(184r) Antioxidants Supplementation to Cell Culture Media and Feed Influences Productivity and Product Quality of Mabs from CHO Cells

High expression of therapeutic proteins in Chinese Hamster Ovary (CHO) cells is crucial in biopharmaceutical processes to meet high product demand and reduce cost of goods. However, high level of recombinant protein synthesis results in high flux through metabolic and protein folding pathways which result in high oxidative stress. This oxidative stress can be characterized by high levels of reactive oxygen species (ROS), which can be detrimental to cellular function, lead to apoptosis, or can modify the desired therapeutic protein. In addition, the biopharmaceutical industry is transitioning away from the use of serum, which has antioxidant properties, and developing serum-free chemically defined media for production. To alleviate oxidative stress, cell culture media is supplemented with various antioxidants, such as glutathione, lipoic acid, retinyl acetate, ascorbic acid, and rosmarinic acid. However, some antioxidants have solubility issues in media, are toxic at high levels, and may interact with each other through similar redox pathways. Determining which antioxidants and their optimal levels in CHO cell media is key to improving cell viability and productivity for bioprocesses.

Results from this study showed that antioxidants supplemented to the production feed increased cell-specific productivity by 1.2-fold. However, antioxidants supplemented to both production media and production feed increased cell-specific productivity by 1.5-fold while inhibiting the cell growth, resulting in lower peak cell density compared to the control. To prevent early growth inhibition, antioxidants were added to the feed for intensified fed-batch reactors and to the production stage perfusion media for continuous manufacturing. This strategy enabled cultures to grow to high cell density first before introduction to the antioxidants that improved productivity. Intensified fed-batch reactors with antioxidant supplemented feed had 1.2-fold increase in titer compared to control, while perfusion bioreactors with antioxidant supplemented perfusion media had 1.3-to-1.7-fold increase in permeate titer throughout the process. In addition, the antioxidant supplementation to media and feed affected quality attributes of the mAb produced, such as increased fragmentation, increased aggregation, and lower mannose and galactosylation. This study provides insight into which antioxidants improve CHO cell culture productivity and/or viability and the strategies of supplementing these antioxidants for optimal effects.