EP4240838A1 - Procédé d'obtention d'hyaluronidase à partir de testicules de bovin - Google Patents

Procédé d'obtention d'hyaluronidase à partir de testicules de bovin

Info

Publication number
EP4240838A1
EP4240838A1 EP21810152.5A EP21810152A EP4240838A1 EP 4240838 A1 EP4240838 A1 EP 4240838A1 EP 21810152 A EP21810152 A EP 21810152A EP 4240838 A1 EP4240838 A1 EP 4240838A1
Authority
EP
European Patent Office
Prior art keywords
obtaining
hyaluronidase
concentration
carried out
hyaluronidase according
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP21810152.5A
Other languages
German (de)
English (en)
Inventor
Monika BESMAN
Magdalena MATWIEJCZYK
Grzegorz CIURA
Filip Porzucek
Konrad BABIJ
Michal LOBOCKI
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Pharmfactor Sp Z OO
Original Assignee
Pharmfactor Sp Z OO
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Pharmfactor Sp Z OO filed Critical Pharmfactor Sp Z OO
Publication of EP4240838A1 publication Critical patent/EP4240838A1/fr
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
    • B01D61/14—Ultrafiltration; Microfiltration
    • B01D61/145—Ultrafiltration
    • B01D61/146—Ultrafiltration comprising multiple ultrafiltration steps
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2474—Hyaluronoglucosaminidase (3.2.1.35), i.e. hyaluronidase
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
    • B01D15/12—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to the preparation of the feed
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/34—Size selective separation, e.g. size exclusion chromatography, gel filtration, permeation
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/36—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving ionic interaction
    • B01D15/361—Ion-exchange
    • B01D15/362—Cation-exchange
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01035—Hyaluronoglucosaminidase (3.2.1.35), i.e. hyaluronidase
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D2311/00—Details relating to membrane separation process operations and control
    • B01D2311/04—Specific process operations in the feed stream; Feed pretreatment
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D2311/00—Details relating to membrane separation process operations and control
    • B01D2311/06—Specific process operations in the permeate stream
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D2311/00—Details relating to membrane separation process operations and control
    • B01D2311/12—Addition of chemical agents
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D2311/00—Details relating to membrane separation process operations and control
    • B01D2311/26—Further operations combined with membrane separation processes
    • B01D2311/2623—Ion-Exchange
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D2311/00—Details relating to membrane separation process operations and control
    • B01D2311/26—Further operations combined with membrane separation processes
    • B01D2311/2676—Centrifugal separation
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D2311/00—Details relating to membrane separation process operations and control
    • B01D2311/26—Further operations combined with membrane separation processes
    • B01D2311/2697—Chromatography

