{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,16]],"date-time":"2026-10-16T21:24:53Z","timestamp":1792185893039,"version":"4.4.0"},"reference-count":47,"publisher":"Wiley","issue":"7","license":[{"start":{"date-parts":[[2025,4,18]],"date-time":"2025-04-18T00:00:00Z","timestamp":1744934400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"content-domain":{"domain":["analyticalsciencejournals.onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Biotech &amp; Bioengineering"],"published-print":{"date-parts":[[2025,7]]},"abstract":"<jats:title>ABSTRACT<\/jats:title><jats:p>Monoclonal antibodies (mAbs) represent a majority of biotherapeutics in the market today. These glycoproteins undergo posttranslational modifications, such as N\u2010linked glycosylation, that influence the structural &amp; functional characteristics of the antibody. Glycosylation is a heterogenous posttranslational modification that may influence therapeutic glycoprotein stability and clinical efficacy, which is why it is often considered a critical quality attribute (CQA) of the mAb product. While much is known about the glycosylation pathways of Chinese Hamster Ovary (CHO) cells and how cell culture chemical modifiers may influence the N\u2010glycosylation profile of the final product, this knowledge is often based on the final cumulative glycan profile at the end of the batch process. Building a temporal understanding of N\u2010glycosylation and how mAb glycoform composition responds to real\u2010time changes in the biomanufacturing process will help build integrated process models that may allow for glycosylation control to produce a more homogenous product. Here, we look at the effect of specific nutrient feed media additives (e.g., galactose, manganese) and feeding times on the N\u2010glycosylation pathway to modulate N\u2010glycosylation of a Herceptin biosimilar mAb (i.e., Trastuzumab). We deploy the N\u2010GLYcanyzer process analytical technology (PAT) to monitor glycoforms in near real\u2010time for bench\u2010scale bioprocesses operated in both fed\u2010batch and perfusion modes to build an understanding of how temporal changes in mAb N\u2010glycosylation are dependent on specific media additives. We find that Trastuzumab terminal galactosylation is sensitive to media feeding times and intracellular nucleotide sugar pools. Temporal analysis reveals an increased desirable production of single and double galactose\u2010occupied glycoforms over time under glucose\u2010starved fed\u2010batch cultures. Comparable galactosylation profiles were also observed between fed\u2010batch (nutrient\u2010limited) and perfusion (non\u2010nutrient\u2010limited) bioprocess conditions. In summary, our results demonstrate the utility of real\u2010time monitoring of mAb glycoforms and feeding critical cell culture nutrients under fed\u2010batch and perfusion bioprocessing conditions to produce higher\u2010quality biologics.<\/jats:p>","DOI":"10.1002\/bit.28999","type":"journal-article","created":{"date-parts":[[2025,4,19]],"date-time":"2025-04-19T01:27:01Z","timestamp":1745026021000},"page":"1710-1723","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":9,"title":["Temporal Galactose\u2010Manganese Feeding in Fed\u2010Batch and Perfusion Bioreactors Modulates UDP\u2010Galactose Pools for Enhanced mAb Glycosylation Homogeneity"],"prefix":"10.1002","volume":"122","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-6531-5028","authenticated-orcid":false,"given":"Aron","family":"Gyorgypal","sequence":"first","affiliation":[{"name":"Department of Chemical and Biochemical Engineering Rutgers The State University of New Jersey Piscataway New Jersey USA"},{"name":"Office of Pharmaceutical Quality Research, CDER, U.S. FDA Silver Spring Maryland USA"},{"name":"Center for Immunology and Inflammatory Diseases, Division of Rheumatology, Allergy and Immunology, Department of Medicine, Massachusetts General Hospital Harvard Medical School Boston MA USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9259-0540","authenticated-orcid":false,"given":"Erica","family":"Fratz\u2010Berilla","sequence":"additional","affiliation":[{"name":"Office of Pharmaceutical Quality Research, CDER, U.S. FDA Silver Spring Maryland USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Casey","family":"Kohnhorst","sequence":"additional","affiliation":[{"name":"Office of Pharmaceutical Quality Research, CDER, U.S. FDA Silver Spring Maryland USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"David N.","family":"Powers","sequence":"additional","affiliation":[{"name":"Office of Pharmaceutical Quality Research, CDER, U.S. FDA Silver Spring Maryland USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3677-6735","authenticated-orcid":false,"given":"Shishir P. S.","family":"Chundawat","sequence":"additional","affiliation":[{"name":"Department of Chemical and Biochemical Engineering Rutgers The State University of New Jersey Piscataway New Jersey USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2025,4,18]]},"reference":[{"key":"e_1_2_9_2_1","doi-asserted-by":"publisher","DOI":"10.1080\/19420862.2019.1608143"},{"key":"e_1_2_9_3_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.clinthera.2018.11.002"},{"key":"e_1_2_9_4_1","doi-asserted-by":"publisher","DOI":"10.4049\/jimmunol.1800896"},{"key":"e_1_2_9_5_1","doi-asserted-by":"publisher","DOI":"10.1007\/s00281-012-0308-x"},{"key":"e_1_2_9_6_1","unstructured":"Braasch K.(2015).Dielectric and Precursor Analysis to Study Metabolic Effects on CHO Cell Viability and Antibody Glycosylation(Vol.13)."},{"key":"e_1_2_9_7_1","doi-asserted-by":"publisher","DOI":"10.1007\/s00449-016-1693-7"},{"key":"e_1_2_9_8_1","volume-title":"Investigation of Process Conditions and Chemical Modulators on CHO\u2010Cell Based Trastuzumab Production","author":"Chaturvedi A.","year":"2022"},{"key":"e_1_2_9_9_1","doi-asserted-by":"publisher","DOI":"10.1080\/19420862.2018.1537533"},{"key":"e_1_2_9_10_1","doi-asserted-by":"publisher","DOI":"10.1002\/bit.26904"},{"key":"e_1_2_9_11_1","doi-asserted-by":"publisher","DOI":"10.1002\/bit.25620"},{"key":"e_1_2_9_12_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1108455108"},{"key":"e_1_2_9_13_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jpba.2021.114379"},{"key":"e_1_2_9_14_1","doi-asserted-by":"publisher","DOI":"10.1093\/glycob\/cwr027"},{"key":"e_1_2_9_15_1","doi-asserted-by":"publisher","DOI":"10.1002\/bit.23075"},{"key":"e_1_2_9_16_1","doi-asserted-by":"publisher","DOI":"10.1039\/d2re00568a"},{"key":"e_1_2_9_17_1","doi-asserted-by":"publisher","DOI":"10.1021\/acs.analchem.1c05396"},{"key":"e_1_2_9_18_1","doi-asserted-by":"publisher","DOI":"10.1002\/bit.10022"},{"key":"e_1_2_9_19_1","doi-asserted-by":"publisher","DOI":"10.18433\/jpps32433"},{"key":"e_1_2_9_20_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jbiotec.2014.08.026"},{"key":"e_1_2_9_21_1","doi-asserted-by":"publisher","DOI":"10.1007\/s00449-018-1996-y"},{"key":"e_1_2_9_22_1","doi-asserted-by":"publisher","DOI":"10.1002\/bit.26315"},{"key":"e_1_2_9_23_1","doi-asserted-by":"publisher","DOI":"10.1002\/bit.25715"},{"key":"e_1_2_9_24_1","unstructured":"King M. 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