Analytical characterization of NOTA-modified somatropins.
Chemical modification of biomolecules like the introduction of metal-chelators into proteins can lead to heterogeneous product formation. The nature and extend of the modification is important in interpreting the biological properties of the bioconjugate, given their possible influence on the pharmacokinetics as well as on the binding affinity to the target. The present study describes the synthesis and analytical characterization of somatropin modified on its lysine's ɛ-amino groups with the acylating chelator S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA). Direct separation and identification techniques (i.e. RP-MS and CE-MS) and peptide mapping after trypsin and chymotrypsin digestion demonstrated that the use of higher amounts of p-SCN-Bn-NOTA during synthesis leads to a complex product composition with higher order substitution degrees (i.e. multiple NOTA-moieties per somatropin molecule), as well as the presence of different position isomers. From the nine lysine (Lys) residues in somatropin, Lys-70 was experimentally found to be the modification hotspot under our synthesis conditions (pH=9.0). This was supported by the in silico calculated lowest pKa value of 8.3 for Lys-70. Based on the crystal structure of somatropin in complex with the extracellular parts of the growth hormone receptor, the Lys-70 residue is positioned outside the binding pockets and will therefore not directly interfere with receptor binding. Gallium chelation by NOTA-somatropin resulted in a 100% complexation. The synthesis of NOTA-somatropin using p-SCN-Bn-NOTA and somatropin under our operational conditions is therefore a suitable synthesis procedure for the production of a target-specific radiopharmaceutical for further investigation toward treatment and visualization of growth hormone-specific cancers.
Drug Quality and Registration (DruQuaR) group, Department of Pharmaceutical Analysis, Faculty of Pharmaceutical Sciences, Ghent University, Harelbekestraat 72, 9000 Ghent, Belgium.
Nathalie
N
Bracke
Drug Quality and Registration (DruQuaR) group, Department of Pharmaceutical Analysis, Faculty of Pharmaceutical Sciences, Ghent University, Harelbekestraat 72, 9000 Ghent, Belgium.
Evelien
E
Wynendaele
Drug Quality and Registration (DruQuaR) group, Department of Pharmaceutical Analysis, Faculty of Pharmaceutical Sciences, Ghent University, Harelbekestraat 72, 9000 Ghent, Belgium.
Matthias
M
D'Hondt
Division of BioAnalytical Chemistry, AIMMS research group BioMolecular Analysis, Faculty of Sciences, VU University, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.
Rob
R
Haselberg
Division of BioAnalytical Chemistry, AIMMS research group BioMolecular Analysis, Faculty of Sciences, VU University, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.
Govert W
GW
Somsen
Center for Molecular Modeling, Ghent University, Technologiepark 903, 9052 Zwijnaarde, Belgium.
Ewald
E
Pauwels
Department of Nuclear Medicine, Ghent University Hospital, 9000 Ghent, Belgium.
Christoph
C
Van de Wiele
Drug Quality and Registration (DruQuaR) group, Department of Pharmaceutical Analysis, Faculty of Pharmaceutical Sciences, Ghent University, Harelbekestraat 72, 9000 Ghent, Belgium. Electronic address: Bart.DeSpiegeleer@UGent.be.
Bart
B
De Spiegeleer
D002614:Chelating Agents / Q000737:chemistry; D003198:Computer Simulation; D003460:Crystallization; D006571:Heterocyclic Compounds / Q000737:chemistry*; D006573:Heterocyclic Compounds, 1-Ring; D019382:Human Growth Hormone / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D017879:Isothiocyanates / Q000737:chemistry*; D010449:Peptide Mapping / Q000379:methods; D019275:Radiopharmaceuticals / Q000138:chemical synthesis / Q000737:chemistry
D002614:Chelating Agents; D006571:Heterocyclic Compounds; D006573:Heterocyclic Compounds, 1-Ring; D017879:Isothiocyanates; D019275:Radiopharmaceuticals; D019382:Human Growth Hormone; C082060:2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid; C048993:1,4,7-triazacyclononane-N,N',N''-triacetic acid
CE–MS; LC–MS; NOTA modification; Peptide mapping; Somatropin