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ValidationofSterilizingGradeFilters
Presentedby
LauraOkhio-Seaman
SartoriusCorporation
2
Thedefinitionofasterilizinggradefilterisonethatwillproduceasterile
effluentafterbeingchallengedbymicroorganismsatachallenge levelof
greaterthanorequalto1x10
7
/cm
2
ofeffectivefiltrationarea.
SterilizingGradeFilters
3
PhysicalTests
Flowrates,differential
pressure,throughput
Sterilizability(SIP,Auto)
Integritytests(bubblepoint,
diffusiveflow)
BiologicalTests
Viability
Bacterialchallengetest
Bioburdenstudies
CompendialTests(USP,EP)
Particlerelease
Oxidizablesubstances
Biosafety
Endotoxin
OtherTests
Non-volatileresidue
Quantitativeandqualitative
extractables analysesin
waterandethanol
Leachables testing
FilterQualificationTests
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Demonstratesthefilterretainsmicroorganismsto
produceasterilefiltrate
Ensuresthefilterdoesnotaltertheproductinan
objectionableway
Ensurestheproductdoesnotadverselyaffectthe
filter compatibility
Ensuresthephysicalparametersoftheprocessdo
notadverselyaffectthefilterortheproduct
ValidationofSterilizingGradeFilters
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FilterQualificationTests
Viability
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FilterQualificationTests
Brevundimonas diminuta (ATCC 19146)
Brevundimonas diminuta (ATCC 19146)
Untilthelate1960s,0.45m-ratedmembranes
wereconsideredsterilizinggradefilters,andwere
usedsuccessfullyinthesterilizingfiltrationofparenterals.
Inthemid-1960sDr.FrancesBowmanobserveda0.45m
sterile-filteredculturemediumtobecontaminatedwitha
micro-organism,subsequentlyshowntopenetrate0.45m-rated
membranesrepeatedlyinsmallnumbers.
Untilthelate1960s,0.45m-ratedmembranes
wereconsideredsterilizinggradefilters,andwere
usedsuccessfullyinthesterilizingfiltrationofparenterals.
Inthemid-1960sDr.FrancesBowmanobserveda0.45m
sterile-filteredculturemediumtobecontaminatedwitha
micro-organism,subsequentlyshowntopenetrate0.45m-rated
membranesrepeatedlyinsmallnumbers.
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ViabilityTest
Evaluationofpotentialbactericidaleffectsoftheproduct
solution
Viabilityverifiedbydirectinoculationintoproductand
makingserialdilutions
Testexposuretimeshouldequalorexceedactualprocess
filtrationtime
Iflessthanaone-logreduction(LRV)isobserved,product
considerednon-bactericidal
Greaterthanone-logreductionindicatesproductis
bactericidalandalternativesshouldbeconsidered
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ViabilityTestExamples
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FilterQualificationTests
BacterialChallenge
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FilterQualificationbySupplier
Bacteria/WaterSuspension
Challenge
Stand.Cond.
Document
Bacteria/ProductSuspension
Process-relatedFilterValidationbyUser/ExternalLab
Challenge
ProcessCond.
Document
BacterialChallengeTest
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Factorspotentiallyaffectingmicrobial
retentioninclude
FilterConstruction(structure,membrane
polymer,poresizedistribution)
Formulationcomponents
Formulationproperties(pH,viscosity)
Processconditions(time,temperature,pressure
differential,flow-rate)
BacterialChallengeTest
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Microbial retention studies onfilter devices:
Brevundimonas diminuta (ATCC19146)hasbeenproven
topenetratea0.45mratedfilter
Spiking ofthedrug product withBrevundimonas
diminuta according toASTM838-05
Challengelevel >10
7
CFUs/cm
2
filtrationarea.
ValidationTestingshouldsimulateworst-case process
conditionse.g.,pressuredifferential,flow-rate,time,
temperature
BacterialChallengeTest
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Challengesuspensionshouldbemono-dispersedtoprovide
worst-casechallenge
Opticalmicroscopyisusedtodetectaggregationand
clumpingofbacteria
Ultrasonictreatmentreducesaggregation
Penetrationof0.45mratedmembranesisindicativeof
single-cellconditionsandthismembraneisusedasa
positivecontrol
BacterialChallengeTest
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Microscopicinvestigation
Criteria:singlecells,motilecells
0.3-
0.4m
0.6-1.0m
worst-case
BacterialChallengeTest
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ASTM838-05(2005)usesB.diminuta asthestandard
challengeorganism
2004AsepticProcessingGuidancesuggeststheuseofnative
bioburdenisolatewhenappropriate
BacterialChallengeTest
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MicrobiologicalchallengetestsusingonelotoflowBubblePoint
(closetomanufacturersminimumspecification)membranesshould
beused.
