细菌内毒素USP上课讲义

BACTERIAL ENDOTOXINS TEST <85USP35*Porti ons of this gen eral chapter have bee n harm oni zed with the corresp onding texts ofthe European Pharmacopoeia and/or the Japanese Pharmacopoeia. Those portions that are not harmonized are marked with symbols ( **) to specify this fact. +这一章节已经和欧洲药典(EP)与日本药典(JP)统一。

没有统一的部分已经用**标记出来。

The Bacterial En dotox ins Test (BET) is a test to detect or qua ntify en dotox ins from Gram-n egative bacteria using amoebocyte lysate from the horseshoe crab (Limulus polyphemus or Tachypleus tride ntatus).细菌内毒素检查法(BET)是通过鲎的阿米巴细胞溶解产物来检测或定量来自革兰氏阴性菌的内毒素。

There are three tech niq ues for this test: the gel-clot tech niq ue, which is based on gel formati on; the turbidimetric tech niq ue, based on the developme nt of turbidity after cleavage of an en doge nous substrate; and the chromoge nic tech niq ue, based on the developme nt of color after cleavage of a syn thetic peptide-chromoge n complex. Proceed by any of the three tech niq ues for the test. In the eve nt of doubt or dispute, the final decisi on is made based upon the gel-clot tech nique uni ess otherwise in dicated in the mono graph for the product being tested. The test is carried out in a manner that avoids en dotox in con tam in ati on.细菌内毒素检查法有3种：凝胶法，基于凝胶的形成；浊度法，BACTERIAL ENDOTOXINS TESTUSP32^Portions of this gen eral chapter have bee n harm oni zed with the corresp onding texts of the Europea n Pharmacopeia an d/or the Japa nese Pharmacopeia. Those portions that are not harm oni zed are marked with symbols (*■♦■) to specify this fact. +This chapter provides a test to detect or qua ntify bacterial en dotox ins that may be prese nt in or on the sample of the article(s) to which the test is applied. It uses Limulus Amebocyte Lysate (LAL) obta ined from the aqueous extracts of circulati ng amebocytes of horseshoe crab ( Limulus polyphemus or Tachypleus tridentatus ) which has been prepared and characterized for use as an LAL Reage nt 余1+There are two types of tech niq ues for this test: the gel-clot tech niq ues, which are based on gel formation, and the photometric techniques. The latter include a turbidimetric method, which is based on the developme nt of turbidity after cleavage of an en doge nous substrate, and a chromoge nic method, which is based on the developme nt of color after cleavage of a syn thetic peptide-chromoge n complex. Proceed by any one of these tech niq ues, uni ess otherwise in dicated in the mono graph. In case of dispute, the final decisi on is based on the gel-clot tech niq ues, uni ess otherwise in dicated in the mono graph.In the gel-clot tech niq ues, the react ion en dpo int is determ ined from diluti ons of the material un der test i n direct comparis on with parallel diluti ons of a reference en dotox in, and qua ntities of endotoxin are expressed in USP Endotoxin Units (USP-EU). [NOTE—One USP-EU is equal to one IU of en dotox in.]Because LAL Reage nts have bee n formulated to be used also for turbidimetric or colorimetric tests, such tests may be used to comply with the requireme nts. These tests require the establishme nt of a sta ndard regressi on curve; the en dotox in content of the test material is determ ined by in terpolati on from the curve. The procedures in clude in cubati on for a preselected time of react ing en dotox in and con trol soluti ons with LAL Reage nt and readi ng of the spectrophotometric light absorba nee at suitable wavele ngths. In the en dpo int turbidimetric procedure the reading is made immediately at the end of the incubation period. In the endpoint colorimetric procedure the react ion is arrested at the end of the preselected time by the additi on of an en zyme react ion-term in at ing age nt prior to the readi ngs. In the turbidimetric and colorimetric kin etic assays the absorba nee is measured throughout the react ion period and rate values are determ ined from those read in gs.APPARATUS AND GLASSWAREDepyrogenate all glassware and other heat-stable materials in a hot-air oven using a validated * 口process. 