who expert committee on biological...

This report contains the collective views of on internorionol group of experts ond does not necessarily represent the decisions or the stotedpoiicy of the Viorld Heolth Organizotion

WHO EXPERT COMMITTEE ON BIOLOGICAL

STANDARDIZATION

Forty-sixth Report

Geneva 1998

WHO Library Cataloguing in Publication Data

WHO Expert Committee on Biological Standardization (1995 : Geneva, Switzerland) WHO Expert Committee on Biological Standardization : forty-sixth report.

(WHO technical report series ; 872)

1 .Biological products - standards I.Title II.Series

ISBN 92 4 120872 4 (NLM Classification: OW 800) ISSN 051 2-3054

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Contents

Introduction

General Development and distribution of International Biological Standards

and Reference Reagents Need for interim standards with an official status conferred by WHO Cytokines Standardization and priorities for action in the field of diagnostics Advice to United Nations agencies on the qzality of vaccines Yellow fever vaccine Reverse-transcriptase activity associated u~ith avian cells Glossary of terms Review of biological standardization Committee meetings

Requirements for biological substances Requirements for cell substrates used for biologicals production Requirements for oral poliomyelitis vaccine Requirements for snake antivenoms Requirements for thromboplastins and plasma used to control oral

anticoagulant therapy Requirements for yellow fever vaccine General requirements for the sterility of biological substances

(amendment, 1995) Guidelines for minimizing the risk of transmitting agents causing

bovine spongiform encephalopathy Guidelines for the production and control of synthetic peptide

vaccines Summary protocol for the batch release of virils vaccines

Antibiotics Gentamicin

Antibodies Anti-borrelia serum Anti-cytomegalovirus immunoglobulin and serum Anti-hepatitis B core antigen serum Anti-hepatitis E virus serum Anti-measles serum Anti-mumps serum Anti-parvovirus B19 IgG serum Anti-pertussis serum Anti-rubella immunoglobulin and serum Anti-streptolysin 0 serum Anti-toxoplasma serum Anti-varicella zoster IgG serum Thyroid-stimulating antibody

iii

Antigens and related substances Pertussis vaccine (whole cell) Poliomyelitis vaccine (inactivated) Live attenuated poliovirus

Blood products and related substances Factors II, VII, IX and X in plasma Factor IXa International working standards in blood coagulation Haemiglobincyanide Mannan-binding protein Plasminogen-activator inhibitor 1 Protein S in plasma Recombinant ferritin Thromboplastin Thromboplastin, rabbit, plain Tissue factor pathway inhibitor Tissue plasminogen activator in plasma Whole blood folate

Cytokines Interferon gamma, human Interleukin-8 Insulin-like growth factor-binding protein 3

Endocrinological and related substances Follicle-stimulating hormone Osteopoietin

Toxins Endotoxin

Annex 1 Laboratories approved by WHO for the production of yellow fever vaccine

Annex 2 Requirements for yellow fever vaccine (Requirements for Biological Substances No. 3, revised 1995)

Annex 3 General requirements for the sterility of biological substances (Requirements for Biological Substances No. 6, revised 1973, amendment 1995)

Annex 4 Summary protocol for the routine batch release of virus vaccines

Annex 5 Biological substances: International Standards and Reference Reagents

Annex 6 Requirements for Biological Substances and other sets of recommendations

Annex 7 Corrigenda: reference materials for anti-interferon alfa and beta sera; interferon gamma, recombinant human; and mumps vaccine (live) 90

WHO Expert Committee on Biological Standardization Geneva, 17-24 October 1995

Members Dr D.H. Calam, National lnstitute for Biological Standards and Control, Potters Bar,

Herts., England (Rapporteur)

Dr S.G. Drozdov, Director, lnstitute of Poliomyelitis and Viral Encephalitides, Mos- cow, Russian Federation

Dr I.D. Gust, Research and Development .Director, CSL Ltd, Parkville, Victoria, Australia

Dr Z. Khan, Principal Scientific Officer, Quality Control Division, National lnstitute of Health, Islamabad, Pakistan

Dr J.G. Kreeftenberg, Bureau for lnternational Cooperation, National lnstitute for Public Health and Environmental Protection, Bilthoven, Netherlands (Vice- Chairman)

Dr J.M.Y. Niqur, Director, National Blood Bank, al-Ashrafieh, Amman, Jordan

Dr F.A. Ofosu, Department of Pathology, McMaster University, Hamilton, Ontario, Canada

Dr J.C. Petricciani, Vice-President, Genetics Institute, Cambridge, MA, USA (Chair- man)

Mr Zhou Hai-jun, Director, National lnstitute for the Control of Pharmaceutical and Biological Products, Temple of Heaven, Beijing, China

Representatives of other organizations Council of Europe Mr J.-M. Spieser, Principal Scientific Officer, Biological Standardization Depart-

ment, European Pharmacopoeia Commission, Council of Europe, Strasbourg, France

lnternational Association of Biological Standardization Professor F. Horaud, Pasteur Institute, Paris, France

lnternational Federation of Clinical Chemistry Dr A. Deom, Liaison Officer with WHO, Responsible Officer, Quality Assurance,

Cantonal Hospital, Geneva, Switzerland

lnternational Federation of Pharmaceutical Manufacturers Associations Dr M , Duchsne, Director, Quality Control and Regulatory Affairs, SmithKline

Beecham Biologicals, Rixensart, Belgium Dr B. Montagnon, Head, Registration, Pasteur Merieux Sera and Vaccines, Marcy

I'Etoile, France

lnternational Society on Thrombosis and Haemostasis Dr T.W. Barrowcliffe, National lnstitute for Biological Standards and Control, Potters

Bar, Herts., England

Secretariat Dr A.F. Bristow, National lnstitute for Biological Standards and Control, Potters Bar,

Herts., England (Temporary Adviser)

Dr V. Grachev, Scientist, Biologicals, WHO, Geneva, Switzerland

Dr E. Griffiths, Chief, Biologicals, WHO, Geneva, Switzerland (Secretary)

Dr G.A. Hansen, Statens Seruminstitut, Copenhagen, Denmark (Temporary Adviser)

Dr M.C. Hardegree, Director, Office of Vaccine Research and Review, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD, USA (Temporary Adviser)

Dr J.E. Idanpaan-Heikkila, Director, Division of Drug Management and Policies, WHO, Geneva, Switzerland

Dr A. Padilla, Scientist, Biologicals, WHO, Geneva, Switzerland Dr G. Siegl, Institute for Microbiology and Immunology, St Gallen, Switzerland

( Temporary Adviser) Dr W.G. van Aken, Medical Director, Central Laboratory of the Netherlands Red

Cross Blood Transfusion Service, Amsterdam, Netherlands (Temporary Adviser)

Dr K. Zoon, Director. Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD, USA (Temporary Adviser)

vii

Introduction

The WHO Expert Committee on Biological Standardization met in Geneva from 17 to 24 October 1995. The meeting was opened on behalf of the Director-General by Dr F. S. Antezana, Assistant Director-General.

Dr Antezana drew attention to the increasing proportion of biological and biotechnological products among new medicines and the role of these in controlling diseases of international importance. He noted that WHO's biological standardization activities and the work of the Expert Committee play a vital role in facilitating the transfer of laboratory science into clinical practice worldwide, by providing a means of assuring the quality, safety and efficacy of new biological products, as well as the reliability of diagnostic tests. He thanked the WHO International Laboratories for Biological Standards for their important contribution to WHO'S biological standardization activities. Although WHO was able to provide only a small part of the cost of the programme, this fact did not reflect the importance of biological standardization or WHO's level of commitment to it. Dr Antezana singled out the National Institute for Biological Standards and Con- trol, Potters Bar, for its important work not only as custodian for WHO of international reference materials but also for its extensive programme of technical development and associated research undertaken in support of international biological reference materials and requirements for biological substances. He thanked the Government of the United Kingdom and its National Biological Standards Board for their generous support of and commitment to this activity. Finally, Dr Antezana informed the Committee that steps were being taken to publicize the activities of the Committee and its decisions in a more timely manner.

General

Development and distribution of International Biological Standards and Reference Reagents

The Committee noted ( ~ ~ ~ 9 5 . 1 8 2 2 ' ) the distribution of international reference materials by the WHO International Laboratories for Bio- logical Standards during 1994 (Table 1). The overall breakdown of

' References prefixed "BSI. . ." and "BLG!. . ." are t: ur~ub! ished ~.vorking documents of the World Health Organization. They are .;ot issued io the genera; public, but a limited number of copies may be avaiiable to professionaliy interested persons on application to Biologicals, World Hea'ih Organization, 121 1 Geneva 27, Smiitzerland.

Table 1 lnternational Biological Standards and Reference Reagents distributed in 1994 to laboratories in WHO regions by the WHO lnternational Laboratories for Biological Standardsa

Number of items distributed by WHO International Laboratories for Biological Standards

Recipient WHO % of total region Amsterdam Copenhagen Potters Bar Weybridge Total for all regions

Africa 36 50 78 8 172 1.6

Americas 96 239 1977 12 2 324 21.2

Eastern Mediterranean

Europe 1101 855 4 564 201 6721 61.4

South-East Asia

Western Pacific

P P

Total 1311 1465 7 920 254 10 950 100.0

"Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam, Netherlands: items distributed during the calendar year 1994. Statens Seruminstitut, Copenhagen, Denmark: items distributed during the calendar year 1994. Nat~onal Institute for Biological Standards and Control, Potters Bar, Herts., England: items distributed between 1 April 1994 and 31 March 1995. Central Veterinary Laboratory, Weybridge, Surrey, England. items distributed during the calendar year 1994.

distribution of these materials was about 70% to national control laboratories and research institutes and about 30% to industry, but proportions differed according to the nature of the materials. The Committee also noted a total expenditure exceeding US$4000000 by the International Laboratories on activities related to biological standardization, including work in the development of reference materials. Information about such expenditures had been requested by the Committee in 1994 and was being presented for the first time. The Committee again emphasized the importance of these activities to the international harmonization of reference materials and requirements for biological substances. It also reaffirmed the need for collaborative studies undertaken for establishing international reference materials to involve laboratories from a wide geographical area and to be truly international.

The Committee was informed of a meeting of the directors of the WHO International Laboratories for Biological Standards. This meeting had been very valuable, and it had been suggested that such a meeting should take place regularly. Among the matters discussed was the basis for and level of the handling charges levied by the laboratories. Although all national control laboratories receive sup- plies of biological reference materials free of charge, the WHO In- ternational Laboratories for Biological Standards now impose a handling charge on other users. It was emphasized that this charge did not, and was not intended to, approach the true cost of preparation, establishment and distribution of these reference materials. The Committee recognized that a balance had to be reached between recovering distribution costs and setting handling charges that would deter laboratories from employing the reference materials. Since the International Laboratories were each free to set their own handling charges, the flexibility necessary to achieve this balance was possible. The Committee emphasized, however, that mechanisms should be available to allow the distribution of international reference materials at minimal or at no cost to users, especially in countries with more limited resources where users might have difficulty in meeting the full handling charge.

In view of problems that had arisen when the WHO International Laboratories were not also the official custodians of certain international reference materials, the Committee recommended that in future, each established reference material should be held by a single centre recognized by WHO. This arrangement would have several advantages, significantly among them that it would be easier for those intending to use a particular reference material to identify the custodian laboratory, and also that potential problems of inhomogeneity of

materials held in different places and possibly subjected to different storage conditions, as well as possible differences in handling charges, would be eliminated.

Need for interim standards with an official status conferred by WHO

The Committee was informed that the speed of development of new biological products, particularly cytokines and related substances, had revealed the need, often before the full programme for establishment of an International Standard could be completed, for the development of interim reference materials with an official status conferred by WHO. This need arose from a combination of regulatory and scientific considerations. At the recent WHO Informal Con- sultation on Cytokine Standards (April 1995), a request had been made to the Committee to consider whether such interim standards with an official status conferred by WHO could be established. This situation had not been envisaged when the 1989 "Guidelines for the preparation, characterization and establishment of International and other Standards and Reference Reagents for biological substances" (WHO Technical Report Series, No. 800, 1990, Annex 4) had been prepared. The Committee agreed that a need for such reference materials existed. In order to shorten the time between the preparation of a candidate material and its distribution, it would usually be sufficient for a limited number of laboratories to examine a characterized preparation and agree to the assignment of a potency expressed as units. The Committee agreed that a report should be submitted by the relevant International Laboratory to the Biologicals unit of WHO, which would either endorse the recommendation and report this action to the Committee at its next meeting or submit the report directly to the Committee for a final decision.

The Committee concluded that reference materials adopted in this way should be designated interim Reference Reagents (as distinct from International Reference Reagents) and be assigned a potency expressed as units. Only when such a material was finally established as an International Standard would the potency be expressed as Inter- national Units. Recognizing that these decisions represented an ex- tension of the existing guidelines, the Committee recommended that the guidelines be reviewed.

Cytokines

The Committee noted the report of the WHO Informal Consultation on Cytokine Standards that had been held in April 1995 (BSl9511824). This was the first meeting of the group constituted as proposed by the Committee in its forty-fifth report (WHO Technical Report Series,

No. 858, 1995, p. 5). Among the topics discussed were the regional production of cytokines, requirements for collaborative studies, issues concerning current reference materials (in particular interferon~), problems of standardization of immunoassays and the clinical consequences of the use of potency values for cytokines.

The Committee was informed that collaborative studies were planned for stem cell factor and interleukin-10, interleukin-l1 and interleukin- 12. In addition, several cytokines had been selected as priorities for action. Since relative priorities can change rapidly in the light of clinical and other information, the Committee agreed with a proposal that decisions on priority should be taken at the annual WHO Infor- mal Consultation on Cytokine Standards and then reported to the Committee.

The Committee was also informed that, because of problems associated with the existence of several established reference materials for interferons alfa, a very large collaborative study was being organized. Because of many projects competing for resources in the cytokine field (see Cytokines, pp. 25-26), and the limited resources available, the Committee reiterated concerns expressed in its previous report (WHO Technical Report Series, No. 858, 1995, p. 5) about large collaborative studies, drew attention to the time and effort that they consumed and again suggested that these resources might be better deployed in smaller studies on more products. The Committee also emphasized the recommendations about numbers of participating laboratories given in the "Guidelines for the preparation, characterization and establishment of International and other Standards and Reference Reagents for biological substances" (WHO Technical Re- port Series, No. 800, 1990: Annex 4).

The Committee fully recognized that the experience gained through the work on interleukin-8 and the extensive work involving other cytokines confirmed the need for a thorough and urgent review of the current procedures for the development, examination and establishment of International Standards and other reference materials. In particular, every effort is necessary to improve the efficiency and effectiveness of biological standardization activities and to ensure that the need for reference materials, once identified, is met in a timely manner.

The Committee strongly urged that the design and aims of the planned collaboratir~e study on interferons alfa should be re- examined in order to reduce its complexity and to concentrate on the main purpose for which it is required. The Committee recommended that only antiviral assays should be included.

The Committee noted that the Informal Consultation on Cytokine Standards had made several recommendations about the use of reference materials for interferons. One recommendation was that the International Reference Preparation of Interferon, Human, Leuko- cyte, coded 69/19, a preparation of interferon alfa, should continue to be used as the primary reference material but should be employed only in antiviral assays. It was proposed that manufacturers of preparations of all types of interferon alfa should continue to calibrate their working standards in terms of this Reference Preparation (691 19) and then use the working standards to calibrate anti-proliferation assays. The Committee endorsed this proposal and suggested that appropriate information for users should be distributed with the preparation. The Committee recognized that use of the Reference Preparation should be reviewed when the results of the planned study on interferons alfa had become available.

In view of the conclusions of the Informal Consultation on Cytokine Standards, the Committee confirmed its previous recommendation that the potency of all new preparations of human interferon beta should be determined relative to the second International Standard for Interferon, Human, Fibroblast, P. The Committee also recommended that any distribution of the first International Standard for Interferon, Human, Recombinant, P,,,,,, should be accompanied by a statement that this preparation is not suitable for relative potency assays.

The consultative group had recommended that the International Standard for Interferon, Human (HuIFN-y), which is a preparation of human gamma interferon, should be discontinued, as stocks were depleted and another suitable standard was available (see p. 26). The next informal consultation was planned for 1996.

Standardization and priorities for action in the field of diagnostics

The Committee was informed that, following the request for information in its forty-fifth report (WHO Technical Report Series, No. 858, 1995, p. 5), three areas of worldwide interest for standardization had been identified in the field of diagnostics:

- standardization of gene-amplification methods for the viral-safety testing of blood and blood products;

- biological reference materials used in the calibration of antibody measurements for microbiological diagnostic procedures;

- development of reference materials for evaluating hepatitis B and C diagnostic kits.

The Committee was also informed of the formation of an informal Working Group to promote the development of reference materials for use in gene-amplification methods for the screening of blood and blood products. Candidate materials for the detection of hepatitis C virus were under evaluation, and a study on proficiency testing for the detection of human immunodeficiency virus (HIV) was planned. A WHO Informal Consultation was planned to assess the need for reference materials and panels of reference materials for the detection of hepatitis B surface antigen and antibodies to hepatitis C virus and HIV in blood and blood products.

The Committee was further informed that an International Work- shop had been held in Copenhagen, organized by the Statens Seruminstitut, to consider the need for reference materials for antibody measurement in microbiological diagnostic procedures. There had been problems with the standardization of serological measurement systems that needed to be addressed. Some of these problems reflected the fact that many international reference materials used for serodiagnostic systems had been established decades ago and the need for better characterized serum standards defining International Units for IgG and IgM antibodies had since been identified.

