培干扰素α1b原液中残留DNA定量PCR检测法的验证  

Validation of a quantitative PCR method for determination of residual DNA in the PEGylated IFNα1b

作　　者：李珺 薛亚慧 杨宇峰 田石华 刘敏 何成 Li Jun;Xue Yahui;Yang Yufeng;Tian Shihua;Liu Min;He Cheng(No.2 Research Laboratory,Shanghai Institute of Biological Products Co.,Ltd.,Shanghai 200051,China)

机构地区：[1]上海生物制品研究所有限责任公司第二研究室,上海200051

出　　处：《国际生物制品学杂志》2024年第2期85-89,共5页International Journal of Biologicals

基　　金：上海市2020年度“科技创新行动计划”生物医药科技支撑专项项目(20S11908800)。

摘　　要：目的验证聚乙二醇修饰的干扰素(培干扰素)α1b原液中残留DNA检测的实时荧光定量PCR(quantitative PCR,qPCR)。方法提取4批培干扰素α1b原液的残留DNA,使用qPCR检测试剂盒进行扩增反应,绘制标准曲线,建立残留DNA的qPCR检测方法,对建立的方法进行线性、重复性、准确性、精密度、定量限和耐用性的验证,并与探针杂交法进行比较。结果qPCR检测在300 pg/μl~30 fg/μl浓度范围内线性关系良好。6次检测相对标准偏差为7.45%;加标回收率为95.67%~129.28%;不同人员检测相对标准偏差小于30%;定量限为30 fg/μl。不同孵育温度条件下检测结果无明显差异,耐用性良好。探针杂交法与qPCR的检测结果均小于中国药典2020年版规定的10 ng/剂。结论qPCR检测培干扰素α1b原液中DNA残留具有良好的线性、重复性、准确性、精密度和耐用性,适用于该项检测。Objective To validate real-time fluorescence quantitative PCR(qPCR)method for detection of residual DNA in polyethylene glycol modified(PEGylated)IFNα1b bulk.Methods Residual DNA was extracted from 4 batches of IFNα1b bulk respectively,and was amplified using qPCR detection kit.Standard curve was drawn,and the qPCR detection method was established.The linearity,repeatability,accuracy,precision,quantification limit and durability of the qPCR method was verified.The qPCR method was compared with the probe hybridization method.Results The qPCR method had good linearity within the concentration range of 300 pg/μl-30 fg/μl.The relative standard deviation(RSD)of 6 test results was 7.45%.The recovery rates for spiking was 95.67%-129.28%.RSD of results by different operators was less than 30%.The minimum quantitative limit was 30 fg/μl.There was no significant difference in the detection results at various incubation temperatures.The results of probe hybridization method and qPCR method were both less than 10 ng/dose,the specification by Chinese pharmacopoeia 2020 edition.Conclusion The qPCR method has good linearity,repeatability,accuracy,precision and durability,and is suitable for detection of residual DNA in PEGylated IFNα1b bulk.

关 键 词：实时荧光定量聚合酶链反应 残留DNA 探针杂交法 方法验证 质量控制 

分 类 号：R392-3[医药卫生—免疫学]