A universal LC-HRMS workflow integrating targeted and untargeted strategies for rapid and comprehensive metabolite profiling of oligonucleotide-based therapeutics.

Metabolite profiling and identification of oligonucleotide-based therapeutics (OBTs) is important in drug discovery and development. Conventional liquid chromatography-high resolution mass spectrometry (LC-HRMS) methods (using ProMass, BioPharma Finder) are effective for targeted analysis of predictable nuclease-derived metabolites but often miss unexpected non-nuclease-mediated ones. This study aimed to develop a universal analytical workflow for comprehensive metabolite profiling of multiple classes OBTs by integrating BioPharma Finder for targeted identification with a Background Subtraction Filter (BSF) for untargeted detection of unexpected biotransformation products. Inclisiran, a GalNAc-conjugated siRNA, was used as a model compound. Samples incubated with rat, monkey, and human liver S9 fractions were analyzed by the LC-HRMS workflow. As a result, a total of 25 (22 predicted +3 unpredicted) inclisiran metabolites were identified across the three species. BioPharma Finder enabled rapid and sensitive identification of 22 predicted metabolites, supported by automated fragment ion assignment. The BSF data processing revealed 22 metabolites not present in control samples, including SS-1GalNAc, SS-2GalNAc, and SS-3GalNAc, which were missed by BioPharma Finder and likely formed via β-N-acetylglucosaminidase-mediated biotransformation. The structures of BSF-detected metabolites were confirmed by comparison with BioPharma Finder-identified metabolites or characterized through manual MS/MS interpretation. Product ion spectra of these metabolites showed weak or absent diagnostic ion (m/z 94.936), a characteristic fragment of phosphorothioate-containing oligonucleotides, suggesting that the product ion filtering of this ion exhibits limited utility in discovering inclisiran metabolites. Overall, the integrated LC-HRMS workflow shows strong potential as a universal platform for biotransformation studies of OBTs.