Hyaluronidase polypeptide and use thereof

Abstract

This article reveals hyaluronidase peptides and their applications.

Description

[Technical Field]

[0001] This invention relates to hyaluronidase polypeptide and its uses. [Previous Technology]

[0002] Hyalurase is a general term for a family of enzymes that catalyze the degradation of hyaluronic acid. Initially, Duran-Reynals described hyalurase as a diffusion factor. Later, it was named hyalurase because it was observed to exhibit potent activity towards hyaluronic acid. Based on their enzymatic mechanisms, these enzymes are classified into three types: hyaluronic acid 4-glycan hydrolase (EC 3.2.1.35), found in testes, lysosomes, and bee venom; hyaluronic acid 3-glycan hydrolase (EC 3.2.1.36), found in extracts; and hyaluronic acid dissociation enzyme (EC 4.2.2.1), found in bacteria.

[0003] Specifically, hyaluronidase (PH-20) in the testes is a glycosylphosphatidylinositol (GPI) anchoring enzyme located on the acrosome of sperm and is an important enzyme that induces fertilization by degrading the thick outer wall of the egg. Furthermore, PH-20 is known to cleave the β(1-4) bonds between D-glucuronic acid and N-acetylglucosamine found in hyaluronic acid, chondroitin, and chondroitin sulfate, which are distributed among glycosaminoglycans in mammalian skin. The general molecular formula of these enzymes is C 2455H 3775N 617O 704S 21, and the molecular weight is 53870.9 g / mol. In the human body, six genes are associated with enzymes including HYAL1, HYAL2, HYAL3, and PH-20 / SPAM1.

[0004] Since the 1950s, the widespread use of hyaluronidase has been thoroughly reviewed. The first application was subcutaneous injection of non-enteric fluids. In addition, these enzymes have been applied in the fields of orthopedic surgery, ophthalmology, plastic surgery, dentistry, oral surgery, gynecology, and otolaryngology, such as for infiltration and blocking anesthesia to increase the diffusion of local anesthetics and steroids, dispersion of accumulated body fluids (e.g., hematomas), prevention of peritoneal adhesions and stone formation, and treatment of infertility.

[0005] Currently available hyaluronidases on the market are extracted from sheep or bovine testicles, as exemplified by Vitrase (ISTA Pharmaceuticals, sheep source) and Amphadase (Amphastar Pharmaceuticals, bovine source). They are commercialized by loading suitable concentrations of untreated hyaluronidase into vials and then freeze-drying them. Commercial products of animal-derived hyaluronidases present numerous problems when applied to various fields because the foreign proteins they contain can cause allergic reactions, and their bioactivity decreases due to reduced stability over time.

[0006] To overcome these problems, recombinant hyaluronidase has been studied. Recombinant proteins can be expressed in various cell types, including E. coli, yeast, insect cells, and animal cells. In particular, the activity of hyaluronidase is affected by glycation during post-translational modification. This is because glycans can affect the antigenicity, structural folding, solubility, and stability of glycoproteins. Based on this viewpoint, animal cell lines are suitable for expression in different cell types, with the most preferred being the Chinese hamster ovary (CHO) cells, whose safety has been ensured, because the post-translational modification process in yeast or insect cells differs from that in mammalian cells.

[0007] The first recombinant hyaluronidase of PH-20 was developed by Halozyme Therapeutic and is sold under the trade name Hylenex. It is under development for various applications such as subcutaneous injection, vitrectomy, and eye diseases. However, the yield or stability of hyaluronidase is still unsatisfactory and the supply is insufficient compared to the demand. Therefore, there is a need for hyaluronidase with improved yield or stability. [Summary of the Invention]

[0008] Technical Issues

[0009] One state of the present invention provides a hyaluronidase polypeptide with excellent stability and improved activity.

[0010] Another aspect of the present invention provides a composition for local delivery comprising a polypeptide according to one aspect of the present invention.

[0011] Another aspect of the present invention provides a drug delivery carrier comprising a polypeptide according to one aspect of the present invention.

[0012] Yet another aspect of the present invention provides a composition for the prevention or treatment of edema, comprising a polypeptide according to one aspect of the present invention.

[0013] Technical Solutions

[0014] One aspect of the present invention relates to a polypeptide, wherein the C-terminal region is derived from the amino acid sequence deletion of wild-type hyaluronidase.

[0015] Another aspect of the present invention relates to a polypeptide comprising a continuous amino acid sequence of 1 to 203 amino acids having at least one segment deleted from the C-terminus of the amino acid sequence of wild-type hyaluronidase.

[0016] Another aspect of the present invention relates to a composition for local application, comprising a polypeptide.

[0017] Yet another aspect of the present invention relates to a drug delivery carrier comprising a polypeptide.

[0018] Another aspect of the present invention relates to a composition for the prevention or treatment of edema, comprising a polypeptide.

[0019] The invention will now be described in detail.

[0020] One aspect of this invention relates to a polypeptide that has 90% or higher sequence homology with a polypeptide whose C-terminal region lacks the amino acid sequence of wild-type hyaluronidase. Wild-type hyaluronidase may be a polypeptide composed of the amino acid sequence of SEQ ID NO: 1.

[0021] The polypeptide according to one state of the present invention has hyaluronidase activity and may have at least one of the following characteristics (1) to (6): (1) Stable at pH 3 to pH 10, specifically, after storage for 4 weeks at a pH range between 3 and 10 or lower, the enzyme activity is 57% or higher of the initial activity, or after storage for 4 weeks at a pH range between pH 3 and below pH 5, the enzyme activity is 32% or higher of the initial activity; (2) Stable at a temperature of -20°C to 45°C, specifically, after storage for 4 weeks at a temperature below 0°C, the enzyme activity is 63% or higher of the initial activity, or after storage for 4 weeks at a temperature of 0°C to 40°C, the enzyme activity is 83% or higher of the initial activity, or after storage for 4 weeks at a temperature of 40°C or higher, the enzyme activity is 52% or higher of the initial activity; (3) High hyaluronidase activity, specifically, the hyaluronidase activity is more than 1 to 3 times that of wild-type hyaluronidase. (4) having a potency that is more than 1 to 3 times that of a 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) having one or more amino acid residues glycosylated.

[0022] Specifically, in an embodiment of the present invention, a polypeptide is prepared by deleting the C-terminal region of the amino acid sequence of sheep-derived wild-type hyaluronidase (CAS No. 488712-31-8). The stability and activity of the polypeptide obtained thereby were observed to be significantly higher than those of wild-type hyaluronidase, and even when used in smaller amounts, it exhibited equivalent effects in drug absorption, drug diffusion promotion, and body fluid reabsorption compared to the polypeptide derived from wild-type hyaluronidase. Therefore, the polypeptide according to a specific embodiment of the present invention can be a hyaluronidase. Specifically, the polypeptide according to a specific embodiment of the present invention can substantially comprise a minimal fragment possessing hyaluronidase activity.

[0023] Specifically, the polypeptide of one specific embodiment of the present invention may have 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, 99.5% or higher, or 99.9% or higher sequence homology with polypeptides comprising a C-terminus of the amino acid sequence of SEQ ID NO: 1. In this regard, the polypeptide of one specific embodiment of the present invention is not a polypeptide composed of the amino acid sequence of SEQ ID NO: 1. Furthermore, the polypeptide of one specific embodiment of the present invention may retain the catalytic activity of wild-type hyaluronidase. Specifically, the activity and / or stability of the polypeptide of one specific embodiment of the present invention may be equal to or greater than the activity and / or stability of wild-type hyaluronidase. Wild-type hyaluronidase may be a polypeptide composed of the amino acid sequence of SEQ ID NO: 1.

[0024] Specifically, the polypeptide according to one specific example of the present invention may be a C-terminal deletion variant, wherein n amino acids from the C-terminus of the amino acid sequence of wild-type hyaluronidase are deleted (where n is a natural number from 1 to 203).

[0025] By way of example, the polypeptide according to one specific embodiment of the present invention may have at least one continuous amino acid sequence of 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 the wild-type hyaluronidase. The polypeptide according to one specific embodiment of the present invention may further have a first amino acid deleted from the N-terminus of the amino acid sequence of the wild-type hyaluronidase. The amino acid sequence of the wild-type hyaluronidase may be the amino acid sequence of SEQ ID NO: 1.

