High cell density growth of High Five suspension cells in
DO-controlled wave-mixed bioreactors
Teddy Beltrametti1, Nicole C. Bögli1, Gerhard Greller2, Regine Eibl1, Dieter Eibl1
1
Zurich University of Applied Sciences, School of Life Sciences and Facility Management, Institute of Biotechnology, Biochemical Engineering and Cell Cultivation Technique,
nicole.boegli@zhaw.ch; Grüental, CH-8820 Wädenswil, www.lsfm.zhaw.ch, www.bioverfahrenstechnik.ch and www.zellkulturtechnik.ch
2 Sartorius Stedim Biotech GmbH, August-Spindler-Str. 11, D-37079 Goettingen, www.sartorius-stedim.com
Background and motivation
In addition to Sf-9 cells from Spodoptera frugiperda, High Five™ (Hi-5) cells represent the most often used host cells in baculovirus expression vector system (BEVS)
based processes aimed at recombinant proteins [1]. In serum-free culture mediums and batch modes non-infected Hi-5 cells already grow to maximum cell densities of
between 8 - 9 x 106 cells mL-1 while reaching doubling times of between 18 and 29 hours. In fact, they can even achieving higher specific protein production rates than Sf9 cells [2]. Preconditions are an optimum cultivation temperature of between 27 and 28 °C, no oxygen limitation, an optimum pH-value of approximately 6.3 and
protection for the cells against shear stress and strong foaming in dynamic cultivation systems [3, 4].
Whereas Weber and Fussenegger [4] and Ries et al. [5] have shown advantageous growth (homogeneous energy dissipation, negligible foaming) of Sf-9 suspension
cells in the disposable BioWave and its successor, the BIOSTAT CultiBag RM, no references for cultivation of Hi-5 cells in wave-mixed bioreactors were found. Therefore
investigations were carried out to determine the process control mode that achieves maximum cell densities for Hi-5 cells in the BIOSTAT CultiBag RM optical, operating
in batch mode with 1 L culture volume. Finally, the possibility of cell growth improvement by applying pH or/and DO control strategies was focused on.
Material and methods
B
A
All experiments were performed in the BIOSTAT CultiBag RM 20 optical from Sartorius Stedim Biotech
(Fig. 1A) with integrated optical pH and DO sensors. The sensor systems consisting of a reusable light
fibre cable and fluorescent indicator were discarded with the bag. A tube guide supports installation of the
light fibre cable (Fig. 1B).
In all growth trials, Hi-5 suspensions cells and Express Five SFM from Gibco Invitrogen were used. Seed
inoculum was generated in single-use shake flasks (Corning) and incubated in an Infors`Ecotron shaker
(27 °C, 100 rpm, 25 mm shaking diameter). The starting parameters for the BIOSTAT CultiBag RM
optical were: 27 °C, 0.2 vvm (sterile filtered air), 6 ° rocking angle and 20 rpm rocking rate. Inoculation
density was always 1 x 106 viable cells mL-1, whereby the BIOSTAT CultiBag RM optical was operated as
follows: non-controlled (only pH- and DO monitored), DO controlled (DO set point 50 %), pH controlled
(set point of 6.3, adjustment of 1 M potassium base) as well as pH and DO controlled. Each control
strategy experiment was performed at least twice. DO control was realized with a cascade by increasing
the rocking rate up to 28 rpm. If required, pure oxygen was added. In addition to the on-line
measurements, daily sampling was carried out for off-line determination of pH-value, cell density, viability
as well as nutrient and metabolite concentrations (glucose, glutamine, lactate, ammonium).
B
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BIOCHEMICAL ENGINEERING AND CELL CULTIVATION TECHNIQUE
Figure 1: (A) Seed inoculum production with the Biostat CultiBag RM 20 optical; (B)
Tube guide and cap with fluorescent indicator.
Results and discussion
In Table 1 maximum values of viable cell density [xmax], growth rate [µmax] and doubling time [td,max] are shown.
The maximum viable cell density ranged of between 6 - 9.35 x 106 cells mL-1 and was reached in 43 to 67.5
hours post inoculation in the DO controlled mode (see also Fig. 2A, blue line). Interestingly, the maximum viable
cell density was achieved up to 24 hours earlier than for the uncontrolled and pH controlled modes.
