Methods for preparing complex multivalent immunogenic conjugates

1. A method for making a complex multivalent immunogenic conjugate, comprising:

reacting a plurality of immunogenic-distinct polysaccharides with a cyanylation agent resulting in a mixture of a plurality of cyanate-activated immunogenic-distinct polysaccharides;

reacting at least one protein with hydrazine, carbohydrazide, hydrazine dichloride, a dihydrazide, or a mixture thereof under conditions sufficient to produce a solution of at least one hydrazide-activated protein; and

contacting the mixture of the plurality of cyanate-activated immunogenic-distinct polysaccharides with the at least one hydrazide-activated protein at a pH of about 6 to about 8 such that the plurality of cyanate-activated immunogenic-distinct polysaccharides simultaneously react with the at least one hydrazide-activated protein resulting in a complex multivalent immunogenic conjugate that includes at least one C—N bond formed between each attached immunogenic-distinct polysaccharide and the protein.

2. The method of claim 1 , wherein the cyanylation agent is selected from 1-cyano-4-dimethylammoniumpyridinium tetrafluorborate, cyanogen bromide, or N-cyano-N,N,N-triethyleammonium tetrafluoroborate.

3. The method of claim 1 , wherein the simultaneous reaction of the plurality of cyanate-activated immunogenic-distinct polysaccharides with the at least one hydrazide-activated protein is effected in a composition that includes the mixture of the plurality of cyanate-activated immunogenic-distinct polysaccharides and the at least one hydrazide-activated protein.

4. The method of claim 1 , wherein the contacting of the mixture of the plurality of cyanate-activated immunogenic-distinct polysaccharides with the at least one hydrazide-activated protein comprises preparing a reaction composition that includes the mixture of the plurality of cyanate-activated immunogenic-distinct polysaccharides with the at least one hydrazide-activated protein.

5. The method of claim 1 , further comprising reacting a second plurality of second immunogenic-distinct polysaccharides with a cyanylation agent resulting in a second mixture of a plurality of second cyanate-activated immunogenic-distinct polysaccharides; and

contacting the second mixture of a plurality of cyanate-activated immunogenic-distinct polysaccharides with the complex multivalent immunogenic conjugate to form at least one C—N bond between each second cyanate-activated immunogenic-distinct polysaccharide and the protein.

6. The method of claim 5 , wherein the reactivity of the second immunogenic-distinct polysaccharides with the cyanylation agent is greater than the reactivity of the first immunogenic-distinct polysaccharides with the cyanylation agent.

7. The method of claim 6 , wherein the first immunogenic-distinct polysaccharide is selected from at least one of Meningococcal group A or Meningococcal group C.

8. The method of claim 6 , wherein the second immunogenic-distinct polysaccharide is selected from at least one of Meningococcal group W135 or Meningococcal group Y.

9. A complex multivalent immunogenic conjugate prepared according to claim 1 .

10. A pharmaceutical composition comprising the complex multivalent immunogenic conjugate of claim 9 and at least one pharmaceutically-acceptable carrier.

11. The method of claim 1 , wherein the plurality of immunogenic-distinct polysaccharides includes at least one pneumococcal polysaccharide, at least one meningococcal polysaccharide, and at least one Haemophilus influenzae type b polysaccharide.

12. The method of claim 1 , wherein the at least one hydrazide-activated protein is soluble at neutral pH.

13. The method of claim 12 , wherein the at least one protein is reacted with hydrazine, carbohydrazide, hydrazine chloride, a dihydrazide or a mixture thereof in the presence of (i) a carbodiimide and (ii) at least one amino acid, at least one peptide, or a mixture of at least one amino acid and at least one peptide.

14. The method of claim 13 , wherein the amino acid is selected from at least one of lysine, arginine, histidine, glycine, serine, threonine, glutamic acid or cysteine.

15. The method of claim 1 , wherein the at least one protein is reacted with hydrazine, carbohydrazide, succinyl dihydrazide, adipic acid dihydrazide or a mixture thereof in the presence of a carbodiimide hydrochloride at a pH of 6 to 7 to obtain a solution of hydrazide-activated protein, and further comprising buffer exchanging the solution of hydrazide-activated protein to a pH of from 10.0 to 11.0.

16. The method of claim 1 , wherein the at least one protein is reacted with hydrazine, carbohydrazide, succinyl dihydrazide, adipic acid dihydrazide or a mixture thereof in the presence of a carbodiimide hydrochloride at a pH of 5.5 to 6.5 to obtain a solution of hydrazide-activated protein, and further comprising buffer exchanging the solution of hydrazide-activated protein to a pH of from 10.0 to 11.0.

17. The method of claim 1 , wherein 2 to 28 cyanate-activated immunogenic-distinct polysaccharides are simultaneously reacted with the at least one hydrazide-activated protein.

18. The method of claim 17 , wherein the immunogenic-distinct polysaccharides are selected from the group consisting of Meningococcal polysaccharides, Pneumococcal polysaccharides, Hemophilus influenzae type b polysaccharide, Vi polysaccharide of Salmonnella typhi and group B Streptococcus polysaccharides.

19. The method of claim 1 wherein the immunogenic-distinct polysaccharides are selected from the group consisting of Meningococcal group A, Meningococcal group C, Meningococcal group W135 and Meningococcal group Y.

20. The method of claim 1 wherein the cyanate-activated immunogenic-distinct polysaccharides are reacted with a single hydrazide-activated protein.

21. The method of claim 1 , wherein the cyanate-activated immunogenic-distinct polysaccharides are reacted with a plurality of different hydrazide-activated proteins.

22. The method of claim 13 , wherein the carbodiimide is 1-[3-(dimethylamino)propyl]-3-ethyl carbodiimide hydrochloride.

23. The method of claim 15 , wherein the carbodiimide is 1-[3-(dimethylamino)propyl]-3-ethyl carbodiimide hydrochloride.

24. The method of claim 1 , wherein a mixture of immunogenic-distinct polysaccharides is reacted with the cyanylation agent.

25. The method of claim 1 , wherein each immunogenic-distinct polysaccharide is initially reacted with the cyanylation agent, and then the resulting individual cyanate-activated immunogenic-distinct polysaccharides are mixed together to form the mixture of cyanate-activated immunogenic-distinct polysaccharides.

26. The method of claim 1 , wherein the complex multivalent immunogenic conjugate comprises a structure having a plurality of immunogenic-distinct polysaccharides conjugated to a single protein construct.

27. The method of claim 1 , wherein the complex multivalent immunogenic conjugate has a structure comprising:

wherein P 1 is a carrier protein; and PS 1 , PS 2 , and PS 3 are each immunogenic-distinct polysaccharides that are covalently attached to P 1 .