Download (rhGH) functional quality characterization by LC/CE-MS and

SOMATROPIN (rhGH) FUNCTIONAL QUALITY
CHARACTERIZATION BY LC/CE-MS AND SAW (SURFACE
ACOUSTIC WAVE) BIOSENSOR TECHNIQUES
Nathalie Bracke1, Evelien Wynendaele1, Rob Haselberg2, Govert W. Somsen2, Sophia Barhdadi1
and Bart De Spiegeleer1,*
1
DruQuaR (Drug Quality & Registration) group, Faculty of Pharmaceutical Sciences, Ghent University,
Harelbekestraat 72, B-9000 Ghent, Belgium. *[email protected]
2 Division of BioAnalytical Chemistry, AIMMS research group BioMolecular Analysis, Faculty of Sciences, VU University,
De Boelelaan 1083, 1081 HV Amsterdam, the Netherlands.
(O. Ref/ 2013-370b)
Somatropin (i.e. recombinant human growth hormone, rhGH) is a biologic drug, approved to
treat growth hormone deficiencies as in pituitary dwarfism. This protein is available on the
market as originator drug, as well as biosimilars, but also as SFFCs (spurious/falselylabelled/falsified/counterfeit). Recently, studies indicate a possible role of hGH in certain
cancers; hence, the oncologic potential of somatropin and analogues/modified proteins is
currently under investigation. Modifications with chelating agents like NOTA allows the
incorporation of radiometals for SPECT/PET-diagnostic or therapeutic purposes. However,
since somatropin has nine potential binding sites (Lys) for NOTA, it is important to characterize
the obtained product under a specified synthesis procedure: direct separation and identification
techniques (i.e. RP-MS and CE-MS) and peptide mapping are used to profile the modified
somatropins (Figure 1).
Figure 1: Analytical characterization of NOTA-modified somatropin. A. MS and MS2 spectra of the
peptide EETQQKSNLELLR with NOTA-modification after tryptic digest of somatropin. The selected
precursor ion is indicated by a black circle. B-C. Analytical results of the substitution degree of NOTAmodified somatropin obtained with RP-C4 HPLC (A) and CE (B). A typical LC chromatogram and CE
electropherogram of the 3:1 NOTA-somatropin sample are given.
Surface acoustic wave (SAW) biosensors allow the user to detect and quantify binding events,
giving information not only on affinity (KD) and kinetics (kon and koff), but also on viscoelastic
effects and conformational changes. SAW thus allows the study of molecular interaction
between an immobilized ligand and flowing analyte without the use of fluorescent or
radiochemical labels. Hence, somatropin and its modified analogues were characterized by their
SAW binding characteristics using hGHAb (Figure 2) and hGHR as immobilized ligands.
Figure 2: Sensorgram of the interaction between immobilized somatropin and hGHAb.
The overall functional quality characterization using SAW is thus a useful additional quality
tool in the development, QC and comparison of new biotechnological drugs and biologics.