无菌检查方案.doc

71 STERILITY TESTS 无菌测试 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 此章节的某些部份与欧洲药典和 或日本药典的相关内容一致 不一致的章节用 符号 来表示 以标明其事实 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 以下程序用来确认药典药品是否按单独记录所列相关无菌测试要求执行 药品的特性允许下 按受检药品无菌测试中的薄膜过滤法来测试药典药品 如果薄膜过滤法不适用 用受检药品 无菌测试中的培养基的直接接种指示方法来测试 除设备路径标明无菌外 所有的仪器全用 培养基的直接接种指示来测试 测试的物品包含于结果观察资料及说明 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 因为无菌测试是一个极其严格的测试 必需确保灭菌程序以求结果的正确诠释 所以对人员 进行相应培训以至合格就显得极为重要 无菌测试在无菌条件下进行 为了达到这样的环境 应改变周围的测试环境至无菌测试环境 应采取预防措施避免污染 并且此措施不影响测试 中有机体的暴露 无菌测试的工作环境应靠在工作区取样来接受定期监控 同时进行适当控 制 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 这些医典中的程序并非由自己确定一批药品的无菌性 或者是已经过灭菌的 它主要是由灭 菌过程和无菌加工程序认证来确定的 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 当通过相应药典中的方法得到的药品中的微生物污染证据 即使另一程序得到不同结果 以 此得到的结果为药品不符合无菌测试要求的最终结果 无菌测试的详细信息见 Compendial Articles的灭菌和无菌保证 1211 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 按以下所述为测试准备培养基 或无水配方 当按生产商和销售商指示重新组成 它们符合 需氧菌 厌氧性生物 和真菌类助长测试要求 按验证方法对培养基进行灭菌 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 Soybean Casein Digest Medium is suitable for the culture of both fungi and aerobic bacteria 以下培养基适用于无菌测试 液态硫乙醇酸盐培养基主要是用来培养厌氧菌 大豆豆蛋白消 化物培养基适用于培养真菌和需氧菌 Fluid Thioglycollate Medium 液态硫乙醇酸盐培养基 L CystineL 胱氨酸0 5 g Sodium Chloride 氯化钠2 5 g Dextrose C6H12O6 H2O 葡萄糖5 5 5 0 g Agar granulated moisture content not exceeding 15 琼脂 颗粒状 湿度不超过 15 0 75 g Yeast Extract water soluble 酵母浸出粉 水溶性 5 0 g Pancreatic Digest of Casein 酪蛋白胰酶消化物15 0 g Sodium Thioglycollate 硫乙醇酸钠0 5 g or Thioglycolic Acid 或者疏乙醋酸0 3 mL Resazurin Sodium Solution 1 in 1000 freshly prepared 新配 制的 1 1000 刃天青钠溶液1 0 mL Purified Water 水1000 mL Mix 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 Fluid Thioglycollate Medium is to be incubated at 32 5 2 5 用纯静水将 L 胱氨酸 氯化钠 葡萄糖 酵母浸出粉 酪蛋白胰酶消化物混合 加热直至形 成溶液 在溶液中溶解硫乙醇酸钠或疏乙醋酸 必要时 加入 1 N 的氢氧化钠 在灭菌后 pH 值会达到 7 1 0 2 如果有必要过滤的话 加热溶液但不用达到沸点 热气通过潮湿的 过滤纸时进行过滤 加入刃天青钠溶液 并搅拌混合 将培养基放入适当的容器中 此容器 可以显示一个培养基表面至深度的比率 培育阶段末时 不超过培养基上半部份的指示剂氧 化层发生颜色变化 用验证方法进行灭菌 如果要存储培养基 将它存储于一个 2 和 25 间的无菌密封容器中 如果超过三分之一的培养基显示粉红色 在水浴或自由流通蒸汽中加 热容器直至粉红色消失 并快速冷却 并防止有菌气体产生并进入容器 液态硫乙醇酸盐培 养基就在 32 5 2 5 下进行培育 Alternative Thioglycollate Medium 可选择疏基醋酸液培养基 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 按液态硫乙醇酸盐培养基所含成份同样准备一份混合物 除去琼脂和刃天青钠溶液 按以上 所示灭菌 使用前允许进行冷却 灭菌后 pH 值为 7 1 0 2 在厌氧条件下进行培育 以求 培育期的持续 Alternative Fluid Thioglycollate Medium is to be incubated at 32 5 2 5 可选择疏基醋酸液培养基应在 32 5 2 5 下进行培育 Soybean Casein Digest Medium 大豆大豆 酪蛋白消化物培养基酪蛋白消化物培养基 Pancreatic Digest of Casein 酪蛋白胰酶消化物17 0 g Papaic Digest of Soybean Meal 大豆粉木瓜蛋白酶水化物3 0 g Sodium Chloride 氯化钠5 0 g Dibasic Potassium Phosphate 磷酸氢二钾2 5 g Dextrose