BioSep: - sonosep

Technical Data Sheet
STS90
Duration: 0.5-1 weeks
Cell density: 2 x 10 6 c/ml
Finish: no more nutrients
Feed
Addition of concentrated nutrients
=> higher product concentration.
Duration: 1-1.5 weeks
Cell density: 2.5 x 10 6 c/ml
Finish: Viability < 50%
Cell retention device
Feed
BATCH
FED - BATCH
PERFUSION
Harvest
Addition of nutrients with
cell retention.
Duration: 1-3 months
Cell density: 20 x 10 6 c/ml
Cell suspension
BioSep:
the advanced acoustic cell retention device
With the progression of the genomics initiative, increasing numbers of proteins will need
to be produced rapidly.
The growing demand for novel proteins has motivated the development of more efficient
and reliable mammalian cell culture production technologies. This currently is resulting in
a spreading use of simpler, more productive processes.
Perfusion is the technology to use, providing:
• high cell density
• high (volumetric) productivity
• cost-effective operation
Fresh Medium Feed
Fig. 1: perfusion set-up with BioSep.
Sedimenting Cell Aggregates
Clarified Culture
Medium
P
P
Recirculating
Cell Suspension
Harvest
In stirred perfusion cultures, high cell densities (over 107 cells ml-1 ) can be achieved by
separating cells from the outflow stream, and retaining them in the reactor (fig. 1) while
fresh medium is added.
The volumetric production in perfusion cultures can be almost two orders of magnitude
higher than in a batch. In some cases the product concentration is increasing up to 5-fold:
➔ the required bioreactor volume can be reduced dramatically (100-fold)!
Because perfusion cultures can last for months, it’s obvious that there are economic
benefits amongst which are reduced labor requirements for bioreactor inoculation and
turnaround.
With the innovative technology of the ultrasonic separation, production costs in
pharmaceutical industry can be dramatically reduced.

Purely based on sound, an
invisible energy mesh is created:
the BioSep, a filter that never
will foul.
Result of the invisible and
harmless energy mesh
Cells appear as evenly
spaced visible vertical lines
in the viewing window of the
BioSep chamber.
They are held by ultrasonic
forces against the upward flow
of the culture medium.
The acoustic forces form a
barrier to the cells, eliminating
the need for mesh or
membrane filters.
Acoustic Energy Field
Transducer
Reflector
The BioSep from AppliSens is the first reliable and economical solution for the realization
of mammalian cell perfusion processes.
The acoustic separation technology of the BioSep can be applied on research, pilot and
production scale.
Perfusion processes using the BioSep acoustic separator typically involve continuous
addition of fresh medium to the bioreactor, while cells are filtered from the harvest
stream by the BioSep chamber and returned to the bioreactor. The BioSep chamber can
directly be mounted onto the bioreactor head plate.
Fresh Feed
clarified
culture medium
concentrated
Cell recycle
harvest
Cell
suspension
Fig. 2: Typical configuration of the acoustic cell retention system.
cell settling
Several modes of operation are available making acoustic perfusion generally applicable
for suspended mammalian and animal cell culture, but also for anchorage dependent cell
lines, or for the perfused culture of plant cells (see literature reference list).
The Biosep separation principle is purely based on gentle acoustically induced loose
aggregation followed by sedimentation. In contrast to other cell separation techniques,
the acoustic energy mesh created within the Biosep constitutes a “virtual”, thus superior
non-contact, non-fouling, non-moving filtration means. The technology allows for up to
thousands of hours of continuous operation. As a result, greatly increased steady state
cell density, productivity, and product quality is obtained.
BioSep acoustic filters are not designed to ultra-purify the harvest stream from any cells.
In contrast, a small escape rate allows for controlled cell bleeding and positively
contributes to the viability of the culture (see publications).
Typical separation efficiency of the BioSep ranges from 90-99%.

