Hyaluronidase polypeptide and its uses

Abstract

The present invention relates to hyaluronidase polypeptides and their uses.

Description

[Technical Field]

[0001] This invention relates to a hyaluronidase polypeptide and its uses. [Background technology]

[0002] Hyaluronidase, a general term for enzymes that break down hyaluronic acid into smaller molecules, was initially known by Duran-Reynals as a diffusion factor. However, it was later observed to exhibit potent activity on hyaluronic acid, leading to the name hyaluronidase. Based on their mechanism of action, these enzymes are classified into hyaluronate 4-glycanohydrolase (EC 3.2.1.35), which is distributed in the testes, lysosomes, and bee venom; hyaluronate 3-glycanohydrolase (EC 3.2.1.36), which is found in leeches; and hyaluronate triase (EC 4.2.2.1), which is found in bacteria.

[0003] In particular, hyaluronidase (PH-20) in the testes is attached to the glycosylphosphatidylinositol anchor (GPI) at the apex of the sperm, and is an important enzyme that breaks down the thick outer wall layer of the egg to induce fertilization. PH-20 is also known to hydrolyze the β(1-4) bond between hyaluronic acid (HA) in glycosaminoglycans present in mammalian skin, and between D-glucuronic acid and N-acetyl-D-glucosamine in chondroitin and chondroitin sulfate. The general molecular formula of this enzyme is C2455H3775N617O704S21, and its molecular weight is 53870.9 g / mol. In humans, six genes, including HYAL1, HYAL2, HYAL3, and PH-20 / SPAM1, are associated with this enzyme.

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

[0005] Currently, commercially available hyaluronidases are extracted from the testicles of sheep (ovine) or cattle (bovine). Examples include Vitrase (ISTA Pharmaceuticals, ovine source) and Amphadase (Amphastar Pharmaceuticals, bovine source). These unprocessed hyaluronidases are dissolved at the appropriate concentration, loaded into vials, and freeze-dried to produce the final product. However, commercially available animal-derived hyaluronidases contain foreign proteins, which can cause allergic reactions. Furthermore, their physiological activity decreases over time due to reduced stability, posing many problems for their application in diverse fields.

[0006] To address these issues, research was conducted on recombinant hyaluronidase. Recombinant proteins can be expressed in a variety of cell types, including E. coli, yeast, insect cells, and animal cells. In particular, in the case of hyaluronidase, glycation, which occurs during the post-translational deformation process, 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 glycation occurs, the post-translational deformation process differs from that of mammals, making animal cells the most suitable expression cell type among various types. Among animal cells, CHO (Chinese Hamster Ovary) cells, which have ensured safety, are the most suitable.

[0007] The first recombinant hyaluronidase for pH20 is marketed under the trade name Hylenex by Halozyme Therapeutic and is under development for various applications including subcutaneous injection, vitrectomy, and ophthalmic disorders. However, hyaluronidase still suffers from low yield and stability, resulting in a supply that far exceeds demand. There is a need for hyaluronidase with improved yield or stability. [Overview of the project] [Problems that the invention aims to solve]

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

[0009] Another example of the present invention is to provide a topical administration composition comprising a polypeptide according to an example of the present invention.

[0010] Another example of the present invention is to provide a drug delivery carrier comprising a polypeptide according to an example of the present invention.

[0011] Another example of the present invention is to provide a composition for the prevention or treatment of edema, comprising a polypeptide according to an example of the present invention. [Means for solving the problem]

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

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

[0014] Another example of the present invention relates to a topical administration composition comprising 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-terminal 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 values from 3 to 10. Specifically, the enzyme activity after storage for 4 weeks at a pH belonging to the range exceeding pH 3 to 10 is 57% or more of the initial activity, or the enzyme activity after storage for 4 weeks at a pH from 3 to less than 5 is 32% or more of the initial activity. (2) A characteristic of being stable at temperatures from -20 to 45 °C. Specifically, the enzyme activity after storage for 4 weeks at a temperature belonging to the range 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 from 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 the range of 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 of 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 this invention, a polypeptide was produced by cleaving the C-terminus of Ovine-derived wild-type hyaluronidase (CAS no. 488712-31-8) along its amino acid sequence. The stability and activity of the produced polypeptide were confirmed, and it was found to exhibit significantly superior stability and activity compared to the aforementioned wild-type hyaluronidase. As a result, it was possible to achieve equivalent drug absorption, drug diffusion enhancement, and body fluid reabsorption enhancement effects using a small amount of protein. Therefore, the polypeptide according to one example of the present invention may be hyaluronidase.

[0021] Specifically, a polypeptide according to an example of the present invention may have 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 to a polypeptide in which 1 to 203 amino acids are deleted from the C-terminus of the amino acid sequence of wild-type hyaluronidase. In this case, 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. Furthermore, 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 equivalent 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.

[0022] Specifically, a polypeptide according to an example of the present invention may be obtained by deleting n amino acids from the C-terminus of the amino acid sequence of wild-type hyaluronidase (wherein n is a natural number between 1 and 203).

