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.