Hyaluronidase polypeptide and its uses

Abstract

The present invention relates to hyaluronidase polypeptides and their uses.

Description

Technical Field

[0001] The present invention relates to hyaluronidase polypeptides and their uses.

Background Art

[0002] Hyaluronidase, which is a general term for enzymes that depolymerize hyaluronic acid, was first known as a spreading factor by Duran-Reynals. Subsequently, it was observed to exhibit strong activity against hyaluronic acid, and thus came to be called hyaluronidase. This enzyme is classified, according to its mechanism of action, into hyaluronate 4-glycanohydrolase (EC 3.2.1.35) distributed in the testis, lysosomes, and bee venom, hyaluronate 3-glycanohydrolase (EC 3.2.1.36) present in the hill, and hyaluronate lyase (EC 4.2.2.1) present in bacteria.

[0003] In particular, hyaluronidase (PH-20) in the testis is attached to the glycosylphosphatidylinositol fixation site (Glycosylphosphatidylinositol, GPI anchor) of the acrosome part of sperm, and is an important enzyme that decomposes the thick outer wall layer outside the egg to cause fertilization. In addition, PH-20 is known to hydrolyze the β(1-4) bond between hyaluronic acid (HA) and chondroitin and D-glucuronic acid and N-acetyl-D-glucosamine present in chondroitin sulfate in glycosaminoglycans present in mammalian skin. The general molecular formula of this enzyme is C2455H3775N617O704S21, and its molecular weight is 53870.9 g / mol. In the case of humans, six genes including HYAL1, HYAL2, HYAL3, and PH-20 / SPAM1 are related to this enzyme.

[0004] Since the 1950s, the widespread use of hyaluronidase has been comprehensively studied. Its first use was subcutaneous injection of infusion solutions. It is also used in infiltration and block anesthesia to increase the diffusion of local anesthetics and steroids in other orthopedic, ophthalmic, plastic, dental, oral surgery, gynecological, and otolaryngological surgeries. It is used to disperse fluid accumulations such as hematomas, prevent peritoneal adhesions, prevent stone formation, and treat infertility, etc.

[0005] Currently marketed hyaluronidase is extracted from the testicles of sheep (ovine) or cows (bovine) for use. Examples include Vitrase (ISTA Pharmaceuticals, ovine source), Amphadase (Amphastar Pharmaceuticals, bovine source), etc. Such unprocessed hyaluronidase is formulated into products by dissolving it at an appropriate concentration, filling it into vials, and freeze-drying. The animal-derived hyaluronidase formulated into products contains foreign proteins and thus may cause allergic reactions. Also, due to the decrease in stability over time, its biological activity decreases, leaving many problems for application in various fields.

[0006] To address such problems, research on recombinant hyaluronidase has been conducted. Recombinant proteins can be expressed in various types of cells such as Escherichia coli, yeast, insect cells, and animal cells. In particular, in the case of hyaluronidase, glycosylation that occurs during the post-translational modification process of the protein affects its activity. This is because glycans can affect the antigenicity, structural folding, solubility, and stability of glycoproteins. From this perspective, in the case of yeast and insect cells where glycosylation occurs, the pattern during the post-translational modification process is different from that in mammals. Among various types of expression cells, animal cells are suitable, and among animal cells, Chinese Hamster Ovary (CHO) cells with ensured safety are the most suitable.

[0007] The recombinant hyaluronidase against the original PH20 is sold under the trade name Hylenex by Halozyme Therapeutic and is being developed for various uses such as subcutaneous injection, vitrectomy, and ophthalmic disorders. However, hyaluronidase still has low yield and stability, and the supply is low compared to the demand. In fact, there is a need for hyaluronidase with improved yield or stability.

Summary of the Invention

Problems to be Solved by the Invention

[0008] One example of the present invention is for providing a hyaluronidase polypeptide with excellent stability and increased activity.

[0009] Another example of the present invention is for providing a composition for topical administration containing the polypeptide according to one example of the present invention.

[0010] Another example of the present invention is for providing a drug delivery carrier containing the polypeptide according to one example of the present invention.

[0011] Another example of the present invention is for providing a composition for preventing or treating edema containing the polypeptide according to one example of the present invention.

Means for Solving the Problems

[0012] One example of the present invention relates to a polypeptide in which the C-terminus is truncated in the amino acid sequence of wild-type hyaluronidase.

[0013] Another example of the present invention relates to a polypeptide in which 1 to 203 amino acids are deleted from the C-terminus in the amino acid sequence of wild-type hyaluronidase.

[0014] Another example of the present invention relates to a composition for topical administration containing the polypeptide.

[0015] Another example of the present invention relates to a drug delivery carrier containing the polypeptide.

[0016] Another example of the present invention relates to a composition for preventing or treating edema containing the polypeptide.

[0017] Hereinafter, the present invention will be described in more detail.

[0018] One example of the present invention relates to a polypeptide having a sequence homology of 90% or more with a polypeptide in which the C-terminus is cleaved with 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.

[0019] The polypeptide according to one example of the present invention has hyaluronidase activity and can have one or more of the following characteristics (1) to (6): (1) A characteristic of being stable at pH 3 to 10. Specifically, the enzyme activity after storage for 4 weeks at a pH belonging to the range of more than pH 3 to 10 is 57% or more of the initial activity, or the enzyme activity after storage for 4 weeks at pH 3 to less than 5 is 32% or more of the initial activity. (2) A characteristic of being stable at a temperature of -20 to 45 °C. Specifically, the enzyme activity after storage for 4 weeks at a temperature belonging to less than 0 °C is 63% or more of the initial activity, the enzyme activity after storage for 4 weeks at a temperature belonging to the temperature range of 0 to 40 °C is 83% or more of the initial activity, or the enzyme activity after storage for 4 weeks at a temperature belonging to 40 °C or more is 52% or more of the initial activity. (3) High hyaluronidase activity. Specifically, the hyaluronidase activity is more than 1-fold to 3-fold compared to wild-type hyaluronidase. (4) Having a titer of more than 1-fold to 3-fold the titer of the polypeptide consisting of the amino acid sequence of SEQ ID NO: 1. (5) Having an activity of 120,000 to 150,000 IU / mg, and (6) One or more amino acid residues being glycosylated.

