# Characterisation of Antisense Oligonucleotides by Ion‐Pair Reversed‐Phase UHPLC‐HRMS: Method development using Design of Experiments

## TL;DR

This paper presents a new method using advanced chromatography and mass spectrometry to analyze and control the quality of antisense oligonucleotide drugs.

## Contribution

A novel IP-RP-UHPLC-HRMS method using Design of Experiments for ASO characterization is developed and optimized.

## Key findings

- DoE enabled optimization of ion-pair reagent concentrations and gradient slope for ASO analysis.
- Four FMV impurities and 13 TFR impurities were identified above 0.1% detection threshold.
- The method achieved a balance between sensitivity and selectivity for ASO characterization.

## Abstract

The quality control of therapeutic antisense oligonucleotides (ASOs) poses significant analytical challenges due to the complexity of their synthesis and degradation processes and the need to ensure the safety and efficacy of active pharmaceutical ingredients (APIs). In this study, an ion‐pair reversed‐phase ultra‐high‐performance liquid chromatography–high‐resolution mass spectrometry (IP‐RP‐UHPLC‐HRMS) method based on Orbitrap technology was developed using fomivirsen (FMV) and tofersen (TFR) as model compounds. A preliminary scouting phase was dedicated to selecting the type of ion‐pair reagent and MS parameters settings (sheath gas, auxiliary gas temperature and S‐lens), based on MS spectrum quality (charge state distribution, presence of adducts and in‐source fragments), MS signal height and chromatographic peak shape. Design of Experiments (DoE) through response surface methodology was employed to evaluate the effects of the following factors in depth: concentration of the ion pair reagent N,N‐diisopropylethylamine and of 1,1,1,3,3,3‐hexafluoroisopropanol in the mobile phase and elution gradient slope. The responses selected were the UV height and baseline width of the main peak of the APIs, as well as the resolutions between selected impurities from their extracted MS chromatograms. A multidimensional space (sweet spot) was defined in which the response requirements were met, enabling multicriteria optimisation and the set‐up of two distinct IP‐RP‐UHPLC‐MS methods for FMV and TFR characterisation, based on shared mobile phase components and MS parameters. All four FMV impurities and 13 out of 15 detected TFR impurities were identified above 0.1%. DoE approach has been demonstrated to be an effective tool for achieving a balance between sensitivity and selectivity in the analysis of ASOs, identifying optimum conditions tailored to oligonucleotide type and ion‐pair agent and paving the way for more structured development processes, as recommended by recent regulatory requirements for pharmaceutical analytical procedure development.