稣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 bee n show n to be free of detectable en dotox in and not to in terfere with thetest. [NOTE—In this chapter, the term f Ube ” in eludes any other receptacle such as a micro-titer well.]PREPARATION OF THE STANDARD ENDOTOXIN STOCK SOLUTION AND STANDARD SOLUTONS The USP Endotoxin RShas a defined potency of 10,000 USP Endotoxin Units (EU) per vial.Con stitute the en tire contents of 1 vial of the RSE with 5 mL of LAL Reage nt Water , mix intermittently for 30 minutes, using a vortex mixer, and use this concentrate for making appropriateserial diluti ons. Preserve the concen trate in a refrigerator for making subseque nt dilutions for notmore than 14 days. Mix vigorously, using a vortex mixer, for not less than 3 minutes before use. Mixeach dilution for not less than 30 seconds before proceeding to make the n ext diluti on. Do not storediluti ons, because of loss of activity by adsorpti on, in the abse nee of support ing data to the contrary.Preparatory Test ingUse an LAL Reage nt of con firmed label sen sitivity.The validity of test results for bacterial en dotox ins requires an adequate dem on strati on that specimens of the article or of solutions, washings, or extracts thereof to which the test is to be applieddo not of themselves in hibit or enhance the react ion or otherwise in terfere with the test. Validati onis accomplished by perform ing the in hibiti on or enhan ceme nt test described un der each of thethree techniques indicated. Appropriate negative controls are included. Validation must be repeated ifthe LAL Reage nt source or the method of manufacture or formulatio n of the article is cha nged.Preparatio n of Sample Soluti onsPrepare sample solutions by dissolving or diluting drugs or extracting medical devices using LALReage nt Water. Some substa nces or preparatio ns may be more appropriately dissolved, diluted, or extracted in other aqueous soluti on s. If n ecessary, adjust the pH of the soluti on (or dilutio nthereof) to be exam ined so that the pH of the mixture of the LAL Reage nt and sample falls within thepH range specified by the LAL Reage nt manu facturer. This usually applies to a product with a pH inthe range of 6.0 to 8.0. The pH may be adjusted using an acid, base, or suitable buffer as recommended by the LAL Reage nt manu facturer. Acids and bases may be prepared from concen trates orsolids with LAL Reage nt Water in containers free of detectable en dotox in.Buffers must be validated to be free of detectable en dotox in and in terferi ng factors.DETERMINATION OF MAXIMUM VALID DILUTION (MVD)The Maximum Valid Dilution is the maximum allowable dilution of a specimen at which the en dotoxin limit can be determ in ed. It applies to injectio ns or to soluti ons for pare nteral admi nistrati on inthe form con stituted or diluted for adm ini strati on, or, where applicable, to the amount of drug byweight if the volume of the dosage form for adm ini strati on could be varied. The general equation to determine MVD is:MVD = (En dotox in limit x Concen trati on of sample solutio n)/( k )where the concen trati on of sample solutio n and 二are as defi ned below. Where the en dotox in limit concen trati on is specified in the in dividual mono graph in terms of volume (in EU per mL), divide the limit by 九,which is the labeled sen sitivity (in EU per mL) of the LAL Reage nt, to obta in the MVD factor. Where the en dotox in limit concen trati on is specified in the in dividual mono graph in terms of weight or Un its of active drug (in EU per mg or in EU per Un it), multiply the limit by the concen trati on (in mg per mL or in Un its per mL) of the drug in the soluti on tested or of the drug con stituted accord ing to the label in struct ions, whichever is applicable, and divide the product of the multiplication by 入,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 LIMITSThe en dotox in limit for paren teral drugs, defi ned on the basis of dose, is equal to K/M,*4* where K is the threshold huma n pyroge nic dose of en dotox in per kg of body weight, and M isequal to the maximum recomme nded huma n dose of product per kg of body weight in a sin gle hour period.The en dotox in limit for pare nteral drugs is specified in in dividual mono graphs in un its such asEU/mL, EU/mg, or EU/Unit of biological activity.GEL-CLOT TECHNIQUESThe gel-clot tech niq ues detect or qua ntify en dotox ins based on clotti ng of the LAL Reage nt in the prese nee of en dotox in. The concen trati on of en dotox in required to cause the lysate to clot un der sta ndard con diti ons is the labeled sen sitivity of the LAL Reage nt. To en sure both the precisi on and validity of the test, tests for confirming the labeled LAL Reage nt sen sitivity and for in terferi ng factors are described un der Preparatory Testi ng for the Gel-Clot Tech niq ues.Preparatory Test ing for the Gel-Clot Tech niq uesTest for Con firmati on of Labeled LAL Reage nt Sen sitivity —— Confirm the labeled sen sitivity using at least 1 vial of the LAL Reage nt lot. Prepare a series of two-fold diluti ons of the USP En dotox in RSin LAL Reage nt Water to give concen tratio ns of 2 入，》，0. 