Reference-Free Thio-Succinimide Isomerization Characterization by Electron-Activated Dissociation

Rationale Isomerism can be an important aspect in pharmaceutical drug development. Identification of isomers can provide insights into drug pharmacology and contribute to better design of drug molecules. The general approaches to differentiate isomers include Fourier-transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR), and circular dichroism. Although proven effective, a commonly used method to differentiate isomers is chromatography coupled with mass spectrometry (MS). Notably, MS is routinely applied in leucine and isoleucine differentiation to facilitate protein sequencing. This work focuses on isomer differentiation of widely applied thio-succinimide structure bridging the antibody backbone and linker-payload of antibody-drug conjugates (ADCs). The hydrolysis of thio-succinimide stabilizes the payload-protein structure while generating a pair of constitutional isomers: thio-aspartyl and thio-isoaspartyl. Methods This paper introduces a hybrid method using ligand binding assay (LBA) and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) to reveal isomerization details of thio-succinimide hydrolysis over time in plasma samples incubated with ADC. By applying two orthogonal dissociation methods, collision-induced dissociation (CID) and electron-activated dissociation (EAD), this pair of isomers showed different MS/MS spectra. This observation enables a unique approach in distinguishing thio-succinimide hydrolysis isomers. Results We observed signature [R1+Thio+57+H]+, [R2+Succ+H2O-57+H]+ and [R2+Succ+H2O-44+2H]2+ product ions that differentiated thio-aspartyl and thio-isoaspartyl isomers using EAD. A newly discovered [R2+ThioSucc+H2O-44+2H]2+ ion also served as additional evidence that further supported our findings. Conclusions This study is a first-to-date identification of thio-succinimide hydrolysis isomers without using synthesized reference materials. This approach should be applicable to all thio-succinimide-linked molecules. Correct identification of thio-succinimide hydrolysis isomers may eventually benefit the development of ADCs in the future.