detection and quantification of gram negative bacterial endotoxin
TRANSCRIPT
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DocumentType DocumentID Version Status PageSOP EUNCL-STE-001.2.2 1.1 1/14
DetectionandQuantificationofGramNegativeBacterialEndotoxinContaminationinNanoparticleFormulationsbyKineticTurbidimetricMicroplate
LALAssay
AUTHORED BY: DATE:
Rainer Ossig 14.04.2016
Matthias Rösslein 01.10.2016
REVIEWED BY: DATE:
Matthias Rösslein 13.07.2016
Matthias Rösslein 01.10.2016
APPROVED BY: DATE:
Matthias Rösslein 13.07.2016
Matthias Rösslein 01.10.2016
DOCUMENTHISTORY
Effective Date Date Revision Required Supersedes
01.10.2016 01.10.2016 13.07.2016
Version Approval Date Description of the Change Author / Changed by
1.0 13.07.2016 All Initial Document Rainer Ossig
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DocumentType DocumentID Version Status PageSOP EUNCL-STE-001.2.2 1.1 2/14
Version Approval Date Description of the Change Author / Changed by
1.1 01.10.2016 Chapter 7 Updated Acceptance criteria now in line with NCI-NCL acceptance criteria
Matthias Rösslein
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TableofContent1 Introduction......................................................................................................................................4
2 PrincipleoftheMethod....................................................................................................................4
3 ApplicabilityandLimitations(Scope)...............................................................................................4
4 RelatedDocuments..........................................................................................................................4
5 EquipmentandReagents..................................................................................................................5
5.1 Equipment.................................................................................................................................5
5.2 Reagents....................................................................................................................................5
5.3 ReagentPreparation..................................................................................................................6
5.3.1 PreparationofPyrotell-TLALReagent...............................................................................6
5.3.2 EndotoxinControlStandardEndotoxinstocksolution.......................................................6
5.3.3 PreparationofEndotoxincalibrationstandards.................................................................7
5.4 AssayControlReactions............................................................................................................7
5.4.1 PreparationofInhibition/EnhancementtestusingaPositiveProductControl(PPC)........7
5.4.2 QualityControls..................................................................................................................8
5.4.3 NegativeControl.................................................................................................................9
6 Procedure.........................................................................................................................................9
6.1 Generalremarks........................................................................................................................9
6.2 PreparationofStudySamples...................................................................................................9
6.3 Flowchart................................................................................................................................10
6.4 MeasurementProcedure........................................................................................................11
6.4.1 Testprocedure.................................................................................................................11
6.4.2 Preparationofthe96-wellmicroplate.............................................................................11
6.5 DataAnalysis...........................................................................................................................12
7 AssayAcceptanceCriteria..............................................................................................................13
8 HealthandSafetyWarnings,CautionsandWasteTreatment.......................................................13
9 Abbreviations.................................................................................................................................13
10 References....................................................................................................................................14
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1 IntroductionThisdocumentdescribesaprotocolforaquantitativedetectionofGramnegativebacterialendotoxininnanoparticlepreparationsusingakineticturbidimetricLimulusAmebocyteLysate(LAL)assay.PrincipleoftheMethod
2 PrincipleoftheMethodThismethodreliesonaninvitroend-productendotoxintestwhichutilizesaLimulusAmebocyteLysate(LAL),anextractofbloodcells(amebocytes)fromthehorseshoecrab.Themethodisdesignedtodetectendotoxinactivityphotometricallywithanautomatedmicroplatereaderincubatingthereactionmixtureatcontrolledtemperatureof37°C.
GramnegativebacterialendotoxincatalyzestheactivationofproenzymeintheLimulusAmebocyteLysate.Bang1observedin1956thattheinfectionofthehorseshoecrabLimuluspolyphemuswithGram-negativebacteriaresultedinintravascularcoagulation,asaresultofareactionbetweenendotoxinandaclottingproteininamebocytesofLimulus2.ThemethodisbasedtheinitialreactionoftheLALwithendotoxin.ALALproenzymeisactivatedinthepresenceofendotoxin.Asaresultofthefollowingcascadeofenzymeactivationstepscoagulationisinitiatedandturbidityofthereactionmixtureincreases.Thedevelopmentofturbidityismeasuredusinganautomatedplatereaderandthetimetoreachaspecificincrementofturbidity(theonsettime)isdetermined.Higherendotoxinconcentrationsgiveshorteronsettimes.ConcentrationofendotoxininasampleiscalculatedfromastandardcurvepreparedbytheonsettimeofknownconcentrationofendotoxinstandardintoLALgradewater.ThismethodreliesonLimulusAmebocyteLysatePYROTELL®–TbyPyroquantDiagnostikGmbH,asubsidiaryofAssociatesofCapeCod,Inc.(ACC)3.DataanalysisisperformedusingMARSsoftware(BMGLabtechGmbH).
TheamountofendotoxinpresentwhichiscalculatedfromastandardcurvepreparedbydilutionofanendotoxinstandardofknownconcentrationsofintoLALgradewater.
3 ApplicabilityandLimitations(Scope)ThisSOPwasdevelopedtodetermineandquantifyendotoxincontaminationofdifferentnanomaterials.ThisSOPwascreatedaccordingtoDINENISO297014adaptedfortheanalysisnanomaterials4.AndaccordingtothesuppliersinstructionfortheuseoftheKineticTurbidimetricMicroplateLALAssayPyrotell-T3.UsePyrotell-Tforinvitrodiagnosticpurposesonly.Donotuseitforthedetectionofendotoxemia3.
4 RelatedDocumentsTable1:
DocumentID DocumentTitleNCLMethodSTE-1.2 DetectionandQuantificationofGramNegativeBacterialEndotoxin
ContaminationinNanoparticleFormulationsbyKineticTurbidityLALAssay
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5 EquipmentandReagents
5.1 Equipment5.1.1 Pyrogen-freemicrocentrifugetubes,1.5mL(e.g.EppendorfBioPure®)
5.1.2 Pyrogen-freepipettesandbarriertipscoveringtherangefrom0.01to1mL(e.g.SarstedtBiosphere®)
5.1.3 Pyrogen-freedispensertips,100µlincrement(e.g.EppendorfBioPure®)
5.1.4 Repeatpipettororeight-chanelpipettor
5.1.5 96wellplate,pyrogen-free(e.g.Costar3596,ACCPYROPLATE®orequivalent)
5.1.6 Disposableendotoxin-freeglassdilutiontubes13×100mm(LonzaN207)or 12x75mm(ACCTB240)orequivalent
5.1.7 Reagentreservoirs(Lonza00190035orequivalent)
5.1.8 Microcentrifuge
5.1.9 Refrigerator,2-8°C
5.1.10 Freezer,-20°C
5.1.11 Vortexmixer
5.1.12 Parafilm®“M”Laboratoryfilm(PechineyPlasticPackaging)
5.1.13 automatedMicroplatereader,temperaturecontrolled37°C,at340nmabsorption(e.g.Novostar®,ClarioStar®,BMGLabtechGmbH)
5.2 Reagents5.2.1 Testnanomaterial
5.2.2 LIMULUSAMEBOCYTELYSATEPYROTELL®–TForTheDetectionAndQuantificationOfGram NegativeBacterialEndotoxins(PYROQUANTDIAGNOSTIK,AssociatesofCapeCod,Inc.(ACC))
5.2.3 ControlStandardEndotoxin(CSE)(PYROQUANTDIAGNOSTIKGmbH,ACC)
5.2.4 Glucashield®(1→3)-ß-D-GlucanInhibitingBuffer(PYROQUANTDIAGNOSTIKGmbH,ACC)
5.2.5 LALReagentWater(PYROQUANTDIAGNOSTIKGmbH,ACC)
5.2.6 Sodiumhydroxide,0,1N,endotoxinfree(e.g.Acila1712200)
5.2.7 Hydrochloricacid0,1N,endotoxinfree(e.g.Acila1712300)
5.2.8 Endotoxinfreewater(e.g.ACCW0051;Acila1715050;orequivalent)
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5.3 ReagentPreparationStoreallprovidedreagentsofthekitat2–8°C.Priortouseallowreagentstoequilibratetoroomtemperature3.
