ABSTRACT 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.

Dariusz J. Janecki

Chi‐Ya Kao‐Scharf

Andreas Hoffmann

Rapid Communications in Mass Spectrometry

RAPID COMMUNICATIONS IN MASS SPECTROMETRY 是一本期刊，其目的是快速发表关于气相离子科学各个方面的原始研究成果和想法；它涵盖了所有相关的科学学科。论文长度没有正式限制（“快速”不是“简短”的同义词），但论文的长度应与所报告结果的重要性和复杂性相称。贡献可能是理论上的或实际的；他们可能涉及方法、技术和应用，或结果的解释；它们可能涵盖直接依赖于对气态离子进行的测量或与此类测量相关的任何科学领域。

Rapid Communications in Mass Spectrometry is a journal aimed at the rapid publication of original research findings and ideas in all aspects of gas-phase ion science. It encompasses all relevant scientific disciplines. There is no formal limit on the length of papers ("rapid" does not equate to "short"), but the length should be commensurate with the significance and complexity of the reported results. Contributions may be theoretical or practical; they may involve methods, techniques, and applications, or interpretations of results. They may cover any scientific field that directly relies on measurements of gas-phase ions or is related to such measurements.

《Rapid Communications in Mass Spectrometry》是一本旨在快速发表气相离子科学各个方面的原创研究成果和想法的期刊。它涵盖所有相关的科学学科。论文长度没有正式限制（“快速”并不等同于“简短”），但应与所报告结果的重要性和复杂性相称。贡献可以是理论性的或实践性的，可能涉及方法、技术和应用，或结果的解释。这些贡献可能直接依赖于气态离子的测量，或与此类测量相关的任何科学领域。

Category	Quartile
化学	3
化学, 生化研究方法	4
化学, 分析化学	4
化学, 光谱学	4

Discovery and Characterization of Unusual O‐Linked Glycosylation of IgG4 Antibody Using LC‐MS

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