title: Impact of IgG2 high molecular weight species on neonatal Fc receptor binding assays.
abstract: A cell-based assay and a solution neonatal Fc receptor (FcRn) binding assay were implemented for the characterization of an IgG2 antibody after observation that different product lots exhibited unexpected differences in FcRn binding in the cell-based format with membrane-bound FcRn. The experiments described here suggest that the apparent differences observed in the FcRn binding across different product lots in the cell-based format can be attributed to the different levels of the higher order high molecular weight species (HMWs) in them. A strong correlation between FcRn binding in the cell-based format and the percentage (%) higher order HMWs suggests that small amounts (∼0.1%) of the latter could cause the enhanced apparent FcRn binding (% relative binding ranging from 50 to 100%) in the format. However, when the binding was assessed with recombinant FcRn in soluble form, avidity effects were minimal and the assay format exhibited less sensitivity toward the differences in higher order HMWs levels across product lots. In conclusion, a solution-based assay may be a more appropriate assay to assess FcRn binding of the dominant species of an Fc-fusion protein or monoclonal antibody if minor differences in product variants such as higher order HMWs are shown to affect the binding significantly.
authors.affiliation: Analytical Sciences, Amgen, Seattle, WA 98119, USA. Electronic address: yulingzhang@chemalum.berkeley.edu.
authors.forename: Yuling
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Regeneron Pharmaceuticals, Rensselaer, NY 12144, USA.
authors.forename: Abhishek
authors.initials: A
authors.lastname: Mathur
authors.affiliation: Functional Biocharacterization, Amgen, Thousand Oaks, CA 91320, USA.
authors.forename: Gwen
authors.initials: G
authors.lastname: Maher
authors.affiliation: Seattle Genetics, Bothell, WA 98021, USA.
authors.forename: Thomas
authors.initials: T
authors.lastname: Arroll
authors.affiliation: Analytical Sciences, Amgen, Seattle, WA 98119, USA.
authors.forename: Robert
authors.initials: R
authors.lastname: Bailey
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000915:Antibody Affinity; D001667:Binding, Competitive; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004355:Drug Stability; D005434:Flow Cytometry; D057809:HEK293 Cells; D015395:Histocompatibility Antigens Class I / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D055767:Immobilized Proteins; D007074:Immunoglobulin G / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016014:Linear Models; D008970:Molecular Weight; D019999:Pharmaceutical Solutions / Q000737:chemistry / Q000378:metabolism; D066329:Protein Aggregates; D011786:Quality Control; D011961:Receptors, Fc / Q000819:agonists* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012995:Solubility; D013050:Spectrometry, Fluorescence
chemical_list: D000911:Antibodies, Monoclonal; D015395:Histocompatibility Antigens Class I; D055767:Immobilized Proteins; D007074:Immunoglobulin G; D019999:Pharmaceutical Solutions; D066329:Protein Aggregates; D011961:Receptors, Fc; D011994:Recombinant Proteins; C100688:Fc receptor, neonatal
keywords: Aggregates; Antibody; FcRn binding; HMWs; SEC