Routine Monitoring of DNA/mRNA Therapeutics With the Alliance™ iS Bio HPLC System and Ultra Wide-Pore GTxResolve™ Premier SEC Column

Importance of the Topic

Gene therapies and mRNA-based drugs require precise monitoring to ensure product safety and efficacy. Size exclusion chromatography (SEC) is essential for detecting aggregates and characterizing size variants in large biomolecules. Advances in column technology and instrumentation are needed to resolve high-molecular-weight nucleic acid therapeutics without compromising resolution.

Objectives and Study Overview

This work evaluated the Alliance iS Bio HPLC System with ultra-wide pore GTxResolve Premier SEC Columns against an ACQUITY Premier UPLC System and a legacy Alliance e2695 HPLC System. The goal was to compare system dispersion, chromatographic resolution, and reproducibility for DNA ladders and a 997-nucleotide EGFP mRNA sample under typical QC conditions.

Methodology and Used Instrumentation

System dispersion was quantified by injecting caffeine under isocratic flow and measuring the 5σ peak volume. DNA ladders (50–1350 bp) and EGFP mRNA were separated on GTxResolve Premier SEC Columns (1000 Å, 4.6 × 300 mm and 7.8 × 300 mm) using buffered mobile phases. Peak width, peak-to-valley resolution, and retention behavior were compared across platforms.

Instrument List

• Alliance iS Bio HPLC System
• ACQUITY Premier UPLC System
• Alliance e2695 HPLC System
• GTxResolve Premier SEC Column, 1000 Å, 4.6 × 300 mm and 7.8 × 300 mm
• Empower 3 Chromatography Data System

Main Results and Discussion

Measured extra-column dispersion volumes were 12.6 µL for ACQUITY Premier, 26.2 µL for Alliance iS Bio, and 92.0 µL for Alliance e2695. The Alliance iS Bio system matched UPLC performance using the smaller 4.6 mm column, whereas the legacy HPLC required a larger 7.8 mm column and higher flow rate to recover resolution. Calibration of DNA ladders demonstrated a linear working range between total exclusion and inclusion limits. In mRNA analysis, monomer and aggregate peaks were well resolved on the Alliance iS Bio system (peak-to-valley ~2.3) versus ~1.6 on the legacy system.

Benefits and Practical Applications

• Low dispersion yields sharper peaks and faster SEC method development
• High-resolution separation of DNA and mRNA species in QC labs
• Reduced solvent consumption by using smaller-diameter columns
• Reproducible detection of low-abundance species with RSD ≤ 5%

Future Trends and Opportunities

Integration of low-dispersion HPLC platforms with real-time data analytics and automation will enhance throughput in QC environments. Development of new ultra-wide pore materials and advanced surface chemistries may extend SEC applications to larger nucleic acid constructs and lipid nanoparticle formulations.

Conclusion

The Alliance iS Bio HPLC System paired with ultra-wide pore GTxResolve Premier SEC Columns delivers low dispersion and reliable performance comparable to UPLC, while reducing operational costs. This configuration is ideal for routine downstream analysis of DNA and mRNA therapeutics in manufacturing and quality control settings.

References

• Le TT Andreadakis A Kumar et al Nature Reviews Drug Discovery 2020 19 305–306
• Goyon A Tang S Fekete et al Anal Chem 2023 95 15017–15024
• Koza SM Reed C Chen W Waters Application Note 720006336 June 2019
• Hong P McConville Waters White Paper 720005723 April 2018
• Hong P Fountain Waters Application Note 720004076 October 2011