Analytical characterization of NOTA-modified somatropins

dc.contributor.authorBracke, Nathalie
dc.contributor.authorWynendaele, Evelien
dc.contributor.authorD’Hondt, Matthias
dc.contributor.authorHaselberg, Rob
dc.contributor.authorSomsen, Govert W.
dc.contributor.authorPauwels, Ewald
dc.contributor.authorVan de Wiele, Christophe
dc.contributor.authorDe Spiegeleer, Bart
dc.date.accessioned2017-03-03T06:07:30Z
dc.date.available2017-03-03T06:07:30Z
dc.date.issued2014-08
dc.description.abstractChemical modification of biomolecules like the introduction of metal-chelators into proteins can lead to heterogeneous product formation. The nature and extend of the modification is important in interpreting the biological properties of the bioconjugate, given their possible influence on the pharmacokinetics as well as on the binding affinity to the target. The present study describes the synthesis and analytical characterization of somatropin modified on its lysine’s ε-amino groups with the acylating chelator S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p- SCN-Bn-NOTA). Direct separation and identification techniques (i.e. RP-MS and CE-MS) and peptide mapping after trypsin and chymotrypsin digestion demonstrated that the use of higher amounts of p-SCN-Bn-NOTA during synthesis leads to a complex product composition with higher order substitution degrees (i.e. multiple NOTA-moieties per somatropin molecule), as well as the presence of different position isomers. From the nine lysine (Lys) residues in somatropin, Lys-70 was experimentally found to be the modification hotspot under our synthesis conditions (pH=9.0). This was supported by the in silico calculated lowest pKa value of 8.3 for Lys-70. Based on the crystal structure of somatropin in complex with the extracellular parts of the growth hormone receptor, the Lys-70 residue is positioned outside the binding pockets and will therefore not directly interfere with receptor binding. Gallium chelation by NOTA-somatropin resulted in a 100% complexation. The synthesis of NOTA-somatropin using p-SCN-Bn-NOTA and somatropin under our operational conditions is therefore a suitable synthesis procedure for the production of a target-specific radiopharmaceutical for further investigation towards treatment and visualization of growth hormonespecific cancers.en_ZA
dc.description.departmentNuclear Medicineen_ZA
dc.description.librarianhb2017en_ZA
dc.description.sponsorshipThis research project was supported by grants from Ghent University (BOF special research fund 01J22510) to BDS and EW and the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen) to MD (101529).en_ZA
dc.description.urihttp://www.elsevier.com/locate/jpbaen_ZA
dc.identifier.citationBracke, N, Wynendaele, E, D’Hondt, M, Haselberg, R, Somsen, GW, Pauwels, E, Van de Wiele, C & De Spiegeleer, B 2014, 'Analytical characterization of NOTA-modified somatropins', Journal of Pharmaceutical and Biomedical Analysis, vol. 96, pp. 1-9.en_ZA
dc.identifier.issn0731-7085 (print)
dc.identifier.issn1873-264X (online)
dc.identifier.other10.1016/j.jpba.2014.03.014
dc.identifier.urihttp://hdl.handle.net/2263/59249
dc.language.isoenen_ZA
dc.publisherElsevieren_ZA
dc.rights© 2014 Elsevier B.V. All rights reserved. Notice : this is the author’s version of a work that was accepted for publication in Journal of Pharmaceutical and Biomedical Analysis. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. A definitive version was subsequently published in Journal of Pharmaceutical and Biomedical Analysis, vol. 96, pp. 1-9, 2014. doi : 10.1016/j.jpba.2014.03.014.en_ZA
dc.subjectNOTA modificationen_ZA
dc.subjectSomatropinen_ZA
dc.subjectPeptide mappingen_ZA
dc.subjectLiquid chromatography–mass spectrometry (LC-MS)en_ZA
dc.subjectCapillary electrophoresis–mass spectrometry (CE-MS)en_ZA
dc.titleAnalytical characterization of NOTA-modified somatropinsen_ZA
dc.typePostprint Articleen_ZA

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