HANDBOOK OF PHARMACEUTICAL BIOTECHNOLOGY

Edited by

SHAYNE COX GAD, PH.D., D.A.B.T.Gad Consulting ServicesCary, North Carolina

WILEY-INTERSCIENCEA John Wiley & Sons, Inc., Publication

InnodataFile Attachment9780470117101.jpg

HANDBOOK OF PHARMACEUTICAL BIOTECHNOLOGY

HANDBOOK OF PHARMACEUTICAL BIOTECHNOLOGY

Edited by

SHAYNE COX GAD, PH.D., D.A.B.T.Gad Consulting ServicesCary, North Carolina

WILEY-INTERSCIENCEA John Wiley & Sons, Inc., Publication

Copyright © 2007 by John Wiley & Sons, Inc. All rights reserved

Published by John Wiley & Sons, Inc., Hoboken, New JerseyPublished simultaneously in Canada

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Library of Congress Cataloging-in-Publication Data:

Printed in the United States of America10 9 8 7 6 5 4 3 2 1

Gad, Shayne C., 1948– Handbook of pharmaceutical biotechnology / Shayne Cox Gad. p. ; cm. Includes bibliographical references and index. ISBN: 978-0-471-21386-4 1. Pharmaceutical biotechnology—Handbooks, manuals, etc. I. Title. [DNLM: 1. Drug Design. 2. Biotechnology. 3. Chemistry, Pharmaceutical. QV 744 G123h 2007] RS380.G33 2007 615′.19—dc22

2006030898

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CONTRIBUTORS

Toshihiro Akaike, Department of Biomolecular Engineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, Japan, Case Studies—Development of Oligonucleotides

Thomas Anchordoquy, School of Pharmacy, University of Colorado at Denver and Health Sciences Center, Denver, Colorado, Basic Issues in the Manufacture of Macromolecules

Aravind Asokan, Division of Molecular Pharmaceutics, School of Pharmacy, and Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, Strategies for the Cytosolic Delivery of Macromolecules: An Overview

Joseph P. Balthasar, University at Buffalo, The State University of New York, Buffalo, New York, Development and Characterization of High-Affi nity Anti-Toptecan IgG and Fab Fragments

Sathy V. Balu-Iyer, University at Buffalo, State University of New York, Amherst, New York, Formulation and Delivery Issues of Therapeutic Proteins

Randal W. Berg, Cancer Research Laboratory Program, London Regional Cancer Program, London, Ontario, Canada; Department of Oncology, The University of Western Ontario, London, Ontario, Canada; and Lawson Health Research Institute, London Health Sciences Centre, London, Ontario, Canada, Pharma-cokinetics of Nucleic-Acid-Based Therapeutics

Isabelle Bertholon, Faculty of Pharmacy, University of Paris XI, Chatenay-Malabry, France, Integrated Development of Glycobiologics: From Discovery to Applications in the Design of Nanoparticular Drug Delivery Systems

v

vi CONTRIBUTORS

Günter Blaich, Abbott Bioresearch Center, Worcester, Massachusetts, Overview: Differentiating Issues in the Development of Macromolecules Compared with Small Molecules

Jeanine I. Boulter-Bitzer, Department of Environmental Biology, University of Guelph, Guelph, Ontario, Canada, Recombinant Antibodies for Pathogen Detection and Immunotherapy

Tania Bubela, Department of Marketing, University of Alberta, Edmonton, Alberta, Canada, Intellectual Property and Biotechnolgy

Brian E. Cairns, University of British Columbia, Vancouver, British Columbia, Canada, Growth Factors and Cytokines

Heping Cao, Peace Technology Development, North Potomac, Maryland, Growth Factors, Cytokines, and Chemokines: Formulation, Delivery, and Pharmacokinetics

María de los Angeles Cortés Castillo, Technology and Health Service Delivery, Pan American Health Organization, Washington, DC, Regulation of Small-Molecule Drugs versus Biologicals versus Biotech Products

Jin Chen, Department of Pharmaceutical Sciences, University at Buffalo, The State University of New York, Buffalo, New York, Development and Character-ization of High-Affi nity Anti-Toptecan IgG and Fab Fragments

Roland Cheung, Division of Molecular Pharmaceutics, School of Pharmacy, and Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, Strategies for the Cytosolic Delivery of Macromolecules: An Overview

Moo J. Cho, Division of Molecular Pharmaceutics, School of Pharmacy, and Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, Strategies for the Cytosolic Delivery of Macromolecules: An Overview

Yong Woo Cho, Asan Institute for Life Sciences, University of Ulsan College of Medicine, Seoul, Korea, PEGylation: Camoufl age of Proteins, Cells, and Nanoparticles Against Recognition by the Body’s Defense Mechanism

Albert H.L. Chow, School of Pharmacy, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong, China, Stability Assessment and Formulation Characterization

Ezharul Hoque Chowdhury, Department of Biomolecular Engineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, Japan, Case Studies—Development of Oligonucleotides

Daan J.A. Crommelin, Department of Pharmaceutics, Utrecht Institute for Phar-maceutical Sciences (UIPS), Utrecht University, The Netherlands, Immunoge-nicity of Therapeutic Proteins; Biosimilars

Mary Jane Cunningham, Houston Advanced Research Center (HARC), The Woodlands, Texas, Toxicogenomics

CONTRIBUTORS vii

Vincenzo De Filippis, Department of Pharmaceutical Sciences, University of Padova, Padova, Italy, Protein Engineering with Noncoded Amino Acids: Appli-cations to Hirudin

Pascal Delépine, EFS Bretagne—Site de Brest, INSERM U613, Brest, France, Assessing Gene Therapy by Molecular Imaging

Binodh DeSilva, Amgen, Inc., Thousand Oaks, California, Analytical Consider-ations for Immunoassays for Macromolecules

José Luis Di Fabio, Technology and Health Service Delivery, Pan American Health Organization, Washington, DC, Regulation of Small-Molecule Drugs versus Biologicals versus Biotech Products

Karen Lynn Durell, Center for Intellectual Property Policy, Faculty of Law, McGill University, Montreal, Quebec, Canada, Intellectual Property and Biotechnolgy

Andrew Emili, Banting and Best Department of Medical Research, Department of Medical Genetics and Microbiology, Donnelly Centre for Cellular and Bio-molecular Research, University of Toronto, Toronto, Ontario, Canada, Enhanced Proteomic Analysis by HPLC Prefractionation

