Bet ppt

Bacterial endotoxin test

1. Bacterial Endotoxin Test

2. The bacterial endotoxins test (BET) is a test to detect or quantify endotoxins from Gram-negative bacteria using amoebocyte lysate from the horseshoe crab (Limulus polyphemus or Tachypleus tridentatus).

3. Endotoxin : ● The toxic activity of LPS was first discovered and termed "endotoxin" by Richard Friedrich Johannes Pfeiffer . which he classified as a toxin that is released by bacteria into the surrounding environment, and endotoxins, which he considered to be a toxin kept "within" the bacterial cell and released only after destruction of the bacterial cell wall . ● Subsequent work showed that release of LPS from gram negative microbes does not necessarily require the destruction of the bacterial cell wall, but rather, LPS is secreted as part of the normal physiological activity of membrane vesicle trafficking in the form of bacterial outer membrane vesicles (OMVs) , which may also contain other virulence factors and proteins .

4. ● T he term 'endotoxin' is mostly used synonymously with LPS, although there are a few molecules secreted by other bacteria that are not related to LPS, such as the so-called delta endotoxin proteins secreted by Bacillus thuringiensis . ● LPS is the major component of the outer membrane of Gram-negative bacteria , contributing greatly to the structural integrity of the bacteria, and protecting the membrane from certain kinds of chemical attack. LPS also increases the negative charge of the cell membrane and helps stabilize the overall membrane structure.

5. ● Lipopolysaccharides ( LPS ) are large molecules consisting of a lipid and a polysaccharide composed of O-antigen , outer core and inner core joined by a covalent bond ; they are found in the outer membrane of Gram-negative bacteria , and elicit strong immune responses in animals . ● Endotoxin of bacteria consist of Lipopolysaccharide ( LPS), which is bound to protein and phospholipid .

6. Consequences of endotoxin contamination • Fever • Headache • Chills • Nausea/Vomiting • Hypotension • Acute lung injury • Miscarriage • Death

7. History of LAL Regulations 1912 Pyrogen test was performed by rabbit test 1941 Rabbit pyrogen test included in usp 12. FDA announced LAL as a biological product FDA described conditions for use of LAL as end product test for human biological and medical devices. Federal register drafted guidelines for use of LAL test for end product testing of human and animal injectable drug products.

8. FDA determine the limit of endotoxin content based on maximum human dose 1983 Federal registers list final guidelines on LAL testing including chrom and turb . USFDA established guidelines for LAL testing of pharmaceutical and medical devices 1991 Interim guidance – Kinetic LAL test kit 1995 USP 23 Bacterial endotoxin test 1998 European pharmacopeia BET 2000 Harmonized BET

9. Principle: Gram-negative bacterial endotoxin catalyzes the activation of a proenzyme in the LAL. The initial rate of activation is determined by the concentration of endotoxin present. The activated enzyme (coagulase) hydrolyzes specific bonds within a clotting protein ( coagulogen) also present in LAL. Once hydrolyzed , the resultant coagulin self- associates and forms a gelatinous clot .

10. ● The coagulation of Limulus amebocyte lysate ( LAL) is brought about by an enzymatic cascade, which consists of three proenzymes , factor C, factor B, and pro-clotting enzyme and one clottable protein, coagulogen. ● LPS activates factor C, which in turn activates factor B and proclotting enzyme, the latter cleaving coagulogen to yield an insoluble gel and releasing a soluble peptide C.

11. ● The resulting clotting enzyme is responsible for anchoring the two peptide units in the coagulogen, forming an insoluble gel. ● The rate of gelation was directly related to the concentration of endotoxin. ● The catalytic nature of each activated enzyme in the coagulation cascade serves in turn to amplify the next step , resulting in a high sensitivity of LAL to LPS.

12. ● The coagulation chain described above is also activated by the (1,3) -β-D- glucan , in this case through the activation of factor G, which is another serine protease pro-enzyme and which also leads to the formation of the gel-forming enzyme which in turn causes the gelation of coagulogen. ● Therefore , the β-D- glucan is considered to be an interfering agent in the LAL test for the measurement of endotoxins.

13. Reagents Used in BET test: Lysate: ● The Danish-born pathologist Frederik B. Bang reported that a bacterial infection of Limulus polyphemus caused fatal intravascular clotting and this clotting could also be induced by a heat-stable derivative of the bacterium. ● In 1964, Levin and Bang demonstrated that extracts of the amebocytes , but not the cell-free hemolymph , would gel in the presence of endotoxin

14. LAL Limulus: Genera of Crab Amebocyte: Crab Blood Cell from which active component is derived Lysate: Component is obtained by separating amebocyte from the plasma and then lysing them

15. ● The reagent (lysate) used in the LAL test is of biological origin and is extracted by the osmotic lysis of amebocytes found in the intracellular fluid of the American horseshoe crab ( Limulus polyphemus ). It is a complex mixture of different enzymes and co-factors that form a clot through a cascade reaction when triggered by the presence of endotoxins in the solution. ● The enzyme activity of this formulation (labeled as lysate sensitivity, λ ) is determined against a Reference Standard Endotoxin (RSE) preparation supplied by the FDA. An assay is performed by preparing a 2-fold dilution series made from 1 EU/mL of RSE. Since the lysate potency is performed in 2-fold dilutions, e.g ., the true sensitivity of a labeled 0.125 EU/mL lysate lot may actually be 0.10 EU/ mL .

