title: Impact of S-sulfocysteine on Chinese hamster ovary cells
Creators: name: Nguyen, Melanie
Publisher: name: UNSPECIFIED
Year: 2025

description: In the biopharmaceutical industry, Chinese hamster ovary (CHO) cells are the primary workhorse for therapeutic protein production (monoclonal antibodies, fusion proteins) in large-scale processes — constantly undergoing optimization to increase the product yield. In this context, optimized cell culture media and feeds are critical and often rely on cysteine (Cys) analogues to overcome the Cys-related stability and solubility issues at neutral pH. Previously, the Cys analogue, S-sulfocysteine (SSC), was successfully applied in cell culture, implying the SSC uptake, metabolization and biological activity, though the latter have not yet been characterized thoroughly. Furthermore, SSC also demonstrated a concentration-dependent toxicity in cells. Therefore, the present study sought to investigate the SSC metabolism and activity in more detail to provide an enhanced understanding and knowledge surrounding its beneficial, non-toxic and toxic characteristics.
In the first part of the study, the CHO-K1 model clone was investigated in the presence of SSC at a non-toxic concentration to understand its metabolism/activity, and underlying mechanism(s) of action. Therefore, a CHO-K1-based fed-batch process (+ pH control), multi-omics and mechanistic approaches were performed, pointing out the following hallmarks: (1) SSC/glutathione (GSH) mixed disulfide formation, (2) glutaredoxin 1- and glutathione reductase (GR)-conveyed reduction and (3) sulfur species liberation, followed by their diversion towards catabolic pathways. The SSC-derived molecules, GSH, taurine and sulfite, were identified as those that are most likely conferring the favorable activity via their antioxidant and protective roles.
In the second part of the study, the CHO-K1 clone was examined using SSC at toxic concentrations to understand the mechanism(s) of toxicity. The CHO-K1 fed-batch process (± pH control) pointed to the SSC toxicity in the pH- surrounding that correlated with an extracellular pH decrease — likely reflecting an intracellular pH drop. The sulfo-glutathione (GS-SO3) mixed disulfide reduction, particularly the GR-conveyed GSH recycling, was strongly hampered at lower pH levels in vitro. Complementary to these findings, CHO-K1 cells experienced an aggravated SSC toxicity upon a GSH decrease triggered by buthionine sulfoximine, while control cells (without SSC treatment) remained vital. These results underline the importance of GSH, though its deficit appeared not to be solely driving the toxicity. Intensified SSC toxicity further correlated with a hindered GS-SO3 reduction and export. Mitigation strategies to prevent the SSC toxicity through the addition of sodium carbonate, sodium glutamate and copper (II) sulfate to the feed were successful.
In the third part of the study, the CHO-K1, CHOZN and CHO-DG44 clones, exhibiting phenotypic heterogeneity, were explored using SSC at wide-ranging doses to provide complementary data. The CHO-DG44 clone showed the lowest SSC sensitivity that was linked to a putative lower SSC uptake capacity, as well as a higher reactive sulfur and oxygen species scavenging capacity. This SSC-related knowledge regarding its dual nature paves the way for further research, and may be transferred to other clones that may respond differently to this modified amino acid.