Glycoconjugation process
View Patent ↗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. A method of making a glycoconjugate comprising a capsular polysaccharide from Group B Streptococcus (GBS) conjugated to a carrier protein, comprising the steps of:
a) reacting said capsular polysaccharide with a stable nitroxyl radical compound and an oxidant 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 to produce an activated capsular polysaccharide,
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 a carrier protein comprising one or more amine groups.
2. The method 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-Iodoacetamido)-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.
3. The method of claim 1 , wherein said oxidant is selected from the group consisting of N-ChloroSuccinimide, N-Bromosuccinimide, N-Iodosuccinimide, 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.
4. The method of claim 1 , wherein the degree of oxidation of the activated capsular polysaccharide ranges from 6 to 14.
5. The method of claim 1 , wherein the carrier protein is CRM197.
6. The method of claim 1 , wherein prior to step a), the saccharide is hydrolyzed to a molecular weight ranging from 100 to 400 kDa.