Monoclonal Antibody Production

Committee on Methods of Producing Monoclonal Antibodies

Institute for Laboratory Animal Research

National Research Council


NATIONAL ACADEMY PRESS
Washington, DC
1999



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Monoclonal Antibody Production Committee on Methods of Producing Monoclonal Antibodies Institute for Laboratory Animal Research National Research Council NATIONAL ACADEMY PRESS Washington, DC 1999

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NATIONAL ACADEMY PRESS 2101 Constitution Avenue, N.W. Washington, DC 20418 NOTICE: The project that is the subject of this report was approved by the Governing Board of the National Research Council, whose members are drawn from the councils of the National Academy of Sciences, the National Academy of Engineering, and the Institute of Medicine. The members of the committee responsible for the report were chosen for their special competences and with regard for appropriate balance. This study was supported by Contract No. N0-OD-4-2139 between the National Academy of Sciences and the National Institutes of Health. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the views of the organizations or agencies that provided support for the project. Additional copies of this report are available from: National Academy Press 2101 Constitution Ave., NW Box 285 Washington, DC 20055 800-624-6242 202-334-3313 (in the Washington metropolitan area) www.nap.edu Copyright 1999 by the National Academy of Sciences Printed in the United States of America

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Committee on Methods of Producing Monoclonal Antibodies PETER A. WARD (Chair), Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan JANE ADAMS, Juvenile Diabetes Foundation, Washington, DC DENISE FAUSTMAN, Immunology Laboratories, Massachusetts General Hospital, Charlestown, Massachusetts GERALD F. GEBHART, Department of Pharmacology, University of Iowa College of Medicine, Iowa City, Iowa JAMES G. GEISTFELD, Laboratory Animal Medicine, Taconic Farms, Germantown, New York JOHN W. IMBARATTO, Cell Culture Manufacturing, Covance Biotechnology Services, Inc., Research Triangle Park, North Carolina NORMAN C. PETERSON, Department of Clinical Studies, University of Pennsylvania, Philadelphia, Pennsylvania FRED QUIMBY, Center for Laboratory Animal Resources, Cornell University Veterinary College, Ithaca, New York ANN MARSHAK-ROTHSTEIN, Department of Microbiology, Boston University School of Medicine, Boston, Massachusetts ANDREW N. ROWAN, Humane Society of the United States, Washington, DC MATTHEW D. SCHARFF, Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York Staff RALPH B. DELL, Director KATHLEEN A. BEIL, Administrative Assistant SUSAN S. VAUPEL, Managing Editor, ILAR Journal MARSHA K. WILLIAMS, Project Assistant NORMAN GROSSBLATT, Editor

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Institute for Laboratory Animal Research Council JOHN VANDEBERG (Chair), Southwest Foundation for Biomedical Research, San Antonio, Texas CHRISTIAN R. ABEE, Department of Comparative Medicine, University of South Alabama, Mobile, Alabama BENNETT DYKE, Southwest Foundation for Biomedical Research, San Antonio, Texas ROSEMARY W. ELLIOTT, Department of Molecular and Cellular Biology, Roswell Park Cancer Institute, Buffalo, New York GERALD F. GEBHART, Department of Pharmacology, College of Medicine, University of Iowa, Iowa City, Iowa HILTON J. KLEIN, Department of Laboratory Animal Resources, Merck Research Laboratories, West Point, Pennsylvania MARGARET LANDI, Department of Laboratory Animal Science, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania CHARLES R. MCCARTHY, Kennedy Institute of Ethics, Georgetown University, Washington, DC HARLEY MOON, Veterinary Medical Research Institute, Iowa State University, Ames, Iowa WILLIAM MORTON, Regional Primate Research Center, University of Washington, Seattle, Washington ROBERT J. RUSSELL, Harlan Sprague Dawley, Inc., Indianapolis, Indiana WILLIAM S. STOKES, Environmental Toxicology Program, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina JOHN G. VANDENBERGH, Department of Zoology, North Carolina State University, Raleigh, North Carolina PETER A. WARD, Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan THOMAS WOLFLE, Annapolis, Maryland JOANNE ZURLO, Center for Alternatives to Animal Testing, Johns Hopkins School of Hygiene and Public Health, Baltimore, Maryland Staff RALPH B. DELL, Director KATHLEEN A. BEIL, Administrative Assistant SUSAN S. VAUPEL, Managing Editor, ILAR Journal MARSHA K. WILLIAMS, Project Assistant

