RESULTS
Effect of plasmid DNA
availability on AAV production at low cell densities
In the present study, we
investigated the improvement of AAV6 production via transient
transfection of suspension-adapted HEK293SF cells by increasing cell
density. The standard AAV production in our laboratory is conducted via
transfection at low cell density with 1 µg of plasmid DNA per mL. First,
we assessed if plasmid DNA availability played an important role during
production in an increased cell density. We compared the AAV6 yield from
productions at 1 and 2 million cells per mL. During transfection,
plasmid DNA was delivered either on a volumetric basis (1 µg/mL) or a
cell basis (1 µg/106 cells). PEI concentration for all
experiments was set at 2 µg per µg of plasmid DNA. Viral vectors were
harvested 48 hours post-transfection (hpt) to determine viral yield in
terms of genome-containing particles (viral genomes, VG). The cell
density and viability profiles during AAV production are shown in Figure
1A. Lower transduction efficiencies were observed in both productions at
2 million cells per mL compared to the control (Figure 1B). A 2.4-fold
increase in viral titer was observed in the transfection of 2 million
cells per mL when DNA was supplied on a cell basis (5.4 ×
109 VG/mL), resulting in a better-than-linear increase
of viral titer when compared to the standard production (2.2 ×
109 VG/mL) (Figure 1C). Productions at 2 million cells
per mL showed the highest cell-specific viral yield (CSVY) only when DNA
was supplied on a cell basis (approximately 3100 VG/cell). The delivery
of DNA as 1 µg/mL in the transfection of 2 million cells per mL caused a
reduction in the CSVY to only 900 VG/cell, resulting in a volumetric
yield of only 1.8 × 109 VG/mL.
Cell-specific production
maintained at a medium cell density
The next step was to evaluate the
viral vector production at a medium cell density (MCD, 4 ×
106 cells/mL at the time of transfection). Again, the
cells were transfected with plasmids on either a volumetric or cell
basis and the viral titer VG/mL was assessed every 24 hours. Figure 2A
shows the cell density and viability profiles during AAV production at
low and medium cell densities. A drop in the transfection efficiency was
observed even when a higher amount of DNA was supplied (Figure 2C).
However, as previously observed, this lower transduction efficiency did
not impact viral titer (Figure 2B, DNA delivered on a cell basis).
Similar to what was observed in the transfection of 2 million cells per
mL, the titer of the MCD production with DNA delivered on a cell basis
was 3.8 times the control, reaching 8.6 × 109VG/mL (Figure 2B). DNA delivery on a
volumetric basis resulted in 4.9 × 109 VG/mL (a
2.2-fold increase from the control). When DNA is supplied on a cell
basis, the CSVY was maintained at around 2,000 VG/cell. On the other
hand, volumetric delivery of DNA resulted in a decrease to 1,200 VG/cell
(Figure 2B). The functional viral titers (enhanced transduction units
(ETU)/mL) are summarized in Figure 2D. An increase in cell density
resulted in a rise of functional titer by 2.5-3.9 times, depending on
the harvest time. At 48 hpt, no statistically significant difference (P
= 0.4261) was observed in the ratio VG/ETU between low- and
medium-cell-density production with 1 µg of DNA per million cells
(Figure 2E).
The cell density effect
is observed at higher densities
To study if cell-based delivery of DNA would be enough to prevent the
cell density effect at a high-cell-density (HCD) production, we
evaluated AAV6 production at 10 million cells per mL. A perfusion-like
mode of operation was devised in 50-mL TubeSpin bioreactors to achieve
the desired cell density. A working volume of 10 mL and medium exchange
of 1 vessel volume per day (VVD) were set. The medium exchange started
24 hours after the seeding of the cells and was maintained past the
transfection to keep providing fresh nutrients to the cells. Cells were
transfected at a density of 10 × 106 cells/mL, with
plasmid DNA delivered on a cell basis, and viral titer was assessed
every 24 hours. A low-cell-density (LCD) control was also prepared in
similar vessels. As expected, the transduction of the high cell-density
production was lower than the control (Figure 3A). Cell densities and
volumetric titers during AAV6 productions are shown in Figure 3B. AAV6
yield peaked at 72 hpt, and the HCD production resulted in 9.3 ×
109 VG/mL, a 4.6-fold increase compared to the LCD
control. Even though the yield was higher than the control, this result
shows that the titer does not increase linearly with the increase in
cell density at the time of transfection. This difference becomes even
more evident at the cell-specific level. At 72 hpt, the LCD batch
control yielded an average of 1,600 VG/cell, while the HCD perfusion
yielded less than 1,000 VG/cell (Figure
5B).
Medium supplementation
improves cell-specific production of genome-containing particles
Nutrients and cell metabolites were analyzed during the production of
AAV6 at low and high cell densities in TubeSpin bioreactors. Even though
the levels of metabolites (lactate and ammonium) would be maintained
low, and levels of glutamine and glutamic acid were kept constant by the
medium exchange, a significant drop in glucose concentration was
observed (Figures 4A and B). At 48 hpt, glucose concentration was 3.2
g/L for LCD, while for HCD, as low as 1.8 g/L. Two other HCD productions
were performed to evaluate the effect of medium supplementation. This
time, the medium was supplemented with either glucose at a final
concentration of 7.2 g/L or 15% Cell Boost 5, which resulted in a
similar concentration of glucose between LCD and HCD production at their
lowest points (Figure 4B).
Figure 5A shows the cell density and
viability profiles during AAV production at high cell density with basal
and supplemented media, and Figure 5B shows the transduction efficiency
24 hours post-transfection. Medium supplementation with Cell Boost 5
resulted in a higher growth rate after transfection, with cell density
reaching 25.5 × 106 cells/mL 96 hpt, while cells in
basal medium reached up to 20.4 × 106 cells/mL. Cells
cultured in the glucose-supplemented medium showed a slower growth rate,
resulting in the transfection being done one day later compared to the
medium supplemented with Cell Boost 5. At 72 hpt, when compared to the
use of the basal medium, the use of supplementation resulted in
increased volumetric titer (1.4- and 1.6-fold increase for glucose and
Cell Boost 5, respectively) and a significant improvement of
cell-specific titer (1.8-fold increase for Cell Boost 5) (Figure 5C).
Medium supplementation alleviated the observed cell density effect in
the HCD production, resulting in CSVY similar to those in the LCD
control, leading to an almost 10-fold increase in viral titer. There was
an increase in functional titer when HCD production was supplemented.
Cell-specific functional titer also increases depending on the
supplementation, but it was 2 times inferior compared to the LCD (Figure
6A). The VG/ETU ratio for LCD production was 34.4, while for HCD, it
reached 79.9 when the medium was supplemented with Cell Boost 5 (Figure
6B).