PATHOGENINAKTIVIERUNG

1. PATHOGENINAKTIVIERUNG Wolfgang R. Mayr Department for Blood Group Serology, University of Vienna, Austria

2. Department for Blood Group Serology, University of Vienna, Austria

3. Department for Blood Group Serology, University of Vienna, Austria

4. Department for Blood Group Serology, University of Vienna, Austria

5. Department for Blood Group Serology, University of Vienna, Austria

6. Department for Blood Group Serology, University of Vienna, Austria

7. Department for Blood Group Serology, University of Vienna, Austria

8. The Safety of Blood Transfusions Has Improved Over Time • Current measures to increase blood safety have been very successful • Donor selection/interviews with medical history • Screening for viral pathogens • HIV-1 & 2 • HBV • HCV • HTLV 1 & 2 • Limited screening for bacterial pathogens - Treponema pallidum (syphilis) • Filtration and gamma irradiation to reduce the number of viable donor leukocytes Department for Blood Group Serology, University of Vienna, Austria

9. 1. Bacteria Introduced during collection 5. Leukocytes 2. Emerging/Unknown Viruses Adverse immune responses and transfusion reactions 4. Known Pathogens 3. Window Period For which no assay is available Limits of detection of current assays (e.g. false negatives) Risks Still Exist in Blood Transfusions Transfusion Recipient Department for Blood Group Serology, University of Vienna, Austria

10. Bacterial Contamination • Risk of bacterial contamination in platelet doses can be as high as 1:2,000 • The mortality rate for platelet-related sepsis is one in four • A prospective study of 3,584 platelet transfusions in 161 bone marrow transplant patients demonstrated risk of symptomatic bacteremia as: • 1 per 16 patients • 1 per 350 transfusions • 1 per 2,100 platelet units • UK SHOT data reported three deaths in UK between 1996 and 1999 as a result of bacterial contamination Department for Blood Group Serology, University of Vienna, Austria

11. Emerging/Unknown Pathogens • It is impossible to know if and when emerging pathogens will threaten the safety of the blood supply • Impact of previously unknown pathogens is demonstrated through HCV and HIV • New viruses continue to emerge at a rate of every 2–3 years with a potentially damaging virus transmitted through blood every 5 years Department for Blood Group Serology, University of Vienna, Austria

12. Window Period (False Negative) • NAT/PCR testing has significantly reduced the window period but it still exists • Collection of blood during the window period is likely the most important source of residual HIV infections Department for Blood Group Serology, University of Vienna, Austria

13. Current Risk of Transfusion Transmitted Virus Department for Blood Group Serology, University of Vienna, Austria

14. Known Pathogens • There are pathogens that are known, but not routinely screened for • Transmission of parasites by transfusion, although currently rare in developed countries, does occur • Donor demographics may bring change in risk level of transfusion-transmitted agents • Example: Incidence of malaria and Chagas’ disease seems to be increasing • Travel to endemic areas increasing • Climate changes • Immigration Department for Blood Group Serology, University of Vienna, Austria

15. Leukocytes • Known to transmit cell-associated infectious agents (e.g. CMV, EBV) • Cause non-hemolytic febrile transfusion reactions (NHFTRs) • Can lead to transfusion-related immunomodulation • Transfusion-associated GvHD (TA GvHD) • Alloimmunization to donor HLA antigens • Limitations of leukodepletion • Pre-storage leukodepletion may not eliminate risk of transfusion-associated GvHD • Degree of leukodepletion not equivalent between leukocyte subsets Department for Blood Group Serology, University of Vienna, Austria

16. Advantages in Prospective Pathogen Inactivation • Inactivates bacteria • The highest risk of contamination • Has the potential to inactivate unknown or emerging pathogens • Reduces many of the risks still inherent in blood transfusions • Window period/false negative risk • Donor selection risk • Inactivates viruses and parasites • Including many of those that are not screened for • Inactivates contaminating donor leukocytes • Important for prevention of TA GvHD Department for Blood Group Serology, University of Vienna, Austria

17. Department for Blood Group Serology, University of Vienna, Austria

18. Department for Blood Group Serology, University of Vienna, Austria

19. pathogen inactivation - pi Techniques psoralen S59: platelets (PLT) and plasma • Cerus/Baxter technology: HelinxR • amino-alkylated psoralen S59 • needs UVA light • photochemical reaction with nucleic acids: intercalation of S59 with pyrimidine base residues, upon UVA: covalent monoadduct, then formation of di-adduct causing cross-linkage Department for Blood Group Serology, University of Vienna, Austria