The Committee thanked the Secretariat for obtaining this information. The Committee agreed that priority should lie in the areas of testing blood and blood products because of their importance for public health. Particular efforts should be directed to testing for hepatitis B and C and HIV and to the establishment of suitable reference materials for these purposes. The Committee again recognized the difficulty of maintaining a balance of priority between different areas, and the dilemma that this presented to the International Labora- tories. The Committee strongly encouraged the Secretariat to streng- then and extend collaboration both within and outside WHO in order to facilitate progress in the area of diagnostic testing.

Advice to United Nations agencies on the quality of vaccines

WHO, taking into account the recommendations and conclusions of the Expert Committee on Biological Standardization, provides advice to United Nations agencies on aspects of good manufacturing practice and compliance with requirements for vaccines intended for use in United Nations programmes. The Committee expressed concern over the possibility for inconsistencies between recommendations for vaccine use and handling in the field and the requirements for those products adopted by the Committee. The Committee emphasized that instructions concerning storage temperature, expiry date and use

should be based on experimental evidence obtained with individual vaccines and should be approved by the relevant national control authority. The Committee therefore emphasized the importance of close liaison between different units of WHO involved in vaccination programmes.

Yellow fever vaccine

The Committee noted (BSl95.1823) that a review had been carried out of the producers of yellow fever vaccine listed in its forty-second report (WHO Technical Report Series, No. 822, 1992, Annex 5). Certain laboratories had been inspected, and others had ceased production. The Committee was informed that, as a result, changes had been made to the list of laboratories approved by WHO for the production of yellow fever vaccine and agreed that the revised list should be annexed to its report (Annex 1).

Reverse-transcriptase activity associated with avian cells

The Committee was informed of a WHO Informal Consultation including certain of its members, representatives of manufacturers and regulatory authorities, and independent scientific experts, held to consider the implications of the detection of reverse-transcriptase activity in some live, attenuated vaccines produced from avian cells. This activity had been detected using a new test which is 106-107 times as sensitive as previous tests. Further work is under way to determine whether this activity is associated with a virus and if so to investigate the ability of such a virus to replicate in human tissue.

Taking into account the outcome of the Consultation, the Committee concluded that the data currently available strongly suggest that existing requirements published by WHO for the manufacture and control of vaccines produced in chicken cells are appropriate. It also concluded that such vaccines should continue to be used to prevent the diseases against which they are directed, since their beneficial effect in reducing the morbidity and mortality associated with those diseases is well established and there is no direct evidence to suggest that they contain an avian virus of medical significance to humans.

Glossary of terms

The Committee noted that a draft glossary of terms used in the texts of requirements for biological substances (BSl95.1793) had been prepared by the Secretariat in response to a request made by the Com- mittee at its forty-first meeting (WHO Technical Report Series, No. 814, 1991, p. 13). The glossary had been intended solely for use in preparing requirements for biological substances and it was hoped that its availability would prevent lengthy discussions in the Commit-

tee about the meaning of terms in the requirements, as well as help in their drafting. The Committee was informed that, following circula- tion of the draft glossary, a number of comments had been received.

The Committee agreed that the availability of such a glossary was a valuable development. However, it expressed concern about a possible conflict between the definitions given there and the perceived meanings of the same terms in other contexts. To minimize any possible confusion, the Committee agreed that the glossary should not be published but should be used only internally. This arrangement would also allow for continuous revision and updating of the glossary without the need for formal amendment. The Committee further agreed that the objective of the glossary should be to achieve harmonization but not necessarily uniformity and that conflict between the definitions given in it and other recognized definitions should be avoided. The Committee recognized that availability of the glossary would imply the need to check and possibly revise existing requirements but accepted that, in view of the magnitude of this task, changes should be made prospectively as requirements are drafted or revised.

The Committee recommended that the Secretariat should attempt to harmonize the glossary definitions with those of the International Conference on Harmonisation and other appropriate bodies and that the Secretariat should be kept informed of possible additions to the glossary. The Committee approved the glossary for use in the formulation of requirements for biological substances and recommended that it should be made available to enquirers on request.

Review of biological standardization

The Committee discussed the need to review the nature, structure and procedures of biological standardization activities in view of far- reaching changes occurring in the pharmaceutical industry (especially the growth of biotechnology)? regulatory processes and the role and funding of the International Laboratories for Biological Standards. The Committee was informed that the governing body of the National Institute for Biological Standards and Control, Potters Bar, i.e. the National Biological Standards Board of the United Kingdom, was about to initiate an international scientific review of the field of biological standardization with the aim of identifying possible future developments and their implications. It was hoped that the final report of the ~ o a r d l would be available during the summer of 1996 and

' Issued as unpublished document Biological standardization and control. A scientific review commissioned by the UK National Biological Standards Board (NBSB). Geneva, World Health Organization, 1997 (WHO/BLG/97.1); available from Distribution and Sales, World Health Organization, 1271 Geneva 27, Switzerland.

that it would be submitted to the Committee, for its information and consideration, at its next meeting. The Committee welcomed and supported this initiative and the opportunity to consider its implications for biological standardization activities. The Committee was further informed that a review of the WHO Division of Drug Man- agement and Policies, including the Biologicals unit, would be carried out by the Executive Board of WHO early in 1996. In view of these activities, the Committee agreed that its own functions and procedures should be considered in depth at its next meeting.

Committee meetings

In view of the heavy agenda at its meetings, the Committee discussed ways in which the efficiency of its proceedings could be improved. The Committee confirmed its previous recommendation that annual progress reports on individual projects should be submitted only in exceptional circumstances. Since all progress reports intended for the Committee have to be circulated to the relevant WHO Expert Advisory Panel and the contents of some do not differ significantly from one year to the next, compliance with this recommendation would simplify the work of both the Secretariat and the full Commit- tee. However, the provision of a simple list of projects from each International Laboratory with an indication of the stage reached for each would be helpful. Since it was intended that the directors of these laboratories should in future meet annually, the Committee suggested that summary status reports should be prepared for these meetings and, further, that an amalgamated summary should be presented by the Secretariat at the meeting of the Expert Committee. The Committee further advised the Secretariat to screen individual progress reports received by it and to process only those complying with the above recommendation.

The Committee further requested the Secretariat to investigate the possibility of distributing documents and reports for its meetings by means of electronic media.

Requirements for biological substances' Requirements for cell substrates used for biologicals production

The Committee noted a draft of proposed Requirements for Cell Substrates used for Biologicals Production (BSl95.1792) prepared by the Secretariat. The document had been circulated to members of

' For a list of all the requirements for biological substances and other sets of recommendations, see Annex 6.

WHO'S Expert Advisory Panel on Biological Standardization and a number of comments had been received. It was also informed that similar guidance documents were being prepared by other bodies. After making some modifications to the draft, the Committee requested that the revised draft should be submitted to the Interna- tional Conference on Harmonisation, appropriate control authorities, and a forthcoming WHO Consultation on the Safety of Biological Products Prepared from Mammalian Cell Substrates, and subse- quently resubmitted to the Expert Advisory Panel for reconsidera- tion. The draft should then be reviewed again by the Committee in 1996. It was recognized that, although the aims of the related initia- tives were broadly similar, the resulting documents would be addressed to different users. However, it was hoped that any differences would be of detail and not substance.

Requirements for oral poliomyelitis vaccine The Committee was informed of the outcome of two meetings held under the auspices of WHO on alternative models .for the neurovirulence test for Type 3 virus contained in the Requirements for Poliomyelitis Vaccine (Oral) (WHO Technical Report Series, No. 800, 1990, Annex 1). In the first model, a study in 14 laboratories on the use of a MAPREC method (mutant analysis by polymerase chain reaction and restriction enzyme cleavage) had given identical results with several samples to those of the existing neurovirulence test. In the second model, encouraging results had been obtained with a neurovirulence test using transgenic mice in place of monkeys. The Committee recognized the great importance of these developments, which represented the first substantial progress after years of effort to find potential alternatives to the current test. However, insufficient data are available at present to permit any amendment to the existing Requirements with respect to the neurovirulence test in monkeys. The Committee nevertheless congratulated those involved in this work and recommended that WHO should monitor fur-ther developments. The Committee specifically endorsed the proposal that the MAPREC procedure should continue to be explored. Further data ~7ould be collected for vaccines produced from Type 3 Sabin virus, with the aim of reviewing the status of the MAPREC test in 1997. Further research on MAPREC analysis of vaccines produced from Type 1 and Type 2 Sabin virus is also needed, to establish the value of the method in assessing the overall quality and consistency of Sabin vaccine.

Requirements for snake antivenoms The Committee was informed of an International Congress on Envenomations and their Treatments held in Paris during June 1995,

following which a WHO Working Group on Envenomations and their Treatments had begun drafting guidelines for the assessment and treatment of venomous bites and stings. These guidelines would include an annex on the production and quality control of antivenoms. The Committee was also informed that venomous bites and stings were a public health problem of increasing significance.

The existing Requirements for Snake Antivenins, adopted in 1970 (WHO Technical Report Series, No. 463, 1971, Annex l ) , now required revision. The Secretariat had proposed that, in view of the above developments, an informal consultation should be held in 1996 to draft a revision of these requirements. The Committee endorsed this proposal and drew attention to the need to include consideration of scorpion and spider antivenoms in the document.

Requirements for thromboplastins and plasma used to control oral anticoagulant therapy

The Committee was informed that the Requirements for Thrombo- plastins and Plasma used to Control Oral Anticoagulant Therapy (WHO Technical Report Series, No. 687,1983, Annex 3) had proved valuable but required revision in view of changes in methodology and practice. At its meeting in June 1995, the Subcommittee for Control of Anticoagulation of the Scientific and Standardization Committee, International Society on Thrombosis and Haemostasis, had formally requested WHO to undertake such a revision. The Secretariat proposed to prepare such a document for the consideration of the Com- mittee in due course. The Committee endorsed this suggestion.

The Committee was further informed of an offer to WHO of a com- puter program designed to enable laboratories to perform the calculations required to determine thromboplastin in accordance with the above-mentioned requirements, and that use of the program was being evaluated. The Committee expressed its thanks for this generous offer.

Requirements for yellow fever vaccine The Committee noted a revised draft (BSl94.1758 Rev. 1) of the Requirements for Yellow Fever Vaccine, which had been prepared by the Secretariat in the light of advice obtained from a number of experts in response to the Committee's request at its forty-fifth meeting (WHO Technical Report Series No. 858,1995, p. 8). After making some modifications, the Committee adopted the revised text as Re- quirements for Yellow Fever Vaccine and agreed that it should be annexed to its report (Annex 2).

General requirements for the sterility of biological substances (amendment, 1995)

The Committee noted the draft revision (BSl95.1794) prepared by the Secretariat of section 5.3, Sterility test for mycoplasmas, in the Gen- eral Requirements for the Sterility of Biological Substances (WHO Technical Report Series, No. 530,1973, Annex 4). The draft revision had been circulated to members of WHO'S Expert Advisory Panel on Biological Standardization and a number of comments had been received. The Committee made some modifications to the draft revision, adopted the text as part of the General Requirements for the Sterility of Biological Substances and agreed that it should be annexed to its report (Annex 3).

Guidelines for minimizing the risk of transmitting agents causing bovine spongiform encephalopathy

The Committee was informed of a WHO Consultation on Public Health Issues related to Human and Animal Transmissible Spongi- form Encephalopathies held in May 1995. At that meeting, it had been proposed that an informal consultation of specialists should be convened by WHO to review current recommendations on minimizing the risk of transmitting agents causing BSE by means of medicinal products and medical devices. Continuing research on various aspects of the diagnosis and pathogenesis of BSE should also be encouraged. The Committee endorsed the organization of such a consultation and agreed that the preparation of suitable recommendations would be valuable.'

Guidelines for the production and control of synthetic peptide vaccines

The Committee was informed that draft guidelines for the production and control of synthetic peptide vaccines had been drawn up by the

' A previously unrecognized form of Creutzfeld-Jakob disease was identified in 10 patients in the United Kingdom following the May 1995 WHO meeting. Recommendations from WHO consultations since held have called for, among other things, the pharmaceutical industry to obtain bovine materials from countries with a BSE-surveillance system in place, and where either no or only sporadic cases of BSE have been reported. A further meeting took place in early 1997 to review WHO recommendations on medicinal products, medical devices, food, and other products of bovine origin. The report oi the meeting nas been issued as unpublished document Report of a WHO Consultation on Mec'icirrai and other Products in Relation to Human and Animal Transmissible Spongiform Encephalopafhies. With the Participation of the Office International des Epizooties (OIE). Geneva, Switzerland, 24-26 March 1997. (WHOlBLGl97.2; available on request from Biologicals, World Health Organization, 121 1 Geneva 27, Switzerland).

Secretariat in consultation with experts and that similar guidelines had also been prepared by the Food and Drug Administration (USA). The Committee recognized a need for these guidelines because of the rapid rise in the availability of materials for clinical trials, often from small producers and academic institutions that lacked experience in implementing quality assurance. The interpretation of data from clinical trials would be compromised if the consistency from batch to batch of the material employed could not be relied upon. At the same time, the guidelines would also be of value as a source of information to regulatory authorities. The Committee welcomed this initiative and strongly encouraged the Secretariat to continue this activity.

Summary protocol for the batch release of virus vaccines

The Committee noted a draft summary protocol (BS195.1795) for the routine batch release of virus vaccines prepared by the Secretariat in an attempt to clarify the documentation required to facilitate the routine distribution of these products. Although examples of summary protocols are sometimes included for guidance in the relevant requirements for biological substances, such protocols are often quite comprehensive and are not necessarily appropriate for the routine batch release documents that accompany each lot of material. The Committee agreed that the purpose of the draft summary protocol was to indicate clearly the type of information that might be included in the routine documentation for lot release. Some countries and international agencies require such documentation to be in conformity with the requirements for the relevant biological substance published by WHO. The Committee recommended substantial modification of the draft summary protocol, adopted the revised text and agreed that it should be annexed to its report (Annex 4).

Antibiotics

Gentamicin

The Committee noted the report (BSl95.1811) on a collaborative study performed by nine laboratories in nine countries on the candidate replacement material for the first International Reference Preparation of Gentamycin (International Nonproprietary Name: Gentamicin). On the basis of the results of the study, it established the preparation coded 921670 as the second International Standard for Gentamicin and assigned a potency of 31020 International Units to

the contents of each ampoule.' The Committee was informed that assignment of potency on this basis would overcome the problems associated with weighing portions of the hygroscopic first Interna- tional Reference Preparation, for which an International Unit was defined in terms of a declared weight of the Reference Preparation.

Anti bodies

Anti-borrelia serum

The Committee was informed that, in response to the request at its forty-fifth meeting (WHO Technical Report Series, No. 858, 1995, p. 10), the Statens Seruminstitut, Copenhagen, had confirmed the need for reference materials for anti-borrelia sera prepared from both acute, early-phase serum (predominantly IgM antibodies) and convalescent-phase serum (predominantly IgG antibodies) and would be seeking to identify suitable candidate materials.

Anti-cytomegalovirus immunoglobulin and serum

The Committee noted (BSl95.1814) the report on a two-part collaborative study on a candidate International Standard for anti- cytomegalovirus immunoglobulin. The Committee also noted that the clinical efficacy of treatment with anti-cytomegalovirus immunoglobulin had not been proved. As the urgent need for such a reference material had therefore not been established, the Committee requested that the results of the collaborative study should be evaluated further and another report submitted. Although the Committee agreed that the proposed International Standard could continue to be distributed by the Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam, it determined that it should not be used to assign potencies to plasma products and is suitable only for validating the consistency of production of immunoglobulin preparations.

The Committee noted that further reference materials are required for evaluating diagnostic kits used for measuring anti- cytomegalovirus antibodies in sera (BSl95.1797). The Committee therefore requested the Statens Seruminstitut, Copenhagen: to obtain pools of sera containing high titres of anti-cytomegalovirus IgM and IgG, respectively, and to arrange collaborative studies.

' The new reference material is designated the "second" International Standard in accordance with the provisions agreed by the Committee at its thirty-seventh meeting (WHO Technical Report Series. No. 760, 1987, p. 16).

Anti-hepatitis B core antigen serum Antibodies to hepatitis B core antigen (anti-HBc) can be detected in both acute and chronic infection with hepatitis B virus. The Commit- tee noted the need for a reference material containing anti-HBc in order to calibrate diagnostic kits used in the microbiological screening of serum (BSl95.1817). Commercial test kits for the detection of anti- HBc are available, but at present there is no international reference material to facilitate the comparison of kits or results obtained in different laboratories.

The Committee was informed that the National Institute for Bio- logical Standards and Control, Potters Bar, had obtained freeze- dried plasma containing both anti-HBc and antibodies to hepatitis B surface antigen (anti-HBs). The Committee requested the Institute to organize a collaborative study in order to assess the suitability of this preparation to serve as a reference material for anti-HBc antibodies. It further requested the Institute to explore the possibility of using the same preparation as a reference material for anti-HBs.

Anti-hepatitis E virus serum The Committee was informed that hepatitis E virus is a major caus- ative agent for enterically transmitted non-A, non-B hepatitis and that many outbreaks have been reported. It was also informed that a reference material is required to aid the comparison of assays for hepatitis E antibodies developed in different laboratories. The Com- mittee therefore requested the National Institute for Biological Stan- dards and Control, Potters Bar, in collaboration with the Centers for Disease Control and Prevention, Atlanta, to obtain a candidate reference material and organize a collaborative study.

