en
Scientific article
English

Impact of the column on effluent pH in cation exchange pH gradient chromatography, a practical study

Published inJournal of Chromatography. A, vol. 1626, 461350
Publication date2020
Abstract

In ionexchange chromatography, the pH gradient mode becomes more and more popular today for the analysis of therapeutic proteins as this mode can provide higher or alternative selectivity to the com- monly used salt gradient mode. Ideally, a linear pH response is expected when performing linear gra- dients. However up to now, only a very few buffer systems have been developed and are commercially available which can perform nearly linear pH responses when flowing through a given column. It is also known that a selected buffer system (mobile phase) can work well on one column but can fail on other column. The goal of this study was to practically evaluate the effects that ionexchange columns (weak and strong exchangers) might have on effluent pH, when performing linear pH gradient separations of therapeutic monoclonal antibodies. To attain this objective, the pH was monitored on-line at the column outlet using a specific setup. To make comprehensive observations of the phenomenon, four different mobile phase conditions and five cation exchange columns (weak and strong exchangers) were employed. The obtained pH responses were systematically compared to responses measured in the absence of the columns. From this work, it has become clear that both the column and mobile phase can have significant effects on pH gradient chromatography and that their combination must be considered when developing a new method. Phase systems (column + mobile phase) providing linear pH responses are indeed the most suitable for separating mAbs with different isoelectric points and, with them, it is possible to elute mAbs across wide retention time ranges and with high selectivity.

Keywords
  • Ionexchange chromatography
  • PH gradient
  • On-line pH meter
  • Monoclonal antibody
  • Charge variants
Citation (ISO format)
FARSANG, Evelin et al. Impact of the column on effluent pH in cation exchange pH gradient chromatography, a practical study. In: Journal of Chromatography. A, 2020, vol. 1626, p. 461350. doi: 10.1016/j.chroma.2020.461350
Main files (1)
Article (Published version)
Identifiers
ISSN of the journal0021-9673
176views
1downloads

Technical informations

Creation06/30/2020 3:54:00 PM
First validation06/30/2020 3:54:00 PM
Update time03/15/2023 10:12:58 PM
Status update03/15/2023 10:12:58 PM
Last indexation02/12/2024 1:15:18 PM
All rights reserved by Archive ouverte UNIGE and the University of GenevaunigeBlack