title: Analytical FcRn affinity chromatography for functional characterization of monoclonal antibodies.
abstract: The neonatal Fc receptor (FcRn) is important for the metabolic fate of IgG antibodies in vivo. Analysis of the interaction between FcRn and IgG in vitro might provide insight into the structural and functional integrity of therapeutic IgG that may affect pharmacokinetics (PK) in vivo. We developed a standardized pH gradient FcRn affinity liquid chromatography method with conditions closely resembling the physiological mechanism of interaction between IgG and FcRn. This method allows the separation of molecular IgG isoforms, degradation products and engineered molecules based on their affinity to FcRn. Human FcRn was immobilized on the column and a linear pH gradient from pH 5.5 to 8.8 was applied. FcRn chromatography was used in comparison to surface plasmon resonance to characterize different monoclonal IgG preparations, e.g., oxidized or aggregated species. Wild-type and engineered IgGs were compared in vitro by FcRn chromatography and in vivo by PK studies in huFcRn transgenic mice. Analytical FcRn chromatography allows differentiation of IgG samples and variants by peak pattern and retention time profile. The method can distinguish: 1) IgGs with different Fabs, 2) oxidized from native IgG, 3) aggregates from monomer and 4) antibodies with mutations in the Fc part from wild-type IgGs. Changes in the FcRn chromatographic behavior of mutant IgGs relative to the wild-type IgG correlate to changes in the PK profile in the FcRn transgenic mice. These results demonstrate that FcRn affinity chromatography is a useful new method for the assessment of IgG integrity.
authors.affiliation: Department of Protein Analytics, Pharma Research and Early Development (pRED), Roche Diagnostics GmbH, Penzberg, Germany. tilman.schlothauer@roche.com
authors.forename: Tilman
authors.initials: T
authors.lastname: Schlothauer
authors.forename: Petra
authors.initials: P
authors.lastname: Rueger
authors.forename: Jan Olaf
authors.initials: JO
authors.lastname: Stracke
authors.forename: Hubert
authors.initials: H
authors.lastname: Hertenberger
authors.forename: Felix
authors.initials: F
authors.lastname: Fingas
authors.forename: Lothar
authors.initials: L
authors.lastname: Kling
authors.forename: Thomas
authors.initials: T
authors.lastname: Emrich
authors.forename: Georg
authors.initials: G
authors.lastname: Drabner
authors.forename: Stefan
authors.initials: S
authors.lastname: Seeber
authors.forename: Johannes
authors.initials: J
authors.lastname: Auer
authors.forename: Stefan
authors.initials: S
authors.lastname: Koch
authors.forename: Apollon
authors.initials: A
authors.lastname: Papadimitriou
mesh_terms: D000818:Animals; D058846:Antibodies, Monoclonal, Murine-Derived / Q000032:analysis* / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D002846:Chromatography, Affinity / Q000379:methods*; D015395:Histocompatibility Antigens Class I / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D007074:Immunoglobulin G / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D008822:Mice, Transgenic; D009154:Mutation; D011961:Receptors, Fc / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D020349:Surface Plasmon Resonance
chemical_list: D058846:Antibodies, Monoclonal, Murine-Derived; D015395:Histocompatibility Antigens Class I; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D011961:Receptors, Fc; C100688:Fc receptor, neonatal
keywords: FcRn; PK; affinity chromatography; antibody; column; degradation; methionine oxidation; neonatal Fc receptor; pH gradient; pharmacokinetics