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Commission on Life Sciences MICHAEL T. CLEGG (Chair), College of Natural and Agricultural Sciences, University of California, Riverside, California PAUL BERG (Vice Chair), Stanford University School of Medicine, Stanford, California FREDERICK R. ANDERSON, Cadwalader, Wickersham & Taft, Washington, DC JOHN C. BAILAR III, Department of Health Studies, University of Chicago, Chicago, Illinois JOANNA BURGER, Division of Life Sciences, Environmental and Occupational Health Sciences Institute, Rutgers University, Piscataway, New Jersey SHARON L. DUNWOODY, School of Journalism and Mass Communication, University of Wisconsin, Madison, Wisconsin DAVID EISENBERG, University of California, Los Angeles, California JOHN L. EMMERSON, Eli Lilly and Co. (ret.), Indianapolis, Indiana NEAL L. FIRST, Department of Animal Science, University of Wisconsin, Madison, Wisconsin DAVID J. GALAS, Chiroscience R&D, Inc., Bothell, Washington DAVID V. GOEDDEL, Tularik, Inc., South San Francisco, California ARTURO GOMEZ-POMPA, Department of Botany and Plant Sciences, University of California, Riverside, California COREY S. GOODMAN, Department of Molecular and Cell Biology, University of California, Berkeley, California HENRY W. HEIKKINEN, Department of Chemistry and Biochemistry, University of Northern Colorado, Greeley, Colorado BARBARA S. HULKA, Department of Epidemiology, University of North Carolina, Chapel Hill, North Carolina HANS J. KENDE, MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, Michigan CYNTHIA J. KENYON, Department of Biochemistry, University of California, San Francisco, California MARGARET G. KIDWELL, Department of Ecology and Evolutionary Biology, University of Arizona, Tucson, Arizona BRUCE R. LEVIN, Department of Biology, Emory University, Atlanta, Georgia OLGA F. LINARES, Smithsonian Tropical Research Institute, Miami, Florida DAVID M. LIVINGSTON, Dana-Farber Cancer Institute, Boston, Massachusetts DONALD R. MATTISON, March of Dimes, White Plains, New York ELLIOT M. MEYEROWITZ, Division of Biology, California Institute of Technology, Pasadena, California ROBERT T. PAINE, Department of Zoology, University of Washington, Seattle, Washington

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RONALD R. SEDEROFF, Department of Forestry, North Carolina State University, Raleigh, North Carolina ROBERT R. SOKAL, Department of Ecology and Evolution, State University of New York at Stony Brook, New York CHARLES F. STEVENS, MD, The Salk Institute for Biological Studies, La Jolla, California SHIRLEY M. TILGHMAN, Department of Molecular Biology, Princeton University, Princeton, New Jersey JOHN L. VANDEBERG, Southwest Foundation for Biomedical Research, San Antonio, Texas RAYMOND L. WHITE, Department of Oncological Sciences, University of Utah, Salt Lake City, Utah Staff MYRON UMAN, Acting Executive Director

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The National Academy of Sciences is a private, nonprofit, self-perpetuating society of distinguished scholars engaged in scientific and engineering research, dedicated to the furtherance of science and technology and to their use for the general welfare. Upon the authority of the charter granted to it by the Congress in 1863, the Academy has a mandate that requires it to advise the federal government on scientific and technical matters. Dr. Bruce M. Alberts is president of the National Academy of Sciences. The National Academy of Engineering was established in 1964, under the charter of the National Academy of Sciences, as a parallel organization of outstanding engineers. It is autonomous in its administration and in the selection of its members, sharing with the National Academy of Sciences the responsibility for advising the federal government. The National Academy of Engineering also sponsors engineering programs aimed at meeting national needs, encourages education and research, and recognizes the superior achievements of engineers. Dr. William A. Wulf is president of the National Academy of Engineering. The Institute of Medicine was established in 1970 by the National Academy of Sciences to secure the services of eminent members of appropriate professions in the examination of policy matters pertaining to the health of the public. The Institute acts under the responsibility given to the National Academy of Sciences by its congressional charter to be an adviser to the federal government and, upon its own initiative, to identify issues of medical care, research, and education. Dr. Kenneth I. Shine is president of the Institute of Medicine. The National Research Council was organized by the National Academy of Sciences in 1916 to associate the broad community of science and technology with the Academy's purposes of furthering knowledge and advising the federal government. Functioning in accordance with general policies determined by the Academy, the Council has become the principal operating agency of both the National Academy of Sciences and the National Academy of Engineering in providing services to the government, the public, and the scientific and engineering communities. The Council is administered jointly by both Academies and the Institute of Medicine. Dr. Bruce M. Alberts and Dr. William A. Wulf are chairman and vice chairman, respectively, of the National Research Council.

