BP_06 - 2001

Monographs

Immunological Products

67-2

ANTISERA

An antiserum for human use that is the subject of an individual monograph in the European Pharmacopoeia orin the British Pharmacopoeia complies with the requirements of the 3rd edition of the European Pharmacopoeiafor Immunosera for Human Use [0084]. These requirements are reproduced below.

The provisions of this monograph apply to the following antisera:

Botulinum Antitoxin*Diphtheria Antitoxin*European Viper Venom Antiserum*Gas-gangrene Antitoxin (Novyi)*Gas-gangrene Antitoxin (Perfringens)*Gas-gangrene Antitoxin (Septicum)*Mixed Gas-gangrene Antitoxin*Tetanus Antitoxin*

*Monograph of the European Pharmacopoeia

Ph Eur ___________________________________________________________________________________________________________

The statements in this monograph are intended to be read in conjunction with the monographs on immunoserafor human use in the Pharmacopoeia. The requirements do not necessarily apply to immunosera which are notthe subject of such monographs.

DEFINITION

Immunosera for human use are purified preparations containing immunoglobulins obtained fromserum of immunised animals. The immunoglobulins have the power of specifically neutralisingvenins or the toxins formed by bacteria or of specifically combining with the bacteria, viruses or otherantigens.

PRODUCTION

Immunosera are obtained from healthy animals immunised by injections of the appropriate toxins ortoxoids, venins, suspensions of micro-organisms or other antigens. During the immunisation theanimals must not be treated with penicillin. The globulins containing the immunising substances maybe obtained from the serum by enzyme treatment and fractional precipitation or by other chemical orphysical methods.

A suitable antimicrobial preservative may be added and is invariably added if the preparations areissued in multidose containers. The final sterile products are distributed aseptically into sterilecontainers which are then closed so as to exclude contamination.

The products may be freeze-dried by a procedure which reduces the water content of the finishedproduct to not more than 1.0 per cent m/m.

Immunosera prepared by enzyme treatment and fractional precipitation are most stable at aboutpH 6. The method of preparation of immunosera is such that the products lose not more than 5 percent of their activity per year at this pH when stored at 20°C and not more than 20 per cent per yearwhen stored at 37°C.

The production method is validated to demonstrate that the product, if tested, would comply withthe test for abnormal toxicity for immunosera and vaccines for human use (2.6.9).

CHARACTERS

Immunosera are almost colourless or very faintly yellow liquids free from turbidity. Freeze-driedimmunosera consist of white or pale-yellow crusts or powders, freely soluble in water to form colour-less or pale-yellow solutions having the same characters as the corresponding liquid preparations.

TESTS

The following requirements refer to liquid immunosera and to the reconstituted freeze-dried preparations.

pH (2.2.3). The pH is 6.0 to 7.0.

Foreign proteins When examined by precipitation tests with specific antisera, only protein from thedeclared animal species is shown to be present.

Total protein Not more than 170 g/l. Carry out the determination of nitrogen by sulphuric aciddigestion (2.5.9) and multiply the result by 6.25.

Albumins Unless otherwise prescribed in the monograph, when examined electrophoretically, theyshow not more than traces of albumins.

Phenol (2.5.15). When the immunosera contain phenol, the concentration is not more than 2.5 g/l.

Sterility (2.6.1). They comply with the test for sterility.

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POTENCY

Carry out a biological assay as indicated in the monograph and express the result in InternationalUnits per millilitre, where appropriate.

STORAGE

Store at a temperature of 5 ± 3°C. Liquid immunosera should not be allowed to freeze.

Expiry date. The expiry date is calculated from the beginning of the test for Potency. It applies toimmunosera stored in the prescribed conditions.

LABELLING

The label states:— the name of the preparation,— the number of International Units per millilitre where applicable,— the batch number or other reference,— the route of administration,— the storage conditions,— the expiry date, except that for containers of 1 ml or less which are individually packed, the

expiry date may be omitted from the label on the container provided it is shown on the packageand the label on the package states that the container must be kept in the package until requiredfor use,

— the name of the animal species of origin,— the name and amount of any antimicrobial preservative or other substance added to the

immunoserum,— a declaration of any substance likely to cause any adverse reaction and any contra-indications to

the use of the product,— for freeze-dried immunosera:— the name or composition and the volume of the reconstituting liquid to be added,— that the immunoserum should be used immediately after reconstitution,— the name and address of the manufacturer.

__________________________________________________________________________________________________________ Ph Eur

67-4

Botulinum Antitoxin

Botulinum Antitoxin complies with the requirements of the 3rd edition of the European Pharmacopoeia [0085].These requirements are reproduced after the heading ‘Definition’ below.

The label may state ‘Bot/Ser’ followed by a letter or letters indicating the type or types present.

When Mixed Botulinum Antitoxin or Botulinum Antitoxin is prescribed or demanded and the typesto be present are not stated, Botulinum Antitoxin prepared from types A, B and E shall be dispensedor supplied.

Ph Eur ___________________________________________________________________________________________________________

DEFINITION

Botulinum antitoxin is a preparation containing antitoxic globulins that have the power of specificallyneutralising the toxins formed by Clostridium botulinum type A, type B or type E, or any mixture ofthese types.

PRODUCTION

It is obtained by fractionation from the serum of horses, or other mammals, that have beenimmunised against Cl. botulinum type A, type B and type E toxins.

IDENTIFICATION

It specifically neutralises the types of Cl. botulinum toxins stated on the label, rendering them harm-less to susceptible animals.

TESTS

It complies with the tests prescribed in the monograph on Immunosera for human use (84).

POTENCY

Not less than 500 I.U. of antitoxin per millilitre for each of types A and B and not less than 50 I.U.of antitoxin per millilitre for type E.

The potency of botulinum antitoxin is determined by comparing the dose necessary to protect miceagainst the lethal effects of a fixed dose of botulinum toxin with the quantity of the standard prepara-tion of botulinum antitoxin necessary to give the same protection. For this comparison a referencepreparation of each type of botulinum antitoxin, calibrated in International Units, and suitablepreparations of botulinum toxins, for use as test toxins, are required. The potency of each test toxinis determined in relation to the specific reference preparation; the potency of the botulinum antitoxinto be examined is determined in relation to the potency of the test toxins by the same method.

International Units of the antitoxin are the specific neutralising activity for botulinum toxin type A,type B and type E contained in stated amounts of the International Standards which consist of driedimmune horse sera of types A, B and E. The equivalence in International Units of the InternationalStandard is stated from time to time by the World Health Organisation.

Selection of animals. Use mice having body masses such that the difference between the lightest andthe heaviest does not exceed 5 g.

Preparation of test toxins. Warning: Botulinum toxin is extremely toxic: exceptional care must be taken inany procedure in which it is employed. Prepare type A, B and E toxins from sterile filtrates ofapproximately 7-day cultures in liquid medium of Cl. botulinum types A, B and E. To the filtrates,add 2 volumes of glycerol, concentrate, if necessary, by dialysis against glycerol and store at orslightly below 0°C.

Selection of test toxins. Select toxins of each type for use as test toxins by determining for mice theL+/10 dose and the LD50, the observation period being 96 h. The test toxins contain at least1000 LD50 in an L+/10 dose.

Determination of test doses of the toxins (L+/10 dose). Prepare solutions of the reference preparations ina suitable liquid such that each contains 0.25 I.U. of antitoxin per millilitre. Using each solution inturn, determine the test dose of the corresponding test toxin.

Prepare mixtures of the solution of the reference preparation and the test toxin such that eachcontains 2.0 ml of the solution of the reference preparation, one of a graded series of volumes of thetest toxin and sufficient of a suitable liquid to bring the total volume to 5.0 ml. Allow the mixtures tostand at room temperature, protected from light, for 60 min. Using four mice for each mixture, injecta dose of 1.0 ml intraperitoneally into each mouse. Observe the mice for 96 h.

The test dose of toxin is the quantity in 1.0 ml of the mixture made with the smallest amount oftoxin capable of causing, despite partial neutralisation by the reference preparation, the death of allfour mice injected with the mixture within the observation period.

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Determination of potency of the antitoxin

Prepare solutions of each reference preparation in a suitable liquid such that each contains0.25 I.U. of anti-toxin per millilitre.

Prepare solutions of each test toxin in a suitable liquid such that each contains 2.5 test doses permillilitre.

Using each toxin solution and the corresponding reference preparation in turn, determine thepotency of the antitoxin. Prepare mixtures of the solution of the test toxin and the antitoxin to beexamined such that each contains 2.0 ml of the solution of the test toxin, one of a graded series ofvolumes of the antitoxin to be examined, and sufficient of a suitable liquid to bring the total volumeto 5.0 ml. Also prepare mixtures of the solution of the test toxin and the solution of the referencepreparation such that each contains 2.0 ml of the solution of the test toxin, one of a graded series ofvolumes of the solution of the reference preparation centred on that volume (2.0 ml) that contains0.5 I.U., and sufficient of a suitable liquid to bring the total volume to 5.0 ml. Allow the mixtures tostand at room temperature, protected from light, for 60 min. Using four mice for each mixture, injecta dose of 1.0 ml intraperitoneally into each mouse. Observe the mice for 96 h.

The mixture that contains the largest volume of antitoxin that fails to protect the mice from deathcontains 0.5 I.U. This quantity is used to calculate the potency of the antitoxin in International Unitsper millilitre.

The test is not valid unless all the mice injected with mixtures containing 2.0 ml or less of thesolution of the reference preparation die and all those injected with mixtures containing more survive.

STORAGE

See Immunosera for human use (84).

LABELLING


The label states the types of Cl. botulinum toxin neutralised by the preparation.__________________________________________________________________________________________________________ Ph Eur

67-6

Diphtheria Antitoxin

Diphtheria Antitoxin complies with the requirements of the 3rd edition of the European Pharmacopoeia [0086].These requirements are reproduced after the heading ‘Definition’ below.

The label may state ‘Dip/Ser’.

Ph Eur ___________________________________________________________________________________________________________

DEFINITION

Diphtheria antitoxin is a preparation containing antitoxic globulins that have the power ofspecifically neutralising the toxin formed by Corynebacterium diphtheriae.

PRODUCTION

It is obtained by fractionation from the serum of horses, or other mammals, that have beenimmunised against diphtheria toxin.

IDENTIFICATION

It specifically neutralises the toxin formed by C. diphtheriae, rendering it harmless to susceptibleanimals.

TESTS


ASSAY

Not less than 1000 I.U. of antitoxin per millilitre for antitoxin obtained from horse serum. Not lessthan 500 I.U. of antitoxin per millilitre for antitoxin obtained from the serum of other mammals.

The potency of diphtheria antitoxin is determined by comparing the dose necessary to protectguinea-pigs or rabbits against the erythrogenic effects of a fixed dose of diphtheria toxin with thequantity of the standard preparation of diphtheria antitoxin necessary to give the same protection.For this comparison a reference preparation of diphtheria antitoxin, calibrated in International Units,and a suitable preparation of diphtheria toxin, for use as a test toxin, are required. The potency of thetest toxin is determined in relation to the reference preparation; the potency of the diphtheriaantitoxin to be examined is determined in relation to the potency of the test toxin by the samemethod.

The International Unit of antitoxin is the specific neutralising activity for diphtheria toxincontained in a stated amount of the International Standard, which consists of a quantity of driedimmune horse serum. The equivalence in International Units of the International Standard is statedby the World Health Organisation.

Preparation of test toxin. Prepare diphtheria toxin from cultures of C. diphtheriae in a liquid medium.Filter the culture to obtain a sterile toxic filtrate and store at 4°C.

Selection of test toxin. Select a toxin for use as a test toxin by determining for guinea-pigs or rabbits the1r/100 dose and the minimal reacting dose, the observation period being 48 h. The test toxin has atleast 200 minimal reacting doses in the 1r/100 dose.

Minimal reacting dose. This is the smallest quantity of toxin which, when injected intracutaneouslyinto guinea-pigs or rabbits, causes a small, characteristic reaction at the site of injection within 48 h.

The test toxin is allowed to stand for some months before being used for the assay of antitoxin.During this time its toxicity declines and the 1r/100 dose may be increased. Determine the minimalreacting dose and the 1r/100 dose at frequent intervals. When experiment shows that the 1r/100 doseis constant, the test toxin is ready for use and may be used for a long period. Store the test toxin inthe dark at 0°C to 5°C. Maintain its sterility by the addition of toluene or other antimicrobialpreservative that does not cause a rapid decline in specific toxicity.

Determination of test dose of toxin (1r/100 dose). Prepare a solution of the reference preparation in asuitable liquid such that it contains 0.1 I.U. of antitoxin per millilitre.

Prepare mixtures of the solution of the reference preparation and of the test toxin such that eachcontains 1.0 ml of the solution of the reference preparation, one of a graded series of volumes of thetest toxin and sufficient of a suitable liquid to bring the total volume to 2.0 ml. Allow the mixtures tostand at room temperature, protected from light, for 15 min to 60 min. Using two animals for eachmixture, inject a dose of 0.2 ml intracutaneously into the shaven or depilated flanks of each animal.Observe the animals for 48 h.

The test dose of toxin is the quantity in 0.2 ml of the mixture made with the smallest amount oftoxin capable of causing, despite partial neutralisation by the reference preparation, a small butcharacteristic erythematous lesion at the site of injection.

Determination of potency of the antitoxin.

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Prepare a solution of the reference preparation in a suitable liquid such that it contains 0.125 I.U. ofantitoxin per millilitre.

Prepare a solution of the test toxin in a suitable liquid such that it contains 12.5 test doses permillilitre.

Prepare mixtures of the solution of the test toxin and of the antitoxin to be examined such thateach contains 0.8 ml of the solution of the test toxin, one of a graded series of volumes of theantitoxin to be examined and sufficient of a suitable liquid to bring the total volume to 2.0 ml. Alsoprepare mixtures of the solution of the test toxin and the solution of the reference preparation suchthat each contains 0.8 ml of the solution of the test toxin, one of a graded series of volumes of thesolution of the reference preparation centred on that volume (0.8 ml) that contains 0.1 I.U. andsufficient of a suitable liquid to bring the total volume to 2.0 ml. Allow the mixtures to stand at roomtemperature, protected from light, for 15 min to 60 min. Using two animals for each mixture, inject adose of 0.2 ml intracutaneously into the shaven or depilated flanks of each animal. Observe theanimals for 48 h.

The mixture that contains the largest volume of antitoxin that fails to protect the guinea-pigs fromthe erythematous effects of the toxin contains 0.1 I.U. This quantity is used to calculate the potencyof the antitoxin in International Units per millilitre.

The test is not valid unless all the sites injected with mixtures containing 0.8 ml or less of thesolution of the reference preparation show erythematous lesions and at all those injected withmixtures containing more there are no lesions.

STORAGE


LABELLING

See Immunosera for human use (84).__________________________________________________________________________________________________________ Ph Eur

67-8

European Viper Venom Antiserum

European Viper Venom Antiserum complies with the requirements of the 3rd edition of the European Pharma-copoeia [0145]. These requirements are reproduced after the heading ‘Definition’ below.

The only poisonous snake native to the British Isles is the adder or common viper, Vipera berus. In ageographical region where other species of snake (including elapids) are found, antisera able toneutralise the venoms of the species of snake indigenous to the region should be used. When thepreparation is intended to neutralise the venom or venoms of one or more snakes other than vipers,the title Snake Venom Antiserum is used.

Ph Eur ___________________________________________________________________________________________________________

DEFINITION

European viper venom antiserum is a preparation containing antitoxic globulins that have the powerof neutralising the venom of one or more species of viper. The globulins are obtained by fractionationof the serum of animals that have been immunised against the venom or venoms.

IDENTIFICATION

It neutralises the venom of Vipera ammodytes, or Vipera aspis, or Vipera berus, or Vipera ursinii or themixture of these venoms stated on the label, rendering them harmless to susceptible animals.

TESTS


ASSAY

Each millilitre of the preparation to be examined contains sufficient antitoxic globulins to neutralisenot less than 100 mouse LD50 of Vipera ammodytes venom or Vipera aspis venom and not less than 50mouse LD50 of the venoms of other species of viper.

The potency of European viper venom antiserum is determined by estimating the dose necessary toprotect mice against the lethal effects of a fixed dose of venom of the relevant species of viper.

Selection of test venoms†. Use venoms which have the normal physicochemical, toxicological andimmunological characteristics of venoms from the particular species of vipers. They are preferablyfreeze-dried and stored in the dark at 5 ± 3°C.

Select a venom for use as a test venom by determining the LD50 for mice, the observation periodbeing 48 h.

Determination of the test dose of venom. Prepare graded dilutions of the reconstituted venom in a 9 g/lsolution of sodium chloride R or other isotonic diluent in such a manner that the middle dilutioncontains in 0.25 ml the dose expected to be the LD50. Dilute with an equal volume of the samediluent. Using at least four mice, each weighing 18 g to 20 g, for each dilution, inject 0.5 mlintravenously into each mouse. Observe the mice for 48 h and record the number of deaths.Calculate the LD50 using the usual statistical methods.

Determination of the potency of the antiserum to be examined. Dilute the reconstituted test venom so that0.25 ml contains the test dose of 5 LD50 (test venom solution).

Prepare serial dilutions of the antiserum to be examined in a 9 g/l solution of sodium chloride R orother isotonic diluent, the dilution factor being 1.5 to 2.5. Use a sufficient number and range ofdilutions to enable a mortality curve between 20 per cent and 80 per cent mortality to be establishedand to permit an estimation of the statistical variation.

Prepare mixtures such that 5 ml of each mixture contains 2.5 ml of one of the dilutions of theantiserum to be examined and 2.5 ml of the test venom solution. Allow the mixtures to stand in awater-bath at 37°C for 30 min. Using not fewer than six mice, each weighing 18 g to 20 g, for eachmixture, inject 0.5 ml intravenously into each mouse. Observe the mice for 48 h and record thenumber of deaths. Calculate the PD50, using the usual statistical methods. At the same time verifythe number of LD50 in the test dose of venom, using the method described above. Calculate thepotency of the antiserum from the expression:

Tv −1

50PD

Tv =number of LD50 in the test dose of venom.

In each mouse dose of the venom-antiserum mixture at the end point there is one LD50 of venomremaining unneutralised by the antiserum and it is this unneutralised venom that is responsible forthe deaths of 50 per cent of the mice inoculated with the mixture. The amount of venom neutralisedby the antiserum is thus one LD50 less than the total amount contained in each mouse dose.Therefore, as the potency of the antiserum is defined in terms of the number of LD50 of venom that

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are neutralised rather than the number of LD50 in each mouse dose, the expression required in thecalculation of potency is Tv–1 rather than Tv.

Alternatively, the quantity of test venom in milligrams that is neutralised by 1 ml or some otherdefined volume of the antiserum to be examined may be calculated.

STORAGE


LABELLING


The label states the venom or venoms against which the antiserum is effective.

†Warning: Because of the allergenic properties of viper venoms, inhalation of venom dust should be avoidedby suitable precautions.

__________________________________________________________________________________________________________ Ph Eur

67-10

Gas-gangrene Antitoxin (Novyi)Gas-gangrene Antitoxin (Oedematiens)

Gas-gangrene Antitoxin (Novyi) complies with the requirements of the 3rd edition of the European Pharmaco-poeia [0087]. These requirements are reproduced after the heading ‘Definition’ below.

The label may state ‘Nov/Ser’.

PreparationMixed Gas-gangrene Antitoxin

Ph Eur ___________________________________________________________________________________________________________

DEFINITION

Gas-gangrene antitoxin (novyi) is a preparation containing antitoxic globulins that have the powerof neutralising the alpha toxin formed by Clostridium novyi (Former nomenclature: Clostridiumoedematiens). It is obtained by fractionation from the serum of horses, or other mammals, that havebeen immunised against Cl. novyi alpha toxin.

IDENTIFICATION

It specifically neutralises the alpha toxin formed by Cl. novyi, rendering it harmless to susceptibleanimals.

TESTS


ASSAY

Not less than 3750 I.U. of antitoxin per millilitre.

The potency of gas-gangrene antitoxin (novyi) is determined by comparing the dose necessary toprotect mice or other suitable animals against the lethal effects of a fixed dose of Cl. novyi toxin withthe quantity of the standard preparation of gas-gangrene antitoxin (novyi) necessary to give the sameprotection. For this comparison a reference preparation of gas-gangrene antitoxin (novyi), calibratedin International Units, and a suitable preparation of Cl. novyi toxin for use as a test toxin arerequired. The potency of the test toxin is determined in relation to the reference preparation; thepotency of the gas-gangrene antitoxin (novyi) to be examined is determined in relation to the potencyof the test toxin by the same method.

The International Unit of antitoxin is the specific neutralising activity for Cl. novyi toxin containedin a stated amount of the International Standard, which consists of a quantity of dried immune horseserum. The equivalence in International Units of the International Standard is stated by the WorldHealth Organisation.


Preparation of test toxin. Prepare the test toxin from a sterile filtrate of an approximately 5-day culturein liquid medium of Cl. novyi. Treat the filtrate with ammonium sulphate, collect the precipitate,which contains the toxin, dry in vacuo over diphosphorus pentoxide R, powder and store dry.

Selection of test toxin. Select a toxin for use as a test toxin by determining for mice the L+ dose and theLD50, the observation period being 72 h. The test toxin has an L+ dose of 0.5 mg or less andcontains not less than 25 LD50 in each L+ dose.

Determination of test dose of toxin (L+ dose). Prepare a solution of the reference preparation in asuitable liquid such that it contains 12.5 I.U. of antitoxin per millilitre.

Prepare a solution of the test toxin in a suitable liquid such that 1 ml contains a precisely knownamount such as 10 mg.

Prepare mixtures of the solution of the reference preparation and the solution of the test toxin suchthat each contains 0.8 ml of the solution of the reference preparation, one of a graded series ofvolumes of the solution of the test toxin and sufficient of a suitable liquid to bring the total volume to2.0 ml. Allow the mixtures to stand at room temperature, protected from light, for 60 min. Using sixmice for each mixture, inject a dose of 0.2 ml intramuscularly into each mouse. Observe the mice for72 h.

The test dose of toxin is the quantity in 0.2 ml of the mixture made with the smallest amount oftoxin capable of causing, despite partial neutralisation by the reference preparation, the death of allsix mice injected with the mixture within the observation period.


Prepare a solution of the reference preparation in a suitable liquid such that it contains 12.5 I.U. ofantitoxin per millilitre.

Prepare a solution of the test toxin in a suitable liquid such that it contains 12.5 test doses permillilitre.

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Prepare mixtures of the solution of the test toxin and the antitoxin to be examined such that eachcontains 0.8 ml of the solution of the test toxin, one of a graded series of volumes of the antitoxin tobe examined and sufficient of a suitable liquid to bring the total volume to 2.0 ml. Also preparemixtures of the solution of the test toxin and the solution of the reference preparation such that eachcontains 0.8 ml of the solution of the test toxin, one of a graded series of volumes of the solution ofthe reference preparation centred on that volume (0.8 ml) that contains 10 I.U. and sufficient of asuitable liquid to bring the total volume to 2.0 ml. Allow the mixtures to stand at room temperature,protected from light, for 60 min. Using six mice for each mixture, inject a dose of 0.2 ml intramuscu-larly into each mouse. Observe the mice for 72 h.

The mixture that contains the largest volume of antitoxin that fails to protect the mice from deathcontains 10 I.U. This quantity is used to calculate the potency of the antitoxin in International Unitsper millilitre.

The test is not valid unless all the mice injected with mixtures containing 0.8 ml or less of thesolution of the reference preparation die and all those injected with mixtures containing a largervolume survive.

STORAGE

See the monograph on Immunosera for human use (84).

LABELLING

See the monograph on Immunosera for human use (84).__________________________________________________________________________________________________________ Ph Eur

67-12

Gas-gangrene Antitoxin (Perfringens)

Gas-gangrene Antitoxin (Perfringens) complies with the requirements of the 3rd edition of the EuropeanPharmacopoeia [0088]. These requirements are reproduced after the heading ‘Definition’ below.

The label may state ‘Perf/Ser’.


Ph Eur ___________________________________________________________________________________________________________

DEFINITION

Gas-gangrene antitoxin (perfringens) is a preparation containing antitoxic globulins that have thepower of specifically neutralising the alpha toxin formed by Clostridium perfringens. It is obtained byfractionation from the serum of horses, or other mammals, that have been immunised against Cl.perfringens alpha toxin.

IDENTIFICATION

It specifically neutralises the alpha toxin formed by Cl. perfringens, rendering it harmless to susceptibleanimals.

TESTS


ASSAY


The potency of gas-gangrene antitoxin (perfringens) is determined by comparing the dose necessaryto protect mice or other suitable animals against the lethal effects of a fixed dose of Cl. perfringenstoxin with the quantity of the standard preparation of gas-gangrene antitoxin (perfringens) necessaryto give the same protection. For this comparison a reference preparation of gas-gangrene antitoxin(perfringens), calibrated in International Units, and a suitable preparation of Cl. perfringens toxin foruse as a test toxin are required. The potency of the test toxin is determined in relation to the refer-ence preparation; the potency of the gas-gangrene antitoxin (perfringens) to be examined is deter-mined in relation to the potency of the test toxin by the same method.

The International Unit of antitoxin is the specific neutralising activity for Cl. perfringens toxincontained in a stated amount of the International Standard, which consists of a quantity of driedimmune horse serum. The equivalence in International Units of the International Standard is statedby the World Health Organisation.


Preparation of test toxin. Prepare the test toxin from a sterile filtrate of an approximately 5-day culturein liquid medium of Cl. perfringens. Treat the filtrate with ammonium sulphate, collect the precipitate,which contains the toxin, dry in vacuo over diphosphorus pentoxide R, powder and store dry.

Selection of test toxin. Select a toxin for use as a test toxin by determining for mice the L+ dose and theLD50, the observation period being 48 h. The test toxin has an L+ dose of 4 mg or less and containsnot less than 20 LD50 in each L+ dose.

Determination of test dose of toxin (L+ dose). Prepare a solution of the reference preparation in asuitable liquid such that it contains 5 I.U. of antitoxin per millilitre.


Prepare mixtures of the solution of the reference preparation and the solution of the test toxin suchthat each contains 2.0 ml of the solution of the reference preparation, one of a graded series ofvolumes of the solution of the test toxin and sufficient of a suitable liquid to bring the total volume to5.0 ml. Allow the mixtures to stand at room temperature, protected from light, for 60 min. Using sixmice for each mixture, inject a dose of 0.5 ml intravenously into each mouse. Observe the mice for48 h.



Prepare a solution of the reference preparation in a suitable liquid such that it contains 5 I.U. ofantitoxin per millilitre.

Prepare a solution of the test toxin in a suitable liquid such that it contains five test doses permillilitre.

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Prepare mixtures of the solution of the test toxin and the antitoxin to be examined such that eachcontains 2.0 ml of the solution of the test toxin, one of a graded series of volumes of the antitoxin tobe examined and sufficient of a suitable liquid to bring the total volume to 5.0 ml. Also preparemixtures of the solution of the test toxin and the solution of the reference preparation such that eachcontains 2.0 ml of the solution of the test toxin, one of a graded series of volumes of the solution ofthe reference preparation centred on that volume (2.0 ml) that contains 10 I.U. and sufficient of asuitable liquid to bring the total volume to 5.0 ml. Allow the mixtures to stand at room temperature,protected from light, for 60 min. Using six mice for each mixture, inject a dose of 0.5 ml intra-venously into each mouse. Observe the mice for 48 h.


The test is not valid unless all the mice injected with mixtures containing 2.0 ml or less of thesolution of the reference preparation die and all those injected with mixtures containing a largervolume survive.

STORAGE


LABELLING


67-14

Gas-gangrene Antitoxin (Septicum)

Gas-gangrene Antitoxin (Septicum) complies with the requirements of the 3rd edition of the European Pharma-copoeia [0089]. These requirements are reproduced after the heading ‘Definition’ below.

The label may state ‘Sep/Ser’.


Ph Eur ___________________________________________________________________________________________________________

DEFINITION

Gas-gangrene antitoxin (septicum) is a preparation containing antitoxic globulins that have thepower of specifically neutralising the alpha toxin formed by Clostridium septicum. It is obtained byfractionation from the serum of horses, or other mammals, that have been immunised against Cl.septicum alpha toxin.

IDENTIFICATION

It specifically neutralises the alpha toxin formed by Cl. septicum, rendering it harmless to susceptibleanimals.

TESTS


ASSAY


The potency of gas-gangrene antitoxin (septicum) is determined by comparing the dose necessary toprotect mice or other suitable animals against the lethal effects of a fixed dose of Cl. septicum toxinwith the quantity of the standard preparation of gas-gangrene antitoxin (septicum) necessary to givethe same protection. For this comparison a reference preparation of gas-gangrene antitoxin(septicum), calibrated in International Units, and a suitable preparation of Cl. septicum toxin for useas a test toxin are required. The potency of the test toxin is determined in relation to the referencepreparation; the potency of the gas-gangrene antitoxin (septicum) to be examined is determined inrelation to the potency of the test toxin by the same method.

The International Unit of antitoxin is the specific neutralising activity for Cl. septicum toxincontained in a stated amount of the International Standard, which consists of a quantity of driedimmune horse serum. The equivalence in International Units of the International Standard is statedby the World Health Organisation.


Preparation of test toxin. Prepare the test toxin from a sterile filtrate of an approximately 5-day culturein liquid medium of Cl. septicum. Treat the filtrate with ammonium sulphate, collect the precipitate,which contains the toxin, dry in vacuo over diphosphorus pentoxide R, powder and store dry.

Selection of test toxin. Select a toxin for use as a test toxin by determining for mice the L+ dose and theLD50, the observation period being 72 h. The test toxin has an L+ dose of 0.5 mg or less andcontains not less than 25 LD50 in each L+ dose.

Determination of test dose of toxin (L+ dose). Prepare a solution of the reference preparation in asuitable liquid such that it contains 5 I.U. of antitoxin per millilitre.


Prepare mixtures of the solution of the reference preparation and the solution of the test toxin suchthat each contains 2.0 ml of the solution of the reference preparation, one of a graded series ofvolumes of the solution of the test toxin and sufficient of a suitable liquid to bring the total volume to5.0 ml. Allow the mixtures to stand at room temperature, protected from light, for 60 min. Using sixmice for each mixture, inject a dose of 0.5 ml intravenously into each mouse. Observe the mice for72 h.



Prepare a solution of the reference preparation in a suitable liquid such that it contains 5 I.U. ofantitoxin per millilitre.


67-15

Prepare mixtures of the solution of the test toxin and the antitoxin to be examined such that eachcontains 2.0 ml of the solution of the test toxin, one of a graded series of volumes of the antitoxin tobe examined and sufficient of a suitable liquid to bring the total volume to 5.0 ml. Also preparemixtures of the solution of the test toxin and the solution of the reference preparation such that eachcontains 2.0 ml of the solution of the test toxin, one of a graded series of volumes of the solution ofthe reference preparation centred on that volume (2.0 ml) that contains 10 I.U. and sufficient of asuitable liquid to bring the total volume to 5.0 ml. Allow the mixtures to stand at room temperature,protected from light, for 60 min. Using six mice for each mixture, inject a dose of 0.5 ml intra-venously into each mouse. Observe the mice for 72 h.


The test is not valid unless all the mice injected with mixtures containing 2.0 ml or less of thesolution of the reference preparation die and all those injected with mixtures containing more survive.

STORAGE


LABELLING


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Mixed Gas-gangrene Antitoxin

Mixed Gas-gangrene Antitoxin complies with the requirements of the 3rd edition of the European Pharmaco-poeia [0090]. These requirements are reproduced after the heading ‘Definition’ below.

The label may state ‘Gas/Ser’.

Ph Eur ___________________________________________________________________________________________________________

DEFINITION

Mixed gas-gangrene antitoxin is prepared by mixing gas-gangrene antitoxin (novyi), gas-gangreneantitoxin (perfringens) and gas-gangrene antitoxin (septicum) in appropriate quantities.

IDENTIFICATION

It specifically neutralises the alpha toxins formed by Clostridium novyi (former nomenclature:Clostridium oedematiens), Clostridium perfringens and Clostridium septicum, rendering them harmless tosusceptible animals.

TESTS


ASSAY

Gas-gangrene antitoxin (novyi), not less than 1000 I.U. of antitoxin per millilitre; gas-gangreneantitoxin (perfringens), not less than 1000 I.U. of antitoxin per millilitre; gas-gangrene antitoxin(septicum) not less than 500 I.U. of antitoxin per millilitre.

Carry out the assay for each component, as prescribed in the monographs on Gas-gangrene antitoxin(novyi) (87), Gas-gangrene antitoxin (perfringens) (88) and Gas-gangrene antitoxin (septicum) (89).

STORAGE


LABELLING


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Tetanus Antitoxin

Tetanus Antitoxin complies with the requirements of the 3rd edition of the European Pharmacopoeia forTetanus Antitoxin for Human Use [0091]. These requirements are reproduced after the heading ‘Definition’below.

The label may state ‘Tet/Ser’.

Ph Eur ___________________________________________________________________________________________________________

DEFINITION

Tetanus antitoxin for human use is a preparation containing antitoxic globulins that have the powerof specifically neutralising the toxin formed by Clostridium tetani.

PRODUCTION

It is obtained by fractionation from the serum of horses, or other mammals, that have beenimmunised against tetanus toxin.

IDENTIFICATION

It specifically neutralises the toxin formed by Cl. tetani, rendering it harmless to susceptible animals.

TESTS


POTENCY

Not less than 1000 I.U. of antitoxin per millilitre when intended for prophylactic use. Not less than3000 I.U. of antitoxin per millilitre when intended for therapeutic use.

The potency of tetanus antitoxin is determined by comparing the dose necessary to protect guinea-pigs or mice against the paralytic effects of a fixed dose of tetanus toxin with the quantity of thestandard preparation of tetanus antitoxin necessary to give the same protection. In countries wherethe paralysis method is not obligatory the lethal method may be used. For this method the number ofanimals and the procedure are identical with those described for the paralysis method but the end-point is the death of the animal rather than the onset of paralysis and the L+/10 dose is used insteadof the Lp/10 dose. For this comparison a reference preparation of tetanus antitoxin, calibrated inInternational Units, and a suitable preparation of tetanus toxin, for use as a test toxin, are required.The potency of the test toxin is determined in relation to the reference preparation; the potency ofthe tetanus antitoxin to be examined is determined in relation to the potency of the test toxin by thesame method.

The International Unit of antitoxin is the specific neutralising activity for tetanus toxin contained ina stated amount of the International Standard which consists of a quantity of dried immune horseserum. The equivalence in International Units of the International Standard is stated by the WorldHealth Organisation.

Selection of animals. If mice are used, the body masses should be such that the difference between thelightest and the heaviest does not exceed 5 g.

Preparation of test toxin. Prepare the test toxin from a sterile filtrate of an approximately 9-day culturein liquid medium of Cl. tetani. To the filtrate add 1 to 2 volumes of glycerol and store slightly below0°C. Alternatively, treat the filtrate with ammonium sulphate, collect the precipitate, which containsthe toxin, dry in vacuo over diphosphorus pentoxide R, powder and store dry, either in sealed ampoulesor in vacuo over diphosphorus pentoxide R.

Determination of test dose of toxin (Lp/10 dose). Prepare a solution of the reference preparation in asuitable liquid such that it contains 0.5 I.U. of antitoxin per millilitre.

If the test toxin is stored dry, reconstitute it using a suitable liquid.Prepare mixtures of the solution of the reference preparation and the test toxin such that each

contains 2.0 ml of the solution of the reference preparation, one of a graded series of volumes of thetest toxin and sufficient of a suitable liquid to bring the volume to 5.0 ml. Allow the mixtures to standat room temperature, protected from light, for 60 min. Using six mice for each mixture, inject a doseof 0.5 ml subcutaneously into each mouse. Observe the mice for 96 h. Mice that become paralysedmay be killed.

The test dose of toxin is the quantity in 0.5 ml of the mixture made with the smallest amount oftoxin capable of causing, despite partial neutralisation by the reference preparation, paralysis in all sixmice injected with the mixture within the observation period.

Determination of potency of the antitoxinPrepare a solution of the reference preparation in a suitable liquid such that it contains 0.5 I.U. ofantitoxin per millilitre.

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Prepare mixtures of the solution of the test toxin and the antitoxin to be examined such that eachcontains 2.0 ml of the solution of the test toxin, one of a graded series of volumes of the antitoxin tobe examined and sufficient of a suitable liquid to bring the total volume to 5.0 ml. Also preparemixtures of the solution of the test toxin and the solution of the reference preparation such that eachcontains 2.0 ml of the solution of the test toxin, one of a graded series of volumes of the solution ofthe reference preparation centred on that volume (2.0 ml) that contains 1 I.U. and sufficient of asuitable liquid to bring the total volume to 5.0 ml. Allow the mixtures to stand at room temperature,protected from light, for 60 min. Using six mice for each mixture, inject into each mousesubcutaneously a dose of 0.5 ml. Observe the mice for 96 h. Mice that become paralysed may bekilled.

The mixture that contains the largest volume of antitoxin that fails to protect the mice fromparalysis contains 1 I.U. This quantity is used to calculate the potency of the antitoxin in Interna-tional Units per millilitre.

The test is not valid unless all the mice injected with mixtures containing 2.0 ml or less of thesolution of the reference preparation show paralysis and all those injected with mixtures containingmore do not.

STORAGE


LABELLING

See Immunosera for human use (84).__________________________________________________________________________________________________________ Ph Eur

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VACCINES

A vaccine for human use that is the subject of an individual monograph in the European Pharmacopoeia or inthe British Pharmacopoeia complies with the requirements of the 3rd edition of the European Pharmacopoeia forVaccines for Human Use [0153]. These requirements are reproduced below.

The provisions of this monograph apply to the following vaccines:

Bacterial vaccinesBacillus Calmette-Guérin Vaccine*Percutaneous Bacillus Calmette-Guérin VaccineCholera Vaccine*Haemophilus Type B Conjugate Vaccine*Meningococcal Polysaccharide Vaccine*Pertussis Vaccine*Pertussis Vaccine (Acellular Component), Adsorbed*Pneumococcal Polysaccharide Vaccine*Tetanus VaccineTyphoid (Strain Ty 21a) Vaccine, Live (Oral)*Typhoid Polysaccharide Vaccine*Typhoid Vaccine*

Bacterial toxoidsAdsorbed Diphtheria Vaccine*Adsorbed Diphtheria Vaccine for Adults and Adolescents*Adsorbed Tetanus Vaccine*

Viral vaccinesInactivated Hepatitis A Vaccine*Hepatitis B Vaccine (rDNA)*Inactivated Influenza Vaccine (Whole Virion)*Inactivated Influenza Vaccine (Split Virion)*Inactivated Influenza Vaccine (Surface Antigen)*Measles Vaccine, Live*Mumps Vaccine, Live*Inactivated Poliomyelitis Vaccine*Poliomyelitis Vaccine, Live (Oral)*Rabies Vaccine*Rubella Vaccine, Live*Tick-borne Encephalitis Vaccine, Inactivated*Varicella Vaccine Live*Yellow Fever Vaccine*

Mixed VaccinesAdsorbed Diphtheria and Tetanus Vaccine*Adsorbed Diphtheria and Tetanus Vaccine for Adults and Adolescents*Adsorbed Diphtheria, Tetanus and Pertussis Vaccine*Hepatitis A (Inactivated) and Hepatitis B (rDNA) Vaccine, Adsorbed*Measles, Mumps and Rubella Vaccine, Live**Monograph of the European Pharmacopoeia

Ph Eur _______________________________________________________________________________________________________________

The statements in this monograph are intended to be read in conjunction with the monographs on vaccines forhuman use in the Pharmacopoeia. The requirements do not necessarily apply to vaccines which are not thesubject of such monographs. For a combined vaccine, where there is no monograph to cover a particularcombination, the vaccine complies with the monograph for each individual component, with any necessarymodifications approved by the competent authority.

DEFINITION

Vaccines for human use are preparations containing antigenic substances capable of inducing aspecific and active immunity in man against an infecting agent or the toxin or the antigen elaboratedby it. They shall have been shown to have acceptable immunogenic activity in man with the intendedvaccination schedule.

Vaccines for human use may contain: organisms inactivated by chemical or physical means thatmaintain adequate immunogenic properties; living organisms that are naturally avirulent or that havebeen treated to attenuate their virulence whilst retaining adequate immunogenic properties; antigensextracted from the organisms or secreted by them or produced by recombinant DNA technology; theantigens may be used in their native state or may be detoxified by chemical or physical means and

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may be aggregated, polymerised or conjugated to a carrier to increase their immunogenicity.Terminology used in monographs on vaccines for human use is defined in chapter 5.2.1.

