Method for controlling streptococcus pneum0niae 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) overlaying the fermentation culture with CO 2 ;   c) lysing the bacterial cells in said fermentation culture; and   d) isolating  Streptococcus pneumoniae  capsular polysaccharides from said fermentation culture;   
       whereby a solution is produced that contains high molecular weight isolated  Streptococcus pneumoniae  capsular polysaccharides wherein said polysaccharides comprise phosphodiester linkages between repeat units. 
     
     
         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 the pH of said fermentation culture is between 6 and 7. 
     
     
         7 . The method of  claim 1 , wherein NaOH is added to the fermentation culture as a base titrant. 
     
     
         8 . The method of  claim 1 , wherein lysing the  Streptococcus pneumoniae  in said resultant fermentation culture of step c) further comprises adding sodium deoxycholate to said fermentation culture. 
     
     
         9 . The method of  claim 1 , wherein the molecular weight of said isolated  Streptococcus pneumoniae  capsular polysaccharide is at least 480 kDa. 
     
     
         10 . The method of  claim 9 , wherein said molecular weight is at least 550 kDa. 
     
     
         11 . The method of  claim 10 , wherein said molecular weight is at least 600 kDa.