L-asparaginase production by Pichia pastoris in batch-fed bioreactors.

Author(s)

Letícia de Almeida Parizotto

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Escola Politécnica (EP/BC)
• Defense date: 2022-06-10

Examining board members

Aldo Tonso; Larissa Pereira Brumano; Viviane Maimoni Gonçalves

• Advisor: Aldo Tonso; Adalberto Pessoa Junior

Abstract

Brazil is highly dependent on the imports of biological medicines, which poses the risk of shortages and price dependence on exchange rate fluctuations. Recently, the lack of supplies has led to disruptions in the stock of the biopharmaceutical L-asparaginase (ASNase), which is used in the treatment of various cancers, particularly Acute Lymphoblastic Leukemia (ALL), one of the main childhood cancers. Although two drugs have been registered since 2018, the supply is unreliable. Moreover, the forms of ASNase approved for clinical use are derived from bacteria, Escherichia coli and Dickeya chrysanthemi (Erwinia chrysanthemi), and all cause immunological reactions in patients and have a half-life of only a few hours. Therefore, thematic projects were established to develop the first steps for the national production of a less immunogenic and more stable ASNase. Collaborating on these projects, this thesis aims to develop the upstream stage of the production process of the recombinant enzyme L-asparaginase from D. chrysanthemi expressed by a Pichia pastoris strain (Glycoswitch®) capable of humanized glycosylation, from cell line development to bench bioreactor cultures. To achieve this goal, a new producing strain was created and improvements were made to the operational strategies of the process. First, two strains of Glycoswitch® were transformed, one auxotrophic (His-) and the other prototrophic (His+) to histidine, characterized in terms of expression of the ansB gene, encoding ASNase, and were studied cultures in complex medium in flasks and in synthetic medium in a 2 L bioreactor. The qPCR analysis showed that the His+ strain had twice the gene expression of the His- strain, which was consistent with the productivity obtained in flasks and in the bioreactor, so it was selected to continue the study. Then, the induction protocol of the bioreactor was changed from an open-loop control for feeding inductor (methanol) in pulses to a closed-loop control with DO-stat, which resulted in a doubling of the volumetric activity. Subsequently, using Quality by Design principles, such as experimental design, application of analytical tools and analysis with mathematical models, a statistical design was performed to investigate the effects of the controlled methanol concentration, dissolved oxygen and temperature on the volumetric activity of ASNase. In the best cultivation condition, 5 g/L methanol, 50% dissolved oxygen and 35 oC, a final volumetric activity of 10700 U/L was achieved with 84 hours of process time, about eight times higher than in an open loop strategy. Furthermore, the predictive model showed that controlling methanol and increasing temperature were critical to this result. The biomass and product yields were obtained by carbon balance and it was observed that the higher temperature resulted in an increase in them, which favoured the formation of active ASNase. Finally, characterization of the recombinant ASNase by MALDI-TOF analysis revealed that it is glycosylated to an epitope that elicits immune responses in patients, reducing its side effects. Thus, a protocol with a high yield of an innovative ASNase compared to other published works was developed, which has the potential to provide treatment with a better quality of life for the patient. (AU)

• FAPESP's process: 17/25065-9 - Production of L-asparaginase by Pichia pastoris in fed-batch bioreactors
• Grantee: Letícia de Almeida Parizotto
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)