Zheng Weifeng, Jiang Lin, Lei Qing, Yang Jun, Gao Xuefeng, Wang Wanru, Zhang Yanli, Kong Tao, Chen Qiaoli, Li Gang
The fourth research department, Lanzhou Institute of Biological Products Co., Ltd, No.888 Yanchang Road, Chengguan District, Lanzhou City, Gansu Province China.
Biol Proced Online. 2019 Sep 1;21:17. doi: 10.1186/s12575-019-0105-1. eCollection 2019.
The presence of residual DNA carried by biological products in the body may lead to an increased oncogenicity, infectivity, and immunomodulatory risk. Therefore, current agencies including WHO, EU, and the FDA limited the accepted amounts of residual DNA (less than 10 ng or 100 pg/dose). Among the methods of detecting residual DNA, qPCR is considered to be the most practical for residual DNA quantitation due to its sensitivity, accuracy, precision, and time-saving.
In this study, the detection capacity of this method was determined by comparing the detected concentration of the commercial kit and the self-designed primer/probe set after the same treatment of the extraction method. Then, a universal sample pretreatment method based on a co-precipitant was optimized. The validation results demonstrated that the method has appropriate specificity, sensitivity, accuracy, and precision according to ICH guidelines. The limit of detection and quantitation reached 3 fg/ul and 0.3 pg/reaction respectively, which satisfies the requirement of limit of residual DNA detection in biologics. Spike recovery (82.3-105.7%) showed that the proposed qPCR assay was accurate and has good extraction efficiency. Moreover, the precision of the method based on intra- and inter-assay was 0.065-0.452% and 0.471-1.312%, respectively.
These results all indicated that the method for determination of residual DNA in biological products expressed from CHO cells is sensitive, accurate and robust.
生物制品携带的残留DNA在体内的存在可能会导致致癌性、感染性和免疫调节风险增加。因此，包括世界卫生组织、欧盟和美国食品药品监督管理局在内的现行机构对残留DNA的可接受量进行了限制（每剂量小于10 ng或100 pg）。在检测残留DNA的方法中，定量聚合酶链反应（qPCR）因其灵敏度、准确性、精密度和省时性，被认为是残留DNA定量最实用的方法。
在本研究中，通过比较商业试剂盒和自行设计的引物/探针组在相同提取方法处理后的检测浓度，确定了该方法的检测能力。然后，优化了一种基于共沉淀剂的通用样品预处理方法。验证结果表明，根据国际人用药品注册技术协调会（ICH）指南，该方法具有适当的特异性、灵敏度、准确性和精密度。检测限和定量限分别达到3 fg/ul和0.3 pg/反应，满足生物制品中残留DNA检测限的要求。加标回收率（82.3 - 105.7%）表明所提出的qPCR检测方法准确且具有良好的提取效率。此外，基于批内和批间的方法精密度分别为0.065 - 0.452%和0.471 - 1.312%。
这些结果均表明，用于测定CHO细胞表达的生物制品中残留DNA的方法灵敏、准确且稳健。
