evaluation on testing results of sterility testing

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Evaluation on Testing Results of Sterility Testing

Benoît RAMOND (Sanofi)

Confidential for restricted use only

Click to edit Master title style Regulations


Pharmacopoeias

Monographs •Harmonized

Chapters (EP/USP/JP)

•Chinese Ph.

Comparison •Chinese Ph.

Vs. Harmonized Chapters

Impact on Tests Performance •Harmonized

Chapters (EP/USP/JP)

•Chinese Ph.

Harmonized Chapters – ICH Q4b

SterilityTesting

ICH Q4b Chinese Pharmacopoeia Commonality Major Difference

USP<71> EP 2.6.1 JP 4.06

1101 – Sterility Tests Both describe membrane filtration & direct inoculation methods using Fluid Thioglycollate Medium & Soybean Casein Digest Medium. Same species for GPT & Suitability testing.

GPT conducted on12mL volume (ChP). Routine use of Positive Controls recommended (ChP).

Click to edit Master title style General Principles


Sterility Test – Compendial methods

Membrane Filtration Direct injection in culture media





Membrane pores size 0.45 µm

Product solution / Filterable

Use of Steritest ® system

Use of Steritest ® pump

Unfilterable product

Direct injection in culture media broth

Volume to be tested ≤ 10% volume of medium

Use of sterile syringes

Use of Steritest ® pump Use of sterile syringes





Incubate Trypcase Soya Broth at 20-25°C for at least 14 days

Perform a negative control at the end of the working session

Perform viable environmental monitoring in operation

Incubate Thioglycollate Broth at 30-35°C for at least 14 days

Inspection of the media each work day


6 6

• Le test de stérilité sous isolateur:

General Principles

4/ Incubate 14 days 5/ Inspection

1/ Filtrate product solution 2/ Rinse 3/ Add Media




Sterility Testing using Steritest® System

Click to edit Master title style Testing Results - Acceptance Criteria


ACCEPTANCE CRITERIA: Media were incubated at the right

temperature,

The total number of samples was compliant with regulation requests,

Quantity of product tobe tested was compliant with regulation requests,

No anomaly in the test performance Respect of the analytical procedure, Culture media, Diluents, Equipment (pump, pipettes, …), Membrane filtration system (e.g. Steritest®), Respect of the contamination control strategy, Respect of the inspection procedure, Data integrity.

Négative control is conform (No Growth).

THB Medium Aspect

TSB Medium Aspect

Click to edit Master title style Testing Results - Inspection Step


Agitate (swirling movement) TSB Medium

Inspection process: Test // Negative control

Observe Turbid aspect +/-

Observe other viability aspects +/-

THB Medium Aspect

Observe Turbid aspect +/-

Observe color of the medium

Observe Resazurine +/-

Observe other viability aspects +/-

TSB Medium Aspect

Observe color of the medium

Never agitate THB (↓ growth anaerobes)

Click to edit Master title style Testing Results - Inspection Step


Yellow translucent

Resazurine +

No growth

Growth Change of color

Viable aggregates +

THB Medium Aspect

Turbid aspect

Change of color

Resazurine -

Orange/Yellow – No Turbid

TSB Medium Aspect

Turbid aspect

No growth

Growth

Viable aggregates +

Click to edit Master title style Testing Results - Examples - THB


Staphylococcus aureus

Pseudomonas aeruginosa

Clostridium Sporogenes

Culture Medium: Thioglycollate Broth strict anaerobic conditions

Inspection: S. aureus Turbid aspect Resazurin + at the top No change in color Growth + P. aeruginosa Turbid aspect Resazurine – No change in color Growth + C. sporogenes Turbid aspect Resazurine – Change in color Growth +

Click to edit Master title style Testing Results - Examples - TSB


Bacillus subtillis

Candida albicans

Aspergillus brasiliensis

Culture Medium: Trypcase Soya Broth

Inspection: B. subtillis Turbid aspect Change in color Growth +

C. albicans Low turbid aspect No change in color Growth +

A. brasiliensis Viable snowy aspect No Change in color Growth +



Staphylococcus aureus

Pseudomonas aeruginosa

Milieu Thio

Milieu Thio

Culture Medium: Thioglycollate Broth anaerobic conditions

Inspection: S. aureus Turbid cloudy aspect Resazurine – No change in color of medium Growth + P. aeruginosa Turbid cloudy aspect Resazurine – No change in color of medium Growth +