*
Example:
MinimumBubblePointValue: 46psi
Releasecriteriaoffiltervendor: 50psi
LowBPRange: 46 50.6psi
*Atornearfiltermanufacturersminimumintegritytestvalue(10%),
1999PDA/FDAJointconference,PDANewsletterDecember1999
BacterialChallengeTest
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Thepurposeofthetestistovalidatetheretentionefficacy
ofaparticularmembranematerial.Asmall47mm
membranedisccanbeused
Tomorecloselysimulateprocessconditionsandto
determinetheintegrityofprocessfilters,asmallarea150
or300cm
2
filterdevicecapsulecanbeused
Fullsized10filtercartridgesrequirelargevolumesof
product(25-30liters)toperformthechallengetesting
FilterMediumvs.Device
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Either0.45or0.2micron-ratedfiltermembranecanbe
usedforrecoverymembranes
Studiesindicatethat0.45micron-ratedmembranesmight
bemoreefficientthan0.2forthispurpose
CelluloseNitrateisthepreferredmembranepolymer
highaffinityforproteins
RecoveryMembraneSelection
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Maximumprocesspressuredifferentials(P)acrossthefilter
shouldbeusedinthechallengestudies
Processflow-rateshouldbeachievedinchallengestudies
Ifpressureandflowratecannotbesimultaneouslyachieved,
theusershoulddeterminewhichismorerelevantanddevelop
arationaletosupportthedecision
Regulatorstendtopreferhigherpressuredataratherthan
flow-rate
PressureDifferentialandFlowRate
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test solution /
Brevundimonas diminuta
suspension
test filter
capsules
analytical
disc filters
peristaltic pump
syringe /
sample collection
venting valve
venting valve
valves
pressure gauge
ManualBCTSet-up
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BCTSet-upforSmallScalePleatedFilters
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BigBertharig
SartoriusAG
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PossibleBCTStrategies
Is
B.diminuta
viableinproductunder
processconditions
?
Determine&DocumentviabilityofB.diminuta
inProductunderProcessConditions
DirectinoculationofB.diminuta
inproductunderprocessconditions
Modifyformulation
(adjustpH,removebactericidal
componentorproductsurrogate)
Useproductfortimeperiodthatthe
challengeorganismisviable
Modifyprocess
(adjusttemperatures,etc.)
ChangefromB.diminuta
Usebacteriaisolatedfrom
formulationorenvironment
VIABLE
NOTVIABLE
PreconditionfilterwithProduct
followedbyMicrobialChallenge
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Is
B.diminuta
viableinproductunder
processconditions
BCTStrategies Method1 (DirectInoculation)
Determine&DocumentviabilityofB.diminuta
inProductunderProcessConditions
DirectinoculationofB.diminuta
inproductunderprocessconditions
VIABLE
DirectinoculationofB.diminuta
inproductunderprocessconditions
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BCTbydirectinoculation
BCTStrategies Method1 (DirectInoculation)
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Is
B.diminuta
viableinproductunder
processconditions
?
Determine&DocumentviabilityofB.diminuta
inProductunderProcessConditions
DirectinoculationofB.diminuta
inproductunderprocessconditions
Modifyformulation
(adjustpH,removebactericidal
componentorproductsurrogate)
Useproductfortimeperiodthatthe
challengeorganismisviable
Modifyprocess
(adjusttemperatures,etc.)
ChangefromB.diminuta
Usebacteriaisolatedfrom
formulationorenvironment
VIABLE
NOTVIABLE
PreconditionfilterwithProduct
followedbyMicrobialChallenge
BCTMethod2 (ProcessModification)
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Temperatureof 40Csupportsmortality
rateofB.diminuta
Checkviabilityoftestbacteriainthe
productsolutionatlowertemperature
Directinoculationintoproductsolution
mightbepossibleat35C
BCTStrategies Method2 (ProcessModification)
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Is
B.diminuta
viableinproductunder
processconditions
?
Determine&DocumentviabilityofB.diminuta
inProductunderProcessConditions
DirectinoculationofB.diminuta
inproductunderprocessconditions
Modifyformulation
(adjustpH,removebactericidal
componentorproductsurrogate)
Useproductfortimeperiodthatthe
challengeorganismisviable
Modifyprocess
(adjusttemperatures,etc.)
