Shortened purification process for the production of capsular Streptococcus pneumoniae polysaccharides

Patent metadata

Field	Value
Publication number	US-9675681-B2
Application number	US-201514633141-A
Country	US
Kind code	B2
Filing date	Feb 27, 2015
Priority date	Mar 23, 2007
Publication date	Jun 13, 2017
Grant date	Jun 13, 2017

Abstract

A shortened process for producing a solution containing substantially purified capsular polysaccharides from a cellular Streptococcus pneumoniae lysate broth is described. Ultrafiltering and diafiltering a clarified S. pneumoniae lysate followed by pH adjustment to less than 4.5, preferably about 3.5, precipitated at least 98% of the protein in the solution without seriously affecting polysaccharide yield. Furthermore, following ultrafiltration and diafiltration and acidification to a pH of less than 4.5, filtration using activated carbon precipitated at least 90% of remaining protein without seriously affecting polysaccharide yield. Exemplary, non-limiting S. pneumoniae serotypes that can be purified using the shortened process of the invention are 1, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F. In one embodiment, the Streptococcus pneumoniae cells are lysed using deoxycholate sodium (DOC), while in another embodiment the lytic agent is a non-animal derived lytic agent such as N-lauryl sarcosine sodium (NLS).

First claim

The invention claimed is:

1. A solution containing substantially purified Streptococcus pneumoniae capsular polysaccharide wherein the percent ratio of protein to polysaccharide(protein/PS) is less than 10%, and wherein said polysaccharide has a molecular weight of at least 275,000 daltons.

2. A solution according to claim 1 wherein the percent ratio of protein/PS is less than 2%.

3. A solution containing substantially purified Streptococcus pneumoniae capsular polysaccharide wherein the percent ratio of nucleic acid to polysaccharide(NA/PS) is less than 5%, and wherein said polysaccharide has a molecular weight of at least 275,000 daltons.

4. A solution according to claim 3 wherein the percent ratio NA/PS is less than 2%.

5. A solution according to claim 1 wherein the percent ratio of NA/PS is less than 5%.

6. A solution according to claim 1 wherein the percent ratio of NA/PS is less than 2%.

7. A solution according to claim 1 or 3 wherein the polysaccharide is serotype 1, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F or 23F Streptococcus pneumoniae capsular polysaccharide.

8. A solution according to claim 7 , wherein the polysaccharide is serotype 6A, and wherein the percent ratio of protein/PS is less than 2% and the percent ratio of NA/PS is less than 2%.

9. A solution according to claim 8 , wherein said polysaccharide has a molecular weight of 640,000 daltons to 670,000 daltons.

10. A solution according to claim 1 , wherein said solution is obtained by a process comprising the steps of:
(a) providing a fermentation broth comprising bacterial cells that produce a selected Streptococcus pneumoniae serotype;
(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) 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 a time sufficient 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.

11. A solution according to claim 1 , wherein said solution is obtained by a 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.

12. A solution containing substantially purified Streptococcus pneumoniae serotype 6A capsular polysaccharides, wherein said capsular polysaccharide has a molecular weight of 640,000 daltons to 670,000daltons.

Assignees

• Wyeth Llc

Inventors

• Yuan Yonghui
• Ruppen Mark
• Sun Wei-Qiang
• Chu Ling
• Simpson John
• Patch James
• Moran Justin Keith
• Fink Pamela S

Classifications

• A61P31/04

  Antibacterial agents · CPC title

• A61P37/04

  Immunostimulants · CPC title

• A61K39/092 Primary

  Streptococcus · CPC title

• C07H1/08

  from natural products · CPC title

• A61K47/646 Primary

  the entire peptide or protein drug conjugate elicits an immune response, e.g. conjugate vaccines · CPC title