1Chapter8ChromatographyDefinitionChromatography

is

a

bioseparation

technique

that

separatescomponentsofamixturebyexploitingtheirdifferentdistributioncoefficientsbetweenamobilephaseandastationaryphase.Asthemixturepassesthroughthestationaryphasewiththemobilephase,componentsmoveatdifferentspeedsduetotheirvaryingadsorption,solubility,andmolecularsize,leadingtoseparation.色譜（層析）分離技術(shù)是一種基于混合物中不同成分在流動相和固定相之間分配系數(shù)差異而實現(xiàn)分離的方法。當(dāng)混合物隨移動相一起通過固定相時，不同組分因其吸附性、溶解度和分子大小或形狀的差異而以不同速度移動，從而實現(xiàn)分離。“chromatographycolumn”（色譜柱）

“stationaryphase”（固定相）

“mobilephase”（流動相）3FormationClassification

(Based

on

separationmechanism):AdsorptionChromatography

GelFiltrationChromatography

PartitionChromatography

4ClassificationClassification

(Based

on

separationmechanism):AdsorptionChromatography

(吸附層析)GelFiltrationChromatography

(凝膠過濾層析)PartitionChromatography

(分配層析)5ClassificationChapter8-1Ion

Exchange

Chromatography6Whatcomestoyour

mind?Introduction7In2008,NobelPrizeinChemistrywas

forthediscoveryandresearchofgreenfluorescentprotein

(GFP).下村修馬丁·沙爾菲錢永健Introduction8AequoreaVictoriaThe

Nobel

Prize

in

Chemistry

2008Introduction9下村修錢永健馬丁Whatarethechallengesand

characteristicsof

these

fluorescent

proteins

fromtheperspectiveofbio-separation,

respectively?How

to

purify

fluorescent

proteins

(such

as

GFP)

using

Chromatography?Introduction10ThinkingWhatistheprincipleofIon

Exchange

Chromatography(IEC)?HowtopurifyGFP

using

IEC?What’stheproblemsofIECandhowtofurtherpurifyGFP?11OverviewFactorsinfluencingseparationeffectApplicationOutline12Overview—Principle13Separationisachievedviathereversibleand

electrostaticinteractionsbetweenchargedmoleculesandthestationaryphase.IonExchangeChromatographyseparatescomponentsbytheirdifferentbindingstrengthstoexchangeresins,duetoreversibleionexchanges.HigherchargeandconcentrationenhancethebindingstrengthinIEC.

14RA+B-

→RB+A-Overview—PrincipleBasic

process15Overview—Process(1)Columnpacking裝柱(2)Equilibration平衡(3)Loading上樣(4)Washing淋洗(5)Elution洗脫(6)Cleaningandregeneration清洗與再生16Overview—ProcessHighCapacity:Offersconcentrationeffect,processeslargevolumesefficiently.Versatility:Suitableforavarietyofapplicationswithshortcolumnlengthsandhighflowrates.EfficientAdsorption:Clearmechanismwithminimalnon-specificadsorptionandhighproductrecovery.Cost-Effective:Widerangeofaffordableionexchangeresinsavailable.Resolution:Lowercomparedtoaffinitychromatography.Overview—Characteristics17ExchangecapacityItreferstotheamountofexchangeableionsthatanionexchangeresincanprovide,anditreflectstheabilityoftheionexchangeresintoexchangeionswithionsinsolution.Totalexchangecapacity:

thetotalamountofexchangeableionsthatanionexchangeresincanprovide,whichisonlyrelatedtothepropertiesoftheionexchangeresinitself.Effectiveexchangecapacity:

the

exchangecapacityofthe

soluteintheactual.Itisnotonlyrelatedtotheionexchangeresinused,butalsohasagreatrelationshipwiththeexperimentalconditions

(e.g.

pH,

ionicstrength…).Overview—Exchange

capacity18StationaryPhase:

IonexchangeresinIncluding:

matrix(carrier),chargegroups(functionalgroups),counterions(exchangeions)固定相—離子交換劑：基質(zhì)（載體），電荷基團(tuán)（功能基團(tuán)），反離子（交換離子）MobilePhase:BuffersolutionIncluding:

equilibrationbuffer,

loading

buffer,

wash

buffer,

elution

buffer,

regenerationbufferOverview—Formation19Hydrophobicresin-polystyreneresin聚苯乙烯樹脂Hydrophilicpolymer纖維素(Cellulose)球狀纖維素(Sephacel)葡聚糖(Dextran)SephadexG-25,

G-50瓊脂糖(Agarose)SepharoseCL-2B，CL-4B1.

