The document discusses the bacterial endotoxin test, which is used to detect or quantify endotoxins from gram-negative bacteria. It defines endotoxins as lipopolysaccharides found in the outer membrane of gram-negative bacteria that are released upon cell lysis. The test utilizes a gel clot formation reaction that occurs when endotoxins interact with amoebocyte lysate from the horseshoe crab. The document outlines the principle, materials, procedures, controls, and interpretation for the gel clot technique of performing the bacterial endotoxin test on pharmaceutical products to ensure quality standards are met.
Endotoxin Testing is performed to ensure that injectable preparations and medical devices are free from pyrogens and safe for human use.
Pyrogens constitute a heterogeneous group of fever causing substances which comprise both microbial and non-microbial substances. The most potent and most widely known are the endotoxins or lipopolysaccharides (LPS), which are cell wall components of gram-negative bacteria. Gram-positive bacteria are also sources of pyrogens, in particular lipoteichoic acid (LTA), as are particles from yeasts and viruses. Non-microbial pyrogens often emanate from production environments. Small particles of packaging materials are a typical example.
Endotoxin Testing is performed to ensure that injectable preparations and medical devices are free from pyrogens and safe for human use.
Pyrogens constitute a heterogeneous group of fever causing substances which comprise both microbial and non-microbial substances. The most potent and most widely known are the endotoxins or lipopolysaccharides (LPS), which are cell wall components of gram-negative bacteria. Gram-positive bacteria are also sources of pyrogens, in particular lipoteichoic acid (LTA), as are particles from yeasts and viruses. Non-microbial pyrogens often emanate from production environments. Small particles of packaging materials are a typical example.
The Bacterial Endotoxin Test BET is a test to quantify Endotoxin from ‘Gram negative bacteria using ‘amoebocyte lysate' extracted from ‘Limulus polyphemus or Tachypleus tridentatus' i.e. horseshoe crab. Three techniques are used for the confirmation of endotoxins, those are the gel clot technique, the turbidimetric technique, and the chromogenic technique. Endotoxins are toxic complexes which is invariably associated with the cell wall of the Gram negative bacteria irrespective of bacterial pathogenicity. Endotoxins are rarely fatal, but they cause fever and hence Endotoxin carrying bacteria are known as ‘Pyrogen'. The test can be done by any of these three techniques. Among all, the gel clot method is chosen to be superior. The test is carried out in a manner to avoid endotoxins contamination. Mostly bacterial Endotoxin test are performed for the inject able pharmaceutical products, e.g. SWFI Sterile Water for Injection , Normal Saline Sodium Chloride Sterile and other injections like Amikacin sulphate, Ascorbic acid, etc. Endotoxin had been authenticated as inflammatory agent, onset clinical diabetes, obesity, nausea, vomiting, diarrhea, fever, etc. Endotoxins are thus very necessary to get removed out from the inject able pharmaceutical products. Faiz Hashmi | Ankush Thakur ""Bacterial Endotoxin Test by Gel-Clot Method"" Published in International Journal of Trend in Scientific Research and Development (ijtsrd), ISSN: 2456-6470, Volume-3 | Issue-3 , April 2019, URL: https://www.ijtsrd.com/papers/ijtsrd22945.pdf
Paper URL: https://www.ijtsrd.com/pharmacy/biotechnology-/22945/bacterial-endotoxin-test-by-gel-clot-method/faiz-hashmi
This presentation gives an overview of : Validation of microbiological methods , Considering some of the limitations and
Key criteria that may be applicable for assessment.
Definition and Objectives of Lyophilization, Advantages & Disadvantages, Basic Principles of Lyophilization, Steps of Lyophilization,
Main Components of Lyophilizer,
Qualification of Lyophilizer,
Development of Lyophilization cycle, Defects of Lyophilizer.
The Bacterial Endotoxin Test BET is a test to quantify Endotoxin from ‘Gram negative bacteria using ‘amoebocyte lysate' extracted from ‘Limulus polyphemus or Tachypleus tridentatus' i.e. horseshoe crab. Three techniques are used for the confirmation of endotoxins, those are the gel clot technique, the turbidimetric technique, and the chromogenic technique. Endotoxins are toxic complexes which is invariably associated with the cell wall of the Gram negative bacteria irrespective of bacterial pathogenicity. Endotoxins are rarely fatal, but they cause fever and hence Endotoxin carrying bacteria are known as ‘Pyrogen'. The test can be done by any of these three techniques. Among all, the gel clot method is chosen to be superior. The test is carried out in a manner to avoid endotoxins contamination. Mostly bacterial Endotoxin test are performed for the inject able pharmaceutical products, e.g. SWFI Sterile Water for Injection , Normal Saline Sodium Chloride Sterile and other injections like Amikacin sulphate, Ascorbic acid, etc. Endotoxin had been authenticated as inflammatory agent, onset clinical diabetes, obesity, nausea, vomiting, diarrhea, fever, etc. Endotoxins are thus very necessary to get removed out from the inject able pharmaceutical products. Faiz Hashmi | Ankush Thakur ""Bacterial Endotoxin Test by Gel-Clot Method"" Published in International Journal of Trend in Scientific Research and Development (ijtsrd), ISSN: 2456-6470, Volume-3 | Issue-3 , April 2019, URL: https://www.ijtsrd.com/papers/ijtsrd22945.pdf
Paper URL: https://www.ijtsrd.com/pharmacy/biotechnology-/22945/bacterial-endotoxin-test-by-gel-clot-method/faiz-hashmi
This presentation gives an overview of : Validation of microbiological methods , Considering some of the limitations and
Key criteria that may be applicable for assessment.
