Residual metals cause variability in methionine oxidation measurements in protein pharmaceuticals using LC-UV/MS peptide mapping

Methionine oxidation has been demonstrated to play an important role in protein stability in vitro and in vivo. It may also cause changes in biological activity and immunogenicity profile of therapeutic proteins. Therefore, it is critical to monitor methionine oxidation in biopharmaceuticals during process and formulation development, as well as long-term stability studies. A common analytical method for methionine oxidation determination is peptide mapping analysis of protein enzymatic digests using UV detection with or without mass spectrometric detection. The quantitation of oxidation is performed based on the UV or extracted ion chromatographic peak areas of the oxidized and non-oxidized peptides. This method was found to be susceptible to significant variability over long-term use. Major factors leading to this variability included presence of low levels of metal ions, especially iron, in the digestion buffer, chromatographic column, LC injector, and other sample contact surfaces. Careful control of metal ion levels generally leads to less variability and long-term consistency of peptide mapping methods for oxidation determination.

► We studied the variability of LC-UV/MS method for analysis of methionine oxidation.
► The variability is mainly due to the residual heavy metals occurred to protein.
► Inclusion of EDTA in the digestion buffer suppresses the artificial oxidation.
► Residual heavy metals in LC columns also induce extra oxidation of peptides.
► Careful control of the heavy metals minimized the variability of LC-UV/MS method.