Investing in Process Development for Increased MSC Production in Stirred Tank Bioreactors
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This document discusses process development for manufacturing mesenchymal stem cells (MSCs) using microcarrier-based suspension culture in bioreactors. It outlines challenges in scaling up MSC production, including maintaining optimal hydrodynamic conditions and controlling dissolved oxygen. A case study demonstrates that MSCs expanded at 50L scale on microcarriers in a bioreactor retain their multipotent characteristics. The document also proposes strategies for optimizing a closed seed train to decrease manufacturing risks and costs compared to traditional planar seeding methods.
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Investing in Process Development for Increased MSC Production in Stirred Tank Bioreactors
- 1. Merck KGaA Darmstadt, Germany Kara Levine, Ph.D. Increased MSC Production in Stirred Tank Bioreactors Investing in process development
- 2. The life science business of Merck KGaA, Darmstadt, Germany operates as MilliporeSigma in the U.S. and Canada. 2
- 3. Cell therapy manufacturing Manufacturing for cell therapies Process development Solid – liquid mixing Gassing Case study Mesenchymal stem/stromal cells Seed train optimization Topics 3
- 5. Current trends Regenerative Medicine Cell Therapies • Leverage inherent capabilities of human cell biology • Potentially curative treatments for diseases that are currently only managed • Chronic treatments replaced with acute therapies Challenges for Industry and Patients • Regulatory uncertainty • Reimbursement uncertainty • Efficient, cost-effective manufacturing Regenerative Medicine Clinical Trials from the Alliance for Regenerative Medicine SOTI 2018 Report 5
- 6. Current trends Regenerative Medicine Cell Therapies • Leverage inherent capabilities of human cell biology • Potentially curative treatments for diseases that are currently only managed • Chronic treatments replaced with acute therapies Challenges for Industry and Patients • Regulatory uncertainty • Reimbursement uncertainty • Efficient, cost-effective manufacturing Regenerative Medicine Clinical Trials from the Alliance for Regenerative Medicine SOTI 2018 Report Successful clinical trials must be translated to marketable treatments 6
- 7. 7
- 8. Transitioning to a Commercialization Platform How do we get from here… … to here? 8
- 9. Transitioning to a Commercialization Platform How do we get from here… … to here? Key Technology Gaps • Understand the science behind the product • Address ways to measure • Identify process influences that effect attributes • Implement standardized and controlled processes 9
- 10. Transitioning to a Commercialization Platform How do we get from here… … to here? Key Technology Gaps • Understand the science behind the product • Address ways to measure • Identify process influences that effect attributes • Implement standardized and controlled processes Critical Needs • Scalable • GMP and closed • Economically viable processes • Clinical-grade or GMP raw materials 10
- 11. Templates Have Driven Successful Commercialization Biopharmaceuticals Selecting and testing raw materials Testing process intermediates Downstream unit operations validated to inactivate and remove adventitious agents 11
- 12. Toward a New Template Cell Therapies Selecting and testing raw materials Testing process intermediates 12
- 13. Toward a New Template Cell Therapies Selecting and testing raw materials Testing process intermediates Closed systems Time from expansion to preservation to maintain viability & potency Effects of hydrodynamics on cells Microcarrier separation Concentration Filling 13
- 16. Challenges and considerations Microcarrier-based Suspension Culture •Microcarrier-based culture is highly complex •Higher hydrodynamic forces than planar culture •Media formulation interacts with other parameters: •Sparging •Oxygen demands •Microcarrier characteristics Schnitzler et al. Biochem Eng J 108 (2016) 3-13. 16
- 17. Challenges and considerations Microcarrier-based Suspension Culture •Microcarrier-based culture is highly complex •Higher hydrodynamic forces than planar culture •Media formulation interacts with other parameters: •Sparging •Oxygen demands •Microcarrier characteristics Schnitzler et al. Biochem Eng J 108 (2016) 3-13. 17
- 18. Defining States of Microcarrier Suspension Nmin Nc Njs Nh State of Suspension No Suspension On-Bottom Off-Bottom Suspension Gradient Large Minimal None Microcarrier Processes Impeller RPM 0 Max 18
- 19. Impact on cell growth Scalability of Microcarrier Suspension 19 Empirical measurements Zwietering correlation Stirred-tank cell culture N: agitation (rpm) S: Zwietering N constant V: kinematic viscosity (m2/s) g: gravitational constant (m2/s) ρs: solid density (kg/m3) ρl: solid density (kg/m3) X: solids loading ((kg solids/kg liquid)x100) dp: particle diameter (m) D: tank diameter (m) 0 2 4 6 8 10 12 CellGrowth(Fold) Days in Culture 100 80 60 40 20 0 ▬Njs ▬Nc / Njs ▬Njs / Nh ▬Nh ▬Nc 0 40 80 120 160 0 5 10 15 20 25 AgitationRate(RPM) Microcarriers (g/L) Nc Njs Nh 0 20 40 60 80 100 0 5 10 15 20 25 AgitationRate(RPM) Microcarriers (g/L) Nc Njs Nh 0 20 40 60 80 100 0 5 10 15 20 25 AgitationRate(RPM) Microcarriers (g/L) Nc Njs Nh 0 40 80 120 160 0 5 10 15 20 25 AgitationRate(RPM) Microcarriers (g/L) Nc Njs Nh