Carrier(matrix)20Overview—FormationPorous

ornon-porousmaterials,

but

the

use

of

non-porousmaterialsshowshigherresolutionandavoidsdiffusioneffects.Inertmaterials,

reducingnon-specificbinding.Highphysicalstability,ensuringthatthecolumnbedvolumeremainsconstantwhenthesaltionconcentrationorpHchanges.Highchemicalstability,ensuringthatthepackingmaterialcanbewashedwithstrongcleaningagentsifnecessary.1.

Carrier(matrix)21Overview—FormationIntroducingchargedgroupsoncarriersCationexchangeresin陽離子交換劑——Negativecharge,exchangeswithcationsAnionexchangeresin陰離子交換劑——Positivecharge,exchangeswithanions2.

Functionalgroups22Overview—FormationAcidicfunctionalgroups-Cationexchangeresin:Strongacidtype:(-SO3H)Mediumacidtype:(-PO3H2,-PO2H)Weakacidtype:(-COOH)Basicfunctionalgroups-Anionexchangeresin:Strongbasetype：季胺基團(tuán)（-N(CH3)3）Mediumbasetype：叔胺（-N(CH3)2）Weakbasetype：仲/伯胺（-NHCH3；-NH2）2.

Functionalgroups23（Q柱，磺酸鹽樹脂）（DEAE柱，季銨鹽樹脂）（CM柱，羧酸樹脂）（ANX柱，二乙胺樹脂）Overview—FormationDiscussion24PleasedesignaIEC-basedpurificationprocessforGFPproducedusing

the

Escherichiacoliexpressionsystem.DiscussionExpressionCell

cultureCell

disruptionClarificationPrecipitation,

DialysisMicro-filtrationIECFormulation25將GFP基因插入大腸桿菌表達(dá)系統(tǒng)超聲波破碎過濾澄清硫酸銨沉淀，透析離子交換色譜（如Sepharose-DEAE）微濾滅菌，裝罐具體色譜條件和過程如何選擇？細(xì)胞培養(yǎng)、誘導(dǎo)表達(dá)、蛋白擴(kuò)增1.Ionexchangeresin2.pH3.Ionic

strength4.Buffer

solution5.Column

size6.Flow

rateFactorsinfluencingseparationeffect261.

Ionexchangeresin——carrierItaffectstheresolution,speed,capacity,andrecovery

rateSmallMolecules:Usesmallerporeionexchangerswhichallowsmallmoleculesfreeaccessintothepores,offeringgreatercontacting

area.LargeMolecules:Optforsmallparticleexchangerswherelargemoleculesarelimitedtosurfaceexchange,asthesesmallparticlesprovidealargersurfacearea.Factorsinfluencingseparationeffect271.

Ionexchangeresin——carrierFactorsinfluencingseparationeffect28Whenseparatingtrypsin,thesmallertheparticlesizeofthecarrier,thehighertheresolution!ChargeCharacteristicsofTargetMolecule:PositivelyCharged:Optforcationexchangers

陽離子交換劑.NegativelyCharged:Chooseanionexchangers

陰離子交換劑.BindingCapabilityofTargetMolecule:LowElectrostaticBinding:Usestrongionexchangers;however,elutionconditionsaremorestringent.HighBindingStrength:Utilizeweakionexchangersformilderelution.1.

