Pharmaceutical (Bio-)Analysis, Institute of Pharmaceutical Sciences, University of Tübingen, Auf der Morgenstelle 8, 72076 Tübingen, Germany.
Instrumental Analytics R&D, Merck KGaA, Frankfurter Str. 250, 64293 Darmstadt, Germany.
J Chromatogr A. 2022 Aug 2;1676:463251. doi: 10.1016/j.chroma.2022.463251. Epub 2022 Jun 14.
To find the best performing column for the analysis of protein-based biopharmaceuticals is a significant challenge as meanwhile numerous modern columns with distinct stationary phase morphologies are available for reversed-phase liquid chromatography. Especially when besides morphology also several other column factors are different, it is hard to decide about the best performing column a priori. To cope with this problem, in the present work 13 different reversed-phase columns dedicated for protein separations were systematically tested by the gradient kinetic plot method. A comprehensive comparison of columns with different morphologies (monolithic, fully porous and superficially porous particle columns), particle sizes and pore diameters as well as column length was performed. Specific consideration was also given to various monolithic columns which recently shifted a bit out of the prime focus in the scientific literature. The test proteins ranged from small proteins starting from 12 kDa, to medium sized proteins (antibody subunits obtained after IdeS-digestion and disulphide reduction) and an intact antibody. The small proteins cytochrome c, lysozyme and β-lactoglobulin could be analysed with similar performance by the best columns of all three column morphologies while for the antibody fragments specific fully porous and superficially porous particle columns were superior. A 450 Å 3,5 µm superficially porous particle column showed the best performance for the intact antibody while a 1.7 µm fully porous particle column with 300 Å showed equivalent performance to the best superficially porous column with thin shell and 400 Å pore size for proteins between 12 and 25 kDa. While the majority of the columns had C4 bonding chemistry, the silica monolith with C18 bonding and 300 Å mesopore size approximated the best performing particle columns and outperformed a C4 300 Å wide-pore monolith. The current work can support the preferred choice for the most suitable reversed-phase column for protein separations.
为了找到分析蛋白质生物制药的最佳性能柱，这是一个重大的挑战，因为目前有许多具有不同固定相形态的现代柱可用于反相液相色谱。特别是当形态之外还有几个其他柱因素不同时，很难预先决定最佳的性能柱。为了解决这个问题，本工作通过梯度动力学图方法系统地测试了 13 种不同的蛋白质分离专用反相柱。对不同形态（整体式、全多孔和表面多孔颗粒柱）、粒径和孔径以及柱长的柱进行了全面比较。还特别考虑了最近在科学文献中略有偏离焦点的各种整体式柱。测试蛋白从小蛋白（起始分子量 12 kDa）、中等大小蛋白（IdeS 消化和二硫键还原后的抗体亚基）到完整抗体。小分子蛋白细胞色素 c、溶菌酶和β-乳球蛋白可以通过所有三种形态柱中最佳柱以相似的性能进行分析，而对于抗体片段，特定的全多孔和表面多孔颗粒柱则具有优势。对于完整抗体，450 Å 3.5 µm 表面多孔颗粒柱表现出最佳性能，而对于分子量在 12 至 25 kDa 之间的蛋白，具有 300 Å 孔径的 1.7 µm 全多孔颗粒柱与最佳表面多孔柱（具有薄壳和 400 Å 孔径）具有相当的性能。虽然大多数柱具有 C4 键合化学性质，但具有 C18 键合和 300 Å 介孔尺寸的硅胶整体柱接近最佳性能的颗粒柱，并优于具有 300 Å 宽孔的 C4 整体柱。本工作可以为蛋白质分离的最适反相柱的选择提供支持。
