This report contains the collective views of an international group of experts and does not necessarily represent the decisions or the stated policy of the World Health Organization
WHO Expert Committee on Biological Standardization
Thi rty-sixth Report
World Health Organization Technical Report Series 745
@ World Health Organization, Geneva 1987
ISBN 92 4 120745 0
'$3 World Health Organization 1987
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CONTENTS
Page General ........................................................................................................... 7
Review of the work of the Expert Committee on Biological Standardization 7 ........................................................ Preparation of standards in ampoules 7
............................................................................................ Vaccine quality 8 ............... Hepatitis B vaccine produced by recombinant DNA techniques 8
Requirements for new biologicals ............................................................... 9 Reference reagents for monoclonal antibodies ........................................... 10 Problems with substitution of hyperimmune animal serum standard prep- arations for human immunoglobulin standard preparations ...................... 10 Handling and testing of biological reference materials of human origin ... 11
....................................... Matrix reagents for immunoassays of hormones 11 ............ Reduction of use of animals in the control of biological products 12
Antibiotics .......................................................................................................... 12
1 . Tobramycin ........................................................................................... 12 2 . Kanamycin B .............................................................................................. 13
Antibodies .......................................................................................................... 13
3 . Anti-Brucella ovis serum ........................................................................ 13 ......................................... 4 . Clostridium botulinum Type B antitoxin. equine 13
........................ 5 . Clostridium perfringens beta and epsilon antitoxin. equine 14
............................................................................................................. Antigens
6 . Acellular pertussis vaccine .......................................................................... .............................. 7 . Purified protein derivative (PPD) of bovine tuberculin
8 . BCG vaccines .............................................................................................. 9 . Diphtheria and tetanus toxoids for flocculation tests .................................
10 . Snake venoms and antivenoms ................................................................... 11 . Measles vaccines (live) ................................................................................ 12 . Hepatitis B vaccine (plasma-derived) .......................................................... 13 . Hepatitis B surface antigen (subtype ad) ....................................................
Blood products and related substances ...............................................................
14 . Tetanus immunoglobulin ............................................................................ ..................................... 15 . Anti-double stranded DNA serum (Anti-dsDNA)
16 . Anti-streptolysin 0. human ........................................................................ 17 . Anti-Toxoplasma IgM serum .....................................................................
. ............................................ . . 18 Blood coagulation factors I1 VII. IX and X 19 . Low molecular weight heparin ................................................................... 20 . Anti-D complete blood-typing serum ......................................................... 21 . Beta, microglobulin (P. m) ..........................................................................
Endocrinological and related substances ............................................................ 22
22 . Insulins ........................................................................................................ ..................................................................................................... . 23 Elcatonin
............................................................................ . 24 Human growth hormone ................................................................................ . 25 Human erythropoietin
............................................................ . 26 Pituitary hormone releasing factors .................................................................. 27 . Human atrial natriuretic factor
........................................................... 28 . Prolactin, human, for immunoassay ........................................ . 29 Luteinizing hormone, bovine, for immunoassay
................................... . 30 Corticotrophin (ACTH), human, for immunoassay
................................... . 31 Parathyroid hormone, bovine, for in vitro bioassay
Miscellaneous ..................................................................................................... 26
....................................................... . 32 Pyrogens: endotoxin and interleukin-l 26
....................................................... 33 . Dimercaprol and Me1 B (melarsoprol) 26 . .................................................................................... 34 Haemiglobincyanide 27
REQUIREMENTS FOR BIOLOGICAL SUBSTANCES
. ...................................................................... 35 Requirements for tuberculins 27 ......................................................... . 36 Requirements for dried BCG vaccine 28 ....................................................... . 37 Requirements for continuous cell lines 28
................................. . 38 Requirements for poliomyelitis vaccine (inactivated) 29 39 . Requirements for antimicrobic susceptibility tests
.................... . 1 Agar diffusion tests using antimicrobic susceptibility discs 29 . ............................................................. 40 Yellow fever virus master seed 30
Annex 1 . Requirements for tuberculins (Revised 1985) ................................... Annex 2 . Requirements for dried BCG vaccine (Revised 1985) ....................... Annex 3 . Requirements for continuous cell lines used for biologicals produc-
tion .................................................................................................... ....................... . Annex 4 Requirements for poliomyelitis vaccine (inactivated)
Annex 5 . Requirements for antimicrobic susceptibility tests .......... 1 . Agar diffusion tests using antimicrobic susceptibility discs
Annex 6 . Production and testing of the WHO yellow fever virus primary ...................................... seed lot 21 3-77 and reference batch 168-73
Annex 7 . Biological substances: international standards, reference prepara- ............................................................. tions, and reference reagents
Annex 8 . Requirements for biological substances and other sets of recom- ........................................................................................ mendations
WHO EXPERT COMMITTEE ON BIOLOGICAL STANDARDIZATION
Genera, 12-18 ~Yovember 1985
Members
Dr D.R. Bangham, Head, Division of Hormones, National Institute for Biologi- cal Standards and Control, London, England
Dr C. Guthrie, Operations Director, Commonwealth Serum Laboratories, Park- ville, Victoria, Australia
Professor T.B. Jabloknva, Head, Laboratory of BCG and Tuberculin, Tarasevic State Research Institute for the Standardization and Control of Medical Biological Preparations, USSR Ministry of Health, Moscow, USSR (Vice- Chairman)
Dr H.W. Krijnen, Director, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam, The Netherlands (Cllairman)
Mr J. Lyng, Head, Laboratory for Biological Standardization, State Serum Institute, Copenhagen, Denmark
Dr H. Mirchamsy, Associate Director, Razi State Institute of Serum and Vaccine Production, Tehran, Islamic Republic of Iran
Dr R. Murata, Honorary Member, National Institute of Health, Tokyo, Japan Dr M.S. Nasution, Director, Perusahaan Umum "Bio Farma", Bandung,
Indonesia Dr W.W. Wright, Senior Scientist, Drug Standards Division, The United States
Pharmacopeia, The National Formulary, Rockville: MD, USA (Rapporteur) Dr Xiang Jian-zhi, Head, Division of Science and Technology, Shanghai Institute
of Biological Products, Shanghai, China
Secretariat
Dr D.H. ~ a l a m , Head, Division of Antibiotics and Chemistry. National Institute for Biological Standards and Control, London, England (Tenlporar~l Adviser)
Dr V. Grachev, Scientist, Biologicals, WHO: Gsneva: Switzerland Dr C. Hardegree, Director, Division of Bacterial Products, OEce of Biologics
Research and Review, Center for Drugs and Biologics, Food and Drug Administration Bethesda, MD? USA (Temporary Adviser)
Professor W . Hennessen, Berne, Switzerland (Tetnporary Adviser) Dr F.H. hieskal, Director, National Research Institute of Health, Addis Ababa,
Ethiopia (Ten~porary Adviser) Dr J.C. Petricciani, Chief, Biologicals, WHO, Geneva, Switzerland (Co-
Secretary) Dr P. Sizaret, Scientist, Biologicals, WHO: Geneva Switzerland (CO-Secretary) Dr D.P. Thomas, Head, Division of Blood Products, Kational Institute for
Biological Standards and Control. London, England (Tetnporarj, Aclviser)
WHO EXPERT COMMITTEE ON BIOLOGICAL STANDARDIZATION
Thirty-sixth Report
The WHO Expert Committee on Biological Standardization met in Geneva from 12 to 18 November 1985. The meeting was opened on behalf of the Director-General by Dr Lu Rushan, Assistant Director-General.
GENERAL
Review of the work of the Expert Committee on Biological Standardization
The Committee reviewed the scope of the items covered in its recent meetings and in its current agenda, and noted that the work could be divided into two broad categories, namely requirements and standards. The Committee also noted that the range of topics had become very broad, and that the items associated with many of the disciplines included in the Committee's terms of reference were becoming increasingly complex. Consequently, problems were arising in drawing enough committee members (from the WHO Expert Advisory Panel on Biological Standardization) who had both the appropriate background to address adequately the items on the agenda and who represented a balanced geographical distribution. The Committtee therefore requested the WHO Secretariat to take appropriate steps to expand the membership of the Expert Advisory Panel, and invited the Director-General to consider increasing the number of experts who participated in the meetings of the Expert Committee on Biological Standardization, by forming subcommittees, or by other appropriate mechanisms.
Preparation of standards in ampoules
The Committee expressed concern that several candidate materials prepared in stoppered vials had been offered to WHO for establishment as international standards. In order to ensure
maximum stability of international standards, it has been the policy of the Committee to establish as standards only those materials that have been prepared in hard-glass ampoules, sealed by fusion of the glass (WHO Technical Report Series, No. 626, 1978, Annex 4). The Committee therefore reaffirmed the instructions given in the same report that only materials prepared in glass ampoules should be accepted for consideration as international standards. It also emphasized that this criterion should be verified before extensive collaborative studies were undertaken and before a candidate material was submitted to the Committee for establishment as a standard. The Committee urged anyone intending to prepare a candidate material to inform the WHO Secretariat in advance, and requested the Secretariat to take steps to ensure that, whenever possible, those intending to provide standard material are aware of the instructions given in the Guidelines for the preparation and establishment of reference materials and reference reagents for biological substances (WHO Technical Report Series, No. 626, 1978, Annex 4).
Vaccine quality
The Committee was informed that the WHO Secretariat was currently drafting a procedure that would allow WHO to evaluate the acceptability of vaccines in general (especially the acceptability of vaccines for use in immunization programmes) organized by WHO or other UN agencies and to check the quality of selected batches of vaccines. The Committee recommended that a working group be convened by WHO to discuss the technical elements of this process.
Hepatitis B vaccine produced by recombinant DNA techniques
The Committee recognized that there had been remarkable progress in the development of vaccines through the use of recombinant DNA techniques and reaffirmed the belief, expressed in its thirty-third report (WHO Technical Report Series, No. 687, 1983, p. 15), that these techniques provided new opportunities for improving the health of large numbers of people in the world, especially in developing countries. For example, recombinant DNA techniques are being used to produce alternatives to the currently available vaccines against hepatitis B virus, as well as for the
production of completely new vaccines that might prevent major parasitic diseases such as malaria and leishmaniasis. The Committee was informed that proposed requirements for hepatitis B vaccines made in yeast by recombinant DNA techniques had been prepared and distributed to the Expert Advisory Panel on Biological Standardization for comments and suggestions; on the basis of these comments, a revision of the proposed requirements had been made which would also be distributed to the Expert Advisory Panel for additional suggestions. The Committee recognized the need to establish WHO requirements for hepatitis B vaccines produced by recombinant DNA techniques, but also stressed the importance of actions by national control authorities in evaluating the safety and efficacy of individual products. The Committee was informed of the possibility that such vaccines might soon be approved by national control authorities and agreed that proposed requirements should be considered for adoption as soon as possible thereafter.
Requirements for new biologicals
The Committee noted that an increasing number of new biologicai products, prepared using recombinant DNA techniques, are at various stages of development. These include vaccines, clotting factors, and endocrine and immunomodulating substances. The Committee acknowledged the usefulness to control authorities and to manufacturers of the availability of guidelines prior to the licensing of new kinds of products or of existing products made by new methods. For example, the guidelines developed by a WHO Consultation on the Quality Control of Biologicals Produced by Recombinant DNA Techniques (Bulletin of the World Health Organization, 61: 897-91 1 (1983)) had been widely used. The Committee recommended that modifications of such guidelines should be considered, when appropriate, taking into account recent experience and technological developments.
The Committee observed that in the past WHO had generally developed proposed requirements for a biological product after it had been approved by a national control authority (usually in the country in which it was produced) and when the biological was of international interest.
The Committee reviewed the issues associated with the development and adoption of WHO requirements for biological products that had not yet been approved by any national control
authority. It recommended that, in general, proposed requirements should be presented to the Committee only after the biological had been approved by a national control authority, usually in the country in which it is produced. The Committee recognized that there was a continuing need for WHO requirements for ensuring reliable manufacturing methods and quality control, especially for the new biotechnology products, and that such requirements should be considered by the Committee in a timely manner. However, the Committee agreed that the responsibility for evaluating the safety and efficacy of new biologicals rests with national control authorities.
Reference reagents for monoclonal antibodies
The Committee recognized that large numbers of monoclonal antibodies are currently being produced, for a variety of purposes, for example, for use in diagnostic and immunopurification procedures. The Committee anticipated that a large number of requests would be forthcoming for WHO to establish international standards for monoclonal antibodies of various types, with many preparations being offered to WHO for such purposes. The Committee agreed that great care should be exercised in selecting the antibodies for which international standards or reagents are needed, and that priority should be given to those that would be of particular value in the calibration of biological products used for diagnosis and treatment.
Problems with substitution of hyperimmune animal serum standard preparations for human immunoglobulin standard preparations
The Committee noted that in one laboratory (Journal of biological standardization, 13: 123 (1985)) inconsistent results had been obtained in potency assays of human rabies immunoglobulins when neutralization tests in mice and cell culture were performed using a common equine anti-rabies serum reference preparation (WHO/BS/ 85.1468). Since human rabies immunoglobulin is frequently used in conjunction with rabies vaccine and since an excess of rabies immunoglobulin might inhibit the development of active immunity, the Committee agreed that this problem should be investigated further. The Committee requested the State Serum Institute, Copenhagen, to arrange for investigative studies that would include
comparisons of the International Standard for Anti-Rabies Serum, Equine, with the International Standard for Rabies Immuno- globulin as well as with other preparations of human rabies immuno- globulin in both cell cultures and mice.
Handling and testing of biological reference materials of human origin The Committee noted that new infectious agents continue to be
recognized. This increases the possibility that certain existing international standards, reference preparations, or reference reagents may contain infectious agents. The Committee therefore emphasized that laboratory workers should take appropriate precautions in handling reference materials derived from human sources. The Committee agreed that all existing international standards and reference preparations of human origin should be tested for hepatitis B surface antigen and LAV/HTLV-I11 antibody.
The Committee also agreed that in the future it would be desirable that reference materials that need to be derived from man should be taken from donors whose sera or plasma have been individually tested and found to be free of the two above-mentioned viruses. The Committee requested the WHO Secretariat to consider the preparation of suitable amendments to the WHO Guidelines for the preparation and establishment of reference materials ,for biological substances (WHO Technical Report Series, No. 626, 1978, p. 101).
Matrix reagents for immunoassaps of hormones
The Committee noted that. in accordance with the request in its thirty-fifth report (Technical Report Series, No. 725, 1985, p. 9), the WHO Secretariat had arranged a meeting of manufacturers of hormone assay kits, and other experts, to discuss the need for the provision of WHO international matrix reagents (WHO/BS/ 84.1462). It had been suggested that in order to improve agreement between assay results obtained with different kits, a preparation of human serum, containing as low amount as possible of the relevant analyte, could be used to dilute a given international standard and then to calibrate each manufacturer's reference standard. The Committee was informed that a joint WHO/International Federation of Clinical Chemistry (IFCC) working group had been formed to study the possible benefit of a common matrix reagent in immunoassays for thyroid stimulating hormone (TSH).
Reduction of use of animals in the control of biological products
The Committee examined the report of the international meeting on the Reduction of Animal Usage in the Development and Control of Biological Products, organized by the International Association of Biological Standardization and held in London, on 23-26 April 1985 (WHO/BS/85.1470). The Committee agreed that possibilities did exist for reducing the use of animals in the control of biological products. The Committee, however, stressed that animal tests were necessary to evaluate the biological activity and safety of biological products. Before the number of animals used could be safely reduced, more data were needed providing evidence of valid correlation between the results of in vitro tests and assays in animals as well as between those tests and the efficacy and safety in man of the biological products. In addition, improvements in animal test and assay methods may be possible, and the Committee encouraged such studies.
The Committee, therefore, requested the WHO Secretariat to keep in mind the possibility of reducing the use of animals when reviewing the Requirements for Biological Substances or preparing new requirements.
SUBSTANCES
ANTIBIOTICS
1. Tobrarnycin
The Committee noted the report of the collaborative assay referred to in its thirty-fourth report (WHO Technical Report Series No. 700, 1984, p. 13) and that, in accordance with the authorization given in the same report, the National Institute for Biological Standards and Control, London, had established the International Standard for Tobramycin and, with the agreement of the participants, had defined the activity of the contents of each ampoule of the International Standard for Tobramycin as 9800 International Units of Tobramycin (WHO/BS/85.1504).
2. Kanamycin B
The Committee noted that the stocks of the International Reference Preparation of Kanamycin B were becoming exhausted (WHO/BS/85.148 1). The Committee also noted that the presence of the impurity kanamycin B in kanamycin is no longer checked by methods involving the measurement of biological activity, but by chromatographic methods. Thus, the purpose for which the preparation was established no longer exists. The Committee therefore discontinued the International Reference Preparation of Kanamycin B.
ANTIBODES
3. An ti-Brucella ovis Serum
The Committee noted that, in accordance with the request in its thirty-third report (WHO Technical Report Series, No. 687, 1983, p. 21), the Central Veterinary Laboratory, Weybridge, England, had obtained material suitable to serve as an international standard for anti-Brucella ovis serum and had arranged a collaborative study among 12 laboratories in which the complement fixation method was used (WHO/BS/85.1485). The Committee noted also that use of the proposed reference material resulted in a substantial reduction in the differences among estimates obtained by the participating laboratories.
The Committee established the material studied as the International Standard for Anti-Brucella ovis Serum, and, on the basis of the results of the collaborative study, assigned an activity of 1000 International Units of Anti-Brucella ovis Serum to the contents of each ampoule.
4. Clostridium botulinum Type B Antitoxin, Equine
The Committee noted that the collaborative study of the preparation proposed as a replacement for the International Standard for Clostridium botulinum Type B Antitoxin, Equine,
referred to in its thirty-fifth report (WHO Technical Report Series No. 725, 1985, p. 15), had been completed (WHO/BS/85.1469). The Committee established the preparation studied, coded BUSB 5004, as the second International Standard for Clostridium botulinum Type B Antitoxin, Equine, and, on the basis of the results of the collaborative study, assigned an activity of 31 International Units of Clostridium botulinum Type B Antitoxin, Equine, to the contents of each ampoule.
5. Clostridium perfringens Beta and Epsilon Antitoxins, Equine
The Committee noted the results of a collaborative study, arranged by the Central Veterinary Laboratory, Weybridge, England, of replacement materials for the International Standard for Clostridium perfringens Beta Antitoxin, Equine, and for the International Standard for Clostridium perfringens Epsilon Antitoxin, Equine, which had included tests for the presence of minute amounts of antibodies to other C. perfringens toxin types (WHO/BS/85.1484). On the basis of the results obtained, the Committee established one of the materials studied (2Cp epsilon At) as the second International Standard for Clostridium perfringens Epsilon Antitoxin, Equine, and assigned an activity of 1020 International Units of Clostridium perfringens Epsilon Antitoxin, Equine, to the contents of each ampoule. Even though the other material studied (2Cp beta At) had been shown to be suitable to serve as a replacement material, the Committee decided that since the stocks of the International Standard for Clostridium perfringens Beta Antitoxin, Equine, would not be exhausted for several years, it should not be replaced at this time.
ANTIGENS
6. Acellular Pertussis Vaccine
The Committee was informed that limited clinical studies of acellular pertussis vaccines had recently been completed. It appears that reference materials of filamentous haemagglutinin (FHA) and
leukocytosis promoting factor (LPF) as well as a reference material of adsorbed acellular pertussis vaccine are needed to determine the degree of detoxification, the antigen contents of non-adsorbed acellular pertussis vaccines, and the potency of adsorbed acellular pertussis vaccines.
The Committee was informed that a collaborative study was in progress, involving eight laboratories from seven countries, in which several test methods were being tried for the testing of three possible reference materials. It is expected that the study will reveal which methods are suitable for testing acellular pertussis vaccines. The Committee agreed that antisera reference preparations might also be needed to compare immunogenicity assay results.
7. Purified Protein Derivative (PPD) of Bovine Tuberculin
The Committee noted that the collaborative study of the proposed international standard for purified protein derivative (PPD) of bovine tuberculin, referred to in its thirty-third report (WHO Technical Report Series, No. 687, 1983, p. 22), was in progress (WHO,'BSl85.1493). Laboratories in eight countries had agreed to participate in the study using assays in either guinea-pigs or cattle, or both.
8. BCG Vaccines
The Committee examined the preliminary results of accelerated thermal degradation studies of the four BCG vaccines, referred to in its thirty-third report (WHO Technical Report Series, No. 687, 1983, p. 22), as candidates for the replacement of the International Reference Preparation of BCG Vaccine and noted that all the preparations studied had shown satisfactory long-term stability (WHO/BS/85.1473). The Committee requested the State Serum Institute, Copenhagen, Denmark, to provide a report of the analysis of the data from the laboratory studies of the candidate vaccines.
The Committee agreed that the process of replacing the International Reference Preparation of BCG Vaccine should be conducted separately from the selection of a suitable primary seed lot for BCG vaccine production, for which field studies were needed.
9. Diphtheria and Tetanus Toxoids for Flocculation Tests
The Committee noted that preparations of diphtheria and tetanus toxoids had been obtained and freeze-dried and that the State Serum Institute, Copenhagen, had agreed to arrange a collaborative study to assess the suitability of these preparations to serve as international reference reagents for the estimation of diphtheria and tetanus toxins and toxoids in Lf units (WHO/BS/85.1475). The Committee also noted that, in addition to flocculation tests, other methods, such as single radial immunodiffusion and rocket immunoelectrophoresis, would be included in the collaborative study.
10. Snake Venoms and Antivenoms
The Committee noted the progress made in the standardization of snake venoms and antivenoms since its thirty-fourth meeting (WHO Technical Report Series, No. 700, 1984, p. 23). Venoms of Notechis scutatus, Bothrops atrox asper, Crotalus atrox, Echis carinatus (Mali), Echis carinatus (Islamic Republic of Iran), Naja naja kaouthia, Vipera russelli and TrimeresurusJEavoviridis have been freeze-dried in ampoules by the National Institute for Public Health and Environmental Hygiene, Bilthoven. Echis carinatus (Islamic Republic of Iran) and TrimeresurusJlavoviridis (Japan) antivenoms have been prepared in vials by the Razi State Institute of Serum and Vaccine Production, Tehran, Islamic Republic of Iran, and in ampoules by the National Institute of Health, Tokyo, Japan, respectively. Collaborative studies of the venom preparations have been arranged by the Liverpool School of Tropical Medicine, Liverpool, England (WHO/BS/85.1492).
11. Measles Vaccine (Live)
The Committee noted that the National Institute for Biological Standards and Control, London, had obtained a preparation of attenuated live measles vaccine (Schwarz strain) suitable to serve as an international reference reagent for the in vitro assay of live measles vaccines (WHO/BS/85.1483). The Committee also noted the
results of a collaborative study in which this preparation was found to be satisfactory for use in the tests for plaque-forming units (PFU) and tissue culture infectious doses (TCID,,) for estimating the infectivity of measles vaccines. The Committee established the preparation studied, coded 821638, as the International Reference Reagent for the Assay of Measles Vaccine (Live), and on the basis of the results of the collaborative study, assigned a titre of 103.7 to the contents of each vial of the International Reference Reagent for the Assay of Measles Vaccine (Live). For practical purposes the titre may be taken to be equivalent to 5000 TCID,, or 5000 PFU.
In view of the fact that the material was freeze-dried in vials, the Committee requested the National Institute for Biological Standards, and Control, London, to conduct long-term stability studies of the material.
12. Hepatitis B Vaccine (Plasma-Derived)
The Committee was informed that a collaborative study, arranged by the National Institute for Biological Standards and Control, London, to assess the suitability of a proposed international standard of plasma-derived hepatitis B vaccine had been completed, and that a report on the study was in preparation. The material studied was an aluminium-hydroxide-adsorbed vaccine containing approximately 20 pg per m1 of plasma-derived hepatitis B surface antigen, as assessed by measurement of the protein content of the aqueous bulk prior to adsorption to adjuvant. As this material cannot be freeze-dried satisfactorily, it is in liquid form. Eleven participants from eight countries contributed data from 21 immunogenicity assays which were carried out in mice or guinea- pigs. The minimum amount required to induce antibodies in 50% of the animals varied widely (100-fold range between laboratories); when potencies of vaccines were calculated relative to the candidate preparation, the results were within a 10-fold range.
Until the report of the collaborative study becomes available and the suitability of the candidate preparation to serve as an international standard for testing the potency of hepatitis B vaccines is confirmed, the Committee agreed that the material should continue to be made available for use as a working reference in immunogenicity studies of plasma-derived hepatitis B vaccines.
I 13. Hepatitis B Surface Antigen (subtype ad)
The Committee noted that a freeze-dried prcparstion of hcpatitis B surface antigen positive serum of subtype crtl had bccn offcrcd by the National Institute for Biological Standards and Control, London, for use as an international standard of hepatitis B surfacc antigen (WHO/BS/85.1476 Rev. 1). The Committee noted also thc results of a collaborative study involving 12 laboratories in which the preparation was used for the standardization of assays for the diagnostic detection of hepatitis B surface antigen. Furthermore, accelerated degradation tests had indicated that the preparation had satisfactory stability.
The Committee therefore established the preparation studied as the International Standard for Hepatitis B Surface Antigen (subtype ad), and on the basis of the results of the collaborative study, assigned an activity of 100 International Units of Hepatitis B Surface Antigen (subtype ad) to the contents of each ampoule.
BLOOD PRODUCTS AND RELATED SUBSTANCES
14. Tetanus Immunoglobulin
The Committee noted that there was a need for an international standard of tetanus immunoglobulin (WHO/BS/85.1472). The Committee also noted that a suitable preparation of tetanus immunoglobulin had been obtained, sealed into ampoules, and freeze-dried, and that the State Serum Institute, Copenhagen, had arranged a collaborative study.
15. Anti-Double-Stranded DNA Serum (Anti-dsDNA)
The Committee noted that there was a need for a reference preparation for the assay of anti-double-stranded DNA serum and that a candidate freeze-dried material (WoI80) had been prepared and studied by the Standardization Committee of the International Union of Immunological Societies (IUIS) (WHO/BS/85.1491). The Committee noted also that the material studied was specific for anti-
double-stranded DNA when tested by different methods with different tissue preparations; it was also shown to have satisfactory stability. Furthermore. the results of collaborative study, in which eight laboratories used immunofluorescence and radioimmunoassay methods, showed that the material was suitable to serve as an international standard.
The Committee therefore established the material studied as the International Standard for Anti-Double-Stranded DNA Serum, and assigned an activity of 100 International Units of Anti-Double- Stranded DNA Serum to the contents of each vial.
In view of the fact that the material was freeze-dried in vials, the Committee requested the Central Laboratory of The Netherlands Red Cross Blood Transfusion Service, Amsterdam, to dissolve the contents of a number of vials of the International Standard for Anti- Double-Stranded DNA Serum, put the dissolved material into the same number of ampoules, freeze-dry the solution, and then conduct long-term studies to compare the stability of the material in the vials with the material in the ampoules.
