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.     

应用于抗体药物捕获的耐碱亲和层析介质性能的比较

目的比较应用于抗体药物捕获的耐碱的4种蛋白A亲和层析介质和2种小分子亲和层析介质的性能,为单抗纯化工艺的选择提供参考。方法利用两种单抗纯品(mAb1和mAb2)测定4种蛋白A亲和层析介质MabSelectSuRe、POROS MabCaptureA、Absolute High Cap、TOYOPEARL AF-rProtein A-650F和2种小分子亲和层析介质Mabsorbent A2P HF、Fabsorbent F1P HF在停留时间分别为4、6、8 min时的动态载量;利用含mAb2的发酵液,从回收率和杂质去除方面比较6种亲和层析介质的纯化效果。结果在5%穿透点,各介质对mAb1和mAb2的动态载量在35~73 g/L之间。小分子亲和层析介质Mabsorbent A2P HF纯化mAb2的回收率为89%,其他5种介质纯化mAb2的回收率均≥96%;6种介质纯化mAb2的宿主细胞蛋白(host cell protein,HCP)在2 000 ppm之内,蛋白A残留量在20 ppm之内。结论结合流速、动态载量、回收率和杂质去除效果等指标,可从6种亲和层析介质中挑选适用于抗体类药物下游纯化工艺的耐碱型蛋白A或小分子亲和介质。     

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.     

光催化再生型活性炭的研制

光催化再生型活性炭的研制是实现吸附饱和活性炭原位再生的重要环节。本文采用溶胶凝胶——再活化法在商品活性炭表面合成TiO2光催化剂，制得新型炭吸附材料一光催化再生型活性炭。以苯酚为模型化合物，考察了该材料的吸附性能和紫外光照射条件下的光催化再生性能。以扫描电子显微镜、低温液氮吸附研究光催化再生型活性炭的表面结构、孔径结构和TiO2的分布状况。结果表明，光催化再生型活性炭保留了原料活性炭的吸附性能，在紫外光照射下即可逐渐恢复其吸附性能。其吸附性能随担载量的增加而降低，再生率随担载量的增加而升高，催化剂担载量为2．0wt％活性炭具有适宜的吸附性能和光催化再生性能。其再生率随再生次数增加而下降。光催化再生型活性炭具有原料炭相似的表面结构和孔径结构。TiO2在活性炭基材料表面呈不均匀分布，主要集中在活性炭的大孔和表面凹陷处。     

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%。     

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，...     

我国耐碱玻璃纤维发展概况

耐碱玻璃纤维(ARGF)是一种新型的轻质、高强、多功能增强无机材料.它在碱性介质中具有良好的抗碱侵蚀的能力,作为增强水泥的无机材料,是非承重水泥构件中钢材和水泥石棉制品中石棉的理想代用品.国外工业发达国家中,尤其是日本、英国,在耐碱玻璃纤维及其制品的生产技术和应用方面发展很快.耐碱玻璃纤维在建筑业、农牧业、土木工程、公路、管道、园林、城市建设、农村能源设施和水利工程等领域得到了日益广泛的应用.     

断裂内含肽介导的亲和介质与亲和标签的应用

应用Cfa断裂内含肽构建了一种新型的亲和纯化系统,并完成对目的蛋白无标签纯化。首先对Cfa断裂内含肽的IN片段与IC片段进行分子改造,通过研究在自由混合条件下的断裂情况,测定了其断裂速率。其次以改造后的IN片段作为配体与环氧活化的琼脂糖微球载体偶联,制备了IN亲和层析介质;IC片段作为亲和标签与目的蛋白GFP融合,并利用IN亲和层析介质实现对目的蛋白进行无标签纯化。结果表明,将IN与IC-GFP片段混合后,30s内即可断裂超过50%,4 h内即可完全断裂;IN亲和层析介质的静态载量为66.78 mg×m L~(-1),动态吸附载量约为30 mg×m L~(-1);纯化后的绿色荧光蛋白(GFP)纯度达到了96.7%,回收率为29.3%。Cfa断裂内含肽的应用为开发新型亲和纯化技术提供了一种潜在的方法。     

