Methods for making polysaccharide-protein conjugates
The present invention provides methods for making polysaccharide-protein conjugates in which polysaccharides, typically from bacteria, are conjugated to a carrier protein by reductive amination under conditions which improve conjugation reaction consistency, increase consumption of protein during conjugation reaction, generate conjugates of higher molecular weight, and/or reduce the levels of free cyanide in the conjugate reaction product. The polysaccharide-protein conjugates obtained using these methods are useful for inclusion in multivalent vaccines.
1. A method for preparing a polysaccharide protein conjugate by reductive amination, the method comprising:
a) reacting an activated polysaccharide with a carrier protein in an aqueous buffer in the presence of a cyanoborohydride salt and nickel to form a conjugate reaction mixture;
b) optionally, adding an additional reducing agent; and
c) purifying the conjugate reaction mixture to remove residual reactant including free cyanide ions.
2. The method of claim 1 , wherein the cyanoborohydride salt is sodium cyanoborohydride.
3. The method of claim 2 , wherein step a) is performed at a pH from 6.0 to 8.5.
4. The method of claim 3 , wherein step a) is performed at a pH of 6.5 to 7.5.
5. The method of claim 1 , wherein the nickel in step a) is in the form of nickel chloride or nickel sulfate.
6. The method of claim 5 , wherein the nickel is present at a concentration of 0.5 mM to 15 mM.
7. The method of claim 6 , wherein the nickel is present at a concentration of 0.5 mM to 5 mM.
8. The method of claim 1 , wherein the nickel in step a) is added before the cyanoborohydride salt.
9. The method of claim 1 , wherein an additional reducing agent in step b) is added.
10. The method of claim 1 , wherein the additional reducing agent comprises borohydride ion.
11. The method of claim 10 , wherein the additional reducing agent is sodium borohydride.
12. The method of claim 1 , wherein the purifying step in c) is by ultrafiltration.
13. The method of claim 1 , wherein a reaction temperature in step a) is at 10-30° C.
14. The method of claim 13 , wherein the reaction temperature in step a) is at 10-22° C.
15. The method according to claim 1 , wherein the polysaccharide is selected from the group consisting of Meningococcal polysaccharides, Pneumococcus polysaccharides, Hemophilus influenzae type b polysaccharide, Vi polysaccharide of Salmonella typhi , and group B Streptococcus polysaccharides.
16. The method of claim 15 , wherein the polysaccharide is a Streptococcus pneumoniae capsular polysaccharide.
17. The method of claim 16 , wherein the Streptococcus pneumoniae capsular polysaccharide is from serotype 3.
18. The method of claim 16 , wherein the Streptococcus pneumoniae capsular polysaccharide is from serotype 6B.
19. The method according to claim 1 , wherein the carrier protein is selected from the group consisting of tetanus toxoid, diphtheria toxoid, and CRM197.
20. The method of claim 19 , wherein the carrier protein is CRM197.
21. The method of claim 18 , wherein the molecular weight of the serotype 6B polysaccharide protein conjugate is from 2000 to 5000 kDa or 3000 to 4000 kDa.