EP5.1.10細(xì)菌內(nèi)毒素測(cè)試使用指南(中英文2)

1、EP 5.1.10 細(xì)菌內(nèi)毒素測(cè)試使用指南（中英文2/2）  2015-12-28 23:00:31|  分類： 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.對(duì)于此藥品的日常檢查，可以取1ml供試液稀釋至20ml（MVD/2四舍五入至最接近的整數(shù)）。但是，如果該測(cè)試結(jié)果為陽(yáng)性，則化驗(yàn)室要稀釋1ml至41.67ml，重復(fù)測(cè)試。如果有爭(zhēng)議，仲裁測(cè)試也必須使用41.67ml的稀釋。3. RISK ASSESSMENT 風(fēng)險(xiǎn)評(píng)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.正如本通論第1部分所述，如果可以排除非內(nèi)毒素?zé)嵩次镔|(zhì)的存在，則原料藥或制劑中不存在細(xì)菌內(nèi)毒素通常可以論述得到結(jié)論不存在熱源成分。為了排除原料藥或制劑中存在非內(nèi)內(nèi)毒素?zé)嵩矗扑]在放行或生產(chǎn)工藝研發(fā)時(shí)使用單核細(xì)胞激活測(cè)試（2.6.30）。如果對(duì)生產(chǎn)工藝進(jìn)行了變更，可能會(huì)影響產(chǎn)品熱源方面的質(zhì)量，則需要重新

6、進(jìn)行單核細(xì)胞激活測(cè)試。這類變更的例子包括使用了不同的原料、不同的生產(chǎn)場(chǎng)所和不同的工藝參數(shù)。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.使用細(xì)菌內(nèi)毒素測(cè)試作為單一熱源測(cè)試的決策要在對(duì)含有非內(nèi)毒素?zé)嵩吹脑纤幓蛑苿┻M(jìn)行謹(jǐn)慎的風(fēng)險(xiǎn)評(píng)估之后方可做出。風(fēng)險(xiǎn)評(píng)估要考慮可能會(huì)導(dǎo)致產(chǎn)品含有不被細(xì)菌內(nèi)毒素測(cè)試檢出的熱源。以下所列項(xiàng)目是

8、風(fēng)險(xiǎn)評(píng)估中要考慮的因素清單，并非完整清單，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).生產(chǎn)工藝（化學(xué)合成、發(fā)酵、生物技術(shù)方法）：對(duì)于發(fā)酵產(chǎn)品，要考慮表達(dá)系統(tǒng)（原核、真核），對(duì)于原核表達(dá)系統(tǒng)，要看是否使用革蘭氏陽(yáng)性或革蘭氏陰性菌。對(duì)于發(fā)培養(yǎng)基成分，要檢查其來(lái)源（合成、動(dòng)物來(lái)源、植物來(lái)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.生物負(fù)載：要考慮革蘭氏陽(yáng)性菌和霉菌可能會(huì)污染原料藥、輔料，或藥品生產(chǎn)用的起始物料和原料藥，以及原料來(lái)源（合成、動(dòng)物來(lái)源、植物來(lái)源）。水的質(zhì)量在總體評(píng)估中起著尤為重要的作用。Capability of the downstream process.It must be verified whether bacterial endotoxin removal steps are part of the downstream process.下游工藝能力：要確認(rèn)下游工藝中是否有細(xì)菌內(nèi)毒素清除步驟。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：在風(fēng)險(xiǎn)評(píng)估中要考慮目標(biāo)種群和給藥途徑（例如，靜脈注射、鞘內(nèi)）。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.可檢出內(nèi)毒素的穩(wěn)定性：要考慮檢出內(nèi)毒素的能力會(huì)受到特定組份的相互反應(yīng)、存貯容器或存貯時(shí)長(zhǎng)、溫度和測(cè)試樣品的處理方式的影響。證明可檢出內(nèi)毒素含量的穩(wěn)定性的程序必須指定其存貯情況、

