Hubbuch Jürgen, Linden Thomas, Knieps Esther, Thömmes Jörg, Kula Maria-Regina
Institut für Enzymtechnologie, Heinrich-Heine Universität Düsseldorf, 52426 Julich, Germany.
J Chromatogr A. 2003 Dec 22;1021(1-2):105-15. doi: 10.1016/j.chroma.2003.08.092.
The impact of different transport mechanism on chromatographic performance was studied by confocal laser scanning microscopy (CLSM) for solutions containing bovine serum albumin (BSA) and monoclonal IgG 2a under different solid- and fluid-phase conditions. During this investigation, a clear influence of the uptake mechanism on the affinity of the respective proteins for the different adsorbents and thus separation performance of the chromatographic process could be observed. For the system SP Sepharose Fast Flow at pH 4.5 pore diffusion could be ascribed to be the dominant transport mechanism for both proteins and the adsorption profiles resembled a pattern similar to that described by the 'shrinking core' model. Under these conditions a significantly higher affinity towards the adsorbent was found for BSA when compared to IgG 2a. With changing fluid- and solid-phase conditions, however, a change of the transport mode for IgG 2a could be detected. While the exact mechanism is still unresolved it could be concluded that both occurrence and magnitude of the now governing transport mechanism depended on protein properties and interaction with the adsorbent surface. For the system SP Sepharose XL at pH 5.0 both parameters leading to the change in IgG 2a uptake were combined resulting in a clear change of the system affinity towards the IgG 2a molecule, while BSA adsorption was restricted to the most outer shell of the sorbent.
利用共聚焦激光扫描显微镜（CLSM），研究了在不同固液相条件下，含牛血清白蛋白（BSA）和单克隆IgG 2a的溶液中不同传输机制对色谱性能的影响。在此研究过程中，可以观察到摄取机制对相应蛋白质与不同吸附剂的亲和力以及色谱过程分离性能有明显影响。对于pH 4.5的SP Sepharose Fast Flow系统，孔扩散可被认为是两种蛋白质的主要传输机制，吸附曲线类似于“收缩核”模型所描述的模式。在这些条件下，与IgG 2a相比，发现BSA对吸附剂的亲和力明显更高。然而，随着固液相条件的变化，可以检测到IgG 2a传输模式的改变。虽然确切机制仍未解决，但可以得出结论，现在起主导作用的传输机制的出现和程度取决于蛋白质性质以及与吸附剂表面的相互作用。对于pH 5.0的SP Sepharose XL系统，导致IgG 2a摄取变化的两个参数相结合，导致系统对IgG 2a分子的亲和力明显变化，而BSA吸附仅限于吸附剂的最外层。
