| Autor: |
Nayak N; LAQV-REQUIMTE, Department of Chemistry, NOVA School of Science and Technology, FCT NOVA, Universidade NOVA de Lisboa, 2829-516 Caparica, Portugal.; Institute on Membrane Technology, National Research Council, ITM-CNR, Via P. Bucci, 17/C, I87030 Rende, Italy., Mazzei R; Institute on Membrane Technology, National Research Council, ITM-CNR, Via P. Bucci, 17/C, I87030 Rende, Italy., Giorno L; Institute on Membrane Technology, National Research Council, ITM-CNR, Via P. Bucci, 17/C, I87030 Rende, Italy., Crespo JG; LAQV-REQUIMTE, Department of Chemistry, NOVA School of Science and Technology, FCT NOVA, Universidade NOVA de Lisboa, 2829-516 Caparica, Portugal., Portugal CAM; LAQV-REQUIMTE, Department of Chemistry, NOVA School of Science and Technology, FCT NOVA, Universidade NOVA de Lisboa, 2829-516 Caparica, Portugal., Poerio T; Institute on Membrane Technology, National Research Council, ITM-CNR, Via P. Bucci, 17/C, I87030 Rende, Italy. |
| Abstrakt: |
This work aims at understanding the attachment mechanisms and stability of proteins on a chromatography medium to develop more efficient functionalization methodologies, which can be exploited in affinity chromatography. In particular, the study was focused on the understanding of the attachment mechanisms of bovine serum albumin (BSA), used as a ligand model, and protein G on novel amine-modified alumina monoliths as a stationary phase. Protein G was used to develop a column for antibody purification. The results showed that, at lower protein concentrations (i.e., 0.5 to 1.0 mg·mL -1 ), protein attachment follows a 1st-order kinetics compatible with the presence of covalent binding between the monolith and the protein. At higher protein concentrations (i.e., up to 10 mg·mL -1 ), the data preferably fit a 2nd-order kinetics. Such a change reflects a different mechanism in the protein attachment which, at higher concentrations, seems to be governed by physical adsorption resulting in a multilayered protein formation, due to the presence of ligand aggregates. The threshold condition for the prevalence of physical adsorption of BSA was found at a concentration higher than 1.0 mg·mL -1 . Based on this result, protein concentrations of 0.7 and 1.0 mg·mL -1 were used for the functionalization of monoliths with protein G, allowing a maximum attachment of 1.43 mg of protein G/g of monolith. This column was then used for IgG binding-elution experiments, which resulted in an antibody attachment of 73.5% and, subsequently, elution of 86%, in acidic conditions. This proved the potential of the amine-functionalized monoliths for application in affinity chromatography. |
