USD3133
Cadence™ Acoustic Separator
Enabling Technology for Continuous Clarification
of Batch Cell Culture
The Cadence Acoustic Separator (CAS) enables the continuous removal of CHO cells and cell debris using
a Cadence single-use acoustic chamber to achieve clarification of harvested cell culture fluid (HCCF) ready
for downstream processing. The acoustophoretic removal is intrinsically scalable and is optimized for the
clarification of high density Chinese Hamster Ovary (CHO) cell lines to provide a robust single-use primary
clarification solution that delivers quality that is equivalent to centrifugation without the scaling, footprint and
cleaning issues common with this solution. The Cadence Acoustic Separator is an easy to use technology
without the volume limitations experienced by depth filtration solutions that lead to large footprint and
buffer consumption or the need for process additives to enhance the performance.
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Optimized for high CHO cell densities in the range 20 - 50 x 106 cells/mL
Performance scales directly from process development to full scale processes
Quality of HCCF unaffected and comparable to centrifugation
Small footprint compared to equivalent centrifugation or depth filter solutions
Reduced depth filtration requirement for secondary polishing
Lower buffer consumption throughout the clarification process
Continuous operation to link with continuous downstream purification including chromatography using
the Cadence BioSMB PD system
Description
Features and Benefits
Advances in fed batch cell culture have led to higher cell
densities of up to 50 x 106 cells/ml and product titers of > 5g/L.
Accompanied by a shift towards the use single-use technology
in cell culture there is a drive to improve the efficiency of the cell
harvest and clarification stage to generate HCCF for capture
chromatography and subsequent downstream processing.
This is further driven by the evolution of continuous processes
where there is a preference for a continuous feed of HCCF
available for direct load to the continuous multicolumn capture
chromatography step using the Cadence BioSMB PD system.
Existing cell culture clarification using either centrifugation or
depth filtration are typically operated in batch mode and require
bulk storage of feed or HCCF during the process.
No adverse effects on the quality of the HCCF following
clarification by the Cadence Acoustic Separator have been
observed for CHO cell culture expressing a monoclonal
antibody (mAb) (Table 1).
The Cadence Acoustic Separator provides a novel scalable
single-use technology for cell culture clarification based on an
acoustophoretic separation. Acoustic wave separation (AWS)
technology involves the use of low frequency acoustic forces to
generate a 3 dimensional standing wave across a flow channel.
Cell culture from a fed batch bioreactor enters the flow channel,
and as the cells pass through the 3D standing wave they are
trapped by the acoustic forces. The trapped cells migrate to
the nodes of the standing wave, and begin to clump together
till such time as their buoyancy decreases and they settle out
of the suspension by gravity.
After separation of cells and cell debris, the permeate from
Cadence Acoustic Separator shows a significant reduction
in turbidity and reduces the area requirements for secondary
clarification using depth filtration and subsequent filtration for
bioburden control.
Table 1
Performance Comparison
Sample mAb
SEC
Description(g/L) agg1 (%)
Feed
Permeate pool
Post filtration DNA
(µg/mL)
HCP
(mg/mL)
1.240.67 7.79 0.96
1.24
0.62
7.11
0.78
1.19
0.66
3.94
0.46
Designed for single-use operation the Cadence Acoustic
Chamber comprises a flow path with 4 acoustophoretic
chambers in series each providing an increasing level of
clarification. The degree of clarification can be monitored
at each stage of the cell separation with the in-line turbidity
detectors.
The efficiency of the clarification for a typical CHO cell culture
(10-20 L) is illustrated in Figure 1.
Figure 1
Clarification performance by stage over time as a percentage
of total starting cell density (TCD) for a typical CHO cell culture
(10 -20 L)
% TCD Reduction
100
90
80
70
60
50
40
30
20
10
0
%TCD vs Time vs Stage
2 hrs
4 hrs
6 hrs
9 hrs
12.5 hrs
Stage 1 Stage 2 Stage 3 Stage 4
Following primary clarification of CHO cell culture using the
Cadence Acoustic Separator the cell culture fluid can be further
polished using an in-line depth filtration stage.
When used in this way, the Cadence Acoustic Separator not
only reduces the solids content of the HCCF to a level suitable
for sterile filtration and chromatographic purification, it has also
been demonstrated to normalise any batch to batch cell culture
variability within a given process, as shown in (Figure 2).
nTurbidity Reduction By Stage
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
Optimization of the clarification process performed at process
development scale can scale directly based upon the optimal
flowrates identified for the primary separation using the Cadence
Acoustic Separator and throughput of the depth filter selection
for secondary polishing. An important design feature of the
Cadence Acoustic Separator system is the ability to replace the
Cadence Acoustic Chamber kit with a single large chamber.
This chamber, for use in the Process Scale Cadence Acoustic
Separator, has an identical flow path geometry and materials of
construction as the chamber kit used in the development scale
separator. This feature supports scale-up method development
and verification during process development rather than having
to conduct additional trials within the GMP environment.
