PDA TR07除热原-1985（中英文）

PDA技术报告----7#第一章DepyrogenationPDA技术报告----7#第一章THISISTHESEVENTHINASERIESOFTECHNICALREPORTS.1-6.Thistechnicalreportdescribesthedifferentmethodsofdepyrogenatingsolutionsanddevices.Sincedepyrogenationisafieldcharacterizedbyrapidtechnologicaladvances,andduetothelimitationsimposedbytimeandspace,amorecomprehensivetreatmentofthemanyareasofendotoxindepyrogenationwasnotpossible.Sufficeittosay,thisreportshouldserveasastimulustoscientistsinvolvedwiththecontrolofendotoxinandwhodesiretoincreasetheirknowledgeofthestructureandfunctionofthebacterialtoxinanddepyrogenation.Thisreport,therefore,documentsthestate-of-the-artofdepyrogenationtotheextentthatthelimitationsstatedabovepermitted.Itisourhopethatitwillalsoencourageresearchinallaspectsofdepyrogenation.本书是这一技术报告丛书中的第七部分，主要阐述了各种除热源的方法及设备。除热源研究是一门快速发展的技术，因时间有限，本书讨论范围未能全面包括除热源的各方面，本书仅作为与研究内毒素控制和希望在内毒素及热源研究领域拓展知识的科研工作者们的互勉。因此，本书是在以上所述范围限制以内，对去除热源前沿方法的研究。我们希望，本书在除热源领域各方面的研究发展中起到推动作用。ThereportwaspreparedbytheDepyrogenationSubcommitteeunderthechairmanshipofFrederickC.Pearson.本书由除热源小组委员会在FrederickC.Pearson.主席的主持下编撰完成。Theinformationcontainedinthisreportshouldbetreatedasaguidelineonly,andnotasaseriesofrequirements.ThistechnicalreportisonlyintendedtoprovideinformationtothePDAmembershipandshouldnotbeconstruedasofficialrecommendationofthePDA.本书编撰目的是为注射用药物协会成员提供相关研究信息，而并非官方推荐文件。因此本书内容仅为参考意见，并非强制要求。研究委员会主席弗洛伊德·本杰明PDA技术报告----7#第一章Introduction简介 1Endotoxin:ItsStructureandFunctionWithRespecttoDepyrogenation内毒素的结构及其功能 1PyrogenRemovalbyUltrafiltration超滤法除热源 15ApplicationofReverseOsmosisinPyrogenRemoval反渗透法在除热源中的应用 28DepyrogenationbyDistillation蒸馏法除热源 37Charge-ModefiedMedia荷电介质 45DepyrogenationbyMicroporousMembraneFilters微孔滤膜除热源 54DepyrogenationbySinteredActivatedCarbon烧结活性炭除热源 70DetoxificationofEndotoxinbyAcidandBase酸碱法去除内毒素毒性 78DepyrogenationbyHydrogenPeroxide过氧化氢法除热源 84InactivationofEndotoxinbyPolymyxinB多粘菌素B去内毒素活性 93DepyrogenationbyLimulusAmebocyteLysate鲎变形细胞溶解物除热源 98DepyrogenationbyDryHeat干热法除热源 101DepyrogenationbyMoistHeat湿热法除热源 109PDA技术报告----7#第一章InactivationofEndotoxinbyEthyleneOxideSterilizationandCobalt60Irradiation环氧乙烷灭菌法及放射性钴-60辐射法去内毒素活性 113PDA技术报告----7#第一章CHAPTER1ENDOTOXIN:ITSSTThischapteraddressesthestructureandchemistryofendotoxinastheyrelatetoveriousmodesofdestroyingorremovingpyrogen.Endotoxinisthemostsignificantpyrogeninthehealthcareinductry.Methodsforitsremovalcanbebasedonsize,molecularweight,charge,andtheendotoxin'shydrophobicLipidAcenter.Inadditiontoremoval,endotoxincanalsobedetoxifiedbyanyofanumberofchemicaltreatmentsthateitherbreaklabilechemicalbondsorblocksitesrequiredforpyrogenicactivity.本章主要介绍内毒素的结构和化学性质，基于这些性质才能讨论破坏或消除热原各种方法。内毒素是在医药健康行业中最常见的一种热原，其去除方法可根据：分子大小，分子量，电荷，内毒素类脂A疏水中心。