ISO TR 4752 2025.docx

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1、ISOTechnicalReportISOTR4752Firstedition2025-06BiotechnologyInventoryofmethodsfordetectionofmicrobiologicalcontaminationinmammaliancellcultureBiotechnologieInventairedesmethodespourIadetectiondelacontaminationmicrobiologiquedanslacultureCeIIulairedemammiferesReferencenumberISO/TR4752:2025(en)COPYRIGH

2、TPROTECTEDDOCUMENTISO2025Allrightsreserved.Unlessotherwisespecified,orrequiredinthecontextofitsimplementation,nopartofthispublicationmaybereproducedorutilizedotherwiseinanyformorbyanymeans,electronicormechanical,includingphotocopying,orpostingontheinternetoranintranet,withoutpriorwrittenpermission.P

3、ermissioncanberequestedfromeitherISOattheaddressbeloworISO,smemberbodyinthecountryoftherequester.ISOcopyrightofficeCP401Ch.deBlandonnet8CH-1214Vernier,GenevaPhone:+41227490111Email:copyrightiso.orgWebsite:www.iso.orgPublishedinSwitzerlandContentsPageForewordivIntroductionv1 Scope12 Normativereferenc

4、es13 Termsanddefinitions14 Generalconcepts34.1 Introduction34.2 Detectionprocess35 Contaminationtestinformation46 Criticalcontrolpointsindetection56.1 Samples56.2 ReagentsandEquipment56.3 Operation66.4 Personnel66.5 Environment6Annex A (informative)Availableandexemplarytestingmethodsforbacteriaandfu

5、ngi7Annex B (informative)AvailableandexemplarytestingmethodsforViruses12Annex C (informative)Availableandexemplarytestingmethodsformycoplasma15Bibliography18ForewordISO(theInternationalOrganizationforStandardization)isaworldwidefederationofnationalstandardsbodies(ISOmemberbodies).TheworkofpreparingI

6、nternationalStandardsisnormallycarriedoutthroughISOtechnicalcommittees.Eachmemberbodyinterestedinasubjectforwhichatechnicalcommitteehasbeenestablishedhastherighttoberepresentedonthatcommittee.Internationalorganizations,governmentalandnon-governmental,inliaisonwithISO,alsotakepartinthework.ISOcollabo

7、ratescloselywiththeInternationalElectrotechnicalCommission(IEC)onallmattersofelectrotechnicalstandardization.TheproceduresusedtodevelopthisdocumentandthoseintendedforitsfurthermaintenancearedescribedintheISO/IECDirectives,Part1.Inparticular,thedifferentapprovalcriterianeededforthedifferenttypesofISO

8、documentshouldbenoted.ThisdocumentwasdraftedinaccordancewiththeeditorialrulesoftheISO/IECDirectives,Part2(seeWWW.iso.org/directives).ISOdrawsattentiontothepossibilitythattheimplementationofthisdocumentmayinvolvetheuseof（八）patent(三).ISOtakesnopositionconcerningtheevidence,validityorapplicabilityofany

9、claimedpatentrightsinrespectthereof.Asofthedateofpublicationofthisdocument,ISOhadnotreceivednoticeof（八）patent(三)whichmayberequiredtoimplementthisdocument.HoweverlImplementersarecautionedthatthismaynotrepresentthelatestinformation,whichmaybeobtainedfromthepatentdatabaseavailableatWWW.iso.org/patents.

10、ISOshallnotbeheldresponsibleforidentifyinganyorallsuchpatentrights.Anytradenameusedinthisdocumentisinformationgivenfortheconvenienceofusersanddoesnotconstituteanendorsement.Foranexplanationofthevoluntarynatureofstandards,themeaningofISOspecifictermsandexpressionsrelatedtoconformityassessment,aswella

11、sinformationaboutISOsadherencetotheWorldTradeOrganization(WTO)principlesintheTechnicalBarrierstoTrade(TBT工seeWWW.iso.org/iso/foreword.htmLThedocumentwaspreparedbyTechnicalCommitteeISO/TC276,Biotechnology,SubcommitteeSCLAnalyticalmethods.Anyfeedbackorquestionsonthisdocumentshouldbedirectedtotheusersn

12、ationalstandardsbody.Acompletelistingofthesebodiescanbefoundatwww.iso.org/members.html.IntroductionCellcultureplaysanextremelyimportantroleinbiotechnologyandlifescienceresearch.Therefore,thereliabilityofscientificdatafromcellcultureisessential.Oneofthemostcommonproblemsincellcultureismicrobiological

