Purity Analysis of Adeno-Associated Virus (AAV) Capsid Proteins using CE-SDS Method

Importance of the topic

Adeno-associated virus (AAV) is a leading vector for gene therapy due to its nonpathogenic nature, low immunogenicity and ability to infect multiple cell types. Accurate purity assessment of its capsid proteins (VP1, VP2, VP3) is critical for product quality and patient safety. Traditional SDS-PAGE methods are labor-intensive and offer limited quantitation and resolution.

Objectives and study overview

This technical note demonstrates a capillary electrophoresis–SDS (CE-SDS) method on the SCIEX PA 800 Plus Pharmaceutical Analysis system to analyze AAV capsid protein purity. The study aims to develop a straightforward sample preparation protocol, optimize separation conditions, evaluate repeatability, sensitivity and linearity, and compare performance across AAV serotypes and titers.

Methodology and instrumentation

Samples of AAV2 and AAV8 were mixed with SDS and 2-mercaptoethanol, incubated at 50 °C, diluted and injected with an online water plug for enhanced sensitivity. Buffer exchange using centrifugal filters enabled analysis of samples with high salt or low titer. Separations were performed on a bare fused-silica capillary (50 µm ID, 30 cm total length) using proprietary SDS-MW buffers. Data were acquired with a PDA detector and 32 Karat Software.

• Instrument: SCIEX PA 800 Plus Pharmaceutical Analysis system
• Detector: Photodiode array (PDA)
• Software: 32 Karat Software 10
• Consumables: EZ-CE Capillary Cartridge, SDS-MW Analysis Kit

Key results and discussion

Optimization identified 1 % SDS and 50 °C incubation as ideal for sharp peak shapes and sensitivity. The method fully resolved VP1 (87 kDa), VP2 (73 kDa) and VP3 (61 kDa). Repeatability studies across eight injections yielded corrected peak area RSDs below 0.7 % for all capsid proteins at titers from 5×10^11 to 1×10^14 genome copies per milliliter (GC/mL). Linearity of absorbance response for VP3 was confirmed over this range with R² = 0.9991. Buffer exchange pretreatment did not alter peak profiles and enabled analysis of low-titer samples after concentration.

Benefits and practical applications

Compared to SDS-PAGE, CE-SDS on the PA 800 Plus system offers automated, high-resolution separations with precise quantitation, robust reproducibility and reduced manual effort. This makes it well suited for quality control and release testing in AAV manufacturing and research laboratories.

Future trends and opportunities

As gene therapy expands, high-throughput, automated CE-SDS workflows will become crucial. Integration with mass spectrometry may provide deeper structural insights. Adaptation to emerging AAV serotypes and hybrid vectors, along with further miniaturization and online buffer exchange, can enhance speed and reduce sample requirements.

Conclusion

The CE-SDS method on the SCIEX PA 800 Plus platform delivers a rapid, sensitive and reproducible approach for AAV capsid protein purity analysis. It addresses the limitations of gel-based techniques and supports rigorous QA/QC standards in gene therapy development.

References

1. Zhang C., Meagher M. M. High-sensitivity capillary electrophoresis–SDS method for viral vector analysis. Analytical Chemistry. 2017;89(6):3285–3292.
2. Quirino J. P. Modern Injection Modes (Stacking) for Capillary Electrophoresis. 2015. doi:10.1002/9783527678129.assep035.
3. Stanford Gene Vector and Virus Core. Minimizing AAV particle loss during processing and analysis. 2020.