改进葡聚糖接枝技术制备高性能层析介质

利用改进的葡聚糖接枝技术,在以环氧氯丙烷为交联剂交联琼脂糖微球骨架的过程中加入葡聚糖溶液,在交联的同时接枝葡聚糖制得葡聚糖接枝型琼脂糖微球Rigose-Dex,再与盐酸2-氯三乙胺（DEAE）反应,获得葡聚糖接枝型高载量弱阴离子交换介质Rigose-Dex DEAE。以牛血清白蛋白（BSA）为模型蛋白,以商品化介质DEAE Sepharose 6FF为对照,系统研究了该葡聚糖接枝型Rigose-Dex DEAE的蛋白吸附性能,并进行了物理性能研究。结果表明,改进后葡聚糖接枝技术的最高接枝量为24.5mg/mL。自制Rigose-Dex DEAE可耐受700cm/h 的线性流速,对BSA的动态饱和载量为127.6mg/mL, 为商品介质DEAE Sepharose 6FF 载量的212%;具有在高流速下快速结合蛋白的能力,上样蛋白溶液在层析柱中停留2min即可基本达到饱和动态载量;重复使用性能好,经120 次在线清洗后,Rigose-Dex DEAE介质的动态载量为原始载量的90.4%。     

葡聚糖接枝型耐碱Protein A介质的层析性能提升研究

Sepharose 4FF微球经环氧活化后与葡聚糖溶液反应,得葡聚糖接枝型琼脂糖微球,再经环氧活化和偶联耐碱型Protein A配基,得葡聚糖接枝型高载量Protein A介质,测定了介质在线清洗稳定性能,并进行了热力学研究. 结果表明,与常规Protein A介质相比,葡聚糖接枝型Protein A介质的最高流速提高约32%,对抗体hIgG的动态载量为60.6 mg/mL,分别为常规介质和MabSelect SuRe介质载量的123%和95%;经40次清洗后,葡聚糖接枝型Protein A介质动态载量为原始载量的92%,远高于常规介质的84%,与MabSelect SuRe稳定性基本一致. 3种介质对抗体的结合均为熵驱动过程,葡聚糖接枝型Protein A介质的吸附热介于MabSelect SuRe和常规Protein A介质之间.     

葡聚糖接枝型Protein A介质的层析性能提升

Sepharose 4FF微球经环氧活化后与葡聚糖溶液反应,得葡聚糖接枝型琼脂糖微球,再经环氧活化和偶联耐碱型Protein A配基,得葡聚糖接枝型高载量Protein A介质,测定了介质在线清洗稳定性能,并进行了热力学研究.结果表明,与常规Protein A介质相比,葡聚糖接枝型Protein A介质的最高流速提高约32%,对抗体h Ig G的动态载量为60.6 mg/m L,分别为常规介质和Mab Select Su Re介质载量的123%和95%;经40次清洗后,葡聚糖接枝型Protein A介质动态载量为原始载量的92%,远高于常规介质的84%,与Mab Select Su Re稳定性基本一致.3种介质对抗体的结合均为熵驱动过程,葡聚糖接枝型Protein A介质的吸附热介于Mab Select Su Re和常规Protein A介质之间.     

应用凝胶过滤筛选纯化甲型肝炎病毒介质

目的　筛选适合甲肝病毒疫苗大规模纯化凝胶过滤层析的介质。方法　采用柱层析法 ,分别以Sepharose 4FF ,Sepharose 6FF及SephacrylS 5 0 0HR三种不同的凝胶过滤介质对经过阴离子交换层析纯化的甲肝病毒样品进行纯化。结果　三种凝胶介质分离甲肝病毒的图谱不相同 ,其中以Sepharose 4FF纯化甲肝病毒 ,抗原回收率达 6 9% ,纯化倍数为 4 4倍。结论　Sepharose 4FF凝胶过滤层析可以用于甲肝灭活疫苗的大规模纯化。     

超大孔聚苯乙烯微球固定蛋白质

采用琼脂糖对孔径为300~500 nm的超大孔聚苯乙烯(MPS)微球进行亲水化修饰,并在修饰后的表面上偶联病毒蛋白颗粒(RV),用于蛋白质的分离纯化实验. 结果表明,经过修饰的MPS层析介质具有优异的流体力学性能,层析过程的最高流速可达1120 cm/h,对病毒蛋白颗粒的固定量为0.638 mg/g,是商品化介质Sepharose 4FF的2倍. 采用RV-MPS介质对病毒蛋白颗粒抗体进行了分离纯化,收率为29.22%,高于Sepharose 4FF的18.92%.     