16. ● LAL manufacturers supply Control Standard Endotoxin (CSE) whose potency is determined against the RSE for every lot of lysate. Each lot-specific combination of LAL and CSE must be characterized according to the method being used and a specific RSE/CSE ratio (EU/ ng value) cannot be assigned until the testing is complete . ● Lyophilized ( unreconstituted ) Limulus Amebocyte Lysate should be stored under refrigeration at 2-8°C. Care should be taken to avoid exposing the lysate to temperatures in excess of 37°C. Lysate which has been exposed to prolonged periods of temperatures above 37°C or to bright light may turn yellow and/or become insoluble . ● Reconstituted lysate may be stored at 2-8°C for 24 hours. For longer storage, reconstituted lysate can be stored at -20°C or colder. Freeze and thaw only once. The lysate should be protected from exposure to light during storage. Use within four weeks after reconstitution.

17. Control Standard Endotoxin: ● CSE is a highly purified lipopolysaccharide (LPS) preparation that is reasonably free of detectable contaminants, particularly protein. It also contains stabilizing fillers like human serum albumin, PEG, and starch, so that it performs reliably and reproducibly when used in control and standard curves. ● A Standard Endotoxin Stock Solution is prepared from a USP Endotoxin Reference Standard that has been calibrated to the current WHO International Standard for Endotoxin. ● Endotoxin is expressed in Endotoxin Units (EU). NOTE: One USP Endotoxin Unit (EU) is equal to one International Unit (IU) of endotoxin.

18. ● Control Standard Endotoxin (CSE) is a widely used standard for endotoxin testing. It is a purified extract from E. coli O113:H10, the same strain used for the United States Pharmacopeia and the European Pharmacopeia Reference Standard Endotoxin (RSE ). ● Each vial when prepared according to the instructions below provides the user with a Control Standard Endotoxin (CSE) whose potency has been established using the current FDA Reference Standard Endotoxin (RSE) and the enclosed lot of lysate. ● The appropriate RSE/CSE ratio and resultant CSE potency is provided on the Certificate of Analysis.

19. Control Standard Endotoxin Preparation: ● Reconstitute by adding 1.0 ml of LAL Reagent Water warmed to room temperature. For example, if the value of the vial is 20 EU, when reconstituted with 1.0 ml water it will yield a concentration of 20 EU/ml. ● Shake vigorously as per COA at high speed on a vortex mixer. ● Lyophilized endotoxin is to be stored at 2–8°C. Reconstituted stock endotoxin is stable for four weeks at 2–8°C. Prior to subsequent use, the solution must be warmed to room temperature and vigorously vortexes for 15 minutes, because the endotoxin tends to attach to glass. ● This endotoxin is provided for the user’s convenience.

20. Preparation of CSE: Step 1 Reconstitute Control Standard Endotoxin (CSE) with 1mL of LAL Reagent Water (LRW). Step 2 Vortex for 15 minutes or as per COA . CSE LRW CSE

21. Step 3 Prepare a solution containing 1.0 EU/ml using the endotoxin potency identified on the Certificate of Analysis (CoA ). 0.1  ml CSE LRW 1.9  ml

22. Step 4 Label tubes with the appropriate endotoxin concentration and add LRW to each. Then prepare a series of endotoxin standards. 1EU/mL 0.5EU/mL 0.25EU/mL 0.125EU/mL 0.06EU/mL 0.5 ml 0.5 ml 0.5 ml 0.5 ml 0.5 ml 0.5 ml 0.5 ml 0.5 ml LRW

23. Confirmation of Labelled Lysate Sensitivity: ● The test for confirmation of lysate sensitivity is to be carried out when a new batch of lysate is used or when there is any change in the test conditions that may affect the outcome of the test . ● Confirm in four replicates the labelled sensitivity, λ, expressed in IU/ml of the lysate prior to use in the test. ● Prepare standard solutions having four concentrations equivalent to 2λ , λ, 0.5λ and 0.25λ by diluting the Standard Endotoxin Stock Solution with water BET.

