生物分离工程双语版

生物分离工程双语版第1页/共72页1IntroductionConsideringproteinpropertiesforbioseparation(1)size(2)charge(3)biologicalaffinity(4)hydrophobicity-solubilityProductsfrommammaliancelltechniques(1)enzyme(2)hormones(3)nonantibodyimmunoregulators(4)monoclonalantibodies(5)tumorspecificantigens第2页/共72页1IntroductionProteinfolding

(1)shortrangedinteraction(secondarystructure)(2)gradualcombinationandreshuffling(重新组合）

(subdomains,domains,andsubunitassemblies)Secondgenerationbiotechnologicalproducts(1)largermolecules(2)morecomplex(3)moreglycosylate(4)moresulphydrylbonds第3页/共72页1IntroductionInitialbioseparationimportance(1)filtration(2)centrifugation(3)chromatographyClassifiedheuristics（论据，规则）(1)unitoperationmethods(2)designstepsequence(3)species(componentsproperties)(4)composition第4页/共72页第5页/共72页第6页/共72页1IntroductionThreefactorsofdesigningbioseparationprocesses(1)purity(2)cost(3)marketMaterialstateandchoiceofseparationmethodsMaterialsecreted(ultrafiltration,centrifugation)Notsecretedmaterial(celldisruption,solid-liquidseparation)Materialinliquid(ultrafiltration,adsorption)Materialinsolid(extractionintoaqueoussolution)第7页/共72页NewandwordsProtactelawsuits诉讼Deleterious有害的，有毒的Vexing烦恼Hierarchic分等级Subdomain亚域Domains蛋白质结构的域Subunit亚单位Endoplasmicreticulum内质网Disulfidebond二硫键Academia学术界Delicate脆弱的Flowchart流程图Cytoplasmic细胞质的Periplasmic外周胞质Osmotic渗透

urea脲Guanidine胍第8页/共72页IIProductexcretionfromthecellorcelldisruptionExcretedproteinadvantage(1)contaminationlevellow(2)easydownstreamprocessingsteps(3)notrequirecelldisruption(4)avoidhandlingviscousfluidsandproteinaserelease(5)facilityextractionofproducts(6)signalpeptidedeterminessecretion第9页/共72页Celldisruption

(1)energy-intensive(强烈）andviolent(2)withoutdestroyinginterestprotein(3)contain(兼容）process(bacteria,yeast,mammalian,plant,andinsectsources)(4)becontrolled(5)bevalidated第10页/共72页ProductsrequiringcelldisruptionVaccines(tetanus(破伤风）,meningitis（脑膜炎)Glucokinase（葡萄糖激酶）Glycerokinase（甘油激酶）Invertase（转化酶）Toxin(enterotoxin)Subcellularcomponents(mitochondrai,chloroplast)Intracellularconstituents(DNA,RNApreparation,andvirus-likeparticles)RecombinantinsulinRecombinantgrowthhormoneProteinAandG.第11页/共72页Ideallargescalecelldisrupter(1)disrupttoughorganisms(2)mechanismofcelldisruptionshouldbewellunderstood(3)sterilizable(4)containable（兼容）andvalidatable(5)amenabletobeingmadeautomatic(6)continuousandcompact（紧凑的）(7)economical第12页/共72页CelldisruptiongoalObjectivefunction(monetary（金钱）

valueofproduct)Smallscalecelldisruption(1)ultrasonic(2)freezethawing(熔化）(3)forcecellsthroughverysmallorificesathighpressure(4)glassorporcelainballmills第13页/共72页LargescalecelldisruptionHomogenizerStructure(positivedisplacementpump,pistons,nozzle)Mechanisms(1)Shear(2)Cavitation（气穴）(3)impingement(冲击）Condition(1)concentration(4-175gdrywt/liter)(2)pressure(30-95MPa)(3)passesnumber(~5time)(4)modeledbyequation(5)wild-typeE.colicellsmoredifficulttodisrupt(heatinductionat42C)第14页/共72页Ln(1/(1-R))=KNPΓ第15页/共72页第16页/共72页Delineatingmechanismsofcelldisruption(1)facilitatecontrolling(2)validating(3)reproducible(4)betterdesignandoptimizeequipment(5)Turbulenteddies(湍流涡旋）

