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Air Sampler Qualification According to ISO 14698 NORM

日期:2019-07-18 19:52
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Air Sampler Qualification According to ISO 14698 NORM

InNovember2007,the ISO TC/209 working group decided to revise thecurrentversion ofthe ISO 14698 norm (Cleanrooms and associatedcontrolledenvironments– Biocontamination Control). This decisioncame fromscientific andindustrial communities willing tointegrateinnovations from the lastdecade and to make the contentof thistext easier to implement in acleanroom’sroutineactivities.

Cleanroominstallationandmonitoring have indeed become an important issuenot onlyinpharmaceutical, cosmetic, and food industries, butalsoinhealthcare establishments and biotech, sinceproductbatchcontamination and/or a human contamination can havehugefinancialor sanitation consequences.

Biologicalcontaminationcontrolof these environments (water, surface, andair), especiallyairquality control, are now necessary to masterand guaranteeproductionprocesses and quality control but also toinsure staffsafety.

Specifictextsgiverecommendations — especially for aerobiocontamination —andmethodsto implement the monitoring and to classify thecleanroomaccordingto an acceptable level of inert andbiologicalcontamination.

ISO14698 NORM
Beyond the ISO 14644 norm (Cleanroomsandassociatedcontrolledenvironments) whichdescribestheairbornecontamination control, the ISO 14698normdealsspecifically withthe microbiological contaminants and howtomanagean efficientcontrol plan. It explains the generalprinciplesandglobalmethodology to evaluate andmonitortheaerobiocontaminationcontrol in such “mastered”environments.Italso specifies therequired methods to guaranteeacoherentmonitoring of the criticalareas, and to apply therightpreventiveand corrective actions incase ofcontamination.

Thefirstpartof the ISO 14698 norm places the emphasis on thedifferentcriteriato take into account in the choice of the bestadaptedsamplingequipment: for example, the type and size ofsearchedparticles, thesensitivity of the viable particles, thesamplingmethod considered,the charge of the controlled environment,theduration of thecollection step, etc.

Theeffectofthe equipment on the environment needs to beconsideredandestimated to make sure that the sample will berepresentative(theexhausted air should not disturb the area nor bere-aspiratedbythe equipment).

Thecontentofthe norm also defines the two main requirements for anyairsamplerused in cleanroooms: 1) a sufficient air flow ratetocollect 1m3 ina “reasonable” time and 2) an adaptedimpactionspeed (around 20m/s) high enough to collect 1 μm diametermicro -organisms butgentle enough to conserve the viabilityandcultivability of thecollected bio-particles — an air flowratethat is too high with animpaction velocity that is too highwouldindeed make the collectionmedia desiccated and would stressthemicroorganisms.

Even ifmostofthe samples are collected from a low charged environment,thenormalso advises to take into account the level ofbasicparticleconcentration of the considered area.

Furthermore,relatedtowork constraints and equipments management incleanrooms, itisnecessary to consider the ergonomic,decontamination, andsettingaspects before making anychoice.

Alltheserecommendationsare applicable to impaction method but alsotoinnovative solutions;this norm has to be considered withothertexts such as ISO 14644,GMP, cGMP, etc. to make sure thattheindustrial process is masteredand the products are of agoodquality.

TRADITIONALMETHODS FOR AIRBORNE PARTICLES SAMPLING
In its last version (2004), the ISO 14698 normpresentstwodifferenttypes of collection methods: 1) passivesampling(settleplates), and2) active sampling equipment.

Passivesamplingisa good way to get a representative result concerningtheproductexposure to the potential contaminated environment inadefined time(one to four hours). But this solution isnotrepresentative of thewhole environment considered. That is whytheactive samplingtechniques are interesting; they can qualifyandquantify thecontaminants in the environment and resultsareexpressed in viableunits/volume of air collected(CFU/m3).

Theimpactiononagar plates is one of these active sampling methods. Itconsistsof:aspirating a defined volume of air (with theairborneparticles),making sure the flow goes through small holes,andimpacting theparticles against a semi-solid collectionmedia(Figure 1).

Theagarplates(collection media) are then incubated up to sevendays,according tothe searched flora, in order to let themicroorganismsgrow. As soonas they are visible, the colonies arevisuallycounted. Instead ofimpacting on agar plates, it is alsopossibleto impact onto liquidor on a filter.

