Adeno-Associated Viral Vector Purification Using a Centrifugal Microfluidic System: Towards Workflow Automation for Low-Volume Sample Processing

Abstract

This paper describes the development of an integrated assay for the purification of Adeno-associated virus (AAV) particles from crude lysate using a microfluidic cartridge and a centrifugal platform that enables pneumatic actuation of liquids during rotation. The cartridge features POROS™ CaptureSelect™ AAVX Affinity Resin as a solid-phase extraction (SPE) matrix and a layout that is compatible with workflow automation and low-volume sample processing. The integrated process takes ~30 to 60 min depending on the input volume and comprises 22 consecutive steps, starting with crude lysate and ending with an AAV sample conditioned for downstream analysis or long-term storage. Numerical simulations, conducted as part of the design validation, reveal uniform flow across the SPE matrix for both in-plane and out-of-plane orientations. The performance of the system is demonstrated using AAV8 with volumes ranging from 50 to 500 µL. The on-chip purification process generally shows higher extraction efficiency than a benchmark procedure performed manually using spin columns. The capacity to remove plasmid DNA is validated using samples with calibrated spike-in concentration that were analyzed by qPCR, demonstrating depletion of >99.99%. Biological activity of purified AAV particles was demonstrated in a transduction assay involving human embryonic kidney cells and recombinant AAV2 expressing green fluorescent protein. The platform’s compatibility with low-volume production addresses gaps in current gene therapy manufacturing pipelines, underscoring its potential to streamline quality assurance protocols, reduce manual intervention, and accelerate scalable AAV production.