title: Continuous Collection of Adeno-Associated Virus from Producer Cell Medium Significantly Increases Total Viral Yield.
abstract: The ability to efficiently produce large amounts of high-titer recombinant adeno-associated virus (AAV) is a prerequisite to the continued success of AAV as a gene therapy tool targeted toward large-animal preclinical studies or human clinical therapeutics. Current manufacturing procedures necessitate laborious and time-consuming purification procedures to obtain AAV particles of sufficient titer and purity for these demanding biomedical applications. The finding that AAV can be harvested and purified from producer cell medium may represent an efficient alternative to purifying AAV from cellular lysates. Here we sought to determine the maximum duration of time, and frequency within which AAV can be harvested from producer cell medium, in order to maximize the yield obtained from a single transfection preparation. Human embryonic kidney 293T cells were transfected with polyethylenimine to produce AAV2/5 expressing green fluorescent protein (GFP), and cellular medium was harvested every 2 days until a maximum duration of 19 days posttransfection. AAV2/5-GFP was released into producer cell medium at a steady state until 7 days posttransfection, at which time titers dropped dramatically. Harvesting medium every two days resulted in the maximum yield of AAV from a single preparation, and the cumulative yield of AAV harvested from the producer cell medium was 4-fold higher than the yield obtained from a traditional purification of AAV from cellular lysates. The AAV2/5 harvested from medium within the 7-day collection time-course mediated high levels of transduction in vivo, comparable to AAV2/5 harvested from cellular lysates. AAV purified from cell lysates showed increasing amounts of empty particles at 5 and 7 days posttransfection, whereas AAV purified from cell medium did not show an increase in the amount of empty particles throughout the 7-day time course. Finally, we extended these findings to AAV2/9, demonstrating that a comparable ratio of AAV2/9 particles are also released for up to 7 days posttransfection.
authors: 1 Department of Translational Science and Molecular Medicine, College of Human Medicine, Michigan State University , Grand Rapids, Michigan.
authors: Matthew J
authors: MJ
authors: Benskey
authors: 1 Department of Translational Science and Molecular Medicine, College of Human Medicine, Michigan State University , Grand Rapids, Michigan.
authors: Ivette M
authors: IM
authors: Sandoval
authors: 1 Department of Translational Science and Molecular Medicine, College of Human Medicine, Michigan State University , Grand Rapids, Michigan.|2 Mercy Health Saint Mary's , Grand Rapids, Michigan.
authors: Fredric P
authors: FP
authors: Manfredsson
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques; D017077:Culture Media, Conditioned; D000229:Dependovirus / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification*; D015870:Gene Expression; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics* / Q000302:isolation & purification*; D057809:HEK293 Cells; D006801:Humans; D008297:Male; D051381:Rats; D013997:Time Factors; D014162:Transfection / Q000379:methods; D019076:Transgenes; D057074:Virus Release
chemical_list: D017077:Culture Media, Conditioned