A New Alternative to Gel Electrophoresis: Higher Resolution and Faster Analysis of Large Nucleic Acids by Rigorously Designed GTxResolveTM 250 Å Slalom Columns

Significance of the Topic

Advances in genetic medicines rely on precise, high‐throughput analysis of large nucleic acids. Conventional agarose or capillary gel electrophoresis methods require long run times and can lack resolution for fragments above 3 kbp. Slalom chromatography using rigorously designed GTxResolve™ 250 Å columns addresses these limitations, enabling faster, high‐resolution separations critical for quality control in cell and gene therapy development.

Aims and Overview of the Study

This application note evaluates the performance of GTxResolve 250 Å Slalom Columns for separating large double‐stranded DNA (dsDNA) species. The study compares slalom chromatography to traditional gel electrophoresis, demonstrates reproducibility across columns and batches, assesses recovery and robustness over 400 injections, and illustrates potential benefits for gene therapy workflows.

Methodology and Instrumentation Used

LC Conditions and Instrumentation:

• Mobile phase: 1× TAE buffer (40 mM Tris‐acetate, 1 mM EDTA, pH 8.3)
• Column: Waters GTxResolve™ 250 Å Slalom Column, 2.5 µm, 4.6 × 300 mm
• System: ACQUITY UPLC I-Class Bio System with QSM H-Class, TUV Detector, Sample Manager FTN
• Flow rate: 1.4 mL/min isocratic at 40 °C, sample 5 °C, injection 1 µL of dsDNA ladder

Main Findings and Discussion

Resolution and Speed:

• Slalom chromatography resolved dsDNA fragments from 2 to 23 kbp in under 6 minutes, versus ≥60 minutes for agarose gels.
• Resolution between 9.4 and 23 kbp improved by 4.5-fold compared to agarose electrophoresis.

Reproducibility and Recovery:

• Five columns from one lot and three different packing batches yielded <2% RSD in retention times and resolution metrics for key dsDNA peaks.
• Optical recovery at 260 nm was 91–95%, indicating minimal non‐specific adsorption.

Robustness:

• After 400 consecutive injections (including periodic spermidine washes), performance metrics (retention time, resolution, backpressure) varied by <1.5%, demonstrating excellent column lifetime.

Benefits and Practical Applications of the Method

• Rapid, high‐resolution separation of mega‐sized nucleic acids (<6 min runs)
• Improved discrimination of fragments >3 kbp
• High sample recovery and minimal secondary interactions
• Reproducible results across columns and batches
• Robust operation with >400 injections without performance loss
• Potential for high‐throughput analysis and downstream fraction collection

Future Trends and Potential Applications

Slalom chromatography may be extended to RNA analysis, small nucleic acid impurities, and hybrid mixed‐mode separations. Integration with automated UHPLC platforms and multi‐omics workflows can streamline plasmid mapping and mRNA product characterization. Further optimization of particle size and column geometry could enhance selectivity for specific nucleic acid conformers and structures.

Conclusion

GTxResolve 250 Å Slalom Columns provide a compelling alternative to agarose gel electrophoresis, delivering rapid, high‐resolution analysis of large nucleic acids with outstanding reproducibility, recovery, and robustness. This technology supports streamlined workflows in cell and gene therapy development by enabling fast restriction mapping, impurity profiling, and precise quantification of large DNA and RNA species.

References

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