8/3/2019 WHO_TRS_530

1/66

This report contains the collective views of an internationalgroup of exper ts and does not necessarily represent the deci-sions or the s ta ted policy of the World Health Organization.

WORLD HEALTH ORGANIZATIONTECHNICAL REPORT SERIES

No. 530

WHO EXPERT COMMITTEE ONBIOLOGICAL STANDARDIZATION

Twenty-fifth Report

WORLD HEALTH ORGANIZATIONGENEVA

1973

8/3/2019 WHO_TRS_530

2/66

World Health Organization 1973Publications of the World Health Organization enjoy copyright protection in accord-ance with the provisions of Pro tocol 2 of the Universal Copyr ight Convent ion. Forrights of reproduction or translation ofWHO publications, in part or in toto, applicationshould be made to the Office of Publications and Translation, World Health Organ-ization, Geneva, Switzerland. The World Health Organization welcomes such appli-cations.The designations employed and the presentation of the material in this publicationdo not imply the expression of any opinion whatsoever on the part of the Director-General of the World Health Organizat ion concerning the legal s tatus of any countryor territory or of its authorities, or concerning the delimitation of its frontiers.The mention of specific companies or of certain manufacturers' products does notimply that they are endorsed or recommended by the World Health Organization inpreference to others of a similar nature that are not mentioned. Errors and omissionsexcepted, the names of proprietary products are distinguished by initial capital letters.

PRINTED IN SWITZERLAND

8/3/2019 WHO_TRS_530

3/66

CONTENTSPHARMACOLOGICAL SUBSTANCES

Antibiotics1. Doxycycline2. Minocycline3. Neomycin .4. Gramicidin S

Hormones, vitamins, enzymes, andmiscellaneous substances5. Glucagon6. Heparin .7. Vitamin D . . . . . . . . . . . . . . . . . .8. Sulfarsphenamine, neoarsphenamine, oxophenarsine9. Mel B (melarsoprol) , dimercaprol , MSb10. Thrombin . . . . . . . . .11. Opacity Reference Preparation . .

IMMUNOLOGICAL SUBSTANCESAntigens12. Diphtheria toxoid, plain13. Yellow fever vaccine.14. Cholera vaccine

Antibodies15. Human immunoglobulin IgE .....16. Diphtheria antitoxin for flocculation test17. Gas-gangrene antitoxins . . . . . . .18. Gas-gangrene antitoxin (Clostridium histolyticumt19. Anti-Salmonella pullorum sera. . . . . . . .

REQUIREMENTS FOR BIOLOGICAL SUBSTANCES20. Requirements for inactivated influenza vaccine21. Requirements for cholera vaccine . . . . . .22. Requirements for rabies vaccine for human use23. General requirements for the steri li ty of biological substances

ANNEXES

Page5666

6778889

91010

10I II I1212

12131313

Annex 1. Requirements for inactivated influenza vaccine (Addendum 1973) 15Annex 2. Requirements for cholera vaccine (Revised 1968) (Addendum 1973) 18Annex 3. Requirements for rabies vaccine for human use . . . . . . 22Annex 4. General requirements for the sterility of biological substances(Revised 1973) . . . . . . . . . . . . . . . . . . . . . . . . . 40Annex 5. Requirements for biolo gical substances and other sets of recommenda tions 62Annex 6. International standards and international reference preparations 64INDEX. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66

3

8/3/2019 WHO_TRS_530

4/66

WHO EXPERT COMMITTEE ON BIOLOGICAL STANDARDIZATIONGeneva, 24-30 April 1973

Members:

Dr H. Cohen , Direc tor, Nat iona l Ins ti tu te of Public Health, Bil thoven, NetherlandsDr J. Desbordes, Director, Microbiology Section,National Public Health Laboratory,Paris, France (Vice-Chairman)Professor S. G. Dzagurov, Director, Tarasevic State Institute for the Standardization

and Control of Biological Preparations, Moscow, USSRProfessor D. G. Evans , Direc tor, Nat iona l Ins ti tu te for Biological S tandards andControl, London, England (Chairman)Dr Sutas Guptarak, Chief, Biological Division, The Government PharmaceuticalOrganization, Bangkok, ThailandDr P. Krag, Director, Department of Biological Standardization, Statens Seruminstitut, Copenhagen, DenmarkDr H. Mirchamsy, Associa te Director, Razi State Insti tute for Serum and VaccineProduction, Teheran, IranDr R. Murata, Director, The Second Department of Bacteriology, National Instituteof Health, Tokyo, JapanDr E. B. Seligmann, Jr , Director, Division of Control Activities, Bureau of Biologics,

Food and Drug Administration, Rockville, Md. , USADr M. Yu, Senior Pathologist , Depar tment of Pathology, Singapore (Rapporteur)

Secretariat:Dr A. S. Outschoorn, Chief Medical Officer, Biological Standardization, WHO,Geneva, Switzerland (Secretary)

4

8/3/2019 WHO_TRS_530

5/66

WHO EXPERT COMMITTEE ONBIOLOGICAL STANDARDIZATION

Twenty-fifth Report

The WHO Expert Committee on Biological Standardization met inGeneva from 24 to 30 April 1973. Dr T. A. Lambo, Assistant DirectorGeneral, opened the Meeting on behalf of the Director-General. Hewelcomed the Members of the Committee and thanked them for comingto Geneva to participate in this Meeting. He drew attention to the longhistory of biological standardization, an activity inherited by WHO fromthe League of Nations Health Organization and the Interim Commission.The WHO Expert Committees on Biological Standardization continue thework carried out previously by the Permanent Commission on BiologicalStandardization of the League and it was interesting to note that in theyear of WHO's 25th anniversary there was also convened the 25th ExpertCommittee on Biological Standardization. He was sure that the discussionsof this Committee would be in keeping with the high standards and traditions maintained over the years.

PHARMACOLOGICAL SUBSTANCES

ANTIBIOTICS1. Doxycycline

In regard to the authorization in the twenty-fourth report 1 for theestablishment of the preparation studied as the international referencepreparation of doxycycline the Committee was informed that it had notyet been possible to establish this preparation. The Committee thereforeendorsed the previous authorization for the National Institute for Biological Standards and Control, London, to establish the material as the International Reference Preparation of Doxycycline on the basis of the resultsof the collaborative assay and to define the international uni t with theagreement of the participants.

1 WId HItlz Org. techno Rep. Ser ., 1972, No. 486, p. 9.5

8/3/2019 WHO_TRS_530

6/66

8/3/2019 WHO_TRS_530

7/66

report,' had been completed. The result had shown that the material wassuitable to serve as the international standard and the Committee thereforeestablished this material as the International Standard for Glucagon,Porcine, for Bioassay. The Committee also noted 2 a proposal for thedefinition of the international unit, which was equivalent to the only knownexisting national unit and which was acceptable to the participants . TheCommittee agreed with this proposal and defined the International Unitfor Glucagon, Porcine, for Bioassay as the activity contained in 4.5302 mgof the International Standard for Glucagon, Porcine, for Bioassay.

6. HeparinThe Committee noted 3 that stocks of the second International Standardfor Heparin were nearly exhausted and agreed that it should be replaced.A preparation of heparin of porcine mucosal origin had been examined ina collaborative study of heparins from different sources 4 and found suitable to replace the current International Standard when this became necessary. The Committee agreed that this preparat ion could serve as thereplacement without the necessity of a further collaborative assay. TheCommittee therefore established the material as the third InternationalStandard for Heparin in replacement of the second international standard.The Committee also noted 3 a proposal on which to base the unitageassigned to the preparat ion from the results of the collaborative study.The Committee was informed that this was acceptable to the participants

and therefore defined the International Unit for Heparin as the activitycontained in 0.005766 mg of the third International Standard for Heparin.7. VitaminD

The Committee considered further the question of the discontinuationof the International Standard for Vitamin D discussed in the twenty-fourthreport.' In response to the request in tha t report," the WHO Secretariathad investigated the possibility of making vitamin D available as a chemical reference substance and action was being taken with a view to providingsuch a reference substance. As it would be advisable also to have availablefor international use a well characterized, stable preparation of vitamin Dthat could be used for biological assays, the Committee agreed that thecurrent International Standard should not be discontinued for the present.

1 Wid Hlth Org. techno Rep. Ser., 1972, No . 486, p. 11.2 Unpublished working document WHO/BS/73.1064.3 Unpublished working document WHO/BS/73.1065.4 Bull. WId Hlth Org., 1970, 42, 129.5 WId Hlth Org: technoRep. Ser. , 1972, No . 486, p. 12.

7

8/3/2019 WHO_TRS_530

8/66

8/3/2019 WHO_TRS_530

9/66

The Committee agreed that there was insufficient evidence at present forthe need for an international reference preparation of thrombin andrequested the WHO Secretariat to collect further information in order toassess the need.11. Opacity Reference Preparation

The Committee noted 1 the results of a study at the Statens Seruminstitut, Copenhagen, in which certain comparisons had been made betweenopacity determinations and est imation by weight of bacterial mass inpertussis vaccine. These studies were made with the International OpacityReference Preparation and a number of samples of rout ine product ionbatches all made at the Statens Seruminstitut from the same strains ofBordetella pertussis by the same procedure. The results showed that betweenbatches there was less variation in bacterial mass per millilitre of vaccinethan in opaci ty uni ts as determined photoelectrically. The Committeeagreed that it would be of interest to undertake further investigations,which might be carried out in individual laboratories, of the relationshipof various parameters of pertussis vaccine, including optical density andweight of bacterial mass used, under differing conditions, with estimates ofimmunizing potency and toxicity. Such determinations may also be usefulfor calibrating photoelectric instruments for measuring optical density ofvaccines.

