Semi-continuous protein fractionating using Affinity Cross-Flow Filtration

Protein purification by means of downstream processing is increasingly important. At the University of Twente a semi-continuous process is developed for the isolation of BSA out of crude protein mixtures. For this purpose an automated Affinity Cross-Flow Filtration, ACFF, process is developed. This process combines the advantages of both affinity adsorption, having high resolution, and microfiltration, for rapid and large continuous processing capacity. The overall process contains 4 sequential steps: affinity binding; purification; dissociation; regeneration. In the first step binding of the desired protein to a tailor-made ligand-immobilized particle is established. This temporary fixation of target proteins on a large entity allows us to wash out the unbound components in the purification step using high flux microfiltration membranes. After washing out the unbound proteins the target protein-ligand immobilized particle complex is dissociated by adding an appropriate salt. In a second filtration cycle the purified protein can be collected as the permeate. In the last step the ligand-immobilized particle is regenerated for the next cycle with buffer. Using this process it was possible to obtain the target protein BSA with a purity of more than 95%.