Chmielowski Rebecca A, Meissner Sandra, Roush David, Linden Thomas O, Glowacki Edward, Konietzko Janelle, Nti-Gyabaah Joseph
Merck and Co., MSD, Biologics Process Development, Process Development and Engineering, Kenilworth, NJ, 07033.
Biotechnol Prog. 2014 May-Jun;30(3):636-45. doi: 10.1002/btpr.1908. Epub 2014 Apr 19.
Clearance of aggregates during protein purification is increasingly paramount as protein aggregates represent one of the major impurities in biopharmaceutical products. Aggregates, especially dimer species, represent a significant challenge for purification processing since aggregate separation coupled with high purity protein recovery can be difficult to accomplish. Biochemical characterization of the aggregate species from the hydrophobic interaction and cation exchange chromatography elution peaks revealed two different charged populations, i.e. heterogeneous charged aggregates, which led to further challenges for chromatographic removal. This paper compares multimodal versus conventional cation exchange or hydrophobic chromatography methodologies to remove heterogeneous aggregates. A full, mixed level factorial design of experiment strategy together with high throughput experimentation was employed to rapidly evaluate chromatographic parameters such as pH, conductivity, and loading. A variety of operating conditions were identified for the multimodal chromatography step, which lead to effective removal of two different charged populations of aggregate species. This multimodal chromatography step was incorporated into a monoclonal antibody purification process and successfully implemented at commercial manufacturing scale.
在蛋白质纯化过程中，清除聚集体变得越来越重要，因为蛋白质聚集体是生物制药产品中的主要杂质之一。聚集体，尤其是二聚体形式，对纯化过程构成了重大挑战，因为聚集体分离以及高纯度蛋白质回收可能难以实现。对来自疏水相互作用和阳离子交换色谱洗脱峰的聚集体进行生化表征，发现了两种不同的带电群体，即异质带电聚集体，这给色谱去除带来了进一步的挑战。本文比较了多模式色谱与传统阳离子交换或疏水色谱方法去除异质聚集体的效果。采用全因子混合水平实验设计策略以及高通量实验，快速评估诸如pH值、电导率和上样量等色谱参数。确定了多模式色谱步骤的各种操作条件，可有效去除两种不同带电群体的聚集体。该多模式色谱步骤被纳入单克隆抗体纯化工艺，并在商业生产规模上成功实施。
