the radius of the sphere. The flux through S is also q/"0, a value independent of the shape of S so long as the charge q is inside. Let us consider the volume enclosed between the two surfaces S and S0 , that has no charge in it. Let us consider the surface shown in Fig. 5. If the E field is like a flow, the net flow out of this box should be zero. That is what we get if by the ‘‘flow’’ from this surface we mean the surface integral of the normal component of E – that is, the flux of E. On the radial faces, the normal component En of the electric field is zero. On the spherical faces, the normal component En is just the magnitude of E – minus for the smaller face and plus for the larger face. The magnitude of E decreases as 1/r2, but the surface area is proportional to r2, so the product is independent of r. The flux of E into face a is just cancelled by the flux out of face b. The total flow out of S1 is zero, which is to say that for this surface Z

E~  d a~ ¼ S1

Z

Z E da cos  ¼ S1

En da ¼ 0

ð30Þ

S1

where E is the electric field vector, da is an infinitesimal element of some surface over which we want to integrate the field, and  is the angle between E and da. If for example the surface were to lie in the x–y plane, then in magnitude

328 da ¼ dx dy. The direction of the vector da is considered perpendicular to the surface at each point on it. For an integral over a closed surface, the direction of da is that of the outward normal. In the simple case were E has a constant magnitude, and makes a constant angle with the surface normal, the integral becomes ES cos , where S is the surface area. The volume enclosed by surface S0 and S can be considered made of several volumes as shown in Fig. 5. Therefore, the flux of E into the volume V through surface S0 is cancelled by the flux of E out of the volume V from surface S. The total flow is then zero (Fig. 6). We can write our conclusions as follows [2]: Z En da ¼ 0 if q is outside S ð31Þ any surface S

Z En da ¼ any surface S

q "0

if q is inside S

ð32Þ

Now let us suppose that there are two charges, a charge q1 at one point and a charge q2 at another point. The electric field whose normal component we integrate for the flux is the field due to both charges. That is, if E1 represents the electric field that would have been produced by q1 alone, and E2 represents the electric field produced by q2 alone, the total electric field is E ¼ E1 þ E2. The flux through any closed surface S is Z

Z

Z

ðE1n þ E2n Þ da ¼ S

E1n da þ S

E2n da

ð33Þ

S

Fig. 5. The flux of E out of the surface S1 is zero.

Fig. 6. The flux of E through a spherical surface containing a point charge q is q/"0. The total flux through the volume V between the two surfaces S0 and S is zero.

329 The flux with both charges present is the flux due to a single charge plus the flux due to the other charge. If both charges are outside S, the flux through S is zero. If q1 is inside S but q2 is outside, then the first integral gives q1/"0 and the second integral gives zero. If the surface encloses both charges, each will give its contribution and we have that the flux is (q1 þ q2)/"0. The general rule is clearly that the total flux out of a closed surface is equal to the total charge inside, divided by "0. This result is an important general law of the electrostatic field, called the Gauss’ law. Gauss’ law Z En da ¼ any closed surface S

Qint "0

ð34Þ

where Qint ¼

X

qi

ð35Þ

inside S

If we describe the location of charges in terms of a charge density
, we can consider that each infinitesimal volume dV contains a ‘‘point’’ charge
dV. The sum over all charges is then the integral Z Qint ¼


dV

ð36Þ

volume inside S

From this derivation we see that Gauss’ law follows from the fact that the exponent in Coulomb’s law is exactly two. A 1/r3 field, or any 1/rn field with n 6¼ 2, would not give Gauss’ law. So the Gauss’ law is just an expression, in a different form, of the Coulomb’s law. The two are quite equivalent so long as we keep in mind the rule that the forces between charges are radial. 4.5.1. Differential form of Gauss’ law Gauss’ law can be thought of as being an integral formulation of the law of electrostatics. We can obtain a differential form (i.e., a differential equation) by using the divergence theorem. The divergence theorem states that for any wellbehaved vector field C(x) defined within a volume V surrounded by the closed surface S, the relation Gauss’ Theorem I Z ~ ~  C~ dV r C  n~ da ¼ S

V

ð37Þ

330 holds between the volume integral of the divergence of C and the surface integral of the outwardly directed normal component of C. The equation in fact can be used as the definition of the divergence. The Gauss’ theorem is demonstrated in Appendix A by applying Gauss’ law to an infinitesimal cubic surface. Gauss’ theorem tell us that the flux of E out of such cube is r  E times the volume dV of the cube. The charge inside of dV, by the definition of
, is equal to
dV, so Gauss’ law gives ~  E~ dV ¼
dV r "0

ð38Þ

~  E~ ¼
r "0

ð39Þ

which is the differential form of the Gauss’ law of electrostatics. The differential form of the Gauss’ law is the first fundamental equation of electrostatics. 4.6. Second law of electrostatics The second law of electrostatics states that the circulation of the electrical field is zero [2]. ~
E~ ¼ 0 r

ð40Þ

For any vector field the circulation around any imaginary closed curve is defined as the average tangential component of the vector multiplied by the circumference of the loop. Circulation ¼ ðaverage tangential componentÞ  ðdistance aroundÞ If we have an arbitrary curve in space and measure the circulation of the electric field around the curve, we will find that it is not, in general, zero. However, it is zero for the Coulomb field. In order to derive the second law of electrostatics we need to use and demonstrate two theorems. The demonstration of these theorems can be found in Appendix A. Theorem 1. The line integral of a scalar field Z ð2Þ  ð1Þ ¼

2

~  d l~ r

1 any curve from 1 to 2

ð41Þ

331 where the function (x,y,z) is a scalar field that assumes the value (x2, y2, z2) and (1) at point (x1, y1, z1).

(2)

at point

Theorem 2. The Stokes’ Theorem I

C~  d l~ ¼ 

Z


~
C~ r

S

 n

da

ð42Þ

where C is a vector field and S is any surface bounded by G. The cross product r
C is a vector whose components we can write by the usual rule for cross product:
 ~
C~ ¼ ry Cz  rz Cy ¼ @Cz  @Cy r x @y @z

ð43Þ


 ~
C~ ¼ rz Cx  rx Cz ¼ @Cx  @Cz r y @z @x

ð44Þ


 ~
C~ ¼ rx Cy  ry Cx ¼ @Cy  @Cx r z @x @y

ð45Þ

The combination r
C is called ‘‘the curl of C’’. After these two theorems have been demonstrated and accepted, it is straightforward to derive the second law of electrostatics: ~
E~ ¼ 0 r

ð46Þ

Let us imagine a closed line from point 1 to point 2 as shown in Fig. 2, where 1 and 2 were named a and b. Since E ¼ r  , Theorem 1 tells us that the integral of the vector r  around any closed loop must be zero: Z

2

~   d l~ ¼ ð2Þ  ð1Þ r

ð47Þ

~   d l~ ¼ ð1Þ  ð2Þ r

ð48Þ

1

Z

1 2

Therefore, I

~   d l~ ¼ r 12

Z

2 1

~   d l~þ r

Z

1

~   d l~ ¼ ð2Þ  ð1Þ þ ð1Þ  ð2Þ ¼ 0 r

2

ð49Þ

332 Using Stokes’ theorem, we can conclude that Z


 ~
r ~  da ¼ 0 r

ð50Þ

over any surface. But if the integral is zero over any surface, the integrand must be zero.
 ~
r ~ ¼ 0 r

ð51Þ

The second law of electrostatics follows directly from Coulomb’s law. 4.7. Equations of the electrostatic potential: Poisson and Laplace equations There are two laws of electrostatics: that the flux of the electric field from a volume is proportional to the charge inside – Gauss’ law, and that the circulation of the electrical field is zero – E is a gradient. These two laws are summarised in the Maxwell equations for electrostatics: ~  E~ ¼
r "0

ð52Þ

~
E~ ¼ 0 r

ð53Þ

In fact, the two equations can be combined into a single equation. From the second equation, we know at once that we can describe the field as the gradient of a scalar ~ E~ ¼ r

ð54Þ

We may completely describe any particular electric field in terms of its potential  . We obtain the differential equation that  must obey by substituting Eq. (54) into Eq. (52), to get ~ r ~ ¼ 
r "0

ð55Þ

The divergence of the gradient of  is the same as r2 operating  : 2 2 2 2 ~ r ~ ¼ r ~ ¼@ þ@ þ@  r @x2 @y2 @z2

ð56Þ

333 so we write Eq. (55) as Poisson equation r2  ¼ 


"0

ð57Þ

The operator r2 is called the Laplacian, and Eq. (57) is called the Poisson equation. In regions of space that lack a charge density, the scalar potential satisfies the Laplace equation: Laplace equation r2  ¼ 0

ð58Þ

The entire subject of electrostatics, from a mathematical point of view, is merely a study of the solutions of the single equation 57. Once  is obtained by solving the Poisson equation we can find E immediately from Eq. (54). 5. Field lines and equipotential surfaces A geometrical description of the electrostatic field will now be given. The two laws of electrostatics, one that the flux is proportional to the charge inside and the other that the electric field is the gradient of a potential, can also be represented geometrically. We shall illustrate this with two examples. First, we take the field of a point charge. Lines in the direction of the field can be drawn – lines which are always tangent to the field. These are called field lines. The lines show everywhere the direction of the electric vector. We also want to represent the magnitude of the vector. We can make the rule that the strength of the electric field will be represented by the ‘‘density’’ of the lines. By the density of the lines we mean the number of lines per unit area through a surface perpendicular to the lines. With these two rules we can have a picture of the electric field. For a point charge, the density of the lines must decrease with 1/r2. But the area of a spherical surface perpendicular to the lines at any radius r increases as r2, so if we keep the same number of lines for all distances from the charge, the density will remain in proportion to the magnitude of the field. We can guarantee that there are the same number of lines at every distance if we insist that the lines are continuous – that once a line is started from the charge, it never stops. In terms of the field lines, Gauss’ law says that lines should start at plus charges and stop at minus charges. The number which leaves a charge q must be equal to q/"0. Now we can find a similar geometrical picture for the potential  . The easiest way to represent the potential is to draw surfaces on which  is constant. We call them equipotential surfaces – surfaces of equal potential. What is the geometrical

334 relationship of the equipotential surfaces to the field lines? The electric field is the gradient of the potential. The gradient is in the direction of the most rapid change of the potential, and is therefore perpendicular to an equipotential surface. If E were not perpendicular to the surface, it would have a component in the surface. The potential would be changing in the surface, but then it would not be an equipotential. The equipotential surfaces must then be everywhere at right angles to the electric field lines. For a point charge all by itself, the equipotential surfaces are spheres centred at the charge (Fig. 7).

Fig. 7. Field lines and equipotential surfaces for a positive point charge.

The electric field lines are directed away from a positive charge and towards a negative charge (Fig. 8).

Fig. 8. Field lines and equipotential surfaces for a pair of point charges (positive charge þ 8, negative charge 8). Image created with the Consortium for Upper-Level Physics Software (CUPS) [4].

6. Electrostatic energy and electric fields from an arbitrary distribution of charges The simple case of a pair of point charges is quite rare. We shall now start to channel this dissertation towards the biological world of proteins, richly populated by charged residues.

335 6.1. Electrostatic energy of an arbitrary distribution of charges We wish now to consider the energy of electrostatic systems. The law of the energy of interaction in electrostatics is very simple. Suppose we have two charges q1 and q2 separated by the distance r12. There is some energy in the system, because a certain amount of work was required to bring the charges together. We have already calculated the work done in bringing two charges together from a large distance. It is q1 q2 4p"0 r12 We also know, from the principle of superposition, that if we have many charges present, the total force on any charge is the sum of the forces from the others. It follows, therefore, that the total energy of system of a number of charges is the sum of the terms due to the mutual interaction of each pair of charges. If qi and qj are any two of the charges and rij is the distance between them, the energy of that particular pair is qi qj 4p"0 rij

ð59Þ

The total electrostatic energy U is the sum of the energies of all possible pairs of charges: U¼

X all pairs

qi qj 4p"0 rij

ð60Þ

It is important to notice that the last equation excludes infinite self-energy terms (i ¼ j), which correspond to the work of assembling a charge into a point. Usually, these Coulombic self-energy terms are of no interest at all, because the use of point charges is precisely done to focus on the charge configurations only. Thus, i ¼ j are usually excluded from the usual definition of electrostatic potential energy for a set of point charges. It should be understood that in actual calculations with point charges the Coulombic self-energy terms should be eliminated from the electrostatic potential energy. If we have a distribution of charge specified by a charge density
, the sum of Eq. (60) is, of course, to be replaced by an integral. As usual, we consider that each volume element dV contains the element of the charge
dV. Then Eq. (60) should be written U¼

1 2

Z all space


ð1Þ
ð2Þ dV1 dV2 4p"0 r12

ð61Þ

336 Notice that the factor 1/2 was introduced because in the double integral over dV1 and dV2 we have counted all pairs of charge elements twice. Next we notice that the integral over dV2 in Eq. (61) is just the potential at (1). That is, Z


ð2Þ dV2 ¼ ð1Þ 4p"0 r12

ð62Þ

so that Eq. (61) can be written as U¼

1 2

Z
ð1Þð1Þ dV1

ð63Þ

Or, since the point (2) no longer appears, we can simply write 1 U¼ 2

Z
 dV

ð64Þ

This equation can be interpreted as follows. The potential energy of the charge
dV is the product of this charge and the potential energy at the same point. The total energy is therefore the integral over 
dV. But there is again the factor 1/2. It is still required because we are counting energies twice. The mutual energy of two charges is the charge of one times the potential at it due to the other. Or, it can be taken as the second charge times the potential at it from the first. Thus, for two-point charges we could write U ¼ q1 ð1Þ ¼ q1

q2 4p"0 r12

ð65Þ

or U ¼ q2 2 ¼ q2

q1 4p"0 r12

ð66Þ

Notice that we could also write 1 U ¼ ½q1 ð1Þ þ q2 ð2Þ 2

ð67Þ

This energy is located in space, where the electric field is. This seems reasonable because we know that when charges are accelerated they radiate electric fields. We would like to say that when light or radio waves travel from one point to another, they carry their energy with them. But there are no charges in waves. So we would like to locate the energy where the electromagnetic field is and not

337 at the charges from which it came. We thus describe the energy, not in terms of the charges, but in terms of the fields they produce. We can, in fact, show that Eq. (64) is numerically equal to (see Appendix A) "0 U¼ 2

Z

E~  E~ dV

ð68Þ

We can then interpret this formula as saying that when an electric field is present, there is located in space an energy whose density (energy per unit volume) is

u¼

"0 ~ ~ "0 E 2 EE ¼ 2 2

ð69Þ

In Appendix A we show that Eq. (68) is consistent with the laws of electrostatics. Eq. (68) is derived using the Poisson equation. PART 2 – MODELLING PROTEIN ELECTROSTATICS IN PROTEINS 7. Perspective and overview Electrostatic interactions in macromolecular systems arise from the following sources: the presence of local charges, the polarisation stemming from the non-spherical distribution of electron density around atoms, the redistribution of electrons caused by local electrical fields (electronic polarisation) and the reorientation of polar groups in the solute and solvent molecules in response to the electric field (orientation polarisation) [5]. The treatment of each of these factors has its own challenges. Can charge distributions be adequately represented by only partial atomic charges at the atom centres? Can electronic polarisation within the macromolecule be ignored or, if not, is it best approximated by point inducible dipoles (PIDs) on the atoms or bonds, or by a continuum dielectric? Can polar group reorientation be treated in any way other then by some form of simulation of motion? Can the very large and environment-specific reorientation of the solvent molecules be represented by a continuum dielectric, or is it necessary to introduce explicit solvent structure? How can each of these approximations be tested? Electrostatic calculations attempt to model these complex and often subtle effects. Electrostatic models should provide insight into the role of electrostatics in macromolecule structure and function, fit appropriate experimental data, and allow us to make predictions about macromolecular structure and function. Arguably, there is no theoretical difficulty in treating all of the electrostatic contributions listed above adequately. Molecular dynamic simulations can provide sufficient sampling of configurational space of both the macromolecules

338 and solvent structures. Charge distributions can be made as elaborate as necessary, in the form of multipole expansions and, electronic polarisation can be treated by polarisation tensors. The difficulty lies in the development of accurate parameterisations of these effects and in the time used by such calculations. Thus, current work in the field of electrostatic simulations is largely concerned with investigating the efficacy of the various possible approximations, both in reproducing experimental observations and in reducing computation time. The fundamentals of classical electrostatics, that allows us to arrive at the Poisson equation, were stated concisely in Part 1 (section 4.7). This equation is the starting point for modelling electrostatic interactions in proteins. The apparent simplicity of such equations, however, can hide the substantial difficulties involved in applying them to complex systems. The problem is particularly acute in studies of proteins and nucleic acids owing to the vast amount of structural information about these macromolecules now available. In contrast to traditional models in which proteins were treated as low-dielectric media spheres and DNA as a charge cylinder, most current questions of interest are asked at the atomic level. The question of how best to represent atomic and molecular properties within the framework of electrostatic theory poses new conceptual as well as numerical difficulties. It is common to encounter the opinion that models based on classical electrostatics have been superseded, or even invalidated, by the advent of computer simulations of atomic motions. A criticism sometimes expressed is that classical electrostatics in not valid on a microscopic scale. Thus, the theory must be applied in a physical meaningful way to the system being studied. Classical electrostatics remains a rigorous and intuitively appealing approach to a wide range of microscopic phenomena. In the following sections we will cover the equations used to model electrostatic interactions in proteins, the different models that have been used to describe the electrostatic interactions in such macromolecules, as well as one of the methods used to find the potential distribution in proteins (see Fig. 9). Such potential maps are a major source of information when trying to correlate protein structure and function and stability. One of the first steps needed to find the potential distribution on proteins is to compute the charge carried by each titratable residue as a function of pH. DelPhi is a widely used program that offers the possibility of finding the charge distribution of a protein [6]. However, this program does not, for example, consider Tyr residues as titratable residues, and at any pH value (from 1 to 14) it considers that Tyr is a neutral amino acid. Unaware of this serious mistake, a large number of scientists still use such program in order to find the charge distribution on a protein as a function of pH. Also, this and other programs, assume that, for example, all Asp residues in a protein have a pKa value of 4, all Glu residues a pKa of 4.5, all His display a pKa of 6.4, all Lys have a pKa of 10.4. As we will see in Appendices A and B, other charged sites and the local environment in the protein may shift the pKa of a

339

Fig. 9. Electrostatic potential distribution on the molecular surface of the lipase/esterase cutinase at pH 8.5, the pH optimum of this enzyme. The arrow points into the active site. Blue colour represents positive potential, white colour neutral potential and red colour negative potential.

given site from its typical value by several pH units. Therefore, it is of crucial importance to use a methodology that takes into account such effects when calculating the protonation state of each titratable residue as a function of pH. We have selected the program TITRA, written by Paulo Martel and Steffen B. Petersen [7,8] for such computations, since the computed pKa values by TITRA have been shown to be in good agreement with the experimental values, for a large number of selective protein [7].

