BioseparationsEngineering生物分离工程

BioseparationsEngineering生物分离工程=$1.32millionpercarats≈$6.6millionpergramUpdatemostexpensivediamondis:2023/1/122HeparinaseI(肝素酶I)=$450/100ng=$4500,000,000/g=$4500millionpergramAbout682times!2023/1/123Itisdifficulttoseparatethem:(2)Complexcomponents

（成分复杂）(1)desiredproductsindilutesuspension（目标产物浓度低）(3)Biologicalproductsrequirehighquality

(生物产品高质量)：*purity（纯度）*sanitation（卫生）*biologicalactivity（生物活性）Howtosolveit?2023/1/124Chapter1Introduction1.1WhatisBioseparation1.2ThecharacteristicsofBioseparation1.3TheunitoperationsinBioseparation1.4TechnicalExamplesinBioseperation1.5EvaluationofBioseparation1.6TheObjectiveoftheCourse2023/1/125WhatisBioseparations?(1)AcollectivetermIncludesallthestepsormethodsrequiredinordertorecoverusefulbiochemicalsorbioproductsfromanykindofindustrialprocesses.(所有从任何工业过程中回收有用的生物化学品或生物产品的方法、步骤)BiochemicalsorBioproducts—chemicalsubstancesorcombinationsofchemicalsubstancesthataremadebylivingthings—rangefrommethanol(甲醇)towholecells.derived2023/1/126Extractedfromwholeplantsandanimals(提取)synthesizedinbioreactorscontainingcellsorenzymes(生物合成)WhatisBioseparations(2)Downstreamprocessingforbiotechnology

Itisparticularlyimportantinbiotechnologywherethedesiredfinalformsoftheproductsareusuallyquitefarremovedfromthestateinwhichtheyarefirstobtainedinthebioreactoraswellasintissuesofanimalsorplants.(产物与其在生物反应器、动植物组织中的状态相去甚远)2023/1/127WhatisBioseparationsWhatisbiotechnology?Definition:Biotechnology(fromtheEuropeanFederationofBiotechnology)isanintegrateduseofbiochemistry,microbiologyandchemicalengineeringinordertoachievethetechnological(industrial)applicationofthecapacitiesofmicrobesandculturedcells.(综合生化、微生物学、化学工程等学科实现微生物及培养细胞的工业应用)2023/1/128WhatisBioseparationsForexample:theproductionofantibiotics,cheese,andwine.Geneengineeringcanmodifybacterialcellstosynthesizecompletelynewsubstances,e.g.,enzymes,hormones,insulinandsoon.Theapplicationmeanstheuseofcarefullyculturedmicroorganisms,animalcellsandplantcellstoproduceproductsusefultohumans.（即指利用培养微生物、动植物细胞来生产对人有用的产品。）2023/1/129EquipmentHardware,bioreactors,softwareandconsumablessupportingbiotechnologyTherapeuticsPharmaceuticalproductsforthecureorcontrolofhumandiseasesincludingantibiotics,vaccines,genetherapyDiagnosticsClinicaltestinganddiagnosisAgriculture/forestryNovelcropsoranimalvarieties,pesticides,fertilizersFoodWiderangeoffoodproducts,beverages,ingredientsEnvironmentWastetreatment,bioremediation,energyproductionChemicalintermediatesReagentsincludingenzymes,DNA/RNA,specialtychemicalsAprotoday'sbiotechnologyindustryWhatisBioseparations2023/1/1210Biotechnologyhasthree-componentcore:Thefirstpartisconcernedwithobtainingthebestbiologicalcatalystforaspecificfunctionorprocess;(geneticengineering)Thesecondpartcreates(byconstructionandtechnicaloperation)thebestpossibleenvironmentforthecatalysttoperform;(fermentation)★Thethirdpart(downstreamprocessing,thatis,bioseparations)isconcernedwiththeseparationandpurificationofanessentialproductorproductsfromafermentationprocess.WhatisBioseparations2023/1/1211Aboveall:Biosepartionsisafractionofbiotechnology;Itconsistsofaseriesofsteps(orprocesses);c.Itsobjectiveistoobtain(recover)usefulbiochemicalsorbioproductsWhatisBioseparations2023/1/1212UpstreamProcessingbiosynthesisBioseparationDownstreamprocessingdownstreamprocessWhatisBioseparations2023/1/1213WhatisBioseparationBioseparationBioproductsVariety(structuresandsizes)Lability(temperature,pH,concentration)MoreunitoperationsWhatisBioseparationsBackto2023/1/1214ThecharacteristicsofBioseparationBioproductioncanbebroadlyclassifiedintothefollowingcategories:Smallmolecules,consistingoffinechemicals,antibiotics,hormones,aminoacids,andvitaminsLargemolecules,consistingofproteins,polysaccharides,andnucleicacids,andparticulateproducts,consistingofcells,spores,liposomes,andsubcellularparticlesororganelles2023/1/1215BioproductExamplesMolecularWeight(Da)TypicalRadiusSmallmoleculesSugars200-6000.5nmAminoacids60-2000.5nmVitamins300-6001-2nmOrganicacids30-3000.5nmLargemoleculesProteins103-1063-10nmpolysaccharides104-1074-20nmNucleicacids103-10102-1,000nmParticlesRibosomes25nmViruses100μmBacteria1μmOrganelles1μmYeastcells4μmAnimalcells10μmThecharacteristicsofBioseparation2023/1/1216SmallBiomoleculesSmallbiomoleculescanbedividedintotwocategories:primarymetabolitesandsecondarymetabolites.Primarymetabolitesincluding:Sugar,Organicalcohols