IMMUNOLOGICAL S U B ST A N C ESANTIGENS

12. Diphtheria Toxoid, PlainIn regard to the reques t in the twenty-fourth report 2 for informationon continued use of purified diphtheria toxoid as a non-adsorbed product,the Committee noted 3 that the Statens Seruminst itut , Copenhagen, incollaboration with the WHO Secretariat, had ascertained that non-adsorbeddiphtheria toxoid was in l imited use. Some laboratories had also agreedon the continuing need for the International Standard for control purposes,particularly for combined vaccines. The Statens Seruminstitut would therefore obtain suitable material that could serve as a replacement for theInternational Standard for Diphtheria Toxoid, Plain, and would arrange acollaborative assay.1 Unpublished working document WHO/BSj72.1061.2 WId Hlth Org . techno Rep. Ser. , 1972, No . 486, p. 14.3 Unpublished working document WHO/BS/72.1060.

9

8/3/2019 WHO_TRS_530

10/66

13. Yellow Fever VaccineThe Committee noted 1 that certain collaborative research studies ofyellow fever vaccines were being arranged by the WHO Secretariat. Theywere designed to develop a more practical method, using cell culturesinstead of mice, for titrating the virus content of such vaccines. The Committee also noted 1 that a further possible outcome of these studies mightbe a suitable preparation of yellow fever vaccine to serve as a referencepreparation. Such material would be useful for the control of yellow fevervaccines and should be considered in relation to the revision of theRequirements for Yellow Fever Vaccine requested in the twenty-secondreport."

14. Cholera VaccineThe Committee considered certain problems in the use of the currentInternational Reference Preparations of Cholera Vaccine (Ogawa) and ofCholera Vaccine (Inaba) in the evaluation of potency of cholera vaccine.In view of the fact that there is recent evidence of a relationship betweenthe results of assays made in mice in one laboratory and protection of manin field trials, the Committee agreed that it might be useful if internationalunits be assigned to these International Reference Preparations.Since, however, in the international collaborative assay for the replacement of the international reference preparations referred to in the twentyfourth report," a wide variation was obtained among laboratories in therelative immunogenicities, the Committee asked the Statens Seruminstitut, Copenhagen, to collect further information to enable a decision tobe made.

ANTffiODIES15. Human ImmunoglobulinIgE

The Committee noted 4 the results of the collaborative studies of apreparation of human immunoglobulin IgE referred to in the twenty-thirdreport." A further preparation of this immunoglobulin had also been madeand a collaborative assay of it had been completed. The results showedthat the latter preparation was suitable to serve as an international reference preparation for the assay of human immunoglobulin IgE preparations. The Committee therefore established the material as the International Reference Preparation of Human Immunoglobulin IgE.

1 Unpublished working document WHO/BS/73.IOn.2 Wid Hltb Org. techno Rep. Ser. , 1970, No. 444, p. 21.3 Wid Hlth Org. techno Rep. Ser., 1972, No. 486, p. 13.4 Unpublished working documentWHO/BS/73.I070.5 Wid Hlth Org. techno Rep. Ser., 1971, No. 463, p, 21.

10

8/3/2019 WHO_TRS_530

11/66

The Committee also noted 1 a proposal , which was agreeable to theparticipants, for a.definition of the international unit, equivalent to the onlyknown national unit. The Committee therefore defined on this basis theInternational Unit for Human Immunoglobulin IgE as the activity contained in 0.006562mg ofthe International Reference Preparation of HumanImmunoglobulin IgE.16. DiphtheriaAntitoxin for Flocculation Test

The Committee noted 2 that, in accordancewith the authorization in thetwenty-fourth report," the material referred to had been established by theStatens Seruminstitut, Copenhagen, as the fifth International ReferencePreparation of Diphtheria Antitoxin for Flocculation Test in replacementof the fourth international reference preparation and that the Institute hadspecified its flocculating activity as 1800 Lf equivalents per ampoule.The Committee agreed that when this international reference preparationis distributed a statement should also be issued to the effect that the relationship of antitoxic activity as determined by in vivo and in vitro methods(in vivo[in vitro ratio) was not stated, since such a rat io depends on themethods of determination and cannot be unequivocally defined.

17. Gas-GangreneAntitoxinsThe various International Standards for Gas-gangrene Antitoxins werefirst established many years ago. The names of these standards wereoriginally assigned according to the practice of the time and are not inconformity with the present principles of bacterial nomenclature; they arealso not named in a uniform manner.The Committee therefore decided that the following InternationalStandards be renamed:

Old nameGas-gangrene antitoxin (perfringens)(Clostridium welchii type Aantitoxin)Gas-gangrene antitoxin (vibrion septiqueiGas-gangrene antitoxin (oedematiens)Gas-gangrene antitoxin (histo1yticus)Gas-gangrene antitoxin (Sorde1li)

New nameGas-gangrene antitoxin (Clostridiumwelchii type A)

Gas-gangrene antitoxin (Clostridiumsepticum)Gas-gangrene antitoxin (Clostridiumoedematiensi

Gas-gangrene antitoxin (Clostridiumhistolyticum)

Gas-gangrene antitoxin (Clostridiumsordellii)

1 Unpublished working document WHO/BS/73.1070.2 Unpublished working document WHO/BSj72.1056.3 Wld Hlth Org. techno Rep. Ser ., 1972, No . 486, p. 17.

11

8/3/2019 WHO_TRS_530

12/66

18. Gas-Gangrene Antitoxin (Clostridium histolyticum)The Committee noted 1 that in accordance with the authorization in thetwenty-fourth report 2, the material referred to had been established by theStatens Seruminstitut, Copenhagen, as the third International Standard forGas-Gangrene Antitoxin (Clostridium histolyticum) in replacement of thesecond international standard and that the Institute had defined the International Unit for Gas-Gangrene Antitoxin (Clostridium histolyticum) asthe activity contained in 0.2 mg of the third International Standard for GasGangrene Antitoxin (Clostridium histolyticum).

19. Anti-Salmonella pullorum SeraThe Committee noted 3 that the collaborative assay of the two preparations of anti-Salmonella pullorum serum referred to in the twenty-fourthreport 4 had been completed. The results showed that the preparationswere suitable to serve as international standards for assaying sera containing

Salmonella pullorum antibodies, one for antibodies of the standard formS and the other for antibodies of the variant form V. The Committeetherefore established the material for the proposed standard S serum as theInternational Standard for Anti-Salmonella pullorum Serum (StandardForm S) and the material for the proposed standard V serum as the International Standard for Anti-Salmonella pullorum Serum (Variant Form V).The Committee also noted 3 a proposal, which was acceptable to theparticipants, for the definition of the international unit for each of thesepreparat ions. The Committee defined, on this basis, the Internat ionalUnit for Anti-Salmonella pullorum Serum (Standard Form S) as the activitycontained in 0.0838 mg of the International Standard for Anti-Salmonellapuflorum Serum (Standard Form S) and the International Unit for AntiSalmonella pullorum (Variant FormV) as the activity contained in 0.0814 mgof the International Standard for Anti-Salmonella pullorum Serum (VariantForm V).

REQUIREMENTSFOR BIOLOGICAL SUBSTANCES

20. Requirements for Inactivated Influenza VaccineThe Committee studied the proposed Addendum 5 to the Requirementsfor Inactivated Influenza Vaccine (Requirements for Biological Substances1 Unpublished working document WHOjBS/72.1055 Rev. 1.2 Wid Hlth Org. techno Rep. Ser., 1972, No. 486, p. 17.3 Unpublished working document WHOjBSj73.1066.4 Wid Hlth Org. technoRep, Ser., 1972, No. 486, p. 18.S Unpublished working document WHOjBSj72.1057 Rev. 1.

12

8/3/2019 WHO_TRS_530

13/66

" -------- " .. -----_ .._-- ---No. 17) 1 prepared by the WHO Secretariat in collaboration with variousexperts. The Addendum was intended to enable specifications for haem-agglutinin content in influenza virus vaccines to be made in terms of theInternational Reference Preparation of Influenza Virus Haemagglutinin(Type A).

The Committee, after making some modifications, adopted the Adden-dum to these requirements and agreed that it should be annexed to thepresent report (see Annex 1).

21. Requirements for Cholera VaccineThe Commit tee studied the proposed Addendum 2 to the revisedRequirements for Cholera Vaccine (Requirements for Biological Sub-stances No.4, Revised 1968) 3 prepared by the WHO Secretariat in collabo-

ration with a number of experts. These modifications were needed becausethe international Reference Preparations of Cholera Vaccine (Ogawa) andof Cholera Vaccine (Inaba) had been replaced since the revised require-ments were formulated.

The Committee, after making some modifications, adopted the Adden-dum to these requirements and agreed that it should be annexed to thepresent report (see Annex 2).

22. Requirements for Rabies Vaccine for Human UseThe Committee studied 4 the proposed Requirements for Rabies Vaccinefor Human Use prepared by the WHO Secretariat in collaboration with a

number of experts. The Committee, a fter making some modifications,agreed that the text of these requirements was satisfactory and that theywould be of value for the control of rabies vaccine for human use producedin different countries.The Committee adopted these requirements and agreed that they shouldbe annexed to the present report (see Annex 3).23. General Requirements for the Sterility of Biological SubstancesThe Committee studied 5 the proposed General Requirements for theManufacture and Control of Sterile Pharmaceutical Preparations andBiological Substances and for Sterility Testing which had been prepared by

the WHO Secretariat in col laboration with a number of experts. These1 WId Hlth Org. techn, Rep. Ser, 1968, No . 384, Annex 2.2 Unpublished working document WHO/BS/72.1059 Rev.I.3 WId Hlth Org. techno Rep. Ser., 1969, No . 413, Annex 1.4 Unpublished working document WHO/BS/72.1058 Rev.I.5 Unpublished working document WHO/BSj73.1062; \VHOjPHARM/73.474.

