This document discusses the preparation of bacterial vaccines. It describes the key steps involved in producing killed bacterial vaccines such as selection of the bacterial strain, growth of bacteria in culture, inactivation of the bacteria through heat or chemicals, standardization, formulation, and storage. The document also discusses the preparation of live attenuated bacterial vaccines, using the BCG vaccine as an example. Key steps for BCG preparation include attenuation of the bacteria by repeated subculture, growth in liquid medium, harvesting and freeze drying the bacteria, and standardization based on viable cell counts.

1. SJM College of Pharmacy,
Chitradurga
Prepare By,
AdarshaBP
Ass Professor(Pharmacognosy)
SJM College of Pharmacy
1
PHARMACEUTICAL
BIOTECHNOLOGY

2. PREPARATION OF BACTERIAL VACCINES:
Killed and attenuated bacterial vaccines are available officially for clinical use.
A. Steps involved in killed bacterial vaccine preparation:
1. Selection of an antigen:
• The exact strain or strains to be incorporated for preparation of bacterial vaccine.
Eg. Cholera vaccine: smooth strains of the two serological types Inaba and Ogawa
• TABC vaccine: O and H antigens in S. typhi and S. paratyphi microorganisms and these
organisms also contains Vi antigen.
• Each strain is carefully checked for freedom from variation and absence of contaminating
organisms.

3. 2. Inoculation into media and incubation:
• Bacteria grown on media those are rich in proteins, vitamins and salts. Media that are often used
in the production of vaccines are Medium 199, Eagle Medium and Minimum Essential Medium.
• The selected strain is inoculated on to a solid or into liquid medium and incubated under
optimum conditions for 1-3 days.
• At the end of this time the cells are harvested from,
 Solid medium by scraping the organism from solid surface with sterile saline and centrifuged to
remove pieces of agar detached during the washing off.
 Liquid medium by centrifugation and after removal of the supernatant fluid, washed free from
broth constituents, which might cause reaction on injection and resuspended in saline.

4. 3. Inactivation of cell suspension:
• The bacterial cell suspension is inactivated by two different methods
• Heat: Low temperatures are essential to avoid damage to the antigens. The usual treatment Is 56
ºC for one hr. As this exposure cannot kill all contaminating organisms particularly Spores, strict
asepsis is necessary throughout manufacture.
• Chemicals: The organisms are killed chemically when heat treatment affects antigenechy (e.g.,
Plague vaccine). Chemical methods are the alternative to the heat method.
 Eg., 0.5% of formalin for plague and pertussis
 Phenol for cholera
 Thiomersol for pertussis
 75% of alcohol for TAB and TABC

5. 4. Standardization:
• The total number of organisms per mL is
determined either directly using Helber cell or
Hemocytometer method, indirectly by an opacity
method such as Brown's tubes or photoelectric
nephelometer.

6. 5. Formulation:
• The standardized cell suspension is formulated into final vaccine by
incorporating some other substances include acidity regulators like sodium
or potassium phosphate, preservatives to avoid contamination in multi dose
vaccines-thiomersol is the commonly used preservative and rarely
formaldehyde or Phenol derivatives are also used.
• Stabilizers are used to help the vaccine maintain its effectiveness during
storage. Eg., MgCl2, MgSO4, Lactose-sorbitol and sorbitol-gelatin.

7. 6. Storage
• Vacines should be stored in the original packaging at +2°C to +8°C and protected
from light All vaccines are sensitive to some extent to heat and cold.
• Heat speeds up the decline in potency of most vaccines, thus reducing their shelf life.
• Effectiveness cannot be guaranteed for vaccines unless they have been stored at the
correct temperature.
• Freezing may cause increased reactogenicity and loss of potency for some vaccines. It
can also cause hairline cracks in the container, leading to contamination of the
contents.

8. B. Attenuated bacterial vaccine:
• Manufacture of satisfactory dead vaccine is not always feasible.
• In some cases the sterilization method damages the antigens while
in others it is possible that immunity stimulating antigens are
produced only when the organisms is multiplying in the body.
• Instead of using pathogenic strain of organisms, weakened
organism is the best alternative for living vaccine preparation which
is safer and have the ability to stimulate antibodies.

