Integrated strategy for biotransformation of antibody-drug conjugates and multidimensional interpretation via high-resolution mass spectrometry.

The characterization of the release mechanism and stability in circulation for novel antibody-drug conjugates (ADCs) has become essential to address the complex variables (linker or payload selection, antibody, conjugating site). Understanding the integrated biotransformation of released catabolites and intact ADCs is necessary to elucidate the mechanism of action and mitigate the premature payload release and formation of inactive ADCs during circulation, which can lead to pharmacokinetic/pharmacodynamic disconnection. Herein, we present a comprehensive strategy for the biotransformation of ADCs from both small- and large-molecule perspectives. Two ADCs with common cleavable linkers were investigated: ADC-1 (maleimidocaproyl glycine-glycine-phenylalanine-glycine deruxtecan) and ADC-2 (maleimidocaproyl valine-citrulline-p-aminobenzyl carbamate monomethyl auristatin E). First, the payload-related catabolites released from lysosome, S9, and tumor cells were identified by sensitive data mining based on high-resolution mass spectrometry to reveal the pharmacologically active components. Second, we demonstrated the biotransformation occurring in intact ADCs using middle-down and bottom-up approaches, which particularly contributed to their instability in incubation. The combined immune capture with subunit or peptide analysis enables a comprehensive evaluation of the structural integrity of ADCs, whereas solely quantitative payload release is insufficient to determine the ADCs' stability. Although subunit analysis can visualize the deconjugation by mass shift directly, the precise and low percentage of biotransformation tended to be imperceptible in intact mass spectra. Therefore, a bottom-up method was developed to analyze three representative peptides conjugated with linker-payload. These multiple dimensional biotransformation approaches provide overall insights into the ADC development. SIGNIFICANCE STATEMENT: A thorough understanding of the release mechanism and stability is pivotal for advancing antibody drug conjugates (ADCs) therapeutics. However, the inherent complexity of ADCs poses significant challenges in biotransformation analysis. We developed an integrated strategy to systematically evaluate ADC biotransformation, encompassing payload release mechanisms and plasma stability assessments with middle-down and bottom-up approaches. This advancement not only clarifies the mechanism of action but also supports the rational design of ADC candidates, such as linker chemistry, payload selection, and antibody engineering.