Characterization of apoptosis in fed‐batch cultures highlights the impact of timely process intervention on improving recombinant protein productivity

Fed-batch processes using Chinese hamster ovary (CHO) cells are primarily employed to manufacture biologics. In these cultures, cells transition from exponential growth to the stationary phase, often resulting in a viability decline. This decline along, with similar reductions in cell growth and productivity is attributed to changes in cellular metabolism, the cell cycle, and apoptosis, potentially caused by nutrient imbalance and the accumulation of inhibitory byproducts. To enhance productivity in fed-batch processes, understanding the interactions among these factors is crucial for identifying intervention strategies to sustain cell culture performance. In this study, we evaluated the dynamics of the cell cycle and apoptosis in a mAb-producing cell line during a fed-batch process. Results showed that the cells transitioned to the G0/G1 phase during the stationary phase. Although viability remained above 80% throughout the culture, early signs of apoptosis were observed as early as Day 5, coinciding with product accumulation. Our investigation revealed that early apoptotic cells were not productive and that removing apoptotic stimuli or adding fresh nutrients did not rescue these cells. This underscores the necessity for early process interventions. Media exchange after Day 7, when cells are in stationary phase, resulted in higher viability, fewer early apoptotic cells, and approximately a 26% increase in productivity compared to conditions with no or earlier media exchange. This study highlights the significance of timely interventions to remove the inhibitory byproducts and replenish the culture with fresh media, based on detailed process characterization.