Bacterial vaccines are suspensions of various degrees of opacity in colourless or almost colourlessliquids, or may be freeze-dried. The concentration of living or inactivated bacteria is expressed interms of International Units of Opacity or, where appropriate, is determined by direct cell count or,for living bacteria, by viable count.

Bacterial toxoids are prepared from toxins by diminishing their toxicity to a non-detectable level orby completely eliminating it by physical or chemical procedures whilst retaining adequateimmunogenic properties. The toxins are obtained from selected strains of micro-organisms. Themethod of production is such that the toxoid does not revert to toxin. Toxoids may be liquid orfreeze-dried. They may be purified and adsorbed. Adsorbed toxoids are suspensions of white or greyparticles dispersed in colourless or pale yellow liquids and may form a sediment at the bottom of thecontainer.

Viral vaccines are prepared from viruses grown in animals, in fertilised eggs, in suitable cell culturesor in suitable tissues or by culture of genetically engineered cells. They are liquids that vary in opacityaccording to the type of preparation or may be freeze-dried. Liquid preparations and freeze-driedpreparations after reconstitution may be coloured if a pH indicator such as phenol red has been usedin the culture medium.

PRODUCTION

General provisions Requirements for production including in-process testing are included inindividual monographs. Where justified and authorised, certain tests may be omitted where it can bedemonstrated, for example by validation studies, that the production process consistently ensurescompliance with the test.

Unless otherwise justified and authorised, vaccines are produced using a seed-lot system. Themethods of preparation are designed to maintain adequate immunogenic properties, to render thepreparation harmless and to prevent contamination with extraneous agents.

Vaccines produced by recombinant DNA technology comply with the monograph Products ofrecombinant DNA technology (0784).

Unless otherwise justified and authorised, in the production of a final lot of vaccine, the number ofpassages of a virus, or the number of subcultures of a bacterium, from the master seed lot shall notexceed that used for production of the vaccine shown in clinical studies to be satisfactory with respectto safety and efficacy.

Vaccines are as far as possible free from ingredients known to cause toxic, allergic or otherundesirable reactions in man. Suitable additives, including stabilisers and adjuvants may beincorporated. Penicillin and streptomycin are not used at any stage of production nor added to thefinal product; however, master seed lots prepared with media containing penicillin or streptomycinmay, where justified and authorised, be used for production.

Where applicable, substances used in the production of vaccines for human use comply with themonograph on Products with risk of transmitting agents of animal spongiform encephalopathies (1483).

Substrates for propagation Substrates for propagation comply with the relevant requirements ofthe Pharmacopoeia (5.2.2, 5.2.3) or in the absence of such requirements with those of the competentauthority. Processing of cell banks and subsequent cell cultures is done under aseptic conditions in anarea where no other cells are being handled. Serum and trypsin used in the preparation of cellsuspensions shall be shown to be free from extraneous agents.

Seed lots The strain of bacterium or virus used in a master seed lot is identified by historical recordsthat include information on the origin of the strain and its subsequent manipulation. No micro-organism other than the seed strain shall be present in a seed lot.

Culture media Culture media are as far as possible free from ingredients known to cause toxic,allergic or other undesirable reactions in man; if inclusion of such ingredients is necessary, it shall bedemonstrated that the amount present in the final lot is reduced to such a level as to render theproduct safe. Approved animal (but not human) serum may be used in the growth medium for cellcultures but the medium used for maintaining cell growth during virus multiplication shall notcontain serum, unless otherwise stated. Cell culture media may contain a pH indicator such asphenol red and approved antibiotics at the lowest effective concentration although it is preferable tohave a medium free from antibiotics during production.

Propagation and harvest The seed cultures are propagated and harvested under defined condi-tions. The purity of the harvest is verified by suitable tests as defined in the monograph.

Control cells For vaccines produced in cell cultures, control cells are maintained and tested asprescribed. In order to provide a valid control, these cells must be maintained in conditions that arerigorously identical with those used for the production cell cultures, including use of the samebatches of media and media changes.

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

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Purification Where applicable, validated purification procedures may be applied.

Inactivation Inactivated vaccines are produced using a validated inactivation process whoseeffectiveness and consistency have been demonstrated. Where there are recognised potentialcontaminants of a harvest, for example in vaccines produced in eggs from healthy, non-SPF flocks,the inactivation process is also validated with respect to the potential contaminants. A test forinactivation is carried out as soon as possible after the inactivation process, unless otherwise justifiedand authorised.

Intermediates Where applicable, the stability of intermediates in given storage conditions shall beevaluated and a period of validity established.

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

Adsorbents. Vaccines may be adsorbed on aluminium hydroxide, aluminium phosphate, calciumphosphate or other suitable adsorbent; the adsorbents are prepared in special conditions which conferthe appropriate physical form and adsorptive properties.

Antimicrobial preservatives. A suitable antimicrobial preservative may be included in sterile andinactivated vaccines and is invariably added if these preparations are issued in multidose containers,unless otherwise stated. If an antimicrobial preservative is used, it shall be shown that it does notimpair the safety or efficacy of the vaccine.

During development studies, the effectiveness of the antimicrobial preservative throughout theperiod of validity shall be demonstrated to the satisfaction of the competent authority.

The efficacy of the antimicrobial preservative is evaluated as described in chapter 5.1.3. If neitherthe A criteria nor the B criteria can be met, then in justified cases the following criteria are applied tovaccines for human use: bacteria, no increase at 24 h and 7 days, 3 log reduction at 14 days, noincrease at 28 days; fungi, no increase at 14 days and 28 days.

Final lot For vaccines for parenteral administration, the final lot is prepared by aseptically distribut-ing the final bulk into sterile tamper-proof containers which, after freeze-drying where applicable, areclosed so as to exclude contamination. For vaccines for administration by a non-parenteral route, thefinal lot is prepared by distributing the final bulk under suitable conditions into sterile, tamper-proofcontainers.

Stability. Maintenance of potency of the final lot throughout the period of validity shall be demon-strated by validation studies; the loss of potency in the recommended storage conditions is assessedand excessive loss even within the limits of acceptable potency may indicate that the vaccine isunacceptable.

Degree of adsorption. During development of an adsorbed vaccine, the degree of adsorption isevaluated as part of the consistency testing. A release specification for the degree of adsorption isestablished in the light of results found for batches used in clinical testing. From the stability datagenerated for the vaccine it must be shown that at the end of the period of validity the degree ofadsorption will not be less than for batches used in clinical testing.

TESTS

Vaccines comply with the tests prescribed in the particular monograph including, where applicable,the following:

Aluminium (2.5.13). Where an aluminium adsorbent has been used in the vaccine, not more than1.25 mg of aluminium (Al) per single human dose, unless otherwise stated.

Calcium (2.5.14). Where a calcium adsorbent has been used in the vaccine, not more than 1.3 mgof calcium (Ca) per single human dose, unless otherwise stated.

Formaldehyde (2.4.18). Where formaldehyde has been used in the preparation of the vaccine, notmore than 0.2 g/l of free formaldehyde is present in the final product, unless otherwise stated.

Phenol (2.5.15). Where phenol has been used in the preparation of the vaccine, not more than2.5 g/l is present in the final product, unless otherwise stated.

Water (2.5.12). For freeze-dried vaccines, not more than 3.0 per cent m/m, unless otherwise stated.

STORAGE

Store protected from light. Unless otherwise stated, the storage temperature is 5 ± 3°C; liquidadsorbed vaccines must not be allowed to freeze.

Expiry date. Unless otherwise stated, the expiry date is calculated from the beginning of the assay. Itapplies to vaccines stored in the prescribed conditions.

LABELLING

The label states:— the name of the preparation,— a reference identifying the final lot,— the recommended human dose and route of administration,

68-4

— the storage conditions,— the expiry date,— the name and amount of any antimicrobial preservative,— the name of any antibiotic, adjuvant, flavour or stabiliser present in the vaccine,— the name of any constituent that may cause adverse reactions and any contra-indications to the

use of the vaccine,— for freeze-dried vaccines:

–the name or composition and the volume of the reconstituting liquid to be added,

–the time within which the vaccine is to be used after reconstitution.__________________________________________________________________________________________________________ Ph Eur

68-5

Bacillus Calmette-Guérin VaccineBCG Vaccine

Bacillus Calmette-Guérin Vaccine complies with the requirements of the 3rd edition of the European Pharma-copoeia for Freeze-dried BCG Vaccine [0163]. These requirements are reproduced after the heading ‘Defini-tion’ below.

The label may state ‘Dried/Tub/Vac/BCG’.

Ph Eur ___________________________________________________________________________________________________________

DEFINITION

Freeze-dried BCG vaccine is a preparation of live bacteria derived from a culture of the bacillus ofCalmette and Guérin (Mycobacterium bovis BCG) whose capacity to protect against tuberculosis hasbeen established.

PRODUCTION

BCG vaccine shall be produced by a staff consisting of healthy persons who do not work with otherinfectious agents; in particular they shall not work with virulent strains of Mycobacterium tuberculosis,nor shall they be exposed to a known risk of tuberculosis infection. BCG vaccine is susceptible tosunlight: the procedures for the preparation of the vaccine shall be so designed that all cultures andvaccines are protected from direct sunlight and from ultraviolet light at all stages of manufacture,testing and storage.

Production of the vaccine is based on a seed-lot system. The production method shall have beenshown to yield consistently BCG vaccines that induce adequate sensitivity to tuberculin in man, thathave acceptable protective potency in animals and are safe. The vaccine is prepared from cultureswhich are separated from the master seed lot by as few subcultures as possible and in any case notmore than eight subcultures. During the course of these subcultures the preparation is not freeze-dried more than once.

BACTERIAL SEED LOTS

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

A suitable batch of vaccine is prepared from the first working seed lot and is reserved for use as thecomparison vaccine. When a new working seed lot is established, a suitable test for delayedhypersensitivity in guinea-pigs is carried out on a batch of vaccine prepared from the new workingseed lot; the vaccine is shown to be not significantly different in activity from the comparison vaccine.

Only a working seed lot that complies with the following requirements may be used for propaga-tion.

Identification The bacteria in the working seed lot are identified as Mycobacterium bovis BCG.

Bacterial and fungal contamination Carry out the test for sterility (2.6.1), using 10 ml for eachmedium. The working seed lot complies with the test for sterility except for the presence ofmycobacteria.

Virulent mycobacteria Examine the working seed lot as prescribed under Tests, using ten guinea-pigs.

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

PROPAGATION AND HARVEST

The bacteria are grown in a suitable medium for not more than 21 days by surface or submergedculture. The culture medium shall contain no substances known to cause toxic or allergic reactions inhuman beings or to cause the bacteria to become virulent for guinea-pigs. The culture is harvestedand suspended in a sterile liquid medium that protects the viability of the vaccine as determined by asuitable method of viable count.

FINAL BULK VACCINE

The final bulk vaccine is prepared from a single harvest or by pooling a number of single harvests. Astabiliser may be added; if the stabiliser interferes with the determination of bacterial concentrationon the final bulk vaccine, the determination is carried out before addition of the stabiliser.

Only final bulk vaccine that complies with the following requirements may be used in the prepara-tion of the final lot.

Virulent mycobacteria Examine as prescribed under Tests.

Bacterial and fungal contamination. Carry out the test for sterility (2.6.1), using 10 ml for eachmedium. The final bulk vaccine complies with the test for sterility except for the presence ofmycobacteria.

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Count of viable units Determine the number of viable units per millilitre by viable count on solidmedium using a method suitable for the vaccine to be examined or by determination of adenosinetriphosphate by a bioluminescence reaction. Carry out the test in parallel on a reference preparationof the same strain.

Bacterial concentration Determine the total bacterial concentration by a suitable method, eitherdirectly by determining the mass of the micro-organisms, or indirectly by an opacity method that hasbeen calibrated in relation to the mass of the organisms; if the bacterial concentration is determinedbefore addition of a stabiliser, the concentration in the final bulk vaccine is established by calculation.The total bacterial concentration is within the limits approved for the particular product.

The ratio of the count of viable units to the total bacterial concentration is not less than thatapproved for the particular product.

FINAL LOT

The final bulk vaccine is distributed into sterile containers and freeze-dried to a moisture contentfavourable to the stability of the vaccine; the containers are closed either under vacuum or under agas that is not deleterious to the vaccine.

Except where the filled and closed containers are stored at a temperature of –20°C or lower, theexpiry date is not later than 4 years from the date of harvest.

Only a final lot that complies with the following requirement for count of viable units and witheach of the requirements given below under Identification, Tests and Assay may be released for use.Provided the test for virulent mycobacteria has been carried out with satisfactory results on the finalbulk vaccine, it may be omitted on the final lot. Provided the test for excessive dermal reactivity hasbeen carried out with satisfactory results on the working seed lot and on five consecutive final lotsproduced from it, the test may be omitted on the final lot.

Count of viable units Determine the number of viable units per millilitre of the reconstitutedvaccine by viable count on solid medium using a method suitable for the vaccine to be examined orby determination of adenosine triphosphate by a bioluminescence reaction. The ratio of the count ofviable units after freeze-drying to that before is not less than that approved for the particular product.

IDENTIFICATION

BCG vaccine is identified by microscopic examination of the bacilli in stained smears demonstratingtheir acid-fast property and by the characteristic appearance of colonies grown on solid medium.

TESTS

Virulent mycobacteria Inject subcutaneously or intramuscularly into each of six guinea-pigs, eachweighing 250 g to 400 g and having received no treatment likely to interfere with the test, a quantityof vaccine equivalent to at least fifty human doses. Observe the animals for at least 42 days. At theend of this period, kill the guinea-pigs and examine by autopsy for signs of infection with tubercu-losis, ignoring any minor reactions at the site of injection. Animals that die during the observationperiod are also examined for signs of tuberculosis. The vaccine complies with the test if none of theguinea-pigs shows signs of tuberculosis and if not more than one animal dies during the observationperiod. If two animals die during this period and autopsy does not reveal signs of tuberculosis repeatthe test on six other guinea-pigs. The vaccine complies with the test if not more than one animal diesduring the 42 days following the injection and autopsy does not reveal any sign of tuberculosis.

Bacterial and fungal contamination The reconstituted vaccine complies with the test for sterility(2.6.1) except for the presence of mycobacteria.

Excessive dermal reactivity Use six healthy white or pale-coloured guinea-pigs, each weighing notless than 250 g and having received no treatment likely to interfere with the test. Inject intradermallyinto each guinea-pig, according to a randomised plan, 0.1 ml of the reconstituted vaccine and of twotenfold serial dilutions of the vaccine and identical doses of the comparison vaccine. Observe thelesions formed at the site of the injection for 4 weeks. The vaccine complies with the test if thereaction it produces is not markedly different from that produced by the comparison vaccine.

Temperature stability Maintain samples of the freeze-dried vaccine at 37°C for 4 weeks.Determine the number of viable units in the heated vaccine and in unheated vaccine as describedbelow. The number of viable units in the heated vaccine is not less than 20 per cent that in unheatedvaccine.

Water (2.5.12). Not more than 3.0 per cent, determined by the semi-micro determination of water.

ASSAY

Determine the number of viable units in the reconstituted vaccine by viable count on solid mediumusing a method suitable for the vaccine to be examined. The number is within the range stated on thelabel. Determine the number of viable units in the comparison vaccine in parallel.

STORAGE

See Vaccines for human use (153).

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LABELLING


The label states:— the minimum and maximum number of viable units per millilitre in the reconstituted vaccine,— that the vaccine must be protected from direct sunlight,— that the vaccine is to be used immediately after broaching the container and any residue is to be

discarded,— the age group for which the vaccine is intended,— the dose for each age group.__________________________________________________________________________________________________________ Ph Eur

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Percutaneous Bacillus Calmette-Guérin VaccinePercut. BCG Vaccine

Definition

Percutaneous Bacillus Calmette-Guérin Vaccine is a suspension of living cells of an authentic strainof the bacillus of Calmette and Guérin with a higher viable bacterial count than Bacillus Calmette-Guérin Vaccine. It is prepared immediately before use by reconstitution from the dried vaccine withan appropriate volume of a suitable sterile liquid.

Production

General considerations Percutaneous Bacillus Calmette-Guérin Vaccine shall be produced by astaff consisting of healthy persons who do not work with other infectious agents; in particular theyshall not work with virulent strains of Mycobacterium tuberculosis, nor shall they be exposed to aknown risk of tuberculosis infection. Percutaneous Bacillus Calmette-Guérin Vaccine is susceptibleto sunlight: the procedures for the preparation of the vaccine shall be so designed that all cultures andvaccines are protected from direct sunlight and from ultraviolet light at all stages of manufacture,testing and storage.

Production of the vaccine is based on a seed-lot system. The production method shall have beenshown to yield consistently BCG vaccines that induce adequate sensitivity to tuberculin in man, thathave acceptable protective potency in animals and are safe. The vaccine is prepared from culturesthat are separated from the master seed lot by as few subcultures as possible and in any case not morethan eight subcultures. During the course of these subcultures the preparation is not freeze driedmore than once.

Bacterial seed lots The strain used to establish the master seed lot is chosen for and maintained topreserve its stability, its capacity to sensitise man and guinea-pigs to tuberculin and to protect animalsagainst tuberculosis, and its relative absence of pathogenicity for man and laboratory animals. Thestrain used shall be identified by historical records that include information on its origin andsubsequent manipulation. A suitable batch of vaccine is prepared from the first working seed lot andis reserved for use as the comparison vaccine. When a new working seed lot is established, a suitabletest for delayed hypersensitivity in guinea-pigs is carried out on a batch of vaccine prepared from thenew working seed lot; the vaccine is shown to be not significantly different in activity from thecomparison vaccine. Only a working seed lot that complies with the following requirements may beused for propagation.

Identification The bacteria in the working seed lot are identified as Mycobacterium bovis BCG.

Bacterial and fungal contamination Carry out the test for sterility, Appendix XVI A, using 10 ml for eachmedium. The working seed lot complies with the test for sterility except for the presence ofmycobacteria.

Virulent mycobacteria Carry out the test as described below.

Sensitisation of guinea-pigs The capacity of the working seed lot to induce sensitivity to tuberculin inguinea-pigs is demonstrated.

Propagation and harvest The bacteria are grown in a suitable medium for not more than 21 daysby surface or submerged culture. The culture medium shall contain no substances known to causetoxic or allergic reactions in human beings or to cause the bacteria to become virulent for guinea-pigs.The culture is harvested and suspended in a sterile liquid medium that protects the viability of thevaccine as determined by a suitable method of viable count.

Final bulk vaccine The final bulk vaccine is prepared from a single harvest or by pooling a numberof single harvests. A stabiliser may be added; if the stabiliser interferes with the determination ofbacterial concentration on the final bulk vaccine, the determination is carried out before addition ofthe stabiliser.


Virulent mycobacteria Carry out the test as described below.

Bacterial and fungal contamination Carry out the test for sterility, Appendix XVI A, using 10 ml for eachmedium. The final bulk vaccine complies with the test for sterility except for the presence ofmycobacteria.

Count of viable units Determine the number of viable units per millilitre by viable count on solidmedium using a method suitable for the vaccine to be examined or by determination of adenosinetriphosphate by a bioluminescence reaction. Carry out the test in parallel on a reference preparationof the same strain.

Bacterial concentration Determine the total bacterial concentration by a suitable method, eitherdirectly by determining the mass of the micro-organisms, or indirectly by an opacity method that hasbeen calibrated in relation to the mass of the organisms; if the bacterial concentration is determined

68-9

before addition of a stabiliser, the concentration in the final bulk vaccine is established by calcula-tion. The total bacterial concentration is within the limits approved for the particular product.

The ratio of the count of viable units to the total bacterial concentration is not less than thatapproved for the particular product.

Final lot The final bulk vaccine is distributed into sterile containers and freeze dried to a moisturecontent favourable to the stability of the vaccine; the containers are closed either under vacuum orunder a gas that is not deleterious to the vaccine.

Except where the filled and closed containers are stored at a temperature of –20° or lower, theexpiry date is not later than 4 years from the date of harvest.

Only a final lot that complies with the following requirement for count of viable units and witheach of the requirements given below may be released for use. Provided the test for Virulentmycobacteria has been carried out with satisfactory results on the final bulk vaccine, it may beomitted on the final lot. Provided the test for excessive dermal reactivity has been carried out withsatisfactory results on the working seed lot and on five consecutive final lots produced from it, thetest may be omitted on the final lot.

Count of viable units Determine the number of viable units per millilitre of the reconstituted vaccineby viable count on solid medium using a method suitable for the vaccine to be examined or bydetermination of adenosine triphosphate by a bioluminescence reaction. The ratio of the count ofviable units after freeze drying to that before is not less than that approved for the particular product.

IdentificationA. When examined microscopically in stained smears, the bacilli exhibit the characteristics of anauthentic strain of the bacillus of Calmette and Guérin.

B. Colonies grown on a suitable solid culture medium have a characteristic appearance.

The vaccine complies with the requirements stated under Vaccines, with the following modifications.

Skin-sensitising potency Prepare a 25-fold dilution of the vaccine using an appropriate sterileliquid. Inject 0.5 ml subcutaneously or intramuscularly into each of two guinea-pigs. Within 4 weeksof injection, inject intracutaneously into each guinea-pig 10 IU of Old Tuberculin, or of TuberculinPurified Protein Derivative, in a volume of 0.1 ml. An inflammatory area of induration and oedemanot less than 5 mm in diameter, irrespective of the area of erythema, is induced within 24 hours.

Excessive dermal reactivity Inject intracutaneously into each of two guinea-pigs, in a volume of0.1 ml, 1, 0.1, 0.01 and 0.001 doses of the vaccine being tested and of the comparison vaccine. Usean appropriate sterile liquid as diluent. The vaccine passes the test if the skin reactions producedwithin 3 weeks do not differ markedly from those produced by the comparison vaccine.

Sterility Complies with the test for sterility, Appendix XVI A, except for the presence ofmycobacteria.

Virulent mycobacteria Prepare a 5-fold dilution of the vaccine using an appropriate sterile liquid.Inject 1 ml intramuscularly into each of six guinea-pigs weighing 250 to 400 g. None of the animalsdies within 42 days or if one dies, a post-mortem examination establishes that it is free fromtuberculosis. If two of the animals die within this period and a post-mortem examination establishesthat both are free from tuberculosis, repeat the test on six further guinea-pigs. None of the secondgroup of animals dies within 42 days or, if one dies, a post mortem examination establishes that it isfree from tuberculosis.

Water Not more than 3.0 %, Appendix IX C.

Storage The vaccine should be protected from light and stored at a temperature below –20°. Thereconstituted vaccine should be used immediately.

Labelling The label states (1) ‘Tub/Vac/BCG (Perc)’; (2) that the vaccine is a living culture of thebacillus of Calmette and Guérin; (3) that any portion of the reconstituted vaccine not used at onceshould be discarded; (4) that the vaccine is for percutaneous administration and must not be given bythe intradermal route.

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Cholera Vaccine

When Cholera Vaccine is issued as a liquid, it complies with the requirements of the 3rd edition of the EuropeanPharmacopoeia for Cholera Vaccine [0154]. These requirements are reproduced after the heading ‘LiquidVaccine’ below.

When Cholera Vaccine is prepared immediately before use by reconstitution from the dried vaccine, the driedvaccine complies with the requirements of the 3rd edition of the European Pharmacopoeia for Freeze-driedCholera Vaccine [0155]. These requirements are reproduced after the heading ‘Dried Vaccine’ below.

The label may state ‘Cho/Vac’ or ‘Dried/Cho/Vac’, as appropriate.

Ph Eur ___________________________________________________________________________________________________________

LIQUID VACCINE [0154]

DEFINITION

Cholera vaccine is a homogeneous suspension of a suitable strain or strains of Vibrio cholerae contain-ing not less than 8 × 109 bacteria in each human dose. The human dose does not exceed 1.0 ml.

PRODUCTION

The vaccine is prepared using a seed-lot system.The vaccine consists of a mixture of equal parts of vaccines prepared from smooth strains of the

two main serological types, Inaba and Ogawa. These may be of the classical biotype with or withoutthe El-Tor biotype. A single strain or several strains of each type may be included. All strains mustcontain, in addition to their type O antigens, the heat-stable O antigen common to Inaba and Ogawa.If more than one strain each of Inaba and Ogawa are used, these may be selected so as to containother O antigens in addition. The World Health Organisation recommends new strains which may beused if necessary, in accordance with the regulations in force in the signatory States of the Conven-tion on the Elaboration of a European Pharmacopoeia. In order to comply with the requirements forvaccination certificates required for international travel, the vaccine must contain not less than 8 ×109 organisms of the classical biotype.

Each strain is grown separately. The bacteria are inactivated either by heating the suspensions (forexample, at 56°C for 1 h) or by treatment with formaldehyde or phenol or by a combination of thephysical and chemical methods.

The production method is validated to demonstrate that the product, if tested, would comply withthe test for abnormal toxicity for immunosera and vaccines for human use (2.6.9) modified asfollows: inject 0.5 ml of the vaccine into each mouse and 1.0 ml into each guinea-pig.

IDENTIFICATION

It is identified by specific agglutination tests.

TESTS

Phenol (2.5.15). If phenol has been used in the preparation, the concentration is not more than 5 g/l.

Antibody production Test the ability of the vaccine to induce antibodies (such as agglutinating,vibriocidal or haemagglutinating antibodies) in the guinea-pig, the rabbit or the mouse. Administerthe vaccine to a group of at least six animals. At the end of the interval of time necessary for maxi-mum antibody formation, determined in preliminary tests, collect sera from the animals and titratethem individually for the appropriate antibody using a suitable method. The vaccine to be examinedpasses the test if each serotype has elicited a significant antibody response.

Sterility (2.6.1). It complies with the test for sterility.

STORAGE


LABELLING


The label states:— the method used to inactivate the bacteria,— the number of bacteria in each human dose.

DRIED VACCINE [0155]

DEFINITION

Freeze-dried cholera vaccine is a preparation of a suitable strain or strains of Vibrio cholerae. Thevaccine is reconstituted as stated on the label to give a uniform suspension containing not less than

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8 × 109 bacteria in each human dose. The human dose does not exceed 1.0 ml of the reconstitutedvaccine.

PRODUCTION

The vaccine is prepared using a seed-lot system.The vaccine consists of a mixture of equal parts of vaccines prepared from smooth strains of the two

main serological types, Inaba and Ogawa. These may be of the classical biotype with or without the El-Tor biotype. A single strain or several strains of each type may be included. All strains must contain, inaddition to their type O antigens, the heat-stable O antigen common to Inaba and Ogawa. If more thanone strain each of Inaba and Ogawa are used, these may be selected so as to contain other O antigens inaddition. The World Health Organisation recommends new strains which may be used if necessary inaccordance with the regulations in force in the signatory States of the Convention on the Elaboration ofa European Pharmacopoeia. In order to comply with the requirements for vaccination certificatesrequired for international travel, the vaccine must contain not less than 8 × 109 organisms of theclassical biotype.

Each strain is grown separately. The bacteria are inactivated either by heating the suspensions (forexample, at 56°C for 1 h) or by treatment with formaldehyde or by a combination of the physical andchemical methods. Phenol is not used in the preparation. The vaccine is distributed into sterilecontainers and freeze-dried to a moisture content favourable to the stability of the vaccine. Thecontainers are then closed so as to exclude contamination.

The production method is validated to demonstrate that the product, if tested, would comply withthe test for abnormal toxicity for immunosera and vaccines for human use (2.6.9) modified as follows:inject 0.5 ml of the vaccine into each mouse and 1.0 ml into each guinea-pig.

IDENTIFICATION

The vaccine reconstituted as stated on the label is identified by specific agglutination tests.

TESTS

The reconstituted vaccine complies with the tests prescribed in the monograph on Cholera vaccine(154).

STORAGE


LABELLING

See Vaccines for human use (153).The label states:

— the method used to inactivate the bacteria,— the number of bacteria in each human dose.__________________________________________________________________________________________________________ Ph Eur

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Adsorbed Diphtheria VaccineAdsorbed Diphtheria Prophylactic

Adsorbed Diphtheria Vaccine complies with the requirements of the 3rd edition of the European Pharmaco-poeia for Diphtheria Vaccine (Adsorbed) [0443]. These requirements are reproduced after the heading‘Definition’ below.

The label may state ‘Dip/Vac/Ads(Child)’.

Ph Eur ___________________________________________________________________________________________________________

DEFINITION

Diphtheria vaccine (adsorbed) is a preparation of diphtheria formol toxoid adsorbed on a mineralcarrier. The formol toxoid is prepared from the toxin produced by the growth of Corynebacteriumdiphtheriae.

PRODUCTION

BULK PURIFIED TOXOID

For the production of diphtheria toxin, from which toxoid is prepared, seed cultures are managed ina defined seed-lot system in which toxinogenicity is conserved and, where necessary, restored bydeliberate reselection. A highly toxinogenic strain of Corynebacterium diphtheriae with known originand history is grown in a suitable liquid medium. At the end of cultivation, the purity of each cultureis tested and contaminated cultures are discarded. Toxin-containing culture medium is separatedaseptically from the bacterial mass as soon as possible. The toxin content (Lf per millilitre) is checkedto monitor consistency of production. Single harvests may be pooled to prepare the bulk purifiedtoxoid. The toxin is purified to remove components likely to cause adverse reactions in humans. Thepurified toxin is detoxified with formaldehyde by a method that avoids destruction of the immuno-genic potency of the toxoid and reversion of the toxoid to toxin, particularly on exposure to heat.Alternatively, purification may be carried out after detoxification.

Only bulk purified toxoid that complies with the following requirements may be used in the prepa-ration of the final bulk vaccine.

Sterility (2.6.1). Carry out the test for sterility using 10 ml for each medium.

Absence of diphtheria toxin Inject subcutaneously at least 500 Lf of purified toxoid in a volume of1 ml into each of five healthy guinea-pigs, each weighing 250 g to 350 g, that have not previouslybeen treated with any material that will interfere with the test. If within 42 days of the injection any ofthe animals shows signs of or dies from diphtheria toxaemia, the toxoid does not comply with thetest. If more than one animal dies from non-specific causes, repeat the test once; if more than oneanimal dies in the second test, the toxoid does not comply with the test.

Irreversibility of toxoid Using the buffer for the final vaccine without adsorbent, prepare a dilutionof the bulk purified toxoid containing the same toxoid concentration as the final vaccine. Divide thedilution into two equal parts. Keep one of them at 5 ± 3°C and the other at 37°C for 6 weeks. Testboth samples by a suitable sensitive assay for active diphtheria toxin such as inoculation on to cellcultures or intradermal injection into guinea-pigs. The toxoid complies with the test if neither sampleproduces any sign of a toxic reaction attributable to diphtheria toxin.

Antigenic purity Not less than 1500 Lf per milligram of protein nitrogen.

FINAL BULK VACCINE

The final bulk vaccine is prepared by adsorption of a suitable quantity of bulk purified toxoid ontohydrated aluminium phosphate, aluminium hydroxide or calcium phosphate; the resulting mixture isapproximately isotonic with blood. Suitable antimicrobial preservatives may be added. Certain anti-microbial preservatives, particularly those of the phenolic type, adversely affect the antigenic activityand must not be used.


Antimicrobial preservative Where applicable, determine the amount of antimicrobial preservativeby a suitable chemical method. The amount is not less than 85 per cent and not greater than 115 percent of the intended amount.


Potency Carry out the test described under Assay.

FINAL LOT

The final bulk vaccine is distributed aseptically into sterile, tamper-proof containers. The containersare closed so as to prevent contamination.

Only a final lot that is satisfactory with respect to each of the requirements given below under

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Identification, Tests and Assay may be released for use. Provided the tests for specific toxicity, freeformaldehyde and antimicrobial preservative and the assay have been carried out with satisfactory resultson the final bulk vaccine, they may be omitted on the final lot.

IDENTIFICATION

Dissolve in the vaccine to be examined sufficient sodium citrate R to give a 100 g/l solution. Maintain at37°C for about 16 h and centrifuge until a clear supernatant liquid is obtained. The clear supernatantliquid reacts with a suitable diphtheria antitoxin, giving a precipitate.

TESTS

Specific toxicity Inject subcutaneously five times the single human dose stated on the label into each offive healthy guinea-pigs, each weighing 250 g to 350 g, that have not previously been treated with anymaterial that will interfere with the test. If within 42 days of the injection any of the animals shows signsof or dies from diphtheria toxaemia, the vaccine does not comply with the test. If more than one animaldies from non-specific causes, repeat the test once; if more than one animal dies in the second test, thevaccine does not comply with the test.

Aluminium When hydrated aluminium phosphate or aluminium hydroxide is used as the adsorbent, thevaccine complies with the test prescribed in the monograph on Vaccines for human use (153).

Calcium When calcium phosphate is used as the adsorbent, the vaccine complies with the testprescribed in the monograph on Vaccines for human use (153).

Free formaldehyde The vaccine complies with the test prescribed in the monograph on Vaccines forhuman use (153).

Antimicrobial preservative Where applicable, determine the amount of antimicrobial preservative by asuitable chemical method. The content is not less than the minimum amount shown to be effective and isnot greater than 115 per cent of the quantity stated on the label.

Sterility (2.6.1). The vaccine complies with the test for sterility.

ASSAY

Carry out one of the prescribed methods for the assay of diphtheria vaccine (adsorbed) (2.7.6).The lower confidence limit (P = 0.95) of the estimated potency is not less than 30 I.U. per single

human dose.

STORAGE


LABELLING


— the minimum number of International Units per single human dose,— where applicable, that the vaccine is intended for primary vaccination of children and is not necessarily

suitable for reinforcing doses or for administration to adults,— the name and the amount of the adsorbent,— that the vaccine must be shaken before use,— that the vaccine is not to be frozen.__________________________________________________________________________________________________________ Ph Eur

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Adsorbed Diphtheria Vaccine for Adults and Adolescents

Adsorbed Diphtheria Vaccine for Adults and Adolescents complies with the requirements of the 3rd edition ofthe European Pharmacopoeia for Diphtheria Vaccine (Adsorbed) for Adults and Adolescents [0646]. Theserequirements are reproduced after the heading ‘Definition’ below..

The label may state ‘Dip/Vac/Ads(Adult)’.

For a vaccine for use in the United Kingdom, the amount of toxoid used is adjusted so that the finalvaccine contains not more than 2.0 flocculation equivalents (2.0 Lf) per dose.

Ph Eur ___________________________________________________________________________________________________________

DEFINITION

Diphtheria vaccine (adsorbed) for adults and adolescents is a preparation of diphtheria formol toxoidadsorbed on a mineral carrier. The formol toxoid is prepared from the toxin produced by the growthof Corynebacterium diphtheriae. It shall have been demonstrated to the competent authority that thequantity of diphtheria toxoid used does not produce adverse reactions in subjects from the age groupsfor which the vaccine is intended.

PRODUCTION



Only bulk purified toxoid that complies with the following requirements may be used in thepreparation of the final bulk vaccine.





FINAL BULK VACCINE

The final bulk vaccine is prepared by adsorption of a suitable quantity of bulk purified toxoid ontohydrated aluminium phosphate, aluminium hydroxide or calcium phosphate; the resulting mixture isapproximately isotonic with blood. Suitable antimicrobial preservatives may be added. Certainantimicrobial preservatives, particularly those of the phenolic type, adversely affect the antigenicactivity and must not be used.




Potency Carry out the test described under Potency.

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FINAL LOT


Only a final lot that is satisfactory with respect to each of the requirements given below underIdentification, Tests and Potency may be released for use. Provided the tests for specific toxicity, freeformaldehyde and antimicrobial preservative and the determination of potency have been carried outwith satisfactory results on the final bulk vaccine, they may be omitted on the final lot.

IDENTIFICATION

Dissolve in the vaccine to be examined sufficient sodium citrate R to give a 100 g/l solution. Maintainat 37°C for about 16 h and centrifuge until a clear supernatant liquid is obtained. The clear super-natant liquid reacts with a suitable diphtheria antitoxin, giving a precipitate. If a satisfactory result isnot obtained with a vaccine adsorbed on aluminium hydroxide, carry out the test as follows. Centri-fuge 15 ml of the vaccine to be examined and suspend the residue in 5 ml of a freshly preparedmixture of 1 volume of a 56 g/l solution of sodium edetate R and 49 volumes of a 90 g/l solution ofdisodium hydrogen phosphate R. Maintain at 37°C for not less than 6 h and centrifuge. The clearsupernatant liquid reacts with a suitable diphtheria antitoxin, giving a precipitate.

TESTS

Specific toxicity Inject subcutaneously five times the single human dose stated on the label intoeach of five healthy guinea-pigs, each weighing 250 g to 350 g, that have not previously been treatedwith any material that will interfere with the test. If within 42 days of the injection any of the animalsshows signs of or dies from diphtheria toxaemia, the vaccine does not comply with the test. If morethan one animal dies from non-specific causes, repeat the test once; if more than one animal dies inthe second test, the vaccine does not comply with the test.

Aluminium When hydrated aluminium phosphate or aluminium hydroxide is used as the adsorbent,the vaccine complies with the test prescribed in the monograph on Vaccines for human use (153).



Antimicrobial preservative Where applicable, determine the amount of antimicrobial preservativeby a suitable chemical method. The content is not less than the minimum amount shown to beeffective and is not greater than 115 per cent of the quantity stated on the label.


ASSAY

Carry out one of the prescribed methods for the assay of diphtheria vaccine (adsorbed) (2.7.6).The lower confidence limit (P = 0.95) of the estimated potency is not less than 2 I.U. per single

human dose.

STORAGE


LABELLING


The label states:— the minimum number of International Units per single human dose,— the name and the amount of the adsorbent,— that the vaccine must be shaken before use,— that the vaccine is not to be frozen.

__________________________________________________________________________________________________________ Ph Eur

68-16

Adsorbed Diphtheria and Tetanus VaccineAdsorbed Diphtheria–Tetanus Prophylactic

Adsorbed Diphtheria and Tetanus Vaccine complies with the requirements of the 3rd edition of the EuropeanPharmacopoeia for Diphtheria and Tetanus Vaccine (Adsorbed).[0444]. These requirements are reproducedafter the heading ‘Definition’ below.

The label may state ‘DT/Vac/Ads(Child)’.

Ph Eur ___________________________________________________________________________________________________________

DEFINITION

Diphtheria and tetanus vaccine (adsorbed) is a preparation of diphtheria formol toxoid and tetanusformol toxoid adsorbed on a mineral carrier. The formol toxoids are prepared from the toxinsproduced by the growth of Corynebacterium diphtheriae and Clostridium tetani, respectively.

PRODUCTION

BULK PURIFIED DIPHTHERIA TOXOID







BULK PURIFIED TETANUS TOXOID

For the production of tetanus toxin, from which toxoid is prepared, seed cultures are managed in adefined seed-lot system in which toxinogenicity is conserved and, where necessary, restored bydeliberate reselection. A highly toxinogenic strain of Clostridium tetani with known origin and historyis grown in a suitable liquid medium. At the end of cultivation, the purity of each culture is testedand contaminated cultures are discarded. Toxin-containing culture medium is collected aseptically.The toxin content (Lf per millilitre) is checked to monitor consistency of production. Single harvestsmay be pooled to prepare the bulk purified toxoid. The toxin is purified to remove components likelyto cause adverse reactions in humans. The purified toxin is detoxified with formaldehyde by amethod that avoids destruction of the immunogenic potency of the toxoid and reversion of toxoid totoxin, particularly on exposure to heat. Alternatively, purification may be carried out after detoxifica-tion.



Absence of tetanus toxin Inject subcutaneously at least 500 Lf of purified toxoid in a volume of1 ml into each of five healthy guinea-pigs, each weighing 250 g to 350 g, that have not previouslybeen treated with any material that will interfere with the test. If within 21 days of the injection any ofthe animals shows signs of or dies from tetanus, the toxoid does not comply with the test. If more

68-17

than one animal dies from non-specific causes, repeat the test once; if more than one animal dies inthe second test, the toxoid does not comply with the test.

Irreversibility of toxoid Using the buffer for the final vaccine without adsorbent, prepare a dilutionof the bulk purified toxoid containing the same toxoid concentration as the final vaccine. Divide thedilution into two equal parts. Keep one of them at 5 ± 3°C and the other at 37°C for 6 weeks. Testboth dilutions by a suitable sensitive assay for active tetanus toxin, such as inoculation into mice orguinea-pigs. The toxoid complies with the test if neither sample produces any sign of a toxic reactionattributable to tetanus toxin.


