Scalable Production and Purification of Adeno-Associated Viral Vectors (AAV)

Abstract

Here we describe methods for the production of adeno-associated viral (AAV) vectors by transient transfection of HEK293 cells grown in serum-free medium in orbital shaken bioreactors and the subsequent purification of vector particles. The protocol for expression of AAV components is based on polyethyleneimine (PEI) mediated transfection of a 2-plasmid system and is specified for production in milliliter-to liter-scales. After PEI and plasmid DNA (pDNA) complex formation the diluted cell culture is transfected without a prior concentration step or medium exchange. Following a three-day batch process, cell cultures are further processed using different methods for lysis and recovery. Methods for the purification of viral particles are described, including iodixanol gradient purification, immunoaffinity chromatography and ultrafiltration, as well as quantitative PCR to quantify vector titer.

Use of Super-Resolution Optical Microscopy To Reveal Direct Virus Binding at Hybrid Core–Shell Matrixes

“…Virus material was obtained in exceptionally high purity (Figure ) with a yield of 60%, along with a clear path toward a scalable process. To date, different studies with POROS material specific for adeno-associated virus (AAV) serotype have been reported in the literature. − In contrast, herein POROS CaptureSelect affinity matrixes specific for AdV5 for selective virus purification are shown. Although the present study focused on the production and purification of AdV5, this process is potentially applicable for other viral vectors due to its scalability.…”

Lentiviral Vectors for T Cell Engineering: Clinical Applications, Bioprocessing and Future Perspectives

“…As they mature, LV vectors bud from the producer cell membrane and are released into the cell culture medium. Consequently, no cell lysis step is required to release the particles as for other non-enveloped vectors such as adeno-associated virus (AAV) vectors [ 38 ].…”