贝伐珠单克隆抗体的HPLC定量检测方法的建立

★★*“”2011ZX09202－301－15＊＊[e-mail][e-mail]*＊＊361009HPLC－ProteinA。Ag-ilentBio－MonolithProteinAColumn5.2mm×4.95mm0.10mLPBS0.12mol·L－1pH7.4A0.5mol·L－1pH2.5B。AB1.0mL·min－125℃280nm。0.078～10.0mg·mL－1Ｒ2＞0.99998.32%～101.7%ＲSD≤1.5%ＲSD≤1.6%0.30μg0.90μgpH±0.2、A±10.0%、±5.0℃、±20.0%ＲSD0.39%～1.0%1.350min18h27ＲSD0.44%。HPLC－ProteinA。ProteinAAＲ917A0254－1793201506－1027－05doi10.16155/j.0254－1793.2015.06.15EstablishmentofanHPLCquantitativemethodforbevacizumab*ZOUYou－tuZHENGHe－dongＲUANKaMAYan－lingCHENXing＊＊SinoBiowayBiomedicineCo.Ltd.Xiamen361009ChinaAbstractObjectiveToestablishanHPLC－ProteinAquantitativemethodforthecontentdeterminationandqual-itycontrolofbevacizumabintheproductionprocess．MethodsAnAgilentBio－MonolithProteinAchromatographiccolumn5.2mm×4.95mm0.10mLwasadoptedwithPBScontaining0.12mol·L－1sodiumchloridepH7.4asthemobilephaseAand0.5mol·L－1aceticacidpH2.5asthemobilephaseB．AfterloadingthesamplethemobilephaseAwasfirstusedforwashingfollowedbyelutionwiththemobilephaseB．Theflowratewas1.0mL·min－1thecolumntemperaturewas25℃andthedetectionwavelengthwas280nm．ＲesultsThestandardcurvewascreatedusingtheconcentrationofbevacizumabasXaxisandthepeakareaasYaxis．Withintheconcentrationrangeof0.078－10.0mg·mL－1thecurvefittingwasgoodＲ2＞0.999．Therecoverywas98.32%－101.7%．TheＲSDofintrabatchprecisionwas≤1.5%andＲSDofinterbatchprecisionwas≤1.6%．TheLOQwas0.3μgandtheDOQwas0.9μg．IntheconditionthatpHvaried±0.2sodiumchlorideconcentrationinthemobilephaseAvaried±10%thecolumntemperaturevaried±5.0℃andtheflowratevaried±20%theＲSDofpeakareawas0.39%－1.01%．Theretentiontimeofbevacizumabinthefermentationbrothwas1.350minwhichwasconsistentwiththereferencestandards．Within18hoursthesamebatchoffermentationliquidwasdetected27timesandＲSDofthepeakareawas0.44%．ConclusionTheresultprovedthattheHPLC－ProteinAmethodissuitableforthequantitativedeterminationandqualitycontrolofbevacizumab．—7201—ChinJPharmAnal2015356KeywordsbevacizumabqualitycontrolofmonoclonalantibodiesProteinAchromatographiccolumnstaphylocco-cusaureusrosenbachProteinArapidquantitativedetectionHPLCmethodvalidation、1。bevacizumabvascularendothelialgrowthfactorVEGFVEGF2－4VEGFVEGFＲ－2VEGF25－6。LowryBCAELISA7。33。8ELISAμg。HPLC19859。Bio－MonolithProteinAColumn。ProteinAAIgGFc10。ProteinApHProteinA。HPLC－ProteinA2。HPLC－ProteinA、、、、、、、。1DIONEXUltiMate3000DADChromeleon7Milli-poreMilli－Q。1013608220140320、ProteinA20140320－1、20140320－31209071、130401112120922214250DACＲOS。22.12.1.1APBSpH7.40.2g、2.9g、7.0g1LB0.5mol·L－1pH2.56mL200mL。2.1.2。