16. Anti-Streptolysin 0, Human
The Committee was informed that the candidate preparation, referred to in its thirty-second report (WHO Technical Report Series, No. 673, 1982, p. 20) intended as a replacement for the International Standard for Anti-Streptolysin 0 , Human, had proved to be unstable and that another serum pool would be collected by the State Serum Institute. Copenhagen, and prepared for consideration as a replacement preparation.
17. Anti-Toxoplasma IgM Serum
The Committee noted that there was a need for an international standard for anti-toxoplasma serum rich in IgM antibodies that would be useful for measuring anti-toxoplasma antibodies of the IgM class (WHO/BS/85.1471). The Committee noted also that a candidate preparation had been obtained and that an international collaborative study had been arranged by the State Serum Institute, Copenhagen.
18. Blood Coagulation Factors 11, VII, IX, and X
The Committee was informed that an international collaborative study had been arranged by the National Institute for Biological Standards and Control, London, with the aim of establishing both plasma and concentrate standards for blood coagulation Factors 11, VII, IX, and X. Freeze-dried plasma and concentrate preparations, in coded ampoules, are being assayed against fresh normal plasma by 28 laboratories in a collaborative study. The concentrate preparations consist of (a) the current International Standard for Factor IX (a concentrate of Factors 11, IX, and X); (b) a new concentrate of Factors 11, IX, and X; (c) a concentrate of Factors 11, VII, IX, and X; and (d) a concentrate of Factor VII. The Committee was also informed that results have been received from most of the laboratories participating in the collaborative study and are being analysed.
19. Low Molecular Weight Heparin
The Committee was informed that a pilot collaborative study of coded preparations of eight different low molecular weight hcparins had been arranged by the National Institute for Biological Standards and Control, London. In the study, all 8 preparations were assayed against the fourth International Standard for Heparin, established in 1984 (WHO Technical Report Series, No. 700, 1984, p. 18), and all of them were shown to have non-parallel dose-response curves to the Standard, regardless of the assay methods used; the potency estimates varied by as much as three times between laboratories using the same method, as well as between different methods. Variations in potency estimates were greatly reduced when the low molecular weight heparins were assayed against comparable common material used as an arbitrary standard. On the basis of these results, the Committee agreed that an international standard for low molecular weight heparin was needed. The Committee was informed also that with the support of the International Committee on Thrombosis and Haemostasis, the National Institute for Biological Standards and Control had arranged a collaborative study, in 25 laboratories, of three ampouled preparations of low molecular weight heparin.
20. Anti-D Complete Blood-Typing Serum
The Committee noted that the material referred to in its thirty- fifth report (WHO Technical Report Series, No. 725, 1985, p. 17), prepared as a candidate international standard for anti-D complete blood-typing serum, had been shown to have unsatisfactory potency and specificity in accelerated degradation tests. The Committee agreed that a chemically modified anti-D complete blood-typing serum would serve the intended purpose, and requested the Central Laboratory of The Netherlands Red Cross Blood Transfusion Service, Amsterdam, to prepare such a modified serum, examine its stability and specificity, and arrange a collaborative study.
21. Beta, Microglobulin @,m)
The Committee noted that the collaborative study referred to in its thirty-fifth report (Technical Report Series, No. 725, 1985, p. 19) had shown that the candidate reference material, a pool of normal human sera, for the assay of beta, microglobulin, was stable. The National Institute for Health and Medical Research (INSERM), Hcipital Edouard Herriot, Lyon, had arranged a second collaborative study in six laboratories in which five methods of assay were used, four based on radioimmunoassay and one on enzyme- linked immunosorbent assay (ELISA) (WHO,'BS/85.1501). The Committee noted also that the results of the second study had confirmed that the material was suitable to serve as an international standard.
The Committee therefore established the preparation coded 801 1213200 as the International Standard for Beta, Microglobulin and assigned an activity of 100 International Units of Beta, Microglobulin to the contents of each vial.
In view of the fact that the material was freeze-dried in vials, the Committee requested the State Serum Institute, Copenhagen, to dissolve the contents of a number of vials of the International Standard for Beta, Microglobulin, put the dissolved material into the same number of ampoules, and then freeze-dry the solution. The Committee also requested the WHO Secretariat to arrange for long- term studies to compare the stability of the material in the vials with that of the material in the ampoules.
ENDOCRINOLOGICAL AND RELATED SUBSTANCES
22. Insulins
The Committee noted that the collaborative study of the ampouled preparations of purified human, porcine, and bovine insulins, referred to in its thirty-fifth report (WHO Technical Report Series No. 725, 1985, p. 20) had been completed. The preliminary results showed that all the three insulin preparations were suitable to serve as an international standard (WHO/BS/85.1502). The Committee was informed that the report on the study would soon be sent to the participants.
In view of the urgent need for international standards of these insulins, the Committee requested the National Institute for Biological Standards and Control, London, to inform the WHO Secretariat of the activity to be assigned to the preparations studied when the participants in the collaborative study had reached agreement so that the Director-General might establish these international standards without delay, as called for in World Health Assembly resolution WHA37.27.
The Committee agreed that, in accordance with the recommendations in its thirty-second report (WHO Technical Report Series, No. 673, 1982 pp. 10-12), when these new international standards have been established national control authorities should be urged to recalibrate their relevant national standards in terms of these new international standards.
23. Elcatonin
The Committee noted that, in accordance with the request in its thirty-fifth report (WHO Technical Report Series, No. 725, 1984, p. 21), a preparation of elcatonin had been obtained and ampouled at the National Institute for Biological Standards and Control, London (WHO/BS/85.1494). The Committee noted also that an international collaborative study of this preparation had been carried out, in which the elcatonin preparation was assayed against the International Reference Preparation of Calcitonin, Salmon, for
Bioassay. A preliminary analysis of the results showed that the preparation was suitable to serve as an international standard.
The Committee therefore established the material in ampoules coded 841614 as the International Standard for Elcatonin, and authorized the National Institute for Biological Standards and Control, with the agreement of the participants in the collaborative study, to define the international unit.
24. Human Growth Hormone
The Committee noted that in addition to the form of human growth hormone (relative molecular mass 22 000) referred to in its thirty-fifth report (WHO Technical Report Series, No. 725, 1985, p. 23), there was also a need for an international standard for the form of human growth hormone that has a relative molecular mass of 20 000 (WHO/BS/85.1487). The Committee noted also that quantities of these two substances, extracted from human pituitary glands, had been offered for this purpose. The Committee requested the National Institute for Biological Standards and Control, London, to arrange collaborative studies.
25. Human Erythropoietin
The Committee noted that an analogue of human erythropoietin was being produced by recombinant DNA procedures using animal cells. It was intended for trial purposes in the treatment of certain types of anaemia (WHO!BSi85.1498). The Committee noted also that there was a need for an international standard of highly purified human erythropoietin since the International Reference Preparation of Erythropoietin, Human, Urinary, for Bioassay, is impure and unsuitable for purposes such as verifying the structure and conformation of the various erythropoietin preparations produced by recombinant DNA techniques. The Committee therefore requested the National Institute for Biological Standards and Control, London, to obtain a suitable preparation of highly purified recombinant DNA analogue of human erythropoietin and to arrange a collaborative study.
26. Pituitary Hormone Releasing Factors
The Committee noted that, in accordance with the request made in its thirty-fifth report (WHO Technical Report Series, No. 725, 1985, p. 21), the National Institute for Biological Standards and Control, London, had obtained and ampouled quantities of 1 4 0 and 1 4 4 human growth hormone releasing factors and of 1 4 1 corticotrophin releasing factor (WHO/BS/85.1499). The Committee noted also that at present it was not clear which form of releasing factor would be used in clinical medicine and for which an international standard would be required. The Committee therefore agreed that until a decision is taken on this matter these preparations should be made available for use as research standards.
27. Human Atrial Natriuretic Factor
The Committee noted that the recently discovered peptide hormone, human atrial natriuretic factor, isolated from human cardiac tissue, had been shown to have potent natriuretic and vasoactive properties (WHO/BS/85.1496). The Committee noted also that synthetic preparations of the 1-28 form of this peptide hormone were undergoing clinical trial in the treatment of hypertension, and that an international standard was needed for diagnostic immunoassays of the hormone in plasma. The Committcc noted further that a quantity of the 1-28 peptide had been obtained and freeze-dried in ampoules. The Committee therefore requested the National Institute for Biological Standards and Control, London, to arrange an international collaborative study.
28. Prolactin, Human, for Immunoassay
The Committee noted that the collaborative study referred to in its thirty-fifth report (WHO Technical Report Series, No. 725, 1985 p. 20), had been completed and that the results were being analysed (WHO/BS/85.1497).
29. Luteinizing Hormone, Bovine, for Immunoassay
The Committee noted that there is a need for an international standard for bovine luteinizing hormone for use in
radioimmunoassays applied in veterinary medicine (WHO/BS/ 85.1474). The Committee noted also that a preparation of highly purified bovine pituitary luteinizing hormone had been obtained, freeze-dried in ampoules. and compared with a research standard for bovine luteinizing hormone in an international collaborative study arranged by the National Institute of Public Health and Environmental Hygiene. Bilthoven. The results of this study, in which in vivo and in vitro bioassays, receptor assays, and immunoassays were used. showed that the material studied was suitable to serve as an international standard.
The Committee therefore established the material studied, coded EHC-bLH-l, as the International Standard for Luteinizing Hormone, Bovine, for Immunoassay, and assigned an activity of 0.025 International Unit of Luteinizing Hormone, Bovine, for Immunoassay, to the contents of each ampoule.
30. Corticotrophin (ACTH), Human, for Immunoassay
The Committee noted that, as requested in its thirtieth report (WHO Technical Report Series, No. 638, 1979, p. 25), a quantity of highly purified corticotrophin (ACTH) had been isolated from human pituitary glands at the National Institute for Biological Standards and Control, London (WHO/BS/82.1366 and WHO/BS/ 85.1489). The Committee noted also that this material had been freeze-dried in ampoules and had been shown to be stable. The Committee was informed that the preparation was being assayed in an international collaborative study to assess its suitability to serve as an international standard. The Committee was informed also that the collaborative study would include the International Standard for Corticotrophin, Porcine, for Bioassay, as well as highly purified porcine ACTH, deamidated and methionine sulfoxide-containing forms of human and porcine ACTH, and phosphorylated human ACTH.
31. Parathyroid Hormone, Bovine, for In Vitro Bioassay
The Committee noted that the international collaborative study of the preparation of bovine parathyroid hormone referred to in its thirty-third report (WHO Technical Report Series, No. 687, 1983,
p. 29) had been completed and the results analysed at the National Institute for Biological Standards and Control, London (WHO/BS/ 85.1490).
The Committee established the preparation studied, in ampoules coded 821632, as the International Standard for Parathyroid Hormone, Bovine, for In Vitro Bioassay, and authorized the National Institute for Biological Standards and Control, with the agreement of the participants in the collaborative study, to define the international unit.
The Committee noted also that the ampouled preparations of human and bovine 1-34 parathyroid hormone peptides referred to in its thirty-fourth report (WHO Technical Report Series, No. 700, 1984, p. 20), which had been included in the collaborative study, will be made available as research standards.
MISCELLANEOUS
32. Pyrogens: Endotoxin and Interleukin-l
The Committee noted that the preliminary studies on the freeze- drying of the preparation of Escherichia coli endotoxin, referred to in its thirty-fourth and thirty-fifth reports (WHO Technical Report Series, No. 700, 1984, p. 21, and No. 725, 1985, p. 24), had proved satisfactory and that a quantity of that preparation had been freeze- dried in ampoules coded 841650 (WHO/BS/85.1495). The Committee noted also that the collaborative study, referred to in its thirty-fourth report (WHO Technical Report Series No. 700, p. 21), was in progress in 36 laboratories in 12 countries.
The Committee noted further that international standards of interleukin-l are needed. The Committee therefore requested the National Institute for Biological Standards and Control, London, to obtain suitable materials and to arrange a collaborative study.
33. Dimercaprol and Me1 B (Melarsoprol)
The Committee noted a proposal to discontinue the International Reference Preparation for Dimercaprol, since the monograph for dimercaprol in the third edition of the International Pharmacopoeia
does not include a toxicity test (WHO/BS/85.1482). The Committee noted also a proposal (WHOIBSj85.1482) to discontinue the International Reference Preparation for Me1 B (Melarsoprol), provided that no monograph including a toxicity test was to be prepared for the International Pharmacopoeia. The Committee further noted that no requests for these two preparations had been received for more than seven years.
The Committee was informed that the third edition of the International Pharmacopoeia would not include a monograph on Me1 B (Melarsoprol).
The Committee therefore discontinued the International Reference Preparation of Dimercaprol and the International Reference Preparation of Me1 B (Melarsoprol).
34. Haemiglobincyanide
The Committee was informed that the haemiglobincyanide solution, coded 40600, prepared by the National Institute of Public Health and Environmental Hygiene, Bilthoven, had been proposed as an international standard for haemiglobincyanide by the International Committee for Standardization in Haematology (ICSH). The Committee was also informed that a collaborative study arranged by the ICSH had been completed and that the solution had been shown to have satisfactory stability. On the basis of the results of the collaborative study, the Committee established the material studied as the fifth International Standard for Haemiglobincyanide, with a content of 588 mg of haemiglobin- cyanide per litre.
REQUIREMENTS FOR BIOLOGICAL SUBSTANCES
35. Requirements for Tuberculins
The Committee noted that since the publication of the Requirements for Tuberculins in 1968 (WHO Technical Report Series, No. 384, 1968, p. 27), a number of developments had taken place. Accordingly, several amendments to the Requirements for
Tuberculins had been prepared, distributed for comment, and further amended in the light of comments received (WHO/BS/85.1463 Rev. 1).
The Committee adopted the revised Requirements for Tuberculins and agreed that they should be annexed to this report (Annex 1).
36. Requirements for Dried BCG Vaccine
The Committee noted that there had been a number of developments in the production and control of BCG vaccine, including the wide use of freeze-dried vaccines prepared from seed lots. Accordingly, several amendments to the Requirements for Dried BCG Vaccine had been prepared, distributed for comment, and further modified in the light of comments received (WHO/BS/ 85.1464 Rev. 1).
The Committee adopted the revised Requirements for BCG Vaccine, with minor modifications, and agreed that they should be annexed to this report (Annex 2).
37. Requirements for Continuous Cell Lines
Requirements for continuous cell lines employed as substrates were established in 1982 as part of the Requirements for Poliomyelitis Vaccine (Inactivated) (WHO Technical Report Series, No. 673, 1982, p. 33). The Committee noted that more experience had been gained in the use of continuous cell lines as substrates for the preparation of biological substances by recombinant DNA techniques (WHO/BS/85.1465 Rev. 2). The Committee agreed that there was a need for general requirements for the characterization of continuous cell lines for use as substrates for the preparation of biological substances.
The Committee noted also that such requirements had been formulated and distributed for comment. After making some minor amendments the Committee adopted the Requirements for Continuous Cell Lines, and agreed that they should be annexed to this report (Annex 3). The Committee agreed also that when requirements for individual products are developed or revised, details of quality control procedures for cell cultures should be written taking these requirements into consideration.
The Committee agreed further that these requirements should supersede those in Part D of the Requirements for Poliomyelitis Vaccine (Inactivated) (WHO Technical Report Series, No. 673, 1982, pp. 70-76) (see Annex 4 of this report).
38. Requirements for Poliomyelitis Vaccine (Inactivated)
In view of the adoption of the Requirements for Continuous Cell Lines (see Annex 3 of this report), the Committee agreed that the Requirements ,for Poliomyelitis Vaccine (Inactivated) (WHO Technical Report Series, No. 673, 1982, Annex 2) should be revised by deleting Part D (and references to Part D in various sections) and by making other amendments. The Committee noted that such amendments to the requirements had been formulated (WHO/BS/ 85.1466 Rev. l). The Committee adopted the amendments and agreed that they should be annexed to this report (Annex 4).
39. Requirements for Antimicrobic Susceptibility Tests 1. Agar Diffusion Tests Using Antimicrobic Susceptibility Discs
The Committee noted that since the publication of a revised list of codes for antibiotic susceptibility discs (WHO Technical Report Series, No. 687, 1983, p. 175), the WHO Secretariat had received a number of requests for the allocation of additional or revised codes for identifying the antiniicrobics contained in susceptibility discs (WHO/BS/85.1503). Ncw antimicrobics had been introduced and WHO had, as part of the programme of devising international nonproprietary names, devised codes for some of them. Since certain Member States may have been obliged to adopt other names, as recognized in the list of International Nonproprietary Names ( I N N ) for Pharmaceutical Substances (Cumulative List 6 , Geneva, WHO, 1982), the Committee agreed that the addition of these other names to Section 1.6 of the Requirements for Antimicrobic Susceptibility Tests would help avoid confusion.
The Committee noted that a list of additions, deletions, and changes of codes and a list of major nonproprietary names used in some countries instead of INN'S had been prepared (WHO/BS/ 85.1503).
After making some minor amendments the Committee adopted these further revisions of the Requirements for Antimicrobic Susceptibility Tests and agreed that they should be annexed to this report (Annex 5).
40. Yellow Fever Virus Master Seed
The Committee noted that for some years the WHO Secretariat had worked towards the establishment of a WHO master seed of Yellow Fever virus to be supplied to manufacturers for the preparation of a working seed from which vaccines could be produced (WHO/BS/83.1430 Rev. 1). The Committee noted also that the Robert Koch Institute, Berlin, had prepared a primary seed (213-77) which, in several laboratories, had been shown to be satisfactory and had been approved by WHO consultants.
The Committee therefore adopted the seed lot (213-77) as the WHO Master Seed for Yellow Fever Vaccine (see Annex 6), emphasizing the importance of adhering to the WHO Requirements for Yellow Fever Vaccine (WHO Technical Report Series, No. 594, 1976) and to the Procedure for Approval by WHO of Yellow Fever Vaccines in Connexion with the Issue of International Vaccination CertiJicates (WHO Technical Report Series, No. 658, 198 1).
ACKNOWLEDGEMENTS
The Expert Committee wishes to acknowledge the valuable contributions made to its deliberations by the following WHO staff members: Dr T. Bektimirov, Chief, Virus Diseases. WHO. Geneva: Dr A.P. Mechkovski. Pharmaceuticals. WHO, Geneva; Dr ~ . ' ~ e r v i k d v , ~ i c r o b i o l o ~ ~ and lmmunolog; Support Services, WHO, Geneva; Mr H. ten Dam, Tuberculosis and Respiratory Infections, WHO, Geneva.
Annex 1
REQUIREMENTS FOR TUBERCULINS
(Requirements for Biological Substances No . 16) (Revised 1985)
Page
....................................................................................................... Introduction ....................................................................................... General considerations
Part A . Manufacturing requirements ................................................................ . ...................................................................................... 1 Definitions
2 . General manufacturing requirements ............................................. 3 . Production control ......................................................................... 4 . Filling and containers ....................................................................
........................................................... 5 . Control of the final product .......................................................................................... . 6 Records .......................................................................................... . 7 Samples ......................................................................................... . 8 Labelling
9 . Distribution and shipping .............................................................. ................................................................ 10 . Storage and expiry date
. ............................................................. Part B National control requirements 1 . General ...........................................................................................
................................................................ 2 . Release and certification Authors ..............................................................................................................
............................................................................................. Acknowledgements .......................................................................................................... References
............ Appendix 1 . Summary protocol for tuberculin production and testing .................................. . Appendix 2 Potency testing of manufacturer's standard
INTRODUCTION
The methods of preparing tuberculin and the ways in which it has been used in the diagnosis and treatment of tuberculosis in animals and man have had a long and varied history since its discovery by Robert Koch in 1890 .
Currently. there is a need for uniformity among tuberculin preparations. with specific requirements for each type of tuberculin according to the purpose for which it is used . Several countries have national requirements for the control of tuberculin preparations. but these preparations vary considerably from country to country . In
1962, the WHO Expert Committee on Biological Standardization stated that: "in view of the present state of knowledge of tuberculins and their widespread use in the diagnosis of human and animal tuberculosis", the WHO Secretariat should be requested "to make arrangements for the formulation of requirements for tuberculins" (l). Thus, in 1968 International Requirements for tuberculins were prepared by WHO. In view of the developments that have taken place during the last 16 years it was felt appropriate to update the Requirements for Tuberculins (2).
GENERAL CONSIDERATIONS
The present requirements are applicable to tuberculins intended chiefly for use in human medicine for the following purposes:
(a) as an aid in the diagnosis of tuberculosis; (b) as a research tool in epidemiological studies, including the
detection of non-specific sensitization to mammalian tuberculin; (c) for tuberculin testing in conjunction with BCG vaccination
and chemoprophylaxis of tuberculosis; (d) for the control of BCG vaccines and vaccination procedures; (e) for tuberculin testing in conjunction with BCG immuno-
therapy.
The requirements are also applicable to tuberculins intended for similar uses in veterinary medicine.
There are many difficulties in the formulation of adequate requirements that would be suitable for international use. These include the choice of international standards for tuberculins and difficulties in specifying the potency of tuberculin preparations in a satisfactory manner. The international standards for tuberculins established by WHO, which have been available for many years, are the International Standard for Old Tuberculin, the International Standard for Purified Protein Derivative (PPD) of Mammalian Tuberculin, and the International Standard for PPD of Avian Tuberculin.
The International Standard for Purified Protein Derivative of Mammalian Tuberculin was established in 1951 (2), and in 1952 the International Unit for PPD of Mammalian Tuberculin was defined as being contained in 0.000028 mg of the standard (3). This definition was based on comparisons with old tuberculin and the
size of the unit was selected as the weight that corresponded to the International Unit for Old Tuberculin. However, in view of the fact that the original assays for defining the International Unit for PPD of Mammalian Tuberculin were done at a time when the interphase adsorption effect was not known to be relevant, in its fourteenth report, the WHO Expert Committee on Biological Standardization (4) considered those assays to be invalid, and decided, therefore, that the existing definition of the international unit should no longer apply. It was further recommended that the expression of the potency of preparations of PPD of mammalian tuberculin should, for the present, be in terms of International Units for Old Tuberculin, on the basis of assays made under defined conditions.
Since PPD of mammalian tuberculin and old tuberculin are of different composition and are now known to have different dose-response relationships, they cannot in principle be compared in a valid assay. The unit of 0.000028 mg for PPD of mammalian tuberculin has been widely used in practice for many years, before, during, and after the period when it was officially valid. The practice of expressing the potency of PPD preparations in terms of units of old tuberculin, as suggested by the WHO Expert Committee on Biological Standardization in 1960 (4), has been followed only to a very limited extent. There is an international standard for each of the three preparations-old tuberculin, PPD of mammalian tuberculin, and PPD of avian tuberculin-and each should have its own international unit. In its twentieth report, the WHO Expert Committee on Biological Standardization therefore re-established the International Unit for PPD of Mammalian Tuberculin as the activity contained in 0.000028 mg of the International Standard for PPD of Mammalian Tuberculin (5).
The potency of tuberculin preparations cannot always be expressed in terms of international units, established by means of a biological assay that would be valid for all conditions of use. This is due to the variation in the results of potency assays arising from the use of different bacterial strains for production purposes, different methods of extraction and purification, and different methods of sensitizing animals used in potency assays. In the present requirements, therefore. the control of potency of tuberculins has been based on the following procedure.
For each tuberculin product issued by a manufacturer, a representative sample is set aside to be used as a laboratory reference preparation. This reference preparation is calibrated by comparing
its activity with that of the relevant International Standard (i.e., the one that it most closely resembles in behaviour) at a particular level of dosage and under the conditions in which it will be used in practice. These conditions include, as well as the level of dosage, the method of making up the preparation, the volume and route of injection, the animal species, and the immunological state of the animals. The conditions chosen might be, for example, human subjects or cattle with natural tuberculous infection, or BCG- vaccinated human subjects, or a particular tuberculin testing technique. The object of this calibration procedure is to determine the amount of the reference preparation that will have a specific biological effect equivalent to that of a given amount of the International Standard under a particular set of conditions. It permits the dose of the reference preparation for that particular set of conditions to be expressed in equivalents of international units.
The control of potency of successive batches can then be carried out by biological assays in guinea-pigs, using the laboratory reference preparation calibrated as described.
The use of these requirements, therefore, necessitates the availability of the International Standards in sufficient quantities to be distributed for the calibration of the various types of reference materials. It will also make it necessary for a manufacturer to establish a reference preparation for each tuberculin product issued.
The control of successive batches of each manufacturer's particular product and each method of manufacture used, by assays with a reference preparation as described above, is advisable to ensure consistency of production between batches. Further, manufacturers should be encouraged to produce tuberculins in large batches.
Considerable difficulty in the control and use of tuberculins is also caused by the phenomenon of interphase adsorption, which occurs to a significant extent under certain circumstances. There is no generally accepted method for dealing with this problem, although the adsorption effect might be considerably reduced by the addition of stabilizers such as Tween 80. Therefore, the present requirements have been formulated so as to ensure that the comparisons of each
A very large batch, RT 23 was acquired by WHO in 1960 and since then it has been used widely, notably in surveys; stock solution or powder of this batch is made available free of charge to national control laboratories.
type of macufacturer's reference preparation with the international standard are made under defined and reproducible conditions related to the particular use of the preparation. In this way, only that portion of the active material that is effective under those particular circumstances would have the potency specified.
Each of the following sections constitutes a recommendation. The parts of each section that are printed in normal type have been written in the form of requirements so that, if a health administration so desires, these parts as they appear may be included in definitive national requirements. The parts of each section that are printed in small type are comments and recommendations for guidance.
Should individual countries wish to adopt these requirements as the basis for their national regulations concerning tuberculin, it is recommended that a clause be included permitting modifications of the manufacturing requirements on the condition that it be demonstrated, to the satisfaction of the national control authority, that such modified requirements ensure that the degree of safety and the potency of tuberculins are at least equal to those provided by the requirements formulated below. It is desirable that the World Health Organization should then be informed of the action taken.
The terms "national control authority" and "national control laboratory" as used in these requirements, always refer to the country in which the tuberculin is manufactured.
PART A: MANUFACTURING REQUIREMENTS
1. DEFINITIONS
1.1 International name and proper name
The international name shall be "Tuberculinum pristinum" followed by the designation "mammalianum" or "avianum" (whichever applies), or "Derivatum proteinosum purijicatum tuberculini" followed by the designation "mammaliani" or "aviani" (whichever applies). The proper name shall be the equivalent of the international name in the language used by the country of origin.
The use of the international name should be limited to tuberculins that satisfy the requirements formulated below.
1.2 Descriptive definition
"Tuberculinum pristinum" (non-purified Tuberculin)' and "Derivatum proteinosum purijicatum tuberculini" (Purified Protein Derivative of Tuberculin (PPD)) are preparations made from the heat-treated soluble products of growth and lysis of tubercle bacilli (Mycobacterium tuberculosis or M . bovis in the case of mammalian tuberculin, or M. avium in the case of avian tuberculin), which reveal delayed hypersensitivity in animals sensitized by micro-organisms of the same species.
Non-purified Tuberculin is a concentrated filtrate of the soluble products of growth and lysis of the tubercle bacillus grown in a fluid medium.