Claude Férec, EFS Bretagne—Site de Brest, INSERM U613, Brest, France, Assessing Gene Therapy by Molecular Imaging

Zoltan Gombos, National Institute for Nanotechnology, University of Alberta, Edmonton, Alberta, Canada, Proteins: Hormones, Enzymes, and Monoclonal Antibodies—Background

Matthew D. Gray, MDG Associates, Inc., Seattle, Washington, RNA Interference: The Next Gene-Targeted Medicine

J. Chris Hall, Department of Environmental Biology, University of Guelph, Guelph, Ontario, Canada, Recombinant Antibodies for Pathogen Detection and Immunotherapy

Pierre C. Havugimana, Banting and Best Department of Medical Research, Department of Medical Genetics and Microbiology, Donnelly Centre for Cel-lular and Biomolecular Research, University of Toronto, Toronto, Ontario, Canada, Enhanced Proteomic Analysis by HPLC Prefractionation

Suzanne Hermeling, Department of Pharmaceutics, Utrecht Institute for Pharma-ceutical Sciences (UIPS); Central Laboratory Animal Institute, Utrecht Univer-sity, The Netherlands, Immunogenicity of Therapeutic Proteins

M.D. Mostaqul Huq, Department of Pharmacology, University of Minnesota Medical School, Minneapolis, Minnesota, Protein Posttranslational Modifi ca-tion: A Potential Target in Pharmaceutical Development

Yukako Ito, Department of Pharmacokinetics, Kyoto Pharmaceutical University, Kyoto, Japan, Pharmacokinetics

Bernd Janssen, Abbott GmbH & Co. KG, Ludwigshafen, Germany, Overview: Differentiating Issues in the Development of Macromolecules Compared with Small Molecules

viii CONTRIBUTORS

Wim Jiskoot, Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences (UIPS), Utrecht University, The Netherlands; Division of Drug Deliv-ery Technology, Leiden/Amsterdam Center for Drug Research (LACDR), Leiden University, Leiden, The Netherlands, Immunogenicity of Therapeutic Proteins; Biosimilars

Beth Junker, Merck Research Laboratories, Rahway, New Jersey, Process Valida-tion for Biopharmaceuticals

David Keast, Site Chief of Family Medicine, Parkwood Hospital, St. Joseph’s Health Care, London, Ontario, Canada; Clinical Adjunct Professor of Family Medicine, University of Western Ontario, London, Ontario, Canada, Unex-pected Benefi ts of a Formulation: Case Study with Erythropoetin

Marian Kelley, Centocor R&D, Inc., Randor, Pennsylvania, Analytical Consider-ations for Immunoassays for Macromolecules

Naoya Kobayashi, Department of Surgery, Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan, Overview of Stem and Artifi cial Cells

James Koropatnick, Cancer Research Laboratory Program, London Regional Cancer Program, London, Ontario, Canada; The University of Western Ontario (Departments of Microbiology and Immunology; Oncology; Physiology and Pharmacology; and Pathology), London, Ontario, Canada; and Lawson Health Research Institute, London Health Sciences Centre, London, Ontario, Canada, Pharmacokinetics of Nucleic-Acid-Based Therapeutics

Krishnanand D. Kumble, Genesis Research, Parnell, Auckland, New Zealand, Microarrays in Drug Discovery and Development

Sarita Kumble, Pictor Limited, Glendowie, Auckland, New Zealand, Microarrays in Drug Discovery and Development

Anne E. Kwitek, Human and Molecular Genetics Center, Department of Physiol-ogy, Medical College of Wisconsin, Milwaukee, Wisconsin, Genetic Markers and Genotyping Analyses for Genetic Disease Studies

Denis Labarre, Faculty of Pharmacy, University of Paris XI, Chatenay-Malabry, France, Integrated Development of Glycobiologics: From Discovery to Applica-tions in the Design of Nanoparticular Drug Delivery Systems

Hung Lee, Department of Environmental Biology, University of Guelph, Guelph, Ontario, Canada, Recombinant Antibodies for Pathogen Detection and Immunotherapy

Kang Choon Lee, Drug Targeting Laboratory, College of Pharmacy, SungKyunKwan University, Jangan-ku, Suwon, Korea, Capillary Separation Techniques

Corinne Lengsfeld, Department of Engineering, University of Denver, Denver, Colorado, Basic Issues in the Manufacture of Macromolecules

Jun Li, School of Chemical Biology and Pharmaceutical Studies, Capital Medical University of Medical Sciences, Beijing, China, Pharmaceutical Bioassay

CONTRIBUTORS ix

Rui Lin, Peace Technology Development, North Potomac, Maryland, Growth Factors, Cytokines, and Chemokines: Formulation, Delivery, and Pharmacokinetics

John C. Lindon, Department of Biomolecular Medicine, Faculty of Medicine, Imperial College, South Kensington, London, United Kingdom, An Overview of Metabonomics Techniques and Applications

Donald E. Mager, Department of Pharmaceutical Sciences, University at Buffalo, The State University of New York, Buffalo, New York, Preclinical Pharmacokinetics

Krishan Maggon, Pharma Biotech R & D Consultant, Geneva, Switzerland, R&D Paradigm Shift and Billion-Dollar Biologics

Mandeep K. Mann, University of British Columbia, Vancouver, British Columbia, Canada, Growth Factors and Cytokines

Wayne Materi, National Institute for Nanotechnology, University of Alberta, Edmonton, Alberta, Canada, Proteins: Hormones, Enzymes, and Monoclonal Antibodies—Background

Razvan D. Miclea, Roswell Park Cancer Institute, Buffalo, New York, Formula-tion and Delivery Issues of Therapeutic Proteins

Jan Moebius, Rudolf-Virchow-Center for Experimental Biomedicine, Würzburg, Germany, Chromatography-Based Separation of Proteins, Peptides, and Amino Acids

Dong Hee Na, College of Pharmacy, Kyungsung University, Nam-ku, Busan, Korea, Capillary Separation Techniques

Nalu Navarro-Alvarez, Department of Surgery, Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan, Overview of Stem and Artifi cial Cells

Michael Oettel, Prof. med. Vet. habil., Jena, Germany, The Promise of Individual-ized Therapy

Andrew V. Oleinikov, Seattle Biomedical Research Group, Seattle, Washington, RNA Interference: The Next Gene-Targeted Medicine

