Introduction

Under bioprocess conditions, CHO cells can be exposed to oxygen microheterogeneity, free radicals generated by cell culture medium components and high oxidative metabolism, which can lead to oxidative stress. Moreover, product quality can also be affected by reactive oxygen species (ROS) production. For these and other reasons, research related to the control of oxidative stress has been of increased interest. In this context, the controlled modulation of oxidative stress can help scientists to improve bioprocesses.
One of the main targets for modulation of oxidative stress is glutathione (GSH). GSH is a tripeptide (γ-L-glutamyl-L-cysteinyl glycine) which is the cofactor of ROS detoxification enzymes, as well as a direct ROS scavenger. Glutathione can also form conjugates with reactive electrophilic compounds to promote their detoxification (Ketterer et al., 1983). It plays a central role in the detoxification of ROS produced in the mitochondria, but also in the regulation of disulfide bond formation in the endoplasmic reticulum (Chakravarthi et al., 2004; Ribas et al., 2014).
A potential relationship between GSH and secreted protein productivity has already been suggested in literature. High producing cell lines have been shown to contain more GSH than low producers and an up-regulated GSH metabolism (Chong et al., 2012; Orellana et al., 2015). In order to reproduce this phenomena, cell engineering has been performed to increase GSH synthesis (Orellana et al., 2017). On the one hand, overexpression of the catalytic subunit of glutamate cysteine ligase (GCLc), the rate limiting enzyme in GSH synthesis, did not lead to increased titers despite higher GSH levels. On the other hand, the overexpression of the regulatory subunit of glutamate cysteine ligase also called the glutamate‐cysteine ligase modifier subunit (GCLm) led to an increase of productivity.
The uncertainty around the actual role of GSH in CHO bioprocessing led us to investigate the role of this metabolite further. Indeed, if the absolute GSH quantity does not explain productivity between two different clones, can it explain productivity differences between two processes with the same clone? What are the other cellular functions that are directly or indirectly impacted by the intracellular levels of GSH? In this context we have modulated intracellular GSH levels using two approaches: the variation of cystine supply through feed medium composition and direct inhibition of the GCL enzyme using buthionine sulfoximine (BSO). Our goal was to understand which pathways are actually affected by lower levels of GSH. To capture the metabolic adaptations to these two variations we investigated the cell phenotype, measured metabolites involved in the central carbon metabolism, and performed proteomic analysis.