29/05/2017 Harmonized Microbial Limits Testing Validation Strategies | IVT - GMP - Microbiology

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HarmonizedMicrobialLimitsTestingValidationStrategies|IVT

By CrystalBoothDec17,201312:41pmPST

PeerReviewed:Microbiology

Abstract
Obtainingsuccessfulvalidationsofcertainrawmaterialsandfinishedproductscanbetricky.
Researchingthematerialsbeforehandisimportant.Byworkingcloselywithdevelopmentscientists
andchemists,settingappropriatespecifications,andperformingresearch,methoddevelopmenttrials
canbestreamlinedtoproduceanadequatemethod.Keyfactorsindevelopingapropermethod
includesomeexperimentationaswellasknowledgeofthepHofthematerial,thewateractivityofthe
material,thewatersolubilityofthematerial,andanyantimicrobialpropertiesofthematerial,toname
afew.Riskassessmentscanbeusedtodeterminewhattotest,howfrequentlytotest,thestability
program,andobjectionablemicroorganismidentification.Cleardocumentationandadequate
scientificallysoundjustificationsarenecessarysothattheanyfuturequestionscanbeeasily
answered.

Introduction
TheHarmonizedMicrobialLimitsTest(HMLT)wasmadeeffectiveinMay2009.Theinternational
harmonizationincludedUnitedStatesPharmacopeia(USP),JapanesePharmacopeia(JP),and
EuropeanPharmacopeia(EP).Companieswereencouragedtorevalidatetheirrawmaterialsand
finishedproductsinordertoobtaincompliancetotheenhancedguidancedocumentspriortothe
implementationdate.Thispaperprovidesinsighttothetestandstrategiesforobtainingsuccessful
validationsofthetest.

Introduction
TheHarmonizedMicrobialLimitTest(HMLT)wasmadeeffectiveinMay2009.Theinternational
harmonizationincludedUnitedStatesPharmacopeia(USP),JapanesePharmacopeia(JP),and
EuropeanPharmacopeia(EP).Companieswereencouragedtorevalidatetheirrawmaterialsand
finishedproductsinordertoobtaincompliancetotheenhancedguidancedocumentspriortothe
implementationdate.Thispaperprovidesinsighttothetestandstrategiesforobtainingsuccessful
validationsofthetest.

ApplicationoftheHarmonizedMicrobialLimitsTest
TheHMLTisusedtoevaluaterawmaterialsandnonsterileproductsforacceptablemicrobialquality.
Itinvolvestheexaminationofnonsterileproductsthatwilldeterminewhetherasubstanceor
preparationcomplieswithestablishedspecificationsformicrobiologicalquality(1).

TheHMLTwasnotoriginallydesignedasaqualitycontroltest.Itwasdesignedasarefereetestto
demonstratecompliancewithmonographrequirements(2).However,manycompaniesusethetest
asameanstodeterminethequalityoftheirfinishedproductsorrawmaterials.Microbiallimittesting
isseenasanattributeofgoodmanufacturingpractice(GMP)andofqualityassurance(3).

Thetestsallowquantitativeenumerationofmesophilicbacteriaandfungithatgrowunderaerobic
conditions(1).Certainmicroorganismscanadverselyimpact(reduceorinactivate)theactivityof
certainproducts(4).Itisimportanttounderstandthemicrobialloadoftherawmaterialsorfinished
productsinordertodetermineiftheproductwillreactthewayitwasintended.

Microorganismscanalsoaffectthehealthofthepatients(4).Knowingtheamountandtypesof
microorganismsinaproductordeviceisimportanttopatientsafety.

TheguidancedocumentslistedinTableIgoverntheMicrobialLimitsTest.Thismaynotbeanall
inclusivelist.
TableI:GoverningDocumentsfortheMicrobialLimitsTest
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TableI:GoverningDocumentsfortheMicrobialLimitsTest

Document
DocumentTitle
Number
USP<61> MicrobiologicalExaminationofNonsterileProducts:MicrobialEnumerationTests
USP<62> MicrobiologicalExaminationofNonsterileProducts:TestsforSpecifiedMicroorganisms
USP MicrobiologicalExaminationofNonsterileProducts:AcceptanceCriteriaforPharmaceutical
<1111> PreparationsandSubstancesforPharmaceuticalUse
EP2.6.12. MicrobiologicalExaminationofNonSterileProducts:MicrobialEnumerationTests
EP2.6.13. MicrobiologicalExaminationofNonSterileProducts:TestforSpecifiedMicroOrganisms
JP4.05 MicrobialLimitTest
ICH
EvaluationandRecommendationofPharmacopoeialTextsforUseintheICHRegionsonMicrobiological
Guidance
ExaminationofNonSterileProducts:MicrobialEnumerationTestsGeneralChapter
QB4

InregardstothedocumentslistedinTableI,USP<61>,EP2.6.12,andJP4.05areharmonizedwith
oneanother,andtheassaysdescribedinthedocumentsareequivalent.Inaddition,USP<62>,EP
2.6.13,andJP4.05areharmonizedwithoneanother,andtheassaysdescribedinthosedocuments
areequivalent.