Ionexchangeresin——functional

groupsFactorsinfluencingseparationeffect29Choosethe

buffer

pHaccordingtothepIoftheprotein.WhenpH=pI,theproteinsurfacechargeiszero.WhenpH<pI,theproteinhasapositivecharge.WhenpH>pI,theproteinhasanegativecharge.2.

pHFactorsinfluencingseparationeffect302.

pHFactorsinfluencingseparationeffect31How

to

choose

ionexchangeresin

and

adjustpHvalueforsampleloadingandelution?3.pH的影響■pH與分辨率32Foranionexchange,samplesareusuallyloadedathigherpH,andelutedbygraduallyloweringthepH.Forcationexchange,samplesareloadedatalowpHandelutedbyincreasingthepH.Note:ThepHvariationrangeshouldnotbetoolarge,otherwiseitmaycausedenaturationoftheprotein.2.

pHFactorsinfluencingseparationeffect33Thehighertheionicstrength,theweakerthebindingforcebetweentheproteinandtheionexchangeresin;conversely,thelowertheionicstrength,thestrongerthebindingforceSampleloading:lowsaltconcentration;Elution:highsaltconcentration3.

IonicStrengthFactorsinfluencingseparationeffect343.

IonicStrengthFactorsinfluencingseparationeffectBefore

loading

to

the

column,

how

to

determined

the

proper

pH

and

Ionic

strength

?35Factorsinfluencingseparationeffect36Chooseabufferthatdoesnotinteractwiththeionexchangeresin.ChooseabufferaccordingtothepHofthesystem.根據(jù)緩沖物質(zhì)pK值，用pK與層析pH接近的物質(zhì)作為緩沖成分。例如，某蛋白等電點為6.5，選用陰離子交換劑為層析介質(zhì)，起始緩沖液pH為8.0，可選擇Tris緩沖液（pK=8.06）。Chooseasuitablebuffer

containing

specific

molecules.氯仿、甲醇、表面活性劑等——增加疏水性蛋白的溶解性；尿素、鹽酸胍等——增加包涵體蛋白的溶解性；酶抑制劑——如：PMSF（苯甲基磺酰氟）,EDTA等。Factorsinfluencingseparationeffect4.

Buffer

Solution37Chosen

basedontheamountofthesample.IECcolumnsaregenerallyshortandthick.TypicalIEC

columnsusedinthelaboratoryare~5-20cmInlarge-scaleseparation,thescaleofseparationcanbeincreasedbyincreasingthediameteroftheIECcolumn.Factorsinfluencingseparationeffect5.

Column

Size38Theflowrateduringchromatographyisrelatedtothesizeofthemoleculesbeingseparated:Forsmallmolecules,theflowratecanbeincreasedappropriatelyduetotheirhighdiffusionrate.Forlargemolecules,theflowrateneedstobereducedappropriatelybecauseoftheirlowerdiffusionrate.。Excessive

flowratecanincreasecolumnpressure.Inparticular,formaterialswithhighviscosity,themaximumflowrateneedstobelimited.Factorsinfluencingseparationeffect6.

Flow

Rate39(1)Columnpacking(2)Equilibration(3)Loading(4)Washing(5)Elution(6)Regeneration40IonexchangeresinColumn

sizeFlow

ratepHIonic

strengthBuffer

solutionDiscussionIECProcess

and

influencing

factors?GFP

purificationDiscussionThemolecularweightofGFPis27kDaanditsisoelectricpointis5.76,anditremainsstableinpH

from

5.5

to

8.

Please

update

the

IEC-based

GFP

purification

process.

IncludetheselectionofIECresin,loadingandelutionconditions,andotherspecificparameters.（設(shè)計純化工藝，寫清楚層析條件）41Discussion42平衡、結(jié)合、洗滌：pH值：pH=7.4（大于PI）離子強(qiáng)度：10mMNaCl（低鹽）洗脫：pH值：pH=5.8離子強(qiáng)度：500

mMNaCl色譜柱：類型：陰離子交換柱（Sepharose-DEAE）柱體積：2

mL

(1~100

mL)流速：2

mL/min（1~10

mL/min）再生：pH值：pH=3（極端pH，兼顧樹脂）離子強(qiáng)度：1

MNaCl（高鹽）最后換成純水清洗ExpressionCell

cultureCell

disruptionClarificationPrecipitation,

DialysisMicro-filtrationIECFormulationDiscussion平衡、結(jié)合、洗滌：pH值：pH=7.4（大于PI）離子強(qiáng)度：10mMNaCl（低鹽）洗脫：pH值：pH=5.5離子強(qiáng)度：500

mMNaCl色譜柱：類型：陰離子交換柱（Sepharose-DEAE）柱體積：2

mL

(1~100

mL)流速：2

mL/min（1~10

mL/min）再生：pH值：pH=3（極端pH，兼顧樹脂）離子強(qiáng)度：1

MNaCl（高鹽）最后換成純水清洗Howcantheseparationprocessofion-exchangechromatography(IEC)betracked,recorded,andrepresented,andwhatresultsmightbeexpected?IEC

