生物技術(shù)藥物課程 R D of Biopharmaceuticals

生物技術(shù)藥物-BiopharmaceuticalDrugsandVaccines

TheBiopharmaceuticalsTheBasicsofBiotechnologyBiotechnology&DrugDiscoveryTheBiopharmaceuticalDrugs&VaccinesBiopharmaceuticaldrugs=Biotechnologydrugs=BiotechdrugsBackgroundofBiopharmaceuticalsMyclassofBiopharmaceuticalDrugs&Vaccineswillfocusondescribingindividualbiotechdrugandvaccine.Iwilltrytoclarifythedrugsintocategoriesbasedontheirpharmacologicalactionsortheirstructuralcharacters.Eachclassofdrugsweredevelopedasnewbiotechnologieswereinventedtomeetmedicaldemands.Iwilltrytolinkthedrugdiscoveriestomedicalpractice,andtheirimpactsontheresearchanddevelopment(R&D)ofnewdrugs.UnderstandofbiotechdrugsoftenneedtohaveknowledgeofMolecularBiology,CellBiology,andGeneralMedicine.Ihopethatyouwillrecallwhatyouhavelearnedbeforetoparticipateactivelyinmyclasses.Biotechdrugsarethedrugsdiscoveredbyverywelldesignedresearchexperiments.Iwillcovertheprinciplesofthoseexperimentstosharewithyouthewaystodesignandcarryoutnewexperimentinyourownthesisresearchprojects.BriefHistoryofTheBiopharmaceuticals1982.Thefirstbiopharmaceuticals,humanrecombinantinsulin,wasapprovedbyFDA.By2000,therewere116biopharmaceuticalsinthemarket,369drugswereinPhaseIIIclinicaltries,2600wereinpre-clinicalandearlyphasesofclinicaltries.Ourcountry,19biotechdrugsareinthemarket,30inclinicaltries.1999,10%revenuesoftotaldrugsaleswerecomingfrombiotechdrugs;2008,thatnumberrosesto~30%.TheBiopharmaceuticalsR&DofBiopharmaceuticalsProteinEngineeringandDrugDevelopmentAnti-senseDNA,RNA,andRibozymedrugsNakedDNAdrugsandTheirDevelopmentTherapeuticAntibodiesBloodSystemBiopharmaceuticalsThrombolyticBiopharmaceuticalsAnti-cancerBiopharmaceuticalsNeuronalBiopharmaceuticalsStemCells&RegenerativeMedicineTissueEngineeringandTheirTherapeuticValueChapter1.R&DofBiopharmaceuticals

ClassI.ClassificationofBiopharmaceuticalDrugsI.RecombinantProteinDrugsII.TherapeuticAntibodiesIII.NucleicAcidDrugsIV.Tissue&CellTherapeuticsI.RecombinantProteinDrugs1.Definition:TheyaremadebytherecombinantDNAtechnology.2.Include:1)Cytokines:Interferons(IFNs),Interleukins(Ils),ColonyStimulationFactors(CSFs),GrowthFactors,TumorNecrosisFactors.2)Hormones:GrowthHormones,Insulin,etc.3)ProteindrugstreatCardiovascularandBlooddisorders:ThrombolyticDrugs,BloodCoagulateFactors,Erythropoietin,Thrombopoietin,VascularendotheliagrowthFactor(VEGF),BloodReplacementReagents:Hemoglobin,4)Proteindrugstreatneuronaldisorders:Nervegrowthfactor(NGF)5)SolubleCytokineReceptors:IL1R,IL4R,6)TargetedToxins:Cytokine-directedtoxins,mAb-directedtoxins.Chapter1.R&DofBiopharmaceuticals

ClassI.ClassificationofBiopharmaceuticalDrugsII.TherapeuticAntibodies1.Definition:MonoclonalAntibodieshavingtherapeuticvalues.2.Include:Non-HodgkinLymphoma:Anti-CD20mAB,1997,Rituxan.(Genentech)InhibitionofEngraftmentRejection:Anti-CD3mAB,1986;anti-CD25mAB,1998.BreastCancer:Anti-HER2/neumAB,1998,Herceptin(Genentech)RheumatoidArthritis:anti-TNFa

mAB,1999FDAapprovalAllergy:anti-IgE

mAB,2003.Xolair(Tanox)Psoriasis:anti-Tcellactivation,migration.2003.Raptiva(Genentech)ColorectalCancer:Anti-EGFRmAB,2004.2.13,Erbitux(Imclone)ColorectalCancer:Anti-VEGFmAB,2004.2.26,Avastin,(Genentech).Chapter1.R&DofBiopharmaceuticals