Testing Results - Examples - THB



Bacillus cereus

Clostridium Sporogenes

Milieu Thio

Milieu Thio

Culture Medium: Thioglycollate Broth anaerobic conditions

Inspection: B. cereus Turbid aspect Resazurine – No change in color of medium Growth + C. sporogenes Turbid aspect Resazurine – No change in color of medium Growth +

Testing Results - Examples - THB


Culture Medium: Trypcase Soya Broth

Inspection: A. brasiliensis Coudy aspect at the bottom No change in color of medium Growth + Neosartorya spp. Very low turbid aspect at the bottom No change in color of medium Growth +


Aspergilus brasiliensis

Neosartorya spp.

Testing Results - Examples - TSB

Click to edit Master title style Testing Results - BacT/Alert - Aspergillus brasiliensis


Strain 1

Examples of growth aspect of Aspergillus brasiliensis after direct Inoculation of iFA plus medium (BacT/ALERT system) showing: - No change of the color of the medium, - No turbid aspect, - Specific growth culture at the bottom of the bottle of medium.

Growth?

+ Growth

Click to edit Master title style Testing Results - BacT/Alert - Propionibacterium acnes


Examples of growth aspect of 5 different strains of Propionibacterium acnes after direct inoculation of iNST medium (BacT/ALERT system) showing:

Strain 1 Strain 2 Strain 4 Strain 5 Strain 3

Low turbid aspect in all

bottle solution

High turbid aspect in all

bottle solution

No turbid aspect No change of

color Low growth at

the bottom

No turbid aspect No change of

color Moderate

growth at the bottom

No turbid aspect / No

change of color Moderate

growth at the bottom

Growth? + Growth

Click to edit Master title style Testing Results - BacT/Alert - Penicillium chrysogenum


Different Growth aspects of 3 different strains of Penicillium chrysogenum from sterility tests by direct inoculation of population less than 100CFU in iFA plus medium (BacT/ALERT system) showing: Strain 1 Strain 2 Strain 3

No turbid aspect No change of color

Specific growth at the bottom





Click to edit Master title style Sterility Testing Failure

In the case of a growth being detected in a sterility test, an investigation should immediately be initiated and the following actions should be taken: Determine product batches potentially

affected, Quarantine the product batch with

growth & other concerned products, Perform a full-scale OOS investigation,

Production investigation: o Batch dossier evaluation, o Current status of equipment used

in production, o Personnel training, o Risk management evaluation, o Deviations & Complaints systems

evaluation, Laboratory investigation:

o Evaluate potential lab error, o Identify the contaminant(s), o Applicability of the criteria from

Pharmacopoeias Both investigation:

o Root cause evaluation.

Click to edit Master title style Sterility Testing Failure

Full-Scale OOS Investigation

1-1 Laboratory Investigation with Invalidation of the initial test result

Review of Production

Interpretation of Results: If no evidence of microbial growth is found, the product to be examined complies with the test for sterility. If evidence of microbial growth is found, the product to be examined does not comply with the test for sterility, unless it can be clearly demonstrated that the test was invalid for causes unrelated to the product to be examined. The test may be considered invalid only if one or more of the following conditions are fulfilled: a. The data of the microbiological monitoring of the sterility testing facility show a fault. b. A review of the testing procedure used during the test in question reveals a fault. c. Microbial growth is found in the negative controls. d. After determination of the identity of the microorganisms isolated from the test, the growth of this species (or these species) may be ascribed unequivocally to faults with respect to the material and or the technique used in conducting the sterility test procedure.

Repetition of the test: If the test is declared to be invalid, it is repeated with the same number of units as in the original test. If no evidence of microbial growth is found in the repeat test, the product examined complies with the test for sterility. If microbial growth is found in the repeat test, the product examined does not comply with the test for sterility.

