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Pyrogen testing as per IP, BP & USP
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Pyrogen testing as per IP, BP & USP

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    Pyrogen testing as per IP, BP & USP Pyrogen testing as per IP, BP & USP Presentation Transcript

    • PYROGEN & ENDOTOXIN TESTING COMPARISON AS PER IP, BP & USP PRESENTED BY- SAURAV BHANDARI DEPT. OF QUALITY ASSURANCE
    • • Pyrogens include any substance capable of eliciting a febrile (or fever) response upon injection or infection • Š Endotoxin is a subset of pyrogens that are strictly of gramnegative bacterial origin; they occur (virtually) nowhere else in nature. Lipopolysaccharide (LPS)is a part of endotoxin, or, endotoxin is the natural complex of LPS occurring in the outer layer of the bilayered gram-negative bacterial cell
    • PYROGEN TESTING
    • • The test involves measurement of the rise in body temperature of rabbits following the intravenous injection of a sterile solution of the substance under examination. It is designed for products that can be tolerated by the test rabbit in a dose not exceeding 10 ml per kg injected intravenously within a period of not more than 10 minutes. Test Animals Use healthy, adult rabbits of either sex, preferably of the same variety, weighing not less than 1.5 kg, fed on a complete and balanced diet and not showing loss of body weight during the week preceding the test. House the animals individually in an area of uniform temperature ( 2º), preferably with uniform humidity, and free from disturbances likely to excite them.
    • PRELIMINARY TEST (SHAM TEST)  If animals are used for first time in pyrogen test or have not been used during the two previous weeks, condition them one to three days before testing the substance being examined, by injecting i.v into them 10 ml per kg of body weight of a pyrogen free saline solution.  Carry out the test in a room where there is no risk of disturbance of exciting the animals and in which the room temperature is within 3 of that of the area where the animals are housed or in which the animals have been kept for at least 18 hrs before the test.
    •  With hold food from the animals overnight and until the test is completed; withhold water during the test.  Record the temperature of the animals beginning at least 90 min. before injection and continuing for 3 hrs after injection of the solution being examined.  Any animal showing a temp. variation of 0.6 or more must not be used in the main test.
    • Main Test • Carry out the test using a group of three rabbits. • Preparation of the sample • Dissolve the substance under examination in, or dilute with, pyrogen-free saline solution or other solution prescribed in the monograph. Warm the liquid under examination to approximately 38.5º before injection.
    • PROCEDURE • Inject the solution(38.5º) under examination slowly into the marginal vein of the ear of each rabbit over a period not exceeding 4 minutes, unless otherwise prescribed in the monograph. • Record the temperature of each animal at half-hourly intervals for 3 hours after the injection. • The difference between the “initial temperature” and the “maximum temperature” which is the highest temperature recorded for a rabbit is taken to be its response. The amount of sample to be injected varies according to the preparation under examination and is prescribed in the individual monograph. The volume of injection is not less than 0.5 ml per kg and not more than 10 ml per kg of body weight.
    • TEST ANIMALS ANIMAL Healthy, adult rabbits of either sex Healthy, adult rabbits of either sex Healthy, mature rabbits of either sex BODY WT Not less than 1.5 kg Not less than 1.5 kg Not less than 2-4 kg CONDITION BEFORE TEST ± 2oC within 3o C Not more than ±3° CONDITION FOR ANIMAL TO USE Do not use animals for pyrogen tests more frequently than once every 48 hours Not used (a) during the preceding 3 days or (b) during the preceding 3 weeks unless the material being examined passed the test. Do not use a rabbit for pyrogen testing more frequently than once every 48 hours 0.6oC or more mean rise exceed 1.2 0.6oC or more FAIL TEST APPARATUS PREPARATION Hot air oven at 250oC for Hot air oven at 250oC for Hot air oven at 250oC for 30 minutes or at 200oC for 30 minutes or at 200oC for not less than 30 minutes 1 hour 1 hour ANIMAL IN INSTRUMENT 1 hour before the test 1 hour before the test Not mentioned
    • INTERPRETATION OF RESULTS I.P • If the sum of the responses of the group of three rabbits does not exceed 1.4o C and if the response of individual rabbit is less than 0.6o C, the preparation under examination passes the test • If exceeds continue the test using five other rabbits • If not more than three of the eight rabbits show individual responses of 0.6oC or more, and if the sum of responses of the group of eight rabbits does not exceed 3.7o C, the preparation under examination passes the test
    • INTERPRETATION OF RESULTS U.S.P • If no rabbit shows an individual rise in temperature of 0.5 or more above its respective control temperature, the product meets the requirements for the absence of pyrogens. • If exceeds continue the test using five other rabbits. • If not more than three of the eight rabbits show individual rises in temperature of 0.5 or more and if the sum of the eight individual maximum temperature rises does not exceed 3.3 , the material under examination meets the requirements for the absence of pyrogens.
