{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,17]],"date-time":"2026-08-17T22:29:20Z","timestamp":1787005760752,"version":"build-2736575974"},"reference-count":60,"publisher":"National Academy of Sciences","issue":"41","license":[{"start":{"date-parts":[[2024,10,4]],"date-time":"2024-10-04T00:00:00Z","timestamp":1728000000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000001","name":"NSF","doi-asserted-by":"publisher","award":["1624684"],"award-info":[{"award-number":["1624684"]}],"id":[{"id":"10.13039\/100000001","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["www.pnas.org"],"crossmark-restriction":true},"short-container-title":["Proc. Natl. Acad. Sci. U.S.A."],"published-print":{"date-parts":[[2024,10,8]]},"abstract":"<jats:p>\n                    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\n                    <jats:sup>13<\/jats:sup>\n                    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\n                    <jats:sup>13<\/jats:sup>\n                    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\n                    <jats:sup>13<\/jats:sup>\n                    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.\n                  <\/jats:p>","DOI":"10.1073\/pnas.2403033121","type":"journal-article","created":{"date-parts":[[2024,10,4]],"date-time":"2024-10-04T13:39:57Z","timestamp":1728049197000},"update-policy":"https:\/\/doi.org\/10.1073\/pnas.cm10313","source":"Crossref","is-referenced-by-count":10,"title":["Comprehensive stable-isotope tracing of glucose and amino acids identifies metabolic by-products and their sources in CHO cell culture"],"prefix":"10.1073","volume":"121","author":[{"given":"Jacqueline E.","family":"Gonzalez","sequence":"first","affiliation":[{"name":"Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE 19716"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8303-4219","authenticated-orcid":false,"given":"Harnish Mukesh","family":"Naik","sequence":"additional","affiliation":[{"name":"Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD 21218"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5382-6557","authenticated-orcid":false,"given":"Eleanor H.","family":"Oates","sequence":"additional","affiliation":[{"name":"Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE 19716"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Venkata Gayatri","family":"Dhara","sequence":"additional","affiliation":[{"name":"Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD 21218"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Brian O.","family":"McConnell","sequence":"additional","affiliation":[{"name":"Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE 19716"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Swetha","family":"Kumar","sequence":"additional","affiliation":[{"name":"Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD 21218"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michael J.","family":"Betenbaugh","sequence":"additional","affiliation":[{"name":"Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD 21218"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0087-2875","authenticated-orcid":false,"given":"Maciek R.","family":"Antoniewicz","sequence":"additional","affiliation":[{"name":"Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE 19716"},{"name":"Department of Chemical Engineering, University of Michigan, Ann Arbor, MI 48109"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"341","published-online":{"date-parts":[[2024,10,4]]},"reference":[{"key":"e_1_3_4_1_2","doi-asserted-by":"publisher","DOI":"10.1007\/s40259-018-0319-9"},{"key":"e_1_3_4_2_2","doi-asserted-by":"publisher","DOI":"10.1016\/B978-0-12-811710-1.00012-4"},{"key":"e_1_3_4_3_2","doi-asserted-by":"publisher","DOI":"10.1007\/s00018-020-03486-y"},{"key":"e_1_3_4_4_2","first-page":"21","article-title":"Mammalian cell-based sensor system","volume":"117","author":"Banerjee P.","year":"2010","unstructured":"P. Banerjee, B. Franz, A. K. Bhunia, Mammalian cell-based sensor system. Adv. Biochem. Eng. Biotechnol. 117, 21\u201355 (2010).","journal-title":"Adv. Biochem. Eng. Biotechnol."},{"key":"e_1_3_4_5_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2012.02.023"},{"key":"e_1_3_4_6_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.coche.2018.10.002"},{"key":"e_1_3_4_7_2","doi-asserted-by":"publisher","DOI":"10.1002\/bit.24858"},{"key":"e_1_3_4_8_2","doi-asserted-by":"publisher","DOI":"10.1002\/biot.200700249"},{"key":"e_1_3_4_9_2","doi-asserted-by":"publisher","DOI":"10.1002\/bit.26259"},{"key":"e_1_3_4_10_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41540-019-0103-6"},{"key":"e_1_3_4_11_2","doi-asserted-by":"publisher","DOI":"10.1002\/bit.26313"},{"key":"e_1_3_4_12_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.mec.2021.e00182"},{"key":"e_1_3_4_13_2","doi-asserted-by":"publisher","DOI":"10.3390\/ijms19020385"},{"key":"e_1_3_4_14_2","doi-asserted-by":"publisher","DOI":"10.1002\/bit.26724"},{"key":"e_1_3_4_15_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.jbiotec.2010.03.018"},{"key":"e_1_3_4_16_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.jbiotec.2011.03.003"},{"key":"e_1_3_4_17_2","doi-asserted-by":"publisher","DOI":"10.1002\/btpr.3193"},{"key":"e_1_3_4_18_2","doi-asserted-by":"publisher","DOI":"10.1002\/bit.23072"