Methods for production of capsular polysaccharide protein conjugates from
The present invention provides a method of producing a polysaccharide-protein conjugate with capsular polysaccharide from Streptococcus pneumoniae serotype 19F conjugated to a carrier protein. The method includes a prolonged incubation step prior to filtration to remove free polysaccharide.
1. A method for the production of a stable Streptococcus pneumoniae serotype 19F polysaccharide-CRM197 carrier protein conjugate, wherein the Streptococcus pneumoniae serotype 19F polysaccharide-CRM197 carrier protein conjugate comprises Streptococcus pneumoniae serotype 19F capsular polysaccharide covalently linked to a CRM197 carrier protein, the method comprising the steps of:
a) performing a conjugation reaction wherein Streptococcus pneumoniae serotype 19F polysaccharide is covalently linked to a CRM197 carrier protein, wherein the conjugation reaction generates a reaction mixture comprising Streptococcus pneumoniae serotype 19F polysaccharide-CRM197 carrier protein conjugates and free Streptococcus pneumoniae serotype 19F polysaccharides;
b) incubating said reaction mixture for 5-7 days at a temperature of 20-25° C. in a buffer at a pH of 6.5-7.5; and
c) performing size separation under conditions that allow removal of the free Streptococcus pneumoniae serotype 19F polysaccharides.
2. The method of claim 1 , wherein said size separation uses a nominal molecular weight cut off membrane of from 100 to 500 kDa whereby the Streptococcus pneumoniae serotype 19F polysaccharide-CRM197 carrier protein conjugate is retained.
3. The method of claim 1 , further comprising d) collecting the Streptococcus pneumoniae serotype 19F polysaccharide-CRM197 carrier protein conjugate.
4. The method of claim 2 , wherein the retained Streptococcus pneumoniae serotype 19F polysaccharide-CRM197 carrier protein conjugate has an average molecular weight of 600 kDa or more.
5. The method of claim 4 wherein the Streptococcus pneumoniae serotype 19F polysaccharide-CRM197 carrier protein conjugate has an average molecular weight of 1000 kDa or more.
6. The method of claim 1 , wherein said buffer is selected from a phosphate buffer, histidine, and TRIS.
7. The method of claim 1 , wherein said size separation is by size-exclusion chromatography, bind/elute chromatography, or wide-pore ultrafiltration.
8. The method of claim 7 , wherein said size separation is by wide-pore ultrafiltration with a membrane having a MWCO of 100 kDa to 300 kDa.