WO2022060235A1 - Method of obtaining hyaluronidase from bovine testes - Google Patents
Method of obtaining hyaluronidase from bovine testes Download PDFInfo
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- WO2022060235A1 WO2022060235A1 PCT/PL2021/050066 PL2021050066W WO2022060235A1 WO 2022060235 A1 WO2022060235 A1 WO 2022060235A1 PL 2021050066 W PL2021050066 W PL 2021050066W WO 2022060235 A1 WO2022060235 A1 WO 2022060235A1
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- Prior art keywords
- obtaining
- hyaluronidase
- concentration
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- hyaluronidase according
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Links
- 108010003272 Hyaluronate lyase Proteins 0.000 title claims abstract description 77
- 102000001974 Hyaluronidases Human genes 0.000 title claims abstract description 77
- 229960002773 hyaluronidase Drugs 0.000 title claims abstract description 77
- 238000000034 method Methods 0.000 title claims abstract description 76
- 210000001550 testis Anatomy 0.000 title claims description 13
- 241000283690 Bos taurus Species 0.000 title claims description 11
- 238000012360 testing method Methods 0.000 title claims description 8
- 238000001914 filtration Methods 0.000 claims abstract description 20
- 238000005185 salting out Methods 0.000 claims abstract description 20
- 238000000108 ultra-filtration Methods 0.000 claims abstract description 16
- 230000001954 sterilising effect Effects 0.000 claims abstract description 12
- 238000004587 chromatography analysis Methods 0.000 claims abstract description 10
- 238000011534 incubation Methods 0.000 claims abstract description 8
- 238000000605 extraction Methods 0.000 claims abstract description 7
- 238000002523 gelfiltration Methods 0.000 claims abstract description 6
- 238000005119 centrifugation Methods 0.000 claims abstract description 5
- 238000001035 drying Methods 0.000 claims abstract description 5
- 238000005342 ion exchange Methods 0.000 claims abstract description 5
- 239000000243 solution Substances 0.000 claims description 29
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 22
- 239000003599 detergent Substances 0.000 claims description 11
- 239000011780 sodium chloride Substances 0.000 claims description 11
- 239000000047 product Substances 0.000 claims description 10
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 9
- WVDDGKGOMKODPV-UHFFFAOYSA-N Benzyl alcohol Chemical compound OCC1=CC=CC=C1 WVDDGKGOMKODPV-UHFFFAOYSA-N 0.000 claims description 9
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 9
- 239000000706 filtrate Substances 0.000 claims description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- 239000012465 retentate Substances 0.000 claims description 7
- 241001494479 Pecora Species 0.000 claims description 6
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 6
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 6
- 239000001166 ammonium sulphate Substances 0.000 claims description 6
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 6
- 239000012466 permeate Substances 0.000 claims description 6
- 239000011148 porous material Substances 0.000 claims description 6
- 239000007858 starting material Substances 0.000 claims description 6
- 238000004659 sterilization and disinfection Methods 0.000 claims description 6
- -1 calcium cations Chemical class 0.000 claims description 5
- 239000000284 extract Substances 0.000 claims description 5
- 239000012528 membrane Substances 0.000 claims description 5
- 238000010521 absorption reaction Methods 0.000 claims description 4
- 239000011575 calcium Substances 0.000 claims description 4
- 229910052791 calcium Inorganic materials 0.000 claims description 4
- 239000002808 molecular sieve Substances 0.000 claims description 4
- 108091005981 phosphorylated proteins Proteins 0.000 claims description 4
- 102000004169 proteins and genes Human genes 0.000 claims description 4
- 108090000623 proteins and genes Proteins 0.000 claims description 4
- 239000008213 purified water Substances 0.000 claims description 4
- 238000003908 quality control method Methods 0.000 claims description 4
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 claims description 4
- 239000006228 supernatant Substances 0.000 claims description 4
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 claims description 3
- 239000005695 Ammonium acetate Substances 0.000 claims description 3
- 238000012369 In process control Methods 0.000 claims description 3
- 239000012506 Sephacryl® Substances 0.000 claims description 3
- 238000013019 agitation Methods 0.000 claims description 3
- 229940043376 ammonium acetate Drugs 0.000 claims description 3
- 235000019257 ammonium acetate Nutrition 0.000 claims description 3
- 235000019445 benzyl alcohol Nutrition 0.000 claims description 3
- 239000000872 buffer Substances 0.000 claims description 3
- 150000001768 cations Chemical class 0.000 claims description 3
- 238000001816 cooling Methods 0.000 claims description 3
- 238000011026 diafiltration Methods 0.000 claims description 3
- 238000010828 elution Methods 0.000 claims description 3
- 238000010965 in-process control Methods 0.000 claims description 3
- 235000013372 meat Nutrition 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 3
- 238000001556 precipitation Methods 0.000 claims description 3
- 238000000746 purification Methods 0.000 claims description 3
- 238000000926 separation method Methods 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 2
- 210000001557 animal structure Anatomy 0.000 claims description 2
- 238000004108 freeze drying Methods 0.000 claims description 2
