Cursory Analysis of Nucleic Acid Components in Adeno-Associated Virus (AAV) Samples Using Anion-Exchange Chromatography (AEX)

Significance of the Topic

Adeno-associated viruses (AAV) are widely used gene therapy vectors. Ensuring the safety and efficacy of AAV-based therapeutics requires rigorous analysis of viral genome integrity and detection of unintended nucleic acid impurities. Unencapsulated or truncated DNA may impact product performance and patient safety, making reliable analytical methods essential in both research and quality control environments.

Objectives and Study Overview

This work demonstrates an anion-exchange chromatography (AEX) approach to identify and characterize nucleic acid components in AAV samples. The primary aims were:

• To establish an AEX method capable of separating intact AAV capsids from free genomic material.
• To confirm the identity of peaks through proteinase K digestion of capsids.
• To evaluate the potential for using this technique in genome integrity assessments.

Methodology

AAV9-CamkIIa-GFP samples were analyzed in both native form and after treatment with proteinase K to digest capsid proteins and release single-stranded DNA (ssDNA). Chromatographic separation was performed on a Protein-Pak Hi-Res Q Column under a linear salt gradient of 0–2300 mM tetramethylammonium chloride over 10 minutes. Dual‐wavelength UV detection at 260 nm and 280 nm enabled distinction between protein‐ and DNA‐rich species based on A260/A280 ratios.

Instrumentation Employed

• ACQUITY UPLC H-Class Bio system
• ACQUITY UPLC Tunable UV Detector (260 nm and 280 nm)
• Protein-Pak Hi-Res Q Column
• Empower 3 chromatography data system

Main Results and Discussion

Native AAV samples exhibited a major peak corresponding to intact capsids (A260/A280 ~1.3). After proteinase K digestion, the capsid peak area decreased markedly, while two new peaks appeared at longer retention times with A260/A280 ~1.8, indicating predominantly ssDNA. Progressive digestion reduced the protein peak further and increased the DNA peaks, confirming release of viral genomes. Control digestions of empty capsids showed no DNA peak increase, supporting the specificity of the assay for encapsulated genomes. Additional charge detection mass spectrometry on collected fractions validated that late-eluting peaks represented ssDNA, including partially annealed fragments.

Benefits and Practical Applications

The described AEX approach offers:

• High sensitivity for detecting unencapsulated or leaked viral genomes.
• Quantitative separation of capsid proteins and free DNA with minimal sample consumption.
• Compatibility with automated UPLC workflows and fraction collection for downstream analyses.

Future Trends and Opportunities

Expanding this methodology could involve:

• Integration with fluorescence or mass spectrometry for enhanced specificity.
• Application to other viral vectors and nucleic acid therapeutics.
• Development of standardized protocols for regulatory‐compliant genome integrity testing.

Conclusion

Anion-exchange chromatography on the Protein-Pak Hi-Res Q Column effectively separates intact AAV capsids from released genomic material. Proteinase K digestion combined with A260/A280 analysis confirms the origin of late‐eluting peaks as ssDNA. This robust, reproducible, and quantitative method holds promise for routine monitoring of viral genome integrity and detection of nucleic acid impurities in AAV-based therapeutics.

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