title: A novel approach to investigate the effect of methionine oxidation on pharmacokinetic properties of therapeutic antibodies.
abstract: Preserving the chemical and structural integrity of therapeutic antibodies during manufacturing and storage is a major challenge during pharmaceutical development. Oxidation of Fc methionines Met252 and Met428 is frequently observed, which leads to reduced affinity to FcRn and faster plasma clearance if present at high levels. Because oxidation occurs in both positions simultaneously, their individual contribution to the concomitant changes in pharmacokinetic properties has not been clearly established. A novel pH-gradient FcRn affinity chromatography method was applied to isolate three antibody oxidation variants from an oxidized IgG1 preparation based on their FcRn binding properties. Physico-chemical characterization revealed that the three oxidation variants differed predominantly in the number of oxMet252 per IgG (0, 1, or 2), but not significantly in the content of oxMet428. Corresponding to the increase in oxMet252 content, stepwise reduction of FcRn affinity in vitro, as well as faster clearance and shorter terminal half-life, in huFcRn-transgenic mice were observed. A single Met252 oxidation per antibody had no significant effect on pharmacokinetics (PK) compared with unmodified IgG. Importantly, only molecules with both heavy chains oxidized at Met252 exhibited significantly faster clearance. In contrast, Met428 oxidation had no apparent negative effect on PK and even led to somewhat improved FcRn binding and slower clearance. This minor effect, however, seemed to be abrogated by the dominant effect of Met252 oxidation. The novel approach of functional chromatographic separation of IgG oxidation variants followed by physico-chemical and biological characterization has yielded the first experimentally-backed explanation for the unaltered PK properties of antibody preparations containing relatively high Met252 and Met428 oxidation levels.
authors.affiliation: a Biochemical and Analytical Research; Large Molecule Research ; Roche Pharma Research and Early Development (pRED); Roche Innovation Center ; Penzberg , Germany.
authors.forename: Jan
authors.initials: J
authors.lastname: Stracke
authors.forename: Thomas
authors.initials: T
authors.lastname: Emrich
authors.forename: Petra
authors.initials: P
authors.lastname: Rueger
authors.forename: Tilman
authors.initials: T
authors.lastname: Schlothauer
authors.forename: Lothar
authors.initials: L
authors.lastname: Kling
authors.forename: Alexander
authors.initials: A
authors.lastname: Knaupp
authors.forename: Hubert
authors.initials: H
authors.lastname: Hertenberger
authors.forename: Andreas
authors.initials: A
authors.lastname: Wolfert
authors.forename: Christian
authors.initials: C
authors.lastname: Spick
authors.forename: Wilma
authors.initials: W
authors.lastname: Lau
authors.forename: Georg
authors.initials: G
authors.lastname: Drabner
authors.forename: Ulrike
authors.initials: U
authors.lastname: Reiff
authors.forename: Hans
authors.initials: H
authors.lastname: Koll
authors.forename: Apollon
authors.initials: A
authors.lastname: Papadimitriou
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism* / Q000493:pharmacokinetics* / Q000627:therapeutic use; D000915:Antibody Affinity; D002846:Chromatography, Affinity; D005260:Female; D015395:Histocompatibility Antigens Class I / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007141:Immunoglobulin Fc Fragments / Q000097:blood / Q000378:metabolism* / Q000627:therapeutic use; D008657:Metabolic Clearance Rate; D008715:Methionine / Q000378:metabolism*; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008822:Mice, Transgenic; D010084:Oxidation-Reduction / Q000187:drug effects; D011485:Protein Binding; D011961:Receptors, Fc / Q000235:genetics / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D000911:Antibodies, Monoclonal; D015395:Histocompatibility Antigens Class I; D007141:Immunoglobulin Fc Fragments; D011961:Receptors, Fc; D008715:Methionine; C100688:Fc receptor, neonatal
keywords: AUC, area under the concentration-time curve; Antibody; ESI-MS, electrospray ionization mass spectrometry; Fab, antigen-binding fragment; Fc, crystallizable fragment; FcRn; FcRn, neonatal Fc receptor; HRP, horseradish peroxidase; IgG, immunoglobulin G; Met, methionine; Met252; Met428; PK, pharmacokinetic; RU, response units; SEC, size exclusion chromatography; SPR, surface plasmon resonance; affinity chromatography; column; degradation; m/z, mass-to-charge ratio; mAb, monoclonal antibody; methionine oxidation; neonatal Fc receptor; pH gradient; pharmacokinetics