Characterization of glutathione proteome in CHO cells and its relationship with productivity and cholesterol synthesis.

Abstract

Glutathione (GSH) plays a central role in the redox balance maintenance in mammalian cells. The study of industrial CHO cell lines have demonstrated a close link between GSH metabolism and clone productivity. However, a deep investigation is still required to understand this correlation and highlights new potential targets for cell engineering. In this study, we have modulated the GSH intracellular content of an industrial cell line under bioprocess conditions in order to further elucidate the role of the GSH synthesis pathway. Two strategies were used : the variation of cystine supply and the direct inhibition of the GSH synthesis using buthionine sulfoximine (BSO). Cysteine supply modulation have revealed a correlation between intracellular GSH and product titer over time. Analysis of metabolites uptake/secretion rates and proteome comparison between BSO-treated cells and non-treated cells has highlighted a slow down of the TCA cycle leading to a secretion of lactate and alanine in the extracellular environment. Moreover, an adaptation of the glutathione related proteome has been observed with a up-regulation of the regulatory subunit of glutamate cysteine ligase and a down-regulation of a specific glutathione transferase subgroup, the Mu family. Surprisingly, the main impact of BSO treatment was observed on a global down-regulation of the cholesterol synthesis pathways. As cholesterol is required for protein secretion, it can be the missing part of the jigsaw to finally elucidate the link between GSH synthesis and productivity.

Engineering redox sensors into CHO cells enables near‐real‐time quantification of intracellular redox in bioprocesses

“…Second, all cell lines were run using the same media, feeds, and bioprocess conditions, which are known to strongly influence the redox environment. Nonetheless, our other findings demonstrate large differences in total GSH content (Figure 4b), a major indicator of antioxidant capability, and quality attributes (Table 2) which are known to be affected by intracellular redox conditions (Ali et al, 2019; Chevallier, Schoof, et al, 2020; Sinharoy et al, 2020).…”

“…Given previous reports linking cellular redox environment and product quality (Ali et al, 2020; Chevallier, Schoof, et al, 2020; Handlogten, Zhu, et al, 2018; van der Reest et al, 2018), we examined whether roGFP2 expression had a downstream effect on protein aggregation, fragmentation, or charge variant profiles. Our results showcase the large product quality differences which can exist between cell lines, particularly with respect to aggregate levels (Table 2; Clone A, 3.6 ± 0.1 %Agg; Clone B, 0.5 ± 0.1 %Agg; Clone C, 42.1 ± 0.4 %Agg).…”

“…Redox reactions are essential for proper cellular function and are present in bioprocess‐critical pathways, including energy metabolism, protein folding, and cellular maintenance (Murphy, 2009; Sinharoy et al, 2021). Intracellular redox is primarily characterized by a balance of reactive oxygen species (ROS) (such as H 2 O 2 and O 2 · − ) produced through cellular metabolism and cellular capacity to detoxify ROS, typically through glutathione (GSH) and other antioxidants (Chevallier, Schoof, et al, 2020). GSH is a thiol‐based antioxidant that reduces damaging ROS as part of a redox cycle (Issels et al, 1988).…”

“…In this cycle, two GSH molecules dimerize into oxidized glutathione (GSSG), detoxifying ROS; GSSG is subsequently reduced enzymatically, restoring the cell's antioxidant capacity. Thus, efficient antioxidant cycling is critical to meet the significant energetic demands of high‐titer processes by maintaining intracellular redox homeostasis (Chevallier, Schoof, et al, 2020; Orellana et al, 2017).…”

High cysteine concentrations in cell culture media lead to oxidative stress and reduced bioprocess performance of recombinant CHO cells

“…Lately, the composition of CDM used for biomanufacturing has been shown to cause oxidative stress thus impacting cell culture performance and product quality. [5][6][7][8][9] In particular, the semi-essential amino acids L-cysteine (Cys)/cystine were shown to function as a source of reactive oxygen species (ROS) formation by reacting with several other CDM ingredients such as iron (Fe 2+ ) due to their highly reactive thiol groups. [8,10,11] In cell culture and even after media preparation, Cys is rapidly oxidized in presence of air or metal ions into L-cystine ((Cys)2) due to its high reactivity.…”