14、處理情況和樣品混和情況。4. REFERENCE MATERIAL 對(duì)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.內(nèi)毒素標(biāo)準(zhǔn)BRP是用作對(duì)照的物品。它根據(jù)WHO國(guó)際內(nèi)毒素標(biāo)準(zhǔn)進(jìn)行了標(biāo)定，其效價(jià)表述為國(guó)際單位內(nèi)毒素/瓶。內(nèi)毒素的國(guó)際單位定義為指定質(zhì)量的國(guó)際標(biāo)準(zhǔn)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，也可以使用另一個(gè)內(nèi)毒素對(duì)照品，但需要已根據(jù)國(guó)際標(biāo)準(zhǔn)內(nèi)毒素或BRP進(jìn)行了標(biāo)定，且其效價(jià)表述為國(guó)際單位內(nèi)毒素。NOTE: 1 International Unit (IU) of endotoxin is equal to 1 Endotoxin Unit (E.U.)注：1國(guó)際單位（IU）內(nèi)毒素等于1內(nèi)毒素單位（EU）。5. WATER FOR BET 細(xì)菌內(nèi)毒素測(cè)試用水Water for BET is ster

17、ile water that is free of detectable levels of endotoxin. Usually it is commercially available and certified.BET測(cè)試用水為無(wú)菌水，內(nèi)毒素低于可檢測(cè)水平。通?？赡苌虡I(yè)采購(gòu)獲取并有證書認(rèn)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.通則2.6.14“細(xì)菌內(nèi)毒素”說(shuō)明三次蒸餾以外的方法也可以用于制備BET檢測(cè)用水。反滲透水的使用情況良好，一些化驗(yàn)員可能傾向于使用蒸餾3次以上的水。不管使用哪種方法，所用的水中細(xì)菌內(nèi)毒素必須低于可檢測(cè)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.在細(xì)菌內(nèi)毒素的測(cè)試中，混合物理想凝膠pH值為6.0-8.0.當(dāng)然，將鱟試劑加入樣品后可能會(huì)使得pH值降低。7. VALIDATION OF THE LYSATE 鱟試劑的驗(yàn)證I

