義翹講堂《重組抗體類型及生產(chǎn)工藝策略》

重組抗體類型及生產(chǎn)工藝策略研發(fā)經(jīng)理北京義翹神州科技股份有限公司SinoBiologicalInc.CONTENT義翹神州簡介1重組抗體簡介2重組抗體生產(chǎn)工藝策略3高通量抗體表達(dá)平臺(tái)4SinoBiologicalInc.義翹神州簡介01CompanyOverview全球布局SinoBiologicalInc.北京總部上海分公司歐洲子公司德國法蘭克福美國子公司賓夕法尼亞州費(fèi)城區(qū)蘇州子公司泰州子公司廣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…主要資質(zhì)Certifiedby:ISO9001ISO13485CNASCompanyOverview一站式科研試劑供應(yīng)商32,000+Genes14,000+Antibodies500+ELISAKits6,500+ProteinsSinoBiologicalInc.End-Usersin90+Countries產(chǎn)品大類文獻(xiàn)引用次數(shù)用戶分布>1700citationsin2020CitationsforSARSCOV2relatedproducts(asof5-31-2021):22publishedpapersin“Cell”15publishedpapersin“Science”8publishedpapersin“Nature”CompanyOverview完善的CRO服務(wù)平臺(tái)RecombinantProteinProductionServicesMammalianCells,InsectCells,E.coliAvailableRecombinantAntibodyProductionServices

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

Anti-idiotypeAntibodyDevelopmentCompanyOverviewSinoBiologicalInc.重組抗體簡介02SinoBiologicalInc.抗體及抗體生理學(xué)AntibodyandDerivativesAbundance:IgG(80%)**>IgM(10%)>IgA>IgD&IgE血液中的抗體形式(人和其他哺乳動(dòng)物)特殊形式高親和力高特異性抗體抗原DOI:10.3389/fimmu.2013.00302SinoBiologicalInc.抗體發(fā)現(xiàn)和重組抗體發(fā)展歷史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.重組抗體的應(yīng)用DiagnosticsTherapeuticsBiomedicalResearchAntibodyandDerivativesWB,anti-COV2NPIF,anti-EGFRIHC,anti-Claudin18.2FCM,anti-CD31ELISA,anti-HER2DOI:

10.2147/BCTT.S175360SinoBiologicalInc.重組抗體形式多樣AntibodyandDerivativesFull-lengthIgG片段抗體FabscFvVHH嵌合抗體抗體融合蛋白scFv-FcVHH-Fc雙/多功能抗體DOI:10.3389/fimmu.2019.02454(1)ExtractionofhybridomamRNA(2)Sequencing(3)denovotranscriptomeassembly(4)SearchforIgh/IgI/IgksequenceDOI:10.1016/j.molimm.2015.01.003SinoBiologicalInc.重組抗體生產(chǎn)工藝策略03HEK293/CHOInsectcellE.coliYeast

低成本，表達(dá)快速，易放大

無PTM,包涵體，重組表達(dá)蛋白分子量上限相對(duì)較低低成本，表達(dá)快速，易放大

高度mannose糖基化PTM

可溶蛋白，高密度培養(yǎng)，正確折疊和一定的PTM

糖基化糖鏈較短，成本相對(duì)較高

分泌表達(dá)，天然PTM，正確折疊

較長培養(yǎng)周期，成本相對(duì)較高SinoBiologicalInc.重組表達(dá)概念和宿主RecombinantAntibodyExpression

分泌蛋白

復(fù)雜的高級(jí)結(jié)構(gòu)(多對(duì)S-S)

需要正確的V區(qū)折疊-抗原結(jié)合需要正確的Fc區(qū)折疊-Fc受體結(jié)合和補(bǔ)體效應(yīng)

需要糖基化以促進(jìn)正確的補(bǔ)體效應(yīng)哺乳動(dòng)物細(xì)胞是重組抗體表達(dá)的合適宿主重組抗體表達(dá)的宿主選擇大腸桿菌或酵母可用于表達(dá)VHH抗體DOI:10.1186/s12934-019-1094-0PTM=翻譯后修飾，包括糖基化，磷酸化，乙?；萐inoBiologicalInc.重組抗體表達(dá)主要過程RecombinantAntibodyExpressionhttps://plueckthun.bioc.uzh.ch/antibody/Introduction/IGGinteractions/index.html抗體序列

雜交瘤

噬菌體庫

單B細(xì)胞測序AI導(dǎo)向的insilicon設(shè)計(jì)表達(dá)載體構(gòu)建瞬時(shí)轉(zhuǎn)染（PEI）HEK293FTDG44SBICompetitor細(xì)胞培養(yǎng)抗體純化功能測試構(gòu)建穩(wěn)定細(xì)胞株（CHO）培養(yǎng)純化工藝優(yōu)化大規(guī)模重組抗體生產(chǎn)抗體發(fā)現(xiàn)