除去除法以外，也可通过破坏不稳定化学键或阻断内毒素活性位点的方式消除内毒素的毒性，已达到除热源的目的。Becauseendotoxinsareassociatedwithgram-negativebacteria(GNB),theyareubiquitousand,likebacteria,arefoundinair,water,andfood.Duetotheirubiquity,relativeheatstability,andabilitytocauseprofoundphysiologicalchangeswhenadministeredparenterally,theirdetectionandcontrolisofparamountconcerntothemanufacturerofparenteralproducts.内毒素存在于革兰氏阴性菌。如细菌一样，内毒素在空气里、水里、食物里，几乎无处不在。因为内毒素普遍存在，且相对热稳定性，非肠道给药时又可引起强烈的生理反应，所以引起了许多非肠道产品厂家的极大重视。Becauseoftimeandspaceconstraints,thefollowingdiscussionisnotintendedtobeacomprehensiveorexhaustivetreatmentofthemanyareasofendotoxindepyrogenation,butrathertoserveasastimulustothoseindividualswhoareinvolvedwiththeroutinecontrolofendotoxinandwouldliketoextendoramplifytheirappreciationofthestructureandfunctionofthisbacterialtoxin.由于时间空间的限制，以下讨论并不会全面详尽地涉及去内毒素方法的各领域，本书仅将与那些致力于控制内毒素方法研究和那些希望不断了解细菌毒素结构和作用研究的同仁共勉。Byandlarge,thepurposeofthismonographistwofold:1.Topresentastate-of-the-artdocumentondepyrogenation.2.Toencouragepotentialresearchanddevelopmentinaspectsofdepyrogenation.综上所述，本论著主要有以下两方面目的：PDA技术报告----7#第一章1、贡献一份前沿的去热源研究方案2、希望对未来更进一步的去热源研发工作起到推动作用Endotoxinsarehigh-molecular-weightcomplexesassociatedwiththeoutermembraneofgramnegativebacteria(GNB),asshowninFigure1.Thesetoxinsareconstantlyshedintotheenvironmentofthebacterium,muchlikethedailysheddingofsuperficiallayersofhumanskin.Whenthebacteriumundergoesautolysis,allendotoxinisreleasedfromthecell.Unpurifiedendotoxinscontainlipid,carbohydrate,andprotein,buthighlypurifiedendotoxinsdonotcontainproteinand,therefore,arereferredtoaslipopolysaccharides(LPS)toemphasizetheirchemicalnature.Becausetheunpurifiedsubstanceisencouteredinthepyrogentestingofin-processandfinishedpharmaceuticalproducts,thetermendotoxinwillbeusedinthefollowingdiscussionunlessotherwisewarranted.内毒素是一种高分子量的化合物，附着于革兰氏阴性菌细胞外膜上，如图1。就如人的表皮每天脱落一样，这些细胞毒素不断脱落到细胞环境中，当菌体发生自溶时，内毒素便完全释放出来。未经纯化的内毒素由类脂、糖类及蛋白质组成，而经纯化的内毒素不含蛋白质，仅由类脂和糖组成，因此又称为脂多糖（LPS以强调其化学性质。由于一般在医药中间体和制剂中热源通常以未经纯化的内毒素形式存在，因此以下讨论中除特别说明，均使用“内图1革兰氏阴性菌细胞膜示意图EndotoxinisolatedfromtheoutermembraneofGNBhasthreedistinctchemicalregions.TheinnermostiscomposedofLipidA,whichislinkedtoacentralpolysaccharidecore.Thecoreis,inturn,linkedtolong,whisker-likeprojections,theO-antigenicsidechains.AnexampleisgiveninFigure2.自革兰氏阴性菌细胞外膜表层分离而得的内毒素由三个特征区域构成：最内层是类脂A，往外是核心多糖，再依次向外连接着晶须状的O-特性链。例如图2。