發(fā)布時(shí)間：2018-06-25 20:32

  本文選題：疏水性電荷誘導(dǎo)層析 + 聚合物接枝�。� 參考：《浙江大學(xué)》2017年博士論文

【摘要】：疏水性電荷誘導(dǎo)層析(Hydrophobic charge-induction chromatography,HCIC)是一種新型抗體分離技術(shù),其配基兼有疏水、靜電、氫鍵等多種相互作用,具有選擇性好、耐鹽性高、洗脫條件溫和以及價(jià)格相對(duì)低廉等特點(diǎn),已有一些成功應(yīng)用實(shí)例。由于配基與抗體結(jié)合力較弱,使得HCIC介質(zhì)在高流速下對(duì)抗體的動(dòng)態(tài)結(jié)合載量較低,而聚合物接枝的離子交換層析介質(zhì)可以顯著提高蛋白的吸附容量、傳質(zhì)速率和動(dòng)態(tài)結(jié)合載量。因此本文以高效抗體分離為目標(biāo),設(shè)計(jì)并制備了兩種聚合物接枝型HCIC介質(zhì),比較蛋白吸附和傳質(zhì)性能,分析層析過(guò)程的固相條件(聚合物接枝、配基密度、接枝密度)和液相條件(pH和添加鹽)的影響,并借助逆體積排阻層析(Inverse size exclusion chromatography,ISEC)、等溫滴定量熱儀(Isothermal titration calorimetry,ITC)和共聚焦激光掃描顯微鏡(Confocal laser scanning microscopy,CLSM)進(jìn)行了微觀(guān)分析,以指導(dǎo)新型介質(zhì)設(shè)計(jì)。主要結(jié)果如下:首先以葡聚糖接枝的瓊脂糖凝膠為基質(zhì),以2-巰甲基咪唑(MMI)為配基,優(yōu)化了配基偶聯(lián)條件,制備了不同配基密度的葡聚糖接枝MMI介質(zhì)。相比非接枝MMI介質(zhì),配基密度提高了 50%左右,達(dá)到2000μmol/g。以hIgG為模型蛋白,考察了系列葡聚糖接枝MMI介質(zhì)的靜態(tài)吸附、吸附動(dòng)力學(xué)和層析柱動(dòng)態(tài)吸附性能。結(jié)果表明,在近中性pH下,葡聚糖接枝介質(zhì)對(duì)蛋白的吸附容量、結(jié)合力、傳質(zhì)速率和動(dòng)態(tài)結(jié)合載量隨著配基密度增加而持續(xù)增加,葡聚糖接枝MMI介質(zhì)相比非接枝MMI介質(zhì)具有更高的蛋白吸附容量、傳質(zhì)速率和動(dòng)態(tài)結(jié)合載量。在pH 7.0～8.9時(shí),飽和吸附容量保持在107.5～110.0 mg/g,受pH影響較小;在100 cm/h線(xiàn)性流速下hIgG動(dòng)態(tài)結(jié)合載量達(dá)到38.3mg/g;在弱酸性條件下,葡聚糖接枝MMI介質(zhì)的蛋白吸附容量和傳質(zhì)速率下降更顯著,有利于蛋白解吸。此外,葡聚糖接枝MMI介質(zhì)吸附能力受鹽濃度的影響較小,但傳質(zhì)速率受鹽濃度的影響較大。其次,針對(duì)葡聚糖接枝介質(zhì)中HCIC配基同時(shí)存在于接枝層和基質(zhì)孔道表面上的局限,利用表面引發(fā)的電子轉(zhuǎn)移再生活化劑的原子轉(zhuǎn)移自由基聚合反應(yīng),制備了不同接枝密度和配基密度的聚甲基丙烯酸縮水甘油酯(GMA)接枝MMI介質(zhì)。結(jié)果表明,高接枝密度和中等配基密度的聚GMA接枝MMI介質(zhì)對(duì)hIgG具有更高的飽和吸附容量和傳質(zhì)速率。相比葡聚糖接枝MMI介質(zhì)以及商業(yè)化HCIC介質(zhì)MEP HyperCel,聚GMA接枝介質(zhì)對(duì)hIgG具有更高動(dòng)態(tài)結(jié)合載量。hIgG動(dòng)態(tài)結(jié)合載量受流速影響較小,線(xiàn)性流速300 cm/h下可達(dá)34.6 mg/g,且具有明顯的pH依賴(lài)吸附和耐鹽吸附特性。此外,考察了從混合蛋白體系(hIgG/HSA=1:4)中分離hIgG和從CHO細(xì)胞培養(yǎng)液中分離單克隆抗體,聚GMA接枝HCIC介質(zhì)均表現(xiàn)出良好的抗體分離和重復(fù)使用性能,50個(gè)周期循環(huán)使用的抗體純度和收率分別穩(wěn)定在98.5～99.8%和 90.2～96.9%。最后,為了從微觀(guān)尺度揭示聚合物接枝MMI介質(zhì)的吸附及傳質(zhì)機(jī)制,利用ISEC考察了介質(zhì)孔結(jié)構(gòu)的影響,利用ITC研究了不同蛋白(hIgG和BSA)的吸附熱,利用CLSM實(shí)時(shí)觀(guān)測(cè)了單個(gè)介質(zhì)顆粒內(nèi)蛋白的吸附、傳質(zhì)及洗脫過(guò)程。ISEC結(jié)果表明,不加鹽時(shí)葡聚糖接枝鏈和聚GMA接枝鏈在基質(zhì)表面形成三維空間分布,有助于提高蛋白吸附容量;高鹽濃度下,接枝鏈出現(xiàn)塌縮,且葡聚糖鏈的塌縮程度更明顯,削弱了鏈傳遞效應(yīng),降低了蛋白傳質(zhì)速率。ITC結(jié)果表明,對(duì)于hIgG,在pH 8時(shí),配基密度和鹽濃度的增加會(huì)顯著增加葡聚糖接枝MMI介質(zhì)吸附hIgG的焓變和熵變,說(shuō)明疏水相互作用增強(qiáng);弱酸條件下,焓變和熵變急劇下降為負(fù)值,說(shuō)明靜電排斥作用主導(dǎo)了蛋白的解吸;對(duì)于BSA,吸附容量隨著鹽濃度的增加而顯著下降,且焓變和熵變值為負(fù),表明主要通過(guò)靜電吸引力吸附BSA。CLSM結(jié)果表明,hIgG和BSA在葡聚糖接枝、聚GMA接枝和非接枝MMI介質(zhì)內(nèi),均體現(xiàn)出均質(zhì)擴(kuò)散控制的傳質(zhì)機(jī)理;hIgG在高配基密度葡聚糖接枝和中等配基密度聚GMA接枝MMI介質(zhì)內(nèi)體現(xiàn)了更高的熒光強(qiáng)度;相比hIgG,BSA達(dá)到MMI介質(zhì)內(nèi)核所需時(shí)間更短;pH4.0時(shí),中等配基密度聚GMA接枝MMI介質(zhì)內(nèi)的熒光強(qiáng)度下降最為明顯;以上結(jié)果與宏觀(guān)吸附、傳質(zhì)及分離性能相匹配。本文從提高HCIC介質(zhì)的蛋白載量出發(fā),制備了兩種新型聚合物接枝HCIC介質(zhì),探討了聚合物接枝、配基密度、接枝密度、pH、添加鹽和蛋白性質(zhì)對(duì)聚合物接枝HCIC介質(zhì)吸附和傳質(zhì)性能的影響,并利用ISEC、ITC、CLSM等手段從微觀(guān)角度揭示了蛋白吸附和傳質(zhì)機(jī)理,結(jié)果顯示孔內(nèi)接枝可以明顯提高HCIC介質(zhì)對(duì)抗體的吸附容量、傳質(zhì)速率及動(dòng)態(tài)載量,顯示出良好的抗體分離應(yīng)用前景。