Anti-measles serum The Committee noted the need for a reference preparation of anti- measles IgM serum to standardize the rapid diagnosis of measles infections by measurement of measles-specific IgM. The second International Standard for Anti-measles Serum is a preparation of convalescent-phase human serum unsuitable for this purpose (BSI 95.1815). The Committee was informed of the possibility of collecting suitable sera as part of an epidemiological study already in progress. The Committee therefore requested the Statens Seruminstitut, Copenhagen, to obtain a suitable candidate reference material and arrange a collaborative study.

Anti-mumps serum

The Committee was informed that, in accordance with the request made at its forty-fourth meeting (WHO Technical Report Series No. 848, 1994, p. l l ) , the Statens Seruminstitut, Copenhagen, had now obtained quantities of both acute, early-phase human serum (predominantly IgM antibodies) and convalescent-phase human serum (predominantly IgG antibodies) to serve as candidate reference materials and that the sera were currently being characterized.

Anti-parvovirus B1 9 IgG serum The Committee noted the report on a collaborative study on a convalescent-phase human serum pool intended as a candidate reference material for anti-parvovirus B19 IgG serum, carried out in nine laboratories in seven countries (BS195.1810). On the basis of the results of the study and the evidence of the stability of the preparation, the Commiltee established the preparation coded 931724 as the first International Standard for Anti-Parvovirus B19 IgG Serum, Human, and assigned a potency of 100 International Units to the contents of each ampoule. The standard is not suitable for use in calibrating assays to detect evidence of recent infection (IgM antibodies).

The Committee noted that during the collaborative study a sample of serum from an individual had demonstrated non-parallelism compared with the candidate International Standard. This observation could be indicative of a general problem in comparing serum samples from individuals with sera from serum pools. The Committee therefore suggested that participants in collaborative studies on antibodies should be encouraged to screen both individual and pooled sera against candidate reference material pools wherever possible and to report any differences observed.

The Committee further noted that the National Institute for Biologi- cal Standards and Control, Potters Bar, had reported the possible need for an anti-parvovirus B19 reference material consisting predominantly of IgM antibodies. It therefore requested the Institute to attempt to collect such a candidate material, although it recognized that it may be difficult to identify suitable individuals to provide donations of serum.

Anti-pertussis serum The Committee noted that a preparation of human anti-pertussis serum (JNIH-10) intended as a candidate reference material had proved reactive in anti-HIV tests (BSi95.1798). The Committee was informed that acellular pertussis vaccines, containing as many as five

different antigens, are currently being introduced. It was also informed of a forthcoming International Symposium to discuss the results of vaccine clinical trials, as well as a WHO Informal Consulta- tion to discuss requirements and reference materials for acellular pertussis vaccines. The Committee recognized the urgent need for such reference materials and requested the Secretariat to take appropriate action as soon as the outcome of the International Symposium was known.

Anti-rubella immunoglobulin and serum

The Committee noted that a preparation of normal human immunoglobulin was undergoing collaborative study (BS195.1827) as a candidate replacement for the second International Reference Preparation of Anti-Rubella Serum, which had likewise been prepared from normal human immunoglobulin (WHO Technical Report Series, No. 858, 1995, p. 9). The Committee again expressed concern about the nomenclature currently in use for such preparations and reiterated that it should be carefully reviewed.

The Committee further noted that an acute, early-phase serum (predominantly IgM antibodies), as well as a preparation of convalescent- phase serum (predominantly IgG antibodies), were available as candidate reference materials for the calibration of diagnostic kits (BSl95.1812 A, B and BS195.1813 A, B), the need for which had been identified previously (WHO Technical Report Series, No. 858, 1995, p. 9). The Committee encouraged the two WHO International Labo- ratories involved to work towards a joint report on the candidate preparations.

Anti-streptolysin 0 serum

The Committee noted that, although the pilot study on the concen- trated human serum that might have served as a candidate replacement for the first International Standard for Anti-Streptolysin 0, Human, had been satisfactory, the manufacturer was no longer able to provide the amount required (BSl95.1825). The Committee noted that collection of an alternative pool of serum had been completed by the Statens Seruminstitut, Copenhagen.

Anti-toxoplasma serum

The Committee noted that collection by the Statens Seruminstitut, Copenhagen, of a pool of convalescent-phase serum containing predominantly IgG antibodies to toxoplasma was well advanced and that sufficient material should be available by the end of 1995 (BSI 95.1800). This-convalescent-phase serum is intended to complement

the third International Standard for Anti-Toxoplasma Serum, Human (WHO Technical Report Series, No. 858, 1995, p. 9), which is an early-phase serum, unlike the first and second International Stan- dards which were convalescent-phase sera.

Anti-varicella zoster IgG serum

The Committee noted the proposal (BSl95.lS26) that the present Brit- ish Standard for anti-varicella zoster IgG serum be considered as a candidate international reference material to supplement the first In- ternational Standard for Varicella Zoster Immunoglobulin. However, it noted that the data for establishing the British Standard had been obtained from a study in British laboratories only. The Committee also noted that this preparation had demonstrated non-parallelism in comparison with other sera and requested that an evaluation of the reasons for this be made. It recommended a further collaborative study in laboratories with broader geographical distribution.

Thyroid-stimulating antibody

The Committee noted the revised draft report on a collaborative study on the proposed International Standard for Thyroid- stimulating Antibody, a preparation of human origin, carried out in nine laboratories in five countries (BSl95.1818). The Committee was informed that the problems identified when the previous draft had been circulated (WHO Technical Report Series, No. 858,1995, p. 11) had now been resolved and appeared to be due to the different natures of the candidate International Standard and the material against which it had been calibrated. On the basis of the results of the study, the Committee established one preparation, coded 901672, as the first International Standard for Thyroid-stimulating Antibody and assigned a potency of 0.1 International Units to the contents of each ampoule. The Committee noted that the Standard is suitable for calibration of bioassays of thyroid-stimulating antibody and receptor assays of thyroid binding. The Committee was informed that the presence of low levels of chorionic gonadotropin in the Standard, which could exhibit activity under some assay conditions, would be indicated in the information distributed with the Standard.

Antigens and related substances Pertussis vaccine (whole cell)

The Committee was informed that demand was continuing for a reference material for whole-cell pertussis vaccine. The candidate replacement for the second International Standard for Pertussis

Vaccine, mentioned in the previous report (WHO Technical Report Series, No. 858, 1995, p. 13), had exhibited satisfactory potency and stability. A collaborative study was planned by the Statens Serum- institut, Copenhagen, using a protocol agreed to by the Food and Drug Administration (USA) and the National Institute for Biological Standards and Control, Potters Bar. The study would incorporate the discontinued first International Standard, which had shown no decrease in potency relative to the second International Standard in a pilot study performed at the Statens Seruminstitut, Copenhagen.

Poliomyelitis vaccine (inactivated)

The Committee was informed that further work on candidate reference materials for inactivated poliomyelitis vaccine had been undertaken since its previous report (WHO Technical Report Series, No. 858,1995, pp. 13-14). A joint WHOIEuropean Pharmacopoeia Com- mission study had revealed significant variation in the results of immunogenicity assays carried out in guinea-pigs compared with those carried out in chickens, and to a lesser extent, rats. More data were expected on in vivo tests using rats and more work on antigen-capture enzyme-linked immunosorbent-assay (ELISA) methods was envisaged. The Committee gave its support to a proposal that WHO and the European Pharmacopoeia Commission should organize further correlation studies of in vivo and in vitro methods, as well as to a proposal that the Food and Drug Administration (USA) should likewise be involved.

Live attenuated poliovirus The Committee noted a report on the stability of the candidate reference material for live attenuated poliovirus (Sabin) Types 1, 2 and 3 (BSl95.1821). The material, which appears to have suitable stability, had been examined in two collaborative studies reported previously to the Committee (BS189.1615 and BSl90.1651). The material had also been included in a further study organized under the auspices of the European Pharmacopoeia Commission. The Committee established the preparation, coded 851659, as the first International Reference Reagent for Live Attenuated Poliovirus (Sabin) Types 1 , 2 and 3. The contents of each ampoule in terms of median cell-culture infective dose (CCID,,) were assigned as:

- 6.610g10 CCID,, (i.e. 4.0 X 106 CCID,,) per m1 for type 1 virus - 5.610glo CCID,, (i.e. 4.0 X 105 CCID,,) per m1 for type 2 virus - 6.210g10 CCID,, (i.e. 1.6 X 106 CCID,,) per m1 for type 3 virus - 6.810glo CCID,, (i.e. 6.3 X 106 CCID,,) per m1 for total virus.

This International Reference Reagent is appropriate for the valida- tion of secondary reference materials or for determination of the infectivity of oral poliovirus vaccine.

The Committee was informed that an extensive 12-month study was being conducted by the European Pharmacopoeia Commission with respect to the batch release system used in the European Union in order to identify a reference material that could serve to determine the relative infectivity of poliomyelitis vaccine. The Committee recommended a review of the possible uses of the above-mentioned International Reference Reagent after the results of the latter study had been obtained.

Blood products and related substances

Factors II, VII, IX and X in plasma The Committee noted the results of a collaborative study on a single preparation of human plasma with respect to factors 11, VII, IX and X. Eleven laboratories studied the preparation with respect to factors 11, IX and X and ten laboratories with respect to factor V11 (BS195.1808 and BSi95.1808 Add.1). The Committee also noted that the material had exhibited good stability. In view of the urgent need for such a reference material, the Committee agreed that it could be distributed as a candidate replacement for the first International Standard for Blood Coagulation Factors 11, VII: IX and X in Human Plasma once the study participants had agreed on assigned potencies. The Commit- tee requested that a single report be submitted to its next meeting to permit the formal establishment of the material as an International Standard. The suitability of the material for the assay of plasma that has undergone viral-inactivation procedures should also be examined.

Factor lXa

The Committee was informed that the collaborative study mentioned in its previous report (WHO Technical Repost Series, No. 858, 1995, p. 15) had confirmed the need for a standard to improve the agree- ment between assays for activated factor IX (factor IXa) (BSI 95.1802). The Committee, while recognizing that problems of stability exist, therefore requested the National Institute for Biological Stan- dards and Control, Potters Bar: to obtain candidate materials of purified human factor IXa and organize a collaborative study.

International working standards in blood coagulation

The Committee was informed of an offer to WHO of part of a batch of 30000 vials of a preparation of purified factor IX concentrate as a

candidate International Standard. The remainder of the batch was intended for use as Food and Drug Administration (USA) and Euro- pean Pharmacopoeia Commission standards. The Committee was in favour of using a large batch of a single preparation to serve different users, as this would facilitate the comparison of measurements, methods and products. A study on this preparation of purified factor IX concentrate was expected to be completed by the end of 1995. Although the Committee was informed that adequate stocks of the second International Standard for Blood Coagulation Factors 11, IX, and X Concentrate, Human, remain and that a replacement is not urgently needed, it nevertheless endorsed the harmonization of the biological standardization activities of WHO and other authorities and requested that further information be submitted at its next meeting.

The Committee was also informed of a preliminary offer to WHO of a batch of 170000 vials of a preparation of purified Factor V111 concentrate, which was currently being characterized.

Haemiglobincyanide The Committee noted the need for a replacement for the fifth Inter- national Standard for Haemiglobincyanide (BSl95.1828). Since the Committee had discussed the material at its forty-fourth meeting (WHO Technical Report Series, No. 848,1994, p. 17), it had become apparent that stocks of the fifth International Standard were exhausted and that another preparation (coded 70600) was already being distributed in its stead. The Committee was informed of data characterizing this preparation but considered that they were insufficient to allow its establishment as an International Standard.

The Committee was also informed of an offer to WHO of a portion of a reference preparation of bovine haemiglobincyanide (coded CRM 522) established as a reference material by the Measurement and Testing Programme of the European Commission. The Committee welcomed the offer and requested the Secretariat to obtain further information and to present a formal proposal for the establishment of an International Standard at its next meeting.

Mannan-binding protein The Committee noted that mannan-binding protein is a recently dis- covered component of human blood that is able to bind to microor- ganisms and viruses and, on the basis of a small clinical trial, may have a role as an anti-infective agent (BSl95.1799). The Committee agreed that the availability of an international reference material would permit the comparison of measurements of mannan-binding activity

obtained by different laboratories and different methods. The Com- mittee therefore requested the Statens Seruminstitut, Copenhagen, to continue the investigation of a candidate material and to organize a collaborative study.

Plasminogen-activator inhibitor 1

The Committee noted the report on a collaborative study of a candidate reference material, consisting of recombinant plasrninogen- activator inhibitor 1 (PAI-1) with human plasma as a carrier, in eight laboratories in seven countries (BSl95.1805). On the basis of the results obtained, the Committee established the material, coded 921 654, as the first International Standard for Plasminogen-Activator Inhibitor 1, Plasma, Human, and assigned potencies of 27.5 Interna- tional Units of neutralizing activity against tissue plasminogen activator and 7.0 International Units of neutralizing activity against urinary plasminogen activator to the contents of each ampoule. The Commit- tee requested that the molar concentration of the material should be stated in the information distributed with the standard.

Protein S in plasma

The Committee noted the results of a collaborative study by 16 laboratories in nine countries to calibrate the proposed International Standard for protein S in plasma (BS/'95.1804). The Committee also noted that the overall mean potencies found niere 0.89 Interna- tional Units per ampoule for total protein S antigen: 0.89 Interna- tional Units per ampoule for free protein S antigen and 0.92 International Units per ampoule for protein S function.

The Committee established the material studied. coded 931590, as the first International Standard for Protein S. Plasma. Because of the similarity of results for the different parameters measured, the Com- mittee assigned a single potency of 0.90 International Units of Protein S to the contents of each ampoule.

The Committee noted that only one laboratory in the study was outside Europe and emphasized the importance of having a more global representation in studies aimed at the establishment of international reference materials.

Recombinant ferritin

The Committee noted that the second International Standard for Ferritin, Human, is widely used in the development of assay kits and for the measurement of serum ferritin (BSl95.1807). The Committee also noted that, because of serious difficulties in obtaining sufficient

quantities of uncontaminated material of human origin, the National Institute for Biological Standards and Control, Potters Bar, and the University of Wales, College of Medicine, Cardiff, had examined one preparation of recombinant ferritin, formulated in different ways, to assess its suitability to serve as a replacement International Standard. On the basis of the results of a small collaborative study, one formulation in plasma appe,ared to be most suitable. A large batch of material had been filled into ampoules and a collaborative study was in progress.

Thromboplastin

The Committee was informed of an offer by the United States Phar- macopeia of a preparation of recombinant human thromboplastin as a candidate replacement for the second International Reference Preparation of Thromboplastin, human, plain, and that this candidate reference material had not been calibrated against the current Inter- national Reference Preparation. The Committee was grateful for the offer but did not wish to take any action at present in view of an ongoing collaborative study.

Thromboplastin, rabbit, plain The Committee noted the report on a collaborative study, organized by the University Hospital, Leiden, carried out in 19 laboratories in 15 countries, on a candidate replacement for the second International Reference Preparation of Thromboplastin, Rabbit, Plain, stocks of which were now depleted (BSl95.1796). The candidate material appeared to be stable. On the basis of the results of the study, the Committee established the preparation, initially identified as 1020 and coded RBTl90, as the third International Reference Reagent for Thromboplastin, Rabbit, Plain, and assigned an International Sensi- tivity Index (ISI) of 1.0 to it.'

Tissue factor pathway inhibitor

The Committee noted that, further to the report of its forty-fifth meeting (WHO Technical Report Series, No. 858, 1995, p. 16), the collaborative study to investigate the assay of tissue factor pathway inhibitor in plasma samples and recombinant preparations had been completed (BSl95.1803). Since the results had not clearly demonstrated the need for an international reference material, the Com- mittee agreed that no further action was necessary unless a need

' The new reference material is designated the "third" International Reference Reagent in accordance with the provisions agreed by the Committee at its thirty-seventh meeting (WHO Technical Report Series, No. 760, 1987, p. 16).

associated with the development of therapeutic products arose, in which case the decision should be reviewed.

Tissue plasminogen activator in plasma

The Committee was informed that the second International Standard for Human Tissue Plasminogen Activator (t-PA) was unsatisfactory in calibrating assays of t-PA in complex biological fluids, such as plasma and cell-culture fluids. Alack of parallelism between the dose-response curves of plasma t-PA and purified t-PA seemed to be involved.

The Committee was informed that the National Institute for Biologi- cal Standards and Control, Potters Bar, had performed a collaborative study to compare the t-PA antigen content of a sample of normal plasma with that of a candidate reference material consisting of normal plasma supplemented with recombinant t-PA. However, the results of the study had indicated that the use of a common reference material did not reduce inter-laboratory variability in assays of normal plasma when compared with the variability resulting when their own in-house reference materials were used by the laboratories participating in the study.

Whole blood folate

The Committee noted the need for an international reference material to standardize the measurement of blood folate by different methodologies, and was informed that a pilot collaborative study carried out by the National Institute for Biological Standards and Control, Potters Bar, and St Bartholome\w's Hospital, London, had been conducted on a haemolysate candidate material, prepared in ascorbic acid as a stabihzer(BSl95.1806). The Committee also noted that some ampoules of this preparation had been subjected to secondary desiccation to allow determination of the optimal lyophilization conditions. Accelerated degradation studies had suggested that secondary desiccation may result in greater long-term stability than freeze-drying alone. The pilot study had shown results encouraging enough to warrant further study with respect to the stability and potential for use in assays of the material prepared in ascorbic acid. Accordingly, a large-scale collaborative study of the candidate material was planned.

