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Scaling Microcarrier-based Expansion Processes for Production of High Quality Cells Mark S. Szczypka BioTech 2016, Wädenswil Switzerland September 5th 2016
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Cell Therapies – Allogeneic donor into patient
– Autologous Cell Therapy patient into same patient
Regenerative Medicine – Engineered tissues – 3D Bio-printing
Gene Delivery Drug Discovery and Testing
– Live cells for pharmaceutical research
Cell-based Medical Technologies
Adult stem cells Primary cells
iPSC ES cells
Cell Therapies
Drug Discovery
Regenerative Medicine
Gene Delivery
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Cell Numbers for Therapeutic Applications and Enabling Technology
Indication Patients/year Cells/year (TD 1e9) (Global) (Global)
Ischemic Stroke 2.00E+06 2.00E+15 Acute Myocardial Infarction
4.00E+06 4.00E+15
Indication Stem Cells/patient Islets for Type I diabetes 1B
TH positive neurons for Parkinson’s
2 T
Allogeneic cell therapy
Regenerative Medicine
Xpansion 2D cell culture reactors Pad Bioreactors
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Building a Simple and Robust Cell Manufacturing Platform for Scalable Expansion of hMSC
Donor tissue and methods used for isolation Cells and Medium Expansion platform
– Substrate for attachment – Bioreactor type
Ancillary technologies and processes – Standardized sterile connections – Holding and process containers – Volume reduction methods – Storage containers
Critical components
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Pall SoloHill Microcarriers for Cell Expansion Six Different highly characterized microcarrier types Used in large scale manufacturing processes for over 30
years Rigid cores allow for easy and rapid dissociation and high
cell yields Support robust growth of many different types of human
and animal cells Excellent performance in serial passage Gamma-irradiated microcarriers in single-use bags
facilitate ease of use Best overall performance in independent studies for
microcarrier selection*
* Rafiq et al. Systematic microcarrier screening and agitated culture conditions improves human mesenchymal stem cell yield in bioreactor Biotech J. 2016.
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Excellent growth and efficient harvest of human mesenchymal stem cells (hMSCs) from Pall SoloHill microcarriers in small scale bioreactors
Robust Growth of hMSCs on Pall SoloHill Microcarriers
Collagen Star-Plus
2.4 B 1.3 B
Bioreactor Harvest
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0 2 4 6 8
Cel
ls/m
L (x
106
)
Days of Growth
Bioreactor Culture
00.10.20.30.40.50.60.70.80.91
0.01.02.03.04.05.06.07.08.09.0
10.0
0 2 3 4 5 6 7
Cel
ls/m
L (x
106
)
Cel
ls/c
m2
(x 1
04)
Days of Growth CollagenStar-Plus
Spinner Control
Medium Exchange
Medium Exchange
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hMSC harvested from microcarriers retain differentiation capacity and surface marker expression
0
20
40
60
80
100
% G
ated
Cel
ls
Marker
Collagen BioreactorT Flask Control
Robust Growth of hMSCs on Pall SoloHill Collagen Coated Microcarriers
Bioreactor
Adipocyte Osteocyte
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hMSC Expansion using PadReactor Platform and Pall SoloHill Microcarriers
Allegro Microcarrier Delivery System
(AMDS-Irradiated microcarriers)
6L PadMini Bioreactor Pad50
3 days 4 days
RoosterBio hMSC (liquid nitrogen stock)
Maintain PDL below 18 to 20 doublings to maintain quality and consistency
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Adherent Cell Culture Solutions Microcarrier Cell Culture
• SoloHill microcarriers, delivered ready-to-use in a single use format
• Sterilized by gamma irradiation • Allows to directly seed
bioreactors – no additional preparation needed
• Low shear mixing supports cell growth on microcarriers
• Superior gas transfer and pH control
• Linear scalable bioreactors (6L to 1000L)
• Downscale bioreactors for PD and small scale GMP batches
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RoosterBio hMSC and Medium
•40-fold increase in viable cell number over 1 week
•RoosterBio media feed (RoosterReplenish) can be used to replace media exchange during bioreactor culture
•Efficient hMSC detachment from microcarriers
Process parameters:
1. Cell thawed & seeded at 10,000 cells/ml or 5M per 500ml culture
2. collagen coated microcarriers
3. Media Feed added on day 3
(Data generated by RoosterBio)
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Small scale microcarrier passage studies
P1 microcarriers Harvest P2 microcarriers
0.0
0.1
0.2
0.3
0.4
0.5
0.6
Cel
ls/m
L (x
106
)
Day 5
Microcarrier (n=1)
Planar culture
Passage 2
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Over twenty-fold expansion was reproducibly achieved in batch-fed microcarrier cultures in PadMini at 6L scale in 4 days
Reproducible and Robust Cell Expansion in 6 Liter PadMini® Bioreactor
0.00
0.10
0.20
0.30
0.40
0.50
0.60
Cel
ls/m
L (x
106
)
Day 4
BR (n = 3)CF10
Expansion Scheme
LN2 Cell Factory
(CF10) PadMini
(6L)
Bioreactor Process Parameters RoosterBio hMSC RoosterBio High Performance media
Seed density 3000 cells/cm2
Microcarrier conc. 10 cm2/mL Medium exchange None Supplement Day 3
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Generation of Billons of hMSC in Pall’s Single use PadReactors in 8 days
0
0.2
0.4
0.6
0.8
1
1.2
0 2 4 6C
ells
/mL
(x 1
06)
Day
PAD50 Reactor
Pad Mini (6 L)
Pad 50 (40L)
Seed Harvest Seed Harvest
Total cells 1.67E+08 1.23E+09 1.11E+09 2.79E+10
PDL 2.9 4.6
3 days 5 days
0.17 B 28 B
Cell Expansion
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PadReactors used in a closed system provide an ideal single-
use bioreactor system for cell expansion Pall SoloHill microcarriers provided an excellent substrate for
growth of human mesenchymal stem cells (hMSC). Cells were harvested from microcarriers using simple
procedures Simplification of processes steps including use of Sterile
microcarriers delivered through the AMDS in single-use bioreactors hMSC were expanded from ~170 million hMSC to 28 billion
cells in 8 days in batch-fed culture on SoloHill microcarriers in Padreactors
Conclusions
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Expansion using PadReactor Platform and Pall SoloHill Microcarriers
6L PadMini Bioreactor Pad50 hMSC
Expedite developmental timelines for cell-based Medical technologies Provide robustness and reproducibility in single-use closed
system Decrease cost of implementation
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Acknowledgements
Pall Life Sciences Dave Splan Jin Liu Alexandria Spruiel Kaitlynn Bayne Grishma Patel Andrew Laskowski
RoosterBio (Cells and Media) Lye-Theng Lock Jon Rowley
Thank You