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Bacterial Endotoxin Test (BET)

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A practicle procedure of Bacterial endotoxin test carried in pharmaceutical industries.

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Bacterial Endotoxin Test (BET)

  1. 1. Bacterial Endotoxin Test SHRIVARDHAN DHEEMAN M.Sc., Microbiology Gurukul Kangri University, Haridwar, U.K. shrivardhandheeman@rediffmail.com www.sites.google.com/site/shrivardhandheeman3/24/2013 1
  2. 2. A presentation on Bacterial Endotoxin Test A part of project report on Analytical Microbiology for Quality Measurement of Pharmaceutical Product in Industry For partial fulfillment of the requirement for the award of M.Sc., Microbiology Submitted 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 Pal Sr. Microbiologist (Executive) Eurolife Healthcare Pvt. Ltd., Roorkee3/24/2013 2
  3. 3. 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 Test3/24/2013 3
  4. 4. 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 cytokines3/24/2013 4
  5. 5. 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 effects3/24/2013 5
  6. 6. Endotoxin and Exotoxin Endotoxin Exotoxin• Lipopolysaccharide complex on the • Made of two component (A and B) outer membrane; lipid A portion is • Cause botulism, diptheria, tetanus toxic. etc. disease.• Cause meningococcemia, fever, death • Highly variable effect on host of etc. different toxin.• Similar effect of all toxin. • Heat sensitive and inactivated at 60-• Heat stable to 250ºC 80ºC• Part of outer membrane of gram • Excreted outside the living cell negative bacteria. • Produced by both gram positive and• Found in only gram negative bacteria; gram negative bacteria. released on bacterial death and some liberated during growth.3/24/2013 6
  7. 7. 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)3/24/2013 7
  8. 8. 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.3/24/2013 8
  9. 9. 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.3/24/2013 9
  10. 10. Endotoxin Sample Proclotting Coagulogen enzyme Gel-clot (insoluble) (Inactive) (Active) Ca++ Procoagulogen 103/24/2013
  11. 11. 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.3/24/2013 11
  12. 12. 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 IV3/24/2013 12
  13. 13. 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).3/24/2013 13
  14. 14. 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.3/24/2013 14
  15. 15. CSE LRW PPC PPC PTC PTC -R1 -R2 -R1 -R2 Product 50 µl product sample 100 µl LRW 50 µl LRW 50 µl CSE NPC NPC NTC NTC 50 µl LRW LAL -R1 -R2 -R1 -R2 100 µl LAL reagent3/24/2013 15
  16. 16. 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.3/24/2013 16
  17. 17. Thank You3/24/2013 17
  18. 18. 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.3/24/2013 18
  19. 19. 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)3/24/2013 19
  20. 20. 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)3/24/2013 20
  21. 21. 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. Endotoxin Limit x Product Concentration MVD = ------------------------------------------------------------------ Lysate sensitivity• Product: Metronidazole IV• Endotoxin limit: 0.5 EU/ml• Product Concentration: 0.35 mg/ml• Lysate sensitivity: 0.125 EU/ml• Calculation: 5 x 0.35 MVD = -------------- 0.125 MVD = 143/24/2013 21
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