Eventhoughthetestsareinternationallyharmonizedwithoneanother,individualmonographsfor
specificrawmaterialsmaynotbeharmonizedinthevariousregions.

Itisessentialtolearntheproductandtheexcipientssothatimportanttestingdecisionscanbemade.
Effectivestrategiestolearnabouttheproductincludecollaboratingwiththeresearchand
development(R&D)team,joiningtheproductteam,andlisteningtothechemistworkingonthe
project.Discoverhowtheproductisused,thetargetaudience,themaximumdose,thedelivery
routes,howtheproductreactsinthebodyorwithotherchemicals,thewatersolubility,thepH,the
wateractivity,andtheantimicrobialproperties,tonameafew.

Knowingtheproductandtheexcipientscanhelpinmakingriskbaseddecisionsonwhichitemsto
testandhowoftentoconductthetest.Someguidancedocumentsgiveinstructionastothe
regulatoryexpectationsfortesting.TableIIlistssomeofthesedocuments(48).

TableII:GuidanceDocumentsListingRegulatoryExpectations.

Document
DocumentTitle
Number
Eachlotofacomponent,drugproductcontainer,orclosurewithpotentialformicrobial
21CFR211.84(d) contaminationthatisobjectionableinviewofitsintendeduseshallbesubjectedto
microbiologicaltestsbeforeuse.
Appropriatewrittenprocedures,designedtopreventobjectionablemicroorganismsindrug
21CFR211.113(a)
productsnotrequiredtobesterile,shallbeestablishedandfollowed.
Thereshallbeappropriatelaboratorytesting,asnecessary,ofeachbatchofdrugproduct
21CFR211.165(b)
requiredtobefreeofobjectionablemicroorganisms.
Thesignificanceofmicroorganismsinnonsterilepharmaceuticalproductsshouldbeevaluated
USPChapter
intermsoftheuseoftheproduct,thenatureoftheproduct,andthepotentialhazardtothe
<1111>
user.
ICHGuidelineQ6A
DecisionTrees6 NA
and8
Individual
NA
Monographs

BasedonCodeofFederalRegulationsTitle21Part211.165,itiswisetotesteverybatchofnon
sterilefinishedproductsthatarerequiredtobefreeofobjectionablemicroorganisms.Ifthetestsare
notconducted,notvalidatedproperly,orthemicroorganismsrecoveredarenotidentifiedthereisa
riskthatobjectionablemicroorganismsmaygoundetected.

Inordertodetermineifanyexcipientsrequirespecifictests,startbyresearchingthemonographs.Ifa
monographexistsforaparticularrawmaterial,testperthatmonograph.Usethemorestringent
monograph(oramixoftheinternationalmonographs)tobeabletouseonetestforglobalmarkets.If
amonographdoesntexist,useariskbasedapproachtodetermineiftestingneedstobeconducted.

Duringtheriskbasedapproach,askthefollowingquestions.

Howmuchofthematerialisusedinthefinishedproduct?
Whatisthepotentialtheamountoftheexcipientaddedwillnegativelyimpactthebioburdenof
thefinishedproduct?
Whatisthenatureoftheexcipient(e.g.,isitplantbased?)?
Isthemanufacturingprocessgoingtoreducethemicrobialload?
Whatisthewateractivity?
Doestheexcipienthavenaturalantimicrobialproperties?

Usethegatheredinformationtoaideinwritingthespecifications.Thespecificationsofthematerial
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Usethegatheredinformationtoaideinwritingthespecifications.Thespecificationsofthematerial
willenablethemethoddevelopmentandvalidationprocesstobemorestreamlined.Inordertosetan
appropriatespecificationforthematerial,startbyresearchingtheproductorexcipient.Ifguidanceor
monographsdonotexist,referenceUSP<1111>.

USP<1111>listsacceptancecriteriafornonsterileproducts.Itliststherecommendedtotalaerobic
microbialcountlimits,thetotalcombinedyeast/moldlimits,andtherecommendedspecified
microorganismtoscreenfor.Dependingontheproductandapplication,acompanymaywantto
screenforadditionalspecifiedmicroorganismsthatarenotlistedinUSP<1111>.Itishighly
recommendedtoidentifyeverymicroorganismrecoveredinordertoevaluatethepresenceor
absenceofobjectionablemicroorganisms.