[0020] Specifically, in the embodiment of the present application, a polypeptide was produced by cleaving the C-terminus with the amino acid sequence of Ovine-derived wild-type hyaluronidase (CAS no. 488712-31-8), and as a result of confirming the stability and activity of the produced polypeptide, it showed significantly better stability and activity compared to the wild-type hyaluronidase, and it was possible to achieve equivalent drug absorption effects, drug diffusion promotion effects, and body fluid reabsorption promotion effects using a small amount of protein. Therefore, the polypeptide according to an example of the present invention may be a hyaluronidase.

[0021] Specifically, the polypeptide according to an example of the present invention may have 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 sequence homology with a polypeptide in which 1 to 203 amino acids are deleted from the C-terminus in the amino acid sequence of wild-type hyaluronidase. At this time, the polypeptide according to an example of the present invention is not a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1. Further, the polypeptide according to an example of the present invention may maintain the catalytic activity of wild-type hyaluronidase. Specifically, the polypeptide according to an example of the present invention may have activity and / or stability equal to or higher 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.

[0022] Specifically, the polypeptide according to an example of the present invention may be one in which n amino acids are deleted from the C-terminus in the amino acid sequence of wild-type hyaluronidase (where n is a natural number from 1 to 203).

[0023] As an example, the polypeptide according to an example of the present invention may have 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 deleted from the C-terminus in the amino acid sequence of wild-type hyaluronidase. The polypeptide according to an example of the present invention may have the first amino acid additionally deleted from the N-terminus in the amino acid sequence of wild-type hyaluronidase. The amino acid sequence of the wild-type hyaluronidase may be the amino acid sequence of SEQ ID NO: 1.

[0024] As an example, the polypeptide according to an example of the present invention may consist of 1 to m or 2 to m amino acids from the N-terminus in the amino acid sequence of wild-type hyaluronidase (where m is a natural number from 315 to 517). The amino acid sequence of the wild-type hyaluronidase may be the amino acid sequence of SEQ ID NO: 1.

[0025] As an example, the polypeptide according to an example 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.

[0026] As an example, the polypeptide according to an example of the present invention may be expressed using animal cells as a host. Thus, the polypeptide according to an example of the present invention may be expressed using animal cells as a host and may be glycosylated through the Post-Translational Modifications (PTM) process during protein expression.

[0027] The polypeptide according to an example of the present invention may be stable at pH 3 to 10. Specifically, the polypeptide according to an example of the present invention may have even less decrease in enzyme activity compared to wild-type hyaluronidase when stored at pH 3 to 10.

[0028] As a result of measuring the enzyme activity while storing the polypeptide according to an example of the present invention in the range of pH 3 to 10 in the embodiment of the present application, the enzyme activity was maintained even at the fourth week of storage.

[0029] For example, the polypeptide according to an example of the present invention has a pH belonging to the range of pH 3 to less than 5, or pH 3 to 4, and as an example, after storage at pH 3 for 4 weeks, the enzyme activity is 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 the initial activity. At this time, the storage temperature of the polypeptide may be 5°C or 37°C.

[0030] For example, the polypeptide according to an example of the present invention has a pH belonging to the range of pH exceeding 3 to 10, pH 4 to 10, or pH 5 to 10, and as an example, after storage at pH 5, pH 7, or pH 10 for 4 weeks, the enzyme activity is 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 the initial activity. At this time, the storage temperature of the polypeptide may be 5°C or 37°C.

[0031] For example, a polypeptide according to an example of the present invention has an enzyme 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 the initial activity after storage at a pH belonging to the range of more than pH 3 to less than 10, more than pH 3 to 9, more than pH 3 to 8, more than pH 3 to 7, pH 4 to less than 10, pH 4 to 9, pH 4 to 8, pH 4 to 7, pH 5 to less than 10, pH 5 to 9, pH 5 to 8, or pH 5 to 7, for example, at pH 5 or pH 7 after storage for 4 weeks. At this time, the storage temperature of the polypeptide may be 5°C or 37°C.

[0032] A polypeptide according to an example of the present invention may be stable under freezing, refrigeration, and high-temperature conditions. Specifically, a polypeptide according to an example of the present invention may have even less reduction in enzyme activity compared to wild-type hyaluronidase when stored under freezing, refrigeration, and high-temperature conditions.

[0033] As a result of measuring the enzyme activity while storing a polypeptide according to an example of the present invention at freezing (for example, -18°C to -20°C), refrigeration (for example, 2 to 8°C), and high-temperature (for example, 40 to 45°C) temperatures in the embodiment of the present application, the enzyme activity was maintained even at the 4th week of storage.

[0034] For example, a polypeptide according to an example of the present invention has an enzyme 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 temperature belonging to the range of freezing temperature, for example, less than 0°C, less than -20 to 0°C, -20 to -10°C, or -20 to -18°C, for example, at -20°C for 4 weeks. At this time, the storage pH of the polypeptide may be 5 or 7.

[0035] For example, a polypeptide according to an example of the present invention has an enzyme 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 temperature belonging to the range of refrigeration temperature, for example, 0 to 40°C, 0 to 10°C, or 2 to 8°C, for example, at 5°C for 4 weeks. At this time, the storage pH of the polypeptide may be 5 or 7.

[0036] For example, a polypeptide according to an example of the present invention has an enzyme 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 initial activity after storage at a temperature belonging to the range of high temperature, for example, 40°C or more, 40 to 50°C, or 40 to 45°C, for example, at 40°C for 4 weeks. At this time, the storage pH of the polypeptide may be 5 or 7.

