Hyaluronidase polypeptide and its applications

Abstract

FIELD: pharmaceutical industry.SUBSTANCE: polypeptide having hyaluronidase activity and its use. A polypeptide having hyaluronidase activity is proposed, in which n consecutive amino acids are deleted from the C-terminus of the polypeptide, consisting of the amino acid sequence SEQ ID NO: 1, where n is a natural number 34, 68, 102, 136, or 170. Also provided are a nucleic acid molecule encoding said polypeptide, an expression vector containing said nucleic acid molecule, and a host cell containing said vector. Also provided are a pharmaceutical composition for local administration, a carrier for drug delivery, and a pharmaceutical composition for preventing or treating edema, containing the said polypeptide.EFFECT: obtaining a polypeptide having an enzymatic activity equal to or exceeding that of mature wild-type PH-20 hyaluronidase, and also having an increased expression level when expressed in CHO cells compared to mature wild-type PH-20 hyaluronidase and being highly stable.22 cl, 7 dwg, 20 tbl, 10 ex

Description

[0001] Field of invention

[0002] The present invention relates to a hyaluronidase polypeptide and its use.

[0003] Previous level of technology

[0004] The term "hyaluronidase" is a general term for a family of enzymes that catalyze the breakdown of hyaluronic acid. Durand-Reynals first described hyaluronidase as a diffusible factor. It was later named hyaluronidase because it was observed to exhibit strong activity against hyaluronic acid. According to their enzymatic mechanism, these enzymes are classified into the following three types: hyaluronate 4-glycanohydrolases (EC 3.2.1.35), common in testes, lysosomes, and bee venoms; hyaluronate 3-glycanohydrolases (EC 3.2.1.36), found in leeches; and hyaluronate lyases (EC 4.2.2.1), found in bacteria.

[0005] In particular, hyaluronidase (PH-20) in the testes is a glycosylphosphatidylinositol (GPI)-anchored enzyme present on the acrosomal portion of the sperm and is an important enzyme inducing fertilization by degrading the thick outer wall of the egg. In addition, PH-20 is known to cleave the β(1-4) bond between D-glucuronic acid and N-acetyl-D-glucosamine, which is present in hyaluronic acid, chondroitin, and chondroitin sulfate among the glycosaminoglycans common in mammalian skin. The general molecular formula of these enzymes is C 2455 H 3775 N 617 O 704 S 21 , and the molecular weight is 53870.9 g / mol. Humans have six genes associated with these enzymes, including HYAL1, HYAL2, HYAL3, and PH-20 / SPAM1.

[0006] Since the 1950s, the wide range of applications of hyaluronidases has been extensively explored. The first application was the subcutaneous injection of parenteral fluids. Furthermore, enzymes have found application in orthopedics, ophthalmology, plastic surgery, dentistry, oral surgery, gynecology, and otolaryngology. These applications include use in infiltration and conduction anesthesia to enhance the diffusion of local anesthetics and steroids, dispersal of biological fluid accumulations such as hematomas, prevention of abdominal adhesions and stones, and treatment of infertility.

[0007] Currently, hyaluronidases extracted from ram or bull testicles are commercially available, such as Vitrase (ISTA Pharmaceuticals, ovine) and Amphadase (Amphastar Pharmaceuticals, bovine). These products have entered mass production, which involves loading the appropriate concentration of crude hyaluronidases into vials and then lyophilizing them. Commercial animal-derived hyaluronidases present numerous challenges in their use in various fields, as the foreign proteins they contain can cause allergic reactions, and their biological activity decreases over time due to reduced stability.

[0008] To overcome these problems, recombinant hyaluronidase was studied. Recombinant proteins can be expressed in various cell types, including E. coli, yeast, insect cells, animal cells, and so on. In the case of hyaluronidases, in particular, their activity is affected by glycosylation, which occurs during the post-translational modification process. This is because glycans can affect the antigenicity, structural folding, solubility, and stability of glycoproteins. From this perspective, animal cells are suitable for expression, with CHO (Chinese hamster ovary) cells being the most suitable. They are highly safe, as yeast or insect cells, in which glycosylation occurs, differ from mammalian cells in terms of the post-translational modification process.

[0009] The first recombinant hyaluronidase PH-20 was developed by Halozyme Therapeutic and sold under the trade name Hylenex, and its various applications in subcutaneous injection, vitrectomy and ophthalmic disorders, etc. are being developed. However, hyaluronidases still have low yield or low stability, and the demand for them greatly exceeds the supply, so there is a need for hyaluronidase with higher yield or stability.

[0010] Detailed description of the invention

[0011] Technical task

[0012] According to one aspect, the present invention is to provide a hyaluronidase polypeptide with excellent stability and increased activity.

[0013] According to another aspect, the present invention provides a composition for topical administration comprising a polypeptide according to an aspect of the present invention.

[0014] According to a further aspect, the present invention is to provide a drug delivery carrier comprising a polypeptide according to an aspect of the present invention.

[0015] According to another aspect, the present invention is to provide a composition for preventing or treating edema comprising a polypeptide according to an aspect of the present invention.

[0016] Technical solution

[0017] According to one aspect, the present invention relates to a polypeptide in which the C-terminal region is deleted from the amino acid sequence of a wild-type hyaluronidase.

[0018] According to another aspect, the present invention relates to a polypeptide comprising a deletion of at least one consecutive amino acid from amino acids 1-203 from the C-terminus of the amino acid sequence of wild-type hyaluronidase.

[0019] According to a further aspect, the present invention relates to a composition for topical administration comprising said polypeptide.

[0020] According to another aspect, the present invention relates to a drug delivery carrier comprising said polypeptide.

[0021] According to another aspect, the present invention relates to a composition for preventing or treating edema, comprising said polypeptide.

[0022] The present invention is described in detail below.

[0023] According to one aspect, the present invention relates to a polypeptide having a sequence homology of 90% or higher with a polypeptide in which the C-terminal region is deleted from the amino acid sequence of wild-type hyaluronidase. The wild-type hyaluronidase may be a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1.

[0024] The said polypeptide according to an aspect of the present invention has hyaluronidase activity and may have at least one of the following characteristics (1) to (6):

[0025] (1) is stable at pH values ​​of 3 to 10, specifically, the enzymatic activity is 57% or higher of the original activity after storage at pH values ​​in the range of above 3 to 10 or less for 4 weeks, or the enzymatic activity is 32% or higher of the original activity after storage at pH values ​​of 3 to less than 5 for 4 weeks,

[0026] (2) stable at -20 to 45°C, specifically, the enzymatic activity is 63% or higher of the original activity after storage at less than 0°C for 4 weeks, the enzymatic activity is 83% or higher of the original activity after storage at 0 to 40°C for 4 weeks, or the enzymatic activity is 52% or higher of the original activity after storage at 40°C or higher for 4 weeks,

[0027] (3) high hyaluronidase activity, in particular, the hyaluronidase activity is more than 1-3 times that of wild-type hyaluronidase,

[0028] (4) the presence of a titer more than 1-3 times higher than the titer of the polypeptide consisting of the amino acid sequence of SEQ ID NO: 1,

[0029] (5) the presence of activity of 120,000-150,000 IU / mg, and

[0030] (6) one or more amino acid residues are glycosylated.

[0031] In more detail, the polypeptide in the Example of the present invention was obtained by deleting the C-terminal region of the amino acid sequence of wild-type ovine hyaluronidase (CAS No. 488712-31-8). It was shown that the polypeptide thus obtained had stability and activity significantly higher than those of wild-type hyaluronidase, and even when used in a smaller amount, demonstrated equivalent drug absorption effects, drug diffusion stimulation effects, and biological fluid reabsorption effects compared to the wild-type enzyme. Thus, the polypeptide according to an embodiment of the present invention can be hyaluronidase.

[0032] In more detail, the polypeptide according to an embodiment of the present invention may have a sequence homology of 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 99.9% or more with a polypeptide comprising a deletion of at least one consecutive amino acid from 1 to 203 amino acids from the C-terminus of the amino acid sequence of wild-type hyaluronidase. In this regard, the polypeptide according to an embodiment of the present invention is not a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1. Additionally, the polypeptide according to an embodiment of the present invention may retain the catalytic activity of wild-type hyaluronidase. In particular, a polypeptide according to an embodiment of the present invention may have an activity and / or stability equal to or greater than that of wild-type hyaluronidase.The wild-type hyaluronidase may be a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1.

[0033] In particular, the polypeptide according to an embodiment of the present invention may be a C-terminal deletion variant in which n amino acids from the C-terminus of the amino acid sequence of wild-type hyaluronidase are deleted (where n is a natural number from 1 to 203).

[0034] As an example, a polypeptide according to an embodiment of the present invention may have a deletion of at least one consecutive amino acid from amino acids 1 to 203, 1 to 170, 1 to 136, 1 to 102, 1 to 68, 34 to 203, 34 to 170, 34 to 136, 34 to 102, 34 to 68, 68 to 203, 68 to 170, 68 to 136, 68 to 102, 34, 68, 102, 136 or 170 amino acids from the C-terminus of the amino acid sequence of wild-type hyaluronidase. A polypeptide according to an embodiment of the present invention may further comprise a deletion of the first amino acid from the N-terminus of the amino acid sequence of wild-type hyaluronidase. The amino acid sequence of wild-type hyaluronidase may be the amino acid sequence of SEQ ID NO: 1.