[0026] For example, the polypeptide according to one specific embodiment of the present invention may consist of an amino acid sequence extending from the N-terminus of the amino acid sequence of wild-type hyaluronidase to the m-th amino acid or from the second amino acid to the m-th amino acid (where m is a natural number from 315 to 517). The amino acid sequence of wild-type hyaluronidase may be the amino acid sequence of SEQ ID NO: 1.

[0027] For example, the polypeptide according to one specific embodiment of the present invention may be composed of the amino acid sequences of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6.

[0028] For example, a polypeptide according to a specific embodiment of the present invention can be expressed using animal cells as a host. In this regard, a polypeptide according to a specific embodiment of the present invention can be expressed in animal cells as a host, and is glycosylated via a post-translational modification (PTM) process during expression.

[0029] The polypeptide according to one specific embodiment of the present invention can be stable at pH 3 to 10. Specifically, compared with wild-type hyaluronidase, the enzyme activity of the polypeptide according to one specific embodiment of the present invention can be reduced when stored at pH 3 to 10.

[0030] In an embodiment of the present invention, it was observed that the polypeptide according to a specific example of the present invention retained its enzymatic activity after being stored at a pH of 3 to 10 for four weeks.

[0031] For example, after storage for 4 weeks at a pH between pH 3 and below pH 5, or in the pH range of 3 to 4 (e.g., pH 3), the polypeptide according to one specific embodiment of the invention may have a total enzymatic activity of 32% or higher, 33% or higher, 34% or higher, 35% or higher, 36% or higher, 37% or higher, 38% or higher, 39% or higher, 40% or higher, 41% or higher, 42% or higher, 43% or higher, 44% or higher, 45% or higher, 46% or higher, 47% or higher, 48% or higher, 49% or higher, 50% or higher, 51% or higher, 52% or higher, 53% or higher, 54% or higher, or 55% or higher. In this case, the polypeptide may be stored at a temperature of 5°C or 37°C.

[0032] For example, after storage for 4 weeks at a pH range (e.g., pH 5, pH 7, or pH 10) between pH 3 and pH 10 or lower, or between pH 4 and pH 10 or between pH 5 and pH 10, the polypeptide according to one specific embodiment of the invention may have a total enzymatic activity of 57% or higher, 58% or higher, 59% or higher, 60% or higher, 61% or higher, 62% or higher, 63% or higher, 64% or higher, or 65% or higher of its initial activity. In this case, the polypeptide may be stored at a temperature of 5°C or 37°C.

[0033] For example, pH values ​​between pH 3 and pH 10, pH 3 and pH 9 or lower, pH 3 and pH 8 or lower, pH 3 and pH 7 or lower, pH 4 or higher and pH 10, pH 4 to 9, pH 4 to 8, pH 4 to 7, pH 5 and pH 10, pH 5 to 9, pH 5 to 8, pH 5 to 7 (e.g., pH 5 or pH 10). 7) After storage for 4 weeks, the polypeptide according to one specific example of the present invention may have 57% or higher, 58% or higher, 59% or higher, 60% or higher, 61% or higher, 62% or higher, 63% or higher, 64% or higher, 65% or higher, 66% or higher, 67% or higher, 68% or higher, 69% or higher, 70% or higher, 71% or higher, 72% or higher, 73% or higher, 74% or higher, 75% or higher, 76% or higher, 77% or higher, 78% or higher, 79% or higher, 80% or higher, 81% or higher, or 82% or higher enzyme activity. In this case, the polypeptide may be stored at a temperature of 5°C or 37°C.

[0034] The polypeptide according to one specific embodiment of the present invention can be stable under freezing, refrigeration and high temperature conditions. Specifically, when stored under freezing, refrigeration and high temperature conditions, the reduction in enzyme activity of the polypeptide according to one specific embodiment of the present invention is less than that of wild-type hyaluronidase.

[0035] In an embodiment of the present invention, it was observed that the polypeptide according to one specific example of the present invention retained its enzyme activity after being stored for 4 weeks under freezing (e.g., -18 to -20°C), refrigeration (e.g., 2 to 8°C), and high temperature (e.g., 40 to 45°C) conditions.

[0036] For example, after storage for 4 weeks at freezing temperatures, such as below 0°C, -20°C to below 0°C, -20°C to -10°C, or -20°C to -18°C (for example, at -20°C), the polypeptide according to one specific embodiment of the invention may have a total activity of 63% or higher, 64% or higher, 65% or higher, 66% or higher, 67% or higher, 68% or higher, 69% or higher, 70% or higher, 71% or higher, 72% or higher, 73% or higher, or 74% of the initial activity. Enzyme activity of 75% or higher, 76% or higher, 77% or higher, 78% or higher, 79% or higher, 80% or higher, 81% or higher, 82% or higher, 83% or higher, 84% or higher, 85% or higher, 86% or higher, 87% or higher, 88% or higher, 89% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher. In this regard, the peptide can be stored at pH 5 or 7.

[0037] For example, after storage for 4 weeks at refrigerated temperatures, such as 0 to 40°C, 0 to 10°C, or 2 to 8°C (e.g., 5°C), the polypeptide according to one specific embodiment of the invention may have a total enzymatic activity of 83% or higher, 84% or higher, 85% or higher, 86% or higher, 87% or higher, 88% or higher, 89% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher of the initial activity. In this regard, the polypeptide may be stored at pH 5 or 7.

[0038] For example, after storage for 4 weeks at high temperatures, such as 40°C or higher, 40 to 50°C, or 40 to 45°C (e.g., 40°C), the polypeptide according to one specific embodiment of the invention may have a total enzymatic activity of 52% or higher, 53% or higher, 54% or higher, 55% or higher, 56% or higher, 57% or higher, 58% or higher, 59% or higher, 60% or higher, 61% or higher, 62% or higher, 63% or higher, 64% or higher, 65% or higher, 66% or higher, 67% or higher, 68% or higher, 69% or higher, 70% or higher, 71% or higher, 72% or higher, 73% or higher, 74% or higher, 75% or higher, 76% or higher, 77% or higher, 78% or higher, 79% or higher. In this regard, the polypeptide may be stored at a pH of 5 or 7.

[0039] The polypeptide according to one specific embodiment of the present invention may have a higher potency than wild-type hyaluronidase. For example, the polypeptide according to one specific embodiment of the present invention may have a potency 1 to 3 times higher, 1 to 2.5 times higher, 1 to 2 times higher, 1 to 1.9 times higher, 1 to 1.8 times higher, 1 to 1.7 times higher, 1.1 to 3 times higher, 1.1 to 2.5 times higher, 1.1 to 2 times higher, 1.1 to 1.9 times higher, 1.1 to 1.8 times higher, 1.1 to 1.7 times higher, 1.2 to 3 times higher, 1.2 to 2.5 times higher, 1.2 to 2 times higher, 1.2 to 1.9 times higher, or 1.2 to 3 times higher than wild-type hyaluronidase. Potency of 1.8 times, 1.2 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.

[0040] For example, the polypeptide according to one specific example of the present invention may have 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 ...50,000 IU / mg, 122,000 to 145,000 IU / mg, 122,000 to 140,000 IU / mg, 122,000 to 140,000 IU / mg, 122,000 to 140,000 IU / mg, 122, Activity at IU / mg, 122,000 to 135,000 IU / mg.

[0041] Another aspect of the present invention relates to a nucleic acid molecule encoding a polypeptide according to a specific embodiment of the present invention, a carrier comprising the nucleic acid molecule, and a cell comprising the carrier. The cell may be selected from the group consisting of: bacteria including Escherichia coli and actinomycetes, yeast, fungi, insect cells, animal cells, mammalian cells, algal cells, and plant cells. Mammalian cells may be selected from the group consisting of: CHO, NSO, HEK293, BHK, Per.C6, MDCK, Vero, MRC, HeLa, IMR, and Sp2 / O. CHO cells may be selected from the group consisting of: CHO-DG44, CHO-DUKX, CHO-S, CHO-K1, and CHO-DP12.

[0042] Another aspect of the present invention relates to a composition for local administration comprising a polypeptide according to a specific embodiment of the present invention. The composition may be adapted for subcutaneous administration. In embodiments of the present invention, when administered locally, the polypeptide according to a specific embodiment of the present invention was observed to exhibit significantly better drug absorption and diffusion-promoting effects compared to wild-type hyaluronidase. Therefore, another specific embodiment of the present invention relates to a drug delivery carrier comprising a polypeptide according to a specific embodiment of the present invention.

