The specific selectivities offered by multimodal ligands drive the increased application of multimodal chromatography in the purification of complex new ”multispecific” antibodies. Together with stronger requirements to remove product-related impurities and robust purification processes, this requires improved understanding of the protein-multimodal ligand interaction mechanism. The goal of the present study is to develop a mechanistic model to predict monoclonal antibody (mAb)-Fab fragment and heterogenous aggregates separation based on the preferential interaction adsorption model developed by Perkin et al. (1997). The fit of the adsorption isotherm to isocratic elutions of mAb, Fab fragment, and aggregates result in a characteristic ‘U’-shaped curve for mAb, Fab, and aggregates retention factors as a function of NaCl salt concentrations. The ‘U’-shaped curve of retention factor is attributed to the mixed interactions of the electrostatic interactions at low salt concentration and the hydrophobic effects at high salt concentration. The retention behavior of mAb, Fab, and aggregates under linear and step gradients were used to estimate the preferential interaction model parameters of each on Capto™ MMC ImpRes multimodal column using isocratic fit parameters as the initial guess value. The proposed mechanistic model successfully simulates the elution peaks for both the regimes of low and high salt concentrations, and can therefore be used for purification process optimization and control for separation of process-related impurities.

Mechanistic modeling of multimodal chromatography for separation of a monoclonal antibody from product-related impurities: Fab fragment and aggregates

ACS Fall 2021

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