Boosting the Liquid Chromatography Separation of Complex Bispecific Antibody Products by Using the Multi-Isocratic Elution Mode

Abstract

In reversed-phase liquid chromatography (RPLC), the selectivity between major species and minor variants of protein biopharmaceutical products is always limited. Unfortunately, the stationary phase chemistry, type of mobile phase (organic modifier and salts) and temperature only have a very limited impact on selectivity. Therefore, instead of using a linear elution gradient, we evaluated a recently developed strategy, named the multi-isocratic elution mode, to improve the chromatographic resolution. In this contribution, a generic workflow involving the use of an Excel spreadsheet is provided for the rapid and successful development of multi-isocratic elution methods, without the need to use HPLC modeling software. This simple strategy was then successfully applied to very complex biopharmaceutical products; these included one reduced mAb-cytokine fusion protein and a mAb-domain-fusion (C-terminal) protein sample, containing numerous minor variants that were poorly separated from the major species. The addition of several isocratic steps during the chromatographic run provides a clear added value in terms of chromatographic selectivity for several variants, simplifying characterization of the sample with advanced MS tools. In addition to these advantages, some of the limitations of the multi-isocratic elution mode were also highlighted; these included the need to use a highly precise pumping device (preferably, a binary pumping system) and the need to prepare highly accurate mobile phases.