Cadence Acoustic Separator

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. 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 10 6 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. 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 mab SEC DNA HCP Description (g/l) agg 1 (%) (µg/ml) (mg/ml) Feed 1.24 0.67 7.79 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. 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.

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 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 Normalized Turbidity 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 23 (68%) 43.8 (58%) 25.1 (68%) 26.5 (65%) nturbidity Reduction By Stage Cell density /10 6.mL -1 (Cell viability) Stage 4 Stage 3 Stage 2 23.5 (82%) 23 (72%) 21 (44%) 40.2 (96%) 22.5 (92%) 23 (69%) 26.6 (58%) Figure 3 Secondary polishing by depth filtration; filter areas per 1000 L as a function of pool turbidity 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 10 6 cells/ml) Primary Separation Technology Depth Cadence Filtration Acoustic Separator Total depth filter area 35 m 2 8 m 2 Depth filter footprint 10 m 2 2 m 2 Pre-use WFI rinse 4200 L 800 L Post-process buffer flush 840 L 160 L Recommended Process Volumes Cadence Maximum Batch Volume 25 liters Acoustic Separator Minimum Batch Volume 3 liters Filter Area (m2) 14 12 10 8 6 4 2 0 0 100 200 300 400 500 600 700 800 Pool Turbidity (NTU) Total Area www.pall.com 3

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: Typical Part Volume Format Grade Area Required Number <25 L Pall Supracap PDK7 0.1 m 2 2 x NP6PDK71 HP depth filter or 2 x Supracap 100 or capsules PDH6 2 x 0.05 m 2 2 x NP6PDH61 <10 L 0.04 m 2 1 x NP6PDK71 1 x Supracap 100 or 1 x 0.05 m 2 1 x NP6PDH61 <5 L 0.002 m 2 1 x NP5PDK51 1 x Supracap 100 or 1 x 0.0025 m 2 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 Kleenpak Supor EKV 375 cm 2 KA1EKVP1G <10 L capsules membrane 0.2 µm <5 L Mini Kleenpak 220 cm 2 KA02EKVP8G capsules 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: Typical Area Part Volume Format Grade Required Number <25 L Pall Supracap HP PDD1 0.05 m 2 1 x NP6PDD11 depth filter 1 x Supracap 100 capsules 0.05 m 2 <10 L 0.02 m 2 1 x NP6PDD11 1 x Supracap 100 0.05 m 2 <5 L 0.01 m 2 1 x NP5PDD11 1 x Supracap 100 0.05 m 2 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 L Kleenpak Supor EKV 375 cm 2 KA1EKVP1G <10 L capsules membrane 0.2 µm <5 L Mini Kleenpak 220 cm 2 KA02EKVP8G capsules Systems and Replacement Parts Part Number CAS-SYS-EU CAS-SYS-US CAS-AC-K1 CAS-TS-S1 Product Specifications Description Cadence Acoustic Separator System, EU Cadence Acoustic Separator System, US Cadence Acoustic Chamber Kit Individual Replacement Turbidity Sensor Component Operating Flow Rate 0 10 L/h (3.6 L/h, typical) Conditions Flow rate scale-up 50 L/h ±10 L/h chamber 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 Max Feed Pressure Chamber Connectors Wetted Materials Material data Chemical Compatibility Weight Shelf Life 30 psi 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 Nominal Flow Rate 60 ml/min Nominal Pressure Drop <70 mbar (<1 psi) pressure per stage drop @ 60 ml/min Hold Up volume per stage 190 ml 4

Component Cadence Acoustic Cadence Acoustic 220 V / 50 Hz Separator Separator EU Version Cadence Acoustic 110V / 60 Hz Separator US Version 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 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 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 Component Turbidity Sensor Materials Polyphenylsulfone (PPS) Assembly Platinum Cured Silicone O-Rings Polycarbonate Turbidity range 0 4400 NTU 5 x turbidity housing with caustic stability 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 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). www.pall.com 5 4/16, PDF, GN16.6491 USD3133