Definitions

  • the subject of the invention is a method of obtaining hyaluronidase and the product obtained thereby.
  • the invention will find use in such fields of medicine as orthopaedics, surgery, ophthalmology, oncology, dermatology and gynaecology, as well as in aesthetic medicine and cosmetology.
  • Hyaluronidase is a white to yellowish amorphous, hygroscopic powder, easily soluble in water and practically insoluble in ethanol, acetone and ether.
  • Hyaluronidase depolymerizes hyaluronic acid - a component of connective tissue, which allows the active substances applied topically to be absorbed faster.
  • the present invention aims to propose a method of obtaining hyaluronidase that does not use hazardous (e.g. flammable) substances or is unscalable due to complexity, financial and equipment requirements of its techniques (in particular affinity chromatography), and at the same time it allows to obtain a stable product with sufficient efficiency and safety (meeting pharmacopeial requirements).
  • hazardous e.g. flammable
  • affinity chromatography affinity chromatography
  • the subject of the present invention is a method of obtaining hyaluronidase comprising sequentially the steps of: a. Comminution b. Extraction c. Ballast salting out d. Filtration e. Concentration I f. Incubation g. Ultrafiltration I h. Concentration II i. Ballast salting out j. Centrifugation k. Chromatography I gel filtration l. Chromatography II ion exchange m. Ultrafiltration II n. Concentration III o. Sterilizing filtration p. Drying
  • the method of obtaining hyaluronidase according to the invention is characterized in that the starting material is animal organs, preferably bovine or sheep testes.
  • the method of obtaining hyaluronidase according to the invention is characterized in that the tissue comminution step is carried out by grinding the tissue in an industrial electrical meat grinder.
  • the method of obtaining hyaluronidase according to the invention is characterized in that the extraction step is carried out in a planetary mixer by mixing the comminuted tissue with a cooled 0.1 M acetic acid solution at a pH of about 3.5.
  • the method of obtaining hyaluronidase according to the invention is characterized in that the ballast salting out step is carried out by precipitation of the phosphorylated proteins in the presence of calcium cations, preferably by adding a 5M CaCI2 solution to obtain a concentration of about 50 mM and adding sodium hydroxide (2M solution) to obtain a pH of about 7.
  • the ballast salting out step is carried out by precipitation of the phosphorylated proteins in the presence of calcium cations, preferably by adding a 5M CaCI2 solution to obtain a concentration of about 50 mM and adding sodium hydroxide (2M solution) to obtain a pH of about 7.
  • the method of obtaining hyaluronidase according to the invention is characterized in that the filtration step is carried out by physical separation of the postextraction tissue on sieve separators with a pore diameter of 1 mm and then transferring the drained extract into a cooling tank and leaving it for at least about 8 hours to salt-out ballast proteins and the solution is clarified by filtration using draining bags with a pore diameter of 1 pm.
  • the method of obtaining hyaluronidase according to the invention is characterized in that the concentration I step is performed by ultrafiltration on tubular filters with a MWCO of 300kDa.
  • the method of obtaining hyaluronidase according to the invention characterized in that the incubation step is performed in the presence of a non-ionic detergent, preferably Tween 20, and sodium chloride with a final concentration of 0.3M, with agitation for at least about 2 hours.
  • a non-ionic detergent preferably Tween 20, and sodium chloride with a final concentration of 0.3M
  • the method of obtaining hyaluronidase according to the invention is characterized in that the ultrafiltration I step is carried out using tubular filters with a MWCO of 300kDa, performing batchwise diafiltration washing the retentate about 6 times with about 5L of water, where hyaluronidase is in the filtrate.
  • the method of obtaining hyaluronidase according to the invention is characterized in that the concentration II step is performed with the use of membrane filters having a MWCO of 50 kDa, concentrating the solution approximately 4 times.
  • the method of obtaining hyaluronidase according to the invention is characterized in that the ballast salting out step is carried out by salting out the concentrated solution with ammonium sulphate in the presence of benzyl alcohol, preferably to an ammonium sulphate concentration of at least about 190 g/L in the presence of about 0.1% benzyl alcohol.
  • the method of obtaining hyaluronidase according to the invention is characterized in that the centrifugation step is performed by spinning in a cup centrifuge for at least about 30 minutes at a rotation frequency of about 4800 rpm, passing the supernatant to further purification steps.
  • the method of obtaining hyaluronidase according to the invention is characterized in that the chromatography I gel filtration step is carried out using a molecular sieve type of bed, preferably Sephacryl, where the mobile phase is purified water.
  • a molecular sieve type of bed preferably Sephacryl
  • the method of obtaining hyaluronidase according to the invention is characterized in that the chromatography II ion exchange step is carried out with a strong cation exchanger type of bed by elution after absorption with a sodium chloride gradient in an ammonium acetate buffer.
  • the method of obtaining hyaluronidase according to the invention is characterized in that the ultrafiltration II step is carried out by filtration using tubular filters with a MWCO of 300kDa with hyaluronidase in the permeate.
  • the method of obtaining hyaluronidase according to the invention is characterized in that the concentration III step is performed with the use of membrane filters with a MWCO of 50 kDA, concentrating the solution approximately 4 times.
  • the method of obtaining hyaluronidase according to the invention is characterized in that the sterilizing filtration step is performed by filtering through a 0.22 pm sterile filter.
  • the method of obtaining hyaluronidase according to the invention is characterized in that the drying step is carried out by freeze drying for the first 24 hours at a condenser temperature of -45°C, shelf temperature of -28°C and atmospheric pressure, then the pressure is reduced to 0.05 mBar, after reaching the set pressure, the shelf temperature is raised to 24°C for 72 hours, then the pressure is reduced to 0.001 mbar and the process is continued for another 24 hours.
  • the method of obtaining hyaluronidase according to the invention is characterized in that it further comprises at least one of the steps selected from the group consisting of in-process control, sterilization, final sterilization, quality control, which steps may be repeated, occur before the first step, in the middle and at the end of the method of obtaining.
