BiotechnologySecond Edition

Volume 3

Bioprocessing

BiotechnologySecond EditionFundamentalsVolume 1 Biological Fundamentals Volume 2 Genetic Fundamentals and Genetic Engineering Volume 3 Bioprocessing Volume 3 Measuring. Modelling. and Control

Special TopicsVolume 9 Enzymes. Biomass, Food and Feed Volume 10 Special Processes Volume 11 Environmental Processes Volume 12 Modern Biotechnology : Legal. Economic and Social Dimensions

ProductsVolume 5 Genetically Engineered Proteins and Monoclonal Antibodies Volume 6 Products of Primary Metabolism Volume 7 Products of Secondary Metabolism Volume 8 Biot ransformat ions

r

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A Multi-Volume Comprehensive Treatise

Second, Completely Revised Edition Edited by H.-J. Rehm and G. Reed in cooperation with A. Puhler and ? Stadler Volume 3

Biotechnology

BioprocessingEdited by G. Stephanopoulos

VCH

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Weinheim New York Base1 - Cambridge Tokyo

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Series E d i t o r s : Prof. Dr. H.-J. R e h m Institut f u r Mikrobiologie Universitat M u n s t e r CorrensstraBe 3 D-48149 M u n s t e r

Dr. G . R e e d 2131 N . S u m m i t Ave. A p a r t m e n t #304 Milwaukee. W I 53202-L347USA

Volume E d i t o r : Prof. Dr. G . S t e p h a n o p o u l o s Massachusetts Institute of Technology C a m b r i d g e , MA 02139 USA

Prof. Dr. A . Puhler Biologie V1 (Genetik)Universitat Bielefeld P.O. B o x 100131 D-33501 Bielefeld

Dr. P. J. W. S t a d l e r Bayer AGVerfahrensentwicklung B i o c h e m i e Leitung Friedrich-Ebert-StraBe 217 D-42096 W u p p e r t a l

This book was carefully produced. Nevertheless. authors. editors and publisher do not warrant the information contained thercin to be free of errors. Readers are advised to keep in mind that statements. data, illustrations, procedural details or other items may inadvertently be inaccuratc.

Published jointly by VCH Verlagsgesellschaft mbH. Weinheim (Federal Republic of Germany) VCH Puhlishers Inc.. New York. NY (USA) Editorial Director: Dr. Hans-Joachim Kraus Editorial Manager: Christa Maria Schultz Copy Editor: Karin Dembowsky Production Director: Maximilian Montkowski Production Manager: Dipl. Wirt.-Ing. (FH) Hans-Jochen Schmitt Library of Congress Card No.: applied for British Library Cataloguing-in-Publication Data: A catalogue record for this book is available from the British Library Die Deutsche Bibliothek CIP-Einheitsaufnahme Biotechnology : a multi volume comprehensive treatise / ed. by H.-J. Rehm and G . Reed. In cooperation with A. Puhler and P. Stadler. 2.. completely rev. ed. -Weinheim; New York; Basel: Cambridge; Tokyo: VCH. NE: Rehm. Hans J. [Hrsg.]~ ~

2.. completely rev. ed. Vol. 3. Bioprocessing / e d . by G . Stephanopoulos IYY3 ISBN 3-527-28313-7(Weinheim) ISBN 1-56081-153-6 (New York) NE: Srephanopoulos. Gregory [Hrsg.]~~

QVC'H Verlagsgesellschaft mbH. D-69451 Weinheim (Federal Republic of Germany), 1993 Printcd on acid-free and low-chlorine paper.All rights reserved (including those o translation into other languages). N o part of this book may be reproduced in any f form - by photoprinting. microfilm, or any other means-nor transmitted or translated into a machine language without written permission from the publishers. Registered names. trademarks. etc. used in this book. even when not specifically marked as such. are not to be considered unprotected by law. Composition and Printing: Zechnersche Buchdruckerei. D-67330 Speyer. Bookbinding: Fikentscher GroBbuchbinderei. D-64205 Darmstadt. Printed in the Federal Republic of Germany

Preface

In recognition of the enormous advances in biotechnology in recent years, we are pleased to present this Second Edition of Biotechnology relatively soon after the introduction of the First Edition of this multi-volume comprehensive treatise. Since this series was extremely well accepted by the scientific community, we have maintained the overall goal of creating a number of volumes, each devoted to a certain topic, which provide scientists in academia, industry, and public institutions with a well-balanced and comprehensive overview of this growing field. We have fully revised the Second Edition and expanded it from ten to twelve volumes in order to take all recent developments into account. These twelve volumes are organized into three sections. The first four volumes consider the fundamentals of biotechnology from biological, biochemical, molecular biological, and chemical engineering perspectives. The next four volumes are devoted to products of industrial relevance. Special attention is given here to products derived from genetically engineered microorganisms and mammalian cells. The last four volumes are dedicated to the description of special topics. The new Biotechnology is a reference work, a comprehensive description of the state-of-the-art, and a guide to the original literature. It is specifically directed to microbiologists, biochemists, molecular biologists, bioengineers, chemical engineers, and food and pharmaceutical chemists working in industry, at universities or at public institutions.

A carefully selected and distinguished Scientific Advisory Board stands behind the series. Its members come from key institutions representing scientific input from about twenty countries. The volume editors and the authors of the individual chapters have been chosen for their recognized expertise and their contributions to the various fields of biotechnology. Their willingness to impart this knowledge to their colleagues forms the basis of Biotechnology and is gratefully acknowledged. Moreover, this work could not have been brought to fruition without the foresight and the constant and diligent support of the publisher. We are grateful to VCH for publishing Biotechnology with their customary excellence. Special thanks are due Dr. Hans-Joachim Kraus and Christa Schultz, without whose constant efforts the series could not be published. Finally, the editors wish to thank the members of the Scientific Advisory Board for their encouragement, their helpful suggestions, and their constructive criticism.

H.-J. Rehm G. Reed A. Piihler P. Stadler

Scientific Advisory Board

Proj Dr. M.J. BekerAugust Kirchenstein Institute of Microbiology Latvian Academy of Sciences Riga, Latvia

Prof Dr. I: K.GhoseBiochemical Engineering Research Centre Indian Institute of Technology New Delhi, India

Prof Dr. J. D. BuLockWeizmann Microbial Chemistry Laboratory Department of Chemistry University of Manchester Manchester, UK

Prof Dr. I. GoldbergDepartment of Applied Microbiology The Hebrew University Jerusalem, Israel

Prof Dr. C. L.CooneyDepartment of Chemical Engineering Massachusetts Institute of Technology Cambridge, MA, USA

Prof Dr. G. GomaDCpartement de Genie Biochimique et Alimentaire Institut National des Sciences AppliquCes Toulouse. France

Prof Dr. H. W DoelleDepartment of Microbiology University of Queensland St. Lucia. Australia

Prof Dr. D. A.HopwoodDepartment of Genetics John Innes Institute Nonvich, UK

Prof Dr. J. DrewsF. Hoffmann-La Roche AG Basel. Switzerland

Prof Dr. E.H.HouwinkOrganon International bv Scientific Development Group Oss, The Netherlands

Prof Dr. A.FiechterInstitut fur Biotechnologie Eidgenossische Technische Hochschule Zurich, Switzerland

Pro$ Dr. A.E.HumphreyCenter for Molecular Bioscience and Biotechnology Lehigh University Bethlehem, PA, USA

VIII

Scientific Advisory Board

Pro$ Dr. I. KarubeResearch Center for Advanced Science and Technology University of Tokyo Tokyo, Japan

Pro$ Dr. K . SchiigerlInstitut fur Technische Chemie Universitat Hannover Hannover, Germany

Prof Dr.M . A. LachanceDepartment of Plant Sciences University of Western Ontario London, Ontario, Canada

Pro$ Dr. l? SensiChair of Fermentation Chemistry and Industrial Microbiology Lepetit Research Center Gerenzano, Italy

Pro$ Dr. Y. LiuChina National Center for Biotechnology Development Beijing. China

ProJ Dr. Y. H . TanInstitute of Molecular and Cell Biology National University of Singapore Singapore

ProJ Dr. 1.F: MartinDepartment of Microbiology University of Leon Leon, Spain

Pro$ Dr. D. ThomasLaboratoire de Technologie Enzymatique Universite de Compikgne Compibgne, France

Prof Dr. B. MattiassonDepartment of Biotechnology Chemical Center University of Lund Lund, Sweden

Pro8 Dr. W VerstraeteLaboratory of Microbial Ecology Rijksuniversiteit Gent Gent, Belgium

Prof Dr. M . RohrInstitut fur Biochemische Technologie und Mikrobiologie Technische Universitat Wien Wien. Austria

Pro$ Dr. E.-L. WinnackerInstitut fur Biochemie Universitat Miinchen Munchen, Germany

Proj Dr. H. SahmInstitut fur Biotechnologie Forschungszentrum Julich Julich, Germany

Contents

Introduction

1 G. Stephanopoulos

I. Nature and Issues of Bioprocessing1 Fermentation - An Overview 7 B. C. Buckland, M. D. Lilly 2 Industrial Animal Cell Culture 23 B. D. Kelley, T.- W. Chiou, M. Rosenberg, D. I. C. Wang 3 Overview of Downstream Processing 39 R. Spears 4 Proteins and Peptides as Drugs: Sources and Methods of Purification 57 S. E. Builder, R. L. Garnick, J. C. Hodgdon, J. R. Ogez

11. Product Formation (Upstream Processing)5 Bioreactors: Description and Modelling 77 J. Nielsen, J. Villadsen 6 Cell Culture Bioreactors 105 A . Sambanis, W.-S. Hu 7 Media for Microbial Fermentations R. L. Greasham 8 Media for Cell Culture 141 R. A . Wove

