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 (pEC values between 9.53 and 9.78) and efficacy (E 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.

Biological Characterisation of Somatropin-Derived Cryptic Peptides

“…During the native MS study, concentrations of 666 nM SDP were used, identical to the concentrations used for somatropin in native MS (Bracke et al 2017). At these SDP concentrations, no signals for a peptide:hGHBp complex were detected.…”

“…The native MS method was essentially similar as previously described (Bracke et al 2017): the non-denaturing MS data of standards (i.e. single peptides/proteins: all SDPs and hGHBp) and complex samples (i.e.…”

“…The PathHunter Cytosolic Tyrosine Kinase (CTK) Functional Assay (JAK2 target) was used for the profiling of the SDPs in agonist and antagonist format (Bracke et al 2017). The cells were seeded in a total volume of 20 µL…”

Thermal sensitivity as a quality control attribute for biotherapeutics: The L‐asparaginase case

“…For the temperature gradient test, the gradient was set at 80°C/h in a range of 20°C to 80°C (with a temperature interval measurement of 5°C). T m is the temperature at which protein possesses 50% of folded fraction, determined by evaluating the CD signal at a wavelength of 222 nm in function of temperature . The maximum point on the first derivative curve corresponds to T m .…”

“…CD spectroscopy, a light absorption spectroscopy which determines the difference in the absorbance of right and left circularly polarized light of a substance, is an interesting technique in pharmaceutical quality evaluation, especially for proteins . It is used to quantify the secondary structure composition of proteins, as well as to measure the ligand interaction .…”