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\begin{document}

\title{High-cell-density production of adeno-associated viral vector serotype 6
by triple transfection in suspension HEK293 cell cultures}



\author[1]{Pablo Moço}%
\author[1]{Xingge Xu}%
\author[1]{Amine Kamen}%
\affil[1]{McGill University}%


\vspace{-1em}



  
  \date{June 14, 2024}


\begingroup
\let\center\flushleft
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\selectlanguage{english}
\begin{abstract}
The use of adeno-associated viruses (AAV) as vectors for gene and cell
therapy has risen considerably in recent years. Consequently, the amount
of AAV vectors required during the validation and clinical trials has
also increased. AAV serotype 6 (AAV6) is well-documented for its
efficiency in transducing different cell types and has been successfully
used in gene and cell therapy protocols. However, the number of vectors
required to effectively deliver the transgene to one single cell has
been estimated at 106 viral genomes (VG). Overall, this means that
large-scale production of AAV6 is needed. Suspension cell-based
platforms are currently limited to low-cell-density productions,
hindering the potential of this production process to increase yields.
Here, we investigate the improvement of the production of AAV6 at higher
cell densities. The production was performed by transient transfection
of HEK293SF cells. When the plasmid DNA is provided on a cell basis, the
production can be carried out at medium cell density without effects on
cell-specific titer or particle functionality, resulting in titers above
1010 VG/mL. Medium supplementation alleviated the cell density effect,
in terms of VG/cell, at high-cell-density productions. On the other
hand, the cell-specific functional titer was not maintained, and further
studies are necessary to understand the observed limitations. The
medium-cell-density production method reported here lays the foundation
for large-scale process operations, potentially solving the current
vector shortage in AAV manufacturing.%
\end{abstract}%



\sloppy


Research Article

\textbf{High-cell-density production of adeno-associated viral vector
serotype 6 by triple transfection in suspension HEK293 cell cultures}

Pablo D. Mo\selectlanguage{ngerman}ço\textsuperscript{1}

Xingge Xu\textsuperscript{1}

Amine A. Kamen\textsuperscript{1}

\textsuperscript{1}Department of Bioengineering, McGill University,
Montreal, Canada

\textbf{Correspondence:} Dr. Amine Kamen, Department of Bioengineering,
McGill University, 3480 University Street \textbar{} McConnell
Engineering Building, Room 363, Montreal, QC, H3A 0E9 Canada.

\textbf{E-mail} : amine.kamen@mcgill.ca

\textbf{Keywords:} Bioprocess engineering, Bioprocess development, Cell
culture, Mammalian cells, Viruses.

\textbf{Abbreviations: AAV} , adeno-associated virus; \textbf{AAV6} ,
AAV serotype 6; \textbf{CDE} , cell density effect; \textbf{CSVY} ,
cell-specific viral yield; \textbf{ETU} , enhanced transducing
units;\textbf{FDA} , U. S. Food and Drug Administration; \textbf{GFP} ,
green fluorescent protein; \textbf{GOI} , gene of interest; \textbf{HCD}
, high cell density; \textbf{LCD} , low cell density, \textbf{MCD} ,
medium cell density; \textbf{PEI} , polyethylenimine; \textbf{VG} ,
viral genomes.



\textbf{ABSTRACT}

The use of adeno-associated viruses (AAV) as vectors for gene and cell
therapy has risen considerably in recent years. Consequently, the amount
of AAV vectors required during the validation and clinical trials has
also increased. AAV serotype 6 (AAV6) is well-documented for its
efficiency in transducing different cell types and has been successfully
used in gene and cell therapy protocols. However, the number of vectors
required to effectively deliver the transgene to one single cell has
been estimated at 10\textsuperscript{6} viral genomes (VG). Overall,
this means that large-scale production of AAV6 is needed. Suspension
cell-based platforms are currently limited to low-cell-density
productions, hindering the potential of this production process to
increase yields. Here, we investigate the improvement of the production
of AAV6 at higher cell densities. The production was performed by
transient transfection of HEK293SF cells. When the plasmid DNA is
provided on a cell basis, the production can be carried out at medium
cell density without effects on cell-specific titer or particle
functionality, resulting in titers above 10\textsuperscript{10} VG/mL.
Medium supplementation alleviated the cell density effect, in terms of
VG/cell, at high-cell-density productions. On the other hand, the
cell-specific functional titer was not maintained, and further studies
are necessary to understand the observed limitations. The
medium-cell-density production method reported here lays the foundation
for large-scale process operations, potentially solving the current
vector shortage in AAV manufacturing.



