生物大分子分离纯化-原理与技术.ppt

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1、生物大分子分离纯化原理与技术,内容提要,1、层析原理与技术2、凝胶过滤层析3、UNOsphere离子交换分离技术4、CHT陶瓷羟基磷灰石分离技术5、疏水相互作用与亲和层析,Life is based on proteins and their interactions,层析原理与操作,层析的起源和原理,起源-1906年，俄国植物学家Tsweet原理-利用物质分配系数不同达到分离 目的,原理与操作,Chromatography,层析,色谱,什么是生物层析,根据生物分子物理化学特性的不同而达到分离 极性：polarity(solubility,volatility)HIC,RP 离子特性：ioni

2、c characteristics(charge)IEX 大小与形状：size/mass(diffusion,sedimentation)GF 结构特征与活性位点：shape(ligand binding,affinity)AC 这些特性的区别使不同蛋白质分子在层析的固定相和流动相的分配不同而达到分离,原理与操作,GelFiltration凝胶过滤,IonExchange离子交换,Hydrophobic Interaction疏水层析反相,Affinity亲和层析,CHT羟基磷灰石,原理与操作,最广泛的蛋白质分离纯化方法 条件温和 维持蛋白质空间结构与活性 基于蛋白质对固定相的不同保留达到分离

3、 蛋白质溶于流动相中，经过装填固定相的柱子分离,层析原理与操作,层析的5个操作步骤,装柱并平衡上样平衡洗脱再生,上样,装填有层析介质的层析柱,分离,分离组分流出,原理与操作,层析图,原理与操作,第一峰,外水体积,Vo,不与柱中填料相互作用直接从柱中流出。这是样品中的未结合物质,Vo,Ve,Vt,蛋白质含量(A280),由UV检测器读出，y轴,基线,洗脱峰，有些可以很好的分离，有些分得不是很好，有部分交叉,有时这里也显示梯度浓度等检测值,时间或体积，x轴,纯化平台的硬件结构 层析工作站层析介质和层析柱,原理与操作,层析系统,DuoFlow中高压层析系统,LP低压层析系统,Profinia全自动亲

4、和层析系统,手动层析组件,DuoFlow中高压层析系统,主要特点：3500psi高压力下梯度模式最大流速可达20ml/min连续波长，4波长同步检测方形X、Y轴组分收集器软件直观容易使用,BioLogic LP 低压层析系统,LP Data View 软件,BioLogic LP 系统,BioFrac 方形组分收集器,主要特点：流速精确，0.01ml/min检测灵敏度更高,0.001AU兼容方形收集器,Profinia 蛋白质纯化系统,主要特点：全自动亲和纯化，包括亲和标签、亲和无标签，抗体等纯化双紫外检测器设计纯化与脱盐串联一步完成结果直接报告目标蛋白的浓度和得率,为什么在层析技术中要使用层

5、析仪,介质的要求实验的精密度和重现性要求时间的要求,纯化平台的硬件结构,层析介质与层析柱，原理与技术,层析介质,离子交换层析介质 Unosphere Q，S MacroPrep High Q,High S,DEAE,CM亲和层析 Profinity IMAC金属螯合介质 Profinity Epoxide环氧亲和介质 Affi-Gel Protein A,Affi-Prep Protein A Affi-Gel蓝胶，DEAE蓝胶，CM蓝胶 Affi-Prep多粘菌素介质 Affi-Gel硼胶 Affi-Gel配体固定化活化介质,凝胶过滤层析介质 Bio-Gel P系列 Bio-Beads S-

