2018-10-26 - chp.org.cn · – Tryptic Soy Broth (TSB): aerobic incubation at 20-25 °C...

2018-10-26

Click to edit Master title style

Issues with the Sterility Test applicationMethod suitability and Routine testing Anja Nuydens

Janssen, Pharmaceutical companies of Johnson & Johnson

Confidential for restricted use only

Click to edit Master title styleContent of the presentation

• Introduction• Sterility test method suitability

• Micro-organisms and Growth media• Amount of product and test articles• Decision flow• Sample preparation• Methodology:

– Membrane filtration– Direct inoculation

• Incubation, reading and subcultures• Optimization steps

• Isolator system• Test for growth-promoting properties• Routine sterility testing• Rapid/alternative sterility test methods• Questions


2018-10-26

Click to edit Master title styleIntroduction

• Goal of the sterility test: detect any microbial growth in a drug product, device, or drug substance claimed to be sterile

• Pharmacopoeial overview:

Industry preference is to have a global harmonized method suitability and routine test approach across all Pharmacopoeia.


<71> Sterility tests 2.6.1 Sterility 4.06 Sterility Test 1101 Sterility Test

Click to edit Master title styleMethod SuitabilityMicro-organisms and Growth media

• Micro-organisms used for method suitability (method qualification):

– Staphylococcus aureus ATCC 6538 (or equivalent)– Pseudomonas aeruginosa ATCC 9027 (or equivalent)– Clostridium sporogenes ATCC 19404 or ATCC 11437 (or equivalent)– Bacillus subtilis ATCC 6633 (or equivalent)– Candida albicans ATCC 10231 (or equivalent)– Aspergillus brasiliensis ATCC 16404 (or equivalent)– In-house environmental isolate

• Inoculation with ≤ 100 CFU (colony forming units)

• Growth media: 2 types :

– Fluid Thioglycollate Medium (FTM): anaerobic incubation at 30-35 °C– Tryptic Soy Broth (TSB): aerobic incubation at 20-25 °C

Confidential for restricted use only 4

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Click to edit Master title styleMethod SuitabilityAmount of product and test articles

• Quantity of product to be tested – According to the pharmacopoeial guidelines– Depending on the characteristics of the product (liquids, antibiotic liquids, solids,

devices and others…)

• Number of test articles (e.g., containers) to be tested– According to the pharmacopeial guidelines– Related to the batch size– Depending on the formulation/administration route (parenteral preparations,

antibiotic solids, opthalmic preparations, non-injectable preparations, devices, bulk solid products…)


Click to edit Master title styleMethod SuitabilityDecision flow

Is the product filterable?

Membrane filtration: antimicrobial properties?

Rinsing ≥ 3 times with 100 mL

Rinsing ≥ 1 time with 100 mL

Direct inoculation: antimicrobial properties?

Further optimization, e.g., media with

neutralizers, increased volume

100 mL FTM and 100 mL TSB


Yes No

Yes No Yes No

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Click to edit Master title styleMethod SuitabilitySample preparation for specific product types (1)

• Aqueous solutions• If necessary, dilute with a chosen sterile diluent• Fluid A• Antimicrobial properties: rinsing ≥ 3 times 100 mL• No antimicrobial properties: rinsing ≥ 1 time 100 mL• Not filterable: direct inoculation method

• Oils and oily solutions• If necessary, dilute with a suitable sterile diluent e.g., isopropyl

myristate• Fluid K and Fluid A• Rinsing ≥ 3 times 100 mL• Not filterable: direct inoculation method



• Solids for injection other than antibiotics• Reconstitute the test article as directed on the label or by the drug

development team• If necessary, dilute with a chosen sterile diluent• Fluid A• Antimicrobial properties: rinsing ≥ 3 times 100 mL• No antimicrobial properties: rinsing ≥ 1 time 100 mL• Not filterable: direct inoculation method

• Antibiotic solids for injection• Reconstitute the test article as directed on the label or by the drug

development team• If necessary, dilute with a chosen sterile diluent• Fluid K and Fluid A• Rinsing ≥ 3 times 100 mL• Not filterable: direct inoculation method