C6H12O6 H2O 葡萄糖2 5 2 3 g Purified Water 水1000 mL Dissolve 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 在水中溶解固体 轻轻加热以形成溶液 将溶液冷却至室温 用 1 N 的氢氧化钠来调节 pH 值 以至在灭菌后其 pH 值达到 7 3 0 2 过滤 如果有必要进行澄清时 将漏斗放入相应 的容器中 并用验证方法来进行灭菌 在一个无菌密封容器中 2 和 25下进行存储 直至 使用 Soybean Casein Digest Medium is to be incubated at 22 5 2 5 大豆酪 蛋白消化物培养基在 22 5 2 5 下进行培育 Media for Penicillins or Cephalosporins 青霉素或头孢菌素培养基青霉素或头孢菌素培养基 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 Soybean Casein 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 NOTE Supplemented lactamase media can also be used in the membrane filtration test 当用受检药品无菌试验测试的培养基直接接种方法来进行无菌试验时 按以下更正液态硫乙 醇酸盐培养基和大豆 酪蛋白消化物培养基的制剂 在无菌情况下 将足够的 内酰胺酶转 移到每种培养基的容器中 来钝化试验样品中的抗生素 用以前化验青霉素或头孢菌素钝化 力的 内酰胺酶制剂来确定需要钝化抗生素的 内酰胺酶的量 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 另外 在另一个完全与无菌试验无关的领域 确定适当量 内酰胺酶混合于培养基中 接 着用验证测试中的方法 用不少于 100cfu 葡萄状球菌 见表1 做为挑战性实验 接种的培 养菌的典型微生物生长应接受观察 以确定 内酰胺酶的浓渡适当 Table 1 Strains of the Test Microorganisms Suitable for Use in the Growth Promotion Test and theValidation Test 表表 1 促生长试验和验证试验中的可用到微生物菌株促生长试验和验证试验中的可用到微生物菌株 Aerobic bacteria 需氧细菌 Staphylococcus aureus 1 金黄色葡萄球 菌 ATCC 6538 CIP 4 83 NCTC 10788 NCIMB 9518 Bacillus subtilis枯草芽孢杆菌ATCC 6633 CIP 52 62 NCIMB 8054 Pseudomonas aeruginosa 2 铜绿假单胞 菌 ATCC 9027 NCIMB 8626 CIP 82 118 Anaerobic bacterium 厌氧细菌 Clostridium sporogenes 3 生孢梭菌 ATCC 19404 CIP 79 3 NCTC 532 or ATCC 11437 Fungi 真菌类 Candida albicans白色念珠菌ATCC 10231 IP 48 72 NCPF 3179 Aspergillus niger黑曲霉ATCC 16404 IP 1431 83 IMI 149007 1 An alternative to Staphylococcus aureus is Bacillus subtilis ATCC 6633 可选择杆状菌 ATCC 6633 代替葡萄球状菌 2 An alternative microorganism is Micrococcus luteus Kocuria rhizophila ATCC 9341 可选择微球菌 ATCC 9341 代替微生物 3 An alternative to Clostridium sporogenes when a nonspore forming microorganism is desired is Bacetroides vulgatus ATCC 8482 当需要不产生孢子的微生物时 可选择普通拟杆菌 ATCC 8482 来代替梭菌 NOTE Seed 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 注释 使用种子批培养保护技术 种子批体系 以至接种所用的可繁殖微生物不超过五代而远离原 始主种子批 Suitability Tests 适用性试验适用性试验 The media used comply with the following tests carried out before or in parallel with the test on the product to be examined 所用培养基和以下试验相符合 它在受检产品测试之或与之同时进行 STERILITY 无菌性无菌性 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 确定每批消过毒的培养基的无菌性 在特殊的培养温度中培育一部份培养基 14 天 微生物 不生长 GROWTH PROMOTION TEST OF AEROBES ANAEROBES AND FUNGI 需氧菌 压氧菌和真菌类助长试验需氧菌 压氧菌和真菌类助长试验 Test each lot of 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 对准备好的每批培养基 和从干粉培养基或混合粉中准备的每批培养基进行试验 表 1 中显示出适宜的微生物类 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 Soybean Casein 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 