Sartorius - November 2021 - Simplifying Adeno Associated Virus - 5

Challenges With Optimizing
Transient rAAV Production
Process optimization for production of
rAAV vectors is often fraught with difficulties,
which include being able to find
the right process factors for plasmid
transfection (HEK293) or baculovirus
infection (Sf9) cells. Determining these
early on can help with identifying critical
process parameters (CPPs) for maintaining
critical quality attributes (CQAs). This
ensures that when the process is scaled,
downstream processing steps, as well as
associated costs and timelines are minimized.
According to many scientists
involved in rAAV manufacturing, with a
systematic approach to process optimization
of suspension culture, titers can
be dramatically improved, but where is it
best to begin? Franziska Bollmann, PhD
Process Technology Consultant for Viral
based Therapeutics at Sartorius states:
" For HEK293, researchers start with cell
line and media. Then, when they have
found the best ones, they look at cell
culture conditions such as pH, DO and
stirring speeds and finally factors that
affect transfection such as plasmid ratio,
total plasmid DNA, and transfection
reagents. " (Table 1)
Optimized Factors for HEK293 cells
Medium
Cell density at transfection [VC/mL]
Dissolved Oxygen [% dO₂]
Stirring speed [rpm]
pH
Cell line
Transfection reagent
Plasmid ratio (pHelper : pVector) [mol]
Transfection reagent to DNA ratio [µL/µg]
Transfection mix volume [%]
Complex time [min]
Total plasmid DNA [µg/mL]
" DOE allows for greater
efficiency as multiple
factors can be assessed
and optimized in parallel,
but since this increases
complexity it is easier
to use a specialist software
such as MODDE®
to set up and run a DOE "
Bollmann explains: " Media can have a
huge impact on titer. For example, additives
in some types of media cause aggregation,
and this can negatively impact
titer. Also, certain metal ions such as iron
in the media can affect the transfection
efficiency. For Sf9 cells which already
grow well in suspension culture, rAAV
process optimization begins with media
and there are less factors to test for.
Since some rAAV serotypes are intracellular,
a chemical lysis step is needed to
harvest them. Many labs test a range of
different lysis reagents at different
concentrations to improve titer after they
have found the best media. "
Franziska Bollmann, PhD
Process Technology Consultant
Viral based Therapeutics
Sartorius
When the use of rAAV in gene therapy
was still in its infancy, much of the process
optimization work was performed in
shake flasks. But is this suitable for
modeling a process that can be transferred
to manufacturing rAAV at scale?
Robin states: " We used to do our process
optimization using shake flasks, but this
meant we could only run six to ten flasks
at one time due to lack of time and incubator
space. Also, we couldn't regulate
gas or stirring speed accurately, so it was
very limited as a model. " Blouin-Tavel
adds: " With shake flasks it is also more
difficult to do a direct comparison with
process factors as there is a lot of variation
we cannot control. "
Optimized Factors for SF9 cells
Medium
Cell density at infection [VC/mL]
Dissolved Oxygen [% dO₂]
Stirring speed [rpm]
pH
Chemical lysis at harvest
Baculovirus MOI (Helper:Vector)
Baculovirus ratio (Helper/Vector)
-
-
-
-
Table 1: Overview of factors being typically optimized for transient production of rAAV
Poor process optimization of rAAV in
suspension culture can lead to challenging
process transfer as a process that works
well in shake flasks will still require validation
in benchtop bioreactors and may
need several engineering runs at production
scale to optimize the cell culture
process for optimal rAAV productivity.
However, this can be costly both in time
and resources and at the manufacturing
scale is also expensive in terms of media,
buffer, and reagent costs.
5 | GENengnews.com
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Sartorius - November 2021 - Simplifying Adeno Associated Virus

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