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JVT
EstablishingaCompleteSetofTarget,Alert,andActionLimitsfor
MicrobialCountsinPurifiedWater|IVT

ByPramoteCholayudthMar8,20131:06pmPST

INTRODUCTION

Purifiedwater(PW)isprobablytheonlyrawmaterialessentially
producedforinternalusebypharmaceuticalandother
healthcarecompanies.ThemicrobiologicalattributeinPWis
hardtocontrolwhencomparedtocontrolusinganonline
conductivitymeterandtotalorganiccarbon(TOC)analyzer.
Althoughrelatedguidelines,includingWorldHealthOrganization
(WHO)andUnitedStatesPharmacopeia(USP),describing
microbiallimitsforPWareavailable,establishinginhouselimits
ispracticallyrequiredtoeffectivelycontrolthemicrobialcontamination.Thispaperisissuedin
additiontotwopreviousJournalofValidationTechnologypapers(EstablishingAlertLimitsfor
MicrobialCountsinPurifiedWaterandApplicationofPoissonDistributioninEstablishingControl
LimitsforDiscreteQualityAttributes)(1,2).Thispaperisintendedtointroducethesamestatistical
method(asdiscussedinthetwoarticles)usingtwodifferentgroupsofvalidationdatatocompute
thetarget,alert,andactionlimits.Readingandunderstandingthetwopreviousarticlesisstrongly
recommended.

AccordingtotheUSFoodandDrugAdministrationGuidetoInspectionsofHighPurityWater
Systems,Thus,inestablishingthelevelofcontaminationallowedinahighpuritywatersystem
usedinthemanufactureofanonsterileproductrequiresanunderstandingoftheuseofthe
product,theformulation(preservativesystem)andmanufacturingprocess.Forexample,antacids,
whichdonothaveaneffectivepreservativesystem,requireanactionlimitbelowthe100cfu/mL
maximum.(3).Therefore,establishingsuchactionlimitswillfulfillthisFDAguideline.

SPECIFICATIONS

PurifiedwaterUSP<1231>hasaspecificationasfollows(4):

QualityAttributes SpecificationLimits

Conductivity 1.3mS/cm(25C)

TotalOrganicCarbon(TOC) 500ppb

MicrobialCount 100cfu/mL

WHOsuggestedmicrobiallimitsatvariousstagesinwaterpurificationsystemareasbelow(5):

MaximumLimits(cfu/mL)SuggestedbyWHO
SamplingPoints
Target Alert Action

Source(Raw)Water 200 300 500

PostCarbonFilter 50 300 500

PreROUnit 20 200 500

PrePolishingUnit* 10 50 100
 PrePolishingUnit* 10 50 100

PurifiedWater 1 10 100

*Maybeelectrodeionization(EDI)unitormixedbedcolumn

BACKGROUND

InaThaigovernmentsWHOgoodmanufacturingpractice(GMP)pharmaceuticalplant,aproject
plannedtostartupinthefirstquarterof2013theauthortakestheresponsibilityforthevalidation
projectsupervision.Purifiedwatersystemisoneofthecriticalsupportingsystemsthatrequire
validation.Designqualification(DQ),installationqualification(IQ),operationalqualification(OQ),
andperformancequalification(PQ)areperformedonthesystem(reverseosmosis[RO]/EDIwith
36pointsofuse)asusual.AccordingtoWHOTechnicalReportSeries(TRS)#970(Annex2)
requirements,validationphaseone,two,andthree(PQphase)willneedtobecarriedoutover
differentsuggestedperiodsoftime(i.e.,twoweekseachforphaseoneandtwoand12monthsfor
phasethree)(6).Theactualtimeforeachofthefirsttwovalidationphases(oneandtwo)isthree
weeks,whichisdeterminedtoprovideasufficientdataofthemicrobialcount.

