FROM CLONE TO CLINIC
and
SPRINGER SCIENCE+BUSINESS MEDIA, B.V.
Library of Congress Cataloging in Publication Data
From clone to clinic I edited by D.J.A. Crommel in and H. Schellekens. p. cm. -- <Developments in blotherapy ; v. 1)
ISBN 978-94-010-5683-0 ISBN 978-94-011-3780-5 (eBook)
DOI 10.1007 /978-94-011-3780-5
1. Pharmaceutical b1otechnology. 2. Monoclonal ant1bodies. 1. Crommelin. D. J. A. (Daan J. A.) II. Schelleken, Huub. III. Series.
[DNLM: 1. Antibodies, Monoclonal--therapeutic use. 2. Biolog1cal Products--standards. 3 . Clon lng, Molecular. 4. DNA, RecoNb1nant. 5. Genetic Engineering . OW 575 F931J RS380.F76 1990 615·.7--dc20 DNLM/DLC for Library of Congres s
ISBN 978-94-010-5683-0
Printed on acid-free paper
AlI Rights Reserved © 1990 by Springer Science+Business Media Dordrecht Origina1ly published by Kluwer Academic Publishers in 1990 Softcover reprint of the hardcover Ist edition 1990
90-5267
No part of the material protected by this copyright notice may be reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording or by any information storage and retrieval system, without written permission from the copyright owner.
Contents
Preface
1. Toxicity testing of rDNA products A.D. Dayan
2. Experiences with the EC "high tech" procedure
IX
Xl
3. The regulation of pharmaceuticals: philosophy and principles J.C. Petricciani 29
4. Quality control of vaccines J.H. Peetermans 29
5. Regulatory affairs and biotechnology in Europe: the CPMP "high tech" and multi­ state procedures
B. Tryzelaar 37
6. Formulating biotechnology products T. Mundorf, M. Bichler and G. Flotzinger 45
7. Development of analytical methods for monitoring the stability of antibody forma­ tion by hybridoma cells in continuous culture systems
J.M. Coco Martin, P. Koolwijk, D.E. Martens, J.W. Oberink, C.A.M. van der Velden-de Groot and E.c. Beuvery 57
PART TWO: MONOCLONAL ANTIBODIES
8. Toward human monoclonal antibodies D.R Burton, M.A. Persson, W.D. Huse and E.S. Golub 67
9. Biodistribution, binding and intemalisation of monoclonal antibodies to human ovarian carcinoma cells
L.G. Poe1s, O.C. Boerman, L. Massuger, R Claessens, U.K. Nlissander, Q. van Hoesse1, C. Thomas, P. Kenemans and P. Jap 73
10. Pharmacokinetics and tissue distribution ofIndium-lll-labe1ed OV -TL 3 F(ab')2 in ovarian cancer patients
L.F.A.G. Massuger, RA.MJ. Claessens, L.G. Poe1s, RH.M. Verheijen, Ch.P.T. Schijf, F.H.M. Corstens and P. Kenemans 83
11. Characterization of human monoclonal antibodies specific for the rabies virus L. Edelman and M. Lafon 89
12. Biochemical and immunological evaluation of an anti-fibrin monoclonal antibody complex containing T2Gls Fab' intended for imaging venous thrombi
P.-H. Lai, J. Brown, L.A. Epps, K. George, A. Sautter, C. Coonley, M.
vi
Plucinsky, S. Buttrum, W.C. Bogard, Jr., RT. Dean and H.I. Berger 95
13. Monoclonal antibodies in radioimmunoscintigraphy. Some hurdles between clone and clinic
D. Blok, R.I.1. Feitsman and E.K.I. Pauwels 105
14. Clinical relevance of the tumor marker CA 15.3 in the management of cancer patients
G.G. Bon, P. Kenemans, C.A. Yedema, G.I. van Kamp, H.W. Nijman and 1. Hilgers 111
15. Practical applications of monoclonal antibodies against polymorphic epithelial mucin in the differential diagnosis of human tumors
J. Hilgers and P. Kenemans 123
16. Selection of monoclonal anti-digoxin antibodies suitable for monitoring of cardiac glycosides
S.N. Tyutyulkova, S.D. Kyurkchiev and I.R Kehayov 135
17. Diagnostic use of anti-modified nucleoside monoclonal antibodies M. Mizugaki, K. Itoh and N. Ishida 143
18. Monoclonal antibodies radiolabeled with different radioisotopes for biodistribution and radioimmunodetection of tumor xenografts in the nude rat
M.W.A. de Jonge, J.G.W. Kosterink, Yu Yan bin, 1.W.M. Bulte, R.A.M. Ken- gen, T.H. The and L. de Leij 149
19. Bispecific monoclonal antibody (BIAB)-retargeted cellular therapy for local treatment of cancer patients
L. de Leij, M.W.A. de Jonge, 1. ter Haar, H. Spakman, E. de Vries, P. Willemse, N.H. Mulder, H. Berendsen, M. Elias, C. Smit Sibinga, W. de Lau, W. Tax and T.H. The 159
20. Enhanced binding of t-PA to fibrin using bispecific monoclonal antibodies R. Bos, M. Otter and W. Nieuwenhuizen 167
21. Recent developments and perspectives on the future of human and murine monoclonal antibodies in the diagnosis and treatment of cancer
R.P. McCabe, M.V. Haspel, J.A. Carrasquillo, RG. Steis, N. Pomato, R. Sub- ramanian, E.M. Paris and M.G. Hanna, Jr. 175
22. Pharmacokinetics and safety of a human IGM antibody, HA-IA R.C. Straube, C.R. Smith, I.E. Allen, C.P. Dating, C. Kilgarriff, 1.B. Cornett and S.D. Bolmer 189
23. Development and clinical experience with humanised monoclonal antibodies G. Hale, M.I.S. Dyer, M.R. Clark and H. Waldmann 195
24. A method for the transformation of hybridoma cell lines with improved efficiency: its use in the production of bispecific monoclonal antibodies
R. Bos, E. van den Berg and W. Nieuwenhuizen 201
25. To an optimal design of an airlift bioreactor for the cultivation of hybridomas D.E. Martens, J. Coco Martin, C.A.M. van der Velden-de Groot, E.C. Beuvery, C.D. de Gooijer and J. Tramper 209
vii
PART THREE: MARKETED PRODUCTS
26. Experience with marketed biotech products: rt-PA L. Nelles and D. Collen 219
27. Clinical trial of recombinant human IL-2 in the treatment of Mycobacterium Avium complex infection
H. Toba, I. Tsuyuguchi, H. Kimura, H. Fujiwara, S. Hanamoto, H. Kawasumi and S. Kishimoto 227
28. Use of recombinant human erythropoietin in anemic dialysis patients I.C. Egrie 233
29. Experience with biosynthetic growth hormone J.M. Wit 239
PART FOUR: NEW PRODUCTS
30. Recombinant follicle stimulating hormone. I. Construction, selection and charac­ terization of a cell line
P. van Wezenbeek, J. Draaijer, F. van Meel and W. Olijve 245
31. Recombinant human follicle stimulating hormone. II. Biochemical and biological characteristics
W. de Boer and B. Mannaerts 253
32. Heterologous expression of human Interleukin-3 R.W. van Leen, PJ. Lemson and J.G. Bakhuis 261
33. Purification of recombinant human Interleukin-3 from Bacillus Licheniformis B. Noordam, R.F.W.C. van Beckhoven, R. van Lambalgen and N.L.M. Persoon 269
34. The acid- and thermolabile interferon alpha: a SUbtype, or a new cell inhibitor? L. Borecky, P. Kontsek, V. Lackovic, I. Mistn'kova and M. Novak 275
35. The IFNy receptor as tool for the discovery of new immunomodulatory drugs L. Ozmen, M. Fountoulakis, D. Stuber and G. Garotta 283
36. Cloned receptors and transfected cell lines in the design of new drugs: muscarinic cholinergic receptors
W. Sadee, S. Maeda, I. Lameh and R.I. Cone 291
PART FIVE: DRUG DELIVERY
37. Structural analysis of carbohydrate chains of native and recombinant-DNA glycoproteins
J.P. Kamerling, K. Hard and J.F.G. Vliegenthart
38. The role of protein structure in surface tension kinetics A.-P. Wei, J.N. Herron and J.D. Andrade
39. Antigen carriers: a success determining factor for subunit vaccines? G.F.A. Kersten, E.C. Beuvery and DJ.A. Crommelin
40. Intranasal delivery of insulin: absorption enhancement by the fusidate derivative STDHF in rabbits and rats and effects on human nasal ciliary movement in vitro
295
305
315
viii
J. Verhoef, W.AJJ. Hennens, MJ.M. Deurloo, S.G. Romeijn, N.G.M. Schipper and P.W.H.M. Merkus 323
41. Improved oral peptide delivery by means ofmucoadhesion C.-M. Lehr, J.A. Bouwstra, J.J. Tukker, J. Verhoef, A.G. de Boer and H.E. Junginger 329
42. Delivery of therapeutic peptides and proteins M. Mackay 335
43. Compatibility studies of a soluble T4 receptor with a microinfusion pump for continuous intravenous therapy
A.L. Shorter, M.B. Seaman, D. Dunleavy, P. Smialkowski and D. Schrader 343
