This presentation reviewed the current strategies and their associated benefits and challenges to scale up the production of adherent stem cells.

1. Copyright 2012 ATMI, Inc. All Rights Reserved.Copyright 2012 ATMI, Inc. All Rights Reserved.
Fabien Moncaubeig
Sr Application Specialist & Cell Culture Lab Associate
Workshop:
Industrialization of a stem cell process – How
to identify the right strategy?
October 21, 2013 – Bethesda, MD
IBC - Cell Therapy Bioprocessing

2. Copyright 2012 ATMI, Inc. All Rights Reserved.
ATMI Lifesciences where we come from
ATMI LIFESCIENCES
Is part of ATMI Inc, a global company market
leader in efficient process solutions for the semi-
conductor and lifesciences industry
ATMI support leading company in the GMP
manufacturing of vaccines, Mabs and protein
production since 10 years
ATMI is expert in clean manufacturing and supply
of integrated technologies for cell culture
ATMI leverage is core competency in cell culture
to develop innovative technologies and efficient
processes

3. Copyright 2012 ATMI, Inc. All Rights Reserved.
Our know how
ATMI approach combines both Product
development , Process development & optimization
ATMI has two Applications & Development centers:
Fully equipped cell culture clean rooms.
45 experienced team including Engineers &
Bioengineers
Tech transfer to your facility or to your CMO
PD Services / Support implementation of our technologies
PD for New Bioreactor development
Application & Development Center
Brussels Minneapolis
Surface 500m² 100m²
Classification Class 10,000 (ISO
class 7)
Non-classified
Biosafety level BL-2 BL-2

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What we want to offer to the cell therapy
industry
Complete solution for cell based product manufacturing
Offer a full solution for cell growth and
harvest, based on the best technologies
availables
Leverage our expertise in cell culture and
cell therapy process development to
streamline industrialization of cell therapy
products
Optimize and enable high volume
manufacturing of cell based therapy
Develop the future solutions

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Different scale-up and manufacturing challenges for
each segment
Autologous
Low dose. cells per patient < 108
High dose. cells per patient > 108
Low # passages possible (Adult SC, chondrocytes, ...)
High # passages possible (hES, iPS,...)
Allogeneic
Patient to Patient variability
Multiple parallel batches
Logisitc
Donor reproducibility
Passage limitations
Targeted lot scale
Downstream and formulation

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Your process = Your product
Has never been so true !
Parameters impacting product (cell) quality:
Shear stress 1
2D VS 3D growth 2
Growth Surface Material 3
Changing production platform may require
clinical trial bridging
1: Biotechnol Bioeng. 2007 Feb 15;96(3):584-95. - Effects of shear stress on 3-D human mesenchymal stem cell construct development in a perfusion bioreactor
system: Experiments and hydrodynamic modeling. - Zhao F, Chella R, Ma T.
2: Tissue Eng Part A. 2009 Jul;15(7):1763-73. doi: 10.1089/ten.tea.2008.0306. - Phenotype and gene expression of human mesenchymal stem cells in alginate
scaffolds. - Duggal S, Frønsdal KB, Szöke K, Shahdadfar A, Melvik JE, Brinchmann JE.
3: Acta Biomater. 2013 Feb 26. pii: S1742-7061(13)00105-0. doi: 10.1016/j.actbio.2013.02.035. - Why the dish makes a difference: Quantitative comparison of
polystyrene culture surfaces. - Zeiger AS, Hinton B, Van Vliet KJ.
Biological challenges to consider during
process development and scale-up
www.solohill.com
www.solohill.com

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Mimic final scale conditions from the early
development steps
Evaluate impact of any process change
Increasing cell culture scale implies streamlining
and optimizing all related operations :
Media preparation
Fluid handling
Harvest
Concentration
Rinsing
Freezing
Filling
Increase scale implies indroducing and validating
new technologies
Technical challenges to consider during
process development and scale-up
Taking everything into consideration

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Business challenges to consider for
process development and scale-up
Supporting product lifecycle
Accelerate time to market, reduce
development time and ressources
Mitigating risk for market approval,
minimize changement
Supporting commercilization ramp up,
offer flexible production
Offer an affordable product, implement a
cost effective manufacturing

