Quantitative determination of capsular polysaccharide, C-polysaccharide, phosphorus of carbohydrate antigens from Streptococcus pneumoniae by quantitative NMR using a single internal standard

Pneumococcal vaccine plays a key role in preventing diseases such as pneumonia and meningitis caused by Streptococcus pneumoniae (S. pneumoniae). Capsular polysaccharide, C-polysaccharide and phosphorus content are important indicators for quality control of polysaccharide antigens in vaccine development and production. In this study, a quantitative ¹H NMR and ³¹P NMR method based on a single internal standard hexamethylphosphoramide (HMPA) was developed to achieve simultaneous determination of capsular polysaccharide, C-polysaccharide and phosphorus content in 6A, 6B, 18C, 19A, 19F and 23F S. pneumoniae polysaccharide antigens. Using the internal reference comparison method, the effect of solubility of polysaccharide on quantitative ¹H NMR determination was investigated. It was found that the determination results of quantitative ¹H NMR were affected by the viscosity and concentration of polysaccharide solution. It was found that high viscosity polysaccharides at 3−15 mg/mL and low viscosity polysaccharides at 5−25 mg/mL were the optimal detection solution concentration range. This “one internal standard three quantitative” NMR method has good precision, specificity and accuracy, and provides a valuable new strategy for the quality control of pneumococcal vaccine.