ESTABLISHINGTARGET,ALERT,ANDACTIONLIMITSUSINGMICROBIALCOUNTDATA
FROMVALIDATIONPHASEONEANDTWO

Thealertandactionlimitsareestablishedfollowingtheprocedures,adoptedfromthesecond
previousarticle,asbelow(2):

Collectmicrobialcountdataforeachtypeofwaterinthesystemoverthesixweektime(i.e.,
amountofdata=sixweeksxatleastfive*days/weekxonewatersample/day=30observed
microbialcountdataperpointofuse:*sevendaysinthisproject).
Computefortheaveragedcount(c).Thisaveragevaluewillbedeterminedasthetargetlimit
foreachtypeofwaterdata.Notethatanindividualresultthatisrecordedas<1forzeroresult
isregardedasonewhencomputedfortheaveragec.
Computefortheupperalertlimitusingc+4 principle.Incaseofactionlimit,thedataequal
toandgreaterthanthecomputedvalueofupperalertlimitarecomputedforaveragevalue
priortocomputingtheupperactionlimit(lowerlimitisnotnecessaryinthiscase).
Justifytheestablishedlimitsbeforeusebyplottingthedatainacchartandadjustthelimits
suchthatthedataareproperlydistributedandfittedwithintheadjustedalertoractionlimits
(Note:acchartisakindofcontrolchartdedicatedtoPoissondistributionwhereinthemean
andstandarddeviationofthedistributionareestimatedbytheaveragedcount(c)andits
squareroot( )respectively,andthecontrolchartlimitsingeneralarec+3 .

AllthemicrobialdataandrelevantcalculatedlimitvaluesaretabulatedinFigure1.Theaverageof
eachwaterdataisusedforconstructionofPoissondistribution(asillustratedinFigures2and3)in
whichonecanimaginehowthemicrobialcountdataforeachtypeofwaterdistribute.Figure4
illustratessummarizationoftarget,alert,andactionlimitsinvolved.Figures5through10illustrate
howthecomputedalertandactionlimitsforwaterinthesystemarejustified.

Figure1:MicrobialCountData(Short).

MicrobialCountData(cfu/mL)

Val.
Day# PurifiedWater/POU#
Phases
Feed ACF Soft RO EDI
Water Water Water Water Water
1 235 36

Val. 1 <1 <1 32 13 <1 <1 <1<1 <1

Phase1
(3
weeks) 2 <1 <1 35 3 <1 <1 <1<1 <1

3 6 <1 73 21 <1 <1 <1<1 <1

420 1...10 12...66 8...26 4...1 <18 <1<1 . <1<1

21 69 28 2 <1 <1 <1 201<1 <1

 21 69 28 2 <1 <1 <1 201<1 <1

22 75 16 7 <1 1 <1 <1<1 <1

23 80 <1 19 <1 <1 <1 <1<1 <1

Val.
Phase2
24 64 4 11 2 4 <1 <1<1 <1
(3
weeks)
25...41 44...<1 3...2 <1...14 <1<1 <14 <1<1 <1<1

42 <1 9 <1 <1 4 41 <1<1 <1

24.2 17.4 11.0 5.1 3.0 1.2

Average(c)

TargetLimit
24 17 11 5 3 1
(Rounded)

43.9 34.1 24.3 14.2 9.9 5.5

UL(c+4 )

ComputedAlert
44 34 24 14 10 6
Limits(Rounded)

Average2(c) 95.3 57.9 39.2 31.0 20.5 15.6

134.3 88.3 64.2 53.3 38.6 31.4

UL2(c+4 )

ComputedAction
134 88 64 53 39 31
Limits(Rounded)

Note:Zeroresultisdenotedas<1andtransformedto1whencomputedforaveragevalue.

POU:pointofuse(36points),ACF:activatedcarbonfilter,RO:reverseosmosis,EDI:
electrodeionization.

1Itishardtoknowwhethertheseindividualmicrobiologicaldataareoutliersornot.Ifoutliers
reallyoccurred,suchafewpercentageoftheoccurrence(onein1,512inthisproject:42
daysx36data/day=1,512data)willhaveverylittlechangeontheaveragevalueand,
subsequently,thealertoractionlimits.Furthermore,whetherornotanoutlierispresent,
priortousejustificationofthelimitsisrequiredattheend.

