Sartorius - November 2021 - Simplifying Adeno Associated Virus - 16

Optimizing rAAV Production
in Sf9 Insect Cells
To determine optimal process conditions
for rAAV8 production in the dualbaculovirus
| Sf9 insect cell platform, a
two-step approach was used. Step One
involved screening factors to determine
which provided the optimal conditions
with the two baculoviruses required for
the rAAV vector production, i.e., the
baculovirus harboring the rAAV genome
(baculovirus vector) and the baculovirus
allowing expression of Rep and Cap proteins
(baculovirus helper). In Step Two,
the best performing factors were selected,
and a DOE was used to test a range
of process factors, including chemical
cell lysis, to determine the optimum
multi-factor combination of CPPs.
Step 1: Optimizing Baculovirus Infection
Conditions
We inoculated Sf9 cell into two different
proprietary media, Sf9 Medim 1, and Sf9
Medium 2. The cells in each different
medium were cultured in spinner flasks
and were inoculated in 24 microbioreactors
2 hours before infection at 27 ˚C,
30% DO, with stirring speed of 750 rpm.
For infection, we varied the Multiplicity
of Infection (MOI) of the Baculovirus
Helper, keeping a constant MOI for the
Baculovirus Vector (MOI 0.1 based on
CSA infectious titer², the VCC at infection
and chemical reagents for cell lysis (see
Table 2). To determine infection
efficacy, we measured rAAV titer and
total particle content using the methods
mentioned previously. We also measured
VCC and cell viability post-infection.
The optimum multi-factor conditions
in this run produced rAAV titers
> 5 × 10¹¹ vg/mL (Figure 5).
Factor
Range
Baculovirus ratio
[MOI Baculovirus Helper : Baculovirus Vector]
Cell density at infection [cells/mL]
Chemical lysis at harvest
Table 2: DOE for optimizing infection conditions
Ratio: 10 (1:0.1); 1 (0.1:0.1);
0.1 (0.01:0.1); 0.01 (0.001:0.1);
0.001 (0.0001:0.1)
1 × 10⁶; 2.5 × 10⁶; 4 × 10⁶
Cell Lysis Reagent 1
and Reagent 2 0.5% v/v
Set-points
5.0e+12
5.0e+11
5.0e+10
5.0e+09
5.0e+08
5.0e+07
5.0e+06
MOI Bac Helper
Figure 5: rAAV vector genome titers versus a range of conditions for baculovirus infection, cell culture
and lysis with Sf9 insect cells in Ambr®
15 micro bioreactors.
We observed that the best AAV titer
results are obtained when the cells were
infected with the baculovirus helper at a
MOI of 0.01 to 0.1.
We used the MODDE®
software to
generate coefficients plots for titer which
showed that MOI ratio, VCC and Lysis
Reagent 2 have the greatest positive
impact (Figure 6).
Step 2: Optimization of Culture
Parameters
As we observed in the dynamic profile
plot from Step One (not shown) that a
further increase of VCC at infection
could potentially increase rAAV titer, we
used the data from the optimized infection
DOE, to select a range of VCCs at
infection (4 × 10⁶ cells/mL - 8 × 10⁶ cells/mL)
and Lysis Reagent concentrations
(0.1% v/v - 1% v/v) for producing the optimum
titer. We inoculated the Sf9 cells
into Sf9 medium 1 and Sf9 Medium 2.
16 | GENengnews.com
Titer [vg/mL]
0.0001
0.0001
0.0001
0.0001
0.001
0.001
0.001
0.01
0.01
0.1
0.1
0.1
0.1
0.1
1
1
1
1
1
1
1
1
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Sartorius - November 2021 - Simplifying Adeno Associated Virus

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