Development of orthogonal chromatographic methods for the purity analysis of therapeutic oligonucleotides

Morgane Lescut
Claire Butré
Arnaud Delobel

Oligonucleotides have been in clinical development for the past 30 years as candidate treatments for rare or genetic diseases. As of October 2020, ten of these molecules provided clear clinical benefit in rigorously controlled trials.

Oligonucleotides are synthetised by a chemical process. Despite extensive purification steps, it is impossible to reach total purity: an oligonucleotide sample does not only contain the oligonucleotide of interest of a given length n but also oligonucleotides of unwanted length, such as n-1 oligonucleotides (i.e. lacking one nucleotide). In order to determine the exact composition of an oligonucleotide sample, three types of high-performance liquid chromatography (HPLC) methods were evaluated: reversed-phase chromatography with ion pairing (IP-RP), hydrophilic interaction liquid chromatography (HILIC) and anion-exchange chromatography (AEX).