Use of Ultrashort Columns for Therapeutic Protein Separations. Part 1: Theoretical Considerations and Proof of Concept,Analytical Chemistry

当前位置： X-MOL 学术 › Anal. Chem. › 论文详情

Our official English website, www.x-mol.net, welcomes your feedback! (Note: you will need to create a separate account there.)

Use of Ultrashort Columns for Therapeutic Protein Separations. Part 1: Theoretical Considerations and Proof of Concept
Analytical Chemistry ( IF 6.7 ) Pub Date : 2020-12-17 , DOI: 10.1021/acs.analchem.0c04082
Szabolcs Fekete ₁ , Balázs Bobály ₂ , Jennifer M. Nguyen ₃ , Alain Beck ₄ , Jean-Luc Veuthey ₁ , Kevin Wyndham ₃ , Matthew A. Lauber ₃ , Davy Guillarme ₁

Affiliation

Due to the particular elution mechanism observed with large solutes (e.g., proteins) in liquid chromatography, column length has less impact in controlling their retention compared to small solutes. Moreover, long columns—in theory—just broaden the peaks of large solutes since a great part of the column only acts as void (extra) volume. Such a theory suggests that using very short columns should result in comparable separation quality versus using long columns and make it possible to perform faster (high-throughput) analyses. Therefore, the elution behavior of various therapeutic monoclonal antibodies and their fragments (25–150 kDa) has been investigated using modern instrumentation and column formats. The possibilities offered by narrow-bore columns packed with state-of-the-art 2.7 μm superficially porous particles with 5, 50, 100, and 150 mm lengths have been compared. In particular, the impact of gradient steepness and column length on separation efficiency was evaluated. Using 5 mm × 2.1 mm columns, it has become possible to separate antibody fragments and antibody–drug conjugate species in less than 30 s. Such fast methods can be very useful for high-throughput screening purposes in biopharmaceutical industries.

中文翻译：

超短柱在治疗性蛋白质分离中的应用。第1部分：理论考量和概念验证

由于在液相色谱法中观察到大溶质（例如蛋白质）具有特殊的洗脱机理，因此与小溶质相比，柱长在控制其保留方面的影响较小。此外，从理论上讲，长色谱柱只会使大溶质的峰变宽，因为该色谱柱的很大一部分仅用作空白体积（多余）。这样的理论表明，使用很短的色谱柱与使用长色谱柱相比，可以得到可比的分离质量，并且可以进行更快的（高通量）分析。因此，已使用现代仪器和色谱柱形式对各种治疗性单克隆抗体及其片段（25–150 kDa）的洗脱行为进行了研究。装有最先进的2.7μm表面多孔颗粒，5、50、100，比较了150毫米的长度。特别是，评估了梯度陡度和柱长对分离效率的影响。使用5 mm×2.1 mm的色谱柱，可以在不到30 s的时间内分离出抗体片段和抗体-药物偶联物。这样的快速方法对于生物制药行业中的高通量筛选目的可能非常有用。

更新日期：2021-01-26

点击分享查看原文

点击收藏

阅读更多本刊最新论文本刊介绍/投稿指南11