Application Note
Influenza Virus Production with Adherent
VERO Cells in PadReactor® Mini Bioreactor
USD3011
Introduction
Single-use solutions are found in many applications as they offer straightforward alternatives to classical glass
or stainless steel systems. These ready-to-use systems require neither cleaning nor sterilization steps and
facilitate the scale-up from bench scale to large scale productions.
Within the range of its PadReactor bioreactor systems, Pall Life Sciences has introduced the PadReactor Mini
bioreactor, as a scale-down model, perfectly suitable for feasibility studies, research, process development and
small volume production of proteins, cells and viruses from both cells in suspension and in adherence on microcarriers (Figure 1). The gentle mixing linked to an efficient gas transfer allows 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 controlled and monitored by the PadReactor Mini controller.
In this application note, we present results for the production of influenza virus with adherent VERO cells in a 16 L
PadReactor Mini system at a working volume of 13 L.
Material and Methods
• PadReactor Mini single-use bioreactor with classical pH and DO probes
• 500 mL Shake Flask
• Adherent VERO cells
• OptiPro medium - serum free (Gibco, Belgium) added by 4 mM Glutamine
• Influenza virus H1N1
• Cytodex-1 (GE Healthcare Bio-Sciences, Sweden) at 3 g/L final concentration
• Glucose and lactate meters
Figure 1
PadReactor Mini system
The cells were seeded at 0.22x106 cells/mL in a 13 L working volume and compared to a 500 mL Shake Flask
(SF – 120 mL working volume). Cell culture set-points for the PadReactor Mini bioreactor were of 7.18-7.22 for
the pH, 50% for the DO with a constant air flow at 35 L/min (equivalent to 0.045 vvm) in the head space and
stirring speed set at 50 RPM (vs. 130 RPM for the SF). Both cultures were infected on day 4 post-inoculation.
Prior to infection, stirring was stopped to allow the microcarriers settlement (15 min) before medium removal.
Viruses were added to the microcarriers containing cells at a 0.01 MOI with fresh medium containing 4 mM
glutamine and 5 µg/mL trypsin final concentrations. During the culture, medium samples were taken to follow the
cell growth, the pH, the metabolites consumption, and the viral production. At the end of the culture, the influenza
virus titers were quantified by TCID50 test and HA assay.
2
Results and Interpretations
4.5
20
4
18
3.5
16
14
3
Medium
exchange +
iinfection
f ti
2.5
12
10
2
8
1.5
6
1
4
0.5
2
0
0
1
2
PadReactor Mini
3
4
5
Day of culture
PadReactor Mini Lac
SF
6
7
Lactate concentration (mM)
Glucose concentration (g/L)
Figure 2
Glucose and lactate concentrations for PadReactor Mini bioreactor and SF control.
0
SF Lac
Cells were seeded at 21,800 cells/mL (equivalent to ~16,000 cells/cm²) and grew in an exponential phase during
4 days to reach 676,000 cells/mL (~50,000 cells/cm²). During that period, the cell viability was above 95%. Also,
during these 4 days, the glucose concentration in the PadReactor Mini system decreased rapidly (below the limit
of detection of the meter) while the lactate was at 18.7 mM (Figure 2). As comparison, the glucose consumption
and the lactate production rates were lower in SF.
Post-infection, cells presented cytopathic effects (Figure 3) with a rapid decrease of the viability. Supernatant
samples were collected at various hours post-infection (hpi) to assess the amount of virus produced. During
the viral production period, glucose decreased rapidly to become negligible on day 7 while the lactate remained
constant (Figure 2).
Table 1
Historic of the influenza samples from the PadReactor Mini bioreactor and SF cultures.
Sample #
Hours post-infection (hpi)
1
2
3
4
5
17
23
41
48
66
www.pall.com/biopharm
3
Figure 3
Microscopic observations of the growth and viral production (cytopathic effect).
Cells prior infection (48 hours post-inoculation)
Cells in PadReactor Mini Bioreactor 24 hpi
Cells in PadReactor Bioreactor Mini 48 hpi
Cells in SF 24 hpi
Cells in SF 48 hpi
The infectious titers as well as the hemagglutinin content of each sample were quantified by respectively TCID50
and HA assay (Figure 4). Based on these two tests, it clearly appeared that the SF production was very low
compared to the bioreactor. The HA contents were negligible except for the two last samples were the value was
2 log HA/100 µL vs. 32 and 64 log HA/100 µL for the two last PadReactor Mini bioreactor samples. Additionally,
the TCID50 titers were 1 Log lower in the SF than in the bioreactor after 48 hours of viral production.
4
Figure 4
Influenza infectious titers (TCID50/mL) and HA content (log HA/100µL) in the different PadReactor Mini bioreactor
and SF samples.
6.38
Log TCID 50/m
L
50/mL
56
5.75
6.00
5.00
4.00
64
6.38
4.90
4 72
4.72
3.43
5.23
4.90
40
32
3.38
24
3.00
16
2.00
1.00
48
Log HA/100 µL
7.00
8
Pad
17 hpi
Pad
23 hpi
Pad
41 hpi
Pad
48 hpi
Pad
66 hpi
SF
17 hpi
SF
23 hpi
SF
41 hpi
SF
48 hpi
SF
66 hpi
0
Influenza sample
Log TCID50
Log H
HA
A titers
Conclusions
We demonstrated the potential of the disposable PadReactor Mini system for adherent cell culture. Here, the agitation speed was set based on preliminary tests performed with Dextran based carriers (see Application Note
USD3012). For a 13 L working volume with 3 g/L of Cytodex-1, we demonstrated that the Cytodex-1 are in good
suspension from 40 RPM. The set-point of 50 RPM, selected to avoid extra sedimentation of microcarriers during
the cell growth, demonstrated efficient mixing for nutrients and oxygen distribution through the liquid meanwhile
reducing excessive shear stress.
In parallel, we highlighted the flexibility of the system to first, enable good cell growth and second, support viral
production. The infectious titers as well as the HA contents are significantly higher in the PadReactor Mini bioreactor than in the SF which renders the system attractive for many viral production applications.
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