Application Note
USD3013
Monoclonal Antibody Production in Suspension
CHO Culture Using Single-Use PadReactor®
Mini Bioreactor
Introduction
Single use solutions are found in many applications as they offer straightforward alternatives to classical glass or
stainless steel bioreactor systems. These ready-to-use systems require neither cleaning nor sterilization steps by
the user, while facilitating the scale-up from bench scale to large scale productions.
Within the range of its PadReactor bioreactors, Pall Life Sciences has introduced the PadReactor Mini bioreactor
(Figure 1), as a scale-down model perfectly suited for feasibility studies, research, process development and
small volume production of cells, proteins or viruses from both suspension and microcarriers cultures. The
gentle mixing combined with efficient gas transfer allows the PadReactor Mini bioreactor to host suspension
and anchorage-dependent cell cultures (e.g. on microcarriers). Critical cell culture parameters such as pH, DO,
and temperature are monitored and controlled by the PadReactor Mini controller.
This application note will present the transfer of a monoclonal antibody (mAb) production process from a classical
glass stirred-tank reactor (STR) to a single-use 16 L PadReactor Mini bioreactor.
Material and Methods for Process Transfer in PadReactor Mini Bioreactor
The mAb production process is based on CHO cells (undisclosed cell strain). The process was applied in 3 systems
for comparison: shake flask (ShF), Applikon◆ glass STR with pitched-blade impeller and microsparger, and
PadReactor Mini system (microsparger), a single-use bioreactor using patented mixing paddle technology.
PadReactor Mini bioreactor was monitored and controlled using a dedicated controller (ez-control from Applikon
adapted for Padreactor bioreactor). STR was controlled with Tryton controller (Pierre Guerin). Shake Flasks were
incubated in INFORS◆ shaker.
Additional equipment used:
• Haemocytometer and trypan blue for cell counting and viability determination;
• Analyzer for Glucose (Roche) and Lactate (Scout) for metabolite follow-up;
• ELISA kit solutions (R&D Biotech) for mAb titration;
Figure 1
PadReactor Mini system
Pre-cultures were done in shake flasks over 3 passages to amplify cells for simultaneous inoculation of the 3 systems.
Cells were kept in culture for 7 days with daily monitoring of cell density, cell viability, metabolites and mAb titer.
2
On day 3, mAb production was activated by sodium butyrate addition.
In all systems temperature was maintained at 37 °C. In the PadReactor Mini bioreactor and the STR, agitation,
aeration and base addition were adapted to maintain a DO of 50% (air sat.) and a pH of 7.20. All culture and
operating conditions are summarized in Table 1.
Table 1
Cell culture and operating conditions
PadReactor Mini
Bioreactor Microsparger
System
Cell type
Inoculation density
Medium and reagents
Glass Stirred-Tank reactor (STR)
Shake Flask
CHO
0.4x106 cells/mL
Working volume
Agitation speed
Temperature
pH setpoint and regulation
Dissolved oxygen setpoint
and regulation
CHO
CHO
0.4x106 cells/mL
0.4x106 cells/mL
Power CHO-2 CD, w/o L-gln, PR or HT (Lonza) + 4mM Glu
Sodium butyrate addition on day 3 (final concentration 2mM)
13 L
3L
350 mL
70 RPM
60 RPM
130 RPM
37 °C
37 °C
37 °C
7.20
7.20
95% Air - 5% CO2
Air/CO2 in overlay
Air/CO2 in overlay
Sodium hydroxide 1M
Sodium hydroxide 1M
> 50%
> 50%
95% Air - 5% CO2
O2 sparging in medium
O2 sparging in medium
Results and Discussion
Cell growth
Cell growth and viability as well as metabolites follow-up were monitored daily. The peak of cell density in the
PadReactor Mini bioreactor was 6.9 x 106 viable cells/mL, reached after 4 days of culture. In the STR the
maximum density achieved was similar, 6.1 x 106 viable cells/mL and is delayed compared to PadReactor Mini
bioreactor culture. In both systems viability stayed near 100% for the first 3 days and decreased to 40% over
the following days (Figure 2). The decrease in cell density and viability corresponds to glucose depletion in the
medium (Figure 3) which occurs after 4 days in the PadReactor Mini bioreactor and after 5 days in the STR.
