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

Cite this

@article{72a24978bd004743a36b169466e575b6,
title = "Downstream Processing of Lentiviral Vectors: Releasing Bottlenecks",
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.",
keywords = "CLINICAL-APPLICATION, MEMBRANE, CELL, RETROVIRAL VECTORS, CHROMATOGRAPHY, FILTRATION, PURIFICATION, STABILITY, GENE-THERAPY VECTORS, ANION-EXCHANGE",
author = "Carrondo, {Manuel Jos{\'e} Teixeira} and Cristina Peixoto and Alves, {Paula Maria} and Coroadinha, {Ana Sofia}",
year = "2012",
month = "1",
day = "1",
doi = "10.1089/hgtb.2012.059",
language = "Unknown",
volume = "23",
pages = "255--263",
journal = "Human Gene Therapy Methods",
issn = "1946-6536",
publisher = "Mary Ann Liebert Inc.",
number = "4",

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TY - JOUR

T1 - Downstream Processing of Lentiviral Vectors: Releasing Bottlenecks

AU - Carrondo, Manuel José Teixeira

AU - Peixoto, Cristina

AU - Alves, Paula Maria

AU - Coroadinha, Ana Sofia

PY - 2012/1/1

Y1 - 2012/1/1

N2 - 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.

AB - 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.

KW - CLINICAL-APPLICATION

KW - MEMBRANE

KW - CELL

KW - RETROVIRAL VECTORS

KW - CHROMATOGRAPHY

KW - FILTRATION

KW - PURIFICATION

KW - STABILITY

KW - GENE-THERAPY VECTORS

KW - ANION-EXCHANGE

U2 - 10.1089/hgtb.2012.059

DO - 10.1089/hgtb.2012.059

M3 - Article

VL - 23

SP - 255

EP - 263

JO - Human Gene Therapy Methods

JF - Human Gene Therapy Methods

SN - 1946-6536

IS - 4

ER -