Evaluation of PERT Assays in Biological Products

生物制品中 PERT 检测的评价

• 批准号: 6545891
• 负责人: KE E STEIN
• 金额: --
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6545891
• 关键词: CHO cells; RNA directed DNA polymerase; Retroviridae disease; animal tissue; bioassay; biological products; communicable disease control; communicable disease transmission; enzyme activity; hybridomas; immunologic substance development /preparation; method development; monoclonal antibody; murine leukemia virus; polymerase chain reaction; public health; virus replication

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

总结：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。 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log 10减少值（LRV），通常在2 - 4 log 10/步之间。 结果表明，TM-PERT方法具有定量、高灵敏度的特点，可在短时间内对大量样品进行分析。 描述这些实验结果的手稿已经发表在Biotechnology Progress（Brorson et al.，Biotechnol Prog 17：188，2001）。 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MS 2模板质量相关。 一份描述这些实验数据的手稿正在《生物学》杂志上出版。 与Genentech Inc.合作，South San弗朗西斯科CA，我们正在使用TM-PERT扩展这些研究，通过稳健的病毒灭活步骤检查工艺变更对细胞培养物和LRV中逆转录病毒表达的影响。 这些研究的结果表明，逆转录病毒表达在细胞系之间的差异可达3 log 10。 我们进一步表明，细胞培养工艺变化（如发酵规模扩大和培养基变化）对逆转录病毒表达的影响较小，而其他变化（如丁酸钠添加和温度变化）对逆转录病毒表达的影响高达2 log 10/ml。 这些实验的结果正在起草一份手稿，提交给《自然生物技术》。正在进行实验，检查低pH值逆转录病毒灭活的耐用性