ChangefromB.diminuta
Usebacteriaisolatedfrom
formulationorenvironment
VIABLE
NOTVIABLE
PreconditionfilterwithProduct
followedbyMicrobialChallenge
BCTStrategiesMethod3 (TimePeriodChallenge)
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BCTStrategies Method3 (TimePeriodChallenge)
BCTbydirectinoculation
Contacttimebacteria/testsolution
max. 30 min.
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Is
B.diminuta
viableinproductunder
processconditions
?
Determine&DocumentviabilityofB.diminuta
inProductunderProcessConditions
DirectinoculationofB.diminuta
inproductunderprocessconditions
Modifyformulation
(adjustpH,removebactericidal
componentorproductsurrogate)
Useproductfortimeperiodthatthe
challengeorganismisviable
Modifyprocess
(adjusttemperatures,etc.)
ChangefromB.diminuta
Usebacteriaisolatedfrom
formulationorenvironment
VIABLE
NOTVIABLE
PreconditionfilterwithProduct
followedbyMicrobialChallenge
BCTStrategiesMethod4 (FormulationModification)
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Is
B.diminuta
viableinproductunder
processconditions
?
Determine&DocumentviabilityofB.diminuta
inProductunderProcessConditions
DirectinoculationofB.diminuta
inproductunderprocessconditions
Modifyformulation
(adjustpH,removebactericidal
componentorproductsurrogate)
Useproductfortimeperiodthatthe
challengeorganismisviable
Modifyprocess
(adjusttemperatures,etc.)
ChangefromB.diminuta
Usebacteriaisolatedfrom
formulationorenvironment
VIABLE
NOTVIABLE
PreconditionfilterwithProduct
followedbyMicrobialChallenge
BCTStrategiesMethod5 (Preconditioning)
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IndirectBCTbyseparatefiltrationof
testsolutionandbacteria
BCTStrategies Method5 (Preconditioning)
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Is
B.diminuta
viableinproductunder
processconditions
?
Determine&DocumentviabilityofB.diminuta
inProductunderProcessConditions
DirectinoculationofB.diminuta
inproductunderprocessconditions
Modifyformulation
(adjustpH,removebactericidal
componentorproductsurrogate)
Useproductfortimeperiodthatthe
challengeorganismisviable
Modifyprocess
(adjusttemperatures,etc.)
ChangefromB.diminuta
Usebacteriaisolatedfrom
formulationorenvironment
VIABLE
NOTVIABLE
PreconditionfilterwithProduct
followedbyMicrobialChallenge
BCTStrategiesMethod6 (BioburdenIsolate)
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BCTwithbioburden isolate
Reproduciblegrowthandsizeofbacteria
isrequired
Growthinrequiredconcentrationfor
BCTmustbepossible
BCTStrategiesMethod6 (BioburdenIsolate)
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FilterQualificationTests
FilterIntegrity
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DestructiveMethods
DestructiveMethods
Non-DestructiveMethods
Directcorrelationofdiffusion,
bubblepoint,and intrusion-limit
valuestobacteria-challengetest.
IntegrityTestCorrelation
Forproducers;appliedto
determinedlimits:
Forproducers;appliedto
determinedlimits:
Bacteria-Challenge-Test
(accordingtoASTM)
Bacteria-Challenge-Test
(accordingtoASTM)
Forusers;appliedtoevery
sterilizingfilterbefore and
aftereachfiltration:
BubblePointTest
DiffusionTest
MultipointDiffusionTest
PressureDecayTest
WaterIntrusionTest(WIT)
Forusers;appliedtoevery
sterilizingfilterbefore and
aftereachfiltration:
BubblePointTest
DiffusionTest
MultipointDiffusionTest
PressureDecayTest
WaterIntrusionTest(WIT)
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IntegrityTestCorrelation
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Bacterial Challenge Test
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BubblePoint
Filtermembraneiswettedwiththeproduct.Pressureisappliedontheupstream
sideofthefilter.Thepressureatwhichastreamofairbubblesisdetected
downstreamofthefilterisknownastheMinimumBubblePointof thefilter.
