Characterizing Oligonucleotides

Significance of the Topic

Oligonucleotide-based drugs have gained momentum due to their ability to treat genetic and hard-to-address diseases. Precise characterization ensures correct sequence identity, purity, and modification integrity critical for safety and efficacy.

Objectives and Article Overview

This guide reviews high-performance chromatographic techniques for thorough analysis of synthetic oligonucleotides. It outlines methods for confirming identity, assessing purity, detecting modifications, and comparing various LC approaches including IP-RPLC, HILIC, AEX, and SEC.

Methodology

Four complementary chromatographic modes are presented:

• Ion-Pairing Reversed-Phase LC (IP-RPLC): Uses triethylamine or alternative alkylamines with C18 stationary phases (130Å and 300Å) for accurate mass confirmation and impurity profiling.
• Anion Exchange (AEX): Exploits charge differences through weak and strong exchangers (Gen-Pak FAX, Protein-Pak HiRes Q) with tunable pH and gradient conditions.
• Hydrophilic Interaction LC (HILIC): Leverages BEH amide phases and ammonium acetate mobile phases for ion-pairing free separations and improved LC–MS performance.
• Size Exclusion Chromatography (SEC): Enables native separation and desalting of oligonucleotides and duplex analysis using 100–300Å columns coupled to UV–MS.

Used Instrumentation

• Waters ACQUITY Premier Oligonucleotide BEH C18 Columns (130Å, 300Å)
• Waters XBridge Premier BEH Amide and ACQUITY Premier BEH Amide Columns (HILIC)
• Gen-Pak FAX and Protein-Pak HiRes Q Anion Exchange Columns
• ACQUITY Premier and XBridge Protein SEC Columns for native LC–UV–MS

Main Results and Discussion

• IP-RPLC delivered robust separation of phosphorothioated siRNA and related impurities, with MaxPeak HPS surfaces enhancing peak shape and reproducibility.
• AEX provided distinct selectivity for charged oligonucleotide species, allowing control over retention via pH and ion-pairing additives.
• HILIC offered a non–ion-pair workflow with high efficiency, benefiting MS compatibility and resolution of modified sequences.
• SEC demonstrated potential for rapid desalting and duplex integrity assessment, separating 5–150 mer oligonucleotides under native conditions.

Benefits and Practical Applications

• Comprehensive identity confirmation through combined retention and accurate mass analysis.
• Purity assessment to detect n-1 shortmers, oxidation, deamination, and depurination products.
• Workflow flexibility to match analytical goals, from routine QC to advanced structural characterization.
• Compatibility with mass spectrometry for detailed fragmentation mapping and modification localization.

Future Trends and Opportunities

Advances in stationary phase design and surface chemistries are expected to further improve separation performance and reduce adsorption. Integration of native LC–MS workflows and automated microfluidic platforms may enable faster, high-throughput characterization. Emerging chromatographic materials and AI-driven method optimization promise streamlined development for next-generation oligonucleotide therapeutics.

Conclusion

A strategic combination of IP-RPLC, AEX, HILIC, and SEC techniques provides a complete toolkit for synthetic oligonucleotide analysis. By leveraging specialized columns and optimized conditions, laboratories can achieve reliable identity, purity, and structural insights essential for therapeutic development and quality control.