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. 2006 Jun 12;49(2-3):161–171. doi: 10.1007/s10616-006-0008-2

Development of a Serum-free Suspension Process for the Production of a Conditionally Replicating Adenovirus using A549 Cells

Robert Longley Jr 1, Laurie Radzniak 1, Marc Santoro 1, Yung-Shyeng Tsao 1, Russell G G Condon 1, Peggy Lio 1, Marcio Voloch 1, Zhong Liu 1,✉
PMCID: PMC3449904

Abstract

Conditionally replicating adenoviruses (CRAVs) are a group of recombinant human adenoviruses genetically engineered to replicate in selected tissues, such as tumors. These viruses could potentially offer significant medicinal benefits, since the restrictive replication of these viral vectors leads to the lysis of target cells without harm to the surrounding tissues. The in vitro propagation and amplification of the CRAV vectors often requires special host cells with deregulated growth control pathways. In order to develop an efficient cell culture process for the scaleable production of a CRAV vector, A549 cells, a human lung carcinoma cell line normally cultured in adherent culture, were adapted to suspension culture. CRAV production was demonstrated with the suspension-adapted A549 cells and a baseline production process was developed in shake flasks. The ability to scale-up virus production was confirmed in stirred tank bioreactors. Molecular characterization of the suspension-adapted A549 cells indicates no significant changes in cellular mechanisms related to adenovirus infection.

Keywords: Adenovirus, Recombinant adenovirus, Gene therapy, A549 cells, Serum-free, Suspension, Conditionally replicating adenoviruses (CRAVs)

