Bacterial Endotoxin Test

SHRIVARDHAN DHEEMAN

M.Sc., Microbiology

Gurukul Kangri University,

Haridwar, U.K.

shrivardhandheeman@rediffmail.com

www.sites.google.com/site/shrivardhandheeman

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A presentation on

Bacterial Endotoxin TestA part of project report on

Analytical Microbiology for Quality Measurement of Pharmaceutical Product in

IndustryFor partial fulfillment of the requirement for the award of

M.Sc., MicrobiologySubmitted to

Prof. G.P. Gupta (H.O.D)Department of Botany and Microbiology

Gurukul Kangri University, Haridwar, U.K. (India)Under Supervision of

Mr. Roki PalSr. Microbiologist (Executive)

Eurolife Healthcare Pvt. Ltd., Roorkee

Bacterial Endotoxin Test

• We will understand:• What is bacterial endotoxin?• Which kind of bacteria produces endotoxin?• What difference between endotoxin and exotoxin?• How pharmaceutical product contaminate with endotoxin?• Why it is require to test?

• We will elaborate:• Principle of bacterial endotoxin test• Bacterial Endotoxin Test

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Bacterial Endotoxin• The term endotoxin was coined by Richard Friedrich

Johannes Pfeiffer.• He considered a toxin kept "within" the bacterial cell and to

be released only after destruction of the bacterial cell wall.• The term endotoxin is used synonymously with the term

lipopolysaccharide.• The key effects of endotoxins on vertebrates are mediated by

their interaction with specific receptors on immune cells such as monocytes, macrophages, dendritic cells, and others. Upon challenge with endotoxin, these cells form a broad spectrum of immune mediators such as cytokines

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Bacteria Produce Endotoxin • Specially Gram negative bacteria produce endotoxin

(pyrogen).(exceptionally Bacillus thuringeinsis, a gram positive bacteria produce delta toxin as endotoxin.)

• The example of endotoxin are lipopolysaccharide (LPS) and O antigen (O specific side chain) found on the outer membrane of various Gram negative bacteria.

• LPS consists of a polysaccharide (sugar) chain and a lipid moiety, known as lipid A, which is responsible for the toxic effects

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Endotoxin and Exotoxin

Endotoxin• Lipopolysaccharide complex on the

outer membrane; lipid A portion is toxic.

• Cause meningococcemia, fever, death etc.

• Similar effect of all toxin.• Heat stable to 250ºC• Part of outer membrane of gram

negative bacteria.• Found in only gram negative bacteria;

released on bacterial death and some liberated during growth.

Exotoxin• Made of two component (A and B)• Cause botulism, diptheria, tetanus

etc. disease.• Highly variable effect on host of

different toxin.• Heat sensitive and inactivated at 60-

80ºC• Excreted outside the living cell• Produced by both gram positive and

gram negative bacteria.

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Contamination of pharmaceutical product with endotoxin

• During water purification for WFI• During production• During DNA isolation (Vaccine production)• During toxoid isolation(Vaccine production)• By pyrogenated vials (I.V./I.M./pediateric)

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Why we test?

• As a essential test for quality control of pharmaceutical product.

• It will be fatal if drug administrated by human.• Monitor product contamination during

handling and processing.• Complies the product standard as GMP, WHO,

HACCP & ISO regulation.

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Principle of bacterial endotoxin test

• The test is based on the observation that when a endotoxin contacts clot protein from circulating amoebocyte of horse shoe crab (Limulus) a gel clot forms.

• The preclotting enzyme activated by bacterial endotoxin (lipopolysaccharide) and calcium to form active clotting enzyme.

• Active clotting enzyme that catalyzes the cleavage of procoagulogen into polypeptide subunit (coagulogen).

• The subunit join by disulfide bond to form a gel-clot. Spectrophotometery is then used to measure the precipitated by the lysate.

