Evaluation of PERT Assays in Biological Products

摘要：小鼠杂杂瘤细胞用于生产单克隆抗体（mab）产生内源性C型逆转录病毒颗粒。监管机构要求证明用于人类的单克隆抗体不含逆转录病毒，并具有足够的安全边际。为了检验基于PCR的检测方法作为下一代病毒安全性评估方法的潜力，我们评估了TaqMan荧光5'核酸酶PCR增强逆转录酶（TM-PERT）检测方法在实验室规模细胞培养样品中测量逆转录酶（RT）活性和通过实验室规模处理步骤模型去除RT的效用。细胞培养收获中含有的RT活性水平（108-1013 pU/mL）大大高于TM-PERT试验的检测极限（106 pU/mL）。细胞培养中RT活性的性质是复杂的，但在澄清的单抗收获中，大部分RT活性似乎包含在大分子质量的病毒粒子中。在实验室规模的色谱运行中，含有单克隆抗体的洗脱液中存在足够的RT活性，可以准确计算其log10还原值（LRV），通常在每步2到4 log10之间。结果表明，TM-PERT法定量准确，灵敏度高，可在短时间内分析大量样品。描述这些实验结果的手稿发表在《生物技术进展》杂志上（broson et al., Biotechnology Prog 17:188, 2001）。

Summary: Murine hybridoma cells used in the production of monoclonal antibodies (mAbs) produce endogenous type C retrovirus particles. Regulatory agencies require a demonstration that mAbs intended for human use are free of retrovirus with an adequate margin of safety. To examine the potential of PCR based assays as next generation assays for viral safety evaluation, we assessed the utility of TaqMan fluorogenic 5'nuclease PCR-Enhanced Reverse Transcriptase (TM-PERT) assays for measuring reverse transcriptase (RT) activity in laboratory-scale cell-culture samples and RT removal by laboratory-scale models of processing steps. The levels of RT activity contained in cell-culture harvests (108-1013 pU/mL) were substantially above the detection limit of the TM-PERT assay (106 pU/mL). The nature of the RT activity from cell-culture was complex, but the bulk of RT activity in clarified mAb harvests appears to be contained in large molecular weight virions. In laboratory-scale chromatographic runs, sufficient RT activity was present in mAb-containing eluates to accurately calculate its log10 reduction value (LRV), typically between 2 and 4 log10 per step. The data indicated that the TM-PERT assay is quantitative, highly sensitive and can be used to analyze a large number of samples in a short period of time. A manuscript describing results from these experiments has been published in Biotechnology Progress (Brorson et al., Biotechnol Prog 17:188, 2001) . To critically examine the performance of the TM-PERT assay in viral safety evaluation, we evaluated the specificity, accuracy, range, precision and robustness of TM-PERT. We found that this assay detects RT activity contained in xenotropic murine leukemia virus (X-MuLV) and CHO cell type C particles and quantifies particle numbers comparably to other assays (e.g., transmission electron microscopy, viral sequence specific TaqMan). Cell culture derived DNA polymerases appeared to contribute only modestly to the assay background. TM-PERT was linear and precise between 107 and 1013 pU/mL, establishing the assay range. The assay was robust in that storage of test articles for 1 week at room temperature or multiple freeze/thaw cycles had little effect on subsequent RT quantification and no interference of the assay by protein or DNA concentrations predicted to be present in cell culture samples was evident. Sporadic background amplification signals present in some assays appeared to correlate with MS2 template quality. A manuscript describing data from these experiments is in press at the journal Biologicals. In collaboration with Genentech Inc., South San Francisco CA , we are extending these studies using TM-PERT by examining the impact of process changes on retrovirus expression in cell culture and LRV by robust virus inactivation steps. Results from these studies have shown that retrovirus expression can vary up to 3 log10 between cell lines. We further showed that cell culture process changes such as fermentation scale-up and media changes have only modest impacts on retrovirus expression, while others such as NaButyrate addition and temperature shifts can impact retrovirus expression up to 2 log10/ml. Results from these experiments are being drafted into a manuscript for submission to Nature Biotechnology. Experiments examining the robustness of low pH retrovirus inactivation are on going