親和層析

色譜純化Vector#1:LCVector#2:HC+SinoBiologicalInc.構(gòu)建設(shè)計(jì)RecombinantAntibodyExpressionSignalPeptide,SP(leadersequence)NameSequenceHumanOSMMGVLLTQRTLLSLVLALLFPSMASMMouseIgKappaMETDTLLLWVLLLWVPGSTGDMouseIgHeavyMGWSCIILFLVATATGVHSBM40MRAWIFFLLCLAGRALASecreconMWWRLWWLLLLLLLLWPMVWAHumanIgKVIIIMDMRVPAQLLGLLLLWLRGARCCD33MPLLLLLPLLWAGALAHumanChymotrypsinogenMAFLWLLSCWALLGTTFGHumantrypsinogen-2MNLLLILTFVAAAVAHumanIL-2MYRMQLLSCIALSLALVTNSAlbumin(HSA)MKWVTFISLLFSSAYSHumaninsulinMALWMRLLPLLALLALWGPDPAAA

常用信號(hào)肽

全長抗體分泌信號(hào)肽亦適用與scFv和VHH等形式片段抗體的表達(dá)

信號(hào)肽的選擇對(duì)抗體產(chǎn)量有一定影響

抗體融合蛋白表達(dá)需要選取切割度高的信號(hào)肽SignalP6.0:https://services.healthtech.dtu.dk/service.php?SignalPGlycanN297FucFucosylatedAbaFucosylatedAbHEK293FUT8-/-HEK293“Miniaturization”–scFvLinkerPurificationTag“Accessories”DOI:10.1371/journal.pone.0116878.g003Linker長度影響scFv的活性(GGGGS)1(GGGGS)3(GGGGS)5(GGGGS)7DOI:10.1080/09168451.2016.1156482抗體表達(dá)構(gòu)建優(yōu)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引入定點(diǎn)偶聯(lián)位點(diǎn):CysMTDOI:10.1073/pnas.0904191106;DOI:10.1021/bc900509sCH1CH3增加抗體穩(wěn)定性突變:hingeregionH229-YDOI:10.1074/jbc.M111.255026;DOI:10.1038/s41598-018-35489-yCH2:L242C,K334CCH3:P343C,A431CDistancebetween?-carbon<4.7?增強(qiáng)抗體穩(wěn)定性:CysMT引入DOI:10.1074/jbc.RA118.005367;DOI:10.1186/s13036-021-00272-7SinoBiologicalInc.RecombinantAntibodyExpression培養(yǎng)條件優(yōu)化抗體序列載體構(gòu)建瞬時(shí)轉(zhuǎn)染（PEI）HEK293FTDG44SBICompetitor細(xì)胞培養(yǎng)培養(yǎng)條件優(yōu)化實(shí)驗(yàn)提高抗體表達(dá)量

選擇20株低表達(dá)量抗體(<20mg/L)

通過使用不同培養(yǎng)基配方和轉(zhuǎn)染試劑排列組合優(yōu)化培養(yǎng)條件

選擇兩代培養(yǎng)基(M1andM2)

選擇兩種配比轉(zhuǎn)染試劑(T1andT2)

相同培養(yǎng)體積，培養(yǎng)周期和溫度

一步ProteinA親和純化抗體M1+T1M1+T2M2+T2AdditivesM2+T2M1+T2WeakGFP+StrongGFP+ProteinAProteinGProteinLSpeciesofOriginStaphylococcus

aureusStreptococcusspp.(GroupCandG)PeptostreptococcusmagnusNative

Size/Sizes40~60kDa40~65kDa76kDaBindingRegionsIgGIgG,HSAIgIgBindingRegionsFcofIgGFcofIgGVL-kappaSinoBiologicalInc.RecombinantAntibodyExpression重組抗體純化策略案例：雙抗純化宿主：HEK293項(xiàng)目要求:1.抗體形式:Fab+HC，Knobs-in-holes2.純度:>95%bySDSandSEChttps://plueckthun.bioc.uzh.ch/antibody/Introduction/IGGinteractions/index.htmltpi.ch/Calculator/ProteinTool抗體親和層析純化等電點(diǎn)（pI）計(jì)算ProteinAIonExchangeGelFiltration共轉(zhuǎn)J鏈-五聚體SinoBiologicalInc.重組IgM抗體表達(dá)不添加J鏈-六聚體純化策略轉(zhuǎn)染策略方法1方法2116.066.245.035.025.018.4116.066.245.035.025.018.4五聚體五聚體116.066.245.035.025.018.4六聚體Kappa輕鏈IgM1.Protein-L親和層析五聚體去除低分子量組分2.分子篩五聚體五聚體含量富集：25%（分子篩前）~99%（分子篩后）其他IgM抗體1.離子交換2.分子篩Pentamer最終抗體純度>95%RecombinantAntibodyExpressionSinoBiologicalInc.新型高通量抗體表達(dá)平臺(tái)04SinoBiologicalInc.高通量重組抗體表達(dá)DOI:10.1016/j.cell.2020.05.025抗體/蛋白序列文庫序列分析高通量引物合成引物文庫建立高通量載體合成抗體/