1. Field of Invention
This invention is directed to reagents and methods of conjugating proteins to carbohydrates and, in particular, the processes and chemicals used in the manufacture of vaccines, and also the vaccines manufactured by the process.
2. Description of the Background
Vaccines that contain protein covalently linked to carbohydrate have proven remarkably successful in inducing an immune response to the carbohydrate moiety. Examples of such vaccines, known as “conjugates” are available for Haemophilus influenzae type b (e.g., ActHib, Hiberix), Neisseria meningiditis types A C W and Y (e.g., Menactra) and S. pneumoniae (e.g., Prevnar, Synflorix). In order for the protein to be linked to the carbohydrate, the latter generally needs to be activated so that it can be reacted with the protein, either directly or via a spacer (Dick, W. E. Jr and Beurret, M. Glyconjugates of bacterial carbohydrate antigens. A survey and consideration of design and preparation factors. In: Conjugate Vaccines (Eds Cruse, J. M. and Lewis, R. E.). Karger, Basel, 1989. One means of activation is through oxidation of the carbohydrate to produce aldehydes, which are then linked to lysines on protein through reductive amination. In other cases, the protein is first functionalized with hydrazides or aminooxy groups, which are subsequently reacted with aldehydes on the carbohydrate (Lees, A. Use of amino-oxy functional groups in the preparation of protein-polysaccharide (PS) conjugate vaccines United States Patent Publication No. 2005/0169941; which is incorporated by reference). Another method for activating polysaccharides is with the use of cyanogen bromide, to form a cyano-ester on the polysaccharide which is subsequently reacted with a spacer molecule such as adipic dihydrazide. The functionalized polysaccharide is then reacted with the protein. Improved methods for cyanating polysaccharides use 1-cyano-4-dimethylaminopyridine tetrafluoroborate (CDAP) (Lees, A., Producing immunogenic constructs using soluble carbohydrates activated via organic cyanylating reagents. U.S. Pat. Nos. 5,651,971; 5,693,326; and 5,849,301). CDAP allows for protein to be linked directly to the polysaccharide. CDAP can also be used to functionalize the polysaccharide with a spacer, which is subsequently linked to the protein. Hydrazide or aminooxy functionalized proteins can also be linked to CDAP activated polysaccharides (U.S. Pat. No. 5,849,301; which is incorporated by reference).