Middle-Up Mass Analysis Of Protease Digested Cetuximab

A complete reversed phase UHPLC-MS workflow has been developed to simplify middle-up mass analysis of an immunoglobulin G antibody. The protocol for purification, proteolysis, reduction, and UHPLC-MS analysis of unlabeled and labeled monoclonal antibodies describes the analysis of proteolyzed and reduced samples. This type of antibody fragment analysis is a much faster assay than peptide mapping for establishing the presence or absence of antibody fragment modifications. In detail, the workflow includes:

• Antibody purification using immobilized protein A
• Antibody proteolysis utilizing IdeS enzyme
• Antibody reduction procedure
• Mass spectrometer calibration
• UV spectrophotometric mAb quantification method
• System suitability test utilizing an intact protein LCMS standard
• RP-UHPLC-MS method for separation and analysis of unlabeled and labeled samples

Explore this complete workflow from the antibody purification, proteolysis, and reduction to its reversed phase UHPLC-MS analysis.