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

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