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INDIAN PHARMACOPOEIA 2007 MONOGRAPHS

General Requirements .....................................................................................................

Monographs .....................................................................................................................Adsorbed Diphtheria, Tetanus and Hepatitis B (rDNA) Vaccine ........................................Adsorbed Diphtheria, Tetanus and Pertussis (Acellular Component) and Haemophilus Type b Conjugate Vaccine ........................................................................Adsorbed Diphtheria, Tetanus and Pertussis (Acellular Component) and Hepatitis B (rDNA) Vaccine ...........................................................................................Adsorbed Diphtheria, Tetanus and Pertussis (Acellular Component), Inactivated Poliomyelitis Vaccine and Haemophilus Type b Conjugate Vaccine .................Adsorbed Diphtheria, Tetanus, Pertussis (Acellular Component) and Inactivated Poliomyelitis Vaccine ...................................................................................Adsorbed Diphtheria, Tetanus, Pertussis and Poliomyelitis (Inactivated) Vaccine ....................Adsorbed Diphtheria, Tetanus, Pertussis, Poliomyelitis (Inactivated) and Haemophilus Type b Conjugate Vaccine ........................................................................Adsorbed Pertussis Vaccine (Acellular Component) .........................................................Adsorbed Pertussis Vaccine (Acellular, Co-purified) .........................................................Bacillus Calmette-Guerin Vaccine (Freeze-Dried) ...............................................................Diphtheria and Tetanus Vaccine (Adsorbed) ........................................................................Diphtheria and Tetanus Vaccine (Adsorbed) for Adults and Adolescents ..............................Diphtheria, Tetanus and Pertussis Vaccine (Adsorbed) .....................................................Diphtheria, Tetanus, Pertussis (Whole Cell), Hepatitis B (rDNA) and Haemophilus Type b Conjugate Vaccine (Adsorbed) ....................................................Diphtheria, Tetanus, Pertussis (Whole Cell) Hepatitis B (rDNA) Vaccine (Adsorbed) ...................................................................................................Diphtheria, Tetanus, Pertussis (Whole Cell) and Haemophilus Type b Conjugate Vaccine (Adsorbed) ......................................................................................Diphtheria Vaccine (Adsorbed) ...........................................................................................Haemophilus Type b Conjugate Vaccine .............................................................................Hepatitis A (Inactivated) and Hepatitis B (rDNA) Vaccine (Adsorbed) .................................Hepatitis B Vaccine (rDNA) .............................................................................................Inactivated Hepatitis A Vaccine (Adsorbed) .......................................................................Inactivated Hepatitis B Vaccine ...........................................................................................

VACCINES AND IMMUNOSERA FOR HUMAN USE

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MONOGRAPHS INDIAN PHARMACOPOEIA 2007

Inactivated Influenza Vaccine (Split Virion) ...........................................................................Inactivated Influenza Vaccine (Surface Antigen) ....................................................................Inactivated Influenza Vaccine (Whole Virion) ....................................................................Japanese Encephalitis Vaccine (Human) ..............................................................................Measles and Rubella Vaccine (Live) .....................................................................................Measles Vaccine (Live) ......................................................................................................Measles, Mumps and Rubella Vaccine (Live) .......................................................................Meningococcal Polysaccharide Vaccine ..............................................................................Mumps Vaccine (Live) ......................................................................................................Pertussis Vaccine ................................................................................................................Pneumococcal Polysaccharide Vaccine ................................................................................Poliomyelitis Vaccine (Inactivated) ......................................................................................Poliomyelitis Vaccine, Live (Oral) .......................................................................................Rabies Vaccine, Human ......................................................................................................Rubella Vaccine (Live) .......................................................................................................Tetanus Vaccine (Adsorbed) ...............................................................................................Tick-borne Encephalitis Vaccine (Inactivated) .....................................................................Tuberculin Purified Protein Derivative ...................................................................................Typhoid (Strain Ty 21a) Vaccine, Live (Oral) ....................................................................Typhoid Polysaccharide Vaccine .........................................................................................Typhoid Vaccine ................................................................................................................Typhoid Vaccine (Freeze Dried) .........................................................................................Varicella Vaccine (Live) .......................................................................................................Viper Venom ........................................................................................................................Yellow Fever Vaccine (Live) ...............................................................................................

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Vaccines : General RequirementsVaccines are preparations of antigenic substances that areadministered for the purpose of inducing in the recipient aspecific and active immunity against the infective agent ortoxin produced by it.

Vaccines may contain living micro-organisms suitably treatedto attenuate their virulence but retain their antigenic potencyor they may consist of pathogenic organisms which havebeen killed or inactivated. Some vaccines consist of antigenicfractions or substances produced by the same pathogenicorganisms but rendered harmless whilst retaining theirantigenic efficiency. Vaccines may be prepared from onespecies only or from a mixture of two or more species.

Vaccines may be prepared by the method described in theindividual monographs or by the general methods given belowor in any other manner provided the identity of the antigens ismaintained and the vaccines are free from microbialcontamination and extraneous agents. Suitable adjuvants maybe added during the preparation but streptomycin, penicillinor other β-lactam antibiotics may not be added at any stage ofmanufacture or in the final vaccine. A suitable bactericide maybe added to sterile and inactivated vaccines. The final productsare distributed aseptically into sterile containers which arethen sealed to exclude extraneous micro-organisms. Unlessotherwise indicated in the monograph, the final vaccine maybe filled into single dose or multiple dose containers butvaccines in multiple dose containers must invariably containa bactericide.

Bacterial Vaccines. Bacterial vaccines are either sterilesuspensions of live or killed bacteria or sterile extracts ofderivatives of bacteria. They may be simple vaccines preparedfrom one species or may be mixed vaccines prepared byblending two or more simple vaccines from different speciesor strains.Bacterial vaccines may be prepared from cultures grown onsuitable solid or liquid media. The whole culture or parts of itmay be used in preparing the vaccine. The identity, antigenicpotency and purity of each bacterial culture must be carefullycontrolled.Vaccines containing killed organisms may be prepared bykilling the organisms by chemical or physical means providedthe antigenic potency of the vaccine is preserved. Vaccinescontaining living bacteria may be prepared from strains whichare avirulent for humans but which stimulate the productionof antibodies active against pathogenic strains of the samespecies. The final vaccines must be free from any substanceknown to cause toxic, allergic or other undesirableimmunological reactions in humans.Bacterial vaccines are suspensions of varying degrees ofopacity in colourless or slightly coloured liquids or they may

be freeze-dried so that the water content is not more than 3.0per cent w/w unless otherwise stated in the individualmonograph. They may be standardized in terms of interopacityunits or, where appropriate, by numbers of living or killedbacteria determined by direct cell count or by viable count.

Bacterial toxoids. Bacterial toxoids are toxins or materialderived therefrom, the toxicity of which has been reduced to avery low level or completely eliminated by chemical or physicalmeans without destroying their immunizing potency. The toxinsare obtained from selected strains of specific micro-organisms,grown in media free from ingredients known to cause toxic,allergic or other undesirable immunological reactions inhumans. Toxoids produced by the action of formaldehyde areknown as formol toxoids.

Bacterial toxoids may be liquid or may be prepared by adsorbingon mineral carriers such as aluminium phosphate, aluminiumhydroxide or any other suitable adsorbent; the adsorbedproduct may be separated, washed and suspended in a salineor other appropriate solution isotonic with blood.

Bacterial toxoids are clear or slightly opalescent liquids,colourless or slightly yellow. Adsorbed toxoids may be whiteor greyish white suspensions or pale-yellow liquids with asediment at the bottom of the container. Freeze-driedpreparations are greyish white or yellowish white powders orpellets.

Viral and rickettsial vaccines. Viral and rickettsial vaccinesare suspensions of viruses or rickettsiae and are preparedfrom infected tissues or blood obtained from artificially infectedanimals, from cultures in fertile eggs, or from cell or tissueculture. Viral vaccines may be live or killed and they may befreeze-dried. Live vaccines are usually prepared usingattenuated strains of the specific organisms. Killed vaccinesmay be inactivated by suitable chemical or physical means.

Mixed Vaccines. Mixed vaccines are mixtures of two or morevaccines. A suitable antibacterial substance may be added toinactivated or live viral and rickettsial vaccines provided thatit has no action against the specific organisms.

Production

General provisions. Requirements for production includingin-process testing are included in individual monographs.Where justified and authorized, certain tests may be omittedwhere it can be demonstrated, for example by validationstudies, that the production process consistently ensurescompliance with the test.

Unless otherwise justified and authorized, vaccines areproduced using a seed-lot system. The methods of preparationare designed to maintain adequate immunogenic properties,to render the preparation harmless and to preventcontamination with extraneous agents.

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Unless otherwise justified and authorized, in the productionof a final lot of vaccine, the number of passages of a virus, orthe number of subcultures of a bacterium, from the masterseed lot shall not exceed that used for production of the vaccineshown in clinical studies to be satisfactory with respect tosafety and efficacy.

Vaccines are as far as possible free from ingredients known tocause toxic, allergic or other undesirable reactions in man.Suitable additives, including stabilizers and adjuvants may beincorporated. Penicillin and streptomycin are neither used atany stage of production nor added to the final product;however, master seed lots prepared with media containingpenicillin or streptomycin may, where justified and authorized,be used for production.

Substrates for propagation. Substrates for propagationcomply with the relevant requirements of the Pharmacopoeiaor in the absence of such requirements with those of thecompetent authority. Processing of cell banks and subsequentcell cultures is done under aseptic conditions in an area whereno other cells are being handled. Serum and trypsin used inthe preparation of cell suspensions shall be shown to be freefrom extraneous agents.

Seed lot. The strain of bacterium or virus used in a masterseed lot is identified by historical records that includeinformation on the origin of the strain and its subsequentmanipulation. No micro-organism other than the seed strainshall be present in a seed lot.

Culture media. Culture media are as far as possible free fromingredients known to cause toxic, allergic or other undesirablereactions in man; if inclusion of such ingredients is necessary,it shall be demonstrated that the amount present in the finallot is reduced to such a level as to render the product safe.Approved animal (but not human) serum may be used in thegrowth medium for cell cultures but the medium used formaintaining cell growth during virus multiplication shall notcontain serum, unless otherwise stated. Cell culture mediamay contain a pH indicator such as phenol red and approvedantibiotics at the lowest effective concentration although it ispreferable to have a medium free from antibiotics duringproduction.

Propagation and harvest. The seed cultures are propagatedand harvested under defined conditions. The purity of theharvest is verified by suitable tests as defined in themonograph.

Control cells. For vaccines produced in cell cultures, controlcells are maintained and tested as prescribed. In order toprovide a valid control, these cells must be maintained inconditions that are rigorously identical with those used forthe production cell cultures, including use of the same batchesof media and media changes.

Control eggs. For live vaccines produced in eggs, controleggs are incubated and tested as prescribed in the monograph.

Purification. Where applicable, validated purificationprocedures may be applied.

Inactivation. Inactivated vaccines are produced using avalidated inactivation process whose effectiveness andconsistency have been demonstrated. Where there arerecognised potential contaminants of a harvest, for examplein vaccines produced in eggs from healthy, non-SPF flocks,the inactivation process is also validated with respect to thepotential contaminants. A test for inactivation is carried outas soon as possible after the inactivation process, unlessotherwise justified and authorised.

Intermediates. Where applicable, the stability of intermediatesin given storage conditions shall be evaluated and a period ofvalidity established.

Final bulk. The final bulk is prepared by aseptically blendingthe ingredients of the vaccine.

Adsorbents. Vaccines may be adsorbed on aluminiumhydroxide, aluminium phosphate, calcium phosphate or othersuitable adsorbent; the adsorbents are prepared in specialconditions which confer the appropriate physical form andadsorptive properties.

Antimicrobial preservative. A suitable antimicrobialpreservative may be included in sterile and inactivatedvaccines and is invariably added if these preparations areissued in multidose containers, unless otherwise stated. If anantimicrobial preservative is used, it shall be shown that itdoes not impair the safety or efficacy of the vaccine and itseffectiveness throughout the period of validity shall bedemonstrated.

Final lot. For vaccines for parenteral administration, the finallot is prepared by aseptically distributing the final bulk intosterile tamper-proof containers which, after freeze-drying whereapplicable, are closed so as to exclude contamination. Forvaccines for administration by a non-parenteral route, the finallot is prepared by distributing the final bulk under suitableconditions into sterile, tamper-proof containers.

Stability. Maintenance of potency of the final lot throughoutthe period of validity shall be demonstrated by validationstudies; the loss of potency in the recommended storageconditions is assessed and excessive loss even within thelimits of acceptable potency may indicate that the vaccine isunacceptable.

Degree of adsorption. During development of an adsorbedvaccine, the degree of adsorption is evaluated as part of theconsistency testing. A release specification for the degree ofadsorption is established in the light of results found forbatches used in clinical testing. From the stability data

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generated for the vaccine it must be shown that at the end ofthe period of validity the degree of adsorption will not be lessthan for batches used in clinical testing.

Tests

Vaccines, reconstituted where necessary, comply with thefollowing requirements unless otherwise stated in theindividual monograph.

Phenol (If present) (2.3.36). Not more than 0.25 per cent w/v.

Thiomersal (If present) (2.3.48). Between 0.005 per cent w/vand 0.02 per cent w/v.

Free formaldehyde (If present) (2.3.20). Maximum 0.02 g/l.

Aluminium (If present) (2.3.9). Not more than 1.25 mg perdose.

Sterility (2.2.11). Unless otherwise stated all vaccines complywith tests for sterility, except that for living bacterial vaccines,growth of the organism from which the vaccine was preparedis permitted (sterility means abesence of becterial and fungalcontaminants except where specified in the individualmonograph).

Abnormal toxicity (2.2.1). Unless otherwise stated, all vaccinescomply with the test for abnormal toxicity, Method B. Invaccines containing phenol as preservative, the test in micemay be inappropriate.

NOTE — The statements given in this general chapter isintended to be read in conjunction with the monographs onthe individual vaccine in this Pharmacopoeia which refer topreparations for human use; they do not necessarily applyto vaccines for use in veterinary practice.

Storage. Liquid vaccines must be stored at a temperaturebetween 2o and 8o and should not be allowed to freeze unlessotherwise specified in the individual monograph. Freeze-driedpreparations must be stored at temperatures below –20o or asspecified in the individual monograph. At higher temperaturesvaccines deteriorate rapidly.

Labelling. The label states (1) for liquid vaccines, the totalnumber of ml in the container and, for dried vaccines, thenumber of doses in the container; (2) unless otherwiseindicated the minimum number of Units per dose or per ml or,for viral vaccines, the minimum viral titre; (3) the dose androute of administration; (4) the name and proportion of anyantibacterial preservative or other auxiliary substances addedto the vaccine; (5) the date after which the vaccine is notintended to be used; (6) the conditions under which it shouldbe stored; (7) for dried vaccines, the liquid to be used forreconstitution and its volume; (8) that the vaccine should beused immediately after reconstitution; (9) unless otherwisedirected, that the vaccine should be shaken well before use;(10) any contraindication to the use of the vaccine.

Adsorbed Diphtheria, Tetanus andHepatitis B (rDNA) VaccineDiphtheria, Tetanus and Hepatitis B (rDNA) Vaccine(Adsorbed) is a combined vaccine composed of: diphtheriaformol toxoid; tetanus formol toxoid; hepatitis B surfaceantigen (HBsAg); a mineral adsorbent such as aluminiumhydroxide or hydrated aluminium phosphate.

The formol toxoids are prepared from the toxins produced bythe growth of Corynebacterium diphtheriae and Clostridiumtetani, respectively.

HBsAg is a component protein of hepatitis B virus; the antigenis obtained by recombinant DNA technology.

Production

General provisions

The production method shall have been shown to yieldconsistently vaccines comparable with the vaccine of provenclinical efficacy and safety in man.

The production method is validated to demonstrate that theproduct, if tested, would comply with the test for abnormaltoxicity for antisera and vaccines, and with the following testfor specific toxicity of the diphtheria and tetanus components:inject subcutaneously 5 times the single human dose statedon the label into each of 5 healthy guinea-pigs, each weighingbetween 250 and 350 g, that have not previously been treatedwith any material that will interfere with the test. If within 42days of the injection any of the animals shows signs of or diesfrom diphtheria toxaemia or tetanus, the vaccine does notcomply with the test. If more than 1 animal dies from non-specific causes, repeat the test once; if more than 1 animaldies in the second test, the vaccine does not comply with thetest.

The content of bacterial endotoxins in the bulk purifieddiphtheria toxoid and tetanus toxoid is determined to monitorthe purification procedure and to limit the amount in the finalvaccine. For each component, the content of bacterialendotoxin is less than the limit approved for the particularvaccine and in any case the contents are such that the finalvaccine contains less than 100 IU per single human dose.

Reference vaccine(s)

Provided valid assays can be performed, monocomponentreference vaccines may be used for the assays on the combinedvaccine. If this is not possible because of interaction betweenthe components of the combined vaccine or because of thedifference in composition between monocomponent referencevaccine and the test vaccine, a batch of combined vaccineshown to be effective in clinical trials or a batch representativethereof is used as a reference vaccine. For the preparation of

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a representative batch, strict adherence to the productionprocess used for the batch tested in clinical trials is necessary.The reference vaccine may be stabilised by a method that hasbeen shown to have no effect on the assay procedure.

Production of the components

The production of the components complies with therequirements of the monographs on Diphtheria Vaccine(Adsorbed), Tetanus Vaccine (Adsorbed) and Hepatitis BVaccine (rDNA).

FINAL BULK VACCINE

The final bulk vaccine is prepared by adsorption, separatelyor together, of suitable quantities of bulk purified diphtheriatoxoid, tetanus toxoid and HBsAg onto a mineral carrier suchas aluminium hydroxide or hydrated aluminium phosphate.Suitable antimicrobial preservatives may be added.

Only a final bulk vaccine that complies with the followingrequirements may be used in the preparation of the final lot.

Antimicrobial preservative. Where applicable, determine theamount of antimicrobial preservative by a suitable chemicalmethod. The content is not less than 85.0 per cent and notgreater than 115.0 per cent of the intended amount.Sterility (2.2.11). Carry out test for sterility using 10 ml of bulkfor each sterility medium.

FINAL LOT

Only a final lot that is satisfactory with respect to the test forosmolality and with respect to each of the requirements givenbelow under Identification, Tests and Assay may be releasedfor use.

Provided the test for antimicrobial preservative and the assaysfor the diphtheria and tetanus components have been carriedout with satisfactory results on the final bulk vaccine, theymay be omitted on the final lot.

Provided the content of free formaldehyde has beendetermined on the bulk purified antigens or on the final bulkand it has been shown that the content in the final lot will notexceed 0.2 g/l, the test for free formaldehyde may be omittedon the final lot.

If an in vivo assay is used for the hepatitis B component,provided it has been carried out with satisfactory results onthe final bulk vaccine, it may be omitted on the final lot.

Osmolality (2.4.23). The osmolality of the vaccine is withinthe limits approved for the particular preparation.

Identification

A. Diphtheria toxoid is identified by a suitable immunochemicalmethod (2.2.14). The following method, applicable to certain

vaccines, is given as an example. Dissolve in the vaccine underexamination sufficient sodium citrate to give a 10 per centw/v solution. Maintain at 37° for about 16 hours and centrifugeuntil a clear supernatant is obtained. The clear supernatantliquid reacts with a suitable diphtheria antitoxin, giving aprecipitate.

B. Tetanus toxoid is identified by a suitable immunochemicalmethod (2.2.14). The following method, applicable to certainvaccines, is given as an example. The clear supernatant liquidobtained during identification test A reacts with a suitabletetanus antitoxin, giving a precipitate.

C. The assay or, where applicable, the electrophoretic profile,serves also to identify the hepatitis B component of thevaccine.

TestsAluminium (2.3.9). Maximum 1.25 mg per single human dose,if aluminium hydroxide or hydrated aluminium phosphate isused as the adsorbent.

Free formaldehyde (2.3.20). Maximum 0.2 g/l.

Antimicrobial preservative. Where applicable, determine theamount of antimicrobial preservative by a suitable chemicalmethod. The content is not less than 85.0 per cent and notgreater than 115.0 per cent of the intended amount.

Sterility (2.2.11). Complies with the test for sterility.

Pyrogens (2.2.8). Complies with the test for pyrogens. Injectthe equivalent of 1 human dose into each rabbit.

Assay

Diphtheria component

Carry out one of the prescribed methods for the assay asstated under Diphtheria Vaccine (Adsorbed).

The lower confidence limit (P = 0.95) of the estimated potencyis not less than 30 IU per single human dose.

Tetanus component

Carry out one of the prescribed methods for the assay asstated under Tetanus Vaccine (Adsorbed).


Hepatitis B component

It complies with the assay of Hepatitis B Vaccine.

Labelling. The label states (1) the minimum number ofInternational Units of diphtheria and tetanus toxoid per singlehuman dose; (2) the amount of HBsAg per single human dose;(3) the type of cells used for production of the HBsAgcomponent; (4) where applicable, that the vaccine is intendedfor primary vaccination of children and is not necessarily

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suitable for reinforcing doses or for administration to adults;(5) the name and the amount of the adsorbent; (6) that thevaccine must be shaken before use; (7) that the vaccine is notto be frozen.

Adsorbed Diphtheria, Tetanus,Pertussis (Acellular Component) andHaemophilus Type B ConjugateVaccineDiphtheria, Tetanus, Pertussis (Acellular Component) andHaemophilus Type b Conjugate Vaccine (Adsorbed) is acombined vaccine composed of: diphtheria formol toxoid;tetanus formol toxoid; individually purified antigeniccomponents of Bordetella pertussis; polyribosylribitolphosphate (PRP) covalently bound to a carrier protein; amineral absorbent such as aluminium hydroxide or hydratedaluminium phosphate. The product may be presented withthe haemophilus type b component in a separate container,the contents of which are mixed with the other componentsimmediately before use.

The formol toxoids are prepared from the toxins produced bythe growth of Corynebacterium diphtheriae and Clostridiumtetani respectively.

The vaccine contains either pertussis toxoid or a pertussis-toxin-like protein free from toxic properties produced byexpression of a genetically modified form of the correspondinggene. Pertussis toxoid is prepared from pertussis toxin by amethod that renders the toxin harmless while maintainingadequate immunogenic properties and avoiding reversion totoxin. The acellular pertussis component may also containfilamentous haemagglutinin, pertactin (a 69 kDa outer-membrane protein) and other defined components ofB. pertussis such as fimbrial-2 and fimbrial-3 antigens. Thelatter two antigens may be copurified. The antigeniccomposition and characteristics are based on evidence ofprotection and freedom from unexpected reactions in the targetgroup for which the vaccine is intended.

PRP is a linear copolymer composed of repeated units of3-β -D-r ibofuranosyl - (1→1)- r ib i to l -5-phosphate[(C10H19O12P)n], with a defined molecular size and derived froma suitable strain of Haemophilus influenzae type b. The carrierprotein, when conjugated to PRP, is capable of inducing aT-cell-dependent B-cell immune response to thepolysaccharide.

ProductionGeneral provisions

The production method shall have been shown to yield

consistently the vaccines comparable with the vaccine ofproven clinical efficacy and safety in man.

If the vaccine is presented with the haemophilus componentin a separate vial, as part of consistency studies the assays ofthe diphtheria, tetanus and pertussis components are carriedout on a suitable number of batches of vaccine reconstitutedfor use. For subsequent routine control, the assays of thesecomponents may be carried out without mixing with thehaemophilus component.

The content of bacterial endotoxins in bulk purified diphtheriatoxoid, tetanus toxoid, pertussis components and PRPconjugate is determined to monitor the purification procedureand to limit the amount in the final vaccine. For eachcomponent, the content of bacterial endotoxins is less thanthe limit approved for the particular vaccine; if the vaccine ispresented with the haemophilus component in a separatecontainer, the contents of the diphtheria, tetanus and pertussisantigens are in any case such that the final vial for thesecomponents contains less than 100 IU per single human dose.

The production method is validated to demonstrate that theproduct, if tested, would comply with the test for abnormaltoxicity for antisera and vaccines.

During development studies and wherever revalidation isnecessary, it shall be demonstrated by tests in animals thatthe vaccine induces a T-cell dependent B-cell immune responseto PRP.

Reference vaccine

Provided valid assays can be performed, monocomponentreference vaccines may be used for the assays on the combinedvaccine. If this is not possible because of interaction betweenthe components of the combined vaccine or because of thedifference in composition between monocomponent referencevaccine and the test vaccine, a batch of combined vaccineshown to be effective in clinical trials or a batch representativethereof is used as a reference vaccine. For the preparation ofa representative batch, strict adherence to the productionprocess used for the batch tested in clinical trials is necessary.The reference vaccine may be stabilised by a method that hasbeen shown to have no effect on the assay procedure.


The production of the components complies with the tests ofthe monographs on Diphtheria Vaccine (Adsorbed), TetanusVaccine (Adsorbed), Pertussis Vaccine (Acellular Component,Adsorbed) and Haemophilus Type b Conjugate Vaccine.

FINAL BULK VACCINE

Different methods of preparation may be used: a final bulkvaccine may be prepared by adsorption, separately or together,of suitable quantities of bulk purified diphtheria toxoid, tetanus

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toxoid, acellular pertussis components and PRP conjugateonto a mineral carrier such as aluminium hydroxide or hydratedaluminium phosphate; or 2 final bulks may be prepared andfilled separately, one containing the diphtheria, tetanus andpertussis components, the other the haemophilus component,which may be freeze-dried. Suitable antimicrobial preservativesmay be added.


Antimicrobial preservative. Where applicable, determine theamount of antimicrobial preservative by a suitable chemicalmethod. The content is not less than 85.0 per cent and notgreater than 115.0 per cent of the intended amount.Sterility (2.2.11). Carry out test for sterility using 10 ml of bulkfor each sterility medium.FINAL LOT

Only a final lot that is satisfactory with respect to the test forosmolality shown below and with respect to each of therequirements given below under Identification, Tests andAssay may be released for use.Provided the tests for absence of residual pertussis toxin,irreversibility of pertussis toxoid and antimicrobial preservativeand the assays have been carried out with satisfactory resultson the final bulk vaccine, they may be omitted on the final lot.Provided the free formaldehyde content has been determinedon the bulk purified antigens or the final bulk and it has beenshown that the content in the final lot will not exceed 0.2 g/l,the test for free formaldehyde may be omitted on the final lot.Osmolality (2.4.23). The osmolality of the vaccine,reconstituted where applicable, is within the limits approvedfor the particular preparation.

pH (2.4.24). The pH of the vaccine, reconstituted if necessary,is within the range approved for the particular product.

Free PRP. Unbound PRP is determined after removal of theconjugate, for example by anion-exchange, size-exclusion orhydrophobic chromatography (2.4.16), ultrafiltration or othervalidated methods. The amount of free PRP is not greater thanthat approved for the particular product.

IdentificationIf the vaccine is presented with the haemophilus componentin a separate vial: identification tests A, B and C are carriedout using the vial containing the diphtheria, tetanus andpertussis components; identification test D is carried out onthe vial containing the haemophilus components.A. Diphtheria toxoid is identified by a suitable immunochemicalmethod (2.2.14). The following method, applicable to certainvaccines, is given as an example. Dissolve in the vaccine underexamination sufficient sodium citrate to give a 10 per cent

w/v solution. Maintain at 37° for about 16 h and centrifugeuntil a clear supernatant is obtained. The clear supernatantreacts with a suitable diphtheria antitoxin, giving a precipitate.B. Tetanus toxoid is identified by a suitable immunochemicalmethod (2.2.14). The following method, applicable to certainvaccines, is given as an example. The clear s u p e r n a t a n tobtained as described in Identification test A reacts with asuitable tetanus antitoxin, giving a precipitate.C. The pertussis components are identified by a suitableimmunochemical method (2.2.14). The following method,applicable to certain vaccines, is given as an example. Theclear supernatant obtained as described in Identificationtest A reacts with a specific antisera to the pertussiscomponents of the vaccine.D. The haemophilus component is identified by a suitableimmunochemical method (2.2.14) for PRP.

TestsIf the product is presented with the haemophilus componentin a separate container: the tests for absence of residualpertussis toxin, irreversibility of pertussis toxoid, aluminium,free formaldehyde, antimicrobial preservative and sterility arecarried out on the container with the diphtheria, tetanus andpertussis components; the tests for PRP content, water (whereapplicable), sterility and pyrogens are carried out on thecontainer with the haemophilus component.

If the haemophilus component is freeze-dried, some tests maybe carried out on the freeze-dried product rather than on thebulk conjugate where the freeze-drying process may affectthe component under test.Absence of residual pertussis toxin and irreversibilityof pertussis toxoid

This test is not necessary for the product obtained by geneticmodification. Use 3 groups each of not less than 5 histamine-sensitive mice. Inject intraperitoneally into each mouse of thefirst group twice the single human dose of the vaccine storedat 2° to 8°. Inject intraperitoneally into each mouse of thesecond group twice the single human dose of the vaccineincubated at 37° for 4 weeks. Inject diluent into the third groupof mice. After 5 days, inject into each mouse 2 mg of histaminebase intraperitoneally in a volume not exceeding 0.5 ml andobserve for 24 h. The test is invalid if 1 or more control micedie following histamine challenge. The vaccine complies withthe test if no animal in the first or second group dies followinghistamine challenge. If 1 mouse dies in either or both of thefirst and second groups, the test may be repeated with thesame number of mice or with a greater number and the resultsof valid tests combined; the vaccine complies with the test if,in both of the groups given the vaccine, not more than 5.0 percent of the total number of mice die following histaminechallenge.

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The histamine sensitivity of the strain of mice used is verifiedat suitable intervals as follows: inject intravenously three-fold dilutions of a reference pertussis toxin preparation inphosphate-buffered saline solution containing 0.2 per centw/v of gelatin and challenge with histamine as above; thestrain is suitable if more than 50 per cent of the animals aresensitised by 50 ng of pertussis toxin and none of the controlanimals injected with only diluent and challenged similarlywith histamine shows symptoms of sensitisation.

PRP. Minimum 80.0 per cent of the amount of PRP stated onthe label. PRP is determined either by assay of ribose (2.7.1) orphosphorus (2.7.1), by an immunochemical method (2.2.14) orby anion-exchange liquid chromatography with pulsed-amperometric detection.

Aluminium (2.3.9). Maximum 1.25 mg per single human dose,if aluminium hydroxide or hydrated aluminium phosphate isused as the adsorbent.Free formaldehyde (2.3.20 ). Maximum 0.2 g/l.Antimicrobial preservative. Where applicable, determine theamount of antimicrobial preservative by a suitable chemicalmethod. The content is not less than 85.0 per cent and notgreater than 115.0 per cent of the intended amount.

Water (2.3.43). Maximum 3.0 per cent for the freeze-driedhaemophilus component.


Pyrogens (2.2.8). Complies with the test for pyrogens. Injectper kg of the rabbit’s mass a quantity of the vaccine equivalentto: 1 mg of PRP for a vaccine with diphtheria toxoid or CRM197 diphtheria protein as carrier; 0.1 mg of PRP for a vaccinewith tetanus toxoid as carrier; 0.025 mg of PRP for a vaccinewith OMP as carrier.

Assay

Diphtheria componentCarry out one of the prescribed methods for the assay asstated under Diphtheria Vaccine (Adsorbed).The lower confidence limit (P = 0.95) of the estimated potencyis not less than the minimum potency stated on the label.Unless otherwise justified and authorised, the minimumpotency stated on the label is 30 IU per single human dose.Tetanus componentCarry out one of the prescribed methods for the assay asstated under Tetanus Vaccine (Adsorbed).The lower confidence limit (P = 0.95) of the estimated potencyis not less than 40 IU per single human dose.Pertussis componentThe vaccine complies with the assay as the stated AdsorbedPertussis Vaccine (Acellular Component).

Labelling. The label states (1) the minimum number ofInternational Units of diphtheria and tetanus toxoid per singlehuman dose; (2) the names and amounts of the pertussiscomponents per single human dose; (3) the number ofmicrograms of PRP per single human dose; (4) the type andnominal amount of carrier protein per single human dose; (5)where applicable, that the vaccine is intended for primaryvaccination of children and is not necessarily suitable forreinforcing doses or for administration to adults; (6) the nameand the amount of the adsorbent; (7) that the vaccine must beshaken before use; (8) that the vaccine is not to be frozen (9)where applicable, that the vaccine contains a pertussis toxin-like protein produced by genetic modification.

Adsorbed Diphtheria, Tetanus andPertussis (Acellular Component) andHepatitis B (rDNA) VaccineDiphtheria, Tetanus, Pertussis (Acellular Component) andHepatitis B (rDNA) Vaccine (Adsorbed) is a combined vaccinecomposed of: diphtheria formol toxoid; tetanus formol toxoid;individually purified antigenic components of Bordetellapertussis; hepatitis B surface antigen; a mineral adsorbentsuch as aluminium hydroxide or hydrated aluminiumphosphate.

The formol toxoids are prepared from the toxins produced bythe growth of Corynebacterium diphtheriae and Clostridiumtetani, respectively.

The vaccine contains either pertussis toxoid or a pertussis-toxin-like protein free from toxic properties, produced byexpression of a genetically modified form of the correspondinggene. Pertussis toxoid is prepared from pertussis toxin by amethod that renders the latter harmless while maintainingadequate immunogenic properties and avoiding reversion totoxin. The vaccine may also contain filamentoushaemagglutinin, pertactin (a 69 kDa outer-membrane protein)and other defined components of B. pertussis such as fimbrial-2 and fimbrial-3 antigens. The latter 2 antigens may becopurified. The antigenic composition and characteristics arebased on evidence of protection and freedom from unexpectedreactions in the target group for which the vaccine is intended.

Hepatitis B surface antigen is a component protein of hepatitisB virus; the antigen is obtained by recombinant DNAtechnology.

Production

General provisions

The production method shall have been shown to yieldconsistently vaccines comparable with the vaccine of proven

A. D. T. AND P. (ACELLULAR COMPONENT) AND HEPATITIS B (rDNA) VACCINE

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clinical efficacy and safety in man.

The content of bacterial endotoxins in the bulk purifieddiphtheria toxoid, tetanus toxoid and pertussis componentsis determined to monitor the purification procedure and tolimit the amount in the final vaccine. For each component, thecontent of bacterial endotoxins is less than the limit approvedfor the particular vaccine.




The production of the components complies with therequirements of the monographs on Diphtheria Vaccine(Adsorbed), Tetanus Vaccine (Adsorbed), Pertussis Vaccine(Acellular Component, Adsorbed) and Hepatitis B Vaccine(rDNA).

FINAL BULK VACCINE

The final bulk vaccine is prepared by adsorption, separatelyor together, of suitable quantities of bulk purified diphtheriatoxoid, tetanus toxoid, acellular pertussis components andhepatitis B surface antigen onto a mineral carrier such asaluminium hydroxide or hydrated aluminium phosphate.Suitable antimicrobial preservatives may be added.



Sterility (2.2.11). Carry out test for sterility using 10 ml of bulkfor each sterility medium.

FINAL LOT


Provided the tests for absence of residual pertussis toxin,irreversibility of pertussis toxoid and antimicrobial preservativeand the assays for the diphtheria, tetanus and pertussiscomponents have been carried out with satisfactory resultson the final bulk vaccine, they may be omitted on the final lot.

Provided the content of free formaldehyde has beendetermined on the bulk purified antigens or on the final bulkand it has been shown that the content in the final lot will notexceed 0.2 g/l, the test for free formaldehyde may be omittedon the final lot.

If an in vivo assay is used for the hepatitis B component,provided it has been carried out with satisfactory results onthe final bulk vaccine, it may be omitted on the final lot.


IdentificationA. Diphtheria toxoid is identified by a suitable immunochemicalmethod (2.2.14). The following method, applicable to certainvaccines, is given as an example. Dissolve in the vaccine underexamination sufficient sodium citrate to give a 10 per centw/v solution. Maintain at 37° for about 16 h and centrifugeuntil a clear supernatant is obtained. The clear supernatantreacts with a suitable diphtheria antitoxin, giving a precipitate.

B. Tetanus toxoid is identified by a suitable immunochemicalmethod (2.2.14). The following method, applicable to certainvaccines, is given as an example. The clear supernatantobtained as described in identification test A reacts with asuitable tetanus antitoxin, giving a precipitate.C. The pertussis components are identified by a suitableimmunochemical method (2.2.14). The following method,applicable to certain vaccines, is given as an example. Theclear supernatant obtained as described in identification testA reacts with a specific antisera to the pertussis componentsof the vaccine.

D. The assay or, where applicable, the electrophoretic profile,serves also to identify the hepatitis B component of thevaccine.

TestsAbsence of residual pertussis toxin and irreversibilityof pertussis toxoid

This test is not necessary for the product obtained by geneticmodification. Use 3 groups each of not less than 5 histamine-sensitive mice. Inject intraperitoneally into each mouse of thefirst group twice the single human dose of the vaccine storedat 2° to 8°. Inject intraperitoneally into each mouse of thesecond group twice the single human dose of the vaccineincubated at 37° for 4 weeks. Inject diluent into the third groupof mice. After 5 days, inject into each mouse 2 mg of histamine

A. D. T. AND P. (ACELLULAR COMPONENT) AND HEPATITIS B (rDNA) VACCINE

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base intraperitoneally in a volume not exceeding 0.5 ml andobserve for 24 h. The test is invalid if 1 or more control micedie following histamine challenge. The vaccine complies withthe test if no animal in the first or second group dies followinghistamine challenge. If 1 mouse dies in either or both of thefirst and second groups, the test may be repeated with thesame number of mice or with a greater number and the resultsof valid tests combined; the vaccine complies with the test if,in both of the groups given the vaccine, not more than5 per cent of the total number of mice die following histaminechallenge.

The histamine sensitivity of the strain of mice used is verifiedat suitable intervals as follows: inject intravenously threefolddilutions of a reference pertussis toxin preparation inphosphate-buffered saline solution containing 0.2 per centw/v of gelatin and challenge with histamine as above; thestrain is suitable if more than 50.0 per cent of the animals aresensitised by 50 ng of pertussis toxin and none of the controlanimals injected with only diluent and challenged similarlywith histamine shows symptoms of sensitisation.

Aluminium (2.3.9). Maximum 1.25 mg per single human dose,if aluminium hydroxide or hydrated aluminium phosphate isused as the adsorbent.



Sterility ( 2.2.11). Complies with the test for sterility.

Pyrogens (2.2.8). Complies with the test for pyrogens. Injectthe equivalent of 1 human dose into each rabbit.

Assay



The lower confidence limit (P = 0.95) of the estimated potencyis not less than the minimum potency stated on the label.

Unless otherwise justified and authorised, the minimumpotency stated on the label is 30 IU per single human dose.

Tetanus component



Pertussis component

The vaccine complies with the assay as stated under AdsorbedPertussis Vaccine (Acellular Component).


The vaccine complies with the assay as stated under HepatitisB Vaccine (rDNA).

Labelling. The label states (1) the minimum number ofInternational Units of diphtheria and tetanus toxoid per singlehuman dose; (2) the names and amounts of the pertussiscomponents per single human dose; (3) the amount of HBsAgper single human dose; (4) the type of cells used for productionof the hepatitis B component; (5) where applicable, that thevaccine is intended for primary vaccination of children and isnot necessarily suitable for reinforcing doses or foradministration to adults; (6) the name and the amount of theadsorbent; (7) that the vaccine must be shaken before use; (8)that the vaccine is not to be frozen (9) where applicable, thatthe vaccine contains a pertussis toxin-like protein producedby genetic modification.

Adsorbed Diphtheria, Tetanus,Pertussis (Acellular Component),Inactivated Poliomyelitis Vaccine andHaemophilus Type b ConjugateVaccineDiphtheria, Tetanus, Pertussis (Acellular Component) andHaemophilus Type b Conjugate Vaccine (Adsorbed) is acombined vaccine composed of: diphtheria formol toxoid;tetanus formol toxoid; individually purified antigeniccomponents of Bordetella pertussis; polyribosylribitolphosphate (PRP) covalently bound to a carrier protein; amineral absorbent such as aluminium hydroxide or hydratedaluminium phosphate. The product may be presented withthe haemophilus type b component in a separate container,the contents of which are mixed with the other componentsimmediately before use.


The vaccine contains either pertussis toxoid or a pertussis-toxin-like protein free from toxic properties produced byexpression of a genetically modified form of the correspondinggene. Pertussis toxoid is prepared from pertussis toxin by amethod that renders the toxin harmless while maintainingadequate immunogenic properties and avoiding reversion totoxin. The acellular pertussis component may also containfilamentous haemagglutinin, pertactin (a 69 kDa outer-

A. D. T. P. (ACELL. COMPONENT), INACTIVATED POLIOMYLITIS VACCINE AND HAEMOPHILUS TYPE B CONJUGATE

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membrane protein) and other defined components of B.pertussis such as fimbrial-2 and fimbrial-3 antigens. The latter2 antigens may be co-purified. The antigenic composition andcharacteristics are based on evidence of protection andfreedom from unexpected reactions in the target group forwhich the vaccine is intended.

PRP is a linear copolymer composed of repeated units of3-β -D-r ibofuranosyl - (1→1)- r ib i to l -5-phosphate[(C10H19O12P)n], with a defined molecular size and derived froma suitable strain of Haemophilus influenzae type b. The carrierprotein, when conjugated to PRP, is capable of inducing a T-cell-dependent B-cell immune response to the polysaccharide.

Production

General provisions


The content of bacterial endotoxins in bulk purified diphtheriatoxoid, tetanus toxoid, pertussis components, purified,inactivated monovalent poliovirus harvests and bulk PRPconjugate is determined to monitor the purification procedureand to limit the amount in the final vaccine. For eachcomponent, the content of bacterial endotoxins is less thanthe limit approved for the particular vaccine and, in any case,the contents are such that the final vaccine contains less than100 IU per single human dose.

The production method is validated to demonstrate that theproduct, if tested, would comply with the following test. Injectsubcutaneously 5 times the single human dose stated on thelabel into each of 5 healthy guinea-pigs, each weighingbetween 250 and 350 g, that have not previously been treatedwith any material that will interfere with the test. If within 42days of the injection any of the animals shows signs of or diesfrom diphtheria, toxaemia or tetanus, the vaccine does notcomply with the test. If more than 1 animal dies from non-specific causes, repeat the test once; if more than 1 animaldies in the second test, the vaccine does not comply with thetest.


As part of consistency studies the assays of the diphtheria,tetanus, pertussis and poliomyelitis components are carriedout on a suitable number of batches of vaccine reconstitutedfor use. For subsequent routine control, the assays of thesecomponents may be carried out without mixing with thehaemophilus component.




The production of the components complies with therequirements of the monographs on Diphtheria Vaccine(Adsorbed), Tetanus Vaccine (Adsorbed), Pertussis Vaccine(Acellular Component, Adsorbed), Poliomyelitis Vaccine(Inactivated) and Haemophilus Type b Conjugate Vaccine.

FINAL BULK VACCINE

The final bulk of the diphtheria, tetanus, pertussis andpoliomyelitis components is prepared by adsorption,separately or together, of suitable quantities of bulk purifieddiphtheria toxoid, bulk purified tetanus toxoid and bulk purifiedacellular pertussis components onto a mineral carrier such asaluminium hydroxide or hydrated aluminium phosphate andadmixture of suitable quantities of purified, monovalentharvests of human polioviruses 1, 2 and 3 or a suitable quantityof a trivalent pool of such monovalent harvests. Suitableantimicrobial preservatives may be added.The final bulk of the haemophilus component is prepared bydilution of the bulk conjugate to the final concentration with asuitable diluent. A stabiliser may be added.


Bovine serum albumin. Determine on the poliomyelitiscomponents by a suitable immunochemical method (2.2.14)during preparation of the final bulk vaccine, before additionof the adsorbent, the amount of bovine serum albumin is suchthat the content in the final vaccine will not be more than 50ng per single human dose.




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FINAL LOT

The final bulk of the haemophilus component is freeze-dried.

Only a final lot that is satisfactory with respect to the test forosmolality shown below and with respect to each of therequirements given below under Identification, Tests andAssay may be released for use.

Provided that the test for absence of residual pertussis toxinand irreversibility of pertussis toxoid, the test for antimicrobialpreservative and the assay have been carried out withsatisfactory results on the final bulk vaccine, they may beomitted on the final lot.

Provided that the free formaldehyde content has beendetermined on the bulk purified antigens and the purifiedmonovalent harvests or the trivalent pool of polioviruses orthe final bulk and it has been shown that the content in thefinal lot will not exceed 0.2 g/l, the test for free formaldehydemay be omitted on the final lot.

Provided that the in vivo assay for the poliomyelitis componenthas been carried out with satisfactory results on the final bulkvaccine, it may be omitted on the final lot.

Osmolality (2.4.23). The osmolality of the vaccine,reconstituted where applicable, is within the limits approvedfor the particular preparation.

Free PRP

Unbound PRP is determined on the haemophilus componentafter removal of the conjugate, for example by anion-exchange,size-exclusion or hydrophobic chromatography (2.4.16),ultrafiltration or other validated methods. The amount of freePRP is not greater than that approved for the particular product.

Identification

Identification tests A, B, C and D are carried out using the vialcontaining the diphtheria, tetanus, pertussis and poliomyelitiscomponents; identification test E is carried out on the vialcontaining the haemophilus component.A. Diphtheria toxoid is identified by a suitable immunochemicalmethod (2.2.14). The following method, applicable to certainvaccines, is given as an example. Dissolve in the vaccine underexamination sufficient sodium citrate to give a 10 per centw/v solution. Maintain at 37° for about 16 hours and centrifugeuntil a clear supernatant is obtained. The clear supernatantreacts with a suitable diphtheria antitoxin, giving a precipitate.

B. Tetanus toxoid is identified by a suitable immunochemicalmethod (2.2.14). The following method, applicable to certainvaccines, is given as an example. The clear supernatantobtained during identification test A reacts with a suitabletetanus antitoxin, giving a precipitate.

C. The pertussis components are identified by suitable

immunochemical methods (2.2.14). The following method,applicable to certain vaccines, is given as an example. Theclear supernatant obtained during identification test A reactswith specific antisera to the pertussis components of thevaccine.

D. The vaccine is shown to contain human polioviruses 1, 2and 3 by a suitable immunochemical method (2.2.14), such asdetermination of D-antigen by enzyme-linked mmunosorbentassay (ELISA).

E. The haemophilus component is identified by a suitableimmunochemical method (2.2.14) for PRP.

Tests

The tests for absence of residual pertussis toxin, irreversibilityof pertussis toxoid, aluminium, free formaldehyde,antimicrobial preservative and sterility are carried out onthe container with the diphtheria, tetanus, pertussis andpoliomyelitis components; the tests for PRP content, water,sterility and pyrogens are carried out on the container withthe haemophilus component.Some tests for the haemophilus component may be carriedout on the freeze-dried product rather than on the bulkconjugate where the freeze-drying process may affect thecomponent under test.

Absence of residual pertussis toxin and irreversibilityof pertussis toxoid

This test is not necessary for the product obtained by geneticmodification. Use 3 groups each of not fewer than 5 histamine-sensitive mice. Inject intraperitoneally into each mouse of thefirst group twice the single human dose of the vaccine storedat 2° to 8°. Inject intraperitoneally into each mouse of thesecond group twice the single human dose of the vaccineincubated at 37° for 4 weeks. Inject diluent into the third groupof mice. After 5 days, inject into each mouse 2 mg of histaminebase intraperitoneally in a volume not exceeding 0.5 ml andobserve for 24 hours. The test is invalid if 1 or more controlmice die following histamine challenge. The vaccine complieswith the test if no animal in the first or second group diesfollowing histamine challenge. If 1 mouse dies in either orboth of the first and second groups, the test may be repeatedwith the same number of mice or with a greater number and theresults of valid tests combined; the vaccine complies with thetest if, in both of the groups given the vaccine, not more than5 per cent of the total number of mice die following histaminechallenge.The histamine sensitivity of the strain of mice used is verifiedat suitable intervals as follows: inject intravenously threefolddilutions of a reference pertussis toxin preparation inphosphate-buffered saline solution containing 0.2 per centw/v of gelatin and challenge with histamine as above; the


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strain is suitable if more than 50.0 per cent of the animals aresensitised by 50 ng of pertussis toxin and none of the controlanimals injected with only diluent and challenged similarlywith histamine show symptoms of sensitisation.PRP. Minimum 80.0 per cent of the amount of PRP stated onthe label. PRP is determined either by assay of ribose ( 2.7.1)or phosphorus ( 2.7.1), by an immunochemical method ( 2.2.14)or by anion-exchange liquid chromatography with pulsed-amperometric detection.Aluminium (2.3.9). Maximum 1.25 mg per single human dose,if aluminium hydroxide or hydrated aluminium phosphate isused as the adsorbent.Free formaldehyde (2.3.20). Maximum 0.2 g/l.Antimicrobial preservative. Where applicable, determine theamount of antimicrobial preservative by a suitable chemicalmethod. The content is not less than 85.0 per cent and notgreater than 115.0 per cent of the intended amount.

Water (2.3.43). Maximum 3.0 per cent for the haemophiluscomponent.Sterility (2.2.11 ). Complies with the test for sterility.Pyrogens (2.2.8). Complies with the test for pyrogens. Injectper kg of the rabbit’s mass a quantity of the vaccine equivalentto: 1 mg of PRP for a vaccine with diphtheria toxoid or CRM197 diphtheria protein as carrier; 0.1 mg of PRP for a vaccinewith tetanus toxoid as carrier; 0.025 mg of PRP for a vaccinewith OMP as a carrier.Assay


Carry out one of the prescribed methods for the assay asstated under Diphtheria Vaccine (Adsorbed).Unless otherwise justified and authorised, the lowerconfidence limit (P = 0.95) of the estimated potency is not lessthan 30 IU per single human dose.

Tetanus componentCarry out one of the prescribed methods for the assay asstated under Tetanus Vaccine (Adsorbed).


Pertussis component

It complies with the assay as stated under Adsorbed PertussisVaccine (Acellular Component).

Poliomyelitis component

D-antigen content

As a measure of consistency of production, determine the D-antigen content for human polioviruses 1, 2 and 3 by a suitable

immunochemical method (2.2.14) using a reference preparationcalibrated in units of D-antigen. For each type, the content,expressed with reference to the amount of D-antigen statedon the label, is within the limits approved for the particularproduct. Poliomyelitis vaccine (inactivated) referencepreparation is calibrated in Units and intended for use in theassay of D-antigen. The Unit and the International Unit areequivalent.

In vivo test

The vaccine complies with the in vivo assay as stated underInactivated Poliomyelitis Vaccine.

Labelling. The label states (1) the minimum number ofInternational Units of diphtheria and tetanus toxoid per singlehuman dose (2) the names and amounts of the pertussiscomponents per single human dose; (3) the nominal amountof poliovirus of each type (1, 2 and 3), expressed in units ofD-antigen per single human dose; (4) the type of cells usedfor production of the poliomyelitis component; (5) the numberof micrograms of PRP per single human dose; (6) the type andnominal amount of carrier protein per single human dose; (7)where applicable, that the vaccine is intended for primaryvaccination of children and is not necessarily suitable forreinforcing doses or for administration to adults; (8) the nameand the amount of the adsorbent; (9) that the vaccine must beshaken before use; (10) that the vaccine is not to be frozen;(11) where applicable, that the vaccine contains a pertussistoxin-like protein produced by genetic modification.