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• Devices with pathways labelled sterile• Aseptically pass ≥ 10 pathway volumes of Fluid D through each

device tested• Collect the fluids in an appropriate sterile vessel• Fluid A• Rinsing ≥ 1 time100 mL

• Sterile Devices• Use the direct inoculation method• Articles can be immersed intact or disassembled• Volume of media should be sufficient to immerse the device

completely• For extremely large devices, immerse the portions of the device that

will be in contact with the patient in a volume sufficient to achieve complete immersion of those portions


Click to edit Master title styleMethod SuitabilityMethodology: Membrane Filtration (1)

• Product Test• Prewetting: ≥ 50 mL of chosen rinsing fluid• Transfer the quantity of product or sample preparation to membrane

and filter• Wash the membrane 2 x 100 mL of chosen rinsing fluid, in case of

pharmaceutical products and (bio)pharmaceutical oils, oily solutions, ointments and creams

• Inoculate 100 mL of the chosen rinsing fluid with ≤ 100 CFU micro-organisms

• Rinse the membrane with 100 mL inoculated rinsing fluid• Close the canister• Transfer FTM or TSB (100 mL) to the canisters:

– FTM: Staphylococcus aureus, Pseudomonas aeruginosa, Clostridium sporogenes, in-house environmental isolate

– TSB: Bacillus subtilis, Candida albicans, Aspergillus brasiliensis, in-house environmental isolate


• Note: for commercial purposes, method suitability is performed on 3 different batches

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• Positive control: in order to check the growing abilities of the micro-organisms and to obtain a reference for growth in unchallenged media

• Inoculate 100 mL of the chosen rinsing fluid with ≤ 100 CFU• Rinse the membrane with 100 mL inoculated rinsing fluid• Close the canister• Transfer FTM or TSB (100 mL) to the canisters:




• Note: the in-house environmental isolate should show growth in at least 1 growth medium (FTM or TSB) for both the product test and positive control


• Negative control: in order to check the aseptic handlings (without product)• Perform the negative control in the same way as the product test without

adding the inoculum and the product. Replace the product with a chosen diluent

• Product control: in order to check the interaction of FTM/TSB with the product and to check the sterility of the product

• Perform the product control in the same way as the product test but without adding the inoculum

• Reference count: in order to check the concentration of the inoculum suspension• Transfer in duplicate the volume of the work dilution used for the

inoculation of the dilution fluid on prefilled TSA petridishes (ø 9 cm)or equivalent (for bacteria and fungi) and perform the spread plate method

• Incubate:– at 30-35 °C: Staphylococcus aureus, Pseudomonas aeruginosa, Clostridium

sporogenes (anaerobic incubation), in-house environmental isolate– at 20-25 °C: Bacillus subtilis, Candida albicans, Aspergillus brasiliensis, in-

house environmental isolate• Reading after at least 3 days


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Click to edit Master title styleMethod SuitabilityMethodology: Direct inoculation (1)

• Product Test• Transfer the quantity of product or sample preparation directly to the

canister or container filled with FTM or TSB• Inoculate with ≤ 100 CFU micro-organisms as follows



• Positive control: in order to check the growing abilities of the micro-organisms and to obtain a reference for growth in unchallenged media

• Transfer the chosen volume of the appropriate culture medium to the canister or container filled with the appropriate culture medium

• Inoculate with ≤ 100 CFU micro-organisms as follows– FTM: Staphylococcus aureus, Pseudomonas aeruginosa, Clostridium

sporogenes, in-house environmental isolate– TSB: Bacillus subtilis, Candida albicans, Aspergillus brasiliensis, in-house

environmental isolate


• Note: the in-house environmental isolate should show growth in at least 1 growth medium (FTM or TSB) for both the product test and positive control

Click to edit Master title styleMethod SuitabilityMethodology: Direct inoculation (2)

• Negative control: in order to check the aseptic handlings (without product )• Perform the negative control in the same way as the product test without

adding the inoculum and the product. Replace the product with a chosen diluent

• Product control: in order to check the interaction of FTM/TSB with the product and to check the sterility of the product

• Perform the product control in the same way as the product test but without adding the inoculum