Therearethreecommonlyusedenumerationmethodslistedinthecompendialchapterstochoose
from.Themethodsincludethemembranefiltrationmethod,theplatecountmethod,andthemost
probablenumber(MPN)method.Whichevermethodisutilizeddependsonfactorssuchasthenature
oftheproductandtherequiredlimitofmicroorganisms.

ThecompendialmethodsarealreadyvalidatedbyscientistsatUSPhowever,thesuitabilityofthe
methodtorecovermicroorganismsiftheyarepresentmustbeestablished.Thisisnotacomplete
validationbutratheraverificationofthesuitabilityofthemethod(2).

Methoddevelopmenttrialsaretheappropriatetimetoexperimentfornewproductsandwillinforma
facilityofthereactiontodiluents,heat,filtration,sonication,shaking,etc.Methoddevelopmenttrials
aretypicallyrecordedinnotebooksandkeptseparatefromthevalidationexercises.

Startmethoddevelopmentbyutilizinginformationabouttheproductthathasbeengatheredfrom
projectteams,scientists,orchemists.Utilizingknownproductinformationwilldecreasetheamountof
manipulationduringthemethoddevelopmenttrials.Forexample,ifaproductisnotsolubleinwater,
tryaddingpolysorbatetothediluentsorusingisopropylmyristateinstead.IfthepHisacidic,itwill
needtobeadjustedtoaneutralrange(i.e.,pHof68)torecovermicroorganisms.Ifaproducthas
antimicrobialactivities,tryaddingneutralizerstothemediaorutilizingthemembranefiltration
method.Samplepreparationdependsonthephysicalcharacteristicsoftheproducttobetested(1).

Thecompendialchaptersoutlineusefulinformationforthedevelopmentprocessaswell:

Iftheproductcontainsantimicrobialactivity,thisshouldbeneutralized.
Ifinactivatorsareused,theirefficacyandtheirabsenceoftoxicityformicroorganismsmustbe
demonstrated.
Commonneutralizingagentsandmethodsincludetheadditionofpolysorbate,theadditionof
lecithin,and/ordilutionmethods(1).

USP<61>statesthatifnosuitableneutralizingmethodcanbefound,itcanbeassumedthatthe
failuretoisolatetheinoculatedmicroorganismsisattributabletothemicrobialactivityoftheproduct
(1).Proceedbyperformingthetestwiththehighestdilutionfactorcompatiblewithmicrobialgrowth
andthespecificacceptancecriterionincaseothermicroorganismsarenotinhibitedbytheproduct
(1).USP<62>continuesontosaythatforagivenproduct,iftheantimicrobialactivitywithrespectto
amicroorganismforwhichtestingisprescribedcannotbeneutralized,thenitistobeassumedthat
theinhibitedmicroorganismwillnotbepresentintheproduct(10).Mostcompaniescontinueinthe
methoddevelopmenttrialsuntilasuitablemethodisidentified.

Dependingonthenatureoftheproductandtherequiredlimitofmicroorganismsallowed,choosethe
appropriatemethodtouseinthemethoddevelopmenttrials.Again,themethodsincludethe
membranefiltrationmethod,theplatecountmethod,andtheMPNmethod.

Forthemembranefiltrationmethod,filtrationmustbeperformedwithfiltersthathaveaporesizenot
greaterthan0.45m(1).Thetypeoffiltermaterialischoseninsuchawaythatthebacteriaretaining
efficiencyisnotaffectedbythecomponentsofthesample(1).Commonfiltermaterialsinclude
cellulose,nylon,andPolyvinylidenefluoride(PVDF).

MembraneFiltrationExample

1.Transferasuitablequantityofthesampleprepared(preferablyrepresenting1gram[g]ofthe
product)tothemembraneandfilterimmediately.Rinsethefilterwithanappropriatediluent(1).
2.ForTotalAerobicMicrobialCounts(TAMC),transferthefiltertosoybeancaseindigestagar.
3.ForTotalYeastMicrobialCounts(TYMC),transferthefiltertosabourauddextroseagar.
4.Incubatealloftheplatesaccordingtothecompendialguidance(1).

Therearetwomethodslistedinthecompendialchaptersfortheplatecountmethods.Theyarethe
pourplatemethodandthesurfacespreadmethod.

