Doctoral Program MIT Portugal Bio-Engineering Systems - Artificial Protein scaffolds for AFFINITY-TRIGGERED bioseparations

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Artificial scaffolds for affinity-triggered bioseparations

Publication . Matos, Manuel João de Almeida Albuquerque Brandão; Roque, Ana; Pina, Ana

Monoclonal antibodies (mAb) are, to this date, the dominant class of biologicals developed by the biopharmaceutical industry. A new trend of growing diversification of other antibody molecular formats is arising with the development of antigen-binding fragments (Fab) and other derived fragments. Despite this growing repertoire of antibodies, the downstream strategies have remained stalled throughout decades, only addressing purification of full-length antibodies. This thesis focused on the development of novel affinity ligands for the purification of not only full-length antibodies, but also of Fab and non-human antibodies. By taking inspiration on a rationally designed Petasis-Ugi library, a synthetic approach was evaluated. The library was screened towards binding of polyclonal human immunoglobulin G (IgG), wherein B1Al2A2 was selected as lead candidate. The broad specificity of B1Al2A2 enabled a purification platform-like behaviour for the isolation of IgG, Fab, Nanobody and immunoglobulin Y, from complex mixtures achieving high purities and yields under mild elution conditions. A different strategy employing biological combinatorial libraries displaying an artificial protein domain scaffold was also tested. The phage pool was screened towards IgG, revealing E6 as a high-affinity binder. E6 was recombinantly expressed in Escherichia coli, purified and exhibited a 154nM affinity for IgG, as determined by Microscale Thermophoresis (MST). Encouraged by these results, the library was screened to select a ligand for Fab. A biopanning depletion scheme was designed to minimize the existence of non-specific binders by inclusion of negative selection steps with closely related impurities to Fab. Computational docking studies were performed to compare binding mode of two of the most promising clones. Hit binder, D5, was selected and was immobilised onto an aldehyde-activated support. The adsorbent showed superior performance in binding Fab at basic pH and was able to efficiently isolate Fab from complex mixtures and to remove unwanted impurities. Overall, the approaches explored throughout this work contribute significantly to the rational design of affinity ligands for immunoglobulins and can pave the way for alternative cost-efficient purification strategies.

2022-01-13Tese de doutoramento

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Fundação para a Ciência e a Tecnologia

Número da atribuição

PD/BD/128251/2016