University Institute of Pharmaceutical Sciences is a flag bearer of excellence in Pharmaceutical education and research in the country. Here is another initiative to make study material available to everyone worldwide. Based on the new PCI guidelines and syllabus here we have a presentation dealing with the concept of Diphtheria vaccine. Thank you for reading. Hope it was of help to you. UIPS,PU team

1. Diphtheria Vaccine
Presented By – Charudatta S. Jog
Guide – Sandip V. Pawar
M.Pharma (Pharmaceutical Analysis)
UIPS, Panjab University,
Chandigarh - 160014
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2. Contents
• Introduction
• History of Diphtheria Vaccine
• Diphtheria Vaccine (Adsorbed)
• Diphtheria Vaccine (Adsorbed) for Adults and
Adolescents
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3. Introduction
• Diphtheria is an infection caused by the bacterium
Corynebacterium diphtheriae.
• Signs and symptoms may vary from mild to severe.
• Usually start 2-5 days after exposure.
• Spread by direct contact and through the air when an
individual coughs or sneezes.
• May also be spread through contaminated objects.
• Diagnosis can often be made based on the appearance of the
throat with confirmation by microbiological culture.
• The disease was first described in the 5th century BC by
Hippocrates.
• The bacterium was identified in 1882 by Edwin Klebs. 3

4.  Signs and Symptoms:-
 Fever of 38℃ (100.4℉) or above
 Chills, fatigue, bluish skin coloration (cyanosis)
 Sore throat, hoarseness, cough, headache
 Difficulty in swallowing and breathing
 Lymphadenopathy
 Within 2-3 days, forms thick gray coating called “pseudomembrane.”
 Complications:-
 Myocarditis, inflammation of nerves,
 Kidney problems, bleeding problems due to low level of platelets,
 Abnormal heart rate
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5. History of Diphtheria Vaccine
 1613, Spain (El Año de los Garrotillos –
The Year of Strangulations)
 1735, swept through New England
 Before 1826, known by different names; In England, Boulogne sore throat
 In 1826, Pierre Bretonneau gave the disease the name diphthérite (from Greek diphtheria “leather”),
describing the appearance of pseudomembrane in the throat.
 In 1883, Edwin Klebs identified the bacterium causing diphtheria and named Klebs-Loeffler bacterium.
 Current nomenclature Corynebacterium diphtheriae.
 Friedrich Loeffler was first person to cultivate C. diphtheriae in 1884.
 Joseph P. O’Dwyer tube for laryngeal intubation in 1885.
 In 1888, Emile Roux and Alexandre Yersin.
 In 1890, Shibasaburo and Emil von Behring – 1st Nobel Prize
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6. Diphtheria Vaccine (Adsorbed)
• Diphtheria Vaccine (Adsorbed) is a preparation of diphtheria formol toxoid
with a mineral adsorbent.
• The formol toxoid is prepared from the toxin produced by the growth of
Corynebacterium diphtheriae.
Production:-
o General provisions:- The maximum number of Lf units per single human
dose of diphtheria vaccine (adsorbed) is 30.
o Bulk purified toxoid:-(BPT)
 Seed cultures are managed in a defined seed-lot system in which
toxinogenecity is conserved and, where necessary, restored by deliberate
reselection.
 A highly toxinogenic strain of Corynebacterium diphtheriae with known
origin and history is grown in a suitable liquid medium.
 At the end of cultivation, the purity of each culture is tested and
contaminated cultures are discarded.
 Toxin containing culture medium is separated aseptically from the bacterial
mass as soon as possible.
 The toxin content (Lf per ml) is checked to monitor consistency of
production. 6