PourPlateMethodExample:

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1.Add1milliliter(mL)ofthesamplepreparationtoduplicatepetridishes.
2.Coverthesamplewith1520mLofmoltenmedia(cooledto~45C).
3.Allowtheplatestosolidifyatroomtemperature.
4.Inverttheplatesandincubateaccordingtothecompendialguidance(1).

SpreadPlateMethodExample:

1.Addmediatosterilepetridishesandallowthemediatosolidify.
2.Addameasuredamountofnotlessthan0.1mLofthepreparedsampletoduplicatesolidified
mediaplates.Spreadthesampleoverthemediasurface.
3.Inverttheplatesandincubateaccordingtothecompendialguidance(1).

AnothermethoddescribedinthecompendialchaptersistheMPNmethod.Theprecisionand
accuracyoftheMPNmethodislessthanthatofthemembranefiltrationmethodortheplatecount
method.Unreliableresultsareobtainedparticularlyfortheenumerationofmold(1).TheMPNmethod
maybeappropriatewithproductswithverylowbioburdenifnoothermethodisavailable(1).

ExampleofMPNMethod:

1.Aseriesofatleastthreeserial10folddilutionsoftheproduct/rawmaterialareprepared.From
eachlevelofdilution,threealiquotsof1gor1mLareusedtoinoculatethreetubeswith9to10
mLofsoybeancaseindigestbroth.Neutralizersmaybeaddedtothemediaifneeded.
2.Incubatealltubesat3035Cfornomorethanthreedays.
3.Ifreadingtheresultsisdifficult,subculturetosoybeancaseindigestagarandincubateatthe
sametemperatureforonetotwodays.
4.UseTable3fromUSP<61>todeterminetheMPNofmicroorganismspergormL(1).

Whichevermethodischosen,itisimperativetoprovethesuitabilityofthetesttorecover
microorganisms.Todothisappropriately,themediautilizedmustbeproperlygrowthpromotedas
describedinthecompendialchapters(USPandUSP).Properpositiveandnegativecontrolsalong
withtiterplatesmustalsobeutilizedthroughouttheprocess.Freshmicroorganismdilutionsmustbe
used(nomorethanfivepassagesfromtheoriginalmasterseedlot)(1,10).Microorganismsmaybe
purchasedfromvendorsinareadytouseformatorpreparedasdescribedinthecompendial
chapters.

USP<62>coversthetestingofthefollowingspecifiedorganisms:

BileTolerantGramNegativebacteria
Escherichiacoli
Salmonella
Pseudomonasaeruginosa
Staphylococcusaureus
Clostridia
Candidaalbicans.

Undereachsection,USPstateshowmuchoftheproductorexcipientistobeexaminedandhowto
incubatewitheachtypeofmediawithproductinordertoisolateanyofthepotentialspecified
microorganismswithinthatproduct.Thesespecifiedmicroorganismchallengesmustbevalidatedto
recovermicrobialgrowthaswell.Thisportionofthemicrobiallimitstestisapresence/absencetest.
Dependingontheproductorexcipient,onemaychoosetovalidateanynumberofthespecified
microorganismsfromUSP<62>.

Itisimportanttostatewhichmicroorganismsarevalidatedinthespecificationsappropriately.Ifa
companysays,ComplywithcurrentUSP<62>,withoutdelineatingwhichofthespecified
microorganismswerevalidated,onecouldassumethatthecompanyvalidatedallofthespecified
microorganismsfromthatchapter.

Tosavetimeandmoneyduringtheinitialmethoddevelopmenttrials,useasubsetofmicroorganisms
totesttheproductreactions.Forexample:

Anantibioticthattargetsgramnegativebacterianeedstobetested.Theproductisreadily
solubleinwater.
TheproductisdilutedinPhosphateBufferpH7.2andthemembranefiltrationmethodischosen.
Becausetheproducttargetsgramnegativebacteria,varyingdilutionsandrinsingagentsare
utilizedwithgramnegativebacteriaforthemethoddevelopmenttrials.
Afteranacceptablemethodhasbeenidentified,thefullcompendialpanelofmicroorganismsare
performedtoassurethatalloftherequiredmicroorganismscanberecoveredaccuratelyutilizing
theemployedmethod.
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theemployedmethod.

Remember,duringmethoddevelopment,becreative!Thisistherighttimetolearntheproductor
excipient:

Howdoesthematerialreacttovaryingscenarios?
Howdoesthematerialdissolve?
Howeasily/accuratelyareinoculatedmicroorganismsrecovered?
DoesthepHneedtobeadjusted?
Whichmethod(membranefiltration,pourplate,orMPN)istherightmethod?
Whichneutralizersareneeded,ifany?
CreativeExample:Onemayneedtofilterthespecifiedmicroorganismchallengesandthenput
thefilterintothemedia.