13

8/3/2019 WHO_TRS_530

14/66

proposed requirements had been prepared for consideration of the WHOExpert Committee on Biological Standardization and the WHO ExpertCommittee on Specifications for Pharmaceutical Preparations.The Committee agreed that the formulation of such requirements bythese two Expert Committees, in liaison, would be useful for the control or'sterility of relevant products in different countries, and made detailedrecommendations that could be considered at a later stage by the ExpertCommittee on Specifications for Pharmaceutical Preparations.In view, however, of the observation in the twenty-fourth report,' andthe need for an early revision of the Requirements for Biological SubstancesNo.6 (General Requirements for the Sterility of Biological Substances) 2,the Committee agreed that the general requirements relevant to the sterilityof immunological biological substances should be considered for adoptionby the Committee at the present time.The Committee therefore studied the material made available and,after making certain modifications, prepared such requirements relatingto immunological biological substances. I t was agreed that the requirements so prepared were satisfactory and that they would be useful for thecontrol of sterility of immunological biological substances produced indifferent countries.The Committee therefore adopted these revised general requirements andagreed that they shouldbe annexed to the present report (see Annex 4).The Committee emphasized that when general requirements for sterilitywere quoted in the requirements for individual biological products in theseries of Requirements for Biological Substances, the revised requirementsshould apply.

ACKNOWLEDGEMENTSThe Committee wishes to record its thanks to the following members of the WHOSecretariat for their special contributionsto its deliberations:

Dr K. Bogel, Veterinary Public Health; Dr P. Bres, Virus Diseases; Dr W. C. Cockburn,Chief, Virus Diseases; Dr M. Gonzalez-Pacheco, Biological Standardization; Mr K. O.Wallen, Chief, Pharmaceuticals; and Dr Y. Watanabe, Bacterial Diseases.

1 Wid Hltb Org. techno Rep. Ser., 1972, No. 486, p. 19.2 Wid Hlth Org. techno Rep. Ser., 1960, No. 200, Annex.

14

8/3/2019 WHO_TRS_530

15/66

Annex 1REQUIREMENTS FOR INACTIVATED INFLUENZA VACCINE

(Requirements for Biological Substances No. 17)Addendum 1973

The Requirements for Inactivated Influenza Vaccine were adopted bythe twent ieth WHO Expert Committee on Biological Standardization(Geneva, 25-30 September 1967).1 In these requirements it was specifiedthat tests for content of virus ant igen in whole virus vaccines be made onboth monovalent bulk and final product, and tha t these tests be made incomparison with the International Reference Preparation of InfluenzaHaemagglutinin (Type A) (established in 1967) 2 or an equivalent referencepreparation approved by the national control authority. The purposewas to enable the content of virus antigen to be expressed in terms ofinternational units.

International collaborative studies made after the establishment of theinternational reference preparation were considered by the twenty-first andtwenty-second Expert Committees on Biological Standardization," Theseshowed that the international reference preparation, which was of Type Avirus haemagglutinin, was also suitable for tests of Type B virus haemagglutinin.s Further studies had also been made on the relation betweenthe immunizing effect, evaluated in various ways, of inactivated influenzavirus vaccines and their haemagglutinin content."

The twenty-first Exper t Committee on Biological Standardizat ionrequested that an addendum be prepared for the Requirements for Inact ivated Influenza Vaccine, taking into consideration the results of thesestudies, since specification of the haemagglutinin content of influenza virusvaccines in in terna tional uni ts was now possible. In formulating thisaddendum account has been taken of opinions and data received from theexperts listed below, whose assistance is gratefully acknowledged.Professor A. de Ba rb ie ri , General Director, Istituto Sieroterapico Milanese" Serafino

Belfanti ", Milan, ItalyDr W. C. Cockburn, Chief, Virus Diseases, WHO, Geneva, SwitzerlandDr H. Cohen, Director, National Institute of Public Health, Bilthoven, Netherlands

1 Wid Hlth Org. techno Rep. Ser ., 1968, No . 384, p. 22.2 Wid Hlth Org. techno Rep. Ser ., 1968, No . 384, p. 15.3 Wid Hlth Org. techno Rep. Ser. , 1969, No . 413, p. 20 ; 1970, No. 444, p. 15.4 Bull. Wid Hlth Org., 1971,45,473-486.5 Wid Hlth Org. techno Rep. Ser ., 1970, No . 444, p. 15.

15

8/3/2019 WHO_TRS_530

16/66

Mr V. F. Davey, Deputy Director (Technical) , Commonwealth Serum Laboratories,Parkville, Victoria, AustraliaDr J. Desbordes, Director, Microbiology Section, National Public Health Laboratory,Paris, FranceProfessor S. G. Dzagurov, Director, Tarasevic State Insti tu te for the Standardization

and Control of Biological Preparations, Moscow, USSRProfessor D. G. Evans, Director, National Institute for Biological Standards and Control,

London, EnglandDr Ch. B. Gerichter, Director, Division of Laboratories, Ministry of Health, Jerusalem,IsraelDr J. J. Graydon, Commonwealth Serum Laboratories, Parkville, Victoria, AustraliaProfessor G. Heymann , Federal Agency for Sera and Vaccines, Frankfurt-am-Main,Federal Republic of GermanyDr A. Krassnigg, Director-General of Public Health, Vienna, AustriaDr W. G. Laver, Microbiology Department, The John Curtin School of MedicalResearch, The Australian National University, Canberra, AustraliaDr R. Murray, Director, Division of Biologics Standards, National Institutes of Health,Bethesda, Md., USAProfessor R. Negri , Chief, Laboratory of Microbiology, Istituto Superiore di Sanita,

Rome, ItalyDr M. Pontecorvo, Director, Biological Division, Istituto Sieroterapico e Vaccinogeno

Toscano" Sclavo ", Siena, ItalyDr G. C. Schild, Director, World Influenza Centre, National Institute for MedicalResearch, Mill Hill , London, EnglandDr J. R. Thayer , Chief Inspecto r, National Biological Standards Laboratory, Canberra,Australia

The following amendments are made to the Requirements for InactivatedInfluenza Vaccine. I t should be noted that, since these requirements applyonly to whole virus vaccines, the present addendum also applies only tosuch vaccines.Amendment 1

In Part A, section 3.5.3 Testfor content of virus antigen, insert after theexisting paragraph the following:

" The material passes the test if the content of haemagglutinin is not lessthan 600 IV in the quantity corresponding to the largest recommendedsingle human dose, provided that not less than two-thirds of such virusantigen is of Type A influenza virus, the remainder being of either Type Aor Type B.

There is now some indication that the protective activity ofinfluenza vaccines is related to their haemagglutinin content.I t is therefore advisable that vaccines should contain as muchhaemagglutinin as has been found from experience to be protective and safe in man.

16

8/3/2019 WHO_TRS_530

17/66

I t is not possible to specify a maximum permissible contentof haemagglutinin for all kinds of vaccine. A con tent grea terthan 600 IV may cause a high frequency of untoward reactions,depending on the degree of purification of the vaccines. Nationalcon trol authori ties should decide on a maximum acceptablecontent of antigen for each kind of vaccine.. .

Amendment 2In Part A, section 8, Labelling, delete "the number of internationalunits or comparable units of haemagglutinin per dose for each strain" andreplace by " the number of international units of haemagglutinin for eachstrain per largest recommended single human dose".

17

8/3/2019 WHO_TRS_530

18/66

Annex 2REQUIREMENTS FOR CHOLERA VACCINE(Requirements for Biological Substances No.4)

Addendum1973

The requirements for cholera vaccine were adopted by a WHO StudyGroup on Requirements for Biological Substances (1958).1 A revised versionof the Requirements was adopted by the twenty-first Expert Committeeon Biological Standardization (1968).2 In the revised requirements it wasspecified that tests of antigenicity be made on each vaccine lot, and thatthese tests be made in comparison with the International Reference Preparations of Cholera Vaccine (Ogawa) and of Cholera Vaccine (Inaba) orsuitable national reference preparations. It was further specified that thenational control authority should provide or approve the reference preparation(s) to be used, the relationship of the preparation(s) to the correspondingInternational Reference Preparations of Cholera Vaccines having beenpreviously established. In the active mouse protection test of antigenicityin the revised requirements, an antigenicity ratio for each serotype, in termsof the respective International Reference Preparation, was specified.At the time the revised requirements for cholera vaccine were adopted,the current International Reference Preparat ions of Cholera Vaccine(Ogawa) and of Cholera Vaccine (Inaba) were those established in 1953.The twentieth Expert Committee on Biological Standardization (1967),3however, requested a collaborative assay of two monovalent choleravaccines whose antigenicity was greater than that of the relevant International Reference Preparations and that resembled in antigenicity thecholera vaccines then being produced. The twenty-fourth Expert Committeeon Biological Standardization (1971)4 established the preparations studiedas the second International Reference Preparations of Cholera Vaccine

(Ogawa) and of Cholera Vaccine (Inaba) in replacement of the first international reference preparations.The Expert Committee also pointed out that the provisions relating tolimits of antigenicity as determined in the active mouse protection test inthe revised requirements for cholera vaccine were no longer applicable andrequested the WHO Secretariat to arrange for suitable modifications to1 Wid Hlth argo techno Rep. Ser., 1959, No. 179, Annex 2.2 Wid Hlth argo techno Rep. Ser., 1969, No. 413, Annex 1.3 Wid Hlth argo techno Rep. Ser., 1968, No. 384, p. 14.4 Wid Hlth argo technoRep. Ser., 1972, No. 486, p. 13.

