Effects of different sterilization methods on the quality of pneumococcal capsular polysaccharides
10.13200/j.cnki.cjb.004697
- VernacularTitle:不同杀菌方式对肺炎球菌荚膜多糖质量的影响
- Author:
Yuelong LI
- Publication Type:Journal Article
- Keywords:
Pneumococcus;
Capsular polysaccharides;
Sterilization;
Sodium deoxycholate(DOC);
Beta-propiolactone(BPL);
Formaldehyde
- From:
Chinese Journal of Biologicals
2026;39(05):572-577
- CountryChina
- Language:Chinese
-
Abstract:
ObjectiveTo compare the effects of different sterilization methods on the quality of pneumococcal capsular polysaccharides and screen the best sterilization method, so as to reduce the content of impurities in purified polysaccharides and improve the safety of polysaccharide vaccines.MethodsAfter fermentation culture, type 5 pneumococcus was sterilized with 0. 1% sodium deoxycholate(DOC), 0. 1% beta-propiolactone(BPL) and 1% formaldehyde at the early and middle logarithmic growth phase, the end of logarithmic phase and plateau phase, as well as the end of plateau phase and decline phase,separately. The morphology of the bacteria was observed by microscope, and the capsular polysaccharide content in the supernatant of fermentation broth at each stage was determined by rate nephelometry to calculate the polysaccharide release rate(based on the polysaccharide content after DOC sterilization). Pneumococci types 5, 7F, 14, and 15B were fermented and cultured, and sterilized using the same sterilization method in the end of plateau phase. Capsular polysaccharides were purified by ultrafiltration concentration, acid precipitation, alcohol precipitation, and secondary ultrafiltration. According to the related methods in Chinese Pharmacopoeia(VolumeⅢ, 2020 edition), the quality indicators such as the content and molecular size distribution of impurity proteins, nucleic acids, total nitrogen, phosphorus, uronic acid, hexosamine, and methylpentose in purified polysaccharides were detected.ResultsDOC sterilization caused complete lysis of the bacteria, while BPL and formaldehyde sterilization could maintain the integrity of the bacteria. In the late stage of fermentation(the end of plateau phase), the polysaccharide release rate of BPL and formaldehyde sterilization reached 60%-80%, which was higher than that in the early and middle logarithmic growth phase(20%-30%) and the end of logarithmic phase and plateau phase(40%-70%). Compared with DOC sterilization, the content of impurity proteins in the four types of purified polysaccharides obtained by BPL and formaldehyde sterilization was significantly reduced. Among them, the content of impurity proteins in type 5 polysaccharide was reduced to 4. 2%(decrease > 30%), and the content of impurity proteins in types 14, 7F and 15B polysaccharides was reduced to 0. 4%-0. 6%, 0. 4%-0. 6% and 0. 3%-0. 4%(decrease of 50%-70%), respectively. The nucleic acid content also decreased. The polysaccharide release rate of BPL sterilization(67%-70%) was higher than that of formaldehyde sterilization(48%-52%). All the quality indicators of the purified polysaccharides met the pharmacopoeia standards.ConclusionBoth BPL and formaldehyde sterilization can effectively reduce the content of impurity proteins in purified capsular polysaccharides and improve the quality. Among them, the polysaccharide release rate of BPL sterilization is better than that of formaldehyde, and it can maintain the integrity of bacteria, which is conducive to simplifying the downstream purification process.
