Development of a suspension serum-free helper-dependent adenovirus production system and assessment of co-infection conditions.
Helper-dependent adenovirus (HDAd), deleted in all viral protein-coding sequences has been designed to reduce immune response and favor long-term expression of therapeutic genes in clinical programs. Its production requires co-infection of E1-complementing cells with helper adenovirus (HAd). Significant progresses have been made in the molecular design of HDAd, but large scale production remains a challenge. In this work, a scalable system for HDAd production is designed and evaluated focusing on the co-infection step. A human embryo kidney 293 (293) derived cell line, the 293SF/FLPe was generated to produce efficiently HDAd while restricting the packaging of HAd. This cell line was adapted to grow in suspension and in serum-free medium. Multiplicity of infection (MOI) of HDAd ranging from 0.1 to 50 was evaluated in presence of HAd at a MOI of 5. Optimal MOIs for HDAd amplification were found in the range of 5-10. HAd contamination was only 1%. These results were validated in a 3 L bioreactor under controlled operating conditions where a higher HDAd yield of 2.6 x 10(9) viral particles (VP)/mL or 3.5 x 10(8) infectious units (IU)/mL of HDAd was obtained.
Meneses-Acosta
Angélica
A
Animal Cell Technology Group, Biotechnology Research Institute, National Research Council, 6100 Royalmount Avenue, Montreal, Quebec, Canada H4P 2R2.
Dormond
Edwige
E
Jacob
Danielle
D
Tom
Roseanne
R
Bernier
Alice
A
Perret
Sylvie
S
St-Laurent
Gilles
G
Durocher
Yves
Y
Gilbert
Rénald
R
Kamen
Amine
A
D000256:Adenoviridae / Q000254:growth & development*; D017498:Adenovirus E1 Proteins / Q000235:genetics; D002452:Cell Count; D002460:Cell Line; D002470:Cell Survival; D016895:Culture Media, Serum-Free; D005822:Genetic Vectors; D006378:Helper Viruses / Q000502:physiology; D006801:Humans; D014161:Transduction, Genetic; D014776:Virus Cultivation / Q000379:methods*
D017498:Adenovirus E1 Proteins; D016895:Culture Media, Serum-Free