Responsibleforcelldisruptionoscillate)(6)shearstress,turbulencebyimpingement第17页/共72页Enzymaticlysiscelldisruption(1)alyticprotease(dissolvetheouterpartofthecellwall(2)endo-β-(l-3)glucanase（葡聚糖酶）

todisintergrateglucannet(3)source（酶的来源）andculturesystemkineticanalysisAunderstandingthesystemBdesigningthesmallandlargescalesystemsCcytophaga（吞噬细胞）systemexhibitshighproteaseactivity,higherconversionandhigherlysisrateatlongertimeDoerskoviasystemshighglucanaselowproteaseactivity,containsomeinhibitorsEaninitiallaginyeastlysisbybothenzymeFsimpletwostepmodel(cellmasssolubilized,proteinhydrolyzedtopeptides)GMishaelis-MentenkineticequationpredictcellwalllysisHproductionoffoodgradesinglecellprotein第18页/共72页NewwordsandvocabularyAmylase淀粉酶Modulate调节Interferons干扰素Viscousfluids粘性流体Signalpeptide信号肽Carboxypeptidase羧肽酶Alkalinephosphatase碱性磷酸酯酶Glyceraldehyde-3-phosphatedehydrogenase甘油醛-3-磷酸脱氢酶Reducednicotinamideadenine还原型尼克酰胺腺嘌呤Dinucleotide二核苷Reductase还原酶Hydrophobic疏水的Hydrophilic亲水的第19页/共72页Newwordsandvocabularies(2)Pathogens病源体Ballmills球磨机Resonance共鸣，回声Vibration振动，摆动Dissipate分散Pinpointing最佳点,针尖Oscillate振荡Candidautilis假丝酵母Extrusion喷出Sapphire兰宝石Jewel宝石Thermocouple热电偶Sleeve袖子，套Hydrodynamic流体力学Hydroxylaseenzyme羟化酶Synergistic增效，协同作用Glucan葡聚糖Morphology第20页/共72页IIIinitialfractionationPrimaryisolationstep(1)adsorption(2)extraction(3)precipitation(4)distillation(5)filtration(6)solventevaporationRequirementSignificantvolumereduction第21页/共72页Interestproductinsolution(1)graduallyremoveimpurities(2)increaseproductconcentration(3)chosemethodsdependsonscaleandorganism(4)washingstagesfollowedbydewateringstepYeastseasytodealwith(1)10µmindiameter(2)easilyflocculated(3)centrifugation(4)filtration(5)cross-flowfiltration第22页/共72页Analysisdisruptedbaker’syeastsuspensions(1)viscosity(2)density(3)sedimentationMeaning(1)minimizedegradationcellwallfollowingrupture(2)minimizecellfragmentproportion(3)restrictviscosityincrease(4)correlationbetweenlabandpilotscalecentrifugesorindustry(5)estimateseparationcostsatanearlystage第23页/共72页Firstcrudeseparationorfinalproductdewatersteps(1)adsorption(2)precipitation(3)membraneseparationAcrossflowfiltrationBmicrofiltrationCultrafiltration(4)liquid-liquidextraction第24页/共72页Aqueoustwo-phasepartition(1)polymer+salt,polymer+polymer(2)easytoscaleup(3)biocompatible(4)rapid(withcentrifugation)(5)purificationandconcentrationofbiologicalproduct(6)cellparticlesandnucleicacidsremoved(7)largevolumereduction第25页/共72页Aadsorption(1)purifyprotein(2)difficulttoremovingparticulateusingpackedorfixedbeds(3)particulateentrappedinvoidsofthebeds(4)excessivepressuredropsanddeformationofadsorbent(5)expandedbed(6)fluidizedbed第26页/共72页Expandedbedadsorption(1)eliminateastepforremovalofcellordebris(2)scaleupsimple(similartopackedbed)(3)combineclarification,concentrationandpurification(4)couldbeusedforviscosefluid(5)highbiomasscontentoffeedstock(6)expandedbedstabilizedbywidedistributionofparticlesize第27页/共72页第28页/共72页第29页/共72页用吸附剂分离的抗生素第30页/共72页NewwordsandvocabularyColloidal（胶状的）Glycan（聚糖）Lysate（溶解物）Aerosol（悬浮微粒,气溶胶）Particulate（微粒）Fluidizedbed（沸腾床）Streptomycin（链霉素）Novobiocin（新生霉素）AnnexinV（膜联蛋白[一类被钙离子活化后可与膜磷脂结合的蛋白）Scout（侦察）Washing（洗涤）Elution（洗脱）第31页/共72页NewwordsandvocabularyReversephasehighpressureliquidphasechromatography(反相HPLC）Circumvented（包围）Hybridoma（杂合细胞，(淋巴细胞)杂交瘤）Penicillin（青霉素）Tetracycline（四环素）CephalosporinC（头孢菌素C）Divinylbenzene（二乙烯苯）Styrene（苯乙烯）第32页/共72页BmembraneseparationFunction:ClarificationandsterilizingProblems(1)Solid-liquidinterfaceadsorption(2)significantsurfacearea(500cm2)Membranematerials