While the DO set point (50 %) remained
constant in the controlled experiments, those in
Tabel 1: Reached viable cell densities, growht rates and doubling times.
uncontrolled mode decreased after the
maximum cell density to 10 % (Fig. 2B). As can
Control strategy
xmax
µmax
t d,max
be clearly seen in Fig. 2C the pH-values were
[x 106 cells mL-1]
[h-1]
[h]
of between 5 and 7.5, indicating that pH-value
control is redundant. The highest lactate
0.043
Uncontrolled
6.91
16.2
-1) was witnessed in
accumulation
(2.5
4.1
g
L
0.041
6.94
17.7
the non-controlled and pH controlled runs, in
0.043
DO controlled
8.18
16.3
which also the lowest maximum viable cell
0.044
9.35
15.6
densities were measured. The glucose was
0.041
pH controlled
6.04
17.9
completely consumed by the end of all the
0.043
7.09
16.1
experiments. Comparable experiments from
0.043
DO and pH controlled
7.95
16.1
Riehl et al. [3] performed in standard stirred cell
0.043
8.67
16.1
culture bioreactors were characterized by
similar maximum cell densities to the BIOSTAT
CultiBag RM.
However, maximum growth rates (0.028 h-1) in the stirred bioreactor were 35 % lower than in the BIOSTAT
CultiBag RM. This finding was also seen in the stirred Mobius CellReady 3 L (Merck Millipore), where the
maximum growth rate was 14 % lower than in the BIOSTAT CultiBag RM and a strong foaming despite the
addition of an antifoam agent.
Conclusions
Figure 2: Growth experiments with Hi-5 suspension cells in the BIOSTAT CultiBag
RM20 optical, uncontrolled (black), DO control (blue), pH and DO control (grey). (A)
Growth and viability courses independent of control strategy: ● Viable cell density, ∆
viability; (B) Courses of DO (-) in dependence on control strategy; (C) on-line pH
measurements (+) in dependence on control strategy. Values in graphs A are average
values including the standard deviation of experiment couples with comparable
strategy of control. Values in graphs B and C represent the typical course of one
experiment.
For Hi-5 suspension cells, DO controlled operation of the BIOSTAT CultiBag RM optical (set point
50 %) is achieved 35 % higher maximum living cell densities in comparison to the uncontrolled mode.
As shown in Figure 2C, on-line control of pH-value works reliably and allows early detection of
complete glucose consumption. An additional pH control with 1 M potassium base resulted in no
significant growth improvement. Consequently, the BIOSTAT CultiBag RM optical (pH on-line
monitoring and DO-control) should be preferred for optimum seed inoculum productions with Hi-5
suspension cells. Here even the addition of acid for pH control and the use of an antifoam agent are no
longer required [6].
References:
[1] Durocher Y., Butler M. (2009): Expression systems for therapeutic glycoprotein production. Current opinion in biotechnology 20:700-707.
[2] Benslimane C., Elias B. C., Hawari J., Kamen A. (2005): Insights into the central metabolism of Spodoptera frugiperda (Sf-9) and Trichoplusia ni BTI-Tn-5B1-4 (Tn-5) insect cells by radiolabling studies. Biotechnology progress 21:78-86.
[3] Rhiel M., Mitchell-Logean C. M., Murhammer D. W. (1997): Comparison of Trichoplusia ni BTI-Tn-5B1-4 (High Five™) and Spodoptera frugiperda Sf-9 insect cell line metabolism in suspension cultures. Biotechnology and bioengineering 55:909-920
[4] Weber W., Fussenegger M. (2009): Insect cell-based recombinant protein production. Cell and tissue reaction engineering: Principles and practice 263-277. Berlin/Heidelberg, Springer-Verlag.
[5] Ries C., John C., Eibl R. (2011): A new scale-down approach for the rapid development of Sf21/BEVS-based processes - a case study, Single-use technology in biopharmaceutical manufacture 208-213. John Wiley & Sons, Hoboken, New Jersey.
[6] Beltrametti T., Bögli N. C., Ries C., Greller G., Eibl R., Eibl D. (2011): Zellkultivierung in einem wellendurchmischten, DO-regulierten Einwegbioreaktor 22-23. BIOForum 1.
Contact:
ZHAW Institute of Biotechnology, Wädenswil
Nicole Bögli
nicole.boegli@zhaw.ch
Sartorius Stedim Biotech GmbH, Goettingen
Gerhard Greller
gerhard.greller@sartorius-stedim.com