6、X介质羟基磷灰石介质（CHT）氟代羟基磷灰石介质（CFT）疏水层析介质 Macro-Prep Methyl HIC Macro-Prep t-Butyl HIC Bio-Beads SM-2吸附剂,层析柱,分析柱 离子交换分析柱：Uno Q,S Aminex分析柱分析单糖、寡糖、有机酸、有机碱，以及肽和核酸有机小分子 羟基磷灰石分析柱：Bio-Scale CHT-I 凝胶过滤分析柱：Bio-Sil,Bio-Silect HPLC 分析柱 反相层析分析柱：Hi-Pore RP304,Hi-Pore RP318,各种层析介质的Cartridges用于条件摸索 UNOsphere MacroPrep

7、 CHT HIC,层析介质结构原理,UnosphereMacroPrep,离子交换层析Q，S，DEAE，CM,疏水层析-CH3,t-Butyl,亲和层析Protein AIDANi多粘菌素,凝胶过滤CHT和CFT,Unosphere Q,SMacroPrep High Q,SMacroPrep DEAE,CM,MacroPrep Methyl HICMacroPrep t-Butyl HIC,Profinity IMACProfinity EpoxideAffi-Gel Protein A,Affi-Prep Protein AAffi-Gel,DEAE,CMAffi-Prep Polymix

8、inAffi-Gel硼胶Affi-Gel配体固定化活化,层析介质及其技术,凝胶过滤层析离子交换层析羟基磷灰石层析疏水层析亲和层析,1.Spherical particles packed into a column,2.Sample applied,3.Buffer mobile phase and sample move through column,molecules diffuse in and out of matrix,4.large molecules leave the column first followed by smaller molecules in order of

9、size,molecules larger than the matrix pores pass straight through.,凝胶过滤,凝胶过滤层析,分离纯化原理,A good gel for gel filtration contains about95%water,凝胶结构,Steric exclusion leads to early elution,按照分子量大小洗脱大分子首先洗脱，最小的分子在最后面洗脱,100 1,000 10,000 100,000 Bio-Gel P2Bio-Gel P4Bio-Gel P6Bio-Gel P10Bio-Gel P30Bio-Gel P6

10、0Bio-Gel P100,1,800,800,4,000,1,000,6,000，用于脱盐,1,500,20,000,2,500,40,000,3,000,60,000,5,000,100,000,100,凝胶过滤层析,凝胶的选择,Bio-Gel P4(800-4,000)for separation digested products from IgG,凝胶过滤层析,凝胶的选择,凝胶过滤层析,Bio-Gel P6(1000-6000),凝胶的选择,凝胶过滤层析,Bio-Gel P10(1500-20,000),凝胶的选择,凝胶过滤层析,Bio-Scale Mini Bio-Gel P6脱盐

11、预装柱,操作方便，可连接在DuoFlow、LP、Profinia和Econo泵上,Bio-Beads S-X介质 多孔的聚苯乙烯二乙烯微球体,Bio-Beads S-X1,Bio-Beads S-X3,600,Bio-Beads S-X8,1,000,Bio-Beads S-X12,400,高分辨率亲脂性分子排阻色谱,2,000,14,000,中草药和天然产物活性成分分离纯化高分辨率，快速纯化酯类、芳香族，多环、杂环化合物,介质可兼容苯、甲苯、二甲苯、四氯化碳、二甲基甲酰胺、酮、二氯甲烷、二氯代苯、全氯乙烯等有机溶剂,凝胶过滤层析,1、三硬脂酸甘油酯，8912、三肉豆蔻酸甘油酯，7293、三月

12、桂酸甘油酯，6454、三辛酸甘油酯，4775、三己酸甘油酯，3936、十六烷，2267、十一烷，156,Bio-Beads S-X8分离效果,高分辨率亲脂性分子排阻色谱Bio-Beads S-X介质,凝胶过滤层析,柱长的选择 分离：30120cm 脱盐：30cm以下 洗脱液 离子强度,pH中性流速，根据层析介质的说明，一般很低样品容量：13CV,凝胶过滤层析,操作参数选择,增加分辨率的方法,检测柱效检测分离度 减少上样体积降低流速使用更小的介质颗粒的介质连接2根层析柱,1.可分离相对分子量从几百到几十万的物质 具有3000个理论塔板的凝胶过滤可将分子量相差2倍的蛋白质完全分开,6000个理论塔