Oncedatahasbeencollectedandareasonablemethodhasbeenidentified,gobackoverthedatato
makesurethebestmethodischosenandcanberepeatedduringthevalidationexercises.Ifa
methodisvalidatedintheUnitedStates,itshouldberepeatableinJapan.

Bythispoint,oneshouldhaveidentifiedamethod,adiluent,adilutionfactor,asamplepreparation,
anyneutralizers,howtoadjustthepH,and,possibly,thewateractivityofthematerial.

Knowingthewateractivityisnotarequirementforthevalidation,butitisausefultool.Wateractivity
isdiscussedinUSP<1112>ApplicationofWaterActivityDeterminationToNonsterilePharmaceutical
Products.Microorganismsmaystillbepresentatlevelsof<0.6aw,buttheywillnotproliferate.The
testaidsinthedecisionsrelatingtothefollowing:

Optimizingproductformulationstoimproveantimicrobialeffectivenessofpreservativesystems
Reducingthedegradationofactivepharmaceuticalingredientswithinproductformulation
susceptibletochemicalhydrolysis
Reducingthesusceptibilityofformulations(especiallyliquids,ointments,lotions,andcreams)to
microbialcontamination
Providingatoolfortherationaleforreducingthefrequencyofmicrobiallimittestingand
screeningforobjectionablemicroorganismsforproductreleaseandstabilitytestingusing
methodscontainedinthegeneralchapters<61>and<62>
Reducingwateractivitytogreatlyassistinthepreventionofmicrobialproliferation(11).

Afterthemethoddevelopmentdatahasbeenevaluated,choosethemethodinwhichthecompendial
microorganismscanberecoveredbyatleast50%ofthepositivecontrols.

Otherpointstoconsider:

Isthemethodonethatanyanalystcaneasilyperformwithouteasilycontaminatingthesample?
Aretherecoverycountsatleast50%ofthepositivecontrols?
Growthshouldnotbeinhibited(reductionbyafactorgreaterthantwo)(1).
DoesthepHneedtobeadjustedforthevalidation?Ifso,thepHwillneedtobeadjustedfor
routinetesting.

Thevalidationexercisesshouldbeperformedundermethodvalidationprotocolsthatarecontrolled
likeothercGMPdocuments.Thevalidationismoreofaverificationofthemethodsuitabilityto
recovermicroorganismsfromthetestingmaterial(2).Typicalanalyticalvalidationsconsiderthe
following:

Accuracy
Precision
Repeatability
IntermediatePrecision
Specificity
DetectionLimit
QuantitationLimit
Linearity
Range.

Alternativemicrobiologicalprocedures,includingautomatedmethods,maybeusedaslongasthese
methodshavebeendemonstratedtobeequivalenttothePharmacopeiamethod(1).Onemayneed

toperformsidebysidecomparisonswiththetraditionalmethodsandalternativemethodtoshow
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toperformsidebysidecomparisonswiththetraditionalmethodsandalternativemethodtoshow
equivalencytothecompendialmethods.

Preparethesampletobetestedaccordingtotheprotocol(whichshouldmatchthemethod
developmentpreparation).Unlessotherwisedirected(e.g.,inamonograph),use10gor10mLofthe
producttobeexaminedtopreparethesamplepreparation(1).

Theamounttobetestedmaybereducedforactivesubstancesif:

Theamountperdosage(tablet,capsule,etc.)islessthanorequalto1mg.
TheamountpergormL(forpreparationsnotpresentedindoseunits)islessthan1mg.

Therefore,theamountofsampletobetestedisnotlessthantheamountpresentin10dosageunits
or10g/10mLoftheproduct

Formaterialsusedasactivesubstanceswherethesamplequantityislimitedorbatchsizeis
extremelysmall(lessthan1000mLor1000g),theamounttestedshallbe1%ofthebatchunlessa
smalleramountisprescribedorjustifiedandauthorized.Forproductswherethetotalnumberof
entitiesinabatchislessthan200,thesamplesizemaybereducetotwounitsoroneunitifthe
samplesizeislessthan100..Samplesshouldbeatrandomfromthebulkmaterialorfromavailable
containersofthepreparation.Toobtaintherequiredquantity,mixthecontentsofasufficientnumber
ofcontainerstoprovidethesample(1).

