title: Mapping the Interactions of Selective Biochemical Probes of Antibody Conformation by Hydrogen-Deuterium Exchange Mass Spectrometry.
abstract: Protein-based pharmaceuticals represent the fastest growing group of drugs in development in the pharmaceutical industry. One of the major challenges in the discovery, development, and distribution of biopharmaceuticals is the assessment of changes in their higher-order structure due to chemical modification. Here, we investigated the interactions of three different biochemical probes (Fabs) generated to detect conformational changes in a therapeutic IgG1 antibody (mAbX) by local hydrogen-deuterium exchange mass spectrometry (HDX-MS). We show that two of the probes target the Fcpart of the antibody, whereas the third probe binds to the hinge region. Through HDX-ETD, we could distinguish specific binding patterns of the Fc-binding probes on mAbX at the amino-acid level. Preliminary surface plasmon resonance (SPR) experiments showed that these domain-selective Fabprobes are sensitive to conformational changes in distinct regions of a full-length therapeutic antibody upon oxidation.
authors.affiliation: Department of Pharmacy, University of Copenhagen, Universitetsparken 2, 2100, Copenhagen, Denmark.
authors.forename: Ulrike
authors.identifier: 0000-0002-8828-3505
authors.initials: U
authors.lastname: Leurs
authors.affiliation: Pharma Technical Development Basel, F. Hoffmann-La Roche Ltd., Grenzacherstrasse 124, 4070, Basel, Switzerland.
authors.forename: Hermann
authors.initials: H
authors.lastname: Beck
authors.affiliation: Pharma Technical Development Penzberg, Roche Diagnostics GmbH, Nonnenwald 2, 82377, Penzberg, Germany.
authors.forename: Lea
authors.initials: L
authors.lastname: Bonnington
authors.affiliation: Pharma Technical Development Penzberg, Roche Diagnostics GmbH, Nonnenwald 2, 82377, Penzberg, Germany.
authors.forename: Ingo
authors.initials: I
authors.lastname: Lindner
authors.affiliation: GE Healthcare Bio-Sciences AB, Björkgatan 30, 75184, Uppsala, Sweden.
authors.forename: Ewa
authors.initials: E
authors.lastname: Pol
authors.affiliation: Department of Pharmacy, University of Copenhagen, Universitetsparken 2, 2100, Copenhagen, Denmark.
authors.forename: Kasper
authors.identifier: 0000-0002-6337-5489
authors.initials: K
authors.lastname: Rand
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000627:therapeutic use; D041961:Deuterium Exchange Measurement; D015195:Drug Design; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry*; D007074:Immunoglobulin G; D013058:Mass Spectrometry / Q000379:methods; D015335:Molecular Probes; D011487:Protein Conformation
chemical_list: D000911:Antibodies, Monoclonal; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D015335:Molecular Probes
keywords: HDX-MS; conformation analysis; structural biology; surface plasmon resonance; therapeutic antibodies