A Multinuclear 1H and 31P NMR Approach for Quantification of Pneumococcal Cell-Wall Polysaccharide in Vaccine Development

Abstract Capsular polysaccharides are the principal constituents of S. pneumoniae capsules and major determinants of bacterial virulence. As key antigenic targets, they represent the foundation for pneumococcal vaccine development. Cell wall polysaccharides (CWPSs) commonly persist as a process-related impurity during fermentation and purification and therefore require reliable quantification throughout vaccine manufacturing. In this work, a multinuclear quantitative NMR strategy based on 1H and 31P detection was developed for the determination of CWPSs in purified pneumococcal polysaccharide preparations. The analytical workflow was evaluated using a representative panel of ten pneumococcal serotypes selected to encompass different polysaccharide structural classes, charge characteristics, and CWPS variants. Both NMR methods were demonstrated to be fit for the purpose, exhibiting specificity, linearity, and accuracy across the investigated concentration range. The complementary use of 1H NMR and 31P NMR enabled reliable CWPS quantification while providing additional structural information and discrimination between CWPS variants. The proposed multinuclear NMR approach represents a robust, selective, and quantitative analytical platform for monitoring CWPS impurities during pneumococcal polysaccharide vaccine development and quality control.