The BioSep chamber is mounted above the bioreactor head plate. The cell suspension
is pumped into the chamber by the recirculation pump. The flow is then split into the
harvest flow and the return flow. The flow rate through the BioSep is controlled by the
harvest pump. The ultrasonic forces in the BioSep aggregate and hold the suspended
cells stationary against the harvest flow, thereby clarifying the harvest stream. The
planar aggregates appear as parallel lines when seen from the side through the viewing
window. Aggregated cells that settle from the resonator are rapidly recycled to the
bioreactor in the return stream where they are dispersed by the impeller.
Conventional cell retention devices include filters, settlers and centrifuges. Regardless of
their design, the filter surfaces are susceptible to fouling. Settling chambers and
centrifuges solely rely on the difference in density between cells and medium.
Settling chambers
require, a large settling area
and long settling times due to
the small difference in density.
This leads to prolonged exposure
of the cells to an uncontrolled
environment.
Centrifugation
the sedimentation process is
enhanced by centrifugal forces
many times the force of gravity.
The separation efficiency
of a centrifuge is a function of a
multitude of operating parameters.
Mechanical systems such as
centrifuges are susceptible to
failure and cells are exposed to
high shear forces.
The BioSep
simple and compact
non-mechanical device
in which only harmless
sound waves are
exploited to separate
the cells from the
suspending medium.
BioSep 10 L BioSep 50 L BioSep 200 L
Cell retention device
Feed
PERFUSION
Harvest
Cell suspension
Compared to technologies such
as filters, centrifuges and
settlers, the BioSep offers an
economic separation technique
in perfusion cultures:
• surprisingly simple
• highly reliable
The BioSep chamber assembly
is entirely solid state and is
unaffected by fouling, rendering
it reliable for thousands of hours
of continuous operation.
The BioSep 10L is designed to
operate at a perfusion harvest
rate between 1 and 10L/day.
The BioSep 50L operation range
is between 5 and 50L/day
The BioSep 200L is designed
for both pilot- and production
scale. The operating range is
between 20L and 200L/day.

The BioSep features
• Cell filtration by ultrasonic
resonance field
• Continuous operation
• Low shear environment
• Simple design
• Compact autoclavable
device
• Compact In-Situ-
Sterilizable device
• Scalable system
The BioSep advantages
• No physical filter surfaces
to foul
• No mechanical parts to fail
(no moving parts)
• Small retention volume
• Rapid turn around
(low hold-up times)
• Increased efficiency
• Wide flow range
• High cell viability
• Consistent culture
environment
• Easy installation
• Easy automation
• Minimal operator
involvement
• Clean-In-Place
The main production systems which are used today for Mab production are stirred tanks.
Homogeneous systems like a stirred tank represent the biggest unit reactor volume
attainable today and with the highest unit production capacities. This capacity can be
increased drastically with the use of a cell retention device.
The BioSep will typically remove between 90 and 99% of the cells in the harvest stream at
a reactor cell concentration of up to 20 million cells/ml. The separation efficiency of the
BioSep system is defined as:
( )
SE = 100 % 1 – Ch
Cb
• SE is the separation efficiency
• Ch is the cell concentration in the harvest
• Cb is the cell concentration in the bioreactor
The separation efficiency of the BioSep system (10L, 50L and 200L) is controlled
by adjusting the acoustic power input to the resonator and the run/stop cycle time ratio
for the BioSep and harvest pump.
Pump control cable
Compressed air
Output
cable
Harvest
Recirculation pump
Feed
Effects on the economics of an acoustic perfused process as compared
to a batch process:
• improved efficiency in medium use (e.g. lower serum concentrations in growth medium),
up to 5 times more efficient. The trapped and returned cells use only nutrients to maintain
their metabolism and for biosynthesis of products.
• a factor 10-20 higher viable cell concentration
• antibody production per reactor volume per day with a factor 10-100 fold higher than
in a batch (high volumetric productivity).
• better on-line control due to steady state condition
• reduced exposure of the products to proteases
• antibody concentration in harvest improved with a factor 2-5 fold compared to batch process
• downstream processing of the secreted product is reduced by one step.