Methodvalidationistypicallyperformedonthreelotsofmaterialtodemonstratetherobustnessofthe
method.Thefirststepofthevalidationisthetotalaerobic/yeastandmoldcountportionofthe
validation(1).Inoculateseparateportionsofthesamplepreparationthatareequivalentto1gor1mL
ofthematerialbeingexamined(1).Theinoculumsaremadewithlessthan100cfuofthe
microorganismsspecifiedinUSP<61>.Theinoculumsmustnotexceed1%ofthevolumeofthe
dilutedproduct(1).

Ifthepourplatemethodisused,inoculatethesamplesothat1mLofthedilutionwillcontain<100
cfuofthechallengedmicroorganism.Otherwise,theplatedsamplemaynotcontaingrowth.

Forthemembranefiltrationmethod,transferthefiltersontotrypticsoyagar(TSA)platesor
sabourauddextroseagar(SDA)platesafterfiltering.IncubatetheTSAplatesfornomorethanthree
daysat3035C.Thisisbelowtheincubationrangeofthreetofivedaysthereby,thisprovesthat
recoverycanbeobtainedbytheminimumofthreedays.Uninoculatedproductplatesshouldbe
incubatedforthethreetofivedayincubationrange(1).

IncubatetheSDAplatesfornotmorethanfivedaysat2025C.Thisisbelowtheincubationrangeof
fivetosevendays,provingthatrecoverycanbeobtainedbytheminimumoffivedays.Uninoculated
productplatesshouldbeincubatedforthethreetofivedayincubationrange(1).

Afterincubation,calculatethenumberofcfupergorpermLofthetestedmaterial.

Fortheplatecountmethod,preparesampleasstatedintheprotocol.Inoculateduplicatesterilepetri
disheswiththeinoculatedproductsample.Coverthedisheswithmedia(orspreadthesampleonto
themediaforthespreadplatemethod).IncubatetheTSAplatesfornomorethanthreedaysat30
35C.Thisisbelowtheincubationrangeofthreetofivedays,provingthatrecoverycanbeobtained
bytheminimumofthreedays.Uninoculatedproductplatesshouldbeincubatedforthethreetofive
dayincubationrange.

IncubatetheSDAplatesfornomorethanfivedaysat2025C.Thisisbelowtheincubationrangeof
fivetosevendays,thusprovingthatrecoverycanbeobtainedbytheminimumoffivedays.Un
inoculatedproductplatesshouldbeincubatedforthethreetofivedayincubationrange.

Selecttheplatescorrespondingtoagivendilutionandshowthehighestnumberofcolonieslessthan
250forTAMCand50forTYMC(1).Takethemeanperculturemediumofthecountsandcalculate
thenumberofcfupergorpermLofproduct(1).

FortheMPNmethod,incubatealltubesfornotmorethanthreedaysat3035C.Subculture,if
necessary,usingtheprocedureshowntobesuitable.Foreachlevelofdilution,recordthenumberof
tubesshowingmicrobialgrowth.DeterminetheMPNofmicroorganismspergormLoftheproductto
beexaminedfromTable3inUSP<61>.

Thespecifiedmicroorganismportionofthevalidationissecondstepofthevalidation(9).Inoculate
separateportionsofthesamplepreparationthatareequivalentto1gor1mLofthematerialbeing
examined(thismaybea1in10dilution)(10)..Theinoculumsaremadewithlessthan100cfuofthe
specifiedmicroorganismsbeingexaminedinUSP<62>.

Incubateeachstepofthespecifiedmicroorganismchallengesusingthelessertimeoftheincubation
range.Forexample,iftheincubationrangeis1824hours,removethevalidationsampleat18
hours.

ThefirststepinUSP<62>isdilutionoftheproductinanenrichmentmediatoencouragelowlevels
ofmicrobialgrowth(2).Thesecondstepistosubculturethemicrobialgrowthsuspensionson
selectiveagartodepressgeneralmicrobialgrowthandallowforthespecifiedmicroorganismsto
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selectiveagartodepressgeneralmicrobialgrowthandallowforthespecifiedmicroorganismsto
grow(2).Thedifferentialmediawasdesignedtodistinguishthecolonymorphologyofthespecified
microorganismstherebyallowingvisualidentification(2).

Formanycompanies,thequestionofhowtotreatclinicalproductsversescommercialproducts
arisesinmeetingrooms.Forcommercialproducts,mostcompaniesagreethattheindustrypractice
istovalidatethreelotsataminimumformethodvalidations.

Whataboutclinicalproducts?Clinicalproductsareadifferentstory.Itisdifficultsometimestohave
threelotsofproductinordertoperformacompletevalidation.Smallamountsofproductareoften
madeforuseinclinics.Formulationschangefrequentlydependingonfieldstudiesanddevelopments
discoveredthroughouttheearlyphasesofaproject.Itisacceptabletoperformmethodvalidationon
onelotforclinicalproducts.Chancesarethecompanywillberedevelopingandrevalidatingwitheach
formulationchangeoftheproduct.Itmaytakeyearsforaproducttoreachcommerciallaunch.

