Production of L-asparaginase by Pichia pastoris in fed-batch bioreactors

Abstract

This doctoral dissertation is part of the FAPESP thematic project called "Production of extracellular L-asparaginase: from bioprospecting to the Engineering of an antileukemic biopharmaceutical" which main aim is to obtain, in industrial scale, the enzyme L-Asparaginase (ASNase), that is applied to the treatment of Acute Lymphoblastic Leukaemia. Its national production development became relevant after the supply break in 2013; since then, the pharmaceutical has been acquired from different suppliers and, currently, the quality of the drug that is distributed by the Unified Health System is questioned. Further than to stimulate the national production, the project also proposes to develop methods that reduce the immunologic reactions caused by the current commercial drugs. One of these methods is the production of ASNase expressed by the gene ansB of Erwinia chrysanthemi in a recombinant Pichia pastoris strain able to perform humanized glycosylation. Additionally, the yeast P. pastoris presents a recombinant system inducted by methanol and, in optimum culture conditions, it reaches high density, which would turn the ASNase production more efficient. Therefore, aiming the optimum culture conditions, this project proposes some operational strategies in bioreactors based on the dissolved oxygen control and the feeding of glycerol and methanol. Moreover, it objectives to implement a closed loop control system for the methanol feeding, because it is a critical production parameter. After improving the culture conditions, the objective is to scale up the process from 2 to 14L bioreactors based on the oxygen volumetric mass transfer coefficient and statistic method validation. Hence, data for the process scale up to industrial production will be available, contributing to the thematic project purpose. (AU)