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8.5 TEST FOR BACTERIAL ENDOTOXINS

8.5 TEST FOR BACTERIAL ENDOTOXINS

      The test for bacterial endotoxins is designed to detect or quantify bacterial endotoxins of gram-negative bacterial origin that may be present in or on the sample to which the test is applied. It uses Limulus Amoebocyte Lysate (LAL) obtained from the aqueous extracts of circulating amoebocytes of horseshoe crab (Limulus polyphemus or Tachypleus tridentatus) which has been prepared and characterized for use as an LAL Reagent or a lysate reagent.

      There are two types of techniques for this test: the gel-clot techniques, which are based on gel formation, and the photometric techniques. The latter include a turbidimetric method, which is based on the development of turbidity after cleavage of an endogenous substrate, and a chromogenic method, which is based on the development of colour after cleavage of a synthetic peptide-chromogen complex. Any one of these techniques for the test may be followed. In the event of doubt or dispute, the final decision is made based on the gel-clot techniques, unless otherwise indicated.

      The test is carried out in a manner that avoids microbial contamination.

Apparatus and Glassware

      Depyrogenate all glassware and other heat-stable materials in a hot-air oven using a validated process. Commonly used time and temperature setting are not less than 30 minutes at 250º or not less than 1 hour at 200º. If employing plastic apparatus, such as multi-well plates and tips for micropipettes, use only that which has been shown to be free of detectable endotoxin and not to interfere with the test.

Preparation of the Solutions

      Preparation of the standard endotoxin stock solution Prepare standard endotoxin stock solution from an endotoxin reference standard that has been calibrated against the International Standard or equivalence. Endotoxin is expressed in Endotoxin Unit (EU). One Endotoxin Unit (EU) is equal to one International Unit (IU) of endotoxin as well.

      Follow the specifications in the package leaflet and on the label for preparation and storage of the standard endotoxin stock solution.

      Preparation of the standard endotoxin solution After vigorously mixing the standard endotoxin stock solution as recommended by the manufacturer, prepare appropriate serial dilutions of this solution using water for bacterial endotoxins test (LAL Reagent Water). Use the solutions as soon as possible to avoid loss of activity.

      Preparation of sample solutions Unless otherwise specified, prepare sample solutions by dissolving or diluting drugs or extracting medical devices using LAL Reagent Water. Some substances or preparations may be more appropriately dissolved, diluted, or extracted in other aqueous solutions. If necessary, adjust the pH of the solution to be examined so that the pH of the mixture of the LAL Reagent and sample solution falls within the specified pH range for the LAL Reagent to be used. This usually applies to a sample solution with a pH in the range of 6.0 to 8.0. The pH may be adjusted using an acid, base, or suitable buffer as recommended by the LAL Reagent manufacturer. Buffers must be validated to be free of detectable endotoxin and interfering factors.

Determination of Maximum Valid Dilution (MVD)

      The Maximum Valid Dilution is the maximum allowable dilution of a sample solution at which the endotoxin limit can be determined. The general equation to determine MVD is:

      Endotoxin limit The endotoxin limit for active substances administered parenterally, defined on the basis of dose, is equal to: K/M, where K is a threshold pyrogenic dose of endotoxin per kilogram of body weight (EU/kg) in a single hour period, and M is equal to maximum recommended dose of product per kilogram of body weight in a single hour period. (Note K is 5 EU/kg for any route of administration other than intrathecal (for which K is 0.2 EU/kg body weight). For radiopharmaceutical products not administered intrathecally the endotoxin limit is calculated as 175/V, where V is the maximum recommended dose in ml. For intrathecally administered radiopharmaceuticals, the endotoxin limit is obtained by the formula 14/V. For formulations (usually anticancer products) administered on a per square metre of body surface area, the formula is K/M, where K = 5 EU/kg and M is the (maximum dose/m2 /hour × 1.80 m2 )/70 kg).

