Comparative Study on Selectivity and Kinetic Performance of Recent Reversed Phase Liquid Chromatography Columns in the Separation of Protein Variants

In this study, the impact of stationary phase chemistry was investigated for the analysis of large biomolecules. For this purpose, widepore state-of the-art C18, C3, diphenyl stationary phases as well as a new superficially porous material possessing a carbon core and nanodiamond-polymer shell (composite material) were tested. To evaluate the similarities and differences between these phases, two samples were considered, including i) oxidatively stressed and reduced recombinant interferon alfa-2A variants as well as ii) light chain and heavy chain fragments of a human IgG1 monoclonal antibody Belimumab. For both samples, the overall selectivity was found to be similar on all the silica-based stationary phases (e.g. α = 1.20 - 1.25 between the two oxidized forms of interferon), while it was somewhat different on the composite material (α = 1.12 between the two oxidized forms of interferon). On the other hand, the kinetic performance of the composite phase was somewhat lower (peak capacity between 70 and 80) than that of silica-based materials (peak capacity higher than 100), except in the presence of a relatively high concentration TFA (0.5% TFA) due to the reduction of ionic interactions. At the end, when separating closely eluting proteins variants in RPLC mode, minor differences in selectivity may be more important than kinetic efficiency and therefore, the composite material might also be of interest for biomolecules characterization.