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Scalable Lentiviral Vector Production Using Perfusion technology


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 10
6 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 (500mL bioreactor vs 1L minibiosep, 3.5L bioreactor vs 10L Biosep ,Applikon ). The first approach uses a basal commercial medium and perfusion mode both pre- and postinduction 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 largescale production and commercial manufacturing.
 

Manceur AP, Kim H, Misic V, et,al.Scalable Lentiviral Vector Production Using Stable HEK293SF Producer Cell Lines.Hum Gene Ther Methods. 2017 Dec;28(6):330-339. doi: 10.1089/hgtb.2017.086. Epub 2017 Nov 21.



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