State Key Laboratory of Chemical Engineering, Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China.
J Chromatogr A. 2012 Jun 29;1244:116-22. doi: 10.1016/j.chroma.2012.04.073. Epub 2012 May 3.
Hydrophobic charge-induction chromatography (HCIC) with 4-mercaptoethyl-pyridine (MEP) as the ligand is a novel technology for antibody purification, however, the separation selectivity still needs to be improved for the applications, especially for the impurity of serum albumin. In this study, with bovine serum immunoglobulin G (IgG) as the model, the purification of IgG from the serum albumin containing feedstock was developed with the commercial HCIC resin MEP HyperCel, focusing on the optimization of operation pH and salt addition. The adsorption isotherms of IgG and bovine serum albumin (BSA) were investigated at different pHs, and the binding and elution behaviors of two proteins in the column were also studied at varying pHs. In addition, the protein-ligand interactions were investigated with some additives in the buffer. It was found that the conditions of pH 6 with 0.1 M NaCl or pH 8 could be used to effectively remove BSA from the MEP resin without the influence on IgG adsorption. Two modes with control of loading or washing buffer were tested to enhance the purification of IgG from BSA containing feedstock, and the purity of IgG was improved to about 95% compared with 62.9% for the control. The results demonstrated that the control of loading pH or the addition of NaCl in the buffer might be an effective method to improve the purification of antibody with the HCIC process.
疏水性电荷诱导层析（HCIC）以 4-巯基乙基吡啶（MEP）为配体，是一种新型的抗体纯化技术，但为了应用，特别是针对血清白蛋白杂质，其分离选择性仍需要进一步提高。在这项研究中，以牛血清免疫球蛋白 G（IgG）为模型，使用商业 HCIC 树脂 MEP HyperCel 从含血清白蛋白的原料中开发 IgG 的纯化方法，重点优化操作 pH 值和加盐条件。在不同 pH 值下研究了 IgG 和牛血清白蛋白（BSA）的吸附等温线，还在不同 pH 值下研究了两种蛋白质在柱中的结合和洗脱行为。此外，还在缓冲液中添加了一些添加剂来研究蛋白质-配体相互作用。结果表明，在 pH 6 时添加 0.1 M NaCl 或在 pH 8 时可以有效去除 MEP 树脂上的 BSA，而不会影响 IgG 的吸附。测试了两种控制加载或洗涤缓冲液 pH 值的模式，以增强从含 BSA 的原料中纯化 IgG，与对照相比，IgG 的纯度提高到约 95%，而对照为 62.9%。结果表明，控制加载 pH 值或在缓冲液中添加 NaCl 可能是改善 HCIC 工艺中抗体纯化的有效方法。
