Sartorius - November 2021 - Simplifying Adeno Associated Virus - 21

Using specialized DOE software such
as Sartorius MODDE®
for example can
help to determine optimal transfection
conditions with HEK293 cells or infection
conditions with Sf9 cells, as well as the
optimal culture conditions to produce
higher rAAV titers.
Adapting DOE for
Academic Laboratories
For process optimization of rAAV, shake
flasks are often the choice for academic
laboratories but is it possible to use them
in a DOE approach? Zuban says: " It is possible
to set up a DOE using shake flasks as
long as the factors being assessed can be
controlled to specific set points. "
According to Enga, in their laboratory, due
to manual handling constraints and incubator
space, the number of shake flasks
they can use is limited to a maximum of 14
in parallel. They select the two optimal cell
lines in a first run, and then find the best
media and transfection reagents in another
run for their rAAV cell culture process.
Using this many flasks for development
runs is expensive as they need to use a
large quantity of raw materials such as
transfection reagents and media. Cécile
Robin, Process Operation Manager for
CPV-Process at the French industrial
integrator CPV in INSERM UMR 1089 at
the University of Nantes adds: " In the past,
when using shake flasks in our laboratory
we could only test one or two factors due
to the limitation of the number of shake
flasks. Running a DOE with shake flasks is
feasible and would reduce the number of
shake flasks needed but would still require
a lot of raw materials and manual handling. "
" Shake flasks can be used in process
optimization of rAAV but it is difficult to
control parameters such as dissolved
oxygen (DO) and pH so the results may
not be accurate or reproducible.
21 | GENengnews.com
However, using an automated mini bioreactor
system such as the Ambr®
15, it is
possible to control set-points for culture
parameters more precisely, " points out
Amélie Boulais, Head of Market Entry
Strategy, Viral based Therapeutics at
Sartorius.
Although running a DOE with shake
flasks is possible, it only allows analysis
of the impact of a few factors whereas
using mini bioreactors systems with DOE
gives more meaningful results, as more
factors and potentially interactions can
be analyzed. Enga states: " Using the
Ambr®
15 system with DOE we can test
many different parameter combinations
because we can set it up to run up to
48 experiments and several different
factors in parallel. "
A Data Scientist in
Every Lab?
If DOE is such a powerful tool, why aren't
all scientists doing rAAV process optimization,
utilizing it? Enga comments:
" This type of software is a new concept
for many bioprocess scientists as we are
generally taught to vary one parameter
and keep everything else constant at the
same time. I think the new generation of
bioprocess scientists will be taught that
looking at multiple factors simultaneously
is better and will start to think this way. "
Zuban agrees saying: " Using a DOE
approach requires most researchers to
think differently as they have to set up
experiments, then analyze the results
and use the results to set up another
DOE. It can look like more steps, but it
actually saves time and is more efficient
in the long run as you are always homing
in on the most critical factors and ranges
to improve productivity. "
" For us running the DOE with the Ambr®
system has been a great help as it has
given us clear guidance on where to go
15
Considerations When
Setting up a DOE
DOE for rAAV process optimization is
an iterative process which involves
screening studies to find the best combination
of media and cell line for example,
followed by optimization studies where
ranges of process parameter such as
plasmid ratios are explored. " Before a
DOE is set up, it is important that scientists
and data analysts discuss the set-up of
the design. For instance, how many
factors are of interest for the study and
what are their appropriate ranges for a
response effect on, for example, titer.
This will lead to the set-up of the first
screening DOE, which with rAAV process
optimization studies in HEK293 is usually
looking at media first to choose the best
media and cell lines for productivity, "
explains Zuban.
Interpreting DOE Data Plots
Using DOE software such as MODDE®
,
the results from a screening study can
be used to produce a variety of plots to
aid interpretation of results, including
observed versus predicted data values, a
summary of fit plot, and a coefficients
plot (Figure 1) of productivity measurements
such as titer, measured by qPCR
in viral genomes (vg)/mL.
next with our process design. But it is
good to have someone in the lab who is
trained in using analytics software to
help interpret the results, " adds Robin.
Boulais concludes: " The great advantage
of using a specific DOE software such as
MODDE®
is that it has guided workflows
with wizards that help scientists through
the steps and interpretation. At Sartorius
we also provide training in DOE and our
software to universities and industry, as
well as consulting on specific projects. "
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Sartorius - November 2021 - Simplifying Adeno Associated Virus

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