title: Selective Oxidation of Methionine and Tryptophan Residues in a Therapeutic IgG1 Molecule.
abstract: Oxidation of methionine and tryptophan are common degradation pathways for monoclonal antibodies and present major analytical challenges in biotechnology. Generally, protein oxidation is detectable in stability and/or stressed samples (e.g., exposed to hydrogen peroxide, UV light, or metal ions). The induced chemical modifications may impact the biological activity of antibodies and may have biological consequences. However, these effects and the contribution of individual protein modifications are difficult to delineate as different amino acids are often oxidized simultaneously and accompanied by other degradants such as aggregates, especially in forced degradation studies. Here, we report a new method to obtain selective oxidation of methionine or tryptophan by using oxidation reagents combined with large excess of free tryptophan or methionine, correspondingly. More specifically, using hydrogen peroxide or tert-butyl hydroperoxide in combination with addition of free tryptophan allowed for selective oxidation of methionine. Conversely, the use of 2,2-azobis(2-amidinopropane) dihydrochloride in combination with free methionine resulted in selective tryptophan oxidation, whereas methionine oxidation was not significantly altered. This novel stress model system may prove to be valuable tool in future mechanistic studies of oxidative degradation of protein therapeutics.
authors.affiliation: Pharmaceutical Development and Supplies, Pharma Technical Development Biologics Europe, F. Hoffmann-La Roche Ltd., Basel, Switzerland.|Division of Pharmaceutical Technology, Department of Pharmaceutical Sciences, University of Basel, Basel, Switzerland.
authors.forename: Emilien
authors.initials: E
authors.lastname: Folzer
authors.affiliation: Analytical Development and Quality Control, Pharma Technical Development Biologics Europe, Roche Diagnostics GmbH, Penzberg, Germany.
authors.forename: Katharina
authors.initials: K
authors.lastname: Diepold
authors.affiliation: Analytical Development and Quality Control, Pharma Technical Development Biologics Europe, Roche Diagnostics GmbH, Penzberg, Germany.
authors.forename: Katrin
authors.initials: K
authors.lastname: Bomans
authors.affiliation: Analytical Development and Quality Control, Pharma Technical Development Biologics Europe, F. Hoffmann-La Roche Ltd., Basel, Switzerland.
authors.forename: Christof
authors.initials: C
authors.lastname: Finkler
authors.affiliation: Pharmaceutical Development and Supplies, Pharma Technical Development Biologics Europe, F. Hoffmann-La Roche Ltd., Basel, Switzerland.
authors.forename: Roland
authors.initials: R
authors.lastname: Schmidt
authors.affiliation: Analytical Development and Quality Control, Pharma Technical Development Biologics Europe, Roche Diagnostics GmbH, Penzberg, Germany.
authors.forename: Patrick
authors.initials: P
authors.lastname: Bulau
authors.affiliation: Division of Pharmaceutical Technology, Department of Pharmaceutical Sciences, University of Basel, Basel, Switzerland.
authors.forename: Jörg
authors.initials: J
authors.lastname: Huwyler
authors.affiliation: Pharmaceutical Development and Supplies, Pharma Technical Development Biologics Europe, F. Hoffmann-La Roche Ltd., Basel, Switzerland.
authors.forename: Hanns-Christian
authors.initials: HC
authors.lastname: Mahler
authors.affiliation: Analytical Development and Quality Control, Pharma Technical Development Biologics Europe, F. Hoffmann-La Roche Ltd., Basel, Switzerland.
authors.forename: Atanas V
authors.initials: AV
authors.lastname: Koulov
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry; D006861:Hydrogen Peroxide / Q000737:chemistry; D007074:Immunoglobulin G / Q000737:chemistry*; D007202:Indicators and Reagents; D008670:Metals / Q000737:chemistry; D008715:Methionine / Q000737:chemistry*; D010084:Oxidation-Reduction; D014364:Tryptophan / Q000737:chemistry*; D014466:Ultraviolet Rays
chemical_list: D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D007202:Indicators and Reagents; D008670:Metals; D014364:Tryptophan; D008715:Methionine; D006861:Hydrogen Peroxide
keywords: mass spectrometry; methionine; protein A binding; protein oxidation; therapeutic proteins; tryptophan