Institute of Pharmaceutical Sciences of Western Switzerland, University of Geneva, CMU-Rue Michel Servet 1, 1211 Geneva 4, Switzerland.
Research and Development Biopharmaceuticals, Solvias AG, Römerpark 2, 4303 Kaiseraugst, Switzerland.
Anal Chem. 2021 Jan 26;93(3):1277-1284. doi: 10.1021/acs.analchem.0c04082. Epub 2020 Dec 17.
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.
由于在液相色谱中大溶质（如蛋白质）的特殊洗脱机制，与小溶质相比，柱长对其保留的控制作用较小。此外，长柱——理论上——只会拓宽大溶质的峰，因为柱的很大一部分仅作为空隙（额外）体积起作用。该理论表明，使用非常短的柱子应该可以获得与使用长柱子相当的分离质量，并使进行更快（高通量）分析成为可能。因此，使用现代仪器和柱格式研究了各种治疗性单克隆抗体及其片段（25-150 kDa）的洗脱行为。比较了填充有最新 2.7 μm 表面多孔颗粒的窄孔柱（5、50、100 和 150 mm 长）提供的可能性。特别是，评估了梯度陡度和柱长对分离效率的影响。使用 5mm×2.1mm 柱子，在不到 30 秒的时间内就可以分离抗体片段和抗体药物偶联物。对于生物制药行业的高通量筛选目的，这种快速方法非常有用。
