USP1227-VALIDATION-OF-MICROBIAL-RECOVERY-FROM-PHAR

1227VALIDATIONOFMICROBIALRECOVERYFROMPHARMACOPEIALARTICLESThischapterprovidesguidelinesforthevalidationofmethodsfortheestimationofthenumberofviablemicroorganisms,forthedetectionofindicatorsorobjectionablemicroorganisms,forthevalidationofmicrobiologicalmethodsusedinantimicrobialeffectivenesstesting,andforthesterilitytestingofPharmacopeialarticles.Itisgenerallyunderstoodthatifaproductpossessesantimicrobialpropertiesbecauseofthepresenceofaspecificpreservativeorbecauseofitsformulation,thisantimicrobialpropertymustbeneutralizedtorecoverviablemicroorganisms.Thisneutralizationmaybeachievedbytheuseofaspecificneutralizer,bydilution,byacombinationofwashinganddilution,orbyanycombinationofthesemethods.ThetestsunderAntimicrobialEffectivenessTesting51,SterilityTests71,andMicrobialLimitTests61requirethevalidationofrecoverymethods.Toensurethattheresultsofthetestsarecredible,neutralizationofantimicrobialpropertiesofthetestsolutionisrequiredbeforeestimatingthenumberofviablemicroorganisms.INFLUENTIALFACTORSSeveralfactorsaffectthemeasurementofatestsolution'santimicrobialactivity,andthesemustbeconsideredinthevalidationdesign.Theyincludethenatureofthemicroorganismsusedaschallengeorganisms,thepreparationoftheinoculumofchallengeorganisms,thespecificconditionsofthetest,andtheconditionsofrecovery.Thesefactorsalsoaffectthevalidationofrecoverymethodsforaqueousornonaqueousproducts,irrespectiveoftheirantimicrobialproperties;thus,alltestmethodsshouldbevalidatedwiththesefactorsinmind.Thenatureofthechallengemicroorganismexertsastrongeffectupontheresponsetotheantimicrobialagent,andsoupontheneutralizationrequiredforrecovery.RepresentedamongtheseorganismsincompendialtestsareGram-positivebacteria,Gram-negativebacteria,yeasts,andmolds.Eachorganismtobeusedinthetestmustbeincludedinthevalidation.Thepreparationoftheinoculumofchallengemicroorganismsalsoaffectsthetestingofproductshavingantimicrobialproperties.Thegrowthandpreparationofthechallengeorganismdeterminesthephysiologicalstateofthecell.Thisstatehasadirectinfluenceontheresultsofanytestofantimicrobialefficacy.Microbialtestsdonotuseindividualcells;rather,populationsofcellsareharvestedforstudy.Thedatageneratedfromthesestudiesarelessvariableifthecellpopulationsarehomogeneous.Liquidculturesorconfluentgrowthsonsolidmediumarebestsuitedforreproducibleculturepreparation.Theconditionsoforganismpreparationandstoragemustbestandardizedfortheneutralizerevaluationandshouldreflecttheconditionsoftheantimicrobialassay.Thespecificconditionsofthetest,includingbuffersused,water,lightconditions,andtemperature,mustbereproducedinthevalidationstudy.Alltestconditionsalsoshouldbestandardizedandperformedinthevalidationstudyexactlyasperformedinthetest.Theconditionsofmicrobialrecoveryareamongthemostcrucialinaccuratelyestimatingthenumberofmicroorganismspresentinatestsolution.Thefirstconsiderationistherecoverymediumusedtosupportthegrowthofsurvivors.Thisconcernisdiscussedindetailbelow.Thesecondconsiderationistheincubationconditions.Optimalconditionsforgrowthmustbepresenttoensurecompletegrowthandreproducibleresults.METHODSOFNEUTRALIZINGANTIMICROBIALPROPERTIESThreecommonmethodsareusedtoneutralizeantimicrobialpropertiesofaproduct:(1)chemicalinhibition,(2)dilution,and(3)filtrationandwashing.ChemicalInhibitionTable1showsknownneutralizersforavarietyofchemicalantimicrobialagentsandthereportedtoxicityofsomechemicalneutralizerstospecificmicroorganisms.However,despitepotentialtoxicity,theconvenienceandquickactionofchemicalinhibitorsencouragetheiruse.Chemicalinhibitionofbactericidesisthepreferredmethodfortheantimicrobialefficacytest.Thepotentialofchemicalinhibitorsshouldbeconsideredinthemembranefiltrationandthedirecttransfersterilitytests.Antibioticsmaynotbesusceptibletoneutralizationbychemicalmeans,butratherbyenzymatictreatment(e.g.,penicillinase).Theseenzymesmaybeusedwhererequired.Table1.SomeCommonNeutralizersforChemicalBiocidesNeutralizerBiocideClassPotentialActionofBiocidesBisulfateGlutaraldehyde,MercurialsNon-SporingBacteriaDilutionPhenolics,Alcohol,Aldehydes,Sorbate—GlycineAldehydesGrowingCellsLecithinQuaternaryAmmoniumCompounds(QACs),Parabens,Bis-biguanidesBacteriaMg+2orCa+2ionsEDTA—PolysorbateQACS,Iodine,Parabens—ThioglycollateMercurialsStaphylococciandSporesThiosulfateMercurials,Halogens,AldehydesStaphylococciDilutionAsecondapproachtoneutralizingantimicrobialpropertiesofaproductisbydilution,becausetheconcentrationofachemicalbactericideexertsalargeeffectonitspotency.Therelationshipbetweenconcentrationandantimicrobialeffectdiffersamongbactericidalagentsbutisconstantforaparticularantimicrobialagent.Thisrelationshipisexponentialinnature,withthegeneralformula:Ct=k,inwhichCistheconcentration;tisthetimerequiredtokillastandardinoculum;kisaconstant;andtheconcentrationexponent,,istheslopeoftheplotoflogtversuslogC.Antimicrobialagentswithhighvaluesarerapidlyneutralizedbydilution,whereasthosewithlowvaluesarenotgoodcandidatesforneutralizationbydilution.MembraneFiltrationAnapproachthatisoftenused,especiallyinsterilitytesting,isneutralizationb