细菌内毒素检测中文翻译.pdf

细菌内毒素检测细菌内毒素检测 Portions of this general chapter have been harmonized with the corresponding texts of the European Pharmacopoeia and or the Japanese Pharmacopoeia Those portions that are not harmonized are marked with symbols to specify this fact 本文中部分内容与欧洲药典和 或日本药典的相应的内容是一致的 那些不一致的内容则用符号 标明 The Bacterial Endotoxins Test BET is a test to detect or quantify endotoxins from Gram negative bacteria using amoebocyte lysate from the horseshoe crab Limulus polyphemus or Tachypleus tridentatus 细菌内毒素测试 BET 是检测或定量革兰氏阴性细菌内毒素的试验 它使用从鲎 美洲鲎或东方鲎 的 循环阿米巴细胞的抽提液中制备的鲎试剂 There are three techniques for this test the gel clot technique which is based on gel formation the turbidimetric technique based on the development of turbidity after cleavage of an endogenous substrate and the chromogenic technique based on the development of color after cleavage of a synthetic peptide chromogen complex Proceed by any of the three techniques for the test In the event of doubt or dispute the final decision is made based upon the gel clot limit test 2s USP 35 unless otherwise indicated in the monograph for the product being tested The test is carried out in a manner that avoids endotoxin contamination 细菌内毒素试验有三种技术 基于凝胶形成的凝胶法 基于一内源性基质断裂后的浊度变化 和显色基质法 基于一个带有生色团的合成肽断裂后颜色的变化 除非文中另有说明 否则可以用三种技术中的任何一 种进行测试 若检测结果存在争议 除非文中另有说明 以凝胶法结果为准 该试验以一种避免内毒素污染 的方式进行 APPARATUS 仪器仪器设备设备 Depyrogenate all glassware and other heat stable materials in a hot air oven using a validated process 1 A commonly used minimum time and temperature is 30 min at 250 If employing plastic apparatus such as microplates and pipet tips for automatic pipetters use apparatus that is shown to be free of detectable endotoxin and does not interfere in the test NOTE In this chapter the term tube includes any other receptacle such as a microtiter well 所有玻璃用具和耐热材料应置于热空气烤箱中有效除热原 通常使用的最少时间和最低温度为 30 分钟 250 如果使用塑料用具 如微孔板和自动加样器配套的吸头之类的 应确认其不含可测内毒素并不会干扰试验 注 在这一章中 管 这个词泛指其它任何容器 如微量孔板的孔 REAGENTS AND TEST SOLUTIONS 试剂和测试方法试剂和测试方法 Amoebocyte Lysate A lyophilized product obtained from the lysate of amoebocytes white blood cells from the horseshoe crab Limulus polyphemus or Tachypleus Solutridentatus This reagent refers only to a product manufactured in accordance with the regulations of the competent authority NOTE Amoebocyte Lysate reacts to some glucans in addition to endotoxins Amoebocyte Lysate preparations that do not react to glucans are available they are prepared by removing the G factor reacting to glucans from Amoebocyte Lysate or by inhibiting the G factor reacting system of Amoebocyte Lysate and may be used for endotoxin testing in the presence of glucans 鲎试剂是一种冻干的产物 它从鲎 美洲鲎或东方鲎 的阿米巴细胞 白血细胞 的溶解产物中获得 本试 剂仅指根据主管当局的规定而生产的产品 注 除了内毒素 鲎试剂也与某些 葡聚糖反应 一些经过处 