Polysaccharide-protein conjugate vaccines
View Patent ↗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 1-cyano-4-dimethylammoniumpyridinium tetrafluoroborate, whereby a solution of a cyanate-activated polysaccharide is obtained;
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 a hydrazide-activated protein is obtained;
raising the 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 cyanate-activated polysaccharide with the hydrazide-activated protein at a pH of from 6 to 8 to yield a conjugate vaccine capable of stimulating an immune response.
2. The method according to claim 1 , wherein the step of reacting the cyanate-activated polysaccharide with the hydrazide-activated protein is conducted in the absence of a blocking agent.
3. 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 .
4. The method according to claim 1 , wherein the protein is selected from the group consisting of tetanus toxoid, diptheria toxoid, CRM 197 , and meningococcal protein.
5. The method of claim 1 , wherein reacting the cyanate-activated polysaccharide with the hydrazide-activated protein is conducted at a temperature of from 0° C. to 5° C. for 36 to 48 hours, followed by an additional 18 to 24 hours at a temperature of from 20° C. to 25° C.
6. The method of claim 1 , wherein the solution of the hydrazide-activated protein is buffer-exchanged at a pH of 10.5.
7. The method of claim 1 , wherein the solution of the hydrazide-activated protein is buffer-exchanged at a pH of 11.
8. The method of claim 1 , wherein the solution of the hydrazide-activated protein is buffer-exchanged with a buffer concentration of 3 mM to 10mM.