纤维素基复合层析基质的功能化与应用基础研究
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摘要
层析技术由于其分离机制多样性和操作的相对简便性,成为新型分离方法和集成化技术研究的焦点。扩张床吸附层析和疏水性电荷诱导层析是新型层析分离和集成化技术的典型代表。
在课题组前期工作的基础上,本文以纤维素基复合层析介质的设计、制备和应用为主线,侧重于基质的功能化,开展了四个方面的工作:其一,纤维素球形基质的物理性能改进,采用合适的手段,实现基质的孔道扩增和密度增大;其二,基质的化学改性,对纤维素基质进行活化处理,实现功能配基的有效偶联;其三,制备功能化的层析介质,包括弱阴离子交换扩张床介质和疏水性电荷诱导层析介质,进行介质的性能评价,研究介质与目标蛋白的相互作用;其四,应用研究,将功能化介质应用于实际的分离过程,包括固定床层析和扩张床吸附。取得的主要结果如下:
纤维素基复合层析基质的物理功能化:以再生纤维素为骨架,碳化钨粉末为增重剂,糊化木薯淀粉为制孔剂,采用“反相悬浮热再生法”和成球后去除淀粉方法,成功制备了纤维素/碳化钨复合微球,可用作层析介质的基质。基质的物理特点在于大孔径和高密度,实现了孔径的可控扩增和密度的可控调节。基质具有良好的球形外观和对数正态粒径分布,平均孔径达到129nm,湿真密度与碳化钨添加量呈线性相关,可达2.4mg/ml,孔结构受增重剂的影响较小。复合层析基质的柱层析行为表明:大孔型基质的洗脱峰形明显改善,柱压小,适合于高流速的固定床和扩张床层析;扩张床内可形成稳定的分级分布,符合扩张床吸附要求;可根据具体需要,调节碳化钨添加比例来优化操作流速和床层扩张率。
纤维素基复合层析基质的化学功能化——阴离子交换配基:采用碱化和偶联两步反应,将纤维素/碳化钨复合层析基质进行阴离子交换的配基功能化,制备成大孔型、高密度的弱阴离子交换扩张床吸附剂(CelI-TuC-DEAE),考察了牛血清白蛋白(BSA)的静态和动态吸附性能。结果表明,静态吸附容量达到97.1mg/ml;流速为500cm/h时(扩张床内扩张率约为2)动态吸附容量为66.6mg/ml;当流速高达900cm/h时,动态吸附容量仍有54.5mg/ml。吸附动力学研究表明,由于介质具有大孔结构,蛋白质扩散传递迅速,与溶液中自由扩散基本处在同一个数量级,验证了基质扩孔的必要性,适用于高流速的扩张床吸附分离。
纤维素基复合层析基质的化学功能化——疏水性电荷诱导配基:采用烯丙基溴(AB)和二乙烯基砜(DVS)活化,偶联4-巯乙基吡啶(MEP)、2-巯基-1-甲基咪唑(MMI)和2-巯基苯并咪唑(MBI),将纤维素/碳化钨复合层析基质进行疏水性电荷诱导配基功能化,制备得到四种大孔径、高密度的疏水性电荷诱导层析(HCIC)介质Cell-TuC-AB-MEP,Cell-TuC-DVS-MEP,Cell-TuC-DVS-MMI和Cell-TuC-DVS-MBI。考察了反应条件对活化和偶联过程的影响,优化了反应条件。烯丙基溴活化密度可达137μmol/ml,偶联后MEP配基密度为90μmol/ml;二乙烯基砜活化密度达到93μmol/ml,配基偶联密度约60μmol/ml。
疏水性电荷诱导层析介质与蛋白质的相互作用:考察了HCIC介质与模型蛋白质(卵黄免疫球蛋白IgY和BSA)的相互作用,从吸附平衡、吸附动力学、层析柱保留因子和穿透曲线等方面探讨了HCIC介质的吸附机理和性能特点。结果表明,溶液pH和盐浓度是影响吸附的关键因素。在pH=5左右,即蛋白质等电点附近,吸附作用达到最大,IgY静态吸附量达到100mg/ml以上;pH升高,吸附容量呈下降趋势;pH下降到蛋白质等电点和介质配基的pK_α值以下,产生静电排斥作用,实现解吸。添加盐可以适度增加介质对蛋白质的吸附。四种介质中吸附量Cell-TuC-DVS-MBI最大,而吸附选择性Cell-TuC-DVS-MMI和Cell-TuC-AB-MEP较好。Cell-TuC-AB-MEP的洗脱条件最为温和,而Cell-TuC-DVS-MBI则需要较苛刻的条件。采用简化了的扩散模型可以拟合吸附动力学过程,发现有效扩散系数相对较小,且随pH和盐浓度变化。蛋白质穿透实验表明,功能化介质可用于固定床或扩张床吸附分离。
疏水性电荷诱导层析介质的应用——卵黄免疫球蛋白的分离:选用Cell-TuC-AB-MEP作为层析介质,从卵黄稀释液中分离免疫球蛋白IgY,考察了层析条件对分离效果的影响。结果表明,采用固定床或扩张床操作模式,均可实现IgY的有效分离。固定床层析时,收率为58.4%,纯化因子2.9,纯度达到了92%;而扩张床吸附层析时,流速高达500cm/h,收率为48.4%,纯化因子2.9,纯度仍有89%。结果验证了所制备的HCIC功能化介质具有良好的IgY吸附特异性,适用于两种层析模式的分离过程。
总之,全文围绕纤维素基复合层析介质的功能化制备、性能表征和应用开展了系列研究,实现了纤维素复合基质的物理性能改善和化学配基功能化,成功制备了多种新型的功能化层析介质,探讨了相应的吸附机理和分离性能,并应用于实际对象的分离,完成了制备-机理-应用的研究路线,为纤维素层析介质的进一步开发奠定了基础。
The chromatography has been developed with new methods and integrated technologies for bioseparation due to varying separation mechanisms and convenient operation modes. Expanded bed adsorption (EBA) and hydrophobic charge induction chromatography (HCIC) are two of important chromatographic techniques. Based on our previous work, a novel macroporous cellulose-tungsten carbide composite matrix was designed and prepared. Then, the composite matrix was functionalized as anion-exchanger for EBA and HCIC adsorbents. The adsorption properties of new adsorbents as well as the application for antibody separation were investigated. The main points of this thesis were listed as follows.
