义翘讲堂《重组抗体类型及生产工艺策略》

重组抗体类型及生产工艺策略研发经理北京义翘神州科技股份有限公司SinoBiologicalInc.CONTENT义翘神州简介1重组抗体简介2重组抗体生产工艺策略3高通量抗体表达平台4SinoBiologicalInc.义翘神州简介01CompanyOverview全球布局SinoBiologicalInc.北京总部上海分公司欧洲子公司德国法兰克福美国子公司宾夕法尼亚州费城区苏州子公司泰州子公司广州分公司日本子公司SinoBiologicalInc.OurPeople500+Employees&Growing30%ofStaffwithMastersorPhDDegreesPrimaryFacility66,000

sq

ft

Lab/OfficeSpace25,000

sq

ft

GMPFacilityCore

BusinessReagents:RecombinantProteins,Antibodies,cDNAClones,ELISAKits…CustomServices:RecombinantExpression,AntibodyDevelopment…主要资质Certifiedby:ISO9001ISO13485CNASCompanyOverview一站式科研试剂供应商32,000+Genes14,000+Antibodies500+ELISAKits6,500+ProteinsSinoBiologicalInc.End-Usersin90+Countries产品大类文献引用次数用户分布>1700citationsin2020CitationsforSARSCOV2relatedproducts(asof5-31-2021):22publishedpapersin“Cell”15publishedpapersin“Science”8publishedpapersin“Nature”CompanyOverview完善的CRO服务平台RecombinantProteinProductionServicesMammalianCells,InsectCells,E.coliAvailableRecombinantAntibodyProductionServices

High-throughputAntibodyProductionServiceLarge-scaleAntibodyProductionServiceSinoBiologicalInc.AntibodyDevelopmentPlatformsRecombinantProductionPlatformsE.coliSystemHEK293/CHOTransientSystemBaculovirus-insectSystemCHO/HEK293StableCellLineDevelopmentMousemAbDevelopment(Hybridoma)RabbitmAbDevelopment(PhageDisplay)SingleB-cellBasedPlatformRabbitpAbDevelopmentAntibodyDevelopmentServicesFastMousemAbDevelopmentRabbitmAbDevelopmentFastRabbitpAbDevelopment

Anti-idiotypeAntibodyDevelopmentCompanyOverviewSinoBiologicalInc.重组抗体简介02SinoBiologicalInc.抗体及抗体生理学AntibodyandDerivativesAbundance:IgG(80%)**>IgM(10%)>IgA>IgD&IgE血液中的抗体形式(人和其他哺乳动物)特殊形式高亲和力高特异性抗体抗原DOI:10.3389/fimmu.2013.00302SinoBiologicalInc.抗体发现和重组抗体发展历史AntibodyandDerivatives/reports/expanding-access-monoclonal-antibodies1770~1890SmallpoxinoculationImmunizedanimalserumcureddiphtheria1900Side-chaintheory+Ab-Agbinding+complementactivation1948BcellinAbgeneration1957Clonalselectiontheory1959~1973MolecularstructureofAbestablished1975Hybridoma1984ChimericAb1985~1990Phagedisplay&Abengineering1994Transgenicmice&fullhumanAb1997Yeastdisplay2000’sBcellsorting2020andbeyondTheriseofAIThestorycontinues…19861stmAbapproved(OKT3,CD3)19941stFabapproved20001stADCapproved20021stfull-humanAbapproved(Humira,TNF)20051stbi-specificAbapproved20141stBiTEapprovedTill2022>135Abtherapeuticsapprovedorinregulatoryreview1997Rituximab(CD20)1998Trastuzumab(HER2)2015Nivolumab(PD1)Pembrolizumab(PD1)2021SotrovimabRegdanvimabCasirivimab+ImdevimabforCOVID-192020AnsuvimabAtoltivimab+Maftivimab+Odesivimab-ebgnforebolainfectionSinoBiologicalInc.重组抗体的应用DiagnosticsTherapeuticsBiomedicalResearchAntibodyandDerivativesWB,anti-COV2NPIF,anti-EGFRIHC,anti-Claudin18.2FCM,anti-CD31ELISA,anti-HER2DOI:

10.2147/BCTT.S175360SinoBiologicalInc.重组抗体形式多样AntibodyandDerivativesFull-lengthIgG片段抗体FabscFvVHH嵌合抗体抗体融合蛋白scFv-FcVHH-Fc双/多功能抗体DOI:10.3389/fimmu.2019.02454(1)ExtractionofhybridomamRNA(2)Sequencing(3)denovotranscriptomeassembly(4)SearchforIgh/IgI/IgksequenceDOI:10.1016/j.molimm.2015.01.003SinoBiologicalInc.重组抗体生产工艺策略03HEK293/CHOInsectcellE.coliYeast

低成本，表达快速，易放大

无PTM,包涵体，重组表达蛋白分子量上限相对较低低成本，表达快速，易放大

高度mannose糖基化PTM

可溶蛋白，高密度培养，正确折叠和一定的PTM

糖基化糖链较短，成本相对较高

分泌表达，天然PTM，正确折叠

较长培养周期，成本相对较高SinoBiologicalInc.重组表达概念和宿主RecombinantAntibodyExpression

分泌蛋白

复杂的高级结构(多对S-S)

需要正确的V区折叠-抗原结合需要正确的Fc区折叠-Fc受体结合和补体效应

需要糖基化以促进正确的补体效应哺乳动物细胞是重组抗体表达的合适宿主重组抗体表达的宿主选择大肠杆菌或酵母可用于表达VHH抗体DOI:10.1186/s12934-019-1094-0PTM=翻译后修饰，包括糖基化，磷酸化，乙酰化等SinoBiologicalInc.重组抗体表达主要过程RecombinantAntibodyExpressionhttps://plueckthun.bioc.uzh.ch/antibody/Introduction/IGGinteractions/index.html抗体序列

