Mechanistic modeling of anti-Langmuirian to Langmuirian behavior of Fc-fusion proteins in cation exchange chromatography

Development of a next-generation chromatographic model, capable of simultaneously meeting academic demands for thermodynamic consistency and industrial requirements in everyday project work, has become a focal point of research. In this study, anti-Langmuirian to Langmuirian (AL-L) elution behavior was observed in cation-exchange chromatographic separation of charge variants of industrial Fc-fusion proteins. To characterize this behavior, the multi-protein Mollerup activity model was integrated into the steric mass action (SMA) model, resulting in a new model named the generalized ion-exchange (nGIEX) isotherm for multi-protein systems. An R2 exceeding 0.95 calibrated by three elution experiments indicates an effective description of the AL-L behavior (dynamic adsorption). Using isotherm sampling, the nGIEX model exhibited sigmoidal AL-L isotherms (static adsorption). Finally, the model's extrapolation capability was externally validated through process optimization, resulting in an optimal two-step elution condition and a yield improvement of the main variant from 25.9 % to 89.1 % within purity specifications (>70 %).