In November 2007, the ISO TC/209 working group decided to revise the current version of the ISO 14698 norm (Cleanrooms and associated controlled environments – Biocontamination Control). This decision came from scientific and industrial communities willing to integrate innovations from the last decade and to make the content of this text easier to implement in a cleanroom’s routine activities.
Cleanroom installation and monitoring have indeed become an important issue not only in pharmaceutical, cosmetic, and food industries, but also in healthcare establishments and biotech, since product batch contamination and/or a human contamination can have huge financial or sanitation consequences.
Biological contamination control of these environments (water, surface, and air), especially air quality control, are now necessary to master and guarantee production processes and quality control but also to insure staff safety.
Specific texts give recommendations — especially for aerobiocontamination — and methods to implement the monitoring and to classify the cleanroom according to an acceptable level of inert and biological contamination.
ISO 14698 NORM
Beyond the ISO 14644 norm (Cleanrooms and associated controlled environments) which describes the airborne contamination control, the ISO 14698 norm deals specifically with the microbiological contaminants and how to manage an efficient control plan. It explains the general principles and global methodology to evaluate and monitor the aerobiocontamination control in such “mastered” environments. It also specifies the required methods to guarantee a coherent monitoring of the critical areas, and to apply the right preventive and corrective actions in case of contamination.
The first part of the ISO 14698 norm places the emphasis on the different criteria to take 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 effect 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 defines the two main requirements for any air sampler used in cleanroooms: 1) a sufficient air flow rate to collect 1m3 in a “reasonable” time and 2) an adapted impaction speed (around 20 m/s) high enough to collect 1 μm diameter micro - organisms but gentle enough to conserve the viability and cultivability of the collected bio-particles — an air flow rate that is too high with an impaction velocity that is too high would indeed make the collection media desiccated and would stress the microorganisms.
Even if most of the samples are collected from a low charged environment, the norm also advises to take into account the level of basic particle concentration of the considered area.
Furthermore, related to work constraints and equipments management in cleanrooms, it is necessary to consider the ergonomic, decontamination, and setting aspects before making any choice.
All these recommendations are applicable to impaction method but also to innovative solutions; this norm has to be considered with other texts such as ISO 14644, GMP, cGMP, etc. to make sure that the industrial process is mastered and the products are of a good quality.
TRADITIONAL METHODS FOR AIRBORNE PARTICLES SAMPLING
In its last version (2004), the ISO 14698 norm presents two different types of collection methods: 1) passive sampling (settle plates), and 2) active sampling equipment.
Passive sampling is a good way to get a representative result concerning the product exposure to the potential contaminated environment in a defined time (one to four hours). But 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/volume of air collected (CFU/m3).
The impaction on agar plates is one of these active sampling methods. It consists of: aspirating a defined volume of air (with the airborne particles), making sure the flow goes through small holes, and impacting the particles against a semi-solid collection media (Figure 1).
The agar plates (collection media) are then incubated up to seven days, according to the searched flora, in order to let the microorganisms grow. As soon as they are visible, the colonies are visually counted. Instead of impacting on agar plates, it is also possible to impact onto liquid or on a filter.
This method is currently considered as the reference and has been used for several decades. Even if it answers most of the ISO norm requirements, the impaction on agar plates still has limits and drawbacks; a long sampling process will desiccate the collection media, a too high air flow rate will stress the micro - organisms and make them lose their cultivability and/or their viability. In addition, the current method gives a result in more than three days (incubation step) and is limited to cultivable flora — and it is well known that the cultivable flora only represents a little part of the microbiology!
In the case of detected microbiological contamination, rapid and precise results are crucial to evaluate its consequences and its potential dissemination and rapidly take the right measures; it is with this goal in mind, that many manufacturers have developed either collection or innovative analysis techniques in the last 15 years.
ALTERNATIVE METHODS, EXAMPLE OF CYCLONIC TECHNOLOGY
These alternative techniques have been tested and optimized in order to go beyond the current results and data, and to better master the aerobiocontamination; the development has been done according to existing norms and texts requirements.
The analysis techniques are called RMM (Rapid Microbiology Methods). The principle is no longer based 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, which is much more close to the reality and corresponds to the ISO 14698 norm.
The two main goals are: 1) to detect rapidly and specifically, and 2) to get rid of the time-consuming incubation step and to focus on specific components of the cell or a specific strain. The probability for contamination to be detected too late is lower and consequently the sanitation risk is better mastered.
