Polysaccharide-protein conjugate vaccines
View Patent ↗Abstract of the Disclosure Methods for synthesis and manufacture of polysaccharide-protein conjugate vaccines at high yield are provided. The methods involve reaction of a hydrazide group on one reactant with an aldehyde or cyanate ester group on the other reactant. The reaction proceeds rapidly with a high conjugation efficiency, such that a simplified purification process can be employed to separate the conjugate product from the unconjugated protein and polysaccharide and other small molecule by-products.
1. A method for preparing a conjugate vaccine, the method comprising:
reacting a polysaccharide with an oxidizing agent, whereby a solution of an aldehyde-activated polysaccharide is obtained;
buffer exchanging the solution of the aldehyde-activated polysaccharide to a pH of from 7 to 8;
reacting a protein with hydrazine or adipic acid dihydrazide in the presence of 1-[3-(dimethylamino)propyl]-3-ethyl carbodiimide hydrochloride at a pH of from 6 to 7, whereby a solution of an hydrazide-activated protein is obtained;
raising a pH of the solution of the hydrazide-activated protein to from 7.0 to 11;
buffer exchanging the solution of the hydrazide-activated protein to a pH of from 10.0 to 11.0;
reacting the aldehyde-activated polysaccharide with the hydrazide-activated protein at a pH of from 6 to 8, whereby a conjugate comprising one or more C═N double bonds is obtained; and
reducing the C═N double bonds of the conjugate to C—N single bonds, whereby a conjugate vaccine capable of stimulating an immune response is obtained.
2. The method according to claim 1 , wherein the oxidizing agent comprises NaIO 4 .
3. The method according to claim 1 , wherein the solution of the aldehyde-activated polysaccharide is buffer exchanged with a HEPES buffer.
4. The method according to claim 1 , wherein the solution of the hydrazide-activated protein is buffer exchanged with a Na 2 CO 3 buffer.
5. The method according to claim I, wherein the aldehyde-activated polysaccharide is reacted with the hydrazide-activated protein at a ratio of from about 1:2 to about 2:1.
6. The method according to claim 1 , wherein reducing comprises reducing with NaBH 4 .
7. 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 Salmonnella typhi , and group B Streptococcus polysaccharides.
8. The method according to claim 1 , wherein the protein is selected from the group consisting of tetanus toxoid, diphtheria toxoid, CRM 197 , and meningococcal protein.
9. A conjugate vaccine, the conjugate vaccine comprising at least one polysaccharide moiety and at least one protein moiety, wherein the polysaccharide moiety is linked to the protein moiety through at least one linking group of the formula
—C(═O)—NH—NH—CH 2 —.
10. The conjugate vaccine of claim 9 , wherein the conjugate vaccine comprises a plurality of polysaccharide moieties and a plurality of protein moieties crosslinked to form a lattice structure by a plurality of linking groups.
11. The conjugate vaccine of claim 9 , wherein the polysaccharide is selected from the group consisting of Meningococcal polysaccharides, Pneumococcus polysaccharides, Hemophilus influenzae type h polysaccharide, Vi polysaccharide of Salmonnellia typhi , and group B Streptococcus polysaccharides.
12. The conjugate vaccine of claim 9 , wherein the protein is selected from the group consisting of tetanus toxoid, diphtheria toxoid, CRM 197 , and meningococcal protein.
13. The conjugate vaccine of claim 9 , wherein the polysaccharide moiety is exclusively linked to the protein moiety through the at least one linking group of the formula
—C(═O)—NH—NH—CH 2 —.
14. The conjugate vaccine of claim 9 , wherein the conjugate vaccine elicits an immune response.
15. The method of claim 1 , wherein the solution of the hydrazide-activated protein is buffer-exchanged at a pH of 10.5.
16. The method of claim 1 , wherein the solution of the hydrazide-activated protein is buffer-exchanged at a pH of 11.
17. The method of claim 1 , wherein the solution of the hydrazide-activated protein is buffer-exchanged with a buffer concentration of 3 mM to 10 mM.
18. The method of claim 1 , wherein the solution of the aldehyde-activated polysaccharide is buffer-exchanged with a buffer concentration of 100 mM to 200 mM.
19. The method of claim 1 , wherein the protein is reacted with hydrazine or adipic acid in the presence of MES.
20. The method of claim 1 wherein the method does not include the use of sodium cyanoborohydride.