1115. A Simple Method for the Purification of Lentiviral Vectors Using a Combination of Ultrafiltration and Anion Exchange Chromatography
Authors: Rachel M. Koldej, Donald S. Anson
Journal: Molecular Therapy (2006)

Ultrafiltration and anion exchange chromatography have both been proposed as useful scalable purification techniques for the production of lentiviral vectors. However, few attempts have been made to develop and optimize integrated processing protocols. Here we present a simple and rapid method for the purification of lentiviruses that combines ultrafiltration and anion exchange chromatography. Virus supernatant is clarified by 0.45 μm filtration and then concentrated and purified via ultrafiltration using a 750 kDa membrane. Approximately 50% of infectious virus is recovered during this step. We are currently attempting to improve recovery by the addition of various agents to the virus. The concentrated virus is then applied to an anion exchange column. Initial investigations examined the suitability of four different resins and showed that Sepharose Q FF resin (GE Healthcare) efficiently bound the virus with average recoveries of 84% of infectious titre. The Q FF column was then incorporated into an FPLC system to allow a more detailed analysis of loading capacity and elution properties. The capacity of the 1 mL column was determined to be approximately 10 mLs of virus (1.15 × 108 NIH3T3 IU, 47 ug p24) with minimal virus in the flow thru. Virus was eluted with a gradient of 0 to 2M NaCl resulting in two distinct virus peaks.The first virus peak contains large amounts of protein resulting in a lower IU/mg of protein ratio than the virus initially applied to the column. SDS-PAGE of this peak suggests that the major contaminating protein is BSA. Currently, we are attempting todetermine if the virus that elutes in this first peak does so because it associates with the BSA or because it is fundamentally different to the virus in the second peak.In contrast, the second virus peak exhibits very high purity with a 10 fold increase in the IU/mg of protein and a 2 fold increase in specific infectivity when compared to the virus initially applied to the column. To simplify the purification process, a step elution protocol that allows the collection of the viral peaks with minimal protein contamination is now being developed and tested. Ultrafiltration and anion exchange chromatography have both been proposed as useful scalable purification techniques for the production of lentiviral vectors. However, few attempts have been made to develop and optimize integrated processing protocols. Here we present a simple and rapid method for the purification of lentiviruses that combines ultrafiltration and anion exchange chromatography. Virus supernatant is clarified by 0.45 μm filtration and then concentrated and purified via ultrafiltration using a 750 kDa membrane. Approximately 50% of infectious virus is recovered during this step. We are currently attempting to improve recovery by the addition of various agents to the virus. The concentrated virus is then applied to an anion exchange column. Initial investigations examined the suitability of four different resins and showed that Sepharose Q FF resin (GE Healthcare) efficiently bound the virus with average recoveries of 84% of infectious titre. The Q FF column was then incorporated into an FPLC system to allow a more detailed analysis of loading capacity and elution properties. The capacity of the 1 mL column was determined to be approximately 10 mLs of virus (1.15 × 108 NIH3T3 IU, 47 ug p24) with minimal virus in the flow thru. Virus was eluted with a gradient of 0 to 2M NaCl resulting in two distinct virus peaks. The first virus peak contains large amounts of protein resulting in a lower IU/mg of protein ratio than the virus initially applied to the column. SDS-PAGE of this peak suggests that the major contaminating protein is BSA. Currently, we are attempting todetermine if the virus that elutes in this first peak does so because it associates with the BSA or because it is fundamentally different to the virus in the second peak. In contrast, the second virus peak exhibits very high purity with a 10 fold increase in the IU/mg of protein and a 2 fold increase in specific infectivity when compared to the virus initially applied to the column. To simplify the purification process, a step elution protocol that allows the collection of the viral peaks with minimal protein contamination is now being developed and tested.