Comparison of CHO Cell Cultivation and mAb Production in the Allegro™ XRS 20
and Conventional Rocker Type Single-Use Bioreactors
Gail Henry, Byron Rees, Charles Golightly, Pall Life Sciences, 5 Harbourgate Business Park, Southampton Rd, Portsmouth, UK, PO6 4BQ
BACKGROUND AND NOVELTY
Lactate was produced at a similar rate in the initial
stages of each culture with the switch from lactate
production to reuse corresponding with the onset of the
stationary phase. As observed from the cell growth data,
this happens approximately 24 hours earlier in the
conventional rocker platform (green lines) compared to
the Allegro XRS 20 system (blue lines). The higher
lactate concentration observed in the XRS bioreactor
cultures correlates with the higher cell density.
1.2
1.0
0.8
0.6
0.4
0.2
0.0
0
0
50
XRS 1
100
XRS 2
150
250
200
300
350
Process time (h)
XRS 3 Conventional 1 Conventional 2
Conventional Rocking Bioreactor
25
N/A
8
42
N/A
10
1.0
0.3 ± 0.1
7.2
40
> 2.0
RESULTS
Ammonium (mmol/L)
10.0
8.0
6.0
4.0
2.0
0.0
0
XRS 1
50
100
XRS 2
150
200
250
300
350
Process time (h)
XRS 3 Conventional 1
Conventional 2
Figure 5
Normalized mAb titer in XRS bioreactor and conventional rocking bioreactors
200
With this fed-batch process, the Pall XRS 20 System
yields a higher maximum viable cell density, for a longer
duration. This is reflected in the end monoclonal
antibody concentration from each reactor: on average
there is a 67% increase in target antibody production
in the Allegro XRS 20 System over the conventional
bioreactor.
175
150
125
100
75
50
25
0
1
2
Conventional rocker
80
60
40
20
0
B
XRS 1
Figure 7
Charged variant profile of the IgG1 antibody in XRS bioreactor and conventional rocking bioreactors
(Dionex WCX10 Chromatography)
Process conditions in the bioreactor can have an impact
on the quality of the therapeutic protein produced.
The end point target antibody from UPLC charged
variant analysis, normalized against the average for
the conventional rocker shows that a similar profile is
observed between both reactor types.
XRS 3
150
200
250
Process time (h)
Conventional 1
300
350
Conventional 2
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100
Relative quantity of correctly
charged mAb (Normalized)
Culture viability
100
2F
G
XRS 2
rm
Fo
XRS 1
50
Glycoform
Conventional Rocker 1
Conventional Rocker 2
XRS 2 XRS 3
100
Figure 2
Percentage viability of XRS bioreactor and conventional rocking bioreactors
The total number of viable cells in the conventional
100%
bioreactor began to steadily decrease shortly after
90%
entering the stationary phase. In the XRS 20 bioreactor
80%
high culture viability was maintained for close to 100
70%
hours. This shows that when operating both bioreactor
60%
systems at the conditions listed in Table 1, there is an
50%
increase in both the maximum cell density and culture
40%
30%
duration with the Allegro XRS 20 System.
20%
10%
0%
0
A
XRS 1 X
XRS 2
150 200 250 300 350
Process time (h)
XRS 3 Conventional 1 Conventional 2
1F
G
50
rm
Fo
0.00E+00
0
0
1F
G
5.00E+06
B
0F
G
-5
an
m
1
n
w
no
nk
U
0F
G
Scale up
to 20 L
3
The antibody quality, in terms of glycans profiling
was unaffected, despite the significant increase in the
quantity produced by the cells in the XRS 20 bioreactor.
No increase in the relative amounts of undesired
post translational modifications such as deamidation,
pyro-glutamate formation or other different glycoforms
was observed. All glycans present are typical for this
biosimilar, with approx. 80% G0F.
Feed
Feed
2
XRS
100
Feed
Feed
1
Figure 6
Glycan profiles of the IgG1 antibody in XRS bioreactor and conventional rocking bioreactors
(XEVO G2 QTOF profiling)
N
G
0G
Figure 1
Cell growth of XRS bioreactor and conventional rocking bioreactors
Cultures from both the conventional rocking bioreactor
3.00E+07
and the Allegro XRS 20 system grew at a similar rate up
2.50E+07
until hour 165 at which point cells in the conventional
device entered the stationary phase. Cell growth
2.00E+07
continued in the XRS 20 bioreactor for a further
1.50E+07
24 hours. This resulted in an average maximum cell
1.00E+07
concentration of 2.36 x 107 viable cells/mL, an increase
of 16% over the conventional rocker average.