Michael Olivier, Human and Molecular Genetics Center, Department of Physiol-ogy, Medical College of Wisconsin, Milwaukee, Wisconsin, Genetic Markers and Genotyping Analyses for Genetic Disease Studies

Aleksandra Pandyra, Cancer Research Laboratory Program, London Regional Cancer Program, London, Ontario, Canada; Department of Microbiology and Immunology, The University of Western Ontario, London, Ontario, Canada, Pharmacokinetics of Nucleic-Acid-Based Therapeutics

Jae Hyung Park, Department of Advanced Polymer and Fiber Materials, College of Environment and Applied Chemistry, Kyung Hee University, Gyeonggi-do, Korea, PEGylation: Camoufl age of Proteins, Cells, and Nanoparticles Against Recognition by the Body’s Defense Mechanism

x CONTRIBUTORS

Ji Sun Park, Asan Institute for Life Sciences, University of Ulsan College of Medicine, Seoul, Korea, PEGylation: Camoufl age of Proteins, Cells, and Nanoparticles Against Recognition by the Body’s Defense Mechanism

Kinam Park, Departments of Pharmaceutics and Biomedical Engineering, Purdue University, West Lafayette, Indiana, PEGylation: Camoufl age of Proteins, Cells, and Nanoparticles Against Recognition by the Body’s Defense Mechanism

Steve Pascolo, Institute for Cell Biology, Department of Immunology, University of Tübingen, Tübingen, Germany, Plasmid DNA and Messenger RNA for Therapy

Shiqi Peng, School of Chemical Biology and Pharmaceutical Studies, Capital Medical University of Medical Sciences, Beijing, China, Pharmaceutical Bioassay

Nicholas J. Pokorny, Department of Environmental Biology, University of Guelph, Guelph, Ontario, Canada, Recombinant Antibodies for Pathogen Detection and Immunotherapy

Vivek S. Purohit, R & D, Eurand, Inc., Vandalia, Ohio, Formulation and Delivery Issues of Therapeutic Proteins

D.M.F. Prazeres, Institute for Biotechnology and Bioengineering, Center for Bio-logical and Chemical Engineering, Instituto Superior Técnico, Lisbon, Portugal, Production and Purifi cation of Adenovirus Vectors for Gene Therapy

Murali Ramanathan, Department of Pharmaceutical Sciences, University at Buffalo, The State University of New York, Buffalo, New York, Preclinical Pharmacokinetics

Raymond M. Reilly, Departments of Pharmaceutical Sciences and Medical Imaging, University of Toronto, Toronto, Ontario, Cananda, The Radiopharma-ceutical Science of Monoclonal Antibodies and Peptides for Imaging and Tar-geted in situ Radiotherapy of Malignancies

Jorge David Rivas-Carillo, Department of Surgery, Okayama University Gradu-ate School of Medicine and Dentistry, Okayama, Japan, Overview of Stem and Artifi cial Cells

Gregory Roth, Abbott GmbH & Co. KG, Ludwigshafen, Germany, Overview: Differentiating Issues in the Development of Macromolecules Compared with Small Molecules

Gabor M. Rubanyi, Cardium Therapeutics, Inc., San Diego, California, Gene Therapy—Basic Principles and the Road from Bench to Bedside

Jochen Salfeld, Abbott GmbH & Co. KG, Ludwigshafen, Germany, Overview: Differentiating Issues in the Development of Macromolecules Compared with Small Molecules

J.A.L. Santos, Institute for Biotechnology and Bioengineering, Center for Biologi-cal and Chemical Engineering, Instituto Superior Técnico, Lisbon, Portugal, Production and Purifi cation of Adenovirus Vectors for Gene Therapy

CONTRIBUTORS xi

Huub Schellekens, Department of Pharmaceutics, Utrecht Institute for Pharma-ceutical Sciences (UIPS); Central Laboratory Animal Institute, Utrecht Univer-sity, The Netherlands, Immunogenicity of Therapeutic Proteins; Biosimilars

Frank-Ranier Schmidt, Sanofi -Aventis Deutschland, Frankfurt am Main, Germany, From Gene to Product: The Advantage of Integrative Biotechnology

John C. Schmitz, VACT Healthcare System, VA Cancer Center, and Yale Cancer Center, Yale University School of Medicine, West Haven, Connecticut, Pharma-cokinetics of Nucleic-Acid-Based Therapeutics

Herbert Schott, Institute of Organic Chemistry, University of Tübingen, Tübin-gen, Germany, Delivery Systems for Peptides/Oligonucleotides and Lipophilic Nucleoside Analogs

R.A. Schwendener, Institute of Molecular Cancer Research, University of Zurich, Zurich, Switzerland, Delivery Systems for Peptides/Oligonucleotides and Lipo-philic Nucleoside Analogs

Gerhard K.E. Scriba, Friedrich-Schiller-University Jena, School of Pharmacy, Jena, Germany, Bioanalytical Method Validation for Macromolecules

Tatiana Segura, Chemical and Biomolecular Engineering Department, University of California, Los Angeles, Los Angeles, California, Formulations and Delivery Limitations of Nucleic-Acid-Based Therapies

Mrinal Shah, Houston Advanced Research Center (HARC), The Woodlands, Texas, Toxicogenomics

Nobuhito Shibata, Department of Biopharmaceutics, Faculty of Pharmaceu-tical Science, Doshisha Women’s College of Liberal Arts, Kyoto, Japan, Pharmacokinetics

Dany Shoham, Begin–Sadat Center for Strategic Studies, Bar Ilan University, Israel, Bioterrorism

Albert Sickmann, Rudolf-Virchow-Center for Experimental Biomedicine, Würzburg, Germany, Chromatography-Based Separation of Proteins, Peptides, and Amino Acids

Alejandro Soto-Gutierrez, Department of Surgery, Okayama University Gradu-ate School of Medicine and Dentistry, Okayama, Japan, Overview of Stem and Artifi cial Cells

Patrick A. Stewart, Department of Political Science, Arkansas State University, State University, Arkansas, Comparability Studies for Later-Generation Prod-ucts—Plant-Made Pharmaceuticals

Remco Swart, LC-Packings—A Dionex Company, Amsterdam, The Netherlands, Chromatography-Based Separation of Proteins, Peptides, and Amino Acids

Kanji Takada, Department of Pharmacokinetics, Kyoto Pharmaceutical Univer-sity, Kyoto, Japan, Pharmacokinetics

