Process for production of purified recombinant cholera toxin B (rCTB) and formulation thereon
The present invention relates to a novel process of production of purified recombinant cholera toxin B (rCTB) which provides protection against diarrhea caused by various bacteria such as Vibrio cholerae and Enterotoxigenic Escherichia coli (ETEC). More particularly, the present invention relates to a process of production of rCTB with significantly higher yield and higher purity. The present invention also relates to a vaccine formulation having synergistic protection against Vibrio cholerae and cross protection against ETEC.
1. A novel process for producing purified recombinant cholera toxin B (rCTB) from Vibrio cholerae and formulating the purified rCtB to obtain a novel vaccine formulation, said process comprising the steps of
fermentating an rCTB producing Vibrio cholerae strain to obtain a fermented culture;
heat inactivating the fermented culture to obtain a harvest of partially purified rCTB;
purifying said harvest to obtain purified rCTB; and
formulating the purified rCTB to produce the novel vaccine formulation,
wherein said fermented culture is subjected to a step of heat inactivating at a temperature range of 65° C.±5° C. for 30±15 mins, followed by centrifugation to pellet out inactivated cells and denatured contaminating proteins; wherein the post centrifugation supernatant contains more than 70% pure rCTB, and wherein this partially purified rCTB is further purified by precipitation and tangential flow filtration to yield more than 90-95% pure rCTB.
2. The process as claimed in claim 1 wherein said yields are in the range of 1.5-2.5 gm/litre and purity of the purified, rCTB is higher than 95% (>95%).
3. The process as claimed in claim 1 wherein said fermentation step comprises of following steps:
(a) inoculating an rCTB producing Vibrio cholerae strain into rCTB production media in a fermenter at predefined conditions;
(b) running the fermenter at a predetermined pH and a predetermined temperature for a predetermined duration of time to obtain the fermented culture.
4. The process as claimed in claim 3 wherein said predefined conditions include addition of feed containing 1M glucose to the fermenter media.
5. The process as claimed in claim 3 wherein said predetermined pH is in the range of 7.2-7.5, said predetermined temperature is in the range of 35−37° C., and said predetermined duration of time ranges from 18-22 hrs.
6. A novel process for producing purified recombinant cholera toxin B (rCTB) from Vibrio cholerae and formulating the purified rCtB to obtain a novel vaccine formulation, said process comprising the steps of
fermentating an rCTB producing Vibrio cholerae strain to obtain a fermented culture;
heat inactivating the fermented culture to obtain a harvest of partially purified rCTB;
purifying said harvest to obtain purified rCTB; and
formulating the purified rCTB to produce the novel vaccine formulation, wherein the purifying step is performed without chromatography, wherein said fermented culture is subjected to a step of heat inactivating at a temperature range of 65° C.±5° C. for 30±15 mins, followed by centrifugation to pellet out inactivated cells and denatured contaminating proteins; wherein the post centrifugation supernatant contains more than 70% pure rCTB, and wherein this partially purified rCTB is further purified by precipitation and tangential flow filtration to yield more than 90-95% pure rCTB.
7. The process as claimed in claim 6 wherein said yields are in the range of 1.5-2.5 gm/litre and purity of the purified, rCTB is higher than 95% (>95%).
8. The process as claimed in claim 6 wherein said fermentation step comprises of following steps:
(c) inoculating an rCTB producing Vibrio cholerae strain into rCTB production media in a fermenter at predefined conditions;
(d) running the fermenter at a predetermined pH and a predetermined temperature for a predetermined duration of time to obtain the fermented culture.
9. The process as claimed in claim 8 wherein said predefined conditions include addition of feed containing 1M glucose to the fermenter media.
10. The process as claimed in claim 8 wherein said predetermined pH is in the range of 7.2-7.5, said predetermined temperature is in the range of 35−37° C., and said predetermined duration of time ranges from 18-22 hrs.