Effect of solution condition on the binding behaviors of monoclonal antibody and fusion protein therapeutics in Protein A chromatography.

Recent development in Protein A chromatography has adopted wash buffers with a wide range of pH to improve the removal of process-related impurities, e.g., host cell proteins (HCPs), from Fc-based target biological products. Thus, it is important to investigate the pH effect on the binding behaviors of target products of various properties on Protein A resins. Here we performed column breakthrough experiments for two monoclonal antibodies (mAbs) and one Fc-fusion protein on two Protein A resins from pH 4 to pH 10, and the experimental data was analyzed using a mechanistic model to obtain isotherm and mass transfer parameters. The two mAbs exhibited relatively high dynamic binding capacity (DBC) at 10% breakthrough of 43 - 67 g/L at pH 6-9 followed by a ∼30% decrease from pH 9 to pH 10; while the Fc-fusion protein reached the highest DBC at 10% breakthrough of 10 - 17 g/L at pH 5 and thereafter the capacity gradually reduced. Model analyses revealed that the two mAbs had higher maximum binding capacity by 2 - 5 fold but weaker binding affinity (12 - 64%) than the Fc-fusion protein from pH 5 to pH 9. For the three molecules, similar patterns of the pH impact were observed on the two Protein A resins with the Jetted A50 resin showing generally higher DBC and stronger binding affinity than the MabSelect SuRe LX resin. Additionally, an inverse relationship between the binding affinity and surface diffusivity was observed for both resins. Besides obtaining the isotherm parameters from the column breakthrough data, a direct batch equilibrium measurement showed comparable trend in these parameters with relatively more scattered values due to the inherent uncertainties to accurately determine the initial slope of the isotherm in highly favorable adsorption conditions. Finally, isothermal titration calorimetry (ITC) results revealed that the measured binding affinity using free Protein A ligand was stronger than that obtained from the breakthrough and isotherm results for the resin, possibly due to the reduced accessibility of the immobilized ligand on resin surface. Overall, this work can facilitate future Protein A ligand design and affinity chromatography process optimization for biomanufacturing.