基于超大孔聚合物微球的混合色谱模式层析介质的制备

以甲基丙烯酸缩水甘油酯与乙二醇二甲基丙烯酸酯共聚的超大孔聚合物微球为基质,采用聚乙烯亚胺和丁基缩水甘油醚先后衍生微球的表面,制备成兼具阴离子交换与疏水相互作用的混合色谱模式层析介质。考察离子交换基团、疏水配基密度对蛋白载量、回收率的影响,结果表明,在离子交换容量0.2~0.5 mol/mL范围内,随着介质离子交换容量的增大,蛋白载量及回收率均呈增加趋势,蛋白载量最高值达40 mg/mL,回收率大于90%。当疏水配基的密度大于0.03 mol/mL时,介质开始表现疏水相互作用。此超大孔混合模式色谱介质在大于2000 cm/h 的流速下依然能保持低于2 MPa 的柱背压,同时在高流速下(2880 cm/h)纯化人血清中的抗体应用中表现良好的分辨率。此介质在高通量分离纯化应用方面具有巨大潜力。     

促卵泡激素的径向色谱纯化工艺

 比较了径向离子交换色谱与经典离子交换色谱在纯化工艺放大过程中的分离效果 ,证实了径向色谱比经典色谱更适合于促卵泡激素 (FSH)的纯化 ,并建立了适合于大规模生产FSH的径向色谱纯化新工艺。纯化后FSH的比活性为 180IU/mg ,活性回收率为 5 6 % ,产品质量符合国家药典规定。     

高效阳离子交换法分离纯化蛋清中的溶菌酶

 建立了用高效阳离子交换分离纯化蛋清中溶菌酶的新方法 ,讨论了纯化的工艺条件。蛋清样品匀浆后 ,用氯化钠初步纯化 ,然后用弱阳离子交换柱XIDACE WCX分离。结果表明 ,被纯化的溶菌酶和杂蛋白得到很好分离。经活性检测 ,溶菌酶过柱后的活性回收率为 10 7% ,蛋白的比活为 15 4 6 7U/mg ,纯化倍数提高了 5 6倍。用尺寸排阻 (SEC)鉴定 ,得到的溶菌酶呈均一性。该法较传统软基质低压离子交换分离速度快 ,纯化效率高。     

高效离子交换膜色谱介质制备及其对蛋白质的分离

制备了一种以纤维素膜为基质,以二乙胺为配基的高效液相离子交换膜色谱介质,并考察了不同柱长、不同洗脱梯度、不同流速下其对几何标准蛋白的分离情况。与高效液相柱色谱相比,这种高效液相膜色谱柱可以更快速的达到蛋白质的分离。即使在较高流速下,柱压仍较低。实验表明,这种高效液相膜分谱柱适合于蛋白质的快速分离。     

高效液相色谱在生物医药研究中的应用第三讲蛋白质的反相高效液相色谱分离

近十年来,高效液相色谱(HPLC)在蛋白质分离纯化方面取得了很大的进展,并得到了广泛的应用。HPLC包括排阻色谱、离子交换色谱、亲和色谱、疏水反应色谱以及反相色谱等,几乎已在蛋白质分离各种传统方法中得到应用。这些方法在蛋白质分离中各有其地位,但到目前为止人们普遍认为反相高效液相色谱(RP-HPLC是分离纯化蛋白质的最有效的方法     

蛋白质制备色谱的研究

研究了蛋白质纯化制备的二种运行模式,其一,在色谱柱超载下纯化制备蛋白质模式,并成功地用离子交换色谱纯化制备了大豆中的胰蛋白酶,用反相液相色谱纯化了细胞色素Ｃ;其二,用溶质－溶质顶替色谱纯化制备了核糖核酸酶－Ａ,并对顶替色谱过程中诸参数进行了讨论和选择。     