13、板的可将分子量相差1.5倍的蛋白质完全分开;建议柱高在80-120cm,直径=H/302.脱盐,凝胶过滤层析,凝胶过滤层析的应用,凝胶过滤层析,凝胶过滤层析的特点：,层析柱规模很大，实验室1.0-2.530-100cm，工艺1020200cm，从而设备成本很高；上样体积少，必须少于柱床体积的3才能达到较好的分离效果，如果大体积样品必须浓缩，从而造成样品的损失和增大工艺的复杂性；工艺时间（生产周期）长，甚至24小时或以上；分辨率低；不需摸索纯化条件，按照分子量区带分离,因此，往往用分子筛分离时只适用于纯化的最后一步去热原，或离子交换上样前的脱盐,离子交换层析,离子交换层析类型及其选择,-O-CH

14、2CH2-NH+,C2H5,C2H5,-N+(CH3)3,-O-CH2-C-O-,O,-SO3-,pI,stability range,stability range,与阳离子交换介质结合,+,-,denaturation,denaturation,pH,10,2,离子交换层析,离子交换层析原理蛋白质滴定曲线,蛋白质净电荷,与阴离子交换介质结合,Products:UNOsphere Q&S,Macro-Prep High Q&S,CM,DEAE,AG resins,Equilibration,SampleApplication,SampleAdsorption,Elution,Regenera

15、tion,离子交换层析,离子交换层析原理,sampleapplicationand wash,elution,equilibration,regeneration,-,-,-,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,-,-,-,-,-,-,-,-,-,-,-,anionexchangerbead,离子交换过程,离子强度 洗脱时多采用离子浓度梯度,常加NaCl,KCl,LiCl等.在不同盐浓度（离子强度）及其不同变化率（梯度）条件下保留行为不同，需对该条件进行摸索。一般的，随离子强度增加，蛋白质保留值减小。,缓冲液与pH

16、 选择适当的缓冲体系，起始浓度要尽可能低些(0.01-0.05M)缓冲液PH值：阳离子低于pI一个pH单位以上 阴离子通常高于pI一个pH单位以上 蛋白质在不同pH下保留行为差别较大，所以许对缓冲体系和操作pH进行摸索，以得到最佳层析条件。,离子交换层析,离子交换层析操作参数选择,pI5.5,+,-,pH,10,2,离子交换层析,缓冲液与pH,pI7.5,阳离子交换,阴离子交换,碱性蛋白，采用阳离子交换,酸性蛋白，采用阴离子交换,蛋白质净电荷,pH4.0,pH9.0,蛋白质稳定性范围,pH 7.0,pH 8.0,pH 8.9,pH 6.0,缓冲液与pH的影响阴离子交换,离子交换层析,离子交换层

17、析操作参数选择,离子交换层析,离子交换层析操作参数选择,盐溶液梯度的影响,Columns MediaBio-Scale Mini High Q Macro-Prep high QBio-Scale Mini High DEAE Macro-Prep DEAEBio-Scale Mini High S Macro-Prep high SBio-Scale Mini UNOsphere Q Macro-Prep CMBio-Scale Mini UNOsphere S Macro-Prep 25 QBio-Scale Mini UNOsphere rapid S Macro-Prep 25 SUN

18、O QUNOsphere QUNO SUNOsphere S UNOsphere rapid S,离子交换层析,离子交换层析产品,50um,25um,120um,80um,10um,100um,更高的选择性和分辨率10%穿透载量:Unosphere Q:150 mg/ml BSA 600 cm/hrUnosphere S:30 mg/ml BIgG 600 cm/hr 高流速，低反压 操作压力:1 week长期:Storage in 0.1 M NaOH RT for 1 year生产效率大大提高,离子交换层析,UNOsphere Q/S性能参数,Column 1.1 x 20 cm Load