(1)hydroxyl-modifiedhydrophilicpolyamide(2)hydrophilicpolyvinylidenefluoride(PVDF)聚偏二乙烯氟化物）(3)nylon-66Interestprotein

(1)insulin(100%recovery)(2)bovineserumalbumin(BSA)98%recovery(3)immunoglobulin第33页/共72页Minimizingproteinloss(1)low-bingingproteinmembrane(2)diluteproteinsolution(3)expensive(4)examineprefiltersandfinalfilters第34页/共72页Removeofvirus(1)thermalinactivation(2)chemicaltreatment(3)denaturant(urea)(4)β-propiolactone（丙内酯）-UVlightexposure(5)membranefiltrationAbaseonsizeBreproducibleCpredictableDcompleteEvalidatableFhighproteinrecovery第35页/共72页NewwordsandvocabularyInsulinBovineserumalbumin（牛血清蛋白）Immunoglobulin（免疫球蛋白）Scale-down（按比例缩减）Discernible（可辩别的）Steadystate（稳态）Asymptotically（渐近的）Low-bindingproteinmembraneβ-propiolactoneUVlight（β-丙内酯-紫外光）Monotonically（无变化的，单调的）Platelet（血小板）ImmunodiseaseHydraulicpermeability（水渗透性）Filtrationresistance（过滤阻力）Purewaterpermeability(PWP)纯水透过性第36页/共72页VocabulariesDomaindisulfidebonds,signalpeptide,turbulenteddiesAqueoustwo-phasepartitioningexpandedbedadsorptionfluidizedbedpackedbedreverse-phasehighpressureliquid-phasechromatographyfloculatebiocompatiblescaled-downsteady-statelangmuirisotherm第37页/共72页Questions1Whichadvantagessecretedproteins?2WhichdifferencesbetweenMammaliancellproteinsandmicroorganismproteins?3Whichmethodsdisruptingcellinlaboratory4Whatisthemostimportantinearlypartofbioseparation?5Whatadvantagehaveexpandedbedadsorptionbycomparingwithpackedbed?6Howtoremovevirusorvirus-likeinproteinsolution?第38页/共72页Chapter2