PDA技术报告----7#第一章图2内毒素脂多糖O-特性链结构图例Ithaslongbeenrecognizedthatendotoxincausesfever.Inmorerecentyears,endotoxinhasalsobeenshowntohaveaprofoundeffectonabroadspectrumofbiologicalactivities,someofwhichwillbediscussedbelow.RecentinvestigationshavefoundthatvirtuallyallbiologicalactivityofendotoxinresidesintheLipidAportionofthemolecule.Whileendotoxinisresponsibleinlargepartfortheantigenicindividualityofgram-negativebacteriaandisthusresponsibleforthousandsofindividualserotypes,anotherportion,thecore,isremarkableuniforminmanyratherdiversegroupsofGNB.Thus,endotoxincontributestoboththeantigenichomogeneityandtheheterogeneityofGNB.Itwasundoubtedlythiscombinationofendotoxin'snovelchemistryanddiversebiologicalpropertiesthatledIvanBennett,arenownedendotoxininvestigator,toconcludethat"endotoxinspossessanintrinsicfascinationthatisnothinglessthanfabulous(1)".众所周知，内毒素可引起发热。然而近几年的研究发现，内毒素有更为广泛的生物活性，接下来我们将要讨论其中部分性质。近期的研究表明，事实上几乎所有的生物活性都存在于类脂A部分。不同的内毒素活性形成了革兰氏阴性菌各种抗原性，因而才有了各种血清型；而另一方面，在不同种群的革兰氏阴性菌中，内毒素的核心多糖却又是同样的。因此，内毒素同时保证了革兰氏阴性菌抗原均质性和抗原异质性。正是由于内毒素这种全新的化学结构和多种多样的生物性质，使得IvanBennett这位著名的内毒素研究者说“内毒素这种物质有着惊人的巨大魅力”。Over30biologicalactivitieshavebeenfoundtobeinducedbytheLipidAmoietyofentotoxin.ApartiallistoftheseactivitiesisgiveninTableI.Althoughonofthemostoftenstudiedeffectsofendotoxinispyrogenicity,endotoxinalsohasthecapacity,insufficientdoses,toactivatethecoagulationsystem,altercarbohydrateandlipidmetabolism,activatecomplement,modifyhemodynamics,causeplateletaggregation,andreleasevasoactiveamines.Inadditiontomanyotherpathophysiologicalaberrations,itcaninducedisseminatedintravascularcoagulation,shock,andultimatelydeath.However,tothoseinthepharmaceuticalindustry,pyrogenicityremainsthemostcriticalaspectofitsbiologicalactivity.迄今为止，已发现的由类脂A部分而形成的内毒素活性已超过30种，其中部分见表1。目前研究最多的内毒素活性是致热源性，然而只要有足够的剂量，内毒素也有很多其他生物活PDA技术报告----7#第一章性，如促进凝固系统、改变糖类和脂类代谢，激活补体，改善血液动力，促进血小板凝固，释放血管活性胺等。除引起许多生理性异常以外，内毒素还可引起弥散性血管内凝血、中风甚至死亡。不过，对于药学领域工作者，内毒素的致热源性依旧是最关键的生物活性。LocalShwartzmanreacBonemarrownecrosiEmbryonicboneresorptionDepressionofbloodpressure血压降低PlateletaggregationHagemanfactoractivation海格Inductionofplasminogenactivator诱导产生纤溶酶原激活物Enhanceddermalreactivitytoepinephrine增强肾上腺素皮肤反应(?)InductionofnonspecificresistancetoinfectionInductionoftolerancetoendotoxin引Inductionofearlyrefractorystatetotemperaturechange引发体温变化的早期耐受(?)Mitogenicactivityforcells促细胞有Inductionofcolonystimulatingfactor催生克隆刺激因子InductionofIgGsynthesisinnewbornmice促进新生小鼠体内lgG合成Inductionofinterferonproduction诱发干扰素生成Inductionoftumor-necrotizingfactor诱发肿瘤坏死因子Inductionofmouseliverpyruvatekinase诱发大鼠肝脏丙酮酸激酶Helperactivityforfriendspleenfocus-forming（？