5^ ,and 0.25入,where 入is as defi ned above. Perform the test on the four sta ndard concen trati ons in quadruplicate and include negative controls. The test for confirmation of lysate sensitivity is to be carried out when a new batch of LAL Reage nt is used or whe n there is any cha nge in the experime ntal con diti ons that may affect the outcome of the test.Mix a volume of the LAL Reage nt with an equal volume (such as 0.1-mL aliquots) of one of the standard soluti ons in each test tube. When sin gle test vials or ampuls containing lyophilized LAL Reage nt are used, add soluti ons directly to the vial or ampul. I ncubate the react ion mixture for a□con sta nt period accord ing to direct ions of the LAL Reage nt manu facturer (usually at 37 ±1 for60 ±2 minutes), avoiding vibration. To test the integrity of the gel, take each tube in turn directly□from the in cubator and in vert it through about 180 in one smooth moti on. If a firm gel hasformed that rema ins in place upon inversion, record the result as positive. A result is n egative if an in tact gel is not formed. The test is not valid uni ess the lowest concen trati on of the sta ndard soluti ons shows a n egative result in all replicate tests.The en dpo int is the last positive test in the series of decreas ing concen trati ons of en dotox in. 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 (S e / f)where Se is the sum of the log endpoint concentrations of the dilution series used, and f is the nu mber of replicate test tubes. The geometric mean en dpo int concen trati on is the measured sen sitivity of the LAL Reage nt (i n EU/mL). If this is not less tha n 0.5 and not more tha n 2 入,the labeled sen sitivity is con firmed and is used in tests performed with this lysate.In terferi ng Factors Test for the Gel-Clot Tech niq ues ——Prepare soluti ons A, B, C, and D as show n in Table 1, and perform the inhibition/enhancement test on the sample solutions at a dilution less tha n the MVD, not containing any detectable en dotox ins, followi ng the procedure in the Test for Con firmatio n of Labeled LAL Reage nt Sen sitivity above. The geometric mea n en dpo int concen trati ons of soluti ons B and C are determ ined using the equatio n in that test.Table 1. Preparati on of Soluti ons for the In hibiti on/Enhan ceme nt Test for Gel-Clot Tech niq uesThis test must be repeated whe n any con diti on that is likely to in flue nee the test results cha nges. The test is not valid uni ess Soluti ons A and D show no react ion and the result of Soluti on C con firms the labeled sen sitivity.If the sensitivity of the lysate determined in the presence of the sample solution under test of Solution B is not less than 0.5 丸and not greater than 2 九，the sample solution does not contain factors which in terfere un der the experime ntal con diti ons used. Otherwise, the sample soluti on to be exam ined in terferes with the test.资料收集于网络，如有侵权请联系网站删除只供学习与交流If the sample under test does not comply with the test at a dilution less than the MVD, repeat the test using a greater diluti on, not exceedi ng the MVD. The use of a more sen sitive lysate permits a greater diluti on of the sample to be exam ined and this may con tribute to the elim in ati on of in terfere nee.Interferenee may be overcome by suitable treatment, such as filtration, neutralization, dialysis, or heati ng. To establish that the chose n treatme nt effectively elimi nates in terfere nee without loss of en dotox ins, perform the assay described below using the preparati on to be exam ined to which USP En dotox in RShas bee n added and which has bee n subjected to the selected treatme nt.Gel-Clot Limit TestThis test is used whe n a mono graph contains a requireme nt for en dotox in limits.Procedure —Prepare Solutions A, B, C, and D as shown in Table 2, and perform the test on these soluti ons follow ing the procedure in the Test for Con firmati on of Labeled LAL Reage nt Sensitivity under Preparatory Testing for the Gel-Clot Techniques.Table 2. Preparation of Solutions for the Gel-Clot Limit TestIn terpretatio n — The test is not valid uni ess both replicates of positive con trol Soluti ons B and C are positive and those of n egative con trol Soluti on D are n egative. The preparati on un der test complies with the test whe n a n egative result is found for both tubes containing Soluti on A. The preparatio n un der test does not comply with the test whe n a positive result is found for both tubes containing Soluti on A.Repeat the test when a positive result is found for 1 tube containing Solution A and a negative result for the other one. The preparati on un der test complies