Method development for determination of free polysaccharide content in Streptococcus pneumoniae type 19A polysaccharide conjugate

Objective To develop a method for determination of free polysaccharide content in Streptococcus pneumoniae type 19A polysaccharide (19APS) conjugate. Methods Tetanus toxoid (TT) solutions of different concentrations were precipitated using 1, 3 and 5 g/L sodium deoxycholate (DOC) (pH7.3), respectively, under certain conditions. The concentration ranges of carrier protein TT precipitated with different concentrations of DOC were determined. The conjugated 19APS (19APS-TT) and free 19APS were separated using 1 g/L DOC, and free PS contents in supernatant were detected. The accuracy and precision of the DOC precipitation method were validated. Results When 19APS-TT was precipitated using 1 and 3 g/L DOC, the TT concentrations should be controlled in ﹤150 and ﹤200 μg/ml, respectively. The DOC precipitation method had good accuracy and precision. The adding recovery rates of PS were 94.0%-107.2%, and the relative standard deviations of the DOC precipitation method were all ﹤4%. Conclusion The method for determination of free polysaccharide content in 19APS-TT is developed successfully. Key words: Streptococcus pneumoniae; Polysaccharides, bacterial; Chemical precipitation