UMMS Affiliation

Department of Biochemistry and Molecular Pharmacology

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Amino Acids, Peptides, and Proteins | Biochemistry, Biophysics, and Structural Biology | Cell and Developmental Biology


Mammalian cells consume large amount of nutrients during growth and production. However, endogenous metabolic inefficiencies often prevent cells to fully utilize nutrients to support growth and protein production. Instead, significant fraction of fed nutrients is diverted into extracellular accumulation of waste by-products and metabolites, further inhibiting proliferation and protein synthesis. In this study, an LC-MS/MS based metabolomics pipeline was used to screen Chinese hamster ovary (CHO) extracellular metabolites. Six out of eight identified inhibitory metabolites, caused by the inefficient cell metabolism, were not previously studied in CHO cells: aconitic acid, 2-hydroxyisocaproic acid, methylsuccinic acid, cytidine monophosphate, trigonelline, and n-acetyl putrescine. When supplemented back into a fed-batch culture, significant reduction in cellular growth was observed in the presence of each metabolite and all the identified metabolites were shown to impact the glycosylation of a model secreted antibody, with seven of these also reducing CHO cellular productivity (titer) and all eight inhibiting the formation of mono-galactosylated biantennary (G1F) and biantennary galactosylated (G2F) N-glycans. These inhibitory metabolites further impact the metabolism of cells, leading to a significant reduction in CHO cellular growth and specific productivity in fed-batch culture (maximum reductions of 27.2% and 40.6% respectively). In-depth pathway analysis revealed that these metabolites are produced when cells utilize major energy sources such as glucose and select amino acids (tryptophan, arginine, isoleucine, and leucine) for growth, maintenance, and protein production. Furthermore, these novel inhibitory metabolites were observed to accumulate in multiple CHO cell lines (CHO-K1 and CHO-GS) as well as HEK293 cell line. This study provides a robust and holistic methodology to incorporate global metabolomic analysis into cell culture studies for elucidation and structural verification of novel metabolites that participate in key metabolic pathways to growth, production, and post-translational modification in biopharmaceutical production.


Amino acid metabolism, Glycolysis, Inhibitory metabolites, Mammalian cell culture, Metabolomics, Process development

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© 2021 Published by Elsevier B.V. on behalf of International Metabolic Engineering Society. This is an open access article under the CC BY-NC-ND license (

DOI of Published Version



Kuang B, Dhara VG, Hoang D, Jenkins J, Ladiwala P, Tan Y, Shaffer SA, Galbraith SC, Betenbaugh MJ, Yoon S. Identification of novel inhibitory metabolites and impact verification on growth and protein synthesis in mammalian cells. Metab Eng Commun. 2021 Sep 6;13:e00182. doi: 10.1016/j.mec.2021.e00182. PMID: 34522610; PMCID: PMC8427323. Link to article on publisher's site

Journal/Book/Conference Title

Metabolic engineering communications

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Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License.