13、contamination.Thisusuallyresultsincatastrophiccelldeathorcontaminationsustainedatlowlevels(sometimesduetotheuseofantibiotics),andthusimpactsthereliabilityofexperimentaldataderivedfromcellculture.Nonetheless,despiteitsprevalenceandimportance,thelackofopendiscussiondiscouragesdevelopmentandlearningofb

14、estpracticestoavoidmicrobialcontaminationincellculture.Microbialcontaminationisaneconomicissueasitaffectsthereproducibilityofscientificdata,theefficiencyofinvitrocellcultureworkandmostimportantly,thesafetyoflaboratoryoperators.Itisthereforenecessarytomonitormicrobialcontaminationthroughafullundersta

15、ndingofthesourceofcontaminationandemploygoodtestingtechniquesbeforeandduringcellculture.Whenculturedcellshavebeencontaminated,firstthevariousaspectsofthecontaminationareidentified,includingthenatureofcontamination,thetimeofcontaminationandtheoperatingenvironment.Contaminantscanincludebacteria,fungi,

16、mycoplasma,virusesandothertypesoforganisms.Inordertodemonstratetheabsenceofmicrobiologicalcontaminationincellculture,itcanbenecessarytoconductaseriesoftestsforlikelyorganismsandsuchanapproachwillbenefitfromriskassessmentofthecelltype,celloriginandreagentsusedfortheirculture.Environmentalmonitoringin

17、areaswherecellsareculturedandstoredcanreducetheriskofmicrobialcontaminationfromtheprocessingenvironmentThisdocumentprovidesaninventoryofmethodsforthedetectionofmicrobiologicalcontaminationinmammaliancellcultureinordertoprovideuserswithanoverviewthatalsoincludesinformationonpro-andcontraindicationsfo

18、rthelistedmethodsinrelationtotheappropriatesampletype.BiotechnologyInventoryofmethodsfordetectionofmicrobiologicalcontaminationinmammaliancellculture1 ScopeThisdocumentprovidesaninventoryofmethodsforthedetectionofmicrobiologicalcontaminationinmammaliancellculture.Thisdocumentincludesconsiderationsfo

19、rtheselectionofmethodstotestthepresenceofcommoncontaminantssuchasbacteria,fungi,virusesandmycoplasma.Thisdocumentisnotapplicabletoprionsandprotists.Thisdocumentisintendedforusebybiomedicalresearchers,biobankoperatorsandothersperformingmammaliancellculture.2 NormativereferencesTherearenonormativerefe

20、rencesinthisdocument.3 TermsanddefinitionsForthepurposesofthisdocument,thefollowingtermsanddefinitionsapply.ISOandIECmaintainterminologydatabasesforuseinstandardizationatthefollowingaddresses: ISOOnlinebrowsingplatform:availableathttps:WWW.iso.org/obp IECElectropedia:availableathttps:WWW.electropedi

21、a.org/1.1accuracyclosenessofagreementbetweenameasuredquantityvalueandthetruequantityvalueofameasurandSOURCE:ISO/IECGuide99:2007,2.13,modifiedNotestoentrydeleted.1.2digitalPCRdPCRprocedureinwhichnucleicacidtemplatesaredistributedacrossmultiplepartitionsofnominallyequivalentvolume,suchthatsomepartitio

22、nscontaintemplateandothersdonot,followedbyPCRamplificationoftargetsequencesanddetectionofspecificPCRproducts,providingacountofthenumberofpartitionswithapositiveandnegativesignalforthetargettemplateNote1toentry:Nucleicacidtargetsequencesareassumedtoberandomlyandindependentlydistributedacrossthepartit

23、ionsduringthepartitioningprocess.Note2toentry:ThecountofpositiveandnegativepartitionsisnormallybasedonendpointdetectionofPCRproductsfollowingthermalcycling,howeverreal-timeqPCRmonitoringofPCRproductaccumulationisadditionallypossibleforsomedPCRplatforms.SOURCE:ISO20395:2019,3.101.3intendeduseintended

24、purposeuseforwhichaproduct,process,orserviceisintendedaccordingtothespecifications,instructionsorinformationormultipleofthemprovidedbythemanufactureroruserSOURCE:ISO23033:2021,3.261.4microbialcontaminationpresenceofunintendedbacteria,fungi,mycoplasma,orvirusesSOURCE:ISO11139:2018,3.1711.5nextgenerat