用一小部份 不超过 100cfu 以下微生物 来接种一部份液态硫乙醇酸盐培养基 以下每 种微生物都分别有一部份培养基 梭菌 假单胞菌和葡萄状球菌 用小数量的 不超过 100cfu 梭菌来嫁接可选择疏基醋酸液培养基 用小数量 不超过 100cfu 的以下微生物 来接种大豆酪蛋白消化物培养基 以下每种微生物使用单独的培养基 黑曲霉菌 枯草芽孢 杆菌和白色念珠菌 在细菌的情况下培育不超过 3 天 在真菌类情况下 培育不超过 5 天 The media are suitable if a clearly visible growth of the microorganisms occurs 如果微生物生长明显出现 培养基适用 STORAGE 存储存储 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 如果已准备培养基存储于未密封容器中 可使用 1 个月 使用的两周时间内进行助长试验 指示剂的颜色符合要求 可使用 1 个月 如果存存储于一个密封的容器中 使用时间的三个 月内进行助长试验 指示剂颜色符合要求 培养基可用 1 年 DILUTING AND RINSING FLUIDS FOR MEMBRANE FILTRATION 薄膜过滤法的稀释液和冲洗液 Fluid A 液体液体 A PREPARATION 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 溶解制剂溶解制剂 将 1g 动物组织胃蛋白酶消化物加入水中制成 1L 如果需要 过滤或者离心直 到溶液澄清 并将其 pH 值调至 7 1 0 2 分配到溶器中 通过验证程序灭菌 PREPARATION FOR PENICILLINS OR CEPHALOSPORINS 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 青霉素和头孢子菌的无菌配制青霉素和头孢子菌的无菌配制 将一定量的 内酰胺酶加入上述所备制剂中 以钝化在 试验样品溶液过滤后薄膜上任何残余抗生素活性 见青霉素或头孢菌素培养基 Fluid D 液体液体 D To 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 每 1L 液体 A 就加入 1mL 聚山梨醇酯 80 将 pH 值调至 7 1 0 2 并入配到容器中 通过 验证程序灭菌 此液体用于含蛋黄素或油脂的药品 或用于贴着 无菌路径 的仪器 Fluid K 液体液体 K Dissolve 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液 3 0g 浓缩牛肉汁 和 10 0g 聚山梨醇酯 80 溶解制成 1L 调节 pH 值 在灭菌后 达到 6 9 0 2 分配到容器中 通过验证程序灭菌 VALIDATION TEST 验证测试 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 按受检产品无菌试验下同样的方法 按以下所述进行试验操作 除以下更改 Membrane Filtration 薄膜过滤法薄膜过滤法 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 在将容器内溶液或者待测容器中的溶液转移至薄膜后 在用来清洗过滤器最后部份的无菌稀 释液里加入 不超过 100cfu 小部份可繁殖微生物的培养基 Direct Inoculation 直接接种直接接种 After transferring the contents of the container or containers to be tested for catgut and other surgical sutures for veterinary use strands to the culture medium add an inoculum of a small number of viable microorganisms not more than 100 cfu to the medium 在将容器内溶液或者待测容器 肠线或其它兽医用的外科缝合用线 中的溶液转移至培养基 后 在培养基中加入 不超过 100 cfu 少量的可繁殖微生物 In both cases use the same microorganisms as those described above under Growth Promotion Test of Aerobes Anaerobes and Fungi Perform a growth promotion test as a positive control Incubate all the containers containing medium for not more than 5 days 在两种情况下 按需氧菌 厌氧性生物 和真菌类的助长试验上面所描述 使用同种微生物 执行助长试验作为积极控制 对含有培养基的所有容器进行不超过五天培育 If clearly visible growth of microorganisms is obtained after the incubation visually comparable to that in the control vessel without product either the product possesses no antimicrobial activity under the conditions of the test or such activity has been satisfactorily eliminated The test for sterility may then be carried out without further modification 如果培养后 与没有产品的控制容器内相比 或者与在试验条件下不含抗微生物活性的产品 相比 