Sartorius - November 2021 - Simplifying Adeno Associated Virus - 14

1,500
1,000
500
-500
-1,000
-1,500
-2,000
Cell Line 1Cell Line 2 Stirring Speed
pH
DO
DO × DO
Cell Line 1
×Stirring
Speed
15 micro bioreactors.
Cell Line 2
×Stirring
Speed
Cell Line 1
×pH
Figure 3: Coefficients plot for rAAV vector genome specific productivity (vg/cell) versus a range of cell culture factors
obtained from production of rAAV8 with suspension HEK293 cells in Ambr®
Cell Line 2
×pH
pH×DO
Step 3: Process Validation
To validate our optimized process factors,
we used the outcome from Steps One
and Two, to generate a DOE to test them
and to assess the effects of a new transfection
reagent, Transfection Reagent 3.
Furthermore, we analyzed the factors
total plasmid DNA in a range of
0.5 - 3 µg/mL and pH from seeding to
6 h post-transfection in a range of 6.9 - 7.5.
At 24 hours before transfection, Cell Line
2 was inoculated into Media 2 at a VCC
of 1.75 × 10⁶ cell/mL in the Ambr®
15
microbioreactors, at 37 ˚C, DO 50%,
stirring speed 400 rpm and pH 7.3
(6 h after transfection until harvest).
For transfection, we used Transfection
Reagent 1 in the ratio Reagent | DNA:
2.5 µL : 1 µg and Transfection Reagent 3
in ratio of Reagent | DNA: 2 µL : 1 µg.
We also used a range of total amounts of
plasmid DNA with a transfection mix
volume of 5% and a complex time of
20 minutes for Transfection Reagent 1
and transfection mix volume of 10%
and a complex time of 15 minutes for
Transfection Reagent 3.
The optimum conditions in this run produced
more than 1 × 10¹⁰ vg/mL in cleared
lysate. We used the MODDE®
software
to analyze the specific productivity and
found that pH and transfection reagent
have the most impact.
In addition, total plasmid amounts lower
than 0.5 µg/mL have a negative effect
whatever the transfection reagent. Using
the software, we then analyzed these
three factors to generate a 4D contour
plot and a Dynamic profile plot (Figure 4)
and found that the optimum multi-factor
combination was Transfection Reagent 3,
low pH (6.9) and high total plasmid
(2.7 - 3.0 µg/mL).
14 | GENengnews.com
Specific Productivity [vg/cell]
https://www.genengnews.com/

Sartorius - November 2021 - Simplifying Adeno Associated Virus

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