Definition and Objectives of Lyophilization, Advantages & Disadvantages, Basic Principles of Lyophilization, Steps of Lyophilization,
Main Components of Lyophilizer,
Qualification of Lyophilizer,
Development of Lyophilization cycle, Defects of Lyophilizer.
Parenterals are the sterile preparation that is directly administered into the circulatory system avoiding the enteral route. And these preparation provide rapid onset of action that is why the administered preparation must be safe.
Stability problem arise from microbial contamination of these products so sterility and stability must be ensured for these preparations.
To ensure their sterility and stability, regulations regarding to quality control through pharmacopeial specifications has great importance.
“Comparability of biotherapeutic products following manufacturing process improvement. Quality aspects and comparability issues”
Focuses on improvements in manufacturing processes of biotherapeutics
Advances in in vitro testing for regulatory compliance in the chemical industryDr Carol Barker-Treasure
This presentation, originally shared at the Chemical Hazards Communication Society in 2018, discusses the regulatory status of in vitro safety assessments for REACH, CLP Regulation and Biocidal Products Regulation, gives an overview of current key methods for skin irritation, genotoxicity and acute toxicity, shows how finished mixtures or products can be tested, how to interpret results and barriers to progress towards an entirely animal-free testing strategy.
Defination,test method, steps, principle, designed to demonstrate the presence or absence of extraneous viable contaminating microorganisms in biological parenterals designed for human use
Explore our infographic on 'Essential Metrics for Palliative Care Management' which highlights key performance indicators crucial for enhancing the quality and efficiency of palliative care services.
This visual guide breaks down important metrics across four categories: Patient-Centered Metrics, Care Efficiency Metrics, Quality of Life Metrics, and Staff Metrics. Each section is designed to help healthcare professionals monitor and improve care delivery for patients facing serious illnesses. Understand how to implement these metrics in your palliative care practices for better outcomes and higher satisfaction levels.
One of the most developed cities of India, the city of Chennai is the capital of Tamilnadu and many people from different parts of India come here to earn their bread and butter. Being a metropolitan, the city is filled with towering building and beaches but the sad part as with almost every Indian city
ICH Guidelines for Pharmacovigilance.pdfNEHA GUPTA
The "ICH Guidelines for Pharmacovigilance" PDF provides a comprehensive overview of the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines related to pharmacovigilance. These guidelines aim to ensure that drugs are safe and effective for patients by monitoring and assessing adverse effects, ensuring proper reporting systems, and improving risk management practices. The document is essential for professionals in the pharmaceutical industry, regulatory authorities, and healthcare providers, offering detailed procedures and standards for pharmacovigilance activities to enhance drug safety and protect public health.
The dimensions of healthcare quality refer to various attributes or aspects that define the standard of healthcare services. These dimensions are used to evaluate, measure, and improve the quality of care provided to patients. A comprehensive understanding of these dimensions ensures that healthcare systems can address various aspects of patient care effectively and holistically. Dimensions of Healthcare Quality and Performance of care include the following; Appropriateness, Availability, Competence, Continuity, Effectiveness, Efficiency, Efficacy, Prevention, Respect and Care, Safety as well as Timeliness.
How many patients does case series should have In comparison to case reports.pdfpubrica101
Pubrica’s team of researchers and writers create scientific and medical research articles, which may be important resources for authors and practitioners. Pubrica medical writers assist you in creating and revising the introduction by alerting the reader to gaps in the chosen study subject. Our professionals understand the order in which the hypothesis topic is followed by the broad subject, the issue, and the backdrop.
https://pubrica.com/academy/case-study-or-series/how-many-patients-does-case-series-should-have-in-comparison-to-case-reports/
2. 3/24/2013 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., Roorkee
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 Test
3/24/2013 3
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 cytokines
3/24/2013 4
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 effects
3/24/2013 5
6. 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.
3/24/2013 6
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. 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. 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
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
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. 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
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
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. 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. 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. 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:
3/24/2013 21
Endotoxin Limit x Product Concentration
MVD = ------------------------------------------------------------------
Lysate sensitivity
5 x 0.35
MVD = --------------
0.125
MVD = 14