Chollangi Srinivas, Parker Ray, Singh Nripen, Li Yi, Borys Michael, Li Zhengjian
Bristol-Myers Squibb, Biologics Development, Global Manufacturing & Supply, Hopkinton, Massachusetts.
Biotechnol Bioeng. 2015 Nov;112(11):2292-304. doi: 10.1002/bit.25639. Epub 2015 Jul 31.
To be administered to patients, therapeutic monoclonal antibodies must have very high purity, with process related impurities like host-cell proteins (HCPs) and DNA reduced to <100 ppm and <10 ppb, respectively, relative to desired product. Traditionally, Protein-A chromatography as a capture step has been the work horse for clearing a large proportion of these impurities. However, remaining levels of process and product related impurities still present significant challenges on the development of polishing steps further downstream. In this study, we have incorporated high throughput screening to evaluate three areas of separation: (i) Harvest treatment; (ii) Protein-A Chromatography; and (iii) Low pH Viral Inactivation. Precipitation with low pH treatment of cell culture harvest resulted in selective removal of impurities while manipulating the pH of wash buffers used in Protein-A chromatography and incorporating wash additives that disrupt various modes of protein-protein interaction resulted in further and more pronounced reduction in impurity levels. In addition, our study also demonstrate that optimizing the neutralization pH post Protein-A elution can result in selective removal of impurities. When applied over multiple mAbs, this optimization method proved to be very robust and the strategy provides a new and improved purification process that reduces process related impurities like HCPs and DNA to drug substance specifications with just one chromatography column and open avenues for significant decrease in operating costs in monoclonal antibody purification.
用于治疗的单克隆抗体在施用于患者时，必须具有非常高的纯度，与宿主细胞蛋白（HCPs）和DNA等工艺相关杂质相对于目标产物分别降至<100 ppm和<10 ppb。传统上，作为捕获步骤的蛋白A层析一直是清除大部分此类杂质的主力方法。然而，剩余的工艺和产品相关杂质水平仍然给下游进一步的精制步骤开发带来重大挑战。在本研究中，我们采用了高通量筛选来评估三个分离领域：（i）收获物处理；（ii）蛋白A层析；以及（iii）低pH病毒灭活。对细胞培养收获物进行低pH处理沉淀可选择性去除杂质，同时通过调节蛋白A层析中使用的洗涤缓冲液的pH值并加入破坏各种蛋白质 - 蛋白质相互作用模式的洗涤添加剂，可进一步且更显著地降低杂质水平。此外，我们的研究还表明，优化蛋白A洗脱后的中和pH值可选择性去除杂质。当应用于多种单克隆抗体时，这种优化方法被证明非常稳健，该策略提供了一种新的、改进的纯化工艺，仅用一根层析柱就能将与工艺相关的杂质如HCPs和DNA降低至原料药规格，并为大幅降低单克隆抗体纯化的运营成本开辟了途径。
