Method development for the separation of monoclonal antibody charge variants in cation exchange chromatography, Part II: pH gradient approach

Abstract

The cation exhange pH gradient approach was evaluated for the characterization of 10 model monoclonal antibodies including panitumumab, natalizumab, cetuximab, bevacizumab, trastuzumab, rituximab, palivizumab, adalimumab, denosumab and ofatumumab. This work shows that retention and resolution can be modelled in cation exchange pH gradient mode, based on only four initial runs (i.e. two gradient times and two mobile phase temperature). Only 6h were required for a complete method optimization when using a 100 mm × 4.6 mm strong cation exchange column. The accuracy of the predictions was excellent, with an average difference between predicted and experimental retention times of about 1%. The 10 model antibodies were successfully eluted in both pH and salt gradient modes, proving that both modes of elution can be considered as multi-product charge sensitive separation methods. For most of the compounds, the variants were better resolved in the salt gradient mode and the peak capacities were also higher in the salt gradient approach. These observations confirm that pH gradient approach may be of lower interest than salt gradient cation exchange chromatography for antibody characterization.

Keywords: Cetuximab; Ion exchange; Method development; Monoclonal antibody; pH gradient.