Methods for preparing complex multivalent immunogenic conjugates
View Patent ↗Methods for preparing complex multivalent immunogenic conjugates that include simultaneously reacting a plurality or immunogenic-distinct polysaccharides with at least one protein to make the complex multivalent immunogenic conjugates. The simultaneous reaction involves reaction of a hydrazide group on one reactant with an aldehyde or cyanate ester group on the other reactant.
1. A method for making a complex multivalent immunogenic conjugate, comprising:
reacting a plurality of immunogenic-distinct polysaccharides with an oxidizing agent resulting in a mixture of a plurality of aldehyde-activated immunogenic-distinct polysaccharides;
reacting at least one protein with hydrazine, carbohydrazide, hydrazine chloride, a dihydrazide or a mixture thereof under conditions sufficient to produce a solution of at least one hydrazide-activated protein;
contacting the mixture of the plurality of aldehyde-activated immunogenic-distinct polysaccharides with the at least one hydrazide-activated protein at a pH of 5 to 8 such that the plurality of aldehyde-activated immunogenic-distinct polysaccharides simultaneously react with the at least one hydrazide-activated protein resulting in a complex multivalent conjugate that includes at least one C═N double bond formed between each attached immunogenic-distinct polysaccharide and the protein; and
reducing the C═N double bonds of the complex multivalent conjugate to C—N bonds resulting in a complex multivalent immunogenic conjugate product.
2. The method of claim 1 , wherein the at least one hydrazide-activated protein is soluble at neutral pH.
3. The method of claim 1 , wherein the simultaneous reaction of the plurality of aldehyde-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 aldehyde-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 aldehyde-activated immunogenic-distinct polysaccharides with the at least one hydrazide-activated protein and the reduction of the C═N double bonds comprises providing, in the presence of sodium borohydride, a composition formed from the mixture of the plurality of aldehyde-activated immunogenic-distinct polysaccharides and the at least one hydrazide-activated protein.
5. The method of claim 2 , 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.
6. The method of claim 5 , wherein the amino acid is selected from at least one of lysine, arginine, histidine, glycine, serine, threonine, glutamic acid or cysteine.
7. 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.
8. 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.
9. The method of claim 1 , wherein 2 to 28 aldehyde-activated immunogenic-distinct polysaccharides are simultaneously reacted with the at least one hydrazide-activated protein.
10. The method of claim 9 , 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.
11. 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.
12. The method of claim 1 wherein the aldehyde-activated immunogenic-distinct polysaccharides are reacted with a single hydrazide-activated protein.
13. The method of claim 1 , wherein the aldehyde-activated immunogenic-distinct polysaccharides are reacted with a plurality of different hydrazide-activated proteins.
14. The method of claim 5 , wherein the carbodiimide is 1-[3-(dimethylamino)propyl]-3-ethyl carbodiimide hydrochloride.
15. The method of claim 7 , wherein the carbodiimide is 1-[3-(dimethylamino)propyl]-3-ethyl carbodiimide hydrochloride.
16. The method of claim 1 , wherein a mixture of immunogenic-distinct polysaccharides is reacted with the oxidizing agent.
17. The method of claim 1 , wherein each immunogenic-distinct polysaccharide is initially reacted with an oxidizing agent, and then the resulting individual aldehyde-activated immunogenic-distinct polysaccharides are mixed together to form the mixture of aldehyde-activated immunogenic-distinct polysaccharides.
18. A method for making a complex multivalent immunogenic conjugate comprising:
(a) contacting at least one first aldehyde-activated immunogenic-distinct polysaccharide with at least one hydrazide-activated protein under conditions sufficient for forming a first conjugate intermediate such that at least one C═N double bond forms between the first immunogenic-distinct polysaccharide and the protein;
(b) contacting at least one second aldehyde-activated immunogenic-distinct polysaccharide with the first conjugate intermediate such that at least one C═N double bond forms between the second immunogenic-distinct polysaccharide and the protein; and
(c) reducing the C═N double bonds to C—N bonds resulting in a complex multivalent immunogenic conjugate product;
wherein the reactivity of the first aldehyde-activated immunogenic-distinct polysaccharide with the hydrazide-activated protein is lower than the reactivity of the second aldehyde-activated immunogenic-distinct polysaccharide with the hydrazide-activated protein.
19. The method of claim 18 , wherein the first aldehyde-activated immunogenic-distinct polysaccharide is selected from at least one of Meningococcal group A or Meningococcal group C.
20. The method of claim 18 , wherein the second aldehyde-activated immunogenic-distinct polysaccharide is selected from at least one of Meningococcal group W135 or Meningococcal group Y.
21. The method of claim 19 , wherein the second aldehyde-activated immunogenic-distinct polysaccharide is selected from at least one of Meningococcal group W135 or Meningococcal group Y.
22. A complex multivalent immunogenic conjugate prepared according to claim 1 .
23. A complex multivalent immunogenic conjugate prepared according to claim 18 .
24. A pharmaceutical composition comprising the complex multivalent immunogenic conjugate of claim 22 and at least one pharmaceutically-acceptable carrier.
25. A pharmaceutical composition comprising the complex multivalent immunogenic conjugate of claim 23 and at least one pharmaceutically-acceptable carrier.
26. 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.
27. 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.
28. The method of claim 18 , wherein the complex multivalent immunogenic conjugate comprises a structure having a plurality of immunogenic-distinct polysaccharides conjugated to a single protein construct.
29. 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 .
30. The method of claim 11 , 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 .
31. The method of claim 18 , 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 .
32. The method of claim 26 , 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 .
33. The method of claim 11 , wherein the complex multivalent immunogenic conjugate comprises a structure having a plurality of immunogenic-distinct polysaccharides conjugated to a single protein construct.
34. The method of claim 26 , wherein the complex multivalent immunogenic conjugate comprises a structure having a plurality of immunogenic-distinct polysaccharides conjugated to a single protein construct.