Firstly, with the method of water-in-oil suspension thermal regeneration and starch addition, a series of cellulose-tungsten carbide composite matrix with different densities and porosities were developed. With cellulose as the skeleton, gelatinized cassava starch solution and tungsten carbide were used as porogenic agent and inert densifier, respectively. The matrix prepared showed good sphericity and a logarithmic symmetrical distribution of particle size. The mean pore diameter could reach 129 nm, while particle wet density could be increased with the addition of tungsten carbide. The results demonstrated that the macroporous matrix could be used in the packed bed and expanded bed with perfect properties, such as fast diffusion, low column pressure and good liquid mixing in the bed. In addition, the density of composite matrix could be controlled by altering the densifier addition to match the requirement of varying fluid velocity and expansion factor in expanded bed.
Secondly, a new anion-exchanger Cell-TuC-DEAE for EBA application was prepared by coupling diethylaminoethyl (DEAE) ligand on the macroporous cellulose-tungsten carbide composite.matrix. With bovine serum albumin (BSA) as model protein, the static and dynamic adsorption properties of Cell-TuC-DEAE were studied. The saturated adsorption capacity could reach 97.1mg/ml, and the dynamic adsorption capacity was 66.6mg/ml at 500cm/h and 54.5 mg/ml at 900cm/h in expanded bed. The results of adsorption kinetic indicated that the protein diffusion was enhances due to large pore in the matrix, which could be used for high fluid velocity in expanded bed.
Thirdly, four HCIC adsorbents were prepared. The macroporous cellulose-tungsten carbide composite matrix was activated with allyl bromide (AB) and divinyl sulfone (DVS), then coupled with three kinds of mercapto heterocyclic groups, 4-mercapto-ethyl-pyridine hydrochloride (MEP), 2-mercapto-1-methyl-imidazole (MMI) and 2-mercapto-benzimidazole (MBI), to form four HCIC adsorbents, Cell-TuC-AB-MEP, Cell-TuC-DVS-MEP, Cell-TuC-DVS-MMI andCell-TuC-DVS-MBI. The preparation conditions were optimized. The content of allyl group could reach 137μmol/ml matrix and the MEP density was 90μmol/ml adsorbents; while the content of vinyl sulfone could reach 93μmol/ml matrix and the ligands density was about 60μmol/ml adsorbents.
