Mechanistic modeling of retention time distribution under high breakthrough conditions for continuous Protein A...

Regulatory authorities strongly recommend using residence time distribution (RTD) to achieve material traceability in continuous bioprocesses for non-adsorption units. For adsorption-based units, such as chromatography, retention time distribution (ReTD) is more suitable than RTD for characterizing material flow. Continuous capture chromatography is widely applied for biopharmaceutical continuous manufacturing. However, the ReTD behavior in these systems is still not fully understood. In this study, an ReTD model combining general rate model and two-component mobile phase modulator Langmuir model was developed for Protein A affinity chromatography under high breakthrough conditions. The model was calibrated using adsorption equilibrium experiments, protein breakthrough curves and elution curves. It was then validated through pulse injection experiments at varying protein loading phase. The results showed good agreement between model predictions and experimental results (R² > 0.945). The exchange mechanism between the solid and liquid phases was further analyzed using confocal laser scanning microscopy images and model simulations, revealing that proteins with stronger binding affinity surpass the bound fraction to bind at the adsorption front while those with weaker affinity would exchange with the surface-bound fractions. Finally, simulations of protein distribution in the column during the interconnected loading step indicate that the exchange effect could broaden the ReTD in continuous chromatography. The model developed lays the groundwork for achieving material traceability and enables non-conforming material diversion strategies to facilitate real-time product release in continuous chromatography processes.