Keepaneyeontheproduct,however.Themomenttheproductmovestocommercialmanufacturing,
athreelotvalidationneedstooccur.

Validationacceptancecriteriaisessentialwhendeterminingifamethodwasproperlyvalidated.
WhenverifyingthesuitabilityofthemembranefiltrationorthePlateCountMethod,ameancountof
anyofthetestorganismsnotdifferingbyafactorgreaterthan2fromthevalueofthecontrolinthe
absenceofproductmustbeobtained(1).Inotherwords,thepercentrecoverybetweenthe
inoculatedproductdilutionsandthepositivecontrolsmustbeatleast50%.Somecompaniesusean
internal70%recoverycriterionthatisacceptablebecauseitismorestringent.

ThecompendialchaptersgiveadditionaldetailtothevalidationcriteriafortheMPNmethod.When
verifyingthesuitabilityoftheMPNMethod,thecalculatedvaluefromtheinoculumsmustbewithin
95%confidencelimitsoftheresultsobtainedwiththecontrol(1).Ifthecriterioncannotbemetfor
oneormoreoftheorganismstestedwithanyofthedescribedmethods,themethodandtest
conditionsthatcomeclosesttothecriteriaareusedtotesttheproduct(1).

Specifiedmicroorganismsmustberecoveredduringthevalidationfortheassaytobevalid.Thetiter
platesmustdemonstratenotmorethan100cfuwasutilizedtoachievethepositiveresults(10).

Aftervalidationsarecomplete,routinetestingmaybegin.Followingvalidationactivities,reportsare
usuallywrittentoapprovethestudiesandmethods(orstandardoperatingprocedures[SOPs])are
writtentolockdownthewaythetestsareroutinelyperformed.

RoutinetestingfortheHMLTwillcontaintheTAMC,theTYMC,andanyspecifiedmicroorganism
challenges.Allrepresentativecoloniesofgrowthobtainedfromanyportionofthetestneedstobe
identifiedtothespecieslevelifpossible.Themicroorganismsneedtoberesearchedanddetermined
iftheyareobjectionablemicroorganisms.

Thereisaregulatoryexpectationthatrecoveredmicroorganismsareidentifiedfromnonsterile
products.Therehavebeenwarninglettersissuedtocompaniesfailingtocomplywiththis
expectation.Productrecallshavealsooccurred.

Inordertodetermineifamicroorganismisobjectionable,USP<1111>providesinformationtoaidein
theriskassessment.Themicroorganismssignificanceshouldbeevaluatedbyresearchingthe
following:

Numberofmicroorganisms(1cfuor1X106cfu)
Microorganismscharacteristics
Useoftheproduct/routeofadministration
Thenatureoftheproduct:Doesitsupportgrowth?Doesithaveantimicrobialpreservatives?
Whatisthewateractivity?
Themethodofapplication
Theintendedrecipient(e.g.,elderly,infants,etc.)
Potentialimpacttopatients
Useofimmunosuppressiveagents
Thepresenceofdisease,wounds,ororgandamage
Doesthemicroorganismhaveahistoryofcausinginfections?

TherearemanysourcestoresearchmicroorganismsontheInternet.Agoodsourceofreferencefor
researchingpotentialobjectionablemicroorganismsistheFDABadBugBook.Thereisnotanall
inclusivelistofobjectionablemicroorganisms.Microorganismsmustbeidentifiedandevaluatedto
determinethepotentialimpacttopatienthealth.

Todeterminethefrequencyoftesting,onecanutilizeariskbasedapproach.Liketheobjectionable
microorganisms,thisisgoingtobeaproductbyproductriskassessment.

ICHQA6,HarmonisedTripartiteGuideline:Specifications:TestProceduresandAcceptanceCriteria
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ICHQA6,HarmonisedTripartiteGuideline:Specifications:TestProceduresandAcceptanceCriteria
forNewDrugSubstancesandNewDrugProducts:ChemicalSubstancesprovidessomeguidance
ondeterminingtheappropriatetestingintervals:

Ingeneral,itisadvisabletotestthedrugproductunlessitscomponentsaretestedbefore
manufactureandthemanufacturingprocessisknown,throughvalidationstudies,nottocarrya
significantriskofmicrobialcontaminationorproliferation(3).