8. Classical continuum electrostatics – the two dielectric media The approaches to model electrostatic interactions in chemical and biochemical systems, either from a purely theoretical or a computational point of view, can be divided in two broad types. The earlier models obviated the atomic level description by treating solute and solvent as homogeneous dielectric media where charges were distributed in a discrete or continuous fashion [1,9–11]. In this way, the treatment of atomic electrostatic interactions was reduced to a problem of classical continuum electrostatics (CE), based on classical electrostatics. These models were justifiable given the lack of atomic-level information about biological molecules at the time they were developed, and the limited computational facilities. With the advent of computers and high-resolution, molecular structure techniques, new methods were introduced for calculations based on simulations at the atomic level, namely Monte Carlo [12] and molecular dynamics (MM) [13,14]. These atomic-level methods became a common practice in chemical–physical studies and were later extended to a wide range of systems of chemical and biological interests [15–18]. The atomic detail of these methods leads to a neglect of CE-based methods, whose less-detailed nature is regarded as a crude approximation. However, the development of fast numerical and computational methods made it possible to achieve a quantitative level in CE

340 calculations and caused a revival in the use of CE methods [19,20]. The work presented in this chapter is primarily based on CE methods. In the most simplistic approach to model electrostatic interactions in proteins, one can assume that charges on a protein interact through a medium characterised by a single dielectric constant, and that all interactions can be described by Coulomb’s law (Part 1, Eq. (9)). However, this approach fails since the protein and the solvent have very different dielectric properties. A more realistic approach is to explicitly consider that the protein and solvent region have different dielectric constants. This means that the interactions can no longer be computed using Coulomb’s law. Instead, the Poisson equation of the system of charges and dielectrics has to be solved [21,22]. In addition, it is reasonable to assume that the protein surrounds itself with an atmosphere of counterions, as described by the Debye–Hu¨ckel theory of electrolytes [1,21,23,24]. In this case the PB equation, usually in its linear form, is solved. When the system has some symmetry, it is often possible to express the solution of either the Poisson or the Poisson–Boltzmann equations in an analytic form. A simple approximation is to consider the protein to be a sphere with the charges placed at a small distance beneath the surface and surrounded by an ionic atmosphere [25]. Although proteins are never perfectly spherical, this model was shown to give satisfactory results in many cases [26], especially when the interactions are corrected according to the solvent accessibility of the residues [27]. Although these simple spherical models do not include atomic detail to any substantial extent, they have the advantage of being analytically solvable and computationally accessible with present-day computers. The dielectric properties of a system are described by the dielectric constant which reflects the reorientation of dipoles under the local electric field. These dipoles are essentially of two types: permanent and induced. Permanent dipoles occur when the distribution of charge over neighbouring atoms is not symmetric. Typical examples are the peptide bond and the water molecule. Induced dipoles arise from electronic polarisation, i.e., from the distortion of electron clouds immersed in an electric field. In liquid water, the relative freedom of the molecules allows a high-dipolar rotation and consequently a high-dielectric constant (78.5 at 298 K). The contribution of electronic polarisation to this overall value is very small,  4. In contrast, permanent dipoles in the protein interior are virtually fixed and the orientation of the induced dipoles leads to a much smaller dielectric constant. Both experiment and theory point to 2–4 for the protein dielectric constant (see section 10.1), where electronic polarisation is considered to be the most important contribution [7]. However, previous studies on the interpretation and modelling of the pKa shifts introduced on particular titratable residues of subtilisin upon mutation of titratable residues has shown that the dielectric constant between charges in a protein could range from 45 to 120 [28]. The resulting dielectric regions can be seen as a cavity (see Fig. 10) with a low-dielectric constant "p (the protein) immersed in a continuous medium

341

Fig. 10. Continuum electrostatic model of a protein in a solution. "p, protein dielectric constant; "s, solvent dielectric constant; I, solvent ionic strength.

with a high-dielectric constant "s (the solvent). In this model, formal charges are assigned to all titratable residues, depending on pH and pKa, and bound ions can be included. In more detailed models, partial charges on all atoms can be included. The charge sources have been divided into two groups, the background charges and the titratable charges. While some of the atomic charges are independent of the protonation state of the molecule (background charges, e.g., partial charges carried by the peptide bond atoms and partial charges carried by non-titratable polar groups, such as the hydroxyl group of serine and threonine, or charges carried by metal ions such as Ca2 þ ), partial atomic charges in the vicinity of the titratable protons of ionic (titratable) residues (Asp, Glu, Lys, Arg, His, Tyr, free Cys, N- and C-terminus) are generally pH dependent, as a consequence of the protonation/deprotonation reactions. In some cases, the contribution of the background charges is not included and the formal charges of the titratable residues are taken as the only electrostatic field sources in the protein. The spatial location of the titratable moieties on the protein derives from the coordinate information obtained from X-ray or NMR studies. In the absence of such information one may have to rely on homology-based modelling. The charges on the solvent molecules, on the other hand, are assumed to be averaged out in the dielectric-based continuum description. However, the polarisation of the molecular surface reflects the orientation of the water molecules throughout the solvent. If there are ions present in the aqueous phase, their distribution will be affected by the protein charges, and in the CE model this effect is normally accounted for through the use of a counterionic charge. The counterions cannot approach the protein more than allowed by their ionic radii, which defines an ionic exclusion boundary. The counterion distribution is usually assumed to be determined simply by the electrostatic potential and the solution ionic strength, I, as in the Debye–Hu¨ckel theory of the solutes [23,24]. We shall now derive the electrostatic equations for dielectrics, namely the Poisson equation for dielectrics. This equation is the starting point for the determination of the electrostatic potential in a protein once we know the distribution of charge in the protein and its dielectric constant has been chosen.

342 We will also gain better insight on why our choice of the uniform dielectric model is a very reasonable choice to account for the electronic polarisation. 9. Equations of electrostatics for dielectrics In Part 1 we considered electrostatic interactions between charges in vacuum, but we did not include the effect that the dielectric constant of the media might have on such interactions. We therefore made no distinction between microscopic fields and macroscopic fields. Air is sufficiently tenuous that the neglect of its dielectric properties causes no great error. Our results so far are applicable there. But much of electrostatics concerns itself with charges and fields in media whose respective electric responses must be taken into account. As mentioned above, we will model the solute protein molecules and the solvent molecules as dielectric media, characterised by a particular dielectric constant. Therefore, we call them dielectrics. Before we enter such discussion, we shall now derive the equations of electrostatics when there are dielectrics. In order to arrive at these equations we need to discuss another of the peculiar properties of matter that arises under the influence of the electric field. 9.1. Macroscopic theory of dielectrics – the polarisation vector P Let us start with a simple but relevant question: Why should a field induce a dipole moment in an atom? We will here give an example to illustrate a possible mechanism. An atom has a positive charge on the nucleus, which is surrounded by negative electrons. In an electric field, the nucleus will be attracted in one direction and the electrons in the other. The orbits or wave patterns of the electrons (or whatever picture is used in quantum mechanics) will be distorted to some extent, as shown in Fig. 11. The centre of gravity of the negative charge will be displaced and will no longer coincide with the positive charge of the nucleus. If we look from a distance, such a neutral configuration is equivalent, to a first approximation, to a little dipole p. It seems reasonable that if the field is not too strong, the amount of induced dipole will be proportional to the field. That is, a small field will displace the charges a little bit and a large field will displace them further – and in proportion

Fig. 11. An atom in an electric field has its distribution of electrons displaced with respect to the nucleus. The centre of positive and negative charges no longer overlap.

343 to the field – unless the displacement gets too large. For the remainder of this chapter, it will be supposed that the dipole moment is exactly proportional to the field. Materials that show a linear response to weak fields eventually show nonlinear behaviour at high-enough field strengths, where the applied electric field no longer induces an electric polarisation proportional to the magnitude of the applied field. Under such conditions the electronic or ionic oscillators are driven to large amplitudes. The linear relation between the polarisation vector and the electric vector (described in Eq. (75)) is no longer valid, and the magnitude of the electric polarisation induced in the medium by the electric field can be expressed in a Taylor series expansion as P ¼ "0 ðE þ 2 E 2 þ 3 E 3 þ    Þ

Non-linear response

P~ ¼ "0 E~

Linear response

where  is the linear susceptibility of the material, and 2 and 3 are the secondorder and third-order non-linear optical susceptibilities, respectively. The first term "0E represents linear effects in which the polarisation of the medium is simply proportional to E. Unless the E field is very large, the coefficients of the higher-order terms are too small to allow high-power terms to influence the polarisation appreciably. Only with the availability of intense, coherent light have these higher-order terms become significant. Interestingly, the non-linear term "02E2 in the case of optical electric fields is responsible for the frequency doubling when light passes through a prism. However, we will not go into detail into this matter. We will now assume that in each atom there are charges q separated by a distance , so that q is the dipole moment per atom. If there are N atoms per unit volume, there will be a dipole moment per unit volume equal to Nq. This dipole moment per unit volume will be represented by a vector, P. Needless to say, it is in the direction of the individual dipole moments, i.e., in the direction of the charge separation  [2]: P~ ¼ Nq~

ð70Þ

In general, P will vary from place to place in the dielectric. However, at any point in the material, P is proportional to the electric field E. The constant of proportionality, which depends on the ease with which the electron is displaced, will depend on the kinds of atoms in the molecule. 9.2. Polarisation charges 9.2.1. Uniform polarisation in the dielectric Let us consider a material in which there is a certain dipole moment per unit volume. Will there be on average any charge density produced by this? Not if P is uniform. If the positive and negative charges being displaced relative to each

344 other have the same average density, the fact that they are displaced does not produce any net charge inside the volume. So, we need to look only at what happens at the surfaces. At one surface the negative charges, the electrons, have effectively moved out a distance . At the other surface they have moved in, leaving some positive charge effectively moved out a distance , as shown in Fig. 12. We will have a surface density of charge, which will be called the surface polarisation charge. This charge can be calculated as follows. If A is the area of the plate, the number of electrons that appears at the surface is the product of A and N, the number per unit volume, and the displacement , which we assume here perpendicular to the surface. The total charge is obtained by multiplying by the electronic charge qe. To get the surface density of the polarisation charge induced on the surface, we divide by A. The magnitude of the surface charge density is pol ¼ Nqe 

ð71Þ

But this is just equal to the magnitude P of the polarisation vector P, pol ¼ P

ð72Þ

The surface density of charge is equal to the polarisation inside the material. The surface charge is, of course, positive on one surface and negative on the other. Now let us assume that our dielectric also has surface charges, which we will call  charge. It should be emphasised that  pol exists only because of  charge. Like in a parallel-plate capacitor, if  charge is removed by discharging the capacitor,  pol will disappear, not by going out on the discharging wire, but by moving back into the dielectric material – by the relaxation of the polarisation inside the material [2]. We are trying to establish an analogy with the surface of the protein, where  charge can be seen as the charge carried by the titratable residues. We can now apply Gauss’ law (see Part 1) to the Gaussian surface S in Fig. 13. The electric field E in the dielectric is equal to the total surface charge density divided by "0. It is clear that  pol and  charge have opposite signs, so E¼

charge  pol "0

ð73Þ

Fig. 12. A dielectric slab in a uniform field. The positive charges displaced the distance  with respect to the negatives.

345

Fig. 13. A dielectric in-between two charged plates.

The field E0 between the surface of the dielectric and the outmost charged surface is higher than the field E. It corresponds to  charge alone. But we are concerned about the field inside the dielectric which, if the dielectric nearly fills the gap, is the field over nearly the whole volume. Using Eq. (72), we can write: E¼

charge  P "0

ð74Þ

This equation does not tell us what the electric field is unless we know what P is. Here, however, we are assuming that P depends on E – in fact, that it is proportional to E. This proportionality is usually written as P~ ¼ "0 E~

ð75Þ

The constant  (Greek ‘‘khi’’) is called the electric susceptibility of the dielectric. Then Eq. (74) becomes E¼

charge 1 "0 ð1 þ Þ

ð76Þ

which gives us the factor 1/(1 þ ) by which the field is reduced. The factor (1 þ ) is a property of the material. It is its dielectric constant. Dielectric constant k¼1þ

ð77Þ

Let us consider something a bit more complicated – the situation in which the polarisation P is not everywhere the same. We shall not get lost! This is our way to understand the Poisson equation for an inhomogeneous medium, the starting equation for finding out the electrostatic potential distribution in proteins. This way we will understand from where this so-spoken equation came about!

346 9.2.2. Non-uniform polarisation in the dielectric If the polarisation is not constant, we would expect in general to find a charge density in the volume, because more charge might come into one side of a small-volume element than leaves it on the other. How can we find out how much charge is gained or lost from a small volume? First, we shall compute how much charge moves across any imaginary surface when the material is polarised. The amount of charge that goes across a surface is just P times the surface area if the polarisation is normal to the surface. Of course, if the polarisation is tangential to the surface, no charge moves across it. This is the same line of thinking as applied in Part 1. Nothing new! Following the same arguments we have already used, it is easy to see that the charge moved across any surface element is proportional to the component of P perpendicular to the surface. In general, Eq. (72) should be written as, pol ¼ P~  n~ ¼ Pn cos 

ð78Þ

where n is the outward unitary vector normal to the surface, and  the angle between the vectors P and n [2]. If we are thinking of an imagined surface element inside the dielectric, Eq. (78) gives the charge moved across the surface but does not result in a net surface charge, because there are equal and opposite contributions from the dielectric on the two sides of the surface. The displacements of the charges can, however, result in a volume charge density. The total charge displaced out of any volume V by the polarisation is the integral of the outward normal component of P over the surface S that bounds the volume (see Fig. 14). An equal excess charge of the opposite sign is left behind. Denoting the net charge inside V by
Qpol we write Z
Qpol ¼ 

P~  n~ da S

Fig. 14. A non-uniform polarisation P can result in a net charge in the body of a dielectric.

ð79Þ

347 We can attribute
Qpol to a volume distribution of charge with the density
pol, and so Z
Qpol ¼


pol dV

ð80Þ

V

Combining the two equations yields Z
vol dV ¼ 

Z V

P~  n~ da

ð81Þ

S

We have a kind of Gauss’ theorem that relates the charge density from polarised materials to the polarisation vector P. Using Eq. (81) with the Gaussian surface of Fig. 13, the surface integral gives P
A, and the charge inside is  pol
A, so we get again that  ¼ P. Just as we did for Gauss’ law of electrostatics, we can convert Eq. (81) to a different form – using Gauss’ mathematical theorem: Z

P~  n~ da ¼ S

Z

~  P~ dV r

ð82Þ

V

we get ~  P~
pol ¼ r

ð83Þ

If there is a non-uniform polarisation, its divergence gives the net density of charge appearing in the material. We emphasise that this is a perfectly real charge density. We shall call it ‘‘polarisation charge’’ only to remind ourselves how it got there. Now we are ready to write the electrostatic equations with the dielectrics, i.e., the Poisson equation for an inhomogeneous medium, the starting equation for finding out the electrostatic potential distribution in proteins (see Fig. 9). 9.3. Poisson equation for a dielectric inhomogeneous medium Now let us combine the above result with the theory of electrostatics. The fundamental equation is (see Part 1, section 4.5) ~  E~ ¼
r "0

ð84Þ

The
here is the density of all electric charges. It is convenient to separate
into two parts. Again we call
pol the charges due to non-uniform polarisation, and call
charge all the rest, usually the charge at known places in space. In the protein

348 world, it is the charge carried by titratable residues and by background charges, as we will allude to incoming sections. Equation (84) then becomes ~  E~ ¼
charge þ
pol r "0

ð85Þ

Since (Eq. (83)) ~  P~
pol ¼ r we get ~ ~ ~  E~ ¼
charge  r  P r "0

ð86Þ

Substituting P by (Eq. (75)) P~ ¼ "0 E~ we get ~ ~ E~ þ P r "0

!

! ~ "
charge E 0 ~ E~ þ ¼r ¼ "0 "0

ð87Þ

equivalent to ~ ½E~ð1 þ Þ ¼
charge r "0

ð88Þ

or Poisson equation for dielectrics in the SI system of units
 ~ kE~ ¼
charge r "0

ð89Þ

where k ¼ 1 þ . These are the equations of electrostatics, in the SI system of units. We have not taken the dielectric ‘‘constant’’, k, out of the divergence. That is because it may not be the same everywhere. If it has everywhere the same value, it can be factored out and the equations are just those of electrostatics with the charge density
charge divided by k. In the form we have given, the equations apply to the general case where different dielectrics may be in different places in the field. Then the equations may be quite difficult to solve. As we saw in Part 1 (section 4.4, Eq. (27)), ~ ð~rÞ E~ ¼ r

ð90Þ

349 So, replacing E by r  (r) in Eq. (89) we get ~ ½kr ~ ð~rÞ ¼ 
charge r "0

ð91Þ

~ ½kr ~ ð~rÞ þ
charge ¼ 0 r "0

ð92Þ

or

There is a matter of some historical importance [2] that should be mentioned here. In the early days of electricity, the atomic mechanism of polarisation was not known and the existence of
pol was not appreciated. The charge
charge was considered to be the entire charge density. In order to write Maxwell’s equations in a simple form, a new vector D (electric displacement vector) was defined to be equal to a linear combination if E and P: D~ ¼ "0 E~ þ P~

ð93Þ

As a result, Eq. (87) was written in an apparently very simple form: ~  D~ ¼
charge r

ð94Þ

This equation can be solved if another equation is given for the relationship between D and E. When Eq. (75) holds, this relationship is D~ ¼ "0 ð1 þ ÞE~ ¼ k"0 E~

ð95Þ

This equation is usually written D~ ¼ "E~

ð96Þ

where " is still another constant for describing the dielectric property of materials. It is called the ‘‘permittivity’’. Now we see why we have "0 in our equations, it is the ‘‘permittivity of empty space’’. Evidently, " ¼ k"0 ¼ ð1 þ Þ"0

ð97Þ

One more point should be emphasised. An equation like (96) is an attempt to describe a property of matter. But matter is extremely complex, and such an equation is in fact not correct (see section 9.1). For instance, if E gets too large, then D is no longer proportional to E. For some substances, the proportionality breaks down even with relatively small fields. Also, the ‘‘constant’’ of proportionality may depend on how fast E changes with time.

