Protein association on multimodal chromatography media.

The phenomenon of protein-protein association on multimodal chromatography resins was described for two different case study examples. The adsorption pattern of single-component solutions of calcium-rich alpha-lactalbumin (aLaCa) and calcium-depleted alpha-lactalbumin (aLa) and their mixtures with bovine serum albumin was determined on a multimodal anion-exchange chromatography medium. In single-component solutions, both aLaCa and aLa exhibited identical adsorption behavior at low resin loadings, whereas at high loadings the adsorption strength of aLa markedly exceeded that of alaCa. In binary mixtures, the adsorption of BSA enhanced at high concentrations of aLa or aLaCa in the adsorbed phase. The unusual adsorption patterns observed were attributed to the tendency of the proteins for molecular association in the adsorbed phase in single and binary solutions. The phenomena was examined for different pH of the solution: pH 6, 7, 8, and different solvent environments: phosphate buffer (PB), bis tris buffer (BT), 100 mM NaCl in BT and bis tris propane buffer (BTP). The strongest effect was observed for PB and for 100 mM NaCl in BT. Its occurrence was also evidenced for other case study example, i.e., adsorption of single-component solutions and binary mixtures of a monoclonal antibody (mAb) and lysozyme (LYZ) on a multimodal cation-exchange chromatography medium. The enhancement of adsorption of mAb was observed at high concentrations of LYZ in the adsorbed phase. To quantify the underlying effects, a mechanistic model was used, which accounted for both protein association and exclusion resulting from attractive and repulsive protein-protein iterations in the adsorbed phase.