[Abstract]:Hydrophobic charge induced chromatography (Hydrophobic charge-induction chromatography, HCIC) is a new type of antibody separation technology. Its ligand has a variety of interactions, such as hydrophobic, electrostatic and hydrogen bonds, with good selectivity, high salt tolerance, mild elution conditions and relatively low price. With weak antibody binding force, the dynamic binding capacity of HCIC medium to the antibody is low at high velocity, and the adsorption capacity, mass transfer rate and dynamic load of the protein can be significantly increased by the polymer graft ion exchange chromatography medium. Therefore, two kinds of polymer graft type HCIC were designed and prepared by high efficiency antibody separation. Medium, the adsorption and mass transfer performance of protein were compared, and the effects of solid state conditions (polymer grafting, ligand density, grafting density) and liquid phase conditions (pH and salt) were analyzed, and the inverse volume exclusion chromatography (Inverse size exclusion chromatography, ISEC), isothermal titration calorimeter (Isothermal titration calorimetry, ITC) and Co Confocal laser scanning microscopy (CLSM) was microanalyzed to guide the design of new medium. The main results were as follows: firstly, the agarose gel grafted by glucan was used as the matrix and 2- mercapto imidazole (MMI) as the ligand. The coupling conditions of the ligand were optimized, and the graft MM with different ligand density was prepared. I medium. Compared with non graft MMI medium, the density of the ligand was increased by 50% and 2000 mol/g. to hIgG as the model protein. The static adsorption, adsorption kinetics and dynamic adsorption properties of a series of glucan graft MMI media were investigated. The results showed that the adsorption capacity, binding force and transmission capacity of the gluconate graft medium on the near neutral pH showed that the adsorption capacity and binding force of the gluconate graft medium on the protein were transmitted. The mass rate and dynamic load increase continuously with the increase of the density of the coordination group. The MMI medium with glucan graft has higher protein adsorption capacity, mass transfer rate and dynamic binding load than non grafted MMI medium. At pH 7 ~ 8.9, the saturated adsorption capacity remains at 107.5 ~ 110 mg/ g, which is less affected by pH; hIgG at 100 cm/h linear flow velocity. Under the condition of weak acid, the adsorption capacity and mass transfer rate of glucan grafted MMI medium decreased more significantly, which was beneficial to protein desorption. In addition, the adsorption capacity of glucan graft MMI medium was less affected by salt concentration, but mass transfer rate was greatly influenced by salt concentration. Secondly, the graft mediate of glucan was applied to the glucan grafting medium. The HCIC ligand in the mass exists at the same time on the surface of the graft layer and the matrix pore. Using the surface induced electron transfer and the atom transfer radical polymerization of the living agent, the MMI medium with different grafting density and the density of polyglycidyl methacrylate (GMA) is prepared. The results show that the high grafting density and the medium distribution are high. The base density poly GMA grafted MMI medium has higher saturated adsorption capacity and mass transfer rate for hIgG. Compared with glucan graft MMI medium and commercial HCIC medium MEP HyperCel, GMA grafting medium has higher dynamic binding load on hIgG.HIgG dynamic binding load is less affected by flow velocity, and