• Reference count: in order to check the concentration of the inoculum suspension• Transfer in duplicate the volume of the work dilution used for the

inoculation of the dilution fluid on prefilled TSA petridishes (ø 9 cm)or equivalent (for bacteria and fungi) and perform the spread plate method

• Incubate:– at 30-35 °C: Staphylococcus aureus, Pseudomonas aeruginosa, Clostridium

sporogenes (anaerobic incubation), in-house environmental isolate– at 20-25 °C: Bacillus subtilis, Candida albicans, Aspergillus brasiliensis, in-

house environmental isolate• Reading after at least 3 days


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Click to edit Master title styleMethod SuitabilityIncubation, Reading and Subcultures

• Incubate for maximum 5 days (Incubation of product control for 14 days)– FTM at 30-35 °C– TSB at 20-25 °C

• Growth must be confirmed microscopically, except for moulds for which growth must be confirmed macroscopically at the least

• Subcultures– When the material renders the medium turbid or if the product shows

interaction with the medium (e.g., particles), so that the presence or absence of microbial growth cannot be determined by visual determination, fluid subcultures must be made. Solid subcultures can be made concurrently if the material of the initial product test still renders the medium of the fluid subculture turbid.

– Incubation:• Fluid subcultures (FTM and TSB): maximum 4 days• Solid subcultures (aerobic and anaerobic TSA): maximum 5 days


FTM and TSB in Steritest canister

Click to edit Master title styleMethod SuitabilityOptimization

• Membrane Filtration:• The quantity of the rinsing fluid can be increased but not more than 500 mL• Different rinsing fluids• Different filter type (e.g., cellulose ester, PVDF, ...)

• Direct inoculation:• For oily liquids: media with addition of emulsifying agents (e.g., polysorbate 80) • Insoluble ointments, oils, non-filterable liquids and creams can be emulsified

with the chosen emulsifying agent (e.g., 1 g/L neutral solution of meat or casein peptone) in a suitable sterile diluent and then transferred to a medium without emulsifying agent

• The use of another container is recommended when there is not enough headspace in the container

• The maximum volume of media will be 500 mL• Fluid subculture:

• Transfer >1 mL but less than 10 mL of the original test medium• Solid subculture:

• 1 mL of the direct inoculation can be transferred on prefilled petridishes ø15 cm or equivalent


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Click to edit Master title styleIsolator system

• Package integrityEvaluation must be done of each new packaging material which will be brought in in the isolator

• Load patternMust be determined and evaluated for each new product that will be tested in the isolator system


La Calhène soft-wall isolator SKAN hard-wall isolator

Click to edit Master title styleTest for growth-promoting properties

• Growth-promoting properties are performed on each incoming batch of media and is performed in the same vials as used during testing to mimic as much as possible the testing environment


FTM TSBMicro-organisms Staphylococcus aureus

Pseudomonas aeruginosaClostridium sporogenesIn-house environmental isolate

Bacillus subtilisCandida albicansAspergillus brasiliensisIn-house environmental isolate

Incubation temperature

30-35 °C 20-25 °C

Incubation times Bacteria: maximum 3 daysFungi: maximum 5 days

Negative control Minimum 14 days

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Click to edit Master title styleRoutine sterility testing

• Performed in a VHP-decontaminated isolator (preferred technology within Janssen)

– Materials are transferred by the use of a transfer isolator– Decontamination with H2O2 (as vaporized hydrogen peroxide; VHP)– Defined load pattern

• Incubation of 14 days with an intermediate reading, if needed subcultures (fluid and/or solid) are made after 14 days

– Incubation temperatures• FTM at 30-35 °C• TSB at 20-25 °C


Click to edit Master title styleRapid/alternative sterility test methods

• Incubation period of 14 days in traditional sterility test can be too long for short-life products or products prepared for immediate use, e.g.,

– Positron Emission tomographic products (PET)– Compounded Sterile Preparations (CSP)– Cell and Gene Therapy Products (CGTP) (e.g., CAR-T)

• Rapid sterility test are preferred to assure fast results and to assure safety of the product in the best achievable way, e.g.,