Sec第39页/共72页CUltrafiltrationExample：antibodiesproduction(1)antibodiesapplicationAdiagnosisBaffinityligand(cytokinesandblood-clottingfactors)(2)membranematerial(cellulosetriacetate)(3)useA50-100-foldconcentrationBminimizingshearforcesCtangentialandframeultrafiltrationsystemDgelpolarization（浓差极化）reducedFacceptablefluxrate第40页/共72页Denaturationduringultrafiltration(1)conformationalchange(2)instability(3)immunogenicity(免疫原性）InfluencingultrafiltrationfactorsAelectrostaticinteraction(attraction,repulsion)BHydrophobicity第41页/共72页NewwordsandvocabularyMandate（命令，要求）Diagnostics诊断学Cytokines细胞因子Bloodclottingfactors（凝血因子）Tangentialplate切向板，错流过滤板Undermine（破坏）Integrity（完整）Trypticβ-caseinpeptidesβ-酪蛋白肽Antihypertensive（抗高血压的）Immunomodulatory免疫调节剂Plasmin血纤维蛋白溶解酶Transmission输送Detrimental有害的Interfacialtension表面张力Emulsionliquidmembrane乳状液膜Carrier载体Diffusivity扩散率第42页/共72页Dcross-flowfiltrationAffinitycross-flowfiltration(1)combiningbioadsorptionandmembraneinasinglestep(2)escortsupportsAchemicallyandmechanicallystableBpossessalargespecificareaCNegligiblenonspecificadsorptionDeasilymodifiabletoincorporateligands(3)highproteinlossinwashingstep第43页/共72页第44页/共72页NewwordsandvocabulariesAffinitycross-flowfiltrationEscort簇Supernatant上清液MonomolecularlayerInnermost内心的，最里面的LangmiurisothermGlutaradehydeSchiffbasesSodiumborohydrideMicroparticles第45页/共72页EEmulsionliquidmembranePenicillinGextractionbyemulsionliquidmembraneApplicationConcentrateweakacids,bases,metalionandbiomaterialPenicillinextraction(1)Carrier:amine(2)pHadjusterNa2CO3atpH5-8,penicillinGstable(3)9timesconcentration第46页/共72页第47页/共72页FExtractionInsulinpurificationprocess(1)extractionbyethanol(2)acidificationatpH2(3)neutralizationwithcalciumcarbonate(4)vacuumconcentration(5)saltprecipitation(6)reprecipitationbyisoelectricpoint(7)gelfiltrationchromatographyorionexchangechromatographyDifficultinformationavailable(1)effectofdifferentprocessvariables(2)sequencingofdifferentsubprocesses(3)scalelevelofoperation第48页/共72页NewwordsandvocabulariesEmulsionliquidmembrane乳化液膜Amine胺Carrier载体Pancreas胰腺Hyperglycemia高血糖Diabetesmellitus糖尿病Porcineorigin猪器官Isoelectricpoint等电点Reverse-phasechromatography反相层析Scarce缺乏的，不足的Proprietary所有权第49页/共72页GprecipitationApplyinghighresolutiontechniqueatearlierstage