[0043] Furthermore, in embodiments of the present invention, when administered topically, the polypeptide according to one specific example of the present invention was observed to exhibit a significantly higher efficacy in promoting the reabsorption of excess body fluid compared to wild-type hyaluronidase. Therefore, another specific example of the present invention relates to a pharmaceutical composition for the prevention or treatment of edema, comprising a polypeptide according to one specific example of the present invention as an active ingredient.

[0044] The composition of the present invention (e.g., a pharmaceutical composition) may further include at least one active ingredient that exhibits equivalent or similar functions.

[0045] Furthermore, according to the prior art known to those skilled in the art, the components of the present invention (e.g., pharmaceutical components) can be formulated and prepared in a single-dose form or encapsulated in a multi-dose container using a pharmaceutically acceptable carrier. As used herein, the term "carrier" refers to a substance that facilitates the incorporation of the relevant material into cells or tissues. Hereinafter, the term "pharmaceutically acceptable" means that it is physiologically acceptable when administered to humans and does not cause gastrointestinal symptoms, allergic reactions (such as dizziness), or similar reactions.

[0046] Any pharmaceutically acceptable carrier may be used, provided that it is typically suitable for use in formulations. Examples of pharmaceutically acceptable carriers include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, talc, magnesium stearate, and mineral oil.

[0047] In addition to the above-mentioned components, the composition according to the present invention (e.g., a pharmaceutical composition) may further include additives such as fillers, anti-aggregating agents, lubricants, humectants, fragrances, emulsifiers, preservatives, etc. In the present invention, the content of additives in the composition is not particularly limited, but can be appropriately adjusted within the range acceptable for typical formulations.

[0048] As used herein, the term "excipient" means any substance that is not itself a therapeutic agent but is used as a carrier or mediator to deliver a therapeutic agent to an individual or to be added to a pharmaceutical composition to improve its operational or storage properties or to allow and promote unit doses of the composition.

[0049] The composition of the present invention (e.g., a pharmaceutical composition) can be formulated into different forms (e.g., sterile injectables) according to the purpose and can be administered via different routes (e.g., local, subcutaneous, intramuscular injections).

[0050] The preferred dosage of the composition (e.g., pharmaceutical composition) according to the present invention may vary within its range depending on the patient's condition, weight, age, sex, health status, dietary composition specificity, formulation characteristics, disease severity, time of administration of the composition, method of administration, period or time interval of administration, excretion rate and form of the drug, but may be appropriately selected by those skilled in the art.

[0051] As used herein, the term "effective dose of a pharmaceutical composition" means the amount of a composition containing an active ingredient sufficient to treat a specific symptom. This can vary depending on the method of preparation, administration, timing and / or route of administration of the pharmaceutical composition, and can be varied based on factors including: the type and extent of response desired by administration of the pharmaceutical composition, the type of individual to whom the medication is administered, the individual's age, weight, general health condition, symptoms or severity of disease, sex, diet, excretion, the composition of other pharmaceutical compositions administered to the individual simultaneously or at different times, and other similar factors well known in the medical field. Furthermore, an expert in this field can easily determine and prescribe an effective dose for the intended treatment.

[0052] The pharmaceutical composition according to the present invention can be administered once a day or divided into several doses. The pharmaceutical composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents. Taking all the above factors into consideration, the pharmaceutical composition of the present invention can be administered in a quantity that achieves maximum efficacy with minimal amount and without side effects.

[0053] Compared to wild-type hyaluronidase, the polypeptide or composition according to one specific example of the present invention has a higher potency and therefore can achieve equivalent efficacy at a lower dose. For example, the polypeptide or composition according to one specific example of the present invention can be administered at 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, or 65% or less of the daily dose of wild-type hyaluronidase. The dose can be based on weight or weight percentage. Furthermore, the total daily dose can be administered in divided doses and continuously or discontinuously as needed.

[0054] Beneficial effects

[0055] With improvements in expression levels and stability in animal cells, the hyaluronidase polypeptide of one specific embodiment of the present invention exhibits enzyme activity equal to or greater than that of mature wild-type PH-20. Therefore, when expressed in CHO cells, the hyaluronidase polypeptide of one specific embodiment of the present invention has an elevated expression level compared to mature wild-type PH-20, and is very stable, thus increasing its industrial applicability for various applications.

[0056] Furthermore, due to its ability to degrade hyaluronic acid, a component of intercellular spaces, hyaluronidase regulates tissue adhesion to promote drug penetration and diffusion, and also promotes the reabsorption of excess body fluids present in tissues, thus its coverage and application areas have gradually expanded. However, animal-derived wild-type PH-20 is highly susceptible to infection from animal-derived substances. The hyaluronidase peptide according to a specific example of the present invention is unlikely to cause infection and is therefore safe. Compared with animal-derived wild-type PH-20, this peptide exhibits high activity at the same dosage and can further increase industrial applicability for various uses.

Implementation Method

[0067] A better understanding of the present invention can be obtained from the following embodiments, which are set forth for the purpose of illustrating the present invention, but should not be construed as limiting the present invention.

[0068] Example 1. Preparation of hyaluronidase

[0069] Hyaluronidase was prepared by truncating the C-terminal extension region of the amino acid sequence of SEQ ID NO: 1 of sheep-derived wild-type hyaluronidase (CAS No. 488712-31-8) by 34 amino acid residues (Examples 1-2), 68 amino acid residues (Examples 1-3), 102 amino acid residues (Examples 1-4), 136 amino acid residues (Examples 1-5), 170 amino acid residues (Examples 1-6), or 204 amino acid residues (Examples 1-7).