24. ● Mix a volume of the Lysate TS with an equal volume (such as 0.1mL aliquots) of one of the Standard Endotoxin Solutions in each test tube. ● Incubate the reaction mixture for a constant period according to the directions of the lysate manufacturer (usually at 37±1° for 60±2 min), avoiding vibration. ● To test the integrity of the gel, take each tube in turn directly from the incubator, and invert it through about 180° in one smooth motion.

25. ● 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 .

26. ● The test is valid when the lowest concentration of the standard solutions shows a negative result in all replicate tests. ● The endpoint is the smallest concentration in the series of decreasing concentrations of standard endotoxin that clots the lysate . ● Determine the geometric mean endpoint by calculating the mean of the logarithms of the endpoint concentrations of the four replicate series and then taking the antilogarithm of the mean value, as indicated in the following formula: geometric mean endpoint concentration = antilog ( ∑ e/f ) where ∑ e is the sum of the log endpoint concentrations of the dilution series used f is the number of replicate test tubes.

27. ● The geometric mean endpoint concentration is the measured sensitivity of the lysate (in EU/mL), this is not less than 0.5 λ and not more than 2 λ . Lysate Sensitivity (λ) = 0.03 EU /ml

28. geometric mean = antilog ( ∑ e/f) = antilog (log (0.03)+log(0.03)+log(0.03)+log(0.03)) -------------------------------------------------------------------- 4 = antilog ( -1.52+ -1.52+ -1.52+ -1.52) ------------------------------------ 4 = antilog (-1.52) = 0.03

29. ● A positive test is characterized by the formation of solid gel which remains intact after inversion. This should be observed in the positive control vial and in the positive sample control vial.   ● A negative test is characterized by the absence of solid clot after inversion. This should be observed in the negative control vial. The lysate may show an increase in turbidity or viscosity. This is considered a negative result.   ● A positive result should be observed with all Inhibit Control vials. The absence of gel formation is indicative of product inhibition. If a negative reaction is observe in the Inhibition Control vial with any sample, then the samples test results are invalid.

30. Spiking Methods used in LAL Testing   ● Double Strength Method or 50:50 Method   ● Hot-Spike Method Double Strength/Dilution Method or 50:50 Method   ● Prepare double the strength test concentration or half of the test dilution of the sample ● Prepare 4 λ of CSE ● Dilute ½ Y (Y = Test Sample Dilution) with equal volume of LRW in Negative Product Control (NPC ) ● D ilute ½ Y (Y = Test Sample Dilution) with equal volume of 4 λ in Positive Product Control (PPC )

31. Example: Product : ABCD Solution Endotoxin limit : NMT 5.56 EU/mL LAL sensitivity (l) = 0.125 EU/mL MVD = 1:44 MVD/2 = 1:22 In order to test the sample at MVD or at Endotoxin Limit, prepare the solution at ½ MVD i.e .1:22 Sample LRW Dilution + 0.1 mL 2.1 mL 1:22

32. NPC = 50 µL LRW + 50µL Sample (1:22) + 100 µL LAL PPC = 50µL Sample (1:22) + 50 µL 4l CSE + 100 µL LAL If then , Endotoxin content of the sample = dilution factor x Lysate Sensitivity = 44 x 0.125 EU/mL = < 5.5 EU/mL

33. Hot-Spike Method   ● Prepare test concentration or test dilution of the sample   ● Prepare 20 λ of CSE   ● Take X product (where X = Test Sample Concentration) or Y (Y = Test Sample Dilution ) in Negative Product Control (NPC)   ● Spike test sample with minimal but accurately measurable concentrated endotoxin solution i.e. 10 µL of 20 λ Example : Product : ACPD Solution Endotoxin limit : NMT 5.56 EU/mL Lysate sensitivity (l) = 0.125 EU/mL MVD = 1:44

34. Sample Preparation Sample LRW Dilution 0.1 mL 2.1 mL 1:22 Solution LRW Dilution 1 mL 1 mL 1:44

35. NPC = 100 µL Sample (1:44) + 100 µL LAL PPC = 100 µL Sample (1:44) + 10 µL 20 λ CSE + 100 µL LAL If NPC = negative and PPC = positive, Then Endotoxin content of the sample = dilution factor x Lysate Sensitivity = 44 x 0.125 EU/mL = < 5.5 EU/mL

36. Interference: ● Interference is defined as a significant difference between the end points of positive water control and positive product control using standard endotoxin. ● This interference could be either inhibition wherein the recovery of endotoxin is below than the expected or enhancement wherein the recovery of endotoxin is higher than expected.

37. Two classes of interference are considered here: 1. inhibition 2. enhancement Inhibition : Inhibition occurs when a material interferes with the ability of the LAL reagent to react with endotoxin, causing underestimation of the amount of endotoxin present. Tests must be properly controlled so that inhibition is detected if it occurs. Appropriate controls provide assurance that negative results are due to absence of endotoxin, not to inhibition.

