Recent Advances in Reversed‐Phase Liquid Chromatography for Therapeutic Oligonucleotide and Conjugate Characterization: From Ion‐Pairing to Green Analytical Approaches
Quang‐Dong Bui, Weronika Hewelt‐Belka, Tiny Deschrijver, Martin Gilar, Sebastiaan EeltinkABSTRACT
Therapeutic oligonucleotides (ONs) have rapidly emerged as a novel class of treatments in the pharmaceutical industry, with over 25 medicines approved in the past decade. Ion‐pair reversed‐phase liquid chromatography coupled with mass spectrometry (IP‐RPLC‐MS) is widely used method for ON characterization. This review critically examines the state‐of‐the‐art developments in IP‐RPLC‐MS for ON analysis, with particular focus on the selection of optimal chromatographic conditions for three key applications: impurity profiling for single‐stranded ON, characterization of novel conjugates (e.g., lipid‐and peptide‐conjugated ONs) and double‐stranded siRNA analysis in non‐denaturing conditions. Finally, recent developments in ion‐pair‐free RPLC are discussed in the context of pharmaceutical and bioanalytical applications.