Hyaluronidase purification method comprising protease removal process

Abstract

The present invention relates to: a hyaluronidase purification method for removing a protease associated with an increase in cleaved hyaluronidase; a hyaluronidase preparation method using same; and hyaluronidase or a variant thereof obtained by using the method. When hyaluronidase is purified using the method according to one aspect, proteases capable of inducing hydrolysis and cleavage of hyaluronidase can be effectively removed, and thus it is possible to prevent an increase in the hydrolyzed / cleaved form content during hyaluronidase storage, and to prepare hyaluronidase having improved stability.

Description

Hyaluronidase purification method including a protease removal process

[0001] The invention relates to a method for purifying hyaluronidase to remove a protease associated with cleaved hyaluronidase increase, 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, there is a need to develop a technology for purifying hyaluronidase with improved stability, in which the content of the cleavage form does not increase even when stored at room temperature.

[0007] One aspect provides a method for purifying hyaluronidase, comprising the step of applying a sample containing hyaluronidase to aminophenylboronate (APB) 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 an increase rate of the content of the hydrolyzed form of hyaluronidase of 30% or less.

[0011] Another aspect provides a hyaluronidase or a variant thereof characterized by having a content of about 10% or less of the hydrolyzed form of hyaluronidase.

[0012] 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.

[0013] 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.

[0014]

[0015] One aspect provides a method for purifying hyaluronidase, comprising the step of applying a sample containing hyaluronidase to aminophenylboronate (APB) chromatography.

[0016] 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).

[0017] The above hyaluronidase may include hyaluronidase or a variant thereof.

[0018] 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.

[0019] The purity of the above hyaluronidase or its variant may be 98% or more, 98.5% or more, 99% or more, or 100%.

[0020] The above hyaluronidase or its variant means an intact form, and may include an intact form from which the hydrolyzed form has been removed.

[0021] 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.

[0022] 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).

[0023] 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.

[0024] 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.

[0025] The above aminophenylboronate (APB) chromatography may use a column containing m-aminophenylboronic acid or a salt thereof.

[0026] The term "m-aminophenylboronic acid" in this specification refers to a compound having the structure of Chemical Formula 1 below. The m-aminophenylboronic acid may be a compound having Cas no. 30418-59-8. Through the strong interaction between the diol group of the aminophenylboronic acid and the -OH group of the protease (specifically, serine protease or cysteine ​​protease), the protease can be strongly bound to the APB column, so the protease can be effectively removed using APB chromatography during the hyaluronidase purification process.

[0027] [Chemical Formula 1]

[0028]

[0029] The term "salt" in this specification refers to a salt in a usable form among substances in which cations and anions are bonded by electrostatic attraction, and typically includes metal salts, salts with organic bases, salts with inorganic acids, salts with organic acids, salts with basic or acidic amino acids, etc. For example, metal salts may include alkali metal salts (sodium salts, potassium salts, etc.), alkaline earth metal salts (calcium salts, magnesium salts, barium salts, etc.), aluminum salts, etc.; salts with organic bases may include salts with triethylamine, pyridine, picoline, 2,6-rutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, N,N-dibenzylethylenediamine, etc.; and salts with inorganic acids may include salts with hydrochloric acid, hydrobromide, nitric acid, sulfuric acid, phosphoric acid, etc. Salts with organic acids may include formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, etc.; salts with basic amino acids may include arginine, lysine, ornithine, etc.; and salts with acidic amino acids may include aspartic acid, glutamic acid, etc. Particularly desirable salts include inorganic salts such as alkali metal salts (e.g., sodium salts, potassium salts, etc.) and alkaline earth metal salts (e.g., calcium salts, magnesium salts, barium salts, etc.) and organic salts such as ammonium salts when the compound has acidic functional groups therein, and salts with inorganic acids such as hydrochloric acid, hydrobromide, nitric acid, sulfuric acid, phosphoric acid, etc. and salts with organic acids such as acetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, methanesulfonic acid, p-toluenesulfonic acid, etc. when the compound has basic functional groups therein.

[0030] The above aminophenylboronic acid may include aminophenylboronic acid or a solvate thereof.

[0031] The term "solvate" in this specification refers to a compound solvated in an organic or inorganic solvent. The solvate may be, for example, a hydrate.

[0032] In one embodiment, applying a sample containing hyaluronidase to the APB chromatography may include: 1) loading a sample containing hyaluronidase into an APB chromatography column; and 2) eluting a product from the column using an elution buffer.

[0033] The APB chromatography column may be a column equilibrated with an equilibration buffer, specifically, a column equilibrated using an equilibration buffer before loading a sample containing hyaluronidase. Accordingly, the method may additionally include a step of equilibrating the APB chromatography column with an equilibration buffer before loading the sample containing hyaluronidase of step '1'.

[0034] The term "equilibration buffer" in this specification refers to a buffer used to equilibrate a column or resin before loading a sample containing a target protein, such as hyaluronidase, onto the column.

[0035] 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).

[0036] The equilibrium buffer may comprise about 300 mM to about 2000 mM of salt, specifically about 300 mM to about 2000 mM, about 300 mM to about 1800 mM, about 300 mM to about 1600 mM, about 300 mM to about 1400 mM, about 300 mM to about 1300 mM, about 300 mM to about 1250 mM, about 300 mM to about 1200 mM, about 400 mM to about 2000 mM, about 400 mM to about 1800 mM, about 400 mM to about 1600 mM, about 400 mM to about 1400 mM, about 400 mM to about 1300 mM, about 400 mM to about 1250 mM, about 400 mM to about 1200 mM, about 500 mM to about 2000 mM, approximately 500 mM to approximately 1800 mM, approximately 500 mM to approximately 1600 mM, approximately 500 mM to approximately 1400 mM, approximately 500 mM to approximately 1300 mM, approximately 500 mM to approximately 1250 mM, approximately 500 mM to approximately 1200 mM, approximately 700 mM to approximately 2000 mM, approximately 700 mM to approximately 1800 mM, approximately 700 mM to approximately 1600 mM, approximately 700 mM to approximately 1400 mM, approximately 700 mM to approximately 1300 mM, approximately 700 mM to approximately 1250 mM, approximately 700 mM to approximately 1200 mM, approximately 900 mM to approximately 2000 mM, approximately 900 mM to approximately 1800 mM, approximately 900 mM to approximately 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, about 1150 mM to about 1250 mM, about 1150 It may contain a salt of 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.