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Preface Monoclonal antibodies (mAb) are used extensively in basic biomedical research, in diagnosis of disease, and in treatment of illnesses, such as infections and cancer. Antibodies are important tools used by many investigators in their research and have led to many medical advances. Producing mAb requires immunizing an animal, usually a mouse; obtaining immune cells from its spleen; and fusing the cells with a cancer cell (such as cells from a myeloma) to make them immortal, which means that they will grow and divide indefinitely. A tumor of the fused cells is called a hybridoma, and these cells secrete mAb. The development of the immortal hybridoma requires the use of animals; no commonly accepted nonanimal alternatives are available. An investigator who wishes to study a particular protein or other molecule selects a hybridoma cell line that secretes mAb that reacts strongly with that protein or molecule. The cells must grow and multiply to form a clone that will produce the desired mAb. There are two methods for growing these cells: injecting them into the peritoneal cavity of a mouse or using in vitro cell-culture techniques. When injected into a mouse, the hybridoma cells multiply and produce fluid (ascites) in its abdomen; this fluid contains a high concentration of anti-body. The mouse ascites method is inexpensive, easy to use, and familiar. However, if too much fluid accumulates or if the hybridoma is an aggressive cancer, the mouse will likely experience pain or distress. If a procedure produces pain or distress in animals, regulations call for a search for alternatives. One alternative is to grow hybridoma cells in a tissue-culture medium; this technique requires some expertise, requires special media, and can be expensive and time-consuming. There has been considerable research on in vitro methods for grow-

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ing hybridomas and these newer methods are less expensive, are faster, and produce antibodies in higher concentration than has been the case in the past. The existence of alternatives to the mouse ascites method raises the question: Is there a scientific need for the mouse ascites method of producing mAb? The American Anti-Vivisection Society (AAVS) petitioned the National Institutes of Health (NIH) on April 23, 1997, to prohibit the use of animals in the production of mAb. On September 18, 1997, NIH declined to prohibit the use of mice in mAb production, stating that "the ascites method of mAb production is scientifically appropriate for some research projects and cannot be replaced." On March 26, 1998, AAVS submitted a second petition, stating that "NIH failed to provide valid scientific reasons for not supporting a proposed ban." The office of the NIH director asked the National Research Council to conduct a study of methods of producing mAb. In response to that request, the Research Council appointed the Committee on Methods of Producing Monoclonal Antibodies, to act on behalf of the Institute for Laboratory Animal Research of the Commission on Life Sciences, to conduct the study. The 11 expert members of the committee had extensive experience in biomedical research, laboratory animal medicine, animal welfare, pain research, and patient advocacy (Appendix B). The committee was asked to determine whether there was a scientific necessity for the mouse ascites method; if so, whether the method caused pain or distress; and, if so, what could be done to minimize the pain or distress. The committee was also asked to comment on available in vitro methods; to suggest what acceptable scientific rationale, if any, there was for using the mouse ascites method; and to identify regulatory requirements for the continued use of the mouse ascites method. The committee held an open data-gathering meeting during which its members summarized data bearing on those questions. A 1-day workshop (Appendix A) was attended by 34 participants, 14 of whom made formal presentations. A second meeting was held to finalize the report. The present report was written on the basis of information in the literature and information presented at the meeting and the workshop. This report has been reviewed by persons chosen for their diverse perspectives and technical expertise in accordance with procedures approved by the National Research Council's Report Review Committee. The purposes of the independent review are to provide candid and critical comments that will assist the authors and the Research Council in making the published report as sound as possible and to ensure that the report meets institutional standards of objectivity, evidence, and responsiveness to the study charge. The contents of the review comments and the draft manuscript remain confidential to protect the integrity of the deliberative process. We wish to thank the following persons for their participation in the review of this report:

