Compositions and methods for preparing
View Patent ↗The present invention relates to immunogenic conjugates comprising S. aureus serotype 5 and 8 capsular polysaccharides conjugated to carrier proteins and methods for their preparation and use. Methods for making the immunogenic conjugates of the invention involve covalent conjugation of the capsular polysaccharides with the carrier proteins using conjugation chemistry involving either 1,1-carbonyl-di-1,2,4-triazole (CDT) or 3-(2-pyridyldithio)-propionyl hydrazide (PDPH).
1. A method of producing an immunogenic polysaccharide protein conjugate comprising an isolated Staphylococcus aureus serotype 8 capsular polysaccharide conjugated to a carrier protein, the method comprising the steps of:
a) reacting an isolated S. aureus serotype 8 capsular polysaccharide with carbonyl ditriazole (CDT) in an organic solvent to produce an activated polysaccharide; and
b) reacting the activated polysaccharide with a carrier protein in an organic solvent to produce a polysaccharide:carrier protein conjugate;
wherein the step of reacting the polysaccharide with CDT comprises determining the water present in the S. aureus serotype 8 capsular polysaccharide and adjusting the CDT concentration to about 1:1 molar ratio of CDT:water in the organic solvent and wherein an immunogenic conjugate comprising a capsular polysaccharide carrier protein conjugate is produced.
2. The method of claim 1 , wherein the method further comprises the steps of lyophilizing the isolated polysaccharide and re-suspending the lyophilized polysaccharide in an organic solvent.
3. The method of claim 1 , wherein the activated isolated polysaccharide is isolated from the activation reaction prior to reacting with the carrier protein.
4. The method of claim 3 , wherein the step of isolating the activated isolated polysaccharide comprises diafiltration.
5. The method of claim 3 , wherein step b) comprises:
i) lyophilizing the isolated activated isolated polysaccharide to produce a lyophilized activated isolated polysaccharide;
ii) lyophilizing the carrier protein to produce a lyophilized carrier protein; and
iii) re-suspending the lyophilized activated isolated polysaccharide and the lyophilized carrier protein in an organic solvent to produce an activated isolated polysaccharide mixed with a carrier protein.
6. The method of claim 5 , wherein prior to lyophilization the carrier protein is diafiltered against NaCl and the w/w ratio of NaCl/carrier protein is adjusted to about 0.5 to about 1.5.
7. The method of claim 1 , wherein the method further comprises dilution of the reaction mixture of step b) into a buffer and maintaining a pH of about 8.8 to about 9.2 for at least 4 hours at about 20° C. to about 26° C.
8. The method of claim 7 , wherein the method comprises dilution of the reaction mixture of step b) into a buffer and maintaining a pH of about 9.0 for at least 4 hours at about 23° C.
9. The method of claim 1 , wherein the method further comprises isolating the S. aureus capsular polysaccharide carrier protein conjugate.
10. The method of claim 1 , wherein the organic solvent is a polar aprotic solvent.
11. The method of claim 10 , wherein the polar aprotic solvent is selected from the group consisting of dimethyl sulfoxide (DMSO), dimethylformamide (DMF), dimethylacetamide, N-methyl-2-pyrrolidone, and hexamethylphosphoramide (HMPA).
12. The method of claim 11 , wherein the organic solvent is dimethyl sulfoxide (DMSO).
13. The method of claim 1 , wherein the CDT concentration is adjusted to about 0.75:1 molar ratio of CDT:water in the organic solvent.
14. The method of claim 13 , wherein CDT concentration is adjusted to about 0.5:1 molar ratio of CDT:water in the organic solvent.
15. The method of claim 1 , wherein the carrier protein is CRM 197 .
16. The method of claim 15 , wherein the activated polysaccharide is reacted with the CRM 197 in a ratio by weight of about 1:1.
17. The method of claim 1 , wherein the isolated S. aureus capsular polysaccharide is mixed with imidazole or triazole prior to mixing with CDT in an organic solvent.
18. The method claim 1 , wherein the method further comprises hydrolyzing the polysaccharide carrier protein conjugate to remove unreacted activation groups.
19. A method of producing an immunogenic polysaccharide protein conjugate comprising an isolated Staphylococcus aureus serotype 5 capsular polysaccharide conjugated to a carrier protein, the method comprising the steps of:
a) reacting an isolated S. aureus serotype 5 capsular polysaccharide with carbonyl ditriazole (CDT) in an organic solvent to produce an activated polysaccharide; and
b) reacting the activated polysaccharide with a carrier protein in an organic solvent to produce a polysaccharide:carrier protein conjugate;
wherein the method further comprises the step of adjusting the water concentration of the reaction mixture comprising S. aureus serotype 5 capsular polysaccharide and CDT in an organic solvent to between about 0.1% and 0.3% and wherein an immunogenic conjugate comprising a capsular polysaccharide carrier protein conjugate is produced.
20. The method of claim 19 , wherein the method further comprises the steps of lyophilizing the isolated polysaccharide and re-suspending the lyophilized polysaccharide in an organic solvent.
21. The method of claim 19 , wherein the activated isolated polysaccharide is isolated from the activation reaction prior to reacting with the carrier protein.
22. The method of claim 21 , wherein the step of isolating the activated isolated polysaccharide comprises diafiltration.
23. The method of claim 21 , wherein step b) comprises:
i) lyophilizing the isolated activated isolated polysaccharide to produce a lyophilized activated isolated polysaccharide;
ii) lyophilizing the carrier protein to produce a lyophilized carrier protein; and
iii) re-suspending the lyophilized activated isolated polysaccharide and the lyophilized carrier protein in an organic solvent to produce an activated isolated polysaccharide mixed with a carrier protein.
24. The method of claim 23 , wherein prior to lyophilization the carrier protein is diafiltered against NaCl and the w/w ratio of NaCl/carrier protein is adjusted to about 0.5 to about 1.5.
25. The method of claim 19 , wherein the method further comprises dilution of the reaction mixture of step b) into a buffer and maintaining a pH of about 8.8 to about 9.2 for at least 4 hours at about 20° C. to about 26° C.
26. The method of claim 25 , wherein the method comprises dilution of the reaction mixture of step b) into a buffer and maintaining a pH of about 9.0 for at least 4 hours at about 23° C.
27. The method of claim 19 , wherein the method further comprises isolating the S. aureus capsular polysaccharide carrier protein conjugate.
28. The method of claim 19 , wherein the organic solvent is a polar aprotic solvent.
29. The method of claim 28 , wherein the polar aprotic solvent is selected from the group consisting of dimethyl sulfoxide (DMSO), dimethylformamide (DMF), dimethylacetamide, N-methyl-2-pyrrolidone, and hexamethylphosphoramide (HMPA).
30. The method of claim 29 , wherein the organic solvent is dimethyl sulfoxide (DMSO).
31. The method of claim 19 , wherein the carrier protein is CRM 197 .
32. The method of claim 31 , wherein the activated polysaccharide is reacted with the CRM 197 in a ratio by weight of about 1:1.
33. The method of claim 19 , wherein the isolated S. aureus capsular polysaccharide is mixed with imidazole or triazole prior to mixing with CDT in an organic solvent.
34. The method claim 19 , wherein the method further comprises hydrolyzing the polysaccharide carrier protein conjugate to remove unreacted activation groups.
35. The method of claim 19 , wherein the step of reacting the serotype 5 polysaccharide with CDT comprises providing about 20-fold molar excess of CDT compared to the polysaccharide.
36. The method of claim 19 , wherein the water concentration is adjusted to 0.2%.