350 Therefore, this kind of equation is kind of approximation, like Hook’s law. It cannot be a deep and fundamental equation. On the other hand, the fundamental equations for E, Eqs. (7) and (8), represent our deepest and most complete understanding of electrostatics. All these equations are valid in the SI system of units. The two systems of electromagnetic units in most common use today are the SI and the Gaussian systems. The SI system has the virtue of overall convenience in practical, large/scale phenomena, especially in engineering applications. The Gaussian system is more suitable for microscopic problems involving the electrodynamics of individual charged particles, etc. Usually, in review papers about electrostatics, Eq. (92) is presented in the Gaussian system of units as Poisson equation for dielectrics in the Gaussian system of units ~  ½"ð~rÞr ~ ð~rÞ þ 4p
ð~rÞ ¼ 0 r

ð98Þ

where "(r) is the dielectric constant of a given system with charge density
(r) at each point r in space. Conversion of equations and amounts between SI units and Gaussian units is discussed in detail in the appendix on Units and Dimensions by Jackson [29]. We started deducting the basic electrostatic equations in Part 1 using the SI systems of units since when they were developed this system of units was most adequate. From now on, since we will be dealing with microscopic systems, we shall use the equations in the Gaussian system of units. In the Gaussian system of units, in practical terms, corresponds to the elimination of most conversion constants. In particular, the SI conversion factor 1/4p"0 does not occur in the Coulomb equation and the factor 1/"0 of the Poisson equation is substituted by the factor 4p (Eq. (98)). Also, the difference between (relative) dielectric constant and electric permittivity (Eq. (98)) disappears. In Table 1 are listed some of the definition of the Lorentz’ force equation, "0, permittivity k and conversion factors in the Gaussian and SI systems of electromagnetic units. Table 1. Definitions of key equations and amounts in two systems of electromagnetic units. Gaussian system of units* Lorentz force equation Poisson equation in vacuum Poisson equation for dielectrics Conversion factors 1/4p"0 Dielectric constant versus permittivity "0 D *

From Refs. [2,29].

SI system of units*

E þ v/c
B Eþv
B r2  (r) ¼ 4p
(r) r2  ¼
/"0 r  ["(r) r  (r)] þ 4p
(r) ¼ 0 r  [k(r) r  (r)] þ
(r)/"0 ¼ 0 Absent in Coulomb equation Present in Coulomb equation "(r) ¼ k(r) "(r) 6¼ k(r) 1 107/4pc2 D ¼ E þ 4pP D ¼ "0E þ P D ¼ "(r)E D ¼ k(r)"0E

351 9.4. Maxwell’s equations in empty space and in dielectric media In Part 1 we considered electrostatic interactions and fields in the presence of charges, but no other ponderable media. We will now write and compare the Maxwell equations of electrostatics in vacuum and in a dielectric medium [2,29].

Maxwell’s equations in vacuum Gaussian system of units r  E0 ¼ 4p
r
E0 ¼ 0

SI system of units r  E0 ¼
/"0 r
E0 ¼ 0

Maxwell’s equations in a dielectric medium Gaussian system of units r  ("E) ¼ 4p
r
("E) ¼ 0

SI system of units r  (kE) ¼
/"0 r
(kE) ¼ 0

If the dielectric medium is not only isotropic but also uniform, we have the following equations: Maxwell’s equations in a isotropic and uniform dielectric medium Gaussian system of units r  E ¼ 4p
/" r
("E) ¼ 0

SI system of units r  E ¼
/k"0 r
(kE) ¼ 0

10. Does the uniform dielectric media account for electronic polarisability? One of the questions we asked at the beginning of this chapter was: Can electronic polarisation within the macromolecule be ignored or, if not, is it best described by PIDs on the atoms or bonds, or by a continuum dielectric? As we just saw in section 9.2, one way to account for electronic polarisability is to incorporate its effects into a dielectric constant – to assume that all charges and permanent dipoles interact with one another as if they were embedded in a medium that has a particular dielectric constant. Until recently, electronic polarisability has usually been neglected in potential energy force fields used in molecular mechanic simulations because the effects cannot be easily reduced to a set of two-body interactions. For example, if a charge on a particular atom polarises the electrons on neighbouring atoms, those electron clouds will also polarise one another, leading to a complex many body interaction. We shall see now and compare three ways to account for electronic polarisability: the already mentioned uniform dielectric model, the induced dipole model and the local dielectric-constant model. A central question concerning the use of a dielectric constant over a region of space involving many atoms asks if using a single, spatially invariant parameter that ignores the atomic nature of matter is valid. This problem will be considered after three microscopic

352 models are presented. The comparison of such models will give us a better insight on why our choice of the uniform dielectric model is a very reasonable choice to amount for the electronic polarisation. 10.1. Uniform dielectric model This model assumes that all nuclei and dipoles are kept in fixed positions and therefore, the dielectric response is determined almost entirely by electronic polarisation. It is assumed that the nuclei will not reorient in the presence of an electric field. The contribution of the elastic displacement of nuclei or of dipoles to the dielectric constant is neglected. Clearly the largest contributions will arise from the electrons with the smallest binding energies, i.e., from valency electrons. The displacement of the electrons is also considered elastic. For all frequencies  which are less that 0 (a particular resonant frequency of an electron bound in an atom) by a sufficient amount, the dielectric constant should be independent of frequency. Thus, for  < < 0 the dielectric constant " should be equal to the static dielectric constant "stat and should satisfy the Maxwell’s relation " ¼ n2 [2,30]. That is, " ¼ "stat ¼ n2

ð99Þ

should hold between the static dielectric constant and the refraction index at frequencies for  < < 0, which is 2 for most polar and non-polar organic liquids. As mentioned in section 8, both experiment and theory point to section 10 for the protein dielectric constant, where electronic polarisation is considered to be the most important contribution. 10.2. Induced dipole model The most common means of representing electronic polarisability at the molecular levels assign PIDs to atoms, bonds or groups [31–33]. In the simplest case, the induced dipole moment  (before represented as p) is presumed to be linearly related to the field by an isotropic polarisability ,  ¼ E. For a collection of charges and PIDs, the field depends on the charges and dipole moments, while each induced dipole moment in turn depends on the field it experiences from the charges and all other dipoles. This leads to a set of simultaneous linear implicit equations for the dipole moments: ~ i ¼ i 

X

~ Þij  ½E~ðqÞij þ E~ð

ð100Þ

j6¼i

where, E(q)ij and E()ij are the electric fields due to the charges and the dipoles, respectively, and the subscripts i and j run over all the charges/dipoles. This matrix equation can be solved analytically only for two-body case because, as

353 pointed out above, electronic polarisation involves a many body interaction that cannot be decomposed into a sum of pairwise interaction. Generally, an iterative procedure is used in which an initial estimate for the dipole moments is substituted into the right side of Eq. (100), giving rise to an improved estimate of the dipole moments. This procedure is repeated until a self-consistent set of fields and dipole moments results [31,33]. The PID model assumes usually that an atom has a uniform polarisability that can be represented by an induced dipole placed at the nucleus. Two difficulties with this model are: (a) atomic polarisabilities taken from experiment or theory on isolated atoms are not necessarily accurate for atoms in molecules [34]; and (b) nearby inducible dipoles can mutually increase each other’s polarisation without limit causing a polarisation catastrophe [35]. The ad-hoc exclusion of interactions between neighbouring atoms has been used to circumvent this problem [36–38]. 10.3. Local dielectric-constant model An alternate way of representing the electronic polarisability treats atoms or groups of atoms as polarisable bodies, each with its own local dielectric constant (LDC) [35,39,40]. The LDC model effectively distributes the dielectric response over the van der Waals volume occupied by the atoms’ electrons. This model make fewer approximations than the other two models, since it assumes neither that the response is uniform throughout space nor that the response arises from infinitesimal dipoles. In the simplest form of the LDC model, each atom is represented as a sphere of constant dielectric, "1. The equivalent point polarisability in the PID model, i, would be [41] ¼

3Vð"i  1Þ 4pð"i þ 2Þ

ð101Þ

where V is the volume of the sphere. Figure 15 schematically illustrates the relationship between the uniform dielectric, PID and LDC representations [35]. The LDC and PID models are equivalent for two special cases: when the atom is in a homogeneous medium or when it is exposed to a uniform field [35]. In general, however, the polarisability response involves higher-order terms then dipoles, and on atomic dimensions the errors in the PID approximation can be quite large [39,41]. The LDC model may also be extended to use non-uniform and anisotropic dielectric distributions [40]. The LDC and PID models shown in Fig. 15 appear microscopic, while the uniform dielectric model appears macroscopic. It can also be seen in this figure that the uniform dielectric and the LDC model differ in the absence of cavities between the atoms in the former and the assumption of the same dielectric constant for each atom.

354

Fig. 15. Schematic diagram illustrating three different models for molecular response to electric fields: The Uniform Dielectric Constant (UDC), Local Dielectric Constant (LDC) and Point Inducible Dipoles (PID). For models, UDC and LDC, the mean-induced dipole density per unit volume at any point P(r) is given by h(r)i/V ¼ ["(r)1]E(r)/4p, where E(r) is the Maxwell field at the point and "(r).

Since atoms in proteins are closely packed and are neither spherical nor static, it is not unreasonable to consider them as filling space. Moreover, the highfrequency dielectric constant of organic liquids depends only weakly on the identity of the solvent molecule. Thus, the use of a single dielectric constant to account for the electronic polarisation response of an entire macromolecule appears to be a very reasonable approximation. It should be emphasised that it is not clear which of the three models is actually most appropriate for applications to biological systems. The PID and LDC models are truly microscopic, but they are numerically complex and the PID model in particular entails a number of questionable assumptions. Moreover, both require knowledge of polarisabilities for a large number of atoms in different molecules and thus involve a significant number of parameters. On the other hand, experimental and theoretical evidence suggest that proteins have an average dielectric response that can be approximated with a dielectric constant of about 4 [42–45], while water has a dielectric constant of approximately 80 at room temperature. Thus, empirically, the protein system can be viewed as uniform and therefore at least two dielectric constants must be used. 11. Mobile ions – the Poisson–Boltzmann equation As we saw in Section 9.3, after having characterised a given system by its dielectric constant "(r) and charge density
(r) at each point r in space, the electrostatic potential  (r) can be determined as the solution of Poisson’s equation for an inhomogeneous medium: Poisson equation for dielectrics in the Gaussian system of units ~  ½"ð~rÞr ~ ð~rÞ þ 4p
ð~rÞ ¼ 0 r

ð102Þ

355 The presence of mobile counterions in solution can be represented implicitly. The chemical potential of each ion is assumed to be uniform throughout the solution. The entropic and electrostatic contributions to the chemical potential of an ion at any point r are kT ln C(r) and q  (r), respectively, where C(r) is the local concentration, q its charge and  (r) is the mean potential. This leads to a Boltzmann’ expression for the ion concentration [35]: Cð~rÞ ¼ Cð1Þ exp½qðrÞ=kT

ð103Þ

where C(1) is the bulk ion concentration. When incorporated into the Poisson equation, this yields the most general of the widely used CE equations, which, after linearization gives the PB equation: Linearized PB equation ~  ½"ð~rÞr ~ ð~rÞ  k02 ð~rÞ"ð~rÞð~rÞ þ 4p
p ð~rÞ ¼ 0 r

ð104Þ

where the charge density
p(r) refers only to the protein charges, and the counterionic term effect is totally contained in the second term of the equation. The parameter k0 is the so-called reciprocal Debye length that assumes the value k0 ¼



8pe2 Na I "out kB T

1=2 ð105Þ

if a point in space is accessible to other ions, and zero otherwise. We use the prime notation to distinguish it from the permittivity k(r) defined in section 9.3. Na is the Avogadro number, e is the proton charge, I the ionic strength, kB the Boltzmann constant and "s the solvent dielectric constant. The ionic strength I of the solution is defined as

I¼

1X 2 cj zj 2 j

with the sum over all ionic species in solution with charge zj, and cj their bulk concentration (ions per volume). The ion exclusion boundary which delimits the region inaccessible to the ions is usually defined as the closest distance that an ionic centre can approach the reference ion, i.e., the boundary lies at one ionic radius from the surface of the reference ion. One of the advantages of the linear form of the PB equation is the linearity of CE (classical CE methods), i.e., the superposition of the potential arising from independent charges. However, when linearity breaks down, higher-order

356 terms have to be introduced (see Eq. (106), non-linear form of the PB equation, NLPBE). ~  ½"ð~rÞr ~ ð~rÞ  k02 ð~rÞ"ð~rÞ sinh½ð~rÞe=kB TkB T=e þ 4p
p ð~rÞ ¼ 0 r sinhðxÞ ¼ x þ

x3 x5 x7 þ þ þ  3! 5! 7!

ðx2 < 1Þ

ð106Þ

There is some controversy about the validity of the NLPBE [23,24,46,47] but this will not be of concern to us here. We will consider here only the case where linearity holds (i.e., the Poisson or LPB equation). The PB equation incorporates electronic and dipole polarisation through " and ion screening through k0 , and it allows shape effects to be modelled through the spatial variation of ", k0 and
. The linearity of the PB equation (104) implies that the superposition principle is still valid (see section 9.1) and that a pairwise decomposition of the interaction of the system also holds. 12. Forces and potentials with dielectrics Let us ask now what would be the Coulombic force between two charges in a dielectric. In a medium of dielectric constant k, all forces will be reduced by this same factor. It means that the Coulombic force equation (9) and the Coulombic potential equation (20) have to be replaced by: In the SI system of units F~ ¼

1 q1 q2 e~12 ¼ F~2 4p"0 k r212

ð107Þ

q 1 4p"0 k r

ð108Þ

ðx, y, zÞ ¼

where k is the dielectric constant of the dielectric material, here assumed to be the same everywhere in the material, like we will assume for molecules such as proteins. or In the Gaussian system of units 1 q1 q2 F~1 ¼ e~12 ¼ F~2 " r212 ðx, y, zÞ ¼

1q " r

where " is the dielectric constant of the dielectric material.

ð109Þ

ð110Þ

357 13. Solving the Poisson–Boltzmann equation with continuum electrostatic models The use of CE methods at the molecular level goes back to the Born model of ionic solvation [9,23] where the Gibbs free energy of solvation is regarded as the electrostatic work (i.e.,
U) of transferring a charged sphere, the ion, from vacuum to a high-dielectric medium, the solvent. This approach arises from an analogy between the microscopic system and a familiar macroscopic model, and seems physically reasonable and intuitive. In fact of the simplicity and relative success of the Born model, it has been widely used and extended in other developments, among the most important being the Debye–Hu¨ckel theory of strong electrolyte solution [1,23,24,46,48]. The model by Debye and Hu¨ckel is essentially the Born model plus an hypothesis concerning the distribution of ions around a reference ion. The theory of electrolytes of Debye and Hu¨ckel allowed the inclusion of the effect of the ion concentration through the formulation of the LPBE (Eq. (104)). The solution of the Poisson equation for dielectrics (Eq. (102)) and the solution of the linearized PB equation (Eq. (104)) can be obtained from analytical or numerical solutions, depending on the complexity of the problem. When the system has some symmetry it is usually possible to express the solution in an analytic form. But symmetry is not a common feature of real proteins. The first introduction of asymmetry in CE models was done by Kirkwood [11] who later generalised the Debye–Hu¨ckel model to include an arbitrary number of charges inside the sphere. The model was later extended to ellipsoids [49]. With the advent of computers, the necessity for analytical solutions became less relevant, and the PB equation can now be solved numerically for molecules with arbitrary shape and charge distribution. The resulting general CE method, which has been widely applied in proteins, is represented in Fig. 10. The solute charges included in the model may vary, but current applications usually include the (partial) charges of all atoms. As in the original Born and Debye–Hu¨ckel models, only two dielectric regions are usually considered: the solvent region, with " equal to the solvent bulk macroscopic constant ( ffi 80 for water at room temperature), and the solute region, with " in the range 2–4, as mentioned in sections 8 and 10.1. The boundary between the two regions can be obtained by using one of the commonly used definitions of molecular surface [51] such as the Connolly molecular surface [51] equivalent to the Richards contact and reentrants surfaces [52]. In most cases, the surface is determined by rolling a spherical probe with the radius of a solvent molecule, e.g., water, on the surface of a molecule. The first application of CE models to proteins was done by Linderstrøm–Lang [53] who modelled a protein molecule as a sphere with its total charge smeared uniformly over the surface. Thus corresponds to assuming that charged groups (in particular titratable sites) are equally likely to lie at any position on the surface, a reasonable assumption considering that at the time the ideas on protein structure were mostly speculative. The model and the corresponding

358 solution for the electrostatic potential are very similar to the ones in the original Debye–Hu¨ckel theory. 13.1. Tanford–Kirkwood continuum model The asymmetric Kirkwood model is better suited to a protein native structure than the Linderstrøm model and can be applied directly to proteins once the charge positions on the protein are known. This was the model used by Tanford and Kirkwood in their theory of protein titration, and is shown in Fig. 16. Tanford and Kirkwood [54] calculated the electrostatic free energy for a set of discrete point charges (as opposed to the smeared charges of the Debye–Hu¨ckel theory) on a spherical surface of radius b and ion exclusion layer a (closest possible approach distance of an ion). Each pair of charges is considered to be placed at the surface of the sphere, which is assumed to form a continuous medium of low-dielectric region, surrounded by solvent with an external dielectric constant "s, and mobile counterions whose Coulombic screening is proportional to square root of ionic strength. The protein is considered as a sphere of a given radius, such that the volume of the sphere is the same as that of the protein. The formula used to compute the interaction energy between two charges is given in Refs. [3,7,8] where the Tanford–Kirkwood (TK) model is further presented. 13.2. Modified Tanford–Kirkwood model A solvent static accessibility parameter [50] for each protein charge site was incorporated by Shire et al. [27] into the TK discrete-charge electrostatic theory. This modification was introduced to overcome the uncertainty of an adjustable charge-burial parameter beneath the dielectric interface, which was required in the original treatment to fit the protein-titration curves [55,56] and to allow for the irregular protein–solvent interface. The use of this parameter has been

Fig. 16. The Tanford–Kirkwood model. The protein is considered spherical. Usually, charges (e.g., qi and qj) are considered to be at the same depth from the surface of the sphere (i.e., the same di) and separated by the experimental (e.g., crystallographic) rij distances. "p, protein dielectric constant; "s, solvent dielectric constant; I, solvent ionic strength; b, protein radius; a, protein radius plus ion exclusion boundary radius.

