Quantification of polysaccharide in Haemophilus influenzae type b conjugate and polysaccharide vaccines by high-performance anion-exchange chromatography with pulsed amperometric detection

Abstract

A sensitive method for the quantification of polysaccharide (PS) in Haemophilus influenzae type b (Hib) conjugate and PS vaccines has been developed. It is based on measurement of the Hib PS subunit after depolymerization of the PS in sodium hydroxide to produce the subunit, which is characterized by chemical composition and 31 P n.m.r. analyses as ribitol-ribose-phosphate. The Hib vaccines were first treated with 0.1 M sodium hydroxide. The Hib PS subunit in the treated vaccines was then analysed directly by high-performance anion-exchange chromatography using a Carbo Pak P A-1 column, and quantified by pulsed amperometric detection. The PS contents of three conjugate vaccines and three PS vaccines from different manufacturers were determined. Their values were in the expected ranges. This method is particularly useful for vaccines with a sugar stabilizer such as lactose which would interfere with the colorimetric orcinol assay currently used for determination of the PS. The method can measure 0.1 μg of PS and its sensitivity is at least 30-fold higher than that of the orcinol assay. It may be used for stability studies of conjugate vaccines since a breakdown as low as 5% of the PS from the PS-protein conjugates would be detected.