Scaling Microcarrier-based Expansion Processes for Production of High Quality Cells

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1 Scaling Microcarrier-based Expansion Processes for Production of High Quality Cells Mark S. Szczypka BioTech 2016, Wädenswil Switzerland September 5 th 2016 This presentation is the work product of Pall Corporation and no portion of this presentation may be copied, published, performed, or redistributed without the express written authority of a Pall corporate officer Pall Corporation.

2 Cell-based Medical Technologies 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 Gene Delivery Live cells for pharmaceutical research Cell Therapies Adult stem cells Primary cells ipsc ES cells Regenerative Medicine Drug Discovery 2

Cell Numbers for Therapeutic Applications and

Indication Patients/year Cells/year (TD 1e 9 )

3 Cell Numbers for Therapeutic Applications and Enabling Technology Allogeneic cell therapy Indication Patients/year Cells/year (TD 1e 9 ) (Global) (Global) Ischemic Stroke 2.00E E+15 Acute Myocardial Infarction 4.00E+06 Regenerative Medicine Indication Islets for Type I diabetes 4.00E+15 Stem Cells/patient 1B TH positive neurons for Parkinson s 2 T Xpansion 2D cell culture reactors 3 Pad Bioreactors

4 Building a Simple and Robust Cell Manufacturing Platform for Scalable Expansion of hmsc Critical components 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 4

5 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

Robust Growth of hmscs on Pall SoloHill Microcarriers Cells/mL (x 10 6 ) 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.

0 0.0 0 2 3 4 5 6 7 Days of Growth Bioreactor Harvest Collagen Star-Plus 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.

6 Robust Growth of hmscs on Pall SoloHill Microcarriers Cells/mL (x 10 6 ) Bioreactor Culture Spinner Control Days of Growth Medium Exchange Cells/cm 2 (x 10 4 ) Days of Growth Bioreactor Harvest Collagen Star-Plus Cells/mL (x 10 6 ) Collagen Star-Plus Medium Exchange 2.4 B 1.3 B Excellent growth and efficient harvest of human mesenchymal stem cells (hmscs) from Pall SoloHill microcarriers in small scale bioreactors 6

Bioreactor Bioreactor T Flask Control 40 20 0 Marker hmsc harvested

7 Robust Growth of hmscs on Pall SoloHill Collagen Coated Microcarriers Adipocyte Osteocyte % Gated Cells Collagen Bioreactor Bioreactor T Flask Control Marker hmsc harvested from microcarriers retain differentiation capacity and surface marker expression 7

hmsc Expansion using PadReactor Platform and Pall SoloHill Microcarriers Allegro Microcarrier Delivery System (AMDS-Irradiated microcarriers)

8 hmsc Expansion using PadReactor Platform and Pall SoloHill Microcarriers Allegro Microcarrier Delivery System (AMDS-Irradiated microcarriers) RoosterBio hmsc (liquid nitrogen stock) 6L PadMini Bioreactor Pad50 3 days 4 days Maintain PDL below 18 to 20 doublings to maintain quality and consistency 8

bioreactors for PD and small scale GMP batches 9 SoloHill microcarriers, delivered ready-to-use in a

9 Adherent Cell Culture Solutions Microcarrier Cell Culture 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 9 SoloHill microcarriers, delivered ready-to-use in a single use format Sterilized by gamma irradiation Allows to directly seed bioreactors no additional preparation needed

10 RoosterBio hmsc and Medium 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 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 (Data generated by RoosterBio) 10

11 Small scale microcarrier passage studies P1 microcarriers Harvest P2 microcarriers 0.6 Passage 2 Cells/mL (x 10 6 ) Microcarrier (n=1) Planar culture 0.0 Day 5 11

12 Reproducible and Robust Cell Expansion in 6 Liter PadMini Bioreactor 0.60 Expansion Scheme Cells/mL (x 10 6 ) BR (n = 3) CF10 LN 2 Cell Factory (CF10) Bioreactor Process Parameters PadMini (6L) RoosterBio hmsc RoosterBio High Performance media Seed density 3000 cells/cm Day 4 Microcarrier conc. Medium exchange 10 cm 2 /ml None Supplement Day 3 Over twenty-fold expansion was reproducibly achieved in batch-fed microcarrier cultures in PadMini at 6L scale in 4 days 12

13 Generation of Billons of hmsc in Pall s Single use PadReactors in 8 days Total cells 3 days 5 days Pad Mini (6 L) Pad 50 (40L) Seed Harvest Seed Harvest 1.67E E E E+10 PDL Cell Expansion 0.17 B 28 B Cells/mL (x 10 6 ) PAD50 Reactor Day 13

14 Conclusions PadReactors used in a closed system provide an ideal singleuse 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 14

15 Expansion using PadReactor Platform and Pall SoloHill Microcarriers Expedite developmental timelines for cell-based Medical technologies Provide robustness and reproducibility in single-use closed system Decrease cost of implementation hmsc 6L PadMini Bioreactor Pad50 15

16 Acknowledgements Pall Life Sciences Dave Splan RoosterBio (Cells and Media) Lye-Theng Lock Jon Rowley Jin Liu Alexandria Spruiel Kaitlynn Bayne Grishma Patel Andrew Laskowski 16

17 Thank You

Application Note USD 3086 (1) Animal Component-Free Star-Plus Microcarriers for Adherent Mammalian Cell Culture

Application Note USD 3086 (1) Animal Component-Free Star-Plus Microcarriers for Adherent Mammalian Cell Culture Application Note USD 386 (1) Animal Component-Free Star-Plus Microcarriers for Adherent Mammalian Cell Culture With the continuing move of the biopharmaceutical and cell therapy industries toward the use