Thismethodiscurrently considered as the reference and has been usedforseveraldecades. Even if it answers most of the ISOnormrequirements, theimpaction on agar plates still has limitsanddrawbacks; a longsampling process will desiccate thecollectionmedia, a too high airflow rate will stress the micro -organismsand make them lose theircultivability and/or theirviability. Inaddition, the currentmethod gives a result in morethan three days(incubation step) andis limited to cultivable flora— and it iswell known that thecultivable flora only represents alittle partof themicrobiology!

In thecaseofdetected microbiological contamination, rapid andpreciseresultsare crucial to evaluate its consequences anditspotentialdissemination and rapidly take the right measures; itiswith thisgoal in mind, that many manufacturers havedevelopedeithercollection or innovative analysis techniques in thelast15years.

ALTERNATIVEMETHODS, EXAMPLE OF CYCLONIC TECHNOLOGY
These alternative techniques have been tested andoptimizedinorderto go beyond the current results and data, andtobettermaster theaerobiocontamination; the development hasbeendoneaccording toexisting norms and texts requirements.

Theanalysistechniquesare called RMM (Rapid Microbiology Methods).Theprinciple is nolonger based on the ability of themicroorganismsto grow on anadapted culture media but based on theactivity orthe content of thecells. It thus gives results inviable unitsinstead of coloniesforming units, which is much moreclose to thereality andcorresponds to the ISO 14698 norm.

Thetwomaingoals are: 1) to detect rapidly and specifically, and 2) togetridof the time-consuming incubation step and to focusonspecificcomponents of the cell or a specific strain.Theprobability forcontamination to be detected too late is lowerandconsequently thesanitation risk is better mastered.

Primarilyusedforthe water and surfaces contamination controls only, theseRMMare nowcompatible with airborne contamination controls withthedevelopmentof an innovative air sampling technology deliveringaliquid sample.This patented cyclonic technology (Coriolis ®,BertinTechnologies),concentrates the airborne particles into aliquidcollection media ata high air flow rate; the principle isdescribedin Figure 2.

Airentersathigh flow rate into a prefilled cone and thecollectionliquidforms a cyclone. The aspirated particles arecentrifuged onthewall of the cone, separated from the air flow, andtracked intotheliquid. Optimal protocol for the tracking of themicroorganismsis300 l/min which allows collecting 3 m3 in tenminutes andmakingthe sample representative of the controlledenvironment. Dueto atangential “impaction” (vs. a vertical one), itis possibletoreach such a high air flow rate without stressing themicro-organisms and with the insurance to collect a widerangeofparticles from 0.5 μm to 20 μm and even more:bacteria,molds,spores, pollens, etc.

Thecyclonictechnologyconforms to ISO 14698 requirements in termsofmicroorganismpreservation, representative samples, use,anddecontamination. Italso provides data on total flora andnoncultivable flora, andresults are available in only few hoursafterthe sampling step. Therapid results are possible due tothecompatibility of the liquidsample not only with culture mediabutalso with alternative methodsof analysis (PCR,immuno-analysis,flow cytometry, ATPbioluminescence).

INNOVATIVEAIR SAMPLER QUALIFICATION ACCORDING TO ISO 14698NORM
Part 1 recommendations of the ISO 14698 norm,theAnnexB(Guidanceonvalidatingairsamplers) can help users to qualifyanyair samplerbygiving advice and protocol information.

MaterialandMethods
This qualification is based on the estimation ofthephysicalandbiological efficiencies of the systemconsidered:thephysicalefficiency is the ability of the air samplertocollectdifferentdiameter particles; the biological efficiencyistheability tocollect viable particles.

AccordingtoannexB, qualification tests should be done in anenvironmentalchamber(closed, sealed, not too large), supplied by ahorizontalflow ofclean air through a bank of HEPA filters, andeasy todecontaminatebetween tests. You also need to choose therightstrains for thebiological counts: Bacillus atrophaeusisrecommended for thephysical efficiency. For thebiologicalefficiency, as far as most ofthe contaminants insidecleanroomscomes from human source (skinespecially), the normproposes towork with Staphylococcusepidermidis.