      Concentration of sample solution

– in mg/ml if the endotoxin limit is specified by mass (EU/mg),

– in mEq/ml if the endotoxin limit is specified by equivalent (EU/mEq),

– in Units/ml if the endotoxin limit is specified by biological unit (EU/Unit),

– in ml/ml if the endotoxin limit is specified by volume (EU/ml)

      λ The labelled lysate sensitivity in the gel-clot techniques (EU/ml) or the lowest point used (EU/ml) in the standard curve of the turbidimetric or chromogenic techniques.

Gel-Clot Techniques 

      The gel-clot techniques detect or quantify endotoxins based on clotting of the LAL Reagent in the presence of endotoxin. The concentration of endotoxin required to cause the lysate to clot under standard conditions is the labelled sensitivity of LAL Reagent. To ensure both the precision and validity of the test, perform the tests for confirming the labelled LAL Reagent sensitivity and for interfering factors as described under Preparatory testing for the gel-clot techniques.

Preparatory testing for the gel-clot techniques

      TEST FOR CONFIRMATION OF LABELLED LAL REAGENT SENSITIVITY Confirm in four replicates the labelled lysate sensitivity λ, expressed in EU/ml, of the lysate solution prior to use in the test. Confirmation of the lysate sensitivity is carried out when a new batch of lysate is used or when there is any change in the experimental conditions which may affect the outcome of the test.

      Prepare standard solutions having four concentrations equivalent to 2λ, 1λ, 0.5λ and 0.25λ by diluting the Standard Endotoxin Stock Solution with LAL Reagent Water. Prepare the lysate solution by dissolving the LAL Reagent with LAL Reagent Water or a suitable buffer. Mix a volume of the lysate solution with an equal volume of one of the standard solutions (usually, 0.1 ml aliquots) in each tube. When single test vials or ampoules containing lyophilized lysate are used, add solutions directly to the vial or ampoule. Incubate the reaction mixture for a constant period according to the recommendations of the lysate manufacturer (usually at 37º±1º for 60±2 minutes), avoiding vibration. To test the integrity of the gel after incubation, invert each tube or container through about 180º 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 either a firm gel is not formed, or if a fragile gel has formed but flows out upon inversion.

      The test is not valid unless the lowest concentration of the standard solutions (0.25λ) shows a negative result in all replicate tests.

      The end-point is the last positive test in the series of decreasing concentrations of endotoxin. Calculate the mean value of the logarithms of the end-point concentration and then the antilogarithm of the mean value using the following equation:

Geometric Mean End-point Concentration = antilog (Σe/f),

where Σe is the sum of the log end-point concentrations of the dilution series used, and f is the number of replicate test-tubes.

      The geometric mean end-point concentration is the measured sensitivity of the lysate solution (EU/ml). If the geometric mean end-point concentration is not less than 0.5λ and not more than 2λ, the labelled lysate sensitivity is confirmed.

      TEST OF INTERFERING FACTORS FOR THE GEL-CLOT TECHNIQUES The test is performed to check for the presence of enhancing or inhibiting factors for the reaction in sample solutions. Prepare solutions A, B, C, and D as shown in Table 1, and use the sample solutions at a dilution less than the MVD, not containing any detectable endotoxins, following the procedure in the Test for confirmation of labelled LAL Reagent sensitivity above. The geometric mean end-point concentrations of solutions B and C are determined using the equation in that test.

      The test must be repeated when any changes are made to the experimental conditions that are likely to influence the result of the test. The test is not valid unless all replicates of Solutions A and D show no reaction and the result of Solution C confirms the labelled lysate sensitivity.

      If the sensitivity of the lysate determined with Solution B is not less than 0.5λ and not greater than 2λ, the sample solution does not contain interfering factors under the experimental conditions used. Otherwise, the sample solution to be examined interferes with the test.

      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. 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, repeat the test for interfering factors using the preparation being examined to which the standard endotoxin has been added and which has been subjected to the selected treatment. 