5.3.1 PreparationofPyrotell-TLALReagentTheassayreagentisprovidedaslyophilizedmixtureofLALlysateItisreconstitutedaccordingtomanufacturer’srecommendations,andcanbeperformedinLALgradewater(LALReagentWater)orGlucashield®Bufferavailablefromthesupplierasseparatecomponents.EUNCLpreferredmethodistheapplicationofGlucashield®bufferwhichallowstoexcludeinterferencefromβ-1,3-Glucanswhichisverycommoninnanomaterialsproducedusingfiltrationsteps.Substancescontainingβ-1,3-Glucansareimportantsourcesoffalse-positivesandasynergisticresponse(i.e.enhancement)isfrequentlyseenwithβ-Glucancontainingsamplesspikedwithendotoxin.TheusageofaGlucashield®bufferisthereforeindicatedwheneverβ-1,3-Glucancontaminationisexpected3.
ReconstitutePyrotell-TLALReagentonlyimmediatelybeforeuse.Addthevolumeasindicatedontheviallabel,whichusuallyis5mL,andisgoodtoperform48singlereactions.Incaseforalargernumberofsamplesmorethanonevialisrequired,poolreconstitutedreagentoftwoorseveralvialsbeforeuse.Exerciseextremecautiontoavoidformationofairbubbles.Donotvortexthereconstitutedlysate.Pipettewithcaution.Mixonlybyverygentlyswiveltoavoidfoaming.Incaseofbubblesallowtoclearbeforeuse.CoverthevialwithParafilmM®whennotinuse.
StorethelyophilizedPyrotell-TLALReagentat-20to8°Cuntilexpirationdateonthelabel.ReconstitutedLALReagentshouldbeusedpromptlyandisstableforupto24hoursat2to8°C.,orcanbestoredatorbelow-20°Corcolderforuptothreemonthsiffrozenimmediatelyafterreconstitution.FreezeandthawthereconstitutedLALReagentonlyonce(ACC)3.
5.3.2 EndotoxinControlStandardEndotoxinstocksolutionE.colilipopolysaccharide(LPS)suppliedbyACCisaUSPcertifiedcontrolstandardendotoxin(CSE)providedasalyophilizedpowder.Preparethestocksolutionof1000EU/mLbyreconstitutionoftheControl-StandardEndotoxin(CSE,E.coliO55:B5Endotoxin3).
Removethemetalsealfromthevial,breakthevacuumbyliftingthestopperjustenoughtoallowairtoenter,andasepticallyremovethestopper.AddLALReagentwaterdirectlyandwithcautiontotheCSEvial.ThefinalvolumeneededforreconstitutionoftheCSEvialshouldbecalculatedforeachlotanddependsonproductpotencydeterminedwithaspecificlotofLALreagentrelativetothecurrentFDAorUSPlotofreference.Thespecificvolumeneededtoreachapotencyof1000EU/mLcanbecalculatedfromtheCustomCertificateofAnalysis for theKineticTurbidimetricMicroplateMethod(providedbythesupplierAssociatesofCapeCod,Inc.3).
During reconstitution andprior to use, the stock solution should be vortexed vigorously for 30-60sec,with5-10minsettlingtimes,overa30-60mintimeframe,andallowedtoequilibratetoroomtemperature.VortextheCSEforatleast30secondseachtimeimmediatelybeforetakinganaliquotforusagetomakeappropriatedilutions.
ThereconstitutedstockCSEsolutionisstablefor4weeksstoredat2-8°C,donotfreezeCSE(ProductInsert CSE Endotoxin E. coli 0113:H10, ACC) 3. Before usage of the stored stock bring to room
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temperatureandmixvigorouslyfor15minutesinordertoreleaseendotoxinthattendstoattachtotheglasssurface.
5.3.3 PreparationofEndotoxincalibrationstandardsIf the Pyrotell-T LAL Endotoxin Detection System (Associates of Cape Cod, Inc.) is being used in amicroplatereaderthedetectionlimit,andthusthelowestpossiblevalueofλis0.005EU/mL3.Adjustthe quantitative range of the assay and the sensitivity of an individual test defined by the lowestendotoxinconcentrationusedtoconstructthestandardcurve.
Labeldisposablepyrogen-freeglassdilutiontubesfortheendotoxindilutions.Prepareaseriousofendotoxinstandard-dilutionsbyadding0.1mLofthepriorendotoxinsolutioninto0.9mLofLALReagentWater.Eachdilutionshouldbevigorouslyvortexedforatleast1minutebeforeproceedingwiththenextstepofthedilutionseries.