359 difficult to justify on physical grounds, but it has nevertheless been effective in improving experimental agreement for protein-titration curves. In this solvent-accessibility (SA) discrete-charge treatment, the fractional solvent accessibility for each group was incorporated into the calculation of the pairwise electrostatic interactive energy Uij [27]. A formalism was adopted that linearly reduced the TK pairwise interactive energy [57,58] at a dielectric interface by the charge pairs’ solvent exposure Uij0 ¼ Uij ð1  SAij Þ

ð111Þ

where SAij is the average accessibility of sites i and j. This approach is usually referred to as the Modified Tanford–Kirkwood (MTK) method. The method has additional, implicit, theoretical difficulties [35,43], but it nevertheless represents an important step in the development of methods that map protein-structural information onto the parameters of the PB equation. Another version of the original TK model can be obtained by placing some of the charges in the outer (solvent) region [59]. Different accessibilities of the various groups reflect the ability of the protein to restrict both solvent interactions and the effective sequestering of counterions. When SAij exceeds 0.95, the interaction energy between the two charge sites is small and neither markedly perturbs the other. For lower values of SAij , the protein prevents access of solvent and mobile counterions to the high local field of the charge sites. Hence, the charge sites with low solvent accessibility are allowed to interact as calculated by the TK formalism. The use of the SAij factor in reducing electrostatic free energy results in a higher-effective Coulombic shielding for solvent-exposed sites. This shielding, which is due to higher-effective LDC, has been interpreted as a local ionic strength [58]. The effects on charge-site interaction mediated by steric constraints on counterion approach are shown in Fig. 17. Figure 17A shows the field calculated for two univalent ions immersed in a uniform dielectric with no mobile ions. In panel B an ion is allowed to approach the midpoint between the two cationic sites, controlling this way the field distribution. Other geometrical restrictions are possible.

Fig. 17. Panel A shows the field calculated for two univalent ions immersed in a uniform dielectric with no mobile ions. In panel B an ion is allowed to approach the midpoint between the two cationic sites, controlling this way the field distribution.

360 13.2.1. Summary While it would appear that any attempt to portray molecular-level interactions in the context of a dielectric formalism should be treated as suspect, it is clear that a continuum model with dielectric boundaries which tries to incorporate the effects of a protein–solvent interface as well as the presence and distribution of mobile ions is preferable to a ‘‘vacuum’’ or uniform CE model. Despite the inherent assumptions and limitations of this type of formalism, the electrostatic treatment, which incorporates a static accessibility modification into the TK discrete-charge dielectric boundary theory, has provided a simple and efficient computational procedure yielding quantitative and qualitative predictions that are in agreement with experimental data. The electrostatic consequences of the peptide dipoles can be included but are usually ignored because of their weak contribution to long-range electrostatic interactions when compared to the formal charge effects. The PB equation incorporates electronic and dipole polarisation through " and ion screening through k0 , and it allows shape effects to be modelled through the spatial variation of ", k0 and
. A more realistic representation of the protein molecule, corresponding to the type of model shown in Fig. 16, usually implies a loss of any geometrical symmetry, meaning that one has to resort to numerical methods to solve the Poisson or Poisson–Boltzmann equations. The method of finite differences is the most common one in protein applications and was used in the work presented in this chapter to solve the linear form of the PB equation. This method is generally referred to as FDPB (finite-difference Poisson–Boltzmann) method. A description of this method is given in section 13.5.3. The choice of a particular CE method is usually the result of a compromise between the atomic detail of the model and the computation time. With the current computer power, a finite difference calculation for any fairly large protein should be feasible. The high detail of some of the methods does not necessarily imply a high accuracy, because a proper parameterisation is necessary (like in Molecular Mechanics, MM, methods). For example, the results differ when partial charge sets from different MM force fields are used, which suggest that a specific parameterisation for each CE method may be the more correct procedure [60]. Another criterion for the choice of a particular CE method should be the electrostatic quantities one is interested in. For example, though the visualisation of the electrostatic potential around the protein can give valuable insight on its function [61–64], its calculation is only meaningful when some molecular detail is included. More simple methods like the TK one can be used to compute electrostatic energies but their electrostatic potentials are not particularly useful, since the spherical approximation makes the method inappropriate to map atomic-level properties. The major use of CE methods is in computing the free-energy difference of processes involving charge changes, which, following Born, Debye and Hu¨ckel, is simply taken as the difference of U between the final and the initial states. The CE potential energy of each state is often called ‘‘electrostatic free energy’’.

361 A particularly important type of interaction which is not included in the CE model is the apolar interaction with the solvent, which gives rise to the so-called hydrophobic. The processes usually considered are ionisation changes such as the ones occurring in redox or titration reactions of protein molecules. The later is of major interest to us here and is discussed in the following sections. For an overview of the applications, see Refs. [26,35,65]. Although this type of model has been mostly pursued for protein applications, it has also been applied to small molecules [60,66–69]. Many times we would like to know the energy associated of a particular interaction express in different units. Appendix B provides us the useful tool that will give us such information. 13.3. Finding the protonation state of proteins prior to solving the PB equation The visualisation of the electrostatic potential distribution in and around macromolecules can give valuable insight on its function and stability. In order to find the potential distribution on the molecular surface of each protein, at different pH values, we will solve the linearized PB equation, LPBE (Eq. (104)), with the FDPB method described in section 13.5.3. Linearized Poisson–Boltzmann equation  ~  ½"ð~rÞr ~  r~   r



 8pe2 Na I "ð~rÞð~rÞ þ 4p
p ð~rÞ ¼ 0 "out ð~rÞkB T

As mentioned in previous sections, in order to solve this equation, i.e., in order to find  (r), we need to characterise the system, thus we need to know the (Fig. 18) – Dielectric constant of the solute (protein, "(r) ¼ "p) – Dielectric constant of the solvent ("out(r) ¼ "s) – Charge density of the protein at each point r in space (
p(r)) – Ionic strength of the solvent (I) – Temperature (T) The location of the charges can be given by the experimentally determined 3D structure of the protein (by X-ray diffraction or NMR). As mentioned earlier in this chapter, the protein’s dielectric constant is usually set to 4 and the solvent (water) dielectric constant is approximately 80 at room temperature. The ionic strength of the solution can be set to any value. In the present work we assume it to be 0.14 M, the physiological ionic strength. The key question now is how can we find the charge, (
p(r)), that each titratable residue will carry at a particular pH value? We will see in the following section that it is not a trivial matter to find the protonation state of each titratable residue. At a first glance, the theoretical task finding the charged state of each titratable residue seems straightforward – given the pKa of the titratable

362

Fig. 18. Solving the Poisson–Boltzmann equation.

residue (available in any biochemistry handbook, see Table 2) it would be a trivial matter to tell whether a given group is charged or not at a particular pH value. However, the situation is far more complicated because the other charged sites and the local environment in the protein may shift the pKa of a given site from its typical value by several pH units (see Tables 3 and 4). In fact, as shown below, even the usual concept of pKa becomes, to some extent, inappropriate. The TITRA program, written by Martel and Petersen, will be the tool used for the calculation of the average protonation state of the titratable sites. The details will be outlined in the following sections. The TITRA program [7,8] is a protein titration program implementing the modified TK sphere model for site–site interactions [7,54] and the Tanford–Roxby iterative mean field approximation [56] for calculation of the average protonation state of the titratable sites. In Refs. [7,8] is discussed how to model the effects of pH on proteins in order to find the charge distribution of a protein at a particular pH value. We will present and discuss some methods from the point of view of their implementation and use, introducing the software tools that we use in order to visualise electrostatic charge and potential distribution in macromolecules. 13.4. Modelling the effects of pH on proteins in order to find the charge distribution of a protein at a particular pH value Enzymes require that the catalytic residues have the appropriate protonation state in the active pH range. Thus, pH is of key importance for enzyme activity.

363 Table 2. pKa’s of titratable groups*. Group

pKa model

Amino acid -COOH Asp (COOH) Glu (COOH) His (imidazole) Amino acid -NH2 Lys ("-NH2) Arg (guanidine) Tyr (OH) Cys (free SH)

Model compounds (pK 3.6 4.0 4.5 6.4 7.8 10.4  12 9.7 9.1

)

Usual range in proteins 2–5.5 5–8 8  10 – 9–12 8–11

*Data from Refs. [71,72].

Table 3. Some highly perturbed pKa’s in proteins*. Enzyme

Residue

pKa

Lysozyme Lysozyme–glycolchitin complex Carbopeptidase A Acetoacetate decarboxylase Chymotrypsin -Lactoalbumin Rhodanese Papain

Glu35 Glu35 Glu27 Lys ("-NH2) IIe-16 ( -NH2) COOH Cys247 His159 Cys25 Asp32

6.5  8.2 7.0 5.9 10.0 7.5 6.5 3.4 3.3 1.5

Pepsin *Data from Refs. [71,72].

Usually, proteins become unstable at extreme pH values, not only because of acid- and base-catalysed reactions but also because of changes in the formal charge states of the titratable groups. Ever since these principles were recognised, there has been great interest in underlying the physical basis of the pH-dependent phenomena in proteins. It is clear that a successful structure-based model for the prediction such phenomena would contribute significantly to our understanding of enzyme mechanisms, protein stability and molecular recognition. The direct result of a pH change is a modification in the equilibrium concentrations of the protonated and deprotonated forms of the titratable sites. The most pronounced consequence of this modification is a corresponding change in the average charge of the titratable sites. Therefore, electrostatic interactions are widely believed to be the primary forces controlling pHdependent phenomena. As a consequence, the development of the PB method for computing detailed electrostatic fields in and around macromolecules has led to a burst of new activity in the theory of pH-dependent phenomena [70].

364 At a first glance, the theoretical task of explaining and predicting these pH-dependent electrostatic changes may seem straightforward – given the pKa of the titratable residue (available in any biochemistry handbook, see Table 2) it would be a trivial matter to tell whether a given group is charged or not at a particular pH value. However, the situation is far more complicated because the other charged sites and the local environment in the protein may shift the pKa of a given site from its typical value by several pH units (see Table 2, Table 3 and Fig. 19, Fig. 20). In fact, as shown below, even the usual concept of pKa becomes, to some extent, inappropriate.

Fig. 19. The titration behaviour of a residue is dependent on the local environment.

Fig. 20. The pK value of each carboxylic group in a dicarboxylic acid will reflect the electrostatic environment.

Following the nomenclature of Bashford and Karplus [73] we will use the following terms: pKmodel – is the pKa of a titrating group in a small model compound, supposedly free from the action of other titrating groups. It can be measured by NMR or other titration methods. pKint – is the pKa of a titrating site with all other groups in the protein neutralised. This quantity depends not only on the residue type but also on its location in the protein. It is pH independent. pKeff – is the pKa displayed by a given group at a given pH by the fully charged protein. This quantity changes with pH throughout the titration due to the mutual interactions between groups. pK1/2 – is the pH at which the residue is half protonated. The protonation equilibrium is fully described by the pKa of the site through the familiar Henderson–Hasselbach equation of the acid–base equilibrium [21]: pKa ¼ pH  log

f 1f

ð112Þ

365 where f is the degree of protonation, i.e., the fraction of molecules that has the site protonated. From this equation, it can be predicted that pKa is the pH value at which the site is half protonated. The pKa value measured in solution for the model compound (pKmodel), typically Gly-X-Gly, where X is the residue in question, reflects an aqueous environment for the residue, considered completely solvent accessible. However, when the titratable residue is transferred from the model compound into a specific site in the protein, new terms contribute to the energetics of its titration [7,70]: – The Born, or desolvation term, represents the free energy change in the protonation reaction du to burying the residue in the protein low dielectric. – ‘‘Background’’ term describes the free energy change coming from the interaction of the residue with the other non-titratable charges in the protein (e.g., peptide-bond dipoles and polar atoms). Together, those two terms account for the difference between pKint and pKmodel. A third energetic term comes from the interaction of the residue with all other titratable residues in the protein. The magnitude of this term is pH dependent. The pKa value resulting from the insertion of the amino acid residue into a neutral protein is usually referred to as the intrinsic pKa, pKint, and may be written as: pK int ¼ pK model þ

1

Genv ð2:3kB TÞ

ð113Þ

where

Genv is the free-energy change due to moving the residue from water into the neutral form of the protein (see Fig. 21).

Genv ¼
Genv ðAÞ 
Genv ðAHÞ

ð114Þ

If we only had one titratable residue in the protein molecule, the protonation equilibrium would be given by Eqs. (112) and (113), with pKa ¼ pKint. However, when other titratable or permanently charged sites exist, the electrostatic interaction between them needs to be considered as well. Thus, the way in which the pKint of a given site is affected by a closely positioned one depends on whether the latter is charged or not. But, conversely, the protonation state of the second group will also depend on the protonation of the first. Another way of stating the problem is to say that a protein with s titratable sites has 2s possible protonation states, and in order to characterise the protonation equilibrium of a

Fig. 21. Thermodynamic cycle to compute the effect of inserting a titratable amino acid (A) in a protein molecule (P).

366 single titratable site we have to specify the populations of each two forms at each of the 2s forms of the protein at each pH value. The probability of each protonation state can be computed [54,73–76] and this task is sometimes referred to as the multiple-state titration problem. Thus, in order to account for the additional interactions that an amino acid residue displays with other charged sites in the protein, an effective pKa is defined: pK eff ¼ pK int þ

1
Ginter 2:3kB T

ð115Þ

where
Ginter is the electrostatic contribution due to the interaction with other charged residues. Since the interaction term is a pH-dependent quantity, the pKeff itself becomes pH dependent and it can no longer be equated with the pH corresponding to half protonation. 13.5. Methods: a practical approach Overview After presenting a view of electrostatic interactions from the point of view of CE and how to model the effect of pH on proteins, it seems appropriate to present and discuss some methods from the point of view of their implementation and use, introducing the software tools that used for calculating and displaying the electrostatic energy and potential in a macromolecule. 13.5.1. Program TITRA – computing the electrostatic interaction energy between charged sites and their protonation state in order to calculate the pKa of each titratable residue The TITRA program, is a protein titration program implementing the TK sphere model for site–site interactions (see Fig. 16) [7,54] and the Tanford–Roxby iterative mean field approximation [56] for calculation of the average protonation state of the titratable sites. In Refs. [7,8] is outlined the general workings of the program. The general flow of the TITRA program is shown in Fig. 22. First, files containing atomic (AA)- or solvent (SA)-exposed area of individual atoms, pKint for each of the titratable sites and TK model parameters are read, and user options and arguments processed. A set of titratable residues and atomic locations for charge placement are selected according to default internal rules and/or information specified in user input files. Values for the site–site coupling function Wij (pairwise electrostatic interaction energy needed in order to calculate the total interaction energy between charges in the protein, described in Refs. [7,8]) are then computed, using the TK formula, for a range of distances specified by the cut-off values, and stored in a table for later use. The pairwise interaction energies Wij between charges i and j, placed at a certain depth under the surface of a sphere of radius b and ion exclusion radius

367

Fig. 22. Flowchart describing the steps within the program TITRA.

a, and at a distance rij from each other (see Fig. 16), are calculated assuming the TK [11,54] model of a protein. As shown in Fig. 16, the positions of m titratable sites are indicated by points. There are interactions between only those points which bear a charge. If they bear charges, these will be point charges embedded in a spherical cavity of dielectric constant "s. The external dielectric constant is "p. The fractional charge of each site is computed at the starting pH value, using the pKint value for that group and Eq. (112). The total electrostatic potential at each group, generated by the remaining groups, is determined using the previously calculated partial charges and Wij coupling terms. Because TITRA uses the MTK model, the interaction terms Wij are further corrected with a scaling factor [27],   Wij0 ¼ Wij 1  SAij

ð116Þ

The solvent accessibility values will be computed by the ACC_RUN program described in the next section. In TITRA there is currently no provision to calculate pKint values from the pKmodel values. Instead, the former have to be provided beforehand when this is

368 found necessary or when there are experimental data indicating a large shift from pKmodel for a particular residue which cannot be explained through interaction with other residues. The user is allowed to edit the pKmodel values for individual residues in one of the TITRA input files. To set pKint equal to pKmodel corresponds to assuming that the titratable sites’ environment is not significantly changed upon inclusion in the protein, which may be a reasonable assumption for solvent-exposed sites. A number of user options may change details of the above-sketched procedure. Energy values may be read from a pre-computed table stored in disk, or a set of site–site coupling constants Wij may be read from a file. The format of the pKint input file allows the values of selected residues to be pre-set or fixed at given pKa or charge values (fixing the charge value of a site creates a background charge, with a pH-independent value) [7]. 13.5.2. Program ACC_RUN ACC_RUN is a simple program that computes contact solvent accessibilities [52]. Each atom is modelled as a collection of evenly distributed points on the surface of a sphere. The atom is considered solvent accessible if a water-probe tangent to one or more of these points does not overlap any other protein atoms (the water probe is usually modelled as a 1.4 A˚ sphere). The solvent accessibility is calculated from the fraction of exposed dots on the surface of each atomic sphere. The program takes as input a PDB file and a water-probe radius value (default value 1.4 A˚), and output solvent-accessibility files for all individual atoms as well as the side chains. The side-chain file contains accessibilities for all side chains, normalised with the standard areas for tripeptides Gly-X-Gly in extended conformation [27], while the atomic accessibility file contains absolute solventexposed atomic areas in A˚2. The program is written in C and runs under SGI IRIX and Linux. The two accessibility files produced by ACC_RUN are required as input for a TITRA calculation. 13.5.3. Solution of the Poisson–Boltzmann equation using finite-difference grid method The PB equation appears to be a good model since it accounts for both the effects of dielectrics and ionic strength. Unfortunately, this equation can be solved analytically only for systems with simple dielectric boundary shapes, such as spheres and planes. In particular, the linear PB solution for a single point charge qi placed in the origin of the coordinate system has the Debye–Hu¨ckel form i ¼

qi expðk0 rÞ "r

ð117Þ

Most molecules of interest have complex shapes, and their conformations may have a significant effect on the resulting electrostatic properties. The alternative to analytical solutions is to use numerical techniques to find an approximate solution.