[0037] 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 300 mM to about 2000 mM of AMS, and the pH may be in the range of about 5.0 to about 9.0.

[0038] The above equilibrium buffer may contain about 300 mM to about 2000 mM of AMS, specifically about 300 mM to about 2000 mM, about 300 mM to about 1800 mM, about 300 mM to about 1600 mM, about 300 mM to about 1400 mM, about 300 mM to about 1300 mM, about 300 mM to about 1250 mM, about 300 mM to about 1200 mM, about 400 mM to about 2000 mM, about 400 mM to about 1800 mM, about 400 mM to about 1600 mM, about 400 mM to about 1400 mM, about 400 mM to about 1300 mM, about 400 mM to about 1250 mM, about 400 mM to about 1200 mM, 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, about 1150 mM to about 1250 mM, about 1150 It may contain AMS of 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.

[0039] 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.

[0040] 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.

[0041] 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 8 to about 30 g / L resin , about 8 to about 25 g / L resin , about 8 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 12 to about 30 g / L resin , about 12 to about 25 g / L resin , about 12 to about 20 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 15 to about 30 g / L resin , about 15 to about 25 g / L resin , or about 15 to about 20 g / L resin It may be loaded at a concentration of

[0042] In one embodiment, the method may include 2) a step of eluting a product from the column using an elution buffer. The step 2) is a step for recovering a product present in the column, and specifically, may be a step of recovering a hyaluronidase from which protease has been removed or a purified product containing hyaluronidase using an elution buffer.

[0043] The above elution step may utilize a gradient elution or isocratic elution method.

[0044] The term "elution buffer" as used in this specification is a buffer used to elute a target protein from a stationary phase.

[0045] 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.

[0046] The above elution buffer may contain about 50 mM to about 500 mM of salt, specifically about 50 mM to about 500 mM, about 50 mM to about 400 mM, about 50 mM to about 300 mM, about 50 mM to about 200 mM, about 50 mM to about 170 mM, about 50 mM to about 150 mM, about 100 mM to about 500 mM, about 100 mM to about 400 mM, about 100 mM to about 300 mM, about 100 mM to about 200 mM, about 100 mM to about 170 mM, about 100 mM to about 150 mM, about 130 mM to about 500 mM, about 130 mM to about 400 mM, about 130 mM to about 300 mM, about 130 mM to about 200 mM, about It may contain salts of 130 mM to about 170 mM, about 130 mM to about 150 mM, about 150 mM to about 500 mM, about 150 mM to about 400 mM, about 150 mM to about 300 mM, about 150 mM to about 200 mM, or about 150 mM to about 170 mM.

[0047] In one embodiment, the elution buffer may comprise sodium phosphate and sodium chloride, specifically, the elution buffer may comprise about 1 mM to about 20 mM of sodium phosphate and about 50 mM to about 500 mM of sodium chloride, and the pH may be in the range of about 5.0 to about 9.0.

[0048] The above elution buffer may contain sodium chloride in an amount of about 50 mM to about 500 mM, specifically about 50 mM to about 500 mM, about 50 mM to about 400 mM, about 50 mM to about 300 mM, about 50 mM to about 200 mM, about 50 mM to about 170 mM, about 50 mM to about 150 mM, about 100 mM to about 500 mM, about 100 mM to about 400 mM, about 100 mM to about 300 mM, about 100 mM to about 200 mM, about 100 mM to about 170 mM, about 100 mM to about 150 mM, about 130 mM to about 500 mM, about 130 mM to about 400 mM, about 130 mM to about 300 mM, about 130 mM to about 200 mM, It may contain sodium chloride of about 130 mM to about 170 mM, about 130 mM to about 150 mM, about 150 mM to about 500 mM, about 150 mM to about 400 mM, about 150 mM to about 300 mM, about 150 mM to about 200 mM, or about 150 mM to about 170 mM.

[0049] The above elution buffer may contain sodium phosphate in an amount of about 1 mM to about 20 mM, specifically about 1 mM to about 20 mM, about 1 mM to about 15 mM, about 1 mM to about 10 mM, about 1 mM to about 7 mM, about 1 mM to about 5 mM, about 3 mM to about 20 mM, about 3 mM to about 15 mM, about 3 mM to about 10 mM, about 3 mM to about 7 mM, about 3 mM to about 5 mM, about 5 mM to about 20 mM, about 5 mM to about 15 mM, about 5 mM to about 10 mM, or about 5 mM to about 7 mM of sodium phosphate.

[0050] 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.

[0051] In one embodiment, the method may further include the step of tracking an APB 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 an APB chromatography column.

[0052] The term "chase buffer" in this specification refers to a buffer used after loading a sample containing a target protein onto a column.

[0053] The above tracking buffer may contain the same composition as the equilibrium buffer.

[0054] The eluent obtained from the above APB chromatography may contain hyaluronidase.

[0055] In the above method, the step of applying the sample to APB chromatography may be for removing or reducing factors capable of inducing the hydrolysis of hyaluronidase in the sample, specifically for removing or reducing protease.

[0056] The terms “protease” or “proteinase” in this specification refer to enzymes that hydrolyze proteins, including enzymes that break down proteins and peptides into smaller peptides and amino acids.

[0057] The above protease may include the cysteine ​​protease family, and specifically may include cysteine ​​protease.

[0058] In one embodiment, the cysteine ​​protease may include legumin, cathepsin B, cathepsin X, and procathepsin L, etc.

[0059] The above protease may include the serine protease family, and specifically may include serine protease.

[0060] In one embodiment, the serine protease family may include trypsin-like protease, chymotrypsin-like protease, thrombin-like protease, and elastase-like protease.

[0061] In one embodiment, the method may further include the step of A) applying a sample containing the hyaluronidase to a first chromatography before applying it to APB chromatography.

[0062] 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.

[0063] In one embodiment, the first chromatography may include cation exchange chromatography (CEX) or mixed-mode chromatography (MMC), and specifically, may include mixed-mode chromatography.

[0064] 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."

[0065] 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.

[0066] The above cation exchange chromatography may utilize a column containing a cation exchange chromatography material.

[0067] 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.

[0068] 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.

[0069] 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."

[0070] The above multi-mode chromatography may utilize a column containing a material having a cation exchange function, and specifically, may utilize a column containing a material having a cation exchange function and a hydrophobic interaction function.

[0071] 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.

[0072] The above multi-mode chromatography may utilize a column containing Eshmuno HCX, and specifically, may contain Eshmuno HCX as the resin of the column.

[0073] 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.

