Bach Julia, Connell-Crowley Lisa
Drug Substance Development, Amgen Inc., 1201 Amgen Court West, Seattle, 98119, Washington.
Biotechnol Bioeng. 2015 Apr;112(4):743-50. doi: 10.1002/bit.25484. Epub 2015 Feb 17.
Protein A chromatography is the most common unit operation used in the manufacture of therapeutic monoclonal antibodies (mAbs) due to its high affinity and specificity for the IgG Fc domain. However, protein A chromatography is often not effective for viral clearance. Typical log reduction values (LRV) for the model retrovirus XMuLV range between 1 and 4 logs, while effective steps such as viral filtration can achieve 5-7 logs of clearance. XMuLV LRVs obtained on protein A are reproducible for a given mAb, but can vary widely for different mAbs, even with the same operating conditions. In order to understand the mechanism of XMuLV clearance on protein A, we have investigated its partitioning on Mabselect SuRe protein A resin and explored how the virus interacts with resin, product, and impurities. The results show that XMuLV has some interaction with the resin backbone and ligand, but also appears to bind to and coelute with the mAb. The interaction with product was further examined by evaluating the effect of feed conditions, loading, and different washes on XMuLV partitioning on the column. Understanding the mechanism of XMuLV removal on a protein A, resin provides insight into the variability and low viral clearance of this step and suggests ways in which the removal of virus by this step can be improved.
由于蛋白A对IgG Fc结构域具有高亲和力和特异性，因此蛋白A层析是治疗性单克隆抗体（mAb）生产中最常用的单元操作。然而，蛋白A层析对病毒清除通常无效。模型逆转录病毒XMuLV的典型对数减少值（LRV）在1至4个对数之间，而诸如病毒过滤等有效步骤可实现5至7个对数的清除。在蛋白A上获得的XMuLV LRV对于给定的mAb是可重复的，但即使在相同的操作条件下，不同的mAb之间也可能有很大差异。为了了解XMuLV在蛋白A上的清除机制，我们研究了它在Mabselect SuRe蛋白A树脂上的分配情况，并探讨了病毒如何与树脂、产品和杂质相互作用。结果表明，XMuLV与树脂骨架和配体有一些相互作用，但似乎也与mAb结合并共同洗脱。通过评估进料条件、上样量和不同洗涤对XMuLV在柱上分配的影响，进一步研究了其与产品的相互作用。了解XMuLV在蛋白A树脂上的去除机制，有助于深入了解该步骤的变异性和低病毒清除率，并提出改进该步骤病毒去除效果的方法。
