Pharmaceutical formulation with improved stability comprising endoglycosidase hydrolase

Abstract

The present invention relates to a pharmaceutical formulation with improved stability, comprising: an endoglycosidase hydrolase; a phosphate-based buffer; a stabilizer comprising sodium chloride, sodium edetate, and calcium chloride; and a nonionic surfactant.

Description

Pharmaceutical formulations with improved stability containing endoglycosidase hydrolase

[0001] The present invention relates to a pharmaceutical formulation with improved stability comprising an endoglycosidase hydrolyzing enzyme.

[0002] Endoglycosidase hydrolase enzymes are a general term for enzymes that hydrolyze and cleave internal glycosidic bonds of polysaccharides, glycoproteins, or glycolipids.

[0003] Hyaluronidase (HDadase), a type of endoglycosidase hydrolytic enzyme, refers to an enzyme that breaks down hyaluronic acid (HA) into smaller molecules. Depending on the mechanism of hydrolysis of hyaluronic acid, hyaluronidase is classified into mammalian type hyaluronidase (EC 3.2.1.35, hyaluronoglucosaminidase), leech type hyaluronidase (EC 3.2.1.36, hyaluronoglucuronidase), and bacterial type hyaluronidase (EC 4.2.2.1, hyaluronate lyase).

[0004] Mammalian hyaluronidase is present in the testes, skin, liver, and placental fluids of the human body and is characterized by hydrolyzing the β-1,4 glycoside bonds between glucuronic acid and glucosamine, which are components of hyaluronic acid, to produce tetrasaccharides, or hydrolyzing chondroitin, chondroitin-4-sulfate, and chondroitin-6-sulfate, which are components of synovial fluid and cartilage in the joints of the body. In particular, hyaluronidase (PH-20) in the testes is attached to the glycosylphosphatidylinositol (GPI) anchor site of the acrosome of sperm and is an important enzyme that causes fertilization by breaking down the thick outer wall layer of the egg.

[0005] Since the 1950s, the extensive use of hyaluronidase has been comprehensively reviewed. Its initial use was the subcutaneous infusion of fluids, and it is also used in infiltration and block anesthesia to increase the diffusion of local anesthetics and steroids in surgeries in orthopedics, ophthalmology, plastic surgery, dentistry, oral surgery, gynecology, and otolaryngology; to disperse fluid accumulations such as hematomas; to prevent peritoneal adhesions; to prevent the formation of stones; and to treat infertility.

[0006] Currently available hyaluronidase is extracted from the testicles of sheep (Ovine) or cattle (Bovine). Examples include Vitrase (ITA Pharmaceuticals, Ovine source) and Amphadase (Amphadase, Amphastar Pharmaceuticals, Bovine source). These products are manufactured by dissolving unprocessed hyaluronidase at an appropriate concentration, filling vials, and freeze-drying them. However, commercialized animal-derived hyaluronidase contains foreign proteins, which can cause allergic reactions. Furthermore, the decline in stability over time leads to a decrease in biological activity, posing significant challenges for its application in various fields.

[0007] To address these issues, research on recombinant hyaluronidase has been conducted. Recombinant proteins can be expressed in various types of cells, including E. coli, yeast, insect cells, and animal cells. In particular, for hyaluronidase, glycosylation occurring during the post-translational modification process affects activity. This is because glycans can influence the antigenicity, structural folding, solubility, and stability of glycoproteins. From this perspective, animal cells are suitable among various expression cell types because the patterns of post-translational modification differ from those of mammals in yeast or insect cells, where glycosylation occurs; specifically, among animal cells, Chinese Hamster Ovary (CHO) cells, which have established safety, are the most suitable.

[0008] Accordingly, there is a need for various formulation studies to improve stability in pharmaceutical preparations containing endoglycosidase hydrolases.

[0009] The present invention aims to provide a pharmaceutical formulation with improved stability, comprising an endoglycosidase hydrolyzing enzyme; a phosphate-based buffer; a stabilizer comprising sodium chloride, sodium edetate and calcium chloride; and a nonionic surfactant.

[0010] However, the technical problems that the present invention aims to solve are not limited to those mentioned above, and other unmentioned problems will be clearly understood by those skilled in the art from the description below.

[0011] The present invention provides a pharmaceutical formulation with improved stability comprising an endoglycosidase hydrolyzing enzyme; a phosphate-based buffer; a stabilizer comprising sodium chloride, sodium edetate and calcium chloride; and a nonionic surfactant.

[0012] The above endoglycosidase hydrolyzing enzyme may be natural human recombinant hyaluronidase PH20 or a variant thereof.

[0013] The content of the sodium edetate may be 3 μg / mL to 300 μg / mL, and the content of the calcium chloride may be 1 μg / mL to 100 μg / mL.

[0014] The above pharmaceutical preparation may additionally contain 1 mg / mL to 30 mg / mL of sucrose or lactose sugar.

[0015] The above pharmaceutical preparation further comprises 0.1 mg / mL to 10 mg / mL of methionine, wherein one or more amino acids selected from the group consisting of arginine, glutamic acid, and histidine may be excluded.

