A case study of the mechanism of unfolding and aggregation of a monoclonal antibody in ion exchange chromatography.

A mechanistic model for describing unfolding of a monoclonal antibody (mAb) in ion exchange chromatography has been developed. The model reproduced retention behavior characteristic for conformational changes of antibodies upon adsorption, including: multi-peak elution, aggregate formation, and recovery reduction. Two competitive paths in the adsorption mechanism of the unfolded protein were assumed: refolding in the adsorbed phase to the native form followed by its desorption, or direct desorption followed by instantaneous aggregation in the liquid phase. The reduction in recovery of the eluted protein was attributed to spreading of the unfolded protein on the adsorbent surface, which enhanced the binding affinity. The model was formulated based on the analysis of retention behavior of a model mAb that was eluted in pH gradients on a strong cation exchange resin. The pH profile was found to be distorted in the presence of the protein, which was ascribed to dissociation of ionizable groups of the protein in the adsorbed phase. Since the protein retention was strongly pH dependent, that phenomenon was also accounted for in mathematical modeling. A series of independent experiments was designed to evaluate the model parameters that quantified the process thermodynamics and kinetics: the Henry constants of the native, unfolded, spread and aggregated forms of the protein along with underlying kinetic coefficients. The model was efficient in reproducing the retention pattern of the protein and the aggregate content in eluting band profiles. After proper calibration, the model can potentially be used to quantify protein unfolding and elution in other ion exchange systems.