Analytical characterization of NOTA-modified somatropins

Bracke, Nathalie; Wynendaele, Evelien; D׳Hondt, Matthias; Haselberg, Rob; Somsen, Govert W.; Pauwels, Ewald; Wiele, Christophe Van de; Spiegeleer, Bart De

doi:10.1016/j.jpba.2014.03.014

Cited by 11 publications

(26 citation statements)

References 61 publications

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“…All NOTA-modified somatropins were in a 2:1 complex when approximately 2-4.5 times molar excess of hGHBp was used ( Figure 1C and 1D), which demonstrated the bindingfunctionality and dimerization capacity of these NOTA-modified somatropins, as well as of the gallium chelated NOTA-modified somatropin towards hGHBp ( Figure 1D) (Figure S3). In general, the observed percentage substitution degree within the NOTA-modified somatropin samples (Figure 2E and Figure S4) are in agreement with previously reported data 8 . The spectra of the standard and sample complex solutions are given in Figure 2.…”

Section: Analytical Size-exclusion Chromatographysupporting

confidence: 92%

“…The synthesis procedure of 1:1 NOTA:somatropin, 3:1 NOTA:somatropin and 10:1 NOTA:somatropin was previously detailed by Bracke et al 8 The data between m/z 3000-4000 of the complex sample and complex standard were further analysed using a Python program. Data were smoothed using a moving average filter (301 points).…”

Section: Synthesis Of Nota-modified Somatropin and Complexation With mentioning

confidence: 99%

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In Vitro Functional Quality Characterization of NOTA-Modified Somatropins

et al. 2017

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Chemical modifications on protein biopharmaceuticals introduce extra variability in addition to their inherent complexity, hence require more comprehensive analytical and functional characterization during their discovery, development, and manufacturing. Somatropin (i.e., recombinant human growth hormone, rhGH) modified with the chelating agent S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA) allows the incorporation of radiometals for research and possible theranostic purposes. We previously demonstrated that this conjugation leads to multiple substitution degrees and positional isomers within the product. In vitro techniques at the molecular and cellular levels were now applied to assess their functional quality: (i) size exclusion chromatography (SEC) demonstrated functional complexation with human growth hormone binding protein (hGHBp) to the different NOTA-modified somatropins as well as to gallium chelated NOTA-functionalities (Ga-10:1 NOTA-somatropin); (ii) native mass spectrometry (MS) offered in-depth information, a substitution degree up to four NOTAs was still functional; (iii) circular dichroism (CD) analysis confirmed the complexation of unmodified and NOTA-modified somatropin to hGHBp; and (iv) a hGHR bioassay demonstrated initiation of the signal transduction cascade, after binding of all investigated products to the receptor presented on cells with a similar potency (pEC values between 9.53 and 9.78) and efficacy (E values between 130 and 160%). We conclude that the NOTA-modified somatropins do not possess a significantly different in vitro functionality profile compared to unmodified somatropin. Techniques such as SEC, MS, and CD, traditionally used in the physicochemical characterization of proteins have a demonstrated potential use in the functionality evaluation not only in drug discovery and development but also in quality control settings.

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Section: Analytical Size-exclusion Chromatographysupporting

confidence: 92%

Section: Synthesis Of Nota-modified Somatropin and Complexation With mentioning

confidence: 99%

In Vitro Functional Quality Characterization of NOTA-Modified Somatropins

et al. 2017

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“…The novelty of the selected SDPs was confirmed by proteolytic digestion (S-carboxymethylation of cysteine residues) of somatropin by trypsin, chymotrypsin, S. aureus V8 protease, PC1, PC2, and IDE were used following the method as previously reported in (Bracke et al 2014). Digestion was performed at 37°C while gently shaking at 300 rpm for the defined period of time in a thermomixer comfort (Eppendorf, Hamburg, Germany).…”

Section: In Vitro Proteolytic Digestions Of Somatropinmentioning

confidence: 99%

“…One hundred microliter samples were taken after 0 h, 4 h, 24 h, 48 h and the reaction was stopped by addition of 20 µL formic acid (FA) to each sample. The samples were analysed by LC-MS as described in the peptide mapping section of reference (Bracke et al 2014).…”

Section: In Vitro Proteolytic Digestions Of Somatropinmentioning

confidence: 99%

Biological Characterisation of Somatropin-Derived Cryptic Peptides

Tack

Bracke

Verbeke

et al. 2018

Int J Pept Res Ther

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Little is known about possible cryptic peptides of the recombinant growth hormone (somatropin). In this study, six synthetic somatropin-derived peptides (SDPs) were selected based on their sequences which correspond to the binding interface of the growth hormone receptor. Their novelty was confirmed by in silico and in vitro proteolytic digestion of somatropin. Chemical characterisation of the SDPs, i.e. identification via LC-MS and purity quantification via HPLC-UV and U(H)PLC-MRM, was first performed. All the SDPs were stable in brain tissue homogenate, liver tissue homogenate and serum (t1/2 > 15 min). The metabolites in brain and serum, formed between 15 min and 120 min, were also identified. The interactions towards the growth hormone receptor (GHR) and the human growth hormone binding protein (hGHBp) were also evaluated using GHR bioassay and native MS. No interaction was detected under the applied conditions. A last part of the study investigated the pharmacokinetics and tissue distribution of two peptides (i.e. SDP167-175 and SDP101-121), selected based on their position within somatropin. A high blood-brain barrier (BBB) influx was observed for SDP101-121, while SDP167-175 showed a negligible BBB influx. Based on the obtained results, the GHR binding of the selected SDPs is very low, requiring structural adaptations for further GHR-binding exploration.

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“…For intact proteins, CE-MS has been most commonly used to characterize and identify potential naturally-occurring isoforms [55][56][57][58] or modifications [59][60][61] of the protein.…”

Section: Top-down Proteomicsmentioning

confidence: 99%