Characterisation of Cetuximab by Different Mass Spectrometric Techniques

Abstract

The structural complexity of therapeutic monoclonal antibodies such as cetuximab requires comprehensive analytical strategies to verify identity, integrity and modification patterns throughout production and storage. In this work, cetuximab was characterised using a combined mass‑spectrometric and electrophoretic approach aimed at evaluating both intact protein features and fragment‑level information. Chemical stressing and enzymatic digestion with trypsin, papain and PNGase F were performed to generate targeted peptide and domain fragments, enabling the assessment of cleavage behaviour, glycan release and sequence‑dependent digestion patterns. SDS‑PAGE provided an initial separation and visualisation of heavy/light chains and digestion products, while MALDI‑ToF mass spectrometry enabled mass profiling of intact subunits and peptide mixtures. Although only single-stage MS analysis was available, theoretical digestion and manual matching allowed preliminary sequence confirmation and evaluation of post‑translational modification effects. Together, these complementary techniques demonstrated their utility in the structural characterisation of cetuximab.