ASTM F2131-2002(2007)e1 使用W-20老鼠基质细胞系的重组体人骨形态形成蛋白-2(rhBMP-2)在实验室条件下的生物活性的标准试验方法
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【英文标准名称】:StandardTestMethodforInVitroBiologicalActivityofRecombinantHumanBoneMorphogeneticProtein-2(rhBMP-2)UsingtheW-20MouseStromalCellLine
【原文标准名称】:使用W-20老鼠基质细胞系的重组体人骨形态形成蛋白-2(rhBMP-2)在实验室条件下的生物活性的标准试验方法
【标准号】:ASTMF2131-2002(2007)e1
【标准状态】:现行
【国别】:
【发布日期】:2002
【实施或试行日期】:
【发布单位】:美国材料与试验协会(US-ASTM)
【起草单位】:F04.42
【标准类型】:(TestMethod)
【标准水平】:()
【中文主题词】:
【英文主题词】:invitrobioactivityassay;recombinanthumanbonemorphogeneticprotein;W-20cellline
【摘要】:AlthoughthetestmethodcanbeusedforassessmentofthebioactivityofcrudepreparationsofrhBMP-2,ithasonlybeenvalidatedforusewithhighlypure(>98%byweightproteinpurity)preparationsofrhBMP-2.1.1Thistestmethoddescribesthemethodusedandthecalculationofresultsforthedeterminationofthein-vitrobiologicalactivityofrhBMP-2usingthemousestromalcelllineW-20clone17(W-20-17).ThisclonewasderivedfrombonemarrowstromalcellsoftheW++mousestrain.1.2Thistestmethod(assay)hasbeenqualifiedandvalidatedbasedupontheInternationalCommitteeonHarmonizationassayvalidationguidelines(withtheexceptionofinterlaboratoryprecision)fortheassessmentofthebiologicalactivityofrhBMP-2.Therelevanceofthisinvitrotestmethodtoinvivoboneformationhasalsobeenstudied.ThemeasuredresponseintheW-20bioassay,alkalinephosphataseinduction,hasbeencorrelatedwiththeectopicbone-formingcapacityofrhBMP-2intheinvivoUseTest(UT).rhBMP-2thatwaspartiallyorfullyinactivatedbytargetedperaceticacidoxidationofthetwomethionineswasusedasatooltocomparetheactivities.OxidationofrhBMP-2withperaceticacidwasshowntobespecificallytargetedtothemethioninesbypeptidemappingandmassspectrometry.ThesemethioninesresideinahydrophobicreceptorbindingpocketonrhBMP-2.Oxidizedsampleswerecomparedalongsideanincubationcontrolandanativecontrol.The62,87,98,and100%oxidizedsampleshadW-20activitylevelsof62,20,7,and5%,respectively.Theincubationandnativecontrolsamplesmaintained100%activity.SampleswereevaluatedintheUTandshowedasimilareffectofinactivationonbone-formingactivity.Thesampleswith62%and20%activityintheW-20assaydemonstratedreducedlevelsofboneformation,similarinlevelwiththereductioninW-20specificactivity,relativetotheincubationcontrol.Littleornoectopicbonewasformedinthe7and5%activerhBMP-2implants.1.3Thus,modificationstotherhBMP-2moleculeinthereceptorbindingsitedecreasetheactivityinboththeW-20andUTassays.ThesedatasuggestthatasinglereceptorbindingdomainonrhBMP-2isresponsibleforbothin-vitroandin-vivoactivityandthattheW-20bioassayisarelevantpredictorofthebone-formingactivityofrhBMP-2.1.4ThevaluesstatedinSIunitsaretoberegardedasstandard.1.5Thisstandarddoesnotpurporttoaddressallofthesafetyconcerns,ifany,associatedwithitsuse.Itistheresponsibilityoftheuserofthisstandardtoestablishappropriatesafetyandhealthpracticesanddeterminetheapplicabilityofregulatorylimitationspriortouse.
【中国标准分类号】:C05
【国际标准分类号】:07_080
【页数】:6P.;A4
【正文语种】:
【原文标准名称】:使用W-20老鼠基质细胞系的重组体人骨形态形成蛋白-2(rhBMP-2)在实验室条件下的生物活性的标准试验方法
【标准号】:ASTMF2131-2002(2007)e1
【标准状态】:现行
【国别】:
【发布日期】:2002
【实施或试行日期】:
【发布单位】:美国材料与试验协会(US-ASTM)
【起草单位】:F04.42
【标准类型】:(TestMethod)
【标准水平】:()
【中文主题词】:
【英文主题词】:invitrobioactivityassay;recombinanthumanbonemorphogeneticprotein;W-20cellline
【摘要】:AlthoughthetestmethodcanbeusedforassessmentofthebioactivityofcrudepreparationsofrhBMP-2,ithasonlybeenvalidatedforusewithhighlypure(>98%byweightproteinpurity)preparationsofrhBMP-2.1.1Thistestmethoddescribesthemethodusedandthecalculationofresultsforthedeterminationofthein-vitrobiologicalactivityofrhBMP-2usingthemousestromalcelllineW-20clone17(W-20-17).ThisclonewasderivedfrombonemarrowstromalcellsoftheW++mousestrain.1.2Thistestmethod(assay)hasbeenqualifiedandvalidatedbasedupontheInternationalCommitteeonHarmonizationassayvalidationguidelines(withtheexceptionofinterlaboratoryprecision)fortheassessmentofthebiologicalactivityofrhBMP-2.Therelevanceofthisinvitrotestmethodtoinvivoboneformationhasalsobeenstudied.ThemeasuredresponseintheW-20bioassay,alkalinephosphataseinduction,hasbeencorrelatedwiththeectopicbone-formingcapacityofrhBMP-2intheinvivoUseTest(UT).rhBMP-2thatwaspartiallyorfullyinactivatedbytargetedperaceticacidoxidationofthetwomethionineswasusedasatooltocomparetheactivities.OxidationofrhBMP-2withperaceticacidwasshowntobespecificallytargetedtothemethioninesbypeptidemappingandmassspectrometry.ThesemethioninesresideinahydrophobicreceptorbindingpocketonrhBMP-2.Oxidizedsampleswerecomparedalongsideanincubationcontrolandanativecontrol.The62,87,98,and100%oxidizedsampleshadW-20activitylevelsof62,20,7,and5%,respectively.Theincubationandnativecontrolsamplesmaintained100%activity.SampleswereevaluatedintheUTandshowedasimilareffectofinactivationonbone-formingactivity.Thesampleswith62%and20%activityintheW-20assaydemonstratedreducedlevelsofboneformation,similarinlevelwiththereductioninW-20specificactivity,relativetotheincubationcontrol.Littleornoectopicbonewasformedinthe7and5%activerhBMP-2implants.1.3Thus,modificationstotherhBMP-2moleculeinthereceptorbindingsitedecreasetheactivityinboththeW-20andUTassays.ThesedatasuggestthatasinglereceptorbindingdomainonrhBMP-2isresponsibleforbothin-vitroandin-vivoactivityandthattheW-20bioassayisarelevantpredictorofthebone-formingactivityofrhBMP-2.1.4ThevaluesstatedinSIunitsaretoberegardedasstandard.1.5Thisstandarddoesnotpurporttoaddressallofthesafetyconcerns,ifany,associatedwithitsuse.Itistheresponsibilityoftheuserofthisstandardtoestablishappropriatesafetyandhealthpracticesanddeterminetheapplicabilityofregulatorylimitationspriortouse.
【中国标准分类号】:C05
【国际标准分类号】:07_080
【页数】:6P.;A4
【正文语种】:
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