– ATP-based– Gas production-based – Fluorescent staining– PCR-based nucleic acid amplification

• Pharmacopoeial and industry guidance:– USP <1223>: Validation of alternative microbiological methods– USP <1071>: Rapid sterility of short-life product (in draft)– Ph. Eur. 5.1.6: Alternative methods for control of microbiological quality– Ph. Eur. 2.6.27: Microbiological examination of cell-based preparations– PDA Technical report 33: Evaluation, validation and implementation of alternative and

rapid microbiological methods


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Click to edit Master title styleQuestions

• Use of ready to use strains (e.g., Bioballs®) during method qualification and for growth-promoting properties

• Questions?


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Click to edit Master title style

无菌检查应用的问题方法适用性和常规检测 Anja Nuyden

Janssen, Pharmaceutical companies of Johnson & Johnson

机密，仅限使用

Click to edit Master title style演示内容

• 简介• 无菌检查方法适用性

• 微生物和生长培养基• 产品和供试品数量• 决策流程• 样品制备• 研究方法：

– 薄膜过滤法– 直接接种

• 孵育、读数和传代培养• 优化步骤

• 隔离器系统• 促生长特性试验• 常规无菌检查• 快速/替代无菌检查方法• 问题


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Click to edit Master title style简介

• 无菌检查的目的：检测声明无菌的制剂、器械或原料药中的任何微生物生长

• 药典概述：

业界倾向于使用具备全球协调方法适用性并兼顾所有药典的常规检测方法


<71>无菌检查 2.6.1 无菌性 4.06 无菌检查 1101 无菌检查

Click to edit Master title style方法适用性微生物和生长培养基

• 用于方法适用性的微生物（方法确认）：

– 金黄色葡萄球菌 ATCC 6538（或同等产品）– 铜绿假单胞菌 ATCC 9027（或同等产品）– 生孢梭菌 ATCC 19404或ATCC 11437（或同等产品）– 枯草芽孢杆菌 ATCC 6633（或同等产品）– 白色念珠菌 ATCC 10231（或同等产品）– 巴西曲霉 ATCC 16404（或同等产品）– 内部环境分离菌

• 接种≤ 100 CFU（菌落形成单位）

• 生长培养基：2种类型：

– 硫乙醇酸盐液体培养基（FTM）：30-35℃下厌氧孵育– 胰蛋白酶大豆肉汤（TSB）：在20-25℃条件下需氧培养

机密，仅限使用 4

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Click to edit Master title style方法适用性产品和供试品的量

• 待测产品数量 – 根据药典指导原则– 取决于产品的特性（液体、抗生素液体、固体、器械和其他）

• 待测供试品（例如容器）数量– 根据药典指导原则– 与批量相关– 取决于制剂/给药途径（胃肠外制剂、抗生素固体、眼科制剂、非注射制剂、器械、散装固体

产品）


Click to edit Master title style方法适用性决策流程

产品是否可过滤？

膜过滤：抗菌特性？

用100 mL冲洗≥ 3次

用100 mL冲洗≥ 1次

直接接种：抗菌特性？

进一步优化，例如使用中和剂、体积

增加的培养基

100 mL FTM和100 mL TSB


是否

是否是否

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Click to edit Master title style方法适用性特定产品类型的样品制备（1）

• 水溶液• 必要时，用选择的无菌稀释液稀释• 冲洗液A• 抗菌特性：冲洗≥ 3次，100 mL• 无抗菌特性：冲洗≥ 1次，100 mL• 不可过滤：直接接种法

• 油和油性溶液• 必要时，用适当的无菌稀释剂（如肉豆蔻酸异丙酯）稀释• 液体K和液体A• 冲洗≥ 3次100 mL• 不可过滤：直接接种法



• 注射用固体（抗生素除外）• 按照标签上或药物开发团队的指示将供试品复溶• 必要时，用选择的无菌稀释液稀释• 冲洗液A• 抗菌特性：冲洗≥ 3次，100 mL• 无抗菌特性：冲洗≥ 1次，100 mL• 不可过滤：直接接种法