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References

  1. Bergelson JM, Cunningham JA, Droguett G, Kurt-Jones EA, Krithivas A, Hong JS, Horwitz MS, Crowell RL, Finberg RW. Isolation of a common receptor for Coxsackie B viruses and adenoviruses 2 and 5. Science. 1997;275:1320–1323. doi: 10.1126/science.275.5304.1320. [DOI] [PubMed] [Google Scholar]
  2. Chu L, Robinson DK. Industrial choices for protein production by large-scale cell culture. Curr Opin Biotechnol. 2001;12:180–187. doi: 10.1016/S0958-1669(00)00197-X. [DOI] [PubMed] [Google Scholar]
  3. Cohen CJ, Shieh JT, Pickles RJ, Okegawa T, Hsieh JT, Bergelson JM. The coxsackievirus and adenovirus receptor is a transmembrane component of the tight junction. Proc Natl Acad Sci USA. 2001;98:15191–15196. doi: 10.1073/pnas.261452898. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cote J, Garnier A, Massie B, Kamen A. Serum-free production of recombinant proteins and adenoviral vectors by 293SF-3F6 cells. Biotechnol Bioeng. 1998;59:567–575. doi: 10.1002/(SICI)1097-0290(19980905)59:5<567::AID-BIT6>3.0.CO;2-8. [DOI] [PubMed] [Google Scholar]
  5. Dobbelstein M. Replicating adenoviruses in cancer therapy. Curr Top Microbiol Immunol. 2004;273:291–334. doi: 10.1007/978-3-662-05599-1_9. [DOI] [PubMed] [Google Scholar]
  6. Farson D, Harding TC, Tao L, Liu J, Powell S, Vimal V, Yendluri S, Koprivnikar K, Ho K, Twitty C, Husak P, Lin A, Snyder RO, Donahue BA. Development and characterization of a cell line for large-scale, serum-free production of recombinant adeno-associated viral vectors. J Gene Med. 2004;6:1369–1381. doi: 10.1002/jgm.622. [DOI] [PubMed] [Google Scholar]
  7. Ferreira TB, Ferreira AL, Carrondo MJT, Alves PM. Effect of re-feed strategies and non-ammoniagenic medium on adenovirus production at high cell densities. J Biotechnol. 2005;119(3):272–280. doi: 10.1016/j.jbiotec.2005.03.009. [DOI] [PubMed] [Google Scholar]
  8. Garnier A, Cote J, Nadeau I, Kamen A, Massie B. Scale-up of the adenovirus expression system for the production of recombinant protein in human 293S cells. Cytotechnology. 1994;15:145–155. doi: 10.1007/BF00762389. [DOI] [PubMed] [Google Scholar]
  9. Hsu KH, Lonberg-Holm K, Alstein B, Cromwell RL. A monoclonal antibody specific for the cellular receptor for the group B coxsackieviruses. J Virol. 1988;62:1647–1652. doi: 10.1128/jvi.62.5.1647-1652.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hu WS, Aunins JG. Large-scale mammalian cell culture. Curr Opin Biotechnol. 1997;8:148–153. doi: 10.1016/S0958-1669(97)80093-6. [DOI] [PubMed] [Google Scholar]
  11. Iyer P, Ostrove JM, Vacante D. Comparison of manufacturing techniques for adenovirus production. Cytotechnology. 1999;30:169–172. doi: 10.1023/A:1008040221630. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Katayose D, Seth P. Chapter 10: development of adenoviral vectors for gene therapy. In: Seth P, editor. Adenoviruses: basic biology to gene therapy. Austin, TX: R.G. Landers Company; 1999. pp. 91–101. [Google Scholar]
  13. McConnell MJ, Imperiale MJ. Biology of adenovirus and its use as a vector for gene therapy. Human Gene Therapy. 2004;15:1022–1033. doi: 10.1089/hum.2004.15.1022. [DOI] [PubMed] [Google Scholar]
  14. Meier O, Greber UF. Adenovirus endocytosis. J Gene Med. 2004;Suppl 1:S152–S163. doi: 10.1002/jgm.553. [DOI] [PubMed] [Google Scholar]
  15. Nettelbeck DM. Virotherapeutics: conditionally replicative adenoviruses for viral oncolysis. Anticancer Drugs. 2003;14:577–584. doi: 10.1097/00001813-200309000-00001. [DOI] [PubMed] [Google Scholar]
  16. Ramachandra M, Rahman A, Zou A, Vaillancourt M, Howe JA, Antelman D, Sugarman B, Demers GW, Engler H, Johnson D, Shabram P. Re-engineering adenovirus regulatory pathways to enhance oncolytic specificity and efficacy. Nat Biotechnol. 2001;19:1035–1041. doi: 10.1038/nbt1101-1035. [DOI] [PubMed] [Google Scholar]
  17. Siegfried JM, Han YH, DeMichele MA, Hunt JD, Gaither AL, Cuttitta F. Production of gastrin-releasing peptide by a non-small cell lung carcinoma cell line adapted to serum-free and growth factor-free conditions. J Biol Chem. 1994;269:8596–8603. [PubMed] [Google Scholar]
  18. Tsao YS, Condon RG, Schaefer EJ, Lindsay DA, Liu Z. Biomass and aggregation analysis of human embryonic kidney 293 suspension cell cultures by particle size measurement. Biotechnol Prog. 2000;16:809–814. doi: 10.1021/bp000088k. [DOI] [PubMed] [Google Scholar]
  19. Tsao YS, Condon RG, Schaefer EJ, Lio P, Liu Z. Development and improvement of a serum-free suspension process for the production of recombinant adenoviral vectors using HEK293 cells. Cytotechnology. 2001;37:189–198. doi: 10.1023/A:1020555310558. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Vincent T, Pettersson RF, Crystal RG, Leopold PL. Cytokine-mediated downregulation of coxsackievirus-adenovirus receptor in endothelial cells. J Virol. 2004;78:8047–8058. doi: 10.1128/JVI.78.15.8047-8058.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Wildner O. Comparison of replication-selective, oncolytic viruses for the treatment of human cancers. Curr Opin Mol Ther. 2003;5:351–361. [PubMed] [Google Scholar]
  22. Wills KN, Maneval DC, Menzel P, Harris MP, Sutjipto S, Vaillancourt MT, Huang WM, Johnson DE, Anderson SC, Wen SF, Bookstein R, Shepard HM, Gregory RJ. Development and characterization of recombinant adenoviruses encoding human p53 for gene therapy of cancer. Hum Gene Ther. 1994;5:1079–1088. doi: 10.1089/hum.1994.5.9-1079. [DOI] [PubMed] [Google Scholar]
  23. Zhang Y, Bergelson JM. Adenovirus receptors. J Virol. 2005;79:12125–12131. doi: 10.1128/JVI.79.19.12125-12131.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]

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