9 Media and Air Sterilization 157 G. K. Raju, C. L . Cooney 10 Oxygen Transfer and Mixing: Scale-Up Implications 185 M. Reuss 11 Oxygen Transfer in Cell Culture Bioreactors 219 J. G. Aunins, H.-J. Henzler 12 Strategies for Fermentation with Recombinant Organisms 283 T. Imanaka 13 Anaerobic Fermentations 295 L . E. Erickson, D. Y . C. Fung, P. Tuitemwong 14 Fermentation Monitoring and Control 319 T. Chattaway, G. A . Montague, A . J. Morris 15 Fermentation Data Analysis for Diagnosis and Control 355 G. Stephanopoulos, K. Konstantinov, U. Saner, T. Yoshida 16 Design of Aseptic, Aerated Fermentors 401 M. Charles, J. Wilson 17 Biotransformations and Enzyme Reactors 427 A. S. Bommarius

127

X

Contents

111. Product Recovery and Purification18 Cell and Cell Debris Removal: Centrifugation and Crossflow Filtration 469 R. V. Datar, C . 4 . Rosen 19 Cell Disruption and Isolation of Non-Secreted Products 505 H . Schiitte, M.-R. Kula 20 In vitro Protein Refolding 527 J . L . Cleland, D. I . C. Wang 21 Liquid-Liquid Extraction (Small Molecules) 557 K . Schiigerl 22 Protein Purification by Liquid-Liquid Extraction 593 B. D. Kelley, T. A . Hatton 23 Protein Separation and Purification 617 J.-C. Janson, L . Ryden 24 Affinity Separations 643 S. Sundaram, M. L . Yarmush 25 Electrokinetic Separations 679 A . J. Grodzinsky, M . L . Yarmush 26 Final Recovery Steps: Lyophilization, Spray-Drying 695 C. F. Gblker

IV. Process Validation, Regulatory Issues27 Analytical Protein Chemistry 717 S. Sundaram, D. M . Yarmush, M . L . Yarmush 28 Biotechnology Facility Design and Process Validation 739 M . G. Beatrice 29 Treatment of Biological Waste 769 D . F. Liberman

Index

789

Contributors

Dr. John G. AuninsBiochemical Process Research and Development Merck & Co., Inc. P.O. Box 2000 Rahway, NJ 07065, USA Chapter I1

Dr. Stuart E. BuilderGenentech, Inc. 460 Point San Bruno Blvd. South San Francisco, CA 94080, USA Chapter 4

Michael G. BeatriceAssociate Director for Policy Coordination and Public Affairs Center for Biologics Evaluation and Research Food and Drug Administration 8800 Rockville Pike, HFM-10 Bethesda, MD 20892, USA Chapter 28

Prof. Dr. Marvin CharlesDepartment of Chemical Engineering Lehigh University Iacocca Hall 111 Research Drive Bethlehem, PA 18015-4791, USA Chapter 16

Dr. Andreas S. BommariusOrganic and Biological Chemistry Research and Development Degussa AG P.O. Box 1345 D-63403 Hanau Federal Republic of Germany Chapter I 7

Dr. Thomas ChattawayZeneca BioProducts P.O. Box 1 Billingham, Cleveland, TS23 1LB England Chapter 14

Dr. Barry C. BucklandBiochemical Process Research and Development Merck & Co., Inc. P.O. Box 2000 Rahway, NJ 07065, USA Chapter I

Dr. Tzyy-Wen ChiouDepartment of Chemical Engineering and Biotechnology Process Engineering Center Massachusetts Institute of Technology Cambridge, MA 02139, USA Chapter 2

XI1

Contributors

Dr. Jeffrey L. ClelandPharmaceutical Research and Development Genentech, Inc. 460 Point San Bruno Blvd. South San Francisco, CA 94080, USA Chapter 20

Dr. Randolph L. GreashamBiochemical Process Research and Development Merck & Co., Inc. P.O. Box 2000 Rahway, NJ 07065, USA Chapter 7

Prof. Dr. Charles L. CooneyDepartment of Chemical Engineering Massachusetts Institute of Technology Cambridge, MA 02139, USA Chapter 9

Prof. Dr. Alan J. GrodzinskyDepartment of Electrical Engineering and Computer Science Massachusetts Institute of Technology Cambridge, MA 02139, USA Chapter 25

Dr. Rajiv V. DatarPall Corporation 30 Sea Cliff Avenue Glen Cove, NY 11542, USA Chapter I8

Prof. Dr. T. Alan HattonDepartment of Chemical Engineering Massachusetts Institute of Technology Cambridge, MA 02139, USA Chapter 22

Prof. Dr. Larry E. EricksonDepartment of Chemical Engineering College of Engineering Durland Hall Kansas State University Manhattan, KS 66506-5102, USA Chapter 13

Dr. Hans-Jiirgen HenzlerVerfahrensentwicklung Biochemie Bayer AG Friedrich-Ebert-StraRe 21 7 D-42096 Wuppertal Federal Republic of Germany Chapter I1

Dr. Daniel Y. C. FungDepartment of Chemical Engineering College of Engineering Durland Hall Kansas State University Manhattan, KS 66506-5102, USA Chapter 13

Dr. James C. HodgdonGenentech, Inc. 460 Point San Bruno Blvd. South San Francisco, CA 94080, USA Chapter 4

Dr. Robert L. GarnickGenentech, Inc. 460 Point San Bruno Blvd. South San Francisco, CA 94080, USA Chapter 4

Prof. Dr. Wei-Shou HuDepartment of Chemical Engineering and Materials Science University of Minnesota Minneapolis, MN 55455, USA Chapter 6

Dr. Christian F. GOlkerVerfahrensentwicklung Biochemie Bayer AG Friedrich-Ebert-Straf3e 21 7 D-42096 Wuppertal Federal Republic of Germany Chapter 26

Prof. Dr. Tadayuki ImanakaDepartment of Biotechnology Faculty of Engineering Osaka University Yamadaoka Suita Osaka 565, Japan Chapter 12

Contributors

XI11

Prof. Dr. Jan-Christer JansonPharmacia BioProcess Technology AB S-75182 Uppsala, Sweden Chapter 23

Dr. Gary A. MontagueDepartment of Chemical and Process Engineering University of Newcastle upon Tyne Newcastle upon Tyne NE1 7RU, England Chapter 14

Dr. Brian D. KelleyGenetics Institute, Inc. One Burtt Road Andover, MA 01810, USA Chapters 2 and 22

Prof. Dr. A. Julian MorrisDepartment of Chemical and Process Engineering University of Newcastle upon Tyne Newcastle upon Tyne NE1 7RU, England Chapter 14

Dr. Konstantin KonstantinovDepartment of Chemical Engineering University of Delaware Newark, DE 19716, USA Chapter 15

Dr. Jens NielsenCenter for Process Biotechnology Department of Biotechnology Technical University DK-2800 Lyngby, Denmark Chapter 5

Prof. Dr. Maria-Regina KulaInstitut fur Enzymtechnologie der Heinrich-Heine-Universitat Diisseldorf im Forschungszentrum Julich D-52425 Julich Federal Republic of Germany Chapter 19

Dr. John R. OgezGenentech, Inc. 460 Point San Bruno Blvd. South San Francisco, CA 94080, USA Chapter 4

Dr. Daniel F. LibermanMassachusetts Institute of Technology Environmental Medical Service Biohazard Assessment Office 18 Vassar Street Cambridge, MA 02139-4307, USA Chapter 29

Gokaraju K. RajuDepartment of Chemical Engineering Massachusetts Institute of Technology Cambridge, MA 02139, USA Chapter 9

Prof. Dr. Malcolm D. LillyAdvanced Centre for Biochemical Engineering Department of Chemical and Biochemical Engineering University College London Torringdon Place London WCIE 7JE, England Chapter I

Prof. Dr. Matthias ReussInstitut fur Bioverfahrenstechnik Universitat Stuttgart Biiblinger Straae 72 D-70199 Stuttgart Federal Republic of Germany Chapter 10

XIV

Contriburors

Dr. Carl-Gustaf RosknABITEC AB Villavagen 36 S-64050 Bjornlunda, Sweden Chapter 18

Dr. Ron SpearsAbbott Laboratories Department 48F/R1/1040 1400 North Sheridan Road North Chicago, IL 60064, USA Chapter 3

Dr. Morris RosenbergBiogen, Inc. 14 Cambridge Center Cambridge, MA 02142, USA Chapter 2

Prof. Dr. Gregory StephanopoulosDepartment of Chemical Engineering Massachusetts Institute of Technology Cambridge, MA 02139, USA Chapter 15

Prof. Dr. Lars RydenDepartment of Biochemistry Biomedical Centre University of Uppsala Box 576 S-75123 Uppsala, Sweden Chapter 23

Srikanth SundaramDepartment of Chemical and Biochemical Engineering Rutgers University Piscataway, NJ 08854-0909, USA Chapters 24 and 27

Prof. Dr. Athanassios SambanisSchool of Chemical Engineering Georgia Institute of Technology Atlanta, GA 30332-0100, USA Chapter 6

Dr. Pravate TuitemwongDepartment of Chemical Engineering College of Engineering Durland Hall Kansas State University Manhattan, KS 66506-5102, USA Chapter 13

Dr. Urs SanerDepartment of Chemical Engineering Massachusetts Institute of Technology Cambridge, MA 02139, USA Chapter 15

Prof. Dr. John VilladsenCenter for Process Biotechnology Department of Biotechnology Technical University DK-2800 Lyngby, Denmark Chapter 5

Prof. Dr. Dr. Karl SchugerlInstitut fur Technische Chemie Universitat Hannover Callinstrane 3 D-30167 Hannover Federal Republic of Germany Chapter 21