18

8/3/2019 WHO_TRS_530

19/66

these requirements. In formulating these modifications, account has beentaken of opinions and data received from the experts listed below, whoseassistance is gratefully acknowledged:Dr D. R. Bangham, Division of Biological Standards, National I nst it ut e for Bio-logical Standards and Control, London, EnglandPro fessor A. de Barbier i, General Director, Istituto Sieroterapico Milanese" SerafinoBelfanti ", Milan, ItalyDr D. Barua, Medical Officer, Bacterial Diseases, WHO, Geneva, SwitzerlandDr H. Cohen, Director, National Institute of Public Health, Bilthoven, NetherlandsDr B. Cvjetanovic, Chief Medical Officer, Bacterial Diseases,WHO, Geneva, SwitzerlandDr V. F. Davey, Deputy Director (Technical), Commonwealth Serum Laboratories,Parkville, AustraliaMr I. Davidson, Central Veterinary Laboratory, Weybridge, Surrey, EnglandDr J. Desbordes, Director, Microbiology Section, National Public Health Laboratory,

Paris, FranceProfessor D. G. Evans , Director, National Institute for Biological Standards and Control,

London, EnglandDr J. C. Feeley, Chief, Bacterial Immunology Section, Bacteriology Branch, Center forDisease Control, Atlanta, Ga., USADr J. J. Graydon, Commonwealth Serum Laboratories, Parkville, AustraliaDr I. J60, " Human" Institute for Serobacteriological Production and Research, Buda-pest, HungaryProfessor A. Lafontaine, Director, Ins ti tu te of Hygiene and Epidemiology, Brussels,BelgiumMr J. W. Lightbown, Division of Biological Standards, National Institute for Biological

Standards and Control, London, EnglandDr M. S. Nasution, Director, PerusahaanNegara "Bio Farma" (Pasteur Institute), Ban-dung, IndonesiaDr R. Netter, Director, Virology Section, National Public Health Laboratory, Paris,

FranceDr F. T. Perkins, Division of Immunological Products Control, National Institute forBiological Standards and Control, London, EnglandDr M. Pittman, 3133 Connecticut Avenue N.W., Washington, D.C., USADr J. D. van Ramshorst, Chief, Department of Biological Standards, National Public

Health Laboratory, Bilthoven, NetherlandsProfessor M. Sa letti, Head, Microbiological Department, Istituto Sieroterapico eVaccinogeno Toscano" Sclavo ", Siena, ItalyDr E. B. Seli gmann, Jr , Chief, Division of Control Activities, Bureau of Biologics,

Food and Drug Administration, Rockville, Md. , USADr J. Spaun, Deputy Director, Department of Biological Standardization, StatensSeruminstitut, Copenhagen, DenmarkDr A. F. B. Standfast , The Lister Institute of Preventive Medicine, Elstree, EnglandDr J. S. Sumpaico, Director of Medical Bureau (Laboratories), Bureau of Research and

Laboratories, Department of Health, Manila, Philippines

19

8/3/2019 WHO_TRS_530

20/66

/Dr J. R. Thayer, Chief Inspector , National Biological Standards Laboratory, Canberra,AustraliaDr W. Aeg, Timmerman, Blauwkapelseweg 29, De Bilt, NetherlandsMr K. Uemura, Chief, Hea lth Sta tist ica l Methodology, WHO, Geneva, SwitzerlandDr Y. Watanabe, Medical Officer, Bacterial Diseases, WHO, Geneva, SwitzerlandDr R. J. Wilson, Chairman and Director, Connaught Laboratories Limited, Ontario,Canada

The requirements for antigenicity in this addendum have been formulatedon the basis that an individual lot of cholera vaccine is acceptable if it isnot significantly lower in antigenicity than the respective InternationalReference Preparations of Cholera Vaccine. Related to the question ofsignificance is that of limits of error of the estimate of the antigenicityratio. In the existing requirements (1968) this question was not dealt with.In the international collaborative assay 1 for the replacement of the international reference preparations of cholera vaccines, the participants hadassayed the antigenicity of the proposed replacements in parallel with theexisting internat ional reference preparations; a wide variation in therelative antigenicities was found. The confidence limits (%), however,varied little between laboratories arid were apparently independent of theestimates of relative antigenicity. The 95% confidence interval for eachserotype, was between 45% and 227% of the relative antigenicity when 3tests were performed and the results combined. In an informal survey of anumber of cholera vaccines produced in different countries, arranged by theWHO Secretariat, it was also found that there was a wide variation in therelative antigenicities.The following amendments are made to the revised Requirements forCholera Vaccine:Amendment 1In Part A, section 1.3 : International standards or referencepreparations and

international unitsReplace the whole section by the following:

" The second International Reference Preparation of CholeraVaccine (Inaba) and the second International Reference Preparation of Cholera Vaccine (Ogawa) were established in 1971. 2Each preparation is dispensed in ampoules and each ampoulecontains freeze-dried material from 5 ml of monovalent vaccineof the particular serotype. These preparations are intended forcalibrating reference preparations that are used in antigenicitytesting (see Part A, section 5.4).

1 Unpublished working document WHO/BS/72.1032 Rev. 1.2 WId Hlth Org, techno Rep. Ser., 1972, No. 486, p. 13.

20

8/3/2019 WHO_TRS_530

21/66

The third International Opacity Reference Preparation(established in 1965)1 is dispensed in ampoules containing 15mlof a suspension of Pyrex-glass par ticles in water (10 IV ofopacity per ml).These reference prepa ra tions are in the custody of th eInternational Laboratory for Biological S tandards , S ta tensSeruminstitut, Copenhagen. Samples ar e distributed free ofcharge, on request, to national control laboratories."

Amendment 2In Part A, section 5.4. 1 : Active mouse protection testReplace the first paragraph by the following:

" T h e antigenicity of th e two serotypes of the vaccine iscompared with that of th e relevant monovalent or divalentreference preparations by immunization o f mice an d subsequentchallenge with a virulent strain of the appropriate serotype ofV. cholerae." Fo r each test the dilutions of the laboratory reference preparation should be such that they correspond in antigenicity tothe dilutions made from the relevant International ReferencePreparation, reconst itut ed with 5 ml of fluid pe r ampoule.Similar dilutions are made of th e test vaccine.

" Fo r the antigenicity test of each serotype th e vaccine andthe relevant reference preparation should be tested in parallel." In some countr ies for each tes t a divalent reference prep-aration is used in th e form that corresponds in antigenicityto a mixtur e of equal volumes of each International ReferencePreparation reconst itut ed with 5 ml of fluid pe r ampoule."

Replace (c) by the following:"(c) Estimate of relative antigenicity. Th e median effectiveimmunization dose (ED.o) of each vaccine is cal culat ed by aconventional method. The antigenicity ratio for each of th erespective challenges (Ogawa an d Inaba) is determined, an d theconfidence limits of the ratio calculated." Th e vaccine passes the test if (i) th e antigenicity of th e

largest recommended human dose is no t significantly less thanthat of 1 ml of th e reconstituted International Reference Preparation of each serotype, as determined by th e antigenicityr at io , a nd (ii) the precision of the test , as determined by theconfidence limits of th e ratio, is acceptable." I f th e vaccine does no t meet these criteria, th e test ma ybe repeated. Th e results of all the tests pe rfo rmed should beused in calculating the final result. "

1 WId H lt h Org . techn, Rep. SeT., 1966, No. 329, p. 21.

21

8/3/2019 WHO_TRS_530

22/66

//

Annex 3REQUIREMENTS FOR RABIES VACCINE FOR HUMAN USE 1

(Requirements for Biological Substances No. 22)

PageIntroduction . 22General considerations 24Part A: Manufacturing requirements

1. Definitions 272. General manufacturing requirements 293. Production control 294. Fi ll ing and containers . 365. Control tests on final product 366. Records. 377. Samples. 378. Labelling 379. Distribution and shipping 3810. Storage and expiry date 38Part B: National control requirements

1. General 382. Release and certification . 39

IntroductionThe WHO monograph Laboratory Techniques in RabiesF which wasfirst published in 1954 and has since been twice revised, includes material

that has been a useful guide for the manufacture and testing of rabiesvaccine. I t was not, howerer, writ ten to serve as international require-ments for the manufacture and control of rabies vaccines. The fifth and1 Prepared by the following members of the WHO Secretariat:Dr M. Abdussalam, Chief, Veterinary Public Health, WHO, Geneva, Switzerland;Dr K. Bogel, Veterinary Public Health, WHO, Geneva, Switzerland; Professor D. G.Evans, Director, National Institute for Biological Standards and Control, London,England (Consultant); Dr M. Kaplan, Director, Office of Science and Technology,WHO, Geneva, Switzerland; Dr R. Netter, Director, Virology Section, National PublicHealth Laboratory, Paris, France (Consultant); Dr A. S. Outschoorn, Chief, BiologicalStandardization, WHO, Geneva, Switzerland; Dr F. T. Perkins, Division of Immu-nological Products Control, National Institute for Biological Standards and Control,London, England (Consultant); Dr E. B. Sel igmann, Jr , Chief, Division of ControlActivities, Bureau of Biologics, Food and Drug Administration, Rockville, Md., USA.2 Kaplan M. M. & Koprowski, H. , ed. (1973) Laboratory techniques in rabies,3rd ed., Geneva, World Health Organization (Monograph Series, No. 23).

