{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T02:31:05Z","timestamp":1760149865186,"version":"build-2065373602"},"reference-count":28,"publisher":"MDPI AG","issue":"3","license":[{"start":{"date-parts":[[2023,9,19]],"date-time":"2023-09-19T00:00:00Z","timestamp":1695081600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Horizon Therapeutics"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Antibodies"],"abstract":"<jats:p>This study presents a novel degradation pathway of a human immunoglobulin G (IgG) molecule featuring a light chain N-terminal asparagine. We thoroughly characterize this pathway and investigate its charge profiles using cation exchange chromatography (CEX) and capillary isoelectric focusing (cIEF). Beyond the well-documented asparagine deamidation into isoaspartic acid, aspartic acid, and succinimide intermediate, a previously unreported clipping degradation pathway is uncovered. This newly identified clipped N-terminal IgG variant exhibits a delayed elution in CEX, categorized as a \u201cbasic variant\u201d, while retaining the same main peak isoelectric point (pI) in cIEF. The influence of temperature and pH on N-terminal asparagine stability is assessed across various stressed conditions. A notable correlation between deamidation percentage and clipped products is established, suggesting a potential hydrolytic chemical reaction underlying the clipping process. Furthermore, the impact of N-terminal asparagine modifications on potency is evaluated through ELISA binding assays, revealing minimal effects on binding affinity. Sequence alignment reveals homology to a human IgG with the germline gene from Immunoglobulin Lambda Variable 6-57 (IGLV6-57), which has implications for amyloid light-chain (AL) amyloidosis. This discovery of the N-terminal clipping degradation pathway contributes to our understanding of immunoglobulin light chain misfolding and amyloid fibril deposition under physiological conditions.<\/jats:p>","DOI":"10.3390\/antib12030059","type":"journal-article","created":{"date-parts":[[2023,9,19]],"date-time":"2023-09-19T23:17:20Z","timestamp":1695165440000},"page":"59","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Characterization of N-Terminal Asparagine Deamidation and Clipping of a Monoclonal Antibody"],"prefix":"10.3390","volume":"12","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-2666-0827","authenticated-orcid":false,"given":"Jing","family":"Zhen","sequence":"first","affiliation":[{"name":"Department of Analytical Sciences, U.S. Technical & Biologics Development, Horizon Therapeutics, Rockville, MD 20850, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jennifer","family":"Lee","sequence":"additional","affiliation":[{"name":"Department of Analytical Sciences, U.S. Technical & Biologics Development, Horizon Therapeutics, Rockville, MD 20850, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yueyang","family":"Wang","sequence":"additional","affiliation":[{"name":"Department of Analytical Sciences, U.S. Technical & Biologics Development, Horizon Therapeutics, Rockville, MD 20850, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Lena","family":"McLaughlin","sequence":"additional","affiliation":[{"name":"Department of Analytical Sciences, U.S. Technical & Biologics Development, Horizon Therapeutics, Rockville, MD 20850, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Fei","family":"Yang","sequence":"additional","affiliation":[{"name":"Department of Analytical Sciences, U.S. Technical & Biologics Development, Horizon Therapeutics, Rockville, MD 20850, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1941-4145","authenticated-orcid":false,"given":"Zhengjian","family":"Li","sequence":"additional","affiliation":[{"name":"Department of Analytical Sciences, U.S. Technical & Biologics Development, Horizon Therapeutics, Rockville, MD 20850, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9271-0218","authenticated-orcid":false,"given":"Jihong","family":"Wang","sequence":"additional","affiliation":[{"name":"Department of Analytical Sciences, U.S. Technical & Biologics Development, Horizon Therapeutics, Rockville, MD 20850, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2023,9,19]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1897","DOI":"10.1021\/bi951526c","article-title":"Isomerization of an aspartic acid residue in the complementarity-determining regions of a recombinant