EP5.1.10细菌内毒素测试使用指南(中英文2)

1、EP 5.1.10 细菌内毒素测试使用指南（中英文2/2）  2015-12-28 23:00:31|  分类： EP For routine tests on this product, it may be expedient to dilute 1 mL of the solution to be examined to 20 mL (MVD/2 rounded to the next lower whole number). However, if this test result is positive the analyst will

2、 have to dilute 1 mL to 41.67mL and repeat the test. A dilution to 41.67 mL is also necessary when the test is performed to settle a dispute.对于此药品的日常检查，可以取1ml供试液稀释至20ml（MVD/2四舍五入至最接近的整数）。但是，如果该测试结果为阳性，则化验室要稀释1ml至41.67ml，重复测试。如果有争议，仲裁测试也必须使用41.67ml的稀释。3. RISK ASSESSMENT 风险评估As stated in section

3、1 of this general chapter, the conclusion is generally justified that the absence of bacterial endotoxins in a substance or product implies the absence of pyrogenic components, provided the presence of non-endotoxin pyrogenic substances can be ruled out. To rule out the presence of non-endotoxin pyr

4、ogens in substance or products, the use of the monocyte-activation test (2.6.30) is recommended at release or during development of the production process; if any changes are made to the production process that could influence the quality of the product regarding pyrogenicity, the monocyte-activatio

5、n test is repeated. Examples of such changes include the use of different raw materials, a different production site and different process parameters.正如本通论第1部分所述，如果可以排除非内毒素热源物质的存在，则原料药或制剂中不存在细菌内毒素通常可以论述得到结论不存在热源成分。为了排除原料药或制剂中存在非内内毒素热源，推荐在放行或生产工艺研发时使用单核细胞激活测试（2.6.30）。如果对生产工艺进行了变更，可能会影响产品热源方面的质量，则需要重新

6、进行单核细胞激活测试。这类变更的例子包括使用了不同的原料、不同的生产场所和不同的工艺参数。The decision to use the test for bacterial endotoxins as the sole pyrogenicity test is to be made after careful evaluation of the risk of the substance or product containing non-endotoxin pyrogens. The risk assessment is made with consideration given to a

7、ny factor that could result in the inclusion of pyrogens not detected by the test for bacterial endotoxins. The items below constitute a non-exhaustive list of factors to be considered in the risk assessment.使用细菌内毒素测试作为单一热源测试的决策要在对含有非内毒素热源的原料药或制剂进行谨慎的风险评估之后方可做出。风险评估要考虑可能会导致产品含有不被细菌内毒素测试检出的热源。以下所列项目是

8、风险评估中要考虑的因素清单，并非完整清单，Production process (chemical synthesis, fermentation, biotechnological method).For products of fermentation, the expression system is to be considered (prokaryotic, eukaryotic) and, for a prokaryotic expression system, whether gram-positive or gram-negative bacteria are use

9、d. Also the culture media components are examined with consideration given to their origin (synthetic, animal, plant).生产工艺（化学合成、发酵、生物技术方法）：对于发酵产品，要考虑表达系统（原核、真核），对于原核表达系统，要看是否使用革兰氏阳性或革兰氏阴性菌。对于发培养基成分，要检查其来源（合成、动物来源、植物来源）。Bioburden.The potential presence of gram-positive bacteria and fungi as contamina

10、nts of the active substance, excipients or starting materials and raw materials used in the production of the medicinal product, and the origin of the raw materials (synthetic, animal, plant) have to be taken into consideration. The quality of the water plays an important role on the overall evaluat

11、ion.生物负载：要考虑革兰氏阳性菌和霉菌可能会污染原料药、辅料，或药品生产用的起始物料和原料药，以及原料来源（合成、动物来源、植物来源）。水的质量在总体评估中起着尤为重要的作用。Capability of the downstream process.It must be verified whether bacterial endotoxin removal steps are part of the downstream process.下游工艺能力：要确认下游工艺中是否有细菌内毒素清除步骤。Safety.The target population and the route of ad

