Production of lentiviral vectors by large-scale transient transfection of suspension cultures and affinity chromatography purification.
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 scaled-up from shake flasks to a 3-L bioreactor from which 10(10) 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.
Segura
María Mercedes
MM
Biotechnology Research Institute, NRC, 6100 Royalmount Avenue, Montreal, Quebec, Canada.
Garnier
Alain
A
Durocher
Yves
Y
Coelho
Helene
H
Kamen
Amine
A
D018929:Cell Culture Techniques / Q000379:methods*; D002460:Cell Line; D002846:Chromatography, Affinity / Q000379:methods; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D007668:Kidney / Q000821:virology*; D016086:Lentivirus / Q000235:genetics* / Q000254:growth & development* / Q000302:isolation & purification; D014162:Transfection / Q000379:methods*; D014776:Virus Cultivation / Q000379:methods*