1. In the name of God 1

2. Summer School Influenza Unit, Pasteur Institute of Iran summer 2011 2

3. Endotoxin Detection by LAL for Parenteral Products 3 by Ali Torabi & Seyedeh Marzieh Hosseini Seyedeh Marzieh Hosseini

4. Summer School Ali Torabi- influenza Unit, Pasteur Institute of Iran 2011 summer 4 Principal Microbiology Tests STERILITY: Detection of live microorganisms PYROGEN: Detection of cell-wall fragments from Gram(-) bacteria. History of a product’s exposure to GNB Pasteur Institute Of Iran

5. Summer School Ali Torabi- influenza Unit, Pasteur Institute of Iran 2011 summer 5 Endotoxins and Pyrogens Pyrogens are fever-inducing agents in humans and animals include endotoxin, gram + cell debris, fungi Endotoxins are components from the outer membrane of gram-negative bacteria Pasteur Institute Of Iran

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7. Summer School Ali Torabi- influenza Unit, Pasteur Institute of Iran 2011 summer 7 Endotoxin Endotoxin - Component from outer membrane of gram-negative bacteria  Environmental (unpurified) endotoxin: complex of protein, carbohydrate & lipid  Purified endotoxin: lipopolysaccharide molecule of polysaccharide and lipid a, without protein Pasteur Institute Of Iran

8. Summer School Ali Torabi- influenza Unit, Pasteur Institute of Iran 2011 summer 8 Purified Endotoxin - Lipopolysaccharide Passes through sterilizing membrane filters Lipid A region causes LAL reaction and potent pharmacological activities associated with endotoxin poorly dispersible in water pyrogenicity correlates with LAL assays Pasteur Institute Of Iran

9. Summer School Ali Torabi- influenza Unit, Pasteur Institute of Iran 2011 summer 9 Why Test for Endotoxins? To protect against adverse reactions (sepsis) in humans and animals FDA and USP guidelines require final product testing on all parenterals and medical devices To safeguard against diminished effectiveness of a product due to endotoxin Pasteur Institute Of Iran

10. Summer School Ali Torabi- influenza Unit, Pasteur Institute of Iran 2011 summer 10 Endotoxicity ENDOTOXIN CAUSES MYELOID TISSUE TO RELEASE INFLAMMATORY MEDIATORS CYTOKINES INDUCE A VARIETY OF TISSUE DAMAGE SHOCK and MULTIPLE ORGAN DYSFUNCTION MAY OCCUR Pasteur Institute Of Iran

11. Summer School Ali Torabi- influenza Unit, Pasteur Institute of Iran 2011 summer 11 How to test for Endotoxins? Rabbit Pyrogen Test FD approved in 1941 LAL / TAL Test gel clot LAL approved in 1973 kinetic LAL in 1987 Cell based assays In Vitro pyrogen assay Pasteur Institute Of Iran

12. Summer School Ali Torabi- influenza Unit, Pasteur Institute of Iran 2011 summer 12 Rabbit Pyrogen Test Measure the change in temperature of 3 rabbits, after intravenous injection of the test solution. Pasteur Institute Of Iran

13. Summer School Ali Torabi- influenza Unit, Pasteur Institute of Iran 2011 summer 13 LAL Limulus - genus of Horseshoe Crab Limulus - genus of Horseshoe Crab Amoebocyte - blood cells Amoebocyte - blood cells Lysate - blood extract Lysate - blood extract LAL is a FDA regulated biological that reacts chemically in the presence of gram negative cell debris. This reaction can be measured and quantified. Pasteur Institute Of Iran

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23. Summer School Ali Torabi- influenza Unit, Pasteur Institute of Iran 2011 summer 23 LAL Discoveries by Bang and Levin Described role of endotoxin in coagulation of Limulus blood Prepared Endotoxin - responsive lysate from Amoebocytes Pasteur Institute Of Iran

24. Summer School Ali Torabi- influenza Unit, Pasteur Institute of Iran 2011 summer 24 LAL Assay LAL is > 100 times more sensitive, specific and accurate Economy of LAL permits more testing Tolerance of rabbits to endotoxin is a concern for FDA LAL activity correlates with pyrogenicity Pasteur Institute Of Iran

25. Summer School Ali Torabi- influenza Unit, Pasteur Institute of Iran 2011 summer 25 History of LAL Regulations 1941 Rabbit Pyrogen test included in USP 1973 FDA announced LAL as a biological product FDA described conditions for use of LAL as end-product test for human biological & medical devices 1980 Federal register drafted guidelines for use of LAL test for end-product testing of human & animal injectable drug products Pasteur Institute Of Iran

