Kokati Venkata Bhaskara Rao et al.

/ Journal of Pharmacy Research 2009, 2(8),1292-1295

Research Article
Available online through
ISSN: 0974-6943
www.jpronline.info
Production of safe and potent whole cell pertussis vaccine
Gaurav Kumar1, Kokati Venkata Bhaskara Rao* 1. N.Chandrasekaran1, Sourabh Sharma2
1 School of Bioscience and Technology, VIT University, Vellore, TN - 632 014, India

2 Triple Vaccine Section, Central Research Institute, Kasauli, HP - 173 204, India

Received on: 27-02-2009; Accepted on: 29-04-2009

ABSTRACT
Present study focused on the production of safe and potent whole cell pertussis vaccine (WCP). Two days old culture of Bordetella
Pertussis strain 10536 was revive in appropriate conditions and inoculated in Verwey medium to prepare seed culture. The seed culture was
transferred to fermentor containing 5 liter sterilized modified Cohen and Wheeler medium (B2 medium). Growth conditions were maintained as
per the requirements. Samples were collected every 12 hours for microscopic examination and pH determination. Bacterial culture was
harvested when pH reach to 8.0. The suspension was inactivated (detoxified) by heating at 560C for 10 minutes in shaker water bath to reduce
the toxicity of the vaccine. To make an effective vaccine various quality control tests were performed during the production like Agglutination
Test, Opacity test, pH test, Purity test, Sterility test, Mouse weight gain test (Potency test) and Mouse protection test (Specific toxicity test).
Opacity of all the batches were range in between 42-57 IOU/ml, Mouse weight gain range in between 66.6–80% and potency range in between
8.03-11.03 IU/dose. Purity, Agglutination, pH and Sterility tests were found in the acceptance range.

Keywords: Whole cell pertussis vaccine; Potency; Specific toxicity; Opacity.

INTRODUCTION
Pertussis (whooping cough) is a highly contagious, acute
infection of upper respiratory tract resulting approximately 295,000
deaths annually worldwide (1, 2). It is caused by the Gram negative
coccobacillus B. pertussis. Pertussis is primarily a disease of chil-
dren, although cases have been reported in all age groups. Disease is
more severe in infants because maternal antibodies are not protec-
tive. Antimicrobial therapy is beneficial if treatment starts within 10
days of onset of disease. Vaccination is a very efficient method to
control pertussis and it had been controlled in many developed coun-
tries by the use of WCP vaccine (3).
WCP vaccine is a safe and highly potent preparation to cure
pertussis. WCP vaccine is a suspension of whole cells of one or more
strains of killed B. pertussis which contain less toxicity and high
potency (4). Production of WCP vaccine was started when Jules
Bordet and Octave Gengou isolate B. pertussis in 1906 (5). Soon after
it first crude WCP vaccine was prepared since then World Health
Organization (WHO) and other health organizations trying to im-
prove the quality of the WCP vaccine. For the production of WCP
vaccine B pertussis is cultivated, centrifuged, resuspended and inac-
tivated.
Initially WCP vaccines was mono component vaccines, but
since 1947 combination vaccines with Diphtheria and Tetanus toxoid
(DTP) are available and used (6). Three doses of DPT vaccine given
intramuscularly at the interval of 4 to 6 weeks, first dose at the age of
4 to 6 weeks, second at the age of 10 to 12 weeks, third at the age of 14
to 16 weeks and a booster dose of 0.5 ml dose at age of 16 to 24
*Corresponding author.
Tel.: + 91-9894350824
Telefax: +91-416-2240411
E-mail: kokatibhaskar@yahoo.co.in
months (7).
Some case studies suggested that few side effects like red-
ness, swelling, fever, vomiting and diarrhoea associated with WCP
vaccine despite of that it found to be quite effective for providing
prophylaxis against pertussis (8, 9, 10). So the present study focused
on the production of safe and potent WCP vaccine by following the
standard methods recommended by WHO and Indian Pharmacopeia
(IP).
MATERIALS AND METHODS
Animals
LACA strain (white mice) of either sex were taken from animal house
of Triple Vaccine Section, Central Research Institute, Kasauli, HP,
India and used throughout the study. They were maintained in the
controlled food and environmental conditions.
Production strains
Freeze dried culture of B. Pertussis strain 10536 was obtained from
Triple Vaccine Division, Central Research Institute, Kasauli, HP, In-
dia.
Revival of the production strain
Freeze dried culture of B. pertussis strain 10536 was revived on Bordet
Gengou (BG) agar slant and incubated at 350C for 48 hours (11).
Preparation of seed culture
The pure growth from BG medium slant was scraped and inoculated
in to seed flask containing 100 ml Verwey medium (12). The flask was
loaded on shaker at a speed of 140 rpm and incubated for 48 hours at
350C.
Mass production
The seed culture was transferred to the fermentor, containing 5 liter
B2 medium (13), previously sterilized at 1210C for 20 minutes. Growth
conditions were maintained as per the requirements such as pH 7.2 +
0.2, speed of stirrer 600 rpm, air flow rate 20 liter per minute and tem-
perature 350C. Samples were collected every 12 hours for microscopic