20. pathogen inactivation - pi Techniques Riboflavin:platelets (PLT) and plasma • Gambro/Cobe BCT technology • Vitamin B2 • needs visible light • photochemical reaction with nucleic acids: cross-linkage by direct sensitiser-substrate interaction based on non-singlet oxygen oxidation of guanosine residues Department for Blood Group Serology, University of Vienna, Austria

21. pathogen inactivation - pi Techniques methylene blue MB: plasma • Springe-DRK, ZLB/SRK, SNBTS,... • phenothiazine • needs UV light • complex reactions involving: - type I reaction (reactive free radicals) - type II reaction (singlet molecular oxygen) • concerns about possible mutageneicity Department for Blood Group Serology, University of Vienna, Austria

22. pathogen inactivation - pi Techniques solvent-detergent SD: plasma • EFS-Bordeaux, Octapharma,Vitex,... • uses two compounds: detergent and solvent • does not need light • very similar to technique used in stable plasma derivatives • needs pooling of plasmas: from 25 to 2000 donations • only inactivates lipid enveloped viruses • affects some plasma proteins (F.VIII, PS, alpha2-antitrypsin) Department for Blood Group Serology, University of Vienna, Austria

23. pathogen inactivation - pi Techniques FRALE S303:Red Blood Cells (RBC) • Cerus/Baxter technology: HelinxR • novel compounds: frangible anchor-linker-effector • does not need light • in principle action similar to S59 (cross-linking of NA) • photochemical reaction with nucleic acids: intercalation of anchor, effector favorably oriented to react with nucleic acids, both being connected by linker; after cross-linking, linker breaks up, effector separates from anchor • effective on all pathogens requiring DNA or RNA Department for Blood Group Serology, University of Vienna, Austria

24. pathogen inactivation - pi Techniques Inactine:Red Blood Cells (RBC) and plasma • V.I. Technologies - Vitex / Pall technology • ethylene imines, PEN110 • does not need light (external energy source), 6 hours at RT • low molecular electrophilic reagents • bind selectively to guanine residues and modify irreversibly nucleic acids • inactivate lipid enveloped viruses (?? as well as non-enveloped viruses); id. for lymphocytes Department for Blood Group Serology, University of Vienna, Austria

25. Helinx Technology for Pathogen Inactivation • Pathogens and leukocytes depend on nucleic acids for replication and/or function • Helinx technology targets and attacks nucleic acids to prevent replication and inhibit function • Helinx technology comprises two compounds: • Amotosalen for platelets & plasma • Formerly called S-59 • S-303 for red cells Department for Blood Group Serology, University of Vienna, Austria

26. Helinx Technology for Pathogen Inactivation • Over one hundred compounds were developed in order to achieve the following optimal properties: • Permanent pathogen and leukocyte inactivation • High safety margins for patients • Retention of therapeutic function of blood cells • Different compounds were chosen for platelets/plasma and red blood cells because of different biological characteristics of blood components • Technology was specifically designed for blood centers’ lab processes Department for Blood Group Serology, University of Vienna, Austria

27. Nucleic Acids Must “UnZip”During Pathogen Replication Replication of Nucleic Acids and Pathogen DNA / RNA Strand Separation Department for Blood Group Serology, University of Vienna, Austria

28. Amotosalen hydrochloride is the formal name of the Helinx compound used for INTERCEPT platelet and plasma inactivation Formerly called S-59 S-303 is the name of the compound used for INTERCEPT red blood cell inactivation We have applied for the formal name for S-303 Helinx Compounds Department for Blood Group Serology, University of Vienna, Austria

29. Amotosalen Mechanism of Action UVA Illumination Amotosalen DNA or RNAof pathogen Docking Permanent Crosslinking Department for Blood Group Serology, University of Vienna, Austria

30. S-303 Mechanism of Action Unreactive By-product S-303 Physiologic pH DNA or RNAof pathogen Docking & Permanent Crosslinking Department for Blood Group Serology, University of Vienna, Austria

31. Helinx Treatment Locks Nucleic Acid and Prevents Replication DNA / RNA No Strand Separation No Replication of Nucleic Acids or Pathogens Department for Blood Group Serology, University of Vienna, Austria

32. Helinx Permanently Crosslinks Both Single- and Double-Stranded Nucleic Acids Helical Regions Single-strandedDNA or RNA Double-strandedDNA or RNA Department for Blood Group Serology, University of Vienna, Austria