FINAL BULK VACCINE

The final bulk vaccine is prepared by adsorption of suitable quantities of bulk purified diphtheriatoxoid and tetanus toxoid onto hydrated aluminium phosphate, aluminium hydroxide or calciumphosphate; the resulting mixture is approximately isotonic with blood. Suitable antimicrobialpreservatives may be added. Certain antimicrobial preservatives, particularly those of the phenolictype, adversely affect the antigenic activity and must not be used.




Potency Carry out the tests described under Assay.

FINAL LOT


Only a final lot that is satisfactory with respect to each of the requirements given below underIdentification, Tests and Assay may be released for use. Provided the tests for specific toxicity, freeformaldehyde and antimicrobial preservative and the assay have been carried out with satisfactoryresults on the final bulk vaccine, they may be omitted on the final lot.

IDENTIFICATION

A. Dissolve in the vaccine to be examined sufficient sodium citrate R to give a 100 g/l solution.Maintain at 37°C for about 16 h and centrifuge until a clear supernatant liquid is obtained. The clearsupernatant liquid reacts with a suitable diphtheria antitoxin, giving a precipitate.

B. The clear supernatant liquid obtained during test A reacts with a suitable tetanus antitoxin, givinga precipitate.

TESTS

Specific toxicity Inject subcutaneously five times the single human dose stated on the label intoeach of five healthy guinea-pigs, each weighing 250 g to 350 g, that have not previously been treatedwith any material that will interfere with the test. If within 42 days of the injection any of the animalsshows signs of or dies from diphtheria toxaemia or tetanus, the vaccine does not comply with the test.If more than one animal dies from non-specific causes, repeat the test once; if more than one animaldies in the second test, the vaccine does not comply with the test.






ASSAY

Diphtheria component Carry out one of the prescribed methods for the assay of diphtheria vaccine(adsorbed) (2.7.6).

The lower confidence limit (P = 0.95) of the estimated potency is not less than 30 I.U. per singlehuman dose.

Tetanus component Carry out one of the prescribed methods for the assay of tetanus vaccine(adsorbed) (2.7.8).

68-18


STORAGE


LABELLING


— the minimum number of International Units of each component per single human dose,— where applicable, that the vaccine is intended for primary vaccination of children and is not

necessarily suitable for reinforcing doses or for administration to adults,— the name and the amount of the adsorbent,— that the vaccine must be shaken before use,— that the vaccine is not to be frozen.

__________________________________________________________________________________________________________ Ph Eur

68-19

Adsorbed Diphtheria and Tetanus Vaccine for Adults and Adole-scents

Adsorbed Diphtheria and Tetanus Vaccine for Adults and Adolescents complies with the requirements of the3rd edition of the European Pharmacopoeia for Diphtheria and Tetanus Vaccine (Adsorbed) for Adults andAdolescents [0647]. These requirements are reproduced after the heading ‘Definition’ below..

The label may state ‘DT/Vac/Ads(Adult)’.

For a vaccine for use in the United Kingdom, the amount of diphtheria toxoid used is adjusted sothat the final vaccine contains not more than 2.0 flocculation equivalents (2.0 Lf) of diphtheriatoxoid per dose.

Ph Eur ___________________________________________________________________________________________________________

DEFINITION

Diphtheria and tetanus vaccine (adsorbed) for adults and adolescents is a preparation of diphtheriaformol toxoid and tetanus formol toxoid adsorbed on a mineral carrier. The formol toxoids areprepared from the toxins produced by the growth of Corynebacterium diphtheriae and Clostridiumtetani, respectively. It shall have been demonstrated to the competent authority that the quantity ofdiphtheria toxoid used does not produce adverse reactions in subjects from the age groups for whichthe vaccine is intended.

PRODUCTION









For the production of tetanus toxin, from which toxoid is prepared, seed cultures are managed in adefined seed-lot system in which toxinogenicity is conserved and, where necessary, restored bydeliberate reselection. A highly toxinogenic strain of Clostridium tetani with known origin and historyis grown in a suitable liquid medium. At the end of cultivation, the purity of each culture is testedand contaminated cultures are discarded. Toxin-containing culture medium is collected aseptically.The toxin content (Lf per millilitre) is checked to monitor consistency of production. Single harvestsmay be pooled to prepare the bulk purified toxoid. The toxin is purified to remove components likelyto cause adverse reactions in humans. The purified toxin is detoxified with formaldehyde by amethod that avoids destruction of the immunogenic potency of the toxoid and reversion of toxoid to

68-20

toxin, particularly on exposure to heat. Alternatively, purification may be carried out afterdetoxification.



Absence of tetanus toxin Inject subcutaneously at least 500 Lf of purified toxoid in a volume of1 ml into each of five healthy guinea-pigs, each weighing 250 g to 350 g, that have not previouslybeen treated with any material that will interfere with the test. If within 21 days of the injection any ofthe animals shows signs of or dies from tetanus, the toxoid does not comply with the test. If morethan one animal dies from non-specific causes, repeat the test once; if more than one animal dies inthe second test, the toxoid does not comply with the test.



FINAL BULK VACCINE

The vaccine is prepared by adsorption of suitable quantities of bulk purified diphtheria toxoid andtetanus toxoid onto hydrated aluminium phosphate, aluminium hydroxide or calcium phosphate; theresulting mixture is approximately isotonic with blood. Suitable antimicrobial preservatives may beadded. Certain antimicrobial preservatives, particularly those of the phenolic type, adversely affect theantigenic activity and must not be used.





FINAL LOT



IDENTIFICATION

A. Dissolve in the vaccine to be examined sufficient sodium citrate R to give a 100 g/l solution.Maintain at 37°C for about 16 h and centrifuge until a clear supernatant liquid is obtained. The clearsupernatant liquid reacts with a suitable diphtheria antitoxin, giving a precipitate. If a satisfactoryresult is not obtained with a vaccine adsorbed on aluminium hydroxide, carry out the test as follows.Centrifuge 15 ml of the vaccine to be examined and suspend the residue in 5 ml of a freshly preparedmixture of 1 volume of a 56 g/l solution of sodium edetate R and 49 volumes of a 90 g/l solution ofdisodium hydrogen phosphate R. Maintain at 37°C for not less than 6 h and centrifuge. The clearsupernatant liquid reacts with a suitable diphtheria antitoxin, giving a precipitate.

B. The clear supernatant liquid obtained during test A reacts with a suitable tetanus antitoxin, givinga precipitate.

TESTS

Specific toxicity Inject subcutaneously five times the single human dose stated on the label intoeach of five healthy guinea-pigs, each weighing 250 g to 350 g, that have not previously been treatedwith any material that will interfere with the test. If within 42 days of the injection any of the animalsshows signs of or dies from diphtheria toxaemia or tetanus, the vaccine does not comply with the test.If more than one animal dies from non-specific causes, repeat the test once; if more than one animaldies in the second test, the vaccine does not comply with the test.



68-21




ASSAY


The lower confidence limit (P = 0.95) of the estimated potency is not less than 2 I.U. per single humandose.



STORAGE


LABELLING


— the minimum number of International Units of each component per single human dose,— the name and the amount of the adsorbent,— that the vaccine must be shaken before use,— that the vaccine is not to be frozen.__________________________________________________________________________________________________________ Ph Eur

68-22

Adsorbed Diphtheria, Tetanus and Pertussis VaccineAdsorbed Diphtheria–Tetanus–Whooping-cough Prophylactic

Adsorbed Diphtheria, Tetanus and Pertussis Vaccine complies with the requirements of the 3rd edition of theEuropean Pharmacopoeia for Diphtheria, Tetanus and Pertussis Vaccine (Adsorbed) [0445]. These require-ments are reproduced after the heading ‘Definition’ below..

The label may state ‘DTPer/Vac/Ads’.

Ph Eur ___________________________________________________________________________________________________________

DEFINITION

Diphtheria, tetanus and pertussis vaccine (adsorbed) is a preparation of diphtheria formol toxoid andtetanus formol toxoid adsorbed on a mineral carrier to which a suspension of inactivated Bordetellapertussis has been added. The formol toxoids are prepared from the toxins produced by the growth ofCorynebacterium diphtheriae and Clostridium tetani, respectively.

PRODUCTION












Absence of tetanus toxin Inject subcutaneously at least 500 Lf of purified toxoid in a volume of1 ml into each of five healthy guinea-pigs, each weighing 250 g to 350 g, that have not previouslybeen treated with any material that will interfere with the test. If within 21 days of the injection any of

68-23

the animals shows signs of or dies from tetanus, the toxoid does not comply with the test. If morethan one animal dies from non-specific causes, repeat the test once; if more than one animal dies inthe second test, the toxoid does not comply with the test.



INACTIVATED B. PERTUSSIS SUSPENSION

Production of the vaccine is based on a seed-lot system. One or more strains of B. pertussis withknown origin and history are used. Strains, culture medium and cultivation method are chosen insuch a way that agglutinogens 1, 2 and 3 are present in the final vaccine. Each strain is grown for24 h to 72 h in a liquid medium or on a solid medium; the medium used in the final cultivation stagedoes not contain blood or blood products. Human blood or blood products are not used in anyculture media. The bacteria are harvested, washed to remove substances derived from the mediumand suspended in a 9 g/l solution of sodium chloride or other suitable isotonic solution. The opacityof the suspension is determined not later than 2 weeks after harvest by comparison with the Interna-tional Reference Preparation of Opacity and used as the basis of calculation for subsequent stages invaccine preparation. The equivalence in International Units of the International Reference Prepara-tion is stated by the World Health Organisation.

Single harvests are not used for the final bulk vaccine unless they have been shown to contain B.pertussis cells with the same characteristics, with regard to growth and agglutinogens, as the parentstrain and to be free from contaminating bacteria and fungi. The bacteria are killed and detoxified incontrolled conditions by means of a suitable chemical agent or by heating or by a combination ofthese two methods. Freedom from live B. pertussis is tested using a suitable culture medium. Thesuspension is maintained at 5 ± 3°C for a suitable period to diminish its toxicity.

FINAL BULK VACCINE

The final bulk vaccine is prepared by adsorption of suitable quantities of bulk purified diphtheriatoxoid and tetanus toxoid onto hydrated aluminium phosphate, aluminium hydroxide or calciumphosphate and admixture of an appropriate quantity of a suspension of inactivated B. pertussis; theresulting mixture is approximately isotonic with blood. The B. pertussis concentration of the final bulkvaccine does not exceed that corresponding to an opacity of 20 I.U. per single human dose. If two ormore strains of B. pertussis are used, the composition of consecutive lots of the final bulk vaccine shallbe consistent with respect to the proportion of each strain as measured in opacity units. Suitableantimicrobial preservatives may be added to the bulk vaccine. Certain antimicrobial preservatives,particularly those of the phenolic type, adversely affect the antigenic activity and must not be used.





FINAL LOT



IDENTIFICATION

A. Dissolve in the vaccine to be examined sufficient sodium citrate R to give a 100 g/l solution.Maintain at 37°C for about 16 h and centrifuge until a clear supernatant liquid is obtained; reservethe precipitate for identification test C. The clear supernatant liquid reacts with a suitable diphtheriaantitoxin, giving a precipitate.

B. The clear supernatant liquid obtained during identification test A reacts with a suitable tetanusantitoxin, giving a precipitate.

C. The pertussis component is identified by agglutination of the bacteria from the resuspended

68-24

centrifugation residue (see identification test A; other suitable methods for separating the bacteriafrom the adsorbent may also be used) by antisera specific to B. pertussis or by the assay of thepertussis component.

TESTS

Specific toxicity Diphtheria and tetanus components Inject subcutaneously five times the singlehuman dose stated on the label into each of five healthy guinea-pigs, each weighing 250 g to 350 g,that have not previously been treated with 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 not comply with the test. If more than one animal dies from non-specific causes,repeat the test once; if more than one animal dies in the second test, the vaccine does not complywith the test.

Pertussis component. Use not fewer than ten healthy mice each weighing 14 g to 16 g for the vaccinegroup 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 and water for at least 2 h before injection andduring the test. Inject each mouse of the vaccine group intraperitoneally with 0.5 ml, containing aquantity of the vaccine equivalent to not less than half the single human dose. Inject each mouse ofthe control group with 0.5 ml of a 9 g/l sterile solution of sodium chloride R, preferably containing thesame amount of antimicrobial preservative as that injected with the vaccine. Weigh the groups ofmice immediately before the injection and 72 h and 7 days after the injection. The vaccine complieswith the test if: (a) at the end of 72 h the total mass of the group of vaccinated mice is not less thanthat preceding the injection; (b) at the end of 7 days the average increase in mass per vaccinatedmouse is not less than 60 per cent of that per control mouse; and (c) not more than 5 per cent of thevaccinated mice die during the test.






ASSAY




If the test is carried out in guinea-pigs, the lower confidence limit (P = 0.95) of the estimatedpotency is not less than 40 I.U. per single human dose; if the test is carried out in mice, the lowerconfidence limit (P = 0.95) of the estimated potency is not less than 60 I.U. per single human dose.

Pertussis component Carry out the assay of pertussis vaccine (2.7.7).The estimated potency is not less than 4 I.U. per single human dose and the lower confidence limit

(P = 0.95) of the estimated potency is not less than 2 I.U. per single human dose.

STORAGE


LABELLING


— the minimum number of International Units of each component per single human dose,— where applicable, that the vaccine is intended for primary vaccination of children and is not

necessarily suitable for reinforcing doses or for administration to adults,— the name and the amount of the adsorbent,— that the vaccine must be shaken before use,— that the vaccine is not to be frozen.

__________________________________________________________________________________________________________ Ph Eur

68-25

Haemophilus Type B Conjugate Vaccine

Haemophilus Type B Conjugate Vaccine complies with the requirements of the 3rd edition of the EuropeanPharmacopoeia [1219]. These requirements are reproduced after the heading ‘Definition’ below.

The label may state ‘Hib/Vac’.

Ph Eur ___________________________________________________________________________________________________________

DEFINITION

Haemophilus type b conjugate vaccine is a liquid or freeze-dried preparation of a polysaccharide,derived from a suitable strain of Haemophilus influenzae type b, covalently bound to a carrier protein.The polysaccharide, polyribosylribitol phosphate, referred to as PRP, is a linear copolymer composedof repeated units of 3-b-D-ribofuranosyl-(1® 1)-ribitol-5-phosphate [(C10H19O12P)n], with a definedmolecular size. The carrier protein, when conjugated to PRP, is capable of inducing a T-cell-dependent B-cell immune response to the polysaccharide.

The vaccine complies with the monograph on Vaccines for human use (153).

PRODUCTION

GENERAL PROVISIONS

The production method shall have been shown to yield consistently haemophilus type b conjugatevaccines of adequate safety and immunogenicity in man. The production of PRP and of the carrierare based on seed-lot systems.


During development studies and wherever revalidation of the manufacturing process is necessary, itshall be demonstrated by tests in animals that the vaccine consistently induces a T-cell-dependent B-cell immune response.

The stability of the final lot and relevant intermediates is evaluated using one or more indicatortests. Such tests may include determination of molecular size, determination of free PRP in theconjugate and the immunogenicity test in mice. Taking account of the results of the stability testing,release requirements are set for these indicator tests to ensure that the vaccine will be satisfactory atthe end of the period of validity.

BACTERIAL SEED LOTS

The seed lots of H. influenzae type b are shown to be free from contamination by examination ofGram-stained smears and by inoculation on suitable media. Several microscopic fields are examinedat high magnification so that at least 10,000 organisms are inspected.

No complex products of animal origin are included in the menstruum used for preservation ofstrain viability, either for freeze-drying or for frozen storage.

It is recommended that PRP produced by the seed lot be characterised using nuclear magneticresonance spectrometry (2.2.33).

H. INFLUENZAE TYPE B POLYSACCHARIDE (PRP)

H. influenzae type b is grown in a liquid medium that does not contain high-molecular-mass poly-saccharides; if any ingredient of the medium contains blood-group substances, the process shall bevalidated to demonstrate that after the purification step they are no longer detectable. The bacterialpurity of the culture is verified by suitable methods. The culture may be inactivated. PRP is separatedfrom the culture medium and purified by a suitable method. Volatile matter, including water, in thepurified polysaccharide is determined by a suitable method such as thermogravimetry (2.2.34); theresult is used to calculate the results of certain tests with reference to the dried substance, asprescribed below.

Only PRP that complies with the following requirements may be used in the preparation of theconjugate.

Identification PRP is identified by an immunochemical method (2.7.1) or other suitable method,for example 1H nuclear magnetic resonance spectrometry (2.2.33).

Molecular-size distribution The percentage of PRP eluted before a given KD value or within arange of KD values, is determined by size-exclusion chromatography (2.2.30); an acceptable value isestablished for the particular product and each batch of PRP must be shown to comply with thislimit. Limits for currently approved products, using the indicated stationary phases, are shown forinformation in Table 1219–1. Where applicable, the molecular-size distribution is also determinedafter chemical modification of the polysaccharide.

Liquid chromatography (2.2.29) with multiple-angle laser light scattering detection may also beused for determination of molecular size distribution.

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

Ribose (2.5.31). Not less than 32 per cent, calculated with reference to the dried substance.

Phosphorus (2.5.18). 6.8 per cent to 9.0 per cent, calculated with reference to the driedsubstance.

Protein (2.5.16). Not more than 1.0 per cent, calculated with reference to the dried substance.Use sufficient PRP to allow detection of proteins at concentrations of 1 per cent or greater.

Nucleic acid (2.5.17). Not more than 1.0 per cent, calculated with reference to the driedsubstance.

Bacterial endotoxins (2.6.14). Not more than 25 I.U. of endotoxin per microgram of PRP.

Residual reagents Where applicable, tests are carried out to determine residues of reagents usedduring inactivation and purification. An acceptable value for each reagent is established for theparticular product and each batch of PRP must be shown to comply with this limit. Where validationstudies have demonstrated removal of a residual reagent, the test on PRP may be omitted.

CARRIER PROTEIN

The carrier protein is chosen so that when the PRP is conjugated it is able to induce a T-cell-dependent immune response. Currently approved carrier proteins and coupling methods are listedfor information in Table 1219-1. The carrier proteins are produced by culture of suitable micro-organisms; the bacterial purity of the culture is verified; the culture may be inactivated; the carrierprotein is purified by a suitable method.

Only a carrier protein that complies with the following requirements may be used in preparation ofthe conjugate.

Table 1219–1Product characteristics and specifications for PRP and carrier protein in currently approved products

Carrier Haemophilus material Conjugation

Type Purity Nominalamount per dose

Type of PRP Nominalamount per dose

Couplingmethod

Procedure

Diphtheriatoxoid

>1500 Lf permilligramof nitrogen

18 µg Size reduced PRPKD: 0.6-0.7, usingcross-linked agarosefor chromatography R

25 µg cyanogenbromideactivation of PRP

activated diphtheria toxoid(D-AH+), cyanogenbromide-activated PRP

Tetanustoxoid

>1500 Lf permilligramof nitrogen

20 µg PRP ≥50% ≤KD :0.30,using cross-linkedagarose forchromatography R

10 µg carbodi-imidemediated

ADH-activated PRP (PRP-cov.-AH) + tetanus toxoid+ EDAC

CRM 197diphtheriaprotein

>90% ofdiphtheriaprotein

25 µg Size reduced PRPDp = 15-35 or10-35

10 µg reductiveamination(one-stepmethod) orN-hydroxy-succinimideactivation

direct coupling of PRP toCRM 197(cyanoborohydrideactivated)

Meningo-coccalgroup B outermembraneprotein(OMP)

outermembraneproteinvesicles;≤ 8% oflipopoly-saccharide

125 µg or250 µg

Size reduced PRPKD>0.6,usingcross-linked agarose forchromatography R orMw> 50 × 103

7.5 µg or 15 µg thioether bond PRP activation by CDiPRP-IM + BuA2 + BrAc =PRP-BuA2-BrAc + thio-activated OMP

Legend

ADH = adipic acid dihydrazideBrAc = bromoacetyl chloride

BuA2 = butane-1,4-diamideCDI = carbonyldi-imidazole

Dp = degree of polymerisationEDAC = 1-ethyl-3-(3-dimethylaminopropyl)carbodi-imide

IM = imidazoliumMw = weight-average molecular weight

Identification The carrier protein is identified by a suitable immunochemical method (2.7.1).

Sterility (2.6.1). Carry out the test using for each medium 10 ml or the equivalent of one hundreddoses, whichever is the less.

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Diphtheria toxoid Diphtheria toxoid is produced as described in Diphtheria vaccine (adsorbed) (443)and complies with the requirements prescribed there for bulk purified toxoid.

Tetanus toxoid Tetanus toxoid is produced as described in Tetanus vaccine (adsorbed) (452) andcomplies with the requirements prescribed there for bulk purified toxoid except that the antigenicpurity is not less than 1500 Lf per milligram of protein nitrogen.

Diphtheria protein CRM 197 It contains not less than 90 per cent of diphtheria CRM 197 protein,determined by a suitable method. Suitable tests are carried out, for validation or routinely, to demon-strate that the product is non-toxic.

OMP (meningococcal group B outer membrane protein complex) OMP complies with thefollowing requirements for lipopolysaccharide and pyrogens.

Lipopolysaccharide. Not more than 8 per cent of lipopolysaccharide, determined by a suitable method.

Pyrogens (2.6.8). Inject into each rabbit 0.25 µg of OMP per kilogram of body mass.

BULK CONJUGATE

PRP is chemically modified to enable conjugation; it is usually partly depolymerised either before orduring this procedure. Reactive functional groups or spacers may be introduced into the carrierprotein or PRP prior to conjugation. As a measure of consistency, the extent of derivatisation ismonitored. The conjugate is obtained by the covalent binding of PRP and carrier protein. Whereapplicable, unreacted but potentially reactogenic functional groups are made unreactive by means ofcapping agents; the conjugate is purified to remove reagents.

Only a bulk conjugate that complies with the following requirements may be used in preparation ofthe final bulk vaccine. For each test and for each particular product, limits of acceptance areestablished and each batch of conjugate must be shown to comply with these limits. Limits applied tocurrently approved products for some of these tests are listed for information in Table 1219–2. For afreeze-dried vaccine, some of the tests may be carried out on the final lot rather than on the bulkconjugate where the freeze-drying process may affect the component being tested.

Table 1219–2 Bulk conjugate requirements for currently approved products

Protein carrierTest

Diphtheria toxoid Tetanus toxoid CRM 197 OMP

Free PRP <37% <20% <25% <15%

Free protein <4% <1%, whereapplicable

<1 % or < 2 %,depending on thecoupling method

not applicable

PRP to protein ratio 1.25–1.8 0.30–0.55 0.3–0.7 0.05–0.1

Molecular size (KD):

cross-linked agarose forchromatography R

95%<0.75 60%<0.2 50% 0.3–0.6 85%<0.3

cross-linked agarose forchromatography R1

0.6-0.7 85%<0.5

PRP The PRP content is determined by assay of phosphorus (2.5.18) or by assay of ribose (2.5.31)or by an immunochemical method (2.7.1).

Protein The protein content is determined by a suitable chemical method (for example, 2.5.16).

PRP to protein ratio Determine the ratio by calculation.

Molecular-size distribution Molecular-size distribution is determined by size-exclusion chromato-graphy (2.2.30).

Free PRP Unbound PRP is determined after removal of the conjugate, for example by size-exclusion or hydrophobic chromatography, ultrafiltration or other validated methods.

Free carrier protein Determine by a suitable method either directly or by deriving the content bycalculation from the results of other tests. The amount is within the limits approved for the particularproduct.

Unreacted functional groups No unreacted functional groups are detectable in the bulk conjugateunless process validation has shown that unreacted functional groups detectable at this stage areremoved during the subsequent manufacturing process (for example, owing to short half-life).

Residual reagents Removal of residual reagents such as cyanide, EDAC(ethyldimethylaminopropylcarbodi-imide) and phenol is confirmed by suitable tests or by validationof the process.

68-28

Sterility (2.6.1). Carry out the test using for each medium 10 ml or the equivalent of one hundreddoses, whichever is the less.

FINAL BULK VACCINE

An adjuvant, an antimicrobial preservative and a stabiliser may be added to the bulk conjugatebefore dilution to the final concentration with a suitable diluent.

Only a final bulk vaccine that complies with the following requirements may be used in prepara-tion of the final lot.

Antimicrobial preservative Where applicable, determine the amount of antimicrobialpreservative by a suitable chemical or physico-chemical method. The content is not less than85 per cent and not greater than 115 per cent of the intended amount.

Sterility (2.6.1). It complies with the test for sterility, carried out using 10 ml for each medium.

FINAL LOTOnly a final lot that is satisfactory with respect to each of the requirements given below under

Identification and Tests may be released for use. Provided the test for antimicrobial preservative hasbeen carried out on the final bulk vaccine, they may be omitted on the final lot.

pH (2.2.3). The pH of the vaccine, reconstituted if necessary, is within the range approved for theproduct.

Free PRP Unbound PRP is determined after removal of the conjugate, for example by anion-exchange, size-exclusion or hydrophobic chromatography, ultrafiltration or other validated methods.The amount of free PRP is not greater than that approved for the particular product.

IDENTIFICATION

The vaccine is identified by a suitable immunochemical method (2.7.1) for PRP.

TESTS

PRP content Not less than 80 per cent of the amount of PRP stated on the label. Determine eitherribose (2.5.31) or phosphorus (2.5.18) or apply an immunochemical method (2.7.1) or by anion-exchange liquid chromatography with pulsed amperometric detection (2.2.29).

Aluminium When aluminium hydroxide is used as the adsorbent, the vaccine complies with the testprescribed in Vaccines for human use (153).

Antimicrobial preservative Where applicable, determine the amount of antimicrobial preservativeby a suitable chemical or physico-chemical method. The content is not less than the minimumamount shown to be effective and not greater than 115 per cent of the quantity stated on the label.

Water (2.5.12). For freeze-dried vaccines, not more than 3.0 per cent.


Pyrogens (2.6.8). It complies with the test for pyrogens. Inject per kilogram of the rabbit’s mass aquantity of the vaccine equivalent to: 1 µg of PRP for a vaccine with diphtheria toxoid or CRM 197diphtheria protein as carrier; 0.1 µg of PRP for a vaccine with tetanus toxoid as carrier; 0.025 µg ofPRP for a vaccine with OMP as a carrier.

STORAGE


LABELLING


The label states:— the number of micrograms of PRP per human dose,— the type and nominal amount of carrier protein per single human dose.

__________________________________________________________________________________________________________ Ph Eur

68-29

Inactivated Hepatitis A Vaccine

1/01

Inactivated Hepatitis A Vaccine complies with the requirements of the 3rd edition of the European Pharmaco-poeia for Hepatitis A Vaccine (Inactivated, Adsorbed) [1107]. These requirements are reproduced after theheading 'Definition' below.

The label may state ‘Hep A/Vac’.

Ph Eur ___________________________________________________________________________________________________________

DEFINITION

Hepatitis A vaccine (inactivated, adsorbed) is a suspension consisting of a suitable strain of hepatitisA virus grown in cell cultures, inactivated by a validated method and adsorbed on a mineral carrier.

The vaccine complies with the monograph on Vaccines for human use (0153).

PRODUCTION

Production of the vaccine is based on a virus seed-lot system and a cell-bank system. The productionmethod shall have been shown to yield consistently vaccines that comply with the requirements forimmunogenicity, safety and stability.


Unless otherwise justified and authorised, the virus in the final vaccine shall not have undergonemore passages from the master seed lot than were used to prepare the vaccine shown in clinicalstudies to be satisfactory with respect to safety and efficacy.

Reference preparation. A part of a batch shown to be at least as immunogenic in animals as a batchthat, in clinical studies in young healthy adults, produced not less than 95 per cent seroconversion,corresponding to a level of neutralising antibody accepted to be protective, after a full-course primaryimmunisation is used as a reference preparation. An antibody level of 20 mI.U./ml determined byenzyme-linked immunosorbent assay is recognised as being protective.

SUBSTRATE FOR VIRUS PROPAGATION

The virus is propagated in a human diploid cell line (5.2.3) or in a continuous cell line approved bythe competent authority.

SEED LOTS

The strain of hepatitis A virus used to prepare the master seed lot shall be identified by historicalrecords that include information on the origin of the strain and its subsequent manipulation.

Only a seed lot that complies with the following requirements may be used for virus propagation.

Identification Each master and working seed lot is identified as hepatitis A virus using specificantibodies.

Virus concentration The virus concentration of each master and working seed lot is determined tomonitor consistency of production.

Extraneous agents The master and working seed lots comply with the requirements for seed lots forvirus vaccines (2.6.16). In addition, if primary monkey cells have been used for isolation of the strain,measures are taken to ensure that the strain is not contaminated with simian viruses such as simianimmunodeficiency virus and filoviruses.

VIRUS PROPAGATION AND HARVEST

All processing of the cell bank and subsequent cell cultures is done under aseptic conditions in anarea where no other cells are being handled. Animal serum (but not human serum) may be used inthe cell culture media. Serum and trypsin used in the preparation of cell suspensions and media areshown to be free from extraneous agents. The cell culture media may contain a pH indicator, such asphenol red, and antibiotics at the lowest effective concentration. Not less than 500 ml of the cellcultures employed for vaccine production is set aside as uninfected cell cultures (control cells).Multiple harvests from the same production cell culture may be pooled and considered as a singleharvest.

Only a single harvest that complies with the following requirements may be used in the preparationof the vaccine. When the determination of the ratio of virus concentration to antigen content hasbeen carried out on a suitable number of single harvests to demonstrate production consistency, itmay subsequently be omitted as a routine test.

Identification The test for antigen content also serves to identify the single harvest.

Bacterial and fungal contamination (2.6.1). The single harvest complies with the test for sterility,carried out using 10 ml for each medium.

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Mycoplasmas (2.6.7). The single harvest complies with the test for mycoplasmas, carried out using1 ml for each medium.

Control cells The control cells of the production cell culture comply with a test for identificationand the requirements for extraneous agents (2.6.16).

Antigen content Determine the hepatitis A antigen content by a suitable immunochemical method(2.7.1) to monitor production consistency; the content is within the limits approved for the particularproduct.

Ratio of virus concentration to antigen content The consistency of the ratio of the concentrationof infectious virus, determined by a suitable cell culture method, to antigen content is established byvalidation on a suitable number of single harvests.

PURIFICATION AND PURIFIED HARVEST

The harvest, which may be a pool of several single harvests, is purified by validated methods. Ifcontinuous cell lines are used for production, the purification process shall have been shown toreduce consistently the level of host-cell DNA.

Only a purified harvest that complies with the following requirements may be used in the prepara-tion of the inactivated harvest.

Virus concentration The concentration of infectious virus in the purified harvest is determined by asuitable cell culture method to monitor production consistency and as a starting point for monitoringthe inactivation curve.

Antigen: total protein ratio Determine the hepatitis A virus antigen content by a suitableimmunochemical method (2.7.1). Determine the total protein by a validated method. The ratio ofhepatitis A virus antigen content to total protein content is within the limits approved for theparticular product.

Bovine serum albumin Not more than 50 ng in the equivalent of a single human dose, determinedby a suitable immunochemical method (2.7.1). Where appropriate in view of the manufacturingprocess, other suitable protein markers may be used to demonstrate effective purification.

Residual host-cell DNA If a continuous cell line is used for virus propagation, the content ofresidual host-cell DNA, determined using a suitable method as described in Products of recombinantDNA technology (0784), is not greater than 100 pg in the equivalent of a single human dose.

Residual chemicals If chemical substances are used during the purification process, tests for thesesubstances are carried out on the purified harvest (or on the inactivated harvest), unless validation ofthe process has demonstrated total clearance. The concentration must not exceed the limits approvedfor the particular product.

INACTIVATION AND INACTIVATED HARVEST

Several purified harvests may be pooled before inactivation. In order to avoid interference with theinactivation process, virus aggregation must be prevented or aggregates must be removed immedi-ately before and/or during the inactivation process. The virus suspension is inactivated by a validatedmethod; the method shall have been shown to be consistently capable of inactivating hepatitis A viruswithout destroying the antigenic and immunogenic activity; for each inactivation procedure, aninactivation curve is plotted representing residual live virus concentration measured at not fewer thanthree points in time (for example, on days 0, 1 and 2 of the inactivation process). If formaldehyde isused for inactivation, the presence of excess free formaldehyde is verified at the end of the inactiva-tion process.

Only an inactivated harvest that complies with the following requirements may be used in thepreparation of the final bulk vaccine.

Inactivation Carry out an amplification test for residual infectious hepatitis A virus by inoculating aquantity of the inactivated harvest equivalent to 5 per cent of the batch or, if the harvest contains theequivalent of 30,000 doses or more, not less than 1500 doses of vaccine into cell cultures of the sametype as those used for production of the vaccine; incubate for a total of not less than 70 days makingnot fewer than one passage of cells within that period. At the end of the incubation period, carry outa test of suitable sensitivity for residual infectious virus. No evidence of hepatitis A virus multiplica-tion is found in the samples taken at the end of the inactivation process. Use infectious virus inoculaconcurrently as positive controls to demonstrate cellular susceptibility and absence of interference.Incubate for a total of not less than 70 days, making not fewer than one passage of cells within thatperiod.

Sterility (2.6.1). The inactivated viral harvest complies with the test for sterility, carried out using10 ml for each medium.

Bacterial endotoxins (2.6.14). Not more than 2 I.U. of endotoxin in the equivalent of a singlehuman dose.

68-31

Antigen content Determine the hepatitis A virus antigen content by a suitable immunochemicalmethod (2.7.1).

Residual chemicals See under Purification and purified harvest.

FINAL BULK VACCINE

The final bulk vaccine is prepared from one or more inactivated harvests. Approved adjuvants,stabilisers and antimicrobial preservatives may be added.

Only a final bulk vaccine that complies with the following requirements may be used in the prepa-ration of the final lot.

Sterility (2.6.1). The final bulk vaccine complies with the test for sterility, carried out using 10 mlfor each medium.

Antimicrobial preservative Where applicable, determine the amount of antimicrobial preservativeby a suitable chemical or physico-chemical method. The amount is not less than 85 per cent and notgreater than 115 per cent of the intended amount.

FINAL LOT

The final bulk vaccine is distributed aseptically into sterile containers. The containers are then closedso as to avoid contamination.

Only a final lot that complies with each of the requirements given below under Identification, Testsand Assay may be released 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, these tests may be omitted on the final lot. If the assay is carried outusing mice or other animals, then provided it has been carried out with satisfactory results on thefinal bulk vaccine, it may be omitted on the final lot.

IDENTIFICATION

The vaccine is shown to contain hepatitis A virus antigen by a suitable immunochemical method(2.7.1) using specific antibodies or by the in vivo assay (2.7.14).

TESTS

Aluminium When hydrated aluminium phosphate or aluminium hydroxide is used as the adsorbent,the vaccine complies with the test prescribed in Vaccines for human use (0153).

Free formaldehyde When formaldehyde has been used for virus inactivation, the vaccine complieswith the test prescribed in Vaccines for human use (0153).

Antimicrobial preservative Where applicable, determine the amount of antimicrobial preservativeby a suitable chemical or physico-chemical method. The amount is not less than the minimumamount shown to be effective and is not greater than 115 per cent of that stated on the label.


ASSAY

The vaccine complies with the assay of hepatitis A vaccine (2.7.14).

STORAGE


LABELLING


The label states the biological origin of the cells used for the preparation of the vaccine.__________________________________________________________________________________________________________ Ph Eur

68-32

Hepatitis A (Inactivated) and Hepatitis B (rDNA) Vaccine

1/01

Hepatitis A (Inactivated) and Hepatitis B (rDNA) Vaccine (Adsorbed) complies with the requirements ofthe 3rd edition of the European Pharmacopoeia for Hepatitis A (Inactivated) and Hepatitis B (rDNA)Vaccine (Adsorbed) [1526]. These requirements are reproduced after the heading ‘Definition’ below.

Ph Eur ___________________________________________________________________________________________________________

DEFINITION

Hepatitis A (inactivated) and hepatitis B (rDNA) vaccine (adsorbed) is a suspension consisting of asuitable strain of hepatitis A virus, grown in cell cultures and inactivated by a validated method, andof hepatitis B surface antigen (HBsAg), a component protein of hepatitis B virus obtained byrecombinant DNA technology; the antigens are adsorbed on a mineral carrier, such as aluminiumhydroxide or hydrated aluminium phosphate.

The vaccine complies with the monograph on Vaccines for human use (0153) and the monograph onProducts of recombinant DNA technology (0784) (for the hepatitis B component).

PRODUCTION

GENERAL PROVISIONS

The two components are prepared as described in the monographs on Hepatitis A vaccine(inactivated, adsorbed) (1107) and Hepatitis B vaccine (rDNA) (1056) and comply with the require-ments prescribed therein.


Reference preparation. The reference preparation is part of a representative batch shown to be at leastas immunogenic in animals as a batch that, in clinical studies in young healthy adults, produced notless than 95 per cent seroconversion, corresponding to a level of neutralising antibody recognised tobe protective, after a full-course primary immunisation. For hepatitis A, an antibody level not lessthan 20 mI.U./ml determined by enzyme-linked immunosorbent assay is recognised as beingprotective. For hepatitis B, an antibody level not less than 10 mI.U./ml against HBsAg is recognisedas being protective.

FINAL BULK VACCINE

The final bulk vaccine is prepared from one or more inactivated harvests of hepatitis A virus and oneor more batches of purified antigen.




FINAL LOTOnly a final lot that complies with each of the requirements given below under Identification, Tests

and Assay may be released 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 the final lot. If the assay of the hepatitis Aand/or the hepatitis B component is carried out in vivo, then provided it has been carried out withsatisfactory results on the final bulk vaccine, it may be omitted on the final lot.

IDENTIFICATION

The vaccine is shown to contain hepatitis A virus antigen and hepatitis B surface antigen by suitableimmunochemical methods (2.7.1), using specific antibodies or by the mouse immunogenicity testsdescribed under Assay.

TESTS

Aluminium When aluminium hydroxide or hydrated aluminium phosphate is used as the absorbent,the vaccine complies with the test prescribed in the monograph on Vaccines for human use (0153).

Free formaldehyde Where applicable, the vaccine complies with the test prescribed in the mono-graph on Vaccines for human use (0153).

Antimicrobial preservative Where applicable, determine the amount of antimicrobial preservativeby a suitable chemical or physico-chemical method. The amount is not less than the minimum

68-33

amount shown to be effective and is not greater than 115 per cent of that stated on the label.


Bacterial endotoxins (2.6.14). Not more than 2 I.U. of endotoxin per human dose.

ASSAY

Hepatitis A component The vaccine complies with the assay of hepatitis A vaccine (2.7.14).

Hepatitis B component The vaccine complies with the assay of hepatitis B vaccine (rDNA)(2.7.15).

STORAGE


LABELLING


The label states:— the amount of hepatitis A virus antigen and hepatitis B surface antigen per container,— the type of cells used for production of the vaccine,— the name and amount of the adsorbent used,— that the vaccine must be shaken before use,— that the vaccine must not be frozen.

__________________________________________________________________________________________________________ Ph Eur

68-34

Hepatitis B Vaccine (rDNA)

revised 1/01

Hepatitis B Vaccine (rDNA) complies with the requirements of the 3rd edition of the European Pharmacopoeia[1056]. These requirements are reproduced after the heading 'Definition' below.

The label may state ‘Hep B/Vac’.

Ph Eur ___________________________________________________________________________________________________________

DEFINITION

Hepatitis B vaccine (rDNA) is a preparation of hepatitis B surface antigen (HBsAg), a componentprotein of hepatitis B virus; the antigen may be adsorbed on a mineral carrier such as aluminiumhydroxide or hydrated aluminium phosphate. The antigen is obtained by recombinant DNAtechnology.

Hepatitis B vaccine (rDNA) complies with the requirements of the monographs on Products ofrecombinant DNA technology (0784) and on Vaccines for human use (0153).

PRODUCTION

GENERAL PROVISIONS

See the monograph on Products of recombinant DNA technology (0784), particularly the sectionsCloning and expression, Cell-bank system, Validation of the cell banks, Validation of the productionprocess and Production consistency.

The vaccine shall have been shown to induce specific, protective antibodies in man. The produc-tion method shall have been shown to yield consistently vaccines that comply with the requirementsfor immunogenicity and safety.


Hepatitis B vaccine (rDNA) is produced by the expression of the viral gene coding for HBsAg inyeast (Saccharomyces cerevisiae) or mammalian cells (Chinese hamster ovary (CHO) cells or othersuitable cell lines), purification of the resulting HBsAg and the rendering of this antigen into animmunogenic preparation. The suitability and safety of the cells are approved by the competentauthority.

