Phase I/II Manufacture of Lentiviral Vectors Under GMP in an Academic Setting

  • Anindya DasguptaEmail author
  • Stuart Tinch
  • Kathleen Szczur
  • Rebecca Ernst
  • Nathaniel Shryock
  • Courtney Kaylor
  • Kendall Lewis
  • Eric Day
  • Timmy Truong
  • William Swaney
Part of the Methods in Molecular Biology book series (MIMB, volume 2086)


In clinical gene transfer applications, lentiviral vectors (LV) have rapidly become the primary means to achieve permanent and stable expression of a gene of interest or alteration of gene expression in target cells. This status can be attributed primarily to the ability of the LV to (1) transduce dividing as well as quiescent cells, (2) restrict or expand tropism through envelope pseudo-typing, and (3) regulate gene expression within different cell lineages through internal promoter selection. Recent progress in viral vector design such as the elimination of unnecessary viral elements, split packaging, and self-inactivating vectors has established a significant safety profile for these vectors. The level of GMP compliance required for the manufacture of LV is dependent upon their intended use, stage of drug product development, and country where the vector will be used as the different regulatory authorities who oversee the clinical usage of such products may have different requirements. As such, successful GMP manufacture of LV requires a combination of diverse factors including: regulatory expertise, compliant facilities, validated and calibrated equipments, starting materials of the highest quality, trained production personnel, scientifically robust production processes, and a quality by design approach. More importantly, oversight throughout manufacturing by an independent Quality Assurance Unit who has the authority to reject or approve the materials is required. We describe here the GMP manufacture of LV at our facility using a four plasmid system where 293T cells from an approved Master Cell Bank (MCB) are transiently transfected using polyethylenimine (PEI). Following transfection, the media is changed and Benzonase added to digest residual plasmid DNA. Two harvests of crude supernatant are collected and then clarified by filtration. The clarified supernatant is purified and concentrated by anion exchange chromatography and tangential flow filtration. The final product is then diafiltered directly into the sponsor defined final formulation buffer and aseptically filled.

Key words

GMP LV Mustang Q chromatography Tangential flow filtration 



We are grateful to Prof. H. Trent Spencer, Department of Pediatrics, School of Medicine, Emory University, Atlanta GA, for his critical review of this book chapter. Our facility is generously supported by the Cincinnati Children’s Hospital Research Foundation and 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.


  1. 1.
    Milone MC, O'Doherty U (2018) Clinical use of lentiviral vectors. Leukemia 32(7):1529–1541CrossRefGoogle Scholar
  2. 2.
    Coffin J (1997) In: Hughes S, Varmus H (eds) 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.
    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
  6. 6.
    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
  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 296:2410–2413CrossRefGoogle 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 302:415–419CrossRefGoogle Scholar
  9. 9.
    Bushman F (2007) Retroviral integration and human gene therapy. J Clin Invest 117(8):2083–2086CrossRefGoogle Scholar
  10. 10.
    Cattoglio C, Pellin D, Rizzi E et al (2010) High definition mapping of retroviral integration sites identifies active regulatory elements in human multipotent hematopoietic progenitors. Blood 116:5507–5517CrossRefGoogle Scholar
  11. 11.
    Raper SE, Chirmule N, Lee FS et al (2003) Fatal systemic inflammatory response syndrome in a ornithine transcarbamylase deficient patient following adenoviral gene transfer. Mol Genet Metab 80:148–158CrossRefGoogle Scholar
  12. 12.
    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
  13. 13.
    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):E234CrossRefGoogle Scholar
  14. 14.
  15. 15.
    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 506:191–205CrossRefGoogle Scholar
  16. 16.
    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
  17. 17.
    Modlich U, Bohne J, Schmidt M et al (2006) Cell-culture assays reveal the importance of retroviral vector design for insertional genotoxicity. Blood 108:2545–2553CrossRefGoogle Scholar
  18. 18.
    Gándara C, Affleck V, Stoll EA (2018) Manufacture of third-generation Lentivirus for preclinical use, with process development considerations for translation to good manufacturing practice. Hum Gene Ther Methods 29(1):1–15CrossRefGoogle Scholar
  19. 19.
    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 22:1269–1279CrossRefGoogle Scholar
  20. 20.
    Valkama AJ, Leinonen HM, Lipponen EM et al (2018) Optimization of lentiviral vector production for scale-up in fixed-bed bioreactor. Gene Ther 25(1):39–46CrossRefGoogle Scholar
  21. 21.
    Merten OW, Hebben M, Bovolenta C (2016) Production of lentiviral vectors. Mol Ther Methods Clin Dev 3:16017. Scholar
  22. 22.
    Tomás HA, Rodrigues AF, Carrondo MJT, Coroadinha AS (2018) LentiPro26: novel stable cell lines for constitutive lentiviral vector production. Sci Rep 8(1):5271. Scholar
  23. 23.
    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(5):89–95CrossRefGoogle Scholar
  24. 24.
    Leath A, Cornetta K (2012) Developing novel lentiviral vectors into clinical products. Methods Enzymol 507:89–108CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Anindya Dasgupta
    • 1
    Email author
  • Stuart Tinch
    • 1
  • Kathleen Szczur
    • 1
  • Rebecca Ernst
    • 1
  • Nathaniel Shryock
    • 1
  • Courtney Kaylor
    • 1
  • Kendall Lewis
    • 1
  • Eric Day
    • 1
  • Timmy Truong
    • 1
  • William Swaney
    • 1
  1. 1.Division of Experimental Hematology and Cancer BiologyCincinnati Children’s Hospital Medical CenterCincinnatiUSA

Personalised recommendations