磁珠分离结合定量PCR法检测单克隆抗体产品中CHO细胞DNA残留量

Determination of residual host cell DNA in monoclonal antibody products by magnetic bead based extraction combined with quantitative PCR

目的 利用磁珠分离法结合定量PCR技术，建立单克隆抗体产品中CHO细胞DNA残留量的检测方法，并进行验证及初步应用。方法通过磁珠分离法提取样品中的残留DNA，再利用Taqman探针法对样品和标准DNA进行定量PCR测定，根据标准曲线对样品中的DNA残留量进行分析。对建立的方法进行种属特异性、准确性及精密性验证，并对5个厂家15批单克隆抗体类产品中的残留DNA进行测定。结果该方法检测CHO细胞DNA残留的最低准确定量浓度可达1×10^-3pg／μl，DNA含量在1×10^-3～10^2 pg／μl范围内曲线线性良好，标准曲线的相关系数为0.994；该方法能特异性地检测CHO细胞DNA，对其他种属的DNA无扩增反应；该方法检测不同DNA加标量样品的DNA回收率相近，均值均在90％以上，3次测定的相对标准偏差均小于20％，92.6％加标样品的DNA回收率在70％～130％之间；该方法检测15批单克隆抗体类产品的DNA残留量均小于100pg／剂量，符合《中国药典》三部（2010版）有关CHO细胞残余DNA的要求。结论磁珠分离法可解决残留DNA检测中样品前处理的技术难点，定量PCR法能够简便、快速、准确地对不同工艺的单克隆抗体产品中CHO细胞DNA残留进行定量测定。

Objective To develop, verify and preliminarily apply a magnetic bead based extraction combined with quantitative PCR （q-PCR） method for determination of residual host cell DNA in monoclonal antibody （McAb） products. Methods The residual host cell DNA in test samples was extracted by magnetic bead based extraction method, and determined by Taqman probe based q-PCR, using standard DNA as control The residual DNA content was analyzed according to the standard curve. The developed method was verified for specificity, accuracy and precision, and used for determination of 15 batches of McAb products from five manufacturers. Results The minimum detection limit of residual CHO cell DNA by the developed method was 1 × 10^-3 pg/μl, while the linear range was 1 × 10^-3 ～ 1× 102 pg/μl, with a correlation coefficient （r value） of 0. 994. The q-PCR method was specific for CHO DNA, which showed no responses to the DNAs of other species. The recovery rates of spiked samples of various concentrations were more than 90%, of which the relatively standard deviations in 3 tests were less than 20%. However, the DNA recovery rates of 92.6% of spike samples were within 70%～130%. All the residual DNA contents in 15 batches of McAb products determined by the developed method were less than 100 pg per dose, which were complied with the requirements for residual CHO cell DNA in Chinese Pharmacopeia （Volume Ⅲ, 2010 edition）. Conclusion The magnetic bead based extraction method successfully solved the technical difficulties of sample pretreatment during residual DNA assay. The q-PCR method was simple, rapid and accurate for quantitation of residual CHO cell DNA in McAb products manufactured by various procedures.