In vitro functional quality characterization of NOTA-modified somatropins
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Date
Authors
Bracke, Nathalie
Yao, Han
Wynendaele, Evelien
Verbeke, Frederick
Xu, Xiaolong
Gevaert, Bert
Maes, Alex
Van de Wiele, Christophe
Sathekge, Mike Machaba
De Saeger, Sarah
Journal Title
Journal ISSN
Volume Title
Publisher
American Chemical Society
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.
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Keywords
Quality control, Substitution degree, Recombinant human growth hormones, Physico-chemical characterization, Native mass spectrometries, Multiple substitutions, Inherent complexity, Human growth hormone, Functional characterization, Size exclusion chromatography, Signal transduction, Proteins, Mass spectrometry, Isomers, Drug products, Dichroism, Chromatography, Chemical modification, Chemical analysis, Chelation, Characterization, Biomaterials, Bioassay, Bins
Sustainable Development Goals
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.