Validation of a new air sampler for rapid environmental monitoring

Transcription

1 PDA Journal of GMP and Validation in Japan Vol. 12, No. 1 (2010) Validation of a new air sampler for rapid environmental monitoring Ms Quitterie Brossard-Desjonqu àeres Bertin Technologies 迅速環境モニタリングの新しいエアー散布ラーのヴァリデーション Ms Quitterie Brossard-Desjonqu àeres Microbial contamination control of air in cleanroom environment has been playing an increasingly important part for pharmaceutical, food and cosmetic industries as well as healthcare establishments. ISO norm describes the microbiological controls methods in cleanrooms and especially those concerning the airborne contamination control. These precautions are necessary to ensure good quality products and safe working conditions. In its last version (2004), the ISO norm presents two dišerent types of collection methods: the passive sampling equipment (settle plates) and the active one. However, these methods are limited to cultivable ora (CFU/m 3 ) and require a long time-to-results. Over the last 15 years, the emergence of Rapid Microbiological Methods (RMM) has tackled those limitations by producing more sensitive and more complete results in real-time and made cleanrooms management less time-consuming and more cost-saving. These RMM are now compatible with airborne contamination controls thanks to the development of a cyclonic air sampling technology Coriolis delivering a liquid sample. This method has been validated according to ISO by Health Protection Agency (UK), and considered to be suitable for routine controls of cleanrooms. Keywords: air microbiology, cleanrooms, airborne biocontamination monitoring, ISO 14698, microbial air sampler, impaction technology, cyclonic technology, RMM, quality control クリーンルーム環境の空気の微生物汚染管理は, ヘルスケア施設と同様に医薬品, 食品, 化粧品産業にとって益々重要な役割を担ってきている ISO 基準は, 大クリーンルーム内の微生物管理法と特に大気中の汚染管理に関連する微生物管理を述べている これらの予防措置は, 良質の製品と安全な労働条件を確実にするのに必要である その最後の ISO 版 (2004) において,ISO 基準は 2 つの異なる捕集方法を提示していて, それらは受動的なサンプリング設備 ( 捕集プレートを安定させるための ) と能動的装置である しかしながら, これらの方法は見出される微生物群 (CFU/m 3 ) を培養することに限定されていて, 結果をだすのに長期間を必要とする この 15 年の間, 迅速微生物測定法 (Rapid Microbiological Methods RMM) の出現は, リアルタイムでより高感度でより完全な結果を出すことによってそれらの限界に取り組み, クリーンルーム管理をより高い時間効率とコスト節減できるようにした これらの RMM は, 現在, 液体サンプルを配供するサイクロン方式の空気サンプリング技術であるコリオリス の開発のおかげの空輸汚染管理に対応している この方法は英国健康保護庁によって ISO によりヴァリデートされてきており, クリーンルームの日常的管理に適していると考えられている Introduction Bertin Technologies, Saint-Quentin-en-Yvelines, France, brossard@bertin.fr In controlled environments where sensitive products are manufactured or sensitive activities take place, microscopic contaminants can lead to killer defects. Microbial contamination control in cleanroom environment, especially air contamination control, has been playing an increasingly important part for pharmaceutical, food and cosmetic industries as well as healthcare establishments. The international standards have been elaborated by the International Organization for Standardization to ensure 8