Fourthly, with model protein, Immunoglobulin of egg yolk (IgY) and BSA, the adsorption mechanism and performance of HCIC adsorbents was discussed. The static adsorption equilibrium, adsorption kinetics, retention factor and protein breakthrough in the column were investigated. The results indicate that the interactions between HCIC adsorbents and protein were determined mainly by pH and salt concentration. At pH 5, the adsorption reached the maximal value and the adsorption capacity was above 100mg/ml. With the increase of pH, the adsorption capacity decreased. When the pH was below the pI of protein and pK_a of ligand, the protein could be desorbed by the protein-ligand electrostatics repulsion. It was also found that the addition of lyotropic salt could enhance the protein adsorption. For all HCIC adsorbents tested, Cell-TuC-DVS-MBI showed highest adsorption capacity, while Cell-TuC-AB-MEP was the least. Cell-TuC-DVS-MMI and Cell-TuC-AB-MEP showed the excellent adsorption selectivity for immunoglobulin. Based on the study of retention factor, the protein could be easily eluted on Cell-TuC-AB-MEP, while harsh elution terms should be used for Cell-TuC-DVS-MBI. The results of adsorption kinetics indicated that the experimental data could be fitted with simplified diffusion model. The total effective diffusion coefficients of HCIC adsorbent were smaller than that of ion exchanger. The protein breakthrough behaviors demonstrated that the adsorbents prepared could be used in packed bed and expanded bed.
Finally, Cell-TuC-AB-MEP was used to separate IgY from egg yolk. For packed bed chromatography, the yield was 58.4%, and the purification factor was 2.9 with the purity of 92%. For expanded bed adsorption, the yield was 48.4% with the purification factor of 2.9 and the purity of 89% for the fluid velocity of 500cm/h. These results indicate that the HCIC adsorbents prepared was suitable for the purification of immunoglobulin both in packed bed and expanded bed.
The thesis focused on the preparation, functionalization and application of novel cellulose-based composite matrix for chromatography, especially for expanded bed adsorption and HCIC. Some important information on the adsorbent preparation, adsorption mechanism and antibody separation were obtained, which would certainly be useful for the developments of new adsorbent and chromatographic methods.
在课题组前期工作的基础上,本文以纤维素基复合层析介质的设计、制备和应用为主线,侧重于基质的功能化,开展了四个方面的工作:其一,纤维素球形基质的物理性能改进,采用合适的手段,实现基质的孔道扩增和密度增大;其二,基质的化学改性,对纤维素基质进行活化处理,实现功能配基的有效偶联;其三,制备功能化的层析介质,包括弱阴离子交换扩张床介质和疏水性电荷诱导层析介质,进行介质的性能评价,研究介质与目标蛋白的相互作用;其四,应用研究,将功能化介质应用于实际的分离过程,包括固定床层析和扩张床吸附。取得的主要结果如下:
纤维素基复合层析基质的物理功能化:以再生纤维素为骨架,碳化钨粉末为增重剂,糊化木薯淀粉为制孔剂,采用“反相悬浮热再生法”和成球后去除淀粉方法,成功制备了纤维素/碳化钨复合微球,可用作层析介质的基质。基质的物理特点在于大孔径和高密度,实现了孔径的可控扩增和密度的可控调节。基质具有良好的球形外观和对数正态粒径分布,平均孔径达到129nm,湿真密度与碳化钨添加量呈线性相关,可达2.4mg/ml,孔结构受增重剂的影响较小。复合层析基质的柱层析行为表明:大孔型基质的洗脱峰形明显改善,柱压小,适合于高流速的固定床和扩张床层析;扩张床内可形成稳定的分级分布,符合扩张床吸附要求;可根据具体需要,调节碳化钨添加比例来优化操作流速和床层扩张率。
纤维素基复合层析基质的化学功能化——阴离子交换配基:采用碱化和偶联两步反应,将纤维素/碳化钨复合层析基质进行阴离子交换的配基功能化,制备成大孔型、高密度的弱阴离子交换扩张床吸附剂(CelI-TuC-DEAE),考察了牛血清白蛋白(BSA)的静态和动态吸附性能。结果表明,静态吸附容量达到97.1mg/ml;流速为500cm/h时(扩张床内扩张率约为2)动态吸附容量为66.6mg/ml;当流速高达900cm/h时,动态吸附容量仍有54.5mg/ml。吸附动力学研究表明,由于介质具有大孔结构,蛋白质扩散传递迅速,与溶液中自由扩散基本处在同一个数量级,验证了基质扩孔的必要性,适用于高流速的扩张床吸附分离。
纤维素基复合层析基质的化学功能化——疏水性电荷诱导配基:采用烯丙基溴(AB)和二乙烯基砜(DVS)活化,偶联4-巯乙基吡啶(MEP)、2-巯基-1-甲基咪唑(MMI)和2-巯基苯并咪唑(MBI),将纤维素/碳化钨复合层析基质进行疏水性电荷诱导配基功能化,制备得到四种大孔径、高密度的疏水性电荷诱导层析(HCIC)介质Cell-TuC-AB-MEP,Cell-TuC-DVS-MEP,Cell-TuC-DVS-MMI和Cell-TuC-DVS-MBI。考察了反应条件对活化和偶联过程的影响,优化了反应条件。烯丙基溴活化密度可达137μmol/ml,偶联后MEP配基密度为90μmol/ml;二乙烯基砜活化密度达到93μmol/ml,配基偶联密度约60μmol/ml。
疏水性电荷诱导层析介质与蛋白质的相互作用:考察了HCIC介质与模型蛋白质(卵黄免疫球蛋白IgY和BSA)的相互作用,从吸附平衡、吸附动力学、层析柱保留因子和穿透曲线等方面探讨了HCIC介质的吸附机理和性能特点。结果表明,溶液pH和盐浓度是影响吸附的关键因素。在pH=5左右,即蛋白质等电点附近,吸附作用达到最大,IgY静态吸附量达到100mg/ml以上;pH升高,吸附容量呈下降趋势;pH下降到蛋白质等电点和介质配基的pK_α值以下,产生静电排斥作用,实现解吸。添加盐可以适度增加介质对蛋白质的吸附。四种介质中吸附量Cell-TuC-DVS-MBI最大,而吸附选择性Cell-TuC-DVS-MMI和Cell-TuC-AB-MEP较好。Cell-TuC-AB-MEP的洗脱条件最为温和,而Cell-TuC-DVS-MBI则需要较苛刻的条件。采用简化了的扩散模型可以拟合吸附动力学过程,发现有效扩散系数相对较小,且随pH和盐浓度变化。蛋白质穿透实验表明,功能化介质可用于固定床或扩张床吸附分离。
疏水性电荷诱导层析介质的应用——卵黄免疫球蛋白的分离:选用Cell-TuC-AB-MEP作为层析介质,从卵黄稀释液中分离免疫球蛋白IgY,考察了层析条件对分离效果的影响。结果表明,采用固定床或扩张床操作模式,均可实现IgY的有效分离。固定床层析时,收率为58.4%,纯化因子2.9,纯度达到了92%;而扩张床吸附层析时,流速高达500cm/h,收率为48.4%,纯化因子2.9,纯度仍有89%。结果验证了所制备的HCIC功能化介质具有良好的IgY吸附特异性,适用于两种层析模式的分离过程。
总之,全文围绕纤维素基复合层析介质的功能化制备、性能表征和应用开展了系列研究,实现了纤维素复合基质的物理性能改善和化学配基功能化,成功制备了多种新型的功能化层析介质,探讨了相应的吸附机理和分离性能,并应用于实际对象的分离,完成了制备-机理-应用的研究路线,为纤维素层析介质的进一步开发奠定了基础。
The chromatography has been developed with new methods and integrated technologies for bioseparation due to varying separation mechanisms and convenient operation modes. Expanded bed adsorption (EBA) and hydrophobic charge induction chromatography (HCIC) are two of important chromatographic techniques. Based on our previous work, a novel macroporous cellulose-tungsten carbide composite matrix was designed and prepared. Then, the composite matrix was functionalized as anion-exchanger for EBA and HCIC adsorbents. The adsorption properties of new adsorbents as well as the application for antibody separation were investigated. The main points of this thesis were listed as follows.