Adsorbed Diphtheria, Tetanus,Pertussis (Acellular Component) andInactivated Poliomyelitis VaccineDiphtheria, Tetanus, Pertussis (Acellular Component) andPoliomyelitis (Inactivated) Vaccine is a combined vaccinecontaining: diphtheria formol toxoid; tetanus formol toxoid;individually purified antigenic components of Bordetellapertussis; suitable strains of human polioviruses 1, 2 and 3grown in suitable cell cultures and inactivated by a validatedmethod; a mineral adsorbent such as aluminium hydroxide orhydrated aluminium phosphate.


The vaccine contains either pertussis toxoid or a pertussis-toxin-like protein free from toxic properties produced byexpression of a genetically modified form of the correspondinggene. Pertussis toxoid is prepared from pertussis toxin by amethod that renders the toxin harmless while maintaining

A. D. T. P. (ACELLULAR. COMPONENT) AND INACTIVATED POLIOMYLITIS VACCINE

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adequate immunogenic properties and avoiding reversion totoxin. The vaccine may also contain filamentoushaemagglutinin, pertactin (a 69 kDa outer-membrane protein)and other defined components of B. pertussis such as fimbrial-2 and fimbrial-3 antigens. The latter 2 antigens may becopurified. The antigenic composition and characteristics arebased on evidence of protection and freedom from unexpectedreactions in the target group for which the vaccine is intended.



The production method is validated to demonstrate that theproduct, if tested, would comply with the test for abnormaltoxicity for antisera and vaccines.

The content of bacterial endotoxins in bulk purified diphtheriatoxoid, tetanus toxoid, pertussis components and purified,inactivated monovalent poliovirus harvests is determined tomonitor the purification procedure and to limit the amount inthe final vaccine. For each component, the content of bacterialendotoxins is less than the limit approved for the particularvaccine and, in any case, the contents are such that the finalvaccine contains less than 100 IU per single human dose.




The production of the components complies with therequirements of the monographs on Diphtheria Vaccine(Adsorbed), Tetanus Vaccine (Adsorbed), Pertussis Vaccine(Acellular Component, Adsorbed) and Poliomyelitis Vaccine(Inactivated).

FINAL BULK VACCINE

The final bulk vaccine is prepared by adsorption onto a mineralcarrier such as aluminium hydroxide or hydrated aluminiumphosphate, separately or together, of suitable quantities of

bulk purified diphtheria toxoid, tetanus toxoid, acellularpertussis components and admixture of suitable quantities ofpurified monovalent harvests of human polioviruses 1, 2 and3 or a suitable quantity of a trivalent pool of such purifiedmonovalent harvests. Suitable antimicrobial preservatives maybe added.


Bovine serum albumin. Determine on the poliomyelitiscomponents by a suitable immunochemical method (2.2.14)after virus harvest and before addition of the adsorbent in thepreparation of the final bulk vaccine, the amount of bovineserum albumin is such that the content in the final vaccine willbe not more than 50 ng per single human dose.



FINAL LOT


Provided the tests for absence of residual pertussis toxin,irreversibility of pertussis toxoid and antimicrobial preservativeand the assays for the diphtheria, tetanus and pertussiscomponents have been carried out with satisfactory resultson the final bulk vaccine, they may be omitted on the final lot.

Provided the free formaldehyde content has been determinedon the bulk purified antigens or on the final bulk and it hasbeen shown that the content in the final lot will not exceed0.2 g/l, the test for free formaldehyde may be omitted on thefinal lot.

Provided that the determination of D-antigen content has beencarried out with satisfactory results during preparation of thefinal bulk before addition of the adsorbent, it may be omittedon the final lot.



Identification

A. Diphtheria toxoid is identified by a suitable immunochemical


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method (2.2.14). The following method, applicable to certainvaccines, is given as an example. Dissolve in the vaccine underexamination sufficient sodium citrate to give a 10 per centw/v solution. Maintain at 37° for about 16 h and centrifugeuntil a clear supernatant is obtained. The clear supernatantreacts with a suitable diphtheria antitoxin, giving a precipitate.

B. Tetanus toxoid is identified by a suitable immunochemicalmethod (2.2.14). The following method, applicable to certainvaccines, is given as an example. The clear supernatantobtained as described in Identification test A reacts with asuitable tetanus antitoxin, giving a precipitate.

C. The pertussis components are identified by a suitableimmunochemical method (2.2.14). The following method,applicable to certain vaccines, is given as an example. Theclear supernatant obtained as described in Identification testA reacts with a specific antisera to the pertussis componentsof the vaccine.

D. The vaccine is shown to contain human polioviruses 1, 2and 3 by a suitable immunochemical method (2.2.14) such asthe determination of D-antigen by enzyme-linked immuno-sorbent assay (ELISA).

Tests

Absence of residual pertussis toxin and irreversibilityof pertussis toxoid

This test is not necessary for the product obtained by geneticmodification. Use 3 groups each of not less than 5 histamine-sensitive mice. Inject intraperitoneally into each mouse of thefirst group twice the single human dose of the vaccine storedat 2° to 8°. Inject intraperitoneally into each mouse of thesecond group twice the single human dose of the vaccineincubated at 37° for 4 weeks. Inject diluent into the third groupof mice. After 5 days, inject into each mouse 2 mg of histaminebase intraperitoneally in a volume not exceeding 0.5 ml andobserve for 24 hours. The test is invalid if 1 or more controlmice die following histamine challenge. The vaccine complieswith the test if no animal in the first or second group diesfollowing histamine challenge. If 1 mouse dies in either orboth of the first and second groups, the test may be repeatedwith the same number of mice or with a greater number and theresults of valid tests combined; the vaccine complies with thetest if, in both of the groups given the vaccine, not more than5.0 per cent of the total number of mice die following histaminechallenge.

The histamine sensitivity of the strain of mice used is verifiedat suitable intervals as follows: inject intravenously three-fold dilutions of a reference pertussis toxin preparation inphosphate-buffered saline solution containing 0.2 per centw/v of gelatin and challenge with histamine as above; thestrain is suitable if more than 50.0 per cent of the animals are

sensitised by 50 ng of pertussis toxin and none of the controlanimals injected with only diluent and challenged similarlywith histamine shows symptoms of sensitisation.

Aluminium (2.3.9). Maximum 1.25 mg per single human doseif aluminium hydroxide or hydrated aluminium phosphate isused as the adsorbent.




Assay



The lower confidence limit (P = 0.95) of the estimated potencyis not less than the minimum potency stated on the label.

Unless otherwise justified and authorised, the minimumpotency stated on the label is 30 IU per single human dose.

Tetanus component



Pertussis component



D-antigen content

As a measure of consistency of production, determine the D-antigen content for human polioviruses 1, 2 and 3 by a suitableimmunochemical method (2.2.14) following desorption using

a reference preparation calibrated in units of D-antigen. Foreach type, the content, expressed with reference to the amountof D-antigen stated on the label, is within the limits approvedfor the particular product. Poliomyelitis vaccine (inactivated)reference preparation is calibrated in Units and intended foruse in the assay of D-antigen. The Unit and the InternationalUnit are equivalent.

In vivo test

The vaccine complies with the in vivo assay as stated underInactivated Poliomyelitis Vaccine.


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Labelling. The label complies with the requirements statedunder Vaccine and also states (1) the minimum number ofInternational Units of diphtheria and tetanus toxoid per singlehuman dose (2) the names and amounts of the pertussiscomponents per single human dose; (3) the nominal amountof poliovirus of each type (1, 2 and 3), expressed in units of D-antigen per single human dose; (4) the type of cells used forproduction of the poliomyelitis component; (5) the number ofmicrograms of PRP per single human dose; (6) the type andnominal amount of carrier protein per single human dose; (7)where applicable, that the vaccine is intended for primaryvaccination of children and is not necessarily suitable forreinforcing doses or for administration to adults; (8) the nameand the amount of the adsorbent; (9) that the vaccine must beshaken before use; (10) that the vaccine is not to be frozen;(11) where applicable, that the vaccine contains a pertussistoxin-like protein produced by genetic modification.

Adsorbed Diphtheria, Tetanus,Pertussis and Poliomyelitis(Inactivated) VaccineDiphtheria, Tetanus, Pertussis and Poliomyelitis (Inactivated)Vaccine (Adsorbed) is a combined vaccine containing:diphtheria formol toxoid; tetanus formol toxoid; an inactivatedsuspension of Bordetella pertussis; suitable strains of humanpolioviruses 1, 2 and 3 grown in suitable cell cultures andinactivated by a validated method; a mineral adsorbent suchas aluminium hydroxide or hydrated aluminium phosphate.


Production

General provisions

The production method shall have been shown to yieldconsistently the vaccines comparable with the vaccine ofproven clinical efficacy and safety in man.

The production method is validated to demonstrate that theproduct, if tested, would comply with the test for abnormaltoxicity for antisera and vaccines, and with the following testfor specific toxicity of the diphteria and tetanus components :inject subcutaneously 5 times the single human dose statedon the label into each of 5 healthy guinea-pigs, each weighingbetween 250 and 350 g, that have not previously been treatedwith any material that will interfere with the test. If within 42days of the injection any of the animals shows signs of or diesfrom diphtheria toxaemia or tetanus, the vaccine does notcomply with the test. If more than 1 animal dies from non-

specific causes, repeat the test once; if more than 1 animaldies in the second test, the vaccine does not comply with thetest.




The production of the components complies with therequirements of the monographs on Diphtheria Vaccine(Adsorbed), Tetanus Vaccine (Adsorbed), Pertussis Vaccine(Adsorbed) and Poliomyelitis Vaccine (Inactivated).

FINAL BULK VACCINE

The final bulk vaccine is prepared by adsorption onto a mineralcarrier such as aluminium hydroxide or hydrated aluminiumphosphate, separately or together, of suitable quantities ofbulk purified diphtheria toxoid and bulk purified tetanus toxoidand admixture of suitable quantities of an inactivatedsuspension of B. pertussis and purified monovalent harvestsof human polioviruses 1, 2 and 3 or a suitable quantity of atrivalent pool of such purified monovalent harvests. Suitableantimicrobial preservatives may be added.


Specific toxicity

Use not less than 5 healthy mice each weighing between14 and 16 g for the vaccine group and for the saline control.Use mice of the same sex or distribute males and females equallybetween the groups. Allow the animals access to food andwater for at least 2 hours before injection and during the test.Inject each mouse of the vaccine group intraperitoneally with0.5 ml, containing a quantity of the vaccine equivalent to notless than half the single human dose. Inject each mouse of thecontrol group with 0.5 ml of a 0.9 per cent sterile solution ofsodium chloride, preferably containing the same amount ofantimicrobial preservative as that injected with the vaccine.Weigh the groups of mice immediately before the injectionand 72 hours and 7 days after the injection. The vaccine

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complies with the test if: (a) at the end of 72 hours the totalmass of the group of vaccinated mice is not less than thatpreceding the injection; (b) at the end of 7 days the averageincrease in mass per vaccinated mouse is not less than60 per cent of that per control mouse; and (c) not more than5 per cent of the vaccinated mice die during the test. The testmay be repeated and the results of the tests combined.

Bovine serum albumin. Determine on the poliomyelitiscomponents by a suitable immunochemical method (2.2.14)during preparation of the final bulk vaccine; before additionof the adsorbent, the amount of bovine serum albumin is suchthat the content in the final vaccine will be not more than 50ng per single human dose.



FINAL LOT


Provided that the tests for specific toxicity and antimicrobialpreservative, and the assays for the diphtheria, tetanus andpertussis components have been carried out with satisfactoryresults on the final bulk vaccine, they may be omitted on thefinal lot.

Provided that the free formaldehyde content has beendetermined on the bulk purified antigens, the inactivated B.pertussis suspension and the purified monovalent harvestsor the trivalent pool of polioviruses or on the final bulk and ithas been shown that the content in the final lot will not exceed0.2 g/l, the test for free formaldehyde may be omitted on thefinal lot.



IdentificationA. Diphtheria toxoid is identified by a suitable immunochemicalmethod (2.2.14). The following method, applicable to certainvaccines, is given as an example. Dissolve in the vaccine underexamination sufficient sodium citrate to give a 10 per centw/v solution. Maintain at 37° for about 16 hours and centrifugeuntil a clear supernatant is obtained. The clear supernatant

reacts with a suitable diphtheria antitoxin, giving a precipitate.

B. Tetanus toxoid is identified by a suitable immunochemicalmethod (2.2.14). The following method, applicable to certainvaccines, is given as an example. The clear s u p e r n a t a n tobtained during identification test A reacts with a suitabletetanus antitoxin, giving a precipitate.

C. The centrifugation residue obtained in identification A maybe used. Other suitable methods for separating the bacteriafrom the adsorbent may also be used. Identify pertussisvaccine by agglutination of the bacteria from the resuspendedprecipitate by antisera specific to B. pertussis or by the assayof the pertussis component prescribed under Assay.

D. The vaccine is shown to contain human polioviruses 1, 2and 3 by a suitable immunochemical method (2.2.14) such asthe determination of D-antigen by enzyme-linked immuno-sorbent assay (ELISA).

Tests





Assay




Tetanus component


If the test is carried out in guinea pigs, the lower confidencelimit (P = 0.95) of the estimated potency is not less than 40 IUper single human dose; if the test is carried out in mice, thelower confidence limit (P = 0.95) of the estimated potency isnot less than 60 IU per single human dose.

Pertussis component

Carry out the assay as stated under Pertussis Vaccine.

The estimated potency is not less than 4 IU per single human

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dose and the lower confidence limit (P = 0.95) of the estimatedpotency is not less than 2 IU per single human dose.


D-antigen content

As a measure of consistency of production, determine the D-antigen content for human polioviruses 1, 2 and 3 by a suitableimmunochemical method (2.2.14) using a reference preparationcalibrated in Units of D-antigen. For each type, the content,expressed with reference to the amount of D-antigen statedon the label, is within the limits approved for the particularproduct. Poliomyelitis vaccine (inactivated) referencepreparation is calibrated in Units and is intended for use inthe assay of D-antigen. The Unit and the IU are equivalent.

In vivo test

The vaccine complies with the in vivo assay as stated underPoliomyelitis Vaccine (Inactivated).

Labelling. The label states (1) the minimum number ofInternational Units of diphtheria and tetanus toxoid per singlehuman dose; (2) the minimum number of International Unitsof pertussis vaccine per single human dose; (3) the nominalamount of poliovirus of each type (1, 2 and 3), expressed inunits of D-antigen per single human dose; (4) the type of cellsused for production of the poliomyelitis component; (5) whereapplicable, that the vaccine is intended for primary vaccinationof children and is not necessarily suitable for reinforcing dosesor for administration to adults; (6) the name and the amount ofthe adsorbent; (7) that the vaccine must be shaken beforeuse; (8) that the vaccine is not to be frozen.

Adsorbed Diphtheria, Tetanus,Pertussis, Poliomyelitis (Inactivated)and Haemophilus Type b ConjugateVaccineDiphtheria, Tetanus, Pertussis, Poliomyelitis (Inactivated) andHaemophilus Type b Conjugate Vaccine (Adsorbed) is acombined vaccine composed of: diphtheria formol toxoid;tetanus formol toxoid; an inactivated suspension of Bordetellapertussis; suitable strains of human polioviruses 1, 2 and 3grown in suitable cell cultures and inactivated by a suitablemethod; polyribosylribitol phosphate (PRP) covalently boundto a carrier protein; a mineral adsorbent such as aluminiumhydroxide or hydrated aluminium phosphate. The product ispresented with the haemophilus component in a separatecontainer, the contents of which are mixed with the othercomponents immediately before use.


PRP is a linear copolymer composed of repeated units of3-β -D-r ibofuranosyl - (1→1)- r ib i to l -5-phosphate[(C10H19O12P)n], with a defined molecular size and derived froma suitable strain of Haemophilus influenzae type b. The carrierprotein, when conjugated to PRP, is capable of inducing a T-cell-dependent B-cell immune response to the polysaccharide.

Production

General provisions


The production method is validated to demonstrate that theproduct, if tested, would comply with the test for abnormaltoxicity for antisera and vaccines, and with the following testfor specific toxicity of the diphtheria and tetanus components:inject subcutaneously 5 times the single human dose statedon the label into each of 5 healthy guinea-pigs, each weighingbetween 250 and 350 g, that have not previously been treatedwith any material that will interfere with the test. If within 42days of the injection any of the animals shows signs of or diesfrom diphtheria toxaemia or tetanus, the vaccine does notcomply with the test. If more than 1 animal dies from non-specific causes, repeat the test once; if more than 1 animaldies in the second test, the vaccine does not comply with thetest.


As part of consistency studies the assays of the diphtheria,tetanus, pertussis and poliomyelitis components are carriedout on a suitable number of batches of vaccine reconstitutedfor use. For subsequent routine control, the assays of thesecomponents may be carried out without mixing with thehaemophilus component.


Provided valid assays can be performed, monocomponentreference vaccines may be used for the assays on the combinedvaccine. If this is not possible because of interaction betweenthe components of the combined vaccine or because of thedifference in composition between monocomponent referencevaccine and the test vaccine, a batch of combined vaccineshown to be effective in clinical trials or a batch representativethereof is used as a reference vaccine. For the preparation ofa representative batch, strict adherence to the productionprocess used for the batch tested in clinical trials is necessary.

A. D., T., P., POLIOMYLITIS (INACTIVATED) AND HAEMOPHILUS TYPE B CONJUGATE VACCINE

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The reference vaccine may be stabilised by a method that hasbeen shown to have no effect on the assay procedure.

Production of components

The production of the components complies with therequirements of the monographs on Diphtheria Vaccine(Adsorbed), Tetanus Vaccine (Adsorbed), Pertussis Vaccine(Adsorbed), Poliomyelitis Vaccine (Inactivated) andHaemophilus Type b Conjugate Vaccine.

FINAL BULK VACCINE

The final bulk of the diphtheria, tetanus, pertussis andpoliomyelitis components is prepared by adsorption,separately or together, of suitable quantities of bulk purifieddiphtheria toxoid, and bulk purified tetanus toxoid onto amineral carrier such as aluminium hydroxide or hydratedaluminium phosphate and admixture of suitable quantities ofan inactivated suspension of B. pertussis and of purified,monovalent harvests of human polioviruses 1, 2 and 3 or asuitable quantity of a trivalent pool of such monovalentharvests. Suitable antimicrobial preservatives may be added.

The final bulk of the haemophilus component is prepared bydilution of the bulk conjugate to the final concentration with asuitable diluent. A stabiliser may be added.

Only final bulk that complies with the following requirementsmay be used in the preparation of the final lot.

Specific toxicity

Use not less than 5 healthy mice each weighing between 14and 16 g, for the vaccine group and for the saline control. Usemice of the same sex or distribute males and females equallybetween the groups. Allow the animals access to food andwater for at least 2 hours before injection and during the test.Inject each mouse of the vaccine group intraperitoneally with0.5 ml, containing a quantity of the vaccine equivalent to notless than half the single human dose. Inject each mouse of thecontrol group with 0.5 ml of a 0.9 per cent sterile solution ofsodium chloride, preferably containing the same amount ofantimicrobial preservative as that injected with the vaccine.Weigh the groups of mice immediately before the injectionand 72 hours and 7 days after the injection. The vaccinecomplies with the test if (a) at the end of 72 hours the totalmass of the group of vaccinated mice is not less than thatpreceding the injection; (b) at the end of 7 days the averageincrease in mass per vaccinated mouse is not less than60 per cent of that per control mouse; and (c) not more than5 per cent of the vaccinated mice die during the test. The testmay be repeated and the results of the tests combined.

Bovine serum albumin. Determine on the poliomyelitiscomponents by a suitable immunochemical method (2.2.14)during preparation of the final bulk vaccine, before addition

of the adsorbent, the amount of bovine serum albumin is suchthat the content in the final vaccine will not be more than 50ng per single human dose.



FINAL LOT

The final bulk of the haemophilus component is freeze-dried.

Only a final lot that is satisfactory with respect to the test forosmolality shown below and with respect to each of therequirements given below under Identification, Tests andAssay may be released for use.

Provided that the tests for specific toxicity and antimicrobialpreservative, and the assays for the diphtheria, tetanus andpertussis components have been carried out with satisfactoryresults on the final bulk vaccine, they may be omitted on thefinal lot.Provided that the free formaldehyde content has beendetermined on the bulk purified antigens, the inactivated B.pertussis suspension and the purified monovalent harvestsor the trivalent pool of polioviruses or on the final bulk and ithas been shown that the content in the final lot will not exceed0.2 g/l, the test for free formaldehyde may be omitted on thefinal lot.Provided that the in vivo assay for the poliomyelitis componenthas been carried out with satisfactory results on the final bulkvaccine, it may be omitted on the final lot.

Osmolality (2.4.23). The osmolality of the vaccine,reconstituted where applicable, is within the limits approvedfor the particular preparation.

Free PRP

Unbound PRP is determined on the haemophilus componentafter removal of the conjugate, for example by anion-exchange,size-exclusion or hydrophobic chromatography (2.4.16),ultrafiltration or other validated methods. The amount of freePRP is not greater than that approved for the particular product.

IdentificationIdentification tests A, B, C and D are carried out using thevial containing the diphtheria, tetanus, pertussis andpoliomyelitis components; identification test E is carriedout on the vial containing the haemophilus component.

A. Diphtheria toxoid is identified by a suitable immunochemicalmethod (2.2.14). The following method, applicable to certain


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vaccines, is given as an example. Dissolve in the vaccine underexamination sufficient sodium citrate to give a 10 per centw/v solution. Maintain at 37° for about 16 hours and centrifugeuntil a clear supernatant is obtained. The clear supernatantreacts with a suitable diphtheria antitoxin, giving a precipitate.

B. Tetanus toxoid is identified by a suitable immunochemicalmethod (2.2.14). The following method, applicable to certainvaccines, is given as an example. The clear s u p e r n a t a n tobtained during identification test A reacts with a suitabletetanus antitoxin, giving a precipitate.

C. The centrifugation residue obtained in identification A maybe used. Other suitable methods for separating the bacteriafrom the adsorbent may also be used. Identify pertussisvaccine by agglutination of the bacteria from the resuspendedprecipitate by antisera specific to B. pertussis or by the assayof the pertussis component prescribed under Assay.

D. The vaccine is shown to contain human polioviruses 1, 2and 3 by a suitable immunochemical method (2.2.14), such asdetermination of D-antigen by enzymelinked immunosorbentassay (ELISA).

E. The haemophilus component is identified by a suitableimmunochemical method (2.2.14) for PRP.

TestsThe tests for specific toxicity, aluminium, free formaldehyde,antimicrobial preservative and sterility are carried out onthe container with diphtheria, tetanus, pertussis andpoliomyelitis components; the tests for PRP content, water,sterility and pyrogens are carried out on the container withthe haemophilus component.

Some tests for the haemophilus component may be carriedout on the freeze-dried product rather than on the bulkconjugate where the freeze-drying process may affect thecomponent under test.

PRP. Minimum 80.0 per cent of the amount of PRP stated onthe label. PRP is determined either by assay of ribose (2.7.1) orphosphorus (2.7.1), by an immunochemical method (2.2.14) orby anion-exchange liquid chromatography with pulsed-amperometric detection.




Water (2.3.43). Maximum 3.0 per cent for the haemophiluscomponent.


Pyrogens (2.2.8). Complies with the test for pyrogens. Injectper kg of the rabbit’s mass a quantity of the vaccine equivalentto 1 mg of PRP for a vaccine with diphtheria toxoid or CRM197 diphtheria protein as carrier; 0.1 mg of PRP for a vaccinewith tetanus toxoid as carrier; 0.025 mg of PRP for a vaccinewith OMP as carrier.

Assay




Tetanus component


If the test is carried out in guinea-pigs, the lower confidencelimit (P = 0.95) of the estimated potency is not less than 40 IUper single human dose; if the test is carried out in mice, thelower confidence limit (P = 0.95) of the estimated potency isnot less than 60 IU per single human dose.

Pertussis component


The estimated potency is not less than 4 IU per single humandose and the lower confidence limit (P = 0.95) of the estimatedpotency is not less than 2 IU per single human dose.


D-antigen content

As a measure of consistency of production, determine the D-antigen content for human polioviruses 1, 2 and 3 by a suitableimmunochemical method (2.2.14) using a reference preparationcalibrated in Units of D-antigen. For each type, the content,expressed with reference to the amount of D-antigen statedon the label, is within the limits approved for the particularproduct. Poliomyelitis vaccine (inactivated) referencepreparation is calibrated in Units and intended for use in theassay of D-antigen. The Unit and the IU are equivalent.

In vivo test

The vaccine complies with the in vivo assay as stated underPoliomyelitis Vaccine (Inactivated).

Labelling. The label states (1) the minimum number ofInternational Units of diphtheria and tetanus toxoid per singlehuman dose; (2) the minimum number of International Unitsof pertussis vaccine per single human dose; (3) the nominalamount of poliovirus of each type (1, 2 and 3), expressed in


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Units of D-antigen per single human dose; (4) the type of cellsused for production of the poliomyelitis component; (5) thenumber of micrograms of PRP per single human dose; (6) thetype and nominal amount of carrier protein per single humandose; (7) where applicable, that the vaccine is intended forprimary vaccination of children and is not necessarily suitablefor reinforcing doses or for administration to adults; (8) thename and the amount of the adsorbent; (9) that the vaccinemust be shaken before use; (9) that the vaccine is not to befrozen.

Adsorbed Pertussis Vaccine (AcellularComponent)Pertussis Vaccine (Acellular Component, Adsorbed) is apreparation of individually prepared and purified antigeniccomponents of Bordetella pertussis adsorbed on a mineralcarrier such as aluminium hydroxide or hydrated aluminiumphosphate.The vaccine contains either pertussis toxoid or a pertussistoxin, like protein free from toxic properties, produced byexpression of a genetically modified form of the correspondinggene. Pertussis toxoid is prepared from pertussis toxin by amethod that renders the latter harmless while maintainingadequate immunogenic properties and avoiding reversion totoxin. The vaccine may also contain filamentoushaemagglutinin, pertactin (a 69 kDa outer-membrane protein)and other defined components of B. pertussis such as fimbrial-2 and fimbrial-3 antigens. The latter 2 antigens may becopurified. The antigenic composition and characteristics arebased on evidence of protection and freedom from unexpectedreactions in the target group for which the vaccine is intended.



Reference vaccine

A batch of vaccine shown to be effective in clinical trials or abatch representative thereof is used as a reference vaccine.For the preparation of a representative batch, strict adherenceto the production process used for the batch tested in clinicaltrials is necessary. The reference vaccine is preferablystabilised by a method that has been shown to have nosignificant effect on the assay procedure when the stabilisedand non-stabilised batches are compared.CHARACTERISATION OF COMPONENTS

During development of the vaccine, the production processshall be validated to demonstrate that it yields consistently

individual components that comply with the followingrequirements; after demonstration of consistency, the testsneed not be applied routinely to each batch.

Adenylate cyclase. Not more than 500 ng in the equivalent of1 dose of the final vaccine, determined by immunoblot analysisor another suitable method.Tracheal cytotoxin. Not more than 2 pmol in the equivalent of1 dose of the final vaccine, determined by a suitable methodsuch as a biological assay or liquid chromatography (2.4.14).Absence of residual dermonecrotic toxin. Inject intradermallyinto each of 3 unweaned mice, in a volume of 0.1 ml, theamount of component or antigenic fraction equivalent to 1dose of the final vaccine. Observe for 48 hours. Nodermonecrotic reaction is demonstrable.

Specific properties. The components of the vaccine areanalysed by one or more of the methods shown below inorder to determine their identity and specific properties (activityper unit amount of protein) in comparison with referencepreparations.

Pertussis toxin

Chinese hamster ovary (CHO) cell-clustering effect andhaemagglutination as in vitro methods; lymphocytosis-promoting activity, histamine-sensitising activity and insulinsecretory activity as in vivo methods. The toxin shows ADP-ribosyl transferase activity using transducin as the acceptor.Filamentous haemagglutininHaemagglutination and inhibition by specific antibody.Pertactin, fimbrial-2 and fimbrial-3 antigens. Reactivity withspecific antibody.

Pertussis toxoidThe toxoid induces in animals production of antibodiescapable of inhibiting all the properties of pertussis toxin.

PURIFIED COMPONENTS

Production of each component is based on a seed-lot system.The seed cultures from which toxin is prepared are managedto conserve or where necessary restore toxinogenicity bydeliberate selection.None of the media used at any stage contains blood or bloodproducts of human origin. Media used for the preparation ofseed lots and inocula may contain blood or blood products ofanimal origin.Pertussis toxin and, where applicable, filamentoushaemagglutinin and pertactin are purified and, after appropriatecharacterisation, detoxified using suitable chemical reagents,by a method that avoids reversion of the toxoid to toxin,particularly on storage or exposure to heat. Other componentssuch as fimbrial-2 and fimbrial-3 antigens are purified either

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separately or together, characterised and shown to be freefrom toxic substances. The purification procedure is validatedto demonstrate appropriate clearance of substances usedduring culture or purification.The content of bacterial endotoxins is determined to monitorthe purification procedure and to limit the amount in the finalvaccine. The limits applied for the individual components aresuch that the final vaccine contains less than 100 IU per singlehuman dose.Before detoxification, the purity of the components isdetermined by a suitable method such as polyacrylamide gelelectrophoresis (PAGE) or liquid chromatography. SDS-PAGEor immunoblot analysis with specific monoclonal or polyclonalantibodies may be used to characterise subunits. Requirementsare established for each individual product.

Only purified components that comply with the followingrequirements may be used in the preparation of the final bulkvaccine.

Sterility (2.2.11). Carry out the test for sterility using for eachmedium a quantity of purified component equivalent to notless than 100 doses.

Absence of residual pertussis toxin

This test is not necessary for the product obtained by geneticmodification. Use a group of not fewer than 5 histamine-sensitive mice each weighing between 18 and 26 g. Inject intoeach mouse the equivalent of 1 human dose intravenously ortwice the human dose intraperitoneally, diluted to not morethan 0.5 ml with phosphate-buffered saline solution containing0.2 per cent w/v of gelatin. Inject diluent into a second groupof control mice. After 5 days, inject 2 mg of histamine baseintraperitoneally in a volume not exceeding 0.5 ml and observefor 24 hours. If no animal dies, the preparation complies withthe test.

The histamine sensitivity of the strain of mice used is verifiedat suitable intervals as follows: inject three-fold dilutions of areference pertussis toxin preparation in phosphate-bufferedsaline solution containing 0.2 per cent w/v of gelatin andchallenge with histamine as above; the strain is suitable ifmore than 50 per cent of the animals are sensitised by 50 ng ofpertussis toxin and none of the control animals injected withonly diluent and challenged similarly with histamine showsymptoms of sensitisation.

A validated test based on the clustering effect of the toxin forChinese hamster ovary (CHO) cells may be used instead ofthe test on mice.

Residual detoxifying agents and other reagents

The content of residual detoxifying agents and other reagentsis determined and shown to be below approved limits unless

validation of the process has demonstrated acceptableclearance.

Antigen content

Determine the antigen content by a suitable immunochemicalmethod (2.2.14) and protein nitrogen by sulphuric aciddigestion (2.2.30) or another suitable method. The ratio ofantigen content to protein nitrogen is within the limitsestablished for the product.

FINAL BULK VACCINE

The vaccine is prepared by adsorption of suitable quantitiesof purified components, separately or together, onto aluminiumhydroxide or hydrated aluminium phosphate. A suitableantimicrobial preservative may be added.




FINAL LOT

Only a final lot that is satisfactory with respect to each of therequirements given below under Identification, Tests andAssay may be released for use. Provided that the tests forabsence of residual pertussis toxin and irreversibility ofpertussis toxoid, antimicrobial preservative, free formaldehydeand the assay have been carried out with satisfactory resultson the final bulk vaccine, these tests may be omitted on thefinal lot.

Identification

Subject the vaccine to a suitable desorption procedure suchas the following: dissolve in the vaccine under examinationsufficient sodium citrate to give a 10 per cent w/v solution;maintain at 37° for about 16 h and centrifuge until a clearsupernatant liquid is obtained. Examined by a suitableimmunochemical method (2.2.14), the clear supernatant liquidreacts with specific antisera to the components stated on thelabel.

Tests

Absence of residual pertussis toxin and irreversibility ofpertussis toxoid

This test is not necessary for the product obtained by geneticmodification. Use 3 groups each of not fewer than 5 histamine-sensitive mice. Inject intraperitoneally into the first group twice

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the single human dose of the vaccine stored at 2° to 8°. Injectintraperitoneally into the second group twice the single humandose of the vaccine incubated at 37° for 4 weeks. Inject diluentinto the third group of mice. After 5 days, inject into eachmouse 2 mg of histamine base intraperitoneally in a volumenot exceeding 0.5 ml and observe for 24 hours. The test isinvalid if 1 or more control mice die following histaminechallenge. The vaccine complies with the test if no animal inthe first or second group dies following histamine challenge.If 1 mouse dies in either or both of the first and second groups,the test may be repeated with the same number of mice or witha greater number and the results of valid tests combined; thevaccine complies with the test if, in both of the groups giventhe vaccine, not more than 5.0 per cent of the total number ofmice die following histamine challenge.

The histamine sensitivity of the strain of mice used is verifiedat suitable intervals as follows: inject intravenously threefolddilutions of a reference pertussis toxin preparation inphosphate-buffered saline solution containing 0.2 per centw/v of gelatin and challenge with histamine as above; thestrain is suitable if more than 50.0 per cent of the animals aresensitised by 50 ng of pertussis toxin and none of the controlanimals injected with only diluent and challenged similarlywith histamine show symptoms of sensitisation.





Assay

The capacity of the vaccine to induce the formation of specificantibodies is compared with the same capacity of a referencepreparation examined in parallel; antibodies are determinedusing suitable immunochemical methods (2.2.14) such asenzyme-linked immunosorbent assay (ELISA). The test onmice shown below uses a three-point model but, aftervalidation, for routine testing a single-dilution method may beused.

Requirement

The capacity to induce antibodies is not significantly (P =0.95) less than that of the reference vaccine.

The following test model is given as an example of a methodthat has been found to be satisfactory.

Selection and distribution of test animalsUse in the test healthy mice (for example, CD1 strain) of thesame stock 4 to 8 weeks old. Distribute the animals in 6 groupsof a number appropriate to the requirements of the assay. Use3 dilutions of the vaccine under examination and 3 dilutions ofa reference preparation and attribute each dilution to a groupof mice. Inject intraperitoneally or subcutaneously into eachmouse 0.5 ml of the dilution attributed to its group.

Collection of serum samples

4 to 5 weeks after vaccination, bleed the mice individuallyunder anaesthesia. Store the sera at -20° until tested forantibody content.

Antibody determination

Assay the individual sera for content of specific antibodies toeach component using a validated method such as the ELISAtest shown below.

ELISAMicrotitre plates (poly(vinyl chloride) or polystyrene asappropriate for the specific antigen) are coated with the purifiedantigen at a concentration of 100 ng per well. After washing,unreacted sites are blocked by incubating with a solution ofbovine serum albumin and then washed. Two-fold dilutionsof sera from mice immunised with test or reference vaccinesare made on the plates. After incubation at 22° to 25° for 1 h,the plates are washed. A suitable solution of anti-mouse IgGenzyme conjugate is added to each well and incubated at 22°to 25° for 1 h. After washing, a substrate is added from whichthe bound enzyme conjugate liberates a chromophore whichcan be quantified by measurement of absorbance. The testconditions are designed to obtain a linear response forabsorbance with respect to antibody content over the rangeof measurement used and absorbance values within the range0.1 to 2.0.A reference antiserum of assigned potency is used in the testand serves as the basis for calculation of the antibody levelsin test sera. A standardised control serum is also included inthe test.

The test is not valid if (a) the value found for the controlserum differs by more than 2 standard deviations from theassigned value; (b) the confidence interval of the potencyestimate is greater than 50.0 per cent to 200.0 per cent.

CalculationThe antibody titres in the sera of mice immunised withreference and test vaccines are calculated and from the valuesobtained the potency of the test vaccine in relation to thereference vaccine is calculated by the usual statistical methods.Labelling. The label states (1) the names and amounts of thecomponents present in the vaccine; (2) where applicable, that

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the vaccine contains a pertussis toxin-like protein producedby genetic modification; (3) the name and amount of theadsorbent; (4) that the vaccine must be shaken before use; (5)that the vaccine is not to be frozen.

Adsorbed Pertussis Vaccine (Acellular,Co-Purified)Pertussis Vaccine (Acellular, Co-Purified, Adsorbed) is apreparation of antigenic components of Bordetella pertussisadsorbed on a mineral carrier such as aluminium hydroxide orhydrated aluminium phosphate.

The vaccine contains an antigenic fraction purified withoutseparation of the individual components. The antigenic fractionis treated by a method that transforms pertussis toxin to toxoid,rendering it harmless while maintaining adequate immunogenicproperties of all the components and avoiding reversion totoxin. The antigenic fraction is composed of pertussis toxoid,filamentous haemagglutinin, pertactin (a 69 kDa outer-membrane protein) and other defined components of B.pertussis such as fimbrial-2 and fimbrial-3 antigens. It maycontain residual pertussis toxin up to a maximum levelapproved by the competent authority. The antigeniccomposition and characteristics are based on evidence ofprotection and freedom from unexpected reactions in the targetgroup for which the vaccine is intended.

Production

General provisions. The production method shall have beenshown to yield consistently vaccines comparable with thevaccine of proven clinical efficacy and safety in man.

Reference vaccine. A batch of vaccine shown to be effectivein clinical trials or a batch representative thereof is used as areference vaccine. For the preparation of a representativebatch, strict adherence to the production process used for thebatch tested in clinical trials is necessary. The reference vaccineis preferably stabilised, by a method that has been shown tohave no significant effect on the assay procedure when thestabilised and non-stabilised batches are compared.

CHARACTERISATION OF COMPONENTS

During development of the vaccine, the production processshall be validated to demonstrate that it yields consistentlyan antigenic fraction that complies with the followingrequirements; after demonstration of consistency, the testsneed not be applied routinely to each batch.

Adenylate cyclase. Not more than 500 ng in the equivalent of1 dose of the final vaccine, determined by immunoblot analysisor another suitable method.

Tracheal cytotoxin. Not more than 2 pmol in the equivalent of1 dose of the final vaccine, determined by a suitable methodsuch as a biological assay or liquid chromatography (2.4.14).

Absence of residual dermonecrotic toxin. Inject intradermallyinto each of 3 unweaned mice, in a volume of 0.1 ml, the amountof antigenic fraction equivalent to 1 dose of the final vaccine.Observe for 48 hours. No dermonecrotic reaction isdemonstrable.Specific properties. The antigenic fraction is analyzed by oneor more of the methods shown below in order to determine theidentity and specific properties (activity per unit amount ofprotein) of its components in comparison with referencepreparations.

Pertussis toxin

Chinese hamster ovary (CHO) cell-clustering effect andhaemagglutination as in vitro methods; lymphocytosis-promoting activity, histamine-sensitising activity and insulinsecretory activity as in vivo methods. The toxin showsADP-ribosyl transferase activity using transducin as theacceptor.

Filamentous haemagglutinin

Haemagglutination and inhibition by specific antibody.

Pertactin, fimbrial-2 and fimbrial-3 antigens. Reactivity withspecific antibody.

Pertussis toxoid

The toxoid induces in animals the production of antibodiescapable of inhibiting all the properties of pertussis toxin.

PURIFIED ANTIGENIC FRACTION

Production of the antigenic fraction is based on a seed-lotsystem. The seed cultures are managed to conserve or, wherenecessary, restore toxinogenicity by deliberate selection.

None of the media used at any stage contains blood or bloodproducts of human origin. Media used for the preparation ofseed batches and inocula may contain blood or blood productsof animal origin.

The antigenic fraction is purified and, after appropriatecharacterisation, detoxified using suitable reagents by amethod that ensures minimal reversion of toxoid to toxin,particularly on or exposure to heat. The purification procedureis validated to demonstrate appropriate clearance ofsubstances used during culture or purification.

The content of bacterial endotoxins is determined to monitorthe purification procedure and to limit the amount in the finalvaccine. The limits applied are such that the final vaccinecontains not more than 100 IU per single human dose.

Before detoxification, the purity of the antigenic fraction is

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determined by a suitable method such as polyacrylamide gelelectrophoresis (PAGE) (2.4.12) or liquid chromatography(2.4.14). SDS-PAGE or immunoblot analysis with specificmonoclonal or polyclonal antibodies may be used tocharacterise subunits. Requirements are established for eachindividual product.

Only a purified antigenic fraction that complies with thefollowing requirements may be used in the preparation of thefinal bulk vaccine.

Sterility (2.2.11). Carry out the test for sterility using for eachmedium a quantity of purified antigenic fraction equivalent tonot less than 100 doses of the final vaccine.

Test for residual pertussis toxin. Use 3 groups of not fewerthan 5 histamine-sensitive mice each weighing between 18and 26 g. Using phosphate-buffered saline containing 0.2 percent of gelatin, prepare a series of dilutions of the purifiedantigenic fraction that have been shown to yield a gradedresponse and attribute each dilution to a separate group ofmice. Inject intraperitoneally into each mouse the dilutionattributed to its group. Inject diluent into a fourth group ofcontrol mice. After 5 days, inject intraperitoneally into eachmouse 1 mg of histamine base in a volume not exceeding 0.5ml. Record the number of animals that die within 24 h ofhistamine challenge. Calculate the weight or volume of apreparation that sensitises 50.0 per cent of the mice injectedusing a suitable statistical method such as probit analysis.The residual activity of pertussis toxin does not exceed thatof batches shown to be safe in clinical studies.

The histamine sensitivity of the strain of mice used is verifiedat suitable intervals as follows: inject threefold dilutions of areference pertussis toxin preparation in phosphate-bufferedsaline solution containing 0.2 per cent w/v of gelatin andchallenge with histamine as described above; the strain issuitable if more than 50 per cent of the animals are sensitisedby 50 ng of pertussis toxin and none of the control animalsinjected with only diluent and challenged similarly withhistamine show symptoms of sensitisation.

A validated test based on the clustering effect of the toxin forChinese hamster ovary (CHO) cells may be used instead ofthe test on mice.

Residual detoxifying agents and other reagents. The contentof residual detoxifying agents and other reagents is determinedand shown to be below approved limits unless validation ofthe process has demonstrated acceptable clearance.

Antigen content. Determine the complete quantitative antigencomposition of the antigenic fraction by suitableimmunochemical methods (2.2.14) and protein nitrogen bysulphuric acid digestion or another suitable method. The ratioof total antigen content to protein nitrogen is within the limitsestablished for the product.

FINAL BULK VACCINE

The vaccine is prepared by adsorption of a suitable quantityof the antigenic fraction onto aluminium hydroxide or hydratedaluminium phosphate. A suitable antimicrobial preservativemay be added.




FINAL LOT

Only a final lot that is satisfactory with respect to each of therequirements given below under Identification, Tests andAssay may be released for use.

Provided that the tests for residual pertussis toxin, reversibilityof toxoid, antimicrobial preservative, free formaldehyde andthe assay have been carried out with satisfactory results onthe final bulk vaccine, these tests may be omitted on the finallot.

Identification

Subject the vaccine to a suitable desorption procedure suchas the following: dissolve in the vaccine under examinationsufficient sodium citrate to give a 10 per cent w/v solution;maintain at 37° for about 16 h and centrifuge until a clearsupernatant is obtained. Examine by a suitable immunochemicalmethod (2.2.14), the clear supernatant reacts with specificantisera to the components in the vaccine.

Tests

Test for residual pertussis toxin. Use 3 groups of not fewerthan 5 histamine-sensitive mice (see under Production) eachweighing between 18 and 26 g. Using phosphate-bufferedsaline containing 0.2 per cent w/v of gelatin, prepare a seriesof dilutions of the vaccine under examination that have beenshown to yield a graded response and attribute each dilutionto a separate group of mice. Inject intraperitoneally into eachmouse the dilution attributed to its group. Inject diluent intoa fourth group of control mice. After 5 days, injectintraperitoneally into each mouse 1 mg of histamine base in avolume not exceeding 0.5 ml. Note the number of animals thatdie within 24 h of histamine challenge. Calculate the weight orvolume of a preparation that sensitises 50 per cent of the miceinjected using a suitable statistical method such as probitanalysis. The residual activity of pertussis toxin does notexceed that of batches shown to be safe in clinical studies.

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Reversibility of toxoid. Carry out the test for residual pertussistoxin described above using the vaccine incubated at 37° for4 weeks in parallel with a sample stored at 2° to 8°. The degreeof reversibility does not exceed that of batches shown to besafe in clinical studies.





Assay


Labelling. The label states (1) the names and amounts of theantigenic components present in the vaccine; (2) the maximumamount of residual pertussis toxin present in the vaccine; (3)the maximum degree of reversion of toxoid to toxin during theperiod of validity; (4) the name and amount of the adsorbent;(5) that the vaccine must be shaken before use; (6) that thevaccine is not to be frozen.

Bacillus Calmette-Guerin Vaccine(Freeze-Dried)Freeze-dried BCG Vaccine is a preparation of live bacteriaderived from a culture of the bacillus of Calmette and Guérin(Mycobacterium bovis BCG) capacity of which to protectagainst tuberculosis has been established.

Vaccine complies with the requirements stated under Vaccineswith the following modifications.


The production method is validated to demonstrate that theproduct, if tested, would comply with the tests for safety andefficacy.

BCG vaccine shall be produced by a staff consisting of healthypersons who do not work with other infectious agents; inparticular they shall not work with virulent strains ofMycobacterium tuberculosis, during the course of productioncycle nor shall they be exposed to a known risk of tuberculosisinfection. BCG vaccine is susceptible to sunlight: theprocedures for the preparation of the vaccine shall be so

designed that all cultures and vaccines are protected fromdirect sunlight and from ultraviolet light at all stages ofmanufacture, testing and storage.

Production of the vaccine is based on a seed-lot system. Theproduction method shall have been shown to yieldconsistently BCG vaccines that induce adequate sensitivityto tuberculin in man, that have acceptable protective potencyin animals and are safe. The vaccine is prepared from cultureswhich are derived from the master seed lot by as fewsubcultures as possible and in any case not more than 12subcultures e.g. If the secondary seed lot is 4 culture passagesremoved from the primary seed lot, the no. of passages fromthe secondary seed lot must not exceed 8.

The capacity of the working seed lot to induce sensitivity totuberculin in guinea-pigs is demonstrated.

If a bioluminescence test or other biochemical method is usedinstead of viable count, the method is validated against theviable count for each stage of the process at which it is used.

SEED LOT

The strain used to establish the master seed lot is chosen forand maintained to preserve its stability, its capacity to sensitiseman and guinea-pigs to tuberculin and to protect animalsagainst tuberculosis, and its relative absence of pathogenicityfor man and laboratory animals. The strain used shall beidentified by historical records that include information on itsorigin and subsequent manipulation.

A suitable batch of vaccine is prepared from the first workingseed lot and is reserved for use as the comparison/ in-housereference vaccine. When a new working seed lot isestablished, a suitable test for delayed hypersensitivity inguinea-pigs is carried out on a batch of vaccine prepared fromthe new working seed lot; the vaccine is shown to be notsignificantly different in activity from the comparison vaccine.

Only a working seed lot that complies with the followingrequirements may be used for propagation.

IdentificationThe bacteria in the working seed lot are identified asMycobacterium bovis BCG using microbiological techniques,which may be supplemented by molecular biology techniques(for example, nucleic acid amplification and restriction-fragment-length polymorphism).

Sterility (2.2.11). Complies with the test for sterility, carriedout using 10 ml for each medium. The working seed lotcomplies with the test for sterility except for the presence ofmycobacteria.

Virulent mycobacteria

Examine the working seed lot as prescribed under Tests, using10 guinea pigs.

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PROPAGATION AND HARVEST

The bacteria are grown in a suitable medium for not more than21 days by surface or submerged culture. The culture mediumshall contain no substances known to cause toxic or allergicreactions in human beings or to cause the bacteria to becomevirulent for guinea-pigs. The culture is harvested andsuspended in a sterile liquid medium that protects the viabilityof the vaccine as determined by a suitable method of viablecount.

Test for purity. Purity is checked by acid fast staining.

FINAL BULK VACCINE

The final bulk vaccine is prepared from a single harvest or bypooling a number of single harvests. A stabiliser may beadded; if the stabiliser interferes with the determination ofbacterial concentration on the final bulk vaccine, thedetermination is carried out before addition of the stabiliser.

Only final bulk vaccine that complies with the followingrequirements may be used in the preparation of the final lot.

Virulent mycobacteria. Examine as prescribed under Tests.

Sterility (2.2.11). Complies with the test for sterility using 10ml for each medium except for the presence of mycobacteria.

Count of viable units

Determine the number of viable units per ml by viable counton solid medium using a method suitable for the vaccine underexamination or by determination of adenosine triphosphateby a bioluminescence reaction. Carry out the test in parallelon a reference preparation of the same strain.

Bacterial concentration

Determine the total bacterial concentration by a suitablemethod, either directly by determining the mass of the micro-organisms, or indirectly by an opacity method that has beencalibrated in relation to the mass of the organisms; if thebacterial concentration is determined before addition of astabiliser, the concentration in the final bulk vaccine isestablished by calculation. The total bacterial concentrationis within the limits approved for the particular product byNational Regulatory Authority.

The ratio of the count of viable units to the total bacterialconcentration is not less than that approved for the particularproduct by National Regulatory Authority.

FINAL LOT

The final bulk vaccine is distributed into sterile containersand freeze-dried to a moisture content favourable to thestability of the vaccine; the containers are closed either undervacuum or under a gas that is not deleterious to the vaccine.

Except where the filled and closed containers are stored at atemperature of -20° or lower, the expiry date is not later than 4years from the date of harvest.

Only a final lot that complies with the following requirementfor count of viable units and with each of the requirementsgiven below under Identification, Tests and Assay may bereleased for use. Provided the test for virulent mycobacteriahas been carried out with satisfactory results on the final bulkvaccine, it may be omitted on the final lot. Provided the testfor excessive dermal reactivity has been carried out withsatisfactory results on the working seed lot and on 5consecutive final lots produced from it, the test may be omittedon the final lot.

Count of viable units

Determine the number of viable units per ml of thereconstituted vaccine by viable count on solid medium usinga method suitable for the vaccine under examination or bydetermination of adenosine triphosphate by a bioluminescencereaction. The ratio of the count of viable units after freeze-drying to that before is not less than that approved for theparticular product.

IdentificationBCG vaccine is identified by microscopic examination of thebacilli in stained smears demonstrating their acid-fast propertyand by the characteristic appearance of colonies grown onsolid medium. Alternatively, molecular biology techniques (likenucleic acid amplification) may be used.

