Genome Sizing Methodology for Genome Integrity Analysis of Adeno-Associated Viruses (AAV)

Importance of the Topic

Adeno-associated virus (AAV) vectors are at the forefront of gene therapy applications thanks to their favorable safety profile and efficient delivery of genetic payloads. Ensuring the correct size and integrity of the encapsidated genome is critical for therapeutic efficacy. Traditional methods for genome size verification, such as denaturing agarose gel electrophoresis and Southern blotting, suffer from low resolution, lengthy workflows, and toxic reagents. The development of rapid, high-resolution analytical techniques is therefore essential for both research and manufacturing quality control of AAV products.

Objectives and Study Overview

This work introduces novel capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) methods for precise sizing and purity assessment of AAV genomes. The goals were to establish workflows capable of separating intact, partial, and small-fragment genomes within minutes; to compare comprehensive versus rapid screening protocols; and to demonstrate applicability across multiple AAV serotypes.

Methodology and Instrumentation

Key steps in the analytical protocol included:

• Benzonase digestion to remove free DNA/RNA impurities outside capsids.
• Ultrafiltration (100 kDa cutoff) to eliminate degraded nucleic acid fragments.
• Proteinase K treatment to release encapsidated genomes.
• Purification of released nucleic acids with a silica-based PCR cleanup kit.
• CE-LIF separation in a gel buffer containing urea, PVP, TBE, and SYBR Green II dye.

Used Instrumentation

• PA 800 Plus Pharmaceutical Analysis System with LIF detector (488 nm excitation, 520 nm emission) and EZ-CE cartridge.
• 32Karat™ Software v10.3 for data acquisition and analysis.
• Auxiliary lab equipment: Amicon centrifugal filters, QIAquick PCR purification kit, benzonase, proteinase K, and RNA size standards.

Main Results and Discussion

• Resolution: Intact genomes (~2.35–4.5 kb), partial fragments (~1.3 kb), and small impurities (0.5–1 kb) were fully resolved within 15 minutes.
• Benzonase Effect: Enzymatic digestion effectively removed external nucleic acids, simplifying interpretation of capsid-derived peaks.
• Workflow Variants: A comprehensive protocol required ~3 hours, whereas a simplified screening procedure delivered qualitative size profiles in ~30 minutes.
• Repeatability: Migration time coefficients of variation were <0.5% over eight consecutive injections.
• Serotype Versatility: Method successfully applied to AAV2, AAV5, and AAV8 samples with comparable performance.
• Potential for Titering: Peak area comparisons between full and empty capsids suggested feasibility of quantitative viral genome titer determination after further validation.

Benefits and Practical Applications

• Significantly faster than gel electrophoresis or Southern blot.
• Higher size resolution and sensitivity for partial genomes and small fragments.
• Reduced use of toxic denaturants and generation of hazardous waste.
• Adaptable to in-process quality checks and release testing in AAV manufacturing.
• Modular workflows that balance speed and depth of analysis.

Future Trends and Potential Applications

Advancements may include integration with automated sample handling, development of higher-throughput capillary arrays, and incorporation of sequence-based confirmation of partial genomes. Extending the approach to other viral vector systems and exploring alternative fluorescent dyes or microfluidic CE formats will further enhance versatility and adoption in both R&D and GMP environments.

Conclusion

The CE-LIF-based genome sizing methodology on the PA 800 Plus system provides a rapid, robust, and high-resolution solution for AAV genome integrity analysis. Its ability to distinguish intact, partial, and small-fragment species in under 30 minutes supports streamlined quality control and accelerates the development and manufacturing of AAV-based gene therapies.

Reference

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3. Viral Quantitative Capillary Electrophoresis for Counting Intact Viruses. Anal Chem. 2011;83(13):5431–5435.
4. Method Optimization and Evaluation for RNA Purity Analysis Using CE-LIF Technology. SCIEX Technical Notes RUO-MKT-02-8017-B. 2018.