聚合物接枝荷电介质的蛋白质吸附和辅助复性研究
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摘要
聚合物接枝的离子交换色谱（IEC）介质比传统非接枝介质具有更高的吸附容量（Q）和传质速率（D），但其高容量吸附和快速传质机理还不清楚。本文围绕聚合物接枝的琼脂糖离子交换介质的性能和应用，开展了系统研究。
首先研究了葡聚糖接枝对γ球蛋白在IEC和混合模式作用色谱（MMC）介质上吸附的影响。结果表明，在IEC中，Q值和D值均随葡聚糖接枝量增加而增加。这是由于带电葡聚糖层提供三维吸附空间有利于蛋白质吸附，葡聚糖层存在静电耦合等作用促进传质。在MMC中，Q值随葡聚糖接枝量增加而降低，而D值与接枝无关。这是由于疏水性较强的葡聚糖链相互结合导致接枝层塌缩，对传质无贡献。而且塌缩的葡聚糖层屏蔽了部分MMC配基，不利于蛋白质吸附。
针对葡聚糖接枝介质中离子交换配基同时存在于接枝层与基质表面上的问题，本文制备了一系列不同离子交换容量（IC，100–1220mmol/L）的聚乙烯亚胺（PEI）接枝介质，以单独研究接枝层在蛋白质吸附中作用。结果表明，存在一个临界IC（cIC），Q值和D值在IC>cIC时快速增大。IC>cIC时，PEI链以其最少位点与琼脂糖连接，灵活的PEI链向孔内空间伸展，且临近的PEI链间距足够近使之彼此能够通过链振荡发生接触。因此，伸展的PEI链提供三维吸附空间，有利于蛋白质吸附；邻近的PEI链可通过链的摆动传递被吸附蛋白质而促进传质，即发生“链传递”作用。PEI接枝介质的Q值对离子强度（IS）的敏感程度低于传统非接枝介质，这是由于蛋白质可利用的孔体积随着IS增加而增加。PEI接枝介质的D值随IS增加先增加后减小。这是由于“链传递”作为一种表面扩散方式，同时依赖于蛋白质的吸附密度和吸附强度，这两个因素均与IS密切相关。在IC>cIC介质中，D值对IS的敏感程度明显大于在ICcIC介质中的快速传质做出贡献。
为拓展PEI接枝介质的应用，将PEI接枝介质应用于同电荷溶菌酶的氧化复性。系统研究了介质性质的影响，发现IC是影响介质辅助复性效果的首要因素，介质比表面也对复性收率有一定影响，但配基结构没有影响。在研究中还发现，Sepharose及以Sepharose为基质的阴离子交换色谱介质可微量吸附带正电的蛋白质，吸附容量可达60μg/mL。这种微量吸附是由于琼脂糖基质上残留的微量酸性基团。提高流动相盐浓度和介质电荷密度（如偶联PEI）可减弱这种吸附作用。
本研究将推动聚合物接枝IEC介质的蛋白质吸附理论发展及其在生物分离中的应用。
Polymer grafted resins for ion exchange chromatography (IEC) have been foundto possess high protein adsorption capacity (Q) as well as very high uptake rate (D),as compared to the traditional IEC resins with ion-exchange groups directly on thematrix surface. However, the mechanism origins of their advantages are not clearlyknown. Herein, we focused on the performance and application of the polymer graftedion exchangers, and the details of this work are summarized as follows.
Firstly, the roles of the grafted dextran layer on the adsorption of γ globulin toIEC resins and mixed-mode chromatography (MMC) resins were investigated. It isshown that, in IEC, both the Q and D values increased with increasing dextran density.It is considered that the binding volume provided by the charged dextran layerbenefitted protein adsorption and the mechanisms (i.e. electrostatic coupling ofdiffusion fluxes) existed in the dextran layer facilitated mass transfer. However, inMMC, the Q value decreased with increasing dextran density, and the D value wasindependent of the dextran density. It is because that the hydrophobic interactionbetween the dextran chains caused the collapse of the dextran layer, which contributedlittle to mass transfer. Additionally, the collapsed dextran layer shield parts of theMMC ligands, giving rise to fewer available binding sites for protein.
