Department of Chemical Sciences, Ariel University, 70400, Ariel, Israel.
Faculty of Chemistry, Weizmann Institute of Science, 76100, Rehovot, Israel.
Sci Rep. 2021 Jun 3;11(1):11697. doi: 10.1038/s41598-021-90966-1.
The research described in this report seeks to present proof-of-concept for a novel and robust platform for purification of antibody fragments and to define and optimize the controlling parameters. Purification of antigen-binding F(ab') fragments is achieved in the absence of chromatographic media or specific ligands, rather by using clusters of non-ionic detergent (e.g. Tween-60, Brij-O20) micelles chelated via Fe ions and the hydrophobic chelator, bathophenanthroline (batho). These aggregates, quantitatively capture the F(ab') fragment in the absence or presence of E. coli lysate and allow extraction of only the F(ab') domain at pH 3.8 without concomitant aggregate dissolution or coextraction of bacterial impurities. Process yields range from 70 to 87% by densitometry. Recovered F(ab') fragments are monomeric (by dynamic light scattering), preserve their secondary structure (by circular dichroism) and are as pure as those obtained via Protein A chromatography (from a mixture of F(ab') and Fc fragments). The effect of process parameters on Ab binding and Ab extraction (e.g. temperature, pH, ionic strength, incubation time, composition of extraction buffer) are reported, using a monoclonal antibody (mAb) and polyclonal human IgG's as test samples.
本报告所述研究旨在为抗体片段的纯化提供一种新颖而强大的平台，并定义和优化控制参数。在没有色谱介质或特定配体的情况下，通过使用非离子型去污剂（例如 Tween-60、Brij-O20）胶束簇螯合 Fe 离子和疏水性螯合剂 bathophenanthroline（batho），实现了抗原结合 F(ab') 片段的纯化。这些聚集体在不存在或存在大肠杆菌裂解物的情况下定量捕获 F(ab') 片段，并允许在 pH 3.8 下仅提取 F(ab') 结构域，而不会伴随聚集体溶解或同时提取细菌杂质。通过密度计，产率范围为 70%至 87%。回收的 F(ab') 片段是单体的（通过动态光散射），保留其二级结构（通过圆二色性），与通过 Protein A 层析法（从 F(ab') 和 Fc 片段的混合物中）获得的一样纯净。报告了工艺参数对 Ab 结合和 Ab 提取的影响（例如温度、pH 值、离子强度、孵育时间、提取缓冲液的组成），使用单克隆抗体（mAb）和多克隆人 IgG 作为测试样品。
