In silico model-based characterization of metabolic response to harsh sparging stress in fed-batch CHO cell cultures.

Mammalian cell culture platform has been successfully implemented for industrial biopharmaceutical production through the advancements in early stage process development including cell-line engineering, media design and process optimization. However, late stage developments such as scale-up, scale-down and large-scale cell cultivation still face many industrial challenges to acquire comparable process performance between different culture scales. One of them is the sparging strategy which significantly affects productivity, quality and comparability. Currently, it is mainly relying on the empirical records due to the lack of theoretical framework and scarcity of available literatures to elucidate intracellular metabolic features. Therefore, it is highly required to characterize the underlying mechanism of physiological changes and metabolic states upon the aeration stress. To this end, initially we cultivated antibody producing CHO cells under mild and harsh sparging conditions and observed that sparging stress leads to the decreased cell growth rate, viability and productivity. Subsequent in silicomodel-driven flux analysis suggested that sparging stress rewires amino acid metabolism towards the enriched H2O2 turnover rate by up-regulated fluxes of amino acid oxidases. Interestingly, many of these H2O2-generating reactions were closely connected with the production of NADH, NADPH and GSH which are typical reducing equivalents. Thus, we can hypothesize that increased amino acid uptake caused by sparging stress contributes to restore redox homeostasis against oxidative stress. The current model-driven systematic data analysis allows us to quickly define distinct metabolic feature under stress condition by using basic cell cultivation datasets.

Production of 2,3-butanediol by a low-acid producing Klebsiella oxytoca NBRF4.

2,3-Butanediol (2,3-BDO) is a value-added chemical with great potential for the industrial production of synthetic rubber, plastic and solvent. For microbial production of 2,3-BDO, in this study, Klebsiella oxytoca NBRF4 was constructed by chemical mutation and screening against NaBr, NaBrO(3) and fluoroacetate. Among metabolic enzymes involved in the production of lactate, acetate and 2,3-BDO, K. oxytoca NBRF4 possessed 1.2 times lower specific activities of lactate dehydrogenase and phosphotransacetylase, and 22% higher specific acetoin reductase activity than the K. oxytoca ATCC43863 control strain. A series of batch fermentations in a defined medium and application of a statistical tool of response surface method led to the determination of optimal culture conditions: 10% dissolved oxygen level, pH 4.3 and 38°C. The actual results of batch fermentation at the optimal conditions using 44 g/L glucose were coincident with the predetermined values: 14.4 g/L 2,3-BDO concentration, 0.32 g/g yield. To increase 2,3-BDO titer, fed-batch fermentation of K. oxytoca NBRF4 was performed by an intermittent feeding of 800 g/L glucose to control its concentration around 5-20 g/L in the culture broth. Finally, 34.2g/L 2,3-BDO concentration and 0.35 g/g yield were obtained without organic acid production in 70 hours of the fed-batch culture, which were 2.4 and 1.2 times higher than those of the batch fermentation using 44 g/L glucose.