25、ionsequencingNGSnon-Sanger-basedhigh-throughputnucleicacidsequencingNote1toentry:Millionsorbillionsofnucleicacidstrandscanbesequencedinparallel,yieldingsubstantiallymorethroughput.Note2toentry:NGS(next-generation-sequencing)isalsowellrecognizedasMPS(massivelyparallelsequencing)intheISO20397series.No

26、te3toentry:MPSorNGScoverslongreadsequencingandshortreadsequencing.SOURCE:ISO/DIS20397-3:2024,3.161.6PCRassayqPCRordPCR(3.2)measurementmethodwithspecifiedoligonucleotideprimers(and,insomecases,aprobeorprobes)thatisusedtoidentifyorquantifyanucleicacidtargetSOURCE:ISO20395:2019,3.231.7reagentsubstanceu

27、sedinchemical/biochemicalanalysisorotherreactionSOURCE:ISO20391-1:2018,3.191.8resolutionsmallestchangeinaquantitybeingmeasuredthatcausesaperceptiblechangeinthecorrespondingindicationNote1toentry:Resolutioncandependon,forexample,ratioofsignalandnoise(internalorexternal.Itcanalsodependonthevalueofaqua

28、ntitybeingmeasured.SOURCE:JCGM200:2012,4.14,modifiedFrictionnotmentionedinNote1toentry.1.9ribosomalRNArRNAnon-codingribonucleicacidcontainedinribosomesNoteltoentryiThenucleotidesequenceofrRNAsubunitscanbeusedtodetectmicroorganisms(e.g.16SrRNA,18SrRNA.3.10sampleoneormorepartstakenfromasystemSOURCE:IS

29、O23033:2021,3.463.11sensitivityquotientofthechangeinanindicationofameasuringsystemandthecorrespondingchangeinavalueofaquantitybeingmeasuredNote1toentry:Sensitivityofameasuringsystemcandependonthevalueofthequantitybeingmeasured.Note2toentry:Thechangeconsideredinavalueofaquantitybeingmeasuredmustbelar

30、gecomparedwiththeresolution.SOURCE:ISO/IECGuide99:2007,4.12,modifiedsensitivityisgivenastheonlypreferredterm.3.12testsamplesmallaliquotofthesamplethatispreparedformeasurementinthemethodofinterestNote1toentry:Generally,testsamplesarerepresentativeofthesampletheyarepreparedfromandaresometimesreferredt

31、oasrepresentativetestSamPle(三)”.SOURCE:ISO20391-2:2019,3.363.13validationconfirmation,throughtheprovisionofobjectiveevidence,thattherequirementsforaspecificintendeduseorapplicationhavebeenfulfilledSOURCE:ISO23033:202L3.544 Generalconcepts4.1 IntroductionContaminationofcellculturecanproduceadverseeff

32、ectsincellphenotypeandquality,aswellasapplicationsinresearchanddevelopmentincludingclinicalstudies,stemcellmanufacturingetc.Diversemethodsandtechniqueshavebeendevelopedforlaboratorymonitoringandtestingofmicrobialcontaminants.Manymicrobialcontaminantsespeciallycoarsecontaminationbybacteriaandfungican

33、leadtosignificantabnormalitiesincellviabilityandappearance,whichcanbevisuallyobservedbythenakedeyeorunderamicroscope.However,manycontaminantssuchaslowlevelsofbacteriaandfungi,mycoplasmaandvirusescannotbedetectedbychangesincellappearanceandcellculturemediumthroughdirectobservation.Thus,appropriatetec

34、hniquesareusedfortestingmicrobialcontaminantsbasedonthecharacteristicsofthemicrobialcontaminantsandlaboratoryfacilities.4.2 DetectionprocessThedetectionofmicrobiologicalcontaminationinmammaliancellculturesiscarriedoutunderasepticconditions.NOTE1Airqualityofthetestingenvironmentcanbedefinedaccordingt

35、oISOstandardsforcertainregulatedapplications.FurtherguidanceisgiveninISO16000-44andISO1105733.Thedetectionofmicrobiologicalcontaminationofmammaliancellsincludingbacteria,fungi,viruses,andmycoplasmadoesnotspecificallyconsidertheevaluationofthelikelihoodofcontaminationbyotherorganismgroupslikeprionsan

36、dprotists.NOTE2Mycoplasmaisabacterium(orgerm)thatcaninfectcellculturesanddifferentpartsofhumanbody.Unlikeotherbacteria,mycoplasmadonothavecellwalls.Theyarealsoverysmallcomparedtootherbacteria.Manyantibioticskillbacteriabyweakeningthosewalls.Sincemycoplasmabacteriadonthavethem,someantibiotics,likepen