ClassI.ClassificationofBiopharmaceuticalDrugsIII.NucleicAcidDrugs1.Definition:Drugsthatareintheformsofnucleicacid.2.Include:Antisenseoligonucleotide.Ribozyme.NakedDNAvaccines.DNAdrugs.Theyaremostlyinearlystagesofclinicaldevelopment.3.R&Ddirections:Delivery,Safety,Efficacy,IPissues.Chapter1.R&DofBiopharmaceuticals

ClassI.ClassificationofBiopharmaceuticalDrugsIV.TissueandCellTherapeutics1.Definition:Drugsthatareintheformsoftissuesorcells.2.Include:Tissues&Cells:Organ/Celltransplantation(Autologous,Allogeneic,Xenogeneic),i.e.,liver,bonemarrow,kidney,neuronstemcell,pancreaticstemcells,hematopoieticstemcells,transplantation.TissueEngineering:Mesenchymalstemcells,Humanembryonicstemcells.ExvivoGeneTherapy:Tcellgenetherapy,Stemcellgenetherapy.Chapter1.R&DofBiopharmaceuticals

ClassI.ClassificationofBiopharmaceuticalDrugsBiopharmaceuticalDrugsandVaccinesAre:TissuesCellsDNARNAProteinsAntibodiesCharacteristicsofBiopharmaceuticalDrugsTheBiopharmaceuticalDrugsAre:Biginmass,andcomplicatedinstructures.Speciesspecific.Immunogenic.Limitingtheirrepeatedusage.Highlypotent,lowinsideeffect,relativelysaferthanconventionaldrugs.Shortinhalf-life,decayquickly.Quicktogenerateeffects.Functionalredundant,multipleeffects.Lowinfunctionalstability,shortinshelf-life,andeasytobecontaminated.Highingeneticinstability.Mastcellbankisimportantformanufacture.Complicatedinmanufacture,Qualitycontrolandqualityassuranceareimportanttoinsuretheconsistencyofthedrugs.InvestigationalDrugApplication(IND)LaboratoryResearch:Discoveryandhypothesis-drivenresearchtoidentifynewdrugs.Lab-scaledrugproduction,andanimalmodeltests.Scale-upproductionandQualitycontrol.Createmastcellbank,ProduceenoughdrugsforPreclinical,QC,PhaseI,IIclinicaltrials.PreclinicalStudies:Potency,Safety,Pharmacokinetics,animaltests.Clinicaltrials:PhaseI:Safetystudies,clinicalpharmacokinectics.PhaseII:Potency,safetystudiesinpatients.PhaseIII:Comparativestudieswithexitingknowndrugs.NewDrugApplication(NDA):TemporarymanufactureandmarketingpermissionfromSFDA.PhaseIV:Continuedclinicalstudiespostmarketing.FinalapprovalformanufactureandmarketingfromSFDA.Biotechnology&DrugDiscoveryHumangenomecontains3billionbasepairs,encodes30~40thousandsgenes.Over10thousandsgenesarenotdiscoveredyet.GenomicsproteomicsTranscriptomicsFunctionalGenomicsDRUGSBiotechnology&DrugDiscoveryGenomicsproteomicsTranscriptomicsFunctionalGenomicsNatural&ChemicalDrugsBiopharmaceuticalDrugsDNA,RNA,Protein,AntibodyDNA,RNA,ProteinTheirrolesinphysiologyandpathologyBiopharmaceuticalProductionSpecificconsiderationsinbiopharmaceuticalproduction:1)Genesareexpressedinforeignhostcellsthatcouldgeneratevariationsinproteinstructure,biologicalfunction,andimmunogenicitycomparedtothenativeprotein.2)Contaminationisintroducedduringproduction,suchas,endotoxinfromE.coli,oncogeneandvirusfrommammaliancellline.3)Variationsinpotencyandpurityexistamongproductionbatchsandlots.BiopharmaceuticalProductionManufactureProteinDrugs1)ProkaryoticProductionSystemA:Advantages:TheproductsaresafeandpotentastheBiologyandMolecularbiologyofbacteriaareverywelldefined.Bacteriagrowtohighdensityfastinverysimpleculturemedium.E.coliexpressionsystemisaverysuccessfulsystemwitheasy-to-cloneexpressioncassetteplasmidsandready-to-useproteinexpressionprotocols.Itiseasytoscaleuptheproductionvolume(Literto10,000literfermentationvolume).B.Disadvantages:ProteinsproducedbyE.coliareintheformsofinclusionbodythatneedtobeprocessedtogeneratesolublebiologicalactiveproteins.AllproteinsintheiraminoterminalhaveanextraMethionine.Proteinsarecontaminatedbyendotoxinthathavetoberemoved.BiopharmaceuticalProductionManufactureProteinDrugs1)EukaryoticProductionSystem(yeast&mammaliancelllines)A:Advantages: Eukaryoticcellshaveperfectpost-translationalmodificationsystem(phosphorylation,glycosylation).Recombinantproteinsaremadewithnormalfirst,second,andtertiarystructures.Theyoftenhavehighbiologicalactivities.B.Disadvantages: Itiscostlyandtechnicallydemanding.Safetyissuesincludepotentialcontaminationofoncogenesandinfectiousvirus(madcowdisease).BiopharmaceuticalProductionProductionofmonoclonalantibodiesAntibodiesaresecretedbyprofessionalmatureBlymphocyte–mastcells.Tomakeantibodyincellculture,immunizedBcellsarefusedwithmyelomacelllinetoformhybridoma.Bothmouseandhumanmyelomacelllinesareusedtomakemouseandhumanantibodiesrespectively.ProductionofNucleicAcidDrugsOligonucleotideissynthesizedbyDNAsynthesizer.NakedDNAvaccinesandgenedrugsaremostlyassembledinplasmidandproducedusingE.colisystems.PharmaceuticalEvaluationofBiotechDrugsRawMaterialsExpressionvectorandhostcells:Originalsources,History,Detailmapofthevectors,Methodoftransformation/transfection,Integrated/episomal,Stability.Sequenceofthegeneinthevectorandhostcells.Expressioncontrolandregulation.

