Bacterial Endotoxins test

Bacterial Endotoxins test-Pharmacopoeial Methods

1. BACTERIAL ENDOTOXINS TESTS D.MAHALAXMI PA-2016-104 Tuesday, April 11, 2017 1

2. DEFINiTION : ENDOTOXINS : these are lipopolysaccharide pyrogenic substances (induces fever) found in cell wall of gram-negative bacteria. Tuesday, April 11, 2017 2

3. BACTERIAL ENDOTOXIN TEST : To detect and quantify the endotoxins from gram-negative bacterial origin using amoebocyte lysate from horseshoe crab(limulus polyphemus and tachypleus tridentatus,tachypleus gigas,carcinoscropius rotundicauda ) Sterilization does not remove any endotoxins as they are heat stable . DEFINiTION : Tuesday, April 11, 2017 3

4. “Why” ???? To protect against adverse reactions (sepsis) in humans and animals FDA and USP guidelines require final product testing on all parenterals and medical devices To safeguard against diminished effectiveness of a product due to endotoxin Tuesday, April 11, 2017 4

5. APPARATUS: Depyrogenated Free of detectable endototxins Should not interfere with test REAGENTS AND TESTS SOLUTIONS: Amoebocyte lysate : Lyophilised product- lysate of amoebocytes -horseshoe crab (limulus polyphemus or tachypleus tridentatus ). Water for Bacterial Endotoxins Test : Use water for injection Lysate TS : Dissolve Amoebocyte lysate in water for BET or in buffer recommended by lysate manufacturer. [ Note:use lysate TS having sensitivity of NLT-0.15 Endotoxin Unit per mL ] Temperature-250 degrees Time-30 min’s Tuesday, April 11, 2017 5

6. Standard endotoxin stock solution USP Endotoxin Reference Standards-calibrated to current WHO international standard for endotoxin . Standard Endotoxin Solution Serial dilutions of SESS with water for BET Sample solution Prepared by dissolving drug pH-6.0-8.0, Use water for BET for preparing buffers PREPARATION OF SOLUTIONS Tuesday, April 11, 2017 6

7. Determination of maximum valid dilution [ mvd ] It is max. allowable dilution at which endotoxin limit can be determined Endotoxin limit -for parentals =K/M where K is threshold pyrogenic dose of endotoxin per Kg of body weight & M is maximum total dose of product per Kg of body weight EU/ mL ,EU/mg ,EU/unit of biological activity Product concentration –mg/ mL [in weight-EU/mg] Endotoxin limit *product concentration Lysate sensitivity MVD= Tuesday, April 11, 2017 7

8. V-Maximum dose in mL For drugs administrated per square meter of body surface (Anticancer drugs)-K is EU/m2 Drugs Radiopharmaceuticals-formula Route of administration K-(EU/mg) 5 0.2 175(EU/V) 14(EU/V) Any –Except intrathecal route Intrathecal Determination of maximum valid dilution [ mvd ] Tuesday, April 11, 2017 8

9. TESTS Techinques Gel formation Turbidity formation-cleavage of an endogenous substrate Colour formation-cleavage of synthetic peptide chromogenic complex chromogenic Gel-cloth turbiditymetric Tuesday, April 11, 2017 9

10. Gel-cloth test Preparatory tests Inhibition/ Enhancement Test Test for confirmation of labelled lysate sensitivity Test for interfering factors Limit test Quantitative test USP Semi-Quantitative test IP,BP,EP Tuesday, April 11, 2017 10

11. Gel-cloth technique The gel-clot technique is used for detecting or quantifying endotoxins based on clotting of the lysate reagent in test tubes in the presence of endotoxin . The minimum concentration of endotoxin required to cause the lysate to clot under standard conditions is the labeled sensitivity of the lysate reactivity . To ensure both the precision and validity of the test perform the tests for confirming the labeled lysate sensitivity and for interfering factors . Tuesday, April 11, 2017 11

12. 1.Preparatory testing 1.Confirmation of labelled lysate sensitivity : Confirm in 4 replicates – labelled sensitivity( ) - [EU/ mL ]. WHEN ???? For new batches of lysate ,change in test conditions 2 1 0.5 0.25 Prepare standard solutions of at least 4 concentrations equivalent to 2λ, λ, 0.5λ and 0.25λ by diluting the standard endotoxin stock solution with water for BET+0.1mL lysate TS Transfer to vials-incubate 37 ± 1° for 60 ± 2 min) To test integrity of gel-invert 180 degrees in one smooth motion Positive Negative Tuesday, April 11, 2017 12

13. Tuesday, April 11, 2017 13

14. Test is valid – low concentration endotoxin standard solution – negative results Geometric endpoint concentration is measured sensitivity of lysate (EU/ mL ) Sigma ‘e’-sum of the log end point used concentrations of dilution series and ‘ f’-no.of replicates used If GMEC - (0.5-2) the labelled sensitivity is confirmed and is used in tests performed with this lysate Geometric mean endpoint concentration = antilog (S e/f) Cntd …. Tuesday, April 11, 2017 14

