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 process for coupling a polysaccharide to a linker, comprising:
combining the polysaccharide with an additional linker comprising a primary amine group in the presence of a reducing agent, wherein the polysaccharide comprises a carbonyl group at the reducing terminus,
reacting the carbonyl group with the primary amine group by reductive amination to form a polysaccharide-additional linker intermediate, wherein the reductive amination is carried out at a pH between 4 and 5,
coupling the polysaccharide-additional linker intermediate to the linker, to form a polysaccharide-linker compound, and
precipitating unreacted linker under aqueous conditions at a pH of less than 5.
2. The process of claim 1 , wherein the polysaccharide comprises a core domain from a lipopolysaccharide of a Gram-negative bacterium, and an O-antigen from a lipopolysaccharide of a Gram-negative bacterium linked to the core domain.
3. The process of claim 2 , wherein the lipopolysaccharide is from a Salmonella bacterium.
4. The process of claim 3 , wherein the lipopolysaccharide is from S. Paratyphi A, S. Typhimurium or S. Enteritidis.
5. The process of claim 1 , wherein the polysaccharide is a bacterial capsular polysaccharide.
6. The process of claim 5 , wherein the bacterial capsular polysaccharide is from N. meningitidis serogroup X.
7. The process of claim 1 , wherein the reductive amination comprises reacting the carbonyl group with Y 1 in the additional linker having the formula Y 1 -L-Y 2 , to form the polysaccharide-additional linker intermediate compound in which the polysaccharide is coupled to the additional linker via a C—N linkage, wherein: Y 1 comprises a primary amine group that can react with the carbonyl group in the polysaccharide; Y 2 comprises a second reactive group; and L is a linking moiety.
8. The process of claim 7 , wherein the Y 1 and Y 2 are both —NHNH 2 groups.
9. The process of claim 7 , wherein L has formula -L′-L 2 -L′-, where L′ is carbonyl and L 2 is a straight chain alkyl with 1 to 10 carbon atoms.
10. The process of claim 8 , wherein L 2 is —(CH 2 ) 4 —.
11. The process of claim 8 , wherein L is carbonyl.
12. The process of claim 7 , wherein Y 2 comprises a primary amine group.
13. The process of claim 7 , wherein Y 2 comprises a —SH group.