Sartorius - November 2021 - Simplifying Adeno Associated Virus - 4

What's Causing the
Bottleneck?
According to Dr. Blouin-Tavel there are
only two main cell lines being used commonly
for rAAV production, adherent
and suspension based human embryonic
kidney (HEK293) and suspension based
Spodoptera frugiperda (Sf9). However,
to date there is no one-size fit all platform
process for scale up. Blouin-Tavel
explains: " We start with a generic process
we have developed from over 20-years
of working with rAAV, but we have to
optimize this generic process each time
we have a new vector with a gene of
interest. " She continues: " It is easier to
produce higher rAAV titers with Sf9 cells
but as some researchers need rAAV produced
in a human cell line we must use
HEK293 too, even though the cell line is
not yet as efficient. This is because some
scientists believe that rAAV derived from
Sf9 and HEK293 cells are not equivalent
and there is minimal information from
current studies to say definitively if it is
the same product3. "
Traditionally, HEK293 cells are cultured
in tissue culture flasks as adherent cells
that can produce enough for pre-clinical
and Phase I trials but are limited in
their ability to scale. These production
processes can be scaled-out but not
properly scaled-up. Blouin-Tavel states:
" We produce rAAV for use in gene therapies
and we must develop a process that
can be used in cGMP platforms.
We have been using cell stack flasks for
10 - 15 years, but this is a very manual
process and is not very efficient or suitable
for large-scale production. Now that
we are producing rAAV for retinal gene
therapy applications we need to produce
around 10¹² viral genomes (vg)/mL and
there are also requests for rAAV-based
therapies to treat neurological conditions,
but this requires titers of 10¹³ vg/mL, so
we really need to adapt our rAAV cell
culture processes to increase our productivity
for these applications. "
4 | GENengnews.com
According to Blouin-Tavel the issue is
scale-up. Producing enough rAAV in 24
cell stacks is possible but since the batch
size is limited to the number of T-flasks
that a scientist can successfully handle
manually, manufacturing is complicated
and time consuming. The lack of scalability
has led to many scientists, like the
team at University of Nantes, to look at
developing suspension culture protocols
using stirred tank reactors (STRs) or
rocking motion bioreactors instead of
traditional shake flasks. These platforms
can produce hundreds of liters of cell
culture, as well as offer greater flexibility,
scalability, and better control of the
culture environment to enable safe,
cost-effective larger scale rAAV production.
Robin concludes: " The goal in our labs
is to produce rAAV titers of >10¹⁰ vg/mL
and we hope in future to reach 10¹⁶ vg/mL
for clinical applications as this will
minimize the process transfer and keep
the scale-up to using 1,000 L STRs for
clinical batches. "
Suspension culture in STRs, especially
of HEK293-based rAAV production,
needs an additional process optimization
step. Robin comments: " HEK293 is an
adherent cell line so it has to be adapted
to suspension culture and in the past,
this meant that it did not produce rAAV
titers which were as high as with Sf9
cells. " Blouin-Tavel adds: " 10 years
ago, to produce large amounts of rAAV,
we would always use Sf9 insect cells
infected by recombinant baculovirus.
Sf9 insect cells grow well naturally in suspension,
and in addition to that infection
with recombinant baculovirus is efficient.
In parallel, to meet the need for large
quantities of rAAV vectors, the production
technology based on the transfection
of HEK293 cells in suspension cells
was also developed. But for many years
this method remained very expensive
because it was less efficient (lower yields
at the same scale) than the reference
technology (transfection of adherent
HEK293 cells).
" Since we are a
pioneering lab that is
experienced with rAAV
vector production,
our services are now in
greater demand, and
we are working with
partners to produce
more research and
pre-clinical grade rAAV
at up to 50 L scale "
Cécile Robin
Process Operation Manager
INSERM UMR 1089
University of Nantes
It was therefore more economical to
produce rAAV at large scale with the
Sf9 cells platform. However, in the past
three-to four years the culture media,
transfection reagents and plasmid systems
used for HEK293 cell suspension
have improved so much that rAAV yields
are becoming comparable to those
produced in Sf9 cells. This means it has
become viable to consider adapting
HEK293 to suspension culture, but it
does still need a lot of process optimization
for each serotype and product to
produce. "
https://www.genengnews.com/

Sartorius - November 2021 - Simplifying Adeno Associated Virus

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