Theendotoxincalibrationstandardsmaybepreparedasdescribedinthefollowingtable(alternativedilutionschemesmaybeused):
Dilutionschemeforpreparationofaseriesofendotoxinstandarddilutions
SampleNominalConcentration
(EU/mL)PreparationProcedure
Int.A* 50* 100µLStockCSE+1900µLLALreagentwater
Cal.1 5.0100µLofInt.Asolution+900µLLALreagentwater
Cal.2 0.5 100µLCal.1+900µLLALreagentwater
Cal.3 0.05 100µLCal.2+900µLLALreagentwater
Cal.4 0.005 100µLCal.3+900µLLALreagentwater
*Thisisanexample;dilutionoftheCSEStocktomakeInt.AsolutionsdependsontheconcentrationofCSEstockandisdeterminedforeachlotofCSEreagent,refertotheCustomCertificateOfAnalysis.NumbersshowninthetableabovearecalculatedbasedonaStockconcentrationof1000EU/mL.
Eachsampleshouldbevigorouslyvortexedforatleastoneminutepriortouse.
5.4 AssayControlReactions
5.4.1 PreparationofInhibition/EnhancementtestusingaPositiveProductControl(PPC)Fortheverificationofresultsitisnecessarytopreparesampleswithadefinedamountofendotoxinstandardtodetermineinhibitionprocessesorinterferenceswiththeproduct.ThenominalendotoxinconcentrationspikedinIECshouldequalthatofastandarddilutionfromthemiddleofthestandard
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curve and should be the same as used in theQuality Controls. For example, 25 µL of a 10EU/mLEndotoxinstandarddilutionareaddedto475µLnanoparticlesuspensionofthesample,resultingina spiked endotoxin concentration of 0.5 EU/mL. For the Quality control, the same amount ofEndotoxinisdilutedinto475µLLALreagentwater.
Dilutionscheme:PreparationofPositiveProductControls(PPC)
SampleNominalConcentration
(EU/mL)PreparationProcedure
Int.A** 100.0* 100µLStockCSE+900µLLALreagentwater
Int.B** 10* 100µLofInt.A+900µLLALreagentwater
IEC 0.5 25µLofInt.B+475µLnanoparticlesuspension***
* Numbers shown in the tableaboveare calculatedbasedona Stock concentrationof1000EU/mL.** IntermediatesolutionsA,B arepreparedonlytomakecontroldilutionsandarenotused inassay.*** Theconcentrationofnanoparticles in IECshouldbeequal tooneassayed for standardcurve.YouwillneedtoprepareanIECforeachdilutionofthenanomaterialassayedinthistest.
Eachsampleshouldbevigorouslyvortexedforat leastoneminutepriortouse.Transfer100μLoftheIECsolutionintothe96-wellplateasdirectedbytheassaytemplate.
5.4.2 QualityControlsDilutionscheme:PreparationofQualityControls
SampleNominalConcentration
(EU/mL)PreparationProcedure
Int.A** 100.0* 100µLStockCSE+900µLLALreagentwater
Int.B** 10.0* 100µLofInt.A+900µLLALreagentwater
QC 0.5 25µLofInt.B+475µLLALreagentwater
* Numbers shown in the tableaboveare calculatedbasedona Stock concentrationof1000EU/mL.** IntermediatesolutionsA,B arepreparedonlytomakecontroldilutionsandarenotused inassay.*** The nominal concentration in QC should equal that of a standard from the middle of thestandardcurveandshouldbethesameasinIEC.
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Eachsampleshouldbevigorouslyvortexedforat leastoneminutepriortouse.Transfer100μLoftheQCsolutionintothe96-wellplateasdirectedbytheassaytemplate.
5.4.3 NegativeControlUseendotoxinfreeLALreagentwaterorrespectivediluentbufferforthesamplesasanegativecontrolreference.
Transfer100μLofeachpreparedAssayControlReactionintothe96-wellplateasdirectedbytheassaytemplate.
6 Procedure
6.1 GeneralremarksMostimportantlymicrobialorendotoxincontaminationofallsamplesandmaterialshavingcontactwiththesampleandallusedtestreagentsmustbeavoidedbycarefulhandlingandtechnique.
