Development of a Serum-free Suspension Process
for the Production of a Conditionally Replicating Adenovirus using A549 Cells
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.
KeywordsAdenovirus Recombinant adenovirus Gene therapy A549 cells Serum-free Suspension Conditionally replicating adenoviruses (CRAVs)
Bergelson, JM, Cunningham, JA, Droguett, G, Kurt-Jones, EA, Krithivas, A, Hong, JS, Horwitz, MS, Crowell, RL, Finberg, RW 1997Isolation of a common receptor for Coxsackie B viruses and adenoviruses 2 and 5Science27513201323PubMedCrossRefGoogle Scholar Chu, L, Robinson, DK 2001Industrial choices for protein production by large-scale cell cultureCurr Opin Biotechnol12180187PubMedCrossRefGoogle Scholar Cohen, CJ, Shieh, JT, Pickles, RJ, Okegawa, T, Hsieh, JT, Bergelson, JM 2001The coxsackievirus and adenovirus receptor is a transmembrane component of the tight junctionProc Natl Acad Sci USA981519115196PubMedCrossRefGoogle Scholar Cote, J, Garnier, A, Massie, B, Kamen, A 1998Serum-free production of recombinant proteins and adenoviral vectors by 293SF-3F6 cellsBiotechnol Bioeng59567575PubMedCrossRefGoogle Scholar Dobbelstein, M 2004Replicating adenoviruses in cancer therapyCurr Top Microbiol Immunol273291334PubMedGoogle Scholar 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 2004Development and characterization of a cell line for large-scale, serum-free production of recombinant adeno-associated viral vectorsJ Gene Med613691381PubMedCrossRefGoogle Scholar Ferreira, TB, Ferreira, AL, Carrondo, MJT, Alves, PM 2005Effect of re-feed strategies and non-ammoniagenic medium on adenovirus production at high cell densitiesJ Biotechnol119272280PubMedCrossRefGoogle Scholar Garnier, A, Cote, J, Nadeau, I, Kamen, A, Massie, B 1994Scale-up of the adenovirus expression system for the production of recombinant protein in human 293S cellsCytotechnology15145155PubMedCrossRefGoogle Scholar Hsu, KH, Lonberg-Holm, K, Alstein, B, Cromwell, RL 1988A monoclonal antibody specific for the cellular receptor for the group B coxsackievirusesJ Virol6216471652PubMedGoogle Scholar Hu, WS, Aunins, JG 1997Large-scale mammalian cell cultureCurr Opin Biotechnol8148153PubMedCrossRefGoogle Scholar Iyer, P, Ostrove, JM, Vacante, D 1999Comparison of manufacturing techniques for adenovirus productionCytotechnology30169172CrossRefGoogle Scholar Katayose, D, Seth, P 1999Chapter 10: development of adenoviral vectors for gene therapySeth, P eds. Adenoviruses: basic biology to gene therapyR.G. Landers CompanyAustin, TX91101Google Scholar McConnell, MJ, Imperiale, MJ 2004Biology of adenovirus and its use as a vector for gene therapyHuman Gene Therapy1510221033PubMedCrossRefGoogle Scholar Meier, O, Greber, UF 2004Adenovirus endocytosisJ Gene MedSuppl 1S152S163CrossRefGoogle Scholar Nettelbeck, DM 2003Virotherapeutics: conditionally replicative adenoviruses for viral oncolysisAnticancer Drugs14577584PubMedCrossRefGoogle Scholar Ramachandra, M, Rahman, A, Zou, A, Vaillancourt, M, Howe, JA, Antelman, D, Sugarman, B, Demers, GW, Engler, H, Johnson, D, Shabram, P 2001Re-engineering adenovirus regulatory pathways to enhance oncolytic specificity and efficacyNat Biotechnol1910351041PubMedCrossRefGoogle Scholar Siegfried, JM, Han, YH, DeMichele, MA, Hunt, JD, Gaither, AL, Cuttitta, F 1994Production of gastrin-releasing peptide by a non-small cell lung carcinoma cell line adapted to serum-free and growth factor-free conditionsJ Biol Chem26985968603PubMedGoogle Scholar Tsao, YS, Condon, RG, Schaefer, EJ, Lindsay, DA, Liu, Z 2000Biomass and aggregation analysis of human embryonic kidney 293 suspension cell cultures by particle size measurementBiotechnol Prog16809814PubMedCrossRefGoogle Scholar Tsao, YS, Condon, RG, Schaefer, EJ, Lio, P, Liu, Z 2001Development and improvement of a serum-free suspension process for the production of recombinant adenoviral vectors using HEK293 cellsCytotechnology37189198CrossRefGoogle Scholar Vincent, T, Pettersson, RF, Crystal, RG, Leopold, PL 2004Cytokine-mediated downregulation of coxsackievirus-adenovirus receptor in endothelial cellsJ Virol7880478058PubMedCrossRefGoogle Scholar Wildner, O 2003Comparison of replication-selective, oncolytic viruses for the treatment of human cancersCurr Opin Mol Ther5351361PubMedGoogle Scholar 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 1994Development and characterization of recombinant adenoviruses encoding human p53 for gene therapy of cancerHum Gene Ther510791088PubMedGoogle Scholar Zhang, Y, Bergelson, JM 2005Adenovirus receptorsJ Virol791212512131PubMedCrossRefGoogle Scholar