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Table:1 (Purity test)
S. No. Batch No. Growth on BG Growth on BA Growth on NA Growth on NB Result
1. 1 Growth Growth No Growth No Growth Pass
2. 2 Growth Growth No Growth No Growth Pass
3. 3 Growth Growth No Growth No Growth Pass

Table – 2 (Agglutination test, pH test, Opacity test)

S. No. Batch No. Production pH at Type of Opacity
Strain harvesting Agglutination Test
1. Batch 1 B. pertussis 10536 8.0 1,2,3 45 IOU/ml
2. Batch 2 B. pertussis 10536 7.9 1,2,3 43 IOU/ml
3. Batch 3 B. pertussis 10536 7.9 1,2,3 47 IOU/ml

Table – 3 (Sterility test)

S. No. Batch No. Growth Growth on Growth on Result
on BG TG Medium SCD Medium
1. 1 No Growth No Growth No Growth Pass
2. 2 No Growth No Growth No Growth Pass
3. 3 No Growth No Growth No Growth Pass

Table – 4 (Specific toxicity test)

S. Sample Weight at Weight at Weight at Total Weight % Weight
No. Injected 0 Day (gm) 3 Day (gm) 7 Day (gm) Gain. (gm) Gain.
1 Saline 150 160 175 25 100%
Batch 1 150 155 170 20 80%
2 Saline 150 180 190 40 100%
Batch 1 150 165 177 27 67.5%
3 Saline 150 170 180 30 100%
Batch 1 150 158 170 20 66.6%

Table – 5 (Potency test)

S. Batch Inoculated No. of Immun. Challenge Survival Death ED50 Potency
No No. dose (ml) mice Dose organism (IU/dose)
1 Working standard 0.5 18 0.0625 10 5 12 6 0.0136
0.5 18 0.0125 10 5 9 9
0.5 18 0.0025 10 5 5 13
Batch 1 0.5 18 0.0625 10 5 14 4 0.0125 8.70
0.5 18 0.0125 10 5 9 9
0.5 18 0.0025 10 5 4 14
2 Working standard 0.5 18 0.0625 10 5 11 7 0.0200
0.5 18 0.0125 10 5 8 10
0.5 18 0.0025 10 5 3 15
Batch 2 0.5 18 0.0625 10 5 14 4 0.0145 11.03
0.5 18 0.0125 10 5 8 10
0.5 18 0.0025 10 5 4 14
3 Working standard 0.5 18 0.0625 10 5 15 3 0.01028
0.5 18 0.0125 10 5 9 9
0.5 18 0.0025 10 5 5 13
Batch 3 0.5 18 0.0625 10 5 13 5 0.01020 8.05
0.5 18 0.0125 10 5 10 8
0.5 18 0.0025 10 5 5 13

Figure - 1 (Specific toxicity test) Figure - 2 (Potency test)

