o Case Study (3): Detection and identification of in vitro metabolites of inclisiran using a novel and universal LC-HRMS method

Introduction: LC-HRMS methods are routinely used for profiling and identification of oligonucleotide (OGN) metabolites, most of which are targeted analyses based on predicted metabolites formed via OGN hydrolysis. However, they often fail to detect uncommon or unpredictable metabolites of OGNs that are formed via phase I and II metabolism. To address this issue, we have developed a novel and universal LC-HRMS method using a combination of an in-house background subtraction filter (BSF) and Biopharma Finder, which can effectively detect and identify all types of ON metabolites. Inclisiran, a marketed siRNA drug that contains a L96 GalNAc moiety, was used as a model compound to validate this LC-HRMS method.

Experimental: Inclisiran was incubated with human, monkey, and rat liver S9 (0, 10, 24 h), followed by LC-HRMS sample analysis. Acquired LC-MS and LC-MS/MS data were processed using background subtraction filter and BioPharma Finer for detecting and identify various types of ON metabolites.

Results: As shown in Fig. 5A, the raw full-scan LC-MS profile of an incubation sample of Inclisiran in liver S9 did not display Inclisiran or its metabolites. The BSF-processed full-scan LC-MS profile effectively removed background noise and endogenous components that were present in both the test and control samples, resulting in a clear Inclisiran metabolite profile with few or no false positives or negatives (Fig. 5B). A major metabolite, SS-3GalNAc, was formed via non-nuclease-mediated metabolism. Its structure (Fig. 5C) was determined via manual interpretation of its MS/MS spectrum (Fig. 5D).

Fig. 5. Inclisiran metabolite profile in monkey liver S9 determined by background subtraction filter 

Conclusions: The results are consistent with the in vivo metabolism data of Inclisiran reported in the literature. This method can be applied to the rapid and comprehensive detection and identification of both predictable and unpredictable metabolites of various types of oligonucleotides.