药物分析杂志ChinJPharmAna)2008.28(10)—1605—■—___一---------------------------------------------------------------------------------------------------------------------------------一------------------------------_..........................................................................................................................................................—11・，■“.......................................................■■GM-CSF/IL-3融合蛋白质量标准的研究和建立1袁力勇，饶春明，李响，韩春梅，郭莹，丁有学，王军志^(中国药品生物制品检定所，北京100050)摘要目的:建立重组GM-CSF/IL-3融合蛋白的质控标准和检测方法•以用于该产品的常规质最控制。方法:用TF・】/MTT细胞增殖试验定量测定GM-CSF/IL-3融合蛋白体外生物学活性;用抗GM-CSF和1L-3的抗体分别进行WesternBlot进行鉴别试验;用C8-HPLC和SDS-PAGE测定蛋白纯度；用胰蛋白酶消化-HPI.C进行肽图分析;用顶空进样气相色谱法检测甲醇残留就;还原型SDS-PAGE测定融合蛋白相对分子质罐；其他原液和成品检测项冃参照2005年版中国药典三部的方法进行'结果:GM-CSF/IL-3融合蛋白的原液及成品与TF-1细胞的反应呈典型的S型曲线，符合四参数方程)=(.4-!))/[1+(x/C)-B)]+0相关系数大于0.99•成品和原液的问收率均大于94%,RSD均小于20%；与GM・CSF和IL-3抗休进行的WesternBlot均为阳性;原液纯度大于95.0%;甲醇残留量小于0.005%•其他项目均符合2005年版中国药典三部要求°结论:建立的重组GM-CSF/IL-3H1合蛋白质控标准和检测方法可以有效控制该制品的质量•并确保制品的安全与有效•已用于该制品的常规检定°关键词：GM-CSF/1L・3；TF-1细胞；WesternBlot;甲醉中图分类号:R9I7文献标识码：A文章编号：0254-1793(2008)10-1605-04StudyoftherequirementsandmethodsforqualitycontrolofrecombinantGM一CSF/IL-3fusionprotein*YUANLi-yong,RAOChun-niing,IJXiang,HANChun-mei,GUOYing,DINGYou-xue,WANGJun-zhi2(NationalInslilutefortheControlofl^iarmaceulicalandBiologicalProductsvBeijing100050.China)AbstractObjective:ToestablishtherequirementsandmethodsforqualitycontrolofrecombinanthumanGM一CSF/IL-3fusionprotein.Methods：TlieMTTcellpniliferationassayofTF-1wasusedtoevaluatethebioactivityofGM-CSF/IL-3in诚proteinpuritywastestedbyCg-HPLC(214nmdetectivewavelength)andSDS-PAGE；WesternBlotwasassayusinganti-GM-CSFandanti-IL-3antibody;Peptidemappingwastestedbytrypsinodigestion/CH*IIPLC；methanolresiduewasdetenninedbyh(?adspacegaschrornatograf)hy;relativemolecularmasswasdeterminedbyreducedSDS-PAGE；otheritemsallwerereferredChPVo)ULResult:I)os(!-responserelationshipofTF-1cellproliferation/MTPstainingassayforbulkanddrugproductsofGM-CSF/IL-3fusionproteinalldisplayedtypicalScurveandwerecompliedwithfour-parameterequationy=(4-1))/{1+(x/C)AB)]+D」hcrcla・tivecoefficientwasmorethan0.99；andtheaveragerecoveryrateforbulkanddnigprexiuctswereallmorethan94%.RSI)werelessthan20%；Westernblotresultswerepositiveusingantil>o<lyofGM-CSFandIL-3individually;proteinpuritywasmorethan95.0%andtheresidualmethanolwaslessthan0.005%；theothertestresultwereallcompliancewithChi'VolIIIof2005edition.Conclusion/Hiespecificationsandanalysismethodsforrecombinant(；M-CSF/IL-3fusionproteinhadlheabilitytoconln)ltherecombinantprotein'squalityandensurelhedrug^ssafelyandstability；ithadalreadybeenusedforGM-CSF/IL-3routineanalysisandlotrelease.Keywords：GM—CSF/IL—3；TF'—1cell；WesternBlot；methanolGM-CSF/IL-3是人粒细胞-巨噬细胞集落刺激因子(GM-CSF)和白细胞介素3(II,-3)2种1IN家86)岛技术发展计划项”资助(2003AA2Z3481)2"通讯作布Tri：(010)67095586：E・mail：wan©z@ni<?pbp・org.cn细胞因子通过一...