Development of a suspension serum-free helper-dependent adenovirus production system and assessment of co-infection conditions

• Published in: Journal of virological methods 2008-03, Vol.148 (1), p.106-114
• Main Authors: Meneses-Acosta, Angélica, Dormond, Edwige, Jacob, Danielle, Tom, Roseanne, Bernier, Alice, Perret, Sylvie, St-Laurent, Gilles, Durocher, Yves, Gilbert, Rénald, Kamen, Amine
• Format: Article
• Language: English
• Subjects: Adenoviridae - growth & development; Adenovirus; Adenovirus E1 Proteins - genetics; Biological and medical sciences; Bioreactor production; Cell Count; Cell Line; Cell Survival; Culture Media, Serum-Free; Fundamental and applied biological sciences. Psychology; Gene therapy; Genetic Vectors; Gutless adenovirus; Helper Viruses - physiology; Helper-dependent adenovirus; Humans; Microbiology; MOI; Serum-free suspension culture; Techniques used in virology; Transduction, Genetic; Virology; Virus Cultivation - methods
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2007.10.017

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 × 10 9 viral particles (VP)/mL or 3.5 × 10 8 infectious units (IU)/mL of HDAd was obtained.