2 Vol. 12, No. 1 (2010) the contamination control in cleanrooms. For instance, the ISO norm ( Cleanrooms and associated controlled environments Biocontamination Control ) presents the conventional air sampling methods which are adopted today by most of the industries over the world. However, the limitations of these conventional air sampling methods such as long time-to-result tend to become a real obstruction. Over the last 15 years, the emergence of Rapid Microbiological Methods (RMM) has tackled those limitations by producing more sensitive and more complete results in real-time and made cleanrooms management less time-consuming and more cost-saving. In November 2007, the ISO TC/209 working group reached upon an agreement to continuously conduct the revision of the ISO 14698, in order to integrate the latest innovations in the ˆeld of contamination control technology, and to facilitate the implementation of the norm in cleanrooms' routine activities. This decision actually creates a very favorable climate for the implementation of RMM and alternative air sampling methods prior to RMM analysis. ISO The standardization ISO TC/209 is split into two standards: the ISO norm ( Cleanrooms and associated controlled environments ) covering the airborne contamination control issues, the ISO norm dealing especially with the microbiological contaminants and how to manage an e cient control plan. ISO explains the general principles and the global methodology to evaluate and monitor the aerobiocontamination in such controlled environments. The ˆrst part of the ISO norm explains the dišerent criteria to be taken into account in the choice of the best adapted sampling equipment: for example, the type and size of searched particles, the sensitivity of the viable particles, the sampling method considered, the charge of the controlled environment, the duration of the collection step etc.. The ešect of the equipment on the environment needs to be considered and estimated to make sure that the sample will be representative (the exhausted air should not disturb the area nor be re-aspirated by the equipment). The content of the norm also deˆnes the two main requirements for any air sampler used in cleanroooms: ˆrstly the air ow rate should be su cient to collect 1 m 3 of airina reasonable time. Secondly the impaction speed should be adapted (around 20 m/s), which means high enough to collect 1 mm diameter microorganisms but gentle enough to conserve the viability and cultivability of the collected bio-particles. Furthermore, regarding work constraints and equipment management in cleanrooms, it is necessary to consider the ergonomics, decontamination and setting aspects before making any choice. Conventional methods for air sampling In its last version (2004), the ISO norm presents two dišerent types of collection methods: the passive sampling equipment (settle plates) and the active one. Passive sampling is a good way to get a representative result concerning the product exposed to the potential contaminated environment in a deˆned time (4 hoursmaximum). However, this solution is not representative of the whole environment considered. That is why the active sampling techniques are interesting: they can qualify and quantify the contaminants in the environment and results are expressed in viable units/volumeofaircollected.evenif the norms request results in viable units/m 3, the current methods usually give results in CFU/m 3 : the impaction on agar plates is one of the active sampling methods: it aspirates a deˆned volume of air (with the airborne particles), leaving the air ow passing through small holes, and then impact the particles against a semi-solid collection media (Figure 1). The agar plates (collection media) are then incubated for several days depending on the searched ora, in order Figure 1: impactionprinciple 9

3 PDA Journal of GMP and Validation in Japan to let the microorganisms grow; as soon as they are visible, the colonies are visually counted. Instead of impacting on agarplates,itisalsopossibletoimpactintoliquidorona ˆlter. The impaction method is currently considered as the reference and is used in most of the cleanrooms. Even if it meets most of the requirements in ISO norms, the impaction on agar plates still has limits and drawbacks: a long sampling process will desiccate the collection media, a too high air ow rate will stress the microorganisms and make them loose their cultivability and/or their viability. Besides, the current method gives a result in more than 3 days due to the incubation step, and is limited to cultivable ora (CFU/m 3 ). It is well known that the cultivable ora only represents a little part of the microbiology! Alternative methods for air sampling In case of detected microbiological contamination, rapid and speciˆc results are crucial to evaluate its consequences and its potential dissemination and to rapidly take the right measures; it is with this goal in mind, that many industrials have developed alternative techniques either for collection or analysis in the last 15 years. These alternative techniques have been tested and optimized in order to go beyond the current results, data and to better master the aerobiocontamination. Their development has been done according to the existing norms. The principle of the analysis techniques RMM (Rapid Microbiology Methods) is not based anymore on the ability of the microorganisms to grow on an adapted culture media but based on the activity or the content of the cells. It thus gives results in viable units instead of colonies forming units, what is much more close to the reality and corresponds to the ISO norm. The two main goals are to detect rapidly and speciˆcally and to get rid of the time-consuming incubation step and to focus on speciˆc components of the cell or a speciˆc strain. With these alternative methods, the probability of early detection of contaminants is much higher and consequently the sanitarian risk is better mastered. Primarily used for the water and surfaces contamination controls only, these RMM are now compatible with air- Figure 2: Cyclonic technology principle borne contamination controls thanks to the development of an innovative air sampling technology delivering a liquid sample. This patented cyclonic technology (Bertin Technologies), called Coriolis, concentrates the airborne particles into a liquid collection media at a high air ow rate; the principle is described on Figure 2. Air enters at high ow rate (300 l/min) into a preˆlled cone and the collection liquid forms a cyclone. The aspirated particles are centrifuged on the wall of the cone, separated from the air ow and tracked into the liquid. The collection of 3 m 3 of air in only 10 minutes is now possible and the sample is more representative of the controlled environment. Thanks to a tangential ``impaction'' (vs. vertical one with traditional air samplers), it is possible to reach such a high air ow rate without stressing the microorganisms, and with the assurance of collecting a wide range of particles from 0.5 mm to20mm: bacteria, molds, spores, pollens, viruses etc.. (Figure 3). The cyclonic technology presented above conforms to ISO requirements in terms of microorganisms preservation, representative samples, use and decontamination. It even goes beyond traditional technologies limits since it provides data on total ora and non cultivable ora. Results are available in only few hours after the sampling step! This rapidity to obtain the results is possible thanks to the compatibility of the Coriolis liquid sample not only with culture media but also with alternative methods of analysis (PCR, immuno-analysis, ow cytometry, ATP biolumines- 10

