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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

2

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

3

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