Method for removing truncated hyaluronidase

Abstract

The present invention relates to: a hyaluronidase purification method for removing truncated hyaluronidase; a method for preparing hyaluronidase using same; and hyaluronidase or a variant thereof obtained by using the method. When hyaluronidase is purified by using the method according to an aspect, truncated forms of hyaluronidase can be effectively removed, and thus the purity and / or yield of hyaluronidase in an intact form is excellent, and the specific activity of purified or produced hyaluronidase is also remarkably excellent, and thus the method can be applied in processes for preparing and / or purifying hyaluronidase.

Description

Method for removing cleaved hyaluronidase

[0001] The invention relates to a method for purifying hyaluronidase to remove cleaved hyaluronidase, a method for producing hyaluronidase using the same, and hyaluronidase or a variant thereof obtained using the said method.

[0002] Hyaluronidase (HDadase) is a general term for enzymes that break down hyaluronic acid (HA) into small molecules. Depending on the mechanism of hydrolysis of hyaluronic acid, it is classified into mammalian type hyaluronidase (Mammalian type, EC 3.2.1.35, hyaluronoglucosaminidase), leech type hyaluronidase (Leeches type, EC 3.2.1.36, hyaluronoglucuronidase), and bacterial type hyaluronidase (Bacterial type, EC 4.2.2.1, hyaluronate lyase).

[0003] In particular, mammalian hyaluronidase is present in the testes, skin, liver, placental fluid, etc., of the human body and is characterized by hydrolyzing the β-1,4 glycoside bond 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 our body.

[0004] 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.

[0005] Conventional hyaluronidase purification methods involve complex and numerous processes, making commercial use difficult. Furthermore, hyaluronidase obtained by conventional methods suffers from low purity, leading to numerous problems such as animal-derived allergies, anaphylactic reactions, and mad cow disease infection. Additionally, there is a problem with the production of hyaluronidase in a fragmented form rather than an intact one.

[0006] Accordingly, the development of technology to effectively remove cleaved hyaluronidase is required.

[0007] One aspect provides a method for purifying hyaluronidase, comprising the step of A) applying a sample containing hyaluronidase to a first chromatography.

[0008] Another aspect provides a method for producing hyaluronidase comprising the above method.

[0009] Another aspect provides a method for improving the removal of impurities in a hyaluronidase purification process, comprising the above method.

[0010] Another aspect provides a hyaluronidase or a variant thereof characterized by having a content of 10% or less of the hydrolyzed form of hyaluronidase.

[0011] All technical terms used herein shall be used in the sense generally understood by those skilled in the art in the relevant field of the present invention, unless otherwise defined. Furthermore, while preferred methods or samples are described herein, similar or equivalents are also included within the scope of the present invention. Additionally, numerical values ​​described herein are deemed to include the meaning of "approximately" unless explicitly stated otherwise. The contents of all publications cited as references in this specification are incorporated into this specification in their entirety by reference.

[0012] In this specification, the terms “about” or “approximately” may be interpreted to mean a value or range within 10%, 5%, 4%, 3%, 2%, or 1% above or below a given value or range.

[0013]

[0014] One aspect provides a method for purifying hyaluronidase, comprising the step of A) applying a sample containing hyaluronidase to a first chromatography.

[0015] In this specification, the term "hyaluronidase (HDadase)" refers to an enzyme that breaks down hyaluronic acid (HA) into small molecules. According to the mechanism of hydrolysis of hyaluronic acid, the hyaluronidase may be classified into mammalian type hyaluronidase (Mammalian type, EC 3.2.1.35, hyaluronoglucosaminidase), leech type hyaluronidase (Leeches type, EC 3.2.1.36, hyaluronoglucuronidase), and bacterial type hyaluronidase (Bacterial type, EC 4.2.2.1, hyaluronate lyase).

[0016] The above hyaluronidase may include hyaluronidase or a variant thereof.

[0017] In this specification, the term "hyaluronidase variant" may also be interpreted to include hyaluronidase in which the amino acid sequence is modified (deleted, inserted, and / or substituted) and / or the glycosylation properties are modified, to the extent that polypeptide function is not affected.

[0018] The purity of the above hyaluronidase or its variant may be 98% or more, 98.5% or more, 99% or more, or 100%.

[0019] The above hyaluronidase or its variant means an intact form, and may include an intact form from which the hydrolyzed form has been removed.

[0020] The above hyaluronidase may be derived from a mammal, or may be a recombinant hyaluronidase expressed by introducing the mammalian-derived hyaluronidase into a microorganism, animal cell, or plant cell.

[0021] In one embodiment, the hyaluronidase may be human hyaluronidase. Humans have six types of hyaluronidase genes, including HyalPS1 (pseudogene), Hyal1, Hyal2, Hyal3, Hyal4, and PH20 / SPAM1. As used herein, the term includes "acid-active" enzymes (e.g., HYAL1) and "neutral-active" enzymes (e.g., PH20).

[0022] In one embodiment, the hyaluronidase may include a precursor hyaluronidase polypeptide and a mature hyaluronidase polypeptide (e.g., with the signal sequence removed), and a form having activity in a truncated form (e.g., a C-terminal truncated form). In one embodiment, the hyaluronidase may include an enzyme that includes or does not include a glycosylphosphatidylinositol (GPI) anchor. In humans, Hyal1 and Hyal2 are expressed in most tissues, and PH20 / SPAM1 (hereinafter PH20) is expressed in the cell membrane and acrosome membrane of sperm. Human PH20 (also referred to as SPAM1) is generally attached to the cell membrane via a glycosylphosphatidylinositol (GPI) anchor. This naturally participates in sperm-egg conjugation and aids in sperm penetration into the cumulus cell layer by degrading hyaluronic acid. In one embodiment, the hyaluronidase may be soluble or may not contain a GPI anchor. For example, the hyaluronidase may contain a C-terminal truncation of one or more amino acid residues to remove all or part of the GPI anchor. In some embodiments, the hyaluronidase may be a hyaluronidase active at a neutral pH. In some embodiments, the hyaluronidase may be a soluble hyaluronidase active at a neutral pH.

[0023] In one embodiment, the hyaluronidase may be recombinant human PH20. The “recombinant human PH20” (abbreviated as “rHuPH20”) means a soluble, neutral pH-active enzyme comprising a truncated human PH20 amino acid sequence. As used herein, the rHuPH20 may be available under CAS registration number 757971-58-7, or commercially available rHuPH20 (HYLENEX®) from Halozyme Therapeutics Inc., or may comprise the same amino acid sequence.

[0024] The first chromatography above may utilize a column comprising a material having a cation exchange function, and specifically, may utilize a column comprising a material having a cation exchange function or a column comprising a material having a cation exchange function and a hydrophobic interaction function.

[0025] In one embodiment, the first chromatography may include cation exchange chromatography (CEX) or mixed-mode chromatography (MMC), and specifically, may include mixed-mode chromatography.

[0026] The term "ion exchange chromatography" in this specification refers to a chromatographic method using an "ion exchange chromatography material." An "ion exchange chromatography material" refers to a fixed high molecular weight solid phase having a covalently bonded charged group as a chromatographic functional group. For total charge neutrality, a non-covalently bonded counterion is bonded to it. An "ion exchange chromatography material" has the ability to exchange similarly charged ions of the surrounding solution with its non-covalently bonded counterion. Depending on the charge of its exchangeable counterion, an "ion exchange chromatography material" is referred to as a "cation exchange chromatography material" or an "anion exchange chromatography material."

[0027] Additionally, depending on the nature of the charged group, "ion exchange chromatography materials" are referred to as cation exchange chromatography materials when they have, for example, a sulfonic acid group (S) or a carboxymethyl group (CM). Depending on the chemical nature of the charged group, "ion exchange chromatography materials" may be further classified into strong or weak ion exchange chromatography materials according to the strength of the covalently bonded charged substituent. For example, strong cation exchange chromatography materials have a sulfonic acid group as a chromatographic functional group, and weak cation exchange chromatography materials have a carboxylic acid group as a chromatographic functional group.

[0028] The above cation exchange chromatography may utilize a column containing a cation exchange chromatography material.

[0029] The above cation exchange chromatography material may include one or more selected from the group consisting of SP Sepharose TM Fast Flow, Sepharose High Performance SP, Sepharose XL, Sepharose TM HT, SOURCE TM 15S, SOURCE TM 30S, RESOURCE TM S, Mono S TM, CM Sepharose Fast Flow, Mini S, SP Sepharose Big Beads, Capto S, Eshmuno CPX, Capto SP ImpRes, Fractogel SO3- (M), etc.

[0030] The above cation exchange chromatography may utilize a column containing Fractogel SO3- (M), and specifically, may contain Fractogel SO3- (M) as the resin of the column.

[0031] The term "Mixed-mode chromatography (MMC)" in this specification refers to a method of separating and purifying analytes by simultaneously using two or more different interactions (e.g., ion exchange + hydrophobic interaction) in a single column / resin, and is also called "multimodal chromatography."

[0032] The above multi-mode chromatography may utilize a column comprising a material having a cation exchange function, and specifically, may utilize a column comprising a material having a cation exchange function and a hydrophobic interaction function.

[0033] The above multi-mode chromatography material may include one or more selected from the group consisting of Capto adhere, Capto MMC, MEP HyperCell, and Eshmuno HCX, etc.

[0034] The above multi-mode chromatography may utilize a column containing Eshmuno HCX, and specifically, may contain Eshmuno HCX as the resin of the column.

[0035] In one embodiment, applying a sample containing hyaluronidase to a first chromatography may include: A-1) loading a sample containing hyaluronidase into a first chromatography column; and A-2) eluting the loaded sample from the column using an elution buffer.

[0036] The first chromatography column may be a column equilibrated with an equilibration buffer, specifically, a column equilibrated using the equilibration buffer before loading a sample containing hyaluronidase. Accordingly, the method may additionally include the step of equilibrating the first chromatography column with the equilibration buffer before loading the sample containing hyaluronidase of step 'A-1)'.

[0037] The term "equilibration buffer" as used herein is a buffer used to equilibrate a column or resin before loading a sample containing a target protein onto the column.

[0038] The above equilibrium buffer may comprise one or more compounds or salts selected from the group consisting of sodium phosphate, sodium chloride, sodium acetate, sodium citrate, sodium carbonate, sodium sulfate, Tris, Tris-hydrogen chloride (Tris-HCl), ammonium sulfate (AMS), MES (2-(N-morpholino)ethanesulfonic acid), MOPS (3-morpholinopropane-1-sulfonic acid), PIPES, potassium sulfate, potassium phosphate, potassium chloride, and HEPES (2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid).

[0039] The equilibrium buffer may comprise about 10 mM to about 500 mM of salt, specifically about 10 mM to about 500 mM, about 10 mM to about 400 mM, about 10 mM to about 350 mM, about 10 mM to about 300 mM, about 10 mM to about 250 mM, about 10 mM to about 200 mM, about 10 mM to about 150 mM, about 10 mM to about 130 mM, about 10 mM to about 120 mM, about 10 mM to about 110 mM, about 10 mM to about 100 mM, about 30 mM to about 500 mM, about 30 mM to about 400 mM, about 30 mM to about 350 mM, about 30 mM to about 300 mM, about 30 mM to about 250 mM, about 30 mM to about 200 mM, about 30 mM to about 150 mM, about 30 mM to about 130 mM, about 30 mM to about 120 mM, about 30 mM to about 110 mM, about 30 mM to about 100 mM, about 50 mM to about 500 mM, about 50 mM to about 400 mM, about 50 mM to about 350 mM, about 50 mM to about 300 mM, about 50 mM to about 250 mM, about 50 mM to about 200 mM, about 50 mM to about 150 mM, about 50 mM to about 130 mM, about 50 mM to about 120 mM, about 50 mM to about 110 mM, about 50 mM to about 100 mM, about 70 mM to about 500 mM, about 70 mM to about 400 mM, about 70 mM to about 350 mM, about 70 mM to about 300 mM, about 70 mM to about 250 mM, about 70 mM to about 200 mM, about 70 mM to about 150 mM, about 70 mM to about 130 mM, about 70 mM to about 120 mM, about 70 mM to about 110 mM, about 70 mM to about 100 mM, about 80 mM to about 500 mM,About 80 mM to about 400 mM, about 80 mM to about 350 mM, about 80 mM to about 300 mM, about 80 mM to about 250 mM, about 80 mM to about 200 mM, about 80 mM to about 150 mM, about 80 mM to about 130 mM, about 80 mM to about 120 mM, about 80 mM to about 110 mM, about 80 mM to about 100 mM, about 90 mM to about 500 mM, about 90 mM to about 400 mM, about 90 mM to about 350 mM, about 90 mM to about 300 mM, about 90 mM to about 250 mM, about 90 mM to about 200 mM, about 90 mM to about 150 mM, about 90 mM to about 130 mM, about 90 mM to about 120 It may comprise a salt of mM, about 90 mM to about 110 mM, about 90 mM to about 100 mM, about 100 mM to about 500 mM, about 100 mM to about 400 mM, about 100 mM to about 350 mM, about 100 mM to about 300 mM, about 100 mM to about 250 mM, about 100 mM to about 200 mM, about 100 mM to about 150 mM, about 100 mM to about 130 mM, about 100 mM to about 120 mM, or about 100 mM to about 110 mM.

[0040] In one embodiment, the equilibrium buffer may comprise sodium phosphate and sodium chloride, specifically, the equilibrium buffer may comprise about 5 mM to about 50 mM of sodium phosphate and about 10 mM to about 500 mM of sodium chloride, and the pH may be in the range of about 4.0 to about 8.0.

