美国药典USP31-71-无菌检查法-双语版.docx

美国药典USP31-NF26无菌检查法71.doc71 STERILITY TESTS 无菌检查法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. 此通则的各部分已经与欧洲药典和/或日本药典的对应部分做了协调。不一致的部分用符号（ ）来标明。 The following procedures are applicable for determining whether a Pharmacopeial article purporting to be sterile complies with the requirements set forth in the individual monograph with respect to the test for sterility. Pharmacopeial articles are to be tested by the Membrane Filtration method under Test for Sterility of the Product to be Examined where the nature of the product permits. If the membrane filtration technique is unsuitable, use the Direct Inoculation of the Culture Medium method under Test for Sterility of the Product to be Examined. All devices, with the exception of Devices with Pathways Labeled Sterile, are tested using the Direct Inoculation of the Culture Medium method. Provisions for retesting are included under Observation and Interpretation of Results.下面这些步骤适用于测定是否某个用于无菌用途的药品是否符合其具体的各论中关于无菌检查的要求。只要其性质许可，这些药品将使用供试产品无菌检查法项下的膜过滤法来检测。如果膜过滤技术是不适合的，则使用在供试产品无菌检查法项下的培养基直接接种法。除了具有标记为无菌通道的设备之外，所有的设备均须使用培养基直接接种法进行检测。在结果的观测与理解项下包含了复验的规定。Because sterility testing is a very exacting procedure, where asepsis of the procedure must be ensured for a correct interpretation of results, it is important that personnel be properly trained and qualified. The test for sterility is carried out under aseptic conditions. In order to achieve such conditions, the test environment has to be adapted to the way in which the sterility test is performed. The precautions taken to avoid contamination are such that they do not affect any microorganisms that are to be revealed in the test. The working conditions in which the tests are performed are monitored regularly by appropriate sampling of the working area and by carrying out appropriate controls.由于无菌检查法是一个非常精确的程序，在此过程中程序的无菌状态必须得到确保以实现对结果的正确理解，因此人员经过适当的培训并取得资质是非常重要的。无菌检查在无菌条件下进行。为了实现这样的条件，试验环境必须调整到适合进行无菌检查的方式。为避免污染而采取的特定预防措施应不会对任何试图在检查中发现的微生物产生影响。通过在工作区域作适当取样并进行适当控制，来定期监测进行此试验的工作条件。These Pharmacopeial procedures are not by themselves designed to ensure that a batch of product is sterile or has been sterilized. This is accomplished primarily by validation of the sterilization process or of the aseptic processing procedures.这些药典规定程序自身的设计不能确保一批产品无菌或已经灭菌。这主要是通过灭菌工艺或者无菌操作程序的验证来完成。When evidence of microbial contamination in the article is obtained by the appropriate Pharmacopeial method, the result so obtained is conclusive evidence of failure of the article to meet the requirements of the test for sterility, even if a different result is obtained by an alternative procedure. For additional information on sterility testing, see Sterilization and Sterility Assurance of Compendial Articles 1211 . 当通过适当的药典方法获得了某物品中微生物污染的证据，这样获得的结果是该物品未能达到无菌检验要求的结论性证据，即便使用替代程序得到了不同的结果也无法否定此结果。 