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

[0024] As an example, a 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 of the amino acid sequence of wild-type hyaluronidase (where m is a natural number between 315 and 517). The amino acid sequence of the wild-type hyaluronidase may also be the amino acid sequence of Sequence ID No. 1.

[0025] As an example, a 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, a polypeptide according to one example of the present invention may be expressed using animal cells as a host. Therefore, a polypeptide according to one example of the present invention may be expressed using animal cells as a host and undergo glycation via a Post-Translational Modifications (PTM) process during protein expression.

[0027] A polypeptide according to one example of the present invention may be stable at pH 3 to 10. Specifically, a polypeptide according to one example of the present invention may exhibit even less reduction in enzyme activity compared to wild-type hyaluronidase when stored at pH 3 to 10.

[0028] In the embodiments of this invention, enzyme activity was measured while storing a polypeptide according to an example of the present invention in the pH range of 3 to 10. The results showed that the enzyme activity was maintained even after 4 weeks of storage.

[0029] For example, a polypeptide according to an example of the present invention may have a pH of less than 3 to 5, or a pH in the range of 3 to 4. For example, after storage at pH 3 for 4 weeks, the enzyme activity may be 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. In this case, the storage temperature of the polypeptide may be 5°C or 37°C.

[0030] For example, a polypeptide according to one example of the present invention may have 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, or 65% or more of the initial activity after 4 weeks of storage at a pH in the range of pH 3 to 10, pH 4 to 10, or pH 5 to 10, for example, pH 5, pH 7, or pH 10. In this case, 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 may have 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, 68% or more, 69% 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 4 weeks of storage at pH 5 or pH 7. In this case, the storage temperature of the polypeptide may be 5°C or 37°C.

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

[0033] In the embodiments of this invention, enzyme activity was measured while storing a polypeptide according to an example of the present invention at freezing temperatures (e.g., -18°C to -20°C), refrigeration temperatures (e.g., 2 to 8°C), and high temperatures (e.g., 40 to 45°C). The results showed that the enzyme activity was maintained even after 4 weeks of storage.

[0034] For example, a polypeptide according to an example of the present invention, after being stored for 4 weeks at a freezing temperature, for example below 0°C, -20 to below 0°C, -20 to -10°C, or a temperature in the range of -20 to -18°C, as an example at -20°C, has 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, and 74% or more of the initial activity. It may be 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. In this case, the storage pH of the polypeptide may be 5 or 7.

[0035] For example, a polypeptide according to one example of the present invention may have 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 4 weeks of storage at a refrigerated temperature, for example, a temperature in the range of 0 to 40°C, 0 to 10°C, or 2 to 8°C, for example, 5°C.

[0036] For example, a polypeptide according to one example of the present invention may have 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 after storage at a high temperature, for example, 40°C or higher for 4 weeks. In this case, 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 of more than 1 to 3 times, more than 1 to 2.5 times, more than 1 to 2 times, more than 1 to 1.9 times, more than 1 to 1.8 times, more than 1 to 1.7 times, 1.1 to 3 times, 1.1 to 2.5 times, 1.1 to 2 times, 1.1 to 1.9 times, 1.1 to 1.8 times, 1.1 to 1.7 times, 1.2 to 3 times, 1.2 to 2.5 times, 1.2 to 2 times, 1.2 to 1.9 times, 1.2 to 1.8 times, 1. It may also have a potency of 2 to 1.7 times, 1.3 to 3 times, 1.3 to 2.5 times, 1.3 to 2 times, 1.3 to 1.9 times, 1.3 to 1.8 times, 1.3 to 1.7 times, 1.4 to 3 times, 1.4 to 2.5 times, 1.4 to 2 times, 1.4 to 1.9 times, 1.4 to 1.8 times, 1.4 to 1.7 times, 1.5 to 3 times, 1.5 to 2.5 times, 1.5 to 2 times, 1.5 to 1.9 times, 1.5 to 1.8 times, or 1.5 to 1.7 times.

[0038] For example, polypeptides according to one example of the present invention include 120,000-150,000 IU / mg, 120,000-145,000 IU / mg, 120,000-140,000 IU / mg, 120,000-135,000 IU / mg, 121,000-150,000 IU / mg, and 121,000-145,000 IU / mg. It may also have activity levels of 121,000-140,000 IU / mg, 121,000-135,000 IU / mg, 122,000-150,000 IU / mg, 122,000-145,000 IU / mg, 122,000-140,000 IU / mg, or 122,000-135,000 IU / mg.

[0039] Another example of the present invention relates to a nucleic acid molecule for 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, fungi, insect cells, animal cells, mammalian cells, algae cells, and plant cells. The mammalian cell may be selected from the group consisting of CHO, NS0, HEK293, BHK, Per.C6, MDCK, Vero, MRC, HeLa, IMR, and Sp2 / 0. The CHO cell 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 topical administration composition containing a polypeptide according to an example of the present invention. The composition may also be for subcutaneous administration. In the embodiments of this application, topical administration of the polypeptide according to an example of the present invention resulted in significantly superior drug absorption and diffusion promotion effects 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] Furthermore, in the embodiments of this application, local administration of a polypeptide according to one example of the present invention resulted in a significantly superior effect in promoting excessive reabsorption of body fluids compared to wild-type hyaluronidase. Therefore, yet another example of the present invention relates to a pharmaceutical composition for the prevention or treatment of edema, comprising a polypeptide according to one example of the present invention as an active ingredient.

