title: Detailed Characterization of Monoclonal Antibody Receptor Interaction Using Affinity Liquid Chromatography Hyphenated to Native Mass Spectrometry.
abstract: We report on the online coupling of FcRn affinity liquid chromatography (LC) with electrospray ionization mass spectrometry (ESI-MS) in native conditions to study the influence of modifications on the interaction of recombinant mAbs with the immobilized FcRn receptor domain. The analysis conditions were designed to fit the requirements of both affinity LC and ESI-MS. The mobile phase composition was optimized to maintain the proteins studied in native conditions and enable sharp pH changes in order to mimic properly IgGs Fc domain/FcRn receptor interaction. Mobile phase components needed to be sufficiently volatile to achieve native MS analysis. MS data demonstrated the conservation of the pseudonative form of IgGs and allowed identification of the separated variants. Native FcRn affinity LC-ESI-MS was performed on a therapeutic mAb undergoing various oxidation stress. Native MS detection was used to determine the sample oxidation level. Lower retention was observed for mAbs oxidized variants compared to their intact counterparts indicating decreased affinities for the receptor. This methodology proved to be suitable to identify and quantify post-translational modifications at native protein level in order to correlate their influence on the binding to the FcRn receptor. Native FcRn affinity LC-ESI-MS can tremendously reduce the time required to assess the biological relevance of the IgG microheterogeneities thus providing valuable information for biopharmaceutical research and development.
authors.affiliation: Division of BioAnalytical Chemistry, Vrije Universiteit Amsterdam , 1081 HV Amsterdam, The Netherlands.
authors.forename: Rabah
authors.identifier: 0000-0001-8459-3138
authors.initials: R
authors.lastname: Gahoual
authors.affiliation: Pharma Technical Development Penzberg, Roche Diagnostics GmbH , Penzberg 82377, Germany.
authors.forename: Anna-Katharina
authors.initials: AK
authors.lastname: Heidenreich
authors.affiliation: Division of BioAnalytical Chemistry, Vrije Universiteit Amsterdam , 1081 HV Amsterdam, The Netherlands.
authors.forename: Govert W
authors.initials: GW
authors.lastname: Somsen
authors.affiliation: Pharma Technical Development Penzberg, Roche Diagnostics GmbH , Penzberg 82377, Germany.
authors.forename: Patrick
authors.initials: P
authors.lastname: Bulau
authors.affiliation: Pharma Technical Development Penzberg, Roche Diagnostics GmbH , Penzberg 82377, Germany.
authors.forename: Dietmar
authors.initials: D
authors.lastname: Reusch
authors.affiliation: Division of BioAnalytical Chemistry, Vrije Universiteit Amsterdam , 1081 HV Amsterdam, The Netherlands.|Center for Proteomics and Metabolomics, Leiden University Medical Center , 2333 ZA Leiden, The Netherlands.
authors.forename: Manfred
authors.initials: M
authors.lastname: Wuhrer
authors.affiliation: Pharma Technical Development Penzberg, Roche Diagnostics GmbH , Penzberg 82377, Germany.
authors.forename: Markus
authors.initials: M
authors.lastname: Haberger
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D010449:Peptide Mapping; D010455:Peptides / Q000032:analysis* / Q000302:isolation & purification; D011485:Protein Binding; D011961:Receptors, Fc / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D000911:Antibodies, Monoclonal; D007141:Immunoglobulin Fc Fragments; D010455:Peptides; D011961:Receptors, Fc; D011994:Recombinant Proteins