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

Navarro-Huerta, Jose Antonio
Nguyen, Jennifer M.
Lauber, Matthew A.
Beck, Alain
Published in Journal of chromatography. 2021, vol. 1657, 462568
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 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.
Keywords Extra-column volumeFast separationIon-exchange chromatographyMonoclonal antibodyUltra-short column
PMID: 34601253
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Research group Sciences analytiques
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NAVARRO-HUERTA, Jose Antonio et al. Ultra-short ion-exchange columns for fast charge variants analysis of therapeutic proteins. In: Journal of chromatography , 2021, vol. 1657, p. 462568. doi: 10.1016/j.chroma.2021.462568 https://archive-ouverte.unige.ch/unige:155482

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Deposited on : 2021-10-18

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