Use of a stable-isotope-labeled reporter peptide and antioxidants for reliable quantification of methionine oxidation in a monoclonal antibody by liquid chromatography/mass spectrometry.

摘要: Rationale Accurate quantification of methionine oxidation in therapeutic proteins by liquid chromatography/mass spectrometry (LC/MS) is challenging due to the potential artifacts introduced during sample preparation and analysis peptide mapping workflow. In this study, a systematic approach for optimization procedure achieve reliable endogenous monoclonal antibodies was developed. Methods The based on usage stable-isotope-labeled reporter peptide, identical sequence tryptic an IgG1 antibody containing residue most prone oxidation. This applied evaluating various desalting procedures, tested nanoLC/MS, microLC/MS UPLC/MS model sensitive oxidation. Results Several steps with LC/MS detection at which protein occurred were identified optimized using reference peptide. Thus, target validated. Conclusions The methodology utilizes applicable could be extended other biotherapeutics once oxidation-prone methionine(s) are identified. Copyright © 2016 John Wiley & Sons, Ltd.