Rapid Determination of Drug-to-Antibody Ratios in Antibody Drug Conjugates Using Ultrafast Microdroplet Digestion Technology

Accurate determination of drug-to-antibody ratios (DARs) is essential for the development, quality control, and performance evaluation of antibody-drug conjugates (ADCs); yet conventional analytical approaches often require extensive sample preparation, long analysis time, and substantial sample consumption. The peak distribution of intact ADCs is highly complex due to inherent glycosylation heterogeneity and variable drug conjugation. By applying enzymatic digestion, ADC can be converted into smaller subunits or deglycosylated species, thereby significantly simplifying the mass spectral profile. This reduction in structural heterogeneity facilitates clearer peak assignment and enables more accurate and reliable DAR quantification. Herein, we report an ultrafast microdroplet digestion-mass spectrometry strategy for rapid DAR characterization of ADCs. Microdroplet enzymatic digestion of antibodies and ADCs occurs within microsecond-time scales during spray ionization, enabling direct online subunit analysis with minimal sample preparation. The method was validated using NIST monoclonal antibody (mAb) conjugated to ADC mimics spanning low to high DAR (0-14) ranges, Cetuximab-derived ADC mimics (DAR[~]5) with complex glycosylation, and the commercial ADC Kadcyla (DAR[~]3.5). Consistent DAR values were obtained across multiple enzymatic workflows (IdeS, EndoS2, and EndoF3) with good reproducibility (%CV typically View larger version (30K): org.highwire.dtl.DTLVardef@23145corg.highwire.dtl.DTLVardef@10ddd88org.highwire.dtl.DTLVardef@14b1370org.highwire.dtl.DTLVardef@1e94ad6_HPS_FORMAT_FIGEXP M_FIG C_FIG