Chemical and Enzymatic Site Specific PEGylation of hGH: The Stability and in vivo Activity of PEG‐N‐Terminal‐hGH and PEG‐Gln141‐hGH Conjugates

Abstract

The use of therapeutic proteins is often impaired by their short in vivo half-lives. PEGylation has been exploited to enhance protein stability and to prolong the pharmacokinetic. The biophysical characterization of two site-specific mono-PEGylated forms of human growth hormone (hGH)--chemically N-terminal PEGylated hGH (PEG-Nter-hGH) and enzymatically Gln141 PEGylated hGH (PEG-Gln141-hGH) via transglutaminase--is outlined here and their pharmacodynamics are compared. The thermal stability of PEG-Nter-hGH was increased with respect to that of hGH and PEG-Gln141-hGH. Pharmacodynamic studies in rats showed that a single injection of the conjugates had a better or comparable potency with respect to a daily hGH on a week schedule in terms of weight gain, femoral length, and tibial diaphysis width.

Site-selective enzymatic chemistry for polymer conjugation to protein lysine residues: PEGylation of G-CSF at lysine-41

“…Several examples of mTGase-mediated polymer conjugation to the protein's Gln residues have been described. [6][7][8][9][10][11] Interestingly, protein modification at the level of Lys residues by mTGase-mediated derivatization is also feasible, but it requires a ligand containing a Gln residue or a glutamine analogue. Being able to incorporate various chemical entities and/or functional groups at the level of protein-bound Gln or Lys residues seems to be an important feature of mTGase use that has enabled a variety of strategies and approaches for protein modification to be developed for basic research purposes and biotechnological applications.…”

Engineering a Long Lasting Tethered, Multimeric Human Growth Hormone Protein to Improve Pharmacokinetic Half-Life and Potency

“…This dosing regime is a burden to patients and decreases adherence and therefore has driven innovative drug development strategies. Site directed PEGylation has been used to improve hGH half-life and reduce immunogenicity [21][22][23]. Our design strategy used targeted PEGylation to control the placement of bi-functional, low molecular weight PEGs on Gln40 and Gln140 residues of hGH.…”

Polymer conjugation of proteins as a synthetic post-translational modification to impact their stability and activity

“…The Pasut group attempted to address this issue while increasing activity and stability through the attachment of PEG chains to hGH at varying locations. 117 Conjugation resulted in significantly increased circulation half-life and significant bone growth in rat models. Thermal stability was measured using CD.…”

“…The Pasut group attempted to address this issue through the attachment of 20kDa PEG chains to hGH at varying locations. 117 The group conjugated the protein at the amine terminus and glutamine at residue 141. In vivo activity was tested by comparing somatic growth produced by the conjugated and native hGH in hypophysectomized rats.…”