Prof. Dr. Daniel I. C. WangDepartment of Chemical Engineering and Biotechnology Process Engineering Center Massachusetts Institute of Technology Cambridge, MA 02139, USA Chapters 2 and 20

Prof. Dip1.-Ing. Horst SchiitteTechnische Fachhochschule Berlin FB 3 , SeestraRe 64 D-13347 Berlin Federal Republic of Germany Chapter 19

John D. WilsonABEC 6390 Hedgewood Drive Allentown, P A 18106, USA Chapter 16

Contributors

xv

Dr. Richard A. WolfeCentral Research Laboratories Department of Cell Culture and Biochemistry Monsanto Company 800 North Lindbergh Blvd. St. Louis, MO 63167, USA Chapter 8

Prof. Dr. Martin L. YarmushDepartment of Chemical and Biochemical Engineering and Center for Advanced Biotechnology Rutgers University Piscataway, NJ 08854, USA Chapters 24, 25 and 27

Dr. David M. YarmushDepartment of Chemical and Biochemical Engineering and Center for Advanced Biotechnology Rutgers University Piscataway, NJ 08854, USA Chapter 27

Prof. Dr. Toshiomi YoshidaIC Biotechnology Faculty of Engineering Osaka University Osaka 558, Japan Chapter 15

Biotechnology Second Edition Edited b y H,-J. Rehm and G. Reed in cooperation with A. Puhler and P. Stadlercopyright OQVC'H Verlagsgesellschaft mbH. D-69451 Weinheim (Federal Republic of Germany), 1993

Introduction

GREGORY STEPHANOPOULOSCambridge, MA 02319, USA

Two major changes have occurred in the field of bioprocessing with respect to the stateof-the-art reviewed in the first edition of Biotechnology. These changes are not due to generic advancements of equipment and processes. They were rather caused by the introduction of two new types of applications in the field, specifically the introduction of microbial recombinant fermentations and cell culture processes for the production of biochemicals and pharmaceutical proteins. In connection with these processes, new methods of production and new operating strategies have been introduced in the upstream as well as the downstream sections of the biotechnological plant. More traditional processes have benefited from these new manufacturing techniques in several ways, such as the use of improved types of equipment, better processing strategies and new methods for bioprocess monitoring and control. The present volume reviews the state-of-theart of bioprocessing as it applies in the case of traditional as well as the newly introduced processes. The structure of the volume follows to some extent the typical arrangement of bioprocessing equipment in a real process. Therefore, the usual distinction of upstream and downstream processing which one encounters in a biotechnological plant is also present in the arrangement of the chapters of this vol-

ume. In addition, we have included a group of chapters providing an overview of fermentation and cell culture processes and a closing group of chapters dedicated to the issues of process validation, measurement and regulation. In keeping with the separation between microbial fermentations and cell culture processes, the first two chapters of Part I provide overviews of these two different types of cell cultivation. These are followed by a chapter providing a general overview of downstream processing. Since the sources and methods of synthesis of pharmaceutical proteins are of considerable importance, an additional chapter dedicated to this topic has also been included to illustrate the rationale for the development of the synthetic manufacturing methodologies that one encounters today. Part II deals exclusively with fermentation and cell culture equipment as a means of biosynthesis of fermentation products and pharmaceutical proteins. It begins with an equipment description and presentation of design equations for microbial fermentors. This chapter reviews the predominant types of industrial fermentors as well as some experimental designs along with the design equations and the main characteristics of these units. Another chapter deals with cell culture bioreactors, their design equations and the predominant

2

Introducrion

types which are presently in use in industry. The remaining chapters in Part I1 review the equipment and operation of other related steps in the way they are practiced in a plant. In this regard, chapters describing media formulation and the rationale of media design for microbial fermentations and cell culture are included. Issues of sterilization of air and fermentation media are considered next. Oxygen transfer and mixing in fermentors and the implications of these processes to the scale-up and control of microbial and cell culture bioreactors are examined in two separate chapters. Following these basic issues of fermentor design and operation, the specifics of particular fermentations are examined such as recombinant microbial fermentations and anaerobic fermentations. The topic of fermentor instrumentation, monitoring and control has been the subject of Volume 4 in this series. It was felt, however, that a review of this most important topic should be included in the present volume for the purpose of completeness. A summary of instrumentation techniques and conventional as well as more advanced control strategies is presented in the corresponding chapter. A new element in the present volume is a concise review of the various methods by which fermentation data can be analyzed and used for the diagnosis and control of fermentors. We hope this chapter will enhance the quality of information which can be obtained from the type of data typically collected in the course of a fermentation or cell culture process. Practical issues of fermentor construction and containment are addressed in a different chapter. The very important area of enzymatic reactions and biotransformations as well as the types of equipment which are used for this purpose are reviewed in the final chapter of Part 11. Part III is devoted to product recovery and purification. These chapters also follow the typical steps that one encounters in a plant. The first chapter addresses processes used for the removal of cells from the fermentation or cell culture medium. Specifically, centrifugation and cross-flow filtration are reviewed as a means of cell removal. Following cell removal, methods for the disruption of cells and isolation of non-secreted products are considered. In this regard, and in connection with micro-

bial recombinant fermentations, the refolding of non-secreted proteins after they have been separated from the cell debris in the form of inclusion bodies is examined. Specifically, in v i m protein refolding and process parameters affecting this step are reviewed in the next chapter. Liquid-liquid extraction as it applies to the separation of small molecules follows. Other extraction processes geared towards the purification of proteins and enzymes are reviewed in another chapter, specifically techniques based on reversed micelles and twophase systems are included. As mentioned earlier, the introduction of new biosynthetic technologies for the production of proteins necessitated the development of purification methods yielding products of very high purity. Chromatographic methods are used primarily for this purpose, and the fundamentals of chromatography as well as chromatographic applications for the purification of biopharmaceuticals are the subject of another chapter. In the same vein, affinity separations and electrokinetic separations have also been employed, and they are the subject of two additional chapters. As a final polishing step, lyophilization and spray-drying are examined in the last chapter of Part 111. Along with the development of the new exciting manufacturing technologies, new requirements for the containment of biologically active material, the satisfactory validation of biotechnological processes, and the means to monitor compliance with regulations have emerged. It is, therefore, fitting to devote Part ZV t o these critical issues. This part includes chapters on analytical protein chemistry devoted to regulatory issues as well as chapters on process validation criteria and methodologies for the treatment of biological waste. There are some topics which have not been included in the present volume. They are in areas which have not yet been fully developed or areas which may be replaced in the future in favor of other competing processes. In the first category belong issues of optimization, in particular those dealing with the optimal structure of a train of equipment and the arrangement of process streams connecting such pieces of equipment. In the second category are processes for handling products from animal organs where the present trend is to replace such

Introduction

3

processes by the better definable and controllable systems based on microbial fermentations or cell culture processes. Depending on the way that these topics develop, they may become subjects of a future edition of Bioprocessing, or will be included in Volume 10 Special Processes. Although the writing style of the various chapters is variable reflecting the individual authors, there are some common features that can be noted throughout the volume. Most chapters begin with a brief introduction of the fundamentals followed by a description of the equipment and processes as practiced in industry. After that, the discussion centers on the presentation of the most common procedures as well as problems and challenges furthering their industrial performance. A significant amount of process data is also included in the form of tables and figures. The reader is encouraged to search through the cited literature which provides information updated to the date of writing the chapter. When reading the present volume, one should bear in mind the very diverse background of the readership that this volume tries to satisfy. The readership comprises the more mathematically oriented as well as those who

favor a more qualitative approach in their field of biotechnological application. Although there was a conscientious effort to maintain a balance between mathematical complexity and process description, it is quite possible that the results may not please everybody. It is, however, the sincere hope of the editor and the authors that the product of their efforts will reach as broad an audience as possible and will satisfy the needs of such audience for a useful and at the same time rigorous treatment of the topic of bioprocessing. The extent to which this ambition is reached will determine the success of this work. In closing, I would like to express my thanks to the authors of all chapters for a very professional and thorough job as well as the large number of reviewers who provided detailed reviews and critiques of the original manuscripts. The latter were instrumental in producing a volume of high quality. The staff of VCH has been extremely helpful in handling many technical and editorial matters and they have our deep appreciation as well. Cambridge, MA, May 1993 G. Stephanopoulos

I. Nature and Issues of Bioprocessing

1 Fermentation: An Overview

BARRYC. BUCKLANDRahway, NJ 07065-0900, USA

MALCOLM LILLY D.London, UK

1 Introduction 8 2 Fermentation Products 8 2.1 Cell as Product 8 2.2 Primary Metabolites 9 2.3 Secondary Metabolites 9 2.4 Enzymes 10 2.5 Therapeutic Proteins 10 2.6 Vaccines 11 2.7 Gums 11 3 Types of Fermentor 12 3.1 Stirred Tank Fermentors 12 3.2 Non-Mechanically Agitated Fermentors 3.3 Facility Design 14 4 Fermentor Operations 15 4.1 Fermentation Media 15 4.2 Mode of Operation 15 4.3 Monitoring and Control 16 4.4 New Measuring Techniques 16 5 Influence of Product Recovery 17 6 Fermentation Process Development 18 6.1 Objectives 18 6.2 Organism Selection 19 6.3 Capital Investment 19 6.4 Regulatory Issues 20 7 Conclusions 21 8 References 21

14

Biotechnology Second Edition Edited b y H,-J. Rehm and G. Reed in cooperation with A. Puhler and P. Stadlercopyright OQVC'H Verlagsgesellschaft mbH. D-69451 Weinheim (Federal Republic of Germany), 1993

8

I Fermentation: An Overview

1 IntroductionMicrobial fermentations are important sources of biological products used in the pharmaceutical, food, and chemical industries. During the last decade, there has been a large increase in the range of commercial products, especially secondary metabolites and recombinant proteins. There have also been significant changes in fermentor and facility design to improve performance and to ensure safe operation. Emphasis is now on the development of processes which are not only cost-effective but also meet the increasing demand by the public and regulatory authorities for greater reliability and reproducibility. This has increased the need for improved monitoring and control. Whereas microbial transformations, like chemical reactions, often reach yields close to the theoretical maximum, many fermentation processes such as those for secondary metabolites are operating at much lower yields. Thus, there is great scope for advances in fermentation processes. Progress will result from an improved understanding of microbial physiology, of the interaction of microorganisms with the physical environment in fermentors and the ability to manipulate metabolic fluxes using molecular biology. In this chapter, we highlight many of these aspects of fermentation, although some of them will be covered moreTab. 1. Some Typical Production Fermentor Sizes~

comprehensively in subsequent chapters of this volume. The level of understanding of each type of fermentation processes is primarily a function of metabolic complexity. These categories of fermentation will be discussed in the next section. The scale of operation varies considerably depending on the type of product and the dose (Tabs. 1 and 2). More recently the scope of fermentation technology has grown to include mammalian cell culture. If a mammalian cell culture line can be adapted to suspension culture, then similar approaches to process improvement can be utillized. Mammalian cells are bigger, more fastidious and more fragile than microbial cells, but the approach to process development can be remarkably similar.

