Determination of N-glycosylation sites and site heterogeneity in a monoclonal antibody by electrospray quadrupole ion-mobility time-of-flight mass spectrometry

This paper presents an improved analytical method for glycosylation structural characterizations of a monoclonal antibody (mAb) using a newly developed quadrupole ion-mobility time-of-flight (ESI-Q-IM-TOF) mass spectrometer. Using this method, high-resolution mass spectra were acquired to produce the overall glycosylation profile of the mAb. Additionally, the light and heavy chains from the reduced antibody were separated in the gas phase by the ion mobility functionality of the instrument, allowing accurate mass measurement of each subunit. Furthermore, the glycan sequences, as well as the glycosylation site, were determined by a two-step sequential fragmentation process using the unique dual-collision-cell design of the instrument, thus providing detailed characterizations of the glycan structures.