EXPERIMENTAL RESULTS ON THE DEVELOPMENT OF HIGHLY PURIFIED HYALURONATE-ENDO- β-N-ACETYLHEXOSAMINIDASE AS A PHARMACEUTICAL SUBSTANCE FOR A DRUG PRODUCT

УДК 616-092.4:615.012:577.15.01

Authors

  • Alexandra M. Shvetsova Research Institute of Clinical and Experimental Lymphology – Branch of the Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia https://orcid.org/0009-0002-7735-6032 Email: aleksa-2904@mail.ru
  • Konstantin I. Ershov Research Institute of Clinical and Experimental Lymphology – Branch of the Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia; Novosibirsk State Medical University, Novosibirsk, Russia https://orcid.org/0000-0003-4139-036X Email: aleksa-2904@mail.ru
  • Maksim A. Korolev Research Institute of Clinical and Experimental Lymphology – Branch of the Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia https://orcid.org/0000-0002-0471-652X Email: kormax@bk.ru
  • Pavel G. Madonov Research Institute of Clinical and Experimental Lymphology – Branch of the Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia; Novosibirsk State Medical University, Novosibirsk, Russia https://orcid.org/0000-0002-1093-8938 Email: pmadonov@yandex.ru

DOI:

https://doi.org/10.31684/25418475-2023-4-68

Keywords:

hyaluronate-endo-β-N-acetylhexosaminidase, pharmaceutical substance, technology development

Abstract

Enzyme preparations, including hyaluronidase, are widely used in medical practice. Most drugs with this active substance on the pharmaceutical market are produced mainly from testicular hyaluronidase, the raw material for which are bovine testes. The technologies of enzyme preparation production are quite well known and diverse. However, the creation of a different method will allow the highly purified hyaluronate-endo-β-N-acetylhexosaminidase with high specific enzymatic activity. Objective: develop a technology and obtain a laboratory batch of the highly purified pharmaceutical substance hyaluronate-endo-β-N-acetylhexosaminidase. Materials and Methods. In the experimental study, preparations of bovine testes extracts were taken to obtain highly purified hyaluronate-endo-β-N-acetylhexosaminidase. In the first stage the protein spectrum of the testes extracts was studied, proteins were fractionally precipitated, and substances of different chemical nature were fractionated by chromatography methods. At the second stage, the development of a method to obtain the pharmaceutical substance hyaluronate-endo-β-N-acetylhexosaminidase was carried out using the methods described further in the work. Results. Based on the results obtained during the experimental study, P-II, a preparation of bovine testes extract produced by Samson-Med LLC (St. Petersburg), was chosen as the initial substance of crude hyaluronidase for further studies. The most optimal is the purification scheme, which includes as the main method affinity chromatography on heparin-sepharose using affinity elution with heparin solution in buffer with a concentration of 3 mg/ml. Conclusions As a result of the experimental study, the optimal scheme was presented to obtain the pharmaceutical substance HEBNA with high enzymatic total activity of 289 (unit of action) was presented, and a laboratory batch of highly purified hyaluronate-endo-β-N-acetylhexosaminidase was obtained.

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Alexandra M. Shvetsova,
Research Institute of Clinical and Experimental Lymphology – Branch of the Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia

Junior Researcher, Research Institute of Clinical and Experimental Lymphology – Branch of the Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia.
E-mail: aleksa-2904@mail.ru
https://orcid.org/0009-0002-7735-6032

Konstantin I. Ershov,
Research Institute of Clinical and Experimental Lymphology – Branch of the Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia; Novosibirsk State Medical University, Novosibirsk, Russia

Cand. Sci. (Biol.), Assistant, Research Institute of Clinical and Experimental Lymphology – Branch of the Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences; Novosibirsk State Medical University, Novosibirsk, Russia.
E-mail: aleksa-2904@mail.ru
https://orcid.org/0000-0003-4139-036X

Maksim A. Korolev,
Research Institute of Clinical and Experimental Lymphology – Branch of the Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia

Dr. Sci. (Med.), Research Institute of Clinical and Experimental Lymphology – Branch of the Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia.
E-mail: kormax@bk.ru
https://orcid.org/0000-0002-0471-652X