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examination and pH determination. media, proper inactivation of the organisms and increase number of
Harvesting bacteria in the vaccine. In the present study fresh growth of B. per-
Culture was harvested when pH reach to 8.0. Culture broth was dis- tussis was cultivated on standardized B2 medium. The bacteria were
tributed in polypropylene centrifuge bottles and centrifuged at 4,000 inactivated by heating at 560C. The quality control testing would give
rpm for 1 hour. Supernatant was discarded and bacterial mass was valuable information about the safety and potency of the prepared
resuspended in sterile physiological saline. Content of each bottle vaccine.
were pooled in sterile blood transfusion bottle (BT bottle). Purity tests for all three batches showed silver color shiny
Purity test growth on BG and BA but no growth on NA and NB. So it concludes
Purity of vaccine was checked on BG agar, Blood agar (BA), Nutrient that there is no contamination in the production medium because B.
agar (NA) and Nutrient broth (NB) tubes. These tubes were incu- pertussis can grow on BG and BA medium not on NA and NB (14).
bated at 350C for five days. All the tubes were examined on 5th day of At the time of harvesting pH of all harvests was find in
incubation (14). between 7.95-8.03, this pH is ideal for harvesting because higher pH
Test for opacity may lead to autolysis of bacterial cells in the production medium.
Prepared vaccine was diluted with distilled water until the opacity is Agglutination test for all three batches shows the presence of agglu-
identical with the 5th International Reference Preparation of Opacity tinins 1, 2, 3 on the cell surface that is required in the WCP vaccine
when compared by eye. The opacity of such suspension is 10 IOU. (20). Opacity of all three batches find in between 42-57 IOU/ml.
By measure the dilution factor, opacity of the sample was calculated Viability and Sterility tests for all three batches showed no
(15). growth on BG, TG, and SCD medium but B. pertussis can grow on BG
Identification of surface antigen (Agglutination Test) and contaminants in TG (for bacteria) and SCD (for fungus) medium
Three drops of vaccine were taken on a clean glass slide. Each drop So it can conclude that there was neither any viable B. pertussis nor
was mixed individually with one drop of monospecific antiserum 1, 2 any contaminant in the inactivated vaccine (17),
and 3. After mixing, slide rocked gently for two minutes. Heavy agglu- For the acceptance of specific toxicity test, at the end of
tination shows the presence of surface antigen 1, 2 and 3 (16). third day the total weight of the mice in the vaccine group should be
Inactivation of bacterial cells same or higher than the initial weight, at the end of seventh days the
Bacterial cells were inactivated by heating at 560C for 10 minutes in average weight gain of mouse in the vaccine groups should not be
shaker water bath. less than 60% of the control groups and not more than 5% of the
Viability and Sterility test inoculated mice die during the test (IP, 1996). In all three batches
1.0 ml of inactivated vaccine was inoculated in Thioglycollate broth weight of mouse in the vaccine groups was found more than 60% of
(TG) medium, Soyabean Casein Digest medium (SCD) and loopful of the control group and no death reported. All three batches pass the
vaccine streaked on BG agar slants. TG and BG tubes were incubated toxicity test (17).
at 350C and SCD medium at 220C for 14 days. All the bottles were For the acceptance of potency test vaccine should posses
examined on 3rd and 14th day of incubation (17). minimum potency level of 4 IU/dose , here potency of all three batches
Specific toxicity test (Mouse weight gain test) find well ahead than 4 IU/dose so all the three batches is potent to use
Two groups of 10 healthy mice having group weight 150 grams were (17).
taken. In test group each mouse was injected intraperitoneally with In the present study all quality control tests were found in
0.5 ml of inactivated test vaccine containing 20X109org/ml. In control acceptance range, so it can concluded that prepared WCP vaccine is
group each mouse was injected with 0.5 ml of normal saline. Each safe and potent to use.
group was weighted on 3rd and 7th days (17). ACKNOWLEDGEMENTS
Potency test (Mouse protection test) The authors wish to thank the Management and Staff of
In this test 15 gm weight mice were used. Three, 5 fold serial dilutions VIT University, Vellore, TN and Central Research Institute, Kasauli,
of the test vaccine and of standard pertussis vaccine (8 IU/ml) were HP for providing necessary facilities to carry out this study.
prepared in normal saline. Three groups each of 18 mice were injected
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Source of support: Nil, Conflict of interest: None Declared

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