Quality control of therapeutic phosphorothioate oligonucleotides by hydrophilic interaction liquid chromatography coupled with ION-MOBILITY QTOF

Therapeutic oligonucleotides are short nucleic acids chemically synthetized. They play a major role in gene regulation and the treatment of various diseases. They target DNA, RNA, proteins, posttranslational protein modifications, carbohydrates, lipids or metabolites. Oligonucleotides are easily in-vivo degraded and need to be modified to improve their pharmacokinetic and pharmacodynamic properties. Phosphorothioate oligonucleotides is the most dominating modification where the oxygen atom of the phosphodiester bond is replaced by a sulfur atom. This result in enhanced resistance against nucleases degradation and thus increased half-life. Currently, the FDA has approved 6 drugs but more than 180 are being tested in clinical trials. There is thus an important need for appropriate analytical techniques to ensure their quality control.
In this study, we compared the selectivity obtained with several hydrophilic interaction liquid chromatography (HILIC with diol, amide or phosphorylcholine functional groups) stationary phases on various oligonucleotides mixtures. Indeed, HILIC represents good alternative to commonly used ion-pair reversed-phase liquid chromatography for the analysis of polar compounds. Moreover, it avoids the use of ion pairing agents, which makes it more compatible with mass spectrometric detection. In this project, we investigated the coupling of HILIC with ion-mobility quadrupole time-of-flight MS detector (IM-QTOF). Ion-mobility provides a third separation dimension to mass spectrometry, as the ions will be separated based on their shape and size. Chromatographic and IMS performances were considered to assess the multidimensional efficacy of each tested system. As conclusion we can say that HILIC-IMS approach brings new potentiality in the quality control of emerging oligonucleotides.