We find gel clot methods in all Pharmacopoeia by 61o2DR8

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									In the name of
     God



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    Summer School
   Influenza Unit,
Pasteur Institute of Iran
              summer 2011




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




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                                                                               Pasteur Institute Of Iran


                   Principal Microbiology Tests

                STERILITY: Detection of live microorganisms
Summer School




                PYROGEN: Detection of cell-wall fragments
                from Gram(-) bacteria. History of a product’s
                exposure to GNB




                  Ali Torabi- influenza Unit, Pasteur Institute of Iran 2011            summer        4
                                                                                 Pasteur Institute Of Iran

                          Endotoxins and Pyrogens

                • Pyrogens are fever-inducing agents in
                  humans and animals
Summer School




                   include endotoxin, gram + cell debris, fungi
                • Endotoxins are components from the
                  outer membrane of gram-negative
                  bacteria



                    Ali Torabi- influenza Unit, Pasteur Institute of Iran 2011           summer        5
Pasteur Institute Of Iran




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                                                                                 Pasteur Institute Of Iran



                                              Endotoxin

                • Endotoxin - Component from outer membrane of
Summer School




                  gram-negative bacteria
                   Environmental (unpurified) endotoxin: complex of protein,
                    carbohydrate & lipid
                   Purified endotoxin: lipopolysaccharide molecule of
                    polysaccharide and lipid a, without protein




                    Ali Torabi- influenza Unit, Pasteur Institute of Iran 2011           summer        7
                                                                               Pasteur Institute Of Iran


                Purified Endotoxin - Lipopolysaccharide

                Passes through sterilizing membrane filters
Summer School




                Lipid A region causes LAL reaction and potent
                pharmacological activities associated with
                endotoxin
                poorly dispersible in water
                pyrogenicity correlates with LAL assays


                  Ali Torabi- influenza Unit, Pasteur Institute of Iran 2011           summer        8
                                                                                  Pasteur Institute Of Iran



                             Why Test for Endotoxins?
                • To protect against adverse reactions (sepsis) in
                  humans and animals
Summer School




                • 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



                     Ali Torabi- influenza Unit, Pasteur Institute of Iran 2011           summer         9
                                                                                 Pasteur Institute Of Iran



                                        Endotoxicity
                • ENDOTOXIN CAUSES MYELOID TISSUE TO RELEASE
Summer School



                  INFLAMMATORY MEDIATORS

                • CYTOKINES INDUCE A VARIETY OF TISSUE
                  DAMAGE

                • SHOCK and MULTIPLE ORGAN DYSFUNCTION MAY
                  OCCUR




                    Ali Torabi- influenza Unit, Pasteur Institute of Iran 2011           summer        10
                                                                                Pasteur Institute Of Iran


                   How to test for Endotoxins?
                • Rabbit Pyrogen Test
                   FD approved in 1941
Summer School




                • LAL / TAL Test
                   gel clot LAL approved in 1973
                   kinetic LAL in 1987
                • Cell based assays
                   In Vitro pyrogen assay




                   Ali Torabi- influenza Unit, Pasteur Institute of Iran 2011           summer       11
                                                                             Pasteur Institute Of Iran


                               Rabbit Pyrogen Test
Summer School




                                                      Measure the change in
                                                      temperature of 3 rabbits,
                                                      after intravenous
                                                      injection
                                                      of the test solution.

                Ali Torabi- influenza Unit, Pasteur Institute of Iran 2011           summer       12
                                                                                Pasteur Institute Of Iran


                                                     LAL
                •Limulus - genus of Horseshoe Crab
                •Amoebocyte - blood cells
Summer School




                •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.

                   Ali Torabi- influenza Unit, Pasteur Institute of Iran 2011           summer       13
Pasteur Institute Of Iran




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                                                                               Pasteur Institute Of Iran



                LAL Discoveries by Bang and Levin
Summer School




                Described role of endotoxin in
                coagulation of Limulus blood

                Prepared Endotoxin - responsive lysate
                from Amoebocytes


                  Ali Torabi- influenza Unit, Pasteur Institute of Iran 2011           summer       23
                                                                                   Pasteur Institute Of Iran


                                              LAL Assay
                • LAL is > 100 times more sensitive,
                  specific and accurate
Summer School




                • Economy of LAL permits more testing
                • Tolerance of rabbits to endotoxin is a
                  concern for FDA
                •   LAL activity correlates with pyrogenicity


                      Ali Torabi- influenza Unit, Pasteur Institute of Iran 2011           summer       24
                                                                                  Pasteur Institute Of Iran


                          History of LAL Regulations
                •   1941                 Rabbit Pyrogen test included in USP
                •   1973
Summer School



                                         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



                     Ali Torabi- influenza Unit, Pasteur Institute of Iran 2011           summer       25
                                                                                Pasteur Institute Of Iran


                      History of LAL Regulations
                • 1983        Federal Register lists final guidelines on
                               LAL testing including chrom and turb
Summer School




                • 1987        USFDA LAL - Test Guideline
                • 1991        Interim Guidance - Kinetic LAL Test
                • 1998          European Pharmacopoeia BET
                • 2000          Harmonized BET
                • 2011          USP 30 Bacterial Endotoxin Test


