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 Eeltink
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Abstract
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.
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