Chemical and Enzymatic Site Specific PEGylation of hGH

Abstract

Several strategies for site-specific PEGylation have been successfully exploited to conjugate poly(ethylene glycol) (PEG) to pharmaceutical proteins. The advantages sought are those of improving efficacy and increasing the half-life of conjugated proteins while achieving a higher degree of homogeneity. Recombinant human growth hormone (hGH) was thus PEGylated exploiting two site-specific strategies: N-terminal PEGylation using the PEG20 kDa-aldehyde polymer and microbial transglutaminase (mTGase) mediated enzymatic PEGylation using PEG20 kDa-NH2. N-Terminal PEGylation of hGH was carried out by covalent attachment of PEG to the α-amine residue of Phe1 that yielded the monoconjugate PEG-Nter-hGH with a mass of 44152.2 Da, as measured by MALDI-TOF mass spectrometry. The mTGase mediated PEGylation, performed in a water/ethanol solution mixture, allowed a PEG coupling reaction only at the level of hGH Gln141, yielding the single monoconjugate PEG-Gln141-hGH with a mass of 44064.9 Da. Circular dichroism studies showed that both conjugation strategies preserved the native-like secondary structures of hGH. It is vital to maintain the structural integrity of hGH if PEGylated hGH is to be used in therapeutic applications. As expected, the pharmacokinetic profile in rats of PEG-Nter-hGH and PEG-Gln141-hGH revealed a significant increase in systemic exposure with respect to unmodified hGH. The conjugates showed a half-life increase of 4.5-fold with respect to hGH. These results demonstrate that both chemical and enzymatic site-selective PEGylation of hGH generates conjugates with a prolonged half-life.

Recent Developments in Protein and Peptide Parenteral Delivery Approaches

“…In vivo studies in rats demonstrated that both PEGylated products significantly improved systemic circulation and exerted a 4.5-fold increase in half-life relative to unmodified hGH. These results indicate that site-specific PEGylation of hGH helps to generate conjugates with prolonged half-life relative to native protein (hGH) [16]. …”

Drug and protein delivery by polymer conjugation

“…To circumvent this possibility, aldehyde groups have been introduced into the HA backbone by periodate oxidation, an approach widely exploited with polysaccharides containing vicinal hydroxyl groups. Mild reactive aldehydes can, in turn, make it possible to obtain a selective N-terminal protein conjugation, as has also been reported with regard to aldehyde-PEGs [71,72]. This strategy was used for the HAylation, for example, of interferon alpha and human growth hormone [73,74].…”

“…For example, when TGase mediated PEGylation of hGH was compared to the well-know N-terminal PEGylation, the two approaches produced comparable results in terms of selectivity and preservation of protein secondary structures and the pharmacokinetic profiles of the mono-conjugates [72].…”

Responsive polymers for biosensing and protein delivery

“…In a recent publication, Browning et al showed 77 that acrylamide-PEG-isocyanate (AAm-PEG-I) linker can be incorporated onto proteins with PEG diacrylamide (PEGDAA) to enhance the hydrolytic stability and half-life of protein drugs. In another study da Silva Freitas et al 78 In one example, Lyon and coworkers 69,80 reported that loosely crosslinked microgels (composed of a random copolymer of N-isopropylacrylamide (NIPAm) and acrylic acid (AAc)) demonstrated a high loading capacity for protein. These kinds of microgels or microgel-based devices can be potentially used as protein drug delivery systems.…”