},{"key":"e_1_3_4_19_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.ymben.2019.03.001"},{"key":"e_1_3_4_20_2","doi-asserted-by":"publisher","DOI":"10.1002\/bit.28403"},{"key":"e_1_3_4_21_2","doi-asserted-by":"publisher","DOI":"10.1007\/s11306-006-0018-2"},{"key":"e_1_3_4_22_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.copbio.2016.10.002"},{"key":"e_1_3_4_23_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.ymben.2016.05.005"},{"key":"e_1_3_4_24_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.ab.2004.09.001"},{"key":"e_1_3_4_25_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.ymben.2012.10.001"},{"key":"e_1_3_4_26_2","doi-asserted-by":"publisher","DOI":"10.1002\/btpr.2131"},{"key":"e_1_3_4_27_2","doi-asserted-by":"publisher","DOI":"10.1007\/978-1-60327-834-8_7"},{"key":"e_1_3_4_28_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41598-018-26262-2"},{"key":"e_1_3_4_29_2","doi-asserted-by":"publisher","DOI":"10.1038\/s12276-018-0060-y"},{"key":"e_1_3_4_30_2","doi-asserted-by":"publisher","DOI":"10.1128\/JB.187.5.1581-1590.2005"},{"key":"e_1_3_4_31_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.ymben.2017.10.007"},{"key":"e_1_3_4_32_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.copbio.2015.08.014"},{"key":"e_1_3_4_33_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.chembiol.2018.09.004"},{"key":"e_1_3_4_34_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41596-019-0204-0"},{"key":"e_1_3_4_35_2","doi-asserted-by":"publisher","DOI":"10.1002\/bit.23269"},{"key":"e_1_3_4_36_2","doi-asserted-by":"publisher","DOI":"10.1002\/btpr.238"},{"key":"e_1_3_4_37_2","doi-asserted-by":"publisher","DOI":"10.1002\/bit.24496"},{"key":"e_1_3_4_38_2","doi-asserted-by":"publisher","DOI":"10.1002\/bit.24983"},{"key":"e_1_3_4_39_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0021-9258(18)64641-1"},{"key":"e_1_3_4_40_2","doi-asserted-by":"publisher","DOI":"10.1016\/0026-0495(88)90037-6"},{"key":"e_1_3_4_41_2","doi-asserted-by":"publisher","DOI":"10.1038\/nchembio.2104"},{"key":"e_1_3_4_42_2","doi-asserted-by":"publisher","DOI":"10.1002\/bit.27661"},{"key":"e_1_3_4_43_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0021-9258(18)41646-8"},{"key":"e_1_3_4_44_2","doi-asserted-by":"publisher","DOI":"10.1080\/15384047.2017.1323586"},{"key":"e_1_3_4_45_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.bbrc.2006.11.111"},{"key":"e_1_3_4_46_2","doi-asserted-by":"publisher","DOI":"10.1186\/1479-7364-4-6-411"},{"key":"e_1_3_4_47_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.bbadis.2012.03.009"},{"key":"e_1_3_4_48_2","first-page":"854","article-title":"Alloisoleucine formation in maple syrup urine disease: Isotopic evidence for the mechanism","volume":"14","author":"Matthews D. E.","year":"1980","unstructured":"D. E. Matthews, E. Ben-Galim, M. W. Haymond, D. M. Bier, Alloisoleucine formation in maple syrup urine disease: Isotopic evidence for the mechanism. Pediatr. Res. 14, 854\u2013857 (1980).","journal-title":"Pediatr. Res."},{"key":"e_1_3_4_49_2","doi-asserted-by":"publisher","DOI":"10.1248\/cpb.34.768"},{"key":"e_1_3_4_50_2","doi-asserted-by":"publisher","DOI":"10.1248\/cpb.33.3887"},{"key":"e_1_3_4_51_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.arr.2016.03.005"},{"key":"e_1_3_4_52_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.bej.2023.109184"},{"key":"e_1_3_4_53_2","doi-asserted-by":"publisher","DOI":"10.1002\/bit.26603"},{"key":"e_1_3_4_54_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.ymben.2010.09.003"},{"key":"e_1_3_4_55_2","doi-asserted-by":"publisher","DOI":"10.1002\/biot.201100052"},{"key":"e_1_3_4_56_2","doi-asserted-by":"publisher","DOI":"10.1002\/bit.27942"},{"key":"e_1_3_4_57_2","doi-asserted-by":"publisher","DOI":"10.1002\/bit.28366"},{"key":"e_1_3_4_58_2","doi-asserted-by":"publisher","DOI":"10.1002\/bit.28417"},{"key":"e_1_3_4_59_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.ymben.2023.02.002"},{"key":"e_1_3_4_60_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.ymben.2021.12.005"}],"container-title":["Proceedings of the National Academy of Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pnas.org\/doi\/pdf\/10.1073\/pnas.2403033121","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2024,10,4]],"date-time":"2024-10-04T13:40:09Z","timestamp":1728049209000},"score":1,"resource":{"primary":{"URL":"https:\/\/pnas.org\/doi\/10.1073\/pnas.2403033121"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,10,4]]},"references-count":60,"journal-issue":{"issue":"41","published-print":{"date-parts":[[2024,10,8]]}},"alternative-id":["10.1073\/pnas.2403033121"],"URL":"https:\/\/doi.org\/10.1073\/pnas.2403033121","relation":{},"ISSN":["0027-8424","1091-6490"],"issn-type":[{"value":"0027-8424","type":"print"},{"value":"1091-6490","type":"electronic"}],"subject":[],"published":{"date-parts":[[2024,10,4]]},"assertion":[{"value":"2024-02-14","order":0,"name":"received","label":"Received","group":{"name":"publication_history","label":"Publication History"}},{"value":"2024-08-06","order":2,"name":"accepted","label":"Accepted","group":{"name":"publication_history","label":"Publication History"}},{"value":"2024-10-04","order":3,"name":"published","label":"Published","group":{"name":"publication_history","label":"Publication History"}}],"article-number":"e2403033121"}}