Cytokines Interferon gamma, human

The Committee noted that stocks of the first International Standard for Interferon, Human (HulFN-y), a preparation of interferon gamma

of human origin, were nearly exhausted (BSl95.1801). The Commit- tee further noted that the first International Standard for Interferon Gamma, Recombinant Human, established in 1994, could be used as the sole International Standard for the calibration of other preparations of interferon gamma used as national or working standards, whether of natural human or recombinant origin. The Committee, following the recommendation of the WHO Informal Consultation on Cytokine Standards (see p. 6), discontinued the first International Standard for Interferon, Human (HuIFN-y) but recommended that a suitable quantity of the remaining stock should be maintained in case it should be required for inclusion in a future collaborative study.

The Committee noted a report on a collaborative study on several preparations of interleukin-8, using a wide range of in vitro bioassays and immunoassays, involving 30 laboratories in 12 countries (BSI 95.1820). The Committee established one of the preparations studied, coded 891520, as the first International Standard for Interleukin-8 and assigned a potency of 1000 International Units to the contents of each ampoule.

Insulin-like growth factor-binding protein 3

The Committee noted the report on a collaborative study on a preparation of recombinant human insulin-like growth factor-binding protein 3, performed by ten laboratories in eight countries (BSl95.1816). The Committee was informed that although the natural substance is glycosylated, at the time the project was initiated the only recombinant material available was non-glycosylated. The collaborative study had shown that the candidate material examined would be of little value, and the Committee decided that the preparation should not be established as an international reference material. However, the Committee requested the National Institute for Biological Standards and Control, Potters Bar, to obtain a suitable preparation of glycosylated material to serve as a candidate reference material and to proceed with a collaborative study.

Endocrinological and related substances

Follicle-stimulating hormone The Committee noted the report on a collaborative study comparing two preparations of recombinant follicle-stimulating hormone (FSH)

and a preparation of purified urinary FSH (urofollitropin) with both the third International Standard for Follicle-stimulating Hormone and Luteinizing Hormone and the first International Standard for Human Pituitary Follicle Stimulating Hormone. The study was carried out by 27 laboratories in 12 countries (BS195.1819). The Commit- tee was informed of the complex relationship between the various reference materials that had been established for the calibration of FSH preparations. One outcome of the collaborative study had been the recognition that the above-mentioned International Standards are not suitable for the calibration of any of the three new preparations and in particular that the third International Standard for Follicle-stimulating Hormone and Luteinizing Hormone is not appropriate for the standardization of urofollitropin. It was also apparent that none of the materials studied was suitable to serve as a standard for the immunoassay of FSH. In view of these results, the Committee established the preparation coded 921512 as the first International Standard for Urofollitropin and assigned a potency of 121 Interna- tional Units to the contents of each ampoule. The Committee also established the preparation coded 921642 as the first International Standard for Follicle-stimulating Hormone, Recombinant, Human and assigned a potency of 138 International Units to the contents of each ampoule. Both International Standards are intended for use in the bioassay, but not immunoassay, of the corresponding FSH preparations.

Because the study had not permitted the establishment of a reference material for the immunoassay of FSH preparations, the Committee requested the National Institute for Biological Standards and Con- trol, Potters Bar, to continue to examine this problem and report to the Committee in due course.

Osteopoietin

The Committee noted that osteopoietin, an osteogenic factor isolated from children's urine, promotes bone regeneration and is of interest as a potential therapeutic agent (BS195.1809). Because of the number of projects competing for priority in the work of the International Laboratories, the Committee wished to avoid a premature commitment of resources. It therefore requested the National Institute for Biological Standards and Control, Potters Bar, to monitor biomedi- cal developments and report to the Committee at an appropriate time regarding the need for an international reference material for osteopoietin.

Toxins

Endotoxin The Committee was informed that the material offered by the United States Pharmacopeia (USP) as a candidate replacement for the first International Standard for Endotoxin for Lirnulus Gelation Tests had been filled into vials. It was pleased to learn that account had been taken of the comments made at its previous meeting (WHO Technical Report Series, No. 858, 1995, p. 20) in designing the collaborative study involving all test methodologies that was about to commence.

0 World Health Organization WHO Technical Report Series, No, 872, 1998

Annex 1 Laboratories approved by WHO for the production of yellow fever vaccine

This list supersedes Annex 5 in WHO Technical Report Series, No. 822,1992.

Bio-Manguinhos Oswaldo Cruz Foundation Rio de Janeiro Brazil

Connaught Laboratories Inc Swiftwater, PA USA

Evans Medical Speke Liverpool England

lnstitute of Poliomyelitis and Viral Encephalitides

Moscow Russian Federation

Pasteur Institute of Dakar Dakar Senegal

Pasteur Merieux Sera and Vaccines Marcy I'Etoile France

Robert Koch lnstitute Berlin Germany

0 World Health Organization WHO Technical Report Series, No. 872. 1998

Annex 2 Requirements for yellow fever vaccine (Requirements for Biological Substances No. 3 , revised 1995)

Introduction

General considerations

Part A. Manufacturing requirements A. 1 Definitions A.2 Certification of the substrain of 17D virus for use in vaccine

production A.3 General manufacturing requirements A.4 Production control A.5 Filling and containers A.6 Control tests on final lot A.7 Records A.8 Samples A.9 Labelling A.10 Distribution and shipping A . l l Storage and expiry date

Part B. National control requirements B . l General B.2 Release and certification

Authors

Acknowledgements

References

Appendix 1 Example, for guidance, of a summary protocol for the production and testing of yellow fever vaccine

Appendix 2 Model certificate for the release of yellow fever vaccine by national control authorities

Appendix 3 Techniques for the potency evaluation of yellow fever vaccine

Introduction

Yellow fever is a viral haemorrhagic fever that, each year, strikes an estimated 200000 persons worldwide and causes an estimated 30000 deaths. The case-fatality rate can reach as high as 20% to 80% in severely ill patients who are hospitalized (l). However, the overall case-fatality rate is lower.

The yellow fever virus is small (35-45nm) and consists of a core containing single-stranded RNA surrounded by a lipoprotein envelope. The yellow fever virus genome has been completely sequenced and has been found to contain 10862 nucleotides (2). The lipoprotein envelope contains a single glycoprotein with type- and group-specific antigenic determinants.

Yellow fever was first distinguished from malaria, dengue and other tropical diseases during a series of epidemics between 1647 and 1649 in Barbados, Cuba, Guadeloupe and Mexico. Recognized yellow fever epidemics have periodically raged since then in parts of the Americas and Africa. In 1900, a commission headed by the American physician Walter Reed confirmed that the disease was transmitted from human to human by the mosquito Aedes negypti, a hypothesis proposed earlier by the Cuban physician Carlos Finlay in 1881 (3).

There are two epidemiological patterns of yellow fever transmission: the urban cycle and the forest cycle (also known as the jungle or sylvan cycle). The two patterns of transmission lead to a clinically identical disease, since the same virus is responsible. In the Americas, the yellow fever virus circulates today by means of an endemic, forest cycle that results in up to several hundred reports of infection in non- immune forest \vorkers per year. In Africa. the virus circulates by means of both urban and forest cycles and periodically breaks out of its endemic pattern to infect large numbers of non-immune persons in the course of major epidemics.

Two live attenuated yellow fever vaccines were developed in the 1930s; the French neurotropic vaccine (FNV), prepared from human virus passaged in mouse brain, and the "17D'' vaccine, prepared from the 17D strain of human virus passaged in embryonated chicken eggs.

Today, 17D vaccine is the only type of yello~v fever vaccine produced, as the use of FNV was found to be associated with a high incidence of encephalitic reactions in children (4). The 17D vaccine was developed by Theiler and Smith in 1937.

More than 200 million doses of 17D vaccine have been administered, and it has proved to be one of the safest vaccines ever developed. This

vaccine has also been shown to be very effective for the control of yellow fever during outbreaks and between epidemics. In 1990, the Global Advisory Group of the Expanded Programme on Im- munization recommended that all countries at risk of yellow fever should incorporate the vaccine in their routine immunization programmes. As of 1992, 16 of 33 such countries in Africa included yellow fever vaccine in their national immunization programmes.

Requirements for yellow fever vaccine (Requirements for Biological Substances No. 3) were first formulated by a WHO Study Group in 1958 (5). The Requirements embodied recommendations made by the first WHO Expert Committee on Yellow Fever Vaccine (6) , and they applied to vaccine prepared from a suitable strain of yellow fever virus. The vaccine was intended to be given by subcutaneous injection. Conformity with these Requirements has been the basis for WHO approval of yellow fever vaccine used for vaccination and revaccination against yellow fever in connection with certification for the purposes of international travel (7), and such approval has been given only to vaccine prepared using seed derived from the 17D strain of yellow fever virus. The Requirements have been used also by national authorities for the control and approval of yellow fever vaccine used in national immunization programmes. In 1969 the twenty- second meeting of the WHO Expert Committee on Biological Standardization agreed that developments in virology in general and in the manufacture and control, of yellow fever vaccine in particular warranted a revision of the existing Requirements, with due consideration of both their national and international application (8). In 1975 the twenty-seventh meeting of the WHO Expert Committee on Bio- logical Standardization formulated revised Requirements for Yellow Fever Vaccine (9). Much experience has been gained with the preparation of yellow fever vaccine since 1975, and a further revision of the Requirements has therefore been prepared.

General considerations

Yellow fever vaccine has been proved to be safe. Only 19 cases of encephalitis temporally associated with yellow fever vaccine prepared from the 17D virus strain have been reported, mostly in children, despite over 200 million doses having been delivered since 1945. Since all but six of these occurred in children immunized at 4 months of age or younger, a review by a panel of experts recommended that yellow fever vaccine should not be routinely given before 6 months of age (10, l l ) .

Formerly, there were some problems associated with under- or over- attenuation of the 17D strain on passage. These problems were resolved by the establishment of a virus seed-lot system. There are a number of substrains in use today for the manufacture of 17D vaccine (12). A detailed study using oligonucleotide mapping and monoclonal antibody analysis of the genetic and antigenic variation among these substrains showed an RNA-sequence homology of 98-loo%, which demonstrates a high degree of genetic and antigenic similarity among substrains (13).

Although all countries now use seed virus free from avian leukosis viruses (ALV), some permit the production of vaccine in embryonated chicken eggs not proved to be free from ALV. This practice may be justified where the cost and difficulty of obtaining ALV-free embryonated eggs would restrict the availability of yellow fever vaccine, particularly in view of epidemiological evidence that shows no relationship between yellow fever vaccination and leukaemia, lym- phoma or other cancers (14). For this reason, the revised Require- ments do not specify ALV-free eggs.

It is important to ensure that new pools of virus seed, whether master or working seed, have levels of neurotropism and viscerotropism within safe limits. The relevant safety test, performed on monkeys, has therefore been retained in the revised Requirements.

One important amendment to the Requirements concerns the inclusion of a test of the thermal stabihty of the vaccine as a requirement instead of as a recommendation (15). Each batch of yellow fever vaccine should accordingly be evaluated by testing for potency before and after storage at 37°C for 2 weeks.

The National Institute for Biological Standards and Control, Potters Bar, distributes the International Reference Preparation of Anti- Yellow-Fever Serum. Such a preparation is needed as a basis for comparison of antibody responses in the monkey neurovirulence test. WHO can provide primary seed virus for the production of yellow fever .vaccine (1 6).

Each of the following sections constitutes a recommendation. The parts of each section that are printed in normal type have been writ- ten in the form of requirements so that, if a national control authority so desires, they may be adopted as they stand as definitive national requirements. The parts of each section printed in small type are comments or recommendations for guidance. To facilitate the licensing and international distribution of vaccines made in accordance with these Requirements, a summary protocol for recording the results of tests is provided as Appendix 1. A model summary protocol

for the routine lot release of virus vaccines is provided as Annex 4 of this volume of the WHO Technical Report Series.

Should individual countries wish to adopt these Requirements as the basis for their national regulations concerning yellow fever vaccine, it is recommended that modifications be made only on condition that the modified requirements ensure at least an equal degree of safety and potency of the vaccine. It is desirable that the World Health Organization should be informed of any such changes.

Part A. Manufacturing requirements

A.I Definitions

A. I . I lnternational name and proper name

The international name shall be "Vaccinum febris Aavae". The proper name shall be the equivalent of the international name in the lan- guage of the country of origin.

The use of the international name should be limited to vaccines that satisfy the requirements formulated below.

A.1.2 Descriptive definition

Yellow fever vaccine shall consist of a freeze-dried preparation of viable, attenuated yellow fever virus (Flavivzrus hominis). The preparation shall satisfy all the requirements formulated below.

A.1.3 lnternational reference materials An lnternational Reference Preparation of Anti-Yellow-Fever Serum is available from the National Institute for Biological Standards and Control, Potters Bar, England. A nonimmune control serum is also available. Samples are distributed free of charge, on request, to national control laboratories.

A. 1.4 Terminology

The following definitions are given for the purpose of these Require- ments only.

Primary or master seed lot. A quantity of virus suspension that has been processed in a single production run and has a uniform composition. It is used for the preparation of secondary (working) seed lots.

Secondary or working seed lot. A quantity of virus suspension that has been processed in a single production run, and is uniform with respect to composition, fully characterized and only one passage from a master seed lot. Material drawn from secondary seed lots is used for inoculating embryonated eggs in the preparation of vaccine.

Single harvest. A quantity of virus suspension, derived from tissues of the same origin that were inoculated with the same working seed lot, that has been collected and processed in a single production run.

Final bulk. The material prepared from one or more single harvests in the vessel from which the final containers are filled.

Filling or final lot. A collection of sealed final containers of finished vaccine that are homogeneous with respect to the risk of contamination during filling and freeze-drying. All the final containers must, therefore, have been filled from a single vessel of final bulk in one working session and lyophilized under standardized conditions in a common chamber.

Plaque-forming unit (PFU). The smallest quantity of a virus suspension that will produce a plaque in monolayer cell cultures.

Median mouse lethal dose (mouse LD,,). The quantity of a virus suspension that will kill 50% of the mice injected with it.

~ . 2 Certification of the substrain of 17D virus for use in vaccine production The seed lot of the substrain of 17D yellow fever virus used in the production of yellow fever vaccine shall be identified by historical records that include information on the origin of the substrain, its method of attenuation and the passage level at which attenuation and immunogenicity were demonstrated by clinical evaluation.

The master and working seed lots of yellow fever virus used in the production of vaccine shall have been shown to be safe and im- munogenic by appropriate laboratory tests (see section A.4 of these Requirements), and the master seed lot shall be shown to be immuno- genic by clinical trials in susceptible humans. Only seed lots that are approved by the national control authority shall be used.

Seed lots that have been certified previously can be used.

A suitable primary seed lot (213-77) is available from WHO (16).

~ . 3 General manufacturing requirements

The general manufacturing requirements contained in Good Manu- facturing Practices for Pharmaceutical (17) and Biological (18) Prod- ucts shall apply to establishments manufacturing yellow fever vaccine, with the following additions: those staff directly involved with the production and testing of yellow fever vaccine shall be shown to be immune to yellow fever.

~ . 4 Production control

A.4. I Control of source materials

A.4.1.1 Tissues for virus production

Virus for the preparation of master and working seed lots shall be grown in the tissue of chicken embryos obtained from a closed, specific-pathogen-free, healthy flock. This flock shall be monitored at regular intervals for agents pathogenic to birds. However, the national control authority may vary these requirements where the cost and difficulty of obtaining such tissue may restrict the availability of the vaccine and where the vaccine produced has been demonstrated to be both safe and effective.

Examples of agents that it may be necessary to monitor include Mycobacterium avium, fowl pox virus, ALV and other avian retroviruses, Newcastle disease virus and other avian parainfluenza viruses, avian en- cephalomyelitis virus, infectious laryngotracheitis virus, avian reticulo- endotheliosis virus, Marek's disease virus, infectious bursa1 disease virus, Haemophilus paragallinarum, Salmonella gallinarum, Salmonella pullorum, Mycoplasma gallisepticum and Mycoplasma synoviae.

In some countries, all birds are bled when a colony is established, and thereafter 5% of the birds are bled each month. The resulting serum samples are screened for antibodies to the relevant pathogens. Any bird that dies is investigated to determine the cause of death.

Virus for the preparation of all vaccine production lots shall be grown in the tissue of chicken embryos obtained from a healthy flock. Moni- toring of the flock or embryos shall include at least tests for exclusion of infection by Salmonella spp., Mycobacterium avium and fowl pox virus. The flock must not have been vaccinated with live Newcastle disease virus vaccine.

In some countries, the national control authority requires that the tissue used for production of vaccine should be shown by suitable tests to be free from ALV.

It is recommended that eggs should be obtained from young birds.

A.4.1.2 Seed-lot system

The production of vaccine shall be based on the master- and working- seed-lot system. All seed lots shall be stored at a temperature of -60°C or below. Primary and secondary seed lots shall not contain any human protein or added serum or antibiotics.

In some laboratories, the master and working seed lots are stored in more than one location.

The master and working seed lots shall be free from ALV, mycoplasmas or other adventitious agents as shown by suitable tests (A.4.1.1 and A.4.1.5).

The inoculum for infecting tissues used in the production of vaccine shall be a working seed lot without intervening passage, in order to ensure that no vaccine shall be manufactured that is more than one passage removed from a seed lot that has passed all safety tests.

