Validation of Bone Allograft Processing Reducing Risk of Disease

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. 1 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 2 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 3 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 4 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 5 Validation of Bone Allograft Processing Comparison of Osteoinductivity: Independent Published Study 4 3 2.27 2 1 0 Graft A Graft B 0 Graft C 3.5 6 NASS Proceedings, 15th Annual Meeting 2000, p38 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 characterized as soft or hard  Soft: ligaments, tendons, fascia, cartilage  Hard: bone  • Cancellous • Cortical 7 Validation of Bone Allograft Processing In terms of mass, bone tissue can be described as having two phases:  Inorganic Organic  8 Validation of Bone Allograft Processing Organic (30%) Cells (2%) Osteoblasts Osteocytes Osteoclasts + Composition of Normal Bone Matrix (98%) Collagen (95%) Non-collagenous proteins (5%) (includes growth factors) Mineral (70%) Hydroxyapatite (95%) + Magnesium Sodium Potassium Fluoride Chloride 9 Validation of Bone Allograft Processing 10 Validation of Bone Allograft Processing Structure of Cortical (Compact) Bone 11 12 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 13 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 14 Validation of Bone Allograft Processing Estimated Theoretical Maximum Viral Burden Demineralized Bone HIV HBV HCV IU/ml Blood IU/cc Bone (0.015ml/cc) IU/ bone dose (15cc) Log IU/ dose (highest value) 15 50 x 10³ 10³ 10 x 10³ 5 x 10² 7.5 x 10³ 10¹ 1.5 x 10² 10² 1.5 x 10³ 3.87 2.18 3.18 Validation of Bone Allograft Processing Maximum Viral Burden: Mineralized Bone Log values HIV-1 Inactive Marrow HTLV-1 HTLV-2 HBV HCV 2.3-5.0 5.3-8.6 1.5-3.5 4.6-6.6 1.5 4.6 1.8-4.3 1.8-4.3 2.8-7.8 4.6-8.6 Active Marrow 16 Validation of Bone Allograft Processing Viral Inactivation - D-MIN® Process Log10 Reduction Step HCL EtOH HIV >5.23 >4.23 None >9.46 3.87 5.59 DHBV (HBV) >3.70 >3.70 N.D. >7.40 2.18 5.22 BVD (HCV) CMV >4.15 >2.92 >3.15 >3.32 1.77 None Polio >5.99 >3.72 2.30 Lyophilization Total MAX Burden Safety Factor 17 >9.07 >6.24 >12.01 3.18 5.89 N/A* N/A* *Insufficient published data to calculate viral burden. Validation of Bone Allograft Processing Standard Bone Allografts Viral Inactivation and Removal Data HIV Step 3 Surfactant BVD DHBV CMV Polio Inactivation 5 Min Step 8 Alcohol Inactivation 5 min Total Inactivation >4.38 >4.59 None >5.09 None >4.38 >4.59 >3.50 >5.09 0.53 >8.76 >9.18 >3.50 >10.18 0 Step 4/2 Removal MuLV BVD BVD HSV Polio 1 min Total Clearance: 6.15 14.91 5.3-8.6 6.31 6.21 15.39 4.6-8.4 6.99 6.21 9.71 1.8-4.3 5.41 6.68 16.86 ND - 8.3 8.3 ND - 18 Theoretical Max. Viral Burden Safety Factor 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 19 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) 20 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 21 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 –  Solid surface cleaning –  Solid surface cleaning – Sporklens Expor (1) Expor (2) > 4.0 > 3.4 > 3.4 22 Validation of Allograft Bone Processing Cleaning Validation to Prevent Cross Contamination Post Cleaning Residuals (mg/sq ft) Lipids Bone Blood Table Bandsaw MedClean 0.90/6.7 1.6/17 0.36/6.9 0.06/0.23 0.05/0.38 0.07/0.18 0.00/0.01 0.00/0.01 0.01/0.02 23