  • the subject of the present invention is the product obtained by the method of obtaining according to the invention.
  • the method of obtaining hyaluronidase according to the invention does not use hazardous (e.g. flammable) substances or technologically complicated and unprofitable techniques (such as, for example, affinity chromatography), and at the same time provides a stable product with sufficiently high efficiency and safety (meeting pharmacopeial standards and requirements).
  • hazardous e.g. flammable
  • affinity chromatography technologically complicated and unprofitable techniques
  • step f it was possible to reduce the precipitation process and increase the purity level of hyaluronidase, which gives better solubility of the finished product
  • step a non-ionic detergent step f
  • the method of obtaining according to the invention is additionally characterized by the simplification of the process, e.g. in terms of technological advancement of stages, exclusion of unprofitable techniques and reduction of equipment requirements, reduction of water losses and indirectly reduction of costs and pollution, exclusion of flammable substances, which in total makes the method according to the invention competitive with methods characterized by higher efficiency.
  • Hyaluronidase will be used in such fields of medicine as orthopaedics, surgery, ophthalmology, oncology, dermatology or gynaecology, e.g. in increasing the absorption of parenteral drugs, improving the effectiveness of local anaesthesia, reducing tissue damage or increasing the activity of anticancer drugs.
  • Hyaluronidase will also find its application in aesthetic medicine and cosmetology in needle mesotherapy, lipolysis and removal of excess hyaluronic acid.
  • Fig. 1 - shows a block diagram of an embodiment of the method of obtaining hyaluronidase according to the invention
  • Fig. 2 - shows the product obtained by the method of obtaining according to the invention
  • Bovine testes were used as a starting material for the process.
  • the starting material (180 kg) was taken from a freezer and ground by an industrial electrical meat grinder into polyethylene containers. The starting material was then manually loaded into a planetary mixer and extraction was performed by mixing it with 540L of cooled 0.1 M acetic acid at pH of 3.5. Person skilled in the art will appreciate a wide variety of mixers that can replace the planetary mixer. One hour later, samples were taken for testing (Tab. 1 "Before ballast salting out"). Next, ballasts were precipitated by salting out of phosphorylated proteins in the presence of calcium cations.
  • the filtrate was disposed and the retentate was incubated in the presence of non-ionic detergent and sodium chloride to remove cell membrane fragments.
  • chloride at a final concentration of 0.3M were added to the concentrated hyaluronidase solution (about 50L) and stirred continuously at room temperature for 2 hours. Thanks to this step, it is possible to separate the hyaluronidase from the cell membrane fragments, which allows it to pass into the permeate during filtration. As a result of removing the cell membrane fragments, the solubility of the finished preparation in aqueous solutions is significantly improved.
  • non-ionic detergents a wide variety of non-ionic detergents.
  • the hyaluronidase solution with detergent and sodium chloride was filtered using polymer tubular filters with a MWCO of 300kDa by performing batchwise diafiltration washing the retentate six times with 5L of purified water. Hyaluronidase was in the filtrate.
  • the result of ultrafiltration of the hyaluronidase solution after incubation with detergent and sodium chloride is presented in Tab. 3 Based on the analysis of the results, it was found that the addition of detergent and sodium chloride allowed hyaluronidase to pass into the permeate, increasing its degree of purity and improving its solubility.
  • concentration was performed on polymer filters with a MWCO of 50 kDa.
  • the solution was concentrated 4 times. The process was completed after reaching the volume of 20 L.
  • the step of salting out the ballast compounds from the retentate was carried out by salting out the concentrated solution with ammonium sulphate to a concentration of 190 g/L of ammonium sulphate in the presence of 0.1% benzyl alcohol. Afterwards, the solution was centrifuged in a cup centrifuge for 30 minutes at a rotation frequency of 4800 rpm, discarding the pellet. The obtained supernatant was purified by gel filtration using a molecular sieve.
  • the present method of obtaining uses Sephacryl where the mobile phase is purified water, but person skilled in the art will appreciate a wide variety of molecular sieve beds.
  • Fractions containing hyaluronidase were adsorbed on a liquid ion exchange chromatography column with a strong cation exchanger. After absorption, elution was performed with a sodium chloride gradient in ammonium acetate buffer.
  • the hyaluronidasecontaining fraction was purified by ultrafiltration on tubular filters (MWCO 300kDa). Then the filtrate was concentrated on membrane filters (MWCO 50kDa). Subsequently, to ensure the safety of the product, sterilizing filtration was performed by filtering the solution with a 0.22 pm sterile filter.
  • the hyaluronidase solution prepared this way was subjected to the lyophilization process. For the first 24 hours at condenser temperature of -45°C, shelf temperature of -28°C and atmospheric pressure. The pressure was then reduced to 0.05 mBar, and after reaching the desired pressure, the shelf temperature was raised to 24°C for another 72 hours. The pressure was then reduced to 0.001 mBar and the process was continued for 24 hours.
  • the starting material, apart from bovine testicles, may also be sheep testicles.
  • the inventor has successfully obtained hyaluronidase from sheep testes using the abovedisclosed method of obtaining according to the invention, but for the sake of clarity and efficiency, the method of obtaining according to the invention is shown above in an embodiment using bovine testicles.
  • bovine testes in particular means bull testicles that have been used in the present embodiment, but does not restrict the scope of the invention as to the age or other representatives of cattle and wild cattle. Same with sheep's testicles.
  • bovine or sheep testes also means in particular mixtures of the above.
  • the above method of obtaining allows obtaining hyaluronidase with a specific activity of about 1500 U/mg.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Water Supply & Treatment (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Analytical Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Coloring Foods And Improving Nutritive Qualities (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