33. INTERCEPT Blood SystemProcessing Steps Platelets Plasma Red Cells Red Blood Cell Collection (with additive) Platelet Collection (with InterSol Solution) Collection (synthetic medium) Plasma Collection Addition of Helinx compound Amotosalen HCl Amotosalen HCl S-303 pH (upon addition) Activation UVA Illumination UVA Illumination Reduction of residual compound Yes Yes Yes Storage (conditions) Standard (Room temp, 5 days) Standard (Frozen, to 1 year) Standard (Refrig, to 35 days) Department for Blood Group Serology, University of Vienna, Austria

34. Pooling of Buffy Coat Plateletswith InterSol Solution InterSol Solution (Platelet Additive Solution) Pooling Container Leuko-depletion Filter Buffy Coat Platelets PL2410 (non PVC) Storage Container Octopus Pooling Set Department for Blood Group Serology, University of Vienna, Austria

35. INTERCEPT Blood System for Platelets Buffy Coat Preparation Buffy Coat Platelets InterSol Solution Pooling Container Leukodepletion Filter 1 2 3 4 Whole Blood Separation Pooling and Addition of InterSol Solution Centrifugation to Pellet Red Blood Cell Separation of Platelets Department for Blood Group Serology, University of Vienna, Austria

36. INTERCEPT Blood SystemAmicus Platelet Preparation Amicus enables theautomatic collection ofone or more therapeutic dose(s)of SDP in 35% plasmaand 65% InterSol Solution. Department for Blood Group Serology, University of Vienna, Austria

37. INTERCEPT Platelet System Department for Blood Group Serology, University of Vienna, Austria

38. INTERCEPT Blood System for Platelets Process Timeline Start Processing Illuminate Removal & Storage Incubate plateletson the CAD for a minimum of 4 hours: SDP6 hours: buffy coat platelets Withinone day of collection 3 J/cm2for~3-6 minutes 1 2 3 Department for Blood Group Serology, University of Vienna, Austria

39. INTERCEPT Blood System for Platelets:Inactivation Data Department for Blood Group Serology, University of Vienna, Austria

40. Inactivation of Currently Tested Pathogens Log Reduction * Tested in animal infection assays Department for Blood Group Serology, University of Vienna, Austria

41. Inactivation of Other Viruses Log Reduction * Tested in animal infection assays ** 35% plasma Department for Blood Group Serology, University of Vienna, Austria

42. Inactivation of Gram-Negative Aerobic Bacteria Log Reduction Department for Blood Group Serology, University of Vienna, Austria

43. Inactivation of Gram-Positive Aerobic Bacteria Log Reduction Department for Blood Group Serology, University of Vienna, Austria

44. Inactivation of Gram-Positive Anaerobic Bacteria Log Reduction * Facultative anaerobes Department for Blood Group Serology, University of Vienna, Austria

45. Inactivation of Protozoa Log Reduction * Used a prototype FRALE compound S-138. Department for Blood Group Serology, University of Vienna, Austria

46. Inactivation of Leukocytes * TUV / IMB approved claims are highlighted Department for Blood Group Serology, University of Vienna, Austria

47. Comparison: T-cell InactivationINTERCEPT vs. Gamma Department for Blood Group Serology, University of Vienna, Austria

48. Pathogens routinely tested in blood centers Pathogens for which there is no screening Broad spectrum aerobic bacteria Leukocytes HTLV-I, HTLV-II and Syphilis agent Protozoa Anaerobic bacteria Summary of Pathogen Inactivation TUV/IMB Approved Inactivation Claims Additional Inactivation Data / Future Claims Department for Blood Group Serology, University of Vienna, Austria

49. Acute (single-dose) Repeated-dose Subchronic Pharmacokinetics Reproductive Genotoxicity Carcinogenicity Safety pharmacology Phototoxicity Vein irritation INTERCEPT Blood System for Platelets: Amotosalen Toxicity Tests Performed Department for Blood Group Serology, University of Vienna, Austria

50. Toxicology Conclusion • The results of these toxicology studies strongly support the safety of the INTERCEPT Blood System for platelets • High Safety Margin demonstrated negligible risk of: • Genotoxicity • Carcinogenicity • Phototoxicity • Reproductive toxicity • Treatment of platelets with this new method of pathogen inactivation is not associated with any significant risk for the patient Department for Blood Group Serology, University of Vienna, Austria