492. Harvest Timing and Media Composition Effects on Lentiviral Vector Production

Abstract

The Indiana University Vector Production Facility (IUVPF), in conjunction with the National Gene Vector Laboratories, has been developing the capability to produce and process a volume of lentiviral vector sufficient for phase I/II clinical trials. To date, the facility has been able to produce vector in Nunc Cell Factories in unprocessed volumes from 120mL to 20L in a single production run. In the course of developing this capability, we have looked at many production parameters and here report on the effects of harvest timing and media composition. The general production scheme had cells seeded into appropriate tissue culture flasks on day 1, cells were transfected by CaPO4 on day 2, DMEM+10% FBS medium (D10) was changed to harvest medium on day 3 followed by harvest of vector supernatant at indicated time points thereafter. Results are primarily reported as relative comparisons within each experiment. We have used p24 capsid protein ELISA as a physical method of estimating titer. For production using transgene vectors containing the GFP gene, the p24 data was supplemented with GFP expression titers determined by limiting dilution of vector on 293 cells. Harvest timings of 12, 24, 48 and 72 and multiple harvests at 12 and 24 hours after the post transfection media change had a range of GFP expression titers from 1.2|[times]|105 to 2.7|[times]|107 TU/mL. The initial 12 hour harvest had the highest titer, 2.7|[times]|107, but multiple harvests at 12 hour intervals resulted in a rapid drop in titer. The 24 hour harvests were found to be relatively consistent, usually only differing by a factor of 2.