Oligonucleotide Analysis: Practical Techniques and Method Development Optimization for HPLC

Significance of the Topic

The rapid rise of oligonucleotide therapeutics and molecular diagnostics has created a critical need for reliable analytical methods to ensure identity, purity and process consistency of synthetic oligos. Applications span from small antisense oligonucleotides and siRNA to large mRNA constructs for vaccines or gene editing. Robust HPLC techniques support quality control, impurity profiling and preparative purification in both research and regulated manufacturing environments.

Study Objectives and Overview

This whitepaper reviews the development and optimization of HPLC methods for synthetic oligonucleotide analysis, focusing on both strong anion-exchange (SAX) and ion-pair reversed-phase (IP-RP) chromatography. The goals include:

• Characterize the impact of stationary-phase pore size on retention, peak shape and dynamic binding capacity.
• Optimize mobile phases, gradients, pH, temperature and organic modifiers for N vs. N–1 resolution.
• Demonstrate preparative scale-up options for purification.
• Evaluate MS-compatible conditions for identity confirmation and impurity analysis.

Methodology and Instrumentation

Analytical workflows employed solid-phase phosphoramidite oligonucleotides (15–1000 nt) analyzed on polymeric PLRP-S and Agilent Bio SAX columns across different pore diameters (100–4000 Å). Both UV detection (260 nm) and high-resolution MS (HFIP/TEA mobile phase) were used. Key parameters:

• Buffers: 20–100 mM Tris or TEAA at pH 7–12, with NaCl gradients for SAX.
• IP-RP mobile phases: TEAA/water–ACN and HFIP/TEA-methanol combinations.
• Temperatures: 30–80 °C to reduce secondary structure and enhance selectivity.
• Gradient ranges tailored for ladder standards and resolution assays (e.g. 6–8 % B in 10 min for 14–21 mers).

Used Instrumentation

• Agilent 1290 Infinity II Bio-LC for analytical separations.
• 1260/1290 preparative LC systems for scale-up fractions.
• UV detector at 260 nm and Agilent QTOF mass spectrometer with negative-ion ESI source.

Main Results and Discussion

• Pore size governs oligo retention and dynamic binding capacity: 1000 Å pores deliver optimal balance of surface area and mass transfer, improving peak sharpness for 25–100 mers.
• SAX chromatography on nonporous Bio SAX particles achieves high-resolution separation of RNA ladders (dT15–dT40) and N/N–1 species with Tris–NaCl gradients at 80 °C.
• IP-RP TEAA methods resolve crude mixtures and resolution standards with sharp peaks; adding HFIP/TEA enables MS compatibility and precise mass confirmation.
• Organic modifiers (up to 15 % ACN) and elevated pH (up to 12) fine-tune selectivity, while temperature increases reduce secondary interactions and improve reproducibility.
• Preparative scale-up from 2.1 mm to 100 mm ID columns supports gram-level purification with consistent chromatographic performance.

Benefits and Practical Applications

• Enhanced resolution of product-related impurities and N-1 or longmer species for QC release.
• Robust column stability (>300 injections) with minimized carryover and consistent retention.
• MS-compatible IP-RP workflows simplify sequence confirmation and impurity identification.
• Flexible scale-up strategies enable both analytical screening and preparative isolation of therapeutic oligonucleotides.

Future Trends and Potential Uses

Emerging demands in gene editing, mRNA vaccines and multiplexed oligo libraries will drive further advances:

• Next-generation superficially porous and core–shell materials for ultra-high throughput.
• Automated method scouting with AI-driven optimization of buffer composition and gradient shapes.
• Integration of HPLC-MS with real-time process analytical technology (PAT) for continuous manufacturing.
• Adaptation of microfluidic HPLC chips for point-of-care oligo analysis.

Conclusion

Optimized HPLC methods employing tailored SAX and IP-RP chemistries provide robust, high-resolution analysis essential for the development, QC and purification of synthetic oligonucleotides. Method parameters including pore size, buffer pH, organic content and temperature can be tuned to deliver reproducible separations, scalable workflows and MS compatibility, meeting the stringent requirements of both research and GMP laboratories.

Reference

Smith J. and Zain R. Annual Review of Pharmacology and Toxicology, 2019;59:605–630.