Developing a scalable framework for mitigating GEV and dissolved CO2 stresses in high-density CHO cell cultures

Intensified fed-batch featuring high-cell-density cultures highlights challenges in bioreactor scale-up and underscores the needs for tailored mitigation, particularly on the reconciliation of the hydrodynamic stress from sparger gas entrance velocity (GEV) and accumulated CO₂ partial pressure (pCO₂). In this study, elevated GEV and pCO₂ were identified as the causes of titer reduction during the scale-up to a 2000 L single-use bioreactor (SUB). To verify this hypothesis, a scaled-down model was established in bench-scale bioreactors equipped with a customized sparger to simulate the high GEV and pCO₂ conditions. The model reproduced the impaired titer performance, showing that both factors inhibited production through an independent mechanism and a culture phase-dependent manner. Proteomic analysis of the two stresses further revealed differentially protein expressions associated with cell proliferation, energy generation and reactive oxygen species induced cellular responses. Lastly, an optimized scale-up strategy balancing effective pCO₂ stripping with controlled GEV stress was implemented through a modified 2000 L SUB with an upgraded sparger design. This work elucidated the interplay between elevated pCO₂ and GEV stresses and culture performance, establishing a systematic framework for modulating GEV and pCO₂ as a universal scale-up tool. The findings advanced scale-up strategies for all mammalian cell cultures, especially for shear stress- and/or pCO₂-sensitive cell lines, while reinforcing mechanistic insights into bioprocess scalability for industrial applications.