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    This document discusses pyrogens and the innate immune response. It describes endotoxins and non-endotoxin pyrogens, and how they trigger an immune response through cytokines and sentinel cells. Traditional tests for pyrogens like the rabbit pyrogen test and bacterial endotoxin test are outlined, as well as their limitations. The development of recombinant factor C as an alternative to the Limulus amebocyte lysate test is then covered, including cloning, initial studies, and inclusion in pharmacopeias.

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    This document discusses pyrogens and the innate immune response. It describes endotoxins and non-endotoxin pyrogens, and how they trigger an immune response through cytokines and sentinel cells. Traditional tests for pyrogens like the rabbit pyrogen test and bacterial endotoxin test are outlined, as well as their limitations. The development of recombinant factor C as an alternative to the Limulus amebocyte lysate test is then covered, including cloning, initial studies, and inclusion in pharmacopeias.

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    © All Rights Reserved
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    14 views16 pages

    Presentazione Endotossine

    Uploaded by

    See Beck
    This document discusses pyrogens and the innate immune response. It describes endotoxins and non-endotoxin pyrogens, and how they trigger an immune response through cytokines and sentinel cells. Traditional tests for pyrogens like the rabbit pyrogen test and bacterial endotoxin test are outlined, as well as their limitations. The development of recombinant factor C as an alternative to the Limulus amebocyte lysate test is then covered, including cloning, initial studies, and inclusion in pharmacopeias.

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    © All Rights Reserved
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    of 16

    Endotossine

    Storia e sviluppo dei sistemi di rilevamento

    24.06.2021 Renato Picchi


    In collaborazione con
    Pyrogens And the Innate Immune Response

    Connecting Pharmaceutical Knowledge ispe.org


    2
    Pyrogens And the Innate Immune Response

    Pyrogens

    Endotoxins Non-endotoxin
    (LPS) pyrogens (NEPS)

    - Components from - Components from


    Gram-negative bacteria Gram-positive bacteria
    e.g. Lipopolysaccharide (LPS) e.g. Lipteichoic Acid (LTA)
    - Yeast and Mold
    - Virus
    - Particle of the environment
    e.g. Rubber, plastic, organic dust,
    Connecting Pharmaceutical Knowledge ispe.org
    Pyrogens And the Innate Immune Response

    cytokines
    IL-1, IL-6, αTNF
    Connecting Pharmaceutical Knowledge ispe.org
    Pyrogens And the Innate Immune Response
    Exogenous
    Pyrogens

    Endotoxins

    Endogenous
    Pyrogens

    Sentinel Cells
    (Macrophage- TLR4)
    Connecting Pharmaceutical Knowledge ispe.org
    PYROGEN AND
    ENDOTOXIN TESTS
    The Rabbit Pyrogen Test (RPT)
    Limitations
    • Animal based

    • Not quantitative

    • Time consuming and expensive

    • Inability to differentiate pyrogens

    • Can't test all products

    Connecting Pharmaceutical Knowledge ispe.org


    Bacterial Endotoxin Test (BET)

    Connecting Pharmaceutical Knowledge ispe.org


    Understanding Limulus Amebocyte Lysate (LAL)

    1977
    1981

    1980 1986
    Connecting Pharmaceutical Knowledge ispe.org
    LAL Limitations
    Season
    Age LAL is biologically
    Sex
    Health sourced and no two
    Habitat lots will react the
    same
    LAL
    •Factor C
    •Factor B
    ? •Proclotting enzyme
    •Coagulogen
    (Pro)Clotting

    FC
    ?
    FB •Factor G,
    FG •Serine protease
    inhibitors 1, 2, and 3
    LAL Reagent Connecting Pharmaceutical Knowledge ispe.org
    RECOMBINANT FACTOR
    C DEVELOPMENT AND
    INITIAL STUDY
    Cloning and Development

    Connecting Pharmaceutical Knowledge ispe.org


    Factor C is the Specific sensor
    LAL: rFC:
    Endotoxin β-1,3-Glucan Endotoxin rFC = exact copy of
    Factor C produced
    synthetically instead
    Factor C of extraction from the
    Limulus blood.
    Factor G
    Factor B
    Recombinant Factor C

    FC (Pro)Clotting
    (Pro)Clotting

    Simple Reaction
    FB
    ? enzyme rFC without cascade

    FG

    Coagulation Color development Fluorescence development rFC reagent is


    gel clot/turbidimetric chromogenic fluorogenic well defined

    Read-out
    Connecting Pharmaceutical Knowledge Read-out ispe.org
    Pharmacopoeia
    • In June 2012 the FDA added rFC to their “Guidance for industry: Pyrogens and Endotoxin Testing:
    Questions and Answers”

    • In 2018 FDA approved first product using rFC for release

    • 2020 China Pharmacopoeia adds a compendial chapter for rFC

    • USP decides not to add rFC to BET chapter from data (LAL manufacturer study)

    • Jan 2021 chapter 2.6.32 rFC for BET went live

    • No need for method suitability (same as LAL)

    • Product suitability needed including NCID and TIF (same as LAL)

    • Japanese Pharmacopoeia to release new chapter Mid 2021

    Connecting Pharmaceutical Knowledge ispe.org


    For a Better World

    Connecting Pharmaceutical Knowledge ispe.org


    Grazie per
    l’attenzione !

    Connecting Pharmaceutical Knowledge ispe.org 16

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