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 single repeating 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 repeating unit 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 vaccines. In addition, the sensitivity of PAD detection allows the HPLC method to quantify degraded free PRP from the conjugates for stability studies of vaccines. Besides the Hib conjugate vaccine, other bacterial PS-conjugate vaccines are being investigated under PLAs or LNDs. Currently, we are exploring HPLC analysis as a method to quantify the PS content of meningococcal A and C conjugate vaccines. Meningococcal A or C PS is first depolymerized in mild acid to their single repeating units, N-acetyl-mannosamie-6-P or sialic acid respectively. The repeating units in the hydrolysates are then subjected to HPLC analyses. When meningococcal A or C PS was hydrolyzed in mild acid and their hydrolysates were subjected to gel-filtration on Sephadex G15 or BioGel P2 chromatography respectively, about 70 -80 % of material in the hydrolysates was found to be single repeating units. Preliminary results indicate that the PS content of meningococcal C-PS conjugates can be quantified by the HPLC method.