[0070] 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 (hereinafter referred to as "PCR") and its expression and activity in CHO-DG44 cells were examined using the pcDNA 3.1 vector. The gene was anchored into CHO-DG44 cells via the pOptiVEC vector. When the CHO-DG44 cell density reached 4 × 10⁶ to 6 × 10⁶ cells per ml, the cells were transfected using plastids constructed by inserting the hyaluronidase cDNA into the pOptiVEC vector using electroporation. After transfection, CHO-DG44 cells were cultured in Power CHO 2CD medium (containing 4 mM L-glutamic acid) and the cell culture was centrifuged at 12,000 rpm for 10 minutes. The supernatant was recovered and subsequently purified as needed using various methods, including affinity chromatography, hydrophobic chromatography, ion exchange chromatography, and similar methods. The purified hyaluronidase was filtered by water displacement during ultrafiltration and microfiltration. The amino acid sequences of the obtained hyaluronidase are given in Table 1. Table 1 name Sequence (N→C) SEQ ID NO: Example 1-1 (CAS No. 488712-31-8) LDFRAPPLIS NTSFLWAWNA PAERCVKIFK LPPDLRLFSV KGSPQKSATG QFITLFYADR LGYYPHIDEK TGNTVYGGIP QLGNLKNHLE KAKKDIAYYI PNDSVGLAVI DWENWRPTWA RNWKPKDVYR DESVELVLQK NPQLSFPEAS KIAKVDFETA GKSFMQETLK LGKLLRPNHL WGYYLFPDCY NHNYNQPTYN GNCSDLEKRR NDDLDWLWKE STALFPSVYL NIKLKSTPKA AFYVRNRVQE AIRLSKIASV ESPLPVFVYH RPVFTDGSST YLSQGDLVNS VGEIVALGAS GIIMWGSLNL SLTMQSCMNL GNYLNTTLNP YIINVTLAAK MCSQVLCHDE GVCTRKQWNS SDYLHLNPMN FAIQTGKGGK YTVPGKVTLE DLQTFSDKFY CSCYANINCK KRVDIKNVHS VNVCMAEDIC IEGPVKLQPS DHSSSQNEAS TTTVSSISPS TTATTVSPCT PEKQSPECLK VRCLEAIANV TQTGCQGVKW KNTSSQSSIQ NIKNQTTY 1 Example 1-2 DFRAPPLISN TSFLWAWNAP AERCVKIFKL PPDLRLFSVK GSPQKSATGQ FITLFYADRL GYYPHIDEKT GNTVYGGIPQ LGNLKNHLEK AKKDIAYYIP NDSVGLAVID WENWRPTWAR NWKPKDVYRD ESVELVLQKN PQLSFPEASK IAKVDFETAG KSFMQETLKL GKLLRPNHLW GYYLFPDCYN HNYNQPTYNG NCSDLEKRRN DDLDWLWKES TALFPSVYLN IKLKSTPKAA FYVRNRVQEA IRLSKIASVE SPLPVFVYHR PVFTDGSSTY LSQGDLVNSV GEIVALGASG IIMWGSLNLS LTMQSCMNLG NYLNTTLNPY IINVTLAAKM CSQVLCHDEG VCTRKQWNSS DYLHLNPMNF AIQTGKGGKY TVPGKVTLED LQTFSDKFYC SCYANINCKK RVDIKNVHSV NVCMAEDICI EGPVKLQPSD HSSSQNEAST TTVSSISPST TATTVSPCTP EKQSPECLKV RCL 2 Examples 1-3 (BMI2004) DFRAPPLISN TSFLWAWNAP AERCVKIFKL PPDLRLFSVK GSPQKSATGQ FITLFYADRL GYYPHIDEKT GNTVYGGIPQ LGNLKNHLEK AKKDIAYYIP NDSVGLAVID WENWRPTWAR NWKPKDVYRD ESVELVLQKN PQLSFPEASK IAKVDFETAG KSFMQETLKL GKLLRPNHLW GYYLFPDCYN HNYNQPTYNG NCSDLEKRRN DDLDWLWKES TALFPSVYLN IKLKSTPKAA FYVRNRVQEA IRLSKIASVE SPLPVFVYHR PVFTDGSSTY LSQGDLVNSV GEIVALGASG IIMWGSLNLS LTMQSCMNLG NYLNTTLNPY IINVTLAAKM CSQVLCHDEG VCTRKQWNSS DYLHLNPMNF AIQTGKGGKY TVPGKVTLED LQTFSDKFYC SCYANINCKK RVDIKNVHSV NVCMAEDICI EGPVKLQPSD HSSSQNEAS 3 Examples 1 - 4 DFRAPPLISN TSFLWAWNAP AERCVKIFKL PPDLRLFSVK GSPQKSATGQ FITLFYADRL GYYPHIDEKT GNTVYGGIPQ LGNLKNHLEK AKKDIAYYIP NDSVGLAVID WENWRPTWAR NWKPKDVYRD ESVELVLQKN PQLSFPEASK IAKVDFETAG KSFMQETLKL GKLLRPNHLW GYYLFPDCYN HNYNQPTYNG NCSDLEKRRN DDLDWLWKES TALFPSVYLN IKLKSTPKAA FYVRNRVQEA IRLSKIASVE SPLPVFVYHR PVFTDGSSTY LSQGDLVNSV GEIVALGASG IIMWGSLNLS LTMQSCMNLG NYLNTTLNPY IINVTLAAKM CSQVLCHDEG VCTRKQWNSS DYLHLNPMNF AIQTGKGGKY TVPGKVTLED LQTFSDKFYC SCYANINCKK RVDIK 4 Examples 1-5 DFRAPPLISN TSFLWAWNAP AERCVKIFKL PPDLRLFSVK GSPQKSATGQ FITLFYADRL GYYPHIDEKT GNTVYGGIPQ LGNLKNHLEK AKKDIAYYIP NDSVGLAVID WENWRPTWAR NWKPKDVYRD ESVELVLQKN PQLSFPEASK IAKVDFETAG KSFMQETLKL GKLLRPNHLW GYYLFPDCYN HNYNQPTYNG NCSDLEKRRN DDLDWLWKES TALFPSVYLN IKLKSTPKAA FYVRNRVQEA IRLSKIASVE SPLPVFVYHR PVFTDGSSTY LSQGDLVNSV GEIVALGASG IIMWGSLNLS LTMQSCMNLG NYLNTTLNPY IINVTLAAKM CSQVLCHDEG VCTRKQWNSS DYLHLNPMNF AIQTGKGGKY T 5 Examples 1-6 DFRAPPLISN TSFLWAWNAP AERCVKIFKL PPDLRLFSVK GSPQKSATGQ FITLFYADRL GYYPHIDEKT GNTVYGGIPQ LGNLKNHLEK AKKDIAYYIP NDSVGLAVID WENWRPTWAR NWKPKDVYRD ESVELVLQKN PQLSFPEASK IAKVDFETAG KSFMQETLKL GKLLRPNHLW GYYLFPDCYN HNYNQPTYNG NCSDLEKRRN DDLDWLWKES TALFPSVYLN IKLKSTPKAA FYVRNRVQEA IRLSKIASVE SPLPVFVYHR PVFTDGSSTY LSQGDLVNSV GEIVALGASG IIMWGSLNLS LTMQSCMNLG NYLNTTLNPY IINVTLAAKM CSQVLCH 6 Examples 1-7 DFRAPPLISN TSFLWAWNAP AERCVKIFKL PPDLRLFSVK GSPQKSATGQ FITLFYADRL GYYPHIDEKT GNTVYGGIPQ LGNLKNHLEK AKKDIAYYIP NDSVGLAVID WENWRPTWAR NWKPKDVYRD ESVELVLQKN PQLSFPEASK IAKVDFETAG KSFMQETLKL GKLLRPNHLW GYYLFPDCYN HNYNQPTYNG NCSDLEKRRN DDLDWLWKES TALFPSVYLN IKLKSTPKAA FYVRNRVQEA IRLSKIASVE SPLPVFVYHR PVFTDGSSTY LSQGDLVNSV GEIVALGASG IIMWGSLNLS LTM 7

[0071] Example 2. Analysis of hyaluronidase stability based on missing sites.

[0072] (1) Compare enzyme activity or content by storage pH based on the missing site.

[0073] The pH stability of six hyaluronidases, excluding the inactive hyaluronidases of Examples 1-7, was analyzed based on the deletion sites in the hyaluronidases prepared in Example 1. The amino acid sequences were expressed in transient cells and the resulting cell cultures were concentrated in the same manner before enzyme activity was measured. The experimental concentration was adjusted to 1,500 IU / mL (=100%), and solutions were prepared by water substitution according to the conditions shown in Table 2. The solutions were stored at 37°C for 4 weeks at pH 3.0, pH 5.0, pH 7.0, or pH 10.0, with titrations performed every two weeks.

[0074] The enzyme activity or content of samples with different deletion sites was analyzed at various time points according to the following protocol: 1) The activity of hyaluronidase with different deletion sites was determined by comparison with the EP STD ratio. 1-1) Preparation of pH 6.4 phosphate buffer: The solution of 2.5 g disodium hydrogen phosphate dodecahydrate, 2.5 g sodium dihydrogen phosphate and 8.2 g sodium chloride in 950 mL of water was adjusted to pH 6.4 with 1 M sodium hydroxide or 1 M hydrochloride, and water was added to form a final volume of 1,000 mL. 1-2) Preparation of diluent: 0.140 g of gelatin reagent was dissolved in a mixture of 100 mL pH 6.4 phosphate buffer and 100 mL of water at 37°C. The diluent should be used within 2 hours after preparation. 1-3) Preparation of substrate solution: 100 mL of water was added little by little to 0.5 g sodium hyaluronate while stirring. Slowly add water until the sodium hyaluronate swells. Continue stirring at 4°C for 12 hours or longer. Store the resulting substrate solution at 4°C and use within 4 days of preparation. 1-4) Preparation of standard solution: Dissolve the EP STD (EDQM) reference standard in diluent to obtain a concentration of approximately 50 IU / mL. Take an exact 3 mL of this solution and place it in a 250 mL volumetric flask, then add diluent to the flask to form an exact volume of 250 mL. Use the resulting solution as the standard solution. 1-5) Preparation of test solution: Adjust the hyaluronidase test solutions with different deletion sites (each with approximately 0.6 IU / mL) to the predetermined pH and then dilute them before use. 2) Manual: Test the standard and test solution according to the following method. 2-1) In a water bath maintained at 37°C, let a 50 mL conical tube containing 7.5 mL of pH 6.4 phosphate buffered saline and 5.0 mL of substrate solution stand. 2-2) Add 2.5 mL of test solution to the conical tube containing pH 6.4 phosphate buffered saline and the test solution, and mix for 1 minute. 2-3) Load all the mixed solution from the conical tube into the Ubbelohde microviscometer (DIN 51 562, Part 2, capillary type MIII, constant: approx. 0.1 mm² / s² or equivalent viscometer). 2-4) Use a second precision timer to record the time taken for the solution to flow from the upper indicator line to the lower indicator line in the Ubbelohde microviscometer. 2-5) Record the time several times over approximately 20 minutes. 2-6) Repeat the above steps three times. 3) Calculation: Calculate the potency (IU / mg) according to the equation.3-1) Reaction Time: T1 + T2 / 2 3-2) ηr -1: {(kxT2) / 0.6915} -1 T1: Time (seconds) for the solution to rise to the upper indicator line in the Ubbelohde microviscometer T2: TT1 T: Time (seconds) for the solution to fall to the lower indicator line in the Ubbelohde microviscometer k: Ubbelohde microviscometer constant (mm² / s²), referring to the specification of the Ubbelohde microviscometer 0.6915: Kinematic viscosity of the substrate solution at 37°C (mm² / s²) 3-3) Activity Calculation: (BT / BR)*(ER / ET)*A BT: Slope of the regression curve plotted for the test solution in coordinates with reaction time on the x-axis and natural logarithm ηr -1 on the y-axis BT: Reaction time on the x-axis and natural logarithm ηr -1 on the y-axis The slope of the regression curve plotted for the standard solution in coordinates -1: ET: concentration of test solution (mg / mL) ER: concentration of standard solution (mg / mL) A: potency of standard solution (IU / mg) Table 2. sample active amino acids at cleavage sites Percentage of regions lacking enzyme (%) Store pH Storage temperature Example 1-1 Hyaluronidase (temporary cells) L - Y Approximately 0% pH 7.0 37℃ Examples 1-2 D - L Approximately 6% Examples 1-3 D - S Approximately 12% Examples 1-4 D - K Approximately 18% Examples 1-5 D - T Approximately 24% Examples 1-6 D - H Approximately 30% Examples 1-7 D - M Approximately 36%

