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1. WO2007109129 - procede de preparation de conjugues immunogeniques multivalents complexes

Note: Texte fondé sur des processus automatiques de reconnaissance optique de caractères. Seule la version PDF a une valeur juridique

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What is claimed is:

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 about 5 to about 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 substantially all of 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 substantially 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 about 6 to about 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 about 10.0 to about 1 1.0.

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 about 5.5 to about 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 about 10.0 to about 1 1.0.

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.

1 1. 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 l-[3-(dimethylamino)propyl]-3-ethyl carbodiimide hydrochloride.

15. The method of claim 7, wherein the carbodiimide is l-(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:
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,

19. The method of claim 18, wherein the cyanylation agent is selected from l-cyano-4-dimethylammoniumpyridinium tetrafluorborate, cyanogen bromide, or N-cyano-h^N/N-triethyleammonium tetrafluoroborate.

20. The method of claim 18, 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.

21. The method of claim 18, 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.

22. The method of claim 18, 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.

23. The method of claim 22, 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.

24. The method of claim 23, wherein the first immunogenic-distinct polysaccharide is selected from at least one of Meningococcal group A or Meningococcal group C.

25. The method of claim 23, wherein the second immunogenic-distinct polysaccharide is selected from at least one of Meningococcal group W135 or Meningococcal group Y.

26. A method for making a complex multivalent immunogenic conjugate, comprising:
reacting a protein with l-amino-2,3-propanediol (ADPO) in the presence of l-[3-(dimethylamino)propyl]-3-ethyl carbodiimide hydrochloride at a pH of from about 5.5 to about 7 resulting in a solution of an ADPO-modified protein;
reacting the ADPO-modified protein with an oxidizing agent resulting in a solution of an aldehyde-activated protein;
contacting a mixture of a plurality of hydrazide-activated immunogenic-distinct polysaccharides with the aldehyde-activated protein at a pH of about 5 to about 8 such that the plurality of hydrazide-activated immunogenic-distinct polysaccharides simultaneously react with at least one aldehyde-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 substantially all of the C=N double bonds of the complex multivalent conjugate to C-N bonds resulting in a complex multivalent immunogenic conjugate product.

27. The method of claim 26, wherein the protein is reacted with ADPO at a pH of about 5.5 to about 6.5.

28. The method of claim 26, wherein the protein is reacted with ADPO at a pH of about 6 to about 7.

29. A method for preparing a hydrazide-activated protein, comprising:
reacting a protein 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.

30. The method of claim 29, wherein the carbodiimide is l-[3-(dimethylamino)propyl]-3-ethyl carbodiimide hydrochloride.

3 1. The method of claim 29, wherein the amino acid is selected from at least one of lysine, arginine, histidine, glycine, serine, threonine, glutamic acid or cysteine.

32. The method of claim 29, wherein the at least one hydrazide-activated protein is substantially soluble at neutral pH.

33. 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 substantially all of 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.

34. The method of claim 33, wherein the first aldehyde-activated immunogenic-distinct polysaccharide is selected from at least one of Meningococcal group A or Meningococcal group C.

35. The method of claim 33, wherein the second aldehyde-activated immunogenic-distinct polysaccharide is selected from at least one of Meningococcal group W135 or
Meningococcal group Y.

36. The method of claim 34, wherein the second aldehyde-activated immunogenic-distinct polysaccharide is selected from at least one of Meningococcal group Wl 35 or
Meningococcal group Y.

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

38. A complex multivalent immunogenic conjugate prepared according to claim 18.

39. A complex multivalent immunogenic conjugate prepared according to claim 26.

40. A complex multivalent immunogenic conjugate prepared according to claim 33.

41. A pharmaceutical composition comprising the complex multivalent immunogenic conjugate of claim 37 and at least one pharmaceutical ly-acceptable carrier.

42. A pharmaceutical composition comprising the complex multivalent immunogenic conjugate of claim 38 and at least one pharmaceutically-acceptable carrier.

43. A pharmaceutical composition comprising the complex multivalent immunogenic conjugate of claim 39 and at least one pharmaceutically-acceptable carrier.

44. A pharmaceutical composition comprising the complex multivalent immunogenic conjugate of claim 40 and at least one pharmaceutically-acceptable carrier.