[0037] A polypeptide according to an example of the present invention may have a higher titer compared to wild-type hyaluronidase. For example, a polypeptide according to an example of the present invention may have a titer that is more than 1-fold to 3-fold, more than 1-fold to 2.5-fold, more than 1-fold to 2-fold, more than 1-fold to 1.9-fold, more than 1-fold to 1.8-fold, more than 1-fold to 1.7-fold, 1.1-fold to 3-fold, 1.1-fold to 2.5-fold, 1.1-fold to 2-fold, 1.1-fold to 1.9-fold, 1.1-fold to 1.8-fold, 1.1-fold to 1.7-fold, 1.2-fold to 3-fold, 1.2-fold to 2.5-fold, 1.2-fold to 2-fold, 1.2-fold to 1.9-fold, 1.2-fold to 1.8-fold, 1.2-fold to 1.7-fold, 1.3-fold to 3-fold, 1.3-fold to 2.5-fold, 1.3-fold to 2-fold, 1.3-fold to 1.9-fold, 1.3-fold to 1.8-fold, 1.3-fold to 1.7-fold, 1.4-fold to 3-fold, 1.4-fold to 2.5-fold, 1.4-fold to 2-fold, 1.4-fold to 1.9-fold, 1.4-fold to 1.8-fold, 1.4-fold to 1.7-fold, 1.5-fold to 3-fold, 1.5-fold to 2.5-fold, 1.5-fold to 2-fold, 1.5-fold to 1.9-fold, 1.5-fold to 1.8-fold, or 1.5-fold to 1.7-fold higher than that of wild-type hyaluronidase.

[0038] For example, a polypeptide according to an example 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.

[0039] Another example of the present invention relates to a nucleic acid molecule encoding a polypeptide according to an example of the present invention, a vector containing the nucleic acid molecule, and a cell containing the vector. The cell may be selected from the group consisting of bacteria including Escherichia coli or actinomycetes, yeast, mold, insect cells, animal cells, mammalian cells, algal cells, and plant cells. The mammalian cells may be selected from the group consisting of CHO, NS0, HEK293, BHK, Per.C6, MDCK, Vero, MRC, HeLa, IMR, and Sp2 / 0. The CHO cells may be selected from the group consisting of CHO-DG44, CHO-DUKX, CHO-S, CHO-K1, and CHO-DP12.

[0040] Another example of the present invention relates to a composition for topical administration containing a polypeptide according to an example of the present invention. The composition may be for subcutaneous administration. As a result of topically administering a polypeptide according to an example of the present invention in the embodiments of the present application, the drug absorption and diffusion promoting effects were significantly superior compared to wild-type hyaluronidase. Therefore, another example of the present invention relates to a drug delivery carrier containing a polypeptide according to an example of the present invention.

[0041] Also, as a result of topically administering a polypeptide according to an example of the present invention in the embodiments of the present application, the excessive body fluid reabsorption promoting effect was significantly superior compared to wild-type hyaluronidase. Therefore, another example of the present invention relates to a pharmaceutical composition for preventing or treating edema containing a polypeptide according to an example of the present invention as an active ingredient.

[0042] The composition of the present invention, for example, the pharmaceutical composition, may additionally contain one or more active ingredients showing the same or similar functions in addition to the above-mentioned active ingredient.

[0043] In addition, the composition according to the present invention, for example, a pharmaceutical composition, can be manufactured in unit dosage form or in a multi-dose container by formulating it using a pharmaceutically acceptable carrier by a method that can be clearly implemented by a person having ordinary knowledge in the technical field to which the invention pertains. In the present invention, the term "carrier" means a compound that facilitates the addition of a compound into cells or tissues, and the term "pharmaceutically acceptable" refers to a composition that is physiologically acceptable and does not usually cause gastrointestinal disorders, allergic reactions such as dizziness, or similar reactions when administered to humans.

[0044] The pharmaceutically acceptable carrier is one that is commonly used in formulation, including but not limited to lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0045] In addition, the composition according to the present invention, for example, a pharmaceutical composition, can additionally contain additives such as fillers, anticoagulants, lubricants, wetting agents, fragrances, emulsifiers, and preservatives in addition to the above components. In the present invention, the content of the additives contained in the composition is not particularly limited and can be appropriately adjusted within the content range used in normal formulation.

[0046] As used herein, the term "excipient" means a substance that is not a therapeutic agent and is used as a carrier or medium for the delivery of a therapeutic agent or added to a pharmaceutical composition. This serves to improve handling and storage characteristics or to allow and facilitate the formation of unit dosages of the composition.

[0047] The compositions according to the present invention, such as pharmaceutical compositions, can be formulated into various forms, such as injections of sterile injection solutions, by ordinary methods according to their respective purposes of use, and can be administered through various routes, including local administration, such as subcutaneous administration or intramuscular injection.

[0048] The preferred dosage of the compositions according to the present invention, such as pharmaceutical compositions, varies depending on the patient's condition and body weight, age, gender, health status, diet and constitution specificity, nature of the formulation, degree of the disease, administration time of the composition, administration method, administration period or interval, excretion rate, and drug form, and can be appropriately selected by a person of ordinary skill in this field.

[0049] As used herein, the term "effective dosage of a pharmaceutical composition" means the amount of the composition of the active ingredient sufficient to treat a specific symptom. This varies depending on the formulation method, administration mode, administration time, and / or administration route of the pharmaceutical composition, and the type and degree of the reaction to be achieved by the administration of the pharmaceutical composition, the type of the individual to be administered, age, body weight, general health status, symptoms and degree of the disease, gender, diet, excretion, drugs used simultaneously or together with the individual at the same time or temporarily, and other components of the composition, and various factors such as similar factors well known in the pharmaceutical field, and a person having ordinary knowledge in the technical field can easily determine and prescribe the dosage effective for the intended treatment.

[0050] The administration of the pharmaceutical composition according to the present invention may be administered once a day or divided into several times. The composition may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. Considering all the above factors, it can be administered in an amount that can obtain the maximum effect with the minimum amount without side effects.

[0051] A polypeptide or composition according to an example of the present invention may have a high titer compared to wild-type hyaluronidase and achieve the same effect with a smaller dosage. For example, a polypeptide or composition according to an example of the present invention may have a daily dosage of 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, or 65% or less of wild-type hyaluronidase. The dosage may be based on weight or weight %. Also, the total daily dosage can be divided and administered continuously or discontinuously as needed.

Advantages of the Invention

[0052] The hyaluronidase polypeptide according to an example of the present invention has increased expression level and stability in animal cells compared to mature wild-type PH20 and has enzyme activity equal to or higher than that of mature wild-type PH20. Therefore, the hyaluronidase polypeptide according to an example of the present invention has the effect of increasing the protein expression level when expressed in CHO cells and having high protein stability compared to mature wild-type PH20, thereby increasing the industrial applicability for various uses.