22

8/3/2019 WHO_TRS_530

23/66

sixth WHO Expert Committees on Rabies 1, 2 recommended that WHOshould take steps to develop requirements for rabies vaccine and expandstudies on tissue culture vaccines. The twenty-first WHO Expert Committee on Biological Standardization 3 agreed that international requirements for rabies vaccine are needed and that it would be feasible toformulate them, especially as an International Reference Preparation ofRabies Vaccine has been established.The following international requirements for rabies vaccine (for humanuse) have been fitted into the framework adopted in the Requirements forBiological Substances Nos. 1 to 21, already published by WHO,4 and indraft ing them, account has been taken of the opinions of consultants, theregulations and requirements for the manufacture and control of rabiesvaccine that have been formulated in a number of countries, as well asinformation from both published and unpublished reports. In addit ion,opinions and data have been received from a number of experts, whoseassistance is acknowledged below:

Dr P. Atanasiu, Pasteur Institute, Paris, FranceProfessor A. de Barbier i, General Director, Istituto Sieroterapico Milanese " SerafinoBelfanti ", }'Iilan, ItalyProfessor Benhassine, Director General, Pasteur Institute, Algiers, AlgeriaDr K. Berger, Director, Federal Vaccine Production Institute, Vienna, AustriaMr Ian Davidson, Central Veterinary Laboratory, Weybridge, EnglandDr J. Desbordes, Director, Microbiology Section, National Public Health Laboratory,Paris, FranceProfessor S. G. Dzagurov, Director, Tarasevic State Institute for the Standardization

and Control of Biological Preparations, Moscow, USSRProfessor G. Edsall, Head, Department of Microbiology, London School of Hygiene and

Tropical Medicine, London, EnglandDr P. Fenje, Connaught Laboratories Limited, Ontario, CanadaProfessor G. Heymann, Federal Agency of Sera and Vaccines, Frankfurt-am-Main,

Federal Republic of GermanyDr Hilary Koprowski, Director, The Wistar Institute, Philadelphia, Pa., USADr M. Majer , Head, Virus Vaccine Production Department, Swiss Serum and VaccineInstitute, Berne, SwitzerlandDr H. Mirchamsy, Razi State Institute for Serum and Vaccine Production, Teheran, IranDr M. Pontecorvo, Director, Biological Division, Istituto Sieroterapico e Vaccinogeno

Toscano" Sclavo ", Siena, ItalyDr J. B. Shrivastav, Director General of Health Services, New Delhi, IndiaDr A. K. Thomas, Director, Central Research Institute, Kasauli, IndiaDr W. Aeg, Timmerman, Blauwkapelseweg 29, De Bilt, NetherlandsD rN . Veeraraghavan, Director, Pasteur Institute of Southern India, Coonoor, India

1 Wld HIth Org. techn . Rep. Ser., 1966, No . 321, p. 12.2 Wld Hlth Org : techno Rep. Ser ., 1973, No . 523, pp. 21 and 45.3 Wld Hlth Org. techno Rep. Ser ., 1969, No . 413, p. 25.4 Fo r a list of references, see WId Hlth Org. techno Rep. Ser., 1973, No . 530, p. 62.

23

8/3/2019 WHO_TRS_530

24/66

General ConsiderationsVarious types of vaccine are described in the fifth report of the WHOExpert Committee on Rabies." Vaccines presently in use in man in mostcountries are derived from nerve tissue, either brain or brain with spinalcord. Vaccines prepared in chick or duck embryos are in use in a fewcountries and vaccine produced in primary hamster kidney-cell cultures hasalso been used. A number of vaccines produced from cell culture are being

studied experimentally but only a few have been used in man. Because ofthe widespread manufacture and use of rabies vaccine from nerve tissueand embryos and the comparatively limited use of cell culture vaccines atthis time, these international requirements for rabies vaccine have beenrestricted to those vaccines that are produced in nerve tissues or in embryos.Furthermore, in view of numerous problems created by the use of rabiesvaccines containing living virus they have also been restricted to inactivatedvaccines.A number of different manufacturing and testing procedures are inuse in various countries. The procedures differ in the rabies virus strainused, the species of animal used for propagation of the virus, the methodof inactivation, the preservatives added, the form in which the vaccine isissued, and the methods for testing potency.The passage histories of the different strains of virus being used forproduction are not well documented. Such a strain should be one knownto produce vaccine that is antigenically active against classical rabies virusstrains." While many strains have been derived from the original Pasteurstrain, others used for production have been isolated recently from man oranimals. Strains used for production should be limited to what is termeda " fixed" strain of virus. I t is desirable that studies be made in order todefine qualitatively and quantitatively the properties of a " fixed" strain.This is one that has a short , stable, and reproducible incubation periodwhen injected intracerebrally into rabbits. However, it can be demonstrated that strains in use at the present time differ considerably in theirability to produce rabies in experimental animals when inoculated by routes

other than intracerebral. The present requirementsrecommend tha t thePasteur strain of rabies" fixed" virus maintained only in rabbits by intracerebral inoculation be used for production. The use of the seed lot systemhas also been specified. I t is recommended, however, that a specific antirabies serum be produced and be made available as an aid to establishingthe identity and purity of the seed virus, since the International Standardfor Anti-Rabies Serum is not made available - and may not be suitable-

1 Wid Hlth Org; techno Rep. Ser., 1966, No. 321, p. 10.2 Classical rabies virus s trains are those belonging to serotype 1 of the rabies subgroup of rhabdoviruses.

24

8/3/2019 WHO_TRS_530

25/66

---- " ------

for this purpose. I t is also desirable that studies be made to establishappropriate genetic markers to characterize the virus used for production.I t has been assumed that the ability of a rabies vaccine to produce highneutralizing antibody titres in man is an indication of its effectiveness inprotection. I t has also been assumed that good protection in experimentalanimals is an indication that a vaccine will be effective in man. Neitherassumption may be entirely correct. In any event, it has not been possible

to specify the minimum level of activity that will afford protection for man.Because of this difficulty, it is important that rabies vaccines be preparedhaving maximum antigenicity in man and experimental animals, togetherwith an acceptable level of adverse reactions. For this reason rabies vaccine must not be diluted to the point where it just satisfies minimum potencyrequirements.Nerve tissue rabies vaccines have been in worldwide use for generations,and experience has indicated that they are generally effective. I t is universally recognized that such vaccines produce some incidence of post-vaccinalcomplications of the central nervous system, but there is some evidencethat the risk is reduced when vaccines are produced in the brains of youngsuckling animals. However, data are lacking on the degree of risk due tothe possible presence in the vaccine of adventitious agents (from mice andrats) that may have survived the inactivation procedures. Duck-embryovaccine has a demonstra ted low risk for central nervous system effects.However, duck-embryo vaccine is somewhat less immunogenic in mousepotency tests than is nerve tissue vaccine and it does no t produce a goodlevel of neutralizing antibody in man. Nevertheless, it has been shown,on a statistical basis, that the number of rabies cases following administration of duck-embryo vaccine is no t greater than the number occurringwhen nerve tissue vaccine is used. Because of its greater safety, duck-embryovaccine is being increasingly used for pre-exposure immunization. Studieshave been made of the effectiveness of duck-embryo vaccine in combinationwith minimal amounts of antirabies human immunoglobulin for postexposure treatment.The development of new rabies vaccines is hindered by the difficultyin evaluating the effectiveness in man. I t has never been possible to determine accurately the degree of risk to an individual following exposure to arabid animal. Because of the nature of the disease, it is virtually impossiblewith rabies vaccine to do control led clinical studies involving an unvaccinated group to determine accurately the degree of effectiveness. Studiesare in progress to develop vaccines free from neurological and allergiceffects and to enhance the antigenicity of vaccines by adjuvants . Whenimproved rabies vaccines are put into general use, these requirementswill require revision.The potency testing of rabies vaccine is of considerable concern. Thepotency test must be capable of differentiating between vaccines of differing

25

8/3/2019 WHO_TRS_530

26/66

activity. Preferably a single test should be applicable to all types of vaccine.The rabies virus strain used for challenge should be one of reproduciblevirulence for the test animal when given intracerebrally. In addition thetest should be reproducible and economical. A common reference pre-paration of vaccine is of considerable aid in evaluating test results. Ideally,such a reference preparation should be protective in animals and found toproduce antibodies in man. The second International Reference Preparationof Rabies Vaccine (established in 1965)1 is available but it has not beentested in man nor examined for suitability for use in man. Classicallyvaccine potency has been based on the wet weight (mg) of brain tissuerequired for protection of 50% of the test animals. With the developmentof purified vaccines that contain reduced amounts of host tissue, and cellculture vaccines, the potency should be expressed on the basis of the dilutionof vaccine (injected in a defined volume) protecting 50% of the test animalsrather than on the basis of the tissue content. Although the minimumpotency ratio to a reference vaccine can be established with either procedureit is not yet possible to determine what ratio represents protection in man.However, for the determination of such a ratio the following considerationsapply. For nerve tissue vaccines the equivalent of at least 2 ml of a 5%tissue suspension has been recommended as the single dose for man." TheInternational Reference Preparation of Rabies Vaccine when reconstitutedfrom the dry form as instructed is equivalent to a 10% brain tissue sus-pension. Hence 1 ml is equivalent to one dose for man. For determiningthe relative potency of any vaccine in animals, the dilutionis made, startingfrom the concentration at which the vaccine is administered to man or theequivalent concentration of the reference vaccine as the case may be. Bythis procedure a single reference vaccine may be used for routine testingof potency of all types of rabies vaccine. This has the additional advantageof providing a common basis for comparing the potency of the classicalnerve tissue vaccines, with which there have been many years of experience,and the potency of the newer types of vaccine.Tests for factors in the vaccine that may induce allergic encephalo-myelitis have not been included in these requirements because no techniqueshave been described that can be relied upon. The degree of reproducibilityof existing procedures has not been evaluated and there is evidence thatconsiderable variation in results can be expected. Existing tests, however,can be used to assess the period during which the factor causing allergicencephalomyelitis develops in the brain of young animals. Studies shouldbe encouraged for improving such tests.Because of the need for large quantities of vaccine where rabies isprevalent, in some countries vaccine is used without fulfilling all of the

1 Wid Hltb Org. techno Rep. Ser., 1966, No. 329, p. 15.2 Wid Hlth Org. techno Rep. Ser., 1966, No. 321, p. 18.

26

8/3/2019 WHO_TRS_530

27/66

requirements for the potency test in order to avoid serious curtailment ofvaccine production. This, however, is not a satisfactory procedure andevery effort should be made to achieve compliance with all the presentrequirements. In cases, however, where the risks of using vaccine thatmay not itself have been tested for potency have to be weighed against theneed for protecting the population at risk against rabies, the decision restswith the national control author ity of the country in which the vaccine isto be used.The use of healthy animals has been specified in these requirements,bUL no provisions are incorporated for tests for extraneous viruses.National control authori ties should, however, pay attention to the prob-lems of ensuring that the animals used are free from infectious agents thatmight contaminate rabies vaccine.