ProcessConstantflowpump

恒流泵Fraction

collector部分收集器IEC

ColumnGFP

solution44Detector

檢測器①

Columnpacking

Equilibration沿壁倒下均勻無氣泡、裂紋PackingEquilibrationIEC

Resins1ml/min0.01mol/L,pH=7.4，PBS

solution

3cmIEC

Process0.01mol/L,pH=7.4，PBS

solution

451.Equilibratetheionexchangechromatographyresinwiththeequilibrationbuffer.IEC

Process46Tips:

EquilibrationbufferIonic

strengthandpH:Ensure

thestabilityoftheproteins.Ensureanappropriatebindingaffinity,andtomaximizethedifferenceinbindingaffinitybetweenthesampleandimpurities.Aovid

includingionsthathaveastrongbindingaffinityfortheionexchangeresin,asthiscansignificantlyreducetheexchangecapacityandseparationefficiency.IEC

Process47②

Sample

Loading用0.01mol/L,pH7.4，PBS緩沖液透析后的GFP溶液Note:Theunboundsamples(flow-through)needtobeanalyzedbySDS.Constantflowpump

IEC

ColumnIEC

Process482.Proteinswithoppositechargesbindtothechargedgroupsoftheionexchangeresin,thusenrichingonthecolumn.Proteinswithnochargeormoleculeswiththesamechargeastheresinwillpassthroughduringloading.IEC

Process49Thesamplemustbefullydissolved,anditisbesttofilteritthrougha0.45μmmembranebeforeloadingontothecolumn.Thesampleconcentrationshouldnotbetoohigh.The

ionicstrengthandpHofthesamplesolutionis

better

to

be

consistentwiththeequilibrationbuffer.Tips

:

Sample

loadingIEC

Process50③

WashingConstantflowpumpIEC

Column0.01mol/L,pH=7.4，PBS

solution

IEC

Process51Fraction

collector④

Elution-1Constantflowpump

IEC

Column0.05mol/L,pH=7.4,PBS

solution

IEC

Process523.

Increase

theionicstrengthwithagradienttodisplaceboundmolecules,asionsinthebuffercompetewithproteinmoleculesforbindingsites.

IEC

Process530.1mol/L,pH6.0，檸檬酸鈉緩沖液④

Elution-2Fraction

collectorConstantflowpump

IEC

ColumnIEC

Process544.Furtherincreasetheionicstrengthtodisplaceproteinswithmorenetcharge(tightlybound).IEC

Process555.Furtherincreasetheionicstrengthtodisplaceproteinswithmorenetcharge(more

tightlybound).IEC

Process56⑤

Cleaningandregeneration1mol/L,pH7.4，NaClConstantflowpump

IEC

ColumnIEC

Process576.Washtheionexchangecolumnwiththefinalbufferofhighionicstrengthtoremoveanyionicallyboundproteinsbeforere-equilibratingthecolumn.58IEC

Process表示什么？59IEC

Process洗脫模式梯度洗脫(線性)梯度洗脫(非線性)分步洗脫增加鹽濃度改變緩沖液pH添加表面活性劑、尿素、鹽酸胍等洗脫方法DiscussionHowcantheseparationprocessofion-exchangechromatography(IEC)betracked,recorded,andrepresented,andwhatresultsmightbeexpected?平衡—上樣—洗滌/洗脫—再生Gradient:

0%to50%elutionbufferover10mL(10CV)DiscussionHowcantheseparationprocessofion-exchangechromatography(IEC)betracked,recorded,andrepresented,andwhatresultsmightbeexpected?平衡—上樣—洗滌—洗脫—再生DiscussionWhatifthereareother

proteinsthathavesimilarchargetoGFPinIEC-elutedsolution?Now,wecanconductthisIECproceduretoimplementyourdesignedplantopurifyGFP.62GFPConclusionKnowledge:Principle

of

IEC

；Factors

influencing

separation

efficiency；The

purification

process.Ability:DesignIEC-based

protein

purificationprocesses,selectappropriateIEC

conditions,predictandanalyzethepotentialresults63

思考題完善基于IEC的GFP純化工藝，選擇合適的IEC分離條件，畫出可能得到的純化結(jié)果，并對結(jié)果進(jìn)行分析；閱讀文獻(xiàn)，整理出青霉素?；傅膬煞N純化工藝路線，明確純化工藝中每一個步驟的具體工藝參數(shù)，并根據(jù)所學(xué)內(nèi)容設(shè)計另一種青霉素?；傅碾x子交換色譜純化過程（如通過不同的色譜柱、樣品純化模式（流穿/洗脫）、結(jié)合/洗脫溶液條件等）。64

思考題65

思考題6667離子交換色譜：GFP組成：固（交換劑-陰/陽）+流影響因素（PH、離子強(qiáng)度…）步驟：平-上樣-洗滌-洗脫-再生純化工藝Chapter8-2Hydrophobic

Interaction

Chromatography（HIC）68Introduction69下村修錢永健馬丁They

all

faced

the

challengesabout

purifying

GFP

from

different

sources.GFPPurificationProcessExpressionCell

cultureCell

disruptionClarificationPrecipitation,

DialysisMicro-filtrationIECFormulation70將GFP基因插入大腸桿菌表達(dá)系統(tǒng)超聲波破碎過濾澄清硫酸銨沉淀，透析離子交換色譜（如Sepharose-DEAE）微濾滅菌，裝罐細(xì)胞培養(yǎng)、誘導(dǎo)表達(dá)、蛋白擴(kuò)增ExpressionCell

cultureCell

disruptionClarificationPrecipitation,

DialysisMicro-filtrationIECFormulation71平衡、結(jié)合、洗滌：pH值：pH=7.4（大于PI）離子強(qiáng)度：10mMNaCl（低鹽）洗脫：pH值：pH=5.5離子強(qiáng)度：500

mMNaCl色譜柱：類型：陰離子交換柱（Sepharose-DEAE）柱體積：2

mL

(1~100

mL)流速：2

mL/min（1~10

mL/min）再生：pH值：pH=3（極端pH，兼顧樹脂）離子強(qiáng)度：1

MNaCl（高鹽）最后換成純水清洗GFPPurificationProcessChromatographyCurve平衡—上樣—洗滌—洗脫—再生GFPGradient:

0%to50%elutionbufferover10mL(10CV)DiscussionWhatifthereareother

proteinsthathavesimilarchargetoGFPinIEC-elutedsolution?GFPOtherproteinsChapter8-2Hydrophobic

Interaction

Chromatography（HIC）74OverviewFactorsinfluencingseparationeffectApplicationOutline75Overview—Principle76Separationisachievedviathereversible

andhydrophobicinteractionsbetweenthe

hydrophobic

region

of

proteins

andthestationaryphase.Proteinsareseparatedbasedonthehydrophobicbindingstrengthwith

the

resin.

Hydrophobicbinding

strengthispositively

relatedtothe

surfacehydrophobicityof

proteins

and

saltconcentration.77Overview—PrincipleRed:

Hydrophilic

amino

acidsYellow:

Hydrophobic

amino

acids

ReversibleinteractionSalt

can

disrupthydrationshellof

proteins

and

enhancethe

hydrophobiceffect.Moleculesareloaded

at

ahighsaltconcentrationsolution,and

eluted

byreducingthesaltconcentration

to

weaken

thehydrophobicinteractions78Overview—PrincipleHIC

separatesbiomoleculesbasedonthedifferenceinhydrophobicinteractions

betweenproteinsandthehydrophobicadsorbentusedasthestationaryphase.Accordingto“saltingout”,inhighsaltconcentrationsolutions,thehydrationlayerofproteinisdisrupted,thus

exposingthehydrophobicpartsandincreasinghydrophobicinteractions.Moleculesareboundtothecolumnusingahighsaltconcentrationsolution,and