Obtainedfromdataequaltoandgreaterthanthosecomputedalertlimits.

Figure2:DeterminingTargetandAlertLimitsUsingPhaseOneandTwoData.
Figure3:DeterminingActionLimitsUsingPhase1and2Data.

Figure4:MicrobialCountLimitsEstablishedBasedonValidationPhaseOneandTwoData.

MaximumLimits(cfu/mL)

Sampling Specification
AlertLimits ActionLimits
Points Limits
Targets
Comp Curve Adj Comp Curve Adj

FeedWater 24 44 39 50 134 126 130 500*

ACFWater 17 34 31 35 88 84 80

SoftWater 11 24 22 25 64 62 60

ROWater 5 14 14 15 53 52 55

EDIWater 3 10 10 10 39 38 35

PurifiedWater 1 6 6 10 31 31 30 100**

c:Averageofmicrobialcountdata,cchartisbasedonPoissondistribution

Comp:computedlimits,Curve:limitsdeterminedfromcurve,Adj:adjustedlimits,thecolumns
 Comp:computedlimits,Curve:limitsdeterminedfromcurve,Adj:adjustedlimits,thecolumns
ingraycoloraretheestablishedtarget,alert,andactionlimits.

*WHOdrinkingwaterstandardandalsoreferredbyUSPasmicrobialqualityofsource
drinkingwaterforpurifiedwatersystem.**USPpurifiedwaterstandard.

Figure5:JustificationofMicrobialAlertandActionLimitsforPurifiedWater.

Figure6:JustificationofMicrobialAlertandActionLimitsforEDIWater.

Figure7:JustificationofMicrobialAlertandActionLimitsforROWater.

Figure8:JustificationofMicrobialAlertandActionLimitsforSoftWater.
Figure8:JustificationofMicrobialAlertandActionLimitsforSoftWater.

Figure9:JustificationofMicrobialAlertandActionLimitsforACFWater.

Figure10:JustificationofMicrobialAlertandActionLimitsforFeedWater.

DISCUSSION

Thepreliminary(computed)alertandactionlimitsrequireestablishmentfromphase1datausing
thesamemethodasdiscussedabove,sotheresultinglimitscanbeusedforcontrollingphasetwo
data.Thentheaccumulateddatauptothelastdayofphasetwoisrecomputedforthetarget(if
required),alert,andactionlimitsforuseinphasethree.Figures11and12illustratethetarget,
alert,andactionlimitsestablishedfromphaseonedataand,afterjustification,usedascontrollimits
forphasetwodata.
Onewillseethatthelimitsconstructedbefore(Figures2and3)andafter(Figures11and12)
Onewillseethatthelimitsconstructedbefore(Figures2and3)andafter(Figures11and12)
phasetwoareslightlydifferent,exceptforthefeedwaterdatalimit.Thisdemonstratesthateachof
PWsystemcomponentsrelevanttomicrobialcontrolisrobustintheirfunction.However,phaseone
andtwoisjustatemporaryperiod,thetruesystemrobustnessistobedemonstratedinphase
threeover12months,whichtakesintoaccounttheworstqualityoffeedwaterduetotheannual
seasonalchanges.

Usingtheaverage+fourstandarddeviationprinciple,upperlimitisbasedonthecriteria
proposedtocompensatethemicrobiologicalmethoderror(1).Nomatterhowthelimits(alertor
action)areestablished(eithercomputationmethodordistributioncurvemethoddescribedbelow),
justificationofthemisessentiallyrequiredthrough,forexample,determiningtheplotteddatatosee
iftheyproperlyfitwithinthelimits.Ifnot,adjust(i.e.,shift)thelimitlineabittoahigherorlower
level(summarizedinFigure4)untilsatisfactoryand,mostimportantly,untilacceptabletothe
qualityassuranceunit.TheadjustedlimitsillustratedinFigures5through10areintendedfor
demonstrationexampleonly:adjustmentwithworstcaseconditionincludedmaybemadefora
morepreventivepurposeofthelimits.Asdiscussedinpreviously,theprincipleofaveragefour
standarddeviationisthespecialcaseformicrobiologicaldataitshouldnotbeappliedtonon
microbiologicaldata(1).

