FIG. 2.
Cell culture growth and rAAV capsid production in 200-L single-use, stirred-tank bioreactors. (A) The minimum working volume (40 L) of serum-free medium was added to the bioreactor and inoculated with 3 × 1010 Sf9 cells. During exponential growth, the cell density doubled from 0.75 × 106/ml to 1.5 × 106/ml in approximately 15 hr (green line and green triangles). Additional medium was added to a total final volume of 200 L. Once the cell density reached 1. 5 × 106/ml, aliquots of BIIC (10 ml of each BIIC) were thawed and added to the bioreactor. Exponential cell growth continued for approximately 74 hr post infection. During the log-phase growth period, cell viability remained >90% (purple line and purple squares). Beyond 74 hr post infection, the cell viability and total cell density declined with a corresponding decrease in oxygen consumption (lower green curve). (B) Western blot detection of the AAV6 capsid proteins. Samples were removed from the bioreactor at the indicated times post infection, and aliquots were fractionated by SDS-PAGE and transferred to PVDF membranes for western blot analysis. The arrow indicates the position of VP3, the most abundant capsid protein, and the first measurable AAV6 capsid proteins were detected in the 48-hr postinfection sample. The values on the bottom represent the relative capsid production normalized to the highest enhanced chemiluminescence signal on the western blot (134 hr post infection). (C) Time course of specific rAAV production. The relative specific production of rAAV was calculated by western blot analysis for capsid amount (B) and viable cell number determined at the specified time points. The value obtained by dividing the normalized amount of capsid protein by viable cell number generated the normalized capsid/cell number values. The x-axis indicates hours after BIIC addition. The left y-axis is a relative scale, 0 to 100, related to either the amount of capsid protein present in the bioreactor or the amount of capsid protein produced per viable cell. The right y-axis indicates the viable cells per milliliter, in millions. Color images available online at www.liebertonline.com/hum