Antibody-drug conjugates (ADCs) present unique challenges for ligand-binding assays primarily due to the dynamic changes of the drug-to-antibody ratio (DAR) distribution in vivo and in vitro. Here, an automated on-tip affinity capture platform with subsequent mass spectrometry analysis was developed to accurately characterize the DAR distribution of ADCs from biological matrices. A variety of elution buffers were tested to offer optimal recovery, with trastuzumab serving as a surrogate to the ADCs. High assay repeatability (CV 3%) was achieved for trastuzumab antibody when captured below the maximal binding capacity of 7.5 μg. Efficient on-tip deglycosylation was also demonstrated in 1 h followed by affinity capture. Moreover, this tip-based platform affords higher throughput for DAR characterization when compared with a well-characterized bead-based method. Graphical Abstract ᅟ.
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