双孔色谱介质的制备和质粒DNA快速色谱纯化

以甲基丙烯酸缩水甘油酯为单体,二甲基丙烯酸乙二酯为交联剂,环己醇与十二醇为有机致孔剂,200 g/L的碳酸钙悬浮液为内水相(超孔致孔剂),用二次乳化法制备了(W/O)/W乳液,通过紫外光引发悬浮聚合生成两类孔型高分子微球(DEA-B).DEA-B孔径为双峰分布,范围分别为50~200 nm和500~5000 nm.其体积平均粒径、湿密度与不含超孔的微孔介质(DEA-M)接近.在流速为0.5 mL/min(150 cm/h)及相同的洗脱条件下,比较了所制备的微孔色谱介质填充的色谱柱与双孔色谱介质填充的色谱柱对5.4 kb质粒DNA的分离效果.结果表明,仅双孔介质色谱柱可以纯化质粒DNA,并且可以在1500 cm/h的高流速下得到色谱纯的质粒DNA,证明该双孔色谱介质可以用于质粒DNA的高速分离.     

在线单柱二维液相色谱法对蛋清中三种活性蛋白的快速分离纯化

基于一种新型的强阳离子交换/疏水色谱(SCX/HIC)双功能色谱柱构建了在线单柱二维液相色谱(2DLC-1C), 分离系统实现了自动控制, 可在线进行样品收集、二维进样和二维色谱分离. 采用SCX-HIC 2DLC-1C, 在120 min内可将8种标准蛋白完全分离, 并成功应用于鸡蛋清中三种活性蛋白质的分离纯化. 结果表明利用在线2DLC-1C技术可在70 min内完成对鸡蛋清中溶菌酶、卵清蛋白和卵转铁蛋白三种活性蛋白的快速分离纯化, 其纯度分别为95%、93%和97%. 该方法减少了样品的处理步骤, 对样品的污染小, 纯化速度快, 操作简单, 易于实现自动化和放大, 对活性蛋白的快速分离制备和工业化生产具有广阔的应用前景.     

亚油酸甲酯硅胶聚合物键合相的制备及对蛋白质的分离

1　引　　言从现代分离科学理论计算得出 ,色谱和电泳是目前所知道最好的两种分离方法 ,但是 ,因受各种因素的限制 ,电泳目前尚不能用于生产规模的生物大分子的分离和纯化。这就是把分离和纯化生物大分子 (包括蛋白质、酶、核酸、多糖等 )的研究重点放在色谱上的原因。在生物技术制取蛋白质的多级纯化过程中 ,液相色谱是一个必需步骤。为了获得生物大分子的快速分离就得从基质到键合基团不断改进色谱柱填料。在本文中介绍的柱填料是键合在大孔硅胶上的乙烯基与亚油酸甲酯和二乙烯基苯共聚形成的。从蛋白质混合样品洗脱曲线看出这一填料具有…     

氨基己酸为配基的新型弱阳离子交换/疏水双功能色谱固定相对蛋白质的分离纯化

以硅胶为基质、氨基己酸为配基制备了一种新型弱阳离子交换/疏水（WCX/HIC）双功能混合模式色谱固定相。该固定相配基具有一定的疏水性且含有羧基,在高盐浓度下表现为HIC的性质,可作为HIC固定相使用;在低盐浓度条件下表现为离子交换的性质,可作为WCX固定相使用。分别考察了该介质在WCX和HIC两种模式下对标准蛋白质的分离性能,并与商品柱进行比较。结果表明,所合成的WCX/HIC双功能固定相在WCX和HIC两种模式下对蛋白质均有较高的分离度和选择性,且分离能力与商品柱相当,两种模式下标准蛋白质的质量和活性回收率均大于93%,表明该柱具有“一柱二用”的功能,适于生物大分子的分离纯化。基于此双功能色谱柱构建的在线单柱二维液相色谱（2DLC-1C）可在60 min内实现8种蛋白质的快速分离。在70 min内完成了对蛋清中溶菌酶的二维纯化,纯度可达到98.3%。该技术中一根色谱柱可当作两根色谱柱使用,对蛋白质组学研究和重组蛋白药物的生产具有重要的应用价值。     

一种从蛇毒中纯化神经生长因子的新工艺

为了能从中华眼镜蛇蛇毒中简单快速地分离纯化神经生长因子（一种治疗各种神经性损伤和神经退行疾病的药物,简称NGF）,采用不同的色谱柱联用的方式对NGF的纯化工艺进行了研究。结果表明,采用DEAE Sepharose F.F.和Sephadex G 50二步柱色谱工艺可以从蛇毒中快速分离得到神经生长因子。经十二烷基硫酸钠聚丙烯酰胺凝胶电泳和反相高效液相色谱分析, 证明所得到的NGF为单一组分,相对分子质量约为 29 000。对8　d龄鸡胚背根神经节采用体外培养法,结果证明,所得NGF具有明显的促进神经纤维     

