Scalable Lentiviral Vector Production Using Stable HEK293SF Producer Cell Lines
| Autor: | Anne-Marie Gélinas, Howard Kim, July Dorion-Thibaudeau, Alice Bernier, Aziza P. Manceur, Sven Ansorge, Vanja Misic, Nadejda Andreev, Rénald Gilbert, Stéphane Lanthier, Sophie Broussau, Sonia Tremblay |
|---|---|
| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Virus Cultivation Transgene Genetic Vectors Cell Culture Techniques Clone (cell biology) Biology Benzoates Applied Microbiology and Biotechnology perfusion Viral vector Green fluorescent protein HEK293SF 03 medical and health sciences Transduction (genetics) Bioreactors Plasmid Transduction Genetic Genetics Humans Research Articles Genetics (clinical) Pharmacology business.industry bioprocessing lentiviral vector Lentivirus HEK 293 cells Cell biology Biotechnology HEK293 Cells 030104 developmental biology Cell culture Doxycycline Molecular Medicine stable producer business |
| Zdroj: | Human Gene Therapy Methods |
| ISSN: | 1946-6544 1946-6536 |
| Popis: | Lentiviral vectors (LV) represent a key tool for gene and cell therapy applications. The production of these vectors in sufficient quantities for clinical applications remains a hurdle, prompting the field towards developing suspension processes that are conducive to large scale production. We describe here a LV production strategy using a stable inducible producer cell line. The HEK293 cell line employed grows in suspension, thus offering direct scalability, and produces a Green Fluorescent Protein (GFP)-expressing lentiviral vector in the 106 Transduction Units (TU)/ml range without optimization. The stable producer cell line, called clone 92, was derived by stable transfection from a packaging cell line with a plasmid encoding the transgene GFP. The packaging cell line expresses all the other necessary components to produce LV upon induction with cumate and doxycycline. First, we demonstrate that LV production using clone 92 is scalable from 20mL shake flasks to 3L bioreactors. Next, we developed two strategies for high yield LV production in perfusion mode using acoustic cell filter technology in 1 to 3L bioreactors. The first approach uses a basal commercial medium and perfusion mode both pre- and post-induction for increasing cell density and LV recovery. The second approach makes use of a fortified medium formulation to achieve target cell density for induction in batch mode, followed by perfusion mode after induction. Using these perfusion-based strategies, the titer was improved to 3.2 x 107 TU/ml. As a result, cumulative functional LV titers were increased by up to 15-fold compared to batch mode, reaching a cumulative total yield of 8 x 1010 TU/L of bioreactor culture. This approach is easily amenable to large-scale production and commercial manufacturing. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|