L'objet de l'invention est un procédé d'obtention d'hyaluronidase comprenant les étapes suivantes de : broyage, extraction, relargage du ballast, filtration, concentration I, incubation, ultrafiltration I, concentration II, relargage du ballast, centrifugation, chromatographie I par filtration sur gel, chromatographie II par échange d'ions, ultrafiltration II, concentration III, filtration stérilisante et séchage. L'invention porte également sur le produit ainsi obtenu.
EP21810152.5A 2020-09-15 2021-09-14 Procédé d'obtention d'hyaluronidase à partir de testicules de bovin Pending EP4240838A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
PL435319A PL244350B1 (pl) 2020-09-15 2020-09-15 Sposób otrzymywania hialuronidazy ze zwierzęcych jąder oraz produkt otrzymany tym sposobem
PCT/PL2021/050066 WO2022060235A1 (fr) 2020-09-15 2021-09-14 Procédé d'obtention d'hyaluronidase à partir de testicules de bovin

Publications (1)

Publication Number Publication Date
EP4240838A1 true EP4240838A1 (fr) 2023-09-13

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ID=78650030

Family Applications (1)

Application Number Title Priority Date Filing Date
EP21810152.5A Pending EP4240838A1 (fr) 2020-09-15 2021-09-14 Procédé d'obtention d'hyaluronidase à partir de testicules de bovin

Country Status (3)

Country Link
EP (1) EP4240838A1 (fr)
PL (1) PL244350B1 (fr)
WO (1) WO2022060235A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR102528707B1 (ko) * 2022-11-01 2023-05-04 한국코러스 주식회사 고순도의 히알루로니다제 정제 방법
CN117723682B (zh) * 2023-11-20 2024-06-11 山东丰金美业科技有限公司 一种交联透明质酸或其盐凝胶交联度的检测方法

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2027759C1 (ru) * 1992-02-03 1995-01-27 Пак Владимир Николаевич Способ получения гиалуронидазы

Also Published As

Publication number Publication date
PL435319A1 (pl) 2022-03-21
PL244350B1 (pl) 2024-01-15
WO2022060235A1 (fr) 2022-03-24

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