Due to their higher acoustic contrast, viable cells are retained by the acoustic filter with
higher efficiency than dead cells and cell debris.
This results in a significantly higher escape rate for non-viable cells.
This effect limits the accumulation of non-viable biomass in the bioreactor.
This effect is beneficial for perfusion strategies:
➔ it will selectively retain the producing cells and remove
the non productive dead cells from the bioreactor.
Cell concentration
(10 6 cell/ml)
45 110
40 100
35 90
30 80
25 70
20 60
15 50
Viable cells
10 40
Total cells
5 30
0
0 100
Economic impact
Viable cells
200 300 400 500 600 700 800 900 1000
20
1100
Time (h)
Results BioSep 50L CHO cell perfusion culture
Average total proteine concentration in harvest 272 µg/ml, courtesy: A.O.A. Miller
Annual production of 1 kilogram Mouse-IgG antibody production using Mouse Hybridoma 2E11
Batch FED-Batch Perfusion at 3 volumes/day
Bioreactor volume (active) 500L 350L 7L
Cell concentration 2 x 10 6 /ml (peak) 2 x 10 6 /ml (average) 20 x 10 6 /ml (steady state)
Runs per year 40 20 4
Duration per run 1 week 2 weeks 10 weeks
Consumption of medium per year 20,000 L 7,000 L 7,000 L
MAb concentration in harvest 50 µg/ml 150 µg/ml 150 µg/ml
MAb conc.[ g/ml]
Cumulative Monoclonal Antibody (g)
Cell Concentration (Cells/mL)
Selective retention
Cell concentration
10 8
10 7
10 6
10 5
Antibody Production
180
160
140
120
100
80
60
40
20
0
0
10
8
6
4
2
0
0
0
Batch
100 200 300 400 500 600 700 800
Time (h)
Batch
Perfusion
200 400 600 800
Time (h)
200 400 600 800
Time (h)
Perfusion
(viable cells)
Perfusion
Successive
Batch Cultures

Technical description BioSep Chamber 10L
Air inlet
BNC
connector
BNC
connector
Recirculation
inlet
Adapter
Return tube
Air inlet
Harvest outlet
Resonator body
Gasket
Cuvette
O-ring
Recirculation inlet
Hexagon screw
Return tube
Harvest outlet
Resonator
body
Gasket
Cuvette
Gasket
O-ring
Hexagon
screw
Mechanical:
Material: Body: SS 316L
Window: Pyrex glass
Gasket: Silicone
O-ring: Silicone
Finish: Interior - Electro polish, Ra < 0.8 µm
Weight: 0.5 kg
Total volume: 24 ml
Resonator volume: 7 ml
Height above headplate: 150 mm
Height: 344 mm
Max. width: 79 mm
Exterior - Electro polish/mechanical polish
Head plate connection: Through a 12 mm diameter (pH/mV) sensor holder
Insertion length: 190 mm
Clarified medium outlet: 4 mm barbed fitting
Medium inlet: 7 mm barbed fitting
Air inlet: 4 mm barbed fitting
Temperature: 130˚C max.
Test pressure (internal): 4 bars
Electrical:
Operating frequency: 2.1 MHz
Power consumption: 10 W max.
BioSep Chamber 50L
Mechanical:
Material: Body: SS 316L
Window: Pyrex glass
Gasket: Silicone
O-ring: Silicone
Finish: Interior - Electro polish, Ra < 0.8 µm
Weight: 1.5 kg
Total volume: 150 ml
Resonator volume: 50 ml
Height above headplate: 177 mm
Height: 344 mm
Exterior - Electro polish/mechanical polish
Max. width: 110 mm (72 mm housing)
Return tube connection: 0.5” Tri-clamp (tube: ø 9.53)
Clarified medium outlet: 6 mm barbed fitting
Medium inlet: 10 mm barbed fitting
Air inlet: 6 mm barbed fitting
Temperature: 130˚C max.
Test pressure (internal): 4 bars
Electrical:
Operating frequency: 2.1-2.15 MHz
Power consumption: 10 W max.