Tests

Virulent mycobacteria

If this test is satisfactory at final bulk stage it can be omitted atthe final lot.

Inject subcutaneously or intramuscularly into each of 6 guinea-pigs, each weighing between 250 and 400 g and having receivedno treatment likely to interfere with the test, a quantity ofvaccine equivalent to at least 50 human doses. Observe theanimals for at least 42 days. At the end of this period, kill theguinea-pigs and examine by autopsy for signs of infectionwith tuberculosis, ignoring any minor reactions at the site ofinjection. Animals that die during the observation period arealso examined for signs of tuberculosis. The vaccine complieswith the test if none of the guinea-pigs shows signs oftuberculosis and if not more than one animal dies during theobservation period. If 2 animals die during this period andautopsy does not reveal signs of tuberculosis repeat the teston 6 other guinea-pigs. The vaccine complies with the test ifnot more than one animal dies during the 42 days followingthe injection and autopsy does not reveal any sign oftuberculosis.

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Sterility (2.2.11). The reconstituted vaccine complies with thetest for sterility except for the presence of mycobacteria.

Excessive dermal reactivity

Use 6 healthy white or pale-coloured guinea-pigs, eachweighing not less than 250 g and having received no treatmentlikely to interfere with the test. Inject intradermally into eachguinea-pig, according to a randomised plan, 0.1 ml of thereconstituted vaccine and of 2 tenfold serial dilutions of thevaccine and identical doses of the comparison vaccine.Observe the lesions formed at the site of the injection for 4weeks. The vaccine complies with the test if the reaction itproduces is not markedly different from that produced by thecomparison vaccine.

Temperature stability

Maintain samples of the freeze-dried vaccine at 37° for 4 weeks.Determine the number of viable units in the heated vaccineand in unheated vaccine as described below. The number ofviable units in the heated vaccine is not less than 20.0 per centof that in unheated vaccine.

Water (2.3.43). Not more than 3.0 per cent, determined by thesemi-micro determination of water.

Assay

Determine the number of viable units in the reconstitutedvaccine by viable count on solid medium or using a suitablevalidated biochemical method for the vaccine underexamination. The number is within the range stated on thelabel. Determine the number of viable units in the comparisonvaccine in parallel.

Labelling. The label states (1) the minimum and maximumnumber of viable units per ml in the reconstituted vaccine; (2)that the vaccine must be protected from direct sunlight; (3)that the vaccine is to be used immediately after broaching thecontainer; (4) the age group for which the vaccine is intended;(5) the dose for each age group; (6) follow instructions asmentioned in the product insert/leaflet.

Diphtheria and Tetanus Vaccine(Adsorbed)Diphtheria and Tetanus Vaccine (Adsorbed) is a preparationof diphtheria formol toxoid and tetanus formol toxoid adsorbedon mineral carrier. The formol toxoids are prepared from thetoxins produced by the growth of Corynebacteriumdiphtheriae and Clostridium tetani, respectively.

The specification for individual component used in formulationis referred in the text of individual monograph.

Production

General provisions

Bulk purified diphtheria and tetanus toxoids

The bulk purified diphtheria and tetanus toxoids are preparedas described in the monographs on Diphtheria vaccine(adsorbed) and Tetanus vaccine (adsorbed) and comply withthe requirements prescribed therein.

Sterility (2.2.11). Carry out test for sterility using 10 ml ofbulk for each sterility medium.Absence of toxin and irreversibility of toxoid

Inject subcutaneously into each of 5 guinea-pigs at least 500 Lfof the non-incubated bulk purified toxoid in a volume of 1 ml,using the same buffer solution as for the final vaccine, withoutadsorbent. Animals that die shall be autopsied and examinedfor symptoms of diphtheria intoxication (red adrenals). Thebulk purified toxoid shall pass the test if no guinea-pig showssymptoms of specific intoxication within six weeks of injectionand if at least 80 per cent of the animals survive the test period.The guinea-pigs shall not have been used previously forexperimental purposes.

Alternatively, a cell-culture test system may be used; in thiscase, the sensitivity of the test shall have been demonstratedto be not less than that of the guinea-pig test, and the testprocedures shall be approved by the National RegulatoryAuthority.

Each bulk purified toxoid shall be tested to ensure thatreversion to toxicity cannot take place on storage. The bulkpurified toxoid shall be diluted in order to obtain the sameconcentration and chemical environment as that present inthe final bulk vaccine, except for the presence of adjuvant.

To determine whether reversion has occurred, diluted toxoidsthat have been stored at 37° for six weeks shall be tested. Thetest employed shall be approved by the National RegulatoryAuthority and should be sufficiently sensitive to detect verysmall amounts of toxin. No toxicity shall be detected.

Intradermal tests in guinea-pigs and cell-culture tests bothare considered to be suitable.

Antigenic purity

Not less than 1500 Lf per mg of protein nitrogen for diphtheriatoxoid and not less than 1000 Lf/mg of protein nitrogen fortetanus toxoid.

FINAL BULK VACICNE

The final bulk vaccine is prepared by adsorption of suitablequantities of bulk purified diphtheria toxoid and tetanus toxoidonto mineral carrier such as hydrated aluminium phosphate,

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aluminium hydroxide; the resulting mixture is approximatelyisotonic with blood. Suitable antimicrobial preservatives maybe added. Antimicrobial preservatives of the phenolic typemust not be used.


IdentificationA. Dissolve sufficient sodium citrate in the vaccine underexamination to give a 10 per cent w/v concentration. Maintainat 37o for about 16 hours and centrifuge. The clear supernatantreacts with a suitable diphtheria antitoxin and yields aprecipitate.

B. The clear supernatant obtained in test A reacts with a suitabletetanus antitoxin and yields a precipitate.

pH (2.4.24). 6.0 to 7.0.

Specific toxicity. Use 5 normal, healthy guinea-pigs weighingbetween 250 and 350 g which have been maintained for atleast 1 week on a uniform, unrestricted diet, and have notbeen previously treated with any material that will interferewith the test. Weigh the animals separately and record theirweights. Inject subcutaneously into each animal 5 times thedose stated on the label. Weigh all the animals at weeklyintervals for 6 weeks. None of the animals shows anysymptoms of diphtheria or tetanus toxaemia or dies fromdiphtheria within 42 days or loses weight at the end of thetest. If more than one animal dies from non-specific causes orloses weight, repeat the test. If an animal dies or loses weightin the second test, the vaccine fails the test.

Assay

Diphtheria toxoidComplies with the test as stated under Diphtheria Vaccine(Adsorbed).

Tetanus toxoidComplies with the test as stated under Tetanus Vaccine(Adsorbed).Antimicrobial preservative. Where applicable, determine theamount of antimicrobial preservative by a suitable chemicalmethod. The amount is not less than 85.0 per cent and notgreater than 115.0 per cent of the intended amount.Free formaldehyde (2.3.20). Maximum 0.2 g/lSterility (2.2.11). Carry out test for sterility using 10 ml ofbulk for each sterility medium.

FINAL LOT

The final bulk vaccine is filled and stored aseptically intosterile containers. The containers are closed so as to preventcontamination.

Only a final lot that is satisfactory with respect to each of therequirements given below under Identification, Tests andAssay may be released for use. Provided the tests for specifictoxicity, free formaldehyde and antimicrobial preservative andthe assay have been carried out with satisfactory results onthe final bulk vaccine, they may be omitted on the final lot.

IdentificationA. Diphtheria toxoid is identified by a suitable immuno-chemical method (2.2.14).Dissolve in the vaccine under examination by adding sufficientsodium citrate to give a 10 per cent w/v solution. Maintain at37° for about 16 hours and centrifuge until a clear supernatantliquid is obtained. The clear supernatant reacts with a suitablediphtheria antitoxin, giving a precipitate or visible floccules.B. Tetanus toxoid is identified by a suitable immuno-chemicalmethod (2.2.14).

The clear supernatant liquid obtained during test A reactswith a suitable tetanus antitoxin, giving a precipitate or visiblefloccules.

Tests


Abnormal toxicity (2.2.1). Complies with the test for abnormaltoxicity

Aluminium (2.3.9.). Not more than 1.25 mg per single humandose when hydrated aluminium phosphate or aluminiumhydroxide is used as the adsorbent.

pH (2.4.24). The pH of the vaccine is within the range approvedfor the product (6.0 to 7.0).


Antimicrobial preservative. Where applicable, determine theamount of antimicrobial preservative by a suitable chemicalmethod. The content is not less than 85.0 per cent and notgreater than 115.0 per cent of the quantity stated on the label.

Assay


Carry out one of the described methods for the assay ofDiphtheria Vaccine (Adsorbed).

Viz a) Intradermal challenge method, b) Lethal challengemethod, c) Antibody induction method, d) Validated serologicalassay in guinea pigs or mice as approved by NationalRegulatory Authority.

Tetanus component

Carry out one of the described methods for the assay ofTetanus Vaccine (Adsorbed) Viz a) Antibody induction

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method; b) Challenge method in guinea pigs/mice; c) Validatedserological assay in guinea pigs or mice as approved byNational Regulatory Authority.

Labelling. The label states (1) the human dose; (2) the minimumLf units per single human dose or the minimum InternationalUnits per single human dose if potency test done by challengemethod; (3) the name and the amount of the adsorbent andpreservative; (4) that the vaccine must be shaken before use;(5) that the vaccine is not to be frozen.

Diphtheria and Tetanus Vaccine(Adsorbed) for Adults and AdolescentsDiphtheria and Tetanus Vaccine (Adsorbed) for Adults andAdolescents is a preparation of diphtheria formol toxoid andtetanus formol toxoid adsorbed on a mineral carrier. The formoltoxoids are prepared from the toxins produced by the growthof Corynebacterium diphtheriae and Clostridium tetani,respectively.

Production

General provisions

Bulk purified diphtheria and tetanus toxoids

The bulk purified diphtheria and tetanus toxoids are preparedas described in the monographs on Diphtheria vaccine(adsorbed) and Tetanus vaccine (adsorbed) and comply withthe requirements prescribed therein.

FINAL BULK VACCINE

The vaccine is prepared by adsorption of suitable quantitiesof bulk purified diphtheria toxoid and tetanus toxoid onto amineral carrier such as hydrated aluminium phosphate oraluminium hydroxide. Suitable antimicrobial preservatives maybe added. Certain antimicrobial preservatives, particularlythose of the phenolic type, adversely affect the antigenicactivity and must not be used.Only final bulk vaccine that complies with the followingrequirements may be used in the preparation of the final lot.

Identification

A. Dissolve sufficient sodium citrate in the vaccine underexamination to give a 10 per cent w/v concentration. Maintainat 37o for about 16 hours and centrifuge. The clear supernatantreacts with a suitable diphtheria antitoxin and yields aprecipitate.

B. The clear supernatant obtained in test A reacts with a suitabletetanus antitoxin and yields a precipitate.

pH (2.4.24 ). 6.0 to 7.0.

Specific toxicity

Inject subcutaneously 5 times the single human dose statedon the label into each of 5 healthy guinea-pigs, each weighingbetween 250 and 350 g, that have not previously been treatedwith any material that will interfere with the test. If within 42days of the injection any of the animals shows signs of or diesfrom diphtheria toxaemia or tetanus, the vaccine does notcomply with the test. If more than 1 animal dies from non-specific causes, repeat the test once; if more than 1 animaldies in the second test, the vaccine does not comply with thetest.

Antimicrobial preservative. Where applicable, determine theamount of antimicrobial preservative by a suitablephysicochemical method. The amount is not less than 85.0per cent and not greater than 115.0 per cent of the intendedamount.


Sterility (2.2.11). Carry out the test for sterility using 10 ml foreach medium.

FINAL LOT

The final bulk vaccine is distributed aseptically into sterile,tamper-proof containers. The containers are closed so as toprevent contamination.

Only a final lot that is satisfactory with respect to each of therequirements given below under Identification, Tests andAssay may be released for use. Provided the tests for freeformaldehyde and antimicrobial preservative and the assayhave been carried out with satisfactory results on the finalbulk vaccine, they may be omitted on the final lot.

Identification

A. Diphtheria toxoid is identified by a suitable immunochemicalmethod (2.2.14). The following method, applicable to certainvaccines, is given as an example. Dissolve in the vaccine underexamination sufficient sodium citrate to give a 10 per centw/v solution. Maintain at 37° for about 16 hours and centrifugeuntil a clear supernatant is obtained. The clear supernatantreacts with a suitable diphtheria antitoxin, giving a precipitate.If a satisfactory result is not obtained with a vaccine adsorbedon aluminium hydroxide, carry out the test as follows.

Centrifuge 15 ml of the vaccine under examination and suspendthe residue in 5 ml of a freshly prepared mixture of 1 volume ofa 56 g/l solution of sodium edetate and 49 volumes of a90 g/l solution of disodium hydrogen phosphate. Maintain at37° for not less than 6 hours and centrifuge. The clearsupernatant reacts with a suitable diphtheria antitoxin, givinga precipitate.

B. Tetanus toxoid is identified by a suitable immunochemicalmethod (2.2.14). The following method, applicable to certain

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vaccines, is given as an example. The clear supernatantobtained during identification test A reacts with a suitabletetanus antitoxin, giving a precipitate.

TestsAluminium (2.3.9). Maximum 1.25 mg per single human dose.


Antimicrobial preservative. Where applicable, determine theamount of antimicrobial preservative by a suitablephysicochemical method. The amount is not less than 85.0per cent and not greater than 115.0 per cent of the intendedamount.


pH (2.4.24). 6.0 to 7.0.

Abnormal toxicity (2.2.1). Complies with the test for abnormaltoxicity.

Assay


Carry out the prescribed method for assay of DiphtheriaVaccine by lethal challenge method described in the assay ofDiphtheria Vaccine (Adsorbed).


Tetanus component



Labelling. The label states (1) the human dose; (2) the minimumnumber of International Units of each component per singlehuman dose, if potency determined by challenge method orthe minimum Lf units per single human dose if test done byantibody induction method; (3) the name and the amount ofthe adsorbent; (4) that the vaccine must be shaken beforeuse; (5) that the vaccine is not to be frozen.

Diphtheria, Tetanus and PertussisVaccine (Adsorbed)Diphtheria, Tetanus and Pertussis Vaccine (Adsorbed) is apreparation of diphtheria formol toxoid, tetanus formol toxoidadsorbed on mineral carrier and a suspension of killedBordetella pertussis organisms. The formal toxoids areprepared from the toxins produced by the growth ofCorynebacterium diphtheriae and Clostridium tetani,

respectively. The Bordetella pertussis suspension is preparedby growth of suitable strains in an appropriate medium, undercontrolled conditions.

The specification for individual component used in formulationis referred in the text of individual monograph.

Production

General provisions

The production method must be validated to demonstratethat the product if tested, would comply with the tests forsafety as described under monographs of Diphtheria Vaccine,Tetanus Vaccine (Adsorbed) and Pertussis Vaccine.

Bulk purified diphtheria and tetanus toxoids, bulk inactivatedB. pertussis suspension

The bulk purified diphtheria and tetanus toxoids andinactivated B. pertussis suspension are prepared as describedin the monograph on Diphtheria Vaccine (Adsorbed), TetanusVaccine (Adsorbed) and Pertussis Vaccine respectively andcomply with the respective requirements.

FINAL BULK VACCINE

The final bulk vaccine is prepared by adsorption of suitablequantities of bulk purified diphtheria toxoid and tetanus toxoidonto hydrated aluminium phosphate or aluminium hydroxideand admixture of an appropriate quantity of a suspension ofinactivated B. pertussis. The B. pertussis concentration of thefinal bulk vaccine does not exceed that corresponding to anopacity of 20 I.U. per single human dose. If two or more strainsof B. pertussis are used, the composition of consecutive lotsof the final bulk vaccine shall be consistent with respect tothe proportion of each strain as measured in opacity units.Suitable antimicrobial preservatives may be added to the bulkvaccine. Antimicrobial preservatives particularly those ofphenolic type which affect the antigenic activity must not beused.


Antimicrobial preservative. Where applicable, determine theamount of antimicrobial preservative by a suitable chemicalmethod. The content is not less than 85.0 percent and notmore than115.0 percent of the intended amount.

Sterility (2.2.11). Carry out test for sterility using 10 ml ofbulk for each sterility medium.

FINAL LOT

The final bulk vaccine is filled and stored aseptically intosterile containers. The containers are closed so as to preventcontamination.

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Only a final lot that is satisfactory with respect to each of therequirements given below under Identification, Tests andAssay may be released for use. Provided the tests for specifictoxicity of diphtheria, tetanus and pertussis components, freeformaldehyde, antimicrobial preservative and the Assay havebeen carried out with satisfactory results on the final bulkvaccine, they may be omitted on the final lot.

Identification

A. Diphtheria toxoid is identified by a suitable immuno-chemical method (2.2.14).

Dissolve in the vaccine under examination by adding sufficientsodium citrate to give a 10 per cent w/v solution. Maintain at37° for about 16 hours and centrifuge until a clear supernatantis obtained; reserve the precipitate for identification test C.The clear supernatant reacts with a suitable diphtheriaantitoxin, giving a precipitate.

B. Tetanus toxoid is identified by a suitable immuno-chemicalmethod (2.2.14).

The clear supernatant obtained during identification test Areacts with a suitable tetanus antitoxin, giving a precipitate.

C. The pertussis component is identified by agglutination ofthe bacteria from the resuspended centrifugation residue (seeidentification test A; other suitable methods for separatingthe bacteria from the adsorbent may also be used) by antiseraspecific to B. pertussis or by the assay of the pertussiscomponent.

TestsSterility (2.2.11). Complies with the test for sterility.

Abnormal toxicity (2.2.1). Each final lot shall be tested forabnormal toxicity by injecting intraperitoneally one humandose , but not more than 0.25 ml into each of the five miceweighing between 17 to 22 g and at least one human dose butnot more than 1.0 ml into each of the two guinea pigs weighingbetween 250 and 350 g. The preparation passes the test ifnone of the animals dies or shows signs of ill health in 7 daysfollowing the injection. If one of the animal dies or shows thesigns of ill health, repeat the test. The preparation passes thetest if none of the animals in the second group dies or showssigns of ill health in the time interval specified.

Specific toxicity

Diphtheria and tetanus components. Inject subcutaneouslyfive times the single human dose stated on the label into eachof five healthy guinea-pigs, each weighing between 250 and350 g, that have not previously been treated with any materialthat will interfere with the test. The animals should be weightedevery week and observations be made. If within 42 days of theinjection any of the animals shows signs of or dies from

diphtheria toxemia or tetanus, the vaccine does not complywith the test. If more than one animal dies from non-specificcauses, repeat the test once; if more than one animal dies inthe second test, the vaccine does not comply with the test.

Pertussis component. Use not less than 10 healthy mice eachweighing between 14 and 16 g for the vaccine group and forthe saline control. Use mice of the same sex or distribute malesand females equally between the groups. Allow the animalsaccess to food and water for at least 2 hours before injectionand during the test. Inject each mouse of the vaccine groupintraperitoneally with 0.5 ml, containing a quantity of thevaccine equivalent to not less than half the single humandose. Inject each mouse of the control group with 0.5 ml of a0.9 per cent sterile solution of sodium chloride, preferablycontaining the same amount of antimicrobial preservative asthat injected with the vaccine. Weigh the mice groupsimmediately before the injection and 72 hours and 7 days afterthe injection. The vaccine complies with the test if: (a) at theend of 72 hours the total mass of the group of vaccinated miceis not less than that preceding the injection; (b) at the end of7 days the average increase in mass per vaccinated mouse isnot less than 60 per cent of that per control mouse; and (c) notmore than 5 per cent of vaccinated mice should die during thetest. The test may be repeated and the results of the testscombined.

Aluminium (2.3.9). Not more than 1.25 mg per single humandose, when hydrated aluminium phosphate or aluminiumhydroxide is used as the adsorbent.

pH (2.4.24). 6.0 to 7.0.


Antimicrobial preservative (2.2.2 ) Where applicable,determine the amount of antimicrobial preservative by asuitable chemical method. The content is not less than 85.0per cent and not more than115.0 per cent of the intendedamount.

Assay


Carry out one of the methods for the assay as stated underDiphtheria Vaccine (Adsorbed).

Tetanus component

Carry out one of the methods for the assay as stated underTetanus Vaccine (Adsorbed).

Pertussis component


Labelling. The label states (1) in case done by challengemethod the minimum number of International Units; if units(as applicable for each component) per single human dose;

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(2) In case done by antibody induction method the minimumnumber of International Units per single human dose ofpertussis component and minimum number of Lf of diphtheriatoxoid and tetanus toxoid; (3) the name and the amount of theadsorbent and preservative; (4) that the vaccine must beshaken before use; (5) that the vaccine is not to be frozen; (6)for vaccine contained in single-dose containers where thespace is too small to accommodate the full name of the vaccine,the abbreviation ‘DTP’ may be used in the label and thecontainer provided that the same code is also stated in thelabel on the package.

Diphtheria, Tetanus, Pertussis (WholeCell), Hepatitis B (rDNA) andHaemophilus Type b ConjugateVaccine (Adsorbed)Diphtheria, Tetanus, Pertussis (Whole cell), Hepatitis B (rDNA)and Haemophilus Type b Conjugate Vaccine (Adsorbed) is acombined vaccine composed of diphtheria formol toxoidcontaining not less than 1,500 Lf, (2.2.16) per mg of proteinnitrogen, purified tetanus formol toxoid containing not lessthan 1,000 Lf, (2.2.16), per mg of protein nitrogen, hepatitis Bsurface antigen and haemophilus type b conjugated to suitableprotein with a mineral adsorbent to which a suspension ofkilled Bordetella pertussis has been added. Mineral adsorbentis a suspension of hydrated aluminium hydroxide, aluminiumphosphate or calcium phosphate, in saline solution or otherappropriate solution isotonic with blood.

The formol toxoids are prepared from the toxin produced bythe growth of Corynebacterium diphtheriae and Clostridiumtetani, respectively, in suitable media. The toxins are convertedto toxoids by treatment with formaldehyde solution bymethods which avoid reversibility of the toxoids.


The polysaccharide, polyribosyl ribitol phosphate, PRP is alinear copolymer composed of repeated units of 3-β-D-ribofuranosyl-(1→1)-ribitol-5-phosphate [(C10H19O12P)n], witha defined molecular size and derived from a suitable strain ofHaemophilus influenzae type b. The carrier protein, whenconjugated to PRP, is capable of inducing a T-cell dependentB-cell immune response to the polysaccharide.

The product may be presented with the haemophiluscomponent in a separate container, the contents of which aremixed with the other components immediately before or duringuse.

The final product contains a suitable antimicrobialpreservative. The antigenic properties of the vaccine areadversely affected by the presence of certain antimicrobialpreservatives particularly those of the phenolic type and someof the quaternary ammonium type and must not be used.

Production

General provisions


If the vaccine is presented with the haemophilus componentin a separate vial, as part of consistency studies the assays ofthe diphtheria, tetanus, pertussis and hepatitis B are carriedout on a suitable number of batches of vaccine reconstitutedas for use. For subsequent routine control, the assays of thesecomponents may be carried out without mixing with thehaemophilus component.

The production method is validated to demonstrate that theproduct, if tested, would comply with the following test forspecific toxicity of the diphtheria and tetanus component:inject subcutaneously 5 times the single human dose statedon the label into each of 5 healthy guinea pigs, each weighingbetween 250 and 350 g, that have not previously been treatedwith any material that will interfere with the test. If within 42days of the injection any of the animals shows signs of or diesfrom diphtheria, toxemia or tetanus, the vaccine does notcomply with the test. If more than 1 animal dies from non-specific causes, repeat the test once; if more than 1 animalshows signs of or dies in the second test, the vaccine doesnot comply with the test.

The stability of the final lot and the relevant intermediates isevaluated using one or more indicator tests. For thehaemophilus component, such tests may include determinationof molecular size, determination of free PRP in the conjugateand kinetics of depolymerisation. Taking account of the resultsof the stability testing, release requirements are set for theseindicator tests to ensure that the vaccine will be satisfactoryat the end of the period of validity.


Provided valid assays can be performed, monocomponentreference vaccines may be used for the assays on the combinedvaccine. If this is not possible because of interaction betweenthe components of the combined vaccine or because of thedifference in composition between monocomponent referencevaccine and the test vaccine, a batch of combined vaccineshown to be effective in clinical trials or a batch representativethereof is used as a reference vaccine. For the preparation ofa representative batch, strict adherence to the production

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process used for the batch tested in clinical trials is necessary.The reference vaccine may be stabilized by a method that hasbeen shown to have no effect on the assay procedure.


The production of the components complies with therequirements of the monographs on Diphtheria Vaccine(Adsorbed), Tetanus Vaccine (Adsorbed), Pertussis Vaccine,Hepatitis B Vaccine (rDNA) and Haemophilus Type bConjugate Vaccine.

FINAL BULK VACCINE

Vaccine with all components in the same container

The final bulk is prepared by adsorption, separately ortogether, of suitable quantities of bulk purified diphtheriatoxoid, bulk purified tetanus toxoid, bulk purified hepatitis Bsurface antigen onto a mineral carrier such as aluminiumhydroxide or hydrated aluminium phosphate, admixture of anappropriate quantity of a suspension of inactivatedB. pertussis component and admixture of a suitable quantityof PRP conjugate; the resulting mixture is approximatelyisotonic with blood. The B. pertussis concentration of thefinal bulk vaccine does not exceed that corresponding to anopacity of 20 IU per single human dose. If 2 or more strains ofB. pertussis are used, the composition of consecutive lots ofthe final bulk vaccine shall be consistent with respect to theproportion of each strain as measured in opacity units. Suitableantimicrobial preservatives may be added.

Vaccine with the haemophilus component in a separatecontainer

The final bulk is prepared by adsorption, separately ortogether, of suitable quantities of bulk purified diphtheriatoxoid, bulk purified tetanus toxoid, bulk purified hepatitis Bsurface antigen onto a mineral carrier such as aluminiumhydroxide or hydrated aluminium phosphate, admixture of anappropriate quantity of a suspension of inactivated B.pertussis component and admixture of a suitable quantity ofPRP conjugate; the resulting mixture is approximately isotonicwith blood. The B. pertussis concentration of the final bulkvaccine does not exceed that corresponding to an opacity of20 IU per single human dose. If 2 or more strains of B. pertussisare used, the composition of consecutive lots of the final bulkvaccine shall be consistent with respect to the proportion ofeach strain as measured in opacity units. The final bulk isfilled separately. Suitable antimicrobial preservatives may beadded. The final bulk of the haemophilus component isprepared by dilution of the bulk conjugate to the finalconcentration with a suitable diluent. A stabilizer may be added.


Antimicrobial preservative. Where applicable, determine theamount of antimicrobial preservative by a suitable chemicalmethod. The amount is not less than 85.0 per cent and notgreater than 115.0 per cent of the intended content.


FINAL LOT

Only a final lot that is satisfactory with respect to the test forosmolality and with respect to each of the requirements givenbelow under Identification, Tests and Assay may be releasedfor use. Provided the tests for specific toxicity of diphtheriatoxoid, tetanus toxoid and pertussis component andantimicrobial preservative and the assays for the diphtheria,tetanus and pertussis components have been carried out withsatisfactory results on the final bulk vaccine, they may beomitted on the final lot. Provided the content of freeformaldehyde has been determined on the bulk purifiedantigens or on the final bulk and it has been shown that thecontent in the final lot will not exceed 0.2 g/l, the test for freeformaldehyde may be omitted on the final lot. If an in vivoassay is used for the hepatitis B component, provided it hasbeen carried out with satisfactory results on the final bulkvaccine, it may be omitted on the final lot.

Free PRP

Unbound PRP is determined after removal of the conjugate,for example by anion exchange, size exclusion or hydrophobicchromatography (2.4.16), ultrafiltration or other validatedmethods. The amount of free PRP is not greater than thatapproved for the particular product.


pH (2.4.24). 6.0 to 7.0.

Description. Whitish turbid liquid in which the mineral carriertends to settle down slowly on keeping.

Identification

Tests A, B, C, D and E may be omitted if test F is carried out.Test F may be omitted if tests A, B, C, D and E are carried out.

A. Diphtheria toxoid. Dissolve sufficient sodium citrate inthe vaccine under examination to give a 10 per cent w/vconcentration. Maintain at 37o for about 16 hours andcentrifuge. Reserve the residue for test C. The clearsupernatant reacts with a suitable diphtheria antitoxin andyields a precipitate.

B. Tetanus toxoid. The clear supernatant obtained in test Areacts with a suitable tetanus antitoxin and yields a precipitate.

C. Pertussis component. To a suspension of the residue


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obtained in test A in saline solution add a suitable Bordetellapertussis antiserum; agglutination indicates presence ofpertussis component.

D. Hepatitis B surface antigen. The suspension of the residueobtained in test A gives a positive reactions when tested bysuitable in-vitro assay.

E. PRP. The suspension of the residue obtained in the test Agives a positive reaction when tested by a suitableimmunochemical method for PRP.

F. The vaccine confers an active immunity in mice and guinea-pigs when administered as directed in the test for Assay.

Tests

If the product is presented with the haemophilus componentin a separate container; the tests for specific toxicity ofdiphtheria toxoid, tetanus toxoid and pertussis component,aluminium, free formaldehyde, antimicrobial preservativeand sterility are carried out on the container with thediphtheria, tetanus, pertussis and hepatitis B components;the tests for PRP content, water (where applicable), sterilityand pyrogens are carried out on the container with thehaemophilus component.

If the haemophilus component is freeze-dried, some tests maybe carried out on the freeze-dried product rather than on thebulk conjugate where the freeze-drying process may affectthe component under test.

PRP. Not less than 80.0 per cent of the amount of PRP statedon the label. PRP is determined either by assay of ribose (2.7.1),or phosphorus (2.7.1), by an immunochemical method (2.2.14)or by anion exchange liquid chromatography with pulsedamperometric detection.

Aluminium (2.3.9). Not more than 1.25 mg per single humandose, if aluminium hydroxide or hydrated aluminium phosphateis used as the adsorbent.




Abnormal toxicity (2.2.1). Each final lot shall be tested forabnormal toxicity by injecting intraperitoneally one humandose, but not more than 0.25 ml into each of five mice weighingbetween 17 and 22 g and at least one human dose but notmore than 1.0 ml into each of two guinea pigs weighingbetween 250 and 350 g. The preparation passes the test ifnone of the animals dies or shows signs of ill health in sevendays following the injection. If one of the animals dies or

shows signs of ill health, repeat the test. The preparationpasses the test if none of the animals in the second group diesor shows signs of ill health in the time interval specified.

Pyrogens (2.2.8). This test is carried out for Haemophilusinfluenzae type b vaccine only if Haemophilus influenzae typeb vaccine is presented as separate lyophilized vial. The vaccinecomplies with the test for pyrogens. Inject per kg of the rabbit’smass a quantity of the vaccine equivalent to: 1 mg of PRP fora vaccine with diphtheria toxoid or CRM 197 diphtheria toxoidas carrier; 0.1 mg of PRP for a vaccine with tetanus toxoid ascarrier protein; 0.025 mg of PRP for vaccine with OMP ascarrier.

Specific toxicity

Diphtheria and tetanus components

Complies with the test as stated under Diphtheria and TetanusVaccine (Adsorbed).

Pertussis component

Complies with the test as stated under Diphtheria, Tetanusand Pertussis Vaccine (Adsorbed).

Assay

Diphtheria toxoid (adsorbed)


Tetanus toxoid (adsorbed)

Complies with the test as stated under assay of Tetanus Vaccine(Adsorbed).

Pertussis vaccine


Hepatitis B surface antigen (adsorbed)

Complies with the test as stated under Hepatitis B Vaccine(Adsorbed).

Storage. When stored under the prescribed conditions thevaccine may be expected to retain potency for not less than 2years from the date on which the potency test for the pertussiscomponent was started.

Labelling. The label states (1) the human dose; (2) Diphtheriaand Tetanus components; (a) in case done by challengemethod, the minimum number of International Units (asapplicable for each component) per single human dose; (b) incase done by antibody induction, the minimum Lf units persingle human dose; (3) pertussis component – IU or IOU persingle human dose; (4) hepatitis B component - mg HBsAgper single human dose; (5) haemophilus conjugate component


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- mg PRP per single human dose; (6) the type and nominalamount of carrier protein per single human dose; (7) the nameand amount of adsorbent and added preservative; (8) that thevaccine must be shaken before use; (9) that the vaccine is notto be frozen.

Diphtheria, Tetanus, Pertussis (WholeCell) and Hepatitis B (rDNA) Vaccine(Adsorbed)Diphtheria, Tetanus, Pertussis and Hepatitis B (rDNA) Vaccine(Adsorbed) is a combined vaccine composed of diphtheriaformol toxoid containing not less than 1,500 Limes flocculationis(Lf) (2.2.16), per mg of protein nitrogen, purified tetanus formoltoxoid containing not less than 1,000 Lf ( 2.2.16), per mg ofprotein nitrogen and hepatitis B surface antigen with a mineraladsorbent to which a suspension of killed Bordetella pertussishas been added. Mineral adsorbent is a suspension ofhydrated aluminium hydroxide, aluminium phosphate orcalcium phosphate in saline solution or other appropriatesolution isotonic with blood.

The formol toxoids are prepared from the toxin produced bythe growth of Corynebacterium diphtheriae and Clostridiumtetani, respectively, in suitable media. The toxins are convertedto toxoids by treatment with formaldehyde solution bymethods, which avoid reversibility of the toxoids.


The final product contains a suitable antimicrobialpreservative. The antigenic properties of the vaccine areadversely affected by the presence of certain antimicrobialpreservatives particularly those of the phenolic type and someof the quaternary ammonium type and must not be used.

Production

General provisions


The production method is validated to demonstrate that theproduct, if tested, would comply with the following test forspecific toxicity of the diphtheria and tetanus component:inject subcutaneously 5 times the single human dose statedon the label into each of 5 healthy guinea pigs, each weighingbetween 250 and 350 g, that have not previously been treatedwith any material that will interfere with the test. If within 42days of the injection any of the animals shows signs of or dies

from diphtheria, toxaemia or tetanus, the vaccine does notcomply with the test. If more than 1 animal dies from non-specific causes, repeat the test once; if more than 1 animalshows signs of or dies in the second test, the vaccine doesnot comply with the test.The stability of the final lot and the relevant intermediates isevaluated using one or more indicator tests. Taking accountof the results of the stability testing, release requirements areset for these indicator tests to ensure that the vaccine will besatisfactory at the end of the period of validity.


Provided valid assays can be performed, monocomponentreference vaccines may be used for the assays on the combinedvaccine. If this is not possible because of interaction betweenthe components of the combined vaccine or because of thedifference in composition between monocomponent referencevaccine and the test vaccine, a batch of combined vaccineshown to be effective in clinical trials or a batch representativethereof is used as a reference vaccine.


The production of the components complies with therequirements of the monographs on Diphtheria Vaccine(Adsorbed), Tetanus Vaccine (Adsorbed), Pertussis Vaccineand Hepatitis B Vaccine (rDNA).

FINAL BULK VACCINE

The final bulk vaccine is prepared by adsorption, separatelyor together, of suitable quantities of bulk purified diphtheriatoxoid, tetanus toxoid, pertussis components and hepatitis Bsurface antigen onto a mineral carrier such as aluminiumhydroxide or hydrated aluminium phosphate. Suitableantimicrobial preservatives may be added. Only a final bulkvaccine that complies with the following requirements may beused in the preparation of the final lot.Antimicrobial preservative. Where applicable, determine theamount of antimicrobial preservative by a suitable chemicalmethod. The amount is not less than 85.0 per cent and notgreater than 115.0 per cent of the intended content.

pH (2.4.24). 6.0 to 7.0.


FINAL LOT

Only a final lot that is satisfactory with respect to the test forosmolality and with respect to each of the requirements givenbelow under Identification, Tests and Assay may be releasedfor use. Provided the tests for specific toxicity of diphtheriatoxoid, tetanus toxoid and pertussis component andantimicrobial preservative and the assays for the diphtheria,

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tetanus and pertussis components have been carried out withsatisfactory results on the final bulk vaccine, they may beomitted on the final lot. Provided the content of freeformaldehyde has been determined on the bulk purifiedantigens or on the final bulk and it has been shown that thecontent in the final lot will not exceed 0.2 g/l, the test for freeformaldehyde may be omitted on the final lot. If an in vivoassay is used for the hepatitis B component, provided it hasbeen carried out with satisfactory results on the final bulkvaccine, it may be omitted on the final lot.



Identification

Tests A, B, C and D may be omitted if test E is carried out. TestE may be omitted if tests A, B C and D are carried out.


B. Tetanus toxoid. The clear supernatant obtained in test Areacts with a suitable tetanus antitoxin and yields a precipitate.

C. Pertussis component. To a suspension of the residueobtained in test A in saline solution add a suitable B. pertussisantiserum; agglutination indicates presence of pertussiscomponent.

D. Hepatitis B surface antigen. The suspension of the residueobtained in test A gives a positive reactions when tested bysuitable in-vitro assay.

E. The vaccine confers an active immunity in mice and guinea-pigs when administered as directed under Assay.

Tests

pH (2.4.24). 6.0 to 7.0.

Aluminium (2.3.9). Not more than 1.25 mg per single humandose, if aluminium hydroxide or hydrated aluminium phosphateis used as the adsorbent.




Abnormal toxicity (2.2.1). Each final lot shall be tested forabnormal toxicity by injecting intraperitoneally one humandose, but not more than 0.25 ml into each of five mice weighingbetween 17 and 22 g and at least one human dose but notmore than 1.0 ml into each of two guinea pigs weighingbetween 250 and 350 g. The preparation passes the test ifnone of the animals dies or shows signs of ill health in sevendays following the injection. If one of the animals dies orshows signs of ill health, repeat the test. The preparationpasses the test if none of the animals in the second group diesor shows signs of ill health in the time interval specified.

Specific toxicity

Diphtheria and tetanus componentsComplies with the test as stated under Diphtheria and TetanusVaccine (Adsorbed).

Pertussis componentComplies with the test as stated under Diphtheria, Tetanusand Pertussis Vaccine (Adsorbed).

Assay

Diphtheria toxoid (adsorbed)Complies with the test as stated under Diphtheria and TetanusVaccine (Adsorbed).

Tetanus toxoid (adsorbed)Complies with the test as stated under assay for TetanusVaccine (Adsorbed).

Pertussis vaccine


Hepatitis B surface antigen (adsorbed)

Complies with the test as stated under Hepatitis B Vaccine(Adsorbed).


Labelling. The label states (1) the human dose (ml); (2)diphtheria and tetanus components, (a) in case done bychallenge method, the minimum number of International Units(as applicable for each component) per single human doseand (b) in case done by antibody induction, the minimum Lfunits per single human dose; (3) pertussis component - IU orIOU per single human dose; (4) hepatitis B component - mgHBsAg per single human dose; (5) the name and amount ofadsorbent and added preservative; (6) that the vaccine mustbe shaken before use; (7) that the vaccine is not to be frozen.

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Diphtheria, Tetanus, Pertussis (WholeCell) and Haemophilus Type bConjugate Vaccine (Adsorbed)Diphtheria, Tetanus, Pertussis and Haemophilus type bConjugate Vaccine (Adsorbed) is a combined vaccinecomposed of diphtheria formol toxoid containing not less than1,500 Limes flocculationis; (Lf), (2.2.16) per mg of proteinnitrogen, purified tetanus formol toxoid containing not lessthan 1,000 Lf, (2.2.16), per mg of protein nitrogen, andHaemophilus type b conjugated to suitable protein with amineral adsorbent to which a suspension of killed Bordetellapertussis has been added. The mineral adsorbent is asuspension of hydrated aluminium hydroxide, aluminiumphosphate or calcium phosphate, in saline solution or otherappropriate solution isotonic with blood.The formol toxoids are prepared from the toxin produced bythe growth of Corynebacterium diphtheriae and Clostridiumtetani, respectively in suitable media. The toxins are convertedto toxoids by treatment with formaldehyde solution bymethods which avoid reversibility of the toxoids.The polysaccharide, polyribosyl ribitol phosphate, PRP is alinear copolymer composed of repeated units of3-β -D-r ibofuranosyl - (1→1)- r ib i to l -5-phosphate[(C10H19O12P)n], with a defined molecular size and derived froma suitable strain of Haemophilus influenzae type b. The carrierprotein, when conjugated to PRP, is capable of inducing a T-cell dependent B-cell immune response to the polysaccharide.The product may be presented with the haemophiluscomponent in a separate container, the contents of which aremixed with the other components immediately before or duringuse.The final product contains a suitable antimicrobialpreservative. The antigenic properties of the vaccine areadversely affected by the presence of certain antimicrobialpreservatives particularly those of the phenolic type and someof the quaternary ammonium type must not be used.

Production

General provisions


If the vaccine is presented with the haemophilus componentin a separate vial, as part of consistency studies, the assaysof the diphtheria, tetanus and pertussis are carried out on asuitable number of batches of vaccine reconstituted for use.For subsequent routine control, the assays of thesecomponents may be carried out without mixing with thehaemophilus component.

The production method is validated to demonstrate that theproduct, if tested, would comply with the following test forspecific toxicity of the diphtheria and tetanus component:inject subcutaneously 5 times the single human dose statedon the label into each of 5 healthy guinea pigs, each weighingbetween 250 and 350 g, that have not previously been treatedwith any material that will interfere with the test. If within 42days of the injection any of the animals shows signs of or diesfrom diphtheria, toxemia or tetanus, the vaccine does notcomply with the test. If more than 1 animal dies from non-specific causes, repeat the test once; if more than 1 animalshows signs of or dies in the second test, the vaccine doesnot comply with the test.

The stability of the final lot and the relevant intermediates isevaluated using one or more indicator tests. For thehaemophilus component, such tests may include determinationof molecular size, determination of free PRP in the conjugateand kinetics of depolymerisation. Taking account of the resultsof the stability testing, release requirements are set for theseindicator tests to ensure that the vaccine will be satisfactoryat the end of the period of validity.


Provided valid assays can be performed, monocomponentreference vaccines may be used for the assays on the combinedvaccine. If this is not possible because of interaction betweenthe components of the combined vaccine or because of thedifference in composition between monocomponent referencevaccine and the test vaccine, a batch of combined vaccineshown to be effective in clinical trials or a batch representativethereof is used as a reference vaccine. For the preparation ofa representative batch, strict adherence to the productionprocess used for the batch tested in clinical trials is necessary.The reference vaccine may be stabilized by a method that hasbeen shown to have no effect on the assay procedure.


The production of the components complies with therequirements of the monographs on Diphtheria Vaccine(Adsorbed), Tetanus Vaccine (Adsorbed), Pertussis Vaccine(Whole Cell) and Haemophilus influenzae Type b ConjugateVaccine.

FINAL BULK VACCINE

Vaccine with all components in the same container

The final bulk is prepared by adsorption, separately ortogether, of suitable quantities of bulk purified diphtheriatoxoid, bulk purified tetanus toxoid, onto a mineral carrier suchas aluminium hydroxide or hydrated aluminium phosphate,admixture of an appropriate quantity of a suspension ofinactivated B. pertussis component and admixture of a suitable

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quantity of PRP conjugate; the resulting mixture isapproximately isotonic with blood. The B. pertussisconcentration of the final bulk vaccine does not exceed thatcorresponding to an opacity of 20 IU per single human dose.If 2 or more strains of B. pertussis are used, the composition ofconsecutive lots of the final bulk vaccine shall be consistentwith respect to the proportion of each strain as measured inopacity units. Suitable antimicrobial preservatives may beadded.

Vaccine with the haemophilus component in a separatecontainer

The final bulk is prepared by adsorption, separately ortogether, of suitable quantities of bulk purified diphtheriatoxoid, bulk purified tetanus toxoid onto a mineral carrier suchas aluminium hydroxide or hydrated aluminium phosphate,admixture of an appropriate quantity of a suspension ofinactivated B. pertussis component and admixture of a suitablequantity of PRP conjugate; the resulting mixture isapproximately isotonic with blood. The B. pertussisconcentration of the final bulk vaccine does not exceed thatcorresponding to opacity of 20 IU per single human dose. If 2or more strains of B. pertussis are used, the composition ofconsecutive lots of the final bulk vaccine shall be consistentwith respect to the proportion of each strain as measured inopacity units. The final bulk is filled separately. Suitableantimicrobial preservatives may be added. The final bulk ofthe haemophilus component is prepared by dilution of thebulk conjugate to the final concentration with a suitable diluent.A stabilizer may be added.


Antimicrobial preservative. Where applicable, determine theamount of antimicrobial preservative by a suitable chemicalmethod. The amount is not less than 85.0 per cent and notgreater than 115.0 percent of the intended content.


FINAL LOT

Where the haemophilus component is in a separate container,the final bulk of the haemophilus component is freeze-dried.

Only a final lot that is satisfactory with respect to the test forosmolality and with respect to each of the requirements givenbelow under Identification, Tests and Assay may be releasedfor use. Provided the tests specific toxicity of diphtheria toxoid,tetanus toxoid and pertussis component and antimicrobialpreservative and the assays for the diphtheria, tetanus andpertussis components have been carried out with satisfactoryresults on the final bulk vaccine, they may be omitted on the

final lot. Provided the content of free formaldehyde has beendetermined on the bulk purified antigens or on the final bulkand it has been shown that the content in the final lot will notexceed 0.2 g/l, the test for free formaldehyde may be omittedon the final lot.

Free PRP. Unbound PRP is determined after removal of theconjugate, for example by anion exchange, size exclusion orhydrophobic chromatography (2.4.16), ultrafiltration or othervalidated methods. The amount of free PRP is not greater thanapproved for the particular product.


pH (2.4.24). 6.0 to 7.0.


Production

Identification

Tests A, B, C and D may be omitted if test E is carried out. TestE may be omitted if tests A, B, C and D are carried out.


B. Tetanus toxoid. The clear supernatant obtained in test Areacts with a suitable tetanus antitoxin and yields a precipitate.C. Pertussis component. To a suspension of the residueobtained in test A in saline solution add a suitable Bordetellapertussis antiserum; agglutination indicates presence ofpertussis component.D. PRP. The suspension of the residue obtained in test Agives a positive reaction when tested by a suitableimmunochemical method for PRP.E. The vaccine confers an active immunity in mice and guineapigs when administered as directed in the test for Potency.

TestsIf the product is presented with the haemophilus componentin a separate container; the tests for specific toxicity ofdiphtheria toxoid, tetanus toxoid and pertussis component,aluminium, free formaldehyde, antimicrobial preservativeand sterility are carried out on the container with thediphtheria, tetanus and pertussis components; the tests forPRP content, water (where applicable), sterility andpyrogens are carried out on the container with thehaemophilus component.

D., T., P., (WHOLE CELL) AND HAEMOPHILUS TYPE CONJUGATE VACCINE (ADSORBED)

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If the haemophilus component is freeze-dried, some tests maybe carried out on the freeze-dried product rather than on thebulk conjugate where the freeze-drying process may affectthe component under test.

PRP. Not less than 80.0 per cent of the amount of PRP statedon the label. PRP is determined either by assay of ribose (2.7.1)or phosphorus (2.7.1), by an immunochemical method (2.2.14)or by anion exchange liquid chromatography with pulsedamperometric detection.





Abnormal toxicity (2.2.1). Each final lot shall be tested forabnormal toxicity by injecting intraperitoneally one humandose , but not more than 0.25 ml into each of the five miceweighing between 17 to 22 g and at least one human dose butnot more than 1.0 ml into each of the two guinea pigs weighingbetween 250 and 350 g. The preparation passes the test ifnone of the animals dies or shows signs of ill health in 7 daysfollowing the injection . If one of the animal dies or shows thesigns of ill health, repeat the test . The preparation passes thetest if none of the animals in the second group dies or showssigns of ill health in the time interval specified.

Pyrogens (2.2.8). This test is carried out for Haemophilusinfluenzae type b vaccine only if Haemophilus influenzaetype b vaccine is presented as separate lyophilized vial. Thevaccine complies with the test for pyrogens. Inject per kg ofthe rabbit’s mass a quantity of the vaccine equivalent to: 1 mgof PRP for a vaccine with diphtheria toxoid or CRM 197diphtheria toxoid as carrier; 0.1 mg of PRP for a vaccine withtetanus toxoid as carrier protein; 0.025 mg of PRP for vaccinewith OMP as carrier.

Specific toxicity

Diphtheria and tetanus components


Pertussis component


Assay

Diphtheria toxoid (adsorbed)


Tetanus toxoid (adsorbed)

Complies with the test as stated under assay of TetanusVaccine (Adsorbed).

Pertussis vaccine



Labelling. The label states (1) the human dose (ml); (2)Diphtheria and Tetanus components (a) in case done bychallenge method the minimum number of international units(as applicable for each component) per single human dose;(b) in case done by antibody induction method, the minimumLf units per single human dose; (3) pertussis component – IUor IOU per single human dose; (4) haemophilus conjugatecomponent - g PRP per single human dose; (5) the type andnominal amount of carrier protein per single human dose; (6)the name and amount of adsorbent and added preservative;(7) that the vaccine must be shaken before use; (9) that thevaccine is not to be frozen.

Diphtheria Vaccine (Adsorbed)Diphtheria Vaccine (Adsorbed) is a preparation of diphtheriaformol toxoid with a mineral adsorbent. The formol toxoid isprepared from the toxin produced by the growth ofCorynebacterium diphtheriae.

Production

General provisions

The maximum number of Lf units per single human doseof diphtheria vaccine (adsorbed) is 30.

Bulk purified toxoid

For the production of diphtheria toxin, from which toxoid isprepared, seed cultures are managed in a defined seed-lotsystem in which toxinogenicity is conserved and, wherenecessary, restored by deliberate reselection. A highlytoxinogenic strain of Corynebacterium diphtheriae withknown origin and history is grown in a suitable liquid medium.At the end of cultivation, the purity of each culture is testedand contaminated cultures are discarded. Toxin-containingculture medium is separated aseptically from the bacterial mass

DIPHTHERIA VACCINE (ADSORBED)

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as soon as possible. The toxin content (Lf per ml) is checkedto monitor consistency of production. Single harvests may bepooled to prepare the bulk purified toxoid. The toxin is purifiedto remove components likely to cause adverse reactions inhumans. The purified toxin is detoxified with formaldehyde bya method that avoids destruction of the immunogenic potencyof the toxoid and reversion of the toxoid to toxin, particularlyon exposure to heat. Alternatively, purification may be carriedout after detoxification.

Only bulk purified toxoid that complies with the followingrequirements may be used in the preparation of the final bulkvaccine.