12、ministration (e.g. intravenous, intrathecal) have to be taken into account in the risk assessment.安全性：在风险评估中要考虑目标种群和给药途径（例如，静脉注射、鞘内）。Stability of the detectable endotoxins.It has to be considered that the ability to detect endotoxins can be affected by interaction with certain components, storage co

13、nditions or storage time, temperature and handling of the test sample. Procedures that demonstrate stability of the detectable endotoxin content have to be established for storing, handling and mixing of samples.可检出内毒素的稳定性：要考虑检出内毒素的能力会受到特定组份的相互反应、存贮容器或存贮时长、温度和测试样品的处理方式的影响。证明可检出内毒素含量的稳定性的程序必须指定其存贮情况、

14、处理情况和样品混和情况。4. REFERENCE MATERIAL 对照物Endotoxin standard BRP is intended for use as the reference preparation. It has been assayed against the WHO International Standard for Endotoxin and its potency is expressed in International Units of endotoxin per vial. The International Unit of endotoxin i

15、s defined as the specific activity of a defined mass of the International Standard.内毒素标准BRP是用作对照的物品。它根据WHO国际内毒素标准进行了标定，其效价表述为国际单位内毒素/瓶。内毒素的国际单位定义为指定质量的国际标准物的活性。For routine purposes, another preparation of endotoxin may be used; provided it has been assayed against the International Standard for Endo

16、toxin or the BRP and its potency is expressed in International Units of endotoxin.日常工作中，也可以使用另一个内毒素对照品，但需要已根据国际标准内毒素或BRP进行了标定，且其效价表述为国际单位内毒素。NOTE: 1 International Unit (IU) of endotoxin is equal to 1 Endotoxin Unit (E.U.)注：1国际单位（IU）内毒素等于1内毒素单位（EU）。5. WATER FOR BET 细菌内毒素测试用水Water for BET is ster

17、ile water that is free of detectable levels of endotoxin. Usually it is commercially available and certified.BET测试用水为无菌水，内毒素低于可检测水平。通常可能商业采购获取并有证书认可。General chapter 2.6.14. Bacterial endotoxins indicates that methods other than triple distillation may be used to prepare water for BET. Reverse osmosi

18、s has been used with good results; some analysts may prefer to distill the water more than 3 times. Whatever method is used, the resultant product must be free of detectable bacterial endotoxins.通则2.6.14“细菌内毒素”说明三次蒸馏以外的方法也可以用于制备BET检测用水。反渗透水的使用情况良好，一些化验员可能倾向于使用蒸馏3次以上的水。不管使用哪种方法，所用的水中细菌内毒素必须低于可检测水平。6.

19、 pH OF THE MIXTURE 混合物的PH值In the test for bacterial endotoxins, optimum gel-clot occurs for a mixture at pH 6.0-8.0. However, the addition of the lysate to the sample may result in a lowering of the pH.在细菌内毒素的测试中，混合物理想凝胶pH值为6.0-8.0.当然，将鲎试剂加入样品后可能会使得pH值降低。7. VALIDATION OF THE LYSATE 鲎试剂的验证I

20、t is important to follow the manufacturers instructions for the preparation of the solutions of the lysate.要根据生产商的指示对制备鲎试剂溶液。The positive end-point dilution factors in gel-clot methods A and B are converted to logarithms. The reason is that if the frequency distribution of these logarithmic values i

21、s plotted, it usually approaches a normal distribution curve much more closely than the frequency distribution of the dilution factors themselves; in fact it is so similar that it is acceptable to use the normal frequency distribution as a mathematical model and to calculate confidence limits with S

22、tudents t-test.将凝胶方法A和B中的阳性终点稀释因子转换成log对数。原因是如果这些对数值的频数分布画出后，通常会比稀释因子本身的频数分布更接近正态分布曲线。由于其非常的相似，因此可以使用正态分布作为数学模型，并采用t检验来计算置信限度。8. PRELIMINARY TEST FOR INTERFEREING FACTORS 干扰因素初步测试Some substances or products cannot be tested directly for the presence of bacterial endotoxins because they are not

