Laboratory of Immunobiochemistry

1. Laboratory of Immunobiochemistry Allergenic Products Advisory Committee, April 8, 2003

2. Allergenic Products Advisory Committee, April 8, 2003 • Lab overview • Staffing • Lot release • Reference replacement • Operational issues • Sheep serum replacement issues • ISO certification report • Research/regulatory update • Endotoxin studies • Cockroach antigens and antibodies

3. Lab overview • Staffing • Lot release • Reference replacement

4. Principal Investigators • Jay E. Slater, MD, Lab Chief – Supervisory Medical Officer (4) • Ronald Rabin, MD - Senior Staff Fellow (2) • Post Doctoral Fellows • Jonny Finlay, PhD - IRTA (2) • Bo Chi, MD - Visiting Associate (<1)

5. Research Technicians • Albert Gam – Biologist (2) • Mona Febus – Microbiologist (3) • Marc Alston – Biologist (2) • Cherry Valerio – Biologist (2) • Katia Dobrovolskaia – Visiting associate (2)

6. LIB staffing 1998-2003

7. “Routine” regulatory activities • Lot release • Reference distribution • Reference maintenance • semiannual checks • replacement

8. Lot release activities • 357 protocols submitted and reviewed • 1 withdrawn • Reference distribution • 2002: 1978 vials in 107 shipments sent to manufacturers

9. Lot release protocols submitted

10. Reference distribution

11. Laboratory of Immunobiochemistry Operational issues

12. Operational issues • Replacement of cat and ragweed antisera • Transition to ISO compliance

13. Ragweed and cat antisera need to be replaced • S2a cat • Released in 1998 • S6 ragweed • Released in 2000 • Replacement programs for both initiated spring 2002

14. Immunization protocol

15. Immunization protocol Immunization doses plasmapheresis

18. Transmissible spongiform encephalopathies • Animal • scrapie (sheep and goats) • chronic wasting disease (mule deer, elk) • transmissible mink encephalopathy • bovine spongiform encephalopathy • feline spongiform encephalopathy

19. Transmissible spongiform encephalopathies • Human • kuru • Creutzfeldt-Jakob disease • classic sporadic • familial • iatrogenic • new variant (a/w bovine TSE) • Gerstmann-Sträussler-Scheincker • fatal familial insomnia • sporadic fatal insomnia

20. Scrapie • In Europe for >250 years • In US since 1947 >1000 flocks • Vertical transmission • Horizontal transmission, presumably by contamination with placenta and blood during lambing season • Long incubation period • No human transmission

21. Prion polymorphisms associate with different susceptibility, especially at codon 171 • QQ (glutamine/glutamine) susceptible • RR (arginine/arginine) resistant • QR (glutamine/arginine) intermediate

22. Scrapie eradication program • Preclinical testing and surveillance • Live animal test (third eyelid biopsy) • May take months to years to turn positive • Tracking of infected and exposed animals • Cleanup strategies: identify and genotype exposed animals • Destroy QQ exposed • QR or RR exposed are tracked but may be slaughtered for human consumption

23. Not believed to pose any risk to humans • No recognized human transmission in three centuries of exposure in Europe • This serum is safe to use because • No documented transmission to humans • Contact with affected sheep was limited • Not in adjoining pens; >30 feet distant • Normal BSL 2 precautions in place for all work with animal sera • However, in order to maintain a serum reagent that is as safe as possible…

24. Current approach… • Begin immunizing two new sheep • Process plasma from ewes 6747 and 7777 now • Conserve current stocks

25. Current approach… • If new sera are available before we run out, sera from 6747 and 7777 will be saved frozen for possible future use • If we have a shortfall, sera from 6747 and/or 7777 will be used until the new sera are available

26. Current approach… • Advantages • Highest degree of safety • Large supplies for future • Disadvantages • Possibility of two serum switches in short period • Time • Expense

28. CBER Laboratory Quality Management Initiative • Seek ISO-17025 compliance for official product testing • ISO – International Organization for Standardization, Geneva, Switzerland • 17025: “General requirements for the competence of testing and calibration laboratories” (1999) • Set of guidelines for labs that do testing and calibration to show operation of a quality system to assure technical competence and production of valid results

29. CBER Laboratory Quality Management Initiative • Establish policy with Center level Quality Manual • Audit for compliance with ISO-17025 • Obtain Test/Lab Accreditation where appropriate • “Accreditation – successful 3rd party audit

30. Why is CBER becoming ISO compliant? • Establish recognized competence, assure the trust and value of our data and processes • We require the manufacturers’ labs to be in GMP compliance • International efforts at harmonization are attempting to equate GMP and ISO requirements • ISO is an internationally recognized standard

