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.     

葡聚糖接枝型高载量金属螯合介质的制备与性能

对琼脂糖凝胶微球进行烯丙基活化,再接枝葡聚糖分子,考察葡聚糖分子量等因素对葡聚糖接枝过程的影响;以葡聚糖接枝琼脂糖凝胶微球为基质,制备亚氨基二乙酸型金属螯合介质,考察葡聚糖接枝过程对金属螯合介质的孔道结构、流通性能和载量等的影响. 结果表明,分子量20~500 kDa的葡聚糖都能均匀分布于琼脂糖凝胶微球内,葡聚糖接枝量随分子量增加而增大,所制的金属螯合介质形貌、粒径及其分布基本不受影响,且具有更好的流通性能,孔道结构比商品介质Ni Sepharose 6FF更丰富. 葡聚糖接枝的金属螯合介质对带组氨酸标签的乳酸脱氢酶和睫状神经营养因子的载量分别达到19和27 mg/mL,较Ni Sepharose 6FF的载量分别提高26.6%和42.0%.     

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。     

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%.     

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。     

基于双流体喷嘴的磁性琼脂糖微球的制备及其蛋白吸附性能探究

针对现有磁性琼脂糖微球(MAM)制备技术工艺流程长、重复性差、难以实现规模化及连续化生产等问题,提出了一种基于双流体喷嘴雾化技术的新型磁性琼脂糖微球制备方法;探究了不同雾化条件下,液滴尺寸变化的规律;在优化后的喷雾条件下,成功制备出球形度高、粒径小、磁响应迅速的磁性琼脂糖微球;将筛分后的微球制备成DEAE阴离子交换剂(DEAE-MAM),并以牛血清白蛋白(BSA)为模型蛋白,探究了不同粒径下的DEAE-MAM的蛋白吸附性能。结果表明,在保证水相性质一致的前提下,喷雾条件通过改变气液比影响液滴尺寸,进而影响微球粒径;粒径最小的DEAE-MAM (d₃₂=36 μm)离子交换容量最大(192.5 μmol/ml),饱和吸附量最高(150.0 mg/ml)。     

胆固醇配基色谱在蛋白质复性中的应用

以三聚氯氰为连接臂,采用两步法将疏水配基胆固醇修饰到交联琼脂糖（Sepharose）介质上,构建了胆固醇配基色谱。以溶菌酶（Lys）为模型蛋白,考察了胆固醇配基色谱的复性效果,并研究了流速、上样质量浓度和上样量对色谱复性效果的影响,确定了溶菌酶复性的最佳方法。研究结果表明,当溶菌酶上样量为1.00 mL,介质含0.25 mg溶菌酶时,在0.02 mL/min操作流速下进行吸附-洗脱复性,其最终蛋白收率和活性收率分别为96.3%和91.4%,有效促进了溶菌酶的复性。     

静电耦合亲和层析纯化人血清白蛋白的研究

人血清白蛋白(Human Serum Albumin,HSA)是人血清中含量最丰富的蛋白，约占血清总蛋白含量的40%～60%。商品化的白蛋白亲和介质(Cibacron Blue F3GA)的配基毒性大、易脱落、载量低，纯化白蛋白回收率较低。本研究制备了一种新型静电耦合亲和介质(DASA-Sepharose,3,5-Diaminobenzoic Acid n-Octyl Succinic Anhydride-Sepharose)，并通过一步层析从人血清中高效纯化白蛋白。DASA-Sepharose介质采用3,5-二氨基苯甲酸间隔臂将正辛基琥珀酸酐亲和配体偶联于琼脂糖微球。DASA-Sepharose间隔臂上的羧基通过静电作用吸附白蛋白，进而与正辛基琥珀酸酐亲和配基协同作用实现静电耦合亲和吸附，在保持亲和吸附高特异性的同时大大提高了吸附载量。采用牛血清白蛋白(Bovine Serum Albumin,BSA)为模型蛋白考察了Na Cl浓度和溶液pH对介质静态吸附载量(Q_m)的影响，NaCl浓度为0.025～0.06 mol/L时Q_m几乎不受影响，...     

