Method for removal of impurities from bacterial capsular polysaccharide based preparations
The present invention relates to an improved process for purification of bacterial capsular polysaccharides, more specifically capsular polysaccharides of gram negative bacteria. The process comprises of concentration and dia filtration of harvest, treatment with anionic detergent and strong alkali followed by centrifugation, diafiltration and cationic detergent based precipitation of bacterial polysaccharides. The process results in significant reduction of endotoxin, protein and nucleic acid impurities thereby providing higher recovery of capsular polysaccharide with the desired O-acetyl levels. Said process is scalable, non-enzymatic, and employs fewer purification steps.
1. A method for isolation of polysaccharide from Neisseria meningitidis selected from serotype A and serotype X, said method comprising the following steps:
a) adding a detergent comprising sodium dodecyl sulfate to a crude polysaccharide solution comprising Neisseria meningitidis selected from serotype A and serotype X, wherein the final concentration of sodium dodecyl sulfate in the solution is 0.1 to less than 1%;
b) adding EDTA and sodium acetate to the solution;
c) subjecting the solution to alcohol precipitation, followed by centrifugation and retention of supernatant;
d) removing the detergent present in the supernatant;
e) concentrating and diafiltrating the polysaccharide;
wherein the obtained purified polysaccharide from Neisseria meningitidis selected from serotype A and serotype X has a recovery of 60-80%,
wherein the removal of detergent is carried out by precipitation of detergent using a potassium salt selected from the group consisting of potassium chloride, potassium acetate, potassium sulfate, potassium carbonate, potassium bicarbonate, potassium phosphate, potassium hydrogen phosphate, potassium dihydrogen phosphate, potassium nitrate, and combinations thereof.
2. The method of claim 1 , wherein
a) purified polysaccharide from Neisseria meningitidis serotype A has O-acetylation levels >2 mMol/gm, protein impurity <3%, nucleic acid impurity <1%, and endotoxin impurity <50 EU/μg; and
b) purified polysaccharide from Neisseria meningitidis serotype X has protein impurity <1%, nucleic acid impurity <1%, and endotoxin impurity <50 EU/μg.
3. The method of claim 1 , wherein the method further comprises the following steps:
a) mixing the polysaccharide solution with a cationic detergent;
b) subjecting the solution to centrifugation and collection of a pellet;
c) dissolving the pellet in alcohol, followed by salt-based precipitation,
wherein the cationic detergent is selected from the group consisting of cetyltrimethylammonium salt, tetrabutylammonium salt, myristyltrimethylammoniurn salt, hexadimethrine bromide, and combinations thereof.
4. The method of claim 3 , wherein the cationic detergent is cetyltrimethylammonium bromide and the concentration of the cationic detergent in the solution is between 0.1% and 4%.
5. The method of claim 4 , wherein the final concentration of cetyltrimethylammonium bromide is in the range of 0.5 to less than 1%.