[0074] The first chromatography column may be a column equilibrated with an equilibration buffer, specifically, a column equilibrated using an equilibration buffer before loading a sample containing hyaluronidase. Accordingly, the method may additionally include a step of equilibrating the first chromatography column with an equilibration buffer before loading the sample containing hyaluronidase of step 'A-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, 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).

[0076] 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 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 100 mM to about 130 mM, about It may contain 100 mM to about 120 mM, or about 100 mM to about 110 mM of salt.

[0077] 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.

[0078] 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.

[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 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.

[0081] The sample containing the above hyaluronidase may be loaded onto a first chromatography column at a concentration of about 5 to about 30 g / Lresin (g protein / L resin volume) per unit volume of resin, specifically about 5 to about 30 g / Lresin, about 5 to about 25 g / Lresin, about 5 to about 20 g / Lresin, about 5 to about 18 g / Lresin, about 5 to about 16 g / Lresin, about 5 to about 15 g / Lresin, about 5 to about 12 g / Lresin, about 5 to about 10 g / Lresin, about 10 to about 30 g / Lresin, about 10 to about 25 g / Lresin, about 10 to about 20 g / Lresin, about 10 to about 18 g / Lresin, about 10 to about 16 g / Lresin, about 10 to about 15 g / Lresin, about 10 to about 12 g / Lresin, about 12 to about 30 g / Lresin, about 12 to about 25 g / Lresin, about 12 to about 20 g / Lresin, about 12 to about 18 g / Lresin, about 12 to about 16 g / Lresin, about 12 to about 15 g / Lresin, about 14 to about 30 g / Lresin, about 14 to about 25 g / Lresin, about 14 to about 20 g / Lresin, about 14 to about 18 g / Lresin, about 14 to about 16 g / Lresin, about 15 to about 30 g / Lresin, about 15 to about 25 g / Lresin, about 14 to about 18 g / Lresin, about 14 to about 16 g / Lresin, about 14 to about 15 g / Lresin, about 15 to about 30 g / Lresin, about 15 to about 25 g / Lresin, It may be loaded at a concentration of about 15 to about 20 g / Lresin, about 15 to about 18 g / Lresin, or about 15 to about 16 g / Lresin.

[0082] 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.

[0083] The above-mentioned elution step may utilize a gradient elution or isocratic elution method, and specifically, may utilize a gradient elution.

[0084] 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.

[0085] 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,It may comprise a salt 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.

[0086] 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.

[0087] 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 in an amount 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.

[0088] 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.

[0089] 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.

[0090] 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.

[0091] 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.

[0092] 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).

[0093] 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.

[0094] 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.

[0095] 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.

[0096] 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.

[0097] 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.

[0098] 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.

[0099] 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.

[0100] The term "chase buffer" in this specification refers to a buffer used after loading a sample containing a target protein onto a column.

[0101] The above tracking buffer may contain the same composition as the equilibrium buffer.

[0102] The eluent obtained from the first chromatography above may contain hyaluronidase.

[0103] In one embodiment, the method may further include the step of B) applying the eluent obtained from the first chromatography to the second chromatography.

[0104] 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.

[0105] The above hydrophobic interaction chromatography may utilize a column containing a hydrophobic interaction chromatography material.

[0106] 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.

[0107] 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.

[0108] 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.

[0109] 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

[0110] 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)'.

[0111] 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.

[0112] 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.

[0113] 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.

[0114] 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.

[0115] 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.

[0116] 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.

[0117] 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.

[0118] The above elution step may utilize a gradient elution or isocratic elution method, and specifically, may utilize isocratic elution.

[0119] 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.

[0120] 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.

[0121] 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.

[0122] 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.

[0123] 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.

[0124] 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.

[0125] 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.

[0126] The above tracking buffer may contain the same composition as the equilibrium buffer.

[0127] The eluent obtained from the second chromatography above may contain hyaluronidase.

[0128] In one embodiment, the method may comprise: A) applying a sample containing hyaluronidase to a first chromatography; B) applying an eluent obtained from the first chromatography to a second chromatography; and C) applying an eluent obtained from the second chromatography to an APB chromatography.

[0129] 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 obtained from host cells or organisms that express / produce hyaluronidase.

[0130] 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 obtained directly from a host cell or organism expressing / producing 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.

[0131] 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.

[0132] 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.

[0133] 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.

[0134] 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.

[0135] 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.

[0136] 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."

[0137] The above method may be intended to remove impurities contained in a sample, specifically to remove forms other than the intact form of hyaluronidase, and / or to improve stability so that the intact form of hyaluronidase is not converted to a hydrolyzed form by removing factors that induce the formation of the hydrolyzed form of hyaluronidase.

[0138] 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."

[0139] The above impurities may include factors that induce the conversion of the intact form of hyaluronidase into a hydrolyzed form, specifically, may include proteases. Additionally, the proteases may include serine proteases or cysteine ​​proteases. Therefore, hyaluronidase from which impurities have been removed through the above method may not be converted from the intact form of hyaluronidase into a hydrolyzed form, may be hardly converted, and / or may have a low level of conversion.

[0140] In addition, the above impurities may include forms other than the intact form of hyaluronidase, and specifically, may include the 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] 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

[0143] 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.

[0144] 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'.

[0145] In one embodiment, the hydrolyzed form of the hyaluronidase may be a form in which the peptide bond in the amino acid sequence of SEQ ID NO. 1 is cleaved by hydrolysis by serine protease and / or cysteine ​​protease and consists of two fragments (a first fragment and a second fragment), and the first fragment and the second fragment may be connected by a disulfide bond.

[0146] 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.

[0147] 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.

[0148] 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

[0149] 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.

[0150] In one embodiment, the eluent obtained from the APB chromatography, the first chromatography, and / or 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.

[0151] In one embodiment, the hyaluronidase or a variant thereof may comprise a hydrolyzed form of hyaluronidase of about 10% or less, about 8% or less, about 7% or less, about 6% or less, about 5% or less, about 4% or less, about 3% or less, or about 2% 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., set to 100%).

[0152] The eluent obtained from the above APB chromatography and / or the hyaluronidase obtained through the above method may have excellent stability. The excellent stability of the hyaluronidase may mean that the intact form of the hyaluronidase is not converted to a hydrolyzed form, or that the level of conversion is significantly low. Therefore, the hyaluronidase has excellent stability, so that when stored at room temperature, a hydrolyzed form is not formed, and / or the formation of a hydrolyzed form is reduced and / or inhibited.

