We have developed an anion-exchange HPLC with pulsed amperometric detection (PAD) to quantify polyribosylribitol phosphate (PRP) content in vaccines containing Hib conjugates (Vaccine 12:700-706,1994). The method is based on specific depolymerization of PRP in sodium hydroxide (0.1 N) to its monomeric unit (ribitol-ribose-phosphate), separation of the monomer from all other components in a vaccine by Dionex's high-pH anion-exchange HPLC column (CarboPac PA1 or PA10), and quantification of the monomer by PAD. This method was successfully used to quantify the PRP content in the final containers of all four US licensed Hib conjugate vaccines and two combination vaccines, COMVAX and Tetramune. Our work suggests that the HPLC method can be used as a general method to quantify the PRP content in all Hib containing vaccines. In addition, the sensitivity of this method allowed Merck to quantify degraded free PRP from Hib conjugate in stability studies of their vaccines (Vaccine 17:1169-1178,1999). Besides the Hib conjugate vaccine, other bacterial polysaccharides (PS) or PS-conjugate vaccines are being investigated. O-acetyl groups are components of many bacterial PS and O-acetyl substitution on PS confers critical epitopes for certain PS such as meningococcal C PS, pneumococcal PS and O-PS of Salmonella paratyphi LPS. Currently, we are exploring HPLC analysis as a method to quantify the O-acetyl content of meningococcal A and C PS and conjugate vaccines with these PS. Meningococcal A PS (0.7 unit of 3-O-acetyl per repeating unit of N-acetyl-mannosamie-6-P) or C PS (1.2 unit of 7/8-O-acetyl per repeating unit of sialic acid) was first treated with mild akali to hydrolyze O-acetyl group. The acetate released in the hydrolysates was then subjected to HPLC analysis and monitored with a conductivity detector. Using sodium acetate in the analysis, ten ng was quantifiable and the assay was linear at least to 200 ng. Preliminary results indicated that O-acetyl content in one ug of meningococcal A PS could be quantified by the HPLC analysis. We plan to develope an HPLC method to quantify O-acetyl content in meningococcal, pneumococcal, and other bacterial PS.