理而制备的鲎试剂不会与 葡聚糖反应 它们是通过除去阿米巴细胞产生的 G 因子或抑制阿米巴细胞反应系 统 G 因子的反应机制实现的 对于含有葡聚糖的样品的检测应用此类试剂 Water for Bacterial Endotoxins Test BET Use Water for Injection or water produced by other procedures that shows no reaction with the lysate employed at the detection limit of the reagent 用于细菌内毒素测试的水 注 在试剂的检测范围内 使用注射用水或不含与鲎试剂反应的物质的水 Lysate TS Dissolve Amoebocyte Lysate in Water for BET or in a buffer recommended by the lysate manufacturer by gentle stirring Store the reconstituted lysate refrigerated or frozen according to the specifications of the manufacturer 将鲎试剂溶解于水中 或在制造商推荐的缓冲液中温和搅拌 然后根据说明书将重组的鲎试剂冷藏或冷冻 1For a validity test of the procedure for inactivating endotoxins see Dry Heat Sterilization under Sterilization and Sterility Assurance of Compendial Articles Use Lysate TS having a sensitivity of not less than 0 15 Endotoxin Unit per mL 为使内毒素进行有效的检验 选择干热灭菌 并保证遵照条例的规定 使用的是一种敏感性每毫升不少于0 15个内毒素 单位的鲎试剂 PREPARATION OF SOLUTIONS 准备的解决方案准备的解决方案 Standard Endotoxin Stock Solution A Standard Endotoxin Stock Solution is prepared from a USP Endotoxin Reference Standard that has been calibrated to the current WHO International Standard for Endotoxin Follow the specifications in the package leaflet and on the label for preparation and storage of the Standard Endotoxin Stock Solution Endotoxin is expressed in Endotoxin Units EU NOTE One USP Endotoxin Unit EU is equal to one International Unit IU of endotoxin 标准内毒素溶液是按 USP 内毒素参考标准制备的 该标准已相当于目前国际内毒素的标准 按照标准内毒素 溶液说明书和和储存标签的说明 内毒素的量用 USP 内毒素单位 USP EU 表示 注 1 一种 USP 内毒素单位 EU 等于一国际单位 国际单位 内毒素 注 1 USP EU 内毒素等于国际单位 1 IU 的内毒素 Standard Endotoxin Solutions After mixing the Standard Endotoxin Stock Solution vigorously prepare appropriate serial dilutions of Standard Endotoxin Solution using Water for BET Use dilutions as soon as possible to avoid loss of activity by adsorption 标准内毒素溶液 在将标准内毒素溶液混合后 将标准内毒素溶液进行适当的连续稀释 尽可能使用稀释剂 以避免因吸附而失去活性 Sample Solutions Prepare the Sample Solutions by dissolving or diluting drugs 2S USP35 using Water for BET Some substances or preparations may be more appropriately dissolved or diluted 2s usp35 in other aqueous solutions If necessary adjust the pH of the solution to be examined or dilution thereof so that the pH of the mixture of the lysate and Sample Solution falls within the pH range specified by the lysate manufacturer usually 6 0 8 0 The pH may be adjusted by use of an acid base or suitable buffer as recommended by the lysate manufacturer Acids and bases may be prepared from concentrates or solids with Water for BET in containers free of detectable endotoxin Buffers must be validated to be free of detectable endotoxin and interfering factors 供试品溶液的制备 用细菌内毒素检查用水溶解或稀释药品或抽提医疗器械来制备供试品溶液 某些物质 或产品用其它水性溶液溶解 稀释或抽提可能更合适 如果有必要 调节待检溶液 或稀释液 的 pH 