反离子对牛血清白蛋白在荷电葡聚糖接枝介质中色谱行为的影响

从前期开发的具有极高的吸附容量及传质速率的二乙氨乙基葡聚糖接枝离子交换介质中选取FF-D50-DexD100和FF-DexD100为典型代表,利用Cl^-、SCN^-、SO₄^2-、HPO₄^2-为模型反离子,以牛血清白蛋白（BSA）为模型蛋白,以商品化介质（Q Sepharose FF、Q Sepharose XL、DEAE Sepharose FF）为对照,在离子强度为0.06mol/L下,系统研究反离子对二乙氨乙基葡聚糖接枝介质的蛋白质吸附与洗脱行为的影响。结果表明,二乙氨乙基葡聚糖接枝介质对不同反离子的偏好性存在差异,且该偏好性差异与基团所处位置（接枝链配基或表面配基）无关。同时,介质偏好性弱的反离子会通过促进二乙氨乙基葡聚糖接枝介质的“链传递”效应加快蛋白质的传质速率,从而提高动态吸附容量。因此,在使用二乙氨乙基葡聚糖接枝介质进行蛋白质色谱柱分离过程中,可在吸附操作中使用HPO₄^2-,在洗脱操作中使用SCN^-来优化分离效果。     

肝素-琼脂糖凝胶6FF的制备及其在抗凝血酶III分离纯化中的应用

以自制琼脂糖凝胶6FF为基质,肝素为配基,利用还原胺化方法制备了肝素-琼脂糖凝胶6FF介质. 考察了肝素偶联反应的影响因素,并对其进行了优化,得到肝素配基的含量为2.7 mg/mL. 用所制介质从人血浆中分离出抗凝血酶III,从血浆开始计算的抗凝血酶III活性回收率为31.76%,与商品Heparin-Sepharose CL-6B的分离效果相当.     

肝素-琼脂糖凝胶6FF的制备及其在抗凝血酶III分离纯化中的应用

以自制琼脂糖凝胶6FF为基质，肝素为配基，利用还原胺化方法制备了肝素-琼脂糖凝胶6FF介质. 考察了肝素偶联反应的影响因素，并对其进行了优化，得到肝素配基的含量为2.7 mg/mL. 用所制介质从人血浆中分离出抗凝血酶III，从血浆开始计算的抗凝血酶III活性回收率为31.76%，与商品Heparin-Sepharose CL-6B的分离效果相当.     

无水体系中环氧氯丙烷活化琼脂糖凝胶制备高载量固定化金属亲和层析介质

在以二甲基亚砜为溶剂的无水体系中,利用环氧氯丙烷对琼脂糖凝胶Sepharose 6 Fast Flow进行活化,并偶联亚氨基二乙酸和Cu2+制备了固定化金属亲和层析介质. 结果表明,该体系中环氧氯丙烷对琼脂糖凝胶的活化效率大幅度提高,在40%(j)环氧氯丙烷、0.02 g/mL NaOH及50℃、反应时间4 h的优化条件下,环氧基活化密度最高达165 mmol/mL,较目前报道的最高值提高50%以上. 最终所制介质的Cu2+螯合密度为128.3 mmol/mL,对BSA的平衡吸附容量达2.05 mmol/L. 以0.5 mol/L咪唑为洗脱剂,被吸附的BSA洗脱率可达90%以上.     

反相悬浮聚合法制备交联葡聚糖凝胶微球及其在脂质体与药物分离中的应用

以葡聚糖4万为原料,环氧氯丙烷为交联剂,甲苯为有机分散相,选用合适的分散剂,利用反相悬浮聚合的原理制备了葡聚糖凝胶微球。以微球的球形度、粒径分布范围以及吸水溶胀度等性能为衡量标准,考察了有机分散相的种类、搅拌速度、实验温度、反应时间以及分散剂的用量对制备效果的影响,确定了最优的制备条件。同时,将所制葡聚糖微球应用于脂质体与药物的分离检测,以测定其凝胶分离性能。结果表明,所制交联葡聚糖凝胶微球具有良好的球形度、分散性以及吸水溶胀性,并具有优良的凝胶过滤分离性能。     

Protein retention in dextran-grafted cation exchange chromatography: The influence of pHs,counterions and polymer structure