杂交瘤

噬菌体库

单B细胞测序AI导向的insilicon设计表达载体构建瞬时转染（PEI）HEK293FTDG44SBICompetitor细胞培养抗体纯化功能测试构建稳定细胞株（CHO）培养纯化工艺优化大规模重组抗体生产抗体发现

亲和层析

色谱纯化Vector#1:LCVector#2:HC+SinoBiologicalInc.构建设计RecombinantAntibodyExpressionSignalPeptide,SP(leadersequence)NameSequenceHumanOSMMGVLLTQRTLLSLVLALLFPSMASMMouseIgKappaMETDTLLLWVLLLWVPGSTGDMouseIgHeavyMGWSCIILFLVATATGVHSBM40MRAWIFFLLCLAGRALASecreconMWWRLWWLLLLLLLLWPMVWAHumanIgKVIIIMDMRVPAQLLGLLLLWLRGARCCD33MPLLLLLPLLWAGALAHumanChymotrypsinogenMAFLWLLSCWALLGTTFGHumantrypsinogen-2MNLLLILTFVAAAVAHumanIL-2MYRMQLLSCIALSLALVTNSAlbumin(HSA)MKWVTFISLLFSSAYSHumaninsulinMALWMRLLPLLALLALWGPDPAAA

常用信号肽

全长抗体分泌信号肽亦适用与scFv和VHH等形式片段抗体的表达

信号肽的选择对抗体产量有一定影响

抗体融合蛋白表达需要选取切割度高的信号肽SignalP6.0:https://services.healthtech.dtu.dk/service.php?SignalPGlycanN297FucFucosylatedAbaFucosylatedAbHEK293FUT8-/-HEK293“Miniaturization”–scFvLinkerPurificationTag“Accessories”DOI:10.1371/journal.pone.0116878.g003Linker长度影响scFv的活性(GGGGS)1(GGGGS)3(GGGGS)5(GGGGS)7DOI:10.1080/09168451.2016.1156482抗体表达构建优化scFv-His-Avi>95%purity(PAGE)Lowyield:<0.5mg/LLowbiotinratio:<0.2Aggregation:>90%oligomer(SEC)scFv-His-spacer-Avi>95%purity(PAGE)Improvedyield:~6mg/LImprovedbiotinratio:0.85Aggregation:~10%oligomer(SEC)scFv-Fc-Avi>95%purity(PAGE)Improvedyield:>25mg/LImprovedbiotinratio:0.9Aggregation:~5%oligomer(SEC)SinoBiologicalInc.Fc改造策略案例RecombinantAntibodyExpression引入定点偶联位点:CysMTDOI:10.1073/pnas.0904191106;DOI:10.1021/bc900509sCH1CH3增加抗体稳定性突变:hingeregionH229-YDOI:10.1074/jbc.M111.255026;DOI:10.1038/s41598-018-35489-yCH2:L242C,K334CCH3:P343C,A431CDistancebetweenß-carbon<4.7Å增强抗体稳定性:CysMT引入DOI:10.1074/jbc.RA118.005367;DOI:10.1186/s13036-021-00272-7SinoBiologicalInc.RecombinantAntibodyExpression培养条件优化抗体序列载体构建瞬时转染（PEI）HEK293FTDG44SBICompetitor细胞培养培养条件优化实验提高抗体表达量

选择20株低表达量抗体(<20mg/L)

通过使用不同培养基配方和转染试剂排列组合优化培养条件

选择两代培养基(M1andM2)

选择两种配比转染试剂(T1andT2)

相同培养体积，培养周期和温度

一步ProteinA亲和纯化抗体M1+T1M1+T2M2+T2AdditivesM2+T2M1+T2WeakGFP+StrongGFP+ProteinAProteinGProteinLSpeciesofOriginStaphylococcus

aureusStreptococcusspp.(GroupCandG)PeptostreptococcusmagnusNative

Size/Sizes40~60kDa40~65kDa76kDaBindingRegionsIgGIgG,HSAIgIgBindingRegionsFcofIgGFcofIgGVL-kappaSinoBiologicalInc.RecombinantAntibodyExpression重组抗体纯化策略案例：双抗纯化宿主：HEK293项目要求:1.抗体形式:Fab+HC，Knobs-in-holes2.纯度:>95%bySDSandSEChttps://plueckthun.bioc.uzh.ch/antibody/Introduction/IGGinteractions/index.htmltpi.ch/Calculator/ProteinTool抗体亲和层析纯化等电点（pI）计算ProteinAIonExchangeGelFiltration共转J链-五聚体SinoBiologicalInc.重组IgM抗体表达不添加J链-六聚体纯化策略转染策略方法1方法2116.066.245.035.025.018.4116.066.245.035.025.018.4五聚体五聚体116.066.245.035.025.018.4六聚体Kappa轻链IgM1.Protein-L亲和层析五聚体去除低分子量组分2.分子筛五聚体五聚体含量富集：25%（分子筛前）~99%（分子筛后）其他IgM抗体1.离子交换2.分子筛Pentamer最终抗体纯度>95%RecombinantAntibodyExpressionSinoBiologicalInc.新型高通量抗体表达平台04SinoBiologicalInc.高通量重组抗体表达DOI:10.1016/j.cell.2020.05.025抗体/蛋白序列文库序列分析高通量引物合成引物文库建立高通量载体合成抗体/