Inhibitionofphosphoenolpyruvatecarbonxykinase促生磷酸烯醇丙酮酸羧激酶LipidAiscomposedofasimpledisaccharideofglucosamine,whichishighlysubstitutedwithamine-linkedandester-linkedlongchainfattyacids(Fig.3).Themostcommonamine-linkedfattyacidisa14-carbon-chainfattyacid,β-hydroxymyristicacid.Ester-linkedfattyacids,whichtendPDA技术报告----7#第一章tobemorevariable,commonlyincludecapric,lauric,myristic,palmitic,andstearicacids.Thesefattyacidsaresaturated,straight-chained,andhaveevennumbersofcarbonatomsintheirchains.Removaloftheeater-linkedfattyacidresiduescanbeaccomplishedbytreatmentwithdilutebase,whichabrogatesthebiologicalactivityofLipidA.Treatmentwith0.25NNaOHfor5minat56℃resultsintheliberationofβ-hydroxymyristicacidwithoutthelossofpyrogenicity,butextendedtreatment(for30-60min)leadstothelossofalltheeater-linkedfattyacidsandadrasticreductioninpyrogenicity(2).Effectivedepyrogenationhasbeenreportedusing0.1NNaONat30℃for72hr(3)or4℃for16hr(4).类脂A以高度脂化的葡萄胺二糖为单位，侧链脂肪酸由胺键或脂键连接（如图3）。其中大多数胺联的侧链14烷基脂肪酸：β-羟基十四烷酸。脂联脂肪酸则较为多变，通常有羊蜡酸、月桂酸、豆蔻酸、硬脂酸、棕榈酸等，大多是有多个碳原子的长链饱和脂肪酸。用碱稀释液处理，去除这些脂联脂肪酸残基，可起到破坏类脂A生物活性的作用。实验证明，在56°C下用0.25N的NaOH处理5分钟，可解离β-羟基十四烷酸侧链，但不破坏致热源性；延长处理时间（30-60分钟可完全断裂所有脂联键，从而显著降低致热源性；而据报道，用0.1N的NaOH在30°C下处理72小时或4°C下处理16小时，可有效除去热源。Thereisaglycosidelinkbetweeneachdisaccharideunitviathecarbon1(C1)ofoneunitandcarbon6(C6)ofanadjacentunit.AlthoughunitsaregenerallylinkedbyaphosphodiesterbondbetweenC1ofonemoleculeandC4ofanadjacentmolecule,theC4phosphateissubstitutedwith10%to20%aminoarabinoseinsomecases(4).在二糖单元之间都有糖苷键连接，连接的方式是一个二糖单元的C1与另一个二糖单元的C6(?)相连。大部分的C1和相邻基团C4之间都是以磷脂键相连，但也有10-20%的C4磷酸基被阿拉伯酸酰胺取代的情况。图3类脂A的结构PDA技术报告----7#第一章Themajorityofgram-negativebacteria(GNB)haveacompleteLPSandarereferredtoassmooth.However,inrecentyears,anumberofbacterialisolateshavebeenobtainedthatlackvariouspolysaccharides,includingO-specificsidechainsandcorecomponents.SuchbacteriaarecalledR-mutants(rough)andareincapableofsynthesizingvariousportionsofcoreLPSorO-specificsidechainpolysaccharide.Dependingontheextentofthemutation,mutantsaredesignatedRa(themostcompletecorepolysaccharide)throughRe(themostdeficient,havingonlyKDO).TherangeofthesemutantscanbeseeninFig.4.PurifiedLPSisolatedfromsuchmutantshasbeeninvaluableindeterminingthechemicalstructureofLPSandtheassociatedfunctionsofLipidA.BecausetheRe595mutantofSalmonellaminnesotacontainsonlyLipidAandKDO,ithasbeenusedextensivelyinexperimentsdesignedtodemonstratethatthebiologicalactivityofLPSisassociatedwithLipidA.大多数革兰氏阴性菌都有完整平滑的脂多糖成分。但是，近几年的研究表明，也存在部分不包含多糖成分（包括O-特异性多糖链和核心多糖）的菌体，我们称这种细菌为R-突变体（非平滑这种细菌本身也无法自主生成脂多糖核心成分及O-特异性多糖链。根据突变的程度，可将突变菌从Ra至Re命名，其中Ra指核心多糖成分完整，Re指仅含KDO（3-脱氧-D-甘露糖-辛酮糖）即缺陷程度最大的一类。突变程度见图4。从这些突变体中分离纯化出的LPS是研究LPS化学结构和类脂A功能的重要依据。沙门氏菌Re595突变体仅含类脂A和KDO，因此这种菌体被广泛运用于验证类脂A是LPS的生物活性中心的实验中。