EP5.1.10細菌內(nèi)毒素測試使用指南〔2/2〕2023-12-2823:00:31|EPForroutinetestsonthisproduct,itmaybeexpedienttodilute1mLofthesolutiontobeexaminedto20mL(MVD/2roundedtothenextlowerwholenumber).However,ifthistestresultispositivetheanalystwillhavetodilute1mLto41.67mLandrepeatthetest.Adilutionto41.67mLisalsonecessarywhenthetestisperformedtosettleadispute.對于此藥品的日常檢查，可以取1ml20ml〔MVD/2四舍五入至最接近的整數(shù)1ml41.67ml，41.67ml的稀釋。RISKASSESSMENT風(fēng)險評估Asstatedinsection1ofthisgeneralchapter,theconclusionisgenerallyjustifiedthattheabsenceofbacterialendotoxinsinasubstanceorproductimpliestheabsenceofpyrogeniccomponents,providedthepresenceofnon-endotoxinpyrogenicsubstancescanberuledout.Toruleoutthepresenceofnon-endotoxinpyrogensinsubstanceorproducts,theuseofthemonocyte-activationtest(2.6.30)isrecommendedatreleaseorduringdevelopmentoftheproductionprocess;ifanychangesaremadetotheproductionprocessthatcouldinfluencethequalityoftheproductregardingpyrogenicity,themonocyte-activationtestisrepeated.Examplesofsuchchangesincludetheuseofdifferentrawmaterials,adifferentproductionsiteanddifferentprocessparameters.1活測試〔2.6.30〕。假設(shè)對生產(chǎn)工藝進展了變更，可能會影響產(chǎn)品熱源方面的質(zhì)不同的生產(chǎn)場所和不同的工藝參數(shù)。Thedecisiontousethetestforbacterialendotoxinsasthesolepyrogenicitytestistobemadeaftercarefulevaluationoftheriskofthesubstanceorproductcontainingnon-endotoxinpyrogens.Theriskassessmentismadewithconsiderationgiventoanyfactorthatcouldresultintheinclusionofpyrogensnotdetectedbythetestforbacterialendotoxins.Theitemsbelowconstituteanon-exhaustivelistoffactorstobeconsideredintheriskassessment.使用細菌內(nèi)毒素測試作為單一熱源測試的決策要在對含有非內(nèi)毒素熱源的原料單，并非完整清單，Productionprocess(chemicalsynthesis,fermentation,biotechnologicalmethod).Forproductsoffermentation,theexpressionsystemistobeconsidered(prokaryotic,eukaryotic)and,foraprokaryoticexpressionsystem,whethergram-positiveorgram-negativebacteriaareused.Alsotheculturemediacomponentsareexaminedwithconsiderationgiventotheirorigin(synthetic,animal,plant).生產(chǎn)工藝〔化學(xué)合成、發(fā)酵、生物技術(shù)方法〕：對于發(fā)酵產(chǎn)品，要考慮表達系統(tǒng)對于發(fā)培育基成分，要檢查其來源〔合成、動物來源、植物來源〕。Bioburden.Thepotentialpresenceofgram-positivebacteriaandfungiascontaminantsoftheactivesubstance,excipientsorstartingmaterialsandrawmaterialsusedintheproductionofthemedicinalproduct,andtheoriginoftherawmaterials(synthetic,animal,plant)havetobetakenintoconsideration.Thequalityofthewaterplaysanimportantroleontheoverallevaluation.〔合成、動物來源、植物來源〕。水的質(zhì)量在總體評估中起著尤為重要的作用。Capabilityofthedownstreamprocess.Itmustbeverifiedwhetherbacterialendotoxinremovalstepsarepartofthedownstreamprocess.下游工藝力氣：要確認下游工藝中是否有細菌內(nèi)毒素去除步驟。Safety.Thetargetpopulationandtherouteofadministration(ravenous,intrathecal)havetobetakenintoaccountintheriskassessment.