{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,5]],"date-time":"2026-08-05T00:58:45Z","timestamp":1785891525003,"version":"3.56.0"},"reference-count":41,"publisher":"MDPI AG","issue":"4","license":[{"start":{"date-parts":[[2025,4,4]],"date-time":"2025-04-04T00:00:00Z","timestamp":1743724800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100016308","name":"BpiFrance","doi-asserted-by":"publisher","award":["DOS0162921\/00"],"award-info":[{"award-number":["DOS0162921\/00"]}],"id":[{"id":"10.13039\/100016308","id-type":"DOI","asserted-by":"publisher"}]},{"name":"University of Tours","award":["DOS0162921\/00"],"award-info":[{"award-number":["DOS0162921\/00"]}]},{"name":"CPER-FEDER","award":["DOS0162921\/00"],"award-info":[{"award-number":["DOS0162921\/00"]}]},{"name":"R\u00e9gion Centre Val de Loire","award":["DOS0162921\/00"],"award-info":[{"award-number":["DOS0162921\/00"]}]},{"name":"LabEx MAbImprove","award":["DOS0162921\/00"],"award-info":[{"award-number":["DOS0162921\/00"]}]},{"name":"Fondation Philippe Maupas","award":["DOS0162921\/00"],"award-info":[{"award-number":["DOS0162921\/00"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pharmaceutics"],"abstract":"<jats:p>Background: Chinese hamster ovary (CHO) cell metabolism is complex, influenced by nutrients like glucose and glutamine and metabolites such as lactate. Real-time monitoring is necessary for optimizing culture conditions and ensuring consistent product quality. Raman spectroscopy has emerged as a robust process analytical technology (PAT) tool due to its non-invasive, in situ capabilities. This study evaluates Raman spectroscopy for monitoring key metabolic parameters and IgG titer in CHO cell cultures. Methods: Raman spectroscopy was applied to five 10 L-scale CHO cell cultures. Partial least squares (PLS) regression models were developed from four batches, including one with induced cell death, to enhance robustness. The models were validated against blind test sets. Results: PLS models exhibited high predictive accuracy (R2 &gt; 0.9). Glucose and IgG titer predictions were reliable (RMSEP = 0.51 g\/L and 0.12 g\/L, respectively), while glutamine and lactate had higher RMSEP due to lower concentrations. Specific Raman bands contributed to the specificity of glucose, lactate, and IgG models. Predictions for viable (VCD) and total cell density (TCD) were less accurate due to the absence of direct Raman signals. Conclusions: This study confirms Raman spectroscopy\u2019s potential for real-time, in situ bioprocess monitoring without manual sampling. Chemometric analysis enhances model robustness, supporting automated control systems. Raman data could enable continuous feedback regulation of critical nutrients like glucose, ensuring consistent critical quality attributes (CQAs) in biopharmaceutical production.<\/jats:p>","DOI":"10.3390\/pharmaceutics17040473","type":"journal-article","created":{"date-parts":[[2025,4,8]],"date-time":"2025-04-08T05:59:00Z","timestamp":1744091940000},"page":"473","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":10,"title":["Monitoring of Nutrients, Metabolites, IgG Titer, and Cell Densities in 10 L Bioreactors Using Raman Spectroscopy and PLS Regression Models"],"prefix":"10.3390","volume":"17","author":[{"ORCID":"https:\/\/orcid.org\/0009-0000-0109-0915","authenticated-orcid":false,"given":"Morandise","family":"Rubini","sequence":"first","affiliation":[{"name":"Centre de Biophysique Mol\u00e9culaire (UPR CNRS 4301), D\u00e9partement Nanom\u00e9dicaments et Nanosondes, UFR de Pharmacie Philippe Maupas, Universit\u00e9 de Tours, 31 avenue Monge, 37 000 Tours, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Julien","family":"Boyer","sequence":"additional","affiliation":[{"name":"Ondalys, 4 Rue Georges Besse, 34 830 Clapiers, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jordane","family":"Poulain","sequence":"additional","affiliation":[{"name":"Ondalys, 4 Rue Georges Besse, 34 830 Clapiers, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ana\u00efs","family":"Berger","sequence":"additional","affiliation":[{"name":"Bioengineering, Bio-S, Technologie Servier, 905 Route de Saran, 45 520 Gidy, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Thomas","family":"Saillard","sequence":"additional","affiliation":[{"name":"Bioengineering, Bio-S, Technologie Servier, 905 Route de Saran, 45 520 Gidy, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Julien","family":"Louet","sequence":"additional","affiliation":[{"name":"Indatech\u2014Chauvin Arnoux Group, 4 Rue George Besse, 34 830 Clapiers, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6627-9660","authenticated-orcid":false,"given":"Martin","family":"Souc\u00e9","sequence":"additional","affiliation":[{"name":"Centre