Metabolite identification and Profiling Methodology - Application for GalNAc Conjugated Oligonucleotides

Antisense oligonucleotides (ASOs) have advanced by becoming more potent and by being modified for the conjugation of ligands to the molecule allowing receptor targeting (and thus better tissue penetration) and with that the needs for the analytical methods for its analysis, especially its metabolism, have also had to advance.
Despite the advancements in ASO chemistry and technology, the phosphorothioate backbone has remained the main site of metabolic cleavage through both endo- and exo-nuclease metabolism, resulting in chain-shortened oligonucleotide metabolites. The conjugation of ligands for receptor targeting however, has greatly increased the number and types of potential metabolites present. The historical conventional extraction and quantification methods are no longer sufficient due to the new chemical modifications.
Here we show how the use of a more unbiased extraction method, a double liquid-liquid extraction, can be utilized in conjunction with a high-resolution accurate-mass mass spectrometer to overcome these challenges. Additional consideration should be made for the evaluation of new ion-pairing reagent combinations which should be evaluated on a case-by-case situation.
We also examined the appropriate method for the relative abundance determination, and under the conditions we developed, metabolite standards should be used to create a scaling factor to account for differences in signal response and allow reasonably accurate quantification calculations. Some literature suggests that a more generalized approach may be used, however, our data indicates the metabolite standards in conjunction with scaling factors to be most appropriate. We also recommend evaluating this on a case-by-case situation until enough evidence suggests it is no longer necessary.
Lastly, we present the metabolite profile and relative abundance data from this updated extraction and quantitation methods from a GalNAc conjugated ASO. To facilitate the description of this data a new system of grouping these metabolites has been devised, where the degree of metabolism of the ligand conjugate portion of the drug determines a metabolite “family” or grouping, where each family consists of all related chain-shortened ASO metabolites. This family system is then used to present the data.