Continuous superporous agarose beds for chromatography and electrophoresis

Continuous agarose beds (monoliths) were prepared by casting agarose emulsions designed to generate superporous agarose. The gel structures obtained were transected by superpores (diameters could be varied in the range 20-200 microns) through which liquids could be pumped. The pore structure and the basic properties of the continuous gel were investigated by microscopy and size exclusion chromatography. The chromatographic behaviour was approximately the same as for beds packed with homogeneous agarose beads with a particle diameter equivalent to the distance between the superpores. In one application, the superporous continuous agarose bed was derivatized with a NAD+ analogue and used in the affinity purification of bovine lactate dehydrogenase from a crude extract. In another application, a new superporous composite gel material was prepared by adding hydroxyapatite particles to the agarose phase. The composite bed was used to separate a protein mixture by hydroxyapatite chromatography. In a third application, the continuous superporous agarose material was used as an electrophoresis gel. Here, a water-immiscible organic liquid was pumped through the superpores to dissipate the joule heat evolved, thus allowing high current densities.     

转Bt基因植物表达产物Cry1Ab蛋白的制备纯化方法研究

以转Bt基因水稻为试材,研究其表达产物Cry1Ab蛋白的提取、分离及纯化的方法。实验结果表明,DEAE-纤维素填料对Bt蛋白有较好捕获效果。根据生物信息学方法预测了目标蛋白和主要共存蛋白的等电点和疏水性差异。合理地选择了阴离子交换色谱与疏水作用色谱组合方法。提取液经DEAE-Sephadex A-50柱层析及Phenyl-Sepharose Fast Flow疏水层析分离后,目标蛋白得到了显著的纯化。考察了疏水层析中用不同洗脱液洗脱Cry1Ab蛋白对活性回收率和纯度的影响,结果表明：以0．25mol／L KSCN作洗脱液对活性影响最小,HIC一步纯化倍数可达8倍,总纯化倍数达100倍。     

Comparison among three anion exchange chromatographic supports to capture erythropoietin from cell culture supernatant Lourdes

Affinity and ion exchange conventional chromatography have been used to capture erythropoietin (EPO) from mammalian cell culture supernatant. Currently, chromatographic adsorbent perfusion is available, however a limited number of applications have been found in the literature. In this work, three anion exchange chromatographic supports (gel, membrane and monolithic) were evaluated in the capture step of the recombinant erythropoietin purification process. The influences of load and flow rate on each support performance were analyzed. Also the purity of the EPO molecules was determined. A productivity analysis, as a decision tool for larger scale implementation, was done. As a conclusion, the evaluated supports are technically suitable to capture EPO with adequate recovery and good purity. However, the monolithic column admits high operating velocity, showing the highest adsorption capacity and productivity.     

3种阴离子交换色谱固定相捕获细胞培养上清液中红细胞生成素的效果比较（英文）

Affinity and ion exchange conventional chromatography have been used to capture erythropoietin(EPO)from mammalian cell culture supernatant.Currently,chromatographic adsorbent perfusion is available,however a limited number of applications have been found in the literature.In this work,three anion exchange chromatographic supports(gel,membrane and monolithic)were evaluated in the capture step of the recombinant erythropoietin purification process.The influences of load and flow rate on each support performance were analyzed.Also the purity of the EPO molecules was determined.A productivity analysis,as a decision tool for larger scale implementation,was done.As a conclusion,the evaluated supports are technically suitable to capture EPO with adequate recovery and good purity.However,the monolithic column admits high operating velocity,showing the highest adsorption capacity and productivity.     