A.4.1.3 Monkey safety test

New master and working seed lots shall be tested for viscerotropism, immunogenicity and neurotropism in a group of 10 test monkeys. For the neurotropism test, the test monkeys inoculated with the virus seed lot shall be compared with a similar group of 10 monkeys injected with a reference virus. The reference virus shall be approved by the national control authority.

The monkeys shall be Mncncn mulntta (i.e. rhesus monkeys) or Macaca fnscicularis (i.e. cynomolgus monkeys) and shall have been demonstrated to be non-immune to yellow fever immediately prior to injection of the seed virus. They shall be healthy and shall not have been previously subjected to intracerebral or intraspinal inoculation. Furthermore, they shall not have been inoculated by any route with neurotropic viruses or antigens related to yellow fever.

The test dose shall consist of 0.25ml containing the equivalent of not less than 5000 and not more than 50000 mouse LD,,, as shown by a titration conducted by the method described in Appendix 3. The test dose shall be injected into one frontal lobe of each monkey under anaesthetic: and the monkeys shall be observed for a minimum of 30 days.

Viscerotropisnz test. The criterion of viscerotropism (indicated by the amount of circulating virus) shall be fulfilled as follows. Sera obtained from each of the test monkeys on the second, fourth and sixth days after injection of the test dose shall be inoculated at dilutions of 1 : 10, 1 : 100 and 1 : 1000 into at least 4 cell-culture vessels (or intracerebrally in 0.031111 aliquots into at least 6 mice) per dilution, as specified in Appendix 3. In no case shall 0.031111 of serum contain more than 500 mouse LD,, or the equivalent in PFU (see section A.6.2) and in no more than one case shall 0.03ml of serum contain more than the equivalent of 100 mouse LD,, (appropriate techniques for potency testing are given in Appendix 3).

Imnzunogenicity test. The criterion of sufficient virus-neutralizing antibody in the sera (immunogenicity) shall be fulfilled as follows. At

least 90% of the test monkeys shall be shown to have become immune within 30 days following injection of the test dose, as determined by examining their sera in the test for neutralization of yellow fever virus described below.

In some countries, it has been shown that, at low dilutions, some sera contain non-specific inhibitors that interfere with this test. The national control authority may require sera to be treated to remove such substances.

Dilutions of 1 : 10, 1 : 40 and 1 : 160 of serum from each test monkey shall be mixed with an equal volume of strain 17D vaccine virus at a dilution that has been shown to yield an optimum number of plaques when assayed according to one of the cell-culture methods given in Appendix 3. These serum-virus mixtures shall be incubated in a water bath at 37 "C for 1 hour and then chilled in an ice-water bath before inoculation of 0.2ml aliquots of each mixture into each of 4 separate cell-culture vessels. The vessels shall be handled according to one of the cell-culture techniques described in Appendix 3. In addition, 10 vessels shall be similarly inoculated with virus as above and an equal volume of a 1:10 dilution of monkey serum known to contain no neutralizing antibodies to yellow fever virus. At the end of the observation period, the mean number of plaques in the vessels receiving virus and non-immune serum shall be compared with the mean number of plaques in the vessels receiving virus and serum from test mo'nkeys. For the immunogenicity test to be satisfied, serum at the 1 : 10 dilution from no more than 10% of the test monkeys shall fail to reduce the mean number of plaques by 50% as compared with the vessels containing non-immune serum.

Neurotropisrn test. Monkeys in the test group shall be compared with 10 monkeys injected with the reference virus with respect to both the clinical evidence of encephalitis and the severity of histological lesions of the nervous system (19,20).

The onset and duration of the febrile reaction should not differ between monkeys injected with the test or reference virus.

The monkeys shall be examined daily for 30 days by personnel famil- iar with the clinical signs of encephalitis in primates.

If necessary, the monkeys may be removed from their cages and examined for signs of motor weakness or spasticity as described elsewhere (20).

Signs of encephalitis, such as paresis, incoordination, lethargy, tremors or spasticity, shall be assigned numerical values for severity by the following grading method. Each day each monkey shall be given a numerical score based on the scale:

1: rough coat, not eating; 2: high-pitched voice, inactive, slow moving;

3: shaky movements, tremors, incoordination, limb weakness; 4: inability to stand, limb paralysis or death.

A monkey that dies receives the score "4" from the day of death until day 30.

The clinical score for a monkey is the average of its daily scores; the clinical score for a group is the arithmetic mean of the individual scores. For the clinical criterion of the neurotropism test to be satisfied, the clinical score of the monkeys injected with the virus being tested shall not exceed the clinical score of the monkeys injected with the reference virus.

The cervical and lumbar enlargements of the spinal cord and specific structures at five levels of the brain shall be examined (20) (see Appendix 1). The cervical and lumbar enlargements shall each be divided equally into six blocks. The blocks shall be dehydrated and embedded in paraffin wax; 15-pm sections shall be cut and stained with gallocyanin. One section, consisting of two hemisections, shall be cut from each block.

Tissue blocks 3-4mm thick shall be taken from the brain by making the following frontal cuts:

Block I: the corpus striatum at the level of the optic chiasma; Block 11: the thalamus at the level of the mamillary bodies; Block 111: the mesencephalon at the level of the superior colliculi; Block I V the pons and cerebellum at the level of the superior olives; Block V the medulla oblongata at the mid-level of the inferior olives.

These blocks shall be dehydrated and embedded in paraffin wax and 15-pm sections shall be cut and stained n-ith gallocyanin. A single section, consisting of two hemisections, shall be cut from each block.

Sections shall be examined microscopically and numerical scores given to each hemisection of the lumbar and cervical cord enlargements and to each anatomical structure (see Appendix 1) within each hemisection of the brain blocks according to the following grading system:

1 (minimal): 1-3 small, focal inflammatory infiltrates. A few neurons may be changed or lost;

2 (moderate): more extensive focal inflammatory infiltrates. Neuronal changes or loss affects not more than one-third of neurons;

3 (severe): neuronal changes or loss of 33-90% of neurons, with moderate focal or diffuse inflammatory infiltration;

4 (overwhelming): more than 90% of neurons are changed or lost, with variable, but frequently severe? inflammatory infiltration.

Each brain block contains several anatomical structures which con- tribute in different ways to the assessment of a test sample. For

example, certain structures differentiate more reproducibly than others between acceptable and unacceptable yellow fever seed lots and vaccines (20). These are called discriminator areas, whereas structures that are more susceptible to yellow fever virus replication are called target areas. Though either rhesus or cynomolgus monkeys are acceptable, the discriminator and target areas are different for the two species. The major difference is that in cynomolgus monkeys the cervical and lumbar cord are target areas whereas in rhesus monkeys they are discriminator areas. The footnotes to the worksheets (Ap- pendix 1) indicate in more detail discriminator and target areas for the two species. The worksheets also lists other anatomical structures that will be present in the brain sections but are not included in the evaluation of a test sample because they are rarely affected (spared areas).

Three separate scores shall be calculated for each monkey: discriminator areas only, target areas only, and discriminator plus target areas. These scores shall be calculated as shown in the example worksheets provided in Appendix 1 (pp. 55-57). Overall mean scores shall also be calculated for each group of monkeys as the arithmetic mean of individual monkey scores for discriminator areas only and for discriminator plus target areas. Both overall mean scores shall be considered in determining virus seed lot acceptability. For the histological criterion of the neurotropism test to be satisfied both overall mean scores for the test monkeys shall not be significantly greater (at the 5 % significance level) than the overall mean scores. for the monkeys injected with reference virus.

Both the clinical and histological criteria of the neurotropism test shall be satisfied for the virus seed lot to satisfy the requirement for neurotropism.

A.4.1.4 Identity test

An identity test shall be performed on at least one container from each master and working seed lot, using one of the techniques described in section A.6.1.

A.4.1.5 Sterility tests on primary and secondary seed lots

Each lot of seed virus in its final containers shall pass the tests described in Part A, sections 5.2 and 5.3, of the revised Requirements for Biological Substances No. 6 (General Requirements for the Steril- ity of Biological Substances) (21)'

' See the 1995 amendment to the General Requirements for the Sterility of Biological Substances, section 5.3, Sterility test for mycoplasmas (Annex 3, p. 70 of this report).

A.4.2 Tests on uninoculated eggs

If monitoring of the flocks supplying embryonated eggs is not under the direct responsibility of the vaccine manufacturer, the following tests shall be performed.

A sample of 2% of, but in any case not less than 20 and not more than 50 uninoculated embryonated eggs from the batch used for vaccine production shall be incubated under the same conditions as the inoculated eggs. At the time of virus harvest, the uninoculated eggs shall be processed in the same manner as the inoculated eggs, and the extract from the control embryos shall be shown to be free from haemagglutinating agents and other extraneous agents by tests approved by the national control authority.

A.4.3 Single harvests

After inoculation and incubation at a controlled temperature, only living, typical chick embryos shall be harvested. The age of embryos at the time of harvest shall be reckoned from the initial introduction of the eggs into the incubator and shall be no more than 12 days. After homogenization and centrifugation, the embryonic extract shall be kept at -60°C or below until further processing.

A.4.3.1 Sterility tests

The embryonic extract that forms a single harvest shall be tested for the presence of bacteria, fungi and mycoplasmas as described in Part A, section 5.2 and 5.3, of the revised Requirements for Biological Substances No. 6 (General Requirements for the Sterility of Biologi- cal Substances) (21).'

A.4.3.2 Virus titration

Live virus content shall be determined by titration in cell culture against a reference preparation of yellow fever virus as described in Appendix 3.

A.4.4 Final bulk

The final bulk shall be prepared from one or several single harvests and shall be submitted to the following tests, unless these tests have already been performed on each single harvest. The final bulk shall in any case be tested for sterility. Samples that are not tested immediately shall be stored at or below -60°C.

' See the 1995 amendment to the General Requirements for the Sterility of Biological Substances, section 5.3, Sterility test for mycoplasmas (Annex 3 , p. 70 of this report)

A.4.4.1 Sterility tests

The requirements concerning tests for bacteria, fungi and mycoplasmas as described in Part A, sections 5.2 and 5.3, of the revised Re- quirements for Biological Substances No. 6 (General Requirements for the Sterility of Biological Substances) (21) shall apply to yellow fever vaccine.'

A.4.4.2 Adventitious agents

Mycobacterium avium shall be tested for by cell-culture methods approved by the national control authority.

In some countries, the national control authority requires that the final bulk should be shown by suitable tests to be free from ALV or other agents.

A.4.4.3 Protein content

The level of protein nitrogen before the addition of any stabilizer shall bc not morc than 0.25mg per human dose.

A.4.4.4 Addition of stabilizers and preservatives

No p-lactam antibiotics shall be added to the virus suspension at this or at any other stage during production. Added stabilizing agents shall be approved by the national control authority.

A. 4.4.5 Virus titration

The live virus content of each final bulk shall be determined by titration in cell culture against a reference preparation of yellow fever virus as described in Appendix 3.

~ . 5 Filling and containers The requirements concerning filling and containers given in Good Manufacturing Practices for Biological Products (18) shall apply to yellow fever vaccine.

Care shall be taken to ensure that the materials of which the container, and if applicable the closure, are made do not adversely affect the virus content of the vaccine under the recommended conditions of storage.

Single- and multiple-dose containers may be used.

The vaccine shall be freeze-dried.

Failure to achieve adequate drying will result in a product that is liable to rapid deterioration even at 0°C.

' See the 1995 amendment to the General Requirements for the Sterility of Biological Substances, section 5.3, Sterility test for mycoplasmas (Annex 3 , p. 70 of this report)

Since yellow fever virus is extremely labile, unless the container is well sealed variations in virus content may occur during storage. The manufacturer should ensure that the seal is satisfactory.

The manufacturer shall provide the national control authority with adequate data to prove the stability of the vaccine under appropriate conditions of storage and shipping.

~ . 6 Control tests on final lot

A.6.1 identity test

An identity test shall be performed on at least one container from each filling lot after reconstitution of the vaccine according to the indications of the manufacturer for preparing the vaccine for human administration. A high-titre, monospecific immune serum known to be free from neutralizing agents that react with other flaviviruses shall be used. Either of two methods may be used for the identity test.

A. 6.1. I Test in mice

Progressive dilutions not greater than fivefold each shall be made of the vaccine in a suitable diluent. Aliquots of each dilution shall be mixed with equal volumes of a reference immune serum and similar aliquots mixed with equal volumes of a reference non-immune serum. The serum-virus mixtures shall be incubated at 37°C and each shall be injected into not less than 6 mice per dilution as described in Appendix 3. All mice shall be observed for 21 days, and all deaths shall be recorded. Only deaths considered to be specifically caused by yellow fever virus infection shall be taken into account. Mice paralysed on day 21 shall be counted as alive. The LD,, for vaccine mixed with immune serum shall be more than ten times the LD,, for vaccine mixed with non-immune serum.

A. 6.1.2 Test in cell cultures (plaque reduction test)

The technique described in section A.4.1.3. I?~.z??1zn1oge17ici test, (pp. 37-38) shall be used, with dilutions of vaccine with immune and non- immune serum. If a 50% reduction in plaque number at the 1 : 10 dilution is not observed for the vaccine mixed with immune serum compared with vaccine mixed with non-immune serum, the vaccine shall be rejected.

A.6.2 Potency test

Three final containers shall be selected at random from each filling lot and shall be individually tested on the same day against a reference preparation of yellow fever vaccine approved by the national control

authority. The containers shall be tested by assay in cell cultures or mice by a technique demonstrated to be sensitive.

Before assay but after reconstitution of the vaccine in the volume and diluent recommended by the manufacturer for preparation for human administration, the vaccine shall stand at a temperature between 20°C and 30°C for 20 minutes before further dilution. This material shall be considered as undiluted vaccine.

The titre of the vaccine shall be not less than 1000 mouse LD,,, or its equivalent in PFU, in the dose recommended by the manufacturer for use in humans. Each laboratory shall establish to the satisfaction of the national control authority the relationship between mouse LD,, and PFU.

Each laboratory using a cell culture assay shall have the approval of the national control authority for this assay. Appropriate techniques for the potency test are given in Appendix 3.

A.6.3 Thermal stability test

Three final containers from the freeze-dried final lot shall be incubated at 37°C for 2 weeks. These containers shall be titrated in parallel with three containers that have been stored at or below the recommended storage temperature. Results should be expressed in infectious units or their equivalent in PFU. A reference reagent for yellow fever virus approved by the national control authority shall be included in each assay. At the end of the incubation period, the geometric mean infectious titre in the incubated final containers shall not have decreased by more than 1.0 log,, unit and shall be at least equal to the required minimum number of infectious units per human dose.

Procedures for carrying out the above test and for the interpretation of results, including the specification of confidence limits, should be approved by the national control authority. A suitable test for determination of virus content (i.e. titre) is described in Appendix 3.

A.6.4 Tests for bacteria and fungi

The requirements concerning tests for bacteria and fungi as described in Part A, section 5.2, of the revised Requirements for Biological Substances No. 6 (General Requirements for the Sterility of Biologi- cal Substances) (21) shall apply to yellow fever vaccine.

A.6.5 Tests for other agents In some countries the national control authority requires that the vaccine should be shown by suitable tests to be free from ALV or other agents. If

these tests have been performed on the final bulk they may be omitted for the final lot.

A.6.6 General safety tests

Each final lot shall be tested for the absence of abnormal toxicity in mice and guinea-pigs by appropriate tests approved by the national control authority.

A.6.7 Residual moisture

The residual moisture in a representative sample of each freeze-dried final lot shall be determined by a method approved by the national control authority. The upper Limit of the moisture content shall be approved by the national control authority on the basis of stability tests.

Generally, moisture levels of 3% or less are considered satisfactory.

A.6.8 Residual ovalbumin

The content of residual ovalbumin shall be limited to a level approved by the national control authority.

A.6.9 Test for endotoxin

Each final lot shall be tested for endotoxin. The test and limits shall be approved by the national control authority.

A.6.10 Inspection of final containers

Every container in each final lot shall be inspected visually, and those showing abnormalities shall be discarded.

~ . 7 Records

The requirements in section 8 of Good Manufacturing Practices for Biological Products (18, pages 27-28) shall apply.

~ . 8 Samples

The requirements in section 9.5 of Good hlanufacturing Practices for Biological Products (18: page 29) shall apply.

~ . 9 Labelling

The requirements in section 7 of Good Manufacturing Practices for Biological Products (18; pages 26-27) shall apply, with the addition of the following:

The label on the carton or the leaflet accompanying the container shall:

- state that the vaccine fulfils Part A of these Requirements; - state the nature of the preparation, specifying the strain of yellow

fever virus in the vaccine, the minimum number of infectious units per human dose, and the origin of the substrate used to prepare the vaccine (i.e. whether the flock was specific-pathogen free (SPF)).

- state the nature and quantity of any residual antibiotic present in the vaccine;

- indicate that the vaccine contains proteins derived from eggs; - indicate that contact of the vaccine with disinfectants is to be

avoided; - indicate that the dose shall be the same for persons of all ages; - ipdicate the volume and nature of the diluent to be added to

reconstitute the vaccine, and specify that only the diluent supplied by the manufacturer should be used;

- state that the vaccine is not recommended for administration to children less than 6 months of age;

- state that the reconstituted vaccine shall be kept at 2-8 "C and in the dark and shall be used or discarded within 1 hour after the container is opened.

A.IO Distribution and shipping

The requirements in Good Manufacturing Practices for Biological Products (18, section 8) shall apply.

A.I I Storage and expiry date

The requirements in Good Manufacturing Practices for Biological Products (18) shall apply.

