PD ISO-TR 4752 2025.docx

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1、PDISO/TR4752:2025BiotechnologyInventoryofmethodsfordetectionofmicrobiologicalcontaminationinmammaliancellculturebsi.NationalforewordThisPublishedDocumentistheUKimplementationofISO/TR4752:2025.TheUKparticipationinitspreparationwasentrustedtoTechnicalCommitteeBTIllBiotechnologies.Alistoforganizationsr

2、epresentedonthiscommitteecanbeobtainedonrequesttoitscommitteemanager.ContractualandlegalconsiderationsThispublicationhasbeenpreparedingoodfaith,howevernorepresentation,warranty,assuranceorundertaking(expressorimplied)isorwillbemade,andnoresponsibilityorliabilityisorwillbeacceptedbyBSIinrelationtothe

3、adequacy,accuracy,completenessorreasonablenessofthispublication.Allandanysuchresponsibilityandliabilityisexpresslydisclaimedtothefullextentpermittedbythelaw.Thispublicationisprovidedasis,andistobeusedattherecipientsownrisk.Therecipientisadvisedtoconsiderseekingprofessionalguidancewithrespecttoitsuse

4、ofthispublication.Thispublicationisnotintendedtoconstituteacontract.Usersareresponsibleforitscorrectapplication.ThispublicationisnottoberegardedasaBritishStandard.TheBritishStandardsInstitution2025PublishedbyBSIStandardsLimited2025ISBN9780539309225ICS07.080CompliancewithaPublishedDocumentcannotconfe

5、rimmunityfromlegalobligations.ThisPublishedDocumentwaspublishedundertheauthorityoftheStandardsPolicyandStrategyCommitteeon30June2025.Amendments/corrigendaissuedsincepublicationDateTextaffectedPDISO/TR4752:2025TechnicalReportISO/TR4752Firstedition2025-06BiotechnologyInventoryofmethodsfordetectionofmi

6、crobiologicalcontaminationinmammaliancellcultureBiotechnologieInventairedesmethodespourIadetectiondelacontaminationmicrobiologiquedanslacultureCeIIulairedemammiferesReferencenumberISO/TR4752:2025(en)COPYRIGHTPROTECTEDDOCUMENTISO2025Allrightsreserved.Unlessotherwisespecified,orrequiredinthecontextofi

7、tsimplementation,nopartofthispublicationmaybereproducedorutilizedotherwiseinanyformorbyanymeans,electronicormechanical,includingphotocopying,orpostingontheinternetoranintranet,withoutpriorwrittenpermission.PermissioncanberequestedfromeitherISOattheaddressbeloworISO,smemberbodyinthecountryofthereques

8、ter.ISOcopyrightofficeCP401Ch.deBlandonnet8CH-1214Vernier,GenevaPhone:+41227490111Email:copyrightiso.orgWebsite:www.iso.orgPublishedinSwitzerlandContentsPageForewordivIntroductionv1 Scope12 Normativereferences13 Termsanddefinitions14 Generalconcepts34.1 Introduction34.2 Detectionprocess35 Contaminat

9、iontestinformation46 Criticalcontrolpointsindetection56.1 Samples56.2 ReagentsandEquipment56.3 Operation66.4 Personnel66.5 Environment6Annex A (informative)Availableandexemplarytestingmethodsforbacteriaandfungi7Annex B (informative)AvailableandexemplarytestingmethodsforViruses12Annex C (informative)

10、Availableandexemplarytestingmethodsformycoplasma15Bibliography18ForewordISO(theInternationalOrganizationforStandardization)isaworldwidefederationofnationalstandardsbodies(ISOmemberbodies).TheworkofpreparingInternationalStandardsisnormallycarriedoutthroughISOtechnicalcommittees.Eachmemberbodyinterest

11、edinasubjectforwhichatechnicalcommitteehasbeenestablishedhastherighttoberepresentedonthatcommittee.Internationalorganizations,governmentalandnon-governmental,inliaisonwithISO,alsotakepartinthework.ISOcollaboratescloselywiththeInternationalElectrotechnicalCommission(IEC)onallmattersofelectrotechnical

12、standardization.TheproceduresusedtodevelopthisdocumentandthoseintendedforitsfurthermaintenancearedescribedintheISO/IECDirectives,Part1.Inparticular,thedifferentapprovalcriterianeededforthedifferenttypesofISOdocumentshouldbenoted.ThisdocumentwasdraftedinaccordancewiththeeditorialrulesoftheISO/IECDire