• Conventional phenotypic microbial identification techniques are generally satisfactory for identification of sterility test isolates. However, in order to use condition (d) as the sole criterion for invalidating a sterility test, it may be necessary to employ sensitive typing techniques, for example, molecular typing with RNA/DNA homology methods:

– Multilocus Comparative Sequencing (MLSA)

– Multilocus Sequence Typing (RAPD & MLST)

• Conventional identification techniques can demonstrate that 2 isolates are not identical, but may not be able to provide unequivocal evidence that two isolates are from the same source.

Click to edit Master title style Specific case – False Positive results?


Yellow translucent

Resazurine +

THB Medium Aspect

Low turbid aspect

No change of color

TSB Medium Aspect

Low turbid aspect

Growth?

Growth?

Click to edit Master title style Specific case – False Positive results?


Yellow translucent

Resazurine +

Growth?

THB Medium Aspect

Low turbid aspect

No change of color

TSB Medium Aspect

Turbid aspect

Growth?

No growth

Non-Viable Particles

Investigation

False Positive Results

Click to edit Master title style Training of the Personnel - Key Point

1/ Isolator used for Sterility Testing: Principle & Functionality, Vapor H2O2 Decontamination Cycle,

2/ Sterility Testing Methods: Filtration Methods (Steritest®), Direct Inoculation, Media, Diluents, Sampling, Tutoring Period,

3/ Inspection & Interpretation of the Results: Principle of the inspection, How and When to Perform Inspection, Acceptance Criteria, Inspection Best Practice Supported by

Practical Exercises with different species (positive controls), Negative Controls & False Positive Cases,

Tutoring Period.




Thank you


无菌检查法检测结果的评估

Benoît RAMOND (Sanofi)


Click to edit Master title style 法规


各国药典

各论

•协调章节 (EP/USP/JP)

•中国药典

对比

•中国药典与

•协调章节

检验结果的影响

•协调章节(EP/USP/JP)

•中国药典

协调章节 – ICH Q4b

无菌检查法

ICH Q4b 中国药典共同点主要区别

USP<71>

EP 2.6.1

JP 4.06

1101 – 无菌检查法都描述了薄膜过滤法和直接接种法，都使用硫乙醇酸盐流体培养基和胰酪大豆胨液体培养基。

都使用同种用于生长促进试验和适用性试验。

ChP中生长促进试验用12mL.

ChP建议常规使用阳性对照 .

Click to edit Master title style 通则


无菌检测法– 药典方法

薄膜过滤法培养基直接接种法





滤膜孔径0.45 µm

供试品溶液/ 可过滤性

使用Steritest ® 系统

使用Steritest ® 泵

不可过滤的产品

直接接种液体培养基

供试品体积 ≤ 培养基体积的10%

使用无菌注射器

使用Steritest ® 泵使用无菌注射器




胰酪大豆胨液体培养基置20-25°C培养不少于14天

后操作阴性对照

操作中有可生长环境的监控

硫乙醇酸盐流体培养基置30-35°C 培养不少于14天

每个工作日检查培养基



6 6

• 无菌检测实验:

通则

4/ 培养14天

5/ 检查

1/ 过滤供试品溶液

2/ 冲洗

3/ 加培养基




无菌检查使用

Steritest® 系统

Click to edit Master title style 检验结果- 可接受标准


验收标准:

培养基置正确温度中培养；

样品总数量符合法规要求；

供试品管数量符合法规要求；

试验无异常现象：

分析步骤

培养基

稀释剂

仪器 (泵, 吸量管, …)

薄膜过滤系统（例如Steritest®）

污染控制策略

检查程序

数据完整性

确认阴性对照（无菌生长）。

THB 培养基

TSB 培养基

Click to edit Master title style 试验结果- 检查步骤


摇动 (漩涡状振摇) TSB 培养基

检查方法: 检验 // 阴性对照

观察浑浊度 +/-

观察其他生长情况 +/-

THB 培养基

观察浑浊度 +/-

观察培养基颜色

观察刃天青 +/-

观察其他生长情况 +/-

TSB 培养基

观察培养基颜色

勿摇动THB (↓ 生长厌氧菌)