Each of the following sections constitutes a recommendation. The partsof each section that are printed in large type have been written in the formof requirements so that, if a health administration so desires, these partsas they appear may be included in definitive national requirements. Thepar ts of each section that are printed in small type are comments andrecommendations for guidance.Should individual countries wish to adopt these requirements as thebasis of their national regulations concerning rabies vaccine, it is recom-mended that a clause be included permitt ing modifications of manufac-turing requirements on the condition that it be demonstrated, to the satis-faction of the national control authority, that such modified requirementsensure a degree of safety and potency of the vaccine at least equal to thoseprovided by the requirements formulated below. I t is desirable that theWorld Health Organization should then be informed of the action taken.The terms " national control authority" and " national control labor-atory " as used in these requirements, always refer to the country in whichthe vaccine is manufactured.

Part A : Manufacturing Requirements1. Definitions1. 1 International name and proper name

The international name shall be " Vaccinum rabiei (for Human Use) ".The proper name shall be the equivalent of the international name in thelanguage of the country of origin.The use of the international name should he limited tovaccines that satisfy the requirements formulated below.

27

8/3/2019 WHO_TRS_530

28/66

1 .2 Descriptive definitionVaccinum rabiei (for Human Use) is a fluid or dried preparation of

rabies" fixed" virus grown in the brains or spinal cords of rabbits, sheep,goats, mice, or rats, or in embryonated eggs, and inactivated by a suitablemethod. The preparations for human use shall satisfy all the requirementsformulated below.1.3 International referencepreparation and international standard

The International Reference Preparation of Rabies Vaccine,established in 1965,1is stored and distributed in ampoules containing 121 mg of freeze-dried material. The vaccine in eachampoule, when reconstitutedwith 8 ml of sterile distilled water,is equivalent to a 10% rabbi t brain t issue suspension. Thisreference preparation is intended for the calibration of nationalreference prepara tions for use in tests of potency of rabiesvaccine (see Part B, section 1).2 After reconstitution the International Reference Preparation may be stored for subsequentanimal immunizations provided that the storage temperatureis below -60C and tha t the period of s to rage is not longerthan one month.The International Standard for Anti-Rabies Serum estab

lished in 1955,3 is stored and distributed in ampoules containing86.6 mg of dried hyperimmune horse serum. The InternationalUnit is defined as the act iv ity conta ined in 1 mg of the International Standard. Each ampoule therefore contains 86.6 IU.This standard is intended for use in the laborato ry assay ofpotency of antirabies immunoglobulin preparations used in man.I t can also be used for the assay of rabies antibodies in manand animals.The reference preparation and the standard are in the custody

of the International Laboratory for Biological Standards,Statens Seruminstitut, Copenhagen. Samples are distributed freeof charge to national control laboratories upon request.1.4 Terminology

Seed lot. A quantity of virus that has been processed together andhas a uniform composition. A seed lot is used for vaccine preparation orfor the preparation of further seed lots.Viral harvest. Tissue harvested from a single animal or from a group ofsuckling animals or a group of embryonated eggs inoculated at the same1 WId Hlth Org: techno Rep. Ser., 1966, No. 329, p. 15.2 Use of the International Reference Preparation for administration to man is no tauthorized. A national reference preparation should no t be considered as suitable for usein man unless it has been approved by the national control authority.3 WId Hlth Org: techno Rep. Ser., 1956, No. 108, p. 11.

28

8/3/2019 WHO_TRS_530

29/66

time and harvested together. The virus in the harvest is without interveningpassage from the seed lot.

Bulk material. A pool of viral harvests before preparation of the finalbulk. Bulkmaterial may be preparedfrom one or a number of viral harvestsand may yield one or more final bulks.

Final bulk. A quantity of vaccine present in a single container fromwhich the final containers are filled.

Filling lot (final lot). A collection of sealed final containers that arehomogeneous with respect to the risk of contamination during filling ordrying. A filling lo t must, therefore, have been filled in one working sessionand (if applicable) have been dried together.2. Generalmanufacturing requirements

The general requirements for manufacturing establishments containedin the revised Requirements for Biological Substances No. 1 (GeneralRequirements for Manufacturing Establishments and Control Labor-atories) 1 shall apply to establishments manufacturing rabies vaccine (forhuman use).3. Productioncontrol3. 1 Control of source materials3.1.1 Strain of virus

The strain of virus used in the production of all seed lots shall be acc fixed" strain and shall be identified by historical records. I t shall havebeen shown to the satisfaction of the national control authori ty to yieldimmunogenic vaccines 'when the virus has been inactivated. In addi tionthe vaccine strain shall be characterized by serological tests and animalinoculation.Records shall be maintained of all tests made periodically for verifi-cation of strain characters. Such tests shall include the titration in animalsof various species and age and by various routes of inoculation as well asserum neutralization tests.

Preferably, the producti on strain should be the Pasteurs train of rabies" fixed" virus maintained only in rabbit pass-age. 2 I t should be capable of producing characteristic paralysiswithin 5 to 7 days in rabbits inoculated intracerebrally.1 Wid Hlth Org. techn, Rep. Ser., 1965, No. 323, p. 11.2 This s train is avai lable to laborator ies on request from the Wor ld Health Organi-zation, 1211 Geneva 27, with the approval of the national authorities.

29

8/3/2019 WHO_TRS_530

30/66

3. I .2 Animals or embryonated eggs for the production of seed virus andvaccineFor vaccine production only healthy animals, or embryos obtained fromhealthy flocks, shall be used. They shall conform to all the requirementsgiven in Part A, section 3.2. I and section 3.2.2 respectively.

Different species of animals may be used for vaccine production or for preparing seed virus. Rabbits (adult or suckling),sheep, goats, suckling mice, suckling rats, and chicken or duckembryonated eggs are used successfully in different countries.

3. I .3 Seed lot systemPreparation of rabies vaccine (for human use) shall be based on the use

of the seed lot system. A seed lot shall not be more than 10 passagesremoved from the characterized strain. Vaccines shall be made from a seedlot without further intervening passage. If a seed lot is used for thepreparation of a further seed lot such further seed lot shall be madewithout intervening passage. Seed lots shall be maintained either in driedor frozen form. If frozen, the seed shall be kept continuously at a temperature below -60C.Seed lots should have been shown, to the satisfaction of the

national control authority, to be capable of yielding vaccinethat meets all of these requirements

3. I .4 Tests on seed lotsThe seed lot, in the dilution used as inoculum for the production ofvaccine shall be tested for bacterial and fungal contamination by appropriatetests according to the requirements of Part A, section 5. 2 of the revisedRequirements for Biological Substances No.6 (General Requirements forthe Sterility of Biological Substances}!Each seed lot shall be identified as rabies virus by methods approved bythe national control authority.A titration of virus content of the seed lot shall be made.

Such titrations may be done by the intracerebral inoculationof suitable dilutions in mice. The mice are observed for 14 days.The virus activity of the seed lo t should be such that all miceare killedwhen so inoculatedwith 0.03-m1 quantities of a dilutionof no t less t han 10-6

3.2 Production precautionsThe general precautions as formulated in the requirements of Part A,section 3, of the revised Requirements for Biological Substances No. 11 Wid Hlth Org. techno Rep. Sen., 1973, No . 530, p. 49.

30

8/3/2019 WHO_TRS_530

31/66

(General Requirements for Manufacturing Establishments and ControlLaboratories) 1 shall apply to the manufacture of rabies vaccine (for humanuse) with the addition of the following:Penicillin and streptomycin preparations shall no t be used at any stageof manufacture of the vaccine.

3.2.1 Vaccinesproduced in animal brain and spinal cordThe animals intended for production shall be kept in quarantine underveterinary supervision for at least 2 weeks prior to inoculation of the seedvirus, except in the case of suckling animals when this requirement shallapply to the mothers. Only animals free from all signs of disease shall beused. Seed virus shall be inoculated intracerebrally. Methods for inocula-tion and harvesting approved by the national control authority shall be used.

While inoculation of virus is always made intracerebrally, thetechnique used varies with the species o f animal. A satisfactoryt echn ique is one that consistently produces paralysis in theanimals inoculated bu t does no t introduce other infection.In order to obtain th e maximum virus t it re, nerve tissuesfrom inoculated animals, apart from suckling animals, should beharvested on an individual basis when the animal is moribundan d estimated to be close to d e ath f r om rabies.

If suckling animals are used, the dose an d date of inoculationshould allow for harvest ing of brain tissue before neuroaller-genic activity becomes demonstrable. This ca n be done for theanimal species an d particular strain used for vaccine produc-tion by immunizing guineapigs with brain material suspendedin complete Freund adjuvant. I t is essential that positive an dnegative controls should be included in the test. On th e basisof th e results of th e test, th e period ca n be assessed dur ingwhich acceptable material can be harvested. Th e time of harvestused by s o me p r o du c tio n laboratories is 8 days for mice, 6days for rabbits , an d 7 to 11 days for rats.

Nerve tissue shall no t be taken from dead animals, whether death isdue to rabies or other causes.Al l animals used in th e production of vaccine should beexamined by autopsy after removal of nerve tissue. If evidence

of tuberculosis or any nerve disease other than rabies is found,th e nerve tissue from th e a ni m al s h ou ld be discarded, or ifnerve tissues have been pooled, t h e p o o l containing nerve tissuef ro m s u ch an a n ima l s h ou ld be discarded. I f there is evidenceof a communicable disease among th e animals, the viral harvestfrom that group should be discarded.

1 Wid Hlth Org: tec1I11. Rep. Ser., 1965, No . 323, p. 11.31

8/3/2019 WHO_TRS_530

32/66

When other than suckling animals are used, the tissue harvested fromeach animal shall be kept separate until completion of the sterility test(Part A, section 3.2.3). When suckling animals are used, .the harvestcomposed of a pool of tissue from a group of animals inoculated at thesame time and harvested together shall similarly be kept separate untilcompletion of the sterility test.3 .2.2 Vaccines produced in embryonated eggs

The eggs shall be derived from healthy flocks free from micro-organismsknown to be pathogenic for man.Such agents include Salmonella pullorum, Mycobacteriumtuberculosis, mycoplasma and avian leucosis viruses. I f eggs are

used from flocks that have no t been shown to be free f rom avianleucosis viruses and mycoplasma, the method of inactivationused should have been shown to the satisfaction of the nationalcontrol authority to be capab le of killing these organisms.

