Genentech Inc., South San Francisco, California.
Thermo Fisher Scientific, Bedford, Massachusetts.
Biotechnol Bioeng. 2019 Apr;116(4):846-856. doi: 10.1002/bit.26866. Epub 2019 Jan 16.
Protein A chromatography is an effective capture step to separate Fc-containing biopharmaceuticals from cell culture impurities but is generally not effective for virus removal, which tends to vary among different products. Previous findings have pointed to the differences in feedstocks to protein A, composed of the products and other cell culture-related impurities. To separate the effect of the feedstock components on virus removal, and understand why certain monoclonal antibody (mAb) products have low virus log reduction values (LRVs) across protein A chromatography, we investigated the partitioning of three types of viruses on Eshmuno® A columns. Using pure mAbs, we found that low LRVs were correlated with the presence of the particular mAb product itself, causing altered partitioning patterns. Three virus types were tested, and the trend in partitioning was the same for retrovirus-like particles (RVLPs) expressed in the cell substrate, and its model virus xenotropic murine leukemia virus (XMuLV), whereas slightly different for murine minute virus. These results were extended from previous observation described by Bach and Connell-Crowley (2015) studying XMuLV partitioning on MabSelect SuRe columns, providing further evidence using additional types of viruses and resin. Other product-specific cell culture impurities in harvested cell culture fluid played a lesser role in causing low LRVs. In addition, using high throughput screening (HTS) methods and Eshmuno® A resin plates, we identified excipients with ionic and hydrophobic properties that could potentially alleviate the mAb-induced LRV reduction, indicating that both ionic and hydrophobic interactions were involved. More excipients of such nature or combinations, once optimized, can potentially be used as load and/or wash additives to improve virus removal by protein A. We have demonstrated that HTS is a valuable tool for this type of screening, whether to gain deeper understanding of a mechanism, or to provide guidance during the optimization of protein A process with improved virus removal.
Protein A 亲和层析是一种有效的捕获步骤，可将含有 Fc 的生物制药从细胞培养杂质中分离出来，但通常对去除病毒无效，不同产品的去除效果差异较大。先前的研究结果表明，Protein A 的进料组成（产品和其他细胞培养相关杂质）存在差异。为了分离进料成分对病毒去除的影响，并了解为什么某些单克隆抗体 (mAb) 产品在 Protein A 亲和层析中具有较低的病毒对数减少值 (LRV)，我们研究了三种类型的病毒在 Eshmuno® A 柱上的分配情况。使用纯 mAb，我们发现低 LRV 与特定 mAb 产品本身的存在相关，导致分配模式发生改变。三种病毒类型进行了测试，对于在细胞基质中表达的逆转录病毒样颗粒 (RVLP) 及其模型病毒嗜性鼠白血病病毒 (XMuLV)，分配趋势与之前 Bach 和 Connell-Crowley (2015) 研究 XMuLV 在 MabSelect SuRe 柱上的分配时的趋势相同，而对于鼠细小病毒略有不同。这些结果扩展了 Bach 和 Connell-Crowley (2015) 之前的观察结果，使用额外类型的病毒和树脂提供了进一步的证据。收获的细胞培养液中的其他特定于产品的细胞培养杂质在导致低 LRV 方面的作用较小。此外，使用高通量筛选 (HTS) 方法和 Eshmuno® A 树脂板，我们鉴定出具有离子和疏水特性的赋形剂，这些赋形剂可能潜在地减轻 mAb 引起的 LRV 降低，表明离子和疏水相互作用都参与其中。一旦优化，这种性质或组合的更多赋形剂可潜在用作负载和/或洗涤添加剂，以通过 Protein A 提高病毒去除率。我们已经证明，HTS 是进行此类筛选的一种有价值的工具，无论是为了更深入地了解机制，还是在优化具有改进病毒去除效果的 Protein A 工艺时提供指导。
