大肠杆菌来源重组霍乱毒素B亚基蛋白细菌内毒素检测方法探讨

Study on method for detection of bacterial endotoxin in recombinant cholera toxin B subunit protein derived from Escherichia coli

目的:通过动态显色法建立大肠杆菌来源的重组霍乱毒素B亚基蛋白质(CTB)细菌内毒素检测方法,探讨如何检测大肠杆菌来源重组蛋白质表达体系细菌内毒素残留量。方法:以大肠杆菌来源的重组CTB为研究对象,按照《中华人民共和国药典》2020年版四部1143细菌内毒素检查法之动态显色法要求,参考《中华人民共和国药典》2020年版四部9099分析方法确认指导原则及《中华人民共和国药典》2020年版四部9101分析方法验证指导原则对其进行品种适应性验证及细菌内毒素含量检测。结果:标准品内毒素含量在0.005~5.0 EU·mL^(-1)范围内,每次标准曲线相关系数(r)均>0.980,标准曲线可靠性验证通过,有效测定范围为0.005~5.0 EU·mL^(-1);4 ng·mL^(-1)供试品浓度添加标准曲线中间浓度已知量的细菌内毒素标准品(0.5 EU·mL^(-1))后,3批样品对应外加细菌内毒素回收率为94%,98%,94%,均在50%~200%范围,且均接近于100%;外加内毒素浓度在标准曲线中间浓度附近3点(1.0,0.5,0.25 EU·mL^(-1))时,4 ng·mL^(-1)供试品中测得的外加内毒素回收率均在50%~200%范围;3批样品多次测定细菌内毒素结果差异较小,同批样品测定结果均在其均值50%~200%范围内,同批样品复管及同批样品3次测定结果变异系数(CV)均<10%。结论:动态显色法适用于重组CTB大肠杆菌表达体系细菌内毒素残留量检测,为大肠杆菌来源重组蛋白质表达体系细菌内毒素残留量检测合适的方法。

Objective:To establish a bacterial endotoxin detection method for recombinant cholera toxin B subunit protein(CTB)derived from Escherichia coli by kinetic chromogenic assay,and discuss how to detect bacterial endotoxin residues in the expression system of the recombinant protein derived from Escherichia coli.Methods:Recombinant CTB derived from Escherichia coli was used as the research object.The requirements of the kinetic chromogenic assay 1143 for bacterial endotoxin in section 4 of“Chinese Pharmacopoeia”2020 edition was referred to.The variety adaptability verification and bacterial endotoxin content detection were carried out by referring to the guidelines for analytical method verification of 9099 and the guidelines for analytical method verification of 9101 in part 4 of“Chinese Pharmacopoeia”2020 edition.Results:The endotoxin content of the standard sample was within the range of 0.005 to 5.0 EU·mL^(-1),and the correlation coefficient(r)of the standard curve was greater than 0.980 each time.The reliability of the standard curve was verified,and 0.005 to 5.0 EU·mL^(-1)was a valid determination range;4 ng·mL^(-1)test sample was added with a known amount of bacterial endotoxin standard(0.5 EU·mL^(-1)),which was the middle of the standard curve.The additional bacterial endotoxin recovery rates of the three batches of samples were 94%,98%and 94%,all within the range of 50%to 200%of pharmacopoeia requirements,and were close to 100%;when the concentration of spiked endotoxin was at three points near the middle of the standard curve(1.0,0.5,0.25 EU·mL^(-1)),the recovery rates measured in the test product(4 ng·mL^(-1))were in the range of 50%~200%.There was little difference in the results of multiple bacterial endotoxin tests among the three batches of samples,and the results of the same batch of samples were all within the range of 50%~200%of their mean value.The CVs of the duplicate tube and the same batch of samples were all<10%.Conclusion:The kinetic colorimetric assay is suitable for the determination of bacterial endotoxin residues in the expression system of recombinant CTB and is suitable for the detection of bacterial endotoxin residues in recombinant protein expression systems derived from Escherichia coli.