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Scale-X™ Bioreactor Seed Amplification Enhancement Application Note
111 1 Introduction
(1) (2) (3-6) (7).
required to prepare a 600 m² fixed-bed bioreactor inoculum using plastic culture vessels (T-bottles and multilayer vessels).
Assumptions: 1) Inoculation density of 3x104 for plastic culture vessels, 1x104 for large-scale nitro 600 bioreactors, 2) harvest yields of 2x105 cells/cm² for plastic culture vessels, and 3) 632 cm²
for each multilayer vessel (MT) surface area.
Figure 2.
Novel cell harvesting module for scale-X carbo systemsConceptual schematic
of the scale-X carbo harvest module.
Left: Scale-X carbo controller (commercial product for GMP and non-GMP cell cultures
).
Right: Concept Cell Harvest module (*now front-end development).
Materials and methods
2.
3.
4.
5.
Figure 3.
Overview of the cell harvesting protocol for the scale-X bioreactorCells are expanded in a scale-X bioreactor for 4-6 days (1
).
On harvest day, rinse the bioreactor (2) and then fill and incubate with enzyme solution for 20-25 min (3).
Subsequently, the bioreactor is transferred to the harvest module while applying vibration and drainage (4
).
Finally, rinse is performed for cell recovery (5
).
Steps 1-3 are performed
using a bioreactor mounted on the scale-X hydro/carbo controller.
For steps 4-5, these bioreactors are transferred to the harvest module
.
outcome
Table 2.
Summary of cell harvest results
.
Micrographs
of cells harvested from a fixed-bed bioreactor (left) and control cells harvested from a T-bottle (right) on day 3 after re-seeding are shown.
B) Cells harvested from a scale-X hydro bioreactor (#4) are used to seed a second hydro bioreactor (#5
).
The trend
of cell density (left axis) and metabolite lactate and glucose concentration (right axis) is shown in two consecutive runs.
Arrows indicate the time
of harvesting and reinoculation to the second bioreactor.
Figure 5.
Process economy for large-scale viral production based on adherent cells, seed amplification chain strengtheningThe overall cost estimate
was calculated by comparing processes using plastic culture vessels and using scale-X carbo 30, inoculated with a 600 m² fixed-bed bioreactor at 1x104 cells/cm².
The results include only the cost
of the seed amplification phase.
The cost
of continuous unit operations was not covered in this study.
The key assumptions used are detailed
in the Materials and Methods section.
Discussion and summary of references: 1.
R.
Baghirzade, Adherent versus suspension based platforms: what is the near future of viral vector manufacturing? Cell Gene Ther.
Insights.
7, 1365–1371 (2021).
2.
T.
Pereira Chilima, Innovative fixed-bed bioreactors unify adherent & suspension processing.
Univercells Technol.
(2021).
3.
D.
M.
Berrie, R.
C.
Waters, C.
Montoya, A.
Chatel, E.
M.
Vela,Development of a high-yield live-virus vaccine production platform using a novel fixed-bed bioreactor.
Vaccine.
38, 3639–3645 (2020).
4.
A.
Chatel, Y.
Ghislain, P.
Puri, T.
Zijdemans, “Scalable Rubella production in scale-X™ bioreactor with MRC-5 cells: Process optimization and scale-up from static flask to scale-X bioreactor with 30 m2 cell growth surface” (2021).
5.
R.
Carvalho, T.
P.
Chilima, C.
Meeüs, E.
Gateau, M.
Ferraiuolo, A.
Noto, “Suspension-based viral vector production in scale-™ fixed-bed bioreactor Shake flask process transferred in a scale-X hydro fixed-bed bioreactor for Adenovirus production with HEK293 cells adapted to suspension.
” (2021).
6.
J.
-C.
Drugmand, A.
Chatel, J.
Castillo, Y.
Dohogne, M.
Gras, F.
Collignon, S.
Kumano, H.
Shiomi, scale-X™ bioreactor for viral vector production: Proof of concept for scalable HEK293 cell growth and adenovirus production, (2020)
7.
Univercells Technologies, The NevoLine™ Upstream platform -Intensified an automated virus manufacturing (2022), (available at scale-X™ carbo fixed-bed bioreactor
111 1 Introduction
(1) (2) (3-6) (7).
Figure 1.