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21,000cm²
What are the technological options for
cell production
Scale/unit
Or
Or any other
suitable SUB
3D environment
Planar technologies 122,400cm²18,000cm² 25,000cm²

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Technology choice is driven by batch size and # patients
50’s10’s 100’s 500’s 5000’s >10000’s
Cells per lot
Patients per year
Allogeneics
Or
Or any
other
suitable
SUB
Future
technologies
Autologous

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Technical and financial considerations
regarding Downstream process scale-up
Choice will be led by
Harvest volumes to process
Time constraint
Concentration factor and final targeted volume
For large volumes, combination of these technologies may be required
100L  Unifuge 1700LATMI or Centrifuge bags  <135ml
Fluidized bed
(i.e:Ksep)
Large scale
centrifuge
(i.e:Unifuge)
Small scale
centrifuge
(i.e:ATMI)
Tangential Flow
Filtration
Flow rate (ml/min) 400 – 9600 3000 500 20 - 12,500
Hold-up volume (ml) 100 - 1600 1700 135 100’s-1000’s
Disposable cost ($)
2000-2500*(Ksep
400)
3000 950 1500-3500$*
Process dvt effort + - - +++
* Jacob Pattasseril, Hemanthram Varadaraju, LyeTheng Lock, and Jon A. Rowley, “Downstream Technology Landscape for Large-Scale
Therapeutic Cell Processing”,BPI, March 2013,

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Now, lets compare the pros and cons of 3
different scale-up approaches using,
Multilayers,
Xpansion,
Microcarriers

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Technical considerations for a scale-up in
multilayer systems
Limited process development:
Multitray systems with headspace  Almost no process dvt required
Compact multitray  Additional optimization of fluid transfer and
harvest protocol required
Scale-up strategy = Scale-out:
Max scale/unit = 25,440 cm² (Cell Factory 40)
Increase number of units
Linear increase of footprint (incubators)
Implementation of automated manipulator system can reduce FTE need
Cell observation can be challenging in the largest scale units (>36 trays)
Cm²/volumetric footprint

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High footprint requirement
Requires high, early, investment in factory / CMO
High FTE cost per batch
Uneven FTE needs
High number of operations = high failure rate
Manufacturing and Business considerations for
a large scale multitray process
0
5
10
15
20
25
30
35
40
45
50
1
11
21
31
41
51
61
71
81
91
101
111
121
131
141
151
161
171
181
191
201
211
221
231
241
251
261
271
281
291
301
311
321
331
FTE Hours required / day
Process B - Hyperstack 36 Process B – Xpansion 200

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Technical considerations for a scale-up in
Xpansion
Linear scalability from 10 (6,120cm²) to 200 plates (122,400cm²)
Scale-up by maintaining linear speed constant
Controlled environment:
pH, DO, Temperature monitoring & control
Cell observation
Process development required to :
Optimize process parameters
Harvest protocol

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Manufacturing and Business considerations for a large
scale Xpansion process
Minimize footprint compared to multilayer
4 Xpansion 200 fits in 1 incubator (42.5 x 34.5 x
87 inches)
Lower cost /batch than multitray systems
Lower FTE requirements
Higher Consumable cost/cm²
Batch Cost distribution
Hyperstack
CoGs
Xpansion

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Manufacturing and Business considerations for a large
scale Xpansion process
Clinical trial phase 3
(300 patients)
FDA
approval
Early commercialization
(1,000 – 25,000 patients)
Commerci
alization
(>50,000)
2014 2015 2016 2017 2018 2019
NEW Facility building
$ 3,732,885
Facility running under capacity Facility running full
capacity
CMO or current facility
NEW Facility building
under
capacity
Facility running full
capacity
$ 3,463,142
CMO or current facility
Hyperstack36Xpansion200
Xpansion enables to decrease investment and delays requirement for new facility

