Skip to main content
PMC home page
Cytotechnology logoLink to Cytotechnology
. 2001 May;35(3):157–164. doi: 10.1023/A:1013148203049

Non-GMP plasmid production for transient transfection in bioreactors

Georg Schmid 1,✉, Ernst-Jürgen Schlaeger 2, Beat Wipf 2
PMCID: PMC3449703  PMID: 22358854

Abstract

We describe a generic plasmid purification process for producing DNA for larger-scale transient transfection. Data on plasmid quality with regard to residual protein, endotoxin content and presence of different plasmid forms is given. The effects of contaminants and plasmid forms on expression levels of TNFRp55 and SEAP are discussed. Transient transfection of serum-free suspension grown mammalian cells represents a suitable approach to provide research quantities of proteins (50–100 mg) within1–2 weeks.

Keywords: endotoxin, HEK293, plasmid purification, SEAP, transient transfection

Full Text

The Full Text of this article is available as a PDF (884.7 KB).

References

  1. Berger J, Hauber J, Hauber R, Gelger R, Cullen BR. Secreted placental alkaline phosphatase: A powerful new quantitative indicator of gene expression in eukaryotic cells. Gene. 1988;66:1–10. doi: 10.1016/0378-1119(88)90219-3. [DOI] [PubMed] [Google Scholar]
  2. Birnboim HC. A rapid alkaline extraction method for the isolation of plasmid DNA. Methods Enzymol. 1983;100:243–255. doi: 10.1016/0076-6879(83)00059-2. [DOI] [PubMed] [Google Scholar]
  3. Boussif O, Lezoualc' HF, Zanta MA, Mergny MD, Scherman D, Demeneix B, Behr J-P. A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: Polyethylenimine. Proc Natl Acad Sci USA. 1995;92:7297–7301. doi: 10.1073/pnas.92.16.7297. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chandra G, Patel P, Kost TA, Gray JG. Large-scale purification of plasmid DNA by fast protein liquid chromatography using a Hi-Load Q Sepharose column. Analyt Biochem. 1992;203:169–172. doi: 10.1016/0003-2697(92)90060-K. [DOI] [PubMed] [Google Scholar]
  5. Colpan M, Riesner D. High-performance liquid chromatography of high-molecular-weight nucleic acids on the macroporous ion exchanger. Nucleogen J Chromatogr. 1984;296:339–353. doi: 10.1016/S0021-9673(01)96428-3. [DOI] [Google Scholar]
  6. Ferreira GNM, Monterio GA, Prazeres DMF, Cabral JMS. Downstream processing of plasmid DNA for gene therapy and DNA vaccine applications. Trends Biotechnol. 2000;18:380–388. doi: 10.1016/S0167-7799(00)01475-X. [DOI] [PubMed] [Google Scholar]
  7. Godfried MH, van der Poll T, Weverling GJ, Mulder JW, Jansen J, van Deventer SJH, Sauerwein HP. Soluble receptors for tumor necrosis factor as predictors of progression to Aids in asymptomatic human immunodeficiency virus type 1 infection. J Infect Dis. 1994;169:739–745. doi: 10.1093/infdis/169.4.739. [DOI] [PubMed] [Google Scholar]
  8. Hines RN, O'Conner KC, Vella G, Warren W. Largescale purification of plasmid DNA by anion-exchange highperformance liquid chromatography. BioTechniques. 1992;12:430–434. [PubMed] [Google Scholar]
  9. Horn NA, Meek JA, Budahazi G, Marquet M. Cancer gene therapy using plasmid DNA: Purification of DNA for human clinical trials. Hum Gen Ther. 1995;6:565–573. doi: 10.1089/hum.1995.6.5-565. [DOI] [PubMed] [Google Scholar]
  10. Kahn DW, Butler MD, Cohen DL, Gordon M, Kahn JW, Winkler ME. Purification of plasmid DNA by tangential flow filtration. Biotechnol Bioeng. 2000;69:101–106. doi: 10.1002/(SICI)1097-0290(20000705)69:1<101::AID-BIT12>3.0.CO;2-1. [DOI] [PubMed] [Google Scholar]