369 Three principal methods have been developed to the point where they can be used to attempt to calculate experimental data. Solutions of the PB equation using finitedifference grid methods treat the protein and solvent as two dielectric continuums but, unlike older TK implementations, allow for the detailed shape of the protein surface. A semi-solvent continuum approach places induced dipoles on a grid for the solvent and on atomic centres within the protein and is therefore termed the protein dipoles/Langevin dipoles (PDLD) method. Free energy perturbation calculations allow some experimental electrostatic quantities to be derived from Molecular Dynamics (MD)andMonte-Carlo (MC)simulations usingexplicit solventmolecule descriptions but usually ignoring electronic polarisation. These methods have been extensively reviewed during the past couple of years. Harvey [43] has provided a full, thoughtful and relatively objective survey of methods together with a description of the background basic theory. Davis and McCammon [77] have given a useful outline of the theoretical basis of each of the contemporary methods. Sharp and Honig [47] have given an excellent if somewhat partisan review, focussing on the FDPB method. Warshel and A˚qvist [78] have championed the PDLD approach and discussed the relationship between the results of calculations and basic electrostatic concepts. Beveridge and Dicapus [79] have reviewed the use of free-energy perturbation calculations. Bashford [80] has outlined the methods and tests that have been made with model systems, as well as the state of the art for their applications to macromolecules. 13.5.3.1. Finite difference approximation – the program DelPhi DelPhi is a software package that calculates the electrostatic potential in and around macromolecules, using a finite-difference solution to the non-linear PB equation [81]. It was developed by Barry Honig and co-workers at Columbia University [42,82–84] and marketed by Biosym Technologies, Inc. [85]. Typical uses for DelPhi include calculating electrostatic potential in and around a protein and displaying isopotential contour maps to gain qualitative information on protein–substrate interactions, determining the effects of site-directed mutagenesis on the pKas of important residues, on binding energies and on catalytic rates. The FDPB method involves mapping the molecule onto a three-dimensional cubical grid, with spacing between grid points of size h (as shown in Fig. 23 in two dimensions). The interior of the solvent-accessible surface is assigned one dielectric, and the exterior is assigned another. A molecule such as a protein has a low-dielectric constant since its dipolar groups are frozen into a hydrogenbonded lattice and cannot reorient in an external electrostatic field. A value near 2 measures its electronic-polarisation response while a value near 4 includes some additional contributions from dipole reorientation. Water, on the other hand, has a very high-dielectric constant (78.5 at 298 K) since its dipoles reorient more freely. Therefore, a protein molecule in aqueous solution yields a system with two very different dielectric media. The PB equation must be satisfied everywhere in the grid, and in particular, at each grid point. If the cube of side h surrounding a grid point is considered, as

370

Fig. 23. Two-dimensional mapping of the molecule on a DelPhi grid. From Ref. [6].

Fig. 24. Cube of side h surrounding the grid point. The black circles are the six surrounding points. Note that associated with each grid point i is a charge qi, a modified Debye–Hu¨ckel reciprocal length k00 , and a potential  1. The dielectric values, however, are associated with the midpoints of the lines between the grid points. The modified Debye–Hu¨ckel parameter, k0 ¼ (")1/2k, is defined for convenience of implementation of the finite-difference formulas. From Ref. [6].

shown in Fig. 24, the derivatives in the equation can be replaced by finite differences over this cube, and the continuous functions  ,
and " can be replaced by their values at the grid points [83]. Using this strategy, a finite-difference formula can be obtained in which the potential at any grid point depends on the charge at the grid point, the value 00 at the grid point, the grid spacing h, and the potential and dielectric values of the six neighbouring grid points [83],
P

 "i i þ 4pq0 =h    0 ¼
P 2 6 0 i¼1 "i þ 0 h 6 i¼1

ð118Þ

where  0 is the potential at each node of the cubic grid with spacing h,  i the potential at each of the six nearest neighbours, "i the dielectric constant at the midpoint of  0 and  i, q0 the charge assigned to the grid node.

371

Fig. 25. Flowchart representing the steps necessary for displaying electrostatic potential maps onto the protein molecular surface. The files needed for the different programs are: protein.acc (side-chain static-accessibility file); protein.atom.acc (atomic accessibility file); protein.tcv (titration curve data); protein.pks (information about each titratable site: Residue_name, Residue_number, pH, pKint, pKeff, Partial_charge) [8]; protein.crg (information about each titratable site: pH, Residue_name, Residue_number, Partial_charge); protein.pdb (coordinates of the residues); delphi.param (solvent, solute and grid parameters); protein.grd (potential map file); protein.frc (optional file: lists the coordinates, charges, potential and field components for a specified set of atoms).

372 13.5.3.2. Program DelPhi – input and output files The input files for DelPhi (see Fig. 25) include a coordinate file (in PDB format), an atomic radii file, an atomic charge file and a parameter file containing various parameters and options that control the program’s behaviour. These include the grid step, its extent and placement relative to the protein molecule, as well as the ionic strength, the dielectric constants for protein and solvent, as well as the maximum number of iterations and boundary conditions. Specification of both charged and non-charged atoms is required because both contribute to the overall protein surface and, in particular, to the definition of the protein–solvent interface. The program outputs a grid file containing potentials for every grid point and a file containing the potential and electrostatic field vectors at the location of each atom in the system. The grid file can be read by Biosym’s viewer program, InsightII [86] and colour-coded equipotential surfaces can be displayed at defined kT/e values. DelPhi charge files can be generated by another application, e.g., TITRA, to allow the display of equipotential surfaces. DelPhi calculation times depend primarily on the total number of grid nodes, but also on the chosen ionic strength and number of point charges of the system, the first two having an effect upon the rate of convergence of the iteration. Setting up the molecular surface dielectric boundary takes very little time, due to the use of an efficient algorithm [87]. The DelPhi computations are not time consuming when compared with, e.g., protein molecular dynamics calculation.

13.5.4. Program Grasp The program Grasp [88] was developed as a consequence of the need for visualising electrostatic potentials at surfaces, in particular, the surface of biological molecules, where the surface is modelled as a solid surface. The program DelPhi, which calculates electrostatic potentials from the PB equation, can be used to obtain quantitative numbers for a variety of biochemical phenomena but visualisation has been limited to qualitative isopotential contouring. The limitation of this approach is that the contours do not highlight local topology or shape. They often extend significant distances away from the surface of the molecule while one expect most of the interactions to be close to the molecule, in fact at the surface of molecules. Whereas DelPhi can give detailed information about the molecular electrostatic signature or shape, it does not permit concurrent viewing of the electrostatic potential and the molecular surface. On the other hand, Grasp allows for the production of a solid surface, colour coded with the local electrostatic potential. Grasp has proven to be an ideal tool for the study of the electrostatics of many families of enzymes, where the details of the molecular surface can be viewed simultaneously with the electrostatic potential features. The flowchart representing the necessary steps for displaying the electrostatic potential maps onto the molecular surface of a protein is displayed in Fig. 25.

373 14. Applications In section 1 we referred to why is it so relevant to model electrostatic interactions and to obtain the electrostatic potential distribution on each atom of a macromolecule or displayed on its molecular surface. All long-range intermolecular forces are thought to be essentially electrostatic in origin. Therefore, the molecular understanding of the initial interaction between a protein and, e.g., its substrate or inhibitor is essentially an understanding of the role of electrostatics in intermolecular interactions, such as molecular recognition. Electrostatic interactions are widely believed to be the primary factors upon which the pH-dependent phenomena are dependent. The protonation state of the catalytic residues and of the residue nearby the active site may influence the charge and potential distribution in the catalytic/binding region of the protein. If a substrate and/or the product(s) of the reaction also carry charge, its strong or weak interaction with the active-site region of the enzyme will depend on the charge/potential of this same region. Ever since these principles were recognised, there has been great interest in uncovering the physical basis of the pH-dependent phenomena in proteins (see Ref. [63] and references therein). The role of electrostatic interaction on enzyme activity, specificity, stability and ion or ligand binding has been partially unravelled by several previous studies (see Ref. [63] and references therein). It is clear that a successful structure-based model for the prediction of such phenomena would contribute significantly to our understanding of enzyme mechanisms, protein stability and molecular recognition. In the following section we will address several applications of electrostaticsderived knowledge and its use. 14.1. Interpretation of electrostatic potential maps displayed on the molecular surface of an enzyme Since the charge carried by a protein will be pH dependent, the electrostatic interaction between the residues in the protein and the electrostatic potential at each location of the protein will be modulated by pH. Enzymatic activity is also pH dependent as well as protein-structural stability. Therefore, it is relevant to correlate the protein’s activity, with its structural stability and electrostatic energy/potential as a function of pH. All over the surface of the protein the effects of titration can be observed. Regions displaying positive potential at pH 4 have become neutral or even carry negative potential at later pH values. This is the result of the titration of the different titratable residues in the protein. As pH goes from acidic to alkaline, the total charge of the protein goes from positive to negative due to the tiltration of C-terminus, Glu, Asp, His, N-terminus, Tyr and Cys (when free), Arg and Lys. That is the reason for the different potential distribution on the molecular surface of a protein as a function of pH.

374 The electrostatic potential distribution as a function of pH on the molecular surface of Fusarium solani pisi cutinase is displayed in Fig. 26. We can observe that when changing pH to very acidic conditions we observe an increasing polarisation of the active-site pocket, which present a more and more positive potential, and when changing pH to more basic conditions the active-site potential becomes more and more negative. The same is observed on the activesite flanking regions. We can also observe that the molecular surface at the bottom of the active-site cleft (pointed by an arrow in Fig. 26) still displays a positive potential at pH 6 due to the presence of the fully or partially positively charged catalytic His residue (blue colour at the edge of the arrow, Fig. 26). It can also be observed that as early as pH 4, a negatively charged residue located just above the arrow (Glu44, pKmodel 4.5) is contributing to a region of negative potential. In the pH ranges where highest activity is reported for F. solani pisi cutinase (pH around 8.5 against tributyrin), the molecular surface at the activesite entrance is displaying negative potential (see map at pH 8.5, Fig. 26). At this pH the catalytic His residue has titrated, therefore it has lost its positive. From pH 8.5 onwards the potential in the active-site environment becomes more and more negative due to the deprotonation of the Tyr residues that are in or very close to the active cleft.

Fig. 26. Electrostatic potential maps displayed on the molecular surface of Fusarium solani pisi cutinase at different pH values: 4, 6, 8.5 and 10. Blue colour represents positive potential and red colour negative potential. The arrow points to the catalytic cleft. The units of the potential energy values are kT/e.

375 The units of the potential-energy values reported in any electrostatic potential maps distribution in the present thesis are kT/e. What is the physical meaning of the kT/e energy levels, where k is the Boltzmann constant? The significance of the  kT/e energy levels comes from the fact that the average thermal energy of the particles in a solvent at temperature T is  kT. Since the electrostatic energy W of a particle experiencing an electrostatic potential  is given by W ¼ q  , the regions where the potential energy level is, in absolute value, above kT are those where the electrostatic energy of charged particle is above the thermal noise, and therefore ready to be electrostatically driven by the action of a protein field. 14.2. Thermal stability, activity and Coulombic electrostatic energy The Coulombic electrostatic energy in kT units of the whole molecule as a function of pH computed by DelPhi as a function of pH for F. solani pisi cutinase is shown in Fig. 27 (using the charge file predicted by TITRA at a particular pH value, as described in the paper by Petersen et al. [63]). The dielectric constant of the protein was set to 4. The shape of the displayed electrostatic energy versus pH profiles in Fig. 27 resembles the Tm versus pH profiles displayed in Figs. 28 and 29. In both the figures there is plateau from pH 6.0 to 8.5 for native cutinase. Electrostatic interactions are thought to have a critical role in defining the thermostability of the studied enzymes. A decrease in Tm is correlated with a reduction of the electrostatic energy. Figure 27 shows that there is a rapid decrease of the electrostatic energy after pH 10, and this is correlated with the titration of the nearby six tyrosine residues present in cutinase. Their deprotonation renders them negatively charged, giving rise to electrostatic repulsion. Also, usually above pH 10 residues like Lys loose their capacity of stabilising the deprotonated/negatively charged Tyr residues since they start titrating and therefore loosing their positive charge. Later on the same happens for Arg residues (pKmodel around 12). From Figs. 27 and 29(b) it can be seen that loss of enzymatic activity above pH 9.5–10 is correlated with loss of structural thermal stability and with an unfavourable increase of the Coulombic energy. The Coulombic electrostatic energy in kT units of the native cutinase at a particular pH value plotted as a function of Tm determined at the same pH value (determined by differential-scanning calorimetry, DSC, by Petersen et al. [63,89]) is displayed in Fig. 30. It can be observed that a positive, thus destabilising, electrostatic energy correlates with the lowest Tm observed (at pH 3). On the other hand, the most negative values of electrostatic energy (thus contributing to the proteins’ structural stability) is correlated with the highest Tm values observed for native cutinase (see Fig. 30). The small variations of the electrostatic energy observed in the pH range from 5.2 to 10.0 correspond to a plateau region of the Tm versus pH plot in the same pH range.

376

Fig. 27. Coulombic electrostatic energy in kT units of the whole molecule has a function of pH computed by DelPhi as a function of pH for native Fusarium solani pisi cutinase (using the charge file predicted by TITRA at a particular pH value as described above). The dielectric constant of the protein was set to 4.

Fig. 28. Changes in thermal stability for native cutinase investigated by CD spectroscopy at pH 4.0, 6.0, 8.5 and 10.0 at a scan rate of 90 C/h [63].

The authors are fully aware that electrostatic interactions alone cannot fully explain the thermal stability of the protein as a function of pH. The hydrogen-bond network as well as the hydrophobic interactions will definitely play an important role for protein stability. However, the correlation observed in Fig. 30 is very significant. We believe that it can be used to predict what changes in Tm the introduction or the removal of salt bridges in native cutinase would

377

Fig. 29. (a) pH-thermal stability profile of Fusarium solani pisi cutinase. Tm determined by differential scanning calorimetry, DSC. (b) pH-activity profile of F. solani pisi cutinase determined by the pH-Stat methodology [63].

bring. However, the Coulombic calculation by DelPhi only reflects the energy necessary to bring the charges present from infinity to their location on the protein using the protein’s dielectric constant. The solvent effects were included only during the charge-file calculation by TITRA, as described in section 13.5.1 (implicit solvent effect).

378

Fig. 30. The Coulombic electrostatic energy in kT units of the native cutinase at a particular pH value plotted as a function of Tm determined at the same pH value determined by differential scanning calorimetry, DSC.

14.3. Engineering the pH optimum of the triglyceride lipase cutinase from F. solani pisi The optimisation of enzymes for particular purposes or conditions remains an important target in virtually all protein-engineering endeavours. In NevesPetersen et al. [63] we have presented a successful strategy for altering the pH optimum of the triglyceride lipase cutinase from F. solani pisi. The computed electrostatic pH-dependent potentials in the active-site environment are correlated with the experimentally observed enzymatic activities. At pH optimum a distinct negative potential is present in all lipases and esterases that we studied so far [62]. This has prompted us to propose the ‘‘The Electrostatic Catapult Model’’ as a model for product release after cleavage of the ester bond [62,63]. The origin of the negative potential is associated with the titration status of specific residues in the vicinity of the active-site cleft. In the case of cutinase, the role of Glu44 was systematically investigated by mutations into Ala and Lys. All charge mutants displayed altered titration behaviour of active-site electrostatic potentials. Typically, the removal of the residue Glu44 pushes the onset of the active-site negative potential towards more alkaline conditions. We therefore predicted more alkaline pH optima, and this was indeed the experimentally observed. The experimentally observed pH optimum of E44K mutant was 10.5 when compared to 8.5 for native cutinase. In Fig. 31 is displayed the effect of carrying out a charge mutation (Glu into Ala or Lys) in the active site of the F. solani pisi cutinase on the electrostatic potential distribution map displayed on the molecular surface of the enzymes. It can clearly be seen that when the glutamic-acid residue (Glu44) is replaced by an

379

Fig. 31. Electrostatic potential maps displayed on the molecular surface of native, E44A and E44K mutant cutinases from Fusarium solani pisi at pH value 8.5. The black arrow indicates location of the active site Ser120O. The green arrow indicates the location of Glu44 on the molecular surface of native cutinase. Blue colour represents positive potential, white colour neutral potential and red colour negative potential. The potential scale used ranged from 5kT/e to þ 5kT/e.

Ala or a Lys, the negative potential observed at the bottom of native cutinase at pH 8.5 is not present at the bottom of the active site of the mutant enzymes. Acknowledgements M.T.N.P. acknowledges the support from the Danish Research Agency, Novo Nordisk A/S, and Novozymes A/S. Appendix A The goal of this appendix is to derive three theorems needed to derive the two laws of electrostatics as presented in Part 1. On the other hand, we will derive Eq. (68) for the energy in the electrostatic field (from Ref. [2]). Theorem 1 – The line integral of r Z

2

ð2Þ  ð1Þ ¼

~ Þ  d l~ ðr

1

Theorem 2 – Gauss’ Theorem Z

C~  n~ da ¼

Z

S

~  C~ dV r V

Theorem 3 – Stokes’ Theorem I

C~  d l~ ¼ 

Z

~
C~Þn da ðr S

380 Equation (3.75) "0 U¼ 2

Z

E~  E~ dV

Vector integral calculus It is relevant to get some understanding of the significance of the derivatives of fields. This way, we will have a better feeling for what a vector-field equation means. We will try to find the meanings of the divergence and curl operations. The interpretation of these quantities is best done in terms of certain vector integrals and equations relating such integrals. We will derive these integral formulas. The equations we shall present in here are really mathematical theorems useful for interpreting the meaning and the content of the divergence and the curl. These mathematical theorems are, for the theory of fields, what the theorem of the conservation of energy is to the mechanics of particles. General theorems like these are important for a deeper understanding of physics. We find them delightful, enlightening! The line integral of r We will take up first an integral involving the gradient. The relation contains a very simple idea: since the gradient represents the rate of change of a field quantity, if we integrate that rate of change, we should get the total change. Suppose we have a scalar field (x, y, z). At any two points (1) and (2), the function will have the values (1) and (2), respectively. We shall use the convenient notation, in which (2) represents the point (x2, y2, z2) and (2) means the same as (x2, y2, z2). If G (gamma) is any curve joining (1) and (2), as in Fig. 32, the following relation is true: Theorem 1 Z

2

ð2Þ  ð1Þ ¼

~ Þ  d l~ ðr

ðA1Þ

1

Fig. 32. The terms used in Eq. (A1). The vector r

is evaluated at the line element dl.

