PMC

IA chromatography profile (step 4 in ). The entire bioreactor contents were applied to the preparative column. The top panel shows the full-scale UV (280 nm) absorbance profile of the flow-through and two elution peaks. Enlargements of both elution peaks are presented in the bottom panels. The particles began eluting at the onset of the pH change. The eluted fractions, 2.5 L from each load and elution, were pooled and concentrated into 0.4 L for subsequent processing. Color images available online at www.liebertonline.com/hum

Gel filtration chromatography (step 5b in ) was used to remove any material that coeluted from the IA chromatography step. Concentrated vector from 100-L vector production was divided into 4 × 0.1 L for gel filtration chromatography (Superdex 200). The UV (280 nm) (red curve) profile is a series of four, symmetrical, sharp peaks that contain the rAAV particles eluting in the void fractions. The four fractions were pooled for subsequent concentration and diafiltration steps. Color images available online at www.liebertonline.com/hum

Determining the dilution of cryopreserved BIIC required to achieve the optimal infection rate of the rAAV Sf9-producing cells. The infection rates of Sf9 cells were determined measuring the cell culture densities (A) and cell diameters (B) as a function of the dilution of BIIC added over time. In a typical experiment, the indicated dilutions of BIIC were added to Sf9 cells (1.5 × 10⁶ cells/ml in 20 ml). Cell density continued at similar rates at the two highest dilutions for 48 hr (A), and the 24-hr lag phase in cell diameter increase (B) occurred also at the two highest BIIC dilutions. Therefore, the criteria for establishing the optimal BIIC dilutions for rAAV production were based on using the least amounts of BIIC that produced sufficient baculovirus to arrest cell growth at ≤5 × 10⁶ cells/ml.

Physical vector characterization. (A) Following CsCl isopycnic centrifugation, gradients were fractionated and assayed for the presence of capsid protein and vector DNA. The graph represents the DNA content of each fraction (solid line), and the columns indicate the relative amount of capsid protein in each fraction. The signal from each fraction was divided by total VP3 signal on the western blot (lower panel). The abscissa denotes the density of each fraction in g/cm³. The ordinates represent the DNA content, as vg/ml (left axis) and relative VP3 amount in each fraction (right axis). The buoyant density of empty particles is approximately 1.32 g/cm³ and that of filled particles is 1.39 g/cm³. Based on the distribution of vector DNA and protein across the gradient, approximately 65% of the capsids contain vector DNA. (B–D) TEM images of rAAV particles. Negatively stained rAAV particles from unfractionated material (B) and high- (C) and low-buoyant (D) density fractions are consistent with the ratio of full and empty particles described in (A). In the unfractionated, undiluted starting material (C), >50% of the particles appear full. (E) Silver-stained polyacrylamide gel of rAAV proteins following IA chromatography and TFF concentration/diafiltration. An aliquot (0.5 μl) of each vector was fractionated by SDS-PAGE, and proteins were visualized by silver staining. The rAAV6-AlkPhos production yielded 1.94 × 10¹⁶ vg from approximately 200 L. The samples and concentrations are listed. Lane 1, molecular mass standards; lane 2, rAAV6-U7smOPT (9 × 10¹³ vg/ml); lane 3, rAAV6-U7smOPT (5.77 × 10¹³ vg/ml); lane 4, rAAV6-AlkPhos (5.18 × 10¹³ vg/ml); lane 5, rAAV6-U7smOPT (7 × 10¹³ vg/ml); lane 6, rAAV6-U7smOPT (9 × 10¹³ vg/ml); lane 7, rAAV6-GFP (6.7 × 10¹³ vg/ml).

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

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