Impact of keto leucine and isoleucine on CHO cell central carbon metabolism and performance in fed-batch and steady-state perfusion

The keto acids of isoleucine and leucine are bioavailable precursors of their branched-chain amino acids in Chinese hamster ovary (CHO) cells, which are used to produce biotherapeutics at industrial scale. In this study, the potential of branched-chain keto acids to improve product yield was evaluated in fed-batch and simulated steady-state perfusion. In fed-batch, combined or individual replacement of isoleucine and leucine at equimolar concentrations with their keto acids moderately increased (+6%) or maintained the cell-specific productivity <i>qP</i>, but this positive impact was counteracted by a reduction in cell growth up to -11%. Higher concentrations of keto acids substantially reduced cell growth (-42%) and <i>qP</i> (-25%). ¹³C-metabolic flux analysis during the growth phase of the fed-batch revealed that this detrimental effect may be associated with impaired glycolysis and TCA cycle activity, along with altered fluxes in anaplerotic reactions, ultimately leading to decreased ATP (-20%) and NADPH (-29%) generation. In steady-state perfusion, keto acid supplementation improved IgG yield up to 100% through (I) reduced bleed rates as a result of lower cell growth and (II) enhanced <i>qP</i>. Untargeted metabolite profiling demonstrated altered levels of various compounds, suggesting pathways that may be associated with the observed improvements. Overall, the findings of this study highlight the potential of novel media components, such as keto isoleucine and keto leucine, to improve yields and efficiency in biopharmaceutical production, thereby contributing to increased sustainability and lower manufacturing costs.