High throughput peptide mapping method for analysis of site specific monoclonal antibody oxidation

• An ultrafast peptide mapping method using a rapid in-solution trypsin digestion was developed to determine site specific oxidation of mAbs.
• In the method, UV peak areas of oxidized and native peptides separated by RP-HPLC were used to estimate % oxidation.
• The method can monitor oxidation of methionine and tryptophan triggered by TBHP, photo-irradiation, or elevated temperatures.
• The method is reproducible, and can be implemented in regular laboratories without mass spectrometers.

Oxidation of therapeutic monoclonal antibodies (mAbs) often occurs on surface exposed methionine and tryptophan residues during their production in cell culture, purification, and storage, and can potentially impact the binding to their targets. Characterization of site specific oxidation is critical for antibody quality control. Antibody oxidation is commonly determined by peptide mapping/LC–MS methods, which normally require a long (up to 24 h) digestion step. The prolonged sample preparation procedure could result in oxidation artifacts of susceptible methionine and tryptophan residues. In this paper, we developed a rapid and simple UV based peptide mapping method that incorporates an 8-min trypsin in-solution digestion protocol for analysis of oxidation. This method is able to determine oxidation levels at specific residues of a mAb based on the peptide UV traces within <1 h, from either TBHP treated or UV light stressed samples. This is the simplest and fastest method reported thus far for site specific oxidation analysis, and can be applied for routine or high throughput analysis of mAb oxidation during various stability and degradation studies. By using the UV trace, the method allows more accurate measurement than mass spectrometry and can be potentially implemented as a release assay. It has been successfully used to monitor antibody oxidation in real time stability studies.