US 12,473,379 B2
Method for obtaining purified bacterial polysaccharides
Rajeev Mhalasakant Dhere, Maharashtra (IN); Swapan Kumar Jana, Maharashtra (IN); and Walmik Karbhari Gaikwad, Maharashtra (IN)
Assigned to Serum Institute of India Private Limited, Pune Maharashtra (IN)
Filed by SERUM INSTITUTE OF INDIA PRIVATE LIMITED, Maharashtra (IN)
Filed on Sep. 2, 2020, as Appl. No. 17/010,084.
Claims priority of application No. 201921036006 (IN), filed on Sep. 6, 2019.
Prior Publication US 2021/0070890 A1, Mar. 11, 2021
Int. Cl. C08B 37/00 (2006.01); B01D 15/32 (2006.01); B01D 21/26 (2006.01); C12P 1/04 (2006.01); A61K 39/00 (2006.01)
CPC C08B 37/0003 (2013.01) [B01D 15/327 (2013.01); B01D 21/262 (2013.01); C12P 1/04 (2013.01); A61K 39/00 (2013.01)] 23 Claims
 
1. A method for obtaining purified Streptococcus pneumoniae polysaccharides, comprising:
a fermentation harvest comprising Streptococcus pneumoniae polysaccharides,
proteins, nucleic acids and cell debris, wherein pH of the fermentation harvest ranges from 5.8 to 6.8;
subjecting the fermentation harvest to centrifugation to separate cell free supernatant;
filtering the cell free supernatant to obtain a concentrated cell free supernatant;
treating the concentrated cell free supernatant with trifluoroacetic acid having a concentration in the range of 2 M to 5 M, at 20° C. to 60° C. and at a pH in the range of 0.1 to 1, to obtain an acid treated mixture, wherein the Streptococcus pneumoniae polysaccharides are separated from the proteins, nucleic acids and cell debris;
subjecting the acid treated mixture to centrifugation to remove precipitated impurities and collecting the supernatant, wherein the precipitated impurities comprise proteins, nucleic acids and cell debris;
incubating the supernatant after removal of the precipitated impurities at a temperature in the range of 20° C. to 50° C. for a time period in the range of 4 hours to 24 hours;
adjusting the pH of the collected supernatant after removal of the precipitated impurities to be in the range of 5.5 to 6.5 with a pH adjusting agent selected from disodium hydrogen phosphate, dipotassium hydrogen phosphate or combinations thereof;
diafiltering to increase a concentration of the Streptococcus pneumoniae polysaccharides in a solution to obtain concentrated Streptococcus pneumoniae polysaccharides; and
filtering the concentrated Streptococcus pneumoniae polysaccharides to obtain purified Streptococcus pneumoniae polysaccharides, wherein the purified Streptococcus pneumoniae polysaccharides have protein content less than 3%, nucleic acid content less than 2%, cell wall polysaccharides (CWPs) content not more than 2 mol %, polydispersity less than 2, molecular size in the range of 10 kDa to 200 kDa (SEC-HPLC), and >10% methyl pentose content.