Evaluation of PERT Assays in Biological Products

用于生产单抗(MAbbs)的小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，供人类使用的单抗不含逆转录病毒，并具有足够的安全边际。这通常是通过小规模进行的验证研究来实现的，以证明制造过程能够去除或灭活几种不同的模型病毒，包括小鼠逆转录病毒。由于目前在这些研究中使用的分析方法(例如，透射电子显微镜、体外感染性分析)昂贵、耗时且不敏感，因此非常需要改进病毒测量和清除/灭活的方法。为了检验基于聚合酶链式反应的检测方法作为新一代病毒安全性评估方法的潜力，我们评估了TaqMan荧光5‘核酸酶聚合酶增强型逆转录酶(TM-PERT)检测方法在实验室规模的细胞培养样品中测量逆转录酶(RT)活性以及通过实验室规模的加工步骤模型去除RT的有效性。细胞培养收获中所含的RT活性水平(108-1013PU/毫升)大大高于TM-PERT分析的检测限(106PU/毫升)。来自细胞培养的RT活性的性质是复杂的，但在澄清的mAb收获中，RT活性的大部分似乎包含在大分子量病毒粒子中。在实验室规模的层析运行中，含有单抗的洗脱液中存在足够的RT活性，以准确计算其log10还原值(LRV)，通常在每一步2至4log10之间。由三个串联柱组成的模型纯化方案纯化的单抗含有接近检测下限的残存RT活性。这些数据表明，TM-PERT分析是一种定量的、高灵敏度的方法，可以在短时间内分析大量样品，非常适合于研究逆转录病毒清除的机制和/或优化纯化过程中逆转录病毒的去除/灭活。描述这些实验结果的手稿已经提交给生物技术进步组织。为了严格检验TM-PERT试验在病毒安全性评估中的性能，我们对TM-PERT的特异性、准确性、范围、精密度和稳健性进行了评估。我们发现，该方法检测异嗜性小鼠白血病病毒(X-MuLV)和C型CHO细胞颗粒中包含的RT活性，并与其他方法(例如，透射电子显微镜、病毒序列特异性TaqMan)相比较地定量颗粒数量。细胞培养衍生的DNA聚合酶似乎对检测背景的贡献不大。TM-PERT在107~1013PU/mL范围内线性关系良好，精密度良好，建立了测定范围。该方法是可靠的，因为测试物品在室温或多次冷冻/解冻循环中储存1周对随后的RT定量几乎没有影响，而且细胞培养样品中预计存在的蛋白质或DNA浓度对该分析没有明显的干扰。在某些检测中出现的零星背景扩增信号似乎与MS2模板质量相关。一份描述这些实验数据的手稿正在准备提交给生物制品公司。在与基因技术公司的合作下，我们正在扩大这些研究的范围，通过强有力的病毒灭活步骤来检查工艺变化对逆转录病毒表达和LRV的影响。这些研究的初步结果表明，细胞培养过程的变化，如发酵放大、pH变化和介质变化，对逆转录病毒的表达只有轻微的影响。检验低pH逆转录病毒灭活的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.