Advantages of using Cadence Acoustic
Separation
The replacement of the primary depth filtration step by a
Cadence Acoustic Separator achieves economic benefits by
reducing the overall operating footprint, reducing the secondary
depth filter area requirements and the associated conditioning
and flush buffers as well as reduced storage and disposal costs.
These become key process drivers as processes enter clinical
manufacture. An example of the anticipated process requirements for a 1000 L CHO cell culture is summarized in Table 2.
Table 2
Anticipated Process Requirements for clarification of a 1000 L
CHO cell harvest (>20 x 106 cells/ml)
Primary Separation Technology
Depth Cadence
Filtration Acoustic Separator
(6
43 8%
)
.8
(
25 58%
.1
)
(
26 68%
.5
)
(
23 65%
.5
)
(8
23 2%
(7 )
2
21 %)
(4
40 4%
)
.2
(9
6
22
.5 %)
(9
23 2%
(6 )
26 9%
)
.6
(5
8%
)
Total depth filter area
Depth filter footprint
Pre-use WFI rinse
Post-process buffer flush
23
Normalized Turbidity Reduction (%)
Figure 2
Scalability
Cell density
Stage 4
/106.mL-1 (Cell
Stage 3
viability)
Stage 2
35 m2
10 m2
4200 L
840 L
8 m2
2 m2
800 L
160 L
Recommended Process Volumes
Cadence Maximum Batch Volume
Acoustic Separator
Minimum Batch Volume
25 liters
3 liters
Figure 3
Secondary polishing by depth filtration; filter areas per 1000 L as
a function of pool turbidity
14
Filter Area (m2)
12
10
ea
l Ar
Tota
8
6
4
2
0
0
100
200
300
400
500
600
Pool Turbidity (NTU)
700
800
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3
Secondary Depth Filter Recommendations
Bioburden Filtration Recommendation – Post PDD1
Selection of the optimal secondary depth filtration products
will require some screening but in a typical fed batch culture
of a CHO-S based cell line expressing a humanised IgG1 mAb,
the following Pall depth filtration grades are recommended for
evaluation:
Post clarification, the HCCF may be optionally stored, filtered to
control the bioburden and stored or filtered to control the bioburden and processed chromatographically. In a typical fed batch
culture of a CHO-S based cell line expressing a humanised IgG1
mAb, the following Pall sterilizing grade filters are recommended:
Volume Format
<25 L
Typical Grade Area Required
Pall Supracap™ PDK7
HP depth filter
or
capsules
PDH6
<10 L
<5 L
0.1 m2
2 x Supracap 100
2 x 0.05 m2
0.04 m2
1 x Supracap 100
1 x 0.05 m2
0.002 m2
1 x Supracap 100
1 x 0.0025 m2
Part
Number
2 x NP6PDK71
or
2 x NP6PDH61
1 x NP6PDK71
or
1 x NP6PDH61
1 x NP5PDK51
or
1 x NP5PDH61
Bioburden Filtration Recommendation – Post PDK7/PDH6
Post clarification, the HCCF may be optionally stored, filtered
to control the bioburden and stored or filtered to control the
bioburden and processed chromatographically. In a typical
fed batch culture of a CHO-S based cell line expressing a
humanised IgG1 mAb, the following Pall sterilising grade filters
are recommended for evaluation:
Supor® EKV Sterilizing-Grade Filter Capsules
Volume Format
Grade
Filter Area Part Number
<25 L
<10 L
<5 L
Kleenpak®
capsules
Mini Kleenpak
capsules
Supor EKV
membrane 0.2 µm
375 cm2KA1EKVP1G
220 cm2KA02EKVP8G
Tertiary Depth Filter Recommendations for PD Scale
Tertiary depth filtration may not be required at small scale
but when used can prevent fouling of subsequent filters and
allow for a reduction in size of the bioburden control filter. In a
typical fed batch culture of a CHO-S based cell line expressing
a humanised IgG1 mAb, the following Pall depth filtration grades
are recommended for evaluation:
VolumeFormat
<25 L
<10 L
<5 L
4
Typical
Area Grade Required
Pall Supracap HP PDD1
depth filter
capsules
0.05 m2
1 x Supracap 100
0.05 m2
0.02 m2
1 x Supracap 100
0.05 m2
0.01 m2
1 x Supracap 100
0.05 m2
Supor EKV Sterilizing-Grade Filter Capsules
Volume Format
Grade
Filter Area Part Number
<25 L
<10 L
<5 L
Kleenpak
capsules
Mini Kleenpak
capsules
Supor EKV
375 cm2KA1EKVP1G
membrane 0.2 µm
220 cm2KA02EKVP8G
Systems and Replacement Parts
Part Number