381 The integral is a line integral, from (1) to (2) along the curve , of the dot product of r – a vector – with dl – another vector which is an infinitesimal element of the curve  [directed away from (1) and towards (2)]. First, we should review what we mean by a line integral. Consider a scalar function f(x, y, z), and the curve  joining two points (1) and (2). We shall mark off the curve at a number of points and join these points by straight-line segments, as shown in Fig. 33. Each segment has the length
si, where i is an index that runs 1, 2, 3, . . .. By the line integral Z

ð2Þ

f dl

ðA2Þ

ð1Þ along 

it is understood the limit of the sum X

fi
li

where fi is the value of the function at the ith segment. The limiting value is what the sum approaches as we add more and more segments. The integral in Eq. (A1) has the same meaning, although it looks different. Instead of f, we have another scalar – the component of r in the direction of
l. If we write (r )t for this tangential component, it is clear that

 ~ ~
l ¼ r 
l~ r

ðA3Þ

t

The integral in Eq. (A1) means the sum of such terms The component of r along a small displacement
s is the rate of change of in the direction of
l. Consider the line segment
l from (1) to point a in Fig. 33. According to the definition,


1


 ~ ¼ ðaÞ  ð1Þ ¼ r 
l~1 1

Fig. 33. The line integral is the limit of a sum.

ðA4Þ

382 Also, we have ~ Þ 
l~2 ðbÞ  ðaÞ ¼ ðr

ðA5Þ

Where, of course, (r )1 means the gradient evaluated at the segment
l1 and (r )2 the gradient evaluated at the segment
l2. If we add Eqs. (A4) and (A5), we get

 ~ ~ ðbÞ  ð1Þ ¼ r 
l~1 þ r 
l~2 1

2

ðA6Þ

We can see that if we keep adding such terms, we get the result ð2Þ  ð1Þ ¼

X
 ~ r 
l~i

ðA7Þ

i

The left-hand side does not depend on how we choose our intervals – if (1) and (2) are kept always the same – so we can take the limit of the right-handed side. We have therefore proved Eq. (A1). This theorem is correct for any curve from (1) to (2) and is independent on how the points a, b, c, . . . are chosen. Gauss’ theorem – the flux from a cube The flux of a vector field Before we consider our next integral theorem – a theorem about the divergence – we would like to generalise a certain idea which has an easily understood physical significance in the case of, e.g., heat flow, to the case where the vector does not represent the flux of anything. For instance, it might be the electric field. Suppose that we have some closed surface S which enclosed the volume V and we would like to find how much heat is flowing out of this surface S. For this purpose we have to define the vector h, which represents the heat that flows through a unit area in a unit time. We shall write da for the area of an element of the surface. The symbol stands for a two-dimensional differential. If, of course, the area happened to be in the xy-plane we would have da ¼ dx dy. Later we shall have integrals over volume and for these it is convenient to consider a differential volume that is a little cube. So, when we write dV we mean dV ¼ dx dy dz. The heat flow out through the surface element da is the area times the component of h perpendicular to da. Defining n as a unit vector pointing outwards at right angles to the surface, the component of h that we want is hn ¼ h~  n~

ðA8Þ

The heat flow through da is then h~  n~ da

ðA9Þ

383 To get the total heat flow through any surface we sum the contributions from all the elements of the surface. In other words, we integrate Eq. (A9) over the whole surface: Total heat flow through S: Z h~  n~ da S

If the vector represents the electric field, E, we can certainly still integrate the normal component of the electric field over an area if we wish. Although it is not the flow of anything, we still call it the ‘‘flux’’. We say Flux of E through the surface S: Z E~  n~ da S

We generalise the word ‘‘flux’’ to mean the ‘‘surface integral of the normal component’’ of a vector. The flux from a cube We now take the special case of a small cube and find an interesting formula for the flux out of it. Consider a cube whose edges are lined up with the axes as in Fig. 34. Let us suppose that the coordinates of the corner nearest the origin are x, y, z. Let
x be the length of the cube in the x direction,
y be the length in the y direction, and
z be the length in the z direction. We wish to find the flux of a vector field C through the surface of the cube. Z  Cx dy dz

Fig. 34. Computation of the flux of C out of a small cube.

384 We shall do this by making a sum of the fluxes through each of the six faces. First, consider the face marked 1 in the figure. The flux outward on this face is the negative of the x component of C, integrated over the area of the face. This flux is Since we are considering a small cube, we can approximate this integral by the value of Cx at the centre of the face – which we call the point (1) – multiplied by the area of the face,
y
z: Flux out of 1: Cx ð1Þ
y
z Similarly, for the flux out of face 2, we write Flux out of 2: Cx ð2Þ ¼ Cx ð2Þ
y
z Now Cx(1) and Cx(2) are, in general, slightly different. If
x is small enough, we can write Cx ð2Þ ¼ Cx ð1Þ þ

@Cx
x @x

There are, of course, more terms, but they will involve (
x)2 and higher powers, and so will be negligible if we consider only the limit of small
x. So, the flux through face 2 is Flux out of 2:   @Cx
x
y
z Cx ð1Þ þ @x Summing the fluxes for faces 1 and 2, we get Flux out of 1 and 2: @Cx
x
y
z @x The derivative should really be evaluated at the centre of face 1, that is, at [x, y þ (
y/2), z þ (
z/2)]. But in the limit of an infinitesimal cube, we make a negligible error if we calculate it at the corner (x, y, z).

385 Applying the same reasoning to each of the other pairs of faces, we have Flux out of 3 and 4: @Cy
x
y
z @y and Flux out of 5 and 6: @Cz
x
y
z @z The total flux through all the faces is the sum of these terms. We find that Z cube

  @Cx @Cy @Cz ~ þ þ
x
y
z C  n~ da ¼ @x @y @z

and the sum of the derivatives in just r  C. Also,
x
y
z ¼
V, the volume of the cube. So we can say that for an infinitesimal cube Z


 ~  C~
V C~  n~ da ¼ r surface

We have shown that the outward flux from the surface of an infinitesimal cube is equal to the divergence of the vector multiplied by the volume of the cube. We now see the ‘‘meaning’’ of the divergence of a vector. The divergence of a vector at the point P is the flux – outgoing ‘‘flow’’ of E – per unit volume, in the neighbourhood of P. We have connected the divergence of C to the flux of C out of each infinitesimal volume. For any finite volume we can use the fact we proved above – that the total flux from a volume is the sum of the fluxes out of each part. We can, that is, integrate the divergence over the entire volume. This gives us the theorem that the integral of the normal component of any vector over any closed surface can also be written as the integral of the divergence of the vector over the volume enclosed by the surface. This theorem is named after Gauss. Gauss’ Theorem Z

C~  n~ da ¼ S

Z

~  C~ dV r V

386 Stokes’ theorem – the circulation around a square The circulation of a vector field We wish now to look at the curl in somewhat the same way we looked at the divergence. We obtained Gauss’ theorem by considering the integral over a surface, although it was not obvious at the beginning that we were going to be dealing with the divergence. It was not at all clear that this would be the result. And with an equal apparent lack of justification, we shall calculate something else about a vector and show that it is related to the curl. This time we calculate what is called the circulation of a vector field. If C is any vector field, we take its component along a curved line and take the integral of this component all the way around a complete loop. The integral is called the circulation of the vector field around the loop. We have already considered a line integral r earlier in this appendix. Now we do the same kind of thinking for any vector field C. Let  be any closed loop in space. The line integral of the tangential component of C around the loop is written as I

I

C~  d l~

Ct dl ¼ 



We should note that the integral is taken all the way around, not from one point to another as we did before. This integral is called the circulation of the vector field around the curve . Playing with the same kind of rational we did for the flux, we can show that the circulation around a loop is the sum of the circulations around two partial loops. Suppose we break up our curve into two loops by joining two points (1) and (2) on the original curve by some line that cuts across as shown in Fig. 35. There are now two loops, 1 and 2. 1 is made up of a, which is that part of the original curve to the left of (1) and (2), plus ab, the ‘‘short cut’’. 2 is made up of the rest of the original curve plus the short cut. The circulation around 1 is the sum of an integral along a and along ab. Similarly, the circulation around 2 is the sum of two parts, one along b and the other along ab. The integral along ab will have, for the curve 2, the opposite sign from what it has for 1, because the direction of travel is opposite – we must

Fig. 35. The circulation of C around the curve i is the line integral of Ct, the tangential component of C. The circulation around the whole loop is the sum of the circulations around the two loops: 1 ¼ a þ ab and 2 ¼ b þ ab.

387 take both our line integrals with the same ‘‘sense’’ of rotation. Following the same kind of argument we used before, we can see that the sum of the two circulations will give just the line integral around the original curve . The parts due to ab cancel. We can continue the process of cutting the original loop into any number of smaller loops. When we add the circulations of the smaller loops, there is always a cancellation of the parts on their adjacent potions, so that the sum is equivalent to the circulation around the original single loop. Now let us divide our original loop into a number of small loops that all lie on the surface we have chosen, as in Fig. 36. No matter what the shape of the surface, if we choose our small loops small enough, we can assume that each of the small loops will enclose an area which is essentially flat. Also, we can choose our small loops so that each is very nearly a square. Now we can calculate the circulation around the big loop  by finding the circulations around all of the little squares and then taking their sum.

Fig. 36. Some surface bounded by the loop  is chosen. The surface is divided into a number of small areas, each approximately a square. The circulation around  is the sum of the circulations around the little loops.

The circulation around a square; Stokes’ theorem How shall we find the circulation for each little square? We could easily make the calculation if it had a special orientation. For example, if it was in one of the coordinate planes. Since we have not assumed anything yet about the orientation of the coordinate axes, we can just as well choose the axes so that the one little square we are concentrating on at the moment lies in the xy-plane, as in Fig. 37.

Fig. 37. Computing the circulation of C around a small square.

388 If our result is expressed in vector notation, we can say that it will be the same no matter what the particular orientation of the plane. We want know to find the circulation of the field C around the little square. It will be easy to do the line integral if we make the square small enough that the vector C does not change much along any one side of the square. The assumption is better the smaller the square, so we are really talking about infinitesimal squares. Starting at point (x, y) – the lower left corner of the figure – we go around in the direction indicated by the arrows. Along the first side (1) the tangential component is Cx(1) and the distance is
x. The rust part of the integral is Cx(1)
x. Along the leg, we get Cy(2)
y. Along the third, we get Cx(3)
x, and along the fourth, Cy4
y. The minus signs are required because we want the tangential component in the direction of travel. The whole line integral is then I

C~  d s~ ¼ Cx ð1Þ
x þ Cy ð2Þ
y þ Cx ð3Þ
x þ Cy ð4Þ
y

Now let us look at the first and third pieces. Together they are ½Cx ð1Þ  Cx ð3Þ
x If we take into account the rate of change of Cx, we write Cx ð3Þ ¼ Cx ð1Þ þ

@Cx
y @x

Since we ultimately think of the limit as
y ! 0, the terms in (
y)2 are neglected. Combining the two previous equations, we find that ½Cx ð1Þ  Cx ð3Þ
y ¼ 

@Cx
x
y @y

The derivative can, to our approximation, be evaluated at (x, y). Similarly, for the other two terms in the circulation, we may write Cy ð2Þ
y  Cy ð4Þ
y ¼

@Cy
x
y @x

The circulation around the square is then 

 @Cy @Cx 
x
y @x @y

389 which is interesting, because the two terms in the parentheses are just the z component of the curl. Also we note that
x
y is the area of the square. So we can write the circulation as
 ~
C~
a r z

But the z component really means the component normal to the surface element. We can therefore write the circulation around a differential square in an invariant vector form: I

 ~
C~ da ¼ r ~
C~  n~
a C~  d s~ ¼ r n

Our result says that the circulation of any vector C around an infinitesimal square is the component of the curl of C normal to the surface, times the area of the square. The circulation around any loop  can now be easily related to the curl of the vector field. We fill the loop with any convenient surface S and add the circulations around a set of infinitesimal squares in this surface. The sum can be written as an integral. Our result is a very useful theorem called Stokes’ theorem. Stokes’ Theorem I Z
 ~ ~ ~
C~ da r C  dl ¼ S



n

where S is any surface bounded by . Energy in the electrostatic field To show that Eq. (68) is consistent with our laws of electrostatics, we begin by introducing into Eq. (64) Z 1
 dV U¼ 2 the relation between
and  that we obtained in Part 1: Poisson equation
¼ "0 r2  We get "0 U¼ 2

Z

r2 

390 Writing out the components of the integrand, we see that 

 @2  @2  @2  r  ¼  þ þ @x2 @y2 @z2 2

   2    2     @ @ @ @ @ @ @ @ @       þ þ ¼ @x @x @x @y @y @y @y @z @z


 ~  r ~  r ~  r ~ ¼r since @ @ @ @ @2   ¼ þ 2 @x @x @x @x @x Our energy integral is then "0 U¼ 2

Z


Z 

 "0 ~ ~ ~  r ~  dV r  r dV  r 2

We can use Gauss’ theorem to change the second integral into a surface integral: Z

Z
 ~  r ~  dV ¼ r vol




 ~   n~ da r

surface

We evaluate the surface integral in the case that the surface goes to infinity (so the volume integrals become integrals over all space), supposing that all the charges are located within some finite distance. The simple way to proceed is to take a spherical surface of enormous radius R whose centre is at the origin of coordinates. We know that when we are very far away from all charges,  varies as 1/R and r  as 1/R2. Since the surface area of the large sphere increases as R2, we see that the surface integral falls off as (1/R)(1/R2)R2 ¼ (1/R) as the radius of the sphere increases. So if we include all space in our integration (R ! 1), the surface integral goes to zero and we have that "0 U¼ 2

Z


all space

Z 
 "0 ~ ~ r  r dV ¼ E~  E~ dV 2 all space

We see that it is possible for us to represent the energy of any charge distribution as being the integral over an energy density located in the field.

391 Appendix B A note on units and dimensions Many times we would like to know the energy associated of a particular interaction express in different units. The goal of this appendix is exactly to provide us the useful tool that will give us such information. If we take the electrostatic interaction energy between two charges in units of charge squared per unit length, the Coulomb equation for the electrostatic potential becomes simply W¼

q1 q2 d

in atomic units (a.u.), being q1 and q2 in units of protonic charge and d in A˚ (angstrom). We then have the relations: Wðkcal=molÞ ¼ 331:842 Wða:u:Þ and Wðkjoule=molÞ ¼ 1388:4269 Wða:u:Þ It is simpler to do all calculations in atomic units and then convert to the units of interest using the above relations. Molecular electrostatic energies and potentials are often expressed respectively in kBT and kBT/e units, kT being an approximate value for the thermal noise of a system. These units depend on the absolute temperature of the system, and therefore are only meaningful when a value for T is given. At T ¼ 298 K we have: WðkB TÞ ¼ 560:780 Wða:u:Þ In atomic units the proton charge e is unitary and kBT are therefore numerically equal to the corresponding kBT/e potential values. When working with pKa calculations, it is also customary to express the electrostatic interaction energies in terms of the induced pKa shift. These units are dimensional and therefore independent of the unit system. At 298 K we have that WðpKa unitsÞ ¼ Wða:u:Þ=½lnð10Þ kB T ¼ 243:499 Wða:u:Þ From the above results we can derive other useful conversions: 1 pKa unit ¼ 1:3628 kcal=mol ¼ 2:303kB T

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397

The development of supportive-care agents for patients with cancer Theresa K. Neumann1 and MaryAnn Foote2,* 1

Associate Director, Clinical Research; 2Director, Medical Writing, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320-1699, USA Abstract. As the population ages, a dramatic increase in the number of cases of cancer is expected and the need for supportive-care agents, those used to ameliorate some of the side effects of cancer or its treatment, becomes more urgent. At present, supportive-care products are available and new agents are being developed with novel mechanisms of action or modifications of existing agents that improve performance. Because of the urgent need for such products, efficient development is required to deliver useful products to patients as rapidly as possible. This chapter uses actual examples to illustrate the stages of drug development, phase 1 through phase 3. Keywords: biotechnology – medical biotechnology, clinical research – phase 1, clinical research – phase 2, clinical research – phase 3, marketing authorization.