20、t is important to follow the manufacturers instructions for the preparation of the solutions of the lysate.要根據(jù)生產(chǎn)商的指示對(duì)制備鱟試劑溶液。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.將凝膠方法A和B中的陽(yáng)性終點(diǎn)稀釋因子轉(zhuǎn)換成log對(duì)數(shù)。原因是如果這些對(duì)數(shù)值的頻數(shù)分布畫出后，通常會(huì)比稀釋因子本身的頻數(shù)分布更接近正態(tài)分布曲線。由于其非常的相似，因此可以使用正態(tài)分布作為數(shù)學(xué)模型，并采用t檢驗(yàn)來(lái)計(jì)算置信限度。8. PRELIMINARY TEST FOR INTERFEREING FACTORS 干擾因素初步測(cè)試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).有些物質(zhì)或制劑不能直接測(cè)試內(nèi)毒素，因?yàn)樗鼈儫o(wú)法與試劑混合，它們不能調(diào)節(jié)pH值至6.0-8.0，或它們的性質(zhì)或活化酶反應(yīng)（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，需要進(jìn)行初步試驗(yàn)來(lái)檢查是否存在干擾因素。如果化驗(yàn)員發(fā)現(xiàn)了干擾因素，則必須證明測(cè)試程序能有效清除它們，并且通過(guò)執(zhí)行這些程序，不會(huì)清除任何細(xì)菌內(nèi)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.初步試驗(yàn)的目的是測(cè)試零假設(shè)（原假設(shè)），即鱟試劑在有原料藥或制劑存在時(shí)的靈敏度，與沒(méi)有原料藥或制劑存在時(shí)靈敏度并無(wú)顯著區(qū)別。在方法A和B中使用了一個(gè)簡(jiǎn)單的標(biāo)準(zhǔn)：當(dāng)鱟試劑在有產(chǎn)品存在時(shí)，其靈敏度至少達(dá)到0.5倍，且不超過(guò)2倍鱟試劑自身的靈敏度，則零假定成立。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.在凝膠方法A和B中干擾因素測(cè)試要求使用原料藥或制劑的樣品，其中不允許檢出內(nèi)毒素，如果要測(cè)試的是一個(gè)全新的產(chǎn)品，這時(shí)理論上是行的通的。因此，在定量方法C、D、E和F中，設(shè)計(jì)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使用了色譜多肽，要使用方法A、B、C和F中不曾使用的試劑，因此在沒(méi)有進(jìn)一步測(cè)試時(shí)，方法A、B、C或F符合干擾因素要求的結(jié)論就不能外推至方法或方法E。9. REMOVAL OF INTERFERING FACTORS 干擾因素的消除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.消除干擾因素的程序一定不能增加或降低受檢原料藥或制劑中的內(nèi)毒素?cái)?shù)量（例如，由于吸附）。正確的檢查方法是使用受檢原料藥或制劑的加標(biāo)樣品來(lái)測(cè)試，也就是說(shuō)，將已知數(shù)量的內(nèi)毒素加入一個(gè)樣品，然后在實(shí)施了清除過(guò)程后，檢測(cè)內(nèi)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.如果受檢產(chǎn)品的性質(zhì)會(huì)導(dǎo)致一種干擾，而不能采用傳統(tǒng)的方法消除（例如，稀釋或離心），有可能通過(guò)對(duì)原料藥制劑進(jìn)行適當(dāng)?shù)奶幚砘蛳♂?，使用無(wú)內(nèi)毒素的同類原料藥或制劑來(lái)確立一個(gè)標(biāo)準(zhǔn)曲線。然后通過(guò)與此標(biāo)準(zhǔn)曲線比較來(lái)進(jìn)行內(nèi)毒素測(cè)試。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.目前已經(jīng)發(fā)現(xiàn)三醋酸纖維素非對(duì)稱膜過(guò)濾超濾方法在多數(shù)情況下是適用的。過(guò)濾器必須經(jīng)過(guò)適當(dāng)驗(yàn)證，因?yàn)樵谟行┣樾蜗?，纖維素衍生物（-D-葡聚糖）可能會(huì)導(dǎo)致假陽(yáng)性結(jié)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.另一個(gè)清除干擾因素的辦法是分兩步實(shí)現(xiàn)的，第一步將干擾樣品中的內(nèi)毒素固定在一個(gè)固定相上，第二步清除干擾物質(zhì)（例如，通過(guò)洗滌）后，在穩(wěn)定的測(cè)試條件下進(jìn)行不受影響的測(cè)試。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.通過(guò)對(duì)BET用水和內(nèi)毒素對(duì)照品在標(biāo)示鱟試劑靈敏度2倍濃度的控制所要達(dá)到的目的是確認(rèn)鱟試劑在測(cè)試的條件下測(cè)試的時(shí)間內(nèi)的活性（對(duì)于方法A和B）。陰性控制的目的是確認(rèn)在BET用水中沒(méi)有可檢出的內(nèi)毒素濃度水平。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.陽(yáng)性控制，其中含有用于測(cè)試相同濃度的受檢樣品，是為了顯示在測(cè)試條件下在測(cè)試時(shí)間內(nèi)沒(méi)有抑制因子。11. READING AND INTERPRETATION OF RESULTS 結(jié)果讀數(shù)和詮釋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，或在其它試劑或鱟試劑在測(cè)試中會(huì)暴露的物料中的微量細(xì)菌內(nèi)毒素，只要其未達(dá)到鱟試劑的靈敏限，就可能會(huì)在檢測(cè)中有被測(cè)到。但是，他們可能會(huì)抬高細(xì)菌內(nèi)毒素在受檢原料藥或制劑溶液中的數(shù)量，正好超出靈敏限導(dǎo)致陽(yáng)性反應(yīng)。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.通過(guò)使用可能找到的最靈敏（或者至少比產(chǎn)品測(cè)試所用鱟試劑更靈敏）的鱟試劑對(duì)水和其它試劑及物料進(jìn)行BET測(cè)試，可以降低上述情況發(fā)生的風(fēng)險(xiǎn)。即使是這樣，發(fā)生“假陽(yáng)性結(jié)果”的風(fēng)險(xiǎn)仍不能完全排除。12. REPLACEMENT OF METHODS PRESCRIBED IN MONOGRAPHS 各論中所述方法的取代方法12-1. REPL

42、ACEMENT BY ANOTHER PH. EUR. METHOD 使用另一歐洲藥典方法取代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.正如凡例中聲明，各論和通論中給定的檢測(cè)方法是根據(jù)可接受的科學(xué)實(shí)踐和現(xiàn)行分析驗(yàn)證建議經(jīng)過(guò)了驗(yàn)證。在通論2.6.14“細(xì)菌內(nèi)毒素”和

44、2.6.30“單核細(xì)胞激活測(cè)試”因而不必要進(jìn)行再驗(yàn)證，其在特定原料藥或制劑在特定分析環(huán)境中使用時(shí)適用性需要考慮者以外。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.測(cè)試程序和方法中所用物料和試劑必須針對(duì)所涉及的測(cè)試所述進(jìn)行驗(yàn)證。至少要使用3個(gè)生產(chǎn)批次來(lái)確認(rèn)沒(méi)有干擾因素存在（并且必要時(shí)，要確認(rèn)消除這些因素的程序）。The necessary information is sought from manufacturers; com