如要获得关于无菌检验的其他信息，见药品的灭菌和无菌保证 MEDIA 培养基Prepare media for the tests as described below, or dehydrated formulations may be used provided that, when reconstituted as directed by the manufacturer or distributor, they meet the requirements of the Growth Promotion Test of Aerobes, Anaerobes, and Fungi. Media are sterilized using a validated process.按照下面描述的方法配制实验用培养基；或者使用脱水培养基，只要根据其制造商或者分销商说明进行恢复之后，其能够符合好氧菌、厌氧菌、霉菌生长促进试验的要求即可。使用经过验证的工艺对培养基进行灭菌操作。The following culture media have been found to be suitable for the test for sterility. Fluid Thioglycollate Medium is primarily intended for the culture of anaerobic bacteria. However, it will also detect aerobic bacteria. SoybeanCasein Digest Medium is suitable for the culture of both fungi and aerobic bacteria.下面的培养基已经被证实适合进行无菌检查。巯基醋酸盐液体培养基主要用于厌氧菌的培养。但其也用于检测好氧菌。大豆酪蛋白消化物培养基适合于培养霉菌和好氧菌。Fluid Thioglycollate Medium 巯基醋酸盐液体培养基L-Cystine L-胱氨酸0.5 gSodium Chloride氯化钠2.5 gDextrose (C6H12O6H2O) 葡萄糖5.5/5.0 gAgar, granulated (moisture content notexceeding 15%)琼脂，呈颗粒状(水分含量不超过15%)0.75 gYeast Extract (water-soluble)酵母提取物(水溶性)5.0 gPancreatic Digest of Casein酪蛋白胰酶消化物15.0 gSodium Thioglycollate巯基乙酸钠0.5 gor Thioglycolic Acid或者巯基乙酸0.3 mLResazurin Sodium Solution (1 in 1000),freshly prepared刃天青钠溶液(1比1000)，新配制1.0 mLPurified Water 纯净水1000 mLMix the L-cystine, sodium chloride, dextrose, yeast extract, and pancreatic digest of casein with the purified water, and heat until solution is effected. Dissolve the sodium thioglycollate or thioglycolic acid in the solution and, if necessary, add 1 N sodium hydroxide so that, after sterilization, the solution will have a pH of 7.1 0.2. If filtration is necessary, heat the solution again without boiling, and filter while hot through moistened filter paper. Add the resazurin sodium solution, mix, and place the medium in suitable vessels that provide a ratio of surface to depth of medium such that not more than the upper half of the medium has undergone a color change indicative of oxygen uptake at the end of the incubation period. Sterilize using a validated process. If the medium is stored, store at a temperature between 2 and 25 in a sterile, airtight container. If more than the upper one-third of the medium has acquired a pink color, the medium may be restored once by heating the containers in a water-bath or in free-flowing steam until the pink color disappears and by cooling quickly, taking care to prevent the introduction of nonsterile air into the container.将L-胱氨酸、氯化钠、葡萄糖、酵母提取物、酪蛋白胰酶消化物与纯净水混合，并加热至实现溶解。将巯基乙酸钠或者巯基乙酸溶解于该溶液，如果需要可再加入1N氢氧化钠，以便在灭菌后该溶液呈pH值7.1 0.2。如需要则过滤，再次加热该溶液但不得煮沸，并趁热以湿润滤纸将该溶液过滤。加入刃天青钠溶液，混匀，并将该培养基置于适当容器中，该容器应为培养基提供特定的面积深度比，以使在培养期末表明氧气摄入的变色部分不超过培养基的上半部分。使用经过验证的工艺进行灭菌。如果需要储存该培养基，将其置于无菌、气密容器中，在2 至25 之间储藏。如果超过上部三分之一的培养基已经呈粉色，可以用以下方法恢复该培养基一次：在水浴锅中或者自由流动蒸气中加热该容器，直至粉色消失，并迅速放凉，须小心防止非无菌空气进入到容器中。Fluid Thioglycollate Medium is to be incubated at 32.5 2.5 .巯基醋酸盐液体培养基将在32.5 2.5 条件下进行培养。