Optimization of a gellan gum support by experimental design for recombinant proteins partition

• Autor(a) principal: Frias, Filipe dos Santos
• Data de Publicação: 2015
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10400.6/5842
• topic: Cromatografia de Troca Iónica; Matriz Cromatográfica; Microesferas de Gelana; Purificação de Proteínas; Técnica de Emulsão Água-Em-Óleo; Domínio/Área Científica::Engenharia e Tecnologia::Engenharia Química

Chromatography is one of the most studied methods, due to its simplicity, versatility and high reproducibility, to separate and purify molecules that can have therapeutic, industrial and biotechnological interest. In recent years, the development of new chromatographic matrices has been continuously increased in order to afford rapid and efficient separations and decrease the use of resources. Gellan gum is a natural anionic exopolysaccharide and, in the presence of divalent cations, has the ability to form thermos-reversible strong gels resistant to temperature and extreme acidic conditions. In this work, it was proposed the preparation of gellan gum microbeads to be used as a stable chromatographic stationary phase. In order to produce the matrix, a low-cost water-in-oil emulsion technique was adopted. To obtain optimal conditions to the bead formulation, experimental design was applied, which allowed the optimization of the experimental conditions and to produce microbeads with the smallest diameter possible. Due to the negative charge of gellan gum, it was possible to study the interactions established with three model proteins (BSA, a-chymotrypsin and lysozyme) and with a therapeutic complex protein, SCOMT. In the model protein assays, MES buffer with pH 6.2 was used, which conferred negative charge to BSA and positive charge to a-chymotrypsin and lysozyme, due to its isoelectric points. Thus, BSA did not bind to the matrix while the other two proteins were retained to the gellan gum, being eluted with an increase of ionic strength. Regarding to prepurified SCOMT sample, a buffer with pH 4.0 was used under equilibrium conditions, conferring positive charge to the protein, thus, it also interacted with the column and was majorly eluted by pH manipulation (by changing the buffer pH to 6.4), allowing the elimination of some protein contaminants. Dynamic binding capacity assay of the gellan gum microbeads was made, in order to characterize this support as a novel chromatographic matrix. The values of DBC of the gellan gum stationary phase to 10 % and 50 % of breakthrough were 2.43 mg/mL and 4.73 mg/mL, respectively. These DBC values are satisfactory when compared to commercial resins used in affinity chromatography, taking into account that protein interaction only occurred at the gellan bead surface. These results indicated that gellan gum microbeads obtained by waterin- oil emulsion technique can be used as an innovative and promising chromatographic support due to its gelling ability and versatility to interact with different biomolecules.