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Bioprocess development for canine adenovirus type 2 vectors

Gene Therapy volume 20, pages 353–360 (2013)Cite this article

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Abstract

Canine adenovirus type 2 (CAV-2) vectors overcome many of the clinical immunogenic concerns related to vectors derived from human adenoviruses (AdVs). In addition, CAV-2 vectors preferentially transduce neurons with an efficient traffic via axons to afferent regions when injected into the brain. To meet the need for preclinical and possibly clinical uses, scalable and robust production processes are required. CAV-2 vectors are currently produced in E1-transcomplementing dog kidney (DK) cells, which might raise obstacles in regulatory approval for clinical grade material production. In this study, a GMP-compliant bioprocess was developed. An MDCK-E1 cell line, developed by our group, was grown in scalable stirred tank bioreactors, using serum-free medium, and used to produce CAV-2 vectors that were afterwards purified using column chromatographic steps. Vectors produced in MDCK-E1 cells were identical to those produced in DK cells as assessed by SDS-PAGE and dynamic light scatering measurements (diameter and Zeta potential). Productivities of ∼109 infectious particles (IP) ml−1 and 2 × 103 IP per cell were possible. A downstream process using technologies transferable to process scales was developed, yielding 63% global recovery. The total particles to IP ratio in the purified product (<20:1) was within the limits specified by the regulatory authorities for AdV vectors. These results constitute a step toward a scalable process for CAV-2 vector production compliant with clinical material specifications.

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Acknowledgements

This work was supported by the Fundação para a Ciência e Tecnologia (FCT)—Portugal (through the projects PTDC/BIO/69452/2006, PTDC/EBB-BIO/119501/2010 and PTDC/EBB-BIO/118615/2010) and the European Commission (BrainCAV HEALTH – HS_2008_222992). Paulo Fernandes acknowledges the FCT for his PhD grant (SFRH/BD/70810/2010). We acknowledge Eng. Marcos Sousa for the technical support in bioreaction. We also acknowledge the Sartorius Stedim Biotech for providing the membranes, the BIA Separations for providing the monolithic columns, the GE Healthcare for providing the core bead prototype matrix and for the technical advice.

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Authors and Affiliations

  1. Animal Cell Technology Unit, Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa (ITQB-UNL), Oeiras, Portugal

    P Fernandes, A S Coroadinha & P M Alves

  2. Instituto de Biologia Experimental e Tecnológica (IBET), Oeiras, Portugal

    P Fernandes, C Peixoto, V M Santiago, A S Coroadinha & P M Alves

  3. Institut de Génétique Moléculaire de Montpellier, CNRS—Universities of Montpellier I and II, Montpellier, France

    E J Kremer

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  1. P Fernandes
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Correspondence to P M Alves.

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Fernandes, P., Peixoto, C., Santiago, V. et al. Bioprocess development for canine adenovirus type 2 vectors. Gene Ther 20, 353–360 (2013). https://doi.org/10.1038/gt.2012.52

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