Henry H.Y. Tong, School of Health Sciences, Macao Polytechnic Institute, Macao, China, Stability Assessment and Formulation Characterization

xii CONTRIBUTORS

Jack T. Trevors, Department of Environmental Biology, University of Guelph, Guelph, Ontario, Canada, Recombinant Antibodies for Pathogen Detection and Immunotherapy

Christine Vauthier, Faculty of Pharmacy, University of Paris XI, Chatenay-Malabry, France, Integrated Development of Glycobiologics: From Discovery to Applications in the Design of Nanoparticular Drug Delivery Systems

Ioannis S. Vizirianakis, Laboratory of Pharmacology, Department of Pharmaceu-tical Sciences, Aristotle University of Thessaloniki, Thessaloniki, Greece, From Defi ning Bioinformatics and Pharmacogenomics to Developing Information-Based Medicine and Pharmacotyping in Health Care

Li-Na Wei, Department of Pharmacology, University of Minnesota Medical School, Minneapolis, Minnesota, Protein Posttranslational Modifi cation: A Potential Target in Pharmaceutical Development

David S. Wishart, National Institute of Nanotechnology, Departments of Biologi-cal Science, Computing Science, and Pharmaceutical Research, University of Alberta, Edmonton, Alberta, Canada, Proteins: Hormones, Enzymes, and Monoclonal Antibodies—Background

Peter Wong, Banting and Best Department of Medical Research, Department of Medical Genetics and Microbiology, Donnelly Centre for Cellular and Biomo-lecular Research, University of Toronto, Toronto, Ontario, Canada, Enhanced Proteomic Analysis by HPLC Prefractionation

Eugene Zabarovsky, Microbiology and Tumor Biology Center, Karolinska Insti-tute, Stockholm, Sweden, Sequencing the Human Genome: Was It Worth It?

Ming Zhao, School of Chemical Biology and Pharmaceutical Studies, Capital Medical University of Medical Sciences, Beijing, China, Pharmaceutical Bioassay

Ying Zheng, Institute of Chinese Medical Sciences, University of Macao, Macao, China, Stability Assessment and Formulation Characterization

CONTENTS

Preface xix

1.1 From Gene to Product: The Advantage of Integrative Biotechnology 1

Frank-Ranier Schmidt

1.2 Sequencing the Human Genome: Was It Worth It? 53 Eugene Zabarovsky

1.3 Overview: Differentiating Issues in the Development of Macromolecules Compared with Small Molecules 89

Günther Blaich, Bernd Janssen, Gregory Roth, and Jochen Salfeld

1.4 Integrated Development of Glycobiologics: From Discovery to Applications in the Design of Nanoparticular Drug Delivery Systems 125

Christine Vauthier, Isabelle Bertholon, and Denis Labarre

1.5 R&D Paradigm Shift and Billion-Dollar Biologics 161 Krishan Maggon

2 From Defi ning Bioinformatics and Pharmacogenomics to Developing Information-Based Medicine and Pharmacotyping in Health Care 201

Ioannis S. Vizirianakis

3.1 Toxicogenomics 229 Mary Jane Cunningham and Mrinal Shah

xiii

xiv CONTENTS

3.2 Preclinical Pharmacokinetics 253 Donald E. Mager and Murali Ramanthan

3.3 Strategies for the Cytosolic Delivery of Macromolecules: An Overview 279

Aravind Asokan, Roland Cheung, and Moo J. Cho

4.1 Basic Issues in the Manufacture of Macromoleucles 297 Corinne Lengsfeld and Thomas Anchordoquy

4.2 Process Validation for Biopharmaceuticals 319 Beth Junker

4.3 Stability Assessment and Formulation Characterization 371 Albert H.L. Chow, Henry H.Y. Tong, and Ying Zheng

4.4 Protein Posttranslational Modifi cation: A Potential Target in Pharmaceutical Development 417

M.D. Mostaqul Huq and Li-Na Wei

4.5 PEGylation: Camoufl age of Proteins, Cells, and Nanoparticles Against Recognition by the Body’s Defense Mechanism 443

Yong Woo Cho, Jae Hyung Park, Ji Sun Park, and Kinam Park

4.6 Unexpected Benefi ts of a Formulation: Case Study with Erythropoetin 463

David Keast

5.1 Capillary Separation Techniques 469 Dong Hee Na and Kang Choon Lee

5.2 Pharmaceutical Bioassay 511 Jun Li, Ming Zhao, and Shiqi Peng

5.3 Analytical Considerations for Immunoassays for Macromolecules 573

Marian Kelley and Binodh DeSilva

5.4 Chromatography-Based Separation of Proteins, Peptides, and Amino Acids 585

Jan Moebius, Remco Swart, and Albert Sickman

5.5 Bioanalytical Method Validation for Macromolecules 611 Gerhard K.E. Scriba

5.6 Microarrays in Drug Discovery and Development 633 Krishnanand D. Kumble and Sarita Kumble

CONTENTS xv

5.7 Genetic Markers and Genotyping Analyses for Genetic Disease Studies 661

Anne E. Kwitek and Michael Olivier

6.1 Proteins: Hormones, Enzymes, and Monoclonal\Antibodies—Background 691

Wayne Materi, Zoltan Gombos, and David S. Wishart

6.2 Formulation and Delivery Issues of Therapeutic Proteins 737 Sathy V. Balu-Iyer, Razvan D. Miclea, and Vivek S. Purohit

6.3 Pharmacokinetics 757 Nobuhito Shibata, Yukato Ito, and Kanji Takada

6.4 Immunogenicity of Therapeutic Proteins 815 Suzanne Hermeling, Daan J.A. Crommelin, Huub Schellekens,

and Wim Jiskoot

6.5 Development and Characterization of High-Affi nity Anti-Topotecan IgG and Fab Fragments 835

Jin Chen and Joseph P. Balthasar

6.6 Recombinant Antibodies for Pathogen Detection and Immunotherapy 851

Nicholas J. Pokorny, Jeanine I. Boulter-Bitzer, J. Chris Hall, Jack T. Trevors, and Hung Lee

6.7 The Radiopharmaceutical Science of Monoclonal Antibodies and Peptides for Imaging and Targeted in situ Radiotherapy of Malignancies 883