4 Vol. 12, No. 1 (2010) Figure 3: STAG 2000, Spinning top aerosol generator (BGI Incorporated, USA) Figure 5: Traditional air sampler Casella slit sampler, Casella (London) Figure 4: Innovative air sampler Coriolis m, BertinTechnologies (Paris) cence). Alternative air sampler qualiˆcation according to ISO The qualiˆcation of the Coriolis air sampler developed by Bertin Technologies (Figure 4), has been conducted by the Health Protection Agency (HPA, UK) according to ISO , Annex B (``Guidance on validating air samplers''). This qualiˆcation indeed lays on the estimation of the physical and biological e ciencies of the system considered: the physical e ciency is the ability of the air sampler to collect particles of dišerent diameters; the biological e ciency is the ability to collect viable particles without denaturating or killing these contaminants. According to this annex B, qualiˆcation tests should be done into an environmental chamber. The right strains should be chosen: Bacillus atrophaeus is recommended for the physical e ciency. For the biological e ciency, as most of the contaminants inside cleanrooms comes from human source (skin especially), the norm proposes to work with Staphylococcus epidermidis. DiŠerent diameters of aerosols are generated, aerosolized and collected by the dišerent samplers: Cascade sampler Casella is used to measure the real diameter of the generated particles. Samples are then analyzed by traditional culture analysis method. Results have been compared to those obtained with the traditional air sampling method (impaction on agar plates with Casella Slit Sampler, Figure 5). Biological e ciency has been measured at 78 with the strain Staphylococcus epidermidis ATCC 14990, common bacteria found in cleanrooms. Physical e ciency obtained with the strain Bacillus atrophaeus NCTC has been evaluated at 61.9 for the 0.8 mm particles, for 4.4 mm particles and 110 for those of 16 mm (Figure 6). With these data, the Coriolis air sampler has been successfully validated according to ISO and is considered totally adapted to cleanrooms controls. The Coriolis air sampler is also able to collect 99 of the current parti- 11

5 PDA Journal of GMP and Validation in Japan Figure 6: Physical e ciency of the Coriolis m cles present in cleanrooms environments (>3 mm). The tests were implemented and coupled with traditional and referent culture analysis method in order to get comparable data; nevertheless the Coriolis air sampler has been developed in order to be coupled with alternative techniques (RMM) that give more complete information in a shorter time. Several studies have been implemented with dišerent industries to couple the Coriolis with PCR, ATPmetry, ELISA techniques... For example Pˆzer US has tested the Coriolis with Chemscan RDI, Microcompass and has compared the results with traditional impaction. They conˆrm the feasibility to introduce Coriolis microbial air sampler for routine controls of cleanrooms. Conclusion ISO norm describes the microbiological controls methods in cleanrooms and especially those concerning the airborne contamination control. These precautions are necessary to ensure good quality products and safe working conditions. This study proves the interest of coupling an innovative sampling method with alternative analysis techniques to get better results in a shorter time. The information provided by these new systems comes to complement with that by the traditional techniques, enabling to get the results in time and anticipate the corrective actions. ThecoupleCoriolis/RMM makes possible the detection and the quantiˆcation of non cultivable ora and also strains di cult to cultivate such as Legionnella sp., Pneumocystis sp. and also pollens, viruses etc... Even if the ISO norm will be reviewed in a few years to be fully adapted to alternative air sampling and analysis methods, far-sighted industrials have already adopted these innovative solutions to better master cleanroom contamination control. References Carvalho and Al. (2008). ``Performance of the Coriolis air sampler, a high-volume aerosol-collection system...'' Aerobiologia, Oct08 HPA report n 41/08 Mrs Sara Speight, Mr Simon Parks Sept 2008 (conˆdential) ISO : 2003, Cleanrooms and associated controlled environments Biocontamination control General principles and methods Mach C. (2006). ``PAT, tougher standards push improved microbial air monitoring'' CleanRooms, oct 2006 May F. K. (1945). ``The Cascade impactor : an instrument for sampling coarse aerosols'' Journal of Scientiˆc instruments, 22, Thibaudon. ``Du Pollen dans vos cellules'' Salles Propres n 55, May