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Technical considerations for a scale-up
with microcarriers 1/2
Very high scale lot size achievable:
6g cytodex1/L x 500L = 1,320m²
Developing a microcarrier process is challenging, requiring time and high expertise
level :
Process development challenges:
Identify scalable cell attachment and growth mixing conditions
Develop scalable harvest protocol (timings)
Maximize cells/ml (carrier density)
Develop a seeding train with bead to bead transfer
Microcarrier key selection criteria to consider:
Density, cm²/g, g/L achievable, coating
Impact on : mixing requirements, cells/ml
Bioreactor challenge:
Low shear while maintaining mixing capacity
Keep aggregates in suspension/movement
www.solohill.com

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Technical considerations for a scale-up
with microcarriers 2/2

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Manufacturing and Business considerations for a large
scale microcarrier-based process
Process development substantially longer and risky
Could delay phase III or early commercialization
Low footprint compared to any planar system
Limited Facility size requirement
Easy future capacity increase
Dramatically lower CoGs

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Summary of technology scale capacity and
associated R&D efforts
Ana S. Simaria, Sally Hassan, Hemanthram Varadaraju, Jon Rowley,
Kim Warren, Philip Vanek, Suzanne S. Farid, Allogeneic cell therapy bioprocess economics and optimization: Single-use
cell expansion technologies, Biotechnologie & Bioengineering, released online on August 16th, 2013

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A dual approach to cover short and long
term needs

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Conclusion
No Universal answer
Multiple factors with specific weight depending on therapy and company status
R&D effort
capacity
Targeted
batch size
Short & long term
Patient number
forecast
Clinical
phase status
Process
constraints
Cell
production
platform

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Conclusion
Controlled
environment
(pH, DO, cell
observation)
Contaminat
ion risk
(#operatio
n/cm²)
Process
dvt
expertise
required
Process
dvt
duration
Cell quality
change risk
(initial
process in
TF)
Max scale
achievable
Required
Footprint
COGS
/cm²
Initial
Investment
required for
commercial
scale
Multitray system (10 or
40)
Compact multitray (36)
Xpansion
Microcarrier + SUB
Hollow fiber
Pros & cons summary table for large scale allogeneic therapies

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Thank you

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Case study Promethera introduction
May 2013 – Bio 2013 - ”Enabling cell therapy : scaling-up allogeneic stem cell manufacturing from
multi-tray stacks to Xpansion™ multiplate bioreactor” – Eric Halioua –Fabien Moncaubeig

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Case study Promethera process
comparaison
May 2013 – Bio 2013 - ”Enabling cell therapy : scaling-up allogeneic stem cell manufacturing from
multi-tray stacks to Xpansion™ multiplate bioreactor” – Eric Halioua –Fabien Moncaubeig
27
EMERGENCE
P0/P1
P3/P4: 4 CS-10
Phase I/II Process
Aseptic process
P4/P5: 2 x 5 CS10
P5R: 2 x 15 CS10
Centrifugation/Filling
Freezing
FORMULATION 3,4x109cells

28. Copyright 2012 ATMI, Inc. All Rights Reserved.
Case study Promethera process
comparaison
May 2013 – Bio 2013 - ”Enabling cell therapy : scaling-up allogeneic stem cell manufacturing from
multi-tray stacks to Xpansion™ multiplate bioreactor” – Eric Halioua –Fabien Moncaubeig
28
EMERGENCE
P0/P1
P3/P4: 4 CS-10
P0/P1
P3/P4: 1 CS10
Phase I/II Process Phase II/III Process
P4/P5: 1 XP100
Aseptic process
P4/P5: 2 x 5 CS10
P5R: 2 x 15 CS10
Centrifugation/Filling
Freezing
FORMULATION
Centrifugation/Filling
Freezing
RECONSTITUTION
3,4x109cells
Closed process
22,1x109cells
P5R: 5 XP200
EMERGENCE

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Case study Promethera – cost
comparaison
May 2013 – Bio 2013 - ”Enabling cell therapy : scaling-up allogeneic stem cell manufacturing from
multi-tray stacks to Xpansion™ multiplate bioreactor” – Eric Halioua –Fabien Moncaubeig