6.2 PreparationofStudySamplesStudysamplesshouldbereconstitutedineitherLALreagentwaterorsterile,pyrogen-freePBS.ThepHof thestudysampleshouldbechecked. Itmaybenecessary toadjust thepHof thesample towithin therange6.0–8.0usingeithersterileendotoxin-freesodiumhydroxideorhydrochloricacid.DonotadjustthepHofunbufferedsolutions.Pyrogen-freeTrisbuffermayalsobeusedtopreparesamplesforendotoxindetectioninplaceofwaterasasamplediluenttoadjustpHofhighlyacidicorbasicsamples.ToavoidsamplecontaminationalwaysmeasurethepHofanaliquotofthepreparedsample. If the samplewas prepared in PBS or other diluent, the diluent alonemust be tested forendotoxin contamination in the assay. The concentration of nanomaterial is unique to eachformulation.Thegoalofthistestistomeasureendotoxinlevelpermgofthetestformulation,whichcommonlyreferstotheactivepharmaceutical ingredient(API),butmayalsobemeasuredinmgoftotalformulationortotalelement(e.g.goldorsilver).ThesampleshouldtestedfromthestockusingseveraldilutionsnotexceedingsocalledMaximumValidDilution(MVD).
To determine the MVD one needs to know three parameters: endotoxin limit (EL), sampleconcentrationandassaysensitivity(λ).ELiscalculatedaccordingtothefollowingformula:EL=K/M,whereKismaximumendotoxinlevelallowedperdose(5EU/kgforallroutesofadministrationexceptfortheintrathecalroute,forwhichKis0.2EU/kg)andMisthemaximumdosetobeadministeredper kg of body weight per single hour (1). Note, estimation of EL for nanomaterials used asradiopharmaceuticalorasmedicaldevicewillbedifferent5.Whenthedoseinformationforthetestnanomaterial isavailablebasedonananimalmodel(e.g. inmouse),onemayuseittoconvertintohumanequivalentdose(HED).Todosotheanimaldoseisdividedbytheconversionfactorspecifictoeachanimalspecies,e.g.12.3formouse.Pleaserefertoguidelinesforotherconversionratios6.Doseforcancertherapeutics isoftenprovidedinmg/m2insteadofmg/kg.Toconvertananimalorhuman dose frommg/m2 to mg/kg the dose in mg/kg is divided by the conversion factor of 37,indicatedaskm(formassconstant).Thekmfactorhasunitsofkg/m2;itisequaltothebodyweightinkgdividedbythesurfaceareainm2.Example74mg/m2/37=2mg/kg6.
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TheMVDisdeterminedaccordingtothefollowingformula:MVD=(ELxsampleconcentration)/λ).Forexample,whennanoparticlesampleconcentrationis10mg/mLandit’smaximumdoseinmouseis 123 mg/kg, the HED is 123/12.3=10mg/kg; EL for all routes except intrathecal is 0.5 EU/mg(5EU/kg/10mg/kg) and MVD is 1000 ((0.5 EU/mg x 10 mg/mL)/0.005EU/mL). In this case, thenanomaterial will be tested directly from stock or at several dilutions not exceeding theMVD of1000,e.g. 10,100and1000timesdilution.Whentheinformationaboutthedoseisunknown,thehighestfinalconcentrationofthetestnanomaterialis1mg/mL,whichisusedtocalculatetheMVD.It is very important to recognize that if the dose, route of administration and/or the sampleconcentrationforthetestnanomaterialchange,theELandMVDwillalsochange.
6.3 Flowchart
Figure1:Briefoutlineoftheworkflow.
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6.4 MeasurementProcedure
6.4.1 TestprocedureTurnonthereaderinstrumentapproximately20-30minutesbeforestartingtheassaytoallowtheinstrumenttowarmup.Settemperatureto37°C.Switchdetectionwavelengthto340nm,andadjustallsettingsaslistedbelow,oruseapreinstalledprogramwhichisaccordingtothesesettings.