After the eggs have been incubated for a suitable period they shall beinoculated with seed virus. After further incubation for a suitable period,the living embryos shall be harvested with aseptic precautions. Embryosinoculated at the same time and harvested together may be pooled and theviral harvest kept separate until completion of the sterility test (Part A,section 3.2.3).3.2.3 Sterility tests of the viral harvest

A sample removed from each viral harvest shall be tested for bacterialand fungal contamination by appropriate tests according to the requirements of Part A, section 5. 2 of the revised Requirements for BiologicalSubstances No. 6 (General Requirements for the Sterility of BiologicalSubstances);' Any viral harvest in which contamination is detected shallbe discarded.

3 .3 Control of bulk material3.3. 1 Pooling of viral harvests

Only viral harvests satisfying the requirements for sterility given inPar t A, section 3.2. 3 of these requirements shall be pooled for bulkmaterial.1 WId Hlth Org: technoRep. Ser., 1973, No . 530, p. 48.

32

8/3/2019 WHO_TRS_530

33/66

3.3.2 Homogenization and virus titrationThe apparatus used for homogenizing the tissues shall be of such adesign as to prevent any escape of aerosols.

Grinding and blending of tissues should be done at as lowa temperature as possible to avoid destruction of virus.Nerve tissue vaccines should be prepared such that a singledose for man in the immunization course is contained in no t

more than 2 ml of a 5% nerve tissue suspension or its equivalent,e.g., 1 ml of a 10% suspensionBulk material shall not be frozen.

A sample should be t ak en fr om the homogenized bulkmaterial prior to inactivation fo r determination of virus titrein mice . The titre of virus in nerve tissue harvests should beno t less than 105 LD 501 per 0.03-ml dose; however, titres of theorder of 106 are achievable.

3.3.3 Time of inactivationInactivation shall be initiated immediately after homogenization andremoval of the sample for determination of virus titre.

3.3.4 InactivationprocedureMethods and agents approved by the national control authori ty shallbe used for inactivation. The method shall be demonstrated to be consistently effective in the hands of the manufacturer. The inactivation processshall also have been shown, to the satisfaction of the national controlauthori ty , to be capable of inactivating mycoplasma as shown by in vitrotests and also, in the case of vaccine produced in embryonated eggs, avianleucosis viruses as shown by tests in tissue culture, or, in the case of vaccineproduced in the brain of suckling animals, any adventitious agent that maybe present as shown by tests using tissue culture or by animal inoculation.

Various methods for inactivating rabies virus have beenused with success. The concentration of the inactivating chemical, temperature, and length of time necessary for inactivationmust be developed for the par ticular type of vaccine beingmanufactured. The most widely used agent is phenol, generallyat a concentration between 0.5% and 1% and at a temperature of20'-30'Cfor several days until completeinactivationhas occurredas shown by th e results of the test specified in section 3.3.5below. Etherized vaccines are produced in some countries bycombining the action of ether and pheno l in th e inactivationprocedure. Beta-propiolactcne (BPL) has also been used. Satisfactory vaccines may be prepared by treating 10% nerve tissue

1 LD 50 is the quantity of virus that kills 50% of mice when injected intracerebrally.33

8/3/2019 WHO_TRS_530

34/66

homogenates at 20C with a concentration of 1 : 3500 toI : 5000 BPL fo r 24 hours or until complete inactivation hasoccur red as shown by the results of the .test specified in section 3.3.5 below.Ul traviolet light i rradiat ion has also been used bu t theequipment required an d the procedures involved make it difficult to prepare vaccine in large volumes. Th e dosage range an dtime of application needed to accomplish complete inactivation

of the virus without reducing antigenicity are critical, bu t whenthe radiation dose is regulated properly, highly antigenicvaccinesmay be prepared. Th e time required for inactivation is shortcompared with chemical methods, and hence the vaccine maybe kept at a low temperature throughout; this also aids in conserving antigenicity. Vaccines produced by this procedure maybe freeze-dried. Fo r best results th e time from inactivation to initiation of the freeze-drying cycle should be kept to a minimum.

3 .3 . 5 Test for effective inactivationA test approved by the national control authority shall be used to testeach bulk material for inactivation of virus prior to the addition of preservatives and other substances.

The test should be per formed with undiluted bulk materialinjected intracerebrally into no t less than 10 mice.In some cases the concen tra tion of inactivating agent ortissue in undiluted bulkmaterial ma y be toxic to the test animals.In this case the test should be performed on final bulk material,which may be diluted, if necessary, b ut n ot more than 1 : 2 .In some countries 2 species o f animal are used, e.g., rabbits

an d mice, for testing effective inactivation. In such cases at least3 rabbi ts should be used. If th e virus was propaga ted in ananimal other than the rabbit , consideration should be given tousing the production species rather than the rabbit.The bulk material passes the test if the product has been shown, to thesatisfaction of the national control authority, to be free from residual livevirus.

3. 4 Preparation and control offinal bulk3.4. I Preservatives and other substances added

In preparing the final bulk, only those preservatives or other substancesapproved by the national control authority shall be added. Such substancesshall have been shown by appropriate tests no t to impair the safety oreffectiveness of the product in the amounts used.If phenol has been used for inactivation, its concentration in the finalbulk shall be such that it will no t exceed 0.25% in the final product.

34

8/3/2019 WHO_TRS_530

35/66

Phenolized vaccines cannot be frozen without destroyingantigenicity and hence should not be freeze-dried unless a protective stabilizer has been added.I f beta-propiolactone has been used for inactivation, the procedure shallbe such that there is no detectable amount ofthe chemical in the final bulk.No antibiotics shall be added to rabies vaccine (for human use).

3.4.2 Potency tests on thefinal bulkIn the case of liquid vaccine a test for potency shall be made on eachfinal bulk unless such a test is made on each filling lot. The test shall beone in which mice are immunized and subsequently challenged with rabiesvirus and shall be made in parallel with a reference vaccine. The challengestrain 1 and reference vaccine as well as the test procedure used shall bethose approved by the national control authority (see Part B, section 1).

Reproducibility of the test depends upon the strain of rabiesvirus used for chal lenge and its maintenance in a large homogeneous working pool kept below -60C. The strain of micemay also affect reproducibility.When the NIH test is used," the potency relative to a reference preparation is determined. An acceptable potency ratio for

a vaccine under test should be no t less than 0.3 of the international reference preparation or its equivalent. The relativepotency is obtained by dividing the mg ED 50 of the referencevaccine by the mg ED 50 of the vaccine under test. When testingvaccines of low tissue content, the NIH procedure may be modified to employ dilutionendpoints rather than endpoints expressedas mg of brain tissue. A single dose for man is equivalent to1 ml of reconstituted International Reference Preparation ofRabies Vaccine (see Part A, section 1.3) or its equivalen t ofa national reference preparation, serial dilutions being madeon this basis. In this case the rel ative po tency is determinedby dividing the reciprocal of the ED 50 dilution of the vaccineunder test by the reciprocal of the ED 50 dilution of the reference vaccine and multiplying, if necessary, by a factor thatcorrects for the dose (human) difference between the test andreference vaccines.When the Habel test is used,2 inclus ion of the referencevaccine (see Part A, section 1.3) would enable the sensitivityof the test system to be monitor ed in the testing of successive

batchesP

1 A suitablechallenge strain, CVS, is available to laboratories on request from theWorldHealth Organization, 1211Geneva27, with the approval of the national authorities.2 See Kaplan, M. M. & Koprowski, H. , ed. (1973) Laboratory techniques in rabies,3rd edition, Geneva, World Health Organization (Monograph Series, No. 23), Part V.3 The reconstituted International Reference Preparation of Rabies Vaccine wheninjected into mice in volumes of 0.25 ml of a strength corresponding to a 0.5% suspensionhas been shown to protect aga inst more than 10000 LD 50 of CVS rabies virus underthe conditions of the test.

35

8/3/2019 WHO_TRS_530

36/66

3 .4 . 3 Sterility testsEach final bulk shall be tested for sterility according to the requirementsgiven in Part A, section 5 of the revised Requirements for BiologicalSubstances No. 6 (General Requirements for the Sterility of BiologicalSubstances)."

4. Filling and containersThe requirements concerning filling and containers given in Part A,section 4 of the revised Requirements for Biological Substances No. 1(General Requirements for Manufacturing Establishments and ControlLaboratories) 2 shall apply with the addition of the following:Containers of dried vaccine shall be hermetically sealed under vacuumor after filling with pure, dry, oxygen-free nitrogen or any other gas notdeleterious to the vaccine. All containers shall be tested for leaks and alldefective containers shall be discarded.

Generally only single-dose containers are used.

5. Control tests on final product5. 1 Identity test

An identity test shall be performed on at least one labelled containerfrom each filling lot by an appropriate method.The test for potency as descr ibed in Part A, section 3.4.2

may serve as an identity test.5.2 Sterility tests

Each filling lot shall be tested for bacterial and mycotic sterility accordingto the requirements given in Part A, section 5, of the revised Requirements for Biological Substances {General Requirements for the Sterilityof Biological Substancesj.!5. 3 Innocuity testsEach filling lot shall be tested for abnormal toxicity by appropriatetests in mice and guineapigs using parenteral injections. The test proceduresshall be those approved by the national control authority.5.4 Potency test of vaccine infinal containers

A test for potency as described in Part A, section 3.4.2, shall be madeon each filling lot in the case of dried vaccine and also in the case of liquid1 Wid Hlth Org; technoRep. Ser., 1973, No. 530, p. 48.2 Wid Hlth Org. techno Rep. Ser., 1966, No. 323, p. 16.

