Webinar: Novel Perfusion Filter and Controller for N-1 Application
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Merck KGaA's life science business, operating as MilliporeSigma, has introduced a novel perfusion filter and controller as part of its BioContinuum seed train platform aimed at enhancing bioprocessing efficiency. The technology promises increased cell density, reduced production lead times, and lower manufacturing costs by allowing for continuous processing and optimized media utilization. A case study demonstrates the successful application of intensified upstream processes that significantly improve cell specific productivity and overall process robustness.
Webinar: Novel Perfusion Filter and Controller for N-1 Application
- 1. The life science business of Merck KGaA, Darmstadt, Germany operates as MilliporeSigma in the U.S. and Canada. Novel Perfusion Filter and Controller for N-1 Application January 2020 Part of the BioContinuum™ Seed Train Platform
- 2. The life science business of Merck KGaA, Darmstadt, Germany operates as MilliporeSigma in the U.S. and Canada
- 3. Part I of the BioContinuum™ Seed Train Platform webinar series: Media and process development for seed train intensification Introduction to intensified upstream processing How combining media specifically designed for seed train, production and harvest intensification, can increase the cell specific productivity (Qp) in the final production stage How applying high cell density cryopreservation can significantly shorten your seed train Previous Webinar 3
- 4. Outline 1 2 3 Trends in Upstream Intensification Overcoming Challenges in Perfusion Introducing Cellicon™ Filter Design 4 Intensified Upstream Case Study
- 5. BioPhorum Operations Group (BPOG) Technology Roadmap Drivers for the switch to intensified bioprocessing Business Drivers Speed Quality Flexibility Cost reduction in new facility build times Compress production lead time by 80% robustness 90% reduction in cost of poor quality reduction in product change over time reduction in cost to manufacture and capital expenditure 90%90%10x70% Key Enabling Technologies Process Intensification Single Use Process Analytics Software & Automation 5
- 6. Manufacturers are moving towards intensified, closed, and continuous processing 2018 | Standard mAb Process Template 2018 - 2020+ | mAb Process Intensification >2025 | Continuous Processing 6
- 7. Perfusion: continuous media addition and waste removal Achieve increased cell density targets 7
- 8. The cell retention device is critical for perfusion operation Key Terms: • VVD: vessel volumes per day of media added • CSPR: cell specific perfusion rate • Bleed: cell mass removed N-1 vs N Perfusion • N-1 (intensified seed train): cells are the product, perfusate is waste • N (production): protein is the product, often a continuous harvest of the perfusate Cellicon™ Perfusion Solution 8
- 9. User research for cell retention technology Overcoming challenges in perfusion Users of existing cell retention technologies express the following needs: - Robust operation over time (low fouling, good reproducibility) - Support of high cell densities and a wide range of media exchange rates - Optimized process control - Harmless for cells (low shear, adequate pumping technology), and shortest cell residence time - Complete, scalable offering: cell retention device availability at all scales and for all applications (N–1 and N, PD and GMP) - Optimum product yield/passage in the N application - Supply chain robustness 9
- 10. Design considerations Solution is driven by user and applications needs Performance Robust High Throughput Low Shear Ready to use in minutes Single Use Pre-sterilized Scalable 3-2000 L Scale TFF Design Pump Type 10
