Manufacturing and Characterization Challenges for Human Stem Cell Products

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1 Manufacturing and Characterization Challenges for Human Stem Cell Products Center for Biomedicine and Genetics Beckman Research Institute of City of Hope Larry A. Couture, Ph.D January, 2009

2 Center for Biomedicine and Genetics Established Jan ,000 square ft. 2 story 10,000 square ft. classified space 3 zones, 12 production rooms class 100 fill suite Products Plasmid DNA Lentivirus Adenovirus Oncolytic vectors mab Recombinant proteins Bacterial products Cell Products Islet cells T-cell CD34+ hesc Neural stem cells

3 The Stem Cell Promise A potential source of transplantable cells to regenerate injured or lost tissues or to restore functions lacking through acquired or inherited disorders. What are Stem Cells? Cells capable of self renewal and the ability differentiate into multiple cell types and tissues in response to biological cues.

4 Are Stem Cells a new Biologic Class The stem cell class: Type Source Potential Clinical Embryonic (hesc) Embryos Totipotent or Pluripotent pending Fetal (NSC) Fetal tissue Multipotent 10 years Adult (CD34+, ASC, MSC) Adult tissue Multipotent 30 years Induced Pluripotent Stem Cells Adult Tissue Totipotent or Pluripotent no All: Capable of self renewal and the ability differentiate into multiple cell types and tissues in response to biological cues. New types and new challenges? Yes A new class? NO

5 Regulation of Stem Cell Therapies Novel biologic therapies comprised of or derived from stem cells will be regulated as human cells, tissues or cellular and tissue- based products (HCT/Ps). HCT/Ps means articles containing or consisting of human cells or tissues that are intended for implantation, transplantation, infusion or transfer into a human recipient.

6 What makes hesc and ipsc unique Safety tumorgenicity Stability genetic Cell culture Source political and social = Characterization and development challenges

7 Development Challenges Source control Derivation Manufacturing Pre-clinical models Characterization safety, purity/identity and stability

8 Undifferentiated hesc Are Not Homogenous Populations hesc H9 hesc H9 SSEA-4 Tra-1-60 Tra-1-81

9 Scalability ROCK Inhibitor-Y27632 H9 p31 No Inhibitor With Inhibitor Inhibitor Removed

10 Defining The Stem Cell Product Undifferentiated hesc MCB Intermediate MCB Differentiated product MCB Differentiation Process (Days to weeks) Tumorgenicity? Safety

11 Characterization Safety, Purity/Identity, Stability Multi-parametric analytical testing Morphologic evaluation Detection of phenotype-specific surface markers Unique biochemical markers Gene and protein expression analyses Epigenetic profiling Cellular impurities profile assessment Biologic activity assessment MHC/HLA expression Tumorgenicity assays

12 Safety Animal testing: Safety Assessment Cell survival post-transplantation Cell migration Cell fate - plasticity; differentiation/ phenotype expression; fusion Tissue integration Tumorigenicity - hyperplastic or unregulated growth What is a relevant animal model? What is the sensitivity of animal model for tumorgenicity? Can hesc spiking experiments address uncommitted cell contaminants?

13 Stability (hesc MCB) hesc P79 TaqI SSEA-4 +/- P29- P29+ P79- P79+ Hep Normal Karyotype TTTTAGGAGGGTTTTGGAAGTTTAGTTAGGTTCGAGGATTAATTTAGTTCGGTTTCGGTTT TTTTGGTTTATTATTTTTATTATTTGGAGGGGGCGAGAAGGCGAAATTCGAAGTTAGGTG TTTCGTTATGGGGAAGGAAGGCGTTTTAAGTCGGGGGTTTGGTGAAATGAGGGTTTGCGA AGGGATTATTTAATTTTTTTTTTTTTTTTTAGTTTTATTTATTAGTTTTGATTTTTGGTTTC p29 SSEA-4 (-) 40% p79 SSEA-4 (-) 45% Liver cells p29 SSEA-4 (-) 58% p79 SSEA-4 (-) 58% p29 SSEA-4 (+) 0% p79 SSEA-4 (+) 0% p29 SSEA-4 (+) 6% p79 SSEA-4 (+) 8% OCT4 promoter methylation correlates with loss of SSEA-4 NANOG promoter methylation correlates with loss of SSEA-4

14 Summary hesc are not a new class of biologics but do present unique characterization and development challenges Existing regulatory paradigms are applicable for now New approaches to demonstrating identity, stability and purity may be necessary Developing suitable Safety tests may prove the most challenging

15 Acknowledgements Manufacturing David Hsu Matthew Luo Lara Ausubel Sylvana Couture Patricia Lopez Christine Knoblauch Cassidy Yang Ross McMahon Jonathan Anderson Ivy Derecho Valerie Quezada Nina Dunphy QA Yasmine Shad David Ceniceros Luis Gutierrez Misty Shakeley Jose Valdez Mosleh Alkhatabeh Mario Marcelo QC Patricia Huang Kunal Jariwala Rafat Khan COH Art Riggs Zunde Wang

16 H9 ES Embryoid Body

17 Derivation and Manufacturing Media for culturing hescs routinely supplemented with bovine serum, growth factors as well as other animal-derived ancillary products Bovine serum is acceptable provided demonstration that source is from herds BSE-free countries. Clinical-grade serum sourced from humans Develop serum-free, chemically defined medium. hesc lines established on non-human feeder cell layers: Fit the FDA definition of xenotransplantation FDA does not intend xenotransplantation requirements to preclude use of hescs in human clinical trials For stem cell products derived from hesc lines raised on non-human feeders it may be necessary to demonstrate that the line is free from infectious agents a that may pose a risk for transmission to recipients Adventitious agent testing is equally important when feeders are comprised of human cells

18 Source control Evaluating human stem cell sources: Informed consent Appropriate screening/testing of donor tissue for communicable disease [Donor Eligibility Rule] Implications of genetic analysis Intrinsic safety concerns based on cell source (adult, fetal, embryonic)

19 Purity (hesc MCB) Immunohistochemistry OCT-3/4 nuclear transcription factor NANOG nuclear transcription factor Alkaline Phosphatase FACS Analysis SSEA-4 Tra-1-60

20 Characterization Demonstrate through analytical and clinical testing: Sterility Purity * Potency Identity Stability * Safety * Efficacy