A.II .I Storage conditions

Before being distributed by the manufacturing establishment, or before being issued from a depot for the maintenance of vaccine reserves, all vaccines shall be kept at all times at a temperature below -20°C.

If possible, storage should be at a temperature below -25°C.

The manufacturer shall recommend conditions of storage and shipping that will ensure the vaccine conforms to the requirements of potency until the expiry date stated on the label.

~.11.2Stability of vaccine and expiry date

The stability of the vaccine shall be established at the recommended storage temperature and calculated from the date of a given satisfactory potency test. The expiry date shall be fixed with the approval of the national control authority and shall be no less than 2 years after the potency test.

Accelerated degradation studies at 37°C may provide useful additional information.

Part B. National control requirements B.I General

The general requirements for control laboratories contained in the Guidelines for National Authorities on Quality Assurance for Bio- logical Products (22) shall apply.

The national control authority shall give directions to manufacturers concerning the yellow fever virus strains to be used in vaccine production and concerning the recommended human dose.

The national control authority should take into consideration information available on strains before deciding on those permitted for vaccine production.

In addition, the national control authority shall provide or approve a reference preparation of live yellow fever virus (see section A.1.3) for tests for virus concentration (see sections A.4.4.5 and A.6.2) and shall specify the virus content required to achieve adequate immunization of humans at the recommended dose.

8.2 Release and certification A vaccine lot shall be released only if it fulfils the national requirements andlor Part A of the present Requirements. A protocol based on the model given in Appendix 1, signed by the responsible official of the manufacturing establishment, shall be prepared and where appropriate submitted to the national control authority in support of a request for release of vaccine for use. For subsequent routine lot release, a less comprehensive document may suffice, a model for which is found in Annex 4 of this volume of the WHO Technical Report Series.

At the request of the manufacturing establishment, the national control authority shall pro,-ide a certificate that states whether the vaccine meets all national requirements andjor Part A of the present Requirements. The certificate shall be based on the model given in Appendix 2.

The purpose of the certificate is to facilitate the exchange of live yellow fever virus vaccine among co~ntrjes.

Authors The draft of these revised Requlreme-rts was prepared by Dr V. Grachev, Scientist, Biologicals; Dr D. Magrath. Chief, Biologicals (1987-1994); and Dr E. Griffiths, Chief, Biologicals. World Health Organization, Geneva, Switzerland.

Acknowledgements Acknowledgements are due to the following experts for their comments and advice on the draft Requirements:

Professor I.D. Gust, Director, Research and Development, CSL Ltd, Victoria, Australia; Dr K.J. Healy, Head, Quality Assurance, CSL Ltd, Victoria, Australia; Professor J. L'age-Stehr, Robert Koch Institute, Berlin, Germany; Mr P. Lemoine, formerly Head, Biological Standardization, lnstitute of Hygiene and Epidemiology, Brussels, Belgium; Dr I. Levenbook, Center for Biologics Evaluation and Research, Division of Product Quality Control, Kensington, MA, USA; Mrs S. Marsden, Viral Division, National lnstitute for Biological Standards and Control, Potters Bar, Herts., England; Dr L. Palkonyay, Chief, Viral Products Division, Bureau of Biologics, Drugs Directorate, Ottawa, Ontario, Canada; Dr F. Reigel, Head, Section for Immunologicals, Swiss Federal Office of Public Health, Bern, Switzerland; MS J. Sokhey, Director, Central Research Institute, Kasauli, India; Dr D. Wood, Division of Virology, National Institute for Biological Standards and Control, Potters Bar, Herts., England.

References 1. The resurgence of deadly yellow fever. Expanded Programme on

Immunization update. Geneva, World Health Organization, March 1992 (unpublished document available on request from Expanded Programme on Immunization, World Health Organization, 121 1 Geneva 27, Switzerland).

2. Rice CU et al. Nucleotide sequence of yellow fever virus: implications for flavivirus gene expression and evaluation. Science, 1985, 229:727-733.

3. Bres PLJ. A century of progress in combating yellow fever. Bulletin of the World Health Organization, 1986, 64:775-786.

4. Rey M et al. Aspects epidemiologiques et cliniques des encephalites consecutives a la vaccination antiamarile (d'apres 248 cas observes dans quatre services hospitaliers de Dakar a la suite de la campagne 1965). [Epidemiological and clinical aspects of encephalitis following yellow fever vaccination (report of'248 cases from four hospital services in Dakar after the 1 965 campaign).] Bulletin de la Societe medicale d'Afrique noire de langue frangaise, 1966, 1 1 :560-574.

5. Requirements for biological substances 3. Requirements for Yellow Fever Vaccine 4. Requirements for Cholera Vaccine. ,Report of a Study Group. Geneva, World Health Organization, 1959 (WHO Technical Report Series, No. 179).

6. Expert Committee on Yellow Fever Vaccine. First Report. Geneva, World Health Organization, 1957 (WHO Technical Report Series, No. 136).

7. International health regulations (1969), 3rd annotated ed. Geneva, World Health Organization, 1983 (Article 66.4).

8. WHO Expert Committee on Biological Standardization. Twenty-second report. Geneva, World Health Organization, 1970: 21 (WHO Technical Report Series, No. 444).

9. Requirements for Yellow Fever Vaccine (Requirements for Biological Substances No. 3, revised 1975). In: WHO Expert Committee on Biological

Standardization. Twenty-seventh report. Geneva, World Health Organization, 1976, Annex 1 (WHO Technical Report Series, No. 594).

10. Meegan JM. Yellow fever vaccine. Geneva, World Health Organization, 1991 (unpublished document WHOIEPIIGENI91.6, available on request from Expanded Programme on Immunization, World Health Organization, 121 1 Geneva 27, Switzerland).

1 1. Drouet A. Meningo-encephalite apres vaccination anti-amarile par la souche 17D: deux observations. [Meningo-encephalitis folloltving yellow fever vaccination using strain 17D: two cases.] Revue de medecine interne, 1993, 14(4):257-259.

12. Galazka AM et al. The immunological basis for immunization of yellow fever. Geneva, World Health Organization, 1993 (unpublished document WHO1 EPIlGENl93.18; available on request from Expanded Programme on Immunization, World Health Organization, 121 1 Geneva 27, Switzerland).

13. Monath TP et al. Ontogeny of yellow fever 17D vaccine. RNA oligonucleotide fingerprint and monoclonal antibody analysis of vaccines produced worldwide. Journal of general virology. 1983, 64:627-637.

14. Waters TD et al. Yellow fever vaccination. avian leukosis virus and cancer risk in man. Science, 1972, 177:76-77.

15. Requirements for Yellow Fever Vaccine (Requirements for Biological Substances No. 3, revised 1975, adde~dum 1987). In: WHO Expert Committee on Bioiogical Standardization. Thirty-eighth Report. Geneva, World Health Organization, 1988, Annex 9 (VVHO Technical Report Series, No. 771).

16. Production and testing of the WHO yellow fever virus primary seed lot 213- 77 and reference batch 168-73. In: WHO Expert Committee on Biological Standardization. Thirty-sixth report. Geneva: World Health Organization, 1987, Annex 6 (WHO Technical Report Series: No. 745).

17. Good manufacturing practices for pharmaceutical products. In: WHO Expert Committee on Specifications for Pharmaceutical Preparations. Thirty-second Report. Geneva, World Health Organization, 1392, Annex 1 (WHO Technical Report Series, No. 823).

18. Good manufacturing practices for biological products. In: WHO Expert Committee on Biological Standara?zati~~-i. Fafly-second re,oort. Geneva, World Health Organization, 1992, Annex l (:',:'?H0 Technical Report Series, No. 822).

19. Fox JP, Penna HA. Behaviour of 17D yell~i:.~ fever virus In rhesus monkeys: relation to substrain, dose and neural or extrar.eural inocujation. American journal of hygiene. 1943, 38: 152-1 72.

20. Levenbook IS: Pelleu LJ! Elisberg BL. The monkey safety iest for neurovirulence of yeiloiv fever vaccines: tke ztility of quantitative clinical evaluation and histologicai examination. ,"oui;r?al of bio1ogica.i standardization, 1987, 15:3G5-313.

21. General Requirements for the Sterility of Bi~l~ogical Substances (Requirements for Biological Substances No. 6, revised 1973). In: WHO Expert Committee on Biological Standardiza iion. Twenty-fiflh Report

Geneva, World Health Organization, 1973, Annex 4 (WHO Technical Report Series, No. 530).

22. Guidelines for national authorities on quality assurance for biological products. In: WHO Expert Committee on Biological Standardization. Forty- second Report. Geneva, World Health Organization, 1992, Annex 2 (WHO Technical Report Series, No. 822).

Appendix 1 Example, for guidance, of a summary protocol for the production and testing of yellow fever vaccine'

Master virus seed lot

Source of 17D substrain

Master virus seed lot no.

Name and address of manufacturer

Passage level

Date of inoculation of embryos

Temperature of incubation

Date of harvest

Age of embryos (at harvest)

Number of containers

Conditions of storage

Production control (A.4)

Monkey safety test (A.4.1.3)

Species

Number inoculated

Master virus seed lot no.

Reference virus lot no.

Date of serology tests before inoculation

Dilution used for the inoculation

Volume and route of inoculation

Date of inoculation

Number of mouse LD,, or PFU inoculated

Date of end of the test

' Based on Requirements for yellow fever vaccine (Requirements for Biological Substances No. 3, revised 1995). In: WHO Expert Committee on Biological Standardization. Forty-sixth Report. Geneva, World Health Organization, 1997, Annex 2 (WHO Technical Report Series, No. 872).

l/iscerotropism test (master virus seed lot)

Specify if mice or cells are used for virus titration.

Result (pass or fail)

lmmunogenicity test (master virus seed lot)

Specify if mice or cells are used for virus titration.


Neurotropism test (master virus seed lot)

Summary clinical results

Date of inoculation:


6

7

8

9

10

Group mean

16

17

18

19

20

Group mean

Histological worksheet

The worksheets below are provided as an example of how the histological score would be calculated for a cynomolgus monkey with lesions graded as shown.

Species: cynomolgus Pathology no: Monkey no:

" Discriminator area for rhesus. b Discriminator area for cynomolgus.

N. ant./med. thalamPb

N. lat. thalamPb

Hypothalamus

Mesencephalon (Block 111)

Colliculi superior

Corpus geniculatum med.

" Target area for rhesus and cynomolgus.

1

0

N. oculomotorius

N. ruber

Substantia nigrac

L

0

0

2

1

0

0

2

R

0

0

Total

0

0

0

0

2

(514 =) 1.25

(114 =) 0.25

1

0

0

0

(412 =) 2.00

1

0

I I1 I11 IV V V1 Total Spinal cord L R L R L R L R L R L R L R L&R

Cervical 2 3 2 3 2 2 2 2 1 2 2 2 11 14 (25/12=) 2.08

Lumbar 2 2 1 2 1 2 1 2 0 1 0 2 5 11 (16112=) enlargemenPd 1.33

a Discriminator area for rhesus. Target area for cynomolgus.

Calculations:

Discriminator areas (globus pallidus, putamen, n. ant./med. thalami, n. lat, thalami):

(0.75 +LOO + 0.75 +1.25) Lesion score = = 0.94

4

Target areas (S. nigra, cervical enlargement, lumbar enlargement):

(2.00 + 2.08 + 1.33) Lesion score = = 1.80

3

Discriminator plus target areas:

0.94 + 1.80 Lesion score = = 1.37

2

Summary histopathology results

Date of inoculation: Species:

area score

Group Group mean mean


Identity test (A.4.1.4)

Comparison of virus titre in presence of normal serum with that in presence of immune serum

Method

Date

Results

Sterility tests (A.4.1.5)

Number of containers tested bacteria fungi mycoplasmas

Date of inoculation

Media used

Observation period

Results

Tests for avian and other viruses

Method

Date

Results

Working virus seed lot Working virus seed lot no.


Passage level



Date of harvest


Number of containers

Conditions of storage

Monkey safety test (A.4.1.3)

Species

Number inoculated

Working virus seed lot no.

Reference virus lot no.

Date of serology tests before inoculation

Dilution used for inoculation

Volume and route of inoculation

Date of inoculation

Number of mouse LD,, or PFU inoculated

Date of end of the test

Viscerotropism test (working virus seed lot)

Specify if mice or cells are used for virus titration. See p. 52 for an example of the viscerotropism test worksheet.


lmmunogenicity test (working virus seed lot)

Specify if mice or cells are used for virus titration. See p. 53 for an example of the immunogenicity test worksheet.


Neurotropism test (working virus seed lot)

Please see pp. 54-58 for examples of the clinical and histological worksheets


Identity test (A.4.1.4)


Method

Date

Results

Sterility tests (A. 4.1 .5)

No. of containers tested

bacteria fungi mycoplasmas

Date of inoculation

Media used

Observation period

Results

Tests for avian and other viruses

Method

Date

Results

Control of single harvests (A.4.3)


Laboratory reference no.

lnformation on source materials

Virus used to inoculate embryos

Derived from master seed virus lot no.

Working virus seed lot, reference no. and source

Passage level of working virus seed lot

Source of eggs

Is the flock under direct control of manufacturer?

Is the flock monitored for compliance with these Requirements?

Results of tests on flock or control tissues

Tests for salmonella

Tests for fowl pox virus

Tests for avian mycobacteria

Tests for other microbial agents (give details)

lnformation on manufacture



Date of harvest


Results of sterility tests

Control of final bulk (A.4.4)

Sterility tests (A.4.4.1)

bacteria fimgi nz~~coplasmas

Date of inoculation

Media used

Observation period

Results

Protein content (A.4.4.3)

Method

Date

Result

Stabilizers and preservatives (A. 4.4.4)

Name of stabilizer

Quantity or percentage

Name of preservative

Quantity or percentage

Date

Virus titration (A .4.4.5)

Method

Date

Result

Control tests on final lot (A.6)

Identity test (A. 6.1 )


Method

Date

Result

Potency test (A.6.2)

Date of test

Reference batch no.

Relationship between titre as determined by inoculation in mice, expressed in LD,, per human dose, andtor the titre as determined in cell culture, expressed in plaque forming units (PFU), per human dose is:

Vaccine Reference samples samples

1 2 3

Virus concentration in each container --p

Mean virus titre per human dose, with 95 % fiducial limits

Thermal stability test (A.6.3)

Method

Date

Results

Sterility tests (A. 6.4)

Date of inoculation

Media used

Observation period

Results

bacteria

Tests for other agents (if performed) (A.6.5)

Method

Date

Results

General safety test (A. 6.6)

Tests in mice

Date of inoculation

No. of mice tested

Volume and route of injection

Observation period

Results (give details of deaths)

Tests in guinea-pigs

Date of inoculation

No. of guinea-pigs tested


Observation period


Residual moisture (A. 6.7)

Method

Date

Result

Residual ovalbumin (A.6.8)

Method

Date

Result

Endotoxin (A. 6.9)

Method

Date

Result

Inspection of final containers (A. 6.1 0)

Method

Date

Result

Submission addressed to national control authority

Name (typed) and signature of head of production laboratory

Date

Certification by person taking overall responsibility for production and control of the vaccine:

I certify that lot no. of yellow fever vaccine satisfies national requirements andlor Part A of the Requirements for Biological

Substances No. 3 (Requirements for Yellow Fever Vaccine, revised 1995).

Signature

Name (typed)

Date

Appendix 2 Model certificate for the release of yellow fever vaccine by national control authorities'

The following final lots of yellow fever vaccine produced by 2 . in 3 , whose numbers appear on the labels of the final containers,

meet all national requirements4 and Part A5 of Requirements for Biologi- cal Substances No. 3 (Requirements for Yellow Fever Vaccine), revised 1999 and comply with Good Manufacturing Practices for Pharmaceutical7 and ~iological' Products.

Lot Date of last Expiry Lot Date of last Expiry no. potency test date no. potency test date

by manu- by manufacturer facturer

As a minimum, t h s certificate is based on examination of the manufacturing protocol.

The Director of the National Control Laboratory (or Authority as appr~priate):~

Name (typed)

Signature

Date

' To be completed by the national control authority of the country where the vaccine has been manufactured, and to be provided by the vaccine manufacturer to importers.

"ame of manufacturer. Country. If any national requirements are not met, specify which one(s) and indicate why release of the lot(s) has nevertheless been authorized by the national control authority. With the exception of provisions on distribution and shipping, which the national control authority may not be in a position to assess. WHO Technical Report Series, No. 872, 1998, Annex 2. WHO Technical Report Series, No. 823, 1992, Annex l. WHO Technical Report Series, No. 822, 1992, Annex 1. Or his or her representative.

Appendix 3 Techniques for the potency evaluation of yellow fever vaccine

Cell-culture technique

Vero cell

Vero-cell seed and a description of a method for Vero-cell cultivation may be obtained from the World Health Organization. Monolayers of Vero cells are prepal-ed in 35-mm Petri dishes, and 4 dishes are inoculated with each virus dilution (0.2ml per dish). After incubation for 1 hour at 36 "C, the virus dilution is replaced by 3 m1 of an overlay medium consisting of Leibovitz medium No. 15 or MEM medium at pH 7.1-7.2, bovine fetal serum (final concentration. 5%); and agarose (final concentration, 0.5%). Five days after inoculation. 0.1 m1 of the overlay medium, with the addition of a 1 : 50 000 dilution of neutral red dye, is added. Plaques are counted on day 6. When plaque- forming titre is calculated, all dilutions should be considered in which the average number of plaques per dish is between 1 and 30.

