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

Therapeutic oligonucleotides are synthetically produced molecules that inherently contain impurities such as truncated sequences of varying lengths, making accurate purity assessment a challenge in their development. High‑performance liquid chromatography (HPLC) offers multiple separation mechanisms, but no single technique provides a complete view of sample composition.

This application note presents the development of three complementary chromatographic methods - ion‑pairing reversed‑phase chromatography (IP‑RP), hydrophilic interaction liquid chromatography (HILIC) and anion‑exchange chromatography (AEX) - applied to model therapeutic oligonucleotides. The approach is illustrated using R&D‑grade Nusinersen, an approved 18‑mer phosphorothioate oligonucleotide with 2’-O-(2‑methoxyethyl) modifications.

The complete experimental methodology and results are detailed in the full application note, available for download.

Three complementary chromatographic approaches

• Ion‑pairing reversed‑phase chromatography (IP‑RP) offers the highest resolution, enabling detailed separation of truncated species from n‑1 to n‑5 and serving as the primary method for impurity profiling
• Hydrophilic interaction liquid chromatography (HILIC) provides an additional selectivity based on polarity, allowing alternative separation patterns and supporting the interpretation of complex profiles
• Anion‑exchange chromatography (AEX) separates oligonucleotides based on charge, requiring high salt conditions and offering a further level of orthogonality for selected impurities

How this supports your oligonucleotide analytical strategy?

Using multiple chromatographic methods based on different separation principles allows a more comprehensive evaluation of oligonucleotide purity and composition.

• Improves impurity profiling by separating oligonucleotides differing by one or several nucleotides
• Enables cross‑validation of results through orthogonal analytical approaches
• Supports method selection depending on oligonucleotide structure and chemical modifications
• Provides reliable identification of species when coupled with mass spectrometry
• Offers complementary solutions when a single chromatographic technique is not sufficient
• Enhances confidence in analytical results through combined interpretation of multiple datasets

This toolbox approach contributes to more robust and reliable purity assessment.