de Biophysique Mol\u00e9culaire (UPR CNRS 4301), D\u00e9partement Nanom\u00e9dicaments et Nanosondes, UFR de Pharmacie Philippe Maupas, Universit\u00e9 de Tours, 31 avenue Monge, 37 000 Tours, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sylvie","family":"Roussel","sequence":"additional","affiliation":[{"name":"Ondalys, 4 Rue Georges Besse, 34 830 Clapiers, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sylvain","family":"Arnould","sequence":"additional","affiliation":[{"name":"Bioengineering, Bio-S, Technologie Servier, 905 Route de Saran, 45 520 Gidy, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Murielle","family":"Verg\u00e8s","sequence":"additional","affiliation":[{"name":"Bioengineering, Bio-S, Technologie Servier, 905 Route de Saran, 45 520 Gidy, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fabien","family":"Chauchard-Rios","sequence":"additional","affiliation":[{"name":"Indatech\u2014Chauvin Arnoux Group, 4 Rue George Besse, 34 830 Clapiers, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Igor","family":"Chourpa","sequence":"additional","affiliation":[{"name":"Centre de Biophysique Mol\u00e9culaire (UPR CNRS 4301), D\u00e9partement Nanom\u00e9dicaments et Nanosondes, UFR de Pharmacie Philippe Maupas, Universit\u00e9 de Tours, 31 avenue Monge, 37 000 Tours, France"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,4,4]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Coulet, M., Kepp, O., Kroemer, G., and Basmaciogullari, S. (2022). Metabolic Profiling of CHO Cells during the Production of Biotherapeutics. Cells, 11.","DOI":"10.3390\/cells11121929"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1085","DOI":"10.1177\/0003702817703270","article-title":"Applications of Raman Spectroscopy in Biopharmaceutical Manufacturing: A Short Review","volume":"71","author":"Buckley","year":"2017","journal-title":"Appl. Spectrosc."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"337","DOI":"10.1002\/btpr.2459","article-title":"Comparison of spectroscopy technologies for improved monitoring of cell culture processes in miniature bioreactors","volume":"33","author":"Racher","year":"2017","journal-title":"Biotechnol. Prog."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"730","DOI":"10.1002\/btpr.2633","article-title":"Development of generic raman models for a GS-KOTM CHO platform process","volume":"34","author":"Webster","year":"2018","journal-title":"Biotechnol. Prog."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"521","DOI":"10.1177\/00037028231160197","article-title":"Development of Raman Calibration Model Without Culture Data for In-Line Analysis of Metabolites in Cell Culture Media","volume":"77","author":"Hara","year":"2023","journal-title":"Appl. Spectrosc."},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Graf, A., Woodhams, A., Nelson, M., Richardson, D.D., Short, S.M., Brower, M., and Hoehse, M. (2022). Automated Data Generation for Raman Spectroscopy Calibrations in Multi-Parallel Mini Bioreactors. Sensors, 22.","DOI":"10.3390\/s22093397"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"26","DOI":"10.1038\/nbt0109-26","article-title":"Quality by design for biopharmaceuticals","volume":"27","author":"Rathore","year":"2009","journal-title":"Nat. Biotechnol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"159","DOI":"10.1016\/j.chemolab.2006.04.024","article-title":"The chemometric analysis of point and dynamic data in pharmaceutical and biotech production (PAT)\u2014Some objectives and approaches","volume":"84","author":"Wold","year":"2006","journal-title":"Chemom. Intell. Lab. Syst."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"1215","DOI":"10.1002\/bit.23023","article-title":"Real time monitoring of multiple parameters in mammalian cell culture bioreactors using an in-line Raman spectroscopy probe","volume":"108","author":"Kenty","year":"2011","journal-title":"Biotechnol. Bioeng."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1355","DOI":"10.1002\/btpr.1590","article-title":"In situ Raman spectroscopy for simultaneous monitoring of multiple process parameters in mammalian cell culture bioreactors","volume":"28","author":"Whelan","year":"2012","journal-title":"Biotechnol. Prog."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"1004","DOI":"10.1002\/btpr.2079","article-title":"Generic Raman-based calibration models enabling real-time monitoring of cell culture bioreactors","volume":"31","author":"Mehdizadeh","year":"2015","journal-title":"Biotechnol. Prog."