[0041] The equilibrium buffer may contain sodium chloride in an amount of about 10 mM to about 500 mM, specifically about 10 mM to about 500 mM, about 10 mM to about 400 mM, about 10 mM to about 350 mM, about 10 mM to about 300 mM, about 10 mM to about 250 mM, about 10 mM to about 200 mM, about 10 mM to about 150 mM, about 10 mM to about 130 mM, about 10 mM to about 120 mM, about 10 mM to about 110 mM, about 10 mM to about 100 mM, about 30 mM to about 500 mM, about 30 mM to about 400 mM, about 30 mM to about 350 mM, about 30 mM to about 300 mM, about 30 mM to about 250 mM, about 30 mM to about 200 mM, about 30 mM to about 150 mM, about 30 mM to about 130 mM, about 30 mM to about 120 mM, about 30 mM to about 110 mM, about 30 mM to about 100 mM, about 50 mM to about 500 mM, about 50 mM to about 400 mM, about 50 mM to about 350 mM, about 50 mM to about 300 mM, about 50 mM to about 250 mM, about 50 mM to about 200 mM, about 50 mM to about 150 mM, about 50 mM to about 130 mM, about 50 mM to about 120 mM, about 50 mM to about 110 mM, about 50 mM to about 100 mM, about 80 mM to about 500 mM, about 80 mM to about 400 mM, about 80 mM to about 350 mM, about 80 mM to about 300 mM, about 80 mM to about 250 mM, about 80 mM to about 200 mM, about 80 mM to about 150 mM, about 80 mM to about 130 mM, about 80 mM to about 120 mM, about 80 mM to about 110 mM, about 80 mM to about 100 mM,About 90 mM to about 500 mM, about 90 mM to about 400 mM, about 90 mM to about 350 mM, about 90 mM to about 300 mM, about 90 mM to about 250 mM, about 90 mM to about 200 mM, about 90 mM to about 150 mM, about 90 mM to about 130 mM, about 90 mM to about 120 mM, about 90 mM to about 110 mM, about 90 mM to about 100 mM, about 100 mM to about 500 mM, about 100 mM to about 400 mM, about 100 mM to about 350 mM, about 100 mM to about 300 mM, about 100 mM to about 250 mM, about 100 mM to about 200 mM, about 100 mM to about 150 mM, about It may contain 100 mM to about 130 mM, about 100 mM to about 120 mM, or about 100 mM to about 110 mM of sodium chloride.

[0042] The equilibrium buffer may contain about 5 mM to about 50 mM of sodium phosphate, specifically about 5 mM to about 50 mM, about 5 mM to about 40 mM, about 5 mM to about 30 mM, about 5 mM to about 25 mM, about 5 mM to about 20 mM, about 10 mM to about 50 mM, about 10 mM to about 40 mM, about 10 mM to about 30 mM, about 10 mM to about 25 mM, about 10 mM to about 20 mM, about 15 mM to about 50 mM, about 15 mM to about 40 mM, about 15 mM to about 30 mM, about 15 mM to about 25 mM, about 15 mM to about 20 mM, about 20 mM to about 50 mM, about 20 mM to about 40 mM, about 20 mM to about 30 mM, about 15 mM to about 25 mM, about 15 mM to about 20 mM, about 20 mM to about 50 mM, about 20 mM to about 40 mM, about 20 mM to about 30 mM, or about 20 mM to about It may contain 25 mM sodium phosphate.

[0043] The pH of the equilibrium buffer may be in the range of about 4.0 to about 8.0, specifically about 4.0 to about 8.0, about 4.0 to about 7.0, about 4.0 to about 6.5, about 4.0 to about 6.0, about 5.0 to about 8.0, about 5.0 to about 7.0, about 5.0 to about 6.5, about 5.0 to about 6.0, about 5.5 to about 8.0, about 5.5 to about 7.0, about 5.5 to about 6.5, about 5.5 to about 6.0, about 6.0 to about 8.0, about 6.0 to about 7.0, or about 6.0 to about 6.5.

[0044] The sample containing the above hyaluronidase has a weight of hyaluronidase of about 5 to about 30 g / L per unit volume of resin. resin It may be loaded onto the first chromatography column at a concentration of (g protein / L resin volume), specifically about 5 to about 30 g / L resin , about 5 to about 25 g / L resin , about 5 to about 20 g / L resin , about 5 to about 18 g / L resin , about 5 to about 16 g / L resin , about 5 to about 15 g / L resin , about 5 to about 12 g / L resin , about 5 to about 10 g / L resin , about 10 to about 30 g / L resin , about 10 to about 25 g / L resin , about 10 to about 20 g / L resin , about 10 to about 18 g / L resin , about 10 to about 16 g / L resin , about 10 to about 15 g / L resin , about 10 to about 12 g / L resin , about 12 to about 30 g / L resin , about 12 to about 25 g / L resin , about 12 to about 20 g / Lresin , about 12 to about 18 g / L resin , about 12 to about 16 g / L resin , about 12 to about 15 g / L resin , about 14 to about 30 g / L resin , about 14 to about 25 g / L resin , about 14 to about 20 g / L resin , about 14 to about 18 g / L resin , about 14 to about 16 g / L resin , about 14 to about 15 g / L resin , about 15 to about 30 g / L resin , about 15 to about 25 g / L resin , about 15 to about 20 g / L resin , about 15 to about 18 g / L resin , or about 15 to about 16 g / L resin It may be loaded at a concentration of

[0045] In one embodiment, the method may include A-2) a step of eluting a product from the column using an elution buffer. The A-2) step is a step for recovering a target product present in the column, and specifically, may be a step of recovering hyaluronidase or a purified product containing hyaluronidase using an elution buffer.

[0046] The above-mentioned elution step may utilize a gradient elution or isocratic elution method, and specifically, may utilize a gradient elution.

[0047] The term "elution buffer" as used in this specification is a buffer used to elute a target protein from a stationary phase.

[0048] The above elution buffer is a buffer used to elute hyaluronidase from a chromatography column, and may use a buffer comprising a component or combination of components suitable for effectively recovering said hyaluronidase while reducing binding between hyaluronidase and a ligand, specifically sodium phosphate, sodium chloride, sodium acetate, sodium citrate, sodium carbonate, sodium sulfate, ammonium sulfate (AMS), Tris, Tris-hydrogen chloride (Tris-HCl), MES (2-(N-morpholino)ethanesulfonic acid), MOPS (3-morpholinopropane-1-sulfonic acid), PIPES, potassium sulfate, potassium phosphate, potassium chloride, HEPES (2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid), Triton X-100, urea, Tween 80, It may include, but is not limited to, one or more compounds or salts selected from the group consisting of LDAO (Lauryldimethylamine oxide), calcium chloride (CaCl2), arginine, EDTA (ethylene-diamine-tetraacetic acid), guanidine, isopropanol, and acceptable salts thereof.

[0049] The above elution buffer may contain about 10 mM to about 2000 mM of salt, specifically about 10 mM to about 2000 mM, about 10 mM to about 1800 mM, about 10 mM to about 1500 mM, about 10 mM to about 1300 mM, about 10 mM to about 1200 mM, about 10 mM to about 1100 mM, about 10 mM to about 1050 mM, about 10 mM to about 1000 mM, about 10 mM to about 800 mM, about 10 mM to about 600 mM, about 10 mM to about 550 mM, about 10 mM to about 500 mM, about 30 mM to about 2000 mM, about 30 mM to about 1800 mM, about 30 mM to about 1500 mM, about 30 mM to about 1300 mM mM, about 30 mM to about 1200 mM, about 30 mM to about 1100 mM, about 30 mM to about 1050 mM, about 30 mM to about 1000 mM, about 30 mM to about 800 mM, about 30 mM to about 600 mM, about 30 mM to about 550 mM, about 30 mM to about 500 mM, about 40 mM to about 2000 mM, about 40 mM to about 1800 mM, about 40 mM to about 1500 mM, about 40 mM to about 1300 mM, about 40 mM to about 1200 mM, about 40 mM to about 1100 mM, about 40 mM to about 1050 mM, about 40 mM to about 1000 mM, about 40 mM to about 800 mM, about 40 mM to about 600 mM, about 40 mM to about 550 mM, about 40 mM to about 500 mM, about 45 mM to about 2000 mM, about 45 mM to about 1800 mM, about 45 mM to about 1500 mM, about 45 mM to about 1300 mM, about 45 mM to about 1200 mM, about 45 mM to about 1100 mM, about 45 mM to about 1050 mM,About 45 mM to about 1000 mM, about 45 mM to about 800 mM, about 45 mM to about 600 mM, about 45 mM to about 550 mM, about 45 mM to about 500 mM, about 50 mM to about 2000 mM, about 50 mM to about 1800 mM, about 50 mM to about 1500 mM, about 50 mM to about 1300 mM, about 50 mM to about 1200 mM, about 50 mM to about 1100 mM, about 50 mM to about 1050 mM, about 50 mM to about 1000 mM, about 50 mM to about 800 mM, about 50 mM to about 600 mM, about 50 mM to about 550 mM, about 50 mM to about 500 mM, about 70 mM to about 1800 mM, about It may contain a salt of 70 mM to about 1500 mM, about 70 mM to about 1300 mM, about 70 mM to about 1200 mM, about 70 mM to about 1100 mM, about 70 mM to about 1050 mM, about 70 mM to about 1000 mM, about 70 mM to about 800 mM, about 70 mM to about 600 mM, about 70 mM to about 550 mM, or about 70 mM to about 500 mM.

[0050] In one embodiment, the elution buffer may comprise sodium phosphate and sodium chloride, specifically, the elution buffer may comprise about 5 mM to about 50 mM of sodium phosphate and about 10 mM to about 2000 mM of sodium chloride, and the pH may be in the range of about 4.0 to about 8.0.

[0051] The above elution buffer may contain sodium chloride in an amount of about 10 mM to about 2000 mM, specifically about 10 mM to about 2000 mM, about 10 mM to about 1800 mM, about 10 mM to about 1500 mM, about 10 mM to about 1300 mM, about 10 mM to about 1200 mM, about 10 mM to about 1100 mM, about 10 mM to about 1050 mM, about 10 mM to about 1000 mM, about 10 mM to about 800 mM, about 10 mM to about 600 mM, about 10 mM to about 550 mM, about 10 mM to about 500 mM, about 30 mM to about 2000 mM, about 30 mM to about 1800 mM, about 30 mM to about 1500 mM, about 30 mM to about 1300 mM, about 30 mM to about 1200 mM, about 30 mM to about 1100 mM, about 30 mM to about 1050 mM, about 30 mM to about 1000 mM, about 30 mM to about 800 mM, about 30 mM to about 600 mM, about 30 mM to about 550 mM, about 30 mM to about 500 mM, about 40 mM to about 2000 mM, about 40 mM to about 1800 mM, about 40 mM to about 1500 mM, about 40 mM to about 1300 mM, about 40 mM to about 1200 mM, about 40 mM to about 1100 mM, about 40 mM to about 1050 mM, about 40 mM to about 1000 mM, about 40 mM to about 800 mM, about 40 mM to About 600 mM, about 40 mM to about 550 mM, about 40 mM to about 500 mM, about 45 mM to about 2000 mM, about 45 mM to about 1800 mM, about 45 mM to about 1500 mM, about 45 mM to about 1300 mM, about 45 mM to about 1200 mM, about 45 mM to about 1100 mM, about 45 mM to about 1050 mM,It may contain sodium chloride of about 45 mM to about 1000 mM, about 45 mM to about 800 mM, about 45 mM to about 600 mM, about 45 mM to about 550 mM, about 45 mM to about 500 mM, about 50 mM to about 2000 mM, about 50 mM to about 1800 mM, about 50 mM to about 1500 mM, about 50 mM to about 1300 mM, about 50 mM to about 1200 mM, about 50 mM to about 1100 mM, about 50 mM to about 1050 mM, about 50 mM to about 1000 mM, about 50 mM to about 800 mM, about 50 mM to about 600 mM, about 50 mM to about 550 mM, or about 50 mM to about 500 mM.

[0052] The above elution buffer may contain about 5 mM to about 50 mM of sodium phosphate, specifically about 5 mM to about 50 mM, about 5 mM to about 40 mM, about 5 mM to about 30 mM, about 5 mM to about 25 mM, about 5 mM to about 20 mM, about 10 mM to about 50 mM, about 10 mM to about 40 mM, about 10 mM to about 30 mM, about 10 mM to about 25 mM, about 10 mM to about 20 mM, about 15 mM to about 50 mM, about 15 mM to about 40 mM, about 15 mM to about 30 mM, about 15 mM to about 25 mM, about 15 mM to about 20 mM, about 20 mM to about 50 mM, about 20 mM to about 40 mM, about 20 mM to about 30 mM, or about 20 mM to about It may contain 25 mM sodium phosphate.

[0053] The pH of the elution buffer may be in the range of about 4.0 to about 8.0, specifically about 4.0 to about 8.0, about 4.0 to about 7.0, about 4.0 to about 6.5, about 4.0 to about 6.0, about 5.0 to about 8.0, about 5.0 to about 7.0, about 5.0 to about 6.5, about 5.0 to about 6.0, about 5.5 to about 8.0, about 5.5 to about 7.0, about 5.5 to about 6.5, about 5.5 to about 6.0, about 6.0 to about 8.0, about 6.0 to about 7.0, or about 6.0 to about 6.5.

[0054] In one embodiment, the method may further include a step of washing a first chromatography column loaded with a sample containing hyaluronidase using a washing buffer, and specifically, may further include a step of washing the column using a washing buffer after the step of loading a sample containing hyaluronidase into the first chromatography column.

[0055] The term "wash buffer" in this specification refers to a buffer that passes through a column after loading a sample containing the target protein and before eluting the target protein. The wash buffer may serve to remove one or more impurities from the resin without substantially eluting the desired target protein.

[0056] The above washing may mean the application of a mobile phase that elutes impurities from the stationary phase but does not elute the target product (hyaluronidase).