[0016] The content of the above nonionic surfactant may be 0.01 mg / mL to 1 mg / mL.

[0017] The above pharmaceutical preparation may have a pH of 5.0 to 8.0.

[0018] The above pharmaceutical preparation may be a liquid preparation.

[0019] The activity of the above natural human recombinant hyaluronidase PH20 or a variant thereof may be 100 unit / mL to 10,000 unit / mL.

[0020] i) if, after storing the above pharmaceutical preparation under refrigeration (temperature of 2-8℃) conditions for 9 months, the change in activity of natural human recombinant hyaluronidase PH20 or its variant is within 20% by weight, or

[0021] ii) After storing the above pharmaceutical preparation for 6 months under conditions of room temperature (temperature of 23-27℃ and relative humidity of 55-65%), the change in activity of natural human recombinant hyaluronidase PH20 or a variant thereof may be within 20% by weight.

[0022] The above phosphate-based buffer may be a phosphate of 0.1 mg / mL to 10 mg / mL.

[0023] The content of the sodium chloride above may be 1 mg / mL to 20 mg / mL.

[0024] The pharmaceutical formulation with improved stability according to the present invention is characterized by comprising all of the following: an endoglycosidase hydrolyzing enzyme; a phosphate-based buffer; a stabilizer comprising sodium chloride, sodium edetate, and calcium chloride; and a nonionic surfactant, thereby having an excellent effect in suppressing the occurrence of foreign matter when prepared as a liquid formulation.

[0025] In particular, when the above sodium edetate and above calcium chloride are included in small amounts, and additionally the sugar of sucrose or lactose and the amino acid of methionine are included, the effect of suppressing foreign matter generation is further enhanced, and as a result of stability tests under long-term and accelerated conditions, the potency (activity), purity, and content can all meet the test standards without the generation of insoluble foreign matter for more than 6 months.

[0026] Figures 1(a) and (b) are graphs showing the results of analyzing foreign substances for formulations 1 to 9 in Example 1 under refrigerated and room temperature conditions, respectively, for 12 days.

[0027] FIG. 2(a) is a graph showing the results of analyzing foreign substances under refrigerated conditions for 7 days for formulations 8 and 10 to 17 in Example 2, and FIG. 2(b) is a graph showing the insoluble fine particles and their content after refrigerated storage for 7 days for formulation 1 (control group), formulation 10, and formulation 11 in Example 2.

[0028] FIG. 3(a) is a graph showing the results of analyzing foreign substances under refrigerated conditions for 21 days for formulations 10 and 18 to 21 in Example 3, and FIG. 3(b) is a graph showing the potency (activity), purity, and content after refrigerated storage for 21 days for formulation 1 (control group), formulation 10, and formulations 18 to 21 in Example 3.

[0029] FIG. 4(a) is a graph showing the results of analyzing foreign substances under refrigerated conditions for 10 days for formulations 10 and 22 to 24 in Example 4, and FIG. 4(b) is a graph showing the potency (activity), purity, and content after refrigerated storage for 10 days for formulations 10, 22, and 24 in Example 4.

[0030] FIG. 5(a) is a graph showing the change in potency (activity), change in purity, and change in content as a stability test for formulation 10 in Example 5 under long-term (refrigerated) and accelerated (room temperature) conditions for 6 months.

[0031] The inventors conducted research to improve stability in pharmaceutical formulations containing endoglycosidase hydrolyzing enzymes (particularly, natural human recombinant hyaluronidase PH20 or a variant thereof), and as a result, secured an optimal formulation and further enhanced the effect of suppressing foreign matter generation when manufacturing into a liquid formulation. Furthermore, based on stability tests under long-term and accelerated conditions, it was confirmed that potency (activity), purity, and content could all meet test standards without the generation of insoluble foreign matter for more than 6 months, thereby completing the present invention.

[0032]

[0033] The present invention will be described in detail below.

[0034]

[0035] Pharmaceutical formulations with improved stability containing endoglycosidase hydrolase

[0036]

[0037] The present invention provides a pharmaceutical formulation with improved stability comprising an endoglycosidase hydrolyzing enzyme; a phosphate-based buffer; a stabilizer comprising sodium chloride, sodium edetate and calcium chloride; and a nonionic surfactant.

[0038]

[0039] First, the pharmaceutical formulation according to the present invention comprises an endoglycosidase hydrolyzing enzyme.

[0040] The above-mentioned endoglycosidase hydrolyzing enzyme is a general term for enzymes that hydrolyze and cleave internal glycosidic bonds of polysaccharides, glycoproteins, or glycolipids, and in particular, may be an enzyme that hydrolyzes internal glycosidic bonds of glycosaminoglycans (GAGs), which are linear polysaccharides among polysaccharides.

[0041] Preferably, the endoglycosidase hydrolyzing enzyme may be natural human recombinant hyaluronidase PH20 or a variant thereof.

[0042] Specifically, regarding the above natural type human recombinant hyaluronidase PH20 or a variant thereof, a human-derived hyaluronidase PH20 known in the art may be used, wherein PH20 is preferably represented by the amino acid sequence of SEQ ID NO. 1, but is not limited thereto.

