GUIDED BY : PRESENTED BY :Mr. Anand K. Patel PARTH PATEL M.Pharm Sem-1 Roll no: 5 DEPARTMENT OF QUALITY ASSURANCE APMC COLLEGE OF PHARMACEUTICAL EDUCATION & RESEACH, HIMMATNAGAR
Pyrogen Testing Rabbit test (sham test) LAL Test (Bacterial Endotoxin Test)Depyrogenation Techniques Physically removal of Pyrogens Inactivation of PyrogensComparison of Pyrogen Test of IP, BP, USP.
A. Rabbit test (sham test)B. LAL Test (Bacterial Endotoxin Test)
The test involves measurement of the rise in body temperature of rabbits following the intravenous injection of a sterile solution of the substance being examined.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.
Selection of animalMaterials:Heated in oven at 250ºc for 30 min & 200ºc for 1 hour.Diluents & Solutions Should be sterile & Pyrogen free.
Recording of TemperatureThermometer or thermistor.Insert the thermometer or temperature sensing probe into the rectum of the test rabbit to a depth of about 5-cm. The depth of insertion is constant for any one rabbit in any one test.
PRELIMINARY TEST: To the rabbit Inject intravenously 10 ml of Pyrogen-free saline solution. Record the temperatures of the animals, beginning at least 90 minutes before injection and continuing for 3 hours after injection of the solution being examined.Any animal showing a temperature variation of 0.6oC or more must not be used in the main test.
Preparation of the sample Procedure:1. Carry out the test using a group of three rabbits.2. Record the temperature of each animal. Not more than 40 minutes immediately preceding the injection of the test dose,3. Record the "initial temperature" of each rabbit.( dT Sh’d not >0.2)4. Do not use any rabbit having a temperature higher than 39.8oC and lower than 38oC .5. Inject the solution being examined slowly into the marginal vein of the ear of each rabbit over a period not exceeding 4 minutes 9
6. The amount of sample to be injected varies according to the preparation being examined and is prescribed in the individual monograph.7. The volume of injection is not less than 0.5 ml per kg and not more than 10 ml per kg of body weight. Records the temperature of each animal at half-hourly intervals for 3 hours after the injection.8. 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. When this difference is negative, the results is counted as a zero response.
If the responses of the group of three rabbits in which individual response is less than 0.6 oC , the preparation being examined passes the test.If the response of any rabbit is 0.6 oC or more, or if the sum of the response of the three rabbits exceeds 1.4oC 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 3.7oC , the preparation being examined passes the test.
It can be replicated and demonstrate the production of fever in humans Detects all kinds of injectable Pyrogen unlike LAL test. DISADVANTAGE: Time consuming Expensive Procedure It is passfail test than assay It cannot be used to test certain drugs that depresses the fever Tolerance to certain class of drugs can develop in rabbits & also biological variations are observed
LAL test = Limulus Amebocyte Lysate Test Test also referred by USP, IP, BP as BET test : Bacterial Endotoxin Test
The addition of a solution containing endotoxins to asolution of the lysate produces turbidity, precipitation or gel.The rate of reaction depends on the concentration ofendotoxin , the pH and the temperature.The reaction requires the presence of certain bivalentcations , a proclotting enzyme system and clottable protein allof which are provided by the lysate.
Mechanism of Action: Endotoxin 1-3-β- D- Glucan Factor C ----> Factor G ----> Factor C* Factor G* Factor B ----> Factor B* PRECLOTTING ENZYME ----> CLOTTING ENZYME COAGULOGEN ----> COAGULIN 15
The following six methods are described in the I.P. 2007 and B.P. 20071) Method A: Gel clot method: Limit test2) Method B: Gel clot method: Semi quantitative test.3) Method C: Turbidimetric Kinetic method4) Method D: Chromogenic kinetic method5) Method E: Chromogenic end- point method6) Method F: Turbidimetric end point method. the final decision is made based upon Method A , Unless otherwise indicated in the monograph:
Depyrogenate all glassware Time and temperature is required 30 minutes and 250 0C Plastic apparatus- which is free from detectable endotoxins1) Water BET2) 0.1M Hydrochloric acid BET3) 0.1M Sodium hydroxide BET4) Tris-chloride buffer pH 7.4 BET. Dissolve 0.6057 g of tris -(hydroxymethyl) methylamine in 30 ml of water BET, add 0.33 ml of hydrochloric acid, dilute to 100 ml with water BET and mix. It gives a negative result under the conditions of the test.
5) Lysate: A lysate of amebocytes from the horseshoe crab, Limulus polyphemus, reconstituted as stated on the label. For each batch, confirm the stated sensitivity as prescribed under Sensitivity of the lysate.6) Lysate solution7) Reference Standard Endotoxin8) The standard endotoxin stock solution Endotoxin is expressed in International Units (IU) 1 IU=1 EU (ENDOTOXIN UNIT).
