title: Quantification of O-acetyl, N-acetyl and phosphate groups and determination of the extent of O-acetylation in bacterial vaccine polysaccharides by high-performance anion-exchange chromatography with conductivity detection (HPAEC-CD).
abstract: The O-acetyl groups in meningococcal A and typhoid Vi polysaccharides (PSs) are functional immunogenic epitopes in humans. To quantify and determine the extent of O-acetylation in these and other bacterial vaccine PSs, anion-exchange HPLC methods have been developed for quantification of O-acetyl, N-acetyl, and phosphate groups in the PSs after these groups were hydrolyzed into anions. The O-acetylation in meningococcal A, C, Y and W-135, pneumococcal 9 V and 18C and typhoid Vi PSs were analyzed. The O-acetyl group was selectively released from a PS as acetate by mild alkaline hydrolysis in 10 or 20 mM NaOH at 37 degrees C until maximum release. The acetate in the hydrolysate was then quantified by high-performance anion-exchange chromatography with conductivity detection (HPAEC-CD) after removal of the PS by filtration with a 10,000 molecular-weight-cut-off membrane. Since the extent of O-acetylation on the PSs depends on bacterial species, strains and growth conditions, the N-acetyl group of amino-sugars, phosphate or monosaccharide components of the PSs were also quantified using HPAEC with conductivity or amperometry detection to determine the molar ratios of the O-acetyl group to these components. The average numbers of O-acetyl molecules in one PS repeating unit of the PSs were obtained from the molar ratios. Besides the O-acetyl determination, the pyruvate component in non-O-acetylated pneumococcal type 4 PS was analyzed by the HPAEC method. The HPAEC method can quantify the O-acetyl content in 0.2 microg of the meningococcal C PS and has a sensitivity at least 10 times higher than that of the colorimetric Hestrin assay. The method can be used for routine analysis of O-acetylation of PSs for quality control of vaccine PSs.
authors.affiliation: Division of Bacterial, Parasitic and Allergenic Products, Center for Biologics Evaluation and Research, FDA, 1401 Rockville Pike HFM-428, Rockville, MD 20852, USA.
authors.forename: George
authors.initials: G
authors.lastname: Kao
authors.forename: Chao Ming
authors.initials: CM
authors.lastname: Tsai
mesh_terms: D000107:Acetylation; D001428:Bacterial Vaccines / Q000737:chemistry* / Q000276:immunology*; D002852:Chromatography, Ion Exchange; D004553:Electric Conductivity; D004563:Electrochemistry; D006868:Hydrolysis; D006898:Hydroxylamines / Q000737:chemistry; D008070:Lipopolysaccharides / Q000737:chemistry* / Q000276:immunology*; D009005:Monosaccharides / Q000737:chemistry; D009345:Neisseria meningitidis / Q000737:chemistry / Q000276:immunology; D010710:Phosphates; D011773:Pyruvates / Q000032:analysis; D012015:Reference Standards; D013296:Streptococcus pneumoniae / Q000737:chemistry / Q000276:immunology
chemical_list: D001428:Bacterial Vaccines; D006898:Hydroxylamines; D008070:Lipopolysaccharides; D009005:Monosaccharides; D010710:Phosphates; D011773:Pyruvates