Ultra-short ion-exchange columns for fast charge variants analysis of therapeutic proteins.

Abstract

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\xa0mm and packed with non-porous 3\xa0µm 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\xa0kDa range). Because of the non-porous nature of the IEX packing material, the column porosity was relatively low (ε\xa0=\xa00.42) 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\xa0min using an IEX column with dimensions of 15\xa0×\xa02.1\xa0mm. 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.