Chapter 10 - Analysis of oligonucleotides by liquid chromatography

Abstract

This chapter first describes the main classes of chemically synthetized oligonucleotides and biologically produced nucleic acids as well as the challenges associated with their therapeutic delivery. The impurities formed during the chemical synthesis or biological production are described. The second part presents the liquid chromatography (LC) analysis of oligonucleotides and nucleic acids. The separation mechanisms, mobile-phase additives, column physicochemical properties, and applications are first presented for ion-pairing reversed-phase liquid chromatography (IPRP) and hydrophilic interaction liquid chromatography (HILIC). These two LC modes are MS-compatible, with IPRP being the reference separation mode for oligonucleotide and the HILIC mode fueled by an increased number of applications published between 2018 and 2021. The application of anion exchange chromatography (AEX), mixed mode chromatography (MMC), and size exclusion chromatography (SEC) for oligonucleotide analysis is reviewed in the next step. Two-dimensional liquid chromatography (2D-LC) applications are discussed at the end. Applications related to the analysis of the heterogeneous components of oligonucleotides therapeutic delivery systems, including lipids and polymers, are included and discussed in the different sections of the chapter.