2.1.310.0mg·mL－1。2.1.4ProteinAProteinA2mol·L－1Tris－HClpH9.0pH7.01.0mLA10ProteinASPQ1.0mLA10。2.2AgilentBio－MonolithProteinA5.2mm×4.95mm0.10mL25℃100%A5min100%A0.5min100%B1.5min1.0mL·min－1280nm。510.0μL210.123min21.350min1。25—8201—ChinJPharmAnal20153561.350min22.99ＲSD0.38%HPLC－ProteinA20101.55ＲSD2.0%11。1HPLC－ProteinA5Fig．1ChromatogramsofbevacizumabdetectedbyHPLC－ProteinArepeat5times1．flowthroughpeak0.123min2．bevacizumabmainpeak1.350nm2.3111。0.123min。1.350min222.99。。2HPLC－ProteinAFig．2ChromatogramsofHPLC－ProteinAforthespecificitytestA．AmobilephaseAB．auxiliarysolutionC．fermentationbrothofblankcelllineD．bevacizumabfermentationbroth1．flowthroughpeak0.123min2．bevacizumabmainpeak1.350nm2.4310。0.30μg0.90μg。2.510.0mg·mL－10.078mg·mL－10.078～10.0mg·mL－18。Ｒ20.99960.078～—9201—ChinJPharmAnal201535610.0mg·mL－1Y=9.4761X－0.0385Ｒ2=0.99961.350min3。3HPLC－ProteinAFig．3ChromatogramsofHPLC－ProteinAforthedetectionofbevacizumabO．bevacizumabfermentationbrothA～H．0.078～10.0mg·mL－1referencestandardsofbevacizumab1．flowthroughpeak0.123min2．bevacizumabmainpeak1.350nm2.6312Pro-teinA3。4。310%15.0、20.0、25.0mg·mL－1110%3。298.3%～101.7%。1Tab．1Ｒecoverytestfordifferentsamplesolutionssampleadded/mgrecovery/%n=3ＲSD/%averagerecovery/%n=91230.0750100.71.50.100099.71.10.1250100.80.920.075098.70.730.100098.60.610.125098.30.450.0750101.10.930.1000101.70.770.1250101.40.74100.498.5101.42.75.0mg·mL－1、2.5mg·mL－1、0.5mg·mL－133、、3ＲSD。3、、3ＲSD。ＲSD0.58%～1.5%ＲSD0.89%～1.6%HPLC－ProteinA。2.82h13927。27ＲSD0.44%。2.9“2.2”3ApH±0.2A±10%±5.0℃±20%3。ＲSD0.39%～1.0%T1.20～1.47—0301—ChinJPharmAnal201535620.0。HPLC－ProteinA。3“2.1”32“2.2”“2.5”33。4HPLC－ProteinA。1HPLC－ProteinA。1.5。HPLC－ProteinA0.078～10.0mg·mL－1Ｒ2＞0.99998.32%～101.7%ＲSD≤1.5%ＲSD≤1.6%18h2h127ＲSD0.44%pH±0.2、A±10%±5.0℃±20%ＲSD0.39%～1.0%。HPLC－ProteinA。10.0g·L－1。HPLC－Pro-teinA。1ＲEICHEＲTJMＲOSENSWEIGCJFADENLBetal．Mono-clonalantibodysuccessesintheclinicJ．NatBiotechnol200523910732PＲESTALGCHENHO’CONNOＲSJetal．Humanizationofananti－vascularendothelialgrowthfactormonoclonalantibodyforthetherapyofsolidtumorsandotherdisordersJ．CancerＲes1997572045933ESCUDIEＲBPLUZANSKAAOＲALEWSKIPetal．Bevaci-zumabplusinterferonalfa－2afortreatmentofmetastaticrenalcellcarcinomaarandomizeddouble－blindphaseⅢtrialJ．Lancet2007370960521034ＲINIBIHALABISOSENBEＲGJEetal．Bevacizumabplusin-terferomalfacomparedwithinterferonalfamonotherapyinpa-tientswithmetastaticrenalcellcarcinomaCALGB90206J．JClinOncol2008263354225YANGJCHAWOＲTHLSHENYＲMetal．Arandomizedtrialofbevacizumabananti－vascularendothelialgrowthfactoranti-bodyf