值以使 鲎试剂和供试品的混合物的 pH 值在鲎试剂生产商指定的 pH 值范围内 这通常适用于 pH 值为 6 0 8 0 之间的 产品 可用酸 碱或鲎试剂生产商推荐的合适的缓冲液调节 pH 值 酸和碱可用原液或固体物加水于无内毒素 的容器中进行制备用于细菌内毒素检查 缓冲液必须经过验证确认无可测的内毒素和干扰因子 DETERMINATION OF MAXIMUM VALID DILUTION MVD 最大有效稀释倍数最大有效稀释倍数 MVD MVD 的确定的确定 The maximum valid dilution is the maximum allowable dilution of a specimen at which the endotoxin limit can be determined Determine the MVD from the following equation MVD endotoxin limit concentration of Sample Solu tion Endotoxin Limit The endotoxin limit for parenteral drugs defined on the basis of dose equals K M 2 where K is a threshold pyrogenic dose of endotoxin per kg of body weight and M is equal to the maximum recommended bolus dose of product per kg of body weight When the product is to be injected at frequent intervals or infused continuously M is the maximum total dose administered in a single hour period The endotoxin limit for parenteral drugs is specified in the individual monograph in units such as EU mL EU mg EU Unit of biological activity etc 最大有效稀释倍数是供试品被允许的最大稀释倍数 在此稀释倍数下可确定内毒素限值 计算 MVD 的等式如下 内毒素限值的建立 依据剂量来定义内毒素限值等于 K M2 K 是每公斤体重的内毒素的阈值 M 等于每公斤 体重的最大推荐剂量 当产品需要频繁注射或连续注射时 M 是在一个小时内注射的最大剂量 非经肠道药 品的内毒素限值在其他文中有给出 以单位表示 如 EU mL EU mg 或 EU 生物活性单位 Concentration of Sample Solution 样品溶液的浓度 mg mL in the case of endotoxin limit specified by weight EU mg 依体重 EU mg 确定的内毒素限值 表表 1 1 凝胶法抑制凝胶法抑制 增强试验的溶液制备增强试验的溶液制备 溶 液 内毒素浓度 加入内毒素的溶液 稀释用液 稀释 倍数 初始内毒素浓度 重复管数 A 无 供试品溶液 4 B 2 供试品溶液 供试品溶液 1 2 4 8 2 1 0 5 0 25 4 4 4 4 C 2 细菌内毒素检查用水 细菌内毒素检查用水 1 2 4 8 2 1 0 5 0 25 2 2 2 2 D 无 细菌内毒素检查用水 2 溶液 A 无可测内毒素的待检供试品溶液 溶液 B 实验干扰对照 溶液 C 鲎试剂灵敏度复核对照 溶液 D 阴性对照 检查用水 Units mL in the case of endotoxin limit specified by unit of biological activity EU Unit mL mL when the endotoxin limit is specified by volume EU mL 供试品溶液浓度内毒素限值 MVD the labeled sensitivity in the Gel Clot Technique EU mL or the lowest concentration used in the standard 2S USP35 curve for the Turbidimetric Technique or Chromogenic Technique Units mL 在生物活性单位 EU Unit 对应的内毒素限值的情况下 mL mL 当内毒素限值按体积 EU mL 来指定时 凝胶法技术的标示敏感度 或用于浊度技术或显色技术标准曲线的最低浓度 GEL CLOT TECHNIQUE The gel clot technique is used for detecting or quantifying endotoxins based on clotting of the lysate reagent in the presence of endotoxin The minimum concentration of endotoxin required to cause the lysate to clot under standard conditions is the labeled sensitivity of the lysate reagent To ensure both the precision and validity of the test perform the tests for confirming the labeled lysate sensitivity and for interfering factors as described in Preparatory Testing mmediately below 凝胶法技术凝胶法技术 凝胶法技术是根据鲎试剂在出现内毒素时会产生凝胶的原理来定量检测内毒素的方法 在标准条件下 能使鲎 试剂产生凝胶的内毒素的最低浓度为鲎试剂的标示灵敏度 为确保该实验的精确性和有效性 应进行标示灵 敏度的复核试验和干扰试验 在 凝胶法预备试验 中介绍了这两个试验 Preparatory Testing Test for