Methods for making polysaccharide-protein conjugates
View Patent ↗The present invention provides methods for making polysaccharide-protein conjugates in which polysaccharides, typically from bacteria, are conjugated to a carrier protein by reductive amination under conditions which improve conjugation reaction consistency, increase consumption of protein during conjugation reaction, generate conjugates of higher molecular weight, and/or reduce the levels of free cyanide in the conjugate reaction product. The polysaccharide-protein conjugates obtained using these methods are useful for inclusion in multivalent vaccines.
1. A method for activating a Streptococcus pneumoniae serotype 3 polysaccharide for conjugation to a CRM 197 carrier protein comprising reacting a serotype 3 polysaccharide with periodate in an aqueous buffer (i) at a pH in the range from 4.3-6.9 and at a temperature in the range from 2-23° C., (ii) at a pH in the range from 4.3-7.7 and at a temperature in the range from 2-10° C., or (iii) at a pH in the range from 4.3-6.2 and at a temperature in the range from 2-35° C., wherein the activated serotype 3 polysaccharide detected after the reaction has a minimal (<20%) reduction in size (molecular weight).
2. The method of claim 1 , wherein the periodate is sodium periodate.
3. The method of claim 1 , wherein the reaction occurs at a pH in the range from 4.3-6.9 and at a temperature in the range from 2-23° C.
4. The method of claim 1 , wherein the reaction occurs at a pH in the range from 4.3-7.7 and at a temperature in the range from 2-10° C.
5. The method of claim 1 , wherein the reaction occurs at a pH in the range from 4.3-6.2 and at a temperature in the range from 2-35° C.
6. An activated serotype 3 polysaccharide prepared according to the method of claim 1 .
7. A serotype 3 polysaccharide protein conjugate prepared with the activated serotype 3 polysaccharide of claim 6 .
8. The conjugate of claim 7 , wherein the protein is selected from the group consisting of tetanus toxoid, diphtheria toxoid and CRM 197 .
9. The conjugate of claim 7 , wherein the protein is CRM 197 .
10. A multivalent pneumococcal conjugate vaccine composition comprising the serotype 3 polysaccharide protein conjugate of claim 7 .
11. The vaccine composition according to claim 10 further comprising Streptococcus pneumoniae polysaccharide protein conjugates wherein the polysaccharide is selected from the following serotypes: 1, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and/or 33F.
12. The vaccine composition of claim 11 , wherein the protein is selected from the group consisting of tetanus toxoid, diphtheria toxoid and CRM 197 .
13. The vaccine composition of claim 11 , wherein the protein is CRM 197 .