31. Why is CBER becoming ISO compliant? (continued) • Implement Laboratory Quality Management policies and practices for official testing activities • To document a high level of training, competence, and proficiency • To establish a consistent product testing process

32. Elements of ISO compliance • Management of • People • Equipment • Documents • Processes

33. Elements of ISO compliance • Management of People • Defined roles • Appropriate training • Demonstrated proficiency • Initial and ongoing • Authorization process

34. Elements of ISO compliance • Management of Equipment • Program for equipment calibration • Maintenance • Process to assure that only calibrated and maintained equipment is used • Assurance of measurement traceability to accepted standards

35. Elements of ISO compliance • Management of Documents • Includes policies, procedures, specifications, equipment manuals, certifications • Must be reviewed, issued and controlled • Approved and issued prior to use

36. Elements of ISO compliance • Management of Processes • Approval of testing materials • Environmental specifications and monitoring • Handling of samples • Validation and suitability • Handling of data (including non-conforming data) • Corrective action, Preventive action systems • Internal Audits • Management review • Recording and handling complaints

37. CBER’s commitment to implementation of a Lab Quality System • Establishment of CBER Quality Board • Development of Quality Assurance structure within CBER • Appointment of Center Level Quality Manager • Hiring Office quality Managers • Appointment and hiring of Division Quality Coordinators • Preparation of CBER Quality Manual • Purchase of Integrated Quality Management computer software

38. What will this mean to LIB? • No substantive protocol revisions • Documentation • Formatting • Substance • Formal separation of research and regulatory equipment • Internal audits • External audits

39. Timetable (tentative) • Implementation in stages over the next 3 years… Seek accreditation in 2005 • Software operational during 2003 • Policies and Quality Manual issued 2003 • Initiation of compliance audits (ongoing) • Training and process development (ongoing)

40. Laboratory of Immunobiochemistry Research/regulatory update

41. Active research projects • PI Slater • Cockroach allergen standardization • Determination of optimal surrogate test • Depletion analysis of CR extracts • Cockroach IgE combinatorial library • Endotoxin in allergen vaccines • PI Rabin • MDR proteins in T cell activation • RSV responses in human tonsil

42. Publications Patterson ML, Slater JE. Characterization and comparison of commercially available German and American cockroach allergen vaccines. Clin Exp Allergy 2002;32:721-727 Sutherland MF, Drew A, Rolland JM, Slater JE, Suphioglu C, O'Hehir RE. Specific monoclonal antibodies and human IgE show Hev b 5 is an abundant allergen in high protein powdered latex gloves. Clin Exp Allergy 2002;32:583-589 Trivedi B, Valerio C,Slater JE. Endotoxin content of standardized allergen vaccines. J Allergy Clin Immunol 2003 (in press).

43. Publications (reviews) Lockey RF, Slater JE, Esch R. Preparation and standardization of allergen vaccines. In Middleton’s Allergy: Principles and Practice, 6th ed. St. Louis: Mosby (in press).

44. Abstracts Rabin RL, Alston MA, Huang H, Slater JE. Cytokine secretion by activated T cells is dependent on multidrug resistance protein-1 (MRP-1). J Allergy Clin Immunol 2003; 111:S153. Valerio C, Slater JE. The effects of lipopolysaccharide (LPS) on immune responses in C57Bl/6 mice. J Allergy Clin Immunol 2003; 111:S166. Slater JE, Valerio C, Trivedi B. Endotoxin in standardized allergen vaccines. J Allergy Clin Immunol 2003; 111:S243. Finlay WJJ, Rabin RL, Slater JE. Analysis of IgE heavy chain V-gene usage in human tonsil. J Allergy Clin Immunol 2003; 111:S313.

45. Endotoxin content of allergen vaccines • Allergenic extracts are not required to undergo evaluation for the presence of pyrogens (21CFR 610.13(b)) • Prior studies confirmed variable endotoxin content (Siraganian, et al. J Allergy Clin Immunol 1979; 64:526-533)

46. Products exempted in 21 CFR 610.13(b) • Blood products • Horse serum • Bacterial, rickettsial and viral vaccines • Toxoids • Toxins • Allergenic extracts

47. LAL gel-clot method

48. LAL gel-clot method

49. ENDOTOXIN (1,3) b–D- glucan Factor C Act. factor C Factor B Act. factor B Act. factor G Factor G Proclotting enzyme Clotting enzyme Coagulogen Coagulin

50. Endotoxin content of allergen vaccines – possible interference • Non-endotoxin (1,3)--D-glucans may induce clotting by an alternative pathway in the standard LAL assays • Proteases (especially in cat and mite extracts) may also induce clotting