©ReGenesys 2013
7th Technical Meeting, Newcastle, 24 September 2013
Pros and cons of different expansion systems for manufacturing of an
allogeneic cell therapy
Jef Pinxteren, Manager and Head R&D
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MultiStem® cell therapy product: a multimodal biologic product
MultiStem® Product Profile• Cell therapy product based on Multipotent Adult Progenitor Cell (MAPC) technology• Developing for “off‐the‐shelf” administration – no tissue matching needed• Expanded product with high production yield (e.g., millions of doses from each donor)• Long storage life – can be kept in frozen form for years• Consistent safety profile• Promotes healing and tissue repair through multiple mechanisms of action• Not a permanent transplant – cells cleared from the body over time
• Expanded, banked product• Obtained from healthy, consenting adult donors
MultiStem® Cell Therapy
• Frozen storage• Administered systemically (IV) or locally (catheter, injection, matrix/implant)
2013
MultiStem®Immunological
IBD / Ulcerative ColitisHematopoietic Stem Cell Transplant / GVHD
Solid Organ TransplantDiabetes
CardiovascularAMI
Congestive Heart FailurePeripheral Artery Disease
NeurologicalStroke
Traumatic Brain InjuryMultiple SclerosisSpinal Cord Injury
OtherBone Allograft
5HT2c Agonists (obesity, other)
IMMUNOLOGICALCARDIOVASCULAR
NEUROLOGICAL
2013‐4 data
2013 Data
FDA approved P2
FDA discussions re: P2/3 designGerman P1 approval
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Overview of the MultiStem® Production Process
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Overview of the MultiStem® Production Process
Lot Release & Product Characterization Testing
Sterility, Potency, Purity and ViabilityStable Cytogenetics
Absence of tumorgenic potential in vivo
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Cell Banking Approach
Isolated MultiStem
1 donor
Isolate MultiStem®
20 PD
13 days
Master Cell Bank
20 million/vial(200 vials)
4.109 cells
1 vial 8 PD
6 days
Working Cell Bank
20 million/vial(200 vials)
1 vial
800.109 cells
12 PD
9 days
Clinical Doses
>40 doses/vial180.106 cells/bag
288.1012 cells~1.6.106 doses
• Requires working in a clean room• Labor intensive and numerous open
events Manual seeding, feeding and harvesting
• Limited process control• Difficulties in achieving high
reproducibility
• Includes xeno media components (e.g., serum)
6
Current cell culture process
7
Images from current MultiStem production process
• High capacity mfg which will allow for manufacturing runs in the 500 billion – 2 trillion cell range (decrease testing costs due to lot size)
• Removal of xeno/human source materials when possible (lowering of regulatory/safety risk)
• Determine optimal storage conditions for final product and generate stability data (no special requirements for storage at clinical site)
• Final container configuration that supports thaw – deliver use (bags, vials or other)
• Automate mfg process (reduce labor costs)• Economy of scale for reagents (reduction in materials costs)
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Future development
Courtesy of Jon Rowley, Lonza
Heavy Lifting Scale Up
Cogs in Scale Up
Courtesy of Jon Rowley, Lonza
• Stem Cell Research and Assay Development MultiStem identity / cell equivalency (distinguish between stem cells) Molecular mechanisms of MultiStem function Immunology Pre-clinical animal studies
• Evaluate novel expansion methods Quantum Cell System (Terumo) Microcarrier/Stirred bioreactor
• Development of xenobiotic free workflow
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The next stage
• 40 Layer Cell Factory, Hyperstack – direct path to scale up with existing technologies Advantages
o Scaled platform from current industry wide experience base Disadvantages
o 2 dimensional platforms with large volume management requirementso Currently untested large volume reduction options
TFF – multiple manufacturers (Pall, Millipore) kSep – promising but untested
• Hollow Fiber Bioreactor; ATMI (closed system stacked pizza platform) Advantages
o Provides scale out capacity with significant advantages Eliminates requirement for large scale volume reduction Closed system with increased safety for sterile breach Significantly reduced labor costs
o Allows in line testing for metabolites, control of gas/glucose in real time Disadvantages
o Limited scalability within current configurationo Better suited for autologous or patient designated approach
• Suspension bioreactor using microcarriers Advantages
o 3D platform with minimal media exposure, likely most economical o Multiple microcarriers with controlled surface coating coming into marketo Corning, BD, GE entering space
Disadvantageso Macro‐aggregates lead to complications in harvest; may be overcome with carrier size (Pluristem)o Difficulty in controlling oxygenation, shear parameters against particle size
Scale Up Manufacturing Options
• Phase III Registration trials must be run using commercial production process
• COGS a very important element of long term competitiveness• 12 – 18 month period required to lock down process
Pre-clinical equivalency and biosafety Process qualification and stability studies Clinical equivalency
• New manufacturing platforms developing with 3 to 6 month generation time!
