Premium
Development of a scalable process for high‐yield lentiviral vector production by transient transfection of HEK293 suspension cultures
Author(s) -
Ansorge Sven,
Lanthier Stéphane,
Transfiguracion Julia,
Durocher Yves,
Henry Olivier,
Kamen Amine
Publication year - 2009
Publication title -
the journal of gene medicine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.689
H-Index - 91
eISSN - 1521-2254
pISSN - 1099-498X
DOI - 10.1002/jgm.1370
Subject(s) - transfection , hek 293 cells , transient (computer programming) , yield (engineering) , suspension (topology) , process (computing) , scalability , viral vector , production (economics) , cell culture , computer science , microbiology and biotechnology , chemistry , materials science , biology , genetics , mathematics , recombinant dna , biochemistry , gene , operating system , macroeconomics , homotopy , economics , pure mathematics , metallurgy
Background Lentiviral vectors (LV) offer several advantages over other gene delivery vectors. Their potential for the integration and long‐term expression of therapeutic genes renders them an interesting tool for gene and cell therapy interventions. However, large‐scale LV production remains an important challenge for the translation of LV‐based therapeutic strategies to the clinic. The development of robust processes for mass production of LV is needed. Methods A suspension‐grown HEK293 cell line was exploited for the production of green fluorescent protein‐expressing LV by transient polyethylenimine (PEI)‐based transfection with LV‐encoding plasmid constructs. Using third‐generation packaging plasmids ( Gag/Pol, Rev ), a vesicular stomatitis virus G envelope and a self‐inactivating transfer vector, we employed strategies to increase volumetric and specific productivity. Functional LV titers were determined using a flow cytometry‐based gene transfer assay. Results A combination of the most promising conditions (increase in cell density, medium selection, reduction of PEI–DNA complexes per cell, addition of sodium butyrate) resulted in significantly increased LV titers of more than 150‐fold compared to non‐optimized small‐scale conditions, reaching infectious titers of approximately 10 8 transducing units/ml. These conditions are readily scalable and were validated in 3‐liter scale perfusion cultures. Conclusions Our process produces LV in suspension cultures and is consequently easily scalable, industrially viable and generated more than 10 11 total functional LV particles in a single bioreactor run. This process will allow the production of LV by transient transfection in sufficiently large quantities for phase I clinical trials at the 10–20‐liter bioreactor scale. Copyright © 2009 John Wiley & Sons, Ltd.