[0043] In particular, unlike its PH20 variant, the above-mentioned natural human recombinant hyaluronidase PH20 is an endogenous protein (enzyme) that has the same amino acid sequence and three-dimensional structure as the PH20 present in humans. Accordingly, it has the advantage of being safe when administered to the human body, as it is less likely to act as an antigen when injected externally.

[0044] Specifically, the activity of the natural human recombinant hyaluronidase PH20 or a variant thereof may be 100 unit / mL to 10,000 unit / mL, preferably 100 unit / mL to 5,000 unit / mL, more preferably 1,000 unit / mL to 3,000 unit / mL, and more preferably 1,000 unit / mL to 2,000 unit / mL, but is not limited thereto.

[0045]

[0046] Next, the pharmaceutical formulation according to the present invention comprises a phosphate-based buffer.

[0047] The above phosphate-based buffer maintains the pH of the pharmaceutical formulation according to the present invention at 5.0 to 8.0, thereby allowing the liquid formulation to be stored under stable conditions.

[0048] Specifically, the phosphate-based buffer may be a phosphate of 0.1 mg / mL to 10 mg / mL, preferably a phosphate of 1 mg / mL to 5 mg / mL, and more preferably a phosphate of 1 mg / mL to 3 mg / mL, but is not limited thereto.

[0049] When a buffer of a different class is used instead of the above-mentioned phosphate-based buffer, there are problems such as reduced stability with respect to pH or excessively high viscosity. In other words, when the pharmaceutical formulation according to the present invention is used for injection, it is unsuitable because there may be various effects such as the possibility of tissue damage at the injection site, pain, and improper distribution of the drug.

[0050]

[0051] Next, the pharmaceutical formulation according to the present invention comprises a stabilizer comprising sodium chloride, sodium edetate, and calcium chloride.

[0052] The above sodium chloride acts as a salt and plays a role in increasing ionic strength and stability.

[0053] Specifically, the content of the sodium chloride may be 1 mg / mL to 20 mg / mL, preferably 1 mg / mL to 10 mg / mL, and more preferably 5 mg / mL to 10 mg / mL, but is not limited thereto.

[0054] In addition, the combination of the sodium edetate and the calcium chloride is included, and in order to suppress the occurrence of unnecessary foreign matter, the content of the sodium edetate and the calcium chloride needs to be controlled to a small amount.

[0055] Specifically, the content of the sodium edetate may be 3 μg / mL to 300 μg / mL (preferably 3 μg / mL to 30 μg / mL, more preferably 6 μg / mL to 15 μg / mL), and the content of the calcium chloride may be 1 μg / mL to 100 μg / mL (preferably 1 μg / mL to 10 μg / mL, more preferably 2 μg / mL to 5 μg / mL).

[0056]

[0057] Optionally, the pharmaceutical formulation according to the present invention may further comprise 1 mg / mL to 30 mg / mL of sugar of sucrose or lactose.

[0058] The above sugar is intended to suppress osmotic stress and reduce viscosity, and is preferably sucrose, but is not limited thereto.

[0059] Specifically, the content of the sugar may be 1 mg / mL to 30 mg / mL, preferably 1 mg / mL to 25 mg / mL, and more preferably 1 mg / mL to 10 mg / mL, but is not limited thereto. In some cases, 0.5 mg / mL to 10 mg / mL of a stabilized protein such as albumin may be additionally included to replace the sugar, but there is a limitation in that the effect of inhibiting foreign matter formation is somewhat reduced.

[0060]

[0061] Optionally, the pharmaceutical formulation according to the present invention may further comprise 0.1 mg / mL to 10 mg / mL of methionine. On the other hand, it is preferable, but not limited to, that one or more amino acids selected from the group consisting of arginine, glutamic acid and histidine be excluded.

[0062] The above methionine is also intended to reduce viscosity and is effective among amino acids in suppressing the occurrence of foreign substances. At this time, other amino acids (arginine, glutamic acid, histidine, etc.) are not desirable for suppressing the occurrence of foreign substances, so they need to be excluded.

[0063] Specifically, the content of the methionine may be 0.1 mg / mL to 10 mg / mL, preferably 1 mg / mL to 5 mg / mL, and more preferably 1 mg / mL to 3 mg / mL, but is not limited thereto.

[0064]

[0065] Next, the pharmaceutical formulation according to the present invention comprises a nonionic surfactant.

[0066] The above nonionic surfactant is intended to prevent aggregation in a liquid formulation and may be a polysorbate-based material, preferably one or more selected from the group consisting of polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80, and more preferably polysorbate 80, but is not limited thereto.

[0067] Specifically, the content of the nonionic surfactant may be 0.01 mg / mL to 1 mg / mL, preferably 0.1 mg / mL to 1 mg / mL, and more preferably 0.1 mg / mL to 0.5 mg / mL, but is not limited thereto.

[0068]

[0069] Meanwhile, the pharmaceutical formulation according to the present invention may have a pH of 5.0 to 8.0, preferably 6.0 (±0.2) to 7.0 (±0.2), and most preferably 7.0 (±0.2), but is not limited thereto.

