Going big, small, and low: semi-prep columns with small particles for polynucleotide applications

Significance of the Topic

Effective purification and analysis of DNA fragments is critical in pharmaceutical and biopharmaceutical industries for quality control, therapeutic oligonucleotide production, and downstream functional assays. High-resolution separations ensure removal of closely related sequence variants and impurities, enabling reliable sample characterization and preparation.

Objectives and Study Overview

This study compares the chromatographic performance of a 21.2×150 mm semipreparative DNAPac RP column packed with 4 µm supermacroporous divinylbenzene particles against standard analytical columns for polynucleotide analysis. Key goals include evaluating separation range, resolution at reduced flow rates, and method scalability between analytical and semipreparative formats.

Methodology and Instrumentation

Ion-pair reversed-phase LC was performed using 100 mM TEAA in water (Mobile Phase A) and acetonitrile (Mobile Phase B). A conventional UHPLC system equipped with a binary pump, autosampler, and UV detector at 260 nm facilitated separations. Instrumentation details:

• Analytical columns: 2.1×50 mm and 2.1×100 mm DNAPac RP, 4 µm particles.
• Semipreparative column: 21.2×150 mm DNAPac RP, 4 µm supermacroporous divinylbenzene.
• Flow rates: analytical 0.2 mL/min; semiprep 4 mL/min (long gradient) and 8 mL/min (short gradient).
• Injection volumes: analytical 1 µL; semiprep 153–306 µL.

Main Results and Discussion

• Both analytical and semiprep columns separated double-stranded DNA from 35 to 10 000 bp with near-baseline resolution in ~14 min on the analytical format.
• Scaling volumetric gradients maintained resolution on the semiprep column despite reduced linear velocity (0.08 mL/min equivalent), with baseline separation of variants.
• High-resolution separation of 21 mer vs 22 mer single-stranded DNA achieved resolution of 2.94, enabling detection and removal of minor sequence variants.
• Semipreparative operation at 8 mL/min maintained pressure below 155 bar and allowed increased sample loading for preparative workflows.

Benefits and Practical Applications

• Separation over a broad molecular weight range on a single column simplifies method development.
• High-resolution purification of oligonucleotides supports therapeutic manufacturing and QA/QC.
• Reduced backpressure and low flow rates enable use on standard UHPLC systems without specialized high-pressure modules.
• Scalable gradients and fraction collection facilitate sample recovery for downstream orthogonal analyses or bioassays.

Future Trends and Opportunities

• Optimization of gradient profiles and column coupling to further decrease analysis time or target specific fragment sizes.
• Integration with mass spectrometry and other detection modalities for detailed variant characterization.
• Automation and high-throughput fraction collection for large-scale oligonucleotide production.
• Application of novel stationary phases or particle technologies to enhance selectivity and speed.

Conclusion

The 21.2×150 mm DNAPac RP semiprep column delivers equivalent separation range to analytical formats while preserving high resolution at reduced linear velocities. This allows efficient, high-capacity purification of DNA fragments on conventional UHPLC systems, supporting diverse analytical and preparative workflows.

References

• Ma K. et al. A versatile reversed phase platform for short, intermediate, and long nucleic acid analysis. HPLC 2023, Düsseldorf, Germany.