37、icillin,wontworkagainstthem.Mycoplasmaisdiscussedseparatelyfrombacteriainthisdocument.Ariskassessmenttoidentifyanylikelycontaminantscanfacilitateregimeestablishmentformicrobiologicalcontaminationdetectioninaparticularcellculture.Thisregimehelpstoaddressanypotentialmicrobiologicalhazardsassociatedwit

38、hthederivation,culturemethods,mediaandstoragehistoryofthecellculturetobetested.Thetestingofcellculturesforbacteria,fungi,viruses,mycoplasmaisusuallyconsideredtobearoutineprocedureforanymammaliancellcultures,unlessspecificconditionsapplytorenderthisunnecessary.Certainorganismsthatarecommoncontaminant

39、sarisingfromthegenerallaboratoryenvironmentandothertypecellculturescanbeconsideredinmicrobialdetectionforanymammaliancellculture.Considerationsforselectionofthetestmeasurementsdependontheintendedpurposeaswellassampleandprocessingfactorsthatintroduceadditionalpotentialformicrobialcontamination.Thesei

40、nclude,butarenotlimitedto,theintendedpurposeforcells,celltypes,cellattributes,potentialeffectsofsampleheterogeneity,andthepresenceofinhibitorysubstancesinsamplestobeusedforthedetection.Thelaboratoryestablishesproceduresforthepreparationofcontrolsandreferencematerials,including,butnotlimitedto,calibr

41、ation,validation,expirydateandhazardidentification.Detailsofrawdata,controlsandresultsarenormallyrecordedanddocumentedforthepurposeofqualitycontrol,review,auditandassessment.NOTE3Suchanapproachisincapableofpredictinganddetectingallformsofcontamination(ie,itcannotassertthatacultureisfreeofcontaminati

42、on).5 ContaminationtestinformationTheusersfirstmakeriskassessmenttodeterminethepossiblecontaminationbeforemicrobialcontaminationtesting.Thisassessmentdependsonthesourceofcells,thehistoryofexposuretopotentialcontamination,theexpectedapplicationpurposeofcellsandtheenvironment.Cellsfromdifferentspecies

43、arelikelytocarrydifferentpotentialmicroorganisms.Theexposurehistoryofthecellcultureistakenintoaccountincludingadetailedassessmentofrawmaterialsandothermaterialswhichcanhavecomeintodirectcontactwiththecellsinquestion.Rawmaterialsofbiologicaloriginareofspecialconcernsincetheycantransmitorganismspresen

44、tintheoriginalsourcematerials.Inparticular,materialsderivedfromhigherriskmammaliantissuesandbodyfluidsareassessedforriskofinfectionofthefinalmaterialsdependingonthelikelihoodofcontaminationanditsinactivationorremovalduringprocessing.Ofspecialconcernareanimalserumandotherrawmaterialsusedwidelytocultu

45、recellsareofspecialconcernastheycanbecomecontaminatedbyvirusespresentindonorswhichcannotbesubjectedtoeffectivedisinfectionorsterilization.Relevantcontaminationdetectioncanbecarriedoutaccordingtothepurposeofcelluse,withrespecttothesafetyoflaboratorypersonnel,thetypeandqualityofdataobtainedfromcellcul

46、ture-basedassays,andthepotentialforongoingapplicationofthetestedcellculturewhereanycontaminantscanbepassedtoothercultures,e.g.asfeedercells,toprovideconditionedcellculturemedia,orextracellularmatrix.Theenvironmentalfactorsofcellpreparationandcellculturecancausecontamination.Possiblecontaminationcanb

47、eassessedbasedonexposurehistory,e.g.animalserumusedtoculturecellscanbecontaminatedbyspecificmicroorganisms.Understandingtheexistingdetectionmethods,theircharacteristics,andtheirdetectioncapabilitiesisimportantinordertomeettheneededdetectionrequirements.Inthecontextoftheriskassessment,theanalyticalla

48、boratoryevaluatestheapplicabilityofexistingtestmethods,andselectstheappropriatemethodforthesamplestobetested.DetailsarelistedinAnnexA,AnneXBandAnneXCforeachtypeofmethod.Fullconsiderationofthetestingapproachincludestypesofcontamination(e.g.specificmicroorganismsorbroadercontaminants),andtestresolution(e.g.llimitsofdetectionandlimitsofquantification)andspecificityoftechniques.Selectionofanappropriatetestingmethodconsidersfactorsincludingtargetmicroorganisms,characteristicsofsamples,andhandli