Chemical modification of membranes with ligands that have affinity and selectivity for the target protein offers potentially advantageous alternatives for protein purification over traditional chromatographic processes based on microbead packed columns. The industrial processes have a clear preference for traditional chromatographic techniques, due to lack of availability of appropriately modified or end-group modifiable membranes. But more importantly, it can be attributed to a lack of understanding of factors that affect the protein binding capacity and selectivity of such membranes. We have been involved with trying to understand the mechanism of action of protein binding that could affect membrane performance in bioseparations (Figure 1). Here we describe a fast, economic, and effective approach to study the protein binding selectivity of affinity membranes. Cellulose acetate membranes (5.0μm) were chemically modified with para-aminobenzamidine (pABA) to obtain an amidine as the end group that binds the active site of the protein of interest (trypsin type serine proteases). A range of ligand densities were obtained on membrane surface by adding different amounts of epichlorohydrin as the spacer arm and resulting amidine was quantified with the glyoxal method. The binding capacity and selectivity for the protein of interest were tested using fluorescently labeled trypsin, lysozyme and pancreatic lipase. This research provides a valuable insight towards the understanding of the efficacy of affinity membranes as bioseparation devices and promotes membrane-based processes in industrial separations.

Protein binding capacity and selectivity of chemically modified cellulose membranes

ACS Fall 2020 Virtual Meeting

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