Shortened purification process for the production of capsular Streptococcus pneumoniae polysaccharides

A process for producing a solution containing substantially purified capsular polysaccharides from a Streptococcus pneumoniae cell lysate comprising serotype 1, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19F, or 23F, the process comprising the steps of:

• (a) providing a fermentation broth comprising bacterial cells that produce Streptococcus pneumoniae serotype 1, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19F, or 23F;
• (b) lysing the bacterial cells in step (a) with a lytic agent, thereby producing a cell lysate comprising cell debris, soluble proteins, nucleic acids, and polysaccharides;
• (c) clarifying the cell lysate of step (b) using centrifugation or filtration to remove cell debris, thereby producing a clarified cell lysate;
• (d) ultrafiltering and diafiltering the clarified cell lysate of step (c) at room temperature at neutral pH in salt free media to remove low molecular weight impurities and increase polysaccharide concentration, thereby producing a salt free retentate;
• (e) lowering the pH of the salt free retentate of step (d) to less than 4.5 to precipitate protein and nucleic acids, thereby forming an acidified retentate solution;
• (f) holding the acidified retentate solution formed in step (e) for at least 2 hours at room temperature to allow settling of the precipitate, followed by filtration or centrifugation of the acidified retentate solution, thereby producing a clarified polysaccharide solution;
• (g) filtering the clarified polysaccharide solution of step (f) through an activated carbon filter;
• (h) ultrafiltering and diafiltering the filtered solution produced by step (g), thereby producing a concentrated purified polysaccharide solution;
• and
• (i) filtering the concentrated purified polysaccharide solution produced by step (h) using a sterile filter;
• whereby a solution containing substantially purified capsular polysaccharides comprising serotype 1, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19F, or 23F is produced.

A process for producing a solution containing substantially purified capsular polysaccharides from a Streptococcus pneumoniae cell lysate comprising serotype 19A, the process comprising the steps of:

• (a) providing a fermentation broth comprising bacterial cells that produce Streptococcus pneumoniae serotype 19A;
• (b) lysing the bacterial cells in step (a) with a lytic agent, thereby producing a cell lysate comprising cell debris, soluble proteins, nucleic acids, and polysaccharides;
• (c) clarifying the cell lysate of step (b) using centrifugation or filtration to remove cell debris, thereby producing a clarified cell lysate;
• (d) ultrafiltering and diafiltering the clarified cell lysate of step (c) at about 4° C. at a pH of about 6 in sodium phosphate buffer to remove low molecular weight impurities and increase polysaccharide concentration, thereby producing a retentate;
• (e) lowering the pH of the retentate of step (d) to less than 4.5 to precipitate protein and nucleic acids, thereby forming an acidified retentate solution;
• (f) holding the acidified retentate solution formed in step (e) for at least 2 hours at about 4° C. to allow settling of the precipitate, followed by filtration or centrifugation of the acidified retentate solution, thereby producing a clarified polysaccharide solution;
• (g) adjusting the pH of the clarified polysaccharide solution of step (f) to about 6, thereby producing a pH-adjusted clarified polysaccharide solution;
• (h) filtering the pH-adjusted clarified polysaccharide solution of step (g) through an activated carbon filter;
• (i) ultrafiltering and diafiltering the filtered solution produced by step (h), thereby producing a concentrated purified polysaccharide solution;
• and
• (j) filtering the concentrated purified polysaccharide solution produced by step (i) using a sterile filter;
• whereby a solution containing substantially purified capsular polysaccharides comprising serotype 19A is produced.