Justificationofusingthedataequaltoandgreaterthanthealertlimitvalueforcomputingtheaction
limitisalsorequired.Thisparticulargroupofdatawillspreadabovetheupperalertlimit.
JustificationismadethroughtheassumptionthatthereisasecondPoissondistributionabovethe
alertlimit.Subsequently,withtheruleofPoissondistribution,theuppertailwillindicatetheaction
limitlocation.

IfonecanconstructaPoissondistributioncurve,usetheMicrosoftExcelfunctionasfollows:

Probability(onyaxis)=Poisson(A,B,False),whereA=0,1,2,3,..(onxaxis),andB=
averagedmicrobialcountvalue.

Thenthelimit(roughly)canalsobedeterminedvisuallyfromthecurveandusedasanalertor
actionlimitdependingonthefullorselecteddataused(alertoractionlimitrespectively)and,
subsequently,followjustificationprocedurepriortouse.

Figure11:DeterminingTargetandAlertLimitsUsingPhase1Data.

Figure12:DeterminingActionLimitsUsingPhase1Data.
CONCLUSION

Inestablishingamicrobialcontrollimit,oneshouldrealizeandunderstandthetypeofdataandits
distribution.Microbialcountisattributedata(discretedata)whereinitsdistributionisbinomialand
Poisson.Realizationofthemicrobialdataasattributeratherthanvariable(normaldistribution)data
willhelpselecttherighttractforcalculationofthecontrollimitsinthepast,normaldistributionof
thedatawasassumed.CalculationofthelimitsusingPoissondistributionparameters(mean=c,
standarddeviation= )issimpleandtheiruseisdiscussedaboveinthispaper.Microbialalert
andactionlimitsshouldbeestablishedforcontrolofpurifiedandotherwatersystems.Targetlimit
willreflectthecentraltendencyofthetotaldata.Combineduseoftheselimitswillprovidean
effectiveandpreventivecontroloftheentiresystem.However,agoodunderstandingofthewater
systemoperationandcontrolisalsorequired.

ACRONYMS

TOC TotalOrganicCarbon
MS Microsoft
RO ReverseOsomosis
EDI Electrodeionization
DQ DesignQualification
IQ InstallationQualification
OQ OperationalQualification
PQ PerformanceQualification
GMP GoodManufacturingPractice
WHO WorldHealthOrganization

REFERENCES

1.P.Cholayudth,"EstablishingAlertLimitsforMicrobialCountsinPurifiedWater,"Journalof
ValidationTechnology13(1),2006.
2.P.Cholayudth,"ApplicationofPoissonDistributioninEstablishingControlLimitsforDiscrete
QualityAttributes,"JournalofValidationTechnology13(3),2007.
3.U.S.FoodandDrugAdministration,GuidetoInspectionsofHighPurityWaterSystems(7/93)
4.USP<1231>WaterforPharmaceuticalPurposes.
5.WHO,SupplementaryTrainingModulesonGoodManufacturingPractice,PresentationFileon
WaterforPharmaceuticalUsePart2:WaterPurificationEngineering.
6.WHO,TechnicalReportSeries#970,FortySixthReport,Annex2:WHOGoodManufacturing
Practices:WaterforPharmaceuticalUse,WorldHealthOrganization,October2011
7.D.H.Besterfield,QualityControl,6thEd.,PrenticeHall,2001.
8.H.M.Wadsworth,K.S.Stephens,A.B.andGodfrey,ModernMethodsforQualityControland
Improvement,2ndEd.,JohnWiley&Sons,2002.

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Tags:GMPMicrobiology,JVT