RESUMEN
Continuous improvements of cell culture media are required in order to ensure high yield and product quality. However, some components can be instable and lead to detrimental effects on bioprocess performances. l-cysteine is an essential amino acid commonly used in cell culture media. Despite its beneficial effect on recombinant protein production, in some cases, this component can be responsible for product microheterogeneity. In this context, alternative components have to be found in order to reduce product variants while maintaining high productivity. In this study, we have assessed the performance of different cysteine and cystine analogs : N-acetyl-cysteine, s-sulfocysteine, N,N'-diacetyl-l-cystine and the N,N'-diacetyl-l-cystine dimethylester (DACDM). Replacement of cysteine by cystine analogs, and especially DACDM, has shown positive impact on charge variants level and recombinant protein coloration level. Moreover, this molecule contributed to the increase of the intracellular glutathione pool, which suggests a close relationship with the oxidative stress regulation.
Asunto(s)
Cisteína , Cistina , Estrés Oxidativo , Aminoácidos , Técnicas de Cultivo de Célula , Medios de Cultivo , Cisteína/metabolismo , Cistina/metabolismo , Glutatión/metabolismoRESUMEN
Glutathione (GSH) plays a central role in the redox balance maintenance in mammalian cells. Previous studies of industrial Chinese hamster ovary cell lines have demonstrated a relationship between GSH metabolism and clone productivity. However, a thorough investigation is required to understand this relationship and potentially highlight new targets for cell engineering. In this study, we have modulated the GSH intracellular content of an industrial cell line under bioprocess conditions 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). Over time of the bioprocess, a correlation between intracellular GSH and product titer has been observed. Analysis of metabolites uptake/secretion rates and proteome comparison between BSO-treated cells and nontreated cells has highlighted a slowdown of the tricarboxylic acid cycle leading to a secretion of lactate and alanine in the extracellular environment. Moreover, an adaptation of the GSH-related proteome has been observed with an upregulation of the regulatory subunit of glutamate-cysteine ligase and a downregulation of a specific GSH transferase subgroup, the Mu family. Surprisingly, the main impact of BSO treatment was observed on a global downregulation of the cholesterol synthesis pathways. As cholesterol is required for protein secretion, it could be the missing piece of the puzzle to finally elucidate the link between GSH synthesis and productivity.
Asunto(s)
Butionina Sulfoximina/metabolismo , Colesterol/metabolismo , Glutatión/metabolismo , Proteoma/metabolismo , Animales , Células CHO/metabolismo , Cricetulus , Proteoma/análisis , ProteómicaRESUMEN
Large scale biopharmaceutical production of biologics relies on the overexpression of foreign proteins by cells cultivated in stirred tank bioreactors. It is well recognized and documented fact that protein overexpression may impact host cell metabolism and that factors associated with large scale culture, such as the hydrodynamic forces and inhomogeneities within the bioreactors, may promote cellular stress. The metabolic adaptations required to support the high-level expression of recombinant proteins include increased energy production and improved secretory capacity, which, in turn, can lead to a rise of reactive oxygen species (ROS) generated through the respiration metabolism and the interaction with media components. Oxidative stress is defined as the imbalance between the production of free radicals and the antioxidant response within the cells. Accumulation of intracellular ROS can interfere with the cellular activities and exert cytotoxic effects via the alternation of cellular components. In this context, strategies aiming to alleviate oxidative stress generated during the culture have been developed to improve cell growth, productivity, and reduce product microheterogeneity. In this review, we present a summary of the different approaches used to decrease the oxidative stress in Chinese hamster ovary cells and highlight media development and cell engineering as the main pathways through which ROS levels may be kept under control.