- 11. Tangential flow filtration basics Crossflow rate >> perfusate rate: minimize surface fouling Membrane surface Perfusate Filtratefluxrate Volume filtered Crossflow 1 Flow range constraints due to limited single-use pumps available2 Impact on design 11
- 12. Broad selection of available membranes ▪ Membrane Materials ▪ Wide Range of Pore Sizes Allows for great flexibility in device design ▪ Control of feed channel dimensions Advantages of a flat sheet design Tangential flow filtration design considerations -5 0 5 10 0 2000 4000 6000 8000 10000 TMP(psi) Throughput (L/m2) Performance of Different Membrane Pore Sizes 0.22 Micron PVDF 0.65 Micron PVDF 1.0 Micron PVDF 5.0 Micron PVDF ✓ Low protein binding membrane selected ✓ Increased pore size = increased throughput by reducing impact of pore fouling ✓ Constant channel length = Scalability ✓ Reduced height = Reduced flow rate ✓ Allows for single-use pump ✓ Consistent dimensions = Robustness! Velocity Channel Height 12
- 13. 13 CelliconTM perfusion filter and controller Delivering on the need o High-capacity, supports high cell densities o Reliable and reproducible, low risk of failure 13 Ready to Use in Minutes Superior Performance Complete, Linearly Scalable Offering o Supplied as a complete assembly (filter, sensors, pump) o Dry (no flushing required) o Gamma irradiated o Sterile weld (PD), sterile connector (pilot & process) 0 2 4 6 8 0 2000 4000 6000 8000 TMP(psi) Throughput (L/m2) 190111 BRX 1 181121 BRX 2 180911 BRX 3 0 1 2 3 4 5 0 2000 4000 6000 8000 TMP(psi) Throughput (L/m2) 3L 50L o Proven linear scalability from 3-50 L o Complete offering from bench to 2000L 0 20 40 60 80 100 120 0 2 4 6 VCD(E6cells/mL) andViability(%) Duration (days) Viable Cell Denisty Viability
- 14. Current and future offerings Complete platform 3 L 50 L 200 L 1000 L 2000 L 1 Complete Offering Device sizes designed to match a range of available bioreactors Easy to manifold for intermediate sizes 2 Linear Scalability All devices have the same channel height and path length, making scale up predictable 3 Scale-Down Option Smallest device size designed for process- development volumes down to 1 L 4 Low-Shear Pump Across the Board Design enables utilization of low-shear pump from bench to manufacturing scales14
- 16. Case study: developing an intensified upstream process The CHOZN® GS cell line fed-batch process was identified as a good foundation for high seed fed-batch PD development • Robustness: Consistent process performance at TPP tube, Ambr15, 3 L and 50 L scale • High PVCD and titer • Stability Perform Intensified Upstream Process Adapt cells to new media and establish working cell bank Determine CSPR requirements of cells in N-1 perfusion process Determine feed strategy of high seed FB process Process Development Steps: • Adapt cells to expansion media • Establish perfused N-1 process utilizing 3 L Mobius® bioreactor, expansion media, and Cellicon™ filter and controller • Determine CSPR requirements • Identify feed strategy for high seed FB process using tubespin • Compare intensified upstream process to established FB control (productivity, product quality) Strategy 16
- 17. Generate working cell bank for CHOZN® GS cells in Cellvento® 4CHO-X expansion media ❑ Receive cells from cell bank frozen in CHO-fusion media ❑ Passage cells in expansion media ensuring media adaption (stable doubling times) ❑ Establish Working Cell Bank: Cells frozen in expansion media Determine CSPR requirements for cell line with Cellvento® 4CHO-X expansion media ❑ Steady state perfusion process established and media rate adjusted until optimal CSPR identified (40 pL/cell/day) Establishing a perfused N-1 process 0.60 0.65 0.70 0.75 0.80 90.0 92.0 94.0 96.0 98.0 100.0 0 1 2 3 4 5 6 SpecificNetGrowthRate (1/day) Viability(%) Passage # Viability Growth Rate Reduced CSPR to 40 pL/cell/day 17
