BIOPHYSICS LETTER FTIR markers of methionine oxidation for early detection of oxidized protein therapeutics Jascindra Ravi • Anna E. Hills • Eleonora Cerasoli • Paulina D. Rakowska • Maxim G. Ryadnov Received: 27 August 2010 / Revised: 25 November 2010 / Accepted: 30 November 2010 / Published online: 13 January 2011 Ó Her Majesty the Queen in Right of Australia as represented by the Commonwealth Scientific and Industrial Research Organisation 2011 Abstract The biological activity of therapeutic proteins is strongly dependent on the stability of their folded state, which can easily be compromised by degradation. Oxida- tion is one of the most common causes of degradation and is typically associated with impairment of the native pro- tein structure. Methionine residues stand out as particularly susceptible to oxidation by reactive oxygen intermediates even under mild conditions. Consequently, methionine oxidation has profound effects on protein activity up to the point of adverse biological responses. Of immediate importance therefore is finding affordable approaches for rapid detection of methionine oxidation before any sub- stantial structural changes can ensue. Herein we report that vibrational bands at 1,044 and 1,113 cm -1 in the mid- infrared region can serve as characteristic markers of methionine oxidation in oxidatively stressed protein ther- apeutics, monoclonal antibodies (IgG1 and its antigen- binding fragment). Such Fourier-transform infrared (FTIR) markers underpin rapid detection assays and hold particular promise for correlation of methionine oxidation with pro- tein structure and function. Keywords Methionine oxidation Á Protein therapeutics (IgG1 and antigen-binding fragment) Á Fourier-transform infrared Á IR marker bands Á Early detection Abbreviations FTIR Fourier-transform infrared LC Liquid chromatography MS Mass spectrometry CD Circular dichroism DSC Differential scanning calorimetry NMR Nuclear magnetic resonance HDX Hydrogen–deuterium exchange ATR Attenuated total reflectance ASN 1-Anilinonaphthalene-8-sulfonate DLS Dynamic light scattering mAb Monoclonal antibody fAb Fragment antigen binding DMSO Dimethyl sulfoxide Introduction Amongst various degradation processes which can adversely affect the activity of protein therapeutics, oxidation can lead to the most profound effects even under mild conditions (Reubsaet et al. 1998). One particular event underpinning the process is the susceptibility of methionine residues to different forms of reactive oxygen (Scheme 1) (Berlett and Stadtman 1997; Carpenter and Manning 2002; Stadtman and Levine 2006; Toennies and Callan 1939). Designed to scavenge environmental oxidants, thereby protecting the activity of a given protein, methionine oxidation can lead to severe effects on protein structure and function (Carpenter and Manning 2002). Establishing approaches that allow detection of methionine oxidation at early stages of protein therapeutics development thus becomes of immediate importance for assessment and monitoring of its impact on protein structure. J. Ravi Á E. Cerasoli Á P. D. Rakowska Á M. G. Ryadnov (&) National Physical Laboratory, Hampton Road, Teddington TW11 0LW, UK e-mail: max.ryadnov@npl.co.uk A. E. Hills Lonza Biologics plc, Bath Road, Slough SL1 4DX, UK 123 Eur Biophys J (2011) 40:339–345 DOI 10.1007/s00249-010-0656-1