2 Fermentation Products2.1 Cell as ProductYeast fermentation for brewing and baking represents the most traditional form of fermentation technology. Techniques and cultures have been developed over the years for production of yeast at a large scale of operation as well as for simplified methods of cell harvesting (for example, by the deliberate enhancement of flocculation). This technology has become increasingly sophisticated, and some of the earliest examples of computer-enhanced fermentation technology were applied toward the more efficient production of yeast (WANG et al., 1977). In the 1970s a number of companies became very interested in the production of single cell

Product Bakers yeast Amino acids Antibiotics Industrial enzymes Recombinant therapeutic proteins

Volume (m3)100-250 100-250 80-200 80-250 0.5-50

Tab. 2. Scale of Operations for Therapeutic Drugs

Dose~ ~~ ~

Annual Production 1OOOOOO Doses0.1-100 g 1-10 kg 100 kg

Immunomodulator or Vaccine Hormone Enzyme Antibiotic

O.OOO1-0.1 mg 1-10 mg 100 mg l g

lo00 kg

2 Fermentation Products

9

protein (SCP), and a whole technology was developed to grow microbial cells on very cheap substrates such as methanol and hydrocarbons. The driving force behind this interest was ultimately the price for other protein products such as soybeans and fish meal. Since the sudden price jump for protein in the early 1970s turned out to be a temporary phenomenon, the interest in SCP took a precipitous nosedive as soon as the protein price returned to normal. The economic question can be summarized by asking whether it is cheaper to generate protein by buying suitable land (for example, in Illinois) and planting soybeans, or is it better to construct and operate a fermentation facility. More recently, the emphasis has switched to generation of a product which can simulate meat (mycoprotein, marketed by Marlow Foods) and thereby compete with the more expensive end of the protein market on issues other than price (e.g., fat content and acceptability by vegetarians). Some of the technology developed proved to have applications in other areas, for example, large-scale DNA removal. Other specific technologies (such as the 1.5 million liter ICI Pressure Cycle Fermentor) have not entered the mainstream. Interest in these innovative technologies will undoubtably resume at some future time, when the need is more urgent to develop chemicals from renewable resources.

per unit volume than a stirred tank fermentor which normally has a height to diameter ratio of < 3 :1. Also, obviously mechanical mixing generates heat.

2.3 Secondary MetabolitesSince the widespread use of antibiotics began (around 1950), this area has represented the most commercially significant fermentation activity. Products such as penicillin, tetracycline, erythromycin, cephalosporins, cephamycins, and clavulanic acid are made at a very large scale in fermentors varying between 50000 to 200000L in volume. In most cases these products serve a vital role in modern medicine, and their sales have continued to increase for decade after decade. Penicillin can now be considered a bulk chemical. To a large extent, the demand for more product has been matched by increases in productivity, and so companies have been able to obtain more and more product from an existing factory by advances made in process development (Fig. 1). More recently, discovery groups have learned to use secondary metabolites as a way of generating leads for other therapeutic areas besides antibiotics. For example, avermectin was discovered by the Merck Research Labo-

2.2 Primary MetabolitesProcess for products such as citric acid, ethanol, and glutamic acid are well developed in view of their historical importance and very large scale of operation. In addition, these are processes which are relatively,simple metabolically and hence tend to be better characterized. Recombinant DNA technology is now used to enhance metabolic fluxes for products such as phenylalanine. Stirred tank fermentors of around 200000 L in scale are generally used. If it is desired to go to greater volumes, then it is simpler to switch to an air-lift type design because of heat transfer limitations. An air-lift would generally have a height to diameter ratio of 10: 1 and, therefore, has much more cooling surface area

l t0 'I I

I

I

I

I

1960 1965 1970 1975 1980 1985 1990Year of Production

Fig. 1. Improvements in the productivity of penicillin fermentations. Arrows indicate the introduction of new strains (courtesy of Gist-Brocades) (redrawn from LILLY,1992).

10

I Fermentation: A n Overview

Historically, industrial enzymes made by fermentation were mainly restricted to those produced extracellularly such as amylases and proteases. In recent years, other bulk enzymes such as lipases have been markteted for industrial and domestic applications. With the advent of mechanical techniques for release of proteins from microorganisms Tab. 3. New Pharmaceutical Products since 1980: on a large scale, intracellular enzymes have Secondary Metabolites found wider application in the food, pharmaceutical, and chemical industries. Of particular Lovastatin (Aspergillus terreus) note are glucose isomerase for production of Avermectin (Streptomyces avermitrlis) Cephamycin C (Nocardia lactamdurans) high-fructose syrups and penicillin acylase for Efrotomycin (Nocardia lactamdurans) removal of the side chain of penicillins to allow FK 506 subsequent manufacture of semi-synthetic penCyclosporin icillins. Other enzymes, such as glucose oxiNew Cephalosporins dase and cholesterol oxidase, are widely used Tniiriamycin (Streptomyces catleya) for clinical analysis. Fermentor sizes for enClavulanic acid (Strepromyces) zyme production generally range from 30 to 220 m3. not yet approved in the USA In the future, it is likely that a large propornow made synthetically tion of enzymes for commercial use will be synthesized using recombinant microorganSecondary metabolite fermentations are isms, This approach has opened up the possivery complex and, despite intensive investiga- bility of producing many different enzymes in tion, have yet to yield many of their secrets substantial quantities, preferably using a small even in the case of such well-known products number of host/vector systems to minimize as penicillin. Progress has been made in indus- development costs. The increasing availability try by a mixture of art and science (random of enzymes will allow their exploitation as pomutation and selection, media development, tent catalysts to introduce chirality and specidevelopment of nutrient feeding strategies, ficity into compounds in chemical processes to and improvements in oxygen transfer (NALLIN make, for instance, drugs and pesticides. et al., 1989). Applications of recombinant DNA technology to improve systematically a well established culture line have generally not 2.5 Therapeutic Proteins been successful. Presumably, at some time in In recent years, the ability to synthesize therthe future, most culture improvement will come from using recombinant DNA technolo- apeutic proteins has improved rapidly. We can gy. However, this area is at a fascinating junc- claim to have moved completely away from ture. Tools via rDNA technology are now the art of fermentation to the science of feravailable which will allow a more complete un- mentation, when we use a well characterized derstanding of these processes, and this in turn host such as Escherichia coli with a chemically will provide further performance improve- defined medium with a known plasrnid and ments or novel products. Despite the lack of promoter. This claim can be less easily made

ratories (MRL) as an anti-parasitic drug. Lovastatin was discovered (also by MRL) as a potent inhibitor of HMGCoA reductase and has subsequently been used as an effective blood cholesterol lowering drug. Cyclosporin and FK506 were discovered as potent immunoregulants and are used as aids in tissue transplant therapies. Thus the 1980s have seen the commercial introduction of many important secondary metabolites (Tab. 3). This is the first decade in which a number of very important secondary metabolites were successfully introduced to the marketplace which are not antibiotics.

fundamental knowledge, progress has often been remarkable with some product titers for secondary metabolites progressing more than a thousand-fold from < 1 mg/L to tens of g/L.