9) STANDARD ENDOTOXIN SOLUTIONS10) PREPARATION OF THE TEST SOLUTIONS Preparation of test solution by dissolving or diluting active substance Adjust the pH of test solution(about 6 to 8) pH adjust with use of acid, base and buffer Buffer must be validated to be free of detectable endotoxin
The Maximum Valid Dilution (MVD) is themaximum allowable dilution of a sample at which theendotoxin limit can be determined.MVD = endotoxin limit × conc. of test solution λλ = the labeled lysate sensitivity in the gel clot, turbidimetric or chromogenic technique.
The Endotoxin limit= K/MK = Threshold pyrogenic dose of endotoxin per kg of body mass in single hour period.M = Maximum dose of product per kg of body mass in a single hour period The value of K is 5.0 EU/kg for injectable preparations except for those administered intrathecally and is 0.2 EU/kg for intrathecal preparations.
The gel-clot technique allows detection or quantification of endotoxins and is based on clotting of the lysate in the presence of endotoxins. The concentration of endotoxins required to cause the lysate to clot under standard conditions is the labelled lysate sensitivity To ensure both the precision and validity of the test, confirm the labelled lysate sensitivity and perform the test for interfering factors as described under Preparatory testing
SENSITIVITY OF THE LYSATE Confirm in 4 replicates the labelled lysate sensitivity(λ) Prepare standard solutions of at least 4 concentrations equivalent to 2λ , λ , 0.5λ and 0.25λ from stock by diluting with water for BET Mix lysate solution with standard in each tube.(1:1) Incubate the reaction mixture at 37 ± 1 °C for 60 ± 2 min (avoiding vibration) Invert it through approximately 180° in one smooth motion.
If a firm gel has formed that remains in place upon inversion, record the result as positive. A result is negative if an intact gel is not formed. The end-point is the last positive result in the series of decreasing concentrations of endotoxin. Calculate Geometric mean end-point concentration =Where, ∑e = Sum of the log end-point concentrations. f = number of replicates. The geometric mean end-point concentration is the measured sensitivity of the lysate solution (IU/ml). If this is not less than 0.5λ and not more than 2λ, the labelled sensitivity is confirmed
Solution Endotoxin Diluents Dilution Initial Number conc./sol. to factor endotoxin of which conc. replicate endotoxin is added A None/Test - - - 4(Sol. Of Prepa.) solution B 2λ/Test Test 1 2λ 4 (Test for solution solution 2 1λ 4 Interference) 4 0.5λ 4 8 0.25λ 4 C 2λ/Water for Water for 1 2λ 2 (+ ve control) BET BET 2 1λ 2 4 0.5λ 2 8 0.25λ 2 D None/Water for - - - 2 (- ve control) BET 26
Prepare solutions A, B, C and D as shown in Table and usethe test solutions at a dilution less than the MVD, notcontaining any detectable endotoxins, operating as describedunder Confirmation of the labelled lysate sensitivity. The geometric mean end-point concentrations of solutions Band C are determined.The test is not valid unless all replicates of solutions A and Dshow no reaction and the result of solution C confirms thelabelled lysate sensitivity
If the sensitivity of the lysate determined with solution B is not less than 0.5λ and not greater than 2λ, the test solution does not contain interfering factors under the experimental conditions used. Otherwise, the solution interferes with the test If the preparation being examined interferes with the test at a dilution less than the MVD, repeat the test for interfering factors using a greater dilution, not exceeding the MVD Interference may be overcome by suitable treatment, such as filtration, neutralization, dialysis or heat treatment.
Solution Endotoxin conc./solution to No. of which endotoxin is added Replicates A None/Test solution 2 B 2λ / Test solution 2 C 2 λ/water for BET 2 D None/Water for BET 2 Prepare solutions A,B,C, and D as shown in table, and perform the test on these solutions by following the procedure described in Confirmation of the labeled lysate sensitivity
Prepare solution A and Solution B (Positive Product control) using a dilution not greater than the MVD and treatments Test for interfering Factors.Interpretation: Ifa positive result is found for one of the test 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. The substance or preparation being examined meets the requirements of the test if the concentration of endotoxin is less than the endotoxin limit stated in the monograph.