19、 buffer:20 mM Tris,pH 8.5Elution Buffer:Load+0.5 NaClLoad:5 mg/ml BSA,Column 1.1 x 20 cm Load buffer:20 mM NaAc,pH 5.0Elution Buffer:Load+0.5 NaClLoad:1.64 mg/ml hIgG,UNOsphere Q:BSA,UNOsphere S:HIgG,高流速，高载量：Unosphere,高流速，低反压：Unosphere,离子交换层析,UNOsphere 更高的生产效率：,更短的工艺时间短时间内处理大量体积样品更小的层析柱，更少的介质更高的产量更短

20、的生产周期生产成本下降,离子交换层析,蛋白质纯化，特别是从原材料或发酵中间体目标蛋白的大量捕获 中间纯化,UNOsphere Q/S应用,4 Process Steps1.Polymerization2.Washing3.Deagg/Sizing4.Base Treatment,MonomerCross-linkerIonomerSurfactantSaltSolventInitiatorHeat,UNOsphere 制造过程,UNOsphere,%cross-linker:S25%Q25%,离子交换层析,UNOsphere Q/S结构,Continuous network of polyme

21、r nodules0.5-1 m,Channels3-5 m,Q：(CH3)3N+S：SO3-,Profinity IMAC：IDA-Ni，Cu，Co,离子交换层析,UNOsphere Q/S结构,Reference:T.Ogawa et.al.Hydroxyapatite Conference 2001,陶瓷羟基磷灰石（CHT）分离技术,合成,左边:Bio-Gel HT/HTP 羟基磷灰石右边:CFT/CHTTM 陶瓷羟基磷灰石 I&II,晶体与陶瓷结构,陶瓷羟基磷灰石（CHT）分离技术,Ca10(PO4)6(OH)25 个带正电荷的Ca离子对（C位点）2 个磷酸三价离子，每个含有带6个负电

22、荷的氧原子（P位点）2 个羟基于其他层析介质不同，CHT的骨架是化学反应的表面,CHT陶瓷羟基磷灰石晶体结构,陶瓷羟基磷灰石（CHT）分离技术,产品类型与参数,陶瓷羟基磷灰石（CHT）分离技术,蛋白质带正电氨基经典的阳离子交换用中性盐溶液(NaCl)或缓冲盐溶液(PO4)洗脱,初级保留机制,陶瓷羟基磷灰石（CHT）分离技术,带负电羧基经典的金属螯合作用，并受离子排阻调节比离子间静电相互作用强1560倍在无PO4 条件下不能被任何浓度NaCl洗脱用PO4洗脱,初级保留机制,陶瓷羟基磷灰石（CHT）分离技术,大部分大分子蛋白质以两种保留机制联合模式结合：Ca亲和与阳离子交换酸性蛋白质的结合，如白蛋

23、白（albumin）以钙亲和作用为主，阳离子交换仅有轻微的作用，这意味着NaCl对白蛋白载量和保留的影响是非常有限的。碱性蛋白质，如IgG以阳离子交换作用为主，其载量和保留受NaCl影响显著,注：对于带负电荷的酸性蛋白质，无论样品是否含有NaCl，都对上样时的吸附载量和保留无影响，这意味着可以将捕获阶段获得的中间体样品无需脱盐处理即可上样到CHT上进行高分辨率中间纯化,混合保留机制,陶瓷羟基磷灰石（CHT）分离技术,Equlibrate:5mM NaPO4 pH 6.5Gradient:5mM300mM NaPO4,Eq:5mM PO4,0.3M NaCl pH 6.5Grad:5mM300m

