Method for preparing recombinant hyaluronidase

Abstract

The present invention relates to a method for preparing hyaluronidase and variants thereof. Specifically, the method is capable of changing the N-glycan level under culture conditions, including controlled glucose concentration in the culture medium and reduced culture temperature and specified culture time, thereby increasing specific activity by 10% or more, and improving quality and yield.

Description

Technical Field

[0001] The present invention relates to a method for preparing a hyaluronidase PH20 variant, which comprises one or more amino acid residue substitutions and optionally comprises a deletion of certain N-terminal and / or C-terminal amino acid residues of the hyaluronidase, in particular a wild-type or mature wild-type PH20 or a wild-type or mature wild-type PH20 amino acid sequence. Background Art

[0002] 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 fluids provided by subcutaneous injection (Muchmore et al., 2012) or intramuscular injection (Krantz et al., 2016), as well as to improve the spread of local anesthetics (Clement et al., 2003). Drugs that have been enhanced for 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 have penetrated into tissues by intravenous injection or hematoma spread. Among the hyaluronidases, a recombinant human PH20 protein active at neutral pH was developed by Halozyme Therapeutics Inc. and sold under the trade name “Hylenex” (Bookbinder et al., 2006).

[0003] Recombinant human PH20 protein has been reported to be expressed in yeast (P. pastoris), DS-2 insect cells and animal cells. The recombinant PH20 protein produced in insect cells and yeast differs from human PH20 in the N-glycosylation pattern during post-translational modification, thus affecting its activity and bringing the risk of side effects in vivo.

[0004] For biopharmaceutical development, product identity and long shelf life are important factors that provide manufacturing flexibility. Various types of microheterogeneity can occur during the manufacturing process due to 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 reduce the pI value, thereby forming acidic variants (Harris RJ et al., 2004). In addition, cleavage of the C-terminal lysine results in a loss of net positive charge and the formation of acidic variants. The formation of basic variants can be caused by deamidation of the C-terminal lysine or glycine, succinimide formation, amino acid oxidation, or sialic acid removal, where sialic acid removal removes additional positive or negative charge, and both types of modifications increase the pI value (Harris RJ et al., 2004).

[0005] Glycosylation is a post-translational process of proteins in cells (eukaryotes) and occurs in the endoplasmic reticulum and the glomerular bodies. Glycosylation is divided into N-glycosylation and O-glycosylation, which differs according to the functional group attached. The process of attaching sugars such as lactose or fucose to the proteins produced in the cell is widely called "glycosylation". When glycans are attached to proteins through glycosylation, the protein undergoes a "folding" process to form a three-dimensional structure. This gives the protein stability, allowing it to remain for a long time without loosening (unfolding). Glycosylation is a key process used to enable communication and information exchange between cells.

[0006] There are two types of reactions for adding glycans to proteins: N-linked or O-linked glycosylation. The two glycosylation processes differ in the mechanisms of glycan synthesis and addition, of which the mechanism and role of N-glycosylation are better known. Glycans added by N-glycosylation are called "N-glycans" and are formed in the endoplasmic reticulum. A series of asparagine-linked glycosylation (ALG) enzymes on dolichol pyrophosphate (PP-Dol) present in the endoplasmic reticulum membrane add N-acetylglucosamine (GlcNAc), mannose (Man), glucose (Glc), etc., ultimately synthesizing Glc3Man9GlcNAc2-PP-Dol, which is a complex glycan in the form of lipid-linked oligosaccharide (LLO). The synthesized LLO is transferred to the N-glycosylated sequence of a peptide containing NxS / T, which is directly translated from the ribosome to the endoplasmic reticulum via a mid-translational translocation mechanism of oligosaccharyltransferases composed of 8 or more subunits. The N-glycans attached to the protein 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, folding is completed with the help of lectin chaperone, calnexin and calreticulin. When all glucose are removed, protein folding is considered complete and the Man8GlcNAc2 glycans attached to it are transferred to the gognathian body. In this regard, there is a quality control process to verify the folding of the glycoprotein again before it is transferred from the endoplasmic reticulum to the gognathian body. When proper folding is not achieved, the process is repeated by allowing a molecule of glucose to enter the calnexin / calreticulin cycle to provide an opportunity to complete folding.

