title: Protein HESylation for half-life extension: synthesis, characterization and pharmacokinetics of HESylated anakinra.
abstract: 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.
authors.affiliation: Department of Pharmacy, Pharmaceutical Technology & Biopharmaceutics, Ludwig-Maximillians-University Munich, Munich, Germany.
authors.forename: Robert
authors.initials: R
authors.lastname: Liebner
authors.affiliation: Department of Pharmacy, Pharmaceutical Technology & Biopharmaceutics, Ludwig-Maximillians-University Munich, Munich, Germany.
authors.forename: Roman
authors.initials: R
authors.lastname: Mathaes
authors.affiliation: I&D Center Carbohydrate Chemistry, Fresenius Kabi Deutschland GmbH, Friedberg, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Meyer
authors.affiliation: I&D Center Carbohydrate Chemistry, Fresenius Kabi Deutschland GmbH, Friedberg, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Hey
authors.affiliation: Department of Pharmacy, Pharmaceutical Technology & Biopharmaceutics, Ludwig-Maximillians-University Munich, Munich, Germany.
authors.forename: Gerhard
authors.initials: G
authors.lastname: Winter
authors.affiliation: Department of Pharmacy, Pharmaceutical Technology & Biopharmaceutics, Ludwig-Maximillians-University Munich, Munich, Germany. Electronic address: ahmed.besheer@cup.uni-muenchen.de.
authors.forename: Ahmed
authors.initials: A
authors.lastname: Besheer
mesh_terms: D000447:Aldehydes / Q000138:chemical synthesis / Q000493:pharmacokinetics; D000586:Amination; D000818:Animals; D000893:Anti-Inflammatory Agents / Q000008:administration & dosage / Q000138:chemical synthesis* / Q000493:pharmacokinetics*; D019540:Area Under Curve; D002151:Calorimetry; D002626:Chemistry, Pharmaceutical; D002850:Chromatography, Gel; D006207:Half-Life; D006895:Hydroxyethyl Starch Derivatives / Q000008:administration & dosage / Q000138:chemical synthesis* / Q000493:pharmacokinetics*; D007275:Injections, Intravenous; D053590:Interleukin 1 Receptor Antagonist Protein / Q000008:administration & dosage / Q000138:chemical synthesis* / Q000493:pharmacokinetics*; D008027:Light; D008297:Male; D008970:Molecular Weight; D010316:Particle Size; D055550:Protein Stability; D017433:Protein Structure, Secondary; D017208:Rats, Wistar; D012542:Scattering, Radiation; D017550:Spectroscopy, Fourier Transform Infrared; D020349:Surface Plasmon Resonance; D013678:Technology, Pharmaceutical / Q000379:methods
chemical_list: D000447:Aldehydes; D000893:Anti-Inflammatory Agents; D006895:Hydroxyethyl Starch Derivatives; D053590:Interleukin 1 Receptor Antagonist Protein; C005556:propionaldehyde
keywords: Anakinra; Binding affinity; HESylation(®); Microcalorimetry; Microscale thermophoresis (MST); Pharmacokinetics