SettingforautomatedtemperaturecontrolledMicroplatereader:
- 340nmabsorbance- 37°Cincubationtemperature- 160–180cycleswith45scycletime
(differentcyclemaybeusedwithtotalmeasurementtime:approximately2h)
Alternatively,differentcycletimeswithadaptednumberofperformedcyclesmaybeusedwithatotalreadingtimeofatleast7200secondstoallowtimeforsampleswithlowamountsofendotoxintodevelop.Note:somelotsofthelysatearelesssensitivethanothers,ifthesensitivityoftheparticularlotislow,thetotalmeasurementtimemayneedtobeadjustedto9000sorlongerinordertoallowthelowestcalibratortodevelop.
6.4.2 Preparationofthe96-wellmicroplateDispense100µLofpreparedendotoxinstandards,differentproductsampledilutions(S),productinhibitionsamples(PPC),Qualitycontrol(QC),andblankEndotoxinfreeLALreagentwaterordiluentbuffer(BW)inthewellsofthe96wellplate.Prepareeachsampleatleastinduplicates.
Thefollowingmatrixcanbeusedasanexampletemplateforpipettingofanassaydesignedfor3test-samplesinthreedifferentdilutions,eachwithaPCC,allreactionsperformedinduplicates.Otherplatedesignsmaybeadvantageoustoapplyfordifferentsampleandcontrolreactionnumbers.
1 2 3 4 5 6 7 8 9 10 11 12
5 0.5 0.05 0.005 S1.1 S1.1 S1.2 S1.2 S1.3 S1.3
5 0.5 0.05 0.005 S1.1PPC
S1.1PPC
S1.2PPC
S1.2PPC
S1.3PCC
S1.3PCC
OC QC BW BW S2.1 S2.1 S2.2 S2.2 S2.3 S2.3
S2.1PPC
S2.1PPC
S2.2PPC
S2.2PPC
S2.3PCC
S2.3PCC
S3.1 S3.1 S3.2 S3.2 S3.3 S3.3
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S3.1PPC
S3.1PPC
S3.2PPC
S3.2PPC
S3.3PCC
S3.3PCC
Shortly before usage reconstitute the necessary LAL Reagent vials with LAL Reagent Water orGlucashield®buffer(accordingtothemanufacturersinstruction),mixonlygently(donotvortex!)asdescribedabove(fordetailsreadunder-ReagentPreparation-).
Use a dispenser to immediately add 100 µL reconstituted LAL- Reagent into each of the reactionwells.Workquicklybutcarefultoavoidcausingbubblesinthewell.Controlallwellsforabsentsofbubbles.
Mix gently for about 15 seconds and start automated reading procedure immediately (reading isperformedwiththemicroplatecoverremoved).
Duringthemeasurementtime,donotdisturbthereactionplate.Thelaboratorybenchsupportingtheopticalreadershouldbefreefromexcessivevibration.
6.5 DataAnalysisThereactiontimeneededfortheappearanceofturbidityisinverselyproportionaltotheamountofpresentendotoxin.Forthedeterminationoftheexactendotoxinamountitisnecessarytocreateastandard curve of at least 3 to 4 different concentrations (e. g. 5 EU/mL, 0.5 EU/mL, 0.05 EU/mL,0.005EU/mL).Basedon the values for theendotoxin standard curvea log/log linear correlation isusedtocalculatevaluesof thecorrespondingEndotoxinconcentration inEU/mLfromthereactiontime. The initial absorbance of each well is used as blank for its subsequent kinetic readings toperformabaselinecorrectionandtodeterminethetimetoreachan increaseof0.100absorbanceunits.Thecorrelationcoefficientabsolutevalueforthestandardcurveshouldbe≥0.980toenableareliable interpolationofunknownsamples. Thedifferentparametersare theabsorptionvalues forthex-range,meanreactiontimeforthey-range,and0.100asthresholdvalue,inordertodeterminethereactiontimefortheincreaseof0.1absorbanceunits.Constructastandardcurvebyregressionofthelogonsettimeagainstthelogendotoxinconcentrationforthestandards.Theequationfortheregression line describes the standard curve. The line equation of the standard curve is used forcalculationofendotoxinconcentrationsofthesamples(includingstandardsandcontrols).Analysisisperformed by the appropriate template of theMARS Data Analysis Software (BMG Labtech). Thesoftware is used to directly calculate the results from the microplate reader of the kineticturbidimetricLALassay.
