FIGURE LEGENDS
Figure 1. Production of AAV6 via triple transient transfection at low cell densities. Cells were transfected at 1 or 2 million cells per mL, and plasmid DNA was delivered on a volumetric basis (VB - 1 µg/mL) or cell basis (CB - 1 µg/106 cells). A) Cell growth kinetics and viability. Black arrows indicate the time of transfection, and dashed arrows indicate the time of viral harvest. B) Transfection efficiency is described as the percentage of cells expressing the transgene (GFP) 24 hours post-transfection. C) Volumetric (VG/mL) and cell-specific viral yields (VG/cell). Ctrl: transfection at 1 × 106 cells/mL; 2M: at 2 × 106cells/mL. Values represent mean ± standard deviation (n=3). ** P< 0.01, * P < 0.05, by ANOVA followed by Dunnett’s test.
Figure 2. Production of AAV6 via triple transient transfection at medium cell densities. Cells were transfected at low cell density (LCD – 1 × 106 cells/mL) as control or at medium cell density (MCD – 4 × 106 cells/mL). Plasmid DNA was delivered on a volumetric basis (VB - 1 µg/mL) or cell basis (CB - 1 µg/106 cells). A) Cell growth kinetics and viability. B) Volumetric (VG/mL) and cell-specific viral yields (VG/cell). Time expressed in hours post-transfection (hpt). C) Transfection efficiency is described as the percentage of cells expressing the transgene (GFP) 24 hpt D) Functional titer (enhanced transducing units (ETU)/mL). E) Ratio between the genomic titer and functional titer (VG/ETU) 48 hpt. Values represent mean ± standard deviation (n=2). **** P < 0.0001, *** P < 0.001, by ANOVA followed by Dunnett’s test and Student’s T-test.
Figure 3. Production of AAV6 via triple transient transfection at high cell density. A) transfection efficiency is described as the percentage of cells expressing the transgene (GFP) 24 hours post-transfection. B) Cell growth kinetics (●) and volumetric viral yield (□). Cells were transfected at low cell density (LCD – 1 × 106 cells/mL) as control or at high cell density (HCD – 10 × 106 cells/mL). Plasmid DNA was delivered on a cell basis (1 µg/106 cells). Values represent mean ± standard deviation (n=3). *** P < 0.001, by Student’s T test.
Figure 4. Metabolic profile of production of AAV6 via triple transient transfection at high cell density. Cells were transfected at low cell density (LCD – 1 × 106 cells/mL) as control or at high cell density (HCD – 10 × 106 cells/mL). Plasmid DNA was delivered on a cell basis (1 µg/106 cells). A) Nutrient and metabolite concentration for production at low and high cell densities using basal or supplemented medium (7.2 g/L final glucose concentration or 15% Cell Boost 5). The dashed line represents the time of transfection. B) Concentration of glucose for productions at low and high cell densities. Time expressed as hours post-transfection (hpt). Values represent mean ± standard deviation (n=3).
Figure 5. Effect of medium supplementation on the production of AAV6. HyCell TransFx-H (basal medium) was supplemented with glucose (7.2 g/L final concentration) or with 15% Cell Boost 5. A) Cell growth kinetics and viability. Black arrows indicate the time of transfection. B) Transfection efficiency is described as the percentage of cells expressing the transgene (GFP) 24 hours post-transfection (hpt). C) Volumetric (VG/mL) and cell-specific viral yields (VG/cell) of low cell-density control and high cell-density productions with basal and supplemented media. Values represent mean ± standard deviation (n=3).
Figure 6. Production of AAV6 at different cell densities. A) Functional titer (ETU/mL) and cell-specific functional titer (ETU/mL) at 72 hpt. B) Ratio between volumetric and functional titer at 72 hpt. LCD: low cell density control; HCD: high cell density. Values represent mean ± standard deviation (n=3). **P < 0.01, * P < 0.05, by ANOVA followed by Dunnett’s test.
Figure 7. Bioreactor production of AAV6 at medium cell density. Cells were transfected at 4 million cells per mL (medium cell density, MCD), and plasmid DNA was delivered on a cell basis (1 µg/106cells). Low-cell-density (LCD, 1 × 106 cell/mL) bioreactor productions shown as control. Time indicated as hours post-transfection (hpt). A) Bioreactor control chart. B) Cell growth kinetics and viability. C) Volumetric titer (VG/mL). D) Cell-specific viral yield (VG/cell). E) Functional titer (ETU/mL). F) Ratio between volumetric and functional titer (VG/ETU). Values represent mean ± standard deviation (n=1 for MCD; n=2 for LCD).
Figure S1. Satellite culture from bioreactor production of AAV6 at medium cell density. Cells were transfected at 4 million cells per mL (medium cell density, MCD), and plasmid DNA was delivered on a cell basis (1 µg/106 cells). Time indicated as hours post-transfection (hpt). A) Cell growth kinetics and viability. B) Volumetric titer (VG/mL). C) Cell-specific viral yield (VG/cell). D) Functional titer (ETU/mL). E) Ratio between volumetric and functional titer (VG/ETU). n=1.