Considering the ionic groups in dextran-grafted resins existed in both thegrafting layer and on the matrix surface, poly(ethylenimine)(PEI) modifiedSepharose FF of10different ionic capacities (ICs,100–1220mmol/L) were preparedto investigate the role of the grafting layer on protein adsorption independently,excluding the influence of ligand distribution. It is found that there was a critical IC(cIC) or PEI density, above which both the Q and D values increased sharply. AtIC>cIC, the PEI chains were bonded to the gel surface at minimum numbers ofcoupling points and the polymers became extended to the pore space with greatflexibility. In addition, adjacent PEI chains became close enough to each other.Consequently, the extended three-dimensional PEI-layer provided greater accessibilityfor protein binding and the―chain delivery‖effect among the adjacent PEI chains bythe swings of the flexible chains facilitated transport of adsorbed protein molecules.The Q values of the PEI-Sepharose FF resins were less sensitive to salt concentrationthan both the commercial non-grafting resins and dextran-grafted ion exchangersreported in literature. It could be interpreted by the increase of pore accessibility with increasing IS. The D values of the PEI-Sepharose resins increased first and thendecreased with increasing IS. It was attributed to the dependencies of the―chaindelivery‖effect on protein capacity and binding strength, both of which are related toIS. Additionally, the PEI-Sepharose resins of ICs>cIC exhibited drastic changes of Dvalues with increasing IS, while those of ICscIC.
In order to expand the application of PEI grafted resins, they were used in thefacilitated refolding of like-charged lysozyme. The effects of like-charged solid phaseproperties on refolding were investigated systematically. The results showed thatcharged group density of charged particles played the most important part in theenhancing effect. The higher the charged group density, the less the charged resinswere required to achieve a desired facilitating effect. As compared to charged groupdensity, the effects of particle size and pore size were secondary. At the same chargedensity, the refolding yield was independent of ligand chemistry. Besides, we haveobserved that positively charged proteins (lysozyme and cytochrome C) are adsorbedon Sepharose gel and Sepharose-based anion-exchangers, in a capacity of10–60μg/mL. Because the trace adsorption is usually ignored in IEC practices, it may tosome extent degrade the product purity and/or recovery in large scale processing. Theresults show that the trace adsorption was caused by the residual negatively chargedgroups, most probably sulfate groups, in the agarose material. The undesirable traceadsorption was reduced by increasing NaCl concentration and/or coupling cationgroups (i.e. PEI).
This work will promote the devolopment of protein adsorption theories andbenefit the potential use of polymer-grated IEC resins in bioseparation processes.
首先研究了葡聚糖接枝对γ球蛋白在IEC和混合模式作用色谱（MMC）介质上吸附的影响。结果表明，在IEC中，Q值和D值均随葡聚糖接枝量增加而增加。这是由于带电葡聚糖层提供三维吸附空间有利于蛋白质吸附，葡聚糖层存在静电耦合等作用促进传质。在MMC中，Q值随葡聚糖接枝量增加而降低，而D值与接枝无关。这是由于疏水性较强的葡聚糖链相互结合导致接枝层塌缩，对传质无贡献。而且塌缩的葡聚糖层屏蔽了部分MMC配基，不利于蛋白质吸附。
针对葡聚糖接枝介质中离子交换配基同时存在于接枝层与基质表面上的问题，本文制备了一系列不同离子交换容量（IC，100–1220mmol/L）的聚乙烯亚胺（PEI）接枝介质，以单独研究接枝层在蛋白质吸附中作用。结果表明，存在一个临界IC（cIC），Q值和D值在IC>cIC时快速增大。IC>cIC时，PEI链以其最少位点与琼脂糖连接，灵活的PEI链向孔内空间伸展，且临近的PEI链间距足够近使之彼此能够通过链振荡发生接触。因此，伸展的PEI链提供三维吸附空间，有利于蛋白质吸附；邻近的PEI链可通过链的摆动传递被吸附蛋白质而促进传质，即发生“链传递”作用。PEI接枝介质的Q值对离子强度（IS）的敏感程度低于传统非接枝介质，这是由于蛋白质可利用的孔体积随着IS增加而增加。PEI接枝介质的D值随IS增加先增加后减小。这是由于“链传递”作为一种表面扩散方式，同时依赖于蛋白质的吸附密度和吸附强度，这两个因素均与IS密切相关。在IC>cIC介质中，D值对IS的敏感程度明显大于在IC
为拓展PEI接枝介质的应用，将PEI接枝介质应用于同电荷溶菌酶的氧化复性。系统研究了介质性质的影响，发现IC是影响介质辅助复性效果的首要因素，介质比表面也对复性收率有一定影响，但配基结构没有影响。在研究中还发现，Sepharose及以Sepharose为基质的阴离子交换色谱介质可微量吸附带正电的蛋白质，吸附容量可达60μg/mL。这种微量吸附是由于琼脂糖基质上残留的微量酸性基团。提高流动相盐浓度和介质电荷密度（如偶联PEI）可减弱这种吸附作用。
本研究将推动聚合物接枝IEC介质的蛋白质吸附理论发展及其在生物分离中的应用。
Polymer grafted resins for ion exchange chromatography (IEC) have been foundto possess high protein adsorption capacity (Q) as well as very high uptake rate (D),as compared to the traditional IEC resins with ion-exchange groups directly on thematrix surface. However, the mechanism origins of their advantages are not clearlyknown. Herein, we focused on the performance and application of the polymer graftedion exchangers, and the details of this work are summarized as follows.
