Designation:F2131−02(Reapproved2012)StandardTestMethodforInVitroBiologicalActivityofRecombinantHumanBoneMorphogeneticProtein-2(rhBMP-2)UsingtheW-20MouseStromalCellLine1ThisstandardisissuedunderthefixeddesignationF2131;thenumberimmediatelyfollowingthedesignationindicatestheyearoforiginaladoptionor,inthecaseofrevision,theyearoflastrevision.Anumberinparenthesesindicatestheyearoflastreapproval.Asuperscriptepsilon(´)indicatesaneditorialchangesincethelastrevisionorreapproval.1.Scope1.1Thistestmethoddescribesthemethodusedandthecalculationofresultsforthedeterminationofthein-vitrobiologicalactivityofrhBMP-2usingthemousestromalcelllineW-20clone17(W-20-17).ThisclonewasderivedfrombonemarrowstromalcellsoftheW++mousestrain.21.2Thistestmethod(assay)hasbeenqualifiedandvali-datedbasedupontheInternationalCommitteeonHarmoniza-tionassayvalidationguidelines3(withtheexceptionofinter-laboratoryprecision)fortheassessmentofthebiologicalactivityofrhBMP-2.Therelevanceofthisinvitrotestmethodtoinvivoboneformationhasalsobeenstudied.ThemeasuredresponseintheW-20bioassay,alkalinephosphataseinduction,hasbeencorrelatedwiththeectopicbone-formingcapacityofrhBMP-2intheinvivoUseTest(UT).rhBMP-2thatwaspartiallyorfullyinactivatedbytargetedperaceticacidoxida-tionofthetwomethionineswasusedasatooltocomparetheactivities.OxidationofrhBMP-2withperaceticacidwasshowntobespecificallytargetedtothemethioninesbypeptidemappingandmassspectrometry.ThesemethioninesresideinahydrophobicreceptorbindingpocketonrhBMP-2.Oxidizedsampleswerecomparedalongsideanincubationcontrolandanativecontrol.The62,87,98,and100%oxidizedsampleshadW-20activitylevelsof62,20,7,and5%,respectively.Theincubationandnativecontrolsamplesmaintained100%ac-tivity.SampleswereevaluatedintheUTandshowedasimilareffectofinactivationonbone-formingactivity.Thesampleswith62%and20%activityintheW-20assaydemonstratedreducedlevelsofboneformation,similarinlevelwiththereductioninW-20specificactivity,relativetotheincubationcontrol.Littleornoectopicbonewasformedinthe7an...
