Determination of Full, Partial and Empty Capsid Ratios for Adeno-Associated Virus (AAV) Analysis

Significance of the Topic

Adeno-associated virus (AAV) vectors are widely used for delivering genetic payloads in gene therapy. The ratio of empty, partial, and full capsids critically impacts efficacy, safety, and manufacturing quality control for AAV-based therapeutics.

Objectives and Study Overview

This technical note presents a capillary isoelectric focusing (cIEF) method for rapid quantification of full, partial, and empty AAV capsids. Analyses were performed across multiple serotypes and results were compared with orthogonal techniques such as anion exchange high performance liquid chromatography (AEX-HPLC).

Methodology and Instrumentation

The cIEF experiments utilized a SCIEX PA 800 Plus Pharmaceutical Analysis System with UV detection at 280 nm and an N-CHO capillary maintained at 20 °C. A master mix was prepared by combining 3 M urea-cIEF gel, wide and narrow pH range ampholytes, cathodic and anodic stabilizers, and internal pI markers. Samples of AAV serotypes 5, 8, 9 and proprietary preparations enriched in empty or full capsids were buffer-exchanged as required, mixed with master mix, and analyzed in under one hour per sample. For comparison, AEX-HPLC employed a CIMac SO3-0.1 column on a Waters ACQUITY UPLC H-Class PLUS system following vendor guidelines.

Main Results and Discussion

• cIEF resolved empty capsids at higher pI values and full capsids at lower pI values, with partial capsid peaks appearing between these regions.
• Mixtures of wide and narrow pH range ampholytes enhanced baseline separation for serotypes with pI differences as small as 0.1, revealing multiple charge variants.
• Repeatability studies on AAV9 showed %RSD < 5% for peak area and < 2% for pI values, demonstrating high method robustness.
• For AAV5, cIEF determined a full/empty ratio of 67/33, closely matching the 69/31 ratio obtained by AEX-HPLC.
• Correction factors based on molar extinction coefficients may be applied to account for UV absorbance of nucleic acids and improve quantitative accuracy.

Benefits and Practical Applications

This cIEF approach delivers rapid, automated analysis of AAV capsid populations with high resolving power, low sample requirements, and broad applicability across serotypes. It supports quality control monitoring of critical quality attributes, vector identification by distinct pI profiles, and timely release testing in gene therapy manufacturing.

Future Trends and Opportunities

Emerging developments may include integration with mass spectrometry techniques for direct mass–charge profiling, novel ampholyte chemistries for further resolution gains, extension to other viral vector platforms, and on-line automation for streamlined QC workflows.

Conclusion

The described cIEF method provides a fast, reproducible, and high-resolution platform for quantifying full, partial, and empty AAV capsids. Its strong agreement with orthogonal assays and ability to resolve closely eluting species make it well suited for routine release testing and process development.

Reference

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