Koch's old tuberculin is no longer in general use since a synthetic medium is usually used in place of the original glycerol broth culture medium. Old tuberculin and crude tuberculin produced on synthetic medium are non-purified tuberculins, as opposed to Purified Protein Derivative of Tuberculin (PPD), which is purified. PPD is prepared from the water-soluble fractions obtained by steaming (to kill the bacteria) and subsequently filtering cultures of the tubercle bacillus grown in a liquid synthetic medium. The active fraction in the filtrate, which is predominantly protein, is usually isolated by precipitation, following which it is washed and redissolved. The preparation may be issued in dried or liquid form.
The generic term "tuberculin7' may be used only to refer to either non-purified tuberculin or PPD.
In view of their potential use in human beings, preparations containing mycobacterial components obtained by processes that do not include heat sterilization should be subject to special requirements, which should be formulated by the national control authorities.
1.3 International standards and international units An International Standard for Non-purified Tuberculin, an
International Standard for Purified Protein Derivative (PPD) of Mammalian Tuberculin, and an International Standard for Purified Protein Derivative of Avian Tuberculin have been established. These preparations are in the custody of the
Formerly called Old Tuberculin.
36
International Laboratory for Biological Standards, State Serum Institute, Copenhagen. and samples are distributed on request to national control authorities and other relevant laboratories. The international standards are intended mainly for the calibration of manufacturers' standards for tuberculins.
1.4 Terminology
Seed lot: A quantity of bacteria processed together and of uniform composition. A seed lot shall be maintained in the dried form. In each manufacturing establishment, a primary seed lot is that from which material is drawn for inoculating media for the preparation of secondary seed lots or single harvests.
Seconda,:,~ seed lot: A working seed lot, not more than four culture passages removed from the primary seed lot and intended for inoculating media for the preparation of single harvests.
Single-cztlture Jlnsk: A flask containing liquid culture medium inoculated with a suitable strain of tubercle bacillus and incubated at a suitable temperature for a suitable period of time. Single-culture flasks shall be prepared from cultures removed from a seed lot by as few culture passages as possible.
Pooled culture jiltrate: The clarified filtrate from a number of steamed single-culture flasks that have been inoculated and processed together.
Concentrated bulk: A quantity of homogeneous material in the form of either concentrated solution or dried powder prepared from one or more pooled culture filtrates.
Final bulk: A finished homogeneous preparation derived from one or more concentrated bulks and present in the container from which the final containers are filled.
Filling lot. A collection of sealed finai containers that have been filled during one working session from a single final bulk and that are uniform with respect to the risk of contamination during filling, freeze-drying (if applicable) and sealing.
2. GENERAL MANUFACTURING REQUIREMENTS
The general manufacturing requirements contained in the revised Requirements for Biological Substances No. l (General Requirements for Manufacturing Establishments and Control Laboratories) (6)
37
shall apply to establishments manufacturing tuberculin, with the addition of the following:
All manufacturing processes up to the killing of the mycobacteria shall take place in a completely separate area using separate equipment .
Precautions should also be taken to protect the personnel against acquiring tuberculosis or developing systemic reactions to tuberculin.
3. PRODUCTION CONTROL
3.1 Control of source materials
3.1.1 Bacterial strains
Strains of Mycobacterium tuberculosis, M. bovis or M. avium used in preparing tuberculin shall have their histories fully documented. Tests shall also be done to identify the species.
Strains selected shall be capable of yielding a product that conforms to the requirements set forth in Part A, section 5, of this document .
One or more strains may be used. In some countries, however, the national control authority prescribes the use of a single particular strain for tuberculin production.
3.1 .2 Seed lot system
The production of tuberculin shall be based on the seed lot system as indicated in Part A, section 1.4. Seed lots prepared from each of the strains approved by the national control authority shall be prepared under conditions satisfying the requirements of Part A, sections 2 and 3.
It is recommended that a large primary seed lot should be set aside under optimum storage conditions as the basic material to which the manufacturer may return for the preparation of further seed lots.
3.1.3 Production culture medium
The medium shall be free from ingredients known to cause toxic or allergic reactions in man or animals. The medium selected shall
be capable of yielding a product that conforms to the requirements set forth in Part A, section 5, of this document.
Several media, differing in composition, are used depending on the class of tuberculin to be made and the strain of organism to be grown. In the synthetic medium used for making tuberculins, the source of nitrogen for bacterial growth is usually asparagine and the sources of carbon are usually glycerol and dextrose. Slight variations in the composition of the medium may affect the properties of the final product and in some countries the national control authority requires that one type of medium of specified composition be used for routine production of tuberculin.
3.2 Production precautions
The general production precautions as formulated in Part A, section 3, of the revised Requirements for Biological Substances No. 1 (General Requirements for Manufacturing Establishments and Control Laboratories) (6) shall apply to the manufacture of tuberculin.
Only personnel concerned with production of tuberculin shall be permitted to enter incubation rooms during the incubation process.
Tuberculin preparations made from different species of mycobacterium shall be processed separately.
If groups of single-culture flasks inoculated at different times with the same bacterial species are kept in the incubator together, they shall be separated and clearly identified.
Culture flasks shall be inspected at intervals during and on completion of incubation, and any flasks showing evidence of abnormal growth shall be discarded.
After completion of incubation the flasks shall be sterilized by steam at not less than 100°C for at least 60 minutes, or by autoclaving at not less than 120 "C for at least 30 minutes.
During the sterilization process the temperature should be continuously recorded.
3.3 Control of pooled culture filtrate
The pooled culture fluid shall be freed from bacteria by filtering the contents of the culture flasks into one or more containers. Further processing of the pooled culture filtrates shall be carried out so as to yield a single homogeneous, clarified, concentrated bulk free from any residual tubercle bacilli.
3.4 Control of concentrated bulk
3.4.1 Concentration procedures
The concentrated bulk is prepared by further processing of the pooled culture filtrate in one or more instalments. The processing shall, however, yield a single homogeneous concentrated bulk. The procedures shall include one that will ensure sterility of the material, and all processing thereafter shall be carried out in such a manner as to maintain the sterility of the product.
Manufacturing procedures commonly used include sterile filtration, precipitation and washing with chemical solvents, heat concentration, and addition of antimicrobial agents in adequate amount, depending on the class of the product and the method of manufacture.
It is desirable to check the sterility of the concentrated bulk by carrying out a sterility test as described in Part A, section 3.5.
3.4.2 Antimicrobial and other substances added
Antimicrobial preservatives or other substances that may be added to the concentrated bulk shall have been shown, to the satisfaction of the national control authority, not to impair the safety and effectiveness of the product.
Care should be taken that any preservative used will remain in an effective concentration at least until the expiry date of the product.
Mercurial derivatives such as merthiolate have been found to cause false positive reactions in tuberculin potency testing.
The test described in Part A, Section 3.4.3, should be done on the samples before the addition of any antimicrobial substances or any substances that may interfere with the performance of the tests.
3.4.3 Test for live mycobacteria
Each concentrated bulk shall be tested for freedom from live mycobacteria by appropriate culture methods. For tests on such materials in the dried form, a solution shall be made containing at least 1 mg of the dried material per m1 of the diluent.
Each concentrated bulk (reconstituted as above if applicable) shall be tested for freedom from live mycobacteria by the
intraperitoneal or subcutaneous inoculation of 5.0 m1 of the concentrated bulk into each of at least two guinea-pigs weighing between 250 g and 400 g.
The test may be performed using the deposit centrifuged from at least 5.0 ml of concentrated bulk and resuspended in a small volume of saline or supernatant. Alternatively, concentration of mycobacteria may be achieved through filtering membranes.
The animals shall be observed for at least 42 days for death or signs of disease. All guinea-pigs that die after the first 24 hours of the test or that show signs of illness shall be examined macroscopically and then the relevant tissues shall be examined both microscopically and culturally for evidence of the presence of the mycobacteria in question. Animals that survive the observation period shall be examined in a similar manner.
The concentrated bulk passes the test if the cultures tested for freedom from live mycobacteria are negative, if all the guinea-pigs remain healthy and survive the observation period, and if none of the animals s h o ~ s signs of the presence of mycobacteria.
I f any animal dies from causes other than tuberculosis before the cnd of thc observation period, the test may be repeated once.
3.4.4 Potency test
Samples shall be taken and assayed for potency after the concentrated bulk has been processed to a stage in which it differs from the final bulk only in strength of active material.
3.4.5 Test for sensitizing eflect
For each type of preparation, at least one sample derived from each concentrated bulk shall be tested in animals for sensitizing effect by a method approved by the national control authority.
The following method has been found satisfactory: Three guinea-pigs. that have not previously been used for any
tests, are inoculated intradermally three times, at intervals of five days. with an appropriate dose of the product under test (such as the nominal equivalent of 500 IU in a volume of 0.1 ml). Fifteen days after the third injection the animals, as well as a control group of guinea-pigs of the same weight, but not having
, had any previous injections, are given an intradermal dose. The reactions of the two groups should not be significantly different.
3.5 Control of final bulk
3.5.1 Sterility
Samples of each final bulk shall be tested for bacterial and mycotic sterility according to the requirements given in Part A, section 5, of Requirements for Biological Substances No. 6 (General Requirements for the Sterility of Biological Substances) (7). For these tests on final bulk in the dried form, a solution shall be made containing at least 1 mg of the dried material per m1 of the diluent.
3.5.2 Potency tests
The final bulk may be tested for potency according to the test described in Part A, section 5.3.2.
4. FILLING AND CONTAINERS
The requirements concerning filling and containers given in Part A, section 4, of the revised Requirements for Biological Substances No. I (General Requirements for Manufacturing Establishments and Control Laboratories) (6) shall apply.
5. CONTROL OF THE FINAL PRODUCT
The final product shall be tested according to the provisions of the following subsections.
The final product may be issued in a concentrated form or as a solution of such a strength that it can be used directly for the tuberculin testing.
If the product is issued in a concentrated form, either the recipient is instructed to make the appropriate dilution of the material immediately before use, or the recipient may process the material further in a manufacturing establishment and prepare a new final bulk for filling into a number of final containers in a form suitable for tuberculin testing.
In the case of a product derived from a new final bulk in a manufacturing establishment other than that in which the tuberculin product originated, the requirements given in Part A, sect!ons 3.5-10 shall apply.
5.1 Identity test
An identity test shall be done on at least one sample for each filling lot.
This material when injected intradermally should provide typical reactions in sensitized guinea-pigs but not in non- sensitized guinea-pigs. The potency test described in Part A, section 5.3, of these requirements may serve as the identity test if performed on material from the final containers.
5.2 Sterility tests
Each filling lot shall be tested for sterility according to the requirements given in Part A, section 5, of Requirements for Biological Substances No. 6 (General Requirements for the Sterility of Biological Substances) (7).
5.3 Control of potency
Specifications of potency shall be based on the appropriate International Standard. Each manufacturing establishment shall use the reference material calibrated against the International Standard as described in Part A, section 5.3.1.
The final product shall be assayed in guinea-pigs, as described in Part A, section 5.3.2, against the reference preparation to ensure that the potency is within limits acceptable to the national control authority.
, When tht potency of the final product is such that no dilution can be made for the purpose of potency testing, estimation against the standard curve can be made for a single concentration, i.e., using the final product directly.
In order to verify the validity of the observations in guinea- pigs, it is desirable to carry out periodic comparisons of the final product with the relevant reference preparation by tests in the species in which the preparation is to be used.
5.3.1 Calibration of reference preparation
For each type of final bulk, a representative homogeneous quantity shall be set aside for reference purposes.
Reference material shall meet all the requirements for tuberculin. In addition, before carrying out the calibration tests, adequate
information shall be obtained relating to the particular product to
be calibrated in order to ensure that appropriate doses are used. Calibration tests in man shall be made subject to the approval of the appropriate national authority.
Calibration of the reference preparation shall be done by a number of tests against the appropriate International Standard,l the tests being carried out in the species in which the tuberculin is to be used.
The purpose of these tests is to assess the amount of proposed reference preparation that will produce the same effect as a given amount of the appropriate International Standard under the specified conditions of carrying out the comparison. The conditions to be specified shall be chosen in relation to the specific purpose for which the preparation is to be used and at the doses concerned.
If a batch of tuberculin has been calibrated against the International Standard (e.g., by using phosphate-buffered saline as a diluent), the reactions it produces may be quite different if a surface-active agent such as Tween 80 is used to prevent adsorption to the container. The reactions not only may be larger but may also differ qualitatively. Calibration in man, therefore, should include such stabilized solutions. Solutions ready for use should be issued in a strength that has been found to be appropriate for use in man.
The procedures for calibration and analysis of the results should be acceptable to the national control authority (see Part B, section 1).
In these tests, dilutions of the International Standard shall be made up in 0.033 molllitre sodium phosphate-buffered saline (at pH 7.0) and dilutions of the reference material shall be made up in the diluent in which the final product is to be used in tuberculin testing of man or animals as the case may be; suitable precautions should be taken to avoid interphase adsorption both for the international and manufacturer's standard.
It may be necessary to recalibrate or replace the reference preparation periodically, depending on its stability.
In certain cases, the reference material may be calibrated against a national standard or other preparation that has been shown to be similar to the International Standard and suitable for use for the purpose by all pertinent tests, including a variety of assay methods of which one shail be in the species in which the preparation is intended for use.
Information on suitable procedures for calibration tests in man may be obtained, on request, from Biologicals, World Health Organization, Avenue Appia, 1211 Geneva 27. Switzerland.
The potency of the reference material shall be specified in equivalents of the relevant international units under each of the sets of conditions of making the comparisons and at the particular concentrations used.
5.3.2 Assay of potency of test tuberculin in guinea-pigs
The final product shall be assayed against the manufacturer's reference material previously calibrated according to the procedure in section 5.3.1 using suitably sensitized guinea-pigs, and shall have a potency within the limits specified by the national control authority for each type of preparation.
For each sample of material under test, an estimate of relative potency and its fiducial limits shall be made by statistical methods using the diameters of the reactions and the logarithms of the doses. All the results obtained shall be included.
As mentioned before (see General Considerations), the results of potency assays can be affected by several factors.
The method should be based on at least a 6-point assay design. For each assay, at least 6 white or albino guinea-pigs should be taken, of not less than 400-g weight. During the period of the test the animals should be maintained under uniform dietary and environmental conditions.
Different methods of sensitization of guinea-pigs can be used. Once a method of sensitization has been selected, strict adherence to it improves the comparability of results between assays. Possible methods of sensitization include:
(a) intramuscular injection of up to 2 mg of heat-killed, dried tubercle bacilli of the strain or strains from which the relevant International Standard was prepared, suspended in sterile liquid paraffin;
(b) 4 intradermal injections at one time, each of 0.1 m1 of suspension containing 0.1 mg of heat-killed, dried tubercle bacilli per m1 of liquid paraffin;
(c) intraperitoneal or intramuscular injection of an aqueous suspension containing 0.0001 mg (moist weight) of living virulent tubercle bacilli;
(d) subcutaneous injection of 0.1 m1 of an aqueous suspension of 0.5 mg (moist weight) of BCG vaccine per ml.
Tuberculin sensitization is sometimes achieved through four intradermal injections, at one time, each of 0.1 ml of an aqueous suspension containing 0.5 mg of BCG vaccine per ml.
When possible, preliminary tests may be carried out with the reference material in order to select the most suitably sensitized animals. The assay should then be made not less than l month and generally not more than 6 months after sensitization and at least 4 weeks after a preliminary test.
At least three dilutions, differing by appropriate steps, of the reference preparation and of the material under test, should be made. The dilutions of both the reference preparation and the material under test should be made in the same way as when the reference preparation was calibrated.
The dilutions should be chosen so that the lesions produced by their injection are not less than 8 mm and not more than 25 mm in diameter at the time chosen for reading the test (generally after 24 hours) and the total reactions of the two preparations are approximately equal.
A separate syringe of a suitable type,' that can dispense the amount to be injected with the desired accuracy, should be used for each of the dilutions. Each syringe should be rinsed twice with the appropriate dilution, and the material in a constant predetermined volume selected from the range 0.1-0.2 m1 should be injected intradermally into each guinea-pig at one of the appropriate sites, allocated by a randomized Latin square (three sites on each side if a 6-point assay is used). After an appropriate interval, the diameters of the reactions produced should be measured by a suitable method.
The material under test is of acceptable potency if its estimated potency is within 80-125% of the declared potency, with fiducial limits (P = 0.95) within the range 64-156%.
If the material fails to meet the requirement for potency, it shall be discarded, or the assay shall be repeated one or more times by the same method using a different group of sensitized animals. The estimates of potency and fiducial limits shall be made using the results of all the assays. The material is of acceptable potency if the combined results of the assays meet the criteria specified above.
5.4 Innocuity test
Samples from each filling lot shall be tested for abnormal toxicity by appropriate tests involving injection into guinea-pigs and mice. The tests shall be those approved by the national control authority.
l "Tuberculin" syringe.
5.5 Content of phenol or other preservatives
If phenol has been used in the preparation, at least one sample derived from each final bulk shall be tested for phenol content, using a method approved by the national control authority. The phenol content shall not exceed 5.5 gllitre.
If other preservatives are used, the national control authority should specify the acceptable concentration.
5.6 Inspection of final containers
Each container in each filling lot shall be inspected visually and those showing abnormalities shall be discarded.
5.7 pH
The pH of the product should be within the range, 6.5-7.5.
6. RECORDS
The requirements given in Part A, section 6 , of the revised Requirements for Biological Substances No. I (General Requirements for Manufacturing Establishments and Control Laboratories) (6) shall apply.
7. SAMPLES
The requirements given in Part A, section 7 , of the revised Requirements for Biological Substances No. l (General Requirements for Manufacturing Establishments and Control Laboratories) (6) shall apply.
8. LABELLING
The requirements for labelling given in Part A, section 8, of the revised Requirements for Biological Substances No. l (General Requirements for Manufacturing Establishments and Control Laboratories) (6) shall apply with the addition of the following:
If the final container is not suitable for labelling, it shall be in a labelled package.
The label on the container shall show in addition:
- the species of the bacillus from which the tuberculin has been made;
-the recommended dose and its nominal equivalence in International Units (as determined by calibration) for the particular species and type of test for which the product is to be used;
-the statement "Use only as instructed".
The leaflet1 in the package shall contain instructions for use of the product and shall include a statement instructing the user on the maximum length of time that the contents of a container may be kept once the container has been opened.
9. DISTRIBUTION AND SHIPPING
The requirements given in Part A, section 9, of the revised Requirements for Biological Substances No. l (General Requirements for Manufacturing Establishments and Control Laboratories) (6) shall apply.
10. STORAGE AND EXPIRY DATE
The requirements given in Part A, section 10, of the revised Requirements for Biological Substances No. 1 (General Requirements for Manufacturing Establishments and Control Laboratories) (6) shall apply.
10.1 Storage conditions
Tuberculin preparations shall be protected from light and stored at all times at a temperature between 2°C and 8°C. Dried preparations may be stored at lower or higher temperatures, but not exceeding 25 "C.
l If a valid biological assay can be made with the relevant International Standard, a statement to that effect can be included at the discretion of the national control authority.
10.2 Expiry date
The expiry date shall relate to the date of the last satisfactory potency test, the date of the potency test being taken as that on which the test animals were inoculated.
The expiry date should be not more than five years from the date of the last potency test for preparations in a concentrated form supplied for further processing before use. For preparations supplied ready for use or those requiring only simple dilution before administration, the expiry date will depend on the type of preparation and the stabilizer employed; experience has shown that expiry dates of 1-2 years from the date of the last satisfactory potency test are realistic.
PART B. NATIONAL CONTROL REQUIREMENTS
1. GENERAL
The general requirements for control laboratories given in Part B of the revised Requirements for Biological Substances No. I (General Requirements for Manufacturing Establishments and Control Laboratories) (6) shall apply.
In addition, the national control authority shall provide sufficient samples of the relevant international standard or a national standard as defined on page 44, footnote 1 , for the calibration of manufacturers' reference material (see Part A, section 5.3.1). The national control authority shall also give directions to manufacturers concerning the calibration of reference material and the method of assay suitable for batches of each type of preparation and for each particular usage.
2. RELEASE AND CERTIFICATION
A lot of tuberculin shall be released only if it fulfils Part A of the present requirements.
A statement signed by the appropriate official of the national control laboratory shall be provided at the request of the manufacturing establishment and shall certify whether the lot of
tuberculin in question meets all national requirements as well as Part A of the present requirements. The certificate shall furthermore state the date of the last satisfactory assay of potency, the lot number, the number under which the lot was released, and the number appearing on the labels of the containers. In addition, a copy of the official national release document shall be attached.
The purpose of the certificate is to facilitate the exchange of tuberculin preparations between countries.
AUTHORS
The Requirements ,for Tuberculins (Revised 1985) were formulated by the following WHO consultants and staff members:
Dr R. Donikian, Merieux Institute, Charbonniere les Bains, France (Consultant) Dr M. Gheorgiu, Chief, BCG Department, Pasteur Institute, Paris, France
(Consultant) Dr T. B. Jablokova, Chief, Laboratory for Standardization and Control of BCG and
Tuberculin, Tarasevii: State Institute for the Standardization and Control of Medical Biological Preparations, Ministry of Health, Moscow, USSR (Consultant)
Miss A. Ladefoged, BCG Department, State Serum Institute, Copenhagen, Denmark (Consultant)
Dr M. Leguenno, Chief, BCG Laboratory, Pasteur Institute, Dakar, Senegal (Consultant)
Mr J. Lyng, Laboratory for Biological Standards, State Serum Institute, Copenhagen, Denmark (Consultant)
Dr A. Pio, Chief, Tuberculosis and Respiratory Infections, World Health Organization, Geneva, Switzerland
Professor J. Ruitenberg, State Institute for Public Health and Environmental Hygiene, Bilthoven, The Netherlands (Consultant)
Dr T. Sawada, Japan BCG Laboratory, Tokyo, Japan (Consultant) Dr P. Sizaret, Scientist, Biologicals, World Health Organization, Geneva, Switzerland Mr H. ten Dam, Tuberculosis and Respiratory Infections, World Health
Organization, Geneva, Switzerland Dr D. Wright, Head, BCG Vaccine Unit, Glaxo Operations UK Ltd., Greenford,
England (Consultant)
ACKNOWLEDGEMENTS
Acknowledgement is due to the following experts for their comments and advice:
Mr D. W. Andrews, Central Veterinary Laboratory, Weybridge, England Mr D.N. Barnett, Central Veterinary Laboratory, Weybridge, England Professor M.P. Beumer, General Secretary International Relations, Ministry of
Public Health and Family Affairs, Brussels, Belgium Dr B. Beys, Belgian Pharmaceutical Association, Brussels, Belgium
Professor H. Brede, President, Paul-Ehrlich Institute, Frankfurt-am-Main, Federal Republic of German)-
Dr I . Di Tommaso. Head, Tuscan Institute for Serotherapy and Vaccine Production (SCLAVO), Siena, Italy
Dr J. Dufaux, Belgian Pharmaceutical Association, Brussels, Belgium Dr D. Calam, National Institute for Biological Standards and Control, London,
England Dr. V.F. Davey, Kew, Australia Dr I. Davidson, Central Veterinary Laboratory, Weybridge, England Dr R. Dobbelaer, Chief Institute of Hygiene and Epidemiology, Brussels, Belgium Mme M. Dufaux, Industrial Pharmacist, Pasteur Institute of Brabant, Brussels,
Belgium Dr A. Engibarov, Chief, Laboratory for Specific Prophylaxis of Tuberculosis,
Medical Academy, Research Institute of Infectious and Parasitic Diseases, Sofia, Bulgaria
Dr I. Furminger, Technical Adviser Biologicals, Glaxo Operations UK Ltd, Greenford, England
Dr C. Guthrie, Operations Director, Commonwealth Serum Laboratories, Parkville, Australia
Professor S. Hollan, Director, National Institute of Haematology and Blood Transfusion, Budapest, Hungary
Dr A. Lafontaine, General Secretary of International Relations, Ministry of Public Health and Family Affairs, Brussels, Belgium
Dr A.M.T. Lee, Central Veterinary Laboratory, Weybridge, England Dr H. Lehmann, Behringwerke AG, Marburg, Federal Republic of Germany Dr P. Lemoine, Chief of Section, Institute of Hygiene and Epidemiology, Brussels,
Belgium Dr D. Levy, Tarasevii: State Institute for the Standardization and Control of Medical
Biological Preparations, Ministry of Health, Moscow, USSR Dr J. Nyabenda, Laboratory of Tuberculins, Pasteur Institute of Brabant, Brussels,
Belgium Dr D.C. Pope, Assistant Director, Bureau of Biologies, Drugs Directorate, Ottawa,
Canada Dr W. Pranter, Behringwerke AG. Marburg. Federal Republic of Germany Dr G. Reber, Behringwerke AGI Marburg, Federal Republic of Germany Professor L. Reichert, Department of Biochemistry, The Albany Medical College of
Union University, Albany, NY, USA Professor G. Swaniker, Faculty of Medicine, Boroko, Papua New Guinea Mr A.J. Taylor, Central Veterinary Laboratory, Weybridge, England
REFERENCES
1. WHO Technical Report Series, No. 259, 1963, p. 16. 2. WHO Technical Report Series, No. 56, 1952. p. 6. 3. WHO Technical Report Series, No. 68, 1953, p. 6. 4. WHO Technical Report Series, No. 222, 1961, p. 14. 5. WHO Technical Report Series, No. 384, 1968, p. 13. 6. WHO Technical Report Series, No. 323, 1966, Annex 1, part A. 7. WHO Technical Report Series, No. 530, 1973, p. 40.
Appendix 1
SUMMARY PROTOCOL FOR PURIFIED TUBERCULIN PRODUCTION AND TESTING
Based on Requirements for Biological Substances No. 16. Requirements for Tuberculins (WHO Technical Report Series,
No. 745, 1987, Annex 1)
Name and address of manufacturer
Lot number and type of tuberculin
Date of manufacture of filling lot
Expiry date
Information on Seed Lot
Species and strain of mycobacteria
Reference number and date of preparation of seed lot
Test for culture purity
Identification of mycobacterium species
Seed lot approved by National Control Authority (~es/no)
Date of approval
Information on Manufacture
Date of reconstitution of seed lot ampoule and inoculation
Number of passages from seed
Medium used
Incubation time of single cultures
Date and method of sterilization of cultures
Date of filtration
2 . Pooled culture filtrate
Date of pooling culture filtrates
Date and method of purification or concentration of culture filtrates
3. Concentrated bulk
Date of completion
Method used for precipitation of tuberculin
Weight
Tests done
3.1 Tcst for abscncc of contaminntion
Datc of inoculation
Mcdia
Iluralion of tcsl
First test Repeat test i f necessary
Results (passlfail)
3.2 Test for absence of live mycobacteria
Amount of tuberculin injected
Number of guinea-pigs injected
Observation period
Weight of animals prior to injection
Mean loss of weight per animal
Number of guinea-pigs still alive at the end of the test
Results of visual inspection of viscera and lymph nodes
Results of histological examination
Dates of inoculation and end of culturing of homogenates of viscera and lymph nodes
Result of culturing
Was a repeat necessary?'
