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.

Fab on a Package: LTCC Microfluidic Devices Applied to Chemical Process Miniaturization.

Microfluidics has brought diverse advantages to chemical processes, allowing higher control of reactions and economy of reagents and energy. Low temperature co-fired ceramics (LTCC) have additional advantages as material for fabrication of microfluidic devices, such as high compatibility with chemical reagents with typical average surface roughness of 0.3154 µm, easy scaling, and microfabrication. The conjugation of LTCC technology with microfluidics allows the development of micrometric-sized channels and reactors exploiting the advantages of fast and controlled mixing and heat transfer processes, essential for the synthesis and surface functionalization of nanoparticles. Since the chemical process area is evolving toward miniaturization and continuous flow processing, we verify that microfluidic devices based on LTCC technology have a relevant role in implementing several chemical processes. The present work reviews various LTCC microfluidic devices, developed in our laboratory, applied to chemical process miniaturization, with different geometries to implement processes such as ionic gelation, emulsification, nanoprecipitation, solvent extraction, nanoparticle synthesis and functionalization, and emulsion-diffusion/solvent extraction process. All fabricated microfluidics structures can operate in a flow range of mL/min, indicating that LTCC technology provides a means to enhance micro- and nanoparticle production yield.