[0042] The composition of the present invention, for example, a pharmaceutical composition, may additionally contain one or more active ingredients that exhibit the same or similar functions as the aforementioned active ingredients.

[0043] Furthermore, the compositions according to the present invention, such as pharmaceutical compositions, may be manufactured in unit volume form by formulation using a pharmaceutically acceptable carrier by a method that can be clearly carried out by a person with ordinary skill in the art to which the invention pertains, or by being manufactured in a multi-volume container. 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" means 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 carriers mentioned above are those commonly used in formulation and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0045] Furthermore, the compositions according to the present invention, for example, pharmaceutical compositions, may additionally contain additives such as fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, and preservatives, in addition to the components mentioned above. 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 formulations.

[0046] In this specification, 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 is added to a pharmaceutical composition. This improves handling and storage properties or allows and facilitates the formation of unit doses of the composition.

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

[0048] The preferred dosage of the composition according to the present invention, for example, a pharmaceutical composition, varies widely depending on the patient's condition and weight, age, sex, health status, dietary constitution specificity, properties of the formulation, severity of the disease, timing of administration of the composition, method of administration, duration or interval of administration, excretion rate, and drug form, and can be appropriately selected by an expert in the art.

[0049] In this specification, the term “effective dose of a pharmaceutical composition” means the amount of the active ingredient in the composition sufficient to treat a particular symptom. This varies depending on the formulation method, administration method, administration time and / or route of administration of the pharmaceutical composition, and is influenced by various factors, including the type and degree of response to be achieved by the administration of the pharmaceutical composition, the type of individual being administered, age, weight, general health status, symptoms and severity of the disease, sex, diet, excretion, and components of other drugs or compositions used simultaneously or concurrently in the individual, as well as similar factors well known in the pharmaceutical field. A person with ordinary skill in the art can easily determine and prescribe an effective dose for the intended treatment.

[0050] The pharmaceutical composition according to the present invention may be administered once a day or in several divided doses. The composition may be administered as a standalone therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. Considering all of 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 higher potency compared to wild-type hyaluronidase and may achieve equivalent effects with a lower dose. For example, a polypeptide or composition according to an example of the present invention may have a daily dose 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 dose may be based on weight or weight percent. Furthermore, the total daily dose can be divided and administered continuously or discontinuously as needed. [Effects of the Invention]

[0052] The hyaluronidase polypeptide according to one example of the present invention exhibits increased expression levels and stability in animal cells compared to mature wild-type PH20, and possesses enzymatic activity equivalent to or greater than that of mature wild-type PH20. Therefore, compared to mature wild-type PH20, the hyaluronidase polypeptide according to one example of the present invention exhibits increased protein expression levels and higher protein stability when expressed in CHO cells, thus increasing its industrial applicability for diverse applications.

[0053] Furthermore, hyaluronidase has the property of breaking down hyaluronic acid, a component of intercellular spaces, thereby regulating tissue binding strength, facilitating drug penetration and diffusion, and promoting the reabsorption of excess bodily fluids in tissues. Therefore, its range of use and application fields are gradually expanding. However, in the case of wild-type PH20 derived from animals, there is a high possibility of infection from the animal-derived substance. A hyaluronidase polypeptide according to this invention is safe with a low risk of infection, and compared to wild-type PH20 derived from animals, it exhibits higher activity for the same amount of protein, further enhancing its industrial applicability for diverse applications. [Brief explanation of the drawing]

[0054] [Figure 1a-1c] This figure shows the results of comparing the pH stability of a polypeptide according to an example of the present invention with that of wild-type hyaluronidase via SDS-PAGE (Figure 1a: 0 weeks of storage, Figure 1b: 2 weeks of storage, Figure 1c: 4 weeks of storage). [Figure 2a-2c] This figure shows the results of comparing the temperature stability of a polypeptide according to an example of the present invention with that of wild-type hyaluronidase via SDS-PAGE (Figure 2a: 0 weeks of storage, Figure 2b: 2 weeks of storage, Figure 2c: 4 weeks of storage; frozen: frozen, refrig.: refrigerated, hightem.: high temperature). [Figure 3] This figure shows the results of plate titer tests under various pH and temperature conditions to confirm whether hyaluronic acid solutions exhibit intrinsic activity. [Figure 4a] This figure shows the results of titer tests for wild-type hyaluronidase under temperature conditions of 20-60°C. [Figure 4b] This figure shows the results of a titer test of a polypeptide according to an example of the present invention under temperature conditions of 20 to 60°C. [Figure 5a] This figure shows the results of titer tests for wild-type hyaluronidase under temperature conditions of 35-40°C. [Figure 5b] This figure shows the results of a titer test of a polypeptide according to an example of the present invention at a temperature of 35-40°C. [Figure 6a] This figure shows the results of titer tests for wild-type hyaluronidase under pH 5-7 conditions. [Figure 6b] This figure shows the results of a titer test of a polypeptide according to an example of the present invention under pH 5-7 conditions. [Figure 7] This figure shows the correlation between the time and blood concentration of a polypeptide according to an example of the present invention and Hirax in SD rats. [Modes for carrying out the invention]

