Understanding ion exchange chromatography adsorption mechanisms under different conditions

• Autor(a) principal: Cardoso, João Carlos Simões
• Data de Publicação: 2016
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10400.6/6986
• topic: Mecanismo de Adsorção; Microcalorimetria de Fluxo; Seroalbumina Bovina; Troca Iónica; Domínio/Área Científica::Engenharia e Tecnologia::Biotecnologia

Nowadays, protein purification is typically the most laborious and more expensive aspect of a biopharmaceutical process. Ion exchange chromatography is probably the most used method in protein purification due to displaying the highest binding capacities for these biomolecules. Protein adsorption onto any surface is a complex process that is controlled by a number of subprocesses with synergistic and antagonistic effects of different types of forces. Investigation of the events involved in protein adsorption promotes knowledge development on protein and surfaces behavior. Flow microcalorimetry (FMC) has proven to be an effective approach to real-time heat signal measurements of adsorption and desorption events occurring inside a chromatographic system. Therefore, it allows a better understanding of the forces that drive the adsorption process without interfering with the system like the majority of used techniques. The present study tries to elucidate and compare the mechanism of protein adsorption, namely bovine serum albumin, onto several commercially available charged resins using the FMC technique. The studied anion exchangers were Toyopearl DEAE 650M, Toyopearl GigaCap Q-650M, and TSKgel SuperQ 5PW. Being ligand attachment technology one of the differences between them. Also, although being a cation exchanger and having the same charge as BSA under the working conditions, TSKgel SP 5PW was used expecting a weaker primary interaction between BSA and the resin in order to evaluate the thermodynamic differences and to try to understand weaker forces of the inherited mechanism. Flow microcalorimetry data coupled with equilibrium binding isotherms illustrated a series of chronological events that occurred during BSA adsorption onto the used resins, including resin and protein dehydration, protein conformational alterations, protein activated jump, chain delivery, and desorption. These events were found to have different magnitudes when comparing the anion exchangers in spite of showing the same heat profile. In “non-binding conditions”, i.e., with TSKgel SP 5PW negative ligands and negative net protein charge, the thermogram profile completely changed. However, in the presence of 50 mM NaCl, the observed peak behavior was similar to those observed with anion exchangers. The studied anion exchangers and TSKgel SP with 50 mM NaCl presented positive heat enthalpies when the protein was flowing through the FMC cell. Hence, the mechanism of adsorption was considered to be entropically driven, which gives much more importance to counter-ion and water molecules role in this complex process. On the other hand, with TSKgel SP in the absence of salt, the adsorption net heat is negative, meaning that in this case adsorption is enthalpically driven. All things considered, this work consisted on another step for the elucidation of the complex protein adsorption process. Theoretical, empirical models and future computational simulations of adsorption equilibrium should account for these complex effects, as well as the primary interactions.