Enrichment of full rAAV capsids in a scalable, reproducible viral vector manufacturing platform

Abstract

Recombinant adeno-associated viruses (rAAV) are the gene transfer vector of choice for many <i>in vivo</i> gene therapies. These vectors are synthetic viral particles which can deliver a therapeutic gene to a patient or patients’ cells to correct a genetic abnormality. These viral vectors can be produced in single-use bioreactors and purified using scalable single-use technologies. We evaluated the use of scalable, single-use filtration and chromatography technologies for downstream purification of an rAAV5 viral vector. In this testing, vector was produced in the Pall iCELLis^® Nano bioreactor by polyethylenimine (PEI) mediated triple-plasmid transfection. The harvest material was clarified using direct flow filtration with a combination of Seitz-P grade depth and 0.2 mm sterilizing grade filters. The product was concentrated using 100 kDa Omega^TM Membrane flat-sheet tangential flow-filtration (TFF) before primary purification with affinity chromatography. Affinity purified vector was polished using Mustang^® Q membrane chromatography to enrich for full capsids. The rAAV5 product was then concentrated and diafiltered to the final formulation using 100 kDa Omega TFF membrane. The final product was sterile filtered using Pall’s Supor^® EKV validated sterilizing-grade filters. This manufacturing process was optimized and evaluated for vector yield, low contaminant profile and full capsid enrichment. We established feasibility of a near complete end-to-end manufacturing process using almost all materials available from Pall Corporation. This process resulted in a theoretical whole process yield of ~25% with a low contaminant profile (host cell protein [HCP] and [DNA]) and a ~5-fold enrichment of full capsids to total capsids. The purification process described here shows potential for a scalable, platformable process for rAAV products.