(1)reducevolume(2)reducechemicalconsumptionAffinityprecipitationusingchitosanasaligandPrincipleSolubleligandbindingtotargetproteinleadtoprecipitate(1)aggregate(2)heterobifunctionalligandsIsolatingtrypsinwithSTIchitosan第50页/共72页第51页/共72页Precipitationfactors(1)saltandcosolvents(supersaturation,seedparticles,crystallization,agglomerationα-lactalbuminseparationWheycomponents:lactose,proteins,saltsandresidualfat(1)1:1bindingcalciumion(α-1A)(2)conformationalchange(unfoldingtransitions,surfacehydrophobicity)(3)selectivelyseparatingα–lactalbumin((α-1A)andβ-lactoglubulinatpHisoelectricpointandabove50℃(4)nonspecificprecipitation第52页/共72页AffinityprecipitationusingligandmodifiedphospholipidasaligandcarrierPrincipleSolubleligandbindingtargetproteintoformprecipitateHeterobifunctionalligand(bindingtargetmoleculeandfacilitateprecipitationofliand-targetprotein)Biotinligandbindingavidin(1)rapidbinding(2)slowhydrophobicinteractionbetweennonpolaralkylsidechainsofphospholipids(3)highyieldandpurityofavidinDrawbacks(1)difficulttoobtainstableandinexpensivebis-ligands,polyligandsandligandcarriers(2)adsorptioncapacitylow(3)nonspecificadsorptiondegreehigh第53页/共72页第54页/共72页AffinityprecipitationmechanismTwomechanism(1)primaryeffectProteincross-linkingandformingnetworkwithligand(2)secondeffectSolubilitydecreaseofligandcarrierduetopH,temperature,andsaltconcentration第55页/共72页STIimmobilizedonPLSasanaffinityagentfortrypsinprecipitation(1)materialAartificialsolutioncontainingBSAandtrypsinBacrudepancreaticextract(2)saltprecipitatedPLSduetoosmoticshock(3)PLSredispersedinsolutionafterremovingsaltbycentrifuge(4)PLShadlargespecificsurfaceareaandligandcouplingcapacity(5)lackofnonspecificadsorption(6)91%trypsincoprecipitatedinartificialsolutionand87%trypsinrecoveredinprecipitateby0.01MNaOH(7)STI-PLSrepeatedthreetimes(8)Purificationfactorwasaboutsix(9)lossofbindingcapacity(93.3%,87.7%,83.3%)(10)80%activityrecoveryandspecificactivitywith1X104U/mg(11)precipitateandredisperse10timesbyaddedsalt第56页/共72页NewwordsandvocabulariesAffinityprecipitation亲和沉淀Heterobifunctionalligand异种双功能试剂Chitosan壳聚糖Oligomeric寡聚体的Soybeantrypsininhibitor大豆蛋白酶抑制剂Chymotrypsin胰凝乳蛋白酶，糜蛋白酶Supersaturation过饱和α-lactalbumin乳白蛋白Wheyprotein乳清蛋白Mammarygland乳腺Trytophan脯氨酸Transitions过渡Turbidity混浊Surfactant表面活性剂Phospholipids磷脂Avidin生物素结合蛋白Biotin生物素Dimyristoylphosphatidylethanolamine二肉豆蔻酰磷酸乙醇胺Octaethyleneglycolmono-n-dodecyl-ether八乙烯醇单十二烷基醚Hastened促进，加速Polymerizedliposome聚合脂质体Redispers再分散Sterichindrance空间位阻第57页/共72页HchromatographicproceduresLargescalepurification(1)yield(2)purity(3)operationscale(4)chromatography(limitedextentininitialstep)Example2.11StaphylococcalenterotoxinBpurificationusingchromatography(1)Twoionexchangesteps(2)Onegelfiltrationstep(3)highlinearflowratesatlowpressure<5kP(4)culturesupernatantA(centrifugation,sterilefiltration,adsorption).Bcontent0.4mg/LCproteincontent11mg/mlDpurity0.004%Evolume400L第58页/共72页Pharmaciacolumn370KSpurification(1)proteincontentfrom11to1.5mg/ml(2)SEBcontentfrom0.00042to0.12mg/ml(3)purificationfactor2000Biologicalmoleculesproductstandards(1)DNA<10pg/dose(2)noviruses(3)pyrogen<300endotoxinsIU/dose第59页/共72页Example2.12Divinylbenzene-polystyreneresinseparation(1)analysisofthermodynamicsusingHPLC(2)linearadsorptionisothermabove293-313K(3)temperature-dependentadsorptionisothermconstantsΔS,ΔH(4)poresizediameter(5)polarityofresin(6)chromatographyisonlyachoiceforindustrialseparation第60页/共72页NewwordsandvocabulariesStaphylococcalenterotoxin葡萄球菌外毒素Methicillin甲氧苯青霉素PharmaciacolumnDose剂量Pyrogen热原Miniscule小字的Divinylbenzenepolystyrenecopolymer苯乙烯-二乙烯苯共聚物Aspartame门冬氨酰苯丙氨酸甲酯（比蔗糖甜200倍的甜味剂）Phenylalanine苯丙氨酸Asparticacid天门冬氨酸Asparagine天门冬素，天冬酰胺酸Adsorptionisotherm吸附等温线Homologues类似物，同族体第61页/共72页IVhigh-resolutionfractionationPreviouspurificationsteps(1)volumereduction(2)clarification(3)adjustmentofcrudeextractChromatographysteps(1)cationexchange(2)anionexchange(3)affinitychromatography(4)hydrophobicchromatography第62页/共72页AchromatographyproceduresAffinitychromatography(1)nonspecificadsorption(2)expensivelabileligand(3)difficultiesofadsorbentsterilization(4)biomimeticligand(5)naturalligandexpensiveanddecreasedactivitywhenimmobilized.(6)pseudo-ligand(7)ligandleakageandproductcontaminationLargescalepurification

(1)removelowlevelsofimpurities(2)reducepyrogen(3)exchangeintobuffersystemssuitableforformulation(4)gelfiltrationchromatographyhaslowproductivity第63页/共72页SEB:Staphylococcalenterotoxin葡萄球菌外毒素第64页/共72页Finalproductsanalysis(1)SDS(singleband)(2)gelfiltration(singlepeak)Developinglarge-scalepurificationstep(1)newmedia(2)equipment(3)optimization第65页/共72页UltrafastHPLC(1)instrumentationAselectivityBsensitivity(2)resinArapidassociationanddissociationBmaterialgeometryplaysignificantrole(3)obtainquantitativeproductrecoveryestimation(4)visualizewidevarietyofhostcellimpuritiesPosttranslationalmodification(deamidation,proteolysisandglycosation)(5)rapidHPLCsignificanceAmonitoringpr