Clemmitt R H, Bruce L J, Chase H A
Department of Chemical Engineering, University of Cambridge, United Kingdom.
Bioseparation. 1999;8(1-5):53-67.
The use of a rapid chromatographic assay to monitor the level of a specific protein during its downstream processing by expanded bed adsorption is described. An expanded bed column (5 cm diameter) has been modified to allow the abstraction of liquid samples at various heights along the bed, in an automated, semi-continuous manner throughout the separation. The withdrawn samples were filtered in-line and the level of the target protein assayed by a rapid on-line chromatographic method. Using this technique it was possible to monitor the development of adsorbate profiles during the loading, washing and elution phases of the application of an unclarified feedstock. The potential of the technique is demonstrated using the separation of histidine tagged glutathione s-transferase (GST-(His)6) from an unclarified Escherichia coli homogenate using an expanded bed of Ni2+ loaded STREAMLINE Chelating. The level of GST-(His)6 in the abstracted homogenate samples was measured using Zn2+ loaded NTA-silica as an affinity chromatographic sensor. The approach described demonstrates potential for the on-line monitoring and control of expanded bed separations and for providing a greater understanding of adsorption/desorption and hydrodynamic processes occurring within the bed.
本文描述了一种利用快速色谱分析方法，在通过膨胀床吸附进行特定蛋白质下游加工过程中监测其水平的方法。一个膨胀床柱（直径5厘米）经过改进，以便能够在整个分离过程中以自动化、半连续的方式，从床层沿不同高度提取液体样品。取出的样品在线过滤，目标蛋白质的水平通过快速在线色谱方法进行测定。利用该技术，可以在应用未澄清原料的加载、洗涤和洗脱阶段监测吸附物分布的变化。通过使用装载Ni2+的STREAMLINE螯合膨胀床从未澄清的大肠杆菌匀浆中分离组氨酸标记的谷胱甘肽S-转移酶（GST-(His)6），证明了该技术的潜力。使用装载Zn2+的NTA-硅胶作为亲和色谱传感器，测定提取的匀浆样品中GST-(His)6的水平。所述方法显示了对膨胀床分离进行在线监测和控制以及更深入了解床层内发生的吸附/解吸和流体动力学过程的潜力。
