Protein HESylation for half-life extension: synthesis, characterization and pharmacokinetics of HESylated anakinra

Robert Liebnera, Roman Mathaes, Martin Meyer, Thomas Hey, Gerhard Winter, Ahmed Besheer

European Journal of Pharmaceutics and Biopharmaceutics
2014 vol: 87 issue: 2 pp: 378-385 doi: 10.1016/j.ejpb.2014.03.010

Half-life extension (HLE) is becoming an essential component of the industrial development of small-sized therapeutic peptides and proteins. HESylation(®) is a HLE technology based on coupling drug molecules to the biodegradable hydroxyethyl starch (HES). In this study, we report on the synthesis, characterization and pharmacokinetics of HESylated anakinra, where anakinra was conjugated to propionaldehyde-HES using reductive amination, leading to a monoHESylated protein. Characterization using size exclusion chromatography and dynamic light scattering confirmed conjugation and the increase in molecular size, while Fourier transform infrared spectroscopy showed that the secondary structure of the conjugate was not affected by coupling. Meanwhile, microcalorimetry and aggregation studies showed a significant increase in protein stability. Surface plasmon resonance and microscale thermophoresis showed that the conjugate retained its nanomolar affinity, and finally, the pharmacokinetics of the HESylated protein exhibited a 6.5-fold increase in the half-life, and a 45-fold increase in the AUC. These results indicate that HESylation(®) is a promising HLE technology.

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Topics: HESylation, Anakinra, Microcalorimetry, Binding affinity, Pharmacokinetics, Monolith – MicroScale Thermophoresis, MST, Proteins, Publications