Process Virology, Purification Development, MS 10, Genentech, Inc., 1 DNA Way, South San Francisco, California, 04080.
Biotechnol Bioeng. 2014 Jan;111(1):95-103. doi: 10.1002/bit.24999. Epub 2013 Aug 16.
Protein A chromatography is widely used as a capture step in monoclonal antibody (mAb) purification processes. Antibodies and Fc fusion proteins can be efficiently purified from the majority of other complex components in harvested cell culture fluid (HCCF). Protein A chromatography is also capable of removing modest levels of viruses and is often validated for viral clearance. Historical data mining of Genentech and FDA/CDER databases systematically evaluated the removal of model viruses by Protein A chromatography. First, we found that for each model virus, removal by Protein A chromatography varies significantly across mAbs, while remains consistent within a specific mAb product, even across the acceptable ranges of the process parameters. In addition, our analysis revealed a correlation between retrovirus and parvovirus removal, with retrovirus data generally possessing a greater clearance factor. Finally, we describe a multivariate approach used to evaluate process parameter impacts on viral clearance, based on the levels of retrovirus-like particles (RVLP) present among process characterization study samples. It was shown that RVLP removal by Protein A is robust, that is, parameter effects were not observed across the ranges tested. Robustness of RVLP removal by Protein A also correlates with that for other model viruses such as X-MuLV, MMV, and SV40. The data supports that evaluating RVLP removal using process characterization study samples can establish multivariate acceptable ranges for virus removal by the protein A step for QbD. By measuring RVLP instead of a model retrovirus, it may alleviate some of the technical and economic challenges associated with performing large, design-of-experiment (DoE)-type virus spiking studies. This approach could also serve to provide useful insight when designing strategies to ensure viral safety in the manufacturing of a biopharmaceutical product.
蛋白 A 层析广泛应用于单克隆抗体（mAb）纯化过程的捕获步骤。抗体和 Fc 融合蛋白可以从收获的细胞培养液（HCCF）中的大多数其他复杂成分中高效地被纯化。蛋白 A 层析还能够去除一定水平的病毒，并且通常经过病毒清除验证。对 Genentech 和 FDA/CDER 数据库的历史数据挖掘系统地评估了蛋白 A 层析对模型病毒的去除效果。首先，我们发现对于每种模型病毒，蛋白 A 层析的去除效果在 mAb 之间差异显著，而在特定的 mAb 产品内保持一致，即使在工艺参数的可接受范围内也是如此。此外，我们的分析还揭示了逆转录病毒和细小病毒去除之间的相关性，逆转录病毒数据通常具有更大的清除因子。最后，我们描述了一种基于过程特征研究样品中存在的逆转录病毒样颗粒（RVLP）水平，用于评估病毒清除对工艺参数影响的多元方法。结果表明，蛋白 A 对 RVLP 的去除效果非常稳健，即在测试范围内没有观察到参数效应。蛋白 A 对 RVLP 的去除稳健性也与 X-MuLV、MMV 和 SV40 等其他模型病毒的去除稳健性相关。这些数据支持使用过程特征研究样品评估 RVLP 的去除效果，可以为 QbD 中蛋白 A 步骤的病毒去除建立多元可接受范围。通过测量 RVLP 而不是模型逆转录病毒，可以减轻进行大型实验设计（DoE）型病毒添加研究相关的一些技术和经济挑战。当设计策略以确保生物制药产品制造过程中的病毒安全性时，这种方法也可以提供有用的见解。