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Endotoxin Sample

Proclotting enzyme

(Inactive)

Proclotting enzyme (Active)

Procoagulogen

Ca++

Coagulogen(insoluble)Gel-clot

Bacterial Endotoxin Test• The test for bacterial endotoxin is used to detect or quantify

endotoxin of gram negative bacterial origin using amoebocyte lysate from horseshoe crab (Limulus polyphemus).

• There are three general technique for this test among which one is most essentially accepted.– Gel clot technique: based on gel formation.– Turbidimetric method: based on development of turbidity after

cleavage of an endogenous substrate.– Chromogenic method: based on the development of color after

cleavage of a synthetic peptide-chromo-gen complex.

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Gel-clot Technique• Apparatus:

– Depyrogenated glassware– Microtitre tubes– Micropipette– Depyrogenated tip

• Equipment:– Heating block

• Material:– CSE: Control Standerd Endotoxin– LAL: Limulus Amoebocyte Lysate– LRW: LAL Reagent Water– Product sample: Metronidazole IV

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Gel-clot Technique

• Procedure:• Set up BET Kit• Switch ON heating block and set on desirable temp.• Reconstitute CSE Vial• Dilute CSE Vial in reference to Lysate sensitivity.• Dilute sample as per MVD.• Add 50 µl product sample (diluted) in four Microtiter tube, two for

Positive Product Control (PPC) and two for Negative Product Control (NPC).

• Add 100 µl LRW in two more Microtitre tube (depyrogenated) for Negative Test Control (NTC).

• Add 50 µl LRW in two more Microtitre tube for Positive Test Contorl (PTC).

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Gel-clot Technique• Add 50 µl CSE form Sol E (0.125 EU/ ml) in both PPC and PTC and

vortex for 15 min.• Add 50 µl LRW in NPC and vortex for 15 min.• Add 100 LAL reagent in each tube.• Palace each tube in heating block and incubate for 1 hrs. at 37˚C• Observe after 1 hrs exactly for best interpretation.

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NPC -R1

NPC -R2

NTC -R1

NTC -R2

CSE LRW

LAL

Product

50 µl CSE

50 µl product sample

100 µl LAL reagent

100 µl LRW

50 µl LRW

50 µl LRW

PPC -R1

PPC -R2

PTC -R1

PTC -R2

Gel-clot Technique

• Observation:• PPC: Gel clot formed• PTC: Gel clot formed• NPC: No gel clot formed• NTC: No gel clot formed

• Interpritation:• PPC and PTC control enriched with CSE. So, it formed gel clot.

• Result:• Product has no detectable endotoxin and complience

USP/IP/BP/IH specification. Endotoxin less then 0.125 EU/ml.05/03/2023 16

ThankYou

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CSE vial Reconstitiution

• Reconstitution of CSE vial:– The CSE vial reconstitute as caliberated against the IU/EU

per vial.– Reconstitution as per direction on the vial with LRW.– Vortex the vial at least 15 min. for well mixing.

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Product Dilution

• Preparation of test solution:– Prepare the sample solution to be test by dissolving or

diluting active substance using LAL reagent water. Dilution factor determined according Maximum Valid Dilution (MVD)

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CSE Dilution

• CSE dilution: (500 EU/ml) (1 ml = 1000 µl)100 µl CSE : 900 µl LRW = 50 EU/ml (Sol A)100 µl Sol A : 900 µl LRW = 5 EU/ ml (Sol B)100 µl Sol B : 900 µl LRW = 0.5 EU/ml (Sol C)100 µl Sol C : 100 µl LRW = 0.25 EU/ml (Sol D)100 µl sol D : 100 µl LRW = 0.125 EU/ml (Sol E)

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Maximum Valid Dilution (MVD)• MVD is the maximum allowable dilution of a sample at which the

endotoxin limit can be determined the MVD using the following formulae.

• Product: Metronidazole IV• Endotoxin limit: 0.5 EU/ml• Product Concentration: 0.35 mg/ml• Lysate sensitivity: 0.125 EU/ml• Calculation:

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Endotoxin Limit x Product ConcentrationMVD = ------------------------------------------------------------------

Lysate sensitivity

5 x 0.35MVD = --------------

0.125

MVD = 14

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