Sterility (2.2.11). Carry out test for sterility using 10 ml ofbulk for each sterility medium.Absence of toxin and irreversibility of toxoid

Inject subcutaneously into each of 5 guinea-pigs at least 500 Lfof the non-incubated bulk purified toxoid in a volume of1 ml, using the same buffer solution as for the final vaccine,without adsorbent. Animals that die shall be autopsied andexamined for symptoms of diphtheria intoxication (redadrenals). The bulk purified toxoid shall pass the test if noguinea-pig shows symptoms of specific intoxication withinsix weeks of injection and if at least 80 per cent of the animalssurvive the test period. The guinea-pigs shall not have beenused previously for experimental purposes.Alternatively, a cell-culture test system may be used; in thiscase, the sensitivity of the test shall have been demonstratedto be not less than that of the guinea-pig test, and the testprocedures shall be approved by the National RegulatoryAuthority.Each bulk purified toxoid shall be tested to ensure thatreversion to toxicity cannot take place on storage. The bulkpurified toxoid shall be diluted in order to obtain the sameconcentration and chemical environment as those present inthe final bulk vaccine, except for the presence of adjuvant.To determine whether reversion has occurred, diluted toxoidsthat have been stored at 37° for six weeks shall be tested. Thetest employed shall be approved by the National RegulatoryAuthority and should be sufficiently sensitive to detect verysmall amounts of toxin. No toxicity shall be detected.Intradermal tests in guinea-pigs and cell-culture tests bothare considered to be suitable.

Cell culture methodUsing the same buffer solution as for the final vaccine, withoutadsorbent, prepare a solution of bulk purified toxoid at 100 Lfper ml. Divide the solution into 2 equal parts. Maintain 1 partat 5° ± 3° and the other at 37° for 6 weeks. Carry out a test inVero cells for active diphtheria toxin using 50 µl per well ofboth samples. The sample should not contain antimicrobial

preservatives and detoxifying agents should be determinedto be below the concentration toxic to Vero cells. Non-specifictoxicity may be eliminated by dialysis.Use freshly trypsinised Vero cells at a suitable concentration,for example 2.5 × 105 per ml and a reference diphtheria toxindiluted in 100 Lf per ml diphtheria toxoid. A suitable referencediphtheria toxin will contain either not less than 100 LD50/mlor 67 to 133 lr/100 in 1 Lf and 25,000 to 50,000 minimal reactingdoses for guinea-pig skin in 1 Lf (diphtheria toxin RP issuitable for use as the reference toxin). Dilute the toxin in 100Lf/ml diphtheria toxoid to a suitable concentration, for example2 × 10-4 Lf per ml. Prepare serial twofold dilutions of the diluteddiphtheria toxin and use undiluted test samples (50 µl perwell). Distribute them in the wells of a sterile tissue cultureplate containing a medium suitable for Vero cells. To ascertainthat any cytotoxic effect noted is specific to diphtheria toxin,prepare in parallel dilutions where the toxin is neutralised by asuitable concentration of diphtheria antitoxin, for example100 IU/ml. Include control wells without toxoid or toxin andwith non-toxic toxoid at 100 Lf per ml on each plate to verifynormal cell growth. Add cell suspension to each well, seal theplates and incubate at 37° for 5 to 6 days. Cytotoxic effect isjudged to be present where there is complete metabolicinhibition of the Vero cells, indicated by the pH indicator ofthe medium. Confirm cytopathic effect by microscopicexamination or suitable staining such as MTT dye. The test isinvalid if 5 × 10-5 Lf per ml of reference diphtheria toxin in 100Lf per ml toxoid has no cytotoxic effect on Vero cells or if thecytotoxic effect of this amount of toxin is not neutralised inthe wells containing diphtheria antitoxin. The bulk purifiedtoxoid complies with the test if no toxicity neutralisable byantitoxin is found in either sample.Antigenic purity. Not less than 1,500 Lf per mg of proteinnitrogen.

FINAL BULK VACCINE

The final bulk vaccine is prepared by adsorption of a suitablequantity of bulk purified toxoid onto a mineral carrier such ashydrated aluminium phosphate or aluminium hydroxide; theresulting mixture is approximately isotonic with blood. Suitableantimicrobial preservatives may be added. Certainantimicrobial preservatives, particularly those of the phenolictype, adversely affect the antigenic activity and must not beused.


Identification

Dissolve sufficient sodium citrate in the vaccine underexamination to give a 10 per cent w/v concentration. Maintainat 37o for about 16 hours and centrifuge. The clear supernatant


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IP 2007

reacts with a suitable diphtheria antitoxin and yields aprecipitate.

pH (2.4.24). 6.0 to 7.0.

Specific toxicity. Use 5 normal, healthy guinea-pigs weighingbetween 250 and 350 g, which have been maintained for atleast 1 week on a uniform, unrestricted diet, and have notbeen previously treated with any material that will interferewith the test. Weigh the animals separately and record theirweights. Inject subcutaneously into each animal 5 times thedose stated on the label. Weigh all the animals at weeklyintervals for 6 weeks. None of the animals shows anysymptoms of diphtheria or tetanus toxaemia or dies fromdiphtheria within 42 days or loses weight at the end of thetest. If more than one animal dies from non-specific causes orloses weight, repeat the test. If an animal dies or loses weightin the second test, the vaccine fails the test.

Potency. Determine by any of the methods of biological assayof Adsorbed Diphtheria Vaccine described.

Biological assay of adsorbed diphtheria vaccine

(a) Intradermal challenge method

The potency of adsorbed diphtheria vaccine is determined bycomparing the dose necessary to protect guinea-pigs againstthe erythrogenic effects of a range of intradermal injections ofdiphtheria toxin with the dose of the Standard preparation ofadsorbed diphtheria toxoid necessary to give the sameprotection. For this comparison, the Standard preparation ofadsorbed diphtheria toxoid and a suitable preparation ofdiphtheria toxin, for use as a challenge toxin, are required.

Standard preparation

The Standard preparation is International standard ofDiphtheria toxoid, adsorbed, or another suitable preparationthe potency of which has been determined in relation to theInternational Standard.

Suggested method

Test animals. Use white guinea-pigs, weighing between 250and 350 g, from the same stock. Distribute the guinea-pigsinto no fewer than six equal groups; use groups containing anumber of animals sufficient to obtain results that fulfill therequirements for a valid Assay prescribed below. The guinea-pigs are all of the same sex or the males and females aredistributed equally among the groups. If the challenge toxinto be used has not been shown to be stable or has not beenadequately standardized, include five guinea-pigs asunvaccinated controls.

Selection of the challenge toxin. Select a preparation ofdiphtheria toxin containing 67 to 133 Limes reactionis/100 (Lr/100) in Limes flocculationis (Lf) and 25,000 to 50,000 minimalreacting doses for guinea-pig skin in 1 Lf. If the challenge

toxin preparation has been shown to be stable, it is notnecessary to verify the activity for every assay.

Preparation of the challenge toxin solutions. Immediately priorto use, dilute the challenge toxin with a suitable diluent toobtain a challenge toxin solution containing about 512x10-4 Lfin 0.2 ml. Dilute a portion of this challenge toxin solution togive a series of five 4-fold dilutions.

Determination of potency of the vaccine. Prepare in salinesolution dilutions of the vaccine under examination and ofthe Standard preparation such that, for each, the dilutionsform a series differing by not more than 2.5 fold steps and inwhich the dilutions of intermediate concentration, wheninjected subcutaneously in 1.0 ml volumes into guinea-pigs,result in an intradermal score of approximately three when theanimals are challenged. Allocate the dilutions, one to each ofthe groups of guinea-pigs, and inject subcutaneously 1.0 mlof each dilution into each guinea-pig in the group to whichthat dilution is allocated. After 28 days shave both flanks ofeach guinea-pig and inject each animal intradermally with 0.2ml of the challenge toxin solution and with 0.2 ml of each ofthe five dilutions thereof in such a way as to minimizeinterference between adjacent sites. If necessary, inject theunvaccinated control guinea-pigs with dilutions containing8x10-5, 4x10-5, 2x10-5, 1x10-5 and 5x10-6 Lf of the challenge toxin.Examine all the injection sites 48 hours after injection of thechallenge toxin and record the incidence of specific diphtheriaerythema. Record also the number of sites free from suchreactions as the intradermal challenge score. Tabulate theintradermal challenge scores for all the animals receiving thesame dilution of vaccine and use those data with a suitabletransformation, such as (score)2 or arcsin [(score/6)2], to obtainan estimate of the relative potency for each of the testpreparations by parallel-line quantitative analysis.

The test is not valid unless (a) for both the preparation underexamination and the Standard preparation, the mean scoreobtained at the lowest dose level is more than three; (b) ifapplicable, the toxin dilution that contains 4x10-5 Lf gives apositive erythema in at least 80.0 per cent of the control guinea-pigs and the dilution that contains 2x10-5 Lf gives no reactionin at least 80 per cent of the guinea-pigs (if these criteria arenot met a different toxin has to be selected); (c) the fiduciallimits of the assay fall between 50.0 and 200.0 per cent of theestimated potency; (d) the statistical analysis shows nodeviation from linearity and parallelism. The test may berepeated but when more than one test is performed the resultsof all valid tests must be combined in the estimate of potency.

The lower fiducial limit of error of the estimated potency is notless than 30 Units per dose.

(b) Lethal challenge method

Test animals. Use healthy, white or light-coloured guinea-

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pigs from the same stock, weighing between 250 and 350 g.Distribute them into six groups of sixteen; and four groups offour. The guinea-pigs should all be of the same sex or themales and females should be distributed equally between thesix groups of sixteen.Challenge toxin. Select a preparation of diphtheria toxincontaining not less than 100 LD50 in 1.0 ml.

Preparation of the challenge toxin solutions. Immediately priorto use, prepare from the challenge toxin by dilution inphosphate buffered saline pH 7.4, or normal saline a challengetoxin containing approximately 100 LD50 in 1.0 ml. Diluteportions of this challenge toxin solution to 2LD50, 1 LD50 and½ LD50 in the same solution

Determination of potency of the vaccine. Prepare in salinesolution three dilutions of the vaccine under examination andthree dilutions of the Standard preparation such that for each,the dilutions form a series differing by not more than 2.5 foldsteps and in which the dilutions of intermediate concentration,when injected subcutaneously in 1.0 ml volumes into guinea-pigs, protect approximately 50 per cent of the animals from thelethal effects of the subcutaneous injection of the quantity ofdiphtheria toxin prescribed for this test. Allocate the sixdilutions, one to each of the six groups of sixteen guinea-pigs,and inject subcutaneously 1.0 ml of each dilution into eachguinea-pig in the groups to which that dilution is allocated.After 28 days inject subcutaneously into each animal in thesix groups of sixteen, 1.0 ml of the challenge toxin solution.Allocate the challenge toxin solution and the three dilutionsmade from it, one to each of the four groups of four guinea-pigs and inject subcutaneously 1.0 ml of each toxin solutioninto each guinea-pig in the group to which that solution isallocated. Examine the guinea pigs twice in a day, removedead animals and kill the animals showing definite signs ofdiphtheria. Count the number of surviving animals 5 dayslater and calculate the potency of the vaccine underexamination relative to the potency of the Standard preparationon the basis of the number of animals that survive in each ofthe six groups of sixteen, using appropriate statistical methods.

The test is not valid unless (a) for the vaccine underexamination and the Standard preparation the 50.0 per centprotective doses lie between the largest and smallest doses ofthe preparations given to the guinea-pigs; (b) the survivorsamong the four groups of guinea-pigs injected with thechallenge toxin and its dilutions indicate that the challengewas approximately 100 LD50 and; (c) statistical analysis showsparallelism, linearity and a significant slope of the dose-response lines. The test may be repeated any number of timesbut when more than one test is performed the results of allvalid tests must be combined in the estimate of potency.

If the lower limit of 95.0 per cent confidence interval of estimatedpotency is less than 30 IU per single human dose then the

limits of the 95.0 per cent confidence interval should be within50 to 200 of the estimated potency.

(c) Antibody induction method

Inject subcutaneously on each of two occasions separatedby an interval of not more than 4 weeks, one-fiftieth of thestated human dose diluted to 1 ml with saline solution, intoeach of 10 normal, healthy guinea-pigs weighing between 250and 350 g. Not earlier than 2 weeks and not later than 3 weeksafter the second injection, collect the serum from each animaland determine the antitoxin content of the serum of each animal,as described under Diphtheria Antitoxin or any other methodapproved by National Regulatory Authority. The geometricmean of the antitoxin contents shall be not less than 2.0 Unitsper ml with reference to the Diphtheria antitoxin standard.

(d) Any other validated serological assay in guinea pigs ormice as approved by National Regulatory Authority

Antimicrobial preservative . Where appl icable,determine the amount of antimicrobial preservative bya suitable chemical method. The amount is not less than85.0 per cent and not greater than 115.0 per cent of theintended amount.


FINAL LOT


Only a final lot that is satisfactory with respect to each of therequirements given below under, Identification, Tests andAssay may be released for use. Provided the tests for specifictoxicity, free formaldehyde and antimicrobial preservative andthe assay have been carried out with satisfactory results onthe final bulk vaccine, they may be omitted on the final lot.

IdentificationDiphtheria toxoid is identified by a suitable immunochemicalmethod. The following method, applicable to certain vaccines,is given as an example. Dissolve in the vaccine underexamination sufficient sodium citrate to give a 10 per cent w/v solution. Maintain at 37° for about 16 hours and centrifugeuntil a clear supernatant liquid is obtained. The clearsupernatant liquid reacts with a suitable diphtheria antitoxin,giving a precipitate.

Tests

Aluminium (2.3.9). Maximum 1.25 mg per single human dose,if aluminium hydroxide or hydrated aluminium phosphate isused as the absorbent.


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pH (2.4.24). 6.0 to 7.0.

Assay

Carry out one of the prescribed methods for the assay ofDiphtheria Vaccine (Adsorbed).


Labelling. The label states (1) the human dose; (2) the minimumLf units per single human dose or the minimum InternationalUnits per single human dose if potency test done by challengemethod; (3) the name and the amount of the adsorbent andpreservative; (4) that the vaccine must be shaken before use;(5) that the vaccine is not to be frozen.

Haemophilus Type b ConjugateVaccineHaemophilus Type b Conjugate Vaccine is a liquid or freeze-dried preparation of a polysaccharide, derived from a suitablestrain of Haemophilus influenzae type b, covalently boundto a carrier protein. The polysaccharide, polyribosylribitolphosphate, referred to as PRP, is a linear copolymer composedof repeated units of 3-β-D-ribofuronosyl-(11)-ribitol-5-phosphate [(C10H19O12P)n], with a defined molecular size. Thecarrier protein, when conjugated to PRP, is capable of inducinga T-cell-dependent B-cell immune response to thepolysaccharide.

Production

General provisions

The production method shall have been shown to yieldconsistently H. influenzae type b conjugate vaccines ofadequate safety and immunogenicity in humans. Theproduction method is validated to demonstrate that the product,if tested, would comply with the test for safety and efficacy ofVaccines. The stability of the final lot and relevantintermediates is evaluated using one or more indicator tests.Such tests may include determination of molecular size,determination of free PRP in the conjugate and the

immunogenicity test on mice. Taking account of the results ofthe stability testing, release requirements are set for theseindicator tests to ensure that the vaccine will be satisfactoryat the end of the period of validity.

SEED LOT

The strain of H. influenzae type b used in preparingHaemophilus type b conjugate vaccine shall be identified bya record of its history, including the source from which it wasobtained and the tests made to determine the characteristicsof the strain. The strain shall have been shown to be capableof producing Type b polysaccharide.

The production of PRP and of the carrier protein is based ondefined seed lot systems. Master seed lot and working seedlot shall be properly characterized and defined. Culturesderived from the working seed shall have the samecharacteristics as of the master seed lot. The sample of cultureof single harvests taken before killing shall be tested forcontamination by examination of Gram-stained smears and byinoculation on suitable media.

H. Influenzae Type b Polysaccharide (PRP)

H. influenzae Type b is grown in a liquid medium that doesnot contain high-molecular-weight polysaccharides; if anyingredient of the medium contains blood-group substances,the process shall be validated to demonstrate that after thepurification step they are no longer detectable. The culturemay be inactivated. PRP is separated from the culture liquidand purified by a suitable method. Volatile matter, includingwater, In the purified polysaccharide is determined by methodssuch as thermogravimetry, Karl Fischer or any other suitablemethod. All chemical analysis shall be based on the dry weightof the polysaccharide, in its salt form.

Only those pools of PRP that comply with the followingrequirements may be used in the preparation of the conjugate.The partially purified PRP shall be stored frozen at or below-20°.

Identification

The PRP is identified by an immunochemical method (2.2.14)or other suitable method (e.g. 1H or 13C NMR spectroscopy).

Molecular size. The percentage of PRP eluted before a givenK0 value or within a range of K0 values, is determined by gelfiltration or high performance size-exclusion chromatography(HPSEC) (2.4.16), either alone or in combination with lightscattering and refractive index detectors (e.g. multiple anglelaser light scattering i.e. MALLS) or any other suitable method.An acceptable value is established for the particular productand each batch of PRP must be shown to comply with thislimit. Limits for currently approved products, using theindicated stationary phases, are shown for information in

HAEMOPHILUS TYPE B CONJUGATE

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Table 1 - Specifications for different components of Haemophilus Type b Conjugate Vaccine.

Polysaccharide Carrier Protein Conjugation

Type of PRP Nominal Type Purity Nominal Coupling Procedureamount amount methodper dose per dose

Polysaccharide 25 µg Diphtheria >1500 Lf per mg 18 µg Cyanogen Activated diphtheria(size reduced) toxoid of protein nitrogen bromide toxoid (D-AH+),K0 60.0 per cent: activation cyanogen bromide0.6-0.7 of PRP activated PRP

Polysaccharide: 10 µg Tetanus >1500 Lf per mg 20 µg Carbodiimide- ADH-activated PRPPRP ≥ 50.0 per toxoid of protein nitrogen mediated (PRP-cov.-AH) +cent ≤ K0: 0.30 coupling tetanus toxoid +

EDAC

Polysaccharide 10 µg CRM 197 >90.0 per cent 25µg Reductive Direct coupling of(size reduced) diphtheria diphtheria amination one- PRP to CRM 197Dp=15-35 or 10-35 protein protein (step method) or (cyanoboro-hydride

N-hydroxy- activated)succinimideactivation

Polysaccharide 15 µg Meningococcal Outer membrane 125 or Thioether PRP activation by(size-reduced) group B outer protein vesicles 250 µg bond CDI PRP-IM +K0 0.3 – 0.6 membrane <8.0 per cent of BuA2 + BrAc =

protein (OMP) lipopolysaccharide PRP-BuA2-BrAc +thioactivated OMP

Abbreviations:ADH = adipic acid dihydrazide Dp = degree of polymerizationBrAc = bromoacetyl chloride EDAC = l-ethyl-3-(3-dimethylaminopropyl) carbodimideBuA2 = butane-1, 4-diamide IM = imidazoliumCDl = carbonyldi-imidazole Mw = weight-average molecular weight.

Table 2 - Requirements on bulk conjugate

Test Protein CarrierSpecifications Diphtheria toxoid Tetanus toxoid CRM 197 OMP

Free Polysaccharide (PRP) <37.0 per cent <20.0 per cent <25.0 per cent <15.0 per cent

Unbound protein <5.0 per cent, <1.0 per cent, <1.0 per cent or Not applicablewhere applicable where applicable <2.0 per cent,

depending on thecoupling method

PRP to protein ratio 1.25-1.75 0.30-0.55 0.3-0.7 0.05-0.1

Molecular size (K0): Cross- 95.0 per cent <0.75 60.0 per cent <0.2 50.0 per cent 85.0 per cent < 0.25linked agarose for 0.3-0.6chromatography RCross-linked agarose for 0.6-0.7 85.0 per cent <0.5chromatography R1


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Tables 1 and 2. Where applicable, the molecular-sizedistribution is also determined after chemical modification ofthe polysaccharide.

A validated determination of the degree of polymerization orof the weight-average molecular weight and the dispersion ofmolecular masses may be used instead of the determination ofmolecular size distribution.

Ribose (2.7.1). Not less than 32.0 per cent, calculated withreference to the dried substance, as estimated by Bial reactionfor pentose, using D-ribose as a standard or any other suitableassay.

Phosphorus (2.7.1). 6.8 per cent to 9.0 per cent, calculatedwith reference to the dried substance.

Protein (2.3.49). Not more than 1.0 per cent, calculated withreference to the dried substance. Use sufficient PRP to allowdetection of 1 per cent of protein (e.g. a minimum of 1 mg ofPRP).

Nucleic acid (2.7.1). Not more than 1.0 per cent, calculatedwith reference to the dried substance by spectroscopy or anyother suitable method.

Bacterial endotoxins (2.2.3 ). Not more than 25 IU of endotoxinper microgram of PRP.

Residual reagents. Where applicable, tests are carried out todetermine residues of reagents used during inactivation andpurification. An acceptable value for each reagent isestablished for the particular product and each batch of PRPmust be shown to comply with this limit. Where validationstudies have demonstrated removal of a residual reagent, thetest on PRP may be omitted.

Carrier protein

The carrier protein is chosen in a way so that when the PRP isconjugated it is able to induce a T-cell-dependent immuneresponse. Currently approved carrier proteins and couplingmethods are listed for information in Table 1. The carrierproteins are produced by culture of suitable microorganisms;the bacterial purity of the culture is verified; the culture maybe inactivated; the carrier protein is purified by a suitablemethod.

Only a carrier protein that complies with the followingrequirements may be used in preparation of the conjugate.

Identification

The carrier protein is identified by a suitable immunochemicalmethod (2.2.14).

Sterility (2.2.11). Carry out the test for sterility using for eachmedium 10 ml or the equivalent of one hundred doses,whichever is less.

Diphtheria toxoid. Diphtheria toxoid is produced as statedunder Diphtheria Vaccine (Adsorbed) and complies with therequirements prescribed there for bulk purified toxoid.

Tetanus toxoid. Tetanus toxoid is produced as stated underTetanus Vaccine (Adsorbed) and complies with therequirements prescribed there for bulk purified toxoid exceptthat the antigenic purity is not less than 1500 Lf per mg ofprotein nitrogen.

Diphtheria protein CRM 197. Suitable tests are carried out,for validation or routinely, to demonstrate that the product isnon-toxic. The protein obtained contains not less than 90.0per cent of diphtheria CRM 197 protein, when prepared byliquid chromatography (2.4.14) or any other suitable method.The carrier protein shall be characterized by a suitable chemicalor physicochemical method like SDS-PAGE, HPLC, isoelectricfocusing, amino acid sequencing, circular dichroism,fluorescence spectroscopy, peptide mapping or massspectroscopy, as appropriate.

OMP (Meningococcal group B outer membrane proteincomplex)

OMP complex of Neisseria meningitidis complies with thefollowing requirements for lipopolysaccharide and pyrogens.

Lipopolysaccharide. Not more than 8.0 per cent oflipopolysaccharide, determined by a suitable method.

Pyrogens (2.2.8). Inject into each rabbit 0.25 µg of OMP per kgbody weight, for determining the pyrogenic effect.

Bulk conjugate

PRP is chemically modified to enable conjugation; it is usuallypartly depolymerised either before or during this procedure.Reactive functional groups or spacers may be introduced intothe carrier protein or PRP prior to conjugation. The conjugateis obtained by the covalent binding of PRP and carrier protein.Where applicable, unreacted but potentially reactogenicfunctional groups are made unreactive by means of cappingagents; the conjugate is purified to remove reagents. Wherevalidation studies have demonstrated removal of a residualreagent (eg. CN, Br etc.), the test on bulk conjugate may beomitted.

Only a bulk conjugate that complies with the followingrequirements may be used in preparation of the final bulkvaccine. For each test and for each particular product, limitsof acceptance are established and each batch of conjugatemust be shown to comply with these limits. Limits applied tocurrently approved products for some of these tests are listedfor information in Table 2.

PRP. The PRP content is determined by assay of phosphorus(2.7.1) or by assay of ribose (2.7.1) or by an immunochemicalmethod (2.2.14) or by any suitable method.


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Protein (2.7.1). The protein content is determined by a suitablechemical method.

PRP to protein ratio. Determine the ratio by calculation.

Molecular size. Molecular-size distribution is determined bygel filtration or size-exclusion chromatography (2.4.16), usinga gel matrix, appropriate to the expected size of the conjugate.

Free PRP. Unbound PRP is determined after removal of theconjugate, for example by size-exclusion or hydrophobicchromatography (2.4.16), ultra-filtration or other validatedmethods.

Free carrier protein. Free carrier protein is determined by asuitable method (which may include deriving the content bycalculation from the results of other tests). The amount iswithin the limits approved for the particular product.

Unreacted functional groups. No unreacted functional groupsare detectable in the bulk conjugate unless process validationhas shown that unreacted functional groups detectable atthis stage are removed during the subsequent manufacturingprocess (for example, owing to short half-life).

Residual reagents. Removal of residual reagents such ascyanide, EDAC (ethyldimethylaminopropylcarbodimide) andphenol is confirmed by suitable tests or by validation of thepurification process.

Sterility (2.2.11). Carry out the tests for sterility using foreach medium 10 ml or the equivalent of one hundred doses,whichever is less.

FINAL BULK VACCINE

An adjuvant, an antimicrobial preservative and a stabilizermay be added to the bulk conjugate before dilution to the finalconcentration with a suitable diluent.

Only a final bulk vaccine that complies with the followingrequirements may be used in preparation of the final lot.



FINAL LOT

The final lots are filled in suitable containers, under stringentaseptic conditions.Only a final lot that is satisfactory with respect to each of therequirements given under Identification, Tests and Assay maybe released for use. Provided the test for antimicrobialpreservative has been carried out on the final bulk vaccine, itmay be omitted on the final lot.

Identification

The vaccine is identified by a suitable immunochemical method(2.2.14) for PRP or the assay serves also to identify the vaccine.

Tests

Sterility (2.2.11). Complies with the test for sterility.Abnormal toxicity (2.2.1). Complies with the test for abnormaltoxicity.Pyrogens (2.2.8). Complies with the test for pyrogens. Injectper kg of the rabbit’s mass a quantity of the vaccine equivalentto 1 µg of PRP for a vaccine with diphtheria toxoid or CRM 197diphtheria protein as carrier; 0.1 µg of PRP for a vaccine withtetanus toxoid as carrier; 0.025 µg of PRP for a vaccine withOMP as carrier.pH (2.4.24). The pH of the vaccine, reconstituted if necessary,is within the range approved for the product (6.5 to 7.5).Aluminium (2.3.9). When aluminium hydroxide or hydratedaluminium phosphate is used as the adsorbent,Antimicrobial preservative. Where applicable, determine theamount of antimicrobial preservative by a suitable chemicalmethod. The content is not less than 85.0 per cent and notgreater than 115.0 per cent of the intended amount.Water (2.3.43). Not more than 3.0 per cent.PRP. Not less than 80.0 per cent of the amount of PRP statedon the label as determined by ribose assay (2.7.1) or byphosphorus assay (2.7.1) or by an immunochemical method(2.2.14) or by any other suitable method like colorimetery orby anion exchange liquid chromatography (2.4.14) with pulsedamperometric detection.Free PRP. Unbound PRP is determined after removal of theconjugate for example by size-exclusion or hydrophobicchromatography (2.4.16), ultra filtration or other validatedmethods.Labelling. The label states (1) the number of micrograms ofPRP per human dose; (2) the type and nominal amount ofcarrier protein per single human dose; (3) for vaccine containedin single-dose containers where the space is too small toaccommodate the full name of the vaccine the abbreviation‘Hib’ may be used in the label on the container provided thatthe same code is also stated in the label on the package.

Hepatitis A (Inactivated) and HepatitisB (rDNA) Vaccine (Adsorbed)

Hepatitis A (Inactivated) and Hepatitis B (rDNA) Vaccine(Adsorbed) is a suspension consisting of a suitable strain ofhepatitis A virus, grown in cell cultures and inactivated by a

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validated method, and of hepatitis B surface antigen (HBsAg),a component protein of hepatitis B virus obtained byrecombinant DNA technology; the antigens are adsorbed ona mineral carrier such as aluminium hydroxide or hydratedaluminium phosphate.

Production

General provisions

The two components are prepared as described in themonographs on Hepatitis A Vaccine (Inactivated, Adsorbed)and Hepatitis B Vaccine (rDNA) and comply with therequirements prescribed therein. The production method isvalidated to demonstrate that the product, if tested, wouldcomply with the test for abnormal toxicity for antisera andvaccines.

Reference preparation

The reference preparation is part of a representative batchshown to be at least as immunogenic in animals as a batchthat, in clinical studies in young, healthy adults, produces notless than 95.0 per cent seroconversion, corresponding to alevel of neutralizing antibody recognized to be protective,after a full-course primary immunization. For hepatitis A, anantibody level not less than 20 mIU/ml determined by enzyme-linked immunosorbent assay is recognized as being protective.For hepatitis B, antibody level not less than 10 mIU/ml againstHBsAg is recognized as being protective.

FINAL BULK VACCINE

The final bulk vaccine is prepared from one or more inactivatedharvests of hepatitis A virus and one or more batches ofpurified antigen of Hepatitis B (rDNA).



Sterility (2.2.11). Carry out test for sterility using 10 ml of bulkfor each sterility medium.FINAL LOT

Only a final lot that complies with each of the requirementsgiven below under Identification, Tests and Assay may bereleased for use. Provided that the tests for free formaldehyde(where applicable) and antimicrobial preservative content(where applicable) have been carried out on the final bulkvaccine with satisfactory results, they may be omitted on thefinal lot. If the assay of the hepatitis A and/or the hepatitis Bcomponent is carried out in vivo, then provided it has been

carried out with satisfactory results on the final bulk vaccine,it may be omitted on the final lot.

IdentificationThe vaccine is shown to contain hepatitis A virus antigen andhepatitis B surface antigen by suitable immunochemicalmethods (2.2.14), using specific antibodies or by the mouseimmunogenicity tests described under assay.

Tests

Aluminium (2.3.9). Maximum 1.25 mg per single human doseif aluminium hydroxide or hydrated aluminium phosphate isused as the adsorbent.




Bacterial endotoxins (2.2.3). Less than 2 IU per human dose.

Assay

Hepatitis A component

Complies with the assay as stated under Inactivated HepatitisA Vaccine (Adsorbed).


Complies with the assay as stated under Hepatitis B Vaccine(rDNA).

Labelling. The label states (1) the amount of hepatitis A virusantigen and hepatitis B surface antigen per container; (2) thetype of cells used for production of the vaccine; (3) the nameand amount of the adsorbent used; (4) that the vaccine mustbe shaken before use; (5) that the vaccine must not be frozen.

Hepatitis B Vaccine (rDNA)Hepatitis B Vaccine (rDNA) is a non-infectious preparationcontaining the purified major surface antigen of Hepatitis Bvirus (HBsAg). This preparation is white or almost whitetranslucent liquid in which the mineral carrier tends to settledown slowly on keeping but is free from foreign particles/floccules.

Production

General provisions

The antigen is manufactured by recombinant DNA technologyby culturing genetically engineered yeast cells or other suitable

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cell lines which carry the gene that codes for major surfaceantigen of the Hepatitis-B virus as approved by the competentauthority. Several physico-chemical steps are employed topurify the Hepatitis-B surface antigen (HBsAg). The vaccinemay contain the product of the S gene (major protein), acombination of the S gene and pre-S2 gene products (middleprotein) or a combination of S gene, the pre-S2 gene, and pre-S1 gene products (large protein). The purity of the antigen isdetermined by comparison with a reference preparation usingliquid chromatography or other suitable methods such as SDS-PAGE with any suitable staining method. The purified antigenis finally adsorbed on aluminium hydroxide or aluminiumphosphate.

The method used for production of the vaccine must havebeen shown to yield a product consistently complying withthe requirements for immunogenicity and safety. It must alsohave been shown to induce specific, protective antibodies inhuman beings.

The production method must be validated to demonstratethat the product if tested, would comply with the tests forsafety and efficacy.

Reference preparation. A part of a batch shown to be at leastas immunogenic as a batch that was used in clinical studiesand approved by National Regulatory Authority anddetermined by any suitable method. For hepatitis B, antibodylevel not less than 10 mIU/ml against HBsAg is recognized asbeing protective..

Characterisation of the substance

Development studies are carried out to characterize theantigen. The complete protein, lipid and carbohydrate structureof the antigen is established. The morphological characteristicsof the antigen particles are established by electron microscopy.The buoyant density of the antigen particles is determined bya physico-chemical method, for example gradientcentrifugation. The antigenic epitopes are characterized. Theprotein fraction of the antigen is characterized in terms of theprimary structure (for example, by determination of the amino-acid composition, by partial amino-acid sequence analysisand by peptide mapping).


Identity, microbial purity, plasmid retention and consistencyof yield are determined at suitable production stages. Ifmammalian cells are used, tests for extraneous agents andmycoplasmas are performed in accordance with tests forextraneous agents in viral vaccines for human use.

PURIFIED ANTIGEN

Only a purified antigen that complies with the followingrequirements may be used in the preparation of the final bulk.

Total protein (2.3.49). The total protein is determined by avalidated method. The content is within the limits approvedfor the specific product.

Antigen content and identification. The quantity andspecificity of HBsAg is determined in comparison with theInternational standard for HBsAg subtype ad or an in-housereference, by a suitable immunochemical method such asradioimmunoassay (RIA), enzyme-linked immunosorbentassay (ELISA), immunoblot (preferably using a monoclonalantibody directed against a protective epitope) or single radialdiffusion. The antigen/protein ratio is within the limits approvedfor the specific product.

The molecular weight of the major band in a sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE)under reduced conditions corresponds to the value expectedfrom the gene sequence and possible glycosylation.

Antigenic purity. The purity of the antigen is determined bycomparison with a reference preparation using liquidchromatography or other suitable methods such as SDS-PAGEwith staining. A suitable method is sensitive enough to detecta potential contaminant at a concentration of 1.0 per cent oftotal protein. Not less than 95.0 per cent of the total proteinconsists of hepatitis B surface antigen.

Composition. The content of proteins, lipids, nucleic acidsand carbohydrates is determined.

Host-cell and vector-derived DNA (2.2.15). If mammalian cellsare used for production, not more than 10 pg of DNA in thequantity of purified antigen equivalent to a single human doseof vaccine.

Caesium. If a caesium salt is used during production, a testfor residual caesium is carried out on the purified antigen. Thecontent is within the limits approved for the specific product.

Sterility (2.2.11). The purified antigen complies with the testfor sterility, carried out using 10 ml for each medium.

Additional tests on the purified antigen may be requireddepending on the production method used: for example, a testfor residual animal serum where mammalian cells are used forproduction or tests for residual chemicals used duringextraction and purification.

FINAL BULK VACCINE

An antimicrobial preservative and an adjuvant may beincluded in the vaccine.




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Only a final lot that complies with each of the requirementsgiven below under Identification, Tests and Assay may bereleased for use. Provided that the tests for free formaldehyde,antimicrobial preservative content and the assay in animals,where applicable, have been carried out on the final bulkvaccine with satisfactory results, they may be omitted on thefinal lot.

Identification

The assay or, where applicable, the electrophoretic profile,also serves to identify the vaccine.

Tests

Aluminium (2.3.9). Maximum 1.25 mg per single human dose,if aluminium hydroxide or hydrated aluminium phosphate isused as the adsorbant.



Pyrogens (2.2.8). Complies with the test for pyrogens. Injectthe equivalent of one human dose into each rabbit.

A validated test for bacterial endotoxins may be used insteadof the test for pyrogens.

Assay. The vaccine complies with the assay of Hepatitis-BVaccine (rDNA) described below.

Potency. The upper fiducial limit (P = 0.95) of the estimatedrelative potency is not less than 1.0.

Determine the potency either in animals (Method A) or by avalidated in vitro procedure (Method B) described below:

Method A (Biological)

The potency of the vaccine under examination is determinedin animals by comparing in given conditions its capacity toinduce specific anti-HBsAg antibodies in mice or guinea-pigswith the same capacity as with the reference standard.

Inject intraperitoneally not less than three doses of suitabledilutions of the vaccine under examination diluted withadjuvant used in the vaccine into groups of a suitable strainof mice, weighing between 15 and 20 g (about 5 weeks old), ofeither sex distributed randomly into several groups of mice.Healthy guinea pigs weighing between 300 and 350g (about 7weeks old) that have not been previously treated with anymaterial that will interfere with the test will also be suitable for

the test. Use animals of the same sex in the test. Inject similargroups of animals with the reference preparation of Hepatitis-B vaccine (r DNA). One group of control animals remainsunvaccinated but is injected intraperitoneally with the samevolume of the diluent alone. Anaesthetize and bleed theanimals 28 to 42 days later, keeping the individual sera separate.Assay the individual sera for specific HBsAg antibodyconcentration by a suitable immunochemical method such asELISA or RIA.

Calculate the result of the assay by standard statisticalmethods (5.7). From the distribution of reaction levelsmeasured on all the sera in the unvaccinated (control group),the maximum reaction level that can be expected to occur in anunvaccinated animal for that particular assay is determined.Any response in the vaccinated animals that exceeds thislevel is by definition seroconversion. The percentage ofanimals showing seroconversion in each group is transformed(for example, probit transformation) and a parallel line model,using the log dose response curve, is applied to the data. Thepotency of the preparation under examination relative to thereference preparation is thus established. The test is not validunless (a) for both the test and reference vaccine, the ED50 liesbetween the smallest and the largest doses given to animals ;(b) the statistical analysis shows no deviation from linearityor parallelism; (c) the fiducial limits of the estimated relativepotency fall between 33.0 and 300.0 per cent of the estimatedpotency.

Method B (In vitro)

The potency of the vaccine under examination is determinedby an in vitro method that has been validated against thebiological test.

Enzyme Linked Immunosorbent Assay (ELISA) usingmonoclonal antibodies specific for protection inducingepitopes of HBsAg have been shown to be suitable. Adequatenumber of dilutions and replicates of the vaccine underexamination and the reference standard are employed in theassay. The data obtained is analyzed by a parallel-line modeland may be suitably transformed for statistical evaluation.Commercially available kits for measuring HBsAg in vitro maybe used provided they are validated to produce equally preciseand accurate results.

The test is not valid unless (a) the statistical analysis showsno deviation from linearity or parallelism; (b) the fiducial limitsof the estimated relative potency fall between 80.0 and 125.0per cent of the estimated potency.

The acceptance criteria are approved for a given referencepreparation by the National Regulatory Authority in the lightof the validation data.

Labelling. The label states (a) the amount of HBsAg per dose;(b) the type of cells used for production of the vaccine; (c) the


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name and amount of the adjuvant; (d) that the vaccine mustbe shaken before use; (e) that the vaccine must not be frozen.

Inactivated Hepatitis A Vaccine(Adsorbed)Hepatitis A Vaccine (Inactivated, Adsorbed) is a liquidpreparation of a suitable strain of hepatitis A virus grown incell cultures, inactivated by a validated method and adsorbedon a mineral carrier. The vaccine is an opalescent suspension.

The vaccine complies with the monograph on Vaccines.

ProductionProduction of the vaccine is based on a virus seed-lot systemand a cell-bank system. The production method shall havebeen shown to yield consistently vaccines that comply withthe requirements for immunogenicity, safety and stability.The production method is validated to demonstrate that theproduct, if tested, would comply with the test for safety andefficacy.Unless otherwise justified and authorised, the virus in thefinal vaccine shall not have undergone more passages fromthe master seed lot than were used to prepare the vaccineshown in clinical studies to be satisfactory with respect tosafety and efficacy.

Reference preparation. A part of a batch shown to be at leastas immunogenic as a batch that, in clinical studies in younghealthy adults, produced not less than 95.0 per centseroconversion, corresponding to a level of neutralisingantibody accepted to be protective, after a full-course ofprimary immunisation is used as a reference preparation. Anantibody level of 20 mIU /ml determined by enzyme-linkedimmunosorbent assay is recognised as being protective.Substrate for virus propagationThe virus is propagated in a human diploid cell line or in acontinuous cell line approved by the competent authority.SEED LOT

The strain of hepatitis A virus used to prepare the master seedlot shall be identified by historical records that includeinformation on the origin of the strain and its subsequentmanipulation.Only a seed lot that complies with the following requirementsmay be used for virus propagation.Description. A clear, colourless or light coloured liquid.

IdentificationEach master and working seed lot is identified as hepatitis Avirus using specific antibodies.

Virus concentration. The virus concentration of each masterand working seed lot is determined to monitor consistency ofproduction.

Extraneous agents (2.7.3). The master and working seed lotscomply with the requirements for seed lots for virus vaccines.In addition, if primary monkey cells have been used forisolation of the strain, measures are taken to ensure that thestrain is not contaminated with simian viruses such as simianimmunodeficiency virus and filoviruses.


All processing of the cell bank and subsequent cell cultures isdone under aseptic conditions in an area where no other cellsare being handled. Animal serum (but not human serum) maybe used in the cell culture media. Serum and trypsin used inthe preparation of cell suspensions and media are shown tobe free from extraneous agents. The cell culture media maycontain a pH indicator, such as phenol red and approvedantibiotics at the lowest effective concentration. Not less than500 ml of the cell cultures employed for vaccine production isset aside as uninfected cell cultures (control cells). Multipleharvests from the same production cell culture may be pooledand considered as a single harvest.

Only a single harvest that complies with the followingrequirements may be used in the preparation of the vaccine.When the determination of the ratio of virus concentration toantigen content has been carried out on a suitable number ofsingle harvests to demonstrate consistency, it maysubsequently be omitted as a routine test.

Identification

The test for antigen content also serves to identify the singleharvest.

Sterility (2.2.11). The single harvest complies with the test forsterility, carried out using 10 ml for each medium.

Mycoplasmas (2.7.4). The single harvest complies with thetest for mycoplasmas carried out using 1ml for each medium.

Control cells. The control cells of the production cell culturecomply with a test for identity and the requirements forextraneous agents.

Antigen content. Determine the hepatitis A antigen contentby a suitable immunochemical method (2.2.14) to monitorproduction consistency; the content is within the limitsapproved for the particular product.

Ratio of virus concentration to antigen content. Theconsistency of the ratio of the concentration of infectiousvirus, as determined by a suitable cell culture method, toantigen content is established by validation on a suitablenumber of single harvests.

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PURIFICATION AND PURIFIED HARVEST

The harvest, which may be a pool of several single harvests,is purified by validated methods. If continuous cell lines areused for production, the purification process shall have beenshown to reduce consistently the level of host-cell DNA. Onlya purified harvest that complies with the following requirementsmay be used in the preparation of the inactivated harvest.Virus concentration. The concentration of infective virus inthe purified harvest is determined by a suitable cell culturemethod to monitor production consistency and as a startingpoint for monitoring the inactivation curve.

Antigen:total protein ratio. Determine the hepatitis A virusantigen content by a suitable immunochemical method (2.2.14).Determine the total protein by a validated method. The ratioof hepatitis A virus antigen content to total protein content iswithin the limits approved for the particular product.

Bovine serum albumin. Not more than 50 ng in the equivalentof a single human dose, determined by a suitableimmunochemical method (2.2.14). Where appropriate in viewof the manufacturing process, other suitable protein markersmay be used to demonstrate effective purification.

Residual host-cell DNA (2.2.15). If a continuous cell line isused for virus propagation, the content of residual host-cellDNA, determined using a suitable method is not greater than100 pg in the equivalent of a single human dose.

Residual chemicals. If chemical substances are used duringthe purification process, tests for these substances are carriedout on the purified harvest (or on the inactivated harvest),unless validation of the process has demonstrated totalclearance. The concentration must not exceed the limitsapproved for the particular product.

INACTIVATION AND INACTIVATED HARVEST

Several purified harvests may be pooled before inactivation.In order to avoid interference with the inactivation process,virus aggregation must be prevented or aggregates must beremoved immediately before and/or during the inactivationprocess. The virus suspension is inactivated by a validatedmethod; the method shall have been shown to be consistentlycapable of inactivating hepatitis A virus without destroyingthe antigenic and immunogenic activity; as part of thevalidation studies, an inactivation curve is plotted representingresidual live virus concentration measured on at least threeoccasions (for example, on days 0, 1 and 2 of the inactivationprocess). If formaldehyde is used for inactivation, the presenceof excess free formaldehyde is verified at the end of theinactivation process.

Only an inactivated harvest that complies with the followingrequirements may be used in the preparation of the final bulkvaccine.

Inactivation. Carry out an amplification test for residualinfectious hepatitis A virus by inoculating a quantity of theinactivated harvest equivalent to 5 per cent of the batch or ifthe harvest contains the equivalent of 30,000 doses or more,not less than 1,500 doses of vaccine into cell cultures of thesame type as those used for production of the vaccine andincubating the cells for at least 28 days. Make two passagesand at the end of incubation carry out a test of suitablesensitivity for residual infectious virus. No evidence ofhepatitis A virus multiplication is found in the samples takenat the end of the inactivation process. Use infective virusinocula concurrently as positive controls to demonstratecellular susceptibility and absence of interference.Sterility (2.2.11). The inactivated viral harvest complies withthe test for sterility, carried out using 10 ml for each medium.

Bacterial endotoxins (2.2.3). Not more than 2 IU of endotoxinin the equivalent of a single human dose.

Antigen content. Determine the hepatitis A virus antigencontent by a suitable immunochemical method (2.2.14).

Residual chemicals. As stated under Purification and PurifiedHarvest.

FINAL BULK VACCINE

The final bulk vaccine is prepared from one or more inactivatedharvests. Approved adjuvants, stabilisers and antimicrobialpreservatives may be added.




FINAL LOT

The final bulk vaccine is distributed aseptically into sterilecontainers. The containers are then closed so as to avoidcontamination.

Only a final lot that complies with each of the requirementsgiven below under Identification, Tests and Assay may bereleased for use. Provided that the tests for free formaldehyde(where applicable) and antimicrobial preservative content(where applicable) and the assay have been carried out on thefinal bulk vaccine with satisfactory results, these tests may beomitted on the final lot. If the assay is carried out using miceor other animals, then provided it has been carried withsatisfactory results on the final bulk vaccine, it may be omittedon the final lot.

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Identification

The vaccine is shown to contain hepatitis A virus antigen bya suitable immunochemical method using specific antibodiesor by the mouse immunogenicity test described under Assay.

Tests

Aluminium (2.3.9). Maximum 1.25 mg per single human dose,if hydrated aluminium phosphate or aluminium hydroxide isused as the adsorbent.

Free formaldehyde (2.3.20). When formaldehyde has beenused for inactivation, the vaccine complies with the testprescribed in Vaccines.




Assay

The vaccine complies with the assay of Hepatitis A vaccine.

The assay is carried out either in vivo, by comparing in givencondition the capacity to induce specific antibodies in micewith the same capacity of a reference preparation or in vitroby an immunochemical determination of antigen content(2.2.14).

In vivo assay

The test on mice shown below is given as an example of amethod that has been found suitable for a given vaccine;other validated methods may also be used.

Selection and distribution of the test animals. Healthy micefrom the same stock, about 5 weeks old and from a strainshown to be suitable should be used in the test. Use animalsof the same sex. Distribute the animals in at least seven equalgroups of a number suitable for the requirements of the assay.

Determination of potency of the vaccine under examination.Using a 0.9 per cent w/v solution of sodium chloride containingthe aluminium adjuvant used for the vaccine, prepare at leastthree dilutions of the vaccine under examination and matchingdilutions of the reference preparation. Allocate the dilutionsone to each of the groups of animals and injectsubcutaneously not more than 0.5 ml of each dilution intoeach animal in the group to which that dilution is allocated.Maintain a group of unvaccinated controls, injectedsubcutaneously with the same volume of diluent. After 28 to32 days, anaesthetise and bleed all animals, keeping theindividual sera separate. Assay the individual sera for specific

antibodies against hepatitis A virus by a suitableimmunochemical method (2.2.14).

Calculations. Carry out the calculations by the usual statisticalmethods (5.7) for an assay with a quantal response.

From the distribution of reaction levels measured on all thesera in the unvaccinated group, determine the maximumreaction level that can be expected to occur in an unvaccinatedanimal for that particular assay. Any response in vaccinatedanimals that exceeds this level is by definition a seroconversion.

Make a suitable transformation of the percentage of animalsshowing seroconversion in each group (for example, a probittransformation) and analyse the data according to a parallel-line log dose-response model. Determine the potency of thetest preparation relative to the reference preparation.

Validity conditions. The test is not valid unless (a) for boththe test and the reference vaccine, the ED50 lies between thesmallest and the largest doses given to the animals; (b) thestatistical analysis shows no significant deviation fromlinearity or parallelism; (c) the fiducial limits of the estimatedrelative potency fall between 33.0 and 300.0 per cent of theestimated potency.

Potency. The upper fiducial limit (P = 0.95) of the estimatedrelative potency is not less than 1.0.

In vitro assay

Carry out an immunochemical determination of antigen content(2.2.14) with acceptance criteria validated against the in vivotest. The acceptance criteria are approved for a given referencepreparation by the National Regulatory Authority in the lightof the validation data.

Labelling. The label states (1) the biological origin of the cellsand; (2) the adjuvant used for the preparation of the vaccine.

Inactivated Hepatitis B VaccineInactivated Hepatitis B Vaccine is a non-infectious inactivatedliquid preparation derived from the surface antigen of HepatitisB virus (HbsAg). This preparation is white or almost whitetranslucent liquid in which the mineral carrier tends to settledown slowly on keeping but is free from foreign particles /floccules.

Production

The antigen is harvested and purified from the plasma of humancarriers of Hepatitis B virus. The surface antigen contains allthe three antigen species (S, Pre-S1, Pre-S2). The individualdonor plasma is shown by sensitive tests to be seronegativefor HIV-I and HIV-2 and for HCV. The plasma pool is tested forfreedom from adventitious viruses and blood borne

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transmissible pathogens by appropriate methods. The purifiedantigen is further inactivated by a validated method, usuallywith formalin or any other inactivating agent, to render thehepatitis B virus harmless. The preparation is also tested forthe residual HBV DNA using a sensitive test approved by thecompetent authority and the level is shown to be less than 1pg HBV DNA per 50 doses.

The method used for production of the vaccine must havebeen shown to yield a product consistently complying withthe requirements of immunogenicity, safety and stability. Theproduction method must also be validated to demonstratethat the product, if tested, would comply with the tests forsafety and efficacy.

Reference preparation. A part of a batch shown to be at leastas immunogenic as a batch that produced in clinical studies inyoung healthy adults not less than 95.0 per centseroconversion, corresponding to a level of neutralizingantibody accepted to be protective (HbsAg antibody titre notless than 10 mIU/ml after a full course of primary immunizationdetermined by enzyme-linked immunosorbent assay (ELISA)is used as a reference preparation.Characterisation of the substance

Development studies are carried out to characterize theantigen. The complete protein, lipid and carbohydrate structureof the antigen is established. The morphological characteristicsof the antigen particles are established by electron microscopy.The buoyant density of the antigen particles is determined bya physico-chemical method (2.4.29), for example gradientcentrifugation. The antigenic epitopes are characterized. Theprotein fraction of the antigen is characterized in terms of theprimary structure (for example, by determination of the amino-acid composition, by partial amino-acid sequence analysisand by peptide mapping).


Identity, microbial purity, plasmid retention and consistencyof yield are determined at suitable production stages.

PURIFIED ANTIGEN

Only a purified antigen that complies with the followingrequirements may be used in the preparation of the final bulk.

