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.
Molecules. 2022 Jul 14;27(14):4496. doi: 10.3390/molecules27144496.
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), 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), 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. Based on this result, protein concentrations of 0.7 and 1.0 mg·mL 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.
本工作旨在了解蛋白质在色谱介质上的附着机制和稳定性，以开发更有效的功能化方法，这些方法可用于亲和色谱。特别是，本研究集中于理解牛血清白蛋白（BSA）作为配体模型和新型胺修饰氧化铝整体柱作为固定相的附着机制，蛋白 G 用于开发抗体纯化柱。结果表明，在较低的蛋白质浓度（即 0.5 至 1.0mg·mL）下，蛋白质附着遵循一级动力学，与整体柱和蛋白质之间存在共价键合一致。在较高的蛋白质浓度（即高达 10mg·mL）下，数据更适合二级动力学。这种变化反映了蛋白质附着的不同机制，在较高浓度下，由于配体聚集，似乎由物理吸附控制，导致形成多层蛋白质。BSA 物理吸附占主导地位的阈值条件在浓度高于 1.0mg·mL 时被发现。基于这一结果，选择蛋白质浓度为 0.7 和 1.0mg·mL 用于蛋白 G 对整体柱的功能化，允许最大附着 1.43mg 蛋白 G/g 整体柱。然后，该柱用于 IgG 结合洗脱实验，结果在酸性条件下，抗体的附着率为 73.5%，随后洗脱率为 86%。这证明了胺功能化整体柱在亲和色谱中的应用潜力。