44. Immunoliposome-mediated delivery of chemotherapeutics U.K. Nassander, G. Stonn, P.A. Steerenberg, W.H. de Jong, G. de Groot, L.G. Poels, Q.G.C.M. van Hoesel and DJ.A. Crommelin 357
45. A novel approach for the selection and detection of cells transfected with adenosine deaminase expression vectors
Q. Shen, V.W. van Beusechem, M.P.W. Einerhand and D. Valerio 367
46. Toward gene therapy in hemophilia A: introduction of factor VIII expression vectors into somatic cells
R.C. Hoeben, M.P.W. Einerhand, SJ. Cramer, E. Briet, H. van Onnondt, D. Valerio and AJ. van der Eb 373
Preface
This book contains a selection of the papers presented at the meeting "Between Clone and Clinic" which was organised in March 1990 in Amsterdam by the dutch Organisation for Applied Research, TNO, and the University of Utrecht. The scope of this meeting was the development of biotechnological pharmaceuticals mainly made by recombinant DNA technology or monoclonal antibody techniques. All aspects concerning the development of the products after host cells producing them are obtained where discussed. The meeting was attended by twohundred specialists from all over the globe, including phar­ macologists, toxicologists, registration experts, Quality Assurence managers, production en­ gineers and physicians. Biotechnological pharmaceuticals are in general large and complex protein molecules. Bringing these products to the market poses other problems than encountered with the classical chemical drugs. The source of biotechnological pharmaceuticals are living cells. The function of cells are depend­ ent on many factors and the stability of production may be a problem. Good Laboratory and Manufactory Practices with Quality Control (GLP and GMP) are of paramount importance and are discussed in a number of papers. The products of the new biotechnology are often highly specific and only active in the human species. Also the side effects can only be studied in the clinical setting. Even when the product is active in animals there is the problem of antigenicity. During treatment the animals will produce antibodies which neutralise the activity. So safety testing may prove difficult. Within the EEC there is a centralised registration of biotechnological pharmaceuticals. Submis­ sion in a single european country within the community is sufficient. This donor country will submit the dossier in Brussels, where an evaluation by EEC experts is made. When approved in Brussels registration in individual countries is greatly facilitated and after 1992 mandatory. Two papers in the book discuss this european registration procedure. Although the techniques for the production of these new biotechnological pharmaceuticals are new, a number of products as OKT-3, insuline, hepatitis B vaccine, interferon, erythropoetine an human growth hormone are now on the market for several years. The problems encountered when developing these products are presented by representatives of the industries involved. Much is expected from the use of monoclonal antibodies alone or as carriers of other agents. To avoid antigenicity a lot of attention has focused on the development of human monoclonal antibodies and a number of papers give a tour d'horizon on this topic. Biotechnology products are by their size and relative instability difficult to get to their target organs. Development of drug delivery systems is urgently needed before most of these new drugs can be used successfully. A number of new opportunities in this area are presented in this book. This was the first time that all these experts convened and discussed the problems of the develop­ ment of biotechnological pharmaceuticals comprehensively. Judging from the quality of the papers in this book the meeting was a success and deserves to be repeated.
Daan Crommelin and Huub Schellekens July 1990
ix
D.BLOK University Hospital Leiden, Division of Nuclear Medicine, Department of Diagnostic Radiology, Rijnsburgerweg 10, 2333 AA Leiden, The Netherlands Co-authors: R. 1. J. FEITSMA and E. K. J. PAUWELS
G. G. BON Free University Hospital, Department of Obstetrics & Gynaecology, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands Co-authors: P. KENEMANS, CA. YEDEMA, G. J. VAN KAMP, H. W. NIJMAN and J. HILGERS
L.BORECKY Institute of Virology, Slovak Academy of Sciences, Dti brawska 9, 84246 Bratislava, Czechos­ lovakia Co-authors: P. KONTSEK, V. LACKOVIC, J. MISTRlKOV A and M. NovAK
R.BOS Gaubius Institute TNO, P.O. Box 612, 2300 AP Leiden, The Netherlands Chapter 20 co-authors: M. OTTER and W. NIEUWENHUIZEN Chapter 24 co-authors: E. V AN DEN BERG and W. NIEUWENHUIZEN
D.R.BURTON Department of Molecular Biology, Scripps Clinic & Research Foundation, 10666 N. Torrey Pines Road, La Jolla, CA 92037, U.S.A. Co-authors: M. A. PERSSON, W. D. HUSE and E. S. GOLUB
J. M. COCO MARTIN National Institute for Public Health and Environmental Protection (RIVM), Department of Inactivated Viral Vaccines, P.O. Box 1,3720 BA Bilthoven, The Netherlands Co-authors: P. KOOLWlJK, D. E. MARTENS, J. W. OBERINK, C. A. M. VAN DER VELDEN-DE GROOT and E. C. BEUVERY
D. J. A. CROMMELIN University of Utrecht, Faculty of Pharmacy, Department of Pharmaceutics, Croesestraat 79, 3522 AD Utrecht, The Netherlands
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A.D. DAYAN DH Department of Toxicology, St Bartholomew's Hospital, Dominion House, 59 Bartholomew Close, London EC1 7ED, U.K.
L. DELEIJ Academic Hospital Groningen, Department of Clinical Immunology, Oostersingel 59, 9713 EZ Groningen, The Netherlands Co-authors: M. W. A. DE JONGE, J. TER HAAR, H. SPAKMAN, E. DE VRIES, P. WIL­ LEMSE, N. H. MULDER, H. BERENDSEN, M. ELIAS, C. SMIT-SIBINGA, W. DE LAU, W. TAX and T. H. THE
M. W. A. DE JONGE University Hospital Groningen, Department of Clinical Immunology, Oostersingel 59, 9713 EZ Groningen, The Netherlands Co-authors: J. G. W. KOSTERINK, YU YAN BIN, J. W. M. BULTE, R. A. M. KENGEN, T. H. THE and L. DE LEIJ
W.DEBOER Organon International BV, Scientific Development Group, P.O. Box 20, 5340 BH Oss, The Netherlands Co-author: B. MANNAERTS
L.EDELMAN Division of Immunology, Institut Pasteur, 25-28, Rue du Dr. Roux, F-75724 Paris Cedex 15, France Co-author: M. LAFON
J. C.EGRIE Amgen, Inc, 1840 DeHavilland Drive, Thousand Oaks, CA 91320, U.S.A.
G.HALE University of Cambridge, Department of Pathology, Tennis Court Road, Cambridge CB2 1QP, U.K. Co-authors: M. J. S. DYER, M. R. CLARK and H. WALDMANN
J. HILGERS Free University Hospital, Department of Obstetrics & Gynaecology, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands Co-author: P. KENEMANS
R.C.HOEBEN University of Leiden, Department of Medical Biochemistry, P.O. Box 9503, 2300 RA Leiden, The Netherlands Co-authors: M. P. W. EINERHAND, S. J. CRAMER, E. BRIET, H. VAN ORMONDT, D. VALERIO and A. J. VAN DER EB
J. P. KAMERLING Utrecht University, Bijvoet Center, Department of Bio-Organic Chemistry, P.O. Box 80.075,
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3508 TB Utrecht, The Netherlands Co-authors: K. HARD and 1. F. G. VLIEGENTHART
G. F. A. KERSTEN National Institute for Public Health and Environmental Protection, Department of Inactivated Viral Vaccines, P.O. Box 1,3720 BA Bilthoven, The Netherlands Co-authors: E. C. BEUVERY and D. J. A. CROMMELIN
P.- H. LAI Centocor, 244 Great Valley Parkway, Malvern, PA 19355, U.S.A. Co-authors: 1. BROLON, L. A. EPPS, K. GEORGE, A. SAUTTER, C. COONLEY, M. PLUCINSKY, S. BUTTRUM, W. C. BOGARD Jr, R. T. DEAN and H. J. BERGER
C.-M.LEHR Center for Bio-Pharmaceutical Sciences, Leiden University, Einsteinweg 5, 2300 RA Leiden, The Netherlands Co-authors: J. A. BOUWSTRA, J. J. TUKKER, 1. VERHOEF, A. G. DE BOER and H. E. JUNGINGER
M.MACKAY Ciba-Geigy Pharmaceuticals, Advanced Drug Delivery Research, Wimblehurst Road, Horsham, West Sussex RH12 4AB, U.K.
