Development of quality control techniques of chemical precursor – recombinant protein with ankyrin repeats for radionuclide imaging of HER2 overexpression in malignant tumors

Introduction. Overexpression of the HER2 receptor is associated with aggressive cancer progression and poor prognosis. Traditional immunohistochemical diagnostics have a number of limitations, including invasiveness, inability to assess tumor heterogeneity, and total spread of the process. A promising alternative is radionuclide imaging using targeted scaffold DARPin G3 proteins. Based on the results of preclinical studies, the drug 99m Tc[Tc]-G3-(G 3 S) 3 C has been proposed for Phase I clinical trials. The experimental drug is a sterile lyophilizate of a chemical precursor in a single vial and must comply with the requirements of OFS 1.11.0005 "Chemical Precursors for Radiopharmaceuticals" and OFS 1.7.1.0007.15 "Medicinal Products Obtained by Recombinant DNA Methods." Aim. The aim of the present study was to develop approaches and techniques for quality control of DARPin G3-(G 3 S) 3 C protein included in the developed composition of chemical precursor lyophilizate lyophilizate for preparation of 99m Tc-containing preparation for radionuclide imaging of HER2 overexpression in malignant tumors Materials and methods. A lyophilizate containing DARPin G3-(G 3 S) 3 C with excipients was prepared for quality control. HPLC with a tandem mass spectrometer equipped with an electrospray ionization source was used to record mass spectra. Vertical electrophoresis with SDS in a heterogeneous buffer system was carried out in a polyacrylamide gel with an acrylamide concentration of 15 % at 110 V for 2 hours. Coomassie B-250 solution was used as the dye solution. Peptide mapping was performed using an HPLC system with a mass spectrometer via a nano-electrospray source. When correlating the amino acid sequence, carbamidomethylation of Cys, deamidation of Asn/Gln and oxidation of Met were counted as variable modifications. UV spectrophotometry was performed to determine the Quantification index at a wavelength of 280 nm. Validation evaluation of the developed method was carried out in accordance with the requirements of OFS.1.1.0012 "Validation of analytical methods". Results and discussion. The authenticity and purity of DARPin G3-(G 3 S) 3 C was confirmed using HPLC-MS. The purity of the protein was close to 100 %. Application of 2 μg of protein for electrophoresis in polyacrylamide gel showed a distinct stain allowing the authenticity of DARPin G3-(G 3 S) 3 C to be established. To determine the homodimer impurity, 10 μg of protein should be applied. The purity of the protein established by the peptide mapping method is 98 %. More than 700 variations of peptide fragments were identified by protein hydrolysate analysis. The value of –10LogP was more than 20. The developed UV-visible spectrophotometry technique meets the validation requirements and allows the quantification of DARPin G3-(G 3 S) 3 C protein in the lyophilizer ±10 % of the nominal content. Conclusion. To determine the "Genuineness" of a new chemical precursor of DARPin G3-(G 3 S) 3 C protein in the lyophilizate for the preparation of 99m Tc-containing preparation for radionuclide imaging of HER2 overexpression in malignant tumors the following methods were proposed: mass spectrometry, peptide mapping, electrophoresis in polyacrylamide gel. For the indicator "Related impurities" – the technique of electrophoresis in polyacrylamide gel. For the indicator "Quantitative determination" the method of UV-spectrophotometry.