Raymond M. Reilly

7.1 Gene Therapy—Basic Principles and the Road from Bench to Bedside 943

Gabor M. Rubanyi

7.2 Plasmid DNA and Messenger RNA for Therapy 971 Steve Pascolo

7.3 Formulations and Delivery Limitations of Nucleic-Acid-Based Therapies 1013

Tatiana Segura

7.4 Pharmacokinetics of Nucleic-Acid-Based Therapeutics 1061 John C. Schmitz, Aleksandra Pandyra, James Koropatnick,

and Randal. W. Berg

xvi CONTENTS

7.5 Case Studies—Development of Oligonucleotides 1087 Ezharul Hoque Chowdhury and Toshihiro Akaike

7.6 RNA Interference: The Next Gene-Targeted Medicine 1109 Andrew V. Oleinikov and Matthew D. Gray

7.7 Delivery Systems for Peptides/Oligonucleotides and Lipophilic Nucleoside Analogs 1149

R.A. Schwendener and Herbert Schott

8.1 Growth Factors and Cytokines 1173 Mandeep K. Mann and Brian E. Cairns

8.2 Growth Factors, Cytokines, and Chemokines: Formulation, Delivery, and Pharmacokinetics 1197

Heping Cao and Rui Lin

9 Protein Engineering with Noncoded Amino Acids: Applications to Hirudin 1225

Vincenzo De Filippis

10.1 Production and Purifi cation of Adenovirus Vectors for Gene Therapy 1261

D.M.F. Prazeres and J.A.L. Santos

10.2 Assessing Gene Therapy by Molecular Imaging 1297 Pascal Delepine and Claude Férec

11 Overview of Stem and Artifi cial Cells 1313 Alejandro Soto-Gutierrez, Nalu Navarro-Alvarez,

Jorge David Rivas-Carrillo, and Naoya Kobayashi

12.1 Regulation of Small-Molecule Drugs Versus Biologicals Versus Biotech Products 1373

María de los Angeles Cortés Castillo and José Luis Di Fabio

12.2 Intellectual Property and Biotechnology 1391 Tania Bubela and Karen Lynne Durell

12.3 Comparability Studies for Later-Generation Products—Plant-Made Pharmaceuticals 1433

Patrick A. Stewart

12.4 Biosimilars 1453 H. Schellekens, W. Jiskoot, and D.J.A. Crommelin

CONTENTS xvii

13.1 The Promise of Individualized Therapy 1463 Michael Oettel

13.2 Enhanced Proteomic Analysis by HPLC Prefractionation 1491 Pierre C. Havugimana, Peter Wong, and Andrew Emili

13.3 An Overview of Metabonomics Techniques and Applications 1503 John C. Lindon

13.4 Bioterrorism 1525 Dany Shoham

Index 1653

xix

PREFACE

This Handbook of Pharmaceutical Biotechnology represents a unique attempt to overview the full range of approaches to discovering, selecting, and producing potentially new therapeutic moieties resulting from biological process. Such moi-eties are the backbone of both the pharmaceutical industry and the prime axis for the advancement of medical science.

The volume is unique in that it seeks to cover possible approaches to the bio-technology drug process as broadly as possible while not just doing so in a superfi -cial manner. Thanks to the persistent efforts of Gladys Mok, these 50 chapters cover all major approaches to the problem of identifying, producing, and formulat-ing new biologically derived therapeutics and were written by leading practitioners in each of these areas.

I hope that this second course of our banquet is satisfying and useful to all those working in or entering the fi eld.

Select fi gures of this title are available in full color at ftp://ftp.wiley.com/public/sci_tech_med/pharmaceutical_biotech/.

S.C. Gad

1

Handbook of Pharmaceutical Biotechnology, Edited by Shayne Cox Gad.Copyright © 2007 John Wiley & Sons, Inc.

1.1FROM GENE TO PRODUCT: THE ADVANTAGE OF INTEGRATIVE BIOTECHNOLOGY

Frank-Ranier SchmidtSanofi -Aventis Deutschland, Frankfurt am Main, Germany

Chapter Contents

1.1.1 Introduction 21.1.2 Production of Organisms and Expression Systems 3 1.1.2.1 Industrially Established Recombinant Expression Systems 3 1.1.2.2 Evaluation of Secretory Expression Systems for

Pharmaceutical Purposes 5 1.1.2.3 Criteria for the Choice of Recombinant Expression Systems 111.1.3 Enhancement of Productivity 13 1.1.3.1 Secretory Recombinant Expression Systems 13 1.1.3.2 Natural Products 13 1.1.3.3 Approaches and Goals for Further Strain Improvement 161.1.4 Biosynthetic Structure Modifi cation 19 1.1.4.1 Combinatorial Biosynthesis 19 1.1.4.2 Precursor Directed Biosynthesis 201.1.5 Fermentation Optimization and Scale-Up 22 1.1.5.1 Reduced Mixing Quality and Enhanced Stress Exposure 22 1.1.5.2 Process Characterization 26 1.1.5.3 Process Optimization 28 1.1.5.4 Physical Scale-Up Parameters 29 1.1.5.5 Development of Fermentation Models and Strategies 311.1.6 Downstream Processing 31 1.1.6.1 Product Recovery and Purifi cation 31 1.1.6.2 Downstream Processing Optimization and Economization 32 1.1.6.3 Downstream Processing Scale-Up 33 1.1.6.4 Downstream Processing of β-Lactam Compounds 34

2 THE ADVANTAGE OF INTEGRATIVE BIOTECHNOLOGY

1.1.7 Postsynthetic Structure Modifi cation 34 1.1.7.1 β-Lactam Side Chain Cleavage 351.1.8 Quality Issues 37 1.1.8.1 Virus and Endotoxin Removal 381.1.9 Conclusion 38 References 38

1.1.1 INTRODUCTION

Biotechnology and biotechnology-based methods are increasing in importance in medical therapies and diagnostics as well as in the discovery, development, and manufacture of pharmaceuticals. Biotechnologically manufactured pharmaceuti-cals will soon reach a market volume of more than $100 billion USD and, thus, some 20% of the total pharmaceutical market. The key step in their manufacture is the conversion of the genetic information into a product with the desired phar-macological activities by an appropriate selection, design, and cultivation of cells and microorganisms harboring the corresponding biosynthetic pathways and physi-ological properties.

