Novel site-specific PEGylated L-asparaginase.

Giovanna Pastore Meneguetti,João Henrique Picado Madalena Santos,Sónia Patrícia Marques Ventura,Sandra Helena Poliselli Farsky,Gisele Monteiro,Carlota De Oliveira Rangel-Yagui,Adriano Marim De Oliveira,Christiano Marcello Vaz Barbosa,Karin Mariana Torres Obreque,Claudia Blanes Angeli,Adalberto Pessoa-Junior,Giuseppe Palmisano,Marco Rito-Palomares

doi:10.1371/journal.pone.0211951

Giovanna Pastore Meneguetti, João Henrique Picado Madalena Santos + Show 11 more

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https://doi.org/10.1371/journal.pone.0211951

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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.

Highlights

PEGylation is one of the most effective approaches to solve intrinsic problems of protein drugs, such as immunogenicity and short half-life
Site-specific PEGylation at N-terminal region is highly influenced by pH, temperature, reaction time and ionic strength [11]
Buffer ionic strength effect on conjugation of polyethylene glycol (PEG) to ASNase was investigated by varying molarity of PBS: 10, 100 and 200 mM were investigated

Summary

Introduction

PEGylation is one of the most effective approaches to solve intrinsic problems of protein drugs, such as immunogenicity and short half-life. It refers to the covalent attachment of polyethylene glycol (PEG) on the protein surface [1]. PEG is a highly water-soluble polymer, with low immunogenicity and approved by the US Agency for Food and Drug Administration (FDA). PEG-protein conjugates have several advantages as increased solubility and stability, prolonged half-life in the body and decreased metabolic degradation by enzymes [2]. PEGylation has become a well-established technology, increasing the therapeutic potential of biopharmaceutics like the L-asparaginase (ASNase) [3].

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