或者与活性已消除产品相比 微生物生长明显示 那么无菌试验可在不修改下执行 If clearly visible growth is not obtained in the presence of the product to be tested visually comparable to that in the control vessels without product the product possesses antimicrobial activity that has not been satisfactorily eliminated under the conditions of the test Modify the conditions in order to eliminate the antimicrobial activity and repeat the validation test 与没有产品的控制容器内相比 或者与在试验条件下含抗微生物活性的产品活性未被消化相 比 如果受检产品的微生物没有明显的生长 那么更改条件 以消除抗微生物活性 并重复 验证测试 This validation is performed a when the test for sterility has to be carried out on a new product and b whenever there is a change in the experimental conditions of the test The validation may be performed simultaneously with the Test for Sterility of the Product to be Examined a 当新产品进行无菌试验时 b 当测试的试验条件出现变化时 验证执行 验证与受检产品 无菌测试可以同时进行 TEST FOR STERILITY OF THE PRODUCT TO BE EXAMINED 受检产品无菌测试 Number of Articles to Be Tested 受检药品数受检药品数 Unless otherwise specified elsewhere in this chapter or in the individual monograph test the number of articles specified in Table 3 If the contents of each article are of sufficient quantity see Table 2 they may be divided so that equal appropriate portions are added to each of the specified media NOTE Perform sterility testing employing two or more of the specified media If each article does not contain sufficient quantities for each medium use twice the number of articles indicated in Table 3 除非此章节或者专论中的其它地方有特别说明 否则按表3 详细列出的药品数进行测试 如 果每种药品的成份足量 见表2 它们将会被分成适当的小份并被加到每份指定的培养基 中 注释 进行无菌试验时 运用两到三种指定培养基 如果每种培养基中的每种药品 量不足 按表3 所示药品用两倍 Table 2 Minimum Quantity to be Used for Each Medium 表表 2 每种培养基所使用的最小量每种培养基所使用的最小量 Quantity per Container 每个容器的量 Minimum Quantity to be Used 除非另有证明及授权 所用的最小量 除非另有证明和授权 Liquids other than anitbiotics 液体 除抗生素外 Less than 1 mL 少于 1mLThe whole contents of each container 每个容器的全部量 1 40 mLHalf the contents of each container but not less than 1 mL 每个容器容量的一半 但不 少于 1mL Greater than 40 mL and not greater than 100 mL 大于 40mL 但不超过 100mL 20 mL Greater than 100 mL 超过 100mL10 of the contents of the container but Quantity per Container 每个容器的量 Minimum Quantity to be Used 除非另有证明及授权 所用的最小量 除非另有证明和授权 not less than 20 mL 容器量的 10 但不超过 20 mL Antibiotic liquids液体抗生素1 mL Other preparations soluble in water or in isopropyl myristate其它可溶于水或透汽性油脂 的 制剂 The whole contents of each container to provide not less than 200 mg 每个容器的全部容量 不少于 200mg Insoluble preparations creams and ointments to be suspended or emulsified 可溶解制剂 悬浮的或浮化的乳脂和药膏 Use the contents of each container to provide not less than 200 mg 每个容器的容量 少于 200 mg Solids固体 Less than 50 mg 少于 50 mgThe whole cont