L-asparaginase production by recombinant Escherichia coli in high cell density cultivation for a pharmaceutical application

• Grant number: 18/24926-3
• Support Opportunities: Scholarships in Brazil - Doctorate
• Start date: May 01, 2019
• End date: November 30, 2020
• Field of knowledge: Engineering - Chemical Engineering
• Principal Investigator: Adalberto Pessoa Junior
• Grantee: Rodrigo Gabriel Simas
• Host Institution: Faculdade de Ciências Farmacêuticas (FCF). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Associated research grant: 13/08617-7 - Production of extracellular L-asparaginase: from bioprospecting to the engineering of an antileukemic biopharmaceutical, AP.TEM
• Associated scholarship(s): 19/12295-1 - Identification of stress factors and their influence on genotype to phenotype changes during recombinant L-Asparaginase production by Escherichia coli, BE.EP.DR

Abstract

Acute Lymphoblastic Leukaemia (ALL) is a haematological cancer, predominantly in children. The enzyme L-Asparaginase (ASNase) is used in all standard protocols to treat this disease, resulting in a near 90% cure rate in children. The Brazilian government provides ASNase free of charge for the ALL treatment, but because ASNase is not manufactured in Brazil, the entire national demand must be met by expensive foreign imports. Fragile dependence on foreign suppliers and the low quality of some of these formulations have fuelled a debate on the urgent need for national production of ASNase to meet the clinical need in Brazil. The main objective of this project is to develop an industrially attractive bioprocess for native Escherichia coli ASNase production in bioreactors, as a necessary phase for process scale up and future industrial production. For this, a high cell density fermentation of E. coli BL21 (DE3) will be developed in 1.0 L bioreactors, focusing on feeding and induction strategies during cultivation. To support development of the process, a combined transcriptome and quantitative proteome analysis will be carried out, at King's College London, in order to monitor genotype to phenotype changes in response to stress conditions during fed-batch cultivation, by using the Proteomics Core Facility (equipped with the latest state-of-the-art mass spectrometry equipment, being a brand new Orbitrap Fusion Lumos Tribrid mass spectrometer coupled to an ultra-high pressure liquid chromatography system (U3000), which presents unrivalled accuracy and sensitivity for protein/peptide identification, post-translation modification analysis and quantification of peptides and proteins in simple or complex biological mixtures). (AU)