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Detailed analysis of membrane adsorber pore structure and protein binding by advanced microscopy.

Detailed analysis of membrane adsorber pore structure and protein binding by advanced microscopy.

J.Wang, F. Dismer, J. Hubbuch and M. Ulbricht,


Journal of Membrane Science, Vol. 320, Pages 456-467

Datum: 2008

Commercial Sartobind® porous cation exchanger membranes, based on stabilized regenerated cellulose and with sulfonic acid (S) or carboxylic acid groups (C), were analysed with respect to their pore structure in dry, slightly swollen and wet state by three microscopic methods, conventional scanning electron microscopy (SEM), environmental SEM (ESEM), and confocal laser scanning microscopy (CLSM). The dehydration behaviour of the membranes was in situ observed at varied vapour pressure in the chamber of the ESEM, indicating some deformations of the macropore structure (largest pore diameters up to 20 μm) and significant changes in dimension and mobility of smaller cellulose fibers within these macropores, both as function of water content of the membrane. The binding of mono-Cy5-labelled lysozyme inside fluoresceine-labelled and unlabelled Sartobind® membranes was monitored by CLSM. The characteristic fluorescence intensity distributions in areas of (146 μm × 146 μm) indicated that protein binding takes place predominately in a layer which is anchored to a fine cellulose fiber network and, to a lower degree, directly to thick cellulose fibers. Due to the limited thickness of this binding layer, a significant fraction of the macropores remained free of protein. Protein binding as function of concentration and incubation times was also monitored by CLSM and discussed related to the binding isotherms for the membrane Sartobind® S and C. Further, a flow-through cell for the in situ monitoring with CLSM of protein binding during the binding step was built, and the results obtained for binding of lysozyme in membranes Sartobind® S indicate this experiment can give very important information on the dynamic behaviour of porous membrane adsorbers during separation: the lateral microscopic resolution in the x, y plane enables the identification of different breakthrough times as function of the location (pore structure), and this information can help to explain possible reasons for axial dispersion (in z-direction) observed in breakthrough analyses of the same separation in a chromatography system. The combination of advanced microscopy with detailed investigations of static and dynamic protein binding will provide a better understanding of the coupling between mass transfer and reversible binding in membrane adsorbers onto separation performance, and it will provide valuable guide-lines for the development of improved membrane adsorbers.