2.4 Enzymes

2 Fermentation Products

11

for a more complicated fungal host such as Saccharomyces cerevisiae and even less for animal cell culture. For the fermentation technologist, the fact that there is a choice of hosts represents a new range of opportunities. For a microbial system one can choose primarily between E.coli or yeast. For a glycosylated protein it is generally necessary to use animal cells as host, but even here there is a choice between cell lines which predominantly attach to surfaces and those which can be grown in suspension culture. Also some cell lines can easily be adapted to serum-free conditions and others cannot. In these examples, decisions made at the initial stage of the project will have a huge impact on the eventual outcome of the manufacturing process because of the highly regulated environment surrounding the development of a biological as a product. A decade ago, it was rare for recombinant microorganisms to be grown on a large scale. However, progress in the 1980s has been rapid, and by 1990 at least six recombinant products had annual sales in excess of one hundred million dollars (LILLY,1992). These were human growth hormone, erythropoietin, alpha-interferon, human insulin, trypsin plasminogen activator, and hepatitis B vaccine. A list of rDNA therapeutic proteins commercialized since 1982 is given in Tab. 4 .Tab. 4. Recombinant DNA Therapeutic Proteins (since 1982)

Tab. 5. Vaccines (since 1980) Hepatitis B (plasma) Hepatitis B (recombinant) Hepatitis A" Combination vaccines (various) HIB conjugate Varicella" Pertussis (acellular)a

not yet approved in USA

Interferon (alpha and gamma) tPA (tissue plasminogen activator) EPO (erythropoietin) Human growth hormone Human insulin Sargramostin (GM-CSF) Filgrastim (rG-CSF) OKT3 (muromonab-CD3)

brane proteins such as HIB conjugate (Tab. 5 ) . As a recent extension to the technology in this area, a vaccine for meningitis has been successfully introduced to the market by the Merck Research Laboratories (PedvaxHIBQ) which is made by chemically conjugating polysaccharides from Haemophilus influenzae type b with an outer membrane protein complex from Neisseria meningitidis. This is one member of a whole new class of vaccines called "conjugate vaccines". Another recent example of a dramatic evolution in the vaccine area is illustrated by the development of a vaccine for hepatitis B. The original vaccine developed in 1980 was made by isolating virus particles from infected human blood and then by purifying the capsid protein followed by formulation with alum. The inactivation for viruses in plasma was performed at three different phases of purification. The recombinant version of the vaccine is made using Saccharomyces cerevisiae as the host. The yeast generates the subunits of the capsid protein which are then assembled to form the desired particle. This allowed vaccine production to move completely away from blood as the raw material toward the inherently safe recombinant yeast process and was the first example of a recombinant protein used as a human vaccine. The product is known as RECOMBIVAX@(Merck & Co., Inc.).

2.6 VaccinesVaccine technology has evolved from using rather crude extracts from microbial cells (for example, Bordetella) as a vaccine to using more highly purified polysaccharides or mem-

2.7 GumsXanthan gum has found an increasingly important role for use in the food industry as well as for applications in tertiary oil extraction. This product is made at a very large scale of operation using fermentors in the 50000 to

12

I Fermentation: An Overview

200000 L size range. This is a very competitive area and economics for production are critical. Technical issues relate to the fascinating problem of improving mass transfer to a very viscous fermentation broth. This is an even greater challenge for some of the newer gums with unusual rheological behavior (e.g., Gellan Gum).

at this scale. Therefore, the large number of new therapeutic protein product candidates, which are typically run at the 100 L to 3000 L scale, have not generated a driving force for innovation in fermentor design in terms of performance except for the need to enhance oxygen transfer rates to meet the demands of E. coli cultures growing to 50 g dry weight per liter or more. Most recent improvements have been driven by good manufacturing practice (GMP) requirements and have resulted in tanks which are very easy to clean and which have good sterility performance. Cleaning is often done in place using CIP (clean in place) technoloA wide range of fermentor types has been gy developed in the food industry. A modern described in the literature. Nevertheless, it re- fermentor design would include spray balls to mains true that the standard of the fermenta- allow for an automatic cleaning cycle. In practice, agitation power/unit volume tion industry is still the aerated agitated tank. There are several reasons for this. First, many decreases with increasing scale of operation, companies installed fermentors of this kind in and the lower agitator power input/volume rethe 1960s and 1970s which still have many sults in longer mixing times and lower oxygen years of useful life and have been upgraded, transfer rates. This is illustrated in Fig. 2 where necessary, with improved agitation, in- which shows that the mixing times (t,) in aerstrumentation, and computer controlled sys- ated stirred fermentors increase with fermentems. Second, stirred tanks, although not nec- tor volume ( V ) according to essarily ideal for a particular fermentation, give good results for many different fermenta- t, =constant * v 0 . 3 tions. This is important, especially for pharmaceutical companies, as several different reaching about 100s in a loom3 vessel (Fig. products may be made in a fermentor during 2). Thus, at a large scale there is likely to be its lifetime. Another benefit arises from the fact that the capital investment in fermentors is normally recuperated from sales of the first A product so that capital costs for subsequent products are greatly reduced. 100

3 Types of Fermentor

A

3.1 Stirred Tank FermentorsAs new fermentation capacity has been installed over the last twenty years, there has been a gradual increase in the size of fermentors for particular products and this trend is likely to continue. It reflects the greater demand for such products, the need to reduce costs to remain competitive, and the improved understanding of fermentor design and operation by biochemical engineers. At the smaller scale (10 L to loo00 L) there is very little incentive for a company to explore alternative designs to the stirred tank fermentors. Energy costs are usually inconsequential

Y

l n

50

.X

c m

3020

10 0.1

I

1

I

I

I

I

0.3

1 3 10 Volume (m3)

30

100

Fig. 2. Mixing times in agitated fermentors. Data taken from EINSELE(1978), (0); JANSEN et al. (1978), (A); and CARLEYSMITH (personal communication), 1-1.

3 Types of Fermentor

13

considerable heterogeneity within the fermentation broth. Liquid additions to the broth will take time to disperse, and nutrient concentration gradients may occur. If the time constant for the rate of oxygen transfer is much shorter than the mixing time, then there will be a vertical dissolved oxygen concentration gradient, as observed by MANFREDINI al. (1983) for et chlorotetracycline and tetracycline production by strains of Streptomyces aureofaciens in a 112 m3 fermentor. It is also possible in viscous fermentations to have radial dissolved oxygen (DOT) gradients in fermentors, especially in the stirrer region (OOSTERHUIS KOSSEN, and 1984). Thus the DOT readings for a large fermentation will depend on the location of the probe. Most companies determine empirically the measured DOT below which, for instance, product formation is reduced. The impact on cultures of the rapid fluctuations in nutrient concentrations, including DOT, which occur in large fermentors, is poorly understood. There has been recent progress in agitator design. Multiple Rushton turbine radial flow impellers (Fig. 3) were the design of choice for

Fig. 4. Axial flow hydrofoil impellers fitted to a 19000 L fermentor.

mycelial fermentations (BUCKLANDet al., 1988). The specific advantages of the hydrofoil axial flow impeller design over the traditional Rushton radial flow impeller design for viscous mycelial fermentations are:0

0

0

0

Improved oxygen transfer per unit power Lower maximum shear rates (thus improving fermentor versatility; i.e., a fermentor can be designed for use with cultures requiring a low shear environment, such as animal cell culture) Improved bulk mixing by elimination of compartmentalized flow resulting from use of multiple radial flow impellers Improved bulk mixing resulting in improved pH control and better control of nutrient feeding at the level of the individual cell. This results in a more homogeneous and stable environment for the microbial cells.

Fig. 3. Radial flow turbine impellers fitted to a 19000 L fermentor.

fermentors from the early 1950s to the early 1980s. Now there is a number of attractive alternatives and these have been reviewed recently (NIENOW, 1992). The biggest design change has been the development of axial flow hydrofoil impellers (Fig. 4) which can give superior performance to the more traditional Rushton radial flow impellers for large-scale viscous

The comparison between radial flow and axial flow impellers is best summarized using the model of BAJPAIand REUSS(1982) (Fig. 5). For a viscous mycelial fermentation, the micromixer region is one of high shear, low apparent viscosity (due to the pseudoplastic rheology of mycelial broths), and high K,a whereas the macromixing region is one of low shear, high apparent viscosity, and low K,a. Presumably, the hydrofoil axial flow impellers radically increase the ratio between the micromixing region and macromixing region. In a

14

I Fermentation: An Overview

,

.

IMACROMIXER Vmocro

input

MICROMIXER "micro

.,Fig. 5. Model of a fermentor as micromixer and macromixer regimes.

large fermentor, the macromixing region becomes much larger than the micromixing region.

In contrast to stirred fermentors, oxygen transfer rates in aerated fermentors are normally low at a small scale and increase with scale. Thus, even where aerated fermentors are used on a large scale, it may be necessary to carry out small-scale development work in stirred fermentors. Similar problems of heterogeneity to those described in Sect. 3.1 can occur in non-mechanically agitated fermentors. For instance, in air-lifts, where there is liquid circulation up one section and down the other the circulation times (tc)may be long. The hydrodynamics of air-lift fermentors have been widely studied (CHISTI, 1989), and many values for circulation times are available. However, far fewer measurements have been made during fermentations where oxygen transfer must also be adequate. CARRINGTON al. (1992) have reet ported circulation times of 9-12 s for a Streptomyces antibiotic fermentation in a 20 m3 airlift fermentor.

3.2 Non-Mechanically Agitated FermentorsSome companies have installed pilot and full-scale aerated tanks, with various internal configurations, to produce a range of microbial metabolites and biomass products. At least one large pharmaceutical company uses predominantly non-mechanically driven tanks, and other companies (including Merck & Co., Inc.) have built very large (250000 L) horizontal stirred tank fermentors. The huge air-lift design fermentor (1 500000 L) pioneered by ICI for SCP has not been widely adopted. The air-lift and non-mechanically driven designs are best suited for very large-scale operation (>2OOOOO L). As scale is increased beyond 200000 L, a mechanically driven tank becomes more and more difficult to design because of heat transfer (cooling) considerations. The knowledge of the performance of such aerated fermentors in these companies has allowed them to be used in the same versatile way as stirred fermentors. Thus, the choice between the two types depends very much on which are already installed in the company and, therefore, its familiarity with that type.

3.3 Facility DesignFor many decades, antibiotics of high quality have been produced at very large multipro-

duct facilities. Because of the ease of final product definition, processes have been continuously improved over the years, and titers have often increased by two orders of magnitude. Process changes are allowed by regulatory authorities so long as there is proof that the drug made by the old and new process is the same. In most cases, the increase in productivity has been accompanied by improvements in final product quality (fewer by-products are being generated). Therefore, society has benefitted in two ways: pharmaceutical companies have been able to reduce prices and increase quality for this type of product. Increases in sales are often matched by rises in productivity without the need for additional fermentation capacity, and consequently many of the production facilities are rather old. The vibrant and fertile process development, which exists for secondary metabolite fermentations (regulated in the USA by CDER, the Center for Drug Evaluation and Research of FDA) long after these have been approved as new pharmaceuticals, results in a steady increase in pro-

4 Fermentor Operations

15

ductivity (see Fig. 1). From 1960 to 1990 penicillin productivity in the factory was increased six-fold. This is in contrast to the biologics area (regulated by CBER) in which the process itself and the equipment used is very tightly defined because of the historical difficulty of a rigorous analysis of the actual final product. As a consequence, it is difficult to make process changes, and the process development environment becomes stagnant. As analytical tools become more and more powerful and with the advent of biologic products which are small enough and pure enough to be well defined, it may be possible for the focus to switch away from regulation of the process to regulation of the final product characterization.

