Digestion-Free\nMiddle-Down Mass Spectrometry Method\nfor Absolute Quantification of Conjugated Payload from Antibody-Drug\nConjugates

Antibody-drug conjugate (ADC) is a therapeutic modality\nthat aims\nto improve payload delivery specificity and reduce systemic toxicity.\nConsidering the complex structure of ADCs, various bioanalytical methods\nby liquid chromatography coupled with mass spectrometry (LC-MS), ligand\nbinding assay (LBA) and hybrid LBA-LC-MS approaches have been established\nfor ADC characterization and quantification. LCMS-based assays enable\ndrug-antibody ratio (DAR) sensitive quantification of the conjugated\npayload. Typically, for quantitative, DAR-sensitive, assessment by\nLC-MS/MS,the conjugated payload is enzymatically liberated and quantified.\nDespite recent advances in ADC bioanalytical methods, the DAR-sensitive\nquantification of noncleavable linker ADCs by LC-MS/MS remains challenging.\nThus, we developed a novel digestion-free middle-down mass spectrometry\n(DF-MDMS) using a collision-induced dissociation approach for absolute\nquantification of conjugated payload from four different ADCs in a\nbiological matrix with minimum sample preparation. These results demonstrate\nthat ADCs with different linker-payload structures can be quantified,\nincluding a noncleavable linker ADC, trastuzumab emtansine. It also\nshows that the assay sensitivity is comparable to the conventional\nADC quantification method by linker-payload cleavage using enzyme,\nwhile the assay dynamic range depends on factors including payload\nionization and dissociation efficiency, DAR and its distribution,\nand species abundance. By demonstrating absolute quantification of\nboth cleavable and noncleavable linker ADCs, this novel middle-down\nADC approach demonstrates its potential application in bioanalysis\nand analytical characterization, especially for early discovery where\nhigh-throughput screening is required as the new approach saves time\nand resources by not requiring enzymatic digestion for cleavable ADCs\nor development of anti-payload antibodies for noncleavable linker\nADCs.