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Doctoral Thesis
DOI
https://doi.org/10.11606/T.9.2021.tde-21122021-144906
Document
Author
Full name
Eduardo Krebs Kleingesinds
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2021
Supervisor
Committee
Pessoa Junior, Adalberto (President)
Azzoni, Adriano Rodrigues
Monteiro, Gisele
Patino, Yolanda Calle
Title in English
Exploring the role of a glycosylated L-asparaginase expressed by a recombinant Pichia pastoris as an antileukemic biopharmaceutical
Keywords in English
Bioprocess
Bioprocesso
Co-cultura
Co-culture
Leucemia
Leukemia
Microambiente tumoral
Proteínas recombinantes
Recombinant proteins
Tumor microenvironment
Abstract in English
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.
Title in English
Explorando o papel de uma L-asparaginase glicosilada expressa por Pichia pastoris recombinante como potencial biofármaco anti-leucêmico
Keywords in English
Bioprocess
Bioprocesso
Co-cultura
Co-culture
Leucemia
Leukemia
Microambiente tumoral
Proteínas recombinantes
Recombinant proteins
Tumor microenvironment
Abstract in English
Segundo a Organização Mundial da Saúde (OMS), em 2020, foram notificados 474 519 novos casos de leucemia em todo o mundo e 311 594 novos óbitos. A importância da Lasparaginase (LASNase) como biofármaco para o tratamento da Leucemia Linfoblástica Aguda (LLA) é reconhecida mundialmente. Neste trabalho, descreve-se o bioprocesso da L-ASNase de Erwinia chrysanthemi expressa extracelularmente por Pichia pastoris com um padrão de glicosilação semelhante ao humano. Inicialmente, foram otimizadas as condições de cultivo em agitador orbital. Em seguida, o processo foi escalonado utilizando um biorreator de bancada de 3L a 35 °C e 1,5% de metanol como um indutor para a produção de L-ASNase. A purificação foi avaliada utilizando ultrafiltração tangencial com cartuchos de diferentes tamanhos de poro (10-100 kDa) seguido por cromatografia de troca catiônica e de exclusão molecular. Foi possível atingir um rendimento final de 54,93% com fator de purificação de 70,93 vezes e os dados obtidos pelo ensaio de proteômica confirmaram a obtenção de uma enzima extremamente pura. Em pH 8,0 e 50 ° C, a enzima mostrou sua atividade ótima. Os parâmetros cinéticos, kM e Vmax, foram de 76,4 µM e 0,065 µmol min-1, respectivamente. Finalmente, foi investigado o impacto concomitante desta L-ASNase recombinante em linhagens de células tumorais marcadas com proteína fluorescente verde (eGFP) (SEMK2, HB119, REH e MV411) quando co-cultivadas com células do fibroblasto da medula óssea (HS5) marcadas com mCherry. Os resultados desta pesquisa sugerem que a linhagem eGFPHb11-9 foi a mais sensível quando tratada com a L-ASNase glicosilada, em contraste com eGFP-REH que foi a linhagem mais resistente. Também foi observada menor eficácia da droga quando as células tumorais foram co-cultivadas com células HS5 do que quando as células tumorais foram cultivadas sozinhas. Portanto, este trabalho abre o caminho para a produção, escalonamento e ensaios pré-clínicos deste promissor novo biofármaco, que pode ajudar a melhorar as taxas de remissão e a qualidade de vida de muitos pacientes com câncer em todo o mundo.
 
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Publishing Date
2021-12-22
 
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