[0075] Test data for pH stability are shown in Table 3 below. Enzyme activity (in IU / mL) in each test is expressed as a percentage of the reference value of 1,500 IU / mL (=100%), which is the initial enzyme activity at week 0. The enzyme activity ratio was calculated using the following equation:

[0076] Enzyme activity ratio (%) = (Enzyme activity at measurement time) / (Initial enzyme activity) * 100 Table 3 Enzyme activity ratio (%) pH 3.0 pH 5.0 pH 7.0 pH 10.0 sample Week 0 Week 2 Week 4 Week 0 Week 2 Week 4 Week 0 Week 2 Week 4 Week 0 Week 2 Week 4 Example 1-1 100.47 61.31 31.62 100.43 72.84 55.30 100.36 73.17 56.03 100.88 63.31 45.92 Examples 1-2 102.00 84.77 55.05 101.17 94.52 80.57 101.47 95.88 81.24 101.25 86.29 64.98 Examples 1-3 102.66 85.48 55.26 101.80 95.02 81.19 101.49 96.22 82.55 102.68 89.95 65.11 Examples 1-4 101.23 84.91 54.76 101.19 94.96 80.65 101.38 95.50 81.73 101.49 88.27 64.19 Examples 1-5 100.46 83.59 52.94 100.15 94.88 77.45 100.60 93.87 78.81 100.67 86.74 62.64 Examples 1-6 100.82 82.32 50.58 100.08 92.61 74.88 99.32 91.36 77.63 99.75 85.76 61.17

[0077] As shown in Table 3, the 4-week pH stability data obtained for hyaluronidases with different deletion sites indicate that the enzyme activity percentage (%) in the pH range of 5.0 to 7.0 is higher than at other pH values. The hyaluronidases of Examples 1-2 to 1-6 exhibit significantly higher pH stability compared to the wild-type hyaluronidase of Example 1-1, with the hyaluronidases of Examples 1-2 to 1-4 being superior to the other hyaluronidases. Values ​​exceeding 100% appear to be measurement errors, which generally arise from larger fluctuations in potency testing of biological products such as vaccines and recombinant proteins (cytokines, monoclonal antibodies, etc.).

[0078] (2) Compare enzyme activity or content based on storage temperature according to the missing sites.

[0079] The temperature stability of the hyaluronidases in Examples 1-1 to 1-6 was analyzed in the same manner as in Example 2(1). Solutions were prepared according to the conditions listed in Table 2 and stored for 4 weeks at freezing temperature (-20°C), refrigeration temperature (5°C), and high temperature (40°C), with potency tests performed every two weeks during this period. The temperature stability of hyaluronidases with different truncated sites was measured for 4 weeks, and the measured values ​​are shown in Table 4. Table 4 Enzyme activity ratio (%) freezing refrigeration high temperature sample Week 0 Week 2 Week 4 Week 0 Week 2 Week 4 Week 0 Week 2 Week 4 Example 1-1 100.22 81.47 62.51 100.54 90.89 82.55 98.13 65.09 51.55 Examples 1-2 101.98 100.74 98.85 101.52 100.81 99.45 98.96 88.92 78.54 Examples 1-3 102.68 100.88 99.22 102.77 101.91 100.23 99.47 89.90 79.63 Examples 1-4 101.07 100.17 98.03 100.32 100.72 99.18 98.16 88.11 77.95 Examples 1-5 100.12 98.82 95.52 100.53 98.70 97.51 99.39 85.31 75.78 Examples 1-6 99.49 96.92 93.12 99.64 96.32 95.63 99.87 82.69 73.12

[0080] As shown in Table 4, the hyaluronidases of Examples 1-2 to 1-6 exhibited greater temperature stability and retained significantly higher enzyme activity ratios (%), especially under frozen and refrigerated storage conditions. The amino acid DS hyaluronidase corresponding to Examples 1-3 was truncated by approximately 12%, showing the highest activity among recombinant hyaluronidases, and was named BMI2004 and used in the experiments.

[0081] Example 3. Stability analysis of wild-type hyaluronidase and BMI2004

[0082] (1) Compare enzyme activity or content based on pH

[0083] The pH stability of BMI2004 prepared in Example 1 and wild-type hyaluronidase was compared. The BMI2004 and wild-type hyaluronidase (manufacturer: BMI Korea, trade name: Hirax, hereinafter referred to as Hirax) used for pH stability testing were 95% or higher in purity and each had a concentration of 1,500 IU / mL (=100%). Solutions were prepared by water substitution according to the conditions in Table 5 and stored at 5°C for 4 weeks at pH 3.0, pH 5.0, pH 7.0, and pH 10.0, with potency tests performed every two weeks during this period. At each time point, the enzyme activity or content of the samples was measured in the same manner as in Example 2(1). Table 5 pH sample Storage temperature 3.0 BMI2004 / Hirax (1,500 IU / mL) 2-8℃ 5.0 2-8℃ 7.0 2-8℃ 10.0 2-8℃

[0084] The measurement results of pH stability analysis are shown in Table 6. The enzyme activity (in IU / mL) in each test is expressed as a percentage of the reference value of 1,500 IU / mL (=100%), which is the initial enzyme activity at week 0. The enzyme activity ratio was calculated according to the following equation:

[0085] Enzyme activity ratio (%) = (Enzyme activity at measurement time) / (Initial enzyme activity) * 100 Table 6 Enzyme activity ratio (%) pH 3.0 pH 5.0 pH 7.0 pH 10.0 sample Week 0 Week 2 Week 4 Week 0 Week 2 Week 4 Week 0 Week 2 Week 4 Week 0 Week 2 Week 4 BMI2004 102.66 85.48 55.26 101.80 95.02 81.19 101.49 96.22 82.55 102.68 89.95 65.11 Hirax 102.04 51.11 31.03 103.39 71.20 50.40 102.33 70.76 50.95 101.88 62.07 41.03