Total protein (2.3.49). The total protein is determined by avalidated method. The content is within the limits approvedfor the specific product.

Antigen content and identification. The quantity andspecificity of HBsAg is determined in comparison with theInternational standard for HBsAg subtype ad or an in-housereference, by a suitable immunochemical method such as radioimmunoassay (RIA), enzyme-linked immunosorbent assay(ELISA), immunoblot (preferably using a monoclonal antibodydirected against a protective epitope) or single radial diffusion.

The antigen/protein ratio is within the limits approved for thespecific product.The molecular weight of the major band in a sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE)under reduced conditions corresponds to the value expectedfrom the gene sequence and possible glycosylation.

Antigenic purity. The purity of the antigen is determined bycomparison with a reference preparation using liquidchromatography or other suitable methods such as SDS-PAGEwith staining. A suitable method is sensitive enough to detecta potential contaminant at a concentration of 1.0 per cent oftotal protein. Not less than 95.0 per cent of the total proteinconsists of hepatitis B surface antigen.

Composition. The content of proteins, lipids, nucleic acidsand carbohydrates is determined.

Sterility (2.2.11). The purified antigen complies with the testfor sterility carried out using 10 ml for each medium.

Additional tests on the purified antigen may be requireddepending on the production method used.

FINAL BULK VACCINE

An antimicrobial preservative and an adjuvant may beincluded in the vaccine.


Antimicrobial preservative. Where applicable, determine theamount of antimicrobial preservative by a suitable chemical orphysico-chemical method. The amount is not less than the85.0 per cent and not greater than 115.0 per cent of that statedon the label.


FINAL LOT

Only a final lot that complies with each of the requirementsgiven below under Identification, Tests and Assay may bereleased for use. Provided that the tests for free formaldehyde,antimicrobial preservative content and the assay in animals,where applicable, have been carried out on the final bulkvaccine with satisfactory results, they may be omitted on thefinal lot.

Identification

The assay or, where applicable, the electrophoretic profile,also serves to identify the vaccine.

Tests

Aluminium (2.3.9). When hydrated aluminium phosphate oraluminium hydroxide is used as the adsorbent, the vaccine

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complies with the test prescribed in the monograph onVaccines.

Test for inactivating agent. The concentration of anyinactivating agent remaining in the final vaccine shall bedetermined by methods approved by the competent authority.The concentration shall not exceed a specified upper limit.



Pyrogens (2.2.8). Complies with the test for pyrogens. Injectthe equivalent of one human dose into each rabbit.

A validated test for bacterial endotoxins (2.2.3) may be usedinstead of the test for pyrogens.

Assay

The upper fiducial limit (P = 0.95) of the estimated relativepotency is not less than 1.0.

Determine the potency by method A (Biological) as describedunder Hepatitis-B vaccine (rDNA).

Labelling. The label states (1) the amount of HBsAg per dose;(2) the name and amount of inactivating agent; (3) the nameand amount of the adjuvant; (4) that the vaccine must beshaken before use; (5) that the vaccine must not be frozen.

Inactivated Influenza Vaccine (SplitVirion)Influenza Vaccine (Split Virion, Inactivated) is a sterile, aqueoussuspension of a strain or strains of influenza virus, type A orB, or a mixture of strains of the two types grown individuallyin eggs derived from specific pathogen free flock or cellcultures, inactivated and treated so that the integrity of thevirus particles has been disrupted without diminishing theantigenic properties of the haemagglutinin and neuraminidaseantigens. The stated amount of haemagglutinin antigen foreach strain present in the vaccine is 15 µg per dose, unlessclinical evidence supports the use of a different amount.

ProductionThe production method is validated to demonstrate that theproduct, if tested, would comply with the test for safety andefficacy.

Choice of vaccine strain

The World Health Organisation reviews the worldepidemiological situation annually and if necessary

recommends new strains corresponding to prevailingepidemiological evidence.

The origin and passage history of virus strains shall beapproved by the National Regulatory Authority.

Substrate for virus propagation

Influenza virus seed to be used in the production of vaccine ispropagated in fertilised eggs from chicken flocks free fromspecified pathogens or in suitable cell cultures, such as chick-embryo fibroblasts or chick kidney cells obtained from chickenflocks free from specified pathogens. For production, the virusof each strain is grown in the allantoic cavity of eggs derivedfrom specific pathogen free flocks.

SEED LOT

The production of vaccine is based on a seed-lot system.Working seed lots represent not more than fifteen passagesfrom the approved reassorted virus or the approved virusisolate. The final vaccine represents one passage from theworking seed lot. The haemagglutinin and neuraminidaseantigens of each seed lot are identified as originating from thecorrect strain of influenza virus by suitable methods.

Only a working virus seed lot that complies with the followingrequirements may be used in the preparation of the monovalentpooled harvest.


Mycoplasmas (2.7.4). Carry out the test for mycoplasmas using10 ml.


An antimicrobial agent may be added to the inoculum. Afterincubation at a controlled temperature, the allantoic fluids areharvested and combined to form a monovalent pooled harvest.An antimicrobial agent may be added at the time of harvest.At no stage in the production, penicillin or streptomycin isused.

MONOVALENT POOLED HARVEST

To limit the possibility of contamination, inactivation is initiatedas soon as possible after preparation. The virus is inactivatedby a method that has been demonstrated on three consecutivebatches to be consistently effective for the manufacturer. Theinactivation process shall have been shown to be capable ofinactivating the influenza virus without destroying itsantigenicity; the process should cause minimum alteration ofthe haemagglutinin and neuraminidase antigens. Theinactivation process shall also have been shown to be capableof inactivating avian leucosis viruses and mycoplasmas. Ifthe monovalent pooled harvest is stored after inactivation, it

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is held at a temperature of 5±3°. If formaldehyde solution isused, the concentration does not exceed 0.2 g/l offormaldehyde at any time during inactivation; ifbetapropiolactone is used, the concentration does not exceed0.1 per cent v/v at any time during inactivation.

Before or after the inactivation procedure, the monovalentpooled harvest is concentrated and purified by high-speedcentrifugation or other suitable method and the virus particlesare disrupted into component subunits by the use of approvedprocedures. For each new strain, a validation test is carriedout to show that the monovalent bulk consists predominantlyof disrupted virus particles.Only a monovalent pooled harvest that complies with thefollowing requirements may be used in the preparation of thefinal bulk vaccine.Haemagglutinin antigen. Determine the content ofhaemagglutinin antigen by an immunodiffusion test (2.2.14),by comparison with a haemagglutinin antigen referencepreparation or with an antigen preparation calibrated againstit. Carry out the test at 20° to 25°.

For some vaccines, the physical form of the haemagglutininparticles prevents quantitative determination byimmunodiffusion after inactivation of the virus. For thesevaccines, a determination of haemagglutinin antigen is madeon the monovalent pooled harvest before inactivation. Theproduction process is validated to demonstrate suitableconservation of haemagglutinin antigen and a suitable traceris used for formulation, for example, protein content.

Neuraminidase antigen. The presence and type ofneuraminidase antigen are confirmed by suitable enzymatic orimmunological methods (2.2.14) on the first three monovalentpooled harvests from each working seed lot.


Viral inactivation. Carry out as described below under Tests.

Chemicals used for disruption. Tests are carried out on themonovalent pooled harvest for the chemicals used fordisruption, the limits being approved by the NationalRegulatory Authority.

FINAL BULK VACCINE

Appropriate quantities of the monovalent pooled harvestsare blended to make the final bulk vaccine.



Sterility (2.2.11). Carry out the test for sterility using 10 ml foreach sterility medium.

FINAL LOT


Only a final lot that is satisfactory with respect to each of therequirements given below under Tests and Assay may bereleased for use. Provided that the test for viral inactivationhas been performed with satisfactory results on eachmonovalent pooled harvest and that the tests for freeformaldehyde, ovalbumin and total protein have beenperformed with satisfactory results on the final bulk vaccine,they may be omitted on the final lot.

Description. The vaccine is a slightly opalescent liquid.

Identification

The assay serves to confirm the antigenic specificity of thevaccine.

TestsViral inactivation. Inoculate 0.2 ml of the vaccine into theallantoic cavity of each of ten fertilised eggs and incubate at33° to 37° for 3 days. The test is not valid unless at least eightof the ten embryos survive. Harvest 0.5 ml of the allantoicfluid from each surviving embryo and pool the fluids. Inoculate0.2 ml of the pooled fluid into a further ten fertilised eggs andincubate at 33° to 37° for 3 days. The test is not valid unless atleast eight of the ten embryoes survive. Harvest about 0.1 mlof the allantoic fluid from each surviving embryo and examineeach individual harvest for live virus by a haemagglutinationtest. If haemagglutination is found for any of the fluids, carryout for that fluid a further passage in eggs and test forhaemagglutination; no haemagglutination occurs.

Total protein (2.3.49). Not more than six times the totalhaemagglutinin content of the vaccine as determined in theassay, but in any case, not more than 100 µg of protein pervirus strain per human dose and not more than a total of 300µg of protein per human dose.

Ovalbumin. Not more than 1 µg of ovalbumin per human dose,determined by a suitable technique using a suitable referencepreparation of ovalbumin.




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Bacterial endotoxins (2.2.3). Not more than 100 IU of endotoxinper human dose.

Assay

Determine the content of haemagglutinin antigen by animmunodiffusion test (2.2.14), by comparison with anappropriate haemagglutinin antigen reference preparation.Carry out the test at 20 to 25°. The confidence interval (P =0.95) of the assay is not greater than 80.0 per cent to 125.0 percent of the estimated content. The lower confidence limit (P =0.95) of the estimate of haemagglutinin antigen content is notless than 80.0 per cent of the amount stated on the label foreach strain.

For some vaccines, quantitative determination ofhaemagglutinin antigen with respect to available referencepreparations is not possible. An immunological identificationof the haemagglutinin antigen and a semi-quantitativedetermination are carried out instead by suitable methods.

Labelling. The label complies with the requirements statedunder Vaccines and also states (a) that the vaccine has beenprepared on eggs; (b) the strain or strains of influenza virusused to prepare the vaccine; (c) the method of inactivation;(d) the haemagglutinin content in µg per virus strain per dose;(e) the season during which the vaccine is intended to protect.

Inactivated Influenza Vaccine (SurfaceAntigen)Influenza Vaccine (Surface Antigen, Inactivated) is a sterile,aqueous suspension of a strain or strains of influenza virus,type A or B, or a mixture of strains of the two types grownindividually in eggs derived from specific pathogen free flocksor cell cultures, inactivated and treated so that the preparationconsists predominantly of haemagglutinin and neuraminidaseantigens, without diminishing the antigenic properties of theseantigens. The stated amount of haemagglutinin antigen foreach strain present in the vaccine is 15 µg per dose, unlessclinical evidence supports the use of a different amount.

ProductionThe production method is validated to demonstrate that theproduct, if tested, would comply with the test for safety andefficacy.


The World Health Organisation reviews the worldepidemiological situation annually and if necessaryrecommends new strains corresponding to prevailingepidemiological evidence.

The origin and passage history of virus strains shall be

approved by the competent authority.


Influenza virus seed to be used in the production of vaccine ispropagated in fertilised eggs from chicken flocks free fromspecified pathogens or in suitable cell cultures, such as chick-embryo fibroblasts or chick kidney cells obtained from chickenflocks free from specified pathogens. For production, the virusof each strain is grown in the allantoic cavity of eggs derivedfrom SPF flocks.

SEED LOT

The production of vaccine is based on a seed-lot system.Working seed lots represent not more than fifteen passagesfrom the approved reassorted virus or the approved virusisolate. The final vaccine represents one passage from theworking seed lot. The haemagglutinin and neuraminidaseantigens of each seed lot are identified as originating from thecorrect strain of influenza virus by suitable methods.Only a working virus seed lot that complies with the followingrequirements may be used in the preparation of the monovalentpooled harvest.Sterility (2.2.11). Carry out the test for sterility using 10 ml foreach medium.Mycoplasmas (2.7.4). Carry out the test for mycoplasmas using10 ml.


An antimicrobial agent may be added to the inoculum. Afterincubation at a controlled temperature, the allantoic fluids areharvested and combined to form a monovalent pooled harvest.An antimicrobial agent may be added at the time of harvest.At no stage in the production penicillin or streptomycin isused.


To limit the possibility of contamination, inactivation is initiatedas soon as possible after preparation. The virus is inactivatedby a method that has been demonstrated on three consecutivebatches to be consistently effective for the manufacturer. Theinactivation process shall have been shown to be capable ofinactivating the influenza virus without destroying itsantigenicity; the process should cause minimum alteration ofthe haemagglutinin and neuraminidase antigens. Theinactivation process shall also have been shown to be capableof inactivating avian leucosis viruses and mycoplasmas. Ifthe monovalent pooled harvest is stored after inactivation, itis held at a temperature of 5±3°. If formaldehyde solution isused, the concentration does not exceed 0.2 g/l offormaldehyde at any time during inactivation; ifbetapropiolactone is used, the concentration does not exceed

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0.1 per cent v/v at any time during inactivation.Before or after the inactivation process, the monovalentpooled harvest is concentrated and purified by high-speedcentrifugation or other suitable method. Virus particles aredisrupted into component subunits by approved proceduresand further purified so that the monovalent bulk consistsmainly of haemagglutinin and neuraminidase antigens.

Only a monovalent pooled harvest that complies with thefollowing requirements may be used in the preparation of thefinal bulk vaccine.Haemagglutinin antigen. Determine the content ofhaemagglutinin antigen by an immunodiffusion test (2.2.14),by comparison with a haemagglutinin antigen referencepreparation or with an antigen preparation calibrated againstit. Carry out the test at 20° to 25°.Neuraminidase antigen. The presence and type ofneuraminidase antigen are confirmed by suitable enzymatic orimmunological methods (2.2.14) on the first three monovalentpooled harvests from each working seed lot.

Sterility (2.2.11). Carry out the test for sterility, using 10ml for each medium.

Viral inactivation. Carry out the test described below underTests.Purity. The purity of the monovalent pooled harvest isexamined by polyacrylamide gel electrophoresis or by otherapproved techniques. Mainly haemagglutinin andneuraminidase antigens shall be present.Chemicals used for disruption and purification. Tests arecarried out on the monovalent pooled harvest for the chemicalsused for disruption and purification, the limits being approvedby the competent authority.

FINAL BULK VACCINE





FINAL LOT


Only a final lot that is satisfactory with respect to each of therequirements given below under Identification, Tests andAssay may be released for use. Provided that the test for viralinactivation has been performed with satisfactory results oneach monovalent pooled harvest and that the tests for freeformaldehyde, ovalbumin and total protein have beenperformed with satisfactory results on the final bulk vaccine,they may be omitted on the final lot.Description. The vaccine is a clear liquid.

IdentificationThe assay serves to confirm the antigenic specificity of thevaccine.

TestsViral inactivation. Inoculate 0.2 ml of the vaccine into theallantoic cavity of each of ten fertilised eggs and incubate at33° to 37° for 3 days. The test is not valid unless at least eightof the ten embryos survive. Harvest 0.5 ml of the allantoicfluid from each surviving embryo and pool the fluids. Inoculate0.2 ml of the pooled fluid into a further ten fertilised eggs andincubate at 33° to 37° for 3 days. The test is not valid unless atleast eight of the ten embryos survive. Harvest about 0.1 ml ofthe allantoic fluid from each surviving embryo and examineeach individual harvest for live virus by a haemagglutinationtest. If haemagglutination is found for any of the fluids, carryout for that fluid a further passage in eggs and test forhaemagglutination; no haemagglutination occurs.Total protein (2.3.49). Not more than 40 µg of protein otherthan haemagglutinin per virus strain per human dose and notmore than a total of 120 µg of protein other than haemagglutininper human dose.Ovalbumin. Not more than 1 µg of ovalbumin per human dose,determined by a suitable technique using a suitable referencepreparation of ovalbumin.Free formaldehyde (2.3.20). Complies with the test for freeformaldehyde as stated under General Requirements forVaccines for Human Use.Antimicrobial preservative. Where applicable, determine theamount of antimicrobial preservative by a suitable chemicalmethod. The content is not less than 85.0 per cent and notgreater than 115.0 per cent of the intended amount.




Assay

Determine the content haemagglutinin antigen by an

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immunodiffusion test (2.2.14), by comparison with anappropriate haemagglutinin antigen reference preparation.Carry out the test at 20° to 25°. The confidence interval(P = 0.95) of the assay is not greater than 80.0 per cent to 125.0per cent of the estimated content. The lower confidence limit(P = 0.95) of the estimate of haemagglutinin antigen content isnot less than 80.0 per cent of the amount stated on the labelfor each strain.

Labelling. The label states (1) that the vaccine has beenprepared on eggs; (2) the strain or strains of influenza virusused to prepare the vaccine; (3) the method of inactivation;(4) the haemagglutinin content in micrograms per virus strainper dose; (5) the season during which the vaccine is intendedto protect.

Inactivated Influenza Vaccine (WholeVirion)Influenza Vaccine (Whole Virion, Inactivated) is a sterile,aqueous suspension of a strain or strains of influenza virus,type A or B, or a mixture of strains of the two types grownindividually in eggs derived from specific pathogen free flocksor cell culture and inactivated in such a manner that theirantigenic properties are retained. The stated amount ofhaemagglutinin antigen for each strain present in the vaccineis 15 µg per dose, unless clinical evidence supports the use ofa different amount.

Production

The production method is validated to demonstrate that theproduct, if tested, would comply with the test for safety andefficacy.


The World Health Organisation reviews the worldepidemiological situation annually and, if necessary,recommends new strains corresponding to prevailingepidemiological evidence.

The origin and passage history of virus strains shall beapproved by the competent authority.


Influenza virus seed to be used in the production of vaccine ispropagated in fertilised eggs from chicken flocks free fromspecified pathogens or in suitable cell cultures, such as chick-embryo fibroblasts or chick kidney cells obtained from chickenflocks free from specified pathogens .For production, the virusof each strain is grown in the allantoic cavity of eggs derivedfrom specific pathogen free flocks.

SEED LOT

The production of vaccine is based on a seed-lot system.Working seed lots represent not more than fifteen passagesfrom the approved reassorted virus or the approved virusisolate. The final vaccine represents one passage from theworking seed lot. The haemagglutinin and neuraminidaseantigens of each seed lot are identified as originating from thecorrect strain of influenza virus by suitable methods.Only a working virus seed lot that complies with the followingrequirements may be used in the preparation of the monovalentpooled harvest.Sterility (2.2.11). Carry out the test for sterility using 10 ml foreach medium.Mycoplasmas (2.7.4). Carry out the test for mycoplasmas using10 ml.


An antimicrobial agent may be added to the inoculum. Afterincubation at a controlled temperature, the allantoic fluids areharvested and combined to form a monovalent pooled harvest.An antimicrobial agent may be added at the time of harvest.At no stage in the production is penicillin or streptomycinused.


To limit the possibility of contamination, inactivation is initiatedas soon as possible after preparation. The virus is inactivatedby a method that has been demonstrated on three consecutivebatches to be consistently effective for the manufacturer. Theinactivation process shall have been shown to be capable ofinactivating the influenza virus without destroying itsantigenicity; the process should cause minimum alteration ofthe haemagglutinin and neuraminidase antigens. Theinactivation process shall also have been shown to be capableof inactivating avian leucosis viruses and mycoplasmas. Ifthe monovalent pooled harvest is stored after inactivation, itis held at a temperature of 5±3°. If formaldehyde solution isused, the concentration does not exceed 0.2 g/l offormaldehyde at any time during inactivation; ifbetapropiolactone is used, the concentration does not exceed0.1 per cent v/v at any time during inactivation.

Before or after the inactivation process, the monovalentpooled harvest is concentrated and purified by high-speedcentrifugation or other suitable method.

Only a monovalent pooled harvest that complies with thefollowing requirements may be used in the preparation of thefinal bulk vaccine.

Haemagglutinin antigen. Determine the content ofhaemagglutinin antigen by an immunodiffusion test (2.2.14),by comparison with a haemagglutinin antigen reference

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preparation or with an antigen preparation calibrated againstit. Carry out the test at 20° to 25°.

Neuraminidase antigen. The presence and type ofneuraminidase antigen are confirmed by suitable enzymatic orimmunological methods (2.2.14) on the first three monovalentpooled harvests from each working seed lot.

Sterility (2.2.11). Carry out the test for sterility using 10 ml foreach medium.Viral inactivation. Carry out the test described below underTests.FINAL BULK VACCINE


Only a final bulk vaccine that complies with the followingrequirements may be used in the preparation of the final lot.Antimicrobial preservative. Where applicable, determine theamount of antimicrobial preservative by a suitable chemicalmethod. The content is not less than 85.0 per cent and notgreater than 115.0 per cent of the intended amount.Sterility (2.2.11). Carry out test for sterility using 10 ml ofbulk for each sterility medium.FINAL LOT


Only a final lot that is satisfactory with respect to each of therequirements given below under Tests and Assay may bereleased for use. Provided that the test for viral inactivationhas been performed with satisfactory results on eachmonovalent pooled harvest and that the tests for freeformaldehyde, ovalbumin and total protein have beenperformed with satisfactory results on the final bulk vaccine,they may be omitted on the final lot.

Description. The vaccine is a slightly opalescent liquid.

IdentificationThe assay serves to confirm the antigenic specificity of thevaccine.

Tests

Viral inactivation. Inoculate 0.2 ml of the vaccine into theallantoic cavity of each of ten fertilised eggs and incubate at33° to 37° for 3 days. The test is not valid unless at least eightof the ten embryos survive. Harvest 0.5 ml of the allantoicfluid from each surviving embryo and pool the fluids. Inoculate0.2 ml of the pooled fluid into a further ten fertilised eggs andincubate at 33° to 37° for 3 days. The test is not valid unless atleast eight of the ten embryos survive. Harvest about 0.1 ml of

the allantoic fluid from each surviving embryo and examineeach individual harvest for live virus by a haemagglutinationtest. If haemagglutination is found for any of the fluids, carryout for that fluid a further passage in eggs and test forhaemagglutination; no haemagglutination occurs.

Total protein (2.3.49). Not more than six times the totalhaemagglutinin content of the vaccine as determined in theassay, but in any case, not more than 100 µg of protein pervirus strain per human dose and not more than a total of 300µg of protein per human dose.

Ovalbumin. Not more than 1 µg of ovalbumin per human dose,determined by a suitable technique using a suitable referencepreparation of ovalbumin.Free formaldehyde (2.3.20). Maximum 0.2 g/l.

Antimicrobial preservative. Where applicable, determine theamount of antimicrobial preservative by a suitable chemicalmethod. The content is not less than the minimum amountshown to be effective and is not greater than 115.0 per cent ofthe quantity stated on the label.




Assay

Determine the content of haemagglutinin antigen by animmunodiffusion test (2.2.14), by comparison with anappropriate haemagglutinin antigen reference preparation.Carry out the test at 20° to 25°. The confidence interval (P =0.95) of the assay is not greater than 80.0 per cent to 125.0 percent of the estimated content. The lower confidence limit (P =0.95) of the estimate of haemagglutinin antigen content is notless than 80.0 per cent of the amount stated on the label foreach strain.

Labelling. The label states (1) that the vaccine has beenprepared on eggs; (2) the strain or strains of influenza virusused to prepare the vaccine; (3) the method of inactivation;(4) the haemagglutinin content in micrograms per virus strainper dose; (5) the season during which the vaccine is intendedto protect.

Japanese Encephalitis Vaccine(Human)Japanese Encephalitis Vaccine for human use is a liquid orfreeze dried preparation of Japanese encephalitis virus grownin approved substrate and inactivated by a validated method.

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Production

General provisions

The vaccine is produced on the basis of virus seed lot systemThe production method shall have been shown to yieldconsistently the vaccines that comply with the tests forimmunogenicity, safety and stability. The production methodis validated to demonstrate that the product, if tested, wouldcomply with the test for safety and efficacy.


The virus is propagated in an approved cell substrate like aVero cell line.

SEED LOT

The strain of Japanese encephalitis virus used shall beidentified by historical records that include information onthe origin of the strain and its subsequent manipulation.

The National Regulatory Authority shall determine theacceptable number of passages from the master virus seed lotto produce working virus seed lots.

Only a working seed lot that complies with the following testsmay be used for virus propagation.

Identification

Each working seed lot is identified as Japanese encephalitisvirus using specific antibodies by an approved method.

Virus concentration. The virus concentration of each workingseed lot is determined by a cell culture method usingimmunofluoresence or any other approved method.

Extraneous agents (2.7.3). The working seed lot complies withthe tests for the virus seed lots.


a) Mouse brain vaccine

The vaccine is prepared by using a seed-lot system. Anapproved strain of virus is grown by inoculating intracerebrallyinto healthy mice. Virus harvests are pooled, concentratedand inactivated by addition of formalin or any other suitableinactivating agent. It may contain a suitable preservative. Thevaccine may be issued in single or multidose containers.

b) Cell culture vaccine

All processing of the cell bank and subsequent cell culturesare done under aseptic conditions in an area where no othercells are handled. Approved animal (but not human) serummay be used in the media, but the final medium for maintainingcell growth during virus multiplication does not contain animalserum; the media may contain human albumin. Serum proteins,

if present are reduced to an acceptable level by suitable methodof purification. Serum and trypsin used in the preparation ofcell suspension and media are shown to be free from infectiousextraneous agents. The cell culture media may contain a pHindicator such as phenol red. Not less than 500 ml of the cellcultures employed for vaccine production are set aside asuninfected cell cultures (control cells). The virus suspensionis harvested on one or more occasions during incubation.Multiple harvests from the same production cell culture maybe pooled and considered as a single harvest.

Virus harvests that comply with the tests given underIdentification and Virus concentration are pooled in thepreparation of the inactivated viral harvest.

Control cells. The control cells of the production cell culturefrom which the single harvest is derived should comply withthe test for identification and with the tests for extraneousagents (2.7.3).

Purification and inactivation

The virus harvest may be concentrated and/or purified bysuitable methods; the virus harvest is inactivated by a validatedmethod at a fixed, well defined stage of the process which maybe before, during or after any concentration or purification.The method shall have been shown to be capable ofinactivating Japanese encephalitis virus without destructionof the immunogenic activity. If formalin is used, theconcentration shall at no time exceed 1:2000.

Only an inactivated viral suspension that complies with thefollowing tests may be used in the preparation of the finalbulk vaccine.

Inactivation. Inactivation is confirmed by carrying out anamplification test for residual infectious Japanese encephalitisvirus. Inoculate a quantity of inactivated viral suspensionequivalent to not less than 25 doses into cell cultures of thesame type as those used for production of the vaccine. Makea passage after 7 days. Maintain the cultures for a furtherperiod of 14 days and then examine the cell cultures forJapanese encephalitis virus using an immunofluorescence test.No Japanese encephalitis virus is detected. Alternatively, 5 mlof each culture fluid is pooled on day 14 and 21 and 0.03 ml isinoculated intracerebrally into each of the 10 mice weighingbetween 12 and 15 g. The mice are observed for 14 days forsymptoms caused by Japanese encephalitis virus, and miceshowing symptoms of Japanese encephalitis virus aresacrificed and virus presence is confirmed byimmunofluorescence test. No Japanese encephalitis virus shallbe detected.

Residual host-cell DNA (2.2.15). The content of residual host-cell DNA, determined using a suitable method, should not begreater than 10 ng per single human dose if cells are used inthe production.


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FINAL BULK VACCINE

The final bulk vaccine is prepared from one or more inactivatedviral suspensions. An approved stabilizer may be added tomaintain the activity of the product. Thiomersal can be usedas preservative.


Formaldehyde (2.3.20). Not more than 0.01 per cent w/v.



FINAL LOT

The final bulk vaccine is distributed aseptically into sterilecontainers. The containers are then sealed so as to preventcontamination.

Only a final lot that complies with each of the tests givenunder Identification, Tests and Assay may be released foruse. Provided that the test for inactivation has been carriedout with satisfactory results on the inactivated virussuspension and the test for bovine serum albumin has beencarried out with satisfactory results on the final bulk vaccine,these tests may be omitted on the final lot.

Identification

The vaccine is shown to contain Japanese encephalitis virusantigen by a suitable immunochemical method using specificantibodies, alternatively, the Assay also serves to identify thevaccine.

Tests

Complies with the test for Inactivation under final Purificationand Inactivation.


Bacterial endotoxins (2.2.3). Less than 25 IU per single humandose.


Bovine serum albumin (for cell culture vaccine). Not morethan 50 ng per single human dose, determined by a suitableimmunochemical method (2.2.14).

Residual host-cell DNA (for continuous cell line vaccines)(2.2.15). Not more than 10 ng per single human dose.

Free formaldehyde (2.3.20). Not more than 0.01 per cent w/v.

Antimicrobial preservative. Determine the amount ofantimicrobial preservative by a suitable chemical method. Thecontent is not less than the minimum amount shown to beeffective and is not greater than 115.0 per cent of that statedon the label.

Biological assay

Potency of Japanese encephalitis virus vaccine is determinedby titrating the neutralizing antibodies produced in theimmunized mice by plaque reduction method or serumneutralization test (SNT) using appropriate cell culture.


The Standard preparation is a freeze-dried Japaneseencephalitis virus vaccine the potency of which has beendetermined in relation to the Japanese encephalitis referencevaccine obtained from the National Institute of Health, Tokyo,Japan.

Suggested method

Preparation of challenge virus suspension

The approved challenge virus strain is stored in freeze-driedform or in liquid form stored below -70° . Prepare a workingpool of the challenge virus strain by inoculating intracerebrally0.03 ml of 100 fold dilution of the standard strain in Hanks’balanced salt solution containing 5 per cent calf serum into asuitable number of 2-day old suckling mice. Sacrifice theanimals after they show characteristic symptoms ofencephalitis and become moribund. Harvest their brainsaseptically, wash them in chilled sterile saline solution toremove blood clots. Homogenize the brains with Hanks’balanced salt solution containing 5 per cent calf serum tomake a 10 per cent emulsion. Centrifuge the emulsion at 2000g for 30 minutes. Dilute the supernatant with Hanks’ balancedsalt solution containing 5 per cent calf serum so as to containabout 200 Plaque-Forming Units (PFU) of the virus per 0.4 ml.

Determination of potency

Prepare appropriate dilutions of the vaccine under examinationand of the Standard Preparation in a suitable medium. Injectintraperitoneally in two doses of 0.5 ml each at 7-day intervalinto at least 20 mice of 4 weeks of age. Bleed each mouse 7days after the second injection, pool the separated serumfrom each group and inactivate the sera by heating at 56° for30 minutes. The inactivated sera may be stored at -20°, ifnecessary.

Dilute the sera appropriately, e.g. 1:40. 1:160, 1:640 etc., mixwith an equal volume of the challenge virus suspension andincubate at 37° for 90 minutes for neutralization. Inoculate themixture into cell cultures and overlay the infected cells with 1per cent agar. After incubation for an appropriate time (about


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48 hours), stain the cells and count the number of plaquesformed on the cultures to obtain the plaque reduction ratesfor the vaccine under examination and the Standardpreparation. Calculate the neutralizing antibody titres for eachgroup using standard statistical methods (5.7). The test is notvalid unless (a) the mean number of plaques obtained with theStandard preparation is between 100 and 150 per dish and (b)the potency of the vaccine under examination is not less thanthat of the Standard preparation.

Labelling. The label states (1) the biological origin of the cellsused for the preparation of the vaccine; (2) the strain of virusused.

Measles and Rubella Vaccine (Live)Measles and Rubella Vaccine (Live) is a freeze-dried preparationof suitable attenuated strains of measles virus and rubellavirus grown in suitable cell cultures.

The vaccine is reconstituted immediately before use to give aclear liquid that may be coloured owing to the presence of apH indicator.

Production

General provisions

The two components are prepared as described in themonographs on Measles vaccine (live) and Rubella vaccine(live) and comply with the tests prescribed therein.


FINAL BULK VACCINE

Virus harvests for each component are pooled and clarified toremove cells. A suitable stabilizer may be added and the pooledharvests diluted as appropriate. Suitable quantities of thepooled harvest for each component are mixed.



FINAL LOT

For each component, a minimum virus concentration forrelease of the product is established such as to ensure, in thelight of stability data, that the minimum concentration statedon the label will be present at the end of the period of validity.

Only a final lot that complies with the tests for minimum virusconcentration of each component for release, with the

following test for thermal stability and with each of the testsgiven below under Identification and Tests and Assay may bereleased for use. Provided that the test for bovine serum albuminhas been carried out with satisfactory results on the final bulkvaccine, it may be omitted on the final lot.

Thermal stability. Maintain samples of the final lot of freeze-dried vaccine in the dry state at 37° for 7 days. Determine thevirus concentration as described under Assay in parallel forthe vaccine held at 37° for 7 days and for vaccine stored at 2°to 8°. For each component, the virus concentration of theheated vaccine is not more than 1.0 log10 lower than that of theunheated vaccine.

IdentificationWhen the vaccine reconstituted as stated on the label is mixedwith antibodies specific for measles virus and rubella virus, itis no longer able to infect cell cultures susceptible to theseviruses. When the vaccine reconstituted as stated on thelabel is mixed with quantities of specific antibodies sufficientto neutralize any one viral components, the second viralcomponent infects susceptible cell cultures.

Tests

Water (2.3.43). Not more than 3.0 per cent, determined by KarlFischer, semi-micro determination of water or by any suitablevalidated method.

Sterility (2.2.11). The reconstituted vaccine complies with thetest for sterility.


Bovine serum albumin. Not more than 50 ng per single humandose, determined by a suitable immunochemical method(2.2.14).

Assay

A. Mix the vaccine with a sufficient quantity of antibodiesspecific for rubella virus.Titrate the vaccine for infectivemeasles virus at least in triplicate, using at least eight cellcultures for each dilution 0.5 log10 step or by a method ofequal precision. Use an appropriate virus reference preparationto validate each assay. The estimated measles virusconcentration is not less than that stated on the label; theminimum measles virus concentration stated on the label isnot less than 1x103 CCID50 per single human dose. The assayis not valid if the confidence limits (P = 0.95) of the logarithmof the virus concentration are greater than ± 0.3.

Measles vaccine (Live) RS is suitable for use as a referencepreparation.

B. Titrate the vaccine for infective rubella virus at least intriplicate, using at least eight cell cultures for each 0.5 log10

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dilution step or by a method of equal precision. Use anappropriate virus reference preparation to validate each assay.The estimated rubella virus concentration is not less than thatstated on the label; the minimum rubella virus concentrationstated on the label are not less than 1x103 CCID50 per singlehuman dose. The assay is not valid if the confidence limits(P = 0.95) of the logarithm of the virus concentration are greaterthan ± 0.3.

Rubella vaccine (Live) RS is suitable for use as a referencepreparation.

Labelling. The label states (1) the strains of virus used in thepreparation of the vaccine; (2) the type and origin of the cellsused for the preparation of the vaccine; (3) the minimum virusconcentration for each component of the vaccine; (4) the timewithin which the vaccine must be used after reconstitution;(5) that the vaccine must not be given to a pregnant womanand that a woman should not become pregnant within twomonths after having the vaccine.

Measles Vaccine (Live)Measles Vaccine (Live) is a freeze-dried preparation of asuitable attenuated strain of measles virus. The vaccine isreconstituted immediately before use, as stated on the label,to give a clear liquid that may be coloured owing to thepresence of a pH indicator.

Production

General provisions

The production of vaccine is based on a virus seed-lot systemand, if the virus is propagated in human diploid cells, a cell-bank system. Unless otherwise justified and authorized, thevirus in the final vaccine shall have undergone no morepassages from the master seed lot than were used to preparethe vaccine shown in clinical studies to be satisfactory withrespect to abnormal toxicity and efficacy; even with authorizedexceptions, the number of passages beyond the level used forclinical studies shall not exceed five.

The production method is validated to demonstrate that theproduct, if tested, would comply with the tests for abnormaltoxicity and efficacy.


The virus is propagated in human diploid cells or in culturesof chick embryo cells derived from a chicken flock free fromspecified pathogens.

SEED LOT

The strain of measles virus used in the production of measlesvaccine shall be identified by historical records that include

information on the origin of the strain and its subsequentmanipulation. Virus seed lots are prepared in large quantitiesand stored at temperatures below -20° if freeze-dried, or below-60° if not freeze-dried.

Only a seed lot that complies with the following tests may beused for virus propagation.

IdentificationThe master and working seed lots are identified as measlesvirus by serum neutralization in cell culture, using specificantibodies.

Virus concentration. The virus concentration of the masterand working seed lots is determined to monitor consistencyof production.

Extraneous agents (2.7.3). The working seed lot complies withthe tests for seed lots.

Neurovirulence (2.7.5). The master/working seed lot complieswith the test for neurovirulence of live virus vaccines. Macacaand Cercopithecus monkeys susceptible to measles virus aresuitable for the test.


All processing of the cell bank and subsequent cell cultures isdone under aseptic conditions in an area where no other cellsare handled. Suitable animal (but not human) serum may beused in the growth medium, but the final medium for maintainingcell growth during virus multiplication does not contain animalserum. Serum and trypsin used in the preparation of cellsuspensions and culture media are shown to be free fromextraneous agents. The cell culture medium may contain a pHindicator such as phenol red and suitable antibiotics at thelowest effective concentration. It is preferable to have asubstrate free from antibiotics during production. Not lessthan 500 ml of the production cell culture is set aside asuninfected cell cultures (control cells). The viral suspensionsare harvested at a time appropriate to the strain of virus beingused.

Only a single harvest that complies with the following testsmay be used in the preparation of the final bulk vaccine.

Identification

The single harvest contains virus that is identified as measlesvirus by serum neutralisation in cell culture, using specificantibody.Virus concentration. The virus concentration in the singleharvest is determined as prescribed under Assay to monitorconsistency of production and to determine the dilution to beused for the final bulk vaccine.Extraneous agents (2.7.3). Complies with the test for extraneousagents.

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Control cells. If human diploid cells are used for production,the control cells comply with the test for identification andextraneous agents.

FINAL BULK VACCINE

Virus harvests that comply with the above tests are pooledand clarified to remove cells. A suitable stabilizer may be addedand the pooled harvests diluted as appropriate.


Sterility (2.2.11). The final bulk vaccine complies with the testfor sterility carried out using 10 ml for each medium.

FINAL LOT

A minimum virus concentration for release of the product isestablished so as to ensure, in the light of stability data, thatthe minimum concentration stated on the label will be presentat the end of the period of validity.

Only a final lot that complies with the tests for minimum virusconcentration for release, with the following requirement forthermal stability and with each of the requirements given belowunder Identification, Tests and Assay may be released foruse. Provided that the test for bovine serum albumin has beencarried out with satisfactory results on the final bulk vaccine,it may be omitted on the final lot.

Thermal stability. Maintain samples of the final lot of freeze-dried vaccine in the dry state at 37° for 7 days. Determine thevirus concentration as described under Assay in parallel forthe vaccine held at 37° for 7 days and for vaccine stored at 2°to 8°. The virus concentration of the heated vaccine is notmore than 1.0 log10 lower than that of the unheated vaccine.

Identification

When the vaccine reconstituted as stated on the label is mixedwith specific measles antibodies, it is no longer able to infectsusceptible cell cultures.

Tests


Sterility ( 2.2.11). The reconstituted vaccine complies withthe test for sterility.

Abnormal toxicity ( 2.2.1). Complies with the test for abnormaltoxicity.


Assay

Titrate the vaccine for infective virus at least in triplicate,using at least five cell cultures for each 0.5 log10 dilution stepor by a method of equal precision. Use an appropriate virusreference preparation to validate each assay. The estimatedvirus concentration is not less than that stated on the label;the minimum virus concentration stated on the label is notless than 1 × 103 CCID50 per human dose. The assay is notvalid if the confidence limits (P = 0.95) of the logarithm of thevirus concentration is greater than ± 0.3.


Labelling. The label states (1) the strain of virus used for thepreparation of the vaccine; (2) the type and origin of the cellsused for the preparation of the vaccine; (3) the minimum virusconcentration; (4) the time within which the vaccine must beused after reconstitution.

Measles, Mumps and Rubella Vaccine(Live)Measles, Mumps and Rubella Vaccine (Live) is a freeze-driedpreparation of suitable attenuated strains of measles virus,mumps virus and rubella virus grown in suitable cell cultures.

The vaccine is reconstituted immediately before use to give aclear liquid that may be coloured owing to the presence of apH indicator.

Production

General provisions

The three components are prepared as described in themonographs on Measles Vaccine (Live), Mumps Vaccine (Live)and Rubella Vaccine (Live) and comply with the testsprescribed therein.The production method is validated to demonstrate that theproduct, if tested, would comply with the test for safety andefficacy.

FINAL BULK VACCINE

Virus harvests for each component are pooled and clarified toremove cells. A suitable stabilizer may be added and the pooledharvests diluted as appropriate. Suitable quantities of thepooled harvest for each component are mixed.Only a final bulk vaccine that complies with the followingrequirements may be used in the preparation of the final lot.Sterility (2.2.11). Carry out the test for sterility using 10 ml foreach medium.

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FINAL LOT

For each component, a minimum virus concentration forrelease of the product is established such as to ensure, in thelight of stability data, that the minimum concentration statedon the label will be present at the end of the period of validity.

Only a final lot that complies with the tests for minimum virusconcentration of each component for release, with thefollowing requirement for thermal stability and with each ofthe requirements given below under Identification and Testsmay be released for use. Provided that the test for bovineserum albumin has been carried out with satisfactory resultson the final bulk vaccine, it may be omitted on the final lot.

Thermal stability. Maintain samples of the final lot of freeze-dried vaccine in the dry state at 37° for 7 days. Determine thevirus concentration as described under Assay in parallel forthe vaccine held at 37° for 7 days and for vaccine stored at 2 to8°. The virus concentration of the heated vaccine is not morethan 1.0 log10 lower than that of the unheated vaccine.

Identification

When the vaccine reconstituted as stated on the label is mixedwith antibodies specific for measles virus, mumps virus andrubella virus, it is no longer able to infect cell culturessusceptible to these viruses. When the vaccine reconstitutedas stated on the label is mixed with quantities of specificantibodies sufficient to neutralize any two viral components,the third viral component infects susceptible cell cultures.

Tests





Assay

A. Mix the vaccine with a sufficient quantity of antibodiesspecific for mumps virus and rubella virus.Titrate the vaccinefor infective measles virus at least in triplicate, using at leasteight cell cultures for each dilution 0.5 log10 step or by a methodof equal precision. Use an appropriate virus referencepreparation to validate each assay. The estimated measlesvirus concentration is not less than that stated on the label;the minimum measles virus concentration stated on the label

is not less than 1x103 CCID50 per single human dose. Theassay is not valid if the confidence limits (P = 0.95) of thelogarithm of the virus concentration are greater than ± 0.3.


B. Mix the vaccine with a sufficient quantity of antibodiesspecific for measles virus and rubella virus. Titrate the vaccinefor infective mumps virus at least in triplicate, using at leasteight cell cultures for each dilution 0.5 log10 step or by a methodof equal precision. Use an appropriate virus referencepreparation to validate each assay. The estimated mumps virusconcentration is not less than that stated on the label; theminimum mumps virus concentration stated on the label is notless than 5 x 103 CCID50 per single human dose. The assay isnot valid if the confidence limits (P = 0.95) of the logarithm ofthe virus concentration are greater than ± 0.3.Mumps vaccine (Live) RS is suitable for use as a referencepreparation.C. Mix the vaccine with a sufficient quantity of antibodiesspecific for mumps virus. Titrate the vaccine for infectiverubella virus at least in triplicate, using at least eight cellcultures for each dilution 0.5 log10 step or by a method ofequal precision. Use an appropriate virus reference preparationto validate each assay. The estimated rubella virusconcentration is not less than that stated on the label; theminimum rubella virus concentration stated on the label is notless than 1x103 CCID50 per single human dose. The assay isnot valid if the confidence limits (P = 0.95) of the logarithm ofthe virus concentration are greater than ± 0.3.


Labelling. The label states (1) the strains of virus used in thepreparation of the vaccine; (2) the type and origin of the cellsused for the preparation of the vaccine; (3) the minimum virusconcentration for each component of the vaccine; (4) the timewithin which the vaccine must be used after reconstitution;(5) that the vaccine must not be given to a pregnant womanand that a woman should not become pregnant within twomonths after having the vaccine.

Meningococcal PolysaccharideVaccineMeningococcal Polysaccharide Vaccine is a freeze-driedpreparation of one or more purified capsular polysaccharidesobtained from one or more suitable strains of Neisseriameningitidis group A, group C, group Y and group W135 thatare capable of consistently producing polysaccharides knownto be safe and effective in man.

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N. meningitidis group A polysaccharide consists of partlyO-acetylated repeating units of N-acetylmannosamine, linkedwith 1α→6 phosphodiester bonds.

N. meningitidis group C polysaccharide consists of partlyO-acetylated repeating units of sialic acid, linked with 2α→9glycosidic bonds.

N. meningitidis group Y polysaccharide consists of partlyO-acetylated alternating units of sialic acid and D-glucose,linked with 2α→6 and 1α→4 glycosidic bonds.

N. meningitidis group W135 polysaccharide consists of partlyO-acetylated alternating units of sialic acid and D-galactose,linked with 2α→6 and 1α→4 glycosidic bonds.

Production

General provisions

Production of the meningococcal polysaccharides is basedon a well defined seed-lot system. The method of productionshall have been shown to yield consistently meningococcalpolysaccharide vaccines of satisfactory immunogenicity andsafety for man.

The production method is validated to demonstrate that theproduct, if tested, would comply with the test of abnormaltoxicity for antisera and vaccines.

SEED LOT

The strains of N. meningitidis used for the master seed lotsshall be identified by historical records that include informationon their origin and by their biochemical, serological,physicochemical or molecular characteristics. Cultures fromthe working seed lot shall have the same characteristics as thestrain that was used to prepare the master seed lot. The strainshave the following characteristics:

a) Colonies obtained from a culture are round, uniform inshape and smooth with a mucous, opalescent, greyishappearance.

b) Gram staining reveals characteristic Gram-negativediplococci in ‘coffee-bean’ arrangement.

c) The oxidase test is positive.

d) The culture utilizes glucose and maltose.

e) Suspensions of the culture agglutinate with specificantisera of known titre.


The working seed lots are cultured on solid media that do notcontain blood-group substances or ingredients of mammalianorigin. The inoculum may undergo one or more subcultures inliquid medium before being used for inoculating the final

medium. The liquid media used and the final medium aresemisynthetic and free from substances precipitated bycetrimonium bromide (hexadecyltrimethylammoniumbromide) and do not contain blood-group substances or high-molecular-mass polysaccharides. The bacterial purity of theculture is verified by microscopic examination of Gram-stainedsmears and by inoculation into appropriate media. The culturesare centrifuged and the polysaccharides precipitated from thesupernatant by addition of cetrimonium bromide. Theprecipitate obtained is harvested and may be stored at orbelow -20° awaiting further purification.

PURIFIED POLYSACCHARIDES

The polysaccharides are purified, after dissociation of thecomplex of polysaccharide and cetrimonium bromide, usingsuitable procedures to remove successively nucleic acids,proteins and lipopolysaccharides. The purification stepconsists of ethanol precipitation of the polysaccharides orpurification with chloroform and n-butanol or by cold phenoltreatment, which are then dried and stored at or below -20°.The loss on drying is determined by thermogravimetry, KarlFischer or any other suitable method and the value is used tocalculate the results of the other chemical tests with referenceto the dried substance.

Only purified polysaccharides that tested comply with thefollowing requirements may be used in the preparation of thefinal bulk vaccine.

Protein (2.7.1). Not more than 10 mg of protein per gram ofpurified polysaccharidefor group A and C organisms and lessthan 50 mg of protein per gram of polysaccharide for group Yand W135 calculated using bovine plasma albumin as areference or other methods approved by National RegulatoryAuthority.

Nucleic acids (2.7.1). Not more than 10 mg of nucleic acids pergram of purified polysaccharide, calculated with reference tothe dried substance.

O-Acetyl groups (2.7.1). Not less than 2 mmol of O-acetylgroups per gram of purified polysaccharide for group A, notless than 1.5 mmol per gram of polysaccharide for group C,not less than 0.3 mmol per gram of polysaccharide for groupsY and W135, all calculated with reference to the driedsubstance.

Phosphorus (2.7.1). Not less than 80 mg of phosphorus pergram of group A purified polysaccharide, calculated withreference to the dried substance.

Sialic acid (2.7.1). Not less than 800 mg of sialic acid per gramof group C polysaccharide and not less than 560 mg of sialicacid per gram of purified polysaccharide for groups Y andW135, all calculated with reference to the dried substance.Use the following reference solutions:


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Group C polysaccharide. A 150 mg/l solution of N-acetylneuraminic acid.

Group Y polysaccharide. A solution containing 95 mg/l of N-acetylneuraminic acid and 55 mg/l of glucose.

Group W135 polysaccharide. A solution containing 95 mg/lof N-acetylneuraminic acid and 55 mg/l of galactose.

Calcium. If a calcium salt is used during purification,determination of calcium is carried out on the purifiedpolysaccharide by a suitable method; the content is withinthe limits approved for the product.

Molecular size. Examine by gel filtration or high performancesize-exclusion chromatography (HPSEC) (2.4.16), usingagarose for chromatography or cross-linked agarose forchromatography either alone or in combination with lightscattering and refractive index detector (e.g. multiple angleLASER light scattering, MALLS) or any other suitable method.Use a column 0.9 m x 15 mm equilibrated with a solvent havingan ionic strength of 0.2 mol/kg and a pH of 7.0 to 7.5. Apply tothe column about 2.5 mg of polysaccharide in a volume ofabout 1.5 ml and elute at about 20 ml/h. Collect fractions ofabout 2.5 ml and determine the content of polysaccharide bya suitable method.

At least 65.0 per cent of group A polysaccharide, 75.0 per centof group C polysaccharide, 80.0 per cent of group Ypolysaccharide and 80.0 per cent of group W135polysaccharide is eluted before a distribution coefficient (K0)of 0.50 is reached. In addition, the percentages eluted beforethis distribution coefficient are within the limits approved forthe particular product.

Identification and serological specificity

The identity and serological specificity are determined by asuitable immunochemical method (2.2.14). Identity and purityof each polysaccharide shall be confirmed; it shall be shownthat there is not more than 1.0 per cent m/m of group-heterologous N. meningitidis polysaccharide.

Pyrogens (2.2.8). Complies with the test for pyrogens. Injecteach of the rabbit with 1ml per kg body weight of solutioncontaining

a) 0.025 µg of polysaccharide for a monovalent vaccine,

b) 0.050 µg of polysaccharide for a bivalent vaccine,

c) 0.075 µg of polysaccharide for a trivalent vaccine,

d) 0.100 µg of polysaccharide for a tetravalent vaccine.

FINAL BULK VACCINE

One or more purified polysaccharides of one or more N.meningitidis groups are dissolved in a suitable solvent thatmay contain a stabilizer.

Only a final bulk vaccine that complies with the followingrequirement may be used in the preparation of the final lot.


