Technology and Process Development (TPD), WuXi Biologics, 288 Fute Zhong Road, Waigaoqiao Free Trade Zone, Shanghai, 200131, China.
Technology and Process Development (TPD), WuXi Biologics, 288 Fute Zhong Road, Waigaoqiao Free Trade Zone, Shanghai, 200131, China.
Protein Expr Purif. 2021 Feb;178:105786. doi: 10.1016/j.pep.2020.105786. Epub 2020 Nov 3.
Protein A, Protein L and KappaSelect affinity resins have been widely used for antibody purification. Elution of antibody bound to these resins is typically achieved by acidic pH. In addition, elution can be moderately adjusted by tuning the salt concentration in mobile phase as hydrophobic interactions play a major role in binding. In this study, we assessed the impact of salt concentration in mobile phase on antibody retention in these three types of affinity chromatography. The data suggest that salt concentration has a bigger impact on retention in the two light chain-binding affinity columns (i.e., Protein L and KappaSelect) than in Protein A column. In particular, lowering salt concentration in mobile phase for Protein L and KappaSelect columns allows elution become feasible at higher pH. In addition, this finding suggests that wash in these two types of column aimed at removing weakly-bound byproducts can also be performed at increased pH by lowering salt concentration in the wash buffer. Rendering wash and elution feasible at higher pH has practical value for cases where the target antibodies are sensitive to stringent conditions.
Protein A、Protein L 和 KappaSelect 亲和层析树脂已广泛用于抗体纯化。这些树脂结合的抗体通常通过酸性 pH 值洗脱。此外，通过调整流动相中的盐浓度，可以适度调节洗脱，因为疏水性相互作用在结合中起主要作用。在这项研究中，我们评估了流动相中的盐浓度对这三种亲和层析中抗体保留的影响。数据表明，盐浓度对两种轻链结合亲和柱（即 Protein L 和 KappaSelect）的保留影响大于 Protein A 柱。特别是，降低流动相中的盐浓度使 Protein L 和 KappaSelect 柱在更高的 pH 值下洗脱成为可能。此外，这一发现表明，旨在去除弱结合副产物的这两种类型柱子的洗涤也可以通过降低洗涤缓冲液中的盐浓度在更高的 pH 值下进行。在目标抗体对严格条件敏感的情况下，在更高 pH 值下实现洗涤和洗脱具有实际价值。
