Identification of GalNAc-Conjugated Antisense Oligonucleotide Metabolites Using an Untargeted and Generic Approach Based on High Resolution Mass Spectrometry

Abstract

Antisense oligonucleotides linked by phosphorothioates are an important class of therapeutics under investigation in various pharmaceutical companies. Antisense oligonucleotides may be coupled to high-affinity ligands (triantennary N-acetyl galactosamine = GalNAc) for hepatocyte-specific asialoglycoprotein receptors (ASGPR) to enhance uptake to hepatocytes and to increase potency. Since disposition and biotransformation of GalNAc-conjugated oligonucleotides is different from unconjugated oligonucleotides, appropriate analytical methods are required to identify main cleavage sites and degradation products of GalNAc conjugated and unconjugated oligonucleotides in target cells. A highly sensitive method was developed to identify metabolites of oligonucleotides using capillary flow liquid chromatography with column switching coupled to a high resolution Orbitrap Fusion mass spectrometer. Detection of GalNAc-conjugated oligonucleotides and their metabolites was achieved by combining full scan MS with two parallel MS experiments, one data-dependent scan and an untargeted MS experiment (all ion fragmentation) applying high collision energy. In the all ion fragmentation scan, a diagnostic fragment originating from the phosphorothioate backbone (OPS-: m/z 94.936) was formed efficiently upon collisional activation. Based on this fragment an accurate determination of metabolites of oligonucleotides was achieved, independent of their sequence or conjugation in an untargeted but highly selective manner. The method was effectively applied to investigate uptake and metabolism of GalNAc-conjugated oligonucleotides in incubations of primary rat hepatocytes; the elucidation of expected and unexpected degradation products was achieved in subnanomolar range.

In vitro metabolism of 2′‐ribose unmodified and modified phosphorothioate oligonucleotide therapeutics using liquid chromatography mass spectrometry

“…As identified by Husser et al (2017), we also confirmed the distinguishable MS/MS product ions (i.e. PO 3 − , H 2 PO 4 − , and PO 2 S − fragment ions) in the PO‐ and PS‐containing oligonucleotides (Figure 3).…”

“…With the high‐quality separation of the method, metabolite identification was performed readily for each compound without effort to avoid assignments due to the overlapping of m/z series from multiple metabolites with similar retention times. Accurate quantifications have been unavailable due to the variance of ionization efficiency for metabolites and the absence of standards for all metabolites (Husser et al, 2017). However, to estimate the relative amounts of each compound in a timely manner, the signal intensities of compounds were optimized using internal standards.…”

“…n –1 metabolite) in the synthesis of ASOs could result in a failed method validation, especially if being used for absolute quantification. However, no metabolism studies were found that have provided chromatograms demonstrating fully resolved peaks of full‐length and n –1 metabolites using LC–MS methods (Beverly, Hartsough, Machemer, Pavco, & Lockridge, 2006; Crooke et al, 2000; Husser et al, 2017; Kim et al, 2019; Thayer et al, 2019). Therefore, we believe that our LC–MS method, which relies on 30 m m DMCHA and 100 m m HFIP to achieve a high‐resolution chromatographic separation of these metabolites, could find widespread utilization in further oligonucleotide analysis.…”

Natural polyphenol assisted delivery of single-strand oligonucleotides by cationic polymers

“…However, chemically modified single-strand oligonucleotides usually possess relatively low membrane permeability, and limited stability during the long-term therapy [ 12 ]. To address those issues, the single-strand oligonucleotides were either conjugated with functional ligands, polymers, and nanoparticles [ 13 , 14 ], or complexed with cationic polymers, liposomes, and nanomaterials [ 15 ] to increase their cell internalization, stability, and transfection efficiency. Cationic polymers with various chemical structures have widely used for intracellular delivery of biomolecules such as genes and proteins [ 16 – 21 ].…”

Spectroscopic diagnostic for the ring-size of carbohydrates in the gas phase: furanose and pyranose forms of GalNAc

“…Moreover, since GalNAc is an excellent ligand of a sialoglycoprotein receptor, it was also used to deliver bioactive antisense oligonucleotides in liver through interaction with membrane hepatocytes. 5 On the other hand, some rare polysaccharides analyzed by NMR spectroscopy have been found to contain GalNAc in a furanose form. For instance, the O-specific polysaccharide produced by Proteus penneri strain 22, responsible for urinary tract infections with subsequent complications, presents a Galf NAc residue linked to the 4-position of a glucuronic acid entity.6 Moreover, the Gram negative bacteria Campylobacter jejuni 11168, responsible for serious gastroenteritis, is able to biosynthesize a chain (containing…”