Exploring the role of a glycosylated L-asparaginase expressed by a recombinant Pichia pastoris as an antileukemic biopharmaceutical

• Ano de defesa: 2021
• Autor(a) principal: Kleingesinds, Eduardo Krebs
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• Idioma: eng
• Instituição de defesa: Biblioteca Digitais de Teses e Dissertações da USP
• Palavras-chave em Português: Bioprocess; Bioprocesso; Co-cultura; Co-culture; Leucemia; Leukemia; Microambiente tumoral; Proteínas recombinantes; Recombinant proteins; Tumor microenvironment
• Link de acesso: https://www.teses.usp.br/teses/disponiveis/9/9134/tde-21122021-144906/

According to the World Health Organization (WHO), in 2020, 474 519 new cases of leukemia were reported around the world, and 311 594 new deaths were reported. The importance of L-asparaginase (LASNase) as a biopharmaceutical to treat Acute Lymphoblastic Leukemia (ALL) is recognized worldwide. This work describes the bioprocessing of L-ASNase from Erwinia chrysanthemi expressed extracellularly by Pichia pastoris with a human-like glycosylation pattern. Firstly, it was optimized the upstream conditions in the orbital shaker flask. Then, it was scaled up using a 3L benchtop bioreactor at batch mode under 35 °C and 1.5% methanol as an inductor for L-ASNase production. The downstream processing was evaluated using crossflow ultrafiltration with different cut-offs (10-100 kDa) followed by cation exchange chromatography and size exclusion chromatography. It was possible to reach a final yield of 54.93% with a purification factor of 70.93 fold and the proteomics data confirmed the attainment of an extremely pure enzyme. At pH 8.0 and 50 °C, the enzyme showed its optimum activity. Kinetic parameters, kM and Vmax, were found to be 76.4 µM and 0.065 µmol min-1, respectively. The thermodynamic study showed that the enzyme irreversible deactivation is well described by first-order kinetics. Finally, using a high throughput fluorescentbased in vitro experimental platform, it was investigated the concomitant impact of this recombinant L-ASNase on enhanced green fluorescent protein (eGFP)-labeled tumor cell lines (SEMK2, HB119, REH, and MV411) when co-cultured with the mCherry-labelled bone marrow fibroblastic stroma cells HS5. The outcomes of this research suggest that the eGFP-Hb11-9 strain was the most sensitive strain when treated with the glycosylated L-ASNase, in contrast with the eGFP-REH that was the most resistant linage. It was also observed lower effectiveness of the drug when tumor cells were co-cultured with stromal cells than when tumor cells were cultured on their own. Hence, this work paves the way for production, scale-up, and pre-clinical trials of this promising novel biopharmaceutical, which may help improve the remission rates and quality of life for many cancer patients around the world.