[0055] The present invention will be described in more detail below with reference to the following examples. However, these examples are merely illustrative of the present invention, and the scope of the present invention is not limited by these examples. [Examples]

[0056] Example 1. Hyaluronidase production Hyaluronidase was produced by cleaving the C-terminus of Ovine-derived wild-type hyaluronidase (CAS no. 488712-31-8) of the amino acid sequence of Sequence ID No. 1, using 34 amino acids (Examples 1-2), 68 amino acids (Examples 1-3), 102 amino acids (Examples 1-4), 136 amino acids (Examples 1-5), 170 amino acids (Examples 1-6), or 204 amino acids (Examples 1-7) as reference points from the C-terminus.

[0057] First, cDNA was synthesized based on the amino acid sequence of wild-type hyaluronidase (CAS no. 488712-31-8). The hyaluronidase gene was amplified using polymerase chain reaction (PCR), and its expression and activity were confirmed in CHO-DG44 cells using the pcDNA3.1 vector. Then, it was inserted into CHO-DG44 cells using the pOtiVEC vector. The number of CHO-DG44 cells was 4-6 × 10⁶. 6 When the cell volume reached cells / mL, CHO-DG44 cells were transformed using electroporation with a plasmid containing hyaluronidase cDNA inserted into a pOptiVEC vector. After plasma injection, CHO-DG44 cells were cultured in Power CHO 2CD (with L-Glutamine 4mM) medium, and the supernatant was collected by centrifugation at 12,000 rpm for 10 minutes. The collected culture medium was purified as needed using various methods such as affinity chromatography, hydrophobic chromatography, and ion exchange chromatography, and the purified hyaluronidase was replaced with water by ultafiltration and microfiltration. The sequences of the produced hyaluronidases are listed in Table 1.

[0058] [Table 1] TIFF0007854527000002.tif250165TIFF0007854527000003.tif205164

[0059] Example 2. Stability confirmation based on hyaluronidase cleavage site (1) Comparison of enzyme activity or content at different storage pH levels based on cleavage site The pH stability of six hyaluronidases, excluding the inactive hyaluronidases from Examples 1-7, was confirmed based on their cleavage sites. After expressing each amino acid sequence in transient cells, the culture medium was concentrated to the same level, and activity was measured. The experimental concentration was set to 1,500 IU / mL (=100%), and solutions were prepared using water substitution under the conditions listed in Table 2. These solutions were stored at 37°C at pH 3.0, pH 5.0, pH 7.0, and pH 10.0 for 4 weeks, and titer tests were performed at each time point every 2 weeks.

[0060] The enzyme activity or content of the sample at each cutting site was tested using the following method:

[0061] 1) Hyaluronidase activity at 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 to pH 6.4 with 1 M sodium hydroxide solution or 1 M hydrochloric acid solution, and add water to make 1,000 mL.

[0063] 1-2) Preparation of diluent: Mix 100 mL of pH 6.4 phosphate buffer with 100 mL of water, then add 0.140 g of gelatin reagent and dissolve at 37°C. Use the diluent within 2 hours.

[0064] 1-3) Preparation of substrate solution: Add 100 mL of water to 0.5 g of sodium hyaluronate little by little while stirring. Gradually add water until the sodium hyaluronate swells. Stir at 4°C for at least 12 hours. Store the substrate solution at 4°C and use within 4 days.

[0065] 1-4) Preparation of standard solution: Dissolve the EP STD (EDQM) standard in the diluent to approximately 50 IU / mL. Take 3 mL of this solution and place it in a 250 mL volumetric flask. Add the diluent to make a total volume of exactly 250 mL to prepare the standard solution.

[0066] 1-5) Preparation of test solution: Dilute the hyaluronidase test solution obtained from the cutting site to approximately 0.6 IU / mL, adjusting the pH according to the conditions, and use.

[0067] 2) Procedure: Test using the standard solution and the test solution according to the following method.

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

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

[0070] 2-3) Ubbelohde Microviscometer (DIN 51 562, Part 2, Calillary type MIII, constant: approx. 0.1 mm) 2 / s 2 Pour the entire volume of the mixed liquid from the Conical Tube into a viscometer (or an equivalent viscometer).

[0071] 2-4) Use a second-second timer to measure the time it takes for the liquid to flow from the upper mark to the lower mark on the Ubbelohde microviscometer.

[0072] 2-5) Measure the time by repeating the process several times for approximately 20 minutes.

[0073] 2-6) The above process is repeated three times for testing.