图4沙门氏突变体中分离出的LPS化学架构Typically,LipidAislinkedtocoreheteropolysaccharidesbyKDO,an8-carbon-sugaracid,whichisuniquetobaterialLPS.Inaqueoussystems,KDO,alongwiththeremainingcorePDA技术报告----7#第一章polysaccharides,actsasasolutecarrierforthelipidicportion.BecauseLipidAislinkedtothecoreLPSbyanacid-labileketosidiclinkagetoKDO,depyrogenationcanbeaccomplishedbyusingacid.Galanos,etal.havepreparedfreeLipidAfromS(wildtype)andRLPS(endotoxinslackingO-specificsidechainsaswellascoredeletions)bymildacidhydrolysisusing0.1Naceticacidat100℃forvariableperiodsoftime(5).BecausefreeLipidAisinsoluble,itisnotpyrogenic.However,whenfreeLipidAcombinedwithbovineserumalbumin,itcausespyrogenicitycomparabletothatofintactendotoxin,therebydemonstratingthattheportionoftheendotoxinmoleculeresponsibleforpyrogenicactivityisLipidA(6).Basedonthisacid-labilelinkagebetweenLipidAandKDO,0.05NHClfor30minat100℃(7)or1.0%glacialaceticacidfor2-3hrat100℃(8)havebeenusedfordepyrogenation.Onedisadvantageofthismethodistheratherharshtemperaturerequirementcomparedtothe56℃requirementreportedforalkalinedepyrogenation.Acid/baseddepyrogenationisdealthwithinChapter8.一般来说，细菌脂多糖中的类脂A通过KDO(一种8碳糖酸)与特异性的核心杂多糖相连。在水溶液环境中，KDO及残余的核心多糖，可对脂类物质起到溶质运载作用。类脂A经由一种酸不稳定性酮苷键与LPS核心多糖的KDO连接，因此可以通过应用这种酸处理的方法除热源。据Galanos等人的实验报道，100°C条件下使用0.1N的醋酸分别处理不同时间，可得到S型（原内毒素）至R型（不含O-抗原和核心多糖）的游离类脂A，而游离的类脂A的不溶性恰是除热源的方法所在。然而，当游离的类脂A与牛血清蛋白结合后，即可引发与原内毒素几乎相同的致热源性，因此可证明，内毒素分子的致热源成分就是类脂A。基于这种解离类脂A和KDO之间连接键酸的不稳定性，可100°C下0.05N的HCL处理30min，或100°C下1%冰醋酸处理2-3小时的条件去除热源。与56°C碱处理法除热源相比，酸法除热源的一个弊端是高温处理，对设备要求较高。本书第八章将详细讨论酸碱除热源法。AnumberofstudieshaveshownthatthecationicantibioticpolymyxinB(PMB)canabrogatetheactivityofLPS(9,10).ThecontroversysurroundingdepyrogenationusingPMBwillbedealtwithindetailinChapter10.WecanbrieflynotethatonestudyfoundPMBbindsstoichiometricallytotheLipidAregionofLPS.AnumberofotherstudieshavesuggestedthatsignificantquantitiesofPMBmustbepresentforpyrogentomodifyLPSactivity,andonereportshowedthattheLALassayisnotinhibitedataconcentrationof3.4mg/mlofPMB.AdiagrammaticrepresentationoftheinteractionbetweenLipidAandPMBisgiveninFig5,asaretheactionsofacidandbaseontheLipidAregion.许多研究表明，阳离子抗菌多粘菌素B(PMB)能够消除LPS活性，详细讨论见第十章。有研究表明，PMB可与LPS的类脂A部分以1:1比例结合；也有许多研究表示用PMB干扰LPS活性法除热源时PMB的使用量必须非常大；另外还有研究提出，PMB浓度达3.4mg/ml时内毒素鲎试剂反应也不会受到抑制作用。图5表明了类脂A和PMB、酸、碱之间的反应图示。PDA技术报告----7#第一章图5PMB、酸、碱与类脂A的相互作用Theaggregationstate(molecularweight)ofendotoxinisacriticalfunctionofbiologicalactivity,asdemonstratedbyLuderitzetal(11)whoworkedwithvarioussaltformsofahighlypurifiedLPSfromSalmonellaabortusequi.TheaggregationofendotoxinisafunctionoftheLipidAmoietytointeracttoformhighermolecularweightaggregates,andoftheelectrostaticnatureofcationiccomponentsassociatedwiththeacidicphosphateandcarboxylgroupsofthemolecule.Monovalentanddivalentinorganiccationsandpolyvalentorganiccations,suchasethanolamine,putrescine,cadaverine,spermine,andspermidine,allofwhichaffectsolubility,canbeassociatedwithunrefinedendotoxin.Luderitzetal(11)demonstratedthatwhenahighlypurifiedLPSwasconvertedtotriethylamine,sodium,orputrescinesalt,respectively,themolecularweightincreasedandwatersolubilitydecreased.Asthemolecularweightincreased,toxicityinrats,rateofclearancefromblood,interactionwithcomplement,andaffinityforcellsalsoincreased,butlethalitydecreasedinmiceasdidpyrogenicityinrabbits.ItisinterestingthatLALactivitydidnotappeartobesignificantlyalteredwithachangeinaggregation,suggestingthatLALmaycontainanendotoxin-disaggregatingagent,whichisactiveduringincubationofthetestsample.TherelationshipbetweenmolecularweightandbiologicalactivitycanbeseeninTableII.Themolecularweightofagivenendotoxinpreparationiscriticaltothebiologicalactivityofthatpreparation.However,substancesthateitherreducesurfacetension(suchasTween20)orsequesterdivalentcations(suchasethylenediaminetetraaceticacid,EDTA)willdecreasemolecularaggregationasmeasuredbyLALactivity.Luderitz等人研究了产自沙门氏菌的纯化LPS的各种盐，他们的研究表明内毒素聚合态是其生物活性的关键因素。内毒素聚合物是由于类脂A部分相互作用，及磷酸、羧酸基团的阳离子部分的静电作用而形成的。单价、二价的无机阳离子和多价有机阳离子都可通过改变可溶性而与未处理的内毒素发生作用，如乙醇胺、腐胺、戊二胺、精胺、亚精胺。Luderitz等的研究表明，如LPS纯化物转化为三乙胺盐、钠盐或丁二胺盐，分子量增加的同时，水溶性将降低，此时，大鼠体内毒性、血液清除率、补体相互作用以及细胞亲和性都有所增加，PDA技术报告----7#第一章但小鼠致死率和兔体内致热源性则有所降低。值得注意的是，多聚并不会显著改变鲎试剂活性，这表明，鲎试剂可能含有一种抗内毒素聚集的成分，这种成分有可能在样品孵化期发生作用。表二列出了内毒素分子量和生物活性之间的关系，特定内毒素生物活性与其分子量密切相关。降低表面张力的物质（如吐温20）或螯合二价阳离子（如EDTA）将降低分子聚集，这种现象可由鲎试剂活性体现。TheLipidAportionofendotoxinhasalsoprovedcentraltothepotentialuseofdepyrogenationbymicroporousmembranefilters,whichcanabsorbbacterialendotoxinbythehydrophobicinteractionoftheLipidAmoietywiththeunchargedpolymersurface.Thisexcitingnewmethodology,discussedinChapter6,ispresentedforthefirsttimeinthismonograph.使用微孔滤膜法除热源也与内毒素的类脂A结构十分相关，因为这种方法是通过不带电荷的滤膜聚合物表面与类脂A的疏水作用吸附细菌内毒素，从而达到去除热源的目的，这种新兴方法将在本书中首次介绍，详见第六章。表2内毒素脂多糖的分子量与生物活性的关系基于内毒素生物分子特性去除热源Inthepresenceofmagnesiumandcalcium,LPSformsbilayersheetsorvesicleswithadiameterontheorderof0.1um.Whenendotoxinpreparationsarereducedbyachelatingagentorsurfactant,rod-shapedsubunitsappear,whichhaveamolecularweightofapproximately20,000daltons,adiameterof8-12angstroms,andalengthof200-700angstroms.Thesestructuresareinterconvertible,ashasbeendemonstratedusingmolecularfiltration(12).StudiesbySweadneretal(13)haveshownthatthevesiclesandbilayersheetswillpassthrougha0.22-ummembranefilter,butwillberetainedbya0.025-umporesize.Theyshowedthatthesmallmicellarformwillpassthrougha0.02-ummembranefilter,butwillberetainedbya1,000,000nominalmolecularweightlimit(nmwl)molecularfilter.WhenLPSisreducedtoitssmallestsubunitbyasurface-activeagent,suchassodiumdeoxycholate,itwillpassthroughamolecularfilterwithanmwlof1,000,000,butwillberetainedbya10,000nmwlmolecularweightfilter.However,fromapracticalpointofview,endotoxincanbeconsideredtohaveamolecularweightof106daltonsinanaqueousenvironmentintheabsenceofsignificantlevelsofdivalentcationsandsurface-activePDA技术报告----7#第一章agents.Thus,thisistheapproximatemolecularaggregatethatismostfrequentlyencounteredinlarge-volumeparenteralsandmedicaldevicerinsesolutions.