Digestion-Free Middle-down Mass Spectrometry Method for Absolute Quantification of Conjugated Payload from ADCs

Antibody-drug conjugate (ADC) is a therapeutic modality that aims to improve payload delivery specificity and reduce systemic toxicity. Considering the complex structure of ADCs, various bioanalytical methods by liquid chromatography coupled with mass spectrometry (LC-MS) and ligand binding assay (LBA) as well as hybrid LBA-LC-MS approaches have been established for ADC characterization and quantification. LCMS-based assays enable Drug-Antibody Ratio (DAR) sensitive quantification of conjugated payload. For quantitative DAR-sensitive assessment by LC-MS/MS, typically the conjugated payload is enzymatically liberated and quantified. Despite recent advances in ADC bioanalytical methods, the DAR-sensitive quantification of non-cleavable linker ADCs by LC-MS/MS remains challenging. Thus, we developed a novel digestion-free middle-down mass spectrometry (DF-MDMS) using collision-induced dissociation approach for absolute quantification of conjugated payload for four different ADCs in biological matrix with minimum sample preparation. These results demonstrate that ADCs with different linker-payload structures can be quantified, including a non-cleavable linker ADC trastuzumab emtansine. It also shows that the assay sensitivity is comparable to conventional ADC quantification method by linker-payload cleavage using enzyme, while assay dynamic range depends on factors including payload ionization and dissociation efficiency, DAR and its distribution, and species abundance. By demonstrating absolute quantification of both cleavable and non-cleavable linker ADCs, this novel middle-down ADC approach demonstrates its potential application in bioanalysis and analytical characterization, especially for early discovery where high-throughput screening is required as the new approach saves time and resources by not requiring enzymatic digestion for cleavable ADCs or development of anti-payload antibodies for non-cleavable linker ADCs.