TW202309296A - Method for producing recombinant hyaluronidase - Google Patents

Abstract

Disclosed is a method for producing hyaluronidase or a variant thereof. Specifically, the method is capable of changing the N-glycan levels under culture conditions including a controlled concentration of glucose in the culture medium and a decreased culture temperature for a specific culture time period, thereby increasing the specific activity by 10% or more and improving the quality and production yield.

Description

Method for producing recombinant hyaluronidase

The present invention relates to a method for producing a hyaluronidase PH20 variant comprising one or more substitutions of amino acid residues, optionally at certain amino-terminal and/or carboxyl groups of hyaluronidase Deletion of terminal amino acid residues, especially wild-type or mature wild-type PH20 or wild-type or mature wild-type PH20 amino acid sequence.

Hyaluronidase is an enzyme that degrades hyaluronic acid present in the extracellular matrix. Hyaluronidase hydrolyzes hyaluronic acid thereby reducing the viscosity of hyaluronic acid in the extracellular matrix and increasing its permeability to tissues (skin) (Bookbinder et al., 2006). Hyaluronidase has been used to enhance the absorption of body fluids delivered by subcutaneous injection (Muchmore et al., 2012) or intramuscular injection (Krantz et al., 2016), and to improve the diffusion of local anesthesia (Clement et al. al., 2003). Drugs that facilitate subcutaneous administration in this way include morphine (Thomas et al., 2009), ceftriaxone (Harb et al., 2010), insulin (Muchmore et al., 2012), and immunoglobulins ( Wasserman et al., 2014). Hyaluronidase can also be used to improve the dispersion of drugs or fluids that penetrate tissue from intravenous injection or hematoma spread. Among hyaluronidases, recombinant human PH20 protein active at neutral pH was developed by Halozyme Therapeutics Inc. and sold under the trade name "Hylenenex" (Bookbinder et al., 2006).

Recombinant human PH20 protein has been reported to be expressed in yeast ( P. pastoris ), DS-2 insect cells and animal cells. Recombinant PH20 proteins produced in insect cells and yeast differ from human PH20 in the N-glycosidation pattern in the process of post-translational modification, thus affecting their activity and bringing the risk of side effects in vivo.

For biopharmaceutical development, product consistency and long shelf life are important factors in providing manufacturing flexibility. During the manufacturing process, various types of microheterogeneity (microheterogeneity) arise from differences in size and charge caused by enzymatic or spontaneous degradation and modification. Various chemical and enzymatic modifications such as deamidation and sialylation increase the net negative charge in the antibody and decrease the pI value, resulting in acidic variants (Harris RJ et al., 2004). In addition, cleavage of the carboxy-terminal lysine results in loss of net positive charge and formation of acidic variants. Formation of basic variants can result from carboxy-terminal lysine or glycine desamidation, succinimide formation, oxidation of amino acids, or removal of sialic acid, which removes additional positive charges or Negatively charged, both types of modification increase the pI value (Harris RJ et al., 2004).

Glycosylation is a post-translational process of proteins in cells (eukaryotes), and occurs in the endoplasmic reticulum and Gorgi bodies. Glycosidation is divided into N-glycosidation and O-glycosidation, which differ according to the functional group attached. The process of attaching sugars such as lactose or fucose to proteins produced in cells is broadly known as "glycosidation". When glycans are attached to proteins through glycosidation, the protein undergoes a process of "folding" to form a three-dimensional structure. This imparts stability to the protein, allowing it to hold for long periods of time without loosening (unfolding). Glycans are a key process used to enable communication and information exchange between cells.

There are two types of reactions that add glycans to proteins: N-linked or O-linked glycosylation. These two glycosidation processes differ in glycan synthesis and addition mechanism, among which, the mechanism and role of N-glycosidation are better known. Glycans added by N-glycosidation are called "N-glycans" and are formed in the endoplasmic reticulum. N-acetylglucosamine ( GlcNAc), mannose (Man), glucose (Glc), etc., finally synthesize Glc3Man9GlcNAc2-PP-Dol, which is a complex glycan in the form of lipid-linked oligosaccharide (LLO). Synthetic LLO is transferred to the N-glycosidation sequence of the peptide containing N-x-S/T, which is directly translated from the ribosome into the internal quality net. N-glycans attached to proteins are removed one by one from the ends of the glycans by glucosidases (α-glucosidase I, Gls1p; α-glucosidase II, Glsp II) present in the endoplasmic reticulum. Since the second and third glucose are removed more slowly, they are folded with the help of lectin chaperones, calnexins, and calreticulin. When all glucose has been excised, protein folding is considered complete and the Man8GlcNAc2 glycans attached to it are transferred to the Golgi body. In this regard, there is a quality control process to revalidate the folding of glycoproteins before their transfer from the endoplasmic reticulum to the Gorgi body. When proper folding is not achieved, the process is repeated by allowing a molecule of glucose to enter the calchaperin/calreticulin cycle to give the opportunity to complete folding.

The above-mentioned initial process of biosynthesis of N-glycans in the endoplasmic reticulum is conserved in almost the same manner in a wide range of organisms from yeast, a simple eukaryotic microorganism, to animals, ie higher organisms. However, glycans transferred to the Gorgi body undergo multiple species-specific glycan modifications, resulting in the formation of completely different types of glycans in yeast, insects, and animals. However, these various glycans usually also share a core position, that is, a structure in which three mannose sugars and two GlcNAcs are linked to the nitrogen of asparagine, which is called "trimannosyl core". The form in which mannose is mainly attached to the three-mannose core is called the "high-mannose type" and is commonly found in yeast and mold. The structure in which multiple mannose is added sequentially to the Gorgi body is also found in Saccharomyces cerevisiae , a well-known yeast. On the other hand, glycoproteins with glycans lacking the deficient mannose type (Pauci-mannose type) are found in insect cells. These are first trimmed in Gorgi bodies by mannosidase IA, IB and IC to form Man5GlcNAc2, N-acetylglucosamine transferase (GNT) I to add a GlcNAc to Man5GlcNAc2, followed by mannosidase II to A mixed structure of GlcNAc added to a trimannose core is formed. Next, the added GlcNAc is cleaved again, forming a glycan structure lacking the mannose type. In animal cells, α(1,6)-fucose is often added to the first GlcNAc linked to an asparagine residue. In animals, GNT II acts on a structure where one GlcNAc is added to the trimannose core and another GlcNAc is added to it, forming a glycan with a two antenna structure. Then, GNT IV and V can be used to form a four-antenna structure, and in some cases, GNT VI, IX or VB can be used to form a six-antenna structure. After addition of GlcNAc to form the antenna backbone, β-galactosyltransferase and α-sialyltransferase present in Gorgi bodies are used to form complex glycan structures where galactose and sialic acid are added to GlcNAc.

N-glycosidation can profoundly affect the folding or activity of proteins. When genetic engineering methods are used in industrial applications to manufacture proteins or their variants that exist in nature, the presence of glycosidation and the structure or form of glycans are likely to depend on Depending on the host cell, recombinant manipulation method, and culture conditions (Schilling, et al., 2002). That is, a difference in the amount of sugar components constituting the glycan or the structure of the glycan occurs depending on the difference in the production conditions in the protein production process. The culture conditions that affect N-glycosidation include the concentration of glucose or glutamine in the medium (Tachibana et al. 1994), the concentration of dissolved oxygen (dissolved oxygen, DO) (Restelli et al. 2006), the pH of the medium (Borys et al. 1993), the concentration of ammonia in the medium (Borys et al. 1994), the culture temperature (Clark et al. 2004), etc.

In general, an enzyme will specifically bind to a substrate to form an enzyme-substrate complex, which acts as a catalyst to reduce the activation energy of the reaction and promote the reaction of the enzyme. Enzymes can distinguish substrates and molecules that compete with substrates, and can specifically bind to substrates due to the distribution of complementary charges, complementary structures, and hydrophilicity and hydrophobicity of the positions where enzymes bind to substrates. To elucidate the binding site of enzymes, a lock-and-key model in which enzymes and substrates are complementary to each other and have geometric shapes has been proposed, but it cannot satisfactorily explain the transition state of enzyme-substrate complexes. According to the induced chimerism model proposed to overcome this problem, as enzymes and substrates continue to interact in the enzyme-substrate complex, they change their structure based on the variable structure of the enzyme protein. During this process, the reaction can be further promoted by the distribution of surrounding charges caused by N-glycans or amino acid residues constituting the active site or by the distribution of hydrophilicity/hydrophobicity.

Furthermore, charge interaction is necessary for the reaction of hyaluronidase, which is an enzyme that hydrolyzes hyaluronic acid as a substrate. Arming et al. revealed that the positively charged arginine in hyaluronidase PH20 is required for the enzymatic activity to bind hyaluronic acid, a substrate with a large amount of negative charges distributed throughout it (Arming et al. 1997). Therefore, it can be inferred that the charge distribution of N-glycans will also affect the enzyme activity. It is important to demonstrate the extent of negatively charged sialic-acid-capping sugar in N-glycans (i.e., sialylation) when hyaluronic acid, a substrate with a large negative charge, is bound to hyaluronidase. degree) will affect the formation of enzyme-substrate complex or the progress of enzyme reaction. To limit the extent of sialylation, the transfer of sialic acid to galactose residues should be limited, desialylation should be performed or the extent of galactosylation should be limited.

Therefore, the productivity and activity of recombinant hyaluronidase PH20 and its variants will be affected by changes in the degree of N-glycans, so for effective mass production in this field, it is necessary to research and develop a production method with high activity and productivity and through control and A method for industrially using hyaluronidase PH20 or its variants by maintaining the level of N-glycans.

Therefore, the present invention is accomplished in view of the above problems. One object of the present invention is to provide a method for cultivating a host cell producing recombinant hyaluronidase PH20 or a variant thereof and the hyaluronidase PH20 or a variant thereof prepared by this method, especially It is a method of manufacturing hyaluronidase PH20 or its variants with improved enzyme activity and productivity.

