{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,1]],"date-time":"2026-06-01T16:01:27Z","timestamp":1780329687037,"version":"3.54.1"},"reference-count":29,"publisher":"MDPI AG","issue":"11","license":[{"start":{"date-parts":[[2026,6,1]],"date-time":"2026-06-01T00:00:00Z","timestamp":1780272000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"State Key Laboratory of Drug Regulatory Science","award":["2024SKLDRS0203"],"award-info":[{"award-number":["2024SKLDRS0203"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Molecules"],"abstract":"<jats:p>N-glycosylation, particularly terminal mannose exposure, is a critical quality attribute affecting macrophage targeting and the clinical efficacy of enzyme replacement therapy for Gaucher disease. This study developed a universal, sensitive, and quantitative method to compare the N-glycan profiles of three recombinant human glucocerebrosidase products from different expression systems: imiglucerase, velaglucerase alfa, and velaglucerase beta. Using 2-aminobenzamide labeling combined with HILIC-UPLC-FLD and high-resolution mass spectrometry, an N-glycan profiling platform was established. A multidimensional calibration system integrating retention time, glucose unit values, and mass-to-charge ratios was constructed, and collision-induced dissociation tandem MS was used to identify isomers and phosphorylated glycans. The method showed good specificity, linearity, precision, and accuracy. Glycan profiling revealed clear product-dependent differences: imiglucerase was enriched in core-fucosylated Man3 structures, velaglucerase alfa was dominated by Man9 and contained more phosphorylated and sialylated glycans, whereas velaglucerase beta showed a highly homogeneous Man5 profile. These findings demonstrate how distinct manufacturing strategies shape glycosylation patterns and provide a basis for biosimilar development and comparability assessment.<\/jats:p>","DOI":"10.3390\/molecules31111904","type":"journal-article","created":{"date-parts":[[2026,6,1]],"date-time":"2026-06-01T15:07:52Z","timestamp":1780326472000},"page":"1904","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Establishment of an N-Glycan Profiling Method for Three ERT Enzymes Used in Gaucher Disease Therapy"],"prefix":"10.3390","volume":"31","author":[{"given":"Jinliang","family":"Chen","sequence":"first","affiliation":[{"name":"School of Life Science, Jilin University, Changchun 130012, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xinyue","family":"Hu","sequence":"additional","affiliation":[{"name":"National Institutes for Food and Drug Control, Beijing 100061, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lyuyin","family":"Wang","sequence":"additional","affiliation":[{"name":"National Institutes for Food and Drug Control, Beijing 100061, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kaixin","family":"Xu","sequence":"additional","affiliation":[{"name":"School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jing","family":"Li","sequence":"additional","affiliation":[{"name":"National Institutes for Food and Drug Control, Beijing 100061, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yingwu","family":"Wang","sequence":"additional","affiliation":[{"name":"School of Life Science, Jilin University, Changchun 130012, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8916-6872","authenticated-orcid":false,"given":"Chenggang","family":"Liang","sequence":"additional","affiliation":[{"name":"National Institutes for Food and Drug Control, Beijing 100061, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,6,1]]},"reference":[{"key":"ref_1","unstructured":"Scriver, C.R., Beaudet, A.L., Sly, W.S., and Valle, D. (2001). Gaucher disease. The Metabolic and Molecular Bases of Inherited Disease, McGraw-Hill."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"6","DOI":"10.1016\/j.ymgme.2004.08.015","article-title":"Gaucher disease: Complexity in a \u201csimple\u201d disorder","volume":"83","author":"Sidransky","year":"2004","journal-title":"Mol. Genet. Metab."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"1263","DOI":"10.1016\/S0140-6736(08)61522-6","article-title":"Phenotype, diagnosis, and treatment of Gaucher\u2019s disease","volume":"372","author":"Grabowski","year":"2008","journal-title":"Lancet"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"447","DOI":"10.1007\/s00431-012-1771-z","article-title":"Revised recommendations for the management of Gaucher disease in children","volume":"172","author":"Kaplan","year":"2013","journal-title":"Eur. J. Pediatr."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1464","DOI":"10.1056\/NEJM199105233242104","article-title":"Replacement therapy for inherited enzyme deficiency\u2014Macrophage-targeted glucocerebrosidase for Gaucher\u2019s disease","volume":"324","author":"Barton","year":"1991","journal-title":"N. Engl. J. Med."},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Wang, L., Xu, K., Lyu, P., Hu, X., and Li, J. (2026). Quantitative and comparative assessment of recombinant human \u03b2-glucocerebrosidase uptake bioactivity using a stable hMMR-expressing CHO cell model. Molecules, 31.","DOI":"10.3390\/molecules31020235"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"1909","DOI":"10.1172\/JCI116409","article-title":"Binding, internalization, and degradation of mannose-terminated glucocerebrosidase by macrophages","volume":"91","author":"Sato","year":"1993","journal-title":"J. Clin. Investig."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1241","DOI":"10.1038\/nbt1252","article-title":"Post-translational modifications in the context of therapeutic proteins","volume":"24","author":"Walsh","year":"2006","journal-title":"Nat. Biotechnol."},{"key":"ref_9","unstructured":"Butler, M. (1987). Animal Cell Technology, Open UP."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"e00071","DOI":"10.1042\/BSR20130081","article-title":"Glycosylation and functionality of recombinant \u03b2-glucocerebrosidase from various production systems","volume":"33","author":"Tekoah","year":"2013","journal-title":"Biosci. Rep."