[0053] In addition, hyaluronidase has the property of decomposing hyaluronic acid, a constituent of the intercellular space, which regulates the tissue binding force, facilitates drug penetration and diffusion, and promotes the reabsorption of excessive body fluid in the tissue. Therefore, the scope of use and application fields are gradually increasing. However, in the case of wild-type PH20 derived from animals, there is a high risk of infection from animal-derived substances. The hyaluronidase polypeptide according to an example of the present invention has a low risk of infection and is safe, has higher activity than the same amount of protein compared to wild-type PH20 derived from animals, and can further enhance the industrial applicability for various uses.

Brief Description of the Drawings

[0054]

Figure 1a-1c

Figure 2a-2c

Figure 3

Figure 4a

Figure 4b

Figure 5a

Figure 5b

Figure 6a

Figure 6b

Figure 7

Mode for Carrying Out the Invention

[0055] Hereinafter, the present invention will be described in more detail by the following examples. However, these examples are merely for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Examples

[0056] Example 1. Production of Hyaluronidase The C-terminus of the amino acid sequence of ovine-derived wild-type hyaluronidase (CAS no. 488712-31-8) with accession number 1 was truncated by 34 (Example 1-2), 68 (Example 1-3), 102 (Example 1-4), 136 (Example 1-5), 170 (Example 1-6), or 204 (Example 1-7) amino acids to produce hyaluronidase.

[0057] First, cDNA was synthesized based on the amino acid sequence of wild-type hyaluronidase (CAS no. 488712-31-8). The wild-type hyaluronidase gene was amplified using polymerase chain reaction (hereinafter referred to as PCR), and its expression and activity were confirmed using the pcDNA3.1 vector in CHO-DG44 cells. Then, it was inserted into CHO-DG44 cells using the pOtiVEC vector. When the cell number of CHO-DG44 cells reached 4 - 6×10 6 cells / mL, CHO-DG44 cells were transformed using the gene injection method (Electroporation) with the plasmid in which the hyaluronidase cDNA was inserted into the pOptiVEC vector. After the transfection, CHO-DG44 cells were cultured in Power CHO 2CD (with L-Glutamine 4 mM) medium. After completion of the culture, the cells were centrifuged at 12,000 rpm for 10 minutes to collect the cell supernatant. The collected culture solution was purified through various methods such as affinity chromatography, hydrophobic chromatography, and ion exchange chromatography when necessary. The purified hyaluronidase was replaced with water through ultrafiltration and microfiltration. The sequence of the produced hyaluronidase is shown in Table 1.

[0058]

Table 1

[0059] Example 2. Confirmation of stability by hyaluronidase cleavage site (1) Comparison of enzyme activity or content according to storage pH by cleavage site Six types of hyaluronidases excluding Example 1-7 without activity among the hyaluronidases produced in Example 1 were confirmed for pH stability by the cleavage site. After expressing each amino acid sequence in Transient cell, the culture solution was concentrated identically and the activity was measured. The experimental concentration was adjusted to 1,500 IU / mL (= 100%), replaced with water, and a solution was prepared according to the conditions described in Table 2. This was stored at 37 °C at pH 3.0, pH 5.0, pH 7.0, and pH 10.0 for 4 weeks, and a titer test was performed at each time point every 2 weeks.

[0060] The enzyme activity or content of the sample by the cleavage site at each time point was tested by the following test method:

[0061] 1) The activity of hyaluronidase by the cleavage site is determined by ratio comparison with EP STD.

[0062] 1-1) Preparation of pH 6.4 phosphate buffer: Weigh 2.5 g of disodium hydrogen phosphate dodecahydrate, 2.5 g of sodium dihydrogen phosphate, and 8.2 g of sodium chloride, dissolve them in 950 mL of water, then adjust the pH to 6.4 with 1 M sodium hydroxide test solution or 1 M hydrochloric acid test solution, and add water to make 1,000 mL.

[0063] 1-2) Preparation of dilution solution: Mix 100 mL of pH 6.4 phosphate buffer and 100 mL of water, then add 0.140 g of gelatin reagent and dissolve it at 37 °C. The dilution solution should be used within 2 hours.

[0064] 1-3) Preparation of substrate solution: While stirring, gradually add 100 mL of water to 0.5 g of sodium hyaluronate little by little. Slowly add water until the sodium hyaluronate swells. Stir at 4 °C for 12 hours or more. The substrate solution should be stored at 4 °C and used within 4 days.

[0065] 1-4) Preparation of standard solution: Use the EP STD (EDQM) standard product to dissolve it in a diluent to make it about 50 IU / mL. Accurately take 3 mL of this solution and put it into a 250 mL volumetric flask, then add the diluent to make it exactly 250 mL to obtain the standard solution.

[0066] 1-5) Preparation of test solution: Adjust the pH of the hyaluronidase test solution according to the conditions to about 0.6 IU / mL and dilute it for use.

[0067] 2) Procedure: Use the standard solution and the test solution to conduct tests by the following method.

[0068] 2-1) After setting the constant temperature water bath to 37°C, put 7.5 mL of pH 6.4 phosphate buffer solution and 5.0 mL of substrate solution into a 50 mL conical tube, mix them, and then place them in the constant temperature water bath and leave them to reach 37°C.

[0069] 2-2) Add 2.5 mL of the test solution to the conical tube containing the pH 6.4 phosphate buffer solution and the substrate solution and mix for 1 minute.

[0070] 2-3) Pour all the mixed liquid in the conical tube into an Ubbelohde microviscometer (DIN 51 562, Part 2, Calillary type MIII, constant: about 0.1 mm 2 / s 2 or a viscometer equivalent to it).

[0071] 2-4) Use a stopwatch to measure the time it takes for the liquid to flow from the upper calibration line to the lower calibration line of the Ubbelohde microviscometer.

[0072] 2-5) Repeat the measurement several times for about 20 minutes.

[0073] 2-6) Repeat the above process 3 times for testing.

[0074] 3) Calculation: Calculate the titer (IU / mg) using the following calculation formula.

