Industrial fed-batch cultivation of mammalian cells is used for the production of therapeutic proteins such as monoclonal antibodies. Besides medium ensuring initial growth, feeding is necessary to improve growth, viability and antibody production. Established processes include a slight acidic main feed and a separate alkaline feed containing l-tyrosine and l-cysteine. Since l-cysteine is not stable at neutral pH, a new derivative, S-sulfocysteine, was tested in neutral pH feeds. In small scale fed-batch processes, the S-sulfocysteine process yielded a comparable maximum viable cell density, prolonged viability and increased titer compared to the two feed system. Bioreactor experiments confirmed the increase in specific productivity. In depth characterization of the monoclonal antibody indicated no change in the glycosylation, or charge variant pattern whereas peptide mapping experiments were not able to detect any integration of the modified amino acid in the sequence of the monoclonal antibody. Finally, the mechanism of action of S-sulfocysteine was investigated, and results pointed out the anti-oxidative potential of the molecule, mediated through an increase in superoxide dismutase enzyme levels and in the total intracellular glutathione pool. Finally, we propose that the increase in specific productivity obtained in the S-sulfocysteine process results from the anti-oxidative properties of the molecule.

The ability of cell culture media components to generate reactive species as well as their sensitivity to oxidative degradation, affects the overall stability of media and the behavior of cells cultured in vitro. This study investigates the influence of thiazolidine molecules, formed from the condensation between cysteine and alpha-ketoacids, on the stability of these complex mixtures and on the performance of cell culture processes aiming to produce therapeutically relevant monoclonal antibodies. Results presented in this study indicate that 2-methyl-1,3-thiazolidine-2,4-dicarboxylic acid and 2-(2-carboxyethyl)-1,3-thiazolidine-2,4-dicarboxylic acid, obtained by condensation of cysteine with pyruvate or alpha-ketoglutarate, respectively, are able to stabilize cell culture media formulations, in particular redox sensitive molecules like folic acid, thiamine, l-methionine (met) and l-tryptophan (trp). The use of thiazolidine containing feeds in Chinese hamster ovary fed-batch processes showed prolonged culture duration and increased productivity. This enhanced performance was correlated with lower reactive species generation, extracellularly and intracellularly. Moreover, an anti-oxidative response was triggered via the induction of superoxide dismutase and an increase in the total glutathione pool, the major intracellular antioxidant. In total, the results confirm that cells in vitro are not cultured in an oxidant-free environment, a concept that has to be considered when studying the influence of reactive species in human diseases. Furthermore, this study indicates that thiazolidines are an interesting class of antioxidant molecules, capable of increasing cell culture media stability and process performance. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:759-770, 2017.