[0057] The above washing buffer may use a buffer containing a component or a combination of components suitable for removing only impurities, including cleaved forms of hyaluronidase within the column, while maintaining the binding between hyaluronidase and the material (resin) within the column; specifically, sodium phosphate, sodium chloride, sodium acetate, sodium citrate, sodium carbonate, sodium sulfate, ammonium sulfate (AMS), Tris, Tris-hydrogen chloride (Tris-HCl), MES (2-(N-morpholino)ethanesulfonic acid), MOPS (3-morpholinopropane-1-sulfonic acid), PIPES, potassium sulfate, potassium phosphate, potassium chloride, HEPES (2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid), Triton X-100, urea, Tween 80, LDAO (Lauryldimethylamine oxide), It may include one or more compounds or salts selected from the group consisting of calcium chloride (CaCl2), arginine, EDTA (ethylene-diamine-tetraacetic acid), guanidine, isopropanol, and acceptable salts thereof, but is not limited thereto.

[0058] The washing buffer may contain about 50 mM to about 600 mM of salt, specifically about 50 mM to about 600 mM, about 50 mM to about 500 mM, about 50 mM to about 450 mM, about 50 mM to about 400 mM, about 50 mM to about 350 mM, about 50 mM to about 300 mM, about 50 mM to about 250 mM, about 50 mM to about 200 mM, about 50 mM to about 150 mM, about 50 mM to about 130 mM, about 80 mM to about 600 mM, about 80 mM to about 500 mM, about 80 mM to about 450 mM, about 80 mM to about 400 mM, about 80 mM to about 350 mM, about 80 mM to about 300 mM, about 80 mM to about 250 mM, About 80 mM to about 200 mM, about 80 mM to about 150 mM, about 80 mM to about 130 mM, about 100 mM to about 600 mM, about 100 mM to about 500 mM, about 100 mM to about 450 mM, about 100 mM to about 400 mM, about 100 mM to about 350 mM, about 100 mM to about 300 mM, about 100 mM to about 250 mM, about 100 mM to about 200 mM, about 100 mM to about 150 mM, about 100 mM to about 130 mM, about 120 mM to about 600 mM, about 120 mM to about 500 mM, about 120 mM to about 450 mM, about 120 mM to about 400 mM, about 120 mM to about 350 mM, about 120 mM to about 300 mM, about 120 mM to about 250 mM, about 120 mM to about 200 mM, about 120 mM to about 150 mM, about 120 mM to about 130 mM, about 130 mM to about 600 mM, about 130 mM to about 500 mM, about 130 mM to about 450 mM,About 130 mM to about 400 mM, about 130 mM to about 350 mM, about 130 mM to about 300 mM, about 150 mM to about 600 mM, about 150 mM to about 500 mM, about 150 mM to about 450 mM, about 150 mM to about 400 mM, about 150 mM to about 350 mM, about 150 mM to about 300 mM, about 200 mM to about 600 mM, about 200 mM to about 500 mM, about 200 mM to about 450 mM, about 200 mM to about 400 mM, about 200 mM to about 350 mM, about 200 mM to about 300 mM, about 250 mM to about 600 mM, about 250 mM to about 500 mM, about 250 mM to about 450 mM, about 200 mM to about 400 mM, about 200 mM to about 350 mM, about 200 mM to about 300 mM, about 250 mM to about 600 mM, about 250 mM to about 500 mM, about 250 mM to about It may contain 450 mM, about 250 mM to about 400 mM, about 250 mM to about 350 mM, about 250 mM to about 300 mM, about 300 mM to about 600 mM, about 300 mM to about 500 mM, about 300 mM to about 450 mM, about 300 mM to about 400 mM, or about 300 mM to about 350 mM of salt.

[0059] In one embodiment, the washing buffer may comprise sodium phosphate and sodium chloride, specifically, the washing buffer may comprise about 5 mM to about 50 mM of sodium phosphate and about 100 mM to about 600 mM of sodium chloride, and the pH may be in the range of about 4.0 to about 8.0.

[0060] The washing buffer may contain about 50 mM to about 600 mM of sodium chloride, specifically about 50 mM to about 600 mM, about 50 mM to about 500 mM, about 50 mM to about 450 mM, about 50 mM to about 400 mM, about 50 mM to about 350 mM, about 50 mM to about 300 mM, about 50 mM to about 250 mM, about 50 mM to about 200 mM, about 50 mM to about 150 mM, about 50 mM to about 130 mM, about 80 mM to about 600 mM, about 80 mM to about 500 mM, about 80 mM to about 450 mM, about 80 mM to about 400 mM, about 80 mM to about 350 mM, about 80 mM to about 300 mM, about 80 mM to about 250 mM, about 80 mM to about 200 mM, about 80 mM to about 150 mM, about 80 mM to about 130 mM, about 100 mM to about 600 mM, about 100 mM to about 500 mM, about 100 mM to about 450 mM, about 100 mM to about 400 mM, about 100 mM to about 350 mM, about 100 mM to about 300 mM, about 100 mM to about 250 mM, about 100 mM to about 200 mM, about 100 mM to about 150 mM, about 100 mM to about 130 mM, about 120 mM to about 600 mM, about 120 mM to about 500 mM, about 120 mM to about 450 mM, about 120 mM to about 400 mM, about 120 mM to about 350 mM, about 120 mM to about 300 mM, about 120 mM to about 250 mM, about 120 mM to about 200 mM, about 120 mM to about 150 mM, about 120 mM to about 130 mM, about 130 mM to about 600 mM, about 130 mM to about 500 mM, about 130 mM to about 450 mM,About 130 mM to about 400 mM, about 130 mM to about 350 mM, about 130 mM to about 300 mM, about 150 mM to about 600 mM, about 150 mM to about 500 mM, about 150 mM to about 450 mM, about 150 mM to about 400 mM, about 150 mM to about 350 mM, about 150 mM to about 300 mM, about 200 mM to about 600 mM, about 200 mM to about 500 mM, about 200 mM to about 450 mM, about 200 mM to about 400 mM, about 200 mM to about 350 mM, about 200 mM to about 300 mM, about 250 mM to about 600 mM, about 250 mM to about 500 mM, about 250 mM to about 450 mM, about 200 mM to about 400 mM, about 200 mM to about 350 mM, about 200 mM to about 300 mM, about 250 mM to about 600 mM, about 250 mM to about 500 mM, about 250 mM to about It may contain 450 mM, about 250 mM to about 400 mM, about 250 mM to about 350 mM, about 250 mM to about 300 mM, about 300 mM to about 600 mM, about 300 mM to about 500 mM, about 300 mM to about 450 mM, about 300 mM to about 400 mM, or about 300 mM to about 350 mM of sodium chloride.

[0061] The washing buffer may contain about 5 mM to about 50 mM of sodium phosphate, specifically about 5 mM to about 50 mM, about 5 mM to about 40 mM, about 5 mM to about 30 mM, about 5 mM to about 25 mM, about 5 mM to about 20 mM, about 10 mM to about 50 mM, about 10 mM to about 40 mM, about 10 mM to about 30 mM, about 10 mM to about 25 mM, about 10 mM to about 20 mM, about 15 mM to about 50 mM, about 15 mM to about 40 mM, about 15 mM to about 30 mM, about 15 mM to about 25 mM, about 15 mM to about 20 mM, about 20 mM to about 50 mM, about 20 mM to about 40 mM, about 20 mM to about 30 mM, about 15 mM to about 25 mM, about 15 mM to about 20 mM, about 20 mM to about 50 mM, about 20 mM to about 40 mM, about 20 mM to about 30 mM, or about 20 mM to about It may contain 25 mM sodium phosphate.

[0062] The pH of the washing buffer may be in the range of about 4.0 to about 8.0, specifically about 4.0 to about 8.0, about 4.0 to about 7.0, about 4.0 to about 6.5, about 4.0 to about 6.0, about 5.0 to about 8.0, about 5.0 to about 7.0, about 5.0 to about 6.5, about 5.0 to about 6.0, about 5.5 to about 8.0, about 5.5 to about 7.0, about 5.5 to about 6.5, about 5.5 to about 6.0, about 6.0 to about 8.0, about 6.0 to about 7.0, or about 6.0 to about 6.5.

[0063] In one embodiment, the method may further include the step of tracking a first chromatography column loaded with a sample containing hyaluronidase using a tracking buffer, and specifically, may further include the step of tracking the column using a tracking buffer after the step of loading a sample containing hyaluronidase into the first chromatography column.

[0064] The term "chase buffer" in this specification refers to a buffer used after loading a sample containing a target protein onto a column.

[0065] The above tracking buffer may contain the same composition as the equilibrium buffer.

[0066] The eluent obtained from the first chromatography above may contain hyaluronidase.

[0067] In one embodiment, the method may further include the step of B) applying the eluent obtained from the first chromatography to the second chromatography.

[0068] The second chromatography may include one or more selected from the group consisting of cation exchange chromatography (CEX), anion exchange chromatography (AEX), affinity chromatography (AC), size exclusion chromatography (SEC), and hydrophobic interaction chromatography (HIC), and specifically may include hydrophobic interaction chromatography.

[0069] The above hydrophobic interaction chromatography may utilize a column containing a hydrophobic interaction chromatography material.

[0070] The above hydrophobic interaction chromatography material may comprise one or more selected from the group consisting of Phenyl Sepharose 6 FF, Capto Phenyl ImpRes, Capto Butyl, and Capto Octyl.

[0071] The above hydrophobic interaction chromatography may use a column containing Phenyl Sepharose 6 FF, and specifically, may include Phenyl Sepharose 6 FF as the resin of the column.

[0072] In one embodiment, applying the eluent obtained from the first chromatography to the second chromatography may include B-1) loading the eluent obtained from the first chromatography into the second chromatography column; and B-2) eluting the loaded sample from the column using an elution buffer.

[0073] The eluent obtained from the first chromatography above has a weight of hyaluronidase of about 5 to about 30 g / L per unit volume of resin. resin It may be loaded onto the second chromatography column at a concentration of (g protein / L resin volume), specifically about 5 to about 30 g / L resin , about 5 to about 25 g / L resin , about 5 to about 20 g / L resin , about 10 to about 30 g / L resin , about 10 to about 25 g / L resin , about 10 to about 20 g / L resin , about 15 to about 30 g / L resin , about 15 to about 25 g / L resin , about 15 to about 20 g / L resin , about 20 to about 30 g / L resin , or about 20 to about 25 g / L resin It may be loaded at a concentration of

[0074] The second chromatography column may be a column equilibrated with an equilibration buffer, specifically, a column equilibrated using an equilibration buffer before loading the eluent obtained from the first chromatography. Accordingly, the method may additionally include the step of equilibrating the second chromatography column with an equilibration buffer before loading the eluent obtained from the first chromatography of step 'B-1)'.

[0075] The above equilibrium buffer may comprise one or more compounds or salts selected from the group consisting of sodium phosphate, sodium chloride, sodium acetate, sodium citrate, sodium carbonate, sodium sulfate, ammonium sulfate (AMS), Tris, Tris-hydrogen chloride (Tris-HCl), MES (2-(N-morpholino)ethanesulfonic acid), MOPS (3-morpholinopropane-1-sulfonic acid), PIPES, potassium sulfate, potassium phosphate, potassium chloride, HEPES (2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid), and AMS.

[0076] The equilibrium buffer may comprise about 500 mM to about 2000 mM of salt, specifically about 500 mM to about 2000 mM, about 500 mM to about 1800 mM, about 500 mM to about 1600 mM, about 500 mM to about 1400 mM, about 500 mM to about 1300 mM, about 500 mM to about 1250 mM, about 500 mM to about 1200 mM, about 700 mM to about 2000 mM, about 700 mM to about 1800 mM, about 700 mM to about 1600 mM, about 700 mM to about 1400 mM, about 700 mM to about 1300 mM, about 700 mM to about 1250 mM, about 700 mM to about 1200 mM, about 900 mM to about 2000 mM, about 900 mM to about 1800 mM, about 900 mM to about 1600 mM, about 900 mM to about 1400 mM, about 900 mM to about 1300 mM, about 900 mM to about 1250 mM, about 900 mM to about 1200 mM, about 1000 mM to about 2000 mM, about 1000 mM to about 1800 mM, about 1000 mM to about 1600 mM, about 1000 mM to about 1400 mM, about 1000 mM to about 1300 mM, about 1000 mM to about 1250 mM, about 1000 mM to about 1200 mM, about 1100 mM to about 2000 mM, about 1100 mM to about 1800 mM, About 1100 mM to about 1600 mM, about 1100 mM to about 1400 mM, about 1100 mM to about 1300 mM, about 1100 mM to about 1250 mM, about 1100 mM to about 1200 mM, about 1150 mM to about 2000 mM, about 1150 mM to about 1800 mM, about 1150 mM to about 1600 mM, about 1150 mM to about 1400 mM, about 1150 mM to about 1300 mM,It may contain salts of about 1150 mM to about 1250 mM, about 1150 mM to about 1200 mM, about 1200 mM to about 2000 mM, about 1200 mM to about 1800 mM, about 1200 mM to about 1600 mM, about 1200 mM to about 1400 mM, about 1200 mM to about 1300 mM, or about 1200 mM to about 1250 mM.

[0077] In one embodiment, the equilibrium buffer may comprise sodium phosphate and AMS, specifically, the equilibrium buffer may comprise about 5 mM to about 50 mM of sodium phosphate and about 500 mM to about 2000 mM of AMS, and the pH may be in the range of about 5.0 to about 9.0.

