Characterization and Performance of the
Allegro™ STR 1000 Single-Use Stirred Tank Bioreactor System
Kerem Irfan, Byron Rees, Tim Barrett, Bojan Markicevic, Camille Segarra; Pall Corporation, 5 Harbourgate Business Park, Southampton Road, Portsmouth, PO6 4BQ, UK
ABSTRACT
The benefits of single-use technologies in both upstream and downstream operations are now widely acknowledged by the biopharmaceutical industry, and have resulted in radical changes to the design and operation of
many pharmaceutical processes. In mammalian cell culture, multi-use, cleanable glass or stainless steel stirred tank reactors (STRs) have been used successfully for growth of suspended cell lines for both small and large
scale systems. However, to achieve the same or better performance from a single-use bioreactor presents a significant challenge to product designers and developers.
This poster describes the studies aimed at characterizing the fluid dynamics of the Allegro STR 1000 bioreactor by measurement and modelling of specific physical parameters known to be critical for mammalian cell
culture. More specifically, oxygen transfer rates, mixing times and carbon dioxide stripping rate.
Our studies have shown that, the Allegro STR 1000 single-use bioreactor can be engineered suitably to provide a practical alternative to stainless steel bioreactors. By evaluating the hydrodynamics of the Allegro STR 1000
bioreactor, we established that the direct drive large impeller could provide power input for fluid volumes up to 1000 L that can support the growth of mammalian cells. This power transfer in conjunction with a customized
cubical biocontainer with integrated baffles produced homogeneous mixing. User experience is also enhanced by providing features that make the installation and use of the bioreactor intuitive and easy.
KEY FEATURES
OXYGEN TRANSFER: DETERMINATION OF kLa
Easy Set-up
Guided biocontainer installation
with integrated instructions
Color tags on all air and fluid
transfer lines
Single operator installation for most
of the process
Integrated tube management system
Integrated Controller
Very intuitive control software
Installation and disassembly
guided by software
Compatible with supervisory
systems via OPC
Hardware
Geometric scale up of the Allegro STR 200
bioreactor
Easy access integrated fluid pumps
Large viewing window for visual inspection
of cultures
High grade stainless steel exterior for easy
cleaning
Integrated heating and cooling jacket
Process Safety
Overpressure sensor integrated into
biocontainer
Smart system helps reduce operator
mistakes
Packaging protects biocontainer and
components during shipping
Figure 4
kLa2080 values at various agitation rates at the maximum airflow of 100 LPMs
50
Error bars represent standard deviation from two probes (n=2) located in the standard
probe inserts. Power input per volume (P/V) was calculated using a measured power
number at 200 L scale
40
kLa2080 (1/Hr)
Mixing System
Excellent mixing achieved with large bottom mounted
direct drive impeller and baffled geometry.
Fluid dynamics comparable to cylindrical stainless
steel reactors
Connectivity
8 liquid inlets from ¼ in. to 1 in. fitted with
Kleenpak™ sterile connectors
Redundant port for additional vent filter
All gas lines fitted with Emflon® II
sterilizing grade gas filters
30
20
At maximum speed, a kLa2080 of 40 h-1 could be achieved.
10
0
15 25 35 45 55 65 75 85 95 105 1
115
15
Agitation Speed (RPM)
295
4
75
P/V (W/m3)
Materials and Methods
The volumetric mass transfer coefficient for oxygen (kLa) using a ring sparger was determined using the
gassing out method with nitrogen and air.
Experiments were conducted using a cell culture media simulant heated to 37 ºC.
kLa values were calculated between a dissolved oxygen (DO) values of 20 and 80% using the equation:
kLa = ln
(
)
DO* - DO20
1
x
DO* - DO80 t80 - t20
Where DO* refers to DO at saturation, and DO20 and D80 refers to 20% and 80% respectively of the DO at saturation.
Sensors
Up to 6 ports for conventional probes
Single-use sensors for pH and DO available
Figure 5
Contour plots for kLa2080 values at various agitation and airflow rates
Two DOE models required to capture change
in curvature with RPM (25 to 65 RPM and
65 to 105 RPM)
EXPERIMENTAL SET-UP
Experimental Goal
The study was conducted to determine the mixing efficiency, kLa O2 and CO2
evacuation rate.