Solution Endotoxin Diluents Dilution Initial Number conc. /solution factor endotoxin of to which conc. replicate endotoxin is added A None/Test Water for 1 - 2 solution BET 2 - 2 4 - 2 8 - 2 B 2λ/Test solution - 1 2λ 2(+ve control) C 2λ/Water for Water for 1 2λ 2 BET BET 2 1λ 2 4 0.5λ 2 8 0.25λ 2 D None/Water for - - - 2
The test is not valid unless the following three conditions are met:1. Both replicates of solution D(negative control) are negative.2. Both replicates of solution B(positive control) are positive3. The geometric mean end-point concentration of solution C is in the range of 0.5 λ to 2 λ. To determine the endotoxin concentration of solution A. calculate the end-point concentration for each replicates series of dilutions by multiplying each end-point dilution factor by λ
The endotoxin concentration in the test solution is the geometric mean end point concentration of the replicates, if the test is conducted with a diluted test solution calculate the concentration of endotoxin in the original solution by multiplying the results by dilution factor. If none of the dilution of the test solution is positive in a valid test, record endotoxin concentration as less than λ . If all dilutions are positive, the endotoxin concentration is recorded as equal to or greater than the greatest dilution factor multiplied by λ
The preparation meets the requirements of the test if the endotoxin concentration is less than that specified in the individual monograph.
1. Turbidimetric technique (Method C and F) This technique is a photometric test to measure the increase in turbidity, Based on the test principle employed classified as, The end –point turnidimetric test (F) The kinetic- turbidimetric test (C)
2.CHROMOGENIC TECHNIQUES (METHODS D &E) This techniques is used to measure the chromophore released from a suitable chromogenic peptide by the reaction of endotoxins with the lysate. Depending on the principle employed , this technique is classified as End- point chromogenic test (E) The kinetic- chromogenic test.(D)
As the use of LAL test became more & more prevalent, the FDA decided that single standardize document was needed to govern all FDA regulated products that were subject to LAL testing, An FDA task force was formed by representatives from1) Center for drug evolution & Research.2) Center for biological evolution & Research.3) Center for devices & Radiologic health.4) Center for veterinary medicine.
Three basic requirementsi. The LAL reagent used in cell validation in process and end product LAL tests must be licensed by CBERii. The product manufacturer must perform an initial Qualification of their LAL test laboratory PERSONNELiii.Inhibition & enhancement tests must be performed on test products do not interfere with the enzymatic LAL endotoxin reaction.
A. Inhibition Occurs when the test recovers less endotoxinCauses:• Chemical nature of the drug product or excipients• Factors that negatively effect serine protease enzyme reaction such as High or low pH• Oxidants or antioxidants• Proteolytic agents• High heat• Chelating agents• Inadequately dispersed purified endotoxin
B. Enhancement: Occurs if more Endotoxins recovered than expectedCauses: Chemical nature of the product Endotoxin contamination present in product Surfactants by increasing surface area of endotoxin.
ADVANTAGE OF LAL TEST In vitro test , More sensitive Easier to perform. Can give Quantitative results. Less time consuming. Less expensive. LIMITATION OF LAL TEST Specific for gram negative pyrogens only. Clotting enzyme is heat labile, pH sensitive. Possible interference Problems.
1) Pharmaceuticals: Parenteral dosage form Large volume Parenterals Small volume Parenterals2) Biologicals In blood products & plasma fractions Vaccines3)Medical Device Nebulizers used in Respiratory therapy4)Diagnosis of disease caused by Gram –negative bacteria5) In food & drinking water6) Others: For validation of dry heat sterilization
INVITRO PYROGEN TEST (IPT): This test exploits the reaction monocytes/macrophages for detection of pyrogens . Human whole blood taken from healthy volunteers is incubated in presence of test sample, pyrogenic contamination initiate the release of “the endogenous pyrogen” Interlukin 1-β determined by ELISA after incubation.
(A)Removal of Pyrogens by physical Methods(1) Dilution(2) Ultra-filtration(3) Reverse osmosis(4) Distillation(5) Adsorption on Charcoal(6) Column Chromatography(7) Charge Modified Media & Electrostatic Attraction(8) Hydrophobic attraction to hydrophobic medium
(1) Dry heat sterilization(2) Moist heat sterilization(3) Use of dilute acids & Bases(4) Oxidation(5) Alkylation
Number of Maximum total peak Minimum total peak Rabbits response (°C) to pass response (°C) to fail the the test test USP BP USP BP 3 1.4 1.15 1.4 2.65 6 - 2.80 - 4.30 8 3.7 - 3.7 - 9 - 4.45 - 5.95 12 - 6.60 - 6.60
Encyclopedia of Pharmaceutical Technology, Volume:13,By James Swarbick & James Boylan. Indian Pharmacopoeia 2007,Vol.-1,Appendice-1. British Pharmacopoeia 2007. United state pharmacopoeia. Remington( The science and practice of pharmacy), Volume-I , Page no.562,832.