APS 990 BioSep Controller for BioSep chamber 10L and 50L
The APS 990 controller consists of a frequency generator and a power amplifier.
The internal control automatically optimises the frequency and amplitude of the output
signal for best separation performance.
The adjustable timers do set the run/stop cycle times for the harvest pump and
the acoustic field. The display indicates the frequency and the LED bar does indicate
the power output in percentages.
Frequency indication
Power knob
APS990 BioSep controller
Mechanical:
Dimensions: W x H x D = 130mm x 130mm x 305mm
Weight: 3.5 kg
Electrical:
Power supply: 110 -240VAC, 50/60 Hz
Power consumption: Max. 150W
Coaxial cable: Length 2 meters
Frequency range: 2.1 – 2.15 MHz
Output power: 10 W max.
Output voltage: 30 Vpp max.
Internal timer: Run time: 1 – 15 minutes
On/off toggle
Stop time: 3 –10 sec.; 5 – 15 minutes
Power indication (%)
Run time knob
Stop time knob
Automatic power/frequency
adjustment
Minimal operator involvement
Simple human interface
• The output power is
increased automatically
during scanning
• Scanning stops at
the resonance peak
Interface I/O port:
• Status output
24 VDC / 100 mA max.
switching load
• Interrupt output
24 VDC / 100 mA max.
switching load
• DC source
max load: 20 mA
• Remote on/off input
max 15 VDC / 4 – 20 mA
Auxiliary port:
• Engaged when aucoustic
field is off
12 VDC
max. load: 1A

The BioSep 200L Acoustic
Perfusion System is a simple,
effective and reliable cell
separation system designed
expressly for cell retention during
perfusion of high-density stirred
suspension cultures.
The system consists of a
resonator chamber with a 20 -
200 L/day harvest rate capacity
and the APS 991 controller.
The chamber, where cell
separation takes place, is
compact and simply installed
on or above the bioreactor head
plate. The system is easy to
operate and provides robust,
continuous operation, whatever
the desired cell culture duration.
Using this device existing batch
or fed-batch reactors can be
conveniently adapted to highproductivity
perfusion.
Scale-up of acoustic perfusion: The BioSep 200L
The 200L acoustic perfusion system consists of a BioSep chamber and the BioSep
controller APS991.
The BioSep chamber assembly is entirely solid state and is essentially unaffected by
fouling, rendering it reliable for thousands of hours of continuous operation.
It is designed to operate at a perfusion harvest rate between 20 and 200L/day.
The separation efficiency of the BioSep 200L system is controlled by adjusting the
power input to the resonator and the run/stop cycle time ratio for the acoustic field
and harvest pump.
Cell concentration
(10 6 cells/ml)
45
40
35
30
25
20
15
10
5
APS991 controller
Resonator chamber
Medium feed
Bioreactor
Total cells
Water bath
Medium
pump
pump control cable
Output cable
Typical configuration of the 200L BioSep acoustic perfusion system.
0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Culture time (days)
Harvest
BioSep 200L CHO perfusion (courtesy 4C, Belgium)
Recirculation
pump
Recirculation
cell suspension
Resonator
support

BioSep Chamber 200L
Mechanical:
Material: Body: 1.4404 SS 316L
Transducer plate: Pyrex glass
Gasket: Silicone
O-ring: Silicone
Finish: Interior - Electro polish, Ra < 0.8 µm
Weight: Approximately 13 kg
Total volume: 1.0 L
Resonator volume: 290 ml
Height: 317 mm
Max. width without sensor port: 193 mm
Diameter housing 158 mm
Clarified medium outlet: 0,5” Triclamp
Medium inlet: 0,5” Triclamp
Return outlet: 0,5” Triclamp
Water bath inlet: 0,5” Triclamp
Water bath outlet: 0,5” Triclamp
Temperature: 130˚C max.
Pressure range (internal): 3.2 barg
Exterior - Electro polish/mechanical polish
Electrical:
Operating frequency range: 2.10 - 2.13 MHz
Power consumption: 100 W max.
APS 991 Controller
Mechanical:
Dimensions: W x H x D = 450 x 150 x 350mm
Weight: 15 kg
Electrical:
Power supply: 110-120/220-240 VAC, 50/60 Hz
Fuses: 3.15 A Slow blow 250V
Power consumption: Max. 570 VA
Coaxial HF cable length: 2 m
Frequency range: 2.10 - 2.19 MHz
Output power: 100 W max.
Output voltage: 100 Vpp max.
Internal Timer: Run time: 10 - 600 s
Stop time: 1 - 60 s
Top
plate
Cuvette
Water
bath inlet
Recirculation inlet
Harvest outlet
Sensor
port
Water bath
outlet
Funnel
Return
outlet

