In the past, we developed an anion-exchange HPLC method to specifically quantify polyribosylribitol phosphate content in vaccines containing Hib conjugates (Vaccine 12:700-706,1994). This HPLC method has been adapted and used by two vaccine manufacturers in quality control of their licensed Hib conjugate vaccines. Because an HPLC analysis provides high sensitivity compared to a classical colorimetric assay, we have further explored using the HPLC method to quantify sugar and non-sugar components in other bacterial polysaccharide and conjugate vaccines. Besides monosaccharides, O-acetyl and phosphate are non-sugar components present in many bacterial polysaccharide (PS) and PS-conjugate vaccines. The O-acetyl group is an important immunogenic and functional epitope in certain PSs such as meningococcal A PS, pneumococcal 9V PS, and Salmonella typhi Vi PS. In order to insure manufacturing consistency of these and many other PSs and their PS-conjugate vaccines, quality control requires O-acetyl and phosphate contents to determined and meet their specifications. Thus, we have developed an HPLC method to quantify the O-acetyl and phosphate contents of bacterial PSs. The PSs were first treated with mild alkali or aqueous HF to hydrolyze O-acetyl and phosphate groups respectively. The acetate or phosphate released in the hydrolysates was then subjected to an anion-exchange HPLC analysis and monitored with a conductivity detector. We have quantified the O-acetyl content of meningococcal A, C, Y, and W-135 PSs, pneumococcal 9V and 18C PSs, and Salmonella typhi Vi PS. The results by the HPLC method were comparable to those obtained by the classical colorimetric assays.