- 18. Establishing feed strategy for high seed fed-batch process Target: Increased titer at same duration compared to 14 day fed-batch control with ending viabilities ≥70% • Evaluate impact of seeding density and feed timing on cell growth/health profiles Background • Current fed-batch process has seeding density of 0.5 million cells/mL • Feed strategy: 5% days 3, 5 & 12, 7.5% days 7 & 10, Glucose feed to 6 g/L Tue Wed Thu Fri Sat Sun Mon Tue Wed Thu Fri Sat Sun Mon Tue D0 D1 D2 D3 D4 D5 D6 D7 D8 D9 D10 D11 D12 D13 D14 0.5E6 SEED - CONTROL (3x) 5% 5% 7.5% 7.5% 5% 2.5E6 SEED (3x) 5% 5% 7.5% 7.5% 5% 5E6 SEED - CONDITION1 (3x) 5% 5% 7.5% 7.5% 5% 5E6 SEED - CONDITION2 (3x) 5% 7.5% 7.5% 5.0% 5% 5E6 SEED - CONDITION3 (3x) 5% 7.5% 7.5% 5.0% 5% 5E6 vc/mL 2.5E6 vc/mLCENTRIFUGE/ RESUSPEND 0.5E6 vc/mL “Control” timing Process development 18
- 19. D14 TITER1 Final Viab IVCC Qp g/L % 109 cells.hr/L pg/cell/day 0.5E6SEED- CONTROL(3x) 3.8±0.1 84±4 4445±232 20±0 2.5E6SEED(3x) 4.9±0.1 79±3 5426±165 22±0 5E6SEED- CONDITION1(3x) 5.0±0.0 65±4 5890±26 21±0 5E6SEED- CONDITION2(2x) 5.1±0.1 73±0 5958±82 21±0 5E6SEED- CONDITION3(3x) 5.4±0.1 68±1 6105±87 21±0 VCD TITER VIABILITY Tubespin results High seed fed-batch resulted in increased titer, PVCD and IVCC but feed strategy still needs improvement RECOMMENDATION: • Use knowledge gained and adapt slightly to try to prolong viability • D0: 5%, D3: 5%, D5: 7.5%, D7: 7.5%, D10: 7.5%, Day 12: 5% • Future: Perform more extensive PD to reduce drop in viability, further increase titer (feed strategy/temp shift) 1 BioHT titer results, proA pending Establishing feed strategy for high seed fed-batch process
- 20. Experimental plan Intensified upstream process N-1 Perfusion 3L High Seed Fed Batch Bioreactor 3L Standard Seed Fed Batch Bioreactor (0.5 e6 cells/mL) (5e6 cells/mL) Batch x 1-2mL 10 - 30 E6 vc/mL WCB Inoculum train 1-2ml 10 - 30 E6 vc/mL Control Condition: Conventional Seed Train WCB Inoculum train Batch x 3L Standard Seed FB reactor (0.5 e6 cells/mL) Experimental Condition: Perfused N-1 Seed Train ❑ Transfer at ~50 million cells/mL 20
- 21. Perfused N-1 0 10 20 30 40 50 60 70 80 90 100 0 1 2 3 4 5 6 7 VCD(E6cells/mL),Viability(%), andDoublingTime(hours) Duration (days) Viability, VCD, and Doubling Time VCD Doubling Time Viability 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 Concentration(mM,g/L) Duration (days) Nutrients/Metabolites Glucose Lactose Glutamine Glutamate Ammonia Process details and results ❑ Bioreactor: Mobius® 3 L bioreactor, 2.2 L WV ❑ Media: Cellvento® expansion media, target 40 pL/cell/day CSPR ❑ Cell Retention Device: Bench-scale Cellicon™ filter and controller at 100 mL/min crossflow ❑ Inoculation Density: 0.65e6 cells/mL ❑ Cells Transferred on Day 6 to Fed-batch Production Bioreactor (~50e6 cells/mL) 21
- 22. Filter performance monitoring Perfused N-1 No filter fouling during N-1 perfusion process Pressure Data Cellicon™ controller: • Automated flow control through flowmeter feedback to pump • Pressure monitoring • Filter performance • Troubleshooting • Display and log of customizable alerts • Data logging through USB port 22
- 23. Intensified upstream process N-1 Perfusion 3L High Seed FB Bioreactor 3L Standard Seed FB Bioreactor (0.5 e6 cells/mL) (5e6 cells/mL) Batch x 1-2mL 10 - 30 E6 vc/mL WCB Inoculum train 1-2mL 10 - 30 E6 vc/mL Control WCB Inoculum train Batch x 3L Standard Seed FB Bioreactor (0.5 e6 cells/mL) Fed-Batch Production Process ❑ Bioreactor: 3L Mobius® bioreactor, 1.5 L WV ❑ Inoculation Density: Control and Standard Density: 0.5e6 cells/mL, High Seed: 5e6 cells/ml ❑ Basel Media: EX-CELL® Advanced ❑ Feed Media: 50:50 Blend of EX-CELL® Advanced and Cellvento® 4Feed ❑ Duration: 14 day process Bioreactor Settings: • Temp: 36.8 ºC • Agitation: 200 rpm • pH: 6.90 +0.1/-0.15 • DO: 50% • Headspace air: 50 mL/min • Sparge: open-pipe *Each run performed in duplicate Experimental plan for production process 23
