Department of Analytical Chemistry, Faculty of Chemistry, Universitat de València, C/ Dr. Moliner 50, 46100, Burjassot, Spain.
School of Pharmaceutical Sciences, University of Geneva, CMU-Rue Michel Servet 1, 1211, Geneva 4, Switzerland; Institute of Pharmaceutical Sciences of Western Switzerland, University of Geneva, CMU-Rue Michel Servet 1, 1211, Geneva 4, Switzerland.
J Chromatogr A. 2021 Nov 8;1657:462568. doi: 10.1016/j.chroma.2021.462568. Epub 2021 Sep 22.
The purpose of this work was to study the potential of recently developed ultra-short column hardware for ion exchange chromatography (IEX). Various prototype and commercial columns having lengths of 5, 10, 15, 20 and 50 mm and packed with non-porous 3 µm particles were systematically compared. Both pH and salt gradient modes of elution were evaluated. Similarly, what has been previously reported for reversed phase liquid chromatography (RPLC) mode, an "on-off" retention mechanism was observed in IEX for therapeutic proteins and their fragments (25-150 kDa range). Because of the non-porous nature of the IEX packing material, the column porosity was relatively low (ε = 0.42) and therefore the volumes of ultra-short columns were very small. Based on this observation, it was important to reduce as much as possible all the sources of extra-column volumes (i.e. injection volume, extra-bed volume, detector cell volume and connector tubing volume), to limit peak broadening. With a fully optimized UHPLC system, very fast separations of intact and IdeS digested mAb products were successfully performed in about 1 min using an IEX column with dimensions of 15 × 2.1 mm. This column was selected for high-throughput separations, since it probably offers the best compromise between efficiency and analysis time. For such ultra-fast separations, PEEK tubing was applied to bypass the column oven (column directly connected) to the optical detector via a zero dead volume connection.
这项工作的目的是研究最近开发的超短柱硬件在离子交换色谱（IEX）中的应用潜力。系统地比较了各种长度为 5、10、15、20 和 50mm 的原型和商业柱，这些柱子均填充有 3µm 的非多孔颗粒。评估了 pH 和盐梯度洗脱模式。同样，对于反相液相色谱（RPLC）模式，先前已经报道过的“开-关”保留机制在 IEX 中也适用于治疗性蛋白质及其片段（25-150kDa 范围）。由于 IEX 填充材料的非多孔性质，柱的孔隙率相对较低（ε=0.42），因此超短柱的体积非常小。基于这一观察结果，重要的是尽可能减少所有柱外体积的来源（即进样体积、柱床外体积、检测器池体积和连接管体积），以限制峰展宽。在完全优化的 UHPLC 系统中，使用尺寸为 15×2.1mm 的 IEX 柱，成功地在大约 1 分钟内实现了完整和 IdeS 消化 mAb 产物的快速分离。由于该柱在效率和分析时间之间提供了最佳的折衷，因此被选择用于高通量分离。对于这种超快分离，应用 PEEK 管绕过柱温箱（直接连接柱），通过零死体积连接将其连接到光学检测器。