[0070] In addition, the pharmaceutical formulation according to the present invention is a liquid formulation and may be used for injection for administration selected from the group consisting of intravenous, subcutaneous, intramuscular, intradermal, and arterial. While such injection use has the advantage of rapidly administering an accurate amount to the human body to affect the entire body with good control, stability and safety must be considered as primary considerations for direct injection into the human body.

[0071] Alternatively, the present invention provides a method for injecting into an individual a pharmaceutical formulation with improved stability comprising an endoglycosidase hydrolase; a phosphate-based buffer; a stabilizer comprising sodium chloride, sodium edetate, and calcium chloride; and a nonionic surfactant. In this case, "individual" refers to a human or non-human primate; or a mammal such as a mouse, rat, dog, cat, horse, and cattle, and may be a subject requiring prevention or treatment of a disease. Additionally, "injection" includes administration selected from the group consisting of intravenous, subcutaneous, intramuscular, intradermal, and arterial.

[0072] A method for preventing or treating a disease can be provided by administering a pharmaceutical preparation according to the present invention by injection in combination with various known drugs, such as protein-based therapeutics, antibody-based therapeutics, nucleic acid-based therapeutics, small molecule-based therapeutics, and cell-based therapeutics.

[0073]

[0074] Thus, i) after storing the pharmaceutical preparation according to the present invention under refrigeration (at a temperature of 2-8°C) for 6 months, the change in activity of natural human recombinant hyaluronidase PH20 or a variant thereof may be within 20% by weight, and preferably within 15% by weight, but is not limited thereto.

[0075] In addition, ii) after storing the pharmaceutical preparation according to the present invention for 6 months under conditions of room temperature (temperature of 23-27℃ and relative humidity of 55-65%), the change in activity of natural human recombinant hyaluronidase PH20 or a variant thereof may also be within 20% by weight, and preferably within 15% by weight, but is not limited thereto.

[0076]

[0077] As reviewed above, the pharmaceutical formulation with improved stability according to the present invention is characterized by comprising all of the following: an endoglycosidase hydrolyzing enzyme; a phosphate-based buffer; a stabilizer comprising sodium chloride, sodium edetate, and calcium chloride; and a nonionic surfactant, thereby having an excellent effect in suppressing the occurrence of foreign matter when prepared as a liquid formulation.

[0078] In particular, when the above sodium edetate and above calcium chloride are included in small amounts, and additionally the sugar of sucrose or lactose and the amino acid of methionine are included, the effect of suppressing foreign matter generation is further enhanced, and as a result of stability tests under long-term and accelerated conditions, the potency (activity), purity, and content can all meet the test standards without the generation of insoluble foreign matter for more than 6 months.

[0079]

[0080] Preferred embodiments are presented below to aid in understanding the present invention. However, the following embodiments are provided merely to facilitate a better understanding of the invention, and the scope of the invention is not limited by the following embodiments.

[0081]

[0082] [Example]

[0083] Example 1: Stability Improvement Test by Optimizing Sodium Edetate and Calcium Chloride Contents

[0084] In order to solve the problem of reduced stability when liquefying natural human recombinant hyaluronidase PH20 (SEQ No. 1), a pharmaceutical formulation was prepared that essentially contains PH20, phosphate, sodium chloride, sodium edetate, and calcium chloride, while optionally containing sucrose / lactose, methionine, and polysorbate 80, as shown in Table 1 below.

[0085] Formulation Type 1 Formulation Type 2 Formulation Type 3 Formulation Type 4 Formulation Type 5 Formulation Type 6 Formulation Type 7 Formulation Type 8 9PH20 1,500 Unit / mL 1,500 Unit / mL 1,500 Unit / mL 1,500 Unit / mL 1,500 Unit / mL 1,500 Unit / mL 1,500 Unit / mL 1,500 Unit / mL 1,500 Unit / mL Phosphate 1.4 mg / mL 1.4 mg / mL 1.4 mg / mL 1.4 mg / mL 1.4 mg / mL 1.4 mg / mL 1.4 mg / mL 1.4 mg / mL - Sodium Chloride 8.5 mg / mL 8.5 mg / mL 8.5 mg / mL 8.5 mg / mL 8.5 mg / mL 8.5 mg / mL 8.5 mg / mL 8.5 mg / mL 9.0 mg / mL Sodium Edetate 0.9 mg / mL 0.9 mg / mL0.9 mg / mL9.9 ug / mL9.9 ug / mL9.9 ug / mL9.9 ug / mL9.9 ug / mL19 ug / mLCalcium chloride0.3 mg / mL0.3 mg / mL0.3 mg / mL3.3 ug / mL3.3 ug / mL3.3 ug / mL3.3 ug / mL3.3 ug / mL7 ug / mLSucrose25 mg / mL25 mg / mL25 mg / mL25 mg / mL25 mg / mL--25 mg / mL-Lactose--------13.3 mg / mLMethionine------1.5 mg / mL1.5 mg / mL-Polysorbate80-0.2 mg / mL0.4 mg / mL-0.2 mg / mL0.2 mg / mL0.2 mg / mL0.2 mg / mL0.1 mg / mLpH7.07.07.07.07.07.07.07.07.0

[0086]

[0087] For formulations 1 to 9, the presence of foreign matter was checked for 12 days under refrigerated (2-8°C) and room temperature (20-30°C) conditions, respectively. The presence of foreign matter was determined based on the insoluble foreign matter method of the European Pharmacopoeia; the insoluble foreign matter test stand was visually observed for 5 seconds against white and black backgrounds with an illuminance meter adjusted to a brightness of 2000-3750 lux, and the results are shown in Tables 2 and 3 and Figures 1(a)-(b).