[0078] The equilibrium buffer may contain about 500 mM to about 2000 mM of AMS, specifically about 500 mM to about 2000 mM, about 500 mM to about 1800 mM, about 500 mM to about 1600 mM, about 500 mM to about 1400 mM, about 500 mM to about 1300 mM, about 500 mM to about 1250 mM, about 500 mM to about 1200 mM, about 700 mM to about 2000 mM, about 700 mM to about 1800 mM, about 700 mM to about 1600 mM, about 700 mM to about 1400 mM, about 700 mM to about 1300 mM, about 700 mM to about 1250 mM, about 700 mM to about 1200 mM, about 900 mM to about 2000 mM, about 900 mM to about 1800 mM, about 900 mM to about 1600 mM, about 900 mM to about 1400 mM, about 900 mM to about 1300 mM, about 900 mM to about 1250 mM, about 900 mM to about 1200 mM, about 1000 mM to about 2000 mM, about 1000 mM to about 1800 mM, about 1000 mM to about 1600 mM, about 1000 mM to about 1400 mM, about 1000 mM to about 1300 mM, about 1000 mM to about 1250 mM, about 1000 mM to about 1200 mM, about 1100 mM to about 2000 mM, about 1100 mM to about 1800 mM, about 1100 mM to about 1600 mM, about 1100 mM to about 1400 mM, about 1100 mM to about 1300 mM, about 1100 mM to about 1250 mM, about 1100 mM to about 1200 mM, about 1150 mM to about 2000 mM, about 1150 mM to about 1800 mM, about 1150 mM to about 1600 mM, about 1150 mM to about 1400 mM, about 1150 mM to about 1300 mM,It may comprise about 1150 mM to about 1250 mM, about 1150 mM to about 1200 mM, about 1200 mM to about 2000 mM, about 1200 mM to about 1800 mM, about 1200 mM to about 1600 mM, about 1200 mM to about 1400 mM, about 1200 mM to about 1300 mM, or about 1200 mM to about 1250 mM of AMS.

[0079] The equilibrium buffer may contain about 5 mM to about 50 mM of sodium phosphate, specifically about 5 mM to about 50 mM, about 5 mM to about 40 mM, about 5 mM to about 30 mM, about 5 mM to about 25 mM, about 5 mM to about 20 mM, about 10 mM to about 50 mM, about 10 mM to about 40 mM, about 10 mM to about 30 mM, about 10 mM to about 25 mM, about 10 mM to about 20 mM, about 15 mM to about 50 mM, about 15 mM to about 40 mM, about 15 mM to about 30 mM, about 15 mM to about 25 mM, about 15 mM to about 20 mM, about 20 mM to about 50 mM, about 20 mM to about 40 mM, about 20 mM to about 30 mM, about 15 mM to about 25 mM, about 15 mM to about 20 mM, about 20 mM to about 50 mM, about 20 mM to about 40 mM, about 20 mM to about 30 mM, or about 20 mM to about It may contain 25 mM sodium phosphate.

[0080] The pH of the equilibrium buffer may be in the range of about 5.0 to about 9.0, specifically about 5.0 to about 9.0, about 5.0 to about 8.0, about 5.0 to about 7.5, about 5.0 to about 7.0, about 6.0 to about 9.0, about 6.0 to about 8.0, about 6.0 to about 7.5, about 6.0 to about 7.0, about 6.5 to about 9.0, about 6.5 to about 8.0, about 6.5 to about 7.5, about 6.5 to about 7.0, about 7.0 to about 9.0, about 7.0 to about 8.0, or about 7.0 to about 7.5.

[0081] In one embodiment, the method may include B-2) a step of eluting a product from the column using an elution buffer. B-2) is a step for recovering a target product present in the column, and specifically, may be a step of recovering hyaluronidase or a purified product containing hyaluronidase using an elution buffer.

[0082] The above elution step may utilize a gradient elution or isocratic elution method, and specifically, may utilize isocratic elution.

[0083] The above elution buffer is a buffer used to elute hyaluronidase from a chromatography column, and may use a buffer comprising a component or combination of components suitable for effectively recovering said hyaluronidase while reducing binding between hyaluronidase and a ligand, specifically sodium phosphate, sodium chloride, sodium acetate, sodium citrate, sodium carbonate, sodium sulfate, ammonium sulfate (AMS), Tris, Tris-hydrogen chloride (Tris-HCl), MES (2-(N-morpholino)ethanesulfonic acid), MOPS (3-morpholinopropane-1-sulfonic acid), PIPES, potassium sulfate, potassium phosphate, potassium chloride, HEPES (2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid), Triton X-100, urea, Tween 80, It may include one or more compounds or salts selected from the group consisting of LDAO (Lauryldimethylamine oxide), calcium chloride (CaCl2), arginine, EDTA (ethylene-diamine-tetraacetic acid), guanidine, isopropanol, AMS, and acceptable salts thereof, but is not limited thereto.

[0084] The above elution buffer may contain about 200 mM to about 600 mM of salt, specifically about 200 mM to about 600 mM, about 200 mM to about 500 mM, about 200 mM to about 400 mM, about 200 mM to about 370 mM, about 300 mM to about 600 mM, about 300 mM to about 500 mM, about 300 mM to about 400 mM, about 300 mM to about 370 mM, about 350 mM to about 600 mM, about 350 mM to about 500 mM, about 350 mM to about 400 mM, about 350 mM to about 370 mM, about 370 mM to about 600 mM, about 370 mM to about 500 mM, or about 370 mM to about 400 mM of salt.

[0085] In one embodiment, the elution buffer may comprise sodium phosphate and AMS, specifically, the elution buffer may comprise about 5 mM to about 50 mM of sodium phosphate and about 200 mM to about 600 mM of AMS, and the pH may be in the range of about 5.0 to about 9.0.

[0086] The above elution buffer may contain about 200 mM to about 600 mM of AMS, and specifically may contain about 200 mM to about 600 mM, about 200 mM to about 500 mM, about 200 mM to about 400 mM, about 200 mM to about 370 mM, about 300 mM to about 600 mM, about 300 mM to about 500 mM, about 300 mM to about 400 mM, about 300 mM to about 370 mM, about 350 mM to about 600 mM, about 350 mM to about 500 mM, about 350 mM to about 400 mM, about 350 mM to about 370 mM, about 370 mM to about 600 mM, about 370 mM to about 500 mM, or about 370 mM to about 400 mM of AMS. there is.

[0087] The above elution buffer may contain about 5 mM to about 50 mM of sodium phosphate, specifically about 5 mM to about 50 mM, about 5 mM to about 40 mM, about 5 mM to about 30 mM, about 5 mM to about 25 mM, about 5 mM to about 20 mM, about 10 mM to about 50 mM, about 10 mM to about 40 mM, about 10 mM to about 30 mM, about 10 mM to about 25 mM, about 10 mM to about 20 mM, about 15 mM to about 50 mM, about 15 mM to about 40 mM, about 15 mM to about 30 mM, about 15 mM to about 25 mM, about 15 mM to about 20 mM, about 20 mM to about 50 mM, about 20 mM to about 40 mM, about 20 mM to about 30 mM, or about 20 mM to about It may contain 25 mM sodium phosphate.

[0088] The pH of the elution buffer may be in the range of about 5.0 to about 9.0, specifically about 5.0 to about 9.0, about 5.0 to about 8.0, about 5.0 to about 7.5, about 5.0 to about 7.0, about 6.0 to about 9.0, about 6.0 to about 8.0, about 6.0 to about 7.5, about 6.0 to about 7.0, about 6.5 to about 9.0, about 6.5 to about 8.0, about 6.5 to about 7.5, about 6.5 to about 7.0, about 7.0 to about 9.0, about 7.0 to about 8.0, or about 7.0 to about 7.5.

[0089] In one embodiment, the method may further include the step of tracking a second chromatography column loaded with an eluent obtained from a first chromatography using a tracking buffer, and specifically, may further include the step of tracking the column using a tracking buffer after the step of loading the eluent obtained from the first chromatography into the second chromatography column.

[0090] The above tracking buffer may contain the same composition as the equilibrium buffer.

[0091] The eluent obtained from the second chromatography above may contain hyaluronidase.

[0092] The sample containing the hyaluronidase described above may comprise cells, their lysates, their culture medium, or pre-treated cells, their lysates, or their culture medium. Specifically, the cells, their lysates, or their culture medium may be derived from host cells or organisms that express / produce hyaluronidase.

[0093] The term “cell culture fluid” in this specification means harvested cell culture fluid (HCCF), cell culture supernatant, or pretreated cell culture fluid / culture supernatant. The cell culture fluid may be derived from a host cell or organism that expresses / produces a target protein (specifically, hyaluronidase). The cell culture fluid may be partially purified or purified by centrifugation and / or filtration, e.g., microfiltration, diafiltration, ultrafiltration, and depth filtration.

[0094] The term "pretreated" in this specification may refer to a series of processes and treatments to improve the efficiency of the chromatography used in the method of the present invention, such as performing one or more adjustments on a sample, for example, buffer exchange, dilution, addition of salts, detergents, chaotropic substances, or organic compounds, pH titration, or filtration, in order to adjust the pH and / or conductivity range and / or buffer capacity to achieve desired chromatographic performance and stabilize the target protein. Accordingly, the pretreated cells, their lysates, or their culture medium may be prepared for the chromatographic process used in the method of the present invention.

[0095] Meanwhile, since the target protein expressed in mammalian cells is typically secreted into the cell culture medium during the culture process, product harvesting at the end of the culture process is achieved by separating the cell culture medium from the cells. The cell separation method must be performed gently to minimize cell disruption in order to avoid an increase in cell debris and the release of other molecules that may affect the quality of the protease and target protein products. Generally, the harvest from mammalian cell cultures undergoes filtration following centrifugation. Therefore, other treatments of the sample prior to purification via the chromatography step may involve concentrating and / or filtering the cell culture medium to a specific target protein concentration, pH range, conductivity, and buffer species concentration.

[0096] In one embodiment, the sample containing the hyaluronidase may include an eluent obtained by applying anion exchange chromatography, and specifically, may include an eluent obtained by applying a cell culture medium or a pre-treated cell culture medium to anion exchange chromatography.

[0097] In one embodiment, the sample containing the hyaluronidase may include an eluent obtained by applying depth filtration, and specifically, may include an eluent obtained by applying depth filtration to a cell culture medium or a pre-treated cell culture medium.

[0098] In one embodiment, the sample containing the hyaluronidase may comprise an eluent obtained by applying host cell protein (HCP) precipitation, and specifically, may comprise an eluent obtained by applying the host cell protein precipitation step to a cell culture medium or a pre-treated cell culture medium.

[0099] The term "Host cell protein (HCP)" as used herein refers to any process-related impurity generated during the metabolic process of a host cell expressing / producing a target protein. As used herein, the term "Host cell protein" may be used interchangeably with "Host cell protein contaminant" and "Host cell protein impurity."

[0100] The above method may be intended to remove impurities contained in a sample, specifically to remove forms other than the intact form of hyaluronidase.

[0101] The term "impurity" as used herein refers to any substance different from the target protein to be purified, recovered, or obtained in the above method. Such impurity may be cell medium components, cell debris, host cell protein (HCP), endotoxins, viruses, lipids, DNA, RNA, leachates from process materials, and aggregates or fragments thereof; product-related substances such as aggregates, charge variants, misfolded molecules, or fragments or variants of the target protein to be purified may also be considered as impurities. As used herein, such impurity may be used interchangeably with "contaminant."

[0102] The above impurities may include forms other than the intact form of hyaluronidase, and specifically, may include the hydrolyzed form of hyaluronidase.

[0103] In one embodiment, the intact form of hyaluronidase may comprise a polypeptide represented by the amino acid sequence of SEQ ID NO. 1 listed in Table 2 below or an amino acid sequence having sequence identity of 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, 99.9% or more, or 100% or more with respect thereto, and specifically, the intact form of hyaluronidase may be a polypeptide represented by the amino acid sequence of SEQ ID NO. 1.

[0104] NameAmino Acid SequenceLengthSequence NumberHyaluronidase (rHuPH20)LNFRAPPVIP NVPFLWAWNA PSEFCLGKFD EPLDMSLFSF IGSPRINATG QGVTIFYVDR LGYYPYIDSI TGVTVNGGIP QKISLQDHLD KAKKDITFYM PVDNLGMAVI DWEEWRPTWA RNWKPKDVYK NRSIELVQQQ NVQLSLTEAT EKAKQEFEKA GKDFLVETIK LGKLLRPNHL WGYYLFPDCY NHHYKKPGYN GSCFNVEIKR NDDLSWLWNE STALYPSIYL NTQQSPVAAT LYVRNRVREA IRVSKIPDAK SPLPVFAYTR IVFTDQVLKF LSQDELVYTF GETVALGASG IVIWGTLSIMRSMKSCLLLD NYMETILNPY IINVTLAAKM CSQVLCQEQG VCIRKNWNSS DYLHLNPDNF AIQLEKGGKF TVRGKPTLED LEQFSEKFYC SCYSTLSCKE KADVKDTDAV DVCIADGVCI DAFLKPPMET EEPQIFY447AA1

[0105]

[0106] The term "sequence identity" as used herein refers to the degree of similarity of amino acid residues or bases between sequences after aligning both sequences to match as closely as possible in a specific comparison region. Sequence identity can be verified according to methods known in the art.

[0107] The hydrolyzed form of the hyaluronidase described above may include a form in which peptide bonds in some regions of the hyaluronidase are cleaved due to hydrolysis. Specifically, the hyaluronidase may be cleaved into two or more fragments due to hydrolysis, and the two or more fragments formed by the cleaving may be connected by disulfided bonds between the fragments. Meanwhile, in this specification, the 'hydrolyzed form' of the hyaluronidase may be used interchangeably with the 'cleaved form'.

[0108] In one embodiment, the hydrolyzed form of the hyaluronidase is a form in which the peptide bond between arginine (R), the 311th amino acid of SEQ ID NO. 1, and serine (S), the 312th amino acid, is cleaved by hydrolysis and consists of two fragments (a first fragment and a second fragment), wherein the first fragment and the second fragment may be connected by a disulfide bond.