- 24. Feed Media: 50:50 blend of EX-CELL® Advanced and Cellvento® 4Feed − Feed percentages based on volume at time of feeding − Feed strategy for high seed based on results of tubespin experiment (modifications to improve VCD) Glucose maintained at 6g/L daily − Increased to 12g/L on Day 5 to skip Day 6 feed − Increased to 10g/L on Day 12 to skip Day 13 feed Feed strategy Fed-batch production process Day 0 3 5 7 10 12 Total Control 0% 5% 5% 7.5% 7.5% 5% 30% pN-1 Standard 0% 5% 5% 7.5% 7.5% 5% 30% pN-1 High 5% 5% 7.5% 7.5% 7.5% 5% 37.5% 24
- 25. Peak VCD achieved 3 days earlier for high seed N, slightly higher actual peak VCD observed Similar trends in VCD and viability across conditions − Within normal process variability High seed process requires further process development − Feed strategy improvements − Temperature shift VCD and viability Fed-batch production process 0 5 10 15 20 25 30 35 - 2 4 6 8 10 12 14 VCDX1E6 Days Viable Cell Density Crtl A Crtl B pN-1 0.5 A pN-1 0.5 B pN-1 5.0 A pN-1 5.0 B 0% 20% 40% 60% 80% 100% 120% - 2 4 6 8 10 12 14 % Days Viability Crtl A Crtl B pN-1 0.5 A pN-1 0.5 B pN-1 5.0 A pN-1 5.0 B 25
- 26. Higher titer trend observed with intensified process − Although further PD for high seed process is required, results are promising to achieve significant productivity improvement Similar titer achieved between control and standard seed perfused N-1 − Utilizing Cellicon™ perfusion technology did not impact cell health or performance Similar specific productivity between conditions − In line with expectations based on historical data Fed-batch production process 0 1 2 3 4 5 6 - 2 4 6 8 10 12 14 g/L Days Titer Ctrl A Ctrl B pN-1 0.5 A pN-1 0.5 B pN-1 5.0 A pN-1 5.0 B 0 5 10 15 20 25 - 2 4 6 8 10 12 14 16 18 20 pg/cell/day Days Specific Productivity Ctrl A Ctrl B pN-1 0.5 A pN-1 0.5 B pN-1 5.0 A pN-1 5.0 B Titer and specific productivity 26
- 27. Glutamine and glutamate consumption trends consistent across conditions − Increases in both observed with feeds Glutamine and glutamate Fed-batch production process 0 1 2 3 4 5 6 7 8 - 2 4 6 8 10 12 14 mM Days Glutamine Crtl A Crtl B pN-1 0.5 A pN-1 0.5 B pN-1 5.0 A pN-1 5.0 B 0 2 4 6 8 10 12 - 2 4 6 8 10 12 14 mM Days Glutamate Crtl A Crtl B pN-1 0.5 A pN-1 0.5 B pN-1 5.0 A pN-1 5.0 B 27
- 28. Glucose consumption similar across conditions − Initial glucose consumption higher in high seed culture due to increased VCD but lower later in process as viability drops Ammonia trends show increase up to day 10-12 and then dropped but notable differences in levels were observed − Unclear why control achieved highest ammonia level − Reduction in ammonia later in culture less pronounced in pN-1 standard seed Glucose and ammonia Fed-batch production process 0 2 4 6 8 10 12 14 - 2 4 6 8 10 12 14 g/L Days Glucose Crtl A Crtl B pN-1 0.5 A pN-1 0.5 B pN-1 5.0 A pN-1 5.0 B 0 1 2 3 4 5 6 - 2 4 6 8 10 12 14 mM Days Ammonia Crtl A Crtl B pN-1 0.5 A pN-1 0.5 B pN-1 5.0 A pN-1 5.0 B 28
- 29. Lactate trend similar with shift from production to consumption correlating with peak VCD − Shift back to production observed at end of run CO2 requirement for pH control consistent with lactate trend − As lactate increases, pH drops and CO2 is not needed − As lactate is consumed, pH increases, requiring CO2 to maintain pH set point Lactate and pCO2 Fed-batch production process 0 20 40 60 80 100 120 140 - 2 4 6 8 10 12 14 pCO2(mmHg) Days pCO2 Crtl A Crtl B pN-1 0.5 A pN-1 0.5 B pN-1 5.0 A pN-1 5.0 B 0 0.5 1 1.5 2 2.5 - 2 4 6 8 10 12 14 g/L Days Lactate Crtl A Crtl B pN-1 0.5 A pN-1 0.5 B pN-1 5.0 A pN-1 5.0 B 29
