{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,21]],"date-time":"2026-09-21T10:01:14Z","timestamp":1789984874167,"version":"4.0.1"},"reference-count":29,"publisher":"MDPI AG","issue":"9","license":[{"start":{"date-parts":[[2026,9,20]],"date-time":"2026-09-20T00:00:00Z","timestamp":1789862400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Innovative Drug Research and Development National Science and Technology Major Project","award":["2026ZD1805800"],"award-info":[{"award-number":["2026ZD1805800"]}]},{"name":"State Key Laboratory of Drug Regulatory Science Project","award":["2025SKLDRS0318"],"award-info":[{"award-number":["2025SKLDRS0318"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pharmaceuticals"],"abstract":"<jats:p>Background\/Objectives: Bispecific antibody\u2013drug conjugates (bispecific ADCs) are among the most rapidly advancing antibody therapeutic modalities, with only one product (BL-B01D1) currently approved for marketing worldwide to date. In this study, we investigated several critical quality attributes (CQAs) of a glyco-site-specific antibody\u2013drug conjugate (ADC) targeting two distinct epitopes of HER2. Methods: We established quality control methods for the assessment of mispaired species, conjugation site, drug-to-antibody ratios (DARs), charge variants, residual free drugs, residual \u03b21,4-galactosyltransferase I (GalT) enzyme, relative binding activity, and cell proliferation inhibition activity. Conclusions: An analytical method suite was developed for the bispecific ADC to comprehensively characterize and appropriately control its critical quality attributes, thereby guiding the research, development, and manufacturing process optimization of the ADC product and ensuring its safety, efficacy, and quality consistency.<\/jats:p>","DOI":"10.3390\/ph19091495","type":"journal-article","created":{"date-parts":[[2026,9,21]],"date-time":"2026-09-21T09:01:38Z","timestamp":1789981298000},"page":"1495","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Critical Quality Attribute Investigation and Analytical Method Establishment for Glyco-Site-Specific Anti-HER2 Bispecific ADC"],"prefix":"10.3390","volume":"19","author":[{"given":"Xiaojuan","family":"Yu","sequence":"first","affiliation":[{"name":"National Institutes for Drug Control, State Key Laboratory of Drug Regulatory Science, NMPA Key Laboratory for Quality Research and Evaluation of Biological Products, NHC Key Laboratory of Research on Quality and Standardization of Biotech Products, Beijing 102629, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"writing-review-editing","vocabulary":"credit"}]},{"given":"Lele","family":"Wang","sequence":"additional","affiliation":[{"name":"Jiangsu Alphamab Biopharmaceuticals Co., Ltd., Suzhou 215125, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"writing-original-draft","vocabulary":"credit"}]},{"given":"Weirong","family":"Cao","sequence":"additional","affiliation":[{"name":"CSPC Megalith Biopharmaceutical Co., Ltd., Shijiazhuang 050035, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"formal-analysis","vocabulary":"credit"}]},{"given":"Yao","family":"Tang","sequence":"additional","affiliation":[{"name":"NMPA Center for Innovation and Research in Regulatory Science, Sichuan Institute for Drug Control, SCMPA Key Laboratory for Quality Monitoring and Risk Assessment of Biological Products, Chengdu 611731, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"writing-review-editing","vocabulary":"credit"}]},{"given":"Tao","family":"Tang","sequence":"additional","affiliation":[{"name":"NMPA Center for Innovation and Research in Regulatory Science, Sichuan Institute for Drug Control, SCMPA Key Laboratory for Quality Monitoring and Risk Assessment of Biological Products, Chengdu 611731, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"writing-review-editing","vocabulary":"credit"}]},{"given":"Yangwei","family":"Fu","sequence":"additional","affiliation":[{"name":"Jiangsu Alphamab Biopharmaceuticals Co., Ltd., Suzhou 215125, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"writing-original-draft","vocabulary":"credit"}]},{"given":"Yalan","family":"Yang","sequence":"additional","affiliation":[{"name":"National Institutes for Drug Control, State Key Laboratory of Drug Regulatory Science, NMPA Key Laboratory for Quality Research and Evaluation of Biological Products, NHC Key Laboratory of Research on Quality and Standardization of Biotech Products, Beijing 102629, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"writing-review-editing","vocabulary":"credit"}]},{"given":"Chuanfei","family":"Yu","sequence":"additional","affiliation":[{"name":"National Institutes for Drug Control, State Key Laboratory of Drug Regulatory Science, NMPA Key Laboratory for Quality Research and Evaluation of Biological Products, NHC Key Laboratory of Research on Quality and Standardization of Biotech Products, Beijing 