Design, modeling, expression, and chemoselective PEGylation of a new nanosize cysteine analog of erythropoietin

Abstract

Background

Recombinant human erythropoietin (rhEPO) is considered to be one of the most pivotal pharmaceutical drugs in the market because of its clinical application in the treatment of anemia-associated disorders worldwide. However, like other therapeutic proteins, it does not have suitable pharmacokinetic properties for it to be administrated at least two to three times per week. Chemoselective cysteine PEGylation, employing molecular dynamics and graphics in in silico studies, can be considered to overcome such a problem.

Methods

A special kind of EPO analog was elicited based on a literature review, homology modeling, molecular dynamic simulation, and factors affecting the PEGylation reaction. Then, cDNA of the selected analog was generated by site-directed mutagenesis and subsequently cloned into the expression vector. The construct was transfected to Chinese hamster ovary/dhfr⁻ cells, and highly expressed clones were selected via methotrexate amplification. Ion-immobilized affinity and size exclusion (SE) chromatography techniques were used to purify the expressed analog. Thereafter, chemoselective PEGylation was performed and a nanosize PEGylated EPO was obtained through dialysis. The in vitro biologic assay and in vivo pharmacokinetic parameters were studied. Finally, E31C analog Fourier transform infrared, analytical SE-high-performance liquid chromatography, zeta potential, and size before and after PEGylation were characterized.

Results

The findings indicate that a novel nanosize EPO31-PEG has a five-fold longer terminal half-life in rats with similar biologic activity compared with unmodified rhEPO in proliferation cell assay. The results also show that EPO31-PEG size and charge versus unmodified protein was increased in a nanospectrum, and this may be one criterion of EPO biologic potency enhancement.

Discussion

This kind of novel engineered nanosize PEGylated EPO has remarkable advantages over rhEPO.

Keywords:

• nanoPEGylated EPO
• cysteine PEGylation
• pharmacokinetic property

Acknowledgements

This project was financially supported by the Pasteur Institute of Iran. The computational resource for running molecular dynamic simulations was provided by the National Institute of Genetic Engineering and Biotechnology (NIGEB). The authors wish to express their deep gratitude to all who provided support during the course of this research.

Disclosure

The authors report no conflicts of interest in this work.