In vitro functional quality characterization of NOTA-modified somatropins

Show simple item record Bracke, Nathalie Yao, Han Wynendaele, Evelien Verbeke, Frederick Xu, Xiaolong Gevaert, Bert Maes, Alex Van de Wiele, Christophe Sathekge, Mike Machaba De Saeger, Sarah De Spiegeleer, Bart 2017-08-25T08:28:35Z 2017-03
dc.description.abstract 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 (pEC50 values between 9.53 and 9.78) and efficacy (Emax 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. en_ZA
dc.description.department Nuclear Medicine en_ZA
dc.description.embargo 2018-03-07
dc.description.librarian hj2017 en_ZA
dc.description.sponsorship Grants from the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen) to B.G. (Grant 121512) and F.V. (Grant 131356), and the PhD research grants from China Scholarship Council (CSC) to H.Y. and X.X. en_ZA
dc.description.uri en_ZA
dc.identifier.citation Bracke, N., Yao, H., Wynendaele, E., Verbeke, F., Xu, X., Gevaert, B., Maes, A., Van de Wiele, C., Sathekge, M., De Saeger, S. & De Spiegeleer, B. 2017, 'In vitro functional quality characterization of NOTA-modified somatropins', Analytical Chemistry, vol. 89, no. 5, pp. 2764-2772. en_ZA
dc.identifier.issn 0003-2700 (print)
dc.identifier.issn 1520-6882 (online)
dc.identifier.other 10.1021/acs.analchem.6b03601
dc.language.iso en en_ZA
dc.publisher American Chemical Society en_ZA
dc.rights This document is the Accepted Manuscript version of a Published Work that appeared in final form in Bioconjugate Chemistry, © 2017 American Chemical Society after peer review and technical editing by the publisher. en_ZA
dc.subject Quality control en_ZA
dc.subject Substitution degree en_ZA
dc.subject Recombinant human growth hormones en_ZA
dc.subject Physico-chemical characterization en_ZA
dc.subject Native mass spectrometries en_ZA
dc.subject Multiple substitutions en_ZA
dc.subject Inherent complexity en_ZA
dc.subject Human growth hormone en_ZA
dc.subject Functional characterization en_ZA
dc.subject Size exclusion chromatography en_ZA
dc.subject Signal transduction en_ZA
dc.subject Proteins en_ZA
dc.subject Mass spectrometry en_ZA
dc.subject Isomers en_ZA
dc.subject Drug products en_ZA
dc.subject Dichroism en_ZA
dc.subject Chromatography en_ZA
dc.subject Chemical modification en_ZA
dc.subject Chemical analysis en_ZA
dc.subject Chelation en_ZA
dc.subject Characterization en_ZA
dc.subject Biomaterials en_ZA
dc.subject Bioassay en_ZA
dc.subject Bins en_ZA
dc.title In vitro functional quality characterization of NOTA-modified somatropins en_ZA
dc.type Postprint Article en_ZA

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