- 238000000227 grinding Methods 0.000 claims description 2
- WVDDGKGOMKODPV-ZQBYOMGUSA-N phenyl(114C)methanol Chemical compound O[14CH2]C1=CC=CC=C1 WVDDGKGOMKODPV-ZQBYOMGUSA-N 0.000 claims description 2
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 2
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 2
- 238000009987 spinning Methods 0.000 claims description 2
- 230000000694 effects Effects 0.000 description 8
- 210000001519 tissue Anatomy 0.000 description 7
- 239000003814 drug Substances 0.000 description 5
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 4
- 239000012634 fragment Substances 0.000 description 4
- 238000001042 affinity chromatography Methods 0.000 description 3
- 210000000170 cell membrane Anatomy 0.000 description 3
- 229920000642 polymer Polymers 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 230000007717 exclusion Effects 0.000 description 2
- 231100001261 hazardous Toxicity 0.000 description 2
- 229920002674 hyaluronan Polymers 0.000 description 2
- 229960003160 hyaluronic acid Drugs 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 238000001356 surgical procedure Methods 0.000 description 2
- 206010002091 Anaesthesia Diseases 0.000 description 1
- FGUUSXIOTUKUDN-IBGZPJMESA-N C1(=CC=CC=C1)N1C2=C(NC([C@H](C1)NC=1OC(=NN=1)C1=CC=CC=C1)=O)C=CC=C2 Chemical compound C1(=CC=CC=C1)N1C2=C(NC([C@H](C1)NC=1OC(=NN=1)C1=CC=CC=C1)=O)C=CC=C2 FGUUSXIOTUKUDN-IBGZPJMESA-N 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004157 Hydrolases Human genes 0.000 description 1
- 108090000604 Hydrolases Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 239000004698 Polyethylene Substances 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 229940042450 amphadase Drugs 0.000 description 1
- 238000001949 anaesthesia Methods 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 229940041181 antineoplastic drug Drugs 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 244000309464 bull Species 0.000 description 1
- 229940043430 calcium compound Drugs 0.000 description 1
- 150000001674 calcium compounds Chemical class 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 229940044700 hylenex Drugs 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 230000004130 lipolysis Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000012792 lyophilization process Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000000694 mesotherapy Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 229920000573 polyethylene Polymers 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 238000000275 quality assurance Methods 0.000 description 1
- 239000010414 supernatant solution Substances 0.000 description 1
- 230000000451 tissue damage Effects 0.000 description 1
- 231100000827 tissue damage Toxicity 0.000 description 1
- 229940054953 vitrase Drugs 0.000 description 1
Classifications
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- 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
-
- 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
- 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
-
- 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
- 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
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- 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
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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- B01D2311/00—Details relating to membrane separation process operations and control
- B01D2311/12—Addition of chemical agents
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- 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
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- 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
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- 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
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Abstract
The subject of the invention is a method of obtaining hyaluronidase comprising the following steps: comminution, extraction, ballast salting out, filtration, concentration I, incubation, ultrafiltration I, concentration II, ballast salting out, centrifugation, chromatography I gel filtration, chromatography II ion exchange, ultrafiltration II, concentration III, sterilizing filtration and drying. The invention also relates to the product obtained thereby.
Description
METHOD OF OBTAINING HYALURONIDASE FROM BOVINE TESTES
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 an enzyme from the group of hydrolases with a molecular weight of 70 kDa and an isoelectric point of pl = 5.2. 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.
There is a rich and varied state of the art regarding the preparation of hyaluronidase. Two well-established methods of obtaining hyaluronidase are the production of recombinant protein in bacterial cells (e.g. Hylenex®) and obtaining it from animal tissue extracts (e.g. Vitrase® and Amphadase®). Nevertheless, the methods of obtaining hyaluronidase available in the state of the art have limitations affecting their effectiveness (including financial) and product safety. Firstly, flammable solvents are routinely used. For example, document EP0005751 discloses the use of acetone. One skilled in the art understands the complications and dangers of using flammable solvents, especially on a large scale. Secondly, many methods of obtaining hyaluronidase make use of complicated and commercially unprofitable affinity chromatography, such as, for example, in document CN104419689. This way, some methods, despite their high yield, may not be industrially applicable in practice due to cost and complexity.
