Advertisement

A Scalable Lentiviral Vector Production and Purification Method Using Mustang Q Chromatography and Tangential Flow Filtration

  • Stuart Tinch
  • Kathy Szczur
  • William Swaney
  • Lilith Reeves
  • Scott R. WittingEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1937)

Abstract

Lentiviral vectors have rapidly become a favorite tool for research and clinical gene transfer applications which seek to permanently introduce alterations in the genome. This status can be attributed primarily to their ability to transduce dividing as well as quiescent cells. When coupled with internal promotor selection to drive expression in one cell type but not another, the ease with which the vectors can be pseudotyped to either restrict or expand tropism offers unique opportunities previously unavailable to the researcher to manipulate the genome. Although LV can be produced from stable packaging cell lines and/or in suspension culture, by and far, most LV vectors are produced using adherent 293 T cells grown in plasticware and production plasmids transiently transfected with either PEI or Calcium Phosphate. The media is usually changed and un-concentrated vector supernatant collected between 24 and 48 h post-transfection. The supernatant may then be purified by Mustang Q chromatography, concentrated by Tangential Flow Filtration, and finally diafiltered into the final formulation buffer of choice. Here we describe a pilot scale method for the manufacture of a Lentiviral vector that purifies and concentrates approximately 6 L of un-concentrated LV supernatant to approximately 150 mL. Typical titers for most vector constructs range between 1 × 108 and 1 × 109 infectious particles per mL. This method may be performed reiteratively to increase total volume or can be further scaled up to increase yield.

Key words

Lentiviral vector production Purification Mustang Q chromatography Tangential flow filtration 

Notes

Acknowledgments

Our facility is generously supported by the Cincinnati Children’s Hospital Research Foundation. Most importantly, we would like to acknowledge the hard work and dedication of the entire staff of the Translational Core Laboratories at Cincinnati Children’s Hospital; without it, our contributions to the gene therapy field would not be possible.

References

  1. 1.
    Otto-Wilhelm M, Hebben M, Bovolenta C (2016) Production of lentiviral vectors. Mol Ther Methods Clin Dev 3:16017.  https://doi.org/10.1038/mtm.2016.17CrossRefGoogle Scholar
  2. 2.
    Coffin J, Hughes S, Varmus H (eds) (1997) Retroviruses. Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar
  3. 3.
    Rosenberg S, Aebersold P, Cornetta K et al (1990) Gene transfer into humans--immunotherapy of patients with advanced melanoma, using tumor-infiltrating lymphocytes modified by retroviral gene transduction. N Engl J Med 323:570–578CrossRefGoogle Scholar
  4. 4.
    Blaese R, Culver K, Miller A et al (1995) T lymphocyte-directed gene therapy for ADA− SCID: initial trial results after 4 years. Science 270:475–480CrossRefGoogle Scholar
  5. 5.
    Cavazzana-Calvo M, Hacein-Bey S, de Saint Basile G et al (2000) Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease. Science 288:669–672CrossRefGoogle Scholar
  6. 6.
    Kohn D, Weinberg K, Nolta J et al (1995) Engraftment of gene-modified umbilical cord blood cells in neonates with adenosine deaminase deficiency. Nat Med 1:1017–1023CrossRefGoogle Scholar
  7. 7.
    Aiuti A, Slavin S, Aker M et al (2002) Correction of ADA-SCID by stem cell gene therapy combined with nonmyeloablative conditioning. Science 296:2410–2413.  https://doi.org/10.1126/science.1070104CrossRefPubMedGoogle Scholar
  8. 8.
    Hacein-Bey-Abina S, Von Kalle C, Schmidt M et al (2003) LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science 302:415–419.  https://doi.org/10.1126/science.1088547CrossRefPubMedGoogle Scholar
  9. 9.
    Bushman F (2007) Retroviral integration and human gene therapy. J Clin Invest 117(8):2083–2086CrossRefGoogle Scholar
  10. 10.
    Schröder A, Shinn P, Chen H et al (2002) HIV-1 integration in the human genome favors active genes and local hotspots. Cell 110(4):521–529CrossRefGoogle Scholar
  11. 11.
    Mitchell R, Beitzel B, Schroder A et al (2004) Retroviral DNA integration: ASLV, HIV, and MLV show distinct target site preferences. PLoS Biol 2(8):E234.  https://doi.org/10.1371/journal.pbio.0020234CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Schambach A, Swaney W, van der Loo J (2009) Design and production of retro- and lentiviral vectors for gene expression in hematopoietic cells. Methods Mol Biol 506:191–205.  https://doi.org/10.1007/978-1-59745-409-4_14CrossRefPubMedGoogle Scholar
  13. 13.
    Modlich U, Bohne J, Schmidt M et al (2006) Cell-culture assays reveal the importance of retroviral vector design for insertional genotoxicity. Blood 108:2545–2553.  https://doi.org/10.1182/blood-2005-08-024976CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Dull T, Zufferey R, Kelly M et al (1998) A third-generation lentivirus vector with a conditional packaging system. J Virol 72(11):8463–8471PubMedPubMedCentralGoogle Scholar
  15. 15.
    Gama-Norton L, Botezatu L, Herrmann S et al (2011) Lentivirus production is influenced by SV40 large T-antigen and chromosomal integration of the vector in HEK293 cells. Hum Gene Ther 22:1269–1279.  https://doi.org/10.1089/hum.2010.143CrossRefPubMedGoogle Scholar
  16. 16.
    Slepushkin V, Chang N, Cohen R et al (2003) Large-scale purification of a lentiviral vector by size exclusion chromatography or Mustang Q ion exchange chromatography. Bioprocess J 2:89–95CrossRefGoogle Scholar
  17. 17.
    Leath A, Cornetta K (2012) Developing novel lentiviral vectors into clinical products. Meth Enzymol 507:89–108.  https://doi.org/10.1016/B978-0-12-386509-0.00005-3CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Stuart Tinch
    • 1
  • Kathy Szczur
    • 1
  • William Swaney
    • 1
  • Lilith Reeves
    • 1
  • Scott R. Witting
    • 1
    • 2
    Email author
  1. 1.Cincinnati Children’s Hospital Medical CenterCincinnatiUSA
  2. 2.Department of PediatricsUniversity of Cincinnati College of MedicineCincinnatiUSA

Personalised recommendations