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Overview of HESylation Based Half-Life Extension

A modified form of plant polysaccharide amylopectin is Hydroxyethyl starch (HES) which is a natural polymer. Hydroxyethylation makes the polysaccharide more water-soluble and prevents degradation due to a-amylase. The conjugation of HES moieties increases the hydrodynamic volume of the modified molecule, thereby making it less clearance susceptible to renal. HESylation, as the name suggests, involves the covalent coupling of HES to the molecule of interest. HES is characterized by a combination of desirable properties like excellent biocompatibility, tunable biodegradability, and high tolerable doses. As a result, HESylation for half-life extension represents a promising alternative to PEGylation technology. HESylation technology showed promising success in the half-life extension of protein therapeutics including erythropoietin and FVIII.

Fig.1 Examples of HESylation chemistry. (van Witteloostuijn, 2016)

Chemical Options of HESylation

• Random HESylation
  Early results for HESylated proteins such as hemoglobin were obtained by a random conjugation step. However, these HES conjugates faced some manufacturing limitations and suffered from toxicity problems. As with PEGylation, random HES linkage resulted in conjugation to a number of lysine residues with poorly controllable stoichiometry. The tolerance of such conjugates was quite low in animal experiments due to the high fraction of covalently linked high molecular weight aggregates.
• Site-Specific HESylation
  Site-directed HESylation was applied to several low molecular weight substances like amphotericin B, peptides, and proteins such as an erythropoietin mimetic peptide, erythropoietin, interferon α-2b, and anakinra. In vivo studies in dogs performed for such conjugates showed that half-life can be tuned by varying the molar mass (MW) and molar substitution (MS). The HES conjugate with high MW and high MS led to a threefold longer half-life compared to that of the commercially available glycosylated form.

  
  

Example of Anakinra HESylation for Half-Life Extension

Anakinra was coupled to HES at the N-terminus by a reductive amination reaction through a single terminal aldehyde group on HES, a highly purified mono-HESylated protein was obtained. The size of HES-conjugate is nearly three times that of native anakinra. The spectrum of HESylated anakinra nearly superimposes with that of the native protein, showing that HESylation did not lead to significant changes in anakinra's secondary structure. The pharmacokinetic properties result shows that plasma concentrations of native and HESylated anakinra demonstrated an exponential declining pattern after i.v. administration, but the elimination of HESylated anakinra was much slower than that of the native protein. The half-life of HESylated anakinra was increased about 6.5 times, as compared to that of native anakinra. This study shows that, while HESylation significantly improves the pharmacokinetic parameters of the model protein, anakinra, it does not negatively affect its structure or binding affinity, and increases its stability, indicating that HESylation is a promising half-life extension technology.

Fig.2 Plasma level of native and HESylated anakinra after single intravenous administration to rats. (Liebner, 2014)

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References

1. van Witteloostuijn, S.B.; et al. Half-life extension of biopharmaceuticals using chemical methods: alternatives to PEGylation. ChemMedChem. 2016, 11(22): 2474-2495.
2. Liebner, R.; et al. Protein HESylation for half-life extension: synthesis, characterization and pharmacokinetics of HESylated anakinra. European Journal of Pharmaceutics and Biopharmaceutics. 2014, 87(2): 378-385.