title: Collision Induced Unfolding of Intact Antibodies: Rapid Characterization of Disulfide Bonding Patterns, Glycosylation, and Structures.
abstract: 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 (∼150 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 "fingerprint plots" 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.
authors.affiliation: Department of Chemistry, University of Michigan , 930 North University Avenue, Ann Arbor, Michigan 48109, United States.
authors.forename: Yuwei
authors.initials: Y
authors.lastname: Tian
authors.affiliation: Department of Chemistry, University of Michigan , 930 North University Avenue, Ann Arbor, Michigan 48109, United States.
authors.forename: Linjie
authors.initials: L
authors.lastname: Han
authors.affiliation: Department of Chemistry, University of Michigan , 930 North University Avenue, Ann Arbor, Michigan 48109, United States.
authors.forename: Adam C
authors.initials: AC
authors.lastname: Buckner
authors.affiliation: Department of Chemistry, University of Michigan , 930 North University Avenue, Ann Arbor, Michigan 48109, United States.
authors.forename: Brandon T
authors.initials: BT
authors.lastname: Ruotolo
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry*; D006031:Glycosylation; D013058:Mass Spectrometry; D015394:Molecular Structure; D058767:Protein Unfolding
chemical_list: D000911:Antibodies, Monoclonal; D004220:Disulfides