The intention of this chapter is to give insights into the typical issues and prob-lems encountered in the manufacture of biopharmaceuticals, to mediate general ideas and current strategies on how to proceed in the design and development of biotechnological processes, and to deliver the immediate theoretical backgrounds necessary for comprehension rather than to give detailed experimental instructions like a manual does. In focusing on gene recombinant proteins and peptidic anti-biotics, the biotechnologically produced pharmaceuticals with the highest market share, representative aspects will be discussed (1) for process development and optimization approaches to increase product yield and process rentability and to ensure a consistent product quality, (2) for experimental approaches to design and to modify the molecular structure of compounds to meet specifi c medical needs, (3) for the replacement of chemical procedures by economically and ecologically advantageous biotechnological processes, (4) for critical issues of product purifi ca-tion, and (5) for specifi c demands in pharmaceutical production to conform to regulatory requirements. Finally, the advantage of an integrative biotechnology is emphasized, which designs the biosynthetic steps of the product in accordance with the requirements of product purifi cation procedures already during early develop-ment stages.1

For further reviews comprehensively illustrating issues of biotechnological pro-duction processes, the reader is referred to further review articles [1–8]. To look up basic subjects of molecular and cellular biology, the reader is referred to textbooks [9, 10].

1 Parts of this chapter were excerpted and modifi ed from previously published reviews of the author [16, 86, 98, 113, 273] by courtesy of Springer-Verlag, Heidelberg.

1.1.2 PRODUCTION ORGANISMS AND EXPRESSION SYSTEMS

Design and development of all microbial production processes start with the selec-tion of appropriate organisms, strains, and expression systems enabling high yields and high quality of a desired product with defi ned pharmacological properties.

1.1.2.1 Industrially Established Recombinant Expression Systems

Industrially established expressions systems for production of the marketed com-pounds are, besides inclusion, body-forming Escherichia coli strains, the yeastSaccharomyces cerevisiae and mammalian cells like CHO- and BHK-cells (Table 1.1-1). These systems were the genetically and physiologically most advanced and therefore mostly applied when recombinant production processes were starting to be developed in the mid-1980s and are now widely accepted by regulatory bodies. E. coli and S. cerevisiae can be grown cheaply and rapidly, are amenable to high cell density fermentations with biomasses of up to 130 g/L, possess short generation times, have high capacities to accumulate foreign proteins, are easy to handle, and are established fermentation organisms.

However, because gene recombinant pharmaceuticals continuously gain an increasing importance in medicine and are expected to help curing diseases that are not yet treatable today, new expression systems have to be exploited enabling the production of such pharmaceuticals with innovative properties that simultane-ously meet key criteria like consistent product quality and cost effectiveness. Of particular interest in this regard are expression systems enabling the secretion of

TABLE 1.1-1. Industrially Used Recombinant Expression Systems

Product Company System

Blood coagulation factors Novo-Nordisk/Bayer/ BHK-Cells (VII, VIII, IX) Centeon Genetics Baxter/ CHO-Cells Centeon/WyethCalcitonin Unigene E. coli/CHO-CellsDNase (cystic fi brosis) Roche CHO-CellsErythropoetin Janssen-Cilag/Amgen/ CHO-Cells Boehringer Darbepoetin Amgen CHO-CellsFollicle stimulating hormone Serono/Organon CHO-Cells (follitropin)Luteinisation hormone Serono CHO-CellsGonadotropin Serono CHO-CellsGlucagon Novo-Nordisk S. cerevisiaeGlucocerebrosidase Genzyme CHO-Cells (Gaucher-disease)Growth hormones Pharmacia & Upjohn/Lilly/ E. coli (somatotropines) Novo-Nordisk/Ferring/ Genentech Serono Mouse Cell Line Serono/Bio-Technology CHO-Cells General Corp

PRODUCTION ORGANISMS AND EXPRESSION SYSTEMS 3

4 THE ADVANTAGE OF INTEGRATIVE BIOTECHNOLOGY

TABLE 1.1-1. Continued

Product Company System

Eutropin (Human growth LG Chemical S. cerevisiae hormone derivative)Growth factors (GCSF u. Novartis/Essex/Amgen/ E. coli GMCSF) Roche Chugai Pharmaceuticals CHO-CellsPlatelet-derived growth Janssen-Cilag S. cerevisiae

factor (PDGF)PDGF-Agonist ZymoGenetics S. cerevisiaeHepatitis B vaccine GlaxoSmithKline S. cerevisiae Rhein Biotech H. polymorphaHirudin Sanofi -Aventis/Novartis S. cerevisiaeInsulin and muteins Sanofi -Aventis/Lilly/Berlin- E. coli ChemieInsulin Bio-Technology General E. coli

Corp Novo-Nordisk S. cerevisiaeInterferon alpha and muteins Roche/Essex/Yamanouchi E. coliInterferon beta Schering E. coli Biogen/Serono CHO-CellsInterferon gamma (mutein) Amgen/Boehringer E. coliInterleukin 2 Chiron E. coliOprelvekin (interleukin Wyeth Human Cell Line 11-agonist) ROMI 8866OP-1 (osteogenic, Curis/Striker E. coli

neuroprotective factor)Tissue plasminogen-activator Genentech/Roche/Boehringer CHO-CellsRecombinant plasminogen- Genentech/Roche/Boehringer E. coli

activatorStem cell factor Amgen CHO-CellsTumor necrosis factor Boehringer E. coli

Note: Overview on the currently worldwide commercialized recombinant pharmaceuticals and the expression systems employed for their production. The substances are not listed strictly alphabetically but are partially grouped according to therapeutic areas. Antibodies, which are mostly manufactured by hybridoma cell line systems, are not considered. Data were extracted from the European patent database Esp@cenet (http://de.espacenet.com) and the IDdb3-database (http://www.iddb3.com). BHK = baby hamster kidney; CHO = chinese hamster ovary. (Taken from Ref. 16 © Springer-Verlag, Heidelberg)

correctly glycosylated and folded proteins into the culture broth. Such secretory systems offer advantages in terms of simple and fast product purifi cation proce-dures and the avoidance of costly cell rupture, denaturation, and refolding pro-cesses (see Section 1.1.4) and thus conform to the requirements of an integrated production process.

Even though animal and plant systems (molecular pharming, [11]) and secretory plant cell culture systems have received a great deal of attention, their commercial feasibility is still under investigation, particularly with respect to their slightly dif-ferent posttranslational modifi cation modus leading to an altered pharmacological behavior and to allergenic properties [12, 13]. Established in the pharmaceutical

industry as production organisms are, besides the above-mentioned systems, further prokaryotic and yeast species as well as fi lamentous fungi, which are already employed for the manufacture of natural products (see Section 1.1.2.2).