[0035] As one example, a polypeptide according to an embodiment of the present invention may consist of an amino acid sequence located from the first amino acid to the m-th amino acid or from the second amino acid to the m-th amino acid from the N-terminus of the amino acid sequence of wild-type hyaluronidase (where m is a natural number from 315 to 517). The amino acid sequence of wild-type hyaluronidase may be the amino acid sequence of SEQ ID NO: 1.

[0036] As one example, a polypeptide according to an embodiment of the present invention may consist of the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6.

[0037] As one example, a polypeptide according to an embodiment of the present invention can be expressed using animal cells as a host. In this regard, the polypeptide according to an embodiment of the present invention can be expressed in animal cells as a host and glycosylated through a post-translational modification (PTM) process during expression.

[0038] The polypeptide according to an embodiment of the present invention may be stable at a pH of 3 to 10. In particular, the enzymatic activity of the polypeptide according to an embodiment of the present invention may decrease to a lesser extent when stored at a pH of 3 to 10 than that of wild-type hyaluronidase.

[0039] In the Example of the present invention, it was found that the polypeptide according to an embodiment of the present invention retained enzymatic activity after four weeks of storage at a pH of 3 to 10.

[0040] For example, the polypeptide according to an embodiment of the present invention may have an enzymatic activity of 32% or more, 33% or more, 34% or more, 35% or more, 36% or more, 37% or more, 38% or more, 39% or more, 40% or more, 41% or more, 42% or more, 43% or more, 44% or more, 45% or more, 46% or more, 47% or more, 48% or more, 49% or more, 50% or more, 51% or more, 52% or more, 53% or more, 54% or more, or 55% or more of its original activity after storage at a pH in the range of pH 3 to less than pH 5, or at a pH of 3 to 4, such as pH 3, for 4 weeks. In this context, the said polypeptide can be stored at a temperature of 5°C or 37°C.

[0041] For example, a polypeptide according to an embodiment of the present invention may have an enzymatic activity of 57% or more, 58% or more, 59% or more, 60% or more, 61% or more, 62% or more, 63% or more, 64% or more, or 65% or more of its initial activity after storage at a pH in the range of more than pH 3 to pH 10 or less, pH from 4 to 10, or pH from 5 to 10, such as pH 5, pH 7, or pH 10, for 4 weeks. In this context, said polypeptide can be stored at a temperature of 5°C or 37°C.

[0042] For example, a polypeptide according to an embodiment of the present invention may have an enzymatic activity of 57% or more, 58% or more, 59% or more, 60% or more, 61% or more, 62% or more, 63% or more, 64% or more, 65% or more, 66% or more, 67% or more, 68% or more, 69% or more, 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, or 82% or more of its original activity after storage at a pH in the range of greater than pH 3 to less than pH 10, from greater than pH 3 to a pH of 9 or less, from more than pH 3 to pH 8 or less, from more than pH 3 to pH 7 or less, from pH 4 or more to less than pH 10, pH from 4 to 9, pH from 4 to 8, pH from 4 to 7, from pH 5 to less than pH 10, pH from 5 to 9, pH from 5 to 8 or pH from 5 to 7, for example pH 5 or pH 7, for 4 weeks. In this context, said polypeptide can be stored at a temperature of 5°C or 37°C.

[0043] The polypeptide according to an embodiment of the present invention can be stable under freezing, refrigerated, and high-temperature storage conditions. In more detail, when stored under freezing, refrigerated, and high-temperature storage conditions, the enzymatic activity of the polypeptide according to an embodiment of the present invention decreases to a lesser extent than that of wild-type hyaluronidase.

[0044] In the Example of the present invention, it was shown that the polypeptide according to an embodiment of the present invention retained enzymatic activity after 4 weeks of storage under frozen conditions (e.g., from -18°C to -20°C), refrigerated storage (e.g., from 2°C to 8°C) and at high temperature (e.g., from 40°C to 45°C).

[0045] For example, the polypeptide according to an embodiment of the present invention may have an enzymatic activity of 63% or more, 64% or more, 65% or more, 66% or more, 67% or more, 68% or more, 69% or more, 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more of the initial activity after storage at a frozen temperature, such as a temperature of less than 0°C, from -20 to less than 0°C, from -20 to -10°C, or from -20 to -18°C, as one example at a temperature of -20°C, for 4 weeks. In this context, said polypeptide can be stored at pH 5 or 7.

[0046] For example, a polypeptide according to an embodiment of the present invention may have an enzymatic activity of 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more of the initial activity after storage at a refrigerated temperature, such as 0 to 40°C, 0 to 10°C, or 2 to 8°C, such as 5°C, for 4 weeks. In this context, said polypeptide can be stored at a pH of 5 or 7.

[0047] For example, the polypeptide according to an embodiment of the present invention may have an enzymatic activity of 52% or more, 53% or more, 54% or more, 55% or more, 56% or more, 57% or more, 58% or more, 59% or more, 60% or more, 61% or more, 62% or more, 63% or more, 64% or more, 65% or more, 66% or more, 67% or more, 68% or more, 69% or more, 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, or 79% or more of the original activity after storage at a high temperature, such as a temperature 40°C or more, from 40 to 50°C, or from 40 to 45°C, for example 40°C, for 4 weeks. In this context, the said polypeptide can be stored at a pH of 5 or 7.

[0048] The polypeptide according to an embodiment of the present invention may have a titer higher than that of wild-type hyaluronidase. For example, a polypeptide according to an embodiment of the present invention may have a titer exceeding greater than 1-3 times, greater than 1-2.5 times, greater than 1-2 times, greater than 1-1.9 times, greater than 1-1.8 times, greater than 1-1.7 times, 1.1-3 times, 1.1-2.5 times, 1.1-2 times, 1.1-1.9 times, 1.1-1.8 times, 1.1-1.7 times, 1.2-3 times, 1.2-2.5 times, 1.2-2 times, 1.2-1.9 times, 1.2-1.8 times, 1.2-1.7 times, 1.3-3 times, 1.3-2.5 times, 1.3-2 times, 1.3-1.9 times, 1.3-1.8 times, 1.3-1.7 times, 1.4-3 times, 1.4-2.5 times, 1.4-2 times, 1.4-1.9 times, 1.4-1.8 times, 1.4-1.7 times, 1.5-3 times, 1.5-2.5 times, 1.5-2 times, 1.5-1.9 times, 1.5-1.8 times or 1.5-1.7 times the titer of wild-type hyaluronidase.

[0049] For example, the polypeptide according to an embodiment of the present invention may have an activity of 120,000 to 150,000 IU / mg, 120,000 to 145,000 IU / mg, 120,000 to 140,000 IU / mg, 120,000 to 135,000 IU / mg, 121,000 to 150,000 IU / mg, 121,000 to 145,000 IU / mg, 121,000 to 140,000 IU / mg, 121,000 to 135,000 IU / mg, 122,000 to 150,000 IU / mg, 122,000 to 145,000 IU / mg, 122,000 to 140,000 IU / mg or 122,000 to 135,000 IU / mg.

[0050] According to another aspect, the present invention relates to a nucleic acid molecule encoding a polypeptide according to an embodiment of the present invention, a vector containing said nucleic acid molecule, and a cell containing said vector. Said cell can be selected from the group consisting of bacteria, including E. coli and actinomycetes, yeast, fungi, insect cells, animal cells, mammalian cells, algae cells, and plant cells. Mammalian cells can be selected from the group consisting of CHO, NS0, HEK293, BHK, Per.C6, MDCK, Vero, MRC, HeLa, IMR, and Sp2 / 0. CHO cells can be selected from the group consisting of CHO-DG44, CHO-DUKX, CHO-S, CHO-K1, and CHO-DP12.

[0051] According to a further aspect, the present invention relates to a composition for topical administration comprising a polypeptide according to an embodiment of the present invention. Said composition can be formulated for subcutaneous administration. In an example of the present invention, it was observed that, upon topical administration, the polypeptide according to an embodiment of the present invention exhibited significantly better effects in stimulating the absorption and diffusion of a drug compared to wild-type hyaluronidase. Thus, another embodiment of the present invention relates to a drug delivery vehicle comprising a polypeptide according to an embodiment of the present invention.

[0052] Furthermore, in the Example of the present invention, it was shown that, upon topical administration, the polypeptide according to an embodiment of the present invention exhibited a significantly more pronounced effect of stimulating the reabsorption of excess biological fluid compared to wild-type hyaluronidase. Thus, another embodiment of the present invention relates to a pharmaceutical composition for the prevention or treatment of edema, comprising the polypeptide according to an embodiment of the present invention as an active ingredient.

[0053] The composition according to the present invention, for example, a pharmaceutical composition, may further contain at least one active ingredient that performs an equivalent or similar function.

[0054] Furthermore, in accordance with standard techniques known to those skilled in the art, the composition of the present invention, for example, a pharmaceutical composition, can be prepared with a pharmaceutically acceptable carrier and received in the form of a single dose or placed in a multi-dose container. As used herein, the term "carrier" means a substance that facilitates the delivery of an object of interest to cells or tissues. In the present invention, the term "pharmaceutically acceptable" means having the property of being physiologically acceptable and not causing gastrointestinal disorders, allergic reactions such as dizziness, or similar reactions when administered to humans.