D. E. MARTENS Department ofInactivated Viral Vaccins, RIVM, P.O. Box 1,3720 BA Bilthoven, The Nether­ lands Co-authors: 1. COCO MARTIN, C. A. M. VANDER VELDEN-DE GROOT, E. C. BEUVERY, C. D. DE GOOIJER and 1. TRAMPER
L. F. A. G. MASSUGER Department of Obstetrics & Gynaecology, University Hospital Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands Co-authors: R. A. M. J. CLAESSENS, L. G. POELS, R. H. M. VERHEUEN, C. P. T. SCHUF, F. H. M. CORSTENS and P. KENEMANS
R.P.MCCABE Organon Teknika Corporation, Biotechnology Research Institute, Cancer Diagnostics and Therapy, 1330A Piccard Drive, Rockville, MD 20850, U.S.A. Co-authors: M. V. HASPEL, 1. A. CARRASQUILLO, R. G. STEIS, N. POMATO, R. SUB­ RAMANIAN, E. M. PARIS and M. G. HANNA Jr
M.MIZUGAKI Tohoku University Hospital, Department of Pharmaceutical Sciences, 1-1 Seiryo-machi, Aoba­ ku, Sendai 980, Japan Co-authors: K. ITOH and N. ISHIDA
T.MUNDORF Present address: Ares Serono, P.O. Box 54, 1211 Geneva 20, Switzerland
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Co-authors: M. BICHLER and G. FLOTZINGER
U. K. NAsSANDER University of Utrecht, Faculty of Pharmacy, Department of Pharmaceutics, Croesestraat 79, 3522 AD Utrecht, The Netherlands Co-authors: G. STORM, P. A. STEERENBERG, W. H. DE JONG, G. DE GROOT, L. G. POELS, Q. G. C. M. VAN HOESEL and D. J. A. CROMMELIN
L.NELLES Catholic University Leuven, Center for Thrombosis and Vascular Research, Herestraat 49, B- 3000 Leuven, Belgium Co-author: D. COLLEN
B.NOORDAM Gist-brocades NV, Department of Protein Chemistry and Enzymology, Wateringseweg 1, 2600 MA Delft, The Netherlands Co-authors: R. F. W. C. VAN BECKHOVEN, R. V AN LAMBALGEN and N. L. M. PERSOON
L.OZMEN Central Research Units, Hoffmann-La Roche, CH-4002 Basel, Switzerland Co-authors: M. FOUNTOULAKIS, D. STOBER and G. GAROTTA
J. H. PEETERMANS SmithKline Biologicals, 89, Rue de l'Institut, B-1330 Rixensart, Belgium
J. G. PETRICCIANI Pharmaceutical Manufacturers Association, Medical and Regulatory Affairs, 1100 15th Street NW, Washington, DC 20005, U.S.A.
L.G.POELS Department of Cell Biology and Histology, St. Radboud Hospital, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands Co-authors: O. C. BOERMAN, L. MASSUGER, R. CLAESSENS, U. K. NAsSANDER, Q. VAN HOESSEL, C. THOMAS, P. KENEMANS and P. JAP
W.SADEE University of California, Department of Pharmacy & Pharmaceutical Chemistry, San Francisco, CA 94143, U.S.A. Co-authors: S. MAEDA, J. LAMEH and R. I. CONE
H. SCHELLEKENS TNO Primate Center, P.O. Box 5815,2280 HV Rijswijk, The Netherlands
Q.SHEN Present address: Tongji Medical University, Hongkong Road 13, Wuhan, The People's Republic of China Co-authors: V. W. VAN BEUSECHEM, M. P. W. EINERHAND and D. VALERIO
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A. L. SHORTER Department of Pharmaceutical Research, SmitKline Beecham Pharmaceuticals, P.O. Box 1539, King of Prussia, PA 19406, U.S.A. Co-authors: M. B. SEAMAN, D. DUNLEAVY, P. SMIALKOWSKI and D. SCHRADER
R. C. STRAUBE Centocor, Inc, 244 Great Valley Parkway, Malvern, PA 19355, U.S.A. Co-authors: C. R. SMITH, I. E. ALLEN, C. P. DATING, C. KILGARRIFF, J. B. CORNETT and S. D. BOLMER
H. TOBA Osaka Prefectural, Habikino Hospital, 7, 3-Chome, Habikino, Habikino City, Osaka 583, Japan Co-authors: I. TSUYUGUCHI, H. KIMURA, H. FUJIWARA, S. HANAMOTO, H. KAWASUMI and S. KISHIMOTO
B. TRYZELAAR Strategic Biotech International, Laan van Vollenhove 3181, 3706 AR Zeist, The Netherlands
S. N. TYUTYULKOV A Chemical Pharmaceutical Research Institute, Kliment Ohridsky la, Sofia 1156, Bulgaria Co-authors: S. D. KYURKCHIEV and I. R. KEHA YOV
H. J. M. V AN DE DONK Laboratory for Control of Bacterial Vaccines, RIVM, P.O. Box 1, 3720 BA Bilthoven, The Netherlands Co-author: I. HEGGER
P. VAN WEZENBEEK Scientific Development Group, Organon International BV, P.O. Box 20, 5340 BH Oss, The Netherlands Co-authors: J. DRAAUER, F. V AN MEEL and W. OLIJVE
R. W. VAN LEEN Royal Gist-brocades NV, Research and Development, P.O. Box 1,2600 MA Delft, The Nether­ lands Co-authors: P. J. LEMSON and J. G. BAKHUIS
J. VERHOEF Center for Bio-Pharmaceutical Sciences, Subdivision of Pharmaceutical Technology & Biophar­ maceutics, P.O. Box 9502, 2300 RA Leiden, The Netherlands Co-authors: W. A. J. J. HERMENS, M. J. M. DEURLOO, S. G. ROMEUN, N. G. M. SCHIP­ PER and F. W. H. M. MERKUS
A.-P. WEI University of Utah, Departments of Bioengineering and Pharmaceutics, Salt Lake City, UT 84112, U.S.A. Co-authors: J. N. HERRON and J. D. ANDRADE
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Toxicity Testing of rDNA Products
A D Dayan
A. GENERAL PLACE OF TOXICITY TESTING IN DEVELOPMENT OF rDNA PHARMACEUTICALS
In the past 15 years the biotechnology revolution has appeared, developed patchily to deliver some of the promised therapies and has matured sufficiently for a general guide to the necessary toxicity testing to be proposed (Graham, 1987; Marshak and Liu, 1988; and compilation of EC procedures by Tryzelaan, 1989).
Certain critical points apply to all toxicity studies and help to determine their nature and extent:
1. Purposes of Toxicity Testing
Studies of this type are primarily general survey procedures done both to detect potentially harmful effects and to exclude actions sought but not detected. They are not necessarily precise, scientific techniques for quantification, nor will they always be appropriate for exploration of the mechanisms of toxic actions, although the nature and pattern of the lesions may give a strong clue to pathogenesis.
Individual procedures (e.g. subacute or reproduction tests) should be identical to the comprehensive set of in vivo and pathological investigations done for a conventional new chemical entity (NCE; Dayan, 1990a) plus special techniques appropriate to the properties and uses of the substance being examined.
2. Range and Duration
Again, as for an NCE, the type and duration of the tests should be adapted to the intended clinical utilisation of the substance.
3. Nature of Toxicity Tests
The detailed content of the tOXICIty tests must be firmly based on the known physiological and pharmacological properties of the substance, whilst making allowance for the general need to detect the unanticipated effects that is the objective of toxicity studies.
To some extent this means that the experiments will often include functional and other 'high dose' pharmacological procedures, or at least the need to explore the latter should always be considered if little is known about the biology of the material under investigation.
D. J. A. Crommelin and H. Schellekens (eds.), From Clone to Clinic, 1-11. © 1990 Kluwer Academic Publishers.
2
Thus the toxicity testing of any rDNA product, whether it belongs generally to the cytokines, or growth factors, if it is a conventional or fragmented antibody, or a living, dead or isolated immunogen for use in vaccination, should follow a strategy pragmatically adapted to the properties and intended therapeutic use of the preparation, and constrained by the practicalities of animal studies.
It is very important that investigations be done in animals, because they provide the best model for the physiological complexities and integrated responses of man, but it is also important that the animals used are capable of showing the appropriate specific biological response to the substance. That may lead to work in only one species, which might, for example, be a rodent or a primate. There is no reason always to do experiments in a subhuman primate just because it is believed to be likely to be "closer to man." Often that is ethically incorrect. Primates are scarce, making the numbers available small, and endemic disease may produce confusing findings. It is sensible to examine several species to find one that is responsive, and to do most work in that supplemented by investigations in one other species (Weissinger, 1989). If only nonsensitive species are available, then work is best done in two convenient species, whilst accepting that hazard prediction then is limited, and that the early clinical studies in man should be particularly cautious.
4. Purposes of Toxicity Testing
The primary objective is to indicate potential toxic hazards so that a clinical decision can be made for the target species (man or animal) about the balance of risk and benefit. Meeting this requirement should satisfy medical and scientific concerns. For pharmaceutical development the next objective is to satisfy specific regulatory requirement, that uneasy balance of scientific empiricism and administrative peccadilos. And the third is to provide a defence against future legal claims for product liability.
In theory, all three objectives should be met by the same body of work, but in practice biological understanding by clinicians, regulators and lawyers is often limited, so additional experimentation may be required to meet all their needs.
B. DETAILED TOXICITY TEST REQUIREMENTS
The specific pattern of testing depends on the stage of development of the material, as the duration and magnitude of likely human exposure (patients at risk, workers in the manufacturing plant and any environmental hazard if there were an "escape" from the plant; see Dayan, Campbell and Jukes, 1989) must be related to the types and duration of the toxicity studies. And, additional experiments may be required to investigate particular adverse effects.
The detailed requirements for different types of product are so disparate, that antibodies and vaccines must be considered separately from cytokines, hormones and other, more conventional rDNA products.
The main types of the latter are described in Table 1. Experience has suggested that general scientific and regulatory concerns can be met as described below, although legalistic anxiety may only be satisfied by additional experimentation.
3
The general types of tests are summarised in Table 2 (and see Graham, 1987; Dayan, 1990b).
Special points to consider in designing the experiments include:
l. Selection and Number of Species
This is difficult because it is important both to select species that respond to the specific actions of the substance, and to do work only in those of which there is sufficient experience, i.e. a rodent, dog, primate or perhaps the pig. The reason for choosing a responsive species is that that properly permits high dose pharmacodynamic actions to be followed as well as other forms of toxicity. In general, many rDNA products produce the majority of their effects in several species, and it is only unusual materials, such as IFNa and IFNg, which show extreme specificity to man and the higher primates (Manning and Deloria, 1987), which have given the contrary impression. However, in planning toxicity studies, it is essential to investigate this point for all new substances.
