Glycoconjugation processes and compositions

Patent metadata

Field	Value
Publication number	US-12447203-B2
Application number	US-202318338961-A
Country	US
Kind code	B2
Filing date	Jun 21, 2023
Priority date	Aug 16, 2012
Publication date	Oct 21, 2025
Grant date	Oct 21, 2025

Abstract

The invention provides eTEC linked glycoconjugates comprising a saccharide covalently conjugated to a carrier protein through a (2-((2-oxoethyl)thio)ethyl)carbamate (eTEC) spacer, immunogenic compositions comprising such glycoconjugates, and methods for the preparation and use of such glycoconjugates and immunogenic compositions.

First claim

The invention claimed is:

1. A method of preparing a glycoconjugate, comprising a S. pneumoniae serotype 33F capsular polysaccharide conjugated to a carrier protein, comprising conjugating activated S. pneumoniae serotype 33F capsular polysaccharide and carrier protein by reductive amination in dimethylsulfoxide (DMSO), wherein said glycoconjugate comprises less than 5% free saccharide compared to the total amount of S. pneumoniae serotype 33F polysaccharide, wherein the carrier protein is CRM 197 and wherein said S. pneumoniae serotype 33F polysaccharide has a degree of O-acetylation between 75-100%.

2. The glycoconjugate of claim 1 , wherein the carrier protein CRM 197 comprises 4 to 16 lysine residues out of 39 covalently linked to the saccharide.

3. The glycoconjugate of claim 1 , wherein the saccharide to carrier protein ratio (w/w) is between 0.4 and 1.7.

4. The glycoconjugate of claim 1 , wherein the polysaccharide has a molecular weight of between 10 kDa and 2,000 kDa, or between 50 kDa and 2,000 kDa.

5. The method of claim 1 , further comprising, before said step of conjugating, reconstituting said activated polysaccharide and said CRM 197 carrier protein in DMSO, each of which had been lyophilized.

6. The method of claim 5 , wherein said activated polysaccharide was lyophilized after compounding with sucrose.

7. The method of claim 6 , wherein said activated polysaccharide was lyophilized after compounding with sucrose using a ratio of 25 grams sucrose per gram activated polysaccharide.

8. The method of claim 1 , wherein said activated polysaccharide was activated by oxidation with sodium periodate.

9. The method of claim 8 , wherein said oxidation was carried out in sodium phosphate buffer, pH 6.

10. The method of claim 1 , wherein said step of conjugating by reductive amination is carried out by addition of sodium cyanoborohydride.

11. The method of claim 10 , wherein said step of conjugating by reductive amination is carried out at 23° C.

12. The method of claim 11 , wherein said step of conjugating by reductive amination is carried out for at least 20 hours.

13. The method of claim 10 , further comprising terminating the reductive amination reaction by addition of sodium borohydride.

14. The method of claim 13 , further comprising diluting the reaction mixture.

15. The method of claim 13 , further comprising filtering the reaction mixture.

16. The method of claim 13 , further comprising concentrating the reaction mixture.

17. The method of claim 13 , further comprising diafiltering the reaction mixture.

18. The method of claim 13 , wherein said step of diafiltering is carried out with a succinate saline buffer.

19. The method of claim 18 , wherein said succinate saline buffer comprises 5 mM succinate and 0.9% saline, pH 6.

20. A glycoconjugate produced by the method of claim 1 , wherein said glycoconjugate comprises S. pneumoniae serotype 33F capsular polysaccharide conjugated to CRM 197 , wherein said glycoconjugate comprises less than 5% free saccharide compared to the total amount of S. pneumoniae serotype 33F polysaccharide, and wherein said S. pneumoniae serotype 33F polysaccharide has a degree of O-acetylation between 75-100%.

Assignees

• Pfizer

Inventors

• Gu Jianxin
• Kim Jin-Hwan
• Prasad Avvari Krishna
• Yang Yu-Ying

Classifications

• A61K2039/6037

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

• A61K47/6415

  Toxins or lectins, e.g. clostridial toxins or Pseudomonas exotoxins · CPC title

• A61K2039/627

  characterised by the linker · CPC title

• A61K2039/6068

  Other bacterial proteins, e.g. OMP · CPC title

• A61K45/06

  Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca · CPC title