[0055] Any pharmaceutically acceptable carrier may be used, provided that it is commonly available for the preparation of the composition. Examples of pharmaceutically acceptable carriers include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0056] In addition to the above ingredient, the composition of the present invention, for example, a pharmaceutical composition, may further contain an additive such as a filler, an antiplatelet agent, a lubricant, a humectant, a flavoring agent, an emulsifying agent, a preservative, etc. In the present invention, the content of the additive in the composition is not specifically limited, but can be appropriately adjusted within the content range acceptable for a typical composition.

[0057] The term "excipient" as used herein means any substance, other than the therapeutic agent itself, used as a carrier or vehicle for delivering the therapeutic agent to a subject or added to a pharmaceutical composition to facilitate handling and storage thereof or to enable and facilitate the preparation of a unit dose of the composition.

[0058] The composition according to the present invention, for example, the pharmaceutical composition, can be prepared in various forms such as sterile injections, etc., according to the purpose and can be administered by various routes such as local administration, subcutaneous, intramuscular injection, etc.

[0059] The preferred dosage of the composition according to the present invention, for example, the pharmaceutical composition, may vary in the dosage range depending on the condition, body weight, age, sex, health status, specific nutritional characteristics and body constitution of the patient, the property of the drug, the severity of the disease, the time of administration of the composition, the route of administration and the period or interval of administration, the excretion rate and the form of the drug, but can be appropriately selected by those skilled in the art.

[0060] The term "effective dosage of a pharmaceutical composition" as used herein means an amount of a composition containing an active ingredient sufficient to treat a specific symptom. It may vary depending on the method of preparing the pharmaceutical composition, the method of administration, the time of administration and / or the route of administration, etc., and may be different according to various factors, including the type and degree of response to be achieved by administering the pharmaceutical composition, the type of individual to whom the administration is carried out, the age, body weight, general health condition of the individual, the symptom or severity of the disease, gender, diet, excretion, ingredients of other drug compositions to be used in the corresponding individual at the same time or at a different time, etc.d, as well as other similar factors well known in the pharmaceutical field, and specialists in the field of technology can easily determine and prescribe an effective dosage for the intended treatment.

[0061] The pharmaceutical composition according to the present invention can be administered once a day or divided into several doses. The pharmaceutical composition according to the present invention can be administered as a single therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with a standard therapeutic agent. Taking into account all of the above factors, the pharmaceutical composition according to the present invention can be administered in an amount such that the maximum effect can be achieved with a minimum amount without side effects.

[0062] Compared to wild-type hyaluronidase, the polypeptide or composition according to an embodiment of the present invention has a higher titer and, therefore, can achieve an equivalent effect at a lower dose. For example, the polypeptide or composition according to an embodiment of the present invention can be administered at a daily dose equal to 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, or 65% or less of the dose of wild-type hyaluronidase. The dose can be based on weight or % by weight. In addition, the total daily dose can be divided and administered continuously or discontinuously as needed.

[0063] Beneficial effects

[0064] By improving the expression level and stability in animal cells, the hyaluronidase polypeptide according to the embodiment of the present invention exhibits enzymatic activity equal to or greater than that of mature wild-type PH-20. Therefore, since the hyaluronidase polypeptide according to the embodiment of the present invention has an increased expression level when expressed in CHO cells compared with mature wild-type PH-20 and is highly stable, there is an effect of enhancing the industrial applicability of its various applications.

[0065] Furthermore, due to the ability to degrade hyaluronic acid, a component of the intercellular space, hyaluronidases regulate tissue adhesion to facilitate the penetration and diffusion of drugs and have the effect of stimulating the reabsorption of biological fluids present in excess in tissues. Therefore, their study and application areas are gradually expanding. However, wild-type PH-20 of animal origin has a high susceptibility to infection by a substance originating from an animal source. The hyaluronidase polypeptide according to an embodiment of the present invention is unlikely to cause infection and is therefore safe. This peptide exhibits high activity at the same dose compared with wild-type PH-20 of animal origin and can further be characterized by increased industrial applicability for various applications.

[0066] Description of graphic materials

[0067] FIGS. 1a-1c are images of SDS-PAGE results showing the stability at different pH of the polypeptide according to an embodiment of the present invention compared with wild-type hyaluronidase (FIG. 1a: week 0 of storage, FIG. 1b: week 2 of storage, FIG. 1c: week 4 of storage).

[0068] FIGS. 2a-2c are SDS-PAGE images showing the stability at different temperatures of the polypeptide according to an embodiment of the present invention compared with wild-type hyaluronidase (FIG. 2a: week 0 of storage, FIG. 2b: week 2 of storage, FIG. 2c: week 4 of storage; fr. means frozen, cold means refrigerated storage, high temp. means high temperature).

[0069] FIG. 3 shows the results of titer determination on plates under conditions of different pH and temperature to check whether the hyaluronic acid solution has its own activity.

[0070] FIG. 4a shows the results of titer determination for wild-type hyaluronidase under conditions of temperature from 20 to 60°C.

[0071] FIG. 4b shows the results of titer determination for a polypeptide according to an embodiment of the present invention under conditions of a temperature of 20 to 60°C.

[0072] FIG. 5a shows the results of titer determination for wild-type hyaluronidase under conditions of 35 to 40°C.

[0073] FIG. 5b shows the results of titer determination for a polypeptide according to an embodiment of the present invention under conditions of a temperature of 35 to 40°C.

[0074] FIG. 6a shows the results of titer determination for wild-type hyaluronidase under pH conditions of 5 to 7.

[0075] FIG. 6b shows the results of titer determination for a polypeptide according to an embodiment of the present invention under pH conditions of 5 to 7.

[0076] FIG. 7 shows graphs of blood levels of a polypeptide according to an embodiment of the present invention and Hirax depending on time in SD rats.

[0077] Implementation of the invention

[0078] The present invention will be better understood in light of the following examples, which are given to illustrate the present invention but should not be construed as limiting it.

[0079] EXAMPLE 1. Obtaining hyaluronidase

[0080] Hyaluronidase was obtained by shortening regions at the C-terminal of the amino acid sequence of SEQ ID NO: 1 of wild-type sheep hyaluronidase (CAS No. 488712-31-8) by 34 amino acid residues (Example 1-2), 68 amino acid residues (Example 1-3), 102 amino acid residues (Example 1-4), 136 amino acid residues (Example 1-5), 170 amino acid residues (Example 1-6), or 204 amino acid residues (Example 1-7).

[0081] First, cDNA based on the amino acid sequence of wild-type hyaluronidase (CAS No. 488712-31-8) was synthesized. The hyaluronidase gene was amplified using polymerase chain reaction (hereinafter referred to as "PCR") and tested for expression and activity in CHO-DG44 cells using the pcDNA 3.1 vector. The gene was introduced into CHO-DG44 cells using the pOptiVEC vector. When CHO-DG44 cells reached a density of approximately 4-6×10 6cells / ml, the cells were transformed with a plasmid constructed by inserting the hyaluronidase cDNA into the pOptiVEC vector using electroporation. After transformation, CHO-DG44 cells were cultured in Power CHO 2CD medium (with 4 mM L-glutamine) and the cell culture was centrifuged at 12,000 rpm for 10 minutes. The supernatant was collected and purified, if necessary, by various methods, including affinity chromatography, hydrophobic chromatography, ion-exchange chromatography, etc. The purified hyaluronidase was filtered by substitution with water during ultrafiltration and microfiltration. The amino acid sequences of the hyaluronidases obtained in this way are listed in Table 1.