[0088] As a result of checking for the occurrence of foreign matter under refrigeration (2-8℃) conditions for 12 days for formulations 1 to 9, as shown in Table 2 and Figure 1(a), almost no foreign matter occurred from immediately before manufacturing up to 5 hours, regardless of the formulation. However, in the case of formulations 1 to 3, a large amount of foreign matter occurred starting from the 1st day due to the excessive content of sodium edetate and calcium chloride. In addition, in the case of formulation 4, a large amount of foreign matter occurred after 12 days as polysorbate 80 was excluded, and in the case of formulations 6 and 7, a large amount of foreign matter occurred after 12 days / 8 days as sugars such as sucrose / lactose were excluded. That is, in the case of formulations 5, 8, and 9, the sodium edetate and calcium chloride content is small, and since they contain all of the sugars such as polysorbate 80 and sucrose / lactose, the effect of inhibiting foreign matter formation can be considered excellent.

[0089] Refrigerated Conditions (2-8℃) Formulation Type 1 Formulation Type 2 Formulation Type 3 Formulation Type 4 Formulation Type 5 Formulation Type 6 Formulation Type 7 Formulation Type 8 Formulation Type 9 Pre-manufacturing XXXXXXXXX 2 hours elapsed XXXXXXXXX 5 hours elapsed XXXXXXXXX 1 day elapsed ○○○△△△X△△ 4 days elapsed ○△○△△△△△X 8 days elapsed ○○△△X△○X△ 12 days elapsed ○○○○△○○△△

[0090] - ○ : Large amount of foreign matter, △ : Small amount of foreign matter, X : No or very small amount of foreign matter

[0091]

[0092] As a result of checking for the occurrence of foreign matter under room temperature (20-30℃) conditions for 12 days for formulations 1 to 9, as shown in Table 3 and Figure 1(b), almost no foreign matter occurred from immediately before manufacturing to 12 days regardless of the formulation.

[0093] Room Temperature Conditions (20-30℃) Formulation 1 Formulation 2 Formulation 3 Formulation 4 Formulation 5 Formulation 6 Formulation 7 Formulation 8 Formulation 9 Pre-manufacturing XXXXXXXXX 2 hours elapsed XXXXXXXXX 5 hours elapsed XXXXXXXXX 1 day elapsed XXXXXXXXX 4 days elapsed XXXXXXXXX 8 days elapsed XXXXXXXXX 12 days elapsed XXXXXXXXX

[0094] - ○ : Large amount of foreign matter, △ : Small amount of foreign matter, X : No or very small amount of foreign matter

[0095]

[0096] Example 2: Stability Improvement Test by Optimizing Sucrose Content and Excluding Other Amino Acids

[0097] In order to solve the problem of reduced stability when liquefying natural human recombinant hyaluronidase PH20 (SEQ No. 1), a pharmaceutical formulation was prepared as shown in Table 4 below, which essentially contains PH20, phosphate, sodium chloride, sodium edetate, and calcium chloride, while optionally containing sucrose / albumin, methionine / arginine / glutamic acid, and polysorbate 80.

[0098] Formulation 8 Formulation 10 Formulation 11 Formulation 12 Formulation 13 Formulation 14 Formulation 15 Formulation 16 Formulation 17PH20 1,500 Unit / mL 1,500 Unit / mL 1,500 Unit / mL 1,500 Unit / mL 1,500 Unit / mL 1,500 Unit / mL 1,500 Unit / mL 1,500 Unit / mL 1,500 Unit / mL Phosphate 1.4 mg / mL 1.4 mg / mL 1.4 mg / mL 1.4 mg / mL 1.4 mg / mL 1.4 mg / mL 1.4 mg / mL 1.4 mg / mL 1.4 mg / mL Sodium Chloride 8.5 mg / mL 8.5 mg / mL 8.5 mg / mL 8.5 mg / mL 8.5 mg / mL 8.5 mg / mL 8.5 mg / mL 8.5 mg / mL 8.5 mg / mL Sodium edetate9.9 ug / mL9.9 ug / mL9.9 ug / mL9.9 ug / mL9.9 ug / mL9.9 ug / mL9.9 ug / mL9.9 ug / mL9.9 ug / mLCalcium chloride3.3 ug / mL3.3 ug / mL3.3 ug / mL3.3 ug / mL3.3 ug / mL3.3 ug / mL3.3 ug / mL3.3 ug / mL3.3 ug / mLSucrose25 mg / mL5 mg / mL10 mg / mL15 mg / mL5 mg / mL10 mg / mL5 mg / mL10 mg / mL-Lactose---------Albumin--------1 mg / mLMethionine1.5 mg / mL1.5 mg / mL1.5 mg / mL1.5 mg / mL1.5 mg / mL1.5 mg / mL1.5 mg / mL1.5 mg / mL 1.5 mg / mL Arginine----5.2 mg / mL 5.2 mg / mL---Glutamic Acid-------4.4 mg / mL 4.4 mg / mL-Polysorbate 800.2 mg / mL 0.2 mg / mL 0.2 mg / mL 0.2 mg / mL 0.2 mg / mL 0.2 mg / mL 0.2 mg / mL 0.2 mg / mL pH 7.07.07.07.07.07.07.07.07.0

[0099]

[0100] The occurrence of foreign matter was checked for 7 days under refrigeration (2-8℃) conditions for formulations 8 and 10 to 17, and the results are shown in Table 5 and Figure 2(a).