Alternative Thioglycollate Medium 替代巯基醋酸盐培养基Prepare a mixture having the same composition as that of the Fluid Thioglycollate Medium, but omitting the agar and the resazurin sodium solution, sterilize as directed above, and allow to cool prior to use. The pH after sterilization is 7.1 0.2. Incubate under anaerobic conditions for the duration of the incubation period.配制与巯基醋酸盐液体培养基成分相同，但省略了琼脂和刃天青钠溶液的混合物，按上述方法灭菌，并在使用前静置至凉。灭菌后pH值为7.1 0.2。在厌氧条件下培养，培养时间同培养期。Alternative Fluid Thioglycollate Medium is to be incubated at 32.5 2.5 . 替代性巯基醋酸盐培养基将在32.5 2.5 条件下进行培养。SoybeanCasein Digest Medium 大豆-酪蛋白消化物培养基Pancreatic Digest of Casein酪蛋白胰酶消化物17.0 gPapaic Digest of Soybean Meal大豆粉木瓜蛋白酶消化物3.0 gSodium Chloride氯化钠5.0 gDibasic Potassium Phosphate磷酸氢二钾2.5 gDextrose (C6H12O6H2O)葡萄糖2.5/2.3 gPurified Water纯净水1000 mLDissolve the solids in the Purified Water, heating slightly to effect a solution. Cool the solution to room temperature, and adjust the pH with 1 N sodium hydroxide so that, after sterilization, it will have a pH of 7.3 0.2. Filter, if necessary to clarify, dispense into suitable containers, and sterilize using a validated procedure. Store at a temperature between 2 and 25 in a sterile well-closed container, unless it is intended for immediate use.将固体物质溶解于纯净水，轻微加热以实现溶解。放凉溶液至室温，并用1N氢氧化钠调整pH值，以便在灭菌后其pH值呈 7.3 0.2。过滤，如需要则使之澄清，分装入适合的容器，并用经过验证的程序消毒。如果不立刻使用，则在2 到25 之间以无菌且密闭良好的容器保存。SoybeanCasein Digest Medium is to be incubated at 22.5 2.5 .大豆-酪蛋白消化物培养基将在22.5 2.5 条件下培养。Media for Penicillins or Cephalosporins 用于青霉素和头孢菌素的培养基Where sterility test media are to be used in the Direct Inoculation of the Culture Medium method under Test for Sterility of the Product to be Examined, modify the preparation of Fluid Thioglycollate Medium and the SoybeanCasein Digest Medium as follows. To the containers of each medium, transfer aseptically a quantity of -lactamase sufficient to inactivate the amount of antibiotic in the specimen under test. Determine the quantity of -lactamase required to inactivate the antibiotic by using a -lactamase preparation that has been assayed previously for its penicillin- or cephalosporin-inactivating power. NOTESupplemented -lactamase media can also be used in the membrane filtration test. 当无菌检查培养基用于供试产品无菌检查项下的培养基直接接种法时，按如下内容变更巯基醋酸盐液体培养基和大豆-酪蛋白消化物培养基的 制备方法。向每一种培养基的容器中，以无菌操作转移足够灭活供试样品中所存在抗生素的 -内酰胺酶。使用此前已经对其青霉素或头孢菌素灭活能力进行了测定的 -内酰胺酶配制品，来测定灭活该抗生素所必需的 -内酰胺酶数量。注意：补充的 -内酰胺酶培养基也可以用于膜过滤试验Alternatively (in an area completely separate from that used for sterility testing), confirm that an appropriate amount of -lactamase is incorporated into the medium, following either method under Validation Test, using less than 100 colony-forming units (cfu) of Staphylococcus aureus (see Table 1) as the challenge. Typical microbial growth of the inoculated culture must be observed as a confirmation that the -lactamase concentration is appropriate. 