动态浊度法检测细菌内毒素含量在重组铜绿假单胞菌外毒素A制备中的应用

Application of bacterial endotoxin content detection by dynamic turbidity method in the preparation of recombinant Pseudomonas aeruginosa exotoxin A

目的:应用动态浊度法监测重组铜绿假单胞菌外毒素A(r EPA)制备各阶段料液中的细菌内毒素含量,为其工艺优化和质量控制提供依据。方法:参考应用《中华人民共和国药典》2015年版三部载录的动态浊度法测定细菌内毒素含量,对该方法的专属性、精密度、重现性、准确性和标准曲线的可靠性进行了验证,并将该方法应用于r EPA制备各阶段料液中细菌内毒素含量的测定及细菌内毒素去除效果的评价。结果:该方法具有较好的专属性、精密度、重现性、准确性。结论:经方法验证显示该方法的专属性、精密度、重现性、准确性等技术参数达到了相关验证要求,结果稳定可靠。现有r EPA制备工艺能稳定、有效地去除细菌内毒素,纯化后r EPA中细菌内毒素含量较低。本研究为r EPA制备过程中细菌内毒素去除效果的评价提供了准确的参考数据。

Objective ： To monitor the bacterial endotoxin content in each phase of recombinant Pseudomonas aeruginosa exotoxin A （rEPA） by dynamic turbidity method, providing a basis for process optimization and quality control. Methods： The content of bacterial endotoxin was determined by the dynamic turbidity method in Chinese Pharmacopoeia （volume III, 2015 edition）. Then, the specificity, precision, reproducibility, accuracy, and reliability of the standard curve of the method were validated. The method was applied to the determination of bacterial endotoxin in each phase of rEPA, and the evaluation of bacterial endotoxin removal. Results： This method had a satisfactory specificity, precision, reproducibility, and accuracy. Conclusion： This method has special properties, precision, reproducibility, and accuracy, and the results are stable and reliable. Existing rEPA preparation process can stably and effectively remove bacterial endotoxin, and lower the content of bacterial endotoxin in rEPA purified. Our study provides an accurate reference data for the evaluation of bacterial endotoxin removal in the process of rEPA preparation.