title: Production of Recombinant Adeno-associated Virus Vectors Using Suspension HEK293 Cells and Continuous Harvest of Vector From the Culture Media for GMP FIX and FLT1 Clinical Vector.
abstract: Adeno-associated virus (AAV) has shown great promise as a gene therapy vector in multiple aspects of preclinical and clinical applications. Many developments including new serotypes as well as self-complementary vectors are now entering the clinic. With these ongoing vector developments, continued effort has been focused on scalable manufacturing processes that can efficiently generate high-titer, highly pure, and potent quantities of rAAV vectors. Utilizing the relatively simple and efficient transfection system of HEK293 cells as a starting point, we have successfully adapted an adherent HEK293 cell line from a qualified clinical master cell bank to grow in animal component-free suspension conditions in shaker flasks and WAVE bioreactors that allows for rapid and scalable rAAV production. Using the triple transfection method, the suspension HEK293 cell line generates greater than 1 × 10(5) vector genome containing particles (vg)/cell or greater than 1 × 10(14) vg/l of cell culture when harvested 48 hours post-transfection. To achieve these yields, a number of variables were optimized such as selection of a compatible serum-free suspension media that supports both growth and transfection, selection of a transfection reagent, transfection conditions and cell density. A universal purification strategy, based on ion exchange chromatography methods, was also developed that results in high-purity vector preps of AAV serotypes 1-6, 8, 9 and various chimeric capsids tested. This user-friendly process can be completed within 1 week, results in high full to empty particle ratios (>90% full particles), provides postpurification yields (>1 × 10(13) vg/l) and purity suitable for clinical applications and is universal with respect to all serotypes and chimeric particles. To date, this scalable manufacturing technology has been utilized to manufacture GMP phase 1 clinical AAV vectors for retinal neovascularization (AAV2), Hemophilia B (scAAV8), giant axonal neuropathy (scAAV9), and retinitis pigmentosa (AAV2), which have been administered into patients. In addition, we report a minimum of a fivefold increase in overall vector production by implementing a perfusion method that entails harvesting rAAV from the culture media at numerous time-points post-transfection.
authors.affiliation: Gene Therapy Center, University of North Carolina, Chapel Hill, North Carolina, USA; Department of Ophthalmology, University of North Carolina, Chapel Hill, North Carolina, USA; Present address: Bamboo Therapeutics, Inc, Chapel Hill, North Carolina, USA.
authors.forename: Joshua C
authors.initials: JC
authors.lastname: Grieger
authors.affiliation: Gene Therapy Center, University of North Carolina, Chapel Hill, North Carolina, USA.
authors.forename: Stephen M
authors.initials: SM
authors.lastname: Soltys
authors.affiliation: Gene Therapy Center, University of North Carolina, Chapel Hill, North Carolina, USA; Department of Pharmacology, University of North Carolina, Chapel Hill, North Carolina, USA. Electronic address: rjs@med.unc.edu.
authors.forename: Richard Jude
authors.initials: RJ
authors.lastname: Samulski
mesh_terms: D061249:Batch Cell Culture Techniques / Q000379:methods*; D019149:Bioreactors; D002448:Cell Adhesion; D016895:Culture Media, Serum-Free; D000229:Dependovirus / Q000235:genetics / Q000254:growth & development* / Q000302:isolation & purification; D005822:Genetic Vectors / Q000302:isolation & purification*; D057809:HEK293 Cells; D006801:Humans; D040281:Vascular Endothelial Growth Factor Receptor-1 / Q000235:genetics* / Q000378:metabolism
chemical_list: D016895:Culture Media, Serum-Free; C501162:FLT1 protein, human; D040281:Vascular Endothelial Growth Factor Receptor-1