Methods for production of capsular polysaccharide protein conjugates from Streptococcus pneumoniae serotype 19F

11883,502

17/544239

December 07, 2021

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.

Merck Sharp & Dohme LLC (Rahway, NJ, US)

Merck Sharp & Dohme LLC (Rahway, NJ, US)

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.

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