Development and Validation of Methods for Assessing the Quality of a Chemical Precursor – a Recombinant Protein Based on Ankyrin Repeats – for EGFR-Targeted Radionuclide Tumor Imaging

Yanovich G. E.¹²
postgraduate student, Department of Pharmaceutical Analysis, engineer

Prach A. A.²
engineer

Kadyrova T. V.¹
PhD in Pharm. Sci., Associate Professor, Department of Pharmaceutical Analysis

Krivoshchekov S. V.¹
PhD in Chem. Sci., Associate Professor, Department of Pharmaceutical Analysis

Shulga A. A.²³
PhD in Biol. Sci., senior researcher, leading researcher

Konovalova E. V.²³
engineer, junior researcher

Ziganshin R. Kh.³
senior researcher

Deev S. M.²³
Doctor of Biol. Sci., leading researcher

Belousov M. V.¹²
Doctor of Pharm. Sci., Professor, Head of the Department of Pharmaceutical Analysis, Professor, Engineering School of Chemical and Biomedical Technologies

Larkina M. S.¹²
Doctor of Pharm. Sci., Professor, Department of Pharmaceutical Analysis, senior researcher

1Siberian State Medical University (SibMed), 2, Moskovsky Trakt, Tomsk, 634050, Russia
2National Research Tomsk Polytechnic University (TPU), 30, Lenin Ave., Tomsk, 634050, Russia
3Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 16/10, Miklukho-Maklaya St., GSP-7, Moscow, 117997, Russia

Gleb E. Yanovich, e-mail: sonne_gleb@mail.ru

The study had no sponsorship.

The authors declare no conflict of interest.

09.05.2026

10.06.2026

Introduction. Overexpression of epidermal growth factor receptor (EGFR) is characteristic of a number of malignant neoplasms, making it an important molecular target for targeted radionuclide imaging. The designed ankyrin repeat protein DARPin (HE)₃-E01 with subnanomolar affinity to EGFR is considered as a promising chemical precursor for the preparation of radiopharmaceuticals labeled with technetium-99m or iodine-123. To be introduced into pharmaceutical practice, a set of validated quality control methods for this protein is required. Objective. To develop and validate methods for identification, purity assessment, and quantification of the DARPin (HE)₃-E01 protein.

Materials and Methods. Identity was confirmed by liquid chromatography–electrospray ionization mass spectrometry (LC-ESI/MS) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Quantification was performed using direct UV spectrophotometry at 280 nm and the bicinchoninic acid (BCA) colorimetric assay. Validation was conducted according to the criteria of specificity, linearity, accuracy (standard addition method), and precision (repeatability, intermediate precision). The two quantitative methods were compared using the F-test and t-test. Results. Mass spectrometric analysis showed complete agreement between the experimental molecular mass (18,260.29 Da) and the theoretically expected value (18,260.25 Da). Electrophoretic determination of molecular weight gave a value of 18.1 kDa, and the protein purity was at least 99%, with impurity content not exceeding 1%. Both quantitative methods demonstrated linearity over the studied concentration range (correlation coefficients of 0.9979 and 0.9997), high accuracy (CV not more than 2% for both methods), and precision (RSD <2%). No statistically significant differences were found between the results of the two methods (p > 0.05). Conclusion. The developed methods for identification and quantification of DARPin (HE)₃-E01 have been successfully validated and can be included in the draft regulatory documentation for quality control of the chemical precursor intended for the preparation of EGFR-targeted radiopharmaceuticals.

DARPin, EGFR, chemical precursor, quality control, method validation

10.29234/2308-9113-2026-14-3-72-87

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