ADSORBED DIPHTHERIA VACCINE (ADV)

MICRO biological assay of vaccinesPRESENTED BY:T. LAKSHMI BHAVANI(2015MPH40023)Under the guidance of: Dr. S. JOSHNA RANISPMVV - TIRUPATHI

M.Pharm.,Ph.DPharmaceutical Analysis1ST YEAR, I-SEM1

CONTENTS Introduction to vaccinesMicrobiological assays

References

Adsorbed diphtheria vaccine (ADV) Rabies vaccine Hepatitis A & B2

INTRODUCTIONA preparation of killed microorganisms, living attenuated organisms, or living fully virulent organisms that is administered to produce or artificially increase the immunity to a particular disease.

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ADSORBED DIPHTHERIA VACCINE (ADV)1.DEFINITION:Diphtheria formol toxoid + Mineral carrier [ which is hydrated Aluminium hydroxide[or] Calcium PO4].

Adsorbed diphtheria vaccine.

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Saline solution or other suitable solution is isotonic with blood.The formol toxoid is prepared from the toxin and produced from the Corynebacterium diphtheriae.Corynebacterium diphtheriae Diphtheria formol toxoid.

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Antigenic properties of ADV is adversely affected by anti microbial preservatives such as: Phenol type and these should not added to the vaccines.[ADV is prepared from diphtheria formol toxoid and it contains not less than 1500Lf/mg of protein nitrogen and mineral carrier].

2.CATEGORY: Immunizing agent.

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A . INTRADERMAL CHALLENGE METHODPrinciple:The potency of adsorbed diphtheria vaccine is determined by comparing the dose is necessary to protect guinea pig against the erythrogenic effect of range of intradermal injection of diphtheria toxin with dose of the standard preparation of adsorbed diphtheria toxin necessary to give the same protection.

3.BIOLOGICAL ASSAY:7

PROCEDURE:Standard preparation:Standard preparation consisting of toxoid adsorbed on a aluminium hydroxide with polygeline.Test animal:White guinea pigWeight between 250-350gm

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Selection of challenge toxin:Selection of preparation of diphtheria toxin containing 67 133Lr / 100 in limes flocculation (Lf) and 25000 50000 minimal reacting doses for guinea pig skin in 1Lf (limes flocculation).Preparation of challenge toxin solution:Dilute challenge toxin with a suitable diluents to obtain a challenge toxin solution containing about 512 10-4Lf in 0.2ml.9

DETERMINATION OF POTENCY OF THE VACCINE0.2ml challenge toxin solution is inject to guinea pig intradermally10

B.LETHAL CHALLENGE METHODTEST ANIMAL:Guinea pig.Weight between 250g 350g.Divide them in to 6 groups of 16 animals.A group containing 5 animals.All guinea pigs have same sex.

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Challenge toxin : Diphtheria toxin containing NLT100 LD50 in 1ml.

Preparation of challenge toxin solution:Challenge toxin + Phosphate buffer saline solution (PH 7.4).Dilute the challenge toxin solution to 2LD50, 1LD50, 1/2LD50 in the same solution.

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Determination of potency of the vaccine: 3 Dilutions of sample and standard vaccine prepared in saline solution each dilution difference by 2.5 fold.Intermediate conc. inject subcutaneously into guinea pigs.This should (intermediate conc) protect 50% animal from lethal effect of subcutaneous injection.

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After 28 days

Test challenge toxin dilution 1ml inject to 4 groups of 5 - guinea pigs - subcutaneously

After 4 days count the number of survival animals Allocate 6 dilutions , one to each of the 6 groups of 16 guinea pigs(1ml)

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Calculate the potency of the vaccine relative to the potency of standard preparation on the basis of number of animals survived in each group of 16 animals.

The test is not valid unless: Vaccine under examination and standard preparation, the 50% protective doses lies between the largest and smallest doses of the preparation given to the guinea-pigs.15

4 groups of 5 guinea pigs injected with the challenge toxin should produce 100LD50.Mortality increases when increases the toxin dose level LR100.The minimum amount of toxin which when combined with 0.01 I.U of standards antitoxin in a volume of 0.2ml causes a local skin reaction that is just visible and indicates the presence of diphtheria toxin.