Result of test (passedlfailed)
3.3 Test of potency
3.3.1 Manufacturer's standardZ
Reference number
Date on which it was calibrated against the international tuberculin standard
Species in which it was calibrated against the WHO tuberculin standard
Relative potency
3.3.2 Test tuberculin
Method of sensitization
Date of reading of reactions
Results
Test tuberculin Manufacturer's standard Dilution No. of guinea-pigs Size of Dilution No. of guinea-pigs Size of of tu- each in- of tu- each in- berculin dividual berculin dividual
reaction reaction
'If yes, give all relevant information on a separate sheet.
2Upon request, provide data on calibration of internal tuberculin standard as described in Appendix 2.
3.3.3. Relative potency (provide a separate sheet with all details of calculation)
Potency of test tuberculin
3.4 Absence of sensitizing properties
Tes t Control
Numbcr of guinca-pigs
Sensitizing dosc given in cach injection
Numbcr of injcclions
lntcrval bctwccn injections
Test dose
Mean sizc of' rcactions in the control and test groups
4. Final hulk
Datc of preparation Composition' Substances added to final bulk
and concentration Protein concentration
4.1 Test for absence of contamination First test Repeat test
if necessary
Number of inoculated tubes
Media
Duration of test
Result (passedlfailed)
l If derived from several parent bulks, give relevant details (serial no., volume of each, etc.) on a separate sheet.
4.2 Test for potency (optional)
If potency test was performed on final bulk, give details on separate sheet following instructions given under 3.3.
5 . Filling lot1
Date of filling
Type and size of containers (sealed ampoules or vials)
Number of containers
Stated number of international units in a single human or animal dose and volume in which it is contained (after reconstitution if lyophilized)
Number of doses per container
Recommended reconstitution fluid (for freeze-dried tuberculins only)
5.1 Test for identity
Method
Results
5.2 Test for absence of contamination
First test Repeat test lj' necevsury
Number of containers tested
Media
Duration of test
Results
5.3 Test for innocuity
Species
Number of animals in each species
Result
l For lyophilized tuberculins, results of tests should be reported on a separate sheet for each lyophilized batch.
5.4 Content of preservative
Type of preservative
Content
5.5 Test for potency
5.5.1 Test
Method of sensitization
Date of reading of reactions
Result:
Test tuberclllin hfanufacturer's standard1 Dilution No. of guinea-pigs Size of Dilution No. of guinea-pigs Size of of tu- cach in- or tu- each in- berculin dividual bcrculin dividual
rcaclion reaction
5.5.2 Relative potency (provide a separate sheet with all details of calculation).
Potency of test tuberculin
Information on Release
Is the lot of tuberculin satisfactory? yes/no
Has the lot been released by the national control authority? yes/no
If yes, give date
Has a certificate been supplied by the national control laboratory? If yes, indicate number of release certificate
'Upon request, provide data on calibration of internal tuberculin standard as described in Appendix 2.
If not which, laboratory would supply such certificate?
Has the lot of tuberculin been tested in man? (answer "not applicable" if the product is not for human use)
Signature of head of production laboratory (name should be typed)
Certification by person taking overall responsibility for control of tuberculin
I certify that Lot No. ... of tuberculin (type ...) satisfies Part A of the WHO Requirements for Tuberculins.
Signature Name (typed)
The protocol must be accompanied by a sample of the label, a copy of the leaflet and, if the vaccine is to be exported, a copy of the release certificate, issued by the national control authority.
Appendix 2
POTENCY TESTING OF MANUFACTURER'S STANDARD
Species in which the test is performed1
Type of sensitization
Age range (if test is performed on human beings)
Reference of WHO tuberculin standard
Reference of the proposed manufacturer's standard
Dates of injection of tuberculin
Time between tuberculin administration and reading of result
'The species in which the calibration is made must be the same as that for which further batches of tuberculin are intended.
Proposed Manufacturer's Standard
WHO Tuberculin Standard
Dilution No. of subjects Diameter Dilution No. of subjects Diameter of tu- of of tu- of berculin reaction' berculin reaction'
Calculation of relativepotcncy (providc a separate sheet with all details)
Relative potency of manufacturer's standard
'If necessary, use an additional sheet of paper to give results of individual reactions.
Annex 2
REQUIREMENTS FOR DRIED BCG VACCINE
(Requirements for Biological Substances No . 11) (Revised 1985)
Page Introduction ....................................................................................................... 60 General considerations ....................................................................................... 61 Part A . Manufacturing requirements ................................................................ 66
1 . Definition ....................................................................................... 66 ............................................. 2 . General manufacturing requirements 67
......................................................................... 3 . Production control 69 4 . Filling and containers .................................................................... 74
................................................................ . 5 Control tests on final lot 75 .......................................................................................... . 6 Records 79
. .......................................................................................... 7 Samples 80 8 . Labelling ......................................................................................... 80
............................................................. 9 . Distribution and transport 80 ................................................................. . 10 Storage and expiry date 81
Part B . National control requirements ............................................................. 82 1 . General ........................................................................................... 82
................................................................ 2 . Release and certification 83 .................................... 3 . Clinical surveillance of the vaccine in man 84
.............................................................................................................. Authors 84 Acknowledgements ............................................................................................. 85 References .......................................................................................................... 86 Appendix 1 . Summary protocol for dried BCG vaccine production and
............................................................................................ testing 87
INTRODUCTION
The formulation of international requirements for the manufacture and control of BCG vaccine was first considered by the WHO Expert Committee on Biological Standardization in its thirteenth report (l) . In its fourteenth report (2). the Committee requested WHO to make arrangements as soon as possible for the formulation of such requirements . These requirements were approved by the WHO Expert Committee on Biological
Standardization at its eighteenth meeting and appeared as Annex 1 to its report (3).
Subsequently, a number of developments took place in the production and control of BCG vaccine. The majority of manufacturers switched to the production of freeze-dried vaccine. Much more became known about the production of freeze-dried vaccines from seed lots, which were shown to yield satisfactory vaccines, and consideration was given to the different routes and methods of administration. Furthermore, careful consideration was also given to the dose of living organisms that causes tuberculin sensitivity without giving rise to untoward reactions.
In the light of these developments, revised requ' irements were drafted in 1978, taking into account the regulations and requirements for manufacture and control of dried BCG vaccine in a number of countries, as well as information from published and unpublished reports. The 1978 revision of the requirements was approved by the WHO Expert Committee on Biological Standardization and appeared as Annex 2 in its thirtieth report (4).
Since then, further experience in production and control has been gained, which has made the current review necessary. It should be added, however, that the main substance of these requirements is still the same as that of the 1978 revision.
GENERAL CONSIDERATIONS
BCG vaccine has been used for many years. In spite of contradictory results obtained in field trials, there is considerable evidence that immunization with BCG vaccine can give a substantial degree of protection against tuberculosis, and that the inconveniences and risks associated with vaccination are very small (5).
Recently, increased emphasis has been given to vaccination as early in life as possible, and BCG vaccination has been included in the WHO Expanded Programme on Immunization.
The formulation of the original international requirements for dried BCG vaccine was complicated by the following: (a) a number of different daughter strains derived from the original strain of the bacillus of Calmette and Guerin are used in vaccine manufacture; (b) a number of different manufacturing and testing procedures are employed; (c) there is no approved laboratory method of assaying
the protective potency of vaccines against tuberculous infection in man; (4 vaccines with different total bacterial content and number of culturable particles are produced; and (e) vaccines intended for administration by different routes are prepared. Although much is known about the characteristics of a good vaccine, several of these difficulties remain.
Scope of these requirements
These revised requirements refer to dried BCG vaccine prepared from strains derived from the bacillus of Calmette and Gukrin. Where BCG vaccine is issued in liquid form, it is entirely under the responsibility of the National Control Authority. In that case, only the relevant parts of these requirements apply. These requirements have been formulated primarily to cover vaccines intended for intradermal and percutaneous administration.
The latest findings from controlled field trials have been taken into consideration in formulating these revised requirements, but there are still a number of characteristics needing further study. In particular, the maximum dose of viable organisms in the various vaccines that can be tolerated without producing an unacceptably high incidence of untoward reactions must be determined by careful studies in the various age groups. Guidance may be given by WHO on these matters, but the studies must be carried out in the communities in which the vaccines are to be used.
BCG strains
Various daughter strains, all derived from the bacillus of Calmette and Gukrin, are at present being used by manufacturing establishments in the preparation of BCG vaccine. While these strains differ from one another to minor degrees when investigated in the laboratory, there is no evidence to suggest that they differ significantly in their power to protect man. There is no strain that can be said to be preferable to others or that is known to be definitely superior to the others. Only seed lots that have been shown to be acceptable by laboratory and clinical tests should be used. Several such seed lots are available through WHO. It is believed that a suitable seed lot of BCG should yield vaccines that give protection in experimental animals, produce a relatively high level of tuberculin
sensitivity in human beings, and have an acceptable (low) frequency of untoward reactions.
Most of the early trials that showed BCG to be effective in man were carried out with liquid vaccines, but there is new strong circumstantial evidence that freeze-dried vaccines are similarly effective.
In view of these considerations, a major controlled trial of some of the most frequently used strains of BCG would be of value. The aim of this trial should be to re-establish the properties of seed lots that are suitable for the preparation of a satisfactory freeze-dried BCG vaccine. National laboratories and international organizations should take up this research problem, using vaccines prepared for administration to human bcings; vaccines prepared from seed lots for which there is littlc information concerning the safety and efficacy should be tested in man only with great caution.
Some manufacturers of dricd vaccine have modified their seed lot strain to make it morc suitablc for their particular production procedure. The seed lots prepared in this way may not retain the same immunogenic properties, and should be used only with the approval of the national control authority.
The selection of the seed lot should be governed by practical criteria relevant to the use of a reference preparation in the laboratory as well as in field studies.
In practice, BCG seed lots may generally be investigated in man only for their properties of producing tuberculin sensitivity and vaccination lesions. The former should be measured by the distribution of tuberculin reactions according to size in persons vaccinated with a given dose of BCG vaccine. A low dose of tuberculin should be employed (e.g., 5 IU of International Standard for Purified Protein Derivative (PPD) of Mammalian Tuberculin, or 2 tuberculin units (TU) of the batch of PPD RT23 with Tween 80).
Field observations should be made in conjunction with laboratory studies in animals. The latter should include protection tests, tests of vaccination lesions, and tests for tuberculin conversion. Immunizing efficacy should be measured in terms of degree of protection afforded to the test animals against a challenge with fully virulent Mycobacterium tuberculosis. Sensitizing efficacy should be measured by the average dose of vaccine that will convert a negative tuberculin reaction in guinea-pigs to a positive one, as well as by the speed with which such conversion is effected. In these animal tests, the inclusion, for comparative purposes, of a reference vaccine
prepared from a seed lot known to be effective in animals and man is recommended. Such a reference vaccine is available from WHO.
Potency tests
There is some evidence that BCG seed lots that have been shown to produce vaccines with protective potency in laboratory animals and tuberculin sensitivity in man will give effective protection against tuberculosis in human beings. However, at the present time there is no established laboratory test that can be used in practice to compare the protective efficacy of different vaccines.
The laboratory tests at present in use and included in these requirements are designed to ensure that new vaccine lots do not differ appreciably from those that have already been shown to be effective in man as regards their ability to induce adequate sensitivity to tuberculin, or from a reference vaccine shown to be safe and effective in man. At present, for batch control purposes, much reliance is placed on tests for the estimation of the total bacterial content and for the number of culturable particles. It is not possible to specify single requirements for the total bacterial content and for the number of culturable particles for all vaccines (6), since different seed lots and methods of manufacture may yield different values for these criteria. Although the number of live bacteria in one human dose may differ depending on the strain used for vaccine production, the vaccines have been shown to be similar and of satisfactory properties as regards their ability to induce adequate sensitivity to tuberculin and their safety in man. It is therefore essential that the national control authorities carry out extensive field trials in man and decide on suitable total bacterial contents and on the number of culturable particles for a particular manufacturer's product. The allowed range in the number of culturable particles should not vary over a range wider than 1-4. In addition, it is necessary to perform animal experiments that give an indication of the safety and efficacy of the vaccines to the satisfaction of the national control authority.
Post-vaccination reactions and complications
Vaccines intended for intradermal injection should be given strictly intradermally and vaccinators should be trained accordingly.
A local reaction at the vaccination site is normal after a BCG vaccination. It may take the form of a nodule, which in many cases
will break down and suppurate. The reaction developing at the vaccination site usually subsides w i t h 2-5 months and in practically all children leaves a superficial scar of 2-10 mm in diameter. The nodule may persist and ulcerate. Swelling of regional lymph nodes may also be seen, and this may be regarded as a normal reaction. Keloid and lupoid reactions may occur at the site of the vaccination. Children with such reactions should not be revaccinated.
Inadvertent subcutaneous injections produce abscess formations and may lead to ugly retracted scars.
Among the major complications, suppurative lymphadenitis has been observed. In the case of certain vaccines, it has been revealed that there is a strong correlation between the incidence of these complications in newborns and the number of culturable particles in the vaccine. Thus, a reduction of the dose for the newborn may reduce these complications to acceptable levels. It is recommended that the dose for babies should be one-half to one-quarter of that for teenage children or adults. It is required that the concentration of the vaccine should be shown to be effective and tolerated in the age groups for which the vaccine is intended (7).
The national health authority should issue guidelines for the treatment of complications.
Presentation and use of the requirements
Each of the following sections constitutes a recommendation. The parts of each section that are printed in normal-size type have been written in the form of requirements. so that. if a health administration so desires, these parts as they appear may be used as definitive national requirements. The parts of each section that are printed in small type concern points on which comments seem desirable.
Should individual countries wish to adopt these requirements as the basis of their national regulations concerning BCG vaccine, it is recommended that a clause should be included permitting modifications of the manufacturing requirements on condition that such modified requirements ensure that the degree of safety and the potency of the vaccine are at least equal to those provided by the requirements formulated below. The World Health Organization should then be informed of the action taken.
l ~ The terms "national control authority" and "national control laboratory", as used in these requirements, always refer to the country in which the vaccine is manufactured.
l PART A: MANUFACTURING REQUIREMENTS
1. DEFINITION
1.1 International name and proper name
The international name shall be "Vaccinum Tuberculosis (BCG) Cryodesiccatum". The proper name shall be the equivalent of the international name in the language of the country of origin.
The use of the international name should be limited to vaccines that satisfy the requirements formulated below.
1.2 Descriptive definition
Vaccinum Tuberculosis (BCG) Cryodesiccatum is a dried preparation containing live bacteria derived from a culture of the bacillus of Calmette and GuCrin, known as BCG, intended for intradermal injection. The preparation shall satisfy all the requirements formulated below. The name of the dried vaccine intended for percutaneous vaccination, shall be "Vaccinum Tuberculosis (BCG) Cryodesiccatum Percutaneum".
Vaccinum Tuberculosis (BCG) Cryodesiccatum is referred to in this document as "dried BCG vaccine".
1.3 International standard and international reference preparation An International Reference Preparation of BCG Vaccine and
an International Standard for Purified Protein Derivative (PPD) of Mammalian Tuberculin have been established. These preparations are in the custody of the International Laboratory for Biological Standards, State Serum Institute, Copenhagen, and samples are distributed on request to national control laboratories. They are intended to be used for the calibration of the respective national or manufacturers' standard materials.
1.4 Terminology
Seed lot: A quantity of bacteria processed together and of uniform composition. A seed lot shall be maintained in the dried form. In each manufacturing establishment, a primary seed lot is that from which material is drawn for inoculating media for the prcparation of secondary seed lots or single harvests.
Secondury seed lot: A working seed lot, prepared from the primary seed lot by as few cultural passages as possible, having the same characteristics as the primary seed lot and intended for inoculating media for the preparation of single harvests.
Single har~lests: A quantity of bacteria harvested from cultures prepared in a single batch of medium and processed together. Single harvests shall be prepared from cultures originating from a seed lot by as few cultural passages as possible, and by not more than 12 passages from the primary seed lot.
Final bulk: The homogeneous finished vaccine present in a single container. It may then be filled either directly or through one or more intermediate containers.
Vaccine lot (vaccine batch): Vaccine in final containers from a single final bulk, subsequently processed together and having therefore, a uniform composition before drying.
Filling lot: A collection of filled final containers that have been filled during one working session from a single final bulk and that are homogeneous with respect to the risk of contamination during filling.
Final lot: A final lot is a collection of sealed final containers from one filling lot, all of which have been dried together in the same vacuum chamber.
2. GENERAL MANUFACTURING REQUIREMENTS
The general manufacturing requirements contained in the revised Requirements for Biological Substances No. l (General Requirements for Manufacturing Establishments and Control Laboratories) (8) shall apply to establishments manufacturing BCG vaccine, with the addition of the following.
Production of BCG vaccine shall take place in completely separate areas, by means of separate equipment. Such areas shall be so situated and ventilated that the hazard of contamination is reduced to a minimum. No animals shall be permitted in the vaccine
production areas. Tests for the control of the vaccine that require cultures to be made of contaminating microorganisms shall be carried out in a completely separate area. If cultures other than for BCG production are seeded in the production area they should be removed immediately for incubation in another area. Tests in which animals are used shall also be carried out in a completely separate area.
For the purposes of these requirements, the processes of vaccine production that must take place in isolated facilities are all operations up to and including the sealing of the vaccine in the final containers. The packaging may be done in a common area.
In some countries, the production of BCG vaccine-although isolated-is carried out in a building in which other work takes place. This should be done only after consultation with, and with the approval of, the national control authority. If production takes place in part of a building, the work carried on in other parts of the building should be of such a nature that there is no possibility of hazard to the BCG vaccine.
No cultures of microorganisms other than strains of BCG approved by the national control authority for vaccine production shall be introduced into the manufacturing areas. In particular, no strains of other mycobacterial species, whether pathogenic or not, shall be permitted in the BCG vaccine production area.
BCG is susceptible to sunlight. Therefore, the procedures for the preparation of the vaccine shall be so designed that all cultures and vaccines are protected from daylight and ultraviolet light at all stages of manufacture, testing, and storage, until the vaccine is issued.
BCG vaccine shall be produced by a staff consisting of healthy persons who do not work with other infectious agents; in particular, they shall not work with virulent strains of M. tuberculosis, nor shall they be exposed to a known risk of tuberculous infection. Precautions shall be taken also to ensure that no worker shall be employed in the preparation of BCG vaccine unless he or she has been shown by medical examination to be free from tuberculosis. The scope and nature of the medical examination shall be at the discretion of the national control authority, but it shall include a radiological examination and shall be repeated at intervals or when there is reason to suspect illness.
The frequency with which the radiological examination should be carried out is at the discretion of the national control authority. It is advisable to keep radiation exposure to a minimum, but the examination should be of suficlent frequency
to detect the appearance of early active tuberculosis. It is estimated that, if workers in BCG vaccine laboratories were given one or two conventional X-ray examinations of the chest each year, not using fluoroscopic methods, and if the best available techniques were employed to minimize the radiation dose, the doses received would be considerably lower than the maximum permissible doses for workers occupationally exposed to radiation that have been set by the International Commission on Radiological Protection (9, 10).
Should an examination reveal signs of tuberculosis or suspected tuberculosis in a worker, he or she shall no longer be allowed to work in the production areas and the rest of the staff shall be examined for possible tuberculous infection. In addition, all cultures1 shall be discarded and the production areas decontaminated. If it is confirmed that the worker has tuberculosis, all vaccine made while he or she was in the production areas shall be discarded.
Suitable clothing shall be worn in the production areas. Persons not normally employed in the production areas shall be
excluded from them unless, after a medical examination, including radiological examination, they are shown to be free from tuberculosis. In particular, persons working with mycobacteria other than the BCG seed strain shall be excluded at all times.
Written descriptions of procedures for the preparation of BCG vaccine shall be submitted for approval to the national control authority. Proposals for modification shall be submitted for approval to the national control authority before their implementation.
3. PRODUCTION CONTROL
3.1 Control of source materials
3.1.1 Seed lot system
The production of vaccine shall be based on the seed lot system (11). A seed lot prepared from a strain approved by the national control authority (see Part B, section 1.1) shall be prepared under conditions satisfying the requirements of Part A, sections 2 and 3.
l In this respect, the freeze-dried seed, in ampoules sealed by fusion of the glass, is not regarded as a culture.
A seed lot should have been shown in field trials in man to induce adequate sensitivity to tuberculin and to be safe; also, it should have been tested for ability to produce protection against tuberculous infection in animals.
It is recommended that a large primary seed lot should be set aside under optimum storage conditions as the basic material to which the manufacturer may return for the preparation of further seed lots. This primary seed lot may be part of a vaccine lot that has been shown in extensive field trials in man to induce adequate sensitivity to tuberculin, with only a low frequency of untoward effects, and, in laboratory experiments in animals, to protect against tuberculous infection.
If a secondary seed lot is being used, the total number of passages for a single production harvest should not exceed 12. For example, if the secondary seed lot is four culture passages removed from the primary seed lot, the number of passages from the secondary seed lot must not exceed eight.
3.1.2 Tests on seed lot
The seed lot used for the production of vaccine shall be tested according to the requirements of Part A, sections 5.1, 5.2, and 5.3. However, the test for absence of virulent mycobacteria, described in Part A, section 3.4.3, shall be made in at least 10 guinea-pigs. The dose of the seed lot to be used in this test shall contain a number of culturable particles equivalent to that injected when a vaccine lot is tested. The guinea-pigs shall be observed for at least 6 months. At the end of the observation period, the animals shall be sacrificed and examined post-mortem for macroscopic evidence of progressive tuberculous disease; similarly, any animals that die before the end of the observation period shall be subjected to post-mortem examination. If none of the animals shows signs of progressive tuberculosis and at least 60% of them survive the observation period, the seed lot shall be considered to be free from virulent mycobacteria.
In addition, it is advisable to test all primary seed lots for protective potency in animals. In such tests, the material, when injected into guinea-pigs and mice in a range of doses under reproducible conditions, should afford a measure of protection, against a range of challenge doses of a fully virulent human strain of M. tuberculosis, not less than that specified by the national control authority in terms of a national reference preparation such as the one described in Part A, section 1.3.
In some countries, an additional test is included to ensure that the first batch of vaccine produced from a new ampoule of the seed lot gives rise to tuberculin sensitivity in guinea-pigs. It is desirable also that each seed lot be tested to demonstrate that its degree of sensitivity to antituberculosis drugs has not changed.
3.1.3 Production culture medium
The production culture medium shall contain no substances known to cause toxic or allergic reactions in human beings. Substances used in that medium shall meet such specifications as the national control authority may prescribe.
3.2 Production precautions
The general precautions formulated in the requirements of Part A, section 3, of the revised Requirements for Biological Substances No. l (General Requirements for Manufacturing Establishments and Control Laboratories) (8) shall apply to the manufacture of BCG vaccine, with the addition of the following.
Effective precautions shall be taken to prevent the accidental mixing of containers from separate lots of vaccine at all stages of production up to the stage at which final lots are labelled.
3.3 Control of single harvests
The cultures in each container shall be examined visually, and any that have grown in an uncharacteristic manner shall not be used for vaccine production.
3.4 Control of final bulk
3.4.1 Final bulk
The final bulk shall be prepared from a single harvest or by pooling a number of single harvests.
3.4.2 Test for absence of contaminating microorganisms
The final bulk shall be tested for bacterial and mycotic contamination in accordance with the requirements given in Part A,
section 5, of Requirements for Biological Substances No. 6 (General Requirements for the Sterility of Biological Substances) (12), and no vaccine lot shall be passed for use unless the final bulk has been shown to be free from such contamination.
3.4.3 Test for absence of virulent nzycobacteria
A sample of the final bulk intended for this test shall be stored at 4 "C for not more than 72 hours after harvest at 4 "C.
At least six guinea-pigs, all of the same sex, each weighing between 250 and 400 g are used.
Some countries prefer to use guinea-pigs that have been shown previously to be tuberculin-negative.
A dose of BCG organisms corresponding to at least 50 human doses of vaccine intended for intradermal injection shall be injected into each guinea-pig by the subcutaneous or intramuscular route.' The animals shall have been maintained on a diet that is free from added substances, such as antibiotics, that might interfere with the test. The guinea-pigs shall be observed for at least 6 weeks. If, during that time, they remain healthy and gain weight, the final bulk shall be considered to be free from virulent mycobacteria.
At the end of the observation period, the animals shall be sacrificed and examined post-mortem for macroscopic evidence of progressive tuberculous disease; similarly, any animals that die before the end of the observation period shall be subjected to a post- mortem examination.
The vaccine lot shall pass the test if none of the guinea-pigs shows evidence of progressive tuberculous disease and if at least two-thirds of them survive the observation period.
Should more than one-third of the guinea-pigs die during the observation period (and freedom from progressive tuberculous disease is verified), the test shall be repeated on at least 6 more guinea-pigs.
If, on the second occasion, more than one-third of the animals fail to survive the observation period, the vaccine lot shall not pass the test.
When a more concentrated vaccine, intended for administration by the percutaneous route, is tested, a dilution factor approved by the national control authority shall be applied so that the mass of BCG injected corresponds to 50 human doses of intradermal vaccine.
Should a vaccine lot fail to satisfy the requirements of this test because animals die from causes other than tuberculosis, the procedure to be followed bj- the manufacturer should be determined with the approval of the national control authority.
If evidence of progressive tuberculous disease is seen, the vaccine lot shall be rejected, all subsequent vaccine lots shall be withheld, and all current vaccine stocks shall be held pending further investigation. The manufacture of BCG vaccine shall be discontinued and it shall not be resumed until a thorough investigation has been made and the cause or causes of the failure determined. Production shall be allowed to resume only upon the approval of the national control authority.
3.4.4 Test ,for bacterial concentration
The bacterial concentration of the final bulk shall be estimated by a method approved by the national control authority and shall havc a value within a range-approved by the national control authority (see Part B, section 1.2).
The estimation of total bacterial content may be made either directly, by determining the dry weight of the organisms, or indirectly, by an opacity method that has been calibrated in relation to the dry weight of the organisms.
It is desirable that only one method of estimation be used for all the vaccine lots produced by a manufacturer.
If an opacity method is used, the International Reference Preparation of Opacity,' or an equivalent reference preparation approved by the national control authority, may be employed in comparative tests.
3.4.5 Test .for number of culturable particles
The number of culturable particles of each final bulk shall be determined by an appropriate method approved by the national control authority.
The medium used in this test should be such that the number of culturable particles may be determined not more than 4 weeks after the medium has been inoculated with dilutions of the vaccine.
l The International Reference Preparation of Opacity is in the custody of the International Laboratory for Biological Standards, National Institute for Biological Standards and Control, Hampstead, London NW3 6RB, England, which supplies samples on request.
There are various methods of determining the number of culturable particles in BCG vaccine, and it is desirable that only one method be used for all the vaccine lots produccd by a manufacturer (6). It is also dcsirablc that thc tcst bc carricd out in parallel with a national rcfcrcncc preparation prcparcd from a strain similar to that uscd for thc production ol' thc vaccinc.
3.4.6 Oxygen uptake test
Some manufacturers use a measurement of oxygen uptake rate (6) as a speedy test on the final bulk vaccine to get some indication of the viability of the batch.