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

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

[0076] 3-2) ηr -1 : {(k × T2 / 0.6915} -1 T1: Time (seconds) to rise to the upper calibration line of the Ubbelohde microviscometer T2: T - T1 T: Time (seconds) to fall to the lower calibration line 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 expressed by comparing the measured activity (IU / mL) at each time point with 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 time of measurement) / (Initial enzyme activity) * 100

[0080] [Table 3]

[0081] As shown in Table 3, the pH stability of hyaluronidase at the cleavage site was confirmed for approximately 4 weeks, and it showed an even higher enzyme activity ratio (%) 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 the pH stability of the hyaluronidases of Examples 1-2 to 1-4 was particularly excellent. Values ​​exceeding 100% were judged to be measurement errors that generally occur in titer tests of biological products such as vaccines and recombinant proteins (cytokines, monoclonal antibodies, etc.) due to large fluctuations in the range of change.

[0082] (2) Comparison of enzyme activity or content at different storage temperatures based on the cutting site The temperature stability of hyaluronidase in Examples 1-1 to 1-6 was confirmed using the same method as in Example 2, Part 1. Solutions were prepared according to the conditions described in Table 2, and stored for 4 weeks at frozen (-20°C), refrigerated (5°C), and high temperature (40°C). Titer tests were performed at each time point every 2 weeks. The temperature stability of hyaluronidase at the cleavage site was confirmed for approximately 4 weeks and is shown in Table 4.

[0083] [Table 4]

[0084] As shown in Table 4, the hyaluronidases of Examples 1-2 to 1-6 exhibited higher temperature stability compared to Example 1-1, and in particular, the enzyme activity ratio (%) of the hyaluronidases of Examples 1-2 to 1-6 was remarkably high under frozen and refrigerated storage conditions. In subsequent examples, the amino acid DS hyaluronidase corresponding to Example 1-3, which showed the highest activity among recombinant hyaluronidases and was cleaved by approximately 12%, was named BMI2004 and used in the experiments.

[0085] Example 3. Confirmation of the stability of wild-type hyaluronidase and BMI2004. (1) Comparison of enzyme activity or content based on pH The pH stability of BMI2004 prepared in Example 1 was compared with that of wild-type hyaluronidase. The BMI2004 and wild-type hyaluronidase (manufacturer: BMI Korea, trade name: Hirax, hereinafter referred to as Hirax) used in the pH stability test were purified to over 95%. The experimental concentration was set to 1,500 IU / mL (=100%), and a solution was prepared by substitution with water under the conditions listed in Table 5. This solution was stored at 5°C at pH 3.0, pH 5.0, pH 7.0, and pH 10.0 for 4 weeks, and titer tests were 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 Example 2 (1).

[0086] [Table 5]

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

[0088] [Table 6]

[0089] As shown in Table 6, the pH-dependent stability of BMI2004 and Hirax was confirmed for approximately 4 weeks. Higher enzyme activity ratios (%) were observed at pH 5.0 and pH 7.0 compared to pH 3.0 and pH 10.0, and from the second week onward, the content (%) of Hirax tended to decrease sharply compared to BMI2004. Values ​​exceeding 100% were considered to be measurement errors that commonly occur in titer testing of biological products such as vaccines and recombinant proteins (cytokines, monoclonal antibodies, etc.) due to large fluctuations. To further test the changes in enzyme activity (or content) for pH-dependent stability confirmation of Hirax and BMI2004, SDS-PAGE (Sodium Dodecyl Sulfate-PolyAcrylamide Gel Electrophoresis) was performed under the conditions shown in Table 5, and the following test methods were used:

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

[0091] 2) Preparation of Running Buffer (1X): Novex TM Use Tris-Glycine SDS Running Buffer (10X) (Invitrogen, Cat No. LC2675). Take exactly 100 mL, place it in a 1,000 mL volumetric flask, add water, and mix until the total volume is exactly 1,000 mL.

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

[0093] 4) Procedure: Perform the following procedure using the sample solution and PageRuler Prestained Protein Ladder standard (Thermo, Cat No. 26616) under the following operating conditions.

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

[0095] 4-2) Secure the washed gel to the Mini Gel Tank and 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 sample solution into the gel.

[0097] 4-4) Connect the power supply to the Mini Gel Tank and, after operating as described below, expand the gel to 90%. Volt: 140V Ampere: 400mA Duration: 60 minutes (The duration can be adjusted depending on the gel's deployment progress.)

[0098] 4-5) Once deployment is complete, separate the gel from the caster and then wash it with water.

[0099] 4-6) Place 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 a Rocker and stain at 30 rpm for 30 minutes.

[0101] 4-8) Once staining is complete, place the gel in a container with destaining solution (Coomassie Brilliant Blue R-250 Destaining Solution, BIO-RAD, Cat No. 1610438) and destain it using a Rocker at 30 rpm, alternating the destaining solution until the gel is destained. Once destaining is partially complete, rinse the gel with water to remove the destaining solution.