[0075] 3-1) Reaction time: T1 + T2 / 2

[0076] 3-2) ηr -1 : {(k × T2 / 0.6915} -1 T1: Time (seconds) to reach the upper calibration mark of the Ubbelohde microviscometer T2: T - T1 T: Time (seconds) to reach the lower calibration mark of the Ubbelohde microviscometer k: Ubbelohde microviscometer constant (mm 2 / s 2 ) Refer to the Ubbelohde microviscometer test report 0.6915: Kinematic viscosity of the substrate solution at 37°C (mm 2 / s 2 )

[0077] 3-3) Activity calculation: (B T / B R ) * (E R / E T ) * A B T : Slope of the regression equation of the test solution obtained by taking the natural logarithm of ηr with the reaction time on the x-axis and ηr on the y-axis -1 B : Slope of the regression equation of the standard solution obtained by taking the natural logarithm of ηr with the reaction time on the x-axis and ηr on the y-axis R : Slope of the regression equation of the standard solution obtained by taking the natural logarithm of ηr with the reaction time on the x-axis and ηr on the y-axis -1 : Slope of the regression equation of the standard solution obtained by taking the natural logarithm of ηr with the reaction time on the x-axis and ηr on the y-axis E T : Concentration of the test solution (mg / mL) E R : Concentration of the standard solution (mg / mL) A: Potency of the standard solution (IU / mg)

[0078]

Table 2

[0079] The results of the pH stability experiment are shown in Table 3 below. The enzyme activity of each test is a percentage value representing the comparison of the measured activity (IU / mL) at each time point based on the initial enzyme activity of 1,500 IU / mL (= 100%) at week 0, and the enzyme activity ratio was calculated by the following formula: Enzyme activity ratio (%) = (Enzyme activity at the measurement time point) / (Initial enzyme activity) * 100

[0080] [Table 3]

[0081] As shown in Table 3, as a result of confirming the pH stability according to the cleavage site of hyaluronidase for about 4 weeks, a higher enzyme activity ratio (%) was shown in the pH range of 5.0 to 7.0. The hyaluronidases of Examples 1-2 to 1-6 had significantly higher pH stability compared to the wild-type hyaluronidase of Example 1-1, and particularly, the pH stability of the hyaluronidases of Examples 1-2 to 1-4 was excellent. Values exceeding 100% were judged as measurement errors that generally occur due to a large variation range in the titer test of biological preparations such as vaccines and recombinant proteins (cytokines, monoclonal antibodies, etc.).

[0082] (2) Comparison of enzyme activity or content according to storage temperature by cleavage site The temperature stability of the hyaluronidases of Examples 1-1 to 1-6 was confirmed in the same manner as in 1 of Example 2. A solution was prepared according to the conditions described in Table 2 above, and this was stored at freezing (-20°C), refrigeration (5°C), and high temperature (40°C) for 4 weeks, and a titer test was performed at each time point every 2 weeks. The stability of hyaluronidase according to the cleavage site at about 4 weeks with respect to temperature was confirmed and shown in Table 4.

[0083] [Table 4]

[0084] As shown in Table 4, the hyaluronidases of Examples 1-2 to 1-6 had higher temperature stability compared to Example 1-1, and particularly, the enzyme activity ratio (%) of the hyaluronidases of Examples 1-2 to 1-6 was maintained significantly higher under freezing and refrigerated storage conditions. In subsequent examples, the amino acid D-S hyaluronidase corresponding to Example 1-3 in which about 12% of the recombinant hyaluronidase with the highest activity was cleaved was named BMI2004 and used in the experiment.

[0085] Example 3. Confirmation of the Stability of Wild-Type Hyaluronidase and BMI2004 (1) Comparison of Enzyme Activity or Content by pH The pH stability of BMI2004 produced in Example 1 was compared with that of wild-type hyaluronidase. BMI2004 and wild-type hyaluronidase (manufacturer; BMIC Korea, product name; Hirax, hereinafter named Hirax) used in the pH stability test were purified to over 95%. The experimental concentration was adjusted to 1,500 IU / mL (=100%), and the solution was prepared by replacing it with water according to the conditions described in Table 5. This was stored at 5°C at pH 3.0, pH 5.0, pH 7.0, and pH 10.0 for 4 weeks, and a titer test was performed at each time point every 2 weeks. The enzyme activity or content of the sample at each time point was confirmed in the same manner as in (1) of Example 2.

[0086] [Table 5]

[0087] The results of the pH stability experiment are shown in Table 6. The enzyme activity of each test is a percentage value expressed by comparing the activity (IU / mL) measured at each time point based on the initial enzyme activity of 1,500 IU / mL (=100%) at week 0. The enzyme activity ratio was calculated by the following formula: Enzyme activity ratio (%) = (enzyme activity at the measurement time point) / (initial enzyme activity) * 100

[0088] [Table 6]

[0089] As shown in Table 6, the stability of BMI2004 and Hirax with respect to pH was confirmed for approximately 4 weeks. They had a higher enzyme activity ratio (%) at pH 5.0 and pH 7.0 compared to pH 3.0 and pH 10.0, and the content (%) of Hirax showed a tendency to decrease rapidly after the second week compared to BMI2004. Values exceeding 100% were judged to be measurement errors commonly occurring with a large variation range in the potency tests of biological preparations such as vaccines and recombinant proteins (cytokines, monoclonal antibodies, etc.). To additionally test the changes in enzyme activity (or content) with respect to the stability confirmation of Hirax and BMI2004 by pH, SDS-PAGE (Sodium Dodecyl Sulfate-PolyAcrylamide Gel Electrophoresis) was performed under the conditions of Table 5 above and tested by the following test methods:

[0090] 1) Preparation of Sample Buffer (5X): Pierce TM Use Lane Marker Reducing Sample Buffer (Thermo Scientific TM , Cat No. 39000).

[0091] 2) Preparation of Running Buffer (1X): Novex TM Use Tris-Glycine SDS Running Buffer (10X) (Invitrogen, Cat No. LC2675). Accurately take 100 mL and put it into a 1,000 mL volumetric flask, add water and mix to make exactly 1,000 mL.