with the test whe n a n egative result is found for both tubes containing Solutio n A in the repeat result. If the test is positive for the preparati on un der test at a diluti on less tha n the MVD, the test may be repeated at a diluti on not greater than the MVD.Gel-Clot AssayThis assay qua ntifies bacterial en dotox ins in sample soluti ons by titrati on to an en dpo int.Procedure — Prepare Solutions A, B, C, and D as shown in Table 3, and test these solutions by follow ing the procedure in the Test for Con firmati on of Labeled LAL Reage ntSensitivity under Preparatory Testing for the Gel-Clot Techniques.Table 3. Preparati on of Solutio ns for the Gel-Clot AssayCalculatio n and In terpretati on —The test is n ot valid uni ess the followi ng con diti ons are met: (1) both replicates of n egative con trol Soluti on D are n egative; (2) both replicates of positive product control Solution B are positive; and (3) the geometric mean endpoint concentration of Solution Cis in the range of 0.5 几to 2几.To determ ine the en dotox in concen trati on of Soluti on A, calculate the en dpo int concen trati on for each replicate series of dilutions by multiplying each endpoint dilution factor by 入.The en dotox in concen trati on in the sample is the geometric mean en dpo int concen trati on of the replicates (see the formula give n in the Test for Con firmatio n of Labeled LAL Reage nt Sen sitivity un der Preparatory Testi ng for the Gel-Clot Tech niq ues ). If the test is con ducted with a diluted samplesoluti on, calculate the concen trati on of en dotox in in the origi nal sample soluti on by multiplying by the dilution factor. If none of the dilutions of the sample solution is positive in a valid assay, report the en dotox in concen trati on as less tha nA (if the diluted sample was tested, less than 入times the lowest dilution factor of the sample.) If all dilutions are positive, the en dotox in concen trati on is reported as equal to or greater tha n the greatest diluti on factor multiplied by 入(e.g., initial dilution factor times 8 times ' in Table 3).The article meets the requireme nts of the test if the concen trati on of en dotox in is less tha n that specified in the in dividual mono graph.PHOTOMETRIC TECHNIQUESThe turbidimetric method measures in creases in turbidity. Depe nding on the test prin ciple used, this technique is classified as either endpoint-turbidimetric or kinetic-turbidimetric. The en dpo in t-turbidimetric tech nique is based on the qua ntitative relati on ship betwee n the concen tratio n of en dotox ins and the turbidity (absorba nee or tran smissi on) of the reacti on mixture at the end of an incubation period. The kinetic-turbidimetric technique is a method to measure either the on set time n eeded to reach a predeterm ined absorba nee of the react ion mixture or the rate of turbidity developme nt.The chromoge nic method measures the chromophore released from a suitable chromoge nic peptide by the reacti on of en dotox ins with the LAL Reage nt. Depe nding on the test prin ciple employed, this tech nique is classified as either en dpo in t-chromoge nic or ki netic-chromoge nic. The en dpo in t-chromoge nic tech nique is based on the qua ntitative relati on ship betwee n the concen tratio n of en dotox ins and the release of chromophore at the end of an in cubati on period. The kin etic-chromoge nic tech nique is a method to measure either the on set time n eeded to reach a predeterm ined absorba nee of the react ion mixture or the rate of color developme nt.All photometric tests are carried out at the in cubati on temperature recomme nded by the LAL Reage nt manu facturer, which is usually 37 ±1 .Preparatory Test ing for the Photometric Tech niq uesTo assure the precisi on or validity of the turbidimetric and chromoge nic tech niq ues, preparatory tests are con ducted to verify that the criteria for the sta ndard curve are valid and that the sample soluti on does not in hibit or enhance the react ion. Revalidati on for the test method is required whe n con diti ons that are likely to in flue nee the test result cha nge.Verificati on of Criteria for the Stan dard Curve —Usi ng the Stan dard En dotox in Solutio n, prepare at least three en dotox in concen trati ons to gen erate the sta ndard curve. Perform the test using at least three replicates of each sta ndard en dotox in concen trati on accord ing to the manu facturer's in struct ions for the LAL Reage nt (with regard to volume ratios, in cubatio n time, temperature, pH, etc.). If the desired range in the kin etic methods is greater tha n two logs, additi onal sta ndards