Alternatively, equal amounts (0.2ml) of a Vero-cell suspension (approximately 6 X 10' cells/ml) and virus dilution in Leibovitz medium No. 15 or MEM medium are placed in each of the 16-mm flat- bottomed wells in sterile trays suitable for cell culture. The trays are sealed and incubated for 4 hours at 36°C. After incubation, 0.4ml of overlay medium, consisting of Leibovitz medium No. 15 or MEM medium, bovine fetal serum (3%). and carbox~~methylcellulose (low viscosity sodium salt) (1.6%). is added to each well. The trays are resealed and incubated at 36 "C for 5 days. On the sixth day, the trays are drained, washed with saline. stained with 1 % naphthalene black or any other suitable stain and thoroughly rinsed with tap water. The plaques are then counted. All dilutions in which the average number of plaques per well is between 1 and 30 should be considered in calculating titre.

PS cell'

This assay technique is a modification of that used by De Madrid & Porterfield (l). Equal amounts (0.2ml) of a PS-cell suspension (approximately 6 X 10' cells/ml) and virus dilution in Leibovitz medium No. 15 (2) are placed in each of the 16-mm flat-bottomed wells

' These cells are latently infected with swine fever virus and their importation is prohibited in certain countries.

in sterile trays suitable for cell culture. The trays are sealed and incubated for 4 hours at 36°C. After incubation, 0.4ml of overlay medium, consisting of Leibovitz medium plus bovine fetal serum (3 %) and carboxymethylcellulose (low-viscosity sodium salt) (1.6%), is added to each well. The trays are resealed and incubated at 36OC for 5 days. On the sixth day, the trays are drained, washed with saline, stained with 1% naphthalene black and thoroughly rinsed with tap water. The plaques are then counted. All dilutions in which the average number of plaques per well is between 1 and 20 should be considered in calculating titre.

Mouse LD,, technique

The mouse LD,, is the quantity of virus suspension estimated to produce fatal, specific encephalitis in 50% of intracerebrally inoculated mice.

Appropriate serial dilutions of the reconstituted vaccine are made in diluent for yellow fever virus (0.75% solution of bovine albumin (fraction V) in phosphate-buffer which has been demonstrated to be free of yellow fever virus inhibitors).

Mice of a strain highly susceptible to yellow fever virus, aged 4-6 weeks, are injected intracerebrally under anaesthesia with 0.03 m1 of vaccine dilution. Groups of at least 6 mice are used for each dilution, and the series of dilutions should result in mortality rates after inoculation spanning the range 0-100%. Inoculation of the mice should be performed immediately after the dilutions have been made. All deaths are recorded during a period of 21 days. Mice dying from unrelated causes are removed from both the numerator and denomi- nator of mortality calculations. Mice paralysed on the twenty-first day are counted as alive.

References 1. De Madrid AT, Porterfield JS. A simple micro-culture method for the study of

group B arboviruses. Bulletin of the World Health Organization, 1969, 40:113-121.

2. Leibovitz A. The growth and maintenance of tissue-cell cultures in free gas exchange with the atmosphere. American journal of hygiene, 1963, 78:173 180.

0 World Health Organization WHO Technical Report Series, No. 872, 1998

Annex 3 General requirements for the sterility of biological substances (Requirements for Biological Substances No. 6, revised 1973, amendment 1995)

Introduction

Since the General Requirements for the Sterility of Biological Sub- stances were revised in 1973 (l): several developments have occurred. It is thus appropriate to modify as follows the existing General Re- quirements for the Sterility of Biological Substances.

Sterility test for mycoplasmas (section 5.3, page 52)

Replace the text of section 5.3 by the following:

"The test for mycoplasmas is carried out by culture in agar and broth or by means of an indicator cell culture and DNA staining.

Where a test for mycoplasmas is prescribed for a master cell bank, for a working cell bank: for a virus seed lot (or bank) or for cell cultures used as controls, the standard culture or the indicator cell-culture method can be used. Where a test for mycoplasmas is prescribed for a virus harvest, bulk vaccine or final lot, the standard culture method is used. The indicator cell-culture method can be used, where necessary, for the screening of media. Both the standard culture and indicator cell-culture methods are given in Appendix 3. Alternative methods may be used provided that they have been validated against the methods described here."

Tests for mycoplasmas (Appendix 3: p. 56)

Replace the text of Appendix 3 by the following:

"Standard culture method

Choice of culture media

The test is carried out with a sufficient number of both solid and liquid media to ensure the growth, under the chosen incubation conditions, of small numbers of organisms that may be present in the product being examined. Liquid media must contain phenol red dye. The

ability of the media to support the growth of at least the following organisms shall be demonstrated:

Mycoplasma orale (vaccines for human use);

Mycoplasma pneumoniae (vaccines for human use);

Mycoplasma hyorhinis (non-avian veterinary vaccines, e.g. strain DBS 1050);

Mycoplasma hyopneumoniae (non-avian veterinary vaccines);

Mycoplasma gallisepticum and Mycoplasma synoviae (where avian material has been used during vaccine production or where the vaccine is intended for use in poultry).

Low-passage test strains are used and are stored frozen or freeze- dried. After cloning, the strains are identified as being of the required species by a suitable method.

In some countries, specific tests for insect mycoplasmas are performed.

Incubation conditions

Divide inoculated media into two equal parts; incubate one in aerobic conditions (an atmosphere of air containing 5-10% carbon dioxide and adequate humidity) and the other in anaerobic conditions (an atmosphere of nitrogen containing 5-10% carbon dioxide and adequate humidity).

Demonstration of nutritive properties of media

Inoculate the media with the appropriate test organisms; use no fewer than 200 and no more than 400 colony-forming units per plate of solid medium and no fewer than 20 and no more than 40 colony-forming units per container of the corresponding liquid medium; use a separate plate and container for each species. Incubate the media under the conditions that will be used for testing the product to be examined (aerobically, anaerobically or both, depending on the requirements of the test organism). The media comply with the test for nutritive properties if there is adequate growth of the test organisms (solid media) and an appropriate colour change (liquid media).

Test for inhibitory substances

Carry out the test for nutritive properties in the presence of the product to be examined. If growth of the test organisms is notably less than that found otherwise, the product contains inhibitory substances that must be neutralized (e.g. by dilution) before the test for mycoplasmas is carried out. The effectiveness of the neutralization process

is checked by repeating the test for inhibitory substances after neutralization.

Test for mycoplasmas

For solid media, use plates 60mm in diameter containing 9ml of medium. Inoculate each of at least 2 plates of each solid medium with 0.2ml of the product to be examined and inoculate at least 2 of each 100ml of liquid medium with 10ml of the product to be examined. If any significant pH change occurs when the product to be examined is added to the liquid media, restore the affected media to their original pH by the addition of a solution of either sodium hydroxide or hydro- chloric acid. Incubate paired media at 35-38 "C, one aerobically and one anaerobically, for 21 days and for the same length of time incubate an uninoculated 100-m1 portion of each liquid medium as a control. On the first, second or third day after inoculation, make a subculture of each inoculated liquid medium b ) ~ inoculating each of two fresh plates of each solid medium with 0.2ml of the product to be examined and incubating at 35-38"C, one aerobically and one anaerobically, for no less than 21 days. Repeat the procedure on the sixth, se-venth or eighth day and again on the thirteenth or fourteenth day of the test. Inspect the liquid media every 2-3 days, and if any colour change occurs prepare a subculture as described above immediately. Inspect solid media once per week.

If any containers of liquid media show bacterial or fungal contamination, repeat the test. If at least 7 days after inoculation, no more than 1 plate of solid medium at each stage of the test has been contaminated with bacteria or fungi, or broken, that plate may be ignored provided that on immediate examination it shows no evidence of mycoplasmal growth. If, at any stage of the test, more than one plate is contaminated with bacteria or fungi, or broken, the test is invalid and must be repeated.

At the end of each incubation period. microscopically examine inoculated solid media for the presence of mycoplasmas. The product passes the test if growth of mycoplasmas has not occurred in any of the inoculated media. If growth of n~ycoplasmas has occurred, the test may be repeated once with twice the amount of inoculum and media; if growth of mycoplasmas does not occur when the test is repeated, the product passes the test.

"Indicator cell-culture method

For the indicator cell-culture method, cell cultures are stained with a fluorescent dye that binds specifically to DNA. Mycoplasmas are

detected by their characteristic particulate or filamentous pattern of fluorescence on cell surfaces and, if contamination is heavy, in sur- rounding areas.

Verification of the substrate

With a Vero-cell substrate, pretest the procedure with an inoculum of no more than 100 colony-forming units (CFU) of a strain growing readily in liquid or solid media and demonstrate its ability to detect potential mycoplasma contaminants such as M. hyorhinis strain DBS 1050 and M. orale strain 1596 or other suitable strains. A different cell substrate may be used, e.g. the vaccine-production cell line, if it has been demonstrated that it provides at least equal sensitivity for the detection of mycoplasma contamination.

Test for mycoplasmas

Take no less than l m l of the material to be examined and use it to inoculate 2 or more indicator cell cultures grown to 50% confluence and representing no less than 25cm2 of cell culture in area; make at least one passage. The product of the last passage is incubated on coverslips placed in suitable containers or on some other suitable surface for the test procedure.

Include in the test a negative (non-inoculated) control and two positive controls inoculated with species such as M. hyorhinis and M. orale. Use an inoculum of not more than 100 CFU for the positive controls.

If for viral suspensions the interpretation of results is obscured by cytopathic effects, the virus may be neutralized by means of a specific antiserum that has no inhibitory effects on mycoplasmas. A cell culture substrate that does not allow growth of the virus may also be used.

Procedure

(a) Seed the culture at a regular density (2 X 104 to 2 X 105 cells per ml, 4 X 103 -2.5 X 104cells/cm2) and incubate at 36 k 1 "Cuntil about 50% confluence is reached. Inoculate the product to be examined and incubate to confluence.

(b) Remove and discard the medium. (c) Rinse the monolayer with phosphate-buffered saline at pH 7.4,

then with a mixture of equal volumes of phosphate-buffered saline at pH 7.4 and methanol, and finally with methanol.

(d) Add methanol and allow to stand for 10 minutes. (e) Remove the methanol and discard.

(f) If the monolayer is to be stained later, dry it completely. (g) If the monolayer is to be stained immediately, rinse it with sterile

water and discard the wash. (h) Add working solution of bisbenzimide or some other suitable

DNA staining agent and allow to stand for 10min. (i) Remove the stain and rinse the monolayer with sterile water. (j) Mount each coverslip with a drop of a mixture of equal volumes of

glycerol and phosphate-citrate buffer solution at pH 5.5; remove excess mountant from the edge of the coverslip by blotting.

(k) Examine by epifluorescence (330d38Onm excitation filter, LP 440nm barrier filter) at 100-400x magnification or greater.

(1) Compare the microscopic appearance of the test cultures with that of the negative and positive controls.

The product to be examined passes the test if there is no evidence of the presence of mycoplasmas in the test cultures inoculated with it.

Reagents

Bisbenzinzide (C,jH,,C13N,0 5H20 (M , 624)) 4- [5-15-(4-methylpip- erazin-l-yl)benzimidazol-2-yl]benzimidazo1-2-y1]phenol trihydro- chloride pentahydrate.

Bisbenzimide stock solution: To prepare stock solution, dissolve 5 mg of bisbenzimide in sterile water and dilute to 100ml with the same solvent. Store in the dark.

Bisbenzimide working solution: To prepare working solution, dilute lop1 of bisbenzimide stock solution to 100ml with phosphate- buffered saline at pH 7.4. Use immediately.

Phosphate-citrate buffer solution, pH 5.5: To prepare buffer, mix 56.85ml of a 2.84% (28.4811) solution of anhydrous disodium hydro- gen phosphate and 43.15ml of a 2.1% (21 gll) solution of citric acid."

Acknowledgements Acknowledgements are due to the following experts for their comments and advice on the draft Requirements:

Dr A. F. Angelo, Mycoplasma Laboratory for the Control of Biological Products, National Institute of Public Health and Environmental Protection, Bilthoven, Netherlands; Dr J. Borvendez, Deputy Director-General, National Institute of Pharmacy, Budapest, Hungary; Dr. I. R. Clement: Director, Regulatory Affairs, Connaught Laboratories, Ontario, Canada; Dr l . D. Gust, Director, Research and Development, CSL Ltd, Victoria, Australia; Dr K. J. Healy, Head, Quality Assurance, CSL Ltd, Victoria, Australia; Professor N. V. Medunitsin, Director, Tarasevich State lnstitute for Standardization and Control of Medical Biological Preparations, Moscow, Russian Federation; Dr R. Pandjaitan, Director, National

Quality Control Laboratory of Drug and Food, Jakarta, Indonesia; Dr L. Palkonyay, Chief, Viral Products Division, Bureau of Biologics, Drugs Directorate, Ottawa, Ontario, Canada.

Reference General Requirements for the Sterility of Biological Substances (Requirements for Biological Substances No. 6, revised 1973). In: WHO Expert Committee on Biological Standardization. Twenty-fifth Report. Geneva, World Health Organization, 1973, Annex 4 (WHO Technical Report Series, No. 530).


Annex 4 Summary protocol for the routine batch release of virus vaccines

Good documentation is an essential part of quality assurance. Its aims are to define the specifications for materials and methods of manufacture and testing and to provide the information necessary for batch release, as well as an audit trail that will permit the investigation of any suspected defective batch. A summary protocol of production and processing and quality control should therefore be prepared for each lot andlor batch of vaccine on the basis of a master record.

The aim of this summary protocol is to indicate the type of information that should be provided to a national control authority for the routine batch release of viral vaccines (inactivated or live), as well as the information that should be included in the release certification provided by a national control authority.

The more comprehensive summary protocol for the production and testing of a virus vaccine that is sometimes included as a part of the Requirements for individual products should be used for the purposes of licence application, as well as for the first submission of the product for batch release and whenever a change is made.

Control of final lot'

International name and proprietary name of vaccine


Lot no. of final product

Potency test

Method

Result

' This information should be used for the release of batches of vaccine during routine production. If the master or working seed lot or cell bank is changed in the production process, the information relating to this change should also be provided.

Reference no. of master seed lot

Reference no. of working seed lot

Tests on final bulk

Sterility tests

Method

Result

Test for chemical used in production (if applicable)

Method

Result

Potency test (if applicable)

Method

Result

Test for preservatives (if applicable)

Method

Result

Tests on final lot

Identity test

Method

Result

Sterility test

Method

Result

Potency test

Method

Result

Test for virus concentration and thermostability (if applicable)

Date of inoculation

Type of cell cultures

Control Heated samples samples (incubated at

maintained at 37 "C for usual storage - days) temperature

1 2 3 1 2 3

Virus concentration in each container (in PFU or CCID,,)

Mean virus titre per human dose: with 95 % fiducial limits

Mean loss in titre due to heat (in log,, units)

Reference preparation used

Identification

Theoretical titre

Actual titre

General safety test

No. of animals tested


Observation period


Test for endotoxin (if applicable)

Method

Result

Pyrogen content (if applicable)

Method

Result

Test for preservatives

Method

Results

Protein content (if applicable)

Method

Result

Adjuvants (if applicable)

Method

Results

Nature and concentration of adjuvant per single human dose

Residual moisture (if applicable)

Method

Result

Inspection of final containers

Method

Result

Submission addressed to national control authority for batch release

Name (typed) and signature of head of production laboratory

Date

Certification by person taking overall responsibility for production and control of the vaccine:

I certify that lot no. of vaccine satisfies national requirements andlor Part A of the Requirements for vaccine in Requirements for Biological Substances No.

Signature

Name (typed)

Date

Model certificate for the release of virus vaccine by national control authorities'

The following lots of vaccine produced by 2 in 3 whose numbers appear on the labels of the final containers,

meet all national requirements4 and Part AS of Requirements for Biologi- cal Substances No. (Requirements for ~ a c c i n e ) ~ and comply with Good Manufacturing Practices for pharmaceutical7 and ~ i o l o ~ i c a l ' Products.

Lot Date of last Expiry Lot Date of last Expiry no. potency test date no. potency test date

by manu- by manufacturer facturer

As a minimum, this certificate is based on examination of the manufacturing protocol.

The Director of the National Control Laboratory (or Authority as appropriate):'

Signature

Name (typed)

Date

' To be completed by the national control authority of the courtr,~ .,,{here the vaccine has been manufactured, and to be provided by the vaccine manufaciurzr to importers. Name of manufacturer. Country. If anv national reauirements are not met, s~ec i f v which one(s) and indicate why release of the lot(sj has neverthhless been authorized by 'the national contioi authority. with' the exception of the provisions on distribution and shipping, which the national control authority may not be in a position to assess. WHO Technical Report Series, No. , [Insert no. of technical report in which Requirements appear.]

' WHO Technical Report Series, No. 823, 1992, Annex l . WHO Technical Report Series No. 822, 1992, Annex l .

g Or his or her representative.

0 World Health Organization WHO Technical Report Series, No. 872. 1998

Annex 5 Biological substances: International Standards and Reference Reagents

A list of International Biological Standards, International Biological Reference Preparations and International Biological Reference Re- agents is issued as a separate publication.' Copies may be obtained from appointed sales agents for WHO publications or from: Distri- bution and Sales, World Health Organization, 1211 Geneva 27, Switzerland.

The Expert Committee made the following changes to the previous list.'