\textbf{INTRODUCTION}

Adeno-associated viruses (AAVs) are small, 25-nm-wide, icosahedral,
non-enveloped viruses with a 4.7-kb-long single-stranded DNA genome
belonging to the family \emph{Parvoviridae} {[}1{]}. AAVs are
non-pathogenic and replication-defective {[}2{]}, depending on
co-infection with helper viruses, such as Adenovirus or Herpes Simplex
virus. First discovered from stocks of adenovirus in the 1960s {[}2,
3{]}, AAVs have recently become a critical gene delivery vector for
treating diseases. Glybera was the first AAV-based gene therapy drug
approved in 2012 by the European Medicines Agency {[}4{]}. The US Food
and Drug Administration (FDA) approved 2 AAV-based gene therapy drugs,
Luxturna, in 2017 {[}5{]} and, Zolgesma, in 2019 {[}6{]}.

To this date, based on phylogenetic analysis, 13 different serotypes and
more than 100 variants of AAV have been identified {[}2, 3, 7-14{]}. Due
to heterogeneity in capsid proteins, each serotype exhibits a distinct
tropism and ability to transduce different cell types {[}15{]}. Serotype
6 has a wide range of target cells and has been shown to successfully
transduce cells in the central nervous system {[}16{]}, human prostate,
breast, and liver cancer cells {[}17{]}, melanocytes {[}18{]}, skeletal
muscle {[}19-21{]}, heart {[}22-24{]}, lung {[}25{]}, and the eye
{[}26{]}. Recently, AAV6 gained popularity due to its ability to
transduce lymphocytes {[}27-29{]} and its use for generating Chimeric
Antigen Receptor T cells {[}30-36{]}.

The number of therapeutic AAV applications being investigated is
steadily increasing, with more than 300 completed or ongoing clinical
trials {[}37{]}. These applications require large amounts of AAV vectors
to validate pre-clinical animal studies and clinical trials. Reported
doses can reach up to 7.5 × 10\textsuperscript{15} VG for targeted
delivery and up to 1.5 × 10\textsuperscript{17} VG for systemic delivery
{[}38{]}. This large requirement in the number of viral vectors implies
that an improvement in current production methods is necessary. This is
especially true for AAV serotype 6, which requires up to
10\textsuperscript{6} VG/cell during the transduction of T cells
{[}27{]}. Recombinant AAV is produced by replacing the viral genes,
\emph{Rep} and \emph{Cap} , with the gene of interest (GOI). Mammalian
cells are transiently transfected with a GOI cassette flanked by the
inverted terminal repeats, a plasmid carrying the \emph{Rep} and
\emph{Cap} functions, and a third plasmid encoding the helper functions
{[}39-42{]}. The transient transfection is generally done using the
cost-effective cationic polymer, Polyethylenimine (PEI) {[}43{]}.
However, adherent cell cultures are not deemed viable for AAV production
at scales that exceed 10\textsuperscript{15} total VGs, making them
unsuitable for late-phase clinical trials and commercial applications of
these viral vectors {[}44{]}. The use of HEK293 cells in suspension for
producing AAV vectors was first described in 2006 {[}45{]}. Despite many
efforts in optimizing production, large-scale production of AAV vectors
is considered a bottleneck in their implementation as a widespread type
of viral vector for gene therapy and cell therapy {[}46{]}, mainly
because current HEK293-based production is done at low cell densities
{[}45, 47-51{]}. Limiting the production of viral vectors to lower cell
densities hinders the potential of this production process to be
intensified {[}52{]}. On the other hand, the production of viral vectors
at higher cell densities tends to be limited by the widely reported cell
density effect (CDE), which results in diminished transfection and
productivity {[}53-55{]}. In this manuscript, we demonstrate that the
production of AAV serotype 6 via transient transfection is not limited
to low-cell-density cultures.

\textbf{MATERIAL AND METHODS}

\textbf{Cell line and culture}

HEK293SF cells were maintained in serum-free suspension cultures at
37°C, 5\% CO\textsubscript{2}, and 75\% relative humidity in a shaker
incubator (Infors, Switzerland) at 135 rpm speed of agitation or
specified otherwise. The basal maintenance and production medium was
HyCell TransFx-H (Cytiva Life Sciences, USA) supplemented with 0.1\% w/v
of Kolliphor P188 (Sigma-Aldrich, USA) and 4 mM GlutaMAX (Gibco, USA).
GlutaMAX was replaced by 6 mM L-Glutamine (Gibco, USA) for experiments
in which nutrients and metabolites were analyzed. The supplemented
medium was prepared by adding either glucose for a final concentration
of 7.2 g/L or 15\% (v/v) Cell Boost 5 (Cytiva Life Sciences, USA) to
achieve a similar glucose concentration.

