Quantitative determination of C-polysaccharide in Streptococcus pneumoniae capsular polysaccharides by use of high-performance anion-exchange chromatography with pulsed amperometric detection

Capsular polysaccharides of Streptococcus pneumoniae are used to formulate polyvalent pneumococcal vaccines. A sensitive method, using high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD), has been developed to quantify the contamination of pneumococcal capsular polysaccharides (PnPs) with the C-polysaccharide (C-Ps). As this polysaccharide is highly immunogenic, and since anti C-Ps antibodies are not protective, the need to monitor and reduce its level is of uppermost importance. The method is based on the quantification by HPAEC-PAD of ribitol, which is released by a two-step hydrolysis of the PnPs using aqueous hydrofluoric acid (HF) followed by trifluoroacetic acid hydrolysis (TFA). This simple method has been shown to provide both qualitative and quantitative information about the purity of polysaccharide preparations.