Glycoconjugation processes and compositions

A method of making a glycoconjugate comprising a saccharide conjugated to a carrier protein through a (2-((2-oxoethyl)thio)ethyl)carbamate (eTEC) spacer, comprising the steps of:

• a) reacting a saccharide with 1,1′-carbonyl-di-(1,2,4-triazole) (CDT) or 1,1′-carbonyldiimidazole (CDI), in an organic solvent to produce an activated saccharide;
• b) reacting the activated saccharide with cystamine or cysteamine or a salt thereof, to produce a thiolated saccharide;
• c) reacting the thiolated saccharide with a reducing agent to produce an activated thiolated saccharide comprising one or more free sulfhydryl residues;
• d) reacting the activated thiolated saccharide with an activated carrier protein comprising one or more α-haloacetamide groups, to produce a thiolated saccharide-carrier protein conjugate;
• and
• e) reacting the thiolated saccharide-carrier protein conjugate with (i) a first capping reagent capable of capping unconjugated α-haloacetamide groups of the activated carrier protein;
• and/or (ii) a second capping reagent capable of capping unconjugated free sulfhydryl residues;
• whereby an eTEC linked glycoconjugate is produced.