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"148","DOI":"10.1016\/j.aca.2015.08.050","article-title":"In-line and real-time prediction of recombinant antibody titer by in situ Raman spectroscopy","volume":"892","author":"Andre","year":"2015","journal-title":"Anal. Chim. Acta"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"566","DOI":"10.1002\/btpr.2035","article-title":"Cross-scale predictive modeling of CHO cell culture growth and metabolites using Raman spectroscopy and multivariate analysis","volume":"31","author":"Berry","year":"2015","journal-title":"Biotechnol. Prog."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"2550","DOI":"10.1002\/bit.26368","article-title":"Developing global regression models for metabolite concentration prediction regardless of cell line","volume":"114","author":"Andre","year":"2017","journal-title":"Biotechnol. Bioeng."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"346","DOI":"10.1016\/j.jpba.2017.06.070","article-title":"On-line prediction of the glucose concentration of CHO cell cultivations by NIR and Raman spectroscopy: Comparative scalability test with a shake flask model system","volume":"145","author":"Kozma","year":"2017","journal-title":"J. Pharm. Biomed. Anal."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"486","DOI":"10.1002\/btpr.2604","article-title":"Real-time monitoring of antibody glycosylation site occupancy by in situ Raman spectroscopy during bioreactor CHO cell cultures","volume":"34","author":"Li","year":"2018","journal-title":"Biotechnol. Prog."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"e3223","DOI":"10.1002\/btpr.3223","article-title":"Raman based chemometric model development for glycation and glycosylation real time monitoring in a manufacturing scale CHO cell bioreactor process","volume":"38","author":"Gibbons","year":"2022","journal-title":"Biotechnol. Prog."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"e2977","DOI":"10.1002\/btpr.2977","article-title":"Analysis of chemometric models applied to Raman spectroscopy for monitoring key metabolites of cell culture","volume":"36","author":"Rafferty","year":"2020","journal-title":"Biotechnol. Prog."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"2575","DOI":"10.1002\/bit.27100","article-title":"A machine-learning approach to calibrate generic Raman models for real-time monitoring of cell culture processes","volume":"116","author":"Tulsyan","year":"2019","journal-title":"Biotechnol. Bioeng."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"406","DOI":"10.1002\/bit.27205","article-title":"Automatic real-time calibration, assessment, and maintenance of generic Raman models for online monitoring of cell culture processes","volume":"117","author":"Tulsyan","year":"2020","journal-title":"Biotechnol. Bioeng."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1231","DOI":"10.1002\/bit.28646","article-title":"Cell culture product quality attribute prediction using convolutional neural networks and Raman spectroscopy","volume":"121","author":"Khodabandehlou","year":"2024","journal-title":"Biotechnol. Bioeng."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"224","DOI":"10.1002\/btpr.2205","article-title":"Quick generation of Raman spectroscopy based in-process glucose control to influence biopharmaceutical protein product quality during mammalian cell culture","volume":"32","author":"Berry","year":"2016","journal-title":"Biotechnol. Prog."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"e3052","DOI":"10.1002\/btpr.3052","article-title":"Raman-based dynamic feeding strategies using real-time glucose concentration monitoring system during adalimumab producing CHO cell cultivation","volume":"36","author":"Domjan","year":"2020","journal-title":"Biotechnol. Prog."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"2300318","DOI":"10.1002\/biot.202300318","article-title":"Raman-controlled pyruvate feeding to control metabolic activity and product quality in continuous biomanufacturing","volume":"19","author":"Romann","year":"2024","journal-title":"Biotechnol. J."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"e3085","DOI":"10.1002\/btpr.3085","article-title":"Tuning monoclonal antibody galactosylation using Raman spectroscopy-controlled lactic acid feeding","volume":"37","author":"Eyster","year":"2021","journal-title":"Biotechnol. Prog."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"127","DOI":"10.1007\/s00449-020-02429-y","article-title":"Feedback control of two supplemental feeds during fed-batch culture on a platform process using inline Raman models for glucose and phenylalanine concentration","volume":"44","author":"Webster","year":"2021","journal-title":"Bioprocess Biosyst. Eng."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"2200184","DOI":"10.1002\/biot.202200184","article-title":"Advancing Raman model calibration for perfusion bioprocesses using spiked harvest libraries","volume":"17","author":"Romann","year":"2022","journal-title":"Biotechnol. J."