[0153] In one embodiment, the eluent to the APB chromatography and / or the hyaluronidase obtained through the method may have excellent stability under temperature conditions of about 10°C to about 35°C. Specifically, the above temperature conditions may be about 10°C to about 35°C, about 10°C to about 30°C, about 10°C to about 27°C, about 10°C to about 25°C, about 15°C to about 35°C, about 15°C to about 30°C, about 15°C to about 27°C, about 15°C to about 25°C, about 20°C to about 35°C, about 20°C to about 30°C, about 20°C to about 27°C, about 20°C to about 25°C, about 23°C to about 35°C, about 23°C to about 30°C, about 23°C to about 27°C, about 23°C to about 25°C, about 25°C to about 35°C, about 25°C to about 30°C, or about 25°C to about 27°C. Accordingly, the hyaluronidase has excellent stability under temperature conditions of about 10°C to about 35°C, so that the formation of the hydrolyzed form is reduced and / or inhibited when stored at room temperature. The eluent obtained from the APB chromatography and / or the hyaluronidase or a variant thereof obtained through the above method may have an increase level (increase rate) of the content of the hydrolyzed form of 30% or less under temperature conditions of about 10°C to about 35°C.

[0154] In one embodiment, the eluent obtained from the APB chromatography and / or the hyaluronidase or variant thereof obtained through the method may have a daily increase rate of the content of the hydrolyzed form over time under storage conditions of about 10°C to about 35°C, which is 30% or less, 20% or less, 15% or less, or 10% or less; more specifically, the daily increase rate may be 0.1% to 30%, 0.1% to 20%, 0.1% to 10%, 0.1% to 5%, 0.5% to 30%, 0.5% to 20%, 0.5% to 10%, 0.5% to 5%, 1% to 30%, 1% to 20%, 1% to 10%, or 1% to 5%.

[0155] In one embodiment, the eluent obtained from the APB chromatography and / or the hyaluronidase or variant thereof obtained through the above method may have a reduced daily increase rate of the content of the hydrolyzed form over storage time compared to the eluent, hyaluronidase, or variant thereof obtained through another method (specifically, a method not using APB chromatography).

[0156] In one embodiment, the daily increase rate of the content of the hydrolyzed form of the eluent obtained from the APB chromatography and / or the hyaluronidase or its variant obtained through the above method over a storage period under a temperature condition of about 10°C to about 35°C may be reduced to a level of 50% or less, 40% or less, 30% or less, or 25% or less of the daily increase rate of the content of the hydrolyzed form of the eluent obtained through another method (specifically, a method not using APB chromatography) over a storage period of the hyaluronidase or its variant obtained through the above method, and specifically may be reduced to a level of 0.1% to 50%, 0.1% to 40%, 0.1% to 30%, 0.1% to 25%, 1% to 50%, 1% to 40%, 1% to 30%, or 1% to 25%.

[0157] In one embodiment, the eluent obtained from the APB chromatography and / or the hyaluronidase or variant thereof obtained through the method may have a rate of change (△%Peak2) of the hydrolyzed form content over a storage period (after 4 days or after 7 days) at a temperature of about 10°C to about 35°C, which may be 2 or less, 1.5 or less, 1 or less, 0.5 or less, or 0.3 or less; specifically, the rate of change (△%Peak2) may be 0.001 to 2, 0.001 to 1.5, 0.001 to 1, 0.001 to 0.5, 0.001 to 0.3, 0.01 to 2, 0.01 to 1.5, 0.01 to 1, 0.01 to 0.5, 0.01 to 0.3, or 0.1 to 2. It may be 0.1 to 1.5, 0.1 to 1, 0.1 to 0.5, or 0.1 to 0.3.

[0158] In one embodiment, the eluent obtained from the APB chromatography and / or the hyaluronidase or variant thereof obtained through the method may have a reduced rate of change (△%Peak2) of the hydrolyzed form content compared to the eluent, hyaluronidase, or variant thereof obtained through another method (specifically, a method not using APB chromatography).

[0159] In one embodiment, the rate of change in the content of the hydrolyzed form (△%Peak2) over a storage period (after 4 days or after 7 days) of the eluent obtained from the APB chromatography and / or the hyaluronidase or its variant obtained through the method at a temperature condition of about 10°C to about 35°C may be reduced to a level of 50% or less, 30% or less, 20% or less, or 10% or less of the rate of change in the content of the hydrolyzed form (△%Peak2) over a storage period (after 4 days or after 7 days) of the eluent, hyaluronidase, or its variant obtained through another method (specifically, a method not using APB chromatography), and specifically 0.001% to 50%, 0.001% to 30%, 0.001% to 20%, or 0.001% to 10%. It may be reduced to a level of 0.01% to 50%, 0.01% to 30%, 0.01% to 20%, 0.01% to 10%, 0.1% to 50%, 0.1% to 30%, 0.1% to 20%, or 0.1% to 10%.

[0160] 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).

[0161] The hyaluronidase or its variant obtained through the above method may have improved stability compared to the hyaluronidase or its variant obtained through another method (a method not using APB chromatography), and specifically, the growth rate (production rate) of the hydrolyzed form of hyaluronidase may be low under temperature conditions of about 10°C to about 35°C.

[0162]

[0163] 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.

[0164] 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.

[0165] 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.

[0166] 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.

[0167] 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 a nucleic acid molecule encoding the hyaluronidase or a variant thereof can be expressed within the host cell. Additionally, it includes integrating a gene sequence encoding the hyaluronidase or a variant thereof 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.

[0168] 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.

[0169] 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.

[0170] 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.

[0171] 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.

[0172]

[0173] 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.

[0174] The above impurities may include factors that induce the conversion of the intact form of hyaluronidase into a hydrolyzed form, specifically, may include proteases. Additionally, the proteases may include serine proteases and / or cysteine ​​proteases. Therefore, hyaluronidase from which impurities have been removed through the above method may not be converted from the intact form of hyaluronidase to the hydrolyzed form, may be hardly converted, and / or may have a low level of conversion.

[0175]

[0176] Another aspect provides a hyaluronidase or a variant thereof characterized by an increase rate of the content of the hydrolyzed form of hyaluronidase of 30% or less. The same parts as described above apply equally to the hyaluronidase or the variant thereof.

[0177] 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.

[0178] 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.