[0101] As a result of checking for the occurrence of foreign matter under refrigeration (2-8°C) conditions for 7 days for formulations 8 and 10 to 17, as shown in Table 5 and Figure 2(a), it was confirmed that formulations 13 to 16 were not effective in suppressing the occurrence of foreign matter because they additionally contained arginine or glutamic acid. In addition, formulation 17 showed that sucrose could be replaced with albumin, but the effect of suppressing the occurrence of foreign matter was seen to be somewhat reduced. That is, for formulations 8 and 10 to 12 (particularly formulations 10 and 11), the sucrose content was optimized to 5 mg / mL to 25 mg / mL (particularly 5 mg / mL to 10 mg / mL), and since only methionine was additionally included while excluding other amino acids (arginine, glutamic acid), the effect of suppressing the occurrence of foreign matter can be seen as excellent.

[0102] Refrigerated Conditions (2-8℃) Formulation 8 Formulation 10 Formulation 11 Formulation 12 Formulation 13 Formulation 14 Formulation 15 Formulation 16 Formulation 17 Before Manufacturing XXXXXXXXXX 1 Day Elapsed △XX△X△△△△ 2 Days Elapsed △XX△△△○△△ 5 Days Elapsed XXX△△△△△△ 7 Days Elapsed XXXX△△△△X

[0103] - ○ : Large amount of foreign matter, △ : Small amount of foreign matter, X : No or very small amount of foreign matter

[0104]

[0105] Meanwhile, the insoluble particulates and their content were checked for Formulation 1 (control group), Formulation 8, Formulation 10, and Formulation 11 after refrigerated storage for 7 days. Insoluble particulates were analyzed using an insoluble particulate analyzer based on the Korean Pharmacopoeia, and the content was analyzed using HPLC, our company's analytical method. The results are shown in Table 6 and Figure 2(b).

[0106] As shown in Table 6 and Figure 2(b), the amount of insoluble microparticles was lowest in Formulation 10, and the content analysis also confirmed levels similar to the expected values. In other words, in the case of Formulation 10, the sucrose content was optimized to 5 mg / mL, and as other amino acids (arginine, glutamic acid) were excluded and only methionine was additionally included, it can be seen that the effect of suppressing foreign matter generation is particularly excellent.

[0107] Analytical Formulation 1 (Control) Formulation 8 Formulation 10 Formulation 11 Insoluble Particles ≥ 10 µm (Particles) 37 4 2 2 5 6 4 25 µm (Particles) ≥ 3 2 11 Content (HPLC Analysis) Area 2 8 8 6 7 3 5 4 5 1 8 9 5 1 4 4 2 9 8 2 4 4 2 0 7 4 Content (ug / mL) 12.4 19.1 18.4 18.3 % Compared to Initial 73% 113% 109% 109%

[0108]

[0109] Example 3: Stability Improvement Test by Excluding Other Amino Acids

[0110] In order to solve the problem of reduced stability when liquefying natural human recombinant hyaluronidase PH20 (SEQ No. 1), a pharmaceutical formulation was prepared that essentially contains PH20, phosphate, sodium chloride, sodium edetate, calcium chloride, sucrose, and polysorbate 80, while optionally containing methionine / histidine, as shown in Table 7 below.

[0111] Formulation 10 Formulation 18 Formulation 19 Formulation 20 Formulation 21PH20 1,500 Unit / mL 1,500 Unit / mL 1,500 Unit / mL 1,500 Unit / mL 1,500 Unit / mL Phosphate 1.4 mg / mL 1.4 mg / mL 1.4 mg / mL 1.4 mg / mL 1.4 mg / mL Sodium Chloride 8.5 mg / mL 8.5 mg / mL 8.5 mg / mL 8.5 mg / mL 8.5 mg / mL Sodium Edetate 9.9 ug / mL 9.9 ug / mL 9.9 ug / mL 9.9 ug / mL 9.9 ug / mL Calcium Chloride 3.3 ug / mL 3.3 ug / mL 3.3 ug / mL 3.3 ug / mL 3.3 ug / mL 3.3 ug / mL Sucrose 5 mg / mL 5 mg / mL 10 mg / mL 5 mg / mL 10 mg / mL Methionine 1.5 mg / mL 1.5 mg / mL 1.5 mg / mL -- Histidine 1.6 mg / mL 1.6 mg / mL 1.6 mg / mL 1.6 mg / mL Polysorbate 800.2 mg / mL 0.2 mg / mL 0.2 mg / mL 0.2 mg / mL 0.2 mg / mL pH 7.07.07.07.07.0

[0112]

[0113] As a result of checking for the occurrence of foreign matter under refrigeration (2-8℃) conditions for 21 days for formulations 10 and 18 to 21, as shown in Table 8 and Figure 3(a), in the case of formulation 10, the effect of suppressing foreign matter occurrence can be seen as excellent as it additionally includes only methionine while excluding other amino acids (histidine).

