Granted Patent B2

US 12,611,451 · App. 18/330,560 · Granted Apr 28, 2026

Inventors: Richard David Kensinger, Jr. (Henryville, PA); Steven L. Hauser (Easton, PA)

Assignee: SANOFI VACCINES US INC.

A61K39/095A61K47/18A61K47/36A61K47/6415A61K47/65A61K2039/55583A61K2039/6037A61K2039/70Y02A50/30

Abstract

Claims (27)

1 . A method of producing a conjugate of a Neisseria meningitidis capsular polysaccharide to a carrier protein, comprising:

a) partially de-O-acetylating the polysaccharide by alkaline hydrolysis to an O-acetylation level ranging from 0.6 μmol/mg polysaccharide to 1.5 μmol/mg polysaccharide;

b) activating the polysaccharide by periodate treatment, thereby converting diols to aldehydes to an extent of at least 20 nmol aldehyde per mg polysaccharide; and

c) conjugating the activated polysaccharide to the carrier protein by reductive amination, wherein the polysaccharide is present in the conjugation reaction at a weight-to-weight ratio of 1:1 to 5:1 relative to the carrier protein, thereby forming the conjugate;

wherein the conjugate is a population comprising double-end-linked conjugated polysaccharides and single-end-linked conjugated polysaccharides which are both directly attached to the carrier protein through a secondary amine,

wherein the polysaccharides of the conjugate to the carrier protein have an O-acetylation level ranging from 0.6 μmol/mg polysaccharide to 1.5 μmol/mg polysaccharide,

wherein the polysaccharide is MenC capsular polysaccharide, and

wherein the carrier protein is tetanus toxoid.

2 . The method of claim 1 , wherein the polysaccharide is present in the conjugation reaction at a weight-to-weight ratio of 1.5:1 to 3:1 relative to the carrier protein.

3 . The method of claim 1 , wherein the de-O-acetylation reduces the initial amount of O-acetylation in the polysaccharide by 40% to 70%.

4 . The method of claim 1 , wherein the activated polysaccharide is at a starting concentration of 20 g/L to 50 g/L in the conjugation reaction.

5 . The method of claim 1 , wherein the polysaccharide is reduced in size to 30 kDa to 150 kDa.

6 . The method of claim 5 , wherein the polysaccharide is reduced in size by acid hydrolysis and/or heat.

7 . The method of claim 5 , wherein the polysaccharide is reduced in size by oxidative cleavage.

8 . The method of claim 1 , further comprising purifying the conjugate by hydrophobic interaction chromatography.

9 . The method of claim 1 , further comprising purifying the conjugate by mixed mode resin chromatography.

10 . The method of claim 1 , wherein the conjugate has a weight average molecular weight ranging from 300 kDa to 1500 kDa.

11 . The method of claim 1 , wherein the conjugate is a population comprising molecules having a molecular weight in the range of 700 kDa to 1400 kDa.

12 . The method of claim 1 , wherein the conjugate is a population comprising molecules having a molecular weight in the range of 800 kDa to 1300 kDa.

13 . The method of claim 1 , wherein the conjugate comprises one or more modifications selected from (i) a primary hydroxyl at the 7 position, (ii) a (2-hydroxy) ethoxy at the reducing end, and (iii) a secondary amine conjugated to the carrier protein, wherein the modifications are present at no less than 25 nmol/mg polysaccharide.

14 . The method of claim 1 , wherein the conjugate has a MenC capsular polysaccharide to carrier protein mass ratio of 0.3 to 1.1.

15 . The method of claim 1 , wherein the conjugate has a MenC capsular polysaccharide to carrier protein mass ratio of 0.4 to 0.8.

16 . The method of claim 1 , wherein the O-acetylation level of the polysaccharide ranges from 0.8 μmol/mg polysaccharide to 1.4 μmol/mg polysaccharide.

17 . The method of claim 5 , wherein the molecular weight is determined by multi-angle light scattering (MALS).

18 . The method of claim 1 , wherein reductive amination comprises reducing imines to amines using a cyanoborohydride, pyridine borane (C 5 H 8 BN), or picoline borane complex (C 6 H 7 N·BH 3 ).

19 . The method of claim 1 , further comprising converting unreacted aldehydes in the conjugate to alcohols with a reducing reagent, wherein the reducing reagent is a borohydride.

20 . The method of claim 1 , wherein periodate is added to a concentration of 1 mM to 4 mM to activate the polysaccharide.

Assignments (2)

Continuity (7)

References Cited (302)