耐碱玻璃纤维砖骨料混凝土性能正交试验研究

以水胶比、粉煤灰掺量、砖骨料取代率、耐碱玻璃纤维体积掺量和耐碱玻璃纤维长度作为试验因素,通过设置五因素三水平正交试验,研究耐碱玻璃纤维二次破碎砖骨料混凝土28 d的立方体抗压强度以及28 d劈裂抗拉强度两种评价指标的变化规律.结果 表明:两种评价指标受水胶比、粉煤灰掺量、耐碱玻璃纤维体积掺量影响较大,二次破碎砖骨料取代率对劈裂抗拉强度影响较小,耐碱玻璃纤维长度对二者影响均不显著,综合考虑配置耐碱玻璃纤维砖骨料混凝土的最佳因素水平方案为水胶比0.4、砖骨料50％、粉煤灰10％、耐碱玻璃纤维体积掺量和长度分别为0.1％和16 mm.此结论可为耐碱玻璃纤维再生砖骨料新型混凝土工程使用提供试验依据.     

Cu~(2+)-IDA金属螯合尼龙-壳聚糖复合膜的制备及其亲和分离性能研究

为获得既具有良好机械强度和化学稳定性又具有大量活性官能团的金属螯合亲和膜介质,以尼龙膜为基膜,采用环氧氯丙烷活化共价偶联壳聚糖,制备尼龙-壳聚糖复合膜,膜上偶联壳聚糖的含量达98.2 mg·(g膜)-1,为配基的固载提供了大量活性位点.再次采用环氧氯丙烷活化复合膜,进而偶联亚氨二乙酸(IDA)、固定化Cu2+,获得金属螯合尼龙-壳聚糖复合型亲和膜,配基Cu2+固载量为5.4μmo1·cm-2.以牛血清白蛋白为目标蛋白,考察亲和膜的分离性能.研究结果表明:牛血清白蛋白在膜上的吸附行为符合Langmuir吸附等温方程,膜对牛血清白蛋白具有较好的亲和吸附效果,吸附容量达1.09 mg·cm-2.该膜具有较长的使用寿命并且容易再生.     

Hydrophobic charge-induction chromatographic resin with 5-aminobenzimidazol ligand: Effects of ligand density on protein adsorption

Hydrophobic charge-induction chromatography (HCIC) is a highly promising technology for antibody separation. HCIC resins ABI-B-6FF were prepared with 5-aminobenzimidazole (ABI) as the functional ligand. The effects of ligand density on the adsorption of human immunoglobulin G (hIgG) and bovine serum albumin were focused. It was found that the adsorption capacity and dynamic binding capacity (DBC) were improved with the increase of ligand density. The adsorption capacity and DBC of hIgG reached 128.07 mg/g gel and 67.63 mg/g gel. The results indicated that ABI-B-6FF resin has a promising potential for the application of antibody purification.     

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.     

Preparation of copolymer-grafted mixed-mode resins for immunoglobulin G adsorption

The mixed-mode resins for protein adsorption have been prepared by a novel strategy, copolymer grafting. Specially, the copolymer-grafted resins CG-MA with two functional groups, 5-amino-benzimidazole (ABI) and methacryloxyethyltrimethyl ammonium chloride (METAC), have been prepared through surfaceinitiated activator generated by electron transfer for atom transfer radical polymerization of METAC and glycidyl methacrylate (GMA), followed by a ring-open reaction to introduce ABI. The charge and hydrophobicity of CG-MA resins could be controlled by manipulating the addition of METAC and GMA/ABI. Besides, METAC and ABI provided positive effects together in both protein adsorption and elution: dynamic binding capacity of human Immunoglobulin G (hlgG) onto CG-M-A resin with the highest ligand ratio of METAC to ABI is 46.8 mg-g 1 at pH 9 and the elution recovery of hlgG is 97.0% at pH 5. The separation experiment showed that purity and recovery of monoclonal antibody from cell culture supernatant are 96.0% and 86.5%, respectively, indicating that copolymer-grafted mixed-mode resins could be used for antibody purification.     

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...     

A simple affinity chromatography method for the separation of gastric proteases from mucous substances

A simple chromatographic method for separating mucous substances contained in the abomasal mucosa from proteases has been developed. The chromatographic technique used controlled porous glass as a matrix, with covalently bound keratin as a ligand. This matrix allowed affinity binding of proteases at pH 7·0. The mucous substances present in calf abomasal mucosa were easily identified in the column eluate. Proteases were eluted from the column with 0·1 ml dm^?3 HCl with good recovery (85%) and high purification.     

