Heterogeneity of Commercial Recombinant Human Growth Hormone (r-hGH) Preparations Containing a Thioether Variant

Lispi, Monica; Datola, Antonio; Bierau, Horst; Ceccarelli, D; Crisci, C.; Minari, Katia; Mendola, D; Regine, Aniello; Ciampolillo, Cinzia; Rossi, Mara; Giartosio, Carlo Emanuele; Pezzotti, Anna Rita; Musto, Raffaella; Jone, Carl S.; Chiarelli, Francesco

doi:10.1002/jps.21774

Cited by 13 publications

(15 citation statements)

References 22 publications

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“…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.…”

Section: Discussionmentioning

confidence: 99%

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IgG1 Thioether Bond Formation in Vivo

Zhang

Schenauer

McCarter

et al. 2013

Journal of Biological Chemistry

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Background: Thioethers have been observed in therapeutic antibodies, with increasing levels upon storage. Results: IgG1 thioether bond formation is naturally occurring, but the formation rate depends on light chain type. Conclusion: Slower thioether bond formation on IgG1 is caused by dehydrogenation impairment through its light chain. Significance: Safety concerns associated with thioether control on therapeutic antibodies are diminished by its natural production.

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Section: Discussionmentioning

confidence: 99%

“…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.…”

mentioning

confidence: 99%

IgG1 Thioether Bond Formation in Vivo

Zhang

Schenauer

McCarter

et al. 2013

Journal of Biological Chemistry

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“…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].…”

Section: Discussionmentioning

confidence: 67%

Preclinical characterization of eleven new Cys-PEGylated hGH mutants

Selis¹,

Genovese

Salis³

et al. 2016

European Journal of Molecular &Amp; Clinical Medicine

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We synthesized and tested for biological activity eleven new PEGylated hGH derivatives. To this aim we used different strategies. A first group of PEGylable Cys derivatives was prepared by mutating into Cys single specific aminoacid residues located either in the connecting loop between helices 1 and 2 (Ile36 and Phe44) or in the connecting loop between helices 3 and 4 (Ile138 and Phe146). A second group of mutants was synthetized by inserting new Cys close to the aforementioned positions. Other PEGylable mutants were prepared by inserting a Gly4-Cys chain either at the N- or at the C-terminus of the hGH molecule. Finally, a Cys residue of the second hGH disulfide bond (Cys182-Cys189) was made available for PEGylation by mutating its companion Cys to open up the disulfide bridge. All these mutants were tested for their ability to affect Nb2 cell proliferation in vitro and showed different effects. Two mutations (Phe44Cys and +Cys47) severely impaired and two (Ile138Cys and Phe146Cys) increased hGH bioactivity. The mutation Cys189Ser was functionally silent whereas the remaining mutations caused a 15-50% decrease in activity. 20kDa-pegylation caused a dramatic decrease in bioactivity in all mutants. The r-hGH-(Ile138Cys)-Cys138-PEG derivative showed the highest activity (15% of wild-type unpegylated hGH) and was selected for pharmacockinetic studies in vivo. It showed a fivefold longer half-life and was significantly more effective than wild-type hGH in causing weight gain when given subcutaneously twice a week to hypophysectomized rats. In conclusion, even when it is directed to residues supposed to have a marginal role in the activity of this hormone, 20 kDa pegylation has detrimental effect on hGH bioactivity. These effects may be counterbalanced by the increase in half-life as it happens in pegylated Ile138Cys-hGH that could represent a promising new long-acting hGH derivative. Focal points:•Bedside: Current therapy of GH deficiency still has the important limitation of being delivered by daily subcutaneous injections and this reduces the compliance of the small pediatric patients. There is, therefore interest in developing long-acting GH derivative such as the new ones that we present here. •Benchside: By investigating the effect of Cys substitutions at aminoacidic positions that had not been investigated in previous studies, we obtained evidence that Ile138 and Phe146 have a role in GH bioactivity in vitro. •Industry: The mono-pegylated hGH mutant described in this work represents a promising candidate for human clinical pharmacology studies as long-acting derivative of h-GH.•Community: By decreasing the frequency of GH injections, long acting GH could decrease the costs of GH treatment and increase medication adherence

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“…Hence, dithiohemiacetal structures in insulin must be considered as potential origin for secondary products, which can form during storage but potentially also after administration of the protein. In this regard it is important to note that a thioether variant of hGH had a significantly reduced affinity to its receptor (23), indicating that the mere removal of a sulfur atom from a disulfide bond can have significant conformational consequences.…”

Section: Nature Of the Photoproductsmentioning

confidence: 99%

“…Evidence for dithiohemiacetal formation was presented also for the photoirradiation of an immunoglobulin, IgG1, in solution (18). Earlier studies on the degradation of human growth hormone (hGH) had documented the formation of a thioether variant and a reduced affinity of such thioether variant of hGH to its respective receptors (23,24). The important question is whether such products are also formed during photoirradiation of protein solids.…”

Section: Introductionmentioning

confidence: 97%