1. Removal of Infectious Prions from Red Cell Concentrates Samuel Coker, PhD Principal Scientist and Technical Director Pall Medical Transmissible Spongiform Encephalopathies Advisory Committee Meeting February 8, 2005

2. Prions Dormancy and Disease: Can vCJD be Transmitted through Blood Transfusion?  Prions can be transmitted to healthy animals through transfusion from infected animals  Two probable cases of vCJD through blood transfusion from an asymptomatic donor  Serious concerns of a second human-to-human wave of vCJD transmission pose a major risk to the blood supply.

3. Approach to Improving the Safety of the Blood Supply: Prion Removal SMART Filter Technology  Remove Prions Associated with Leukocytes  Remove Prions Present in Plasma  Removes both Prion Types (Normal + Abnormal)  Without a Monoclonal Antibody  Without a Ligand for Abnormal Prion  With Proprietary Surface Modification (biocompatible) CE Mark Spring 2005

4. Exogenous-In vitro Spiking TestsEndogenous Infection Western Blot Assay Bioassay Brain samples from scrapie infected hamsters Homogenized sample contains high titre of infectious prions Homogenate is spiked into blood/component Prion reduction process Detection of residual infectious prion 50µL of SIHBH injected intracerebrally 100-200 hamsters are injected Blood samples are collected into CPD during clinical phase of disease Blood separated into plasma and red cells Prion removal process Test Methods to Validate Prion Reduction Process

5. Preparation of Red Cell Concentrates from Whole Blood Obtained from Scrapie Infected Hamsters Scrapie Infected Hamsters Normal Hamsters Intracerebral Injection Hard spin centrifugation Remove supernatant plasma Add red cell additive solution Prion removal filter BRAIN 10% brain homogenate

6. Removal of Infectious Prion from Red Cell Concentrate Endogenously Infected with Scrapie Before Filtration After Filtration Recovered from Filter and Concentrated 500x Western Blots After Proteinase K Digestion

7. Animal Model of Blood Transfusion - Intracerebral Injection of Red Cells into Normal Healthy Hamsters 1.50 µL of pre and post filtration red cell concentrates were injected intracerebrally into healthy hamsters. 2.Hamsters were monitored for 300 days for any clinical signs of scrapie infection. 50 µL 20 Recipients

8. Results of Endogenous Infectivity Study Filtered Red Cell Concentrate Control – UnFiltered Red Cell Concentrate Western Blot of Brain Homogenates After Proteinase K Digestion 3 out of 18 hamsters were infected with scrapie 0 out of 20 Infectious Prions PrP SC X = Clinical signs of scrapie

9. Model of Human vCJD Removal of vCJD using Mouse Adapted Model of human vCJD Infectious Prion (mvCJD)

10. Prion Reduction Filter Filtered Red Cell Concentrate Red Cell Concentrate Containing Infectious Prions BRAIN 10% brain homogenate Exogenous Infectivity Study: Addition of 10% (w/v) of Mouse-Adapted vCJD Brain Homogenates into Red Cell Concentrate in Additive Solution

11. Spiked red cells concentrate Infectious mvCJD agent in Red Cell Concentrate before and after Filtration with “Pall Leukotrap® Affinity Prion Reduction Filter” Before Filtration (10 7-8 LD 50 /mL) After Filtration ( Below Limit of Detection)

12. PART 4 – Leukocyte Reduction And Quality of Red Cells After Filtration Measurement of Residual Leukocytes, Haemolysis and Cell Deformability

13. Residual leukocytes in red cell concentrates in SAGM after filtration with with prion reduction filter

14. Supernatant haemoglobin in red cell concentrates in SAGM after filtration with prion reduction filter and storage at 4°C for 42 days. Each data point represents the mean  standard deviation of N=40.

15. Supernatant haemoglobin in red cell concentrates in CPDA-1 after filtration with prion reduction filter and storage at 4°C for 35 days. Each data point represents the mean  standard deviation of N=14.

16. Osmotic gradient deformability profiles of red blood cells from red cell concentrates in SAGM measured before and after filtration with prion reduction filter

17. Summary Results - Leukocyte Reduction, Red Cell Haemolysis and Deformability  Reduction of leukocytes below AABB/FDA guideline of 5 x 10 6 wbc/unit.  No change in red cell deformability profiles in response to osmotic stress - suggests normal in vivo survival.  Mean supernatant haemoglobin levels in red cell concentrates were below AABB/FDA guideline of 1.0% at end of storage.

18. SUMMARY  EXOGENOUS INFECTIVITY: Prion reduction filter significantly removed different strains of infectious prion including mouse adapted model of human form of vCJD from red cell concentrates.  ENDOGENOUS INFECTIVITY: Infectious prions in red cell concentrates obtained from hamsters infected endogenously with scrapie were removed below the limit of detection of our Western blot assay.

19. CONCLUSION  CONCURRENT PRION AND LEUKOCYTE REDUCTION Prion reduction below limit of detection Leukocyte reduction below 1 x 10 5 per unit. The Pall Leukotrap® Affinity Reduction Filter may help improve blood safety and availability by reducing the risk of transmission of human vCJD through blood transfusion