title: Combination of baculovirus-mediated gene delivery and packed-bed reactor for scalable production of adeno-associated virus.
abstract: The production of recombinant adeno-associated virus (rAAV) commonly requires plasmid cotransfection, which hinders its mass production. Herein we describe the development of a novel process for rAAV production by combining the advantages of baculovirus-mediated gene delivery and BelloCell bioreactor (a novel packed-bed reactor for animal cell culture; CESCO Bioengineering, Hsinchu, Taiwan). We constructed three baculoviral vectors: Bac-LacZ carries the lacZ gene flanked by AAV inverted terminal repeats, Bac-RC harbors AAV rep and cap genes, and Bac-Helper carries helper genes derived from adenovirus. Cotransduction of HEK-293 cells with these three baculoviruses resulted in successful production of rAAV, and the protein and rAAV yield did not decrease with Bac-RC passage for up to four passages. By adjusting the dose ratio of Bac-LacZ to Bac-RC, adding sodium butyrate, and transferring the production process to the BelloCell-500-AP (500 ml), which allowed for high-density culture and effective baculovirus-mediated transduction of HEK-293 cells, the maximal specific rAAV yield reached approximately 3.8 x 10(4) vector genome (VG) or 247 infectious viral particles (IVP) per cell, which corresponded to approximately 1 x 10(14) VG or 8.5 x 10(11) IVP per reactor run. The yield was comparable or superior to those obtained with other production systems. Baculoviral transduction is simple and cost-effective and the BelloCell-500-AP offers high-density culture of HEK-293 cells. Altogether, the combination of baculoviral transduction and BelloCell reactor culture provides a novel and economically viable approach for rAAV production.
authors.affiliation: Department of Chemical Engineering, National Tsing Hua University, Hsinchu 300, Taiwan.
authors.forename: Kuo-Shiang
authors.initials: KS
authors.lastname: Huang
authors.forename: Wen-Hsin
authors.initials: WH
authors.lastname: Lo
authors.forename: Yao-Chi
authors.initials: YC
authors.lastname: Chung
authors.forename: Yiu-Kay
authors.initials: YK
authors.lastname: Lai
authors.forename: Chi-Yuan
authors.initials: CY
authors.lastname: Chen
authors.forename: Szu-Ting
authors.initials: ST
authors.lastname: Chou
authors.forename: Yu-Chen
authors.initials: YC
authors.lastname: Hu
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D019149:Bioreactors / Q000191:economics; D002460:Cell Line; D000229:Dependovirus / Q000235:genetics / Q000254:growth & development*; D018014:Gene Transfer Techniques; D006801:Humans; D007313:Insecta / Q000166:cytology; D011994:Recombinant Proteins / Q000235:genetics; D014776:Virus Cultivation / Q000379:methods; D014779:Virus Replication
chemical_list: D011994:Recombinant Proteins