Table 1 Preparation of Solutions for the Inhibition/Enhancement Test for Gel-Clot Techniques

      Gel-clot limit test Based on the formation of a firm gel in the presence of endotoxin at above labelled LAL Reagent sensitivity, this method tests whether a sample solution contains endotoxin not greater than the endotoxin limit.

      PROCEDURE Prepare Solutions A, B, C, and D as shown in Table 2, and perform the test on these solutions following the procedure in the Test for confirmation of labelled LAL Reagent sensitivity under Preparatory testing for the gel-clot techniques.

      INTERPRETATION The test is not valid unless both replicates of positive control Solutions B and C are positive and those of negative control Solution D are negative. The sample complies with the test when a negative result is found for both tubes containing Solution A. The sample does not comply with the test when a positive result is found for both tubes containing Solution A.

      Repeat the test when a positive result is found for one tube containing Solution A and a negative result for the other one. The sample complies with the test when a negative result is found for both tubes containing Solution A in the repeat result. If the test is positive for the sample at a dilution less than the MVD, the test may be repeated at a dilution not greater than the MVD.

      Gel-clot assay The test measures endotoxin concentrations of sample solution by titration to an end-point of gel formation.

      PROCEDURE Prepare Solutions A, B, C, and D as shown in Table 3, and test these solutions by following the procedure in the Test for confirmation of labelled LAL reagent sensitivity under Preparatory testing for the gel-clot techniques.

      CALCULATION AND INTERPRETATION The test is not valid unless the following conditions are met: (1) both replicates of negative control Solution D are negative; (2) both replicates of positive product control Solution B are positive; and (3) the geometric mean end-point concentration of Solution C is in the range of 0.5λ to 2λ. 

Table 2 Preparation of Solutions for the Gel-Clot Limit Test

Table 3 Preparation of Solutions for the Gel-Clot Assay​

To determine the endotoxin concentration of Solution A, calculate the end-point concentration for each replicate series of dilutions by multiplying each end-point dilution factor by λ.

      The endotoxin concentration in the sample is the geometric mean end-point concentration of the replicates (see the equation given in the Test for confirmation of labelled LAL reagent sensitivity under Preparatory testing for the gel-clot techniques). If the test is conducted with a diluted sample solution, calculate the concentration of endotoxin in the original sample solution by multiplying by the dilution factor.

      If none of the dilutions of the sample solution is positive in a valid assay, report the endotoxin concentration as less than λ (or, if the diluted sample was tested, less than λ × the lowest dilution factor of the sample). If all dilutions are positive, the endotoxin concentration is reported as equal to or greater than the greatest dilution factor multiplied by λ.

      The sample meets the requirements of the test if the endotoxin concentration is less than that specified in the individual monograph.

Photometric Techniques

      Photometric techniques which include turbidimetric and chromogenic require the establishment of a standard regression curve and the endotoxin content of the sample is determined by interpolation from the curve. The procedures include incubation for a preselected time of reacting endotoxin and control solutions with the LAL Reagent and reading of the spectrophotometric light absorbance at suitable wavelengths.

      The turbidimetric technique measures the endotoxin concentrations of sample solution based on the measurement of turbidity change accompanying gel formation of the LAL Reagent. The technique is classified as either endpoint-turbidimetric or kinetic-turbidimetric. The endpoint-turbidimetric technique is based on the quantitative relationship between the concentration of endotoxins and the turbidity (absorbance or transmission) of the reaction mixture at the end of an incubation period. The kinetic-turbidimetric technique is based on the quantitative relationship between the concentration of endotoxins and either the time needed to reach a predetermined turbidity of the reaction mixture or the rate of turbidity development.

      The chromogenic method measures the endotoxin concentrations of sample solutions based on the measurement of chromophore released from a suitable chromogenic peptide by the reaction of endotoxins with the LAL Reagent. This technique is classified as either end-point-chromogenic or kinetic-chromogenic. The end-point-chromogenic technique is based on the quantitative relationship between the concentration of endotoxins and the release of chromophore at the end of an incubation period. The kinetic-chromogenic technique is based on the quantitative relationship between the concentration of endotoxins and either the time needed to reach a predetermined absorbance (or transmittance) of the reaction mixture or the rate of colour development.