Exploration of overloaded cation exchange chromatography for monoclonal antibody purification

Cation exchange chromatography using conventional resins, having either diffusive or perfusive flow paths, operated in bind-elute mode has been commonly employed in monoclonal antibody (MAb) purification processes. In this study, the performance of diffusive and perfusive cation exchange resins (SP-Sepharose FF (SPSFF) and Poros 50HS) and a convective cation exchange membrane (Mustang S) and monolith (SO(3) Monolith) were compared. All matrices were utilized in an isocratic state under typical binding conditions with an antibody load of up to 1000 g/L of chromatographic matrix. The dynamic binding capacity of the cation exchange resins is typically below 100 g/L resin, so they were loaded beyond the point of anticipated MAb break through. All of the matrices performed similarly in that they effectively retained host cell protein and DNA during the loading and wash steps, while antibody flowed through each matrix after its dynamic binding capacity was reached. The matrices differed, though, in that conventional diffusive and perfusive chromatographic resins (SPSFF and Poros 50HS) demonstrated a higher binding capacity for high molecular weight species (HMW) than convective flow matrices (membrane and monolith); Poros 50HS displayed the highest HMW binding capacity. Further exploration of the conventional chromatographic resins in an isocratic overloaded mode demonstrated that the impurity binding capacity was well maintained on Poros 50HS, but not on SPSFF, when the operating flow rate was as high as 36 column volumes per hour. Host cell protein and HMW removal by Poros 50HS was affected by altering the loading conductivity. A higher percentage of host cell protein removal was achieved at a low conductivity of 3 mS/cm. HMW binding capacity was optimized at 5 mS/cm. Our data from runs on Poros 50HS resin also showed that leached protein A and cell culture additive such as gentamicin were able to be removed under the isocratic overloaded condition. Lastly, a MAb purification process employing protein A affinity chromatography, isocratic overloaded cation exchange chromatography using Poros 50HS and anion exchange chromatography using QSFF in flow through mode was compared with the MAb's commercial manufacturing process, which consisted of protein A affinity chromatography, cation exchange chromatography using SPSFF in bind-elute mode and anion exchange chromatography using QSFF in flow through mode. Comparable step yield and impurity clearance were obtained by the two processes.     

Preparative isolation of angiotensin-converting enzyme from human lung.

Angiotensin-converting enzyme from human lung was purified to apparent homogeneity using a five-step purification procedure consisting of ammonium sulfate precipitation, ion-exchange chromatography on DEAE Sephadex A-50, gel permeation on Sephadex G-200, chromatofocusing on a polybuffer exchange (PBE 94) column and high-performance liquid chromatographic gel permeation on a Bio-Sil TSK-250 column. This procedure gave an approximately 700-fold purification with a 20% yield compared to a 550-fold purification and a 1% yield with an affinity chromatography-based procedure. The 20-fold greater yield of the five-step procedure offers a major advantage for preparative use in the structural characterization of angiotensin-converting enzyme.     

Faster isolation of recombinant factor VIII SQ, with a superporous agarose matrix.

A superporous agarose matrix was compared with a corresponding homogenous matrix in the isolation of recombinant factor VIII SQ (r-VIII SQ) by immunoaffinity chromatography. As a reference, the commercially available Sepharose FastFlow, used for a similar purification in the industry, was also evaluated. Breakthrough curves are described for flows between 50 and 400 cm/h with pre-purified r-VIII SQ and with cell culture broth. The superporous gel gave the best performance and a 1000-fold purification was obtained in a one-step procedure. The superporous matrix made it possible to increase the throughput about four-fold, presumably due to its better mass transfer properties. The importance of the ligand distribution profile is discussed based upon immunofluorescence microscopy data.     

DNA removal on different chromatography supports used for EPO purification by spike studies

Chromatographic techniques are used in the purification step of human recombinant erythropoietin production process to obtain a reliable product with high purity. Anion-exchange chromatography supports have proved high efficient in removing contaminants such as DNA. For that reason, the DNA removal was determined by spike studies, on three anion-exchange chromatographic supports: gel, membrane, and monolithic column, which is used in intermediate purification stage. This study showed that membrane and monolith columns have very good results in the removal of contaminants at this step. Log removal values (LRV) greater than 3.5 were obtained from DNA spike clearance studies. Monolithic column was determined as the best technological proposal, with more than 4 LRV, 7.72?mg DNA per milliliter of adsorbent and 85% protein recovery in nonspike run. The results of this study may be used as a guide in the selection of commercially available chromatography supports for intermediate purification steps in recombinant protein production.