Characterization of therapeutic oligonucleotides by liquid chromatography

Marketed therapies in the pharmaceutical landscape are rapidly evolving and getting more diverse. Small molecule medicines have dominated in the past while antibodies have grown dramatically in recent years. However, the failure of traditional small and large molecules in accessing certain targets has led to increased R&D efforts to develop alternative modalities. Therapeutic oligonucleotides (ONs) can accurately be directed against their ribonucleic acid (RNA) target and represent a promising approach in previously untreated diseases. Established automated synthesis of ONs coupled with chemical improvements and the advance of new drug delivery technologies has recently brought ONs to a heightened level of interest. The first part of the present review describes the different classes of oligonucleotides, namely antisense oligonucleotide (ASO), small interfering RNA (siRNA), microRNA (miRNA), aptamer and immunostimulatory ON, with a focus on their delivery systems relevant for future analytical characterization. The second part reviews the typical impurities in therapeutic ON products. The third part discusses the use of historical methods anion exchange chromatography (AEX), ion-pair reversed phase liquid chromatography (IP-RP), mixed-mode chromatography (MMC) and recent analytical methodologies of hydrophilic interaction liquid chromatography (HILIC), two-dimensional liquid chromatography (2D-LC) mass spectrometry for the characterization of ASO and siRNA modalities. The effects of physicochemical properties of RPLC columns and ion-pair agents on ON separation are specifically addressed with possible future directions for method development provided. Finally, some innovative analytical developments for the analysis of siRNAs and their delivery materials to pave the way toward the use of multi-attribute methods in the near future are discussed. (C) 2020 Elsevier B.V. All rights reserved.