Monitoring activation sites on polysaccharides by GC-MS.

A method has been developed to determine the location and order of activation for potential saccharide antigens used in conjugate vaccine development. Saccharides were monitored for activation by sodium periodate oxidation and subsequent analysis by gas chromatography-mass spectrometry (GC-MS). Pneumococcal serotype polysaccharides 7F and 18C were evaluated as polysaccharides containing multiple potential sites for activation. Sialyllactose was used as a model oligosaccharide compound to evaluate oxidation of terminally linked sialic acids and reducing sugar residues. Oxidized saccharides were analyzed by monosaccharide composition and/or linkage analysis to elucidate specific activation of cis versus trans diols, as well as diols containing primary versus secondary alcohols, at specified levels of periodate. Samples (100-500 microg) were sequentially oxidized, reduced, methanolyzed, and derivatized in a single reaction vial for routine analysis by GC-MS.

Determination of saccharide content in pneumococcal polysaccharides and conjugate vaccines by GC-MSD.

A simple and sensitive gas chromatographic method was designed for quantitative analysis of Streptococcus pneumoniae capsular polysaccharides, activated polysaccharides, and polysaccharide conjugates. Pneumococcal serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F polysaccharide or conjugate were subjected to methanolysis in 3N hydrochloric acid in methanol followed by re-N-acetylation and trimethylsilylation. Derivatized samples were chromatographed and detected using gas chromatography with mass selective detector. Gas chromatographic results were compared with colorimetric values with agreement of 92 to 123% over the range of all samples tested. Monosaccharides released during methanolysis included hexoses, uronic acids, 6-deoxy-hexoses, amino sugars, and alditols. Quantitative recovery of monosaccharides was achieved for all serotypes by the use of a single methanolysis, derivatization, and chromatography procedure. Response factors generated from authentic monosaccharide standards were used for quantitation of pneumococcal polysaccharides and conjugates with confirmation of peak assignments by retention time and mass spectral analysis. This method allows saccharide quantitation in multivalent pneumococcal vaccine intermediates and final drug products with low-level detection (10 pg) and peak purity.