85 BACTERIAL ENDOTOXINS TEST Portions of this general chapter have been harmonized with the corresponding texts of the European Pharmacopeia and/or the Japanese Pharmacopeia. Those portions that are not harmonized are marked with symbols ( ) to specify this fact. This chapter provides a test to detect or quantify bacterial endotoxins that may be present in or on the sample of the article(s) to which the test is applied. It uses Limulus Amebocyte Lysate (LAL) obtained from the aqueous extracts of circulating amebocytes of horseshoe crab (Limulus polyphemus or Tachypleus tridentatus) which has been prepared and characterized for use as an LAL Reagent. 1 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 color after cleavage of a synthetic peptide-chromogen complex. Proceed by any one of these techniques, unless otherwise indicated in the monograph. In case of dispute, the final decision is based on the gel-clot techniques, unless otherwise indicated in the monograph. In the gel-clot techniques, the reaction endpoint is determined from dilutions of the material under test in direct comparison with parallel dilutions of a reference endotoxin, and quantities of endotoxin are expressed in USP Endotoxin Units (USP-EU). [NOTE—One USP-EU is equal to one IU of endotoxin.] Because LAL Reagents have been formulated to be used also for turbidimetric or colorimetric tests, such tests may be used to comply with the requirements. These tests require the establishment of a standard regression curve; the endotoxin content of the test material is determined by interpolation from the curve. The procedures include incubation for a preselected time of reacting endotoxin and control solutions with LAL Reagent and reading of the spectrophotometric light absorbance at suitable wavelengths. In the endpoint turbidimetric procedure the reading is made immediately at the end of the incubation period. In the endpoint colorimetric procedure the reaction is arrested at the end of the preselected time by the addition of an enzyme reaction-terminating agent prior to the readings. In the turbidimetric and colorimetric kinetic assays the absorbance is measured throughout the reaction period and rate values are determined from those readings.
APPARATUS AND GLASSWARE Depyrogenate all glassware and other heat-stable materials in a hot-air oven using a validated process. 2 Commonly used minimum time and temperature settings are 30 minutes at 250 . If employing plastic apparatus, such as microplates and pipet tips for automatic pipetters, use only that which has been shown to be free of detectable endotoxin and not to interfere with the test. [NOTE—In this chapter, the term “tube” includes any other receptacle such as a micro-titer well.]
PREPARATION OF THE STANDARD ENDOTOXIN STOCK SOLUTION AND STANDARD SOLUTIONS The USP Endotoxin RS has a defined potency of 10,000 USP Endotoxin Units (EU) per vial. Constitute the entire contents of 1 vial of the RSE with 5 mL of LAL Reagent Water 3 , mix intermittently for 30 minutes, using a vortex mixer, and use this concentrate for making appropriate serial dilutions. Preserve the concentrate in a refrigerator for making subsequent dilutions for not more than 14 days. Mix vigorously, using a vortex mixer, for not less than 3 minutes before use. Mix each dilution for not less than 30 seconds before proceeding to make the next dilution. Do not store dilutions, because of loss of activity by adsorption, in the absence of supporting data to the contrary. Preparatory Testing Use an LAL Reagent of confirmed label sensitivity. The validity of test results for bacterial endotoxins requires an adequate demonstration that specimens of the article or of solutions, washings, or extracts thereof to which the test is to be applied do not of themselves inhibit or enhance the reaction or otherwise interfere with the test. Validation is accomplished by performing the inhibition or enhancement test described under each of the three techniques indicated. Appropriate negative controls are included. Validation must be repeated if the LAL Reagent source or the method of manufacture or formulation of the article is changed. Preparation of Sample Solutions 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 (or dilution thereof) to be examined so that the pH of the mixture of the LAL Reagent and sample falls within the pH range specified by the LAL Reagent manufacturer. This usually applies to a product 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. Acids and bases may be prepared from concentrates or solids with LAL Reagent Water in containers free of detectable endotoxin. 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 specimen at which the endotoxin limit can be determined. It applies to injections or to solutions for parenteral administration in the form constituted or diluted for administration, or, where applicable, to the amount of drug by weight if the volume of the dosage form for administration could be varied. The general equation to determine MVD is: MVD = (Endotoxin limit × Concentration of sample solution)/( ) where the concentration of sample solution and are as defined below. Where the endotoxin limit concentration is specified in the individual monograph in terms of volume (in EU per mL), divide the limit by , which is the labeled sensitivity (in EU per mL) of the LAL Reagent, to obtain the MVD factor. Where the endotoxin limit concentration is specified in the individual monograph in terms of weight or Units of active drug (in EU per mg or in EU per Unit), multiply the limit by the concentration (in mg per mL or in Units per mL) of the drug in the solution tested or of the drug constituted according to the label instructions, whichever is applicable, and divide the product of the multiplication by , to obtain the MVD factor. The MVD factor so obtained is the limit dilution factor for the preparation for the test to be valid.
ESTABLISHMENT OF ENDOTOXIN LIMITS The endotoxin limit for parenteral drugs, defined on the basis of dose, is equal to K/M, 4 where K is the threshold human pyrogenic dose of endotoxin per kg of body weight, and M is equal to the maximum recommended human dose of product per kg of body weight in a single hour period. The endotoxin limit for parenteral drugs is specified in individual monographs in units such as EU/mL, EU/mg, or EU/Unit of biological activity.
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 labeled sensitivity of the LAL Reagent. To ensure both the precision and validity of the test, tests for confirming the labeled LAL Reagent sensitivity and for interfering factors are described under Preparatory Testing for the Gel-Clot Techniques. Preparatory Testing for the Gel-Clot Techniques Test for Confirmation of Labeled LAL Reagent Sensitivity— Confirm the labeled sensitivity using at least 1 vial of the LAL Reagent lot. Prepare a series of two-fold dilutions of the USP Endotoxin RS in LAL Reagent Water to give concentrations of 2 , , 0.5 , and 0.25 , where is as defined above. Perform the test on the four standard concentrations in quadruplicate and include negative controls. The test for confirmation of lysate sensitivity is to be carried out when a new batch of LAL Reagent is used or when there is any change in the experimental conditions that may affect the outcome of the test. Mix a volume of the LAL Reagent with an equal volume (such as 0.1-mL aliquots) of one of the standard solutions in each test tube. When single test vials or ampuls containing lyophilized LAL Reagent are used, add solutions directly to the vial or ampul. Incubate the reaction mixture for a constant period according to directions of the LAL Reagent manufacturer (usually at 37 ± 1 for 60 ± 2 minutes), 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. 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 test is not valid unless the lowest concentration of the standard solutions shows a negative result in all replicate tests. The endpoint is the last positive test in the series of decreasing concentrations of endotoxin. Calculate the mean value of the logarithms of the endpoint concentration and then the antilogarithm of the mean value using the following equation: Geometric Mean Endpoint Concentration = antilog (e / f) where e is the sum of the log endpoint concentrations of the dilution series used, and f is the number of replicate test tubes. The geometric mean endpoint concentration is the measured sensitivity of the LAL Reagent (in EU/mL). If this is not less than 0.5 and not more than 2 , the labeled sensitivity is confirmed and is used in tests performed with this lysate. Interfering Factors Test for the Gel-Clot Techniques— Prepare solutions A, B, C, and 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, following the procedure in the Test for Confirmation of Labeled LAL Reagent Sensitivity above. The geometric mean endpoint concentrations of solutions B and C are determined using the equation in that test.
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