CHO宿主细胞DNA残留检测试剂盒(PCR-Taqman探针法)的方法学验证

Method validation of CHO host cell residual DNA quantitative kit(PCR-Taqman probe)

目的:中国仓鼠卵巢(Chinese hamster ovary,CHO)宿主细胞DNA残留检测(PCR-Taqman探针法)方法的验证。方法:利用磁珠分离法结合Taqman探针定量PCR技术,对自主研制的CHO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、参考品DNA标定等验证。特别针对残留定量限、蛋白浓度、pH值、缓冲体系等关键影响因素,以及生产工艺各中间品进行回收率考察,验证试剂盒对复杂样品的检测性能。选取采用不同工艺的多个重组蛋白产品,通过独立三方协作标定,开展试剂盒的适用性研究。结果:DNA标准曲线在3 fg·μL-1～300 pg·μL-1范围内,线性良好(R2> 0. 99);对5个浓度梯度检测的准确度偏差均<10%;定量限为0. 5 fg·μL-1;内部参考品DNA标定结果为10μg·m L-1;精密度良好(RSD≤15%);大鼠、小鼠、大肠杆菌、人类基因组DNA对检测无干扰。另外,对于样品中DNA含量在1pg·m L-1～10 ng·m L-1、pH值在4～9,采用磷酸缓冲体系、Tris缓冲体系、醋酸缓冲体系、柠檬酸缓冲体系等多种基质样品,以及生产工艺中间品的检测回收率均在70%～130%,RSD均<15%。3个独立实验室间协标检测数据RSD均<30%。结论:自主研发CHO宿主细胞DNA残留检测试剂盒(荧光探针法)特异性强、灵敏度高、准确度好,能够满足复杂基质条件下各种重组制品的宿主DNA残留检测要求。

Objective： To validate a detection method （PCR-Taqman probe） for residual DNA of Chinese hamster ovary （CHO） host cells. Methods： By using the method of magnetic beads separation combined with Taqman probe quantitative PCR technology, the detection method validation including linear and range, accuracy, precision, specificity, quantitative limit and reference DNA calibration was carried out for self-developed CHO host cell residual DNA quantitative kit （PCR-Taqman probe）. In particular, recovery test was carried out for various DNA residues, protein concentration, pH value, commonly used buffer systems and intermediate products in the production process to verify the recovery performance of complicated samples by the kit. Recombinant protein products from different sources and production processes were selected and calibrated collaboratively to study the applicability of the kit. Results ： The standard curve of DNA was in the range of 3 fg· μL-1 to 300 pg·μL-1with good linearity （R2 〉 0. 99）. The deviation of the mean from the true value was less than 10% at five different concentrations. The quantitative limit was 0.5 fg·μL- 1. The DNA calibration result of the internal reference sample was 10 ·μg-mL-1. Good precision （RSD 〈~ 15% ） was obtained. The detection was not interfered by the genome DNA of rats, mice, E. coli and human species. In addition, the detection recovery rate was in the range of 70% to 130% with RSD less than 15% for the sample DNA content ranging from 1 pg·mL-1 to 10 ng·mL-1 , pH ranging from 4 to 9, different substrates such as phosphoric acid buffer system, Tris buffer system, acetic acid buffer system and citric acid buffer system, as well as intermediate products in the production process. The RSD of the three collaborative laboratories was less than 30%. Conclusion： The self-developed DNA CHO host cell residual DNA quantitative kit （PCR-Taqman probe） has good specificity, sensitivity and accuracy, and can meet the requirements of host DNA residue detection for various recombinant products under complicated matrix conditions.