Convention requires multidose studies in two species, as for a synthetic chemical.
2. Routine and Additional Investigations
As a survey, a broad range or clinical and laboratory tests is required plus any special measure of effect, e.g. fever, enumeration of lymphocyte subsets, assay hormone levels etc.
It is very important to look for antibody formation in multidose studies, and to characterise the nature and effect (neutralising, altered kinetics etc) of any found.
Such an immune response will make further testing of little or no value, and continued dosing may cause severe and misleading tissue damage, if immune complexes are formed.
3. Dose Levels
It is customary to employ three dose levels plus a vehicle only control group.
The top dose may be chosen on the basis that it is the maximum tolerated, or that it is a multiple of that intended for man, e.g. 100 to 200 x. Lower doses should be logarithmically spaced. That is the conventional advice for a straightforward NCE. It may or may not be applicable to an rDNA product, depending on species responsiveness and the planned treatment of man.
4. Duration of Dosing
As for a conventional chemical, this should be related to the duration of human exposure, provided that an immune response is not produced. If an antibody develops, continued dosing may well not give useful information.
4
5. Imperative of Kinetics and Metabolism
As a guide to man, the basic kinetics and disposition of the substance should be examined. A change in clearance is also an excellent guide to the appearance of an antibody.
Comparison of the kinetics of the compound and that of the biological response is also important as an aid to understanding the mechanism underlying certain types of toxicity ("high dose" pharmacological effects), and as guide to a monitoring strategy in the clinical studies.
As proteins or large polypeptides, the majority of rDNA products are likely to be taken up by the liver and after glomerular filtration, by renal tubule cells, and to be catabolised there by conventional proteases. These phenomena can be checked in vitro by the use of tissue homogenates if the disposition data suggest the need for such investigations.
In all the kinetic and related studies it is important to know what the analytical techniques is measuring. In other words, is the method responding to the intact molecule, to the biological activity or to an epitope or fragment, as interpretation of the diverse types of information may give quite different impressions of the behaviour of the substance.
6. What Toxicity Testing Cannot Exclude
Biological surveillance techniques of this type are not appropriate for quality control purposes as they are inefficient and insensitive indicators of the presence of impurities (low or high molecular weight, e.g. producing cell proteins, process chemicals etc), contaminating organisms and endotoxin, or of DNA or other hypothetical biological and even oncogenic products.
C. GENERAL CONCLUSIONS ABOUT TOXICITY TESTING rDNA PRODUCTS
The most effective general strategy, both for the manufacturer seeking an efficient development programme, and the clinician, who needs appropriate safety and efficacy information, is to follow the breadth of the studies done for a conventional new chemical entity, but to adapt them to the special nature and biological activities of each product, by making a case-by-case assessment of its activities and intended uses. Scientific flexibility is essential if studies are to be adapted to explore real problems rather than trying to force all the experimentation into a common, inappropriate and therefore confusing mould.
D. ANTIBODIES
The need for empiricism and focused safety testing is even more apparent in considering antibodies and fragment of them - poly or monoclonal, Fab, Fc etc, as well as new developments, such as humanised and chimeric molecules.
All these materials are likely to be antigenic to some degree in any species available for testing, so the need is to devise a pattern of short-term investigations that will reveal predictable hazards, whilst affording a reasonable opportunity to detect unanticipated actions.
A general scheme is suggested in Table 3. The importance of testing general systemic toxicity and autoimmunity is self-evident. The need for special studies of acute pharmacodynamic effects, and of possible anti-idiotype formation (which may lead, for example, to receptor stimulation or other abnormalities of regulatory control) may appear hypothetical, but the antibody-receptor combination, and the 'internal image' antibody may have powerful biological actions, which cannot be ignored.
A valuable scheme of this type for testing antibodies has been proposed by the UKCCCR (1986), which can readily be fitted into the broader requirements of the CPMP Guidelines and the FDA 'Points to Consider' (this volume and Tryzelaar, 1989).
As genetic engineering leads both to more extensive and more subtle modifications of antibodies for diagnostic and therapeutic use the toxicologist will have to adapt pragmatically to the detection and assessment of discrete but more powerful physiological effects, including, for example, following specific forms of immunosuppression, hormonal receptor stimulation or antagonism etc. This is an excellent example of the need to integrate toxicity testing with physiological studies and the investigation both of dynamic functional and terminal pathological processes.
The anticipated occurrence of the outer antibody in man (human anti mouse antibody against monoclonal preparation, HAMA response) may not be exactly mimicked in animals, but its occurrence shows the need for some extended study in the laboratory.
E. VACCINES
The toxicity testing of a candidate vaccine, or the bioassay and safety clearance of an established product, are probably amongst the most specialised and most difficult tasks for the toxicologist, because of the range of materials employed and the diversity and complexity of the harmful actions to be excluded. As examples, vaccines may be living or dead, natural or modified organisms, fractions of the latter or relatively purified antigens, presented with or without antigens etc. And, the immunising material may be derived from a "naturally" modified pathogen (by passage in an unnatural system), one altered by deliberate, even site directed mutagenesis, or it may come from genetically engineered insertion of a nucleotide sequence in a convenient carrier host, which is administered alive, dead or as an extracted and more or less purified antigen. Vaccines are commonly administered to healthy individuals to produce a longer lived change in immune responsiveness, a further severe challenge to any attempt to predict safety. A valuable general account of vaccine development is given in Kohler and Lo Verde (1988) and Lindberg, Norrby and Wigzell (1988).
The complexity covered by the term "vaccine" means that subdivision is essential, and that the strategy of toxicity testing can only be based on case by case assessment designed according to our understanding of the vaccine substrate's origin and properties.
6
The needs and objectives of the toxicologist, as for any complex biological preparation, are:-
1. To understand the nature, composition and intended uses of the preparation (including any special features of the recipient populations. These factors include information about the state of the vaccine as a living or dead organism or isolated antigen, and any evidence suggesting cross reactivity between the immunogen and normal body constituents.
2. To be aware of any evidence about the nature of the immune state produced by the vaccine in comparison with the following spontaneous infection, e.g. aberrant humoral (and which Ig isotype?) or cellular response to antigens on the immunogen.
3. To take account of any suggestions that there may be an immune reaction against any normal structure in the host, i.e. an induced auto­ immune reaction.
4. To appreciate the normal pattern of damage produced by the spontaneous infection, i.e. to be familiar with the virulence of the infective agent in terms of dysfunction or structural damage.
5. And, to understand the dynamics and pattern of damage that may be produced by any adjuvant or other excipient in the vaccine.
From all this information it should be possible to devise a series of experiments, pragmatically closely adapted to each candidate vaccine to explore:
a) The nature and duration of the local and systemic immune response. this does require an appropriate responsive species, which may be a particular problem for a live agent, owing to the fastidious nature of many organisms, which is dependent on cellular receptors and the extracellular milieu.
b) The virulence of a living organism in terms both of its propensity to cause damage in the host by local or systemic infection, or the likelihood that it may spread to non-injected subjects, so infecting the community. Lateral spread of that type may be associated with reversion to loss of the modified vaccine characteristics and so to reversion to the dangerous wild type of virulence.
c) The degree of local or distant reaction to the adjuvant and other compounds of the complete preparation.
d) Any likelihood of producing an auto-immune reaction either by molecular mimicry pf a host antigen, or by haptenisation or uncovering of an endogenous epitope.
The nature and extent of the appropriate experimental studies in vitro and in vivo, and the corresponding investigations in man, can only be described in relation to particular vaccines and agents.
7
A far more difficult safety consideration is the notorious ability of certain immunogenic agents to excite serious and even life threatening reactions in a small proportion of recipients (Wilson, 1967). This is probably best known for vaccinia, for which the incidence of severe paralysis and other forms of extensive damage to the nervous system reached up to about 1 in 20-40,000 adult recipients, and after the recent swine flu immunisation campaign in the USA (Greenstreet, 1984).
The causes of such idiosyncratic responses are not known, but their occurrence, sometimes with an alarmingly high frequency, is a strong warning that certain agents or antigens may carry a serious risk, perhaps by interacting with factors in the individual, and that the use of vaccinia (present volume) as a convenient carrier of other antigens should be regarded with caution until the factor responsible for such a devastating idiosyncratic reaction can be identified and excluded. A formal risk benefit analysis is required before employing such agents for the direct or indirect (spread from other species) immunisation of man.
F. CONCLUSIONS
The toxicity testing of all biological preparations involves a complex process of adapting convenient, conventional laboratory procedure to the individual properties and uses of specific preparations.
The types and nature of in vivo and in vitro experiments must reflect the individual activities, whether known or anticipated, of each material and its pharmacological actions.
They must also be sufficiently broad as to be capable of revealing indirect toxic actions (e.g. auto-immune phenomena) and all the unanticipated activities common to every preclinical study.
The toxicologist working on such materials must adopt methods responsive to classical target organ damage, to dynamic reactive processes, and to functional disorders. All the work has to be done under circumstances in which the test animal or system shows rapidly changing responses, which may magnify, nullify or direct a toxic reaction, or they may themselves be the harmful effect. Although biological reactivity, which is usually the very reason for administering a biological therapy, may sharply limit the scope of toxicity testing, the latter is always an important opportunity for the toxicologist to display physiological understanding and pathological skills in detecting and analysing action, and so relating toxic risks to clinical benefits.