The vaccine may contain the product of the S gene (major protein), a combination of the S geneand pre-S2 gene products (middle protein) or a combination of the S gene, the pre-S2 gene and pre-S1 gene products (large protein).

Reference preparation. The reference preparation is part of a representative batch shown to be at leastas immunogenic in animals as a batch that, in clinical studies in young, healthy adults, produced notless than 95 per cent seroconversion, corresponding to a level of HBsAg neutralising antibodyrecognised to be protective, after a full-course primary immunisation. An antibody level not less than10 mI.U./ml is recognised as being protective.

CHARACTERISATION OF THE SUBSTANCE

Development studies are carried out to characterise the antigen. The complete protein, lipid andcarbohydrate structure of the antigen is established. The morphological characteristics of the antigenparticles are established by electron microscopy. The mean buoyant density of the antigen particles isdetermined by a physico-chemical method, such as gradient centrifugation. The antigenic epitopesare characterised. The protein fraction of the antigen is characterised in terms of the primarystructure (for example, by determination of the amino-acid composition, by partial amino-acidsequence analysis and by peptide mapping).

CULTURE AND HARVEST

Identity, microbial purity, plasmid retention and consistency of yield are determined at suitableproduction stages. If mammalian cells are used, tests for extraneous agents and mycoplasmas areperformed in accordance with Tests for extraneous agents in viral vaccines for human use (2.6.16).

PURIFIED ANTIGEN

Only a purified antigen that complies with the following requirements may be used in the preparationof the final bulk vaccine.

Total protein The total protein is determined by a validated method. The content is within thelimits approved for the specific product.

Antigen content and identification The quantity and specificity of HBsAg is determined incomparison with the International Standard for HBsAg subtype ad or an in-house reference, by asuitable immunochemical method (2.7.1) such as radio-immunoassay (RIA), enzyme-linked

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immunosorbent assay (ELISA), immunoblot (preferably using a monoclonal antibody directedagainst a protective epitope) or single radial diffusion. The antigen/protein ratio is within the limitsapproved for the specific product.

The molecular weight of the major band revealed following sodium dodecyl sulphate poly-acrylamide gel electrophoresis (SDS-PAGE) performed under reducing conditions corresponds tothe value expected from the known nucleic acid and polypeptide sequences and possible glycosyla-tion.

Antigenic purity The purity of the antigen is determined by comparison with a reference prepara-tion using liquid chromatography or other suitable methods such as SDS-PAGE with staining by acidblue 92 and silver. A suitable method is sensitive enough to detect a potential contaminant at aconcentration of 1 per cent of total protein. Not less than 95 per cent of the total protein consists ofhepatitis B surface antigen.

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

Host-cell- and vector-derived DNA If mammalian cells are used for production, not more than10 pg of DNA in the quantity of purified antigen equivalent to a single human dose of vaccine.

Caesium If a caesium salt is used during production, a test for residual caesium is carried out on thepurified antigen. The content is within the limits approved for the specific product.

Sterility (2.6.1). The purified antigen complies with the test for sterility, carried out using 10 ml foreach medium.

Additional tests on the purified antigen may be required depending on the production methodused: for example, a test for residual animal serum where mammalian cells are used for production ortests for residual chemicals used during extraction and purification.

FINAL BULK VACCINE

An antimicrobial preservative and an adjuvant may be included in the vaccine.Only a final bulk vaccine that complies with the following requirements may be used in the prepa-

ration of the final lot.



FINAL LOT

Only a final lot that complies with each of the requirements given below under Identification, Testsand Assay may be released 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 the final lot. If the assay is carried out invivo, then provided it has been carried out with satisfactory results on the final bulk vaccine, it maybe omitted on the final lot.

IDENTIFICATION

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

TESTS

Aluminium When aluminium hydroxide or hydrated aluminium phosphate is used as the adsorbent,the vaccine complies with the test prescribed in the monograph on Vaccines for human use (0153).

Free formaldehyde Where applicable, the vaccine complies with the test prescribed in the mono-graph on Vaccines for human use (0153).

Antimicrobial preservative Where applicable, determine the amount of antimicrobial preservativeby a suitable chemical or physico-chemical method. The amount is not less than the minimumamount shown to be effective and is not greater than 115 per cent of that stated on the label.


Pyrogens (2.6.8). The vaccine complies with the test for pyrogens. Inject the equivalent of onehuman dose into each rabbit.

ASSAY

The vaccine complies with the assay of hepatitis B vaccine (rDNA) (2.7.15).

STORAGE


LABELLING


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The label states:— the amount of HBsAg per container,— the type of cells used for production of the vaccine,— the name and amount of the adsorbent used,— that the vaccine must be shaken before use,— that the vaccine must not be frozen.

__________________________________________________________________________________________________________ Ph Eur

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Inactivated Influenza Vaccine (Whole Virion)

Inactivated Influenza Vaccine (Whole Virion) complies with the requirements of the 3rd edition of theEuropean Pharmacopoeia. [0159]. These requirements are reproduced after the heading ‘Definition’ below.

The label may state ‘Flu/Vac’.

When Inactivated Influenza Vaccine or Influenza Vaccine is prescribed or demanded and the form isnot stated, Inactivated Influenza Vaccine (Whole Virion), Inactivated Influenza Vaccine (SplitVirion) or Inactivated Influenza Vaccine (Surface Antigen) may be dispensed or supplied.

Ph Eur ___________________________________________________________________________________________________________

DEFINITION

Influenza vaccine (whole virion, inactivated) is a sterile, aqueous suspension of a strain or strains ofinfluenza virus, type A or B, or a mixture of strains of the two types grown individually in fertilisedhens’ eggs and inactivated in such a manner that their antigenic properties are retained. The statedamount of haemagglutinin antigen for each strain present in the vaccine is 15 µg per dose, unlessclinical evidence supports the use of a different amount.

The vaccine is a slightly opalescent liquid.

PRODUCTION


CHOICE OF VACCINE STRAIN

The World Health Organisation reviews the world epidemiological situation annually and if necessaryrecommends new strains corresponding to prevailing epidemiological evidence.

Such strains are used in accordance with the regulations in force in the signatory states of theConvention on the Elaboration of a European Pharmacopoeia. It is now common practice to usereassorted strains giving high yields of the appropriate surface antigens. The origin and passagehistory of virus strains shall be approved by the competent authority.


Influenza virus seed to be used in the production of vaccine is propagated in fertilised eggs fromchicken flocks free from specified pathogens (5.2.2) or in suitable cell cultures (5.2.4), such as chick-embryo fibroblasts or chick kidney cells obtained from chicken flocks free from specified pathogens(5.2.2). For production, the virus of each strain is grown in the allantoic cavity of fertilised hens’ eggsfrom healthy flocks.

VIRUS SEED LOT

The production of vaccine is based on a seed-lot system. Working seed lots represent not more thanfifteen passages from the approved reassorted virus or the approved virus isolate. The final vaccinerepresents one passage from the working seed lot. The haemagglutinin and neuraminidase antigens ofeach seed lot are identified as originating from the correct strain of influenza virus by suitablemethods.

Only a working virus seed lot that complies with the following requirements may be used in thepreparation of the monovalent pooled harvest.

Bacterial and fungal contamination Carry out the test for sterility (2.6.1), using 10 ml for eachmedium.

Mycoplasmas (2.6.7). Carry out the test for mycoplasmas, using 10 ml.


An antimicrobial agent may be added to the inoculum. After incubation at a controlled temperature,the allantoic fluids are harvested and combined to form a monovalent pooled harvest. An antimicrob-ial agent may be added at the time of harvest. At no stage in the production is penicillin orstreptomycin used.

MONOVALENT POOLED HARVEST

To limit the possibility of contamination, inactivation is initiated as soon as possible after prepara-tion. The virus is inactivated by a method that has been demonstrated on three consecutive batchesto be consistently effective for the manufacturer. The inactivation process shall have been shown tobe capable of inactivating the influenza virus without destroying its antigenicity; the process shouldcause minimum alteration of the haemagglutinin and neuraminidase antigens. The inactivationprocess shall also have been shown to be capable of inactivating avian leucosis viruses andmycoplasmas. If the monovalent pooled harvest is stored after inactivation, it is held at a temperature

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of 5 ± 3°C. If formaldehyde solution is used, the concentration does not exceed 0.2 g/l of CH2O atany time during inactivation; if betapropiolactone is used, the concentration does not exceed 0.1 percent V/V at any time during inactivation.

Before or after the inactivation process, the monovalent pooled harvest is concentrated and purifiedby high-speed centrifugation or other suitable method.

Only a monovalent pooled harvest that complies with the following requirements may be used inthe preparation of the final bulk vaccine.

Haemagglutinin antigen Determine the content of haemagglutinin antigen by an immunodiffusiontest (2.7.1), by comparison with a haemagglutinin antigen reference preparation or with an antigenpreparation calibrated against it(1). Carry out the test at 20°C to 25°C.

Neuraminidase antigen The presence and type of neuraminidase antigen are confirmed by suitableenzymatic or immunological methods on the first three monovalent pooled harvests from each work-ing seed lot.

Sterility (2.6.1). Carry out the test for sterility, using 10 ml for each medium.

Viral inactivation Carry out the test described below under Tests.

FINAL BULK VACCINE

Appropriate quantities of the monovalent pooled harvests are blended to make the final bulk vaccine.Only a final bulk vaccine that complies with the following requirements may be used in the

preparation of the final lot.

Antimicrobial preservative Where applicable, determine the amount of antimicrobial preservativeby a suitable chemical method. The content is not less than 85 per cent and not greater than 115 percent of the intended amount.


FINAL LOT


Only a final lot that is satisfactory with respect to each of the requirements given below under Testsand Assay may be released for use. Provided that the test for viral inactivation has been performedwith satisfactory results on each monovalent pooled harvest and that the tests for free formaldehyde,ovalbumin and total protein have been performed with satisfactory results on the final bulk vaccine,they may be omitted on the final lot.

IDENTIFICATION

The assay serves to confirm the antigenic specificity of the vaccine.

TESTS

Viral inactivation Inoculate 0.2 ml of the vaccine into the allantoic cavity of each of ten fertilisedeggs and incubate at 33°C to 37°C for 3 days. The test is not valid unless at least eight of the tenembryos survive. Harvest 0.5 ml of the allantoic fluid from each surviving embryo and pool the fluids.Inoculate 0.2 ml of the pooled fluid into a further ten fertilised eggs and incubate at 33°C to 37°C for3 days. The test is not valid unless at least eight of the ten embryos survive. Harvest about 0.1 ml ofthe allantoic fluid from each surviving embryo and examine each individual harvest for live virus by ahaemagglutination test. If haemagglutination is found for any of the fluids, carry out for that fluid afurther passage in eggs and test for haemagglutination; no haemagglutination occurs.

Total protein Not more than six times the total haemagglutinin content of the vaccine as deter-mined in the assay, but in any case, not more than 100 µg of protein per virus strain per human doseand not more than a total of 300 µg of protein per human dose.

Free formaldehyde It complies with the test for free formaldehyde prescribed in the monograph onVaccines for human use (153).

Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservativeby a suitable chemical method. The content is not less than the minimum amount shown to beeffective and is not greater than 115 per cent of the quantity stated on the label.

Ovalbumin Not more than 1 µg of ovalbumin per human dose, determined by a suitable techniqueusing a suitable reference preparation of ovalbumin.



ASSAY

Determine the content of haemagglutinin antigen by an immunodiffusion test (2.7.1), by comparisonwith a haemagglutinin antigen reference preparation or with an antigen preparation calibrated againstit(1). Carry out the test at 20°C to 25°C. The confidence interval (P = 0.95) of the assay is not greater

68-39

than 80 per cent to 125 per cent of the estimated content. The lower confidence limit (P = 0.95) ofthe estimate of haemagglutinin antigen content is not less than 80 per cent of the amount stated onthe label for each strain.

STORAGE


LABELLING


The label states:— that the vaccine has been prepared on eggs,— the strain or strains of influenza virus used to prepare the vaccine,— the method of inactivation,— the haemagglutinin content in micrograms per virus strain per dose,— the season during which the vaccine is intended to protect.

1Reference haemagglutinin antigens are availablefrom the National Institute for BiologicalStandards and Control, Blanche Lane, South Mimms, Potters Bar, Hertfordshire EN6 3QG,United Kingdom.

__________________________________________________________________________________________________________ Ph Eur

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Inactivated Influenza Vaccine (Split Virion)

Inactivated Influenza Vaccine (Split Virion) complies with the requirements of the 3rd edition of the EuropeanPharmacopoeia [0158]. These requirements are reproduced after the heading ‘Definition’ below.

The label may state ‘Flu/Vac/Split’.

When Inactivated Influenza Vaccine or Influenza Vaccine is prescribed or demanded and the formis not stated, Inactivated Influenza Vaccine (Whole Virion), Inactivated Influenza Vaccine (SplitVirion) or Inactivated Influenza Vaccine (Surface Antigen) may be dispensed or supplied.

Ph Eur ___________________________________________________________________________________________________________

DEFINITION

Influenza vaccine (split virion, inactivated) is a sterile, aqueous suspension of a strain or strains ofinfluenza virus, type A or B, or a mixture of strains of the two types grown individually in fertilisedhens’ eggs, inactivated and treated so that the integrity of the virus particles has been disruptedwithout diminishing the antigenic properties of the haemagglutinin and neuraminidase antigens. Thestated amount of haemagglutinin antigen for each strain present in the vaccine is 15 µg per dose,unless clinical evidence supports the use of a different amount.

The vaccine is a slightly opalescent liquid.

PRODUCTION







VIRUS SEED LOT








To limit the possibility of contamination, inactivation is initiated as soon as possible after prepara-tion. The virus is inactivated by a method that has been demonstrated on three consecutive batchesto be consistently effective for the manufacturer. The inactivation process shall have been shown tobe capable of inactivating the influenza virus without destroying its antigenicity; the process shouldcause minimum alteration of the haemagglutinin and neuraminidase antigens. The inactivationprocess shall also have been shown to be capable of inactivating avian leucosis viruses andmycoplasmas. If the monovalent pooled harvest is stored after inactivation, it is held at a temperature

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of 5 ± 3°C. If formaldehyde solution is used, the concentration does not exceed 0.2 g/l of CH2O atany time during inactivation; if betapropiolactone is used, the concentration does not exceed 0.1 percent V/V at any time during inactivation.

Before or after the inactivation procedure, the monovalent pooled harvest is concentrated andpurified by high-speed centrifugation or other suitable method and the virus particles are disruptedinto component subunits by the use of approved procedures. For each new strain, a validation test iscarried out to show that the monovalent bulk consists predominantly of disrupted virus particles.


Haemagglutinin antigen Determine the content of haemagglutinin antigen by an immunodiffusiontest (2.7.1), by comparison with a haemagglutinin antigen reference preparation or with an antigenpreparation calibrated against it( 1). Carry out the test at 20°C to 25°C.

For some vaccines, the physical form of the haemagglutinin particles prevents quantitativedetermination by immunodiffusion after inactivation of the virus. For these vaccines, a determinationof haemagglutinin antigen is made on the monovalent pooled harvest before inactivation. Theproduction process is validated to demonstrate suitable conservation of haemagglutinin antigen and asuitable tracer is used for formulation, for example, protein content.




Chemicals used for disruption Tests are carried out on the monovalent pooled harvest for thechemicals used for disruption, the limits being approved by the competent authority.

FINAL BULK VACCINE





FINAL LOT



IDENTIFICATION


TESTS


Total protein Not more than six times the total haemagglutinin content of the vaccine as deter-mined in the assay, but in any case, not more than 100 µg of protein per virus strain per human doseand not more than a total of 300 µg of protein per human dose.



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Ovalbumin Not more than 1 µg of ovalbumin per human dose, determined by a suitabletechnique using a suitable reference preparation of ovalbumin.

Sterility It complies with the test for sterility (2.6.1).


ASSAY

Determine the content of haemagglutinin antigen by an immunodiffusion test (2.7.1), bycomparison with a haemagglutinin antigen reference preparation or with an antigen preparationcalibrated against it(1). Carry out the test at 20°C to 25°C. The confidence interval (P = 0.95) ofthe assay is not greater than 80 per cent to 125 per cent of the estimated content. The lowerconfidence limit (P = 0.95) of the estimate of haemagglutinin antigen content is not less than 80 percent of the amount stated on the label for each strain.

For some vaccines, quantitative determination of haemagglutinin antigen with respect toavailable reference preparations is not possible. An immunological identification of thehaemagglutinin antigen and a semi-quantitative determination are carried out instead by suitablemethods.

STORAGE


LABELLING



1Reference haemagglutinin antigens are availablefrom the National Institute for BiologicalStandards and Control, Blanche Lane, South Mimms, Potters Bar, Hertfordshire EN6 3QG,Great Britain.

__________________________________________________________________________________________________________ Ph Eur

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Inactivated Influenza Vaccine (Surface Antigen)

Inactivated Influenza Vaccine (Surface Antigen) complies with the requirements of the 3rd edition of theEuropean Pharmacopoeia [0869]. These requirements are reproduced after the heading ‘Definition’ below.

The label may state ‘Flu/Vac/SA’.

When Inactivated Influenza Vaccine or Influenza Vaccine is prescribed or demanded and the form isnot stated, Inactivated Influenza Vaccine (Whole Virion), Inactivated Influenza Vaccine (SplitVirion) or Inactivated Influenza Vaccine (Surface Antigen) may be dispensed or supplied.

Ph Eur ___________________________________________________________________________________________________________

DEFINITION

Influenza vaccine (surface antigen, inactivated) is a sterile, aqueous suspension of a strain or strains ofinfluenza virus, type A or B, or a mixture of strains of the two types grown individually in fertilisedhens’ eggs, inactivated and treated so that the preparation consists predominantly of haemagglutininand neuraminidase antigens, without diminishing the antigenic properties of these antigens. Thestated amount of haemagglutinin antigen for each strain present in the vaccine is 15 µg per dose,unless clinical evidence supports the use of a different amount.

The vaccine is a clear liquid.

PRODUCTION







VIRUS SEED LOT








To limit the possibility of contamination, inactivation is initiated as soon as possible after prepara-tion. The virus is inactivated by a method that has been demonstrated on three consecutive batchesto be consistently effective for the manufacturer. The inactivation process shall have been shown tobe capable of inactivating the influenza virus without destroying its antigenicity; the process shouldcause minimum alteration of the haemagglutinin and neuraminidase antigens. The inactivationprocess shall also have been shown to be capable of inactivating avian leucosis viruses and

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mycoplasmas. If the monovalent pooled harvest is stored after inactivation, it is held at a tempera-ture of 5 ± 3°C. If formaldehyde solution is used, the concentration does not exceed 0.2 g/l ofCH2O at any time during inactivation; if betapropiolactone is used, the concentration does notexceed 0.1 per cent V/V at any time during inactivation.

Before or after the inactivation process, the monovalent pooled harvest is concentrated andpurified by high-speed centrifugation or other suitable method. Virus particles are disrupted intocomponent subunits by approved procedures and further purified so that the monovalent bulkconsists mainly of haemagglutinin and neuraminidase antigens.


Haemagglutinin antigen Determine the content of haemagglutinin antigen by an immunodiffusiontest (2.7.1), by comparison with a haemagglutinin antigen reference preparation or with an antigenpreparation calibrated against it(1). Carry out the test at 20°C to 25°C.




Purity The purity of the monovalent pooled harvest is examined by polyacrylamide gel electro-phoresis or by other approved techniques. Mainly haemagglutinin and neuraminidase antigens shallbe present.

Chemicals used for disruption and purification Tests are carried out on the monovalent pooledharvest for the chemicals used for disruption and purification, the limits being approved by thecompetent authority.

FINAL BULK VACCINE





FINAL LOT



IDENTIFICATION


TESTS


Total protein Not more than 40 µg of protein other than haemagglutinin per virus strain per humandose and not more than a total of 120 µg of protein other than haemagglutinin per human dose.



Ovalbumin Not more than 1 µg of ovalbumin per human dose, determined by a suitable techniqueusing a suitable reference preparation of ovalbumin.

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Sterility It complies with the test for sterility (2.6.1).


ASSAY

Determine the content of haemagglutinin antigen by an immunodiffusion test (2.7.1), by comparisonwith a haemagglutinin antigen reference preparation or with an antigen preparation calibrated againstit(1). Carry out the test at 20°C to 25°C. The confidence interval (P = 0.95) of the assay is not greaterthan 80 per cent to 125 per cent of the estimated content. The lower confidence limit (P = 0.95) ofthe estimate of haemagglutinin antigen content is not less than 80 per cent of the amount stated onthe label for each strain.

STORAGE


LABELLING



1Reference haemagglutinin antigens are availablefrom the National Institute for BiologicalStandards and Control, Blanche Lane, South Mimms, Potters Bar, Hertfordshire EN6 3QG,United Kingdom.__________________________________________________________________________________________________________ Ph Eur

68-46

Measles Vaccine, Live

Measles Vaccine, Live complies with the requirements of the 3rd edition of the European Pharmacopoeia forMeasles Vaccine (Live) [0213]. These requirements are reproduced after the heading ‘Definition’ below.

The label may state ‘Meas/Vac(Live)’.

Ph Eur ___________________________________________________________________________________________________________

DEFINITION

Measles vaccine (live) is a freeze-dried preparation of a suitable attenuated strain of measles virus.The vaccine is reconstituted immediately before use, as stated on the label, to give a clear liquid thatmay be coloured owing to the presence of a pH indicator.

PRODUCTION

The production of vaccine is based on a virus seed-lot system and, if the virus is propagated inhuman diploid cells, a cell-bank system. The production method shall have been shown to yieldconsistently live measles vaccines of adequate immunogenicity and safety in man. Unless otherwisejustified and authorised, the virus in the final vaccine shall have undergone no more passages fromthe master seed lot than were used to prepare the vaccine shown in clinical studies to be satisfactorywith respect to safety and efficacy; even with authorised exceptions, the number of passages beyondthe level used for clinical studies shall not exceed five.



The virus is propagated in human diploid cells (5.2.3) or in cultures of chick-embryo cells derivedfrom a chicken flock free from specified pathogens (5.2.2).

SEED LOT

The strain of measles virus used shall be identified by historical records that include information onthe origin of the strain and its subsequent manipulation. To avoid the unnecessary use of monkeys inthe test for neurovirulence, virus seed lots are prepared in large quantities and stored at temperaturesbelow –20°C if freeze-dried, or below –60°C if not freeze-dried.


Identification The master and working seed lots are identified as measles virus by serum neutralisa-tion in cell culture, using specific antibodies.

Virus concentration The virus concentration of the master and working seed lots is determined tomonitor consistency of production.

Extraneous agents (2.6.16). The working seed lot complies with the requirements for seed lots.

Neurovirulence (2.6.18). The working seed lot complies with the test for neurovirulence of livevirus vaccines. Macaca and Cercopithecus monkeys susceptible to measles virus are suitable for thetest.


All processing of the cell bank and subsequent cell cultures is done under aseptic conditions in anarea where no other cells are handled. Suitable animal (but not human) serum may be used in thegrowth medium, but the final medium for maintaining cell growth during virus multiplication doesnot contain animal serum. Serum and trypsin used in the preparation of cell suspensions and culturemedia are shown to be free from extraneous agents. The cell culture medium may contain a pHindicator such as phenol red and suitable antibiotics at the lowest effective concentration. It ispreferable to have a substrate free from antibiotics during production. Not less than 500 ml of theproduction cell culture is set aside as uninfected cell cultures (control cells). The viral suspensions areharvested at a time appropriate to the strain of virus being used.

Only a single harvest that complies with the following requirements may be used in the preparationof the final bulk vaccine.

Identification The single harvest contains virus that is identified as measles virus by serum neutral-isation in cell culture, using specific antibodies.

Virus concentration The virus concentration in the single harvest is determined as prescribed underAssay to monitor consistency of production and to determine the dilution to be used for the finalbulk vaccine.

Extraneous agents (2.6.16). The single harvest complies with the tests for extraneous agents.

Control cells If human diploid cells are used for production, the control cells comply with a test foridentification. They comply with the tests for extraneous agents (2.6.16).

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FINAL BULK VACCINE

Virus harvests that comply with the above tests are pooled and clarified to remove cells. A suitablestabiliser may be added and the pooled harvests diluted as appropriate.

Only a final bulk vaccine that complies with the following requirement may be used in the prepara-tion of the final lot.

Bacterial and fungal contamination The final bulk vaccine complies with the test for sterility(2.6.1), carried out using 10 ml for each medium.

FINAL LOT

A minimum virus concentration for release of the product is established such as to ensure, in the lightof stability data, that the minimum concentration stated on the label will be present at the end of theperiod of validity.

Only a final lot that complies with the requirements for minimum virus concentration for release,with the following requirement for thermal stability and with each of the requirements given belowunder Identification and Tests may be released for use. Provided that the test for bovine serumalbumin has been carried out with satisfactory results on the final bulk vaccine, it may be omitted onthe final lot.

Thermal stability Maintain samples of the final lot of freeze-dried vaccine in the dry state at 37°Cfor 7 days. Determine the virus concentration as described under Assay in parallel for the heatedvaccine and for unheated vaccine stored at 5 ± 3°C. The virus concentration of the heated vaccine isnot more than 1.0 log10 lower than that of the unheated vaccine.

IDENTIFICATION

When the vaccine reconstituted as stated on the label is mixed with specific measles antibodies, it isno longer able to infect susceptible cell cultures.

TESTS

Bacterial and fungal contamination The reconstituted vaccine complies with the test for sterility(2.6.1).

Bovine serum albumin Not more than 50 ng per single human dose, determined by a suitableimmunochemical method (2.7.1).


ASSAY

Titrate the vaccine for infective virus at least in triplicate, using at least five cell cultures for each 0.5log10 dilution step or by a method of equal precision. Use an appropriate virus reference preparationto validate each assay. The estimated virus concentration is not less than that stated on the label; theminimum virus concentration stated on the label is not less than 1 × 103 CCID50 per human dose.The assay is not valid if the confidence interval (P = 0.95) of the logarithm of the virus concentrationis greater than ± 0.3.

STORAGE


LABELLING


The label states:— the strain of virus used for the preparation of the vaccine,— the type and origin of the cells used for the preparation of the vaccine,— the minimum virus concentration,— that contact with disinfectants is to be avoided,— the time within which the vaccine must be used after reconstitution.

__________________________________________________________________________________________________________ Ph Eur

69-1

Measles, Mumps and Rubella Vaccine, Live

Measles, Mumps and Rubella Vaccine, Live complies with the requirements of the 3rd edition of the EuropeanPharmacopoeia for Measles, Mumps and Rubella Vaccine (Live)[1057]. These requirements are reproducedafter the heading ‘Definition’ below.

The label may state ‘MMR/Vac(Live)’.

Ph Eur _______________________________________________________________________________________________________________

DEFINITION

Measles, mumps and rubella vaccine (live) is a freeze-dried preparation of suitable attenuated strainsof measles virus, mumps virus and rubella virus.

The vaccine is reconstituted immediately before use, as stated on the label, to give a clear liquidthat may be coloured owing to the presence of a pH indicator.

PRODUCTION

The three components are prepared as described in the monographs on Measles vaccine (live) (213),Mumps vaccine (live) (538) and Rubella vaccine (live) (162) and comply with the requirementsprescribed therein.


FINAL BULK VACCINE

Virus harvests for each component are pooled and clarified to remove cells. A suitable stabiliser maybe added and the pooled harvests diluted as appropriate. Suitable quantities of the pooled harvest foreach component are mixed.



FINAL LOT

For each component, a minimum virus concentration for release of the product is established such asto 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 requirements for minimum virus concentration of eachcomponent for release, with the following requirement for thermal stability and with each of therequirements given below under Identification and Tests may be released for use. Provided that thetests for bovine serum albumin and, where applicable, for ovalbumin have been carried out withsatisfactory results on the final bulk vaccine, they 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°Cfor 7 days. Determine the virus concentration as described under Assay in parallel for the heatedvaccine and for unheated vaccine stored at 5 ± 3°C. For each component, the virus concentration ofthe heated vaccine is not more than 1.0 log10 lower than that of the unheated vaccine.

IDENTIFICATION

When the vaccine reconstituted as stated on the label is mixed with antibodies specific for measlesvirus, mumps virus and rubella virus, it is no longer able to infect cell cultures susceptible to theseviruses. When the vaccine reconstituted as stated on the label is mixed with quantities of specificantibodies sufficient to neutralise any two viral components, the third viral component infectssusceptible cell cultures.

TESTS



Ovalbumin If the mumps component is produced in chick embryos, the vaccine contains not morethan 1 µg of ovalbumin per single human dose, determined by a suitable immunochemical method(2.7.1).


ASSAY

A. Mix the vaccine with a sufficient quantity of antibodies specific for mumps virus. Titrate the

69-2

vaccine for infective measles virus at least in triplicate, using at least five cell cultures for each 0.5 log10dilution step or by a method of equal precision. Use an appropriate virus reference preparation tovalidate each assay. The estimated measles virus concentration is not less than that stated on the label;the minimum measles virus concentration stated on the label is not less than 1 × 103 CCID50 per singlehuman dose. The assay is not valid if the confidence interval (P = 0.95) of the logarithm of the virusconcentration is greater than ± 0.3.

B. Mix the vaccine with a sufficient quantity of antibodies specific for measles virus. Titrate the vaccinefor infective mumps virus at least in triplicate, using at least five cell cultures for each 0.5 log10 dilutionstep or by a method of equal precision. Use an appropriate virus reference preparation to validate eachassay. The estimated mumps virus concentration is not less than that stated on the label; the minimummumps virus concentration stated on the label is not less than 5 × 103 CCID50 per single human dose.The assay is not valid if the confidence interval (P = 0.95) of the logarithm of the virus concentration isgreater than ± 0.3.

C. Mix the vaccine with a sufficient quantity of antibodies specific for mumps virus. Titrate the vaccinefor infective rubella virus at least in triplicate, using at least five cell cultures for each 0.5 log10 dilutionstep or by a method of equal precision. Use an appropriate virus reference preparation to validate eachassay. The estimated rubella virus concentration is not less than that stated on the label; the minimumrubella virus concentration stated on the label is not less than 1 × 103 CCID50 per single human dose.The assay is not valid if the confidence interval (P = 0.95) of the logarithm of the virus concentration isgreater than ± 0.3.

STORAGE


LABELLING


The label states:— the strains of virus used in the preparation of the vaccine,— where applicable, that chick embryos have been used for the preparation of the vaccine,— the type and origin of the cells used for the preparation of the vaccine,— the minimum virus concentration for each component of the vaccine,— that contact with disinfectants is to be avoided,— the time within which the vaccine must be used after reconstitution,— that the vaccine must not be given to a pregnant woman and that a woman must not become

pregnant within 2 months after having the vaccine.__________________________________________________________________________________________________________ Ph Eur

69-3

Meningococcal Polysaccharide Vaccine

Meningococcal Polysaccharide Vaccine complies with the requirements of the 3rd edition of the EuropeanPharmacopoeia [0250]. These requirements are reproduced after the heading ‘Definition’ below.

The label may state ‘Neimen/Vac’.

Ph Eur ___________________________________________________________________________________________________________

DEFINITION

Meningococcal polysaccharide vaccine is a freeze-dried preparation of one or more purified capsularpolysaccharides obtained from one or more suitable strains of Neisseria meningitidis group A, group C,group Y and group W135 that are capable of consistently producing polysaccharides.

N. meningitidis group A polysaccharide consists of partly O-acetylated repeating units of N-acetyl-mannosamine, linked with 1a® 6 phosphodiester bonds.

N. meningitidis group C polysaccharide consists of partly O-acetylated repeating units of sialic acid,linked with 2a® 9 glycosidic bonds.

N. meningitidis group Y polysaccharide consists of partly O-acetylated alternating units of sialic acidand D-glucose, linked with 2a® 6 and 1a® 4 glycosidic bonds.

N. meningitidis group W135 polysaccharide consists of partly O-acetylated alternating units of sialicacid and D-galactose, linked with 2a® 6 and 1a® 4 glycosidic bonds.

The polysaccharide component or components stated on the label together with calcium ions andresidual moisture account for over 90 per cent of the mass of the preparation.

Meningococcal polysaccharide vaccine complies with the monograph on Vaccines for human use(153).

PRODUCTION

Production of the meningococcal polysaccharides is based on a seed-lot system. The method ofproduction shall have been shown to yield consistently meningococcal polysaccharide vaccines ofsatisfactory immunogenicity and safety for man.


SEED LOTS

The strains of N. meningitidis used for the master seed lots shall be identified by historical records thatinclude information on their origin and by their biochemical and serological characteristics.

Cultures from the working seed lot shall have the same characteristics as the strain that was used toprepare the master seed lot. The strains have the following characteristics:— colonies obtained from a culture are rounded, uniform in shape and smooth with a mucous,

opalescent, greyish appearance,— Gram staining reveals characteristic Gram-negative diplococci in ‘coffee-bean’ arrangement,— the oxidase test is positive,— the culture utilises glucose and maltose,— suspensions of the culture agglutinate with suitable specific antisera.


The working seed lots are cultured on solid media that do not contain blood-group substances oringredients of mammalian origin. The inoculum may undergo one or more subcultures in liquidmedium before being used for inoculating the final medium. The liquid media used and the finalmedium are semisynthetic and free from substances precipitated by cetrimonium bromide(hexadecyltrimethylammonium bromide) and do not contain blood-group substances or high-molecular-mass polysaccharides.

The bacterial purity of the culture is verified by microscopic examination of Gram-stained smearsand by inoculation into appropriate media; several fields are observed at high magnification so that atleast 10,000 organisms are examined.

The cultures are centrifuged and the polysaccharides precipitated from the supernatant by additionof cetrimonium bromide. The precipitate obtained is harvested and may be stored at –20°C awaitingfurther purification.

PURIFIED POLYSACCHARIDES

The polysaccharides are purified, after dissociation of the complex of polysaccharide andcetrimonium bromide, using suitable procedures to remove successively nucleic acids, proteins andlipopolysaccharides.

The final purification step consists of ethanol precipitation of the polysaccharides which are then

69-4

dried and stored at –20°C. The loss on drying is determined by thermogravimetry (2.2.34) and thevalue is used to calculate the results of the other chemical tests with reference to the dried substance.

Only purified polysaccharides that comply with the following requirements may be used in thepreparation of the final bulk vaccine.

Protein (2.5.16). Not more than 10 mg of protein per gram of purified polysaccharide, calculatedwith reference to the dried substance.

Nucleic acids (2.5.17). Not more than 10 mg of nucleic acids per gram of purified polysaccharide,calculated with reference to the dried substance.

O-Acetyl groups (2.5.19). Not less than 2 mmol of O-acetyl groups per gram of purified poly-saccharide for group A, not less than 1.5 mmol per gram of polysaccharide for group C, not less than0.3 mmol per gram of polysaccharide for groups Y and W135, all calculated with reference to thedried substance.

Phosphorus (2.5.18). Not less than 80 mg of phosphorus per gram of group A purified poly-saccharide, calculated with reference to the dried substance.

Sialic acid (2.5.23). Not less than 800 mg of sialic acid per gram of group C polysaccharide and notless than 560 mg of sialic acid per gram of purified polysaccharide for groups Y and W135, all calcu-lated with reference to the dried substance. Use the following reference solutions:

Group C polysaccharide: a 150 mg/l solution of N-acetylneuraminic acid R.

Group Y polysaccharide: a solution containing 95 mg/l of N-acetylneuraminic acid R and 55 mg/l ofglucose R.

Group W135 polysaccharide: a solution containing 95 mg/l of N-acetylneuraminic acid R and 55 mg/lof galactose R.

Calcium If a calcium salt is used during purification, a determination of calcium is carried out onthe purified polysaccharide; the content is within the limits approved for the product.

Distribution of molecular size Examine by size-exclusion chromatography (2.2.30), using agarosefor chromatography R or cross-linked agarose for chromatography R. Use a column about 0.9 m long and16 mm in internal diameter equilibrated with a solvent having an ionic strength of 0.2 mol/kg and apH of 7.0 to 7.5. Apply to the column about 2.5 mg of polysaccharide in a volume of about 1.5 mland elute at about 20 ml/h. Collect fractions of about 2.5 ml and determine the content of poly-saccharide by a suitable method.

At least 65 per cent of group A polysaccharide, 75 per cent of group C polysaccharide, 80 per centof group Y polysaccharide and 80 per cent of group W135 polysaccharide is eluted before a distribu-tion coefficient (KD) of 0.50 is reached. In addition, the percentages eluted before this distributioncoefficient are within the limits approved for the particular product.

Identification and serological specificity The identity and serological specificity are determinedby a suitable immunochemical method (2.7.1). Identity and purity of each polysaccharide shall beconfirmed; it shall be shown that there is not more than 1 per cent m/m of group-heterologous N.meningitidis polysaccharide.

Pyrogens (2.6.8). The polysaccharide complies with the test for pyrogens. Inject into each rabbit perkilogram of body mass 1 ml of a solution containing 0.025 µg of purified polysaccharide per millilitre.

FINAL BULK VACCINE

One or more purified polysaccharides of one or more N. meningitidis groups are dissolved in a suitablesolvent that may contain a stabiliser. When dissolution is complete, the solution is filtered through abacteria-retentive filter.



FINAL LOT

The final bulk vaccine is distributed aseptically into sterile containers. The containers are then closedso as to avoid contamination.

Only a final lot that is satisfactory with respect to each of the requirements prescribed below underIdentification, Tests and Assay may be released for use.

CHARACTERS

A white or cream-coloured powder or pellet, freely soluble in water.

IDENTIFICATION

Carry out an identification test for each polysaccharide present in the vaccine by a suitableimmunochemical method (2.7.1).

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TESTS

Distribution of molecular size Examine by size-exclusion chromatography (2.2.30). Use a columnabout 0.9 m long and 16 mm in internal diameter equilibrated with a solvent having an ionic strengthof 0.2 mol/kg and a pH of 7.0 to 7.5. Apply to the column about 2.5 mg of each polysaccharide in avolume of about 1.5 ml and elute at about 20 ml/h. Collect fractions of about 2.5 ml and determine thecontent of polysaccharide by a suitable method.

For a divalent vaccine (group A + group C), use cross-linked agarose for chromatography R. Thevaccine complies with the test if:— 65 per cent of group A polysaccharide is eluted before KD = 0.50,— 75 per cent of group C polysaccharide is eluted before KD = 0.50.

For a tetravalent vaccine (group A+ group C + group Y + group W135), use cross-linked agarose forchromatography R1 and apply a suitable immunochemical method (2.7.1) to establish the elutionpattern of the different polysaccharides. The vaccine complies with the test if KD for the principal peakis:— not greater than 0.70 for group A and group C polysaccharide,— not greater than 0.57 for group Y polysaccharide,— not greater than 0.68 for group W135 polysaccharide.



Pyrogens (2.6.8). It complies with the test for pyrogens. Inject per kilogram of the rabbit’s mass 1 mlof a solution containing:— 0.025 µg of polysaccharide for a monovalent vaccine,— 0.050 µg of polysaccharide for a divalent vaccine,— 0.10 µg of polysaccharide for a tetravalent vaccine.

ASSAY

Carry out an assay of each polysaccharide present in the vaccine.

For a divalent vaccine (group A +group C), use measurement of phosphorus (2.5.18) to determine thecontent of polysaccharide A and measurement of sialic acid (2.5.23) to determine the content of poly-saccharide C. To determine sialic acid, use as reference solution a 150 mg/l solution of N-acetyl-neuraminic acid R.

For a tetravalent vaccine (group A + group C + group Y + group W135) a suitable immunochemicalmethod (2.7.1) is used with a reference preparation of purified polysaccharide for each group.

The vaccine contains not less than 70 per cent and not more than 130 per cent of the quantity ofeach polysaccharide stated on the label.

STORAGE


LABELLING


The label states:— the group or groups of polysaccharides (A, C, Y or W135) present in the vaccine,— the number of micrograms of polysaccharide per human dose.__________________________________________________________________________________________________________ Ph Eur

69-6

Mumps Vaccine, Live

Mumps Vaccine, Live complies with the requirements of the 3rd edition of the European Pharmacopoeia forMumps Vaccine (Live) [0538]. These requirements are reproduced after the heading ‘Definition’ below.

The label may state ‘Mump/Vac(Live)’.

Ph Eur ___________________________________________________________________________________________________________

DEFINITION

Mumps vaccine (live) is a freeze-dried preparation of a suitable attenuated strain of mumps virus.The vaccine is reconstituted immediately before use, as stated on the label, to give a clear liquid thatmay be coloured owing to the presence of a pH indicator.

