Q Sepharose^TM XL强阴离子交换色谱快速纯化猪心中肌红蛋白和细胞色素C

以Q SepharoseTM XL强阴离子交换色谱结合Sephadex G-75分子排阻色谱快速提取纯化猪心中肌红蛋白（Myoglobin,Mb）和细胞色素C（Cyt C）。在pH值为7.6-8.5范围内,Cyt C吸附在Q SepharoseTMXL强阴离子交换柱上,而Mb会缓慢通过。所以选择QSepharoseTMXL强阴离子交换柱的上样pH值为7.9,洗脱pH值为7.2。电泳检测证明,经过QSepharoseTMXL柱初步纯化所得Mb和Cyt C的产率分别为89.7%和93.2%;再经过Sephadex G-75分子排阻色谱可得到纯的Mb和Cyt C,其产量分别为77.43mg.（kg猪心）-1和86.42mg.（kg猪心）-1。使用QSepha-roseTMXL强阴离子交换色谱能从猪心中快速制备纯的Mb和Cyt C。     

Q Sepharose^TM-XL强阴离子交换色谱快速纯化精氨酸激酶及其多克隆抗体的研究

利用QSepharose^TM-XL强阴离子交换色谱从虾的精氨酸激酶粗提液中纯化出精氨酸激酶。以其免疫大白兔．然后利用QSepharose^TM-XL强阴离子交换色谱从高免疫兔血清中纯化精氨酸激酶的多克隆抗体。采用SDS-PAGE检测精氨酸激酶及其抗体的纯度,并进行了活性测定。结果表明．利用QSepharose^TM-XL强阴离子交换色谱纯化出的精氨酸激酶及其多克隆抗体纯度高、活性强。建立了快速高效、操作方便而又适合实际应用的制备精氨酸激酶及其多克隆抗体的方法．可为精氨酸激酶及食品过敏的相关研究提供帮助。     

以双水相萃取法从猪心中快速制备细胞色素C

把化工上常用的双水相萃取法应用于生物分离,从猪心中纯化具有药用价值的细胞色素C。以14％聚乙二醇和12％硫酸铵两相系统对粗提液进行萃取,细胞色素C全部浓缩于盐相。对盐相进行去盐后,经人造沸石快速吸附与洗脱、三氯醋酸沉淀等制得细胞色素C半成品,以弱酸性阳离子交换柱进行精制。聚丙烯酰胺凝胶电泳、可见吸收光谱扫描等定性定量分析证明了此法所得产品纯度好,提取率也较高。与传统方法比较,此法工艺简单,成本低,耗时少,尤其适用于大规模生产。     

全原子分子动力学模拟反相色谱分离细胞色素C的过程

采用全原子分子动力学方法模拟了反相色谱分离蛋白质的吸附和洗脱过程。采用表面键合C4烷基链的硅胶基质作为反相色谱介质和细胞色素C作为蛋白质模型,模拟蛋白质在反相色谱分离过程中的构象变化。结果显示:在吸附过程中,蛋白质在释放出表面结合水分子的同时置换出色谱介质表面的水分子。与其在溶液中的天然构象相比,吸附态蛋白质的构象发生改变。在洗脱过程中,随着溶剂从水切换到甲醇,甲醇取代水分子包覆在介质和蛋白质表面,将蛋白质从介质表面置换下来。分子模拟的结果再现了有关反相色谱"优先水化"的吸附机制,并从分子水平上展现了吸附和洗脱过程中蛋白质、色谱介质和溶剂之间相互作用,对反相色谱介质设计和过程优化提供了参考。     

阴离子交换层析法纯化聚唾液酸工艺

以大肠杆菌发酵所产聚唾液酸粗品(含蛋白质、核酸、无机盐和色素等杂质)为纯化对象,通过静态吸附和梯度洗脱实验选择了分离效果较好的阴离子交换介质Q-琼脂糖凝胶FF,对聚唾液酸的纯化工艺条件进行优化. 得到最佳洗脱条件为：洗脱液为pH 7.2的NaCl-0.02 mol/L磷酸钠缓冲液体系,流速0.8 mL/min,洗脱线性梯度方程为CNaCl=0.0017VEluent (CNaCl为NaCl浓度,VEluent为洗脱液体积),层析柱体积46 mL时最大进样量4.0 mL. 该条件下聚唾液酸回收率在86.0%以上,纯化后样品中的蛋白质含量从1.9%降低至0.04%,纯度在98%以上. 紫外吸收光谱和高效凝胶过滤色谱分析表明,聚唾液酸产品组分均一,重均分子量为303 kDa.     

