Sartorius - November 2021 - Simplifying Adeno Associated Virus - 25

To optimize culture parameters, DOE
was used. The following parameters
(Table 1) were investigated to find the
best operating conditions for high
rAAV titer.
Other cultivation factors were set
as constant including DO at 30%,
proprietary pH and stirring speeds
for 96 hours at 27 ˚C with harvest
time points of 72 h and 96 h. Cells
were infected at a range of VCDs
using a recombinant baculovirus
ratio of 1:1 and a range of proprietary
multiplicity of infection (MOI) ratios
of Baculovirus Helper | Baculovirus
Vector.
Parameter
Viable cell density (VCD)
at transfection
Media
Ratios of Baculovirus Helper |
Baculovirus Vector
Type of lysis reagents
Abbreviation
Cell
-
rat
Lys
Sf9 Medium 1
Low
Lysis reagent 1
Sf9 Medium 2
High
Lysis reagent 2
Table 1: Parameters tested to determine optimum operating conditions for high rAAV titer
Design of Experiments
The MODDE®
software guides the
experimental setup. As an outcome
a D-optimal design with 24 experiments
was selected and the runs
performed. In a next step the guided
analysis workflow of the software was
used to analyze the DOE data. Coefficient
and contour plots were then
used for exploring the investigated
space to define optimal conditions.
DOE Analysis
The DOE model is represented by the
coefficient plot (Figure 1). The higher
the bar, the greater the influence the
tested parameter has on viral titer. The
analysis clearly shows that Baculovirus
Helper | Baculovirus Vector ratio and
cell density had the largest effects on
rAAV titer (Figure 1).
Titer 120 h
01.5
1
0.5
-0.5
-1
-1.5
Sf9 Medium 1
Sf9 Medium 2
Titer 120 h (N=22, DF=16, R2=0.99), Confidence = 0.95
Ratio
Cell Densitiy
Lysis Reagent 1Lysis Reagent 2Ratio×Ratio
Figure 1: Coefficient plot for titer versus a range of cell culture factors obtained from transient production
of rAAV8 with Sf9 cells in Ambr®
15 micro bioreactors.
25 | GENengnews.com
Titer [vg/mL]
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Sartorius - November 2021 - Simplifying Adeno Associated Virus

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