Sartorius - November 2021 - Simplifying Adeno Associated Virus - 23

Optimization Studies
Titer qPCR
[vg/mL]
From screening studies of rAAV process
development, the best performing cell
lines and media are taken forward for
optimization. Step one in this workflow is
to optimize cell culture factors such as
pH, stir speed and DO. Step two is to
determine factors affecting transfection
efficiency. According to Robin, Step two
screening studies at the University of
Nantes involve using different transfection
reagents, ratios of plasmids to vector,
ratios of transfection reagent to total
DNA, different viable cell concentrations
(VCCs) at transfection, and different
amounts of total plasmid DNA.
The results from an optimization study
can be used to again produce a plot of
observed versus predicted data values
and a summary of fit plot. An example of
these plots for viral titer from an rAAV
process development study at the
University of Nantes is shown in (Figure 2).
Robin says: " To further optimize rAAV
process development, we used the
results from a transfection optimization
to set up a culture optimization DOE,
where we test the best multi-factor
combinations of for example media, cell
Transfection reagents = TR1
3
3e+10
3e+10
2.5
1.5e+10
1e+10
5e+09
2
2.5e + 10
3e + 10
3.5e + 10
Transfection reagents = TR3
1.5
2e + 10
1.5e + 10
1
1e + 10
0.5
77.1
7.27.3
pH
7.4
77.1
7.27.3
pH
7.47.5
5e +09
Figure 3: 4D response contour plots for rAAV titer to determine the optimum pH and plasmid amount
with different transfection reagents for transfection efficiency to produce rAAV2/8 with HEK293 in
Ambr®
15 micro bioreactors.
line, transfection reagent and plasmid
ratio with ranges of culture parameters
including stirring speed, DO, and pH. "
To find the optimum operating ranges
for a combination of factors, scientists
can produce a contour plot.
Figure 3 is an example of one for titer
from a rAAV process development study
at the University of Nantes.
Zuban comments: " The contour plot on
the right shows that high titer in orange
and red is associated with a specific
transfection reagent, low pH and high
total plasmid amounts. This plot also
shows in blue where the plasmid quantity
is limiting.
Benefits of a DOE Approach
" Using a DOE approach, we have seen that a specific combination
of culture media, plasmid ratio and cell line will give
us the best productivity for our rAAV production process.
It has also shown us how important plasmid amounts are
too. " According to Enga, going forward, they will continue
to use DOE software as it: 1, helps them extrapolate results,
2, provides trends to follow to find which parameters are
critical and 3, helps them combine several factors easily to
perform less experiments. " Before when we just varied one
or two parameters, we were doing lots more process runs, "
Enga explains.
Enga concludes: " Using DOE in combination with the
Ambr®
Ambr®
15 system, means we can find the right process conditions
and improve our productivity much faster. Investing
the time in training to use the MODDE®
software with the
15 system, we can set up the DOE easily and run 24
parallel experiments in one run. We can analyze the results
quickly. So now instead of the three-four months that it
used to take using shake flasks and just looking at one to two
parameters each time, we can do our process optimization
in just one month, saving us time and resources in our rAAV
cell culture development work. "
23 | GENengnews.com
Total Plasmid DNA [μg/mL]
https://www.genengnews.com/

Sartorius - November 2021 - Simplifying Adeno Associated Virus

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