Considering the above limitations of the available state of the art, 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).
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
Preferably, the method of obtaining hyaluronidase according to the invention is characterized in that the starting material is animal organs, preferably bovine or sheep testes.
Preferably, 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.
Preferably, 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.
Preferably, 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.
Preferably, 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.
Preferably, 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.
Preferably, 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.
Preferably, 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.
Preferably, 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.
Preferably, 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.
Preferably, 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.
Preferably, 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.
Preferably, 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.
Preferably, 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.
Preferably, 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.
Preferably, 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.
Preferably, 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.
Preferably, 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 present invention has many advantages. First of all, 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). In addition, thanks to the ballast salting out step (step c), it was possible to reduce the precipitation process and increase the purity level of hyaluronidase, which gives better solubility of the finished product, and thanks to the incubation step with a non-ionic detergent (step f), it was possible to significantly improve the solubility of the finished preparation in aqueous solutions.
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.
The invention will be illustrated closer by the following preferred non-limiting embodiments with reference to the accompanying drawing in which:
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
Embodiment
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. Person skilled in the art will appreciate the possibility of precipitating phosphorylated proteins in the presence of calcium cations by adding various calcium compounds, however, in the method of obtaining according to the invention, a 5M CaCb solution was specifically added to obtain a concentration of 50mM and a 2M sodium hydroxide solution was added to obtain a pH of 7. This step allowed to shorten the ballast salting out process and increased the purity level of hyaluronidase, which in turn translates into better solubility of the finished product. After that, separation was carried out by physically separating the post-extraction tissue using sieve separators with a pore diameter of 1 mm. The filtrate was directed to a disc centrifuge. Person skilled in the art will appreciate that these may be other centrifuges as the purpose is to remove fine fragments of organic tissue. The resulting supernatant was placed in a mixer with a cooling jacket and left at 8°C for about 12 hours for the ballast proteins to precipitate (without agitation). The solution was then filtered by decanting the clear supernatant solution, pouring the cloudy residual solution onto a set of draining bags with a pore size of 1pm. The filtrate after the bags was sent for analysis (Tab. 1 "After ballast salting out").
Table 1 Effect of ballast salting out on specific activity and yield.
Then the solution was concentrated by ultrafiltration on polymer tubular filters with a MWCO of 300kDa. The process was carried out until a 10-fold reduction in the volume of the retentate was obtained. The results of concentrating the extract containing hyaluronidase are shown in Table 2. Based on the results, it can be seen that in the case of filtration (MWCO 300kDa) of a solution of hyaluronidase without detergent and sodium chloride, only less than 10% of hyaluronidase passes into the permeate. This means that the vast majority stays in the retentate. The opposite is true for protein - most of it goes to the permeate (filtrate). This results in a significant increase in the degree of purification (from 65.3 U/mg to 321 U/mg).
Tab. 2 Effect of extract concentration (without detergent and NaCI) on the activity and specific activity of hyaluronidase
The filtrate was disposed and the retentate was incubated in the presence of non-ionic detergent and sodium chloride to remove cell membrane fragments. In this embodiment,
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. Person skilled in the art will appreciate a wide variety of non-ionic detergents. Next, 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.
Tab. 3 Effect of ultrafiltration of hyaluronidase solution after incubation with detergent and sodium chloride
Then 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. Next, 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.
Person skilled in the art understands the need for quality assurance or control and will modify the above method of obtaining hyaluronidase as needed to add one or more steps required or indicated by local or international guidelines or regulations. In particular, these may be one or more steps of in-process control, one or more sterilization or final sterilization steps, one or more quality control steps. These steps may occur at all stages of the method of obtaining the hyaluronidase according to the invention, also before the first step and after the last step. Additional steps may also follow one another.
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. The term 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. The term 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.
Claims
1. 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
2. The method of obtaining hyaluronidase according to claim ^ characterized in that the starting material is animal organs, preferably bovine or sheep testes.
3. The method of obtaining hyaluronidase according to any one of claims 1 - 2, characterized in that the tissue comminution step is carried out by grinding the tissue in an industrial electrical meat grinder.
4. The method of obtaining hyaluronidase according to any of claims 1 - 3, 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.
5. The method of obtaining hyaluronidase according to any one of claims 1 -
4, 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 CaCb solution to obtain a concentration of about 50 mM and adding sodium hydroxide (2M solution) to obtain a pH of about 7.
