Hanna Nguyen

Adeno-associated viruses are commonly used viral vectors in gene therapy due to their relatively low immunogenicity and cytotoxicity in vivo. Recombinant AAVs (rAAVs) are naked viruses made up of a protein shell, or capsid, that encapsulates a therapeutic DNA fragment. One of the major issues in the production of rAAVs is the presence of product-related impurities such as empty capsids, partial capsids, or capsids with contaminating DNA. These impurities increase the risk of immmunotoxicity and lower gene transduction. Analytical methods to detect empty/full capsids include transmission electron microscopy, analytical ultracentrifugation, quantitative polymerase chain reaction, and anion exchange high performance liquid chromatography. Unfortunately, these are off-line methods that can be time-consuming and labor intensive, and some cannot identify partial or empty capsids. We set out to address these shortcomings by using thermoplastic in-plane nanopore devices to determine the fill status of capsids via resistive pulse sensing (RPS). Empty and full capsids differ in their resistivity due to payload differences, which allow the use of the polarity of the rAAV RPS electrical signal and/or electrophoretic mobility when two in-plane pores are placed in series to determine the fill status of rAAVs in near real-time. We fabricate our nanofluidic device consisting of dual in-plane nanopore sensors in cyclic olefin polymer (COP) via thermal nanoimprint lithography that was subsequently bonded with a cyclic olefin copolymer (COC) cover plate via thermal fusion bonding. Thermoplastics fabrication techniques are amenable to mass production even when producing nanoscale structures. This RPS method is the first in-line method capable of determining the fill status of capsids to allow for process corrections and quality assurance during the production of rAAVs.