title: Effect of individual Fc methionine oxidation on FcRn binding: Met252 oxidation impairs FcRn binding more profoundly than Met428 oxidation.
abstract: The long serum half-lives of mAbs are conferred by pH-dependent binding of IgG-Fc to the neonatal Fc receptor (FcRn). The Fc region of human IgG1 has three conserved methionine residues, Met252, Met358, and Met428. Recent studies showed oxidation of these Met residues impairs FcRn binding and consequently affects pharmacokinetics of therapeutic antibodies. However, the quantitative effect of individual Met oxidation on Fc-FcRn binding has not been addressed. This information is valuable for defining critical quality attributes. In the present study, two sets of homodimeric site-directed IgG1 mutations were generated to understand how individual Fc Met oxidation affects FcRn binding. The first approach used Met to Leu mutants to block site-specific Met oxidation. In the other approach, Met to Gln mutants were designed to mimic site-specific Met oxidation. Both mutagenesis approaches show that either Met252 or Met428 oxidation alone significantly impairs Fc-FcRn binding. Met252 oxidation has a more deleterious effect on FcRn binding than M428 oxidation, whereas Met428 oxidation has a bigger destabilization effect on the thermal stability. Our results also show that Met358 oxidation does not affect FcRn binding. In addition, our study suggests that Met to Gln mutation may serve as an important tool to understand Met oxidation.
authors.affiliation: Late Stage Pharmaceutical Development, Genentech, South San Francisco, California, 94080.
authors.forename: Xuan
authors.initials: X
authors.lastname: Gao
authors.forename: Junyan A
authors.initials: JA
authors.lastname: Ji
authors.forename: Karthik
authors.initials: K
authors.lastname: Veeravalli
authors.forename: Y John
authors.initials: YJ
authors.lastname: Wang
authors.forename: Taylor
authors.initials: T
authors.lastname: Zhang
authors.forename: William
authors.initials: W
authors.lastname: Mcgreevy
authors.forename: Kai
authors.initials: K
authors.lastname: Zheng
authors.forename: Robert F
authors.initials: RF
authors.lastname: Kelley
authors.forename: Michael W
authors.initials: MW
authors.lastname: Laird
authors.forename: Jun
authors.initials: J
authors.lastname: Liu
authors.forename: Mary
authors.initials: M
authors.lastname: Cromwell
mesh_terms: D001665:Binding Sites; D015395:Histocompatibility Antigens Class I / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry* / Q000378:metabolism*; D007074:Immunoglobulin G / Q000737:chemistry / Q000378:metabolism; D008715:Methionine / Q000737:chemistry* / Q000378:metabolism*; D010084:Oxidation-Reduction; D011961:Receptors, Fc / Q000737:chemistry* / Q000378:metabolism*; D020349:Surface Plasmon Resonance
chemical_list: D015395:Histocompatibility Antigens Class I; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D011961:Receptors, Fc; D008715:Methionine; C100688:Fc receptor, neonatal
keywords: HPLC; calorimetry; critical quality attributes; monoclonal antibody; neonatal Fc receptor; oxidation; protein binding; serum half-life; stability; surface plasmon resonance