317. Simplified Concentration and Purification of Lentivirus Pseudotypes
Authors: Robert H. Kutner, Brian DaLeo, Jakob Reiser
Journal: Molecular Therapy (2006)

Lentivirus (LV)-based gene delivery vectors have several advantages such as being able to incorporate other viral glycoproteins into their membrane, but a drawback exists with a lack of standard scaleable techniques to generate purified and concentrated virus stocks. A number of protocols are available to concentrate and purify lentiviral vector pseudotypes on a small scale including centrifugation approaches, precipitation methods as well as ultrafiltration, tangential flow filtration and chromatography-based procedures. Many of these methods are time-consuming and difficult to scale up while others are dependent on sophisticated equipment. We have established a facile purification protocol based on anion exchange membrane chromatography for LV pseudotypes bearing the VSV-G, amphotropic Moloney murine leukemia virus or lyssavirus-derived glycoproteins produced by transient transfection using second or third generation LV packaging systems or stable packaging cell lines. In this protocol, virus-containing cell culture supernatants are captured onto chromatography membranes bearing functional quarternary (Q) ammonium groups, eluted with high salt, then desalted, and further concentrated. Our results show that these purification procedures are useful for concentrating LV pseudotypes with a minimal loss of infectivity using syringe-adaptable ion exchange membrane cartridges. We expect these approaches to facilitate the preparation of lentiviral pseudotypes for routine laboratory-scale operations and for large-scale preclinical and clinical protocols. Lentivirus (LV)-based gene delivery vectors have several advantages such as being able to incorporate other viral glycoproteins into their membrane, but a drawback exists with a lack of standard scaleable techniques to generate purified and concentrated virus stocks. A number of protocols are available to concentrate and purify lentiviral vector pseudotypes on a small scale including centrifugation approaches, precipitation methods as well as ultrafiltration, tangential flow filtration and chromatography-based procedures. Many of these methods are time-consuming and difficult to scale up while others are dependent on sophisticated equipment. We have established a facile purification protocol based on anion exchange membrane chromatography for LV pseudotypes bearing the VSV-G, amphotropic Moloney murine leukemia virus or lyssavirus-derived glycoproteins produced by transient transfection using second or third generation LV packaging systems or stable packaging cell lines. In this protocol, virus-containing cell culture supernatants are captured onto chromatography membranes bearing functional quarternary (Q) ammonium groups, eluted with high salt, then desalted, and further concentrated. Our results show that these purification procedures are useful for concentrating LV pseudotypes with a minimal loss of infectivity using syringe-adaptable ion exchange membrane cartridges. We expect these approaches to facilitate the preparation of lentiviral pseudotypes for routine laboratory-scale operations and for large-scale preclinical and clinical protocols.