9. Advantages of attenuated vaccines:
• Mimics the route of entry of many pathogens and stimulate the mucosal immune response.
• Can be administered orally or nasally avoiding the risk associated with contaminated needles and
need for a professional healthcare infrastructure.
• In comparison to highly purified subunit vaccines, they are relatively cheap to produce and
administer, and do not require sophisticated downstream processing or formulation with adjuvant.
• The immunity is stronger and more lasting because the organism multiplies in the tissue.
• As a result of multiplication, smaller dose can be given.
• Often reduce the need for booster vaccinations,
• The attenuation process often preserves replication and quantities of pathogen antigen are
synthesized and accumulate in the host.

10. Disadvantages:
i) The bacteria may spread to other individuals.
i) It is more difficult to attenuate than to inactivate the organism.
i) There is no inactivation process great care must be taken to prevent
contamination
iv) The organism is still alive, can sometimes recover its virulence and cause
disease in the
v) Live organisms are grown in large quantities in culture vessels, contaminating
organises may invade the culture and become incorporated into the vaccine.

11. Preparation of live bacterial vaccines:
• The methods used to prepare dead and living bacterial
vaccines are essentially the same except that for living
preparations:
There is no sterilization or inactivation stage
The viability of the cells must be maintained
Standardization is on the basis of viable count
• Examples of attenuated bacterial vaccines:

12. BCG Vaccine:
• This is a suspension of living cells of a strain of Mycobacterium tuberculosis known as ihe
• bacillus of Calmette & Guerin. Calmette and Guerin were French bacteriologists. Ther
• investigated attenuated vaccines because Calmette had worked under Pateur who hai
• been very successful with this type of preparation in anthrax and rabies.
Source of microorganism:
• A bovine (cattle) strain was selected in the hope that it would be safer for man than the human
variety.
• The TICE strain used in the BCG Vaccine preparation was developed at the University of Illinois
from a strain originated at the Pasteur Institute.

13. Method of attenuation:
• It was grown on a medium containing ox bile as this had been found to
reduce virulence of organisms.
• It was sub-cultured on this medium for 13 years and had been sub cultured
231 times by which time it had become safer to administer to human and
was stable.
Medium for growth of the organism:
• Glycerin, asparagine, citric acid, potassium phosphate, magnesium sulfate
and iron, ammonium citrate are the components for the growth of TICEO
strain.

14. Preparation
• Particularly strict regulations are laid down for the manufacture of attenuated vaccine.
• These includes
 Use of a completely self contained laboratory suit in which no living organisms except the BCG strain are allowed
 Superlative air-conditioning
 Regular X-ray examination of staff to prevent contamination with virulent human tubercle bacilli.
Preparation of liquid vaccine
• Before use the strain is rigorously checked for antigenecity and freedom from pathogenecity.
• It is then grown on a liquid medium for not more than 14 days at 37 C, older cultures being less efficient antigens.
Then the organisms are separated by cententrifugation, washed and suspended in a vehicle that preserves
antigenecity and viability for as long as possible.
• The resulting liquid vaccine has been replaced by a freeze-dried form because the former has serious
disadvantages:

15. b) Preparation of freeze dried vaccine:
• The freeze-drying preparation consisted of dextran 8.3%,
glucose 7.5% and triton 339 0.025%. The primary freeze-
drying and secondary drying (over P2O5) conditions were
controlled so as to leave final residual moisture of
approximately 1% in the frreeze dried vaccine.
• The ampoules were sealed in vacuo (about 0.05 mm. Hg).

16. Standardization:
In case of BCG vaccine viable count techniques like pour plate
technique, drop technique, spread plate technique, roll tube
technique and membrane filtration are followed to determine the
number of living cells present per mL of sample. BCG vaccine
contains 8x 10 colony forming units (CFU).
Storage:
Freeze-dried BCG vaccines may be kept frozen at -15 ºC to -25 °C