Slalom-Aided Anion Exchange Chromatography for Enhanced Analysis of Plasmid DNA Topological Impurities

Importance of Topic

The precise analysis of plasmid DNA isoforms supercoiled open circular and linear is a cornerstone in gene therapy and biopharmaceutical manufacturing. Differences in topology influence transfection efficiency product potency and regulatory acceptance. Conventional anion exchange chromatography often struggles to resolve these conformers consistently especially across varying plasmid sizes. Introducing a slalom chromatography mechanism in conjunction with traditional AEX enhances selectivity based on DNA elasticity under flow thereby offering improved resolution of topological impurities.

Study Objectives and Overview

This application note aimed to investigate the synergistic effect of slalom aided anion exchange chromatography on a Protein Pak Hi Res Q column for resolving plasmid DNA isoforms. Three DNA constructs pBR322 (4.361 kbp) ΦX174 RF I/RF II (5.386 kbp) and a pCMV Cas9 plasmid (7.037 kbp) were selected. By systematically varying salt gradient and flow rate the study evaluated retention mechanisms and optimized conditions to achieve baseline separation of supercoiled open circular and linear forms using a single gradient method.

Methodology

Mobile phase A consisted of 20 mM Tris pH 9.0 with 5 percent urea while mobile phase B included 1.0 M NaCl in the same buffer. A linear 7.5 mL sodium chloride gradient was applied at flow rates from 0.1 to 0.8 mL per minute. The column temperature was maintained at 35 °C and UV detection was performed at 260 nm. Retention was evaluated in terms of peak resolution window and volume normalized separation profiles.

Used Instrumentation

• ACQUITY Premier UPLC system with Binary Solvent Manager Flow Through Needle Sample Manager and Column Manager
• ACQUITY Premier eLambda PDA detector with titanium flow cell
• Protein Pak Hi Res Q column 4.6 mm x 100 mm 5 micron
• Empower 3 software for control and data acquisition

Main Results and Discussion

• pBR322 separations improved progressively with flow rate achieving baseline resolution of supercoiled open circular and linear forms at 0.8 mL per minute.
• ΦX174 RF I and RF II isoforms showed symmetrical well resolved peaks at 0.8 mL per minute confirming flow dependent slalom effects even in absence of significant linear component.
• The 7 kbp Cas9 plasmid reached optimal separation of all three forms at an intermediate flow of 0.3 mL per minute whereas higher rates caused co elution of linear and supercoiled species.
• These observations demonstrate that slalom retention driven by DNA elasticity under shear complements electrostatic anion exchange selectivity and that flow rate tuning can selectively enhance separation performance.

Practical Benefits and Applications

The dual mode AEX slalom approach streamlines method development eliminating the need for multiple buffer additives or complex gradient changes. A single optimized gradient with adjustable flow rate provides high resolution analysis of diverse plasmid constructs facilitating accurate impurity profiling in gene therapy workflows and QC laboratories.

Future Trends and Opportunities

The integration of slalom mechanisms in high pressure small particle stationary phases offers potential for automation and deeper insight into DNA conformation under flow. Emerging column chemistries and advanced pressure rated hardware may extend this strategy to even larger nucleic acid vectors viral genomes and RNA therapeutics. Coupling with mass spectrometry or multi wavelength detection could further enhance analytical depth.

Conclusion

Slalom aided anion exchange chromatography on a Protein Pak Hi Res Q column enables robust baseline separation of plasmid DNA topological isoforms across a range of sizes using a unified salt gradient. Flow rate serves as a powerful variable to balance electrostatic and hydrodynamic retention leading to precise control of selectivity. This methodology offers a streamlined versatile platform for characterizing plasmid based therapeutics ensuring product quality and regulatory compliance.

References

• X Piao et al Supercoiled DNA Percentage A Key In Process Control of Linear DNA Template for mRNA Drug Substance Manufacturing Molecular Therapy Nucleic Acids 35 102223 2024 doi 10.1016 j omtn 2024 102223
• C G Huber Micropellicular Stationary Phases for High Performance Liquid Chromatography of Double Stranded DNA Journal of Chromatography A 806 3 30 1998 doi 10.1016 S0021 9673 97 01124 2
• M A Strege and A L Lagu Anion Exchange Chromatography of DNA Restriction Fragments J Chromatogr A 555 109 124 1991 doi 10.1016 S0021 9673 01 87171 5
• C R Smith et al Separation of Topological Forms of Plasmid DNA by Anion Exchange HPLC Shifts in Elution Order of Linear DNA Journal of Chromatography B 854 121 127 2007 doi 10.1016 j jchromb 2007 04 005
• N Pavlin et al Analytical Separation of Plasmid DNA Isoforms Using Anion Exchanging Chromatographic Monoliths with 6 micron Channels Electrophoresis 44 1967 1977 2023 doi 10.1002 elps.2023.00031
• J Hirabayashi and K ichi Kasai Effects of DNA Topology Temperature and Solvent Viscosity on DNA Retardation in Slalom Chromatography Journal of Chromatography A 893 115 122 2000 doi 10.1016 S0021 9673 00 00693 2
• F Gritti Retention Mechanism in Slalom Chromatography Perspectives on the characterization of Large DNA and RNA Biopolymers in Cell and Gene Therapy Journal of Chromatography A 1743 465691 2025 doi 10.1016 J CHROMA.2025.465691
• F Gritti Ultra High Pressure Slalom Chromatography Application to the Characterization of Large DNA and RNA Samples Relevant in Cell and Gene Therapy Journal of Chromatography A 1738 465487 2024 doi 10.1016 j.chroma.2024.465487