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In recent decades, there has been a trend toward using larger biological molecules as new active pharmaceutical ingredients (APIs) instead of the classical small organic API molecules. More recently, this trend has shifted from very large biomolecules toward intermediate-sized APIs, such as oligonucleotide therapeutics. Because of their fundamental role in gene regulation, therapeutic oligonucleotides can be directed against their specific ribonucleic acid (RNA) targets, representing a promising customized approach for the treatment of hitherto incurable diseases. There are several FDA-approved oligonucleotide-based therapeutics and many more are awaiting approval. The complicated synthesis and degradation pathways of oligonucleotides, involving sophisticated new chemical modifications, generate hundreds of impurities, in contrast to classical small APIs, which typically contain only around three to five well-defined impurities (Fig. 1). Therefore, this new class of putative drugs entails challenging separation tasks: for example, a small mass change such as 1 Da must be distinguished in a 10,000 Da parent molecule for purposes of both quantification and purification and at extremely high resolution. All therapeutic oligonucleotides must be chemically modified before entering the body. One such modification is the phosphorothioate (PS) modification, which generates diastereomers: for a 20-nucleotide-long PS oligonucleotide, this exceeds half a million diastereomers. In this review, we will examine recently published ion-pair liquid chromatographic separation strategies to meet current challenges in oligonucleotide separations. Ion-exchange chromatography will be briefly discussed based on its merits for large-scale purification. The review focuses on studies combining theory and practice and aiming at the analysis and preparative separation necessary for performing reliable quality control as well as purification. All relevant aspects of the separation systems will be discussed, including the stationary phase, pore size, mobile phase, and ion-pairing reagents. We will also discuss how the properties of the oligonucleotide and its impurities can be exploited to increase separation selectivity. A particular focus will be on the adsorption of ion-pairing reagent and the electrostatic surface potential it generates, allowing for interaction with the highly charged oligonucleotides. Furthermore, the effects of various gradient modes to decrease the electrostatic potential and thereby elute oligonucleotides will be covered. |
