Browsing by Author "Rouqueiro, Rodrigo Anselmo"
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- Protein A chromatography designed for improved antibody capturePublication . Rouqueiro, Rodrigo Anselmo; Cabral, Ana Cristina Mendes Dias; Cruz, CarlaProtein A affinity chromatography has been the most used purification method for therapeutic antibodies in industrial processes, once it selectively purifies exploring molecular recognition and is an easy methodology. However, this type of chromatography has some costassociated drawbacks and upstream titer limitations. This, coupled to the demand for high purity and quality requirements from regulatory agencies and to the increasingly growth of monoclonal antibodies importance in therapeutic market, led to some work with the purpose of getting a better performance of engineered protein A ligands. Nowadays, derivate from the wild type Staphylococcal protein A (with five different antibody-binding domains, A, B, C, D, E), engineered variants are present in the market. All the domains consist in three a-helices, which bind to the CH2 region in the Fc part of the antibody and one of the most popular engineered variants is the tetrameric Z form, a mutant from the B-domain. The present work is part of a major aim where we intent to develop a Protein A monolithic column that shows better performance for the capture of antibodies from cell culture supernatant. Thus, two different main goals are studied: the thermodynamic evaluation of matrix properties (2,5% QA monolith) by analyzing its response to the adsorption of a model protein (Bovine serum albumin (BSA)); and the capacity assessment of an engineered ligand for antibody capture. The first goal was achieved using a Flow Microcalorimeter (FMC) and it is subdivided in two parts: first, multiple injections of different volumes with the same BSA concentration were performed submitting the column to different protein surface concentrations; and later, multiple consecutive injections were performed to understand what happens under overloading conditions and to observe the point at which the monolith stops to adsorb. The second aim is subdivided in three different objectives: interaction study of protein A novel ligand, presenting eight binding domains, with an IgG antibody; optimization of different ligand immobilization procedures; and comparison of the novel ligand with the commercial already existent engineered Z form (four domains, MabSelect SuRe), in terms of antibody isolation potential. Two prototypes variants of the novel ligand with 8 B binding domains were used. One of them is marked with a poly-histidine tag (B8(RH4)) and the other is tagged with cysteine-lysine-cysteine-lysine tail (B8 cys-lys-cys-lys). These two are thought, as previously mentioned, to be used primarily in protein A chromatography, namely in the direct capture of monoclonal antibodies due to the high specificity of the antibody-protein A bind.