Antioxidant effect of thiazolidine molecules in cell culture media improves stability and performance

Abstract

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.

Impact of S-sulfocysteine on fragments and trisulfide bond linkages in monoclonal antibodies

“…In the second condition, cysteine was allowed to interact with pyruvate in the feed, allowing formation of the thiazolidine molecule 2-methyl-1,3-thiazolidine-2,4-dicarboxylic acid (CP). 13,14 This condensation product has demonstrated its efficacy in stabilizing cell culture media formulations containing cysteine, 15,16 and has been described as a tool to reduce trisulfide bond formation in IgG. 9 It was used in this study as positive control.…”

“…The potential mechanisms explaining this increase in productivity were described elsewhere. 16,17 To demonstrate that the observed effect is not specific to the clone, the process or the cell culture media platform chosen for this study, similar experiments were performed using different processes, cell lines producing other mAbs and fed-batch platforms. The overall specific productivity obtained is presented in Fig.…”

Interaction of cell culture process parameters for modulating mAb afucosylation

“…Stability issues with cell culture media have been previously associated with HTST treatment (Floris et al, 2018) or with extended storage in the cold or at room temperature (Hossler, McDermott, Racicot, & Fann, 2013;Kuschelewski, Schnellbaecher, Pering, Wehsling, & Zimmer, 2017;McCoy, Costa, & Morris, 2015). These extensive investigations identified a number of root causes for media F I G U R E 3 (a) Effect of pCO 2 model, media hold, and Mn supplementation on afucosylation of mAb at time of harvest (Day 12) in a full-factorial DOE study.…”

Elucidating the Impact of CHO Cell Culture Media on Tryptophan Oxidation of a Monoclonal Antibody Through Gene Expression Analyses

“…Further studies would be necessary to fully elucidate the mechanism of cysteine's effect and decipher any interactions among the four factors in lowering Trp‐ox.Collectively, the four factors used in the CDM synergistically regulated the expression of the genes described above, which in turn may play significant roles in lowering the Trp‐ox through controlling oxidative stress and balancing copper and glutathione pools without impacting cell growth and mAb productivity under CDM conditions. The impact of the glutathione pool, or more generally, the intracellular redox status of the cells, on CHO cell growth and productivity has been described in the literature . The results of our transcriptome analyses, to our knowledge, are the first indication that a proper glutathione pool may also directly affect mAb modifications without impacting CHO cell growth.…”