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. 2001 Nov;37(3):189–198. doi: 10.1023/A:1020555310558

Development and improvement of a serum-free suspension process for the production of recombinant adenoviral vectors using HEK293 cells

Yung-Shyeng Tsao 1, Russell Condon 1, Eugene Schaefer 1, Peggy Lio 1, Zhong Liu 1
PMCID: PMC3449791  PMID: 19002922

Abstract

Human Embryonic Kidney 293 (HEK293) cells were adapted into a serum-free suspension medium through steps of gradual serum weaning for the production of adenoviral (AdV) gene therapy vectors. The presence of sodium heparin in the medium formulation reduced cell clumping dramatically in suspension culture. The adapted cells were ready to grow either in serum-containing medium as an attached culture or in serum-free medium in suspension culture. A scalable production process was developed in shake flasks and was then evaluated in stirred tank bioreactors. This process includes a growth phase in batch-mode followed by a production phase involving medium perfusion and supplementation. Fortification with calcium chloride post viral inoculation resulted in an increase in virus production by at least one fold. Addition of stimulating agents such as sodium butyrate, N-acetyl-L-cysteine (NAC), dimethyl sulfoxide(DMSO), or ethyl alcohol post infection was shown to further improve virus production in a dose-dependent manner. The serum-free suspension process described here should be suitable for the manufacturing of other E1-deleted AdV vectors and could potentially be used for the production of recombinant proteins by HEK293 cells.

Keywords: Adenovirus, Dimethyl sulfoxide (DMSO), Ethyl alcohol, Gene therapy, Human Embryonic Kidney 293 (HEK293) cells, N-acetyl-L-cysteine, Recombinant adenoviral vector, Serum-free suspension culture, Sodium butyrate

