目的 NS0宿主细胞残留DNA检测(定量PCR-Taqman探针法)方法的验证。方法 针对小鼠骨髓瘤细胞(NS0)基因组重复序列设计合成多对引物、探针,通过实验优选最佳的引物探针组合;应用所建立的前处理试剂盒(磁珠法)对供试品中宿主细胞残留DNA进行富集纯化;根据《国际人用药品注册技术协调会(ICH)》及《中国药典》通则9101的要求,应用所建立的NS0宿主细胞DNA残留检测试剂盒进行线性与范围、准确度、精密度、专属性、定量限的方法学验证。应用NS0细胞生产的单克隆抗体工艺中间品及原液,验证所建立试剂盒对实际生产样品的检测性能,并组织5个独立实验室进行协作标定。结果 NS0细胞DNA检测(Taqman法)正向引物序列: CCCCTTCAGCTCCTTGGGTA、反向引物序列:GCCTGGCAAATACAGAAGTGG、荧光探针序列: FAM-AGGGCCCCCAATGGAGGAGCT-TAMRA;NS0细胞DNA标准曲线在3 fg·μL^-1～300 pg·μL^-1内,线性良好(r²＞0.99);对6个浓度梯度检测的准确度偏差均＜15%;定量限为3 fg·μL^-1;内部参考品 DNA 标定结果为30 μg·mL^-1;精密度良好(RSD≤30%);能特异性地检测NS0细胞DNA,对大肠杆菌DNA、人类基因组 DNA(人肾上皮293T细胞)、CHO(中国仓鼠卵巢)细胞DNA无扩增反应。另外,对于生产工艺中间品及原液的检测回收率均在70%～130%,RSD均＜15%。5个独立实验室间协标检测数据RSD均＜30%。结论 自主研发NS0宿主细胞DNA残留检测试剂盒(定量PCR-Taqman探针法)特异性强、灵敏度高、准确度好,能够满足NS0宿主 DNA 残留检测要求。

OBJECTIVE To validate a PCR-Taqman probe method for detection of residual DNA of NS0 host cells. METHODS Multiple pairs of primers and probes were designed and synthesized for the NS0 genome repeat sequence, and the optimal primer probe combination was selected through experiments. Pretreatment kit (magnetic bead method) was used to enrich and purify the host cell DNA residue in the sample.According to the requirements of ICH and China Pharmacopoeia general principle No. 9101, validation of the detection method including linearity and range, accuracy,precision, specificity, quantitative limit and reference DNA calibration was carried out for self-developed residual CHO host cell DNA quantitation kit (PCR-Taqman probe).Using the intermediate product and drug substance of the monoclonal antibody process produced by NS0 cells, the test performance of the kit was verified, and five independent laboratories were organized to coordinate the calibration. RESULTS NS0 cell DNA detection (Taqman method) forward primer sequence was CCCCTTCAGCTCCTTGGGTA, reverse primer sequence was GCCTGGCAAATACAGAAGTGG, and probe sequence was FAM-AGGGCCCCCAATGGAGGAGCT-TAMRA. The standard curve of DNA was in the range of 3 fg·μL^-1 to 300 pg·μL^-1 with good linearity (r²＞0. 99). The deviation of the mean from the true value was less than 15% at six different concentrations. The quantitative limit was 3 fg·μL^-1.The DNA calibration result of the internal reference sample was 30 μg·mL^-1. Good precision (RSD≤30%) was obtained. The q-PCR method was specific for CHO DNA,which showed no responses to the DNA of E. coli, human genome DNA(kidney epithelium 293T cells), and CHO genome DNA(Chinese hamster ovary cells). In addition, the detection recovery rate was in the range of 70% to 130% with RSD less than 15% for the intermediate products and drug substance in the production process. The RSD of the five collaborative laboratories was less than 30%. CONCLUSION The self-developed residual NS0 host cell DNA quantitation kit (PCR-Taqman probe) has good specificity, sensitivity and accuracy,and can meet the requirements of host DNA residue detection.