Cell and gene therapies have the potential to treat cancer, genetic disorders, and other life-threatening diseases, yet their high cost and manufacturing variability limit patient access. A major challenge is the lack of real-time monitoring of key metabolic variables in static cell culture systems. Dissolved oxygen (DO), dissolved carbon dioxide (dCO2), and glucose regulate cellular respiration and metabolic balance, but static G-Rex workflows typically rely on incubator setpoints and intermittent off-line sampling, offering limited insight into dynamic culture behavior. To address this gap, we evaluated 10 to 12 day human embryonic kidney (HEK) cell cultures in static G-Rex systems to assess the feasibility of continuous DO and dCO2 monitoring under production-relevant conditions. HEK cells were selected as a model system due to their well-characterized metabolic and respiratory profiles. Using real-time DO sensors and a rate-based approach to estimate dCO2, we demonstrated proof-of-concept for continuous, 24/7 monitoring over extended culture durations. Continuous monitoring provided more detailed insight into culture dynamics than traditional methods. These findings support integration of process analytical technology strategies into static G-Rex workflows, enabling greater automation, improved process consistency, and more informative batch records to support regulatory review and scalable, affordable manufacturing.
