Novel site-specific PEGylated L-asparaginase


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.



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Science & Technology - Other Topics


Meneguetti, GP; Santos, JHPM; Obreque, KMT; Barbosa, CMV; Monteiro, G; Farsky, SHP; de Oliveira, AM; Angeli, CB; Palmisano, G; Ventura, SPM; Pessoa, A; Rangel-Yagui, CD

our authors


This research was supported by grants from the State of Sao Paulo Research Foundation (FAPESP-Brazil, processes number 2013/08617-7 and 2016/22065-5), the National Council for Scientific and Technological Development (CNPq-Brazil), the Coordination of Improvement of Higher Education Personnel (CAPES-Brazil, Project 001) and Technological Research Institute Foundation (FIPT, Brazil). This work was also developed within scope of the project CICECO-Aveiro Institute of Materials, POCI-01-0145-FEDER-007679 (FCT Ref. UID/CTM/50011/2013), financed by national funds through the FCT/MEC and when appropriate co-financed by FEDER under the PT2020 Partnership Agreement. The authors are grateful for the financial support of the Portuguese Foundation for Science and Technology (FCT) for the doctoral grant of SFRH/BD/102915/2014 of Joao H. P. M. Santos. S. P. M. Ventura acknowledges FCT for the contract IF/00402/2015.

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