Production of adeno-associated virus (AAV) serotypes by transient transfection of HEK293 cell suspension cultures for gene delivery.
Adeno-associated virus (AAV) is being used successfully in gene therapy. Different serotypes of AAV target specific organs and tissues with high efficiency. There exists an increasing demand to manufacture various AAV serotypes in large quantities for pre-clinical and clinical trials. A generic and scalable method has been described in this study to efficiently produce AAV serotypes (AAV1-9) by transfection of a fully characterized cGMP HEK293SF cell line grown in suspension and serum-free medium. First, the production parameters were evaluated using AAV2 as a model serotype. Second, all nine AAV serotypes were produced successfully with yields of 10(13)Vg/L cell culture. Subsequently, AAV2 and AAV6 serotypes were produced in 3-L controlled bioreactors where productions yielded up to 10(13)Vg/L similar to the yields obtained in shake-flasks. For example, for AAV2 10(13)Vg/L cell culture (6.8×10(11)IVP/L) were measured between 48 and 64h post transfection (hpt). During this period, the average cell specific AAV2 yields of 6800Vg per cell and 460IVP per cell were obtained with a Vg to IVP ratio of less than 20. Successful operations in bioreactors demonstrated the potential for scale-up and industrialization of this generic process for manufacturing AAV serotypes efficiently.
Vaccine Program, Human Health Therapeutics Portfolio, National Research Council of Canada, 6100 Royalmount Avenue, Montreal, QC, Canada H4P2R2.
Parminder Singh
PS
Chahal
Vaccine Program, Human Health Therapeutics Portfolio, National Research Council of Canada, 6100 Royalmount Avenue, Montreal, QC, Canada H4P2R2.
Erica
E
Schulze
Vaccine Program, Human Health Therapeutics Portfolio, National Research Council of Canada, 6100 Royalmount Avenue, Montreal, QC, Canada H4P2R2.
Rosa
R
Tran
Vaccine Program, Human Health Therapeutics Portfolio, National Research Council of Canada, 6100 Royalmount Avenue, Montreal, QC, Canada H4P2R2.
Johnny
J
Montes
Vaccine Program, Human Health Therapeutics Portfolio, National Research Council of Canada, 6100 Royalmount Avenue, Montreal, QC, Canada H4P2R2. Electronic address: amine.kamen@gmail.com.
Amine A
AA
Kamen
D019149:Bioreactors / Q000821:virology*; D016895:Culture Media, Serum-Free; D000229:Dependovirus / Q000254:growth & development* / Q000302:isolation & purification; D057809:HEK293 Cells; D006801:Humans; D014162:Transfection; D014776:Virus Cultivation / Q000379:methods
D016895:Culture Media, Serum-Free
Bioreactor; Gene therapy; Large-scale transient transfection; Manufacturing; Process; Production