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Sobering thoughts on Manufacturing
Cell Factories, Hyperstack
Large Scale Volume Reduction
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Microcarriers
• 12 microcarriers were tested for cell attachment in dishes• The best 3 were tested in small 125 ml spinner flasks• The best one was used for expansion in 3 liter bioreactor
12,000 cm² culture surface 2.4 liter of medium used
Primary Screen: 12 Secondary Screen: 3 Proof of concept: 1
Quantum System• An automated integrated cell culture system
Bioreactor, incubator, media and waste management
• Controlled cell expansion All steps can be programmed and monitored
• Decrease in labor• Closed system
Reduction in contamination potential
• CE marked and FDA listed device
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Quantum
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Hollow fiber technology
• Self-contained bench top unit using a hollow fiber bioreactor Small device footprint (48.3 cm x 58.4 cm x 50 cm) Contains ≈ 10,000 fibers/bioreactor Total surface is 2.1 m² (3255 in²) Equivalent of 280 T75 flasks or one 40-stack Smaller surface to volume ratio requires media
perfusion Metabolites and gas easily diffuse across fiber
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Quantum cell expansion set
Bioreactor
Tubing organizer
Waste bag
Harvest bagGas transfer module
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Graphical user interface
• Graphical user interface makes operation very straightforward • Reduced risk of errors• Reduced labor
Release and harvest of adherent cells takes 20 minutes Potential to run more cell expansion processes simultaneously
• Key steps in the process are automated e.g. Cell loading, feeding,... Possibility to adjust procedure and save as a custom task
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Automation
• MultiStem cells were seeded in parallel on the Quantum and in standard culture conditions on cell culture plastic
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Cell equivalency: Quantum vs plastic
• Growth on the Quantum is monitored and feed rates may beadjusted based on glucose and lactate levels
Harvest populations were tested on a set of QC assays
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Cell equivalency: Quantum vs plastic
Flow cytometryCytogenetic analysis on CGH arrays
DifferentiationInhibition of T cell proliferation
Marker Criterium Standard culture Quantum
CD13 > 90% 97.51% 99.45%
CD34 < 2% 0.00% 0.00%
CD45 < 2% 0.05% 0.03%
CD49c > 90% 94.47% 98.71%
CD90 > 90% 99.91% 99.92%
CD105 > 90% 99.53% 95.76%
HLA I < 25% 5.39% 6.20%
HLA II < 2% 0.01% 0.01%
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Reproducibility
# cells seeded (x106) # cells havested (x106) Run time (h)
8.45 752 146
7.28 782 148
8.57 753 144
3 independent runs from the same donor generate the same harvest yields
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MultiStem isolation from bone marrow
With prior density centrifugation Whole bone marrow load
Whole BM load on Quantum as good as seeding BMMNC on
cell culture plastic
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Cell equivalency of MultiStem isolation: Quantum vs plastic
Angiogenesis assay
Differentiation
Flow cytometry
Inhibition of T cell proliferation
Marker Criterion Plastic Quantum
CD13 > 90% 99.65% 98.93%
CD34 < 2% 0.19% 1.08%
CD45 < 2% 0.42% 0.75%
CD49c > 90% 98.99% 99.07%
CD90 > 90% 98.19% 99.17%
CD105 > 90% 98.82% 99.11%
HLA I < 25% 17.95% 14.11%
HLA II < 2% 0.07% 0.10%
Cells from 3 donors tested
• Donor 1 3 consecutive runs on the Quantum starting from bone marrow in run 1 Cells cultured on regular plastic
• Donor 2 2 runs starting from the same seeding stock (run 1 & 2) Run with different type of coating which was not successful (run 3) Cells cultured on regular plastic
• Donor 3 Isolation on the Quantum Isolation on plastic MSC isolated on plastic
• HUVEC as control
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Microarray analysis
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Microarray analysis - Dendrogram
