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Cadence™ Acoustic Separator

Enabling Technology for Continuous Clarification of Batch Cell Culture

The Cadence Acoustic Separator 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.

  • 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

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.

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.



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Description

Enabling Technology for Continuous Clarification of Batch Cell Culture

The Cadence Acoustic Separator 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.

  • 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

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.

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.

Specifications

Product Specifications

 
Component Specifications Parameter Specification
Operating Conditions Flow Rate 0 – 10 L/h (3.6 L/h, typical)
Flow rate scale-up chamber 50 L/h ±10 L/h
Pressure Range 0 to 2 barg (0 to 30 psig)
Feed Fluid Temperature 0 °C to 40 °C (32 °F to 104 °F)
Operating Temperature 0 °C to 40°C (32 °F to 104 °F)
     
     
Cadence Acoustic Chamber Max Feed Pressure 30 psi
Connectors 1/8” hosebarb
Wetted Materials Barbs – Polysulfone (PS)
Housing - Polyphenylsulfone (PPS)
Transducer Film – Polyether etherketone (PEEK)
O-rings - Platinum Cured Silicone)
Reflector - Borosilicate Glass
Material data USP Class VI
Chemical Compatibility 8 hours in 0.5M NaOH (ambient temp)
Weight  <0.5 kg (<1 lb)
Shelf Life 1 year
Nominal Flow Rate 60 mL/min
Nominal Pressure Drop per stage  <70 mbar (<1 psi) pressure drop @ 60 mL/min
Hold Up volume per stage 190 mL
     

 
Component Specifications Parameter Specification
Cadence Acoustic Separator Cadence Acoustic Separator EU Version 220 V / 50 Hz
Cadence Acoustic Separator US Version 110V / 60 Hz
RF Power Output  0 - 100 W per channel

Maximum Power Consumption 

1.8 kW
Sonic Channels  5
Sonic Frequency 1.9 - 2.2 MHz
Pump Flow Rate (L/S 16 tubing); 30 mL/min – 160 mL/min (L/S 14 tubing); 1 mL/min – 40 mL/min
Footprint (W/o Pump head) D 559 mm x W 546 mm x H 327 mm (22.0” D x 21.5” W x 12.875” H)
Footprint (W/Pump head) D 603 mm x W 546 mm x H 327 mm (23.75” D x 21.5” W x 12.875” H)
Pumps per Controller 5
Certification UL 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
Regulatory Information The Cadence Acoustic Separator meets national and international standards including UL 61010-1, CAN/CSA C22.2# 61010-1, CENELEC EN 61010- 1, CENELEC EN 61326-1, FCC 47CFR 15B clA
     

 
Component Specifications Parameter Specification
Turbidity Sensor Assembly Materials Polyphenylsulfone (PPS)
Platinum Cured Silicone O-Rings
Polycarbonate
Turbidity range 0 – 4400 NTU
  5 x turbidity housing with caustic stability
Operating Temperature 2 – 40 °C
     
Performance

Features and Benefits

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).

Table 1

Performance Comparison

 
Sample Description mAb (g/L) SEC agg1 (%) (g/L) DNA (μg/mL) HCP (mg/mL)
Feed 1.24 0.67 D7.79 0.96
Permeate pool 1.24 0.62 7.11 0.78
Post filtration 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)

Figure 1

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).

Figure 2

Figure 2

Figure 3

Secondary polishing by depth filtration; filter areas per 1000 L as a function of pool turbidity

Figure 3

Scalability

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 Filtration Cadence Acoustic Separator
Total depth filter area 35 m2 8 m2
Depth filter footprint 10 m2 2 m2
Pre-use WFI rinse 4200 L 800 L
Post-process buffer flush 840 L 160 L
 

Recommended Process Volumes

 
Cadence Acoustic Separator Maximum Batch Volume 25 liters
Minimum Batch Volume 3 liters
 

Secondary Depth Filter Recommendations

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:

 
Volume Format Grade Typical Area Required Part Number
<25 Pall Supracap™ HP depth filter capsules PDK7 or PDH6 0.1 m2 2 x Supracap 100 2 x 0.05 m2 2 x NP6PDK71 or 2 x NP6PDH61
<10 0.4 m2 1 x Supracap 100 1 x 0.05 m2 1 x NP6PDK71 or 1 x NP6PDH61
<5 0.002 m2 1 x Supracap 100 1 x 0.0025 m2 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 Kleenpak® capsules Supor EKV membrane 0.2 μm 375 cm2 KA1EKVP1G
<10
<5 Mini Kleenpak capsules 220 cm2 KA02EKVP8G
 

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:

 
Volume Format Grade Typical Area Required Part Number
<25 Pall Supracap HP depth filter capsules PDD1 0.05 m2 1 x Supracap 100 0.05 m2 1 x NP6PDD11
<10 0.02 m2 1 x Supracap 100 0.05 m2 1 x NP6PDD11
<5 0.01 m2 1 x Supracap 100 0.05 m2 1 x NP5PDD11
 

Bioburden Filtration Recommendation – Post PDD1

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:

Supor EKV Sterilizing-Grade Filter Capsules

 
Volume Format Grade Filter Area Part Number
<25 Kleenpak capsules Supor EKV Supor EKV 375 cm2 KA1EKVP1G
<10
<5 Mini Kleenpak capsules 220 cm2 KA02EKVP8G
 
Additional Information

Amgen: Industry must look to next-generation continuous cell harvesting tech (Click here)



Ordering Information

Systems and Replacement Parts

 
Part Number Description
SYS-US Cadence Acoustic Separator, Universal System
AC-K1 Cadence Acoustic Chamber Kit
TS-S1 Individual Replacement Turbidity Sensor
 
Application
Systems