13、ctives,Part2(seeWWW.iso.org/directives).ISOdrawsattentiontothepossibilitythattheimplementationofthisdocumentmayinvolvetheuseof（八）patent(三).ISOtakesnopositionconcerningtheevidence,validityorapplicabilityofanyclaimedpatentrightsinrespectthereof.Asofthedateofpublicationofthisdocument,ISOhadnotreceivedn

14、oticeof（八）patent(三)whichmayberequiredtoimplementthisdocument.HoweverlImplementersarecautionedthatthismaynotrepresentthelatestinformation,whichmaybeobtainedfromthepatentdatabaseavailableatWWW.iso.org/patents.ISOshallnotbeheldresponsibleforidentifyinganyorallsuchpatentrights.Anytradenameusedinthisdocu

15、mentisinformationgivenfortheconvenienceofusersanddoesnotconstituteanendorsement.Foranexplanationofthevoluntarynatureofstandards,themeaningofISOspecifictermsandexpressionsrelatedtoconformityassessment,aswellasinformationaboutISOsadherencetotheWorldTradeOrganization(WTO)principlesintheTechnicalBarrier

16、stoTrade(TBT工seeWWW.iso.org/iso/foreword.htmLThedocumentwaspreparedbyTechnicalCommitteeISO/TC276,Biotechnology,SubcommitteeSCLAnalyticalmethods.Anyfeedbackorquestionsonthisdocumentshouldbedirectedtotheusersnationalstandardsbody.AcompletelistingofthesebodiescanbefoundatWWW.iso.org/members.html.Introd

17、uctionCellcultureplaysanextremelyimportantroleinbiotechnologyandlifescienceresearch.Therefore,thereliabilityofscientificdatafromcellcultureisessential.Oneofthemostcommonproblemsincellcultureismicrobiologicalcontamination.Thisusuallyresultsincatastrophiccelldeathorcontaminationsustainedatlowlevels(so

18、metimesduetotheuseofantibiotics),andthusimpactsthereliabilityofexperimentaldataderivedfromcellculture.Nonetheless,despiteitsprevalenceandimportance,thelackofopendiscussiondiscouragesdevelopmentandlearningofbestpracticestoavoidmicrobialcontaminationincellculture.Microbialcontaminationisaneconomicissu

19、easitaffectsthereproducibilityofscientificdata,theefficiencyofinvitrocellcultureworkandmostimportantly,thesafetyoflaboratoryoperators.Itisthereforenecessarytomonitormicrobialcontaminationthroughafullunderstandingofthesourceofcontaminationandemploygoodtestingtechniquesbeforeandduringcellculture.Whenc

20、ulturedcellshavebeencontaminated,firstthevariousaspectsofthecontaminationareidentified,includingthenatureofcontamination,thetimeofcontaminationandtheoperatingenvironment.Contaminantscanincludebacteria,fungi,mycoplasma,virusesandothertypesoforganisms.Inordertodemonstratetheabsenceofmicrobiologicalcon

21、taminationincellculture,itcanbenecessarytoconductaseriesoftestsforlikelyorganismsandsuchanapproachwillbenefitfromriskassessmentofthecelltype,celloriginandreagentsusedfortheirculture.Environmentalmonitoringinareaswherecellsareculturedandstoredcanreducetheriskofmicrobialcontaminationfromtheprocessinge

22、nvironment.Thisdocumentprovidesaninventoryofmethodsforthedetectionofmicrobiologicalcontaminationinmammaliancellcultureinordertoprovideuserswithanoverviewthatalsoincludesinformationonpro-andcontraindicationsforthelistedmethodsinrelationtotheappropriatesampletype.PDISO/TR4752:2025BiotechnologyInventor

23、yofmethodsfordetectionofmicrobiologicalcontaminationinmammaliancellculture1 ScopeThisdocumentprovidesaninventoryofmethodsforthedetectionofmicrobiologicalcontaminationinmammaliancellculture.Thisdocumentincludesconsiderationsfortheselectionofmethodstotestthepresenceofcommoncontaminantssuchasbacteria,f

24、ungi,virusesandmycoplasma.Thisdocumentisnotapplicabletoprionsandprotists.Thisdocumentisintendedforusebybiomedicalresearchers,biobankoperatorsandothersperformingmammaliancellculture.2 NormativereferencesTherearenonormativereferencesinthisdocument.3 TermsanddefinitionsForthepurposesofthisdocument,thef

