Rapid assessment of oxidation via middle-down LCMS correlates with methionine side-chain solvent-accessible surface area for 121 clinical stage monoclonal antibodies

MAbs. 2017 May/Jun;9(4):646-653. doi: 10.1080/19420862.2017.1290753. Epub 2017 Feb 14.


Susceptibility of methionine to oxidation is an important concern for chemical stability during the development of a monoclonal antibody (mAb) therapeutic. To minimize downstream risks, leading candidates are usually screened under forced oxidation conditions to identify oxidation-labile molecules. Here we report results of forced oxidation on a large set of in-house expressed and purified mAbs with variable region sequences corresponding to 121 clinical stage mAbs. These mAb samples were treated with 0.1% H2O2 for 24 hours before enzymatic cleavage below the hinge, followed by reduction of inter-chain disulfide bonds for the detection of the light chain, Fab portion of heavy chain (Fd) and Fc by liquid chromatography-mass spectrometry. This high-throughput, middle-down approach allows detection of oxidation site(s) at the resolution of 3 distinct segments. The experimental oxidation data correlates well with theoretical predictions based on the solvent-accessible surface area of the methionine side-chains within these segments. These results validate the use of upstream computational modeling to predict mAb oxidation susceptibility at the sequence level.

Keywords: Chemical liability; chemical stability; developability; forced degradation; mAbs; methionine; oxidation; solvent accessible surface area.

MeSH terms

  • Antibodies, Monoclonal / chemistry*
  • Chromatography, Liquid / methods
  • HEK293 Cells
  • Humans
  • Hydrogen Peroxide / chemistry*
  • Mass Spectrometry / methods*
  • Methionine / chemistry*
  • Models, Molecular*
  • Oxidation-Reduction


  • Antibodies, Monoclonal
  • Methionine
  • Hydrogen Peroxide