抗CD52单克隆抗体外源DNA残留量检测方法的建立及验证

Establishment and validation of a method for determination of residual DNA in anti-CD52 monoclonal antibody

目的建立检测抗CD52单克隆抗体原液中残余DNA的方法,并对该方法进行验证。方法利用CHO Host Cell DNA Extraction&Amplification Kit,采用qPCR法检测抗CD52单克隆抗体中宿主细胞残留DNA含量,并进行方法的专属性、线性、准确度、精密度和耐用性验证。结果该方法可以准确定量检测CHO细胞残留DNA含量。专属性验证试验中对照制剂无特异扩增曲线,而CHO细胞上清有明显的扩增曲线;5次试验中标准曲线相关系数R^2均≥0.98,表明该方法线性良好;准确度验证试验中所有试验的加标回收率均在70%~130%范围内;精密度验证试验中所有试验检测结果的相对标准偏差(RSD)均不大于30%;耐用性验证试验中,Proteinase K不同消化温度下检测结果的CV为0.85%,不同稀释倍数的供试品加标回收率在70%~130%范围内,不同稀释倍数外源DNA检测结果CV为4.71%。结论该方法专属性、准确度、精密度、线性和耐用性均符合要求,可采用该试剂盒qPCR法检测外源DNA残留量。

Objective To establish and validate a method for determination of residual host cell DNA in anti-CD52 monoclonal antibody. Methods The residual exogenous DNA was determined by qPCR using CHO Host Cell DNA Extraction Amplification Kit. The method was verified for specificity, linearity, accuracy, precision and durability.Results The residual DNA of CHO cells was accurately quantified by the developed method. No specific amplification curve was obtained by specificity validation test on control reagent, while obvious amplification curve in that on CHO cell supernatant. All the correlation coefficients（R^2） in five tests were not less than 0. 98, indicating high linearity of the method. All the spiked recovery rates in accuracy validation test were 70% ~ 130%. All the relative standard deviations（RSDs）of results of precision validation test were not more than 30%. However, in durability test, the CV of test results of samples digested with protease K at various temperatures was 0. 85%, while the spike recovery rates of samples at various dilutions were 70% ~ 130%, and the CV of determination results of exogenous DNA at various dilutions was 4. 71%.Conclusion The specificity, accuracy, precision, linearity and durability of the method met the relevant requirements.The method was suitable for determination of residual exogenous DNA of anti-CD52 monoclonal antibody by qPCR.