用于重组抗体生产的细胞大规模培养技术

ChinaBiotechnology2013337103-111*＊＊050015近年来，用于单抗药物生产的动物细胞大规模培养技术发展迅速。此领域的技术进展集中在个性化培养基开发，工艺条件优化等方面。本文总结了用于提高重组抗体表达水平的常用方法，以及细胞培养工艺对抗体药物“关键质量属性”(聚体、降解、糖基化修饰、电荷变异等)的诸多影响。此外，细胞培养工艺在产业化过程面临着工艺放大与技术转移，定性研究与工艺验证等实际问题。未来大规模细胞培养工艺的开发，将进一步借助动物细胞的组学研究成果和新兴的“过程分析技术”。重组抗体细胞大规模培养培养基优化工艺开发Q5112013-04-072013-05-16*“973”2012CB724502＊＊[e-mail]1。1986OKT3TM100mg/L。。1～2×107cell/mL200mg/L/day700mg/L/D3～5g/L13g/L220000LLonzaPortsmouth/NH—facility32×108cell/ml25～40g/LPerciviaXDTM4。5。、“”。1pHDODCO2、、、、、、、。“”。58mmol/L、382mOsm/kg、5．1mmol/L6。1。1．1EnbrelTMHerceptinTMＲituxanTMSynagisTMfedbatch、20000L。、。、29-1112-13。1．2prefusionDOI10.13523/j.cb.20130716ChinaBiotechnologyVol．33No．7201317-8Table1TheparameterandcontrolforanimalcellcultureprocesspHDODCO232～38℃6．8～7．220%～60%5%～25%40～180mmHg270～330mOsm/kgpHDODCO2PI/PIDCO2、0～10g/L0～10mmol/L0～10mmol/L0～10mmol/L、HPLCHPLC0～50mmol/L1～10mmol/L、、ATP105～108/ml＞70%0．5～5pg/cell-HPLC1、SDS-PAGE2、0．1～10g/LG0、G1F、G2F、Man5、ELISA3、proteinAHPLC4、Westernblot5SEC-HPLC6HPLCiCIEF7、IEF8、CEX9、HPLCNote1Highperformanceliquidchromatography2Sodiumdodecylsulfatepolyacrylamidegelelectrophoresis3Enzyme-linkedImmunosorbentassay4Proteinaaffinitychromatography5Proteinimmunoblot6Sizeexclusionchromatography7Imagedcapillaryisoelectricfocusing8Isoelectricfocusing9Cationexchangechromatography“”、14-15。16-17。ＲeoProTMＲemicadeTMSimulectTMSimponiTMStelaraTM。18。。controlledfedprefusion。。19。2。“”。、spintube。BioLectorTMSimCellTMMicro-24TMambrTM20pH、DO、。SimcellTM180650μl21。Micro-24TM5ml3LpHDO22。2．1。PF、ADCFCD23。＜20/L、、24。NS0、CHO。。“”、。。2．1．1componenttitration4012013337。70“”。、25、。2．1．2mediablending。DOEexperimentdesignexpert26。。2．1．3spentmediaanalysis。、、27-28。2．1．4stoichiometricanalysis、。、、2．4g/L29-30。2．1．5rationalculturemediadesign。。。、。CＲOSigmaBD31-34。2．1．6systematicapproaches、、。35-362．2“”biphasiccultureprocess“”“”、pH2。“”metabolicshift。2．32。。NaBuDMSO。2Table2Thesummaryofmethodsandcompositionsusedforimprovingrecombinantantibodyexpressionincellcultureprocess、、、111237、30385AMPATP39、、40Q10、、414243PBS、44454647G0/G14849pH385051、、。5253、、、。54－57DMSOG158G159606162、、、63501ChinaBiotechnologyVol．33No．720133、、、、、、64。“”FDA、EMEAcriticalqualityattributes65。2。365-67Table3Thechangesofrecombinantantibodyqualityattributeincellcultureprocessandimpactonantibodydrugclinicalefficacy、H2L、H、L。NS0、sp/0NeuGc、Gal-a13-Gal。NANAG0/G1/G2CDCFUT、Man5ADCCNA3．1、、。。30%。、pHDO、Cu2+、、、68。6970pH、6071。。4672-73。72、、74。、pH72-7375。3．2IgGASN297N。ADCD、CDC3。76-77。、DO、、pH、pCO2、、78。3．2．1glyslationglyslation79。3．2．2highmannoseformsMan5。