1 Materials and Methods

To investigate the effect of forcing overexpression of two functional genes on cell viability and MAb production in CHO-DXB11 cells, we cloned hamster TAUT and human ALT1 cDNA, introduced their expression plasmids into MAb-producing DXB11 cells and DXB11 host cells, and isolated all high-growth cells. We then selected TAUT-overexpressing cell lines (MAb1/DXB11/TAUT) and characterized them with null plasmid-transfected cell lines (MAb1/DXB11/null) and parent cells (MAb1/DXB11) as controls under various conditions (Table 1).

Table 1 Effects of TAUT and ALT1 overexpression in CHO-DXB11 cells
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2 Results and Discussions

On the seventh day of spinner culture, the TAUT-overexpressing cell lines produced significantly higher yields (p < 0.01) and significantly less lactate (p < 0.05).

Next, we compared the performance of a high-yield MAb1/DXB11/TAUT cell line (T10) to the parent cell line in long-term bioreactor culture. The T10 cells showed “super-immortality”, that is, they maintained >80% viability up to the 32nd day of culture, while the parent cells became biologically degraded.

T10 cells also showed enhanced consumption of glutamine (>200% higher than parent cells) independently of taurine. Glutamine is a major energy source and is a biosynthetic precursor for cell growth that is required as a carbon source for the TCA cycle, so enhanced glutamine consumption may give a survival advantage. We found that glutathione pathway genes were upregulated in T10 cells, so we concluded that their enhanced glutamine consumption was likely to have prolonged their survival. Furthermore, when we attributed the higher product yield of T10 cells not only to prolonged viability but also to the activated consumption of glutamine, their increased viability was consistent with the suppression of TNF-α mRNA and partial up-regulation of bcl2 mRNA seen in T10 cells on the 13th day of culture. This was the day when a difference in viability became noticeable in 1-L bioreactor culture. Moreover, there was increased yield throughout the culture period.

Although T10 cells produced higher yields of MAb1 (2.9 g/L on Day 32), the MAb1 yield was still limited by the growth capacity of the cells. We therefore tried modifying the cells further, with the aim of achieving even higher yield in a shorter time. We therefore forced the overexpression of ALT1, an enzyme that catalyzes reversible transamination between alanine and 2-oxoglutarate to form pyruvate and glutamate. We did this because alanine, a substrate for ALT1, accumulates late in the culture period, and we hypothesized that the accumulation of alanine in vivo might trigger the enzyme function of overexpressed ALT1 causing enhancement of the MAb yield. We found that the independent overexpression of ALT1 significantly increased MAb1 yields (p < 0.01). We also found that co-overexpression of both ALT1 and TAUT (T10/ALT1) significantly increased MAb1 yields compared to T10 cells (p < 0.01). This co-overexpression strategy was also effective in 1-L bioreactor culture: the yield from MAb1-producing T10/ALT1 cells was 5.3 g/L on Day 21, which is a higher yield in a shorter time than we obtained from T10 cells (2.9 g/L on Day 32).

We also established new ALT1-overexpressing host cells (DXB11/TAUT/ALT1) and used them to establish MAb2-producing cells. The yield from these MAb2-producing cells in a 1-L bioreactor was 5.2 g/L on Day 17, demonstrating enhanced production of a different antibody.

We concluded that co-overexpression of ALT1 and TAUT shows promise as a new strategy for enhancing cell culture performance and increasing MAb yield.