5型肺炎球菌荚膜多糖无乙醇无苯酚纯化工艺的建立

Develop an alcohol-free and phenol-free purification process for capsular polysaccharide of Streptococcus pneumoniae type 5

目的建立一种无乙醇无苯酚的5型肺炎球菌荚膜多糖纯化工艺,并进行工艺验证。方法5型肺炎球菌发酵培养液采用脱氧胆酸钠法去除蛋白质,再利用层析法去除核酸等杂质,并对去除蛋白质工艺、层析工艺分别进行优化。采用优化后的方法纯化3批5型肺炎球菌发酵培养液,并对新工艺进行验证。结果去除蛋白质工艺的最适参数为:脱氧胆酸钠体积分数为0.5%,pH 4.3;层析工艺的最佳条件为:氯化钠浓度200 mmol/L,pH 8.0,上样流速150 cm/h,多糖质量浓度0.50 mg/mL。在上述工艺条件下,纯化的荚膜多糖各项指标均符合《欧洲药典》9.0版标准。结论该工艺稳定、可靠,可用于大规模生产,相比于传统的乙醇苯酚纯化工艺具有先进性。

Objective To develop and validate an alcohol-free and phenol-free purification process for capsular polysaccharide of Streptococcus pneumoniae type 5.Methods Protein was removed by sodium deoxycholate from Pneumococcus type 5 fermentation broth media,and nucleic acid was removed by a chromatography performance,by which two processes were respectively optimized.Three batches of fermentation broth were purified by the optimized methods,and carried out in verification of the new process.Results The optimal parameters for protein removal process were with concentration of 0.5%sodium deoxycholate and pH 4.3.The optimum conditions for chromatography process were with concentration of 200 mmol/L sodium chloride and pH 8.0,sample flow rate of 150 cm/h and polysaccharide concentration of 0.50 mg/mL.Under the above conditions,the quality for purified capsular polysaccharide met the standard of European Pharmacopoeia Version 9.0.Conclusion The developed purification process was suitable to large-scale production,with stable and reliable.It had advantages in comparison to the traditional purification process by using ethanol and phenol.