Man580。3．2．3fucosylationFUT881。kifunensine82。3．2．4sialylationCHONS0NeuGc。CO28361。3．2．5galactosylation、、CHO84。60120133373．3、、C/、85。Cu2+Zn2+C86-87。SerAsn。Asn88Ser89。4。。。processcharacterization、processvalidation7。“”“”。4．1、、90-91。、DO、pH、、。＞1000Lmixingtime、。。、、、、92-93。4．2FDA“”qualitybydesignQbDcriticalqualityattributesCQAs。CQAs。。“”scaledown“”failuremodeandeffectsanalysis、、pH、DO94-95。“”。FDAEMEA35“”BLA96。5。、、、97。。“QbD”“”processanalyticaltechnologies。、、98-99。。1ChartrainMChuL．DevelopmentandproductionofcommercialtherapeuticmonoclonalantibodiesinmammaliancellexpressionsystemsAnoverviewofthecurrentupstreamtechnologies．CurrentPharmaceuticalBiotechnology200896447-467．2HuangYMHuWＲustandiEetal．Maximizingproductivityofchocell-basedfed-batchcultureusingchemicallydefinedmediaconditionsandtypicalmanufacturingequipment．BiotechnologyProgress20102651400-1410．3．．2013335132-138．LiuBN．Theprogressoftherapeuticantibodydrugandtheindustrialkey－technologyofantibodyproduction．ChinaBiotechnoloy2013335132-138．4DePalmaA．Enhancementofcellculturetechniques．GeneticEngineering＆BiotechnologyNews20092918．5．．2013336111-116．LiuBN．Thetechnologyprogressofantibody－producingcelllinedevelopment．ChinaBiotechnoloy2013336111-116．701ChinaBiotechnologyVol．33No．720136XingZLiZChowVetal．Identifyinginhibitorythresholdvaluesofrepressingmetabolitesinchocellcultureusingmultivariateanalysismethods．BiotechnologyProgress2008243675-683．7LiFVijayasankaranNShenAYetal．Cellcultureprocessesformonoclonalantibodyproduction．MAbs201025466-479．8WhitfordCJaW．BioreactorchaptertwoBioreactorcontrol．BioprocessIntSupplement2007．9HermesPACastroCD．Afullydefinedfed-batchrecombinantNS0cultureprocessformonoclonalantibodyproduction．BiotechnologyProgress20102651411-1416．10BurkyJEWessonMCYoungAetal．Protein-freefed-batchcultureofnon-gsNS0celllinesforproductionofrecombinantantibodies．BiotechnologyandBioengineering2007962281-293．11CheeFWDTinKWKTangGLetal．Impactofdynamiconlinefed-batchstrategiesonmetabolismproductivityandn-glycosylationqualityinchocellcultures．BiotechnologyandBioengineering2005892164-177．12KhattakSFXingZKentyBetal．Feeddevelopmentforfed-batchchoproductionprocessbysemisteadystateanalysis．BiotechnologyProgress2010263797-804．13DeAlwisDMDuttonＲLScharerJetal．Statisticalmethodsinmediaoptimizationforbatchandfed-batchanimalcellculture．BioprocessBiosystEng2007302107-113．14JardinBAMontesJLanthierSetal．Highcelldensityfedbatchandperfusionprocessesforstablenon-viralexpressionofsecretedalkalinephosphataseseapusinginsectcellsComparisontoabat