The object of the present invention is not limited to the above. Other purposes not described herein will be clearly understood by those skilled in the art from the following description.

In the present invention, when the host cells used to produce recombinant hyaluronidase PH20 or its variants are cultivated under specific culture conditions, the N-glycosidation characteristics of the produced recombinant hyaluronidase PH20 or its variants, furthermore, The enzymatic activity of the produced hyaluronidase PH20 or its variants will be significantly improved.

Specifically, it was found that by controlling the degree of sialylation, galactosylation and/or mannosylation in N-glycans, especially the degree of sialylation, the activity of hyaluronidase PH20 or its variants produced can be effectively enhanced, and It was found that the enzymatic activity and productivity of the hyaluronidase PH20 or its variants according to the present invention can be significantly improved by culturing for a specific time after changing the culturing temperature.

Specifically, the method for manufacturing hyaluronidase PH20 or its variants according to the present invention comprises:

(1) Cultivate host cells expressing recombinant hyaluronidase PH20 or its variants at a culture temperature of 35°C to 38°C to an integral viable cell density of ^20x106 to ^120x106 cells x day/ml ;as well as

(2) Lowering the culture temperature to 28°C to 34°C, and then cultivating the host cells for 2 to 18 days while maintaining the culture temperature according to at least one method selected from the group consisting of:

(a) culturing the host cells while maintaining a residual glucose concentration in the culture medium between 0.001 grams per liter (g/L) and 4.5 g/L during the culturing period; and

(b) Culturing the host cells while maintaining the pH of the medium at 6.8 to 7.2.

The PH20 or its variant produced by the production method according to the present invention is characterized in that the sialylation amount of the N-glycan of the produced PH20 or its variant is 1 to 38%, so that the enzyme activity will be significantly improved, but not As a limitation, this value is an experimental value with a margin of error of 10%. The reason is that when setting the culture conditions, there will be differences depending on conditions such as the equipment used for the culture and the operator's proficiency in operation. Therefore, considering these differences, the numerical values set in the present invention should be interpreted in a broad sense. rather than in a narrow sense. The host cells can be cultured by batch culture (batch culture), repeated batch culture (repeated batch culture), fed-batch culture (fed-batch culture), repeated fed-batch culture (repeated fed-batch culture) , continuous culture (continuous culture) and perfusion culture (perfusion culture) group consisting of one or more methods.

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used.

The present invention is based on the discovery that the degree of sialylation, galactosylation and/or mannosylation, in particular the degree of sialylation of N-glycans, is important when using genetic engineering methods to produce hyaluronidase PH20 or variants thereof for industrial applications. The enzyme activity and productivity of hyaluronidase PH20 or its variants are very critical.

Specifically, it was found that when the sialylation amount of N-glycans of PH20 or its variants is 1 to 38%, preferably 1 to 30%, more preferably 1.5 to 28%, most preferably 2 to 25% , the enzyme activity and productivity of hyaluronidase PH20 or its variants will be improved to an unexpectedly high level.

More specifically, it was found that when the degree of galactosylation of N-glycans of PH20 or its variants was 1 to 68%, the amount of sialylation thereof was 1 to 38%, and the amount of mannosylation thereof was 40 to 63%, the Preferably when its galactosylation amount is 5 to 60%, its sialylation is 1 to 30%, and its mannosylation amount is 42 to 62%, more preferably when its galactosylation amount is 10 to 56%, its When the amount of sialylation is 1.5 to 28% and the amount of mannosylation is 44 to 61%, the optimum is when the amount of galactosylation is 15 to 50%, the amount of sialylation is 2 to 25%, and the amount of mannosylation is From 47 to 60%, the enzymatic activity and productivity of Hyaluronidase PH20 or its variants improved to an unexpectedly high level.

The above numerical values are obtained from the experimental results of the example shown in Table 1, and are obtained with a 95% confidence interval, and the numerical values may also include an error of 10%. This is because there are differences in the measurement of the sugar (glucose) level in protein, which depends on conditions such as equipment used in the experiment, enzyme reaction time, test temperature, and the proficiency of the tester. The difference between them, the glucose level measured in the present invention should be interpreted in a broad sense rather than a limited sense.

In the present invention, the galactosylation ratio (%) of N-glycans is the sum of the ratios (%) of glycans containing galactose at their terminals among N-glycans, such as G1, G1F, G1F', G2, G2F, A1, A1F, A2, and A2F, the sialylation ratio (%) is the sum of the ratios (%) of N-glycans containing sialic acid at their terminals among N-glycans, such as A1, A1F, A2 and A2F, the mannosylation ratio (%) is the sum of N-glycans containing mannose at their terminals among N-glycans, such as M4G0F, M5, M5G0, M6, M7 and M8.

More specifically, the method for producing hyaluronidase PH20 or variants thereof with a sialylation amount of 1 to 38% in N-glycans according to the present invention comprises:

(1) Cultivate host cells expressing recombinant hyaluronidase PH20 or its variants at a culture temperature of 35°C to 38°C to an integral viable cell density of ^20x106 to ^120x106 cells x day/ml ;as well as

(2) Lowering the culture temperature to 28°C to 34°C, and then cultivating the host cells for 2 to 18 days while maintaining the culture temperature according to at least one method selected from the group consisting of:

(a) culturing the host cells while maintaining a residual glucose concentration in the culture medium between 0.001 grams per liter (g/L) and 4.5 g/L during the culturing period; and

(b) Culturing the host cells while maintaining the pH of the medium at 6.8 to 7.2.

The above values are experimental values with an error range of 10%. The reason is that when setting the culture conditions, there will be differences depending on conditions such as the equipment used for the culture and the operator's proficiency in operation. Therefore, considering these differences, the numerical values set in the present invention should be interpreted in a broad sense. rather than in a narrow sense.

The lowering of the culture temperature from step (1) to step (2) is carried out when the integrated viable cell density reaches ^20x106 to ^120x106 cells x days/ml, preferably ^40x106 to ^100x106 cells x days/ml, More preferably, it is 60x10 ⁶ to 80x10 ⁶ cells x day/ml, but not limited thereto.

In addition, the culture period of step (2) may be 2 to 18 days, preferably 3 to 16 days, more preferably 4 to 14 days, but not limited thereto.

For example, in the case of using a fed-batch culture method in the production of PH20 or variants thereof according to the present invention, the productivity of PH20 or variants thereof can be maximized and the produced PH20 or variants thereof The enzymatic activity of the variants can be significantly improved by inoculum cultivation at 35°C to 38°C at 35°C to 38°C immediately before the main culture until the cell concentration reaches a certain level, followed by inoculation below The main culture by inoculation is carried out at a reduced temperature of 35° C. for 2 to 18 days, preferably 3 to 16 days, more preferably 4 to 14 days.

The seeding concentration of the main cultured cells may be 1×10 ⁵ cells/ml or higher, preferably 5×10 ⁵ cells/ml or higher, more preferably 1×10 ⁶ cells/ml or higher, but not limited thereto.

By culturing using a combination of fed-batch culture and perfusion culture in the production of PH20 or variants thereof according to the present invention, the productivity of PH20 or variants thereof can be maximized and the enzymes of PH20 or variants thereof produced The activity can be significantly improved.

In the present invention, the remaining glucose concentration in the culture medium is maintained at 0.001 g/L to 4.5 g/L, preferably 0.01 to 4.0 g/L, more preferably 0.1 to 3.5 g/L, but the present invention Not limited to this.

The expression "the residual glucose concentration in the culture medium is maintained at 0.001 g/L to 4.5 g/L, preferably 0.01 to 4.0 g/L, more preferably 0.1 to 3.5 g/L" used herein , means that when the residual glucose concentration in the culture medium measured immediately is lower than 0.001 g/L to 4.5 g/ When setting the reference concentration of L, preferably 0.01 to 4.0 g/L, more preferably 0.1 to 3.5 g/L, the glucose stock solution is added to the culture medium to obtain the corresponding reference concentration during cultivation.

In the present invention, it is obvious to those skilled in the art that the reference concentration of the remaining glucose concentration in the culture medium can be set at 0.001 g/L to 4.5 g/L, preferably 0.01 to 4.0 g/L, more preferably 0.1 to 3.5 g/L, the reference concentration can be changed appropriately during the culture period. For example, the reference concentration of residual glucose in the culture medium on the first and second days of culture is 2 g/L, the reference concentration is reduced to 1.5 g/L on the third to fifth days of culture, and then the reference concentration Set it to be lower than 1.5 g/L, such as 1.0 g/L.

Hyaluronidase PH20 or its variants produced by the method according to the present invention and having a predetermined degree of sialylation, galactosylation and/or mannosylation at the N-glycan moiety have an activity of 10,000 units/ml in a hyaluronidase medium (unit/mL), preferably 11,000 units/mL, or more preferably 12,000 units/mL or higher, but not limited thereto.

In addition, hyaluronidase PH20 or its variants produced by the method according to the present invention and having predetermined sialylation, galactosylation and/or mannosylation on N-glycans are wild-type human PH20 produced by conventional methods, and Compared with the activity of wild-type human PH20 produced by conventional methods, the enzyme activity is increased by 10% or more, preferably 12% or more, more preferably 15% or more, but the present invention is not limited thereto.

Preferably, in the method for producing hyaluronidase PH20 or its variants according to the present invention, in step (1) and/or step (2), the culture of host cells can be selected from one of the following groups or Under multiple conditions: (i) adding ammonia to the medium or increasing the ammonia concentration in the medium to 5 mM or more; (ii) adding a protein selected from glutamic acid, glucosamine, uridine, glucosamine and butyric acid one or more of the group consisting of sodium is added to the medium; and (iii) galactose and manNAc are not added to the medium, but the present invention is not limited thereto.

More specifically, in the method for manufacturing hyaluronidase PH20 or its variants according to the present invention, the cultivation of host cells in step (1) and/or step (2) can be carried out by adding ammonia to Medium, raising the ammonia concentration to 5 mM or more, adding glutamine, not adding galactose and manNAc, adding uridine or glucosamine and adding sodium butyrate, but the method is not limited to this.

