FIG. 3.

Down-stream processing (DSP). (A) Process flow diagram. The upstream production component, A, represents cell growth, expansion, and infection. Subsequent steps are numbered 1 to 7. The details are described in the text. Except for the volumes where indicated, the steps used for processing 200 L are derived from smaller-scale cultures with adjustments made for filter area or capsule filter lengths. TFF, tangential flow filtration. Gel exclusion chromatography (step 5b) was an optional step depending on vector administration. Step 6, the final TTF concentration/diafiltration procedure, was performed prior to the final sterile filtration and vialing step (step 7). (B) Vector recovery following each DSP step. The amount of capsid antigen was determined by western blot at each sampling point analyzed quantitatively to determine efficiency of recovery during the DSP. The capsid proteins in the starting material (step A) represent 100% of the rAAV. Line 1: “step” refers to the process in the DSP corresponding to the diagram in (A). Line 2: “%” represents the percentage of vector particle recovered determined by using volume-adjusted samples to accommodate concentration or dilution steps in the process. Lines 3 and 4: “Volume” indicates either the process volume at each step (in liters) or the amount of the process volume applied to the PAGE for the western blot analysis (in microliters), respectively. Steps 1 through 3 were performed on the entire bioreactor volume and required no adjustments for dilution or concentration. The capsid level apparently decreased by 15% during the homogenization and nuclease treatment steps, but the single largest change occurred during the first filtration step (step 3a). The second capsule filtration resulted in lower vector loss (step 3b). IA chromatography caused relatively minor vector loss, approximately 8% overall. The IA eluted fraction was concentrated and diafiltrated using TFF without vector loss (step 6). The last step (step 7) represents the final sterile filtration and vialing of the product. (C) Histogram of rAAV recovered following each DSP step. The graph represents the values obtained from the western blot analysis described in (B). Color images available online at www.liebertonline.com/hum