Sartorius - November 2021 - Simplifying Adeno Associated Virus - 18

N14
N12
N17
4e+11
N13
N21
N4
N11
N6
N22
N7
N3
2e+11
N1
N2
N8
N10
N15
N9
N18
N5
N23
N19 N20
N24
N16
1e+11
Sf9 Medium 1
Sf9 Medium 2
Cell Density
Figure 7: Effect of cell density and media on the rAAV8 production yield in Sf9 cells using the Ambr®
microbioreactors. The concentration of DNA-containing particles was determined by qPCR in cleared lysate.
15
Conclusion
Our goal was to rapidly identify optimal process factors for
producing rAAV vectors with HEK293 and Sf9 in suspension
culture. We have shown that the Ambr®
15 microbioreactor
system can be used as a scale-down model in a step-wise
approach to identify multi-factor CPPs for optimizing
conditions of plasmid transfection (HEK293) and baculovirus
infection (Sf9), followed by process optimization. When used
with MODDE®
DOE software, this system enables rapid,
high-throughput process improvement and optimization
to produce HEK293 suspension titers of >1 × 10¹⁰ vg/mL in
cleared lysate. With HEK293 cells, we developed a suspension
process that matches our adherent cell production process
and identified pH, plasmid amount and Transfection Reagent
3 as multi-factor interacting CPPs that increase rAAV8 titer.
Based on our findings, we identified a set-point of low pH (6.9)
and high total plasmid (2.7 - 3.0 µg/mL) as being optimal for
high rAAV productivity. With Sf9 cells, the optimization of
production has allowed us to obtain titers of >4 × 10¹¹ g/mL in
cleared lysate, and we determined that Sf9 Medium 2, VCC
at infection and Lysis Reagent 2 are interacting multi-factor
CPPs and identified the set-point for VCC as 8 × 10⁶ cells/mL
and 1% Lysis Reagent 2.
We have achieved our process optimization with just three
sets of experiments (HEK293) and two sets of experiments
(Sf9). Using our current laboratory set-up, each experimental
run was completed in one month, and as we are becoming
more familiar with the system and DOE software, we aim to
reduce each run to two-weeks. This will be a significant time
saving compared to performing process optimization with
shake flasks where a run can take several months to complete.
18 | GENengnews.com
vg/mL
4.00e+06
4.00e+06
4.00e+06
4.00e+06
4.00e+06
4.00e+06
5.33e+06
5.33e+06
5.33e+06
6.00e+06
6.00e+06
6.00e+06
6.00e+06
6.00e+06
6.67e+06
6.67e+06
8.00e+06
8.00e+06
8.00e+06
8.00e+06
8.00e+06
8.00e+06
8.00e+06
8.00e+06
https://www.genengnews.com/

Sartorius - November 2021 - Simplifying Adeno Associated Virus

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Sartorius - November 2021 - Simplifying Adeno Associated Virus - 18
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