[0109] In one embodiment, the first fragment and the second fragment of the hydrolyzed form of hyaluronidase may comprise a polypeptide represented by an amino acid sequence of SEQ ID NO. 2 or 3 listed in Table 2 below, or an amino acid sequence having sequence identity of 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, 99.9% or more, or 100% or more with respect thereto, and specifically may be composed of a polypeptide represented by the amino acid sequence of SEQ ID NO. 2 or 3.

[0110] Name Amino Acid Sequence Length Sequence Number 1st Fragment LNFRAPPVIP NVPFLWAWNA PSEFCLGKFD EPLDMSLFSF IGSPRINATG QGVTIFYVDR LGYYPYIDSI TGVTVNGGIP QKISLQDHLD KAKKDITFYM PVDNLGMAVI DWEEWRPTWA RNWKPKDVYK NRSIELVQQQ NVQLSLTEAT EKAKQEFEKA GKDFLVETIK LGKLLRPNHL WGYYLFPDCY NHHYKKPGYN GSCFNVEIKR NDDLSWLWNE STALYPSIYL NTQQSPVAAT LYVRNRVREA IRVSKIPDAK SPLPVFAYTR IVFTDQVLKF LSQDELVYTF GETVALGASG IVIWGTLSIMR311AA2 2nd Fragment SMKSCLLLD NYMETILNPY IINVTLAAKM CSQVLCQEQG VCIRKNWNSS DYLHLNPDNF AIQLEKGGKF TVRGKPTLED LEQFSEKFYC SCYSTLSCKE KADVKDTDAV DVCIADGVCI DAFLKPPMET EEPQIFY136AA3

[0111] In one embodiment, the hydrolyzed form of the hyaluronidase may be a form in which the 25th amino acid (cysteine) of the first fragment and the 5th amino acid (cysteine) of the second fragment are connected by a disulfide bond.

[0112] In one embodiment, the eluent obtained from the first chromatography, the eluent obtained from the second chromatography, and / or the hyaluronidase obtained through the method may comprise an intact form and a hydrolyzed form. Accordingly, the obtained hyaluronidase may be a mixture of the intact form and the hydrolyzed form.

[0113] The eluent obtained from the first chromatography, the eluent obtained from the second chromatography, and / or the hyaluronidase obtained through the method may comprise a hydrolyzed form of hyaluronidase in an amount of about 10% or less, about 8% or less, about 7% or less, about 6% or less, about 5% or less, or less than about 5% based on the total weight (or the total sum of all forms of hyaluronidase, including whole, oxidized, and cleaved forms, etc., is set to 100%), specifically in an amount of about 0.1 to about 10%, about 0.1 to about 8%, about 0.1 to about 7%, about 0.1 to about 6.5%, about 0.1 to about 6%, about 0.1 to about 5.5%, or about based on the total weight (or the total sum of all forms of hyaluronidase, including whole, oxidized, and cleaved forms, etc., is set to 100%). 0.1 to about 5%, about 0.1 to about 4.5%, about 0.1 to about 4%, about 0.1 to about 3.5%, about 0.1 to about 3%, about 0.1 to about 2.5%, about 0.1 to about 2.2%, about 0.1 to about 2%, about 0.1 to about 1.9%, about 0.1 to about 1.8%, about 0.1 to about 1.5%, about 0.1 to about 1.2%, about 0.1 to about 1.0%, about 0.1 to about 0.9%, about 0.1 to about 0.8%, about 0.1 to about 0.7%, about 0.1 to about 0.6%, about 0.3 to about 10%, about 0.3 to about 8%, about 0.3 to about 7%, About 0.3 to about 6.5%, about 0.3 to about 6%, about 0.3 to about 5.5%, about 0.3 to about 5%, about 0.3 to about 4.5%, about 0.3 to about 4%, about 0.3 to about 3.5%, about 0.3 to about 3%, about 0.3 to about 2.5%, about 0.3 to about 2.2%, about 0.3 to about 2%, about 0.3 to about 1.9%, about 0.3 to about 1.8%, about 0.3 to about 1.5%, about 0.3 to about 1.2%, about 0.3 to about 1.0%, about 0.3 to about 0.9%, about 0.3 to about 0.8%, about 0.3 to about 0.7%, about 0.3 to about 0.6%, about 0.4 to about 10%, about 0.4 to about 8%, about 0.4 to about 7%, about 0.4 to about 6.5%, about 0.4 to about 6%, about 0.4 to about 5.5%, about 0.4 to about 5%, about 0.4 to about 4.5%, about 0.4 to about 4%, about 0.4 to about 3.5%, about 0.4 to about 3%, about 0.4 to about 2.5%, about 0.4 to about 2.2%, about 0.4 to about 2%, about 0.4 to about 1.9%, about 0.4 to about 1.8%, about 0.4 to about 1.5%, about 0.4 to about 1.2%, about 0.4 to about 1.0%, about 0.4 to about 0.9%, about 0.4 to about 0.8%, about 0.4 to about 0.7%, about 0.4 to about 0.6%, about 0.5 to about 10%, about 0.5 to about 8%, about 0.5 to about 7%, about 0.5 to about 6.5%, about 0.5 to about 6%, about 0.5 to about 5.5%, about 0.5 to about 5%, about 0.5 to about 4.5%, about 0.5 to about 4%, about 0.5 to about 3.5%, about 0.5 to about 3%, about 0.5 to about 2.5%, About 0.5 to about 2.2%, about 0.5 to about 2%, about 0.5 to about 1.9%, about 0.5 to about 1.8%, about 0.5 to about 1.5%, about 0.5 to about 1.2%, about 0.5 to about 1.0%, about 0.5 to about 0.9%, about 0.5 to about 0.8%, about 0.5 to about 0.7%, about 0.5 to about 0.6%, about 0.6 to about 10%, about 0.6 to about 8%, about 0.6 to about 7%, about 0.6 to about 6.5%, about 0.6 to about 6%, about 0.6 to about 5.5%, about 0.6 to about 5%, about 0.6 to about 4.5%, about 0.6 to about 4%, about 0.6 to about 3.5%, about 0.6 to about 3%, about 0.6 to about 2.5%, about 0.6 to about 2.2%, about 0.6 to about 2%, about 0.6 to about 1.9%, about 0.6 to about 1.8%, about 0.6 to about 1.5%, about 0.6 to about 1.2%, about 0.6 to about 1.0%, about 0.6 to about 0.9%, about 0.6 to about 0.8%, or about 0.6 to about 0.7%, about 1.0 to about 10%, about 1.0 to about 8%, about 1.0 to about 7%, about 1.0 to about 6.5%, about 1.0 to about 6%, about 1.0 to about 5.5%, about 1.0 to about 5%, about 1.0 to about 4.5%, about 1.0 to about 4%, about 1.0 to about 3.5%, about 1.0 to about 3%, about 1.0 to about 2.5%, about 1.0 to about 2.2%, about 1.0 to about 2%, about 1.0 to about 1.9%, about 1.0 to about 1.8%, about 1.0 to about 1.5%, about 1.0 to about 1.2%, about 1.5 to about 10%, about 1.5 to about 8%, about 1.5 to about 7%, about 1.5 to about 6.5%, about 1.5 to about 6%, about 1.5 to about 5.5%, about 1.5 to about 5%, about 1.5 to about 4.5%, about 1.5 to about 4%, about 1.5 to about 3.5%, about 1.5 to about 3%, about 1.5 to about 2.5%, about 1.5 to about 2.2%, about 1.5 to about 2%, about 1.5 to about 1.9%, about 1.5 to about 1.8%, about 1.8 to about 10%, about 1.8 to about 8%, about 1.8 to about 7%, about 1.8 to about 6.5%, about 1.8 to about 6%, about 1.8 to about 5.5%, about 1.8 to about 5%, about 1.8 to about 4.5%, about 1.8 to about 4%, about 1.8 to about 3.5%, about 1.8 to about 3%, about 1.8 to about 2.5%, about 1.8 to about 2.2%, about 1.8 to about 2%, or about 1.8 to about 1.It may contain 9% of a hydrolyzed form of hyaluronidase.

[0114] The eluent obtained from the first chromatography, the eluent obtained from the second chromatography, and / or the hyaluronidase obtained through the method may comprise at least about 70% of the intact form of hyaluronidase based on the total weight (or the total sum of all forms of hyaluronidase, including intact, oxidized, and cleaved forms, etc., is set to 100%), specifically, about 70% to about 95%, about 70% to about 90%, about 70% to about 85%, about 70% to about 84%, about 70% to about 83.5%, about 70% to about 83%, about 70% to about 82.5%, about 70% to about 82%, and about based on the total weight (or the total sum of all forms of hyaluronidase, including intact, oxidized, and cleaved forms, etc., is set to 100%). 70% to about 81.5%, about 70% to about 81%, about 70% to about 80.5%, about 70% to about 80%, about 70% to about 79.5%, about 70% to about 79%, about 70% to about 78.5%, about 72% to about 95%, about 72% to about 90%, about 72% to about 85%, about 72% to about 84%, about 72% to about 83.5%, about 72% to about 83%, about 72% to about 82.5%, about 72% to about 82%, about 72% to about 81.5%, about 72% to about 81%, about 72% to about 80.5%, about 72% to about 80%, about 72% to about 79.5%, about 72% to about 79%, about 72% to about 78.5%, about 73% to about 95%, about 73% to about 90%, about 73% to about 85%, about 73% to about 84%, about 73% to about 83.5%, about 73% to about 83%, about 73% to about 82.5%, about 73% to about 82%, about 73% to about 81.5%, about 73% to about 81%, about 73% to about 80.5%, about 73% to about 80%, about 73% to about 79.5%, about 73% to about 79%, about 73% to about 78.5%, about 74% to about 95%, about 74% to about 90%, about 74% to about 85%, about 74% to about 84%, about 74% to about 83.5%, about 74% to about 83%, about 74% to about 82.5%, about 74% to about 82%, about 74% to about 81.5%, about 74% to about 81%, about 74% to about 80.5%, about 74% to about 80%, about 74% to about 79.5%, about 74% to about 79%, about 74% to about 78.5%, about 76% to about 95%, about 76% to about 90%, About 76% to about 85%, about 76% to about 84%, about 76% to about 83.5%, about 76% to about 83%, about 76% to about 82.5%, about 76% to about 82%, about 76% to about 81.5%, about 76% to about 81%, about 76% to about 80.5%, about 76% to about 80%, about 76% to about 79.5%, about 76% to about 79%, about 76% to about 78.5%, about 78% to about 95%, about 78% to about 90%, about 78% to about 85%, about 78% to about 84%, about 78% to about 83.5%, about 78% to about 83%, about 78% to about 82.5%, about 78% to about 82%, about 78% to about 81.5%, about 78% to about 81%, about 78% to about 80.5%, about 78% to about 80%, about 78% to about 79.5%, about 78% to about 79%, about 78% to about 78.5%, about 80% to about 95%, about 80% to about 90%, about 80% to about 85%, about 80% to about 84%, about 80% to about 83.5%, about 80% to about 83%, about 80% to about 82.5%, about 80% to about 82%, about 80% to about 81.5%, about 80% to about 81%, about 80% to about 80.5%, about 81.5% to about 95%, about 81.5% to about 90%, about 81.5% to about 85%, about 81.5% to about 84%, about 81.5% to about 83.5%, about 81.5% to about 83%, about 81.5% to about 82.5%, about 81.5% to about 82%, about 82% to about 95%, about 82% to about 90%, about 82% to about 85%, about 82% to about 84%, about 82% to about 83.5%, about 82% to about 83%, about 82% to about 82.5%, about 83% to about 95%, about 83% to about 90%, about 83% to about 85%, about 83% to about 84%, about 83% to about 84%, about 83% to about It may contain an intact form of hyaluronidase of 83.5%, about 83.5% to about 95%, about 83.5% to about 90%, about 83.5% to about 85%, or about 83.5% to about 84%.

[0115] In one embodiment, the level of the hydrolyzed form of hyaluronidase contained in the eluent obtained from the first chromatography, the eluent obtained from the second chromatography, and / or the hyaluronidase obtained through the method can be confirmed using liquid chromatography such as RP-HPLC (Reversed-phase high performance liquid chromatography) and RP-UPLC (Reversed-phase ultra performance liquid chromatography), and specifically, can be confirmed through the content of Peak 2 (%Peak 2) of the RP-HPLC and / or RP-UPLC analysis results.

[0116] In one embodiment, the level of the intact form of hyaluronidase contained in the eluent obtained from the first chromatography, the eluent obtained from the second chromatography, and / or the hyaluronidase obtained through the method can be confirmed using liquid chromatography such as RP-HPLC and RP-UPLC, and specifically, can be confirmed through the content of Peak 4 (%Peak 4) of the RP-HPLC and / or RP-UPLC analysis results.

[0117] In one embodiment, the method may further include conventional steps performed for purifying and obtaining a target protein such as hyaluronidase. It may further include a virus inactivation step and a filtration step (e.g., depth filtration, microfiltration, nanofiltration, virus filtration, ultrafiltration, and / or diafiltration).

[0118] The eluent, recovered product, or purified product obtained through the above method may have impurities removed more effectively than the eluent, recovered product, or purified product obtained through another method (a method not using MMC), and specifically, the content of the truncated form of hyaluronidase may be low or similar, while the purity, yield, and / or inactivity of the intact form of hyaluronidase may be increased, thereby obtaining an eluent, recovered product, or purified product.

