Department of Chemical Engineering, Carnegie Mellon University, Pittsburgh, Pennsylvania, USA.
Department of Chemical Engineering, The Pennsylvania State University, State College, Pennsylvania, USA.
Biotechnol Prog. 2020 Nov;36(6):e3041. doi: 10.1002/btpr.3041. Epub 2020 Jul 24.
As upstream product titers increase, the downstream chromatographic capture step has become a significant "downstream bottleneck." Precipitation becomes more attractive under these conditions as the supersaturation driving force increases with the ever-increasing titer. In this study, two precipitating reagents with orthogonal mechanisms, polyethylene glycol (PEG) as a volume excluder and zinc chloride (ZnCl ) as a cross linker, were examined as precipitants for two monoclonal antibodies (mAbs), one stable and the other aggregation-prone, in purified drug substance and harvested cell culture fluid forms. Manual batch solubility and redissolution experiments were performed as scouting experiments. A high throughput (HTP) liquid handling system was used to investigate the design space as fully as possible while reducing time, labor, and material requirements. Precipitation and redissolution were studied by systematically varying the concentrations of PEG and ZnCl to identify combinations that resulted in high yield and good quality for the stable mAb; PEG concentrations in the range 7-7.5 wt/vol% together with 10 mM ZnCl gave a yield of 97% and monomer contents of about 93%. While yield for the unstable mAb was high, quality was not acceptable. Performance at selected conditions was further corroborated for the stable mAb using a continuous tubular precipitation reactor at the laboratory scale. The HTP automation system was a powerful tool for locating desired (customized) conditions for antibodies of different physicochemical properties.
随着上游产物滴度的增加，下游的色谱捕获步骤已成为一个显著的“下游瓶颈”。在这些条件下，由于过饱和度驱动力随滴度的不断增加而增加，沉淀变得更具吸引力。在这项研究中，两种具有正交机制的沉淀试剂——聚乙二醇（PEG）作为体积排除剂和氯化锌（ZnCl ）作为交联剂——被用作两种单克隆抗体（mAb）的沉淀剂，一种是稳定的，另一种是易于聚集的，分别在纯化的药物物质和收获的细胞培养液形式中进行研究。手动分批溶解度和重溶解实验作为探索性实验进行。高通量（HTP）液体处理系统用于尽可能全面地研究设计空间，同时减少时间、劳动力和材料需求。通过系统地改变 PEG 和 ZnCl 的浓度来研究沉淀和重溶解，以确定对稳定 mAb 产生高收率和良好质量的组合；PEG 浓度在 7-7.5wt/vol%范围内，同时使用 10mM ZnCl 可获得 97%的收率和约 93%的单体含量。虽然不稳定 mAb 的收率很高，但质量不可接受。使用实验室规模的连续管式沉淀反应器对稳定 mAb 在选定条件下的性能进行了进一步验证。HTP 自动化系统是一种强大的工具，可用于定位具有不同物理化学性质的抗体的所需（定制）条件。
