Evaluation of PERT Assays in Biological Products

用于生产单克隆抗体（mAb）的鼠杂交瘤细胞产生内源性C型逆转录病毒颗粒。 监管机构要求证明拟用于人的mAb不含逆转录病毒，并具有足够的安全裕度。 这通常通过小规模验证研究来实现，以证明生产工艺能够去除或灭活几种不同的模型病毒，包括鼠逆转录病毒。 由于目前在这些研究中使用的测定法（例如TEM，体外感染性测定法）昂贵、耗时且不敏感，因此非常需要改进病毒测量和清除/灭活的测量方法。 为了检查基于PCR的检测方法作为下一代病毒安全性评价检测方法的潜力，我们评估了TaqMan荧光5 '核酸酶PCR-增强逆转录酶（TM-PERT）检测方法用于测量实验室规模细胞培养样品中逆转录酶（RT）活性和通过实验室规模处理步骤模型去除RT的效用。 细胞培养收获物中所含的RT活性水平（108 - 1013 pU/mL）显著高于TM-PERT测定的检测限（106 pU/mL）。来自细胞培养物的RT活性的性质是复杂的，但澄清mAb收获物中的大部分RT活性似乎包含在大分子量病毒体中。 在实验室规模的色谱运行中，含mAb的洗脱液中存在足够的RT活性，以准确计算其log10减少值（LRV），通常在2 - 4 log10/步之间。 使用由三个连续柱组成的模型纯化方案纯化的单克隆抗体含有接近检测限的一些残留RT活性。 数据表明，TM-PERT检测，因为它是定量的，高灵敏度，可用于分析大量的样品在短时间内，是理想的适合研究逆转录病毒清除机制和/或优化逆转录病毒的清除/灭活纯化过程。 描述这些实验结果的手稿已提交给Biotechnology Progress。 为了严格检查TM-PERT检测试剂盒在病毒安全性评价中的性能，我们评价了TM-PERT的特异性、准确度、范围、精密度和耐用性。我们发现，该测定检测异嗜性鼠白血病病毒（X-MuLV）和CHO细胞C型颗粒中所含的RT活性，并定量与其他测定（例如，透射电子显微术，病毒序列特异性TaqMan）。 细胞培养物衍生的DNA聚合酶似乎仅对测定背景有适度贡献。 TM-PERT在107和1013 pU/mL之间具有线性和精密度，确立了测定范围。 该试验具有耐用性，因为供试品在室温下储存1周或多次冻融循环对后续RT定量几乎没有影响，并且细胞培养物样品中预测存在的蛋白质或DNA浓度对试验无明显干扰。 在某些检测中存在的零星背景扩增信号似乎与MS2模板质量相关。 描述这些实验数据的手稿正在准备提交给生物制品。我们与Genentech合作，通过稳健的病毒灭活步骤检查工艺变更对逆转录病毒表达和LRV的影响，扩展了这些研究。 这些研究的初步结果表明，细胞培养工艺的变化，如发酵规模扩大，pH值变化和培养基变化对逆转录病毒表达只有适度的影响。 检查低pH逆转录病毒灭活稳健性的实验正在进行中。

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. This is usually achieved by validation studies, performed at small scale, to demonstrate that the manufacturing process is capable of removing or inactivating several different model viruses, including a murine retrovirus. Because the assays currently used in these studies (e.g. TEM, in vitro infectivity assays) are expensive, time consuming and insensitive, improvements in the way viral measurement and clearance/inactivation are measured are highly desirable. 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. Monoclonal antibody purified using a model purification scheme consisting of three serial columns contained some residual RT activity near the limit of detection. The data indicated that the TM-PERT assay, because it is quantitative, highly sensitive and can be used to analyze a large number of samples in a short period of time, is ideally suited to investigate mechanisms of retrovirus clearance and/or to optimize retrovirus removal/inactivation in purification processes. A manuscript describing results from these experiments has been submitted to Biotechnology Progress. 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 preparation for submission to Biologicals. In collaboration with Genentech, we are extending these studies by examining the impact of process changes on retrovirus expression and LRV by robust virus inactivation steps. Preliminary results from these studies have shown that cell culture process changes such as fermentation scale-up, pH shifts and media changes have only modest impacts on retrovirus expression. Experiments examining the robustness of low pH retrovirus inactivation are on going.