CHROMATOGRAPHIC PURIFICATION OF BOVINE TESTICULAR HYALURONIDASE

DOI: https://doi.org/10.29296/25877313-2019-04-05
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Issue: 
4
Year: 
2019

I.V. Melnikova Post-graduate Student, Department of Biotechnology, St. Petersburg State Chemical-Pharmaceutical University E-mail: yana.melnikova@pharminnotech.com A.D. Yashtubaeva Master Student, Department of Biotechnology, St. Petersburg State Chemical-Pharmaceutical University N.V. Glazova Ph.D. (Chem.), Associate Professor, Department of Biotechnology, St. Petersburg State Chemical-Pharmaceutical University

Hyaluronidase is an enzyme that breaks down the acidic mucopolysaccharides which are the basis of connective tissue. Integration and analysis of the literature data of different authors, that presented in the article [1], and comparative analysis of the properties of hyaluronidase from different sources, that was carried out at the Department of Biotechnology SPCPU [2], showed that hyaluronidases isolated from various sources possess different physicochemical characteristics such as molecular weight, the range of enzymatic activities, stability etc. Therefore, the choice of raw materials determines the approaches to hyaluronidase isolation and purification. In this investigation the bovine testes were chosen as a raw material, since currently only this raw material is used to produce hyaluronidase on an industrial scale. Studies of ion exchange sorption of hyaluronidase from the bovine testes showed that the enzyme is selectively sorbed on macroporous sulfoca-tionite KU-23 [3, 4]. The use of this chromatographic method made it possible to replace the unsustainable conventional method of isolating hyaluroni-dase using organic solvents. However, ionite KU-23 is currently commercially unavailable, that’s why the selection of modern sorbent for the isolation and purification of hyaluronidase is a crucial task. The aim of this investigation was to develop a chromatographic procedure of hyaluronidase purification from test solution with the use of modern macroporous sorbents. Materials and methods. The object of research – hyaluronidase in active pharmaceutical ingredient «Lidaza», which is manufactured by «Sam-son-Med”. The object of research is a test solution of hyaluronidase with a protein concentration 10 mg / ml and pH 4.0-4.5. Methods: method for deter-mination of protein with the use of biuret reagent [5]; method for determination of hyaluronidase activity in solution [6]; method for preparation of sorbents [7, 8]; method of chromatographic procedure under static and dynamic condition. Conclusion. The study of chromatographic purification of hyaluronidase under static and dynamic conditions with the use of macroporous sorbents revealed that the optimal sorbent is Nuvia™ S (Bio-Rad Laboratories, Inc). That sorbent provides for the maximum of the protein and hyaluroni-dase activity yield. Hereafter Nuvia™ S can be used for isolation and purification of hyaluronidase from the orchic extract. New technology can replace conventional technology that uses organic solvents.

Keywords: 
hyaluronidase
macroporous sorbents
purification
chromatographic method

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References: 
  1. Mel'nikova Ya.V., Glazova N.V. Svojstva i biologicheskaya rol' gialuronidaz iz razlichnyh istochnikov syr'ya // Sb. materialov VIII Vseross. nauch. konf. studentov i aspirantov s mezhdunarodnym uchastiem «Molodaya Farmaciya – potencial budushchego» (Sankt-Peterburg, 23.04.18–24.04.18). SPb.: SPHFU. 2018. S. 259–263.
  2. Zainkova N.V. Poluchenie i sravnitel'nyj analiz svojstv gialuronidaz iz razlichnyh istochnikov: Avtoref. dis. … kand. biol. nauk: 03.00.04. SPb. 1998. 142 s
  3. Igonina L.M. Hromatograficheskoe vydelenie i izuchenie svojstv fermenta gialuronidazy iz semennikov krupnogo rogatogo skota: Avtoref. dis. …kand. him. nauk: 03.00.04. SPb. 1996. 134 s.
  4. Igonina L.M., Glazova N.V., El'kin G.E. et al. Sorption of hyaluronidase to macroporous sorbents // Applied Biochemistry and Microbiology. V. 998. № 5. 442–445
  5. Gosudarstvennaya Farmakopeya XIII. T. 1. M.: FEHMB. 2015. 1469 s.
  6. Patent № 2417097 (RF). Farmacevticheskaya kompoziciya, soderzhashchaya gialuronidazu i liposomy dlya naruzhnogo primeneniya / V.L. Bagirova, N.V. Glazova, M.D. Dul'kis, V.N. Ivanov. 27.04.11.
  7. GOST 20298-74. Smoly ionoobmennye. Kationity. Tekhnicheskie usloviya (s Izmeneniyami N 1-5). Vzamen GOST 13505-68, GOST 5.1428-72. Vved. 1976-01-01. M.: Izdatel'stvo standartov. 1991. 13 s.
  8. UNOsphere™ S and Q, Macro-Prep®, Nuvia™ S and Q High-Capacity Ion Exchange Media Instruction manual. Bio-Rad Laboratories Inc.
  9. Patent № 2658605 (RF). Sposob polucheniya gialuronidazy iz semennikov krupnogo rogatogo skota / N.V. Glazova, A.M. Elikhanov, O.V. Chajka, Ya.V. Mel'nikova, O.G. Dudnik. 21.06.18.