There are two components to the TIPS process: the first part involves determining the optimal time point for harvesting the BIIC, and the second part determines the ratios of BIIC to Sf9 cells for producing rAAV. The time point for harvesting the BIIC was developed using small-scale, shake flasks (0.02
L). Initially, low-passage Sf9 cell cultures were infected with Bac-GFP [containing the green fluorescent protein (GFP) transcription cassette regulated with the AcMNPV p10 late promoter (Urabe et al., 2002
)], and a time course of infection was established by measuring cell viability, diameter, and density, and green fluorescence. Uninfected cells are approximately 14
μm in diameter; within 24
hr post infection, cell diameter increased to 17.4
μm, and at 48
hr post infection, the diameter increased to 19
μm, with cell viability remaining at 90%. Cells were harvested at 48
hr post infection, because the cell viability diminished beyond 48
The yield of rAAV produced in Sf9 cells depends on the cell density at the time of infection (Negrete et al., 2007b
). In cultures with cell densities up to about 5
cells/ml, the rAAV produced per cell is independent of density; the medium can support the cell metabolism and the rAAV produced per cell is unaffected. Beyond 5
cells/ml, the specific yield of rAAV diminishes and the increased biomass compromises the downstream processing. Therefore, the ratios of BIIC to Sf9 cells were determined over a 4-log range of BIIC:producer cell ratios, i.e
, and 10–5
. Cells were monitored for 3 days post-BIIC inoculation for cell density, viability, and diameter.
The effect of baculovirus infection on cell growth was evaluated by measuring cell density after inoculation with BIIC (). The density of cells in cultures inoculated with the lowest BIIC dilution did not double (10–2
), and at the highest dilution (10–5
) the cells did not arrest. However, cultures inoculated with BIIC dilutions of either 10–3
continued to grow for 72
hr. Cell-diameter increase correlates with baculovirus infection, thereby providing another measure of the infection rate. The lag time in cell-diameter increase occurred with the two highest BIIC dilutions, indicating that the infection () was delayed, whereas the cell density continued to increase geometrically (). By 72
hr post inoculation, the average cell diameter had reached similar values regardless of the amount of BIIC added. Based on these results, 10–4
BIIC:Sf9 producer cell ratio caused cell-cycle arrest at the optimum cell density and was used for the large-scale productions.
FIG. 1. 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 (more ...)
TIPS for larger volume rAAV production
Based on the findings described above, rAAV was produced in larger volume cultures using BIIC. A series of production runs in volumes ranging from 10 to approximately 200
L was performed (summarized in ). Initially, rAAV was produced in the rocking platform bioreactor in a set of five runs: two 10-L, two 20-L, and one 27-L culture volume. Changes in cell viability and cell diameter were relatively uniform among the five production runs. The average yield of purified rAAV for the rocking platform productions was 15,400 (±7,050) vg/cell, with specific yields ranging between 7.3
rAAV particles/cell. The one relatively poor production resulted from the addition of suboptimal amounts of BIIC.
Based on the conditions established in the small- and mid-scale production runs, the process was transferred to100-L and 200-L stirred-tank, single-use bioreactors. The procedure for 100-L and 200-L single-use bioreactors was similar, starting with the minimum working volume of medium for both bioreactors, which coincidentally was 40
L. After temperature and dO were equilibrated, 3
Sf9 cells were added to achieve the initial cell density of 7.5
/ml. The cells were expanded by repetitive twofold dilutions with fresh medium while maintaining the density between 1 and 2
cells/ml. Once the final working volume was reached and the cell density reached 1.6 to 1.8
cells/ml, the BIIC aliquots were thawed and added to the culture (t
0). The cells continued to grow and cell density to increase. As the thawed BIIC revived, the two baculoviruses were released into the medium. Measuring the increase in cell diameter indicates the infection rate. shows typical culture values for the 200-L production. The cell growth rate was logarithmic during the expansion and continued until approximately t
hr post infection, when the baculovirus infection caused cell-cycle arrest, indicating that the majority of the cells were infected with at least one baculovirus. Shortly thereafter, the slope of the cell viability curve became negative. The cell viability at harvest depended on the vector: Sf9 cells producing rAAV-cU7 remained at higher viability levels than cells producing rAAV-GFP vectors. This difference was serotype-independent and may be due to the expressed transgene affecting baculovirus–cell interactions. Capsid proteins first appeared about 36
hr post infection and continued increasing until harvesting at 134
hr post infection (). The rAAV produced reflects the cell viability and cell density; however, specific production, i.e
., the rAAV yield per cell, leveled off after 120
hr post infection ().