45.000 psi
POWER
CORD
SARTOCHECK 3
INLET TUBING FROM
PRESSURE SOURCE
BP TEST
Pressure increase
45.000 PSI
OUTLET TUBING
N.I.S.T. TRACEABLE
REFERENCE
GAUGE
BLIND END CAP
ProductWetIntegrityTesting
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PBPmin=WBPminxCorrectionFactor PBPmin=WBPminxCorrectionFactor
FlushwithWater
Testa,b,c
Calculateaverage,WBP
avg
FlushwithProduct
Testa,b,c
Calculateaverage,PBP
avg
a
b c
ProductSpecificIntegrityTest BubblePoint
46 psi
46 46 psi psi
PBPavg/WBPavg
PBPavg/WBPavg
e.g.
e.g.
36 psi/49 psi
36 psi/49 psi
x
x
PBPmin = 46 psi x 0.73
PBPmin = 33.79 psi
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DiffusiveFlow
Dependsmainlyonthesolubilityofthetestgasinthewettingfluidinaddition
totemperatureandtestpressure.Thefiltermembraneporesare wettedwith
fluidandagaspressurelessthantheBPisapplied.Duetodifferentialpressure,
gasdiffusesthroughthefluidintheporesandisquantifiedas downstreamflow
inmL/min.
45.000 psi
POWER
CORD
SARTOCHECK 3
INLET TUBING FROM
PRESSURE SOURCE
BP TEST
Pressure increase
45.000 PSI
OUTLET TUBING
N.I.S.T. TRACEABLE
REFERENCE
GAUGE
BLIND END CAP
NOTE: BEAKER AND I NVERTED
BURETTE MAY BE SUBSTITUTED
WITH A N.I.S.T. TRACEABLE MASS
FLOWMETER OR ROTAMETER.
GRADUATED
BURETTE
ProductWetIntegrityTesting
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DFL
PW
=DFL
WW
xCorrectionFactor DFL
PW
=DFL
WW
xCorrectionFactor
FlushwithWater
Testa,b,cwithMTP
WW
Calculateaverage,DF
WW
FlushwithProduct
Testa,b,c withTP
PW
Calculateaverage,DF
PW
ProductSpecificIntegrityTest DiffusiveFlow
15 ml/min
15 15 ml/min ml/min
DF
PW
/DF
WW
DF
PW
/DF
WW
e.g.
e.g.
16ml/min/9ml/min
16
16ml/min ml/min
/9
/9ml/min ml/min
x
x
DFL
PW
= 15 ml/min x 1.77
DFL
PW
= 26.5 ml/min
a b c
TP
PW
=MTP
WW
xPBP
avg
/WBP
avg
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IntegrityTestFailure
TR#26includesaTroubleShootingGuide
incaseofIntegrityTestFailures:
Determineswhenafilterhastobeclassifiedasfailed
Filter fails first time Measurements & Actions
Filter fails second time Wetting with solvent
Filter fails third time = Filter failed
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Guidance
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FinalGuidancereleasedon
September29,2004,which
replacesthe1987Guidance
Describestheintegritytest
requirementsofliquidandair
filters,aswellasthevalidation
requirementsofliquidfilters
FDAGuidanceonAsepticProcessing CGMP
46
FDA
PostapprovalChangeGuidance
forchemicalentities(Revision).
Describeswhichchangesin
filtrationstepsareconsidered
moderate(CBE30)ormajor
(PostApprovalSupplement)
changes.
47
FDA
Warninglettersandinspection
guidesarealwaysgoodto
review learnfromthe
mistakesandfromthetraining
ListingsarepostedonFDAwebsite
asprescribedbytheFOIAct.
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ECcGMPGuidance,
RevisionofAnnex1.
Describessterilizationby
filtration,integritytest
needs,somevalidation
topics.
Importantdocumenttoknow
forexporting.
ECGuidetoGMPofSterileMedicinalProducts
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ReleasedISOGuidance,part2.on
filtration.
Describesfiltrationrequirements,
validationandintegritytest
needsthoroughlyforliquid
andairfilters.
Agooddocumenttohave,with
thoroughdescriptions.
ISO:APofHealthCareProducts Filtration
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PDA
PDATechnicalReport#26.
Themostthoroughand
descriptivedocumenton
thetopicofliquid
sterilizingfiltration.
Thisdocumentisamusthave,
mustreadandmust
understandasitisusedby
QCandregulators
worldwide.
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ViabilityTesting
BacterialChallengeTesting
ChemicalCompatibility
Analysis ofExtractables
Product IntegrityTesting
PlantandProcessSurveys
SystemsandIntegrityTester
Validation
ProcessRelated ValidationStudies
Regulatory
Requirement
Industry
Requirement
Filter
Validation
Conclusion
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ValidationofSterilizingGradeFilters
END
END