Modulating cell culture oxidative stress reduces protein glycation and acidic charge variant formation

Controlling acidic charge variants is critical for an industrial bioprocess due to the potential impact on therapeutic efficacy and safety. Achieving a consistent charge variant profile at manufacturing scale remains challenging and may require substantial resources to investigate effective control strategies. This is partially due to incomplete understanding of the underlying causes for charge variant formation during the cell culture process. To address this gap, we examined the effects of four process input factors (temperature, iron concentration, feed media age, and antioxidant (rosmarinic acid) concentration) on charge variant profile. These factors were found to affect the charge profile by modulating the cell culture oxidative state. Process conditions with higher acidic peaks corresponded to elevated supernatant peroxide concentration, intracellular reactive oxygen species (ROS) levels, or both. Changes in glycation level were the primary cause of the charge heterogeneity, and for the first time, supernatant peroxide was found to positively correlate with glycation levels. Based on these findings, a novel mathematical model was developed to demonstrate that the rate of acidic species formation was exponentially proportional to the concentrations of supernatant peroxide and protein product. This work provides critical insights into charge variant formation during the cell culture process and highlights the importance of modulating of cell culture oxidative stress for charge variant control.