• 注射用抗生素固体• 按照标签上或药物开发团队的指示将供试品复溶• 必要时，用选择的无菌稀释液稀释• 液体K和液体A• 冲洗≥ 3次100 mL• 不可过滤：直接接种法


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• 带有标记无菌通路的器械• 在无菌条件下，将≥ 10个通路体积的液体D通过各受试器械• 在适当的无菌容器中收集液体• 冲洗液A• 冲洗≥ 1次，100 mL

• 无菌器械• 使用直接接种方法• 物品可完整浸入或拆卸• 培养基的体积应足以使器械完全浸入• 对于极大的器械，将与患者接触的器械部分浸入足以使这些部分完全浸入的

体积中


Click to edit Master title style方法适用性方法：膜过滤（1）

• 产品检测• 预润湿：≥ 50 mL所选冲洗液• 将产品或样品制备物量转移至膜和过滤器中• 在药品和（生物）药用油、油性溶液、软膏和乳膏的情况下，用100 mL所选

冲洗液冲洗膜2次• 用≤ 100 CFU微生物接种100 mL所选冲洗液• 用100 mL接种的冲洗液冲洗膜• 关闭罐• 将FTM或TSB（100 mL）转移至罐中：

– FTM: 金黄色葡萄球菌、铜绿假单胞菌、生孢梭菌、内部环境分离菌– TSB: 枯草芽孢杆菌、白色念珠菌、巴西曲霉、内部环境分离菌


• 注释：出于商业目的，对3个不同批次实施方法适用性

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• 阳性对照：为了检查微生物的生长能力，并获得在无挑战培养基中生长的参考。• 用≤ 100 CFU接种100 mL所选冲洗液• 用100 mL接种的冲洗液冲洗膜• 关闭罐• 将FTM或TSB（100 mL）转移至罐中：



• 注释：对于产品检测和阳性对照，内部环境分离菌应在至少1种生长培养基（FTM或TSB）中显示生长


• 阴性对照：为了检查无菌处理（无产品）• 用与产品检测相同的方法进行阴性对照，不添加接种物和产品。用所

选稀释液替换产品

• 产品对照：为了检查FTM/TSB与产品的相互作用并检查产品的无菌性• 按照与产品检测相同的方法实施产品对照，但不添加接种物

• 参比计数：为了检查接种物混悬液的浓度• 将用于将稀释液接种在预充式TSA培养皿（约9 cm）或同等产品（细

菌和真菌）上的工作稀释液体积以一式两份转移，并执行涂布平板法。• 孵育：

– 在30-35℃下：金黄色葡萄球菌、铜绿假单胞菌、生孢梭菌（厌氧培养）、内部环境分离菌

– 在20-25 °C下：枯草芽孢杆菌、白色念珠菌、巴西曲霉、内部环境分离菌

• 至少3天后读数


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Click to edit Master title style方法适用性方法：直接接种（1）

• 产品检测• 将产品或样品制备物量直接转移至填充FTM或TSB的罐或容器中• 接种≤ 100 CFU微生物，如下所示


• 阳性对照：为了检查微生物的生长能力，并获得在无挑战培养基中生长的参考。• 将所选体积的适当培养基转移至填充适当培养基的罐或容器中• 接种≤ 100 CFU微生物，如下所示



• 注释：对于产品检测和阳性对照，内部环境分离菌应在至少1种生长培养基（FTM或TSB）中显示生长

Click to edit Master title style方法适用性方法：直接接种（2）

• 阴性对照：为了检查无菌处理（无产品）• 用与产品检测相同的方法进行阴性对照，不添加接种物和产品。用所

选稀释液替换产品

• 产品对照：为了检查FTM/TSB与产品的相互作用并检查产品的无菌性• 按照与产品检测相同的方法实施产品对照，但不添加接种物

• 参比计数：为了检查接种物混悬液的浓度• 将用于将稀释液接种在预充式TSA培养皿（约9 cm）或同等产品（细

菌和真菌）上的工作稀释液体积以一式两份转移，并执行涂布平板法。• 孵育：

– 在30-35℃下：金黄色葡萄球菌、铜绿假单胞菌、生孢梭菌（厌氧培养）、内部环境分离菌

– 在20-25 °C下：枯草芽孢杆菌、白色念珠菌、巴西曲霉、内部环境分离菌

• 至少3天后读数


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Click to edit Master title style方法适用性孵育、读数和传代培养

