Novel site-specific PEGylated L-asparaginase

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.

Fig 1. Reaction conditions to produce monoPEGylated L-asparaginase (monoPEG-ASNase).

(A) Electrophoresis (SDS-PAGE) showing the influence of PBS buffer ionic strength on N-terminal PEGylation with pH 7.5 (PEG:ASNase ratio of 25:1, 2 kDa mPEG-NHS): Column 1- ASNase (control), column 2- reaction in 10 mM PBS, column 3- reaction in 100 mM of PBS, column 4- reaction in 200 mM PBS, and column 5- Molecular weight (BioRad). (B) Percentage of PEGylation at different PEG:ASNase ratios, 25:1 and 50:1, in 100 mM of PBS pH 7.5, 30 min of time reaction. (C) Percentage of PEGylation at different pH values (6.0, 6.5, 7.0, 7.5 or 8.0) in 100 mM of PBS, PEG:ASNase ratio of 25:1 and 30 min of reaction time. (D) Percentage of PEGylation on different reaction times (15 to 90 minutes) in 100 mM of PBS, pH 7.5, PEG:ASNase ratio of 25:1. Grey bars—polypegylated ASNase, white bars—monoPEGylated ASNase, dotted bars—free ASNase. Percentage of PEGylation was based on gel analysis by band intensity.

Fig 2. Enzymatic behavior of monoPEG-ASNase compared to ASNase (control) and polyPEGylated form.

(A) Enzymatic kinetics of ASNase. (B) Enzymatic kinetics of monoPEG-ASNase. (C) Enzymatic kinetics of polyPEG-ASNase. Data analysis and statistical analysis (F-test) were done using GraphPad Prism 5.0 software. All trials were in triplicates and error bars represent the standard deviation.

Fig 3. PEG attachment influence on native enzyme.

(A) Enzymatic activity versus storage time at 4°C for ASNase control, monoPEG-ASNase and polyPEG-ASNase. (B) Dynamic light scattering profiles of unreacted ASNase, mono and polyPEGylated ASNase. The hydrodynamic radius were 7.53 nm, 9.85 nm and 11.75 nm, respectively.

Fig 4. MALDI-TOF of free ASNAse and monoPEG-ASNase (with 2kDa and 10kDa PEG).

(A) N-terminal peptide LPNITILATGGTIAGGGDSATK.(S) at m/z 2028.1809 and (B) lysine peptide SVNYGPLGYIHNGK.(I), at m/z 1518.7. Samples were acquired in duplicate. ASNase; blue line–monoPEG-ASNase 2kDa; light and dark green line—monoPEG-ASNase 10kDa; light blue line and orange.

Fig 5. Electrophoresis gel (native-PAGE) showing the proteolytic degradation /resistance of native ASNase and PEGylated forms.

ASNase, monoPEG-ASNase and polyPEG-ASNase in presence of asparagine endopeptidase (AEP) and cathepsin B (CTSB) after 84 h at 37°C, stained with CBB (top gel) and stained with ferric chloride (bottom gel). Column 1- Protease-free ASNase, column 2- ASNase with CTSB, column 3- ASNase with AEP, column 4- protease-free monoPEG-ASNase, column 5- monoPEG-ASNase with CTSB, column 6- monoPEG-ASNase with AEP, 7- protease-free polyPEG-ASNase, column 8- polyPEG-ASNase with CTSB and column 9- polyPEG-ASNase with AEP.

Fig 6. Cytotoxicity of monoPEG-ASNase in MOLT-4 and REH cells.

Assays performed at 48 h and 72 h, with cells alone (control), without enzyme (PBS) and at different concentrations of enzymatic activity (0.01, 0.05, 0.1, 0.3 or 0.6 U.mL^-1). Grey bars—free ASNase and white bars—monoPEGylated ASNase. Error bars represent the standard deviation. Likelihood of significance less than 0.05 (*), less than 0.01 (**) and less than 0.001 (***) when compared with the control.