Although the Langmuir isotherm is frequently used for mechanistic modelling in Protein A (ProA) chromatography, it must be empirically manipulated to capture the relationship between pH and monoclonal antibody (mAb) retention during loading, washing and elution. And while empirical isotherm expressions incorporating the pH-dependence of retention have been developed by others, they cannot provide mechanistic insight into chromatographic behavior. We have considered the titratable residues within the primary mAb-ProA binding site as well as the maximum mAb-ProA binding stoichiometry within a given resin to propose a new, fundamental isotherm. We have demonstrated the model’s practicability and feasibility via experiment and simulation for a broad range of breakthrough loading and pH step elution and gradient elution schemes with a model mAb and MabSelect SuRe media. Besides the isotherm parameters, the main input to the chromatographic simulations is the pH profile used. The correlation between gradient experiments and simulations is good, supporting routine industrial and academic application. Further, we demonstrate that the chromatogram for a complete loading, washing and elution cycle can be accurately described with a single set of parameters. The isotherm also provides a framework for parsing mass transport details from binding details in the analysis of chromatograms and provides mechanistic insight into the energetics of binding as a function of pH.

A first-principles affinity complex titration isotherm for protein A chromatography

ACS Fall 2021

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