Next Steps in AAV Characterization via Light Scattering Measuring AAV Critical Quality Attributes

Significance of the Topic

Adeno-associated virus (AAV) vectors have emerged as leading gene delivery tools owing to their low immunogenicity, broad tropism, and capacity for stable transgene expression. Accurate quantification of AAV critical quality attributes (CQAs) such as capsid concentration, genome packaging efficiency, and aggregate content is essential for ensuring safety, efficacy, and regulatory compliance in gene therapy development.

Goals and Overview of the Study

This application note demonstrates a comprehensive workflow for AAV characterization using size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS), refractive index and UV detectors, complemented by batch dynamic light scattering (DLS) and asymmetric flow field-flow fractionation (AF4-MALS). The method aims to deliver absolute molar mass and size information, independently quantify protein and DNA components, differentiate empty and full capsids, and detect high molecular weight aggregates in a single analytical sequence.

Methodology and Instrumentation

The main technique employs SEC-MALS with a typical setup including an HPLC system, DAWN multi-angle light scattering detector and Optilab refractive index detector, along with UV280 absorption. Protein conjugate analysis software interprets dual concentration signals to calculate capsid and genome molar masses and derive particle titers (CFull, CEmpty, Vg/Cp). Batch DLS/SLS provides rapid assessments of hydrodynamic radius and weight-average molar mass for both empty (Rh≈14.7 nm, Mw≈3.67 MDa) and full (Rh≈15.6 nm, Mw≈6.78 MDa) AAV populations. AF4-MALS using an Eclipse AF4 channel further resolves large aggregates and oligomers.

Main Results and Discussion

SEC-MALS analysis yielded precise quantification of total capsid counts, empty/full ratios, and aggregate levels without reliance on calibration standards. Protein conjugate analysis accurately separated contributions of capsid protein and encapsidated DNA, enabling computation of genome copies per particle. Batch DLS differentiated monomeric capsids from small oligomers, while AF4-MALS revealed high molecular weight aggregates that may elute in the void volume of SEC. Blank injections confirmed minimal sample retention on separation media.

Benefits and Practical Applications

• Conformation-independent molar mass determination without external standards
• Simultaneous quantification of capsid protein and genomic payload
• Differentiation of empty and full particles for potency evaluation
• Aggregate detection to support formulation and stability studies
• High-throughput batch DLS as a screening tool prior to detailed MALS analysis

Future Trends and Applications

Advances in light scattering detectors and software algorithms are expected to enhance sensitivity and throughput in viral vector characterization. Integration of orthogonal separation techniques such as AF4 or ion exchange chromatography may provide deeper insights into capsid heterogeneity, post-translational modifications, and impurity profiles. Automation and inline data processing will streamline CQA monitoring in both research and manufacturing environments.

Conclusion

This application demonstrates that SEC-UV-MALS-dRI, when combined with batch DLS and AF4-MALS, offers a robust, multi-attribute platform for comprehensive characterization of AAV vectors. The workflow simplifies AAV quality assessment by delivering critical metrics—capsid concentration, genome packaging efficiency, and aggregate content—in a single, conformation-independent analysis.