Low- and medium-cell-density cultures (1 and 4 ×
10\textsuperscript{6}cells/mL, respectively, at the time of transfection
- TOT) were done in 125-mL polycarbonate shake flasks (TriForest, USA)
with a working volume of 25 mL. High-cell-density (10 ×
10\textsuperscript{6} cells/mL at TOT) transfection was performed in
50-mL TubeSpin bioreactors (TPP, Switzerland) with a working volume of
10 mL and 180 rpm. For achieving and maintaining high cell densities,
from 48 hours post-seeding, a perfusion-like mode of operation was
followed with medium exchange of 1 vessel volume per day (VVD) both
before and after transfection, where cells were centrifuged and
resuspended in fresh medium.

The bioreactor production was performed in a 3-L vessel (Applikon
Biotechnology, The Netherlands) equipped with a double marine impeller,
a pH sensor, a temperature sensor, a dissolved oxygen (DO) concentration
sensor, a micro sparger with 100-nm pore size, and a capacitance probe
for monitoring of cell culture characteristics, such as cell growth. The
pH, temperature, and DO parameters were set at 7.15 ± 0.05, 37°C, and
40\% oxygen saturation, respectively. The HEK293SF cells from shake
flasks were inoculated into the bioreactor at 4 ×
10\textsuperscript{6}cells/mL in a fresh culture medium. The
transfection was conducted immediately after cell inoculation with
plasmid delivered on a cell basis (1 \selectlanguage{greek}µ\selectlanguage{english}g/10\textsuperscript{6} cells).

\textbf{Plasmids}

The following plasmids were used to produce adeno-associated viral
vectors: (1) pAdDeltaF6 was a gift from James M. Wilson (Addgene plasmid
\#112867; http://n2t.net/addgene:112867; RRID:Addgene\_112867), (2)
pRep2Cap6 (provided by Dr. Samulski, University of North Carolina, USA),
(3) pAAV-CAG-GFP was a gift from Edward Boyden (Addgene plasmid \#37825;
http://n2t.net/addgene:37825; RRID:Addgene\_37825) and (4)
pX601-AAV-saCas9-sgRNA, modified from
pX601-AAV-CMV::NLS-SaCas9-NLS-3xHA-bGHpA;U6::BsaI-sgRNA, a gift from
Feng Zhang (Addgene plasmid \#61591; http://n2t.net/addgene:61591;
RRID:Addgene\_61591).

\textbf{Transient transfection}

Recombinant AAV6 particles expressing GFP driven by the CAG promoter
were produced via triple transient transfection of HEK293SF cells as
previously described {[}56{]}. In brief, the transgene plasmid
pAAV-CAG-GFP, the plasmid pRep2Cap6, and the helper plasmid pAdDeltaF6
were co-transfected into HEK293SF cells in a ratio of 1:1:1 after
complexation with 25-KDa linear polyethylenimine (PEI, Polysciences,
USA) at a ratio of 1:2 (DNA:PEI). Plasmid DNA and PEI were diluted
separately in HyCell TransFx-H in a volume corresponding to 2.5\% of the
volume of cells to be transfected. For experiments where the DNA was
delivered on a volumetric basis, 1 \selectlanguage{greek}µ\selectlanguage{english}g of plasmid was added per
millilitre of cell culture. For delivering DNA on a cell basis, 1 \selectlanguage{greek}µ\selectlanguage{english}g of
plasmid was added per million viable cells. Diluted DNA and PEI were
mixed and incubated at room temperature for 15 minutes. The DNA-PEI
cocktail was then added to the cells.

\textbf{Determination of transfection efficiency}

For evaluation of transfection efficiency, cells were collected 24 hours
post-transduction and fixed with a solution of 2\% paraformaldehyde.
Cells were analyzed using a BD Accuri C6 flow cytometer (BD Biosciences,
USA), and EGFP expression was analyzed using BD Accuri C6 Plus Analysis
Software (BD Biosciences, USA).

\textbf{Harvest of produced viral vectors}

Viral particles were harvested from the cell culture broth as previously
described {[}47, 56{]}. Briefly, the cells were harvested and lysed by
adding a 10X Lysis Buffer (20 mM MgCl\textsubscript{2}, 1\% Triton X-100
in 500 mM Tris-buffered solution). Benzonase was added to a final
concentration of 5 U/mL to digest host-cell DNA and unpacked virus DNA.
After 1 hour of incubation at 37\selectlanguage{ngerman}°C with agitation,
MgSO\textsubscript{4}was added to a final concentration of 37.5 mM to
prevent AAV aggregation and binding to cellular components. After 30
minutes of incubation at 37°C with agitation, the lysate was clarified
via centrifugation at 10,000 × g for 15 minutes. The clarified viral
lysate was stored in a -80°C freezer.