the linear velocity is 300 cm/h up to 34.6 mg/g. In addition, the separation of hIgG from the mixed protein system (hIgG/HSA=1:4) and the separation of monoclonal antibodies from the culture medium of CHO cells were investigated. The GMA graft HCIC media showed good antibody separation and reuse performance, and the purity and yield of the 50 cycles used for the 50 cycles were stable, respectively. At the end of 98.5 ~ 99.8% and 90.2 ~ 96.9%., in order to reveal the adsorption and mass transfer mechanism of the polymer graft MMI medium from the microscale, the effect of the pore structure of the medium was investigated by ISEC. The adsorption heat of different proteins (hIgG and BSA) was studied by ITC. The adsorption, mass transfer and elution of the protein in a single medium particle were observed by CLSM in real time. .ISEC results show that the three-dimensional spatial distribution of glucan graft chain and poly GMA graft chain is formed on the surface of the matrix without salt, which helps to improve the protein adsorption capacity. Under high salt concentration, the graft chain appears collapsing, and the collapse degree of the glucan chain is more obvious, which weakens the chain transfer effect and reduces the mass transfer rate.ITC results to the hIg G, at pH 8, the increase in the density and salt concentration of the ligand significantly increases the enthalpy and entropy change of the adsorbed hIgG by the dextran graft MMI medium, indicating the enhancement of the hydrophobic interaction. Under the weak acid conditions, the enthalpy change and entropy change sharply decrease to negative values, indicating that the electrostatic repellent dominates the desorption of the protein; for BSA, the adsorption capacity increases with the increase of salt concentration. With the decrease, the enthalpy change and the entropy change value are negative, it is shown that the adsorption of BSA.CLSM mainly through electrostatic attraction shows that hIgG and BSA show the mass transfer mechanism of homogeneous diffusion control in the grafting of dextran, GMA graft and non grafted MMI medium; hIgG is embodied in the grafting of high ligand density glucan and the medium density poly GMA graft MMI medium. Compared with hIgG, the time required for BSA to reach the MMI medium is shorter than that of the MMI medium; when pH4.0, the fluorescence intensity in the medium density GMA graft MMI medium is the most obvious; the above results are matched with the macro adsorption, mass transfer and separation performance. In this paper, two new types of polymerization are prepared from the increase of the protein load of the HCIC medium. Grafted HCIC medium, the effects of polymer grafting, density, grafting density, pH, salt and protein properties on the adsorption and mass transfer properties of the polymer grafted HCIC medium were investigated. The mechanism of protein adsorption and mass transfer was revealed by means of ISEC, ITC, CLSM and other means. The results showed that the grafting in the hole could improve the antagonism of the HCIC medium. The adsorption capacity, mass transfer rate and dynamic load of the body showed good prospects for antibody separation.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：O631.3;TQ460.1

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