[0082] TABLE 1

[0083] Name More (N → C) SEQ ID NO: Пример 1-1 (CAS № 488712-31-8) LDFRAPPLIS NTSFLWAWNA PAERCVKIFK LPPDLRLFSV KGSPQKSATG QFITLFYADR LGYYPHIDEK TGNTVYGGIP QLGNLKNHLE KAKKDIAYYI PNDSVGLAVI DWENWRPTWA RNWKPKDVYR DESVELVLQK NPQLSFPEAS KIAKVDFETA GKSFMQETLK LGKLLRPNHL WGYYLFPDCY NHNYNQPTYN GNCSDLEKRR NDDLDWLWKE STALFPSVYL NIKLKSTPKA AFYVRNRVQE AIRLSKIASV ESPLPVFVYH RPVFTDGSST YLSQGDLVNS VGEIVALGAS GIIMWGSLNL SLTMQSCMNL GNYLNTTLNP YIINVTLAAK MCSQVLCHDE GVCTRKQWNS SDYLHLNPMN FAIQTGKGGK YTVPGKVTLE DLQTFSDKFY CSCYANINCK KRVDIKNVHS VNVCMAEDIC IEGPVKLQPS DHSSSQNEAS TTTVSSISPS TTATTVSPCT PEKQSPECLK VRCLEAIANV TQTGCQGVKW KNTSSQSSIQ NIKNQTTY 1 Пример 1-2 DFRAPPLISN TSFLWAWNAP AERCVKIFKL PPDLRLFSVK GSPQKSATGQ FITLFYADRL GYYPHIDEKT GNTVYGGIPQ LGNLKNHLEK AKKDIAYYIP NDSVGLAVID WENWRPTWAR NWKPKDVYRD ESVELVLQKN PQLSFPEASK IAKVDFETAG KSFMQETLKL GKLLRPNHLW GYYLFPDCYN HNYNQPTYNG NCSDLEKRRN DDLDWLWKES TALFPSVYLN IKLKSTPKAA FYVRNRVQEA IRLSKIASVE SPLPVFVYHR PVFTDGSSTY LSQGDLVNSV GEIVALGASG IIMWGSLNLLS LTMQSCMNLG NYLNTTLNPY IINVTLAAKM CSQVLCHDEG VCTRKQWNSS DYLHLNPMNF AIQTGKGGKY TVPGKVTLED LQTFSDKFYC SCYANINCKK RVDIKNVHSV NVCMAEDICI EGPVKLQPSD HSSSQNEAST TTVSSISPST TATTVSPCTP EKQSPECLKV RCL 2 Пример 1-3 (BMI2004) DFRAPPLISN TSFLWAWNAP AERCVKIFKL PPDLRLFSVK GSPQKSATGQ FITLFYADRL GYYPHIDEKT GNTVYGGIPQ LGNLKNHLEK AKKDIAYYIP NDSVGLAVID WENWRPTWAR NWKPKDVYRD ESVELVLQKN PQLSFPEASK IAKVDFETAG KSFMQETLKL GKLLRPNHLW GYYLFPDCYN HNYNQPTYNG NCSDLEKRRN DDLDWLWKES TALFPSVYLN IKLKSTPKAA FYVRNRVQEA IRLSKIASVE SPLPVFVYHR PVFTDGSSTY LSQGDLVNSV GEIVALGASG IIMWGSLNLS LTMQSCMNLG NYLNTTLNPY IINVTLAAKM CSQVLCHDEG VCTRKQWNSS DYLHLNPMNF AIQTGKGGKY TVPGKVTLED LQTFSDKFYC SCYANINCKK RVDIKNVHSV NVCMAEDICI EGPVKLQPSD HSSSQNEAS 3 Пример 1-4 DFRAPPLISN TSFLWAWNAP AERCVKIFKL PPDLRLFSVK GSPQKSATGQ FITLFYADRL GYYPHIDEKT GNTVYGGIPQ LGNLKNHLEK AKKDIAYYIP NDSVGLAVID WENWRPTWAR NWKPKDVYRD ESVELVLQKN PQLSFPEASK IAKVDFETAG KSFMQETLKL GKLLRPNHLW GYYLFPDCYN HNYNQPTYNG NCSDLEKRRN DDLDWLWKES TALFPSVYLN IKLKSTPKAA FYVRNRVQEA IRLSKIASVE SPLPVFVYHR PVFTDGSSTY LSQGDLVNSV GEIVALGASG IIMWGSLNLS LTMQSCMNLG NYLNTTLNPY IINVTLAAKM CSQVLCHDEG VCTRKQWNSS DYLHLNPMNF AIQTGKGGKY TVPGKVTLED LQTFSDKFYC SCYANINCKK RVDIK 4 Пример 1-5 DFRAPPLISN TSFLWAWNAP AERCVKIFKL PPDLRLFSVK GSPQKSATGQ FITLFYADRL GYYPHIDEKT GNTVYGGIPQ LGNLKNHLEK AKKDIAYYIP NDSVGLAVID WENWRPTWAR NWKPKDVYRD ESVELVLQKN PQLSFPEASK IAKVDFETAG KSFMQETLKL GKLLRPNHLW GYYLFPDCYN HNYNQPTYNG NCSDLEKRRN DDLDWLWKES TALFPSVYLN IKLKSTPKAA FYVRNRVQEA IRLSKIASVE SPLPVFVYHR PVFTDGSSTY LSQGDLVNSV GEIVALGASG IIMWGSLNLLS LTMQSCMNLG NYLNTTLNPY IINVTLAAKM CSQVLCHDEG VCTRKQWNSS DYLHLNPMNF AIQTGKGGKY T 5 Пример 1-6 DFRAPPLISN TSFLWAWNAP AERCVKIFKL PPDLRLFSVK GSPQKSATGQ FITLFYADRL GYYPHIDEKT GNTVYGGIPQ LGNLKNHLEK AKKDIAYYIP NDSVGLAVID WENWRPTWAR NWKPKDVYRD ESVELVLQKN PQLSFPEASK IAKVDFETAG KSFMQETLKL GKLLRPNHLW GYYLFPDCYN HNYNQPTYNG NCSDLEKRRN DDLDWLWKES TALFPSVYLN IKLKSTPKAA FYVRNRVQEA IRLSKIASVE SPLPVFVYHR PVFTDGSSTY LSQGDLVNSV GEIVALGASG IIMWGSLNLLS LTMQSCMNLG NYLNTTLNPY IINVTLAAKM CSQVLCH 6 Пример 1-7 DFRAPPLISN TSFLWAWNAP AERCVKIFKL PPDLRLFSVK GSPQKSATGQ FITLFYADRL GYYPHIDEKT GNTVYGGIPQ LGNLKNHLEK AKKDIAYYIP NDSVGLAVID WENWRPTWAR NWKPKDVYRD ESVELVLQKN PQLSFPEASK IAKVDFETAG KSFMQETLKL GKLLRPNHLW GYYLFPDCYN HNYNQPTYNG NCSDLEKRRN DDLDWLWKES TALFPSVYLN IKLKSTPKAA FYVRNRVQEA IRLSKIASVE SPLPVFVYHR PVFTDGSSTY LSQGDLVNSV GEIVALGASG IIMWGSLNLS LTM 7

[0084] EXAMPLE 2. Analysis of hyaluronide stability in accordance with the deletion site

[0085] (1) Comparison of enzymatic activity or content depending on pH during storage according to the deletion site

[0086] Among the hyaluronidases obtained in Example 1, six hyaluronidases, excluding the hyaluronidase in Examples 1-7 with no activity, were analyzed for stability at different pH values ​​according to the deletion site. Each amino acid sequence was expressed in transiently transfected cells, and the resulting cell cultures were similarly concentrated before measuring the enzymatic activity. The concentration for the experiment was adjusted to 1500 IU / mL (equal to 100%), solutions were prepared according to the conditions shown in Table 2 by substituting with water, and they were stored at 37°C for 4 weeks under conditions of pH 3.0, pH 5.0, pH 7.0, or pH 10.0, performing titer determination every two weeks.

[0087] Samples with different deletion sites were analyzed for enzymatic activity or content at each time point according to the following protocols:

[0088] 1) The activity of hyaluronidases with different deletion sites was determined as a proportion in comparison with the European Pharmacopoeia standard (EP STD).

[0089] 1-1) Preparation of phosphate buffered saline, pH 6.4: A solution of 2.5 g sodium hydrogen phosphate dodecahydrate, 2.5 g sodium dihydrogen phosphate and 8.2 g sodium chloride in 950 ml water was adjusted with 1 M sodium hydroxide or 1 M hydrochloride to pH 6.4 and water was added to make a final volume of 1000 ml.

[0090] 1-2) Preparation of diluted solution: 0.140g of gelatin reagent was dissolved in a mixture of 100ml of phosphate buffer saline with pH 6.4 and 100ml of water at 37°C. The diluted solution should be used within 2 hours after preparation.

[0091] 1-3) Preparation of the substrate solution: 100 ml of water was added to 0.5 g of sodium hyaluronate in small portions while stirring. Water was added slowly until the sodium hyaluronate swelled. Stirring was continued at 4°C for 12 hours or more. The resulting substrate solution was stored at 4°C and should be used within 4 days of preparation.

[0092] 1-4) Preparation of standard solution: The EP STD (EDQM) standard sample was dissolved in a diluted solution to obtain a concentration of approximately 50 IU / mL. An accurately measured 3 mL of this solution was added to a 250 mL volumetric flask, into which the diluted solution was then added to bring the volume to an exact 250 mL. The resulting solution was used as the standard solution.

[0093] 1-5) Preparation of test solution: The test solutions of hyaluronidases with different deletion sites, each approximately 0.6 IU / mL, were adjusted to predetermined pH values ​​and then diluted before use.

[0094] 2) Guidance: The standard and test solutions were tested according to the following procedure.

[0095] 2-1) A 50 ml conical tube was placed in a water bath maintained at 37°C, in which 7.5 ml of phosphate buffer saline with pH 6.4 and 5.0 ml of the substrate solution were mixed.

[0096] 2-2) 2.5 ml of the test solution was added to a conical tube containing phosphate buffer saline with pH 6.4 and the substrate solution and stirred for 1 minute.

[0097] 2-3) The entire mixture of solutions from the conical tube was loaded into an Ubbelohde type microviscometer (DIN 51 562, Part 2, capillary type MIII, constant: approximately 0.1 mm 2 / With 2 , or equivalent viscometer).

[0098] 2-4) Using a stopwatch, the time it took for the solution in the Ubbelohde type microviscometer to travel the distance from the upper indicator line to the lower indicator line was recorded.

[0099] 2-5) The time was recorded several times for about 20 minutes.

[0100] 2-6) The above procedure was repeated three times.

[0101] 3) Calculations: Titers (IU / mg) were calculated using the following formula.

[0102] 3-1) Reaction time: T1+ T2 / 2

[0103] 3-2) ηr -1 : {(k × T2) / 0.6915} -1

[0104] T1 represents the time it takes for the solution in the Ubbelohde type microviscometer to rise to the upper indicator line (seconds);

[0105] T2 is T - T1

[0106] T represents the time it takes for the solution in the Ubbelohde type microviscometer to fall to the lower indicator line (seconds);

[0107] k represents the constant of the Ubbelohde type microviscometer (mm 2 / With 2 ), see the specification of the Ubbelohde type microviscometer;

[0108] 0.6915 is the kinematic viscosity of the substrate solution at 37°C (mm 2 / With 2 ).

