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.