38. Enhancement : Enhancement is interference that increases the sensitivity of the assay, resulting in overestimation of the endotoxin concentration of the sample. Enhancement is much less dangerous than inhibition. It could result in the inability to release a product that should have passed, but it does not result in a threat to public health and safety.

39. Interference: Causes and Solutions pH : The LAL reaction consists of a series of enzymatic reactions in which serine proteases, each with a pH optimum, cleave their respective substrates. Consequently, it is critical that the pH of the reaction mixture of product and LAL reagent be in the range specified in the product insert. The harmonized pharmacopeia endotoxins test require pH is in the range of 6.0 - 8.0. However, it is quite possible that the pH of the product alone will be outside the 6.0 - 8.0 range, but that the pH of the reaction mixture will be within it because of the buffering capacity of the LAL reagent .

40. The pH of the reaction mixture may be outside the specified range for undiluted product, but the pH of a dilution of product (not to exceed the MVD) can be within the range. For example, if the pH of the reaction mixture is out of range for an undiluted product with an MVD of 1:200, but it meets specification at 1:20, then the product can be validated and tested at 1:40 or 1:50. There is no need to adjust the pH If pH problems are not overcome by dilution, use Tris Buffer to reconstitute the LAL reagent as stipulated in the product insert and then check pH of the reaction mixture.

41. In samples for which the buffer does not resolve the problem, the pH of the sample (or of a sample dilution) can be adjusted by adding a solution of acid or base ( HCl or NaOH ). Prepare solutions from concentrated HCl or NaOH pellets with LAL reagent water (LRW). Before conducting any LAL tests, perform titrations to determine the appropriate volume and concentration of acid or base solution to bring the pH into range. The volume of added NaOH or HCl should not change the sample volume by more than 10%.

42. Divalent Cations : Divalent cations influence both the reactivity of endotoxin and the LAL reaction. Cations are attracted to and neutralize the negative charge of endotoxin, allowing increased aggregation size and decreasing activity/potency, which is observed as inhibition. Divalent cations are also required for optimal LAL sensitivity, but excess concentrations inhibit the reaction. Dilution is the usual solution to this problem. When interference by salts (or other small molecules) cannot be overcome by dilution, endotoxin can be separated from interfering substances by ultrafiltration .

43. Chelating Agents : Materials which bind (chelate) divalent cations , such as EDTA (ethylene diamine tetraacetic acid) and heparin, can reduce the aggregation state of endotoxin. This results in increased reactivity, which is observed as enhancement. Once again, interference is usually overcome by dilution of the sample. Extractables : Extractables from plastics can interfere with LAL tests. Water soluble substance extracted from polypropylene tubes that was highly inhibitory.

44. Lipids and Liposomes: Some drug products are dissolved in oils, such as sesame oil, while others are enclosed in liposomes. Perhaps surprisingly, some products dissolved in oils can be diluted in water (LRW) and tested in the normal way, as endotoxin may partition into the aqueous phase during dilution. Verify (and validate) this by spiking the product with endotoxin and demonstrate recovery. In other cases, a surfactant may be used to disperse a lipid, e.g. 0.01% sodium desoxycholate (which is slightly inhibitory). Piluso and Martinez(11) report that sodium dodecyl sulfate (SDS) or CHAPS may be used to disrupt liposomes to facilitate their testing.

45. Other Strategies for Overcoming Interference Ultrafiltration can be used to separate lower molecular weight interfering substances from endotoxin in the sample. One product, the Sartorius Ultrasart ® D-20, in which the membrane has a 20 kD molecular weight cutoff , is designed specifically for this purpose and has been used effectively in laboratory.

46. The Three Essentials Steps for Endotoxin Testing ● Establish endotoxin limits for pharmaceutical and medical devices. ● Establish procedures for validating the use of the BET in your laboratory. ● Establish procedures for conducting routine testing.

47. Establishment of endotoxin limits ● The endotoxin limit for active substances administered parenterally , defined on the basis of dose, is equal to: EL = K/M K = threshold pyrogenic dose of endotoxin per kilogram of body mass in a single hour period, M =maximum recommended dose of product per kilogram of body mass in a single hour period. ● The endotoxin limit for active substances administered parenterally is specified in units such as IU/ml, IU/mg, IU/Unit of biological activity, etc.,

48. ● Note: The endotoxin limit depends on the product and its route of administration ● For intravenous route: K = 5 IU endotoxin per kg body weight ● For intrathecal route K = 0.2 IU endotoxin per kg body weight ● For radiopharmaceutical products not administered intrathecally , the endotoxin limit is calculated as 175 EU/V, where V is the maximum recommended dose in mL ● For intrathecally administered radiopharmaceuticals, the endotoxin limit is obtained by the formula 14 EU/V ● For formulations (usually anticancer products) administered on a per square meter of body surface, the formula is K/M, where K = 100 EU/m2 and M is the maximum dose/m2

49. Example: Product: Piperacillin and tazobactam for Injection 2.25 g Endotoxin limit calculation: K = 5 EU/kg M= 64.28 mg/kg/ hr (4500mg per 70kg/ hr ) = 4500mg/70kg/ hr = 64.28 mg/kg/ hr BET Limit = K/M = 5 EU/kg/ hr = 0.077 EU/mg ≈ 0.07 EU/mg 64.28 mg/kg/ hr BET Limit = 0.07 EU/mg.

