Virus Production From Bench Scale To Industrial Scale:
New Technologies That Intensify, Simplify Virus Manufacturing
And Increase Overall Process Efficiency
A. Onraedt , P. Lefebvre , M. Kremer (Pall Life Sciences)
SUMMARY
Contributing to the complexities of developing viral vaccines are the availability of purpose built process technologies to manufacture virus. Current upstream and downstream technologies either are scaled-out laboratory tools, including 2D growth
surfaces for adherent cells, or are derived from other biotech processes, where the size of the molecule or its shear sensitivity are not as critical as in virus manufacturing. The streamlined viral manufacturing platform that will be discussed is based
on technologies that are specifically developed for large-scale manufacture of virus for vaccine or gene therapy applications. The result is a process that simplifies virus manufacturing and reduces manufacturing footprint. It also increases the process
efficiency and reduces cost-of-goods versus traditional approaches.
For the adherent culture of the host cell line, the iCELLis® fixed-bed bioreactor from Pall Life Sciences is designed to achieve high cell densities and good virus productivity by combining controlled growth conditions and a large growth surface area
in a small reactor volume. This has been demonstrated on a wide range of viruses including MVA, Bovine Herpes, Paramyxovirus, Influenza, Adenovirus, rAAV and more in various production cell lines, including VERO, HEK293 and MDBK.
Scale-up of the cell culture and virus production from benchtop iCELLis bioreactors to up to 500 m² has been realized. Specific virus productivity in the iCELLis bioreactors at benchtop and industrial scale was similar or better than that achieved in
static culture or roller bottles. In combination with Pall’s virus downstream processing solution, the iCELLis bioreactor offers a versatile platform for industrial scale virus production.
TRANSFER AND SCALE-UP OF A HEK293 CELL CULTURE
PROCESS FOR PRODUCTION OF ADENOVIRUS
PALL VIRUS MANUFACTURING PLATFORM
Sequence of automated, single-use technologies optimized for high virus yield and quality, process robustness and
optimized process economics:
Figure 2
The iCELLis nano bioreactor system
(from 0.53 to 4 m2)
Cell debris rremoval
emoval
with V100P depth filter
Final sterilizing filtration
Further formulation & filling
Hardware
iCELLis nano bioreactor (from 0.53 to 4 m2 )
iCELLis 500 bioreactor (133 m2)
Metabolites were analyzed with a Bioprofile-100
bio-analyzer (Nova Biomedical, MA, USA)
Biological Material
HEK293 cells were grown in serum-supplemented
medium
Purification with Mustang®
AEX and Cadence™ SU TFF
ICELLIS BIOREACTOR
Linear velocity of medium through the fixed-bed (cm/s)
Fixed-bed height (2, 4 or 10 cm)
RESULTS
The single-use iCELLis bioreactor containes a compact fixed-bed with carriers made of medical grade polyester. This
matrix provides a large growth surface area in a relatively small volume: the iCELLis design provides up to 500 m2 in only
25 liters of fixed-bed. Commercially available iCELLis bioreactors range in size from 0.53 to 500 m2 with two compaction
densities of carriers (Table 1).
Table 1
Commercially available iCELLis bioreactor size specifications
Fixed-Bed (FB)
Compaction Density
FB Height (cm)
iCELLis Nano Bioreactor
(Process Development)
FB Volume (L) FB Surface Area (m2)
96 g/L
(C 1)
13 m2/L
144 g/L
(C 1.5)
19.5 m2/L
2
4
10
2
4
10
0.04
0.08
0.2
0.04
0.08
0.2
0.53
1.06
2.67
0.8
1.6
4.0
Table 2
Results of HEK293 cell culture four days after inoculation in small- and large-scale iCELLis bioreactors.
Average
Average
Fixed Bed
Fixed Bed
Cell Density
Total
Bioreactor
Surface
Height
Fixed Bed
Reached
Cells per
Scale
Area (m2)
(cm)
Volume (L)
(cells/cm2)
Bioreactor
iCELLis 500 Bioreactor
(Large-Scale Production)
FB Volume (L) FB Surface Area (m2)
iCELLis nano
iCELLis 500
5
10
25
5
10
25
Analysis of glucose and lactate (Figure 3) at both scales in comparison to a 5-tray Cell Factory control indicated that cell
metabolism was comparable between small and large scale iCELLis bioreactors and the standard 2-D process.
66
133
333
100
200
500
Figure 1
The iCELLis 500 control system and bioreactor
1.06
133
4
4
0.08
10
2.8 to 3.8 x 105
2.7 to 3.4 x 105
5
30
4
25
3
5CS
2
nano (1,06 m2)
large (133 m2)
1
0
Outer shell, top section
with probes and ports
0
0.5
1
1.5
2
2.5
Time (days)
3
3.5
4
4.5
20
5CS
15
nano (1,06 m2)
10
large (133 m2)
5
0
0
0.5
Fixed-bed retaining ring
Fixed-bed filled with
500 m2 macrocarriers
1
1.5
2
2.5
Pump housing
Impeller
Pump housing ring
Outer shell, bottom section
Double-jacket
Evenly distributed medium circulation is powered by a built-in magnetic mixer, ensuring low shear stress. The cell culture
medium flows through the fixed-bed from the bottom to the top and returns to the bottom of the bioreactor as a waterfall,
resulting in a high mass transfer co-efficient (kLa).