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References

  1. Amalfitano A. Next-generation adenoviral vectors: new and improved. Gene. Ther. 1999;6:1643–1645. doi: 10.1038/sj.gt.3301027. [DOI] [PubMed] [Google Scholar]
  2. Anderson W.F. Human gene therapy. Nature. 1998;392:25–30. doi: 10.1038/32801. [DOI] [PubMed] [Google Scholar]
  3. Arts J., Lansink M., Grimbergen J., Toet K.H., Kooistra T. Stimulation of tissue-type plasminogen activator gene expression by sodium butyrate and trichostatin A in human endothelial cells involves histone acetylation. Biochem. J. 1995;310:171–176. doi: 10.1042/bj3100171. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bazzoni G., Beltran Nunez A., Mascellani G., Bianchini P., Dejana E., Del Maschio A. Effect of heparin, dermatan sulfate, and related oligo-derivatives on human polymorphonuclear leukocyte functions. J. Lab. Clin. Med. 1993;121:268–275. [PubMed] [Google Scholar]
  5. Benihoud K., Yeh P., Perricaudet M. Adenovirus vectors for gene delivery. Curr. Opin. Biotechnol. 1999;10:440–447. doi: 10.1016/S0958-1669(99)00007-5. [DOI] [PubMed] [Google Scholar]
  6. Berg D.T., McClure D.B., Grinnell B.W. High-level expression of secreted proteins from cells adapted to serum-free suspension culture. Biotechniques. 1993;14:972–978. [PubMed] [Google Scholar]
  7. Collins S.J., Ruscetti F.W., Gallagher R.E., Gallo R.C. Terminal differentiation of human promyelocytic leukemia cells induced by dimethyl sulfoxide and other polar compounds. Proc. Natl. Acad. Sci. USA. 1978;75:2458–2462. doi: 10.1073/pnas.75.5.2458. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. Dee K.U., Shuler M.L., Wood H.A. Inducing single-cell suspension of BTI-TN5B1-4 insect cells: I. the use of sulfated polyanions to prevent cell aggregation and enhance recombinant protein production. Biotechnol. Bioeng. 1997;54:191–205. doi: 10.1002/(SICI)1097-0290(19970505)54:3<191::AID-BIT1>3.0.CO;2-A. [DOI] [PubMed] [Google Scholar]
  10. Dee K.U., Wood H.A., Shuler M.L. Inducing single-cell suspension of BTI-TN5B1-4 insect cells: II. the effect of sulfated polyanions on baculovirus infection. Biotechnol. Bioeng. 1997;54:206–220. doi: 10.1002/(SICI)1097-0290(19970505)54:3<206::AID-BIT2>3.0.CO;2-J. [DOI] [PubMed] [Google Scholar]
  11. Eldor A., Weksler B.B. Heparin and dextran sulfate antagonize PGI2 inhibition of platelet aggregation. Thromb. Res. 1979;16:617–628. doi: 10.1016/0049-3848(79)90206-8. [DOI] [PubMed] [Google Scholar]
  12. Fiore M., Degrassi F. Dimethyl sulfoxide restores contact inhibition-induced growth arrest and inhibits cell density-dependent apoptosis in hamster cells. Exp. Cell Res. 1999;251:102–110. doi: 10.1006/excr.1999.4542. [DOI] [PubMed] [Google Scholar]
  13. Frank W.O., Rayyes A.N., Washington A., Holt P.R. Effect of acute ethanol administration upon hepatic regeneration. J. Lab. Clin. Med. 1979;93:402–413. [PubMed] [Google Scholar]
  14. 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]
  15. Gorman C.M., Howard B.H., Reeves R. Expression of recombinant plasmids in mammalian cells is enhanced by sodium butyrate. Nucleic Acids Res. 1983;11:7631–7648. doi: 10.1093/nar/11.21.7631. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Graham F.L. Adenovirus vectors for high-efficiency gene transfer into mammalian cells. Immunol. Today. 2000;21:426–428. doi: 10.1016/S0167-5699(00)01676-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Graham F.L., Smiley J., Russell W.C., Nairn R. Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J. Gen. Virol. 1977;36:59–74. doi: 10.1099/0022-1317-36-1-59. [DOI] [PubMed] [Google Scholar]
  18. Greber U.F., Willetts M., Webster P., Helenius A. Stepwise dismantling of adenovirus 2 during entry into cells. Cell. 1993;75:477–486. doi: 10.1016/0092-8674(93)90382-Z. [DOI] [PubMed] [Google Scholar]
  19. Hu W.S., Aunins J.G. Large-scale mammalian cell culture. Curr. Opin. Biotechnol. 1997;8:148–153. doi: 10.1016/S0958-1669(97)80093-6. [DOI] [PubMed] [Google Scholar]
  20. Irvine Scientific, Inc. 1997. Product Description and Applications (PN 99269 Rev2 1/97) IS 293 (Catalog No. 99269).
  21. Iyer P., Ostrove J.M., 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]
  22. Kallos M.S., Behie L.A., Vescovi A.L. Extended serial passaging of mammalian neural stem cells in suspension bioreactors. Biotechnol. Bioeng. 1999;65:589–599. doi: 10.1002/(SICI)1097-0290(19991205)65:5<589::AID-BIT12>3.0.CO;2-S. [DOI] [PubMed] [Google Scholar]
  23. Kameneva M.V., Antaki J.F., Watach M.J., Borovetz H.S., Kormos R.L. Heparin effect on red blood cell aggregation. Biorheology. 1994;31:297–304. doi: 10.3233/bir-1994-31307. [DOI] [PubMed] [Google Scholar]
  24. Katayose D., Seth P. Development of adenoviral vectors for gene therapy. In: Seth P., editor. Adeno-viruses: Basic Biology to Gene Therapy. Austin, TX: R.G. Landers Company; 1999. pp. 91–101. [Google Scholar]
