Dissecting cell death pathways in fed-batch bioreactors

Chinese hamster ovary (CHO) cells are widely used for production of biologics including therapeutic monoclonal antibodies. Cell death in CHO cells is a significant factor in biopharmaceutical production, impacting both product yield and quality. Apoptosis has previously been described as the major form of cell death occurring in CHO cells in bioreactors. However, these studies were undertaken when less was known about non-apoptotic cell death pathways. Here, we report the occurrence of non-apoptotic cell death in an industrial antibody-producing CHO cell line during fed-batch culture. Under standard conditions, crucial markers of apoptosis were not observed despite a decrease in viability towards the end of the culture; only by increasing stress within the system did we observe caspase activation indicative of apoptosis. In contrast, markers of parthanatos and ferroptosis were observed during standard fed-batch culture, indicating that these non-apoptotic cell death pathways contribute to viability loss under these conditions. These findings pave the way for targeting non-conventional cell death pathways to improve viability and biologic production in CHO cells. Antibody-producing CHO cells decline in viability towards the end of fed-batch culture, which can have negative impacts on product yield and quality. Previous studies stated that apoptosis was the main cause of cell death. This study investigated more recently characterized non-apoptotic forms of cell death during fed-batch culture. Markers of apoptosis could not be observed under standard conditions. However, markers of two non-apoptotic pathways, parthanatos and ferroptosis, were associated with viability loss. Only by increasing the impeller speed could markers of apoptosis be observed. These findings provide a framework to improve biologics production by targeting non-apoptotic death pathways in CHO cells. image