23、miscible with the reagents, they cannot be adjusted to pH 6.0-8.0 or they inhibit or activate enzymatic reaction (such as -D-glucans).有些物质或制剂不能直接测试内毒素，因为它们无法与试剂混合，它们不能调节pH值至6.0-8.0，或它们的性质或活化酶反应（例如-D-葡聚糖）。Therefore a preliminary test is required to check for the presence of interfering factors;

24、when these are found the analyst must demonstrate that the procedure to remove them has been effective and that by applying this procedure, any bacterial endotoxins present have not been removed.因此，需要进行初步试验来检查是否存在干扰因素。如果化验员发现了干扰因素，则必须证明测试程序能有效清除它们，并且通过执行这些程序，不会清除任何细菌内毒素。The object of the preliminary

25、 test is to test the null hypothesis that the sensitivity of the lysate in the presence of the substance or product to be examined does not differ significantly from the sensitivity of the lysate in the absence of the product. A simple criterion is used in methods A and B: the null hypothesis is acc

26、epted when the sensitivity of the lysate in the presence of the product is at least 0.5 times and not more than twice the sensitivity of the lysate by itself.初步试验的目的是测试零假设（原假设），即鲎试剂在有原料药或制剂存在时的灵敏度，与没有原料药或制剂存在时灵敏度并无显著区别。在方法A和B中使用了一个简单的标准：当鲎试剂在有产品存在时，其灵敏度至少达到0.5倍，且不超过2倍鲎试剂自身的灵敏度，则零假定成立。The test for in

27、terfering factors in gel-clot methods A and B requires the use of a sample of the substance or product in which no endotoxins are detectable. This presents a theoretical problem when an entirely new product has to be tested. Hence, a different approach was designed for quantitative methods C, D, E a

28、nd F.在凝胶方法A和B中干扰因素测试要求使用原料药或制剂的样品，其中不允许检出内毒素，如果要测试的是一个全新的产品，这时理论上是行的通的。因此，在定量方法C、D、E和F中，设计了不同的方法。Note that methods D and E, which used a chromogenic peptide, require reagents that are absent in methods A, B, C and F, and hence compliance of methods A, B, C or F with the requirements for interfering

29、factors cannot be extrapolated to method D or method E without further testing.注意，方法D和E使用了色谱多肽，要使用方法A、B、C和F中不曾使用的试剂，因此在没有进一步测试时，方法A、B、C或F符合干扰因素要求的结论就不能外推至方法或方法E。9. REMOVAL OF INTERFERING FACTORS 干扰因素的消除The procedures to remove interfering factors must not increase or decrease (for example, by a

30、dsorption) the amount of endotoxin in the substance or product to be examined. The correct way of checking this is to apply the procedures to a spiked sample of the substance or product to be examined, that is, a sample to which a known amount of endotoxin has been added, and then to measure the rec

31、overy of the endotoxin after the removal process has been conducted.消除干扰因素的程序一定不能增加或降低受检原料药或制剂中的内毒素数量（例如，由于吸附）。正确的检查方法是使用受检原料药或制剂的加标样品来测试，也就是说，将已知数量的内毒素加入一个样品，然后在实施了清除过程后，检测内毒素的回收率。Methods C and D. 方法C和DIf the nature of the product to be examined results in an interference that cannot be remove

32、d by classical methods (e.g. dilution or centriguation), it may be possible to determine the standard curve in the same type of substance or product freed from endotoxins by appropriate treatment or by dilution of the substance or product. The endotoxins test is then carried out by comparison with t

33、his standard curve.如果受检产品的性质会导致一种干扰，而不能采用传统的方法消除（例如，稀释或离心），有可能通过对原料药制剂进行适当的处理或稀释，使用无内毒素的同类原料药或制剂来确立一个标准曲线。然后通过与此标准曲线比较来进行内毒素测试。Ultrafiltration with cellulose triacetate asymmetric membrane filters has been found to be suitable in most cases. The filters must be properly validated, because under some

34、 circumstance cellulose derivatives (-D-glucans) can cause false positive results.目前已经发现三醋酸纤维素非对称膜过滤超滤方法在多数情况下是适用的。过滤器必须经过适当验证，因为在有些情形下，纤维素衍生物（-D-葡聚糖）可能会导致假阳性结果。Another option to remove interfering factors is a 2-step procedure in which 1) endotoxin within the interfering sample is fixed on a solid