安全性：在風(fēng)險評估中要考慮目標種群和給藥途徑〔例如，靜脈注射、鞘內(nèi)〕。Stabilityofthedetectableendotoxins.Ithastobeconsideredthattheabilitytodetectendotoxinscanbeaffectedbyinteractionwithcertaincomponents,storageconditionsorstoragetime,temperatureandhandlingofthetestsample.Proceduresthatdemonstratestabilityofthedetectableendotoxincontenthavetobeestablishedforstoring,handlingandmixingofsamples.量的穩(wěn)定性的程序必需指定其存貯狀況、處理狀況和樣品混和狀況。REFERENCEMATERIAL比照物EndotoxinstandardBRPisintendedforuseasthereferencepreparation.IthasbeenassayedagainsttheWHOInternationalStandardforEndotoxinanditspotencyisexpressedinInternationalUnitsofendotoxinpervial.TheInternationalUnitofendotoxinisdefinedasthespecificactivityofadefinedmassoftheInternationalStandard.BRPWHO國際內(nèi)毒素標準進展了標定，其效價表述為國際單位內(nèi)毒素/瓶。內(nèi)毒素的國際單位定義為指定質(zhì)量的國際標準物的活性。Forroutinepurposes,anotherpreparationofendotoxinmaybeused;providedithasbeenassayedagainsttheInternationalStandardforEndotoxinortheBRPanditspotencyisexpressedinInternationalUnitsofendotoxin.BRP進展了標定，且其效價表述為國際單位內(nèi)毒素。NOTE:1InternationalUnit(IU)ofendotoxinisequalto1EndotoxinUnit(E.U.)注：1國際單位〔IU〕1內(nèi)毒素單位〔EU〕。WATERFORBET細菌內(nèi)毒素測試用水WaterforBETissterilewaterthatisfreeofdetectablelevelsofendotoxin.Usuallyitiscommerciallyavailableandcertified.BET測試用水為無菌水，內(nèi)毒素低于可檢測水平。通常可能商業(yè)選購獵取并有證書認可。Generalchapter2.6.14.BacterialendotoxinsindicatesthatmethodsotherthantripledistillationmaybeusedtopreparewaterforBET.Reverseosmosishasbeenusedwithgoodresults;someanalystsmayprefertodistillthewatermorethan3times.Whatevermethodisused,theresultantproductmustbefreeofdetectablebacterialendotoxins.2.6.14“細菌內(nèi)毒素”說明三次蒸餾以外的方法也可以用于制備BET檢測用3次以上的水。不管使用哪種方法，所用的水中細菌內(nèi)毒素必需低于可檢測水平。pHOFTHEMIXTUREPH值Inthetestforbacterialendotoxins,optimumgel-clotoccursforamixtureatpH6.0-8.0.However,theadditionofthelysatetothesamplemayresultinaloweringofthepH.pH6.0-8.0.固然，將鱟試劑參與pH值降低。VALIDATIONOFTHELYSATE鱟試劑的驗證Itisimportanttofollowthemanufacturer’sinstructionsforthepreparationofthesolutionsofthelysate.要依據(jù)生產(chǎn)商的指示對制備鱟試劑溶液。Thepositiveend-pointdilutionfactorsingel-clotmethodsAandBareconvertedtologarithms.Thereasonisthatifthefrequencydistributionoftheselogarithmicvaluesisplotted,itusuallyapproachesanormaldistributioncurvemuchmorecloselythanthefrequencydistributionofthedilutionfactorsthemselves;infactitissosimilarthatitisacceptabletousethenormalfrequencydistributionasamathematicalmodelandtocalculateconfidencelimitswithStudent’st-test.AB中的陽性終點稀釋因子轉(zhuǎn)換成log對數(shù)。緣由是假設(shè)這些對數(shù)值的頻數(shù)分布畫出后，通常會比稀釋因子本身的頻數(shù)分布更接近正態(tài)分布曲線。由于其格外的相像，因此可以使用正態(tài)分布作為數(shù)學(xué)模型，并承受t檢驗來計算置信限度。