7. ……Contd
 Single harvests may be pooled to prepare the bulk purified
toxoid.
 The toxin is purified to remove components likely to cause
adverse reactions in humans.
 The purified toxin is detoxified with formaldehyde by a
method that avoids destruction of the immunogenic 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 preparation of the final bulk
vaccine.
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8. o Absence of toxin and irreversibility of toxoid:-
Inject s.c. into each of 5 guinea pigs at
least 500Lf of non-incubated BPT in
vol. of 1ml using same buffer as for
final vaccine, without adsorbent
Animals that die shall be autopsied and
examined for symptoms of diphtheria
intoxication (red adrenals)
The bulk purified toxoid shall pass
the test if no guinea pig shows
symptoms of specific intoxication
within 6 weeks of injection and if at
least 80% of animals
All test procedures shall be
approved by National
Regulatory Authority.
Each bulk purified toxoid shall be
tested to ensure that reversion to
toxicity cannot take place on storage.
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9. Cell Culture method
 Antigenic purity:- Not less than 1,500 Lf per mg of protein
nitrogen.
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10. Final Bulk Vaccine
 Identification:- Sodium citrate 10%w/v concentration.
Maintain at 37℃ for about 16 hours + centrifuge clear
supernatant reacts with a suitable diphtheria antitoxin and yields
precipitate.
 pH:- 6.0 to 7.0
 Specific toxicity
 Potency:- determine by any of methods of biological assay of
Adsorbed Diphtheria Vaccine.
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11. Biological assay of adsorbed diphtheria
vaccine
1. Intradermal challenge method:-
• Principle:- The potency of adsorbed diphtheria vaccine is
determined by comparing the dose necessary to protect
guinea pigs against the erythrogenic effects of a range of
intradermal injections of diphtheria toxin with the dose of
the Standard preparation of adsorbed diphtheria toxoid
necessary to give the same protection.
• Standard preparation:- Is International standard of Diphtheria
toxoid, adsorbed, or another suitable preparation the potency of
which has been determined in relation to International standard.
• Suggested method:-
i. Test animals:- white guinea pig (250-350g)
- 6 groups
- the guinea pigs are all of same sex 11

12. ……Contd
ii. Selection of the challenge toxin:-
 Select a preparation of diphtheria toxin containing 67 to
133 Limes reactionis/100 (Lr/100) in Limes flocculationis
(Lf) and 25,000 to 50,000 minimal reacting doses for
guinea pig skin in 1 Lf.
 If challenge toxin preparation has been shown to be stable,
it is not necessary to verify activity for every assay.
iii. Preparation of challenge toxin solutions:-
 Immediately prior to use, dilute the challenge toxin with a
suitable diluent to obtain a challenge toxin solution
containing about 512×10-4 in 0.2ml.
 Dilute a portion of this challenge toxin solution to give a
series of five 4-fold dilutions.
iv. Determination of potency of the vaccine:-
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13. 2. Lethal challenge method:-
i. Test animals:- healthy, white or light coloured guinea pig
(250-350g), 6 groups of sixteen; four groups of four.
- should be of same sex.
ii. Challenge toxin :- select a preparation of diphtheria toxin
containing not less than 100 LD50 in 1.0ml.
iii. Preparation of the challenge toxin solutions:- Immediately
prior to use, dilute the challenge toxin with a phosphate
buffered saline pH 7.4 or normal saline a challenge toxin
containing approx. 100 LD50 in 1.0ml.
 Dilute portions of this challenge toxin solution to 2LD50,
1LD50 and ⅟2 LD50 in the same solution.
iv. Determination of potency of the vaccine:-
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14. 3. Antibody induction method:-
 Inject s.c. on each of each of each of two occasions
separated by an interval of NMT 4 weeks, ⅟15 of stated
human dose diluted to 1ml with saline solution, into each
of 10 normal, healthy guinea pigs (250-350g).
 Not earlier than 2 weeks and not later than 3 weeks after
second injection, collect the serum from animal and
determine the antitoxin content of the serum of each
animal, as described under Diphtheria antitoxin or any
other method approved by National Regulatory Authority.
 The geometric mean of the antitoxin contents shall be not
less than 2.0 units per ml with reference to Diphtheria
antitoxin standard.
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15. Final Lot
• Identification
• Tests
- Aluminium
- Free formaldehyde
- Antimicrobial preservative
- Sterility
- Abnormal toxicity
- pH
- Assay
- Labelling 15

16. Diphtheria Vaccine (Adsorbed) for Adults
and Adolescents
• Production
• General provisions
• Bulk purified diphtheria toxoid
Final Bulk Vaccine
 Identification
 pH
 Specific toxicity
 Assay
 Antimicrobial preservative
 Sterility
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17. Final Lot:-
 Identification
 Tests:-
- pH
- Aluminium
- Free formaldehyde
- Antimicrobial preservative
- Sterility
- Abnormal toxicity
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