Adeno-associated virus (AAV) particles are intensively being investigated for therapeutic use in gene therapy due to low immunogenicity and ability for long-term gene expression in vivo. Luxturna and Zolgensma are two recently FDA approved drugs that utilize AAV technology for treatment of inherited retinal dystrophy and spinal muscular atrophy respectively. The AAV viral vector belongs to the parvovirus family and is comprised of a protein capsid with encapsulated ssDNA. The protein capsid is composed of three capsid proteins VP1, VP2 and VP3 with expected ratio of 1:1:10 respectively. The proteins are packaged into a stable icosahedral structure with diameter ~25 nm. The encapsulated ssDNA is typically up to ~4.8 kbp and are the therapeutic payloads that are critical to treating various diseases. During development, testing strategies for the viral protein stoichiometry, capsid product purity, and ssDNA loading efficacy are used to monitor and assess AAV efficacy. The loading efficacy is observed as the percentage of capsids containing ssDNA (full capsids). Ineffective capsids may contain no ssDNA (empty capsids) or DNA that is lower molecular weight (partial-filled capsids).
Methods to analyze empty/full AAV capsid content include transmission electron microscopy (TEM), Analytical Ultracentrifugation (AUC), charge-based chromatographic techniques, and UV Absorbance spectroscopy, with each displaying advantages and drawbacks. While TEM and AUC measurements display high accuracy, the methods have high cost and time requirements. Charge-based separation techniques are faster (~30 min per sample) but can display serotype specificity. UV absorption spectroscopy requires purified samples to ensure accurate empty/full characterization due to potential interference when determining A280/A260 ratio. As such, there is a need for an accurate and high-throughput characterization system for empty/full measurement that requires low AAV volume input. Here we describe a microfluidic capillary electrophoresis method