102629, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"conceptualization","vocabulary":"credit"},{"role":"writing-review-editing","vocabulary":"credit"},{"role":"writing-original-draft","vocabulary":"credit"}]},{"given":"Yan","family":"Li","sequence":"additional","affiliation":[{"name":"NMPA Center for Innovation and Research in Regulatory Science, Sichuan Institute for Drug Control, SCMPA Key Laboratory for Quality Monitoring and Risk Assessment of Biological Products, Chengdu 611731, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"writing-review-editing","vocabulary":"credit"}]},{"given":"Lan","family":"Wang","sequence":"additional","affiliation":[{"name":"National Institutes for Drug Control, State Key Laboratory of Drug Regulatory Science, NMPA Key Laboratory for Quality Research and Evaluation of Biological Products, NHC Key Laboratory of Research on Quality and Standardization of Biotech Products, Beijing 102629, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"writing-review-editing","vocabulary":"credit"}]}],"member":"1968","published-online":{"date-parts":[[2026,9,20]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Gao, X., Tao, L., Hao, W., Wu, X., Zhu, F., Lv, S., and Zhang, Y. (2026). Comparative efficacy of antibody-drug conjugates and chemotherapy for malignant tumors: A systematic review and meta-analysis. Front. Oncol., 15.","DOI":"10.3389\/fonc.2025.1697340"},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"D\u00edaz-Rodr\u00edguez, E., Gandullo-S\u00e1nchez, L., Oca\u00f1a, A., and Pandiella, A. (2021). Novel ADCs and strategies to overcome resistance to anti-HER2 ADCs. Cancers, 14.","DOI":"10.3390\/cancers14010154"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"948","DOI":"10.1016\/j.ccell.2019.05.010","article-title":"A biparatopic HER2-targeting antibody-drug conjugate induces tumor regression in primary models refractory to or ineligible for HER2-targeted therapy","volume":"35","author":"Li","year":"2019","journal-title":"Cancer Cell"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"130","DOI":"10.1016\/j.trecan.2019.12.010","article-title":"Trastuzumab emtansine: Mechanisms of action and resistance, clinical progress, and beyond","volume":"6","author":"Pandiella","year":"2020","journal-title":"Trends Cancer"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1965","DOI":"10.1016\/j.apsb.2024.01.009","article-title":"Bispecific antibody drug conjugates: Making 1+1>2","volume":"14","author":"Gu","year":"2024","journal-title":"Acta Pharm. Sin. B"},{"key":"ref_6","first-page":"116561","article-title":"Multi-specific antibody drug conjugate: The next frontier for solid tumors","volume":"235","author":"Hao","year":"2026","journal-title":"J. Pharm. Biomed. Anal."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"S527","DOI":"10.1016\/j.annonc.2024.08.741","article-title":"Evaluation of the safety and efficacy of JSKN003 in patients with advanced HER2-positive (IHC 3+) solid tumors (excluding breast cancer)","volume":"35","author":"Shen","year":"2024","journal-title":"Ann. Oncol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"20833","DOI":"10.1074\/jbc.M111183200","article-title":"Structure-based design of beta 1,4-galactosyltransferase I (beta 4Gal-T1) with equally efficient N-acetylgalactosaminyltransferase activity: Point mutation broadens beta 4Gal-T1 donor specificity","volume":"277","author":"Ramakrishnan","year":"2002","journal-title":"J. Biol. Chem."},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Hafeez, U., Parakh, S., Gan, H.K., and Scott, A.M. (2020). Antibody-drug conjugates for cancer therapy. Molecules, 25.","DOI":"10.3390\/molecules25204764"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"4361","DOI":"10.1039\/C8CS00676H","article-title":"Cleavable linkers in antibody-drug conjugates","volume":"48","author":"Bargh","year":"2019","journal-title":"Chem. Soc. Rev."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"51037","DOI":"10.18632\/oncotarget.17558","article-title":"Structural basis of a novel heterodimeric Fc for bispecific antibody production","volume":"8","author":"Wei","year":"2017","journal-title":"Oncotarget"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1284","DOI":"10.1039\/D5CB00096C","article-title":"A biparatopic HER2-targeting ADC constructed via site-specific glycan conjugation exhibits superior stability, safety, and efficacy","volume":"6","author":"Xue","year":"2025","journal-title":"RSC Chem. Biol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"331","DOI":"10.2147\/OTT.S357326","article-title":"An innovative site-specific anti-HER2 antibody-drug conjugate with high homogeneity and improved therapeutic index","volume":"15","author":"Hui","year":"2022","journal-title":"OncoTargets Ther."},{"key":"ref_14","unstructured":"(2026, September 04). A-Mab: A Case Study in Bioprocess Development, Available online: https:\/\/casss.org\/page\/a-mab-case-study."