PRODUCTION

The production of vaccine is based on a virus seed-lot system and, if the virus is propagated inhuman diploid cells, a cell-bank system. The production method shall have been shown to yieldconsistently live mumps vaccines of adequate immunogenicity and safety in man. Unless otherwisejustified and authorised, the virus in the final vaccine shall have undergone no more passages fromthe master seed lot than were used to prepare the vaccine shown in clinical studies to be satisfactorywith respect to safety and efficacy.



The virus is propagated in human diploid cells (5.2.3) or in chick-embryo cells or in the amnioticcavity of chick embryos derived from a chicken flock free from specified pathogens (5.2.2).

SEED LOT

The strain of mumps virus used shall be identified by historical records that include information onthe origin of the strain and its subsequent manipulation. To avoid the unnecessary use of monkeys inthe test for neurovirulence, virus seed lots are prepared in large quantities and stored at temperaturesbelow –20°C if freeze-dried, or below –60°C if not freeze-dried.


Identification The master and working seed lots are identified as mumps virus by serum neutralisa-tion in cell culture, using specific antibodies.

Virus concentration The virus concentration of the master and working seed lots is determined toensure consistency of production.


Neurovirulence (2.6.18). The working seed lot complies with the test for neurovirulence of livevirus vaccines. Macaca and Cercopithecus monkeys are suitable for the test.


All processing of the cell bank and subsequent cell cultures is done under aseptic conditions in anarea where no other cells are handled. Suitable animal (but not human) serum may be used in theculture media. Serum and trypsin used in the preparation of cell suspensions and culture media areshown to be free from extraneous agents. The cell culture medium may contain a pH indicator suchas phenol red and suitable antibiotics at the lowest effective concentration. It is preferable to have asubstrate free from antibiotics during production. Not less than 500 ml of the production cellcultures is set aside as uninfected cell cultures (control cells). If the virus is propagated in chickembryos, 2 per cent but not less than twenty eggs are set aside as uninfected control eggs. The viralsuspensions are harvested at a time appropriate to the strain of virus being used.


Identification The single harvest contains virus that is identified as mumps virus by serum neutral-isation in cell culture, using specific antibodies.

Virus concentration The virus concentration in the single harvest is determined as prescribed underAssay to monitor consistency of production and to determine the dilution to be used for the finalbulk vaccine.


Control cells or eggs If human diploid cells are used for production, the control cells comply with atest for identification; the control cells and the control eggs comply with the tests for extraneousagents (2.6.16).

69-7

FINAL BULK VACCINE

Single harvests that comply with the above tests are pooled and clarified to remove cells. A suitablestabiliser may be added and the pooled harvests diluted as appropriate.



FINAL LOT


Only a final lot that complies with the requirements for minimum virus concentration for release,with the following requirement for thermal stability and with each of the requirements given belowunder Identification and Tests may be released for use. Provided that the tests for bovine serumalbumin and, where applicable, for ovalbumin have been carried out with satisfactory results on thefinal bulk vaccine, they 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°C for7 days. Determine the virus concentration as described under Assay in parallel for the heated vaccineand for unheated vaccine stored at 5 ± 3°C. 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 mixed with specific mumps antibodies, it is nolonger able to infect susceptible cell cultures.

TESTS



Ovalbumin If the vaccine is produced in chick embryos, it contains not more than 1 µg of ovalbuminper single human dose, determined by a suitable immunochemical method (2.7.1).


ASSAY

Titrate the vaccine for infective virus at least in triplicate, using at least five cell cultures for each 0.5log10 dilution step or by a method of equal precision. Use an appropriate virus reference preparation tovalidate each assay. The estimated virus concentration is not less than that stated on the label; theminimum virus concentration stated on the label is not less than 5 × 103 CCID50 per human dose. Theassay is not valid if the confidence interval (P = 0.95) of the logarithm of the virus concentration isgreater than ± 0.3.

STORAGE


LABELLING


The label states:— the strain of virus used for the preparation of the vaccine,— that the vaccine has been prepared in chick embryos or the type and origin of cells used for the

preparation of the vaccine,— the minimum virus concentration,— that contact with disinfectants is to be avoided,— the time within which the vaccine must be used after reconstitution.__________________________________________________________________________________________________________ Ph Eur

69-8

Pertussis VaccineWhooping-cough Vaccine

When Pertussis Vaccine is issued as a plain vaccine, it complies with the requirements of the 3rd edition of theEuropean Pharmacopoeia for Pertussis Vaccine [0160]. These requirements are reproduced after the heading‘Plain Vaccine’ below.

When Pertussis Vaccine is issued as an adsorbed vaccine, it complies with the requirements of the 3rd edition ofthe European Pharmacopoeia for Pertussis Vaccine (Adsorbed) [0161]. These requirements are reproducedafter the heading ‘Adsorbed Vaccine’ below.

The label may state ‘Per/Vac’ or ‘Per/Vac/Ads’, as appropriate.

When Pertussis Vaccine is prescribed or demanded and the form is not stated, either the plain or theadsorbed vaccine may be dispensed or supplied.

Ph Eur ___________________________________________________________________________________________________________

PLAIN VACCINE [0160]

DEFINITION

Pertussis vaccine is a sterile saline suspension of inactivated whole cells of one or more strains ofBordetella pertussis.

PRODUCTION

Production of the vaccine is based on a seed-lot system. One or more strains of B. pertussis withknown origin and history are used. Strains, culture medium and cultivation method are chosen insuch a way that agglutinogens 1, 2 and 3 are present in the final vaccine. Each strain is grown for 24 hto 72 h in a liquid medium or on a solid medium; the medium used in the final cultivation stage doesnot contain blood or blood products. Human blood or blood products are not used in any culturemedia. The bacteria are harvested, washed to remove substances derived from the medium andsuspended in a 9 g/l solution of sodium chloride or other suitable isotonic solution. The opacity of thesuspension is determined not later than 2 weeks after harvest by comparison with the Interna-tional Reference Preparation of Opacity and used as the basis of calculation for subsequent stages invaccine preparation. The equivalence in International Units of the International Reference Prepara-tion is stated by the World Health Organisation.


FINAL BULK VACCINE

Suitable quantities of the inactivated single harvests are pooled to prepare the final bulk vaccine.Suitable antimicrobial preservatives may be added. The bacterial concentration of the final bulkvaccine does not exceed that corresponding to an opacity of 20 I.U. per single human dose. If two ormore strains of B. pertussis are used, the composition of consecutive lots of the final bulk vaccine shallbe consistent with respect to the proportion of each strain as measured in opacity units.

Only a final bulk vaccine that complies with the following requirements may be used in the prepara-tion of the final lot.




FINAL LOT



IDENTIFICATION

Identify pertussis vaccine by agglutination of the bacteria in the vaccine by antisera specific to B.pertussis.

69-9

TESTS

Specific toxicity Use not fewer than ten healthy mice each weighing 14 g to 16 g for the vaccinegroup 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 and water for at least 2 h before injection andduring the test. Inject each mouse of the vaccine group intraperitoneally with 0.5 ml, containing aquantity of the vaccine equivalent to not less than half the single human dose. Inject each mouse ofthe control group with 0.5 ml of a 9 g/l sterile solution of sodium chloride R, preferably containing thesame amount of antimicrobial preservative as that injected with the vaccine. Weigh the groups ofmice immediately before the injection and 72 h and 7 days after the injection. The vaccine complieswith the test if: (a) at the end of 72 h the total mass of the group of vaccinated mice is not less thanthat preceding the injection; (b) at the end of 7 days the average increase in mass per vaccinatedmouse is not less than 60 per cent of that per control mouse; and (c) not more than 5 per cent of thevaccinated mice die during the test.

Free formaldehyde When the bacteria are inactivated with formaldehyde, the vaccine complies withthe test prescribed in the monograph on Vaccines for human use (153).



POTENCY

Carry out the assay of pertussis vaccine (2.7.7).The estimated potency is not less than 4 I.U. per single human dose and the lower confidence limit


STORAGE


LABELLING


The label states:— the minimum number of International Units per single human dose,— that the vaccine must be shaken before use,— that the vaccine is not to be frozen.

ADSORBED VACCINE [0161]

DEFINITION

Pertussis vaccine (adsorbed) is a sterile saline suspension of inactivated whole cells of one or morestrains of Bordetella pertussis, to which hydrated aluminium phosphate, aluminium hydroxide orcalcium phosphate has been added.

PRODUCTION

Production of the vaccine is based on a seed-lot system. One or more strains of B. pertussis withknown origin and history are used. Strains, culture medium and cultivation method are chosen insuch a way that agglutinogens 1, 2 and 3 are present in the final vaccine. Each strain is grown for24 h to 72 h in a liquid medium or on a solid medium; the medium used in the final cultivation stagedoes not contain blood or blood products. Human blood or blood products are not used in anyculture media. The bacteria are harvested, washed to remove substances derived from the mediumand suspended in a 9 g/l solution of sodium chloride or other suitable isotonic solution. The opacityof the suspension is determined not later than 2 weeks after harvest by comparison with the Interna-tional Reference Preparation of Opacity and used as the basis of calculation for subsequent stages invaccine preparation. The equivalence in International Units of the International Reference Prepara-tion is stated by the World Health Organisation.


FINAL BULK VACCINE

Suitable quantities of the inactivated single harvests are pooled to prepare the final bulk vaccine.Hydrated aluminium phosphate, aluminium hydroxide or calcium phosphate is added to the cellsuspension. Suitable antimicrobial preservatives may be added. The bacterial concentration of thefinal bulk vaccine does not exceed that corresponding to an opacity of 20 I.U. per single human dose.

69-10

If two or more strains of B. pertussis are used, the composition of consecutive lots of the final bulkvaccine shall be consistent with respect to the proportion of each strain as measured in opacity units.

Only a final bulk vaccine that complies with the following requirements may be used in thepreparation of the final lot.




FINAL LOT



IDENTIFICATION

Dissolve in the vaccine to be examined sufficient sodium citrate R to give a 100 g/l solution. Maintainat 37°C for about 16 h and centrifuge to obtain a bacterial precipitate. Other suitable methods forseparating the bacteria from the adsorbent may also be used. Identify pertussis vaccine by agglutina-tion of the bacteria from the resuspended precipitate by antisera specific to B. pertussis or by the assayprescribed under Potency.

TESTS

Specific toxicity Use not fewer than ten healthy mice each weighing 14 g to 16 g for the vaccinegroup 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 and water for at least 2 h before injection andduring the test. Inject each mouse of the vaccine group intraperitoneally with 0.5 ml, containing aquantity of the vaccine equivalent to not less than half the single human dose. Inject each mouse ofthe control group with 0.5 ml of a 9 g/l sterile solution of sodium chloride R, preferably containing thesame amount of antimicrobial preservative as that injected with the vaccine. Weigh the groups ofmice immediately before the injection and 72 h and 7 days after the injection. The vaccine complieswith the test if: (a) at the end of 72 h the total mass of the group of vaccinated mice is not less thanthat preceding the injection; (b) at the end of 7 days the average increase in mass per vaccinatedmouse is not less than 60 per cent of that per control mouse; and (c) not more than 5 per cent of thevaccinated mice die during the test.



Free formaldehyde When the bacteria are inactivated with formaldehyde, the vaccine complies withthe test prescribed in the monograph on Vaccines for human use (153).



POTENCY

Carry out the assay of pertussis vaccine (2.7.7).The estimated potency is not less than 4 I.U. per single human dose and the lower confidence limit


STORAGE


LABELLING


The label states:— the minimum number of International Units per single human dose,— the name and the amount of the adsorbent,— that the vaccine must be shaken before use,— that the vaccine is not to be frozen.

__________________________________________________________________________________________________________ Ph Eur

69-11

Adsorbed Pertussis Vaccine (Acellular Component)

Adsorbed Pertussis Vaccine (Acellular Component) complies with the requirements of the 3rd edition of theEuropean Pharmacopoeia for Pertussis Vaccine (Acellular, Component, Adsorbed) [1356]. These require-ments are reproduced after the heading ‘Definition’ below.

Ph Eur ___________________________________________________________________________________________________________

DEFINITION

Pertussis vaccine (acellular, component, adsorbed) is a preparation of individually prepared andpurified antigenic components of Bordetella pertussis adsorbed on a mineral carrier such as aluminiumhydroxide or hydrated aluminium phosphate.

The vaccine contains either pertussis toxoid or a pertussis-toxin-like protein free from toxicproperties, produced by expression of a genetically modified form of the corresponding gene.Pertussis toxoid is prepared from pertussis toxin by a method that renders the latter harmless whilemaintaining adequate immunogenic properties and avoiding reversion to toxin. The vaccine may alsocontain filamentous haemagglutinin, pertactin (a 69 kDa outer-membrane protein) and other definedcomponents of B. pertussis such as fimbrial-2 and fimbrial-3 antigens. The latter two antigens may becopurified. The antigenic composition and characteristics are based on evidence of protection andfreedom from unexpected reactions in the target group for which the vaccine is intended.

PRODUCTION

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

Where a genetically modified form of B. pertussis is used for production consistency and geneticstability shall be established in conformity with the requirements of the monograph Products ofrecombinant DNA technology (784).

Reference vaccine. A batch of vaccine shown to be effective in clinical trials or a batch representativethereof is used as a reference vaccine. For the preparation of a representative batch, strict adherenceto the production process used for the batch tested in clinical trials is necessary. The referencevaccine is preferably stabilised by a method that has been shown to have no significant effect on theassay procedure when the stabilised and non-stabilised batches are compared.

CHARACTERISATION OF COMPONENTS

During development of the vaccine, the production process shall be validated to demonstrate that ityields consistently individual components that comply with the following requirements; after demon-stration of consistency, the tests need not be applied routinely to each batch.

Adenylate cyclase Not more than 500 ng in the equivalent of one dose of the final vaccine, deter-mined by immunoblot analysis or another suitable method.

Tracheal cytotoxin Not more than 2 pmol in the equivalent of one dose of the final vaccine,determined by a suitable method such as a biological assay or liquid chromatography (2.2.29).

Absence of residual dermonecrotic toxin Inject intradermally into each of three unweaned mice,in a volume of 0.1 ml, the amount of component or antigenic fraction equivalent to one dose of thefinal vaccine. Observe for 48 h. No dermonecrotic reaction is demonstrable.

Specific properties The components of the vaccine are analysed by one or more of the methodsshown below in order to determine their identity and specific properties (activity per unit amount ofprotein) in comparison with reference preparations.

Pertussis toxin. Chinese hamster ovary (CHO) cell-clustering effect and haemagglutination as in vitromethods; lymphocytosis-promoting activity, histamine-sensitising activity and insulin secretoryactivity as in vivo methods. The toxin shows ADP-ribosyl transferase activity using transducin as theacceptor.

Filamentous haemagglutinin. Haemagglutination and inhibition by specific antibody.

Pertactin, fimbrial-2 and fimbrial-3 antigens. Reactivity with specific antibody.

Pertussis toxoid. The toxoid induces in animals production of antibodies capable of inhibiting all theproperties of pertussis toxin.

PURIFIED COMPONENTS

Production of each component is based on a seed-lot system. The seed cultures from which toxin isprepared are managed to conserve or where necessary restore toxinogenicity by deliberate selection.

None of the media used at any stage contains blood or blood products of human origin. Mediaused for the preparation of seed lots and inocula may contain blood or blood products of animalorigin.

69-12

Pertussis toxin and, where applicable, filamentous haemagglutinin and pertactin are purified and,after appropriate characterisation, detoxified using suitable chemical reagents, by a method thatavoids 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 separately or together, characterised andshown to be free from toxic substances. The purification procedure is validated to demonstrateappropriate clearance of substances used during culture or purification.

The content of bacterial endotoxins (2.6.14) is determined to monitor the purification procedureand to limit the amount in the final vaccine. The limits applied for the individual components aresuch that the final vaccine contains not more than 100 I.U. per single human dose.

Before detoxification, the purity of the components is determined by a suitable method such aspolyacrylamide gel electrophoresis (PAGE) or liquid chromatography. SDS-PAGE or immunoblotanalysis with specific monoclonal or polyclonal antibodies may be used to characterise subunits.Requirements are established for each individual product.

Only purified components that comply with the following requirements may be used in thepreparation of the final bulk vaccine.

Sterility (2.6.1). Carry out the test for sterility using for each medium a quantity of purifiedcomponent equivalent to not less 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 five histamine-sensitive mice each weighing 18 g to 26 g.Inject into each mouse the equivalent of one human dose intravenously or twice the human doseintraperitoneally, diluted to not more than 0.5 ml with phosphate-buffered saline solution containing2 g/l of gelatin. Inject diluent into a second group of control mice. After 5 days, inject 2 mg ofhistamine base intraperitoneally in a volume not exceeding 0.5 ml and observe for 24 h. If no animaldies, the preparation complies with the test.

The histamine sensitivity of the strain of mice used is verified at suitable intervals as follows: injectthreefold dilutions of a reference pertussis toxin preparation in phosphate-buffered saline solutioncontaining 2 g/l of gelatin and challenge with histamine as above; the strain is suitable if more than50 per cent of the animals are sensitised by 50 ng of pertussis toxin and none of the control animalsinjected with only diluent and challenged similarly with histamine show symptoms of sensitisation.

A validated test based on the clustering effect of the toxin for Chinese hamster ovary (CHO) cellsmay be used instead of the test in mice.

Residual detoxifying agents and other reagents The content of residual detoxifying agents andother reagents is determined and shown to be below approved limits unless validation of the processhas demonstrated acceptable clearance.

Antigen content Determine the antigen content by a suitable immunochemical method (2.7.1) andprotein nitrogen by sulphuric acid digestion (2.5.9) or another suitable method. The ratio of antigencontent to protein nitrogen is within the limits established for the product.

FINAL BULK VACCINE

The vaccine is prepared by adsorption of suitable quantities of purified components, separately ortogether, onto aluminium hydroxide or hydrated aluminium phosphate. A suitable antimicrobialpreservative may be added.


Antimicrobial preservative Where applicable, determine the amount of antimicrobial preservativeby a suitable chemical or physico-chemical method. The amount is not less than 85 per cent and notgreater than 115 per cent of the intended content.


FINAL LOT

Only a final lot that is satisfactory with respect to each of the requirements given below underIdentification, Tests and Assay may be released for use. Provided that the tests for absence of residualpertussis toxin, irreversibility of toxoid, antimicrobial preservative, free formaldehyde and the assayhave been carried out with satisfactory results on the final bulk vaccine, these tests may be omitted onthe final lot.

IDENTIFICATION

Subject the vaccine to a suitable desorption procedure such as the following: dissolve in the vaccineto be examined sufficient sodium citrate R to give a 10 g/l solution; maintain at 37°C for about 16 hand centrifuge until a clear supernatant liquid is obtained. Examined by a suitable immunochemicalmethod (2.7.1), the clear supernatant liquid reacts with specific antisera to the components stated onthe label.

TESTS

Absence of residual pertussis toxin This test is not necessary for the product obtained by geneticmodification. Inject intraperitoneally into a group of not fewer than five histamine-sensitive mice (see

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under Production) twice the single human dose. Inject diluent into a second group of control mice.After 5 days, inject 2 mg of histamine base intraperitoneally in a volume not exceeding 0.5 ml andobserve for 24 h. The vaccine complies with the test if the test animals do not show sensitisation tohistamine.

Irreversibility of toxoid This test is not necessary for the product obtained by genetic modification. Carryout the test for absence of residual pertussis toxin described above, using the vaccine incubated at37°C for 4 weeks in parallel with a sample stored at 2°C to 8°C. The vaccine complies with the test ifnone of the animals in either group dies due to histamine sensitisation.

Antimicrobial preservative Where applicable, determine the amount of antimicrobial preservativeby a suitable chemical or physico-chemical method. The amount is not less than the minimumamount shown to be effective and is not greater than 115 per cent of the quantity stated on the label.

Aluminium When aluminium hydroxide or hydrated aluminium phosphate has been used as theadsorbent, the vaccine complies with the test prescribed in Vaccines for human use (153).

Free formaldehyde When formaldehyde has been used in the preparation of the vaccine, itcomplies with the test prescribed in Vaccines for human use (153).


ASSAY

The capacity of the vaccine to induce the formation of specific antibodies is compared with the samecapacity of a reference preparation examined in parallel; antibodies are determined using suitableimmunochemical methods (2.7.1) such as enzyme-linked immunosorbent assay (ELISA). The test inmice shown below uses a three-point model but, after validation, for routine testing a single-dilutionmethod may be used.

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

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

Selection and distribution of test animals. Use in the test healthy mice (for example, CD1 strain) of thesame stock 4 to 8 weeks old. Distribute the animals in six groups of a number appropriate to therequirements of the assay. Use three dilutions of the vaccine to be examined and three dilutions of areference preparation and attribute each dilution to a group of mice. Inject intraperitoneally orsubcutaneously into each mouse 0.5 ml of the dilution attributed to its group.

Collection of serum samples. 4 to 5 weeks after vaccination, bleed the mice individually underanaesthesia. Store the sera at –20°C until tested for antibody content.

Antibody determination. Assay the individual sera for content of specific antibodies to each componentusing a validated method such as the ELISA test shown below.

ELISA test. Microtitre plates (poly(vinyl chloride) or polystyrene as appropriate for the specificantigen) are coated with the purified antigen at a concentration of 100 ng per well. After washing,unreacted sites are blocked by incubating with a solution of bovine serum albumin and then washed.Two-fold dilutions of sera from mice immunised with test or reference vaccines are made on theplates. After incubation at 22°C to 25°C for 1 h, the plates are washed. A suitable solution of anti-mouse IgG enzyme conjugate is added to each well and incubated at 22°C to 25°C for 1 h. Afterwashing, a substrate is added from which the bound enzyme conjugate liberates a chromophorewhich can be quantified by measurement of absorbance (2.2.25). The test conditions are designed toobtain a linear response for absorbance with respect to antibody content over the range of measure-ment used and absorbance values within the range 0.1 to 2.0.

A reference antiserum of assigned potency is used in the test and serves as the basis for calculation ofthe antibody levels in test sera. A standardised control serum is also included in the test.

The test is not valid if:— the value found for the control serum differs by more than two standard deviations from the

assigned value,— the confidence interval of the potency estimate is greater than 50 per cent to 200 per cent.

Calculation. The antibody titres in the sera of mice immunised with reference and test vaccines arecalculated and from the values obtained the potency of the test vaccine in relation to the referencevaccine is calculated by the usual statistical methods.

STORAGE


LABELLING


The label states:— the names and amounts of the components present in the vaccine,

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— where applicable, that the vaccine contains a pertussis toxin-like protein produced by geneticmodification,

— the name and amount of the adsorbent,— that the vaccine must be shaken before use,— that the vaccine is not to be frozen.__________________________________________________________________________________________________________ Ph Eur

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Pneumococcal Polysaccharide Vaccine

Pneumococcal Polysaccharide Vaccine complies with the requirements of the 3rd edition of the EuropeanPharmacopoeia [0966]. These requirements are reproduced after the heading ‘Definition’ below.

The label may state ‘Pneumo/Vac’.

Ph Eur _______________________________________________________________________________________________________________

DEFINITION

Pneumococcal polysaccharide vaccine consists of a mixture of equal parts of purified capsular poly-saccharide antigens prepared from suitable pathogenic strains of Streptococcus pneumoniae whosecapsules have been shown to be made up of polysaccharides that are capable of inducing satisfactorylevels of specific antibodies in man. It contains the twenty-three immunochemically different capsularpolysaccharides listed in the Table 966-1.

The vaccine is a clear, colourless liquid.

PRODUCTION

Production of the vaccine is based on a seed-lot system for each type. The production method shallhave been shown to yield consistently pneumococcal polysaccharide vaccines of adequate safety andimmunogenicity in man.

The production method is validated to demonstrate that the product, if tested, would comply withthe test for abnormal toxicity for immunosera and vaccines for human use (2.6.9) modified as followsfor the test in guinea-pigs: inject ten human doses into each guinea-pig and observe for 12 days.

MONOVALENT BULK POLYSACCHARIDES

The bacteria are grown in a suitable liquid medium that does not contain blood-group substances orhigh-molecular-mass polysaccharides. The bacterial purity of the culture is verified and the culture isinactivated with phenol. Impurities are removed by such techniques as fractional precipitation,enzymatic digestion and ultrafiltration. The polysaccharide is obtained by fractional precipitation,washed, and dried in a vacuum to a residual moisture content shown to be favourable to the stabilityof the polysaccharide. The residual moisture content is determined by drying under reduced pressureover diphosphorus pentoxide or by thermogravimetric analysis and the value obtained is used tocalculate the results of the tests shown below with reference to the dried substance. The monovalentbulk polysaccharide is stored at a suitable temperature in conditions that avoid the uptake ofmoisture.

Only a monovalent bulk polysaccharide that complies with the following requirements may be usedin the preparation of the final bulk vaccine. percentage contents of components, determined by themethods prescribed below, are shown in the Table 966-1.

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Table 966–1 Percentage contents of components of monovalent bulk polysaccharides

Molecular Type*

Proteins Nucleic acids

Total Nitrogen

Phosphorus Molecular size (KD) Uronic acids

Hexos- amines

Methyl-pentoses

O-acetyl Groups

** ***

1 ≤2 ≤2 3.5–6 0–1.5 ≤0.15 ≤45 ≤1.8

2 ≤2 ≤2 0–1 0–1.0 ≤0.15 ≤15 ≤38

3 ≤5 ≤2 0–1 0–1.0 ≤0.15 ≤40

4 ≤3 ≤2 4–6 0–1.5 ≤0.15 ≤40

5 ≤ 7.5 ≤2 2.5–6.0 ≤2 ≤60 ≤12 ≤20

6B ≤2 ≤2 0–2 2.5–5.0 ≤50 ≤15

7F ≤5 ≤2 1.5–4.0 0–1.0 ≤0.20 ≤13

8 ≤2 ≤2 0–1 0–1.0 ≤0.15 ≤25

9N ≤2 ≤l 2.2–4 0–1.0 ≤0.20 ≤20 ≤28

9V ≤2 ≤2 0.5–3 0–1.0 ≤0.45 ≤15 ≤13

10A ≤7 ≤2 0.5–3.5 1.5–3.5 ≤0.65 ≤12

11A ≤3 ≤2 0–2.5 2.0–5.0 ≤0.40 ≤9 12F ≤3 ≤2 3–5 0–1.0 ≤0.25 ≤25

14 ≤5 ≤2 1.5–4 0–1.0 ≤0.30 ≤20

15B ≤3 ≤2 2–5 2.0–4.5 ≤0.55 ≤15

17F ≤2 ≤2 0–0.5 0–3.5 ≤0.45 ≤20

18C ≤3 ≤2 0–1 2.4–4.9 ≤0.15 ≤14

19A ≤2 ≤2 0.6–3.5 3.0–7.0 ≤0.45 ≤15 ≤20

19F ≤3 ≤2 1.4–3.5 3.0–5.5 ≤0.20 ≤12.5 ≤20

20 ≤2 ≤2 0.5–2.5 1.5–4.0 ≤0.60 ≤12

22F ≤7 ≤2 0–2 0–1.0 ≤0.55 ≤15 ≤25

23F ≤2 ≤2 0–1 3.0–4.5 ≤0.15 ≤37

33F ≤2.5 ≤2 0–1 0–1.0 ≤0.50

Protein (2.5.16).

Nucleic acids (2.5.17).

Total nitrogen (2.5.9).

Phosphorus (2.5.18).

Molecular size Determine by size-exclusion chromatography (2.2.30) using cross-linked agarose forchromatography R or cross-linked agarose for chromatography R1.

Uronic acids (2.5.22).

Hexosamines (2.5.20).

Methylpentoses (2.5.21).

O-Acetyl groups (2.5.19).

Identification (2.7.1). Confirm the identity of the monovalent bulk polysaccharide by doubleimmunodiffusion or electroimmunodiffusion (except for polysaccharides 7F, 14 and 33F), usingspecific antisera.

Specificity No reaction occurs when the antigens are tested against all the antisera specific for theother polysaccharides of the vaccine, including factor sera for distinguishing types within groups. Thepolysaccharides are tested at a concentration of 50 µg/ml using a method capable of detecting0.5 µg/ml.

FINAL BULK VACCINE

The final bulk vaccine is obtained by aseptically mixing the different polysaccharide powders. Theuniform mixture is aseptically dissolved in a suitable isotonic solution so that one human dose of0.50 ml contains 25 µg of each polysaccharide. An antimicrobial preservative may be added. Thesolution is sterilised by filtration through a bacteria-retentive filter.

Only a final bulk vaccine that complies with the following requirements may be used in thepreparation of the final lot.


Sterility (2.6.1). The final bulk vaccine complies with the test for sterility, using 10 ml for eachmedium.

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FINAL LOT

The final bulk vaccine is distributed aseptically into sterile, tamper-proof containers.Only a final lot that is satisfactory with respect to each of the requirements given below under

identification, tests and assay may be released for use. Provided that the tests for phenol and for anti-microbial preservative have been carried out with satisfactory results on the final bulk vaccine, they maybe omitted on the final lot. When consistency of production has been established on a suitable numberof consecutive batches, the assay may be replaced by a qualitative test that identifies each poly-saccharide, provided that an assay has been performed on each monovalent bulk polysaccharide used inthe preparation of the final lot.

IDENTIFICATION

The assay serves also to identify the vaccine.

TESTS

pH (2.2.3). The pH of the vaccine is 4.5 to 7.4.

Antimicrobial preservative Where applicable, determine the amount of antimicrobial preservative bya suitable chemical method. The content is not less than the minimum amount shown to be effectiveand is not greater than 115 per cent of the quantity stated on the label.

Phenol (2.5.15). Not more than 2.5 g/l.


Pyrogens (2.6.8). It complies with the test for pyrogens. Inject per kilogram of the rabbit’s mass 1 mlof a dilution of the vaccine containing 2.5 µg/ml of each polysaccharide.

ASSAY

Determine the content of each polysaccharide by a suitable immunochemical method (2.7.1), usingantisera specific for each polysaccharide contained in the vaccine, including factor sera for types withingroups, and purified polysaccharides of each type as standards.

The vaccine contains not less than 70 per cent and not more than 130 per cent of the quantity statedon the label for each polysaccharide. The confidence interval (P = 0.95) of the assay is not greater than80 per cent to 120 per cent.

STORAGE


LABELLING


The label states:— the number of micrograms of each polysaccharide per human dose,— the total amount of polysaccharide in the container.__________________________________________________________________________________________________________ Ph Eur

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Inactivated Poliomyelitis Vaccine

Inactivated Poliomyelitis Vaccine complies with the requirements of the 3rd edition of the European Pharma-copoeia for Poliomyelitis Vaccine (Inactivated) [0214]. These requirements are reproduced after the heading‘Definition’ below.

The label may state ‘Pol/Vac(Inact)’.

Ph Eur ___________________________________________________________________________________________________________

DEFINITION

Poliomyelitis vaccine (inactivated) is a liquid preparation of suitable strains of human polioviruses 1,2 and 3 grown in suitable cell cultures and inactivated by a validated method. It is a clear liquid thatmay be coloured owing to the presence of a pH indicator.

Poliomyelitis vaccine (inactivated) complies with the monograph Vaccines for human use (0153).

PRODUCTION

The production method shall have been shown to yield consistently vaccines of acceptable safety andimmunogenicity in man.

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, production complieswith the requirements indicated below.

Unless otherwise justified and authorised, the virus in the final vaccine shall not have undergonemore passages from the master seed lot than was used to prepare the vaccine shown in clinical studiesto be satisfactory with respect to safety and efficacy.



The virus is propagated in a human diploid cell line (5.2.3), in a continuous cell line (5.2.3) or inprimary, secondary or tertiary monkey kidney cells.

Primary, secondary or tertiary monkey kidney cells The following special requirements for thesubstrate for virus propagation apply to primary, secondary or tertiary monkey kidney cells.

Monkeys used in the preparation of kidney cell cultures for production and control of the vaccine. Theanimals used are of a species approved by the competent authority, in good health and, unlessotherwise justified and authorised, have not been previously employed for experimental purposes.Kidney cells used for vaccine production and control are derived from monitored, closed colonies ofmonkeys bred in captivity, not from animals caught in the wild; a previously approved seed lotprepared using virus passaged in cells from wild monkeys may, subject to approval by the competentauthority, be used for vaccine production if historical data on safety justify this.

Monitored, closed colonies of monkeys. The monkeys are kept in groups in cages. Freedom from extran-eous agents is achieved by the use of animals maintained in closed colonies that are subject tocontinuous and systematic veterinary and laboratory monitoring for the presence of infectious agents.The supplier of animals is certified by the competent authority. Each monkey is tested serologically atregular intervals during a quarantine period of not less than 6 weeks imposed before entering thecolony and then during its stay in the colony.

The monkeys used are shown to be tuberculin-negative and free from antibodies to simian virus 40(SV40) and simian immunodeficiency virus. If Macaca sp. monkeys are used for production, themonkeys are also shown to be free from antibodies to herpesvirus B (cercopithecine herpesvirus 1)infection. Human herpesvirus 1 has been used as an indicator for freedom from herpesvirus Bantibodies on account of the danger of handling herpesvirus B (cercopithecine herpesvirus 1).

Monkeys from which kidneys are to be removed are thoroughly examined, particularly for evidenceof tuberculosis and herpesvirus B (cercopithecine herpesvirus 1) infection. If a monkey shows anypathological lesion relevant to the use of its kidneys in the preparation of a seed lot or vaccine, it isnot to be used nor are any of the remaining monkeys of the group concerned unless it is evident thattheir use will not impair the safety of the product.

All the operations described in this section are conducted outside the area where the vaccine isproduced.

Monkey cell cultures for vaccine production. Kidneys that show no pathological signs are used forpreparing cell cultures. Each group of cell cultures derived from a single monkey forms a separateproduction cell culture giving rise to a separate single harvest.

The primary monkey kidney cell suspension complies with the test for mycobacteria (2.6.2);disrupt the cells before carrying out the test.

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If secondary or tertiary cells are used, it shall be demonstrated by suitable validation tests that cellcultures beyond the passage level used for production are free from tumorigenicity.

SEED LOTS

Each of the three strains of poliovirus used shall be identified by historical records that includeinformation on the origin of the strain and its subsequent manipulation.

Only a working seed lot that complies with the following requirements may be used for viruspropagation.

Identification Each working seed lot is identified as human poliovirus 1, 2 or 3 by virus neutralisa-tion in cell culture using specific antibodies.

Virus concentration The virus concentration of each working seed lot is determined to define thequantity of virus to be used for inoculation of production cell cultures.

Extraneous agents The working seed lot complies with the requirements for seed lots for virusvaccines (2.6.16). In addition, if primary, secondary or tertiary monkey kidney cells have been usedfor isolation of the strain, measures are taken to ensure that the strain is not contaminated withsimian viruses such 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 given below under Virus propagation and harvestfor single harvests produced in such cells.


All processing of the cell bank and subsequent cell cultures is done under aseptic conditions in anarea where no other cells or viruses are being handled. Approved animal serum (but not humanserum) may be used in the cell culture media. Serum and trypsin used in the preparation of cellsuspensions and media are shown to be free from extraneous agents. The cell culture media maycontain a pH indicator such as phenol red and approved antibiotics at the lowest effective concentra-tion. Not less than 500 ml of the cell cultures employed for vaccine production is set aside asuninfected cell cultures (control cells); where continuous cell lines in a fermenter are used forproduction, 200 × 106 cells are set aside to prepare control cells; where primary, secondary or tertiarymonkey kidney cells are used for production, a cell sample equivalent to at least 500 ml of the cellsuspension, at the concentration employed for vaccine production, is taken to prepare control cellcultures.

Only a single harvest that complies with the following requirements may be used in the preparationof the vaccine. The tests for identification and bacterial and fungal contamination may be carried outinstead on the purified, pooled monovalent harvest. After demonstration of consistency of productionat the stage of the single harvest, the test for virus concentration may be carried out instead on thepurified, pooled monovalent harvest.

Control cells The control cells of the production cell culture comply with a test for identification (ifa cell-bank system is used for production) and with the requirements for extraneous agents (2.6.16;where primary, secondary or tertiary monkey kidney cells are used, the tests in cell cultures arecarried out as shown below under Test in rabbit kidney cell cultures and Test in cercopithecus kidney cellcultures).— Test in rabbit kidney cell cultures. Test a sample of at least 10 ml of the pooled supernatant fluid

from the control cultures for the absence of herpesvirus B (cercopithecine herpesvirus 1) andother viruses in rabbit kidney cell cultures. The dilution of supernatant in the nutrient medium isnot greater than 1:4 and the area of the cell layer is at least 3 cm2 per millilitre of inoculum. Setaside one or more containers of each batch of cells with the same medium as non-inoculatedcontrol cells. Incubate the cultures at 37°C and observe for at least 2 weeks. The test is not validif more than 20 per cent of the control cells are discarded for non-specific, accidental reasons.

— Test in cercopithecus kidney cell cultures. Test a sample of at least 10 ml of the pooled supernatantfluid from the control cultures for the absence of SV40 virus and other extraneous agents byinoculation onto cell cultures prepared from the kidneys of cercopithecus monkeys, or other cellsshown to be at least as sensitive for SV40, by the method described under Test in rabbit kidneycell cultures. The test is not valid if more than 20 per cent of the control cell cultures arediscarded for non-specific, accidental reasons.

Identification The single harvest is identified as containing human poliovirus 1, 2 or 3 by virusneutralisation in cell cultures using specific antibodies.

Virus concentration The virus concentration of each single harvest is determined by titration ofinfectious virus in cell cultures.

Bacterial and fungal contamination The single harvest complies with the test for sterility (2.6.1),carried out using 10 ml for each medium.

Mycoplasmas (2.6.7). The single harvest complies with the test for mycoplasmas, carried out using10 ml.

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Test in rabbit kidney cell cultures Where primary, secondary or tertiary monkey kidney cells areused for production, test a sample of at least 10 ml of the single harvest for the absence of herpesvirusB (cercopithecine herpesvirus 1) and other viruses in rabbit kidney cell cultures as described for thecontrol cells.

Test in cercopithecus kidney cell cultures Where primary, secondary or tertiary monkey kidneycells are used for production, test a sample of at least 10 ml of the single harvest for the absence ofSV40 virus and other extraneous agents. Neutralise the sample by a high-titre antiserum against thespecific type of poliovirus. Test the sample in primary cercopithecus kidney cell cultures or cells thathave been demonstrated to be at least as susceptible for SV40. Incubate the cultures at 37°C andobserve for 14 days. At the end of this period, make at least one subculture of fluid in the same cellculture system and observe both primary cultures and subcultures for an additional 14 days.

PURIFICATION AND PURIFIED MONOVALENT HARVEST

Several single harvests of the same type may be pooled and may be concentrated. The monovalentharvest or pooled monovalent harvest is purified by validated methods. If continuous cell lines areused for production, the purification process shall have been shown to reduce consistently thecontent of substrate-cell DNA to not more than 100 pg per single human dose.

Only a purified monovalent harvest that complies with the following requirements may be used forthe preparation of the inactivated monovalent harvest.

Identification The virus is identified by virus neutralisation in cell cultures using specific antibodiesor by determination of D-antigen.

Virus concentration The virus concentration is determined by titration of infectious virus.

Specific activity The ratio of the virus concentration or the D-antigen content, determined by asuitable immunochemical method (2.7.1), to the total protein content (specific activity) of thepurified monovalent harvest is within the limits approved for the particular product.

INACTIVATION AND INACTIVATED MONOVALENT HARVEST

Several purified monovalent harvests of the same type may be mixed before inactivation. To avoidfailures in inactivation caused by the presence of virus aggregates, filtration is carried out before andduring inactivation; inactivation is started within a suitable period, preferably not more than 24 h andin any case not more than 72 h, of the prior filtration. The virus suspension is inactivated by avalidated method that has been shown to inactivate poliovirus without destruction of immuno-genicity; during validation studies, an inactivation curve with at least four points (for example, time 0,24 h, 48 h, and 96 h) is established showing the decrease in concentration of live virus with time. Ifformaldehyde 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 with the following requirements may be usedin the preparation of a trivalent pool of inactivated monovalent harvests or a final bulk vaccine.

Test for effective inactivation After neutralisation of the formaldehyde with sodium bisulphite(where applicable), verify the absence of residual live poliovirus by inoculation on suitable cellcultures of two samples of each inactivated monovalent harvest, corresponding to at least 1500human doses. Take one sample not later than three-quarters of the way through the inactivationperiod and the other at the end. Inoculate the samples in cell cultures such that the dilution ofvaccine in the nutrient medium is not greater 1:4 and the area of the cell layer is at least 3 cm2 permillilitre of inoculum. Set aside one or more containers with the same medium as non-inoculatedcontrol cells. Observe the cell cultures for at least 3 weeks. Make not fewer than two passages fromeach container, one at the end of the observation period and the other 1 week before; for thepassages, use cell culture supernatant and inoculate as for the initial sample. Observe the subculturesfor at least 2 weeks. No sign of poliovirus multiplication is present in the cells. At the end of theobservation period, test the susceptibility of the cell culture used by inoculation of live poliovirus ofthe same type as that present in the inactivated monovalent harvest.