26. Summer School Ali Torabi- influenza Unit, Pasteur Institute of Iran 2011 summer 26 History of LAL Regulations 1983 Federal Register lists final guidelines on LAL testing including chrom and turb 1987 USFDA LAL - Test Guideline 1991 Interim Guidance - Kinetic LAL Test 1998 European Pharmacopoeia BET 2000 Harmonized BET 2011 USP 30 Bacterial Endotoxin Test Pasteur Institute Of Iran

27. Summer School Ali Torabi- influenza Unit, Pasteur Institute of Iran 2011 summer 27 Other Regulatory Documents USP monographs Manufacturer’s Package Insert Internal SOP AAMI guidelines other Pharmacopoeia Pasteur Institute Of Iran

28. Summer School Ali Torabi- influenza Unit, Pasteur Institute of Iran 2011 summer 28 New Harmonized BET First Microbiological test harmonized by ICH Allows use of endotoxin-specific LAL must test for glucans if suspected in your sample Includes kinetic LAL methods Pasteur Institut Of Iran

29. Gel Clot & Kinetics – The Basics 29 Pasteur Institute Of Iran

30. 30 Clotting Reaction For Activated LAL Endotoxin  - Glucan Pro-Clotting Enzymes Clotting Enzyme Substrate CoagulogenCoagulin Gel M ++ pH=7.2 Pasteur Institute Of Iran

31. Summer School Ali Torabi- influenza Unit, Pasteur Institute of Iran 2011 summer 31 LAL Testing Gel Clot(Limit Test) Gel Clot(Semi-Quantitative) Endpoint Chromogenic Endpoint Turbidimetric Kinetic Chromogenic Kinetic Turbidimetric Pasteur Institute Of Iran

32. Summer School Ali Torabi- influenza Unit, Pasteur Institute of Iran 2011 summer 32 Summary of Gel Clot Test Endpoint sought by 180 inversion of sample tube Positive = Firm Gel Negative = Flow down side of tube Pasteur Institute Of Iran

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34. 34 Gel Clot Reagent In general In general  Lowest reagent cost  Simple reporting (+-)  Found in all Pharmacopoeia  Good stability  Simple technique Pasteur Institute Of Iran

35. Summer School Ali Torabi- influenza Unit, Pasteur Institute of Iran 2011 summer 35 Sources of gel clot test problems Use of wrong EU/ng ratio use of improper reaction or dilution tubes Incubating temperature too hot or too cold Improper dilutions Improper handling of lysate Pasteur Institute Of Iran

36. Summer School Ali Torabi- influenza Unit, Pasteur Institute of Iran 2011 summer 36 Gel Clot Disadvantages Limits test Least sensitive Most time consuming Poor trending options Variability, subjectivity Pasteur Institute Of Iran

37. Summer School Ali Torabi- influenza Unit, Pasteur Institute of Iran 2011 summer 37 LAL and Glucans No current LAL on the market is totally unreactive to glucans Reactivity tied to extraction method Variability between lots Best way to test today is to use glucan blocker Pasteur Institute Of Iran

38. Summer School Ali Torabi- influenza Unit, Pasteur Institute of Iran 2011 summer 38 LAL - Test Interference Inhibition: Recovery < Expected Amount Enhancement: Recovery > Expected Amount Non-interference: Recovery = Expected Amount Pasteur Institute Of Iran

39. Summer School Ali Torabi- influenza Unit, Pasteur Institute of Iran 2011 summer 39 Calculate EL, MVD, MVC Make sample dilutions between undiluted and MVD Screen these dilutions to determine non- interfering test concentration Once a non-interfering sample dilution is determined perform validation on three lots of this product Product Screen (Inh / Enh) Pasteur Institute Of Iran

40. Summer School Ali Torabi- influenza Unit, Pasteur Institute of Iran 2011 summer 40 Measure and document pH of sample + LAL  If no pH adjustment is made then, the validation data serves as documentation for routine testing, and pH measurements will not have to be performed routinely  If pH adjustments are made then routine testing should include pH measurement and documentation. Product Screen (Inh / Enh) Pasteur Institute Of Iran

41. 41 Eliminating Causes Of Interference PROBLEM pH Endotoxin loss Mg depletion LAL-RM SOLUTION Buffered LAL Reagent In-plate spiking Permissible Dilution Endotoxin-Specific buffer Pasteur Institute Of Iran

42. Summer School Ali Torabi- influenza Unit, Pasteur Institute of Iran 2011 summer 42 Current Trends in LAL Testing Continued move toward kinetic testing – over 55% of all LAL tests performed today Compliant software Database trending Robotics In-vitro pyrogen test Pasteur Institute Of Iran

43. LAL Applications Institute Pasteur Institute Of Iran 43

44. Summer School Ali Torabi- influenza Unit, Pasteur Institute of Iran 2011 summer 44 LAL Applications Pharmaceuticals Medical Devices Water testing Dialysis testing Cell culture Pasteur Institute Of Iran

45. Summer School Ali Torabi- influenza Unit, Pasteur Institute of Iran 2011 summer Thanks 45