Application of capillary ion electrophoresis and ion chromatography for the determination of O-acetate groups in bacterial polysaccharides.
Many bacterial polysaccharides possess O-linked acetate groups as constituents of their repeating units which often can serve as immunological determinants. It is therefore important to develop analytical methods for process monitoring as well as product characterization when such O-acetylated polysaccharides are used as components of vaccines. This is the case in a polysaccharide conjugate vaccine under development for treatment of diseases caused by Streptococcus pneumoniae. An ion chromatographic (IC) method utilizing suppressed conductivity detection (SCD) was developed to quantitatively measure O-acetate groups in the capsular polysaccharides from S. pneumoniae types 18C and 9V following hydrolytic release of O-acetate from the polysaccharide backbones using 2 mM sodium hydroxide. IC was carried out using an OmniPac PAX-500 column and 0.98 mM NaOH in 2% methanol as the mobile phase. Capillary ion electrophoresis (CIE) with indirect photometric detection was evaluated as an alternative method. The CIE method utilized a 72 cm x 75 microns I.D. fused-silica capillary and an electrolyte composed of 5 mM potassium hydrogenphthalate, 0.5 mM tetradecyltrimethylammonium bromide, and 2 mM sodium tetraborate, pH 5.88. A comparison of CIE and IC-SCD in terms of reproducibility, accuracy, linearity, and sensitivity will be presented.
Department of Virus and Cell Biology, Merck Research Laboratories, West Point, PA 19486.
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D000085:Acetates / Q000032:analysis*; D002240:Carbohydrate Sequence; D002853:Chromatography, Liquid / Q000379:methods*; D004586:Electrophoresis / Q000379:methods*; D006868:Hydrolysis; D007477:Ions; D008969:Molecular Sequence Data; D011135:Polysaccharides, Bacterial / Q000737:chemistry*; D015203:Reproducibility of Results; D013056:Spectrophotometry, Ultraviolet; D013296:Streptococcus pneumoniae / Q000737:chemistry
D000085:Acetates; D007477:Ions; D011135:Polysaccharides, Bacterial