Introduction Patients who have cancer – and particularly those receiving chemotherapy or radiation therapy – frequently have a variety of comorbid conditions. These comorbid conditions may be due to their primary disease (e.g., anemia secondary to malignancy), but often they are the result of chemotherapy or radiation therapy. Common treatment-related conditions include extreme fatigue, pain, alopecia, nausea and vomiting, malnutrition and cachexia, mucositis and stomatitis, anemia, thrombocytopenia, and neutropenia. These manifestations of cancer and/or its therapy are often major contributors to morbidity and mortality from the disease and are known to reduce a patient’s quality of life. Examples of drugs developed to treat cancer-related symptoms or ameliorate side effects from chemotherapy include bisphosphonates, hematopoietic growth factors, and antiemetics. Pamidronate, a bisphosphonate, was approved for the treatment of hypercalcemia of malignancy, but it later showed utility in decreasing skeletal-related events. Hematopoietic growth factors, such as filgrastim (r-metHuG-CSF) and epoetin alfa (rHuEPO), have been shown to correct chemotherapy-induced neutropenia and anemia of cancer, respectively. Ondansetron, an antiemetic, was approved for postoperative nausea and vomiting in adults, but it is also an effective treatment in the prevention and treatment of chemotherapy-induced nausea and vomiting. This chapter will focus on drug development of biotech products and recent development of selective new supportive-care agents and not on the conditions themselves. For more information, interested readers should refer to any one of *Corresponding author: Tel: þ 1 805 447 4925. Fax: þ 1 805 498 5593. E-mail: [email protected] BIOTECHNOLOGY ANNUAL REVIEW VOLUME 9 ISSN 1387-2656 DOI: 10.1016/S1387-2656(03)09011-2

ß 2003 ELSEVIER SCIENCE BV ALL RIGHTS RESERVED

398 the many excellent textbooks that discuss the practical aspects of treating these manifestations [1]. Drug development All new biologics follow the same highly regulated process to attain marketing approval. Drug development has become more global in the past decade, particularly under the aegis of the International Conference on Harmonization (ICH) [2]. With the adoption of the Common Technical Document (CTD), the uniformity of drug applications worldwide will be strengthened. Although the New Drug Application (NDA) and Biologic License Application (BLA) processes have common ground with the European Union’s documents (Clinical Trial Application [CTA] and Marketing Authorisation Application [MAA]), the process in the United States will be used to illustrate the steps in drug and biologic development. Many regulatory documents are required for drug development and are briefly described. Investigational New Drug application A new biologic cannot be tested in humans in the United States without an Investigational New Drug (IND) application. This application requires Food and Drug Administration (FDA) approval to permit the interstate shipment of drug products. The IND process ensures that humans are not exposed to undue risks from investigational products by requiring quality controls in manufacturing; extensive testing in animals; and review of the clinical protocol, investigator’s brochure, and informed consent document by institutional review boards (IRBs); the entire package is reviewed by the FDA. The contents of the IND are listed in Table 1. As the testing of a drug or biologic proceeds through the development process, the IND may be amended; the investigational plan and protocols may be revised; study sites may change; the investigator’s brochure is updated to reflect new and significant information; new protocols are submitted; and manufacturing or product characteristics may change. Serious adverse events reported by patients or their healthcare professional must be communicated to the FDA on a timely basis and in annual IND updates. Table 1. Components of an investigational new drug application. Introduction General investigational plan Investigator’s brochure Clinical protocol(s) Chemistry, manufacturing, and controls Nonclinical pharmacology and toxicology data Previous human experience

399 The initial clinical protocol is a part of the original IND submission and as a program matures, other clinical protocols are filed to the IND. A clinical protocol is a plan for the specific scientific study of a drug or biologic in humans. The goal of a clinical protocol usually is to collect data to support label claims (i.e., prescribing information). Protocols differ by the phase of drug development (phases 1, 2, 3, or postmarketing phases 3b or 4) or by use (compassionate). Not all INDs are held by drug sponsors – Investigator INDs are possible. Other INDs include Treatment INDs and Compassionate-use INDS. These latter INDs are submitted to the FDA to facilitate the availability of investigational drugs for critically ill patients who have failed all other treatment options or for whom no treatments are available. These atypical INDs do not replace the full IND. Instead, they are filed as additions to the original IND submission. New Drug Application/Biologic License Application A new drug cannot be marketed in the United States without an NDA/BLA approval. NDAs were reviewed by the FDA’s Center for Drug Evaluation and Research (CDER), and BLAs were reviewed by the FDA’s Center for Biologics Evaluation and Research (CBER), but new regulations may change this process. Some biologics are reveiwed by CDER, rather than CBER, and some biologics require an NDA rather than a BLA. NDAs and BLAs are huge documents, often containing hundreds of thousands of pages of data. Well-designed clinical development programs that allow determination of safe and efficacious dose and schedule and which typically include a comparative treatment as standard of care are required. Clinical trials submitted as part of the NDA or BLA to gain marketing approval are routinely conducted in a sequential fashion, phase 1 through phase 3 (Table 2). Postmarketing trials (i.e., phase 3b or phase 4) are trials done after gaining regulatory approval to market a product and serve to collect further safety information and assess other potential indications. Table 2. Description of phase 1, 2, and 3 clinical studies. Phase

No. of patients

Purpose

Comments

1

Few, maybe less than 25

2

More, maybe 25–100

Establish preliminary safety risks and obtain pharmacokinetic and pharmacodynamic data Further explore safety, provide data on early indicators of efficacy, provide sufficient data to design phase 3 trials

3

Many, often several hundred to several thousand

Healthy volunteers can be used to test drugs, but patients are usually used to test biologics. Need a proper control group, double-blind drug administration, and proper randomization of patients to treatment groups. Design is crucial because the endpoints supported by the data make up the label and marketing claims.

Confirm efficacy and further characterize safety

400 The remainder of the chapter will discuss the science and decision-making used in the development of support-care agents in oncology and use examples from our experiences. For each drug, we discuss the basic mechanism of action, phase 1 results, phase 2 proof-of-concept studies, and phase 3 comparative studies done to obtain a marketing label. Pegfilgrastim Background information Neutropenia and infection are potential serious complications of cancer chemotherapy, and the risk of infection is directly related to the depth and duration of neutropenia [3]. The severity of neutropenia depends on the intensity of the chemotherapy regimen, as well as on host- and disease-related factors. Fever may be the only manifestation of infection because underlying immunosuppression may obscure the classic signs and symptoms. Delay in initiating subsequent cycles of chemotherapy or decrease in the dose of chemotherapy, or both may be required because of severe neutropenia. Such delay may compromise an otherwise effective chemotherapy. Filgrastim, with properties comparable to the endogenous protein, was licensed for amelioration of chemotherapy-induced neutropenia in 1991. Treatment of severe neutropenia with filgrastim reliably increases neutrophils and can prevent febrile neutropenia. Filgrastim, however, must be injected daily for up to 14 consecutive days. Although filgrastim has been shown to be an effective supportive-care agent, it was thought that a product that could be administered less frequently, ideally once per cycle, would be more acceptable to patients, their caregivers, and healthcare providers and that it might allow for more flexible administration. A drug candidate with a similar safety and efficacy profile, but with a longer half-life, was desirable. Pegfilgrastim is a sustained-duration formulation of filgrastim that has been developed by covalent attachment of a polyethylene glycol (PEG) molecule to the filgrastim molecule (Table 3). Table 3. Comparison of filgrastim and pegfilgrastim (Amgen data on file). Characteristic

Filgrastim

Pegfilgrastim

Number of amino acids Cell source Glycosylation Pegylation Cmax

175 E. coli None No 1.65
0.80 ng/mL for single 75-mg/kg dose 5.5
1.8 for single 75-mg/kg dose 14.3
4.3 (0–24 h, ng/l h) for single 75-mg/kg dose

175 E. coli None Yes 43.6
20 ng/mL for single 30-mg/kg dose 9.50
3.51 for single 30-mg/kg dose 887
336 (0–1, ng/l h) for single 30-mg/kg dose

tmax (h) Area under the curve

401 Pegfilgrastim received marketing approval in the United States in 2002 and is under review for approval in the EU for the amelioration of chemotherapyinduced neutropenia. Mechanism of action Endogenous granulocyte colony-stimulating factor (G-CSF) is usually detectable in serum, and its concentration increases during bouts of infection [4]. G-CSF maintains neutrophil production during steady-state conditions. Filgrastim reduces neutrophil maturation time from five days to one day, leading to the rapid release of mature neutrophils from the bone marrow into the blood, and increases the circulating half-life of neutrophils [5]. Filgrastim enhances neutrophil chemotaxis by increasing the binding of fMLP (formyl-methionylleucyl-phenylalanine) [6] and increases neutrophil superoxide production in response to chemoattractants [7]. Filgrastim has a half-life of 3–4 h and needs to be administered daily [8]. One way to produce a product that is cleared less rapidly is to add a PEG molecule to the product, since PEG-modification of proteins has been shown to sustain the duration of action by reducing renal clearance of the protein and by decreasing rates of cellular uptake and proteolysis [9]. Using information from X-ray crystallography studies of filgrastim [10], it was found that adding a 20-kd PEG molecule to the amino terminal residue did not change the mechanism of action, namely the binding of the molecule to the G-CSF receptor on myeloid cells. Pegfilgrastim has decreased plasma clearance and increased plasma half-life, thus sustaining the duration of the pharmacologic effect. The target product profile for a pegylated form of filgrastim required several important criteria. The ideal candidate molecule needed to provide adequate hematologic support with only a single injection per chemotherapy cycle rather than with up to 14 days of daily injections of filgrastim. This administration schedule would provide convenience to patients, caregivers, and healthcare providers and it would promote compliance. The product could not induce any undesirable side effects that would exceed the safety profile of filgrastim, particularly sustained bone pain. Lastly, the product needed to be given as a subcutaneous injection. After careful preclinical testing of several candidates, the first pegylated form of filgrastim was tested in phase 1 and phase 2 proof-of-concept studies. The clearance mechanism of pegfilgrastim is thought to be mediated by neutrophils bearing the G-CSF receptor. This proposed mechanism of action is based in part on results obtained from a phase 2 study in patients with nonsmall-cell lung cancer in which the pharmacodynamic and pharmacokinetic profiles were compared in the same patients before chemotherapy (i.e., hematologic steady state) and after chemotherapy (myelosuppressive state) [11].

402 Phase 1 studies Phase 1 studies were conducted to establish safety, optimal drug dosage, and potential efficacy, including pharmacodynamic and pharmacokinetic properties [12]. For each cohort, eight normal volunteers received a single subcutaneous injection of pegfilgrastim at doses ranging from 30 to 300 mg/kg. Blood samples for serum drug concentrations, complete blood counts, and CD34 þ cell counts were collected at specified intervals to fully characterize the pharmacodynamic and pharmacokinetic profiles. The safety profile of pegfilgrastim was consistent with the known effects of filgrastim with observed adverse events that included moderate bone pain, headache, and reversible changes in platelet counts, liver enzyme concentrations, and uric acid concentrations with no clinical sequelae. The results of this study confirmed findings that pegfilgrastim exhibits nonlinear pharmacokinetics. As predicted, the serum clearance decreased as the dose increased. The mean terminal half-life was independent of dosage and ranged from 46 to 62 h. The magnitude and duration of absolute neutrophil counts and CD34 þ cell counts was dosage dependent (Table 4) [12]. This study also confirmed the agent was active and warranted further clinical development. Phase 2 studies Once activity was confirmed in volunteers, studies in patients with cancer receiving chemotherapy were necessary. In an open-label, dose-escalation study, 13 patients were randomized to receive a single subcutaneous injection of pegfilgrastim or five daily injections of filgrastim before chemotherapy. After a washout period, the patients received chemotherapy, followed by a single injection of pegfilgrastim or daily injections of filgrastim starting approximately 24 h after completion of chemotherapy. This study was designed to evaluate the safety and pharmacodynamic and pharmacokinetic profiles of several dosages of pegfilgrastim and compare pegfilgrastim with filgrastim both with and without chemotherapy. Table 4. Summary statistics for noncompartmental pharmacokinetic variables, week 1 of cycle 1. Only data from 14 patients are included because one patient had extremely low concentrations. These data have been omitted. Data courtesy of Amgen, Thousand Oaks, California. Variable

Tmax (h)

Cmax (ng/ml)

t1/2 (h)

CL/F (ml/h/kg)

n Mean SD

14 86.1 22.8

14 8.9 5.1

7 32.6 11.8

7 3.7 0.9

Tmax, time to maximum concentration; Cmax, maximum serum concentration; t1/2, terminal half-life; CL/F, relative clearance; SD, standard deviation.

403 A single subcutaneous injection of pegfilgrastim administered before chemotherapy produced the expected increase in absolute neutrophil count (ANC) and a subsequent rapid decrease in serum drug concentration, while a single subcutaneous injection of pegfilgrastim administered after chemotherapy maintained serum drug concentration longer because of a reduction in ANC, suggesting a neutrophil receptor-mediated mechanism of clearance. This finding suggested that serum pegfilgrastim levels are self-regulating, i.e., as ANC recovered to normal levels, serum concentration of pegfilgrastim decreased. The safety data from this trial suggested no difference in adverse events and, in particular, no difference in the incidence and severity of bone pain. The study provided preliminary evidence that a single injection of pegfilgrastim provides similar hematologic support as filgrastim in patients undergoing chemotherapy. Based on these data, a phase 2 program was initiated in patients with breast cancer. This program would form the basis for the pivotal phase 3 trial and was designed to allow selection of optimal dose and directly compare efficacy with filgrastim. Doxorubicin–docetaxel combination chemotherapy was chosen because it was a promising regimen that produced significant myelosuppression. The duration of severe neutropenia in the absence of growth factor support was reported to be between five and seven days, with an incidence of grade 4 neutropenia approaching 90%. Patients with high-risk stage II, III, and IV breast cancer were enrolled into a randomized, multicenter trial [13]. The key endpoints included duration of grade 4 neutropenia, incidence of febrile neutropenia, pharmacokinetics, and safety. The results of this study showed that a single injection of pegfilgrastim at a dose of 100 mg/kg/cycle was equivalent to filgrastim in supporting neutrophil recovery in patients with breast cancer who were receiving multiple cycles of chemotherapy. Other phase 2 studies comparing filgrastim with pegfilgrastim were conducted in patients with non-Hodgkin’s lymphoma to confirm the efficacy and safety for a broad range of tumor and chemotherapy settings (Amgen data on file). Phase 3 studies In the phase 3 program, the same patient population, chemotherapy regimen, and study endpoints were used as in the phase 2 studies [14,15]. These studies were double-blind, noninferiority trials to test the hypothesis that there was no difference between a single injection of pegfilgrastim administered as 100 mg/kg or as a 6-mg fixed dose and multiple 5-mg/kg injections of filgrastim. ANC profile, time to ANC recovery, and safety were assessed. The results from both trials showed that a single fixed dose or a by-weight dose produced a similar duration of severe neutropenia, ANC profile, and time to ANC recovery compared with daily injections of filgrastim. In addition to ANC recovery, the serum concentrations of pegfilgrastim showed clearance similar to what

404 was described in the nonsmall-cell lung cancer study, which further confirms the cell-mediated clearance. Safety profiles were similar between treatment groups. Results of the weight-based study showed a statistically significant decrease in the overall incidence of febrile neutropenia in favor of pegfilgrastim. A similar trend, not statistically significant possibly due to lower sample size, was noted in the fixed-dose study. Taken together, these trials confirmed that a single injection of pegfilgrastim per chemotherapy cycle was safe and effective in treating chemotherapy-induced neutropenia and that once-per-cycle treatment with sustained-duration pegfilgrastim has significant advantages over standard filgrastim. Summary A clear understanding of the native G-CSF molecule and an ability to use pegylation to extend half-life facilitated the development of pegfilgrastim. Potential benefits of the longer-acting pegfilgrastim molecule to patients with chemotherapy-induced neutropenia include fewer injections, increased patient compliance, and decreased burden on healthcare providers. Darbepoetin alfa Background Anemia is common in patients with cancer, and it is an important contributor to the morbidity associated with cancer and its treatment. Anemia in patients with cancer is usually manifested by fatigue [16]. Anemia in patients with cancer is best managed by treatment of the underlying cause, when possible; however, treatment is often not successful and can exacerbate anemia. Epoetin alfa acts like the endogenous protein and is licensed to treat anemia of cancer, human immunodeficiency virus (HIV) infection, and chronic renal failure, and for perisurgical use. Many studies have been published concerning the treatment of anemia and its sequelae in patients with cancer [17–20]. These studies have shown that treatment with epoetin alfa reduces the requirement for red blood cell transfusions and improves quality of life in patients with a broad range of nonmyeloid tumors and chemotherapeutic agents [17, 18, 21, 22]. Darbepoetin alfa is the recombinant product of a gene produced through site-directed mutagenesis of the erythropoietin gene that increases the glycosylation of the resultant protein. Darbepoetin alfa binds to the erythropoietin receptor and stimulates erythropoiesis by the same mechanism as endogenous erythropoietin and rHuEPO, and it has increased potency due to its extended serum residence time [23]. Darbepoetin alfa is currently approved for marketing in the United States for the amelioration of anemia associated with cancer and its treatment.

405 Mechanism of action Endogenous erythropoietin is the hormone that stimulates the production of red cells from the erythroid precursor cells in the bone marrow. Erythropoietin functions as a growth factor, binding to receptors on erythroid-progenitor cells and stimulating the mitotic activity of erythrocyte burst-forming and colony-forming units and early precursor cells (proerythroblasts) [24]. Although the precise location for the production of erythropoietin in the kidney tubule is not fully understood, it has been suggested that tubular or interstitial cells function as the main site for localization of the hormone and its mRNA [24]. Preliminary work has also identified the liver as the major site (>90%) of erythropoietin production for the fetus [25] and shows that some erythropoietin is produced in the adult human brain, but not enough to have an effect on systemic amounts [26]. The recombinant proteins epoetin alfa and darbepoetin alfa have the same mechanism of action as the endogenous protein, increasing red blood cell count by causing committed erythroid progenitor cells to proliferate and differentiate into normoblasts, thus keeping the body’s red blood cell mass at the optimal level [27–29]. The binding affinity of darbepoetin alfa is lower than that of epoetin alfa or natural erythropoietin, but the longer half-life of darbepoetin has been shown to increase in vivo biologic activity [23, 29]. Phase 1 studies Pharmacokinetic data are available from 15 evaluable patients with a variety of nonmyeloid tumor types who were enrolled into an open-label study [30]. Patients received injections of darbepoetin alfa 2.25 mg/kg/week immediately before receiving chemotherapy, continuing through three cycles of chemotherapy that were given at least three weeks apart. Blood samples were collected for pharmacokinetic analysis periodically during week 1 of chemotherapy cycles 1 and 3. Analyses of the results suggested that the drug is slowly absorbed after subcutaneous injection, reaching a peak concentration approximately 85 h later, and that it had a low relative clearance and long terminal half-life. Mean hemoglobin response (defined as a change from baseline, in the absence of a red blood cell transfusion) was 2.3 g/dl after three cycles of chemotherapy. The mean change in hemoglobin concentrations appeared similar to historical results using epoetin alfa [17, 31] and additional development of darbepoetin alfa was undertaken. Phase 2 studies Phase 2 studies of darbepoetin alfa were initiated to determine dose, schedule, and to compare darbepoetin alfa to epoetin alfa and placebo. Several phase 2 trials have shown a dose response to darbepoetin alfa in patients receiving multicycle chemotherapy for the treatment of solid tumors [32, 33] and in patients

