Winters Dwight, Tran Mai, Yoo Daniel, Walker Kenneth W
Amgen Research, Therapeutic Discovery, Amgen, Inc., One Amgen Center, Thousand Oaks, CA, 91320-1789, USA.
Amgen Research, Therapeutic Discovery, Amgen, Inc., One Amgen Center, Thousand Oaks, CA, 91320-1789, USA.
Protein Expr Purif. 2020 Jan;165:105497. doi: 10.1016/j.pep.2019.105497. Epub 2019 Sep 7.
Affinity purification, such as Protein A (ProA) followed by size exclusion chromatography (SEC) remains a popular method to obtain research scale proteins. With the need for higher throughput protein production increasing for discovery research, there is substantial interest in the automation of complex protein purification processes, which often start with a ProA step followed by SEC. However, the harsh elution conditions from ProA based chromatography can destabilize some proteins resulting in particulates, which in turn can cause column fouling and potential cross-contamination of subsequent purifications. We modified both Bio Rad NGC and ÄKTA Pure systems to run a three-column process (ProA to buffer exchange to SEC) enabling automated tandem affinity to SEC purification while minimizing the risk of SEC column fouling and subsequent cross-contamination. The intervening buffer exchange column, unlike the final SEC column, can be rapidly regenerated using harsh methods between runs, and these automated systems are capable of processing up to six samples per day without user intervention.
亲和纯化，如先进行蛋白A（ProA）亲和纯化再进行尺寸排阻色谱（SEC），仍然是一种获得研究规模蛋白质的常用方法。随着发现研究对高通量蛋白质生产的需求不断增加，人们对复杂蛋白质纯化过程的自动化产生了浓厚兴趣，这些过程通常从ProA步骤开始，然后是SEC。然而，基于ProA的色谱法苛刻的洗脱条件可能会使一些蛋白质不稳定，从而产生颗粒，进而导致柱污染以及后续纯化过程中的潜在交叉污染。我们对伯乐公司的NGC系统和安玛西亚公司的ÄKTA Pure系统进行了改进，以运行三柱流程（从ProA到缓冲液置换再到SEC），实现自动化串联亲和到SEC纯化，同时将SEC柱污染和后续交叉污染的风险降至最低。中间的缓冲液置换柱与最后的SEC柱不同，运行之间可以使用苛刻的方法快速再生，并且这些自动化系统每天能够处理多达六个样品，无需用户干预。