Confirmation of Labeled Lysate Sensitivity Confirm in four replicates the labeled sensitivity expressed in EU mL of the lysate prior to use in the test The test for confirmation of lysate sensitivity is to be carried out when a new batch of lysate is used or when there is any change in the test conditions that may affect the outcome of the test Prepare standard solutions having at least four concentrations equivalent to 2 0 5 and 0 25 by diluting the USP Endotoxin RS with Water for BET 凝胶法预备试验凝胶法预备试验 鲎试剂灵敏度复核试验 进行 4 次灵敏度复核试验 在鲎试剂检验前 用表示 EU mL 当使用新批号的鲎试剂或当试验条件发生了可能影响试验结果的变化时 须进行鲎试剂标示灵敏度复核试验 用细菌内毒素检查用水将 USP 内毒素参考标准品溶解制成一个 2 倍稀释系列 即 2 0 5 和 0 25 四个浓 度的内毒素标准品溶液 定义如上 Mix a volume of the Lysate TS with an equal volume such as 0 1 mL aliquots of one of the Standard Endotoxin Solutions in each test tube When single test vials or ampuls containing lyophilized lysate are used add solutions directly to the vial or ampul Incubate the reaction mixture for a constant period according to the directions of the lysate manufacturer usually at 37 1 for 60 2 min 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 considered valid when the lowest concentration of the standard solutions shows a negative result in all replicate tests 将一定量的鲎试剂和等量 如 0 1mL 的各浓度内毒素标准溶液分别加入每支检测试管中混合 如果使用装 有冻干鲎试剂的单次试管或安瓿 可将溶液直接加入试管或安瓿中 根据鲎试剂生产商的建议将反应混合物 恒温孵育一段时间 通常是 37 1 60 2 分钟 避免振动 将每支试管轮流从恒温箱中直接取出并轻轻翻 转 180 观察结果 如果内容物形成坚实凝胶并在试管翻转后不从管壁滑脱 则记为阳性 如果未形成完整的 凝胶 则结果为阴性 最低浓度的标准品溶液的所有重复管都为阴性结果时 试验才为有效试验 The endpoint is the smallest concentration in the series of decreasing concentrations of standard endotoxin that clots the lysate Determine the geometric mean endpoint by calculating the mean of the logarithms of the endpoint concentrations of the four replicate series and then taking the antilogarithm of the mean value as indicated in the following formula geometric mean endpoint concentration antilog e f where Se 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 lysate 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 终点浓度是指系列递减的内毒素浓度中最后一个呈阳性结果的浓度 按下式计算反应终点浓度的对数平均值 然后求此平均值的反对数 终点浓度的几何平均值 antilog e f 式中 e 为所用稀释系列的终点浓度的对数值之和 f 为重复试管数 终点浓度几何平均值为鲎试剂灵敏度的 测定值 EU mL 如果该值在 0 5 2 之间 则该批鲎试剂的标示灵敏度符合规定 可以用于试验 Test for Interfering Factors Usually prepare solutions A 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 operating as described for Test for Confirmation of Labeled Lysate Sensitivity The geometric mean endpoint concentrations of Solutions B and C are determined using the formula described in the Test for Confirmation of Labeled Lysate Sensitivity The test for interfering factors must be repeated when any condition changes that is likely to influence the result of the test IRA 1 Apr 2011 The test is considered valid when all