  25. Malorni W., Rivabene R., Santini M.T., Donelli G. N-acetylcysteine inhibits apoptosis and decreases viral particles in HIV-chronically infected U937 cells. FEBS Lett. 1993;327:75–78. doi: 10.1016/0014-5793(93)81043-Y. [DOI] [PubMed] [Google Scholar]
  26. Mastrangelo A.J., Zou S., Hardwick J.M., Betenbaugh M.J. Antiapoptosis chemicals prolong productive lifetimes of mammalian cells upon Sindbis virus vector infection. Biotechnol. Bioeng. 1999;65:298–305. doi: 10.1002/(SICI)1097-0290(19991105)65:3<298::AID-BIT7>3.0.CO;2-S. [DOI] [PubMed] [Google Scholar]
  27. Mather J.P., Moore A., Shawley R. Optimization of growth, viability, and specific productivity for expression of recombinant proteins in mammalian cells. Methods Mol. Biol. 1997;62:369–382. doi: 10.1385/0-89603-480-1:369. [DOI] [PubMed] [Google Scholar]
  28. Nadeau I., Garnier A., Cote J., Massie B., Chavarie C., Kamen A. Improvement of recombinant protein production with the human adenovirus /293S expression system using fed-batch strategies. Biotechnol. Bioeng. 1996;51:613–623. doi: 10.1002/(SICI)1097-0290(19960920)51:6<613::AID-BIT1>3.0.CO;2-K. [DOI] [PubMed] [Google Scholar]
  29. Nakano M.Y., Greber U.F. Quantitative microscopy of fluorescent adenovirus entry. J. Struct. Biol. 2000;129:57–68. doi: 10.1006/jsbi.1999.4201. [DOI] [PubMed] [Google Scholar]
  30. Peshwa M.V., Kyung Y.S., McClure D.B., Hu W.S. Cultivation of mammalian cells as aggregates in bioreactors: effect of calcium concentration on spatial distribution of viability. Biotechnol. Bioeng. 1993;41:179–187. doi: 10.1002/bit.260410203. [DOI] [PubMed] [Google Scholar]
  31. Reches A., Eldor A., Salomon Y. The effects of dextran sulfate, heparin and PGE1 on adenylate cyclase activity and aggregation of human platelets. Thromb. Res. 1979;16:107–116. doi: 10.1016/0049-3848(79)90274-3. [DOI] [PubMed] [Google Scholar]
  32. Robbins P.D., Ghivizzani S.C. Viral vectors for gene therapy. Pharmacol. Ther. 1998;80:35–47. doi: 10.1016/S0163-7258(98)00020-5. [DOI] [PubMed] [Google Scholar]
  33. Rochon Y.P., Simon S.I., Lynam E.B., Sklar L.A. A role for lectin interactions during human neutrophil aggregation. J. Immunol. 1994;152:1385–1393. [PubMed] [Google Scholar]
  34. Russell W.C. Update on adenovirus and its vectors. J. Gen. Virol. 2000;81:2573–2604. doi: 10.1099/0022-1317-81-11-2573. [DOI] [PubMed] [Google Scholar]
  35. Saito H., Kagawa T., Tada S., Tsunematsu S., Guevara F.M., Watanabe T., et al. Effect of dexamethasone, dimethylsulfoxide and sodium butyrate on a human hepatoma cell line PLC/PRF/ 5. Cancer Biochem. Biophys. 1992;13:75–84. [PubMed] [Google Scholar]
  36. Schmid S.I., Hearing P. Adenovirus Assembly. In: Seth P., editor. Adenoviruses: Basic Biology to Gene Therapy. Austin, TX: R.G. Landers Company; 1999. pp. 85–89. [Google Scholar]
  37. Schoofs G., Monica T.J., Ayala J., Horwitz J., Montgomery T., Roth G., et al. A high-yielding serum-free, suspension cell culture process to manufacture recombinant adenoviral vectors for gene therapy. Cytotechnology. 1998;28:81–89. doi: 10.1023/A:1008021428969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Shabram P.W., Giroux D.D., Goudreau A.M., Gregory R.J., Horn M.T., Huyghe B.G., et al. Analytical anion-exchange HPLC of recombinant type-5 adenoviral particles. Hum. Gene. Ther. 1997;8:453–465. doi: 10.1089/hum.1997.8.4-453. [DOI] [PubMed] [Google Scholar]
  39. Shenk T. Group C adenoviruses as vectors for gene therapy. In: Kaplitt M.G., Loewy A.D., editors. Viral Vectors: Gene Therapy and Neuroscience Applications. New York: Academic Press; 1995. pp. 43–54. [Google Scholar]
  40. Shenk T. Adenoviridae: The viruses and their replication. In: Fields B.N., Knipe D.M., Howley P.M., editors. Fields Virology. 3rd edn. Philadelphia: Lippincott - Raven Publishers; 1996. pp. 2111–2148. [Google Scholar]
  41. Spier R.E. Factors limiting the commercial application of animal cells in culture. Cytotechnology. 1997;23:113–117. doi: 10.1023/A:1007975904301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Stanley M.J., Liebersbach B.F., Liu W., Anhalt D.J., Sanderson R.D. Heparan sulfate-mediated cell aggregation. Syndecans-1 and-4 mediate intercellular adhesion following their transfection into human B lymphoid cells. J. Biol. Chem. 1995;270:5077–5083. doi: 10.1074/jbc.270.45.26986. [DOI] [PubMed] [Google Scholar]
  43. Tsao D., Shi Z.R., Wong A., Kim Y.S. Effect of sodium butyrate on carcinoembryonic antigen production by human colonic adenocarcinoma cells in culture. Cancer Res. 1983;43:1217–1222. [PubMed] [Google Scholar]
  44. Tsao Y.S., Condon R.G., Schaefer E.J., Lindsay D.A., 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]
  45. Wands J.R., Carter E.A., Bucher N.L., Isselbacher K.J. Inhibition of hepatic regeneration in rats by acute and chronic ethanol intoxication. Gastroenterology. 1979;77:528–531. [PubMed] [Google Scholar]

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