Scalable lentiviral vector production using stable HEK293SF producer cell lines

From National Research Council Canada

Download	View accepted manuscript: Scalable lentiviral vector production using stable HEK293SF producer cell lines (PDF, 1 MB)
DOI	Resolve DOI: https://doi.org/10.1089/hgtb.2017.086
Author	Search for: Manceur, Aziza P.¹; Search for: Kim, Howard; Search for: Misic, Vanja; Search for: Andreev, Nadejda; Search for: Dorion-Thibaudeau, July¹; Search for: Lanthier, Stéphan¹; Search for: Bernier, Alice¹; Search for: Tremblay, Sonia¹; Search for: Gélinas, Anne-Marie¹; Search for: Broussau, Sophie¹; Search for: Gilbert, Rénald¹; Search for: Ansorge, Sven¹
Name affiliation	National Research Council of Canada. Human Health Therapeutics
Format	Text, Article
Journal title	Human Gene Therapy Methods
ISSN	1946-6536 1946-6544
Subject	lentiviral vector; stable producer; HEK293SF; perfusion; bioprocessing
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 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 10<sup>6</sup> 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 10<sup>7</sup> 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 10<sup>10</sup> TU/L of bioreactor culture. This approach is easily amenable to large-scale production and commercial manufacturing.
Publication date	2017-08-21
Publisher	Mary Ann Liebert, Inc.
Language	English
Peer reviewed	Yes
NPARC number	23002127
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Record identifier	a9ae2bd6-61d3-4eaf-bc53-d0f9ce7c86e2
Record created	2017-08-24
Record modified	2020-06-04

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Date modified:: 2020-09-28