antibody to human IgE: Identification and effect on binding affinity","volume":"35","author":"Cacia","year":"1996","journal-title":"Biochemistry"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"432","DOI":"10.4161\/mabs.24458","article-title":"Comparison of the in vitro and in vivo stability of a succinimide intermediate observed on a therapeutic IgG1 molecule","volume":"5","author":"Ouellette","year":"2013","journal-title":"mAbs"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"1871","DOI":"10.1021\/acs.analchem.5b04276","article-title":"LC-MS\/MS-Based Monitoring of In Vivo Protein Biotransformation: Quantitative Determination of Trastuzumab and Its Deamidation Products in Human Plasma","volume":"88","author":"Bults","year":"2016","journal-title":"Anal. Chem."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"383","DOI":"10.1038\/s41598-019-57184-2","article-title":"Characterization of IgG1 Fc Deamidation at Asparagine 325 and Its Impact on Antibody-dependent Cell-mediated Cytotoxicity and Fc\u03b3RIIIa Binding","volume":"10","author":"Lu","year":"2020","journal-title":"Sci. Rep."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"949","DOI":"10.1093\/glycob\/cwr027","article-title":"High-mannose glycans on the Fc region of therapeutic IgG antibodies increase serum clearance in humans","volume":"21","author":"Goetze","year":"2011","journal-title":"Glycobiology"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"1145","DOI":"10.1007\/s11095-007-9241-4","article-title":"Accumulation of succinimide in a recombinant monoclonal antibody in mildly acidic buffers under elevated temperatures","volume":"24","author":"Chu","year":"2007","journal-title":"Pharm. Res."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"1686","DOI":"10.1021\/ac802708s","article-title":"Direct identification and quantification of aspartyl succinimide in an IgG2 mAb by RapiGest assisted digestion","volume":"81","author":"Huang","year":"2009","journal-title":"Anal. Chem."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"94","DOI":"10.1016\/0003-9861(90)90014-P","article-title":"Formation of cyclic imide-like structures upon the treatment of calmodulin and a calmodulin peptide with heat","volume":"276","author":"Martin","year":"1990","journal-title":"Arch. Biochem. Biophys."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"3168","DOI":"10.1021\/ac702245c","article-title":"Separation and characterization of an IgG2 antibody containing a cyclic imide in CDR1 of light chain by hydrophobic interaction chromatography and mass spectrometry","volume":"80","author":"Jones","year":"2008","journal-title":"Anal. Chem."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"2714","DOI":"10.1021\/ac0617870","article-title":"18O labeling method for identification and quantification of succinimide in proteins","volume":"79","author":"Xiao","year":"2007","journal-title":"Anal. Chem."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"3509","DOI":"10.1002\/jps.21655","article-title":"Succinimide formation at Asn 55 in the complementarity determining region of a recombinant monoclonal antibody IgG1 heavy chain","volume":"98","author":"Yan","year":"2009","journal-title":"J. Pharm. Sci."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"5912","DOI":"10.1021\/ac200750u","article-title":"Accurate determination of succinimide degradation products using high fidelity trypsin digestion peptide map analysis","volume":"83","author":"Yu","year":"2011","journal-title":"Anal. Chem."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"9","DOI":"10.1016\/j.biologicals.2020.06.008","article-title":"A cross-industry forum on benchmarking critical quality attribute identification and linkage to process characterization studies","volume":"67","author":"Demmon","year":"2020","journal-title":"Biologicals"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"1145","DOI":"10.4161\/mabs.29883","article-title":"In vitro and in vivo modifications of recombinant and human IgG antibodies","volume":"6","author":"Liu","year":"2014","journal-title":"mAbs"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"3194","DOI":"10.1016\/j.xphs.2019.05.023","article-title":"Cyclization of N-Terminal Glutamic Acid to pyro-Glutamic Acid Impacts Monoclonal Antibody Charge Heterogeneity Despite Its Appearance as a Neutral Transformation","volume":"108","author":"Liu","year":"2019","journal-title":"J. Pharm. Sci."