25、ollowingtermsanddefinitionsapply.ISOandIECmaintainterminologydatabasesforuseinstandardizationatthefollowingaddresses: ISOOnlinebrowsingplatform:availableathttps:WWW.iso.org/obp IECElectropedia:availableathttps:WWW.electropedia.org/1.1accuracyclosenessofagreementbetweenameasuredquantityvalueandthetru

26、equantityvalueofameasurandSOURCE:ISO/IECGuide99:2007,2.13,modifiedNotestoentrydeleted.1.2digitalPCRdPCRprocedureinwhichnucleicacidtemplatesaredistributedacrossmultiplepartitionsofnominallyequivalentvolume,suchthatsomepartitionscontaintemplateandothersdonot,followedbyPCRamplificationoftargetsequences

27、anddetectionofspecificPCRproducts,providingacountofthenumberofpartitionswithapositiveandnegativesignalforthetargettemplateNote1toentry:Nucleicacidtargetsequencesareassumedtoberandomlyandindependentlydistributedacrossthepartitionsduringthepartitioningprocess.Note2toentry:Thecountofpositiveandnegative

28、partitionsisnormallybasedonendpointdetectionofPCRproductsfollowingthermalcycling,howeverreal-timeqPCRmonitoringofPCRproductaccumulationisadditionallypossibleforsomedPCRplatforms.SOURCE:ISO20395:2019,3.101.3intendeduseintendedpurposeuseforwhichaproduct,process,orserviceisintendedaccordingtothespecifi

29、cations,instructionsorinformationormultipleofthemprovidedbythemanufactureroruserSOURCE:ISO23033:202L3.261.4microbialcontaminationpresenceofunintendedbacteria,fungi,mycoplasma,orvirusesSOURCE:ISO11139:2018,3.1711.5nextgenerationsequencingNGSnon-Sanger-basedhigh-throughputnucleicacidsequencingNote1toe

30、ntry:Millionsorbillionsofnucleicacidstrandscanbesequencedinparallel,yieldingsubstantiallymorethroughput.Note2toentry:NGS(next-generation-sequencing)isalsowellrecognizedasMPS(massivelyparallelsequencing)intheISO20397series.Note3toentry:MPSorNGScoverslongreadsequencingandshortreadsequencing.SOURCE:ISO

31、/DIS20397-3:2024,3.161.6PCRassayqPCRordPCR(3.2)measurementmethodwithspecifiedoligonucleotideprimers(and,insomecases,aprobeorprobes)thatisusedtoidentifyorquantifyanucleicacidtargetSOURCE:ISO20395:2019,3.231.7reagentsubstanceusedinchemical/biochemicalanalysisorotherreactionSOURCE:ISO20391-1:2018,3.191

32、8resolutionsmallestchangeinaquantitybeingmeasuredthatcausesaperceptiblechangeinthecorrespondingindicationNote1toentry:Resolutioncandependon,forexample,ratioofsignalandnoise(internalorexternal).Itcanalsodependonthevalueofaquantitybeingmeasured.SOURCE:JCGM200:2012,4.14,modifiedFrictionnotmentionedinN

33、ote1toentry.1.9ribosomalRNArRNAnon-codingribonucleicacidcontainedinribosomesNoteltoentryiThenucleotidesequenceofrRNAsubunitscanbeusedtodetectmicroorganisms(e.g.16SrRNA,18SrRNA.3.10sampleoneormorepartstakenfromasystemSOURCE:ISO23033:2021,3.463.11sensitivityquotientofthechangeinanindicationofameasurin

34、gsystemandthecorrespondingchangeinavalueofaquantitybeingmeasuredNote1toentry:Sensitivityofameasuringsystemcandependonthevalueofthequantitybeingmeasured.Note2toentry:Thechangeconsideredinavalueofaquantitybeingmeasuredmustbelargecomparedwiththeresolution.SOURCE:ISO/IECGuide99:2007,4.12,modifiedsensiti

35、vityisgivenastheonlypreferredterm.3.12testsamplesmallaliquotofthesamplethatispreparedformeasurementinthemethodofinterestNote1toentry:Generally,testsamplesarerepresentativeofthesampletheyarepreparedfromandaresometimesreferredtoasrepresentativetestSamPle(三)”.SOURCE:ISO20391-2:2019,3.363.13validationco

36、nfirmation,throughtheprovisionofobjectiveevidence,thattherequirementsforaspecificintendeduseorapplicationhavebeenfulfilledSOURCE:ISO23033:202L3.544 Generalconcepts4.1 IntroductionContaminationofcellculturecanproduceadverseeffectsincellphenotypeandquality,aswellasapplicationsinresearchanddevelopmenti