The term "hyaluronidase PH20 or its variants" as used herein refers to an enzyme that degrades hyaluronic acid located in the extracellular matrix.

The term "hyaluronidase PH20" or "PH20" as used herein is interpreted to include both wild-type PH20 and its mature form, and the term "hyaluronidase PH20 or a variant of PH20" means one or more amino acid residues Substitution, deletion or insertion of bases and optional PH variants including deletion of certain amino-terminal and/or carboxy-terminal amino acid residues in the amino acid sequence of "hyaluronidase PH20" or "PH20", But not limited to this.

The hyaluronidase according to the present invention refers to hyaluronidase derived from animals or microorganisms such as Streptomyces and having hyaluronidase activity, wherein "hyaluronidase PH20" or "PH20" is derived from animals or microorganisms such as Streptomyces , preferably derived from humans, cattle or sheep.

Human-derived "hyaluronidase PH20" or "PH20 variants" according to the present invention are exemplified in International Patent Publication No. 2020/022791 and U.S. Patent No. 9,447,401, but are not limited thereto, and are construed to include any Substitution, deletion and insertion of multiple amino acid residues and optionally included in the amino acid sequence of "hyaluronidase PH20" or "PH20" The truncation of amino acid residues at the amino terminal and/or carboxyl terminal ( truncation) and hyaluronidase or variants with hyaluronidase enzyme activity.

Examples of host cells for expressing hyaluronidase protein used herein include animal cells, yeast, actinomycetes, and insect cells, but are not limited thereto.

Animal cells are preferably mammalian cells, more preferably, commonly used animal culture cells, such as CHO cells, HEK cells, COS cells, 3T3 cells, myeloma cells, BHK cells, HeLa cells and Vero cells, specifically preferably using in abundantly expressed CHO cells. Furthermore, in order to produce a desired protein, it is preferable to use cells suitable for introducing a desired gene, such as dhfr-CHO cells (Proc. Natl. Acad. Sci. USA (1980) 77, 4216-4220) or CHO K-1 Cells (which are DHFR gene knockout CHO cells) (Proc. Natl. Acad. Sci. USA (1968) 60, 1275) or CHO K-1 cells (Proc. Natl. Acad. Sci. USA (1968) 60, 1275 ). Specifically, the CHO cells are preferably DG44, DXB-11, K-1 or CHO-S cell lines, and the introduction of the vector into the host cells is carried out using methods such as the calcium phosphate method, the DEAE dextran method or the lipoprotein method.

Examples of yeast include Saccharomyces sp. , Hansenula sp. , Kluyveromyces , Pichia sp., etc., and actinomycetes include, for example, Streptomyces , but are not limited thereto.

In the method for manufacturing hyaluronidase PH20 or its variants according to the present invention, the culture of the host cells in step (1) and/or step (2) can be selected from batch culture, repeated batch culture, feeding Batch culture, repeated feeding batch culture, continuous culture and perfusion culture can be carried out by one or more methods of the group, but not limited thereto.

Batch culture is a culture method for proliferating cells that does not add fresh medium or discharge culture solution during the culture process. Continuous culture is a culture method in which medium is continuously added and discharged during the culture process. Furthermore, continuous culture includes perfusion culture. Feed-fed batch culture falls between batch and continuous culture, also known as "semi-batch culture" and involves continuous or sequential addition of medium during the culture process. This culture method prevents discharge of cells even if the culture solution is continuously discharged. In the present invention, any culture method can be used, preferably feed-batch culture or continuous culture, particularly preferably feed-batch culture.

As described above, in the hyaluronidase PH20 or its variants produced by the method of the present invention, compared with those produced by the general method, especially compared with the specific activity of wild-type human PH20, the specific activity of the enzyme can be increased by 10% or More. This increase in enzyme activity is due to changes in the N-glycosidation properties and/or charge modification of the hyaluronidase PH20 or its variants produced by the method of the present invention.

In particular, enzyme activity is increased according to changes in sialylation, galactosylation and/or mannosylation among N-glycosidation patterns. These N-glycosidation characteristics, especially sialylation, galactosylation and/or mannosylation, can be adjusted by the production method according to the invention.

In the present invention, examples of the covalent bond between glucose and protein include N-glycosidic bond and O-glycosidic bond in which N-acetyl-D-glucosamine is covalently linked to the constituent protein Asparagine residues (N-glycoside-linked glycans), N-acetyl-D-galactosamine covalently linked to serine or threonine residues in O-glycosidic bonds (O- Glycoside-linked glycans), the type of covalent bond between glucose and protein in the glycoprotein of the present invention is not particularly limited, glycoproteins comprising one or both of N-glycoside-linked glycans and O-glycoside-linked glycans In the glycoprotein according to the present invention.

In this aspect, the present invention is directed to hyaluronidase PH20 or its variants, which have a sialylation amount of 1 to 38%, preferably 1 to 30%, more preferably 1.5 to 28% in the amount of N-glycans %, the best is 2 to 25%.

More specifically, the present invention is directed to hyaluronidase PH20 or its variants, which have a galactosylation amount of 1 to 68% of the N-glycan content and a sialylation content of 1 to 38% of the N-glycan content. And the amount of mannosylation in the amount of N-glycans is 40 to 63%, preferably the amount of galactosylation in the amount of N-glycans is 5 to 60%, and the amount of sialylation in the amount of N-glycans is 1 to 63%. 30% and the amount of mannosylation in the amount of N-glycans is 42 to 62%, more preferably the amount of galactosylation in the amount of N-glycans is 10 to 56%, and the amount of sialylation in the amount of N-glycans is 1.5 to 28% and 44 to 61% mannosylation on the N-glycan content, optimally 15 to 50% galactosylation on the N-glycan content, sialylation on the N-glycan content The amount is from 2 to 25% and the amount of mannosylation on the amount of N-glycans is from 47 to 60%.

The above numerical values are obtained from the experimental results of the exemplary embodiments shown in Table 1, and are obtained from numerical values with a 95% confidence interval, which also includes an error of 10%. This is because there is a difference in the measurement of the glucose content in the protein, which depends on conditions such as the equipment used in the experiment, the enzyme reaction time, the test temperature, the proficiency of the tester, etc., so the differences between laboratories are considered , the glucose content measured in the present invention should be interpreted in a broad sense rather than a limited meaning. In addition, the charge modification of PH20 or its variants can be determined by isoelectric focusing.

The hyaluronidase PH20 or its variants having a specific amount of sialylation and/or galactosylation and mannosylation in the amount of N-glycans according to the present invention are preferably produced by the production method according to the present invention, but are not limited thereto , and obviously hyaluronidase PH20 or variants thereof can be produced by other methods modified by those skilled in the art.

In the present invention, the medium used for culturing cells to express glycoproteins is preferably but not limited to serum-free medium, and DMEM/F12 medium (a combination of DMEM and F12 medium) can be used as the basic medium. In addition, commercially available serum-free media such as HycellCHO medium, ActiPro medium (HyClone, USA), CD OptiCHO ^TM medium, CHO-S-SFM II medium or CD CHO medium (Gibco, USA), IS CHO-V ^TM medium (Irvine Scientific, USA), EX-CELL ^® Advanced CHO fed batch medium (Sigma-Aldrich, USA), etc., as the basic medium, but the present invention is not limited thereto.

In the present invention, the feed medium used for culturing cells to express glycoproteins is a serum-free medium, for example, Cell Boost ^TM 1, Cell Boost ^TM 2, Cell Boost ^TM 3, Cell Boost ^TM can be used 4. Cell Boost ^TM 5, Cell Boost ^TM 6, Cell Boost ^TM 7a/7b (HyClone, USA), CD CHO EfficientFeed ^TM A AGTTM, CD CHO EfficientFeed ^TM B AGTTM, CD CHO EfficientFeed ^TM C AGTTM, CD CHO EfficientFeed ^TM A plus AGTTM, CD CHO ^{EfficientFeedTM} B plus AGTTM, CD CHO ^{EfficientFeedTM} C plus ^AGTTM (Gibco, USA), BalanCD ^® CHO Feed 4 (Irvine Scientific, USA), EX-CELL ^® Advanced CHO Feed (Sigma-Aldrich, USA) ), CHO-U Feed Mix U1B7/ CHO-U Feed Mix U2B13 (Kerry, USA) as feed medium, but the present invention is not limited thereto.

The terms "feed medium" and "concentrated nutrient medium" as used herein refer to a medium composed of specific nutrients or various nutrients such as amino acids, vitamins, salts, trace elements, lipids and glucose, and can be Concentrated product of basal medium. The composition and concentration of the manufactured feed culture can vary depending on the cells to be cultured. In addition, commercially available feed media can be used, such as Cell Boost series supplement medium (HyClone, USA), EfficientFeed supplement medium, GlycanTune feed medium (Gibco, USA), BalanCD CHO feed medium (Irvine Scientific, USA), Cellvento® CHO Cell Culture Feed Medium (Merck, USA), EX-CELL® Advanced CHO Feed Medium (Sigma-Aldrich, USA) and the like were used as the feed medium, but the present invention is not limited thereto.

The term "plant-derived hydrolyzate" as used herein refers to a product extracted from pea, cottonseed, wheat gluten, soybean, etc. and does not contain animal-derived ingredients, and contains a large amount of amino acids , peptides, vitamins, carbohydrates, nucleotides, minerals and other ingredients can also be manufactured to contain various ingredients and ingredient concentrations according to the cells to be cultured. In addition, commercially available plant-derived hydrolysates such as HyPep™ 7404, UltraPep™ Cotton, HyPep™ 7504, HyPep™ 4601N (Kerry, USA), Cotton 100, Cotton 200, Phytone™ and Soy 100 (Gibco , USA), as a supplement, but the present invention is not limited thereto.

Additives used to increase or decrease the amount of N-glycans of the glycoproteins used in the present invention are generally ingredients known to be involved in protein glycosylation. In particular, in order to limit the amount of sialylation, the transfer of sialic acid to galactose residues should be limited, desialylation should be performed or the amount of galactosylation should be limited. This supplement added to the medium at a predetermined concentration when culturing cells contains components that are precursors of glycosylation such as N-acetyl-D-mannosamine, glucose, mannose, glutamic acid, and galactose , ammonia and butyric acid.

