Dariusz J. Janecki, Chi-Ya Kao-Scharf, Andreas Hoffmann
{"title":"使用LC-MS发现和表征IgG4抗体异常o -链糖基化。","authors":"Dariusz J. Janecki, Chi-Ya Kao-Scharf, Andreas Hoffmann","doi":"10.1002/rcm.9969","DOIUrl":null,"url":null,"abstract":"<div>\n \n \n <section>\n \n <h3> Background</h3>\n \n <p>Consensus is that immunoglobulin IgG4 contains only N-linked glycosylation. The analysis of several batches of commercial biopharmaceutical product Dupixent using top-down intact mass spectrometry revealed that this IgG4 features a small amount of O-linked glycosylation in the Fab region. This is the first report of an O-linked glycosylation in an IgG4 antibody.</p>\n </section>\n \n <section>\n \n <h3> Methods</h3>\n \n <p>Monoclonal antibody solutions were subjected to cation exchange (CEX) and reverse phase (RP) chromatography and/or additional preconcentration/fractionation methods to prepare samples for subsequent analysis. Advanced MS analysis and fragmentation techniques (HCD, ETD, and EThcD) were employed to localize the O-linked glycosylation as well as elucidate the structure of the glycan(s).</p>\n </section>\n \n <section>\n \n <h3> Results</h3>\n \n <p>O-linked glycosylation in the IgG4 dupilumab was discovered by intact-MS. The probable location was narrowed down to four sites in the CH1 domain, and the structure of the O-linked glycan was determined to be of Core 1 type. The relative quantities of the modifications were low, but the glycosylation was consistently detected in several batches of Dupixent.</p>\n </section>\n \n <section>\n \n <h3> Conclusions</h3>\n \n <p>We discovered a rare glycosylation modification on dupilumab, an IgG4 antibody. The O-linked glycosylation was characterized and localized in the Fab region.</p>\n </section>\n </div>","PeriodicalId":225,"journal":{"name":"Rapid Communications in Mass Spectrometry","volume":"39 5","pages":""},"PeriodicalIF":1.8000,"publicationDate":"2024-12-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11635057/pdf/","citationCount":"0","resultStr":"{\"title\":\"Discovery and Characterization of Unusual O-Linked Glycosylation of IgG4 Antibody Using LC-MS\",\"authors\":\"Dariusz J. Janecki, Chi-Ya Kao-Scharf, Andreas Hoffmann\",\"doi\":\"10.1002/rcm.9969\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div>\\n \\n \\n <section>\\n \\n <h3> Background</h3>\\n \\n <p>Consensus is that immunoglobulin IgG4 contains only N-linked glycosylation. The analysis of several batches of commercial biopharmaceutical product Dupixent using top-down intact mass spectrometry revealed that this IgG4 features a small amount of O-linked glycosylation in the Fab region. This is the first report of an O-linked glycosylation in an IgG4 antibody.</p>\\n </section>\\n \\n <section>\\n \\n <h3> Methods</h3>\\n \\n <p>Monoclonal antibody solutions were subjected to cation exchange (CEX) and reverse phase (RP) chromatography and/or additional preconcentration/fractionation methods to prepare samples for subsequent analysis. Advanced MS analysis and fragmentation techniques (HCD, ETD, and EThcD) were employed to localize the O-linked glycosylation as well as elucidate the structure of the glycan(s).</p>\\n </section>\\n \\n <section>\\n \\n <h3> Results</h3>\\n \\n <p>O-linked glycosylation in the IgG4 dupilumab was discovered by intact-MS. The probable location was narrowed down to four sites in the CH1 domain, and the structure of the O-linked glycan was determined to be of Core 1 type. The relative quantities of the modifications were low, but the glycosylation was consistently detected in several batches of Dupixent.</p>\\n </section>\\n \\n <section>\\n \\n <h3> Conclusions</h3>\\n \\n <p>We discovered a rare glycosylation modification on dupilumab, an IgG4 antibody. 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Discovery and Characterization of Unusual O-Linked Glycosylation of IgG4 Antibody Using LC-MS
Background
Consensus is that immunoglobulin IgG4 contains only N-linked glycosylation. The analysis of several batches of commercial biopharmaceutical product Dupixent using top-down intact mass spectrometry revealed that this IgG4 features a small amount of O-linked glycosylation in the Fab region. This is the first report of an O-linked glycosylation in an IgG4 antibody.
Methods
Monoclonal antibody solutions were subjected to cation exchange (CEX) and reverse phase (RP) chromatography and/or additional preconcentration/fractionation methods to prepare samples for subsequent analysis. Advanced MS analysis and fragmentation techniques (HCD, ETD, and EThcD) were employed to localize the O-linked glycosylation as well as elucidate the structure of the glycan(s).
Results
O-linked glycosylation in the IgG4 dupilumab was discovered by intact-MS. The probable location was narrowed down to four sites in the CH1 domain, and the structure of the O-linked glycan was determined to be of Core 1 type. The relative quantities of the modifications were low, but the glycosylation was consistently detected in several batches of Dupixent.
Conclusions
We discovered a rare glycosylation modification on dupilumab, an IgG4 antibody. The O-linked glycosylation was characterized and localized in the Fab region.
期刊介绍：
Rapid Communications in Mass Spectrometry is a journal whose aim is the rapid publication of original research results and ideas on all aspects of the science of gas-phase ions; it covers all the associated scientific disciplines. There is no formal limit on paper length ("rapid" is not synonymous with "brief"), but papers should be of a length that is commensurate with the importance and complexity of the results being reported. Contributions may be theoretical or practical in nature; they may deal with methods, techniques and applications, or with the interpretation of results; they may cover any area in science that depends directly on measurements made upon gaseous ions or that is associated with such measurements.
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