Pavel G. Madonov,
Research Institute of Clinical and Experimental Lymphology – Branch of the Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia; Novosibirsk State Medical University, Novosibirsk, Russia

Dr. Sci. (Med.), Associate Professor, Research Institute of Clinical and Experimental Lymphology – Branch of the Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences; Novosibirsk State Medical University, Novosibirsk, Russia.
Email: pmadonov@yandex.ru
https://orcid.org/0000-0002-1093-8938

References

  • Barsukov A.K., Kozhevnikova O.V., Khokhryakova A.V. Separation and purification of bovine testicular hyaluronidase. Prikladnaya biokhimiya i mikrobiologiya=Applied Biochemistry and Microbiology. 2003; 39(6): 625. (In Russ.)
  • Vidal Directory. Medicinal preparations in Russia. Moscow, 2022:1120.
  • Dygai A.M., Zyuzkov G.N., Zhdanov V.V. et al. Effect of transplantation of peripheral blood mononuclear cells obtained using granulocyte colony-stimulating factor and hyaluronidase on hematopoietic tissue regeneration in myelosuppression. Kletochnye tekhnologii v biologii i meditsine = Cell Technologies in Biology and Medicine (Bull. Exp. Biol. Med.). 2009; 3:136-142. (In Russ.)
  • Dygai A.M., Zyuzkov G.N., Zhdanov V.V. Regenerative medicine: in search of the «elixir of life». SCIENCE First Hand. 2013; 3(51): 24-31.
  • Dygai A.M., Pershina O.V., Krupin V.A., Ermolaeva L.A., Ermakova N.N., Shilova M.A., Madonov P.G., Kinsht D.N., Skurikhin E.G. Study of antifibrotic activity of modified and native hyaluronidase in pneumofibrosis. Sibirskij Nauchnyj Medicinskij Zhurnal = Siberian Scientific Medical Journal. 2017; 37(4): 5–10.
  • Laemmli U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970; 227(5259): 680-5. https://doi.org/10.1038/227680a0
  • Guntenhöner M.W., Pogrel M.A., Stern R. A substrate-gel assay for hyaluronidase activity. Matrix. 1992; 12(5): 388-96. https://doi.org/10.1016/S0934-8832(11)80035-1
  • Scopes R. Methods of protein purification. Moscow. 1985: 385.
  • Dawson R., Elliott D., Elliott W., Jones K. Handbook of Biochemist. Moscow. 1991: 544.
  • Bradford M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976; 72: 248-254. https://doi.org/10.1006/abio.1976.9999. PMID: 942051.
  • Lalancette C., Dorval V., Leblanc V., Leclerc P. Characterization of an 80-kilodalton bull sperm protein identified as PH-20. Biol Reprod. 2001; 65(2): 628-36. https://doi.org/10.1095/biolreprod65.2.628. PMID: 11466235.
  • Morin G., Lalancette C., Sullivan R., Leclerc P. Identification of the bull sperm p80 protein as a PH-20 ortholog and its modification during the epididymal transit. Mol Reprod Dev. 2005; 71(4): 523-34. https://doi.org/10.1002/mrd.20308. PMID: 15892045
  • Methodology for measuring hyaluronidase activity. LSR-009264/09-171109 for Lidaza substance Samson-Med LLC.
  • http://www.uniprot.org/
    • Том 32 № 4 (2023): Бюллетень медицинской науки

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    2023-12-22

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    Shvetsova A. M., Ershov K. I., Korolev M. A., Madonov P. G. EXPERIMENTAL RESULTS ON THE DEVELOPMENT OF HIGHLY PURIFIED HYALURONATE-ENDO- β-N-ACETYLHEXOSAMINIDASE AS A PHARMACEUTICAL SUBSTANCE FOR A DRUG PRODUCT : УДК 616-092.4:615.012:577.15.01 // Bulletin of Medical Science, 2023. Vol. 32, № 4. P. 68–80 DOI: 10.31684/25418475-2023-4-68. URL: https://newbmn.asmu.ru/bmn/article/view/635.

    Issue

    Vol. 32 No. 4 (2023): Bulletin of Medical Science

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    ORIGINAL PAPERS

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