    • INTERPRETATION OF RESULTS B.P. Number of Rabbits Material passes if summed response doesn’t exceed Material fails if summed response exceeds 3 1.15 2.65 6 2.80 4.3 9 4.45 5.95 12 6.60 6.60
    • LAL (Limulus Amebocyte Lysate)  LAL (Limulus Amebocyte Lysate) test is used for detecting endotoxin  based on clotting reaction of horseshoe crab lysate by endotoxin  The specimen is incubated with LAL of a known senstivity. Formation of a gel clot is positive for endotoxin.  Types of LAL test  Gel Clot  Turbidimetric  Colorimetric  Equipment used in test must be endotoxin free
    • The test is applied using a lysate derived from the hemolymph cells or amoebocytes of the horseshoe crab, Limulus polyphemus. Other species of horseshoe crab namely Tachypleus gigas also yield amoebocyte lysate having similar activity. • The following methods can be used to monitor the endotoxin concentration in a product official in the Pharmacopoeia and to determine whether the product complies with the limit specified in the monograph. • Method A. Gel-Clot Limit Test Method • Method B. Semi-quantitative Gel-Clot Method • Method C. Kinetic Turbidimetric Method • Method D. Kinetic Chromogenic Method • Method E. End-Point Chromogenic Method
    • • Before carrying out the test for endotoxins in the preparation under examination it is necessary to verify • (a) in the case of gel-clot methods, the sensitivity of the lysate; • (b) in the case of quantitative methods, the linearity of the standard curve; • (c) the absence of interfering factors, which inhibit or enhance the reaction or otherwise interfere with the test on the preparation under examination.
    • Special Reagents The Endotoxin Reference Standard (ERS) is the freeze-dried, purified endotoxin of Escherichia coli, which is calibrated in Endotoxin Units (EU) by comparison with the International Standard. The Endotoxin Reference Standard (ERS) or any other suitable preparation the activity of which has been determined in relation to the ERS or the International Standard using a gel-clot or other suitable method is maintained by the Central Drugs Laboratory, Kolkata.
    • Sensitivity of the lysate Confirm the labelled sensitivity of each new batch of lysate prior to use in the test using at least one vial of each batch of lysate. Prepare a series of dilutions of CSE to give concentrations of 2l, l, 0.5l and 0.25l, where l is the labelled sensitivity of the lysate in EU per ml. Perform the test as given under Method on these four standard concentrations in duplicate and include a negative control consisting of water BET.
    • Test for interfering factors There is possibility of interference with the bacterial endotoxins test by certain factors. Dilution of the test preparation with water BET is the easiest method for overcoming inhibition. The allowable dilution level or Maximum Valid Dilution (MVD) is dependent on the concentration of the product, the endotoxin limit for the product and the lysate sensitivity. It is calculated by the following expression:
    • Method A. Gel-Clot Limit Test Method Preparation of test solutions Unless otherwise prescribed, prepare the solutions and dilutions with water BET. If necessary, adjust the pH of the solution under examination to 6.0 to 8.0 using sterile 0.1M hydrochloric acid BET , 0.1M sodium hydroxide BET or a suitable buffer prepared with water BET . Prepare the sample solution at any dilution at or below MVD. Use water BET as negative control (NC) and two positive controls. One of the positive controls consists of the CSE at a concentration of 2 λ and the other consists of the test solution spiked with CSE to give a concentration of 2 λ (PPC). Methods A and B depend on the formation of a firm gel when a solution containing bacterial endotoxins is incubated after mixing with the lysate. 19
    • Interpretation of results The product under examination complies with the bacterial endotoxin test if the positive product control is positive and the negative control as well as the test solutions are negative. The test is not valid if the positive product control is negative or if the negative control is positive. The product under examination meets the requirements of the test if the endotoxin content is less than the endotoxin limit stated in the individual monograph. Retests If a positive result is found for one of the test solution duplicates and a negative result for the other, the test may be repeated as described above. The results of the retest should be interpreted as for the initial test. 20
    • The kinetic turbidimetric method is a photometric assay measuring the increase in turbidity caused by the reaction of the endotoxin with the lysate. The kinetic turbidimetric assay is a method measuring either the time (onset time) needed to reach a predetermined absorbance of the reaction mixture or the rate of turbidity development. The kinetic chromogenic method is a photometric assay measuring the colour developed by the chromophore released from a chromogenic substrate by the reaction of the endotoxin with the lysate. The kinetic chromogenic assay is a method measuring either the time (onset time) needed to reach a predetermined absorbance of the reaction mixture or the rate of colour development. The end-point chromogenic method is a photometric assay measuring the colour developed by the chromophore released from a chromogenic substrate by the reaction of the endotoxin with the lysate. The end-point assay is a method measuring the colour intensity at the end of an incubation period after the reaction is stopped by the addition of a 21 suitable acid.
    • LAL Assay Gel clot Colorimetric Turbidometric Semi-quantitative Quantitative Quantitative Simple, Least expensive Requires incubating plate or tube reader Requires incubating plate or tube reader Manually read and recorded Can be automated Can be automated Sensitive down to 0.03 EU/ml Sensitive down to .005 EU/ml Sensitive down to .001 EU/ml *
    • Limitations –LAL assay • Disturbed by endotoxin-binding components like blood components lipids • difficult to correlate with rabbit test • False-positive for cellulose and many herbal preparations 23