或者（在与无菌试验所用场所彻底隔离的区域中），按照验证试验项下的任意一种方法，使用少于100个菌落（cfu）的金黄色葡萄球菌（见表1）作为验证菌，来确认适当数量的 -内酰胺酶已经被整合到该培养基中。必须观测到接种后培养物中出现典型微生物生长，才能确认 -内酰胺酶浓度是适当的。Table 1. Strains of the Test Microorganisms Suitable for Use in the Growth Promotion Test and theValidation Test 表1 适合用于生长促进试验和验证试验中的试验微生物的菌株Aerobic bacteria好氧菌Staphylococcus aureus 1 金黄色葡萄球菌ATCC 6538, CIP 4.83, NCTC 10788, NCIMB 9518Bacillus subtilis枯草芽孢杆菌ATCC 6633, CIP 52.62, NCIMB 8054Pseudomonas aeruginosa 2 绿脓杆菌ATCC 9027, NCIMB 8626, CIP 82.118Anaerobic bacterium厌氧菌Clostridium sporogenes 3 产芽胞梭状芽胞杆菌ATCC 19404, CIP 79.3, NCTC 532 or ATCC 11437Fungi霉菌Candida albicans白色念珠菌ATCC 10231, IP 48.72, NCPF 3179Aspergillus niger黑曲霉ATCC 16404, IP 1431.83, IMI 1490071 An alternative to Staphylococcus aureus is Bacillus subtilis (ATCC 6633).可替代金黄色葡萄球菌的是枯草杆菌(ATCC 6633) 2 An alternative microorganism is Micrococcus luteus (Kocuria rhizophila), ATCC 9341. 替代微生物是藤黄微球菌（Kocuria rhizophila），ATCC 9341。 3 An alternative to Clostridium sporogenes, when a nonspore-forming microorganism is desired, is Bacetroides vulgatus (ATCC 8482). 当需要不形成芽孢微生物时，产芽胞梭状芽胞杆菌的替代微生物是Bacetroides vulgatus NOTESeed-lot culture maintenance techniques (seed-lot systems) are used so that the viable microorganisms used for inoculation are not more than five passages removed from the original master seed lot. 注意：采用适宜的菌种保藏技术，以确保用于接种的微生物的传代次数不超过5代Suitability Tests 适合性试验The media used comply with the following tests, carried out before, or in parallel, with the test on the product to be examined.所使用的培养基须符合下列试验，这些试验应在检验供试产品之前或者同时进行。STERILITY 无菌状态Confirm the sterility of each sterilized batch of medium by incubating a portion of the media at the specified incubation temperature for 14 days. No growth of microorganisms occurs.通过在指定培养温度下将一部分培养基培养14天，来确认每一批已灭菌培养基的无菌状态。不得出现微生物生长。GROWTH PROMOTION TEST OF AEROBES, ANAEROBES, and FUNGI 好氧菌、厌氧菌、霉菌的生长促进试验Test each lot of ready-prepared medium and each batch of medium prepared either from dehydrated medium or from ingredients 1 . Suitable strains of microorganisms are indicated in Table 1.检查每一批已经配制好的培养基和每一批用脱水培养基或配料制备的培养基 1 。适当微生物菌株见表1。Inoculate portions of Fluid Thioglycollate Medium with a small number (not more than 100 cfu) of the following microorganisms, using a separate portion of medium for each of the following species of microorganism: Clostridium sporogenes, Pseudomonas aeruginosa, and Staphylococcus aureus. Inoculate portions of Alternative Fluid Thioglycollate Medium with a small number (not more than 100 cfu) of Clostridium sporogenes. Inoculate portions of SoybeanCasein Digest Medium with a small number (not more than 100 cfu) of the following microorganisms, using a separate portion of medium for each of the following species of microorganism: Aspergillus niger, Bacillus subtilis, and Candida albicans. Incubate for not more than 3 days in the case of bacteria and not more than 5 days in the case of fungi.