Cell wall polysaccharides (C-poly) are impurities present in purified pneumococcal polysaccharide (PnPs) that can lower the immunogenicity of pneumococcal conjugate vaccines (PCV) and potentially cause an undesired response. Despite recent studies conducted with NMR and size-exclusion chromatography (SEC), it remains a challenge to identify and quantitate C-poly at the low levels present in multivalent PCV. In this study, we have utilized chemical degradation followed by mass spectrometry (MS) to generate a unique oligosaccharide marker for C-poly to be able to quantitate it in mixtures of PnPs.
C-poly was subjected to several chemical degradation conditions by varying the choice of acid/base, its concentration, reaction time and reaction temperature to partially degrade it to a unique oligosaccharide. Hydrophilic interaction liquid chromatography (HILIC) coupled to negative electrospray ionization mass spectrometry ((-) ESI MS) was utilized to detect the potential C-poly markers. Confirmation of a unique oligosaccharide marker for C-poly was determined by subjecting other PnPs to the same degradation conditions and comparing these samples' mass spectra to one another.
After C-poly was subjected to hydrolysis with 1.5 M sodium hydroxide (NaOH), 2.5M NaOH, 1M trifluoroacetic acid (TFA), 1 M acetic acid (CH3COOH), 1.5 M hydrochloric acid (HCI), and 1 M HCI, no unique markers were identified. However, methanolysis with 3M hydrochloric acid in methanol shows great potential as the degradation reagent to identify a unique marker. The potential marker was ions of m/z 306, which eluted at a retention time of 2.1 mins. Unfortunately, this marker was observed in three of the eleven serotypes tested.
One of the limitations of this method is that C-poly may exist in the PnPs standard in various
concentrations which would impact our ability to identify a unique marker for C-poly. Therefore, the marker could be unique, however, further tests to confirm this will be MS/MS and additional hydrolysis conditions may be tested.

Nathan Edwards

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