50. Establish endotoxin limit For Medical Devices ● Level of endotoxin on medical devices are obtained by an extraction procedure. This involves soaking a number of devices in an extracting fluid normally Lal reagent water EL = K X N/V K= 20 EU/ device, for cerebrospinal device K= 2.15 EU/device N = Number of device: if batch size is 30-100 test 3 samples, batch size is more than 100 test 3% and up to maximum of 10 V = Total volume of extraction solution

51. Example: Product: Coronary stents Number of devices (N) : 3 K: 20 EU/ device Rinse Volume (V) : 60 ml EL = K X N/V = 20 x 3/ 60 = 1 EU/ mL

52. Maximum Valid Dilution (MVD ): Maximum valid dilution is the maximum allowable dilution of a sample at which the endotoxin limit can be determined. It applies to injections or to solution for parenteral administration in the form of constituted or diluted for administration, or, where applicable, to the amount of drug by weight if the volume of the dosage form for administrated could be varied. MVD = (endotoxin limit × concentration of Sample Solution )/( λ ) The endotoxin limit for parenteral drugs is specified in the individual monograph in units such as EU/mL, EU/mg, EU/Unit of biological activity

53. Concentration of Sample Solution: mg/mL : in the case of endotoxin limit specified by weight (EU/mg ) Units/mL : in the case of endotoxin limit specified by unit of biological activity (EU/Unit ) mL/mL : when the endotoxin limit is specified by volume (EU/mL ). λ : the labeled sensitivity in the Gel-Clot Technique (EU/mL) or the lowest concentration used in the standard curve for the Turbidimetric Technique or Chromogenic Technique

54. Example Product name : Gentamycin Sulphate Concentration : 40 mg/mL Endotoxin Limit : 0.71 EU/mg Lysate Sensitivity ( λ ) : 0.125 EU/mL MVD =40 mg/mL x 0.71 EU/mg ------------------------------- 0.125 EU/mL = 1: 227 ≈ 224

55. PRODUCT INHIBITION:   ● The Limulus Amebocyte Lysate reaction is enzyme mediated and, as such, has an optimal pH range, and specific salt and divalent cation requirements. ● Occasionally test samples may alter these optimal conditions to an extent that the lysate is rendered insensitive to endotoxin. ● Negative results with samples which inhibit the LAL test do not necessarily indicate the absence of endotoxin . Initially , each type of sample should be screened for product inhibition .

56. ● Prepare a series of two-fold dilutions of endotoxin in LAL Reagent Water and a similar series of endotoxin dilutions using sample as diluent. Assay each series in parallel using standard procedures. At the end of the incubation period, record positive and negative results and calculate the geometric mean endpoint for both series of endotoxin dilutions. ● Products are said to be free of product inhibition if the geometric mean endpoint of endotoxin in product is within 1/2 to 2 times the labeled lysate sensitivity. See the following example.

57. Product Inhibition Testing: Sr.No 0.25 0.125 0.06 0.03 1 + + - - 2 + + + - 3 + + - - 4 + + - - geometric mean endpoint = 0.10 EU/ml non-inhibitory Water for Injection: Labelled Lysate sensitivity: 0.125 EU/mL

58. Sr.No 0.25 0.125 0.06 0.03 1 + - - - 2 + + - - 3 + + - - 4 + + - - geometric mean endpoint = 0.15 EU/ml non-inhibitory in Product A

59. in Product B: Sr.No 0.25 0.125 0.06 0.03 1 - - - - 2 - - - - 3 + - - - 4 + - - - geometric mean endpoint = 0.15 EU/ml inhibitory

60. ● The easiest way to overcome product inhibition is through dilution. ● The dilution factor must be taken into account when calculating the total endotoxin concentration in a test sample. ● As a quick screen to determine a non-inhibitory dilution of product, prepare a series of increasing dilutions of the product containing an endotoxin spike equal in concentration to twice the lysate sensitivity. ● Assay each spiked product dilution using standard procedures. Positive results indicate when product inhibition has been overcome.

61. Methods - Bacterial Endotoxin Test Gel Clot Technique: based on gel formation. Turbidimetric Method: based on development of turbidity after cleavage of an endogenous substrate . Chromogenic Method: based on development of colour after cleavage of synthetic peptide chromogen complex .

