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Protein Attachment Mechanism for Improved Functionalization of Affinity Monolith Chromatography (AMC)
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 mec...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9323659/ https://www.ncbi.nlm.nih.gov/pubmed/35889369 http://dx.doi.org/10.3390/molecules27144496 |
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author | Nayak, Nayan Mazzei, Rosalinda Giorno, Lidietta Crespo, João G. Portugal, Carla A. M. Poerio, Teresa |
author_facet | Nayak, Nayan Mazzei, Rosalinda Giorno, Lidietta Crespo, João G. Portugal, Carla A. M. Poerio, Teresa |
author_sort | Nayak, Nayan |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-9323659 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-93236592022-07-27 Protein Attachment Mechanism for Improved Functionalization of Affinity Monolith Chromatography (AMC) Nayak, Nayan Mazzei, Rosalinda Giorno, Lidietta Crespo, João G. Portugal, Carla A. M. Poerio, Teresa Molecules Article 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. MDPI 2022-07-14 /pmc/articles/PMC9323659/ /pubmed/35889369 http://dx.doi.org/10.3390/molecules27144496 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Nayak, Nayan Mazzei, Rosalinda Giorno, Lidietta Crespo, João G. Portugal, Carla A. M. Poerio, Teresa Protein Attachment Mechanism for Improved Functionalization of Affinity Monolith Chromatography (AMC) |
title | Protein Attachment Mechanism for Improved Functionalization of Affinity Monolith Chromatography (AMC) |
title_full | Protein Attachment Mechanism for Improved Functionalization of Affinity Monolith Chromatography (AMC) |
title_fullStr | Protein Attachment Mechanism for Improved Functionalization of Affinity Monolith Chromatography (AMC) |
title_full_unstemmed | Protein Attachment Mechanism for Improved Functionalization of Affinity Monolith Chromatography (AMC) |
title_short | Protein Attachment Mechanism for Improved Functionalization of Affinity Monolith Chromatography (AMC) |
title_sort | protein attachment mechanism for improved functionalization of affinity monolith chromatography (amc) |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9323659/ https://www.ncbi.nlm.nih.gov/pubmed/35889369 http://dx.doi.org/10.3390/molecules27144496 |
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