should be in cluded to bracket each log in crease withi n the range of the sta ndard curve. Theabsolute value of the correlati on coefficie nt, | r|, must be greater tha n or equal to 0.980 for the range of en dotox in concen trati ons in dicated by the manu facturer of the LAL Reage nt.In terferi ng Factors Test for the Photometric Tech niq ues —— Select an en dotox in concen trati on at or n ear the middle of the en dotox in sta ndard curve. Prepare Soluti ons A, B, C, and D as show n in Table 4. Perform the test on Solutions A, B, C, and D at least in duplicate following the in struct ions for the LAL Reage nt used (with regard to volume of sample and LAL Reage nt, volume ratio of sample to LAL Reage nt, in cubati on time, etc.).Table 4. Preparati on of Soluti ons for the In hibiti on/Enhan ceme nt Test for PhotometricTech niq uesen dotox in at a concen tratio n equal to or n ear the middle of the sta ndard curve.c Soluti on C: the sta ndard en dotox in at the concen trati ons used in the validati on of themethod described in Verificati on of Criteria for the Stan dard Curve un der PreparatoryTesti ng for the Photometric Tech niq ues (positive con trol series).d Solution D: LAL Reage nt Water (n egative con trol).Calculate the mean recovery of the added en dotox in by subtract ing the mean en dotox in concen trati on in the soluti on (if any) from that containing the added en dotox in. In order to be con sidered free of in terferi ng factors un der the con diti ons of the test, the measured concen tratio n of the en dotox in added to the sample soluti on must be within 50% to 200% of the known added en dotox in concen trati on after subtract ion of any en dotox in detected in the soluti on without added en dotox in.When the en dotox in recovery is out of the specified ran ges, the in terferi ng factors must be removed as described in the Interfering Factors Test for the Gel-ClotTechniques under Preparatory Testing for the Gel-Clot Techniques. Repeating the Interfering Factors Test for the Gel-Clot Techniques validates the treatment.Procedure for the Photometric Tech niq uesFollow the procedure described in the In terferi ng Factors Test for the PhotometricTechniques under Preparatory Testing for the Photometric Techniques.Calculati on for the Photometric Tech niq uesCalculate the en dotox in concen trati on of each of the replicates of test Soluti on A using the sta ndard curve gen erated by positive con trol series C. The test is not valid uni ess the follow ing con diti ons are met: (1) the results of con trol series C comply with the requireme nts for validati on defi ned un der Verificatio n of Criteria for the Stan dard Curve un der Preparatory Testi ng for the Photometric Tech niq ues; (2) the en dotox in recovery, calculated from the concen trati on found in Soluti on B after subtract ing the en dotox in concen trati on found in Soluti on A is within 50 to 200%; and (3) the result of negative control series D does not exceed the limit of the blank value required in the descriptio n of the LAL Reage nt used.In terpretati on of Results from the Photometric Tech niq uesIn photometric assays, the preparati on un der test complies with the test if the mean en dotox in concen trati on of the replicates of Soluti on A, after correct ion for diluti on and concen trati on, is less tha n the en dotox in limit for the product.1LAL Reage nt reacts with some P -gluca ns in additi on to en dotox ins. Some preparati onsthat are treated will not react with 0 -glucans and must be used for samples that contain glucans. * 2For a validity test of the procedure for in activati ng en dotox ins, see Dry-Heat Sterilization under Sterilization and Sterility Assuranee of Compendial Articles 〈1211). Use an LAL Reage nt havi ng a sen sitivity of not less tha n 0.15 En dotox in Unit per mL. *3Sterile Water for Injectio n or other water that shows no react ion with the specific LALReage nt with which it is to be used, at the limit of sen sitivity of such reage nt. *K is 5 USP-EU/kg for any route of admi nistratio n other tha n in trathecal (for which K is 0.2 USP-EU/kg body weight). For radiopharmaceutical products n ot admi nistered in trathecally the 资料收集于网络，如有侵权请联系网站删除只供学习与交流en dotox in limit is calculated as 175/ V, where V is the maximum recomme nded dose in mL. For in trathecally admi nistered radiopharmaceuticals, the en dotox in limit is obta ined by the formula 14/V. For formulations (usually anticancer products) administered on a per square meter of body surface, the formula is K/M, where K = 5 EU/kg and M is the (maximum dose/m 2/hour X1.80m2)/70 Kg. ♦。