METHODS FOR PRODUCTION OF CAPSULAR POLYSACCHARIDE PROTEIN CONJUGATES FROM STREPTOCOCCUS PNEUMONIAE SEROTYPE 19F
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 polysaccharide-protein conjugate comprising Streptococcus pneumoniae serotype 19F capsular polysaccharide covalently linked to a carrier protein from a mixture comprising polysaccharide-protein conjugate and free polysaccharide, the method comprising the steps of:
a) incubating said mixture for a minimum of 6 hours at a temperature ranging from 2-30° C. in a buffer having a pH in the range of 5.0 to 9.0; and
b) performing size separation under conditions that allow removal of free polysaccharide.
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 polysaccharide-protein conjugate is retained in the retentate.
3 . The method of claim 1 , further comprising
c) collecting the polysaccharide-protein conjugate.
4 . The method of claim 1 , wherein the carrier protein is CRM 197 .
5 . The method of claim 2 , wherein the retained polysaccharide-protein conjugate has an average molecular weight of 600 kDa or more.
6 . The method of claim 5 wherein the polysaccharide-protein conjugate has an average molecular weight of 1000 kDa or more.
7 . The method of claim 1 , wherein said buffer is selected from a phosphate buffer, histidine, and TRIS.
8 . The method of claim 1 , wherein said buffer has a pH in the range of pH 5.8 to 7.0.
9 . The method of claim 8 , wherein said buffer has a pH of 7.0.
10 . The method of claim 1 , wherein the incubation temperature is in the range of 4-25° C.
11 . The method of claim 1 , wherein said size separation is by size-exclusion chromatography, bind/elute chromatography, or wide-pore ultrafiltration.
12 . The method of claim 11 , wherein said size separation is by wide-pore ultrafiltration with a membrane having a MWCO of 100 kDa to 300 kDa.
13 . The method of claim 1 , wherein the incubation is for at least 12 hours.
14 . The method of claim 13 , wherein the incubation is for 108 to 132 hours.
15 . The method of claims 1 , further comprising formulating the polysaccharide-protein conjugate with one or more additional polysaccharide-protein conjugates from a different serotype.
16 . The method of claim 15 , further comprising formulating with an adjuvant.
17 . A serotype 19F polysaccharide protein conjugate produced by the method of claims 1 .
18 . A method for measuring dose and/or potency in a pneumococcal conjugate vaccine manufacturing lot comprising performing an immunoassay for serotype 19F polysaccharide protein conjugate in said manufacturing lot using the 19F conjugate of claim 17 as a reference standard.