Separation of Epimers of a Cleavable ADC Drug Linker Using SLIM-Ion Mobility Mass Spectrometry

Antibody-drug conjugates (ADCs) represent a rapidly evolving class of potent biopharmaceuticals, combining the selective targeting of monoclonal antibodies with the cytotoxic power of small-molecule drugs. These targeted cancer therapies are reliant on cleavable linkers, often peptide-based, to connect cytotoxic drugs to monoclonal antibodies. Cathepsin-sensitive dipeptide linkers such as Valine-Citrulline are commonly used in current therapeutic ADCs. These linkers are designed for controlled drug release by tumor-associated enzymes like cathepsin B. Critically, the stereochemical configuration of components, such as citrulline, within these linkers dictates their enzymatic cleavability and biological activity. For instance, L-citrulline can be cleaved by cathepsin B, while D-citrulline is not. High chiral purity ensures that the linker is recognized and cleaved efficiently by specific enzymes, enhancing the effectiveness of the drug-delivery system. Beyond the linker, the stereoisomerism of the drug payload itself (in the case of a chiral drug payload) also profoundly impacts ADC efficacy and safety. Ion mobility-mass spectrometry (IM-MS) is an emerging molecular characterization technique that offers a powerful orthogonal dimension of separation by differentiating ions based on their size, shape, and charge, prior to MS analysis. This technique provides collision cross-section (CCS) measurements, which are invaluable for resolving isobaric and isomeric compounds that cannot be differentiated by mass alone. In this work, traveling wave IM spectrometry (TWIMS) enabled by the structures for lossless ion manipulation (SLIM) architecture was evaluated for the separation of isobaric epimers of a cleavable Val-Cit drug linker (DL). SLIM coupled with quadrupole time-of-flight (qTOF) MS was used to separate not only the epimers around the cleavable citrulline center of the DL (compounds A and B) but also the coeluting epimers from the other chiral centers of the drug payload (compounds C and D). A novel aspect of this approach lies in its ability to selectively exploit different adducts of the same charge state to enable the separation of distinct stereoisomeric or isomeric pairs, which are otherwise difficult to separate. Separation of the epimers was optimized by varying the traveling wave frequency, amplitude, and gas pressure in the SLIM chamber. Using this novel approach, citrulline-containing DL's, compounds A-D, were all baseline resolved with a single set of TWIMS parameters. This work underscores the critical role of high-resolution SLIM-IM-MS in resolving subtle structural differences in chiral ADC components, providing essential insights into their development and quality control.