The suitability of the most prominent secretory systems among these organisms from the viewpoint of an integrative process design for the manufacture of recom-binant proteins will be evaluated in the next section by discussing their potential productivity and their physiological properties.

1.1.2.2 Evaluation of Secretory Expression Systems for Pharmaceutical Purposes

Escherichia Coli. As E. coli lacks fundamental prerequisites for effi cient secretion, the marketed pharmaceuticals (Table 1.1-1) manufactured by E. coli-systems are mostly produced as inclusion bodies. Due to the membrane structure, the low chaperone and foldase level and the high periplasmatic protease concentration E. coli-secretion systems allow only comparably low product yields, making them suitable only for compounds marketed in small quantities like orphan drugs. Genentech (San Francisco, CA), for instance, has patented a secretory E. coli-system for the preparation of human growth hormone [14]. The secretory potential of E. coli is indicated by exceptional high product titers in the range of several grams per liter, which were reached in a system developed for secretion of hirudin using the alpha-cyclodextringlykosysl-transferase signal sequence as a leader und secretor mutants defi cient in their membrane structure [15]. Titers of human-insulin-like-growth-factor or human-epidermal-growth-factor were reported to be as high as 900 mg/L and 325 mg/L, respectively [references in 16]. Most of the reached and published data, however, refers to processes leading to a periplasmatic product concentration (e.g., 2 g/L of a human antibody fragment, 700 mg/L of a monoclonal antibody) or stays below 100 mg/L, a value that generally is not considered to be in a competitive and economic range. Efforts are thus undertaken to condition E. coli-strains to effi cient secreters. The main strategies to enhance secretion effi ciency [17–19] comprise (1) employment of well-characterized secretion pathways like the alpha-hemolysin system [20] or components of such pathways like effi cient signal sequences from effl ux proteins [21] or outer membrane proteins [22], for instance, the maltose binding protein [23] or the TolC-protein [24]; (2) variation of the signal peptide; (3) cocloning of and coexpression of chaperones and foldases [25–27]; (4) enhancement of gene expression by employment of strong promotors and effi cient transcription termination sequences; (5) generation of protease defi cient mutants; (6) generation of cell wall lacking or cell wall defi cient mutants [28, 29]; and (7) modulation of the protein primary structure that was found to exert a strong infl uence on productivity and secretion effi ciency by infl uencing protease resistance, folding effi ciency, and the tendency to form inclusion bodies. Details of the strategies for the design and development of secretory E. coli strains as well as for the controlled soluble cytoplasmatic expression of recombinant proteins may be taken from general reviews [17, 30–32].

Alternative Prokaryotic Expression Systems. In addition to conditioning E. coli-strains to effi cient secreters, alternative species, which are considered to inherently possess a superior secretion capacity, are tried to be established as expression

PRODUCTION ORGANISMS AND EXPRESSION SYSTEMS 5

6 THE ADVANTAGE OF INTEGRATIVE BIOTECHNOLOGY

systems. Comparably high product yields of 2 g/L and 1 g/L were reported for production of human calcitonin by Staphylococcus carnosus [33] and of proinsulin by Bacillus subtilis [34], an organism that is continuously characterized and improved as a cell factory for pharmaceutical proteins [8]. Bacillus megaterium,which is thought to be as effi cient as B. subtilis, is currently developed as a secretory expression system by a Collaborative Research Center (SFB) of the German Research Community (DFG). For Ralstonia eutropha (formerly Alcaligenes eutrophus), employed at ICI and Monsanto for polyhydroxyalkanoate production at a scale of several 100 m3 and genomically completely sequenced, 1,2 g/L of secreted organophosphohydrolase, a model enzyme proned to form inclusion bodies in E. coli, were reported [35]. R. eutropha displays a more effi cient carbohydrate metabolism than E. coli and is easily amenable to high cell density fermentations with biomass concentration of more than 150 g/L dry weight. This permits a lower specifi c productivity that in turn reduces the inclinement to form inclusion bodies and thus enables a more effi cient secretion. Rhodococcus, Corynebacterium,Mycobacterium, actinomycetes, and streptomycetes [36] are also considered to be potentially suitable for the development of effi cient secretion systems. A comparative study with recombinant alpha-amylase demonstrated that fi nal yields as well as enzyme activity were considerably higher when produced by Streptomyces lividans,by which it was completely secreted than by E. coli in which it was concentrated periplasmatically [37]. The yields reported so far, however, are still below cost-effi cient ranges. A system developed by Hoechst/Aventis for insulin production yielded around 100 mg/L, and the yields of correctly folded human CD4-receptor sites are in the range of 200 mg/L. Attached as signal proteins were the prepeptide of the alpha-amylase inhibitor from S. tendae (tendamistat) and the signal sequence of a protease inhibitor (LTI) from S. longisporus. In the course of these studies, it was found that the choice of the linker and its length strongly infl uences secretion effi ciency. The comparably low yields, however, demonstrate that still a lot of fundamental research is necessary to render streptomyces systems competitive. (Strategies and examples for enhancement of recombinant protein expression in S. lividans and the current status of the genetic and physiological development are given in Refs. 38 and 39.) A general focus of research will be the detailed exploration of the twin arginine translocation (TAT) pathway, which has been recently discovered in addition to the conventional prokaryotic secretory (sec) pathways and enables the export of proteins with cofactors in a fully folded conformation [40, 41]. It evidently plays a more important role in Streptomyces species [42] but might also be useable in other species.

As the potential and capacity of prokaryotes for prosttranslational modifi cation appear to be quite limited and the knowledge about the pathways is quite scarce (reviews on bacterial protein glycosylation see Refs. 43 and 44), the employability of most of the known prokaryotes usually is restricted to the preparation of proteins that are naturally not glycosylated, such as insulin, hirudins, or somatotropins, or to natively glycosylated proteins that are pharmacologically also active without glycosylation, like various cytokines (tumor necrosis factor, interleukines, inter-ferones). For production of proteins that are pharmacologically active only with an appropriate modifi cation pattern, eukayotic cell systems are more suitable.

