Digestion-Free Middle-Down Mass Spectrometry Method for Absolute Quantification of Conjugated Payload from Antibody-Drug Conjugates.

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), ligand binding assay (LBA) and hybrid LBA-LC-MS approaches have been established for ADC characterization and quantification. LCMS-based assays enable drug-antibody ratio (DAR) sensitive quantification of the conjugated payload. Typically, for quantitative, DAR-sensitive, assessment by LC-MS/MS,the conjugated payload is enzymatically liberated and quantified. Despite recent advances in ADC bioanalytical methods, the DAR-sensitive quantification of noncleavable linker ADCs by LC-MS/MS remains challenging. Thus, we developed a novel digestion-free middle-down mass spectrometry (DF-MDMS) using a collision-induced dissociation approach for absolute quantification of conjugated payload from four different ADCs in a biological matrix with minimum sample preparation. These results demonstrate that ADCs with different linker-payload structures can be quantified, including a noncleavable linker ADC, trastuzumab emtansine. It also shows that the assay sensitivity is comparable to the conventional ADC quantification method by linker-payload cleavage using enzyme, while the 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 noncleavable 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 noncleavable linker ADCs.