406 with cancer who were not receiving chemotherapy but were nevertheless anemic [34]. Results from another phase 2 study suggest that darbepoetin alfa can be administered as infrequently as once every three weeks and still maintain hemoglobin concentrations [35]. The initial studies with darbepoetin alfa suggested the possibility of developing schedules that result in a more rapid benefit to a greater proportion of patients with anemia who are receiving chemotherapy [32, 36]. Dose loading may be both more efficacious and more cost effective. In one study, 127 patients were randomized to receive either epoetin alfa 40,000 U with escalations to 60,000 U for nonresponders, or darbepoetin alfa at 4.5 mg/kg/week until hemoglobin concentration  12 g/dl, followed by doses of 1.5, 3.0, or 4.5 mg/kg/week on a variety of schedules [33]. Overall, after four weeks of treatment, mean change in hemoglobin was 80% greater in the groups receiving darbepoetin alfa than in the group receiving epoetin alfa. By the end of the study, the mean change in hemoglobin was 30% greater in patients receiving darbepoetin alfa compared with patients receiving epoetin alfa. Loading doses of darbepoetin alfa for four weeks followed by a lower dose and/or a less frequent administration schedule (every two weeks or every three weeks) appear to be safe and may decrease the time to response and increase the proportion of patients benefiting from therapy compared with current approaches using rHuEPO. Phase 3 studies A phase 3, multicenter, double-blind, placebo-controlled study evaluated darbepoetin alfa compared with placebo in anemic patients with cancer receiving chemotherapy [37]. Endpoints were red blood cell transfusions and hemoglobin concentration, adverse events, antibody formation to darbepoetin alfa, hospitalizations, Functional Assessment of Cancer Therapy (FACT) Fatigue score, and disease outcome. Three hundred fourteen patients with lung cancer receiving chemotherapy were randomly assigned to receive darbepoetin alfa or placebo administered weekly for 12 weeks. Darbepoetin alfa reduced the proportion of patients requiring a transfusion and the number of RBC units transfused, increased the proportion of patients with a hemoglobin response, and the proportion of patients with improvement in the FACT Fatigue score. A trend towards fewer hospitalization days for patients receiving darbepoetin alfa was seen. Patients receiving darbepoetin alfa had a longer median progression-free and overall survival. Adverse events were comparable between the groups. No antibodies to darbepoetin alfa were detected. Based on these data, darbepoetin alfa received marketing approval for treatment of patients with cancer receiving chemotherapy. Summary The development of darbepoetin alfa is an example of drug development based on knowledge of the physical properties of the recombinant and native

407

Fig. 1. Biochemical and biological properties of rHuEPO and rHuEPO analogs containing four and five N-linked carbohydrate chains [23]. Used with permission of British Journal of Cancer.

proteins (Fig. 1). Using this knowledge of the physical properties of the longacting darbepoetin alfa, it is possible to examine various doses and schedules to optimize treatment of the anemia of cancer for individual patients. Potential benefits may include reduced number of injections and faster amelioration of anemia. Keratinocyte Growth Factor Background Chemotherapy and radiotherapy, alone or in combination, kill rapidly proliferating tumor cells and, consequently, often damage rapidly dividing normal cells of the gastrointestinal tract. Damage or destruction of the normal cells in the gastrointestinal tract causes mucositis that can be a dose-limiting side effect of chemotherapy and/or radiotherapy. Mucositis compromises the integrity of the protective mucosal barrier and may limit a patient’s ability to speak, eat, or swallow. Existing oral mucositis treatment focuses on alleviating symptoms, which requires substantial healthcare resources. This increase in resources is particularly true in the setting of bone marrow or stem cell transplantation [38–40]. Treatments that address the pathogenesis of oral mucositis rather than symptomatic relief could improve the health-related quality of life of patients with cancer who are receiving mucositis-inducing treatment. Keratinocyte growth factor (KGF) is a natural ligand for the KGF receptor that is found on nearly all epithelial cells, including those lining the digestive tract. KGF stimulates the proliferation and differentiation of the epithelium, including that of the gastrointestinal tract. Recombinant human forms of KGF (rHuKGF) are under development. Because rHuKGF increases the proliferation of the epithelium, the increased thickness may provide protection from the damaging effects of radiation and chemotherapy.

408 Mechanism of action Keratinocyte growth factor, a member of the fibroblast growth factor (FGF) family, was originally isolated from cultured human embryonic fibroblasts [41, 42]. Unlike other members of this family, however, KGF exhibits strict specificity of action for epithelial cells and has no direct effects on other types of cells [43, 44]. KGF stimulates cell proliferation, as evidenced by incorporation of 3 H-thymidine into the DNA of epithelial cells [43, 45–49]. Early preclinical testing of rHuKGF showed that systemic administration in animals caused the proliferation and thickening of epithelial tissues throughout the gastrointestinal tract [50]. In addition, pretreatment with rHuKGF appeared to markedly reduce damage to the mucosal lining of the oral and lower gastrointestinal tracts in animals given chemotherapy or radiation [51] (Fig. 2). Theoretically rHuKGF has the potential to stimulate epithelial tumors, but there are no data to support this theory. Phase 1 studies Phase 1 studies were done in healthy volunteers and patients with cancer. Dose-escalation studies in healthy volunteers used a single intravenous dose or daily dosing for three consecutive days at dosages up to 20 mg/kg/day. After intravenous administration at both 10 and 20 mg/kg/day, serum rHuKGF concentrations declined rapidly (i.e., by 50- to 100-fold) after the initial 30 min and reached a plateau between 1 and 6 h [52] (Table 5). After the plateau, a terminal half-life of approximately 3 h was seen. No accumulation was evident after three days of dosing, and the pharmacokinetics of rHuKGF were dose linear over the range of dosages. The biologic activity was measured using buccal Table 5. Summary of pharmacokinetic variables in normal human volunteers on days 1 and 3. rHuKGF was administered intravenously for three days. Numbers are reported as mean (SD). Data courtesy of Amgen Inc., Thousand Oaks, California. Parameter

V0 (mL/kg) AUC (pg h/mL) t1/2 (h) CL (mL/h/kg) Vss (mL/kg)

rHuKGF 20 mg/kg/day Day 1 (n ¼ 6)

Day 3 (n ¼ 5)

89.1 (42.6) 36,700 (11,900) 3.31 (0.59) 596 (194) 1890 (51)

79.9 (41.8) 41,900 (2000) 6.21 (2.69) 558 (222) 1620 (1110)

AUC, area under the curve, 0–1; CL, clearance; t1/2, half-life associated with terminal phase; V0, volume of distribution at time 0; Vss, volume of distribution at steady state.

409

Fig. 2. Biologic activity of rHuKGF can be quantified by measuring the proliferation of epithelial cells. Antibodies to BrdUrd (Accurate Chemical & Scientific Corporation, Westbury, NY) bind to Ki67-stained cells (Ki67 is a nuclear marker found in proliferating epithelial cells). The effect of rHuKGF on surviving intestinal crypts of mice after irradiation and bone marrow transplantation. Panel A, no rHuKGF; panel B, rHuKGF for three days before irradiation. C Farrell, personal communication.

mucosa biopsy samples to evaluate the presence of mitotic activity and Ki67 immunohistochemistry, biomarkers of proliferation. At dosages of 10 and 20 mg/kg/day, a statistically significant increase in mitotic figures was evident, and Ki67 staining was significant at dosages of 5–20 mg/kg/day. A phase 1 study was done in patients with metastatic colorectal cancer who were receiving high-dose chemotherapy with autologous stem cell transplantation. rHuKGF serum concentration declined rapidly after an intravenous bolus administration of 60 mg/kg/day (i.e., approximately 50-fold over the initial 30 min) [53]. Serum concentration reached a plateau between 1 and 4 h after administration and then declined further, with a terminal half-life of approximately 3–4 h. rHuKGF was detectable in the serum up to 36 h after administration. These phase 1 studies show that rHuKGF is well tolerated and biologically active only when administered at dosages  10 mg/kg/day for three days as an intravenous infusion.

Phase 2 studies Since the mucositis is difficult to grade and has a large subjective component, it will be necessary to have readily measurable endpoints in clinical studies and to train the investigators to a uniform standard for grading mucositis. Several phase 2 studies were done with patients with cancer receiving chemotherapy and/or radiotherapy, using doses and schedules chosen based on phase 1 acute

410 toxicity data and preliminary evidence of efficacy [54, 55]. The phase 2 program included studies in patients with hematologic malignancies who were receiving stem cell transplantation and in patients with advanced colorectal or head-andneck cancer. In one phase 2, randomized, placebo-controlled study, rHuKGF reduced the duration of grade 3/4 oral mucositis and improved the quality of life of patients with hematologic malignancies who were receiving autologous stem cell transplants [55]. Patients administered 60 mg/kg rHuKGF three days before and three days after transplantation had a significant reduction in the duration of grade 3/4 oral mucositis, required fewer days of intravenously administered opioid analgesics, and had improved health-related quality-of-life assessments (i.e., ability to swallow, eat, drink, talk, and sleep) than patients who received placebo. Transient, asymptomatic increases in serum amylase and lipase occurred more frequently in rHuKGF-treated patients than in patients receiving placebo. Another phase 2, randomized, placebo-controlled study was done in patients with advanced colorectal cancer [54]. Patients were randomly assigned to receive two cycles of either rHuKGF 40 mg/kg/day or placebo by intravenous bolus on days 1–3, followed by chemotherapy. Incidence of grade 2–4 mucositis was 78% in placebo patients compared with 32% of rHuKGF-treated patients. Other endpoints including duration of mucositis were reduced in patients receiving rHuKGF (3.4 days) compared with patients receiving placebo (10.2 days). Asymptomatic increases in serum amylase and lipase were seen in patients receiving rHuKGF, but without sequelae. These studies supported the hypothesis that rHuKGF acts on the pathogenesis of oral mucositis and can prevent mucositis and associated symptoms in patients receiving mucositis-inducing chemotherapy. Phase 3 studies Based on promising phase 2 results, a phase 3 trial in the setting of stem cell transplantation is underway. This trial will attempt to confirm the promising phase 2 results. Summary The development of rHuKGF is an example of how knowledge of the pathogenesis of a comorbid condition lead to a specifically targeted therapy to attempt to prevent the underlying cause of this toxicity. To date, phase 1 and phase 2 studies have shown that rHuKGF has biologic activity with acceptable acute toxicity. Large randomized studies, however, will determine the magnitude of the benefit and determine whether there are any chronic toxicities.

411 Osteoprotogerin Background Bone is a common site for metastasis for patients with breast, lung, prostate, or renal cancers [56–58]. Lytic bone lesions, caused primarily by increased osteoclastic activity, can lead to pathologic fractures, spinal collapse, hypercalcemia, and pain. A medicine that could inhibit the activity of osteoclasts could have clinical utility in preventing these sequelae. Osteoprotegerin (OPG) (meaning ‘‘to protect bone’’) is a member of the tumornecrosis factor receptor (TNFR) superfamily and acts to reduce bone resorption by inhibiting differentiation and activation of osteoclasts. OPG is an endogenous protein; a recombinant product (rHuOPG) in early clinical development. Several nonbiologic approaches have been used to treat bone metastasis, these include the bisphosphonates, a class of compounds based on the naturally occurring pyrophosphates. These small-molecule drugs have been used to reduce skeletal-related events and bone pain [59]. Bisphosphonates act by complexing with bone mineral. They prevent tumor cells from adhering to the bone, prevent or restrict osteoclast-mediated bone resorption, and inhibit matrix metalloproteinases. These compounds may inhibit angiogenesis and reduce the level of growth factors involved in bone resorption. A recently approved bisphosphonate called zoledronic acid has been shown to be significantly superior to the standard, pamidronate in the treatment of hypercalcemia of malignancy. In a randomized, double-blind trial comparing intravenous zoledronic acid with intravenous pamidronate in patients with hypercalcemia of malignancy, patients receiving zoledronic acid had a significantly higher response rate in the correction of serum calcium, a faster onset of effect, and longer duration of action. Zolendronic acid was well tolerated at a single dose of 4 mg. One advantage of zolendronic acid over other marketed products is that the medicine is administered as a 5-min infusion compared with the 2-h infusions required for pamidronate [60]. Mechanism of action Bone resorption can be inhibited by three mechanisms: reducing the activation frequency of basic multicellular units, reducing the bone resorptive activity of mature osteoclasts, or increasing the rate of osteoclastic apoptosis. OPG reduces the terminal differentiation of osteoclasts and thus affects the pool of mature osteoclasts and also reduces the activity of mature osteoclasts [61]. OPG is important in bone metabolism and has been shown to be a potent inhibitor of bone resorption in vivo, acting as a decoy receptor to bind and inactivate OPG ligand [62] (Fig. 3). OPG ligand is required for osteoclast differentiation [61, 63]. Additionally, OPG opposes the bone resorptive activity of parathyroid hormone, PTH, interleukin (IL-1), and (TNF) [64], the main mediators of

412

Fig. 3. Proposed mechanism of action of OPG (osteoprotegerin). OPG ligand (OPGL) is produced by osteoblasts in response to bone resorptive agents such as parathyroid hormone (PTH), vitamin D, interleukin (IL)-1b, and tumor necrosis factor (TNF)-a. OPGL, either on cell surfaces or in solution, interacts with its receptor, osteoclast differentiation and activation receptor (ODAR), on osteoclast precursors to promote differentiation or on mature osteoclasts to cause activation. OPG binds to and inactivates OPGL either in solution or on exposed cell surfaces. In the absence of OPG, OPGL binds to ODAR to increase osteoclast numbers and to increase bone resorption. Used with permission of Amgen Inc., Thousand Oaks, California.

cancer-related bone diseases. OPG and OPG ligand are present in the systemic circulation of adult humans [65] and have been found to regulate the differentiation of osteoclasts from precursors in human peripheral blood [66]. Recombinant human OPG (rHuOPG) has been shown to be bone antiresorptive in postmenopausal women [67]. Phase 1 studies Several phase 1 studies are underway to characterize the safety and potential efficacy of targeted rHuOPG therapy in patients with lytic bone metastases or to examine several routes of administration in healthy postmenopausal women. Phase 2 and phase 3 studies Because phase 1 studies have not been completed, no phase 2 or phase 3 studies actively studying rHuOPG administration to patients with cancer are being done. Summary The early development of rHuOPG illustrates that the drug development process is orderly and proceeds in a stepwise fashion. The potential hope for rHuOPG as a viable biologic to provide amelioration of symptoms related to bone metastasis is an exciting area of research. More research is needed to fully understand the utility of this drug in this supportive-care setting.

413 Discussion The need for supportive-care agents to treat patients with cancer continues. As the population ages, a dramatic increase in the number of cases of cancer is expected. It has been estimated that by 2020, 20 million new patients will be diagnosed with cancer [68]. These data illustrate the need for the continual development of improved therapeutic and supportive-care agents. At present, supportive-care products are available to treat a number of comorbid conditions associated with cancer and morbidities associated with cancer treatments. New agents are being developed with novel mechanisms of action or modifications of existing agents that improve performance. Because of the urgent need for such products, efficient development is required to deliver useful products to patients as rapidly as possible. Although we have discussed several examples of successfully developed or promising supportive-care products, many agents entering clinical development will not be successful. An example of this is megakaryocyte growth and development factor (MGDF). In preclinical studies [69–71], this product increased platelet counts with mechanism similar to the endogenous protein, thrombopoetin. Initial clinical studies showed increased platelet counts confirming the preclinical studies [72–74]. The rare but potentially serious adverse events of antibody formation to MGDF leading to severe and potentially chronic thrombocytopenia, however, terminated the clinical program. Analysis of all aspects of this drug development suggests that the ideal candidate for a platelet mobilizer would be one that mimics the endogenous protein but does induce antibody formation. We hope that the examples provided in this chapter will be useful to investigators as they develop the next generation of supportive-care products. Acknowledgments Dr Neumann and Dr Foote are employees of Amgen Inc., the manufacturer of Epogen, Neupogen, Neulasta, and Aranesp; and the developer of rHuKGF, PEG-rHuMGDF, and OPG. References 1. 2. 3. 4. 5.

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Note Added in Proof Pegfilgrastim received marketing approval in the EU in September 2002 for use in chemotherapy-induced neutropenia.

419

Index of authors Azevedo, A.M. 199 Cabral, J.M.S. 199 Derion, T. 249 Fonseca, L.P. 199 Foote, M. 269, 397 Giffin, M. 279 Hecker, S.J. 269 Ilag, L.L. 1 Lawen, A. 151 Martins, V.C. 199 Mazanet, R. 285

McLeish, S. 279 Molowa, D.T. 285 Neumann, T.K. 397 Neves-Petersen, M.T. 315 Ng, J.H. 1 Paik, S. 259 Petersen, S.B. 315 Prazeres, D.M.F. 199 Preston, C. 269 Velkov, T. 151 Vojinovic´, V. 199

421

Keyword index

b-alanine 151 -amino acids 151 applications 199 arrays 1 bacterial cell culture 285 benzhydroxamic acid 199 biosensors 199 biotechnology medical biotechnology 259, 269, 285, 303, 397 calcium 199 cell assays 1 chips 1 clinical research BLA 269 IND 269 phase 1 397 phase 2 269, 397 phase 3 259, 269, 397 PMA 249 publication of trials 303 use of contract research organization (CRO) 269 clinical trial 259 cancer clinical trial 259 combinatorial biosynthesis 151 compound I 199 compound II 199 cyclosporin A 151 cyclosporin synthetase 151 data management 249 Debye–Hu¨ckel 315 Deming Prize 279 diagnostics 1 dielectric constant 315 drug discovery 1 drug screening 1 E. coli 285 electrophoresis 1 electrostatic potential distribution 315 expression profiling 1 ferulic acid 199

genomic markers 259 glycans 199 good publication practices 303 heme proteins 199 horseradish peroxidase 199 hydrogen peroxide 199 -hydroxy acids 151 immunoassays 1 immunosuppresant 151 indole acetic acid 199 industry trends 279 informed consent 249 IVD 249 in vitro biosynthesis 151 in vitro diagnostic 249 lab-on-a-chip 1 laws of electrostatics 315 mammalian cell culture 285 marketing authorization 397 Maxwell equations of electrostatics 315 medical device 249 micro total analysis systems 1 microarray 1, 259 microfluidics 1 modular enzymes 151 molecular surface 315 monitoring 249 multi-functional enzymes 151 nanolithography 1 nanotechnology 1 N-methylation 151 non-ribosomal code 151 non-ribosomal peptide synthetase 151 panel parent 249 patterning 1 peptide antibiotics 151 peptide assembly 151 peptolides 151 40 -phosphopantetheine 151 photolithography 1 physiological role 199 pKa 315

422 plant peroxidases 199 Poisson equation 315 Poisson–Boltzmann equation 315 polyketide synthases 151 precision 249 precursor-directed biosynthesis 151 predictive markers 259 premarket approval 249 preoperative chemotherapy trial 259 protein 1 electrostatics 315 manufacturing 285 proteomics 1

reporter systems 199 reproducibility study 249

quality control 279

uniform requirements for biomedical journals 303 unlinked specimens 249

regulatory agency (ies) CPMP 269 EMEA 269 FDA 249, 269

SDZ 214-103 151 secondary metabolites 151 soft lithography 1 structure 199 Tanford–Kirkwood model 315 thiotemplate mechanism 151 tissue array 1 titratable residues 315 total quality management 279 transgenics 285

Vancouver conventions 303 variability 249