This document discusses the production of various types of vaccines. It begins by explaining what vaccines are and how they work to stimulate immunity. It then provides classifications of official vaccines, including bacterial killed, bacterial attenuated, rickettsial killed, and viral killed or attenuated vaccines. The document proceeds to discuss the specific production methods for several vaccines, including diphtheria antitoxin, tetanus toxoid, cholera, BCG, typhus, and polio vaccines. Production may involve growing bacteria or viruses in eggs, animals, or cell cultures and treating them with chemicals or heat to kill or attenuate the pathogens while retaining antigenicity.

1. Dr. Pulipati Sowjanya
Professor & Head
Dept. of Pharmaceutical Biotechnology
Vignan Pharmacy College
Vadlamudi, Guntur (Dt)
1

2. INTRODUCTION
2
Vaccines are medical marvels. Basically, they
are purified preparations that can stimulate
body's immune system to produce antibodies
that can provide future, long-term protection
from any exposure to a specific type of
microbe.
A vaccine is a biological preparation that
improves immunity to particular disease.
Edward Jenner

3. INTRODUCTION (Cont..)
3
A vaccine typically contains an agent that resembles a
disease-causing microorganism and is often made from weakened
or killed forms of the microbe, its toxins or one of its surface
proteins.
This agent stimulates the body's immune system to
recognize the foreign agent, destroy it, and "remember" it, so that
the immune system can more easily recognize and destroy any of
these microorganisms that it later encounters.

4. 1. BACTERIAL KILLED:
a) Cholera d) Typhoid-paratyphoid A&B
b) Pertusis (TAB)
c) Plague e) Typhoid-paratyphoid A,B&C
(TABC)
2.BACTERIALATTENUATED (LIVE):
a) Bacillus calmette guerin (BCG)
3.RICKETTSIAL KILLED: a) Typhus
4.VIRAL KILLED:
a) Influenza c) Poliomyelitis
b) Measles d) Rabies
5.VIRAL ATTENUATED:
a) Measles c) Yellow fever
b) Smallpox d) Poliomyelitis
CLASSIFICATION OF OFFICIAL VACCINES
4

5. 5
Antigen-containing
preparations (Vaccines)
Antibody-containing
preparations (Sera)
Stimulate active immunity Give passive immunity
Patient produces antibodies Patient receives antibodies
Immunity develops slowly Immunity produced quickly
Long lasting effect Temporary effect
Used for long-term prophylaxis
Used for short-term prophylaxis
and therapeutically
Difference Between Vaccines & Sera

6. TYPES OF VACCINES
❑ These are different forms of vaccines
❑Simple vaccines Eg: Plague (Pasteurella pestis)
➢ Mixed vaccines
➢ Univalent vaccine
➢ Polyvalent vaccines
❑Vaccines are administered by the parental route except
poliomyelitis vaccine which is administered by the oral route.
6

7. DIPHTHERIA
7
Causative organism:
➢ “Corynebacterium diphtheriae”
Selection of Animal:
➢ Horse
➢ Goat
➢ Injecting the inoculums and
collection of more blood is possible
with horse.
IMMUNE SERA

8. Diphtheria (cont….)
8
The neck muscles of horse are injected with
toxoid for a few minutes to several months.
The first dose is not more than 5ml and
increased to 600ml to get satisfactory antitoxin
titre
8 litres of blood was withdrawn from
jugular veins into the bottles containing anti
coagulant solution.
Repeat the bleeding twice for the next 8
days after that give rest for 10 days.
PREPARATION

9. 9
Give a short course of antigen to
stimulate further antibody
Repeat 3 bleedings, give rest, course of
antigen and continue bleedings till animal
stops producing a satisfactory antitoxin
titer (usually 4 to 5 courses)
Store the collected blood in refrigerator
till cells are settled
Plasma is siphoned off & calcium
chloride is added to induce clotting
Clot is separated from the serum by
filtration

10. Serum + ammonium sulphate
Gamma globulin fraction pptd & is separated and discarded
Add ammonium sulphate
Beta globulin fraction + antitoxin is precipitated
Liquid portion with albumin is separated by filter press
Precipitate is transferred from filter cloth to cellulose film
bags
Suspend in tanks of chlorinated running water for dialysis
Ammonium sulphate passes out, remove it and antitoxin passes
back into the solution. Adjust isotonicity
1. Concentration by Fractional Precipitation
10

11. 2. Concentration by Proteolytic Digestion
Diluted serum+pepsin, adjust pH to 4 and incubate at 37°C for 2 days
The albumin is completely digested and pdt will pass through a dialysing
membrane
The gamma-globulin fraction is partly digested to dialysable compounds
and precipitated by PH
The beta-globulin is split into two fragments one of which has antitoxic
activity
The liquid is filtered to remove the pptd gamma-globulin
To the filtrate add ammonium sulphate and heat at 55°C for 1 hr
The inactive fragment of β-globulin is denatured and pptd. It is filtered off
and add more ammonium sulphate to ppt active fragment
This is separated, dialysed, adjusted to isotonicity and preserved
Sunday, October 18, 2020 Department of Biotechnology, Vignan Pharmacy College, Vadlamudi 1411