Firstly, with the method of water-in-oil suspension thermal regeneration and starch addition, a series of cellulose-tungsten carbide composite matrix with different densities and porosities were developed. With cellulose as the skeleton, gelatinized cassava starch solution and tungsten carbide were used as porogenic agent and inert densifier, respectively. The matrix prepared showed good sphericity and a logarithmic symmetrical distribution of particle size. The mean pore diameter could reach 129 nm, while particle wet density could be increased with the addition of tungsten carbide. The results demonstrated that the macroporous matrix could be used in the packed bed and expanded bed with perfect properties, such as fast diffusion, low column pressure and good liquid mixing in the bed. In addition, the density of composite matrix could be controlled by altering the densifier addition to match the requirement of varying fluid velocity and expansion factor in expanded bed.
Secondly, a new anion-exchanger Cell-TuC-DEAE for EBA application was prepared by coupling diethylaminoethyl (DEAE) ligand on the macroporous cellulose-tungsten carbide composite.matrix. With bovine serum albumin (BSA) as model protein, the static and dynamic adsorption properties of Cell-TuC-DEAE were studied. The saturated adsorption capacity could reach 97.1mg/ml, and the dynamic adsorption capacity was 66.6mg/ml at 500cm/h and 54.5 mg/ml at 900cm/h in expanded bed. The results of adsorption kinetic indicated that the protein diffusion was enhances due to large pore in the matrix, which could be used for high fluid velocity in expanded bed.
Thirdly, four HCIC adsorbents were prepared. The macroporous cellulose-tungsten carbide composite matrix was activated with allyl bromide (AB) and divinyl sulfone (DVS), then coupled with three kinds of mercapto heterocyclic groups, 4-mercapto-ethyl-pyridine hydrochloride (MEP), 2-mercapto-1-methyl-imidazole (MMI) and 2-mercapto-benzimidazole (MBI), to form four HCIC adsorbents, Cell-TuC-AB-MEP, Cell-TuC-DVS-MEP, Cell-TuC-DVS-MMI andCell-TuC-DVS-MBI. The preparation conditions were optimized. The content of allyl group could reach 137μmol/ml matrix and the MEP density was 90μmol/ml adsorbents; while the content of vinyl sulfone could reach 93μmol/ml matrix and the ligands density was about 60μmol/ml adsorbents.
Fourthly, with model protein, Immunoglobulin of egg yolk (IgY) and BSA, the adsorption mechanism and performance of HCIC adsorbents was discussed. The static adsorption equilibrium, adsorption kinetics, retention factor and protein breakthrough in the column were investigated. The results indicate that the interactions between HCIC adsorbents and protein were determined mainly by pH and salt concentration. At pH 5, the adsorption reached the maximal value and the adsorption capacity was above 100mg/ml. With the increase of pH, the adsorption capacity decreased. When the pH was below the pI of protein and pK_a of ligand, the protein could be desorbed by the protein-ligand electrostatics repulsion. It was also found that the addition of lyotropic salt could enhance the protein adsorption. For all HCIC adsorbents tested, Cell-TuC-DVS-MBI showed highest adsorption capacity, while Cell-TuC-AB-MEP was the least. Cell-TuC-DVS-MMI and Cell-TuC-AB-MEP showed the excellent adsorption selectivity for immunoglobulin. Based on the study of retention factor, the protein could be easily eluted on Cell-TuC-AB-MEP, while harsh elution terms should be used for Cell-TuC-DVS-MBI. The results of adsorption kinetics indicated that the experimental data could be fitted with simplified diffusion model. The total effective diffusion coefficients of HCIC adsorbent were smaller than that of ion exchanger. The protein breakthrough behaviors demonstrated that the adsorbents prepared could be used in packed bed and expanded bed.
Finally, Cell-TuC-AB-MEP was used to separate IgY from egg yolk. For packed bed chromatography, the yield was 58.4%, and the purification factor was 2.9 with the purity of 92%. For expanded bed adsorption, the yield was 48.4% with the purification factor of 2.9 and the purity of 89% for the fluid velocity of 500cm/h. These results indicate that the HCIC adsorbents prepared was suitable for the purification of immunoglobulin both in packed bed and expanded bed.
The thesis focused on the preparation, functionalization and application of novel cellulose-based composite matrix for chromatography, especially for expanded bed adsorption and HCIC. Some important information on the adsorbent preparation, adsorption mechanism and antibody separation were obtained, which would certainly be useful for the developments of new adsorbent and chromatographic methods.
引文
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