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Production of lentiviral vectors by large-scale transient transfection of suspension cultures and affinity chromatography purification

From National Research Council Canada

DOIResolve DOI: https://doi.org/10.1002/bit.21467
AuthorSearch for: 1; Search for: 1; Search for: 1; Search for: ; Search for: 1
Affiliation
  1. National Research Council of Canada. NRC Biotechnology Research Institute
FormatText, Article
Subjectbiotechnology; biotechnology; cells; chromatography; heparin; lentivirus; plasmids; transfection; gene therapy; lentiviral vectors; large-scale production; serum-free; suspension cells; 293 EBNA-1; purification; heparin affinity chromatography
Abstract
The use of lentiviral vectors as gene delivery vehicles has become increasingly popular in recent years. The growing interest in these vectors has created a strong demand for large volumes of vector stocks, which entails the need for scaleable vector manufacturing procedures. In this work, we present a simple and robust process for the production of lentiviral vectors using scaleable production and purification methodologies. Lentivirus particles were produced by transient transfection of serum-free suspension- growing 293 EBNA-1 cells with four plasmids encoding the vector components using linear polyethylenimine (PEI) as transfection reagent. This process was successfully scaledup from shake flaks to a 3-L bioreactor from which 10¹º IVP were recovered. In addition, an affinity chromatography protocol designed for purification of bioactive oncoretroviral vectors has been adapted in this work for the purification of VSV-G pseudotyped lentiviral vectors. Using heparin affinity chromatography, lentiviral particles were concentrated and purified directly from the clarified supernatants. During this step, a recovery of 53% of infective lentiviral particles was achieved while removing 94% of the impurities contained in the supernatant.
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In
  • Biotechnology and Bioengineering 98, no. 4 (1 November 2007): 789–799.
NoteDA - 20070427IS - 0006-3592 (Print)LA - ENGPT - JOURNAL ARTICLE
LanguageEnglish
NRC number47804
NRCC 47804
NPARC number3539335
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Record created2009-03-01
Record modified2020-05-10
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