8
References
Dayan A D. 1990a. Toxicity Testing. In Development of New Medicines, ed. J O'Grady and 0 Kolar. MacMillan, London.
Dayan A D. 1990b. Toxicity and Safety. In Hider R (ed.) Production, Characterisation and Formulation of Protein Drugs. Royal Pharmaceutical Society, London, in press.
Dayan A D, Campbell P N, Jukes T H. eds. 1989. Hazards of Biotechnology, Real or Imaginary? J. Chern. Techno!. Biotechnol. 43, no 4 (special issue).
Graham C E. ed. 1987. Preclinical safety of Biotechnology Products Intended for Human Use. Progr. Clin. BioI. Res. 235, 1-213.
Greenstreet R L. 1984. Estimation of the Probability that Guillain-Barre Syndrome was caused by the Swine Flu Vaccine: US Experience (1976-77) Med. Sci. Law, 24 61-7.
Kohler H, Lo Verde P T. 1988. Vaccines: New Concepts and Developments. Longman, Harlow.
Lindberg A A, Norrby E. Wigzell H. Eds. 1988. Nobel Symposium on The Vaccines of the Future. Vaccine, 6., 75-205.
Manning G, Deloria L B. 1986. The Pharmacology and Toxicology of the Interferons. An overview. Ann. Rev. Pharmacol. Toxicol. 26, 455-515.
Marshak D, Liu D T. eds. 1988. Therapeutic Peptides and Proteins. Banbury Report 29. Cold Spring Harbor Laboratory, Jackson.
Tryzelaar B. 1989. Regulatory Affairs and Biotechnology in Europe. Biotherapy, 1, 179-196.
UKCCCR. 1986. Monoclonal Antibody Testing. Br. J. Cancer, 54.
Weissinger J. 1989. Nonclinical Pharmacologic and Toxicologic Considerations for Evaluating Biologic Products. Reg. Tox. Pharmacol., 10, 255-263.
Wilson G S. 1967. The Hazards of Immunisation. Athlone Press, University of London.
T a
b le
* Test Type Purpose
effects of single high dose
target organs, timing, dose-response, reversibility
uncertain scientific value satisfies craven bureaucrats
parental, fetal or post-natal development explored
essential kinetics and metabolism. Possible interactions with co-therapies.
local effects at site of application, including actions on blood cells
focused study of special actions, e.g. immunotoxicity, endocrine effects, cell populations etc.
*General content of investigations in each type of test should be similar to those conventionally done for a new chemical entity.
Table 3 General Scheme for Toxicity Testing Antibodies
Test
basic kinetics and disposition, including clearance (especially smaller fragments)
follow any development of host antibody to test substance. ? need to check for anti-idiotype antibody.
1 species, i.v. dosing. 1 or a few doses and follow for 7-14 days. Assess survival, behaviour and general laboratory and pathological measures plus any indicator of specific actions.
check reactivity against panel of normal human tissues (stomach, thyroid, muscle, kidney, liver, lung etc) by immunofluorescence.
local actions at the site of administration.
II
H.J.M.van de Donk I.Hegger
The European organizations relevant to drug licensing are listed in fig.l. The council of Europe is the oldest organization, including almost all Western European countries. The EFTA comprises the 5 Nordic countries and Austria and is a very succesfull organization with respect to free trade. Finally, the EC, also called the 12 is now the predominant organization.
UROPEAN ORGANIZATIONS----------------------, fig.!.
Council of Europe
European Community
The Council of Europe is a non-military and non-economic organization. It is non-economic as the OECD is already taking care of economic affairs in an even wider concept. It is non-military as neutral countries are associated. The council was founded in 1949 and its greatest achievement is the foundation of the European Convention on Human Rights. Moreover, 83 treaties on cultural, judicary and medical affairs have been concluded. With respect to drugs licensing the Council has earned its merits by supporting the European Pharmacopoeia.
The EFTA has contributed to the arising of the PIC (Pharmaceutical Inspection Convention). The associated countries recognize each others
13
D. J. A. Crommelin and H. Schellekens (eds.), From Clone to Clinic, 13-19. © 1990 Kluwer Academic Publishers.
l4
inspections and provide each other with inspection reports. A large number of countries are associated, even more intend to become a member.
From 1992 a similar situation will exist in the EC as well. Fig.2. shows that the European Communities actually consist of three communities. The first (ECSC) was founded in 1951 by the French Minister Robert Schuman.
UROPEAN COMMUNITIES:------------------------~ fig.2.
European Atomic Energy Community
The original intention was to create an opportunity to rebuild the German industry in collaboration with some other West-European countries to balance the feared Sovjet domination. In 1957 the EEC was founded, which became heavily involved in agricultural affairs; one year later the EAEC was founded. These three communities merged in 1965 and share the institutions indicated in fig.3.
C----------------------------------------~ fig.3.
National parliaments
European Parliament (budget, dismiss Comm)
European Commission (right of initiative)
The national parliaments still have the highest authority as they control the ministers. Within the EC the Council of Ministers represents the highest authority as they decide on the directives, the community laws. Each directive mentions in its final article a date after which the directive supersedes national laws. Yet some 70 directives have been finalized, but only less than a dozain of them have been fully implemented by all member states. The European Parliament, elected directly by the European citizens, decides amongst others on the budget and it can dismiss Commissioners. The Commissioners form the executive of the community and have the right of initiative. There is a delicate balance of power; each institution has to collaborate in order to accomplish achievements. The most important directives concluded so far related to drugs are listed in
15
89/381 on blood products
The first on proprietary preparations formed the initial step towards harmonization and described the information that needs to be included in the licensing file. In 1975 two directives were made; one on standards for analytical, toxicological, pharmacological and clinical test methods and one on the mUlti-state procedure. This procedure allows manufactures, once obtained a license in one member state and having submitted an application in two other member states to start a concertation procedure in Brussels. Alternatively a member state may start this procedure. In 1987 the high-tech procedure directive was made, succeeded in 1989 by directives on immunological medicinal products (vaccines, toxins, serums and allergens) and on bloodproducts. The latter two contain obligations for manufacturers; batch to batch consistency has to be established and procedures and test methods have to be validated. There is a provision for batch release, however once a member state has released a batch, the other member states may not delay access to their markets. The batch release has to be completed witbin 60 days by the competent authority. For bloodproducts instructions for competent authorities for the prevention of transmission of infection diseases (with references to EP and WHO) and for promotion of community self­ sufficiency are included. Directives and Notes to Applicants (EC guidelines) can be ordered for at the addresses mentioned in fig.5.
16
Delegation of the Commission of the
European Communities
European Communities
Tel.: 352 499 281 Telex Pubof LU 1324 b
The CPMP (fig.6.) executes the multi-state and high-tech procedure.
ICENSING in EUROPE------------------------~
High - Tech Procedure
fig.6.
The latter is obligatory for products made by DNA recombination, controlled expression of genes and cell fusion (monclonal antibodies). For products representing a major innovation the choice is up to the manufacturer. The high-tech procedure starts once the product is submitted to the first competent authority in the EC. This authority will then act as a rapporteur; it is the communication channel to the manufacturer, it makes an in-depth evaluation of the licensing file and sends the evaluation to the other member states. Within 210 days the procedure must have been completed, including implementation at national level, excluding periods of time when the clock has been stopped to give the manufacturer a chance to answer questions. The European Commission installed a Working Party on Biotechnology and Pharmacy (fig.7.) to facilitate licensing of biotech/high-tech products.
17
Drafting guidelines (Notes to Applicants)
Evaluation of Dossiers
Commenting draft Directives
The WP started four years ago drafting Notes to Applicants using documents already available in countries like the UK, Germany and the Netherlands. Nowadays over half the time available is spent on predigesting of licensing applications for the CPMP. The chemical/pharmaceutical part (production and quality control) of the dossier is evaluated by the WP, the result is generally acknowledged by the CPMP. The WP has been invited to comment on draft directives, but has not been able to devote much time to this matter. The WP produced four Notes to Applicants, listed in fig.8.
OTES to APPLICANTS-------------------------, fig.8.
Preclinical Studies (1988)
The drafts are sent via the CPMP to the European Federation of Pharmaceutical Industry Associations, EP, WHO and FDA. Relevant comments are incorporated if the WP decides so. Recently plans have been launched to establish an EEC Medicines Agency (EMA). The main tasks of this agency will be: - The coordination of evaluation of dossiers. - The generation of assessment reports. - The execution of a pharmacovigilance program. - The supply of technical support to the member states. - Advising manufacturers. This agency will start from the first of January 1993.
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Batch definition
Although it is not obligatory to send a full dossier to competent authorities of member states where the manufacturer does not intend to market the product, it is very helpful when all the member states receive the full dossiers and not just the expert reports. Original photographs of gels and chromatograms are absolutely necessary. Results of testing on five consecutively produced batches should be submitted, similar to WHO requirement for vaccines. A batch should be clearly defined, especially in the case of continuous culture. When a culture results in several batches data should be provided, proving that early and late harvests lead to indentical batches. With respect to source materials (fig.10) the life expectancy of the seed lot has to be established.
ROBLEMS Source Materials------------------, fig.IO.