[0119] In one embodiment, the eluent, recovered material, or purified material obtained through the method may exhibit an impurity content reduced by about 50% or more compared to the eluent, recovered material, or purified material obtained through another method (a method not using CEX or MMC). Specifically, the impurity ratio of the eluent, recovered material, or purified material obtained through the method may be about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 85% or more, about 87% or more, about 50% to 90%, about 50% to 87%, about 50% to 85%, about 50% to 80%, about 60% to 90%, about 60% to 87%, about 60% to 85%, about 60% to 80%, about 60% to 85%, about 60% to 80%, about It may be reduced by 70% to 90%, about 70% to 87%, about 70% to 85%, about 70% to 80%, about 80% to 90%, about 80% to 87%, or about 80% to 85%.

[0120]

[0121] Another aspect provides a method for producing hyaluronidase, comprising a method for purifying the hyaluronidase. The same parts as described above apply equally to the method.

[0122] In one embodiment, the method may comprise: 1) a step of culturing a host cell expressing hyaluronidase or a variant thereof; 2) a step of obtaining a sample containing hyaluronidase or a variant thereof from the cultured host cell or a culture medium thereof; and 3) a step of removing impurities from the sample using a method for purifying the hyaluronidase.

[0123] The above method can yield a product with reduced impurity content (specifically, cleaved or hydrolyzed forms of hyaluronidase), and the purity, yield, and / or inactivity of the hyaluronidase produced by the above method may be significantly superior.

[0124] The host cell expressing the above hyaluronidase or a variant thereof may include a transformant into which a gene encoding hyaluronidase or a variant thereof or an expression vector containing the same has been introduced.

[0125] In this specification, the term "transformation" means introducing a gene encoding the hyaluronidase or a variant thereof, or an expression vector containing such a gene, into a host cell so that the nucleic acid molecule encoding the hyaluronidase or its variant can be expressed within the host cell. Additionally, it includes integrating the gene sequence encoding the hyaluronidase or its variant into a specific location on the chromosome of the host cell to achieve the expression and secretion of the hyaluronidase or its variant. The transformed gene or polynucleotide encoding the hyaluronidase or its variant includes all of these, regardless of whether they are inserted into or located outside the chromosome of the host cell, as long as they can be expressed within the host cell. Furthermore, the nucleic acid molecule may include DNA and RNA in the form of a sequence encoding the hyaluronidase or its variant, and it does not matter in what form it is introduced as long as it can be introduced into the host cell and expressed.

[0126] The term "transformer" in this specification may be a host cell into which a gene encoding the hyaluronidase or a variant thereof, or an expression vector containing the same, can be introduced. The host cell suitable for introduction may be a prokaryotic cell such as Escherichia coli, Bacillus subtilis, Streptomyces sp., Pseudomonas sp., Proteus mirabilis, or Staphylococcus sp. In addition, it may be fungi such as Aspergillus sp., yeasts such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces sp., or Neurospora crassa, other lower eukaryotic cells, or cells of higher eukaryotes such as plants or insects. In addition, it may be mammalian cells, specifically monkey kidney cells 7 (COS7), NSO cells, SP2 / 0, Chinese hamster ovary (CHO) cells, W138, baby hamster kidney (BHK) cells, MDCK, myeloma cell lines, HeLa cells, HuT 78 cells, or HEK293 cells, but is not limited thereto.

[0127] The transformation method of the present invention comprises any method of introducing nucleic acid into an organism, cell, tissue, or organ, and can be performed by selecting a suitable standard technique according to the host cell known in the art. Specifically, it includes, but is not limited to, electroporation, protoplasmic fusion, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, stirring using silicon carbide fibers, Agrobacterium-mediated transformation, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, lithium acetate-DMSO method, lipofectamine, and drying / inhibition-mediated transformation methods.

[0128] In this specification, the term "culture" means growing microorganisms or cells under appropriately artificially controlled environmental conditions. In the present invention, the method of producing hyaluronidase or a variant thereof by culturing a host cell expressing the hyaluronidase or a variant thereof can be carried out using methods widely known in the art.

[0129] Methods for recovering hyaluronidase or variants or products thereof from the above transformant or culture / culture medium may be methods known in the art, such as centrifugation, filtration, anion exchange chromatography, crystallization, and HPLC, but are not limited to these examples.

[0130]

[0131] Another aspect is to provide a method for improving the removal of impurities in a hyaluronidase purification process, comprising the above method. The same parts as described above also apply to the above method.

[0132] The above method has significantly superior removal efficiency of impurities (specifically, cleaved or hydrolyzed forms of hyaluronidase), so using the above method can significantly improve impurity removal efficiency.

[0133]

[0134] Another aspect provides a hyaluronidase or a variant thereof characterized by having a content of 10% or less of the hydrolyzed form of hyaluronidase. The same parts as described above apply equally to the hyaluronidase or the variant thereof.

[0135] In one embodiment, the hyaluronidase or a variant thereof may be in the form of a mixture in which the intact form and the hydrolyzed form of hyaluronidase are mixed.

[0136] In one embodiment, the hyaluronidase or a variant thereof may be prepared, purified, and / or obtained using the hyaluronidase purification method and / or the hyaluronidase preparation method of this specification.

[0137] The hydrolyzed form of the hyaluronidase described above may include a form in which peptide bonds in some regions of the hyaluronidase are cleaved due to hydrolysis. Specifically, the hyaluronidase may be cleaved into two or more fragments due to hydrolysis, and the two or more fragments formed by the cleaving may be connected by disulfided bonds between the fragments. Meanwhile, in this specification, the 'hydrolyzed form' of the hyaluronidase may be used interchangeably with the 'cleaved form'.

[0138] In one embodiment, the hydrolyzed form of the hyaluronidase is a form in which the peptide bond between arginine (R), the 311th amino acid of SEQ ID NO. 1, and serine (S), the 312th amino acid, is cleaved by hydrolysis and consists of two fragments (a first fragment and a second fragment), wherein the first fragment and the second fragment may be connected by a disulfide bond.

[0139] In one embodiment, the first fragment and the second fragment of the hydrolyzed form of hyaluronidase may comprise a polypeptide represented by an amino acid sequence of SEQ ID NO. 2 or 3 listed in Table 2, or an amino acid sequence having sequence identity of 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, 99.9% or more, or 100% or more with respect thereto, and specifically may be composed of a polypeptide represented by the amino acid sequence of SEQ ID NO. 2 or 3.

[0140] In one embodiment, the hyaluronidase or a variant thereof may comprise a hydrolyzed form of hyaluronidase in an amount of about 10% or less, about 8% or less, about 7% or less, about 6% or less, about 5% or less, less than about 5%, about 0.5 to about 6.5%, or about 1.5 to about 3.5% based on the total weight (or the total sum of all forms of hyaluronidase, including whole, oxidized, and cleaved forms, etc., is set to 100%), specifically in an amount of about 0.1 to about 10%, about 0.1 to about 8%, about 0.1 to about 7%, about 0.1 to about 6.5%, about 0.1 to about 6%, about 0.1 to about 5.5%, or about based on the total weight (or the total sum of all forms of hyaluronidase, including whole, oxidized, and cleaved forms, etc., is set to 100%). 0.1 to about 5%, about 0.1 to about 4.5%, about 0.1 to about 4%, about 0.1 to about 3.5%, about 0.1 to about 3%, about 0.1 to about 2.5%, about 0.1 to about 2.2%, about 0.1 to about 2%, about 0.1 to about 1.9%, about 0.1 to about 1.8%, about 0.1 to about 1.5%, about 0.1 to about 1.2%, about 0.1 to about 1.0%, about 0.1 to about 0.9%, about 0.1 to about 0.8%, about 0.1 to about 0.7%, about 0.1 to about 0.6%, about 0.3 to about 10%, about 0.3 to about 8%, about 0.3 to about 7%, About 0.3 to about 6.5%, about 0.3 to about 6%, about 0.3 to about 5.5%, about 0.3 to about 5%, about 0.3 to about 4.5%, about 0.3 to about 4%, about 0.3 to about 3.5%, about 0.3 to about 3%, about 0.3 to about 2.5%, about 0.3 to about 2.2%, about 0.3 to about 2%, about 0.3 to about 1.9%, about 0.3 to about 1.8%, about 0.3 to about 1.5%, about 0.3 to about 1.2%, about 0.3 to about 1.0%, about 0.3 to about 0.9%, about 0.3 to about 0.8%, about 0.3 to about 0.7%, about 0.3 to about 0.6%, about 0.4 to about 10%, about 0.4 to about 8%, about 0.4 to about 7%, about 0.4 to about 6.5%, about 0.4 to about 6%, about 0.4 to about 5.5%, about 0.4 to about 5%, about 0.4 to about 4.5%, about 0.4 to about 4%, about 0.4 to about 3.5%, about 0.4 to about 3%, about 0.4 to about 2.5%, about 0.4 to about 2.2%, about 0.4 to about 2%, about 0.4 to about 1.9%, about 0.4 to about 1.8%, about 0.4 to about 1.5%, about 0.4 to about 1.2%, about 0.4 to about 1.0%, about 0.4 to about 0.9%, about 0.4 to about 0.8%, about 0.4 to about 0.7%, about 0.4 to about 0.6%, about 0.5 to about 10%, about 0.5 to about 8%, about 0.5 to about 7%, about 0.5 to about 6.5%, about 0.5 to about 6%, about 0.5 to about 5.5%, about 0.5 to about 5%, about 0.5 to about 4.5%, about 0.5 to about 4%, about 0.5 to about 3.5%, about 0.5 to about 3%, about 0.5 to about 2.5%, about 0.5 to About 2.2%, about 0.5 to about 2%, about 0.5 to about 1.9%, about 0.5 to about 1.8%, about 0.5 to about 1.5%, about 0.5 to about 1.2%, about 0.5 to about 1.0%, about 0.5 to about 0.9%, about 0.5 to about 0.8%, about 0.5 to about 0.7%, about 0.5 to about 0.6%, about 0.6 to about 10%, about 0.6 to about 8%, about 0.6 to about 7%, about 0.6 to about 6.5%, about 0.6 to about 6%, about 0.6 to about 5.5%, about 0.6 to about 5%, about 0.6 to about 4.5%, about 0.6 to about 4%, about 0.6 Up to about 3.5%, about 0.6 to about 3%, about 0.6 to about 2.5%, about 0.6 to about 2.2%, about 0.6 to about 2%, about 0.6 to about 1.9%, about 0.6 to about 1.8%, about 0.6 to about 1.5%, about 0.6 to about 1.2%, about 0.6 to about 1.0%, about 0.6 to about 0.9%, about 0.6 to about 0.8%, or about 0.6 to about 0.7%, about 1.0 to about 10%, about 1.0 to about 8%, about 1.0 to about 7%, about 1.0 to about 6.5%, about 1.0 to about 6%, about 1.0 to about 5.5%, about 1.0 to about 5%, about 1.0 to about 4.5%, about 1.0 to about 4%, about 1.0 to about 3.5%, about 1.0 to about 3%, about 1.0 to about 2.5%, about 1.0 to about 2.2%, about 1.0 to about 2%, about 1.0 to about 1.9%, about 1.0 to about 1.8%, about 1.0 to about 1.5%, about 1.0 to about 1.2%, about 1.5 to about 10%, about 1.5 to about 8%, about 1.5 to about 7%, about 1.5 to about 6.5%, about 1.5 to about 6%, about 1.5 to about 5.5%, about 1.5 to about 5%, about 1.5 to about 4.5%, about 1.5 to about 4%, about 1.5 to about 3.5%, about 1.5 to about 3%, about 1.5 to about 2.5%, about 1.5 to about 2.2%, about 1.5 to about 2%, about 1.5 to about 1.9%, about 1.5 to about 1.8%, about 1.8 to about 10%, about 1.8 to about 8%, about 1.8 to about 7%, about 1.8 to about 6.5%, about 1.8 to about 6%, about 1.8 to about 5.5%, about 1.8 to about 5%, about 1.8 to about 4.5%, about 1.8 to about 4%, about 1.8 to about 3.5%, about 1.8 to about 3%, about 1.8 to about 2.5%, about 1.8 to about 2.2%, about 1.8 to about 2%, or about 1.8 to about 1.It may contain 9% of a hydrolyzed form of hyaluronidase.

[0141] In one embodiment, the intact form of hyaluronidase may comprise a polypeptide represented by the amino acid sequence of SEQ ID NO. 1 listed in Table 1 below or an amino acid sequence having sequence identity of 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, 99.9% or more, or 100% or more with respect thereto, and specifically, the intact form of hyaluronidase may be a polypeptide represented by the amino acid sequence of SEQ ID NO. 1.

