For the difficult task of separating closely related protein variants, multimodal (MM) chromatography may impart selective advantages over traditional IEX or HIC processes due to the combined electrostatic and hydrophobic modes of interaction. In this study, a series of antibody Fab fragments containing various combinations of surface hydrophobicity and electrostatic potential were generated to evaluate the relative importance of these local surface properties on the protein's chromatographic behavior. The Fab fragment variants were designed in silico to create the desired surface properties, generated by site-directed mutagenesis, expressed transiently in HEK293 cells and purified by affinity chromatography. Gradient column experiments were carried out with the resulting Fab fragment variants on traditional cation exchange and MM materials with several mobile phase modifiers exploring the effects of both fluid phase conditions and ligand chemistry on the selectivity of the MM systems. Additionally, new protein surface characterization protocols based on molecular dynamic simulations were tested and refined using this rich data set. This work sets the stage for the a priori design of a MM-based purification step for separations of Fab fragment variants.

Investigations on Fab fragment variants in multimodal chromatographic systems

247th National Meeting (2014)

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