FINAL LOT

The final bulk vaccine is distributed aseptically into sterilecontainers. The containers are then closed so as to avoidcontamination. Only a final lot that is satisfactory with respectto each of the requirements prescribed below underIdentification, Tests and Assay may be released for use.

IdentificationCarry out an identification test for each polysaccharide presentin the vaccine by a suitable immunochemical method (2.2.14).



Pyrogens (2.2.8). Complies with the test for pyrogens. Injecteach of the rabbit with 1ml per kg body weight of solutioncontaining

a) 0.025 µg of polysaccharide for a monovalent vaccine,

b) 0.050 µg of polysaccharide for a bivalent vaccine,

c) 0.075 µg of polysaccharide for a trivalent vaccine,

d) 0.100 µg of polysaccharide for a tetravalent vaccine.

Water (2.3.43). Not more than 3.0 per cent, of moisture contentby thermogravimetery, Karl Fischer or any other suitablemethod.Molecular size. Examine by gel filtration or size-exclusionchromatography (2.4.16). Use a column about 0.9 m long and16 mm in internal diameter equilibrated with a solvent havingan ionic strength of 0.2 mol/kg and a pH of 7.0 to 7.5. Apply tothe column about 2.5 mg of each polysaccharide in a volumeof about 1.5 ml and elute at about 20 ml/h. Collect fractions ofabout 2.5 ml and determine the content of polysaccharide bya suitable method.Use cross-linked agarose for chromatography and apply asuitable immunochemical method (2.2.14) to establish theelution pattern of the different polysaccharide(s). The vaccinecomplies with the test if:a) 65.0 per cent of Group A polysaccharide is eluted before

K0 of 0.50,

b) 75.0 per cent of Group C polysaccharide is eluted beforeK0 of 0.50,

c) 80.0 per cent of Group Y & W135 polysaccharide is elutedbefore K0 of 0.50.


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For a tetravalent vaccine (group A + group C + group Y andgroup W135), use cross linked agarose for chromatographyR1 and apply a suitable immunochemical method (2.2.14) toestablish the elution pattern of the different polysaccharides.The vaccine complies with the test if K0 for the principal peakis

a) not greater than 0.70 for group A and group Cpolysaccharide,

b) not greater than 0.57 for group Y polysaccharide,

c) not greater than 0.68 for group W135 polysaccharide.

Assay

Carry out an assay as stated under each polysaccharidepresent in the vaccine.

For a divalent vaccine (group A + group C), use measurementof phosphorus (2.7.1) to determine the content ofpolysaccharide A and measurement of sialic acid (2.7.1) todetermine the content of polysaccharide C. To determine sialicacid, use as reference solution a 150 mg/l solution of N-acetylneuraminic acid.

For a tetravalent vaccine (group A + group C + group Y +group W135) a suitable immunochemical method (2.2.14) isused with a reference preparation of purified polysaccharidefor each group.

The vaccine contains not less than 70.0 per cent and not morethan 130.0 per cent of the quantity of each polysaccharidestated on the label.

Labelling. The label states (1) the group or groups ofpolysaccharides (A, C, Y or W135) present in the vaccine; (2)the number of µg of polysaccharide per human dose.

Mumps Vaccine (Live)Mumps Vaccine (Live) is a freeze-dried preparation of a suitableattenuated strain of mumps virus. The vaccine is reconstitutedimmediately before use to give a clear liquid that may becoloured owing to the presence of a pH indicator.

Production

General provisions

The production of vaccine is based on a virus seed-lot systemand, if the virus is propagated in human diploid cells, a cell-bank system. The production method shall have been shownto yield consistently live mumps vaccines of adequateimmunogenicity and safety in man.

Unless otherwise justified and authorised, the virus in thefinal vaccine shall have undergone no more passages fromthe master seed lot than were used to prepare the vaccine

shown in clinical studies to be satisfactory with respect tosafety and efficacy even with authorized exceptions, thenumber of passages beyond the level used for clinical studiesshall not exceed five.

The production method is validated to demonstrate that theproduct, if tested, would comply with the tests for safety andefficacy.


The virus is propagated in human diploid cells or in primarycultures of chick embryo cells derived from a chicken flockfree from specified pathogens.

SEED LOT

The strain of mumps virus used shall be identified by historicalrecords that include information on the origin of the strainand its subsequent manipulation. To avoid unnecessary useof monkeys in the test for neurovirulence, Virus seed lots areprepared in large quantities and stored at temperatures below-20° if freeze-dried, or below -60° if not freeze-dried.

Only a seed lot that complies with the following tests may beused for virus propagation.

Identification

The master and working seed lots are identified as mumpsvirus by serum neutralisation in cell culture, using specificantibodies.

Virus concentration. The virus concentration of the masterand working seed lots is determined to ensure consistency ofproduction.Extraneous agents (2.7.3). The working seed lot complies withthe tests for seed lots.

Neurovirulence (2.7.5). The master/working seed lot complieswith the test for neurovirulence of live virus vaccines. Macacaand Cercopithecus monkeys are suitable for the test.


All processing of the cell bank and subsequent cell cultures isdone under aseptic conditions in an area where no other cellsare handled. Suitable animal (but not human) serum may beused in the growth media. Serum and trypsin used in thepreparation of cell suspensions and culture media are shownto be free from extraneous agents. The cell culture mediummay contain a pH indicator such as phenol red and suitableantibiotics at the lowest effective concentration. It is preferableto have a substrate free from antibiotics during production.Not less than 500 ml of the production cell culture is set asideas uninfected cell culture (control cells). The viral suspensionsare harvested at a time appropriate to the strain of virus beingused.

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Only a single harvest that complies with the followingrequirements may be used in the preparation of the final bulkvaccine.

Identification

The single harvest contains virus that is identified as mumpsvirus by serum neutralization in cell culture, using specificantibodies.

Virus concentration. The virus concentration in the singleharvest is determined as prescribed under Assay to monitorconsistency of production and to determine the dilution to beused for the final bulk vaccine.

Sterility (2.2.11). Single harvest complies with sterility testshould be processed further.

Control cells. The control cells comply with a test forextraneous agents (2.7.3).

FINAL BULK VACCINE

Single harvests that comply with the above tests are pooledand clarified to remove cells. A suitable stabiliser may be addedand the pooled harvests diluted as appropriate.Only a final bulk vaccine that complies with the followingrequirements may be used in the preparation of the final lot.Sterility (2.2.11). The final bulk vaccine complies with the testfor sterility, carried out using 10 ml for each medium.

FINAL LOT

A minimum virus concentration for release of the product isestablished such as to ensure, in the light of stability data,that the minimum concentration stated on the label will bepresent at the end of the period of validity.Only a final lot that complies with the tests for minimum virusconcentration for release, with the following requirement forthermal stability and with each of the requirements given belowunder Identification and Tests and Assay may be released foruse. Provided that the test for bovine serum albumin has beencarried out with satisfactory results on the final bulk vaccine,it may be omitted on the final lot.Thermal stability. Maintain samples of the final lot of freeze-dried vaccine in the dry state at 37° for 7 days. Determine thevirus concentration as described under Assay in parallel forthe vaccine held at 37° for 7 days and for vaccine stored at 2°to 8°. The virus concentration of the vaccine exposed to 37°for 7 days is not more than 1.0 log10 lower than that of theunheated vaccine.

IdentificationWhen the vaccine is reconstituted as stated on the label ismixed with specific mumps antibodies, it is no longer able toinfect susceptible cell cultures.

TestsWater (2.3.43). Not more than 3.0 per cent, determined by KarlFischer, semi-micro determination of water or by any suitablevalidated method.Sterility ( 2.2.11). Complies with the test for sterility.Abnormal toxicity (2.2.1). Complies with the test for abnormaltoxicity.


Assay

Titrate the vaccine for infective virus at least in triplicate,using at least five cell cultures for each 0.5 log10 dilution stepor by a method of equal precision. Use an appropriate virusreference preparation to validate each assay. The estimatedvirus concentration is not less than that stated on the label;the minimum virus concentration stated on the label is notless than 5 x 103 CCID50 per human dose. The assay is notvalid if the confidence limits (P = 0.95) of the logarithm of thevirus concentration is greater than 0.3.Mumps vaccine (Live) RS is suitable for use as a referencepreparation.Labelling. The label states (1) the strain of virus used for thepreparation of the vaccine; (2) the type and origin of the cellsused for the preparation of the vaccine; (3) the minimum virusconcentration and; (4) the time within which the vaccine mustbe used after reconstitution.

Pertussis VaccinePertussis Vaccine is a sterile saline suspension of inactivatedwhole cells of one or more strains of Bordetella pertussis.

Production

General provisions

Inactivated B. pertussis suspension

Production is based on a seed-lot system. One or more strainsof B. pertussis with known origin and history are used. Strains,culture medium and cultivation method are chosen in such away that agglutinogens 1, 2 and 3 are present in the finalvaccine. Each strain is grown for 24 to 72 hours in a liquidmedium or on a solid medium; the liquid medium used in thefinal cultivation stage does not contain blood or bloodproducts. Human blood or blood products are not used in anyculture media. The bacteria are harvested, washed to removesubstances derived from the medium and suspended in a0.9 per cent w/v solution of sodium chloride or other suitable

PERTUSSIS VACCINE

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isotonic solution. The opacity of the suspension is determinednot later than 2 weeks after harvest by comparison with thereference preparation of Opacity and used as the basis ofcalculation for subsequent stages in vaccine preparation.Single harvests are not used for the final bulk vaccine unlessthey have been shown to contain B. pertussis cells with thesame characteristics with regard to growth and agglutinogens,as the parent strain and to be free from contaminating bacteriaand fungi. The bacteria are killed and detoxified in controlledconditions by means of a suitable chemical agent or by heatingor by a combination of these methods. Freedom from live B.pertussis is tested using a suitable culture medium. Thesuspension is maintained at 5 + 3° for a suitable period todiminish its toxicity.

FINAL BULK VACCINE

Suitable quantities of the inactivated single harvests are pooledto prepare the final bulk vaccine. Suitable antimicrobialpreservatives may be added. The bacterial concentration ofthe final bulk vaccine does not exceed that corresponding toan opacity of 20 IU per single human dose. If 2 or more strainsof B. pertussis are used, the composition of consecutive lotsof the final bulk vaccine shall be consistent with respect tothe proportion of each strain as measured in opacity units.


Antimicrobial preservative. Where applicable, determine theamount of antimicrobial preservative by a suitable chemicalmethod. The amount is not less than 85.0 per cent and notgreater than 115.0 per cent of the intended amount.

Sterility (2.2.11). Carry out test for sterility using 10 ml ofbulk for each sterility medium.FINAL LOT


Only a final lot that is satisfactory with respect to each of therequirements given below under Identification, Tests andAssay may be released for use. Provided the tests for specifictoxicity, free formaldehyde and antimicrobial preservative andthe assay have been carried out with satisfactory results onthe final bulk vaccine, they may be omitted on the final lot.

Identification

Identify pertussis vaccine by agglutination of the bacteria inthe vaccine by antisera specific to B. pertussis.

TestsSpecific toxicity

Use not less than 10 healthy mice each weighing between 14

to 16 g for the vaccine group and for the saline control. Usemice of the same sex or distribute males and females equallybetween the groups. Allow the animals access to food andwater for at least 2 hours before injection and during the test.Inject each mouse of the vaccine group intraperitoneally with0.5 ml, containing a quantity of the vaccine equivalent to notless than half the single human dose. Inject each mouse of thecontrol group with 0.5 ml of a 0.9 per cent w/v sterile solutionof sodium chloride, preferably containing the same amountof antimicrobial preservative as that injected with the vaccine.Weigh the groups of mice immediately before the injection, 72hours and 7 days after the injection. The vaccine complieswith the test if (a) at the end of 72 h the total mass of the groupof vaccinated mice is not less than that preceding the injection;(b) at the end of 7 days the average increase in mass pervaccinated mouse is not less than 60.0 per cent of that percontrol mouse; and (c) not more than 5.0 per cent of thevaccinated mice die during the test. The test may be repeatedand the results of the tests combined.


Antimicrobial preservative. Where applicable, determine theamount of antimicrobial preservative by a suitable chemicalmethod. The content is not less than 85.0 per cent and notmore than 115.0 per cent of the intended amount.Sterility (2.2.11). Complies with the test for sterility.Assay

Carry out the assay of Pertussis Vaccine as described below :

Biological assay of pertussis vaccine

The potency of PertussisVaccine is determined by comparisonof the dose necessary to protect mice against the effects of alethal dose of Bordetella pertussis challenge culture,administered intracerebrally, with the dose of a referencepreparation, calibrated in International Units, required to givethe same level of protection. For this comparison, the Standardpreparation of Pertussis vaccine & a suitable strain of B.pertussis (e.g.18323, to be used as challenge strain), arerequired.

Reference preparation

The reference preparation is an International standard ofPertussis vaccine, consisting of a freeze dried vaccine oranother suitable preparation, calibrated in comparison toInternational standard, from time to time. The InternationalUnit is the activity contained in a stated amount of theInternational standard, which consists of a quantity of driedpertussis vaccine. The equivalence in International Units ofthe International Standard is stated by the World HealthOrganization.

Use healthy mice of a suitable strain, weighing between 13and 16 g from the same stock. Distribute the mice randomly, in

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six to eight groups of not less than 16 and not more than 24and four groups of 10. The mice should all be of the same sexor the males and females should be distributed equally betweenthe groups. Half of the groups of 16 to 24 should receive thereference preparation and the other half should receive thevaccine under examination. The four groups of 10 each shouldbe used for the LD50 titration of challenge suspension.

Use at least, three dilutions of the reference vaccine and similardilutions of the vaccine under examination. In each case thedilutions are so selected that the dilution protecting 50 percent of the mice (ED50) is as near as possible to middle of thedilution range. For example, suggested dilutions are (1/8, 1/40and 1/200 of the human dose of the vaccine under examination)and (0.5 IU, 0.1 IU and 0.02 IU or any other suitablestandardized dilutions, of the reference preparation), each dosebeing contained in a volume, not exceeding 0.5 ml. For eachdilution use 16 to 24 mice and inject intraperitoneally, intoeach mouse one dose of the dilution.

Select a suitable strain of B. pertussis (e.g. 18323), capable ofcausing the death of mice within 14 days of intracerebralinjection. Make two subcultures after reviving the strain on asuitable medium (e.g. B.G. medium) and suspend the harvestedgrowth in a solution containing 1 per cent w/v of caseinhydrolysate (e.g. casamino acid) and 0.85 per cent w/v ofsodium chloride and having a pH of 7.0 to 7.2 or in anothersuitable solution. Determine the opacity of the suspension byusing 5th International reference preparation for opacity (10OU) and/or spectophotometerically. Alternatively, aliquots ofchallenge suspension frozen in liquid nitrogen with a suitablepreservative like 10 per cent DMSO may be used, to avoidheterogenicity. After 14 to 17 days of immunization, injectintracerebrally, a dose of 0.02 to 0.03 ml of the challengedilution randomly, into each immunized mouse. The challengeshould contain, approximately 1,00,000 organisms and 100 to1000 LD50 per dose, in a volume of not more than 0.03 ml. In thesame way, inject 4 groups of 10 control mice each, for LD50titration of challenge preparation, prepared by a series ofdilutions, from the dilution selected for challenge. Thechallenge should be completed within 2 to 2.5 hours ofpreparation. Exclude any mouse from consideration, that dieswithin 3 days of challenge. Count the number of mice survivingin each of the groups, after 14 days. On the basis of the numbersof animals surviving in each of the groups of 16 to 24 mice,calculate the potency of vaccine under examination, againstthe potency of reference preparation. Seed a suitable highestdilution of the challenge suspension, into each of two B.Gmedium plates, before and after challenge. Incubate the platesat 37° for 48 to 72 hours and calculate the number of colonyforming units (CFUs).

Calculate the potency of the vaccine by Probit analysis andLD50 of the challenge suspension by Reed and MunchMethod.

The test is not valid unless: a) for both the vaccine underexamination and the reference preparation, the ED50 (protectivedose), lies between the largest and the smallest doses givento the mice; b) the number of animals, which die in the fourgroups of 10 injected with the challenge suspension and itsdilutions indicate that the challenge dose contains 100 to 1000LD50 and 1 LD50 contains not more than 300 colony formingunits; c) and the statistical analysis shows no deviation fromlinearity or parallelism, in terms of significance of the scope ofdose response curve; d) the vaccine passes the requirementsfor potency, if the test results of a statistically valid assayshow that the estimated potency of the vaccine is not lessthan 4.0 IU per single human dose and the lower fiducial limit(P = 0.95) of estimated potency is not less than 2.0 IU.

The test may be repeated once, but when more than one testis performed the results of all valid tests must be combined, inthe estimate of potency.

Labelling. The label states (1) the minimum number ofInternational Units per single human dose; (2) that the vaccinemust be shaken before use; (3) that the vaccine is not to befrozen.

Pneumococcal Polysaccharide VaccinePneumococcal Polysaccharide Vaccine consists of a mixtureof equal parts of purified capsular polysaccharide of variousserotype antigens prepared from suitable pathogenic strainsof Streptococcus pneumoniae in different desiredcombinations whose capsules have been shown to be madeup of polysaccharides that are capable of inducing satisfactorylevels of specific antibodies in man. It contains upto 23immunochemically different capsular polysaccharides listedin the Table 1.

Production

General provisions

Production of the vaccine is based on a well defined seed-lotsystem for each type. The production method shall have beenshown to yield consistently pneumococcal polysaccharidevaccines of acceptable immunogenicity and safety in man.

The production method is validated to demonstrate that theproduct, if tested, would comply with the tests for abnormaltoxicity of vaccines for human use, modified as follows forthe tesonn guinea-pig; inject 10 human dose into each guinea-pig and observe for 12 days.

Monovalent bulk polysaccharides

The bacteria are grown in a suitable liquid medium that doesnot contain blood-group substances or high-molecular-masspolysaccharides. The bacterial purity of the culture is verified

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and the culture is inactivated with phenol. Impurities areremoved by such techniques as fractional precipitation,enzymatic digestion and ultrafiltration. The polysaccharide isobtained by fractional precipitation, washed, and dried in avacuum to a residual moisture content shown to be favourableto the stability of the polysaccharide. The residual moisturecontent is determined by drying under reduced pressure overdiphosphorus pentoxide or by thermogravimetric analysis andthe value obtained is used to calculate the results of the testsshown below with reference to the dried substance. Themonovalent bulk polysaccharide is stored at a suitabletemperature in conditions that avoid the uptake of moisture.Only a monovalent bulk polysaccharide that complies withthe following requirements may be used in the preparation ofthe final bulk vaccine. Percentage contents of components,

determined by the methods prescribed below, are shown inthe Table 1. The purified polysaccharides comply with the following testsas applicable:

Protein (2.7.1). Comply with the test for protein.

Nucleic acids (2.7.1). Comply with the test for nucleic acids.

Total nitrogen (2.3.30). Comply with the test for total nitrogen.

Phosphorus (2.7.1). Comply with the test for phosphorus.

Uronic acids (2.7.1). Comply with the test for uronic acids.

Hexosamine (2.7.1). Comply with the test for hexosamine.

Methylpentoses (2.7.1). Comply with the test formethylpentoses.

Table 1- Specifications on monovalent bulk polysaccharides (per cent contents):

Molecular Proteins Nucleic Total Phosphorus Molecular size K0 Uronic Hexo- Methyl- O-acetylType* acids Nitrogen CL-4B** CL-2B*** acids samines pentoses Groups

1 < 2 < 2 3.5-6 0-1.5 < 0.15 ≥ 45 ≥ 1.82 < 2 < 2 0-1 0-1.0 < 0.15 ≥ 15 ≥ 383 < 5 < 2 0-1 0-1.0 < 0.15 ≥ 404 < 3 < 2 4-6 0-1.5 < 0.15 ≥ 405 < 7.5 < 2 2.5-6.0 < 2 < 0.60 ≥ 12 ≥ 206B < 2 < 2 0-2 2.5-5.0 < 0.50 ≥ 157F < 5 < 2 1.5-4.0 0-1.0 < 0.20 ≥ 138 < 2 < 2 0-1 0-1.0 < 0.15 ≥ 259N < 2 < 1 2.2-4 0-1.0 < 0.20 ³ 20 ≥ 289V < 2 < 2 0.5-3 0-1.0 < 0.45 ³ 15 ≥ 1310A < 7 < 2 0.5-3.5 1.5-3.5 < 0.65 ≥ 1211A < 3 < 2 0-2.5 2.0-5.0 < 0.40 ≥ 912F < 3 < 2 3-5 0-1.0 < 0.25 ≥ 2514 < 5 < 2 1.5-4 0-1.0 < 0.30 ≥ 2015B < 3 < 2 1-3 2.0-4.5 < 0.55 ≥ 1517A or 17F < 2 < 2 0-1.5 0-3.5 < 0.45 ≥ 2018C < 3 < 2 0-1 2.4-4.9 < 0.15 ≥ 1419A < 2 < 2 0.6-3.5 3.0-7.0 < 0.45 ≥ 12 ≥ 2019F < 3 < 2 1.4-3.5 3.0-5.5 < 0.20 ≥ 12.5 ≥ 2020 < 2 < 2 0.5-2.5 1.5-4.0 < 0.60 ≥ 1222F < 2 < 2 0-2 0-1.0 < 0.55 ≥ 15 ≥ 2523F < 2 < 2 0-1 3.0-4.5 < 0.15 ≥ 3733F < 2.5 < 2 0-2 0-1.0 < 0.50

* The different types are indicated using the Danish nomenclature** Cross linked agarose for chromatography R*** Cross linked agarose for chromatography R1

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O-Acetyl groups (2.7.1). Comply with the test for O-acetylgroups.Sterility (2.2.11). Comply with the test for sterility.Molecular size. Molecular size is determined by gel filtrationor high performance size-exclusion chromatography (HPSEC)(2.4.16) using cross linked Agarose for chromatography R orchromatography Agarose for chromatograph R1, either aloneor Multiple angle light laser scattering (MALLS) or any othersuitable method.

IdentificationConfirm the identity of the monovalent bulk polysaccharideby immunochemical method (2.2.14)(except for polysaccharides7F, 14 and 33F).

Specificity

For establishing the specificity, no reaction should occur,when the antigens are tested against all the antisera specificfor the other polysaccharides of the vaccine, including factorsera for distinguishing types within groups. Thepolysaccharides are tested at a concentration of 50 µg/ml usinga method capable of detecting 0.5 µg/ml.FINAL BULK VACCINE

The final bulk vaccine is obtained by aseptically mixing thedifferent polysaccharide powders. The uniform mixture isaseptically dissolved in a suitable isotonic solution so thatone human dose of 0.5 ml contains 25 µg of eachpolysaccharide. An antimicrobial preservative may be added.The solution is sterilized by filtration through a bacteria-retentive filter.Only a final bulk vaccine that complies with the followingrequirements may be used in the preparation of the final lot.



FINAL LOT

The final bulk vaccine is distributed and filled aseptically intosterile containers (vials or ampoules). Only a final lot that issatisfactory with respect to each of the requirements givenbelow under Identification, Tests and Assay may be releasedfor use. Provided that the tests for phenol and for antimicrobialpreservative have been carried out with satisfactory resultson the final bulk vaccine, they may be omitted on the final lot.When consistency of production has been established on asuitable number of consecutive batches, the assay may bereplaced by a qualitative test that identifies eachpolysaccharide, provided that an assay has been performed

on each monovalent bulk polysaccharide used in thepreparation of the final lot.

IdentificationThe assay also serves to identify the vaccine.


Abnormal Toxicity (2.2.1). Complies with the test for abnormaltoxicity with the following modifications.Inject 10 human doses each in two guinea pigs weighingbetween 250 and 350 g by intraperitoneal route and observefor 12 days,Pyrogens (2.2.8). Complies with the test for pyrogens. Injecteach of the rabbit with 1 ml of a dilution of the vaccinecontaining 2.5 µg/ml of each polysaccharide.Antimicrobial preservative. Where applicable, determine theamount of antimicrobial preservative by a suitable chemicalmethod. The content is not less than 85.0 per cent and notgreater than 115.0 per cent of the intended amount.Phenol (2.3.36). Not more than 2.5 g/l.

pH (2.4.24). 4.5 to 7.4.

Assay

Determine the content of each polysaccharide by a suitablebiochemical, physicochemical or immunochemical method(2.2.14), using antisera specific for each polysaccharidecontained in the vaccine, including factor sera for types withingroups, and purified polysaccharides of each type asstandards.The vaccine contains not less than 70.0 per cent and not morethan 130.0 per cent of the quantity stated on the label for eachpolysaccharide. The confidence interval (P = 0.95) of the assayis not less than 80.0 and not more than 120.0 per cent of theestimated content.Labelling. The label states (1) the number of µg of eachpolysaccharide per human dose; (2) the total amount ofpolysaccharide in the container.

Poliomyelitis Vaccine (Inactivated)Poliomyelitis Vaccine (Inactivated) is a liquid preparation ofsuitable strains of human polioviruses 1, 2 and 3 grown insuitable cell cultures and inactivated by a validated method.


The production method should consistently yield vaccinesof acceptable safety and immunogenicity in man.

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Production of the vaccine is based on a virus seed-lot system.Cell lines are used according to a cell-bank system. If primary,secondary or tertiary monkey kidney cells are used, productioncomplies with the requirements indicated below.

Unless otherwise justified and authorised, the virus in thefinal vaccine shall not have undergone more passages fromthe master seed lot than was used to prepare the vaccineshown in clinical studies to be satisfactory with respect tosafety and efficacy.



The virus is propagated in a human diploid cell line (2.7.2), ina continuous cell line (2.7.2) or in primary, secondary or tertiarymonkey kidney cells.

Primary, secondary or tertiary monkey kidney cells. Thefollowing special requirements for the substrate for viruspropagation apply to primary, secondary or tertiary monkeykidney cells.

Monkeys used in the preparation of kidney cell cultures forproduction and control of the vaccine. The animals used areof a species approved by the competent authority, in goodhealth and, unless otherwise justified and authorised, havenot been previously employed for experimental purposes.Kidney cells used for vaccine production and control arederived from monitored, closed colonies of monkeys bred incaptivity, not from animals caught in the wild; a previouslyapproved seed lot prepared using virus passaged in cells fromwild monkeys may, subject to approval by the competentauthority, be used for vaccine production if historical data onsafety justify this.

Monitored, closed colonies of monkeys. The monkeys arekept in groups in cages. Freedom from extraneous agents isachieved by the use of animals maintained in closed coloniesthat are subject to continuous and systematic veterinary andlaboratory monitoring for the presence of infectious agents.The supplier of animals is certified by the competent authority.Each monkey is tested serologically at regular intervals duringa quarantine period of not less than 6 weeks imposed beforeentering the colony and then during its stay in the colony.

The monkeys used are shown to be tuberculin-negativeand free from antibodies to simian virus 40 (SV40) andsimian immunodeficiency virus. If Macaca spp. monkeys areused for production, the monkeys are also shown to be freefrom antibodies to herpesvirus B (Cercopithecine herpesvirus1) infection. Human herpesvirus 1 has been used as anindicator for freedom from herpesvirus B antibodies onaccount of the danger of handling herpesvirus B

(Cercopithecine herpesvirus 1).Monkeys from which kidneys are to be removed are thoroughlyexamined, particularly for evidence of tuberculosis andherpesvirus B (Cercopithecine herpesvirus 1) infection. If amonkey shows any pathological lesion relevant to the use ofits kidneys in the preparation of a seed lot or vaccine, it is notto be used nor are any of the remaining monkeys of the groupconcerned unless it is evident that their use will not impair thesafety of the product.

All the operations described in this section are conductedoutside the area where the vaccine is produced.

Monkey cell cultures for vaccine production. Kidneys thatshow no pathological signs are used for preparing cell cultures.Each group of cell cultures derived from a single monkey formsa separate production cell culture giving rise to a separatesingle harvest.

The primary monkey kidney cell suspension complies withthe test for mycobacteria ; disrupt the cells before carryingout the test.

If secondary or tertiary cells are used, it shall be demonstratedby suitable validation tests that cell cultures beyond thepassage level used for production are free from tumorigenicity.

SEED LOT

Each of the three strains of poliovirus used shall be identifiedby historical records that include information on the origin ofthe strain and its subsequent manipulation.

Only a working seed lot that complies with the followingrequirements may be used for virus propagation.

Identification

Each working seed lot is identified as human poliovirus 1, 2 or3 by virus neutralisation in cell culture using specificantibodies.

Virus concentration. The virus concentration of eachworking seed lot is determined to define the quantityof virus to be used for inoculation of production cellcultures.

Extraneous agents (2.7.3). The working seed lot complies withthe requirements for seed lots for virus vaccines. In addition,if primary, secondary or tertiary monkey kidney cells havebeen used for isolation of the strain, measures are taken toensure that the strain is not contaminated with simian virusessuch as simian immunodeficiency virus, simian virus 40,filoviruses and herpesvirus B (Cercopithecine herpesvirus 1).A working seed lot produced in primary, secondary or tertiarymonkey kidney cells complies with the requirements givenbelow under Virus Propagation and Harvest for single harvestsproduced in such cells.


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All processing of the cell bank and subsequent cell cultures isdone under aseptic conditions in an area where no other cellsor viruses are being handled. Approved animal serum (but nothuman serum) may be used in the cell culture media. Serumand trypsin used in the preparation of cell suspensions andmedia are shown to be free from extraneous agents. The cellculture media may contain a pH indicator such as phenol redand approved antibiotics at the lowest effective concentration.Not less than 500 ml of the cell cultures employed for vaccineproduction is set aside as uninfected cell cultures (controlcells); where continuous cell lines in a fermenter are used forproduction, 200 × 106 cells are set aside to prepare controlcells; where primary, secondary or tertiary monkey kidneycells are used for production, a cell sample equivalent to atleast 500 ml of the cell suspension, at the concentrationemployed for vaccine production, is taken to prepare controlcell cultures.

Only a single harvest that complies with the followingrequirements may be used in the preparation of the vaccine.The tests for Identification and Sterility may be carried outinstead on the purified, pooled monovalent harvest. Afterdemonstration of consistency of production at the stage ofthe single harvest, the test for virus concentration may becarried out instead on the purified, pooled monovalent harvest.

Control cells. The control cells of the production cell culturecomply with a test for Identification (if a cell-bank system isused for production) and with the requirements for extraneousagents, where primary, secondary or tertiary monkey kidneycells are used, the tests in cell cultures are carried out asshown below under Test in Rabbit Kidney Cell Cultures andTest in Cercopithecus Kidney Cell Cultures).

Test in rabbit kidney cell cultures. Test a sample of at least 10ml of the pooled supernatant fluid from the control culturesfor the absence of herpesvirus B (Cercopithecine herpesvirus1) and other viruses in rabbit kidney cell cultures. The dilutionof supernatant in the nutrient medium is not greater than 1:4and the area of the cell layer is at least 3 cm2 per ml of inoculum.Set aside one or more containers of each batch of cells withthe same medium as non-inoculated control cells. Incubatethe cultures at 37° and observe for at least 2 weeks. The test isnot valid if more than 20 per cent of the control cells arediscarded for non-specific, accidental reasons.

Test in Cercopithecus kidney cell cultures. Test a sample ofat least 10 ml of the pooled supernatant fluid from the controlcultures for the absence of SV40 virus and other extraneousagents by inoculation onto cell cultures prepared from thekidneys of cercopithecus monkeys, or other cells shown to beat least as sensitive for SV40, by the method described underTest in Rabbit Kidney Cell Cultures. The test is not valid ifmore than 20 per cent of the control cell cultures are discarded

for non-specific, accidental reasons.

Identification

The single harvest is identified as containing human poliovirus1, 2 or 3 by virus neutralisation in cell cultures using specificantibodies.

Virus concentration. The virus concentration of each singleharvest is determined by titration of infectious virus in cellcultures.

Sterility (2.2.11). The single harvest complies with the test forsterility, carried out using 10 ml for each medium.

Mycoplasmas (2.7.4). The single harvest complies with thetest for mycoplasmas, carried out using 10 ml.

Test in rabbit kidney cell cultures. Where primary, secondaryor tertiary monkey kidney cells are used for production, test asample of at least 10 ml of the single harvest for the absence ofherpesvirus B (Cercopithecine herpesvirus 1) and other virusesin rabbit kidney cell cultures as described for the control cells.

Test in Cercopithecus kidney cell cultures. Where primary,secondary or tertiary monkey kidney cells are used forproduction, test a sample of at least 10 ml of the single harvestfor the absence of SV40 virus and other extraneous agents.Neutralise the sample by a high-titre antiserum against thespecific type of poliovirus. Test the sample in primarycercopithecus kidney cell cultures or cells that have beendemonstrated to be at least as susceptible for SV40. Incubatethe cultures at 37° and observe for 14 days. At the end of thisperiod, make at least one subculture of fluid in the same cellculture system and observe both primary cultures andsubcultures for an additional 14 days.

PURIFICATION AND PURIFIED MONOVALENTHARVEST

Several single harvests of the same type may be pooled andmay be concentrated. The monovalent harvest or pooledmonovalent harvest is purified by validated methods. Ifcontinuous cell lines are used for production, the purificationprocess shall have been shown to reduce consistently thecontent of substrate-cell DNA to not more than 500 pg persingle human dose.

Only a purified monovalent harvest that complies with thefollowing requirements may be used for the preparation of theinactivated monovalent harvest.

Identification

The virus is identified by virus neutralisation in cell culturesusing specific antibodies or by determination of D-antigen.

Virus concentration. The virus concentration is determinedby titration of infectious virus.


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Specific activity. The ratio of the virus concentration or the D-antigen content, determined by a suitable immunochemicalmethod (2.2.14). to the total protein content (specific activity)of the purified monovalent harvest is within the limits approvedfor the particular product.

INACTIVATION AND INACTIVATED MONOVALENTHARVEST

Several purified monovalent harvests of the same type maybe mixed before inactivation. To avoid failures in inactivationcaused by the presence of virus aggregates, filtration is carriedout before and during inactivation; inactivation is startedwithin a suitable period, preferably not more than 24 h and inany case not more than 72 h, of the prior filtration. The virussuspension is inactivated by a validated method that has beenshown to inactivate poliovirus without destruction ofimmunogenicity; during validation studies, an inactivationcurve with at least four points (for example, time 0, 24, 48, and96 h) is established showing the decrease in concentration oflive virus with time. If formaldehyde is used for inactivation,the presence of an excess of formaldehyde at the end of theinactivation period is verified.

Only an inactivated monovalent harvest that complies withthe following requirements may be used in the preparation ofa trivalent pool of inactivated monovalent harvests or a finalbulk vaccine.

Test for effective inactivation. After neutralisation of theformaldehyde with sodium bisulphite (where applicable), verifythe absence of residual live poliovirus by inoculation onsuitable cell cultures of two samples of each inactivatedmonovalent harvest, corresponding to at least 1500 humandoses. Take one sample not later than three-quarters of theway through the inactivation period and the other at the end.Inoculate the samples in cell cultures such that the dilution ofvaccine in the nutrient medium is not greater then ¼ and thearea of the cell layer is at least 3 cm2 per ml of inoculum. Setaside one or more containers with the same medium as non-inoculated control cells. Observe the cell cultures for at least3 weeks. Make not fewer than two passages from eachcontainer, one at the end of the observation period and theother 1 week before; for the passages, use cell culturesupernatant and inoculate as for the initial sample. Observethe subcultures for at least 2 weeks. No sign of poliovirusmultiplication is present in the cell cultures. At the end of theobservation period, test the susceptibility of the cell cultureused by inoculation of live poliovirus of the same type as thatpresent in the inactivated monovalent harvest.Sterility (2.2.11). The inactivated monovalent harvest complieswith the test for sterility, carried out using 10 ml for eachmedium.

D-antigen content. The content of D-antigen determined by a

suitable immunochemical method (2.2.14) is within the limitsapproved for the particular preparation.

FINAL BULK VACCINE

The final bulk vaccine is prepared directly from the inactivatedmonovalent harvests of human polioviruses 1, 2 and 3 or froma trivalent pool of inactivated monovalent harvests. If atrivalent pool of inactivated monovalent harvests is used, atest for effective inactivation is carried out on this pool insteadof on the final bulk vaccine. A stabiliser and an antimicrobialpreservative may be added.




Inactivation. Before addition of any antimicrobial preservative,a sample of at least 1500 ml or, for a purified and concentratedvaccine, the equivalent of 1500 doses is tested for residuallive poliovirus in cell cultures, as described for the inactivatedmonovalent harvest. If the final bulk vaccine is prepared froma trivalent pool of inactivated monovalent harvests, the testfor inactivation is carried out on that pool rather than on thefinal bulk vaccine.

FINAL LOT

Only a final lot that complies with each of the requirementsgiven below under Identification, Tests and Assay may bereleased for use. Provided that the tests for free formaldehydeand antimicrobial preservative and the in vivo assay havebeen performed with satisfactory results on the final bulkvaccine, they may be omitted on the final lot. Provided thatthe test for bovine serum albumin has been performed withsatisfactory results on the trivalent pool of inactivatedmonovalent harvests or on the final bulk vaccine, it may beomitted on the final lot.

Identification

The vaccine is shown to contain human polioviruses 1, 2 and3 by a suitable immunochemical method such as thedetermination of D-antigen by enzyme-linked immunosorbentassay (ELISA).

Tests


Antimicrobial preservative. Where applicable, determine theamount of antimicrobial preservative by a suitable chemical


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method. The content is not less than 85.0 per cent and notgreater than 115.0 per cent of the intended amount.

Protein content (2.3.49). Not more than 10 µg of proteinnitrogen per human dose.



Bacterial endotoxins ( 2.2.3). Not more than 5 IU per humandose.

Assay

D-antigen content. As a measure of consistency of production,determine the D-antigen content for human polioviruses 1, 2and 3 by a suitable immunochemical method (2.2.14) using anappropriate reference preparation calibrated in D-antigen units.For each type, the content, expressed with reference to theamount of D-antigen stated on the label, is within the limitsapproved for the particular product.

In vivo test. The capacity of the vaccine to induce the formationof neutralizing antibodies is determined in-vivo by one of thefollowing methods:

Test in chicks or guinea-pigs. Prepare a suitable series of atleast three dilutions of the vaccine under examination using asuitable buffered saline solution. Inject 0.5 ml of the dilutionsintramuscularly into groups of ten 3-week-old chickens orgroups of ten guinea-pigs, each weighing between 250 and350 g, using a separate group for each dilution of vaccine.Bleed the animals on the fifth or sixth day after the injectionand separate the sera. Examine the sera for the presence ofneutralising antibody, at a dilution of 1 in 4, to each of thehuman polioviruses 1, 2 and 3. Mix 100 CCID50 of virus withthe dilution of serum and incubate at 37° for 4 h 30 min to 6 h.Keep at 5 ± 3° for 12 to 18 h. Inoculate the mixtures into cellcultures for the detection of unneutralised virus and read theresults up to 7 days after inoculation. For each group of animals,note the number of sera which have neutralising antibodyand calculate the dilution of the vaccine giving an antibodyresponse in 50.0 per cent of the animals. Carry out in parallel acontrol test using a suitable reference preparation.

The vaccine complies with the test if a dilution of 1 in 100 ormore produces an antibody response for each of the threetypes of virus in 50.0 per cent of the animals.

Test on rats. A suitable in vivo assay method consists ofintramuscular injection into the hind limb(s) of not fewer than3 dilutions of the vaccine under examination and a referencevaccine, using for each dilution a group of 10 specificpathogen-free rats of the suitable strain. Use of 4 dilutions isoften necessary to obtain valid results for all 3 serotypes. The

number of animals per group must be sufficient to obtainresults that meet the validity criteria; groups of 10 rats areusually sufficient although valid results may be obtained withfewer animals per group. The weight of individual animal mustnot vary by more than 10.0 per cent from the group mean. Aninoculum of 0.5 ml per rat is used. The dose range is chosensuch that a dose response to all 3 poliovirus types is obtained.Bleed the animals after 20 to 22 days. Neutralising titres againstall 3 polivirus types are measured separately using 100 CCID50of the Sabin strains as challenge viruses. Vero or Hep2 asindicator cells, and neutralization conditions of 3 h at 35° to37° followed by 18 h at 2° to 8°. Results are read followingfixation and staining after 7 days of incubation at 35°. For avalid antibody assay, the titre of each challenge virus must beshown to be within the range of 10 to 1000 CCID50 and theneutralizing antibody titre of a control serum must be within 2twofold dilutions of the geometric mean titre of the serum.The potency is calculated by comparison of the preparationof responders for the vaccine under examination and thereference vaccine by the probit method or, after validation,using a parallel-line model. For the probit method it isnecessary to establish a cut-off neutralising antibody titre foreach poliovirus type to define a responder. Due tointerlaboratory variation, it is not possible to define cut-offvalues that could be applied by all laboratories. Rather, thecut-off values are determined for each laboratory based on aminimum series of 3 tests with the reference vaccine. The mid-point on a log 2 scale of the minimum and maximum geometricmean titres of the series of 3 or more tests is used as the cut-off value. For each of the 3 poliovirus types, the potency ofthe vaccine is not significantly less than that of the referencepreparation. The test is not valid unless (1) for both the testand reference vaccines the ED50 lies between the smallest andthe largest doses given to the animals; (2) the statisticalanalysis shows no significant deviation from linearity orparallelism; (3) the fiducial limits of the estimated relativepotency fall between 25.0 per cent and 400.0 per cent of theestimated potency.

Labelling. The label states (1) the types of polioviruscontained in the vaccine; (2) the nominal amount of virus ofeach type (1, 2 and 3), expressed in units of D-antigen persingle human dose; (3) the cell substrate used to prepare thevaccine.

Poliomyelitis Vaccine, Live (Oral)Oral Poliomyelitis Vaccine is a preparation of approved strainsof live attenuated poliovirus type 1, 2 or 3 grown in in vitrocultures of approved cells, containing any one type or anycombination of the three types of Sabin strains, prepared in aform suitable for oral administration.

POLIOMYELITIS VACCINE, LIVE (ORAL)

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Production

General provisions

The vaccine strains and the production method shouldconsistently yield vaccines that are both immunogenic andsafe in man.

The production of vaccine is based on a virus seed-lot system.Cell lines are used according to a cell-bank system. If primarymonkey kidney cells are used, production complies with therequirements indicated below. Unless otherwise justified andauthorised, the virus in the final vaccine shall not haveundergone more than two passages from the master seed lot.


The virus is propagated in human diploid cells (2.7.2) or incontinuous cell lines (2.7.2) or in primary monkey kidney cells(including serially passaged cells from primary monkey kidneycells). Continuous cell lines are approved by the competentauthority.

Primary monkey cells. The following special requirementsfor the substrate for virus propagation apply to primarymonkey cells.

Monkeys used for preparation of kidney cell cultures and fortesting of virus. If the vaccine is prepared in monkey kidneycell cultures, animals of a species approved by the competentauthority, in good health, and not previously employed forexperimental purposes shall be used.

The monkeys shall be kept in well-constructed and adequatelyventilated animal rooms in cages spaced as far apart aspossible. Adequate precautions shall be taken to preventcross-infection between cages. Not more than two monkeysshall be housed per cage and cage-mates shall not beinterchanged. The monkeys shall be kept in the country ofmanufacture of the vaccine in quarantine groups for a periodof not less than 6 weeks before use. A quarantine group is acolony of selected, healthy monkeys kept in one room, withseparate feeding and cleaning facilities, and having no contactwith other monkeys during the quarantine period. If at anytime during the quarantine period the overall death rate of ashipment consisting of one or more groups reaches 5 per cent(excluding deaths from accidents or where the cause wasspecifically determined not to be an infectious disease),monkeys from that entire shipment shall continue in quarantinefrom that time for a minimum of 6 weeks. The groups shall bekept continuously in isolation, as in quarantine, even aftercompletion of the quarantine period, until the monkeys areused. After the last monkey of a group has been taken, theroom that housed the group shall be thoroughly cleaned anddecontaminated before being used for a fresh group. If kidneysfrom near-term monkeys are used, the mother is quarantinedfor the term of pregnancy.

Monkeys from which kidneys are to be removed shall beanaesthetised and thoroughly examined, particularly forevidence of tuberculosis and cercopithecid herpesvirus 1 (Bvirus) infection.

If a monkey shows any pathological lesion relevant to the useof its kidneys in the preparation of a seed lot or vaccine, itshall not be used, nor shall any of the remaining monkeys ofthe quarantine group concerned be used unless it is evidentthat their use will not impair the safety of the product.

All the operations described in this section shall be conductedoutside the areas where the vaccine is produced.

The monkeys used shall be shown to be free from antibodiesto simian virus 40 (SV40) and simian immunodeficiency virus.If Macaca spp. are used for production, the monkeys shallalso be shown to be free from antibodies to cercopithecidherpesvirus 1 (B virus). Human herpesvirus has been used asan indicator for freedom from B virus antibodies on accountof the danger of handling cercopithecid herpesvirus 1 (B virus).Monkey kidney cell cultures for vaccine production. Kidneysthat show no pathological signs are used for preparing cellcultures. If the monkeys are from a colony maintained forvaccine production, serially passaged monkey kidney cellcultures from primary monkey kidney cells may be used forvirus propagation, otherwise the monkey kidney cells are notpropagated in series. Virus for the preparation of vaccine isgrown by aseptic methods in such cultures. If animal serum isused in the propagation of the cells, the maintenance mediumafter virus inoculation shall contain no added serum.Each group of cell cultures derived from a single monkey orfrom fetuses from no more than ten near-term monkeys isprepared and tested as an individual group.

SEED LOT

The strains of poliovirus used shall be identified by historicalrecords that include information on the origin and subsequentmanipulation of the strains.

Working seed lots are prepared by a single passage from amaster seed lot and at an approved passage level from theoriginal Sabin virus. Virus seed lots are prepared in largequantities and stored at a temperature below -60°.

Only a virus seed lot that complies with the followingrequirements may be used for virus propagation.

Identification

Each working seed lot is identified as poliovirus of the giventype, using specific antibodies.

Virus concentration. Determined by the method describedbelow, the virus concentration is the basis for the quantity ofvirus used in the neurovirulence test.


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Extraneous agents (2.7.3). If the working seed lot is producedin human diploid cells (2.7.2) or in continuous cell lines (2.7.2)it complies with the requirements for seed lots for virusvaccines. If the working seed lot is produced in primarymonkey cells, it complies with the requirements given belowunder Propagation and Harvest and Monovalent PooledHarvest and with the tests in adult mice, suckling mice andguinea-pigs given under Tests for extraneous agents in viralvaccines for human use.Working seed lot shall be free from detectable DNA sequencesfrom simian virus 40 (SV40)

Neurovirulence (2.7.6). Each master and working seed lotcomplies with the test for neurovirulence of poliomyelitisvaccine (oral) in monkeys. Furthermore, the seed lot shall ceaseto be used in vaccine production if the frequency of failure ofthe monovalent pooled harvests produced from it is greaterthan predicted statistically. This statistical prediction iscalculated after each test on the basis of all the monovalentpooled harvests tested; it is equal to the probability of falserejection on the occasion of a first test (i.e. 1 per cent), theprobability of false rejection on retest being negligible. If thetest is carried out only by the manufacturer, the test slides areprovided to the control authority for assessment.Genetic markers. Each working seed lot is tested for itsreplicating properties at temperatures ranging from 36° to 40°as described under Monovalent Pooled Harvest.PROPAGATION AND HARVEST

All processing of the cell-banks and subsequent cell-culturesis done under aseptic conditions in an area where no othercells are handled. Approved animal (but not human) serummay be used in the media, but the final medium for maintainingcell growth during virus multiplication does not contain animalserum. Serum and trypsin used in the preparation of cellsuspensions and media are shown to be free from liveextraneous agents. The cell-culture medium may contain a pHindicator such as phenol red and approved antibiotics at thelowest effective concentration. It is preferable to have asubstrate free from antibiotics during production. Not lessthan 5 per cent and not more than 1000 ml of the cell culturesemployed for vaccine production are set aside as uninfectedcell cultures (control cells); special requirements, given below,apply to control cells when the vaccine is produced in primarymonkey cells The virus suspension is harvested not later than4 days after virus inoculation. After inoculation of theproduction cell culture with the virus working seed lot,inoculated cells are maintained at a fixed temperature, shownto be suitable, within the range 33° to 35°; the temperature ismaintained constant to ±0.5°; control cell cultures aremaintained at 33° to 35° for the relevant incubation periods.Only a single virus harvest that complies with the followingrequirements may be used in the preparation of the monovalent

pooled harvest.

Virus concentration. The virus concentration of virusharvests is determined as prescribed under Assay to monitorconsistency of production and to determine the dilution to beused for the final bulk vaccine.

Extraneous agents ( 2.7.3 ). Complies with tests for extraneousagents.Control cells. The control cells of the production cell culturefrom which the virus harvest is derived comply with a test foridentity and with the requirements for extraneous agents or,where primary monkey cells are used, as shown below.Primary monkey cells. The following special requirementsapply to virus propagation and harvest in primary monkeycells.

Cell cultures. On the day of inoculation with virus seed, eachcell culture is examined for degeneration caused by an infectiveagent. If, in this examination, evidence is found of the presencein a cell culture of any extraneous agent, the entire group ofcultures concerned shall be rejected.

On the day of inoculation with the virus working seed lot, asample of at least 30 ml of the pooled fluid removed from thecell cultures of the kidneys of each single monkey or fromfetuses from not more than ten near-term monkeys is dividedinto two equal portions. One portion of the pooled fluid istested in monkey kidney cell cultures prepared from the samespecies, but not the same animal, as that used for vaccineproduction. The other portion of the pooled fluid is, wherenecessary, tested in monkey kidney cell cultures from anotherspecies so that tests on the pooled fluids are done in cellcultures from at least one species known to be sensitive toSV40. The pooled fluid is inoculated into bottles of these cellcultures in such a way that the dilution of the pooled fluid inthe nutrient medium does not exceed 1 in 4. The area of the cellsheet is at least 3 cm2 per ml of pooled fluid. At least one bottleof each kind of cell culture remains uninoculated to serve as acontrol. If the monkey species used for vaccine production isknown to be sensitive to SV40, a test in a second species isnot required. Animal serum may be used in the propagation ofthe cells, provided that it does not contain SV40 antibody, butthe maintenance medium after inoculation of test materialcontains no added serum except as described below.The cultures are incubated at a temperature of 35° to 37° andare observed for a total period of at least 4 weeks. During thisobservation period and after not less than 2 weeks’ incubation,at least one subculture of fluid is made from each of thesecultures in the same cell culture system. The subcultures arealso observed for at least 2 weeks.

Serum may be added to the original culture at the time ofsubculturing, provided that the serum does not contain SV40antibody.


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Fluorescent-antibody techniques may be useful for detectingSV40 virus and other viruses in the cells.

