Protein A affinity chromatography is the dominant capture step in monoclonal antibody manufacture. Since it is the first unit operation in a succession of chromatography steps, process understanding using a mechanistic model can be efficacious. Although mechanistic models based on Langmuir isotherms are widely used, the physical significance of the model parameters is not always considered in detail. In this work, we have calibrated such a model using complete breakthrough curves in the presence of various mobile-phase modulators at different pH values. Both modified Langmuir and colloidal-based models accommodating pH effects were incorporated in the model. The isotherm models were coupled with the general rate model, including pore and surface diffusion effects. The dynamic binding capacity was shown to be impacted by the load concentration (0.5-10 mg/mL), kosmotropic salts (sulfates, phosphates, and citrates) and organic phase modulators (ethanol, isopropanol). The maximum binding capacity within the Langmuir isotherm and the protein-protein repulsion within the colloidal isotherm led to significant differences in the protein-surface binding affinity extracted from the models under these conditions. The parameters obtained from breakthrough curves were used to model the effects on product recovery of wash and elution buffers for mobile phase modulators and pH. The pH buffer titration on the Protein A column is one of the challenges in modeling elution peak shape and thus pool volume. This was overcome by modeling the column with a buffer titration curve run without protein load. The impact of the removal of host-cell protein and protein aggregate with mobile phase modulators and its pH was found to be important. Overall, this work demonstrates that mechanistic models can provide broad insight into protein binding behavior inside the column and hence utilize the knowledge to develop an optimal process.

Impact of mobile phase modulators on capacity and impurity clearance in protein A chromatography: Insights from mechanistic models

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.