},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Beck, A., Nowak, C., Meshulam, D., Reynolds, K., Chen, D., Pacardo, D.B., Nicholls, S.B., Carven, G.J., Gu, Z., and Fang, J. (2022). Risk-Based Control Strategies of Recombinant Monoclonal Antibody Charge Variants. Antibodies, 11.","DOI":"10.3390\/antib11040073"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"460","DOI":"10.1016\/j.jpba.2019.05.058","article-title":"Comparative charge-based separation study with various capillary electrophoresis (CE) modes and cation exchange chromatography (CEX) for the analysis of monoclonal antibodies","volume":"174","author":"Kahle","year":"2019","journal-title":"J. Pharm. Biomed. Anal."},{"key":"ref_18","first-page":"951","article-title":"Antibody characterization using novel ERLIC-MS\/MS-based peptide mapping","volume":"10","author":"Zhen","year":"2018","journal-title":"mAbs"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1712","DOI":"10.1002\/jps.23529","article-title":"Asparagine deamidation dependence on buffer type, pH, and temperature","volume":"102","author":"Pace","year":"2013","journal-title":"J. Pharm. Sci."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"299","DOI":"10.1182\/blood-2016-10-743997","article-title":"Clarifying immunoglobulin gene usage in systemic and localized immunoglobulin light-chain amyloidosis by mass spectrometry","volume":"129","author":"Kourelis","year":"2017","journal-title":"Blood"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"2101","DOI":"10.1182\/bloodadvances.2020003671","article-title":"IGVL gene region usage correlates with distinct clinical presentation in IgM vs non-IgM light chain amyloidosis","volume":"5","author":"Sidana","year":"2021","journal-title":"Blood Adv."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"43","DOI":"10.1186\/s40164-021-00236-z","article-title":"Genetic pathogenesis of immunoglobulin light chain amyloidosis: Basic characteristics and clinical applications","volume":"10","author":"Xu","year":"2021","journal-title":"Exp. Hematol. Oncol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"1005","DOI":"10.1038\/s41591-018-0070-2","article-title":"A human anti-IL-2 antibody that potentiates regulatory T cells by a structure-based mechanism","volume":"24","author":"Trotta","year":"2018","journal-title":"Nat. Med."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"10664","DOI":"10.1039\/C8CC04396E","article-title":"Immunoglobulin light chain amyloid aggregation","volume":"54","author":"Misra","year":"2018","journal-title":"Chem. Commun."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"3521","DOI":"10.1021\/bi061716v","article-title":"Structural characterization of the partially folded intermediates of an immunoglobulin light chain leading to amyloid fibrillation and amorphous aggregation","volume":"46","author":"Qin","year":"2007","journal-title":"Biochemistry"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"774","DOI":"10.1111\/j.1471-4159.2012.07707.x","article-title":"N-Terminal pyroglutamate formation of A\u03b238 and A\u03b240 en-forces oligomer formation and potency to disrupt hippocampal long-term potentiation","volume":"121","author":"Schlenzig","year":"2012","journal-title":"J. Neurochem."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"7072","DOI":"10.1021\/bi900818a","article-title":"Pyroglutamate formation influences solubility and amyloi-dogenicity of amyloid peptides","volume":"48","author":"Schlenzig","year":"2009","journal-title":"Biochemistry"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"48","DOI":"10.1186\/s13024-016-0115-2","article-title":"Immunotherapy targeting pyroglutamate-3 A\u03b2: Prospects and challenges","volume":"11","author":"Cynis","year":"2016","journal-title":"Mol. Neurodegener."}],"container-title":["Antibodies"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2073-4468\/12\/3\/59\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T20:53:40Z","timestamp":1760129620000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2073-4468\/12\/3\/59"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,9,19]]},"references-count":28,"journal-issue":{"issue":"3","published-online":{"date-parts":[[2023,9]]}},"alternative-id":["antib12030059"],"URL":"https:\/\/doi.org\/10.3390\/antib12030059","relation":{},"ISSN":["2073-4468"],"issn-type":[{"type":"electronic","value":"2073-4468"}],"subject":[],"published":{"date-parts":[[2023,9,19]]}}}