62. Gel-clot Method: ● The gel-clot method is the simplest and most widely used LAL test. The reaction in the test tube is essentially the same as that in nature when a horseshoe crab is injured. ● The gel-clot technique is used for detecting or quantifying endotoxins based on clotting of the lysate reagent 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 reagent. ● Gel-clot reagent is labelled with a sensitivity(A.) which is the lowest endotoxin concentration to cause a clot to form under standard conditions.

63. ● The accepted error of the method is plus or minus two fold dilution. Therefore, a reagent with a sensitivity of 0.125 EU/ mI may not clot at this concentration in some tests. However, it should always clot at 0.25 EU/ mI. Similarly, the reagent may clot at 0.125EU/ mI and at 0.06EU/ mI , but it should never clot at 0.03 EU/ mI. Because of the twofold error of the method, positive controls and positive product controls (spiked sample) are always at a concentration of 2 λ (twice the labeled sensitivity ). ● The gel-clot test yields a binary result, either positive or negative. A tube is scored as positive (+) if the clot withstands 180° inversion without breaking. All other conditions are scored as negative (-), even if the clot almost remains intact but then collapses.

64. ● The endotoxin concentration of the original sample is calculated by multiplying the reagent sensitivity by the dilution factor at the endpoint. ● Gel-Clot reagent is available in a range of sensitivities. In order to decide which sensitivity to use, a number of factors must be considered. ● First , determine the endotoxin levels or limits to be detected. Clearly the reagent must be sufficiently sensitive to at least detect the limits. Also consider the type of sample and, if possible, the likelihood of it interfering with the test. A greater sensitivity will give increased scope for dilution to overcome interferences . ● Ultimately , the sample must be tested to assure that it does not interfere with the test at a dilution at which the endotoxin limit can be detected.

65. ● The gel-clot method includes a limit test (qualitative) and an endotoxin standard series is prepared using four concentrations of CSE that bracket the lysate sensitivity (λ). ● It is run at least once a day with the first set of tests and repeated if there is any change in lysate lot, endotoxin lot, or test conditions during the day. Please refer to Appendix C: 1987 FDA Guidelines or the USP <85> Bacterial Endotoxins Test for detailed procedures 5 .   ● The gel-clot test is valid when the end-point of the endotoxin standard series is within a + two-fold dilution of the labeled lysate sensitivity (indicating the inherent 2-fold error/variability in the test). ● The test is performed in duplicate. In all gel-clot assays, the PPC must always be positive (form a clot) to be valid.

66. Test for Interfering Factors: ● Usually prepare solutions (A–D) as shown in Table 1, and perform the inhibition/ enhancement test on the Sample Solutions at a dilution less than the MVD, not containing any detectable endotoxins, operating as described for Test for Confirmation of Labeled Lysate Sensitivity. ● The geometric mean endpoint concentrations of Solutions B and C are determined using the formula described in the Test for Confirmation of Labeled Lysate Sensitivity. ● The test is considered valid when all replicates of Solutions A and D show no reaction and the result of Solution C confirms the labeled sensitivity. If the sensitivity of the lysate determined in the presence of Solution B is not less than 0.5 λ and not greater than 2 λ , the Sample Solution does not contain factors that interfere under the experimental conditions used. If the sample under test does not comply with the test at a dilution less than the MVD, repeat the test using a greater dilution, not exceeding the MVD .

67. ● The use of a more sensitive lysate permits a greater dilution of the sample to be examined, and this may contribute to the elimination of interference. Interference may be overcome by suitable treatment such as filtration, neutralization, dialysis, or heating. ● To establish that the chosen treatment effectively eliminates interference without loss of endotoxins, perform the assay described above using the preparation to be examined to which Standard Endotoxin has been added and which has then been submitted to the chosen treatment . ● The test for interfering factors must be repeated when any condition changes that is likely to influence the result of the test.

68. Solution Endotoxin Concentration/ Solution to Which Endotoxin Is Added Diluent Dilution Factor Endotoxin Concentration Number of Replicates A a None/ Sample Solution — — — 4 B b 2 λ / Sample Solution Sample Solution 1 2 λ 4 2 1 λ 4 4 0.5 λ 4 8 0.25 λ 4 C c 2 λ / Water for BET Water for BET 1 2 λ 2       2 1 λ 2       4 0.5 λ 2       8 0.25 λ 2 D d None/ Water for BET — — — 2 Table 1. Preparation of Solutions for the Inhibition/Enhancement Test for Gel-Clot Techniques

69. Solution A: A Sample Solution of the preparation under test that is free of detectable endotoxins . Solution B : Test for interference . Solution C : Control for labeled lysate sensitivity . Solution D : Negative control of Water for BET .

