Size-Exclusion Chromatography Analysis of Adeno-Associated Virus (AAV) Preparations Using a 450 Å Diol-Bonded BEH Column and Fluorescence Detection

Importance of the Topic

The rapid growth of gene therapy has increased the demand for robust methods to evaluate the quality of adeno-associated virus (AAV) vectors.
Size-exclusion chromatography (SEC) enables non-denaturing separation of AAV monomers, aggregates, and fragments and provides critical information on product purity and stability.
Monitoring high and low molecular weight species in AAV preparations is essential to ensure safety and efficacy during process development and quality control.

Objectives and Study Overview

This study aimed to optimize an SEC method for various AAV serotypes (1, 2, 5, 6, 8, and 9) using a diol-bonded bridged ethylene hybrid (BEH) column with 450 Å average pore size and intrinsic fluorescence detection.
The goals included resolving monomeric AAV from high molecular weight species (HMWS) such as dimers and multimers, as well as detecting low molecular weight fragments (LMWS), while minimizing sample volume and concentration requirements.

Methodology

SEC separation was performed under non-denaturing conditions on a 7.8 × 300 mm XBridge Protein BEH SEC column (450 Å, 3.5 µm).
A phosphate buffer mobile phase (20 mM sodium phosphate, pH 6.6) with varying KCl concentrations optimized for each serotype was used at a flow rate of 0.6 mL/min.
Intrinsic protein fluorescence (excitation 280 nm, emission 350 nm) provided sensitive detection of capsid species.
Multi-angle light scattering (MALS) and refractive index (RI) detectors were employed during method development to confirm molecular weight assignments.
Auto•Blend Plus Technology within Empower software facilitated systematic optimization of pH and ionic strength.

Instrumentation

• ACQUITY UPLC H-Class Bio System with FLR detector
• XBridge Protein BEH SEC column, 450 Å, 3.5 µm, 7.8 × 300 mm
• Wyatt microDAWN MALS detector
• Waters ACQUITY Refractive Index Detector
• Empower 3 software with Auto•Blend Plus

Main Results and Discussion

The BEH 450 Å column successfully separated AAV monomer from dimeric and multimeric HMWS under optimized buffer conditions.
Intrinsic fluorescence enabled detection of low abundance species with injection volumes below 10 µL and capsid concentrations around 1012 to 5 × 1012 capsids/mL.
Serotype-specific KCl concentrations (150 to 400 mM) were required to maximize peak shape and recovery of higher order species.
AAV9 exhibited the most pronounced LMW fragment peak, while other serotypes showed minimal fragmentation.
The addition of 10 µg/mL L-tryptophan to the SEC protein standard mix provided a reliable column permeation volume marker under fluorescence detection.
SEC resolution above 100 nm is limited; larger aggregates require complementary techniques such as dynamic light scattering and nanoparticle tracking analysis.

Benefits and Practical Applications

• Non-denaturing separation preserves native capsid structure.
• Fluorescence detection achieves high sensitivity at low sample loads.
• Tailored buffer conditions enhance aggregate recovery across serotypes.
• Quick analysis time (~25 minutes) accelerates process monitoring.
• Method can support quality control and process development in AAV manufacture.

Future Trends and Potential Applications

The integration of full-factorial experimental designs may further refine buffer composition and column conditions.
Hybrid methods combining SEC with orthogonal techniques (light scattering, AUC) can improve characterization of large aggregates beyond 100 nm.
Advances in column materials with wider pore distributions could broaden the applicability to other viral vectors and nanoparticle-based therapeutics.
Enhanced software automation and AI-driven optimization might reduce method development time and increase reproducibility.

Conclusion

This study demonstrates that a BEH SEC column with 450 Å pore size coupled to intrinsic fluorescence detection provides effective separation and quantification of AAV monomers, aggregates, and fragments under non-denaturing conditions.
The method offers robust performance across multiple AAV serotypes with minimal sample requirements and can be a valuable tool in gene therapy analytics.

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