Therecoveryrateofpositiveproductcontrol(PPC)andQualitycontrols(QC)iscalculatedbydividingthe measured spiked endotoxin concentration by the nominated one to determine potentialinhibitionorenhancementreactionsofthesampleingredientsattherespectiveconcentrationofthetestedsample.
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7 AssayAcceptanceCriteria1. Linearregressionalgorithmisusedtoconstructthestandardcurve.Precision(%CV)and
accuracy(PDFT)ofeachcalibrationstandardandqualitycontrolshouldbewithin25%.2. Atleastthreecalibrationstandardsshouldbeavailableforassaytobeconsidered
acceptable.3. Thecorrelationcoefficientofthestandardcurvemustbeatleast0.980.4. Ifqualitycontrolsfailtomeetacceptancecriteriondescribedin7.1,runshouldberepeated.5. Ifstandardcurvefailstomeetacceptancecriteriondescribedin7.1–7.3,therunshouldbe
repeated.6. Precisionofthestudysampleshouldbewithin25%.7. Precisionofinhibition/enhancementcontrolshouldbewithin25%.8. Spikerecoveryindicativeoftheaccuracyoftheinhibition/enhancementcontrolshould
between50and200%[4].Spikerecoverylessthan50%isindicativeofinhibition;thatabove200%isindicativeofeitherendotoxincontaminationorenhancement.
9. Ifsampleinterferenceisdetected,theassayresultsforthissampleareinvalid.Othertestsshouldbeconsideredasdiscussedinreference5.
8 HealthandSafetyWarnings,CautionsandWasteTreatmentUsePyrotell-Tforinvitrodiagnosticpurposesonly.Donotuseitforthedetectionofendotoxemia.Thetoxicityofthereagenthasnotbeendetermined;thus,cautionshouldbeexercisedwhenhandlingPyrotell-T.
Informyourselfaboutthecontentandsamplematerialandallrelevantsafetyissuesconcerningthesamplesbeforeunpackingandhandlingofanyreceivedsample.
Alwayswearadequatepersonalprotectiveequipment,inparticularprotectivelaboratorycoat,glovesandsafetyglassesandtakeallnecessaryprecautionstoprotectyourselfandothers.Glovesmustbeinspectedpriortouse.Usepropergloveremovaltechnique(withouttouchingglove'soutersurface)toavoidskincontactwiththeproducts.Userespiratoryprotectionwheneveradvisable.Openthesamplevialsonlyundersterileconditionsofthesterileworkbenchinordertoavoidsamplespillingandcontamination.Takeallnecessaryprecautionstoavoidanyfurthersamplespillingincaseofdamagedsamplecontainer.Wastedisposalhastobeproceededinaproperformusingmeansadequateforthematerialspecifications,incompliancewiththelaboratoryregulationsandgeneralregulatoryconditionsaccordingtoapplicablelegalregulations.
9 AbbreviationsAPI activepharmaceuticalingredient
BW blankwater
CV coefficientofvariation
EL EndotoxinLimit
EU endotoxinunit
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DocumentType DocumentID Version Status PageSOP EUNCL-STE-001.2.2 1.1 14/14
HCl hydrochloricacid
LAL LimulusAmebocyteLysate
MVD MaximumValidDilution
NaOH sodiumhydroxide
PBS phosphatebufferedsaline
PES polyethersulfone
PPC positiveproductcontrol
RT roomtemperature
S sample
10 References1:Bang,F.B.AbacteriladiseaseofLimuluspolyphemus.Bull.JohnsHopkinsHosp.98:325(1956)
2:Levin,J.,Bang.F.B.ClottableproteininLimulus:itslocalisationandkineticsofitscoagulationbyendotoxin.Thromb.Diath.Haemorrh.19:186(1968)
3:USP34-NF29.<85>.BacterialEndotoxins.Rockville,MD:UnitedStatesPharmacopeia,2011,Volume1,78-81.
4:FDAGuidanceforIndustryandReviewersEstimatingtheSafeStartingDoseinClinicalTrialsforTherapeuticsinAdultHealthyVolunteers.December2002.
5:USFDA.GuidanceforIndustry.PyrogenandEndotoxinstesting:Questionsandanswers,2012.