24、M PO4,0.3M NaCl,IgG MAb,BSA,混合保留机制,陶瓷羟基磷灰石（CHT）分离技术,单克隆抗体，多克隆抗体纯化重组疫苗抗体片段重组蛋白质酶核酸:DNA/RNA(单双链)膜蛋白质,羟基磷灰石应用,陶瓷羟基磷灰石（CHT）分离技术,优点独特的分离机制高再生能力 高分辨率容易规模放大容易进行方法开发高机械稳定性高化学稳定性层析柱寿命长易于将HT工艺直接转移到CHT/CFT上CFT 比CHT更耐酸,颗粒大小：20um、40um和80um,陶瓷羟基磷灰石（CHT）分离技术,Type I由于具有更高的表面积，I型具有更高的蛋白载量，而且具有更大的保留.Type II由于具有大孔结构，I

25、I 型具有更高的核酸载量，能分离单链和双链DNA，和超螺旋与非超螺旋的DNA白蛋白不能与II型结合，对于一些含白蛋白的抗体样品，II型分离纯化更有利.大分子重组疫苗的中间纯化和大规模制备,如何选择类型,陶瓷羟基磷灰石（CHT）分离技术,CHT纯化或去除DNA的机理,陶瓷羟基磷灰石（CHT）分离技术,分离纯化原理与操作参数选择,羟基磷灰石（CHT）,影响蛋白质色谱行为的操作参数：CHT类型 NaCl浓度 PB缓冲液浓度梯度 双梯度洗脱 PB缓冲液梯度含不同NaCl浓度 缓冲液pH值,双梯度洗脱模型先以0-500mM NaCl，再以0-500mM 磷酸钾,不同类型CHT陶瓷羟基磷灰石在不同pH下不

26、同蛋白质的保留时间（min）,羟基磷灰石（CHT）,不同蛋白质分离纯化的Protocol：IgG,羟基磷灰石（CHT）,不同蛋白质分离纯化的Protocol：球形蛋白质，质粒,羟基磷灰石（CHT）,不同蛋白质分离纯化的Protocol：酸性蛋白质,羟基磷灰石（CHT）,双梯度洗脱,羟基磷灰石（CHT）,0-0.5MNaCl,0-0.4MNaPO4,玉米种子中纯化转基因抗体IgG,图1 CHT-I柱纯化1-AT。柱尺寸：752mm(10ml)；上样量：5.0ml；Buffer A：10mM磷酸钠缓冲液（pH7.0）；Buffer B：400mM磷酸钠缓冲液（pH7.0）；梯度：0-5%B和5-3

27、0%B各5个柱体积，30-100%B 10个柱体积；流速：2.5 ml/min。分部收集组分为1.5ml，收集如下：收集组分I-VIII分别为：运行组分（图上方轴）12-17、23-24、42-49、49-50、51-53、54-58、69-71和74-77。绿条框指示为1-AT洗脱液。,CHT纯化转基因羊奶中的重组人1-抗胰蛋白酶,羟基磷灰石（CHT）,PI,其中，1-AT，pI5.27 乳白蛋白，pI5.16-乳球蛋白,pI=4.77,5.274.775.16,Market trend of therapeutic antibodies,Twenty approved antibody t

28、herapeutics currently available for the treatment of various diseases-Reopro,Rituxan,Herceptin,Remicade,Synagis,Avastin,Erbitux,Xolair,Raptiva,Lucentis,Tysabri,etc.The antibody therapeutics market is expected to grow by about 30%annually reaching excess of$22 billion by 2007.Reference:Antibody Thera

29、peutics:Protein Development,Market Trends,and Strategic Issues,Revised edition http:/,Production of Monoclonals,FermentationHarvest,Recovery,DownstreamProcessing,Bulk DrugSubstance,Monoclonals Downstream Process A Generic Approach,Critical contaminants in downstream process development,Retention of

30、aggregates,Aggregates tend to bind CHT more strongly than“monomeric”IgG.This indicates that the binding characteristics of the individual IgG molecules in an aggregate are additive.The larger the aggregate,the stronger the binding.Aggregate removal in NaCl gradients at low concentrations of phosphat