HU
VEC
Don
or 3
MSC
Don
or 2
run
3
Don
or 3
pla
stic
Don
or 2
pla
stic
Don
or 1
pla
stic
Don
or 3
run
1
Don
or 1
run
1
Don
or 1
run
3
Don
or 1
run
2
Don
or 2
run
1
Don
or 2
run
2
• Hollow fiber’s large surface area maximizes cell expansion in a minimal amount of space
• MultiStem cells can be isolated and expanded on the Quantum• MultiStem cells show the same growth kinetics and retain all cell
characteristics • Automation: ease of use and better control• Decrease in labor: 2 hours of lab work to generate an average of
800 million cells• Expensive clean rooms not required
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Conclusions
• MultiStem and MSC derived from same donor bone marrow • Differences are of epigenetic origin• Further describing MultiStem identity
Transcriptome miRnome Methylome Proteome
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Omics screenings : MultiStem vs MSC
3.8x109
3.3x1014
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• PCA to monitor cell equivalency of different cell culture methods
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Transcriptome : comparability after culture manipulation
Plastic expanded Quantum expanded
MSC
HUVEC
First Principal Component
Second
Prin
cipal Com
pone
nt
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MultiStem versus MSC : Gene Set Enrichment analysis
MultiStem
MSC
MSC more mature?
Telomerase activitymaintenance
Proliferation
• Telomerase activity is preserved in XF-MS
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Telomerase activity
y i o y i o y i o0
1.0107
2.0107
3.0107
MSC MultiStem Xeno‐freeMultiStem
Telo
mer
ase
activ
ity (c
opy
n°)
• Identify markers by comparing differential expression between Multistemand MSC Extract ‘key’ miRNAs Consistent effect in 3 donors Develop multiplex qPCR as xeno-free equivalency assays
• Gene Set Enrichment Analysis Identify mRNA targets of miRNA Gene Set enrichment / Pathway analysis
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Application of miRNA expression data
miRNA screening:
Positive MultiStem markers Negative MultiStem markers
Using two different platforms, array and PCR-based, we are currently exploring the possibility of using miRNA profiles to confirm the uniqueness of our product.
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“omics screening”
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miRNA marker screening strategy
qPCR Array
Total targets 755 1400
Detected targets 207 847
Differential expression 160 140
Donor independent, FC > 1.5 67 32
18 510
MS > MSC
33 116
MSC > MS
qPCR array qPCR array
2: MultiStem marker identification
Positive markers Negative markers
1: miRNome + data analysis
4 : Use panel of top markers in cell comparability testing
3 : Validate 16 miRs by qPCR in independent donors top markers
• miRNA marker expression is maintained during : Regular expansion Xeno-free expansion Bioreactor expansion
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miRNA expression qPCR
Rel
ativ
e ex
pres
sion
0.0
0.1
0.2
0.3
0.4
0.5
Y O Y O
MS XF
CES MSC
donor A donor B
Y O Y O
MS XF
CES MSC Y O Y O
MS XF
CES MSCR
elat
ive
expr
essi
on0.0
0.2
0.4
0.6
0.8
1.0
Y O Y O
MS XF
CES MSCY O Y O
MS XF
CES MSC Y O Y O
MS XF
CES MSC
donor A donor B
Genome-wide DNA methylation analysis
Find epigenetic markers for distinguishing MultiStem from competing productsand proof epigenetic stability during expansions
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“omics screening”
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Methylome : Multi-layered epigenetic regulation of MultiStem expansion capacity
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Application of ‘omics’ data in product development
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Acknowledgments
ReGenesys: Bart Vaes, David Craeye, Annelies Bogaert, Aline Visser, Saartje Walbers, Peter Sterkendries, Marian Crabbé, Liesbeth Vandenpoel, Kristel Gijbels, Ellen Van Houtven, Lien Timmerman
Athersys: Bob Deans, Tony Ting, Jon Rowley, Willie Mays
www.regenesys.eu