12. • The toxoids used as
vaccines are toxins
that are incubated with
formalin
• This completely destroys the toxic properties without loss of
antigenic properties
• Formalin does this by acting directly on the toxic groups
(probably free amino groups) without affecting the
antigenically active sites
TOXOIDS
12

13. Preparation of toxoid
1. Suitable strain on liquid medium (not made with broth from
horse muscle)
2. Incubate till toxin production reached satisfactory level.
3. Bulk of organisms are removed on paper pulp (to prevent
clogging of bacterial proof filters)
4. Filtrate is sterilized using fibrous pads or ceramic candles.
Conversion to toxoid
1. Add Formaldehyde and incubate at 37°C(2-3weeks) [toxicity
is removed]
2.This is unpurified form (called as Anatoxin) & Purified one are
called Purified formal toxoid.(PFT)
13

14. TETANUS TOXOID
CAUSATIVE ORGANISM:
➢ “Clostridium tetani”
➢ Anaerobic organism that contains
exotoxin.
14

15. Toxin is converted to toxoid by adding
formalin and incubate at 37 ̊ C for 2-3
weeks is known as formal toxoid.
To formal toxoid add charcoal to remove
impurities.
Filter the solution and treat the filtrate with alum.
This reacts with bicarbonates, phosphates and
protein impurities in the toxoid to produce a ppt
containing aluminium hydroxide and phosphate.
The toxoid is adsorbed on to the mineral carrier.
The precipitate is washed and suspended in saline
containing bactericide.
15

16. PURIFIED TOXOID ALUMINIUM PHOSPHATE
The precipitate is separated by filtration and suspended in saline solution.
The toxoid precipitates as aluminium phosphate toxoid.
Add aluminium sulphate and cadmium chloride as protein precipitants
Toxoid is treated with magnesium hydroxide to precipitate colour,
phosphates and proteins.
16

17. It is a killed bacterial vaccine
CAUSATIVE ORGANISM:
➢ Vibrio cholerae
Conversion into vaccines
➢ Treat the organism with chemical
bactericide like
▪ Formalin
▪ Phenol
▪ Thiomersal
17

18. CHOLERA ( Cont…)
18
The suspension of vibrio
cholera organism is killed
by either heating or treating
with bactericide.
0.5% formalin and 7%
alcohol are used as
bactericides to develop the
killed strain .
The organism is suspended
in a medium containing the
preservative and freeze dry
the product.

19. BCG (Bacillus Calmette Guerin)
It is a live attenuated vaccine
Causative organism:
➢ Mycobacterium tuberculae
Preparation:
➢ It is a suspension of live micro organism.
➢ The organism is attenuated to inhibit the pathogenicity and
retains the antigenicity.
➢ The culture grown for not more that 14 days.
19

20. Bacillus Calmette Guerin (Cont....)
The organism is grown in an appropriate
medium (LJ).
It should be retain the antigenicity as long
as possible .
Freeze dry the product.
When the organism grown in the medium
it forms clumps.
This can be avoided by addition of
polyoxyethylene ether into the medium.
20

21. 21
Virulent rickettsiae are injected into the yolk
sacs of embryonated eggs and incubate for
7days.
These are collected under aseptic conditions
and suspended in saline
Add Formaldehyde solution so that to get a
concentration of about 0.2 to 0.5%.
It is purified with ether or trichloro trifluoro
ethane.
PREPARATION:
CAUSATIVE ORGANISM: Rickettsia prowazeki
21

22. 22

23. Cultivation of viruses in vaccine production
Since viruses are intracellular parasites they will
grow only within living cells.
1. Free – Living Animals
2. Fertile Eggs
3. Tissue Culture
23

24. Free-Living Animals
Very few vaccines are made using free-living animals.
The products are good antigens but the method is
inconvenient and costly.
❖Typhus - Lungs of rodents
❖Rabies – Brains of sheep or rabbits
❖Poliomyelitis – Kidneys of monkeys
24

25. Fertile Eggs
Advantage:
Easier to keep the product free from contamination
Precautions:
1. Strict asceptic conditions
2. Repeated passage of virus from egg to egg must be avoided.
3. Viruses grown in the yolk sac or embryo are separated by
grinding to prevent traces get protein get into the vaccine.
4. The eggs must be candled to confirm the embryos are alive.
25