[0086] As shown in Table 6, the stability of BMI2004 and Hirax was observed over a period of approximately 4 weeks, depending on pH. Higher enzyme activity percentages (%) were measured at pH 5.0 and pH 7.0 than at pH 3.0 and pH 10.0. After the second week, the content (%) of Hirax tended to decrease rapidly compared to BMI2004. Values ​​exceeding 100% appear to be measurement errors, which generally arise from larger fluctuations in potency testing of biological products such as vaccines and recombinant proteins (cytokines, monoclonal antibodies, etc.). To further test changes in enzyme activity (or content) for confirmation of the stability of Hirax and BMI2004 at pH, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed under the conditions specified in Table 5 above and according to the following protocol: 1) Preparation of sample buffer (5X): Pierce™ Lane Marker Reducing Sample Buffer (Thermo Scientific™, catalog number 39000). 2) Preparation of operating buffer (1X): Novex™ Tris-Glycine SDS Running Buffer (10X) (Invitrogen, catalog number LC2675). Accurately measure 100 mL of this solution and place it in a 1,000 mL volumetric flask, then add diluent to the flask to bring the total volume to 1,000 mL. 3) Preparation of test solutions: Modify the Hirax and BMI2004 reference standards with water as needed. Accurately measure approximately 20 μL of each solution and 5 μL of sample buffer (5X) and mix them in an EP tube. 4) Manual: Perform the following analysis on the test solution and PageRuler Prestained Protein Ladder reference standard (Thermo, catalog number 26616). 4-1) After removing the comb, clean the Novex TMWedgewell™ 8~16% Tris-Glycine Gel (Invitrogen, catalog number XP08160BOX) or equivalent gel sheet with water to replace the buffer. 4-2) Mount the washed gel into a Mini Gel Tank. In the Mini Gel Tank, completely fill the cathode compartment with operating buffer (1X) while filling the anode compartment with operating buffer (1X) to approximately 2 / 3 full. 4-3) Load 7 μL of PageRuler Prestained Protein Ladder standard and 25 μL of test solution into the gel.4-4) Connect the microgel tank to the power supply and operate as follows, then allow the sample to reach 90% gel length: Volts: 140 V Amps: 400 mA Time: 60 minutes (time may be adjusted depending on operating conditions). 4-5) After operation, separate the gel from the mold and wash with water. 4-6) Immerse the washed gel in a dish containing staining reagent (Coomassie Brilliant Blue R-250 Staining Solution, BIO-RAD, catalog number 1610436). 4-7) Place the dish on a shaker and shake at 30 rpm for 30 minutes. 4-8) After staining, transfer the gel to a dish containing destaining solution (Coomassie Brilliant Blue R-250 Destaining Solution, BIO-RAD, catalog number 1610438), and place the dish on a shaker and shake at 30 rpm until the gel is destained, replacing the destaining solution as needed. When destaining has progressed to a certain extent, immerse the gel in water to remove the destaining solution. 4-9) Observe the gel under a white light lamp while removing the destaining solution.

[0087] The test results are depicted in Figures 1a to 1c. As shown in Figures 1a to 1c, it should be understood that, based on whether other bands are generated over time under the water displacement state as shown in the SDS-PAGE results, BMI2004 is more stable than Hirax for pH over time.

[0088] (2) Compare enzyme activity or content based on storage temperature

[0089] The temperature stability of BMI2004 and Hirax prepared in Example 1 was compared. The purity of BMI2004 and Hirax used for temperature stability testing was 95% or higher, and each had a concentration of 1,500 IU / mL (=100%). Solutions were prepared with water according to the conditions in Table 7 and stored at freezing temperature (-20°C), refrigeration temperature (5°C), and high temperature (40°C) for 4 weeks, with potency tests performed every two weeks during this period. Table 7 Storage temperature sample Store pH ≤-20℃ Hirax / BMI2004 (1,500 IU / mL) pH 5.0 2 to 8℃ pH 5.0 ≥40℃ pH 5.0

[0090] The temperature stability of Hirax and BMI2004 was measured over four weeks, and the measured values ​​are summarized in Table 8 and plotted in Figure 2. The highest content (%) was detected at freezing temperatures of -20°C or lower. After the second week, the content (%) of BMI2004 remained significantly higher than that of Hirax under all temperature conditions. Table 8 Enzyme activity ratio (%) freezing refrigeration high temperature sample Week 0 Week 2 Week 4 Week 0 Week 2 Week 4 Week 0 Week 2 Week 4 BMI2004 102.68 100.88 99.22 102.77 101.91 100.23 99.47 89.90 79.63 Hirax 102.76 80.24 59.75 101.42 91.51 82.83 102.19 66.40 40.15

[0091] In another stability analysis based on temperature, the changes in the content of Hirax and BMI2004 at different temperatures were measured in the same manner as in Example 3. For this purpose, SDS-PAGE was performed under the conditions in Table 7. The results of the SDS-PAGE are depicted in Figures 2a to 2c. As shown in Figures 2a to 2c, it should be understood that BMI2004 is more stable than Hirax due to the formation of other bands at different initial positions than the main band under aqueous conditions over time during freezing, refrigeration, and high temperatures.

[0092] Example 4. Optimal enzyme activity based on temperature

[0093] The disk potency analysis in this embodiment is designed based on the disk analysis method in the microbial analysis of antibiotics. Specifically, a Peni cylinder containing a predetermined amount of hyaluronidase is placed on a solid agarose disk containing hyaluronic acid to allow the hyaluronidase to diffuse within the agarose disk. Through this diffusion, the hyaluronic acid is degraded by the hyaluronidase, and the remaining undegraded hyaluronic acid is precipitated by cetylpyridinium chloride to form a clear halo. In this embodiment, the enzyme activity of hyaluronidase can therefore be determined by the size of the halo produced by using the disk in this simple method. Under various conditions, the disk potency analysis is performed as follows: 1) Preparation of hyaluronic acid solution: 0.2 g of hyaluronic acid is completely dissolved in 100 mL of water, and the pH is adjusted to 7.0 ± 0.1 using hydrochloric acid and sodium hydroxide. 2) Preparation of 1.5% agarose: 1.5 g of agarose (SIGMA, catalog number A9539) is added to 100 mL of water and dissolved using microwave. 3) Preparation of 10% hexadecylpyridinium chloride: Dissolve 10 g of hexadecylpyridinium chloride (SIGMA, catalog number C0732) in 100 mL of water. 4) Test solution: Adjust the Hirax and BMI2004 reference standards with water according to conditions. 5) Manual analysis: Perform the following analysis on the test solution. 5-1) Warm the hyaluronic acid solution at 37°C for approximately 20 minutes before mixing with 1.5% agarose. 5-2) After cooling 100 mL of 1.5% agarose to approximately 60°C, add 100 mL of hyaluronic acid solution and mix by stirring. 5-3) Pour the mixture into a petri dish (SPL, catalog number 10050) to a thickness of approximately 3 mm. 5-4) After the agarose gel has completely solidified, place a peni cylinder (KisanBio, catalog number KS-P0161) at a 90° angle on the concentric circles of the petri dish. Take 20 μL of the test solution and load it into a Peni cylinder, then incubate for 18 to 20 hours at the temperatures listed in the tables. 5-5) After the reaction is complete, remove the Peni cylinder and add 3 mL of 10% cetylpyridinium chloride. A clear halo is observed after 20 minutes. 5-6) The halo is measured precisely to a diameter (mm) of 0.5 mm or less.

[0094] (1)Confirmation of the activity of hyaluronic acid solution

[0095] To test whether the hyaluronic acid solution exhibited its own activity, potency tests were performed under various pH and temperature conditions before analyzing optimal enzyme activity. Tests were conducted at pH 4.0, pH 7.0, or pH 10.0, and at 20°C, 25°C, 30°C, 35°C, and 40°C. The test results are depicted in Figure 3. As shown in Figure 3, the hyaluronic acid solution was observed to lack its own activity.

[0096] (2) Comparison of enzyme activities under temperature conditions of 20 to 40℃

[0097] The purity of BMI2004 and Hirax used in this analysis of enzyme activity was 95% or higher, and each was adjusted with water to a concentration of 1,500 IU / mL (=100%). Solutions were prepared according to the conditions in Table 9 and titer analysis was performed at 20°C, 25°C, 30°C, 35°C, and 40°C. Table 9 Store pH Storage temperature sample Condition 1 pH 4.0 20 to 40℃ Hirax / BMI2004 (1,500 IU / mL) Condition 2 pH 7.0 20 to 40℃ Condition 3 pH 10.0 20 to 40℃

[0098] The analytical results are depicted in Figures 4a and 4b. The enzyme activities of Hirax and BMI2004 were examined at 20 to 40°C using plate titer analysis. Therefore, the halos were larger at 35°C and 40°C. To quantify enzyme activity, the diameters of the halos in Figures 4a and 4b were measured, and the measured values ​​are shown in Table 10 below. In Table 10, the diameter (mm) has an error deviation of ±0.5 mm. Table 10 temperature sample pH 4.0 pH 7.0 pH 10.0 20℃ Hirax 8 mm 10 mm 11 mm BMI2004 8 mm 12 mm 13 mm 25℃ Hirax 9 mm 10 mm 11 mm BMI2004 9 mm 13 mm 13 mm 30℃ Hirax 9 mm 13 mm 14 mm BMI2004 10 mm 13 mm 15 mm 35℃ Hirax 10 mm 15 mm 18 mm BMI2004 11 mm 18 mm 21 mm 40℃ Hirax 10 mm 15 mm 18 mm BMI2004 11 mm 18 mm 21 mm

[0099] As shown in Table 10, the comparison of enzyme activities at 20 to 40°C indicates that the activity of BMI2004 is higher than that of Hirax.