Usually 3&120 mg (semi-dry weight) of the final bulk is tested in a Warburg apparatus, and the respiration rate is measured over an hour or more. Other methods of measuring oxygen uptake include polarographic methods.
3.4.7 Substances added to theJinal bulk
Substances used in preparing the final bulk, or added to it, shall meet such specifications as the national control authority may prescribe.
Substances added to improve the efficiency of the freeze-drying process or to aid the stability of the dried product shall be sterile and of high and consistent quality, and shall be used at suitable concentrations in the vaccine.
4. FILLING AND CONTAINERS
The requirements concerning filling and containers given in Part A, section 4, of the Requirements for Biological Substances No. 1 (General Requirements for Manufacturing Establishments and Control Laboratories) (8) shall apply, with the addition of the following.
It shall have been demonstrated to the satisfaction of the national control authority that the containers are made from materials that have no deleterious effect on the vaccine.
The containers should be in a form that renders the process of reconstitution as simple as possible. Their packaging should be such that the reconstituted vaccine is protected from daylight. It is desirable that the capacity of the final containers be large enough for the full volume of the reconstituting fluid to be added.
Single-dose and multipledose containers may be used; the latter should be of a type that will not encourage storage of the reconstituted vaccine. If vials are used, special attention should be paid to the requirements of Part A, section 8.
All containers of the final vaccine shall be sterilized before filling. Provision shall be made for adequate and continuous mixing of
the final bulk during filling into the final containers. As soon as possible after harvesting, the vaccine shall be filled into
containers and freeze-dried. Containers shall be hermetically sealed, either under vacuum or
after filling with pure (especially oxygen-free), dry nitrogen or any gas not deleterious to the vaccine. It shall be shown to the satisfaction of the national control authority that the vaccines stored under a gas have satisfactory stability.
All containers sealed under vacuum shall be tested for leaks not less than 2 months after sealing, and all defective containers shall be discarded.
In some countries, the test for leaks is repeated after further storage. The most convenient time to do this is immediately before distribution.
5. CONTROL TESTS ON FINAL LOT
Tests on the final lot shall be performed after reconstitution. The fluid supplied or recommended for reconstitution shall be used, unless such fluid would interfere with any of the tests, in which case some other suitable fluid shall be used. The vaccine shall be reconstituted to the concentration at which it is to be used for injection into human beings.
An exception may be made in the case of the test for absence of virulent mycobacteria (Part -4. section 5.3.1), when a higher concentration of reconstituted vaccine may be necessary.
5.1 Identity test
An identity test shall be performed on samples of the vaccine from each final lot.
The identity of each final lot of vaccine shall be verified by the morphological appearance of the bacilli in stained smears and by the characteristic appearance of the colonies grown on solid media.
5.2 Test for absence of contaminating microorganisms
Samples from each final lot shall be tested for bacterial and mycotic contamination according to the requirements given in Part A, section 5, of Requirements for Biological Substances No. 6 (Requirements for the Sterility of Biological Substances) (12).
5.3 Safety tests
5.3.1 Test for absence of virulent mycobacteria
If the test for the absence of virulent mycobacteria, applied to the final bulk, is unsatisfactory, it shall be repeated with a sample of a final lot (see Part A, section 3.4.3).
This test may be applied to each final lot and omitted for the final bulk.
5.3.2 Test for skin reactions in guinea-pigs
Some manufacturers include a test of the reactivity OS each vaccine lot when injected intradermally into guinca-pigs. Thc injections consist oS0.I ml ofundilutcd vaccinc' and 0.1 ml cach of vaccine dilutions 1 : 10 and 1 : 100. At Icast 4 guinca-pigs arc used, all of the same sex and wcighing not lcss than 250 g cach. If female guinea-pigs are uscd, lhcy should not bc prcgnanl.
The lesions produced are observed for at least 4 weeks. The reactions caused should be compared with those caused by a reference preparation approved by the national control authority and that has been shown to induce in man adequate sensitivity to tuberculin with a low frequency of untoward effects.
The same dilutions of the reference preparation and vaccine should be injected into the same guinea-pigs at randomly selected sites.
5.4 Test for total bacterial content
The total bacterial content of the reconstituted vaccine shall be estimated for each vaccine lot by a method approved by the national control authority, and shall have a value within a range approved by the national control authority (see Part B, section 1.2).
Percutaneous vaccine should be diluted (see Part A, section 3.4.3).
76
The estimation of total bacterial content may be made either directly. b!- determining the dry weight of organisms, or indirect],., by an opacity method that has been calibrated in relation to the dry weight of the organisms.
It is desirab!e that one method of estimation should be adhered to for all the vaccine lots produced by a manufacturer.
5.5 Test for viability
5.5.1 Test for number of culturable particles
The number of culturable particles of each final lot shall be determined by an appropriate method approved by the national control authority (see Part A, section 3.4.5). The viable count shall have a value within a range approved by the national control authority (see Part B, section 1.2). By comparison with the results of the test for number of culturable particles carried out on final bulk, as described in Part A , section 3.4.5, the percentage survival on freeze-drying may be calculated and this value should be not less than one approved by the national control authority.
Thc usc of a common culture medium may be necessary in ordcr to avoid discrepant results between laboratories.
Thc survival rate usually varies between 75% and 20%. Clumping occurring after reconstitution of vaccines may result in low survival rate, since it is likely that seeding of one clump of bacilli will give only one colony.
Care should be taken to avoid the presence of large aggregates in BCG vaccines.
5.5.2 Rapid test for viability
The bioluminescence reaction occurring in fireflies depends upon the presence of adenosine triphosphate (ATP), luciferin luciferase, oxygen, and magnesium ions. This reaction can be easily reproduced in vitro by mixing these components. If all components except ATP are present in excess, the amount of light emitted is proportional to the amount of ATP.
Since ATP is present in all living cells and is immediately destroyed when the cell dies. '4TP is a reliable marker for living cells.
Studies (13) haw shown that measurement of ATP using the bioluminescence reaction can be used to estimate the viable count of freeze-dried BCG vaccine within minutes, as accurately as other time-consuming methods, once the mean content of ATP per culturable particle has been estimated for a given vaccine strain.
5.6 Stability test
Each final lot shall be tested for its degree of stability by a method approved by the national control authority.
The test shall involve the determination of the number of culturable particles before and after the samples have been held at appropriate temperatures and for appropriate periods.
An accelerated degradation test shall be carried out by taking samples of the vaccine and incubating them at 37 "C for 28 days. The percentage decrease in the number of culturable particles is then compared with that of samples of the same vaccine stored at 4°C. The number of culturable particles in the vaccine after heating shall be not less than 20% of that in the refrigerated vaccine. The absolute value shall be approved by the national control laboratory. The same test shall also be performed with a reference preparation. One method of determining the number of culturable particles shall be adhered to, as suggested in Part A, section 3.4.5.
The purpose of including a reference preparation is to have a check on the quality of the medium used for the determination of the number of culturable particles. It is not intended to adjust the count of the vaccine by comparison with the reference preparation.
All manufacturers shall keep their product for the permitted storage period and shall determine the number of culturable particles from time to time to demonstrate that the number is being maintained at an adequate level.
In some countries, the stability test is carried out only after the vaccine has been stored for 3-4 weeks after drying, since it is considered that the degree of stability during the first 3 weeks may not be related to the long-term stability of the product.
As a guide to stability, some manufacturers of dried BCG vaccine determine the residual water content of the final vaccine, since failure to achieve a certain degree of desiccation results in an unstable product. However, such a test cannot be regarded as an alternative to tests involving the determination of the number of culturable particles.
5.7 Production consistency
The manufacturer shall show to the satisfaction of the national control authority that the method of manufacture ensures a consistent product.
The degree of consistency in producing satisfactory final lots is an important factor in judging the efficacy and safety of a particular manufacturer's product.
The data that shall be considered in determining the consistency of production shall include the results obtained with consecutive vaccine lots when tested as described in Part A, section 5, for example, by the test for total bacterial content (Part A, section 5.4), the test for number of culturable particles (Part A, section 5.9, and the stability test (Part A, section 5.6).
In addition, the examination of morphological appearance of the bacteria in stained smears (Part A, section 5.1) or in unstained microscopic preparations may be used to determine the consistency of bacterial aggregation.
A specified number of consecutive vaccine lots shall have been satisfactorily prepared before any vaccine from a given manufacturer, or resulting from a new method of manufacture, is released. In subsequent routine production, if a specified proportion of vaccine lots or a specified number of consecutive vaccine lot fails to meet the requirements, the manufacture of BCG vaccine shall be discontinued and not be resumed until a thorough investigation has been made and the cause or causes of the failures determined to the satisfaction of the national control authority.
6. RECORDS
The requirements given in Part A. section 6, of the revised Requirenze?zts for Biological Slibsta~zces ,Vo. 1 (General Reqztirements for Manufactztring Establish17te?zts and Control Laboratories) (8) shall apply, with the addition of the following:
Written records shall be kept of all seed lots. all cultures intended for vaccine production, all single harvests. all final bulk vaccines, and all vaccine in the final containers produced by the manufacturing establishments, including all tests irrespective of their results.
The records shall be of a type approved by the national control authority. An example of a suitable protocol is given in Appendix I, on pages 87-92.
7. SAMPLES
The requirements given in Part A, section 7, of the revised Requirements for Biological Substances No. l (General Requirements for Manufacturing Establishments and Control Laboratories) (8) shall apply.
It is desirable that samples should be retained for at least one year after the expiry date for all the vaccine in the final lot.
8. LABELLING
The requirements given in Part A, section 8 of the revised Requirements for Biological Substances No. l (General Requirements for Manufacturing Establishments and Control Laboratories) (8) shall apply, with the addition of the following:
The label printed on or affixed to each container shall show the volume and nature of the reconstituting fluid. Also, this label, or the label on the carton enclosing several final containers, or the leaflet accompanying the containers, shall contain the following additional information:
-the fact that the vaccine fulfils the requirements of this document;
- instructions for use of the vaccine and information concerning contraindications and the reactions that may follow vaccination;
-the conditions recommended during storage and transport, with information on the reduced stability of the vaccine if exposed to temperatures higher than that stated on the label;
- warnings that the vaccine must be protected from daylight and that, when reconstituted, the vaccine should be kept on ice; and
- a statement that, after a final container has been opened, the vaccine must be used as soon as possible, and that any opened container remaining at the end of the immunization session (maximum four hours) should be discarded.
9. DISTRIBUTION AND TRANSPORT
The requirements given in Part A, section 9, of the revised Requirements for Biological Substances No. 1 (General Requirements
for Manufacturing Establishments and Control Laboratories) (8) shall apply.
It is the responsibility of the manufacturer to use means of transport likely to maintain the potency of the vaccine.
10. STORAGE AND EXPIRY DATE
The statement concerning storage temperature and expiry dates, appearing on the label and leaflet as required in Part A, section 8, shall be based on experimental evidence obtained from the tests indicated in Part A, section 5.6, and shall be submitted for approval to the national control authority.
10.1 Storage conditions
Before being distributed by the manufacturing establishment, or before being issued from a depot for the storage of vaccine, all vaccincs in their final containers shall be kept constantly at a temperature below 8 "C and shall be protected from daylight.
Precautions should also be taken to maintain the vaccine, during transport and up to the time of use, at the temperature and under the storage conditions recommended by the manufacturer.
10.2 Expiry date
The date after which dried BCG vaccine may not be used shall be determined in relation to the experimental evidence referred to in Part A, section 5.6, and with the approval of the national control authority. The date shall be not more than 12 months after the date of issue, provided that this is not more than 24 months from the date of the last satisfactory test for culturable particles, referred to in Part A, section 5.5, and provided that the vaccine has been stored continuously at the specified storage temperature and protected from daylight. In any event, the expiry date shall be not more than 4 years from the date of harvest. Each manufacturer shall test the stability of the vaccine to ensure that it satisfies these conditions.
PART B. NATIONAL CONTROL REQUIREMENTS
1. GENERAL
The general requirements for control laboratories, given in Part B of the revised Requirements for Biological Substances No. l (General Requirements for Manufacturing Establishments and Control Laboratories) (8), shall apply.
In addition, the national control authority shall provide a reference vaccine (see Part A, section 1.3) or approve one used by a manufacturer, and shall give directions concerning the use of the reference vaccine in specified tests. The national control authority shall also give directions to manufacturers concerning the BCG strain to be used in vaccine production, the total quantity of bacteria, the number of culturable particles, and the stability required of the vaccine, and shall specify the requirements to be fulfilled by the manufacturer in accordance with the provisions of Part A of this document, including those for consistency of quality in respect of the points referred to in Part A, section 5.7.
1.1 BCG strain
The strain of BCG (maintained in the form of a seed lot) used in the production of vaccine shall be derived from the original strain maintained by Calmette and GuCrin and shall be identified by historical records. On the basis of cultures and biochemical and animal tests,l the BCG seed lot shall show characteristics that conform to those of BCG and generally differ from those of other mycobacteria. The seed lot shall show consistency in the morphological appearance of colonies on serial subculture. It shall also have been shown to yield vaccines that, upon administration by intradermal injection to children and adults, induce adequate sensitivity to tuberculin, with a low frequency of untoward effects. In addition, the seed lot shall have been shown to give adequate protection against tuberculosis in experimental animals in tests for protective potency such as are referred to in Part A, section 3.1.2.
l In investigating various BCG strains, it may be advisable to test their infectivity in animals, including hamsters.
1.2 Concentration of BCG vaccine
The concentration of BCG vaccine varies with different vaccines and is dependent on a number of factors, such as the strain of BCG used and the method of manufacture. It is therefore essential, for each manufacturer as well as for each different method of manufacture, for the optimum potency of vaccine to be ascertained by trials in tuberculin-negative subjects (newborn babies, older children, and adults) to determine the response to vaccination in respect of the induction of adequate sensitivity to tuberculin, the production of acceptable local skin lesions, and the occurrence of a low frequency of untoward reactions. As a result of such trials, the national control authority shall give directions to the manufacturer concerning the total bacterial content and the number of culturable particles required for the vaccine.
If a manufacturer changes his procedure of preparing BCG vaccine, and if the national control authority considers that the change might affect the final product, it may be necessary to conduct further trials in order to determine the optimum content of BCG organisms in the new product.
2. RELEASE AND CERTIFICATION1
A lot of dried BCG vaccine shall be released only if it fulfils Part A of the present requirements.
A statement signed by the appropriate official of the national control laboratory shall be provided at the request of the manufacturing establishment and shall certify that the lot of vaccine in question meets all national requirements as well as Part A of the present requirements. The certificate shall also state the date of the harvest, the date of the last satisfactory test for number of culturable particles, and the lot number. In addition, a copy of the official national release document shall be attached.
The purpose of the certificate is to facilitate the exchange of dried BCG vaccine between countries.
l Where there is no national control authority, a manufacturer may request advice and help from: Chief, Biologicals, World Health Organization, 1211 Geneva 27, Switzerland.
3. CLINICAL SURVEILLANCE OF THE VACCINE IN MAN
The national control authorities shall satisfy themselves that adequate control of BCG vaccine has been achieved, by arranging for studies in children to be made at regular intervals on some of the final lots prepared.
Such studies should be made, in at least 100 tuberculin- negative persons per year, and records should be obtained of the degree of sensitivity to tuberculin induced (distribution of tuberculin reactions by size)' with a defined dose of tuberc~l in,~ local skin lesions (nature and size of BCG vaccine reaction), and the occurrence of untoward vaccination reactions. It is desirable that such tests should be performed in parallel on two or more vaccine lots in the same population group, one of the vaccine lots being preferably a reference vaccine.
The frequency of testing batches will depend on the number of batches of vaccine produced, but, in any case, at least one batch each year should be tested. The age groups of children in whom the vaccine is tested should be the same as those in which the vaccine will be eventually used.
If a batch of vaccine is to be exported, it must be ascertained in which age group it will be used in the importing country; the vaccine should be then tested accordingly.
AUTHORS
The Requirements for Dried BCG Vuccine (Revised 1985) were prepared by the following WHO consultants and staff members:
Dr R. Donikian, Mtrieux Institute, Marcy I'Etoile, Charbonnitre les Bains, France Dr M. Gheorghiu, Chief, BCG Department, Pasteur Institute, Paris, France Dr T.B. Jablokova, Chief, Laboratory for Standardization and Control of BCG and
Tuberculin, Tarasevii: State Institute for the Standardization and Control of Medical Biological Preparations, Moscow, USSR
' In some countries, the proportion of cases showing a negative reaction to tuberculin before BCG vaccination, but giving a positive result after vaccination, is called the "tuberculin conversion rate". Unless positive and negative reactions are carefully defined, however, such a rate may not include certain cases in which a weak reaction to tuberculin is changed after BCG vaccination into a strong reaction.
An intradermal test with a dose of tuberculin equivalent to 5 IU of tuberculin PPD is suitable. A description of an appropriate method and a design for a study to assess BCG vaccines in man are available on application to Chief, Tuberculosis and Respiratory Infections, World Health Organization, 121 1 Geneva 27, Switzerland.
Miss A. Ladefoged? BCG Department, State Serum Institute, Copenhagen, Denmark Dr M. Leguenno, Chief, BCG Laboratory. Pasteur Institute, Dakar, Senegal Mr J. Lyng, Laboratory for Biological Standards, State Serum Institute,
Copenhagen, Denmark Dr A. Pio, Chief. Tuberculosis and Respiratory Infections, World Health
Organization, Geneva, Switzerland Dr T. Sawada, Japan BCG Laboratory, Tokyo, Japan Dr P. Sizaret, Scientist, Biolo@cals, World Health Organization, Geneva, Switzerland Mr H. ten Dam, Tuberculosis and Respiratory Infections, World Health
Organization, Geneva, Switzerland Mr D. Wright, Head, BCG Vaccine Unit, Glaxo Operations UK Ltd, Greenford,
England
ACKNOWLEDGEMENTS
Acknowledgement is due to the following experts for their comments and advice: Professor H. Brede, President, Paul Ehrlich Institute, Frankfurt-am-Main, Federal
Republic of Germany Professor M.P. Beumer, Ministry of Public Health and Family Affairs, Brussels,
Belgium Dr D. Calam, National Institute for Biological Standards and Control, London,
England Dr V.F. Davey, Kew, Victoria, Australia Dr I. Di Tommaso, Head, Tuscan Institute for Serotherapy and Vaccine Production
(SCLAVO), Siena, Italy Dr R. Dobbelaer, Chief Institute of Hygiene and Epidemiology, Brussels, Belgium Dr D. Draghici, Chief BCG Laboratory, Cantacuzino Institute, Bucharest, Romania Mme M. Dufaux, Industrial Pharmacist, Pasteur Institute of Brabant, Brussels,
Belgium Dr A. Engibarov, Chief. Laboratory for Specific Prophylaxis of Tuberculosis,
Medical Academy, Research Institute of Infectious and Parasitic Diseases, Sofia, Bulgaria.
Dr C. Guthrie, Operations Director. Commonwealth Serum Laboratories, Parkville, Victoria, Australia
Professor S. Hollan, Director, National Institute of Haematology and Blood Transfusion, Budapest, Hungary
Dr A. Lafontaine, Ministry of Public Health and Family Affairs, Brussels, Belgium Dr H. Lehmann, Behringwerke AG, Marburg, Federal Republic of Germany Dr P. Lemoine, Section Chief, Institute of Hygiene and Epidemiology, Brussels,
Belgium Dr L. Lugosi, Head, BCG Department, National Institute of Hygiene, Budapest,
Hungary Dr M. S. Nasution, Director, Pasteur Institute, Bandung, Indonesia Dr J. Nyabenda, Tuberculin Laboratory, Pasteur Institute of Brabant, Brussels,
Belgium Dr D.C. Pope, Assistant Director, Bureau of Biologies, Drugs Directorate, Virus
Building, Ottawa, Canada Dr W. Pranter, Behringwerke AG, Marburg, Federal Republic of Germany
Dr J. Pruchova, Head, Immunological Department, State Institute for the Control of Drugs, Prague, Czechoslovakia
Dr G. Reber, Behringwerke AG, Marburg, Federal Republic of Germany Professor L. Reichert, Department of Biochemistry, The Albany Medical College of
Union University, Albany, NY, USA Professor E. Ruitenberg, State Institute for Public Health and Environmental
Hygiene, Bilthoven, Netherlands Dr D. Rymkiewicz, Chief, Vaccines and Sera Control Department, National Institute
of Hygiene, Warsaw, Poland Professor G. Swaniker, Boroko, Papua New Guinea Mr A.J. Taylor, Central Veterinary Laboratory, Weybridge, England Dr J . van Ramshorst, Milsbeek, Netherlands
REFERENCES
1. WHO Technical Report Series, No. 187, 1960. 2. WHO Technical Report Series, No. 222, 1961. 3. WHO Technical Report Series, No. 329, 1966. 4. WHO Technical Report Series, No. 638, 1979. 5. WHO Technical Report Series, No. 195, 1960. 6. In vitro assays of BCG products. Unpublished working document, WHO/TB/
Technical guidel77.9, 1977. 7. WHO Technical Report Series, No. 552, 1974. 8. WHO Technical Report Series, No. 323, 1966. 9. INTERNATIONAL COMMISSION on RADIOLOGICAL PROTECTION. Report of Committee
II on permissible dosefor internal radiation. London, Pergamon Press, 1959 (ICRP Publication 2).
10. UNITED NATIONS SCIENTIFIC COMMITTEE on the EFFECT of ATOMIC RADIATION. Ionizing radiation: levels and effects. Report. New York, United Nations, 1972.
I l. Guld, J. Use of the seed lot system. International Symposium on BCG vaccine, Frankfurt 1970. Symposium Series Zmmunobiological Standard, 17, 143, Easel, Karger, 1972.
12. WHO Technical Report Series, No. 530, 1973. 13. GHEORGHIU M. ET AL. Methode rapide de contrBle sur le terrain de la viabilite
du vaccin BCG par dosage de I'ATP. Journal of biological standardization 12: 257-264, (1984).
Appendix 1
SUMMARY PROTOCOL FOR DRIED BCG VACCINE PRODUCTION AND TESTING
Based on Requirenzent.~ jbr Biological Substances No. 11. Rey uirements jbr Dried BCG Vaccine
(WHO Technical Report Series, No. 745, 1987)
Identification of Final Lot
Name and address or manuhclurcr
Lot no. and type of vaccine Intradermal/Percutaneous/Other
Date of manufacture of final 101
Seed Lot
1. Information on seed lot
Reference of seed lot
Origin of seed lot
Date of preparation of seed lot
Date of receipt of seed lot (if applicable)
Date of reconstitution of seed lot ampoule
2. Tests on seed lot (If these data have been submitted before, completion of this paragraph is not necessary.)
(a) Identity test
Has the seed lot been identified as BCG? yes/no
(b) Test for absence of contaminating microorganisms
Date of start of test
Date of end of test
Results
First test Repeat test if necessary
(c) Test for absence of virulent mycobacteria
Dose injected
No. of guinea-pigs given injection
Weight range of guinea-pigs
Observation period
Health of animals during test
Weight gains (losses)
Results (passedlfailed)
Seed lot approved
Date of approval
Information on Manufacture
1. Single harvest
No. of passages from primary seed
Medium
No. and volume of containers inoculated
Date of inoculation
Date of harvest
Results of visual inspection
2. Final bulk
Date of preparation
No. of single harvests included
First test Repeat Lest (a) Absence of co?~tcrr~ii~~ation if necessary
Quantity tested
Media
Duration of test Results
(b) Absence of virulent m?lcobacteria
No. of human doses injected per guinea-pig
No. of guinea-pigs given injection
Observation period
Health of animals during test
Weight gains (losses)
Results (passed/failed)
First test Repeat test if necessary
(c) Nature of substances added to final bulk andfinal concentration
3. Freeze-drying
Type and size of containers
No. of doses per container
Method used for sealing the vacuum-sealinglsealing under gas
containers
No. of containers in the final lot
Information on Final Product Controls
Recommended reconstitution fluid
Volume of reconstitution fluid per final container
1. Identity test
Type of test
Results
2. Absence of contamination
No. of containers tested
Media
Duration of test
Results
First test Repeat test i f necessary
3. Safety tests
(a) Absence of virulent mycobacteria First test Repeat test ( i f test not performed on final bulk) i f necessary
No. of human doses injected per guinea-pig
Dilution factor applied if percutaneous vaccine
No. of guinea-pigs
Observation period
Health of animals during test
Weight gains (losses)
Results (passedlfailed)
(b) Skin reaction test
Number of guinea-pigs
Dilutions injected
Observation period
Mean diameter of lesions (for each dilution)
4. Total bacterial content
Method of estimation Result (per ml)
5. Tests for viability
Vaccine Reference
(a) Culturable particles
Medium
Before After lyophilization lyophilizution
No. of containers tested
Mean count of culturable particles per ml
Mean survival rate (%)
(b) ATP content (optional)
Mean survival rate (%j
6. Test of thermostability Vials
Unheated Heated
No. of containers tested
Culturable particles in each container per m1
Mean survival (%)
Information on Release
Is the batch satisfactory?
Has the lot been released by the national control authority?
If yes, date
Has a certificate been supplied by the national control laboratory? yes/no
Name and signature of head of laboratory
Certification by person taking overall responsibility for production of the vaccine:
I certify that lot No. of BCG X-accine statisfies Part A of the WHO Requirements for BCG Vaccine.
Date
Signature
Name typed
The protocol must be accompanied by a sample of the label, a copy of the leaflet, and if the vaccine is to be exported, by a copy of the national control release certificate.
Information on the Manufacturer's product
When was the vaccine last tested in humans? Summary of results:
No. of children examined
Age group
Vaccination-testing interval
Mean postvaccination tuberculin reaction
Mean vaccination lesion size
Mean percentage of lymphadenitis
Annex 3
REQUIREMENTS FOR CONTINUOUS CELL LINES USED FOR BIOLOGICALS PRODUCTION
(Requirements for Biological Substances No. 37)
Page ....................................................................................................... Introduction 93
....................................................................................... General considerations 94 Part A. Manufacturing requirements ................................................................. 96 . . .................................................................................... 1. Defin~tlons 96
2. General manufacturing requirements .......................................... 97 3. Identification and characteristics of cell line ............................... 97
3.1 Cell seed and manufacturer's working cell bank (MWCB) ... 97 3.2 Tests for adventitious agents ................................................. 98
......................................................... 3.3 Tests for tumorigenicity 100 3.4 Identity test ............................................................................ 101
Authors .............................................................................................................. 102 Acknowledgen~ents ......................................................................................... 102 References .......................................................................................................... 103 Appendix I. Summary protocol for continuous cell lines used for biologicals
production .................................................................................... 103
INTRODUCTION
Since WHO requirements for continuous cell lines used as substrates in the production of inactivated virus vaccines were established in 1983 as part of Requiren~ents for Poliomyelitis Vaccine (Inactivated) (1). there have been several technical advances concerning the characterization of continuous cell lines. In addition, more experience has been gained in the use of continuous cell lines for the preparation of inactivated vaccines and as substrates for the preparation of biological substances by recombinant DNA techniques. Consequently. there is a need to provide general requirements for the characterization and use of continuous cell lines for the preparation of inacti~ ated iral I acclnes and other biological substances for use in man. Spec~fic requirements for individual products will be developed or revised as appropriate and it will be particularly important to consult WHO requirements for individual products .for details on quality control procedures related to cell cultures.