37、ncludingclinicalstudies,stemcellmanufacturingetc.Diversemethodsandtechniqueshavebeendevelopedforlaboratorymonitoringandtestingofmicrobialcontaminants.Manymicrobialcontaminantsespeciallycoarsecontaminationbybacteriaandfungicanleadtosignificantabnormalitiesincellviabilityandappearance,whichcanbevisual

38、lyobservedbythenakedeyeorunderamicroscope.However,manycontaminantssuchaslowlevelsofbacteriaandfungi,mycoplasmaandvirusescannotbedetectedbychangesincellappearanceandcellculturemediumthroughdirectobservation.Thus,appropriatetechniquesareusedfortestingmicrobialcontaminantsbasedonthecharacteristicsofthe

39、microbialcontaminantsandlaboratoryfacilities.4.2 DetectionprocessThedetectionofmicrobiologicalcontaminationinmammaliancellculturesiscarriedoutunderasepticconditions.NOTE1AirqualityofthetestingenvironmentcanbedefinedaccordingtoISOstandardsforcertainregulatedapplications.FurtherguidanceisgiveninISO160

40、00-44andISO1105733.ISO2025-AllrightsreservedThedetectionofmicrobiologicalcontaminationofmammaliancellsincludingbacteria,fungi,viruses,andmycoplasmadoesnotspecificallyconsidertheevaluationofthelikelihoodofcontaminationbyotherorganismgroupslikeprionsandprotists.NOTE2Mycoplasmaisabacterium(orgerm)thatc

41、aninfectcellculturesanddifferentpartsofhumanbody.Unlikeotherbacteria,mycoplasmadonothavecellwalls.Theyarealsoverysmallcomparedtootherbacteria.Manyantibioticskillbacteriabyweakeningthosewalls.Sincemycoplasmabacteriadonthavethem,someantibiotics,likepenicillin,wontworkagainstthem.Mycoplasmaisdiscusseds

42、eparatelyfrombacteriainthisdocument.Ariskassessmenttoidentifyanylikelycontaminantscanfacilitateregimeestablishmentformicrobiologicalcontaminationdetectioninaparticularcellculture.Thisregimehelpstoaddressanypotentialmicrobiologicalhazardsassociatedwiththederivation,culturemethods,mediaandstoragehisto

43、ryofthecellculturetobetested.Thetestingofcellculturesforbacteria,fungi,viruses,mycoplasmaisusuallyconsideredtobearoutineprocedureforanymammaliancellcultures,unlessspecificconditionsapplytorenderthisunnecessary.Certainorganismsthatarecommoncontaminantsarisingfromthegenerallaboratoryenvironmentandothe

44、rtypecellculturescanbeconsideredinmicrobialdetectionforanymammaliancellculture.Considerationsforselectionofthetestmeasurementsdependontheintendedpurposeaswellassampleandprocessingfactorsthatintroduceadditionalpotentialformicrobialcontamination.Theseinclude,butarenotlimitedto,theintendedpurposeforcel

45、ls,celltypes,cellattributes,potentialeffectsofsampleheterogeneity,andthepresenceofinhibitorysubstancesinsamplestobeusedforthedetection.Thelaboratoryestablishesproceduresforthepreparationofcontrolsandreferencematerials,including,butnotlimitedto,calibration,validation,expirydateandhazardidentification

46、Detailsofrawdata,controlsandresultsarenormallyrecordedanddocumentedforthepurposeofqualitycontrol,review,auditandassessment.NOTE3Suchanapproachisincapableofpredictinganddetectingallformsofcontamination(ie,itcannotassertthatacultureisfreeofcontamination).5 ContaminationtestinformationTheusersfirstmak

47、eriskassessmenttodeterminethepossiblecontaminationbeforemicrobialcontaminationtesting.Thisassessmentdependsonthesourceofcells,thehistoryofexposuretopotentialcontamination,theexpectedapplicationpurposeofcellsandtheenvironment.Cellsfromdifferentspeciesarelikelytocarrydifferentpotentialmicroorganisms.T

48、heexposurehistoryofthecellcultureistakenintoaccountincludingadetailedassessmentofrawmaterialsandothermaterialswhichcanhavecomeintodirectcontactwiththecellsinquestion.Rawmaterialsofbiologicaloriginareofspecialconcernsincetheycantransmitorganismspresentintheoriginalsourcematerials.Inparticular,materialsderivedfromhigherriskmammaliantissuesandbodyfluidsareassessedforriskofinfectionofthefinalmaterialsdependingonthelikelihoodofcontaminationanditsinactivationorremovalduringprocess