A further sample of at least 10 ml of the pooled fluid is testedfor cercopithecid herpesvirus 1 (B virus) and other viruses inrabbit kidney cell cultures. Serum used in the nutrient mediumof these cultures shall have been shown to be free frominhibitors of B virus. Human herpesvirus has been used as anindicator for freedom from B virus inhibitors on account of thedanger of handling cercopithecid herpesvirus 1 (B virus). Thesample is inoculated into bottles of these cell cultures in sucha way that the dilution of the pooled fluid in the nutrientmedium does not exceed 1 in 4. The area of the cell sheet is atleast 3 cm2 per ml of pooled fluid. At least one bottle of the cellcultures remains uninoculated to serve as a control.

The cultures are incubated at a temperature of 35° to 37°and observed for at least 2 weeks.

A further sample of 10 ml of the pooled fluid removed from thecell cultures on the day of inoculation with the seed lot virusis tested for the presence of extraneous agents by inoculationinto human cell cultures sensitive to measles virus.

The tests are not valid if more than 20 per cent of the culturevessels have been discarded for non-specific accidentalreasons by the end of the respective test periods.

If, in these tests, evidence is found of the presence of anextraneous agent, the single harvest from the whole group ofcell cultures concerned is rejected.

If the presence of cercopithecid herpesvirus 1 (B virus) isdemonstrated, the manufacture of oral poliomyelitis vaccineshall be discontinued and the competent authority shall beinformed. Manufacturing shall not be resumed until a thoroughinvestigation has been completed and precautions have beentaken against any reappearance of the infection, and thenonly with the approval of the competent authority.If these tests are not done immediately, the samples of pooledcell-culture fluid shall be kept at a temperature of -60° or below,with the exception of the sample for the test for B virus, whichmay be held at 4°, provided that the test is done not more than7 days after it has been taken.Control cell cultures. On the day of inoculation with the virusworking seed lot 25 per cent (but not more than 2500 ml) of thecell suspension obtained from the kidneys of each singlemonkey or from not more than ten near-term monkeys is takento prepare uninoculated control cell cultures. These controlcell cultures are incubated in the same conditions as theinoculated cultures for at least 2 weeks and are examined duringthis period for evidence of cytopathic changes. The tests arenot valid if more than 20 per cent of the control cell cultureshave been discarded for non-specific, accidental reasons. Atthe end of the observation period, the control cell cultures are

examined for degeneration caused by an infectious agent. Ifthis examination or any of the tests required in this sectionshows evidence of the presence in a control culture of anyextraneous agent, the poliovirus grown in the correspondinginoculated cultures from the same group shall be rejected.

Tests for haemadsorbing viruses. At the time of harvest orwithin 4 days of inoculation of the production cultures withthe virus working seed lot, a sample of 4 per cent of the controlcell cultures is taken and tested for haemadsorbing viruses.At the end of the observation period, the remaining controlcell cultures are similarly tested. The tests are made as describedin (2.7.3), Tests for extraneous agents in viral vaccines forhuman use.

Tests for other extraneous agents. At the time of harvest, orwithin 7 days of the day of inoculation of the productioncultures with the working seed lot, a sample of at least 20 ml ofthe pooled fluid from each group of control cultures is takenand tested in two kinds of monkey kidney cell culture, asdescribed above.

At the end of the observation period for the original controlcell cultures, similar samples of the pooled fluid are taken andthe tests referred to in this section in the two kinds of monkeykidney cell culture and in the rabbit cell cultures are repeated,as described above under Cell cultures.

If the presence of Cercopithecid herpesvirus 1 (B virus) isdemonstrated, the production cell cultures shall not be usedand the measures concerning vaccine production describedabove must be undertaken.

The fluids collected from the control cell cultures at the timeof virus harvest and at the end of the observation period maybe pooled before testing for extraneous agents. A sample of 2per cent of the pooled fluid is tested in each of the cell culturesystems specified.

Single harvests

Tests for neutralised single harvests in monkey kidney cellcultures. A sample of at least 10 ml of each single harvest isneutralised by a type-specific poliomyelitis antiserum preparedin animals other than monkeys. In preparing antisera for thispurpose, the immunising antigens used shall be prepared innon-simian cells.

Half of the neutralised suspension (corresponding to at least5 ml of single harvest) is tested in monkey kidney cell culturesprepared from the same species, but not the same animal, asthat used for vaccine production. The other half of theneutralised suspension is tested, if necessary, in monkeykidney cell cultures from another species so that the tests onthe neutralised suspension are done in cell cultures from atleast one species known to be sensitive to SV40.


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The neutralised suspensions are inoculated into bottles ofthese cell cultures in such a way that the dilution of thesuspension in the nutrient medium does not exceed 1 in 4. Thearea of the cell sheet is at least 3 cm2 per ml of neutralisedsuspension. At least one bottle of each type of cell cultureremains uninoculated to serve as a control and is maintainedby nutrient medium containing the same concentration of thespecific antiserum used for neutralisation.Animal serum may be used in the propagation of the cells,provided that it does not contain SV40 antibody, but themaintenance medium, after the inoculation of the test material,contains no added serum other than the poliovirus neutralisingantiserum, except as described below.The cultures are incubated at a temperature of 35° to 37° andobserved for a total period of at least 4 weeks. During thisobservation period and after not less than 2 weeks’ incubation,at least one subculture of fluid is made from each of thesecultures in the same cell-culture system. The subcultures arealso observed for at least 2 weeks.Serum may be added to the original cultures at the time ofsubculturing, provided that the serum does not contain SV40antibody.

Additional tests are made for extraneous agents on a furthersample of the neutralised single harvests by inoculation of 10ml into human cell cultures sensitive to measles virus.

Fluorescent-antibody techniques may be useful for detectingSV40 virus and other viruses in the cells.

The tests are not valid if more than 20 per cent of the culturevessels have been discarded for non-specific accidentalreasons by the end of the respective test periods.

If any cytopathic changes occur in any of the cultures, thecauses of these change are investigated. If the cytopathicchanges are shown to be due to unneutralised poliovirus, thetest is repeated. If there is evidence of the presence of SV40 orother extraneous agents attributable to the single harvest,that single harvest is rejected.


Monovalent pooled harvests are prepared by pooling a numberof satisfactory single harvests of the same virus type.Monovalent pooled harvests from continuous cell lines maybe purified. Each monovalent pooled harvest is filtered througha bacteria-retentive filter.Only a monovalent pooled harvest that complies with thefollowing requirements may be used in the preparation of thefinal bulk vaccine.

IdentificationEach monovalent pooled harvest is identified as poliovirus ofthe given type, using specific antiserum.

Virus concentration

The virus concentration is determined by the methoddescribed below and serves as the basis for calculating thedilutions for preparation of the final bulk, for the quantity ofvirus used in the neurovirulence test and to establish andmonitor production consistency.

Genetic markers. A ratio of the replication capacities of thevirus in the monovalent pooled harvest is obtained over atemperature range between 36° and 40° in comparison withthe seed lot or a reference preparation for the marker tests andwith appropriate rct/40- and rct/40+ strains of poliovirus ofthe same type. The incubation temperatures used in this testare controlled to within ±0.1°. The monovalent pooled harvestpasses the test if, for both the virus in the harvest and theappropriate reference material, the titre determined at 36° is atleast 5.0 log greater than that determined at 40°. If growth at40° is so low that a valid comparison cannot be established, atemperature in the region of 39.0° to 39.5° is used, at whichtemperature the reduction in titre of the reference materialmust be in the range 3.0 to 5.0 log of its value at 36°; theacceptable minimum reduction is determined for each virusstrain at a given temperature. If the titres obtained for one ormore of the reference viruses are not concordant with theexpected values, the test must be repeated.

Neurovirulence (2.7.6). Each monovalent pooled harvestcomplies with the test for neurovirulence of poliomyelitisvaccine (oral). If the test is carried out only by the manufacturer,the test slides are provided to the competent authority forassessment. The TgPVR21 transgenic mouse model providesa suitable alternative to the monkey neurovirulence test forneurovirulence testing of types 1, 2 or 3 vaccines once alaboratory qualifies as being competent to perform the testand the experience gained is to the satisfaction of thecompetent authority. The test is carried out using a standardoperating procedure approved by the competent authority. Asuitable procedure (Neurovirulence test of type 1, 2 or 3 livepoliomyelitis vaccines (oral) in transgenic mice susceptibleto poliovirus) is available from WHO, Quality and Safety ofBiologicals, Geneva.

Primary monkey cells. The following special requirementsapply to monovalent pooled harvests derived from primarymonkey cells.

Retroviruses. The monovalent pooled harvest is examinedusing a reverse transcriptase assay. No indication of thepresence of retrovirus is found.

Test on rabbits. A sample of the monovalent pooled harvest istested for cercopithecid herpesvirus 1 (B virus) and otherviruses by injection of not less than 100 ml into not fewer than10 healthy rabbits each weighing between 1.5 and 2.5 kg. Eachrabbit receives not less than 10 ml and not more than 20 ml, of


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which 1 ml is given intradermally at multiple sites, and theremainder subcutaneously. The rabbits are observed for atleast 3 weeks for death or signs of illness.

All rabbits that die after the first 24 h of the test and thoseshowing signs of illness are examined by autopsy, and thebrain and organs removed for detailed examination to establishthe cause of death.

The test is not valid if more than 20.0 per cent of the inoculatedrabbits show signs of intercurrent infection during theobservation period. The monovalent pooled harvest passesthe test if none of the rabbits shows evidence of infectionwith B virus or with other extraneous agents or lesions of anykind attributable to the bulk suspension.

If the presence of B virus is demonstrated, the measuresconcerning vaccine production described above under Cellcultures are taken.

Test on guinea-pigs. Administer to not less than five guinea-pigs, each weighing between 350 and 450 g, 0.1 ml of themonovalent pooled harvest by intracerebral injection and 0.5ml by intraperitoneal injection. Measure the rectal temperatureof each animal on each working day for 6 weeks. At the end ofthe observation period carry out autopsy on each animal.

In addition, administer to not fewer than five guinea-pigs 0.5ml by intraperitoneal injection and observe as described abovefor 2 to 3 weeks. At the end of the observation period, carryout a passage from these animals to not fewer than five guinea-pigs using blood and a suspension of liver or spleen tissue.Measure the rectal temperature of the latter guinea-pigs for 2to 3 weeks. Examine by autopsy all animals that, after the firstday of the test, die or are killed because they show disease orshow for three consecutive days a body temperature higherthan 39°; carry out histological examination to detect infectionwith Marburg virus; in addition, inject a suspension of liver orspleen tissue or of blood intraperitoneally into not fewer thanthree guinea-pigs. If any signs of infection with Marburg virusare noted, confirmatory serological tests are carried out onthe blood of the affected animals. The monovalent pooledharvest complies with the test if not fewer than 80.0 per centof the guinea-pigs survive to the end of the observation periodand remain in good health and no animal shows signs ofinfection with filoviruses virus.

FINAL BULK VACCINE

The final bulk vaccine is prepared from one or moresatisfactory monovalent pooled harvests and may containmore than one virus type. Suitable flavouring substances andstabilisers may be added.



FINAL LOT

Only a final lot that complies with the following requirementfor thermal stability and is satisfactory with respect to each ofthe requirements given below under Identification, Tests andAssay may be released for use.

Thermal stability. Expose samples of the final lot at 37° for48 hours. Determine the total virus concentration as describedunder Assay in parallel for the heated vaccine and for unheatedvaccine. The estimated difference between the total virusconcentration of the unheated and heated vaccines is notgreater than 0.5 log10 infectious virus units (CCID50) per singlehuman dose.

Identification

The vaccine is shown to contain poliovirus of each type statedon the label, using specific antibodies.


Assay

Titrate for infectious virus at least in triplicate using the methoddescribed below. Use an appropriate virus referencepreparation to validate each assay. If the vaccine containsmore than one poliovirus type, titrate each type separately,using appropriate type-specific antiserum (or preferably amonoclonal antibody) to neutralise each of the other typespresent.

For a trivalent vaccine, the estimated mean virus titres mustbe: not less than 1 × 106.0 infectious virus units (CCID50) persingle human dose for type 1; not less than 1 × 105.0 infectiousvirus units (CCID50) for type 2; and not less than 1 × 105.8

infectious virus units (CCID50) for type 3.

For monovalent or divalent vaccine, the minimum virus titresare decided by the competent authority.

Method. Groups of eight to twelve flat-bottomed wells in amicrotitre plate are inoculated with 0.05 ml of each of theselected dilutions of virus followed by a suitable cellsuspension of the Hep-2 (Cincinnati) line. The plates areincubated at a suitable temperature. Examine the cultures ondays 7 to 9.The assay is not valid if (a) the confidence interval (P = 0.95)of the logarithm of the virus concentration is greater than±0.3; (b) the virus concentration of the reference preparationdiffers by more than 0.5 log CCID50 from the assigned value.Labelling. The label states (1) the types of polioviruscontained in the vaccine; (2) the minimum amount of virus ofeach type contained in one single human dose; (3) the cellsubstrate used for the preparation of the vaccine; (4) that thevaccine is not to be injected.


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Rabies Vaccine, HumanRabies Vaccine for Human use is a freeze-dried or liquid(adsorbed) preparation of a suitable approved, strain of fixedrabies virus grown in an approved cell culture/ embryos ofduck/chicken and inactivated by a validated method.

The freeze-dried vaccine is reconstituted immediately beforeuse as stated on the label to give a clear or slightly opalescentsolution/suspension. It may be coloured owing to the presenceof a pH indicator.

The vaccine complies with the General Requirements ofVaccines for Human Use.

Production

General provisions

The vaccine is produced on the basis of virus seed lot systemand if a cell line is used for virus propagation, a cell-banksystem shall be followed. The production method shall havebeen shown to yield consistently vaccines that comply withthe requirements for immunogenicity, safety and stability.Unless otherwise justified and authorized, the virus in thefinal vaccine shall not have undergone more passages fromthe master seed lot than was used to prepare the vaccineshown in clinical studies to be satisfactory with respect tosafety and efficacy.The production method is validated to demonstrate that theproduct, if tested, would comply with the test for safety andefficacy.

Substrate for virus propagationThe virus is propagated in any suitable approved cell substratelike a human diploid cell line (2.7.2), a continuous cell line, orin duck embroys or in cultures of chicken embryos derivedfrom a flock certified as free from specified pathogens(2.7.7).

SEED LOT

The strain of rabies virus used shall be identified by historicalrecords that include information on the origin of the strainand its subsequent manipulation.

Working seed lots are prepared by not more than five passagesfrom the master seed lot.

Only a working seed lot that complies with the followingrequirements may be used for virus propagation.

Identification

Each working seed lot is identified as rabies virus using specificantibodies by an approved method.

Virus concentration. The virus concentration of each workingseed lot is determined by a cell culture method using

immunofluoresence or any other approved method.

Extraneous agents (2.7.3). The working seed lot complies withthe requirements for the virus seed lots.


All processing of the cell bank and subsequent cell culturesare done under aseptic conditions in an area where no othercells are handled. Approved animal (but not human) serummay be used in the media, but the final medium for maintainingcell growth during virus multiplication does not contain animalserum; the media may contain human albumin. Serum proteins,if present are reduced to an acceptable level by a suitablemethod of purification. Serum and trypsin used in thepreparation of cell suspension and media are shown to be freefrom infectious extraneous agents. The cell culture media maycontain a pH indicator such as phenol red and approvedantibiotics at the lowest effective concentration. Not less than500 ml of the cell cultures employed for vaccine productionare set aside as uninfected cell cultures (control cells). Thevirus suspension is harvested on one or more occasionsduring incubation. Multiple harvests from the same productioncell culture may be pooled and considered as a single harvest.When vaccine is prepared in embryonated eggs, the eggproteins are minimized by an appropriate method of purification.The eggs are inoculated with virus seed by the yolk sac route.The infected sterile living embryos are harvested, minced andemulsified in suitable diluent, and stabilizer with asepticprecautions. Emulsions are centrifuged, supernatants arecollected and stored as raw virus harvest at a suitabletemperature.Viral harvests that comply with the following requirements arepooled in the preparation of the inactivated viral harvest.

Identification

The single harvest contains virus that is identified as rabiesvirus using specific antibodies by an approved method.

Virus concentration. Titrate for infective virus in cell culturesor by any other approved method. The titre is used to monitorconsistency of production.

Control cells. The control cells of the production cell culturefrom which the single harvest is derived should comply with atest for identification and with the requirements for extraneousagents (2.7.3).

Control eggs. Control eggs shall be tested for freedom fromhaemagglutinating agents, and other extraneous agents.

PURIFICATION AND INACTIVATION

The virus harvests may be concentrated and/or purified bysuitable methods; the virus harvest is inactivated by a validatedmethod at a fixed, well defined stage of the process which may

RABIES VACCINE, HUMAN

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be before, during or after any concentration or purification.The method shall have been shown to be capable ofinactivating rabies virus without destruction of theimmunogenic activity. If betapropiolactone is used, theconcentration shall at no time exceed 1:3500.

Cell culture vaccines

Only an inactivated viral suspension that complies with thefollowing requirements may be used in the preparation of thefinal bulk vaccine.

Inactivation. Inactivation is confirmed by carrying out anamplification test for residual infectious rabies virus, not morethan 4 days after inactivation or on the sample frozen afterinactivation and stored at -70º. Inoculate a quantity ofinactivated viral suspension equivalent to not less than 25vaccine doses into cell cultures of the same type as thoseused for production of the vaccine. Make a passage after 7days. Maintain the cultures for a further period of 14 days andthen examine the cell cultures for rabies virus using an immunefluorescence test. No rabies virus is detected. Alternatively, 5ml of each culture fluid is pooled on days 14 and 21 and 0.03 mlis inoculated intracerebrally into each of the 10 mice weighing12 to 15 g. The mice are observed for 14 days for symptomscaused by rabies virus, and mice showing symptoms of rabiesare sacrificed and virus presence is confirmed byimmunoflurescence test or tested for live virus in cell cultureby immunofluorescence test or method of equal sensitivity.No rabies virus should be detected.Residual host-cell DNA (2.2.15). If a continuous cell line isused for virus propagation, the content of residual host-cellDNA, determined using a suitable method, should not begreater than 10 ng per single human dose.

Embryonated egg vaccineOnly concentrated viral suspensions that comply with thetest for sterility, antigen content and endotoxin requirementsmay be used for preparation of bulk for inactivation.Inactivation is confirmed by carrying out Mice InoculationTest for residual infectious rabies virus, not more than fourdays after inactivation or on the sample frozen 4 days afterinactivation are stored at –70°. Inoculate intracerebrally 0.03ml into each of 20 mice weighing between 12 and 15 g. Themice are observed for 14 days for symptoms caused by rabiesvirus and mice showing symptoms of rabies are sacrificed andvirus presence is confirmed by an immunoflurescence test ortested for live virus in cell culture by immunofluorescencetest or method of equal sensitivity. No rabies virus should bedetected.

FINAL BULK VACCINE

The final bulk vaccine is prepared from one or more inactivatedviral suspensions. An approved stabilizer may be added to

maintain the activity of the product during and after freeze-drying. Thiomersal can be used as preservative.


Antigen content. Determine the antigen content by a suitableapproved in vitro or in vivo method. The content should bewithin the limits approved for the particular product.


FINAL LOT

The final bulk vaccine is distributed aseptically into sterilecontainers and can be freeze-dried in case of lyophilizedproducts. The containers are then sealed so as to preventcontamination and the introduction of moisture.

Only a final lot that complies with each of the requirementsgiven below under Identification, Tests and Assay may bereleased for use. Provided that the test for inactivation hasbeen carried out with satisfactory results on the inactivatedvirus suspension and the test for bovine serum albumin hasbeen carried out with satisfactory results on the final bulkvaccine, these tests may be omitted on the final lot.

IdentificationThe vaccine is shown to contain rabies virus antigen by asuitable immunochemical method using specific antibodies,alternatively, the Assay also serves to identify the vaccine.

TestsInactivation. Inoculate a quantity equivalent to not less than25 human doses of vaccine into cell cultures of the same typeas those used for production of the vaccine. Make a passageafter 7 days. Maintain the culture for a further 14 days andthen examine the cell culture for rabies virus using animmunofluorescence test. No rabies virus is detected.Alternatively, inject 0.03ml of the vaccine intracerebrally intoeach of the 10 mice weighing between 12 and 15g. Neithersymptoms of disease in the central nervous system nor deathoccurs in any of the animal within 14 days. If the inactivationtest is already performed on inactivated virus used for finallot, it may be omitted from the test on final lot.


Bacterial endotoxins (2.2.3). Less than 25 IU per single humandose.

Pyrogens (2.2.8). Complies with the test for pyrogens. Unlessotherwise justified and authorized, inject into each rabbit asingle human dose of the vaccine diluted to ten times itsvolume.

Water (2.3.43). Not more than 3.0 per cent determined by anapproved method.


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Abnormal toxicity (2.2.1). Complies with the test for abnormaltoxicity.Accelerated degradation. The potency determined by methoddescribed under “Assay” of a sample of the preparation underexamination after storage at 37° for 4 weeks is not less than 2.5units per single human dose. This may not be mandatory forlot release, once the consistency of the product is approvedby National Regulatory Authority.Bovine serum albumin (for cell culture vaccine). Not morethan 50 ng per single human dose, determined by a suitableimmunochemical method (2.2.14).Aluminium content (for gel absorbed vaccine) (2.3.9). Notmore than 1.25 mg per single human dose.Ovalbumin (for egg based vaccines). Not more than 1 µg ofovalbumin per human dose, determined by a suitable techniqueusing a suitable reference preparation of ovalbumin.Residual host-cell DNA (for continuous cell line vaccines)(2.2.15). Should not be greater than 10 ng per single humandose.Antimicrobial preservative. Where applicable, determine theamount of antimicrobial preservative by a suitable chemicalmethod. The content is not less than 85.0 per cent and notgreater than 115.0 per cent of the intended amount.

Assay

The Standard preparation is the international standard oranother suitable preparation, the potency of which has beendetermined in relation to the International standard. Thepotency of rabies vaccine is determined by comparing thedose necessary to protect mice against a lethal intracerebraldose of rabies vaccine necessary to provide the sameprotection. For this comparison, a reference preparation ofrabies vaccine, calibrated in international units, and a suitablepreparation of rabies virus for use as the challenge preparationare necessary.

The international unit is the activity contained in a statedquantity of the international standard. The equivalence ininternational units of the international standard is stated bythe World Health Organization.

The test described below uses a parallel-line model with atleast three points for the vaccine under examination and thereference preparation.

Test animals. Use mice of a suitable strain, drawn from a uniformstock three to four weeks old, weighing between 11 and 15 g.Distribute the mice into six groups of at least 16 mice each andfour groups of 10 mice each and must be of the same sex or thesexes must be equally distributed among the groups.

Throughout the test all mice that die before the fifth day afterchallenge are excluded from the test and all mice that die with

signs of rabies between the fifth and fourteenth day afterchallenge are counted as failing to resist the challenge.

The strain of mice suitable for the test is such that when 0.03ml containing 5 to 50 LD50 of the challenge virus suspensionis injected intracerebrally per mouse there is 100 per centmortality.

Standard challenge virus suspension. A working pool of thechallenge virus strain is prepared by injecting intracerebrally0.03ml of a 10 fold dilution of the CVS strain of rabies virus in2 per cent v/v sterile inactivated normal horse serum in waterfor injection or another suitable diluent approved by thecompetent authority into a suitable number of test animals.The animals when moribund after showing characteristic signsof rabies are sacrificed and their brains harvested aseptically.They are then washed in chilled saline solution to removeblood clots. A 10 per cent suspension of the brains is preparedin a suitable diluent approved by the competent authority andthoroughly homogenised. After centrifuging lightly, thesupernatant liquid is distributed into sterile vials and freezedried. The sealed and freeze-dried supernatant liquid containingvials are stored at -20°. When stored under prescribedconditions the virus titre of the freeze-dried preparation maybe expected to be maintained for not less than 3 years.Alternatively, the washed brains are homogenised in a suitablediluent approved by the competent authority to give 10 percent suspension. It is then centrifuged lightly, distributed intosterile ampoules or sterile plastic vials and sealed. The sealedampoules or plastic vials can be stored at –60º or below. Whenstored under prescribed conditions, the virus titre may beexpected to be maintained for not less than one year. Storagetime needs to be validated by the manufacturer.

Virus titre of the challenge virus. Prepare ten fold serialdilutions of the standard challenge virus suspension. Usingthe four groups of 10 mice each, inject 0.03 ml of the virussuspension intracerebrally into each mouse, using a differentgroup for each suspension. Observe the mice for 14 days.Calculate the virus titre of the standard challenge virussuspension in LD50 per dose of 0.03 ml by standard statisticalmethods.

Determination of potency of the vaccine. Reconstitute thestandard preparation with a suitable diluent. Prepare at leastthree 5-fold serial dilutions of the solution of the standardpreparation and three 5-fold serial dilutions of the vaccineunder examination. For both, the standard preparation andthe preparation under examination, the serial dilutions shouldbe prepared in such a way that the lowest dilution protectsmore than 50 per cent of the injected mice. Allocate one dilutionto each of the six groups of 16 mice each. Injectintraperitoneally each mouse in each group with dilutions ofthe vaccine and reference preparation and repeat the injections.After 7 days, prepare identical dilutions of the vaccine and


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reference preparation and repeat the injections.

After a further 7 days, inject each vaccinated mouseintracerebrally with 0.03 ml of the standard challenge virussuspension such that on the basis of preliminary titration,0.03 ml contains between 5 to 50 LD50. Observe the mice for 14days and record the number of mice surviving the challengein each group. Calculate the potency of the preparation underexamination by standard statistical methods.

The vaccine complies with the test if the estimated potency isnot less than 2.5 IU per single human dose.

The test is not valid unless (a) for both the preparation underexamination and the standard preparation, the 50 per centprotective dose lies between the largest and smallest dosesgiven to the mice; (b) there is not deviation from linearity orparallelism of the dose response lines, the confidence limit (P= 0.95) are not less than 25.0 per cent and not more than 400per cent of the estimated potency; (c) the titre of the challengevirus suspension lies between 5 to 50 LD50.

Labelling. The label states the biological origin of the cellsused for the preparation of the vaccine.

Rubella Vaccine (Live)Rubella Vaccine (Live) is a freeze-dried preparation of a suitableattenuated strain of rubella virus. The vaccine is reconstitutedimmediately before use to give a clear liquid or may be colouredowing to the presence of a pH indicator.

Production

General provisions

The production of vaccine is based on a virus seed-lot systemand a cell-bank system. The production method shall havebeen shown to yield consistently live rubella vaccines ofadequate immunogenicity and safety in man. Unless otherwisejustified and authorised, the virus in the final vaccine shallhave undergone no more passages from the master seed lotthan were used to prepare the vaccine shown in clinical studiesto be satisfactory with respect to safety and efficacy.



The virus is propagated in human diploid cells (2.7.2).

SEED LOT

The strain of rubella virus used in the production of rubellavaccine shall be identified by historical records that include

information on the origin of the strain and its subsequentmanipulation.To avoid unnecessary use of monkeys in the test forneurovirulence virus seed lots are prepared in large quantitiesand stored at temperatures below -20° if freeze-dried, or below-60° if not freeze-dried.Only a seed lot that complies with the following tests may beused for virus propagation.

IdentificationThe master and working seed lots are identified as rubellavirus by serum neutralisation in cell culture, using specificantibodies.Virus concentration. The virus concentration of the masterand working seed lots is determined to ensure consistency ofproduction.Extraneous agents (2.7.3). The working seed lot complies withthe tests for seed lots.Neurovirulence (2.7.5). The master/working seed lot complieswith the test for neurovirulence of live virus vaccines. Macacaand Cercopithecus monkeys are suitable for the test.PROPAGATION AND HARVEST

All processing of the cell bank and subsequent cell cultures isdone under aseptic conditions in an area where no other cellsare handled. Suitable animal (but not human) serum may beused in the growth medium, but the final medium for maintainingcell growth during virus multiplication does not contain animalserum. Serum and trypsin used in the preparation of cellsuspensions and culture media are shown to be free fromextraneous agents. The cell culture medium may contain a pHindicator such as phenol red and suitable antibiotics at thelowest effective concentration. It is preferable to have asubstrate free from antibiotics during production. Not lessthan 500 ml of the production cell culture is set aside asuninfected cell culture (control cells). The temperature ofincubation is controlled during the growth of the virus. Thevirus suspension is harvested, on one or more occasions,within 28 days of inoculation. Multiple harvests from the sameproduction cell culture may be pooled and considered as asingle harvest.

Only a single harvest that complies with the followingrequirements may be used in the preparation of the final bulkvaccine.

IdentificationThe single harvest contains virus that is identified as rubellavirus by serum neutralization in cell culture, using specificantibodies.Virus concentration. The virus concentration in the singleharvest is determined as prescribed under Assay to monitor

RUBELLA VACCINE (LIVE)

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consistency of production and to determine the dilution to beused for the final bulk vaccine.

Extraneous agents (2.7.3). The single harvest complies withthe tests for extraneous agents.

Control cells. The control cells comply with a test foridentification and with the tests for extraneous agents (2.7.3).

FINAL BULK VACCINE

Single harvests that comply with the above tests are pooledand clarified to remove cells. A suitable stabilizer may be addedand the pooled harvests diluted as appropriate.



A minimum virus concentration for release of the product isestablished such as to ensure, in the light of stability data,that the minimum concentration stated on the label will bepresent at the end of the period of validity.

Only a final lot that complies with the tests for minimum virusconcentration for release, with the following requirement forthermal stability and with each of the requirements given belowunder Identification and Tests may be released for use.Provided that the test for bovine serum albumin has beencarried out with satisfactory results on the final bulk vaccine,it may be omitted on the final lot.

Thermal stability. Maintain samples of the final lot of freeze-dried vaccine in the dry state at 37° for 7 days. Determine thevirus concentration as described under Assay in parallel forthe vaccine held at 37° for 7 days and for vaccine stored at 2°to 8°. The virus concentration of the heated vaccine is notmore than 1.0 log10 lower than that of the unheated vaccine.

Identification

When the vaccine reconstituted as stated on the label is mixedwith specific rubella antibodies, it is no longer able to infectsusceptible cell cultures.

Tests





Assay

Titrate the vaccine for infective virus at least in triplicate,using at least five cell cultures for each 0.5 log10 dilution stepor by a method of equal precision. Use an appropriate virusreference preparation to validate each assay. The estimatedvirus concentration is not less than that stated on the label;the minimum virus concentration stated on the label is notless than 1 × 103 CCID50 per human dose. The assay is notvalid if the confidence limits (P=0.95) of the logarithm of thevirus concentration is greater than ± 0.3.


Labelling. The label states (1) the strain of virus used for thepreparation of the vaccine; (2) the type and origin of the cellsused for the preparation of the vaccine; (3) the minimum virusconcentration; (4) the time within which the vaccine must beused after reconstitution; (5) that the vaccine must not begiven to a pregnant woman and that a woman must not becomepregnant within two months.

Tetanus Vaccine (Adsorbed)Tetanus Vaccine (Adsorbed) is a preparation of tetanus formoltoxoid adsorbed on mineral carrier. The formol toxoid isprepared from the toxin produced by the growth of Clostridiumtetani.

Production

General provisions

The maximum number of Lf per single human dose of tetanusvaccine is 25.

The production method is validated to demonstrate that theproduct, if tested, would comply with the following test.

BULK PURIFIED TOXOID

For the production of tetanus toxin, from which toxoid isprepared, seed cultures are managed in a defined seed-lotsystem in which toxinogenicity is conserved and, wherenecessary, restored by deliberate reselection. A highlytoxinogenic strain of Clostridium tetani with known originand history is grown in a suitable liquid medium. At the end ofcultivation, the purity of each culture is tested andcontaminated cultures are discarded. Toxin-containing culturemedium is collected aseptically. The toxin content (Lf per ml)is checked to monitor consistency of production. Singleharvests may be pooled to prepare the bulk purified toxoid.

TETANUS VACCINE (ADSORBED)

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The toxin is purified to remove components likely to causeadverse reactions in humans. The purified toxin is detoxifiedwith formaldehyde by a method that avoids destruction of theimmunogenic potency of the toxoid and reversion of toxoid totoxin, particularly on exposure to heat. Alternatively,purification may be carried out after detoxification.

Only bulk purified toxoid that complies with the followingrequirements may be used in the preparation of the final bulkvaccine.


Absence of tetanus toxin. Inject subcutaneously at least 500Lf of purified toxoid in a volume of 1 ml into each of fivehealthy guinea-pigs, each weighing 250 to 350 g, that havenot previously been treated with any material that will interferewith the test. If within 21 days of the injection any of theanimals shows signs of or dies from tetanus, the toxoid doesnot comply with the test. If more than one animal dies fromnon-specific causes, repeat the test once; if more than oneanimal dies in the second test, the toxoid does not complywith the test.

Irreversibility of toxoid. Using the buffer for the final vaccine,prepare a dilution of the bulk purified toxoid containing thesame toxoid concentration as the final vaccine. Divide thedilution into two equal parts. Keep one of them at 2° to 8° andthe other at 37° for 6 weeks. Test both dilutions by a suitablesensitive assay for active tetanus toxin, such as inoculationinto mice or guinea-pigs. The toxoid complies with the test ifneither sample produces any sign of a toxic reaction attribut-able to tetanus toxin.

Antigenic purity. Not less than 1,000 Lf per mg of proteinnitrogen.

FINAL BULK VACCINE

The final bulk vaccine is prepared by adsorption of a suitablequantity of bulk purified toxoid onto a mineral carrier such ashydrated aluminium phosphate or aluminium hydroxide; theresulting mixture is approximately isotonic with blood. Suitableantimicrobial preservatives may be added. Certain antimicrobialpreservatives, particularly those of the phenolic type,adversely affect the antigenic activity and must not be used.Only final bulk vaccine that complies with the followingrequirements may be used in the preparation of the final lot.Specific toxicity. Inject five times the dose stated on the labelsubcutaneously or intraperitoneally into each of five guinea-pigs. None of the guinea-pigs shows any symptoms of, ordies from, tetanus within 21 days. If more than one animal diesfrom non-specific causes within this period repeat the test.None of the second group of animals shows symptoms of

tetanus or dies from tetanus or any other cause within 21days.



pH (2.4.24). 6.0 and 7.0


Abnormal toxicity (2.2.1). Complies with the test forabnormal toxicity for antisera and vaccine.

FINAL LOT


Only a final lot that is satisfactory with respect to each of therequirements given below under Identification, Tests andAssay may be released for use. Provided the tests forantimicrobial preservative and the assay have been carriedout with satisfactory results on the final bulk vaccine, theymay be omitted on the final lot.

Identification

Tetanus toxoid is identified by a suitable immunochemicalmethod (2.2.14). The following method, applicable to certainvaccines, is given as an example. Dissolve in the vaccine underexamination sufficient sodium citrate to give a 10 per centsolution. Maintain at 37° for about 16 hours and centrifugeuntil a clear supernatant liquid is obtained. The clearsupernatant liquid reacts with a suitable tetanus antitoxin,giving a precipitate.

Tests





Abnormal toxicity (2.2.1). Complies with the test for abnormaltoxicity for antisera and vaccine.


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Potency of tetanus component

Assay. Determine by either of the following methods.

(1) Inject subcutaneously on each of two occasions separatedby an interval of not more than 4 weeks, one-tenth of thestated human dose diluted to 1 ml with saline solution intoeach of 9 normal, healthy guinea-pigs weighing between 250and 350 g. Not more than 2 weeks after the second injection,collect the serum from each animal and carry out the biologicaltest for tetanus antitoxin, described under Tetanus antitoxinor any other method approved by National RegulatoryAuthority.Sera of at least 6 guinea-pigs out of 9 should contain not lessthan 0.5 Unit of tetanus antitoxin per ml.(2) Carry out the biological assay of adsorbed Tetanus Vaccine(Adsorbed) described below.If the lower limit of the 95.0 per cent confidence interval ofestimated potency is less than 40 IU per single human dosethen the limits of the 95.0 per cent confidence interval of theestimater of potency shall be within 50 to 200 per cent of theestimated potency unless the lower limit of the 95.0 per centconfidence interval of the estimated potency is greater than40 IU per single human dose.

Biological assay of adsorbed tetanus vaccine

The potency of adsorbed tetanus vaccine is determined bycomparing the dose of the vaccine required to protect guinea-pigs or mice from the paralytic effects of a subcutaneousinjection of tetanus toxin with the dose of the Standardpreparation needed to give the same protection. For thiscomparison, the Standard preparation of adsorbed tetanustoxoid and a suitable preparation of tetanus toxin, for use as achallenge toxin, are necessary.


The Standard preparation is International standard for Tetanustoxoid, adsorbed or another suitable preparation, the potencyof which has been determined in relation to the Internationalstandard.Suggested method

(a) Test on guinea-pigs

Test animals. Use healthy guinea-pigs from the same stockand weighing between 250 and 350 g. Distribute them into sixgroups of sixteen each. The guinea-pigs should all be of thesame sex or the males and females should be distributed equallybetween the groups. If the challenge toxin to be used has notbeen shown to be stable or has not been adequatelystandardised, include four further groups of five guinea-pigsto serve as unvaccinated controls.Challenge toxin. Select a preparation of tetanus toxincontaining not less than 50 times the 50 per cent paralytic

dose per ml. If the challenge toxin preparation has been shownto be stable, it is not necessary to verify the paralytic dose forevery assay.

Preparation of the challenge toxin solution. Immediately priorto use, prepare from the challenge toxin by dilution withphosphate buffered saline pH 7.4 a challenge toxin solutioncontaining fifty times the 50 per cent paralytic dose per ml. Ifnecessary, dilute portions of this challenge toxin solution16-, 50- and 160-fold with the same buffer solution.

Determination of potency. Prepare in saline solution threedilutions of the vaccine under examination and three dilutionsof a solution of the Standard preparation such that, for each,the dilutions form a series differing by not more than 2.5-foldsteps and in which the dilutions of intermediate concentration,when injected subcutaneously in 1-ml volumes into guinea-pigs, protect approximately 50 per cent of the animals from theparalytic effects of the subcutaneous injection of the quantityof tetanus toxin prescribed for this test. Allocate the sixdilutions one to each of the six groups of sixteen guinea-pigsand inject subcutaneously 1.0 ml of each dilution into eachguinea-pig in the group to which that dilution is allocated.After 28 days inject each animal subcutaneously with 1.0 mlof the challenge toxin solution containing fifty times the 50per cent paralytic dose. If necessary, allocate the challengetoxin solution and the three dilutions made from it one to eachof the four groups of five guinea-pigs and injectsubcutaneously 1.0 ml of each toxin solution into each guinea-pig in the group to which that toxin solution is allocated.Examine the guinea-pigs twice daily, remove and kill all animalsshowing definite signs of tetanus paralysis. Count the numberof guinea-pigs without paralysis 5 days after injection of thechallenge toxin and calculate the potency of the vaccine underexamination relative to the potency of the Standard preparationon the basis of the number of animals without paralysis ineach of the six groups of sixteen, using standard statisticalmethods.

The test is not valid unless (a) for both the vaccine underexamination and the Standard preparation, the 50 per centprotective doses lie between the largest and smallest doses ofthe preparations given to the guinea-pigs; (b) if applicable,the number of paralysed animals among the four groups offive injected with the challenge toxin solution and its dilutionsindicate that the challenge was approximately 50 times the 50per cent paralytic dose; (c) the fiducial limits of assay liebetween 50.0 per cent and 200.0 per cent of the estimatedpotency; (d) the statistical analysis shows no deviations fromlinearity or parallelism. The test may be repeated any numberof times but when more than one test is performed the resultsof all valid tests must be combined in the estimate of potency.

(b) Test on mice

Test animals. Use healthy mice from the same stock, weighing


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between 14 and 20 g. Distribute them into six groups of sixteeneach. If the challenge toxin to be used has not been shown tobe stable or has not been adequately standardised, includefour further groups of six mice to serve as unvaccinatedcontrols. The mice should all be of the same sex or the malesand females should be distributed equally among the groups.

Challenge toxin. Select a preparation of tetanus toxincontaining not less than 100 times the 50 per cent paralyticdose per ml.

Preparation of the challenge toxin solutions. Immediately priorto use prepare from the challenge toxin by dilution withphosphate buffered saline pH 7.4 a challenge toxin solutioncontaining fifty times the 50 per cent paralytic dose in each 0.5ml. If necessary, dilute portions of this challenge toxin solution16-, 50- and 160-fold with the same buffer solution.

Determination of potency. Prepare in saline solution threedilutions of the vaccine under examination and three dilutionsof a solution of the Standard preparation such that, for each,the dilutions form a series differing by not more than 2.5-foldsteps and in which the dilutions of intermediate concentration,when injected subcutaneously in 0.5 ml volumes into mice,protect approximately 50 per cent of the animals from theparalytic effects of the subcutaneous injection of the quantityof tetanus toxin prescribed for this test. Allocate the sixdilutions one to each of the six groups of sixteen mice andinject subcutaneously 0.5 ml of each dilution into each mousein the group to which the dilution is allocated. After 28 daysinject each animal subcutaneously with 0.5 ml of the challengetoxin solution containing fifty times the 50 per cent paralyticdose. If necessary, allocate the challenge toxin solution andthe three dilutions made from it one to each of the four groupsof six mice and inject subcutaneously 0.5 ml of each toxinsolution into each mouse in the group to which that toxinsolution is allocated. Count the number of mice withoutparalysis 4 days after injection of the challenge toxin andcalculate the potency of the vaccine under examination relativeto the potency of the Standard preparation on the basis of thenumbers of animals without paralysis in each of the six groupsof sixteen, using standard statistical methods.

The test is not valid unless (a) for both the vaccine underexamination and the Standard preparation, the 50 per centprotective doses lie between the largest and smallest doses ofthe preparations given to the mice; (b) if applicable, the numberof paralysed animals among the four groups of six injectedwith the challenge toxin solution and its dilutions indicatethat the challenge was approximately 50 times the 50 per centparalytic dose; (c) the fiducial limits of the assay lie between50.0 per cent and 200.0 per cent of the estimated potency; (d)the statistical analysis shows no deviation from linearity orparallelism. The test may be repeated any number of times butwhen more than one test is performed the results of all valid

tests must be combined in the estimate of potency.

(c) Determination of antibodies in guinea-pigs

Preparation of serum samples. For preparation of serumsamples, the following technique has been found suitable.Invert the tubes containing blood samples 6 times and allowto stand at 37° for 2 h, then at 4° for 2 hours, centrifuge at roomtemperature at 800 g for 20 min. transfer the serum to steriletubes and store at a temperature below -20°. At least 40.0 percent yield of serum is obtained by this procedure.

Determination of antibody titre. The ELISA and ToBI testsshown below are given as examples of immunochemicalmethods that have been found suitable for the determinationof antibody tire.

Determination of antibody titre in guinea-pig serum byenzyme-linked immunosorbent assay (ELISA). Dilutions oftest and reference sera are made on ELISA plates coated withtetanus toxoid. A positive guinea-pig serum control and anegative guinea-pig serum control are included on each plateto monitor the assay performance. Peroxidase-conjugatedrabbit or goat antibody directed against guinea-pig-IgG isadded followed by a peroxidase substrate. Optical density ismeasured and the relative antibody titre is calculated usingthe usual statistical methods.

Reagents and equipment

ELISA plates: 96 wells, columns 1-12, rows A-H.

Clostridium tetani guinea-pig antiserum (for vaccines-humanuse) reference preparation (positive control serum).

Peroxidase conjugate. Peroxidase-conjugated rabbit or goatantibody directed against guinea-pig IgG.

Tetanus toxoid.

Carbonate coating buffer pH 9.6. Dissolve 1.59 g of anhydroussodium carbonate and 2.93 g of sodium hydrogen carbonatein 1000 ml of water. Distribute into 150 ml bottles and steriliseby autoclaving at 121° for 15 min.

Phosphate buffered saline pH 7.4 (PBS). Dissolve with stirring80.0 g of sodium chloride, 2.0 g of potassium dihydrogenphosphate, 14.3 g of disodium hydrogen phosphate dihydrateand 2.0 g of potassium chloride in 1000 ml of water. Store atroom temperature to prevent crystallisation. Dilute to 10 timesits volume with water before use.Citric acid solution. Dissolve 10.51 g of citric acid in 1000 mlof water and adjust the solution to pH 4.0 with a 400 g/l solutionof sodium hydroxide.Washing buffer. PBS containing 0.5 g/l of polysorbate 20.Diluent block buffer. PBS containing 0.5 g/l of polysorbate 20and 25 g/l of dried skimmed milk.Peroxidase substrate. Shortly before use, dissolve 10 mg of


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diammonium 2,2'-azinobis(3-ethylbenzothiazoline-6-sulphonate) (ABTS) in 20 ml of citric acid solution.Immediately before use add 5 µl of strong hydrogen peroxidesolution.

Method

The description below is given as an example of a suitableplate lay-out but others may be used. Wells 1A-H are fornegative control serum and wells 2A-H and 3A-H are forpositive control serum for assay monitoring. Wells 4-12A-Hare for test samples.

Coat each well of the ELISA plates with 100 µl of tetanustoxoid solution (0.5 Lf/ml in carbonate coating buffer).

Allow to stand overnight at 4° in a humid atmosphere. Toavoid interference from temperature gradient, do not stackmore than 4 plates high. On the following day, wash the platesthoroughly with washing buffer. Block the plates by additionof 100 µl of diluent block buffer to each well. Incubate in ahumid atmosphere at 37° for 1 h. Wash the plates thoroughlywith washing buffer. Place 100 µI of diluent block buffer ineach well of the plates, except those of row A. Prepare suitabledilutions of negative control serum, positive control serum(from about 0.01 IU/ml) and test sera. Allocate the negativecontrol serum to column 1, positive control serum to columns2 and 3 and test sera to columns 4-12 and add 100 µI of eachserum to the first 2 wells of the column to which it is allocated.Using a multi channel micropipette, make twofold serialdilutions from row B down the plate to row H by transferring100 µl to the following well. Discard 100 µl from the last row sothat all wells contain 100 µI. Incubate at 37° for 2 h. Washthoroughly with washing buffer. Prepare a suitable dilution (a1 in 2000 dilution has been found suitable) of peroxidaseconjugate in diluent block buffer and add 100 µI to each well.Incubate at 37° in a humid atmosphere for 1 h. Wash the platesthoroughly with washing buffer. Add 100 µI of peroxidasesubstrate to each well. Allow to stand at room temperature,protected from light, for 30 min. Read the plates at 405 nm inthe same order as addition of substrate was made.

Determination of antibody titre in guinea-pig serum by toxin-or toxoid-binding inhibition (ToBI). Tetanus toxin or toxoid isadded to serial dilutions of test and reference sera; the serum/antigen mixtures are incubated overnight. To determineunbound toxin or toxoid, the mixtures are transferred to anELISA plate coated with tetanus antitoxin. Peroxidase-conjugated equine anti-tetanus IgG is added followed by aperoxidase substrate. Optical density is measured and theantibody titre is calculated using the usual statistical methods.A positive control serum and a negative control serum areincluded on each plate to monitor assay performance.

Reagents and equipment

Round-bottomed, rigid polystyrene microtitre plates.

Flat-bottomed ELISA plates.

Tetanus toxin or tetanus toxoid.

Clostridium tetani guinea-pig antiserum (for vaccines-humanuse) reference preparation.

Equine anti-tetanus IgG.

Peroxidase-conjugated equine anti-tetanus IgG.

Carbonate buffer pH 9.6. Dissolve 1.5 g of anhydrous sodiumcarbonate, 2.39 g of sodium hydrogen carbonate and 0.2 g ofsodium azide in 1000 ml of water, adjust to pH 9.6 and autoclaveat 121° for 20 min.

Sodium acetate buffer pH 5.5. Dissolve 90.2 g of anhydroussodium acetate in 900 ml of water, adjust to pH 5.5 using asaturated solution of citric acid monohydrate and dilute to1000 ml with water.

Phosphate buffered saline pH 7.2 (PBS). Dissolve 135.0 g ofsodium chloride, 20.55 g of disodium hydrogen phosphatedihydrate and 4.80 g of sodium dihydrogen phosphatemonohydrate in water and dilute to 15 litres with the samesolvent. Autoclave at 100° for 60 min.

Diluent buffer. PBS containing 5 g/l of bovine albumin and 0.5g/l of polysorbate 80.

Block buffer. PBS containing 5 g/l of bovine albumin.

Tetramethylbenzidine solution. 6 g/l solution oftetramethylbenzidine in alcohol. The substance dissolveswithin 30-40 min at room temperature.

Peroxidase substrate. Mix 90 ml of water, 10 ml of sodiumacetate buffer pH 5.5, 1.67 ml of tetramethylbenzidine solutionand 20 µI of strong hydrogen peroxide solution.

Washing solution. Tap water containing 0.5 g/l of polysorbate80.

Method

Block the round-bottomed polystyrene microtitre plates byplacing in each well 150 µI of block buffer. Cover the plateswith a lid or sealer. Incubate in a humid atmosphere at 37° for1 h. Wash the plates thoroughly with washing solution. Place100 µI of PBS in each well. Place 100 µl of reference guinea-pig tetanus antitoxin in the first well of a row. Place 100 µl ofundiluted test sera in the first well of the required number ofrows. Using a multichannel micropipette, make it two fold serialdilutions across the plate (up to column 10), by transfer of 100µI to the following well. Discard 100 µl from the last column sothat all wells contain 100 µl. Prepare 0.1 Lf/ml solution of tetanustoxin or toxoid using PBS a diluent. Add 40 µl of this solutionto all wells except those column 12. The wells of row 11 are apositive control. Add 40 µl of PBS to the wells of column 12(negative control). Shake the plates gently and cover themwith lids. Coat the ELISA plates: immediately before use make


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a suitable dilution of equine anti-tetanus IgG in carbonatebuffer pH 9.6 and add 100 µl to all wells. Incubate the 2 seriesof plates overnight in a humid atmosphere at 37°. To avoidtemperature gradient effects, do not stack more than 4 platehigh. Cover the plates with lids. On the following day, washthe ELISA plates thoroughly with washing solution. Blockthe plates by placing in each well 125 µl of block buffer. Incubateat 37° in a humid atmosphere for 1 h. Wash the platesthoroughly with washing solution. Transfer 100 µl of the pre-incubation mixture from the polystyrene plates to thecorresponding wells of the ELISA plates, starting with column12 and then from 1 to 11. Cover the plates with a lid. Incubateat 37° in a humid atmosphere for 2 h. Wash the ELISA platesthoroughly with washing solution. Make a suitable dilution(a 1 in 4000 dilution has been found suitable) of the peroxidase-conjugated equine anti-tetanus IgG in diluent buffer. Add100 µl of the dilution to each well and cover the plates with alid. Incubate at 37° in a humid atmosphere for 1.5 h. Wash theELISA plates thoroughly with washing solution. Add 100 µlof peroxidase substrate to each well. A blue colour develops.Incubate the plates at room temperature. Stop the reaction ata given time (within 10 min) by the addition of 100 µl of 2 Msulphuric acid to each well in the same order as the additionof substrate. The colour changes from blue to yellow. Measurethe absorbance (2.4.7) at 450 nm immediately after addition ofthe sulphuric acid or maintain the plates in the dark untilreading.(d) Any other validated serological assay in guinea-pigs/miceapproved by National Regulatory Authority.Labelling. The label states (1) the human dose (ml); (2) theminimum units per single human dose or the minimumInternational Units per single human dose if potency test doneby challenge method or both; (3) the name and the amount ofthe adsorbent and preservative; (4) that the vaccine must beshaken before use; (5) that the vaccine is not to be frozen.

