(204g) BSA Purification with Continuous Annular Chromatography

Bovine serum albumin (BSA) is an indispensable ingredient of the test kits and a major compound of the tissue culture medium. Recently, serum albumins are experiencing an increase interest from the standpoint of preparative chromatographic separations. Conventional preparative elution chromatography is a batch process. In order to overcome the batch process drawbacks, continuous chromatography is enjoying an important share in the preparative chromatography applications. The continuous annular chromatography (CAC) is a potential and promising process intensified technology that allows large-scale continuous preparative chromatographic separation and purification of multi-component mixtures. Improvement of the purification of BSA via CAC is the focus of this work. In chromatographic simulation and modeling studies, the complexity of the problem increases if the usually assumed equilibrium-dispersive condition, which neglects all transient resistances, is not invoked. However when mass transfer resistances are abundant the assumption of equilibrium-dispersive condition might become inconsistent with the system of interest. In the modeling phase of this work, the non-equilibrium methodology [1] is applied to present CAC modeling, so as to understand the contributions of mass transfer resistances on the elution profiles, and thereby providing the efficient and reliable simulation profiles. It was concluded that in order to reach a realistic CAC dynamics portrait the inclusion of non-equilibrium constraints in the modeling studies, as separate identities, is essential. In the experimental phase, bovine blood is collected from Eskisehir (Turkey) slaughterhouse and bovine serum is prepared in situ by successive de-fibrinating, centrifuging, filtration steps. The so prepared serum was stored in a deepfreeze at ?20 C. Prior to CAC treatment, bovine serum is thawed at room temperature, and filtered through a series of filters, using 0.2 µm poliethersulphon (PES) catridge filter as the final filter. For further concentration and purification of BSA, 500 KDa membrane-based tangential flow ultrafiltration is carried out. The bovine serum filtrate is then introduced to the CAC system of our design for continuous chromatographic purification where highly cross-linked spherical agarose (GE Healthcare) is filled to the annulus. The eluents was 20μM Tris-HCI buffer solution (pH 7). Both HPLC and FTIR characterization studies indicated that CAC is a very effective continuous chromatographic technology for obtaining up to 97 % BSA purity. It was demonstrated that careful selection of operation parameters is needed for an efficient high level purification and recovery of BSA where the presently used CAC model of ours might save considerable time for the optimization of chromatographic purification strategy during the employment of CAC apparatus. Reference: [1] Özdural, A.R., Alkan, A., Kerkhof, P.A.J.M., ?Modeling chromatographic columns: Non-equilibrium packed-bed adsorption with non-linear adsorption isotherms?, Journal of Chromatography A, 1041, 77-85, 2004.