acidsandketones,Vitamins,AminoAcids,isonethatisformedduringtheprimarygrowthphaseoftheorganism.Secondarymetabolitesincluding:antibioticsareproducedatornearthebeginningofthestationaryphase.ThecharacteristicsofBioseparation2023/1/1217SmallBiomoleculesAminoAcidAminoAcidAlanine(丙胺酸)AlaLeucine(亮氨酸)LeuArginine(精氨酸)ArgLysine(赖氨酸)LysAsparagine(天冬酰胺酸)AsnMethionine(蛋氨酸)MetAsparticAicd(天冬氨酸)AspPhenylalanine(苯丙氨酸)PheCysteine(半胱氨酸)CysProline(脯氨酸)ProGlutamicAcid(谷氨酸)GluSerine(丝氨酸)SerGlutamine(谷氨酸盐)GlnThreonine(苏氨酸)ThrGlycine(氨基乙酸)GlyTryptophan(色氨酸)TrpHistidine(组氨酸)HisTyrosine(酪氨酸)TyrIsoleucine(异亮氨酸)IleValine(缬氨酸)ValAminoAcidandtheirAbbreviationsThecharacteristicsofBioseparation2023/1/1218SmallmoleculesPrimarymetabolitesSecondarymetabolitesThecharacteristicsofBioseparation2023/1/12192023/1/1220ProteinsThecharacteristicsofBioseparationLargeBiomolecules2023/1/1221ThecharacteristicsofBioseparationProteinsLargeBiomolecules2023/1/1222NucleicacidsandOligonucleotides腺嘌呤鸟嘌呤胞嘧啶尿嘧啶胸腺嘧啶ThecharacteristicsofBioseparationLargeBiomolecules2023/1/1223ThecharacteristicsofBioseparationNucleicacidsandOligonucleotidesLargeBiomolecules2023/1/1224PolysaccharidesThecharacteristicsofBioseparationLargeBiomolecules2023/1/1225★lowconcentrationofthetargets(目标产物低浓度)Theconcentrationofthetargetsisverylowinfermentationliquor.Forexample,penicillin:4.2%,gentamycin(庆大霉素):0.2%.Moreover,theconcentrationofthetargetsinsomeanimal’stissuecultureisonlyabout5~50g/ml.★Biological(orchemical)activity(活性)Bioproductsaresoldfortheirbiological(orchemical)activity:Methanol:solventactivity;ethanol:neurologicalactivity;penicillin:antibacterialactivity;Taxol(红豆杉醇):anticanceractivity,andsoon.ThecharacteristicsofBioseparation2023/1/1226TabletypicalproductsfromfermentationThecharacteristicsofBioseparation2023/1/1227★Fermentationbrothbeingacomplicatedmixture(complexcomponents)(发酵液成分复杂)Thefermentationbrothisthecomplicatedmixtureofmanycomponents(solubleorinsoluble),notonlyincludingmacromoleculessuchasproteins,nucleicacid,polysaccharidesbutalsoincludingintactorganisms,fragmentedmycelia,otherinsolublefractionsofmediumingredients,residualsubstrates,etc.ThecharacteristicsofBioseparation2023/1/1228★Poorstability(稳定性差)Duetochemicaldegradationormicrobialdegradation,theproductscanbeinactivatedbytemperature,pHorvariousenzymes.★HighpurityandstrictsanitationcriterionAlmostallproductsareusedasmedicine,biologicalreagentsorfood,theirpuritymustmeettheneedofpharmacopoeia(药典),reagentcriterionorfoodcriterionetc.Allabovemakesbioseparationsmuchmoredifficultthanchemicalseparations!!ThecharacteristicsofBioseparation2023/1/1229Thediversityofproductsspawns(resultsin)thebroadspectrumofseparationmethodsused(2)CharacteristicsofBioseparations★Broadspectrumofseparationmethods（产品的多样性导致分离方法的多样性）★mostbioseparationmethodscomefromseparationsofchemicals

