title: Downstream processing of lentiviral vectors: releasing bottlenecks.
abstract: Lentiviral vectors (LVs) hold great potential as gene delivery vehicles. However, the manufacturing and purification of these vectors still present major challenges, mainly because of the low stability of the virus, essentially due to the fragility of the membrane envelope. The main goal of this work was the establishment of a fast, scalable, and robust downstream protocol for LVs, combining microfiltration, anion-exchange, and ultrafiltration membrane technologies toward maximization of infectious LVs recovery. CIM(®) (Convective Interaction Media) monolithic columns 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 in the literature. These recoveries, combined with the results obtained after optimization of the remaining downstream purification steps, resulted in overall infectious LV yields of 36%. Moreover, the inclusion of a Benzonase step allowed a removal of approximately 99% of DNA impurities. The entire downstream processing strategy herein described was conceived based on disposable and easily scalable technologies. Overall, CIM DEAE columns have shown to be a good alternative for the purification of LVs, since they allow faster processing of the viral bulks and enhanced preservation of virus biological activity, consequently, increasing infectious vector recoveries.
authors.affiliation: Instituto de Biologia Experimental e Tecnológica (IBET), 2781-901 Oeiras, Portugal.
authors.forename: Vanessa
authors.initials: V
authors.lastname: Bandeira
authors.forename: Cristina
authors.initials: C
authors.lastname: Peixoto
authors.forename: Ana F
authors.initials: AF
authors.lastname: Rodrigues
authors.forename: Pedro E
authors.initials: PE
authors.lastname: Cruz
authors.forename: Paula M
authors.initials: PM
authors.lastname: Alves
authors.forename: Ana S
authors.initials: AS
authors.lastname: Coroadinha
authors.forename: Manuel J T
authors.initials: MJ
authors.lastname: Carrondo
mesh_terms: D002498:Centrifugation; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004706:Endodeoxyribonucleases / Q000378:metabolism; D004722:Endoribonucleases / Q000378:metabolism; D005822:Genetic Vectors / Q000302:isolation & purification* / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D016086:Lentivirus / Q000235:genetics*
chemical_list: D004706:Endodeoxyribonucleases; D004722:Endoribonucleases; C021835:Serratia marcescens nuclease