Aumann Lars, Morbidelli Massimo
ETH Zurich, Institute for Chemical and Bioengineering, Department of Chemistry and Applied Biosciences, Hoenggerberg, HCI, 8093 Zurich, Switzerland.
Biotechnol Bioeng. 2007 Dec 1;98(5):1043-55. doi: 10.1002/bit.21527.
Biomolecules are often purified via solvent gradient batch chromatography. Typically suitable smooth linear solvent gradients are applied to obtain the separation between the desired component and hundreds of impurities. The desired product is usually intermediate between weakly and strongly adsorbing impurities, and therefore a central cut is required to get the desired pure product. The stationary phases used for preparative and industrial separations have a low efficiency due to strong axial dispersion and strong mass transfer resistances. Therefore a satisfactory purification often cannot be achieved in a single chromatographic step. For large scale productions and for very valuable molecules, countercurrent operation such as the well known SMB process, is needed in order to increase separation efficiency, yield and productivity. In this work a novel multicolumn solvent gradient purification process (MCSGP-process) is introduced, which combines two chromatographic separation techniques, which are solvent gradient batch and continuous countercurrent SMB. The process consists of several chromatographic columns, which are switched in position opposite to the flow direction. Most of the columns are equipped with a gradient pump to adjust the modifier concentration at the column inlet. Some columns are interconnected, so that non pure product streams are internally, countercurrently recycled. Other columns are short circuited and operate in batch mode. As a working example the purification of an industrial stream containing 46% of the hormone Calcitonin is considered. It is found that for the required purity the MCSGP unit achieves a yield close to 100% compared to a maximum value of a single column batch chromatography of 66%.
生物分子通常通过溶剂梯度间歇色谱法进行纯化。通常应用合适的平滑线性溶剂梯度来实现所需组分与数百种杂质之间的分离。所需产物通常处于弱吸附杂质和强吸附杂质之间，因此需要进行中心切割以获得所需的纯产物。由于强烈的轴向扩散和强大的传质阻力，用于制备和工业分离的固定相效率较低。因此，在单个色谱步骤中通常无法实现令人满意的纯化。对于大规模生产和非常有价值的分子，需要采用逆流操作，如著名的模拟移动床（SMB）工艺，以提高分离效率、产率和生产率。在这项工作中，引入了一种新型的多柱溶剂梯度纯化工艺（MCSGP工艺），它结合了两种色谱分离技术，即溶剂梯度间歇法和连续逆流SMB法。该工艺由几个色谱柱组成，这些色谱柱在与流动方向相反的位置进行切换。大多数色谱柱配备有梯度泵，以调节柱入口处的改性剂浓度。一些色谱柱相互连接，以便非纯产物流在内部进行逆流循环。其他色谱柱则短路并以间歇模式运行。作为一个工作示例，考虑对含有46%降钙素激素的工业流进行纯化。结果发现，对于所需的纯度，MCSGP装置的产率接近100%，而单柱间歇色谱法的最大值为66%。