- 20. Gassing Strategy Gas Input Gas Input Overlay Gassing Openpipe Sparging Micro- sparging Gas Input Gas diffusion & Shear effect 20
- 21. MSC cell growth may also be impacted by non-optimal gassing Gassing Strategy Gas Input Gas Input Overlay Gassing Openpipe Sparging Micro- sparging 0 2 4 6 8 10 12 CellGrowth(Fold) Day Openpipe-Unoptimized Overlay 140 120 100 80 60 40 20 0 Note: Openpipe control started on day 6 (overlay prior to day 6) Gas Input Gas diffusion & Shear effect 21
- 22. Cell O2 consumption can be modeled without cells or media Dissolved Oxygen Control Mock media-1X PBS, 1g/L Pluronic®, 37.5ppm Antifoam Simulated MSC Oxygen Uptake Rate (OUR): 98fmol/cell/hr* Simulate OUR Manual N2=Modeled O2 Consumption by Cells * Pattappa, G, et al. The metabolism of Human Mesenchymal Stem Cells During Proliferation and Differentiation, Journal of Cellular Physiology, 226: 2562-2570, 2011 CellGrowth 22
- 23. Optimized PID and minimized flow rates for effective control Variable Gassing Strategies 0 1 2 3 4 5 6 7 8 9 0 10 20 30 40 50 60 70 80 90 100 0.0 0.2 0.4 0.6 0.8 1.0 FlowRate(sL/hr) dOorXO2% Duration (days) 80% dO O2 Concentration Flow Rate 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0 10 20 30 40 50 60 70 80 90 100 3.33 3.53 3.73 3.93 FlowRatesL/hr dOorXO2% Duration (days) 50% dO O2 Concentration Flow Rate Overlay Gassing Open Pipe Sparging 23
- 24. MSC cell growth and metabolites did not vary between conditions Comparing Gassing Strategies CellGrowth Feed 24
- 25. MSC cell surface marker expression did not vary between conditions Comparing Gassing Strategies 0 20 40 60 80 100 CD90 CD73 CD105 CD146 CD34 HLA %ofCells 25
- 26. MSC cell growth did not vary, slight increase of glucose consumption Comparing DO Set-points CellGrowth Feed 26
- 27. MSC cell surface marker expression did not vary based on DO set-point Comparing DO Set-points 0 20 40 60 80 100 CD90 CD73 CD105 CD146 CD34 HLA %ofCells 20% 50% 80% 20% DO 50% DO 80% DO 27
- 29. Case study
- 30. Process optimization is an iterative process of parameter evaluation Case Study – bmMSC, 50 L Performance 30
- 31. Improved run resulted in more than two-fold process improvement 31
- 32. MSCs expanded at 50 L scale retain functional attributes Seed train Post-50 L AdipocytesChondrocytesOsteoblasts Multipotency 32
- 33. MSCs expanded at 50 L scale retain functional attributes Seed train Post-50 L AdipocytesChondrocytesOsteoblasts Multipotency Post-50 L 99% Inhibition Seed train 98% Inhibition ActivatedNon-Activated Lawson et al. Biochem Eng J 120 (2017) 49-62. Immune Modulation Inhibition of T cell growth Activation marker expression 33
- 34. Seed Train Strategies for bioreactor culture
- 35. Typical Seed Train Strategies for Production of MSCs Seed Train Production Planar Process •Manually Driven •Open process •Labor intensive Seed Train Production Bioreactor Process •Greater control •Partially closed •Production Scale 35
- 36. Optimizing seed train can decrease manufacturing risk, time & cost Closed Seed Train for Production of MSCs Seed Train Production Closed Process Risk Control Timelines Costs 36
- 37. Direct Thaw of Cryopreserved MSCs into N-1 Seed Bioreactor Seed Train Production Direct Thaw into n-1 Bioreactor • Planar seed train remains a common step in MSC production • Risks of direct thaw into the bioreactor may include: • Increased exposure to shear • Cryo carry-over into production 37
- 38. Cell-microcarrier attachment is not impacted by direct thaw into the bioreactor Bioreactor Day 1 Attachment Counts and Calcein Staining Direct Thaw into BRXPlanar Seed Train Planar Seed Direct Thaw 38
- 39. Direct Seed of MSCs on Microcarriers into the Production Bioreactor •Modeled 3 50L microcarrier based MSC production process •Verified feasibility using a scaled down system based in 3L STR bioreactors Seed Train Production Closed Scale Up of Bioreactor Process N-1 Reactor Seeds Production 39
- 40. Comparable yields can be achieved in less time with an optimized seed train Typical vs. Closed Bioreactor Processing •Cells grown in αmem media + human platelet lysate •Microcarrier density constant at 15 g/L •Identical feed strategies Closed Process Typical Process TotalCells 40
- 41. Surface markers are unchanged following implementation of a closed process Flow Cytometry: MSCs Produced in a Typical or Closed process 41
- 42. Cell therapy manufacturing: Where are we? Closing GOAL Cost effective, scalable processes to reproducibility produce the product 42
- 43. Gene Editing and Novel Modalities Product Development & Services Carlsbad - US Virus Manufacturing Gene Editing, Cell Models & Cell Line Development Cell & Gene Therapy Manufacturing Tools St. Louis - US Bedford, MA - US Glasgow – UK 43
- 44. The vibrant M is a trademark 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. © 2017 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved.