Optimisation of the separation of therapeutic phosphorothioate oligonucleotides by hydrophilic interaction liquid chromatography and capillary zone electrophoresis.

Introduction:
Therapeutic oligonucleotides are short nucleic acids chemically synthetized that play a major role in gene regulation and the treatment of various diseases. They can 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 pharmacodynamics properties. Phosphorothioate oligonucleotides (PS ON) 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.
Goals:
Since many therapeutic oligonucleotides are arriving on the global market, there is an important need for appropriate analytical techniques to ensure their quality control. In this work, we optimized hydrophilic interaction liquid chromatography and capillary zone electrophoresis methods to detect PS ON impurities.
Material and methods:
Two complex mixtures were used to optimize the separation methods. Firstly, a mixture containing short PS ON of identical sequence varying by the position of the phosphodiester bonds will be analyzed. Secondly, a mixture of 20 PS ON of different lengths and thus different number of PS linkages will be used.
Results and discussions:
In hydrophilic interaction liquid chromatography, the stationary phase composition and the mobile phase composition and gradient were carefully optimized. In capillary zone electrophoresis, the pH and molarity of the background electrolyte will be studied. The final developed methods were compared in terms of peak efficiency, resolution and analysis time. The advantages and disadvantages related to each technique will be discussed.
Conclusions:
We demonstrated that hydrophilic interaction liquid chromatography and capillary electrophoresis are suitable techniques to differentiate closely related PS ON and could easily be applied for the quality control of those emerging medicines.