Kong Darryl Yc, Gerontas Spyridon, McCluckie Ross A, Mewies Martin, Gruber David, Titchener-Hooker Nigel J
Department of Biochemical Engineering University College London UK.
Ipsen Limited Wrexham UK.
J Chem Technol Biotechnol. 2018 Jul;93(7):1959-1965. doi: 10.1002/jctb.5411. Epub 2017 Oct 9.
Poorly packed chromatography columns are known to reduce drastically the column efficiency and produce broader peaks. Controlled bed compression has been suggested to be a useful approach for solving this problem. Here the relationship between column efficiency and resolution of protein separation are examined when preparative chromatography media were compressed using mechanical and hydrodynamic methods. Sepharose CL-6B, an agarose based size exclusion media was examined at bench and pilot scale. The asymmetry and height equivalent of a theoretical plate (HETP) was determined by using 2% v/v acetone, whereas the void volume and intraparticle porosity (ϵ ) were estimated by using blue dextran. A protein mixture of ovalbumin (chicken), bovine serum albumin (BSA) and γ'- globulin (bovine) with molecular weights of 44, 67 and 158 kDa, respectively, were used as a 'model' separation challenge.
Mechanical compression achieved a reduction in plate height for the column with a concomitant improvement in asymmetry. Furthermore, the theoretical plate height decreased significantly with mechanical compression resulting in a 40% improvement in purity compared with uncompressed columns at the most extreme conditions of compression used.
The results suggest that the mechanical bed compression of Sepharose CL-6B can be used to improve the resolution of protein separation. © 2017 The Authors. published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.
众所周知，装填不佳的色谱柱会大幅降低柱效并产生更宽的峰。有人提出控制床层压缩是解决这一问题的有效方法。本文研究了使用机械和流体动力学方法压缩制备型色谱介质时，柱效与蛋白质分离分辨率之间的关系。在实验室规模和中试规模下对基于琼脂糖的尺寸排阻介质Sepharose CL-6B进行了研究。使用2% v/v丙酮测定理论塔板的不对称性和理论塔板高度等效值（HETP），而使用蓝色葡聚糖估算空隙体积和颗粒内孔隙率（ϵ）。分别使用分子量为44、67和158 kDa的卵清蛋白（鸡）、牛血清白蛋白（BSA）和γ'-球蛋白（牛）的蛋白质混合物作为“模型”分离挑战物。
机械压缩使色谱柱的塔板高度降低，同时不对称性得到改善。此外，在使用的最极端压缩条件下，与未压缩的色谱柱相比，机械压缩使理论塔板高度显著降低，纯度提高了40%。
结果表明，Sepharose CL-6B的机械床层压缩可用于提高蛋白质分离的分辨率。© 2017作者。由John Wiley & Sons Ltd代表化学工业协会出版。
