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

.

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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

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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

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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

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Validation of Bone Allograft Processing

Comparison of Osteoinductivity: Independent Published Study

4 3.5

3 2.27

2 1 0 Graft A Graft B 0 Graft C NASS Proceedings, 15 th Annual Meeting 2000, p38

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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

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Validation of Bone Allograft Processing In terms of mass, bone tissue can be described as having two phases:

  Inorganic Organic

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Validation of Bone Allograft Processing Organic (30%) Cells (2%)

Osteoblasts Osteocytes Osteoclasts

+ Matrix (98%)

Collagen (95%) Non-collagenous proteins (5%) (includes growth factors)

Composition of Normal Bone Mineral (70%) Hydroxyapatite (95%) +

Magnesium Sodium Potassium Fluoride Chloride

10

Validation of Bone Allograft Processing

11

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

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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

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Validation of Bone Allograft Processing

Estimated Theoretical Maximum Viral Burden Demineralized Bone

HIV 50 x 10 ³ HBV 10 ³ HCV 10 x 10 ³ IU/ml Blood IU/cc Bone (0.015ml/cc) IU/ bone dose (15cc) Log IU/ dose (highest value) 5 x 10 ² 7.5 x 10 ³ 3.87

10 ¹ 1.5 x 10 ² 2.18

10 ² 1.5 x 10 ³ 3.18

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Validation of Bone Allograft Processing

Maximum Viral Burden: Mineralized Bone

Log values HIV-1 Inactive Marrow Active Marrow 2.3-5.0

5.3-8.6

HTLV-1 1.5-3.5

4.6-6.6

HTLV-2 1.5

4.6

HBV 1.8-4.3

1.8-4.3

HCV 2.8-7.8

4.6-8.6

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Validation of Bone Allograft Processing Viral Inactivation - D-MIN ® Process Step HCL EtOH Lyophilization Total MAX Burden Safety Factor HIV Log 10 Reduction DHBV (HBV) BVD (HCV) CMV >5.23

>3.70

>4.15 >2.92

>4.23

>3.70

>3.15 >3.32

None >9.46

3.87

5.59

N.D.

>7.40

2.18

5.22

1.77

>9.07 >6.24 >12.01

3.18

5.89

None N/A* Polio >5.99

>3.72

2.30

N/A* *Insufficient published data to calculate viral burden.

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Step 3 Step 8 Validation of Bone Allograft Processing Standard Bone Allografts Viral Inactivation and Removal Data HIV BVD DHBV CMV Polio Surfactant Inactivation 5 Min Alcohol Inactivation 5 min Total Inactivation >4.38

>4.38

>8.76

>4.59

>4.59

>9.18

None >3.50

>3.50

>5.09

>5.09

>10.18

None 0.53

0 Step 4/2 Removal MuLV BVD 1 min Total Clearance: Theoretical Max. Viral Burden Safety Factor 6.15

14.91

5.3-8.6

6.31

6.21

15.39

4.6-8.4

6.99

BVD 6.21

9.71

1.8-4.3

5.41

HSV 6.68

16.86

ND Polio 8.3

8.3

ND -

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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

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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)

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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

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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

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Validation of Allograft Bone Processing

Cleaning Validation to Prevent Cross Contamination Post Cleaning Residuals (mg/sq ft) Table Bandsaw MedClean Lipids 0.90/6.7

1.6/17 0.36/6.9

Bone 0.06/0.23

0.05/0.38

0.07/0.18

Blood 0.00/0.01

0.00/0.01

0.01/0.02