Staby Arne, Sand Maj-Britt, Hansen Ronni G, Jacobsen Jan H, Andersen Line A, Gerstenberg Michael, Bruus Ulla K, Jensen Inge Holm
Novo Nordisk A/S, Health Care Discovery and Development, Protein Separation, Hagedornsvej 1, DK-2820 Gentofte, Denmark.
J Chromatogr A. 2005 Mar 25;1069(1):65-77. doi: 10.1016/j.chroma.2004.11.094.
A comparative study was performed on heparin resins and strong and weak cation exchangers to investigate the pH dependence, efficiency, binding strength, particle size distribution, static and dynamic capacity, and scanning electron microscopy pictures of chromatographic resins. The resins tested include: Heparin Sepharose FF, SP Sepharose FF, CM Sepharose FF, Heparin Toyopearl 650 m, SP Toyopearl 650 m, CM Toyopearl 650 m, Ceramic Heparin HyperD M, Ceramic S HyperD 20, and Ceramic CM HyperD F. Testing was performed with four different proteins: anti-FVII Mab (IgG), aprotinin, lysozyme, and myoglobin. Dependence of pH on retention was generally very low for proteins with high isoelectric point (pI), though some decrease of retention with increasing pH was observed for CM Ceramic HyperD F and S Ceramic HyperD 20. Binding of anti-FVII Mab with pI < 7.5 was observed on several resins at pH 7.5. Efficiency results show the expected trend of increasing dependence of the plate height with increasing flow rate of Ceramic HyperD resins followed by Toyopearl 650 m resins and the highest flow dependence of the Sepharose FF resins corresponding to their pressure resistance. Determination of particle size distribution by two independent methods, coulter counting and SEM, was in good agreement. Binding strength of cation-exchange resins as a function of ionic strength varies depending on the protein. Binding and elution at high salt concentration may be performed with Ceramic HyperD resins, while binding and elution at low salt concentration may be performed with model proteins on heparin resins. Employing proteins with specific affinity for heparin, a much stronger binding is observed, however, some cation exchangers may still be good substitutions for heparin resins. Dynamic capacity at 10% breakthrough compared to static capacity measurements and dynamic capacity displays that approximately 40-80% of the total available capacity is utilized during chromatographic operation depending on flow rate. A general good agreement was obtained between results of this study and data obtained by others. Results of this study may be used in the selection of resins for testing during protein purification process development.
对肝素树脂以及强、弱阳离子交换剂进行了一项比较研究，以考察色谱树脂的pH依赖性、效率、结合强度、粒径分布、静态和动态容量以及扫描电子显微镜图像。所测试的树脂包括：肝素琼脂糖凝胶FF、SP琼脂糖凝胶FF、CM琼脂糖凝胶FF、肝素东曹650m、SP东曹650m、CM东曹650m、陶瓷肝素HyperD M、陶瓷S HyperD 20以及陶瓷CM HyperD F。使用四种不同的蛋白质进行测试：抗FVII单克隆抗体（IgG）、抑肽酶、溶菌酶和肌红蛋白。对于等电点（pI）较高的蛋白质，pH对保留时间的依赖性通常非常低，不过对于CM陶瓷HyperD F和S陶瓷HyperD 20，观察到随着pH升高保留时间有所下降。在pH 7.5时，在几种树脂上观察到pI < 7.5的抗FVII单克隆抗体的结合。效率结果显示出预期的趋势，即陶瓷HyperD树脂的板高对流速的依赖性随着流速增加而增大，其次是东曹650m树脂，而琼脂糖凝胶FF树脂对流速的依赖性最高，这与其耐压性相对应。通过库尔特计数和扫描电子显微镜这两种独立方法测定的粒径分布结果吻合良好。阳离子交换树脂的结合强度作为离子强度的函数，因蛋白质而异。高盐浓度下的结合和洗脱可使用陶瓷HyperD树脂进行，而低盐浓度下的结合和洗脱可使用肝素树脂上的模型蛋白质进行。使用对肝素有特异性亲和力的蛋白质时，观察到结合更强，然而，一些阳离子交换剂仍可能是肝素树脂的良好替代品。与静态容量测量相比，10%穿透率时的动态容量以及动态容量显示，根据流速不同，在色谱操作过程中约40 - 80%的总可用容量被利用。本研究结果与其他人获得的数据总体吻合良好。本研究结果可用于在蛋白质纯化工艺开发过程中选择用于测试的树脂。
