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

• We describe the synthesis, characterization and PK of HESylated anakinra.
• HESylated anakinra maintained its bioactivity as shown by SPR and MST.
• HESylated anakinra is more stable than anakinra as shown by DSC and stress tests.
• HESylation significantly improved the pharmacokinetics of anakinra.

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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