4 Fermentor OperationsA fermentor is usually surrounded by a web of auxiliary equipment: seed fermentors, nutrient feed tanks, pH control tanks, antifoam tanks, an area for media preparation, and one or more medium sterilization systems. Obviously, the types of operations involved are a function of the type of process being run. Viewed simplistically, the fermentation step can be seen as a single unit operation. In practice, there are a multiple of operations which need to be tightly choreographed to result in a successful run: these include the seed train, medium preparation, and nutrient feeding. Everything has to be ready at exactly the right time. Maintaining asepsis is usually the single biggest and ongoing challenge, especially for a long-cycle secondary metabolite fermentation. Bacteriophage infections can also be a problem (PRIMOSE, 1990).

these makes the fermentation more complicated to operate and is probably only practical for a computer-controlled facility (BUCKLAND,1984) with excellent alarm scanning capabilities to give instant warning of any process perturbation. Efforts to generate satisfactory chemically defined media for secondary metabolites have been less successful. The desired biochemical environment has proven more difficult to define rigorously and empirically derived media using complex ingredients work remarkably well. The slow release of nutrients such as protein and phosphate from a material such as soybean meal often provides an excellent cell environment for high levels of product expression. For this type of medium, sterilization itself is an important variable, because the act of heating the medium changes its composition (CORBETT, 1980). For this reason, continuous sterilization is often preferred: it is usually cheaper to operate, control is much more precise than for batch sterilization, and scale-up is much easier than for batch sterilization (JAIN and BUCKLAND, 1988).

4.2 Mode of OperationWith the exception of sewage and industrial effluent treatment, very few industrial fermentations are operated as continuous-flow systems. Continuous operation in large fermentors has only been justified for low-cost products where the market is highly-competitive. Continuous culture is a useful developmental tool allowing investigation of the effects of limitation of growth and product formation by supply of individual nutrients. The information gained can be used to optimize feeding regimes for fed-batch fermentations. It is equally true that few fermentations are operated in a truly batch mode. Intermittent or continuous feeding of nutrients may be performed for several reasons. First, the quantities of nutrients at the start of the fermentation may be restricted to avoid the oxygen demand during the growth phase exceeding the oxygen transfer capacity of the fermentor or because high concentrations are inhibitory or cause undesirable precipitates. Second, nutrient feeding regulates the metabolism of the

4.1 Fermentation MediaFor recombinant cultures producing proteins, there is a growing trend toward tailoring a chemically defined (and soluble) medium for a particular host. This makes possible the use of filter sterilization and also allows for the tight control of the cell environment using nutrient feeding and pH control. The need for

16

I Fermenlation: An Overview

organism and is one of the most important ways to enhance product formation. Finally, there are many fermentations where the nutrients required during the growth and product formation phases may be different. Because of the large volumes of nutrients which may be added during the fermentation, the fermentor will be only partly full at the start, and the impact of this on its operations needs to be understood. In some cases, fed-batch fermentations may be extended by harvesting part of the broth on one or more occasions with the vessel being refilled through nutrient feeding.

indication gained from on-line process monitoring is extremely valuable. In practice, we have found that the single most powerful tool available comes from on-line monitoring of vent gases using mass spectrometry (BUCKLAND et al., 1985). Subtle changes in oxygen uptake rate, carbon dioxide evolution rate, and respiratory quotient can be immediately detected and alarms activated.

4.4 New Measuring TechniquesDuring the last decade, a range of new measuring techniques have become available. Some of these allow better characterization of the fluid dynamical behavior in reactors of pilot or production scale (LOBBERT, 1992). Determination of biomass concentrations, particularly when undissolved nutrients are present, still poses problems. HARRISet al. (1987) reported the possibility of using dielectric permittivity. There were some problems with early commercial devices, but the latest versions seem promising, particularly for the measurement of yeasts and mycelial organisms (FEHRENBACH al., 1992) even in the preset ence of undissolved nutrients. The magnitude of the signal with non-mycelial bacteria is lower, but recently it has been used successfully to monitor cultures of Pseudomonas putida. The development of image analysis techniques for measurement of the morphology of filamentous microorganisms over the last few years (PACKER and THOMAS,1990) now allows the influence of the fermentation conditions on morphology to be determined more precisely. Furthermore, it is possible to measure biomass concentration and the extent of cell vacuolization during fermentations of fungi, such as Penicillium chrysogenurn (PACKER et al., 1992). Progress has been made with the automation of sampling and subsequent measurement

4.3 Monitoring and ControlComputer process control has had a fundamental impact on the way a fermentation facility can be operated. Besides all the obvious benefits of tighter process control, alarm scanning capabilities are invaluable, and capabilities such as cascade control of dissolved oxygen (DO) (Fig. 6) allow for tighter control of the cell environment. In Japan, at Yamanouchi Pharmaceutical Company, this has been taken one step further (EIKI et al., 1992) toward lights-out production using computer control combined with very extensive automation. Many operations (including transfer steps) are carried out unattended at night and during the weekend. For example, the raw media stocked in the outdoor silos are automatically weighed and transferred into the interior material hoppers. Harvesting of the batch is initiated unattended very early in the morning. After broth transfer, the fermentor is automatically washed with alkaline hot water. By the time the operators come to the plant, these jobs are almost complete. A production manager continually needs information to indicate whether the various processes are progressing normally. Any usefulDO Measurement

RPM Measurement

changes in agitator speed.

5 Influence of Product Recovery

17

of metabolite concentration by analytical techniques such as HPLC and GLC. A novel approach has been the use of near infrared (NIR) spectroscopy, which may be adapted for online measurement using a fiber optic module (HAMMOND BROOKES, and 1992). The authors describe the use of NIR for determination of antibiotics (Fig. 7).

portant to understand the impact of the fermentation conditions on the subsequent processing operations (FISH and LILY, 1984). Major improvements can be made by correct selection of the organism, culture medium, and growth conditions. Many problems in purification can best be addressed by changing fermentation conditions. Some examples are summarized below:0

I rZ

0

HPLC

Fig. 7 Correlation plot for at-line antibiotic meas. urement using near infrared (NIR) spectroscopy and high performance liquid chromatography (HPLC) (HAMMOND BROOKES, and 1992).

A recent extension has been the enhancement of automation for sample preparation using robotics. This approach has been taken, because many of the most useful components to measure are intracellular or cell-associated. In one example, samples are automatically taken from the fermentor, accurately weighed, mixed with a calculated amount of methanol, centrifuged, and the clarified methanol transferred to HPLC for detailed analysis (REDAet al., 1991).

0

0

In a recombinant fermentation, if an exotic ingredient is added (for example, neomycin to help reduce contamination or to maintain plasmid stability), the burden will be on the producer to prove that this material is not in the final product. It is better to find a way to avoid its use in fermentation. In a recombinant fermentation, a very common problem is one of amino acid clipping (for example, by an amino peptidase) which may occur under certain conditions. This can present a severe problem to the manufacturer. It may then be necessary to separate a protein containing 189 amino acids from one containing 188 amino acids. This is a major challenge and best solved by a better understanding and control of the fermentation so that the amino acid clipping does not occur in the first place. In an enzyme fermentation, it proved to be much easier to harvest the host culture (Pseudomonas aeruginosa) by filtration using a chemically defined medium composition. Under certain culture conditions, one amino acid can be substituted for another (e.g., norleucine for methionine).

5 Influence on Product RecoveryThere are many fermentation products, e.g., therapeutic proteins, where the cost of product recovery and purification exceed those for fermentation. Particularly in these cases, it is im-

In these examples in our own process development work it has often proved easier to solve a purification problem at the fermentation stage.

18

1 Fermentation: An Overview

6 Fermentation Process Development6 . I ObjectivesThe objective of process development is the production of sufficient new or modified product to meet market demand in the quickest time possible, meeting all safety and quality requirements and by a cost-effective and reliable process. The role of biochemical engineers working on process development is to translate the process from the laboratory to full-scale production using knou-ledge provided by the biological scientists in the laboratory and their own expertise in process development and scale-up. The various stages of process development are summarized in Fig. 8. Following identification of a new product candidate, a commitment to begin process development is made by the company. In parallel to the process development, there are other sequences which may need to take place. For instance, for therapeutic products it is necessary to provide materials for clinical trials. It may be possible to provide sufficient material for the pharmacological and toxicological studies which make up the pre-clinical trials by repeated use of the laboratory procedure. However, a pilot-scale or large-scale process must have evolved in time for the clinical trials which require large quantities of product. By the start of phase I11 trials it is essential that the process is well defined and understood as much as possible. Once

phase 111 trials have commenced, it becomes difficult to make further process changes. In fact, the Center for Biological Evaluation and Research (CBER), FDA, prefers phase 111 material t o be made in the final manufacturing facility using manufacturing staff. There are many advantages gained by a company which does effective process development. First of all, especially for a biologic, it allows for the reproducible production of highly purified product which is a sine qua non for modern pharmaceutical manufacturing. Second, the impact on capital expenditure can be huge, for example, by quadrupling process performance both for new products and to meet increasing sales without having to build new factories. Third, there is an obvious benefit by reducing production cost thus releasing money for further investments by the company. Finally, the time between starting development of a process and introduction of the product to the market is minimized. This may result in reduction of development costs through, for example, lower interest charges and earlier sales income and resulting extension of sales under patent protection. Successful scale-up of fermentations requires a good understanding of the interactions between microorganisms and the chemical and physical environments in the fermentor. The biochemical engineer endeavors to control the chemical environment by monitoring the fermentation and feeding nutrients intermittently or continuously. It is more difficult to control the physical environment during translation of scale as both agitation and aeration conditions change. Their impact on

0

Time: Years

w 5-1 1

-1Identification of New Product Candidate

Material for Clinical Trials and Other Requirements Process Development

Approval Continue to Improve Process

k

Test New Equipmentr

Specify Factory Equipmentr

Factory Start-up

Fig. 8. Stages in process development.