eluted

byreducingthesaltconcentration

to

weaken

thehydrophobicinteractions79Overview—PrincipleOverview—CharacteristicsHIC

isa

supplementtootherproteinseparationmethods

basedonproteincharge,size,andligand-receptor

recognition,andiswidelyusedinproteinpurification.It

isanidealdownstreamstepforsamplesafterammoniumsulfateprecipitationorionexchangechromatography,

which

containshighsalt

concentrationstobedirectlyloadedontotheHIC.Duringtheseparation,thesampleispurifiedandelutedinsmallvolumes,makingitpossibletodirectlyloadtheconcentratedsampleontogelfiltrationchromatographyorother

chromatographyafterbufferexchange.80Overview—FormationHIC

resinconsistsoftwoparts:carrier(matrix)

and

functionalgroups

(ligand).81Hydrophobicresin-polystyreneresin聚苯乙烯樹脂Hydrophilicpolymer纖維素(Cellulose)球狀纖維素(Sephacel)葡聚糖(Sephadex)SephadexG-25和G-50瓊脂糖(Sepharose)SepharoseCL-6B821.

Carrier(matrix)Overview—Formation瓊脂糖類凝膠仍是應(yīng)用最廣泛的疏水介質(zhì)ThehydrophobicligandsusedinHICaremainlyalkyl

(烷基)andaromatic

(芳香基)groups.ThealkylgroupsareusuallyshorterthanC8,whilethearomaticgroupsareoftenphenyl

(苯基)groups.

R:

Hydrophobicligands，M:

Carrier2.

Functionalgroups(ligand)83Overview—Formation（A）丁基Butyl（B）辛基Octyl（C）苯基Phenyl（D）新戊基Pentyl

2.

Functionalgroups(ligand)Commonly-used84Overview—FormationSome

commercialHIC

resins.1.

ButylSepharose4FastFlow正丁烷基，疏水性最弱，適合含脂族配體的生物分子。2.OctylSepharose4FastFlow正辛烷基，疏水性中等，適合各種蛋白的分離純化。⒊PhenylSepharose6FastFlow苯基Phenyl，疏水性最強(qiáng)，載量高，適合含芳香族配體的生物分子的預(yù)處理，2.

Functionalgroups(ligand)85Overview—FormationDiscussion86ExpressionCell

disruptionClarificationPrecipitation,

DialysisMicro-filtrationIECFormulation微濾OtherproteinsDiscussion87ExpressionCell

disruptionClarificationPrecipitation,

DialysisMicro-filtrationIECHICFormulation將GFP基因插入大腸桿菌表達(dá)系統(tǒng)超聲波破碎過濾澄清硫酸銨沉淀，透析DEAE,

pH=7.4,低鹽上樣，高鹽洗脫微濾疏水作用色譜濃縮，滅菌裝瓶色譜條件和過程如何選擇？1.

Stationaryphase

(HIC

Resin).2.Mobile

phase.3.Chromatographicconditions.88Factorsinfluencingseparationeffect1.HIC

ResinFactors:

①carrier,

②ligand

type,

③ligand

density.Carriers

usedinHICresinaremainlypolysaccharides(suchasagarose瓊脂糖)andsyntheticpolymers(suchaspolyethylene聚乙烯andpolyvinylalcohol聚乙烯醇).89Factorsinfluencingseparationeffect大分子——無孔小粒徑填料小分子——多孔填料1.HIC

ResinFactors:

①carrier,

②ligand

type,

③ligand

density.Carriers

usedinHICresinaremainlypolysaccharides(suchasagarose瓊脂糖)andsyntheticpolymers(suchaspolyethylene聚乙烯andpolyvinylalcohol聚乙烯醇).Ligand

type

includes

alkyl烷基andaromatic芳香基groups.Generally,thebindingcapacity(hydrophobicity)increaseswithincreasingalkylchainlength.90Factorsinfluencingseparationeffect91這四種不同的配基條件是:苯基(高取代)，苯基(低取代)，辛基和丁基。Ligand

Type92Ligand

density

(substitutiondegree):

It

affectsthebindingcapacity.Increasing

ligand

densityresultinmorebindingsitesforproteins,therebyincreasingthebindingcapacity.However,whenitreachesacertainvalue,thesterichindrancelimit

the

further

increase

of

bindingcapacity.And

multiple

ligandsboundtoeachprotein,resultinginoverlystrongbindingthatisdifficulttoelute.93FactorsinfluencingseparationeffectLow

ligand

densityModerate

ligand

densityHigh

ligand

densityLow

binding

capacityHard

to

eluteMaximumBindingand

easyto

eluateFactors:1)Ion

typeandionic

strength.2)pH.3)Additives

(添加劑).942.Mobile

phaseFactorsinfluencingseparationeffectSodium/ammoniumsulfatecanpromoteligand-protein

interactioninHIC.