These observations show that cell growth and metabolites evolution in the PadReactor Mini bioreactor exhibits
a behavior equal to or better than the classical STR.
Cell Growth and V
iability Monitoring in Padreactor Mini and STR
Viability
100
9.0
90
8.0
80
7.0
70
6.0
60
5.0
50
4.0
40
3.0
30
2.0
20
1.0
10
0.0
0
1
2
4
3
Culture duration (Day)
Padreactor - Cell density
5
6
STR - Cell density
7
0
V
Viability
iability (%)
Cell concentration
entration (million cell/mL))
1
10.0
Figure 3
Glucose and Lactate concentrations follow-up in the
PadReactor Mini bioreactor and STR.
Glucose and Lactate concentration (mM)
Figure 2
Viable cell density and viability monitoring in the
PadReactor Mini bioreactor and STR.
Cell Growth and V
iability Monitoring in Padreactor Mini and STR
Viability
50.0
45.0
40.0
35.0
30.0
25.0
20.0
15.0
10.0
5.0
0.0
0
1
2
3
4
5
Culture du
duration
ration (Day)
Padreactor - Glucose
6
7
STR - Glucose
www.pall.com/biopharm
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Monoclonal Antibody production
mAb titer was measured from daily sampling by ELISA method. Follow-up shows a constant increase in titer up
to day 6 in both the PadReactor Mini bioreactor and the STR, reaching 182 and 188 mg/L respectively (Figure 4).
The cell specific productivity, defined as mAb titer divided by total cell concentration (viable and non-viable), was
calculated for each system. By comparing the maximum reached in each system (Figure 4) we see that the
PadReactor Mini bioreactor and the STR had very similar specific productivities. The shake flask performance,
used only for final point reference is below the PadReactor Mini bioreactor and the STR. This is most probably
due to the absence of culture condition control in this system.
Monoclonal Ab titers measured by ELIS
ELISA
A
250
Monoclona
Monoclonall Ab spec prod
Mabcell PA
PAD
D
MAb titer (mg/ml)
200
30
Mabcell STR
25
Mabcell SHF
150
20
15
100
10
50
0
5
0
1
2
3
4
5
Culture duration (Day)
Padreactor - mAb titer
6
7
1
2
3
0
Mab specific productivity (mg/million cell)
Figure 4
Volumetric production of CHO cells in PadReactor Mini bioreactor and STR (trends) and maximum mAb cell
specific productivity peak (bars). Error bars represent ELISA method error.
STR - mAb titer
Conclusion
In this study, a CHO-based monoclonal antibody production was transposed without any productivity loss
from classical glass stirred tank reactor to PadReactor Mini bioreactor. The growth results suggest that culture
conditions are more favorable thanks to the paddle-driven agitation. This demonstrates the suitability of the
disposable PadReactor Mini system for suspension cell culture and bioproductions.
Compared to classical STR, the PadReactor Mini bioreactor comes pre-sterilized and allows reduction
of preparation time combined with ease of use.
In addition, results generated in the small scale PadReactor Mini bioreactor are comparable to previous results
in larger volume bioreactors, from 25 to 1000 L PadReactors systems1,2.
This bioreactor offers a good platform for developing processes at benchtop scale before scale-up.
References
1. Collignon F., CHO cells cultivation and antibodies production in a new 25 L disposable bioreactor –
October 2007.
2. Castillo J, Physical Characterization of a Cube-Shaped, Single-Use Bioreactor from ATMI and MAb Production
at the 1000L Scale, Poster ESACT 2011.
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The information provided in this literature was reviewed for accuracy at the time of publication. Product data may
be subject to change without notice. For current information consult your local Pall distributor or contact Pall directly.
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© 2015, Pall Corporation. Pall,
, and PadReactor are trademarks of Pall Corporation. ® indicates a
trademark registered in the USA and TM indicates a common law trademark. Filtration.Separation.Solution.
is a service mark of Pall Corporation. ◆Applikon is a trademark of Applikon Biotechnology BV and Infors is a
trademark of Infors AG.
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