Development of downstream process of humanized L-asparaginase and its characterization

• Grant number: 17/20384-9
• Support Opportunities: Scholarships in Brazil - Doctorate
• Start date: January 01, 2019
• End date: August 31, 2021
• Field of knowledge: Engineering - Chemical Engineering - Chemical Process Industries
• Principal Investigator: Adalberto Pessoa Junior
• Grantee: Eduardo Krebs Kleingesinds
• 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): 19/06919-2 - In vivo test of humanized L-asparaginase free and nano-encapsulated in liposomes, BE.EP.DR

Abstract

The search for new biopharmaceuticals is mobilizing academic, industrial and governmental sectors in the development of biotechnological processes that enable the production on an industrial scale of active principles that act against a certain disorder and minimize its side effects, as is the case of the antileukemic biopharmaceutical L-asparaginase. Acute Lymphoblastic Leukemia (ALL) is a disorder of the autoimmune system that is characterized by overproduction of mutated lymphoblasts and has become incapable of synthesizing the amino acid L-asparagine. The action of the enzyme L-asparaginase consists of its selective catalytic action in order to promote the deamination of the amino acid L-asparagine circulating in the blood in aspartic acid and ammonia. In the absence of this amino acid, essential for lecocyclic lymphoblasts, the synthesis of DNA, RNA and proteins is compromised leading to cellular apoptosis. Healthy cells, on the other hand, are not affected by being self-sufficient in the synthesis of this amino acid. The high incidence of undesirable immunogenic manifestations in patients medicated with commercially available L-Asparaginase formulations reflects the need for improvement in the ALL treatment protocol. The elucidation of the physico-chemical and kinetic properties of this enzyme, as well as the search for new and alternative sources, optimized conditions of cultivation and implementation of adequate purification methodologies, will contribute with new perspectives for the national production of a potential biopharmaceutical that does not present the limitations of current formulations. In recently developed works in our laboratory we started to produce L-Asparaginase II expressed by Erwinia chrysanthemi in vector Pichia pastoris, and the main objective of this project is to establish the best strategy for purifying the enzyme to evaluate its potential as a biopharmaceutical. (AU)

Scientific publications

(5)

(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)

PARIZOTTO, LETICIA DE ALMEIDA; KLEINGESINDS, EDUARDO KREBS; PEDROTTI DA ROSA, LUIZA MANFRINATO; EFFER, BRIAN; LIMA, GUILHERME MEIRA; HERKENHOFF, MARCOS EDGAR; LI, ZHAOPENG; RINAS, URSULA; MONTEIRO, GISELE; PESSOA, ADALBERTO; et al. Increased glycosylated L-asparaginase production through selection of Pichia pastoris platform and oxygen-methanol control in fed-batches. Biochemical Engineering Journal, v. 173, SEP 2021. (17/20384-9, 18/15041-8, 17/25065-9, 13/08617-7, 19/02583-0, 19/02657-3)

TORRES-OBREQUE, KARIN; KLEINGESINDS, EDUARDO KREBS; SANTOS, JOAO H. P. M.; CARRETERO, GUSTAVO; RABELO, JHENIFFER; CONVERTI, ATTILIO; MONTEIRO, GISELE; PESSOA JR, ADALBERTO; RANGEL-YAGUI, CARLOTA O.. PEGylation versus glycosylation: effect on the thermodynamics and thermostability of crisantaspase. PREPARATIVE BIOCHEMISTRY & BIOTECHNOLOGY, v. N/A, p. 11-pg., 2023-09-11. (16/22065-5, 17/20384-9, 13/08617-7, 18/25994-2)

EFFER, BRIAN; LIMA, GUILHERME MEIRA; CABARCA, SINDY; PESSOA, ADALBERTO; FARIAS, JORGE G.; MONTEIRO, GISELE. L-Asparaginase from E. chrysanthemi expressed in glycoswitch: effect of His-Tag fusion on the extracellular expression. PREPARATIVE BIOCHEMISTRY & BIOTECHNOLOGY, v. 49, n. 7, APR 2019. (17/20384-9, 16/25896-5, 15/07749-2, 13/08617-7, 16/15787-4)

KLEINGESINDS, EDUARDO KREBS; PARIZOTTO, LETICIA DE ALMEIDA; EFFER, BRIAN; MONTEIRO, GISELE; LONG, PAUL F.; ARROYO-BERDUGO, YOANA; BEHRENDS, VOLKER; ESPOSITO, MARIA TERESA; CALLE, YOLANDA; PESSOA-JR, ADALBERTO. Downstream process and evaluation of the concomitant impact of a recombinant glycosylated L-asparaginase on leukemic cancer cells and the bone marrow tumor microenvironment. Process Biochemistry, v. 131, p. 11-pg., 2023-06-09. (19/06919-2, 13/08617-7, 15/07749-2, 18/03734-9, 17/25065-9, 17/20384-9, 18/15104-0)

EFFER, BRIAN; KLEINGESINDS, EDUARDO KREBS; LIMA, GUILHERME MEIRA; COSTA, IRIS MUNHOZ; SANCHEZ-MOGUEL, IGNACIO; PESSOA, ADALBERTO; SANTIAGO, VERONICA FEIJOLI; PALMISANO, GIUSEPPE; FARIAS, JORGE G.; MONTEIRO, GISELE. Glycosylation of Erwinase results in active protein less recognized by antibodies. Biochemical Engineering Journal, v. 163, p. 11-pg., 2020-11-15. (13/08617-7, 15/07749-2, 17/20384-9, 18/15104-0, 16/15787-4, 16/25896-5)

Academic Publications

(References retrieved automatically from State of São Paulo Research Institutions)

KLEINGESINDS, Eduardo Krebs. Exploring the role of a glycosylated L-asparaginase expressed by a recombinant Pichia pastoris as an antileukemic biopharmaceutical. 2021. Doctoral Thesis - Universidade de São Paulo (USP). Conjunto das Químicas (IQ e FCF) (CQ/DBDCQ) São Paulo.