Quality by Design in continuous bioprocessing: Investigating the impact of critical process parameters of...

Given the complexities of continuous bioprocessing, it is critical to thoroughly investigate the process parameters unique to multi-column chromatography (MCC) and their potential impacts. However, existing studies have focused on either loading densities or residence time at steady states only, and their combined impact on critical quality attributes (CQAs) especially during transient phases were less known. In this study, we investigated the impact of critical process parameters during both steady-state and transient phases (start-up, close-down, and intermediate perturbation) through full factorial design. Our findings revealed that MCC operation with static control results in a maximum delta UV variation (breakthrough percentage from 1^st loading column) of 30 % between the start-up phase and steady state. Additionally, eluate concentration showed a slight increase (with a maximum difference of 1.9 mg/mL, equivalent to 25 % deviation) when transitioning from the start-up phase to steady state but significantly decreased (with a maximum difference of 8.2 mg/mL, equivalent to 93 % deviation) when shifting from steady state to the close-down phase. The monomer purity during start-up and steady-state phases remained above 97 % while the close-down phase eluate purity ranged from 57 % to 98 %, depending on the operating conditions. Nevertheless, the variation in product quality can be potentially mitigated by product diversion or pooling with high-purity eluates from steady-state, highlighting the robustness of MCC operation in maintaining consistent product quality. Therefore, our study underscores the critical need to comprehensively understand the process dynamics of MCC operation at both steady state and transient phases.