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, CO2 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 CO2 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 CO2.

Description

[0001]This application claims benefit of priority to U.S. Provisional Application No. 61 / 138,570, which was filed on 18 Dec. 2008, and which is incorporated herein by reference in its entirety.FIELD OF THE INVENTION[0002]The invention relates to methods for increasing the molecular weight of isolated Streptococcus pneumoniae capsular polysaccharides having a phosphodiester linkage between saccharide repeat units.BACKGROUND[0003]In the preparation of multivalent conjugate pneumococcal vaccines directed to the prevention of invasive diseases caused by the organism Streptococcus pneumoniae (also known as pneumococcus), selected Streptococcus pneumoniae serotypes are grown to supply polysaccharides needed to produce the vaccine. The cells are grown in fermentors with lysis induced at the end of the fermentation by addition of sodium deoxycholate or an alternate lysing agent. The lysate broth is then harvested for downstream purification and the recovery of the capsular polysaccharide wh...

AI Technical Summary

Benefits of technology

[0006]Improved methods for the recovery of high molecular weight capsular polysaccharides from cellular Streptococcus pneumoniae lysates containing serotypes having a phosphodiester linkage between saccharide repeat units are provided. In one method, CO2 is supplied to a fermentation culture of a Streptococcus pneumoniae serotype containing a phosphodiester linkage between saccharide repeat units. Accordingly, in one embodiment of the invention, the method includes the steps of: 1) preparing a fermentation culture of Streptococcus pneumoniae bacterial cells that produce capsular polysaccharides comprising a phosphodiester linkage between repeat units; 2) supplying CO2 to the fermentation culture; 3) lysing the bacterial cells in the fermentation culture; and 4) isolating Streptococcus pneumoniae capsular polysaccharides from the fermentation culture, where a solution containing high molecular weight isolated Streptococcus pneumoniae capsular polysaccharides containing phosphodiester linkages between repeat units is produced.

[0007]In a particular embodiment, fermentation cultures of Streptococcus pneumoniae bacterial cells that produce polysaccharide serotypes 19A, 6A, 19F, 6B, and combinations thereof are prepared. In another particular embodiment, supplying CO2 to the fermentation culture includes adding bicarbonate ion (HCO3−) to the fermentation culture, for example, adding NaHCO3 (sodium bicarbonate) to the fermentation culture. In a further embodiment, supplying CO2 to the fermentation culture includes adding carbonate ion (CO32−) to the fermentation culture, for example, adding Na2CO3 (sodium carbonate) to the fermentation culture. In another embodiment, supplying CO2 to the fermentation culture includes a first addition of NaHCO3 and a second addition of Na2CO3. In yet another embodiment, supplying CO2 to the fermentation culture includes overlaying the fermentation culture with CO2. In another embodiment, the molecular weight of the isolated Streptococcus pneumoniae capsular polysaccharide is at least 700 kDa. In another embodiment, a solution containing high molecular weight isolated Streptococcus pneumoniae capsular polysaccharides in which the polysaccharides comprise phosphodiester linkages between repeat units is provided, where the solution is produced by the method described above.

[0008]In another embodiment of the present invention, a method is provided for producing a solution containing high molecular weight isolated Streptococcus pneumoniae serotype 19A capsular polysaccharides. The method includes the steps of: 1) preparing a fermentation culture of Streptococcus pneumoniae bacterial cells that produce serotype 19A capsular polysaccharides; 2) supplying CO2 to the fermentation culture; 3) lysing the bacterial cells in the fermentation culture; and 4) isolating Streptococcus pneumoniae serotype 19A capsular polysaccharides from the fermentation culture; whereby a solution containing high molecular weight isolated Streptococcus pneumoniae serotype 19A capsular pol...

Problems solved by technology

However, such traditional processing methods result in lower molecular weight polysaccharide (<450 kDa) for serotypes having a phosphodiester linkage between saccharide repeat units (e.g., 6A, 6B, 19A, and 19F).

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