(大多数生物分离方法来源于化学品的分离方法)ThecharacteristicsofBioseparation2023/1/1230classificationUsedforallconventionalchemicalsUsedinBioseparationsPhysicalseparationsEquilibriumcontrolledseparationsRatecontrolledseparations7221371810(2)CharacteristicsofBioseparationsThistableshowsthat80%ofalltheseparationmethodsforconventionalchemicalsarepracticalinbiotechnology.

(大约80%的化工分离方法可应用于生物分离技术)ThecharacteristicsofBioseparation2023/1/1231★TheseseparationsareelaborateandexpensiveBothdilutefeedandproductswithhighpuritymaketheseparationsbeelaborateandexpensive.－Moreequipmentandlaborthanallotherpartsoftheprocesscombinedareneeded.－Costingasubstantialfractionofthetotalcostofthefinalproduct.Thisfractioncanbeashighas90%ormore!ThecharacteristicsofBioseparation2023/1/1232☆Yeastbiomass,penicillinG,andcertainenzymeprocessingcostsarepercentagesofthesellingpriceswhichare20%,20-30%and60-70%respectively.☆Productsexpressedbygeneticengineering,theirprocessingcostscanbeashighasabout85~90%oftheirsellingprices.☆Theprocessingcostofasparaginase(天冬酰胺酶)usedaspharmaceuticalsisabout85%ofitssellingpricesbecauseofitshighpurity.ThecharacteristicsofBioseparation2023/1/1233PricevsProduction红细胞生成素长春碱红豆杉醇洋地黄果胶酶ThecharacteristicsofBioseparation2023/1/1234Therecoveryandthepurificationprocessesmustbewellconceivedandwelldesigned!Important!

Organicmolecules,$5-$10/g

Biopolymers,$1-$5/g

Food&beverageadditives,<$1/g

Biopharmaceuticals>$20,000/gHowtolowerthem?（因此必须仔细考虑和设计产品的回收和纯化过程）ThecharacteristicsofBioseparation2023/1/1235So,Beforedesigning,whatweshouldaskandanswerare:Whatisthevalueoftheproduct?

(产品价值)Whatisanacceptableproductquality?

(产品质量)Whereistheproductandimpuritiesineachprocessstream?

(产物及杂质在加工过程中出现位置)ThecharacteristicsofBioseparation2023/1/1236Whataretheunusualphysicochemicalpropertiesoftheproductandtheprincipalimpurities?

(产物及主要杂质独特的物化性质是什么？)Whataretheeconomicsofvariousalternativeseparations?

(各种分离方法的经济性比较)Carefulconsiderationofthesequestionscanhelpustodeterminetheoptimalseparatingprocesses.ThecharacteristicsofBioseparation2023/1/1237ImprovetheefficiencyofbioseperationReducethestepsofoperationsReducethetimeofoperationprocessReducecostImprovepurityKeepactivityThecharacteristicsofBioseparationAboveall:Backto2023/1/1238

Removetheinsolubles(不溶物的去除）Filtration[过滤]Centrifugation[离心]Celldisruption(necessarywhenthedesiredproductisintracellular[细胞破碎]Relativelylittleproductconcentrationorimproveofproductqualityoccurs

[产物浓度和质量得到了提高]TheunitoperationsinBioseparation2023/1/1239

Isolationofproducts(产物分离)