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TOXOIDS : Toxoids are modified toxins that have toxigenicity but retained the antigenicity. Toxoids are usually prepared by treating toxins with 0.3% formalin called formal.Examples: D.T , T.T LIMES FLOCCULATION :The amount of toxin, mixed with 1 I.U of antitoxin gives a flocculation.Filter the medium with fibrous pads or ceramic candles.

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DEFINITION :Rabies vaccine is a suspension of a suitable strain of fixed rabies virus grown in suitable approved cell culture and inactivated by a suitable method.The vaccine is prepared immediately before use by reconstitution from the dried vaccine with a suitable sterile liquid.RABIES VACCINE

Dried vaccine + sterile liquid Suspension.18

Principle:The potency of rabies vaccine is determined by comparing a lethal intracerebral dose of a rabies virus with the dose of the standard preparation of rabies necessary to give for same protection.Standard preparation: Freeze dried preparation.BIOLOGICAL ASSAY OF RABIES VACCINE19

Test animals: White mice weight 11gm 15gm.6 groups of 16 animals , 4 groups of 10 animals.These two groups are used for titration of LD50 challenge suspension.Injected 0.03ml/mice intracerebrally.

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STANDARD CHALLENGE VIRUS SUSPENSION21

Prepare 3-10 fold dilution of standard challenge virus suspension. 0.03 ml inject intra cerebrally to a groups of 10 mice.

Observe for 14 days

Count the number of mice surviving in each group

Calculate the virus titre of standard challenge virus suspension by statistical method.DETERMINATION OF CHALLENGE VIRUS (Determination of virus titre of the challenge virus) 22

DETERMINATION OF POTENCY OF THE VACCINE

Prepare 3-5 fold serial dilution of standard and test solution of vaccines

Separate mice in 6 groups of 16 each

Inject 0.03 ml intra peritoneally and after 7 days prepare same solution and inject

Both standard and test should prepared in such away

After 7 days inject 0.03 ml standard virus suspension to vaccinated miceObserve if for 14 days and calculate its potency by statistical method.23

Lowest dilution should protect the 50% of the animalHighest dilution protect less than 50% of the animal

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RABIES ANTISERUM

Rabies antiserum is a preparation containing the specific globulin or its derivatives obtained by purification of hyper immune serum or plasma of healthy horses or other animals having the specific activity of neutralising the rabies virus.

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BIOLOGIAL ASSAY OF RABIES ANTISERUM:Principle:The potency of rabies antiserum is determined by comparing a lethal intra cerebral dose of rabies virus with the dose of standard preparation of rabies antiserum necessary to give the same protection.PROCEDUREStandard preparation:Standard preparation is dried serum or other preparation, the potency of which has been determined in relation to international standard.

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Test animals:Mice, 10g-14g animal same sex.Test virus:Any suitable strain rabies virus of known potency, such as the cvs strain may be used.Test dose of virus:20-1000 LD50 intra cerebral injection to each mouse.

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DETERMINATION OF TEST DOSE OF VIRUSVirus dilution of equal quantity of 2%v/v solution of heat inactivated horse serum in water.

Maintain at 370C at one hour.

Prepare 10 fold dilution in a 2%v/v solution of heat inactivated normal horse serum inject into mouse.

The test is not valid unless the quantity of virus used lies between 20 -1000LD50s.28

DETERMINATION OF POTENCY OF RABIES ANTISERUMPrepare 2 fold dilution of std. Preparation and test preparation with 2%v/v heat inactivated normal horse serum in water.

To each dilution add a quantity of suspension of test virus.

Keep the mixture at 370C for 1 hr.

Inject 0.03ml intra cerebrally to mice.

Observe mice for 14 days

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Mice dying before 5th day after inoculation of virus are eliminate from test , all the mice dying between 5th to 14th day after showing signs of rabies.

Count the number of mice surviving

Calculate the potency of test preparation by standard statistical method.

The preparation pass the test it found to have 80 units/ml.The preparation or the test also same as the standard.

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ASSAY OF HEPATITIS A VACCINEAssay can be carried out by either in vivo or in vitro. In vivo: By comparing the given conditions its capacity to induce specific antibodies in mice with the same capacity of a reference preparation. In vitro: By immunochemical determination of Ag content.

