1. Cellular Therapies for Repair and Regeneration of Joint Surfaces— Product Characterization and Testing Malcolm Moos Jr., M.D., Ph.D. Medical Officer CBER/OCTGT/DCGTmoos@cber.fda.gov

2. Source Process Product Testing

3. “Device”“Biologic” Well-characterized Well-characterized Precisely defined materials Precisely defined materials Precisely defined structure Precisely defined structure Tests that measure critical product parameters reliably Tests that measure critical product parameters reliably Data defining these parameters Data defining these parameters Poorly characterized Poorly characterized Heterogeneous Heterogeneous Complex molecular entities Complex molecular entities Inadequate analytical procedures Inadequate analytical procedures Insufficient scientific basis correlating measurable properties with biological activity Insufficient scientific basis correlating measurable properties with biological activityReleaseTestingProcesscontrol

4. “Device”“Biologic” Well-characterized Well-characterized Precisely defined materials Precisely defined materials Precisely defined structure Precisely defined structure Tests that measure critical product parameters reliably Tests that measure critical product parameters reliably Data defining these parameters Data defining these parameters Poorly characterized Poorly characterized Heterogeneous Heterogeneous Complex molecular entities Complex molecular entities Inadequate analytical procedures Inadequate analytical procedures Insufficient scientific basis correlating measurable properties with biological activity Insufficient scientific basis correlating measurable properties with biological activityReleaseTestingProcesscontrol

5. Source Process Release Testing

6. Strategic considerations “Measure twice, cut once”: “Measure twice, cut once”: Up-front investment in each area may prevent costly waste of time and resources on sub-optimal process or variable/ineffective product Up-front investment in each area may prevent costly waste of time and resources on sub-optimal process or variable/ineffective product Better characterization makes many things easier Better characterization makes many things easier

7. Source Control Anatomic site Anatomic site Pathologic involvement Pathologic involvement Cellular homogeneity/inhomogeneity Cellular homogeneity/inhomogeneity

9. Freshly Isolated Chondrocytes In vitro culture is not required HACDC in regenerating muscle

10. Product Testing Is guided by detailed understanding of the product and the manufacturing process Is guided by detailed understanding of the product and the manufacturing process Should ensure consistency of process and product Should ensure consistency of process and product Should predict in vivo activity Should predict in vivo activity

11. Product Testing —An example of new alternatives Terminal markers may not predict biological function Terminal markers may not predict biological function Characterization of functional chondroprogenitors Characterization of functional chondroprogenitors Qualification by meaningful biological responses Qualification by meaningful biological responses

12. Islet cell transplantation in diabetic mice Live/dead Staining (%) 0204080 100 60 OCR/DNA (%) 20 40 80 100 60 0 Theoretical correlation Oxygen Consumption Rate and dye exclusion are uncorrelated (K. Papas, University of Minnesota Department of Surgery, in preparation)

13. Normalized Oxygen Consumption Rate, OCR/DNA (nmol/minmgDNA) Oxygen Consumption Rate, OCR (nmol/min) Fractional Viability (%) Viable Islet Equivalents, VIEQ Response to Rat Islet Transplants in Diabetic Balb/C Mice (Anti-CD4) 2 / 0 / 02 / 0 / 0 1 / 0 / 01 / 0 / 0 2 / 0 / 02 / 0 / 0 2 / 0 / 02 / 0 / 0 6/ 0 / 0 4/ 0 / 0 3 / 0 / 03 / 0 / 0 3 / 0 / 03 / 0 / 0 1 / 0 / 11 / 0 / 1 0 / 1 / 20 / 1 / 2 1 / 0 / 11 / 0 / 1 2 / 0 / 02 / 0 / 0 1 / 0 / 01 / 0 / 0 0 / 1 / 00 / 1 / 0 1 / 0 / 01 / 0 / 0 3 / 0 / 03 / 0 / 0 2 / 0 / 02 / 0 / 0 3 / 0 / 03 / 0 / 0 all cure mixed all fail 3 / 0 /0 2 / 0 / 02 / 0 / 0 1 / 0 / 21 / 0 / 2 1 / 0 / 01 / 0 / 0 0 4 5 6 050100150200250300350400450 100 800 700 600 500 400 300 200 100 0 0504020106030807090 0 / 1 / 10 / 1 / 1 0 / 0 / 40 / 0 / 4 0 / 0 / 60 / 0 / 6 0 / 0 / 30 / 0 / 3 0 / 0 / 20 / 0 / 2 1 2 3 0 / 0 / 30 / 0 / 3 0 / 0 / 20 / 0 / 2

14. Cure rates and days to cure in mice receiving 1,000 human IE according to ATP/DNA 0 20 40 60 80 100 010203040 Days after transplant Cure rate (%) ATP < 110 pmol/µg DNA pmol/µg DNA ATP > 110 pmol/µg DNA P=0.008 (B. Hering, University of Minnesota Department of Surgery, with permission)

15. The nude mouse model Dell’Accio et al, A&R, 2001

16. The Shmoo —Al Capp, ca. 1948

20. Instructions go both ways Donor Recipient

21. Donor-Host Interactions? Cell product Articular surface Subchondral bone Remnants of calcified cartilage Cover Synovial Joint Fluid

22. Competence Factors gene expression Biological Response P

23. β-actin ALK-1 BMP-2 Col2a1 FGFR-3 Population doublings FI 2 4 10 12 Safranin O Molecular profile of the stable chondrocyte

24. Histology score Gene score -5-3135 0 1 2 3 Relation of Gene score to Stable Cartilage Formation Relation of Gene score to Stable Cartilage Formation Stable cartilage

25. Space—the final frontier? Thomas and Moos, 2004 (unpublished)

26. Joint-specific growth factor Enzyme Overlapgrowth factor activation joint surface specification Molecular Boundaries in Tissue Formation 400x Thomas and Moos, 2005 (submitted)

27. Thought Questions Acceptance, selection, or instruction of starting material Acceptance, selection, or instruction of starting material Consider competence factors sufficient to define chondroprogenitors (rather than characteristics of terminal differentiation) Consider competence factors sufficient to define chondroprogenitors (rather than characteristics of terminal differentiation) Distribution of characteristics in 3D may be critical Distribution of characteristics in 3D may be critical Qualification of potential tests against appropriate biological responses in vivo Qualification of potential tests against appropriate biological responses in vivo

28. Criteria for obtaining starting tissueCriteria for obtaining starting tissue Characteristics of functional chondroprogenitor cellsCharacteristics of functional chondroprogenitor cells Analytical methods to determine these characteristicsAnalytical methods to determine these characteristics Qualification of these tests in preclinical modelsQualification of these tests in preclinical models Potency assaysPotency assays Special issues for cells contained in natural or artificial matricesSpecial issues for cells contained in natural or artificial matrices Questions for Discussion