High-Resolution Separations of Single and Double-Stranded Nucleic Acids Using Strong Anion-Exchange Chromatography

Significance of the Topic

Reliable separation and characterization of single and double-stranded nucleic acids are critical for gene therapy, vaccine development, and regulatory compliance in biopharmaceutical workflows. High-resolution chromatographic techniques enable accurate size-based analysis, quality control, and facilitate downstream automated studies.

Objectives and Study Overview

This application note describes an optimized strong anion-exchange (AEX) method using the Protein-Pak Hi Res Q Column to separate nucleic acid standards ranging from 10-mer single-stranded DNA to 15 000 base pair double-stranded DNA. The study aims to evaluate resolution, recovery, and carryover, offering a scalable alternative to gel electrophoresis.

Methodology and Used Instrumentation

The separation employs a Tris buffer (20 mM, pH 9.0) with 5 % (w/v) urea and a sodium chloride gradient to modulate ionic strength. Key parameters include a 0.5 mL/min flow rate, 35 °C column temperature, and injection volumes of 1–2 µL. Instrumentation comprises:

• ACQUITY Premier LC System with Binary Solvent Manager and Flow-Through Needle Sample Manager
• Protein-Pak Hi Res Q Column (4.6 × 100 mm, 5 µm)
• ACQUITY Premier eLambda PDA Detector with 5 mm titanium flow cell
• Empower 3 software for system control and data acquisition

Main Findings and Discussion

The method resolved ssDNA ladders (10–60 mer and 20–100 mer) with discrete, well-shaped peaks, demonstrating clear size-dependent elution. For the 1 Kb Plus dsDNA ladder, 18 fragments from 100 bp to 15 000 bp were baseline resolved, except minor co-elutions at 300/400 bp and 1 kb/1.5 kb. Recovery studies showed >105 % overall recovery and low %RSD (<1 %), indicating negligible carryover. Inclusion of urea sharpened peaks and deepened valleys by minimizing secondary structure effects.

Benefits and Practical Applications

• High-resolution separation of ssDNA and dsDNA across a broad size range
• Enhanced selectivity through urea-supplemented NaCl gradient
• Reproducible, scalable, and automatable chromatography workflow
• Alternative to electrophoresis with improved quantitation and fraction collection
• Critical support for gene therapy and vaccine quality control and regulatory submissions

Future Trends and Applications

Advances may include integration with mass spectrometry for structural analysis, extension to RNA and modified oligonucleotides, high-throughput multiplexed separations, and alignment with evolving regulatory expectations for nucleic acid therapeutics.

Conclusion

The optimized AEX method on the Protein-Pak Hi Res Q Column delivers robust, high-resolution separation of nucleic acids from 10 mer to 15 kbp, with excellent recovery and minimal carryover. It offers a practical and scalable alternative to gel-based techniques, enhancing analytical capabilities in biopharmaceutical development.

References

1. Sun X, Setrerrahmane S, Li C, Hu J, Xu H. Nucleic acid drugs: recent progress and future perspectives. Signal Transduction and Targeted Therapy. 2024;9(1):1–31.
2. U.S. Food and Drug Administration. Cellular & Gene Therapy Guidances. 2025.
3. Yamakawa H, Higashino K, Ohara O. Sequence-dependent DNA separation by anion-exchange HPLC. Analytical Biochemistry. 1996;240(2):242–250.
4. Finny AS, Reidy C, Addepalli B, Lauber M. High-resolution size exclusion chromatography of megadalton-sized DNA vectors and plasmids. Waters Application Note. 2025.