Animal component free meningococcal polysaccharide fermentation and seedbank development

Abstract

Animal-free meninge fermentation media and process is developed based upon use of a chemically defined medium. To improve polysaccharide production, fed-batch fermentation is examined using different feed solutions and feeding strategies. A feed solution containing glucose, amino acids, and trace metal elements produces Group A polysaccharide at approximately 3 times the level observed with batch fermentation. This process is used successfully to produce polysaccharides of N. meningitidis serotypes A, C, Y and W-135 and is run reproducibly at the 20L scale and can be scaled to 400L or more.

1 . A fermentation composition, comprising compounds in a ratio, by weight, of 2.5±10% sodium phosphate, dibasic, 5-30±10% soy peptone, 5±10% monosodium glutamate, 0.103±10% potassium chloride, 0.732±10% magnesium sulfate, 0.016±10% L-Cysteine, and 11.250±10% glucose, and wherein the composition does not comprise NH 4 Cl. 
     
     
         2 . The composition according to  claim 1 , wherein the composition is aqueous. 
     
     
         3 . The composition according to  claim 1 , wherein the soy peptone is HSP-A®. 
     
     
         4 . A fermentation composition, comprising compounds in a ratio, by weight, of 10±10% glucose, 5-30±10% soy peptone, 5.8±10% Sodium Chloride, 1±10% potassium sulfate, 4±10% potassium phosphate, dibasic, 5-6±10% L-Glutamic Acid, 0.3±10% L-Arginine, 0.5±10% L-Serine, 0.23±10% L-Cysteine, 0.19±10% Magnesium Chloride, 0.021±10% Calcium chloride, and 0.002±10% Ferrous Sulfate, and wherein the composition does not comprise NH 4 Cl. 
     
     
         5 . The composition according to  claim 4 , wherein the composition is aqueous. 
     
     
         6 . The composition according to  claim 4 , wherein the soy peptone is HSP-A®. 
     
     
         7 . A fermentation composition, comprising compounds in a ratio, by weight, of 10±10% glucose, 5-30±10% soy peptone, 5.8±10% Sodium Chloride, 1±10% potassium sulfate, 4±10% potassium phosphate, dibasic, 0.19±10% Magnesium Chloride, 0.021±10% Calcium chloride, and 0.002±10% Ferrous Sulfate, and wherein the composition does not comprise NH 4 Cl. 
     
     
         8 . The composition according to  claim 7 , wherein the composition is aqueous. 
     
     
         9 . The composition according to  claim 7 , wherein the soy peptone is HSP-A®. 
     
     
         10 . A method of fermenting  Neisseria , comprising fermenting  Neisseria  in a fermentation composition wherein the fermentation composition does not comprise NH 4 Cl. 
     
     
         11 . The method according to  claim 10 , wherein the fermentation composition is according to  claim 1 . 
     
     
         12 . The method according to  claim 10 , wherein the fermentation composition is according to  claim 4 . 
     
     
         13 . The method according to  claim 10 , wherein the fermentation composition is according to  claim 7 . 
     
     
         14 . A method of fermenting  Neisseria , wherein the  Neisseria  are fermented in multiple batches wherein at least one fermentation is in a fermentation composition of  claim 1 . 
     
     
         15 . A method of fermenting  Neisseria , wherein the  Neisseria  are fermented in multiple batches wherein at least one fermentation is in a fermentation composition of  claim 4 . 
     
     
         16 . A method of fermenting  Neisseria , wherein the  Neisseria  are fermented in multiple batches wherein at least one fermentation is in a fermentation composition of  claim 7 . 
     
     
         17 . A method of fermenting  Neisseria , comprising:
 inoculating a vial of  Neisseria  to a first flask containing a fermentation composition;   cultivating the  Neisseria  in the first flask;   transferring the  Neisseria  from the first flask to a plurality of second flasks containing a fermentation composition;   fermenting the contents of the second flasks;   transferring the contents of the second flasks to a fed-batch fermentor containing a fermentation composition;   fermenting the contents of the fed-batch fermentor with a fermentation composition, wherein at least one fermentation composition is of  claim 1 .   
     
     
         18 . A method of fermenting  Neisseria , comprising:
 inoculating a vial of  Neisseria  to a first flask containing a fermentation composition;   cultivating the  Neisseria  in the first flask;   transferring the  Neisseria  from the first flask to a plurality of second flasks containing a fermentation composition;   fermenting the contents of the second flasks;   transferring the contents of the second flasks to a fed-batch fermentor containing a fermentation composition;   fermenting the contents of the fed-batch fermentor with a fermentation composition, wherein at least one fermentation composition is of  claim 4 .   
     
     
         19 . A method of fermenting  Neisseria , comprising:
 inoculating a vial of  Neisseria  to a first flask containing a fermentation composition;   cultivating the  Neisseria  in the first flask;   transferring the  Neisseria  from the first flask to a plurality of second flasks containing a fermentation composition;   fermenting the contents of the second flasks;   transferring the contents of the second flasks to a fed-batch fermentor containing a fermentation composition;   fermenting the contents of the fed-batch fermentor with a fermentation composition, wherein at least one fermentation composition is of  claim 7 .   
     
     
         20 . A method of producing  Neisseria  polysaccharide, the method comprising:
 fermenting  Neisseria  in a fermentation composition wherein the fermentation composition does not comprise NH 4 Cl, and   harvesting  Neisseria  polysaccharide.