[0007] The initial process of N-glycan biosynthesis in the endoplasmic reticulum is conserved in almost the same way in a wide range of organisms from yeast (a simple eukaryotic microorganism) to animals (i.e., higher organisms). However, the glycans transferred to the homozygous body undergo various species-specific glycan modifications, resulting in the formation of completely different types of glycans in yeast, insects, and animals. However, these various glycans also generally share a core position, i.e., a structure in which three mannoses and two GlcNAcs are attached to the nitrogen of asparagine, which is called a "trimannosyl core". The form in which mannose is mainly attached to the trimannosyl core is called a "high-mannose type" and is commonly found in yeast and molds. The structure in which multiple mannoses are continuously added to the homozygous body is also found in Saccharomyces cerevisiae, a well-known yeast. On the other hand, glycoproteins with glycans of the deficient mannose type (Pauci-mannose type) are found in insect cells. These are first trimmed in the Homozygous body by mannosidases IA, IB and IC to form Man5GlcNAc2, N-acetylglucosamine transferase (GNT) I is used to add one GlcNAc to Man5GlcNAc2, and then mannosidase II is used to form a mixed structure in which a GlcNAc is added to the trimannose core. Then, the added GlcNAc is cleaved again to form a glycan structure of the deficient mannose type. In animal cells, α(1,6)-fucose is often added to the first GlcNAc linked to the asparagine residue. In animals, GNT II acts on a structure in which one GlcNAc is added to the trimannose core and another GlcNAc is added to it, forming a glycan with a two antenna structure. GNT IV and V can then be used to form a tetra-antenna structure, and in some cases, GNT VI, IX or VB can be used to form a hexa-antenna structure. After the addition of GlcNAc to form the antenna backbone, β-galactosyltransferase and α-sialyltransferase present in the Golgi apparatus are used to form a complex glycan structure with galactose and sialic acid added to the GlcNAc.

[0008] N-glycosylation can profoundly affect protein folding or activity. When a protein or its variant existing in nature is prepared by genetic engineering methods for industrial applications, the presence of glycosylation and the structure or form of glycans are likely to vary depending on the host cell, the recombinant manipulation method and the culture conditions (Schilling, et al., 2002). In other words, differences in the amount of sugar components constituting glycans or the structure of glycans may occur depending on the production conditions during protein production. Culture conditions that affect N-glycosylation include the concentration of glucose or glutamine in the culture medium (Tachibana et al. 1994), the concentration of dissolved oxygen (DO) (Restelli et al. 2006), the pH of the culture medium (Borys et al. 1993), the concentration of ammonia in the culture medium (Borys et al. 1994), the culture temperature (Clark et al. 2004), etc.

[0009] In general, enzymes specifically bind to substances to form enzyme-substance complexes, which act as catalysts to reduce the activation energy of the reaction and promote the reaction of the enzyme. Enzymes can distinguish between substances and molecules that compete with substances, and can specifically bind to substances due to the distribution of complementary charges, complementary structures, and hydrophilicity and hydrophobicity at the position where the enzyme binds to the substance. In order to clarify the binding position of the enzyme, a lock-and-key model has been proposed in which the enzyme and the substance are complementary to each other and have a geometric shape, but it cannot satisfactorily explain the transition state of the enzyme-substance complex. According to the induced chimeric model proposed to overcome this problem, as the enzyme and the substance continue to interact in the enzyme-substance complex, they change their structure based on the variable structure of the enzyme protein. In this process, the reaction can be further promoted by the distribution of surrounding charges caused by the N-glycans or amino acid residues that constitute the active site or by the distribution of hydrophilicity / hydrophobicity.