ProductionThecellbank.Mastcellbank&productioncellbank.Theirhistoryofestablishment,culturemethodandmedium,cellpassagehistory,cellstoragecondition,etc.Limitednumberofhostcellpassages.Monitorcontamination,stabilityofproduction,propertiesofhostcell/vectorafterculture.Perfusionculturesystem.Monitorandcontrolofcell’sphenotypeandgenotype,stabilityofthevectoranditsproduct,contamination,expressionlevel,etc.3)PurificationProteinIsolationMethods:ReversePhaseChromatography,GelFiltration,IronExchange,AffinityChromatography,etc.Detectionofcontaminants:Endotoxin,Hostcellproteins,Mediumprotein,DNA,Proteinaggregation/degradation,Aminoacidexchange/replacement.Detectionofcontaminants:bacteria,yeast,fungus,microplasma,virus.PharmaceuticalEvaluationofBiotechDrugsQualityControl&QualityAssuranceinBiopharmaceuticalsIdentity:MolecularMass:MethodsofGelfiltration,Massspectrophotometer,SDS.IsoelectricPoint(pI):IsoelectricFocusingelectrophoresis.MolecularUV-Visibleabsorptionspectroscopy.

Aminoacidsequenceanalysis,aminoterminal,carboxyterminalsequenceanalysisPeptidemapping:AnalysisofproteindegradationproductsbySDS,HPLC,MassSpec,CapillaryElectrophoresis.ImmunogenicProperties:Radioimmunoassay(RIA),Enzyme-linkedimmunosorbentassay(ELISA).2)Purity:>95%Proteinpurity:PAGE,SDS,MassSpec,HPLC,Proteinquantification:Spectrophotometry,Bradfordmethod(CoomassieBrilliantbluesolution).Quantificationofcontaminants:Protein,DNA,endotoxin,bacteria,etc.3)Potency:Animalmodel:Establishedbasedonthebiologicalactivityofthedrug.Cell-basedassay:Cellproliferationassay,etc.Biochemicalassay:Enzymeactivityassay,etc.QualityControl&QualityAssuranceinBiopharmaceuticalsPre-clinicalStudyofTheBiopharmaceuticalsTheGoal:Definetheinitialdosageandthedoseescalationprotocol.Definethepotentialtoxicspecificityanditsreversibility.Definetheparametersmonitoringclinicaltrial.Pre-clinicalStudyofTheBiopharmaceuticalsEstablishProperAnimalModel:Biotechdrugsoftenhavespecies-specificandimmunogenicactivity.Choosetwotypesofanimalsifpossible,butnotnecessary.Manyhumandiseasesnowhaveanimalmodels.Theywereestablishedbytra