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.