Firstly, the roles of the grafted dextran layer on the adsorption of γ globulin toIEC resins and mixed-mode chromatography (MMC) resins were investigated. It isshown that, in IEC, both the Q and D values increased with increasing dextran density.It is considered that the binding volume provided by the charged dextran layerbenefitted protein adsorption and the mechanisms (i.e. electrostatic coupling ofdiffusion fluxes) existed in the dextran layer facilitated mass transfer. However, inMMC, the Q value decreased with increasing dextran density, and the D value wasindependent of the dextran density. It is because that the hydrophobic interactionbetween the dextran chains caused the collapse of the dextran layer, which contributedlittle to mass transfer. Additionally, the collapsed dextran layer shield parts of theMMC ligands, giving rise to fewer available binding sites for protein.
Considering the ionic groups in dextran-grafted resins existed in both thegrafting layer and on the matrix surface, poly(ethylenimine)(PEI) modifiedSepharose FF of10different ionic capacities (ICs,100–1220mmol/L) were preparedto investigate the role of the grafting layer on protein adsorption independently,excluding the influence of ligand distribution. It is found that there was a critical IC(cIC) or PEI density, above which both the Q and D values increased sharply. AtIC>cIC, the PEI chains were bonded to the gel surface at minimum numbers ofcoupling points and the polymers became extended to the pore space with greatflexibility. In addition, adjacent PEI chains became close enough to each other.Consequently, the extended three-dimensional PEI-layer provided greater accessibilityfor protein binding and the―chain delivery‖effect among the adjacent PEI chains bythe swings of the flexible chains facilitated transport of adsorbed protein molecules.The Q values of the PEI-Sepharose FF resins were less sensitive to salt concentrationthan both the commercial non-grafting resins and dextran-grafted ion exchangersreported in literature. It could be interpreted by the increase of pore accessibility with increasing IS. The D values of the PEI-Sepharose resins increased first and thendecreased with increasing IS. It was attributed to the dependencies of the―chaindelivery‖effect on protein capacity and binding strength, both of which are related toIS. Additionally, the PEI-Sepharose resins of ICs>cIC exhibited drastic changes of Dvalues with increasing IS, while those of ICs
In order to expand the application of PEI grafted resins, they were used in thefacilitated refolding of like-charged lysozyme. The effects of like-charged solid phaseproperties on refolding were investigated systematically. The results showed thatcharged group density of charged particles played the most important part in theenhancing effect. The higher the charged group density, the less the charged resinswere required to achieve a desired facilitating effect. As compared to charged groupdensity, the effects of particle size and pore size were secondary. At the same chargedensity, the refolding yield was independent of ligand chemistry. Besides, we haveobserved that positively charged proteins (lysozyme and cytochrome C) are adsorbedon Sepharose gel and Sepharose-based anion-exchangers, in a capacity of10–60μg/mL. Because the trace adsorption is usually ignored in IEC practices, it may tosome extent degrade the product purity and/or recovery in large scale processing. Theresults show that the trace adsorption was caused by the residual negatively chargedgroups, most probably sulfate groups, in the agarose material. The undesirable traceadsorption was reduced by increasing NaCl concentration and/or coupling cationgroups (i.e. PEI).
This work will promote the devolopment of protein adsorption theories andbenefit the potential use of polymer-grated IEC resins in bioseparation processes.
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