Purity Analysis of Adeno-Associated Virus (AAV) Capsid Proteins using CE-LIF Technology

Significance of the Topic

Adeno-associated virus (AAV) vectors are fundamental tools in gene therapy, and accurate purity assessment of their capsid proteins is critical for ensuring product safety, potency, and consistency.
Conventional SDS-PAGE techniques often lack the sensitivity and throughput required for low-titer in-process samples, motivating the adoption of capillary electrophoresis methods with enhanced detection.

Objectives and Study Overview

This work describes the development and validation of a highly sensitive capillary electrophoresis sodium dodecyl sulfate method combined with fluorescence labeling and laser-induced fluorescence detection (CE-SDS-LIF) for quantifying AAV capsid proteins VP1, VP2, and VP3 at titers as low as 1×10^10 genome copies per milliliter (GC/mL).

Methodology

• Sample labeling using 3-2 furoyl quinoline carboxaldehyde dye and potassium cyanide under controlled heating to derivatize primary amines on capsid proteins.
• Denaturation and alkylation of AAV samples with SDS and N-ethylmaleimide to maintain protein integrity and prevent disulfide reshuffling.
• Optional buffer exchange or desalting to reduce salt content, improving stacking injection performance and sensitivity.
• Capillary electrophoresis separation employing stacking injection with a water plug for on-line sample concentration.

Instrumentation Used

• PA800 Plus Pharmaceutical Analysis CE system equipped with laser-induced fluorescence detector (488 nm excitation, 600 nm emission).
• SDS-MW analysis kit for CE-SDS separations, including proprietary gel buffer and wash solutions.
• EZ-CE capillary cartridge with bare fused-silica capillary (50 μm ID, 30 cm total length).
• 32 Karat software for method control, data acquisition, and analysis.

Key Findings and Discussion

• The CE-SDS-LIF method achieved baseline resolution of VP1, VP2, and VP3 with consistent detection down to 1×10^10 GC/mL.
• Migration time repeatability exhibited RSD values below 0.5% for all three proteins across AAV2 and AAV8 serotypes at multiple concentrations.
• Corrected peak area repeatability remained under 5% RSD at high titers (1×10^12 and 1×10^13 GC/mL) and below 10% RSD at the lowest titer tested (1×10^10 GC/mL).
• Linearity of VP3 peak area versus concentration yielded an R2 of 0.9989 over the range from 1×10^10 to 1.6×10^14 GC/mL, demonstrating excellent quantitative performance.
• Comparison with UV/PDA detection confirmed that fluorescence labeling and LIF detection greatly improved sensitivity and baseline stability for low-titer samples.

Benefits and Practical Applications

• Ultra-high sensitivity supports in-process monitoring of AAV manufacturing at low concentration levels.
• Rapid and straightforward sample preparation in under one hour enhances laboratory throughput.
• Automated separation and quantitation reduce manual labor and improve reproducibility.
• Method compatibility with multiple serotypes and standard CE consumables enables broad adoption in QC and R&D.

Future Trends and Applications

Integration of microfluidic CE platforms, multiplexed fluorescence detection for simultaneous profiling of multiple viral vectors, and incorporation into real-time process analytical technologies are expected to further accelerate gene therapy development and ensure robust quality control.

Conclusion

The CE-SDS-LIF workflow with FQ fluorescence labeling provides a robust, sensitive, and reproducible approach for AAV capsid purity analysis, addressing critical needs for low-titer in-process testing in gene therapy manufacturing.

References

1. https://med.stanford.edu/gvvc/AAV.html
2. SCIEX Technical Note Purity Analysis of Adeno-Associated Virus AAV Capsid Proteins using CE-SDS Method
3. Zhang C; Meagher MM. Analytical Chemistry 2017, 89, 3285–3292
4. Quirino J. Modern Injection Modes (Stacking) for CE, 2015
5. SCIEX Technical Note Using Fluorescent Labels to Increase the Sensitivity of IgG Purity and Heterogeneity Assay