Microbiological assay of vaccines(adsorbed diptheria vaccine,rabies vaccine,Hepatitis-A&B vaccines)

1. MICRO biological assay of vaccines
PRESENTED BY:
T. LAKSHMI BHAVANI
(2015MPH40023)
Under the guidance of:
Dr. S. JOSHNA RANI
SPMVV - TIRUPATHI
M.Pharm.,Ph.D
Pharmaceutical Analysis
1ST YEAR, I-SEM
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2. CONTENTS
 Introduction to vaccines
Microbiological assays
 References
• Adsorbed diphtheria vaccine (ADV)
• Rabies vaccine
• Hepatitis A & B
2

3. INTRODUCTION
A 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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4. ADSORBED DIPHTHERIA VACCINE (ADV)
1.DEFINITION:
Diphtheria formol toxoid
+ Mineral carrier
[ which is hydrated
Aluminium hydroxide[or]
Calcium PO4].
Adsorbed diphtheria
vaccine.
4

5.  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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6. 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.
6

7. A . INTRADERMAL CHALLENGE METHOD
Principle:
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:
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8. PROCEDURE:
Standard preparation:
Standard preparation consisting of toxoid adsorbed on a
aluminium hydroxide with polygeline.
Test animal:
 White guinea pig
 Weight between 250-350gm
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9. 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.
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10. DETERMINATION OF POTENCY OF THE VACCINE
Assay limit falls between 50% and 200% of the estimated potency.
After 48 hrs, record the incidence of specific diphtheria erythema
After 28 days shave the both flanks of each guinea pig
Inject guinea pig subcutaneously (5 numbers)
Vaccine is dilute with the saline solution
0.2ml challenge toxin solution is inject to guinea pig intradermally
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11. B.LETHAL CHALLENGE METHOD
TEST 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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12. 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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13. 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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14. 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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15.  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.
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16. 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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17. 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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18. 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.
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19. 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.
BIOLOGICALASSAY OF RABIES VACCINE
19

20. 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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21. Centrifuge and separated liquid is distributed into sterile ampoules and
stored at 2 – 80.
Homogenized brain with 10% digested casein hydrolysate (PH 7.2)
Wash it with saline solution to remove blood clot
Harvest the brain aseptically
Showing characteristic symptom of encephalitis are sacrified
Intracerebrally 0.03ml of 10 fold dilution standard strain horse serum to the
test animal
Standard challenge virus suspension is prepared by injecting
STANDARD CHALLENGE VIRUS SUSPENSION
21

22. 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)
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23. 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 mice
Observe if for 14 days and calculate its potency by statistical method.
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24. Lowest dilution should protect the 50% of the animal
Highest dilution protect less than 50% of the animal
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25. 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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26. 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.
PROCEDURE
Standard 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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27. 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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28. DETERMINATION OF TEST DOSE OF VIRUS
Virus 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.
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29. DETERMINATION OF POTENCY OF RABIES
ANTISERUM
Prepare 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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30. 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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31. ASSAY OF HEPATITIS –A VACCINE
Assay 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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32. INVIVO ASSAY
Selection 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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33. 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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34. 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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35. CALCULATIONS
Carry 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.
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36. VALIDITY CONDITIONS
This 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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37. 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

38. ASSAY OF HEPATITIS –B VACCINE
Assay 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.
38

39. INVIVO ASSAY
Selection 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.
39

40. DETERMINATION OF POTENCY OF VACCINE TO
BE EXAMINED
Using 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

41. 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.
41

42. CALCULATIONS
Carry 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.
42

43. VALIDITY CONDITIONS
This 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.
43

44. 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 ASSAY
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45. REFERENCES
1. Indian pharmacopeia 2007 ; volume : 3.
2. European pharmacopeia 2010 ; volume :3.
3. Woodberry T et al.. Prime boost vaccination
strategies: CD8 T cell numbers, protection and Th1
bias. J Immunol 2003; 170: 2599–2604. ; PubMed
; ChemPort
4. Author stream.com
5. Google search
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