Use of chelating agents to improve the resolution and consistency of cation-exchange chromatography of monoclonal antibodies

Abstract

Analytical cation-exchange chromatography (CEX) is widely used to profile the charge heterogeneity of therapeutic monoclonal antibodies (mAbs). However, the consistency of CEX profiles of a mAb can be significantly reduced by metal ion impurities from sample, mobile phase or leachates from the stainless steel components of the pumping system. In this work, we have developed a new CEX method that dynamically removes metal ions during sample analysis by incorporating the use of chelating agents (1–5mM) in HPLC mobile phases. Among four different chelating agents that were evaluated, EDTA and oxalic acid showed excellent capability of removing metal ions and provided consistent CEX chromatograms for mAb1. Furthermore, the use of oxalic acid in mobile phases not only improved the reproducibility of CEX chromatograms, but also increased the resolution of charge isoforms. Oxalic acid appears capable of binding to mAbs and reducing the positive surface charge density, resulting in a modulation of chromatographic separation. Due to this modulation effect, the CEX resolution was dependent on the concentration of the chelating agent. Optimal resolution for mAb1 was obtained with 2mM of oxalic acid. The oxalic acid modulated CEX method was shown to be capable of monitoring the degradation of mAb1. We further qualified this method according to International Committee on Harmonization (ICH) guidelines and demonstrated that the oxalic acid modulated CEX method is precise and robust at different chromatographic conditions and is suitable for use in a development and/or GMP setting.