Technical Data Sheet BioSep:

STS90 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:

BATCH • high cell density
Duration: 0.5-1 weeks • high (volumetric) productivity
6
Cell density: 2 x 10 c/ml • cost-effective operation
Finish: no more nutrients

Feed

Clarified Culture Harvest

Medium P

FED - BATCH P
Addition of concentrated nutrients
=> higher product concentration.
Recirculating
Duration: 1-1.5 weeks Cell Suspension
Fresh Medium Feed
Cell density: 2.5 x 10 6 c/ml
Finish: Viability < 50%

Cell retention device Harvest

Feed Cell suspension

Fig. 1: perfusion set-up with BioSep. Sedimenting Cell Aggregates

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.

PERFUSION The volumetric production in perfusion cultures can be almost two orders of magnitude
Addition of nutrients with higher than in a batch. In some cases the product concentration is increasing up to 5-fold:
cell retention. ➔ the required bioreactor volume can be reduced dramatically (100-fold)!
Duration: 1-3 months
Cell density: 20 x 10 6 c/ml 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 The BioSep from AppliSens is the first reliable and economical solution for the realization
invisible energy mesh is created: of mammalian cell perfusion processes.
the BioSep, a filter that never The acoustic separation technology of the BioSep can be applied on research, pilot and
will foul. 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.
Result of the invisible and
harmless energy mesh
clarified
culture medium harvest

Cell
Fresh Feed
suspension

cell settling

Cells appear as evenly

spaced visible vertical lines concentrated
Cell recycle
in the viewing window of the
BioSep chamber.
They are held by ultrasonic
forces against the upward flow Fig. 2: Typical configuration of the acoustic cell retention system.

of the culture medium.

The acoustic forces form a
Several modes of operation are available making acoustic perfusion generally applicable
barrier to the cells, eliminating
for suspended mammalian and animal cell culture, but also for anchorage dependent cell
the need for mesh or
lines, or for the perfused culture of plant cells (see literature reference list).
membrane filters.

Acoustic Energy Field 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.

Transducer
Reflector 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
Cell retention device Harvest
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 Feed Cell suspension
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 PERFUSION
centrifuges solely rely on the difference in density between cells and medium.

Settling chambers Centrifugation The BioSep

require, a large settling area the sedimentation process is simple and compact Compared to technologies such
and long settling times due to enhanced by centrifugal forces
non-mechanical device as filters, centrifuges and
the small difference in density. many times the force of gravity.
settlers, the BioSep offers an
This leads to prolonged exposure The separation efficiency in which only harmless
of the cells to an uncontrolled of a centrifuge is a function of a economic separation technique
sound waves are
environment. multitude of operating parameters. in perfusion cultures:
Mechanical systems such as exploited to separate • surprisingly simple
centrifuges are susceptible to
the cells from the • highly reliable
failure and cells are exposed to
high shear forces. suspending medium.

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.

BioSep 10 L BioSep 50 L BioSep 200 L

The BioSep features The main production systems which are used today for Mab production are stirred tanks.
• Cell filtration by ultrasonic Homogeneous systems like a stirred tank represent the biggest unit reactor volume
resonance field attainable today and with the highest unit production capacities. This capacity can be
• Continuous operation increased drastically with the use of a cell retention device.
• Low shear environment
• Simple design The BioSep will typically remove between 90 and 99% of the cells in the harvest stream at
• Compact autoclavable a reactor cell concentration of up to 20 million cells/ml. The separation efficiency of the
device BioSep system is defined as:
• Compact In-Situ-

( )
Sterilizable device • SE is the separation efficiency
Ch
• Scalable system SE = 100 % 1 – • Ch is the cell concentration in the harvest
Cb • 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

The BioSep advantages
• No physical filter surfaces Compressed air
Harvest
to foul
• No mechanical parts to fail Recirculation pump

(no moving parts) Output

cable
Feed
• 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

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 Selective retention
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.