6 Fermentation Process Development

19

various fermentation parameters is shown diagrammatically in Fig. 9. For instance, if agitation is low, mixing will be poor. If both agitation and aeration are low, then oxygen transfer will be poor and the culture will be oxygenlimited. Thus it is possible to define the operating boundaries within which the fermentation should be maintained. These boundaries are, of course, broad regions across which the operating problem increases in magnitude. The biochemical engineer needs to know how these boundaries shift during translation of scale so that fermentations can be operated without encountering these problems. This may not be possible for highly viscous fermentations such as those producing xanthan gums.

the fermentation conditions. Substantial improvements are made as a result of changes in the seed train, mixing and aeration conditions, and feeding of nutrients. This is illustrated in Fig. 1 which shows the increases in productivity for penicillin fermentations at Gist-Brocades over the last three decades. It highlights the fact that improvements to fermentation processes are often made for many years after the product is first marketed (in this case for 50 years after the product was first marketed!).

6.2 Organism SelectionMany different microorganisms are already used for industrial fermentation processes. Nevertheless, this represents only a small proportion of the wide range of microorganisms around the world most of which, particularly in subtropical and tropical regions, have not been characterized. At the same time the number of vectors described for recombinant microorganisms is growing rapidly. The proliferation of choice is both exciting and challenging. In the case of recombinant organisms to produce proteins, it is beneficial to select a small number of hosts, vectors, and expression systems for new microbial processes to minimize the amount of work required and the time taken to develop each new process. This also requires evaluation of the advantages and disadvantages of hosts such as Escherichia coli and Saccharomyces cerevisiae and the location of the desired protein, i.e., intracellular, periplasmic, or extracellular.

I

Cell Damage

.c m .-

Limitation Mixing Aeration

Fig. 9. Operating boundaries for fermentation scaleup (redrawn from LILLY, 1983).

Both agitation and aeration affect the distribution of nutrients, including oxygen, and relative fluid velocities in the fermentation broth, which in turn impact on culture morphology, broth rheology and product formation. It is important, therefore, to understand the interactions between these. For instance, increase in agitation rates can lead to greater fragmentation of mycelia and reduced penicillin production in Penicillium chrysogenum fermentations (SMITH al., 1990). et During the initial scale-up and subsequent process development, there may be large increases in product yields or productivity. Each new strain which is introduced requires the biochemical engineer to modify appropriately

6.3 Capital InvestmentWhile process development is going on, it is also necessary to plan ahead for commercial operation and to estimate the capital investment required. The sequence shown in Fig. 8 implies that new equipment will be required. This will depend on whether or not there is existing fermentation capacity and recovery equipment available in the production plant. Although in recent years some companies have installed new fermentors, many new products are introduced without the need for new

20

I Fermentation: An Overview

plants. Whenever possible, companies rely on improvements in titers of existing products to release sufficient fermentation capacity in time for introduction of the next new product. If this can be done, it minimizes the capital investment required for a new product. This approach does rely on the fact that by choosing in the past agitated aerated tanks or aerated towers, companies have very flexible if not ideal types of reactor.

6.4 Regulatory IssuesSecondary metabolites, such as antibiotics, can be well characterized analytically, and this has resulted in an environment in which process changes can be made, i.e., minor changes can be made on an ongoing basis as long as there is proof that product made by the new process is the same as that made by the old. The main challenge results from the fact that certain fungal and actinomycetes cultures can produce a remarkable array of very similar metabolites. Analytical techniques and purification techniques need to be very good, both to identify and remove minor levels of these similar metabolites. By contrast, it is more difficult to characterize fully a protein molecule, and many of the same regulations developed historically for products such as vaccines (where the final product can be much harder to define) have been applied to recombinant proteins. In these examples, the process itself is much more rigorously defined which has resulted in a stagnant process development environment once a product is far advanced in clinical trials. In a recent talk, BADEK (1992) made the interesting comment that, in order to improve product quality, changes have to be made to a process.Tab. 6 . ComparisonDrugs versus Biologicals Drugs

These changes are inherently difficult to make for production of a biologic which is dominated by quality assurance and the emphasis is placed on installing safeguards to make sure that as few deviations as possible occur. In addition, the process definition submitted to the regulatory authorities includes a detailed description of the equipment to be used as part of the Establishment License Amendment (Tab. 6 ) . This has resulted in highly validated facilities, which are inherently less adaptable for other product candidates. Consequently, costs for recombinant proteins, both to operate a modern facility and also to construct a factory, are much higher than for secondary metabolites. In 1992 it costs as much to build a 3 000 L scale biologics facility as it does to build a standard 200000 L scale antibiotics production facility. In addition to design for good manufacturing practice (GMP), there has also been an emphasis in recent years on containment of waste streams from a facility using recombinant cultures. These environmental restrictions have recently been considerably relaxed in the USA, as the evidence grows that the common microbial hosts used for recombinant DNA technology are indeed very safe and certainly present no credible threat to the environment. In fact, often these hosts represent the safest cultures being used in the pharmaceutical companies, because they have been deliberately adapted so that they will not survive in the general environment.

-

AreaRelease specs Process specs Establishment license Focus Change control

Biologicals Yes Specific Yes Drug substance Rigid

Yes General No Drug product Flexible

8 References

21

7 ConclusionsFor some decades, the fermentation industry has made a major contribution to society through the provision of healthcare and food products, and the treatment of wastes. There is a growing demand by the regulatory authorities and the general public that products for healthcare and food products should be single enantiomers rather than the racemic mixtures often used in the past. Although some chemical and physical methods have been developed for separation of enantiomers, the biological route is increasingly attractive for this purpose. The impact of rDNA technology is now being seen in the commercialization of therapeutic proteins. In the future, many enzymes will be made with recombinant organisms, and improvements in the synthesis of primary and secondary metabolites through metabolic engineering are likely. However, the excitement which these new developments have quite rightly caused should not lead to an underestimate of the major contribution which traditional secondary metabolite fermentations will make to the market into the next century. For reasons explained earlier, in the past the emphasis during process development has been on yield improvement. While this will remain of prime importance, other criteria such as process reliability and reproducibility, safety and minimization of waste streams must be satisfied. All of these depend on the application of good biochemical engineering to the design, development and operations of fermentation processes in the future.

8 References

BADER, (1992), Evolution in fermentation facility F. design from antibiotics to recombinant proteins, in: Harnessing Biotechnology f o r the 21st Century (LADISCH,M., BOSE, A., Eds.), pp. 228231, Washington, DC: American Chemical Society. BAJPAI,R. K., REUSS,M. (1982), Coupling of mixing and microbial kinetics for evaluation of the performance of bioreactors, Can. J. Chem. Eng. 60, 384-392. BUCKLAND, C. (1984), The translation of scale in B. fermentation processes: The impact of computer process control, Bio/Technology 2, 875-884. BUCKLAND, C., BRIX,T., FASTERT,H., GBEB. WONYO, K., HUNT, G., JAIN, D. (1985), Bio/ Technology 3 , 982-988. BUCKLAND, C., GBEWONYO, DIMASI, D., B. K., HUNT, G., WESTERFIELD, NIENOW, W. G., A. (1988), Improved performance in viscous mycelial fermentations of agitator retrofitting, Biotechnol. Bioeng. 31, 737-742. R., CARRINGTON, DIXON, K., HARROP, A. J. (1992), Oxygen in industrial air agitated fermentors, in: Harnessing Biotechnology for the 21st M. Century (LADISCH, R., BOSE,A., Eds.), pp. 325-333, Washington, DC: American Chemical Society. CHISTI, Y. (1989), Airlift Reactors, Amsterdam: M. Elsevier. CORBETT, (1980), Preparation, sterilization and K. design of media, in: Fungal Biotechnology (SMITH,J. E., BERRY, R., KRISTIANSEN, D. B., Eds.), pp. 25-41, New York: Academic Press. EIKI, H., KISHI, I., GOMI,T., OGAWA, (1992), M. Lights Out production of cephamycins in automated fermentation facilities, in: Harnessing BioM. technology for the 21st Century (LADISCH, R., BOSE,A., Eds.), pp. 223-227, Washington, DC: American Chemical Society. EINSELE, (1978), Scaling up bioreactors, Process A. Biochem., July, 13-14. R., M., FEHRENBACH, COMBERBACH, PETRE, J. 0. (1992), On-line biomass monitoring by capaciAcknowledgements tance Biotechnol. 23, 303-3 14. The authors wish to thank Drs. MAIGETTER, FISH,N. measurement, J.D. (1984), The interactions M., LILLY,M. MASUREKAR and MONAGHAN numerous for between fermentation and protein recovery, Bio/ helpful editorial comments. Technology 2, 623-628. The authors also wish to thank Professor N. HAMMOND, V., BROOKES, K. (1992), Near inS. I. W . KOSSEN (Gist-Brocades) and Dr. S. CARfrared spectroscopy - powerful technique for atline and on-line analysis of fermentations, in: LEYSMITH (Smithkline Beecham) for data Harnessing Biotechnology for the 21st Century shown in Figs. 1 and 2, respectively. (LADISCH, R., BOSE,A., Eds.), pp. 325-333, M. Washington, DC: American Chemical Society. HARRIS,C. M., TODD, R. W., BUNGARD, J., S.

