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A Toolbox and Blueprint for Comparability Testing in Process
Development
Robert Deans, Athersys Inc
ISCT NA Regional Meeting
9.9.13
Where is Cell Comparability Critical in the Development Path?
• Analytical tools support consistency in product manufacturing and process change towards commercialization
• Donor comparability is key whether comparing master cell banks or large donor sets
• Large scale manufacturing moves towards extended population doublings and need to assess safety and potency
• Ensuring distinction between competing products in adherent stem cell space – enforcing and growing IP boundaries
• Building a Regulatory strategy for Implementing major process changes (xeno-free media; alternate bioreactor work; driving down COGS)
Critical Product Attributes
• Potency demonstrated in pre-clinical disease model with hypothesis for mechanistic pathway supported by in vitro surrogates– Angiogenesis in vivo linked to expression of angiogenic factors
• Cell size, stability effective with route of delivery and devices
• Cytogenetic stability
• Replicative capacity and scalability for manufacturing capable of meeting commercial needs
• Phenotype and characterization meeting intellectual property definitions and distinction from competitors
• Cautious eye towards biological properties (eg., tissue migration) that may not manifest ex vivo
Cell Equivalency Under Current Process
Cell equivalency assays
Growth kinetics – telomerase activity
Viability, attachment post thaw
Defined cell population in size, granularity
Acceptable expression of CXCL5, VEGF & IL8
Valid flow cytometric profile - identity and purity
Normal karyotype
qPCR marker expression
Acceptable differentiation to osteo, adipo, chondro
Activity in T cell proliferation assay
Activity in angiogenesis assay Automated quantitation of vascular tube formation
assay
Advantaged Expansion Profile for MultiStem vs. MSC
5
Cell Expansion over TimeHuman Cells Isolated From Same Donor
Doublings
Days
MultiStem (40x)~15-20 microns
MSC (40x)~25-30+ microns
*MultiStem Different in Size than MSC and other Bone Marrow MNCs
MSC (red)
MultiStem (green)
Lymphocytes
Monocytes
6
MSC
Lew
is
pretrea
ted M
SC L
ewis
Multi
Stem
0
10
20
30
ce
ll s
ize
µm
Flow Cytometric Analysis Of HumanMultiStem By Forward / Side Scatter Plot
Microscopic Size Comparisonof Rat MultiStem and MSC
AVAILABLE AS ATHERSYS STUDY REPORT
CONFIDENTIAL
• Telomerase activity is preserved in XF-MS
7
Telomerase activity
y i o y i o y i o0
1.0107
2.0107
3.0107
MSC MultiStem Xeno-freeMultiStem
Te
lom
era
se
activi
ty (
co
py n
°)
MultiStem and MSC have Different Angiogenic Factor Profiles
MSC147 P4
MultiStem147 P4
MSC149 P6
MultiStem149 P6
MSC153 P6
MultiStem153 P6
• Angiogenic Factor Immunoblot
– Capture Assay using 60 angiogenic factor mAB
• Comparison of activity from MSC (top) and MultiStem(bottom)
– MSC / MultiStem from same donor bone marrow
– Repeated across three donors
8
Distinctive Transcription Profiles for MultiStem and MSC – Can Be Applied to Distinguish HCT/P Products
9
Blinded analysis of gene expression by an independent lab demonstrates that:(1) MSC gene expression is tightly correlated with other MSC samples;(2) MultiStem gene expression is tightly correlated with other MultiStem samples; and(3) MSC and MultiStem gene expression patterns are distinctive.
X-axis represents the correlation value.