PRELIMINARYTESTFORINTERFEREINGFACTORS干擾因素初步測試Somesubstancesorproductscannotbetesteddirectlyforthepresenceofbacterialendotoxinsbecausetheyarenotmisciblewiththereagents,theycannotbeadjustedtopH6.0-8.0ortheyinhibitoractivateenzymaticreaction(suchasβ-D-glucans).pH6.0-8.0，或它們的性質(zhì)或活化酶反響〔例如β-D-葡聚糖〕。Thereforeapreliminarytestisrequiredtocheckforthepresenceofinterferingfactors;whenthesearefoundtheanalystmustdemonstratethattheproceduretoremovethemhasbeeneffectiveandthatbyapplyingthisprocedure,anybacterialendotoxinspresenthavenotbeenremoved.菌內(nèi)毒素。Theobjectofthepreliminarytestistotestthenullhypothesisthatthesensitivityofthelysateinthepresenceofthesubstanceorproducttobeexamineddoesnotdiffersignificantlyfromthesensitivityofthelysateintheabsenceoftheproduct.AsimplecriterionisusedinmethodsAandB:thenullhypothesisisacceptedwhenthesensitivityofthelysateinthepresenceoftheproductisatleast0.5timesandnotmorethantwicethesensitivityofthelysatebyitself.〔原假設(shè)即AB中使用了一個簡潔的標準：當鱟試劑在有產(chǎn)品存在時，其靈敏度至少到達0.5倍，且2倍鱟試劑自身的靈敏度，則零假定成立。Thetestforinterferingfactorsingel-clotmethodsAandBrequirestheuseofasampleofthesubstanceorproductinwhichnoendotoxinsaredetectable.Thispresentsatheoreticalproblemwhenanentirelynewproducthastobetested.Hence,adifferentapproachwasdesignedforquantitativemethodsC,D,EandF.AB中干擾因素測試要求使用原料藥或制劑的樣品，其中不允許C、D、EF中，設(shè)計了不同的方法。NotethatmethodsDandE,whichusedachromogenicpeptide,requirereagentsthatareabsentinmethodsA,B,CandF,andhencecomplianceofmethodsA,B,CorFwiththerequirementsforinterferingfactorscannotbeextrapolatedtomethodDormethodEwithoutfurthertesting.留意，方法DE使用了色譜多肽，要使用方法A、B、CF中不曾使用的試劑，因此在沒有進一步測試時，方法A、B、CF符合干擾因素要求的結(jié)論就E。REMOVALOFINTERFERINGFACTORS干擾因素的消退Theprocedurestoremoveinterferingfactorsmustnotincreaseordecrease(forexample,byadsorption)theamountofendotoxininthesubstanceorproducttobeexamined.Thecorrectwayofcheckingthisistoapplytheprocedurestoaspikedsampleofthesubstanceorproducttobeexamined,thatis,asampletowhichaknownamountofendotoxinhasbeenadded,andthentomeasuretherecoveryoftheendotoxinaftertheremovalprocesshasbeenconducted.〔例〕。正確的檢查方法是使用受檢原料藥或制劑的加標樣品來測試，內(nèi)毒素的回收率。MethodsCandD.CDIfthenatureoftheproducttobeexaminedresultsinaninterferencethatcannotberemovedbyclassicalmethods(e.g.dilutionorcentriguation),itmaybepossibletodeterminethestandardcurveinthesametypeofsubstanceorproductfreedfromendotoxinsbyappropriatetreatmentorbydilutionofthesubstanceorproduct.Theendotoxinstestisthencarriedoutbycomparisonwiththisstandardcurve.〔或離心測試。Ultrafiltrationwithcellulosetriacetateasymmetricmembranefiltershasbeenfoundtobesuitableinmostcases.Thefiltersmustbeproperlyvalidated,becauseundersomecircumstancecellulosederivatives(β-D-glucans)cancausefalsepositiveresults.