Analysis of Oligonucleotides by Size-Exclusion Chromatography: SEC-UV, SEC- MS, SEC-MALS

The field of therapeutic oligonucleotides has expanded significantly in recent years, with multiple RNA-based drugs approved for applications such as gene silencing. These molecules require careful characterisation, particularly regarding size distribution, the presence of impurities (such as n-1, n-2 variants), and multimeric species that can affect activity and safety. Analytical challenges arise due to their sensitivity to conditions that may cause dissociation. Size-Exclusion Chromatography (SEC), performed under mild aqueous conditions, offers a suitable approach for analysing these biomolecules. This application note presents the development of SEC methods coupled to UV, MALS and MS detection for comprehensive characterisation. The full technical details and results are available in the complete application note.

KEY HIGHLIGHTS OF THE METHOD

• SEC separation is performed under mild aqueous conditions, enabling analysis of oligonucleotides in their native state.
• PBS mobile phase provides improved peak shape and chromatographic resolution compared to other buffer conditions.
• SEC-MALS enables absolute molecular weight determination independent of elution time, including molecular mass distribution and polydispersity.
• SEC-MS allows identification of oligonucleotide fragments (n-1, n-2, etc.) and determination of exact molecular masses using ammonium acetate as mobile phase.
• Combined SEC-UV, SEC-MALS and SEC-MS approaches enable evaluation of purity, multimer formation and impurity profiling.

BENEFITS FOR YOUR ANALYTICAL PROGRAMME

The combination of SEC techniques provides complementary information for the comprehensive characterisation of therapeutic oligonucleotides, supporting both development and quality control activities.

• Enables monitoring of size distribution and detection of impurities such as truncated oligonucleotides.
• Supports evaluation of multimeric species, which can impact product activity and safety.
• Provides reliable molecular weight determination using SEC-MALS without dependence on calibration.
• Allows routine purity assessment using SEC-UV with optimised chromatographic conditions.
• Delivers advanced structural insights through SEC-MS for detailed impurity identification.

Together, these approaches support robust identity and purity assessment of therapeutic oligonucleotides.

TYPICAL APPLICATIONS

• Characterisation of therapeutic oligonucleotides including siRNA, ASOs, miRNA and aptamers.
• Monitoring of oligonucleotide size distribution during development and quality control.
• Detection and evaluation of multimeric species and aggregates.
• Identification of oligonucleotide fragments and impurities using MS-based approaches.