In the iCELLis bioreactors, the following parameters can be measured and controlled: pH, DO, biomass (using a biomass
probe), temperature, gas flow rate, agitation, pressure and medium recirculation/perfusion rate.
The unique waterfall oxygenation together with gentle agitation, biomass immobilization and process control provides a
favored growth environment that can increase the cell specific productivity of the desired protein or virus.
3
3.5
4
4.5
Time (days)
VIRUS PRODUCTION RESULTS IN ICELLIS
Table 3
Results for different viruses from various cell lines at different bioreactor scales.
Surface Productivity
Total Virus
iCELLis SA.
(Virus Titer per cm2)
Extrapolated to
Cells
Virus
Tested (m2)
iCELLis
Control
Units
500 m2 iCELLis
CEF
MVA
0.07*
3.5E+06
1.0E+07
pfu/cm2
1.7E+13 pfu
MDBK
Bovine
4
2.2E+07
1.3E+07
pfu/cm2
1.1E+14 pfu
Herpes
20*
1.7E+07
8.5E+13 pfu
Virus
66
3.3E+07
1.6E+14 pfu
A549
rAAV
0.53
5.0E+08
3.0E+08
vg/cm2
2.6E+15 vg
Adenovirus
2.67
9.8E+09
9.6E+09
TCID50/cm2
4.9E+16 TCID50
Vero
Influenza
4
3.8E+06
1.3E+06
TCID50/cm2
1.9E+13 TCID50
20*
2.5E+06
1.3E+13 TCID50
Paramyxovirus 2.67
6.4E+05
5.7E+04
TCID50/cm2
3.2E+12 TCID50
2.9E+08
5.3E+07
Vp/cm2
1.5E+15 vp
Undisclosed
40*
Confidential
Lytic Virus
133
660*
L
Fixed-bed support
3.5 x 109
4.1 x 1011
Figure 3
Comparability of Glucose and Lactate Profiles of HEK293 culture in the iCELLis 133 m2 bioreactor (Blue), iCELLis
T
2
nano bioreactor 1.06 m (Green) and 5-tray Cell Factory (Red).
Glucose (g/L)
Virus pr
production
oduction in iCELLis 500
Cell Culture Parameters
An existing process using HEK293 cells for the production
of adenovirus was first transferred from multi-tray systems
to an iCELLis nano bioreactor (0.53 m2, 40 mL of fixed-bed)
by keeping equivalent cell culture parameters:
Temperature, pH and DO (% saturation with air)
Multiplicity of infection (pfu/cell)
Time of infection
Cell seeding density (cells/cm2 and cells/mL)
Culture duration
Additional experiments were performed with lower cell
densities at inoculation in order to reduce the number of
preculture steps at large scale. The following parameters were
also optimized for cell growth and virus productivity:
Compaction of carriers inside the fixed-bed (96 g/L or
144 g/L)
Lactate (mM)
Virus analytics
with BLI (Octet®, Blitz®)
EXPERIMENTAL APPROACH
Scale Up of Control
Process Required
to Match iCELLis 500
780 CF-10
310 L MC (6g/L)
240 L MC (6g/L)
460 L MC (6g/L)
1300 CF-10
810 CF-10
540 L MC (6g/L)
360 L MC (6g/L)
8900 CF-10
4300 CF-10
794 CF-10
(serum-free)
1238 CF-10 (+serum)
*Not a standard bioreactor size. Please refer to table 1 where all commercially available bioreactor sizes are specified
CONCLUSION: VIRUS PRODUCTION INTENSIFICATION AND SIMPLIFICATION
This summary of experiments demonstrates that the fixed-bed design of the iCELLis bioreactor enables high cell densities to be achieved and maintained in both small and large bioreactor volumes. Different processes have been easily scaled up
by keeping cell culture conditions and process parameters identical to the standard 2-D cell culture process. The iCELLis bioreactor can be inoculated at a very low cell density, leading to a dramatic simplification of seed train operations and a
significant reduction of development timelines. In conclusion, large biomass amplification and excellent virus productivities, combined with the advantages of a fully closed single-use system with low shear stress, make the iCELLis fixed-bed
bioreactor a simple and straightforward solution for industrial production of viruses. In combination with Pall's virus downstream processing solution, it brings an industrial virus production platform with a reduced footprint that is fully automatable
for better control and that brings overall process economics.
Contact: 800.717.7255 (USA and Canada) • 1.516.484.5400 (Outside USA and Canada) • www.pall.com/bioreactors • www.pall.com/vaccines • E-mail: vaccines@pall.com
© 2015, Pall Corporation. Pall,
, Blitz, Cadence, iCELLis, Mustang, and Octet are trademarks are trademarks of Pall Corporation. ® indicates a trademark registered in the USA. 3/15, GN15.6201