title: In Vitro Functional Quality Characterization of NOTA-Modified Somatropins.
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 (pEC50values between 9.53 and 9.78) and efficacy (Emaxvalues 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.
authors.affiliation: Drug Quality and Registration (DruQuaR) Group, Department of Pharmaceutical Analysis, Faculty of Pharmaceutical Sciences, Ghent University , B-9000 Ghent, Belgium.
authors.forename: Nathalie
authors.initials: N
authors.lastname: Bracke
authors.affiliation: Drug Quality and Registration (DruQuaR) Group, Department of Pharmaceutical Analysis, Faculty of Pharmaceutical Sciences, Ghent University , B-9000 Ghent, Belgium.
authors.forename: Han
authors.initials: H
authors.lastname: Yao
authors.affiliation: Drug Quality and Registration (DruQuaR) Group, Department of Pharmaceutical Analysis, Faculty of Pharmaceutical Sciences, Ghent University , B-9000 Ghent, Belgium.
authors.forename: Evelien
authors.initials: E
authors.lastname: Wynendaele
authors.affiliation: Drug Quality and Registration (DruQuaR) Group, Department of Pharmaceutical Analysis, Faculty of Pharmaceutical Sciences, Ghent University , B-9000 Ghent, Belgium.
authors.forename: Frederick
authors.initials: F
authors.lastname: Verbeke
authors.affiliation: Drug Quality and Registration (DruQuaR) Group, Department of Pharmaceutical Analysis, Faculty of Pharmaceutical Sciences, Ghent University , B-9000 Ghent, Belgium.
authors.forename: Xiaolong
authors.initials: X
authors.lastname: Xu
authors.affiliation: Drug Quality and Registration (DruQuaR) Group, Department of Pharmaceutical Analysis, Faculty of Pharmaceutical Sciences, Ghent University , B-9000 Ghent, Belgium.
authors.forename: Bert
authors.initials: B
authors.lastname: Gevaert
authors.affiliation: AZ Groeninge, Department of Nuclear Medicine, Catholic University Leuven , B-8500 Kortrijk, Belgium.
authors.forename: Alex
authors.initials: A
authors.lastname: Maes
authors.affiliation: Department of Radiology and Nuclear Medicine, Ghent University , B-9000 Ghent, Belgium.
authors.forename: Christophe
authors.initials: C
authors.lastname: Van de Wiele
authors.affiliation: Department of Nuclear Medicine, Steve Biko Academic Hospital, University of Pretoria , Pretoria, 0002, South Africa.
authors.forename: Mike
authors.initials: M
authors.lastname: Sathekge
authors.affiliation: Laboratory of Food Analysis, Department of Bioanalysis, Faculty of Pharmaceutical Sciences, Ghent University , B-9000 Ghent, Belgium.
authors.forename: Sarah
authors.identifier: 0000-0002-2160-7253
authors.initials: S
authors.lastname: De Saeger
authors.affiliation: Drug Quality and Registration (DruQuaR) Group, Department of Pharmaceutical Analysis, Faculty of Pharmaceutical Sciences, Ghent University , B-9000 Ghent, Belgium.
authors.forename: Bart
authors.identifier: 0000-0002-6103-3565
authors.initials: B
authors.lastname: De Spiegeleer
mesh_terms: D001681:Biological Assay; D002850:Chromatography, Gel; D002942:Circular Dichroism; D005708:Gallium / Q000737:chemistry; D006571:Heterocyclic Compounds / Q000737:chemistry*; D006573:Heterocyclic Compounds, 1-Ring; D019382:Human Growth Hormone / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification
chemical_list: D006571:Heterocyclic Compounds; D006573:Heterocyclic Compounds, 1-Ring; D008565:Membrane Proteins; D011994:Recombinant Proteins; C405010:delta-hGHR; D019382:Human Growth Hormone; C048993:1,4,7-triazacyclononane-N,N',N''-triacetic acid; D005708:Gallium