[0092] 3) Preparation of test solution: Replace the Hirax and BMI2004 standards with water and adjust according to the conditions for use. Accurately take about 20 μL of this and 5 μL of Sample Buffer (5X) and put them into an EP tube and mix.

[0093] 4) Operating method: Operate with the test solution and PageRuler Prestained Protein Ladder standard (Thermo, Cat No. 26616) under the following operating conditions.

[0094] 4-1) Novex TM Wedgewell TM Remove the comb with one 8 - 16% Tris - Glycine Gel (Invitrogen, Cat No. XP08160BOX) or equivalent gel, and then wash the storage solution of the gel with water.

[0095] 4-2) Fasten the washed gel in the Mini Gel Tank. Fill the Cathode region of the Mini Gel Tank completely with Running Buffer (1X), and fill the Anode region to about 2 / 3.

[0096] 4-3) Inject 7 μL of PageRuler Prestained Protein Ladder standard and 25 μL of the test solution into the gel.

[0097] 4-4) Connect the power supply to the Mini Gel Tank. After operating as follows, develop the gel until 90% of it is developed. Volt: 140V Ampere: 400 mA Time: 60 minutes (The time can be changed according to the development status of the gel.)

[0098] 4-5) After the development is completed, separate the gel from the caster and then wash it with water.

[0099] 4-6) Put the washed gel into a container containing the staining solution (Coomassie Brilliant Blue R - 250 Staining Solution, BIO - RAD, Cat No. 1610436).

[0100] 4-7) Place the container on the Rocker and stain it at 30 rpm for 30 minutes.

[0101] 4-8) When the staining is completed, place it in a container containing a destaining reagent (Coomassie Brilliant Blue R-250 Destaining Solution, BIO-RAD, Cat No.1610438) and destain it while alternating the destaining reagent with a Rocker at 30 rpm until the gel is destained. When partial destaining is completed, place it in water and wash the destaining solution.

[0102] 4-9) When the destaining solution is washed, observe the gel over a white light.

[0103] The experimental results are shown in FIGS. 1a to 1c. As shown in FIGS. 1a to 1c, from the presence or absence of generation of other bands over time in the state of being replaced with water in the SDS-PAGE results, it can be interpreted that Bore BMI2004 has higher stability against pH than Hirax.

[0104] (2) Comparison of enzyme activity or content depending on storage temperature The pH stability of BMI2004 produced in Example 1 was compared with that of wild-type hyaluronidase Hirax. BMI2004 and Hirax used in the temperature stability test were purified to 95% or more, and the experimental concentration was adjusted to 1,500 IU / mL (=100%). The solution was prepared by replacing it with water according to the conditions described in Table 7, and this was stored at freezing (-20°C), refrigeration (5°C), and high temperature (40°C) for 4 weeks, and a titer test was performed at each time point every 2 weeks.

[0105]

Table 7

[0106] The stability of Hirax and BMI2004 depending on temperature was confirmed for about 4 weeks and shown in Table 8 and FIG. 2 below. It showed the highest content (%) at a freezing temperature of -18°C or lower, and the content (%) of BMI2004 was maintained significantly higher than that of Hirax under all temperature conditions after the second week.

[0107]

Table 8

[0108] Additional tests on the content change with respect to the stability test by temperature of Hirax and BMI2004 were carried out in the same manner as in Example 3 above, and SDS-PAGE was performed under the conditions of Table 7 above.

[0109] The SDS-PAGE experimental results are shown in Figure 2. As shown in Figure 2, in the state replaced with water, in terms of the generation of other bands other than the initial main band position over time under freezing, refrigeration, and high-temperature conditions, it can be interpreted that BMI2004 has higher stability against storage conditions than Hirax.

[0110] Example 4. Optimal enzyme activity by temperature In this example, the Plate titer test was devised based on the cylinder plate method among the microbiological titer test methods for antibiotics. Specifically, if a penicillin cylinder filled with a certain amount of hyaluronidase is placed on a solid agarose Plate containing hyaluronic acid, the hyaluronidase will diffuse. Through such diffusion, hyaluronic acid is decomposed by hyaluronidase, and the undegraded hyaluronic acid is precipitated by cetylpyridinium chloride to form a transparent circle. Therefore, in this example, the enzyme activity of hyaluronidase can be confirmed from the size of the circle generated through a simple method using a Plate. The Plate titer test was tested by the following test method under each condition:

[0111] 1) Preparation of hyaluronic acid solution: 0.2 g of hyaluronic acid is put into water and completely dissolved, then after making it 100 mL, hydrochloric acid and sodium hydroxide are added to adjust the pH to 7.0 ± 0.1.

[0112] 2) Preparation of 1.5% agarose: 1.5 g of agarose (SIGMA, Cat No. A9539) is put into 100 mL of water and dissolved using a microwave.

[0113] 3) Preparation of 10% cetylpyridinium chloride: Dissolve 10 g of cetylpyridinium chloride (SIGMA, Cat No. C0732) in 100 mL of water.

[0114] 4) Test solution: Replace the Hirax and BMI2004 standards with water and adjust according to the conditions for use.

[0115] 5) Operating procedure: Test with the test solution under the following operating conditions.

[0116] 5-1) Warm the hyaluronic acid solution at 37 °C for about 20 minutes before mixing with 1.5% agarose.

[0117] 5-2) When 100 mL of 1.5% agarose has cooled to about 60 °C, add 100 mL of the hyaluronic acid solution and stir to mix.

[0118] 5-3) Pour it into a Petri Dish (SPL, Cat No. 10050) to a thickness of about 3 mm.

[0119] 5-4) When the agarose gel has completely solidified, place a penicillin cylinder (KisanBio, Cat No. KS-P0161) on the Petri Dish concentric circle at 90°. After taking 20 μL of the test solution and dispensing it into the penicillin cylinder, react it in an incubator under the temperature conditions described in each table for 18 - 20 hours.

[0120] 5-5) After the reaction is complete, remove the penicillin cylinder, add 3 mL of 10% cetylpyridinium chloride, and check for a clear circle after about 20 minutes.

[0121] 5-6) Accurately measure the diameter (mm) of the circle to 0.5 mm or less.