31、e generally provides even better separation than size exclusion chromatography,at higher flow rates,and much higher capacity.,The effect of pH on aggregate separation,15mM,10mM,pH 7.5,pH 6.5,Sodium chloride gradient at constant 5mM NaPO4,1.5M NaCl,0.5M PO4,protein A purified IgG on CHT type I 20 m,T

32、he effect of PO4 on aggregate separation,1M NaCl,0.5M PO4,15mM,10mM,25mM PO4,Indicated phosphate concentration maintained across the sodium chloride gradient,protein A purified IgG on CHT type I 20 m,DOE to Optimize Resolution of Aggregates,Contour plot of pH and PO4 on aggregate resolution,Resoluti

33、on factor=2tB-tA/(WA+WB)where t and W correspond to the retention time and peak width at baseline respectively.Reference:PK Ng et.al.Journal of Chromatography A,1142(2007)13-18,Analysis of CHT fractions,HPSEC of CHT fractionsBio-Silect 400-550 L sample,0.8 mL/min50mM HEPES,1.0M NaCl,2M urea,pH 7.2,C

34、HT pool from 0.5M PO4 cleaning,Native IgG pool from NaCl gradient,IgG pool from protein A,NativeIgG ElutionZone,Retention of protein A,The elution zones for free*protein A and free IgG overlap to varying degrees.However it is important to remember that protein A is not“free”under most conditions:it

35、is affinity-complexed with IgG.As with IgG aggregates,the binding characteristics of IgG and protein A are apparently additive in the complex.When elution is conducted with NaCl at a constant low concentration of phosphate,protein A-IgG complexes elute much later and well separated from free IgG.Pro

36、tein A-IgG complexes elute mostly in the 0.5M NaPO4 cleaning step.Greater than 90%reduction is possible in a single step.,Retention of free protein A vs IgG,Phosphate gradient elution at different concentrations of NaCl,CHT,type I,40m Green protein A,Gray IgG,0.0M NaCl,0.3M NaCl,Retention of free pr

37、otein A vs IgG,NaCl gradient elution at different phosphate concentrations,CHT,type I,20m Green protein A,Gray IgG,5mM PO4,20mM,10mM,Retention of DNA,The binding of DNA is dominated by metal affinity with CHT calcium.This also applies to other phosphorylated contaminants such as endotoxins.Phosphate

38、 concentrations of 200400 mM are required for elution.When IgG is eluted in a NaCl gradient at low phosphate concentration,DNA reduction of more than 3 logs is possible.Endotoxin reduction under the same conditions is more than 4 logs.,Retention of DNA on CHT,+0.50M NaCl,+1.0M NaCl,0.80M PO4,+0.25M

39、NaCl,10mM PO4,+0.00M NaCl,Sheared salmon sperm DNA CHT type I,40 micron,600 cm/hr,pH 7.0,Retention of DNA/RNA on CHT,Removal of viruses across CHT,Retention of endotoxins,Endotoxins are highly acidic due to a high content of phosphoryl and carboxyl residues.Both should have strong affinity for CHT c

40、alcium.Elution in the absence of phosphate should not occur.Some endotoxins have amino groups which may cation exchange with CHT phosphates at low ionic strength.Endotoxins occur in a range of aggregation states,into the millions of Daltons.Most of the charges are internalized because of their assoc

41、iation with the hydrophobic lipid A region.Both size and charge shielding may affect retention.Endotoxins are frequently complexed with proteins and other cell wall components that may also influence retention,Removal of host cell proteins(HCP),HCP are unique for each cell line.Hundreds to thousands

42、 of HCPs derived from cell or cell debris are present in fermentor fluid.Bulk of HCPs is removed across protein A chromatography.Mixed mode interaction with CHT can offer unique opportunity for removal of residual HCPs.,The effect of NaCl and phosphate on CHOP clearance,CHT fractionation of contamin

