Downstream Processing of Lentiviral Vectors: Releasing Bottlenecks

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Abstract

AbstractLentiviral vectors (LVs) hold great potential as gene delivery vehicles. However, the manufacturing and purificationof these vectors still present major challenges, mainly because of the low stability of the virus, essentially dueto the fragility of the membrane envelope. The main goal of this work was the establishment of a fast, scalable, androbust downstream protocol for LVs, combining microfiltration, anion-exchange, and ultrafiltration membranetechnologies toward maximization of infectious LVs recovery. CIM (Convective Interaction Media) monolithiccolumns with diethylaminoethanol (DEAE) anion exchangers were used for the purification of clarified LV supernatants,allowing infectious vector recoveries of 80%, which is 10% higher than the values currently reported inthe literature. These recoveries, combined with the results obtained after optimization of the remaining downstreampurification steps, resulted in overall infectious LV yields of 36%. Moreover, the inclusion of a Benzonasestep allowed a removal of approximately 99% of DNA impurities. The entire downstream processing strategyherein described was conceived based on disposable and easily scalable technologies. Overall, CIM DEAE columnshave shown to be a good alternative for the purification of LVs, since they allow faster processing of the viral bulksand enhanced preservation of virus biological activity, consequently, increasing infectious vector recoveries.
Original languageUnknown
Pages (from-to)255-263
JournalHuman Gene Therapy Methods
Volume23
Issue number4
DOIs
Publication statusPublished - 1 Jan 2012

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