Manufacturing of recombinant adeno-associated viruses using mammalian expression platforms.
Manufacturing practices for recombinant adeno-associated viruses (AAV) have improved in the last decade through the development of new platforms in conjunction with better production and purification methods. In this review, we discuss the advantages and limitations of the most popular systems and methods employed with mammalian cell platforms. Methods and systems such as transient transfection, packaging and producer cells and adenovirus and herpes simplex virus are described. In terms of best production yields, they are comparable with about 104-105vector genomes produced per cell but transient transfection of HEK293 cells is by far the most commonly used. For small-scale productions, AAV can be directly purified from the producing cell lysate by ultracentrifugation on a CsCl or iodixanol-step gradient whereas large-scale purification requires a combination of multiple steps. Micro/macrofiltration (i.e. including tangential flow filtration and/or dead-end filtration) and chromatography based-methods are used for large-scale purification. Purified AAV products must then be quantified and characterized to ensure quality. Recent purification methods and current analytical techniques are reviewed here. Finally, AAV technology is very promising, but manufacturing improvements are still required to meet the needs of affordable, safe and effective AAV vectors essential for licensing of gene therapy clinical protocols.
Département de génie chimique, Université Laval, Québec, QC, Canada.|National Research Council Canada, Montréal, QC, Canada.
Marc-André
MA
Robert
National Research Council Canada, Montréal, QC, Canada.
Parminder S
PS
Chahal
Département de génie chimique, Université Laval, Québec, QC, Canada.|National Research Council Canada, Montréal, QC, Canada.
Alexandre
A
Audy
Department of Bioengineering, McGill University, Montréal, QC, Canada.
Amine
A
Kamen
National Research Council Canada, Montréal, QC, Canada.
Rénald
R
Gilbert
Département de génie chimique, Université Laval, Québec, QC, Canada.
Bruno
B
Gaillet
D000229:Dependovirus / Q000235:genetics* / Q000302:isolation & purification; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000096:biosynthesis*; D057809:HEK293 Cells; D006801:Humans; D014162:Transfection; D014776:Virus Cultivation / Q000379:methods*
Gene therapy; HEK293; Large-scale manufacturing; Mammalian cells; Recombinant adeno-associated viruses