Collision Induced Unfolding of Intact Antibodies: Rapid Characterization of Disulfide Bonding Patterns, Glycosylation, and Structures

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• 作者：Yuwei Tian ; Linjie Han ; Adam C. Buckner ; Brandon T. Ruotolo
• 刊名：Analytical Chemistry
• 出版年：2015
• 出版时间：November 17, 2015
• 年：2015
• 卷：87
• 期：22
• 页码：11509-11515
• 全文大小：421K
• ISSN：1520-6882

文摘

Monoclonal antibodies (mAbs) are among the fastest growing class of therapeutics due to their high specificity and low incidence of side effects. Unlike most drugs, mAbs are complex macromolecules (鈭?50 kDa), leading to a host of quality control and characterization challenges inherent in their development. Recently, we introduced a new approach for the analysis of the intact proteins based on ion mobility-mass spectrometry (IM-MS). Our protocol involves the collision induced unfolding (CIU) of intact antibodies, where collisional heating in the gas-phase is used to generate unfolded antibody forms, which are subsequently separated by IM and then analyzed by MS. Collisional energy is added to the antibody ions in a stepwise fashion, and 鈥渇ingerprint plots鈥?are created that track the amount of unfolding undergone as a function of the energy imparted to the ions prior to IM separation. In this report, we have used these fingerprints to rapidly distinguish between antibody isoforms, possessing different numbers and/or patterns of disulfide bonding and general levels of glycosylation. In addition, we validate our CIU protocols through control experiments and systematic statistical evaluations of CIU reproducibility. We conclude by projecting the impact of our approach for antibody-related drug discovery and development applications.