[0142] In one embodiment, the hyaluronidase or a variant thereof may comprise at least about 70% of the intact form of hyaluronidase based on the total weight (or the total sum of all forms of hyaluronidase, including the intact form, oxidized form, cleaved form, etc., is set to 100%), and specifically, based on the total weight (or the total sum of all forms of hyaluronidase, including the intact form, oxidized form, cleaved form, etc., is set to 100%), about 70% to about 95%, about 70% to about 90%, about 70% to about 85%, about 70% to about 84%, about 70% to about 83.5%, about 70% to about 83%, about 70% to about 82.5%, about 70% to about 82%, about 70% to about 81.5%, about 70% to about 81%, about 70% to about 80.5%, about 70% to about 80%, about 70% to about 79.5%, about 70% to about 79%, about 70% to about 78.5%, about 72% to about 95%, about 72% to about 90%, about 72% to about 85%, about 72% to about 84%, about 72% to about 83.5%, about 72% to about 83%, about 72% to about 82.5%, about 72% to about 82%, about 72% to about 81.5%, about 72% to about 81%, about 72% to about 80.5%, about 72% to about 80%, about 72% to about 79.5%, about 72% to about 79%, about 72% to about 78.5%, about 73% to about 95%, about 73% to about 90%, about 73% to about 85%, about 73% to about 84%, about 73% to about 83.5%, about 73% to about 83%, about 73% to about 82.5%, about 73% to about 82%, about 73% to about 81.5%, about 73% to about 81%, about 73% to about 80.5%, about 73% to about 80%, about 73% to about 79.5%, about 73% to about 79%, about 73% to about 78%.5%, about 74% to about 95%, about 74% to about 90%, about 74% to about 85%, about 74% to about 84%, about 74% to about 83.5%, about 74% to about 83%, about 74% to about 82.5%, about 74% to about 82%, about 74% to about 81.5%, about 74% to about 81%, about 74% to about 80.5%, about 74% to about 80%, about 74% to about 79.5%, about 74% to about 79%, about 74% to about 78.5%, about 76% to about 95%, about 76% to about 90%, about 76% to about 85%, about 76% to about 84%, about 76% to about 83.5%, about 76% to about 83%, about 76% to about 82.5%, about 76% to about 82%, about 76% to about 81.5%, about 76% to about 81%, about 76% to about 80.5%, about 76% to about 80%, about 76% to about 79.5%, about 76% to about 79%, about 76% to about 78.5%, about 78% to about 95%, about 78% to about 90%, about 78% to about 85%, about 78% to about 84%, about 78% to about 83.5%, about 78% to about 83%, about 78% to about 82.5%, about 78% to about 82%, about 78% to about 81.5%, about 78% to about 81%, about 78% to about 80.5%, about 78% to about 80%, about 78% to about 79.5%, about 78% to about 79%, about 78% to about 78.5%, about 80% to about 95%, about 80% to about 90%, about 80% to about 85%, about 80% to about 84%, about 80% to about 83.5%, about 80% to about 83%, about 80% to about 82.5%, about 80% to about 82%, about 80% to about 81.5%, about 80% to about 81%, about 80% to about 80.5%, about 81.5% to about 95%, about 81.5% to about 90%, about 81.5% to about 85%, about 81.5% to about 84%, about 81.5% to about 83.5%, about 81.5% to about 83%, about 81.5% to about 82.5%, about 81.5% to about 82%, about 82% to about 95%, about 82% to about 90%, about 82% to about 85%, about 82% to about 84%, about 82% to about 83.5%, about 82% to about 83%, about 82% to about 82.5%, about 83% to about 95%, about 83% to about 90%, about 83% to about 85%, about 83% to about 84%, about 82% to about 83.5%, about 83% to about 95%, about 83% to about 90%, about 83% to about 85%, about 83% to about 84%, about 83% to about 83.5%, about 83.5% to about It may contain 95%, about 83.5% to about 90%, about 83.5% to about 85%, or about 83.5% to about 84% of an intact form of hyaluronidase.

[0143] The above hyaluronidase or its variant may have impurities removed more effectively than hyaluronidase or its variant obtained through another method (a method not using MMC), specifically, the content of the cleaved form of hyaluronidase may be low or similar, while the purity, yield, and / or inactivity of the intact form of hyaluronidase may be increased.

[0144] In one embodiment, the hyaluronidase or variant thereof obtained through the above method may exhibit an impurity content reduced by about 50% or more compared to the hyaluronidase or variant thereof obtained through another method (a method not using CEX or MMC), and specifically, the impurity ratio of the hyaluronidase or variant thereof obtained through the above method is about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 85% or more, about 87% or more, about 50% to 90%, about 50% to 87%, about 50% to 85%, about 50% to 80%, about 60% to 90%, about 60% to 87%, about 60% to 85%, about 60% to 85%, based on the impurity ratio of the hyaluronidase or variant thereof obtained through another method (a method not using CEX or MMC). It may be reduced by about 60% to 80%, about 70% to 90%, about 70% to 87%, about 70% to 85%, about 70% to 80%, about 80% to 90%, about 80% to 87%, or about 80% to 85%.

[0145]

[0146] Another aspect is to provide a composition comprising the above-mentioned hyaluronidase or a variant thereof. The same parts as described above also apply to the above composition.

[0147] In one embodiment, the composition may include an intact form and a hydrolyzed form of hyaluronidase.

[0148] When hyaluronidase is purified using a method according to one aspect, the cleavage form of the hyaluronidase can be effectively removed, resulting in excellent purity and / or yield of the intact hyaluronidase, and significantly excellent inactivity of the purified or produced hyaluronidase, so it can be applied in the hyaluronidase manufacturing and / or purification process.

[0149] Figure 1 is a diagram showing the hyaluronidase yield, purity, and impurity removal efficiency according to the concentration of sodium chloride added to the buffer in the MMC process.

[0150] The following experimental examples will be explained in more detail. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

[0151]

[0152] [Experimental Method]

[0153]

[0154] 1. RP-UPLC - %Peak2, %Peak3, and %Peak4 Measurement

[0155] The purity of the sample was tested by reversed phase-ultra performance liquid chromatography (RP-UPLC) based on the hydrophobicity of the material. In reversed phase-ultra performance liquid chromatography, the material is classified into five types (Peak1, Peak2, Peak3, Peak4, Peak5) and determined as a percentage of the amount of each. Among these, %Peak2 defines the hydrolyzed form (cleaved form) of hyaluronidase, %Peak3 defines the oxidized form of hyaluronidase, and %Peak4 defines the intact form of hyaluronidase.

[0156] Specifically, the above RP-UPLC is an Acquity UPLC under a temperature condition of 80 ± 2 ℃. ®A BEH300 C4 (1.7 μm, 2.1 x 100 mm) column was used, and the sample was injected at a rate of 0.2 mL / min. Mobile phase A (MP A) used Milli-Q containing 0.1% TFA (Trifluoroacetic Acid), and mobile phase B (MP B) used acetonitrile (ACN) containing 0.1% TFA; mobile phases A and B were operated under the following gradient program conditions. The absorbance of the eluent was monitored at 280 nm and analyzed using Empower software.

[0157] Run Time 28 min Gradient Step Linear (Curve setting 6 on Waters UPLC) Gradient Program Time (min) % MP A % MP B Initial 85 15 2.00 85 15 3.00 70 30 23.00 40 60 25.00 59 5 26.00 85 15 28.00 85 15

[0158]

[0159] 2. SE-UPLC - %HMW Measurement

[0160] The purity of the sample was examined by size exclusion chromatography (SE-HPLC). In size exclusion chromatography, the percentages of protein (hyaluronidase) monomers, high molecular weight species (HMW), and low molecular weight species (LMW) are determined. In size exclusion chromatography, the presence of HMW indicates protein aggregation, and the presence of LMW indicates protein fragments.

[0161] Specifically, the SE-HPLC used a TSKgel Super SW2000 SWXL (4 μm / 4.6 mm X 300 mm) column at a temperature of 25 ± 4°C, and the sample was injected at a rate of 0.2 mL / min. The mobile phase consisted of a solution containing 100 mM sodium phosphate and 200 mM sodium chloride with a pH of 6.8, and the sample was eluted for 30 minutes using an isocratic gradient of 0.2 mL / min. The absorbance of the eluent was monitored at 280 nm and analyzed using Empower software.

[0162]

[0163] 3. Analysis of Host Cell Proteins (HCP)

[0164] To analyze the host cell protein content of the sample, a sandwich immunoassay was performed using polyclonal antibodies generated against HCP in CHO cell lines based on the ECL-MSD (electrochemiluminescent-Meso Scale Discovery) assay format.

[0165] Specifically, as antibodies against HCP, the capture antibody was biotinized, and the detection antibody was labeled with a sulfo-tag. First, an MSD streptavidin-coated ECL plate was blocked using a blocking solution. A sample containing HCP was diluted using HCP assay buffer (PBS containing 1.0% BSA and 1.5% Tween-20), and the diluted sample was loaded onto the blocked plate. Subsequently, an antibody solution containing the capture antibody and the detection antibody was added to the wells loaded with the sample, and the plate was incubated for 4–5 hours. Afterward, the plate was washed, MSD read buffer was added, and the sample was analyzed using an MSD Imager.

[0166]

[0167] 4. Turbidimetric assay - Measurement of Specific Activity (IU / mg)

[0168] The enzyme activity of the sample was examined by turbidimetric assay. The degree of hyaluronidase activity is determined by turbidimetric analysis.

[0169] Specifically, each hyaluronidase sample formulation was prepared to a concentration of approximately 150 U / mL using sample dilution buffer (145 mM sodium chloride, 10 mM sodium phosphate, 10 mM L-methionine, 0.02% polysorbate 80, and 0.1% human albumin, pH 7.00 ± 0.05), loaded into a 96-well plate, and serially diluted using assay buffer (20 mM sodium phosphate, 77 mM sodium chloride, and 0.01% BSA, pH 7.00 ± 0.05). An HA substrate solution [300 mM sodium phosphate containing 0.03% HA (Hyaluronic Acid), pH 3.70 ± 0.05] was additionally loaded into each well, and the plate was incubated at 37 °C for approximately 45 minutes. Subsequently, a detection agent capable of detecting substrates degraded by hyaluronidase (24 mM sodium acetate, 79 mM acetic acid, and 0.1% BSA, pH 3.75 ± 0.05) was loaded into each well and incubated for about 10 minutes. The plate was loaded into a SpectraMax instrument, and the activity level of each sample was calculated using the SoftMax program.

[0170]

[0171] Example 1: Resin screening suitable for hyaluronidase purification process

[0172] For the purpose of the present invention, the following experiments were performed to screen resin materials suitable for effectively removing the hydrolyzed form in the hyaluronidase purification process.

[0173] Specifically, to search for a resin capable of removing the hyaluronidase hydrolysis form among resins of different modes (AEX, CEX, MMC, or HIC), the following resins were used as resins for each mode.

[0174] 1) Anion Exchange Chromatography (AEX): POROS XQ

[0175] 2) Cation exchange chromatography (CEX): Fractogel SO3 -

[0176] 3) Mixed-mode chromatography (MMC): Eshmuno HCX

[0177] 4) Hydrophobic Interaction Chromatography (HIC): Capto Butyl ImpRes

[0178] Meanwhile, the above-mentioned multi-mode chromatography utilized a column containing a material having cation exchange and hydrophobic interaction functions.

[0179] All processes were carried out at room temperature at 200 cm / h with a column height of 18-22 cm, with the loading, chase, and elution steps carried out at 100 cm / h. First, each column was equilibrated using 5 CV (column volume) of equilibration buffer under the following conditions.

[0180] 1) Anion exchange chromatography: 20 mM sodium phosphate, 50 mM NaCl, pH 7.5

[0181] 2) Cation exchange chromatography: 20 mM sodium phosphate, 50 mM NaCl, pH 6.0

[0182] 3) Multi-mode chromatography: 20 mM sodium phosphate, 50 mM NaCl, pH 6.0

[0183] 4) Hydrophobic interaction chromatography: 20 mM sodium phosphate, 1200 mM AMS, pH 7.0

[0184] Next, samples titrated to the same salt concentration and pH (samples pretreated using AEX) were loaded into each column (AEX: 13 g / L resin , CEX / MMC: 10 g / L resin , HIC: 20 g / L resin Afterwards, a tracking step was performed with the equilibrium buffer 3 CV.

[0185] Next, hyaluronidase was recovered from each column using an elution buffer under the following conditions, and the results were compared.

[0186] 1) Anion exchange chromatography: 20 mM sodium phosphate, 50 to 500 mM NaCl, pH 7.5

[0187] - A: 20 mM sodium phosphate, 50 mM NaCl, pH 7.5

[0188] - B: 20 ​​mM sodium phosphate, 500 mM NaCl, pH 7.5

[0189] - Gradient elution: B 0% → B 100%, 20 CV (Concentration gradient is performed in elution buffer 20 times the column volume so that the NaCl concentration increases from 50 mM to 500 mM)

[0190] 2) Cation exchange chromatography: 20 mM sodium phosphate, 50 to 500 mM NaCl, pH 6.0

[0191] - A: 20 mM sodium phosphate, 50 mM NaCl, pH 6.0

[0192] - B: 20 ​​mM sodium phosphate, 500 mM NaCl, pH 6.0

[0193] - Gradient elution: B 0% → B 100%, 20 CV (Concentration gradient is performed in elution buffer 20 times the column volume so that the NaCl concentration increases from 50 mM to 500 mM)

[0194] 3) Multi-mode chromatography: 20 mM sodium phosphate, 50 to 1000 mM NaCl, pH 6.0

[0195] - A: 20 mM sodium phosphate, 50 mM NaCl, pH 6.0

[0196] - B: 20 ​​mM sodium phosphate, 1000 mM NaCl, pH 6.0

[0197] - Gradient elution: B 0% → B 100%, 20 CV (Concentration gradient is performed in elution buffer 20 times the column volume so that the NaCl concentration increases from 50 mM to 1000 mM)

[0198] 4) Hydrophobic interaction chromatography: 20 mM sodium phosphate, 370 mM AMS, pH 7.0 (isocratic elution).

[0199]

[0200] As a result of the above experiment, it was confirmed that when CEX and MMC (CEX+HIC) resins were applied compared to other resins, %peak 2 (hydrolyzed form) decreased by approximately 12.9-13.6%p (loading 15.8% → release 2.2-2.9%). In addition, it was confirmed that the specific activity of hyaluronidase increased by approximately 35-38% (loading 71,105 IU / mg → release 95,930-98,107 IU / mg) (Table 4).

[0201] ClassificationEnzymeYield(%)- pH 5.0Specific Activity(IU / mg)- pH 5.0HCP(ppm)HCPLRVRP-UPLCSE-HPLC%Peak 2 (Hydrolyzed)%Peak 3 (Oxidized)%Peak 4 (Main)%HMW Loading Sample (Pilot AEX Eluate)N / A71,105[4,249]140,488N / A15.89.165.64.0AEX93.079,88354,0100.417.49.164.74.2CEX70.595, 9307,7401.32.29.982.43.4MMC79.098,1079,4121.22.910.681.50.3HIC94.681,9716,9621.315.99.467.00.7

[0202] Based on the above results, it can be seen that applying CEX or MMC (CEX+HIC) resin among various types of resins can improve the purification purity and activity of hyaluronidase, and that MMC exhibits superior purification efficiency.