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J. Donald Capra, Oklahoma Medical Research Foundation, Oklahoma City Philip Carter, North Carolina State University, Raleigh Joseph Chandler, Maine Biotechnology Services, Inc., Portland Jon W. Gordon, Mt. Sinai School of Medicine, New York, NY Coenraad Hendricksen, National Institute of Public Health and the Environment, Bilthoven, The Netherlands Dave Hill, Oncogene Research Products, Cambridge, MA Charles A. Janeway, Yale University School of Medicine, New Haven, CT Neil S. Lipman, Memorial Sloan-Kettering, New York, NY Uwe Marx, University of Leipzig, Leipzig, Germany Henry Metzger, National Institute of Arthritis and Musculoskeletal and Skin Diseases, Bethesda, MD William E. Paul, National Institute of Allergy and Infectious Diseases, Bethesda, MD Jon Richmond, Home Office, United Kingdom Alan Stall, PharMingen, San Diego, CA Peter Theran, Massachusetts Society for the Prevention of Cruelty to Animals, Boston Jonathan W. Uhr, University of Texas Southwestern Medical Center, Dallas, TX Ellen S. Vitetta, University of Texas Southwestern Medical Center, Dallas, TX The list shows the diversity and background of the reviewers, again attesting to the rigor of the process of producing this report. Although the persons listed have provided many constructive comments and suggestions, responsibility for the final content of this report rests solely with the authoring committee and the National Research Council. To the committee members, reviewers, and staff, I extend my deepest appreciation. Members of the committee devoted precious weekends, evenings, and work hours and endless energy to meet short deadlines. The reviewers also worked under short deadlines, and their efforts greatly improved the logic, coherence, and comprehensibility of our report. I am grateful for the guidance and support provided by the Institute for Laboratory Animal Research staff throughout the process. Kathleen Beil provided timely and important communications to the committee in arranging travel and lodging and in report production. Ralph Dell's focus on the topic and his management of the review and publication were of inestimable value. Norman Grossblatt's editing made the report eminently more readable—a feature that will be appreciated by readers. PETER A. WARD, CHAIR COMMITTEE ON METHODS OF PRODUCING MONOCLONAL ANTIBODIES

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Contents     Executive Summary   1     Introduction   5     Generation of Hybridomas: Permanent Cell Lines Secreting Monoclonal Antibodies   8     In Vitro Production of Monoclonal Antibody   12     Batch Tissue-Culture Methods   12     Semipermeable-Membrane-Based Systems   14     Scientific Needs for Mouse Ascites Production of mAb   16     Summary of Advantages and Disadvantages of In Vitro and In Vivo Methods   22     Advantages of In Vitro Methods   22     Disadvantages of In Vitro Methods   23     Advantages of Mouse Ascites Method   24     Disadvantages of Mouse Ascites Methods   24     Large-Scale Production of Monoclonal Antibodies   25     Monoclonal Antibody Production for Diagnostic and Therapeutic Purposes   25     In Vivo and In Vitro Methods for Commercial Production of mAb   26

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    In Vivo Production   28     In Vitro Production   30     Regulatory Requirements   31     Animal-Welfare Issues Related to the Ascites Method for Producing Monoclonal Antibodies   33     Availability of Data   33     Animal-Welfare Issues Related to Ascites Method   34     Methods for Measuring Pain or Distress in Laboratory Rodents   34     Priming   36     Ascites   37     Harvesting Ascitic Fluid   39     Feeder Cell Harvesting and Serum Supplements for In Vitro Hybridoma Culture   43     Summary of Animal Welfare Issues   43     Conclusions and Recommendations   45     References   48 Appendix A   Workshop on Methods of Producing Monoclonal Antibodies   55 Appendix B   Biographical Sketches of Authoring Committee   57