[0122] (1) Confirmation of the activity of the hyaluronic acid solution Before conducting the optimal enzyme activity test, plate titer tests were performed under various pH and temperature conditions to confirm whether the hyaluronic acid solution exhibits self-activity. Experiments were conducted by adjusting the temperature to 20°C, 25°C, 30°C, 35°C, and 40°C under pH 4.0, pH 7.0, or pH 10.0 conditions, and the experimental results are shown in Figure 3. As shown in Figure 3, it was confirmed that the hyaluronic acid solution does not exhibit self-activity.

[0123] (2) Comparison of enzyme activities under temperature conditions of 20 - 40°C Hirax and BMI2004 used in the enzyme activity test were more than 95% purified. They were adjusted to a concentration of 1,500 IU / mL (= 100%) and replaced with water, and solutions were prepared according to the conditions described in Table 9 below. Plate titer tests were performed on these solutions at each temperature condition of 20°C, 25°C, 30°C, 35°C, 40°C, and 60°C.

[0124]

Table 9

[0125] The experimental results are shown in Figures 4a and 4b. As a result of confirming the enzyme activities of Hirax and BMI2004 by plate titer tests under temperature conditions of 20 - 60°C, the size of the circles was shown to be large at 35°C and 40°C. To quantify the enzyme activity, the diameters of the circles in Figures 4a and 4b were measured and shown in Table 10 below. The error range of the diameter (mm) in Table 10 is ±0.5 mm.

[0126]

Table 10

[0127] As shown in Table 10, as a result of comparing the enzyme activities under temperature conditions of 20 - 40°C, the activity of BMI2004 was shown to be higher than that of Hirax.

[0128] (3) Comparison of enzyme activities under temperature conditions of 35 - 40°C For additional testing under temperature conditions subdivided based on the activity comparison of Hirax and BMI2004 confirmed in (2) of Example 4 above, a solution was prepared according to the conditions described in Table 11 below, and a plate titer test was conducted at each temperature condition of 35°C, 37°C, and 40°C.

[0129]

Table 11

[0130] The experimental results are shown in FIGS. 5a and 5b. As a result of confirming the enzyme activities of Hirax and BMI2004 at 35°C, 37°C, and 40°C by a plate titer test, the size of the circle was shown to be the largest at 37°C, and the diameter of the circle is shown in Table 12 below. The error range of the diameter (mm) is ±0.5 mm.

[0131]

Table 12

[0132] As shown in Table 12, as a result of comparing the enzyme activities under the temperature conditions of 35°C, 37°C, and 40°C, the activity of BMI2004 was shown to be even higher than that of Hirax.

[0133] Example 5. Optimal Enzyme Activity by pH Hirax and BMI2004 used in the optimal enzyme activity test were purified to 95% or more, and adjusted to a concentration of 1,500 IU / mL (=100%) and replaced with water, and a solution was prepared according to the conditions described in Table 13 below. The enzyme activity was measured by the test method of Example 4, and a plate titer test was conducted at each pH under the condition of 37°C where the size of the circle was shown to be the largest based on the activity results by temperature confirmed in (3) of Example 4.

[0134]

Table 13

[0135] The experimental results are shown in FIGS. 6a and 6b. As a result of confirming the enzyme activity of Hirax and BMI2004 at 37°C with respect to pH 5.0 - 10.0 by a plate titer test, a tendency was shown that the size of the circles was particularly large at pH 7.0 - 10.0, and a comparison of the circle diameters is shown in Table 14 below. The error range of the diameter (mm) is ±0.5 mm.

[0136]

Table 14

[0137] As shown in Table 14 above, as a result of comparing the enzyme activity of BMI2004 replaced with water with respect to pH 3.0 - 10.0 at 37°C, BMI2004 had superior enzyme activity compared to Hirax at pH 5.0 or higher, and showed excellent activity particularly at pH 7.0 or higher.

[0138] Example 6. Confirmation of the titer of hyaluronidase The titers of hyaluronidase according to an example of the present invention and conventional Hirax were measured in the same manner as in (1) of Example 2 and shown in Table 15.

[0139]

Table 15

[0140] As shown in Table 15, BMI2004 showed an activity of about 122,433 - 134,678 IU per mg of protein, and Hirax showed an activity of about 80,538 - 81,396 IU per mg of protein. Therefore, the hyaluronidase according to an example of the present invention can achieve equivalent activity even with about 63% of the protein amount compared to conventional Hirax.

[0141] Example 7. Drug absorption and diffusion promoting effect of hyaluronidase (1) In order to confirm the effect of promoting the absorption and diffusion of drugs by hyaluronidase according to an example of the present invention, a drug absorption and diffusion promotion test using Trypan Blue was conducted.

[0142] Specifically, after mixing Hirax and BMI2004 with 0.2% Trypan blue solution respectively and administering them subcutaneously to BALB / c nude mice once, the degree of Trypan blue diffusion was compared. Based on the conditions described in Table 16 below and the titers described in Table 15, all drugs were prepared to 10 IU / mL or 100 IU / mL and administered in 0.05 mL portions each, and the area (π mm 2 ) diffused at each time point was confirmed.

[0143]

Table 16

[0144] As shown in Table 16 above, the area diffused for 2.5 to 20 minutes was confirmed. The area diffused by Hirax and BMI2004 was significantly increased compared to Saline, which was the negative control group, starting from 5 minutes after administration.

[0145] Example 8. Drug Absorption and Diffusion Promotion Effect of Hyaluronidase (2) To confirm the effect of hyaluronidase according to an example of the present invention in promoting drug absorption and diffusion, a drug absorption and diffusion promotion test was conducted using an Akinesia Model.

[0146] Specifically, Cynomolgus Monkey was used to confirm the increased drug penetration using the Akinesia Model. BMI2004 and Hirax described in Table 15 were used as test substances, and Saline was used as the negative control group. Lidocaine and Bupivacaine were used as anesthetics mixed with the specimens. The administration was 2 mL each around the right and left eyes (Peribulbar) of 6 monkeys, and the movement was observed.

[0147] After administering an anesthetic together with a test substance, the time until the pupil movement disappeared (anesthesia required time; Time to Akinesia) was measured, and the time from when the pupil was anesthetized until the pupil movement was observed after the anesthesia wore off (anesthesia duration; Duration of Akinesia) was measured and shown in Table 17.