[0179] The above hyaluronidase or its variant may have excellent stability under temperature conditions of about 10°C to about 35°C. Specifically, the above temperature conditions may be about 10°C to about 35°C, about 10°C to about 30°C, about 10°C to about 27°C, about 10°C to about 25°C, about 15°C to about 35°C, about 15°C to about 30°C, about 15°C to about 27°C, about 15°C to about 25°C, about 20°C to about 35°C, about 20°C to about 30°C, about 20°C to about 27°C, about 20°C to about 25°C, about 23°C to about 35°C, about 23°C to about 30°C, about 23°C to about 27°C, about 23°C to about 25°C, about 25°C to about 35°C, about 25°C to about 30°C, or about 25°C to about 27°C. Accordingly, the above hyaluronidase has excellent stability under temperature conditions of about 10°C to about 35°C, so the formation of a hydrolyzed form when stored at room temperature may be reduced and / or inhibited.

[0180] The above hyaluronidase or its variant may have an increase level (increase rate) of the content of the hydrolyzed form of about 10°C to about 35°C under temperature conditions of about 10°C to about 35°C of about 30°C or less.

[0181] In one embodiment, the hyaluronidase or a variant thereof may have a daily increase rate of the content of the hydrolyzed form over time under storage conditions of about 10°C to about 35°C of about 30% or less, about 20% or less, about 15% or less, or about 10% or less, and more specifically, the daily increase rate may be 0.1% to 30%, 0.1% to 20%, 0.1% to 10%, 0.1% to 5%, 0.5% to 30%, 0.5% to 20%, 0.5% to 10%, 0.5% to 5%, 1% to 30%, 1% to 20%, 1% to 10%, or 1% to 5%.

[0182] In one embodiment, the hyaluronidase or a variant thereof may have a reduced daily increase rate of the content of the hydrolyzed form over storage time compared to the hyaluronidase or a variant thereof obtained through another method (specifically, a method not using APB chromatography).

[0183] In one embodiment, the daily increase rate of the content of the hydrolyzed form of the hyaluronidase or its variant over time of storage under a temperature condition of about 10°C to about 35°C may be reduced to about 50% or less, about 40% or less, about 30% or less, or about 25% or less of the daily increase rate of the content of the hydrolyzed form of the hyaluronidase or its variant over time of storage obtained through another method (specifically, a method not using APB chromatography), and specifically may be reduced to 0.1% to 50%, 0.1% to 40%, 0.1% to 30%, 0.1% to 25%, 1% to 50%, 1% to 40%, 1% to 30%, or 1% to 25%.

[0184] In one embodiment, the hyaluronidase or a variant thereof may have a rate of change (△%Peak2) of the hydrolyzed form content over a storage period (after 4 days or after 7 days) at a temperature of about 10°C to about 35°C, which is about 2 or less, about 1.5 or less, about 1 or less, about 0.5 or less, or about 0.3 or less; specifically, the rate of change (△%Peak2) may be 0.001 to 2, 0.001 to 1.5, 0.001 to 1, 0.001 to 0.5, 0.001 to 0.3, 0.01 to 2, 0.01 to 1.5, 0.01 to 1, 0.01 to 0.5, 0.01 to 0.3, 0.1 to 2, 0.1 to 1.5, 0.1 to 1, It may be 0.1 to 0.5, or 0.1 to 0.3.

[0185] In one embodiment, the hyaluronidase or its variant may have a reduced rate of change (△%Peak2) of the hydrolyzed form content compared to the hyaluronidase or its variant obtained by another method (specifically, a method not using APB chromatography).

[0186] In one embodiment, under a temperature condition of about 10°C to about 35°C, the rate of change in the content of the hydrolyzed form (△%Peak2) over a storage period (after 4 days or after 7 days) of the hyaluronidase or its variant may be reduced to about 50% or less, about 30% or less, about 20% or less, or about 10% or less of the rate of change in the content of the hydrolyzed form (△%Peak2) over a storage period (after 4 days or after 7 days) of the hyaluronidase or its variant obtained by another method (specifically, a method not using APB chromatography), and specifically 0.001% to 50%, 0.001% to 30%, 0.001% to 20%, 0.001% to 10%, 0.01% to 50%, 0.01% to 30%, It may be reduced to a level of 0.01% to 20%, 0.01% to 10%, 0.1% to 50%, 0.1% to 30%, 0.1% to 20%, or 0.1% to 10%.

[0187] 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'.

[0188] 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.

[0189] 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.

[0190] 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, about 4% or less, about 3% or less, about 2.5% or less, about 2% or less, about 1.5% or less, about 1.4% or less, or about 1.3% or less 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 0.1 to 10%, 0.1 to 8%, 0.1 to 7%, 0.1 to 6%, 0.1 to 5%, 0.1 to 4%, 0.1 to 3%, 0.1 to 2.5%, 0.1 to 2%, or 0.1 to 1.5%, 0.1 to 1.4%, 0.1 to 1.3%, 0.5 to 10%, 0.5 to 8%, 0.5 to 7%, 0.5 to 6%, 0.5 to 5%, 0.5 to 4%, 0.5 to 3%, 0.5 to 2.5%, 0.5 to 2%, 0.5 to 1.5%, 0.5 to 1.4%, 0.5 to 1.3%, 1.0 to 10%, 1.0 to 8%, 1.0 to 7%, 1.0 to 6%, 1.0 to 5%, 1.0 to 4%, 1.0 to 3%, 1.0 to 2.5%, 1.0 to 2%, 1.0 to 1.5%, 1.0 to 1.4%, 1.0 to 1.3%, 1.2 to 10%, 1.2 to 8%, 1.2 to 7%, 1.2 to 6%, 1.2 to 5%, 1.2 to 4%, 1.2 to 3%, 1.2 to 2.5%, 1.2 to 2%, 1.2 to 1.5%, 1.2 to 1.4%, 1.2 to 1.3%, 1.3 to 10%, 1.3 to 8%, 1.3 to 7%, 1.3 to 6%, 1.3 to 5%, 1.3 to 4%, 1.3 to 3%, 1.3 to 2.5%, 1.3 to 2%, 1.3 to 1.5%, or 1.3 to 1.4% It may contain a hydrolyzed form of hyaluronidase.

