Engineering Oxidative Stress Resistance in CHO Cell Factories

Oxidative stress is a phenomenon created by an imbalance in the amount of Reactive Oxygen Species (ROS) created within a cell, and the ability of its defence mechanisms to effectively deal with ROS. Oxidative stress is extremely deleterious to the cell, and is known to cause damage to DNA, proteins and lipids (Turrens, 2003). Mitochondria are the cell’s predominant producer of ROS (Murphy, 2009), but it has also been shown that increased protein folding in the Endoplasmic Reticulum (ER) results in an increase in ROS levels (Malhotra, 2008), an issue particularly pertinent as developers move towards hard-to-express proteins. As well as many enzymes dedicated to the eradication of ROS, such as caspases, peroxidases and superoxide dismutases (SODs) the cell maintains a glutathione pool to buffer the increase of ROS (Lu, 2009).
Design of Experiment models were designed and implemented using the growth, productivity and ROS content data from batch experiments in order to design anti-oxidant supplementation strategies. Two rounds of fed-batch screening were performed and a feeding strategy identified that improved the growth and ROS burden of three cell lines producing the same recombinant MAb product.
A directed evolution strategy was employed to engineer oxidative stress resistant host cell lines through chronic exposure to Hydrogen Peroxide. Following transfection with a recombinant MAb product, the novel engineered cell line consistently outperformed the original cell line in terms of growth and ROS content, in both transient and stable transfection processes. Doubling time of stably transfected evolved cell line was reduced to 23 hours, a substantial time frame reduction.
A link between ROS level reduction and improvement in cell line performance was demonstrated, with further investigation needed to unpick the mechanistic underpinnings of the oxidative stress resistance as well as to attempt to address the imbalance of improvements in growth compared to productivity.