The growing biopharmaceutical industry has reached a level of maturity that allows for the monitoring of numerous key variables for both process characterization and outcome predictions. Our study highlights the utility of genetically encoded redox biosensors to guide metabolic engineering or intervention strategies aimed at optimizing cell viability, growth, and productivity. Medium replenishment restored, albeit partially, the intracellular redox homeostasis. Under batch culture conditions, a significant and progressive oxidation of the bio-sensor occurred when CHO-K1-hGM-CSF cells entered the late-log phase. The redox reporter cell lines showed a sensitive and reversible response to different redox stimuli (reducing and oxi-dant reagents). The hGM-CSF production was not affected by the expression of the biosensor. Growth kinetics and cellular metabolism (glucose consumption, lactate and ammonia production) of the redox reporter cells were comparable to those of the parental cell lines. Flow cytometry was selected as the read-out technique for rxYFP signal given its high-throughput and statistical robustness.
In this study, we validate the use of cytosolic rxYFP on two cell lines widely used in biomanufacturing processes, namely, CHO-K1 cells expressing the human granulocyte macrophage colony-stimulating factor (hGM-CSF) and HEK-293.
Recently, different redox-sensitive variants of green fluorescent proteins (e.g., rxYFP, roGFP2, and rxmRuby2) have been engineered and proved suitable to detect, in a non-invasive manner, perturbations in the pool of reduced and oxidized glutathi-one, the major low molecular mass thiol in mammals. Thus, monitoring redox changes in real time and in situ is deemed essential for optimizing the production of recombinant proteins. Exogenous (i.e., nutrients, deterioration of media components, xenobiotics) and endogenous factors (i.e., metabolism, growth) may alter the redox homeo-stasis of cells.
Cellular functions such as DNA replication and protein translation are influenced by changes in the intracellular redox milieu.
0 kommentar(er)
