Abstract
Affinity capture represents an important step in downstream processing of proteins and it is conventionally performed through a chromatographic process. The performance of this step highly depends on the type of matrix employed. In particular, resin beads and convective materials, such as membranes and monoliths, are the commonly available supports. The present work deals with non-competitive binding of bovine serum albumin (BSA) on different chromatographic media functionalized with Cibacron Blue F3GA (CB). The aim is to set up the development of the purification process starting from the lab-scale characterization of a commercially available CB resin, regenerated cellulose membranes and polymeric monoliths, functionalized with CB to identify the best option. The performance of the three different chromatographic media is evaluated in terms of BSA binding capacity and productivity. The experimental investigation shows promising results for regenerated cellulose membranes and monoliths, whose performance are comparable with those of the packed column tested. It was demonstrated that the capacity of convective stationary phases does not depend on flow rate, in the range investigated, and that the productivity that can be achieved with membranes is 10 to 20 times higher depending on the initial BSA concentration value, and with monoliths it is approximately twice that of beads, at the same superficial velocity.
Highlights
Selectivity, versatility and efficiency make chromatography a widely used technique for protein purification and separation, due to the great number of possible chemical interactions that intervene between the target biomolecule and the stationary phase and to the availability of many adsorbent materials
It was demonstrated that the capacity of convective stationary phases does not depend on flow rate, in the range investigated, and that the productivity that can be achieved with membranes is 10 to 20 times higher depending on the initial bovine serum albumin (BSA) concentration value, and with monoliths it is approximately twice that of beads, at the same superficial velocity
An experimental study regarding BSA capture was performed through affinity chromatography An experimental study regarding BSA capture was performed through affinity chromatography using different chromatographic media functionalized with Cibacron Blue F3GA ligand
Summary
Selectivity, versatility and efficiency make chromatography a widely used technique for protein purification and separation, due to the great number of possible chemical interactions that intervene between the target biomolecule and the stationary phase and to the availability of many adsorbent materials. Among the different kind of chromatographic separations usually needed for recovery and purification of a particular molecule, the main step in downstream processing is represented by the affinity capture. It allows the selective and efficient recovery of the target molecule thanks to a specific and reversible interaction between a ligand, immobilized on the support material, and the product itself. Columns packed with chromatographic resins exhibit a good performance in terms of binding capacity, thanks to their high surface area, but the functionalized materials are usually very expensive and several limitations, Membranes 2020, 10, 1; doi:10.3390/membranes10010001 www.mdpi.com/journal/membranes
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