Adsorption-ion-exchange[离子交换吸附]Membraneseparation[膜分离]Extraction[萃取]Solventextraction[溶剂萃取]Reversedmicelleextraction[反微团萃取]Supercriticalfluidextraction[超临界流体萃取]Two-aqueousphaseextraction[双水相萃取]这些分离过程不具备特异性，只是进行初分TheunitoperationsinBioseparation2023/1/1240

Purificationofproducts(产品的纯化)Chromatography[色谱]Electrophoresis[电泳]

Precipitation[沉淀]具有产物的高选择性和杂质的去除性TheunitoperationsinBioseparation2023/1/1241

Polishing(产品的精制)Crystallization[结晶]Drying[干燥]TheunitoperationsinBioseparation2023/1/1242StageObjective(s)TypicalUnitOperationsSeperationofinsolublesRemoveorcollectcells,celldebris,orotherparticulatesFiltration,sedimentation,Extraction,AdsorptionReducevolume(dependsonunitoperation)IsolationofproductRemovematerialshavingpropertieswidelydifferentfromthosedesiredinproductExtraction,adsorption,Ultrafiltration,precipitationReducevolume(dependsonunitoperation)PurificationRemoveremainingimpurities,whichtypicallyaresimilartothedesiredproductinchemicalfunctionalityandphysicalpropertiesChromatography,affinitymethods,crystallization,fractionalprecipitationPolishingRemoveliquidsConverttheproducttocrystallineform(notalwayspossible)Drying,crystallizationTheunitoperationsinBioseparationBackto2023/1/1243TechnicalExamplesinBioseperationDownstreamprocessesforproductionofantibiotic2023/1/1244TechnicalExamplesinBioseperationProductionofrecombinanthumanalpha-1-antitrypsin(抗胰蛋白酶)2023/1/1245RecoveryofanextracellularenzymeNutrientmediumFermentationMicroorganismsSelf-cleaningclarifierCellmassSelf-cleaningclarifierResidualsolidsConcentratione.g.ultrafiltrationorvacuumevaporationPrecipitationFilterpressDryingEnzymepowderLipidSterilizationLiquidenzymeTechnicalExamplesinBioseperation2023/1/1246RecoveryofintracellularenzymesNutritivemediumFermentationMicroorganismsSelf-cleaningclarifier(cellharvesting)CelldisintegrationDilution(ifpossible)Continuousliquid-liquidCross-flowextractionwithaqueoustwo-phasesystemsSelf-cleaningclarifier(separationofcelldebris)ConcentrationandenrichmentPurificationConcentration/precipitationTechnicalExamplesinBioseperation2023/1/1247TechnicalExamplesinBioseperation2023/1/1248BioreactorfluidwithcellsChromatographyCelldisruptionCentrifugalextractionDialysisCleaningproinsulinPrecipitationDialysisSulfonationExtractionPrecipitationChromatographyCelldebrissupernatantB-ChainA-ChainRecoveryandpurificationofhumaninsulinTechnicalExamplesinBioseperation2023/1/1249BioreactorfluidpretreatmentSterileultrafiltrationFiltrationorcentrifugationTwo-stageultrafiltrationUltrafiltrationPrecipitationPolishingultrafiltrationRotaryfilterFormulationDryingFormulationCellsFilteraidprecoatLiquidsorsolidsCoolto4-5℃FilteraidLiquidproductSolidproductDownstreamprocessingforproductionofliquidorsolidproteaseTechnicalExamplesinBioseperation2023/1/1250BioreactorfluidwithcellsFiltrationExtractionpH2-3BackextractionFiltrationCrystallizationIonexchangecentrifugationDryingKeroseneandtertiaryamineWateradsorptionT=5℃CaCl2andpolyelectrolyteProductfiltrateActivatedcarbonSpentsolventDownstreamprocessingofcitricacidTechnicalExamplesinBioseperation2023/1/1251Example:(p33)Youhavebeengiventheassignmenttopurifytheantibioticerythromycin(红霉素).YouhaveatyourdisposaltheMerckIndex,whichhastheinformationonerythromycinshowninFigureE1.2.Whatdoyouthinkarethemostlikelyunitoperationsthatshouldbeusedfortheisolationandpurificationoferythromycin?TechnicalExamplesinBioseperation2023/1/1252Solution:ThepropertiesoferythromycinLimite