43、ants,Protein A-purified human IgG1 CHT type I,20 micron,300 cm/hr,20mM NaPO4,pH 6.540CV linear gradient to 1M NaCl(20mM PO4)Clean with 0.5M NaPO4,Monomer,Aggregate(tetramer),dnaetoxpa,Largeraggregates,2-Step Platform Purification,protein A/CHT,Elute protein A with 0.1M glycine*or arginine*0.05M NaCl

44、,pH 3.8(no citrate or EDTA).Hold for viral inactivation.Raise pH to 6.5 by addition of 0.5M NaPO4 pH 10.5,1%v:v.Equilibrate CHT to 5mM NaPO4,pH 6.5Run optimized CHT fractionation conditions*Glycine and arginine concentration can be raised to 1-2M to reduce aggregation.,2-Step platform(Chimeric IgG),

45、Case 1,OM PPA PCHTAggregate%IgG-40 1Protein A ng-162 6DNA ng 9.9x105 3.8x104 12Endotoxin EU 2.6x103 5.0 x102 0.05IgG%100 25*45*OM:original materialPPA:IgG pool from protein APCHT:native IgG pool from CHT*low recovery PPA due to aggregation;PCHT due to aggregate removal,Three Step Platform Purificati

46、on,conditions,Elute protein A with 0.1M glycine,0.05M NaCl,pH 2.5(no citrate or EDTA).Raise pH to 3.8 by addition of 1M Tris pH 8.5,4.9%v:v.Hold for viral inactivation(see reference)Raise pH to 7.0 by addition of 1M Tris pH 8.5,2.3%v:v.This will yield a conductivity of 9mS.Equilibrate UNOsphere Q co

47、lumn to 0.07M Tris,0.1M glycine,pH 7.0,flow-through modeReduce pH to 6.5 by addition of 0.5M monobasic NaPO4(pH 4.1),1%v:v.Equilibrate CHT with 5mM NaPO4,pH 6.5Load,wash,20 CV linear gradient to 1.5M NaCl*Clean with 0.5M NaPO4*screening conditions,increase to 10mM if IgG does not elute,3-Step Platfo

48、rm purification,summary,UNOsphere SUPrA,CHT,UNOsphere Q,4,1,2,3,1,2,3,4,5,2,4,5,0,S,HPSECBio-Silect SEC 400-550mM HEPES0.5M NaCl2M urea,pH 7,3 Step-Platform Purification,summary,Aggregate removal by CHTMore than 99%reduction by HPSECLeached protein A removal by CHT90%removal by Cygnus ELISAreduced f

49、rom 55 to 5 ng/mLDNA removal by CHT 3 logs reduction by PCRreduced to 1ng/mL by picogreenEndotoxin removal by CHTSpiked with LPS from E.coli,Salmonella,and P.aeruginosaremoved 7 x 104 EU by LALfinal concentration,IgG pool,1EU/mL,基于生物分子表面疏水性不同而达到分离的技术疏水作用来自于分子的水化结构，高浓度盐溶液破坏生物分子的水化结构，使蛋白质内部的疏水基团暴露于分子外

50、表面，从而可与疏水介质结合不同离子的疏水性Anions:SO42-Cl-Br-NO3-ClO4-I-SCN-Cations:Mg2+Li+Na+K+NH4+蛋白质以高浓度盐溶液结合与疏水层析柱上，以低浓度盐溶液洗脱,原理,疏水层析,MacroPrep Methyl HICMacroPrep t-Butyl HIC,产品,疏水层析,生物分子表面大都有或强或弱的疏水区域，在不同环境下，与各种疏水介质产生不同强弱的结合高离子强度可加强疏水性跟离子交换相反，高盐吸附，低盐洗脱；洗脱样品又 可直接或稍加稀释后加上其他层析柱，作为连接层析 步骤的桥梁，取代盐析沉淀技术比反相层析的配体密度低很多，无须有机溶