[0100] (3) Comparison of enzyme activities under temperature conditions of 35 to 40℃

[0101] Based on the results of the activity comparison between Hirax and BMI2004 measured in Example 4(2), additional tests were performed under specific temperature conditions. For this purpose, solutions were prepared according to the conditions listed in Table 11 below, and platelet potency analysis was performed at 35°C, 37°C, and 40°C. Table 11 Store pH Storage temperature sample Condition 1 pH 4.0 35℃ Hirax / BMI2004 (1,500 IU / mL) 37℃ 40℃ Condition 2 pH 7.0 35℃ 37℃ 40℃ Condition 3 pH 10.0 35℃ 37℃ 40℃

[0102] The test results are shown in Figures 5a and 5b. The enzyme activities of Hirax and BMI2004 were tested at 35°C, 37°C, and 40°C using plate titer analysis. Therefore, the halo was largest at 37°C. The diameter of the halo is shown in Table 12 below. The diameter (mm) has an error deviation of ±0.5 mm. Table 12 sample 35℃ 37℃ 40℃ Hirax BMI2004 Hirax BMI2004 Hirax BMI2004 pH 4.0 10 mm 12 mm 10 mm 13 mm 10 mm 12 mm pH 7.0 16 mm 19 mm 17 mm 20 mm 16 mm 19 mm pH 10.0 18 mm 21 mm 19 mm 22 mm 18 mm 21 mm

[0103] As shown in Table 12, the comparison of enzyme activities at 35°C, 37°C and 40°C indicates that the activity of BMI2004 is higher than that of Hirax.

[0104] Example 5. Optimal enzyme activity based on pH

[0105] The purity of Hirax and BMI2004 used in the analysis of optimal enzyme activity was 95% or higher, and each was adjusted with water to a concentration of 1,500 IU / mL (=100%). Solutions were prepared according to the conditions in Table 13. Enzyme activity was measured according to the analysis in Example 4. Based on the activity results measured at the temperature according to Example 4 (3), pH-based plate titer analysis was performed at 37°C, under which the maximum halo appeared. Table 13 Store pH Storage temperature sample Condition 3 pH 5.0 37 ℃ Hirax / BMI2004 (1,500 IU / mL) Condition 4 pH 6.0 Condition 5 pH 7.0 Condition 6 pH 8.0 Condition 7 pH 9.0 Condition 8 pH 10.0

[0106] The analytical results are depicted in Figures 6a and 6b. As a result of the disc potency analysis of the enzyme activities of Hirax and BMI2004 at 37°C under conditions of pH 5.0 to 10.0, the halos were particularly large at pH 7.0 to 10.0. A comparison of the halo diameters is shown in Table 14 below. Diameters (mm) have an error deviation of ±0.5 mm. Table 14 sample 37 ℃ Hirax BMI2004 pH 5.0 14 mm 16 mm pH 6.0 17 mm 18 mm pH 7.0 19 mm 20 mm pH 8.0 19 mm 20 mm pH 9.0 19 mm 20 mm pH 10.0 20 mm 21 mm

[0107] As can be seen in Table 14, the comparison of enzyme activities at 37°C under pH conditions from 5.0 to 10.0 indicates that the activity of BMI2004 is superior to that of Hirax at pH 5.0 or higher, and is particularly superior at pH 7.0 or higher.

[0108] Example 6. Analysis of the potency of hyaluronidase

[0109] The titers of hyaluronidase and the known enzyme Hirax according to a specific example of the present invention were measured in the same manner as in Example 2(1), and the measured values ​​are shown in Table 15. Table 15 Sample number BMI 2004 (IU / mg) Hirax (IU / mg) 1 124,497 81,396 2 133,870 81,528 3 134,678 80,653 4 122,433 80,538 average value 128,870 81,029

[0110] As shown in Table 15, BMI2004 exhibits an activity of approximately 122,433 to 134,678 IU per mg of protein, while Hirax exhibits an activity of approximately 80,538 to 81,528 IU per mg of protein. Therefore, even when the hyaluronidase according to a specific embodiment of the present invention is used in an amount of approximately 63% of the amount of conventional Hirax, equivalent activity can be obtained.

[0111] Example 7. The promoting effect of hyaluronidase on drug absorption and diffusion (1)

[0112] To determine the effect of hyaluronidase on promoting drug absorption and diffusion according to a specific embodiment of the present invention, trypan blue was used to analyze the drug absorption and diffusion promotion.

[0113] Specifically, Hirax and BMI2004 were each mixed with 0.2% trypan blue solution and injected subcutaneously once into Balb / c nude mice, and the diffusion of trypan blue in these mice was monitored. Based on the potency listed in Table 15, all drugs were prepared to have a concentration of 10 IU / mL or 100 IU / mL under the conditions in Table 16 and administered at a dose of 0.05 mL. At each time point, the diffusion area (π mm²) was measured. Table 16 Sample (IU / ml) Peptide dosage (ug / mL) 2.5 minutes 5 minutes 10 minutes 15 minutes 20 minutes brine 0 77.38± 15.86 84.61± 13.92 98.11± 7.83 117.67± 10.35 121.84± 11.48 Hirax (10 IU / mL) Approximately 0.123 74.45± 22.40 112.84± 15.01* 146.69± 29.36** 165.98± 18.49* 189.90± 41.85** Hirax (100 IU / mL) Approximately 1.234 84.25± 15.81 133.90± 14.52*** 149.27± 27.41** 184.80± 37.10** 215.12± 20.60*** BMI2004 (10 IU / mL) Approximately 0.078 82.70± 22.95 109.65± 12.70* 153.17± 15.30*** 176.85± 14.08** 187.63± 36.40** BMI2004 (100 IU / mL) Approximately 0.776 87.02± 17.51 126.70± 16.00*** 140.23± 16.95*** 180.98± 29.93*** 218.14± 26.85*** 1) Mean ± SD 2) *p < 0.05, **p < 0.01, ***p < 0.001, compared with the saline treatment group using SPSS (one-way ANOVA, LSD test)

[0114] As shown in Table 16, the diffusion area was measured from 2.5 to 20 minutes. Five minutes after administration, Hirax and BMI2004 significantly increased the diffusion area compared to the negative control group saline.

[0115] Example 8. The promoting effect of hyaluronidase on drug absorption and diffusion (2)

[0116] To determine the promoting effect of hyaluronidase on drug absorption and diffusion according to a specific embodiment of the present invention, an analysis of drug absorption and diffusion promotion was performed using a motor inertia model.

[0117] Specifically, the cynomolgus monkey was used as a model of motor incapacitation to test the ability to enhance drug penetration. The test materials were BMI2004 and Hirax listed in Table 15, while saline was used as a negative control group. Lidocaine and bupivacaine were used as anesthetics to be mixed with the samples. The samples were injected into 6 monkeys at a dose of 2 mL each via the right and left periorbital routes.

[0118] After administering the anesthetic along with the same sample, the time until pupillary movement ceases (time to reach immobility) and the time from pupillary anesthesia to the observation of pupillary movement (duration of immobility) are measured, and the measured values ​​are shown in Table 17. Table 17 sample Peptide dosage (ug / mL) Model (monkey) Eye Time (in minutes) at which exercise inability is reached Duration of inability to exercise (minutes) brine 0 02 Left No movement No movement is not possible 03 right No movement is not possible No movement is not possible 05 Left No movement is not possible No movement is not possible 06 right No movement No movement Hirax (500 IU / 0.2 mL) Approximately 6.17 μg / 0.2 mL 01 Left 4 27 02 right 4 27 04 Left 2 17 05 right 2 32 average value 3.0 25.8 SD 1.2 6.3 BMI2004 (500 IU / 0.2 mL) Approximately 3.88 μg / 0.2 mL 01 right 1 32 03 Left 2 twenty four 04 right 2 17 06 Left 1 41 average value 1.5 28.5 SD 0.6 10.3 1) Drugs; lidocaine 2% (0.9 mL), bupivacaine 0.5% (0.9 mL) and preparation materials (0.2 mL)

[0119] As shown in Table 17, the saline solution in the negative control group did not exhibit an akinetic effect. However, when administered together with an anesthetic, the peptide according to one specific embodiment of the present invention enhanced the penetration of the anesthetic and thus produced an akinetic effect equivalent to that of the known peptide Hirax, demonstrating its ability to induce drug absorption and diffusion. Furthermore, the peptide according to one specific embodiment of the present invention exhibited equivalent activity to the known Hirax at approximately 63% of the protein dose.

