Desenvolvimento de plataforma de expressão em Escherichia coli visando à produção de biossimilar da anakinra.

Abstract

Different prokaryotic expression systems produce biopharmaceuticals, especially E. coli, a very well-characterized species with low cost, high productivity, fast production cycles, and ideal for producing small proteins that do not require post-translational modifications. The extracellular secretion of these biomolecules into E. coli culture medium offers significant advantages over intracellular strategies. Chief among these advantages is the simplified recovery and purification process, significantly reducing production costs. Furthermore, extracellular secretion can prevent the target protein from accumulating in the cytosol, as inclusion bodies, increasing this macromolecule's stability and biological activity. Efficient methods that allow the translocation of biomolecules, overcoming the outer membrane barrier, are still poorly available and challenging on an industrial scale. Therefore, in this project we proposed the development of national platforms to produce the biopharmaceutical anakinra (interleukin 1 receptor antagonist) and the enzyme enterokinase in strains of E. coli, aiming at the future development of a biosimilar medicine. In this sense, a plasmid used in the transformation of the E. coli strain BL21 (DE3) was designed, which was expressed in the adapted NZCYM and M9 culture media, in a shaker incubator (18°C and 37°C, respectively) at 200 rpm for 48h. Tests of culture medium additives with 0.03% SDS and 1% Triton X-100 were conducted. Expression was evaluated using SDS-PAGE and Western Blotting. For anakinra, the expressed protein, the adapted M9 medium was standardized. The macromolecule was purified from the contents of the periplasm using affinity chromatography on a nickel column, obtaining the biopharmaceutical with a high degree of purity. For enterokinase, under the conditions tested, it was impossible to confirm the enzyme's expression. To achieve the ideal concentration for cell cultivation, aiming to scale the production process at an industrial level, some improvements are necessary, such as standardization testing of temperature, time, cultivation media, and bacterial strains. Furthermore, in vivo and in vitro assays of anakinra are essential to evaluate the pharmacological effect of the protein.