Sterility (2.6.1). The inactivated monovalent harvest complies with the test for sterility, carried outusing 10 ml for each medium.

D-antigen content. The content of D-antigen determined by a suitable immunochemical method(2.7.1) is within the limits approved for the particular preparation.

FINAL BULK VACCINE

The final bulk vaccine is prepared directly from the inactivated monovalent harvests of humanpolioviruses 1, 2 and 3 or from a trivalent pool of inactivated monovalent harvests. If a trivalent poolof inactivated monovalent harvests is used, a test for effective inactivation is carried out on this poolinstead of on the final bulk vaccine. A stabiliser and an antimicrobial preservative may be added.



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Antimicrobial preservative Where applicable, determine the amount of antimicrobial preservativeby a suitable chemical or physicochemical method. The amount is not less than the 85 per cent andnot greater than 115 per cent of the intended amount.

Inactivation Before addition of any antimicrobial preservative, a sample of at least 1500 ml or, for apurified and concentrated vaccine, the equivalent of 1500 doses is tested for residual live poliovirus incell cultures, as described for the inactivated monovalent harvest. If the final bulk vaccine is preparedfrom a trivalent pool of inactivated monovalent harvests, the test for inactivation is carried out on thatpool rather than on the final bulk vaccine.

FINAL LOT

Only a final lot that complies with each of the requirements given below under Identification, Testsand Assay may be released for use. Provided that the tests for free formaldehyde and antimicrobialpreservative and the in vivo assay have been performed with satisfactory results on the final bulkvaccine, they may be omitted on the final lot. Provided that the test for bovine serum albumin hasbeen performed with satisfactory results on the trivalent pool of inactivated monovalent harvests oron the final bulk vaccine, it may be omitted on the final lot.

IDENTIFICATION

The vaccine is shown to contain human polioviruses 1, 2 and 3 by a suitable immunochemicalmethod (2.7.1) such as the determination of D-antigen by enzyme-linked immunosorbent assay(ELISA).

TESTS

Free formaldehyde When formaldehyde has been used for inactivation, the vaccine complies withthe test prescribed in the monograph on Vaccines for human use (0153).

Antimicrobial preservative Where applicable, determine the amount of antimicrobial preservativeby a suitable chemical or physicochemical method. The amount is not less than the minimumamount shown to be effective and is not greater than 115 per cent of that stated on the label.

Protein nitrogen content (Lowry method). Not more than 10 µg of protein nitrogen per humandose.



Bacterial endotoxins (2.6.14). Not more than 5 I.U. per human dose.

ASSAY

D-antigen content As a measure of consistency of production, determine the D-antigen content forhuman polioviruses 1, 2 and 3 by a suitable immunochemical method (2.7.1) using a referencepreparation calibrated in European Pharmacopoeia D-antigen units. For each type, the content,expressed with reference to the amount of D-antigen stated on the label, is within the limits approvedfor the particular product.

Poliomyelitis vaccine (inactivated) BRP is calibrated in European Pharmacopoeia units and intendedfor use in the assay of D-antigen. The European Pharmacopoeia unit and the International Unit areequivalent.

In vivo test Determine the potency in vivo by one of the following methods:

Test in chicks or guinea-pigs. Prepare a suitable series of at least three dilutions of the vaccine to beexamined using a suitable buffered saline solution. Inject 0.5 ml of the dilutions intramuscularly intogroups of ten 3-week-old chickens or groups of ten guinea-pigs, each weighing 250 g to 350 g, usinga separate group for each dilution of vaccine. Bleed the animals on the fifth or sixth day after theinjection and separate the sera. Examine the sera for the presence of neutralising antibody, at adilution of 1 in 4, to each of the human polioviruses 1, 2 and 3. Mix 100 CCID50 of virus with thedilution of serum and incubate at 37°C for 4 h 30 min to 6 h. Keep at 5 ± 3°C for 12 h to 18 h.Inoculate the mixtures into cell cultures for the detection of unneutralised virus and read the resultsup to 7 days after inoculation. For each group of animals, note the number of sera which haveneutralising antibody and calculate the dilution of the vaccine giving an antibody response in 50 percent of the animals. Carry out in parallel a control test using a suitable reference preparation.

The vaccine complies with the test if a dilution of 1 in 100 or more produces an antibody responsefor each of the three types of virus in 50 per cent of the animals.

Test in rats. A suitable in vivo method for the determination of immunogenicity consists in theintramuscular injection of three dilutions of the vaccine to be examined and a reference vaccine,using a group of not fewer than ten rats for each dilution. The range of dilutions is chosen such thatall the animals are expected to give a detectable serum antibody response. The neutralising titres aremeasured and the geometric mean for each type of virus is calculated. The potency is calculated by

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comparison of the regression curves for the vaccine to be examined and the reference vaccine. For eachof the three types, the potency is not significantly less than that of the reference preparation.

STORAGE


LABELLING


The label states:— the types of poliovirus contained in the vaccine,— the nominal amount of virus of each type (1, 2 and 3), expressed in Ph. Eur. units of D-antigen, per

single human dose,— the cell substrate used to prepare the vaccine.__________________________________________________________________________________________________________ Ph Eur

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Poliomyelitis Vaccine, Live (Oral)

Poliomyelitis Vaccine, Live (Oral) complies with the requirements of the 3rd edition of the European Pharma-copoeia for Poliomyelitis Vaccine (Oral) [0215]. These requirements are reproduced after the heading ‘Defini-tion’ below.

The label may state ‘Pol/Vac(Oral)’.

For vaccine presented in single doses where the individual container is too small to accommodate theabbreviation ‘Pol/Vac(Oral)’, the code ‘OPV’ may be stated on the label on the container providedthat the code ‘OPV’ is also stated on the label on the package.

Ph Eur ___________________________________________________________________________________________________________

DEFINITION

Oral poliomyelitis vaccine is a preparation of approved strains of live attenuated poliovirus type 1, 2or 3 grown in in vitro cultures of approved cells, containing any one type or any combination of thethree types of Sabin strains, prepared in a form suitable for oral administration.

The vaccine is a clear liquid that may be coloured owing to the presence of a pH indicator.

PRODUCTION

The vaccine strains and the production method shall have been shown to yield consistently vaccinesthat are both immunogenic and safe 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 primary monkey kidney cells are used, production complies with the requirementsindicated below. Unless otherwise justified and authorised, the virus in the final vaccine shall nothave undergone more than two passages from the master seed lot.



The virus is propagated in human diploid cells (5.2.3), in continuous cell lines or in monkey kidneycells. Continuous cell lines are approved by the competent authority.

Primary monkey cells The following special requirements for the substrate for virus propagation apply toprimary monkey cells.

Monkeys used for preparation of kidney cell cultures and for testing of virus. If the vaccine is prepared inmonkey kidney cell cultures, animals of a species approved by the competent authority, in goodhealth, and not previously employed for experimental purposes shall be used.

The monkeys shall be kept in well-constructed and adequately ventilated animal rooms in cagesspaced as far apart as possible. Adequate precautions shall be taken to prevent cross-infectionbetween cages. Not more than two monkeys shall be housed per cage and cage-mates shall not beinterchanged. The monkeys shall be kept in the country of manufacture of the vaccine in quarantinegroups for a period of not less than 6 weeks before use. A quarantine group is a colony of selected,healthy monkeys kept in one room, with separate feeding and cleaning facilities, and having nocontact with other monkeys during the quarantine period. If at any time during the quarantine periodthe overall death rate of a shipment consisting of one or more groups reaches 5 per cent (excludingdeaths from accidents or where the cause was specifically determined not to be an infectious disease),monkeys from that entire shipment shall continue in quarantine from that time for a minimum of 6weeks. The groups shall be kept continuously in isolation, as in quarantine, even after completion ofthe quarantine period, until the monkeys are used. After the last monkey of a group has been taken,the room that housed the group shall be thoroughly cleaned and decontaminated before being usedfor a fresh group. If kidneys from near-term monkeys are used, the mother is quarantined for theterm of pregnancy.

Monkeys from which kidneys are to be removed shall be anaesthetised and thoroughly examined,particularly for evidence of tuberculosis and cercopithecid herpesvirus 1 (B virus) infection.

If a monkey shows any pathological lesion relevant to the use of its kidneys in the preparation of aseed lot or vaccine, it shall not be used, nor shall any of the remaining monkeys of the quarantinegroup concerned be used unless it is evident that their use will not impair the safety of the product.

All the operations described in this section shall be conducted outside the areas where the vaccineis produced.

The monkeys used shall be shown to be free from antibodies to simian virus 40 (SV40) and simianimmunodeficiency virus. If Macaca spp. are used for production, the monkeys shall also be shown tobe free from antibodies to cercopithecid herpesvirus 1 (B virus). Human herpesvirus has been used asan indicator for freedom from B virus antibodies on account of the danger of handling cercopithecidherpesvirus 1 (B virus).

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Monkey kidney cell cultures for vaccine production. Kidneys that show no pathological signs are used forpreparing cell cultures. If the monkeys are from a colony maintained for vaccine production, seriallypassaged monkey kidney cell cultures from primary monkey kidney cells may be used for viruspropagation, otherwise the monkey kidney cells are not propagated in series. Virus for the preparationof vaccine is grown by aseptic methods in such cultures. If animal serum is used in the propagation ofthe cells, the maintenance medium after virus inoculation shall contain no added serum.

Each group of cell cultures derived from a single monkey or from fetuses from no more than tennear-term monkeys is prepared and tested as an individual group.

VIRUS SEED LOTS

The strains of poliovirus used shall be identified by historical records that include information on theorigin and subsequent manipulation of the strains.

Working seed lots are prepared by a single passage from a master seed lot and at an approvedpassage level from the original Sabin virus. Virus seed lots are prepared in large quantities and storedat a temperature below -60°C.

Only a virus seed lot that complies with the following requirements may be used for virus propaga-tion.

Identification Each working seed lot is identified as poliovirus of the given type, using specificantibodies.

Virus concentration Determined by the method described below, the virus concentration is thebasis for the quantity of virus used in the neurovirulence test.

Extraneous agents (2.6.16). If the working seed lot is produced in human diploid cells (5.2.3) or incontinuous cell lines, it complies with the requirements for seed lots for virus vaccines. If the workingseed lot is produced in primary monkey cells, it complies with the requirements given below underVirus Propagation and Harvest and Monovalent Pooled Harvest and with the tests in adult mice,suckling mice and guinea-pigs given under 2.6.16. Tests for extraneous agents in viral vaccines for humanuse.

Neurovirulence (2.6.19). Each master and working seed lot complies with the test forneurovirulence of poliomyelitis vaccine (oral). Furthermore, the seed lot shall cease to be used invaccine production if the frequency of failure of the monovalent pooled harvests produced from it isgreater than predicted statistically. Reference preparations of the three types of poliovirus at the SabinOriginal +2 passage level are available on application to Biologicals, WHO, Geneva, Switzerland.This statistical prediction is calculated after each test on the basis of all the monovalent pooledharvests tested; it is equal to the probability of false rejection on the occasion of a first test (i.e. 1 percent), the probability of false rejection on retest being negligible. If the test is carried out only by themanufacturer, the test slides are provided to the control authority for assessment.

Genetic markers Each working seed lot is tested for its replicating properties at temperatures rang-ing from 36°C to 40°C as described under Monovalent Pooled Harvest.


All processing of the cell-banks and subsequent cell-cultures is done under aseptic conditions in anarea where no other cells are handled. Approved animal (but not human) serum may be used in themedia, but the final medium for maintaining cell growth during virus multiplication does not containanimal serum. Serum and trypsin used in the preparation of cell suspensions and media are shown tobe free from live extraneous agents. The cell-culture medium may contain a pH indicator such asphenol red and approved antibiotics at the lowest effective concentration. It is preferable to have asubstrate free from antibiotics during production. Not less than 5 per cent and not more than1000 ml of the cell cultures employed for vaccine production are set aside as uninfected cell cultures(control cells); special requirements, given below, apply to control cells when the vaccine is producedin primary monkey cells The virus suspension is harvested not later than 4 days after virus inocula-tion. After inoculation of the production cell culture with the virus working seed lot, inoculated cellsare maintained at a fixed temperature, shown to be suitable, within the range 33°C to 35°C; thetemperature is maintained constant to ± 0.5°C; control cell cultures are maintained at 33°C to 35°Cfor the relevant incubation periods.

Only a single virus harvest that complies with the following requirements may be used in thepreparation of the monovalent pooled harvest.

Virus concentration The virus concentration of virus harvests is determined as prescribed underAssay to monitor consistency of production and to determine the dilution to be used for the finalbulk vaccine.

Extraneous agents (2.6.16).

Control cells The control cells of the production cell culture from which the virus harvest is derivedcomply with a test for identity and with the requirements for extraneous agents (2.6.16) or, whereprimary monkey cells are used, as shown below.

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Primary monkey cells The following special requirements apply to virus propagation and harvest inprimary monkey cells.

Cell cultures. On the day of inoculation with virus seed, each cell culture is examined for degenerationcaused by an infective agent. If, in this examination, evidence is found of the presence in a cellculture of any extraneous agent, the entire group of cultures concerned shall be rejected.

On the day of inoculation with the virus working seed lot, a sample of at least 30 ml of the pooledfluid removed from the cell cultures of the kidneys of each single monkey or from fetuses from notmore than ten near-term monkeys is divided into two equal portions. One portion of the pooled fluidis tested in monkey kidney cell cultures prepared from the same species, but not the same animal, asthat used for vaccine production. The other portion of the pooled fluid is, where necessary, tested inmonkey kidney cell cultures from another species so that tests on the pooled fluids are done in cellcultures from at least one species known to be sensitive to SV40. The pooled fluid is inoculated intobottles of these cell cultures in such a way that the dilution of the pooled fluid in the nutrient mediumdoes not exceed 1 in 4. The area of the cell sheet is at least 3 cm2 per millilitre of pooled fluid. Atleast one bottle of each kind of cell culture remains uninoculated to serve as a control. If the monkeyspecies used for vaccine production is known to be sensitive to SV40, a test in a second species is notrequired. Animal serum may be used in the propagation of the cells, provided that it does not containSV40 antibody, but the maintenance medium after inoculation of test material contains no addedserum except as described below.

The cultures are incubated at a temperature of 35°C to 37°C and are observed for a total period ofat least 4 weeks. During this observation period and after not less than 2 weeks’ incubation, at leastone subculture of fluid is made from each of these cultures in the same cell culture system. Thesubcultures are also observed for at least 2 weeks.

Serum may be added to the original culture at the time of subculturing, provided that the serumdoes not contain SV40 antibody.

Fluorescent-antibody techniques may be useful for detecting SV40 virus and other viruses in thecells.

A further sample of at least 10 ml of the pooled fluid is tested for cercopithecid herpesvirus 1 (Bvirus) and other viruses in rabbit kidney cell cultures. Serum used in the nutrient medium of thesecultures shall have been shown to be free from inhibitors of B virus. Human herpesvirus has beenused as an indicator for freedom from B virus inhibitors on account of the danger of handlingcercopithecid herpesvirus 1 (B virus). The sample is inoculated into bottles of these cell cultures insuch a way that the dilution of the pooled fluid in the nutrient medium does not exceed 1 in 4. Thearea of the cell sheet is at least 3 cm2 per millilitre 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°C to 37°C and observed for at least 2 weeks.A further sample of 10 ml of the pooled fluid removed from the cell cultures on the day of inocula-

tion with the seed lot virus is tested for the presence of extraneous agents by inoculation into humancell cultures sensitive to measles virus.

The tests are not valid if more than 20 per cent of the culture vessels have been discarded for non-specific accidental reasons by the end of the respective test periods.

If, in these tests, evidence is found of the presence of an extraneous agent, the single harvest fromthe whole group of cell cultures concerned is rejected.

If the presence of cercopithecid herpesvirus 1 (B virus) is demonstrated, the manufacture of oralpoliomyelitis vaccine shall be discontinued and the competent authority shall be informed.Manufacturing shall not be resumed until a thorough investigation has been completed and precau-tions have been taken against any reappearance of the infection, and then only with the approval ofthe competent authority.

If these tests are not done immediately, the samples of pooled cell-culture fluid shall be kept at atemperature of –60°C or below, with the exception of the sample for the test for B virus, which maybe held at 4°C, provided that the test is done not more than 7 days after it has been taken.

Control cell cultures. On the day of inoculation with the virus working seed lot 25 per cent (but notmore than 2.5 litres) of the cell suspension obtained from the kidneys of each single monkey or fromnot more than ten near-term monkeys is taken to prepare uninoculated control cell cultures. Thesecontrol cell cultures are incubated in the same conditions as the inoculated cultures for at least 2weeks and are examined during this period for evidence of cytopathic changes. The tests are not validif more than 20 per cent of the control cell cultures have been discarded for non-specific, accidentalreasons. At the end of the observation period, the control cell cultures are examined for degenerationcaused by an infectious agent. If this examination or any of the tests required in this section showsevidence of the presence in a control culture of any extraneous agent, the poliovirus grown in thecorresponding inoculated cultures from the same group shall be rejected.

Tests for haemadsorbing viruses. At the time of harvest or within 4 days of inoculation of the productioncultures with the virus working seed lot, a sample of 4 per cent of the control cell cultures is takenand tested for haemadsorbing viruses. At the end of the observation period, the remaining control cellcultures are similarly tested. The tests are made as described in 2.6.16. Tests for extraneous agents inviral vaccines for human use.

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Tests for other extraneous agents. At the time of harvest, or within 7 days of the day of inoculation ofthe production cultures with the working seed lot, a sample of at least 20 ml of the pooled fluid fromeach group of control cultures is taken and tested in two kinds of monkey kidney cell culture, asdescribed above.

At the end of the observation period for the original control cell cultures, similar samples of thepooled fluid are taken and the tests referred to in this section in the two kinds of monkey kidney cellculture and in the rabbit cell cultures are repeated, as described above under Cell cultures.

If the presence of cercopithecid herpesvirus 1 (B virus) is demonstrated, the production cellcultures shall not be used and the measures concerning vaccine production described above must beundertaken.

The fluids collected from the control cell cultures at the time of virus harvest and at the end of theobservation period may be pooled before testing for extraneous agents. A sample of 2 per cent of thepooled fluid is tested in each of the cell culture systems specified.

Single harvests.

Tests for neutralised single harvests in monkey kidney cell cultures. A sample of at least 10 ml of eachsingle harvest is neutralised by a type-specific poliomyelitis antiserum prepared in animals other thanmonkeys. In preparing antisera for this purpose, the immunising antigens used shall be prepared innon-simian cells.

Half of the neutralised suspension (corresponding to at least 5 ml of single harvest) is tested inmonkey kidney cell cultures prepared from the same species, but not the same animal, as that usedfor vaccine production. The other half of the neutralised suspension is tested, if necessary, in monkeykidney cell cultures from another species so that the tests on the neutralised suspension are done incell cultures from at least one species known to be sensitive to SV40.

The neutralised suspensions are inoculated into bottles of these cell cultures in such a way that thedilution of the suspension in the nutrient medium does not exceed 1 in 4. The area of the cell sheet isat least 3 cm2 per millilitre of neutralised suspension. At least one bottle of each type of cell cultureremains uninoculated to serve as a control and is maintained by nutrient medium containing thesame concentration of the specific antiserum used for neutralisation.

Animal serum may be used in the propagation of the cells, provided that it does not contain SV40antibody, but the maintenance medium, after the inoculation of the test material, contains no addedserum other than the poliovirus neutralising antiserum, except as described below.

The cultures are incubated at a temperature of 35°C to 37°C and observed for a total period of atleast 4 weeks. During this observation period and after not less than 2 weeks’ incubation, at least onesubculture of fluid is made from each of these cultures in the same cell-culture system. Thesubcultures are also observed for at least 2 weeks.

Serum may be added to the original cultures at the time of subculturing, provided that the serumdoes not contain SV40 antibody.

Additional tests are made for extraneous agents on a further sample of the neutralised singleharvests by inoculation of 10 ml into human cell cultures sensitive to measles virus.

Fluorescent-antibody techniques may be useful for detecting SV40 virus and other viruses in thecells.

The tests are not valid if more than 20 per cent of the culture vessels have been discarded for non-specific accidental reasons by the end of the respective test periods.

If any cytopathic changes occur in any of the cultures, the causes of these change are investigated.If the cytopathic changes are shown to be due to unneutralised poliovirus, the test is repeated. Ifthere is evidence of the presence of SV40 or other extraneous agents attributable to the single harvest,that single harvest is rejected.


Monovalent pooled harvests are prepared by pooling a number of satisfactory single harvests of thesame virus type. Monovalent pooled harvests from continuous cell lines may be purified. Each mono-valent pooled harvest is filtered through a bacteria-retentive filter.


Identification Each monovalent pooled harvest is identified as poliovirus of the given type, usingspecific antibodies.

Virus concentration The virus concentration is determined by the method described below andserves as the basis for calculating the dilutions for preparation of the final bulk, for the quantity ofvirus used in the neurovirulence test and to establish and monitor production consistency.

Neurovirulence (2.6.19). Each monovalent pooled harvest complies with the test for neurovirulenceof poliomyelitis vaccine (oral). If the test is carried out only by the manufacturer, the test slides areprovided to the competent authority for assessment.

Genetic markers A ratio of the replication capacities of the virus in the monovalent pooled harvestis obtained over a temperature range between 36°C and 40°C in comparison with the seed lot or a

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reference preparation for the marker tests and with appropriate rct/40- and rct/40+ strains ofpoliovirus of the same type. The incubation temperatures used in this test are controlled to within± 0.1°C. The monovalent pooled harvest passes the test if, for both the virus in the harvest and theappropriate reference material, the titre determined at 36°C is at least 5.0 log greater than thatdetermined at 40°C. If growth at 40°C is so low that a valid comparison cannot be established, atemperature in the region of 39.0°C to 39.5°C is used, at which temperature the reduction in titre ofthe reference material must be in the range 3.0 to 5.0 log of its value at 36°C; the acceptable mini-mum reduction is determined for each virus strain at a given temperature. If the titres obtained forone or more of the reference viruses are not concordant with the expected values, the test must berepeated.

Primary monkey cells The following special requirements apply to monovalent pooled harvests derivedfrom primary monkey cells.Test in rabbits. A sample of the monovalent pooled harvest is tested for cercopithecid herpesvirus 1 (Bvirus) and other viruses by injection of not less than 100 ml into not fewer than 10 healthy rabbitseach weighing 1.5 kg to 2.5 kg. Each rabbit receives not less than 10 ml and not more than 20 ml, ofwhich 1 ml is given intradermally at multiple sites, and the remainder subcutaneously. The rabbitsare observed for at least 3 weeks for death or signs of illness.

All rabbits that die after the first 24 h of the test and those showing signs of illness are examined byautopsy, and the brain and organs removed for detailed examination to establish the cause of death.

The test is not valid if more than 20 per cent of the inoculated rabbits show signs of intercurrentinfection during the observation period. The monovalent pooled harvest passes the test if none of therabbits shows evidence of infection with B virus or with other extraneous agents or lesions of any kindattributable to the bulk suspension.

If the presence of B virus is demonstrated, the measures concerning vaccine production describedabove under Cell cultures are taken.

Test in guinea-pigs. Administer to not fewer than five guinea-pigs, each weighing 350 g to 450 g,0.1 ml of the monovalent pooled harvest by intracerebral injection and 0.5 ml by intraperitonealinjection. Measure the rectal temperature of each animal on each working day for 6 weeks. At the endof the observation period carry out autopsy on each animal.

In addition, administer to not fewer than five guinea-pigs 0.5 ml by intraperitoneal injection andobserve as described above for 2 to 3 weeks. At the end of the observation period, carry out a passagefrom these animals to not fewer than five guinea-pigs using blood and a suspension of liver or spleentissue. Measure the rectal temperature of the latter guinea-pigs for 2 to 3 weeks. Examine by autopsyall animals that, after the first day of the test, die or are killed because they show disease or show forthree consecutive days a body temperature higher than 39°C; carry out histological examination todetect infection with Marburg virus; in addition, inject a suspension of liver or spleen tissue or ofblood intraperitoneally into not fewer than three guinea-pigs. If any signs of infection with Marburgvirus are noted, confirmatory serological tests are carried out on the blood of the affected animals.The monovalent pooled harvest complies with the test if not fewer than 80 per cent of the guinea-pigs survive to the end of the observation period and remain in good health and no animal showssigns of infection with Marburg virus.

FINAL BULK VACCINE

The final bulk vaccine is prepared from one or more satisfactory monovalent pooled harvests andmay contain more than one virus type. Suitable flavouring substances and stabilisers may be added.



FINAL LOT

Only a final lot that complies with the following requirement for thermal stability and is satisfactorywith respect to each of the requirements given below under Identification, Tests and Assay may bereleased for use.

Thermal stability Maintain samples of the final lot at 37°C for 48 h. Determine the total virusconcentration as described under Assay in parallel for the heated vaccine and for unheated vaccine.The estimated difference between the total virus concentration of the unheated and heated vaccinesis not greater than 0.5 log10 infectious virus units (CCID50) per single human dose.

IDENTIFICATION

The vaccine is shown to contain poliovirus of each type stated on the label, using specific antibodies.

TESTS

Bacterial and fungal contamination The vaccine complies with the test for sterility (2.6.1).

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ASSAY

Titrate for infectious virus at least in triplicate using the method described below. Use an appropriatevirus reference preparation to validate each assay. If the vaccine contains more than one poliovirus type,titrate each type separately, using appropriate type-specific antiserum (or preferably a monoclonalantibody) to neutralise each of the other types present.

For a trivalent vaccine, the estimated mean virus titres must be: not less than 1 × 106.0 infectious virusunits (CCID50) per single human dose for type 1; not less than 1 × 105.0 infectious virus units (CCID50)for type 2; and not less than 1 × 105.5 infectious virus units (CCID50) for type 3.

For monovalent or divalent vaccine, the minimum virus titres are decided by the competent authority.

Method. Groups of eight to twelve flat-bottomed wells in a microtitre plate are inoculated with 0.1 ml ofeach of the selected dilutions of virus followed by a suitable cell suspension of the Hep-2 (Cincinnati)line. The plates are incubated at a suitable temperature. Examine the cultures on days 7 to 9. The assayis not valid if the confidence interval (P = 0.95) of the logarithm of the virus concentration is greaterthan ± 0.3.

STORAGE


LABELLING


The label states:— the types of poliovirus contained in the vaccine,— the minimum amount of virus of each type contained in one single human dose,— the cell substrate used for the preparation of the vaccine,— that the vaccine is not to be injected.__________________________________________________________________________________________________________ Ph Eur

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Rabies Vaccine

corrected 1/01

Rabies Vaccine complies with the requirements of the 3rd edition of the European Pharmacopoeia for RabiesVaccine For Human Use Prepared In Cell Cultures [0216]. These requirements are reproduced after theheading ‘Definition’ below.

The label may state ‘Rab/Vac’.

Ph Eur ___________________________________________________________________________________________________________

DEFINITION

Rabies vaccine for human use prepared in cell cultures is a freeze-dried preparation of a suitablestrain of fixed rabies virus grown in cell cultures and inactivated by a validated method.

The vaccine is reconstituted immediately before use as stated on the label to give a clear liquid thatmay be coloured owing to the presence of a pH indicator.

The vaccine complies with the requirements of the monograph on Vaccines for human use (0153).

PRODUCTION

The production of the vaccine is based on a virus seed-lot system and, if a cell line is used for viruspropagation, a cell-bank system. The production method shall have been shown to yield consistentlyvaccines that comply with the requirements for immunogenicity, safety and stability. Unlessotherwise justified and authorised, the virus in the final vaccine shall not have undergone morepassages from the master seed lot than was used to prepare the vaccine shown in clinical studies to besatisfactory with respect to safety and efficacy; even with authorised exceptions, the number ofpassages beyond the level used for clinical studies shall not exceed five.



The virus is propagated in a human diploid cell line (5.2.3), in a continuous cell line approved by thecompetent authority or in cultures of chick-embryo cells derived from a flock free from specifiedpathogens (5.2.2).

SEED LOTS

The strain of rabies virus used shall be identified by historical records that include information on theorigin of the strain and its subsequent manipulation.

Working seed lots are prepared by not more than five passages from the master seed lot.Only a working seed lot that complies with the following tests may be used for virus propagation.

Identification Each working seed lot is identified as rabies virus using specific antibodies.

Virus concentration The virus concentration of each working seed lot is determined by a cellculture method using immunofluorescence, to ensure consistency of production.

Extraneous agents (2.6.16). The working seed lot complies with the requirements for virus seedlots. If the virus has been passaged in mouse brain, specific tests for murine viruses are carried out.


All processing of the cell bank and subsequent cell cultures are done under aseptic conditions in anarea where no other cells are handled. Approved animal (but not human) serum may be used in themedia, but the final medium for maintaining cell growth during virus multiplication does not containanimal serum; the media may contain human albumin complying with the monograph on Humanalbumin solution (0255). Serum and trypsin used in the preparation of cell suspensions and media areshown to be free from infectious extraneous agents; trypsin complies with the monograph on Trypsin(0694). The cell culture media may contain a pH indicator such as phenol red and approvedantibiotics at the lowest effective concentration. Not less than 500 ml of the cell cultures employedfor vaccine production are set aside as uninfected cell cultures (control cells). The virus suspension isharvested on one or more occasions during incubation. Multiple harvests from the same productioncell culture may be pooled and considered as a single harvest.

Only a single harvest that complies with the following requirements may be used in the preparationof the inactivated viral harvest.

Identification The single harvest contains virus that is identified as rabies virus using specificantibodies.

Virus concentration Titrate for infective virus in cell cultures; the titre is used to monitorconsistency of production.

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Control cells The control cells of the production cell culture from which the single harvest is derivedcomply with a test for identification and with the requirements for extraneous agents (2.6.16).

PURIFICATION AND INACTIVATION

The virus harvest may be concentrated and/or purified by suitable methods; the virus harvest isinactivated by a validated method at a fixed, well defined stage of the process which may be before,during or after any concentration or purification. The method shall have been shown to be capable ofinactivating rabies virus without destruction of the immunogenic activity. If betapropiolactone isused, the concentration shall at no time exceed 1:3500.

Only an inactivated viral suspension that complies with the following requirements may be used inthe preparation of the final bulk vaccine.

Inactivation Carry out an amplification test for residual infectious rabies virus immediately afterinactivation or using a sample frozen immediately after inactivation and stored at –70°C. Inoculate aquantity of inactivated viral suspension equivalent to not less than 25 doses of vaccine into cellcultures of the same type as those used for production of the vaccine. Make a passage after 7 days.Maintain the cultures for a further 14 days and then examine the cell cultures for rabies virus usingan immunofluorescence test. No rabies virus is detected.

Residual host-cell DNA If a continuous cell line is used for virus propagation, the content ofresidual host-cell DNA, determined using a suitable method as described in Products of recombinantDNA technology (0784), is not greater than 100 pg per single human dose.

FINAL BULK VACCINE

The final bulk vaccine is prepared from one or more inactivated viral suspensions. An approvedstabiliser may be added to maintain the activity of the product during and after freeze-drying.


Glycoprotein content Determine the glycoprotein content by a suitable immunochemical method(2.7.1), for example, single-radial immunodiffusion, enzyme-linked immunosorbent assay or anantibody-binding test. The content is within the limits approved for the particular product.


FINAL LOT

The final bulk vaccine is distributed aseptically into sterile containers and freeze-dried to a moisturecontent shown to be favourable to the stability of the vaccine. The containers are then closed so as toavoid contamination and the introduction of moisture.

Only a final lot that complies with each of the requirements given below under Identification, Testsand Assay may be released for use. Provided that the test for inactivation has been carried out withsatisfactory results on the inactivated viral suspension 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 finallot.

IDENTIFICATION

The vaccine is shown to contain rabies virus antigen by a suitable immunochemical method (2.7.1)using specific antibodies, preferably monoclonal; alternatively, the assay serves also to identify thevaccine.

TESTS

Inactivation Inoculate a quantity equivalent to not less than 25 human doses of vaccine into cellcultures of the same type as those used for production of the vaccine. Make a passage after 7 days.Maintain the cultures for a further 14 days and then examine the cell cultures for rabies virus usingan immunofluorescence test. No rabies virus is detected.



Bacterial endotoxins (2.6.14). Not more than 25 I.U. per single human dose.

Pyrogens (2.6.8). The vaccine complies with the test for pyrogens. Unless otherwise justified andauthorised, inject into each rabbit a single human dose of the vaccine diluted to ten times its volume.


ASSAY

The potency of rabies vaccine is determined by comparing the dose necessary to protect mice againstthe effects of a lethal dose of rabies virus, administered intracerebrally, with the quantity of a refer-ence preparation of rabies vaccine necessary to provide the same protection. For this comparison a

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reference preparation of rabies vaccine, calibrated in International Units, and a suitable preparation ofrabies virus for use as the challenge preparation are necessary.

The International Unit is the activity contained in a stated quantity of the International Standard. Theequivalence in International Units of the International Standard is stated by the World HealthOrganisation.

The test described below uses a parallel-line model with at least three points for the vaccine to beexamined and the reference preparation. Once the analyst has experience with the method for a givenvaccine, it is possible to carry out a simplified test using a single dilution of the vaccine to be examined.Such a test enables the analyst to determine that the vaccine has a potency significantly higher than therequired minimum but will not give full information on the validity of each individual potency deter-mination. The use of a single dilution allows a considerable reduction in the number of animalsrequired for the test and must be considered by each laboratory in accordance with the provisions of theEuropean Convention for the Protection of Vertebrate Animals used for Experimental and otherScientific Purposes.

Selection and distribution of the test animals. Use in the test healthy female mice about 4 weeks old, eachweighing 11 g to 15 g, and from the same stock. Distribute the mice into six groups of a size suitable tomeet the requirements for validity of the test and, for titration of the challenge suspension, four groupsof five.

Preparation of the challenge suspension. Inoculate mice intracerebrally with the CVS strain of rabies virusand when the mice show signs of rabies, but before they die, sacrifice them, remove the brains andprepare a homogenate of the brain tissue in a suitable diluent. Separate gross particulate matter bycentrifugation and use the supernatant liquid as the challenge suspension. Distribute the suspension insmall volumes in ampoules, seal and store at a temperature below –60°C. Thaw one ampoule of thesuspension and make serial dilutions in a suitable diluent. Allocate each dilution to a group of five miceand inject intracerebrally into each mouse 0.03 ml of the dilution allocated to its group. Observe themice for 14 days. Calculate the LD50 of the undiluted suspension using the number in each group that,between the fifth and fourteenth days, die or develop signs of rabies.

Determination of potency of the vaccine to be examined. Prepare three fivefold serial dilutions of the vaccineto be examined and three fivefold serial dilutions of the reference preparation. Prepare the dilutionssuch that the most concentrated suspensions may be expected to protect more than 50 per cent of theanimals to which they are administered and the least concentrated suspensions may be expected toprotect less than 50 per cent of the animals to which they are administered. Allocate the six dilutionsone to each of the six groups of mice and inject intraperitoneally into each mouse 0.5 ml of the dilutionallocated to its group. After 7 days, prepare three identical dilutions of the vaccine to be examined andof the reference preparation and repeat the injections. Seven days after the second injection, prepare asuspension of the challenge virus such that, on the basis of the preliminary titration, 0.03 ml containsabout 50 LD50. Inject intracerebrally into each vaccinated mouse 0.03 ml of this suspension. Preparethree suitable serial dilutions of the challenge suspension. Allocate the challenge suspension and thethree dilutions one to each of the four groups of five control mice and inject intracerebrally into eachmouse 0.03 ml of the suspension or one of the dilutions allocated to its group. Observe the animals ineach group for 14 days and record the number in each group that die or show signs of rabies in theperiod 5 days to 14 days after challenge.

The test is not valid unless: for both the vaccine to be examined and the reference preparation the50 per cent protective dose lies between the largest and smallest doses given to the mice; the titration ofthe challenge suspension shows that 0.03 ml of the suspension contained not less than 10 LD50; thestatistical analysis shows a significant slope and no significant deviations from linearity or parallelism ofthe dose-response lines; the fiducial limits of error (P = 0.95) are not less than 25 per cent and notmore than 400 per cent of the estimated potency.

The vaccine complies with the test if the estimated potency is not less than 2.5 I.U. per human dose.

STORAGE


LABELLING


The label states the biological origin of the cells used for the preparation of the vaccine.__________________________________________________________________________________________________________ Ph Eur

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Rubella Vaccine, Live

Rubella Vaccine, Live complies with the requirements of the 3rd edition of the European Pharmacopoeia forRubella Vaccine (Live) [0162]. These requirements are reproduced after the heading ‘Definition’ below.

The label may state ‘Rub/Vac(Live)’.

Ph Eur ___________________________________________________________________________________________________________

DEFINITION

Rubella vaccine (live) is a freeze-dried preparation of a suitable attenuated strain of rubella virus. Thevaccine is reconstituted immediately before use, as stated on the label, to give a clear liquid that maybe coloured owing to the presence of a pH indicator.

PRODUCTION

The production of vaccine is based on a virus seed-lot system and a cell-bank system. The productionmethod shall have been shown to yield consistently live rubella vaccines of adequate immunogenicityand safety in man. Unless otherwise justified and authorised, the virus in the final vaccine shall haveundergone no more passages from the master seed lot than were used to prepare the vaccine shown inclinical studies to be satisfactory with respect to safety and efficacy.



The virus is propagated in human diploid cells (5.2.3).

SEED LOT

The strain of rubella virus used shall be identified by historical records that include information on theorigin of the strain and its subsequent manipulation. To avoid the unnecessary use of monkeys in thetest for neurovirulence, virus seed lots are prepared in large quantities and stored at temperaturesbelow –20°C if freeze-dried, or below –60°C if not freeze-dried.


Identification The master and working seed lots are identified as rubella virus by serum neutralisationin cell culture, using specific antibodies.

Virus concentration The virus concentration of the master and working seed lots is determined toensure consistency of production.


Neurovirulence (2.6.18). The working seed lot complies with the test for neurovirulence of live virusvaccines. Macaca and Cercopithecus monkeys are suitable for the test.


All processing of the cell bank and subsequent cell cultures is done under aseptic conditions in an areawhere no other cells are handled. Suitable animal (but not human) serum may be used in the growthmedium, but the final medium for maintaining cell growth during virus multiplication does not containanimal serum. Serum and trypsin used in the preparation of cell suspensions and culture media areshown to be free from extraneous agents. The cell culture medium may contain a pH indicator such asphenol red and suitable antibiotics at the lowest effective concentration. It is preferable to have asubstrate free from antibiotics during production. Not less than 500 ml of the production cell culturesis set aside as uninfected cell cultures (control cells). The temperature of incubation is controlledduring the growth of the virus. The virus suspension is harvested, on one or more occasions, within 28days of inoculation. Multiple harvests from the same production cell culture may be pooled andconsidered as a single harvest.


Identification The single harvest contains virus that is identified as rubella virus by serum neutralisa-tion in cell culture, using specific antibodies.

Virus concentration The virus concentration in the single harvest is determined as prescribed underAssay to monitor consistency of production and to determine the dilution to be used for the final bulkvaccine.


Control cells The control cells comply with a test for identification and with the tests for extraneousagents (2.6.16).

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FINAL BULK VACCINE

Single harvests that comply with the above tests are pooled and clarified to remove cells. A suitablestabiliser may be added and the pooled harvests diluted as appropriate.



FINAL LOT


Only a final lot that complies with the requirements for minimum virus concentration for release,with the following requirement for thermal stability and with each of the requirements given belowunder Identification and Tests may be released for use. Provided that the test for bovine serum albuminhas been carried out with satisfactory results on the final bulk vaccine, it may be omitted on the finallot.

Thermal stability Maintain samples of the final lot of freeze-dried vaccine in the dry state at 37°C for7 days. Determine the virus concentration as described under Assay in parallel for the heated vaccineand for unheated vaccine stored at 5 ± 3°C. 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 mixed with specific rubella antibodies, it is nolonger able to infect susceptible cell cultures.

TESTS




ASSAY

Titrate the vaccine for infective virus at least in triplicate, using at least five cell cultures for each0.5 log10 dilution step or by a method of equal precision. Use an appropriate virus reference prepara-tion to validate each assay. The estimated virus concentration is not less than that stated on the label;the minimum virus concentration stated on the label is not less than 1 × 103 CCID50 per human dose.The assay is not valid if the confidence interval (P = 0.95) of the logarithm of the virus concentration isgreater than ± 0.3.