The Requirements presented in this document are intended to supersede Part D of Requirements for Poliomyelitis Vaccine (Inactivated) (l).
GENERAL CONSIDERATIONS
Several types of cell have been employed as substrates for the preparation of inactivated viral vaccines. These include: (a) cells derived directly by primary culture (or by a low number of passages) of normal animal tissues (e.g., monkey kidney and rabbit kidney cells); (b) diploid cell lines of human and monkey origin (e.g., WI38, MRCS and FRhL-2 cells) which have a finite capacity for serial propagation; and (c) continuous cell lines (e.g., VERO and C H 0 cells). Diploid cell lines have certain disadvantages for the production of some biologicals in that their growth characteristics make them difficult to adapt to large-scale industrial production. Moreover, they have a finite life span and may produce relatively low yields of some vaccine viruses or other biological products.
Many current approaches to the development of inactivated viral vaccines employing modifications of conventional methods or new biotechniques, including recombinant DNA methods and controlled gene expression, involve the use of continuous cell lines. The advantage of such cell lines is that they grow relatively rapidly, providing high yields of monolayer, or, in some cases, suspension cultures.
Continuous cell lines may have biochemical, biological, and genetic abnormalities. In particular, they may produce transforming proteins and may contain viruses and potentially oncogenic DNA. In some cases, the cells may cause tumours when inoculated into animals. It is therefore important to ensure that biological products derived from continuous cell lines are highly purified. Generally, the purification procedures will result in the extensive removal of cellular DNA, other cellular components, and adventitious agents. Procedures that extensively degrade or denature DNA might also be considered.
The data required for the characterization of any continuous cell line to be used for production of biologicals include: (a) information on its origin, derivation and passage history; (b) information on its growth and morphological characteristics; (c) results of tests for adventitious agents; (d) distinguishing features, such as biochemical,
immunological and cytogenetic patterns which allow the cells to be clearly recognized among other cell lines; and (e ) results of tests for tumorigenicity, if this information is not already known.
Special considerations for control will apply to products derived from cells that contain known viral genomes (e.g., Namalva cells) or from cells modified by recombinant DNA technology.
It is important to establish a complete characterization of the continuous cell line so that appropriate tests for purity of the final product can be included. For example, if a cell line contains an endogenous virus, tests to ensure the absence of biologically active virus could be incorporated as one of the requirements for products derived from that cell line.
There has been considerable discussion internationally on general criteria for the acceptability of products (e.g., hormones, blood products, inactivated viral vaccines) prepared from transformed mammalian cells. A consensus has emerged on the desirability of achieving a high degree of purification of the product involving extensive removal or destruction of DNA of cell substrate origin. Manufacturers considering the use of transformed cells should be aware of the need to develop and authenticate efficient methods for the purification of the product and to establish sensitive methods for detection of cellular DNA as an essential element of any product development programme.
If transformed cells are being considered for the preparation of experimental live vaccines, careful consideration must be given to factors such as the possible incorporation of cellular DNA into the virions; considerable discussion will be required to assess the acceptability of such products.
Specific requirements for purity as well as other quality control procedures will be incorporated in WHO requirements for the individual products.
Each of the following sections constitutes a recommendation. The parts of each section that are printed in normal type have been written in the form of requirements so that, if a health administration so desires, these parts as they appear may be included in definitive national requirements. The parts of each section that are printed in small type are comments and recommend. tions for guidance.
Should individual countries wish to adopt these requirements as the basis of their national regulations concerning the use of continuous cell lines in the preparation of biological products, it is
recommended that a clause should be included permitting modifications of manufacturing requirements on the condition that it can be demonstrated, to the satisfaction of the national control authority, that such modified requirements ensure that the degree of characterization of the continuous cell line is at least equal to those provided by the requirements formulated below. The World Health Organization should then be informed of the action taken.
The terms "national control authority" and "national control laboratory", as used in these requirements, always refer to the country in which the vaccine or other biological is manufactured.
PART A. MANUFACTURING REQUIREMENTS
1. DEFINITIONS
Cell seed: A quantity of cells of human or animal origin stored frozen at - 70 "C or below in aliquots of uniform composition, one or more of which would be used for the production of a manufacturer's working cell bank.
Manufacturer's working cell bank ( M WCB): A quantity of cells derived from one or more ampoules of the cell seed and of uniform composition, stored frozen at - 70 "C or below in aliquots, one or more of which would be used for production purposes.
In normal practice a cell seed is expanded by serial sub- culture up to a passage number (or population doubling, as appropriate) selected by the manufacturer, at which point the cells are combined into one pool and preserved cryogenically to form the MWCB. One or more of the ampoules from such a pool would be used for production purposes.
Production cell culture: A collection of cell cultures being used for biological production that have been derived from one or more ampoules of the MWCB.
Adventitious agents: Contaminating microorganisms of the cell line, including bacteria, fungi, mycoplasmas, and endogenous and exogenous viruses.
2. GENERAL MAKUFACTURIhG REQUIREMENTS
The general requirements contained in the revised Requirements for Biological Slrbstances -Vo. l iGenernl Requirements for Manufacturing Establislunents and Control Laboratories) (2, page l l ) shall apply but with the provision that during the production of a given biological no cell cultures other than those approved by the national control authority for the production of the appropriate biological shall be introduced or handled in the production area.
3. IDENTIFICATION AND CHARACTERISTICS O F CELL LINE
3.1 Cell seed and manufacturer's working cell bank (MWCB)
The use of continuous cell lines for the manufacture of biological products shall be based on the cell seed system. A continuous cell line shall be subcultured to a point at which it is convenient to prepare a cell seed. Thc passage level, or population doubling, of the cell seed should bc as low as possible.
Thc ccll sccd used for the production of biologicals shall be that approvcd by and registered with the national control authority. The continuous cell line from which the cell seed and the MWCB have been derived shall have been characterized with respect to genealogy and growth characteristics at passage levels (or population doublings, as appropriate) equivalent to those of the cell seed, MWCB, and the cell cultures used for production. In addition, information on specific immunological markers and storage conditions will be of value. Also the cell line shall have been tested in animals, eggs, and cell culture for detectable adventitious agents. Virus susceptibility and capacity for interferon production may be other useful markers for cell identification. In addition, cytogenetic data may be useful for cell identification as well as for assessing the genetic stability of the cell line used for production purposes. However, a complete cytogenetic characterization of the cell line is not recommended because of the limited usefulness of such information.
These data shall be made available to the national control authority.
3.2 Tests for adventitious agents
Tests in the following sections are intended to identify any endogenous or exogenous agents that may be present in the cell line. Special attention should be given to tests for agents known to be present in a latent state in the species from which the cells were derived (e.g., SV-40 virus in rhesus monkeys).
3.2.1 Tests in animals and eggs
The tests in animals and eggs for adventitious agents that can induce pathology shall include the inoculation by the intramuscular route of each of the following groups of animals with cells from the MWCB propagated to or beyond the maximum passage level (or population doubling, as appropriate) used for production, using at least 107 viable cells divided equally among the animals in each group:
- 2 litters of suckling mice, comprising at least 10 animals, less than 24 h old;
- 10 adult mice; - 5 guinea-pigs; and - 5 rabbits.
The test in rabbits for the presence of B virus in the cell line may be replaced by a test in rabbit kidney cell cultures.
At least 106 viable cells shall also be injected into the allantoic cavity, the chorioallantoic membrane, and yolk sac of each of 10 embryonated chicken eggs 9-1 1 days old.
Also at least 106 viable cells shall be injected intracerebrally into each of 10 adult mice to test for the presence of lymphocytic choriomeningitis virus.
The animals shall be observed for at least 4 weeks and the embryonated chicken eggs shall be examined after not less than 5 days of incubation. Any animals that are sick or show any abnormality shall be investigated to establish the cause of illness. The allantoic fluids shall be tested with guinea-pig and chick or other avian red cells for the presence of haernagglutinins.
The cells are suitable for production if at least 80% of the animals or eggs inoculated with the cells remain healthy and survive the observation period and none of the animals or eggs shows evidence of the presence in the cell cultures of any adventitious agent.
3.2.2 Sterility tests Tests for bacteria, fungi and mycoplasmas shall be performed
according to the Gerzercrl Requirements for tlze Sterility of Biological Substances (3).
3.2.3 Morphological tests
The cells from the M WCB propagated to the maximum passage level used for production, or a level beyond it, shall be examined and characterized by light and clcctron microscopy to establish their morphological features. Comparisons of these data with those derived from cells of a passagc Icvel close to that of the MWCB will be of value in assessing the stability of the cell line and in determining the presence of potential microbial contaminants.
The cells, propagated to at least 10 population doublings beyond the maximum level used for production, shall also be tested for the presence of endogenous viruses. Suitable methods include induction with agents such as idoxuridine (IUDR) and the examination of electron micrographs for virus particles.
3.2.4 Tests on cell cultures
(1) Intact cells should be cultivated along with a range of other cell systems, including cells derived from man, and examined for morphological changes. Disrupted cells should be inoculated onto a similar set of cell culture systems and examined as above.
(2) Cell culture fluids should be examined for the presence of cytopathic agents in cell cultures of various species, including human cells, and at the end of the observation period the cell culture fluids should be tested for haemagglutinating viruses.
3.2.5 Tests for retroviruses
In addition to the morphological tests described in section 3.2.3, assays for viral reverse transcriptase should be performed on cells that have been induced with chemicals such as idoxuridine (IUDR).
3.2.6 Other tests
Additional studies may be required to characterize and identify the adventitious agents found in any of the tests required in section 3.2.
3.3 Tests for tumorigenicity
3.3.1 In vivo tests
Cells from the MWCB, propagated to a t least 10 population doublings beyond the maximum passage level used for production, shall be examined for tumorigenicity in a test approved by the national control authority. The test should involve a comparison between the continuous cell line and a suitable positive control reference preparation (e.g., HeLa or Hep 2 or FL cells).l A negative control is not essential but desirable. For that purpose non- tumorigenic diploid cell lines such as WI-38 or MRC-5 may be used.
Animal systems that have been shown to be suitable for this test include:
(a) athymic mice (Nu/Nu genotype); or (b) newborn mice, rats or hamsters that have been treated with
antithymocyte serum or globulin; or (c) thymectomized and irradiated mice that have been
reconstituted (T-, B +) with bone marrow from healthy mice. Whichever animal system is selected, the cell line and the reference
cells are injected into separate groups of 10 animals each. In both cases, the inoculum for each animal is 107 cells suspended in a volume of 0.2m1, and the injection may be by either the intramuscular or subcutaneous route. In the case of newborn animals (system b) the animals are treated with 0.1 m1 of antithymocyte serum or globulin on days 0, 2, 7, and 14 after birth. A potent serum or globulin is one that suppresses the immune mechanisms of the growing animals to the extent that the subsequent inoculum of 107 positive reference cells regularly produces tumours and metastases. Animals in all tests are observed for 21 days for evidence of nodule formation at the site of injection. If nodules appear, they are measured in two perpendicular dimensions, the measurements being recorded regularly to determine whether there is progressive growth of the nodule.
At the end of the observation period all animals, including the reference group(s), are sacrificed and examined for gross and microscopic evidence of the proliferation of inoculated cells at the
l Information concerning the sources of suitable positive control reference cells may be obtained from Chief, Biologicals, World Health Organization, 121 1 Geneva 27, Switzerland.
site of injection and in cther organs (e.g., lymph nodes, lungs, kidneys, and liver).
In a valid test, progressively growing tumours should be produced in at least 9 of 10 animals injected with the positive reference cells.
If the continuous cell line has already been documented to be tumorigenic. or if the class of cells to which it belongs (e.g., hybridomas) is tumorigenic, the national control authority may not require additional tumorigenicity tests.
Tests for the oncogenic potential of cellular DNA and crude cell lysates in animal systems may also be useful in establishing the basic characteristics of the cells.
3.3.2 In vitro tests
Two in vitro tests that have been found to correlate well with tumorigenicity are: (a) colony formation in soft agar gels; and (b) production of invasive cell growth following inoculation onto organ cu1tures.l These may be used to characterize more fully the cell lines that show no evidence of tumorigenicity in animal tests (section 3.3.1) or when the results are equivocal.
In vitro tests may be considered sufficient to characterize the cells for tumorigenicity by some national control authorities.
A matter of particular concern is the possibility that the cells used in the production of biologicals may contain activated oncogenes. For that reason, assays of cell transformation with DNA derived from the cell line (at the passage level used for production) should be performed in order to determine whether or not activated oncogenes can be detected in the continuous cell line. The standard 3T3 assay system should be used for these tests; but additional tests should also be considered as new techniques are developed for the detection of a broader range of oncogenes.
3.4 Identity test
The MWCB shall be identified by a method approved by the national control authority. Methods for identity testing include biochemical (e.g., isoenzyme analyses), immunological (e.g., histocompatibility antigen assays), and cytogenetic marker tests.
l Information concerning details of these test procedures can be obtained from Chief, Biologicals, World Health Organization, 121 1 Geneva 27, Switzerland.
AUTHORS
The Requirements for Continuous Cell Lines usedfor Biologicals Production were formulated by the following WHO consultants and staff members:
Dr F. Assaad, Director, Division of Communicable Diseases, WHO, Geneva, Switzerland
Dr J. Furesz, Director, Bureau of Biologics. Drug Directorate, Ottawa, Canada (Consultant)
Dr V. Grachev, Scientist, Biologicals, WHO, Geneva, Switzerland Professor F. Horaud, Medical Virology, Pasteur Institute, Paris, France (Consultant) Professor L.O. Kallings, Director, National Bacteriological Laboratory, Stockholm,
Sweden (Consultant) Dr V.R. Kalyanaraman, Director, Pasteur Institute of India, Coonoor, India
(Consultant) Dr B. Montagnon, Merieux Institute, Charbonnieres les Bains, France (Consultant) Dr R. Netter, Director-General, National Laboratory of Health, Ministry of Social
Affairs and National Solidarity, Paris, France (Consultant) Dr J. Petricciani, Director, Blood and Blood Products, Office of Biologics, Center for
Drugs and Biologics, Food and Drug Administration, Bethesda, MD, USA (Consultant)
Dr G.C. Schild, National Institute for Biological Standards and Control, London, England (Consultant)
Dr P. Sizaret, Scientist, Biologicals, WHO, Geneva, Switzerland Dr H. J . van de Donk, State Institute for Public Health and Environmental Hygiene,
Bilthoven, Netherlands (Consultant)
ACKNOWLEDGEMENTS
~ Acknowledgements are due to the following experts for their comments and advice:
Mr I. Davidson, Central Veterinary Laboratories, Weybridge, England Dr B. Elisberg, Ofice of Biologics Research and Review, Center for Drugs and
Biologics, Bethesda, MD, USA Dr E. Esber, Ofice of Biologics Research and Review, Center for Drugs and
Biologics, Bethesda, MD, USA Dr C. Guthrie, Commonwealth Serum Laboratories, Parkville, Victoria, Australia Dr P. Lemoine, Institute of Hygiene and Epidemiology, Brussels, Belgium Dr G. Mann, London School of Hygiene and Tropical Medicine, London, England Dr B.C. Meyer, Ofice of Biologics Research and Review, Center for Drugs and
Biologics, Bethesda, MD, USA Dr P. Noguchi, Office of Biologics Research and Review, Center for Drugs and
Biologics, Bethesda, MD, USA Professor Oon Chong Jin, National University of Singapore, Singapore General
Hospital, Singapore Dr P. Parkman, Ofice of Biologics Research and Review, Center for Drugs and
Biologics, Bethesda, MD, USA
Dr L. Peyron, Mkrieux Institute, Charbonnikres les Bains, France Dr J.C. Vincent-Falquet. Merieux Institute, Charbonnikres les Bains, France Dr Yuan-Yuan Chu, Ofice of Biologics Research and Review, Center for Drugs and
Biologics, Bethesda: MD? USA Professor A.J. Zuckerman, London School of Hygiene and Tropical Medicine,
London, England
REFERENCES
1. WHO Technical Report Series, No. 673, 1982. 2. WHO Technical Report Series, No. 323, 1966. 3. WHO Tcchnical Report Series, No. 530, 1973.
Appendix 1
SUMMARY PROTOCOL FOR TESTING CONTINUOUS CELL LINES
Based on Requirements for Biological Substances No. 37. Requirements for Continuous Cell Lines Used for Biologicals Production (WHO Technical Report Series, No. 745, 1987,
Annex 3)
General Information
Name and address of manufacturer
Name and indentification of continuous cell line
Brief information on the derivation of the continuous cell line
Brief information on the source of cells from which cell seed was made
Brief information on passage history of the continuous cell line
Growth characteristics:
Doubling time
Media used
Nature and concentration of serum used
Nature and concentration of antibiotic(s) used, if any
Cell Seed
Origin and short history of the cell seed
Authority that approved the cell seed
Total number of ampoules of cells stored
Describe the preparation of the cell seed and indicate the number of ampoules frozen and measures taken to ensure that a homo- geneous population of cells is dispensed into each ampoule
The passage level (or population doubling) of the cell seed
Storage conditions
Percentage of total cell seed ampoules that were tested
Growth characteristics
Morphological characteristics
Immunological markers
Cytogenetic data
Biochemical data
Results of other tests of identity
Results of tests for adventitious agents ,
Results of tests for tumorigenicity
Capacity for interferon production (if determined)
Viral susceptibility
Manufacturer's Working Cell Bank (hlWCB)
Date the MWCB was established
Quantity of cells stored
The passage level of the h1IVCB
Storage conditions
Percentage of total MWCB ampoules that were tested
Tests for adventitious agents
TL'S~S in sucklitzg mice
No. and weight of animals
Quantity injected (intramuscular)
Observation period
Results of injection (pathological changes, no. survived, etc.)
Tests in adult rnice
No. and weight of animals
Quantity injected (intramuscular)
Observation period
Results of injection (pathological changes, no. survived, etc.)
Tests in guinea-pigs
No. and weight of animals
Quantity injected (intramuscular)
Observation period
Results of injection (pathological changes, no. survived, etc.)
Tests in rabbits
No. and weight of animals
Quantity injected (intramuscular)
Observation period
Results of injection (pathological changes, no. survived. etc.)
Tests in eggs
No. and age of embryonated chicken eggs
Quantity of cells injected
Observation period
Results of injection (pathological changes, no. survived, etc.)
Tests in adult mice
No. and weight of animals -
Quantity injected (intracerebral) - -
Observation period
Results of injection (pathological changes, no. survived, etc.)
Tests for bacteria, fungi and mycoplasma
Media used
Results
Morphological test
Examination by light microscopy
Examination by electron microscopy (if this test has not been done on the cell seed)
Tests on cell cultures
Percentage of MWCB tested
Test performed on cell system
Period of observation of cell cultures
Results
Tc.s/ ,/br huc~rnugglurirzatit~g viru.re,r
Pcrccnlagc or M WCB tcstcd
Mcthod
Rcsults
Tests for retroviruse.~
Percentage of M WCB tested
Method
Results
Identity tests
Method used
Results
Methods
Results
Annex 4
REQUIREMENTS FOR POLIOMYELITIS VACCINE (INACTIVATED)
(Requirements for Biological Substances No. 2)
Addendum 1985
An informal consultation held in Geneva from 28 April to 1 May 1985 discussed the requirements for continuous cell lines that might be used in the manufacture of biologicals such as inactivated virus vaccines. The opportunity was taken to review the requirements for continuous cell lines that had been formulated as Part D of the Requirements for Poliomyelitis Vaccine (Inactivated) (WHO Technical Report Series, No. 673, 1982, Annex 2, p. 40), as well as the relevant sections of Part A. No attempt was made to review the entire Requirements.
Amendments
Part D (pages 70-76)
In view of the development of general requirements for continuous cell lines used for the manufacture of biologicals including inactivated virus vaccines, Part D should be deleted in its entirety.
Part A, section 3.1.2.2 Cell banks used for providing cell cultures
In view of the deletion of Part D, the reference to Part D at the end of the section should be removed.
Part A, section 3.2 Control cells
In view of the deletion of Part D, the reference to Part D, section 3.2 should be removed. Tests on control cells as referred to in Part C, section 3.2 are still appropriate for continuous cell lines and that reference remains.
Part A, section 3.4.3 Treatmenf before inactivatiorz
In view of the deletion of Part D. the reference to Part D should be removed.
Part A, section 3.4.3.1 Purzfication of nzonovalent pools
In view of the deletion of Part D, a revision of this section was made to ensure that purification includes a consideration of cellular DNA; as a result, the whole of section 3.4.3.1. is replaced by the following:
Each filtered monovalent pool of virus grown in a continuous cell line shall be purified before inactivation. The purification procedure should aim at the extensive removal of DNA of cell origin. The amount of DNA in the monovalent pool shall be accurately measured and shall be shown to be consistently reduced to low levels by the purification process. The national control authority shall approve an upper limit of acceptability of the DNA content in the monovalent pool.
A method acceptable in some countries is to clarify the virus suspension by filtration, to concentrate the virus by ultrafiltration and, thereafter, collect the virus peak after passing it through a sepharose gel filtration column. Further purification is achieved by passing the virus through a DEAE ion-exchange column. Other purification procedures, such as passing the preparation through an immobilized DNA-ase column or immuno-adsorption column, may be considered.
The purified pool shall be shown to contain no more than 0.1 pg of protein per D-antigen unit of poliomyelitis virus.
The examination for viral peptides by polyacrylamide gel electrophoresis and sucrose density gradient analysis have been shown to be useful. Countercurrent electrophoresis has been used for the detection of bovine serum proteins, Improved methods of analysis for the purity of vaccine, however, should be developed.
Addition of a new section to Part A
In view of the deletion of Part D, a new section 3.5.4 should be inserted in the Requirements for Poliomyelitis Vaccine (Inactivated) on page 59 as follows:
3.5.4 Purity tests
Only those monovalent pools of virus grown in continuous cell lines that comply with the requirements given in Part A, section 3.4.3.1 shall be blended to form a trivalent bulk.
Evidence has been accumulated to show that the amount of residual cellular DNA in the final product can be reduced to 10 pg or less per dose.
Annex 5
REQUIREMENTS FOR AIVTIMICROBIC SUSCEPTIBILITY TESTS
1. AGAR DIFFUSION TESTS USING ANTIMICROBIC SUSCEPTIBILITY DISCS
(Requirements for Biological Substances No. 26) (Revised 1981,' 1982,2 19853)
Addendum
Part A, section 1.6 Codes (page 151)' Change the list of codes as follows:
Add amoxicillinlclavulanic acid AMC apalcillin APL clavulanic acid CLA enoxacin ENX etharnbutol EB ethionamide E A roxithromycin R XT sulbenicillin SBC sulfacitine RE teicoplanin TEC ticarcillinlclavulanic acid TCC
Delete fradiornycin sinefungin (p. 178)2
sulfisomazole
Explanations' F JAN for neornycin SF INN and USAN lists: no
longer marketed Z USAN for sulfafurazole
Change flavornycin FL Change entry to: barnberrnycin (INN) imipemide (p. 176)' IPM Change entry to: irnipenem (INN) leucornycin LU Change entry to: kitasarnycin (INN) rnethenarnine mandelate M Delete rnandelate: other salts used nitrofuzone (p . 177)' FC Change entry to: nitrofural (INN) sulfarnethoxine MO Change entry to: sulfamonornethoxine
thiosporin TS (INN)
Change entry to: sulfomyxin (INN)
Additional information National nonproprietary names. Some national nonproprietary names differing significantly from the international nonproprietary name have been assigned to antibiotics listed in this section. For the convenience of users of the list, these names taken from Cumulative List no.6 of International Nonproprietary Names (INN) for Pharmaceubcal Substances are given on p. 112 with the INN. The Curnulative List also gives INN in Latin, Russian, French and Spanish. Spelling differences (e.g. sulphaphenazole. solfafenazolo for sulfafenazole (INN)) are not given.
' WHO Technical Reporl Series, No. 673. 1982 WHO Technical Report Series, No. 687, 1983
" WHO Technical Report Series. No. 745, 1987 See key on page I I2 for explanation of abbreviations
National name acrosoxacin arndinocillin cefaprin CO-trirnoxazole dernethylchlortetracyclinurn floxacillin fradiornycin furacilinurn furadoninurn hexarnine hexarnethylentetrarninurn laevornycetinurn rnethylenecycline rnoxalactarn nifurazolidonurn nitrofurazone
nitrofurrnethonurn norsulfazolurn paucirnycinurn penicillin G penicillin V pirnaricine rifarnpin sulfadirnezinurn sulfaisodirnidinurn sulfarnethazine sulfarnethoxine sulphasomidine sulfisoxazole
Origin BAN USAN DCF BAN NFN USAN JP USSRP USSRP BAN HPlPH USSRP DCF USAN NFN BAN/ JPIUSP NFN USSRP NFN USP USAN DCF US AN USSRP NFN USP JAN BAN USAN
INN rosoxacin rnecillinarn cefapirin sulfarnethoxazolef trimithoprim derneclocycline flucloxacillin neornycin nitrofural nitrofurantoin methenarnine methenamine chlorarnphenicol rnetacycline latamoxef furazolidone nitrofural
furaltadone sulfathiazole paromomycin benzylpenicillin phenoxymethyipenicillin natarnycin rifarnpicin sulfadirnidine sulfisomidine sulfadirnidine sulfarnonornethoxine sulfisornidine sulfafurazole
' BAN = British Approved Name DCF = Denomination commune franqaise HP = Pharrnacopoea Hungarica JAN = Japanese Accepted Name JP = Pharmacopoeia of Japan NFN = Nordic Pharmacopoeia Council name PH = Pharrnacopoea Helvetica USAN = United States Adopted Name USP = United States Pharmacopeia USSRP = State Pharmacopoeia of the USSR
Annex 6
PRODUCTION AND TESTIKG OF THE WHO YELLOW FEVER VIRUS PRIMARY SEED LOT 213-77
AND REFERENCE BATCH 168-73
Dr P. Brks (former Chief, Virus Diseases, WHO, Geneva)
and Professor M. Koch
Robert Koch Institute, Berlin (West)
Page 1. Introduction ................................................................................................... 113 2. Information on the Robert Koch Institute seed lot 112-69 from which the
WHO primary seed lot 213-77 and reference batch 168-73 were derived ..... 115 3. Production and testing of the WHO primary seed lot 213-77 ....................... 121 4. Information on production and testing of manufacturer's secondary seed lot
and vaccine to be derived from the WHO primary seed lot ......................... 122 5. Production and testing of the WHO reference batch 168-73 ........................ 124 6. References ...................................................................................................... 125 7. Appendix ........................................................................................................ 126
During the past several years WHO has tried, albeit unsuccessfully, to obtain a seed material of a low passage level suitable for the preparation of yellow fever 17D vaccine. Such material is needed to serve as a WHO primary seed and as a WHO emergency secondary seed for the preparation of large batches of vaccine, and to prepare a WHO reference vaccine. Finally, it was concluded that a vaccine derived from passage levels up to 239 would not differ (in terms of safety and efficacy for man) from seed material of a lower passage level, provided that the seed material met the WHO Requirements for Yellow Fever Vaccine (l).
