Salt gradient separation of nucleic acid components in Adeno-Associated Virus using anion exchange chromatography

Importance of Topic

Gene therapy vectors based on adeno-associated virus (AAV) are critical for treating a variety of diseases, from genetic disorders to cardiovascular conditions. Reliable assessment of the viral genome content and integrity within AAV capsids is essential for ensuring vector potency, identity, and safety. Anion exchange chromatography (AEX) offers a fast, sensitive approach to quantify encapsidated DNA and evaluate genome integrity after enzymatic digestion.

Goals and Overview of Study

This study aimed to develop and validate a salt-gradient AEX method for:

• Rapid quantification of encapsulated AAV6 single-stranded DNA (ssDNA) before capsid digestion.
• Assessment of genome integrity by separating released ssDNA, partial DNA fragments, and oversized DNA after Proteinase K digestion.

Methodology

Sample preparation involved an AAV6 vector at 1×10¹³ genome copies per mL. For content analysis, serial dilutions (1:1, 1:4, 1:5, 1:10) were directly injected. For integrity studies, capsids were digested with Proteinase K for up to 15 minutes before DNA extraction.

An AEX separation used a ProPac 3R SAX column (4.0×100 mm, 3 µm) with a ternary mobile phase: 100 mM Tris-HCl (A), 0.8 M NaClO₄ (B), and water (C). A linear gradient increased salt concentration from 20 % A/0 % B to 20 % A/80 % B over 16 minutes at 0.3 mL/min, 50 °C column temperature, and 10 °C autosampler. Detection was performed by UV at 260/280 nm (DAD) and fluorescence (Ex 280 nm, Em 330 nm, sensitivity 8).

Použitá instrumentace

• Thermo Scientific Vanquish Flex Binary system
• Vanquish Quaternary Pump F and Diode Array Detector HL
• Vanquish Fluorescence Detector C (Dual-PMT)
• ProPac 3R SAX column (P/N 43203-104068)
• Chromeleon 7.3.1 chromatography data system

Main Results and Discussion

Calibration over four dilution levels yielded a linear response (r² = 0.99979) with well-resolved peaks. The elution order correlated with DNA length: shorter ssDNA eluted earlier, while larger fragments showed delayed retention due to stronger ionic interactions.

After Proteinase K digestion, three distinct peaks at 13.2, 13.8, and 14.2 minutes corresponded to ssDNA, partial DNA fragments, and oversized DNA, respectively. UV absorbance at 260 nm and 3D spectra confirmed peak identities. Fluorescence detection highlighted a decrease in capsid protein signal and a concurrent increase in DNA signal with digestion time. Agarose gel electrophoresis validated chromatographic peak assignments.

Optimization studies showed NaClO₄ as the preferred eluent salt. Temperature evaluation indicated optimal separation at 50 °C, reflecting the temperature-dependent conformation of ssDNA.

Benefits and Practical Applications

The described AEX method offers:

• Rapid genome quantification with minimal sample preparation (<1 hour).
• High sensitivity and peak resolution for accurate titer determination.
• Effective integrity assessment of released DNA fragments.

These features support quality control workflows in AAV vector production for research and clinical applications.

Future Trends and Opportunities

Emerging directions include integration of mass spectrometry for direct genome and capsid protein profiling, automation of sample digestion and injection, and development of novel stationary phases to further enhance resolution of complex viral genomes. Miniaturized and high-throughput formats will accelerate screening of therapeutic vectors.

Conclusion

An optimized salt-gradient AEX method on a ProPac SAX column enables precise quantification and integrity assessment of AAV6 genomes. The approach reduces analysis time, improves resolution of DNA species, and supports robust quality control in gene therapy vector development.

References

[1] Martinez-Fernandez de la Camara C. et al. Accurate Quantification of AAV Vector Genomes by Quantitative PCR. Genes. 2021;12(4):601.
[2] Mayginnes J.P. et al. Quantitation of Encapsidated Recombinant AAV DNA in Crude Lysates by qPCR. J. Virol. Methods. 2006;133(2):183-193.
[3] Genomic DNA Isolation Kits Technical Resources. Thermo Fisher Scientific.
[4] Vesnaver G., Breslauer K.J. Contribution of ssDNA Order to Duplex Thermodynamics. PNAS. 1991;88:3569-3573.