15. Procedure is same for remaining all tests as that of confirmation of lysate procedure . Tuesday, April 11, 2017 15

16. 2.Test for interfering factors Tuesday, April 11, 2017 16

17. Cntd …. Perform inhibition & enchancemant test on the sample solutions at dilution less than MVD Repeated when any conditional changes influence the results of test. The test is valid – 1. All replicates of solution A & D –no reaction 2.solution C-confirms the labelled sensitivity 3.solution B-0.5-2 labelled sensitivity-indicates no interfers If test not complied use greater dilution not exceeding MVD Interference can be removed by neutralisation,dialysis , filtering and dialysis. Tuesday, April 11, 2017 17

18. 2.Limit test Tuesday, April 11, 2017 18

19. Cntd …. Test is valid- 1. two replicates solutions of B&C-positive 2.two replicates solutions of D-negative 3.two replicates solutions of A-negative(comply the test) If positive in the case of A than repeat the test If test doesn’t complied with dilution less than MVD, repeat the test using greater dilution not exceeding MVD. Tuesday, April 11, 2017 19

20. 3.Quantitative test Tuesday, April 11, 2017 20

21. Cntd …. INTERPRETATION: The test quantifies the bacterial endotoxins in sample solution The test is valid- 1.Both replicates of negative control solution D are negative 2.Both replicates of positive product control solution B are positive 3.GMEC concentration of solution C-0.5-2 The endotoxin concentration in the sample solution is endpoint concentration of replicates,so endpoint concentration can be calculate by multiplying the endpoint dilution factor by labelled sensitivity. The test is considered as valid when endotoxin concentration in the both replicates is less than that specified in individual monograph. Tuesday, April 11, 2017 21

22. Photometric quantitative technique Turbidimetric technique Chromogenic technique Endpoint- turbidimetric assay Kinetic- turbidimetric assay Endpoint- chromogenic assay Kinetic- chromogenic assay IP,BP,EP IP,BP,EP IP,BP EP Tuesday, April 11, 2017 22

23. 1.Turbidimetric technique Measures the reactant turbidity The endpoint – turbidimetric assay is based on the quantitative relationship between the concentration of endotoxins and turbidity (absorbance or transmission) of the reaction mixture at the end of incubation period. The kinetic- turbidimetric assay is a method to measure the either the time needed to reach a predetermined absorbance or transmission of a reaction mixture or rate of turbidity development,this test is carried out at incubation temperature recommended by lysate manufacture (37 ± 1°) . Tuesday, April 11, 2017 23

24. 2.Chromogenic technique Measures the chromophore released from the suitable chromogenic peptide by the reaction of endotoxins with lysate . The endpoint- chromogenic assay is based on the quantitative relationship between the concentration of endotoxins and release of chromophore at the end of an incubation period. The kinetic- chromogenic assay is a method to measure either time (onset time) needed to reach a predetermined absorbance of a reaction mixture or a rate of color development- (37 ± 1°) . Tuesday, April 11, 2017 24

25. Mechanism involved in chromogenic technique: Tuesday, April 11, 2017 25

26. 3.Preparatory testing 1 .To assure precision or validity of turbidimetric and chromogenic tests. 2 .To assure criteria for standard curve is statisfied and test solution doesn’t interfere with test. 3 .Any changes in experimental conditions influence –results of test . ??? Tuesday, April 11, 2017 26

27. 1.Assurance of criteria for standard curve: Prepare atleast 3 endotoxins concentrations to generate standard curve using standard endotoxins solutions. Perform test using atleast 3 replicates of each standard endotoxin solutions. If desired range >2 in kinetic methods than additional standards included to bracket –each log increase in the range of standard curve. The absolute value of ‘r’ should be = or > than 0.980. 2.Testing for interfering factors: select an endotoxin concentration at or near the middle of standard endotoxin curve. Prepare solutions A,B,C,&D as per below table .perform the tests on atleast 2 replicates of these solutions. Tuesday, April 11, 2017 27

28. Tuesday, April 11, 2017 28

29. Calculation: 1 . Calculate - mean recovery of the added endotoxin by subtracting the mean endotoxin concentration in the solution from the solution containing the added endotoxin concentration. 2. The test solution - free of interfering factors the measured concentration of the endotoxin added to the test solution is - 50-200 per cent of the known added endotoxin concentration, after subtraction of any endotoxin detected in the solution without added endotoxin . 3 . When the endotoxin recovery is out of the specified ranges, the interfering factors must be removed as described in the section Gel-clot technique, under ( i ) Preparatory testing, (ii) Testforinterferingfactors . Tuesday, April 11, 2017 29

30. Interpretation: The preparation being examined complies with the test if the mean endotoxin concentration of the replicates of solution A, after correction for dilution and concentration, is less than the endotoxin limit for the product. Tuesday, April 11, 2017 30

31. References: Tuesday, April 11, 2017 31

32. Tuesday, April 11, 2017 32