[0120] Example 9. Analysis of the ability of hyaluronidase to promote the reabsorption of excess body fluids

[0121] To confirm the promoting effect on the reabsorption of excess body fluid, an edema model was used. In this embodiment, the edema model was constructed by artificially inducing lymphedema. The test materials were BMI2004 and Hirax listed in Table 15, while saline was used as a negative control group. Samples and dosages are given in Table 18 below. Table 18 sample Lymphedema Dosage ( / site) way Animal number brine + - SC 6 Hirax + 100 IU (approximately 1.234 ug) SC 6 BMI2004 + 100 IU (approximately 0.776 ug) SC 6

[0122] Due to its accuracy and ease of use, lymphedema was induced in the tail. A 2 mm wide circumferential incision was made in the skin 1 cm from the base of the mouse tail. Approximately 4 mm² of the ventral side was left uncut. To test the efficacy in reducing edema, the tail diameter (mm) was measured at various time points from before the first administration (0 hours) to day 15 of induction, as listed in Table 19 below. A second administration was given after 24 hours, and a third administration after 48 hours. In this test, the tail diameter (mm) was measured using a caliper at a point 10 mm below the defect. Table 19 sample brine Hirax BMI2004 0 hours 4.55±0.43 4.53±0.49 4.53±0.38 1 hour 4.57±0.43 4.39±0.49 4.57±0.65 2 hours 4.48±0.36 4.45±0.40 4.50±0.37 4 hours 4.52±0.38 4.41±0.38 4.48±0.51 24 hours 4.56±0.34 4.57±0.39 4.55±0.45 25 hours 4.68±0.32 4.48±0.35 4.53±0.44 26 hours 4.56±0.32 4.43±0.38 4.45±0.47 28 hours 4.58±0.37 4.42±0.44 4.46±0.48 48 hours 4.65±0.39 4.49±0.41 4.47±0.38 49 hours 4.56±0.27 4.22±0.37 4.26±0.37 50 hours 4.58±0.25 4.13±0.38 4.09±0.36 52 hours 4.59±0.25 4.16±0.47 4.18±0.36

[0123] As shown in Table 19, administration of BMI2004 reduced lymphedema, demonstrating the protein's promoting effect on the reabsorption of excess body fluid. Furthermore, the polypeptide according to one specific example of the present invention exhibited activity equivalent to conventional Hirax at approximately 63% of the protein dose.

[0124] Example 10. Analysis of in vivo stability of hyaluronidase

[0125] To determine in vivo stability, hyaluronidase according to one specific example of the present invention was administered intravenously and its pharmacokinetics were monitored. In this embodiment, SD rats were used as test animals. Hirax and BMI2004 were administered to each animal via intravenous infusion over a total dose of approximately 180,000 IU over a period of 30 minutes. Blood samples were collected before administration, 15 minutes after the start of infusion (during infusion), 30 minutes after the start of infusion (end of infusion), and at 31, 33, 36, 40, 45, 60, 75, 90 minutes and 2.5 hours, 4.5 hours, 24.5 hours, and 48.5 hours after the start of infusion.

[0126] The pharmacokinetic variables of the polypeptide and Hirax according to one specific embodiment of the present invention are given in Table 20. The blood concentration of the polypeptide over time is depicted in Figure 7. As shown in Figure 7 and Table 20, Hirax degrades too rapidly to measure its in vivo half-life. In contrast, the polypeptide according to one specific embodiment of the present invention was observed to have a half-life of approximately 0.272 hours (approximately 16.3 minutes). Due to its longer half-life than Hirax, the polypeptide according to one specific embodiment of the present invention may exhibit greater efficacy than Hirax by remaining in the body for a longer period. Furthermore, they differ in terms of in vivo blood concentration. The blood concentration of Hirax peaks within 0.25 hours (15 minutes), but the blood concentration of BMI2004 peaks 0.5 hours (30 minutes) after administration. The polypeptide exhibits the potential to deliver greater efficacy than Hirax because its blood concentration increases more slowly and remains in vivo for a longer period than Hirax. Table 20 Test materials Dosage (IU / kg) t max (h) t 1 / 2 (h) CL (mL / h / kg) Hirax 180000 0.250 NC NC BMI2004 180000 0.500 0.272 418 [Simplified Explanation of the Diagram]

[0057] [Figure 1a] to [Figure 1c] are SDS-PAGE diagrams showing the pH stability of the polypeptide according to a specific example of the present invention compared with wild-type hyaluronidase (Figure 1a: Week 0 of storage, Figure 1b: Week 2 of storage, Figure 1c: Week 4 of storage).

[0058] [Figures 2a] to [Figures 2c] are SDS-PAGE diagrams showing the temperature stability of the peptide according to a specific example of the present invention compared with wild-type hyaluronidase (Figure 2a: Week 0 of storage, Figure 2b: Week 2 of storage, Figure 2c: Week 4 of storage; frozen: frozen, refrigerated: refrigerated, high temperature: high temperature).

[0059] [Figure 3] shows the results of the plate potency analysis under different pH and temperature conditions to verify whether the hyaluronic acid solution exhibits its own activity.

[0060] [Figure 4a] shows the results of titer analysis for wild-type hyaluronidase at temperatures ranging from 20 to 60 °C.

[0061] [Figure 4b] shows the results of potency analysis of a polypeptide according to a specific example of the present invention at temperatures ranging from 20 to 60°C.

[0062] [Figure 5a] shows the results of potency analysis of wild-type hyaluronidase at temperatures of 35 to 40°C.

[0063] [Figure 5b] shows the results of potency analysis of a polypeptide according to a specific example of the present invention at a temperature of 35 to 40°C.

[0064] [Figure 6a] shows the results of titer analysis for wild-type hyaluronidase under pH conditions from 5 to 7.

[0065] [Figure 6b] shows the results of potency analysis of a polypeptide according to a specific example of the present invention under pH conditions of 5 to 7.

[0066] [Figure 7] shows a graph of the blood content of the polypeptide and Hirax according to a specific example of the present invention in SD rats over time.

Claims

1. A polypeptide having hyaluronidase activity, wherein n consecutive amino acids are deleted from the C-terminus of the polypeptide consisting of the amino acid sequence of SEQ ID NO: 1, and the first amino acid is deleted from the N-terminus of the polypeptide consisting of the free amino acid sequence of SEQ ID NO: 1, wherein n is a natural number from 34 to 170.

2. The polypeptide as requested in item 1, where n is a natural number from 34 to 68.

3. The polypeptide as requested in claim 1, wherein n is a natural number of 34, 68, 102, 136 or 170.

4. The polypeptide of claim 1, wherein the polypeptide comprises an amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO:

5.

5. The polypeptide of claim 1, wherein the polypeptide consists of the amino acid sequence of SEQ ID NO:

3.

6. The polypeptide of claim 1, wherein the polypeptide is glycosylated.

7. A nucleic acid molecule encoding a polypeptide as claimed in any one of claims 1 to 6.

8. A vector comprising a nucleic acid molecule as claimed in claim 7.

9. A cell comprising the carrier as claimed in claim 8.

10. The cells in claim 9, wherein the cell line is selected from the group consisting of: animal cells, algal cells and plant cells.

11. The cells of claim 10, wherein the animal cell line is selected from the group consisting of mammalian cells and insect cells.

12. The cells of claim 11, wherein the mammalian cell line is selected from the group consisting of: CHO, NSO, HEK293, BHK, Per.C6, MDCK, Vero, MRC, HeLa, IMR and Sp2 / 0.

13. The cells of claim 9, wherein the cell line is selected from the group consisting of bacteria, yeast and fungi.

14. Use of a composition for manufacturing a drug delivery carrier for topical administration, wherein the composition comprises a polypeptide as claimed in any one of claims 1 to 6.

15. As claimed in claim 14, wherein the drug delivery carrier is adapted for subcutaneous or intramuscular administration.

16. A drug delivery carrier comprising a polypeptide as claimed in any one of claims 1 to 6.

17. Use of a composition for manufacturing a pharmaceutical product for the prevention or treatment of edema, wherein said composition comprises a polypeptide as claimed in any one of claims 1 to 6.

18. As claimed in claim 17, wherein the pharmaceutical product is intended for local administration.

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