• 最长孵育5天（产品对照孵育14天）– 30-35 ℃下的FTM– 20-25 ℃下的TSB

• 必须在显微镜下确认生长，但至少在显微镜下确认生长的模具除外

• 传代培养– 当材料使培养基混浊或产品与培养基（例如颗粒）显示相互作用时，因此不能通

过目视测定确定微生物生长或不生长，必须进行液体传代培养。如果初始产品检测的材料仍使液体传代培养基混浊，则可同时进行固体传代培养。

– 温育：• 液体传代培养（FTM和TSB）：最长4天• 固体传代培养（需氧和厌氧TSA）：最长5天


Steritest罐中的FTM和TSB

Click to edit Master title style方法适用性优化

• 膜过滤法：• 冲洗液的量可以增加但不超过500 mL• 不同的冲洗液• 不同的过滤器类型（例如纤维素酯、PVDF、...）

• 直接接种：• 油性液体：添加乳化剂（如聚山梨酯80）的培养基 • 不溶性软膏、油、不可过滤液体和乳膏可用选择的乳化剂（例如，1 g/L

的中性肉汁或酪蛋白胨溶液）在适当的无菌稀释剂中乳化，然后转移至不含乳化剂的培养基中

• 当容器中的顶空空间不足时，建议使用另一个容器• 培养基的最大体积为500 mL

• 液体传代培养：• 转移> 1 mL但小于10 mL的原始试验培养基

• 固体传代培养：• 1 mL直接接种物可转移至预充式培养皿ø15 cm或同等产品


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Click to edit Master title style隔离器系统

• 包装完整性必须对每种新包装材料进行评价，这些新包装材料将引入隔离器中

• 负荷模式必须对隔离器系统中将要检测的每种新产品进行测定和评价


La Calhène软壁隔离器 Skan硬壁隔离器

Click to edit Master title style促生长特性试验

• 对每批进料的培养基进行促生长特性，并在与检测期间使用的相同小瓶中进行，以尽可能模拟检测环境


FTM TSB微生物金黄色葡萄球菌

铜绿假单胞菌梭状芽胞杆菌内部环境分离菌

枯草芽孢杆菌白色念珠菌巴西曲霉菌内部环境分离菌

培养温度 30-35 °C 20-25 °C

温育时间细菌：最长3天真菌：最长5天

阴性对照品最短14天

2018-10-26

Click to edit Master title style常规无菌检查

• 在VHP净化隔离器中执行（Janssen内的首选技术）– 通过使用转移隔离器转移材料– 用H2O2去污（以蒸发过氧化氢；VHP）– 定义的负荷模式

• 用中间读数孵育14天，如果需要，在14天后进行传代培养（液体和/或固体）– 培养温度

• 30-35 ℃下的FTM• 20-25 ℃下的TSB


Click to edit Master title style快速/替代无菌检查方法

• 传统无菌检查中14天的孵育时间对短期产品或使用前即刻制备的产品而言可能会太长，例如： – 正电子发射断层扫描产品（PET）– 混合的无菌制剂（CSP）– 细胞和基因治疗产品（CGTP）（例如CAL-T）

• 首选快速无菌检查，以确保快速结果，并以最佳可达到的方式确保产品的安全性，例如： – 基于ATP的方法– 基于气体产生的方法 – 荧光染色– 基于PCR的核酸扩增

• 药典和行业指南：– USP <1223>: 替代微生物方法的验证– USP <1071>: 较短保质期产品的快速无菌性（草案中）– pH: Eur.5.1.6: 控制微生物质量的替代方法– pH: Eur.2.6.27: 基于细胞的制剂的微生物检查– PDA技术报告33：替代和快速微生物方法的评价、验证和实施


2018-10-26

Click to edit Master title style问题

• 在方法确认和促生长特性期间使用即用菌（例如Bioball®）

• 问题？


2018-10-26 - chp.org.cn · – Tryptic Soy Broth (TSB): aerobic incubation at 20-25 °C...

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