replicates of Solutions A and D show no reaction and the result of Solution C confirms the labeled sensitivity 凝胶法干扰试验凝胶法干扰试验 按表 1 制备溶液 A B C 和 D 并用不超过 MVD 稀释的不含有可检测到的内毒素供试品溶液 进行抑制 增强试验 操作方法同上述鲎试剂标示灵敏度的复核项下 溶液 B 和 C 的终点浓度几何平均值的 计算与该检测中使用的等式相同 当任何有可能影响检测结果的条件改变时应重新进行试验 如果该试验中 溶液 A 和 D 所有重复管都为阴性结果 而且溶液 C 的结果在鲎试剂灵敏度复核范围内 则认为此次试验有效 If the sensitivity of the lysate determined in the presence of Solution B is not less than 0 5 and not greater than 2 the Sample Solution does not contain factors that interfere 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 perform the assay described above using the preparation to be examined to which Standard Endotoxin has been added and which has then been submitted to the chosen treatment 如果用溶液 B 所测得的试剂灵敏度在 0 5 2 0 之间 则供试品溶液在所使用的试验条件下没有干扰 否则 供试品溶液对试验有干扰 如果供试品在小于 MVD 的稀释倍数下对试验有干扰 将供试品溶液进行不超过 MVD 的进一步稀释后 再重复 干扰试验 使用高灵敏度的鲎试剂可对供试品进行更大倍数的稀释 这有助于消除干扰 干扰可用适当的方法来排除 例如中和 透析或加热 为了证明所选择的处理方法有效消除了干扰而不会使 内毒素失去活性 要使用已添加的标准内毒素且已经过选定的方法处理制备的供试品溶液进行下述试验 Table 3 Table 3 凝胶法定量试验的供试品溶液的制备凝胶法定量试验的供试品溶液的制备 溶液 内毒素浓度 加入内毒素的溶液 稀释用液 稀释倍数 内毒素初始浓度 重复管数 A 无 供试品溶液 细菌内毒素检查用水 1 2 4 8 2 2 2 B 2 供试品溶液 1 2 2 C 2 细菌内毒素检查用水 细菌内毒素检查用水 1 2 4 8 2 1 0 5 0 25 2 2 2 2 D 无 细菌内毒素检查用水 2 溶液 A 以不超过 MVD 稀释的供试品溶液 且已通过凝胶法干扰试验 其随后的稀释也不能超过最大有效稀释倍数 用细菌内 毒素检查用水将已通过干扰试验的四管供试品溶液再进行 1 2 4 和 8 倍稀释 也可用其它适当的稀释倍数 溶液 B 含浓度为 2 的标准内毒素的溶液 A 产品阳性对照 溶液 C 分别含浓度为 2 0 5 和 0 25 的标准内毒素的两个细菌内毒素检查用水系列 溶液 D 细菌内毒素检查用水 阴性对照 Limit Test Procedure Prepare Solutions A B C and D as shown in Table 2 and perform the test on these solutions following the procedure above for Preparatory Testing Test for Confirmation of Labeled Lysate Sensitivity 凝胶法限量试验凝胶法限量试验 方法 按表2制备溶液 A B C 和 D 按照凝胶法预试验项下的鲎试剂标示灵敏度复核试验步骤进行试验操 作 表 2 凝胶法限量试验的溶液制备 溶液 内毒素浓度 添加了内毒素的溶液 重复管数 A 无 稀释的供试品溶液 2 B 2 稀释的供试品溶液 2 C 2 细菌内毒素检查用水 2 D 无 细菌内毒素检查用水 2 用不超过最大有效稀释倍数和凝胶法预试验项下的干扰试验中所述的处理方法来制备溶液 A 和阳性产品对照溶液 B 阳性对照 溶液 B 和 C 中含有相当于 2 浓度的标准内毒素 阴性对照溶液 D 为细菌内毒素检查用水 Interpretation The test is considered valid when both replicates of Solutions B and C are positive and those of Solution D are negative When a negative result is found for both replicates of Solution A the preparation under test complies with the test When a positive result is found for both replicates of Solution A the preparation under test does not comply with the test When a positive result is found for one replicate of Solution A and a negative result is found for the other repeat the test In the repeat test the preparation under test complies with the test if a negative result is found for both replicates of Solution A The preparation does not comply with the test if a positive result is found for one or both replicates of Solution A However if the preparation does not comply with the test at a dilution less than