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INVIVO ASSAYSelection and distribution of test animals: Healthy mice of same stock about 5weeks old and from a strain shown to be suitable.

Use animals of same sex

Animals are divided into 7 groups.

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DETERMINATION OF POTENCY OF VACCINE TO BE EXAMINED:Using a 0.9%w/v solution of NaCl R containing the Al Adjuvant used for vaccine.

Prepare at least 3 dilutions to be examined & matching dilutions of reference preparation.

Allocate the dilutions one to each group of animals.

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Inject SC NMT 0.5 ml of each dilution into each animal in the group.

Maintain a group of unvaccinated controls injected SC with the same volume of diluents.

After 28-32 days anaesthetise and bleed all animals, keeping individual sera separate.

Assay the individual sera for specific Ab against Hepatitis A virus by suitable Immunochemical method.

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CALCULATIONSCarry out calculations by usual statistical methods for an assay with a quantal response from the distribution of reaction levels measured on all the sera in the unvaccinated group, determine the maximum reaction that can be excepted to occur in an unvaccinated animals that exceeds this level is by defintion a seroconversion.Make a suitable transformation of percentage of animal showing seroconversion in each group and analyse the data according to a parallel line log dose- response model. Determine the potency of test preparation relative to reference preparation. 35

VALIDITY CONDITIONSThis test is not valid unless: For both the test & reference vaccine, the ED50 lies between the smallest and largest doses given to animals.The statistical analysis shows no significant deviation from linearity or parallelism.The confidence limits are NLT 33% and NMT 300%.Potency requirements:The Upper confidence limit (P = 0.95) of estimated relative potency is NLT 1.0.

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INVITRO ASSAY

Carry out the immunochemical determination of Ag content with acceptance criteria validated against in vivo test . The acceptance criteria are approved for a given reference preparation by competent authority in the light of validation data.37

ASSAY OF HEPATITIS B VACCINEAssay can be carried out by either in vivo or in vitro In vivoBy comparing the given conditions its capacity to induce specific antibodies in mice with the same capacity of a reference preparation.

In vitro By immunochemical determination of Ag content.

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INVIVO ASSAYSelection and distribution of test animals: Healthy mice of same stock about 5weeks old and from a strain shown to be suitable.

Use animals of same sex

Animals are divided into 7 groups.

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DETERMINATION OF POTENCY OF VACCINE TO BE EXAMINEDUsing a 0.9%w/v solution of NaCl R containing the Al adjuvant used for the vaccine.

Prepare at least 3 dilutions to be examined and matching dilutions of reference preparation.

Allocate the dilutions one to each group of animals.40

Inject I.P. NMT 0.5 ml of each dilution in to each animal in group.

Maintain a one group of unvaccinated controls injected I.P. with the same of diluents.

After 28-32 days anaesthetise and bleed all animals, keeping individual sera separate.

Assay the individual sera for specific Ab against Hepatitis B virus by suitable immunochemical method.

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CALCULATIONSCarry out calculations by usual statistical method for an assay with a quantal response from the distribution of reaction levels measured on all the sera in the unvaccinated group, determine the maximum reaction that can be excepted to occur in an unvaccinated animals that exceeds this level is by defintion a seroconversion.Make a suitable transformation of percentage animal showing seroconversion in each group and analyse the data according to a parallel line log dose-response model. Determine the potency of test preparation relative to reference preparation.

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VALIDITY CONDITIONSThis test is not valid unless: For both the test & reference vaccine, the ED50 lies between the smallest and largest doses given to animals.The statistical analysis shows no significant deviation from linearity or parallelism.The confidence limits are NLT 33% and NMT 300%.Potency requirements:The Upper confidence limit (P = 0.95) of estimated relative potency is NLT 1.0.

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Carry out the immunochemical determination of Ag content with acceptance criteria validated against in vivo test .The acceptance criteria are approved for a given reference preparation by competent authority in the light of validation data.INVITRO ASSAY44

REFERENCESIndian pharmacopeia 2007 ; volume : 3.European pharmacopeia 2010 ; volume :3.WoodberryT et al.. Prime boost vaccination strategies: CD8 T cell numbers, protection and Th1 bias. J Immunol 2003; 170: 25992604.;PubMed ;ChemPortAuthor stream.com Google search 45

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