The need for high quality materials that are animal origin-free and compatible with a limited number of downstream processing steps will increase as cell therapies achieve clinical success. Large scale manufacturing necessitates transition from planar culture to technologies such as stirred tank bioreactors where culture of suspension cells or adherent-dependent cells on microcarriers is enabled. This webinar will discuss challenges and solutions to the elimination of animal-derived components from cell culture processes, with focus on human mesenchymal stromal/stem cells (hMSCs). Fetal bovine serum in particular is associated with regulatory, supply, and consistency challenges, yet a wide range of performance has been observed between different serum-free media formulations for expansion of hMSCs in planar formats. Moreover, a positive performance in static culture is not necessarily predictive of that under agitated conditions with microcarriers, highlighting ongoing challenges to the generation of a fully chemically-defined and scalable cell culture medium. Through use of pharma-grade basal media manufactured with advanced milling technology and EMPROVE® raw materials, as well as transition to serum-free supplementation and process development activities, the robust expansion of hMSCs across platforms has been achieved. Presented by Aletta Schnitzler, Senior Scientist on 5/5/16

1. Aletta C. Schnitzler, Ph.D.
May 5, 2016
Challenges and Innovations
ONLY FOR U.S. AND CANADA AUDIENCE

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1. Clinical and commercial considerations for cell therapies
2. Cell culture media manufacturing
3. Media supplements for MSCs
4. Serum-free media
 Performance in planar systems
 Performance in microcarrier systems
5. Large-scale bioreactor culture
Topics

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Cell Therapy Is an Active Area for Clinical Trials
Source: Alliance for Regenerative Medicine 2015 Annual Data Report

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Cell Therapy Bioprocessing
Systems and
Tools
Customer
Collaborations
Media and
Reagents
Preferred partner for cell therapy
process development services. A
specific set of operating parameters
must be generated for each cell,
microcarrier and medium combination.
Extensive process know-how in
scalable volume controlled systems,
for consistent, predictable cell
expansion, and maintenance of cell
phenotype and function.
High quality raw materials meeting
GMP standards that support cell
therapy manufacturing.
A comprehensive regulatory
information package for each
formulation.

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Innovation is required to achieve commercial success
Cell Therapy Manufacturing Considerations
Clinical Success
Commercial successes
Scalable, GMP, Closed, Economically Viable Processes
Pharma grade OR GMP raw materials (media, supplements, buffers, enzymes)
Innovation
TODAY
Collect &
isolate
Culture &
expand
Manipulate
Harvest &
concentrate
Formulate
and fill

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Autologous Therapies: One to One
AUTOLOGOUS = SCALE-OUT
Trends and details
Autologous manufacturing
• Customized to patient
• 1 patient = 1 batch
Key Needs
• Automated closed manufacturing
• Pharma grade or cGMP raw materials
• Faster & cost-effective release testing

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Allogeneic Therapies: One to Many
ALLOGENEIC = SCALE-UP
Trends and details
Allogeneic manufacturing
• Off the shelf therapy
• 1 dose MSCs ~100 x106 cells
Key Needs
• Scalable expansion systems up to 200 - 1000L
• Downstream processing
• Chemically defined media / serum replacement

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Regulatory Challenges – Materials and Reagents

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Cell Culture Medium
The Combination of raw materials and
the production process is mandatory for
excellent dry powder media performance
Raw Material Quality
Key for optimal media
performance in cell
culture applications:
• Formulation
• Raw material selection
• Documentation
Production Process
Directly linked with
lot-to-lot consistency and
cellular performance:
• Milling and Mixing process
• Homogeneity
• Particle size distribution

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Advanced Manufacturing - Milling
High Quality Basal Media
• Fully scalable non-animal origin production line
• Controlled particle size and production temperature
• Full GMP (several FDA and EMEA Audits)
• Inert cold milling process (liquid Nitrogen)
Mill (Ø mm) 40 100 400
Batch size (kg) 0.01-1 0.5-10 100-200

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Advanced Manufacturing – High particle size uniformity
High Quality Basal Media
A B C
A: Our media is a homogeneous powder
B/C: Commercial CDM powder showing crystals and
non-homogeneous blending
Lab scale
Mid scale
Large Scale batch 1
Large Scale batch 2