Click to edit Master title style 试验结果- 检查步骤


黄色、透明

刃天青 +

无菌生长

生长颜色改变

生长数量+

THB 培养基

浑浊度

颜色改变

刃天青 -

橙色/黄色 – 无浑浊

TSB 培养基

浑浊度

无菌生长

生长

生长数量+

Click to edit Master title style 试验结果- 举例 – THB


金黄色葡萄球菌铜绿假单胞菌生孢梭菌

培养基:

硫乙醇酸盐流体培养基完全厌氧环境

检查:

金黄色葡萄球菌浑浊度

刃天青 + 顶部位置

无颜色变化

生长 +

铜绿假单胞菌浑浊度

刃天青 –

无颜色变化

生长 +

生孢梭菌浑浊度

刃天青 –

颜色变化

生长 +

Click to edit Master title style 试验结果 - 举例 - TSB


枯草芽孢杆菌白色念珠菌巴西曲霉

培养基:

胰酪大豆胨液体培养基

检查:

枯草芽孢杆菌

浑浊度

颜色改变

生长 +

白色念珠菌

低浑浊度

无颜色变化

生长 +

巴西曲霉

生长雪花状

无颜色变化

生长 +



金黄色葡萄球菌铜绿假单胞菌

Milieu

Thio

Milieu

Thio

培养基:

硫乙醇酸盐流体培养基厌氧环境

检查:

金黄色葡萄球菌

浑浊絮状物

刃天青 –

培养基颜色无变化

生长 +

铜绿假单胞菌

浑浊絮状物

刃天青 –


生长 +

试验结果- 举例 – THB


枯草芽孢杆菌生孢梭菌

Milieu

Thio

Milieu

Thio

培养基:

硫乙醇酸盐流体培养基厌氧环境

检查:

枯草芽孢杆菌

浑浊度

刃天青 –

培养基无颜色变化

生长 +

生孢梭菌

浑浊度

刃天青 –

培养基无颜色变化

生长 +

试验结果- 举例 – THB



培养基:

胰酪大豆胨液体培养基

检查:

巴西曲霉

底部絮状物


生长 +

新萨托菌种

底部有非常浅的浑浊


生长 +

巴西曲霉新萨托菌

试验结果- 举例 – TSB


Click to edit Master title style 试验结果- 细菌计数/Alert - 巴西曲霉


菌种 1

iFA培养基直接接种法接入巴西曲霉（细菌计数/ALERT 系统），生长情况如下：

- 培养基颜色无变化,

- 不浑浊,

- 只生长在培养基瓶的底部.

生长?

+ 生长

Click to edit Master title style 试验结果- 细菌计数/Alert - 疮疱丙酸杆菌


iNST 培养基直接接种法接入疮疱丙酸杆菌5种不同菌种（细菌计数/ALERT 系统），生长情况如下：

菌种 1 菌种 2 菌种 4 菌种 5 菌种 3

整瓶溶液低浑浊

整瓶溶液高浑浊

无浑浊

无颜色变化

底部少量生长

无浑浊

无颜色变化

底部中等生长

无浑浊

无颜色变化

底部中等生长

生长? + 生长

Click to edit Master title style 试验结果- 细菌计数/Alert - 产黄青霉菌

无菌实验中，iFA 培养基直接接种法接入小于100CFU的3个不同菌种的产黄青霉菌 (细菌计数/ALERT 系统)，不同的生长情况如图：

菌种 1 菌种 2 菌种 3

无浑浊无颜色变化

特定生长在底部






Click to edit Master title style 无菌试验失败

如果无菌试验中检测到有菌生长，应立即启动调查，步骤如下：

判断产品批次是否可能受到影响；

隔离有菌生产的产品批次，以及其他有关的产品；

进行全面的OOS调查,

产品调查:

o 评估批记录；

o 所用仪器的当前状态；

o 人员培训；

o 评估风险管理；

o 评估偏差和投诉体系。

实验室调查:

o 评估潜在的实验室错误；

o 识别污染物；

o 药典标准的适用性。

两者都调查:

o 根本原因评估。

确定OOS结果分析员立即通知QC 经理

1.实验室调查

1-1实验室调查

回顾无菌试验数据：确认试验程序是否正确执行。（例如取样和样品制备，仪器功能或资质，人员培训，试剂，培养基等）

识别微生物实验室根本原因

其他分析试验和/或检验产品的影响

评估，如阳性

行动计划和跟进

初次试验有效

调查报告

调查报告

批次失败调查

初次试验无效

识别实验室错误

进行重复试验

结论

新评估

1-产品：

产品回顾

• 评估批记录

• 仪器（验证，资质，校准）

• 评估人员培训

• 风险评估

• 评估偏差

1-2/ 额外实验室试验

重新取样方案

重新取样试验

无菌试验：不符合

产品发现根本原因

全面OOS调查

Click to edit Master title style 无菌试验失败

Full-Scale OOS Investigation

1-1 Laboratory Investigation with

Invalidation of the initial test result Review of Production

Interpretation of Results: If no evidence of microbial growth is found, the product to be examined complies with the test for sterility. If evidence of microbial growth is found, the product to be examined does not comply with the test for sterility, unless it can be clearly demonstrated that the test was invalid for causes unrelated to the product to be examined. The test may be considered invalid only if one or more of the following conditions are fulfilled: a. The data of the microbiological monitoring of the sterility testing facility show a fault. b. A review of the testing procedure used during the test in question reveals a fault. c. Microbial growth is found in the negative controls. d. After determination of the identity of the microorganisms isolated from the test, the growth of this species (or these species) may be ascribed unequivocally to faults with respect to the

material and or the technique used in conducting the sterility test procedure.

Repetition of the test: If the test is declared to be invalid, it is repeated with the same number of units as in the original test. If no evidence of microbial growth is found in the repeat test, the product examined complies with the test for sterility. If microbial growth is found in the repeat test, the product examined does not comply with the test for sterility.

• 传统的表型微生物鉴定技术对无菌试验菌株的鉴定通常是令人满意的。然而，为了用条件(d)作为使无菌试验无效的唯一标准，可能有必要采用灵敏的分型技术，例如RNA/DNA同源的分子分型：

– 多位点比较测序 (MLSA)

– 多位点序列分型(RAPD &

MLST)

• 常规鉴定技术可以证明两个分离物不相同，但是可能无法提供明确的证据证明两个分离物来自同一来源。

产品回顾

全面OOS调查

1-1初次试验结果无效的实验室调查

结果判定：

如果发现没有微生物生长的证据，供试品符合无菌试验规定。

如果发现微生物生长的证据，供试品不符合无菌试验规定，除非清晰证明不是供试品引起的试验无效。当以下条件有一项或多项符合时，可以认为试验无效：

a. 无菌试验仪器的微生物监测数据显示错误

b. 回顾试验的检测步骤显示错误

c. 阴性对照发现微生物生长

d. 确定从试验中分离的微生物之后，这个或这些菌种可明确的归因于无菌试验流程中所用的材料和/或技术。

重复试验：

如果试验宣布无效，重复试验应与初次试验使用相同的单位数量。

如果重复试验发现没有微生物生长的证据，供试品符合无菌试验要求。

如果重复试验发现有微生物生长的证据，供试品不符合无菌试验要求。


Click to edit Master title style 特例– 假阳性结果?


黄色、透明

刃天青 +

THB 培养基

低浑浊度

颜色无变化

TSB 培养基

低浑浊度

生长?

生长?

Click to edit Master title style 特例– 假阳性结果?

黄色、透明

刃天青 +

生长?

THB 培养基

低浑浊度

无颜色变化

TSB 培养基

浑浊度

生长?

无生长

非生长粒子

调查

假阳性结果


Click to edit Master title style 人员培训- 要点

1/ 用于无菌试验的隔离器:

原理和功能,

蒸汽 H2O2 净化循环,

2/ 无菌试验方法:

过滤法(Steritest®)

直接接种

培养基，稀释液

取样

培训周期

3/ 检查和结果判断:

检查的原则

如何以及何时进行检查

验收标准

最佳的检查操作是经过实际的练习，用不同的菌种（阳性结果）、阴性结果和假阳性结果的例子

培训周期



谢谢


evaluation on testing results of sterility testing

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