Fresh feed
Recirculation pump
• speed ±3 x harvest flow
Fresh feed
Clarified culture
medium
Harvest pump
• variable speed with remote
control input
• variable speed with remote
control input and prime rate
reverse action
Clarified culture
medium
Concentrated
cell recycle
Concentrated
cell recycle
Harvest
Cell
suspension
Harvest
Prime rate
reverse pump
Cell
suspension
Pumps and other hardware to complete a perfusion set-up
The harvest pump is a variable speed pump. The pump is stopped (remotely) by the
BioSep controller during field off times, facilitating the settling of aggregates. This means
that the harvest flow is stopped for a few seconds, arresting the flow in the chamber.
The stop time of a few seconds will affect the overall harvest flow rate by a small
amount. This pump can be controlled directly by a connecting cable between the BioSep
controller and the pump.
In cultures of sticky cells or too large aggregates, the variable speed pump with prime
rate reverse action (code Z288010020) is a recommended option.The pump has the
capability to reverse the harvest flow at full pump speed (prime), during field off time.
Sticky cells can adhere to the glass of the resonator chamber. By reversing the flow at
full speed during field off times (for 3 sec) these cells will get a small push back into the
bioreactor. Reversing the flow every 10 min will result in less adherence of cells in the
BioSep chamber.
A typical BioSep set-up
BioSep 10L: • holder to fit resonance chamber into the headplate
• suction tube for recirculation loop
• recirculation pump
• harvest pump (variable speed / remote controlled)
• fresh medium inlet and pump (e.g. controlled via level controller)
BioSep 50L: • diptube for return of cells from chamber. (The diptube should be
provided with the proper headplate connector and a 0.5” TC to install
the resonance chamber).
• suction tube for recirculation loop
• recirculation pump
• harvest pump (variable speed / remote controlled)
• fresh medium inlet and pump (e.g. controlled via level controller)
Various adapters are available to fit the BioSep 50L to any type or brand of bioreactor.
Adapter M18-0.5”SC-9.53 Adapter PG13.5-0.5”SC-9.53 Adapter D27-0.5”SC-9.53

BioSep 200L: Scale-up of acoustic perfusion
There are several options for connecting the return outlet to the reactor,
depending on the chosen way of sterilization.The BioSep chamber is
placed on a support above the reactor preferably straight above the
return tube in the top plate to ensure smooth return of the recirculation
medium preventing sedimentation of cells in the tubing.
Additional hardware for a BioSep 200L set-up:
• BioSep chamber and controller
• BioSep support
• Pumps, pump head, pump tubing (harvest, recirculation, feed)
• Water bath (temperature control of resonance chamber)
• Contained additions
)
• Sensors (pH, DO, T) Optional
• Safety clamps pump tubing
Example P&ID for a fully contained BioSep in S.I.P.
References:
1. H. Bierau, A. Perani, M. Al-Rubeai, A.N. Emery. A comparison of intensive cell culture bioreactors operating with Hybridomas
modified for inhibited apoptotic response. Journal of biotechnology 62, 195-207, 1998. 2. Gorenflo, V.M., Smith, L, Dedinsky,
B., Persson, B. and Piret, J.M. Scale-up and Optimization of an Acoustic Filter for 200 L/day Perfusion of a CHO Cell Culture.
Biotechnology and Bioengineering, vol 80, no. 4, nov. 2002 3. Miller, A.O.A. Combing cell culture & process operation.
Sonoperfusion allows direct feed with expanded-bed chromatography. GEN Vol. 21, p29, 2001. 4. Pui, P.W.S., Trampler,
F., Sonderhoff, S.A., Groeschl, M., Kilburn, D.G. and Piret, J.M. “Batch and Semi-Continuous Aggregation and Sedimentation of
Hybridoma Cells by Acoustic Resonance Fields”, Biotechnol. Prog. 11: 146-152, 1995. 5. Ryll, T., Dutina, G., Reyes, A., Gunson,
J., Krummen, L., Etcheverry, T., Performance of small-scale CHO perfusion cultures using an acoustic cell filtration device for cell
retention: Characterization of separation efficiency and impact of perfusion on product quality, Biotechnol Bioeng 69: 440-449,
2000. 6. Trampler, F., Sonderhoff, S.A., Pui, P.W.S., Kilburn, D.G. and Piret, J.M. “Acoustic Cell Filter for High Density Perfusion
Culture of Hybridoma Cells”, Bio/Technol. 12: 281-284, 1994. 7. S.M.Woodside, B.D. Bowen, J.M. Piret. Mammalian cell retention
devices for stirred perfusion bioreactor. Cytotechnology 28, 163-175, 1998. 8. Zhang, J., A. Collins, M. Chen, I. Knyazev and
R. Gentz “High-density perfusion culture of insect cells with a BioSep ultrasonic filter”, Biotechnol Bioeng 59: 351-359, 1998.
A 100L working volume bioreactor with
a BioSep 200L chamber and support.
The BioSep technology
is patented (US 5.626.767)