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Advanced Manufacturing - Compaction
High Quality Basal Media
0 0 0 0
0 0 0
0 0
0
0
0
0 000
00
Compaction
•Is a granulation technology
•No water is added to the process
•Works with compression force only
•Fixes homogeneity in place
•Process Parameters:
 Design of compression unit
 Applied force
 Media formulation

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Advanced Manufacturing - Compaction
High Quality Basal Media
3x Elevated Dissolution Rate Low Dust Formation Flowability
Process efficiencies – Time saving, cost saving, safety and handling improvement
Reduced dissolution time Reduced
contamination risk
Reduced transport and handling cost
60 sec (Endpoint 22 min)
Powder Medium
16 sec (Endpoint 7 min)
Compacted Medium
Lower Bulk Volume

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EMPROVE®
High Quality Basal Media
98%
αMEM
Raw Material Grades
% by weight
Emprove Compendial For CCM
99%
DMEM
Raw Material Grades
% by weight
Emprove Compendial For CCM

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Expansion of Mesenchymal Stem Cells
• SAFC fetal bovine serum, GMP quality level
• PLTMax® human platelet lysate, currently research grade. GMP coming soon.
Passage
FBS C1
SAFC Lot 1
SACF Lot 2
FBS
hPL
Supplementation is required for non-immortalized cells

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Performance across platforms
Expansion of Mesenchymal Stem Cells

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Platelet lysate supports large-scale process
Expansion of Mesenchymal Stem Cells
• MSCs retain function after expansion at large
scale in αMEM + platelet lysate
Differentiation
Identity
Function
Analytics

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Variable performance of commercialized media in planar systems
Technical Challenges with Serum-free Media
XF1 SF1 SF2 SF3XF5XF2 XF3 XF4 LS1 LS2 DMEM
10% FBS
• Many serum-free and xeno-free media for MSC expansion are coming to market; however, highly
variable performance in both planar and microcarrier systems is observed.

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Technical Challenges with Serum-free Media
• Cell surface marker expression is generally well-supported.
SF1
SF2
XF1
XF5
XF2
XF3
XF4
LS1
DMEM 10% FBS

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Differentiation potential is maintained
Serum-free Media can Support Key Features
P8 bmMSC expanded in XF1
medium prior to differentiation
• Multi-lineage differentiation potential is maintained by serum-free formulations.
• XF1 medium also had best growth and cell surface marker expression.
Osteogenesis
Adipogenesis
Chondrogenesis
XF1 XF5XF2 XF3 XF4

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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.
Solid -Liquid Mixing, North American Mixing Forum
Figure 5. States of solid suspension. A) On bottom, B) Off-bottom, C) Uniform suspension

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Performance in planar culture is not predictive of suspension culture
Technical Challenges with Serum-free Media
• XF1 medium performed best in planar culture.
LS1 SF2 SF1XF2 XF1αMEM
PL
DMEM
FBS
DMEM FBS
αMEM PL
Xeno-free and/or defined
Animal-origin or undefined
LS1
SF2
SF1
XF2
XF1
• Good performance in planar culture, does not predict
success in microcarrier-based systems.
• Xeno-free / defined formulations generally perform poorly
in microcarrier-based systems.

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Technical Challenges with Serum-free Media
10% FBS15% XFSup1
p6
p8
• FBS and platelet lysate typically support 10-14 cumulative
population doublings in 4 passages in planar format.
• XFSup1 does not fully support MSC growth in αMEM
alone.

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From planar to suspension systems
Technical Challenges with Serum-free Media
• XFSup1 rescues performance of XF1, a defined medium, in microcarrier-based
suspension culture.
Planar Microcarrier
XF1 + 15% XFSup1
αMEM+10% FBS
XF1 alone
αMEM
FBS
XF1
XFSup1: 0% 5% 10% 15%

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Summary
Process
Understanding
Cell
Function
Quality
Materials
• Emprove®
• Advanced manufacturing
• Non-animal origin
• Optimize control parameters
• Hydrodynamics
• Role of media components
• Cell identity
• Differentiation potential
• Immune modulation
Linking function to clinical efficacy!
How do we get from here… … to here?
Best Practices in Cell Therapy Manufacturing,
J. Carmen. (2013) The Cell Culture Dish

26. Senior Scientist
Cell Therapy Bioprocessing
MilliporeSigma
Bedford, MA, USA
aletta.schnitzler@emdmillipore.com