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In vitro functional quality characterization of NOTA-modified somatropins

Nathalie Bracke, Han Yao UGent, Evelien Wynendaele UGent, Frederick Verbeke UGent, Xiaolong Xu UGent, Bert Gevaert, Alex Maes, Christophe Van De Wiele UGent, Mike Sathekge, Sarah De Saeger UGent, et al. (2017) ANALYTICAL CHEMISTRY. 89(5). p.2764-2772
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 (4-isS-2-(4-isothiocyanatobenzy1)-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(50) values between 9.53 and 9.78) and efficacy (E-max 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.
Please use this url to cite or link to this publication:
author
organization
year
type
journalArticle (original)
publication status
published
subject
keyword
GROWTH-HORMONE RECEPTOR, NATIVE MASS-SPECTROMETRY, FDA DRUG APPROVALS, OPTICAL BIOSENSORS, PROSTATE-CANCER, ANTIBODY, COMPLEXES, DISCOVERY, INCB018424, ACTIVATION
journal title
ANALYTICAL CHEMISTRY
Anal. Chem.
volume
89
issue
5
pages
2764 - 2772
Web of Science type
Article
Web of Science id
000395964400017
ISSN
0003-2700
1520-6882
DOI
10.1021/acs.analchem.6b03601
language
English
UGent publication?
yes
classification
A1
copyright statement
I have transferred the copyright for this publication to the publisher
id
8517462
handle
http://hdl.handle.net/1854/LU-8517462
date created
2017-04-11 10:23:30
date last changed
2017-07-06 08:18:25
@article{8517462,
  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 (4-isS-2-(4-isothiocyanatobenzy1)-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(50) values between 9.53 and 9.78) and efficacy (E-max 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.},
  author       = {Bracke, Nathalie and Yao, Han and Wynendaele, Evelien and Verbeke, Frederick and Xu, Xiaolong and Gevaert, Bert and Maes, Alex and Van De Wiele, Christophe and Sathekge, Mike and De Saeger, Sarah and De Spiegeleer, Bart},
  issn         = {0003-2700},
  journal      = {ANALYTICAL CHEMISTRY},
  keyword      = {GROWTH-HORMONE RECEPTOR,NATIVE MASS-SPECTROMETRY,FDA DRUG APPROVALS,OPTICAL BIOSENSORS,PROSTATE-CANCER,ANTIBODY,COMPLEXES,DISCOVERY,INCB018424,ACTIVATION},
  language     = {eng},
  number       = {5},
  pages        = {2764--2772},
  title        = {In vitro functional quality characterization of NOTA-modified somatropins},
  url          = {http://dx.doi.org/10.1021/acs.analchem.6b03601},
  volume       = {89},
  year         = {2017},
}

Chicago
Bracke, Nathalie, Han Yao, Evelien Wynendaele, Frederick Verbeke, Xiaolong Xu, Bert Gevaert, Alex Maes, et al. 2017. “In Vitro Functional Quality Characterization of NOTA-modified Somatropins.” Analytical Chemistry 89 (5): 2764–2772.
APA
Bracke, Nathalie, Yao, H., Wynendaele, E., Verbeke, F., Xu, X., Gevaert, B., Maes, A., et al. (2017). In vitro functional quality characterization of NOTA-modified somatropins. ANALYTICAL CHEMISTRY, 89(5), 2764–2772.
Vancouver
1.
Bracke N, Yao H, Wynendaele E, Verbeke F, Xu X, Gevaert B, et al. In vitro functional quality characterization of NOTA-modified somatropins. ANALYTICAL CHEMISTRY. 2017;89(5):2764–72.
MLA
Bracke, Nathalie, Han Yao, Evelien Wynendaele, et al. “In Vitro Functional Quality Characterization of NOTA-modified Somatropins.” ANALYTICAL CHEMISTRY 89.5 (2017): 2764–2772. Print.