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Scale-up and manufacturing of clinical-grade self-inactivating γ-retroviral vectors by transient transfection

Gene Therapy volume 19, pages 246–254 (2012)Cite this article

Abstract

The need for γ-retroviral (gRV) vectors with a self-inactivating (SIN) design for clinical application has prompted a shift in methodology of vector manufacturing from the traditional use of stable producer lines to transient transfection-based techniques. Herein, we set out to define and optimize a scalable manufacturing process for the production of gRV vectors using transfection in a closed-system bioreactor in compliance with current good manufacturing practices (cGMP). The process was based on transient transfection of 293T cells on Fibra-Cel disks in the Wave Bioreactor. Cells were harvested from tissue culture flasks and transferred to the bioreactor containing Fibra-Cel in the presence of vector plasmid, packaging plasmids and calcium-phosphate in Dulbecco's modified Eagle's medium and 10% fetal bovine serum. Virus supernatant was harvested at 10–14 h intervals. Using optimized procedures, a total of five ecotropic cGMP-grade gRV vectors were produced (9 liters each) with titers up to 3.6 × 107 infectious units per milliliter on 3T3 cells. One GMP preparation of vector-like particles was also produced. These results describe an optimized process for the generation of SIN viral vectors by transfection using a disposable platform that allows for the generation of clinical-grade viral vectors without the need for cleaning validation in a cost-effective manner.

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Acknowledgements

We thank the following laboratories of the CCHMC Translational Core Laboratory (TCL): (i) Translational Trials Development and Support Laboratory (TTDSL) for titration of vectors using quantitative polymerase chain reaction, and (ii) the CCHMC Viral Vector Core for vector titration using flow cytometry. The Vector Production Facility in the TCL is supported by Institutional funds from the Cincinnati Children's Research Foundation. AS and CB thank the German Academic Exchange Service (DAAD), the European Union (FP7 program PERSIST) and the German Ministry for Research (BMBF project PIDNET.IFB-Tx) for their support. AJT acknowledges the Medical Research Council, The Wellcome Trust and Great Ormond Street Children's Charity for their support. Finally, we thank Dr Carolyn Lutzko for critically reviewing the manuscript.

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Authors and Affiliations

  1. Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA

    J C M van der Loo, E Grassman, A Terwilliger, T Higashimoto, D L Nordling & P Malik

  2. Siteman Cancer Center, Barnes Jewish Hospital, Washington University St Louis School of Medicine, St Louis, MO, USA

    W P Swaney

  3. Division of Experimental Hematology, Hannover Medical School, Hannover, Germany

    A Schambach & C Baum

  4. Centre Immunodeficiency, UCL Institute of Child Health, London, UK

    A J Thrasher

  5. Department of Immunology, Great Ormond Street Hospital NHS Trust, London, UK

    A J Thrasher

  6. Division of Hematology/Oncology, Children's Hospital Boston, Harvard Medical School, Boston, MA, USA

    D A Williams

  7. Indiana University School of Medicine, Indianapolis, IN, USA

    L Reeves

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  1. J C M van der Loo
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Correspondence to J C M van der Loo.

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van der Loo, J., Swaney, W., Grassman, E. et al. Scale-up and manufacturing of clinical-grade self-inactivating γ-retroviral vectors by transient transfection. Gene Ther 19, 246–254 (2012). https://doi.org/10.1038/gt.2011.102

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  • DOI: https://doi.org/10.1038/gt.2011.102

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