title: Functional assessment of antibody oxidation by native mass spectrometry.
abstract: Oxidation of methionine (Met) residues is one of several chemical degradation pathways for recombinant IgG1 antibodies. Studies using several methodologies have indicated that Met oxidation in the constant IgG1 domains affects in vitro interaction with human neonatal Fc (huFcRn) receptor, which is important for antibody half-life. Here, a completely new approach to investigating the effect of oxidative stress conditions has been applied. Quantitative ultra-performance liquid chromatography mass spectrometry (MS) peptide mapping, classical surface plasmon resonance and the recently developed FcRn column chromatography were combined with the new fast-growing approach of native MS as a near native state protein complex analysis in solution. Optimized mass spectrometric voltage and pressure conditions were applied to stabilize antibody/huFcRn receptor complexes in the gas phase for subsequent native MS experiments with oxidized IgG1 material. This approach demonstrated a linear correlation between quantitative native MS and IgG-FcRn functional analysis. In our study, oxidation of the heavy chain Met-265 resulted in a stepwise reduction of mAb3/huFcRn receptor complex formation. Remarkably, a quantitative effect of the heavy chain Met-265 oxidation on relative binding capacity was only detected for doubly oxidized IgG1, whereas IgG1 with only one oxidized heavy chain Met-265 was not found to significantly affect IgG1 binding to huFcRn. Thus, mono-oxidized IgG1 heavy chain Met-265 most likely does not represent a critical quality attribute for pharmacokinetics.
authors.affiliation: a Pharma Technical Development Penzberg; Roche Diagnostics GmbH ; Penzberg , Germany.
authors.forename: Markus
authors.initials: M
authors.lastname: Haberger
authors.forename: Anna-Katharina
authors.initials: AK
authors.lastname: Heidenreich
authors.forename: Tilman
authors.initials: T
authors.lastname: Schlothauer
authors.forename: Michaela
authors.initials: M
authors.lastname: Hook
authors.forename: Jana
authors.initials: J
authors.lastname: Gassner
authors.forename: Katrin
authors.initials: K
authors.lastname: Bomans
authors.forename: Michelle
authors.initials: M
authors.lastname: Yegres
authors.forename: Adrian
authors.initials: A
authors.lastname: Zwick
authors.forename: Boris
authors.initials: B
authors.lastname: Zimmermann
authors.forename: Harald
authors.initials: H
authors.lastname: Wegele
authors.forename: Lea
authors.initials: L
authors.lastname: Bonnington
authors.forename: Dietmar
authors.initials: D
authors.lastname: Reusch
authors.forename: Patrick
authors.initials: P
authors.lastname: Bulau
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000378:metabolism; D002853:Chromatography, Liquid; D015395:Histocompatibility Antigens Class I / Q000378:metabolism*; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry / Q000378:metabolism*; D013058:Mass Spectrometry / Q000379:methods*; D010084:Oxidation-Reduction; D010449:Peptide Mapping; D011961:Receptors, Fc / Q000378:metabolism*; D020349:Surface Plasmon Resonance
chemical_list: D000911:Antibodies, Monoclonal; D015395:Histocompatibility Antigens Class I; D007074:Immunoglobulin G; D011961:Receptors, Fc; C100688:Fc receptor, neonatal
keywords: critical quality attributes; native mass spectrometry; neonatal Fc receptor; oxidation; protein degradation; recombinant antibodies