So,

Themostcommonlyusedsalts:

Na2SO4

and

(NH4)2SO4AnionssuchasClO4-andI-withsmallionicradiiandlowchargedensitycanweakenthehydrophobicinteraction,

whichcan

be

usedduringelution.

Factorsinfluencingseparationeffect2.Mobile

phase

—

①Iontypeandionicstrength952.流動相組成

1）離子種類及強(qiáng)度96Ion

type

matters!Load

thesampleatasaltconcentrationslightlybelowthesalting-outpoint.在略低于鹽析點的鹽濃度上樣吸附Graduallyreducetheionicstrengthofthemobilephaseduringelution.洗脫時逐漸降低流動相的離子強(qiáng)度97Factorsinfluencingseparationeffect2.Mobile

phase

—

①Iontypeandionicstrength98FactorsinfluencingseparationeffectIonic

strength

matters!99FactorsinfluencingseparationeffectThechoiceofpHmustbecompatiblewithproteinstabilityandactivity.ThepHcanaffectthesurfacechargeof

theproteins.Generally,apHvalueclosetotheprotein'sisoelectricpoint(pI)ischosentoreduceelectrostaticinteractionsbetweenproteins,whichisbeneficialforimprovingseparationperformance.100Factorsinfluencingseparationeffect2.Mobile

phase

—

②pHAdditivescanbeusedtoimproveselectivity/resolution.Theyaretypicallyusedtopromoteproteinsolubilizationandalterproteinconformationtofacilitateelutionwhensampleandhydrophobicinteractionchromatographypackingmaterialsaretightlybound.Factorsinfluencingseparationeffect2.Mobile

phase

—

③Additives101Factors:

Temperature,

Flow

rate,

etc.1023.

ChromatographicconditionsFactorsinfluencingseparationeffectHydrophobicbindingforceincreaseswithincreasingtemperature.Generally,thehighertheflowrate,theworsetheseparationefficiency.3.

ChromatographicconditionsFactorsinfluencingseparationeffect103DiscussionThemolecularweightofGFPis27kDaanditsisoelectricpointis5.76,anditremainsstableinpH

from

5.5

to

8.

Please①updatetheIEC&HIC-basedGFPpurificationprocess.IncludetheselectionofHICresin,loadingandelutionconditions,andotherspecificparameters.②Drawthepotentialchromatographycurve.（設(shè)計純化工藝，寫清楚層析條件，畫出層析曲線）104IECHICDiscussionExpressionCell

disruptionClarificationPrecipitation,

DialysisMicro-filtrationIECHICFormulation平衡、結(jié)合、洗滌：pH值：pH=7.4（大于PI）離子強(qiáng)度：10mMNaCl（低鹽）洗脫：pH值：pH=5.8離子強(qiáng)度：10mMNaCl,

1

M

(NH4)2SO4

色譜柱：類型：陰離子交換柱（Sepharose-DEAE）柱體積：2

mL

(1~100

mL)流速：2

mL/min（1~10

mL/min）再生：pH值：pH=3（極端pH，兼顧樹脂）離子強(qiáng)度：1

MNaCl（高鹽）最后換成純水清洗Discussion106ExpressionCell

disruptionClarificationPrecipitation,

DialysisMicro-filtrationIECHICFormulation平衡、結(jié)合、洗滌：pH值：pH=6.0（穩(wěn)定）鹽：10mMNaCl,

