ORIGINAL RESEARCH article
Front. Bioeng. Biotechnol.
Sec. Bioprocess Engineering
Impact of keto leucine and isoleucine on CHO cell central carbon metabolism and performance in fed-batch and steady-state perfusion
Provisionally accepted
- 1Merck Life Science Germany GmbH, Darmstadt, Germany
- 2Technische Universitat Darmstadt Fachbereich Chemie, Darmstadt, Germany
- 3Metalytics Inc., Cary, NC, United States
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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 branched-chain keto acids' potential to improve the product yield was evaluated in fed-batch and simulated, steady-state perfusion. In fed-batch, equimolar replacement of isoleucine and/or leucine by their keto acids moderately increased (+6%) or maintained the cell-specific productivity qP, but the positive impact was counteracted by a reduction of cell growth up to -11%. Higher concentrations of keto acids substantially reduced cell growth (-42%) and qP (-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 as well as altered fluxes in anaplerotic reactions, ultimately leading to decreased ATP (-20%) and NADPH (-29%) generation. In steady-state perfusion, the keto acid supplementation improved the IgG yield up to 100% through (I) reduced bleed rates as a result of lower cell growth and (II) enhanced qP. 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.
Keywords: Bleed reduction, Branched-chain keto acids, Isoleucine, Leucine, Metabolic Flux Analysis, Metabolomics, Steady-state perfusion
Received: 18 Sep 2025; Accepted: 07 Jan 2026.
Copyright: © 2026 Reifenberg, Benjamin, Le Mignon and Zimmer. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence: Aline Zimmer
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