- 30. SEC analysis shows consistent product quality across conditions − Small amount of fragment appears in one of the high seed, perfused N-1 − Not observed in duplicate Relatively consistent charge profiles observed between conditions − Slightly higher acidic peaks in high seed, perfused N-1 cultures − Resulting in slightly lower neutral and basic peaks − Correlated to lower pH toward end of culture − Resulting from increase in lactate level SEC and charge analysis Fed-batch production process 0 10 20 30 40 50 60 70 80 90 100 Ctrl A Ctrl B pN-1 0.5A pN-1 0.5B pN-1 5.0A pN-1 5.0B PeakArea(%) SEC Analysis Aggregates Monomer Fragments 0 10 20 30 40 50 60 Ctrl A Ctrl B pN-1 0.5A pN-1 0.5B pN-1 5.0A pN-1 5.0B PeakArea(%) Charge Analysis Acidic Neutral Basic 30
- 31. Consistent glycan profile observed across conditions Glycan analysis fed-batch production process Fed-batch production process MAJOR PEAKS (only main peaks are considered in calculating %area) Area (%) Structure G0F-N G0 G0F Man5 G1a G1b G1Fa G1Fb G2F Time 10.9 11.5 13.2 14.0 14.6 15.1 16.3 16.7 19.7 Ctrl A 1.2 5.2 55.9 2.6 1.5 0.6 21.4 7.9 3.7 Ctrl B 1.1 5.3 56.0 2.4 1.4 0.6 21.4 8.1 3.7 pN-1 0.5A 1.0 5.1 55.5 2.4 1.5 0.6 21.9 8.3 3.8 pN-1 0.5B 0.9 4.9 54.5 2.2 1.4 0.7 22.8 8.6 4.1 pN-1 5.0A 1.6 5.2 54.7 3.1 1.7 0.6 21.4 8.0 3.8 pN-1 5.0B 1.5 5.1 55.4 3.0 1.6 0.6 21.1 8.0 3.7 0.0 10.0 20.0 30.0 40.0 50.0 60.0 Area(%) Peaks Glycan Analysis Ctrl A Ctrl B pN-1 0.5A pN-1 0.5B pN-1 5.0A pN-1 5.0B 31
- 32. A perfused N-1 process using a CHOZN® GS cell line was developed using the new Cellicon™ filter and controller along with the new Cellvento® 4CHO-X expansion media − The N-1 bioreactor reached 50 million cells/mL in 6 days while maintaining logarithmic growth − The Cellicon™ filter and controller was easy to use with its plug-n-play design and its gentle processing of cells; this enabled quick process development to successfully transfer a traditional upstream batch/fed-batch process to an intensified process (perfused N-1 to fed-batch N) Cells from the perfused N-1 bioreactor, utilizing the Cellicon™ filter were used to seed a fed-batch production reactor at both standard density (0.5e6) and high density (5e6) − Growth, metabolism and product quality trends were comparable to the control process − The intensified upstream process resulted in greater cell mass production in a shorter time, and showed potential for significantly higher titers to be achieved Conclusions Intensified upstream process 32
- 33. Part III of the BioContinuum™ Seed Train Platform webinar series: A Cost Analysis and Evaluation of Perfused Seed Train Scenarios Through Process Modeling Different variations of perfused seed trains Potential benefits from high seed fed batch vs. traditional fed batch Impact of perfused seed trains in single bioreactor vs. multiple bioreactor scenarios Impact of perfused seed trains in multiple production/seed train ratio scenarios Upcoming webinar February 6, 2020 33
- 34. Acknowledgements Cellicon™ filter and controller Team Cell Culture Sciences Team Analytical Sciences Team Cell Culture Performance Team BioContinuum™ Upstream Team Brandon Medeiros Hiral Gami Kimberly Mann Patricia Kumpey Rebecca Hood Doug Rank Derek Silva Kristina Cunningham Yana Ostrovskiy Shawn Bates Joe Muldoon Joe Sinagra Jochen Sieck Mona Bausch Amy Wood Mikhail Kozlov
- 35. Thank You! © 2020 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved. BioContinuum, The Vibrant M, Millipore, Cellicon, Mobius, EX-CELL, and Cellvento are trademarks of Merck KGaA, Darmstadt, Germany or its affiliates. All other trademarks are the property of their respective owners. Detailed information on trademarks is available via publicly accessible resources.