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Background One of the potent immune modulators that is within the scope of medicine interest for over 50 years is bovine thymus extract called thymomodulin. The role of thymus gland and its secreted compounds with pleiotropic activity are undisputed in proper functioning of the immune system. Thymus extract contains numerous peptides including thymosins. 1 Immunomodulatory activity of thymomodulin and the synthetic analogues of thymosins as well as their use in supportive therapy in different disorders have been documented in immune dysregulation.2 Available on the market are drug formulations containing synthetic peptides as an active ingredient as well as numerous dietary supplements with thymus derived components. Rapidly developing supplements sales market is placing increasing emphasis on a qualitative approach to the control of such products. In this study report we present the biochemical characteristics of the biologically active bovine thymus extract to promote further standardizations of preparations.Methods Thymus extract was obtained from Biofac A/S, Kastrup, Denmark. Biochemical composition of the preparation was determined including protein (Kjeldahl method), sugar (Luff-Schoorl reagent), and lipid content (Soxhlet test). Absorbance and fluorescence properties were assessed using SpectraMax iD3 (Molecular Devices) apparatus. Protein pattern of thymus extract was assessed by liquid chromatography on HiLoad Superdex 75 16/600 column (Cytiva, Sweden) and SDS-PAGE. Targeted analysis of thymosin b4 presence was determined by ELISA and untargeted peptide composition by mass spectrometry followed by database identification using Mascot Server (version 2.8, Matrixscience). Biological activity of the thymomodulin was assessed using ELISA for quantification of cytokines released by macrophages derived from Human monocytic THP-1 cell line (American Tissue Culture Collection, TIB-202).Results High solubility in aqueous solutions (up to 450 mg/mL) and protein content of 40% is typical for tested thymomodulin material. Size exclusion chromatography profile shows prevalence of proteins and peptides below 17 kDa of molecular weight, which is in agreement with SDS-PAGE analysis. Targeted ELISA confirmed presence of thymosin β4, but it was undetected by untargeted mass spectrometry, unlike other bovine and thymus proteins that can serve as standardization factors. Thymomodulin stimulated cytokine production (TNF α and IL-1β) in THP-1 derived macrophage model.Conclusions The preliminary standards for further testing of thymomodulin were set up. As far as presented results can be used as a foundation for development of quality control methods, further experiments are needed to confirm batch to batch diversity and to establish acceptability criteria for immunomodulatory composition of medicinal products containing thymus extract.Acknowledgements This work was supported by the European Union from the Lubelskie Regional Programme RPLU.01.02.00-06-0015/20. Authors thank Aleksandra Antos, Dariusz Lenart, Magdalena Ozga, Agnieszka-Topolska-Woś, and Marcin Woś for technical support in data acquisition and methodology.References Hannappel E, Iavarone F, Castagnola M. Thymosin fraction 5 re-evaluated after 35 years by high-resolution mass spectrometry. Expert Opin Biol Ther. 2018;18(sup1):199–203.Hymos A, Grywalska E, Klatka J, Klatka M, Korona-Głowniak I, Rolinski J. ThymicPeptides reverse immune exhaustion in patients with reactivated human alphaherpesvirus1 infections. Int J Mol Sci. 2020 Mar 30;21(7):2379.