Monitoring Physiological Response to ROS in CHO Fed-Batch Culture

• Justin Stauffer, Chemical Engineering, University of Delaware

• Mark Blenner, Chemical and Biomolecular Engineering, University of Delaware

Reactive oxygen species (ROS) are natural byproducts of cell processes. At low concentrations, ROS are beneficial to the cell through oxidative signaling, but at high concentrations ROS can lead to DNA damage and cell death. Cells regulate ROS levels by producing Glutathione, which reacts with and reduces ROS to non-harmful compounds, such as water. In Chinese Hamster Ovary (CHO) cell fed-batch culture, the cells remain in the same flask throughout, allowing ROS to build up over time. When ROS levels become too high to manage, the risk of DNA damage and early culture death increases, potentially affecting the yield and quality of antibody products. Current methods of monitoring ROS levels in CHO fed batch are expensive assays that utilize fluorescence. By exploring the relationship between ROS and Glutathione levels in fed-batch culture, the development of a cheaper polymer to measure ROS levels is ongoing. ROS and Glutathione levels were monitored in two fed-batch runs, one of which was exposed to physiological conditions, and one which was spiked to known concentrations of Hydrogen Peroxide, a potent ROS. Cultures were monitored using ROS and Glutathione assays to obtain relative fluorescence levels over time. Through multiple rounds of testing, signs of a reactive relationship were observed. Cells produced more Glutathione as ROS levels spiked, which was followed by a decrease in ROS levels as the Glutathione is consumed. A future fed-batch run will be conducted with continuous spiking of Hydrogen Peroxide and similar monitoring of intracellular species. This will determine the ROS levels CHO cells can manage over time and their cumulative tolerance for oxidative stress.