4.1. Intact Mass Analysis

Measurement of the intact ADC, also known as a top-down approach, can provide information on DAR, glycosylation, and biotransformations, although it lacks site-specific information. The advantage of top-down analysis is that little sample preparation is required, allowing a more high-throughput analysis compared to other MS-based techniques. However, this sensitivity is compromised due to the large size of the ADC (>150 kDa), which, along with its inherent heterogeneity, results in the MS signal being spread across numerous charge states. For this reason, a high-resolution mass spectrometer, such as a time-of-flight (TOF), Orbitrap, or FTICR mass spectrometer is employed for detection of intact ADCs. It is useful to deconvolute the mass spectra to the single-charge domain to identify the various drug forms present. Liquid chromatography is accomplished using a reversed-phase column (e.g., PLRP) with a standard mobile phase comprised of 0.1% formic acid in water and acetonitrile. While structural information from an intact mass analysis is not site-specific, it can provide important structural information to characterize the ADC. For example, Xu et al. were able to monitor the change in DAR in plasma over 21 days after administration of Anti-MUC16 TDC [162]. He et al. monitored the DAR for trastuzumab emtansine 7 days post-dose and cytotoxic drug loss via maleimide exchange through intact mass analysis using a quadrupole-TOF mass spectrometer [163].