Overview of large-scale adherent bioreactor seed amplification chain requirements using plastic culture vessels
The figure above depicts the continuous stepsOverview of large-scale adherent bioreactor seed amplification chain requirements using plastic culture vessels
required to prepare a 600 m² fixed-bed bioreactor inoculum using plastic culture vessels (T-bottles and multilayer vessels).
Assumptions: 1) Inoculation density of 3x104 for plastic culture vessels, 1x104 for large-scale nitro 600 bioreactors, 2) harvest yields of 2x105 cells/cm² for plastic culture vessels, and 3) 632 cm²
for each multilayer vessel (MT) surface area.
Figure 2.
Novel cell harvesting module for scale-X carbo systems
of the scale-X carbo harvest module.
Left: Scale-X carbo controller (commercial product for GMP and non-GMP cell cultures
).
Right: Concept Cell Harvest module (*now front-end development).
Materials and methods
Table 1.
Bioreactor culture conditions
1.Bioreactor culture conditions
2.
3.
4.
5.
Figure 3.
Overview of the cell harvesting protocol for the scale-X bioreactor
).
On harvest day, rinse the bioreactor (2) and then fill and incubate with enzyme solution for 20-25 min (3).
Subsequently, the bioreactor is transferred to the harvest module while applying vibration and drainage (4
).
Finally, rinse is performed for cell recovery (5
).
Steps 1-3 are performed
using a bioreactor mounted on the scale-X hydro/carbo controller.
For steps 4-5, these bioreactors are transferred to the harvest module
.
outcome
Table 2.
Summary of cell harvest results
Figure 4.
Reoccupation of harvested cells
in T-bottles and fixed-bed bioreactors.
A) Cells harvested from all bioreactor runs are re-seeded in T-flasks for growth curve and morphological assessmentReoccupation of harvested cells
in T-bottles and fixed-bed bioreactors.
.
Micrographs
of cells harvested from a fixed-bed bioreactor (left) and control cells harvested from a T-bottle (right) on day 3 after re-seeding are shown.
B) Cells harvested from a scale-X hydro bioreactor (#4) are used to seed a second hydro bioreactor (#5
).
The trend
of cell density (left axis) and metabolite lactate and glucose concentration (right axis) is shown in two consecutive runs.
Arrows indicate the time
of harvesting and reinoculation to the second bioreactor.
Figure 5.
Process economy for large-scale viral production based on adherent cells, seed amplification chain strengthening
was calculated by comparing processes using plastic culture vessels and using scale-X carbo 30, inoculated with a 600 m² fixed-bed bioreactor at 1x104 cells/cm².
The results include only the cost
of the seed amplification phase.
The cost
of continuous unit operations was not covered in this study.
The key assumptions used are detailed
in the Materials and Methods section.
Discussion and summary of references: 1.
R.
Baghirzade, Adherent versus suspension based platforms: what is the near future of viral vector manufacturing? Cell Gene Ther.
Insights.
7, 1365–1371 (2021).
2.
T.
Pereira Chilima, Innovative fixed-bed bioreactors unify adherent & suspension processing.
Univercells Technol.
(2021).
3.
D.
M.
Berrie, R.
C.
Waters, C.
Montoya, A.
Chatel, E.
M.
Vela,Development of a high-yield live-virus vaccine production platform using a novel fixed-bed bioreactor.
Vaccine.
38, 3639–3645 (2020).
4.
A.
Chatel, Y.
Ghislain, P.
Puri, T.
Zijdemans, “Scalable Rubella production in scale-X™ bioreactor with MRC-5 cells: Process optimization and scale-up from static flask to scale-X bioreactor with 30 m2 cell growth surface” (2021).
5.
R.
Carvalho, T.
P.
Chilima, C.
Meeüs, E.
Gateau, M.
Ferraiuolo, A.
Noto, “Suspension-based viral vector production in scale-™ fixed-bed bioreactor Shake flask process transferred in a scale-X hydro fixed-bed bioreactor for Adenovirus production with HEK293 cells adapted to suspension.
” (2021).
6.
J.
-C.
Drugmand, A.
Chatel, J.
Castillo, Y.
Dohogne, M.
Gras, F.
Collignon, S.
Kumano, H.
Shiomi, scale-X™ bioreactor for viral vector production: Proof of concept for scalable HEK293 cell growth and adenovirus production, (2020)
7.
Univercells Technologies, The NevoLine™ Upstream platform -Intensified an automated virus manufacturing (2022), (available at scale-X™ carbo fixed-bed bioreactor
