Key Laboratory of Biomass Chemical Engineering of Ministry of Education, Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China.
J Chromatogr B Analyt Technol Biomed Life Sci. 2013 Oct 1;936:33-41. doi: 10.1016/j.jchromb.2013.07.029. Epub 2013 Aug 7.
Mixed-mode chromatography has been focused as a cost-effective new technique for antibody purification. In this study, four mixed-mode resins with N-benzyl-N-methyl ethanol amine, 2-benzamido-4-mercaptobutanoic acide, 4-mercapto-ethyl-pyridine and phenylpropylamine as the ligands were tested and the multi-functional interactions between ligand and protein were discussed. Immunoglobulin G (IgG), bovine serum albumin (BSA) and the binary mixture of BSA and IgG were used as the model feedstock to compare the separation behaviors by pH gradient elution. The comparison analysis showed mixed-mode resin with N-benzyl-N-methyl ethanol amine as the ligand had the best ability to separate IgG and BSA. The results indicated that for four resins tested ionic interaction might play the dominant role in the separation of IgG and BSA while the hydrophobic interactions and hydrogen bonding have some subsidiary effects. The pH stepwise elution and sample loading were optimized to improve the IgG purification from serum albumin containing feedstock. High purity (92.3%) and high recovery (95.6%) of IgG were obtained. The results indicated that mixed-mode chromatography would be a potential option for antibody purification with the control of loading and elution conditions.
混合模式色谱已被视为一种具有成本效益的新型抗体纯化技术。在这项研究中，测试了四种带有 N-苄基-N-甲基乙醇胺、2-苯甲酰胺-4-巯基丁酸、4-巯基乙基吡啶和苯丙胺作为配体的混合模式树脂，并讨论了配体与蛋白质之间的多功能相互作用。免疫球蛋白 G (IgG)、牛血清白蛋白 (BSA) 和 BSA 与 IgG 的二元混合物被用作模型进料，以比较 pH 梯度洗脱的分离行为。比较分析表明，带有 N-苄基-N-甲基乙醇胺作为配体的混合模式树脂具有分离 IgG 和 BSA 的最佳能力。结果表明，对于测试的四种树脂，离子相互作用可能在 IgG 和 BSA 的分离中起主导作用，而疏水性相互作用和氢键则具有一些次要作用。通过优化 pH 分步洗脱和样品加载来改善从含白蛋白的原料中纯化 IgG。从血清白蛋白中获得了高纯度 (92.3%) 和高回收率 (95.6%) 的 IgG。结果表明，通过控制加载和洗脱条件，混合模式色谱将成为抗体纯化的一种潜在选择。