[0191] In one embodiment, the eluent obtained from the APB chromatography and / or the hyaluronidase or variant thereof obtained through the method may have a hydrolyzed form content (%Peak2) of about 3% or less, about 2.5% or less, about 2% or less, about 1.5% or less, about 1.4% or less, or about 1.3% or less after a storage period (after 4 days or 7 days) under a temperature condition of about 10°C to about 35°C; specifically, the hyaluronidase or variant thereof may have a hydrolyzed form content (%Peak2) of 0.1 to 3%, 0.1 to 2.5%, or 0.1% after a storage period (after 4 days or 7 days) under a temperature condition of about 10°C to about 35°C based on the total weight (or the total sum of all forms of hyaluronidase, including whole form, oxidized form, and cleavage form, etc., set to 100%). Up to 2%, 0.1 to 1.5%, 0.1 to 1.4%, 0.1 to 1.3%, 0.5 to 3%, 0.5 to 2.5%, 0.5 to 2%, 0.5 to 1.5%, 0.5 to 1.4%, 0.5 to 1.3%, 1.0 to 3%, 1.0 to 2.5%, 1.0 to 2%, 1.0 to 1.5%, 1.0 to 1.4%, 1.0 to 1.3%, 1.2 to 3%, 1.2 to 2.5%, 1.2 to 2%, 1.2 to 1.5%, 1.2 to 1.4%, 1.2 to 1.3%, 1.3 to 3%, 1.3 to 2.5%, It may be 1.3 to 2%, 1.3 to 1.5%, or 1.3 to 1.4%.

[0192] 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.

[0193] The above hyaluronidase or its variant may have improved stability compared to hyaluronidase or its variant obtained by another method (a method not using APB chromatography), and specifically, the above hyaluronidase may have excellent stability, so that the formation of a hydrolyzed form is reduced or / or inhibited when stored at room temperature.

[0194] In one embodiment, the hyaluronidase or its variant may have a reduced protease content compared to the hyaluronidase or its variant obtained through another method (a method not using APB chromatography), specifically, it may be reduced to a level of about 90% or less, about 70% or less, about 50% or less, about 40% or less, about 30% or less, about 20% or less, or about 10% or less based on the protease contained in the hyaluronidase or its variant obtained through another method (a method not using APB chromatography).

[0195]

[0196] Another aspect is to provide a hyaluronidase or a variant thereof characterized by having a content of about 10% or less of the hydrolyzed form of hyaluronidase.

[0197] The same as the content described above applies equally to the hyaluronidase or its variants.

[0198] 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.

[0199] 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.

[0200] 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, about 4% or less, about 3% or less, about 2.5% or less, about 2% or less, about 1.5% or less, about 1.4% or less, or about 1.3% or less 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 0.1 to 10%, 0.1 to 8%, 0.1 to 7%, 0.1 to 6%, 0.1 to 5%, 0.1 to 4%, 0.1 to 3%, 0.1 to 2.5%, 0.1 to 2%, or 0.1 to 1.5%, 0.1 to 1.4%, 0.1 to 1.3%, 0.5 to 10%, 0.5 to 8%, 0.5 to 7%, 0.5 to 6%, 0.5 to 5%, 0.5 to 4%, 0.5 to 3%, 0.5 to 2.5%, 0.5 to 2%, 0.5 to 1.5%, 0.5 to 1.4%, 0.5 to 1.3%, 1.0 to 10%, 1.0 to 8%, 1.0 to 7%, 1.0 to 6%, 1.0 to 5%, 1.0 to 4%, 1.0 to 3%, 1.0 to 2.5%, 1.0 to 2%, 1.0 to 1.5%, 1.0 to 1.4%, 1.0 to 1.3%, 1.2 to 10%, 1.2 to 8%, 1.2 to 7%, 1.2 to 6%, 1.2 to 5%, 1.2 to 4%, 1.2 to 3%, 1.2 to 2.5%, 1.2 to 2%, 1.2 to 1.5%, 1.2 to 1.4%, 1.2 to 1.3%, 1.3 to 10%, 1.3 to 8%, 1.3 to 7%, 1.3 to 6%, 1.3 to 5%, 1.3 to 4%, 1.3 to 3%, 1.3 to 2.5%, 1.3 to 2%, 1.3 to 1.5%, or 1.3 to 1.4% It may contain a hydrolyzed form of hyaluronidase.

[0201] In one embodiment, the content of the hydrolyzed form (%Peak2) of the hyaluronidase or its variant may be about 3% or less, about 2.5% or less, about 2% or less, about 1.5% or less, about 1.4% or less, or about 1.3% or less after a storage period (after 4 days or 7 days) under a temperature condition of about 10°C to about 35°C. Specifically, the content of the hydrolyzed form (%Peak2) of the hyaluronidase or its variant may be 0.1 to 3%, 0.1 to 2.5%, 0.1 to 2%, 0.1 to 1.5%, or 0.1 to 1.4%, 0.1 to 1.3%, 0.5 to 3%, 0.5 to 2.5%, 0.5 to 2%, 0.5 to 1.5%, 0.5 to 1.4%, 0.5 to 1.3%, 1.0 to 3%, 1.0 to 2.5%, 1.0 to 2%, 1.0 to 1.5%, 1.0 to 1.4%, 1.0 to 1.3%, 1.2 to 3%, 1.2 to 2.5%, 1.2 to 2%, 1.2 to 1.5%, 1.2 to 1.4%, 1.2 to 1.3%, 1.3 to 3%, 1.3 to 2.5%, 1.3 to 2%, 1.3 to 1.5%, 1.2 to 1.4%, 1.2 to 1.3%, 1.3 to 3%, 1.3 to 2.5%, 1.3 to 2%, 1.3 to 1.5%, or It can be 1.3 to 1.4%.

[0202]

[0203] Another aspect is to provide a composition comprising hyaluronidase or a variant thereof. The same parts as described above apply equally to said composition.

[0204] The above composition may include an intact form and a hydrolyzed form of hyaluronidase.

[0205] The above composition may not contain protease or may contain little to no protease.

[0206] In one embodiment, the composition may have a reduced protease content compared to a composition containing hyaluronidase or a variant thereof obtained through another method (a method not using APB chromatography), specifically, it may be reduced to a level of 90% or less, 70% or less, 50% or less, 40% or less, 30% or less, 20% or less, or 10% or less based on the protease contained in a composition containing hyaluronidase or a variant thereof obtained through another method (a method not using APB chromatography).

[0207] When hyaluronidase is purified using a method according to one aspect, proteases that can induce hydrolysis and cleavage of hyaluronidase can be effectively removed, thereby preventing an increase in the content of hydrolyzed / cleaved forms during storage of hyaluronidase, and thus enabling the production of hyaluronidase with improved stability.

[0208] Figure 1 is a diagram showing the level of change in %Peak2 as a result of treating hyaluronidase with a protease inhibitor.