45 110 Cell concentration

40 100 108

C ell C on cen t rat ion (C ells/m L )

35 90
P e r f u s io n
107
C e l l c o n c e n t ra t i o n

(v ia b le c e lls )
30 80
(106 cell/ml)

25 70 106
B a tc h

20 60
105
15 50 0 100 200 300 400 500 600 700 800
V i a bl e c e l l s Ti m e ( h)
10 40
To t a l c e l l s Antibody Production
5 30
V i a bl e c e l l s 180
0 20 Batch
160
0 100 200 300 400 500 600 700 800 900 1000 1100 Pe r f u s i o n
140

MA b c onc . [ g/ m l ]
Time (h) 120
100
Results BioSep 50L CHO cell perfusion culture
80
Average total proteine concentration in harvest 272 μg/ml, courtesy: A.O.A. Miller
60
40
20
0
0 200 400 600 800
Economic impact
Time (h)
C u m u la ti v e M o n oc lo na l A n ti bo d y ( g)

Annual production of 1 kilogram Mouse-IgG antibody production using Mouse Hybridoma 2E11

Batch FED-Batch Perfusion at 3 volumes/day 10

Bioreactor volume (active) 500L 350L 7L 8

P e r f u s io n
Cell concentration 2 x 10 6/ml (peak) 2 x 10 6/ml (average) 20 x 10 6/ml (steady state) 6

Runs per year 40 20 4 4

Duration per run 1 week 2 weeks 10 weeks 2 Successive

B a t c h C u lt u re s
Consumption of medium per year 20,000 L 7,000 L 7,000 L 0
0 200 400 600 800
MAb concentration in harvest 50 μg/ml 150 μg/ml 150 μg/ml Ti m e ( h)
Technical description BioSep Chamber 10L

Mechanical:

Material: Body: SS 316L

Window: Pyrex glass

Gasket: Silicone
Air inlet Harvest outlet
O-ring: Silicone

Finish: Interior - Electro polish, Ra < 0.8 μm

Resonator body Exterior - Electro polish/mechanical polish

Gasket Weight: 0.5 kg

BNC
connector Total volume: 24 ml
Cuvette
Resonator volume: 7 ml
O-ring
Height above headplate: 150 mm

Height: 344 mm
Recirculation inlet
Max. width: 79 mm

Hexagon screw Head plate connection: Through a 12 mm diameter (pH/mV) sensor holder

Insertion length: 190 mm

Return tube
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

Air inlet Harvest outlet Gasket: Silicone

O-ring: Silicone

Finish: Interior - Electro polish, Ra < 0.8 μm

Resonator
body Exterior - Electro polish/mechanical polish

Weight: 1.5 kg

Gasket Total volume: 150 ml

BNC
connector Resonator volume: 50 ml
Cuvette
Height above headplate: 177 mm
Recirculation
inlet Gasket Height: 344 mm

Max. width: 110 mm (72 mm housing)

Return tube connection: 0.5” Tri-clamp (tube: ø 9.53)

O-ring
Adapter
Clarified medium outlet: 6 mm barbed fitting

Return tube Medium inlet: 10 mm barbed fitting

Hexagon
screw 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 Automatic power/frequency
The APS 990 controller consists of a frequency generator and a power amplifier. adjustment
The internal control automatically optimises the frequency and amplitude of the output
signal for best separation performance. Minimal operator involvement
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 Simple human interface
the power output in percentages. • The output power is
increased automatically
during scanning
• Scanning stops at
the resonance peak

Power indication (%)

Frequency indication Run time knob

Power knob Stop time knob

On/off toggle

APS990 BioSep controller

Interface I/O port:
Mechanical:
• Status output
Dimensions: W x H x D = 130mm x 130mm x 305mm
24 VDC / 100 mA max.
Weight: 3.5 kg
switching load
Electrical:

Power supply: 110 -240VAC, 50/60 Hz

• Interrupt output
Power consumption: Max. 150W
24 VDC / 100 mA max.
Coaxial cable: Length 2 meters
switching load
Frequency range: 2.1 – 2.15 MHz

Output power: 10 W max.