36

8/3/2019 WHO_TRS_530

37/66

vaccine if such a test was not made on the final bulk. Before the test ismade, dried vaccine shall be reconstituted to the form in which it is to beused in man.5.5 Stability test for freeze-dried vaccine

A test for potency as described in PartA, section 3.4.2, shall be madeon each filling lot after storage of samples for 4 weeks at 37C and to besatisfactory the lot shall show no loss in potency.5.6 Residual moisture tests onfreeze-dried vaccine

In the case of dried vaccine it is advisableto test for res idualmoisture as a guide to the opt imum content for the stabi li ty ofthe product.With some vaccines it is possible to dry the product to lessthan 1% residual moisture without impairing its stability andpotency. However, depending on the type of stabilizer pres-ent, higher values may be accepted by the national controlauthority.

5.7 Inspection offinal containersEach container in each filling lot shall be inspected and those showingabnormalities shall be discarded.

6. RecordsThe requirements given in Part A, section 6, of the revised Require-ments for Biological Substances No.1 (General Requirements for Manu-

facturing Establishments and Control Laboratories) 1 shall apply.7. Samples

The requirements given in Part A, section 7, of the revised Require-ments for Biological Substances No. 1 (General Requirements for Manu-facturing Establishments and Control Laboratories) 2 shall apply.8. Labelling

The requirements given in Part A, section 8, of the revised Require-ments for Biological Substances No.1 (General Requirements for 'Manu-facturing Establishments and Control Laboratories) 2. shall apply with theaddition of the following:1 WId Hlth Org. techn, Rep . Ser ., 1966, No. 323, p. 17.2 WId Hlth Org . techn. Rep. Ser ., 1966, xo . 323, p. 18.

37

8/3/2019 WHO_TRS_530

38/66

The leaflet accompanying the package shall include the followinginformation:the tissue and animal species in which the vaccine was prepared;the method used for inactivating the virus;if the vaccine is in the dried form, a statement that, after its recon-stitution it shall be used as soon as possible or stored at 50 3C

and discarded at the end of the day.9. Distribution and shipping

The requirements given in Part A, section 9, of the revised Require-ments for Biological Substances No.1 (General Requirements for Manu-facturing Establishments and Control Laboratories) 1 shall apply.

10. Storage and expiry dateThe requirements given in Part A, section 10, of the revised Require-ments for Biological Substances No.1 (General Requirements for Manu-

facturing Establishments and Control Laboratories) 2 shall apply.10. 1 Storage conditions

Rabies vaccine (for human use) shall be stored at a temperature of53C.10.2 Expiry date

The date after which liquid vaccine may not be used shall be not morethan 6 months after passing the last test for potency. The date after whichdried vaccine may not be used shall be no t more than 18 months afterpassing the last test for potency.

Part B : National Control Requirements1. General

The general requirements for control laboratories contained in Part B ofthe revised Requirements for Biological Substances No.1 (General Require-ments for Manufacturing Establishments and Control Laboratories) 2 shallapply.1 WId Hlth Org; techno Rep. Ser., 1966, No . 323, p, 18.2 WId Hlth Org; techno Rep. Ser., 1966, No . 323, p. 19.

38

8/3/2019 WHO_TRS_530

39/66

The national control authority shall give directions to manufacturersconcerning the strain of rabies virus 3 to use for production of vaccine.The national control authority shall provide or approve the strain forchallenge 1 and the reference vaccine for use in the potency test (Part A,section 3.4.2).2. Release and certification

A vaccine lot shall be released only if it fulfils Part A of these require-ments.A statement signed by the appropriate official of the national controllaboratory shall be provided at the request of the manufacturing estab-lishment and shall certify whether or not the lot of vaccine in questionmeets all national requirements aswell asPartA of the present requirements.The certificate shall further state the date of the last satisfactory test forpotency, the lot number, the number under which the lot was released, andthe number appeasing on the labels of the containers. In addition, a copyof the official national release document shall be attached.The purpose of the certificate is to facilitate the exchange ofrabies vaccine (for human use) between countries.

1 The Pasteur strain for vaccine production and the CVS virus are available to labor-atories on request from the WorldHealthOrganization, 1211Geneva 27, withthe approvalof the national authorities.

39

8/3/2019 WHO_TRS_530

40/66

Annex 4GENERAL REQUIREMENTS FOR THE

STERILITY OF BIOLOGICAL SUBSTANCES(Requirements for Biological Substances No. 6)

(Revised 1973) 1

PageIntroduction. . . . . 41General considerations 41Part A. Manufacturing requirements

1. Terminology . . . . . . . . . . . 432. General precautions against microbialcontamination in manufacture . . 443. General precautions against microbialcontamination from materials used formanufacture. . . . . . . . . . 474. General precautions against microbialcontamination in sterility testing 475. Sterility tests . . . . . . . . . . . 485.1 Sampling . . . . . . . . . . 48

5. 2 Sterility tests for bacteria and fungi 495.3 Sterility test for mycoplasmas. . . 525. 4 Sterility test for viruses . . . . . 525.5 Tests for specific micro-organisms 526. Records . . . . . . . . 537. Additional samples. . . . . . . . . . 53

Part B. National control requirements1. General. . . . . . . . . . . . . . . 532. Release and certification . . . . . . . 53

Appendix 1. Media for the detection of aerobic andanaerobic bacteria and fungi 54Appendix 2. Procedure for sterility test using

membrane filtration 55Appendix 3. Tests for mycoplasma. 56Appendix 4. Acknowledgements . . 57

1 These revised requirements have been derived from the document entitled" Sterilityand Sterility Testing of Pharmaceutical Preparations and Biological Substances" (unpublished workingdocumentWHOjBSf73.1062; WHOjPHARMj73.474)which was preparedfor consideration of the WHO Expert Committee on Biological Standardization and of theWHO Expert Committee on Specifications for Pharmaceutical Preparations; additionalinformat ion was obtained from a numbe r of other sources. The names of those whoprepared the original unpublished working document and the names of those who havesubmitted suggestions and comments to date on the document are given in Appendix 4 on p. 57 et seq.40

8/3/2019 WHO_TRS_530

41/66

IntroductionGeneral requirements for the sterility of biological substances (Requirements for Biological Substances No.6) were formulated by a WHO StudyGroup in 1959.1 These general requirements were applicable to any biological product from which the exclusion of microbial contamination isimperative, and they have been quoted in all the sets of requirements for

individual biological substances that have since been formulated. Thetwenty-fourth Expert Committee on Biological Standardization 2 agreedthat in view of recent developments I II methods of sterility testing of biological products and the improvements in control measures that were nowfeasible, the general requirements for sterility should be revised. This taskwas therefore undertaken. Since the preparation of a set of amendments oralterations would be an unsatisfactory way of revising the requirements, itwas decided to provide a complete document embodying all the requirements concerned. Where provisions formulated by the earlier StudyGroup were retained unchanged they have been embodied as such in thepresent revised requirements.In preparing these revised requirements account has been taken of theopinions of consultants, the relevant regulations and requirements thathave been formulated in a number of countries, as well as informationfrom both published and unpublished reports. In addition, opinions anddata relevant to these requirements have been received from a number ofexperts to whom grateful acknowledgement is made (see Appendix 4,page 57).

GeneralConsiderationsThe requirements in this document apply to all immunological biological substances, i.e., vaccines and sera, that must be sterile and are used foradministration to man. Many of the provisions, however, may be used forthe control of sterility of other biological preparations, or of blood andblood products, with suitable modifications, where necessary.1 WId Hlth Org. technoRep. Ser., 1960, No. 200. Themembers of the StudyGroup were:Dr M. Weis Bentzon, Statens Seruminstitut, Copenhagen, Denmark; Dr P. H. Bonnel,Centre de Transfusion - Reanimation de l'Armee, Seine, France (Rapporteur) ; Dr P. deGoes, Institute of Microbiology, Rio de Janeiro, Brazil; Dr J\L Pittman, Division ofBiologics Standards, National Institutes of Health, Bethesda, Md., USA (Chairman);Dr G. Penso, Laboratory of Microbiology, Istituto Superiore di Sanita, Rome, Italy;Dr R. H. Regamey, Institut d'Hygienc de l'Universite, Geneva, Switzerland; Dr Sumiatno,Pasteur Institute, Bandung, Indonesia (Vice-Chairman); Dr J . O 'H . Tobin, BiologicalStandards Control Laboratory, Medical Research Council, Hampstead, London, England(Rapporteur); Dr G. V. Vygodchikov, N. F. Gamaleja Institute of Epidemiology andMicrobiology, Moscow, USSR, Secretariat: Dr B. K. Bhattacharya, Medical Officer,Biological Standardization, WHO; Dr N. K. Jerne , Chief Medical Officer, BiologicalStandardization, \VHO, acted as Secretary.2 WId Hith Org. techno Rep. Ser., 1972, No . 486, p. 19.

41

8/3/2019 WHO_TRS_530

42/66

The Study Group on General Requirements for the Sterility of Biological Substances 1 (in 1959) pointed out tha t confidence in the sterility ofbiological preparations depended on two important considerations; first,adequate control tests for the sterility of biological preparations in theirfinal containers, using a sufficient number of random samples, and secondlythe use of suitable precautions with respect to source materials andmanufacturing procedures.' The Study Group considered available informat ion on sampling procedures adopted in several countries and alsodiscussed a suggested schedule for sampling of final containers to ensure anacceptably low probability of the release of contaminated final containers.

Theoretically, sterility may be defined as the absence of all microorganisms capable of multiplying.s Sterility tests employing a reasonablenumber of samples are capable of detecting contamination only in a lotwith a high percentage of contaminated units. Accordingly, it is notpossible to detect, on the basis of acceptable confidence limits, a low percentage of contaminated units in a homogeneous lot of final product. Ifthe finished product is a single final container of bulk material, then thecontrol measures are applicable on the basis of an assumed homogeneityof material in such a single final container. Sterility, therefore, cannot beassured in the control laboratory, but must be built into the product duringprocessing. Experience in many countries over the years has confirmedthat greater reliance must be placed