biosep · centrifuges solely rely on the difference in density between cells and medium. ... the...

Duration: 0.5-1 weeks

Cell density: 2 x 10 6 c/ml

Finish: no more nutrients

Addition of concentrated nutrients

=> higher product concentration.

Duration: 1-1.5 weeks

Cell density: 2.5 x 10 6 c/ml

Finish: Viability < 50%

Addition of nutrients with

cell retention.

Duration: 1-3 months

Cell density: 20 x 10 6 c/ml

BioSep: the advanced acoustic cell retention deviceSTS90

Technical Data Sheet

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

Harvest

RecirculatingCell Suspension

Sedimenting Cell Aggregates

Fresh Medium Feed

Clarified CultureMedium P

P

PERFUSION

Harvest

Cell suspension

Cell retention device

Feed

FED - BATCH

Feed

BATCH

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.

Fig. 1: perfusion set-up with BioSep.

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.

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%.

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.

Acoust ic Energy F ie ld

TransducerRef lector

Fresh Feed

concentratedCell recycle

cell settling

Cell suspension

harvestclarified

culture medium

Fig. 2: Typical configuration of the acoustic cell retention system.

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.

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.

BioSep 10 L BioSep 50 L BioSep 200 L

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.

PERFUSION

Harvest

Cell suspension

Cell retention device

Feed

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:

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.

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.

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

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

Outputcable

Compressed air

Pump control cable

Harvest

Recirculation pump

Feed

Selective retentionDue 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.

Economic impact

Cell concentration

Antibody Production

00

200 400 600 800

20

40

60

80

100

120

140

160

180

Time (h)

Batch

Perfusion

MA

b c

on

c.[

g/m

l]

00

200 400 600 800

2

Time (h)

Successive Batch Cultures

Perfusion

Cu

mu

lati

ve

Mo

no

clo

na

l A

nti

bo

dy

(g

)

4

6

8

10

0105

106

107

108

100 200 300 400 500

Batch

Time (h)

Ce

ll C

on

ce

ntr

ati

on

(C

ell

s/m

L)

600 700 800

Perfusion(v iable cel ls )

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 106/ml (peak) 2 x 106/ml (average) 20 x 106/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

Results BioSep 50L CHO cell perfusion cultureAverage total proteine concentration in harvest 272 µg/ml, courtesy: A.O.A. Miller

45 110

Time (h)

Ce

ll c

on

ce

ntr

ati

on

(10

6 c

ell

/ml)

V iable cel ls

Total cel ls

Viable cel ls

40 100

35 90

30 80

25 70

20 60

15 50

10 40

5 30

0

0 100

20

200 300 400 500 600 700 800 900 1000 1100

Technical description BioSep Chamber 10L

Mechanical:

Material: Body: SS 316L

Window: Pyrex glass

Gasket: Silicone

O-ring: Silicone

Finish: Interior - Electro polish, Ra < 0.8 µm

Exterior - Electro polish/mechanical polish

Weight: 0.5 kg

Total volume: 24 ml

Resonator volume: 7 ml

Height above headplate: 150 mm

Height: 344 mm

Max. width: 79 mm

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



Weight: 1.5 kg

Total volume: 150 ml


Height above headplate: 177 mm

Height: 344 mm

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


Test pressure (internal): 4 bars

Electrical:

Operating frequency: 2.1-2.15 MHz


BNCconnector

Harvest outlet

Resonator body

Harvest outlet

Gasket

Cuvette

O-ring

O-ring

Return tube

Recirculation inlet

Hexagon screw

Air inlet

BNCconnector

Resonatorbody

Gasket

Gasket

Hexagonscrew

Cuvette

Return tube

Recirculationinlet

Air inlet

Adapter

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

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.

APS990 BioSep control ler

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

Stop time: 3 –10 sec.; 5 – 15 minutes

Power indication (%)

Run time knob

On/off toggle

Stop time knobPower knob

Frequency indication

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.

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 high-

productivity perfusion.

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

45

Culture t ime (days)

Ce

ll c

on

ce

ntr

ati

on

(10

6 c

ell

s/m

l)

Total cel ls40

35

30

25

20

15

10

5

0

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Typical configuration of the 200L BioSep acoustic perfusion system.

Output cable

Resonator chamber

Bioreactor

APS991 controller

Harvest

pump control cable

Recirculationpump

Resonatorsupport

Recirculationcell suspensionWater bath

Medium feed

Medium pump

BioSep Chamber 200L

Mechanical:

Material: Body: 1.4404 SS 316L

Transducer plate: Pyrex glass

Gasket: Silicone

O-ring: Silicone



Weight: Approximately 13 kg

Total volume: 1.0 L


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


Pressure range (internal): 3.2 barg

Electrical:

Operating frequency range: 2.10 - 2.13 MHz


APS 991 Control ler

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

Harvest outlet

Water bathoutlet

Water bath inlet

Sensorport

Funnel

Returnoutlet

Recirculation inlet

Topplate

Cuvette

Recirculation pump

• speed ±3 x harvest flow

Harvest pump

• variable speed with remote

control input

• variable speed with remote

control input and prime rate

reverse action

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)

Fresh feed

Concentratedcell recycle

Cell suspension

HarvestClarified culture

medium

Fresh feed

Concentratedcell recycle

Cell suspension

HarvestClarified culture

medium

Prime ratereverse pump

Adapter M18-0.5”SC-9.53 Adapter PG13.5-0.5”SC-9.53 Adapter D27-0.5”SC-9.53

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

A 100L working volume bioreactor witha BioSep 200L chamber and support.

The BioSep technology

is patented (US 5.626.767)

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

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.

Example P&ID for a fully contained BioSep in S.I.P.

)

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: [email protected]

Internet: www.applikon.com

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

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 Control ler

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 addit ional 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)

biosep · centrifuges solely rely on the difference in density between cells and medium. ... the...

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