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"105785","DOI":"10.1016\/j.jece.2021.105785","article-title":"Real-time analysis of multicomponent dissolved inorganic carbon in the air-sea exchanging process using gas-liquid Raman spectroscopy","volume":"9","author":"Guo","year":"2021","journal-title":"J. Environ. Chem. Eng."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"659","DOI":"10.1002\/btpr.2635","article-title":"Monitoring mAb cultivations with in-situ raman spectroscopy: The influence of spectral selectivity on calibration models and industrial use as reliable PAT tool","volume":"34","author":"Santos","year":"2018","journal-title":"Biotechnol. Prog."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"203","DOI":"10.1016\/S0008-6215(00)85652-9","article-title":"Laser-raman spectra of d-glucose and sucrose in aqueous solution","volume":"81","author":"Mathlouthi","year":"1980","journal-title":"Carbohydr. Res."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"225","DOI":"10.1016\/0008-6215(80)90002-6","article-title":"Laser-raman spectra of d-fructose in aqueous solution","volume":"78","author":"Mathlouthi","year":"1980","journal-title":"Carbohydr. Res."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"17","DOI":"10.1016\/j.chemphys.2014.08.003","article-title":"L-glutamine: Dynamical properties investigation by means of INS, IR, RAMAN, 1H NMR and DFT techniques","volume":"443","author":"Bator","year":"2014","journal-title":"Chem. Phys."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"879","DOI":"10.1007\/s00216-006-1047-4","article-title":"Comparison of multivariate calibration models for glucose, urea, and lactate from near-infrared and Raman spectra","volume":"387","author":"Ren","year":"2007","journal-title":"Anal. Bioanal. Chem."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"9701","DOI":"10.1021\/acsomega.1c07206","article-title":"Detection of Glutamate Encapsulated in Liposomes by Optical Trapping Raman Spectroscopy","volume":"7","author":"Masui","year":"2022","journal-title":"ACS Omega"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"1187","DOI":"10.1016\/j.saa.2010.12.079","article-title":"Raman spectra of amino acids and their aqueous solutions","volume":"78","author":"Zhu","year":"2011","journal-title":"Spectrochim. Acta A Mol. Biomol. Spectrosc."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"2861","DOI":"10.1002\/jps.20895","article-title":"Raman spectroscopy of protein pharmaceuticals","volume":"96","author":"Wen","year":"2007","journal-title":"J. Pharm. Sci."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"6977","DOI":"10.1039\/c3an01341c","article-title":"The challenge of applying Raman spectroscopy to monitor recombinant antibody production","volume":"138","author":"Ashton","year":"2013","journal-title":"Analyst"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"308","DOI":"10.1002\/btpr.2430","article-title":"Mammalian cell culture monitoring using in situ spectroscopy: Is your method really optimised?","volume":"33","author":"Andre","year":"2017","journal-title":"Biotechnol. Prog."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"401","DOI":"10.1002\/bit.26476","article-title":"Types of cell death and apoptotic stages in Chinese Hamster Ovary cells distinguished by Raman spectroscopy","volume":"115","author":"Rangan","year":"2018","journal-title":"Biotechnol. Bioeng."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"1","DOI":"10.2225\/vol16-issue3-fulltext-2","article-title":"Advances in improving mammalian cells metabolism for recombinant protein production","volume":"16","author":"Altamirano","year":"2013","journal-title":"Electron. J. Biotechnol."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"e1700499","DOI":"10.1002\/biot.201700499","article-title":"Impact of CHO Metabolism on Cell Growth and Protein Production: An Overview of Toxic and Inhibiting Metabolites and Nutrients","volume":"13","author":"Pereira","year":"2018","journal-title":"Biotechnol. J."}],"container-title":["Pharmaceutics"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4923\/17\/4\/473\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,9]],"date-time":"2025-10-09T17:10:30Z","timestamp":1760029830000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4923\/17\/4\/473"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,4,4]]},"references-count":41,"journal-issue":{"issue":"4","published-online":{"date-parts":[[2025,4]]}},"alternative-id":["pharmaceutics17040473"],"URL":"https:\/\/doi.org\/10.3390\/pharmaceutics17040473","relation":{},"ISSN":["1999-4923"],"issn-type":[{"value":"1999-4923","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,4,4]]}}}