Stability of Seed Lot (extended cell bank)
Sterility of buffers (bioburden <lOO/ml)
Absence of viruses (e.g. bovine serum)
Material for Immunization (MoAbs)
The stability of seed lot should be determined by cultivating the cell beyond the normal production level and checking items like plasmid copynumber, yield and product characterization. Buffers used ougth to be sterile, although an upper limit for the bioburden « 100 germs/ml) may be acceptable provided a sterile filtration is performed just before filling. Source materials should be free of infectious agents, specially bovine sera. The origin of material used for immunization of animals in order to raise monoclonal antibodies should be clarified; records of patients or volunteers donating this material should be kept. Criteria for rejection should be specified with respect to subsequent production stages (fig.ll.).
19
Criteria for rejection
steps
Repeated use of the same column in different production stages should be avoided. Procedures used to make products derived from mammalian cells should be validated for the reduction of several viruses with different chemical/physical behaviour (fig.12.). The residual DNA should not exceed the level of 10-100 pg/dose.
ROBLEMS Final Bulk----------------------~ fig.12.
several viruses
Host Protein < 20 ng/dose
Yield
Even though in case of extended or lifelasting treatment this level should be further reduced. Host proteins should not exceed the level of approximately 20 ng/dose; to be tested for by an ELISA or other suitable method. The potency test should be validated with respect to accuracy and precision. When an in-house reference is used it should be calibrated against an international reference preparation or if non-existent a lot tested clinically should be used. The calibration should be submitted to statistic analysis. And finally, the yield of the product should remain within set limits. The increasing collaboration in Europe is quite fascinating; colleagues are learning to know each other, we notice that different countries emphasize on different matters but steadily we grow to a common approach. It will take many more years before our goal is accomplished.
THE REGULATION OF PHARMACEUTICALS: PHILOSOPHY AND PRINCIPLES
JOHN C. PETRICCIANI, M.D.
Introduction
The regulation of both drugs and biologicals has a relatively short history, punctuated by the need to ensure that medicines which are provided to the public are both safe and effective. The regulatory systems that are now in place throughout the developed world evolved in response to specific situations that arose in the past in which there was a clear public demand for tighter controls. The current systems are continuing to change as new needs are recognized that cannot be easily accommodated within the present frameworks.
Advances in the biomedical sciences as well as in technology during the 1980s have led us to the threshold of producing many new medicines, and to a new era in the treatment of diseases. As an example, a variety of cell growth factors have been identified, their genes have been cloned, and now they can be manufactured in suff ic ien t 1 Y large quantities, using sophisticated genetic engineering techniques, to make them economically feasible as products. These and similar advances in other areas have opened the door to therapeutic approaches that have been out of reach until now. Other equally exciting products derived from other new techniques are being evaluated in the laboratory. Some have already reached the clinic. This strong growth in research and development offers the prospect for major advances in the treatment and diagnosis of disease that will have a profound effect on our ability to save lives and improve the quality of life worldwide. These new products also pose challenges to the manufacturer, the clinician, and the regulator.
A significant issue is whether or not the current approach to the regulation of new medicines should be modified to manage issues generated by biotechnology products. One might reasonably quest ion whether regulatory programs with their roots in the early part of the twentieth century are actually capable of dealing with the issues and problems of the 1990s and beyond.
The following sections review the history and philosophy that lie behind the regulation of medicines, and several illustrations will be given to show how regulatory principles have been applied. The further
21
D. J. A. Crommelin and H. Schellekens (eds.), From Clone to Clinic, 21-28. © 1990 Kluwer Academic Publishers.
22
History of Regulation in the USA
Biologicals
From the beginning, the regulation of biologicals has evolved in response to crises. It has also relied heavily on science and scientists in the development of policy and decisions in response to those crises. History provides a number of specific and key examples. The first incident involving a biological product which attracted federal interest occurred at the beginning of this century during a diphtheria epidemic in St. Louis in which 10 of 11 children inoculated with diphtheria antitoxin died - not of diphtheria, but of a tetanus contaminant in the antiserum. This tragedy resulted in the 1901 enactment of the Federal Biologics Control Law.
No similar incidents occurred until 1955 when the prospects seemed bright for controlling poliomyelitis with the new Salk vaccine. In that same year, the potential for creating a biologic disaster of major proportions in national and global immunization programs was dramatized by a case, commonly referred to as the Cutter incident, in which a number of children contracted polio after receiving the Salk vaccine. This episode reemphasized the necessity for maintaining strict control of biological products by the manufacturer and the nationRI regulatory agpnry (NRA). 11 was out of that failure that the Division of Biologics Standards (DBS) of the National Institutes of Health (NIH) was created to provide greater assurance of the safety of biological products. The DBS was then transferred to the Food and Drug Administration (FDA) in 1972 partly because of the perceived need for the organization to function in an environment more oriented towards regulation.
Drugs
The initial regulation of drugs in the United States dates back to 1906 when the Pure Food and Drug Act was passed overwhelmingly by both houses of Congress primarily in response to the sale of contaminated food products. But the safety of patent medicines was also questioned, and the regulation of drugs was included in the Act.
Thirty-two years later, a pediatric sulfanilamide syrup, in which diethylene glycol was used as the solvent, went onto the market without testing for potential toxicity. The subsequent death of about 100 children led to the enactment of the 1938 Food, Drug and Cosmetic Act. The emphasis of the new law, as in the case of biologicals, was on assuring the safety of pharmaceutical products.
It became very clear in 1962 that safety is an elusive goal and that totally unexpected adverse events can occur. The thalidomide incident, in which limb malformations occurred in many children born to mothers who had taken the drug during pregnancy, occurred at that time. Even though most cases occurred in Europe because the drug had not yet been approved in the USA, the incident led to a further strengthening and
23
expansion of the regulatory role of the FDA. This brief sketch of the history of the regulation of medicines in the
United States shows that the current regulatory framework grew out governmental responses to a series of crises. The practical impact of those human tragedies has been not only increased regulation, but also an attitude of general conservatism on the part of NRAs in most developed countries.
The Framework of Regulation
Philosophy
The basic philosophy underlying the regulation of medicines, including those derived through biotechnology, is that government has an obligation and responsibility to protect the public from unreasonable risks. As already pointed out, it was for these reasons that NRAs were established and grew to their current level of importance in the introduction of new drugs into medical practice. Enlightened regulation aims at achieving a high level of protection of the public, while at the same time not stif] ing medical advances, biomedical research, and product development. Even under the best of circumstances, that is difficult to achieve, especially in an atmosphere where political considerations playa role. Nevertheless, most regulatory systems have been responsive to the need for public protection as well as to the medical need by that same public for new thelapeutic agents.
There are three basic questions that should be considered any time a regulatory role is being proposed or is being re-evaluated. First, why undertake a regulatory activity? What is it supposed to accomplish that cannot be accompl ished in any other way? Second, who should be responsible for the regulatory activity? Is it an international, a national, a local, or a private responsibility? And third, when should an area be regulated? Are there specific levels of danger or concern?
The answer to "why" is relatively straightforward in the sense that most people would agree that there should be minimal standards associated with any therapeutic intervention, and the way to achieve uniform compliance is through some type of regulation. The "who" question, however, opens up a significant number of considerations that have become more acute recently with the emergence of the European Community (EC). The "when" question raises perhaps even more difficult issues on which there can be legitimate differences of opinion especially when the scientific data bases are incomplete. Nevertheless, a consideration of the basic issues of why, who, and when can provide very useful insights into alternative approaches to regulation.
Principles
Once those questions have been answered, and a decision has been reached to proceed with regulation along certain lines, there are four basic operating principles that should be applied to serve the overall interest of society. First, in an ideal situation, the regulators
24
should be capable biomedical scientists conversant with the issues surrounding the products for which they are responsible. Alternately, if the regulators are not biomedical scientists themselves, they must have access to unbiased experts who can guide them in making decisions. There could be nothing worse for the public or for industry than to have a regulatory system in which the decision-makers did not depend on scientific expertise.
Second, scientific information should flow freely between the manufacturers and the regulators during product development. For example, once the preliminary human studies have been completed and a drug appears to have potential as an approved product, the NRA and drug sponsor should discuss the data and plans for further studies. This should help to ensure that the design of the major clinical trials is acceptable and that, if the test results are favorable, marketing approval would be likely. Such early sc ientif ic interaction offers potential advantages in facilitating later regulatory reviews.
Third, expert biomedical advice should be sought by regulators to assist in making decisions, even if the regulators are scientists themselves. Usually, NRAs do not have as much internal expertise as might be needed to address every biomedical question that might arise during the review of a new product. Because NRAs need the best advice available, they should make every effort to obtain it from "outside" experts.
And fourth, there should be a continuing broad and open scientific interchange in emerging areas such as biotechnology so that issues can be identified and resolved through consensus. Because both the science and production methods for new medicines, including biotechnology products, are evolving rapidly, any new regulatory requirements should take a parallel evolutionary course, rather than being rigid and based entirely on past experiences with other unrelated products.
Clinical research, by its very nature, carries unknown risks, and this is as true for studies of biotechnology products as it is with other medicines. The drug and biological development systems that have evolved in most countries rely on two assumptions. First, based on laboratory and animal studies, there is a potential benefit to patients from an experimental product; and second, there may be risks associated with the use of a product. There is a delicate balance between the value of studying the effectiveness of a new medicine in human beings and the possibility of incurring risks in those studies.