Primarily used for the water and surfaces contamination controls only, these RMM are now compatible with airborne contamination controls with the development of an innovative air sampling technology delivering a liquid sample. This patented cyclonic technology (Coriolis ®, Bertin Technologies), concentrates the airborne particles into a liquid collection media at a high air flow rate; the principle is described in Figure 2.
Air enters at high flow rate into a prefilled cone and the collection liquid forms a cyclone. The aspirated particles are centrifuged on the wall of the cone, separated from the air flow, and tracked into the liquid. Optimal protocol for the tracking of the microorganisms is 300 l/min which allows collecting 3 m3 in ten minutes and making the sample representative of the controlled environment. Due to a tangential “impaction” (vs. a vertical one), it is possible to reach such a high air flow rate without stressing the micro - organisms and with the insurance to collect a wide range of particles from 0.5 μm to 20 μm and even more: bacteria, molds, spores, pollens, etc.
The cyclonic technology conforms to ISO 14698 requirements in terms of microorganism preservation, representative samples, use, and decontamination. It also provides data on total flora and non cultivable flora, and results are available in only few hours after the sampling step. The rapid results are possible due to the compatibility of the liquid sample not only with culture media but also with alternative methods of analysis (PCR, immuno-analysis, flow cytometry, ATP bioluminescence).
INNOVATIVE AIR SAMPLER QUALIFICATION ACCORDING TO ISO 14698 NORM
Part 1 recommendations of the ISO 14698 norm, the Annex B (Guidance on validating air samplers) can help users to qualify any air sampler by giving advice and protocol information.
Material and Methods
This qualification is based on the estimation of the physical and biological efficiencies of the system considered: the physical efficiency is the ability of the air sampler to collect different diameter particles; the biological efficiency is the ability to collect viable particles.
According to annex B, qualification tests should be done in an environmental chamber (closed, sealed, not too large), supplied by a horizontal flow of clean air through a bank of HEPA filters, and easy to decontaminate between tests. You also need to choose the right strains for the biological counts: Bacillus atrophaeus is recommended for the physical efficiency. For the biological efficiency, as far as most of the contaminants inside cleanrooms comes from human source (skin especially), the norm proposes to work with Staphylococcus epidermidis.
The first step of the experiment is to generate aerosols for the chosen strains — done with a nebulizer with disks or a spinning head.
Aerosols containing Bacillus atrophaeus spores were diffused in the chamber air either by injecting suspensions of B. atrophaeus in various concentrations (0-7%) of potassium iodide in a solution containing 80% ethanol or by spraying B. atrophaeus (10 cfu ml-') in distilled water into the STAG 2000 (Spinning Top Aerosol Generator - BGI Incorporated, USA) or the Collision Atomizer.
Different diameters of particles are thus generated in order to estimate the physical efficiency of the air sampler; Cascade sampler Casella was used to measure the real diameter of the generated particles. To be qualified, the air sampler was placed at 1m from the aerosols generator. Samples were then analyzed by traditional culture. In the case of the Coriolis air sampler, the qualification was done by an independent agency — Health Protection Agency (HPA), UK. Results have been compared to those obtained with the traditional air sampling method. HPA qualification of the Coriolis: 0.2ml of the liquid samples from the Coriolis were taken and spread on TSA plates and incubated at 37°C for 18 hours.
Biological efficiency has been measured at 78% with the strain Staphylococcus epidermidis ATCC 14990, common bacteria found into cleanrooms. Physical efficiency obtained with the strain Bacillus atrophaeus NCTC 10073 has been evaluated at 61.9% for the 0.8 μm particles, 100.4% for 4.4 μm particles and 110% for those of 16 μm.
The cyclonic technology air sampler has been successfully validated in view of ISO 14698 and is adapted to cleanroom controls. It also confirms high performance with 62% physical efficiency for less than 1 μm and 100% from 4 μm — thus able to collect 99% of the current particles present into cleanroom environments.
ISO 14698 norm describes the microbiological controls methods in cleanrooms and especially those concerning the airborne contamination control; in this way the industrial would better master the environmental conditions and anticipate potential contamination events. These precautions are necessary to insure good quality products and safety working conditions.
This study shows the interest of coupling innovative sampling method and alternative analysis techniques and thus the importance for such evolution to be integrated into the normative texts.
The information provided by those new systems comes to complement those from the traditional techniques and allows getting the results earlier and anticipating the corrective actions.
The use of the new methods of high performance enlarges the competence field of scientists in terms of mastering biocontamination risks and implementing corrective actions. Even if the ISO norm will be reviewed and published in only several years, it is interesting to begin using such innovations and study how they can give unedited data to the airborne biological contamination control arena.
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