22

I Fermenfation: A n Overview

LOVITT, W., MORRIS, G., KELL,D. G. NIENOW, W. (1992), New agitators vs. Rushton R. J. A. turbines: A critical comparison of transport phe(19871, Dielectric permittivity of microbial suspensions at radio frequencies: a novel method for nomena, in: Harnessing Biotechnology f o r the real-time estimation of microbial biomass, En21st Century (LADISCH,M., BOSE, A., Eds.), pp. 193-196, Washington, DC: American Chemizyme Microb. Technol. 9, 181-186. cal Society. JAIN,D., BUCKLAND, C. (1988), Scale-up of the B. EfroLomycin fermentation using a computer con- OOSTERHUIS, M. G., KOSSEN, N. W. F. (1984), N. trolled pilot plant, Bioprocess Eng. 3, 31-36. Dissolved oxygen concentration profiles in a production-scale bioreactor, Biotechnol. Bioeng. 26, JANSEN, . H., SLOTT,S . , GURTTER, (1978), P H. Determination of mixing times in large-scale fer546-550. mentors using radioactive isotopes, Proc. 1st PACKER, . L., THOMAS, R. (1990), MorphoH C. logical measurements on filamentous microorEur. Congr. Biotechnol., Part 11, 80-82. ganisms by fully automated image analysis, BioLILLY, D. (1983), Problems in process scale-up, M. technol. Bioeng. 35, 870-881. in: Bioactive Microbial Products 2 (WINSTANH LILLY,M. LEY, J., NISBET, . J., Eds.), pp. 79-89, Lon- PACKER, . L., KESHAVARZ-MOORE, D. L D., THOMAS, R. (1992), Estimation of cell C. don: Academic Press. volume and biomass of Penicillium chrysogenum LILI.Y, D. (1992), Biochemical engineering reM. search; its contribution to the biological indususing image analysis, Biotechnol. Bioeng. 39, tries, Trans. Inst. Chem. Eng. 70, Part C, 3-7. 384-391. L ~ ~ B H E RA., (1992), Advanced measuring tech- PRIMROSE, B. (1990), Controlling bacteriophage T S. niques for mixing and mass transfer in bioreacinfections in industrial bioprocesses, Adv. Biotors, in: Harnessing Biotechnology f o r the 2lst chem. Eng. 43, 1-10. M. , I. Century (LADISCH, R., Bost, A., Eds.), pp. REDA,K. D., TH IENM. P., FEYGIN, , MARCIN, C. S., CHARTRAIN, M., GREASHAM, L. M. R. 178-182, Washington DC: American Chemical Society. (1991), Automatic whole broth multi-fermenter MANFRFDINI, CAVALLERA, MAR I NIL., sampling, J. Znd. Microbiol. 7, 215-220. R., V., , SMITH, . J., LILLY, D., Fox, R. I. (1990), The J M. DONATI, (1983), Mixing and oxygen transfer G. effect of agitation on the morphology and peniin conventional stirred fermentors, Biotechnol. cillin production of Penicillium chrysogenum, Bioeng. 25, 3115-3132. NALLIN OMSTEAD, M., KAPLAK, BUCKLAND, Biotechnol. Bioeng. 35, 101 1-1023. L., C. B. C. (1989), Fermentation development and WANG, H., COONEY, L., WANG, D. I. C. process improvement, in: Zvermectin and Aba(1977), Computer-aided baker's yeast fermentation, Biotechnol. Bioeng. 19, 69. W. mectin (CAMPBELL, C., Ed.), pp. 33-54.

2 Industrial Animal Cell Culture

BRIAND. KELLEYAndover, MA 01810, USA

TZYY-WEN CHIOUCambridge, MA 02139, USA

MORRIS ROSENBERGCambridge, MA 02142, USA

DANIELI. C. WANGCambridge, MA 02139, USA 1 Introduction 24 2 Historical Perspective 24 3 Current Applications 26 3.1 Cell Types 27 3.2 Types of Bioreactors 27 3.3 Operating Strategies 28 3.4 Purification of Animal Cell-Derived Products 29 3.5 Regulatory and Safety Issues 29 4 Problems Motivating Current Research Interests 30 4.1 Cell Biology 30 4.1.1 Host Cell Development 30 4.1.2 Media Formulation 3 1 4.1.3 Genetic Engineering Approaches 31 4.1.4 Glycoprotein Microheterogeneity 32 4.2 Bioeactor Engineering 32 4.2.1 Oxygenation of Bioreactors 32 4.2.2 Agitation and an Understanding of Shear Damage 33 4.2.3 High Cell Density Bioreactors 34 4.2.4 Application of Biosensors 34 5 Future Applications 35 6 Conclusions 36 7 References 36

Biotechnology Second Edition Edited b y H,-J. Rehm and G. Reed in cooperation with A. Puhler and P. Stadlercopyright OQVC'H Verlagsgesellschaft mbH. D-69451 Weinheim (Federal Republic of Germany), 1993

24

2 Industrial Animal Cell Culture

1 IntroductionRecombinant proteins may be produced by either bacterial, yeast, or animal cell cultures, and the choice of host is determined by several factors. If the quantitiy of total protein required is quite large, hosts such as bacteria or yeast have an advantage because of their rapid growth and higher expression levels. Appropriate blood clearance rates and solubility of glycoproteins for parenteral therapeutics may require post-translational modifications similar to the native structures, which may not be provided by bacterial sources. The principles of refolding bacterially-derived proteins are becoming better understood, but not all proteins can be refolded at high concentrations with acceptable recoveries. These and other issues, including additional post-translational modifications, the presence of endotoxins, medium and bioreactor cost, and questions of viral contamination all figure into the choice of host for protein production. The current status of industrial research and development reflects the difficulty of this choice, as products are often produced by both prokaryotic and eukaryotic hosts during initial feasibility studies, before the final host selection is made. Animal cells may now be cultured to very large volumes (up to 10000 L) t o provide the necessary quantity of protein for new markets. This increase in scale brings the economics of animal cell production into focus, stressing previously less important questions such as the cost of medium, bioreactor choice, and maximum sustained cell density. These problems all motivate current research in animal cell culture, much of which will be summarized in the chapters in this book. This review will examine the historical perspective of cell culture, providing a basic understanding of animal cell culture within the development of biochemical engineering. Descriptions of the processes used to manufacture current products will provide a snapshot of the state of the art as it is practiced today, and will emphasize limitations of existing technology, equipment, and understanding. The basic questions which drive industrial and academic research will be addressed individually, with summaries of recent developments and solution methodologies. Fi-

nally, we will predict where animal cell culture is heading and what its position in the health care industry will be, by extrapolation of progress in both animal cell culture and competing technologies.

2 Historical PerspectiveThe history of industrial animal cell culture is a fascinating interplay of medicine, biology, and engineering. Cell culture products have progressed from vaccines for both human and animal diseases, to monoclonal antibodies and interferons, and now to recombinant proteins from genetically engineered cells. The first processes employed primary cells; later, normal cells maintained as a cell strain were approved, and now, transformed cell lines which have infinite life spans are used. Cell culture and propagation were initially conducted with roller bottles, then microcarriers, and now, many cell lines are cultured in stirred tank bioreactors. Cell lines are now being adapted to adverse culture conditions, including serumfree media, lack of anchorage support, and elevated ammonia and lactate levels. Table l gives a brief overview of the development of these products and various cell types used in industry. Modern industrial cell culture began in the mid-l950s, with the use of animal cells for vaccine research and development (SPIER,1991). Industrial animal cell culture was first developed for the production of polio vaccines through propagation of normal diploid cells derived from tissue explants (BUTLER,1987). These primary cells were trypsinized and grown to a confluent monolayer on roller bottles. The virus was inoculated into the confluent culture and harvested several days post infection. Vaccines could not be obtained by any other method, and so animal cell culture produced the inactivated or attenuated viruses necessary for vaccination. Because of the small amounts of material required, there was little pressure then t o scale-up the process beyond roller bottle capabilities. Important principles of media preparation and formulation were developed during this stage, especially with re-

2 Historical Perspective

25

Tab. 1. Development of Mammalian Cell Culture for Industrial Use 1949 Virus growth demonstrated in cell culture (ENDERS) Salk polio vaccine produced in primary cells 1954 Chemically defined medium developed (EAGLE) 1955 Human diploid cell strain WI-38 established (HAYFLICK) 1962 BHK cells first grown in suspension (CAPSTICK al.) et Continuous VERO cell line established (YASUMURA) Foot-and-mouth disease vaccine produced from BHK cell line 1964 1964-69 Vaccines for rabies, rubella, and mumps developed using WI-38 Microcarrier culture developed (VAN WEZEL) 1967 Large-scale suspension culture developed for FMD vaccine production 1970s Monoclonal antibody technology discovered (KOHLER and MILSTEIN) 1975 Interferon derived from continuous Namalwa cell line licensed 1986 Polio and rabies vaccines produced from VERO cell line First therapeutic monoclonal antibody (OKT3) licensed 1987 1988-89 Recombinant products from continuous CHO cell lines licensed (tPA, EPO)

gard to understanding the basic metabolic requirements of the cell (EAGLE,1959). The susceptibility of human diploid cell strains to viral infection and propagation directed processes to be developed which avoided primary cell culture (HAYFLICK al., et 1963). These cell strains, while having finite life spans in culture unlike transformed, continuous cell lines, could be frozen and stored bef