(b) Use a peristaltic pump to fill the medium into the STR (see
Note 8).
7. Fill up the thermal jacket with VE water and start heating the
medium to 37
C via the external loop (see Note 9).
8. Start stirring at 110 rpm.
9. Fill up the base tube (dead volume) manually with 1 M NaOH
(see Note 10) and start pH regulation.
3.1.6 Cell Expansion
in the STR
Microcarrier (e.g., Cytodex 1) may provide the growth surface
required for the adherently growing cells in the STR.
1. Prepare and sterilize the microcarrier Cytodex 1 (3 g/L)
(GE Healthcare, Uppsala, Sweden) according to the manufacturer’s recommendations.
2. Split the DMEM medium:
(a) for conditioning in the bioreactor vessel (350 mL),
(b) for cell inoculation (150 mL).
3. Harvest cells from T-175 flask as described in Subheading
3.1.2.
4. Inoculate at the following cell density: 5 Â 10
3 cell/cm
2 .
5. Allow cells to adhere to the microcarrier (4–7 h) at 70 rpm
constant agitation rate.
3.1.7 Nanoplex
Production in the STR
1. A stable conductivity signal from the dielectric spectroscopy
indicates the time point at which the cells (e.g., Vero) completed their attachment and spreading on the microcarrier. This
represents the initial time point of infection.
2. To ensure a single infection cycle, infect the cells with a high
MOI: ~30 TCID 50 /cell. Lower MOIs may lead to several
infection cycles (see Note 11).
3. Optimal TOH is reached ~40 h after a global maximum of the
permittivity signal, which represents the detachment of the
cells from the microcarrier, rather than a maximum of syncytial
abundance. Stop the reactor run and start harvesting.
3.2 Downstream
Processing
3.2.1 Harvest of the Viral
Nanoplex-Containing
Supernatant and Primary
Clarification
Before starting to harvest, make sure you have prepared the following steps 1–10 to have the clarification equipment ready at the
point of harvest (step 11).
1. Connect the tubes to the Schott flasks (with outlet for the feed;
min. Volume 1 L) and place the depth filter capsule with the
correct orientation (flow direction is indicated on the capsule)
into the closed system.
2. Steam-sterilize the capsule, tubes, flasks and liquids at 121
C
for 20 min (autoclaving).
Viral Nanoplex Vaccine Production
233
Note 8).
7. Fill up the thermal jacket with VE water and start heating the
medium to 37
C via the external loop (see Note 9).
8. Start stirring at 110 rpm.
9. Fill up the base tube (dead volume) manually with 1 M NaOH
(see Note 10) and start pH regulation.
3.1.6 Cell Expansion
in the STR
Microcarrier (e.g., Cytodex 1) may provide the growth surface
required for the adherently growing cells in the STR.
1. Prepare and sterilize the microcarrier Cytodex 1 (3 g/L)
(GE Healthcare, Uppsala, Sweden) according to the manufacturer’s recommendations.
2. Split the DMEM medium:
(a) for conditioning in the bioreactor vessel (350 mL),
(b) for cell inoculation (150 mL).
3. Harvest cells from T-175 flask as described in Subheading
3.1.2.
4. Inoculate at the following cell density: 5 Â 10
3 cell/cm
2 .
5. Allow cells to adhere to the microcarrier (4–7 h) at 70 rpm
constant agitation rate.
3.1.7 Nanoplex
Production in the STR
1. A stable conductivity signal from the dielectric spectroscopy
indicates the time point at which the cells (e.g., Vero) completed their attachment and spreading on the microcarrier. This
represents the initial time point of infection.
2. To ensure a single infection cycle, infect the cells with a high
MOI: ~30 TCID 50 /cell. Lower MOIs may lead to several
infection cycles (see Note 11).
3. Optimal TOH is reached ~40 h after a global maximum of the
permittivity signal, which represents the detachment of the
cells from the microcarrier, rather than a maximum of syncytial
abundance. Stop the reactor run and start harvesting.
3.2 Downstream
Processing
3.2.1 Harvest of the Viral
Nanoplex-Containing
Supernatant and Primary
Clarification
Before starting to harvest, make sure you have prepared the following steps 1–10 to have the clarification equipment ready at the
point of harvest (step 11).
1. Connect the tubes to the Schott flasks (with outlet for the feed;
min. Volume 1 L) and place the depth filter capsule with the
correct orientation (flow direction is indicated on the capsule)
into the closed system.
2. Steam-sterilize the capsule, tubes, flasks and liquids at 121
C
for 20 min (autoclaving).
Viral Nanoplex Vaccine Production
233