26. 26

27. 1. SELECTION OF SUITABLE TISSUE
Viruses grow only in primate cells- monkey kidney has
become the most widely used. (Eg: in poliomyelitis, measles)
• Attenuated measles: chick embryo.
• Cow pox: calf embryo skin.
• Tissues should be free from living microbes.
1.Maintainence of cleanliness of cells by continuous subculture.
2.Establishment of growth and maintenance of metabolism under
artificial conditions.
3.Organ or tissue removed is cut up or minced and treated usually
with trypsin, to disperse the cells.
TISSUE CULTURE
27

28. TWO TYPES OF CULTURE
(A) suspended cell culture
❖ Suspended in liquid medium & maintains cell metabolism &
very little or no proliferation.
(B) Fixed cell (monolayer) culture
❖Few cells are maintained in medium to settle on one side of large
flat sided bottle.
❖On incubation they attach to glass & multiply into uniform layer
one cell thick and spread over the lower side of bottle.
❖Higher yield of virus per cell, as multiplication occurs. Easier to
change medium
28

29. 1. Balanced salt solution –Optimum PH & osmotic pressure
2. Nutrients: aminoacids, growth factors, dextrose, protein
hydrolysates etc. Serum & proteins must be excluded
3. A PH Indicator phenol red to show the state of cell
metabolism.
4. Antibiotics, both antibacterial and antifungal to prevent growth
of the contaminants.
5. Heavy metals are toxic to culture so in low concentration.
2.MEDIA NEEDED TO MAINTAIN THESE CULTURES
29

30. • After the suspended cells have become adjusted to medium or
monolayer has fully developed seed virus is added and incubated.
• Here it is rocked to prevent accumulation of high concentration of
harmful metabolites on the surface of tissue cells & to ensure free
exchange of oxygen and CO2.
• Virus invades cells, multiplies and is released into medium at the
end of incubation.
• After suspended cells are allowed to settle, is siphoned off
aseptically.
3. CULTIVATION OF VIRUSES IN THE CELLS
30

31. POLIOMYELITIS
❑ There are 3 distinct antigenic types of poliomyelitis virus
Type-I, Type-II, Type-III.
❑ The 3 types of virus are grown separately in cell culture of monkey
kidney tissue.
❑ Rhesus monkeys are used. They are quarantined and checked for
communicable disease.
31

32. Inactivated Vaccine: It is known as Salk-type after the American
virologist who first developed it.
After the virus suspension has been harvested it is tested for
1. The correct strain of poliomyelitis virus
2. The virus titre
3. Free from viral, bacterial and fungal contaminants.
➢ The viral suspension is passed through filters to remove tissue cells
and bacteria.
➢ Inactivated by treating with 0.01% formaldehyde under controlled PH
and temp with magnetic stirrer.
➢ This process takes around 6 days.
➢ Neutralize the formaldehyde with sodium metabisulphite & add
thiomersal as bactericide.
32
POLIOMYELITIS

33. Attenuated (Oral) Vaccine: It is known as Sabin-type after the American
virologist who first developed it.
It is manufactured in same way as Salk-type except that-
➢Attenuated strains, prepared by rapid passages through tissue cultures
of monkey kidney cells, are used.
➢There is no inactivation stage
➢In addition to testing for freedom from extraneous bacteria, moulds and
viruses, special tests are necessary, because the virus in the
vaccine is living, to confirm the absence of virulent poliomyelitis virus.
33
POLIOMYELITIS

34. SMALL POX
CAUSATIVE ORGANISM:
➢ Variola virus
PREPARATION:
Living cow pox virus are used.
Vaccine is usually obtained from lesions produced on the skin of living
mammals Eg: healthy calves and sheep.
Inoculation
The flanks and abdomen are scrubbed, disinfected shaved rescrubbed and
redisinfected.Then in special rooms, the shaved areas are-
(a) scarified, i.e lightly scratched with comb like device, without
drawing blood.
(b) inoculated by rubbing into scratches.
34Department of Biotechnology, Vignan Pharmacy College, Vadlamudi

35. SMALL POX (CONT.….)
35
Incubation:
During next 4-5 days vesicles development
along the lines of scarification.
Through out this period inoculated areas are kept aseptically
clean.
Harvesting:
• Animals are killed, exanguinated and washed.
• Contents of vesicles are (lymph) removed by curretage i.e
by scraping with Volkmann's spoon
• Pooled material is homogenized.
• Postmortem the animal to confirm absence of diseases.

36. SMALL POX (CONT.….)
Purification:
1.Done by grinding with equal volume of glycerin & storing
at -10ºC for long time.
2.Lymph extracted with protein solvent
Eg: trichlorofluoroethane
3. Phenol 0.4% & incubated at 22ºC
4. Glycerin 40% & peptone 1% at -10ºC.
5. Brilliant green to mark the area of application of vaccine.
36

37. 37