[0109] 3-3) Activity calculation: (B T / B R ) × (E R / E T ) × A

[0110] B T represents the slope angles of the regression curves on the graphs of the test solutions on the coordinate plane with the reaction time on the x-axis and the natural logarithm ηr -1 along the y-axis;

[0111] B R is the slope of the regression curve on the graph of a standard solution on a coordinate plane with the reaction time on the x-axis and the natural logarithm ηr -1 along the y-axis;

[0112] E T represents the concentration of the test solution (mg / ml);

[0113] E R represents the concentration of the standard solution (mg / ml);

[0114] A represents the titer of standard solution (IU / mg).

[0115] TABLE 2

[0116] Sample Activity Amino acids at the cleavage site Proportion of deleted region of the enzyme (%) pH during storage Storage temp. Example 1-1 Hyaluronidase (temporarily transfected cells) LY approximately 0% pH 7.0 37°C Example 1-2 DL approximately 6% Example 1-3 DS approximately 12% Example 1-4 DK approximately 18% Example 1-5 DT approximately 24% Example 1-6 DH approximately 30% Example 1-7 DM approximately 36%

[0117] The stability test data at different pH values ​​are shown in Table 3 below. The enzymatic activity (IU / mL) in each test is expressed as a percentage of the reference value of 1500 IU / mL (equal to 100%), which represents the initial enzymatic activity at week 0. The ratio of enzymatic activities was calculated using the following formula:

[0118] Ratio of enzyme activities (%) = (enzyme activity at the time of measurement) / (initial enzyme activity) × 100

[0119]

[0120] As shown in Table 3, the pH stability data obtained for the hyaluronidases with different deletion sites over 4 weeks showed higher enzymatic activity ratios (%) in the pH range of 5.0 to 7.0 than at other pH values. The hyaluronidases of Examples 1-2 to 1-6 showed remarkably high pH-dependent stability compared with the wild-type hyaluronidase of Example 1-1, and the hyaluronidases of Examples 1-2 to 1-4 showed the best results compared with the other hyaluronidases. The values ​​exceeding 100% were apparently due to measurement errors, which usually arise due to the large fluctuation in the titer determination results of biological products such as vaccines and recombinant proteins (cytokines, monoclonal antibodies, etc.).

[0121] (2)Comparison of enzymatic activity or content depending on storage temperatures according to the deletion site

[0122] The hyaluronidases of Examples 1-1 to 1-6 were subjected to stability analysis at various temperatures in the same manner as in part (1) of Example 2. The solutions were prepared according to the conditions specified in Table 2 and stored at a freezing temperature (-20°C), a refrigerator temperature (5°C), and a high temperature (40°C) for 4 weeks, during which titers were determined every two weeks. The stability of the hyaluronidases with different shortening sites was measured at various temperatures for 4 weeks; the measurement results are shown in Table 4.

[0123] TABLE 4

[0124] Ratio of enzymatic activities (%) Freezing In the refrigerator High temperature Sample Week 0 Week 2 Week 4 Week 0 Week 2 Week 4 Week 0 Week 2 Week 4 Example 1-1 100,22 81,47 62,51 100,54 90,89 82,55 98,13 65,09 51,55 Example 1-2 101,98 100,74 98,85 101,52 100,81 99,45 98,96 88,92 78,54 Example 1-3 102,68 100,88 99,22 102,77 101,91 100,23 99,47 89,90 79,63 Example 1-4 101,07 100,17 98,03 100,32 100,72 99,18 98,16 88,11 77,95 Example 1-5 100,12 98,82 95,52 100,53 98,70 97,51 99,39 85,31 75,78 Example 1-6 99,49 96,92 93,12 99,64 96,32 95,63 99,87 82,69 73,12

[0125] As shown in Table 4, the hyaluronidases from Examples 1-2 to 1-6 exhibited significant stability at different temperatures and maintained remarkably high enzymatic activity ratios (%), especially under freezing and refrigerated storage conditions. The DS amino acid hyaluronidase corresponding to Example 1-3, which was shortened by approximately 12% and showed the highest activity among the recombinant hyaluronidases, was named BMI2004 and used in the experiments.

[0126] EXAMPLE 3. Stability analysis of wild-type hyaluronidase and BMI2004

[0127] (1)Comparison of enzymatic activity or content depending on pH

[0128] The stability at different pH values ​​was compared between BMI2004 obtained in Example 1 and wild-type hyaluronidase. BMI2004 and wild-type hyaluronidase (manufacturer: Korea BMI, trade name: Hirax, hereinafter referred to as Hirax) used in the stability tests at different pH values ​​had a purity of 95% or higher, and the concentration of each was 1500 IU / mL (equal to 100%). The solutions were prepared according to the conditions in Table 5 by substituting with water and stored at 5°C under the conditions of pH 3.0, pH 5.0, pH 7.0, and pH 10.0 for 4 weeks, with titration testing performed every two weeks. At each time point, the samples were measured for enzymatic activity or content in the same manner as in part (1) of Example 2.

[0129] TABLE 5

[0130] pH Sample Storage temp. 3,0 BMI2004 / Hirax (1500 IU / ml) 2-8°C 5,0 2-8°C 7,0 2-8°C 10,0 2-8°C

[0131] The results of the stability analysis at different pH values ​​are shown in Table 6. The enzymatic activity (IU / mL) in each test is expressed as a percentage of the reference value of 1500 IU / mL (equal to 100%), which is the initial enzymatic activity at week 0. The ratio of enzymatic activities was calculated using the following formula:

[0132] Ratio of enzyme activities (%) = (enzyme activity at the time of measurement) / (initial enzyme activity) × 100

[0133]

[0134] As shown in Table 6, the stability of BMI2004 and Hirax at different pH values ​​was maintained for about 4 weeks. Higher ratios of enzymatic activities (%) were obtained at pH 5.0 and pH 7.0 than at pH 3.0 and pH 10.0. After week 2, the Hirax content (%) tended to decrease rapidly compared with BMI2004. Values ​​greater than 100% were apparently due to measurement errors, which usually occur due to the large fluctuation in the results of titer determination of biological products such as vaccines and recombinant proteins (cytokines, monoclonal antibodies, etc.). To further test the change in enzymatic activity (or content) to confirm the stability of Hirax and BMI2004 as a function of pH, SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) was performed under the conditions specified in Table 5 above and according to the following protocol:

[0135] 1) Preparation of sample buffer (5×): Pierce™ Lane Marker sample buffer (Thermo Scientific™, Cat. No. 39000) was used.

[0136] 2) Preparation of electrode buffer (1×): Novex™ Tris-glycine SDS electrode buffer (10×) (Invitrogen, Cat. No. LC2675) was used. An accurately measured 100 mL of this solution was transferred to a 1000 mL volumetric flask, which was then filled with water to make the exact volume 1000 mL.

[0137] 3) Preparation of test solution: Hirax and BMI2004 standard samples were modified with water according to the conditions. Approximately 20 µL of each solution and 5 µL of sample buffer (5×) were accurately taken and mixed in an Eppendorf tube.

[0138] 4) Manual: The test solutions and the protein molecular weight reference standard PageRuler Prestained Protein Ladder (Thermo, Cat. No. 26616) were analyzed as follows.

[0139] 4-1) After removing the comb from the Novex™ Wedgewell™ 8-16% Tris-glycine buffer gel (Invitrogen, cat. XP08160BOX) or equivalent gel plate, it was washed with water to replace the buffer.

[0140] 4-2) The washed gel was placed in the Mini Gel Tank. In the Mini Gel Tank, the cathode compartment was completely filled with electrode buffer (1×), and the anode compartment was approximately 2 / 3 filled with electrode buffer (1×).

[0141] 4-3) 7 μl of PageRuler Prestained Protein Ladder protein molecular weight standard and 25 μl of test solution were applied to the gel.

[0142] 4-4) The Mini Gel Tank was connected to a power source and the settings were set as follows; separation was then performed until the samples had passed 90% of the gel length:

[0143] Voltage: 140V

[0144] Current: 400mA

[0145] Time: 60 minutes (time may vary depending on the conditions of the separation).

[0146] 4-5) After separation was completed, the gel was removed from the cassette and washed with water.

[0147] 4-6) The washed gel was immersed in a tray filled with a staining reagent (Coomassie Brilliant Blue R-250 staining solution, BIO-RAD, cat. no. 1610436).

[0148] 4-7) The tray was placed on a shaker and shaken at 30 rpm for 30 minutes.

[0149] 4-8) After staining was completed, the gel was transferred to a tray filled with staining solution (Coomassie Brilliant Blue R-250 staining solution, BIO-RAD, cat. no. 1610438), placed on a shaker, and shaken at 30 rpm until the stain was washed out of the gel, replacing the staining solution with fresh one. After reaching a certain degree of stain removal, the gel was immersed in water to remove the staining solution.

[0150] 4-9) After removing the staining solution from it, the gel was observed under a white lamp.

[0151] The test results are shown in FIG. 1a-1c. As shown in FIG. 1a-1c, from the SDS-PAGE results, based on whether another band is formed over time in the sample in the water-substituted state, it can be understood that BMI2004 was more stable at different pH over time than Hirax.

