Quantitative assessment of C-polysaccharide in capsular polysaccharides of Streptococcus pneumoniae by 31PNMR

Capsular polysaccharides of Streptococcus pneumoniae are key components of commercially available anti-pneumococcal vaccines; meanwhile C-polysaccharide is considered an impurity. World Health Organization recommends a strict control over the presence of this biomolecule due to the possibility of introducing an undesired response. An alternative way for assessing this impurity is focused on detect the phosphocholine residues by means of quantitative 1H-NMR. This could be tricky due to the amounts of this substituent may vary generating two C-polysaccharides forms. In this work we propose an improved quantitative NMR methodology based on 31P-NMR for the quantification of C-polysaccharide on capsular polysaccharide preparations. The technique also focuses on phosphocholine but, conversely to above-mentioned methods, allows to discriminate between phosphocholine linked in different positions. The methodology was run on samples of eleven vaccine serotypes, including seven with phosphate groups. From a rational acceptance criterion of 10 wt%, the method allows to quantified from 30 μg of the impurity in 3 mg of total polysaccharide (1 wt%) with a signal/noise ratio of 16:1. Repeatability and intermediate precision evaluation showed a relative standard deviation of 3.33 % and 8.34 % respectively. Additionally, the method provides information about structural identity of phosphate contained in capsular polysaccharides and C-polysaccharide species. This constitutes a new contribution from the NMR that highlights the power of these techniques for assessing imperative parameters in carbohydrate-based vaccines.