High-resolution separation and quantification of dual-AAV drug products by density gradient equilibrium analytical ultracentrifugation

Adeno-associated virus (AAV) vectors are widely used in gene therapy, but their limited packaging capacity (~4.7 kb) restricts delivery of large therapeutic genes. Dual-AAV drug products (DPs), which split large transgenes across two vectors, offer a promising solution but they introduce a critical analytical challenge of precisely quantifying each vector within the DP. Here, we developed a density gradient equilibrium analytical ultracentrifugation (DGE-AUC) method for high-resolution separation and quantitation of highly similar vectors in dual-AAV DPs. An analytical Quality by Design (aQbD) approach employing Design of Experiments (DoE) was used to optimize critical method parameters, including rotor speed and initial salt density, to maximize resolution and quantification accuracy. RbCl was identified as the optimal density-forming salt, generating a shallow equilibrium density gradient that enabled clear resolution of the 3' and 5' Full AAV capsids. The optimized method demonstrated high precision, excellent linearity, low limits of detection and quantification, and accurate quantification across defined dual-AAV mixtures. The method also enables monitoring of vector-specific stability and preferential loss of individual vectors due to stress and is adaptable for multi-vector AAV DPs. These results establish RbCl DGE-AUC as a robust, low-sample-consuming method for component-specific characterization of dual-AAV DPs and support analytical development of emerging multi-vector gene therapies.