the MVD the test may be repeated using a greater dilution not exceeding the MVD 结果判断 当阳性对照溶液 B 和 C 中所有重复管均为阳性结果以及阴性对照溶液 D 所有重复管均为阴性结果 时 试验方为有效 如果溶液 A 的两支重复管均为支重复管均为阳性时 供试品不符合规定 如果溶液 A 中一管为阳性结果另一管为阴性结果 则需重复此试验 重复试验中 如果溶液 A 的两管均为阴 性结果 则该供试品符合规定 如果用小于 MVD 的倍数稀释供试品其检测结果为阳性 则该试验可重复进行 但稀释倍数不得大于 MVD Quantitative Test Procedure The test quantifies bacterial endotoxins in Sample Solutions by titration to an endpoint Prepare Solutions A B C and D as shown in Table 3 and test these solutions by following the procedure in Preparatory Testing Test for Confirmation of Labeled Lysate Sensitivity 凝胶法凝胶法定量定量试验试验 方法 通过滴定法对供试品溶液中的细菌内毒素进行定量测定 制备表3所示的A B C和D溶液 按照凝胶 法预试验项下的鲎试剂标示灵敏度复核试验步骤进行试验操作 Calculation and Interpretation The test is considered valid when the following three 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 endpoint concentration of Solution C is in the range of 0 5 to 2 To determine the endotoxin concentration of Solution A calculate the endpoint concentration for each replicate bymultiplying each endpoint dilution factor by l The endotoxin concentration in the Sample Solution is the endpoint concentration of the replicates 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 l if the diluted sample wastested report as less than l times 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 l e g initial dilution factor times eight times l in Table 3 The preparation under test meets the requirements of the test if the concentration of endotoxin in both replicates is less than that specified in the individual monograph 计算与说明 当试验符合以下条件时才为有效试验 1 阴性对照溶液 D 的两支重复管均为阴性结果 2 样 品阳性对照溶液 B 的两支重复管均为阳性结果 3 溶液 C 的终点浓度的几何平均值在 0 5 2 范围内 要计算溶液 A 的内毒素浓度 可将每一终点对应的稀释倍数乘以 计算出每个稀释系列的每支重复管的终点浓 度 供试品溶液的内毒素浓度即是各重复管的终点浓度的几何平均值 公式用 凝胶法预试验 项下鲎试剂 灵敏度复核试验中的公式 如果试验中用的是稀释过的供试品溶液 计算其原始供试品溶液的内毒素浓度时 可以用原始浓度乘以各自的稀释倍数 如果供试品溶液在有效试验中结果均为阴性 内毒素浓度应记为小于 如果是稀释的样品 其检测结果浓度记为小于 乘以供试品的最小稀释倍数 如果所有结果为阳性 内毒 素浓度应记为等于或大于最大稀释倍数乘以 例 表 3 中初始稀释倍数乘以 8 乘以 如果内毒素浓度小于各品种正文中规定的浓度时 供试品符合规定 表表 4 4 光度技术抑制光度技术抑制 增强试验的溶液制备增强试验的溶液制备 溶液 内毒素浓度 加入内毒素的溶液 重复管数 A 无 供试品溶液 不少于 2 B 标准曲线的中点浓度 供试品溶液 不少于 2 C 至少 3 个浓度 为最低浓度 细菌内毒素检查用水 D 无 细菌内毒素检查用水 不少于 2 溶液 A 稀释倍数不超过 MVD 的供试品溶液 溶液 B 已添加浓度等于或接近标准曲线的中点浓度的内毒素 与溶液 A 有相同稀释倍数的供试品溶液 溶液 C 光度技术预试验项下 标准曲线的可靠性试验 中所述的验证方法所用标准内毒素溶液 阳性对照系 列 溶液 D 细菌内毒素检查用水 阴性对照 PHOTOMETRIC QUANTITATIVE TECHNIQUES Turbidimetric Technique This technique is a photometric assay measuring increases in reactant turbidity On the basis of the particular assay principle employed this technique may be classified as either an endpoint turbidimetric assay or a kinetic turbi dimetric assay The endpoint turbidimetric assay is based on the quantitative relationship between the concentration of endotoxins and the turbidity absorbance or transmission of th