Downstream processing and production results
The downstream process flow diagram is represented schematically in . Single-use components were used for most of the processing steps, except for the homogenizer and preparative chromatography systems, which are reusable and require cleaning and sanitization between productions. Following homogenization (, step 1), the mixture of cell lysate and medium was returned to the bioreactor for nuclease treatment (, step 2). Three filter membranes were used for clarification: a 1.2-μm depth filter, then a two-stage 0.8-μm/0.2-μm capsule filter (, step 3). The vector recovery after each downstream processing step was analyzed by PAGE and western blots (). The major vector loss occurs during the first filtration step (, compare steps 2 and 3a). Approximately 55% of the vector capsid is retained, i.e., lost, during the initial clarification step. Although the vector recovered after each subsequent steps was more efficient (), the cumulative recovery of steps 4, 5, and 6 was about 50%.
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, (more ...)
IA chromatography produced the greatest-fold purification in the downstream process (, step 4). By using a chromatography skid specifically configured for this process, the entire bioreactor contents were pumped through a 20-cm-diameter by 6–7-cm-bed-height preparative column at 0.4
L/min. The UV (280
nm) absorbance, pH, and conductivity were monitored and recorded. Due to the relatively small column dimensions, approximately 100
L of treated culture were applied per bind–wash–elute cycle. After each cycle, the column was washed with PBS until the UV absorbance returned to baseline values, and then the rAAV was eluted using 0.05 M
citrate (pH 3.0). The eluted vector appears as spikes in the full-scale chromatograph (, top panel) and as similar symmetric peaks in the enlarged chromatograph (, bottom panels).
FIG. 4. IA chromatography profile (step 4 in ). The entire bioreactor contents were applied to the preparative column. The top panel shows the full-scale UV (280nm) absorbance profile of the flow-through and two elution peaks. Enlargements of both (more ...)
A polishing step, when necessary, involved gel filtration using column chromatography with Superdex 200 (, step 5b). Obtaining high-resolution fractionation limited the volume of vector solution applied to the column. Therefore, processing the entire quantity of vector required dividing the 0.4
L of concentrated vector solution into fourths and repeating the chromatography procedure for each aliquot. The chromatography profile () demonstrates the reproducibility of this final purification step, and the vector DNA analysis demonstrated near-quantitative recovery from the gel filtration process (data not shown).
The solubility and recovery of rAAV were improved by a series of treatments: adding a surfactant did not improve vector recovery, whereas treating the lysed culture with nuclease and then adding NaCl (final concentration, 0.4 M) resulted in minor improvements in rAAV solubility (data not shown).
shows the results of 10 large-scale rAAV production runs using BIIC at 10
L, and 200
L. The average rAAV yield per cell for all scales was 1.76
) vg particles/cell. Vectors produced using rocking platform cultures averaged 1.54
) vg particles/cell, and in stirred tank 100- and 200-L bioreactors, the yields were 1.98
) vg particles/cell. Strikingly consistent production values were obtained in all formats and volumes, with the most reliable results obtained in the two 200-L productions yielding final purified rAAV of 1.89
(±282) vg particles/cell for a total of 3.22
. Overall, 7.15
vg rAAV particles were obtained from 880
L of total culture volume (). The efficiency of vector recovery indicated that approximately 20% of the rAAV in the biomass was recovered in the final purified product.
The protein components of the vector particles were analyzed using PAGE and western blots. The DNA content was assessed quantitatively using two techniques: qPCR and fluorescent dye binding to extracted virion DNA. Both methods provided similar results. The ratio of empty to filled capsid particles was estimated using cesium chloride isopycnic gradients. Following centrifugation, the gradients were fractionated and aliquots were analyzed for the presence of capsid proteins (PAGE/western blots) and for DNA (qPCR) (). The signal from the VP3 western blot in each fraction was divided by the sum of VP3 signal in all fractions (, lower panel). Capsid proteins were detected in two regions of the gradient: fractions with densities of 1.41, 1.39, and 1.37
and two fractions with densities of 1.33 and 1.32
. The capsids in the three denser fractions represented 65% of the total capsids in the gradient () and, according to qPCR analysis, contain the vector genome. The morphology and empty/filled composition of the capsids were examined by negative staining and electron microscopy (EM). The EM image of unfractionated vector showed densely populated fields in the grid with apparently mostly filled particles, i.e
., excluding the molybdate staining solution (), whereas the EM images of particles in the 1.39
() and 1.32
() fractions showed filled particles and empty particles, respectively. Therefore, two independent analytical methods indicate that the majority of the capsids are filled. The products of seven different rAAV production runs are shown in by silver-stained PAGE. An aliquot (0.5
μl) of the vector following IA chromatography and concentration and diafiltration using tangential flow filtration was processed.
FIG. 6. 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 (more ...)