蛋白质生化分离技术课件

BioseparationofproteinsBioseparationofproteins1Chapter1Introduction1IntroductionBiomaterialsolutionproperties:(1)Dilutesolution(2)Componentscomplicate(3)Unstable(4)BatchesdifferenceBioseparationrequirements(1)Highpurity(2)Largescale(3)Economical(4)BiocompatibleChapter1Introduction1I2Newwordsandvocabulary1cellfusion2interestprotein3biocompatible4purification5biologicalmaterial6plantcellculture7fermentation8strategiesNewwordsandvocabulary1cel3IITheNeedforbioseparationProcessrequirements(1)Safety(2)Quality(3)Amount(4)PreventingPosttranslationalchange(5)LowcostWhatwillbioseparationdo?(1)Modificationofexistingprocedures(2)DevelopingnewandnovelbioseparationtechnologyDifficultyofgeneralizedalgorithmprocessesandsubtledifferenceofdifferentproteinsIITheNeedforbioseparationP4NewwordsandvocabularyProtocol(方案）Algorithm(法则）Generalized(通用的，一般化的）Invaluable(有价值的）Inactivate(失活的）Therapeutic(治疗的)Viable(可行的）NewwordsandvocabularyProtoc5IIIClassificationofbioseparationsteps(1)Bioseparationprocedures(1)Removalofinsolubles(2)Isolationofproduct(3)Purification(4)PolishingIIIClassificationofbiosepar6IIIClassificationofbioseparationsteps(2)Bioseparationunitoperation(1)Celldisruption(2)Centrifugation(3)Chromatography(4)Drying(5)Evaporation(6)Extraction(7)Filtration(8)Membraneseparation(9)PrecipitationIIIClassificationofbiosepar7IIIClassificationofbioseparationsteps(3)Qualityrequirements:Productsfreefrom:

(1)detergent(2)endotoxins(3)proteases(4)immunogenicsubstance(5)microbiologicalcontamination(6)modifiedproteins(7)nucleidacids(8)oligonucleotide(9)enterotoxinsIIIClassificationofbiosepar8IIIClassificationofbioseparation

steps(4)Therapeuticusagerequirements:(1)Potency(2)Identity(3)Abnormaltoxicity(4)Nucleicacids(5)Homogeneity(6)Absenceofconflictingactivity

Processrequirements:(1)Maximizeproductyieldininitialstep(2)Limitthenumberofsteps(3)IntensificationIIIClassificationofbiosepar9蛋白质生化分离技术课件10IIIClassificationofbioseparation

steps(5)Newwordsandvocabulary

High-resolutionfractionaton(高分辩率分级）Harvesting(收获，收割）Clarification（澄清）Enrichment（富集）Hazardous（危险）Integrate（集成）Intensification（集成，加强）Expandedbedadsorption（扩张床）Homogenate（匀浆液）Fermentationbroth（发酵液）IIIClassificationofbiosepar11IVUpstreamanddownstreamprocessing(1)

