Pharmaceutical Research

, Volume 30, Issue 5, pp 1311–1327 | Cite as

Fluorogenic Tagging Methodology Applied to Characterize Oxidized Tyrosine and Phenylalanine in an Immunoglobulin Monoclonal Antibody

  • Shuxia Zhou
  • Olivier Mozziconacci
  • Bruce A. Kerwin
  • Christian Schöneich
Research Paper



Metal-catalyzed oxidation (MCO) of proteins is of primary concern in the development of biotherapeutics as it represents a prominent degradation pathway with potential undesired biological and biotherapeutic consequences.


We developed a fluorogenic derivatization methodology to study the MCO of IgG1 using a model oxidation system, CuCl2/L-ascorbic acid.


Besides the oxidation of Met, Trp and His residues, we detected significant oxidation of Phe and Tyr in IgG1.


The fluorogenic derivatization method provides an alternative approach for the rapid detection of oxidized Tyr and Phe as their benzoxazole derivatives by fluorescence spectrometry and size exclusion chromatography coupled to fluorescence detection.


ABS fluorogenic tagging/derivatization immunoglobulin G (IgG) monoclonal antibody (mAb) metal catalyzed oxidation (MCO) protein degradation reverse phase liquid chromatography with mass spectrometry (RPLC-MS) 





4-(aminomethyl) benzenesulfonic acid








electrospray ionization – mass spectrometry


fourier transform ion cyclotron resonance mass spectrometry




immunoglobulin G


monoclonal antibody


metal-catalyzed oxidation


size exclusion chromatography


Acknowledgments and Disclosures

This research was supported by Amgen Incorporation. The authors thank Dr. Nadya Galeva from the Mass Spectrometry Laboratory at University of Kansas for performing MS measurements on the FT-ICR instrument.


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Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Shuxia Zhou
    • 1
  • Olivier Mozziconacci
    • 1
  • Bruce A. Kerwin
    • 2
  • Christian Schöneich
    • 1
  1. 1.Department of Pharmaceutical ChemistryUniversity of KansasLawrenceUSA
  2. 2.Department of Process and Product DevelopmentAmgen Inc.SeattleUSA

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