0.125 0.100 0.075 0.050 0.025 0
ATH113
ATH114
ATH102
ATH104
ATH107
ATH103
ATH106
ATH105
ATH117
ATH116
ATH115
ATH110
ATH111
ATH112
MultiStem
MSC
AVAILABLE AS ATHERSYS STUDY REPORT
Distinctive Protein Expression – Representative Examples
10
MultiStem MSC
MultiStem MSC
Caveolin
Prostacyclin Synthase
Target Marker Detection using FITC (green) FluoresenceBlue Nuclear Counterstain, Red Actin Filament Counterstain
AVAILABLE AS ATHERSYS STUDY REPORT
Scale-Up vs Scale-Out Manufacturing Options
• 40 Layer Cell Factory, Hyperstack– direct path to scale up with existing
technologies in 2-D format
• Suspension bioreactor using microcarriers– 3-D footprint targeting large scale stirred tank
bioreactors mimicking MAb technology
• Disposable patient designated options– Quantum Hollow Fiber Bioreactor (Terumo)
– Xpansion Bioreactor (ATMI)
Heavy Lifting Scale UP
Courtesy of Jon Rowley, Lonza
Prohibitive Development Costs for Large Scale Manufacturing
• Individual companies cannot afford to perform large scale process development
• Catch-22 – bioprocessing industry is only now recognizing market opportunity for investment in cell therapy tools
• Industry consortia (ISCT, ARM) being organized for public domain development tools emphasizing downstream processing
Major Process Changes
15
Quantum
Cell Expansion System from CaridianBCT
• An automated integrated cell culture system
Bioreactor, incubator, media and waste management
• Controlled, reproducible cell expansion
• Decrease labor
• Self-contained bench top unit using a hollow fiber bioreactor
16
• MultiStem® was seeded in parallel on the Quantum and in standard culture conditions on cell culture plastic
• Growth is monitored and feed rates are adjusted based on glucose and lactate levels
• QC assays
• Growth rates and viability at harvest and after freeze/thawing
• Cytogenetics
• Flow cytometric profile
• Differentiation potential
• Immunomodulatory properties
QC
17
• Identical growth rates
• Viability at Quantum harvest: 93.9 % ± 1.6 %
• Viability after freeze/thawing: 90.1 % ± 2.2 %
Growth and viability
Quantum
18
Growth
With density centrifugation Whole BM load
Whole BM load on Quantum as good as seeding BMMNC on
cell culture plastic
19
Quantum
Isotype
Marker
• Panel of 22 markers was tested
Flow cytometric profile
Marker CriteriumStandardculture Quantum
CD3 < 1% 0.03% 0.14%
CD13 > 95% 97.51% 99.45%
CD19 < 1% 0.04% 0.03%
CD34 < 1% 0.00% 0.00%
CD40 < 1% 0.07% 0.03%
CD44 > 95% 98.51% 97.86%
CD45 < 1% 0.05% 0.03%
CD49c > 95% 94.47% 98.71%
CD49d N/A 11.34% 2.79%
CD54 N/A 6.18% 10.60%
CD56 < 1% 0.08% 0.08%
CD58 N/A 65.90% 72.68%
CD73 > 95% 97.85% 99.38%
CD80 < 1% 0.02% 0.01%
CD86 < 1% 0.38% 0.09%
CD90 > 95% 99.91% 99.92%
CD105 > 95% 99.53% 95.76%
CD117 N/A 28.49% 27.75%
CD140b < 1% 0.07% 0.10%
CD146 N/A 97.55% 92.15%
HLA I < 25% 5.39% 6.20%
HLA II < 1% 0.01% 0.01%
No significant differences for the flow cytometric markers and no pass/fail
criteria are exceeded
CD13HLA II CD49c
Standard conditions
Isotype
Marker
20
• Cytogenetic analysis on Agilent CGH arrays
Cytogenetics
No significant differences in karyotype
21
• Multistem® was differentiated to osteoblasts and adipocytes
• Histochemistry
Differentiation potential
22
Angiogenesis assay
L07 CES
L09 CES
23
• Quantification of differentiation
• qPCR
• Flow cytometry
Differentiation potential
CONFIDENTIAL 24
Maintenance of biological properties
Epigenetics and Profiling Comparability Testing
TranscriptomeProteome
miRNAGene methylation
MultiStem: A Distinctive ProductMultivariate analysis of Gene Expression Profile
Violet - MSCGreen - MesoangioblastsRed - MAPC (Verfaillie lab)Blue - MultiStem
26
MSC / likeMAPC / MultiStem
Violet - MSCGreen - MesoangioblastsRed - MAPC (Verfaillie lab)Blue - MultiStemOrange - ESC
ESC
MAPC / MultiStem
MSC / like
CONFIDENTIAL
• Key miRNA markers (MS vs MSC)
27
miRNAs as markers
MultiStem upregulated miRNAs(MS markers)
MultiStem downregulated miRNAs(MSC markers)
qPCR profiling Microarray profiling qPCR profiling Microarray profiling
MS markers
miR-1
miR-2
miR-3
miR-4
miR-5
miR-6
miR-7
miR-8
miR-9
miR-10
MSC markers
miR-a
miR-b
miR-c
miR-d
miR-e
miR-f
high stringency : FC > 1,5 in all three donors
low stringency : FC> 1,5 in at least two donors
Stability of Gene Methylation Profiles at Early and Late Expansion Stages
• Methylation profile of early (~20 PD) and late (~40 PD) MultiStem cultures depicted as the ratio of early/late methylation levels for 1536 CpGislands
• x-axis represents the Gene ID corresponding to the alphabetical listing of the genes associates with the CpG islands as shown in supplemental data
• Average ratio for two independent MultiStem lines, BMC2 and BMC 3 was 1.08+0.31 and 1.25+0.48, respectively
28
See Boozer, S, Lehman N, Lakshmipathy et al. Global Characterization and Genomic Stability of Human MultiStem, A Multipotent Adult Progenitor Cell. J Stem Cells 2009: 4(1).
