细菌内毒素USP之欧阳法创编.docx

细菌内毒素USP之欧阳法创编.docx

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细菌内毒素USP之欧阳法创编

BACTERIALENDOTOXINSTEST<85>USP35

时间：

2021.03.09

创作：

欧阳法

Portionsofthisgeneralchapterhavebeenharmonizedwiththecorrespondingtextsofthe EuropeanPharmacopoeia and/orthe JapanesePharmacopoeia.Thoseportionsthatarenotharmonizedaremarkedwithsymbols（

）tospecifythisfact.

这一章节已经和欧洲药典（EP）与日本药典（JP）统一。

没有统一的部分已经用

标记出来。

TheBacterialEndotoxinsTest（BET）isatesttodetectorquantifyendotoxinsfromGram-negativebacteriausingamoebocytelysatefromthehorseshoecrab（Limuluspolyphemus or Tachypleustridentatus）.

细菌内毒素检查法（BET）是通过鲎的阿米巴细胞溶解产物来检测或定量来自革兰氏阴性菌的内毒素。

Therearethreetechniquesforthistest:

thegel-clottechnique,whichisbasedongelformation;theturbidimetrictechnique,basedonthedevelopmentofturbidityaftercleavageofanendogenoussubstrate;andthechromogenictechnique,basedonthedevelopmentofcoloraftercleavageofasyntheticpeptide-chromogencomplex.Proceedbyanyofthethreetechniquesforthetest.Intheeventofdoubtordispute,thefinaldecisionismadebaseduponthegel-clottechniqueunlessotherwiseindicatedinthemonographfortheproductbeingtested.Thetestiscarriedoutinamannerthatavoidsendotoxincontamination.

细菌内毒素检查法有3种：

凝胶法，基于凝胶的形成；浊度法，

BACTERIALENDOTOXINSTESTUSP32

PortionsofthisgeneralchapterhavebeenharmonizedwiththecorrespondingtextsoftheEuropeanPharmacopeiaand/ortheJapanesePharmacopeia.Thoseportionsthatarenotharmonizedaremarkedwithsymbols（

）tospecifythisfact.

Thischapterprovidesatesttodetectorquantifybacterialendotoxinsthatmaybepresentinoronthesampleofthearticle（s）towhichthetestisapplied.ItusesLimulusAmebocyteLysate（LAL）obtainedfromtheaqueousextractsofcirculatingamebocytesofhorseshoecrab（Limuluspolyphemus or Tachypleustridentatus）whichhasbeenpreparedandcharacterizedforuseasanLALReagent.

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Therearetwotypesoftechniquesforthistest:

thegel-clottechniques,whicharebasedongelformation,andthephotometrictechniques.Thelatterincludeaturbidimetricmethod,whichisbasedonthedevelopmentofturbidityaftercleavageofanendogenoussubstrate,andachromogenicmethod,whichisbasedonthedevelopmentofcoloraftercleavageofasyntheticpeptide-chromogencomplex.Proceedbyanyoneofthesetechniques,unlessotherwiseindicatedinthemonograph.Incaseofdispute,thefinaldecisionisbasedonthegel-clottechniques,unlessotherwiseindicatedinthemonograph.

Inthegel-clottechniques,thereactionendpointisdeterminedfromdilutionsofthematerialundertestindirectcomparisonwithparalleldilutionsofareferenceendotoxin,andquantitiesofendotoxinareexpressedinUSPEndotoxinUnits（USP-EU）. [NOTE—OneUSP-EUisequaltooneIUofendotoxin.]

BecauseLALReagentshavebeenformulatedtobeusedalsoforturbidimetricorcolorimetrictests,suchtestsmaybeusedtocomplywiththerequirements.Thesetestsrequiretheestablishmentofastandardregressioncurve;theendotoxincontentofthetestmaterialisdeterminedbyinterpolationfromthecurve.TheproceduresincludeincubationforapreselectedtimeofreactingendotoxinandcontrolsolutionswithLALReagentandreadingofthespectrophotometriclightabsorbanceatsuitablewavelengths.Intheendpointturbidimetricprocedurethereadingismadeimmediatelyattheendoftheincubationperiod.Intheendpointcolorimetricprocedurethereactionisarrestedattheendofthepreselectedtimebytheadditionofanenzymereaction-terminatingagentpriortothereadings.Intheturbidimetricandcolorimetrickineticassaystheabsorbanceismeasuredthroughoutthereactionperiodandratevaluesaredeterminedfromthosereadings.

APPARATUSANDGLASSWARE

Depyrogenateallglasswareandotherheat-stablematerialsinahot-airovenusingavalidatedprocess.

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 Commonlyusedminimumtimeandtemperaturesettingsare30minutesat250

.Ifemployingplasticapparatus,suchasmicroplatesandpipettipsforautomaticpipetters,useonlythatwhichhasbeenshowntobefreeofdetectableendotoxinandnottointerferewiththetest. [NOTE—Inthischapter,theterm“tube”includesanyotherreceptaclesuchasamicro-titerwell.]

PREPARATIONOFTHESTANDARDENDOTOXINSTOCKSOLUTIONANDSTANDARDSOLUTIONS

The USPEndotoxinRS hasadefinedpotencyof10,000USPEndotoxinUnits（EU）pervial.Constitutetheentirecontentsof1vialoftheRSEwith5mLofLALReagentWater

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mixintermittentlyfor30minutes,usingavortexmixer,andusethisconcentrateformakingappropriateserialdilutions.Preservetheconcentrateinarefrigeratorformakingsubsequentdilutionsfornotmorethan14days.Mixvigorously,usingavortexmixer,fornotlessthan3minutesbeforeuse.Mixeachdilutionfornotlessthan30secondsbeforeproceedingtomakethenextdilution.Donotstoredilutions,becauseoflossofactivitybyadsorption,intheabsenceofsupportingdatatothecontrary.