  11. Lahijani R, Hulley G, Soriano G, Horn NA, Marquet M. High-yield production of pBR322-derived plasmids intended for human gene therapy by employing a temperature-controllable point mutation. Hum Gene Ther. 1996;7:1971–1980. doi: 10.1089/hum.1996.7.16-1971. [DOI] [PubMed] [Google Scholar]
  12. Loetscher H, Pan YCE, Lahm H-W, Gentz R, Brockhaus M, Tabuchi H, Lesslauer W. Molecular cloning and expression of the human 55kd tumor necrosis factor receptor. Cell. 1990;61:351–359. doi: 10.1016/0092-8674(90)90815-V. [DOI] [PubMed] [Google Scholar]
  13. McClung JK, Gonzales RA. Purification of plasmid DNA by fast protein liquid chromatography on Superose 6 preparative grade. Anal Biochem. 1989;177:378–382. doi: 10.1016/0003-2697(89)90069-9. [DOI] [PubMed] [Google Scholar]
  14. Merion M, Warren W. Purification of supercoiled plasmids from crude cell lysates using high performance anion exchange chromatography. BioTechniques. 1989;7:60–67. [PubMed] [Google Scholar]
  15. Mitzner S, Schneidewind J, Falkenhagen D, Loth F, Klinkmann H. Extracorporeal endotoxin removal by immobilized polyethylenimine. Artif Organs. 1993;17:775–781. doi: 10.1111/j.1525-1594.1993.tb00630.x. [DOI] [PubMed] [Google Scholar]
  16. Montbriand PM, Malone RW. Improved method for the removal of endotoxin from DNA. J Biotechnol. 1996;44:43–46. doi: 10.1016/0168-1656(95)00091-7. [DOI] [PubMed] [Google Scholar]
  17. Prazeres DMF, Ferreira GNM, Monteiro GA, Cooney CL, Cabral JMS. Large-scale production of pharmaceutical-grade plasmid DNA for gene therapy: problems and bottlenecks. Trends Biotechnol. 1999;17:169–174. doi: 10.1016/S0167-7799(98)01291-8. [DOI] [PubMed] [Google Scholar]
  18. Raymond GJ, Bryant PK, III, Nelson A, Johnson JD. Large-scale isolation of covalently closed circular DNA using gel filtration chromatography. Anal Biochem. 1988;173:125–133. doi: 10.1016/0003-2697(88)90169-8. [DOI] [PubMed] [Google Scholar]
  19. Sayers JR, Evans D, Thomson JB. Identification and eradication of a denatured DNA isolated during alkaline lysis-based plasmid purification procedures. Anal Biochem. 1996;241:186–189. doi: 10.1006/abio.1996.0397. [DOI] [PubMed] [Google Scholar]
  20. Schlaeger E-J, Christensen K. Transient gene expression in mammalian cells grown in serum-free suspension culture. Cytotechnology. 1999;30:71–83. doi: 10.1023/A:1008000327766. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Schlaeger E-J, Christensen K & Kitas EA (1999) Scale-up of transient gene expression in mammalian cells grown in serum-free suspension culture. Presentation at the 4th Gene Delivery & Cellular Protein Expression Conference, Lake Tahoe, CA.
  22. Schorr J, Moritz P, Weber M & Schleef M (1997) Nucleic acids for therapeutic applications: Pharmaceutical quality with industrial scale production, Poster at: Gene Therapy Meeting, Cold Spring Harbor, NY, USA.
  23. Schumpp B, Schlaeger E-J. Optimization of culture conditions for high cell density proliferation of HL60 human promyelocytic leukemia cells. J Cell Science. 1990;97:639–647. doi: 10.1242/jcs.97.4.639. [DOI] [PubMed] [Google Scholar]
  24. Wils P, Escriou V, Warnery A, Lacroix F, Lagneaux D, Ollivier M, Cruzet J, Mayaux J-F, Scherman D. Efficient purification of plasmid DNA for gene transfer using triple-helix affinity chromatography. Gene Ther. 1997;4:323–330. doi: 10.1038/sj.gt.3300388. [DOI] [PubMed] [Google Scholar]

Articles from Cytotechnology are provided here courtesy of Springer Science+Business Media B.V.

RESOURCES