Analytical characterization of NOTA-modified somatropins

Area of Science:

• Bioconjugation Chemistry
• Radiopharmaceutical Development
• Protein Engineering

Background:

• Chemical modification of biomolecules, such as proteins, can result in heterogeneous products.
• Understanding the extent and nature of modification is crucial for interpreting bioconjugate properties, including pharmacokinetics and target binding affinity.

Purpose of the Study:

• To synthesize and characterize somatropin modified with the acylating chelator p-SCN-Bn-NOTA.
• To identify the modification sites and degree of substitution on somatropin.
• To evaluate the suitability of the modified somatropin for radiopharmaceutical applications.

Main Methods:

• Synthesis of somatropin modified with p-SCN-Bn-NOTA.
• Analytical characterization using reversed-phase mass spectrometry (RP-MS) and capillary electrophoresis-mass spectrometry (CE-MS).
• Peptide mapping following trypsin and chymotrypsin digestion.
• In silico pKa calculations and analysis of somatropin crystal structure.

Main Results:

• Higher amounts of p-SCN-Bn-NOTA led to complex product mixtures with varying degrees of substitution and positional isomers.
• Lysine-70 (Lys-70) was identified as the primary modification site, supported by its lowest calculated pKa value (8.3).
• Lys-70 is located outside the growth hormone receptor binding pocket, minimizing interference with receptor binding. Gallium chelation by NOTA-somatropin achieved 100% complexation.

Conclusions:

• The developed synthesis procedure for NOTA-somatropin is suitable for producing target-specific radiopharmaceuticals.
• This modified somatropin holds potential for further investigation in the treatment and visualization of growth hormone-specific cancers.