\textbf{AAV genome quantification by droplet digital PCR}

Genome-containing viral particles were titrated via ddPCR {[}57{]}.
Viral DNA from samples was extracted using a High Pure Viral Nucleic
Acid Extraction kit (Roche Diagnostics, Switzerland). The following
primers targeting the transgene were used: EGFP,
5'-CTGCTGCCCGACAACCAC-3' (forward) and 5'-TCACGAACTCCAGCAGGAC-3'
(reverse); SaCas9, 5'-GGCCAGATTCAGGATGTGCT-3' (forward) and 5'-
CATCATCCCCAGAAGCGTGT-3' (reverse). The primers were purchased from
Integrated DNA Technologies (USA). The thermocycling temperature
programming for EGFP was: preincubation at 95°C/15 min for denaturation.
40 cycles of 94°C/30 s, 53°C/30 s and 72°C/1 min, and final extension at
72°C for 5 min. For SaCas9 was: preincubation at 95°C/15 min for
denaturation. 40 cycles of 94°C/30 s, 60°C/1 min and 72°C/30 s, and
final extension at 72°C for 5 min. The plates were scanned on a QX100
droplet reader (Bio-Rad, USA), and the analysis was carried out with
QuantaSoft software (Bio-Rad, USA). Quantification was performed as
previously described {[}58{]}. Cell-specific viral yield (CSVY) was
determined by dividing the titer by the cell density at the time of
transfection.

\textbf{Functional virus titer via gene transfer assay}

Suspension-adapted HEK293SF cells were transduced with serial dilutions
of AAV6-GFP and co-infected with an E1, E3-deleted adenovirus serotype 5
(\selectlanguage{english}[?]E\textsubscript{1}E\textsubscript{3}Ad5) at an MOI of 5 infectious
particles/cell. At 24 hours post-transduction, the cells were harvested
and fixed in 2\% paraformaldehyde at 4degC for 30 min. Cells were analyzed
using a BD Accuri C6 flow cytometer (BD Biosciences, USA), and EGFP
expression was analyzed using BD Accuri C6 Plus Analysis Software (BD
Biosciences, USA). The linear range of quantification was established
between 2\%--20\% of GFP-positive cells {[}59{]}.

\textbf{Analysis of medium nutrients and metabolites}

Culture samples were collected every 24 h, centrifuged for removal of
cell pellet, and the supernatant was analyzed in a BioProfile FLEX2
Analyzer (Nova Biomedical, USA) to determine the following nutrients and
metabolites: glutamine, glutamic acid, glucose, lactate, and ammonium.

\textbf{Statistical analysis}

Results are expressed as mean +- standard deviation. Student's t-test was
used to measure the statistical significance between two groups. One-way
ANOVA followed by Dunnett's post-hoc test was used to measure the
statistical difference between three or more groups. \emph{P}\textless{}
0.05 was considered to indicate a statistically significant difference.
Asterisks are used to indicate \emph{P} values as follows: *, \emph{P}
\textless{} 0.05; **, \emph{P} \textless{} 0.01; ***,\emph{P}
\textless{} 0.005, ****, \emph{P} \textless{} 0.0005.

\textbf{RESULTS}

\textbf{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 \selectlanguage{greek}µ\selectlanguage{english}g 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 \selectlanguage{greek}µ\selectlanguage{english}g/mL) or a cell basis (1 \selectlanguage{greek}µ\selectlanguage{english}g/10\textsuperscript{6}
cells). PEI concentration for all experiments was set at 2 \selectlanguage{greek}µ\selectlanguage{english}g per \selectlanguage{greek}µ\selectlanguage{english}g 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 \selectlanguage{ngerman}× 10\textsuperscript{9} VG/mL), resulting in a
better-than-linear increase of viral titer when compared to the standard
production (2.2 × 10\textsuperscript{9} 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 \selectlanguage{greek}µ\selectlanguage{english}g/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 \selectlanguage{ngerman}× 10\textsuperscript{9}
VG/mL.

\textbf{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 × 10\textsuperscript{6} 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 × 10\textsuperscript{9}VG/mL (Figure 2B). DNA delivery on a
volumetric basis resulted in 4.9 × 10\textsuperscript{9} 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 \selectlanguage{greek}µ\selectlanguage{english}g of DNA per million cells
(Figure 2E).

\textbf{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 \selectlanguage{ngerman}× 10\textsuperscript{6} 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 ×
10\textsuperscript{9} 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).

\textbf{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 ×
10\textsuperscript{6} cells/mL 96 hpt, while cells in basal medium
reached up to 20.4 × 10\textsuperscript{6} 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).