Tick-borne Encephalitis Vaccine(Inactivated)Tick-borne Encephalitis Vaccine (Inactivated) is a liquidpreparation of a suitable strain of tick-borne encephalitis virusgrown in cultures of chick-embryo cells or other suitable cellcultures and inactivated by a suitable, validated method.


The vaccine complies with the General Requirements ofVaccines for Human Use.

Production of the vaccine is based on a virus seed lot system.The production method shall have been shown to yield

consistently vaccines comparable with the vaccine of provenclinical efficacy and safety in man. The virus in the final vaccineshall not have undergone more passages from the master seedlot than the virus in the vaccine used in clinical trials.

The production method is validated to demonstrate that theproduct, if tested, would comply with the test for abnormaltoxicity and immunogenicity.


The virus is propagated in chick embryo cells prepared fromeggs derived from a chicken flock free from specified pathogens(2.7.7) or in other suitable cell cultures.

SEED LOT

The strain of virus used is identified by historical records thatinclude information on the origin of the strain and itssubsequent manipulation. Virus seed lots are stored at orbelow -60°.

Only a seed lot that complies with the following requirementsmay be used for virus propagation.

IdentificationEach seed lot is identified as containing the vaccine strain oftick-borne encephalitis virus by a suitable immunochemicalmethod, preferably using monoclonal antibodies.

Virus concentration. The virus concentration of each seedlot is determined by titration in suitable cell cultures to monitorconsistency of production.

Extraneous agents (2.7.3). Each seed lot complies with therequirements for extraneous agents in viral vaccines for humanuse; the tests in cell cultures are carried out in human andsimian cells only.


All processing of the cell cultures if performed under asepticconditions in an area where no other cells are being handled.Serum and trypsin used in the preparation of cell suspensionsand media used must be shown to be free from extraneousagents. The cell culture media may contain a pH indicatorsuch as phenol red and approved antibiotics at the lowesteffective concentration. At least 500 ml of the cell culturesemployed for vaccine production is set aside as uninfectedcell cultures (control cells).

Only a single harvest that complies with the followingrequirements may be used in the preparation of the inactivatedharvest.

Identification

The single harvest is shown to contain tick-borne encephalitisvirus by a suitable immunochemical method, preferably using

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monoclonal antibodies, or by virus neutralization in cellcultures.

Sterility (2.2.11). Complies with the test for sterility carriedout using 10 ml for each medium.

Mycoplasma (2.7.4). Complies with the test for mycoplasmascarried out using 1 ml for each medium.

Control cells. The control cells comply with the tests forextraneous agents (2.7.3). If the vaccine is produced using acell-bank system, the control cells comply with a test foridentification.

Virus concentration. Determine the virus concentration bytitration in suitable cell culture to monitor consistency ofproduction.

Inactivation

To avoid interference, viral aggregates are removed by filtrationimmediately before the inactivation process. The virussuspension is inactivated by a validated method; the methodshall have been shown to be consistently capable ofinactivating tick-borne encephalitis virus without destroyingthe antigenic and immunogenic activity; as part of thevalidation studies, an inactivation curve is plotted representingresidual live virus concentration measured on not fewer thanthree occasions. If formaldehyde is used for inactivation, thepresence of an excess of free formaldehyde is verified at theend of the inactivation process.Only an inactivated harvest that complies with the followingrequirements may be used in the preparation of the final bulkvaccine.

Residual infective virus. Inoculate a quantity of the inactivatedharvest equivalent to not less than ten human doses of vaccinein the final lot into primary chicken fibroblast cell cultures, orother cells shown to be at least as sensitive to tick-borneencephalitis virus with not less than 3 cm2 of cell sheet per mlof inoculum. Incubate at 37 ± 1° for 14 days. No cytopathiceffect is detected at the end of the incubation period. Collectthe culture fluid and inoculate 0.03 ml intracerebrally into eachof not fewer than ten mice about 4 weeks old. Observe themice for 14 days. They show no evidence of tick-borneencephalitis virus infection.

Purification

Several inactivated single harvests may be pooled beforeconcentration and purification by suitable methods, preferablyby continuous-flow, sucrose density-gradient centrifugation.Only a purified, inactivated harvest that complies with thefollowing requirements way be used in the preparation of finalbulk vaccine.Sterility ( 2.2.11). Complies with the test for sterility, carriedout using 10 ml for each medium.

Specific activity. Determine the antigen content of the purified,inactivated harvest by a suitable immunochemical method(2.2.14). Determine the total protein content by a suitablemethod. The specific activity, calculated as the antigen contentper unit mass of protein, is within the limits approved for thespecific product.

FINAL BULK VACCINE

The final bulk vaccine is prepared from one or more purified,inactivated harvests.



FINAL LOT

Only a final lot that is satisfactory with respect to each of therequirements given below under Identification, Tests andAssay may be released for use. Provided that the tests for freeformaldehyde, bovine serum albumin (where applicable) andpyrogens and the assay have been carried out satisfactoryresults on the final bulk vaccine, they may be omitted on thefinal lot.

Identification

The vaccine is shown to contain tick-borne encephalitis virusantigen by a suitable immunochemical method using specificantibodies or by the mouse immunogenicity test describedunder Assay.

Tests



Bovine serum albumin. If bovine serum albumin has beenused during production, the vaccine contains not more than50 ng per single human dose, determined by a suitableimmunochemical method (2.2.14).


Pyrogens (2.2.8). Complies with the test for pyrogens. Injectinto each rabbit, per kg of body mass, one dose of vaccine.

Assay

The potency is determined by comparing the dose necessaryto protect a given proportion of mice against the effects of alethal dose of tick-borne encephalitis virus, administeredintraperitoneally, with the quantity of a reference preparation

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of tick-borne encephalitis vaccine necessary to provide thesame protection. For this comparison an approved referencepreparation and a suitable preparation of tick-borneencephalitis virus from an approved strain for use as thechallenge preparation are necessary.

The following is cited as an example of a method that has beenfound suitable for a given vaccine.

Selection and distribution of test animals. Use healthy miceweighing between 11 and 17 g derived from the same stock.Distribute the mice into not less than six groups of a suitablesize to meet the requirements for validity of the test; for titrationof the challenge suspension, use not fewer than four groupsof ten mice. Use mice of the same sex or distribute males andfemales equally between groups.

Determination of potency of the vaccine. Prepare not lessthan three suitable dilutions of the vaccine under examinationand of the reference preparation; in order to comply withvalidity criteria four to five dilutions will usually be necessary.Prepare dilutions such that the most concentrated suspensionis expected to protect more than 50 per cent of the animals andthe least concentrated suspension less than 50.0 per cent.Allocate each dilution to a different group of mice and injectsubcutaneously into each mouse 0.2 ml of the dilution allocatedto its group. Seven days later make a second injection usingthe same dilution scale. 14 days after the second injectionprepare a suspension of the challenge virus containing notless than 100 LD50 in 0.2 ml. Inject 0.2 ml of this virussuspension intraperitoneally into each vaccinated mouse. Toverify the challenge dose, prepare a series of not fewer thanthree dilutions of the challenge virus suspension at not greaterthan one-hundredfold intervals. Allocate the challengesuspension and the four dilutions, one to each of the fivegroups of ten mice, and inject intraperitoneally into each mouse0.2 ml of the challenge suspension or the dilution allocated toits group. Observe the animals for 21 days after the challengeand record the number of mice that die in the period between7 and 21 days after the challenge.

Calculations. Calculate the results by the usual statisticalmethods for an assay with quantal responses (5.7).

Validity criteria. The test is not valid unless (1) theconcentration of the challenge virus is not less than 100 LD50;(2) for both the vaccine under examination and the referencepreparation the 50.0 per cent protective dose (PD50) liesbetween the largest and smallest doses given to the mice; (3)the statistical analysis shows a significant slope and nosignificant deviation from linearity and parallelism of the dose-response lines; (4) the fiducial limits (P = 0.95) are not lessthan 33.0 per cent and not more than 300.0 per cent of theestimated potency.

Potency requirement. Include all valid tests to estimate the

mean potency and the fiducial limits (P = 0.95) for the meanpotency; compute weighed means with the inverse of thesquared standard error as weights. The vaccine complies withthe test if the estimated potency is not less than that approvedby the competent authority, based on data from clinical efficacytrials.

Labelling. The label states (1) the strain of virus used inpreparation; (2) the type of cells used for production of thevaccine.

Tuberculin Purified Protein DerivativeTuberculin PPD; Tuberculin Purified Protein Derivative forHuman Use

Tuberculin Purified Protein Derivative is a preparation madefrom the heat-treated products of growth and lysis of one ormore strains of Mycobacterium tuberculosis that revealdelayed hypersensitivity in animals sensitised by a micro-organism of the same species.

Production

It is prepared from the water-soluble fraction obtained byheating in free-flowing steam or in an autoclave andsubsequently filtering cultures of the mycobacteria grown ina suitable liquid medium. The active fraction in the filtrate,which is predominantly protein, is separated by precipitation,washed and redissolved. The preparation is free frommycobacteria. An antimicrobial preservative that does not giverise to false positive reactions, such as 0.5 per cent w/v ofphenol, and a suitable stabiliser may be added. Phenol is notadded to preparations that are to be freeze-dried. The finalsterile product is distributed into sterile glass containers whichare then sealed so as to prevent microbial contamination oralternatively it is freeze-dried and the containers subsequentlysealed.** To ensure availability of a preparation of uniform potency, TuberculinPurified Protein Derivative is produced and issued by the StatensSerum Institute ,Denmark as a powder to be reconstituted as statedon the label.

The preparation may be issued either as a sterile liquid or as afreeze-dried product. If issued as a liquid, it is in a ready-to-use form and 0.1 ml constitutes one intradermal dosecontaining appropriate number of Units. If issued as a freeze-dried product, it should yield a ready-to-use preparation whenreconstituted as per manufacturer’s instructions.

Description. A colourless or pale, straw-coloured liquid, ordry cream-coloured powder, or pellet.

The preparation, reconstituted if necessary as stated on thelabel, complies with the following requirements.

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Identification

A. When progressively increasing doses are injectedintradermally into specifically sensitised guinea-pigs, reactionsoccur at the points of injection, varying from erythema tonecrosis. When similar injections are administered to non-sensitised guinea-pigs no such reactions occur.

B. The potency test described below serves as a test foridentity if it is performed on material from the final containers.

Tests

pH (2.4.24). 6.5 to 7.5.

Phenol (if present) (2.3.36). Not more than 0.5 per cent w/v.

Sterility (2.2.11). Complies with the tests for sterility.

Potency. Carry out the biological assay of tuberculin purifiedprotein derivative described below.

Tuberculin Purified Protein Derivative in freeze-dried formcomplies with the following additional requirements.

Live mycobacteria

A. Inject 5.0 ml intraperitoneally or subcutaneously into eachof two guinea-pigs weighing between 300 and 400 g. Observethe animals for not less than 42 days. Kill the animals andcarry out an autopsy. No guinea-pig shows signs of infectionwith mycobacteria.

B. Carry out tests for live mycobacteria in the preparationunder examination using suitable culture media. No growth ofmycobacteria should occur.

Sensitising effect. Inject intradermally into each of threeguinea-pigs three times, at intervals of 5 days, a dose of thepreparation under examination containing about 500 Units ina volume of 0.1 ml. Two to three weeks after the third injectioninoculate the same dose intradermally into these animals andinto a control group of three guinea-pigs of the same weightbut that have not had any previous injections of tuberculin.The reactions of the two groups are not significantly differentafter 48 to 72 hours.

Toxicity. Inject subcutaneously into 2 healthy guinea-pigs,weighing not less than 250 g and which have not previouslybeen treated with any material which will interfere with thetest, 0.5 ml of a solution containing 1,00,000 Units per ml. Noharmful effects are produced within 7 days.

Biological assay

The potency of tuberculin purified protein derivative isdetermined by comparing the dose necessary to reveal delayedhypersensitivity in guinea-pigs or other animalshypersensitised with mycobacteria of the same type as thatused in the preparation of the tuberculin purified protein

derivative with the dose of the appropriate StandardPreparation necessary to give the same effect.

The estimated potency is not less 80 per cent and not morethan 125 per cent of the stated potency. The fiducial limits oferror are not less than 64 per cent and not more than 156 percent of the stated potency.

Standard Preparation

The Standard Preparation is the 1st International Standard forTuberculin, purified protein derivative (PPD), mammalian,established in 1951, consisting of PPD derived from culturesof M. tuberculosis (supplied in ampoules containing 5,00,000Units) or another suitable preparation the potency of whichhas been determined in relation to the International Standard.

Method

Sensitise 12 guinea-pigs each weighing not less than 400 g bythe intramuscular injection of a total of about 0.5 ml of asuspension in a suitable mineral oil with or without emulsifierand containing 0.1 mg per ml of heat-inactivated, driedmycobacteria of the same type as that used in the preparationof tuberculin. Use phosphate-buffered saline pH 7.4containing 0.005 per cent w/v of polysorbate 80 to preparethe dilutions of the Standard preparation and of thepreparation under examination and to reconstitute freeze-driedpreparations containing no stabiliser. Not less than 1 monthand not more than 6 months later carry out the followingtest :

Shave the flanks to provide space for at least three reactionson each side, but not more than a total of 12 injection sites peranimal. Use at least three doses of the Standard preparationand at least three doses of the preparation under examination,the highest dose being about 10 times as strong as the lowest.Dilute the preparations so that the lesions produced are 8 to25 mm in diameter. Allocate the doses to the available sites ina random manner, in a Latin square design. Inject each doseintradermally in the same volume (0.1 or 0.2 ml) at the sites towhich it has been allocated. Measure the diameters of thelesions after 24 to 48 hours and calculate the result of the testusing standard statistical methods on the basis that the lesiondiameters are directly proportional to the logarithm of theconcentration of the tuberculin.

Storage. Store in light-resistant containers at a temperaturebetween 2° and 8°. It should not be allowed to freeze.

Labelling. The label states (1) the number of Units per dose of0.1 ml or per ml or per mg; (2) the total volume in the container(for liquid preparation); (3) the nature and quantity of thereconstituting liquid (for the freeze-dried preparation); (4) thename and proportion of any added substances; (5) the speciesor strain used; (6) the storage conditions; (7) the date afterwhich the contents are not intended to be used; (8) that care

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should be taken to avoid inhaling the powder (for the freeze-dried preparation).

Typhoid (Strain Ty 21a) Vaccine, Live(Oral)Typhoid Vaccine (Live, Oral, strain Ty 21a) is a freeze-driedpreparation of the live Salmonella typhi strain Ty 21a grownin a suitable medium. When presented in capsules, the vaccinecomplies with the tests stated under Capsules.

Production


The main characteristic of the strains is the defect of theenzyme uridine diphosphate-galactose-4 epimerase. Theactivities of galactopermease, galactokinase and galactose-1-phosphate uridyl-transferase are reduced by 50 to 90 per cent.Whatever the growth conditions, the strain does not containVi antigen. The strain agglutinates to anti-O:9 antiserum onlyif grown in medium containing galactose. It contains theflagellar H:d antigen and does not produce hydrogen sulphideon Kligler iron agar. The strain is nonvirulent for mice. Cells ofstrain Ty 21a lyse if grown in the presence of 1.0 per cent ofgalactose.

SEED LOT

The vaccine is prepared using a seed-lot system. The workingseed lots represent not more than one subculture from themaster seed lot. The final vaccine represents not more thanfour subcultures from the original vaccine on which were madethe laboratory and clinical tests showing the strain to besuitable.

Only a master seed lot that complies with the followingrequirements may be used in the preparation of working seedlots.

Galactose metabolism

In a spectrophotometric assay, no activity of the enzymeuridine diphosphate-galactose-4-epimerase is found in thecytoplasm of strain Ty 21a compared to strain Ty 2.

Biosynthesis of lipopolysaccharide

Lipopolysaccharides are extracted by the hot-phenol methodand examined by size-exclusion chromatography (2.4.16). StrainTy 21a grown in medium free of galactose shows only therough (R) type of lipopolysaccharide.

Serological characteristics

Strain Ty 21a grown in a synthetic medium without galactosedoes not agglutinate to specific anti-O:9 antiserum. Whatever

the growth conditions, strain Ty 21a does not agglutinate toVi antiserum. Strain Ty 21a agglutinates to H:d flagellarantiserum.

Biochemical markers

Strian Ty 21a does not produce hydrogen sulphide on Kligleriron agar. This property serves to distinguish Ty 21a fromother galactose-epimerase-negative S. typhi strains.

Cell growth

Strain Ty 21a cells lyse when grown in the presence of 1.0 percent of galactose.


The bacteria from the working seed lot are multiplied in apreculture, subcultured once and are then grown in a suitablemedium containing 0.001 per cent of galactose at 30° for 13 to15 hours. The bacteria are harvested. The harvest must befree from contaminating micro-organisms.

Only a single harvest that complies with the followingrequirements may be used for the preparation of the freeze-dried harvest.

pH (2.4.24). 6.8 to 7.5.

Optical density

The optical density of the culture, measured at 546 nm, is 6.5to 11.0. Before carrying out the measurement, dilute the cultureso that a reading in the range 0.1 to 0.5 is obtained and correctthe reading to take account of the dilution.

Identification

Culture bacteria on an agar medium containing 1.0 per cent ofgalactose and bromothymol blue. Light blue, concavecolonies, transparent due to lysis of cells, should be found.No yellow colonies (galactose-fermenting) should be found.

FREEZE-DRIED HARVEST

The harvest is mixed with a suitable stabilizer and freeze-driedby a process that ensures the survival of at least 10.0 per centof the bacteria and to a water content shown to be favourableto the stability of the vaccine. No antimicrobial preservative isadded to the vaccine.

Only a freeze-dried harvest that complies with the followingtests may be used for the preparation of the final bulk.

Identification

Culture bacteria are examined on an agar medium containing1.0 per cent of galactose and bromoethymol blue. Light blue,concave colonies, transparent due to lysis of cells, should befound. No yellow colonies (galactose-fermenting) should befound.

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Number of live bacteria

Not less than 1 x 1011 live S. typhi strain Ty 21a per gram.

Water (2.4.43). 1.5 to 4.0 per cent, determined by the semi-micro determination of water or any other validated method.

FINAL BULK VACCINE

The final bulk vaccine is prepared by aseptically mixing one ormore freeze-dried harvests with a suitable sterile excipient.

Only a final bulk that complies with the following requirementmay be used in the preparation of the final lot.

Number of live bacteria. Not less than 40 × 109 live S. typhistrain Ty 21a per gram.

FINAL LOT

The final bulk vaccine is distributed under aseptic conditionsinto capsules with a gastro-resistant shell or into suitablecontainers.

Only a final lot that is satisfactory with respect to Identification,Tests and Number of live bacteria will be released for use,except that in the determination of number of live bacteriaeach dosage unit must contain not less than 4 x 109 livebacteria.

IdentificationCulture bacteria from the vaccine under examination on anagar medium containing 1.0 per cent of galactose andbromothymol blue. Light blue, concave colonies transparentdue to lysis of cells, should be found. No yellow colonies(galactose-fermenting) should be found.

TestsMicrobial contamination (2.2.9). Carry out the test usingsuitable selective media. Determine the total viable count usingthe plate-count method. The number of contaminating micro-organisms per dosage unit is not greater than 102 bacteria and20 fungi. No pathogenic bacterium, particularly Escherichiacoli, Staphylococcus aureus, Pseudomonas aeruginosa, andno Salmonella other than strain Ty 21a are found.

Water (2.4.43). 1.5 to 4.0 per cent.

Number of live bacteria

Carry out the test using not less than five dosage units.Homogenise the contents of the dosage units in a 0.9 per centw/v solution of sodium chloride at 4° using a mixer in a coldroom with sufficient glass beads to emerge from the liquid.Immediately after homogenization prepare a suitable dilutionof the suspension using cooled diluent and inoculate brainheart infusion agar, incubate at 36 ± 1° for 20 to 36 hours. Thevaccine contains not less than 2 x 109 live S. typhi Ty 21abacteria per dosage unit.

Labelling. The label states (1) the minimum number of livebacteria per dosage unit; (2) that the vaccine is for oral useonly.

Typhoid Polysaccharide VaccineTyphoid Polysaccharide Vaccine is a preparation of purifiedVi capsular polysaccharide obtained from Salmonella typhiTy2 strain or some other suitable strain of known origin andhistory that has the capacity to produce Vi polysaccharide,and which have been characterized by suitable biochemical,physicochemical, serological or molecular methods.

Capsular polysaccharide is a partly 3-O-acetylated repeatedunits of 2-acetylamino-2-deoxy-D-galactopyranuronic acidwith á-(1?4)-linkages.

Production

General provisions

The production of Vi polysaccharide is based on a definedseed-lot system. The method of production shall have beenshown to yield consistently Vi polysaccharide typhoidvaccines of adequate immunogenicity and safety in man. Theproduction method is validated to demonstrate that the product,if tested, would comply with the tests for abnormal toxicity.

SEED LOT

The strain of S. typhi used for the master seed lot shall beidentified by historical records that include information on itsorigin and by its biochemical and serological characteristics.Cultures from the working seed lot shall have the samecharacteristics as the strain that was used to prepare the masterseed lot, shall be demonstrated along with adequatedocumentation.

Only a strain that has the following characteristics may beused in the preparation of the vaccine: (a) stained smears froma culture are typical of S. typhi; (b) the culture utilises glucosewithout production of gas; (c) colonies on agar are oxidase-negative; (d) a suspension of the culture agglutinatesspecifically with an appropriate Vi antiserum or colonies formhaloes on an agar plate containing a suitable Vi antiserum.


The working seed lot is cultured on a solid medium, whichmay contain blood-group substances, or a liquid medium; theinoculum obtained is transferred to a liquid medium, which isused to inoculate the final medium. The liquid medium usedand the final medium are semi-synthetic, free from substancesthat are precipitated by cetrimonium bromide and do notcontain blood-group substances or high-molecular-mass

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polysaccharides, unless it has been demonstrated that theyare removed by the purification process. The bacterial purityof the culture is verified by microscopic examination of Gram-stained smears and by inoculation into appropriate media.The culture is then inactivated at the beginning of thestationary phase by the addition of formaldehyde. Bacterialcells are eliminated by centrifugation; the polysaccharide isprecipitated from the culture medium by addition ofhexadecyltrimethylammonium bromide (cetrimoniumbromide). The precipitate is harvested and may be stored ator below -20° before purification.

Purified Vi Polysaccharide

The polysaccharide is purified, after dissociation of thepolysaccharide/cetrimonium bromide complex, using suitableprocedures to eliminate successively nucleic acids, proteinsand lipopolysaccharides. The polysaccharide is precipitatedin its salt form and dried at 5+3°; the powder obtainedconstitutes the purified Vi polysaccharide. The loss on dryingis determined by thermogravimetry, Karl Fischer or any othersuitable method and is used to calculate the results of thechemical tests shown below with reference to the driedsubstance.

Only those pools of purified Vi polysaccharide that complywith the following requirements may be used in the preparationof the final bulk:Protein (2.7.1). Not more than 10 mg per gram ofpolysaccharide, calculated with reference to the driedsubstance.Nucleic acids (2.7.1). Not more than 20 mg per gram ofpolysaccharide, calculated with reference to the driedsubstance.O-Acetyl groups (2.7.1). Not less than 2 mmol per gram ofpolysaccharide, calculated with reference to the driedsubstance.

Molecular size. Examine by gel filtration or size-exclusionchromatography (2.4.16) using cross-linked agarose forchromatography. Use a column 0.9 m long and 16 mm in internaldiameter equilibrated with a solvent having an ionic strengthof 0.2 mol per kg and a pH of 7.0 to 7.5. Apply about 5 mg ofpolysaccharide in a volume of 1 ml to the column and elute atabout 20 ml/h. Collect fractions of about 2.5 ml. Determine thepoint corresponding to K0 = 0.25 and make two poolsconsisting of fractions eluted before and after this point.Determine O-acetyl groups on the two pools. Not less than 50per cent of the polysaccharide is found in the pool containingfractions eluted before K0 = 0.25.

IdentificationCarry out an identification test using a suitableimmunochemical method (2.2.14).

Bacterial endotoxins (2.2.3). Determine by a suitable method,the content should not be more than 150 IU per mg ofpolysaccharide.

FINAL BULK VACCINE

One or more batches of purified Vi polysaccharide aredissolved in a suitable solvent, which may contain anantimicrobial preservative, so that the volume correspondingto one dose contains 25 µg of polysaccharide and the solutionis isotonic with blood (250 mosm/kg to 350 mosm/kg).

Only a final bulk vaccine that complies with the followingrequirements may be used in the preparation of the final lot;


Antimicrobial preservative. Where applicable, determine theamount of antimicrobial preservative by a suitable chemicalmethod. The content is not less than 85.0 per cent and notgreater than 115.0 per cent of the intended amount. If phenolhas been used in the preparation, the content is not more than2.5 g/l.

FINAL LOT

The final bulk vaccine is distributed and filled aseptically intosterile containers. The containers are closed so as to preventcontamination.

Only a final lot that is satisfactory with respect to each of therequirements prescribed below under Identification, Tests andAssay and with the requirements for Bacterial endotoxins maybe released for use. Provided the tests for free formaldehydeand antimicrobial preservative have been carried out on thefinal bulk vaccine, they may be omitted on the final lot.

The vaccine contains minimum of 25 µg purified Vi-Polysaccharide per dose of 0.5 ml.

Identification

Carry out an identification test using a suitableimmunochemical method (2.2.14).

Tests

Sterility (2.2.11). Complies with the tests for sterility.


pH (2.4.24). 6.5 to 7.5.

O-Acetyl groups (2.7.1). 0.085 (± 25 per cent) µmol per dose(25 µg of polysaccharide).

Test solution. Place 3 ml of the vaccine in each of three tubes(two reaction solutions and one correction solution).

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Reference solutions. Dissolve 0.150 g of acetylcholinechloride in 10 ml of water (stock solution containing 15 g/l ofacetylcholine chloride). Immediately before use, dilute 0.5 mlof the stock solution to 50 ml with water (working dilutioncontaining 150 µg/ml of acetylcholine chloride). In ten tubes,place in duplicate (reaction and correction solutions) 0.1 ml,0.2 ml, 0.5 ml, 1.0 ml and 1.5 ml of the working dilution.

Prepare a blank using 3 ml of purified water.

Make up the volume in each tube to 3 ml with water. Add 0.5ml of a mixture of 1 volume of water and 2 volumes of dilutehydrochloric acid to each of the correction tubes and to theblank. Add 1.0 ml of alkaline hydroxylamine solution to eachtube. Allow the reaction to proceed for exactly 2 min and add0.5 ml of a mixture of 1 volume of water and 2 volumes ofdilute hydrochloric acid to each of the reaction tubes. Add0.5 ml of a 20 per cent w/vl solution of ferric chloride in 0.2Mhydrochloric acid to each tube, stopper the tubes and shakevigorously to remove bubbles.

Measure the absorbance of each solution at 540 nm using theblank as the compensation liquid. For each reaction solution,subtract the absorbance of the corresponding correctionsolution. Draw a calibration curve from the correctedabsorbance for the five reference solutions and thecorresponding content of acetylcholine chloride and read fromthe curve the content of acetylcholine chloride in the testsolution for each volume tested. Calculate the mean of thetwo values.

1 mol of acetylcholine chloride (181.7 g) is equivalent to 1 molof O-acetyl (43.05 g).



If phenol has been used in the preparation, the content is notmore than 2.5 g/l.

Assay

Determine Vi polysaccharide content by a suitableimmunochemical method (2.2.14) using a reference purifiedpolysaccharide. The estimated amount of polysaccharide perdose is 80.0 per cent to 120.0 per cent of the content stated onthe label. The fiducial limits of error (P = 0.95) of the estimatedamount of polysaccharide are not less than 80.0 per cent andnot more than 120.0 per cent.

Labelling. The label states (1) the number of micrograms ofpolysaccharide per human dose (25 µg); (2) the total quantityof polysaccharide in the container.

Typhoid VaccineTyphoid Vaccine is a sterile suspension of inactivatedSalmonella typhi containing not less than 5 x 108 and notmore than 1 x 109 bacteria per human dose. The human dosedoes not exceed 1.0 ml.

Production

The vaccine is prepared using a seed-lot system from asuitable strain of S. typhi such as, Ty 2. The final vaccinerepresents not more than 3 subcultures from the strain onwhich were made the laboratory and clinical tests that showedit to be suitable. The bacteria are inactivated by acetone, byformaldehyde, by phenol or by heating or by a combinationof the last two methods.

The production method is validated to demonstrate that theproduct, if tested, would comply with the test for abnormaltoxicity (2.2.1) modified to the extent that 0.5 ml of the vaccineis injected subcutaneously or intramuscularly orintraperitoneally into each mouse and 1.0 ml into each guinea-pig.

Identification

It is identified by specific agglutination.

Phenol (2.3.36). If phenol has been used in the preparation,the concentration is not more than 0.5 per cent w/v.

Antigenic power. When injected into susceptible laboratoryanimals, it elicits anti-O, anti-H and, to a lesser extent, anti-Viagglutinins.


Labelling. The label states (1) the method used to inactivatethe bacteria; (2) the number of bacteria per human dose.

Typhoid Vaccine (Freeze Dried)Freeze Dried Typhoid Vaccine is a freeze-dried preparation ofinactivated Salmonella typhi. The vaccine is reconstituted asstated on the label to give a uniform suspension containingnot less than 5 x 108 and not more than 1 x 109 bacteria perhuman dose. The human dose does not exceed 1.0 ml of thereconstituted vaccine.

Production

The vaccine is prepared from a seed-lot system from a suitablestrain of S. typhi, such as Ty 2. The final vaccine representsnot more than 3 subcultures from the strain on which weremade the laboratory and clinical tests that showed it to besuitable. The bacteria are inactivated either by acetone or by

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formaldehyde or by heat. Phenol is not used in the preparation.The vaccine is distributed into sterile containers and freeze-dried to a moisture content favourable to the stability of thevaccine.

The production method is validated to demonstrate that theproduct, if tested, would comply with test for abnormal toxicity(2.2.1), modified to the extent that 0.5 ml of the vaccine isinjected subcutaneously or intramuscularly orintraperitoneally into each mouse and 1.0 ml into each guinea-pig.

Identification

The vaccine reconstituted as stated on the label is identifiedby specific agglutination.

Antigenic power. When injected into susceptible laboratoryanimals, the reconstituted vaccine elicits anti-O, anti-H and,to a lesser extent, anti-Vi agglutinins.

Sterility (2.2.11). The reconstituted vaccine complies with thetests for sterility.

Water. Not more than 5.0 percent.

Labelling. The label states (1) the method used to inactivatethe bacteria; (2) the number of bacteria per human dose; (3)that the vaccine should be used within 8 hours ofreconstitution.

Varicella Vaccine, LiveVaricella Vaccine (Live) is a freeze-dried preparation of a suitableattenuated strain of Herpesvirus varicellae.

Production

General provisions

The production of vaccine is based on a virus seed-lot systemand a cell-bank system. The production method shall havebeen shown to yield consistently live varicella vaccines ofadequate immunogenicity and safety in man. The virus in thefinal vaccine shall not have been passaged in cell culturesbeyond the 38th passage from the original isolated virus.



The virus is propagated in human diploid cells (2.7.2).

SEED LOT

The strain of varicella virus shall be identified as being suitableby historical records which shall include information on the

origin of the strain and its subsequent manipulation. The virusshall at no time have been passaged in continuous cell lines.Seed lots are prepared in the same kind of cells as those usedfor the production of the final vaccine. To avoid theunnecessary use of monkeys in the test for neurovirulence,virus seed lots are prepared in large quantities and stored attemperatures below -20°, if freeze-dried, or below -60°, if notfreeze-dried.


Identification

The master and working seed lots are identified as varicellavirus by serum neutralisation in cell culture, using specificantibodies.

Virus concentration. The virus concentration of the masterand working seed lots is determined as prescribed under Assayto monitor consistency of production.

Extraneous agents (2.7.3). Complies with the requirements forseed lots for live virus vaccines; a sample of 50 ml is taken forthe test in cell cultures.

Neurovirulence (2.7.5). Complies with the test forneurovirulence of live virus vaccines.


All processing of the cell bank and subsequent cell cultures isdone under aseptic conditions in an area where no other cellsare handled. Approved animal (but not human) serum may beused in the media. Serum and trypsin used in the preparationof cell suspensions and media are shown to be free fromextraneous agents. The cell culture medium may contain a pHindicator such as phenol red and approved antibiotics at thelowest effective concentration. It is preferable to have asubstrate free from antibiotics during production. 5.0 per cent,but not less than 50.0 ml, of the cell cultures employed forvaccine production is set aside as uninfected cell cultures(control cells). The infected cells constituting a single harvestare washed, released from the support surface and pooled.The cell suspension is disrupted by sonication.

Only a virus harvest that complies with the followingrequirements may be used in the preparation of the final bulkvaccine.

Identification

The virus harvest contains virus that is identified as varicellavirus by serum neutralisation in cell culture, using specificantibodies.

Virus concentration. The concentration of infective virus invirus harvests is determined as prescribed under assay tomonitor consistency of production and to determine the

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dilution to be used for the final bulk vaccine.

Extraneous agents (2.7.3). Use 50 ml for the test in cell cultures.

Control cells. The control cells of the production cell culturefrom which the single harvest is derived comply with a test foridentity and with the requirements for extraneous agents(2.7.3).

FINAL BULK VACCINE

Virus harvests that comply with the above tests are pooledand clarified to remove cells. A suitable stabiliser may be addedand the pooled harvests diluted as appropriate.



FINAL LOT

The final bulk vaccine is distributed aseptically into sterile,tamper-proof containers and freeze-dried to a moisture contentshown to be favourable to the stability of the vaccine. Thecontainers are then closed so as to prevent contaminationand the introduction of moisture.

Only a final lot that is satisfactory with respect to each of therequirements given below under Identification, Tests andAssay may be released for use. Provided that the test forbovine serum albumin has been carried out with satisfactoryresults on the final bulk vaccine, it may be omitted on the finallot.

Identification

When the vaccine reconstituted as stated on the label is mixedwith specific Herpesvirus varicellae antibodies, it is no longerable to infect susceptible cell cultures.

Tests

Sterility (2.2.11) Complies with the test for sterility.


Bovine serum albumin. Not more than 0.5 µg per human dose,determined by a suitable immunochemical method (2.2.14).

Water (2.3.43). Not more than 3.0 per cent, determined by thesemi-micro determination of water.

Assay

Titrate for infective virus, using at least ten cell cultures foreach fourfold dilution or by a technique of equal precision.Use a suitable virus reference preparation to validate eachassay. The virus concentration is not less than the minimumstated on the label.

Labelling. The label states (1) the strain of virus used for thepreparation of the vaccine; (2) the type and origin of the cellsused for the preparation of the vaccine; (3) that contact withdisinfectants is to be avoided; (4) the minimum virusconcentration; (5) that the vaccine is not to be administeredto pregnant women; (6) the time within which the vaccinemust be used after reconstitution.

Viper VenomDaboia VenomViper Venom is the dried secretion obtained from the poisonglands of Viperae russelli and other species of Viperae (Fam.Viperidae).

Viper Venom contains not less than 50 Mouse Units per mg.

Production

Immediately after extraction, the poisonous secretion is driedfrom the frozen state. The dried venom is pooled, mixed,dissolved in ice-cold water for injection and then filteredthrough a bacteria-proof filter to give a stock solution. Furtherdilutions of the stock solution are made with water forinjection under aseptic conditions to give solutions with therequired number of Mouse Units per ml. These solutions arethen distributed in single dose sterile glass containers, driedfrom the frozen state and sealed at a pressure not exceeding2.75 kPa.

Description. An almost white or very light yellow, dry powderwhich when mixed with water yields a clear solution withsome insoluble residue.

Identification

A. Produces almost immediate coagulation of blood andcitrated human plasma.

B. Mix the soluble fraction from at least 0.6 mg with 1 ml ofpolyvalent antisnake venom serum and incubate the mixtureat 37o for 30 minutes. Inject 0.5 ml of the mixture intravenouslyinto a group of mice weighing between 18 and 20 g. Observethe animals for 24 hours; no animal dies.

Tests

Sterility (2.2.11). Complies with tests for sterility.

Assay. Carry out the biological assay of snake venomdescribed below:

Biological assay of snake venom

Dissolve a quantity of the freeze-dried venom equivalent to50 Mouse Units in 25 ml of saline solution. Inject 0.5 ml

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intravenously into each of 10 mice weighing between 18 and20 g and observe the animals for 24 hours. Not less than 3 andnot more than 8 of the mice die in 2 to 24 hours. If the numberof deaths is not within this range, change the dilution of thevenom suitably.

Express the result in terms of number of Mouse Units per mg.

NOTE — The quantity in mg of the venom which will kill in2 to 24 hours not less than 3 and not more than 8 micerepresents one Mouse Unit.

Storage. Store in single dose, light-resistant containers.

Labelling. The label states (1) the number of Mouse Units percontainer; (2) the volume of water for injection to be used forreconstitution.

Yellow Fever Vaccine (Live)Yellow Fever Vaccine (Live) is a freeze-dried preparation ofthe 17D strain of yellow fever virus grown in fertilised heneggs.

Production

General provisions

The production of vaccine is based on a virus seed-lot system.The production method shall have been shown to yieldconsistently the yellow fever vaccine (live) of acceptableimmunogenicity and safety for man.


Reference preparation. In the test for neurotropism, a suitablebatch of vaccine known to have satisfactory properties inman is used as the reference preparation.


Virus for the preparation of master and working seed lots andfor all vaccine batches is grown in the tissues of chick embryosfrom a flock free from specified pathogens (2.7.7).

SEED LOT

The 17D strain shall be identified by historical records thatinclude information on the origin of the strain and itssubsequent manipulation. Virus seed lots are prepared in largequantities and stored at a temperature below -60°. Master andworking seed lots shall not contain any human protein oradded serum.

Unless otherwise justified and authorised, the virus in thefinal vaccine shall be between passage levels 204 and 239from the original isolate of strain 17D. A working seed lot shall

be only one passage from a master seed lot. A working seedlot shall be used without intervening passage as the inoculumfor infecting the tissues used in the production of a vaccinelot, so that no vaccine virus is more than one passage from aseed lot that has passed all the safety tests.


Identification

The master and working seed lots are identified as containingyellow fever virus by serum neutralisation in cell culture, usingspecific antibodies.

Extraneous agents (2.7.3). Each working seed lot complieswith the test for extraneous agents.


All processing of the fertilised eggs is done under asepticconditions in an area where no other infectious agents or cellsare handled at the same time. Two per cent but not less thantwenty and not more than fifty eggs are set aside as uninfectedcontrol eggs. After inoculation and incubation at a controlledtemperature, only living and typical chick embryos areharvested. The age of the embryo at the time of virus harvestis reckoned from the initial introduction of the egg into theincubator and shall not be more than 12 days. Afterhomogenisation and clarification by centrifugation, the extractof embryonic pulp is tested as described below and kept at-70° or colder until further processing. Virus harvests thatcomply with the prescribed tests may be pooled. No humanprotein is added to the virus suspension at any stage duringproduction. If stabilisers are added, they shall have been shownto have no antigenic or sensitising properties for man.

Only a single harvest that complies with the following testsmay be used in the preparation of the final bulk vaccine.

Identification

The single harvest contains virus that is identified as yellowfever virus by serum neutralisation in cell culture, usingspecific antibodies.

Extraneous agents (2.7.3). Complies with the tests forextraneous agents.

Control eggs. Complies with the tests for extraneous agents(2.7.3).

Virus concentration. In order to calculate the dilution forformulation of the final bulk, each single harvest is titrated asdescribed under Assay.

FINAL BULK VACCINE

Single harvests that comply with the tests prescribed aboveare pooled and clarified again. A test for protein nitrogen

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content is carried out. A suitable stabiliser may be added andthe pooled harvests diluted as appropriate.

Only a final bulk vaccine that complies with the followingtests may be used in the preparation of the final lot.Sterility (2.2.11). Carry out test for sterility using 10 ml of bulkfor each sterility medium.Protein nitrogen content (2.3.30). The protein nitrogencontent, before the addition of any stabiliser, is not more than0.25 mg per human dose.

FINAL LOT

The final bulk vaccine is distributed aseptically into sterile,tamper-proof containers and freeze-dried to a moisture contentshown to be favourable to the stability of the vaccine. Thecontainers are then closed so as to prevent contaminationand the introduction of moisture.

Only a final lot that is satisfactory with respect to thermalstability and each of the tests given under Identification, Testsand Assay may be released for use. Provided that the test forovalbumin has been performed with satisfactory results onthe final bulk vaccine, it may be omitted on the final lot.

Thermal stability. Maintain samples of the final lot of freeze-dried vaccine in the dry state at 37° for 14 days. Determine thevirus concentration as described under Assay in parallel forthe heated vaccine and for unheated vaccine. The differencein the virus concentration between unheated and heatedvaccine does not exceed 1.0 log10, and the virus concentrationof the heated vaccine is not less than the number of TCID50 orplaque-forming units (PFU) equivalent to 1 × 103 mouse LD50per human dose.

Identification

When the vaccine reconstituted as stated on the label is mixedwith specific yellow fever virus antibodies, there is a significantreduction in its ability to infect susceptible cell cultures.

Tests

Sterility (2.2.11). Complies with the test for sterility.Ovalbumin. Not more than 5 µg of ovalbumin per human dose,determined by a suitable immunochemical method (2.2.14).Abnormal toxicity (2.2.1). Complies with the test for abnormaltoxicity.Bacterial endotoxins (2.2.3). Not more than 5 IU of bacterialendotoxin per human dose.Water (2.3.43). Not more than 3.0 per cent, determined by thesemi-micro determination of water.

Assay

Titrate for infective virus in cell cultures. Use an appropriatevirus reference preparation to validate each assay.

The virus concentration is not less than the equivalent inTCID50 or PFU of 1 × 103 mouse LD50 per human dose. Therelationship between mouse LD50 and TCID50 or PFU isestablished by each laboratory and approved by the competentauthority.The method shown below, or another suitable technique, maybe used to determine the mouse LD50.Mouse LD50. The statistically calculated quantity of virussuspension that is expected to produce fatal specificencephalitis in 50 per cent of mice of a highly susceptiblestrain, 4 to 6 weeks of age, after intracerebral inoculation.Appropriate serial dilutions of the reconstituted vaccine aremade in diluent for yellow fever virus (0.75 per cent solutionof bovine albumin in phosphate-buffered saline pH 7.4, orany other diluent that has been shown to be equivalent formaintaining the infectivity of the virus).

Mice of a highly susceptible strain, 4 to 6 weeks of age, areinjected intracerebrally under anaesthesia with 0.03 ml of thevaccine dilution. Groups of not less than eight mice are usedfor each dilution; the series of dilutions is chosen so as tocover the range 0 to 100.0 per cent mortality of the mice.Injection of the mice is performed immediately after thedilutions have been made. The mice are observed for 21 daysand all deaths are recorded. Only survivors and deaths causedby typical yellow fever infections are counted in thecomputations. Mice paralysed on the twenty-first day ofobservation are counted as survivors.

Tests in monkeys for Yellow Fever Vaccine

Each master and working seed lot complies with the followingtests in monkeys for viraemia (viscerotropism),immunogenicity and neurotropism.

The monkeys shall be Macaca spp. susceptible to yellowfever virus and shall have been shown to be non-immune toyellow fever at the time of injecting the seed virus. They shallbe healthy and shall not have received previously intracerebralor intraspinal inoculation. Furthermore, they shall not havebeen inoculated by other routes with neurotropic viruses orwith antigens related to yellow fever virus. Not fewer than tenmonkeys are used for each test.

Use a test dose of 0.25 ml containing the equivalent of notless than 5000 mouse LD50 and not more than 50,000 mouseLD50, determined by a titration for infectious virus and usingthe established equivalence between virus concentration andmouse LD50 (see under Assay). Inject the test dose into onefrontal lobe of each monkey under anaesthesia and observethe monkeys for not less than 30 days.

Viraemia (Viscerotropism). Viscerotropism is indicated by theamount of virus present in serum. Take blood from each of thetest monkeys on the second, fourth and sixth days after


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inoculation and prepare serum from each sample. Prepare 1:10,1:100 and 1:1000 dilutions from each serum and inoculate eachdilution into a group of at least six cell culture vessels used forthe determination of the virus concentration. The seed lotcomplies with the test if none of the sera contains more thanthe equivalent of 500 mouse LD50 in 0.03 ml and at most oneserum contains more than the equivalent of 100 mouse LD50 in0.03 ml.Immunogenicity. Take blood from each monkey 30 days afterthe injection of the test dose and prepare serum from eachsample. The seed lot complies with the test if at least 90.0 percent of the test monkeys are shown to be immune, asdetermined by examining their sera in the test for neutralisationof yellow fever virus described below.It has been shown that a low dilution of serum (for example,1:10) may contain non-specific inhibitors that influence thistest; such serum shall be treated to remove inhibitors. Mixdilutions of at least 1: 10, 1:40 and 1: 160 of serum from eachmonkey with an equal volume of 17D vaccine virus at a dilutionthat will yield an optimum number of plaques with the titrationmethod used. Incubate the serum-virus mixtures in a water-bath at 37° for 1 h and then cool in iced water; add 0.2 ml ofeach serum-virus mixture to each of four cell-culture platesand proceed as for the determination of virus concentration.Inoculate similarly ten plates with the same amount of virusplus an equal volume of a 1:10 dilution of monkey serum knownto contain no neutralising antibodies to yellow fever virus. Atthe end of the observation period, compare the mean numberof plaques in the plates receiving virus plus non-immune serumwith the mean number of plaques in the plates receiving virusplus dilutions of each monkey serum. Not more than 10 percent of the test monkeys have serum that fails to reduce thenumber of plaques by 50.0 per cent at the 1:10 dilution.Neurotropism. Neurotropism is assessed from clinicalevidence of encephalitis, from incidence of clinicalmanifestations and by evaluation of histological lesions, incomparison with ten monkeys injected with the referencepreparation. The seed lot is not acceptable if either the onsetand duration of the febrile reaction or the clinical signs ofencephalitis and pathological findings are such as to indicatea change in the properties of the virus.Clinical evaluation. The monkeys are examined daily for 30days by personnel familiar with clinical signs of encephalitisin primates (if necessary, the monkeys are removed from theircage and examined for signs of motor weakness or spasticity).The seed lot is not acceptable if in the monkeys injected withit the incidence of severe signs of encephalitis, such asparalysis or inability to stand when stimulated, or mortality isgreater than for the reference vaccine. These and other signsof encephalitis, such as paresis, in-coordination, lethargy,tremors or spasticity are assigned numerical values for theseverity of symptoms by a grading method. Each day each

monkey in the test is given a score based on the scale:Grade 1 — rough coat, not eating,

Grade 2 — high-pitched voice, inactive, slow moving,

Grade 3 — shaky, tremors, unco-ordinated, limb weakness,

Grade 4 — inability to stand, limb paralysis or death (a deadmonkey receives a daily score of 4 from the dayof death until day 30).

A clinical score for a particular monkey is the average of itsdaily scores; the clinical score for the seed lot is the mean ofthe individual monkey scores. The seed lot is not acceptableif the mean of the clinical severity scores for the group ofmonkeys inoculated with it is significantly greater (P = 0.95)than the mean for the group of monkeys injected with thereference preparation. In addition, special consideration isgiven to any animal showing unusually severe signs whendeciding on the acceptability of the seed lot.

Histological evaluation. Five levels of the brain are examinedincluding :

Block 1 — the corpus striatum at the level of the opticchiasma,

Block II — the thalamus at the level of the mamillary bodies,

Block III — the mesencephalon at the level of the superiorcolliculi,

Block IV — the pons and cerebellum at the level of thesuperior olives,

Block V — the medulla oblongata and cerebellum at thelevel of the mid-inferior olivary nuclei.

Cervical and lumbar enlargements of the spinal cord are eachdivided equally into six blocks; 15 µm sections are cut fromthe tissue blocks embedded in paraffin wax and stained withgallocyanin. Numerical scores are given to each hemisectionof the cord and to structures in each hemisection of the brainas listed below. Lesions are scored as follows:

Grade 1. Minimal: 1 to 3 small focal inflammatory infiltrates.Degeneration or loss of a few neurons.

Grade 2. Moderate: 4 or more focal inflammatory infiltrates.Degeneration or loss of neurons affecting not more than onethird of cells.

Grade 3. Severe: moderate focal or diffuse inflammatoryinfiltration. Degeneration or loss of up to two third of theneurons.

Grade 4. Overwhelming: variable but often severe inflammatoryreaction. Degeneration or loss of more than 90.0 per cent ofneurons.

It has been found that inoculation of yellow fever vaccineinto the monkey brain causes histological lesions in different


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anatomical formations of the central nervous system withvarying frequency and severity (I. S. Levenbook et al., Journalof Biological Standardization, 1987, 15, 305-313). Based onthese two indicators, the anatomical structures can be dividedinto target, spared and discriminator areas. Target areas arethose which show more severe specific lesions in a majorityof monkeys irrespective of the degree of neurovirulence ofthe seed lot. Spared areas are those which show only minimalspecific lesions and in a minority of monkeys. Discriminatorareas are those where there is a significant increase in thefrequency of more severe specific lesions with seed lots havinga higher degree of neurovirulence. Discriminator and targetareas for Macaca cynomolgus and Macaca rhesus monkeysare shown in the table below:

Table 1. The discriminator and target areas for monkey.

Type of monkey Discriminator areas Target areas

Macaca cynomolgus globus pallidus substantia nigraputamen anterior/median thalamicnucleus lateralthalamic nucleus

Macaca rhesus caudate nucleus substantia nigraglobus pallidus cervicalputamen anterior/ lumbarmedian thalamic enlargementnucleus lateralthalamic nucleuscervical enlargementlumbar enlargementenlargement

Scores for discriminator and target areas are used for the finalevaluation of the seed lot. The individual monkey score iscalculated from the sum of individual target area scores ineach hemisection divided by the number of areas examined. Aseparate score is calculated similarly for the discriminator areas.

Mean scores for the test group are calculated in two ways: (1)by dividing the sum of the individual monkey discriminatorscores by the number of monkeys and (2) by dividing the sumof the individual monkey target and discriminator scores bythe number of monkeys. These two mean scores are takeninto account when deciding on the acceptability of the seedlot. The seed lot is not acceptable if either of the mean lesionscores is significantly greater (P = 0.95) than for the referencepreparation.

Labelling. The label states (1) the strain of virus used inpreparation; (2) that the vaccine has been prepared in chickembryos; (3) the minimum virus concentration; (4) that contact


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