Polymer-grafted ion exchange adsorbents were of great interest for the development of high-performance protein chromatography in biopharmaceutical and related fields. In this work, protein retention was systematically investigated in ion exchange chromatography packed respectively with dextran-grafted cation exchange adsorbents containing sulphopropyl(SP) ligand, SP Sepharose XL and Capto S, and non-grafted cation exchange adsorbent, SP Sepharose FF, using five proteins. With an increase of buffer p Hs, retention factors of proteins decreased among all the adsorbents, demonstrating the dominant role of electrostatic interaction for protein binding on cation exchange adsorbents. The evidences further revealed that the scattered positive charges on the surface of protein molecules, rather than net charge of protein molecule, determined protein retention on cation exchange adsorbent. Likely, counterions including NH~(4+), K~+, Na~+ and Mg~(2+) exhibited distinct influence on protein retention. It was well ascribed to solvent-mediated indirect ion–macromolecule interactions and direct ion–macromolecule interactions. Compared with SP Sepharose FF, polymer structure in dextran-grafted cation exchange adsorbents ultimately brought about different ligand distributions and smaller pore sizes, thereby regulating protein retention in cation exchange chromatography. By comparing the retention of myoglobin and β-lactoglobulin B in SP Sepharose XL and Capto S, we reasonably speculated that the enhancement of nonelectrostatic interaction caused by reducing the space arm length was a major reason for an increasing retention factor of myoglobin in Capto S. The results in this research help us understand adsorption mechanism of protein in polymer-grafted adsorbents and give scientific guidance for the development of chromatographic materials.     

Protein adsorption onto diethylaminoethyl dextran modified anion exchanger: Effect of ionic strength and column behavior

Our previous studies on bovine serum albumin(BSA) adsorption to diethylaminoethyl dextran(DEAE dextran,DexD, grafting-ligand) and DEAE(D, surface-ligand) modified Sepharose FF resins found that all the grafted resins(FF-DexD and FF-D-DexD) exhibited extremely fast uptake rate(effective diffusivity, D_e, D_e/D_O 1.4),which was six times greater than the ungrafted resins(D_e/D_O 0.3). In this work, the influence of ionic strength(IS) on 6 typical DEAE dextran-grafted resins was investigated. Bath adsorption equilibria and kinetics, breakthrough, and linear gradient elution experiments were conducted. Commercial DEAE Sepharose FF was used for comparison. It is found that protein adsorption capacities on DEAE dextran-FF resins and the commercial resin decreased with increasing IS, but DEAE dextran-FF resins exhibited much higher capacity sensitivity to salt concentration. Besides, steeper decrease of adsorption capacities could be obtained at higher graftingligand or surface-ligand density. It is worth noting that the facilitating role of surface-ligand to the "chain delivery" effect was weakened after adding salt, leading to the less improvement in uptake rate by increasing surface-ligand density at higher IS. Although the uptake rates of the DEAE dextran-FF resins increased first and then decreased with increasing IS, they kept the extremely high level of De values(D_e/D_O 1.1) at the their working/binding IS range. Moreover, the DEAE dextran-FF resin displayed much higher adsorption capacities and De values than commercial ungrafted resin in their working condition. Furthermore, the column results of DEAE dextran-FF resins presented higher dynamic binding capacities than and similar elution ISs with DEAE Sepharose FF to achieve similar(or even higher) recoveries suggest the excellent chromatographic column performance of the DEAE dextran-FF resins. Finally, both high recovery and purity of BSA and γ-globulin could be easily achieved using the typical DEAE dextran-FF column, FF-D60-DexD160, to separate their binary mixtures,by step gradient elution. The research has provided new insights into the practical application of the series of DEAE-dextran grafted resins in protein chromatography and proved their superiority.     

Implications from protein uptake kinetics onto dextran-grafted Sepharose FF coupled with ion exchange and affinity ligands

Our previous studies have reported the presence of "chain delivery" effects of protein adsorption onto ion exchangers with polymer-grafted ion-exchange groups,such as dextran-grafted and poly(ethylenimine)-modified Sepharose gels.However,it is unclear if the "chain delivery" occurs on affinity adsorption with specific interactions.This work is designed to address this issue.A dextran-grafted Sepharose gel was prepared,and then the matrix was modified using diethylaminoethyl,a typical ion-exchange group,or octapeptide (FYCHWQDE),an affinity ligand for human immunoglobulin G (hIgG) to prepare ion-exchange or affinity adsorbents,respectively.Results of hIgG adsorption showed that the uptake rate represented by the effective diffusivity of hIgG onto the dextran-grafted ion exchangers was obviously enhanced by the dextran grafting,indicating the presence of "chain delivery" of the bound proteins on the charged groups on the dextran chains.By contrast,the effective diffusivity of hIgG changed little as ligand density increased on the dextran-grafted FYCHWQDE adsorbents.Their adsorption capacities decreased and effective diffusivities were not accelerated by the dextran grafting.Thus,this work clarified that grafted dextran could not accelerate hIgG uptake rate on the affinity resins,or in other words,chain delivery did not occur on the specific interaction-based affinity adsorption.     

