Methods for making saccharide-protein glycoconjugates

Patent metadata

Field	Value
Publication number	US-10113009-B2
Application number	US-201615150302-A
Country	US
Kind code	B2
Filing date	May 9, 2016
Priority date	Sep 14, 2011
Publication date	Oct 30, 2018
Grant date	Oct 30, 2018

Abstract

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.

First claim

We claim:

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.

Assignees

• Glaxosmithkline Biologicals Sa

Inventors

• Saul Allan James
• Micoli Francesca

Classifications

• C08B37/0063

  Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan · CPC title

• C08B37/0081

  Reaction with amino acids, peptides, or proteins · CPC title

• A61K2039/6037

  Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT] · CPC title

• C07K16/1285

  from Corynebacterium (G) · CPC title

• C08B37/006

  Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof · CPC title