Characterisation of a Novel Growth Hormone Variant Comprising a Thioether Link between Cys182 and Cys189

Abstract

A novel variant of recombinant human growth hormone (r-hGH), isolated from biopharmaceutical preparations produced in E. coli, was identified and characterised. This variant contains a nonreducible thioether bridge near the C terminus between Cys182 and Cys189 and was characterised using various analytical techniques. As previous work by Cunningham and Wells (1993) highlighted the involvement of several residues in this part of the sequence in the binding and affinity of the molecule to its receptor, the presence of this modified intramolecular link may have important implications with regard to the biological behaviour of the molecule. Furthermore, as the conversion of a disulfide into a thioether was previously reported for a therapeutic monoclonal antibody (Tous et al., 2005), this may imply that disulfide bridges located in this part of the molecule have a generic susceptibility to thioether formation. This in turn is relevant to the biopharmaceutical industry for monitoring the integrity of disulfide bridges near the protein C terminus. The present study exhibits a state of the art physicochemical investigation for the unequivocal elucidation of a novel structure involving peptide mapping with mass spectrometry and de novo peptide sequencing. Changes in the higher order structure of the molecule were highlighted by near UV circular dichroism and molecular modelling.

IgG1 Thioether Bond Formation in Vivo

“…The thioether (or lanthionine) linkage has been reported in recombinant monoclonal antibodies (16) and recombinant human growth hormone (17,18) as well as in wheat proteins treated with alkaline pH and at a high temperature (6). Although the thioether bond occurs naturally in certain peptide and protein antibiotics and in body organs and tissues (5), its presence in a normal endogenous human protein had not been reported.…”

“…Recently, thioether linkages have been found to form in therapeutic recombinant proteins during production and storage, including monoclonal antibodies (16) and human growth hormone (17,18). Among the 14 disulfide bonds in IgG1, as shown in Fig.…”

Preclinical characterization of eleven new Cys-PEGylated hGH mutants

“…Consistent with these findings we found that substitutions at residues 182 and 189 had a small or negligible effect on biological activity. In contrast with these findings, it has been reported that a byproduct with a thioether link between Cys 182 and Cys 189 that is formed during r-hGH production in bioreactors [26,27], has a significantly decreased receptor affinity and bioactivity in comparison with unmodified hGH [28]. However, this kind of molecular modification is expected to have opposite effects as compared with the bridge opening that we made and to increase the rigidity of the C terminus of GH that is considered one of the most mobile portions of the molecule in normal conditions [27].…”

Advances and challenges in analytical characterization of biotechnology products: Mass spectrometry-based approaches to study properties and behavior of protein therapeutics

“…It has been recently shown that accurate mass measurement in some cases may provide a distinction between an innovator mAb product and its biosimilar version (Xie et al 2010). Mass measurement also allows the heterogeneity of the protein preparation to be assessed, revealing the presence of various protein isoforms with different PTMs (Bondarenko et al 2009)or contaminating protein species (Datola et al 2007). In the case of multi-unit protein therapeutics (such as mAbs or antibody-based products) mass measurements following reduction of disulfide bonds and alkylation of cysteine residues can be used to confirm the structure of individual subunits (Chelius et al 2010).…”