Development and application of a detection method for monosaccharide composition of pneumococcal capsular polysaccharides
10.13200/j.cnki.cjb.004633
- VernacularTitle:肺炎球菌荚膜多糖单糖组成检测方法的建立及应用
- Author:
Xiangyu ZHOU
1
Author Information
1. Fosun Adgenvax (Chengdu) Biopharmaceutical Co., Ltd., Chengdu 610200, Sichuan Province, China
- Publication Type:Journal Article
- Keywords:
Streptococcus pneumonia;
Capsular polysaccharides;
Polysaccharide hydrolysis;
Monosaccharide composition;
Anion-exchange chromatography;
Pulsed amperometric detection;
Pre-column derivatization;
High-performance liquid chromatography(HPLC)
- From:
Chinese Journal of Biologicals
2026;39(01):85-92
- CountryChina
- Language:Chinese
-
Abstract:
Objective To develop a method for detecting the monosaccharide composition of different serotypes of pneumococcal capsular polysaccharides(PnPs), in order to provide a reliable detection method for the quality control of pneumococcal PnPs vaccines and polysaccharide-protein conjugate vaccines.Methods According to the structural differences of different serotypes of PnPs, trifluoroacetic acid(TFA), hydrofluoric acid(HF) + TFA, and anhydrous hydrogen chloride/methanol(HCl/Meth) + TFA were used for hydrolysis to release the monosaccharide components, respectively. The high performance anionexchange chromatography with pulsed amperometric detection(HPAEC-PAD) without derivatization and 1-phenyl-3-methyl-5-pyrazolone(PMP) pre-column derivatization high-performance liquid chromatography(HPLC) were used for detection to compare the differences in the separation performance for acidic monosaccharides, neutral monosaccharides and alkaline monosaccharides between different detection methods. Different hydrilysis methods(TFA, HF + TFA, HCl/Meth + TFA, and HCl) were used to hydrolyze monosaccharides, and the stability of each monosaccharide component was evaluated by PMP pre-column derivatization HPLC.Results The three hydrolysis methods could effectively break the glycosidic bonds of different serotypes of PnPs and release various monosaccharide components. The PMP derivatization combined with a C18 column exhibited the best separation performance, enabling baseline separation of 10 neutral, acidic, and alkaline monosaccharides, suitable for the comprehensive analysis of all serotypes. In the HPAEC-PAD method, PA10 column had good separation performance for most monosaccharides, but some glycosamines were not well separated. MA1 column could specifically detect ribitol and glycerol components which cannot be derivatized, while could not elute uronic acids. HF and TFA treatments had little effect on the recovery of most monosaccharides. However, HCl/Meth treatment could easily lead to the degradation of hexosamine,while HCl treatment alone leaded to the degradation of neutral monosaccharides and uronicacids.Conclusion PMP pre-column derivatization HPLC has good separation performance and strong universality, and is a reliable method to detect the monosaccharide composition of different serotypes of PnPs. HPAEC-PAD method has unique advantages in detecting specific components such as ribitol. The combination of the two forms a complete method system for the quality control of pneumococcal polysaccharide conjugate vaccines.