Yeasts. Besides possessing complex posttranslational modifi cation pathways, they offer the advantage to be neither pyrogenic nor pathogenic and to secrete more

effi ciently. Species established in industrial production procedures are Saccharomyces cerevisiae, Kluyveromyces lactis, Pichia pastoris, and Hansenula polymorpha,which will be dealt with more in detail in this section. Whereas S. cerevisiae is the best genetically characterized eukaryotic organism at all and still is the prevalent yeast species in pharmaceutical production processes (Table 1.1-1), P. pastoris, fi rst employed by Phillips Petroleum for single-cell-protein production, is currently the most frequently used yeast species for heterologous protein expression in general. Whereas only just a few proteins were expressed by Pichia species at the beginning of the last decade [45], the expression of more than 400 proteins have been meanwhile reported now [46, 47]. P. pastoris is considered to be superior to any other known yeast species with respect to its secretion effi ciency and permits the production of recombinant proteins without intense process development. The highest yields were reported for murine collagen (15 g/L), tetanus toxin fragment C (12 g/L compared with 1 g/L in S. cerevisiae), human serum albumin (10 g/L compared with 3 g/L in K. lactis and to 90 and 150 mg/L in S. cerevisiae), and human interleukin 2 (10 g/L). The highest reported yields for H. polymorpha relate to phytase (13,5 g/L) and to hirudin (g/L range) and for K. lactis to human serum albumin (3 g/L, see above). Even though S. cerevisiae offers a high secretory potential as evidenced by some 9-g/L secreted Aspergillus niger glucose oxidase [references in 16], such data document a general inferior secretory capacity, the reasons for which are numerous. For the methylotrophic species Hansenula and Pichia and the lactose using K. lactis, natively strong promoters are available that derive from the methanol and lactose assimilating pathways and their enzymes (e.g., alcohol- and methanol oxidase, lactose permease, galactosidase). As the enzymes of these pathways account for up to 30% of the total protein content, the metabolic effi ciency with respect to the secreted protein is signifi cantly higher as documented by Buckholz and Gleeson [48]: Only one or a few gene copies are suffi cient in P. pastoris to gain the same yields as with 50 gene copies in S. cerevisiae.Furthermore, proteins with a molecular mass of above 30 kD are retained in the cytoplasma of S. cerevisiae, whereas H. polymorpha effi ciently secretes proteins with a molecular mass of up to 150 kD, like the glucoamylase of Aspergillus niger.[Detailed overviews on the physiological properties of methylotrophic yeasts and K. lactis with respect to their use for recombinant protein production is given in Refs. 46, 47, and 49–53]. Further reasons for the differing secretion rates among the species are the specifi c proteolytic activities and the specifi c degrees and patterns of glycosylation. Besides having an impact on the protein’s fi nal pharmacological activity, glycosylation also exercises an infl uence on the folding and secretion effi ciency. Among the discussed species, S. cerevisiae was shown to possess, besides a higher enzymatic activity in the secretion vesicles that leads to a reduced portion of intact secreted proteins [54], also the highest glycosylation capacity leading to a hyperglycosylation of the protein and a reduced secretion rate. Both the degree and the pattern of glycosylation are dependent on the genetic background of the species and strains employed as well as on the sequences of the expressed protein and adjacent regions. By employment of the natively highly glycosylated alpha-mating type factor as a secretion signal, the extent of the glycosylation of the product can be diminished or completely avoided as shown for human interleukin 6 [55]. NovoNordisk reported leader sequence-dependent insulin yields in S. cerevisiae [56] and in S. cerevisiae and P. pastoris [57]: The sequence and therewith the degree of glycosylation of the leader infl uences the

PRODUCTION ORGANISMS AND EXPRESSION SYSTEMS 7

8 THE ADVANTAGE OF INTEGRATIVE BIOTECHNOLOGY

effi ciency of the multistage cleavage and folding processes as well as the insulin glycosylation rate and secretability. Further enhancement of the secretion effi ciency can be achieved by (1) mutating secretion enhancer genes [58], (2) suppressing secretion blocking functions, and (3) reducing proteolytic activities in secretion vesicles [54]. So-called supersecreter strains of S. cerevisiae have, for instance, been generated by inactivation of the PMR1 (SSC1) function and suppression of the secretion blocking ypt1-1 gene: the yields of non-glycosylated human pro-urokinase [59], of human serum albumin, and of human plasminogen-activator have been augmented to a factor of up to 10 [60]. The traditional approaches pursued for enhancement of gene expression are gene amplifi cation, employment of strong promoters, and enhancement of the transcription and translation rate. A high amplifi cation of the gene copy number [45, 46, 61] usually is achievable with episomal vectors, which however do not reach the mitotic stability of integrative systems like the transposon (e.g., Ty-element) mediated embedment of reiterative, dispers repetitive sequences in S. cerevisae. Transcription rates were reported to be enhanced up to 100 times through cotransformation with transcription activators and enhancers, which evidently are limiting factors for overexpression of foreign proteins [62, 63]. Translation effi ciency can be enhanced by preventing an accelerated degradation of transcripts and the yeast typical random transcription termination through modulation of the recognition sequences. Prevention of the random transcription termination led to an increase of tetanus toxin fragment C yields in S. cerevisiae by a factor 2000–3000 to 1 g/L and 3% of the total soluble protein fraction [64].

Despite their physiologically advantageous properties and natively high expres-sion and secretion capacity, just one industrial application is reported for each of the alternative yeast species: H. polymorpha is employed for hepatitis B vaccine production at Rhein-Biotech (Düsseldorf, Germany), K. lactis for bovine prochy-mosin production in a 40-m3 scale at Gist-Brocades (Delft, Netherlands), and P. pastoris for production of recombinant carboxypeptidase B and trypsin at Roche (Basel, Switzerland). For pharmaceutical application, it has to be considerered that the methylotrophic yeasts in contrast to S. cerevisiae and K. lactis are not used in the production of foodstuffs and therewith have no GRAS (generally regarded as safe) status according to the U.S. Food and Drug Administration (FDA) criteria and have to be grown in expensive explosion-proofed equipments when the above-mentioned native induction systems are used. The discussed properties of the mentioned yeast species are compiled in Table 1.1-2 for comparison. The employ-

TABLE 1.1-2. Typical Sequence of Biotechnological Production Process Steps

Step Method/Approach

Selection/design/engineering/ Criteria: pharmacological activity and properties development of an of the compound, productivity, process appropriate species/strain/ behavior, suitability for downstream processing

expression system steps, spectrum and pharmacological activity of side products to be removed, experimental experience with the respective system, biological and medical safety, acceptance by regulatory bodies