串联阴阳离子交换色谱分离牛初乳中乳铁蛋白与IgG

脱脂牛初乳用浓度为6 mol/L的盐酸调节其pH至4.0,以5 000 r/m in离心分离20 m in除去酪蛋白制得乳清,缓慢滴加浓度为1 mol/L的氢氧化钠回调pH至6.8;处理后的乳清经截留相对分子质量为50 000的组分并超滤稀释后,再经过阴阳离子交换串联色谱,乳铁蛋白(Lf)吸附于CM Sepharose Fast F low,免疫球蛋白G(Immunoglobu lin G,IgG)吸附于DEAE Sepharose Fast F low;阳离子交换柱用c(NaC l)=0.27 mol/L、0.85 mol/L的水溶液阶跃洗脱,得到色谱纯度为96.6%的Lf产品,阴离子交换柱用c(NaC l)=17 mmol/L、51 mmol/L的水溶液阶跃洗脱,得到色谱纯度为95%的IgG产品。     

Separation and Purification of Sinigrin and Gluconapin from Defatted Indian Mustard Seed Meals by Macroporous Anion Exchange Resin and Medium Pressure Liquid Chromatography

Defatted seed meals, the byproducts of Indian mustard seed oil industry, which are just used as animal feedstock and nitrogen fertilizers, possess various nutrients and phytochemical compounds, such as sinigrin and gluconapin. The present study demonstrated a novel, low-cost, and efficient method for the co-production of sinigrin and gluconapin from defatted Indian mustard seed meals by D261 strong basic anion-exchange macroporous resin and medium pressure liquid chromatography (MPLC). The adsorption/desorption capacities of eight adsorbents characterized by BET, IR, and EDS were screened. 26.4 g defatted Indian mustard seed meals, which contained about 1210.3 mg sinigrin and 696.6 mg gluconapin, could produce 2059.6 mg of glucosinolate-rich extract of 53.67% sinigrin and 31.30% gluconapin after separation by D261 resin. Then, 949.40 mg of 97% sinigrin and 568.40 mg of 95% gluconapin could be obtained after the extracts were purified by MPLC, with the recovery of 76.03% of sinigrin and 77.38% of gluconapin. The products were assessed by analytical HPLC and characterized by UV, MS, ¹H NMR, and ¹³C NMR. In conclusion, this method is practical and environmentally friendly, and it is a low-cost process to make full use of Indian mustard seeds.     

Separation of Gallium and Indium Isotopes by Cation and Anion Exchange Chromatography

Ion exchange chromatography was applied to study chemical isotope effects of gallium and indium in ligand exchange reactions. A strongly acidic cation and a strongly basic anion exchange resin were used as a solid phase, and aqueous HCl as a liquid phase. On the cation exchanger, the light isotope ⁶⁹Ga was enriched at the front part of the elution band and the heavy isotope ⁷¹Ga at the end part. Instead, the light ¹¹³In isotope was enriched at the end part, and the heavy isotope ¹¹⁵In at the front part. The isotope separation factor ? is equal to 3.3?×?10^–5 for gallium and 2.0?×?10^–4 for indium. On the anion exchanger, the heavy gallium isotope was enriched at the front part, whereas the heavy indium isotope at the end part of the band, with ? equal to ～10^–3 and 1.7?×?10^–4, respectively. This pattern of enrichment is caused by stronger Ga³⁺–OH₂ than Ga³⁺–Cl^? bond, and by inverse order of bond strength for indium. In the displacement method, gallium and indium on anion exchanger also show opposite enrichment of their isotopes, but the ? values (1.5?×?10^–2 for gallium and 5?×?10^–3 for indium) are greater than those found in the elution method, probably due to much higher concentrations of the metals.     