[0114] Refrigerated Conditions (2-8℃) Formulation 10 Formulation 18 Formulation 19 Formulation 20 Formulation 21 Before Manufacturing XXXXX 2 Days Elapsed X△X△△ 4 Days Elapsed X○X○○ 7 Days Elapsed X○○○○ 10 Days Elapsed △○○○○ 14 Days Elapsed △○○○○ 21 Days Elapsed △△○○○

[0115] - ○ : Large amount of foreign matter, △ : Small amount of foreign matter, X : No or very small amount of foreign matter

[0116]

[0117] Meanwhile, the potency (activity), purity, and content of formulation 1 (control group), formulation 10, and formulations 18 to 21 were checked after refrigerated storage for 21 days, and the results are shown in Table 9 and Figure 3(b).

[0118] As shown in Table 9 and Figure 3(b), it was confirmed that stability was ensured for all formulations 10 and 18 to 21, with potency (active) (103-113%), purity (99%), and content (118-119%) meeting the standards.

[0119] Analytical Formulation 1 (Control) Formulation 10 Formulation 18 Formulation 19 Formulation 20 Formulation 21 Potency (Activity) Potency (unit / mL) 1,127 1,538 1,699 1,645 1,672 1,699 % Compared to Initial 72% 103% 113% 110% 111% 113% Purity (HPLC Analysis) 100% 99% 99% 99% 99% 99% Content (HPLC Analysis) Content (ug / mL) 12.4 18.1 18.0 17.9 18.1 18.0 % Compared to Initial 73% 118% 118% 118% 119% 118%

[0120]

[0121] Example 4: Test of stability improvement through optimization of polysorbate 80 content and pH adjustment

[0122] In order to solve the problem of reduced stability when liquefying natural human recombinant hyaluronidase PH20 (SEQ No. 1), a pharmaceutical formulation was prepared by optimizing the polysorbate 80 content and adjusting the pH, as shown in Table 10 below, which essentially includes PH20, phosphate, sodium chloride, sodium edetate, calcium chloride, sucrose, methionine, and polysorbate 80.

[0123] Formulation 10 Formulation 22 Formulation 23 Formulation 24PH20 1,500 Unit / mL 1,500 Unit / mL 1,500 Unit / mL 1,500 Unit / mL Phosphate 1.4 mg / mL 1.4 mg / mL 1.4 mg / mL 1.4 mg / mL Sodium Chloride 8.5 mg / mL 8.5 mg / mL 8.5 mg / mL 8.5 mg / mL Sodium Edetate 9.9 ug / mL 9.9 ug / mL 9.9 ug / mL 9.9 ug / mL Calcium Chloride 3.3 ug / mL 3.3 ug / mL 3.3 ug / mL 3.3 ug / mL Sucrose 5 mg / mL 5 mg / mL 5 mg / mL 5 mg / mL Methionine 1.5 mg / mL 1.5 mg / mL 1.5 mg / mL 1.5 mg / mL Polysorbate 800.2 mg / mL 0.2 mg / mL 0.8 mg / mL 0.8 mg / mL pH 7.06.07.06.0

[0124]

[0125] As a result of checking for the occurrence of foreign matter under refrigeration (2-8℃) conditions for 12 days for formulations 10 and 22 to 24, as shown in Table 11 and Fig. 4(a), in the case of formulation 10, by maintaining the content of polysorbate 80 at 0.01 mg / mL to 0.5 mg / mL and maintaining the pH at 7.0, almost no foreign matter occurred until 7 days had passed, and only a small amount of foreign matter occurred even after 10 days, so it can be considered that the effect of suppressing foreign matter occurrence is excellent.

[0126] Refrigeration Conditions (20-30℃) Formulation 10 Formulation 22 Formulation 23 Formulation 24 Pre-manufacturing XXXX 2 days elapsed X△X△ 7 days elapsed X○○○ 10 days elapsed △○○○

[0127] - ○ : Large amount of foreign matter, △ : Small amount of foreign matter, X : No or very small amount of foreign matter

[0128]

[0129] Meanwhile, the potency (activity), purity, and content of formulations 10, 22, and 24 were checked after refrigerated storage for 10 days, and the results are shown in Table 12 and Figure 4(b).

[0130] As shown in Table 12 and Figure 4(b), all formulations 10, 22, and 24 were confirmed to have stability with potency (active) (108-112%), purity (100%), and content (118-119%) meeting the standards.