In 1977, the Robert Koch Institute, Berlin (West), generously offered to prepare a batch of yellow fever virus from its seed lot 1 12- 69 whch had been prepared in leukosis-free eggs and shown to be
free from fowl leukosis virus; this batch was of the 237th passage level.
In 1978, two WHO officials visited the Robert Koch Institute to examine the laboratories and found them to be satisfactory as per the Requirements for manufacturing establishments and control laboratories (2).
1.1 WHO yellow fever 17D vaccine primary seed lot 213-77
The data presented in this annex were collected over a number of years and record the history of the production of the WHO primary seed of the 17D yellow fever virus to be used in the production of vaccine. The data show that the WHO primary seed:
-consists of the 237th passage of strain 17D, substrain 204 of the yellow fever virus;
- meets the Requirements for Yellow Fever Vaccine ( I ) - is made at approved premises; -is free from avian leukosis virus; - may be used to prepare a vaccine that is well tolerated by man,
gives good antibody responses, and is stable.
The WHO material consists of
- 300 ampoules of 0.5 m1 of freeze-dried 17D virus to be used as WHO primary seed lot;
- 500 ampoules of 0.5 m1 of the same production batch, which could be used as secondary seed lot in emergencies.
Manufacturers using this seed must bear the reponsibility for testing their working seeds and vaccines, and for the use of their vaccines.
It must be noted that the manufacture of vaccine will involve two further passages from the WHO primary seed lot and at each passage level (working seed and one batch of vaccine) the virus must be tested for neurotropism, viscerotropism, and immunogenicity in monkeys (rhesus or cynomolgus) by the manufacturer and two external control laboratories (3).
The working seed lot should be tested by the manufacturer in 50 subjects to measure the antibody response. Then, once the vaccine is ready, it should also be tested in man. Initially, the tests should be carried out in a few subjects; if the results are satisfactory, the
number of subjects should be gradually increased. Throughout the tests the subjects should be observed carefully.
As most of the yellow fever vaccine is used in countries with high ambient temperatures, the vaccine should be shown to be stable in the freeze-dried form at a high ambient temperature (37 "C) for at least 2 weeks.
1.2 WHO yellow fever 17D vaccine reference preparation for potency testing 168-73
Documents 'l l and 12 provide information on WHO reference batch 168-73 prepared at the Robert Koch Institute. This may be used by manufacturers and control authorities to compare their own reference preparations with the WHO preparation.
The WHO material consists of 600 ampoules of 0.5 m1 of freeze- dried 17D virus lot 168-73 at the 237th passage and is intended to be used as the WHO reference batch.
1.3 Procurement
The primary seed and reference batch may be obtained by vaccine manufacturers and controllers upon request from Chief, Biologicals, World Health Organization, 121 1 Geneva 27, Switzerland. All material is in flame-sealed ampoules under vacuum and is kept in liquid nitrogen at the Robert Koch Institute. It is free from avian leukosis virus.
2. INFORMATION ON THE ROBERT KOCH INSTITUTE SEED LOT 112-69 FROM WHICH THE WHO PRIMARY
SEED LOT 21377 AND REFERENCE BATCH 168-73 WERE DERIVED
2.1 Genealogy of primary seed lot RKI 112-69
Figs. 1, 2, 3, and 4 trace the passage history of yellow fever virus strains used in the world for the production of vaccine since the series isolated in tissue cultures in 1933 through the Colombia 88 strain at passage 228 after which the passage of the virus has been more accurately recorded. The main function of the seed lot system is to avoid further passages of the virus and thus reduce the likelihood of change in its immunogenicity or neurotropism.
Fig. 1. History of the seed virus used for the preparation of yellow fever vaccine a
18 passages in cultures containing mouse embryo tissues, Tyrode solution, and normal monkey serum.
..... . .
Sub- cultures
......... ........ ....... ...... ........ ....... ....... ...... ...... ....... 50 passages in cultures containing whole chick embryo tissues, Tyrode solution, and normal monkey serum.
...... ....... ..... ....
December 1933. Tissue cultures initiated with the virus from the serum of a monkey infected with the unmodified Asibi strain of yellow fever virus.
136 passages in cultures containing whole chick embryo tissues from which head and spinal cord had been removed, Tyrode solution, and normal monkey or human serum (not inactivated).
17 passages in cultures containing chick embryo tissues from which head and spinal cord had been removed, Tyrode solution, and normal human *rum (not inactivated).
1 chick embryo passage for preparation of vaccine lot No. N.Y.75.
4 passages in cultures containing chick embryo tissuesfrom which head and spinal cord had been removed, Tyrode solution, and human serum.
1 passage in chick embryo for preparation of Colombia vaccine No. 70.
1 passage in chick embryo for preparation of Colombia vaccine Nos. 88 and
\ l passage in chick embryo for preparation of seed virus for vaccine production in New York.
W H O 861160
'Source: SAWYER, W.A. ET AL. American journal of hygiene, 40: 35 (1944).
116
Fig. 2. Derivation of s u b s t r a i n s of 17D virus a
S;;;)"re , 1 , ORIGINAL 7 7 D
320 1 Passages made in:
NEW YORK BRAZIL
340 1 0 0 lubstrain designation
1 I Passed in tissue culture
1 Passed in chick embryo
'Source Fox, J P & P E N N A , H A AmencanlournaI of hygiene, 38: 152 (1943)
The genealogy of the WHO primary seed lot can be traced back to the strain AB617 at passage 234. supplied to the Robert Koch Institute by the Royal Institute for the Tropics, Amsterdam, Netherlands; this strain, in turn, can be traced back to Colombia 88 at passage 228 (Fig. 3). AB617 virus was derived from AB616 virus obtained from the Rocky Mountain Laboratory, Hamilton, MT,
243 sc = 0 EP
f 2 5 0 ~ .
17 DD,
35 EP+ 40 EPT
I I
3 0 5 s ~ - . I
76 E P ~ 80 E P ~
395 sc-
to ( 1 7 DD,)
pEzzzl
- 9 3 S
Fig. 4. Passage history of seed lots derived from substrain 17DD
In vitro Egg subcultures passages
243 0
BRAZIL SENEGAL
USA in 1967. The Bureau of Biologies (now Ofice of Biologicals), Bethesda, MD, USA, accurately recorded the passage of this strain from the 1 7 0 204 series, through Colombia vaccine numbers 70 and 90, to lot 145-3 which was sent to the Rocky Mountain Laboratory in January 1941. It is in the records, therefore, that the AB61 7 virus received at the Robert Koch Institute was at the 234th passage.
Batch 83-66 was prepared at the Robert Koch Institute in ordinary eggs; it represents passage 235; none of this material remains (Fig. 5).
Batch 112-69, was prepared from batch 83-66 in eggs free from avian leukosis virus (ALV); it represents passage 236; there are only 30 ampoules (0.5 ml) remaining. This is regarded as the Robert Koch Institute's (RKI) primary seed lot.
Batch 160-72, prepared in ALV-free eggs, is at present used as secondary seed lot at passage 237.
Vaccine is produced at passage 238 in ALV-free eggs.
Fig.5.Genealogy of WHO primary seed lot 213-77 and WHO reference batch 168-73
Pasnage level
237 RKI seclndary SL 47 Gtches WHO primary SL WHO reference batch 160-72 of vaccine
I I 213;77 I 1 168-13 1
I I
a 238 23 batches 202-76 209-76 212-77 Manufacturer's P
of vaccine secondary SL 2 (21 3--A-80)
I 239 ~anufacturer's
vaccine (213-8115-82)
- - Virus derived for WHO RKI Robert Koch Institute SL Seed lot
2.2 Production and testing of primary seed lot RKI 112-69
Protocol of production. An examination of the conditions of production and tests carried out at the Robert Koch Institute showed that the primary seed lot RKI 112-69 complies with WHO requirements for Yellow Fever Vaccine (Revised 1975) (1).
Absence of avian leukosis virus. Although batch 83-66 (passage 235) was made in normal fertile eggs, batch 1 12-69 (passage 236) was made in leukosis-free eggs, but was not tested for ALV. All further batches prepared from 112-69 were also prepared in leukosis-free eggs, and those that were tested were found to be free from avian leukosis virus, i.e., batch 160-72 and those derived from it: 202-76, 209-76, 212-77 and 213-77. The tests were carried out at the Robert Koch Institute, The Houghton Poultry Research Station, Huntingdon, England, and the Bureau of Biologics (now Ofice of Biologicals), Food and Drug Administration, Bethesda, MD, USA.
Immunogenicity andsafetj, in man. Forty-seven batches of vaccine prepared from seed 112-69 were given to 4373 persons. There were no untoward reactions and in all 15 persons studied for antibody responses the vaccine was found to have satisfactory potency.
2.3 Production and testing of secondary seed lot RKI 160-72
Genealogy. As may be seen from Fig. 5, the Robert Koch Institute's secondary seed lot 160-72 is derived from the primary seed lot RKI 112-69 and is a sister batch of the WHO primary seed lot 213177 and of the WHO reference batch 168-73.
Protocol of production. Tests carried out at the Robert Koch Institute showed that the secondary seed lot complied with WHO requirements (l).
Absence of leukosis virus. This lot was prepared in ALV-free eggs (Valo) and was shown to be free from extraneous viruses, as were vaccine batches 202-76,209-76 and 212-77 prepared from it (Fig. 5).
Immunogenicity and safety in man. Twenty-three batches of vaccine made from 160-72 were shown to be satisfactory in 1476 human subjects and 28 vaccinees gave satisfactory antibody responses to vaccines prepared from this seed.
3. PRODUCTION AND TESTING OF THE WHO PRIMARY SEED LOT 213-77
Genealogy. The seed lot 213-77 was prepared at passage 237 special for WHO at the Robert Koch Institute from their primary seed lot -1 12-69. It is proposed as the WHO primary (master) seed lot for yellow fever vaccine preparation. This is a sister batch of RKI 160-72 secondary seed lot (Fig. 5).
Protocol of production. The Robert Koch Institute's protocol for production and testing established that the seed lot 213-77 was satisfactory for viscerotropism and neurotropism in rhesus monkeys as per WHO requirements (2) (Document 1). The potency of the virus was 153 000 LD,,/mll and 4.3 X 106 PFU/m12.
The seed lot 213-77 was also tested in a second laboratory (Document 2). In view of the difficulty in obtaining rhesus monkeys
l LD,, = median lethal dose. PFU = plaque-forming units.
(Macaca mulatta), a test in cynomolgus monkeys (Macaca fascicularis) was carried out by the external control laboratory (Bureau of Biologics, Food and Drug Administration, Bethesdz, MD, USA) after it had been demonstrated that rhesus 2nd cynomolgus monkeys have similar sensitivities to yellow fever virus. It was shown that the seed 213-77 was satisfactory in monkeys for neurotropism, viscerotropism, and immunogenicity.
Absence of avian leukosis virus. Eggs (Valo) used for the preparation of the WHO seed lot 213-77 were certified as being free from avian leukosis viruses and other contaminants. Tests carried out at the Robert Koch Institute and at the Bureau of Biologics showed that this passage was free from avian leukosis virus (Documents 3 and 4).
Absence of other extraneous viral agents. Extensive tests for extraneous agents were not carried out on the WHO primary seed lot 213-77 to save material but were performed on a secondary (manufacturer's) seed lot 213/A/80 and gave satisfactory results (see below).
Immunogenicity and safety in man. A secondary (manufacturer's) seed lot (213/A/80) and vaccine batch (213/B2/81) at the 239th passage level were derived from the WHO primary seed lot 213-77. They were both found to be satisfactory with regard to immunogenicity and safety in humans (see next section).
4. INFORMATION ON PRODUCTION AND TESTING OF A MANUFACTURER'S SECONDARY SEED LOT
AND VACCINE BATCHES DERIVED FROM THE WHO PRIMARY SEED LOT
In order to obtain more complete information on the WHO primary seed lot 213-77, a secondary seed lot (213/A/80) was prepared and tested at the Robert Koch Institute under the same conditions as those found in manufacturers' laboratories. Five vaccine batches were also derived from it in order to test the vaccine at passage 239.
4.1 Manufacturer's secondary (working) seed lot derived from WHO primary seed lot
Protocol of production. Tests carried out at the Robert Koch Institute showed that the secondary seed lot 213/A/80 complied with
WHO Requirements for Yellow Fever Vaccine (Revised 1975) (2) (Document 5). A further test in cynomolgus monkeys was done at the Bureau of Biologics, Bethesda, MD, USA, the results of which were satisfactory (Document 6). The potency of the batch was 21 1 600 LD,,/ml.
Testing for other extraneous viral agents. The secondary seed lot 213/A/80 was tested at the Robert Koch Institute using a monoclonal antibody to neutralize the virus before inoculating human embryo lung cells and secondary cynomolgus monkey kidney cells. No extraneous viral agents were detected. However, 30-40% of the cynomolgus kidney cells showed a positive fluorescence when stained with FITC1-labelled yellow fever virus antiserum, a finding that does not invalidate the significance of this test.
Testing in humans. The seed lot 213/A/80 was administered to 16 persons who were kept under close observation. No untoward reactions were noted and all showed seroconversion (Document 7).
4.2 Vaccine batches derived from manufacturer's secondary (working) seed lot
Five vaccine batches (213/Bl-5/82 at the 239th passage level (Fig. 5) were derived at the Robert Koch Institute from secondary seed lot 213/A/80 (manufacturer's secondary seed lot level) and submitted to tests recommended for vaccine batches in WHO requirements (I).
Potency tests. The geometric means of virus titration in mice and pig kidney (PS) cells are given for each vaccine batch in Table 1 of Document 8.
Identity tests. The testing of the five vaccine batches is recorded in Table 2 of Document 8. Virus growth was suppressed by low dilutions of a specific antiserum and breakthroughs occurred at higher dilutions, as could be expected.
Innocuity tests. The five vaccine batches were tested for bacterial sterility and also for innocuity in guinea-pigs and mice according to WHO requirements and passed the tests (Document 8, Table 2).
Testing in humans. The vaccine lot 213jB2j8 1 was tested in 10 human subjects and none of the vaccinees experienced side-effects or impairment of well-being after vaccination. They all sero-
FITC = Fluorescein isothiocyanate.
converted with titres in the same range as those produced by a reference vaccine of the Robert Koch Institute and other vaccines produced at the same institute (Document 9).
Thermostability. Thermostability tests were carried out with the five vaccine batches after 7 days at 37 "C and 14 days at room temperature without any added stabilizing substance; the results were satisfactory (Document 10).
4.3 Genetic stability of virus at passages 238 and 239
Studies carried out at the Division of Vector-Borne Viral Diseases, Centers for Disease Control, Fort Collins, CO, USA, showed that a monoclonal antibody prepared against Connaught 17D vaccine virus clearly distinguished between vaccines derived from the 17D-204 and the 17DD substrains in a neutralization test (Figs. 2, 3, 4). Vaccines from Connaught, Wellcome and Robert Koch Institute were neutralized to equal titre by the monoclonal antibody, whereas 17D-derived vaccines (Dakar, Brazil and Colombia) were not neutralized (or were neutralized at a very low antibody titre). The 17D vaccines were also clearly different antigenically from the parent Asibi virus.
When tested by means of oligonucleotide finger-print assays, the seed lot 21 3/A/80 and vaccine 21 3/B2/81 were found to be identical to the 17D Connaught reference strain. This provides additional information regarding the stability and identity of these materials.
5. PRODUCTION AND TESTING OF THE WHO REFERENCE BATCH 168-73
Genealogy. WHO reference batch 168-73 derived at the Robert Koch Institute from the primary seed lot RKI 112-69 is a sister batch of the WHO primary seed lot 213-77 and of the RKI secondary seed lot 160-72 (Fig. 5). It is a 237th passage of the 17D yellow fever virus substrain 204 (Fig. 3).
Composition. Batch 168-33 comprises 600 ampoules, each filled with 0.5 m1 of chick embryo juice infected with 17D yellow fever virus, freeze-dried and flame-sealed in vacuum. It was offered to WHO as a reference preparation. It will be available to yellow fever vaccine manufacturers and controllers for calibration of their own reference preparations.
Protocol of production. Batch 168-73 was found satisfactory for viscerotropism and neurotropism in tests carried out in rhesus monkeys at the Robert Koch Institute according to WHO requirements (Document 11). A further testing was carried out in cynomolgus monkeys at the Bureau of Biologies (Document 12). The geometric mean of the potency titration of 3 ampoules was 82 885 mouse LD,, per 0.5 m1 (human dose) and 1.5 X 107 PFU/ml in PS cells. The total nitrogen content was 0.25 mg per human dose.
Immunogenicity and safety in man. Batch 168-73, part of which is also the Robert Koch Institute's reference preparation, was used to vaccinate 75 staff members of the Institute. No adverse reactions were observed and all the 37 sera tested by haemagglutination inhibition for yellow fever antibodies gave satisfactory seroconversion titres (Document 13).
6. REFERENCES
1. WHO Technical Report Series, No. 594, 1976. 2. WHO Technical Report Series, No. 323, 1966. 3. WHO Technical Report Series, No. 658, 1981, p. 46.
7. Appendix
DOCUMENT 1. SUMMARY PROTOCOL OF YELLOW FEVER VACCINE PRODUCTION
WHO PRIMARY SEED LOT 213-77
Name and address of manufacturing laboratory: Robert Koch Institute
Laboratory reference number of lot: 21 3-77
Date when the processing was completed: 22 February 1978
Information on Manufacture
1. Virus used to inoculate tissues for the manufacture of the lot:
(a) strain and substrain: (b) passage level: (c) source and reference number:
(d) remarks:
2. Date of inoculation of embryos: 3. Date of harvesting: 4. Age of embryos: 5. Temperature of incubation: 6. Results of sterility tests: 7. Number of containers prepared: 8. Conditions of storage:
17D 204 236 Robert Koch Institute
1 12-69 first passage in leukosis-
free eggs (Valo) 4 February 1977 7 February 1977 12 days 38.5 "C sterile 800 ampoules of 0.5 m1 in liquid nitrogen
Information on Safety Tests
1. Monkey safety test (a) Date of inoculation
of monkeys: 1 February 1978 (b) Dilution of, the reconstituted
seed lot used for the inoculum: none
Record of monkeys
Monkey
-
Weight Postinoculation viracmia Maximum titre of Days post- Signs ol encapha- (kg) (Pi-U/ml)lO circulating virus inoculation whorl litis and /or
- .. . . . . (mouse LDS, temperature paralysis
day day day per 0.03 ml) exceeded 40°C
Death Ant~body titre
Haernagglutina- Neutralization lion ( ~ n mice)'
inhibition
Mouse LD,, in virus challenge dose: 194 766 per 0.03 ml No plaques detected at 1:10 dilution.
(c) Virus titre (LD,, or PFU) inoculated in each monkey: 41 803 mouse LDS,
( d ) Record of monkeys: (see table on p. 127) (e) Identity test Normal serum 1:3 Date of test:
in mice: 7145 mouse LDS, l February 1978 Immune serum 1 : 3 < 10 mouse LD,,
2. Sterility test Date of test: 24 February 1977
(a) Number of containers examined: 10 ampoules (b) Number showing contamination: none
.............................................................................. (c) Remarks
3. Guinea-pig safety test Date of test: 23 February 1977
(a) Number of animals injected: two animals (b) Number of animals showing reaction: none (c) Remarks ..............................................................................
...... 4. Other safety tests
5. Remarks:
Signature of Director of Manufacturing Laboratory
....................................................................
residual moisture 0.39%
(Signed) Professor Date: 19 June 1981 Dr M.A. Koch
DOCUMENT 2. VIRAEMIA, IMMUNOGENICITY, AND NEUROTROPISM TESTS IN CYNOMOLGUS
MONKEYS FOR WHO PRIMARY SEED LOT 213-77 AT THE BUREAU OF BIOLOGICS, BETHESDA, MD, USA
Test date: 11 October 198 1 Titre of inoculum (0.25 ml)
Tissue culture PFU 2 176 000 Mouse LD,, 773 1
Viraemia
Log,, TCID,; per ml Mouse LD,. per 0.03 ml
Day 2 Day 4 Day 6 Day 2 Day 4 Day 6
Number of positive monkeys 12/12 6/12 0112 12/12 9/12 0112
Maximum titre 4.1 1.9 < 1:O 40.09 3.89 - Geometric mean tltre
(positive only) 2.48 1.57 - 4.30 0.96 - Standard deviation
(l0!3,0) 0.842 0.372 0.591 0.312 - -
lmmunogenicity (30 day)
Mouse neutralization testb 50% plaque
neutralization test (titre = log,, per ml) Number of monkeys
Pass Fail Neither
Conversion 12/12 8/12 0112 4/12 Geometric mean titre 2.69 Not applicable Standard deviation 0.294 Not applicable
(log,,)
Clinical disease
Observation
No. of monkeys sick No. of monkeys with severe encephalitis Average severity per monkey per day Standard deviation
'TCID = Tissue culture infectious dose 'Challenge dose (LD,): 49.09.
DOCUMENT 3: COMPLEMENT-FIXATION TEST FOR AVIAN LEUKOSIS VIRUS AT THE ROBERT KOCH
INSTITUTE AND LOHMANN TIERZUCHT LABORATORIES FOR WHO PRIMARY SEED LOT 213-77
Sample Serum Percentage of haemolysis at 1:2 and 1:4 dilution of antigen
---
Day 8, Day 22, Day 23, passage 2 a passage 5 passage 5"
Seed lot Anti-yellow fever 100 100 100 213-77 rhesus monkey
serum dilution 1:10
Positive Anti-yellow fever control rhesus monkey
serum dilution 1:10
AMVc Anti-yellow fever NT rhesus monkey
serum dilution 1:10
RSVe Prague Anti-yellow fever NT rhesus monkey
serum dilution 1:10
Negative Anti-yellow fever 100 control rhesus monkey
serum dilution 1:10
Positive normal rhesus control monkey
AMV normal rhesus NT 0 0 monkey
RSV Prague normal rhesus NT 0 monkey
Negative normal rhesus 100 100 100 control monkey
Test performed at the Robert Koch lnstttute Test performed at the laborator~es of Lohmann Tlerzucht, Cuxhaven, Federal Republtc of Germany AMV = Av~an rnyeloblastosls vtrus
* NT = not tested RSV = Rous sarcoma vlrus
DOCUMENT 4. COMPLEAIEKT-FIXATION TEST AND REVERSE TRANSCRIPTASE ASSAY AT THE BUREAU O F BIOLOGICS, BETHESDA, >ID, USA FOR WHO PRIMARY SEED LOT 213-77 AND SECONDARY SEED LOT 213-A-80
Prom letters dated 1 and 10 February 1982 from the Bureau of Biologies, Food and Drug Administration, Bethesda, MD, USA, to
the Robert Koch Institute, Berlin (West)
The seed viruses and appropriate positive and negative controls were passaged three times in primary chick embryo cell cultures for a total incubation of 20 days. The tissue culture fluids from the three passages as well as cell pack antigens from the final harvest were tested by complement fixation for avian leukosis virus (COFAL). Both the primary seed No. 213-77 and the secondary seed No. 213-A-80 are negative for avian leukosis viruses.
The testing for reverse transcriptase activity on the yellow fever vaccine seed pools 213-77 and 213-A-80 has been completed. Not unexpectedly, both are negative, confirming the COFAL test results that I sent you last week.
DOCUMENT 5. SUMMARY PROTOCOL O F YELLOW FEVER VACCINE PRODUCTION: MANUFACTURER'S
WORKING SEED LOT (213-A-80)
Secondary Seed Lot
Name and address of manufacturing laboratory: Robert Koch Institute
Laboratory reference number of lot: 213-A-80
Date when the processing was completed: 7 May 1980
Information on manufacture
1. Virus used to inoculate tissues for the manufacture of the lot:
(a) strain and substrain: 17D 204
(b) passage level: 237 (c) source and reference number: WHO primary seed lot
213-77 (d) remarks: second passage in leukosis-
free eggs (Valo) 2. Date of inoculation of embryos: 15 February 1980 3. Date of harvesting: 18 February 1980 4. Age of embryos: 12 days 5. Temperature of incubation: 38.5 "C 6. Results of sterility tests: sterile 7. Number of containers prepared: 860 ampoules of 0.5 m1 8. Conditions of storage: over liquid nitrogen
Information on safety Tests
1. Monkey safety test (a) Date of inoculation
of monkeys: 16 April 1980 (b) Dilution of the reconstituted
seed lot used for the inoculum: none
(c ) Virus titre (LD,, or PFU) inoculated in each monkey: 14 968 mouse LDS,
(d) Record of monkeys: (see table on p. 133) (e ) Identity test Normal serum Date of test:
in mice: 1:3 638 LD,, 16 April 1980 Immune serum 1:3 10 LDSo
Record of monkeys .
Monkey Weight Postinoculation viracniia Days postinoculation Signs of encephalitis Death Antibody litre (kg) (PFU1ml)lO ' when andlor paralysis
lornporalure exceeded 40 'C Haemaggiutination Nculrai~zation'
day 2 day 4 day 6 inhibition
1 4.45 0.2 1.5 0" - No No 1 280 1 :40 2 3.75 21.5 0.1 0 1 No No 1 280 1 :40 3 4.46 0 0 0 1 No No 80 1:40 4 4.95 5.9 8.1 0 - No No 1 280 1 :40 5 3.65 0.4 7.9 0 - No No 1 280 1:160 6 4.0 0.9 1.8 0 - No No 640 1:160 7 3.76 0.9 0.9 0.2 11 No No 1 280 1 :40 8 4.5 0.9 1.9 0 11 No No 1 280 1 :40 9 4.45 1.2 0.7 0 - No No 1 280 1 :40
10 3.57 2.4 16.7 0 No No 640 1 :40 11 4.6 7.6 8.5 0 - No No 1 280 1:160
Plaque reduction Lest at 90% level. a No plaques delectcd at a 1:10 dilution ol scrum.
