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

Oligonucleotides have been in clinical development for approximately 30 years. Anti-sense oligonucleotides and aptamers were initially developed, followed by siRNAs over the past 15 years. During that lengthy period, numerous clinical trials were performed and thousands of oligonucleotides underwent studies for a possible market introduction. 

Oligonucleotides are short RNA chains from 20 to 35 nucleotides. The general structure of oligonucleotides is based on three molecules linked by covalent bonds: a nitrogen base, a sugar and a phosphate group that form a nucleotide. However, therapeutic oligonucleotides can also be subtly modified.

This application note presents the results obtained for the development of different HPLC methods on model therapeutic oligonucleotides, including R&D-grade Nusinersen, an approved 18-mer phosphorothioate oligonucleotide with 2’-O-(2-methoxyethyl) modifications.

Three techniques of liquid chromatography (IP-RP, AEX and HILIC) were developed for the purpose of studying phosphorothioate oligonucleotides. Among these three methods, reversed-phase liquid chromatography with ion-pairing provided better results than the two others. Indeed, the chromatogram obtained in IP-RP allowed a better separation of the sample components (i.e. the separation of the different lengths of oligonucleotides composing the sample). Moreover, this method can be coupled to mass spectrometry for identification purposes, or for the specific quantification of co-eluting species. However, the use of HILIC and AEX could be beneficial as orthogonal methods, or if IP-RP does not provide satisfactory results on particular samples.

Quality Assistance thanks Kaneka Eurogentec for providing oligonucleotide samples used during this internal R&D projet.