在有镁离子或钙离子的环境中，LPS会形成双分子层或直径约0.1微米的囊泡。当有螯合剂或表面活性剂存在时，内毒素会降解，形成一种直径约8-12A、长约200-700A分子量大约20000Da的杆状亚基，有研究说明这些结构之间可互相转化。Sweadner等研究表明，内毒素形成的双分子层和囊泡可通过0.025-0.22微米的滤膜，亚基微粒可通过1000000nmwl（标准分子量限制）--0.02微米的滤膜，如使用表面活性剂（如脱氧胆酸钠）将LPS还原成最小亚基，则可通过范围为10000-1000000nmwl。然而事实上，如果在没有一定含量的二价阳离子或表面活性剂存在的前提下，一般认为内毒素在水溶液中的分子量为106Da，这也是大多数注射剂中和药品生产设备清洗液中内毒素一般存在的聚合度。Basedonmolecularsizeofendotoxin,distillation,reverseosmosis,andultrafiltration(UF)havebeenusedforvariousapplicationsofdepyrogenation.UFmembranesarecomposedofpolymerstructureswithporesizelargerthanreverseosmosismembranes,butsmallerthanthoseinmicroporousfilters.Poresinthelatterareratedinmicrometers,whereasintheformertheyareratedinnominalmolecularweight.ThereareanumberofdepyrogenationapplicationsofUF.ThesearediscussedmorefullyinChapter2.ROmembranesarealsoexclusionmembranes,butbecauseoftheirsmallpores,havebeenusedforremovalofendotoxinfromwater.Evenlowmolecularweightsubstances,suchassalts,areexcludedwiththesemembranes,whichlimitstheiruseinthepharmaceuticalindustry.ROisdiscussedinChapter3.基于内毒素分子大小的除热源法有蒸馏、反渗、超滤等。其中超滤膜是一种聚合结构，其孔径大于以标称分子量衡量的反渗膜而小于以微米衡量的微孔滤膜。超滤法在除热源中的应用很多，详见第二章。反渗膜也是一种排阻过滤膜，但是由于孔径十分小，仅用于去水中内毒素，因为反渗膜可以截留盐类这样分子量很小的物质，所以在医药工业中应用有限。有关反渗膜除热源的讨论详见第三章。Distillation,whichisdealtwithinChapter4,isthetraditionalmethodfordepyrogenationofwaterandoneoftwoapprovedmethodsforthemanufactureofnonpyrogenicwater(WaterforInjection).Basedonthelargemolecularweightofendotoxinandthesmallmolecularweightofwater,onecaneasilyappreciatetheeffectivenessofdistillationinrenderingsourcewaternonpyrogenic.第四章讨论的蒸馏法是一种传统除热源方法，是目前两种药典认可的注射用水除热源法的其中之一。利用内毒素分子量大于水分子量的原理，可以轻松高效地对水源进行除热源。Inadditiontoremovalofendotoxinbasedonmolecularweight(size)orhydrophobicinteractionoftheLipidAportionwithahydrophobicsurface,endotoxinalsocontainsanetnegativecharge,whichcanbeusedforabsorptiontoapositivelychargedsurface.Chapter5coverscharge-modifiedfilters.Theabsorptiveremovalofbacterialendotoxinbyactivatedcarbonwasestablishedduringthe1940s,andisdealtwithinChapter7.除了利用分子量和利用类脂A疏水性去除内毒素的方法，还可利用内毒素带负电荷的性质，进行正电荷吸附，从而去除内毒素。第五章将讨论这种带电滤膜法。第七章则将阐述一种自PDA技术报告----7#第一章20世纪40年代便发展起来的另一种吸附去内毒素的方法：活性炭吸附法。Althoughitprobablyhaslittlepotentialvaluetothepharmaceuticalindustry,depyrogenationofvariouspreparationsutilizingthespecificinteractionbetweenendotoxinandLALhasbeenreported.However,themechanismofendotoxinremovalisnotwellunderstoodandhasbeenattributedtodetoxification.ThisissueisdiscussedinChapter11.利用内毒素和鲎试剂特异性反应去除热源的方法虽然利用价值不大，但在医药领域已常见报道，不过其中机理尚未明确，详细讨论见第十一章。