Development and Validation of an NMR-Based Identity Assay for Bacterial Polysaccharides

• Published in: Analytical biochemistry 2000-03, Vol.279 (2), p.226-240
• Main Authors: Abeygunawardana, Chitrananda, Williams, Thomas C., Sumner, James S., Hennessey, John P.
• Format: Article
• Language: English
• Subjects: bacterial polysaccharide; Bacterial Vaccines - analysis; Bacterial Vaccines - chemistry; Carbohydrate Sequence; Deuterium Oxide; Evaluation Studies as Topic; Hydrogen - chemistry; identity assay; Magnetic Resonance Spectroscopy - methods; Magnetic Resonance Spectroscopy - statistics & numerical data; method development; Molecular Sequence Data; NMR; Pneumococcal Vaccines; Polysaccharides, Bacterial - analysis; Polysaccharides, Bacterial - chemistry; Reproducibility of Results; Serotyping; Streptococcus pneumoniae - chemistry; Streptococcus pneumoniae - classification; Temperature; validation
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1006/abio.1999.4470

A method utilizing NMR spectroscopy has been developed to confirm the identity of bacterial polysaccharides used to formulate a polyvalent pneumococcal polysaccharide vaccine. The method is based on 600 MHz proton NMR spectra of individual serotype-specific polysaccharides. A portion of the anomeric region of each spectrum (5.89 to 4.64 ppm) is compared to spectra generated for designated reference samples for each polysaccharide of interest. The selected region offers a spectral window that is unique to a given polysaccharide and is sensitive to any structural alteration of the repeating units. The similarity of any two spectral profiles is evaluated using a correlation coefficient (ρ), where ρ ≥ 0.95 between a sample and reference profile indicates a positive identification of the sample polysaccharide. This method has been shown to be extremely selective in its ability to discriminate between serotype-specific polysaccharides, some of which differ by no more than a single glycosidic linkage. Furthermore, the method is rapid and does not require extensive sample manipulations or pretreatments. The method was validated as a qualitative identity assay and will be incorporated into routine quality control testing of polysaccharide powders to be used in preparation of the polyvalent pneumococcal vaccine PNEUMOVAX 23. The specificity and reproducibility of the NMR-based identity assay is superior to the currently used colorimetric assays and can be readily adapted for use with other bacterial polysaccharide preparations as well.