Sartorius - November 2021 - Simplifying Adeno Associated Virus - 11

Optimization of Transient
Production of rAAV in
Suspension Culture
Process optimization for rAAV production
in suspension culture is usually performed
in shake flasks and benchtop
bioreactors (2 L - 5 L) for scale-up to pilot
and manufacturing scale STRs. Due to
the high cost of reagents, consumables,
and labor, this format rapidly becomes
too expensive and labor intensive to
determine Critical Process Parameters
(CPPs). As a proof-of-concept study, we
wanted to establish optimal working protocols
in a small-scale STR for culturing
suspension-based HEK293 and Sf9
cells to produce rAAV at titer levels of
>1 × 10¹⁰ vg/mL. With the goal of increasing
throughput during development, we
used the Ambr®
15 cell culture system
from Sartorius (Figure 1) with its singleuse
(SU) micro bioreactors (15 mL max
working volume) in conjunction with
MODDE®
Experiment) planning and analysis of
results. We chose the Ambr®
software for DOE (Design of
15 system
as each micro bioreactor reproducibly
mimics features and process control
variables (such as pH, dissolved oxygen
[DO], temperature, and stirring rate)
seen in larger STRs. The system can
screen multiple parameters in parallel, to
identify factors that significantly affect
Critical Quality Attributes (CQAs) such
as viral titer. When used with MODDE®
software, the system can enable a rapid
DOE approach to help choose which
process parameters to assess and can
identify multiple interacting factors to
determine optimal set-points for CPPs
and improve process understanding.
Optimizing rAAV Production
Parameters With HEK293
Suspension Cells
To determine optimal process conditions
for rAAV serotype 8 production in
HEK293 suspension cells by plasmid
transfection, we used a three-step
approach. Step One involved screening
a range of culture factors including
commercially available media, cell lines
and transfection reagents to determine
which produce optimal transfection conditions
and to determine CPPs and their
set-points. In Step Two, we selected the
best performing transfection factors, and
used a DOE to test a range of culture
parameters. In Step Three, we tested the
optimized conditions to validate them
with an additional transfection reagent.
Figure 1: Ambr®
15 cell culture multi-parallel microbioreactor system
for process optimization studies of rAAV in suspension culture
11 | GENengnews.com
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Sartorius - November 2021 - Simplifying Adeno Associated Virus

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