Novel site-specific PEGylated L-asparaginase

Author(s):Show less -

Meneguetti, Giovanna Pastore ^[1] ; Picado Madalena Santos, Joao Henrique ^{[1, 2]} ; Torres Obreque, Karin Mariana ^[1] ; Vaz Barbosa, Christiano Marcello ^[3] ; Monteiro, Gisele ^[1] ; Poliselli Farsky, Sandra Helena ^[3] ; de Oliveira, Adriano Marim ^[4] ; Angeli, Claudia Blanes ^[5] ; Palmisano, Giuseppe ^[5] ; Marques Ventura, Sonia Patricia ^[2] ; Pessoa-Junior, Adalberto ^[1] ; Rangel-Yagui, Carlota de Oliveira ^[1]

Affiliation

^[1] Univ Sao Paulo, Dept Biochem & Pharmaceut Technol, Sao Paulo - Brazil

^[2] Univ Aveiro, Dept Chem, CICECO Aveiro Inst Mat, Aveiro - Portugal

^[3] Univ Sao Paulo, Dept Clin & Toxicol Anal, Sao Paulo - Brazil

^[4] Technol Res Inst, Dept Bionanomanufacture, Sao Paulo - Brazil

^[5] Univ Sao Paulo, Inst Biomed Sci, Dept Parasitol, Sao Paulo - Brazil

• Document type: Journal article
• Source: PLoS One; v. 14, n. 2 FEB 12 2019.
• Web of Science Citations: 4

Abstract

L-asparaginase (ASNase) from Escherichia coli is currently used in some countries in its PEGylated form (ONCASPAR, pegaspargase) to treat acute lymphoblastic leukemia (ALL). PEGylation refers to the covalent attachment of poly(ethylene) glycol to the protein drug and it not only reduces the immune system activation but also decreases degradation by plasmatic proteases. However, pegaspargase is randomly PEGylated and, consequently, with a high degree of polydispersity in its final formulation. In this work we developed a site-specific N-terminus PEGylation protocol for ASNase. The monoPEG-ASNase was purified by anionic followed by size exclusion chromatography to a final purity of 99%. The highest yield of monoPEG-ASNase of 42% was obtained by the protein reaction with methoxy polyethylene glycol-carboxymethyl N-hydroxysuccinimidyl ester (10kDa) in 100 mM PBS at pH 7.5 and PEG: ASNase ratio of 25:1. The monoPEG-ASNase was found to maintain enzymatic stability for more days than ASNase, also was resistant to the plasma proteases like asparaginyl endopeptidase and cathepsin B. Additionally, monoPEG-ASNase was found to be potent against leukemic cell lines (MOLT-4 and REH) in vitro like polyPEG-ASNase. monoPEG-ASNase demonstrates its potential as a novel option for ALL treatment, being an inventive novelty that maintains the benefits of the current enzyme and solves challenges. (AU)

• FAPESP's process: 16/22065-5 - N-terminal pegylation of proteins and purification by aqueous two-phase systems
• Grantee: Carlota de Oliveira Rangel Yagui
• Support Opportunities: Regular Research Grants
• FAPESP's process: 13/08617-7 - Production of extracellular L-asparaginase: from bioprospecting to the engineering of an antileukemic biopharmaceutical
• Grantee: Adalberto Pessoa Junior
• Support Opportunities: Research Projects - Thematic Grants