High-Resolution Size Exclusion Chromatography of Megadalton-Sized DNA Vectors and Plasmids Using Waters GTxResolve 2000 Å SEC Columns

Significance of the Topic

The accurate characterization of megadalton-sized DNA vectors and plasmids is essential for advancing cell and gene therapy workflows. High-resolution size exclusion chromatography (SEC) preserves native biomolecular conformations, enables precise sizing of aggregates, monomers, and structural variants, and supports reliable quality control of complex therapeutic modalities.

Objectives and Study Overview

This application note evaluates the performance of Waters GTxResolve 2000 Å SEC MaxPeak Premier 3 µm columns for separating large DNA constructs and high-molecular-weight proteins. Key goals include:

• Benchmarking resolution against a commercial 2000 Å SEC column
• Assessing column-to-column and batch-to-batch reproducibility
• Demonstrating out-of-the-box readiness without extensive conditioning

Methodology and Instrumentation

Chromatographic separations were performed on an ACQUITY Premier UPLC system equipped with a Binary Solvent Manager, Flow-Through Needle Sample Manager, Column Manager, and eLambda PDA detector using a 5 mm titanium flow cell. A Waters GTxResolve 2000 Å SEC 3 µm column (4.6 × 150 mm) and Agilent Bio SEC-5 2000 Å column (4.6 × 150 mm, 5 µm) were compared. The mobile phase was Dulbecco’s PBS, 1×, at 35 °C column temperature, 5 °C sample temperature, 0.1 mL/min flow rate, and UV detection at 260 nm.

Main Results and Discussion

• Resolution: GTxResolve columns achieved an average USP resolution of 1.79 for plasmid aggregates vs monomer and 5.93 for plasmid vs φX174 DNA, improving separation by ~50% versus comparison columns.
• Column-to-column reproducibility: Five columns from one packing batch exhibited retention time %RSD below 1% and resolution RSD below 2%.
• Batch-to-batch consistency: Three columns from different packing batches showed RT %RSD of 2.8–3.2% for plasmid and viral peaks.
• Thyroglobulin test: High-molecular-weight protein analysis confirmed consistent monomer/dimer separation, peak symmetry, and plate counts across multiple columns and batches.
• Out-of-the-box performance: Back-to-back injections without conditioning yielded dimer and monomer peak area changes under 6% and RRT variation below 0.03, demonstrating immediate usability.

Benefits and Practical Applications

The GTxResolve 2000 Å SEC columns offer:

• Superior resolution for DNA vectors, plasmids, and large proteins
• Minimal secondary interactions via hydrophilic MaxPeak HPS surfaces and HO-PEO bonding
• High reproducibility supporting rapid QC of CGT products
• Scalability from discovery to manufacturing environments

Future Trends and Possibilities

Advancements in SEC packing chemistries and hardware inertness will drive broader adoption for emerging modalities such as lipid nanoparticles, mRNA constructs, and viral vectors. Integration with multi-angle light scattering and mass spectrometry detection may further enrich aggregate profiling and structural analyses.

Conclusion

Waters GTxResolve 2000 Å SEC MaxPeak Premier 3 µm columns deliver high-resolution separations of megadalton DNA vectors and large proteins, outperforming conventional wide-pore SEC columns. Their robust column-to-column and batch-to-batch reproducibility, combined with out-of-the-box readiness, makes them a valuable tool for accelerating analytical workflows in gene and cell therapy development.

References

• M. Youssef et al. Enabling mRNA Therapeutics: Current Landscape and Challenges in Manufacturing. Biomolecules. 2023;13(10):1497. doi:10.3390/BIOM13101497
• T. Li et al. CRISPR Cas9 Therapeutics: Progress and Prospects. Signal Transduction and Targeted Therapy. 2023;8(1):1–23. doi:10.1038/s41392-023-01309-7
• C. Holladay et al. Gene Therapy in Molecular Cellular and Tissue Engineering. New England Journal of Medicine. 2018;381(5):56-1–56-35. doi:10.1056/NEJMRA1706910
• A. S. Finny et al. Efficient Profiling of Lipid Nanoparticle Formulations Using Waters GTxResolve 2000 Å SEC Column MaxPeak Premier 3 µm. Waters Application Note. 2025;720008797
• K. J. Camacho et al. Bridged Ethylene Polyethylene Oxide Surfaces to Improve Packing Materials for Widepore SEC. Journal of Separation Science. 2024;47(20):e202400541. doi:10.1002/jssc.202400541