Development of a bispecific antibody-homodimer separation process by cation exchange chromatography based on...

During the recombinant production of IgG-like bispecific antibodies (bsAbs), homodimer formation, which stems from unbalanced chain expression and incorrect chain pairing, presents a major purification challenge. Despite its utility in addressing such charge-based separations, the development of cation exchange chromatography processes remains inherently complex due to the interplay of multiple parameters (e.g., salt concentration, loading conditions) that govern protein-resin interactions. For the separation of bsAb and homodimer, a mechanistic model of cation exchange chromatography was employed to predict the elution behavior, thereby facilitating the optimization of process conditions. The calibration processes and validation results of the extended Langmuir and steric mass action (SMA) two adsorption models were compared. The latter was selected for process optimization due to its streamlined calibration workflow, enabling efficient prediction of elution behavior. The optimal eluent concentration was determined to be 120 mM NaCl. Under this condition, the target bsAb yield reached 96.4 %, and homodimer 1 content was reduced from 26.4 % to 0.5 %. Deviations between the predicted and experimental results for both yield and impurity content were confined within 1.5 %, demonstrating the high accuracy and reliability of the established chromatographic mechanistic model for guiding process optimization.