STORAGE


LABELLING


The label states:— the strain of virus used for the preparation of the vaccine,— the type and origin of the cells used for the preparation of the vaccine,— the minimum virus concentration,— that contact with disinfectants is to be avoided,— the time within which the vaccine must be used after reconstitution,— that the vaccine must not be given to a pregnant woman and that a woman must not become

pregnant within 2 months after having the vaccine.__________________________________________________________________________________________________________ Ph Eur

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Tetanus Vaccine

Definition Tetanus Vaccine is prepared from tetanus toxin produced by the growth of Clostridiumtetani. The toxin is converted to tetanus formol toxoid by treatment with Formaldehyde Solution.

Production

Bulk purified toxoid For the production of tetanus toxin, from which toxoid is prepared, seedcultures are managed in a defined seed-lot system in which toxinogenicity is conserved and, wherenecessary, restored by deliberate reselection. A highly toxinogenic strain of Clostridium tetani withknown origin and history is grown in a suitable liquid medium. At the end of cultivation, the purity ofeach culture is tested and contaminated cultures are discarded. Toxin-containing culture medium iscollected aseptically. The toxin content (Lf per ml) is checked to monitor consistency of production.Single harvests may be pooled to prepare the bulk purified toxoid. The toxin is purified to removecomponents likely to cause adverse reactions in humans. The purified toxin is detoxified withFormaldehyde Solution by a method that avoids destruction of the immunogenic potency of thetoxoid and reversion of toxoid to toxin, particularly on exposure to heat. Alternatively, purificationmay be carried out after detoxification.


Sterility Carry out the test for sterility, Appendix XVI A, using 10 ml for each medium.

Absence of tetanus toxin Inject subcutaneously at least 500 Lf of purified toxoid in a volume of 1 mlinto each of five healthy guinea-pigs, each weighing 250 g to 350 g, that have not previously beentreated with any material that will interfere with the test. If within 21 days of the injection any of theanimals shows signs of or dies from tetanus, the toxoid does not comply with the test. If more thanone animal dies from non-specific causes, repeat the test once; if more than one animal dies in thesecond test, the toxoid does not comply with the test.

Irreversibility of toxoid Using the buffer for the final vaccine, prepare a dilution of the bulk purifiedtoxoid containing the same toxoid concentration as the final vaccine. Divide the dilution into twoequal parts. Keep one of them at 2° to 8° and the other at 37° for 6 weeks. Test both dilutions by asuitable sensitive assay for active tetanus toxin, such as inoculation into mice or guinea-pigs. Thetoxoid complies with the test if neither sample produces any sign of a toxic reaction attributable totetanus toxin.

Antigenic purity Not less than 1000 Lf per mg of protein nitrogen.

Final bulk vaccine The final bulk vaccine is prepared by addition of suitable antimicrobialpreservatives. Certain antimicrobial preservatives, particularly those of the phenolic type, adverselyaffect the antigenic activity and must not be used.

Only final bulk vaccine that complies with the following requirements may be used in the preparationof the final lot.

Antimicrobial preservative Where applicable, determine the amount of antimicrobial preservative by asuitable chemical method. The amount is not less than 85% and not greater than 115% of theintended amount.

Sterility Carry out the test for sterility, Appendix XVI A, using 10 ml for each medium.


Final lot The final bulk vaccine is distributed aseptically into sterile, tamper-evident containers. Thecontainers are closed so as to prevent contamination.

Only a final lot that is satisfactory with respect to each of the requirements given below may bereleased for use. Provided the tests for specific toxicity, free formaldehyde and antimicrobialpreservative and the determination of potency have been carried out with satisfactory results on thefinal bulk vaccine, they may be omitted on the final lot.

The vaccine complies with the requirements stated under Vaccines, with the following modifications.

Identification Flocculates when mixed under appropriate conditions with tetanus antitoxin.

Specific toxicity Inject five times the dose stated on the label subcutaneously or intraperitoneallyinto each of five guinea-pigs. None of the guinea-pigs shows any symptoms of, or dies from, tetanuswithin 21 days. If more than one animal dies from non-specific causes within this period repeat thetest. None of the second group of animals shows symptoms of tetanus or dies from tetanus or anyother cause within 21 days.

Sterility Complies with the test for sterility, Appendix XVI A.

Potency Inject into each of no fewer than nine guinea-pigs either a single quantity containing fivetimes the dose stated on the label or two quantities, separated by an interval of not more than 4weeks, each containing one tenth of the dose stated on the label; some of the guinea-pigs may receivethe single dose and the remainder two doses. Not later than 6 weeks after the single injection or, if

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two injections have been given, not later than 2 weeks after the second injection, bleed the guinea-pigs and examine the sera of the guinea-pigs for antitoxin using the biological test for tetanus antitoxindescribed below. The serum of each of no fewer than two thirds of the guinea-pigs contains not lessthan 0.05 Unit of tetanus antitoxin per ml or, alternatively, the serum of each of no fewer than onethird of the guinea-pigs contains not less than 0.5 Unit per ml.

Labelling The label states ‘Tet/Vac/FT’.

When Tetanus Vaccine is prescribed or demanded and the form is not stated, Adsorbed TetanusVaccine may be dispensed or supplied.

BIOLOGICAL TEST FOR TETANUS ANTITOXIN

The potency of tetanus vaccine (or the tetanus component of a mixed vaccine) is tested by biologicalassay, Appendix XIV, by assessing the ability of the vaccine to stimulate the production of tetanusantitoxin in guinea-pigs to which the vaccine has been administered as prescribed in the test forPotency. The sera of the guinea-pigs are examined for antitoxin by comparing their ability to protectmice from the paralytic effects of a fixed dose of tetanus toxin with the ability of the StandardPreparation of tetanus antitoxin to give the same protection. For this comparison the StandardPreparation of tetanus antitoxin and a suitable preparation of tetanus toxin, for use as a test toxin, arerequired. The Standard Preparation is used to determine an appropriate dose of the test toxin whichis subsequently used in tests of the neutralising properties of the serum of sera being tested.

Standard Preparation

The Standard Preparation is the 3rd British Standard for Tetanus antitoxin, established in 1963,consisting of dried serum, or another suitable preparation the potency of which has been determinedin relation to the British Standard.

Suggested method

PREPARATION OF TEST TOXIN Prepare tetanus toxin from a sterile filtrate of an 8 to 10 days cultureof Clostridium tetani. Test toxin may be prepared by adding this filtrate to glycerol in the proportion of1 volume of the filtrate to 1 or 2 volumes of glycerol. This solution of tetanus toxin is stored at orbelow 0°. Test toxins may also be prepared in stable form by saturating the filtrate with ammoniumsulphate, collecting the resulting precipitate, drying it over phosphorus pentoxide and reducing it to afine powder. The powder so obtained is preserved in the dry condition at a low temperature, either insealed ampoules, or over phosphorus pentoxide at a pressure of 2 kPa.

DETERMINATION OF THE DOSE OF TEST TOXIN First determine the Limes paralyticum/200(Lp/200) dose of the test toxin. This is the smallest quantity of the toxin which, when mixed with0.005 Unit of the Standard preparation and injected into mice causes tetanic paralysis within 4 days.The severity of the tetanic paralysis to be regarded as the end point is such that the paralysis is readilyrecognised but not sufficiently extensive to cause significant suffering.

Prepare a solution of the Standard Preparation by dissolving the contents of one ampoule in 1 mlof water for injections and adding sufficient saline solution to produce a total volume of 23 ml. Dilute aportion of the solution 200-fold with saline solution so that 0.5 ml contains 0.025 Unit of tetanusantitoxin. Accurately measure or weigh a quantity of the test toxin and dilute with, or dissolve in,saline solution. Prepare mixtures such that each contains 0.5 ml of the Standard Preparation (0.025Unit), one of a series of graded volumes of the solution of test toxin and sufficient saline solution togive a total volume of 2.5 ml. Allow the mixtures to stand at room temperature, protected from light,for at least 1 hour. After this time inject 0.5 ml of each mixture subcutaneously into mice, four micebeing used for each mixture, and observe the mice for four days.

The mixture that contains the smallest amount of toxin sufficient to cause tetanic paralysis withinthe 4-day period after injection contains five times the Lp/200 dose of the test toxin.

NEUTRALISATION TESTS WITH GUINEA-PIG SERA Prepare two dilutions of the serum obtained fromeach guinea-pig, the first containing 0.5 ml of undiluted serum and 1.0 ml of saline solution and thesecond 0.5 ml of a 10-fold dilution of guinea-pig serum and 1.0 ml of saline solution. Prepare a fresh200-fold dilution of the Standard Preparation and use to make a series of further dilutions contain-ing, respectively, 0.3, 0.4, 0.5, 0.6 and 0.7 ml and sufficient saline solution to give a final volume of1.5 ml. Lastly, prepare a fresh solution of the test toxin such that 1.0 ml contains five times theLp/200 dose as determined previously and add 1 ml of this solution to each dilution of the guinea-pigsera and of the Standard Preparation. Allow the mixtures so prepared to stand at room temperatureand protect from light for at least an hour. After this time inject 0.5 ml of each mixturesubcutaneously into mice, two or three mice being used for each mixture. Survival of the miceinjected with mixtures made with undiluted serum to the fourth day after injection, either withoutparalysis or with paralysis comparable with that observed in the mice injected with the mixtureconsidered in the determination of the dose of test toxin to contain five Lp/200 doses of toxin,indicates a concentration of antitoxin in the undiluted serum of not less than 0.05 Unit per ml.Survival of the mice injected with mixtures made with serum diluted 10-fold to the fourth day after

69-36

injection, either without paralysis or with paralysis comparable with that observed in the miceinjected with the mixture considered in the determination of the dose of test toxin to contain five Lp/200 doses of the test toxin, indicates a concentration of antitoxin in the undiluted serum of not lessthan 0.5 Unit per ml.

The test is not valid unless (a) the mice injected with mixtures containing 0.5 ml of the StandardPreparation develop paralysis comparable with that observed in the mice injected with the mixtureconsidered in the determination of the dose of test toxin to contain five Lp/200 doses of toxin, (b) themice injected with mixtures containing 0.3 ml and 0.4 ml of the Standard Preparation developparalysis earlier in the observation period and (c) the mice injected with mixtures containing 0.6 mland 0.7 ml of the Standard Preparation do not develop paralysis. Record the number of sera contain-ing more than 0.05 Unit per ml and the number containing more than 0.5 Unit per ml.

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Adsorbed Tetanus Vaccine

Adsorbed Tetanus Vaccine complies with the requirements of the 3rd edition of the European Pharmacopoeiafor Tetanus Vaccine (Adsorbed) [0452]. These requirements are reproduced after the heading ‘Definition’below.

The label may state ‘Tet/Vac/Ads’.

When Tetanus Vaccine is prescribed or demanded and the form is not stated, Adsorbed TetanusVaccine may be dispensed or supplied.

Ph Eur ___________________________________________________________________________________________________________

DEFINITION

Tetanus vaccine (adsorbed) is a preparation of tetanus formol toxoid adsorbed on a mineral carrier.The formol toxoid is prepared from the toxin produced by the growth of Clostridium tetani.

PRODUCTION





Absence of tetanus toxin Inject subcutaneously at least 500 Lf of purified toxoid in a volume of1 ml into each of five healthy guinea-pigs, each weighing 250 g to 350 g, that have not previouslybeen treated with any material that will interfere with the test. If within 21 days of the injection any ofthe animals shows signs of or dies from tetanus, the toxoid does not comply with the test. If morethan one animal dies from non-specific causes, repeat the test once; if more than one animal dies inthe second test, the toxoid does not comply with the test.



FINAL BULK VACCINE

The final bulk vaccine is prepared by adsorption of a suitable quantity of bulk purified toxoid ontohydrated aluminium phosphate, aluminium hydroxide or calcium phosphate; the resulting mixture isapproximately isotonic with blood. Suitable antimicrobial preservatives may be added. Certain anti-microbial preservatives, particularly those of the phenolic type, adversely affect the antigenic activityand must not be used.





FINAL LOT


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IDENTIFICATION

Dissolve in the vaccine to be examined sufficient sodium citrate R to give a 100 g/l solution. Maintain at37°C for about 16 h and centrifuge until a clear supernatant liquid is obtained. The clear supernatantliquid reacts with a suitable tetanus antitoxin, giving a precipitate.

TESTS

Specific toxicity Inject subcutaneously five times the single human dose stated on the label into eachof five healthy guinea-pigs, each weighing 250 g to 350 g, that have not previously been treated with anymaterial that will interfere with the test. If within 21 days of the injection any of the animals shows signsof or dies from tetanus, the vaccine does not comply with the test. If more than one animal dies fromnon-specific causes, repeat the test once; if more than one animal dies in the second test, the vaccinedoes not comply with the test.




Antimicrobial preservative Where applicable, determine the amount of antimicrobial preservative bya suitable chemical method. The content is not less than the minimum amount shown to be effectiveand is not greater than 115 per cent of the quantity stated on the label.


POTENCY

Carry out one of the prescribed methods for the assay of tetanus vaccine (adsorbed) (2.7.8).The lower confidence limit (P = 0.95) of the estimated potency is not less than 40 I.U. per single

human dose.

STORAGE


LABELLING


The label states:— the minimum number of International Units per single human dose,— the name and the amount of the adsorbent,— that the vaccine must be shaken before use,— that the vaccine is not to be frozen.__________________________________________________________________________________________________________ Ph Eur

69-39

Tick-borne Encephalitis Vaccine, Inactivated

Tick-borne Encephalitis Vaccine, Inactivated complies with the requirements of the 3rd edition of theEuropean Pharmacopoeia for Tick-borne Encephalitis Vaccine (Inactivated) [1375]. These requirements arereproduced after the heading ‘Definition’ below.

Ph Eur ___________________________________________________________________________________________________________

DEFINITION

Tick-borne encephalitis vaccine (inactivated) is a liquid preparation of a suitable strain of tick-borneencephalitis virus grown in cultures of chick-embryo cells or other suitable cell cultures andinactivated by a suitable, validated method.

PRODUCTION

Production of the vaccine is based on a virus seed-lot system. The production method shall havebeen shown to yield consistently vaccines comparable with the vaccine of proven clinical efficacy andsafety in man. Unless otherwise justified and authorised, the virus in the final vaccine shall not haveundergone more passages from the master seed lot than the virus in the vaccine used in clinical trials.



The virus is propagated in chick embryo cells prepared from eggs derived from a chicken flock freefrom specified pathogens (5.2.2) or in other suitable cell cultures (5.2.3).

SEED LOTS

The strain of virus used is identified by historical records that include information on the origin of thestrain and its subsequent manipulation. Virus seed lots are stored at or below –60°C.


Identification Each seed lot is identified as containing the vaccine strain of tick-borne encephalitisvirus by a suitable immunochemical method (2.7.1), preferably using monoclonal antibodies.

Virus concentration The virus concentration of each seed lot is determined by titration in suitablecell cultures to monitor consistency of production.

Extraneous agents (2.6.16). Each seed lot complies with the requirements for extraneous agents inviral vaccines for human use; the tests in cell cultures are carried out in human and simian cells only.For neutralisation of the vaccine virus, the use of monoclonal antibodies is preferable.


All processing of the cell cultures is performed under aseptic conditions in an area where no othercells are being handled. Serum and trypsin used in the preparation of cell suspensions and mediaused must be shown to be free from extraneous agents. The cell culture media may contain a pHindicator such as phenol red and approved antibiotics at the lowest effective concentration. At least500 ml of the cell cultures employed for vaccine production is set aside as uninfected cell cultures(control cells).

Only a single harvest that complies with the following requirements may be used in the preparationof the inactivated harvest.

Identification The single harvest is shown to contain tick-borne encephalitis virus by a suitableimmunochemical method (2.7.1), preferably using monoclonal antibodies, or by virus neutralisationin cell cultures.

Bacterial and fungal contamination (2.6.1). The single harvest complies with the test for sterility,carried out using 10 ml for each medium.

Mycoplasmas (2.6.7). The single harvest complies with the test for mycoplasmas carried out using1 ml for each medium.

Control cells The control cells comply with the tests for extraneous agents (2.6.16). If the vaccine isproduced using a cell-bank system, the control cells comply with a test for identification.

Virus concentration Determine the virus concentration by titration in suitable cell cultures tomonitor consistency of production.

INACTIVATION

To avoid interference, viral aggregates are removed by filtration immediately before the inactivationprocess. The virus suspension is inactivated by a validated method; the method shall have beenshown to be consistently capable of inactivating tick-borne encephalitis virus without destroying theantigenic and immunogenic activity; as part of the validation studies, an inactivation curve is plotted

69-40

representing residual live virus concentration measured on not fewer than three occasions. Ifformaldehyde is used for inactivation, the presence of an excess of free formaldehyde is verified at theend of the inactivation process.

Only an inactivated harvest that complies with the following requirements may be used in thepreparation of the final bulk vaccine.

Residual infective virus Inoculate a quantity of the inactivated harvest equivalent to not less thanten human doses of vaccine in the final lot into primary chicken fibroblast cell cultures, or other cellsshown to be at least as sensitive to tick-borne encephalitis virus, with not less than 3 cm2 of cell sheetper millilitre of inoculum. Incubate at 37 ± 1°C for 14 days. No cytopathic effect is detected at theend of the incubation period. Collect the culture fluid and inoculate 0.03 ml intracerebrally into eachof not fewer than ten mice about 4 weeks old. Observe the mice for 14 days. They show no evidenceof tick-borne encephalitis virus infection.

PURIFICATION

Several inactivated single harvests may be pooled before concentration and purification by suitablemethods, preferably by continuous-flow, sucrose density-gradient centrifugation.

Only a purified, inactivated harvest that complies with the following requirements may be used inthe preparation of the final bulk vaccine.

Sterility (2.6.1). The purified, inactivated harvest complies with the test for sterility carried out using10 ml for each medium.

Specific activity Determine the antigen content of the purified, inactivated harvest by a suitableimmunochemical method (2.7.1). Determine the total protein content by a suitable method. Thespecific activity, calculated as the antigen content per unit mass of protein, is within the limitsapproved for the specific product.

FINAL BULK VACCINE

The final bulk vaccine is prepared from one or more purified, inactivated harvests.Only a final bulk vaccine that complies with the following requirement may be used in the prepara-

tion of the final lot.


FINAL LOTOnly a final lot that is satisfactory with respect to each of the requirements given below under

Identification, Tests and Assay may be released for use. Provided that the tests for free formaldehyde,bovine serum albumin (where applicable) and pyrogens and the assay have been carried out withsatisfactory results on the final bulk vaccine, they may be omitted on the final lot.

IDENTIFICATION

The vaccine is shown to contain tick-borne encephalitis virus antigen by a suitable immunochemicalmethod (2.7.1) using specific antibodies or by the mouse immunogenicity test described under Assay.

TESTS

Aluminium When hydrated aluminium phosphate or aluminium hydroxide has been used as anadjuvant, the vaccine complies with the test prescribed in the monograph on Vaccines for human use(153).

Free formaldehyde (2.4.18). Not more than 0.1 g/l.

Bovine serum albumin If bovine serum albumin has been used during production, the vaccinecontains not more than 50 ng per single human dose, determined by a suitable immunochemicalmethod (2.7.1).


Pyrogens (2.6.8). The vaccine complies with the test for pyrogens. Inject into each rabbit, perkilogram of body mass, one dose of vaccine.

ASSAY

The potency is determined by comparing the dose necessary to protect a given proportion of miceagainst the effects of a lethal dose of tick-borne encephalitis virus, administered intraperitoneally,with the quantity of a reference preparation of tick-borne encephalitis vaccine necessary to providethe same protection. For this comparison an approved reference preparation and a suitable prepara-tion of tick-borne encephalitis virus from an approved strain for use as the challenge preparation arenecessary.

The following is cited as an example of a method that has been found suitable for a given vaccine.

Selection and distribution of test animals. Use healthy mice weighing 11 g to 17 g and derived from thesame stock. Distribute the mice into not fewer than six groups of a suitable size to meet the require-

69-41

ments for validity of the test; for titration of the challenge suspension, use not fewer than four groups often mice. Use mice of the same sex or distribute males and females equally between groups.

Determination of potency of the vaccine. Prepare not fewer than three suitable dilutions of the vaccine tobe examined and of the reference preparation; in order to comply with validity criteria four to fivedilutions will usually be necessary. Prepare dilutions such that the most concentrated suspension isexpected to protect more than 50 per cent of the animals and the least concentrated suspension lessthan 50 per cent. Allocate each dilution to a different group of mice and inject subcutaneously intoeach mouse 0.2 ml of the dilution allocated to its group. 7 days later make a second injection using thesame dilution scale. 14 days after the second injection prepare a suspension of the challenge viruscontaining not less than 100 LD50 in 0.2 ml. Inject 0.2 ml of this virus suspension intraperitoneally intoeach vaccinated mouse. To verify the challenge dose, prepare a series of not fewer than three dilutionsof the challenge virus suspension at not greater than one-hundredfold intervals. Allocate the challengesuspension and the four dilutions, one to each of the five groups of ten mice, and inject intra-peritoneally into each mouse 0.2 ml of the challenge suspension or the dilution allocated to its group.Observe the animals for 21 days after the challenge and record the number of mice that die in theperiod between 7 days and 21 days after the challenge.

Calculations. Calculate the results by the usual statistical methods for an assay with quantal responses(for example, 5.3. Statistical analysis of results of biological assays and tests).

Validity criteria. The test is not valid unless:— the concentration of the challenge virus is not less than 100 LD50,— for both the vaccine to be examined and the reference preparation the 50 per cent protective dose

(PD50) lies between the largest and smallest doses given to the mice,— the statistical analysis shows a significant slope and no significant deviation from linearity and

parallelism of the dose-response lines,— the fiducial limits of error (P = 0.95) are not less than 33 per cent and not more than 300 per cent

of the estimated potency.

Potency requirement. Include all valid tests to estimate the mean potency and the fiducial limits (P =0.95) for the mean potency; compute weighted means with the inverse of the squared standard error asweights. The vaccine complies with the test if the estimated potency is not less than that approved bythe competent authority, based on data from clinical efficacy trials.

STORAGE


LABELLING


The label states:— the strain of virus used in preparation,— the type of cells used for production of the vaccine.__________________________________________________________________________________________________________ Ph Eur

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Typhoid Polysaccharide Vaccine

1/01Typhoid Polysaccharide Vaccine complies with the requirements of the 3rd edition of the European Pharmaco-poeia [1160]. These requirements are reproduced after the heading ‘Definition’ below.

The label may state ‘Typhoid/Vi/Vac’.

Ph Eur ___________________________________________________________________________________________________________

DEFINITION

Typhoid polysaccharide vaccine is a preparation of purified Vi capsular polysaccharide obtained fromSalmonella typhi Ty2 strain or some other suitable strain that has the capacity to produce Vi poly-saccharide.

Capsular Vi polysaccharide consists of partly 3-O-acetylated repeated units of 2-acetylamino-2-deoxy-D-galactopyranuronic acid with a-(1® 4) linkages.

PRODUCTION

The production of Vi polysaccharide is based on a seed-lot system. The method of production shallhave been shown to yield consistently typhoid polysaccharide vaccines of adequate immunogenicityand safety in man.


SEED LOTS

The strain of S. typhi used for the master seed lot shall be identified by historical records that includeinformation on its origin and by its biochemical and serological characteristics. Cultures from theworking seed lot shall have the same characteristics as the strain that was used to prepare the masterseed lot.

Only a strain that has the following characteristics may be used in the preparation of the vaccine:(a) stained smears from a culture are typical of enterobacteria; (b) the culture utilises glucose withoutproduction of gas; (c) colonies on agar are oxidase-negative; (d) a suspension of the cultureagglutinates specifically with a suitable Vi antiserum or colonies form haloes on an agar plate contain-ing a suitable Vi antiserum.

CULTURE AND HARVEST

The working seed lot is cultured on a solid medium, which may contain blood-group substances, or aliquid medium; the inoculum obtained is transferred to a liquid medium which is used to inoculatethe final medium. The liquid medium used and the final medium are semi-synthetic, free fromsubstances that are precipitated by cetrimonium bromide and do not contain blood-group substancesor high-molecular-mass polysaccharides, unless it has been demonstrated that they are removed bythe purification process.

The bacterial purity of the culture is verified by microscopic examination of Gram-stained smearsand by inoculation into appropriate media; several fields are observed at high magnification so that atleast 10,000 organisms are examined. The culture is then inactivated at the beginning of the sta-tionary phase by the addition of formaldehyde. Bacterial cells are eliminated by centrifugation; thepolysaccharide is precipitated from the culture medium by addition of hexadecyltrimethylammoniumbromide (cetrimonium bromide). The precipitate is harvested and may be stored at –20°C beforepurification.

PURIFIED Vi POLYSACCHARIDE

The polysaccharide is purified, after dissociation of the polysaccharide/cetrimonium bromidecomplex, using suitable procedures to successively eliminate nucleic acids, proteins and lipopoly-saccharides. The polysaccharide is then precipitated as the calcium salt in the presence of ethanol anddried at 2°C to 8°C; the powder obtained constitutes the purified Vi polysaccharide. The loss ondrying is determined by thermogravimetry (2.2.34) and is used to calculate the results of thechemical tests shown below with reference to the dried substance.

Only a purified Vi polysaccharide that complies with the following requirements may be used in thepreparation of the final bulk vaccine.

Protein (2.5.16). Not more than 10 mg per gram of polysaccharide, calculated with reference to thedried substance.

Nucleic acids (2.5.17). Not more than 20 mg per gram of polysaccharide, calculated with referenceto the dried substance.

O-Acetyl groups (2.5.19). Not less than 2 mmol per gram of polysaccharide, calculated withreference to the dried substance.

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Molecular size Examine by size-exclusion chromatography (2.2.30), using cross-linked agarose forchromatography R. Use a column 0.9 m long and 16 mm in internal diameter equilibrated with asolvent having an ionic strength of 0.2 mol/kg and a pH of 7.0 to 7.5. Apply about 5 mg of poly-saccharide in a volume of 1 ml to the column and elute at about 20 ml/h. Collect fractions of about2.5 ml. Determine the point corresponding to KD = 0.25 and make two pools consisting of fractionseluted before and after this point. Carry out an assay for O-acetyl groups on the two pools (2.5.19).Not less than 50 per cent of the polysaccharide is found in the pool containing fractions eluted beforeKD = 0.25.

Identification Carry out an identification test using a suitable immunochemical method (2.7.1).

Bacterial endotoxins (2.6.14, Method D). Not more than 150 I.U. per microgram of poly-saccharide.

FINAL BULK VACCINE

One or more batches of purified Vi polysaccharide are dissolved in a suitable solvent, which maycontain an antimicrobial preservative, so that the volume corresponding to one dose contains 25 µg ofpolysaccharide and the solution is isotonic with blood (250 mosmol/kg to 350 mosmol/kg).

Only a final bulk vaccine that complies with the following tests may be used in the preparation ofthe final lot.


Antimicrobial preservative Where applicable, determine the amount of antimicrobial preservativeby a suitable physico-chemical method. The amount is not less than 85 per cent and not greater than115 per cent of the intended amount.

FINAL LOT

The final bulk vaccine is distributed aseptically into sterile tamper-proof containers that are thenclosed so as to prevent contamination.

Only a final lot that is satisfactory with respect to each of the requirements prescribed below underIdentification, Tests and Assay may be released for use. Provided the tests for free formaldehyde andantimicrobial preservative have been carried out on the final bulk vaccine, they may be omitted onthe final lot.

CHARACTERS

A clear, colourless liquid, free from visible particles.

IDENTIFICATION

Carry out an identification test using a suitable immunochemical method (2.7.1).

TESTS

pH (2.2.3). The pH of the vaccine is 6.5 to 7.5.

O-Acetyl groups 0.085 µmol (± 25 per cent) per dose (25 µg of polysaccharide).

Test solution. Place 3 ml of the vaccine in each of three tubes (one correction solution and tworeaction solutions).

Reference solutions. Dissolve 0.150 g of acetylcholine chloride R in 10 ml of water R (stock solutioncontaining 15 g/l of acetylcholine chloride). Immediately before use, dilute 0.5 ml of the stocksolution to 50 ml with water R (working dilution containing 150 µg/ml of acetylcholine chloride). Inten tubes, place in duplicate (reaction and correction solutions) 0.1 ml, 0.2 ml, 0.5 ml, 1.0 ml and1.5 ml of the working dilution.

Prepare a blank using 3 ml of water R.Make up the volume in each tube to 3 ml with water R. Add 0.5 ml of a mixture of 1 volume of

water R and 2 volumes of dilute hydrochloric acid R to each of the correction tubes and to the blank.Add 1.0 ml of alkaline hydroxylamine solution R to each tube. Allow the reaction to proceed for exactly2 min and add 0.5 ml of a mixture of 1 volume of water R and 2 volumes of dilute hydrochloric acid Rto each of the reaction tubes. Add 0.5 ml of a 200 g/l solution of ferric chloride R in 0.2M hydrochloricacid to each tube, stopper the tubes and shake vigorously to remove bubbles.

Measure the absorbance (2.2.25) of each solution at 540 nm using the blank as the compensationliquid. For each reaction solution, subtract the absorbance of the corresponding correction solution.Draw a calibration curve from the corrected absorbances of the five reference solutions and thecorresponding content of acetylcholine chloride and read from the curve the content of acetylcholinechloride in the test solution for each volume tested. Calculate the mean of the two values.

1 mole of acetylcholine chloride (181.7 g) is equivalent to 1 mole of O-acetyl (43.05 g).

Free formaldehyde It complies with the test for free formaldehyde prescribed in Vaccines for humanuse (0153).

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Antimicrobial preservative Where applicable, determine the amount of antimicrobial preservative bya suitable physico-chemical method. The content is not less than the minimum amount shown to beeffective and not more than 115 per cent of the content stated on the label. If phenol has been used inthe preparation, the content is not more than 2.5 g/l (2.5.15).


Bacterial endotoxins (2.6.14, Method D). Not more than 3750 I.U. per human dose.

ASSAY

Determine Vi polysaccharide by a suitable immunochemical method (2.7.1), using a reference purifiedpolysaccharide. The measured amount of Vi polysaccharide per dose is 80 per cent to 120 per cent ofthe content stated on the label. The fiducial limits of error (P = 0.95) of the measured amount ofpolysaccharide are not less than 80 per cent and not more than 120 per cent.

STORAGE


LABELLING


The label states:— the number of micrograms of polysaccharide per human dose (25 µg),— the total quantity of polysaccharide in the container.__________________________________________________________________________________________________________ Ph Eur

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Typhoid Vaccine

When Typhoid Vaccine is issued as a liquid, it complies with the requirements of the 3rd edition of theEuropean Pharmacopoeia for Typhoid Vaccine [0156]. These requirements are reproduced after the heading‘Liquid Vaccine’ below.

When Typhoid Vaccine is prepared immediately before use by reconstitution from the dried vaccine, the driedvaccine complies with the requirements of the 3rd edition of the European Pharmacopoeia for Freeze-driedTyphoid Vaccine [0157]. These requirements are reproduced after the heading ‘Dried Vaccine’ below.

The label may state ‘Typhoid/Vac’ or ‘Dried/Typhoid/Vac’, as appropriate.

Ph Eur ___________________________________________________________________________________________________________

LIQUID VACCINE [0156]

DEFINITION

Typhoid vaccine is a sterile suspension of inactivated Salmonella typhi containing not less than 5 ×108 and not more than 1 × 109 bacteria (S. typhi) per human dose. The human dose does not exceed1.0 ml.

PRODUCTION

The vaccine is prepared using a seed-lot system from a suitable strain, such as Ty 2( 1), of S. typhi.The final vaccine represents not more than three subcultures from the strain on which were made thelaboratory and clinical tests that showed it to be suitable. The bacteria are inactivated by acetone, byformaldehyde, by phenol or by heating or by a combination of the last two methods.

The production method is validated to demonstrate that the product, if tested, would comply withthe test for abnormal toxicity for immunosera and vaccines for human use (2.6.9) modified asfollows: inject 0.5 ml of the vaccine into each mouse and 1.0 ml into each guinea-pig.

IDENTIFICATION

It is identified by specific agglutination.

TESTS

Phenol (2.5.15). If phenol has been used in the preparation, the concentration is not more than 5 g/l.

Antigenic power When injected into susceptible laboratory animals, it elicits anti-O, anti-H and, toa lesser extent, anti-Vi agglutinins.


STORAGE


LABELLING


The label states:— the method used to inactivate the bacteria,— the number of bacteria per human dose.

1This strain is issued by the World Health Organisation Collaborating Centre for Reference andResearch on Bacterial Vaccines, Human Serum and Vaccine Institute, Szallas Utea 5, H-1107,Budapest, Hungary.

DRIED VACCINE [0157]

DEFINITION

Typhoid vaccine, freeze-dried is a freeze-dried preparation of inactivated Salmonella typhi. Thevaccine is reconstituted as stated on the label to give a uniform suspension containing not less than5 × 108 and not more than 1 × 109 bacteria (S. typhi) per human dose. The human dose does notexceed 1.0 ml of the reconstituted vaccine.

PRODUCTION

The vaccine is prepared using a seed-lot system from a suitable strain, such as Ty 2(1), of S. typhi.The final vaccine represents not more than three subcultures from the strain on which were made thelaboratory and clinical tests that showed it to be suitable. The bacteria are inactivated either byacetone or by formaldehyde or by heat. Phenol is not used in the preparation. The vaccine isdistributed into sterile containers and freeze-dried to a moisture content favourable to the stability ofthe vaccine. The containers are then closed so as to exclude contamination.

The production method is validated to demonstrate that the product, if tested, would comply with

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the test for abnormal toxicity for immunosera and vaccines for human use (2.6.9) modified as follows:inject 0.5 ml of the vaccine into each mouse and 1.0 ml into each guinea pig.

IDENTIFICATION

The vaccine reconstituted as stated on the label is identified by specific agglutination.

TESTS

The reconstituted vaccine complies with the tests prescribed in the monograph on Typhoid vaccine(156).

STORAGE


LABELLING

See Vaccines for human Use (153).

The label states:— the method used to inactivate the bacteria,— the number of bacteria per human dose,— that the vaccine should be used within 8 h of reconstitution.

1This strain is issued by the World Health Organisation Collaborating Centre for Reference andResearch on Bacterial Vaccines, Human Serum and Vaccine Institute, Szallas Utea 5, H-1107,Budapest, Hungary.__________________________________________________________________________________________________________ Ph Eur

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Typhoid (Strain Ty 21a) Vaccine, Live (Oral)

Typhoid (Strain Ty 21a) Vaccine, Live (Oral) complies with the requirements of the 3rd edition of theEuropean Pharmacopoeia for Typhoid Vaccine (Live, Oral, Strain Ty 21a) [1055]. These requirements arereproduced after the heading ‘Definition’ below.

The label may state ‘Typhoid/Vac(Oral)’.

Ph Eur ___________________________________________________________________________________________________________

DEFINITION

Typhoid vaccine (live, oral, strain Ty 21a) is a freeze-dried preparation of live Salmonella typhi strainTy 21a grown in a suitable medium. When presented in capsules, the vaccine complies with themonograph on Capsules (16).

PRODUCTION


The main characteristic of the strain is the defect of the enzyme uridine diphosphate-galactose-4-epimerase. The activities of galactopermease, galactokinase and galactose-1-phosphate uridyl-transferase are reduced by 50 per cent to 90 per cent. Whatever the growth conditions, the straindoes not contain Vi antigen. The strain agglutinates to anti-O:9 antiserum only if grown in mediumcontaining galactose. It contains the flagellar H:d antigen and does not produce hydrogen sulphideon Kligler iron agar. The strain is nonvirulent for mice. Cells of strain Ty 21a lyse if grown in thepresence of 1 per cent of galactose.

BACTERIAL SEED LOTS

The vaccine is prepared using a seed-lot system. The working seed lots represent not more than onesubculture from the master seed lot. The final vaccine represents not more than four subculturesfrom the original vaccine on which were made the laboratory and clinical tests showing the strain tobe suitable.

Only a master seed lot that complies with the following requirements may be used in the prepara-tion of working seed lots.

Galactose metabolism In a spectrophotometric assay, no activity of the enzyme uridine diphos-phate-galactose-4-epimerase is found in the cytoplasm 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. Strain Ty 21a grown in medium free of galactoseshows only the rough (R) type of lipopolysaccharide.

Serological characteristics Strain Ty 21a grown in a synthetic medium without galactose does notagglutinate to specific anti-O:9 antiserum. Whatever the growth conditions, strain Ty 21a does notagglutinate to Vi antiserum. Strain Ty 21a agglutinates to H:d flagellar antiserum.

Biochemical markers Strain Ty 21a does not produce hydrogen sulphide on Kligler iron agar. Thisproperty serves to distinguish Ty 21a from other galactose-epimerase-negative S. typhi strains.

Cell growth Strain Ty 21a cells lyse when grown in the presence of 1 per cent of galactose.

BACTERIAL PROPAGATION AND HARVEST

The bacteria from the working seed lot are multiplied in a preculture, subcultured once and are thengrown in a suitable medium containing 0.001 per cent of galactose at 30°C for 13 h to 15 h. Thebacteria are harvested. The harvest must be free from contaminating micro-organisms.

Only a single harvest that complies with the following requirements may be used for the prepara-tion of the freeze-dried harvest.

pH The pH of the culture is 6.8 to 7.5.

Optical density The optical density of the culture, measured at 546 nm, is 6.5 to 11.0. Beforecarrying out the measurement, dilute the culture so that a reading in the range 0.1 to 0.5 is obtainedand correct the reading to take account of the dilution.

Identification Culture bacteria on an agar medium containing 1 per cent of galactose andbromothymol blue. Light blue, concave colonies, transparent due to lysis of cells, are formed. Noyellow colonies (galactose-fermenting) are found.

FREEZE-DRIED HARVEST

The harvest is mixed with a suitable stabiliser and freeze-dried by a process that ensures the survivalof at least 10 per cent of the bacteria and to a water content shown to be favourable to the stability ofthe vaccine. No antimicrobial preservative is added to the vaccine.

Only a freeze-dried harvest that complies with the following tests may be used for the preparationof the final bulk.

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Identification Culture bacteria are examined on an agar medium containing 1 per cent of galactoseand bromothymol blue. Light blue, concave colonies, transparent due to lysis of cells, are formed. Noyellow colonies (galactose-fermenting) are found.

Number of live bacteria Not fewer than 1 × 1011 live S. typhi strain Ty 21a per gram.

Water (2.5.12). 1.5 per cent to 4.0 per cent, determined by the semi-micro determination of water.

FINAL BULK VACCINE

The final bulk vaccine is prepared by aseptically mixing one or more freeze-dried harvests with asuitable sterile excipient.

Only a final bulk that complies with the following requirement may be used in the preparation of thefinal lot.

Number of live bacteria Not fewer than 40 × 109 live S. typhi strain Ty 21a per gram.

FINAL LOT

The final bulk vaccine is distributed under aseptic conditions into capsules with a gastro-resistant shellor into suitable containers.

Only a final lot that is satisfactory with respect to each of the requirements given below underIdentification, Tests and Number of live bacteria may be released for use, except that in the determina-tion of the number of live bacteria each dosage unit must contain not fewer than 4 × 109 live bacteria.

IDENTIFICATION

Culture bacteria from the vaccine to be examined on an agar medium containing 1 per cent of galactoseand bromothymol blue. Light blue, concave colonies, transparent due to lysis of cells, are formed. Noyellow colonies (galactose-fermenting) are found.

TESTS

Contaminating micro-organisms (2.6.12, 2.6.13). Carry out the test using suitable selective media.Determine the total viable count using the plate-count method. The number of contaminating micro-organisms per dosage unit is not greater than 102 bacteria and 20 fungi. No pathogenic bacterium,particularly Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and no salmonella other thanstrain Ty 21a are found.

Water (2.5.12). 1.5 per cent to 4.0 per cent, determined on the contents of the capsule or of thecontainer by the semi-micro determination of water.

NUMBER OF LIVE BACTERIA

Carry out the test using not fewer than five dosage units. Homogenise the contents of the dosage unitsin a 9 g/l solution of sodium chloride R at 4°C using a mixer in a cold room with sufficient glass beads toemerge from the liquid. Immediately after homogenisation prepare a suitable dilution of the suspensionusing cooled diluent and inoculate brain heart infusion agar; incubate at 36 ± 1°C for 20 h to 36 h. Thevaccine contains not fewer than 2 × 109 live S. typhi Ty 21a bacteria per dosage unit.