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"771","DOI":"10.1016\/0006-291X(90)92388-G","article-title":"Human acid \u03b2-glucosidase: Glycosylation is required for catalytic activity","volume":"168","author":"Grace","year":"1990","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"467","DOI":"10.1093\/glycob\/cwm008","article-title":"Effect of mannose chain length on targeting of glucocerebrosidase for enzyme replacement therapy of Gaucher disease","volume":"17","author":"Hughes","year":"2007","journal-title":"Glycobiology"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"24","DOI":"10.1093\/glycob\/cwp138","article-title":"Characterization of gene-activated human acid-\u03b2-glucosidase: Crystal structure, glycan composition, and internalization into macrophages","volume":"20","author":"Brumshtein","year":"2010","journal-title":"Glycobiology"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"15599","DOI":"10.1016\/S0021-9258(18)55439-9","article-title":"Kifunensine, a potent inhibitor of the glycoprotein processing mannosidase I","volume":"265","author":"Elbein","year":"1990","journal-title":"J. Biol. Chem."},{"key":"ref_15","first-page":"377","article-title":"Chinese hamster ovary cell mutants with multiple glycosylation defects for production of glycoproteins with minimal carbohydrate heterogeneity","volume":"9","author":"Stanley","year":"1989","journal-title":"Mol. Cell. Biol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"100448","DOI":"10.1016\/j.jbc.2021.100448","article-title":"Genetic glycoengineering in mammalian cells","volume":"296","author":"Narimatsu","year":"2021","journal-title":"J. Biol. Chem."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"713","DOI":"10.1038\/nchembio.437","article-title":"A systematic approach to protein glycosylation analysis: A path through the maze","volume":"6","author":"Bones","year":"2010","journal-title":"Nat. Chem. Biol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"205","DOI":"10.1080\/19420862.2015.1117719","article-title":"Glycan analysis of therapeutic glycoproteins","volume":"8","author":"Zhang","year":"2016","journal-title":"mAbs"},{"key":"ref_19","first-page":"32","article-title":"A review of glycan analysis requirements","volume":"28","author":"Fournier","year":"2015","journal-title":"BioPharm Int."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"113896","DOI":"10.1016\/j.ab.2020.113896","article-title":"More comprehensive standards for monitoring glycosylation","volume":"612","author":"Guo","year":"2021","journal-title":"Anal. Biochem."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"167","DOI":"10.4161\/19420862.2014.986000","article-title":"Comparison of methods for the analysis of therapeutic immunoglobulin G Fc-glycosylation profiles\u2014Part 1: Separation-based methods","volume":"7","author":"Reusch","year":"2015","journal-title":"mAbs"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"732","DOI":"10.1080\/19420862.2015.1045173","article-title":"Comparison of methods for the analysis of therapeutic immunoglobulin G Fc-glycosylation profiles\u2014Part 2: Mass spectrometric methods","volume":"7","author":"Reusch","year":"2015","journal-title":"mAbs"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"23","DOI":"10.1016\/j.jpha.2019.11.008","article-title":"Comparing different domains of analysis for the characterisation of N-glycans on monoclonal antibodies","volume":"10","author":"Carillo","year":"2020","journal-title":"J. Pharm. Anal."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"403","DOI":"10.1016\/j.jchromb.2009.12.013","article-title":"Separation of 2-aminobenzamide labeled glycans using hydrophilic interaction chromatography columns packed with 1.7 \u03bcm sorbent","volume":"878","author":"Ahn","year":"2010","journal-title":"J. Chromatogr. B"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.ab.2007.12.012","article-title":"HPLC-based analysis of serum N-glycans on a 96-well plate platform with dedicated database software","volume":"376","author":"Royle","year":"2008","journal-title":"Anal. Biochem."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"1214","DOI":"10.1093\/bioinformatics\/btn090","article-title":"GlycoBase and autoGU: Tools for HPLC-based glycan analysis","volume":"24","author":"Campbell","year":"2008","journal-title":"Bioinformatics"},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Mastrangeli, R., Satwekar, A., and Bierau, H. (2023). Innovative metrics for reporting and comparing the glycan structural profile in biotherapeutics. Molecules, 28.","DOI":"10.3390\/molecules28083304"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"2418","DOI":"10.1016\/j.bbrc.2017.12.101","article-title":"Four unreported types of glycans containing mannose-6-phosphate are heterogeneously attached at three sites (including newly found Asn 233) to recombinant human acid alpha-glucosidase that is the only approved treatment for Pompe disease","volume":"495","author":"Park","year":"2018","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"100368","DOI":"10.1016\/j.jbc.2021.100368","article-title":"Structural analysis of carbohydrate binding by the macrophage mannose receptor CD206","volume":"296","author":"Feinberg","year":"2021","journal-title":"J. Biol. Chem."}],"container-title":["Molecules"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1420-3049\/31\/11\/1904\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,6,1]],"date-time":"2026-06-01T15:08:19Z","timestamp":1780326499000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1420-3049\/31\/11\/1904"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,6,1]]},"references-count":29,"journal-issue":{"issue":"11","published-online":{"date-parts":[[2026,6]]}},"alternative-id":["molecules31111904"],"URL":"https:\/\/doi.org\/10.3390\/molecules31111904","relation":{},"ISSN":["1420-3049"],"issn-type":[{"value":"1420-3049","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,6,1]]}}}