                   Ali Torabi- influenza Unit, Pasteur Institute of Iran 2011           summer        26
                                                                                 Pasteur Institute Of Iran


                    Other Regulatory Documents

                • USP monographs
Summer School




                • Manufacturer’s Package Insert
                • Internal SOP
                • AAMI guidelines
                • other Pharmacopoeia


                    Ali Torabi- influenza Unit, Pasteur Institute of Iran 2011           summer       27
                                                                                Pasteur Institut Of Iran



                            New Harmonized BET

                • First Microbiological test harmonized by
                  ICH
Summer School




                • Allows use of endotoxin-specific LAL
                   must test for glucans if suspected in your sample

                • Includes kinetic LAL methods




                   Ali Torabi- influenza Unit, Pasteur Institute of Iran 2011         summer       28
                 Pasteur Institute Of Iran




Gel Clot & Kinetics –
     The Basics



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                                                  Pasteur Institute Of Iran




Clotting Reaction For Activated LAL

   Endotoxin                   b - Glucan



   Pro-Clotting                      Clotting
     Enzymes                         Enzyme        Substrate




                                   M++ pH=7.2
                  Coagulogen                    Coagulin Gel


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                                                                                  Pasteur Institute Of Iran


                                           LAL Testing
                •   Gel Clot(Limit Test)
                •   Gel Clot(Semi-Quantitative)
Summer School




                •   Endpoint Chromogenic
                •   Endpoint Turbidimetric
                •   Kinetic Chromogenic
                •   Kinetic Turbidimetric


                     Ali Torabi- influenza Unit, Pasteur Institute of Iran 2011           summer       31
                                                                               Pasteur Institute Of Iran



                     Summary of Gel Clot Test

                Endpoint sought by 180 inversion of
                sample tube
Summer School




                  Positive = Firm Gel
                  Negative = Flow down side of tube




                  Ali Torabi- influenza Unit, Pasteur Institute of Iran 2011           summer       32
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                              Pasteur Institute Of Iran




           Gel Clot Reagent
• In general
     Lowest reagent cost
     Simple reporting (+-)
     Found in all
      Pharmacopoeia
     Good stability
     Simple technique

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                                                                                  Pasteur Institute Of Iran


                   Sources of gel clot test problems
                • Use of wrong EU/ng ratio
                • use of improper reaction or dilution tubes
Summer School




                • Incubating temperature too hot or too cold
                • Improper dilutions
                • Improper handling of lysate




                     Ali Torabi- influenza Unit, Pasteur Institute of Iran 2011           summer       35
                                                                              Pasteur Institute Of Iran


                           Gel Clot Disadvantages

                Limits test
Summer School




                Least sensitive
                Most time consuming
                Poor trending options
                Variability, subjectivity

                 Ali Torabi- influenza Unit, Pasteur Institute of Iran 2011            summer       36
                                                                              Pasteur Institute Of Iran



                                  LAL and Glucans

                No current LAL on the market is totally
                unreactive to glucans
Summer School




                Reactivity tied to extraction method
                Variability between lots
                Best way to test today is to use glucan blocker




                 Ali Torabi- influenza Unit, Pasteur Institute of Iran 2011           summer        37
                                                                                Pasteur Institute Of Iran



                         LAL - Test Interference

                • Inhibition:
Summer School




                                              Recovery < Expected Amount
                • Enhancement:
                             Recovery > Expected Amount
                • Non-interference:
                                               Recovery = Expected Amount

                   Ali Torabi- influenza Unit, Pasteur Institute of Iran 2011           summer       38
                                                                                Pasteur Institute Of Iran



                  Product Screen (Inh / Enh)
                • Calculate EL, MVD, MVC
                • Make sample dilutions between undiluted and
Summer School




                  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


                   Ali Torabi- influenza Unit, Pasteur Institute of Iran 2011           summer       39
                                                                                    Pasteur Institute Of Iran


                   Product Screen (Inh / Enh)
                • Measure and document pH of sample +
                  LAL
Summer School




                     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.




                       Ali Torabi- influenza Unit, Pasteur Institute of Iran 2011               summer          40
                           Pasteur Institute Of Iran




Eliminating Causes Of Interference

PROBLEM          SOLUTION

pH                Buffered LAL Reagent

Endotoxin loss    In-plate spiking

Mg depletion      Permissible Dilution

LAL-RM            Endotoxin-Specific buffer


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                                                                                Pasteur Institute Of Iran

                  Current Trends in LAL Testing

                • Continued move toward kinetic testing
                   – over 55% of all LAL tests performed today
Summer School




                • Compliant software
                • Database trending
                • Robotics
                • In-vitro pyrogen test



                   Ali Torabi- influenza Unit, Pasteur Institute of Iran 2011               summer          42
              Pasteur Institute Of Iran




LAL Applications



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                                                                                  Pasteur Institute Of Iran



                                 LAL Applications

                • Pharmaceuticals
Summer School




                • Medical Devices
                • Water testing
                • Dialysis testing
                • Cell culture

                     Ali Torabi- influenza Unit, Pasteur Institute of Iran 2011           summer       44
Summer School




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

								
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