Mixed-Beds of Strong and Weak Anion Exchange Resins for Protein Separations with Step-Induced pH Gradients

A new way of performing pH gradient-based protein separations using a mixed-bed column comprising a small-pore weak base resin and a large-pore strong base resin and a step change in pH at the column entrance is introduced. The approach effectively decouples intracolumn generation of pH gradients, which ensues from ionic interactions with the weak base resin, from protein separation, which ensues from interactions with the strong base resin ligands, resulting in a process that is more easily predictable, less prone to the effects of protein loading on the pH gradient, less susceptible to hydrophobic interactions between protein and resin surface, and more flexible. The approach is demonstrated successfully using a tertiary amine resin as the weak base component together with a range of commercial strong anion exchangers with quaternary amine functionality. A predictive model, based on the potentiometric titration of the weak base resin, is in excellent agreement with experimentally determined pH profiles obtained using mixed-bed columns and amine buffers with pH steps from 9 to 7. The separation of protein charge variants with the mixed-bed concept is demonstrated using these columns for a deamidated monoclonal antibody mixture, which yielded highly homogeneous fractions.     

阴离子交换高效液相色谱法同时分离和分析三种氨基羧酸

建立了利用阴离子交换高效液相色谱同时分离分析甘氨酸、亚氨基二乙酸和氨三乙酸的方法。色谱柱为Sax强阴离子交换柱,用0.01 mol/L磷酸二氢钾缓冲液(pH=2.2)为流动相,紫外检测器波长为210 nm,对样品进行分析,并用外标法对这三种氨基羧酸进行定量。结果表明甘氨酸、亚氨基二乙酸和氨三乙酸分别在0.1024~2.0484 g/L、0.1006~2.0119 g/L、0.0050~0.3009g/L范围内线性关系良好(r>0.9995)。此法具有灵敏度高,线性相关系数和重现性好,流动相成本较低等特点。     

Optimal Integration of Directly Combined Hydrophobic Interaction and Ion Exchange Chromatography Purification Processes

Hydrophobic interaction and ion exchange chromatography are basic steps in purification of fermentative biopharmaceuticals. An optimization by statistical design of experiments requires a huge amount of feed. An alternative approach is the combination of model parameter determination using small scale experimental model parameter determination (1‐mL columns) and rigorous process modeling. Applicability for the prediction of the separation of a fermentation mixture of CHO mammalian cell culture is validated and hence IgG is purified from cell culture supernatant. Hydrophobic interaction chromatography directly combined with ion exchange chromatography is optimized. Any direct integration of those two main unit operations in purification processes is a methodological first step towards total process optimization. The potential for cost reduction and overall yield improvement is demonstrated and this leads to the conclusion that single step optimization is a feigned and not a real optimum.     

从阴树脂氯化母液中回收氧化锌技术

介绍了氯化母液中回收制取氧化锌的具体办法。     

Predicting Retention and Resolution of Protein Charge Variants in Mixed-Beds of Strong and Weak Anion Exchange Resins with Step-Induced pH Gradients

A model is developed to predict protein retention and resolution in mixed-bed columns containing a mixture of a small-pore weak base resin and a large-pore strong base resin. Using amine buffers, the weak base resin generates smooth pH gradients when the column is subjected to pH steps, while the strong base resin interacts with the proteins effecting the separation. The model describes simultaneously ion exchange interaction on the weak base resin and protein adsorption and transport on the strong base resin. The separation of a monoclonal antibody (mAb) from a charge variant whose pI is about 0.2 pH units lower is studied experimentally with the anion exchange resins AG 4-X4 and SOURCE 30Q as the weak and strong base components, respectively. The properties of each resin are characterized independently and model parameters are obtained experimentally for each. Model predictions of pH gradient-based separations in columns containing different proportions of the two resins and at different buffer concentrations are found to be in agreement with experimental results. The model can thus be used as a process design tool to predict combinations of buffer and mixed-bed compositions, column lengths, and mobile phase velocities that would yield a desired separation performance.