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Improving the cellular capacity of Chinese hamster ovary (CHO) cells to produce large amounts of therapeutic proteins remains a major challenge for the biopharmaceutical industry. In previous studies, we observed strong correlations between the performance of CHO cells and expression of two transcription factors (TFs), MYC and XBP1s. Here, we have evaluated the effective of overexpression of these two TFs on CHO cell productivity. To address this goal, we generated an EPO-producing cell line (CHOEPO) using a targeted integration approach, and subsequently engineered it to co-overexpress MYC and XBP1s (a cell line referred to as CHOCXEPO). Cells overexpressing MYC and XBP1s increased simultaneously viable cell densities and EPO production, leading to an enhanced overall performance in cultures. These improvements resulted from the individual effect of each TF in the cell behaviour (i.e., MYC-growth and XBP1s-productivity). An evaluation of the CHOCXEPO cells under different environmental conditions (temperature and media glucose concentration) indicated that CHOCXEPO cells increased cell productivity in high glucose concentration. This study showed the potential of combining TF-based cell engineering and process optimisation for increasing CHO cell productivity.

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Chinese Hamster Ovary (CHO) cells are the main expression system for production of therapeutic recombinant (r-) proteins. The increased demand for these therapeutics has boosted the development of more robust production processes. Use of optimised feed/media has dramatically improved the performance of CHO cells in culture. However, such progress in biomass synthesis and r-protein production often come with an increased accumulation of lactate. In this study, we present a combined feeding strategy that uses galactose and lactate to replace glucose in CHO cell cultures. Replacement of glucose by galactose and lactate sustained cell growth and r-protein production in CHO cells. This strategy supported a better-balanced and more efficient metabolism, observed by an overall decreased consumption of carbon sources and amino acids, associated with an increased ATP production per C-mol consumed. Our results provide new insights of CHO cell metabolism in glucose-free media based on galactose and lactate. and glutamate, respectively, led to a significantly decreased rate of lactate production (C. Altamirano et al., 2000). However, with the implementation of this strategy, cell growth was dramatically decreased. In a subsequent study, a biphasic culture strategy using glucose and 4 galactose as primary and secondary carbon sources was designed to overcome the growth limitations observed in wholly galactose-based cultures. While the initial phase was characterised by a rapid cell growth with high rates of glucose consumption and lactate production, the second phase, when glucose was depleted, was characterised by the consumption of both galactose and lactate (produced in the initial phase) (Altamirano et al., 2006; Sun et al., 2013). To understand this phenomenon, Wilkens et al., (2011) studied lactate metabolism in CHO cell batch cultures growing in glucose and galactose using a metabolic flux analysis approach. These authors suggested that the lactate switch (i.e., from production to consumption) occurred when cytosolic pyruvate concentration was insufficient for cells to support their energy requirements, and that lactate uptake emerges as an alternative to incorporate carbon into the TCA cycle and improve energy metabolism (Wilkens et al., 2011). Similar observations were made in two different CHO cell lines (Gray et al., 2012; Sun et al., 2013) and in a mouse myeloma cell line (NS0) (Mulukutla et al., 2012) that underwent a lactate switch. Whilst galactose supplementation had little impact on r-protein production in CHO cell cultures, it did alter r-protein glycosylation (

Piscirickettsia salmonis is a facultative Gram-negative intracellular bacterium that produces piscirickettsiosis, disease that causes a high negative impact in salmonid cultures. The so-far-unidentified nutritional requirements have hindered its axenic culture at laboratory and industrial scales for the formulation of vaccines. The present study describes the development of a defined culture medium for P. salmonis. The culture medium was formulated through rational design involving auxotrophy test and statistical designs of experiments, considering the genome-scale metabolic reconstruction of P. salmonis reported by our group. The whole optimization process allowed for a twofold increase in biomass and a reduction of about 50% of the amino acids added to the culture medium. The final culture medium contains twelve amino acids, where glutamic acid, threonine and arginine were the main carbon and energy sources, supporting 1.65 g/L of biomass using 6.5 g/L of amino acids in the formulation. These results will contribute significantly to the development of new operational strategies to culture this bacterium for the production of vaccines.