[0102] 4-9) Once the decolorizing solution has been washed off, observe the gel under a white light.

[0103] The experimental results are shown in Figures 1a to 1c. As shown in Figures 1a to 1c, the SDS-PAGE results, based on the presence or absence of other bands over time in the water-substituted state, can be interpreted as indicating that Bohr BMI2004 has higher pH stability compared to Hirax.

[0104] (2) Comparison of enzyme activity or content based on storage temperature BMI2004 manufactured in Example 1 temperature The stability was compared with wild-type hyaluronidase Hirax. The BMI2004 and Hirax used in the temperature stability test were purified to over 95%, and the experimental concentration was set to 1,500 IU / mL (=100%). Solutions were prepared by substituting with water under the conditions listed in Table 7, and these were stored for 4 weeks at frozen (-20°C), refrigerated (5°C), and high temperature (40°C). Titer tests were performed at each time point every 2 weeks.

[0105] [Table 7]

[0106] The temperature stability of Hirax and BMI2004 was confirmed over approximately four weeks and is shown in Table 8 and Figure 2 below. 20 The highest content (%) was observed at freezing temperatures below °C, and from the second week onward, the content (%) of BMI2004 remained significantly higher than that of Hirax under all temperature conditions.

[0107] [Table 8]

[0108] Additional tests for content changes in temperature-dependent stability tests of Hirax and BMI2004 were performed in the same manner as in Example 3, and SDS-PAGE was performed under the conditions shown in Table 7.

[0109] Figure 2 shows the results of the SDS-PAGE experiment. a~Figure 2c As shown in Figure 2. a~Figure 2c As shown, BMI2004 can be interpreted as having higher stability to storage conditions compared to Hirax, in terms of the generation of other bands besides the initial main band position over time under freezing, refrigeration, and high-temperature conditions when replaced with water.

[0110] Example 4. Optimal enzyme activity by temperature In this embodiment, the plate titer test was devised based on the cylindrical plate method, one of the microbiological titer testing methods for antibiotics. Specifically, when a penicylation containing a certain amount of hyaluronidase is placed on a solid agarose plate containing hyaluronic acid, the hyaluronidase diffuses. Through this diffusion, the hyaluronic acid is broken down by the hyaluronidase, and the unbroken hyaluronic acid precipitates with cetylpyridinium chloride, forming clear spheres. Therefore, in this embodiment, the enzymatic activity of hyaluronidase can be confirmed from the size of the spheres produced through a simple method using a plate. The plate titer test was performed under each condition by the following test method:

[0111] 1) Preparation of hyaluronic acid solution: Dissolve 0.2g of hyaluronic acid in water until completely dissolved, then add hydrochloric acid and sodium hydroxide to make a total volume of 100mL and adjust the pH to 7.0±0.1.

[0112] 2) Preparation of 1.5% agarose: Dissolve 1.5g of agarose (SIGMA, Cat No. A9539) in 100mL of water using a microwave.

[0113] 3) Preparation of 10% cetylpyridinium chloride: Prepare by adding 10g of cetylpyridinium chloride (SIGMA, Cat No. C0732) to 100mL of water.

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

[0115] 5) Procedure: Test the sample solution under the following operating conditions.

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

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

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

[0119] 5-4) Once the agarose gel has completely solidified, place the penicylinder (KisanBio, Cat No. KS-P0161) on the concentric circles of the Petri Dish at a 90° angle, take 20 μL of the sample solution and dispense it into the penicylinder, then incubate for 18-20 hours in an incubator that matches the temperature conditions listed in each table.

[0120] 5-5) Once the reaction is complete, remove the penicylyl, add 3 mL of 10% cetylpyridinium chloride, and observe for a clear ball after approximately 20 minutes.

[0121] 5-6) Measure the diameter of the circle (mm) accurately to within 0.5 mm.

[0122] (1) Confirmation of the activity of the hyaluronic acid solution Before performing the optimal enzyme activity test, plate titer tests were conducted under various pH and temperature conditions to confirm whether the hyaluronic acid solution exhibited intrinsic activity. Experiments were performed at pH 4.0, pH 7.0, or pH 10.0 conditions, with temperatures adjusted to 20°C, 25°C, 30°C, 35°C, and 40°C. The experimental results are shown in Figure 3. As shown in Figure 3, it was confirmed that the hyaluronic acid solution did not exhibit intrinsic activity.

[0123] (2) Comparison of enzyme activity under temperature conditions of 20-40°C The Hirax and BMI2004 used in the enzyme activity test were purified to over 95%, and were combined at a concentration of 1,500 IU / mL (=100%), replaced with water, and a solution was prepared according to the conditions listed in Table 9 below. This solution was then heated at 20°C, 25°C, 30°C, and 35°C. and 40 ℃ Plate titer tests were performed under various temperature conditions.

[0124] [Table 9]

[0125] The experimental results are shown in Figures 4a and 4b. 20 ~40 The enzyme activity of Hirax and BMI2004 was confirmed by plate titer testing under °C temperature conditions. The results showed that the size of the circles was larger at 35°C and 40°C. To quantify the enzyme activity, the diameters of the circles in Figures 4a and 4b were measured and are shown in Table 10 below. The error range for the diameter (mm) in Table 10 is ±0.5 mm.