35、phase, and 2) after removal of the interfering substance (e.g. by washing ) the endotoxin is detected unimpaired under suitable testing conditions.另一个清除干扰因素的办法是分两步实现的，第一步将干扰样品中的内毒素固定在一个固定相上，第二步清除干扰物质（例如，通过洗涤）后，在稳定的测试条件下进行不受影响的测试。10. THE PURPOSE OF THE CONTROLS 控制的目的The purpose of the control ma

36、de up with water for BET and the reference preparation of endotoxin at twice the concentration of the labeled lysate sensitivity is to verify the activity of the lysate at the time and under the conditions of the test (for method A and B). The purpose of the negative control is to verify the absence

37、 of t a detectable concentration of endotoxin in the water of BET.通过对BET用水和内毒素对照品在标示鲎试剂灵敏度2倍浓度的控制所要达到的目的是确认鲎试剂在测试的条件下测试的时间内的活性（对于方法A和B）。阴性控制的目的是确认在BET用水中没有可检出的内毒素浓度水平。The positive control, which contains the product to be examined at the concentration used in the test, is intended to show the absenc

38、e of inhibiting factors at the time and under the conditions of the test.阳性控制，其中含有用于测试相同浓度的受检样品，是为了显示在测试条件下在测试时间内没有抑制因子。11. READING AND INTERPRETATION OF RESULTS 结果读数和诠释Minute amounts of the bacterial endotoxin in the water for BET, or in any other reagent or material to which the lysate is exp

39、osed during the test, may escape detection as long as they do not reach the sensitivity limit of the lysate. However, they may raise the amount of bacterial endotoxin in the solution containing the substance or product to be examined to just above the sensitivity limit and cause a positive reaction.

40、在BET用水中，或在其它试剂或鲎试剂在测试中会暴露的物料中的微量细菌内毒素，只要其未达到鲎试剂的灵敏限，就可能会在检测中有被测到。但是，他们可能会抬高细菌内毒素在受检原料药或制剂溶液中的数量，正好超出灵敏限导致阳性反应。The risk of this happening may be reduced by testing the water for BET and the other reagents and materials with the most sensitive lysate available, or at least one that is more sensitive t

41、han the one used in the test on the product. Even then, the risk of such a false positive result cannot be ruled out completely.通过使用可能找到的最灵敏（或者至少比产品测试所用鲎试剂更灵敏）的鲎试剂对水和其它试剂及物料进行BET测试，可以降低上述情况发生的风险。即使是这样，发生“假阳性结果”的风险仍不能完全排除。12. REPLACEMENT OF METHODS PRESCRIBED IN MONOGRAPHS 各论中所述方法的取代方法12-1. REPL

42、ACEMENT BY ANOTHER PH. EUR. METHOD 使用另一欧洲药典方法取代As stated in the General Notice, the test methods given in monographs and general chapters have been validated in accordance with accepted scientific practice and current recommendations on analytical validation. The methods described in general ch

43、apters 2.6.14. Bacterial endotoxins and 2.6.30. Monocyte-activation test therefore do not have to be re-validated per se, other than in consideration of their use for a specific substance or product in a specific analytical environment.正如凡例中声明，各论和通论中给定的检测方法是根据可接受的科学实践和现行分析验证建议经过了验证。在通论2.6.14“细菌内毒素”和

44、2.6.30“单核细胞激活测试”因而不必要进行再验证，其在特定原料药或制剂在特定分析环境中使用时适用性需要考虑者以外。The procedure and the materials and reagents used in the method must be validated as described for the test concerned. The absence of interfering factors (and, if necessary, the procedure for removing them) is verified on samples of at least 3 production batches.测试程序和方法中所用物料和试剂必须针对所涉及的测试所述进行验证。至少要使用3个生产批次来确认没有干扰因素存在（并且必要时，要确认消除这些因素的程序）。The necessary information is sought from manufacturers; com