Additions

Antibiotics

Gent amicin 31 020 IUIampoule Second International Standard 1995

Antibodies

Anti-Parvovirus B19 100IU/ampoule First International IgG Serum, Human Standard 1995

Thyroid-stimulating 0.1 IUIampoule First International Antibody Standard 1995

Antigens and related substances

Live Attenuated Poliovirus (Sabin)

First International Reference Reagent 1995

Type 1 6.6 logl, CCID,, (i.e. 4.0 X 106 CCID,,)/ml

' Biological substances: lnternational Standards and Reference Reagents, 1990. Geneva, World Health Organization, 1991.

"reviously published in Weekly epidemiological record, 1996, 71 :105-108.

Type 2 5.610glo CCID,, (i.e. 4.0 X lo5 CCID,,)iml

Type 3 6.210gl, CCIDS, (i.e. 1.6 X 106 CCID,,)/ml

Total virus 6.8 log,, CCID,, content (i.e. 6.3 X 106 CC1~,,)/ml

These substances are held and distributed by the International Laboratory for Biological Standards: National Institute for Biological Standards and Control, Potters Bar, Herts. EN6 3QG, England.

Blood products and related substances

Protein S, Plasma 0.90 IUiampoule

Plasminogen- Activator Inhibitor 1, Plasma, Human

First International Standard 1995


Tissue- 27.5 IUIampoule plasminogen- activator- neutralizing activity

Urinary- 7.0 IUiampoule plasminogen- activator- neutralizing activity

These substances are held and distributed by the !nternational Laboratory for Biological Standards, Nationai Institute fcr Bl3iogjcal Standards and Control, Potters Bar, Herts. EN6 3QG, England.

Thromboplastin, International Third International Rabbit, Plain Sensitivity Reference Reagent

Index: 1.0 1995

This substance is held and distributed by the lniernational Laboratory for Biological Standards, Central Laboratory of the Netherlar!ds Red Cross Blood Transfusion Service, Plesmanlaan 125, Amsterdam, Netherlands.

Cytokines

Interleukin-8 1000 IUiampoule First International Standard 1995

Endocrinological and related substances

Follicle-stimulating 138 IUIampoule First International Hormone, Standard 1995 Recombinant, Human

Urofollitropin 121 IUIampoule First International Standard 1995

These substances are held and distributed by the International Laboratory for Biological Standards, National Institute for Biological Standards and Control, Potters Bar, Herts. EN6 3QG, England.

Discontinuation Interferon, Human (HuIFN- y)


O World Health Organization WHO Technical Report Series, No. 872, 1998

Annex 6 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 con- cerned with biological substances are formulated by international groups of experts and are published in the Technical Report Series of the World Health organization,' as listed here.

Requirements

1. General Requirements for Manufacturing Establishments and Control Laboratories

Revised 1965, TRS 323 (1966) Replaced by "Good manufacturing practices for biological products", TRS 822 (1992) and "Guidelines for national authorities on quality assurance for biological products", TRS 822 (1992)

2. Requirements for Poliomyelitis Vaccine (Inactivated) Revised 1981, TRS 673 (1987) Addendum 1985, TRS 745 (1987)

3. Requirements for Yellow Fever Vaccine Revised 1975, TRS 594 (1976) Addendum 1987, TRS 771 (1988) Revised 1995, TRS 872 (1998)

4. Requirements for Cholera Vaccine Revised 1968, TRS 413 (1969) Addendum 1973, TRS 530 (1973)

' Abbreviated here as TRS.

5. Requirements for Smallpox Vaccine Adopted 1966, TRS 323 (1966)

6. General Requirements for the Sterility of Biological Substances Revised 1973, TRS 530 (1973) Amendment 1995, TRS 872 (1998)

7. Requirements for Poliomyelitis Vaccine, Oral Revised 1989, TRS 800 (1990)

S.& Requirements for Diphtheria, Tetanus, Pertussis and Combined 10. Vaccines

Revised 1989, TRS 800 (1990)

9. Requirements for Procaine Benzylpenicillin in Oil with Aluminium Monostearate

Revised 1966, TRS 361 (1967) Discontinued 1989, TRS 800 (1990)

11. Requirements for Dried BCG Vaccine Revised 1985, TRS 745 (1987) Amendment 1987, TRS 771 (1988)

12. Requirements for Measles Vaccine (Live) Revised 1987, TRS 771 (1988) Replaced by Requirements No. 47

13. Requirements for Anthrax Spore Vaccine (Live, for Veterinary Use)

Adopted 1966, TRS 361 (1967)

14. Requirements for Human Immunoglobulin Adopted 1966, TRS 361 (1967) Replaced by Requirements No. 27

15. Requirements for Typhoid Vaccine Adopted 1966, TRS 361 (1967)

16. Requirements for Tuberculins Revised 1985, TRS 745 (1987)

17. Requirements for Influenza Vaccine (Inactivated) Revised 1990, TRS 814 (1991)

18. Requirements for Immune Sera of Animal Origin Adopted 1968, TRS 413 (1969)

19. Requirements for Rinderpest Cell Culture Vaccine (Live) and Rinderpest Vaccine (Live)

Adopted 1969, TRS 444 (1970)

20. Requirements for Brucella abortus Strain 19 Vaccine (Live, for Veterinary Use)

Adopted 1969, TRS 444 (1970) Addendum 1975, TRS 594 (1976)

21. Requirements for Snake Antivenins Adopted 1970, TRS 463 (1971)

22. Requirements for Rabies Vaccine for Human Use Revised 1980, TRS 658 (1981) Amendment 1992, TRS 840 (1994)

23. Requirements for Meningococcal Polysaccharide Vaccine Adopted 1975, TRS 594 (1976) Addendum 1980, TRS 658 (1981)

24. Requirements for Rubella Vaccine (Live) Adopted 1976, TRS 610 (1977) Addendum 1980: TRS 658 (1981) Replaced by Requirements No. 47

25. Requirements for Br~icelln melitensis Strain Rev. 1 Vaccine (Live, for Veterinary Use)

Adopted 1976, TRS 610 (1977)

26. Requirements for Antimicrobic Susceptibility Tests I. Agar Diffusion Tests Using Antimicrobic Susceptibility Discs

Revised 1981, TRS 673 (1982) Addendum 1982, TRS 687 (1983) Addendum 1985, TRS 745 (1987) Addendum 1987, TRS 771 (1988) Addendum 1989, TRS 800 (1990) Addendum 1990, TRS 814 (1991) Addendum 1991, TRS 822 (1992) Discontinued 1991, TRS 822 (1992)

27. Requirements for the Collection, Processing and Quality Control of Blood, Blood Components and Plasma Derivatives

Revised 1992, TRS 840 (1994)

28. Requirements for Influenza Vaccine (Live) Adopted 1978: TRS 638 (1979)

29. Requirements for Rabies Vaccine for Veterinary Use Adopted 1980, TRS 658 (1981) Amendment 1992, TRS 840 (1994)

30. Requirements for Thromboplastins and Plasma Used to Control Oral Anticoagulant Therapy

Revised 1982, TRS 678 (1983)

31. Requirements for Hepatitis B Vaccine Prepared from Plasma Revised 1994, TRS 858 (1995)

32. Requirements for Rift Valley Fever Vaccine Adopted 1981, TRS 673 (1982)

33. Requirements for Louse-Borne Human Typhus Vaccine (Live)

Adopted 1982, TRS 687 (1983)

34. Requirements for Typhoid Vaccine (Live, Attenuated, Ty 21a, Oral)

Adopted 1983, TRS 700 (1984)

35. Requirements for Rift Valley Fever Vaccine (Live, Attenuated) for Veterinary Use

Adopted 1983, TRS 700 (1984)

36. Requirements for Varicella Vaccine (Live) Revised 1993, TRS 848 (1994)

37. Requirements for Continuous Cell Lines Used for Biologicals Production

Adopted 1985, TRS 745 (1987)

38. Requirements for Mumps Vaccine (Live) Adopted 1986, TRS 760 (1987) Replaced by Requirements No. 47

39. Requirements for Hepatitis B Vaccines Made by Recombinant DNA Techniques in Yeast

Adopted 1986, TRS 760 (1987) Replaced by Requirements No. 45

40. Requirements for Rabies Vaccine (Inactivated) for Human Use Produced in Continuous Cell Lines

Adopted 1986, TRS 760 (1987) Amendment 1992, TRS S40 (1994)

41. Requirements for Human Interferons Made by Recombinant DNA Techniques

Adopted 1987, TRS 771 (1988)

42. Requirements for Human Interferons Prepared from Lymphoblastoid Cells

Adopted 1988, TRS 786 (1989)

43. Requirements for Japanese Encephalitis Vaccine (Inactivated) for Human Use

Adopted 1987, TRS 771 (1988)

44. Requirements for Haemorrhagic Fever with Renal Syndrome (HFRS) Vaccine (Inactivated)

Adopted 1993, TRS 848 (1994)

45. Requirements for Hepatitis B Vaccines Made by Recombinant DNA Techniques

Adopted 1988, TRS 786 (1989)

46. Requirements for Hnernophilzis Type b Conjugate Vaccines Adopted 1990, TRS 814 (1991)

47. Requirements for Measles: Mumps and Rubella Vaccines and Combined Vaccine (Live)

Adopted 1992, TRS 840 (1994) Note, TRS 848 (1994)

48. Requirements for Vi Polysaccharide Typhoid Vaccine Adopted 1992, TRS 840 (1994)

49. Requirements for Hepatitis A Vaccine (Inactivated) Adopted 1994, TRS 858 (1995)

Requirements for Immunoassay Kits [unnumbered] Adopted 1980, TRS 658 (1981)

Other documents

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)

TRS 565 (1975)

Development of national assay services for hormones and other substances in community health care

TRS 565 (1975)

Report of a WHO Working Group on the Standardization of Human Blood Products and Related Substances

TRS 610 (1977)

Guidelines for quality assessment of antitumour antibiotics TRS 658 (1981)

The national control of vaccines and sera TRS 658 (1981) Replaced by "Guidelines for national authorities on quality assurance for biological products", TRS 822 (1992)

Procedure for approval by WHO of yellow fever vaccines in connexion with the issue of international vaccination certificates

TRS 658 (1981)

A review of tests on virus vaccines TRS 673 (1982)

Standardization of interferons (reports of WHO informal consultations)

TRS 687 (1983) TRS 725 (1985) TRS 771 (1988)

Production and testing of the WHO yellow fever virus primary seed lot 213-77 and reference batch 168-73

TRS 745 (1987)

Report of a WHO Meeting on Hepatitis B Vaccines Produced by Recombinant DNA Techniques

TRS 760 (1987)

Procedure for evaluating the acceptability in principle of vaccines proposed to United Nations agencies for use in immunization programmes, revised 1988

TRS 786 (1989)

Guidelines for the preparation, characterization and establishment of international and other standards and reference reagents for biological substances, revised 1989

TRS 800 (1990)

Guidelines for assuring the quality of pharmaceutical and biological products prepared by recombinant DNA technology

TRS 814 (1991)

Good manufacturing practices for biological products TRS 822 (1992)

Guidelines for national authorities on quality assurance for biological products

TRS 822 (1992)

Guidelines for assuring the quality of monoclonal antibodies for use in humans

TRS 822 (1992)

Laboratories approved by WHO for the production of yellow fever vaccine, revised 1995

TRS 872 (1998)

Regulation and licensing of biological products in countries with newly developing regulatory authorities

TRS 858 (1995)

Summary protocol for the routine batch release of virus vaccines TRS 872 (1998)


Annex 7 Corrigenda: reference materials for anti-interferon alfa and beta sera; interferon gamma, recombinant human; and mumps vaccine (live)

Anti-interferon alfa and beta sera The International Reference Reagents for Anti-Interferon Alfa Se- rum and Anti-Interferon Beta Serum, established in 1994, are held and distributed by:

National Institute of Allergy and Infectious Diseases Anti-Viral Research Branch 6003 Executive Boulevard Solar Building, Rm. 3A22 Rockville, MD 20892 USA.

The address given for these reference materials in WHO Technical Report Series, No. 858, 1995, p. 93 should be corrected accordingly.

Interferon gamma, recombinant human The International Standard for Interferon Gamma, Recombinant Human, established in 1994, is held and distributed by:

National Institute of Allergy and Infectious Diseases Anti-Viral Research Branch 6003 Executive Boulevard Solar Building, Rm. 3A22 Rockville, MD 20892 USA.

The address given for this reference material in WHO Technical Report Series, No. 858, 1995, p. 95 should be corrected accordingly.

Mumps vaccine (live) The first International Reference Reagent for Mumps Vaccine (Live) has an assigned content of 4.6 log,, (40 000) infectious units of mumps virus in each ampoule. The value given in WHO Technical Report Series, No. 858, 1995, p. 93 should be corrected accordingly.

World Health Organization Technical Report Series Recent reports: No.

828 (1992) Evaluation of certain food additives and naturally occurring toxicants Thirty-ninth report of the Joint FAOIWHO Expert Committee on Food Additives (57 pages)

829 (1993) Evaluation of recent changes in the financing of health services Report of a WHO Study Group (79 pages)

830 (1993) The control of schistosomiasis Second report of the WHO Expert Committee (93 pages)

831 (1993) Rehabilitation after cardiovascular diseases, with special emphasis on developing countries Report of a WHO Expert Committee (130 pages)

832 (1993) Evaluation of certain veterinary drug residues in food Fortieth report of the Joint FAOiWHO Expert Committee on Food Additives

(68 pages) 833 (1993) Health promotion in the workplace: alcohol and drug abuse

Report of a WHO Expert Committee (39 pages) 834 (1993) WHO Expert Committee on Specifications for Pharmaceutical

Preparations Thirty-third report (35 pages)

835 (1993) Aging and working capacity Report of a WHO Study Group (55 pages)

836 (1993) WHO Expert Committee on Drug Dependence Twenty-eighth report (50 pages)

837 (1993) Evaluation of certain food additives and contaminants Forty-first report of the Joint FAOIWHO Expert Committee on Food Additives (61 pages)

838 (1993) Increasing the relevance of education for health professionals Report of a WHO Study Group on Problem-Solving Education for the Health Professions (33 pages)

839 (1993) Implementation of the Global Malaria Control Strategy Report of a WHO Study Group on the Implementation of the Global Plan of Action for Malaria Control 1993-2000 (62 pages)

840 (1994) WHO Expert Committee on Biological Standardization Forty-third report (223 pages)

841 (1994) Cardiovascular disease risk factors: new areas for research Report of a WHO Scientific Group (59 pages)

842 (1994) Nursing beyond the year 2000 Report of a WHO Study Group (25 pages)

843 (1994) Assessment of fracture risk and its application to screening for postmenopausal osteoporosis Report of a WHO Study Group (134 pages)

844 (1994) Prevention of diabetes mellitus Report of a WHO Study Group (108 pages)

845 (1994) Information support for new public health action a t district level Report of a \ W O Expert Committee (35 pages)

846 (1994) Fluorides and oral health Report of a \$'H0 Expert Committee on Oral Health Status and Fluoride Use (12 pages)

847 (1994) Chemotherapy of lepros!- Report of a I$'HO Study Group (29 pages)

* Prices in developing countries are 7096 of those listed here.

(1994) WHO Expert Committee on Biological Standardization Forty-fourth report (94 pages) (1995) Control of foodhorne trematode infections Report of a WHO Study Group (165 pages) (1995) The use of essential drugs Sixth report of the WHO Expert Committee (144 pages) (1995) Evaluation of certain veterinary drug residues in food Forty-second report of the Joint FAOIWHO Expert Committee on Food Additives (50 pages) (1995) Onchocerciasis and its control Report of a WHO Expert Committee on Onchocerciasis Control (111 pages) (1995) Epidemiology and prevention of cardiovascular diseases in elderly people Report of a WHO Study Group (72 pages) (1995) Physical status: the use and interpretation of anthropometry Report of a WHO Expert Committee (462 pages) (1995) Evaluation of certain veterinary drug residues in food Forty-third report of the Joint FAOIWHO Expert Committee on Food Additives (65 pages) (1995) WHO Expert Committee on Drug Dependence Twenty-ninth report (21 pages) (1995) Vector control for malaria and other mosquito-borne diseases Report of a WHO Study Group (97 pages) (1995) WHO Expert Committee on Biological Standardization Forty-fifth report (108 pages) (1995) Evaluation of certain food additives and contaminants Forty-fourth report of the Joint FAOIWHO Expert Committee on Food Additives (62 pages) (1996) Nursing practice Report of a WHO Expert Committee (37 pages) (1996) Integration of health care delivery Report of a WHO Study Group (74 pages) (1996) Hypertension control Report of a WHO Expert Committee (89 pages) (1996) WHO Expert Committee on Specifications for Pharmaceutical Preparations Thirty-fourth report (200 pages) (1996) Evaluation of certain veterinary drug residues in food Forty-fifth report of the Joint FAOIWHO Expert Committee on Food Additives (66 pages) (1996) Control of hereditary diseases Report of a WHO Scientific Group (89 pages) (1996) Research on the menopause in the 1990s Report of a WHO Scientific Group (114 pages) (1997) The use of essential drugs Seventh report of the WHO Expert Committee (80 pages) (1997) Evaluation of certain food additives and contaminants Forty-sixth report of the Joint FAOIWHO Expert Committee on Food Additives (77 pages) (1997) Improving the performance of health centres in district health systems Report of a WHO Study Group (70 pages) (1997) Promoting health through schools Report of a WHO Expert Committee on Comprehensive School Health Education and Promotion (99 pages)

(1997) Medical methods for termination of pregnancy Report of a WHO Scientific Group (116 pages)

who expert committee on biological...

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