Sensitive AAV Capsid Protein Impurity Analysis by CE Using Easy to Label Fluorescent Chromeo Dye P503

Significance of the Topic

Adeno-associated viruses (AAVs) are widely utilized vectors in gene therapy due to their high stability, non-pathogenic nature, and customizable serotype-specific tropism. Accurate quantitation and purity assessment of AAV capsid proteins are essential to ensure product safety, efficacy, and regulatory compliance. Traditional UV-based assays lack the sensitivity needed to detect low-abundance impurities at clinically relevant concentrations, motivating the development of more sensitive and reliable analytical methodologies.

Objectives and Overview

This study aimed to establish a streamlined, sensitive capillary electrophoresis (CE) method coupled with laser-induced fluorescence (LIF) detection for the quantitative analysis of AAV8 capsid protein impurities. By employing a simple two-step denaturation and fluorescent labeling using Chromeo P503, the protocol seeks to minimize sample handling, eliminate buffer exchange steps, and achieve detection limits compatible with therapeutic AAV formulations.

Methodology and Instrumentation

AAV8 samples were sourced at 1×10^13 genomic copies per milliliter and diluted to target concentrations of 1.1×10^10 and 1.1×10^9 GC/mL. The protocol comprised:

1. Denaturation: Mixing 5 µL of diluted AAV8 with CE-SDS sample buffer and DTT, heated at 60 °C for 10 minutes.
2. Fluorescent Labeling: Addition of 0.5 µL of 1 mg/mL Chromeo P503 dye, heated at 60 °C for 20 minutes.
3. Post-Label Dilution: Samples brought to volume with water to final concentrations suitable for CE-LIF analysis.

Instrumentation:

• A PA 800 Plus CE system with LIF detector (488 nm excitation, 600 nm emission filter).
• SCIEX CE-SDS MW kit (PN 390953) and Performance Test Mix (PN 726022).
• 32 Karat software V10 for data acquisition and analysis.

Main Results and Discussion

The optimized CE-SDS LIF approach enabled clear separation and quantitation of capsid proteins VP1, VP2, and VP3 with signal-to-noise ratios exceeding detection thresholds at 1.1×10^9 GC/mL. Comparison with UV absorbance revealed a significant sensitivity improvement, detecting impurities at levels undetectable by conventional methods. Buffer composition was found to influence assay sensitivity: a 50 mM phosphate buffer (pH 8) provided higher peak intensities and improved labeling efficiency compared to PBS. Heat-stress experiments confirmed the method’s capability to detect structural degradation, as indicated by altered electropherogram profiles after thermal challenge.

Benefits and Practical Applications

• Enhanced sensitivity for trace-level capsid impurity detection in therapeutic AAV formulations.
• Streamlined sample preparation with no requirement for buffer exchange or dye cleanup.
• Compatibility with existing CE-SDS kits, facilitating rapid implementation in quality control laboratories.
• Applicability to stability studies, process development, and lot release testing in gene therapy manufacturing.

Future Trends and Opportunities

Anticipated developments include integration with automated microfluidic platforms to further reduce sample consumption and enhance throughput. Advances in fluorescent dyes may allow multiplexed detection of capsid variants and process contaminants. Coupling CE-LIF with orthogonal techniques such as mass spectrometry could provide comprehensive molecular characterization of AAV products.

Conclusion

The described CE-SDS LIF method using Chromeo P503 labeling delivers a sensitive, user-friendly workflow for AAV capsid purity assessment. It achieves quantitation limits down to 5 ng/mL, streamlines sample handling, and supports critical quality control requirements in gene therapy vector production.

References

1. Agbandjie-McKenna M. et al. Molecular Therapy Methods & Clinical Development 2017, 6, 171–182.
2. Bothner B. et al. Journal of Virology 2013, 87(24), 13150–13160.
3. Active Motif. Chromeo P503 Product Insert.
4. SCIEX Technical Note RUO-MKT-02-10086-A. Purity Analysis of AAV Capsid Proteins using CE-LIF Technology.
5. Wright JF. Biomedicines 2014, 2, 80–97.