70. Limit Test: Procedure: Prepare Solutions A, B, C, and D as shown in Table 2, and perform the test on these solutions following the procedure above for Preparatory Testing, Test for Confirmation of Labeled Lysate Sensitivity .   Table 2. Preparation of Solutions for the Gel-Clot Limit Test     Solution * Endotoxin Concentration/ Solution to Which Endotoxin Is Added     Number of Replicates A None/Diluted Sample Solution 2 B 2 λ /Diluted Sample Solution 2 C 2 λ /Water for BET 2 D None/Water for BET 2 * Prepare Solution A and the positive product control Solution B using a dilution not greater than the MVD and treatments as described for the Test for Interfering Factors in Preparatory Testing . The positive control Solutions B and C contain the Standard Endotoxin Solution at a concentration corresponding to twice the labeled lysate sensitivity. The negative control Solution D consists of Water for BET.

71. Interpretation: ● The test is considered valid when both replicates of Solutions B and C are positive and those of Solution D are negative. ● When a negative result is found for both replicates of Solution A, the preparation under test complies with the test. ● When a positive result is found for both replicates of Solution A, the preparation under test does not comply with the test . ● When a positive result is found for one replicate of Solution A and a negative result is found for the other, repeat the test. ● In the repeat test, the preparation under test complies with the test if a negative result is found for both replicates of Solution A. The preparation does not comply with the test if a positive result is found for one or both replicates of Solution A. However, if the preparation does not comply with the test at a dilution less than the MVD, the test may be repeated using a greater dilution, not exceeding the MVD.

72. Troubleshooting Guide for Gel Clot Assay Endotoxin Mixing: ● Endotoxins that are used as controls with LAL tests are very large, highly purified molecules that have unique properties affecting their behaviour in aqueous solutions. A portion of the molecule is very water-soluble and another part is not. For this reason, endotoxin molecules in solution tend to aggregate. When a solution containing endotoxin is withdrawn, such as when making serial dilutions for endotoxin testing, the endotoxin must be well dispersed or the portion withdrawn may not contain the proper amount of endotoxin. Thorough mixing of endotoxin and accurate transfer throughout a dilution series are best accomplished by observing the points noted below .

73. 1. Make endotoxin dilutions in increments no greater than 1:10. This will increase the chance of obtaining the correct amount of endotoxin in the withdrawn portion and decrease the extent to which it will have to be dispersed when it is diluted . 2 . Adhere strictly to the agitation and vortex mixing times specified for each step in the endotoxin dilution instructions. Vortex mixing should be done at the “high” speed setting in order to disperse endotoxin aggregates. The aliquot for subsequent dilution should be withdrawn immediately after mixing, and the endotoxin sample should be thoroughly mixed just prior to use. NOTE : It is not recommended to vortex for more than 30 minutes in one 8 hour period to avoid damage to the endotoxin and loss of potency . B. Pipetting Accuracy Pipetting accuracy is exceptionally important during every phase of LAL testing. Ensure you are adding the proper amounts of material by utilizing high quality graduated pipettes and pipette tips and the proper technique for the pipette you have chosen. Pipettes should be recalibrated annually at a minimum.

74. C . Conditions for Incubation of LAL Test Tubes: • Check the temperature of the water bath or heating block. • The LAL reaction with endotoxin is temperature dependent and the specified standard conditions for the test require one hour incubation at 37°C ±1°C . • The critical temperature is that of the material inside the reaction tube. An accurate check can be made by placing a calibrated thermometer inside a 75 x 10 mm test tube containing just enough water to cover the thermometer bulb.

75. • Place the tube and thermometer in a test tube incubation rack or a heating block and observe the temperature over a one-hour period. Check the location of the water bath or heating block. • While the gel is forming, it is quite fragile and may be irreversibly broken by mechanical shock or vibration. Small motions, including closing a door or drawer in your work bench or a centrifuge running can cause sufficient vibration to affect the development of the clots. • Generally speaking, any motion of the incubating test tubes must be regarded as a potential problem source. Agitating or circulating water baths must not be used for this reason.

76. D . Test Results A positive result is a firm gel that maintains its integrity through inversion to 180°. When reading test results after the one-hour incubation period, gently withdraw each tube and invert it 180° in one smooth motion. Do not stop at 45° or 90° to look before continuing inversion, and be mindful of adjacent tubes when removing them from the heat block or tube rack . E. Test Reagent Handling Before reconstitution, the PYROGENT™ Gel Clot LAL reagent is very stable but should be stored in the dark at 2 – 8°C (note expiration date). The lysate reagent, when reconstituted, is a buffered solution containing enzymes and other proteins from the Limulus amebocyte . Preparation, handling and storage are critical and should be approached keeping the following points in mind:

77. 1. The water used to rehydrate the PYROGENT™ Gel Clot LAL reagent should be LAL Reagent Water (LRW ). Water for Injection (WFI) is generally not acceptable as it is not tested to the proper endotoxin concentrations for endotoxin content nor has it been tested for assay performance compatibility. Furthermore, LRW manufactured by other endotoxin detection companies may not be compatible with the LAL gel clot reagents. If it is necessary to use reagent water other than a Lonza product, it must be thoroughly validated prior to use in testing samples. 2. Pipetting technique must be aseptic and apyrogenic . Even when rehydration water is pyrogen -free, it can easily become contaminated unless appropriate precautions are observed. Aseptic and apyrogenic techniques consist of precautions to avoid the entry of gross amounts of bacterial and/or endotoxin contamination. A great deal of endotoxin may be present on surfaces (hands, glassware, etc.) even when few or no viable bacteria are found. For this reason, labware must be treated to remove endotoxin and handled carefully to prevent contamination.