用盐析与疏水层析相偶合快速分离提纯猪胰激肽释放酶

将疏水层析技术用于从猪胰脏中分离纯化激肽释放酶,建立了一种简便、快速的分离提纯方法：将粗品溶解后经过硫酸铵沉淀处理,然后经过Butyl Sepharose FF疏水层析后得到目标蛋白,分析其纯度大于500U／mg,盐析和疏水两步纯化的收率大于85．0％,同时比较了Phenyl Sepharose FF,Octyl Sepharose FF和Butyl Sepharose FF三种疏水介质分离纯化胰K的效果,本实验工艺与传统工艺相比,具有操作简单、快速、回收率和纯化倍数高等优点,有望成为一种从动物组织中快速分离纯化药用蛋白质的有效技术平台。     

用于单克隆抗体纯化的仿生多肽超大孔PGMA微球制备及其性能

以仿生多肽配基FYEILHC为亲和配基、以葡聚糖修饰的聚甲基丙烯酸缩水甘油酯[Dextran-poly(glycidyl methacrylate), Dextran-PGMA]超大孔微球为基质，制备用于单克隆抗体纯化的仿生多肽超大孔PGMA微球，在环氧氯丙烷中滴加2 mol/LNaOH使其表面衍生出环氧基，在表面修饰FYEILHC；用扫描电镜表征微球表面形貌，用AKTA蛋白纯化系统考察了Dextran-PGMA微球和琼脂糖微球对抗体的动态吸附量随线性流速的变化。结果表明，偶联FYEILHC后Dextran-PGMA微球仍能保持其大孔结构，在923 cm/h线性流速下，其对抗体的动态吸附量仅下降约8%，而琼脂糖微球的动态吸附量则迅速下降25%。表明在较高流速下，抗体在Dextran-PGMA微球上的传质性能较好。吸附?用0.1 mol/L NaOH原位清洗重复40次后，Dextran-PGMA微球对抗体的动态吸附量约为(21?1) mg/mL，表明微球具有良好的化学稳定性；血清中回收的抗体纯度为95.0%，表明仿生多肽亲和介质具有从复杂生物样品中纯化抗体的巨大潜力，可满足高流速、高通量抗体分离纯化需求。     

单分散多孔聚合物微球的制备及其反相色谱应用

用分散聚合的方法制得单分散微米级聚苯乙烯微球(PS),以此聚苯乙烯微球作为种子,以邻苯二甲酸二丁酯为溶胀剂,苯乙烯为单体,二乙烯基苯为交联剂,甲苯为致孔剂,采用种子溶胀聚合的方法制得粒径分布较窄的多孔高交联的聚苯乙烯-二乙烯基苯微球(PS-DVB)。研究了交联剂与致孔剂的加入量对微球形貌、粒径及孔结构参数的影响。结果表明,所得多孔微球球形圆整,库尔特测得平均粒径为5.067~5.520μm,粒径分布窄,D90/D10为1.23~1.56,孔结构可控,并以此多孔微球作为反相色谱填料基质,理论塔板数每米可达6 000~15 000,可以用作高效液相色谱(HPLC)填料。     

System development for size exclusion chromatography of starch hydrolysates

A system for the aqueous size exclusion chromatography (SEC) of starch hydrolysates has been developed. Sepharose CL is the chromatographic support material, and aqueous sodium hydroxide solution is the eluent. Various factors affecting the resolution of the proposed system are discussed. Support materials having small particle size and narrow particle size distribution are necessary for maximizing separation efficiency. The ionic strength of the eluent, however, has a negligible effect on separation efficiency. The fractionation range of the system has been broadened by connecting in series columns containing two different Sepharose-CL gel types according to the bimodal pore size distribution concept. Well-characterized sodium polystyrene sulfonate and dextran standards are used to calibrate this SEC system. The Coll–Prusinowski calibration procedure leads to a linear calibration curve over a wide molecular weight domain.