Peterka M, Jarc M, Banjac M, Frankovic V, Bencina K, Merhar M, Gaberc-Porekar V, Menart V, Strancar A, Podgornik A
BIA Separations d.o.o., Teslova 30, SI-1000 Ljubljana, Slovenia.
J Chromatogr A. 2006 Mar 17;1109(1):80-5. doi: 10.1016/j.chroma.2005.08.057. Epub 2005 Sep 12.
Monoliths are attractive stationary phases for purification of large biomolecules like proteins because of their flow-unaffected properties. Isolation of histidine containing proteins to high purity can be efficiently performed using metal-chelate interactions within a single chromatographic step. In this work, we investigated properties of commercial metal-chelate methacrylate monoliths-Convective Interaction Media (CIM). Analytical CIM disk monolithic columns and CIM 8 ml monolithic columns were used for purification of tumor necrosis factor-alpha (TNF-alpha) analog LK-801 and green fluorescence protein with 6 histidine tag (GFP-6His). In both cases, purity over 90% was achieved. Dynamic binding capacity at 10% of breakthrough was around 17-18 mg/ml for LK-801 and around 30 mg/ml for GFP-6His. Adsorption isotherm revealed that the maximal capacity is achieved at protein concentration above 60 microg/ml. Dynamic binding capacity and resolution were found to be flow unaffected.
整体柱因其不受流动影响的特性，是用于纯化蛋白质等大型生物分子的有吸引力的固定相。利用单一色谱步骤中的金属螯合相互作用，可以高效地将含组氨酸的蛋白质分离至高纯度。在这项工作中，我们研究了商业金属螯合甲基丙烯酸酯整体柱——对流相互作用介质（CIM）的特性。使用分析型CIM圆盘整体柱和CIM 8 ml整体柱纯化肿瘤坏死因子-α（TNF-α）类似物LK-801和带有6个组氨酸标签的绿色荧光蛋白（GFP-6His）。在这两种情况下，均实现了纯度超过90%。对于LK-801，10%穿透率时的动态结合容量约为17 - 18 mg/ml，对于GFP-6His约为30 mg/ml。吸附等温线表明，在蛋白质浓度高于60 μg/ml时达到最大容量。发现动态结合容量和分离度不受流动影响。