Society has adopted a system in which such studies are done in a reasonable and stepwise fashion, and in which risk is minimized to the extent possible. This is commonly referred to as the "phase" concept, which is simply a controlled incremental increase in the number of humans participating as subjects in the evaluation of a new medicine. The exposure of larger and larger numbers of people to experimental products is based on the evaluation of the data at each preceding stage. The conclusion that the new product continues to be acceptable, depends on weighing both the potential benefits as well as the risks in the context of the disease for which it is intended.
The regulatory process is intended to address reasonable concerns for safety, while at the same time not placing such a burden on research
25
that product development is stifled. The extent to which that ideal is met depends on cooperative efforts and understanding between the research conununity and the NRA. In general, that means regulatory decisions usually have the support of the scientific community in government, academia, and industry.
NRA and Industry Responsibilities
As already mentioned, the basic reason for the existence of NRAs is to provide protection to the public. On the one hand, the public is not protected if NRAs take their responsibilities casually or if they are overly permissive in their regulatory activities. On the other hand, it is equally true that the public is not being well served by inappropriate over-regulation of the kind that Dr. David Baltimore has described as unlimited prudent behavior. Fear of making wrong decisions, or of accepting even the smallest theoretical risks, should not be the driving force in evaluating new and improved products. The aim of NRAs should be to achieve a high level of protection of public health, without inhibiting research and product development.
NRAs have a responsibility to identify and define outstanding scientific and medica I issues for which answers are required in order for them to make decisiuns. It does not serve the interest of health, in any part of the world, for regulatory authorities to delay decisions by failing to ask specific and relevant questions that can be answered. Ir. ~(Ltltl()n, tf",,~ NRAs with laboratnry faci it les and the appropriate resources also have an obligation to use some of those resources to independently pursue answers to the questions, and to provide some of the data on which decisions can be made. Industry, of course, has a responsibility to provide scientific data to show that the manufacturing process consistently yields a safe and effective product with well­ defined characteristics. In addition, industry should try to address specific questions that are identified by an NRA as necessary for its evaluation of the new medicine.
Reasonable regulation is in everyone's best interest, including the research-based pharmaceutical industry which has invested heavily in the development of new and useful medicines many of which are being derived from biotechnology. Ultimately, conunon interests lie in providing safe and effective products to the public with a minimum of delay from the preclinical studies to the clinical research st age, and finally to marketing. Changes in regulations should be made to the extent that the current regulatory framework imposes unnecessary roadblocks in that pathway.
When approaching changes in regulatory processes, it is imperative that there be an atmosphere of openness, and a willingness to listen to the concerns of those who are outside of the NRA. A constructive resolution of regulatory issues requires a regulatory environment that is responsible, while at the same time, supportive of research and product development. In addition, NRAs need to be responsive to the public demand for moving new and better drugs onto the market in the shortest possible time, where they can do the most good for the most people. In that context, the regulatory process should not simply focus
26
Differences Among NRAs
It should also of safe and
An example of differences in the efficiency of NRAs comes from a comparison of the review and approval time for new drugs. Twenty-three new drugs were approved in 1989 by the FDA, with an average review time of 32.5 months, which was about the same length of time as it has taken since 1981. Eighteen of the 23 new drugs already had been approved in one or more foreign countries that have approval systems comparable in quality to that of the United States. This does not necessarily mean that FDA review was slower; companies often submit approval applications first to foreign countries.
It is possible, however, to compare the time of U.S. approval with that of the first approval outside the U.S. For example, in 1988, a comparison shows a significant United States lag. For the 16 products first approved abroad, the average review time in the country of first approval was 11.7 months, compared with an average review time in the United States for those same products of 29.7 months. In other words, it took 2.5 times as long to go through the FDA system as it did in the country of first approval. Clearly, the U.S. review and approval system can be made more efficient.
The lesson here is that even though the basic principles and philosophy of regulation are accepted in most countries, their translation into actual practice can be very different. The central question that arises is not whether approvals are slower or faster in one country compared to another, but whether the FDA model is really necessary, or even desirable, to provide protection to the public.
As the EC moves closer to decisions on the structure and process that will be used for new drug reviews and approvals, it will be extremely important to make benefit/risk assessments of the various alternatives. In that regard, the data just presented should give decision-makers pause with regard to the risks inherent in attempting to develop a centralized bureaucracy along the lines of the FDA. Clearly, the individual European drug regulatory systems that are now in place have served their countries well in terms of making reasonable assessments of the safety and efficacy of new drugs. There have been no repeats of either the Cutter or the thalidomide incidents. The real risk, then, of moving directly to a centralized system in Europe is one of bogging down a process while at the same time not adding any measurable benefit in terms of increased safety.
Public Attitudes Towards Regulation
AIDS has brought into extremely sharp focus the global concern for the rapid approval of new drugs. More spec if ically, AIDS has forced a reassessment of the type and amount of data needed by the FDA to approve
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a new drug. The overall goal of society is to get new drugs that work onto the
market in the shortest possible time so that they become available to treat people who are suffering from a variety of diseases. In doing that, there must be an orderly process to generate data to determine whether a new drug is safe and effective. Anything that damages our ability to do that is self-defeating. At the same time, the system should not be so paternalistic and restrictive that it denies patients timely access to effective drugs. Because of AIDS, a new balance is being struck in which the impact that unnecessarily long regulatory review times have on patient morbidity and mortality is being taken into consideration along with the benefit/risk assessment. The dangers of releasing drugs too quickly are well-known. What is now becoming much better understood is that significant and unnecessary harm to patients can be the outcome of an approval procedure that bends too heavily towards safety through slow and deliberate reviews.
Conclusion
During this past century, the road to new drug discovery -has been long and frequently filled with both positive and negative surprises. But there has been remarkable progress overall. The key to the development and introduction of these new medicines has been discovery. The evolution of drug discovery methods and techniques has been subject to many twists and turns. Research has been getting incL-easingly expensive as our knowledge base has increased and as the remaining medical problems to be solved turn out to be the most difficult. The result is that pharmaceutical companies in the United States will invest a record $8.2 billion in 1990. This financial commitment to discovering new drugs to treat the most difficult diseases now surpasses the biomedical research outlays of the NIH ($6.8 billion in 1989), and it represents the leading source of biomedical research and development in the world. The average cost of discovering and developing a new drug was recently estimated to be about $200 million.
The health of the pharmaceut ical industry is not a luxury, and it should not be taken for granted. Rather, it should be protected and nurtured in the same way that new drugs which result from industry's efforts protect society from diseases. Incentives must continue to be provided for industry to invest more in research and development because it is out of that process that new cures will be found for some of the most serious diseases that still confront mankind. Efforts to achieve short-term financial economies by governments imperil the long-term benefits that will only be found through the strong commitment to research and development that a healthy pharmaceutical industry can provide. The industry's track record on performance in the publ ic interest is clear, and the future is equally bright if governments do not over-regulate the science and the economics of the industry.
Biotechnology has provided challenges to regulatory systems, but the philosophy and the basic operating principles of regulation remain applicable. The more significant impact on the regulation of medicines
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has come from the political arena and patients themselves--at least in the U.S.A. The emergence of a unified Europe, and the efforts being made to approach the regulation of drugs and biologicals in a uniform manner within the EC, has led to a much broader movement to harmonize requirements among the EC, Japan, and the U.S.A. At the same time, AIDS has resulted in patient activism in the U.S.A. that has caused a significant amount of regulatory re-thinking and change: the expedited review and approval of new drugs for life-threatening or seriously debilitating diseases; and early broad access to experimental drugs that show promise for those diseases.
We are in the midst of a regulatory reassessment in which a new balance is being struck where there is still a respect of good science, but informed choice and human dignity are being given appropriate increased weight. Broader global movements towards regulatory systems that are much more compatible with each other will be of significant benefit to the patients who need new medicines, to the NRAs who review the data, and to the industry that does the research, development, and production.
QUALITY CONTROL OF VACCINES
INTRODUCTION
Rapid progress during the last decade in the research and development of recombinant DNA biologicals opens the way to numerous applications. In parallel with new product development arises the need for adequate regulation. Authorities rapidly responded by issuing "guidelines", "points to consider", requirements and directives for this class of biologicals in general and for vaccines in particular (Ref. 1 to 7).
The quality assessment of each product has to be considered on a case-by-case basis and the manufacturer in collaboration with the national control authorities has to develop the quality control procedures and specifications to be used at the different stages of development and routine production. These documents should be based on the guidelines of the authorities and on the existing requirements for classical vaccines. There exists a profound difference with the quality control approach of chemical entities because of the biological nature of production, the need for biological tests and the possible hazards arising from the starting materials and the manufacturing processes. The principal factors involved in the quality assessement of recombinant DNA vaccines and specifications related to their routine quality control will be discussed. The experience with the quality control of recombinant DNA Hepatitis B vaccine, the sole licensed genetically engineered vaccine until now, has been extensively used in the preparation of this paper.
1. PRINCIPAL FACTORS INVOLVED IN THE QUALITY ASSESSMENT OF RECOMBINANT DNA VACCINES
The quality of a vaccine is determined by its safety, purity and efficacy. Safety and purity comprise absence of extraneous infectious agents and acceptable levels of toxic materials, host cell components, cellular DNA and endotoxins. Faithful expression and identity of the antigen and satisfactory immunogenicity and potency should result in the expected efficacy.
