Pharmaceutical Biotechnology Unit III
LECTURE-23
GENERAL METHOD OF PREPARATION OF IMMUNITY RELATED PRODUCTS Cont…
1. PREPARATION OF BACTERIAL VACCINES
A. Killed bacterial vaccine preparation
(Example: Cholera vaccine)
i. Selection of an antigen
Each strain is carefully checked for freedom from variation and absence of contaminating
organisms.
ii. Inoculation into media and incubation
Bacteria grown in media rich in proteins, vitamins and salts
Selected strain is inoculated onto a solid or liquid medium
Incubated under optimum conditions for 1-3 days
At the end, cells are harvested from:
Solid medium- by scraping the organism from solid surface with sterile saline centrifuged
to remove pieces of agar washing off
Liquid medium- by centrifugation washed free from broth constituents
iii. Inactivation of cell suspension
Cell suspension can be inactivated by:
Heat: 56ºC for one hour
Chemicals:
0.5% formalin for plague and pertussis,
Phenol for cholera,
Thiomersol for pertussis,
75% alcohol for TAB and TABC
iv. Standardization
The total number of organisms per mL is determined by any one of the following methods:
Direct: Helber cell or hemocytometer method
Indirect: Opacity method such as Brown’s tube or photoelectric merthod
v. Formulation
By incorporating some other substances including
Acidity regulators: Sodium or potassium phosphate
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Pharmaceutical Biotechnology Unit III
Preservatives: Thiomersol
Stabilizers: Formaldehyde or phenol
vi. Storage:
Stored in original packing at optimum temp. (mostly 2-8ºC) and protected from light
All vaccines are sensitive to some extent to heat and cold as,
Heat may speed up the decline in potency
Freezing may cause increased reactogenicity, loss of potency and hairline cracks in the
container, leading to content contamination,
B. Attenuated bacterial vaccine preparation
Steps involved in preparation of attenuated bacterial vaccine are same as for killed bacterial
vaccine.
The only difference is that there is no sterilization or inactivation stage
Following strict regulations are laid down for the manufacture of attenuated vaccine:
Use of completely self contained laboratory suit in which no living organism except the desired
one are allowed
Superlative air conditioning
Regular X-ray examination of staff to prevent contamination from virulent bacilli
Example: BCG vaccine
2. PREPARATION OF VIRAL VACCINES
Presently existing viral vaccines categorized in to types:
a) Whole virus vaccine: Live (smallpox vaccine) and inactivated (Typhus vaccine)
Inactivated whole virus vaccine: Virus is developed through the normal virus culture techniques
like, Tissue culture (Polio salk), Eggs (Influenza), Mouse brain (rabies semple vaccine)
Live whole virus vaccine: Manufactured from attenuated strains that are almost or totally
devoid of pathogenicity but capable of prompting a protective immune response.
b) Subunit vaccines: Prepared from purified viral antigens
Methods of production
a) Cultivation of virus using free living animals
b) Fertile eggs
c) Tissue cultures
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Pharmaceutical Biotechnology Unit III
**Preparation of viral or bacterial vaccines includes all the steps as discussed in general method of
preparation (Lecture 22) and can be summarized as following figure-1.
3. PREPARATION OF TOXOIDS
Toxoids are the immunological preparations gives against disease caused by some toxigenic strains.
Ability of bacteria to produce toxins is called toxigenicity.
There are two types of toxins:
Endotoxins: Cell associated toxins, produced from gram negative bacteria
Exotoxins: Extracellular and diffusible, associated with the cell wall of gram positive bacteria.
3.1 Toxoids formulation
Toxins are converted into toxoid either by chemical or enzymatic treatment.
Formulation is accelerated by treating with variety of reagents: formalin, iodine, ascorbic acid,
kettones etc.
Mixture is maintained at 37ºC at pH range 6-9 for several weeks.
Treatment destroys their toxic properties with no significant loss of antigenic activity.
Some of the official toxoids are:
Diphtheria vaccine or diphtheria toxoid
Tetanus toxoid (TT)
** Preparation steps of toxoids are same as mentioned in general method of preparation in lecture 22.
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Pharmaceutical Biotechnology Unit III
4. PREPARATION OF ANTITOXINS
Antitoxins are antibodies that are used to protect the body by the process of passive immunization.
Antitoxins have the capacity to neutralize a particular toxin.
Manufactured by certain animals, plants, and bacteria
Prepared within organisms but can be inserted into other organisms comprising humans.
4.1 Preparation technique
Technique includes injecting an animal with a safe amount of a particular toxin.
Then the animal’s body makes the antitoxin needed to neutralize the toxin.
After a period of several weeks to months of active immunization, the blood is collected from the
animal.
Immunoglobulins are When the antitoxins is attained from the blood, it is purified and separated
into specific components that are then injected into a human or other animal, prompting passive
immunity.
Examples of antitoxin and antibody containing preparations:
i. Antitoxic antibodies: Those neutralize Exotoxins
Botulinum antitoxin
Diphtheria antitoxin
Gas-gangrene antitoxin
Tetanus antitoxin
ii. Antibacterial antibodies: Those neutralize endotoxin containing bacteria
Leptospira antiserum
iii. Antiviral antibodies: Those neutralize virus
Rabies antiserum
iv. Human normal immunoglobulin injection: Contains the purified antibodies from human plasma
Bhawana Kapoor
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