Method for controlling streptococcus pneumoniae polysaccharide molecular weight using carbon dioxide

Abstract

The present invention provides improved methods for producing a solution containing high molecular weight isolated Streptococcus pneumoniae capsular polysaccharides having phosphodiester linkages between saccharide repeat units. In certain methods, CO 2 is supplied to a fermentation culture of Streptococcus pneumoniae bacterial cells that produce capsular polysaccharide serotypes containing phosphodiester linkages between saccharide repeat units. Exemplary Streptococcus pneumoniae serotypes containing a phosphodiester linkage between saccharide repeat units include serotypes 6A, 6B, 19A, and 19F. Supplying CO 2 to the fermentation culture includes adding bicarbonate ions to the fermentation culture, adding carbonate ions to the fermentation culture, adding mixtures of bicarbonate and carbonate ions to the fermentation culture, and overlaying the fermentation culture with CO 2 .

1 . A method for producing a solution containing high molecular weight isolated  Streptococcus pneumoniae  capsular polysaccharides wherein said polysaccharides comprise phosphodiester linkages between repeat units, the method comprising:
 a) preparing a fermentation culture of  Streptococcus pneumoniae  bacterial cells that produce capsular polysaccharides comprising a phosphodiester linkage between repeat units;   b) supplying CO 2  to said fermentation culture;   c) lysing the bacterial cells in said fermentation culture; and   d) isolating  Streptococcus pneumoniae  capsular polysaccharides from said fermentation culture;   
     whereby a solution containing high molecular weight isolated  Streptococcus pneumoniae  capsular polysaccharides wherein said polysaccharides comprise phosphodiester linkages between repeat units is produced. 
   
   
       2 . The method of  claim 1 , wherein said  Streptococcus pneumoniae  capsular polysaccharides are serotype 19A. 
   
   
       3 . The method of  claim 1 , wherein said  Streptococcus pneumoniae  capsular polysaccharides are serotype 6A. 
   
   
       4 . The method of  claim 1 , wherein said  Streptococcus pneumoniae  capsular polysaccharides are serotype 19F. 
   
   
       5 . The method of  claim 1 , wherein said  Streptococcus pneumoniae  capsular polysaccharides are serotype 6B. 
   
   
       6 . The method of  claim 1 , wherein supplying CO 2  to said fermentation culture comprises adding bicarbonate ion (HCO 3   − ) to the fermentation culture. 
   
   
       7 . The method of  claim 6 , wherein adding HCO 3   −  to the fermentation culture comprises adding NaHCO 3 . 
   
   
       8 . The method of  claim 1 , wherein supplying CO 2  to said fermentation culture comprises adding carbonate ion (CO 3   2− ) to the fermentation culture. 
   
   
       9 . The method of  claim 8 , wherein adding CO 3   2−  to the fermentation culture comprises adding Na 2 CO 3 . 
   
   
       10 . The method of  claim 9 , further wherein the pH of said fermentation culture is between 6.0 and 6.6. 
   
   
       11 . The method of  claim 1 , wherein supplying CO 2  to said fermentation culture comprises a first addition of NaHCO 3  and a second addition of Na 2 CO 3 . 
   
   
       12 . The method of  claim 1 , wherein supplying CO 2  to said fermentation culture comprises overlaying the fermentation culture with CO 2 . 
   
   
       13 . The method of  claim 1 , wherein lysing the  Streptococcus pneumoniae  in said fermentation culture comprises adding sodium deoxycholate to said fermentation culture. 
   
   
       14 . The method of  claim 1 , wherein the molecular weight of said isolated  Streptococcus pneumoniae  capsular polysaccharide is at least 480 kDa. 
   
   
       15 . A solution containing high molecular weight isolated  Streptococcus pneumoniae  capsular polysaccharides wherein said polysaccharides comprise phosphodiester linkages between repeat units, wherein said solution is produced by the method of  claim 1 .