Abstract: The invention is a novel chemical coupling methodology for the synthesis of a stable polysaccharide-protein conjugates as the immunogenic component for vaccines. A covalent bond is formed between polysaccharide and protein in the dry state in the absence of water and oxygen. A polysaccharide antigen is covalently linked to the protein by activating the polysaccharide with periodate to introduce aldehyde groups into the polysaccharide, lyophilizing an aqueous mixture of a protein and activated polysaccharide, sealing the dry lyophilized mixture in a vessel under vacuum or inert gas and then incubating the sealed vessel at an elevated temperature.

Abstract: It has been discovered that facile glycation of proteins can be achieved by colyophilization of a protein with a reducing sugar, subjecting the lyophilized mixture to a vacuum (10 to 50 millitor) and incubating at an elevated temperature (50 to 100° C.) for 1 to 24 h. A stable ketoamine derivative is formed with amino groups in the protein and no advanced glycation end products (browning reaction) are observed, as is the case with aqueous glycation procedures. Another novel feature is that the in vacuo glycation reaction takes place with the protonated amine and not the deprotonated amine as is believed to be the case for aqueous glycation reactions. Advantage can be taken of the in vacuo glycation reaction to achieve facile covalent cross-linking of proteins by lyophilizing protein or proteins with compounds containing two or more reducing sugars separated by a linker.