Engineering of Chinese hamster ovary cells for co-overexpressing MYC and XBP1s increased cell proliferation and recombinant EPO production

Abstract

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.

Enhancing Antibody-Specific Productivity: Unraveling the Impact of XBP1s Overexpression and Glutamine Availability in SP2/0 Cells

“…In contrast, the abundance of glutamine positively affected both transfectants, resulting in a similar growth rate of 0.9 day −1 , almost 24% higher than TAFE. While at low [Gln], h-XBP1s overexpression did not significantly influence μ, as all clones exhibited similar growth rates (∼0.7 day −1 ), aligning with the findings in CHO cells overexpressing XBP1s cultured with low glutamine concentration (2 mM) [ 27 ].…”

“…In CHO cells, where the endogenous XBP1s factor is weakly expressed [ 31 ], overexpressing this transcription factor has proven to increase specific productivity by 5.4-fold for monoclonal antibodies [ 44 ], and by 4.5, 2.1, 5, and 2-fold for VEGF, SAMY, SEAP, and total protein, respectively [ 31 ]. Our research group has contributed to the evidence in CHO cells, showing only slight impacts of XBP1s overexpression over erythropoietin-specific productivity [ 27 ]. In B-cells, it has been shown that the mechanism behind these improvements involves the induction of genes facilitating protein translocation into the ER, protein folding, and trafficking in the secretory pathway [ 30 ], and expansion of the endoplasmic reticulum in CHO-K1-derived cell lines when XBP1s is overexpressed [ 31 ].…”

“…Even more, XBP1s plays a crucial role in protein production and immunoglobulin secretion, as its deficiency leads to a 50% reduction in secreted antibodies and an inability to effectively manage the propagation of infections caused by polyomavirus [ 24 , 25 ], while the knockdown of XBP1s expression in the liver demonstrated reduced cholesterol levels [ 26 ]. Furthermore, overexpression of XBP1s leads to the increase in recombinant proteins in CHO cells, NS0 cells, and HEK cells [ 27 , 28 , 29 ]. It has been suggested that overexpression of XBP1s in B-cell lines induces the expression of genes involved in enhancing the ER processing capacity, such as protein translocation into the ER, protein folding, trafficking in the secretory pathway [ 30 ], expansion of the ER, and finally increasing the overall production capacity [ 31 ].…”

Rosmarinic acid enhances CHO cell productivity and proliferation through activation of the unfolded protein response and the mTOR pathway

“…Lastly, intracellular and extracellular IgG levels were not significantly different from control cultures (Figure 7C,D), possibly since the cells were still in the early exponential phase (48 h post-inoculation) and protein production was insignificant at this time point. Thus, XBP1 splicing may be a more reli-able marker for productivity enhancement, since XBP1 splicing has been reported to correlate with antibody production, [105,106] likely through enhancement of molecular chaperone translation and protein folding and secretion. Note that molecular chaperone overexpression is indispensable for folding non-native, difficult-to-fold proteins.…”

CHOsynthetic promoters improve expression and product quality of biotherapeutic proteins

“…Moreover, promoter squelching might affect the overexpression of multiple target genes if their expression is driven by promoters using the same TRFEs. XBP-1s is a transcription activator involved in regulation of both protein secretion and the unfolded protein response and it is well studied in rProtein expression, 24,26,27 therefore in this study we sought to finely control the overexpression level of XBP-1s, using synthetic promoters of varying strengths and determine the impact on DTE mAb expression in CHO cells. The synthetic promoters contain TFREs for transcription factors that are abundant in CHO and are therefore not likely to be limited in their capacity to drive expression.…”