[0131] Analytical Formulation 10 Formulation 22 Formulation 24 Potency (Active) Potency (unit / mL) 1,657 1,627 1,673 % compared to initial 110% 108% 112% Purity (HPLC Analysis) 100% 100% 100% Content (HPLC Analysis) Content (ug / mL) 18.1 17.9 18.2 % compared to initial 119% 118% 119%

[0132]

[0133] Example 5: Long-term / accelerated stability test for Formulation 10

[0134] Long-term and accelerated stability tests were performed on 10,000 vials of Formulation 10 manufactured at a GMP facility. Long-term stability was performed under refrigerated conditions (2-8°C, no humidity conditions), and accelerated stability was performed under room temperature conditions (23-27°C, 55-65% RH). The long-term and accelerated stability tests were conducted at cycles of 1, 3, and 6 months, and insoluble foreign matter, potency (activity), purity, and content were analyzed as test items. The results are shown in Table 13.

[0135] Long-term Stability (2-8℃) pH Insoluble Foreign Matter Insoluble Particulates Potency (Activity) Purity (HPLC) Content (HPLC) ≥ 10 um ≥ 25 um 0 months 7.0 No foreign matter 1,441 7 1,681 Unit / mL 99% 15.4 ug / mL 1 month 7.1 No foreign matter 1,647 9 1,698 Unit / mL 100% 15.5 ug / mL 3 months 7.0 No foreign matter 1,395 8 1,601 Unit / mL 100% 17.0 ug / mL 6 months 7.0 No foreign matter 277 5 1,629 Unit / mL 99% 16.1 ug / mL Accelerated Stability (23-27℃ 55-65% RH) pH Insoluble Foreign Matter Insoluble Particulates Potency (Activity) Purity (HPLC) Content (HPLC) ≥ 10 um ≥ 25 0 months 7.0 No foreign matter 1,441 71,681 Unit / mL 99% 15.4 ug / mL 1 month 7.1 No foreign matter 1,416 91,736 Unit / mL 100% 15.6 ug / mL 3 months 7.0 No foreign matter 777 41,659 Unit / mL 100% 16.8 ug / mL 6 months 7.0 No foreign matter 281 31,641 Unit / mL 99% 15.9 ug / mL

[0136]

[0137] As shown in Table 13, Formulation 10 was confirmed to meet the test standards for insoluble foreign matter, potency (activity), purity, and content for 6 months under both refrigerated and room temperature conditions. In other words, Formulation 10 can be seen as having further enhanced the effect of suppressing foreign matter generation, and as a result of stability tests under long-term and accelerated conditions, it met the test standards for potency (activity), purity, and content without the generation of insoluble foreign matter for more than 6 months.

[0138]

[0139] The foregoing description of the present invention is for illustrative purposes only, and those skilled in the art will understand that other specific forms can be easily modified without altering the technical spirit or essential features of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims

1. Endoglycosidase hydrolyzing enzyme; Phosphate-based buffer; A stabilizer comprising sodium chloride, sodium edetate, and calcium chloride; and A pharmaceutical formulation with improved stability containing a nonionic surfactant.

2. In Paragraph 1, A pharmaceutical preparation characterized in that the above-mentioned endoglycosidase hydrolyzing enzyme is natural human recombinant hyaluronidase PH20 or a variant thereof.

3. In Paragraph 1, A pharmaceutical preparation characterized in that the content of the sodium edetate is 3 μg / mL to 300 μg / mL and the content of the calcium chloride is 1 μg / mL to 100 μg / mL.

4. In Paragraph 1, The above pharmaceutical preparation is characterized by further comprising 1 mg / mL to 30 mg / mL of sucrose or lactose sugar.

5. In Paragraph 1, The above pharmaceutical preparation is characterized by further comprising 0.1 mg / mL to 10 mg / mL of methionine, wherein one or more amino acids selected from the group consisting of arginine, glutamic acid, and histidine are excluded.

6. In Paragraph 1, A pharmaceutical preparation characterized in that the content of the above-mentioned nonionic surfactant is 0.01 mg / mL to 1 mg / mL.

7. In Paragraph 1, The above pharmaceutical preparation is characterized by having a pH of 5.0 to 8.

0.

8. In Paragraph 1, A pharmaceutical preparation characterized in that the above pharmaceutical preparation is a liquid preparation.

9. In Paragraph 2, A pharmaceutical preparation characterized in that the activity of the above-mentioned natural human recombinant hyaluronidase PH20 or a variant thereof is 100 unit / mL to 10,000 unit / mL.

10. In Paragraph 2, i) if, after storing the above pharmaceutical preparation under refrigeration (temperature of 2-8℃) conditions for 6 months, the change in activity of natural human recombinant hyaluronidase PH20 or its variant is within 20% by weight, or ii) A pharmaceutical preparation characterized in that, after storing the above pharmaceutical preparation for 6 months under conditions of room temperature (temperature of 23-27℃ and relative humidity of 55-65%), the change in activity of natural human recombinant hyaluronidase PH20 or a variant thereof is within 20% by weight.

11. In Paragraph 1, A pharmaceutical preparation characterized in that the above-mentioned phosphate-based buffer is a phosphate of 0.1 mg / mL to 10 mg / mL.

12. In Paragraph 1, A pharmaceutical preparation characterized by having a sodium chloride content of 1 mg / mL to 20 mg / mL.

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