An affinity adsorbent derived from aminopropyl silica for serine protease chromatography

A silica-based adsorbent for affinity chromatography of serine protease was prepared by bonding p-aminobenzamidine (pABZ) to aminopropyl silica. Silanization of silica with both γ-aminopropyltrimethoxysilane and γ-amino-propyltriethoxysilane under anhydrous conditions led to a monolayer density of primary amino groups. The aminopropyl silica was converted to primary hydroxylcontaining silica via diazotization, and consequently activated with p-nitrophenyl chloroformate and immobilized with ligand pABZ. The resultant silica–pABZ has a ligand density of 13.8 μmol g^?1. Pyridine was indicated by the data to increase the reactivity of the chloroformate toward the hydroxyl group more efficiently than 4-dimethylaminopyridine. Two stages of adsorption were found in batch adsorption of trypsin with an equilibrium adsorption isotherm of the Langmuir type. When the chromatographic column packed with this silica–p ABZ was operated under a higher flow rate (2·33 cm³ min^?1) and with 3 g dm^?3 of crude urokinase as the influence, the yield was 55%. Both the flow rate and the concentration of the crude protein were shown, by measuring the dynamic binding capacity from chromatographic experiments, to be the factors which influenced the chromatographic efficiency.     

ISEC法在评价色谱介质孔径结构方面的应用

层析是生物大分子分离纯化的一种重要手段,层析介质作为层析过程的关键的一部分受到了广泛的关注。层析介质的孔径结构对其分离纯化的效果有着显著的影响,因此探究层析介质的孔径结构至关重要。介绍了测定色谱介质孔径结构常用的方法,如：压汞法、氮气吸附法、小角X射线散射法、电镜扫描法（SEM和TEM）和逆体积排阻色谱法(ISEC)。重点阐述了ISEC法的基本原理和常用的探针类型,总结了ISEC法应用中的优点及不足,介绍了ISEC法的发展历史及其在国内外的研究现状。     

Preparation of methacrylamide grafted and dye-ligand immobilized PET fibers: Studies of adsorption and purification of lysozyme

In this study, novel affinity chromatographic fibers was prepared from methacrylamide grafted poly(ethylene terephthalate), PET-g-pMAA, using benzoylperoxide as an initiator. A dye ligand (i.e., Procion Brown) as a ligand was then covalently immobilized on the different amount of pMAAm grafted PET fibers, (PET-g-pMAAm-PB). The fibers were characterized by surface area measurement, infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), and scanning electron microscopy (SEM). Adsorptive properties of the composite fibers were tested using a model protein (i.e., lysozyme). To achieve these purposes, the influence of pH, ionic strength, initial lysozyme concentration, and temperature on adsorption system has been investigated and evaluated. A maximum lysozyme adsorption PET-g-pMAAm-PB fiber was obtained as 43.9 mg g⁻¹ at pH 7.5. The experimental equilibrium data obtained for lysozyme adsorption onto PET-g-pMAAm-PB fibers fitted well to the Langmuir isotherm model. The result of kinetic analyzed for lysozyme adsorption onto affinity fibers showed that the second-order rate equation was favorable. The purity of the eluted lysozyme, as determined by HPLC, was 84% with recovery 73% for PET-g-pMAAm-PB fiber. Experiments on regeneration and dynamic adsorption were also performed. It appears that PET-g-pMAAm-PB fibers can be applied for lysozyme separation without causing any denaturation. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2008     

人免疫球蛋白G酶解制备Fab和Fc片段及其分离纯化

为了制备抗体Fab和Fc片段,采用木瓜蛋白酶酶解人免疫球蛋白G(IgG),通过优化酶解条件和色谱法分离过程,得到了纯度较高的Fab片段和Fc片段。考察了酶解pH、酶加入量、添加半胱氨酸和酶解时间等对IgG酶解过程的影响,优化了酶解条件,提高酶解效率,IgG转化率大于98%。酶解产物通过Protein A亲和色谱法和DEAE阴离子交换色谱法进行了纯化,分离得到Fc片段和Fab片段,收率分别为72.6%和40.1%。经SEC-HPLC分析,Fc片段纯度达95.7%,Fab片段纯度达96.6%。