Withacceptablescientificjustification,itmaybepossibletoproposenomicrobiallimittestingforsolid
oraldosageformsorskiplottesting.

ICHQ6ADecisionTreeNumber6:MicrobiologicalQualityAttributesofDrugSubstancesand
Excipientswalksthroughaseriesofquestionsaboutthedrugsubstanceandexcipientstoaidinthe
riskbaseddecisionsaboutthefrequencyofroutinetesting.

ICHQ6ADecisionTreeNumber8:MicrobiologicalAttributesofNonSterileDrugProductsisanother
decisiontree.Itwalksthroughaseriesofquestionsaboutthedrugproducttoaideintheriskbased
decisionsaboutthefrequencyofroutinetesting.

Stabilitytestingisperformedundervarioustemperaturesandhumiditysetpoints.Microbiallimits
testingperformedforstabilitytestingistypicallyperformedatinitial,sixmonths,12months,24
months,etc.Whendesigningtheprogram,takeintoaccounttheproductformulations,strengths,and
packagingconfigurations.Itmaybeappropriatetoleveragebracketingormatrixingstabilitytesting
designsasallowedunderICHQ1D.Bracketingormatrixingcangreatlyreducethetestsamples
neededforthestabilityprogram.

Conclusion
Learnaboutthematerialbeforebeginning.Setappropriatespecificationssothatthemethodcanbe
appropriatelytargeted.Forinternationalcompliance,usethemoststringentinternationalmethodora
mixtureofthemethods.

Whendevelopingmethods,becreative!Learnasmuchaspossiblefromdevelopmentscientists,
chemists,research,andprojectteamstoaideinthemethoddevelopmenttrials.Utilizethe
informationtotargetthemethoddevelopmenttrialsinordertosavetimeandmoney.Onceasuitable
methodhasbeenidentified,performatrialasonewoulddoduringthevalidationwithallofthe
microorganismstodemonstratethemethodwillworkproperly.Performthesetrialsinanotebookand
keepexcellentnotesaboutthetrials.BesuretotestthepHandchoosethebestsuitablemethodfor
theapplication.Dontunderestimatethevalueofknowingthewateractivity.

Duringvalidation,writeamethodvalidationprotocolutilizingthemethoddevelopedinthetrialruns.
Becertainthatthemethodisperformedthesamewayeachtimeandthatitisrepeatable.Forclinical
products,aonelotvalidationissufficientduetovariousclinicalconstraints.Forcommercialproducts,
aminimumofathreelotvalidationshouldbeperformed.

Createacleanvalidationpackageforregulatoryaudits.Duringroutinetesting,lockdownthemethod
sothatitisperformedthesamewayeverytime.Bracketingormatrixingcanbeutilizedindecreasing
thevolumeofstabilitytesting(12).

Riskbasedapproachesshouldbeutilizedininvestigatingobjectionablemicroorganismsandin
determiningthetestingfrequencies.Cleardocumentationandjustificationsshouldbemaintainedso
thatdecisionscanbeclearlyunderstoodinthefuture.

References

1.USPChapter<61>,MicrobiologicalExaminationofNonsterileProducts:MicrobialEnumeration
Tests.
2.S.Sutton"DoesInternationalHarmonizationoftheUSPMicrobialLimitsTestsRequireRe
ValidationofFinishedProductTests?",JournalofValidationTechnology16(3),2009.
3.ICHQ6A,Specifications:TestProceduresforNewDrugSubstancesandNewDrugProducts:
ChemicalSubstances.
4.USPChapter<1111>,MicrobiologicalExaminationofNonsterileProducts:AcceptanceCriteria
forPharmaceuticalPreparationsandSubstancesforPharmaceuticalUse.
5.CodeofFederalRegulations,Title21,FoodandDrugs(GovernmentPrintingOffice,Washington,
DC),Part211.84(d).
6.CodeofFederalRegulations,Title21,FoodandDrugs(GovernmentPrintingOffice,Washington,
DC),Part211.113(a).
7.CodeofFederalRegulations,Title21,FoodandDrugs(GovernmentPrintingOffice,Washington,
DC),Part211.165(b).
8.ICHGuidelineQ6A,DecisionTrees6and8.

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9.USPChapter<62>,MicrobiologicalExaminationofNonsterileProducts:TestsforSpecified
Microorganisms.
10.USPChapter<1112>,ApplicationofWaterActivityDeterminationToNonsterilePharmaceutical
Products.
11.ICHQ2(R1),ValidationofAnalyticalProcedures:TextandMethodology.
12.ICHQ1D,BracketingandMatrixingDesignsforStabilityTestingofNewDrugSubstancesand
Products.

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