[0148]

Table 17

[0149] As shown in Table 17 above, no Akinesia effect was shown in Saline, which is the negative control group. However, when the peptide according to an example of the present invention was administered together with an anesthetic, the penetration power of the anesthetic was improved and a pupil paralysis effect was shown, and it had an effect of inducing drug absorption and diffusion by showing an Akinesia effect equivalent to that of conventional Hirax. In addition, the peptide according to an example of the present invention showed equivalent activity at a protein dosage of about 63% less than that of conventional Hirax.

[0150] Example 9. Confirmation of promotion of excessive body fluid reabsorption by hyaluronidase In order to confirm the effect that hyaluronidase according to an example of the present invention promotes excessive body fluid reabsorption, a test was conducted using an edema model (Edema Model). In this example, C57BL / 6 mice were used to artificially induce lymph edema and confirm the effect in a lymphedema model. As the test substances, Hirax and BMI2004 described in Table 15 were used, and as the negative control group, Saline was used. The specimens and dosages were carried out as described in Table 18 below.

[0151]

Table 18

[0152] Lymphedema induction was performed on the tail, which is accurate and easy to measure. Skin was incised circularly with a width of 2 mm at a point 1 cm away from the base of the mouse's tail. At this time, 4 mm in the ventral side direction was left without incision. 2 To confirm the edema reduction effect, on the 15th day after induction, as shown in Table 19 below, the diameter (mm) of the tail was measured at each time point from before administration (0 hour) to after the first administration, and the second administration was performed 24 hours later, and the third administration was performed 48 hours later. The test measured the diameter (mm) of the tail using a caliper at a point 10 mm from the defect site.

[0153]

Table 19

[0154] As shown in Table 19 above, it was confirmed that administration of BMI2004 reduced lymphedema and promoted the reabsorption of excessive body fluids. In addition, the peptide according to an example of the present invention showed equivalent activity at a protein dosage of about 63% compared to conventional Hirax.

[0155] Example 10. Confirmation of the in vivo stability of hyaluronidase To confirm the stability in vivo of hyaluronidase according to an example of the present invention, it was intravenously administered to confirm pharmacokinetics. In this example, SD rats were used as test animals, and Hirax and BMI2004 were each administered intravenously by infusion method for 30 minutes to a dose of about 180,000 IU. Blood sampling was performed before administration, 15 minutes after the start of administration (mid-infusion), 30 minutes after the start of administration (at the end of infusion), and at 31, 33, 36, 40, 45, 60, 75, 90 minutes, 2.5 hours, 4.5 hours, 24.5 hours, and 48.5 hours.

[0156] Table 20 shows the pharmacokinetic variables of the polypeptide according to an example of the present invention and Hirax, and Fig. 7 shows the blood concentration of the polypeptide over time. As can be seen from Fig. 7 and Table 20, in the case of Hirax, it is decomposed rapidly to the extent that the in vivo half-life cannot be measured, whereas the peptide according to an example of the present invention has a longer half-life than Hirax at about 0.272 hours (about 16.3 minutes) and stays in the body longer, indicating the possibility of showing a greater effect than Hirax. Also, there is a difference in the in vivo blood concentration. In the case of Hirax, the peak is reached within 0.25 hours (15 minutes), whereas BMI2004 has a slower increase in blood concentration than Hirax at 0.5 hours (30 minutes) and stays in the body for a longer time, indicating the possibility of showing an even greater effect than Hirax.

[0157] [Table 20] [Sequence Listing]

Claims

1. A polypeptide having hyaluronidase activity, which is obtained by deleting 1 to 203 amino acids from the C-terminus of the polypeptide consisting of the amino acid sequence of SEQ ID NO:

1.

2. The polypeptide according to claim 1, wherein 34 to 170 amino acids are deleted from the C-terminus of the amino acid sequence of SEQ ID NO:

1.

3. The polypeptide according to claim 1, wherein 34 to 68 amino acids are deleted from the C-terminus of the amino acid sequence of SEQ ID NO:

1.

4. The polypeptide according to claim 1, wherein 34, 68, 102, 136, or 170 amino acids are deleted from the C-terminus of the amino acid sequence of SEQ ID NO:

1.

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

6. The polypeptide according to claim 1, which consists 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.

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

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

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

10. The polypeptide according to claim 1, wherein the enzyme activity after storage for 4 weeks at a pH belonging to the range of more than pH 3 to 10 is 57% or more of the initial activity.

11. The polypeptide according to claim 1, wherein the enzyme activity after storage for 4 weeks at a pH of 3 to less than 5 is 32% or more of the initial activity.

12. The polypeptide according to claim 1, wherein the enzyme activity after storage for 4 weeks at a temperature belonging to less than 0°C is 63% or more of the initial activity.

13. The polypeptide according to claim 1, wherein the enzyme activity after storage for 4 weeks at a temperature belonging to the range of 0 to 40°C is 83% or more of the initial activity.

14. The polypeptide according to claim 1, wherein the enzyme activity after storage for 4 weeks at a temperature belonging to 40°C or higher is 52% or more of the initial activity.

15. The polypeptide according to claim 1 has a titer exceeding 1-fold to 3-fold the titer of the polypeptide consisting of the amino acid sequence of SEQ ID NO:

1.

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

17. A nucleic acid molecule encoding the polypeptide according to any one of claims 1 to 16.

18. A vector containing the nucleic acid molecule of claim 17.

19. A cell containing the vector of claim 18.

20. The cell according to claim 19 is selected from the group consisting of bacteria, yeast, mold, insect cells, animal cells, mammalian cells, algal cells, and plant cells.

21. The mammalian cell according to claim 20 is selected from the group consisting of CHO, NS0, HEK293, BHK, Per.C6, MDCK, Vero, MRC, HeLa, IMR, and Sp2 / 0.

22. A composition for topical administration containing the polypeptide according to any one of claims 1 to 16.

23. The composition according to claim 22 is for subcutaneous administration or intramuscular injection.

24. A carrier for drug delivery containing the polypeptide according to any one of claims 1 to 16.

25. A composition for preventing or treating edema containing the polypeptide according to any one of claims 1 to 16.

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