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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References
Dormond E, Perrier M, Kamen A . From the first to the third generation adenoviral vector: what parameters are governing the production yield? Biotechnol Adv 2009; 27: 133–144.
Kremer EJ, Boutin S, Chillon M, Danos O . Canine adenovirus vectors: an alternative for adenovirus-mediated gene transfer. J Virol 2000; 74: 505–512.
Perreau M, Kremer EJ . Frequency, proliferation, and activation of human memory T cells induced by a nonhuman adenovirus. J Virol 2005; 79: 14595–14605.
Soudais C, Boutin S, Kremer EJ . Characterization of cis-acting sequences involved in canine adenovirus packaging. Mol Ther 2001; 3: 631–640.
Bru T, Salinas S, Kremer EJ . An update on canine adenovirus type 2 and its vectors. Viruses 2010; 2: 2134–2153.
Soudais C, Laplace-Builhe C, Kissa K, Kremer EJ . Preferential transduction of neurons by canine adenovirus vectors and their efficient retrograde transport in vivo. Faseb J 2001; 15: 2283–2285.
Falkner E, Appl H, Eder C, Losert UM, Schoffl H, Pfaller W . Serum free cell culture: the free access online database. Toxicol In Vitro 2006; 20: 395–400.
Armendariz-Borunda J, Bastidas-Ramirez BE, Sandoval-Rodriguez A, Gonzalez-Cuevas J, Gomez-Meda B, Garcia-Banuelos J . Production of first generation adenoviral vectors for preclinical protocols: amplification, purification and functional titration. J Biosci Bioeng 2011; 112: 415–421.
Eglon M, McGrath B, O'Brien T . HPLC purification of adenoviral vectors. Methods Mol Biol 2010; 594: 395–408.
Eglon MN, Duffy AM, O'Brien T, Strappe PM . Purification of adenoviral vectors by combined anion exchange and gel filtration chromatography. J Gene Med 2009; 11: 978–989.
Forcic D, Brgles M, Ivancic-Jelecki J, Santak M, Halassy B, Barut M et al. Concentration and purification of rubella virus using monolithic chromatographic support. J Chromatogr B Analyt Technol Biomed Life Sci 2011; 879: 981–986.
Iyer G, Ramaswamy S, Asher D, Mehta U, Leahy A, Chung F et al. Reduced surface area chromatography for flow-through purification of viruses and virus like particles. J Chromatogr A 2011; 1218: 3973–3981.
Genzel Y, Fischer M, Reichl U . Serum-free influenza virus production avoiding washing steps and medium exchange in large-scale microcarrier culture. Vaccine 2006; 24: 3261–3272.
Tree JA, Richardson C, Fooks AR, Clegg JC, Looby D . Comparison of large-scale mammalian cell culture systems with egg culture for the production of influenza virus A vaccine strains. Vaccine 2001; 19: 3444–3450.
Genzel Y, Behrendt I, Konig S, Sann H, Reichl U . Metabolism of MDCK cells during cell growth and influenza virus production in large-scale microcarrier culture. Vaccine 2004; 22: 2202–2208.
Genzel Y, Ritter JB, Konig S, Alt R, Reichl U . Substitution of glutamine by pyruvate to reduce ammonia formation and growth inhibition of mammalian cells. Biotechnol Prog 2005; 21: 58–69.
Chillon M, Kremer EJ . Trafficking and propagation of canine adenovirus vectors lacking a known integrin-interacting motif. Hum Gene Ther 2001; 12: 1815–1823.
Nadeau I, Kamen A . Production of adenovirus vector for gene therapy. Biotechnol Adv 2003; 20: 475–489.
Ferreira TB, Ferreira AL, Carrondo MJ, Alves PM . Effect of re-feed strategies and non-ammoniagenic medium on adenovirus production at high cell densities. J Biotechnol 2005; 119: 272–280.
Altaras NE, Aunins JG, Evans RK, Kamen A, Konz JO, Wolf JJ . Production and formulation of adenovirus vectors. Adv Biochem Eng Biotechnol 2005; 99: 193–260.
Kamen A, Henry O . Development and optimization of an adenovirus production process. J Gene Med 2004; 6: 184–192.
Schoehn G, El Bakkouri M, Fabry CM, Billet O, Estrozi LF, Le L et al. Three-dimensional structure of canine adenovirus serotype 2 capsid. J Virol 2008; 82: 3192–3203.
Segura MM, Kamen AA, Garnier A . Overview of current scalable methods for purification of viral vectors. Methods Mol Biol 2011; 737: 89–116.
Silva AC, Peixoto C, Lucas T, Kuppers C, Cruz PE, Alves PM et al. Adenovirus vector production and purification. Curr Gene Ther 2010; 10: 437–455.
Lusky M . Good manufacturing practice production of adenoviral vectors for clinical trials. Hum Gene Ther 2005; 16: 281–291.
Peixoto C, Ferreira TB, Sousa MF, Carrondo MJ, Alves PM . Towards purification of adenoviral vectors based on membrane technology. Biotechnol Prog 2008; 24: 1290–1296.
Goerke AR, To BC, Lee AL, Sagar SL, Konz JO . Development of a novel adenovirus purification process utilizing selective precipitation of cellular DNA. Biotechnol Bioeng 2005; 91: 12–21.
Klonjkowski B, Gilardi-Hebenstreit P, Hadchouel J, Randrianarison V, Boutin S, Yeh P et al. A recombinant E1-deleted canine adenoviral vector capable of transduction and expression of a transgene in human-derived cells and in vivo. Human Gene Ther 1997; 8: 2103–2115.
Ferreira TB, Perdigao R, Silva AC, Zhang C, Aunins JG, Carrondo MJ et al. 293 cell cycle synchronisation adenovirus vector production. Biotechnol Prog 2009; 25: 235–243.
Soudais C, Skander N, Kremer EJ . Long-term in vivo transduction of neurons throughout the rat CNS using novel helper-dependent CAV-2 vectors. FASEB J 2004; 18: 391–393.
Segura MM, Monfar M, Puig M, Mennechet F, Ibanes S, Chillon M . A real-time PCR assay for quantification of canine adenoviral vectors. J Virol Methods 2010; 163: 129–136.
Salinas S, Bilsland LG, Henaff D, Weston AE, Keriel A, Schiavo G et al. CAR-associated vesicular transport of an adenovirus in motor neuron axons. PLoS pathogens 2009; 5: e1000442.
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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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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DOI: https://doi.org/10.1038/gt.2012.52
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