Method for controlling
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 serotype 19A capsular polysaccharides wherein said polysaccharides comprise phosphodiester linkages between repeat units, the method comprising:
(a) preparing a fermentation culture of Streptococcus pneumoniae serotype 19A bacterial cells that produce capsular polysaccharides comprising a phosphodiester linkage between repeat units;
(b) supplying CO 2 to said fermentation culture of step (a) by first adding 10 to 50 mM NaHCO 3 and subsequently adding 0.1 to 2.0 mM Na 2 CO 3 and fermenting the CO 2 -supplied fermentation culture at a pH of 6.0 to 7.0;
(c) lysing the bacterial cells in the resultant fermentation culture of step (b); and
(d) isolating Streptococcus pneumoniae serotype 19A capsular polysaccharides from the resultant lysed cell fermentation culture of step (c);
wherein the molecular weight of each of said isolated Streptococcus pneumoniae serotype 19A capsular polysaccharides is at least 480 kDa, and
whereby a solution containing high molecular weight isolated Streptococcus pneumoniae serotype 19A capsular polysaccharides comprising phosphodiester linkages between repeat units is produced.
2. The method of claim 1 , wherein the pH of said fermentation culture is between 6.0 and 6.6.
3. The method of claim 1 , wherein lysing the Streptococcus pneumoniae in said fermentation culture comprises adding sodium deoxycholate to said resultant fermentation culture of step (c).