琼脂糖-DEAE离子交换介质的配基密度和孔径对BSA吸附的影响

离子交换色谱是蛋白质分离纯化的有效方法之一,配基密度和介质孔径是影响蛋白质吸附的关键因素。采用3种不同琼脂糖浓度的凝胶为基质,具有不同的平均孔径,分别偶联上阴离子交换配基DEAE,通过调控反应条件,包括反应温度、反应时间、碱浓度和DEAE浓度,得到了不同配基密度和介质孔径的系列DEAE离子交换介质。考察了牛血清白蛋白（BSA）的静态和动态吸附性能,发现随配基密度增加或介质孔径减小,BSA饱和吸附容量有所增大;对于吸附动力学,介质孔径显著影响有效扩散系数。结果表明,配基密度和介质孔径共同决定了蛋白质的吸附性能,介质孔径主导蛋白质的孔内扩散,而配基密度则影响配基-蛋白质间的相互作用。     

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.     

Surface DEAE groups facilitate protein transport on polymer chains in DEAE‐modified‐and‐DEAE‐dextran‐grafted resins

Sepharose FF was modified with diethylaminoethyl‐dextran (DEAE‐dextran, DexD) and/or DEAE (D) to fabricate three types of ion exchangers FF‐DexD (grafting‐ligand resin), FF‐D (surface‐ligand resin), and FF‐D‐DexD (mixed‐ligand resin), for protein adsorption equilibria and kinetics study. It was found that both adsorption capacity and uptake rate (effective diffusivity, D_e) were significantly enhanced by grafting DEAE‐dextran. Notably, the D_e values on FF‐DexD and FF‐D‐DexD (D_e/D₀ > 1.4) were six times greater than those on FF‐D (D_e/D₀ < 0.3). More importantly, the increase of surface‐ligand density greatly enhanced uptake kinetics on FF‐D‐DexD. The results indicate that the surface ligands assisted the transport of bound proteins on polymer chains in the mixed‐ligand resins. That is, surface ligands worked as “transfer stations” between two neighboring chains, resulting in enhanced transport of bound proteins on chains. The research thus disclosed the unique role of surface ligands in facilitating protein uptake kinetics onto polymer‐grafted ion‐exchangers. © 2016 American Institute of Chemical Engineers AIChE J, 62: 3812–3819, 2016     

Adsorption Performance of Proteins to CM Sepharose FF and DEAE Sepharose FF Adsorbents

Adsorption equilibrium and kinetics were studied for the binding of proteins to CM Sepharose FF and DEAE Sepharose FF. The influence of temperature, pH, viscosity, initial concentration and the volume of adsorbents on the adsorption characteristics was investigated in detail. The results showed that the isotherms of lysozyme to CM Sepharose FF were well described by a Langmuir-type correlation. The two phase resistance model describing the dynamic adsorption process of lysozyme, papain, BSA to CM Sepharose FF was presented, and the pore diffusion coefficients were determined by using this model and the dynamic adsorption data.     

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.     

蛋白质在膨胀床吸附层析剂的静态和动态吸附性能

以牛血清白蛋白 (BSA)为目标蛋白 ,考察膨胀床用离子交换树脂StreamlineDEAE的静态和动态吸附性能 ,并和离子交换树脂DEAESepharoseFF进行比较 ,实验发现两者的静态吸附性能相似 ,而动态吸附性能差别较大。根据动态吸附数据计算出液膜扩散系数和孔内扩散系数。     

一步离子交换层析从Cohn组分V上清液中分离人血清白蛋白

传统的血浆低温乙醇沉淀工艺中Cohn组分V上清液由于其乙醇浓度高(体积浓度40％),进一步回收残余蛋白困难而被作为废弃组分.本研究探索了采用一步层析从Cohn组分V上清中回收入血清白蛋白的方法.首先以牛血清白蛋白(BSA)为模型蛋白,比较了三种不同类型介质在不同乙醇-水溶液中的吸附容量.疏水介质在乙醇-水溶液中对BSA...     

长白白眉蝮蛇蛇毒类凝血酶的纯化及质量检测

长白白眉蝮蛇原毒经DEAE Sepharose FF ,CM Sepharose FF和G Sepharose CL 6B层析后 ,制得长白白眉蝮蛇蛇毒类凝血酶精品。该产品经质量检测完全符合卫生部颁发的质量标准 ,该工艺适合于大规模纯化长白白眉蝮蛇蛇毒类凝血酶。