Anion-Exchange Chromatography for Determining Empty and Full Capsid Contents in Adeno-Associated Virus

Significance of the Topic

Recombinant AAV vectors are widely adopted for gene therapy but their efficacy and safety depend on the ratio of genome-containing (full) to empty capsids. Accurate, rapid, and low-sample-volume methods for quantifying this ratio are essential to ensure consistent product quality, optimize dosing, and minimize immune reactions or reduced transduction efficiency.

Objectives and Study Overview

• Develop and optimize an anion-exchange chromatography (AEX) method to separate and quantify empty and full AAV capsids.
• Compare performance across multiple AAV serotypes (AAV1, AAV2, AAV5, AAV6, AAV8, AAV9).
• Assess effects of mobile phase pH, buffer type, salt species, and divalent cations on resolution and peak performance.
• Demonstrate linear quantification of empty-to-full capsid ratios using fluorescence detection.

Methodology and Instrumentation

• Chromatographic System: Waters ACQUITY UPLC H-Class Bio System
• Stationary Phase: Protein-Pak Hi Res Q, 4.6×100 mm, 5 μm particle size
• Mobile Phase Composition: gradients of bis-tris propane (pH 9–10) with tetramethylammonium chloride or other salts, optional Mg²⁺ inclusion
• Detection: Fluorescence (Ex 280 nm/Em 350 nm) and dual-wavelength UV at 260 nm/280 nm
• Data Analysis: Empower 3 software

Main Results and Discussion

• Full capsids elute later than empty ones, reflecting higher net negative charge from encapsulated DNA.
• UV 260/280 nm ratio confirms DNA content differences; fluorescence detection offers superior sensitivity, enabling sub-microliter injections.
• Buffer pH (optimal at pH 9) and salt type (tetramethylammonium chloride) critically influence resolution; Bis-tris propane buffer improved peak stability.
• Inclusion of 2 mM Mg²⁺ had minimal impact on AAV8 separation but may benefit other serotypes.
• Optimized method achieved linear response for empty/full mixtures across a wide ratio range using a fluorescence response factor.
• Retention times vary among serotypes, indicating the need for minor method adjustments when switching between AAV1–AAV9.

Benefits and Practical Applications of the Method

• High-resolution separation of empty and full AAV capsids in under 30 minutes
• Reduced sample volume requirements compared to AUC and spectrophotometry
• Robust, reproducible, and amenable to standard LC platforms
• Linear quantification enabling process monitoring, quality control, and batch release

Future Trends and Potential Applications

• Extension of AEX methods to emerging AAV serotypes and engineered capsids
• Integration with mass spectrometry (e.g., CDMS) for combined charge and mass analysis
• High-throughput, multi-parametric platforms combining AEX with orthogonal techniques
• Application in process development, formulation screening, and regulatory assays for gene therapy products

Conclusion

An optimized anion-exchange chromatography method using a Protein-Pak Hi Res Q column provides rapid, sensitive, and reproducible separation and quantification of empty and full AAV capsids. This approach addresses limitations of traditional ultracentrifugation and spectrophotometric assays, offering a robust tool for gene therapy vector characterization and quality control.

References

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