PreparatoryTesting

UseanLALReagentofconfirmedlabelsensitivity.

Thevalidityoftestresultsforbacterialendotoxinsrequiresanadequatedemonstrationthatspecimensofthearticleorofsolutions,washings,orextractsthereoftowhichthetestistobeapplieddonotofthemselvesinhibitorenhancethereactionorotherwiseinterferewiththetest.Validationisaccomplishedbyperformingtheinhibitionorenhancementtestdescribedundereachofthethreetechniquesindicated.Appropriatenegativecontrolsareincluded.ValidationmustberepeatediftheLALReagentsourceorthemethodofmanufactureorformulationofthearticleischanged.

PreparationofSampleSolutions

PreparesamplesolutionsbydissolvingordilutingdrugsorextractingmedicaldevicesusingLALReagentWater.Somesubstancesorpreparationsmaybemoreappropriatelydissolved,diluted,orextractedinotheraqueoussolutions.Ifnecessary,adjustthepHofthesolution（ordilutionthereof）tobeexaminedsothatthepHofthemixtureoftheLALReagentandsamplefallswithinthepHrangespecifiedbytheLALReagentmanufacturer.ThisusuallyappliestoaproductwithapHintherangeof6.0to8.0.ThepHmaybeadjustedusinganacid,base,orsuitablebufferasrecommendedbytheLALReagentmanufacturer.AcidsandbasesmaybepreparedfromconcentratesorsolidswithLALReagentWaterincontainersfreeofdetectableendotoxin.Buffersmustbevalidatedtobefreeofdetectableendotoxinandinterferingfactors.

DETERMINATIONOFMAXIMUMVALIDDILUTION（MVD）

TheMaximumValidDilutionisthemaximumallowabledilutionofaspecimenatwhichtheendotoxinlimitcanbedetermined.Itappliestoinjectionsortosolutionsforparenteraladministrationintheformconstitutedordilutedforadministration,or,whereapplicable,totheamountofdrugbyweightifthevolumeofthedosageformforadministrationcouldbevaried.ThegeneralequationtodetermineMVDis:

MVD=（Endotoxinlimit×Concentrationofsamplesolution）/（

）

wheretheconcentrationofsamplesolutionand 

 areasdefinedbelow.Wheretheendotoxinlimitconcentrationisspecifiedintheindividualmonographintermsofvolume（inEUpermL）,dividethelimitby 

whichisthelabeledsensitivity（inEUpermL）oftheLALReagent,toobtaintheMVDfactor.WheretheendotoxinlimitconcentrationisspecifiedintheindividualmonographintermsofweightorUnitsofactivedrug（inEUpermgorinEUperUnit）,multiplythelimitbytheconcentration（inmgpermLorinUnitspermL）ofthedruginthesolutiontestedorofthedrugconstitutedaccordingtothelabelinstructions,whicheverisapplicable,anddividetheproductofthemultiplicationby 

toobtaintheMVDfactor.TheMVDfactorsoobtainedisthelimitdilutionfactorforthepreparationforthetesttobevalid.

ESTABLISHMENTOFENDOTOXINLIMITS

Theendotoxinlimitforparenteraldrugs,definedonthebasisofdose,isequalto K/M,

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 where K isthethresholdhumanpyrogenicdoseofendotoxinperkgofbodyweight,and M isequaltothemaximumrecommendedhumandoseofproductperkgofbodyweightinasinglehourperiod.

TheendotoxinlimitforparenteraldrugsisspecifiedinindividualmonographsinunitssuchasEU/mL,EU/mg,orEU/Unitofbiologicalactivity.

GEL-CLOTTECHNIQUES

Thegel-clottechniquesdetectorquantifyendotoxinsbasedonclottingoftheLALReagentinthepresenceofendotoxin.TheconcentrationofendotoxinrequiredtocausethelysatetoclotunderstandardconditionsisthelabeledsensitivityoftheLALReagent.Toensureboththeprecisionandvalidityofthetest,testsforconfirmingthelabeledLALReagentsensitivityandforinterferingfactorsaredescribedunder PreparatoryTestingfortheGel-ClotTechniques.

PreparatoryTestingfortheGel-ClotTechniques

TestforConfirmationofLabeledLALReagentSensitivity— Confirmthelabeledsensitivityusingatleast1vialoftheLALReagentlot.Prepareaseriesoftwo-folddilutionsofthe USPEndotoxinRS inLALReagentWatertogiveconcentrationsof2

0.5

and0.25

where 

 isasdefinedabove.Performthetestonthefourstandardconcentrationsinquadruplicateandincludenegativecontrols.ThetestforconfirmationoflysatesensitivityistobecarriedoutwhenanewbatchofLALReagentisusedorwhenthereisanychangeintheexperimentalconditionsthatmayaffecttheoutcomeofthetest.

MixavolumeoftheLALReagentwithanequalvolume（suchas0.1-mLaliquots）ofoneofthestandardsolutionsineachtesttube.WhensingletestvialsorampulscontaininglyophilizedLALReagentareused,addsolutionsdirectlytothevialorampul.IncubatethereactionmixtureforaconstantperiodaccordingtodirectionsoftheLALReagentmanufacturer（