Characterisation of small RNA-based therapeutics and their process impurities by fast and sensitive liquid chromatography high resolution mass spectrometry

Oligonucleotides offer a powerful class of new therapeutic modalities, which requires the support of robust and sensitive analytical methods. The primary structure of RNA-based therapeutic drugs is considered a critical quality attribute by regulatory agencies, and must be empirically confirmed to ensure quality, safety and efficacy, together with the analysis of the 5′ and 3′ termini, and any site-specific modifications. This study highlights the use of amine-based ion pair reversed-phase LC coupled to a high-resolution MS and MS/MS method (HRMS) for the characterisation of small RNA-based molecules, single stranded antisense oligonucleotides (ss-ASOs), designed with different chemical modifications from second and third generation categories, including backbone modifications, sugar modifications at the 2’ position and base modifications. The study evaluates the applicability of an alternative ion pairing reagent, with moderate hydrophobicity, and the use of a mild ESI source, with the aim to reduce the formation of ion pair-adducts and in-source induced impurities. The developed method allows for sequence verification and confident identification of low abundant impurities with high sensitivity and high mass accuracy at the intact and sequencing level. Intact analysis allowed for the detection and quantification of full-length products with different sizes and purity values, and the detection of multiple impurities and degradants, even without being fully chromatographically resolved, with high sensitivity. Most common impurity found in analysed RNA-based ss-ASOs was the presence of the phosphodiester (PO) conversion, with fractional abundances from 9.2 % to 1.2 %, followed by failure sequence shortmers.