Minimizelossofthevaluablebiologicalproduct(1)Locationandformofproteinsinthecell(2)Separationofcellulardebrisandinclusionbodies（Increasingthenumberofpassesthroughthehomogenizer）(3)Separatingabiologicalproductbydifferentprotocols(4)Understandingupstreamanddownstreamconditions(harvestingtimeandcellwallstrengthandproteases)IVUpstreamanddownstreampro12蛋白质生化分离技术课件13蛋白质生化分离技术课件14IVUpstreamanddownstreamprocessing(2)Biologicalprocessdevelopmentobjective:(1)Producttothemarketinquickesttimepossible(2)meetingallpossiblesafetyrequirements(3)processcosteffective(4)reliable(5)safetyfactorsparticularlyimportantduringscale-upIVUpstreamanddownstreampro15IVUpstreamanddownstreamprocessing(3)Enhancethequantityandquality(1)continuousmediasterilization(2)enhancemasstransfer(3)gasanalysisbymassspectrometry(4)computerizeddata(expertsystem)(5)searchfornoveltechniquesAmicrogravityseparation(eliminateshearstress)BmolecularimprintingpolymersCfoamsseparationIVUpstreamanddownstreampro16IVUpstreamanddownstreamprocessing(4)Separationunderspacemicrogravity(1)higherthroughputsforelectrophoreticseparation(2)twophasepartitioningundermicrogravity(mixinganddemixing)(3)crystallizationofproteinsundermicrogravity(needweakinteractingforcewhenproteinformingcrystallization)(4)advantagesandexpensecarefullyanalyzedongroundbioprocessingfordifferentapplicationIVUpstreamanddownstreampro17IVUpstreamanddownstreamprocessing(4)NewWordsandvocabuloryCoarse-gradecentrifuge(粗糙级别的离心）Harsher（苛刻的）Intractably(不听话的）Debris(碎片）Endogeneous(内源性的）Dextransucrase(葡聚糖蔗糖酶）Highlight(突出）Copewith(应对）Trypsin（胰蛋白酶）Lysozyme(溶菌酶）Containerless无模板的Shearstress剪切力Thermodynamic(热力学）IVUpstreamanddownstreampro18VSomefactorsinfluencingbioseparation(1)AProcessMonitoring(1)monitorstructureandbiologicalactivityduringcontinuousprocess(2)insitusensors(3)flowinjectionanalysis(4)synchronizedpeak-switchinghighpressureliquidchromatography(5)capillaryelectrophoresis(6)ionizationmassspectrometry(7)rapidprocessmonitoring(8)poorqualityproduct(a)furtherpurification(b)identifyanddiscardtheproductVSomefactorsinfluencingbio19VSomefactorsinfluencingbioseparation(2)BBioseparationeconomics(1)atleast50%oftotalcost(2)spendsonlyabout5%ofeffort(3)reducethedegreeofwasteandemissions(product:waste=1:3250)(4)processmodification(a)molecularbiology(hybridgene,fusionprotein,charged-fusiontechnique(b)fermentationparametersVSomefactorsinfluencingbio20VSomefactorsinfluencingbioseparation(3)CProteinrefoldingandinclusionbodies(1)(1)aggregationformationpathway(2)specificbiteinvolvedinaggregation(3)minimizingaggregation(a)eliminatingdiffusion(b)covalentmodification(c)Chaperoneshsp10and60(4)processingaggregatesadvantages(a)initialpurificationstraightforward(50%)(b)ion-exchangeto90%productpurityeasily(c)proteasedonotattackinclusionbodies(d)inclusionbodiessolubilized,refoldedVSomefactorsinfluencingbio21蛋白质生化分离技术课件22VSomefactorsinfluencingbioseparation(4)CProteinrefoldingandinclusionbodies(2)(1)Intentionallydenatureaprotein(usingdenaurants(SDS-Page))(2)chemicalmodification(a)extractionofproteinstoorganicsolventsbyPEGmodified(b)solubilityofproteinsintoaqueoussolutionbyadditionofchargedgroupstoproteinsurfaces(3)interfacialadsorptionofproteins(a)understandingofnon-specificproteininteractions(b)delicatebalanceofthevanderwaals,electrostatichydrophobicandhydrationVSomefactorsinfluencingbio23VSomefactorsinfluencingbioseparation(5)CProteinrefoldingandinclusionbodies(3)Recoveryprocessconsider:(1)mildcondition(2)minimizeprocesstime(3)avoidextremepHandtemperatureconditions(4)avoidexposuretoair-waterinterfacesandshear(5)minimizedexposuretononpolarsolventsandhydrophobicadsorbents(6)beefficientVSomefactorsinfluencingbio24VSomefactorsinfluencingbioseparation(6)CProteinrefoldingandinclusionbodies(4)Processeconomicsandrenaturation(1)removingdetergent(a)ionicdetergents(ionexchangecolumn)(b)longhydrophobicsidechaindetergents(adsorptiononahydrophobicresin)(c)detergentsbealsoremovedbysolventextractionVSomefactorsinfluencingbio25Vsomefactorsinfluencingbioseparation(7)CProteinrefoldingandinclusionbodies(5)Futuresaleestimation(1)ReallyprofitablecompaniesinUSAmgenBiogenGenentechGenzymeVsomefactorsinfluencingbio26VSomefactorsinfluencingbioseparation(8)CProteinrefoldingandinclusionbodies(6)(2)DevelopingproductsrisksPitfallandunpredictability(a)drugdevelopment(b)clinicaltrials(c)FDAadvisorycommitteereviews(d)FDAapproval(e)Competition(f)Marketsize(g)DrugPrice(h)MarketpenetrationVSomefactorsinfluencingbio27VSomefactorsinfluencingbioseparation(9)Cproteinrefoldingandinclusionbodies(7)Particulardrugproduction(1)processeconomics(2)innovativemethod(a)multiuseflexiblefacility(b)shareafacilitywithothercompanies(c)reducedesigning,building,runningcostVSomefactorsinfluencingbio28VSomefactorsinfluencingbioseparation(10)CProteinrefoldingandinclusionbodies(8)