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D. J. A. Crommelin and H. Schellekens (eds.), From Clone to Clinic, 29-35. © 1990 Kluwer Academic Publishers.
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Compared to drugs, the quality assessment of vaccines has to be approached in a different way. It has to take into account the biological nature of the manufacturing process resulting in the need to establish batch-to-batch reproducibility, the biological testing methods showing high variations in the obtained results and confidence limits. Even more important, it has to cope with the difficult detection of extraneous infectious agents originating from the starting materials and the hazards arising from the fermentation and cell culture processes and from the purification steps. Therefore, final product testing is not sufficient. It has to be completed by Current Good Manufacturing Procedures (CGMP) and "in process" tests by the genetic analysis of the expression system and by extensive testing for specificity and characterization of the obtained antigen.
The basis of each biological manufacture is the development of satisfactory seed stocks as starting materials. For genetically engineered microorganisms, such as bacteria and yeasts, the seed lot is a uniform harvest obtained by the multiplication of a master seed. The manufacturer's working cell bank is also obtained from an engineered master cell bank. The cells may be from mammalian or from insect or1g1n. Manipulated viruses such as potential vaccine candidates showing more stable attenuation markers obtained by the modification of genetic information of viruses, live attenuated viral vectors and live attenuated microorganisms carrying in their genetic material the information for the expression of selected antigens are another approach for the development of recombinant vaccines. Their production principle is based on working seed lots of the infectious agents and manufacturers' working cell banks used for their multiplication. Vaccine virus seed lots and manufacturers' working cell banks have to be extensively tested for purity, stability, identity, according to the requirements of regulatory authorities. Validation tests for the removal and/or inactivation of potential extraneous agents, DNA and contaminants, complete the quality assessment of the seed stocks.
Another group of starting materials comprises the components of the growth medium of the fermentation and the cell culture production runs. Medium ingredients of biological origin such as serum, trypsin and albumin need to be tested for extraneous infectious agents, while Pharmacopoeia specifications can cover the tests on chemically defined ingredients. Specifications and test methods have to be developed for materials used during the purification process. Monoclonal antibodies used in immuno-affinity purification processes have to be tested following the specific requirements for this group of biologicals.
2. MONITORING OF FERMENTATION AND PURIFICATION PROCESSES.
During fermentation cycles, the working seed will multiply in order to provide the biomass necessary for industrial production of the vaccine. The multiplication must be carefully determined so that the end product is obtained after a constant number of cycles, irrespective of the fermentation volume used.
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As the manufacturing of biotechnology products comprises several steps, adequate tests and controls must be carried out at each of these production steps according to specifications based on the principles discussed above. The aim of quality assessment during production, also called "in-process" control, is to guarantee that the final products prepared in different production runs have identical quality. Detailed standard operating procedures for each of the steps ensure reproducibility, consistency, and good manufacturing practice. Furthermore, all methods must be validated during product development. Tests are carried out at each stage in order to determine effectiveness in ensuring optimal yields, elimination of contaminants, and especially the maximal recovery of the active ingredient.
2.1. Monitoring of Fermentation
An important first step in biotechnical production is fermentation. In addition to the use of media with a standardized composition, the careful monitoring of temperature during the fermentation cycle and the strict application of the seed lot principle, certain tests have to be carried out during the cycle. Such controls cover monitoring of pH, dissolved oxygen, optical density, as well as the microscopical characteristics of the colonies at the seed level and at the end of fermentation. Additional controls include sterility testing of the fermentation medium, microbiological purity of the harvest, plasmid retention of the host cell at the end of each fermentation run, and the stability of the host cell characteristics. The limits for the above parameters are determined during the development phases of the product and must be strictly adhered to in each fermentation cycle.
2.2. Purification Procedures
The most important and difficult procedure is purification. This is a multistep procedure combining centrifugation, precipitation, ultrafiltration, chromatography, and other separation methods. Each step must be validated during development. Purification should eliminate the contaminants from the host cell and fermentation medium, and should remove reagents or additives used during the extraction/ purification process within acceptable limits. Cell and vector DNA must be reduced to the picogram level.
Efficient purification will result in a purified concentrated product, which must first be assessed to fully characterize the antigen obtained; secondly, routine control tests must be carried out on each lot of purified, concentrated vaccine.
3. SPECIFICITY AND CONFIRMATION OF IDENTITY OF THE EXPRESSED ANTIGEN
The specificity of the expressed HBsAg is determined as follows : the stability of the seed lot is checked during storage and at the end of consecutive fermentation runs. The number of viable cells of the working seed remains between acceptable limits during storage.
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Fermentations carried out after different time periods give constant results meeting the specifications of the "in-process" tests and the final product analysis.
The genetic stability should be proven by nucleotide sequencing of the coding region of the constructed product and of the adjacent segments at the seed lot level, and also at the end of a fermentation cycle. Finally, restriction endonuclease mapping of the recombinant vector at the end of representative fermentation cycles should give constant patterns.
Several useful methods for obtaining information on the chemical structure of the antigen are listed in the general guidelines already mentioned. The preferred methods include SDS-PAGE analysis followed by Coomassie Blue and silver staining or Western blot with polyclonal and monoclonal antibodies. The determination of the amino acid composition gives values comparable with the data obtained for the natural product or with the amino acid sequence predicted from the nucleotide sequence. Peptide mapping after cleavage with enzymes of different specificity will enable reconstruction of the predicted amino acid sequence. Finally, N/C terminal sequence determination will complete the information required on the correct expression of the antigen.
The quality of the immune response to the purified antigen is the last and most important point to check. The overall immune response, seroconversion, identification of the relevant epitopes, protective efficacy of the induced antibodies and duration of immunity are studied by inoculation of laboratory animals, challenge experiments in vaccinated chimpanzees and clinical trials in human volunteers.
4. ROUTINE QUALITY CONTROL ON PRODUCTION LOTS
The routine quality control of vaccine lots is carried out at five different consecutive production steps : raw and starting materials, "in process" monitoring, at the stage of the purified concentrated vaccine pool, on the final bulk vaccine and on the final containers. The most important control on the starting materials is the test to be carried out on control cell cultures for the absence of contaminants and viruses. This test is carried out on part of the substrate or host cells. During incubation of the control cells, microscopic monitoring takes place and specific tests in media, cell cultures and laboratory animals on samples taken during and at the end of the observation period should prove the purity of the substrate.
The fermentation of "engineered" microorganisms and the cell culture of antigen producing transformed cells is monitored by standard parameters of the multiplication cyles : the purity of the inoculum, the identity of the host cells during the whole incubation period, the consistency of the expressing system, the quantity of the crude antigen, the limits of microbiological contaminants. Physical parameters such as pH, oxygen concentration, cell density should be kept within predetermined close limits. In case antigens are harvested by disruption of the producing cells, careful monitoring of the different parameters and conditions of this process is necessary.
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After a series of purification steps, the concentrated purified antigens are submitted to series of tests in order to show freedom of extraneous agents, microbiological sterility, effective inactivation (if applicable). The protein content can be determined by several methods such as UV spectrophotometry, Kjeldahl nitrogen determination, Lowry assay or amino acid analysis. Amino acid analysis however only yields valuable information for low molecular weight peptides (not more than 16 Kilo daltons). Each of these methods need accurate validation by well-defined standards.
The antigen activity is determined by titration methods for live viruses or microorganisms. The antigenic activity can be quantified by adequate immunological tests such as RIA or ELISA. The ratio antigen to protein is a valuable indicator of consistency and immunogenicity of the product. The identity testing of the purified antigen can be completed by gel electrophoresis (SDS-PAGE) followed by staining of the separated bands with Coomassie Blue or silver staining. The bands after electrophoresis can also be identified by Western Blot techniques using specific poly- or monoclonal antibodies.
The quantitative determination of trace impurities is in most cases very difficult. Chromatographic profile methods useful for the separation of recombinant proteins and impurities are reverse-phase (RP), ion-exchange (IE) and size-exclusion (SE) HPLC. Other methods involve SDS-PAGE, Western blots using polyclonal antibodies against host cell proteins, immunoassays such as ELISA and slot-blot. Special problems may arise for antigens which aggregate in particles wherein impurities are most likely to be entrapped also, and where for determination, the particles have to be disrupted while keeping the proteins in solution. In some cases, the impurities may be separated or elute in the profiles of the antigen. In these difficult cases, a combination of methods has to be used. Correct validation by incorporating small quantities of several pure proteins in the antigen preparation to be tested or by running them in parallel is necessary.
DNA from the host cell can be determined by dot-blot hybridization while total DNA may be estimated using the recently developed biosensor technology. If DNA is present in the product at significant quantities, it is important to evaluate the molecular size. Tests for components of the growth media and purification processes have to be developed for each case. The endotoxin content is determined by the classical LAL test.
Most of the routine testing on the final bulk vaccine and/or on the final containers are identical to those carried out on classical vaccines: sterility according to compendia1 requirements, endotoxin content using the routine LAL method which is also applicable to vaccines containing Al(OH3) as adjuvant, pH, limits of the filled volumes, identity and concentration of antiseptics, adjuvants and inactivating substances. Absence of abnormal toxicity or general safety is carried out in mice and guinea-pigs.
The identity of the final product can be tested by SDS-PAGE on the antigen eluted from the adjuvant, followed by staining or Western blotting using specific antibodies. In some cases, immunological identity tests can be carried out directly on the adsorbed antigen.
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The potency test