Confidential
CONFIDENTIAL 29
Distribution of methylation cores
CONFIDENTIAL 30
Analysis overview
‘MultiStem miRNAs’ ‘MSC miRNAs’
Predicted miRNA targets
‘MultiStem mRNAs’ ‘MSC mRNAs’
OVERLAP ?
mRNAs that are predicted to be a
target, based on the presence of
conserved seed-sequences in the 3’-
UTR (via TargetScan)
Xeno-free Workflow
32
• Identical growth as in MS medium when cells are isolated in TheraPEAK™ on Synthemax coatings and subsequently transferred to CellBIND coatings
Surface Coatings are Critically Linked to Media Formulation
days
PD
0 10 20 30 40 500
5
10
15
20
25
MS
TP Synthemax
TP Synthemax -> CellBIND
TP CellBIND
MS IVTTheraPEAK
Morphology 9 days after seeding
CONFIDENTIAL 33
2D-DIGE workflow
Derivatise withCy3
Derivatise withCy5
Spots too dim to view by eye
Overlayimages
Mix and separate on one single
2D gel
Isoelectric focusingSDS-PAGE
E Cy3Image A
E Cy2Image C
DeCyder 2-D DifferentialAnalysis Software
Control Test
False-coloured pattern of differential spots is visible
Derivatise withCy2
Acquire images fromthe gel
E Cy5Image B
Cy3-labelledsample
Cy5-labelledsample
Cy2-labelledsample
Internal standard
CONFIDENTIAL 34
Gel 1: Control 1 Cy3 + Test 1 Cy5 + Internal standard Cy2Gel 2: Control 1 Cy5 + Test 1 Cy3 + Internal standard Cy2
Gel 3: Control 2 Cy3 + Test 2 Cy5 + Internal standard Cy2Gel 4: Control 2 Cy5 + Test 2 Cy3 + Internal standard Cy2
Gel 5: Control 3 Cy3 + Test 3 Cy5 + Internal standard Cy2Gel 6: Control 3 Cy5 + Test 3 Cy3 + Internal standard Cy2
CONFIDENTIAL 35
-PCA analysis: on pH 4-7, all spots, filtered only on: “spots have to be present in >75% of the spot maps”
PC
2
4.8
4.6
4.4
4.2
4
3.8
3.6
3.4
3.2
3
2.8
2.6
2.4
2.2
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
-0.2
-0.4
-0.6
-0.8
-1
-1.2
-1.4
-1.6
-1.8
-2
-2.2
-2.4
-2.6
-2.8
-3
-3.2
-3.4
-3.6
-3.8
-4
-4.2
-4.4
-4.6
-4.8
PC1876543210-1-2-3-4-5-6-7-8
MultiStem medium
TheraPEAK medium
Donor SVG/2
Donor L11
Secretome Following Immune Priming: Stem Cells Translational Medicine (Burrows et al)
Acknowledgments
Bart VaesDavid Craeye, Annelies Bogaerts, Silvie Cloosen, Saskia van Cruchten Saartje Walbers, Peter Sterkendries, Marian CrabbéKristel Gijbels, Ellen Van Houtven
www.regenesys.eu
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