[0203]

[0204] Example 2: Establishment of MMC Process Conditions

[0205] To establish optimal conditions for MMC execution when applying MMC in the hyaluronidase purification process, the following experiments were conducted.

[0206] Specifically, the above MMC process was carried out at room temperature at 200 cm / h with a column height of 18-22 cm, and the loading, chase, wash, and elution steps were carried out at 100 cm / h. First, equilibration was performed by washing the MMC column with an equilibration buffer of 5 CV under the following conditions.

[0207] Condition 1) 20 mM sodium phosphate, 300 mM NaCl, pH 6.0

[0208] Condition 2) 20 mM sodium phosphate, 50 mM NaCl, pH 6.0

[0209] Condition 3) 20 mM sodium phosphate, 100 mM NaCl, pH 6.0

[0210] Next, samples adjusted to the same salt concentration and pH (samples pretreated using AEX) were loaded onto the MMC column (15 g / L resin Afterwards, a tracking step was performed with the equilibrium buffer 3 CV.

[0211] Next, for multimode chromatography #2 and #3, a washing step was performed using a wash buffer under the following conditions.

[0212] - 20 mM sodium phosphate, 300 mM NaCl, pH 6.0

[0213] Next, hyaluronidase was recovered from the column through the elution step described in Example 1, and the result (AEX-MMC) was compared.

[0214] Specific experimental conditions are listed in Table 5 below.

[0215] Resin SequenceMMC (Eshmuno HCX) Experimental ConditionStep[NaCl (MMC), AMS (HIC)] mMLoadRatio(g / L resin )LoadWash 1ElutionLoad (HDC-24A17 (Pilot AEX Eluate))AEX-MMC-HIC #1MMC300N / A60015HIC1,200N / A37020AEX-MMC-HIC #2MMC5030060015HIC1,200N / A37020AEX-MMC-HIC #3MMC10030060015HIC1,200N / A37020

[0216] Meanwhile, in the above experiment, the eluent of MMC was additionally applied to HIC based on the method described in Example 1, and hyaluronidase was recovered from it to further confirm the results (AEX-MMC-HIC).

[0217] Based on the experimental results above, it was confirmed that 'Condition 3' exhibited a significantly superior purification effect when considering %peak 2 and HCP removal efficiency. Specifically, the %peak 2 of the final purified product decreased by 14.0%p (start material 15.8% → final purified product 1.8%), and the specific activity increased by 51% (start material 71,105 IU / mg → final purified product 107,967 IU / mg), confirming that the purity of the purified product was significantly improved (Tables 6, 7 and Fig. 1).

[0218] Resin SequenceSTEPResultsEnzyme Yield(%) - pH 5.0SpecificActivity(IU / mg)- pH 5.0HCP(ppm)HCPLRV(Log reduction value)Step3-column (Assuming AEX 78%)Loading sample (Pilot AEX Eluate)N / AN / A71,105[4,249]140,488N / AAEX-MMC-HIC #1MMC67.453.0100,862210,345-0.2HIC100.7105,98835,3290.8AEX-MMC-HIC #2MMC85.769.592,84214,2821.0HIC103.899,1457841.3AEX-MMC-HIC #3MMC91.775.1103,235137,2550.0HIC104.9107,9675,2201.4

[0219] Resin SequenceSTEPResultsRP-UPLCSE-HPLC%Peak 2 (Hydrolyzed)%Peak 3 (Oxidized)%Peak 4 (Main)%HMWLoading Sample (Pilot AEX Eluate)15.89.165.64.0AEX-MMC-HIC #1MMC0.910.084.52.8HIC0.610.485.60.7AEX-MMC-HIC #2MMC6.110.678.50.7HIC5.611.079.01.4AEX-MMC-HIC #3MMC1.810.783.31.3HIC1.811.383.51.2

[0220]

[0221] 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. A) A step comprising applying a sample containing hyaluronidase to a first chromatography, Method for purifying hyaluronidase.

2. The method of claim 1, wherein the first chromatography comprises cation exchange chromatography (CEX) or mixed-mode chromatography (MMC).

3. The method of claim 2, wherein the cation exchange chromatography utilizes a column comprising a cation exchange chromatography material.

4. The method of claim 2, wherein the cation exchange chromatography uses a column comprising Fractogel SO3- (M).

5. The method of claim 2, wherein the multi-mode chromatography utilizes a column comprising a material having a cation exchange function.

6. The method of claim 2, wherein the multi-mode chromatography utilizes a column comprising a material having a cation exchange function and a hydrophobic interaction function.

7. The method of claim 2, wherein the multi-mode chromatography utilizes a column containing Eshmuno HCX.

8. In claim 1, the one applied to the first chromatography is, A-1) A step of loading a sample containing hyaluronidase into a first chromatography column; and A-2) Step of eluting the product from the column using an elution buffer A method that includes 9. The method of claim 8, wherein the first chromatography column is a column equilibrated with an equilibration buffer.

10. The method of claim 9, wherein the equilibrium buffer comprises about 10 mM to about 500 mM of salt.

11. The method of claim 9, wherein the equilibrium buffer comprises about 10 mM to about 500 mM of sodium chloride.

12. The method of claim 9, wherein the equilibrium buffer comprises about 80 mM to about 200 mM of sodium chloride.

13. The method of claim 9, wherein the equilibrium buffer comprises about 5 mM to about 50 mM of sodium phosphate.

14. The method of claim 9, wherein the pH of the equilibrium buffer is in the range of about 4.0 to about 8.

0.

15. In claim 8, the sample containing the hyaluronidase is about 5 to about 30 g / L resin A method of loading at a concentration of (g protein / L resin volume).

16. The method of claim 8, wherein the elution buffer comprises about 10 mM to about 2000 mM of salt.

17. The method of claim 8, wherein the elution buffer comprises about 10 mM to about 2000 mM of sodium chloride.

18. The method of claim 8, wherein the elution buffer comprises about 5 mM to about 50 mM of sodium phosphate.

19. The method of claim 8, wherein the pH of the elution buffer is in the range of about 4.0 to about 8.

0.

20. In claim 8, the method A step of washing a first chromatography column loaded with a sample containing hyaluronidase using a washing buffer. A method that additionally includes 21. The method of claim 20, wherein the washing buffer comprises about 100 mM to about 600 mM of salt.

22. The method of claim 20, wherein the washing buffer comprises about 50 mM to about 600 mM of sodium chloride.

23. The method of claim 20, wherein the washing buffer comprises about 5 mM to about 50 mM of sodium phosphate.

24. The method of claim 20, wherein the pH of the washing buffer is in the range of about 4.0 to about 8.

0.

25. In claim 8, the method A step of tracking a first chromatography column loaded with a sample containing hyaluronidase using a tracking buffer. A method that additionally includes 26. In Claim 1, the method B) A step of applying the eluent obtained from the first chromatography to the second chromatography. A method that additionally includes 27. The method of claim 26, wherein the second chromatography comprises hydrophobic interaction chromatography.

28. The method of claim 27, wherein the hydrophobic interaction chromatography uses a column comprising Phenyl Sepharose 6 FF.

29. In claim 26, the method applied to the second chromatography is, B-1) A step of loading the eluent obtained from the first chromatography into a second chromatography column; and B-2) Step of eluting the product from the column using an elution buffer A method that includes 30. The method of claim 29, wherein the second chromatography column is a column equilibrated with an equilibration buffer.

31. The method of claim 30, wherein the equilibrium buffer comprises about 500 mM to about 2000 mM of salt.

32. The method of claim 30, wherein the equilibrium buffer comprises about 500 mM to about 2000 mM of AMS.

33. The method of claim 30, wherein the equilibrium buffer comprises about 5 mM to about 50 mM of sodium phosphate.

34. The method of claim 30, wherein the pH of the equilibrium buffer is in the range of about 5.0 to about 9.

0.

35. The method of claim 29, wherein the elution buffer comprises about 100 mM to about 600 mM of salt.

36. The method of claim 29, wherein the elution buffer comprises about 100 mM to about 600 mM of AMS.

37. The method of claim 29, wherein the elution buffer comprises about 5 mM to about 50 mM of sodium phosphate.

38. The method of claim 29, wherein the pH of the elution buffer is in the range of about 5.0 to about 9.

0.

39. In claim 29, the method A step of tracking a second chromatography column loaded with an eluent obtained from a first chromatography using a tracking buffer. A method that additionally includes 40. The method of claim 1, wherein the sample containing the hyaluronidase comprises an eluent obtained by applying anion exchange chromatography.

41. The method of claim 1, wherein the sample containing the hyaluronidase comprises a product obtained by applying depth filtration.

42. The method of claim 1, wherein the sample containing the hyaluronidase comprises a product obtained by applying host cell protein (HCP) precipitation.

43. The method of claim 1, wherein the method is for removing impurities contained in the sample.

44. The method of claim 43, wherein the impurity comprises a hydrolyzed form of hyaluronidase.

45. A method according to any one of claims 1 to 44, wherein the hyaluronidase obtained through the method comprises an intact form and a hydrolyzed form.

46. ​​The method of claim 45, wherein the intact form of the hyaluronidase is a polypeptide represented by the amino acid sequence of SEQ ID NO. 1 or an amino acid sequence having at least 80% sequence identity therewith.

47. The method of claim 45, wherein the hydrolyzed form of the hyaluronidase comprises a form in which the peptide bonds of a portion of the hyaluronidase are hydrolyzed and cleaved.

48. The method of claim 45, wherein the hydrolyzed form of the hyaluronidase comprises two or more fragments formed by cleaving an intact form of hyaluronidase.

49. The method of claim 48, wherein two or more fragments formed by cutting are connected through disulfide bonds between the fragments.

50. In claim 45, the hydrolyzed form of the hyaluronidase is 1) A first fragment which is a polypeptide represented by the amino acid sequence of SEQ ID NO. 2 or an amino acid sequence having at least 80% sequence identity therewith, and 2) A second fragment which is a polypeptide represented by the amino acid sequence of SEQ ID NO. 3 or an amino acid sequence having at least 80% sequence identity therewith. A method that includes 51. The method of claim 50, wherein the hydrolyzed form of the hyaluronidase is a form in which the 25th amino acid (cysteine) and the 5th amino acid (cysteine) of the second fragment are connected by a disulfide bond.

52. The method of claim 45, wherein the hyaluronidase obtained through the method comprises a hydrolyzed form of 10% or less based on the total weight (or the total sum of all forms of hyaluronidase including whole form, oxidized form, and cleaved form, etc.).

53. The method of claim 45, wherein the hyaluronidase obtained through the method comprises at least 70% of an intact form based on the total weight (or the sum of all forms of hyaluronidase including intact form, oxidized form, cleaved form, etc.).

54. A method for producing hyaluronidase comprising the method of any one of claims 1 to 53.

55. A method for improving the removal of impurities in a hyaluronidase purification process, comprising the method of any one of claims 1 to 53.

56. Hyaluronidase or a variant thereof, characterized by having a content of 10% or less of the hydrolyzed form of hyaluronidase.

57. The hyaluronidase or a variant thereof according to claim 56, wherein the hyaluronidase or a variant thereof is obtained using the method of claims 1 to 53.

58. The hyaluronidase or a variant thereof according to claim 56, wherein the hydrolyzed form of the hyaluronidase comprises a form in which the peptide bonds of a portion of the hyaluronidase are hydrolyzed and cleaved.

59. The hyaluronidase or a variant thereof according to claim 56, wherein the hydrolyzed form of the hyaluronidase comprises two or more fragments formed by cleaving an intact form of hyaluronidase.

60. Hyaluronidase or a variant thereof, wherein the two or more fragments formed by cleavage are connected through disulfide bonds between the fragments.

61. In claim 56, the hydrolyzed form of the hyaluronidase is 1) A first fragment which is a polypeptide represented by the amino acid sequence of SEQ ID NO. 2 or an amino acid sequence having at least 80% sequence identity therewith, and 2) A second fragment which is a polypeptide represented by the amino acid sequence of SEQ ID NO. 3 or an amino acid sequence having at least 80% sequence identity therewith. Hyaluronidase or a variant thereof, comprising 62. Hyaluronidase or a variant thereof according to claim 61, wherein the hydrolyzed form of the hyaluronidase is a form in which the 25th amino acid (cysteine) and the 5th amino acid (cysteine) of the second fragment are connected by a disulfide bond.

63. A hyaluronidase or a variant thereof according to claim 56, wherein the content of the hydrolyzed form of the hyaluronidase is about 0.5 to about 6.5% based on the total weight (or the total sum of all forms of hyaluronidase including whole form, oxidized form, and cleaved form, etc.).

64. A hyaluronidase or a variant thereof according to claim 56, wherein the content of the hydrolyzed form of the hyaluronidase is about 1.5 to about 3.5% based on the total weight (or the total sum of all forms of hyaluronidase including whole form, oxidized form, and cleaved form, etc.).

65. The hyaluronidase or a variant thereof according to claim 56, wherein the hyaluronidase or a variant thereof comprises hyaluronidase in its intact form.

66. A hyaluronidase or a variant thereof, wherein the intact form of the hyaluronidase comprises a polypeptide represented by the amino acid sequence of SEQ ID NO. 1 or an amino acid sequence having at least 80% sequence identity with respect thereto.

67. A hyaluronidase or a variant thereof according to claim 65, wherein the content of the intact form of the hyaluronidase is about 70% or more based on the total weight (or the total sum of all forms of hyaluronidase including the intact form, oxidized form, and cleaved form, etc.).

Images