2. Sterility test Date of test: 7 March 1980
(a) Number of containers examined: 10 ampoules (6) Number showing contamination: none
.............................................................................. (c) Remarks 3. Guinea-pig safety test Date of test:
17 April 1980 (a) Number of animals injected: 5 animals (b) Number of animals showing reaction: none
.............................................................................. (c) Remarks 4. Other safety tests ......................................................................... 5. Remarks: residual moisture 0.29%
Signature of Director (Signed) Professor Date: 19 June 1980
l of Manufacturing Dr M.A. Koch Laboratory
l
DOCUMENT 6. VIRAEMIA, IMMUNOGENICITY, AND NEUROTROPISM TESTS IN CYNOMOLGUS MONKEYS FOR MANUFACTURER'S WORKING SEED (213-A-80) AT
THE BUREAU OF BIOLOGICS, BETHESDA, MD, USA
Test date: 11 October 1980 Titre of inoculum (0.25 ml) -
Tissue culture PFU 1 792 000 Mouse LD,, 6332
Vlraemia
Log,, TCID,, per ml Mouse LD,, per 0.03 ml
Day 2 Day 4 Day 6 Day 2 Day 4 Day 6
Number of positive monkeys 12112 10112 0112 11112 9112 1/12
Maximum titre 3.7 2.5 1.0 50.35 23.01 0.5 Geometric mean titre
(positive only) 2.48 1.50 - 4.70 1.00 - Standard deviation
(log,,) 0.948 0.558 - 0.640 0.617 -
lmmunogenicity (30 day)
Mouse neutralization test'
50% plaque neutralization test Number of monkeys (titre = log,.lml)
Pass Fail Neither
Conversion 12/12 Geometric mean titre 2.69 Standard deviation (log,.) 0.362
7/12 0112 5/12 Not applicable Not applicable
Clinical disease
Observation
No. of monkeys sick No. of monkeys with severe encephalitis Averrage severity per monkey per day Standard deviation
' Challenge dose (LD,,): 49 09
DOCUMENT 7. IMMUNOGENICITY OF 17D YELLOW FEVER VACCINE SEED LOT 213-A-80
PASSAGE LEVEL 238"
No. Patient Sex Haemagglutination inhibition titreb
Four weeks after vaccination
H.G. f H.G. m C.A. f U.S. f I.R. f
M.A. m C.R. f L.G. m M.F. m R.€. m C.E. m P.B. m P.F. f C.O. f M.M. f A.B. f
Vaccination with 1500 PFU subcutaneously. All subjects were seronegative prior to vaccination.
DOCUMENT 8. TESTING OF FIVE VACCINE BATCHES AT 239TH PASSAGE LEVEL DERIVED FROM
SECONDARY SEED LOT 213-A-80 AND WHO PRIMARY SEED LOT 213-77
Table 1. Potency titrations
No. of doses Lot No. per ampoule PFUlmla
Titration in a stable line of pig kidney (PS) cells according to WHO procedure For each of 5 dilutions 8 mice were inoculated.
Table 2. ldentity and innocuity test
Identity test Innocuity test
Serum Reaction in Reaction in dilution" PFUlml guinea-pigsb mice
None
None
None
None
None
None
None
None
None
None
NS = normal serum; IS = immune serum Five animals.
DOCUMENT 9. IMhluPiOGENICITY IN MAN OF VACCINE 213-B2-81 (239TH PASSAGE) DERIVED
FROM WHO PRIMARY SEED LOT 213-77 AND 213-A-80 MANUFACTURER'S WORKING SEED
Haemagglutination inhibition titre Neutralization test'
No. Patient Sex Before Four weeks after Before Four weeks
vaccination vaccination vaccination after vaccination
1. CH.E f 0 1:160 0 1:85 2. R.K. m 0 1: 80 0 1 :50 3. M.6 . m 0 1320 0 1:24 4. C.W. f 0 1:160 0 1 :24 5. H.R. m 0 1:160 0 1 :40 6. H.J.B. m 0 1: 80 0 1:23 7. B.B. f 0 1: 20 1:lO 1:46 8. L.B. f 0 1: 40 0 1 :22 9. U.H. f 0 1: 40 0 1 :27
10. H.P. f 0 1: 40 0 1 :22
Titres expressed as the dilution giving 90% inhibition in a plaque reduction test.
DOCUMENT 10. THERMOSTABILITY OF 17D YELLOW FEVER VACCINES PREPARED FROM
WHO SECONDARY SEED 213/A/80
Vaccine No. Titre of vaccine Titre after 7 days Titre after 14 days kept at - 20°C at 37 ' C at room temperature
DOCUMENT 11. SUMMARY PROTOCOL OF YELLOW FEVER VACCINE PRODUCTION: WHO REFERENCE BATCH 168-73
Name and address of manufacturing laboratory: Robert Koch Institute
Laboratory reference number of lot: 168-73
Date when the processing was completed: 12 October 1977
Record of monkeys
Monkey Weight (kg)
Postinoculation viraemia
Day Mouse LD,, Days postinoculation per 0 03 ml when temperature
exceeded 40 O C
4 < l 0 None 2 4 10 None 2 4 10 None 2.4 < l 0 None 2 < 10 None
Died after ~noculation 6 4 10 None 2 < 10 None 2 < l 0 None 2 1 1 0 None
Signs of encephalitis Death Neutralization titrea andlor paralysis
No neutralization titre of monkeys before inoculation; neutralization tested only with sera at 1:20. Mouse LD,. in virus challenge dose: 52 656 per 0.03 rnl
Information on Manufacture
l . Virus used to inoculate tissues for the manufacture of the lot
(a) strain and substrain: 17D 204 (b) passage level: 236 (c) source and reference number: Robert Koch Institute
1 12-69 (d) remarks: first passage in leukosis-
free eggs (Valo) 2. Date of inoculation of embryos: 29 January 1973 3. Date of harvesting: 1 February 1973 4. Age of embryos: 12 days 5. Temperature of incubation: 38.5 "C 6. Results of sterility tests: sterile 7. Number of containers prepared: 829 ampoules of 0.5 m1
and 100 ampoules of 1.0 m1
8. Conditions of storage: 7 0 "C
Information on Safety Tests
l . Monkey safety test (a) Date of inoculation
of monkeys: 9 June 1977 (b) Dilution of the reconstituted
seed lot used for the inoculum: 1 :2
(c) Virus titre (LD,, or PFU) inoculated in each monkey: 6874 mouse LD,,
(geometric mean of titres at beginning and end of test)
(d) Record of monkeys: See table on p. 138 (e) Identity test Normal serum: Date of test:
in mice: 3855 LD,, 21 September 1977 Immune serum: < 10 LD,,
2. Sterility test Date of test: 21 February 1973
(a) Number of containers examined: 10 ampoules (b) Number showing contamination: none
.............................................................................. (c) Remarks 3. Guinea-pig safety test Date of test:
19 February 1973 (a) Number of animals injected: 2 animals (b) Number of animals showing reaction: none
.............................................................................. (c) Remarks 4. Other safety tests: residual moisture 0.25%
....................................................................................... 5. Remarks
Signature of Director (signed) Dr H. Voss Date: 23 January of Manufacturing Director and 1978 Laboratory Professor
DOCUMENT 12. VIRAEMIA, IMMUNOGENICITY AND NEUROTROPISM TESTS IN CYNOMOLGUS MONKEYS FOR WHO REFERENCE BATCH 168-73 AT THE BUREAU
OF BIOLOGICS, BETHESDA, MD, USA
Test date: 22 November 1980 Titre of inoculum (0.25 ml)
Tissue culture PFU 208 000 Mouse LD,, 15 594
Viraemia
Log,, TCID,,lml Mouse LD,./0.03 ml
Number of positive monkeys 12/12 7/12 0112 10112 7/12 0112
Maximum titre 4.3 3.7 <1.0 29.65 2.30 -
Geometric mean titre (positive only)
- 2.95 2.26 - 2.48 0.837 Standard deviation - 1.013 1.104 - 0.684 0.389 ( b 3 3
lmmunogenicity (30 day)
Mouse neutralization testa 50% plaque
neutralization test Number of monkeys (titre = log,Jml)
Pass Fail Neither
Conversion Geometric mean titre Standard deviation
(log,.)
8/12 0112 4/12 Not applicable
Not applicable -
Clinical disease
Observation
No. of monkeys sick No. of monkeys with severe encephalitis Average severity per monkey per day Standard deviation
Challenge dose (LD,). 41 12
DOCUMEKT 13. IMMUNOGENICITY IN MAN OF WHO REFERENCE BATCH 168-73
Immunogenicity of batch 168-73 (passage level 237 prepared from batch 112-69): tests performed between August 1978 and March 1979
p~
Patient's Sex Post-vaccination Patient's Sex Post-vaccination initials haemagglutination initials haemagglutination
inhibition titre' inhibition titre'
L.W. W.R. H.C. G.R. W.W. M-H.A. M.R. K.M. S.M. L.I. S.G. B.D. S.G. S.J. S.K. P.J. F.R. K.A.
U.N. F.R. L.W. F. W. H.M. S.M. K.M.
E.H-A. W.M. B.J. V.W. C.U. D.U. N.W. H.Ch. H.A.
W.Ch. W.E. L.K.
All sera tested had no haemagglutination inhibition antibody before vaccination.
141
Annex 7
BIOLOGICAL SUBSTANCES: INTERNATIONAL STANDARDS, REFERENCE PREPARATIONS,
AND REFERENCE REAGENTS
The list of international biological standards, international biological reference preparations, and international reference reagents previously included as annexes to the reports of the WHO Expert Committee on Biological Standardization is issued as a separate publication. Copies may be obtained from the agents shown on the back cover of this report, or may be ordered from: World Health Organization, Distribution and Sales Service, 121 1 Geneva 27, Switzerland.
A new edition of this booklet will be published in 1987l and will include the following changes, compared with the 1984 list, as a result of the discussions of this Expert Committee:
ADDITIONS
Antibiotics Tobramycin 9800 IU/ampoule Second International
Standard 1985
(This substance is held and distributed by the International Laboratory for Biological Standards, National Institute for Biological Standards and Control, London NW3 6RB, England.)
Antibodies
Anti-Brucella ovis 1000 IU/ampoule First International Serum Standard 1985
Clostridium 1020 IU/ampoule Second International perfringens Standard 1985
Epsilon Antitoxin, Equine
(These substances are held and distributed by the International Laboratory for Biological Standards, Central Veterinary Laboratory, Weybridge, England.)
l Biological substances: international standards and reference reagents, 1986. Geneva, World Health Organization (in press).
Clostridium 3 1 IU/'ampoule Second International botulinum Standard 1985
Type B antitoxin, Equine
(This substance is held and distributed by the International Laboratory for Biological Standards, State Serum Institute, 80 Amager Boulevard, 2300 Copenhagen S, Denmark.)
Antigens
Hepatitis B 100 IU/ampoule First International Surface Antigen Standard 1985 (subtype a 4
(This substance is held and distributed by the International Laboratory for Biological Standards, National Institute for Biological Standards and Control, London NW3 6RB, England.)
Blood products and related substances
Anti-Double 100 IU/vial First International Stranded DNA Standard 1985 Serum
(This substance is held and distributed by the International Laboratory for Biological Standards, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Plesmanlaan 125; Amsterdam. Setherlands.)
Beta, Microglobulin 100 1U:'vial First International ( P m > Standard 1985
(This substance is held and distributed b!- the International Laboratory for Biological Standards, State Serum Institute. SO Amager Boulevard. 2300 Copenhagen S, Denmark.)
Endocrinological and related substances
Elcatonin IU/ampoule First International to be defined Standard 1985
Parathyroid IU/ampoule First International Hormone, Bovine, to be defined Standard 1985 for in vitro Bioassay
(These substances are held and distributed by the International Laboratory for Biological Standards, National Institute for Biological Standards and Control, London NW3 6RB, England.)
Luteinizing 0.025 IU/ampoule First International Hormone Bovine, Standard 1985 for Immunoassay
(This substance is held and distributed by the International Laboratory for Biological Standards, Central Veterinary Laboratory, Weybridge, England.)
Reference reagents
Measles Vaccine Titre = 103.7/vial First International (Live) Reference Reagent
1985
(This substance is held and distributed by the International Laboratory for Biological Standards, National Institute for Biological Standards and Control, London NW3 6RB, England.)
Miscellaneous substances
Haemiglobincyanide 588 mg/litre Fifth International Standard 1985
(This substance is held and distributed by the National Institute of Public Health and Environmental Hygiene, Bilthoven, Netherlands.)
DISCONTINUED
Antibiotics Kanamycin B First International
Reference Preparation 1964
Dimercaprol First International Reference Preparation 1954
Me1 B (Melarsoprol) First International Reference Preparation 1954
Annex 8
REQUIREMENTS FOR BIOLOGICAL SUBSTANCES AND OTHER SETS
OF RECOMMENDATIONS
The specification of requirements to be fulfilled by preparations of biological substances is necessary in order to ensure that these products are safe, reliable, and potent prophylactic or therapeutic agents. International recommendations on requirements are intended to facilitate the exchange of biological substances between different countries and to provide guidance to workers responsible for the production of these substances as well as to others who may have to decide upon appropriate methods of assay and control. Recommended requirements and sets of recommendations concerned with biological substances formulated by international groups of experts and published in the Technical Report Series of the World Health Organization are listed hereunder: No. Year of
publication
178 1959 Requirements for Biological Substances: * 1. General Requirements for Manufacturing Establishments and
Control Laboratories * 2. Requirements for Poliomyelitis Vaccine (Inactivated)
179 1959 Requirements for Biological Substances: * 3. Requirements for Yellow Fever Vaccine * 4. Requirements for Cholera Vaccine
180 1959 Requirements for Biological Substances: * 5. Requirements for Smallpox Vaccine
200 1960 Requirements for Biological Substances: * 6. General Requirements for the Sterility of Biological Substances
237 1962 Requirements for Biological Substances: * 7. Requirements for Poliom>elitis Vaccine (Oral)
274 1964 WHO Expert Committee on Biological Standardization: * 8. Requirements for Pertussis Vaccine * 9. Requirements for Procaine Benzylpenicillin in Oil with Aluminium
Monostearate 293 1964 WHO Expert Committee on Biological Standardization:
* 10. Requirements for Diphtheria Toxoid and Tetanus Tosoid 323 1966 WHO Expert Group:
Requirements for Biological Substances (Revised 1965): 1 . General Requirements for Manufacturing Establishments and
Control Laboratories
* Replaced by revised Requirements.
No. Year of publicat~on * 2. Requirements for Poliomyelitis Vaccine (Inactivated)
5. Requirements for Smallpox Vaccine * 7. Requirements for Poliomyelitis Vaccine (Oral)
329 1966 WHO Expert Committee on Biological Standardization: * 11. Requirements for Dried BCG Vaccine
12. Requirements for Measles Vaccine (Live) and Measles Vaccine (Inactivated)
361 1967 WHO Expert Committee on Biological Standardization: * 9. Requirements for Procaine Benzylpenicillin in Oil with Aluminium
Monostearate (Revisions adopted 1966) * 13. Requirements for Anthrax Spore Vaccine (Live-for Veterinary
Use) * 14. Requirements for Human Immunoglobulin * 15. Requirements for Typhoid Vaccine
384 1968 WHO Expert Committee on Biological Standardization: * 16. Requirements for Tuberculins * 17. Requirements for Inactivated Influenza Vaccine
413 1969 WHO Expert Committee on Biological Standardization: t 4. Requirements for Cholera Vaccine (Revised 1968) * 18. Requirements for Immune Sera of Animal Origin
444 1970 WHO Expert Committee on Biological Standardization: 19. Requirements for Rinderpest Cell Culture Vaccine (Live) and
Rinderpest Vaccine (Live) t 20. Requirements for Brucella abortus Strain 19 Vaccine ( L i v e f o r
Veterinary Use)
444 1970 WHO Expert Committee on Biological Standardization: * Development of a National Control Laboratory for Biological
Substances (A guide to the provision of technical facilities)
463 1971 WHO Expert Committee on Biological Standardization: 21. Requirements for Snake Antivenins
486 1972 WHO Expert Committee on Biological Standardization: * 7. Requirements for Poliomyelitis Vaccine (Oral) (Revised 1971)
530 1973 WHO Expert Committee on Biological Standardization: 4. Requirements for Cholera Vaccine (Revised 1968) (Addendum
1973) 6. General Requirements for the Sterility of Biological Substances
(Revised 1973) * 17. Requirements for Inactivated Influenza Vaccine (Addendum 1973) * 22. Requirements for Rabies Vaccine for Human Use
* Replaced by revised Requirements. t Refer also to subsequent Addendum.
No. Year of publication
565 1975 WHO Expert Committee on Biological Standardization: Recommendations for the Assessment of Binding-Assay Systems (Including Immunoassay and Receptor Assay Systems) for Human Hormones and their Binding Proteins (A guide to the formulation of requirements for reagents and assay kits for the above assays and notes on cytochemical bioassay systems) Development of national assay services for hormones and other substances in community health care
594 1976 WHO Expert Committee on Biological Standardization: 3. Requirements for Yellow Fever Vaccine (Revised 1975)
20. Requirements for Brlicella abortus Strain 19 Vaccine (Live-for Veterinary Use) (Specification of tests used in the Requirements) (Addendum 19 75)
t 23. Requirements for Meningococcal Polysaccaride Vaccine
610 1977 WHO Expert Committee on Biological Standardization: Report of a WHO Working Group on the Standardization of Human Blood Products and Related Substances
610 1977 WHO Expert Committee on Biological Standardization: 1 23. Requirements for Meningococcal Polysaccharide Vaccine
(Addendum 1976) t 24. Requirements for Rubella Vaccine (Live)
25. Requirements for Brucella melitensis Strain Rev. 1 Vaccine (Live -for Veterinary Use)
7 26. Requirements for Antibiotic Susceptibility Tests. I. Agar Diffusion Tests using Antibiotic Susceptibility Discs
626 1978 WHO Expert Committee on Biological Standardization: 17. Requirements for Inactik-ated Influenza Vaccine (Addendum 1977)
t 23. Requirements for Meningococcal Polysaccharide Vaccine (Addendum 1977. incorporating Addendum 1976)
27. Requirements for the Collection, Processing. and Quality Control of Human Blood and Blood Products
626 1978 WHO Expert Committee on Biological Standardization: Guidelines for the Preparation and Establishment of Reference Materials for Biological Substances
638 1979 WHO Expert Committee on Biological Standardization: t 8 and 10. Requirements for Diphtheria Toxoid, Pertussis Vaccine,
Tetanus Toxoid: and Combined Vaccines (Revised 1978) * 11. Requirements for Dried BCG Vaccine (Revised 1978)
17. Requirements for Influenza Vaccine (Inactivated) (Revised 1978) 28. Requirements for Influenza Vaccine (Live)
* Replaced by revised Requirements. t Refer also to subsequent Addendum.
No. Year of publication
658 1981 WHO Expert Committee on Biological standardization: * 7. Requirements for Poliomyelitis Vaccine (Oral) (Addendum 1980)
Procedure for Approval by WHO of Yellow Fever Vaccines in Connexion with the Issue of International Vaccination Certificates
t 8 and 10. Requirements for Diphtheria Toxoid, Pertussis, Vaccine, Tetanus Toxoid, and Combined Vaccines (Addendum 1980)
22. Requirements for Rabies Vaccine for Human Use (Revised 1980) 23. Requirements for Meningococcal Polysaccharide Vaccine
(Addendum 1980) 29. Requirements for Rabies Vaccine for Veterinary Use
* 31. Requirements for Hepatitis B Vaccine * 26. Requirements for Antibiotic Susceptibility Tests (Suggested
changes 1980) 24. Requirements for Rubella Vaccine (Live) (Addendum 1980)
* 30. Requirements for Thromboplastins and Plasma used to Control Oral Anticoagulant Therapy
Guidelines for Quality Assessment of Antitumour Antibiotics The National Control of Vaccines and Sera Requirements for Immunoassay Kits
673 1982 WHO Expert Committee on Biological Standardization: t 2. Requirements for Poliomyelitis Vaccines (Inactivated) (Revised
1981) f 8 and 10. Requirements for Diphtheria Toxoid, Pertussis Vaccine,
Tetanus Toxoid, and Combined Vaccines (Addendum 1981) t 26. Requirements for Antimicrobic Susceptibility Tests (Revised 1981)
32. Requirements for Rift Valley Fever Vaccine A Review of Tests on Virus Vaccines
687 1983 WHO Expert committee on Biological Standardization: 7. Requirements for Poliomyelitis Vaccine (Oral) (Revised 1982)
30: Requirements for Thromboplastins and Plasma used to Control Oral Anticoagulant Therapy (Revised 1982)
33. Requirements for Louse-Borne Human Typhus Vaccine (Live) The Standardization of lnterferons
t 26. Requirements for Antimicrobic Susceptibility Tests (Addendum 1982)
700 1984 WHO Expert Committee on Biological Standardization: t 8 and 10. Requirements for Diphtheria Toxoid, Pertussis Vaccine,
Tetanus Toxoid, and Combined Vaccines (Addendum 1983) 35. Requirements for Rift Valley Fever Vaccine (Live, Attenuated)
for Veterinary Use 34. Requirements for Typhoid Vaccine (Live Attenuated, Ty 21a,
Oral)
* Replaced by revised Requirements. t Refer also to subsequent Addendum.
148
725 1985 WHO Expert Committee on Biological Standardization: 8 and 10. Requirements [or Diphtheria Toxoid, Pertussis Vaccines,
Tetanus Tosoid, and Combined Vaccines (Addendum 1984) 31. Requirements for Hepatitis B Vaccine prepared from Human
Plasma (Re~rised 1984j 36. Requirements for Varicella Vaccine (Live)
Informal Consultation on the Standardization of Interferons
745 1987 WHO Expert Committee on Biological Standardization: 2. Requirements for Poliomyelitis Vaccine (Inactivated) (Addendum
1985) 1 1. Requirements for Dried BCG Vaccine (Revised 1985) 16. Requirements for Tuberculins (Revised 1985) 26. Requirements for Antimicrobic Susceptibility Tests
1. Agar Diffusion Tests Using Antimicrobic Susceptibility Discs (Addendum 1985)
37. Requirements for Continuous Cell Lines Used for Biologicals Production
Rece No.
WORLD HEALTH ORGANIZATION TECHNICAL REPORT SERIES
'nt reports:
(1983) New approaches to health education in primary health care Report of a WHO Expert Committee (44 pages) ................................. (1983) Prevention of liver cancer Report of a WHO Meeting (30 pages) ................................................. (1983) Gestational trophoblastic diseases Report of a WHO Scientific Group (81 pages) .................................... (1983) Viral vaccines and antiviral drugs Report of a WHO Scientific Group (72 pages) .................................... (1983) Research for the reorientation of national health systems Report of a WHO Study Group (71 pages) ......................................... (1983) Smoking control strategies in developing countries Report of a WHO Expert Committee (92 pages) ................................. (1983) Evaluation of certain food additives and contaminants Twenty-seventh report of the Joint FAOIWHO Expert Committee on
.................................................................... Food Additives (47 pages) (1984) Cardiomyopathies Report of a WHO Expert Committee (68 pages) ................................. (1984) Mental health care in developing countries: a critical appraisal of research findings Report of a WHO Study Group (59 pages) ......................................... (1984) Chemistry and specifications of pesticides Eighth report of the WHO Expert Committee on Vector Biology and
................................................................................ Control (46 pages) (1984) WHO Expert Committee on Biolagical Standardization
............................................................. Thirty-fourth report (75 pages) (1984) The leishmaniases Report of a WHO Expert Committee (140 pages) ............................... (1984) Lymphatic filariasis Fourth report of the WHO Expert Committee on Filariasis (1 12 pages) (1984) Road tratlic accidents in developing countries
................................................. Report of a WHO Meeting (36 pages) (1984) WHO Expert Committee on Specifications for Pharmaceutical Preparations
............................................................ Twenty-ninth report (54 pages) (1984) The role of food safety in health and development Report of a Joint FAOIWHO Expert Committee on Food Safety (79 pages) .............................................................................................. (1984) The uses of epidemiology in the study of the elderly Report of a WHO Scientific Group on the Epidemiology of Aging (84 pages) ..............................................................................................
SW. fr.
4.-
4.-
7.-
6.-
7 .-
8.-
(1984) Recommended health-based occupational exposure limits for respiratory irritants Report of a WHO Study Group (154 pages) ..................................... (1984) Education and training of nurse teachers and managers with special regard to primary health care
............................. Report of a WHO Expert Committee (54 pages) (1984) W H O Expert Committee on Rabies
........................................... .................. Seventh report (104 pages) .. (1984) Evaluation of certain food additives and contaminants Twenty-eighth report of the Joint FAO/WHO Expert Committee on
.................................................................... Food Additives (44 pages) (1984) Advances in malaria chemotherapy
.................................. Report of a WHO Scientific Group (218 pages) (1984) Malaria control as part of primary health care
......................................... Report of a WHO Study Group (73 pages) (1984) Prevention methods and programmes for oral diseases
................................. Report of a WHO Expert Committee (46 pages) (1985) Identification and control of work-related diseases
................................. Report of a WHO Expert Committee (71 pages) (1985) Blood pressure studies in children Report of a WHO Study Group (36 pages) ......................................... (1985) Epidemiology of leprosy in relation to control Report of a WHO Study Group (60 pages) ......................................... (1985) Health manpower requirements for the achievement of health for all by the year 2000 through primary health care Report of a WHO Expert Committee (92 pages) .............. : .................. (1985) Environmental pollution control in relation to development
................................. Report of a WHO Expert Committee (63 pages) (1985) Arthropod-borne and rodent-borne vim1 diseases Report of a WHO Scientific Group (1 16 pages) ................................ (1985) Safe use of pesticides Ninth report of the WHO Expert Committee on Vector Biology and Control (60 pages) .............................................................................. (1985) Viral haemorrhagic fevers Report of a WHO Expert Committee (126 pages) .............................. (1985) The use of essential drugs Second report of the WHO Expert Committee on the Use of Essential Drugs (50 pages) ................................................................................. (1985) Future use of new imaging technologies in developing countries Report of a WHO Scientific Group (67 pages) .................................... (1985) Energy and protein requirements Report of a Joint FAO/WHO/UNU Expert Consultation (206 pages) (1985) W H O Expert Committee on Biological Standardization
................................................... (1985) Thirty-fifth report (140 pages) (1985) Sudden cardiac death Report of a WHO Scientific Group (25 pages) .................................... (1985) Diabetes mellitus Report of a WHO Study Group (1 13 pages) ....................................... (1985) The control of schistosomiasis Report of a WHO Expert Committee (1 13 pages) ...............................
(1985) WHO Expert Committee on Drug Dependence ......................................................... Twenty-second report (3 1 pages).
(1986) Dementia in later life: research and action Report of a WHO Scientific Group (74 pages) .................................... (1986) Young people's health-a challenge for society Report of a WHO Study Group on Young People's Health and
.................................... "Health for All by the Year 2000" (1 17 pages) (1986) Community prevention and control of cardiovascular diseases Report of a WHO Expert Committee (62 pages) ................................. (1986) Evaluation of certain food additives and contaminants Twenty-ninth report of the Joint FAO/WHO Expert Committee on
.................................................................... Food Additives (59 pages) (1986) Recommended health-based limits in occupational exposure to selected mineral dusts (silica, coal)
......................................... Report of a WHO Study Group (82 pages) (1986) WHO Expert Committee on Malaria Eighteenth Report (104 pages) ............................................................ (1986) WHO Expert Committee on Venereal Diseases and Trepo- nematoses Sixth Report (141 pages) ..................................................................... (1986) Resistance of vectors and reservoirs of disease to pesticides Tenth report of the WHO Expert Committee on Vector Biology and
............................................................................... Control (87 pages) (1 986) Regulatory mechanisms for nursing training and practice: meeting primary health care needs Report of a WHO Study Group (71 pages) ........................................ (1986) Epidemiology and control of African trypanosomiasis Report of a WHO Expert Committee (127 pages) ............................... (1986) Joint FAOIWHO Expert Committee on Brucellosis Sixth Report (132 pages) ...................................................................... (1987) WHO Expert Committee on Drug Dependence Twenty-third Report (64 pages) ............................................................
