(174ar) Simple Chip Assay for Determination of Percentage Full Payload Adeno-Associated Virus Capsids

Biomanufacturing output has struggled to meet rising clinical demand for adeno-associated virus (AAV), a promising gene therapy vector. AAV has several advantages over other vector delivery platforms. However, manufacturing of AAV vectors suffers from low efficiency as well as a lack of robust and standardized process analytical technology (PAT) sensors and systems for process monitoring. One of the most commonly defined critical quality attributes (CQA) for AAV production is the percentage of full capsids, i.e. percentage of capsids that contains the intended therapeutic genome. Despite it’s importance, there is not a gold standard method for determining percent full capsids. Many AAV manufacturers use a combination of methods that all have their own drawbacks, either analytical or logistical in nature. Here we present a fast (30 minute), chip-based analytical method for at-line quantification of the percent full AAV capsid titer that minimizes end user manual input. We combined the CRISPR AAV Evaluation (CRAAVE) assay and the D4 (Dispense, Dissolve, Diffuse, Detect) assay, a washing-free immunosandwich assay, for total vg, total capsid, and percent full titers all-in-one. The combined assays show high sensitivity, specificity, and flexibility on multiple genome constructs and capsids. The assays are chip-based with reagents pre-deposited for quick off-the-shelf use where assay prep involves simply adding prepared samples. We envision this new cost-effective PAT tool could be a viable at-line analytical solution for AAV manufacturing.