Comprehensive stable-isotope tracing of glucose and amino acids identifies metabolic by-products and their sources in CHO cell culture.

Autor:	Gonzalez JE; Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE 19716., Naik HM; Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD 21218., Oates EH; Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE 19716., Dhara VG; Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD 21218., McConnell BO; Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE 19716., Kumar S; Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD 21218., Betenbaugh MJ; Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD 21218., Antoniewicz MR; Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE 19716.; Department of Chemical Engineering, University of Michigan, Ann Arbor, MI 48109.
Jazyk:	angličtina
Zdroj:	Proceedings of the National Academy of Sciences of the United States of America [Proc Natl Acad Sci U S A] 2024 Oct 08; Vol. 121 (41), pp. e2403033121. Date of Electronic Publication: 2024 Oct 04.
DOI:	10.1073/pnas.2403033121
Abstrakt:	Mammalian cell culture processes are widely utilized for biotherapeutics production, disease diagnostics, and biosensors, and hence, should be optimized to support robust cell growth and viability. However, toxic by-products accumulate in cultures due to inefficiencies in metabolic activities and nutrient utilization. In this study, we applied comprehensive 13 C stable-isotope tracing of amino acids and glucose to two Immunoglobulin G (IgG) producing Chinese Hamster Ovary (CHO) cell lines to identify secreted by-products and trace their origins. CHO cells were cultured in media formulations missing a single amino acid or glucose supplemented with a 13 C-tracer of the missing substrate, followed by gas chromatography-mass spectrometry (GC-MS) analysis to track labeled carbon flows and identify by-products. We tracked the sources of all secreted by-products and verified the identity of 45 by-products, majority of which were derived from glucose, leucine, isoleucine, valine, tyrosine, tryptophan, methionine, and phenylalanine. In addition to by-products identified previously, we identified several metabolites including 2-hydroxyisovaleric acid, 2-aminobutyric acid, L-alloisoleucine, ketoisoleucine, 2-hydroxy-3-methylvaleric acid, desmeninol, and 2-aminobutyric acid. When added to CHO cell cultures at different concentrations, certain metabolites inhibited cell growth while others including 2-hydroxy acids, surprisingly, reduced lactate accumulation. In vitro enzymatic analysis indicated that 2-hydroxy acids were metabolized by lactate dehydrogenase suggesting a possible mechanism for lowered lactate accumulation, e.g., competitive substrate inhibition. The 13 C-labeling assisted metabolomics pipeline developed and the metabolites identified will serve as a springboard to reduce undesirable by-products accumulation and alleviate inefficient substrate utilization in mammalian cultures used for biomanufacturing and other applications through altered media formulations and pathway engineering strategies. Competing Interests: Competing interests statement:Current employment of authors: J.E.G. is currently affiliated with Takeda Pharmaceutical Company; H.M.N. is currently affiliated with IQVIA; E.H.O. is currently affiliated with Genentech; V.G.D. is currently affiliated with Pfizer; B.O.M. is currently affiliated with Amgen; S.K. is currently affiliated with Sanofi.
Databáze:	MEDLINE
Externí odkaz:	Zobrazit plný text záznamu