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

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• 作者：Angé ; lica Meneses-Acosta ; Edwige Dormond ; Danielle Jacob ; Roseanne Tom ; Alice Bernier ; Sylvie Perret ; Gilles St-Laurent ; Yves Durocher ; Ré ; nald Gilbert ; Amine Kamen
• 关键词：Adenovirus ; Helper-dependent adenovirus ; Gutless adenovirus ; Bioreactor production ; Serum-free suspension culture ; MOI ; Gene therapy
• 刊名：Journal of Virological Methods
• 出版年：2008
• 出版时间：March 2008
• 年：2008
• 卷：148
• 期：1-2
• 页码：106-114
• 全文大小：674 K

文摘

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 × 10⁹ viral particles (VP)/mL or 3.5 × 10⁸ infectious units (IU)/mL of HDAd was obtained.