Department of Therapeutic Discovery, Amgen Research, 1120 Veterans Blvd, South San Francisco, CA 94080, United States; Current address: Janssen Research and Development, 3210 Merryfield Row, San Diego, CA 92121, United States.
Department of Therapeutic Discovery, Amgen Research, 1120 Veterans Blvd, South San Francisco, CA 94080, United States.
J Chromatogr B Analyt Technol Biomed Life Sci. 2020 Jan 1;1136:121849. doi: 10.1016/j.jchromb.2019.121849. Epub 2019 Nov 12.
There is a great need for high-throughput protein purification to produce protein molecules for research and therapeutics. Although there have been significant advancements made in automated multi-step chromatography and preparative in-process design-of-experiment (DOE) capabilities in commercial fast performance liquid chromatography (FPLC) instruments, almost all commercial FPLCs rely on a binary buffer mixing system, which hinders automated buffer preparation. Nevertheless, current-generation FPLCs are equipped with a quaternary mixer designed for limited in-line buffer preparation and preparative pH scouting DOE experiments. We decided to leverage the quaternary mixing capability by extending and re-programming AkTA Avant's quaternary valve into an automated in-process buffer preparation system to simplify automated purification requiring complex washing steps. We accomplished this by using two extra inlet valves, a sample valve, and versatile valve to split inputs of the quaternary valve into software-selectable stock solutions of pH buffers, salts, eluents, and additives. We also devised a new flow scheme to perform automated two-step chromatography using only one versatile valve. This was accomplished by using only stock parts and software to facilitate reproduction. To demonstrate the versatility and capability of the system, we purified a transmembrane protein that requires a detergent to stay soluble and needs an in-column, high-salt washing step to achieve high purity.
高通量蛋白质纯化对于研究和治疗用蛋白质分子的生产非常重要。虽然自动化多步层析和制备过程中实验设计（DOE）能力在商业快速液相色谱（FPLC）仪器中已经取得了重大进展，但几乎所有商业 FPLC 都依赖于二元缓冲液混合系统，这阻碍了自动化缓冲液制备。然而，目前的 FPLC 配备了一个四元混合器，用于有限的在线缓冲液制备和制备 pH 探测 DOE 实验。我们决定利用四元混合能力，通过扩展和重新编程 AkTA Avant 的四元阀，将其转换为自动化的过程中缓冲液制备系统，以简化需要复杂洗涤步骤的自动化纯化。我们通过使用两个额外的入口阀、一个样品阀和一个多功能阀，将四元阀的输入分成软件可选的 pH 缓冲液、盐、洗脱液和添加剂的储备溶液来实现这一点。我们还设计了一种新的流动方案，仅使用一个多功能阀即可执行自动化两步层析。这是通过仅使用储备部件和软件来实现的，以方便复制。为了展示系统的多功能性和能力，我们纯化了一种需要去污剂才能保持可溶性的跨膜蛋白，并且需要柱内高盐洗涤步骤才能达到高纯度。