[0010] Furthermore, charge interactions are essential for the reaction of hyaluronidase, an enzyme that hydrolyzes hyaluronic acid as a substance. Arming et al. revealed that positively charged arginine in hyaluronidase PH20 is essential for the enzyme activity to bind hyaluronic acid, a substance having a large amount of negative charges distributed therein (Arming et al. 1997). Therefore, it can be inferred that the charge distribution of N-glycans also affects the enzyme activity. It is important to demonstrate that when hyaluronic acid, a substance having a large amount of negative charges, binds to hyaluronidase, the degree of negatively charged sialic-acid-capping sugars in N-glycans (i.e., the degree of sialylation) affects the formation of the enzyme-substance complex or the progress of the enzyme reaction. In order to limit the degree of sialylation, the transfer of sialic acid to galactose residues should be limited, desialylation should be performed, or the degree of galactosylation should be limited.

[0011] Therefore, the productivity and activity of recombinant hyaluronidase PH20 and its variants will be affected by changes in the level of N-glycans. Therefore, for effective mass production in this field, it is necessary to study and develop a method for preparing hyaluronidase PH20 or its variants that have high activity and productivity and can be used in industry by controlling and maintaining the level of N-glycans. Summary of the invention

[0012] Therefore, the present invention is completed in view of the above-mentioned problems. One object of the present invention is to provide a method for culturing host cells for preparing recombinant hyaluronidase PH20 or its variants and hyaluronidase PH20 or its variants prepared by this method, especially a method for preparing hyaluronidase PH20 or its variants with improved enzyme activity and productivity.

[0013] The purpose of the present invention is not limited to the above contents. Other purposes not described here will be clearly understood by those skilled in the art from the following description.

[0014] In the present invention, when the host cells used to prepare recombinant hyaluronidase PH20 or its variants are cultured under specific culture conditions, the N-glycosylation properties of the produced recombinant hyaluronidase PH20 or its variants, and further, the enzyme activity of the produced hyaluronidase PH20 or its variants, will be significantly improved.

[0015] 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 the produced hyaluronidase PH20 or its variants can be effectively improved, and it was found that by culturing for a specific time after changing the culture temperature, the enzyme activity and productivity of the hyaluronidase PH20 or its variants according to the present invention can be significantly improved.

[0016] Specifically, the method for preparing hyaluronidase PH20 or a variant thereof according to the present invention comprises:

[0017] (1) Cultivate host cells expressing recombinant hyaluronidase PH20 or its variants at a culture temperature of 35°C to 38°C until the integral viable cell density is 20x10 6 Up to 120x10 6 cells x day / mL; and

[0018] (2) lowering the culture temperature to 28° C. to 34° C., and then culturing the host cells for 2 to 18 days while maintaining the culture temperature according to at least one method selected from the following:

[0019] (a) culturing the host cell while maintaining the 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

[0020] (b) culturing the host cells while maintaining the pH of the medium at 6.8 to 7.2.

[0021] The PH20 or its variant prepared by the preparation method according to the present invention is characterized in that the amount of sialylation of the N-glycan of the produced PH20 or its variant is 1 to 38%, so that the enzyme activity is significantly improved, but not limited to this, this value is an experimental value with an error range of 10%. The reason is that when setting the culture conditions, differences will occur depending on the conditions such as the equipment used for culture and the operating proficiency of the experimenter, so considering these differences, the values ​​set by the present invention should be interpreted as a broad sense, rather than a narrow sense. The host cell can be carried out by one or more methods selected from batch culture, repeated batch culture, fed-batch culture, repeated fed-batch culture, continuous culture and perfusion culture. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The above and other objects, features and other advantages of the present invention will be more clearly understood from the following embodiments with reference to the accompanying drawings, in which:

[0023] Figure 1 The present invention discloses the analysis results of the enzyme activity, isoelectric focusing pattern and N-glycan amount of wild-type human hyaluronidase PH20 and its variants, wherein

[0024] Figure 1 A reveals the enzymatic activity of wild-type human hyaluronidase PH20 and its variants,

[0025] Figure 1 B reveals the results of isoelectric focusing of each sample, and

[0026] Figure 1 C reveals the amount of N-glycans.