BioSep
Z099001010 BioSep Chamber 10 L/day
Z099005010 BioSep Chamber 50 L/day
Z099020010 BioSep Chamber 200L/day
Z099020020 BioSep Chamber 200L/day with sensor ports
BioSep Controller
Z299005020 BioSep Controller APS 990 (10-50L/day) 110-240VAC
Z299025010 BioSep Controller APS 991 (200L/day) 110-240VAC
Z299025011 Computer interface board APS 991 (optional)
BioSep additional hardware
Z199001310 Adapter PG13.5-0.5”SC-9.53
Z199001810 Adapter M18-0.5”SC-9.53
Z199002710 Adapter D27-0.5”SC-9.53
Z199020010 Basic support BioSep 200L H=2.0m
Z199020050 Hardware basic P&ID BioSep 200L
Z199020060 Hardware contained P&ID BioSep 200L
Z230001210 Thermo-circulator BioSep 200L (220-240V)
Z230001220 Thermo-circulator BioSep 200L (110-120V)
BioSep pumps
Z188000010 Pumphead Easyload (10L & 50L)
Z188000030 Pumphead Easyload L/S II (200L)
Z188001410 Tubing norprene 15m, size 14 (harvest 10L)
Z188001610 Tubing norprene 15m, size 16 (recirculation 10L, harvest 50L)
Z188001810 Tubing norprene 15m, size 18 (recirculation 50L)
Z188003510 Tubing norprene 15m, size 35 (recirculation and harvest 200L)
Z288001710 Pump drive fixed speed 17rpm 230V (recirculation 10L/50L)
Z288010010 Pumpdrive var.speed 1-100rpm 230V (recirculation 50L and harvest 10L/50L/200L)
Z288010020 Pumpdrive var.speed prime rate reverse 1-100rpm 230V (harvest 10L/50L/200L)
Z288060010 Pumpdrive var.speed 6-600rpm 230V (recirculation 200L)
Z188141410 Safety clamp for tubing size 14
Z188161610 Safety clamp for tubing size 16
Z188353510 Safety clamp for tubing size 35 (200L)
The BioSep acoustic cell retention system:
•a non-fouling perfusion device with increased separation capacity
and improved reliability
• making large-scale perfusion an increasing viable option for cell culture processes
STS90 - VZXV122902 - Subject to modifications - Printed by Applikon Dependable Instruments bv - The Netherlands 10.02
Ordering information
Applikon Dependable Instruments bv
AppliSens
De Brauwweg 13
P.O. Box 149, 3100 AC Schiedam
The Netherlands
Phone: +31 10 298 35 85
Fax: +31 10 437 96 48
E-mail: applisens@applikon.com
Internet: www.applikon.com