[0152] (2)Comparison of enzymatic activity or content depending on storage temperatures

[0153] The stability of BMI2004 obtained in Example 1 and Hirax at various temperatures was compared. BMI2004 and Hirax used in the temperature stability test had a purity of 95% or higher, and the concentration of each was 1500 IU / mL (equal to 100%). Solutions were prepared using water according to the conditions in Table 7 and stored at freezing temperature (-20°C), refrigerated temperature (5°C), and high temperature (40°C) for 4 weeks, during which titer determinations were performed every two weeks.

[0154] TABLE 7

[0155] Storage temp. Sample pH during storage ≤-20°C Hirax / BMI2004 (1500 IU / ml) pH 5.0 2-8°C pH 5.0 ≥40°C pH 5.0

[0156] The stability of Hirax and BMI2004 was measured depending on temperature for 4 weeks, and the measurement results are summarized in Table 8 and shown in FIG. 2. The highest content (%) was found at freezing temperatures of -20°C or lower. After week 2, the content (%) of BMI2004 remained at a significantly higher level than Hirax under all temperature conditions.

[0157]

[0158] In a further stability analysis depending on temperature, the changes in the contents of Hirax and BMI2004 were measured at different temperatures in the same way as in Example 3. In this regard, SDS-PAGE was performed under the conditions shown in Table 7. The results of the SDS-PAGE are shown in FIG. 2a to FIG. 2c. As shown in FIG. 2a to FIG. 2c, from the formation of other bands located differently from the initial position of the main band over time at freezing, refrigerator, and high temperature temperatures under the same aqueous environment conditions, it was clear that BMI2004 was more stable than Hirax.

[0159] EXAMPLE 4. Optimum enzymatic activity depending on temperature

[0160] The plate titer assay in this Example was developed based on the cylinder plate assay from the arsenal of microbiological methods for determining antibiotic activity. Specifically, cylinders (Peni cylinders) containing a specified amount of hyaluronidase were placed on plates of solidified agarose containing hyaluronic acid to allow the hyaluronidase to diffuse within the agarose plates. This diffusion caused the hyaluronic acid to be cleaved by hyaluronidase, and the remaining undigested hyaluronic acid was precipitated with cetylpyridinium chloride to form distinct rings. In this Example, it was possible to determine the enzymatic activity of hyaluronidase based on the size of the ring formed using this simple plate assay. Under each condition, the plate titer assay was performed as follows:

[0161] 1) Preparation of hyaluronic acid solution: 0.2 g of hyaluronic acid was completely dissolved in 100 ml of water and the pH was adjusted with hydrochloric acid and sodium hydroxide to a pH of 7.0±0.1.

[0162] 2) Preparation of 1.5% agarose: 1.5 g of agarose (SIGMA, Cat. No. A9539) was added to 100 ml of water and dissolved in a microwave oven.

[0163] 3) Preparation of 10% cetylpyridinium chloride: 10 g of cetylpyridinium chloride (SIGMA, Cat. No. C0732) was dissolved in 100 ml of water.

[0164] 4) Test solution: Hirax and BMI2004 standard samples were adjusted with water according to the conditions.

[0165] 5) Manual: The test solutions were analyzed as follows:

[0166] 5-1) The hyaluronic acid solution was heated at 37°C for about 20 minutes before mixing with 1.5% agarose.

[0167] 5-2) After 100 ml of 1.5% agarose was cooled to about 60°C, 100 ml of hyaluronic acid solution was added and mixed with a stirrer.

[0168] 5-3) The mixture was poured into a Petri dish (SPL, cat. no. 10050) to a layer thickness of approximately 3 mm.

[0169] 5-4) After the agarose gel had completely solidified, a peni cylinder (KisanBio, Cat. No. KS-P0161) was placed at a 90° angle on the concentric circle of the Petri dish. A 20 μL aliquot of the test solution was added to the cylinder, and the mixture was incubated for 18-20 hours in an incubator maintained at the temperature specified in each table.

[0170] 5-5) After the reaction was complete, the cylinder was removed and 3 ml of 10% cetylpyridinium chloride was added. After 20 minutes, the formation of distinct rings was observed.

[0171] 5-6) The diameter (in mm) of the ring was measured with an accuracy of 0.5 mm or less.

[0172] (1) Confirmation of the activity of hyaluronic acid solution

[0173] Before the optimal enzymatic activity analysis, titer determinations were performed on plates at various pH values ​​and temperature conditions to check whether the hyaluronic acid solutions exhibited intrinsic activity. The analysis was carried out at 20°C, 25°C, 30°C, 35°C, and 40°C under conditions of pH 4.0, pH 7.0, or pH 10.0. The results of the determination are shown in FIG. 3. As shown in FIG. 3, the hyaluronic acid solutions did not exhibit intrinsic activity.

[0174] (2)Comparison of enzymatic activity under temperature conditions of 20-40°C

[0175] BMI2004 and Hirax used in this enzyme activity assay were 95% pure or higher, and each was adjusted to a concentration of 1500 IU / mL (equal to 100%) after adjustment with water. Solutions were prepared according to the conditions in Table 9, and titer determinations were performed on plates at 20°C, 25°C, 30°C, 35°C, and 40°C.

[0176] TABLE 9

[0177] pH during storage Storage temp. Sample Condition 1 pH 4.0 20-40°C Hirax / BMI2004 (1500 IU / ml) Condition 2 pH 7.0 20-40°C Condition 3 pH 10.0 20-40°C

[0178] The test results are shown in FIG. 4a and FIG. 4b. The enzymatic activity of Hirax and BMI2004 was tested by the plate titer method at 20-40°C. As a result, large rings appeared at 35°C and 40°C. To quantify the enzymatic activity, the diameter of the rings in FIG. 4a and FIG. 4b was measured, and the measurement results are shown in Table 10 below. In Table 10, the diameters (in mm) had a measurement error of ±0.5 mm.

[0179] TABLE 10

[0180] Pace. Sample pH 4.0 pH 7.0 pH 10.0 20 Hirax 8 mm 10 mm 11 mm BMI2004 8 mm 12 mm 13 mm 25 Hirax 9 mm 10 mm 11 mm BMI2004 9 mm 13 mm 13 mm 30 Hirax 9 mm 13 mm 14 mm BMI2004 10 mm 13 mm 15 mm 35 Hirax 10 mm 15 mm 18 mm BMI2004 11 mm 18 mm 21 mm 40 Hirax 10 mm 15 mm 18 mm BMI2004 11 mm 18 mm 21 mm

[0181] As shown in Table 10, the comparison of enzymatic activity at 20-40°C showed that the activity of BMI2004 was higher than that of Hirax.

[0182] (3)Comparison of enzymatic activity under temperature conditions of 35-40°C

[0183] Based on the comparison results between Hirax and BMI2004 in terms of activity measured in Part (2) of Example 4, additional tests were conducted under conditions with smaller temperature ranges. Solutions were prepared in accordance with the conditions listed in Table 11 below and analyzed using titer plates at 35°C, 37°C, and 40°C.

[0184] TABLE 11

[0185] pH during storage Storage temp. Sample Condition 1 pH 4.0 35°C Hirax / BMI2004 (1500 IU / ml) 37°C 40°C Condition 2 pH 7.0 35°C 37°C 40°C Condition 3 pH 10.0 35°C 37°C 40°C

[0186] The test results are shown in FIG. 5a and FIG. 5b. The enzymatic activity of Hirax and BMI2004 was tested by titer plate determination at 35°C, 37°C, and 40°C. As a result, the largest rings appeared at 37°C. The ring diameters are shown in Table 12 below. The diameters (in mm) had a measurement error of ±0.5 mm.

[0187] TABLE 12

[0188] Sample 35°C 37°C 40°C Hirax BMI2004 Hirax BMI2004 Hirax BMI2004 pH 4.0 10 mm 12 mm 10 mm 13 mm 10 mm 12 mm pH 7.0 16 mm 19 mm 17 mm 20 mm 16 mm 19 mm pH 10.0 18 mm 21 mm 19 mm 22 mm 18 mm 21 mm

[0189] As shown in Table 12, the comparison of the enzymatic activity at 35°C, 37°C and 40°C indicated that the activity of BMI2004 was higher than that of Hirax.

[0190] EXAMPLE 5. Optimum enzymatic activity depending on pH

[0191] Hirax and BMI2004 used in the optimal enzyme activity assay had a purity of 95% or higher, and the concentration of each was 1500 IU / mL (equal to 100%) after adjustment with water. The solutions were prepared according to the conditions in Table 13. The enzyme activity was measured using the assay in Example 4. Based on the activity determination results at various temperatures in Part (3) of Example 4, a titer plate determination was performed depending on pH under the condition of 37°C, which produced the largest rings.

[0192] TABLE 13

[0193] pH during storage Storage temp. Sample Condition 3 pH 5.0 37°C Hirax / BMI2004 (1500 IU / ml) Condition 4 pH 6.0 Condition 5 pH 7.0 Condition 6 pH 8.0 Condition 7 pH 9.0 Condition 8 pH 10.0

[0194] The analysis results are shown in FIG. 6a and FIG. 6b. According to the titer determination results on the Hirax and BMI2004 enzymatic activity plates at 37°C under pH 5.0-10.0 conditions, the largest rings were formed at pH 7.0-10.0. A comparison of the ring diameters is shown in Table 14 below. The diameter (in mm) had a measurement error of ±0.5 mm.