      All photometric tests are carried out at the incubation temperature recommended by the LAL Reagent manufacturer, which is usually 37º±1º.

      Preparatory testing for the photometric techniques To assure the precision or validity of the turbidimetric and chromogenic techniques, preparatory tests are conducted to verify that the criteria for the standard curve are valid and that the sample solution does not inhibit or enhance the reaction. Revalidation for the test method is required when conditions that are likely to influence the test result change.

      VERIFICAITON OF CRITERIA FOR THE STANDARD CURVE Using the Standard Endotoxin Solution, prepare at least three endotoxin concentrations to generate the standard curve. Perform the test using at least three replicates of each standard endotoxin concentration according to the manufacturer’s instructions for the LAL Reagent (with regard to volume ratios, incubation time, temperature, pH, etc.). If the desired range in the kinetic methods is greater than two logs, additional standards should be included to bracket each log increase within the range of the standard curve.

      The absolute value of the correlation coefficient, |r|, must be greater than or equal to 0.980 for the range of endotoxin concentrations set up.

      INTERFERING FACTORS TEST FOR THE PHOTOMETRIC TECHNIQUES Select an endotoxin concentration at or near the middle of the endotoxin standard curve. Prepare Solutions A, B, C, and D as shown in Table 4. Perform the test on Solutions A, B, C, and D at least in duplicate following the instructions for the LAL Reagent used (with regard to volume of sample and LAL Reagent, volume ratio of sample to LAL Reagent, incubation time, etc.).

      Calculate the mean recovery of the added endotoxin by subtracting the mean endotoxin concentration in the solution (if any) from that containing the added endotoxin. In order to be considered free of interfering factors under the conditions of the test, the measured concentration of the endotoxin added to the sample solution must be within 50 per cent to 200 per cent of the known added endotoxin concentration after subtraction of any endotoxin detected in the solution without added endotoxin.

      When the endotoxin recovery is out of the specified ranges, the interfering factors must be removed as described in the Test of interfering factors for the gelclot techniques under Preparatory testing for the gel clot techniques. The efficiency of the treatment is verified by repeating the Test of interfering factors for the gelclot techniques.

Table 4 Preparation of Solutions for the Inhibition/Enhancement Test for Photometric Techniques

      Procedure for the photometric techniques Follow the procedure described in the Test of interfering factors for the photometric techniques under Preparatory testing for the photometric techniques.

      Calculation for the photometric techniques Calculate the endotoxin concentration of each of the replicates of test Solution A using the standard curve generated by positive control series C. The test is not valid unless the following conditions are met: (1) the results of control series C comply with the requirements for validation defined under Verification of criteria for the standard curve under Preparatory testing for the photometric techniques; (2) the endotoxin recovery, calculated from the concentration found in Solution B after subtracting the endotoxin concentration found in Solution A is within 50 to 200 per cent; and (3) the result of negative control series D does not exceed the limit of the blank value required in the description of the LAL Reagent used.

      Interpretation of results from the photometric techniques In photometric assays, the preparation under test 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.

      Reagents

      AMOEBOCYTE LYSATE (LAL) is a lyophilized product obtained for amoebocyte lysate from horseshoe crab. The reagent refers only to a product manufactured in accordance with the regulations of the competent authority. Amoebocyte lysate reacts with some βglucans in addition to endotoxins. Some preparations which do not react with glucans are available; they are prepared by removing from amoebocyte lysate the G factor, which reacts with glucans, or by inhibiting the G factor reacting system of amoebocyte lysate. These preparations may be used for endotoxin testing in the presence of glucans.

      LAL REAGENT WATER (water for bacterial endotoxin test) is Sterile Water for Injection or other types of water that show no reaction with the LAL Reagent used, at the detection limit of the reagent.  

APPENDICES • 8.5 TEST FOR BACTERIAL ENDOTOXINS
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หมายเหตุ / Note : TP II 2011 PAGE 522-527