Glycoconjugation process
The present disclosure relates generally to methods of preparing glycoconjugates containing a saccharide conjugated to a carrier protein by use of stable nitroxyl radical related agent/oxidant as an oxidizing agent, to immunogenic compositions comprising such glycoconjugates, and to methods for the use of such glycoconjugates and immunogenic compositions.
1. An immunogenic composition comprising a glycoconjugate comprising a Pn-serotype 12F capsular polysaccharide conjugated to a carrier protein, wherein the amount of free Pn-serotype 12F polysaccharide in the composition is less than 35% after 120 days from when it was prepared, and wherein said glycoconjugate is prepared by a process comprising:
a) reacting a Pn-serotype 12F capsular polysaccharide with a stable nitroxyl radical compound and an oxidant, to produce an activated capsular polysaccharide, wherein said oxidant is a molecule bearing a N-halo moiety which selectively oxidizes primary alcohols in the presence of a nitroxyl radical compound to generate aldehyde groups, wherein said stable nitroxyl radical compound is a molecule bearing a TEMPO or a PROXYL (2,2,5,5-tetramethyl-1-pyrrolidinyloxy) moiety, having the ability to selectively oxidize primary alcohols in the presence of an oxidant, to generate aldehyde groups without affecting secondary hydroxyl groups; and
b) reacting the activated capsular polysaccharide with the carrier protein comprising one or more amine groups.
2. The immunogenic composition of claim 1 , wherein the amount of free Pn-serotype 12F polysaccharide is less than 30% after 120 days from when it was prepared.
3. The immunogenic composition of claim 1 , wherein said nitroxyl radical compound is selected from the group consisting of TEMPO, 2,2,6,6-Tetramethyl-4-(methylsulfonyloxy)-1-piperidinooxy, 4-Phosphonooxy-TEMPO, 4-Oxo-TEMPO, 4-Methoxy-TEMPO, 4-Isothiocyanato-TEMPO, 4-(2-lodoacetamido)-TEMPO free radical, 4-Hydroxy-TEMPO, 4-Cyano-TEMPO, 4-Carboxy-TEMPO, 4-(2-Bromoacetamido)-TEMPO, 4-Amino-TEMPO, and 4-Acetamido-2,2,6,6-tetramethylpiperidine 1-oxyl.
4. The immunogenic composition of claim 1 , wherein said oxidant is selected from the group consisting of N-Chlorosuccinimide, N-Bromosuccinimide, N-lodosuccinimide, Dichloroisocyanuric acid, 1,3,5-trichloro-1,3,5-triazinane-2,4,6-trione, Dibromoisocyanuric acid, 1,3,5-tribromo-1,3,5-triazinane-2,4,6-trione, Diiodoisocyanuric acid and 1,3,5-triiodo-1,3,5-triazinane-2,4,6-trione.
5. The immunogenic composition of claim 1 , wherein the carrier protein is a toxin from tetanus, diphtheria, pertussis, Pseudomonas, E. coli, Staphylococcus or Streptococcus.
6. The immunogenic composition of claim 1 , wherein the carrier protein is CRM 197 .
7. The immunogenic composition of claim 1 further comprising a pharmaceutically acceptable excipient, carrier, or diluent.
8. The immunogenic composition of claim 1 , further comprising an additional antigen.
9. The immunogenic composition of claim 8 , wherein the additional antigen comprises a protein antigen or a glycoconjugate of a capsular polysaccharide derived from S. pneumonia.
10. The immunogenic composition of claim 9 , wherein the additional antigen comprises a glycoconjugate of a capsular polysaccharide selected from Pn-serotypes 1, 4, 5, 6A, 6B, 7F, 8, 9V, 11A, 14, 15B, 18C, 19A, 19F, 22F, and 23F capsular polysaccharides.
11. The immunogenic composition of claim 8 , wherein the additional antigen comprises a protein antigen or a glycoconjugate of a capsular polysaccharide derived from N. meningitidis.
12. The immunogenic composition of claim 11 , wherein the additional antigen comprises a glycoconjugate of a capsular polysaccharide selected from serotypes A, C, W135 and Y capsular polysaccharides.
13. The immunogenic composition of claim 11 , wherein the additional antigen comprises a glycoconjugate of serotypes X capsular polysaccharide.
14. The immunogenic composition of claim 8 , wherein the additional antigen comprises a glycoconjugate of a capsular polysaccharide derived from Group B Streptococcus (GBS).
15. The immunogenic composition of claim 14 , wherein the additional antigen comprises a glycoconjugate of a capsular polysaccharide selected from GBS serotypes Ia, Ib, II, III, IV, V, VI, VII and VIII.
16. The immunogenic composition of claim 1 , further comprising an adjuvant.
17. The immunogenic composition of claim 16 , wherein the adjuvant is an aluminum-based adjuvant.
18. The immunogenic composition of claim 17 , wherein the aluminum-based adjuvant is selected from the group consisting of aluminum phosphate, aluminum sulfate, and aluminum hydroxide.
19. A method of ameliorating a bacterial infection, disease or condition in a subject, comprising administering to the subject an immunologically effective amount of an immunogenic composition of claim 1 .
20. The method of claim 19 , wherein the infection, disease or condition is associated with S. pneumoniae bacteria.
21. A method of inducing a protective immune response in a subject, comprising administering to the subject an immunologically effective amount of an immunogenic composition of claim 1 .