[0195] TABLE 14

[0196] Sample 37°C Hirax BMI2004 pH 5.0 14 mm 16 mm pH 6.0 17 mm 18 mm pH 7.0 19 mm 20 mm pH 8.0 19 mm 20 mm pH 9.0 19 mm 20 mm pH 10.0 20 mm 21 mm

[0197] As can be seen from Table 14, the comparison of the enzymatic activity at 37°C under pH 5.0-10.0 conditions showed that the activity of BMI2004 was higher than that of Hirax at pH 5.0 or more, and was especially noticeably higher at pH 7.0 or more.

[0198] EXAMPLE 6. Titragialuronidase assay

[0199] For the hyaluronidase according to the embodiment of the present invention and the standard enzyme Hirax, titer determination was carried out in the same manner as in part (1) of Example 2, and the measurement results are shown in Table 15.

[0200] TABLE 15

[0201] Sample number BMI2004 (IU / mg) Hirax (IU / mg) 1 124497 81396 2 133870 81528 3 134678 80653 4 122433 80538 Average 128870 81029

[0202] As shown in Table 15, BMI2004 exhibited an activity of approximately 122,433 to 134,678 IU / mg protein, while Hirax exhibited an activity of approximately 80,538 to 81,528 IU / mg protein. Therefore, the hyaluronidase according to the embodiment of the present invention could achieve equivalent activity even when used in an amount of approximately 63% of that of standard Hirax.

[0203] EXAMPLE 7. Stimulating effect of hyaluronidase on the absorption and diffusion of a drug (1)

[0204] To confirm the stimulating effect of hyaluronidase according to an embodiment of the present invention on the absorption and diffusion of a drug, an absorption and diffusion stimulation assay of a drug was performed using trypan blue.

[0205] More specifically, Hirax and BMI2004 were each mixed with 0.2% trypan blue solution and administered subcutaneously once to Balb / c nude mice, which were monitored for trypan blue diffusion. Based on the titer values ​​listed in Table 15, all drugs were prepared at a concentration of either 10 IU / mL or 100 IU / mL under the conditions listed in Table 16 and administered at a dose of 0.05 mL. Diffusion areas (π×mm) were measured at each time point. 2 ).

[0206] TABLE 16

[0207] Sample (IU / ml) Peptide dose (µg / ml) 2.5 min 5 min 10 min 15 min 20 min Saline solution 0 77,38± 15,86 84,61± 13,92 98,11± 7,83 117,67± 10,35 121,84± 11,48 Hirax (10 IU / ml) About 0.123 74,45± 22,40 112,84± 15,01* 146,69± 29,36** 165,98± 18,49* 189,90± 41,85** Hirax (100 IU / ml) Approximately 1,234 84,25± 15,81 133,90± 14,52*** 149,27± 27,41** 184,80± 37,10** 215,12± 20,60*** BMI2004 (10 IU / ml) About 0.078 82,70± 22,95 109,65± 12,70* 153,17± 15,30*** 176,85± 14,08** 187,63± 36,40** BMI2004 (100 IU / ml) About 0.776 87,02± 17,51 126,70± 16,00*** 140,23± 16,95*** 180,98± 29,93*** 218,14± 26,85***

[0208] 1) Mean±standard deviation (SD)

[0209] 2) *p < 0.05, **p < 0.01, ***p < 0.001 when compared with the saline group as analyzed by SPSS (one-way analysis of variance (ANOVA), least significant difference (LSD) test).

[0210] As shown in Table 16, diffusion areas were measured after 2.5–20 minutes. Starting from 5 minutes after administration, Hirax and BMI2004 significantly increased diffusion areas compared to the negative control with saline.

[0211] EXAMPLE 8. Stimulating effect of hyaluronidase on the absorption and diffusion of a drug (2)

[0212] To confirm the stimulating effect of hyaluronidase according to an embodiment of the present invention on the absorption and diffusion of a drug, an absorption and diffusion analysis of a drug was performed using an akinesia model.

[0213] More specifically, cynomolgus macaques were used as a model of akinesia to test their ability to enhance drug permeation. The test substances were BMI2004 and Hirax, listed in Table 15, with saline serving as a negative control. Lidocaine and bupivacaine were used as anesthetics and mixed with the samples. Each sample was injected at a dose of 2 ml into six monkeys via the right and left parabulbar routes.

[0214] After the administration of anesthetics together with the samples, the time until the pupil movement stopped (time to akinesia) and the time from pupil anesthesia to the observation of pupil movement (akinesia duration) were measured, and the measurement results are shown in Table 17.

[0215] TABLE 17

[0216] Sample Peptide dose (µg / ml) Model (monkey) Eye Time to akinesia (min) Duration of akinesia (min) Saline solution 0 02 Left Akinesia is absent Akinesia is absent 03 On the right Akinesia is absent Akinesia is absent 05 Left Akinesia is absent Akinesia is absent 06 On the right Akinesia is absent Akinesia is absent Hirax (500 IU / 0.2 ml) approximately 6.17 mcg / 0.2 ml 01 Left 4 27 02 On the right 4 27 04 Left 2 17 05 On the right 2 32 Average 3,0 25,8 Standard deviation 1,2 6,3 BMI2004 (500 IU / 0.2 ml) approximately 3.88 mcg / 0.2 ml 01 On the right 1 32 03 Left 2 24 04 On the right 2 17 06 Left 1 41 Average 1,5 28,5 Standard deviation 0,6 10,3

[0217] 1) Drug: Lidocaine 2% (0.9 ml), Bupivacaine 0.5% (0.9 ml) and Dosage Compound (0.2 ml)

[0218] As shown in Table 17, the negative control with saline solution did not exhibit an akinesia effect, whereas when administered together with anesthetics, the polypeptide according to an embodiment of the present invention enhanced the penetration of anesthetics and thus caused an akinesia effect equivalent to that of the standard Hirax peptide, demonstrating its ability to induce drug absorption and diffusion. Furthermore, the polypeptide according to an embodiment of the present invention exhibited equivalent activity at a protein dose of approximately 63% of the standard Hirax dose.

[0219] EXAMPLE 9. Analysis of the ability of hyaluronide to stimulate reabsorption of excess biological fluid

[0220] To confirm the stimulating effect on the reabsorption of excess biological fluid, an edema model was used. In this example, the edema model was created by artificially inducing lymphedema. The test substances were BMI2004 and Hirax, listed in Table 15, and saline served as a negative control. Samples and doses are listed in Table 18 below.

[0221] TABLE 18

[0222] Sample Lymphatic edema Dose (per site) Route of administration Number of animals Saline solution + - PC 6 Hirax + 100 IU (approximately 1.234 mcg) PC 6 BMI2004 + 100 IU (approximately 0.776 mcg) PC 6

[0223] Lymphedema induction was performed in the tail for reasons of accuracy and simplicity. A 2 mm wide annular skin incision was made 1 cm from the base of the mouse's tail. Approximately 4 mm 2The ventral side was left uncut. To test the edema-reducing effect, as shown in Table 19 below, the tail diameter (mm) was measured at each time point from pre-dose (0 hour) to day 15 of induction after the first dose. The second dose was administered after 24 hours and the third dose after 48 hours. In this test, the tail diameter (mm) was measured using a caliper at a point 10 mm from the injury site.

[0224] TABLE 19

[0225] Sample Saline solution Hirax BMI2004 0 h 4,55±0,43 4,53±0,49 4,53±0,38 1 hour 4,57±0,43 4,39±0,49 4,57±0,65 2 hours 4,48±0,36 4,45±0,40 4,50±0,37 4 hours 4,52±0,38 4,41±0,38 4,48±0,51 24 hours 4,56±0,34 4,57±0,39 4,55±0,45 25 hours 4,68±0,32 4,48±0,35 4,53±0,44 26 hours 4,56±0,32 4,43±0,38 4,45±0,47 28 hours 4,58±0,37 4,42±0,44 4,46±0,48 48 hours 4,65±0,39 4,49±0,41 4,47±0,38 49 hours 4,56±0,27 4,22±0,37 4,26±0,37 50 hours 4,58±0,25 4,13±0,38 4,09±0,36 52 hours 4,59±0,25 4,16±0,47 4,18±0,36

[0226] As shown in Table 19, administration of BMI2004 reduced lymphedema, demonstrating the stimulating effect of this protein on the reabsorption of excess biological fluids. Furthermore, the polypeptide according to the embodiment of the present invention demonstrated equivalent activity at a protein dose of approximately 63% compared to standard Hirax.

[0227] EXAMPLE 10. In vivo hyaluronidase stability analysis

[0228] To confirm the in vivo stability of hyaluronidase according to an embodiment of the present invention, it was administered intravenously and the pharmacokinetic parameters were monitored. In this Example, SD rats were used as test animals. The animals were administered each of Hirax and BMI2004 at a total dose of approximately 180,000 IU over 30 minutes by intravenous infusion. Blood samples were collected before administration, 15 minutes after the start of the infusion (mid-infusion), 30 minutes after the start of the infusion (end of the infusion), and 31, 33, 36, 40, 45, 60, 75, 90 minutes and 2.5 hours, 4.5 hours, 24.5 hours and 48.5 hours after the start of the infusion.