• DC source
Output voltage: 30 Vpp max.
max load: 20 mA
Internal timer: Run time: 1 – 15 minutes

Stop time: 3 –10 sec.; 5 – 15 minutes

• Remote on/off input
max 15 VDC / 4 – 20 mA

Auxiliary port:
• Engaged when aucoustic
field is off
12 VDC
max. load: 1A
 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.
pump control cable

APS991 controller
Output cable Harvest

Resonator chamber
Recirculation Recirculation
Water bath pump cell suspension

Medium feed

The BioSep 200L Acoustic Medium

Perfusion System is a simple, pump

effective and reliable cell

separation system designed Bioreactor
expressly for cell retention during
Resonator
perfusion of high-density stirred support
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 Typical configuration of the 200L BioSep acoustic perfusion system.
on or above the bioreactor head
plate. The system is easy to
45
operate and provides robust, To t a l c e l l s
40
continuous operation, whatever
the desired cell culture duration. 35
C e l l c o n c e n t ra t i o n

Using this device existing batch 30

(106 cells/ml)

or fed-batch reactors can be 25

conveniently adapted to high-
20
productivity perfusion.
15

10

0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
C u l t u r e t i m e ( d ay s )

BioSep 200L CHO perfusion (courtesy 4C, Belgium)

BioSep Chamber 200L

Mechanical:

Material: Body: 1.4404 SS 316L

Harvest outlet
Transducer plate: Pyrex glass

Gasket: Silicone

O-ring: Silicone

Finish: Interior - Electro polish, Ra < 0.8 μm

Top
Exterior - Electro polish/mechanical polish plate
Weight: Approximately 13 kg
Cuvette
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

Sensor
Medium inlet: 0,5” Triclamp port
Water
Return outlet: 0,5” Triclamp bath inlet
Water bath
Water bath inlet: 0,5” Triclamp outlet

Water bath outlet: 0,5” Triclamp

Funnel
Temperature: 130˚C max.
Recirculation inlet Return
Pressure range (internal): 3.2 barg
outlet
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: 2m

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
 Clarified culture Pumps and other hardware to complete a perfusion set-up
medium Harvest
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
Cell that the harvest flow is stopped for a few seconds, arresting the flow in the chamber.
suspension
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
Fresh feed
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

Concentrated
capability to reverse the harvest flow at full pump speed (prime), during field off time.
cell recycle 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.

Recirculation pump A typical BioSep set-up

• speed ±3 x harvest flow BioSep 10L: • holder to fit resonance chamber into the headplate
• suction tube for recirculation loop
Harvest pump • recirculation pump
• variable speed with remote • harvest pump (variable speed / remote controlled)
control input • fresh medium inlet and pump (e.g. controlled via level controller)
• variable speed with remote
BioSep 50L: • diptube for return of cells from chamber. (The diptube should be
control input and prime rate
provided with the proper headplate connector and a 0.5” TC to install
reverse action
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)
Clarified culture
medium Harvest

Prime rate
reverse pump

Various adapters are available to fit the BioSep 50L to any type or brand of bioreactor.

Fresh feed Cell

suspension

Concentrated
cell recycle

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
A 100L working volume bioreactor with
• Sensors (pH, DO, T) Optional a BioSep 200L chamber and support.
• 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
The BioSep technology
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. is patented (US 5.626.767)
 BioSep Ordering information

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 Applikon Dependable Instruments bv

• making large-scale perfusion an increasing viable option for cell culture processes 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

STS90 - VZXV122902 - Subject to modifications - Printed by Applikon Dependable Instruments bv - The Netherlands 10.02 Internet: www.applikon.com