\textbf{Validation of medium-cell-density production at bioreactor
scale}

Since the MCD production resulted in a significant improvement in titer
and no loss in functionality, we decided to evaluate the scalability of
this process. For that, we conducted the production in a 3-L bioreactor,
where the cells were seeded at 4 × 10\textsuperscript{6} cells/mL in
fresh medium and were immediately transfected with plasmids delivered on
a cell basis. Whole broth culture was sampled at 8-16h intervals. A
satellite culture, with a volume of around 25 mL taken from the
bioreactor post-transfection, was evaluated in parallel in a shake flask
(Figure S1). At 24 hpt, a transfection efficiency of 36.9\% was
observed, similar to the production in shake flasks (33.8\%, Figure 2C).
Figure 7 shows cellular and production kinetics in the bioreactor,
demonstrating that MCD production is feasible at a bioreactor scale.
Low-cell-density 3-L bioreactor productions previously completed in our
laboratory were used as control. In these LCD runs, AAV6 vectors
packaging a genome without fluorescent marker were produced; thus, it
was impractical to assess their functional titer. Cell density in the
MCD bioreactor reached its maximum at 59 hpt with 9.5 ×
10\textsuperscript{6} viable cells/mL. Vector production peaked around
60 hpt with a titer of 5.9 × 10\textsuperscript{10} VG/mL, a
\textasciitilde{}4-fold increase compared to control bioreactors (Figure
7C). At this point, the cell-specific titer was around 15,000 VG/cell, a
similar value to the one obtained by LCD production in bioreactors
(Figure 7D). Regarding the functional titer, the bioreactor yielded 7.8
× 10\textsuperscript{9} ETU/mL 48 hours post-transfection (Figure 7E).
As a result, the VG/ETU ratio was as low as 4.6 at 48 hpt, plateauing
around 10 at subsequent time points (Figure 7F).

\textbf{DISCUSSION}

In Figure 1, we observe that plasmid DNA availability plays an essential
role in the maintenance of VG titer when cell density at the time of
transfection is increased from 1 to 2 million cells per mL. In
low-cell-density transient transfection, plasmid DNA is usually
delivered on a volumetric basis {[}45, 47-50{]}. Like our study,
Grieger, Soltys and Samulski {[}48{]} also observed a titer increase
during production when cell density and plasmid concentration were
doubled. The need for a higher plasmid DNA concentration was also
observed for productions at 4 × 10\textsuperscript{6} cells/mL (medium
cell density, MCD). A 4-fold increase in the number of cells at the time
of transfection resulted in an almost linear increase in viral titer
(VG/mL) only when plasmid DNA was delivered per million cells (Figure
2B). Moreover, the cell-specific yield was maintained during MCD
production, with plasmid DNA concentration maintained on a per-cell
basis (Figure 2B). Yields between 1,500 and 3,000 VG/cell were obtained,
similar to those reported by Chahal, Schulze, Tran, Montes and Kamen
{[}47{]}. The improvement in titer was also observed for functional
particles (ETU). The functional titer of the MCD production with plasmid
DNA delivered on a cell basis was maintained constant (Figure 2D),
resulting in a difference of up to 3.9 times compared to the LCD control
at 96 hpt. At LCD, the slight decrease in transducing units, but not in
genome-containing particles, could be explained by the loss in
functionality of the viral vectors. AAV vectors are thermally stable,
but their transduction efficiency decreases when maintained at 37°C and
as the pH decreases {[}60{]}. Extending the culture to 96 hpt can
decrease intracellular and extracellular pH due to the accumulation of
metabolites, such as ammonia and lactate {[}61, 62{]}.

The VG/ETU ratio measures viral functionality, showing the proportion of
genome-containing viruses competent to transduce a target cell. At 48
hpt, the MCD production had a mean VG/ETU ratio of 67.1 (Figure 2E).
Although this value is higher than reported in the literature {[}47,
48{]}, there was no statistically significant difference from our LCD
control. As shown in Figure 7, the production of AAV6 at medium cell
density was successfully performed at a 3-L bioreactor. The cells were
inoculated into the bioreactor in fresh medium at the desired cell
density of 4 × 10\textsuperscript{6} cells/mL to prevent detrimental
effects from the spent medium. At larger scales, this could be achieved,
for example, by medium exchange using Tangential Flow Depth Filtration
(TFDF) as a cell retention device {[}63, 64{]}. This bioreactor
production resulted in 30- and 7.5-fold increase in VG/mL and VG/cell
titers, respectively, compared to previously reported production at a
similar scale {[}47{]}. Surprisingly, the viral vector yield on the
bioreactor was around 10-fold higher than the small-scale satellite
culture. Similarly, the LCD bioreactor production also showed a higher
yield than the small-scale experiments. This enhanced production in the
bioreactors could be associated with better-regulated culture
conditions, such as dissolved oxygen and pH. Again, no loss in
functional titer was observed, with a maximum value of 7.8 ×
10\textsuperscript{9} ETU/mL achieved 48 hours post-transfection and a
VG/ETU ratio of 4.6 (Figure 7D and E). These results show an improvement
from previous reports {[}47, 48{]}.

