Sartorius - November 2021 - Simplifying Adeno Associated Virus - 12

Step 1: Testing Different Cell Lines,
Media, and Transfection Reagents
We inoculated two proprietary HEK293
cell lines, Cell Line 1, and Cell Line 2 into
two different proprietary media, Media 1,
and Media 2. At 24 hours before transfection,
the cells were inoculated with
different viable cell concentrations
(VCCs) (1 × 10⁶ cell/mL - 2.5 × 10⁶ cells/mL)
in the Ambr®
15 microbioreactors at 37 ˚C,
pH 7.3, 50% DO, with a stirring speed of
400 rpm. For transfection, we tested
two proprietary transfection reagents,
Transfection Reagent 1 and Transfection
Reagent 2, ratios of plasmid to vector
(from 1:3 - 3:1), ratios of transfection
reagent to total DNA (1:1 - 4:1), and different
amounts of total plasmid DNA (1 µg/mL -
5 µg/mL). We used a transfection mix
volume of 5 % and a complex time of
20 minutes for both transfection reagents.
To ensure sufficient lysis of the cells for
measuring rAAV in the supernatant, we
froze the bulk at harvest (cells with
supernatant) and thawed them in a 37 °C
water bath. We repeated this procedure
three times and then centrifuged the cell
lysate to remove cell debris from the
supernatant. To determine productivity,
we measured rAAV vector genome
vg/mL titer in the supernatant by
qPCR. Other parameters we measured
included transfection complex size,
VCC and cell viability post-transfection.
This run produced rAAV titers of
<5 × 10⁹ vg/mL which is below our goal
titer and indicates that the process
needs further optimization. To determine
which factors had a negative and positive
impact on rAAV productivity, we
used MODDE®
software to analyze the
results and we identified that the cell line
and the transfection reagent as well as
the plasmid ratio had a significant effect
on the AAV titer. These results served
as a basis for the second optimization
experiment.
Step 2: Optimization of rAAV Production
Parameters
To optimize rAAV production parameters,
we used the data from Step One to
select the factors which produced the
optimum transfection efficacy. We took
these forward for a new DOE experimental
plan. Cells were cultured in shake flask in
each different media. 24 hours before
transfection, the cells were inoculated at
a VCC of 1.75 × 10⁶ cell/mL in the Ambr®
15
microbioreactors, using a range of pH,
DO, and stirring speed set points (Table 1).
For transfection, we used Transfection
Reagent 1 in the ratio, Reagent | DNA:
2.5 µL : 1 µg. We also used a low helper
plasmid to vector plasmid ratio (1:3) and
total plasmid DNA (3 µg/mL) with a
transfection mix volume of 5% and a
complex time of 20 minutes.
To determine the effect of different
factors, we measured rAAV vg titer using
qPCR as previously discussed. As there
can be slight difference in cell density at
the time of transfection due to the different
culture condition, we normalized the
viral genome titer to the transfected cell
number, yielding the specific productivity
vg/cell. We also measured total particle
number by ELISA in particles/mL, as well
as transfection complex size, VCC and
cell viability post-transfection.
This run produced rAAV titers
>8.5 × 10⁹ vg/mL in cleared lysate which
is below our goal titer of >1 × 10¹⁰ vg/mL.
We used MODDE®
Factor
Range
Stirring speed (rpm)
pH
DO (%)
Cell Line
Media
Table 1: DOE for optimizing process parameters
6.9 - 7.3
30 - 70
Cell Line 1; Cell Line 2
Media 1; Media 2
Set-points
400; 800
software to produce
coefficients plots for Cell viability (figure
2), product titer (not shown) and specific
productivity (figure 3). The coefficients
plot for cell viability (Figure 2) shows
that pH has a negative correlation to
cell viability at transfection but a positive
correlation to cell viability at harvest.
This indicates that by manipulating pH
values we could obtain better results
both at transfection and harvest steps.
We observed interaction terms for cell
line and media, meaning that Cell Line 1
produces optimum results in Media 1
and Cell Line 2 provides the best results
in Media 2. The choice of the cell line has
a significant effect on the cell viability at
transfection, either positive or negative
depending on the specific cell line. In
summary, we found that pH and cell line
were Critical Process Parameters (CPPs)
for cell viability.
The specific productivity coefficients
plot (Figure 3) indicates that pH
correlates positively with AAV productivity
and should be maintained at 7.3. Furthermore,
we saw that the choice of the cell
line also has a significant effect on the
virus productivity, which can be either
positive or negative. Media and stir
speed only had a limited impact on the
specific productivity.
12 | GENengnews.com
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Sartorius - November 2021 - Simplifying Adeno Associated Virus

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