Methods for making saccharide-protein glycoconjugates
The invention provides a process for the reductive amination of a carbonyl group at the reducing terminus of a polysaccharide, wherein the reductive amination is carried out at a pH between 4 and 5. The invention also provides a process for preparing a conjugate of a polysaccharide and a carrier molecule, comprising the steps of: (a) coupling the polysaccharide to a linker, to form a polysaccharide-linker compound in which the free terminus of the linker is an ester group; and (b) reacting the ester group with a primary amine group in the carrier molecule, to form a polysaccharide-linker-carrier molecule conjugate in which the linker is coupled to the carrier molecule via an amide linkage. The invention also provides a process for reducing contamination of a polysaccharide-linker compound with unreacted linker, comprising a step of precipitating unreacted linker under aqueous conditions at a pH of less than 5. The invention also provides polysaccharide-linker-carrier molecule conjugates and intermediate compounds obtained or obtainable by these processes.
1. A polysaccharide-linker intermediate of the formula:
wherein OAg is an O-antigen-core from a lipopolysaccharide which has been coupled using a carbonyl group at the reducing terminus.
2. The polysaccharide-linker intermediate of claim 1 , wherein the lipopolysaccharide is from a Salmonella bacterium.
3. The polysaccharide-linker intermediate of claim 2 , wherein the lipopolysaccharide is from Salmonella serogroups A, B or D.
4. A polysaccharide antigen conjugate prepared by a process comprising the steps of:
(a1) reacting a carbonyl group at a reducing terminus of a polysaccharide with Y 1 of an additional linker of Formula 1,
wherein Formula 1 is Y 1 -L-Y 2 ,
wherein L is a linking moiety, Y 1 and Y 2 are each —NHNH 2 groups;
(a2) reacting Y 2 of the additional linker with a linker, to form a polysaccharide-additional linker-linker compound in which the free terminus of the linker is an ester group; and
(b) reacting the ester group of the linker with a primary amine group in a carrier molecule, to form a polysaccharide-additional linker-linker-carrier molecule conjugate in which the linker is coupled to the carrier molecule via an amide linkage.
5. The polysaccharide antigen conjugate of claim 4 , wherein the polysaccharide comprises an O-antigen from a lipopolysaccharide.
6. The polysaccharide antigen conjugate of claim 5 , wherein the polysaccharide also comprises a core domain.
7. The polysaccharide antigen conjugate of claim 5 , wherein the lipopolysaccharide is from a Gram negative bacterium.
8. The polysaccharide antigen conjugate of claim 6 , wherein the lipopolysaccharide is from a Gram negative bacterium.
9. The polysaccharide antigen conjugate of claim 7 , wherein the lipopolysaccharide is from a Salmonella bacterium.
10. The polysaccharide antigen conjugate of claim 8 , wherein the lipopolysaccharide is from a Salmonella bacterium.
11. The polysaccharide antigen conjugate of claim 4 , wherein the carrier molecule is CRM 197 .
12. The polysaccharide antigen conjugate of claim 4 , wherein the additional linker is adipic acid dihydrazide (ADH).
13. The polysaccharide antigen conjugate of claim 4 , wherein the polysaccharide comprises an O-antigen and a core from a lipopolysaccharide from a Salmonella bacterium, wherein the carrier molecule is CRM 197 , wherein the additional linker is adipic acid dihydrazide (ADH), wherein the linker is adipic acid N-hydroxysuccinimide diester (SIDEA) and wherein the additional linker is coupled using a carbonyl group at the reducing terminus of the polysaccharide.
14. A process for preparing a conjugate of a polysaccharide and a carrier molecule comprising the steps of:
(a1) reacting a carbonyl group at a reducing terminus of the polysaccharide with Y 1 of an additional linker of Formula 1,
wherein Formula 1 is Y 1 -L-Y 2 ,
wherein L is a linking moeity, Y 1 and Y 2 are each —NHNH 2 groups;
(a2) reacting Y 2 with the linker SIDEA, to form a polysaccharide-additional linker-linker compound in which the free terminus of the linker is an ester group; and
(b) reacting the ester group of the linker with a primary amine group in a carrier molecule, to form a polysaccharide-additional linker-linker-carrier molecule conjugate in which the linker is coupled to the carrier molecule via an amide linkage.
15. The process of claim 14 , wherein the polysaccharide comprises an O-antigen from a lipopolysaccharide.
16. The process of claim 15 , wherein the polysaccharide also comprises a core domain.
17. The process of claim 15 , wherein the lipopolysaccharide is from a Gram negative bacterium.
18. The process of claim 16 , wherein the lipopolysaccharide is from a Gram negative bacterium.
19. The process of claim 17 , wherein the lipopolysaccharide is from a Salmonella bacterium.
20. The process of claim 18 , wherein the lipopolysaccharide is from a Salmonella bacterium.
21. The process of claim 14 , wherein the carrier molecule is CRM 197 .
22. The process of claim 14 , wherein the additional linker is adipic acid dihydrazide (ADH).
23. The process of claim 14 , wherein the polysaccharide comprises an O-antigen and a core from a lipopolysaccharide from a Salmonella bacterium, wherein the carrier molecule is CRM 197 , wherein the additional linker is adipic acid dihydrazide (ADH), wherein the linker is adipic acid N-hydroxysuccinimide diester (SIDEA) and wherein the additional linker is coupled using a carbonyl group at the reducing terminus of the polysaccharide.