When productions were conducted at cell densities other than 1 ×
10\textsuperscript{6} cell/mL, the transfection efficiency, measured by
the expression of the transgene 24 hours post-transfection, decreased to
about 30\% independently of the cell density (Figure 1B, Figure 2C, and
Figure 3A). However, the percentage of cells expressing the transgene
did not correlate directly with production efficiency. Others observed
this same phenomenon during the production of viral-like particles via
transient transfection of HEK293SF suspension cells {[}65{]}. Hildinger,
Baldi, Stettler and Wurm {[}66{]} concluded that the transfection
efficiency reduction resulted from DNA being provided on a volumetric
basis. However, increasing the plasmid DNA availability in our study did
not significantly improve transduction efficiency (Figure 1B, Figure 2C,
and Figure 3A), as measured by the detection of the product of one of
the three plasmids, which corroborates previous findings {[}47{]}.
Increased energetic demand is reported to occur at higher cell densities
and to allow recovery from transfection, an event known to be cytotoxic
{[}67{]}. Transfection efficiency did not improve during production in a
perfusion-like mode, even when the medium was supplemented (Figure 5B).
The transfection efficiency was even lower at a higher glucose
concentration. Lavado-García, Jorge, Cervera, Vázquez and Gòdia {[}67{]}
partially explained the low transfection efficiency as a result of
downregulated pathways involved in lipid biosynthesis and nuclear
transportation of intracellular proteins. The transfection step, per se,
is a known bottleneck in the production of AAV vectors. A recent study
from our group showed that the proportion of cells that produce viral
particles is as low as 7\%, despite high transduction efficiency
{[}68{]}.

Recently, the improvement of AAV8 production at high-cell density with a
medium exchange strategy was reported {[}64{]}. Conversely, the cell
density effect was confirmed during our high-cell-density productions
(Figures 3B and 5). CDE refers to the diminished production at high cell
densities resulting from decreased cell-specific productivity {[}55,
69{]}. This effect has been previously documented for the generation of
AAV using other production systems, such as insect cells {[}53, 59,
70-72{]}. During production, 25 to 30\% of the AVV6 vectors are released
in the supernatant {[}47{]}. Because of the high cell density and the
elevated shaking speed to properly oxygenate the bioreactors used, it is
possible that the cells were under increased sheer stress and could have
released more vectors into the supernatant, which were removed during
the medium exchange. This could explain the sudden drop in viral titer
at 96 hpt in the HCD production (Figure 3B), which is not expected since
AAV vectors are considered very stable at a wide range of temperatures
{[}60, 73{]}. The same drop, however, was not observed when the medium
was supplemented (Figure 5C).

The cell density effect is thought to occur mainly due to metabolic
limitations caused by low concentrations of nutrients or the
accumulation of inhibitory metabolites {[}53, 55, 74{]}. During the
production at HCD, a perfusion-like mode was employed to provide enough
nutrients to support high cell density and remove inhibitory
metabolites; however, the conditions selected were insufficient to
prevent the CDE (Figure 3). The glucose level dropped significantly at
HCD, even though the medium was fully exchanged daily. For that reason,
the basal medium, HyCell TransFx-H, which contains around 6 g/L of
glucose, was supplemented with either glucose or Cells Boost 5. The
amount of supplement added was based on the cell-specific consumption
rate of glucose so that a minimum of 2 g/L of glucose was available at
any given time {[}75{]}, mimicking the observed glucose concentrations
in the LCD productions. Medium supplementation with glucose alone did
not restore the cell-specific productivity of AAV6 (Figure 5C). The CDE
was alleviated, and the cell-specific yield (VG/cell) was restored only
when a medium supplemented with 15\% Cell Boost 5 was used (Figure 5C),
indicating that other critical nutrients were limiting viral vector
yield. Besides glucose, other components of the Cell Boost 5 supplement
are thought to contribute to alleviating the CDE. While producing
adenoviral vectors using the HEK293SF cells, Shen, Voyer, Tom and Kamen
{[}76{]} emphasized that the composition of the medium used is crucial
to support high yields at HCD. However, the complexity of culture media
and supplements, such as Cell Boost 5, which can contain hundreds of
different components at variable concentrations, complicates the
understanding of individual nutrients' effect on viral vector
productions {[}55{]}. An optimized cell culture medium to produce AAV
vectors could improve titers and vector quality, as seen and suggested
for other viral vectors {[}61, 67, 75, 76{]}.