[0209] Figure 2 is a diagram showing the %Peak2 change level of hyaluronidase purified by a process with added APB chromatography.

[0210] 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.

[0211]

[0212] [Experimental Method]

[0213] 1. RP-UPLC - %Peak2, %Peak3, and %Peak4 Measurement

[0214] 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.

[0215] 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.

[0216] 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

[0217]

[0218] Example 1: Identification of factors inducing an increase in the hydrolyzed form of hyaluronidase

[0219] The object of the present invention is to develop a hyaluronidase purification process to prevent an increase in the level of the hydrolyzed form of hyaluronidase when stored at room temperature.

[0220] Accordingly, to explore inducing factors that increase the level of the truncated form of hyaluronidase, the following experiment was performed.

[0221] Specifically, purified hyaluronidase was treated with a cocktail containing various protease inhibitors (including Aprotinin, Bestatin, E-64, Leupeptin, and Pepstatin A), and the level of change in %Peak2 according to the storage period at 25°C was determined. In addition, as a control group, only DMSO, the solvent of the protease inhibitor cocktail, was treated (DSMO), and a control group was not treated at all (Control). Meanwhile, %Peak2 of RP-UPLC defines the hydrolyzed form (cleaved form) of hyaluronidase.

[0222] As a result of the above experiment, it was confirmed that %Peak2 continuously increased with storage time in the control group, whereas %Peak2 hardly increased in the experimental group treated with a protease inhibitor (Fig. 1).

[0223] Based on the above results, it can be seen that the increase in the level of cleavage forms of hyaluronidase during room temperature storage is due to protease that was not completely removed during the purification process.

[0224]

[0225] Example 2: Development of a process capable of suppressing the increase of cleaved forms of hyaluronidase during room temperature storage

[0226] As confirmed in Example 1 above, it was confirmed that the cleaved form of hyaluronidase may increase during storage due to proteases (specifically, serine proteases or cysteine ​​proteases) that are not completely removed during the hyaluronidase production / purification process.

[0227] Therefore, in order to develop a method to effectively remove residual protease, APB chromatography containing aminophenylboronate (APB) resin was introduced into the hyaluronidase purification process.

[0228] Meanwhile, the above APB resin is a resin in which m-aminophenylboronic acid is bound as a ligand, and can provide selectivity through the affinity between the diol group of boronic acid and the substance. In the above method, it is expected that only hyaluronidase can be selectively separated through the affinity binding between the -OH group of the boronic acid of the APB resin and the -OH group of the protease by applying APB chromatography.

[0229] Specifically, to verify the removal efficiency of proteases (specifically, serine proteases or cysteine ​​proteases) through APB chromatography, a sample pretreated with AEX (AEX eluent) was sequentially subjected to mixed-mode chromatography (MMC), hydrophobic interaction chromatography (HIC), and APB chromatography under the following conditions.

[0230] Meanwhile, the above-mentioned multi-mode chromatography utilized a column containing a material having cation exchange and hydrophobic interaction functions.

[0231] First, the above chromatography 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, each column was equilibrated using an equilibration buffer 5 CV under the following conditions.

[0232] 1) Multi-mode chromatography: 20 mM sodium phosphate, 100 mM NaCl, pH 6.0

[0233] 2) Hydrophobic interaction chromatography: 20 mM sodium phosphate, 1200 mM AMS, pH 7.0

[0234] 3) APB chromatography: 20 mM sodium phosphate, 500 mM or 1200 mM AMS, pH 7.0

[0235] Next, samples appropriate to the same salt concentration and pH were loaded into each column (MMC: 10 g / L resin , HIC: 20 g / L resin , APB: 8-20 g / L resin ). Afterwards, a tracking step was performed with equilibrium buffer 2 to 3 CV.

[0236] Next, in the case of multimode chromatography, a washing step was performed using a wash buffer under the following conditions.

[0237] 1) Multi-mode chromatography: 20 mM sodium phosphate, 420 mM NaCl, pH 6.0

[0238] Next, hyaluronidase was recovered from each column using an elution buffer under the following conditions.

[0239] 1) Multi-mode chromatography: 20 mM sodium phosphate, 750 mM NaCl, pH 6.0 (isocratic elution).

[0240] 2) Hydrophobic interaction chromatography: 20 mM sodium phosphate, 370 mM AMS, pH 7.0 (isocratic elution).

[0241] 3) APB chromatography: 5 mM sodium phosphate, 150 mM NaCl, pH 7.0 (isocratic elution).

[0242] Next, the hyaluronidase purified through the above process was stored at 25°C (room temperature) for 4 days, and the level of change in %Peak2 according to the storage period was checked. Meanwhile, as a control, hyaluronidase obtained by sequentially applying AEX-MMC-HIC without applying APB chromatography was used.

[0243] As a result of the above experiment, it was confirmed that %Peak2 continuously increased with storage period in the control group, whereas %Peak2 hardly increased in the experimental group in which the APB chromatography process was introduced (Fig. 2).

[0244] Additionally, the hyaluronidase purified through the above process was stored for 7 days under low temperature (2 to 8 ℃) and room temperature (15 to 26 ℃) conditions, and the level of change in %Peak2 (hydrolyzed) (RP-UPLC analysis) according to the storage period was checked, and it was confirmed that %Peak2 did not increase under low temperature and room temperature storage conditions (Table 4).

[0245] Storage Temperature Storage Period 0 days 1 day 3 days 7 days Low temperature (2~8℃) 1.3 1.3 1.3 1.3 Room temperature (15~26℃) 1.3 1.3 1.3 1.3

[0246] Based on the above results, it can be seen that the increase in the level of the cleavage form of hyaluronidase can be suppressed by removing protease through APB chromatography, and that hyaluronidase with significantly improved stability can be obtained by using a method incorporating the above process.

[0247]

[0248] 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 step comprising applying a sample containing hyaluronidase to aminophenylboronate (APB) chromatography, Method for purifying hyaluronidase.

2. The method of claim 1, wherein the APB chromatography uses a column containing aminophenylboronic acid (m-Aminophenylboronic acid).

3. In claim 1, the application to the APB chromatography is, 1) a step of loading a sample containing hyaluronidase onto an APB chromatography column; and 2) Step of eluting the product from the column using an elution buffer A method that includes 4. The method of claim 3, wherein the APB chromatography column is a column equilibrated with an equilibration buffer.

5. The method of claim 4, wherein the equilibrium buffer comprises about 300 mM to about 2000 mM of salt.

6. The method of claim 4, wherein the equilibrium buffer comprises about 300 mM to about 2000 mM of AMS.

7. The method of claim 4, wherein the equilibrium buffer comprises about 5 mM to about 50 mM of sodium phosphate.

8. The method of claim 4, wherein the pH of the equilibrium buffer is in the range of about 5.0 to about 9.

0.

9. In claim 3, 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).

10. The method of claim 3, wherein the elution buffer comprises about 50 mM to about 500 mM of salt.

11. The method of claim 3, wherein the elution buffer comprises about 50 mM to about 500 mM of sodium chloride.

12. The method of claim 3, wherein the elution buffer comprises about 1 mM to about 20 mM of sodium phosphate.

13. The method of claim 3, wherein the pH of the elution buffer is in the range of about 5.0 to about 9.

0.

14. In claim 3, the method A step of tracking an APB chromatography column loaded with a sample containing hyaluronidase using a tracking buffer. A method that additionally includes 15. The method of claim 1, wherein the step of applying the sample to APB chromatography is for removing or reducing protease in the sample.

16. The method of claim 15, wherein the protease comprises serine protease and / or cysteine ​​protease.

17. In claim 1, the method A) A step of applying the sample containing the above hyaluronidase to a first chromatography before applying it to APB chromatography. A method that additionally includes 18. The method of claim 17, wherein the first chromatography comprises cation exchange chromatography (CEX) or mixed-mode chromatography (MMC).

19. In claim 17, 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 20. The method of claim 19, wherein the first chromatography column is a column equilibrated with an equilibration buffer.

21. The method of claim 20, wherein the equilibrium buffer comprises about 10 mM to about 500 mM of salt.

22. The method of claim 20, wherein the equilibrium buffer comprises about 10 mM to about 500 mM of sodium chloride.

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

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

0.

25. In claim 19, 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).

26. The method of claim 19, wherein the elution buffer comprises about 10 mM to about 2000 mM of salt.

27. The method of claim 19, wherein the elution buffer comprises about 10 mM to about 2000 mM of sodium chloride.

28. The method of claim 19, wherein the elution buffer comprises about 5 mM to about 50 mM of sodium phosphate.

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

0.

30. In claim 19, 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 31. The method of claim 30, wherein the washing buffer comprises about 100 mM to about 600 mM of salt.

32. The method of claim 30, wherein the washing buffer comprises about 50 mM to about 600 mM of sodium chloride.

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

34. The method of claim 30, wherein the pH of the washing buffer is in the range of about 4.0 to about 8.

0.

35. In claim 19, 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 36. In claim 17, the method B) A step of applying the eluent obtained from the first chromatography to the second chromatography. A method that additionally includes 37. The method of claim 36, wherein the second chromatography comprises hydrophobic interaction chromatography (HIC).

38. In claim 36, 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 39. The method of claim 38, wherein the second chromatography column is a column equilibrated with an equilibration buffer.

40. The method of claim 39, wherein the equilibrium buffer comprises about 500 mM to about 2000 mM of salt.

41. The method of claim 39, wherein the equilibrium buffer comprises about 500 mM to about 2000 mM of AMS.

42. The method of claim 39, wherein the equilibrium buffer comprises about 5 mM to about 50 mM of sodium phosphate.

43. The method of claim 39, wherein the pH of the equilibrium buffer is in the range of about 5.0 to about 9.

0.

44. The method of claim 38, wherein the elution buffer comprises about 100 mM to about 600 mM of salt.

45. The method of claim 38, wherein the elution buffer comprises about 100 mM to about 600 mM of AMS.

46. ​​The method of claim 38, wherein the elution buffer comprises about 5 mM to about 50 mM of sodium phosphate.

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

0.

48. In claim 38, 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 49. The method of claim 1, wherein the sample containing the hyaluronidase comprises an eluent obtained by applying anion exchange chromatography.

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

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

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

53. The method of claim 52, wherein the impurity comprises protease.

54. A method according to any one of claims 1 to 53, wherein the hyaluronidase obtained through the method has excellent stability.

55. A method according to any one of claims 1 to 53, wherein the hyaluronidase obtained through the method has excellent stability under temperature conditions of about 10°C to about 35°C.

56. A method according to any one of claims 1 to 53, wherein the hyaluronidase obtained through the method comprises an intact form and a hydrolyzed form.

57. A method according to any one of claims 1 to 53, wherein the hyaluronidase obtained through the method is not converted from an intact form to a hydrolyzed form.

58. The method of claim 56, 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.

59. The method of 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.

60. The method of claim 56, wherein the hydrolyzed form of the hyaluronidase comprises two or more fragments formed by cleaving an intact form of hyaluronidase.

61. A method according to claim 60, wherein two or more fragments formed by cutting are connected through disulfide bonds between the fragments.

62. 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. A method that includes 63. The method of claim 62, 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.

64. A method for producing hyaluronidase comprising the method of any one of claims 1 to 63.

65. A method for improving the removal of impurities in a hyaluronidase purification process, comprising the method of any one of claims 1 to 63.

66. A method according to claim 65, wherein the impurity comprises a protease.

67. Hyaluronidase or a variant thereof, characterized in that the increase rate of the content of the hydrolyzed form of hyaluronidase is 30% or less.

68. The hyaluronidase or a variant thereof according to claim 67, wherein the hyaluronidase or a variant thereof is obtained using the method of claims 1 to 63.

69. The hyaluronidase or a variant thereof according to claim 67, wherein the hyaluronidase or a variant thereof has excellent stability under temperature conditions of about 10°C to about 35°C.

70. The hyaluronidase or a variant thereof according to claim 67, wherein the hyaluronidase or a variant thereof has an increase rate of 30% or less in the content of the hydrolyzed form under temperature conditions of about 10°C to about 35°C.

71. Hyaluronidase or a variant thereof according to claim 67, 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.

72. The hyaluronidase or a variant thereof according to claim 67, wherein the hydrolyzed form of the hyaluronidase comprises two or more fragments formed by cleaving an intact form of hyaluronidase.

73. Hyaluronidase or a variant thereof according to claim 72, wherein two or more fragments formed by cleavage are connected through disulfide bonds between the fragments.

74. In claim 67, 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 75. Hyaluronidase or a variant thereof according to claim 74, 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.

76. Hyaluronidase or a variant thereof, characterized in that the content of the hydrolyzed form of hyaluronidase is about 10% or less.

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