Validation of Bone Allograft Processing: Reducing Risk of Disease Transmission via Conventional Agents While Maintaining

1. Validation of Bone Allograft Processing:Reducing Risk of Disease Transmission via Conventional Agents While Maintaining Graft Performance TSE Advisory Committee Meeting June 26, 2002 Gaithersburg, MD Richard Russo Osteotech, Inc.

2. Validation of Bone Allograft Processing Risk reduction is a multi-functional process, built on several factors: • Donor testing and screening • Donor deferral and tissue discard policies • Appropriate segregation and quarantine procedures • Adequate cleaning and disinfection between batches • Processing to inactivate and/or remove pathogens

3. Validation of Bone Allograft Processing Potential pathogens in tissue: • Bacteria, yeasts, mold and fungi • Viruses • TSE Presentation focus: • Viruses in standard non-demineralized grafts Principal viruses of concern: • HIV 1 + 2, HTLV 1+2, HBV, HCV

4. Validation of Bone Allograft Processing Performance • Primary purpose of bone graft is to support bone formation across a defect due to disease, trauma, or reconstructive surgery • Not all processing has same effect on bone graft performance • Failure of graft procedure typically leads to revision surgery • Limitation of capacity of graft to support bone formation leads to increased morbidity and risk to patient

5. Validation of Bone Allograft Processing:Treatments Affecting Performance • Preprocessing Storage • Temperature • Multiple Freeze-Thaw Cycles • Sterilization Procedures • UV Radiation • Gamma • E-beam • Ethylene Oxide • Solvents (GnHC1, Acid-Alcohol, EDTA) • Sonication (> 20,000 cycles/sec.) • Peracetic Acid • Hydrogen Peroxide • Concentration and time dependent • Strong base, e.g, NaOH Reviewed by Russell & Block, Orthopedics Vol. 22 (5); 524, 1999

6. Validation of Bone Allograft Processing Comparison of Osteoinductivity: Independent Published Study NASS Proceedings, 15th Annual Meeting 2000, p38

7. Validation of Bone Allograft Processing Risk reduction via processing begins with characterization of tissues and assessment of risk • Each type of tissue presents its own profile • Musculoskeletal tissues can be characterizedas soft or hard • Soft: ligaments, tendons, fascia, cartilage • Hard: bone • Cancellous • Cortical

8. Validation of Bone Allograft Processing In terms of mass, bone tissue can be described as having two phases: • Inorganic • Organic

9. Organic (30%) Mineral (70%) Cells (2%) Osteoblasts Osteocytes Osteoclasts + Matrix (98%) Collagen (95%) Non-collagenous proteins (5%) (includes growth factors) Hydroxyapatite (95%) + Magnesium Sodium Potassium Fluoride Chloride Validation of Bone Allograft Processing Composition of Normal Bone

10. Validation of Bone Allograft Processing

11. Validation of Bone Allograft Processing Structure of Cortical (Compact) Bone

13. Validation of Bone Allograft Processing Maximum Viral Burden in Bone: General Considerations • No published reports that quantitative viral burden in bone • Extrapolate viral burden from values reported for blood: data exist for HIV, HTLV, HBV, and HCV • Bone is divided into discrete compartments • Conservative bone allografts “dose” • 90cc mineralized tissue • Calculation of blood volumes • Calculation of theoretical maximum viral burden

14. Validation of Bone Allograft Processing Viral Clearance Process Validation: General Approach • Spike bone with known amount of virus • Relevant panel • Demonstrate quantitative recovery • Proceed through process step • Quantitatively assay viral inactivation • Calculate difference between theoretical maximum viral burden and log reduction value of process • Viral clearance must be at least 3 logs greater than maximum viral burden to provide acceptable level of safety

15. Validation of Bone Allograft Processing Estimated Theoretical Maximum Viral BurdenDemineralized Bone

16. Validation of Bone Allograft Processing Maximum Viral Burden: Mineralized Bone Log values

17. Validation of Bone Allograft ProcessingViral Inactivation - D-MIN® Process Log10 Reduction *Insufficient published data to calculate viral burden.

18. Validation of Bone Allograft ProcessingStandard Bone AllograftsViral Inactivation and Removal Data

19. Validation of Bone Allograft Processing • Current process validation guidance documents and virology perspectives can be successfully adapted to viral clearance process validation for bone allografts • Bone allograft performance can be maintained while producing a significant SAL using a conservatively calculated theoretical maximum viral burden

20. Validation of Bone Allograft Processing • Center for Biologics Evaluation and Research. “Points to Consider in the Characterization of Cell Lines Used to Produce Biologicals” (Food and Drug Administration) 1993 • Center for Biologics Evaluation and Research. “Points to Consider in Manufacture and Testing of Monoclonal Antibody Products for Human Use” (Food and Drug Administration) 1997 • Committee for Proprietary Medicinal Products Ad Hoc Working Party on Biotechnology/Pharmacy and Working Party on Safety Medicines, Note for Guidance. “Validation of Virus Removal and Inactivation Procedures”, Biologicals 1991: 19-247-251 • Committee for Proprietary Medicinal Products: EEC Council Directive 89/381: “Medicinal Products Derived from Human Plasma” (Revised Draft 1995)

21. Validation of Bone Allograft Processing • Committee for Proprietary Medicinal Products: 1995 Revised CPMP Guidelines. Virus Validation Studies: The design, contribution and interpretation of studies validating the inactivation and removal of viruses (revised) • ICH Viral Safety Document: QSA Viral Safety Evaluation of Biotechnology Products Derived from Cell Lines of Human or Animal Origin: 1998

22. Validation of Allograft Bone Processing Prion Inactivation by Cleaning Procedures • Spike Samples – Hamster Scrapie Homogenate • Evaluation by prion Western Blot Assay • Solid surface cleaning with existing procedures Log Reduction Factors • Solid surface cleaning – Sporklens > 4.0 • Solid surface cleaning – Expor (1) > 3.4 • Solid surface cleaning – Expor (2) >3.4

23. Validation of Allograft Bone Processing Cleaning Validation to Prevent Cross Contamination Post Cleaning Residuals (mg/sq ft)