[0229] The pharmacokinetic parameters of the polypeptide according to an embodiment of the present invention and Hirax are shown in Table 20. The dynamics of the polypeptide levels in the blood are depicted in FIG. 7. As can be seen in FIG. 7 and Table 20, Hirax was degraded too quickly for the half-life to be measured in vivo. In contrast, the polypeptide according to an embodiment of the present invention was observed to have a half-life of approximately 0.272 hours (approximately 16.3 minutes). Having a longer half-life than Hirax, the polypeptide according to an embodiment of the present invention has the potential to exhibit a more pronounced effect than Hirax, persisting in the body for a longer time. Additionally, the enzymes differed in their blood levels in vivo. For Hirax, blood levels peaked within 0.25 hours (15 minutes), while for BMI2004, blood levels peaked at 0.5 hours (30 minutes) after administration.This polypeptide demonstrated the potential to exert a more pronounced effect than Hirax, as its blood levels increased more slowly and it was maintained in vivo for a longer period of time than Hirax.

[0230] TABLE 20

[0231] Test substance Dose (IU / kg) tmax (h) t1 / 2 (h) CL (ml / h / kg) Hirax 180000 0,250 Neg. counter Neg. counter BMI2004 180000 0,500 0,272 418

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[0485] <insdseq>

[0486] <INSDSeq_length>347< / INSDSeq_length>

[0487] <INSDSeq_moltype>AA< / INSDSeq_moltype>

[0488] <INSDSeq_division>PAT< / INSDSeq_division>

[0489] <INSDSeq_feature-table>

[0490] <insdfeature>

[0491] <INSDFeature_key>REGION< / INSDFeature_key>

[0492] <INSDFeature_location>1..347< / INSDFeature_location>

[0493] <INSDFeature_quals>

[0494] <insdqualifier id="q11">

[0495] <INSDQualifier_name> note< / INSDQualifier_name>

[0496] <INSDQualifier_value> Hyaluronidase (Example

[0497] 1-6)< / INSDQualifier_value>

[0498] <NonEnglishQualifier_value> Hyaluronidase (Example

[0499] 1-6)< / NonEnglishQualifier_value>

[0500] < / insdqualifier>

[0501] < / INSDFeature_quals>

[0502] < / insdfeature>

[0503] <insdfeature>

[0504] <INSDFeature_key>source< / INSDFeature_key>

[0505] <INSDFeature_location>1..347< / INSDFeature_location>

[0506] <INSDFeature_quals>

[0507] <insdqualifier>

[0508] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[0509] <INSDQualifier_value>protein< / INSDQualifier_value>

[0510] < / insdqualifier>

[0511] <insdqualifier id="q12">

[0512] <INSDQualifier_name> organism< / INSDQualifier_name>

[0513] <INSDQualifier_value> synthetic construct< / INSDQualifier_value>

[0514] <NonEnglishQualifier_value> synthetic

[0515] design< / NonEnglishQualifier_value>

[0516] < / insdqualifier>

[0517] < / INSDFeature_quals>

[0518] < / insdfeature>

[0519] < / INSDSeq_feature-table>

[0520] <INSDSeq_sequence>DFRAPPLISNTSFLWAWNAPAERCVKIFKLPPDLRLFSVKGSPQKSATG

[0521] QFITLFYADRLGYYPHIDEKTGNTVYGGIPQLGNLKNHLEKAKKDIAYYIPNDSVGLAVIDWENWRPTWA

[0522] RNWKPKDVYRDESVELVLQKNPQLSFPEASKIAKVDFETAGKSFMQETLKLGKLLRPNHLWGYYLFPDCY

[0523] NHNYNQPTYNGNCSDLEKRRNDDLDWLWKESTALFPSVYLNIKLKSTPKAAFYVRNRVQEAIRLSKIASV

[0524] ESPLPVFVYHRPVFTDGSSTYLSQGDLVNSVGEIVALGASGIIMWGSLNLSLTMQSCMNLGNYLNTTLNP

[0525] YIINVTLAAKMCSQVLCH< / INSDSeq_sequence>

[0526] < / insdseq>

[0527] < / sequencedata>

[0528] <sequencedata sequenceidnumber="7">

[0529] <insdseq>

[0530] <INSDSeq_length>313< / INSDSeq_length>

[0531] <INSDSeq_moltype>AA< / INSDSeq_moltype>

[0532] <INSDSeq_division>PAT< / INSDSeq_division>

[0533] <INSDSeq_feature-table>

[0534] <insdfeature>

[0535] <INSDFeature_key>REGION< / INSDFeature_key>

[0536] <INSDFeature_location>1..313< / INSDFeature_location>

[0537] <INSDFeature_quals>

[0538] <insdqualifier id="q13">

[0539] <INSDQualifier_name> note< / INSDQualifier_name>

[0540] <INSDQualifier_value> Hyaluronidase (Example

[0541] 1-7)< / INSDQualifier_value>

[0542] <NonEnglishQualifier_value> Hyaluronidase (Example

[0543] 1-7)< / NonEnglishQualifier_value>

[0544] < / insdqualifier>

[0545] < / INSDFeature_quals>

[0546] < / insdfeature>

[0547] <insdfeature>

[0548] <INSDFeature_key>source< / INSDFeature_key>

[0549] <INSDFeature_location>1..313< / INSDFeature_location>

[0550] <INSDFeature_quals>

[0551] <insdqualifier>

[0552] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[0553] <INSDQualifier_value>protein< / INSDQualifier_value>

[0554] < / insdqualifier>

[0555] <insdqualifier id="q14">

[0556] <INSDQualifier_name> organism< / INSDQualifier_name>

[0557] <INSDQualifier_value> synthetic construct< / INSDQualifier_value>

[0558] <NonEnglishQualifier_value> synthetic

[0559] design< / NonEnglishQualifier_value>

[0560] < / insdqualifier>

[0561] < / INSDFeature_quals>

[0562] < / insdfeature>

[0563] < / INSDSeq_feature-table>

[0564] <INSDSeq_sequence>DFRAPPLISNTSFLWAWNAPAERCVKIFKLPPDLRLFSVKGSPQKSATG

[0565] QFITLFYADRLGYYPHIDEKTGNTVYGGIPQLGNLKNHLEKAKKDIAYYIPNDSVGLAVIDWENWRPTWA

[0566] RNWKPKDVYRDESVELVLQKNPQLSFPEASKIAKVDFETAGKSFMQETLKLGKLLRPNHLWGYYLFPDCY

[0567] NHNYNQPTYNGNCSDLEKRRNDDLDWLWKESTALFPSVYLNIKLKSTPKAAFYVRNRVQEAIRLSKIASV

[0568] ESPLPVFVYHRPVFTDGSSTYLSQGDLVNSVGEIVALGASGIIMWGSLNLSLTM< / INSDSeq_sequen

[0569] ce>

[0570] < / insdseq>

[0571] < / sequencedata>

[0572]

[0573] <---

Claims

1. A polypeptide having hyaluronidase activity, in which n consecutive amino acids are deleted from the C-terminus of the polypeptide consisting of the amino acid sequence of SEQ ID NO: 1, where n is a natural number of 34, 68, 102, 136 or 170.

2. The polypeptide of claim 1, wherein the first amino acid from the N-terminus of the amino acid sequence SEQ ID NO: 1 is additionally deleted.

3. The polypeptide according to claim 1, consisting of the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO:

6.

4. The polypeptide according to claim 1, which is glycosylated.

5. The polypeptide according to claim 1, which is stable at pH from 3 to 10.

6. The polypeptide according to claim 1, which is stable at a temperature from -20 to 45°C.

7. The polypeptide of claim 1, having an enzymatic activity that is 57% or higher of the initial activity after storage under pH conditions in the range of greater than pH 3 to pH 10 or less for 4 weeks.

8. The polypeptide of claim 1, having an enzymatic activity of 32% or greater than the initial activity after storage at pH conditions in the range of pH 3 or greater to less than pH 5 for 4 weeks.

9. The polypeptide of claim 1, having an enzymatic activity that is 63% or higher of its initial activity after storage at a temperature of less than 0°C for 4 weeks.

10. The polypeptide of claim 1, having an enzymatic activity that is 83% or higher of its initial activity after storage at a temperature of 0 to 40°C for 4 weeks.

11. The polypeptide of claim 1, having an enzymatic activity that is 52% or higher of its initial activity after storage at a temperature of 40°C or more for 4 weeks.

12. The polypeptide of claim 1, having an activity in the range of more than once to three times or less than the activity of a polypeptide consisting of the amino acid sequence of SEQ ID NO:

1.

13. The polypeptide according to claim 1, having an activity of 120,000 to 150,000 IU / mg.

14. A nucleic acid molecule encoding a polypeptide according to any one of claims 1-13.

15. An expression vector containing a nucleic acid molecule according to claim 14.

16. A host cell for expressing a polypeptide having hyaluronidase activity, containing the vector according to claim 15.

17. The cell of claim 16, selected from the group consisting of bacteria, yeast, fungi, insect cells, animal cells, mammalian cells, algal cells, and plant cells.

18. The cell of claim 17, wherein the mammalian cells are selected from the group consisting of CHO, NS0, HEK293, BHK, Per.C6, MDCK, Vero, MRC, HeLa, IMR, and Sp2 / 0.

19. A pharmaceutical composition of hyaluronidase for topical administration, comprising a polypeptide according to any one of claims 1-13 and a pharmaceutically acceptable carrier.

20. A drug delivery vehicle comprising a polypeptide according to any one of claims 1-13 and a drug.

21. A pharmaceutical composition for the prevention or treatment of edema, comprising a polypeptide according to any one of claims 1-13 and a pharmaceutically acceptable carrier.

22. A pharmaceutical composition according to claim 21, prepared with the possibility of subcutaneous administration or intramuscular injection.

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