Figure 1
Experimental set up
Measurement Probe Locations
For the mixing study, pH probes were located in eight different locations.
For kLa O2 and CO2 evacuation rate determination two probes in the standard
probe inserts were used (P1 & P2).
Media Simulant
A cell culture media simulant was used for all experiments:
– Bicarbonate buffer, Pluronic◆ F68, Antifoam A
CO2 IS EFFICIENTLY STRIPPED OUT
MIXING PERFORMANCE
Figure 2
Mixing times at 1000 L volume for various agitation speeds
Mixing Time (Seconds)
40
Error bars represent standard deviation from three replicate runs (n=3) in which the
mixing time was averaged from 8 probes within a single run. Power input per volume
(P/V) was calculated using a measured power number at 200 L scale
35
30
25
20
Mixing was homogenous across the bioreactor
Mixing times below 14 sec can be achieved at maximum
agitation speed.
15
10
5
0
31.1
25
4
17.8
65
Agitation Speed (RPM)
75
P/V (W/m3)
13.5
105
295
Materials and Methods
Mixing studies were conducted by performing a single shot addition of 4 M NaOH and then measuring
the change in pH of 1000 L of cell culture media simulant at ambient temperature.
No significant difference was seen in the mixing time measured between the eight probe positions.
Mixing time was defined as the time required to reach 100 ± 5 % of the final pH value.
Figure 3
Evolution of simulated tracer concentration within the Allegro STR 1000 bioreactor
Simulations carried out at 100 RPM
CO2 Evacuation (mol/L/day)
Figure 6
CO2 evacuation rate for various agitation speeds at the maximum air flow rate of 100 LPM
Error bars represent standard deviation from two probes (n=2) located in the standard
probe inserts. Power input per volume (P/V) was calculated using a measured power
number at 200 L scale
7
6
5
4
3
2
1
0
15 25 35 45 55 65 75 85 95 105 115
115
Agitation Speed (RPM)
4
75
295
P/V (W/m3)
Materials and Methods
The partial pressure of CO2 (pCO2) level in the media simulant was measured indirectly using pH probes,
due to the superior probe response time. In the pH range pH 6.5 to 8 the pCO2 is proportional to the cell
culture simulant pH as follows:
pCO2 = (pH–pHinitial) x log(pCO2initial)
1000 L of media simulant was heated up to 37 ºC and brought down to pH 6.5 using CO2 gas and
maintained stable for a duration of 2 minutes. A pCO2 probe recorded the initial pCO2. Air was then
sparged through the ring sparger of the bioreactor at the desired flowrate and the change in pH measured.
Figure 7
Contour plots for CO2 evacuation rates at various agitation and airflow rates
A central composite face-centred design was used to generate the DOE
model. Values above 0.28 and 2.50 mol/L/day are adequate for fed-batch
and perfusion cultures, respectively (Goudard et al, 2011).
Computational results showed
quick and homogenous
mixing with no dead zones
The model showed good
agreement with experimental
results
Materials and Methods
Computational fluid dynamics (CFD) was carried out using ANSYS Fluent at a fill volume of 1000 L
A turbulent k-epsilon model was used to generate CFD results
A tracer was patched at a similar location to the experimental runs
Contact: 800.717.7255 (USA and Canada) • 1.516.484.5400 (Outside USA and Canada) • www.pall.com/biopharm • E-mail: [email protected]
CO2 can efficiently be stripped using the sparger alone
For a 3 x 107 cells.mL-1 CHO cell culture, an agitation of 25 RPM
is sufficient to strip CO2 (Goudard et al, 2011)
REFERENCES
Goudar, C.T., Piret, J.M. & Konstantinov, K.B., 2011. Estimating Cell Specific Oxygen Uptake and Carbon Dioxide Production Rates for Mammalian
Cells in Perfusion Culture. Biotechnology Progress, 27(5), pp.1347–1357.
© 2014, Pall Corporation. Pall,
, Allegro, Emflon and Kleenpak are trademarks of Pall Corporation. ® indicates a trademark registered in the USA. uPluronic is a trademark of BASF Corporation.
11/14, GN14.5967