(157b) Establishing a Multiscale Workflow to Study Fc-Dependent Binding in Protein a Chromatography

Protein A chromatography remains the industry standard for monoclonal antibody (mAb) capture. Variability in monoclonal antibody (mAb) Fc regions can influence binding interactions with protein A resins, leading to changes in yield, dynamic binding capacity, and overall process robustness. To systematically investigate how Fc-region differences affect protein A chromatography performance, we have established a comprehensive, multiscale workflow integrating wet-lab experiments, molecular simulations and mechanistic modeling.

Our workflow employs molecular simulations, including but not limited to MM-GBSA and FEP simulations, to probe how variations in the Fc region influence interaction strength and binding stability with the protein A ligands. These insights are used to support observed trends in process performance, including differences in adsorption and elution behavior.

At the process level, we use the ACT isotherm to quantify the effects of Fc region differences on the binding capacity Qmax and affinity Keq changes at different pHs via experimental breakthrough curves and elution profiles. This allows quantitative comparison of chromatographic behaviors across mAbs with diverse Fc-region features.

To further isolate and investigate Fc contributions, we incorporate enzymatic digestion using papain to generate mAb fragments. The binding characteristics of Fc-containing fragments are evaluated using several commercially available protein A resins, enabling a more direct assessment of Fc-specific interactions independent of Fab-mediated effects.

This integrative approach provides a robust framework for understanding and predicting how Fc-region variability influences protein A chromatography, supporting platform development and improved manufacturability across a range of antibody formats.