Description
CAS-SYS-EU
CAS-SYS-US
CAS-AC-K1
CAS-TS-S1
Cadence Acoustic Separator System, EU
Cadence Acoustic Separator System, US
Cadence Acoustic Chamber Kit
Individual Replacement Turbidity Sensor
Product Specifications
Component
SpecificationsParameter
Operating
Flow Rate
Conditions
Flow rate scale-up
chamber
Pressure Range
Feed Fluid Temperature
Operating Temperature
Cadence Acoustic Max Feed Pressure
Chamber
Connectors
Wetted Materials
0 to 2 barg (0 to 30 psig)
0 °C to 40 °C (32 °F to 104 °F)
0 °C to 40°C (32 °F to 104 °F)
30 psi
Weight
Shelf Life
Nominal Flow Rate
Nominal Pressure Drop per stage
Hold Up volume per stage
60 mL/min
<70 mbar (<1 psi) pressure
drop @ 60 mL/min
190 mL
Material data
Chemical Compatibility
1 x NP6PDD11
1 x NP5PDD11
50 L/h ±10 L/h
1/8” hosebarb
Barbs – Polysulfone (PS)
Housing - Polyphenylsulfone (PPS)
Transducer Film – Polyether
etherketone (PEEK)
O-rings - Platinum Cured Silicone
Reflector - Borosilicate Glass
USP Class VI
8 hours in 0.5M NaOH
(ambient temp)
<0.5 kg (<1 lb)
1 year
Part
Number
1 x NP6PDD11
Specification
0 – 10 L/h (3.6 L/h, typical)
Component
SpecificationsParameter
Cadence Acoustic Cadence Acoustic
Separator
Separator EU Version
Cadence Acoustic Separator US Version
RF Power Output
Maximum Power
Consumption
Sonic Channels
Sonic Frequency
Pump Flow Rate
Specification
220 V / 50 Hz
Component
SpecificationsParameter
Turbidity Sensor Materials
Assembly
110V / 60 Hz
Specification
Polyphenylsulfone (PPS)
Platinum Cured Silicone
O-Rings
Polycarbonate
0 - 100 W per channel
1.8 kW
5
1.9 - 2.2 MHz
(L/S
16 tubing); 30 mL/min –
160 mL/min
(L/S 14 tubing); 1 mL/min –
40 mL/min
Footprint
D 559 mm x W 546 mm x
(W/o Pump head)H 327 mm (22.0” D x 21.5” W
x 12.875” H)
Footprint
D 603 mm x W 546 mm x
(W/Pump head)
H 327 mm (23.75” D x 21.5”
W x 12.875” H)
Pumps per Controller
5
CertificationUL 61010-1, CAN/CSA C22.2#
61010-1
CENELEC EN 61010-1,
CENELEC EN 61326-1,
FCC 47CFR 15B clA
Weight
18 kg (40 lb)
Configurations
4 stages of CAC20, 1 stage of
CAC50
Operating Temperature 2 – 40 °C
Turbidity range
Operating Temperature
0 – 4400 NTU
5 x turbidity housing with
caustic stability
2 – 40 °C
Regulatory InformationThe Cadence Acoustic
Separator meets national and
international standards including
UL 61010-1, CAN/CSA C22.2#
61010-1, CENELEC EN 610101, CENELEC EN 61326-1, FCC
47CFR 15B clA
Visit us on the Web at www.pall.com/allegro
E-mail us at allegro@pall.com
Corporate Headquarters
Port Washington, NY, USA
+1.800.717.7255 toll free (USA)
+1.516.484.5400 phone
biopharm@pall.com e-mail
European Headquarters
Fribourg, Switzerland
+41 (0)26 350 53 00 phone
LifeSciences.EU@pall.com e-mail
Asia-Pacific Headquarters
Singapore
+65 6389 6500 phone
sgcustomerservice@pall.com e-mail
International Offices
Pall Corporation has offices and plants throughout the world in: Argentina, Australia, Austria, Belgium, Brazil,
Canada, China, France, Germany, India, Indonesia, Ireland, Italy, Japan, Korea, Malaysia, New Zealand, Norway,
Philippines, Poland, Russia, Singapore, South Africa, Spain, Sweden, Switzerland, Taiwan, Thailand, United
Kingdom, and Vietnam. Distributors in all major industrial areas of the world. To locate the Pall office or distributor
nearest you, visit www.pall.com/contact.
The information provided in this literature was reviewed for accuracy at the time of publication. Product data
may be subject to change without notice. For current information consult your local Pall distributor or contact
Pall directly.
© 2016, Pall Corporation. Pall,
, Cadence, Kleenpak, Supor, and Supracap are trademarks of Pall
Corporation. ® indicates a trademark registered in the USA. TM indicates a common law trademark.
Filtration.Separation.Solution. is a service mark of Pall Corporation. Acoustic Wave Separation is an
exclusive technology licensed to Pall by FloDesign Sonics (FDS).
4/16, PDF, GN16.6491
www.pall.com
USD3133
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