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"291","DOI":"10.1016\/j.biologicals.2016.06.005","article-title":"Determination of critical quality attributes for monoclonal antibodies using quality by design principles","volume":"44","author":"Alt","year":"2016","journal-title":"Biologicals"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"617","DOI":"10.1093\/protein\/9.7.617","article-title":"Knobs-into-holes\u2019 engineering of antibody CH3 domains for heavy chain heterodimerization","volume":"9","author":"Ridgway","year":"1996","journal-title":"Protein Eng."},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Ducry, L. (2013). Antibody-Drug Conjugates Methods in Molecular Biology, Humana Press.","DOI":"10.1007\/978-1-62703-541-5"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"2354","DOI":"10.1007\/s11095-012-0800-y","article-title":"Pharmacokinetic considerations for antibody drug conjugates","volume":"29","author":"Lin","year":"2012","journal-title":"Pharm. Res."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"789","DOI":"10.1038\/nrc1977","article-title":"Topoisomerase I inhibitors: Camptothecins and beyond","volume":"6","author":"Pommier","year":"2006","journal-title":"Nat. Rev. Cancer"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1086","DOI":"10.1080\/00498254.2018.1531158","article-title":"Comprehensive preclinical pharmacokinetic evaluations of trastuzumab deruxtecan (DS-8201a), a HER2-targeting antibody-drug conjugate, in cynomolgus monkeys","volume":"49","author":"Nagai","year":"2019","journal-title":"Xenobiotica"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1039","DOI":"10.1111\/cas.12966","article-title":"Bystander killing effect of DS-8201a, a novel anti-human epidermal growth factor receptor 2 antibody-drug conjugate, in tumors with human epidermal growth factor receptor 2 heterogeneity","volume":"107","author":"Ogitani","year":"2016","journal-title":"Cancer Sci."},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Diepold, K., Bomans, K., Wiedmann, M., Zimmermann, B., Petzold, A., Schlothauer, T., Mueller, R., Moritz, B., Stracke, J.O., and M\u00f8lh\u00f8j, M. (2012). Simultaneous assessment of Asp isomerization and Asn deamidation in recombinant antibodies by LC-MS following incubation at elevated temperatures. PLoS ONE, 7.","DOI":"10.1371\/journal.pone.0030295"},{"key":"ref_23","unstructured":"U.S. Food and Drug Administration (2025, December 22). Drug Approval Package: Ontruzant (trastuzumab-ttb): Chemistry Review(s), Available online: https:\/\/www.accessdata.fda.gov\/drugsatfda_docs\/nda\/2019\/761100Orig1s000ChemR.pdf."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"105529","DOI":"10.1016\/j.pep.2019.105529","article-title":"Removal of half antibody, hole-hole homodimer and aggregates during bispecific antibody purification using MMC ImpRes mixed-mode chromatography","volume":"167","author":"Tang","year":"2020","journal-title":"Protein Expr. Purif."},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Esnault, C., Schrama, D., Houben, R., Guy\u00e9tant, S., Desgranges, A., Martin, C., Berthon, P., Viaud-Massuard, M.C., Touz\u00e9, A., and Kervarrec, T. (2022). Antibody-drug conjugates as an emerging therapy in oncodermatology. Cancers, 14.","DOI":"10.3390\/cancers14030778"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"343176","DOI":"10.1016\/j.aca.2024.343176","article-title":"Imaged capillary isoelectric focusing method development for charge variants of high DAR ADCs","volume":"1328","author":"Leng","year":"2024","journal-title":"Anal. Chim. Acta"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"117105","DOI":"10.1016\/j.jpba.2025.117105","article-title":"Strategies for accurate quantitation of free payloads in antibody-drug-conjugates: Application for a payload with a labile lactone group","volume":"266","author":"Fang","year":"2025","journal-title":"J. Pharm. Biomed. Anal."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"51","DOI":"10.1016\/j.jchromb.2019.04.012","article-title":"A method to directly analyze free-drug-related species in antibody-drug conjugates without sample preparation","volume":"1116","author":"Mulagapati","year":"2019","journal-title":"J. Chromatogr. B"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"44246","DOI":"10.1038\/srep44246","article-title":"Proteomic analysis of host cell protein dynamics in the culture supernatants of antibody-producing CHO cells","volume":"7","author":"Park","year":"2017","journal-title":"Sci. Rep."}],"container-title":["Pharmaceuticals"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1424-8247\/19\/9\/1495\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,21]],"date-time":"2026-09-21T09:10:59Z","timestamp":1789981859000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1424-8247\/19\/9\/1495"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,9,20]]},"references-count":29,"journal-issue":{"issue":"9","published-online":{"date-parts":[[2026,9]]}},"alternative-id":["ph19091495"],"URL":"https:\/\/doi.org\/10.3390\/ph19091495","relation":{},"ISSN":["1424-8247"],"issn-type":[{"value":"1424-8247","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,9,20]]}}}