[0126] [Table 10]

[0127] As shown in Table 10, when comparing enzyme activity under temperature conditions of 20-40°C, BMI2004 showed even higher activity than Hirax.

[0128] (3) Comparison of enzyme activity under 35-40°C temperature conditions Based on the activity comparison of Hirax and BMI2004 confirmed in Example 4(2) above, solutions were prepared under subdivided temperature conditions to conduct additional tests. These solutions were then subjected to plate titer tests at temperatures of 35°C, 37°C, and 40°C.

[0129] [Table 11]

[0130] The experimental results are shown in Figures 5a and 5b. The enzyme activity of Hirax and BMI2004 at 35°C, 37°C, and 40°C was confirmed by plate titer testing. The largest circle size was observed at 37°C, and the diameter of the circles is shown in Table 12 below. The error range for diameter (mm) is ±0.5 mm.

[0131] [Table 12]

[0132] As shown in Table 12, when comparing enzyme activity under temperature conditions of 35°C, 37°C, and 40°C, BMI2004 showed even higher activity compared to Hirax.

[0133] Example 5. Optimal enzyme activity by pH The Hirax and BMI2004 used in the optimal enzyme activity test were purified to over 95% purity. They were combined at a concentration of 1,500 IU / mL (=100%), replaced with water, and the solution was prepared under the conditions described in Table 13 below. Enzyme activity was tested using the method of Example 4, and plate titer tests were performed at each pH under the condition of 37°C, where the largest circle size was observed based on the activity results at temperature confirmed in (3) of Example 4.

[0134] [Table 13]

[0135] The experimental results are shown in Figures 6a and 6b. The enzyme activity of Hirax and BMI2004 at 37°C for pH 5.0–10.0 was confirmed by plate titer testing. The results showed a tendency for the size of the circles to be particularly large at pH 7.0–10.0, and a comparison of the circle diameters is shown in Table 14 below. The error range for diameter (mm) is ±0.5 mm.

[0136] [Table 14]

[0137] As shown in Table 14 above, pH at 37°C 5.0~ When comparing enzyme activity at pH 10.0, BMI2004, substituted with water, showed superior enzyme activity compared to Hirax at pH 5.0 and above, and particularly excellent activity at pH 7.0 and above.

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

[0139] [Table 15]

[0140] As shown in Table 15, BMI2004 showed an activity of approximately 122,433 to 134,678 IU per mg of protein, while Hirax showed an activity of approximately 80,538 to 134,678 IU per mg of protein. 81,528 The IU value was shown. Therefore, hyaluronidase according to one example of the present invention can achieve equivalent activity with approximately 63% less protein compared to conventional Hirax.

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

[0142] Specifically, Hirax and BMI2004 were mixed in a 0.2% Trypan blue solution and administered as a single subcutaneous dose to BALB / c nude mice, after which the degree of Trypan blue diffusion was compared. Based on the conditions described in Table 16 and the potency described in Table 15, all drugs were prepared to 10 IU / mL or 100 IU / mL and administered in 0.05 mL doses, and the area (π mm²) of diffusion at each time point was measured. 2 ) was confirmed.

[0143] [Table 16] 1) Mean ± SD 2)*p<0.05, **p<0.01, ***p<0.001 compared to saline treated group by SPSS(one-way ANOVA, LSD test)

[0144] As shown in Table 16 above, the area diffused for 2.5 to 20 minutes was examined. From 5 minutes after administration, the diffused area was significantly increased with Hirax and BMI2004 compared to the negative control group, Saline.

[0145] Example 8. Drug absorption and diffusion promoting effect of hyaluronidase (2) To confirm the effect of hyaluronidase according to an example of the present invention on promoting drug absorption and diffusion, drug absorption and diffusion enhancement tests were conducted using an akinesia model.

[0146] Specifically, Cynomolgus monkeys were used to confirm the increased drug penetration using the Akinesia Model. BMI2004 and Hirax, listed in Table 15, were used as test substances, and Saline was used as the negative control group. Lidocaine and Bupivacaine were used as anesthetics prepared by mixing with the samples. Administration involved administering 2 mL each to the peribulbar area of ​​the right and left eyes of six monkeys, and observing their movements.

[0147] After administering the anesthetic agent together with the test substance, the time until pupil movement ceased (Time to Akinesia) was measured, and the time from when the pupil was anesthetized until the anesthesia wore off and pupil movement was observed (Duration of Akinesia) was also measured and is shown in Table 17.

[0148] [Table 17] 1)Drug; Lidocaine 2% (0.9mL), Bupivacaine 0.5% (0.9mL) and the dosing article (0.2mL)

[0149] As shown in Table 17 above, the Akinesia effect was not observed in Saline, the negative control group, but according to one example of the present invention... Poly When the peptide was administered together with an anesthetic, the penetration of the anesthetic was enhanced, resulting in a palsy effect on the pupil. It exhibited an akinesia effect equivalent to that of conventional Hirax, and was able to induce drug absorption and diffusion. Furthermore, according to an example of the present invention... Poly The peptide showed comparable activity with approximately 63% less protein dosage than conventional Hirax.

