1. 19 September 2006 Gaithersburg, MD David Peretz M.Sc., D.Sc. Senior Scientist CHIRON Submission To FDA TSE Advisory Committee Meeting

2. 2 TSEAC 19 Sep 06 Review vCJD assay development progress Review assay performance data Review proposed validation process Presentation Outline

3. 3 TSEAC 19 Sep 06 Issues and Implications…  BiologyUnknown levels and prevalence of prions in vCJD donations  Disease modelsQuestionable relevance of rodent models  Sample Availability< 30 human vCJD samples available, and small volumes  Confirmation Test None available in vitro  Sensitivity goal Nucleic acid test model not applicable  IncidenceDeclining incidence raises question about continued testing  RealismAtypical infectious disease test Challenges in Assay Development

4. 4 TSEAC 19 Sep 06 Development Assumptions and Rationale Analyte Specificity Assay was developed to detect human PrP Sc presuming this to be both the cause and biochemical marker of the disease (Prusiner et al. Science 1982, Bueler et al. Cell 1993, Legname et al. Science 2004) Assay Sensitivity Sensitivity requirements based on rodent studies, human transmission to primates and transgenic mice, using the following criteria: (Bolton et al. Arch. Biochem. Biophes. 1987, Taguchi et al. Am. J. Patho. 2003, Brown et al. J. Lab. Clin. Med. 2001) 1LD 50 = 0.1pg = 4.3 attomole Syrian hamster PrP Sc 1LD 50 = 0.1-10 nl 10% CJD brain homogenate Initial Analytical Studies Spiking experiments with animal and human tissues into plasma Detection limit of endogenously animal infected blood Clinical Validation Studies Establish clinical sensitivity and specificity Confirm with an assay which is as sensitive

5. 5 TSEAC 19 Sep 06 Assay Format Transfer Supernatant Dissociation and ConditionCapture PrP Sc in plasma ELISA - CaptureELISA - Detection Level of Selection > 1000 fold

6. 6 TSEAC 19 Sep 06 CHIRON Analytical Sensitivity for Capture and Detection of vCJD Human PrP Sc Spiked in plasma Complete AssayrPrP ELISA alone Performance Exceeds Benchmarks and Approaches the Limit of Detection Required for Blood Screening 1pg PrP=43amol

7. 7 TSEAC 19 Sep 06 Initial Specificity of Prion Assay on Normal Human Plasma Samples

8. 8 TSEAC 19 Sep 06 Proposed Pre-Clinical Confirmation Process 1.PSR-1 Selects PrP Sc and Infectivity  Pull-down material is PK resistant, demonstrated  Pull-down material is infectious, ongoing study 2.Proof of Principle  Detection of PrP Sc in sheep plasma, ongoing study  100 disease samples  100 Australian negatives 3.Biology of PrP Sc in Blood  Time course in Syrian hamster, ongoing study  300 animals

9. 9 TSEAC 19 Sep 06 Proposed Clinical Validation Process 1.Clinical Specificity a.Donor samples 5000 UK (high prevalence) 5000 US (low prevalence) b.Hospitalized patients 100 non-neurodegenerative (non-ND) patients 100 ND patients c.Interfering substances Bilirubin, Rheumatoid factor, Human anti-mouse antibodies, etc 2. Clinical Sensitivity All available vCJD plasma samples >40 Symptomatic sCJD plasma samples 3. Validation Commercial validated assay with similar/better sensitivity Research assay with similar/better sensitivity

10. 10 TSEAC 19 Sep 06 Recommendation/Proposal With currently available data, rodent infectivity studies appear unlikely to add value, given: 1.Persuasive evidence for transmission of vCJD by blood transfusion 2.Proven correlation between PrP Sc and infectivity in multiple tissues 3.The variability of prion biology between species  PrP Sc levels vary (Brown et al. J. Lab. Clin. Med. 2001)  Levels of infectivity vary (Brown et al. J. Lab. Clin. Med. 2001)  Incubation period can exceed life span (Race et al. J. Virol. 2001) 4.An assay which reliably demonstrated PrP Sc detection in disease progression models 5.An assay which was specific in separating vCJD positives from normal human PrP 6.An assay with surrogate specificity validated with a confirmatory test for PrP Sc Since assay validation will require defined standards: 1.Encourages the allocation of government funding to support generation of surrogate reference materials and a confirmatory assay 2.Surrogate reference material could be blood derived from a time-course in animals 3.Confirmatory assay could be WB or PMCA or cell culture or another technique 4.Intent is for manufacturers to calibrate their assays against the confirmatory assay

11. 11 TSEAC 19 Sep 06 David Peretz, MSc, DSc, Senior Scientist, R&D W. Andrew Heaton, MD, VP/Chief Medical Officer Alisha J. McReynolds, Associate, Regulatory Affairs Rainer Ziermann, PhD, Director, Scientific Affairs The CHIRON Team