The culture of most animal cells mainly uses medium containing serum. However, since serum-containing media are complex compositions and their chemical composition is unknown, it is difficult to design media suitable for protein production. Serum-free media or media containing a small amount of serum are mainly used due to possible negative effects of serum on isolation or purification and issues related to cost and reproducibility. Since the concentration of glucose as a carbon source in serum-free medium is very low, glucose will be added to the culture medium as the main carbon source to maintain cell growth and produce high concentrations of target proteins, and glutamine can be further added Acid is added thereto for cultivation. In particular, in order to increase the in vivo half-life in the production of protein pharmaceuticals, the degree of sialylation should be increased. For this reason, the glucose and glutamic acid in the medium should be maintained at a specific concentration or higher to avoid depletion, and the pH of the medium should also be maintained at a specific level. The glucose and glutamine concentrations measured in the medium refer to the concentrations of glucose and glutamine remaining after consumption by the cells. In addition, a promoter that improves the activity of an enzyme involved in an increase in the degree of sialylation or a repressor that suppresses desialylation can be used. In addition, by increasing the precursors of N-glycans or controlling the active promoters or culture conditions of related enzymes, the degree of galactosylation can be increased, and thus the degree of sialylation can also be increased.

However, in the production of hyaluronidase PH20 or its variants, the degree of N-glycans cannot be controlled by the above-mentioned method, but when produced by the method according to the present invention, hyaluronidase PH20 or its variants can be produced with a desired degree of glycans and high enzyme activity. Hyaluronidase PH20 or its variants.

The production method according to the present invention may further include isolating and purifying the produced hyaluronidase PH20 or its variants.

The separation and purification of hyaluronidase PH20 or its variants according to the present invention is preferably performed without using affinity binding but using the ionic bond and/or hydrophobic interaction properties of hyaluronidase PH20 or its variants, but the present invention Not limited to this.

Specifically, the separation and purification of hyaluronidase PH20 or its variants according to the present invention is preferably carried out without using affinity chromatography but using hydrophobic interaction chromatography and ion exchange chromatography, such as cationic Exchange chromatography and/or anion exchange chromatography, but the present invention is not limited thereto.

In addition, the separation and purification of hyaluronidase PH20 or its variants according to the present invention is aimed at removing acid hyaluronidase PH20 or its variants with low enzymatic activity, and the removal of acid hyaluronidase PH20 or its variants is preferably performed using performed by ion exchange chromatography.

The catalytic reaction rate of the enzyme needs to be analyzed to determine the industrial applicability of the enzyme. Enzyme reactions can be divided into enzyme reactions with active sites of fixed reactivity and enzyme reactions with multiple active sites with different reactivity. It is known that the rate of the catalytic reaction of an enzyme with a single active site of fixed reactivity, such as hyaluronidase, follows the Michaelis-Menten kinetic equation.

Michaelis-Menten enzyme kinetics is based on the following premise: Assuming that the enzyme reaction is a two-step reaction system including reversible and irreversible reactions, a complex of enzyme (E)-substrate (S) is formed in the reversible reaction [ ES], in an irreversible reaction the ES complex dissociates and produces the product (P). In this case, k _f , k _r and k _cat are the rate constants of the reactions in each direction (Alan Fersht (1977). Enzyme structure and mechanism).

For enzymatic reactions, it is assumed that the process by which the enzyme reacts with the substrate to produce the ES complex rapidly reaches an equilibrium or pseudo-steady state, assuming that the enzyme's concentration is sufficiently reduced by performing the reaction to maintain a sufficiently high substrate concentration. The concentration satisfies d[ES]/dt≒0. Since kinetic formulas assuming a rapid equilibrium or pseudo-steady state are derived in the same way, a pseudo-steady state in which the substrate concentration is initially higher than the enzyme concentration will be assumed in most experiments.

When using such assumptions as "the amount of enzyme is constant before and after the reaction" and "when the chemical reaction reaches chemical equilibrium, the reaction rate when the product is obtained is equal to the rate when the product is decomposed again", the reaction rate of the final product can be obtained by The following Mie (Michaelis-Menten) kinetic equation said. In this case, K _M = (k _r + k _cat ) / k _f and V _max = k _cat [E] ₀ .

The Lineweaver-Burk equation was used for the experimental analysis of enzyme reaction rates using the Michaelis-Menten kinetic equation. This equation shows the relationship between the reciprocal 1/V of the experimentally measured reaction rate and the reciprocal 1/[S] of a given substrate concentration in the experiment. The statistical verification that this equation is a linear equation indicates that the enzyme reaction follows the Michaelis-Menten kinetic equation, and K _M and V _max can be calculated using this equation.

Enzymes that catalyze chemical reactions have a transition state after the active site is bound to a substrate, and the activation energy used to reach a high-energy transition state is reduced through multiple bonds with the substrate. The equilibrium constant for reaching this transition state is proportional to k _cat /K _M . Here, 1/K _M is an index combining the degree of "generating an enzyme-substrate complex by combining an enzyme and a substrate" and the degree of "maintaining an enzyme-substrate complex without decomposing", and k _cat is derived from the enzyme - The equilibrium constant at which the substrate complex obtains a product. Therefore, k _cat /K _M can be said to be an indicator of how much product can be obtained from the substrate and the enzyme, that is, the catalytic efficiency of the enzyme.

The industrial availability of hyaluronidase is directly proportional to its enzymatic efficiency. In particular, the enzymatic efficiency of hyaluronidase plays an important role when the enzyme is co-injected subcutaneously with polymeric pharmacologically active substances such as monoclonal antibodies. In the case of variants according to the invention having a higher k _cat /K _M compared to wild-type PH20, when the hyaluronidase contained in the polymeric pharmacologically active substance is administered subcutaneously, the hyaluronic acid present therein is rapidly decomposed , so the superior effect of rapid dispersion of pharmacologically active substances can be obtained. In addition, when the variant according to the present invention has a larger k _cat than the wild-type PH20, the maximum reaction rate V _max will increase at the same enzyme concentration, thereby providing the ability to decompose a larger amount of hyaluronic acid during the same period and to combine pharmacological effects. Excellent effect of active substances dispersed in a wider area.

Therefore, in order to determine the enzyme properties of the PH20 variants according to the present invention, the enzyme reaction rate of each variant was analyzed, and its V _max (maximum enzyme reaction rate), K _m (in the case of 50% V _max ) was compared in Example 4. Substrate concentration), k _cat (substrate turnover rate) and k _cat /K _m (enzyme catalytic efficiency). These results show that the PH20 variants according to the invention are superior to wild type PH20.

example

Hereinafter, the present invention is described in detail with reference to Examples. However, it is obvious to those skilled in the art that these examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention.

Example 1, the relationship between hyaluronidase activity, N-glycans, and isoelectric focusing pattern

The activity, isoelectric focusing pattern and N-glycan extent of wild human hyaluronidase PH20 and its variant HM46 are revealed in FIG. 1 . Although the activity units of the two hyaluronidases differed by more than twofold, there was not much difference in the range of isoelectric points or the degree of N-glycans.

Experiments were performed to determine the relationship between N-glycans, isoelectric focusing patterns and activity in hyaluronidase PH20. During the purification of the hyaluronidase PH20 variant, the basic fraction (fraction 1) and the acidic fraction (fraction 2) can be separated and each fraction can be used as a sample. Figure 2 shows samples treated with PNGase F to remove all N-glycans, sialidase A to remove terminal sialic acid, and sialidase A and galactase to remove terminal saliva The results of isoelectric focusing mode and enzyme activity analysis of acid and galactose samples. The two fractions showed differences in the range of isoelectric points, with the acidic fraction showing reduced enzyme activity. When treated with PNGase F, both fractions were inactive and had similar isoelectric focusing patterns. These results show that the degree of N-glycans is closely related to enzyme activity. Furthermore, by the phenomenon that the isoelectric focusing pattern or enzyme activity of the acidic part is similar to that of the basic part when the sialic acid located at the terminal is removed, it was found that the content of the sialic acid located at the terminal and the enzyme activity There is a relationship between. However, the basic fraction with terminal low sialic acid content showed improved enzymatic activity of hyaluronidase compared to the acidic fraction with high sialic acid content.

The relationship between the enzyme activity and the amount of N-glycans was further elucidated based on the results of evaluating wild-type and various mutant hyaluronidases derived from different cultured cells.

Table 1 Hyaluronidase type preparation relative activity N-glycan content Galactosylation (%) Sialylation (%) Mannosylation (%) wild-type human PH20 temporary performance training 100% 32.9 15.2 57.2 wild-type human PH20 Cell line culture 100% 42.5 21.7 54.8 HM46 temporary performance training 167% 37.1 7.4 48.1 HM46 Cell line colony test culture #1 115% 46.9 18.6 48.9 HM46 Cell Line Propagation Test Culture #2 Purified Fraction #1 161% 40.6 16.4 54.4 HM46 Cell line colony test culture #2 Purified fraction #2 86% 42.1 28 56.8 HM46 Cell line test culture #1 158% 49.6 14.4 48.1 HM46 Cell line test culture #2 186% 31.2 5.5 48.3 HM46 Cell line test culture #3 152% 30.6 4 48.7 HM46 Cell line test culture #4 174% 15.8 4.5 52.8 HM46 Cell Line Culture #1 170% 14.8 2.8 49.9 HM46 Cell Line Culture #2 161% 16.4 2.5 47.9 HM46 Cell Line Culture #3 162% 17.9 4.4 49.1 HM46 Cell Line Culture #4 158% 18.5 3.8 48.1 Sheep PH20 temporary performance training 193% 35.5 7.9 58.3 Bonobo PH20 temporary performance training 72% 38.7 18.8 55.5

Relative activity: activity expressed as a percentage of (activity of sample)/(activity of wild human PH20).

Galactosylation percentage (%): the sum of the content percentage (%) of N-glycans containing galactose at the end of G1, G1F, G1F', G2, G2F, A1, A1F, A2 and A2F.

Sialylation percentage (%): the sum of the content percentages (%) of N-glycans containing sialic acid at the end of A1, A1F, A2 and A2F.

Mannosylation percentage (%): the sum of the content percentages (%) of N-glycans containing mannose at the end of M4G0F, M5, M5G0, M6, M7, M8 and M9.

The sequences of wild-type human PH20 and HM46 are disclosed in International Patent Publication No. 2020/022791.

Ovine PH20 and Bonobo PH20 samples were prepared by the method shown in Example 10, and the sequences are disclosed in Table 3.

As shown in Table 1, various types of hyaluronidase PH20 and mutant proteins were produced and the amount of N-glycan was investigated. To this end, cultures using various cell sources were performed, such as temporary expression cultures produced by transfecting ExpiCHO cells with recombinant genes using ExpiFectamine CHO reagent (Gibco, USA) at each culture, using Cell line clone culture of cell line clones produced by recombinant genes and cell line culture of selected single strains with high productivity. Here, the cell line culture is a culture in which a high-productivity cell line is selected from a mammalian cell line transfected with a recombinant gene using a selectable marker, and is performed using multiple cell lines, and is intended to produce Single cell line. Cell lines include a research cell bank (RCB), a production master cell bank (MCB) and a working cell bank (WCB). In addition, in the culture method, experimental culture Variations in activity and N-glycans were found even here in order to optimize production.

Cell strain test culture #1 is a 2-liter (L) batch culture using HycellCHO medium (HyClone, USA) without adjusting the culture temperature at 37°C. Cell line test culture #2 purified fraction #1 and cell line test culture #2 purified fraction #2 are the fractions separated and purified from the harvested cell culture fluid by anion exchange chromatography, wherein the harvested cells Culture medium was used in 8 L batches containing EX-CELL® Advanced CHO Feed Batch Medium (Sigma-Aldrich, USA) with Cotton 100UF (Gibco) and BalanCD® CHO Feed 4 (Irvine Scientific, USA) feed The batch method was incubated at 37°C, and purified fraction #1 was the low salt eluting fraction and purified fraction #2 was the high salt eluting fraction. Cell line test culture #1 was prepared by using EX-CELL® Advanced CHO Feed Batch Medium (Sigma-Aldrich, USA) with Cotton 100UF (Gibco) and BalanCD® CHO Feed 4 (Irvine Scientific , USA) by culturing at a culture temperature of 37°C, and then adjusting the culture temperature to 32°C when the integrated viable cell density reached a predetermined level. Cell line test culture #2 was obtained by using EX-CELL® Advanced CHO Feed Batch Medium (Sigma-Aldrich, USA) with Cotton 100UF (Gibco, USA) and CD CHO EfficientFeed ^TM B plus in a 10 L batch The feed-batch method of AGTTM (Gibco, USA) was obtained by culturing at a culture temperature of 37°C, and then adjusting the culture temperature to 32°C when the integrated viable cell density reached a predetermined level, followed by culturing for 12 days. Cell line test culture #3 was obtained by using EX-CELL® Advanced CHO Feed Batch Medium (Sigma-Aldrich, USA) with Cotton 100UF (Gibco, USA) and CD CHO EfficientFeedTM B plus AGTTM in a 50 L batch The fed-batch method (Gibco, USA) was obtained by culturing at a culture temperature of 37°C and then adjusting the culture temperature to 32°C when the integrated viable cell density reached a predetermined level. Cell line test culture #4 was obtained by using EX-CELL® Advanced CHO Feed Batch Medium (Sigma-Aldrich, USA) in a 10 L batch with Cotton 100UF (Gibco, USA) and CD CHO EfficientFeedTM B plus The fed-batch method of AGTTM (Gibco, USA) was cultured at a culture temperature of 37°C, and then the culture temperature was adjusted to 32°C when the integrated viable cell density reached a predetermined level, and the cells were cultured for 13 days to obtain . Cell line culture #1, cell line culture #2, cell line culture #3 and cell line culture #4 were obtained by supplementing EX-CELL® Advanced CHO fed batch medium (Sigma-Aldrich, USA) with Cotton 100UF (Gibco, USA) and CD CHO EfficientFeedTM B plus AGTTM (Gibco, USA) fed batch method to cultivate research cell bank (RCB) and production master cell bank at a culture temperature of 37°C (MCB) and working cell bank (WCB), and then adjust the culture temperature to 32°C when the integrated viable cell density reaches a predetermined level, and culture the cells for 12 days. Based on these conditions, experiments for the culture conditions of Examples 2 to 5 were performed to establish a culture method for maintaining the degree of N-glycans.

It can be seen from Table 1 that the galactosylation degree of wild human hyaluronidase PH20 and its variants and mammalian hyaluronidase PH20 in N-glycans should be 15 to 50%, and the sialylation degree should be 2.5 to 28%. and the degree of mannosylation should be from 47 to 60%, and when applying a 95% confidence interval to the test results, the degree of lactosylation in the N-glycan moiety should be from 1 to 68%, the degree of sialylation should be from 1 to 38%, and The degree of mannosylation should be from 40 to 63% to confer industrially useful hyaluronidase activity on it. This value may also contain an error of 10%, because there are differences in the measurement of the level of glucose in the protein, which depends on conditions such as the equipment used in the experiment, the reaction time of the enzyme, the temperature of the experiment, the proficiency of the experimenter, etc., Therefore, considering the differences between laboratories, the glucose level measured in the present invention should be interpreted in a broad sense rather than a limited meaning.

Furthermore, in particular, considering the relationship between activity and sialylation, it can be seen that only when sialylation is limited to about 30% or less can an activity with an activity higher than wild-type human PH20 be produced and thus be useful in industry on hyaluronidase. The above values may be derived from experimental results and may include an error of 10%.

High-quality hyaluronidase maintaining N-glycan levels was confirmed by temporal expression culture, and cell lines were produced by selecting individual strains with high productivity for stable commercial production. All cell line test cultures #2, #3, #4 and cell line culture #1, #2, #3, #4 are disclosed in Table 1, which are cultivated by using the results of Examples 2, 3, 4, and 5 method, which exhibited an enzyme expression level of 10,000 units/mL or more, thereby proposing the possibility of producing high-quality hyaluronidase with high efficiency and low cost.

Example 2, culture dependent on additives

Hyaluronidase PH20 variants were seeded at ^2x106 cells/ml in supplemented with or without Cotton 200UF (Gibco, USA) and 20 mM N-acetyl-D-mannosamine (NZP, Netherlands) or 50 mM galactose (Pfanstiehl, USA) in each of three Erlenmeyer flasks containing EX-CELL® Advanced CHO fed batch medium (Sigma-Aldrich, USA) in an _incubator at 37°C and 8% CO Culture was performed by batch culture, followed by fed batch culture at a reduced temperature of 32°C when the integral viable cell density (IVCD) reached the adjusted range. CD CHO EfficientFeed ^™ B plus AGT™ medium (Gibco, USA) was supplied daily as feed medium at 1.88% of the initial culture volume in the flask. Cell samples were collected daily from the cell culture medium to measure viable cell density, cell viability, pH and lactic acid levels. After the culture was terminated, centrifugation was performed at 2,000 rpm for 10 minutes to obtain a culture supernatant. The activity of samples incubated under the above conditions was determined by HPLC and turbidity analysis, and the protein pattern and N-glycan level were observed by isoelectric focusing and glycosylation analysis. Media supplemented with 50 mM galactose exhibited a 24% increase in galactosylation and a 2% decrease in activity compared to media not supplemented with 50 mM galactose, compared to media not supplemented with N-acetyl-D-mannose Glycosamine-based medium, supplemented with 20 mM N-acetyl-D-mannosamine, exhibited a 35% increase in sialylation and a 20% decrease in activity (Fig. 3, Fig. 4, Fig. 5, Fig. 6).

Example 3, depending on the culture during the culture

Cells expressing the hyaluronidase PH20 variant were seeded at ^2x106 cells/ml in EX-CELL® Advanced CHO-fed batch medium (Sigma-Aldrich, USA) in a Sartorius 200 L bioreactor. On the second day of culture, feed batches were performed using Cotton 200UF (Gibco, USA) and CD CHO EfficientFeedTM B plus AGTTM medium (Gibco, USA), which is a concentrated nutrient medium (Gibco, USA), as feeding medium. Formula culture, the pH is 7.2 ± 0.4 and the DO is 40% to cultivate at the rate of 47 rpm. Initial culture was performed at 37°C, and when the integrated viable cell density reached the changing range, the temperature was adjusted to 32°C for fed-batch culture. CD CHO EfficientFeedTM B plus AGTTM medium (Gibco, USA) was used as feed medium at a daily rate of 1.88% of the initial culture volume in a 200 L bioreactor. For the culture period, the culture was terminated on day 19 after culture when the cell viability dropped to 40% or less. Collect multiple cell samples from the cell culture solution every day, measure the viable cell density, cell viability, integrated viable cell density and ammonium ion level, and use a depth filter to harvest the cultured solution. The activity of samples incubated under the above conditions was determined by HPLC and turbidity analysis, and the protein pattern and N-glycan level were observed by isoelectric focusing and glycosylation analysis. With the increase of culture days, the degree of galactosylation and sialylation decreased and the activity increased (Figure 7, Figure 8, Figure 9, Figure 10).

Example 4, Cultivation under controlled glucose concentration in the medium

Cells expressing the hyaluronidase PH20 variant were seeded at ^2x106 cells/ml in EX-CELL® Advanced CHO-fed batch medium (Sigma-Aldrich, USA) in a Sartorius 2 L bioreactor. On the second day of culture, Cotton 200UF (Gibco, USA) and CD CHO EfficientFeedTM B plus AGTTM medium (Gibco, USA), which is a concentrated nutrient medium (Gibco, USA), were used as feeding medium to carry out feeding batches. Formula culture, the pH is 7.2 ± 0.4 and the DO is 40% to cultivate at the rate of 120 rpm. Initial culture was performed at 37°C, and when the integrated viable cell density reached the changing range, the temperature was adjusted to 32°C, followed by fed-batch culture. CD CHO ^{EfficientFeedTM} B plus AGTTM medium (Gibco, USA) was supplied as feed medium at 1.88% of the initial culture volume in a 2 L bioreactor per day. Multiple cell samples were collected daily from the cell culture medium to measure viable cell density, cell viability, pH, osmolarity, and glucose and lactate concentrations.

The concentration of glucose in the medium is measured daily and controlled from the point when the concentration of glucose contained in the culture supernatant does not exceed a reference concentration of 2, 4 or 6 g/L as the glucose is consumed by cell growth concentration of glucose. When the measured glucose concentration is 2, 4 or 6 g/L or lower (which is the respective standard concentration), add a certain amount of 200 g/L glucose stock solution for a maximum time of 3 hours to reach the standard concentration . When the measured glucose concentration is 2, 4 or 6 g/L or more, no stock solution is added to maintain the standard concentration. Generally speaking, during the culture of mammalian cells, the effect of glucose content changes on cell growth is negligible within 3 hours.

In this case, the condition of maintaining 2 g/L as the reference concentration is called 1 g/L (±1 g/L) concentration condition, and the condition of maintaining 4 g/L as the reference concentration is called 3 g/L ( ±1 g/L) concentration condition, the condition of maintaining 6 g/L as the reference concentration is called 5 g/L (±1 g/L) concentration condition. For example, the condition of maintaining the glucose concentration at 1 g/L (±1 g/L) means that the lower limit of the control range of the glucose concentration is set at 0 g/L and the upper limit of the control range of the glucose concentration is set at 2 g/L, It also indicates that in actual culture, when the measured glucose concentration in the culture supernatant reaches the lower limit of 0 g/L, 200 g/L of glucose stock solution is further added to make the maximum glucose concentration reach 2 g/L, And, when the glucose concentration in the culture supernatant reached the upper limit of 2 g/L, no 200 g/L glucose stock solution was added. Therefore, under two concentration conditions, that is, 1 g/L (±1 g/L) concentration and 3 g/L (±1 g/L) concentration, the concentration condition of 2 g/L corresponds to 1 g/L ( The upper limit of the concentration condition of ±1 g/L) corresponds to the lower limit of the concentration condition of 3 g/L (±1 g/L), so the two conditions are the conditions for completely different actions, so they are not considered to overlap each other.

After the culture was terminated, the culture supernatant was obtained by centrifugation at 4°C and 10,000 rpm for 60 minutes. The activity of samples incubated under the above conditions was determined by HPLC and turbidity analysis, and the protein pattern and N-glycan level were observed by isoelectric focusing and glycosylation analysis. The results revealed that the degree of galactosylation and sialylation increased and the activity decreased as the concentration of glucose in the culture supernatant increased ( FIG. 11 , FIG. 12 , FIG. 13 and FIG. 14 ).

Example 5, Cultivation at Controlled pH

Cells expressing the hyaluronidase PH20 variant were seeded at ^2x106 cells/ml in EX-CELL® Advanced CHO-fed batch medium (Sigma-Aldrich, USA) in a Sartorius 2 L bioreactor. On the second day of culture, feed was performed using Cotton 200UF (Gibco, USA) and CD CHO EfficientFeed ^™ B plus AGT™ medium (Gibco, USA), which is a concentrated nutrient medium (Gibco, USA), as feed media. Batch culture was carried out at a DO rate of 40% at a rate of 120 rpm. Initial culture was performed at 37°C, and when the integrated viable cell density reached the changing range, the temperature was adjusted to 32°C, followed by fed-batch culture. CD CHO ^{EfficientFeedTM} B plus AGTTM medium (Gibco, USA) was supplied as feed medium at 1.88% of the initial culture volume in a 2 L bioreactor per day. The culture temperature was adjusted, and the culture was divided and further cultured according to four pH conditions, namely, pH 6.8±0.1, pH 7.0±0.1, pH 7.2±0.1, and pH 7.4±0.1, to find the Improved conditions. The pH control range of the culture was set according to these four conditions using a general bioreactor including a pH control function. For example, pH 7.0±0.1 means that the lower limit of the pH control range is pH 6.9, and the upper limit of the pH control range is pH 7.1. In actual cultivation, when the pH of the medium reaches the lower limit of pH 6.9, alkali is added to increase the pH, and when the pH of the medium reaches the upper limit of pH 7.1, carbon dioxide is added to lower the pH. Therefore, among the two conditions of pH 6.8±0.1 and pH 7.0±0.1, the condition of pH 6.9 corresponds to the upper limit of pH 6.8±0.1 and corresponds to the lower limit of pH 7.0±0.1. Therefore, the two conditions are considered non-overlapping with each other. Culture until the culture day when the cell viability is 40% or lower, collect multiple cell samples from the cell culture medium every day, and measure the viable cell density, cell viability, pH and integrated viable cell density. After the culture was terminated, the culture supernatant was obtained by centrifugation at 4°C and 10,000 rpm for 60 minutes. The activity of samples incubated under the above conditions was determined by HPLC and turbidimetric analysis, and the protein pattern was determined by isoelectric focusing. The pH of the medium with the highest activity and the lowest sialylation degree was observed at pH 7.0±0.1, which is an improved condition compared to general conditions (Table 2, Figure 15, Figure 16, Figure 17).

Table 2 N-glycan pattern pH 7.2±0.4 (general conditions) pH 6.8±0.1 pH 7.0±0.1 pH 7.2±0.1 pH 7.4±0.1 Galactosylation (%) 39.6 49.4 37.0 42.1 47.6 Sialylation (%) 14.6 12.7 6.3 13.9 20.9 Mannosylation (%) 48.4 43.3 50.5 50.3 49.3 Total defucosylation (%) 53.4 46.2 51.2 56.2 57.2 Defucosylation (%) 7.7 5.9 4,2 8.9 10.7 Relative specific activity (%) 100% 92% 115% 104% 98%

Example 6, Purification of hyaluronidase using animal cell culture supernatant

Step 1: Treat supernatant with buffer exchange/surfactant

Use a 30 kDa MWCO membrane filter to control conductivity and pH through UF/DF, and readjust the condition of the culture solution to the equilibrium condition of the first anion exchange column. Treat the readjusted solution with an appropriate concentration of solvent/surfactant to inactivate the virus, and react at room temperature for about 60 minutes.

Step 2: Primary Anion Exchange (Q Sepharose Fast Flow) Column Chromatography

The filtered protein solution is passed through a primary anion exchange column to capture hyaluronidase through the anion exchange resin and eluted from the column at high salt concentration. The column was equilibrated with tromethamine buffer (pH 8.0 and salt concentration 30 mM) before loading. After loading, flush the column with the same buffer (primary flush). After the primary flush, the column was flushed a second time with the same buffer (pH 8.0, but with a salt concentration of 60 mM and a higher conductivity than in the primary flush). After the second wash, elution of the desired protein (hyaluronidase) was performed using an appropriate buffer with a pH of 8.0 and a salt concentration of 200 mM, as shown in FIG. 18 .

Step 3: Secondary Anion Exchange (Capto Q) Column Chromatography

The filtered protein was passed through a secondary anion exchange column to remove acidic hyaluronidase variants using anion resin exchange. Before loading, equilibrate the column with Bistris buffer (pH 6.0 and no salt). After loading, flush the column with the same Bistris buffer (primary flush). After the primary wash, the column was flushed with the same Bistris buffer (with a salt concentration of 20 mM, which has a higher conductivity compared to the salt concentration in the primary wash). After the second wash, the desired protein (hyaluronidase) was eluted using Bistris buffer with a certain salt concentration, as shown in FIG. 19 .

Step 4: Cation Exchange (Capto MMC) Column Chromatography

The reconstituted protein solution was passed through a cation exchange column to remove acidic hyaluronidase variants using the cation exchange resin and eluted from the column at high salt concentration. Before loading, equilibrate the column with pH 5.5 citrate buffer with a salt concentration of 80 mM. After loading, flush the column with the same citrate buffer (primary flush). After the primary flush, flush the column with an appropriate pH 7.5 Bistris buffer. After the second wash, elution of the desired protein (hyaluronidase) was carried out using Bistris buffer pH 8.0 with a salt concentration of 400 mM, as shown in Figure 20.

Step 5: Nanofiltration/Formulation

After the cation exchange column step, the protein solution containing the desired hyaluronidase was filtered through a 1 micron filter and subjected to nanofiltration. The nanofiltered protein solution was concentrated to a high concentration of 10 milligrams per milliliter (mg/mL) and reconditioned by UF/DF using an 8 kDa MWCO membrane filter to pH 7.0 histidine buffer containing 145 mM salt fluid exchange.

Example 7, Analysis of Enzyme Activity of Hyaluronidase

The enzymatic activity of hyaluronidase PH20 and other hyaluronidases was measured using the nephelometric method as described below.

Turbidimetry is a method that uses absorbance to measure the precipitate produced during the mixing of hyaluronic acid and albumin (BSA). When hyaluronic acid is hydrolyzed by PH20, the absorbance will decrease during mixing with albumin. In general, this process proceeds as follows. Hyaluronidase PH20 (Sigma) was diluted to 1, 2, 5, 7.5, 10, 15, 20, 30, 50 or 60 units/mL and placed in each tube. The purified protein samples were dissolved in enzyme dilution buffer (20 mM Tris·HCl pH 7.0, 77 mM NaCl, 0.01% (w/v) bovine serum albumin) and diluted 100X, 300X, 600X, 1200X or 2400X in each tube. Dilute the 3 mg/mL hyaluronic acid solution 10x to 0.3 mg/mL in other tubes to adjust the volume in each tube to 180 microliters (μL). Mix 60 μL of enzyme with diluted hyaluronic acid solution and react at 37°C for 45 minutes. After the reaction, 50 μL of the reacted enzyme and 250 μL of acidic albumin solution were added to each well of the 96-well plate and shaken for 10 minutes, and the absorbance was measured at 600 nm using a spectrophotometer. The activity unit of the sample is obtained using the test result of the standard whose activity unit is known and the test result of the sample.

Example 8, Isoelectric Focusing Analysis of Hyaluronidase

Isoelectric focusing of hyaluronidase was analyzed using a precast gel (pH 3-7, Invitrogen) and a buffer solution for isoelectric focusing. The purified hyaluronidase sample was loaded on the pregel, and the gel was subjected to electrophoresis at 100V for 1 hour, at 200V for 1 hour, and at 500V for 30 minutes using an electrophoresis apparatus manufactured by Novex Corporation. After the electrophoresis was completed, the gel was washed with pure water, the protein was fixed with 12% TCA solution, stained with Coomassie Blue R-250 staining solution and bleached with acetic acid-methanol solution to analyze the protein bands presented on the gel.

Example 9, Analysis of N-glycan extent of hyaluronidase

The N-glycan level of hyaluronidase was analyzed by labeling N-glycan samples, separated by treating hyaluronidase with PNGase F (N-glycosidase F), 2-AB (2-aminobenzamide), and then Ultra-high performance liquid chromatography was performed by using ACQUITY UPLC Glycan BEH Amide column (Waters). The purified hyaluronidase samples were desalted and reacted with PNGase F at 37°C for 16 to 18 hours to separate N-glycans therefrom. N-glycans were labeled with 2-AB, followed by reaction at 65°C for 3 hours, and excess 2-AB was removed therefrom. Labeled N-glycan samples were separated by HPLC with a 72%-20% acetonitrile gradient. The separated samples were detected with a fluorescent detector (FLD) to analyze the level of N-glycans. Classify each isolated N-glycan and determine the percentage of galactosylation by summing the N-glycans (G1, G1F, G1F', G2, G2F, A1, A1F, A2, A2F, etc.) containing galactose at the end (%) level, the N-glycans (A1, A1F, A2, A2F, etc.) containing sialic acid at their ends are added to determine the percentage of sialylation (%), and the N-glycans containing mannose at their ends (M4G0F, M5, M5G0, M6, M7, M8, M9, etc.) were added together to determine the percentage (%) of mannose.

Example 10, Preparation of animal-derived hyaluronidase and analysis of N-glycan levels

(1) Production of bonobo and sheep hyaluronidase PH20 genes

In order to study the N-glycan level and activity of hyaluronidase PH20 derived from animals such as anthropoid (ie, bonobo) and artiodactyla (ie, sheep), hyaluronidase PH20 was prepared as follows. cDNA was synthesized from the wild-type gene and inserted into the Xho I and Not I restriction enzyme positions of the pcDNA3.4-TOPO vector. For expression in ExpiCHO cells, the message peptide of one of human growth hormone, human serum albumin, or human Hyal1 was used as the message peptide instead of the natural message peptide of PH20. For protein purification using HisTrap columns, the His-tag DNA sequence is located at the 3' end of the PH20 cDNA. Each sequence was determined using DNA sequencing. Table 3 discloses the sequences of hyaluronidases derived from animals.

table 3 Hyaluronidase from Animals amino acid sequence Sheep PH20 LDFRAPPLISNTSFLWAWNAPAERCVKIFKLPPDLRLFSVKGSPQKSATGQFITLFYADRLGYYPHIDEKTGNTVYGGIPQLGNLKNHLEKAKKDIAYYIPNDSVGLAVIDWENWRPTWARNWKPKDVYRDESVELVLQKNPQLSFPEASKIAKVDFETAGKSFMQETLKLGKLLRPNHLWGYYLFPDCYNHNYNQPTYNGNCSDLEKRRNDDLDWLWKESTALFPSVYLNIKLKSTPKAAFYVRNRVQEAIRLSKIASVESPLPVFVYHRPVFTDGSSTYLSQGDLVNSVGEIVALGASGIIMWGSLNLSLTMQSCMNLGNYLNTTLNPYIINVTLAAKMCSQVLCHDEGVCTRKQWNSSDYLHLNPMNFAIQTGKGGKYTVPGKVTLEDLQTFSDKFYCSCYANINCKKRVDIKNVHSVNVCMAEDICIEGPVKLQPSDH Bonobo PH20 LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINVTGQDVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDIYKNRSIELVQQQNVQLNLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFVYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEESQIFY

(2) PH20 performance of bonobo and sheep hyaluronidase

Performance was performed using the ExpiCHO performance system. When the number of ExpiCHO cells reached 6x10 ⁶ cells/ml, ExpiFectamine CHO reagent was used to transfect ExpiCHO cells with plastids in which hyaluronidase PH20 cDNA was inserted into pcDNA3.4-TOPO vector. The cell culture medium used here is ExpiCHO expression medium (100~500ml). After transfection, ExpiCHO cells were cultured while shaking at 130 rpm for 6 days. During this period, the cells were cultured at 37°C for 1 day, followed by a further 5 days at a reduced temperature of 32°C. When the culture was completed, the cells were centrifuged at 10,000 rpm for 30 minutes to obtain a cell supernatant.

(3) Purification of bonobo and hyaluronidase PH20

AKTA prime system (GE Healthcare Systems) was used to purify the animal-derived hyaluronidase recombinant protein produced in ExpiCHO cells with a His-tag attached to its carboxy-terminus by two-step column chromatography. The pI of bonobo PH20 is 6, so Q Sepharose with anion exchange chromatography is used, and the pI of sheep PH20 is 8 or more, so a Capto S column with cation exchange chromatography is used for one-step purification. Each protein was purified in two steps using a HisTrap HP column for His-Tag affinity chromatography.

For protein purification using Q Sepharose columns, buffer A (20 mM sodium phosphate, pH 7.5) and buffer B (20 mM sodium phosphate, pH 7.5, 0.5 M NaCl) were prepared. Proteins were bound to Q Sepharose columns, and the column was washed with 5 CVs of Buffer A to remove non-specific binding proteins, followed by 5 CVs of Buffer B at a concentration gradient from 0 to 100% Wash down to elute the protein.

For protein purification using Capto S columns, buffer A (20 mM sodium phosphate, 15 mM NaCl, pH 6.0) and buffer B (20 mM sodium phosphate, 500 mM NaCl, pH 6.0) were prepared. The pH and conductivity of the medium were adjusted to be the same as buffer A, and the medium was filtered through a membrane with a pore size of 0.22 μm. Protein samples were bound to a Capto S column, and the column was washed with 3 CV of buffer A to remove non-specifically bound proteins. Wash the column with 4CV of buffer B to elute the protein.

To use HisTrap HP column for protein storage, prepare buffer A (20 mM sodium phosphate, 500 mM NaCl, pH 7.5) and buffer B (20 mM sodium phosphate, 500 mM NaCl, 500 mM imidazole, pH 7.5). The protein sample was bound to the HisTrap HP column, and the column was washed with 7 CV of 7% buffer B to remove non-specific binding proteins, followed by 3 CV of 40% buffer B to elute the target protein. The column extract was dialyzed against dialysis buffer (20 mM sodium phosphate, 100 mM NaCl, pH 7.0).

(4) Bonobo and sheep PH-20 hyaluronidase analysis

Activity analysis was performed in the same manner as in Example 7, and N-glycan level analysis was performed in the same manner as in Example 9. The results are disclosed in Table 1.

Example 11, Enzyme Kinetic Analysis of Variants Depending on the Degree of N-Glycans

To analyze the enzyme kinetics of the variants according to the invention, the enzyme activity was measured by the Morgan-Elson method (Takahashi, T. et al (2003) Anal. Biochem. 322:257-263). The Morgan-Elson method is a colorimetric method that uses p-dimethylaminobenzaldehyde (DMAB) (ie Ehrlich reagent) to detect N-acetyl-D-glucosamine ( The red substance (at 545 nm) produced by the reaction of the reducing end of GlcNAc). Diluted to 0.25, 0.50, 0.75, 1.00 or 1.25 mM N-acetyl- D-glucosamine (GlcNAc, Sigma) was reduced in each test tube by tetraborate treatment, followed by the addition of DMAB to induce a colorimetric reaction. After the reaction, the absorbance was measured at 545 nm to prepare a standard reaction curve of GlcNAc. Hyaluronic acid as a substrate was diluted to 0.54, 0.65, 0.87, 1.23 or 2.17 μM in dilution buffer solution in each test tube, hyaluronidase was added to each test tube, followed by reaction at 37°C for 5 minutes and at 100°C C for 5 minutes to complete the enzyme reaction. The product was reduced by treatment with tetraborate and DMAB was added to induce a colorimetric reaction. The absorbance was measured at 545 nm after the reaction, and the enzyme activity was measured using the above-mentioned standard reaction curve of GlcNAc. This method was used to analyze the enzyme kinetics of the wild-type PH20 of SEQ ID NO: 1 and the PH20 variants according to the invention. As a result, it was found that the Lineweaver-Burk curve was linear, which indicated that the PH20 variant according to the present invention followed the Michaelis-Menten enzyme kinetic formula.

Table 4 discloses the analysis of enzyme kinetics of Fraction #1 and Fraction #2 obtained from cell line colony test culture #2 among the samples in Table 1 of Example 1. These results showed that when the degree of galactosylation and mannosylation fell in a similar range and the degree of sialylation was low, the enzymatic activity of recombinant hyaluronidase was increased due to the high catalytic efficiency (kcat/Km) of the enzyme.

The experimental results confirmed that the activity of enzymes with the same amino acid structure may be affected by changes in glycosylation, more specifically, by changes in the degree of sialylation. Therefore, this means that in an attempt to manufacture industrially useful hyaluronidase by mass-producing wild PH20 hyaluronidase or its variants using recombinant methods, an enzyme with greater industrial applicability can be developed by controlling the degree of sialylation.

Table 4 Hyaluronidase type preparation relative activity N-glycan content _KM (µM) k _cat (1/sec) k _cat /K _M (sec /µM) Galactosylation (%) Sialylation (%) Mannosylation (%) HM46 Cell Line Propagation Test Culture #2 Purified Fraction #1 161% 40.6 16.4 54.4 1.11±0.07 47.6 ±3.8 42.7±0.7 HM46 Cell line colony test culture #2 Purified fraction #2 86% 42.1 28.0 56.8 1.05±0.15 33.9 ±4.9 32.4±0.1

According to the method for producing recombinant hyaluronidase PH20 protein or its variants of the present invention, recombinant hyaluronidase PH20 protein or its variants with high productivity and high enzyme activity can be produced, so the mass production of recombinant hyaluronidase PH20 protein or its variants can be realized and supply.

Although specific configurations of the present invention have been described in detail, those skilled in the art should understand that the embodiments are for illustrative purposes only and should not be construed as limiting the scope of the present invention. Therefore, the substantive scope of the present invention is defined by the scope of the patent application and its equivalent scope.

references

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2. Douglas B. Muchmore, M.D., and Daniel E. Vaughn, Ph.D. (2012). Accelerating and Improving the Consistency of Rapid-Acting Analog Insulin Absorption and Action for Both Subcutaneous Injection and Continuous Subcutaneous Infusion Using Recombinant Human Hyaluronidase. J. Diabetes Sci. and Technol. 6(4): 764-72

3. E. M. Krantz. (1980). Low-dose intramuscular ketamine and hyaluronidase for induction of anaesthesia in non-premedicated children. S. Afr. Med. J. 58(4):161-2.

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11. Veronica Restelli, Ming-Dong Wang, Norman Huzel, Martin Ethier, Helene Perreault, Michael Butler. (2006). The effect of dissolved oxygen on the production and the glycosylation profile of recombinant human erythropoietin produced from CHO cells. Biotechnol. Bioeng . 94:481-494.

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13. M. C. Borys, D. I. Linzer, E. T. Papoutsakis. (1994). Ammonia affects the glycosylation patterns of recombinant mouse placental lactogen-I by Chinese hamster ovary cells in a pH-dependent manner. Biotechnol. Bioeng. 43:505-514.

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none.

The above and other purposes, features and other advantages of the present invention will be more clearly understood from the following embodiments with reference to the accompanying drawings, wherein:

Figure 1 reveals the analysis results of the enzyme activity, isoelectric focusing pattern and N-glycan content of wild human hyaluronidase PH20 and its variants, wherein Figure 1A reveals the enzyme activity of wild human hyaluronidase PH20 and its variants, Figure 1B reveals the results of isoelectric focusing for each sample, and Figure 1C reveals N-glycan amounts.

Figure 2 reveals the results of comparative analysis of enzyme activity and isoelectric focusing patterns between purified hyaluronidase PH20 variants treated with PNGase F, sialidase A, and sialidase A+galactase, wherein Figure 2A reveals the results of isoelectric focusing for each sample, Figure 2B reveals enzyme activity, and Figure 2C reveals N-glycan amounts.

Figure 3 reveals the changes in cell growth, cell viability, pH and lactic acid concentration depending on the culture conditions supplemented with N-acetyl-D-mannosamine (ManNAc) or galactose for cells producing hyaluronidase PH20 variants the result of which Figure 3A reveals changes in cell growth, Figure 3B reveals changes in cell viability, Figure 3C reveals changes in pH, and Figure 3D reveals changes in lactate concentration.

Fig. 4 is a graph showing the activity of harvested cell culture fluids for production of hyaluronidase PH20 variants depending on culture conditions added with N-acetyl-D-mannosamine (ManNAc) or galactose.

Fig. 5 discloses the results of isoelectric focusing analysis of harvested cell culture fluids for production of hyaluronidase PH20 variants depending on culture conditions with addition of N-acetyl-D-mannosamine (ManNAc) or galactose.

Figure 6 reveals the results of the N-glycan structure analysis of the harvested cell culture medium for the production of hyaluronidase PH20 variants depending on the culture conditions with the addition of N-acetyl-D-mannosamine (ManNAc) or galactose result.

Figure 7 reveals the changes in cell growth, cell viability and ammonia concentration of cells producing hyaluronidase PH20 variants as a function of days in culture, wherein Figure 7A reveals changes in cell growth, Figure 7B reveals changes in cell viability, Figure 7C reveals changes in integrated viable cell density, and Figure 7D reveals the ammonia concentration.

Fig. 8 is a graph showing the specific activity of the harvested cell culture fluid used for the production of the hyaluronidase PH20 variant according to the days of cell culture.

FIG. 9 reveals the results of isoelectric focusing of the harvested cell culture medium used for the production of hyaluronidase PH20 variants according to the days of cell culture.

Figure 10 reveals the results of the activity analysis and N-glycan structure of the harvested cell culture fluid used for the production of hyaluronidase PH20 variants with the number of days of cell culture.

Figure 11 reveals that for cells producing hyaluronidase PH20 variants, changes in cell growth depending on glucose concentration conditions, cell viability, pH, osmotic pressure, glucose concentration, and lactic acid concentration, wherein Figure 11A reveals changes in cell growth, Figure 11B reveals changes in cell viability, Figure 11C reveals the change in pH, Figure 11D reveals changes in osmotic pressure, Figure 11E reveals changes in glucose concentration, and Figure 1 IF reveals changes in lactate concentration.

Figure 12 is a graph revealing the specific activity of harvested cell culture fluid for production of hyaluronidase PH20 variants as a function of glucose concentration.

Figure 13 reveals the results of isoelectric focusing of harvested cell culture fluids used for production of hyaluronidase PH20 variants depending on glucose concentration.

Figure 14 reveals the results of N-glycan structure analysis of harvested cell culture fluids used for production of hyaluronidase PH20 variants depending on glucose concentration.

Figure 15 reveals the results of changes in cell growth, cell viability, pH and integrated viable cell density depending on the pH level for cells producing hyaluronidase PH20 variants, wherein Figure 15A reveals changes in cell growth, Figure 15B reveals changes in cell viability, Figure 15C reveals the change in pH, and Figure 15D reveals changes in integrated viable cell density.

Figure 16 is a graph showing the specific activity of the harvested cell culture fluid of cells used to produce hyaluronidase PH20 variants depending on the pH level.

Figure 17 shows the results of isoelectric focusing of the harvested cell culture fluids used for the production of hyaluronidase PH20 variants depending on the pH level.

Figure 18 is a purification chromatogram obtained by primary anion exchange resin chromatography during the purification of hyaluronidase PH20 variants.

Fig. 19 is a purification chromatogram obtained by secondary anion exchange resin chromatography during the purification process of hyaluronidase PH20 variant.

Figure 20 is a purification chromatogram obtained by cation exchange resin chromatography during the purification process of hyaluronidase PH20 variant.

Claims (16)

A method for producing recombinant hyaluronidase PH20 or a variant thereof, comprising: (1) cultivating a host cell expressing recombinant hyaluronidase PH20 or a variant thereof at a culture temperature of 35°C to 38°C until the host cell Integral viable cell density (Integral viable cell density) reaches 20x10 ⁶ to 120x10 ⁶ cells x day/ml; and (2) lower the culture temperature to 28°C to 34°C, and then maintain the culture temperature while The host cells are cultured for 2 to 18 days: (a) wherein the residual glucose concentration in the medium is maintained at 0.001 g/L to 4.5 g/L during the culture period; and/or (b) wherein the pH of the medium is maintained at 6.8 to 7.2; wherein the sialylation amount of an N-glycan of the PH20 or its variant is 1 to 38%, and wherein the hyaluronidase activity in the culture medium is 10,000 units/ml or higher. The method according to claim 1, wherein the galactosylation amount of the N-glycan of the PH20 or its variants produced is 1 to 68%, the sialylation amount is 1 to 38%, and a mannosylation The amount is 40 to 63%. The method according to claim 1, wherein the sialylation amount of the N-glycan of the PH20 or its variants produced is 1 to 30%. The method according to claim 1, wherein the specific activity of the produced PH20 or its variant is at least 10% higher than that of wild human PH20. The method as described in claim 1, wherein in step (1) and/or step (2), the culture of the host cell is selected from batch culture, repeated batch culture, feeding batch culture, repeated feeding One or more methods of the group consisting of batch culture, continuous culture and perfusion culture. The method according to claim 1, wherein the culture of the host cell in step (1) and/or step (2) is carried out under one or more conditions selected from the group consisting of: (i) medium The ammonia concentration is maintained at 5 mM or more; (ii) one or more substances selected from the group consisting of glutamic acid, glucosamine, uridine, glucosamine and sodium butyrate are added to the medium; and (iii) a condition in which galactose and manNAc are not added to the medium. The method according to claim 1, wherein the PH20 variant comprises substitution of one or more amino acid residues at the amino terminal and/or carboxyl terminal in the amino acid sequence of the wild PH20, and optionally comprises A truncation of one or more amino acid residues. The method according to claim 1, wherein the host cell is animal cell, yeast, actinomycete or insect cell. The method as described in claim 1, further comprising isolating and purifying the produced PH20 or its variants. The method as claimed in claim 1, wherein the isolation and purification of the PH20 or its variants is performed using the ionic bond and/or hydrophobic interaction properties of the PH20 or its variants instead of affinity binding. The method according to claim 10, wherein the isolation and purification of the PH20 or its variants are performed using hydrophobic interaction chromatography and ion exchange chromatography instead of affinity chromatography. The method according to claim 9, wherein acidic PH20 or variants thereof are removed. The method according to claim 12, wherein the acidic hyaluronidase PH20 or its variants are removed by ion exchange chromatography. A PH20 or a variant thereof, wherein the galactosylation amount of an N-glycan of the PH20 or its variant is 1 to 68%, the sialylation amount is 1 to 38%, and the mannosylation amount is 40 to 63% %. PH20 or its variant as described in claim 14, wherein the sialylation amount of the N-glycan is limited to 1 to 30%. The PH20 or its variant as described in Claim 14, wherein the PH20 or its variant is produced by the method as described in any one of Claims 1 to 13.

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