器必需經(jīng)過適當驗證，由于在有些情形下，纖維素衍生物〔β-D-葡聚糖〕可能會導(dǎo)致假陽性結(jié)果。Anotheroptiontoremoveinterferingfactorsisa2-stepprocedureinwhich1)endotoxinwithintheinterferingsampleisfixedonasolidphase,and2)afterremovaloftheinterferingsubstance(e.g.bywashing)theendotoxinisdetectedunimpairedundersuitabletestingconditions.〔例如，通過洗滌〕后，在穩(wěn)定的測試條件下進展不受影響的測試。THEPURPOSEOFTHECONTROLS把握的目的ThepurposeofthecontrolmadeupwithwaterforBETandthereferencepreparationofendotoxinattwicetheconcentrationofthelabeledlysatesensitivityistoverifytheactivityofthelysateatthetimeandundertheconditionsofthetest(formethodAandB).ThepurposeofthenegativecontrolistoverifytheabsenceoftadetectableconcentrationofendotoxininthewaterofBET.BET用水和內(nèi)毒素比照品在標示2倍濃度的把握所要到達的目的是確認鱟試劑在測試的條件下測試的時間內(nèi)的活性〔AB〕。BET用水中沒有可檢出的內(nèi)毒素濃度水平。Thepositivecontrol,whichcontainstheproducttobeexaminedattheconcentrationusedinthetest,isintendedtoshowtheabsenceofinhibitingfactorsatthetimeandundertheconditionsofthetest.測試時間內(nèi)沒有抑制因子。READINGANDINTERPRETATIONOFRESULTS結(jié)果讀數(shù)和詮釋MinuteamountsofthebacterialendotoxininthewaterforBET,orinanyotherreagentormaterialtowhichthelysateisexposedduringthetest,mayescapedetectionaslongastheydonotreachthesensitivitylimitofthelysate.However,theymayraisetheamountofbacterialendotoxininthesolutioncontainingthesubstanceorproducttobeexaminedtojustabovethesensitivitylimitandcauseapositivereaction.BET用水中，或在其它試劑或鱟試劑在測試中會暴露的物料中的微量細菌內(nèi)陽性反響。TheriskofthishappeningmaybereducedbytestingthewaterforBETandtheotherreagentsandmaterialswiththemostsensitivelysateavailable,oratleastonethatismoresensitivethantheoneusedinthetestontheproduct.Eventhen,theriskofsucha‘falsepositiveresult’cannotberuledoutcompletely.〔或者至少比產(chǎn)品測試所用鱟試劑更靈敏的鱟試劑BET測試，可以降低上述狀況發(fā)生的風(fēng)險。即使是這樣，發(fā)生“假陽性結(jié)果”的風(fēng)險仍不能完全排解。REPLACEMENTOFMETHODSPRESCRIBEDINMONOGRAPHS各論中所述方法的取代方法12-1.REPLACEMENTBYANOTHERPH.EUR.METHOD使用另一歐洲藥典方法取代AsstatedintheGeneralNotice,thetestmethodsgiveninmonographsandgeneralchaptershavebeenvalidatedinaccordancewithacceptedscientificpracticeandcurrentrecommendationsonanalyticalvalidation.Themethodsdescribedingeneralchapters2.6.14.Bacterialendotoxinsand2.6.30.Monocyte-activationtestthereforedonothavetobere-validatedperse,otherthaninconsiderationoftheiruseforaspecificsubstanceorproductinaspecificanalyticalenvironment.分析驗證建議經(jīng)過了驗證。在通論2.6.14“細菌內(nèi)毒素”和2.6.30“單核細胞激時適用性需要考慮者以外。Theprocedureandthematerialsandreagentsusedinthemethodmustbevalidatedasdescribedforthetestconcerned.Theabsenceofinterferingfactors(and,ifnecessary,theprocedureforremovingthem)isverifiedonsamplesofatleast3productionbatches.3〔的程序〕。Thenecessaryinformationissou