Wide-ranging polynucleotide separation capabilities using Reversed Phase particles with variable pore geometry

Importance of Topic

Nucleic acids such as DNA and RNA are central to modern research, diagnostics and therapeutics, including gene editing, vaccine development and oligonucleotide drugs. Accurate separation and characterization of these biopolymers is essential for ensuring product purity, identifying variants and supporting downstream biological applications.

Objectives and Study Overview

This study evaluates a reversed phase chromatography particle with supermacroporous pore geometry and 4 micrometer polymer composition. The goal is to separate polynucleotides from tens to thousands of nucleotides in length, assess the effects of mobile phase composition, temperature and pH, and demonstrate method scale up from analytical to semi-preparative formats for sample purification and recovery.

Instrumentation

• Thermo Scientific DNAPac RP columns, 4 micrometer particle size, formats: 2.1x50 mm, 2.1x100 mm and 21.2x150 mm
• Vanquish H chromatography system with binary pump, autosampler and UV detector at 260 nm
• Chromeleon chromatography data system (versions 7.2.10 and 7.3)

Methodology

Ion pairing reversed phase chromatography was performed using triethylamine acetate, hexylamine acetate and hexafluoroisopropanol as ion pairing agents. Gradient elution profiles were optimized for oligonucleotides of varying length. Separations were conducted at temperatures ranging from 30 to 60 C and mobile phase pH values of 7.9 and 9.9. Semi-preparative scaling was achieved by applying volumetric gradients based on column bed volume.

Main Results and Discussion

The polymeric supermacroporous particle exhibited a broad pore size distribution from 50 to 2500 angstroms, enabling efficient separation of oligonucleotides up to 10000 base pairs. High selectivity was observed for single stranded and double stranded nucleic acid mixtures, including siRNA, guide RNA and mRNA. Ion pairing reagent selection and mobile phase pH tuning improved resolution of stereoisomers. Analytical methods translated directly to semi-preparative scale with preserved or enhanced peak resolution and increased sample concentration in collected fractions.

Benefits and Practical Applications

• High resolution across a wide range of nucleotide lengths from 10 to 10000 nt
• Robust performance under varied mobile phase composition, pH and temperature conditions
• Seamless scaling from analytical to preparative applications for purification and sample recovery
• Enhanced mass transfer and enrichment due to supermacroporous particle structure

Future Trends and Opportunities

Further development of pore architectures may enable analysis of even larger nucleic acids and complex biomolecular conjugates. Integration with mass spectrometry and high throughput automation is anticipated to accelerate method development. Advanced data analytics and artificial intelligence may optimize separation parameters and support quality control in therapeutic oligonucleotide manufacturing.

Conclusion

The DNAPac RP supermacroporous reversed phase particle provides a versatile and scalable platform for high resolution separation of nucleic acids. Its broad pore distribution, chemical stability and compatibility with diverse mobile phases make it well suited for research, quality control and preparative purification of oligonucleotide based products.

Reference

• K Musunuru et al Patient Specific In Vivo Gene Editing to Treat a Rare Genetic Disease New England Journal of Medicine 2025
• J Maurer Malburet C Francois Heude M Guillarme D Evaluation of ion pairing reversed phase liquid chromatography for the separation of large RNA molecules Journal of Chromatography A 1740 465574 2025
• K Ma et al A versatile reversed phase platform for short intermediate and long nucleic acid analysis HPLC 2023 Dusseldorf Germany