Ammonium production indicates a similar trend for all
cultures during the exponential growth phase. However,
there is a higher rate of ammonia reuptake in the
cultures grown in the XRS system at the switch to
stationary, and this trend continues until harvest. The
net effect is lower ammonium in the XRS bioreactor
culture compared to the conventional rocking system.
12.0
Normalized maximum mAb titer
Table 1
Summary of bioreactor and process conditions
XRS 20 Bioreactor
Start Agitation (5 L)
• Rate (CPM)
25
• X Angle (Deg)
5
• Y Angle (Deg)
5
Agitation (14 – 20 L)
• Rate (CPM)
35
• X Angle (Deg)
5
• Y Angle (Deg)
15
Aeration Rate (L/min)
1.0
Seed Density (106 Cells/mL)
0.3 ± 0.1
pH Set Point
7.2
DO Set Point (%)
40
Glucose Concentration (g/L)
> 2.0
14.0
Relative quantity (%)
In this poster we demonstrate increased performance of the Allegro XRS 20 system compared to a conventional
single axis rocking bioreactor, for a CHO cell line producing a monoclonal antibody, in fed-batch. It has been
established in previous optimization studies that this cell line favors conditions where the mixing time is low
and the kLa is high. A conventional rocking type bioreactor was set up in parallel and operated to give mixing
times and oxygen transfer rates as close as possible to the Allegro XRS 20 system. Samples were taken
throughout each bioprocess to determine cell growth, antibody production, metabolite profiles and product
quality.
4 L of CD FortiCHOu (Life Technologies) medium was aseptically transferred to each biocontainer.
Each bioreactor was seeded at 0.3 ± 0.1 viable cells/mL from shake flask
Culture conditions were applied as detailed in Table 1.
Culture volumes were scaled up to 14 L when cells grew to 3.0 ± 1.0 x 106 cells/mL.
Initial feed of 1.2 L of EfficientFeedu C (Life Technologies) added to each culture when the viable cell
density reached 8.0 ± 2.0 x 106 cells/mL.
Four further bolus additions of 1.2 L were added at 24 hour intervals from the time of the initial feed.
Concentrated glucose solution was added when required to maintain each culture above 2 g/L of glucose.
Each bioreactor culture was inspected visually for foam accumulation. Antifoam C was added when required.
Viable
Viable cell density (cells/mL)
1.4
Figure 4
Ammonium concentration for XRS bioreactor and conventional rocking bioreactors
EXPERIMENTAL APPROACH
Feed
Figure 3
Lactate concentration for XRS bioreactor and conventional rocking bioreactors
Lactate concetration (g/L)
The Allegro XRS 20 bioreactor system is a single-use bioreactor system suitable for applications ranging from
general life sciences research to seed train operations as well as small scale production at the 2 to 20 liter
scale. It features a 3D rectangular biocontainer that is rocked across two independent axes perpendicular
to one another.
These features allow for significant improvements in mixing and kLa when compared with a conventional
rocking bioreactor that features a 2D “pillow” biocontainer that rocks across a single axis.
The Allegro XRS 20 controller is able to control agitation of the biocontainer over a wide range of conditions,
from gentle agitation required for shear sensitive cultures, to more vigorous conditions generating higher
oxygen transfer rates and decreased mixing times needed for the high producing processes seen in the
biopharmaceutical industry today. Control of pH and DO is fully automated by the optical sensors supplied
with the biocontainer. Gassing strategy is managed by three independent mass flow controllers and fluid
additions can be made via three integrated pumps. All operations are managed through a user friendly
touchscreen interface.
Given these benefits, a cell culture comparison test between this new rocking design and a conventional,
single axis rocking bioreactor was performed to evaluate the cell culture performance.
80
60
40
20
0
1
2
1
Conventional rocker
2
3
XRS
CONCLUSION
We have demonstrated that the Allegro XRS 20 System is ideally suited to the cultivation of CHO cells, and
compares favorably to the rocker system for this process. This is likely due to the unique design features built
into this system. Therefore, the results confirm expectations, shown by improved cell titers and increased
antibody production without compromising on product quality.
© 2015, Pall Corporation. Pall,
, and Allegro are trademarks of Pall Corporation. ® indicates a trademark registered in the USA. uFortiCHO and EfficientFeed are trademarks of Life Technologies.
5/15, GN15.6277