Process for preparation of polysaccharides
A novel alcohol and CTAB free process for purification of capsular polysaccharides, wherein the C-polysaccharide, protein, nucleic acid content of the purified polysaccharide is substantially reduced The said process is cost efficient and less laborious.
1. A cost effective, alcohol- & cetyltrimethylammonium bromide (CTAB)-free chromatography based method for removal of C-polysaccharide from an antigenic polysaccharide which comprises:
(a) lysing bacterial cells in a fermentation medium with deoxycholate thereby producing an aqueous cell lysate containing a polysaccharide solution and solid cell debris;
(b) clarifying the aqueous cell lysate by separating the solid cell debris from the polysaccharide solution;
(c) concentrating the polysaccharide solution by ultrafiltration using a 100 kDa molecular weight cutoff membrane to remove low molecular weight contaminants and to form a solution of concentrated polysaccharide;
(d) treatment of the solution of concentrated polysaccharide with a nuclease;
(e) precipitation of impurities from the solution of concentrated polysaccharide using ammonium sulphate or sodium deoxycholate treatment;
(f) diafiltration of the solution of concentrated polysaccharide using a 100 kDa molecular weight cutoff membrane;
(g) subjecting the solution of concentrated polysaccharide to a multi-step chromatography to produce a purified polysaccharide solution, wherein the multi-step chromatography comprises hydrophobic interaction chromatography (HIC) followed by ion exchange chromatography (IEX);
(h) concentration and diafiltration of the purified polysaccharide solution; and
(i) sterile filtration of the purified polysaccharide solution;
wherein the purified polysaccharide solution shows a 1- to 5-fold reduction of type-specific C-polysaccharide contamination relative to a purified polysaccharide solution obtained by subjecting the solution of concentrated polysaccharide to a single step HIC or ion exchange chromatography; and
wherein the antigenic polysaccharide is a polysaccharide from S. pneumoniae serotype 18C.
2. The method according to claim 1 , wherein the purified polysaccharide solution shows protein contamination less than 1% and nucleic acid contamination less than 1%.
3. The method according to claim 1 , wherein the method requires at least 80% less time and at least 90% less cost than CTAB/Alcohol based methods.
4. The method according to claim 1 , wherein the hydrophobic interaction chromatography support is selected from the group consisting of butyl-agarose, phenyl-agarose, octyl-agarose, phenyl-sepharose and organic polymer resin functionalized with a group selected from butyl, phenyl or ether.
5. The method according to claim 1 , wherein the hydrophobic interaction chromatography column is run in the pH range of 5 to 8.
6. The method according to claim 1 , wherein the hydrophobic interaction chromatography uses a buffer of sodium or potassium phosphate containing 2 M to 5 M sodium chloride salt.
7. The method according to claim 1 , wherein the ion exchange chromatography is an anion exchange chromatography.
8. The method according to claim 7 , wherein the anion exchange chromatography is performed on an anion exchange resin selected from the group consisting of DEAE cellulose, MonoQ, Capto Q, Eshmino Q, Gigacap Q 650M, Nuvia-Q, Cellufine Q-h, MiniQ, Source 15Q and 30Q, Q, DEAE Sepharose Fast Flow, Q Sepharose high Performance, QAE SEPHADEX™ and FAST Q SEPHAROSE™ (GE Healthcare), UNOsphere Q, Macro-Prep DEAE and Macro-Prep High Q from Biorad, Ceramic HyperD Q, ceramic HyperD DEAE, Toyopearl SuperQ-650S, 650M and 650C, QAE-550C and 650S, DEAE-650M.
9. The method according to claim 8 , wherein the anion exchange chromatography is performed on an anion exchange resin Gigacap Q 650M.
10. The method according to claim 7 , wherein the anion exchange column is eluted at 10 to 60% of 1 M NaCl in phosphate buffer.
11. The method according to claim 10 , wherein the anion exchange column is eluted at any one or more of 10%, 15%, 20%, 25%, 30%, 45% and 60% of 1 M NaCl in phosphate buffer.
12. The method according to claim 11 , wherein the anion exchange column is eluted at a linear or step gradient of 1 M NaCl in phosphate buffer.
13. The method according to claim 10 , wherein the anion exchange column is carried out using phosphate buffer ranging from 10 mM to 300 mM.
14. The method according to claim 7 , wherein the anion exchange chromatography is run in the pH range of 5 to 8.