Rapid Determination of Drug-To-Antibody Ratios Using Ultrafast Microdroplet Digestion Technology

Abstract 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. Following offline desalting and buffer exchange, ADC samples were subjected to automated online microdroplet-assisted enzymatic digestion coupled with flow-injection mass spectrometry. This approach integrates enzymatic processing and mass spectrometric detection without chromatographic separation, enabling a 1 min FIA-MS acquisition cycle per sample. 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 <5%). The approach provides rapid online subunit-level DAR characterization while retaining an offline sample preparation requirement.