Detection and Quantification of Gram Negative Bacterial Endotoxin ...

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:



APPROVED BY: DATE:



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


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


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


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


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)


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


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



*Thisisanexample;dilutionoftheCSEStocktomakeInt.AsolutionsdependsontheconcentrationofCSEstockandisdeterminedforeachlotofCSEreagent,refertotheCustomCertificateOfAnalysis.NumbersshowninthetableabovearecalculatedbasedonaStockconcentrationof1000EU/mL.

Eachsampleshouldbevigorouslyvortexedforatleastoneminutepriortouse.

5.4 AssayControlReactions

5.4.1 PreparationofInhibition/EnhancementtestusingaPositiveProductControl(PPC)Fortheverificationofresultsitisnecessarytopreparesampleswithadefinedamountofendotoxinstandardtodetermineinhibitionprocessesorinterferenceswiththeproduct.ThenominalendotoxinconcentrationspikedinIECshouldequalthatofastandarddilutionfromthemiddleofthestandard


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)



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



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.


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.


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.


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


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.


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


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.