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Scientific article
English

Empirical correction of non-linear pH gradients and a tool for application to protein ion exchange chromatography

Published inJournal of Chromatography. A, vol. 1651, 462320
Publication date2021
Abstract

This concept article reports a practical solution to improve the linearity of effluent pH response as observed in pH gradient cation exchange chromatography (CEX). When performing pH gradient CEX, it is not easy to develop buffer systems that will universally provide pH response proportional with the mobile phase (buffer) composition. It is an especially challenging pursuit when exploring MS compatible buffers (e.g. ammonium-acetate, ammonium-carbonate). In addition to "non-proportional" behavior from the mobile phase composition, the chromatographic column itself will sometimes impose an unpredictable impact on the effluent pH. Here, we propose a simple approach based on the on-line measurement of effluent pH response, conversion of pH to mobile phase volume fraction (φ) and then generation of the inverse response function in the time domain. In the end, when setting the inverse function as the gradient program instead of a linear gradient, an improved - ideally linear - pH response can be produced. A simple Excel tool was developed to assist analysts with this correction procedure, and it has been made available by download for public use.

Keywords
  • pH gradient
  • Ion exchange chromatography
  • Monoclonal antibody
  • Protein biopharmaceuticals
  • Charge variants
Citation (ISO format)
FEKETE, Szabolcs et al. Empirical correction of non-linear pH gradients and a tool for application to protein ion exchange chromatography. In: Journal of Chromatography. A, 2021, vol. 1651, p. 462320. doi: 10.1016/j.chroma.2021.462320
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Article (Published version)
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Identifiers
ISSN of the journal0021-9673
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Technical informations

Creation07/05/2021 1:48:00 PM
First validation07/05/2021 1:48:00 PM
Update time03/16/2023 12:51:52 AM
Status update03/16/2023 12:51:52 AM
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