Hybrid mode of hydrophobic interaction chromatography of monoclonal antibodies and related biomolecules: Influence of elution conditions on chromatographic performance using poly (alkyl aspartamide) silica columns.

A hybrid mode of hydrophobic interaction chromatography (HHIC) is an emerging analytical technique for the separation of biomolecules under non-denaturing conditions that combines elements of conventional hydrophobic interaction and reversed-phase chromatography. This article explores the impact of mobile phase composition such as salt concentration and organic modifier on the separation of therapeutic monoclonal antibodies and related large biomolecules using poly (alkyl aspartamide) silica HIC columns. The initial mobile phase salt concentration had a significant impact on the separation of a mixture of large biomolecules demonstrating that the relationship of elution and salt concentration was more complex than in conventional HIC. In general, the earlier eluting components exhibited greater retention at higher salt concentration as is typical of HIC separations. Conversely, the later eluting components showed greater retention at lower initial salt concentration. This differential is useful for improving the overall separation by widening the elution window for components of a mixture. In addition, no significant unfolding of the proteins was detected by intrinsic fluorescence or electrospray mass spectrometry. The impact of linear velocity and gradient steepness was also evaluated.