Thefirststepof the experiment is to generate aerosols for thechosenstrains —done with a nebulizer with disks or aspinninghead.

AerosolscontainingBacillusatrophaeussporeswerediffused in the chamber aireither by injectingsuspensionsofB.atrophaeusinvariousconcentrations (0-7%) of potassiumiodide in asolutioncontaining80% ethanol or by sprayingB.atrophaeus (10cfu ml-')indistilled water into theSTAG 2000 (Spinning TopAerosol Generator-BGI Incorporated, USA)or the CollisionAtomizer.

Different diameters of particles are thus generated inordertoestimate the physical efficiency of the airsampler;Cascadesampler Casella was used to measure the realdiameter ofthegenerated particles. To be qualified, the air samplerwas placedat1m from the aerosols generator. Samples were thenanalyzedbytraditional culture. In the case of the Coriolis airsampler,thequalification was done by an independent agency —HealthProtectionAgency (HPA), UK. Results have been compared tothoseobtained withthe traditional air sampling method. HPAqualificationof theCoriolis: 0.2ml of the liquid samples from theCoriolis weretakenand spread on TSA plates and incubated at 37°Cfor 18hours.

RESULTS
Biological efficiency has been measured at 78%withthestrainStaphylococcus epidermidis ATCC 14990, commonbacteriafoundintocleanrooms. Physical efficiency obtained withthestrainBacillusatrophaeus NCTC 10073 has been evaluated at 61.9%forthe0.8 μmparticles, 100.4% for 4.4 μm particles and 110% forthoseof16μm.

The cyclonic technology air sampler has beensuccessfullyvalidatedin view of ISO 14698 and is adapted tocleanroom controls.It alsoconfirms high performance with 62%physical efficiency forlessthan 1 μm and 100% from 4 μm — thus ableto collect 99% ofthecurrent particles present into cleanroomenvironments.

CONCLUSION
ISO 14698 norm describes the microbiologicalcontrolsmethodsincleanrooms and especially thoseconcerningtheairbornecontamination control; in this way theindustrialwouldbettermaster the environmental conditionsandanticipatepotentialcontamination events. These precautionsarenecessary toinsuregood quality products and safetyworkingconditions.

This study shows the interest of coupling innovativesamplingmethodand alternative analysis techniques and thus theimportancefor suchevolution to be integrated into thenormativetexts.

The information provided by those new systems comestocomplementthose from the traditional techniques and allowsgettingtheresults earlier and anticipating the correctiveactions.

The use of the new methods of high performanceenlargesthecompetence field of scientists in termsofmasteringbiocontamination risks and implementingcorrectiveactions. Even ifthe ISO norm will be reviewed andpublished in onlyseveral years,it is interesting to begin usingsuch innovations andstudy howthey can give unedited data to theairbornebiologicalcontamination controlarena.

References

·                         Barthet MC. (2008). Qualité de l’air: les analysess’affinent.Enjeuxn°285, Juin 2008

·                         Benbough, et al. (1992). Determination of thecollectionefficiencyof a microbial air sampler. Journal ofAppliedBacteriology 1993,74, 170-173

·                         Carvalho, et al. (2008). Performance of the Coriolis airsampler,ahighvolume aerosol-collection system … “Aerobiologia,Oct2008

·                         Decker, et all. (1969). Advances in large-volumeairsampling.Contamination Control August, 13-17

·                         Desjonquères (2008). Stage général ASPEC-Contrôlesmicrobiologiquesen salles propres

·                         HPA report n° 41/08 –Sara Speight, Simon Parks –Sept2008(confidential) • ISO 14698-1:2003, Cleanroomsandassociatedcontrolled environments – Biocontamination control–Generalprinciples and methods

·                         Mach C. PAT, tougher standards push improvedmicrobialairmonitoring. CleanRooms, Oct 2006

·                         May F.K. (1945). The Cascade impactor : an instrumentforsamplingcoarse aerosols. Journal of Scientificinstruments,22,187-195

·                         Thibaudon. Du Pollen dans vos cellules. Salles Propresn°55,May2008

 

Q. Desjonquères, S. Hamdi, Bertin Technologies, Bertin Systems Department, Saint-Quentin-en-Yvelines, France. www.bertin.fr
 Air Sampler Qualification According toISO 14698 NORM

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