Althoughnotdealtwithasaseparatetopicinthismonograph,depyrogenationbyglasswarewashingprocessvalidationhasbeenaddressedbyFeldsineandco-workers(14).用玻璃清洗法去除热源已见报道（Feldsine等但本书并未将其作为单独章节详述。Inbroadterms,depyrogenationcanbeaccomplishedbyeitheroftwogeneralapproaches--removalorinactivation.Treatmentofendotoxinwithacid,base,orPMBareexamplesofdepyrogenationbyinactivation.ThesemodeswerepreviouslyaddressedinthetextbecausetheiractionisrelatedtospecificportionsoftheLipidAcoreregion,whichisalsobeingdealtwith.Variousproceduresfordepyrogenationbyremovalhavealsobeendiscussed.Thesearepredicatedonsuchbiomolecularcharacteristicsofendotoxinaselectrostaticcharge,hydrophobicity,molecularweight,andaggregation.Inadditiontotheseapproaches,threegeneralcategoriesofdepyrogenationbyinactivationremaintobediscussed,i.e.,oxidation,alkylation,and"sterilization".总体上来说，热源可以通过脱除和灭活两种方法去除。灭活除热源法有酸处理、碱处理和PMB法，如前所述，这些方法均是与类脂A特异性反应而产生作用。而基于内毒素生物特性，如带电荷、疏水性、分子量及聚合等性质的脱除法除热源也已讨论过。除此之外，以下还有三种以灭活为原理的除热源法：氧化法、烷基化法和灭菌法。Oxidation氧化法Themoststudiedformofoxidativedepyrogenationistheuseofhydrogenperoxide,whichisdiscussedinChapter9.WhileanumberofinvestigatorshavestudiedthedepyrogenatingcapacityofH2O2,NovitskyandCase,asdiscussedinthatchapter,werethemostrecenttoextendthefindingsofearlierworkers.UsingsterileWaterforInjectionand10ng/mlofendotoxin,theseinvestigatorsclearlydemonstratedthatthedestructionofendotoxinbyH2O2istime,pH,andconcentrationdependent.Usingaslittleas2.7%H2O2at65℃for1hr,anapproximately90%reductionofendotoxinwasdemonstrated.WhentheH2O2wasincreasedto27%,virtually100%destructionofendotoxinoccurredwithin1hr.PDA技术报告----7#第一章在氧化法除热源中过氧化氢使用最多，详见第九章。在众多做此项研究的学者当中，Novitsky和Case的研究最近期，他们拓展了前人的研究结果，根据他们的研究，使用无菌注射用水和10ng/ml的内毒素为实验材料，结果显示，过氧化氢对内毒素的破坏作用取决于处理时间、pH值和过氧化氢浓度。在65°C条件下使用2.7%的过氧化氢处理1小时，约有90%的内毒素被破坏；而当将过氧化氢浓度提升至27%时，在1小时内几乎所有的内毒素都被破坏。Theauthorsrecognizethepotentialuseofthismodeofdepyrogenationforpyrogen-freeclosures,implantablemedicaldevices,andgelatinoussubstances.However,themethodisoflimitedutilityinthepharmaceuticalindustry.Studiesindicatethatpotassiumpermanganate,sodiumperiodateandhypochlorousacidcanalsobeusedforoxidativedepyrogenation.Theprecisenatureofoxidativedestructionofendotoxinisnotknown.作者强调了氧化法除热源在无热源密封、植入式医疗设备和凝胶物质中的应用前景。然而氧化法除热源在制药领域使用比较少。研究表明，高锰酸钾、高碘酸钠和次氯酸都可用于氧化法除热源，不过确切的作用机理尚不明确。Alkylation烷基化法Alkylativeinactivationofendotoxinisaprocessinwhichendotoxincanbetreatedbyanalkylatingagent,resultinginlossinpyrogenicity.Schenckandworkersdemonstrateda100-folddecreaseinpyrogenicitywhenendotoxinwastreatedwithaceticanhydride(15).Thesamegroupreporteda100-to1000-folddecreasewhenendotoxinwastreatedwithsuccinicanhydride.Themechanismbehindthis