1

M

(NH4)2SO4

（高鹽）體積：10倍柱體積洗脫：pH值：pH=7.4鹽：10mMNaCl（低鹽）梯度：0-50%洗脫液（10倍柱體積）色譜柱：類型：苯基低取代（Sepharose-Phenyl）柱體積：2

mL

(1~100

mL)流速：2

mL/min（1~10

mL/min）再生：低鹽/純水清洗，加入表面活性劑Discussion107GFP

Purification-1108109GFP

Purification-2110GFP

Purification-3111GFP

Purification-4112GFP

Purification-5113GFP

Purification-6114GFP

PurificationDiscussionExpressionCell

disruptionClarificationDialysis

&

Micro-filtrationIECHICConcentration

&

Buffer

changeFormulation115將GFP基因插入大腸桿菌表達(dá)系統(tǒng)高壓勻漿破碎加入絮凝劑，板框過濾機(jī)過濾透析換液，微濾過膜離子交換色譜（如Sepharose-DEAE）滅菌，裝罐疏水作用色譜（如Sepharose-phenyl）切向流超濾（TFF,

MWCO=10

kDa）Discussion固定相：瓊脂糖填料。配基類型：使用Butyl或Phenyl作為疏水固定相配基。（先試butyl，不行再換phenyl）配基取代程度：50%。中度取代可以平衡蛋白與固定相之間的相互作用，避免過強(qiáng)的疏水作用損害GFP結(jié)構(gòu)。流動相：氯化鈉或硫酸銨。上樣時，可以選擇1M至3M的鹽濃度范圍?？梢酝ㄟ^線性梯度降低鹽濃度至0.1M進(jìn)行洗脫。pH應(yīng)選擇GFP穩(wěn)定的范圍，通常在pH7.0至8.0之間。添加劑：為了提高GFP純化的分辨率，可以在流動相中添加一些穩(wěn)定蛋白結(jié)構(gòu)的添加劑，例如尿素（1-2M）、非離子型表面活性劑（例如0.01-0.1%的TritonX-100）等。這些添加劑可以減少非特異性吸附，提高GFP的純化效果。116ConclusionKnowledge:Principle

of

HIC

；Factors

influencing

separation

efficiency；The

purification

process.Ability:DesignIEC&HIC-based

protein

purificationprocesses,selectappropriateIEC

conditions,predictandanalyzethepotentialresults117

思考題118根據(jù)視頻內(nèi)容整理GFP工業(yè)生產(chǎn)純化工藝，明確每一個步驟的具體純化工藝參數(shù)；畫出可能得到的IEC和HIC的層析曲線。閱讀文獻(xiàn)，整理出文獻(xiàn)中GFP的完整純化工藝路線（包括上游生產(chǎn)），并寫出HIC步驟的具體工藝參數(shù)119疏水作用色譜：GFP載體、配體（C4、C8、苯…）影響因素（配體/密度、鹽、PH…）平-上樣-洗滌-洗脫-再生Chapter8-3AffinityChromatography120The

Nobel

Prize

in

Physiology

or

Medicine

2018Cancer

ImmnotherapyTumorcellscancontrolthe"brake"ofTcells-ImmuneCheckpoint.Introduction121Cancer

Immunotherapy踩下油門松開剎車PD-1inhibitors(Opdivo)K藥How

to

prepare

and

purify

PD-1/PD-L1

antibodies？Introduction122PD-L1inhibitors(Keytruda)O藥CentrifugationMembrane

SeparationPrecipitationIEC、HICDoesthepuritymeettheproduct

requirements

of

the

antibodies?Howtofurtherimprovethepurity

or

simplify

the

purification

process?Affinity

ChromatographyIntroduction123ThinkingWhat’stheprincipleofaffinity

chromatography?Which

type

of

ligand-receptor

pair

can

we

use

to

enable

affinity

separation

for

PD-L1

antibodies?How

toreducethecostoftumorimmunotherapyproducts?124Outline1.

Overview2.

Ligand

type3.

Application

&

Discussion125Overview—PrincipleSeparationisachievedthroughthereversible

andspecificbindingbetweenligand-receptormolecules.126Affinitychromatographyisan

adsorptionchromatographythatreliesontherecognitionabilityofbiologicalmoleculesand

the

complementarybindingpartners(ligands).Comparedtoothertechniques,ithashighselectivity,andsomesubstancescanachieveaproductpurityofover90%withjustonestepofaffinitychromatography,whichgreatlysimplifiesthepurificationprocess.Affinitychromatographyiswi