[0150] Example 9. Confirmation of excessive promotion of body fluid reabsorption by hyaluronidase. To confirm the effect of hyaluronidase according to an example of the present invention on promoting the reabsorption of excess body fluids, a test was conducted in an edema model. In this example, Lymph edema was artificially induced using C57BL / 6 mice, and the effect was confirmed in a Lymphedema Model. Hirax and BMI2004, listed in Table 15, were used as the test substances, and Saline was used as the negative control group. The samples and dosages were as shown in Table 18 below.

[0151] [Table 18]

[0152] Lymphedema induction was performed in the tail, where measurement is accurate and easy. A 2mm wide ring-shaped incision was made in the skin 1cm away from the base of the mouse's tail, with a 4mm ventral side. 2 The extent of the edema reduction was left intact without being gouged out. To confirm the edema reduction effect, on day 15 of induction, the tail diameter (mm) was measured at each time point from before administration (0 hours) to after the first administration, as shown in Table 19 below. The second administration was given 24 hours later, and the third administration 48 hours later. In the test, the tail diameter (mm) was measured using a caliper at a point 10 mm from the defect site.

[0153] [Table 19]

[0154] As shown in Table 19 above, we confirmed that administration of BMI2004 reduces lymphedema and promotes the reabsorption of excess body fluids. Furthermore, according to an example of the present invention Poly The peptide showed comparable activity to conventional Hirax at approximately 63% of the protein dosage.

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

[0156] Table 20 shows the pharmacokinetic variables of a polypeptide according to an example of the present invention and Hirax, and Figure 7 shows the blood concentration of the polypeptide over time. As can be seen from Figure 7 and Table 20, in the case of Hirax, it is degraded so rapidly that its half-life in the body cannot be measured, whereas according to an example of the present invention... Poly The peptide has a longer half-life of approximately 0.272 hours (approximately 16.3 minutes) than Hirax, remaining in the body longer and potentially exhibiting a greater effect than Hirax. Differences were also observed in blood concentration; while Hirax reached its peak within 0.25 hours (15 minutes), BMI2004 showed a slower increase in blood concentration over 0.5 hours (30 minutes) and remained in the body for an even longer period, suggesting a potentially greater effect than Hirax.

[0157] [Table 20] [Sequence List]

Claims

1. A polypeptide having 34 to 170 amino acids deleted from the C-terminus of the polypeptide consisting of the amino acid sequence of SEQ ID NO: 1, and possessing hyaluronidase activity.

2. 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.

3. 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.

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

1.

5. The polypeptide according to claim 1, wherein the polypeptide comprises 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.

6. The polypeptide according to claim 1, wherein the polypeptide is glycated.

7. The polypeptide according to claim 1, wherein the polypeptide is stable at pH 3 to 10.

8. The polypeptide according to claim 1, wherein the polypeptide is stable at temperatures of -20 to 45°C.

9. The polypeptide according to claim 1, wherein the polypeptide has an enzyme activity of 57% or more of its initial activity after being stored for 4 weeks at a pH in the range of pH 3 to 10.

10. The polypeptide according to claim 1, wherein the polypeptide has an enzyme activity of 32% or more of its initial activity after being stored for 4 weeks at a pH of 3 to less than 5.

11. The polypeptide according to claim 1, wherein the polypeptide exhibits enzyme activity of 63% or more of its initial activity after being stored for four weeks at a temperature below 0°C.

12. The polypeptide according to claim 1, wherein the polypeptide exhibits enzyme activity of 83% or more of its initial activity after being stored for 4 weeks at a temperature in the range of 0 to 40°C.

13. The polypeptide according to claim 1, wherein the polypeptide has an enzyme activity of 52% or more of its initial activity after being stored for 4 weeks at a temperature of 40°C or higher.

14. The polypeptide according to claim 1, wherein the polypeptide has a titer of more than 1 to 3 times that of the polypeptide comprising the amino acid sequence of SEQ ID NO:

1.

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

16. A nucleic acid molecule for encrypting a polypeptide according to any one of claims 1 to 15.

17. A vector comprising the nucleic acid molecule of claim 16.

18. A cell comprising the vector of claim 17.

19. The cell according to claim 18, wherein the cell is selected from the group consisting of bacteria, yeast, fungi, insect cells, animal cells, mammalian cells, algae cells, and plant cells.

20. The cells according to claim 19, 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.

21. A composition for topical administration comprising a polypeptide according to any one of claims 1 to 15.

22. The composition according to claim 21, wherein the composition is for subcutaneous administration or intramuscular injection.

23. A drug delivery carrier comprising a polypeptide according to any one of claims 1 to 15.

24. A composition for the prevention or treatment of edema, comprising a polypeptide according to any one of claims 1 to 15.

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