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. 2017 Dec;28(6):330-339.
doi: 10.1089/hgtb.2017.086. Epub 2017 Nov 21.

Scalable Lentiviral Vector Production Using Stable HEK293SF Producer Cell Lines

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Free PMC article

Scalable Lentiviral Vector Production Using Stable HEK293SF Producer Cell Lines

Aziza P Manceur et al. Hum Gene Ther Methods. .
Free PMC article

Abstract

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 toward developing suspension processes that are conducive to large-scale production. This study describes 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, the study demonstrated that LV production using clone 92 is scalable from 20 mL shake flasks to 3 L bioreactors. Next, two strategies were developed for high-yield LV production in perfusion mode using acoustic cell filter technology in 1-3 L 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 × 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 × 1010 TU/L of bioreactor culture. This approach is easily amenable to large-scale production and commercial manufacturing.

Keywords: HEK293SF; bioprocessing; lentiviral vector; perfusion; stable producer.

Conflict of interest statement

No competing financial interests exist

Figures

<b>Figure 1.</b>
Figure 1.
Schematic of the cumate-inducible system and stability of clone 92. (A) In the packaging cells and thus also clone 92, transcription of Rev and the envelope protein (VSV-G) is under the control of the tetracycline and cumate switches. Addition of doxycycline and cumate in the culture medium is required to induce the production of LV. Cumate prevents binding of the cumate repressor (CymR) to the cumate operator (CuO). Doxycycline promotes binding of the reverse tetracycline transactivator (rtTA2s-M2) to the tetracycline promoter (TR5). (B) To test the stability of clone 92, the cells were maintained in suspension culture without selective pressure for 10 weeks. After 2, 6, and 10 weeks of culture, a portion of the cells were induced at a concentration of 1.0 × 106 cells/mL by addition of doxycycline and cumate. The amount of LVs produced at 48 h post induction was analyzed by gene transfer assay (GTA) in triplicate (error bars show the standard deviation).
<b>Figure 2.</b>
Figure 2.
Results obtained in 3 L bioreactor runs operated in batch mode. (A) Viable cell count (black line) and viability (gray line) measured in four independent bioreactor runs (batches 1–4) operated in batch mode using SFM4TransFx293 medium. (B) Functional titer kinetics for four batch runs up to 3 days post induction. (C) Functional LV titers at 3 dpi for four independent batch productions and related shake flask controls. The internal control was obtained by sampling an aliquot from the bioreactor shortly after induction. This aliquot was then transferred into and kept in a shake flask during the LV production phase. The external control was performed in parallel in shake flask throughout the experiment during both the growth phase and LV production phase. (D) Total functional titer produced per 1 L batch culture for four batch runs.
<b>Figure 3.</b>
Figure 3.
Results obtained in shake flasks. (A) Viable cell count (black line) and viability (gray line) between −4 dpi and 3 dpi upon daily media replacement (DMR) at 0–100% in SFM4TransFx293 medium. Medium replacement took place on −3 dpi, −2 dpi, −1 dpi, and 0 dpi in duplicate shake flasks. (B) LV titer was measured by GTA at 3 dpi for samples in duplicate shown in panel (A) (error bars show the standard deviation). (C) Viable cell count and viability of cells in HyCell™ TransFx-H media supplemented with 3.5 g/L of Cell Boost 5™ Supplement (CB5; squares) or without CB5 (circles). For each LV producer cell suspension, DMR was performed at 50% with fresh media, either supplemented with CB5 or not (two independent shake flasks for both conditions) every 24 h following induction. (D) The functional titer (TU/mL) was assayed for each of the harvests shown in panel (C) for both conditions by GTA (error bars show the standard deviation).
<b>Figure 4.</b>
Figure 4.
Schematic showing the setup used in perfusion mode. Cells are transferred into the bioreactor vessel using the inoculation bottle at 0.25–0.35E06 cells/mL. Cell-free daily harvests and samplings are performed using the harvest bottle and the associated sampler under sterile conditions (cells are retained using an acoustic filter and are recirculated into the bioreactor while the supernatant is harvested). The sampler on the left-hand side is used to sample cells daily to measure cell density and viability. Medium is kept at 4°C and pumped into the vessel so that 0.5–1 volume of medium per reactor volume vessel is added over 24 h; the exact volume of media added is further monitored using a scale and a level detector. Oxygen (pO2), temperature, and pH are monitored with sterilizable probes. The pH is kept between 7.05 and 7.1 through the addition of CO2 via the surface or the addition of a base (NaHCO3). Peristaltic pumps are also shown on the schematic (reversed triangle in a circle).
<b>Figure 5.</b>
Figure 5.
Cell viability and titers measured in four independent perfusion cultures. (A) Viable cell counts (VCC) and (B) cell viability prior to and after induction of virus production at 1–3.5 L scale. (C) Functional LV titer (TU/mL) was assayed from each of the daily harvests (perfusion filtrate collected on ice) from the four independent runs using the GTA assay. (D) The cumulative titers were calculated for 1 L harvests for all the runs.

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