Non-affinity purification of a nanobody by void-exclusion anion exchange chromatography and multimodal weak cation exchange chromatography
Non-affinity purification of a nanobody by void-exclusion anion exchange chromatography and multimodal weak cation exchange chromatography
复制标题
通过空隙排除阴离子交换色谱和多模式弱阳离子交换色谱非亲和纯化纳米抗体
DOI：
10.1016/j.seppur.2019.05.068
复制
发表时间：
2019-10-15
影响因子：
8.6
通讯作者：
Xian, Mo
中科院分区：
文献类型：
--
作者：
Fan, Xiying;Zhou, Yuhang;Xian, Mo
据报道，多模式色谱树脂是抗体纯化中经典吸附剂的极好替代品。在这项研究中，整合了空隙排阻阴离子交换色谱（VEAX）和正电性多模式色谱（Capto MMC）用于无标签纳米抗体（2D 5）纯化。系统地表征了VEAX在初始2D 5分离中的潜在用途。VEAX实现了宿主细胞蛋白（HCP）减少89.9%，DNA减少99.6%，内毒素减少99.9%，周质蛋白聚集体减少100%，2D 5回收率接近100%，纯度从3.5%提高到25.0%。随后，Capto MMC层析与直接蛋白上样无缝结合用于进一步纯化。该步骤的回收率为80.5%。整个两步纯化平台产生2D 5，其中HCP为16 ppm，DNA为127 ppb，内毒素为35 EU/mL，总蛋白回收率为80.5%。2D 5的纯度为98.5%。此外，该方法对2D 5的亲和力没有影响。同时，所建立的方法还用于纯化抗PD-L1（KPU）的纳米抗体，纯度为95.8%，回收率> 99%，HCP为55 ppm，DNA为289 ppb，内毒素为128 EU/mL。本研究首次报道了一种用于纯化无标签纳米抗体的简化、无缝和非亲和纯化平台，为常规亲和层析抗体纯化策略提供了一种有竞争力的替代方案。
Multimodal chromatography resins have been reported to be excellent alternatives to classical absorbents in the purification of antibodies. In this study, void-exclusion anion exchange chromatography (VEAX) and electropositive multimodal chromatography (Capto MMC) were integrated for tag-free nanobody (2D5) purification. VEAX was systematically characterized for its potential use in the initial 2D5 separation. VEAX achieved a 89.9% reduction in host cell proteins (HCPs), a 99.6% reduction in DNA, a 99.9% reduction in endotoxins, and a 100% reduction in aggregates from periplasmic proteins, with a 2D5 recovery of near 100% and a purity enhancement from 3.5% to 25.0%. Subsequently, Capto MMC chromatography was seamlessly incorporated with direct protein loading for further purification. The recovery of this step was 80.5%. The entire two-step purification platform yielded 2D5 with 16 ppm of HCPs, 127 ppb of DNA, 35 EU/mL of endotoxins, with a total protein recovery of 80.5%. The purity of 2D5 was 98.5%. Moreover, this method had no effect on the affinity of 2D5. Meanwhile, the established method was also used to purify a nanobody against PD-L1 (KPU) with 95.8% purity, > 99% recovery, 55 ppm of HCPs, 289 ppb of DNA and 128 EU/mL of endotoxins. For the first time, this study reports a simplified, seamless and non-affinity purification platform for the purification of a tag-free nanobody, which provides a competitive alternative for the conventional affinity chromatography antibody purification strategy.