Engineering Carbon and Energy Metabolism for CHO Cells for Improved Productivity and Extended Lifespan

Several studies have shown that lactate's specific production rate and concentration inhibit the production of the recombinant protein in mammalian cell cultures. In a previous work we showed that lactate production was associated to a fast consumption of sugar leading to a high pyruvate synthesis rate. MFA indicates that pyruvate entry into the TCA cycle was limiting, causing pyruvate accumulation inside the cell which leads to lactate production [1].

Improvement in specific protein productivity has been observed after introducing specific changes in key points of the central carbon metabolism through cell engineering. Expression of the yeast pyruvate carboxylase enzyme provided the cell an alternative pathway for pyruvate to enter the TCA cycle [2]. Over expression of the enzyme malate dehydrogenase II which catalyzes a limiting step in the TCA cycle also improved the cycle's flux [3]. Manipulation of sugar transport leaded to a reduced carbon source consumption and lower lactate synthesis [4]. Finally, a knock out of the lactate dehydrogenase gene reduced the specific rate of lactate production [5].

In this work we aim at improving cell metabolism through cell engineering by reducing lactate formation, pyruvate accumulation and increasing pyruvate flux towards energy metabolism of a cell line. These changes will be achieved by transfecting CHO DP-12 cells producing Human IgG with different genes involved in the central carbon metabolism that were selected based on the empirical evidence of other studies. After transfecting the cells with the an alternative sugar transporter we observed in the selected clones a reduced flux in the glycolitic pathway which lead to decreased lactate synthesis. By transfecting the cells with the PYC or MDHII gene there is further improvement in the metabolism due to a reduction in pyruvate accumulation which in turn decreases lactate production. Metabolic flux redistribution is studied in all the new engineered cell lines through metabolic flux analysis to compare carbon and amino acid utilization in the engineered cells with respect to the wildtype under normal culture conditions.

Engineered cells exhibit a lower production of lactate, increased fluxes in the TCA cycle, and a redistribution in their metabolism. Cells with an improved metabolism are capable of achieving higher cell densities in culture with an extended lifespan and show an increase in recombinant protein production. 

[1] Wilkens, C.A., Altamirano, C., Gerdtzen, Z.P., Biotechnol. Bioprocess Eng (2011, accepted)

[2] Irani, N., Wirth, M., van Den Heuvel, J., Wagner, R., Biotechnol Bioeng, 66(4):238-46 (1999)

[3] Chong, W.P., Reddy, S.G., Yusufi, F.N., Lee, D.Y., Wong, N.S., Heng, C.K., Yap, M.G., Ho, Y.S., J Biotechnol, 147(2):116-21 (2010)

[4] Wlaschin, K.F., Hu, W-S., J Biotechnol, 131(2):168-76 (2007)

[5] Chen, K., Liu, Q., Xie, L., Sharp, P.A., Wang, D.I., Biotechnol Bioeng, 72(1):55-61 (2001)