6. The method of obtaining hyaluronidase according to any one of claims 1 -
5, 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.
7. The method of obtaining hyaluronidase according to any one of claims 1 - 6, characterized in that the concentration I step is performed by ultrafiltration on tubular filters with a MWCO of 300kDa.
8. The method of obtaining hyaluronidase according to any one of claims 1 - 7, 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.
9. The method of obtaining hyaluronidase according to any one of claims 1 -
8, 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.
10. The method of obtaining hyaluronidase according to any of the claims 1 -
9, 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.
11. The method of obtaining hyaluronidase according to any of claims 1 - 10, 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.
12. The method of obtaining hyaluronidase according to any of claims 1 - 11 , 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.
13. The method of obtaining hyaluronidase according to any one of claims 1 -
12, 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.
14. The method of obtaining hyaluronidase according to any one of claims 1 -
13, 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.
15. The method of obtaining hyaluronidase according to any one of claims 1 - 14 , 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.
16. The method of obtaining hyaluronidase according to any of the claims 1 - 15, 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.
17. The method of obtaining hyaluronidase according to any of claims 1 - 16, characterized in that the sterilizing filtration step is performed by filtering through a 0.22 pm sterile filter.
18. The method of obtaining hyaluronidase according to any of claims 1 - 17, 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.
19. The method of obtaining hyaluronidase according to any one of claims 1 - 18, 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.
20. Product obtained by the method of obtaining according to claims 1 - 19.
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| Application Number | Priority Date | Filing Date | Title |
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| EP21810152.5A EP4240838A1 (en) | 2020-09-15 | 2021-09-14 | Method of obtaining hyaluronidase from bovine testes |
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| PL435319A PL244350B1 (en) | 2020-09-15 | 2020-09-15 | Method for producing hyaluronidase and a product obtained using this method |
| PLP.435319 | 2020-09-15 |
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| WO2022060235A1 true WO2022060235A1 (en) | 2022-03-24 |
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| EP (1) | EP4240838A1 (en) |
| PL (1) | PL244350B1 (en) |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RU2027759C1 (en) * | 1992-02-03 | 1995-01-27 | Пак Владимир Николаевич | Method of hyaluronidase preparing |
-
2020
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2021
- 2021-09-14 EP EP21810152.5A patent/EP4240838A1/en active Pending
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RU2027759C1 (en) * | 1992-02-03 | 1995-01-27 | Пак Владимир Николаевич | Method of hyaluronidase preparing |
Non-Patent Citations (3)
| Title |
|---|
| KAYA MUSTAFA OGUZHAN ET AL: "A new affinity method for purification of bovine testicular hyaluronidase enzyme and an investigation of the effects of some compounds on this enzyme", JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY, vol. 30, no. 4, 6 November 2014 (2014-11-06), GB, pages 524 - 527, XP055879123, ISSN: 1475-6366, DOI: 10.3109/14756366.2014.949253 * |
| LYON M ET AL: "A rapid purification of bovine testicular hyaluronidase by chromatography on dermatan sulphate-substituted 1,6-diaminohexane-sepharose 4B", BIOCHEMICAL JOURNAL, vol. 199, no. 2, 1 November 1981 (1981-11-01), GB, pages 419 - 426, XP055879124, ISSN: 0264-6021, Retrieved from the Internet <URL:https://watermark.silverchair.com/bj1990419.pdf?token=AQECAHi208BE49Ooan9kkhW_Ercy7Dm3ZL_9Cf3qfKAc485ysgAAA-4wggPqBgkqhkiG9w0BBwagggPbMIID1wIBADCCA9AGCSqGSIb3DQEHATAeBglghkgBZQMEAS4wEQQMsjiLRdSpj-AapllbAgEQgIIDoUYRp2kggZ7czoxVgzl7T0ePcIEdL5Cd1u-VhQrmApiee2w3Bx-NHL5LUkFB7lrJPM0neyFiDWmFJiLlcRuxntHui3> DOI: 10.1042/bj1990419 * |
| MICHAEL F MEYER ET AL: "The soluble hyaluronidase from bull testes is a fragment of the membrane-bound PH-20 enzyme", FEBS LETTERS, ELSEVIER, AMSTERDAM, NL, vol. 413, no. 2, 28 August 1998 (1998-08-28), pages 385 - 388, XP071235857, ISSN: 0014-5793, DOI: 10.1016/S0014-5793(97)00936-8 * |
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| PL244350B1 (en) | 2024-01-15 |
| EP4240838A1 (en) | 2023-09-13 |
| PL435319A1 (en) | 2022-03-21 |
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