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.

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

4. Manufacturing for
cell therapies

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

14. Process development

15. POLL QUESTION 2
15

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

28. Process Development Summary
28

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.