As discussed above, supplementing the medium with Cell Boost 5
alleviated the cell density effect in terms of genome-containing
particles; however, the same was not observed for functional particles.
Whereas there was an increase in functional yield when the medium was
supplemented, the cell-specific functional titer (ETU/cell) was not
fully maintained, resulting in higher VG/ETU ratios compared to the LCD
control (Figure 6). One possible explanation is the misassembly of the
viral capsid, which contains a stoichiometry of 1:1:10 (VP1:VP2:VP3). If
this proportion is altered, the efficacy of the AAV to deliver the
transgene decreases {[}77{]}. Due to nuclear localization signals, the
VP1 and VP2 proteins play a crucial role in transduction, specifically
in nuclear transportation {[}78{]}. The N-terminus of VP1 also contains
a phospholipase A2 domain responsible for viral escape from the endosome
{[}79-82{]}. Our results highlight the importance of assessing viral
transduction efficacy by measuring biologically active vectors {[}83{]}
as part of optimizing AAV vector production. This measurement is
sometimes overlooked by authors {[}49, 51{]}, even though the quality
attributes of a viral vector, including functional titer, are an
essential part of the potency tests recommended by the FDA {[}84{]}.

In summary, we demonstrated that it is possible to produce AAV6 via
triple transient transfection at medium cell density without losing
cell-specific productivity or functional titer. For that, the plasmid
DNA must be provided on a cell basis. Our medium-cell-density
productions achieved titers at the order of 10\textsuperscript{10} VG/mL
of crude lysate. The yields obtained by this method represent a
significant increase compared to a well-established production protocol
{[}47{]}. The medium-cell-density method for AAV vector production
described in this manuscript was shown to be efficient at a 3-L
bioreactor scale. It could be used as the foundation for large-scale
production processes, potentially contributing to solving the current
vector shortage in AAV manufacturing. The cell density effect could be
alleviated at a higher cell density, resulting in similar cell-specific
productivity (VG/cell) when a perfusion-like operation was conducted
with a supplemented medium. However, the cell-specific functional
productivity (ETU/cell) was reduced, highlighting the importance of
evaluating both viral genomes and transducing units during bioprocess
optimization. Further research is necessary to fully understand how the
cell density effect results in reduced functional titer and how to
alleviate it fully.

\textbf{ACKNOWLEDGMENT}

PDM is financially supported by a fellowship from the Faculty of
Engineering at McGill University and a doctoral scholarship from the
Fonds de Recherche du Québec -- Santé (FRQS). XX is financially
supported by a doctoral scholarship from Fonds de Recherche du Québec --
Nature et technologies (FRQNT). AAK is partially funded through Canada
Research Chair CRC-240394. The funding sources were not involved in the
study design, the collection, analysis and interpretation of data, the
writing of the report or the decision to submit the article for
publication. The authors would like to thank Cristina Silva for her
support.

\textbf{DATA AVAILABILITY STATEMENT:}

The data that support the findings of this study are available from the
corresponding author upon reasonable request.

\textbf{AUTHOR CONTRIBUITIONS}

Conceptualization: PDM, AAK; Investigation: PDM, XX; Formal analysis:
PDM; Writing - original draft: PDM; Writing -- review \& editing: PDM,
XX, AAK; Supervision: AAK, Funding acquisition: AAK

\textbf{DECLARATION OF COMPETING INTEREST}

The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to
influence the work reported in this paper.

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\textbf{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 \selectlanguage{greek}µ\selectlanguage{english}g/mL) or
cell basis (CB - 1 \selectlanguage{greek}µ\selectlanguage{english}g/10\textsuperscript{6} 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 \selectlanguage{ngerman}×
10\textsuperscript{6} cells/mL; 2M: at 2 ×
10\textsuperscript{6}cells/mL. Values represent mean ± standard
deviation (n=3). ** \emph{P}\textless{} 0.01, * \emph{P} \textless{}
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 ×
10\textsuperscript{6} cells/mL) as control or at medium cell density
(MCD -- 4 × 10\textsuperscript{6} cells/mL). Plasmid DNA was delivered
on a volumetric basis (VB - 1 \selectlanguage{greek}µ\selectlanguage{english}g/mL) or cell basis (CB - 1
\selectlanguage{greek}µ\selectlanguage{english}g/10\textsuperscript{6} 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 \selectlanguage{ngerman}± standard deviation (n=2). **** P \textless{}
0.0001, *** P \textless{} 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 (\selectlanguage{english}*) and volumetric viral yield (\selectlanguage{english}#). Cells were
transfected at low cell density (LCD -- 1 x 10\textsuperscript{6}
cells/mL) as control or at high cell density (HCD -- 10 x
10\textsuperscript{6} cells/mL). Plasmid DNA was delivered on a cell
basis (1 \selectlanguage{greek}µ\selectlanguage{english}g/10\textsuperscript{6} cells). Values represent mean \selectlanguage{ngerman}±
standard deviation (n=3). *** P \textless{} 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 × 10\textsuperscript{6} cells/mL) as control or at
high cell density (HCD -- 10 × 10\textsuperscript{6} cells/mL). Plasmid
DNA was delivered on a cell basis (1 \selectlanguage{greek}µ\selectlanguage{english}g/10\textsuperscript{6} 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 \selectlanguage{ngerman}± 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). **\emph{P} \textless{} 0.01, * \emph{P}
\textless{} 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
\selectlanguage{greek}µ\selectlanguage{english}g/10\textsuperscript{6}cells). Low-cell-density (LCD, 1 \selectlanguage{ngerman}×
10\textsuperscript{6} 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 \selectlanguage{greek}µ\selectlanguage{english}g/10\textsuperscript{6} 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.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

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 \selectlanguage{greek}µ\selectlanguage{english}g/mL) or
cell basis (CB - 1 \selectlanguage{greek}µ\selectlanguage{english}g/10\textsuperscript{6} 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 \selectlanguage{ngerman}×
10\textsuperscript{6} cells/mL; 2M: at 2 ×
10\textsuperscript{6}cells/mL. Values represent mean ± standard
deviation (n=3). ** \emph{P}\textless{} 0.01, * \emph{P} \textless{}
0.05, by ANOVA followed by Dunnett's test.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

Figure 2. Production of AAV6 via triple transient transfection at medium
cell densities. Cells were transfected at low cell density (LCD -- 1 \selectlanguage{ngerman}×
10\textsuperscript{6} cells/mL) as control or at medium cell density
(MCD -- 4 × 10\textsuperscript{6} cells/mL). Plasmid DNA was delivered
on a volumetric basis (VB - 1 \selectlanguage{greek}µ\selectlanguage{english}g/mL) or cell basis (CB - 1
\selectlanguage{greek}µ\selectlanguage{english}g/10\textsuperscript{6} 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 \selectlanguage{ngerman}± standard deviation (n=2). **** P \textless{}
0.0001, *** P \textless{} 0.001, by ANOVA followed by Dunnett's test and
Student's T-test.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\textbf{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 \selectlanguage{ngerman}×
10\textsuperscript{6} cells/mL) as control or at high cell density (HCD
-- 10 × 10\textsuperscript{6} cells/mL). Plasmid DNA was delivered on a
cell basis (1 \selectlanguage{greek}µ\selectlanguage{english}g/10\textsuperscript{6} cells). Values represent mean \selectlanguage{ngerman}±
standard deviation (n=3). *** P \textless{} 0.001, by Student's T test.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

\textbf{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 \selectlanguage{ngerman}× 10\textsuperscript{6} cells/mL) as control
or at high cell density (HCD -- 10 × 10\textsuperscript{6} cells/mL).
Plasmid DNA was delivered on a cell basis (1
\selectlanguage{greek}µ\selectlanguage{english}g/10\textsuperscript{6}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 \selectlanguage{ngerman}±
standard deviation (n=3).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

\textbf{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 \selectlanguage{ngerman}± standard deviation (n=3).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image6/image6}

\end{center}
\end{figure}

\textbf{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
\selectlanguage{ngerman}± standard deviation (n=3). ** \emph{P} \textless{} 0.01, *
\emph{P}\textless{} 0.05, by ANOVA followed by Dunnett's test.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image7/image7}

\end{center}
\end{figure}

\textbf{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
\selectlanguage{greek}µ\selectlanguage{english}g/10\textsuperscript{6} cells). Low-cell-density (LCD, 1 \selectlanguage{ngerman}×
10\textsuperscript{6} 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).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image8/image8}

\end{center}
\end{figure}

\textbf{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 \selectlanguage{greek}µ\selectlanguage{english}g/10\textsuperscript{6} 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.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-1/Figure-1}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-2/Figure-2}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-3/Figure-3}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-4/Figure-4}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-5/Figure-5}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-6/Figure-6}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-7/Figure-7}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-S1/Figure-S1}

\end{center}
\end{figure}

\selectlanguage{english}
\FloatBarrier
\end{document}

