Downstream process for purifying polysaccharides
The present invention relates to a novel process for purifying bacterial polysaccharide. It is an efficient and scalable process for removing impurities from Neisseria meningitidis serogroup C (Men-C) polysaccharide which is capable of being used as such in a derivatized form or linked to other molecules, for the preparation of vaccines, more particularly conjugate vaccines for N. meningitidis infection.
1. A process for purifying Men C-Polysaccharide, wherein said process comprises the steps of:
(a) centrifugation of the fermented harvest to clarify the fermented broth;
(b) concentrating the fermented supernatant by ultrafiltration;
(c) deacetylation and incubating said concentrated supernatant of step (b) at a temperature ranging from 70° C. to 80° C.;
(d) collecting the supernatant of step (c) and subjecting to diafiltration and concentration;
(e) purification of diafiltered supernatant of step (d) by chromatography;
(f) diafiltration and concentration to obtain purified polysaccharides; and
(g) sterile filtration of said purified polysaccharides,
wherein the said purification process is completed within 6±1 hours and wherein the purification process is completed without the use of a detergent.
2. The process as claimed in claim 1 wherein said step of centrifugation of the fermented harvest is carried at 4500×g to 5000×g.
3. The process as claimed in claim 1 wherein said step of deacetylation is carried with NaOH solution at a concentration which ranges from 1 to 1.5 M.
4. The process as claimed in claim 1 wherein the incubation in the deacetylation step is performed with a total incubation time ranging from 1 hour 30 mins to 2 hours 30 min.
5. The process as claimed in claim 1 wherein the diafiltration in the collecting step after the deacetylation step is carried out 20 to 25 times with Milli-Q water followed by 8 to 10 times with 25 mM Tris HCl buffer.
6. The process as claimed in claim 1 wherein said chromatography used in the purification step is hydrophobic interaction chromatography (HIC) Phenyl sepharose 6 fast flow.
7. The process as claimed in claim 1 wherein the diafiltration and concentration to obtain purified polysaccharides step is carried with 100 KDa PES membrane (0.1 m 2 ), with 6 to 8 volumes of Milli-Q water.