Multifunctional Microfluidic Chip for Rapid Vector Genome and Empty Capsid Quantitation in AAV Vector Production

Performance Period: 9/1/2021 to 8/31/2023

Gene therapy holds transformative potential for the treatment of numerous genetic disorders. Adeno associated viral (AAV) vectors are the most promising gene therapy delivery method and have seen a meteoric rise over the past decade in the biopharmaceutical industry. AAV production, however, lacks innovative process analytical technology (PAT) needed for continued success. A prominent analytical challenge is the characterization of the ratio of empty-to-full AAV capsids, the determination of which is required by the FDA. To this end, we propose a dualfunctional microfluidic chip utilizing CRISPR-based DNA quantification and a washing-free sandwich assay for at-line capsid quantification in a miniaturized format, delivering fast, reliable, and accurate determination of the empty-to-full capsid ratio and total vector genome (vg) on a single device. The CRISPR technology will quantify vg titer, and the sandwich assay will measure capsid titer. Together, the two miniature assays will yield an empty-to-full ratio with a much faster turnaround time compared to the existing methods. This technology has the potential to be adopted as a novel go-to characterization method for simultaneous detection of nucleic acid and protein targets on the same device and could be a vanguard for future on-line AAV PAT and CRISPR diagnostics.