followingguidelinefordrugproduction(1)segregationwhenflowpathcross(2)stringentvalidation(3)Carefulschedulingtooptimizetheplantutilization(4)discussionondesign(a)fermentation(b)primaryrecoverystep(c)Purificationstep(d)fillingstep(e)FlowpathsVSomefactorsinfluencingbio29VSomefactorsinfluencingbioseparation(11)CProteinrefoldingandinclusionbodies(9)Qualitycontrol(1)pressurefromregulatoryagencies(2)publicawareness(3)expandedandfiercecompetition(4)safetyfactors(5)specificactionandminimalsideeffectrequirementsVSomefactorsinfluencingbio30蛋白质生化分离技术课件31VSomefactorsinfluencingbioseparation(12)CProteinrefoldingandinclusionbodies(10)Proteinstabilityinfinishedformulation(1)surfactantsorstabilizers(sugars,aminoacidsandfattyacid)(2)pH(3)antioxidants(containingsulfuraminoacids)(4)cryoprotectants(minimizingfreezingloss)(5)lyoprotectants(minimizingfreezy-dryingloss)VSomefactorsinfluencingbio32Newwordsandvocabulary(1)Synchronized（同步的）Variant(变量）Conformation(构象）Variance(变异）Feedstocks(给料）Construction(构建）Polypeptide(多肽）Hypothetical(假定）native(未触动的，天然的）Mitochondrial(线料体）Malate(苹果酸盐）Newwordsandvocabulary(1)Sy33Newwordsandvocabulary(2)Dehydrogenase(脱氢酶）Invitro(体外）Refractile(能折射的）Refolded（再折叠）Underscored（在…下划线）Intentionally（故意地）Review（评论，审阅）Sodiumdodecylsulfatepolyacrylamidegelelectrophoresis(SDS)RodlikeDNAtopoisomerase（DNAtopoisomerase)Estrogenreceptor(雌激素受体)Fructosyltransferase(果糖基转移酶）Newwordsandvocabulary(2)De34Newwordsandvocabulary(3)Supersaturation（超饱和）Nicking（基因巧合，切割）Deamidation(脱酰胺）Alleviate（缓和，减轻）Tankage（桶槽容量）Behooves(适宜，应该）Erythropoietin(促红细胞生成素）Pitfalls(缺陷）abound(大量存在的）Segregation(隔离）Newwordsandvocabulary(3)Su35Newwordsandvocabulary(4)Premium(额外费用）Filling(填补）Beboundto(一定要）Fierce(残酷的）Reluctance(勉强）Parenteral(注射用的，非肠道的）Proneto(倾向于）Colonystimulatingfactor(集落刺激因子）Newwordsandvocabulary(4)Pr36Question1Whydidsomebioseparationexperimentswerecarriedoutundermicrogravityorzerogravityinspace?2Whyintentionallydenatureaproteininsomecasesduringbioseparation?3Howstabilizeproteinsinfinishedformulation?4Whatadvantagesprocessintegrationhas?5concept:inclusion,renaturation,inactivation,integration,fuseprotein,aggregation?Question1Whydidsomebiosep37BioseparationofproteinsBioseparationofproteins38Chapter1Introduction1IntroductionBiomaterialsolutionproperties:(1)Dilutesolution(2)Componentscomplicate(3)Unstable(4)BatchesdifferenceBioseparationrequirements(1)Highpurity(2)Largescale(3)Economical(4)BiocompatibleChapter1Introduction1I39Newwordsandvocabulary1cellfusion2interestprotein3biocompatible4purification5biologicalmaterial6plantcellculture7fermentation8strategiesNewwordsandvocabulary1cel40IITheNeedforbioseparationProcessrequirements(1)Safety(2)Quality(3)Amount(4)PreventingPosttranslationalchange(5)LowcostWhatwillbioseparationdo?(1)Modificationofexistingprocedures(2)DevelopingnewandnovelbioseparationtechnologyDifficultyofgeneralizedalgorithmprocessesandsubtledifferenceofdifferentproteinsIITheNeedforbioseparationP41NewwordsandvocabularyProtocol(方案）Algorithm(法则）Generalized(通用的，一般化的）Invaluable(有价值的）Inactivate(失活的）Therapeutic(治疗的)Viable(可行的）NewwordsandvocabularyProtoc42IIIClassificationofbioseparationsteps(1)Bioseparationprocedures(1)Removalofinsolubles(2)Isolationofproduct(3)Purification(4)PolishingIIIClassificationofbiosepar43IIIClassificationofbioseparationsteps(2)Bioseparationunitoperation(1)Celldisruption(2)Centrifugation(3)Chromatography(4)Drying(5)Evaporation(6)Extraction(7)Filtration(8)Membraneseparation(9)PrecipitationIIIClassificationofbiosepar44IIIClassificationofbioseparationsteps(3)Qualityrequirements:Productsfreefrom:

(1)detergent(2)endotoxins(3)proteases(4)immunogenicsubstance(5)microbiologicalcontamination(6)modifiedproteins(7)nucleidacids(8)oligonucleotide(9)enterotoxinsIIIClassificationofbiosepar45IIIClassificationofbioseparation

steps(4)Therapeuticusagerequirements:(1)Potency(2)Identity(3)Abnormaltoxicity(4)Nucleicacids(5)Homogeneity(6)Absenceofconflictingactivity

Processrequirements:(1)Maximizeproductyieldininitialstep(2)Limitthenumberofsteps(3)IntensificationIIIClassificationofbiosepar46蛋白质生化分离技术课件47IIIClassificationofbioseparation

steps(5)Newwordsandvocabulary

High-resolutionfractionaton(高分辩率分级）Harvesting(收获，收割）Clarification（澄清）Enrichment（富集）Hazardous（危险）Integrate（集成）Intensification（集成，加强）Expandedbedadsorption（扩张床）Homogenate（匀浆液）Fermentationbroth（发酵液）IIIClassificationofbiosepar48IVUpstreamanddownstreamprocessing(1)

Minimizelossofthevaluablebiologicalproduct(1)Locationandformofproteinsinthecell(2)Separationofcellulardebrisandinclusionbodies（Increasingthenumberofpassesthroughthehomogenizer）(3)Separatingabiologicalproductbydifferentprotocols(4)Understandingupstreamanddownstreamconditions(harvestingtimeandcellwallstrengthandproteases)IVUpstreamanddownstreampro49蛋白质生化分离技术课件50蛋白质生化分离技术课件51IVUpstreamanddownstreamprocessing(2)Biologicalprocessdevelopmentobjective:(1)Producttothemarketinquickesttimepossible(2)meetingallpossiblesafetyrequirements(3)processcosteffective(4)reliable(5)safetyfactorsparticularlyimportantduringscale-upIVUpstreamanddownstreampro52IVUpstreamanddownstreamprocessing(3)Enhancethequantityandquality(1)continuousmediasterilization(2)enhancemasstransfer(3)gasanalysisbymassspectrometry(4)computerizeddata(expertsystem)(5)searchfornoveltechniquesAmicrogravityseparation(eliminateshearstress)BmolecularimprintingpolymersCfoamsseparationIVUpstreamanddownstreampro53IVUpstreamanddownstreamprocessing(4)Separationunderspacemicrogravity(1)higherthroughputsforelectrophoreticseparation(2)twophasepartitioningundermicrogravity(mixinganddemixing)(3)crystallizationofproteinsundermicrogravity(needweakinteractingforcewhenproteinformingcrystallization)(4)advantagesandexpensecarefullyanalyzedongroundbioprocessingfordifferentapplicationIVUpstreamanddownstreampro54IVUpstreamanddownstreamprocessing(4)NewWordsandvocabuloryCoarse-gradecentrifuge(粗糙级别的离心）Harsher（苛刻的）Intractably(不听话的）Debris(碎片）Endogeneous(内源性的）Dextransucrase(葡聚糖蔗糖酶）Highlight(突出）Copewith(应对）Trypsin（胰蛋白酶）Lysozyme(溶菌酶）Containerless无模板的Shearstress剪切力Thermodynamic(热力学）IVUpstreamanddownstreampro55VSomefactorsinfluencingbioseparation(1)AProcessMonitoring(1)monitorstructureandbiologicalactivityduringcontinuousprocess(2)insitusensors(3)flowinjectionanalysis(4)synchronizedpeak-switchinghighpressureliquidchromatography(5)capillaryelectrophoresis(6)ionizationmassspectrometry(7)rapidprocessmonitoring(8)poorqualityproduct(a)furtherpurification(b)identifyanddiscardtheproductVSomefactorsinfluencingbio56VSomefactorsinfluencingbioseparation(2)BBioseparationeconomics(1)atleast50%oftotalcost(2)spendsonlyabout5%ofeffort(3)reducethedegreeofwasteandemissions(product:waste=1:3250)(4)processmodification(a)molecularbiology(hybridgene,fusionprotein,charged-fusiontechnique(b)fermentationparametersVSomefactorsinfluencingbio57VSomefactorsinfluencingbioseparation(3)CProteinrefoldingandinclusionbodies(1)(1)aggregationformationpathway(2)specificbiteinvolvedinaggregation(3)minimizingaggregation(a)eliminatingdiffusion(b)covalentmodification(c)Chaperoneshsp10and60(4)processingaggregatesadvantages(a)initialpurificationstraightforward(50%)(b)ion-exchangeto90%productpurityeasily(c)proteasedonotattackinclusionbodies(d)inclusionbodiessolubilized,refoldedVSomefactorsinfluencingbio58蛋白质生化分离技术课件59VSomefactorsinfluencingbioseparation(4)CProteinrefoldingandinclusionbodies(2)(1)Intentionallydenatureaprotein(usingdenaurants(SDS-Page))(2)chemicalmodification(a)extractionofproteinstoorganicsolventsbyPEGmodified(b)solubilityofproteinsintoaqueoussolutionbyadditionofchargedgroupstoproteinsurfaces(3)interfacialadsorptionofproteins(a)understandingofnon-specificproteininteractions(b)delicatebalanceofthevanderwaals,electrostatichydrophobicandhydrationVSomefactorsinfluencingbio60VSomefactorsinfluencingbioseparation(5)CProteinrefoldingandinclusionbodies(3)Recoveryprocessconsider:(1)mildcondition(2)minimizeprocesstime(3)avoidextremepHandtemperatureconditions(4)avoidexposuretoair-waterinterfacesandshear(5)minimizedexposuretononpolarsolventsandhydrophobicadsorbents(6)beefficientVSomefactorsinfluencingbio61VSomefactorsinfluencingbioseparation(6)CProteinrefoldingandinclusionbodies(4)Processeconomicsandrenaturation(1)removingdetergent(a)ionicdetergents(ionexchangecolumn)(b)longhydrophobicsidechaindetergents(adsorptiononahydrophobicresin)(c)detergentsbealsoremovedbysolventextractionVSomefactorsinfluencingbio62Vsomefactorsinfluencingbioseparation(7)CProteinrefoldingandinclusionbodies(5)Futuresaleestimation(1)ReallyprofitablecompaniesinUSAmgenBiogenGenentechGenzymeVsomefactorsinfluencingbio63VSomefactorsinfluencingbioseparation(8)CProteinrefoldingandinclusionbodies(6)(2)DevelopingproductsrisksPitfallandunpredictability(a)drugdevelopment(b)clinicaltrials(c)FDAadvisorycommitteereviews(d)FDAapproval(e)Competition(f)Marketsize(g)DrugPrice(h)MarketpenetrationVSomefactorsinfluencingbio64VSomefactorsinfluencingbioseparation(9)Cproteinrefoldingandinclusionbodies(7)Particulardrugproduction(1)processeconomics(2)innovativemethod(a)multiuseflexiblefacility(b)shareafacilitywithothercompanies(c)reducedesigning,building,runningcostVSomefactorsinfluencingbio65VSomefactorsinfluencingbioseparation(10)CProteinrefoldingandinclusionbodies(8)

followingguidelinefordrugproduction(1)segregationwhenflowpathcross(2)stringentvalidation(3)Carefulschedulingtooptimizetheplantutilization(4)discussionondesign(a)fermentation(b)primaryrecoverystep(c)Purificationstep(d)fillingstep(e)FlowpathsVSomefactorsinfluencin