在部分巯基醋酸盐液体培养基上接种少量（不超过100cfu）下列微生物，每一种微生物均使用单独一部分培养基：产芽胞梭状芽胞杆菌、绿脓杆菌、金黄色葡萄球菌。 在部分替代巯基醋酸盐液体培养基上接种少量（不超过100cfu）产芽胞梭状芽胞杆菌。 在部分大豆-酪蛋白消化物培养基上接种少量（不超过100cfu）下列微生物，每一种微生物均使用单独一部分的培养基：黑曲霉、枯草芽孢杆菌、白色念珠菌。细菌培养时间不超过3天，霉菌培养时间不超过5天。The media are suitable if a clearly visible growth of the microorganisms occurs.如果出现清晰可见的微生物生长，则该培养基是适合的。STORAGE 保存If prepared media are stored in unsealed containers, they can be used for 1 month, provided that they are tested for growth promotion within 2 weeks of the time of use and that color indicator requirements are met. If stored in tight containers, the media can be used for 1 year, provided that they are tested for growth promotion within 3 months of the time of use and that the color indicator requirements are met. 如果配制好的培养基保存于未密闭的容器中，只要在使用时间的2周内对其进行了生长促进试验并且符合颜色指示剂的要求，它们就可以使用1个月。如果保存在密闭的容器中，只要在使用时间的3个月内对其进行了生长促进试验并且符合颜色指示剂的要求，则该培养基可以使用1年。DILUTING AND RINSING FLUIDS FOR MEMBRANE FILTRATION 用于膜过滤的稀释和冲洗液Fluid A 液体APREPARATION 配制品Dissolve 1 g of peptic digest of animal tissue in water to make 1 L, filter or centrifuge to clarify, if necessary, and adjust to a pH of 7.1 0.2. Dispense into containers, and sterilize using a validated process.将1g动物组织胃蛋白酶消化物溶于1L水中，如果需要则通过滤或离心使其澄清，再调节pH值至7.1 0.2。分装入容器中，并用经过验证的工艺灭菌。PREPARATION FOR PENICILLINS OR CEPHALOSPORINS用于青霉素或头孢菌素的配制品Aseptically add to the above Preparation, if necessary, a quantity of sterile -lactamase sufficient to inactivate any residual antibiotic activity on the membranes after the solution of the test specimen has been filtered (see Media for Penicillins or Cephalosporins).在供试样品溶液已经过滤（见用于青霉素或头孢菌素的培养基）之后，如果需要，向上述配制品中，以无菌操作加入数量足够灭活滤膜上残余抗生素活性的 -内酰胺酶。Fluid D 液体DTo each L of Fluid A add 1 mL of polysorbate 80, adjust to a pH of 7.1 0.2, dispense into containers, and sterilize using a validated process. Use this fluid for articles containing lecithin or oil, or for devices labeled as “sterile pathway.”向每升液体A中，加入1mL聚山梨酯80，调节pH值至7.1 0.2，分装入容器中，并使用经过验证的工艺灭菌。此液体用于含有卵磷脂或油脂的物品，或用于标为 “无菌通道”的设备。Fluid K 液体KDissolve 5.0 g of peptic digest of animal tissue, 3.0 g of beef extract, and 10.0 g of polysorbate 80 in water to make 1 L. Adjust the pH to obtain, after sterilization, a pH of 6.9 0.2. Dispense into containers, and sterilize using a validated process. 将5.0g动物组织胃蛋白酶消化物、3.0g牛肉提取物、10.0g聚山梨酯80溶解于1L水中。调节pH值，以便使pH值在灭菌后呈6.9 0.2。分装入容器中，并使用经过验证的工艺灭菌。VALIDATION TEST 验证试验Carry out a test as described below under Test for Sterility of the Product to be Examined using exactly the same methods, except for the following modifications.按照下面供试产品无菌检查项下的描述，使用除了下面变更之外完全相同的方法，进行试验。Membrane Filtration 膜过滤After transferring the content of the container or containers to be tested to the membrane, add an inoculum of a small number of viable microorganisms (not more than 100 cfu) to the final portion of sterile diluent used to rinse the filter.在将一个或多个供试容器中的内容物转移到滤膜之后，在最后一次的冲洗液中加入少量（不超过100cfu）试验菌.Direct Inoculation 直接接种