78. 3. Rehydrated PYROGENT™ Gel Clot LAL reagent may be stored for as long as 24 hours, but it must be kept at 2 – 8°C. The lysate should not be pre-incubated in the LAL test tubes before adding samples to them, as this may cause sensitivity loss in the PYROGENT™ Gel Clot LAL reagent . If rehydrated lysate must be stored for a longer period (up to four weeks), it must be kept frozen at –10°C or below and checked to ensure the lysate is frozen solid in the tubes. Many frost-free freezers have surface heating cycles that may periodically warm articles in contact with the heated surfaces. Repeated freezing and thawing of the lysate will result in deterioration well before four weeks have passed. 4. Lyophilized E. coli endotoxin should be stored at 2 – 8°C. Endotoxin solutions are generally very stable upon storage except when highly diluted. This is related to the solubility characteristics discussed earlier. Rehydrated E. coli endotoxin should be stored refrigerated only at 2 – 8°C and not be frozen. The stock solution kept for up to four weeks. Further dilutions of E. coli endotoxin may begin to lose potency after a few days.

79. The storage stability of dilute endotoxins may be related to the cleanliness of the glassware in which they are kept, as endotoxin can bind on irregular or dirty glass surfaces. Since some types of plastic have been shown to have an affinity for endotoxin, the use of plastic containers to prepare and store endotoxin must be approached with caution. The precautions for technique, water and pipettes discussed previously (for the lysate rehydration) also apply to endotoxin rehydration . 5. Diluents other than water or saline may also cause inhibition. When all water controls behave as expected in the LAL test but a specific sample fails to give positive results when prepared to contain the specified endotoxin concentrations, true inhibition has been recognized 6. Handling of samples is as critical as the handling of reagents, and cleanliness is important to ensure the use of aseptic and apyrogenic techniques. Always check the product for inhibition as some products, especially members of the small volume parenteral family, may contain substances at great enough concentrations to inhibit the LAL gelation reaction .

80. In addition, the pH of the sample to be tested should be checked and should be in the range of 6.0 – 8.0. A pH outside of this range is one of the most common causes of product inhibition in the LAL test . The PYROGENT™ Gel Clot LAL reagent does have a certain amount of buffering capacity, but it must be assured that a product, when added to the PYROGENT™ Gel Clot LAL reagent, does not exceed that capacity. An easy way to determine whether or not a given product will require pH adjustment is simply add a volume of the product in question to an equal volume of rehydrated PYROGENT™ Gel Clot LAL reagent and check the pH to determine if it falls between 6.0 and 8.0. Remember that because of PYROGENT™ Gel Clot LAL reagent’s buffering capacity, there is no need to adjust the pH of WFI or Saline for Injection USP. If a pH adjustment is necessary, Lonza Tris Buffer ( Lonza Catalog No: S50-642) is available for this use. Tris Buffer is a convenient way to adjust pH when the buffering capacity of the sample will allow it.

81. Category II: Problem Sources • The majority of Category II problems is caused by failure to observe proper handling technique, failure to use adequate equipment preparation procedures, and/or use of contaminated water in the test. • The extreme sensitivity of LAL to endotoxin is regarded as its greatest virtue, but this sensitivity can also be a source of problems when careless technique is used. • Care in preparation and handling of PYROGENT™ Gel Clot LAL reagent and test samples is essential. Any surface or substance that could come into contact with either the LAL or the sample must be free of endotoxins. This need was discussed previously and is re-emphasized here. Remember that endotoxin is virtually everywhere in nature. As a rule, when testing parenteral pharmaceutical products, if a firm gel appears in the LAL test, it must be assumed that endotoxin is present. • Use of a negative control as part of the test will help you determine if positive results are obtained due to endotoxin in the test sample or to inadvertent contamination. • During inhibition/enhancement testing, another indication of contamination may appear when you have prepared a series of dilutions of endotoxin in a product and another series of endotoxin in LRW. If the endpoint of the LAL assay of endotoxin in product is more than a two-fold dilution lower in concentration than the endpoint of the water-endotoxin dilution series, contamination of the product might be suspected. Further testing is necessary to exclude enhancement .