Exploring Proteoforms of the IgG2 Monoclonal Antibody Panitumumab Using Microchip Capillary Electrophoresis-Mass Spectrometry

The IgG2 type monoclonal antibody panitumumab is an anti-epidermal growth factor receptor (EGFR) drug used for the treatment of EGFR-expressing, chemotherapy resistant, metastatic colorectal carcinoma. In this study, panitumumab drug product was first analysed using size exclusion chromatography coupled to mass spectrometry for rapid identity testing. The experimental data led to the identification of two panitumumab isoforms with several prominent forms remaining unidentified, despite apparently low sample complexity. Microchip capillary electrophoresis-mass spectrometry (CE-MS) was subsequently utilised for a more detailed characterization. It was observed that panitumumab is subject to partial N-terminal pyroglutamate formation. Incomplete conversion is uncharacteristic for N-terminally exposed glutamines and in case of panitumumab gives rise to forms which show successive mass offsets of 17 Da, respectively. If not separated before mass spectrometric analysis, e.g. by capillary electrophoresis, such near isobaric species coalesce into single MS peaks, which subsequently hampers or prevents their assignment. With a total of 42 panitumumab isoforms assigned by CE-MS, these observations highlight a potential pitfall of commonly applied rapid identity testing workflows and demonstrate that even low complexity biopharmaceuticals can require separation strategies which offer high separation selectivity for species close in mass.