STORAGE


LABELLING


The label states:— the minimum number of live bacteria per dosage unit,— that the vaccine is for oral use only.__________________________________________________________________________________________________________ Ph Eur

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Varicella Vaccine, Live

Varicella Vaccine, Live complies with the requirements of the 3rd edition of the European Pharmacopoeia forVaricella Vaccine (Live) [0648]. These requirements are reproduced after the heading ‘Definition’ below.

The label may state ‘Var/Vac(Live)’.

Ph Eur ___________________________________________________________________________________________________________

DEFINITION

Varicella vaccine (live) is a freeze-dried preparation of the OKA attenuated strain of Herpesvirusvaricellae. The vaccine is reconstituted immediately before use, as stated on the label, to give a clearliquid that may be coloured owing to the presence of a pH indicator.

PRODUCTION

The production of vaccine is based on a virus seed-lot system and a cell-bank system. The produc-tion method shall have been shown to yield consistently live varicella vaccines of adequateimmunogenicity and safety in man. The virus in the final vaccine shall not have been passaged in cellcultures beyond the 38th passage from the original isolated virus.



The virus is propagated in human diploid cells (5.2.3).

VIRUS SEED LOT

The strain of varicella virus shall be identified as OKA by historical records which shall includeinformation on the origin of the strain and its subsequent manipulation. The virus shall at no timehave been passaged in continuous cell lines. Seed lots are prepared in the same kind of cells as thoseused for the production of the final vaccine. To avoid the unnecessary use of monkeys in the test forneurovirulence, virus seed lots are prepared in large quantities and stored at temperatures below –20°C, if freeze-dried, or below –60°C, if not freeze-dried.


Identification The master and working seed lots are identified as varicella virus by serum neutralisa-tion in cell culture, using specific antibodies.

Virus concentration The virus concentration of the master and working seed lots is determined asprescribed under assay to monitor consistency of production.

Extraneous agents (2.6.16). The working seed lot complies with the requirements for seed lots forlive virus vaccines; a sample of 50 ml is taken for the test in cell cultures.

Neurovirulence (2.6.18). The working seed lot complies with the test for neurovirulence of livevirus vaccines.


All processing of the cell bank and subsequent cell cultures is done under aseptic conditions in anarea where no other cells are handled. Approved animal (but not human) serum may be used in themedia. Serum and trypsin used in the preparation of cell suspensions and media are shown to be freefrom extraneous agents. The cell culture medium may contain a pH indicator such as phenol red andapproved antibiotics at the lowest effective concentration. It is preferable to have a substrate free fromantibiotics during production. 5 per cent, but not less than 50 ml, of the cell cultures employed forvaccine production is set aside as uninfected cell cultures (control cells). The infected cells constitut-ing a single harvest are washed, released from the support surface and pooled. The cell suspension isdisrupted by sonication.

Only a virus harvest that complies with the following requirements may be used in the preparationof the final bulk vaccine.

Identification The virus harvest contains virus that is identified as varicella virus by serumneutralisation in cell culture, using specific antibodies.

Virus concentration The concentration of infective virus in virus harvests is determined asprescribed under assay to monitor consistency of production and to determine the dilution to be usedfor the final bulk vaccine.

Extraneous agents (2.6.16). Use 50 ml for the test in cell cultures.

Control cells The control cells of the production cell culture from which the single harvest is derivedcomply with a test for identity and with the requirements for extraneous agents (2.6.16).

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FINAL BULK VACCINE

Virus harvests that comply with the above tests are pooled and clarified to remove cells. A suitablestabiliser may be added and the pooled harvests diluted as appropriate.

Only a final bulk vaccine that complies with the following requirements may be used in the prepara-tion of the final lot.

Bacterial and fungal contamination Carry out the test for sterility (2.6.1) using 10 ml for eachmedium.

FINAL LOT

The final bulk vaccine is distributed aseptically into sterile, tamper-proof containers and freeze-dried toa moisture content shown to be favourable to the stability of the vaccine. The containers are thenclosed so as to prevent contamination and the introduction of moisture.

Only a final lot that is satisfactory with respect to each of the requirements given below underidentification, tests and assay may be released for use. Provided that the test for bovine serum albuminhas been carried out with satisfactory results on the final bulk vaccine, it may be omitted on the finallot.

IDENTIFICATION

When the vaccine reconstituted as stated on the label is mixed with specific Herpesvirus varicellaeantibodies, it is no longer able to infect susceptible cell cultures.

TESTS


Bovine serum albumin Not more than 0.5 µg per human dose, determined by a suitableimmunochemical method (2.7.1).


ASSAY

Titrate for infective virus, using at least ten cell cultures for each fourfold dilution or by a technique ofequal precision. Use a suitable virus reference preparation to validate each assay. The virus concentra-tion is not less than 2.0 × 103 PFU per human dose.

STORAGE


LABELLING


The label states:— the strain of virus used for the preparation of the vaccine,— the type and origin of the cells used for the preparation of the vaccine,— that contact with disinfectants is to be avoided,— the minimum virus concentration,— that the vaccine is not to be administered to pregnant women,— the time within which the vaccine must be used after reconstitution.__________________________________________________________________________________________________________ Ph Eur

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Yellow Fever Vaccine, Live

Yellow Fever Vaccine, Live complies with the requirements of the 3rd edition of the European Pharmacopoeiafor Yellow Fever Vaccine (Live) [0537]. These requirements are reproduced after the heading ‘Definition’below.

The label may state ‘Yel/Vac’.

Ph Eur ___________________________________________________________________________________________________________

DEFINITION

Yellow fever vaccine (live) is a freeze-dried preparation of the 17D strain of yellow fever virus grownin fertilised hen eggs. The vaccine is reconstituted immediately before use, as stated on the label, togive a clear liquid.

PRODUCTION

The production of vaccine is based on a virus seed-lot system. The production method shall havebeen shown to yield consistently yellow fever vaccine (live) of acceptable immunogenicity and safetyfor man.

The production method is validated to demonstrate that the product, if tested, would comply withthe test for abnormal toxicity for immunosera and vaccines for human use (2.6.9) modified as followsfor the test in guinea-pigs: inject ten human doses into each guinea-pig and observe for 21 days.

Reference preparation. In the test for neurotropism, a suitable batch of vaccine known to havesatisfactory properties in man is used as the reference preparation.


Virus for the preparation of master and working seed lots and of all vaccine batches is grown in thetissues of chick embryos from a flock free from specified pathogens (5.2.2).

SEED LOTS

The 17D strain shall be identified by historical records that include information on the origin of thestrain and its subsequent manipulation. Virus seed lots are prepared in large quantities and stored ata temperature below –60°C. Master and working seed lots shall not contain any human protein oradded serum.

Unless otherwise justified and authorised, the virus in the final vaccine shall be between passagelevels 204 and 239 from the original isolate of strain 17D. A working seed lot shall be only onepassage from a master seed lot. A working seed lot shall be used without intervening passage as theinoculum for infecting the tissues used in the production of a vaccine lot, so that no vaccine virus ismore than one passage from a seed lot that has passed all the safety tests.


Identification The master and working seed lots are identified as containing yellow fever virus byserum neutralisation in cell culture, using specific antibodies.

Extraneous agents (2.6.16). Each working seed lot complies with the following tests:

Bacterial and fungal sterility

Mycoplasmas

Mycobacteria

Avian viruses

Test in adult mice (intraperitoneal inoculation only)

Test in guinea-pigs

Tests in monkeys Each master and working seed lot complies with the following tests in monkeysfor viraemia (viscerotropism), immunogenicity and neurotropism.

The monkeys shall be Macaca sp. susceptible to yellow fever virus and shall have been shown to benon-immune to yellow fever at the time of injecting the seed virus. They shall be healthy and shallnot have received previously intracerebral or intraspinal inoculation. Furthermore, they shall not havebeen inoculated by other routes with neurotropic viruses or with antigens related to yellow fevervirus. Not fewer than ten monkeys are used for each test.

Use a test dose of 0.25 ml containing the equivalent of not less than 5000 mouse LD50 and notmore than 50,000 mouse LD50, determined by a titration for infectious virus and using theestablished equivalence between virus concentration and mouse LD50 (see under Assay). Inject thetest dose into one frontal lobe of each monkey under anaesthesia and observe the monkeys for notless than 30 days.

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Viraemia (Viscerotropism). Viscerotropism is indicated by the amount of virus present in serum. Takeblood from each of the test monkeys on the second, fourth and sixth days after inoculation andprepare serum from each sample. Prepare 1:10, 1:100 and 1:1000 dilutions from each serum andinoculate each dilution into a group of at least six cell culture vessels used for the determination ofthe virus concentration. The seed lot complies with the test if none of the sera contains more than theequivalent of 500 mouse LD50 in 0.03 ml and at most one serum contains more than the equivalentof 100 mouse LD50 in 0.03 ml.

Immunogenicity. Take blood from each monkey 30 days after the injection of the test dose andprepare serum from each sample. The seed lot complies with the test if at least 90 per cent of the testmonkeys are shown to be immune, as determined by examining their sera in the test for neutralisa-tion of yellow fever virus described below.

It has been shown that a low dilution of serum (for example, 1:10) may contain non-specificinhibitors that influence this test; such serum shall be treated to remove inhibitors. Mix dilutions of atleast 1: 10, 1:40 and 1: 160 of serum from each monkey with an equal volume of 17D vaccine virusat a dilution that will yield an optimum number of plaques with the titration method used. Incubatethe serum-virus mixtures in a water-bath at 37°C for 1 h and then cool in iced water; add 0.2 ml ofeach serum-virus mixture to each of four cell-culture plates and proceed as for the determination ofvirus concentration. Inoculate similarly ten plates with the same amount of virus plus an equalvolume of a 1:10 dilution of monkey serum known to contain no neutralising antibodies to yellowfever virus. At the end of the observation period, compare the mean number of plaques in the platesreceiving virus plus non-immune serum with the mean number of plaques in the plates receiving virusplus dilutions of each monkey serum. Not more than 10 per cent of the test monkeys have serum thatfails to reduce the number of plaques by 50 per cent at the 1:10 dilution.

Neurotropism. Neurotropism is assessed from clinical evidence of encephalitis, from incidence ofclinical manifestations and by evaluation of histological lesions, in comparison with ten monkeysinjected with the reference preparation. The seed lot is not acceptable if either the onset and durationof the febrile reaction or the clinical signs of encephalitis and pathological findings are such as toindicate a change in the properties of the virus.

Clinical evaluation. —The monkeys are examined daily for 30 days by personnel familiar withclinical signs of encephalitis in primates (if necessary, the monkeys are removed from their cage andexamined for signs of motor weakness or spasticity). The seed lot is not acceptable if in the monkeysinjected with it the incidence of severe signs of encephalitis, such as paralysis or inability to standwhen stimulated, or mortality is greater than for the reference vaccine. These and other signs ofencephalitis, such as paresis, inco-ordination, lethargy, tremors or spasticity are assigned numericalvalues for the severity of symptoms by a grading method. Each day each monkey in the test is given ascore 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 dead monkey receives a daily score of 4 fromthe day of death until day 30).

A clinical score for a particular monkey is the average of its daily scores; the clinical score for the seedlot is the mean of the individual monkey scores. The seed lot is not acceptable if the mean of theclinical severity scores for the group of monkeys inoculated with it is significantly greater (P = 0.95)than the mean for the group of monkeys injected with the reference preparation. In addition, specialconsideration is given to any animal showing unusually severe signs when deciding on theacceptability of the seed lot.

Histological evaluation.— Five levels of the brain are examined including:

Block I — the corpus striatum at the level of the optic chiasma,

Block II — the thalamus at the level of the mamillary bodies,

Block III — the mesencephalon at the level of the superior colliculi,

Block IV — the pons and cerebellum at the level of the superior olives,

Block V — the medulla oblongata and cerebellum at the level of the mid-inferior olivary nuclei.

Cervical and lumbar enlargements of the spinal cord are each divided equally into six blocks; 15 µmsections are cut from the tissue blocks embedded in paraffin wax and stained with gallocyanin.Numerical scores are given to each hemisection of the cord and to structures in each hemisection ofthe brain as 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 neuronsaffecting not more than one third of cells.

Grade 3. Severe: moderate focal or diffuse inflammatory infiltration. Degeneration or loss of up totwo thirds of the neurons.

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Grade 4. Overwhelming: variable but often severe inflammatory reaction. Degeneration or loss ofmore than 90 per cent of neurons.

It has been found that inoculation of yellow fever vaccine into the monkey brain causes histologicallesions in different anatomical formations of the central nervous system with varying frequency andseverity (I. S. Levenbook et al., Journal of Biological Standardization, 1987, 15, 305-313). Based onthese two indicators, the anatomical structures can be divided into target, spared and discriminatorareas. Target areas are those which show more severe specific lesions in a majority of monkeysirrespective of the degree of neurovirulence of the seed lot. Spared areas are those which show onlyminimal specific lesions and in a minority of monkeys. Discriminator areas are those where there is asignificant increase in the frequency of more severe specific lesions with seed lots having a higherdegree of neurovirulence. Discriminator and target areas for Macaca cynomolgus and Macaca rhesusmonkeys are shown in the table.

Type of monkey Discriminator areas Target areas

Macaca cynomolgus globus pallidus substantia nigraputamenanterior/median

thalamic nucleuslateral thalamic nucleus

Macaca rhesus caudate nucleus substantia nigraglobus pallidus cervical

enlargement

putamen lumbarenlargement

anterior/medianthalamic nucleus

lateral thalamic nucleuscervical enlargementlumbar enlargement

Scores for discriminator and target areas are used for the final evaluation of the seed lot. Theindividual monkey score is calculated from the sum of individual target area scores in each hemisec-tion divided by the number of areas examined. A separate score is calculated similarly for thediscriminator areas.

Mean scores for the test group are calculated in two ways: (1) by dividing the sum of the individualmonkey discriminator scores by the number of monkeys and (2) by dividing the sum of the individualmonkey target and discriminator scores by the number of monkeys. These two mean scores are takeninto account when deciding on the acceptability of the seed lot. The seed lot is not acceptable ifeither of the mean lesion scores is significantly greater (P = 0.95) than for the reference preparation.


All processing of the fertilised eggs is done under aseptic conditions in an area where no otherinfectious agents or cells are handled at the same time. 2 per cent but not less than twenty and notmore than fifty eggs are set aside as uninfected control eggs. After inoculation and incubation at acontrolled temperature, only living and typical chick embryos are harvested. The age of the embryo atthe time of virus harvest is reckoned from the initial introduction of the egg into the incubator andshall be not more than 12 days. After homogenisation and clarification by centrifugation, the extractof embryonic pulp is tested as described below and kept at –70°C or colder until further processing.Virus harvests that comply with the prescribed tests may be pooled. No human protein is added tothe virus suspension at any stage during production. If stabilisers are added, they shall have beenshown to have no antigenic or sensitising properties for man.


Identification The single harvest contains virus that is identified as yellow fever virus by serumneutralisation in cell culture, using specific antibodies.


Control eggs (2.6.16). The control eggs comply with the tests for extraneous agents.

Virus concentration In order to calculate the dilution for formulation of the final bulk, each singleharvest is titrated as described under Assay.

FINAL BULK VACCINE

Single harvests that comply with the tests prescribed above are pooled and clarified again. A test for

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protein nitrogen content is carried out. A suitable stabiliser may be added and the pooled harvestsdiluted as appropriate.



Protein nitrogen content The protein nitrogen content, before the addition of any stabiliser, is notmore than 0.25 mg per human dose.

FINAL LOT

The final bulk vaccine is distributed aseptically into sterile, tamper-proof containers and freeze-driedto a moisture content shown to be favourable to the stability of the vaccine. The containers are thenclosed so as to prevent contamination and the introduction of moisture.

Only a final lot that is satisfactory with respect to thermal stability and each of the requirementsgiven below under Identification, Tests and Assay may be released for use. Provided that the test forovalbumin has been performed with satisfactory results on the final bulk vaccine, it may be omittedon the final lot.

Thermal stability Maintain samples of the final lot of freeze-dried vaccine in the dry state at 37°Cfor 14 days. Determine the virus concentration as described under Assay in parallel for the heatedvaccine and for unheated vaccine. The difference in the virus concentration between unheated andheated vaccine does not exceed 1.0 log10, and the virus concentration of the heated vaccine is not lessthan the number of plaque-forming units (PFU) equivalent to 1 × 103 mouse LD50 per human dose.

IDENTIFICATION

When the vaccine reconstituted as stated on the label is mixed with specific yellow fever virusantibodies, there is a significant reduction in its ability to infect susceptible cell cultures.

TESTS


Ovalbumin Not more than 5 µg of ovalbumin per human dose, determined by a suitable immuno-chemical method (2.71).

Bacterial endotoxins (2.6.14). Not more than 5 I.U. of bacterial endotoxin per human dose.


ASSAY

Titrate for infective virus in cell cultures. Use an appropriate virus reference preparation to validateeach assay.

The virus concentration is not less than the equivalent in PFU of 1 × 103 mouse LD50 per humandose. The relationship between mouse LD50 and PFU is established by each laboratory and approvedby the competent authority.

The method shown below, or another suitable technique, may be used to determine the mouseLD50.

Suggested method for determination of the mouse LD50

Mouse LD50. The statistically calculated quantity of virus suspension that is expected to produce fatalspecific encephalitis in 50 per cent of mice of a highly susceptible strain, 4 to 6 weeks of age, afterintracerebral inoculation.

Appropriate serial dilutions of the reconstituted vaccine are made in diluent for yellow fever virus (a7.5 g/l solution of bovine albumin R in phosphate-buffered saline pH 7.4 R, or any other diluent that hasbeen shown to be equivalent for maintaining the infectivity of the virus).

Mice of a highly susceptible strain, 4 to 6 weeks of age, are injected intracerebrally under anaes-thesia with 0.03 ml of the vaccine dilution. Groups of not fewer than six mice are used for eachdilution; the series of dilutions is chosen so as to cover the range 0 to 100 per cent mortality of themice. Injection of the mice is performed immediately after the dilutions have been made. The miceare observed for 21 days and all deaths are recorded. Only survivors and deaths caused by typicalyellow fever infections are counted in the computations. Mice paralysed on the twenty-first day ofobservation are counted as survivors.

STORAGE


LABELLING


The label states:— the strain of virus used in preparation,

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— that the vaccine has been prepared in chick embryos,— the minimum virus concentration,— that contact with disinfectants is to be avoided,— the period of time within which the vaccine is to be used after reconstitution.__________________________________________________________________________________________________________ Ph Eur

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DIAGNOSTIC PREPARATIONS

Schick Test ToxinDefinition Schick Test Toxin is the preparation used in the Schick test to determine susceptibility todiphtheria. It is prepared from a toxigenic strain (a well-characterised PW-8 subculture issatisfactory) of Corynebacterium diphtheriae. It contains a suitable antimicrobial preservative.

Production Schick Test Toxin is prepared from a sterile filtrate of a culture in liquid medium of theorganism. The toxin may be purified. It is then diluted with a sterile aqueous buffer solution of pH7.2 to 7.6 that will render the preparation isotonic with blood so that the test dose is contained in0.1 ml or 0.2 ml.

Schick Test Toxin is distributed in sterile containers which are sealed so as to exclude micro-organisms. When stored at 25° for two months Schick Test Toxin retains its potency.

Characteristics A clear, colourless or very pale straw-coloured liquid.

Identification Causes a local reaction when injected into the skin of a healthy, white guinea-pig orrabbit that has not been previously treated with any material that will interfere with the test but failsto cause this reaction when mixed with a sufficient quantity of Diphtheria Antitoxin.

Alkalinity pH, 7.2 to 7.6, Appendix V L.


PotencyA. Combining power Prepare two mixtures of the preparation being examined such that in one mixtureone test dose (0.1 ml or 0.2 ml as stated on the label) is mixed with 1/750 of IU of DiphtheriaAntitoxin and in the other mixture one test dose is mixed with 1/1250 of 1 IU of DiphtheriaAntitoxin. Allow both mixtures to stand at room temperature for 30 to 60 minutes and inject each,on separate flanks, into the depilated skin of a white guinea-pig that weighs not less than 500 g or of awhite rabbit that weighs not less than 2.50 kg. Examine the guinea-pig or rabbit 2 days after injec-tion. No reaction occurs at the site injected with the mixture containing 1/750 of 1 IU of DiphtheriaAntitoxin. A reaction of the type known as a positive Schick reaction occurs at the site injected withthe mixture containing 1/1250 of 1 IU of Diphtheria Antitoxin.

B. Erythrogenic activity The estimated potency is 0.5 to 2.0 IU per test dose (0.1 ml or 0.2 ml asstated on the label) when determined in guinea-pigs or rabbits by comparing the effects ofintradermal injections of serial dilutions of one test dose of the preparation being examined withthose of corresponding dilutions of the Standard Preparation of Diphtheria (Schick) test toxin asfollows.

Depilate two healthy, white guinea-pigs or rabbits in such a way that each has an area of denudedskin sufficient for 16 discrete injection sites, two columns of four sites on each flank. Prepare twosolutions of the preparation being examined and two solutions of the Standard Preparation such that,for both preparations, one solution contains toxin at one fifth of the concentration of the toxin in theother solution and both evoke erythematous lesions of apposite size when injected intradermally in0.2-ml volumes into the depilated skin of guinea-pigs or rabbits. Solutions of the preparation beingexamined prepared by diluting 1 volume of the preparation with 2 volumes of diluent and 1 volumeof the preparation with 14 volumes of diluent, respectively, and solutions of the Standard Preparationcontaining 1/3 and 1/15 of 1 IU in each 0.2 ml are likely to satisfy these requirements. Inject 0.2 mlof each of the four solutions so prepared, in turn, intradermally at each of four sites on each guinea-pig or rabbit. Distribute the four injections of each dilution among the 16 available sites in eachanimal in a Latin square design.

Measure the longitudinal and transverse axes of the resulting lesions at the injection sites 2 daysafter the injections have been made. Using for each lesion the geometric mean of the measurements,calculate by regression analysis the erythrogenic activity of one test dose in IU of Schick test toxin.

Standard Preparation The Standard Preparation is the 1st International Standard for Diphtheria(Schick) test toxin, established in 1954, consisting of purified toxin with bovine albumin and phos-phate buffer salts, or another suitable preparation, the potency of which has been determined inrelation to the International Standard.

Storage Schick Test Toxin should be stored at a temperature of 2° to 8°.

Labelling The label states (1) the total volume in the container; (2) whether the test dose iscontained in 0.1 ml or 0.2 ml of the preparation; (3) the name and proportion of the antimicrobialpreservative; (4) the date after which the preparation is not intended to be used; (5) the conditionsunder which the preparation should be stored.

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Schick ControlDefinition Schick Control is Schick Test Toxin that has been heated at a temperature not lowerthan 70° and not higher than 85° for not less than five minutes. It may be used in conjunction withSchick Test Toxin in order to exclude reactions due to non-specific substances. It is prepared fromthe same batch of Schick Test Toxin as that with which it is to be used.


Inactivation Inject 0.2 ml into the depilated skin of a healthy white guinea-pig. Examine the guinea-pig 2 days after injection. No erythematous reaction occurs at the injection site.

Storage Schick Control should be kept with the Schick Test Toxin with which it is to be used andstored at a temperature of 2° to 8°.

Labelling The label states the batch of Schick Test Toxin from which the Schick Control wasprepared.

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Old Tuberculin

Old Tuberculin complies with the requirements of the 3rd edition of the European Pharmacopoeia for OldTuberculin For Human Use [0152]. These requirements are reproduced after the heading ‘Definition’ below.

Ph Eur ___________________________________________________________________________________________________________

DEFINITION

Old tuberculin for human use consists of a filtrate, concentrated by heating, containing the solubleproducts of the culture and lysis of one or more strains of Mycobacterium bovis and/or Mycobacteriumtuberculosis that is capable of demonstrating a delayed hypersensitivity in an animal sensitised tomicro-organisms of the same species.

Old tuberculin for human use in concentrated form is a transparent, viscous, yellow or brown liquid.

PRODUCTION

GENERAL PROVISIONS

The production of old tuberculin is based on a seed-lot system. The production method shall havebeen shown to yield consistently old tuberculin of adequate potency and safety in man. A batch ofold tuberculin, calibrated in International Units by the method described under Assay and for whichadequate clinical information is available as to its activity in man, is set aside to serve as a referencepreparation.

The International Unit is the activity of a stated quantity of the International Standard. Theequivalence in International Units of the International Standard is stated by the World HealthOrganisation.

SEED LOTS

The strains of mycobacteria used shall be identified by historical records that include information ontheir origin and subsequent manipulation.

The working seed lots used to inoculate the media for the production of a concentrated harvestshall not have undergone more than four subcultures from the master seed lot.

Only seed lots that comply with the following requirements may be used for propagation.

Identification The species of mycobacterium of the master and working seed lots is identified.

Bacterial and fungal contamination. Carry out the test for sterility (2.6.1), using 10 ml for eachmedium. The working seed lot complies with the test for sterility except for the presence ofmycobacteria.


The bacteria are grown in a liquid medium which may be a glycerolated broth or a synthetic medium.Growth must be typical for the strain. The culture is inactivated by a suitable method, such astreatment in an autoclave (121°C for not less than 30 min) or in flowing steam at a temperature notless than 100°C for at least 1 h. The culture liquid, from which the micro-organisms may or may nothave been separated by filtration, is concentrated by evaporation, usually to one-tenth of its initialvolume. The preparation is free from live mycobacteria. The concentrated harvest is shown tocomply with the test for mycobacteria (2.6.2) before addition of any antimicrobial preservative orother substance that might interfere with the test. Phenol (5 g/l) or another suitable antimicrobialpreservative that does not give rise to false positive reactions may be added.

Only a concentrated harvest that complies with the following requirements may be used in thepreparation of the final bulk tuberculin.

pH (2.2.3). The pH of the concentrated harvest is 6.5 to 8.

Glycerol Where applicable, determine the glycerol content of the concentrated harvest. The amountis within the limits approved for the particular product.

Antimicrobial preservative Where applicable, determine the amount of antimicrobial preservativeby a suitable chemical or physico-chemical method. The content is not less than 85 per cent and notmore than 115 per cent of the intended amount. If phenol has been used in the preparation, theconcentration is not more than 5 g/l (2.5.15).

Sensitisation Carry out the test described under Tests.

Sterility (2.6.1). The concentrated harvest complies with the test for sterility, carried out using10 ml for each medium.

Potency Determine the potency as described under Assay.

FINAL BULK TUBERCULIN

The concentrated harvest is diluted aseptically.

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Only a final bulk tuberculin that complies with the following requirement may be used in thepreparation of the final lot.

Sterility (2.6.1). The final bulk tuberculin complies with the test for sterility, carried out using 10 mlfor each medium.

FINAL LOT

The final bulk tuberculin is distributed aseptically into sterile containers which are then closed so asto prevent contamination.

Only a final lot that is satisfactory with respect to each of the requirements given below underIdentification, Tests and Assay may be released for use.

The following tests may be omitted on the final lot if they have been carried out at the stagesindicated:

live mycobacteria concentrated harvest

sensitisation concentrated harvest

toxicity concentrated harvest or final bulk tuberculin

antimicrobial preservative final bulk tuberculin.

IDENTIFICATION

Inject increasing doses of the preparation to be examined intradermally into healthy, white or pale-coloured guinea-pigs, specifically sensitised (for example, as described under Assay). A reactionvarying from erythema to necrosis is produced at the site of the injection. Similar injectionsadministered to non-sensitised guinea-pigs do not stimulate a reaction. The assay may also serve asidentification.

TESTS

Old tuberculin for human use in concentrated form (³ 100,000 I.U./ml) complies with each of the testsprescribed below; the diluted product complies with the tests for antimicrobial preservative and sterility.

Toxicity. Inject a quantity equivalent to 50,000 I.U. subcutaneously into each of two healthy guinea-pigs weighing 250 g to 350 g and which have not been subjected to any treatment likely to interferewith the test. Observe the animals for 7 days. No adverse effect is produced.

Sensitisation. Use three guinea-pigs that have not been subjected to any treatment likely to interferewith the test. On three occasions at intervals of 5 days, inject intradermally into each guinea-pigabout 500 I.U. of the preparation to be examined in a volume of 0.1 ml. Two to three weeks after thethird injection, administer the same dose intradermally to the same animals and to a control group ofthree guinea-pigs of the same mass that have not previously received injections of tuberculin. After24 h to 72 h, the reactions in the two groups of animals are not substantially different.

Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservativeby a suitable chemical or physico-chemical method. The content is not less than the minimumamount shown to be effective and not more than 115 per cent of the amount stated on the label. Ifphenol has been used in the preparation, the concentration is not more than 5 g/l (2.5.15).

Live mycobacteria (2.6.2). It complies with the test for mycobacteria.


ASSAY

The potency of old tuberculin is determined by comparing the reactions produced by the intradermalinjection of increasing doses of the preparation to be examined into sensitised guinea-pigs with thereactions produced by known concentrations of the reference preparation.

Prepare a suspension containing a suitable amount (0.1 mg to 0.4 mg per millilitre) of heat-inactivated, dried mycobacteria in mineral oil with or without emulsifier; use mycobacteria of a strainof the same species as that used in the preparation to be examined. Sensitise not fewer than six pale-coloured guinea-pigs weighing not less than 300 g by injecting intramuscularly or intradermally atotal of about 0.5 ml of the suspension, divided between several sites if necessary.

Carry out the test after the period of time required for optimal sensitisation which is usually 4 to 8weeks after sensitisation. Depilate the flanks of the animals so that it is possible to make at least threeinjections on each side and not more than a total of twelve injection points per animal. Use at leastthree different doses of the reference preparation and at least three different doses of the preparationto be examined. For both preparations, use doses such that the highest dose is about ten times thelowest dose. Choose the doses such that when they are injected the lesions produced have a diameterof not less than 8 mm and not more than 25 mm. In any given test, the order of the dilutions injectedat each point is chosen at random in a Latin square design. Inject each dose intradermally in aconstant volume of 0.1 ml or 0.2 ml. Measure the diameters of the lesions 24 h to 48 h later andcalculate the results of the test by the usual statistical methods, assuming that the diameters of thelesions are directly proportional to the logarithm of the concentration of the preparation.

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The estimated potency is not less than 80 per cent and not more than 125 per cent of the statedpotency. The fiducial limits of error (P = 0.95) are not less than 64 per cent and not more than156 per cent of the stated potency.

STORAGE

Store protected from light.

LABELLING

The label states:— the number of International Units per millilitre,— the species of mycobacterium used to prepare the product,— the name and quantity of any antimicrobial preservative or other substance added to the prepara-

tion,— the expiry date,— where applicable, that old tuberculin is not to be injected in its concentrated form but diluted so

as to administer not more than 100 I.U. per dose.__________________________________________________________________________________________________________ Ph Eur

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Tuberculin Purified Protein Derivative

Tuberculin P.P.D.

Tuberculin Purified Protein Derivative complies with the requirements of the 3rd edition of the EuropeanPharmacopoeia for Tuberculin Purified Protein Derivative for Human Use [0151]. These requirements arereproduced after the heading ‘Definition’ below.

Ph Eur ___________________________________________________________________________________________________________

DEFINITION

Tuberculin purified protein derivative (tuberculin PPD) for human use is a preparation obtained byprecipitation from the heated products of the culture and lysis of Mycobacterium bovis and/orMycobacterium tuberculosis and capable of demonstrating a delayed hypersensitivity in an animalsensitised to micro-organisms of the same species.

Tuberculin PPD is a colourless or pale-yellow liquid; the diluted preparation may be a freeze-driedpowder which upon dissolution gives a colourless or pale-yellow liquid.

PRODUCTION

GENERAL PROVISIONS

The production of tuberculin PPD is based on a seed-lot system. The production method shall havebeen shown to yield consistently tuberculin PPD of adequate potency and safety in man. A batch oftuberculin PPD, calibrated in International Units by method A described under Assay and for whichadequate clinical information is available as to its activity in man, is set aside to serve as a referencepreparation.

The International Unit is the activity of a stated quantity of the International Standard. Theequivalence in International Units of the International Standard is stated by the World HealthOrganisation.

SEED LOTS

The strains of mycobacteria used shall be identified by historical records that include information ontheir origin and subsequent manipulation.

The working seed lots used to inoculate the media for production of a concentrated harvest shall nothave undergone more than four subcultures from the master seed lot.

Only seed lots that comply with the following requirements may be used for propagation.

Identification The species of mycobacterium of the master and working seed lots is identified.

Bacterial and fungal contamination Carry out the test for sterility (2.6.1), using 10 ml for eachmedium. The working seed lot complies with the test for sterility except for the presence ofmycobacteria.


The bacteria are grown in a liquid synthetic medium. Growth must be typical for the strain. Theculture is inactivated by a suitable method such as treatment in an autoclave (121°C for not less than30 min) or in flowing steam at a temperature not less than 100°C for at least 1 h and filtered. Theactive fraction of the filtrate, consisting mainly of protein, is isolated by precipitation, washed and re-dissolved. The preparation is free from mycobacteria. The concentrated harvest is shown to complywith the test for mycobacteria (2.6.2) before addition of any antimicrobial preservative or othersubstance that might interfere with the test. Phenol (5 g/l) or another suitable antimicrobialpreservative that does not give rise to false positive reactions may be added; a suitable stabiliserintended to prevent adsorption on glass or plastic surfaces may be added. The concentrated harvestmay be freeze-dried. Phenol is not added to preparations that are to be freeze-dried.

Only a concentrated harvest that complies with the following requirements may be used in thepreparation of the final bulk tuberculin PPD.

Antimicrobial preservative Where applicable, determine the amount of antimicrobial preservativeby a suitable chemical or physico-chemical method. The content is not less than 85 per cent and notmore than 115 per cent of the intended amount. If phenol has been used in the preparation, theconcentration is not more than 5 g/l (2.5.15).

Sensitisation Carry out the test described under Tests.

Sterility (2.6.1). The concentrated harvest, reconstituted if necessary, complies with the test forsterility, carried out using 10 ml for each medium.

Potency Determine the potency as described under Assay.

FINAL BULK TUBERCULIN PPD

The concentrated harvest is diluted aseptically, after reconstitution if necessary.

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Only a final bulk tuberculin PPD that complies with the following requirement may be used in thepreparation of the final lot.

Sterility (2.6.1). The final bulk tuberculin PPD complies with the test for sterility, carried out using10 ml for each medium.

FINAL LOT

The final bulk tuberculin PPD is distributed aseptically into sterile containers which are then closedso as to prevent contamination. It may be freeze-dried.

Only a final lot that is satisfactory with respect to each of the requirements given below underIdentification, Tests and Assay may be released for use.

The following tests may be omitted on the final lot if they have been carried out at the stagesindicated:

live mycobacteria concentrated harvest

sensitisation concentrated harvest

toxicity concentrated harvest or final bulk tuberculin PPD

antimicrobial preservative final bulk tuberculin PPD.

IDENTIFICATION

Inject increasing doses of the preparation to be examined intradermally into healthy, white or pale-coloured guinea-pigs, specifically sensitised (for example as described under Assay). A reactionvarying from erythema to necrosis is produced at the site of the injection. Similar injectionsadministered to non-sensitised guinea-pigs do not stimulate a reaction. The assay may also serve asidentification.

TESTS

Tuberculin purified protein derivative for human use in concentrated form (³ 100,000 I.U./ml) complies witheach of the tests prescribed below; the diluted product complies with the tests for pH, antimicrobial preservativeand sterility.

pH (2.2.3). The pH of the preparation, reconstituted if necessary as stated on the label, is 6.5 to 7.5.

Toxicity Inject subcutaneously 50,000 I.U. of the preparation to be examined into each of twohealthy guinea-pigs weighing 250 g to 350 g and which have not been subjected to any treatmentlikely to interfere with the test. Observe the animals for 7 days. No adverse effect is produced.

Sensitisation Use three guinea-pigs that have not been subjected to any treatment likely to interferewith the test. On three occasions at intervals of 5 days, inject intradermally into each guinea-pigabout 500 I.U. of the preparation to be examined in a volume of 0.1 ml. Two to three weeks after thethird injection, administer the same dose intradermally to the same animals and to a control group ofthree guinea-pigs of the same mass that have not previously received injections of tuberculin. After24 h to 72 h, the reactions in the two groups of animals are not substantially different.

Antimicrobial preservative Where applicable, determine the amount of antimicrobial preservativeby a suitable chemical or physico-chemical method. The content is not less than the minimumamount shown to be effective and not more than 115 per cent of the amount stated on the label. Ifphenol has been used in the preparation, the concentration is not more than 5 g/l (2.5.15).

Live mycobacteria (2.6.2). It complies with the test for mycobacteria.


ASSAY

Use method A or, where the preparation contains 1 I.U. to 2 I.U., use method B.

Method A

The potency of tuberculin PPD is determined by comparing the reactions produced by theintradermal injection of increasing doses of the preparation to be examined into sensitised guinea-pigswith the reactions produced by known concentrations of the reference preparation.

Prepare a suspension containing a suitable amount (0.1 mg to 0.4 mg per millilitre) of heat-inactivated, dried mycobacteria in mineral oil with or without emulsifier; use mycobacteria of a strainof the same species as that used in the preparation to be examined. Sensitise not fewer than six pale-coloured guinea-pigs weighing not less than 300 g by injecting intramuscularly or intradermally atotal of about 0.5 ml of the suspension, divided between several sites if necessary. Carry out the testafter the period of time required for optimal sensitisation which is usually 4 to 8 weeks after sensitisa-tion. Depilate the flanks of the animals so that it is possible to make at least three injections on eachside but not more than a total of twelve injection points per animal. Prepare dilutions of the prepara-tion to be examined and of the reference preparation using isotonic phosphate-buffered saline (pH6.5 to 7.5) containing 50 mg/l of polysorbate 80 R. If the preparation to be examined is freeze-driedand does not contain a stabiliser, reconstitute it using the liquid described above. Use at least threedifferent doses of the reference preparation and at least three different doses of the preparation to be

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examined. For both preparations, use doses such that the highest dose is about ten times the lowestdose. Choose the doses such that when they are injected the lesions produced have a diameter of notless than 8 mm and not more than 25 mm. In any given test, the order of the dilutions injected ateach point is chosen at random in a Latin square design. Inject each dose intradermally in a constantvolume of 0.1 ml or 0.2 ml. Measure the diameters of the lesions 24 h to 48 h later and calculate theresults of the test by the usual statistical methods, assuming that the diameters of the lesions aredirectly proportional to the logarithm of the concentration of the preparation.


Method B

The potency of tuberculin PPD is determined by comparing the reactions produced by theintradermal injection of the preparation to be examined into sensitised guinea-pigs with the reactionsproduced by known concentrations of the reference preparation.

Prepare a suspension in mineral oil with or without emulsifier and containing a suitable amount(0.1 mg to 0.4 mg per millilitre) of heat-inactivated, dried mycobacteria; use mycobacteria of a strainof the same species as that used in the preparation to be examined. Sensitise not fewer than six pale-coloured guinea-pigs weighing not less than 300 g by injecting intramuscularly or intradermally atotal of about 0.5 ml of the suspension, divided between several sites if necessary. Carry out the testafter the period of time required for optimal sensitisation which is usually 4 to 8 weeks after sensitisa-tion. Depilate the flanks of the animals so that it is possible to make at least three injections on eachside but not more than a total of twelve injection points per animal. Prepare dilutions of the referencepreparation using isotonic phosphate-buffered saline (pH 6.5 to 7.5) containing 50 mg/l of polysorbate80 R. Use at least three different doses of the reference preparation such that the highest dose isabout ten times the lowest dose and the median dose is the same as that of the preparation to beexamined. In any given test, the order of the dilutions injected at each point is chosen at random in aLatin square design. Inject the preparation to be examined and each dilution of the reference prepa-ration intradermally in a constant volume of 0.1 ml or 0.2 ml. Measure the diameters of the lesions24 h to 48 h later and calculate the results of the test by the usual statistical methods, assuming thatthe areas of the lesions are directly proportional to the logarithm of the concentration of the prepara-tion to be examined. (This dose relationship applies to this assay and not necessarily to other testsystems.)


STORAGE

Store protected from light.

LABELLING

The label states:— the number of International Units per container,— the species of mycobacterium used to prepare the product,— the name and quantity of any antimicrobial preservative or other substance added to the

preparation— the expiry date,— where applicable, that old tuberculin is not to be injected in its concentrated form but diluted so

as to administer not more than 100 I.U. per dose.__________________________________________________________________________________________________________ Ph Eur

Documents

BP_06 - 2001