[0027] FIG. 2 shows the results of comparative analysis of enzyme activity and isoelectric focusing patterns between purified hyaluronidase PH20 variant fractions treated with PNGase F, sialidase A, and sialidase A+galactosidase, wherein

[0028] Figure 2A The results of isoelectric focusing of each sample are revealed.

[0029] Figure 2B Revealing enzyme activity, and

[0030] Figure 2C Reveals N-glycan amounts.

[0031] Figure 3 Disclosed are results of changes in cell growth, cell viability, pH, and lactate concentration depending on culture conditions of adding N-acetyl-D-mannosamine (ManNAc) or galactose for cells producing hyaluronidase PH20 variants, wherein

[0032] Figure 3 A reveals changes in cell growth,

[0033] Figure 3 B reveals changes in cell viability,

[0034] Figure 3 C reveals changes in pH, and

[0035] Figure 3 D reveals changes in lactate concentration.

[0036] Figure 4 Graphs showing the activity of harvested cell culture fluid for the preparation of hyaluronidase PH20 variants depending on culture conditions with the addition of N-acetyl-D-mannosamine (ManNAc) or galactose.

[0037] Figure 5 The results of isoelectric focusing analysis of harvested cell culture fluids for the preparation of hyaluronidase PH20 variants depending on the culture conditions of the addition of N-acetyl-D-mannosamine (ManNAc) or galactose are disclosed.

[0038] Figure 6The results of N-glycan structural analysis of harvested cell culture fluids used to prepare hyaluronidase PH20 variants depending on culture conditions of the addition of N-acetyl-D-mannosamine (ManNAc) or galactose are disclosed.

[0039] Figure 7 Revealing changes in cell growth, cell viability and ammonia concentration of cells producing hyaluronidase PH20 variants as a function of culture days, wherein

[0040] Figure 7 A reveals changes in cell growth,

[0041] Figure 7 B reveals changes in cell viability,

[0042] Figure 7 C reveals changes in the integrated viable cell density, and

[0043] Figure 7 D reveals the ammonia concentration.

[0044] Figure 8 is a graph showing the specific activity of the cell culture fluid harvested for the preparation of hyaluronidase PH20 variants over the number of days of cell culture.

[0045] Fig. 9 The results of isoelectric focusing of cell culture fluid harvested over the number of days of cell culture for preparing hyaluronidase PH20 variants are disclosed.

[0046] Fig.10 The results of activity analysis and N-glycan structure of cell culture fluid harvested over the number of days of cell culture for preparing hyaluronidase PH20 variants are disclosed.

[0047] Fig.11 The invention discloses a cell for preparing a hyaluronidase PH20 variant, which is dependent on changes in cell growth under glucose concentration conditions, cell viability, pH, osmotic pressure, glucose concentration, and lactate concentration, wherein

[0048] Fig.11 A reveals changes in cell growth,

[0049] Fig.11 B reveals changes in cell viability,

[0050] Fig.11 C reveals changes in pH,

[0051] Fig.11 D reveals changes in osmotic pressure,

[0052] Fig.11 E reveals changes in glucose concentration, and

[0053] Fig.11 F reveals the changes in lactate concentration.

[0054] Fig.12 Graph showing the specific activity of harvested cell culture fluid used to prepare hyaluronidase PH20 variants depending on glucose concentration.

[0055] Fig.13 The results of isoelectric focusing of harvested cell culture fluid for the preparation of hyaluronidase PH20 variants are revealed depending on the glucose concentration.

[0056] Fig.14 The results of N-glycan structural analysis of harvested cell culture fluids used to prepare hyaluronidase PH20 variants are revealed depending on glucose concentration.

[0057] Fig.15 Disclosed are results of changes in cell growth, cell viability, pH, and integrated viable cell density depending on pH levels for cells producing hyaluronidase PH20 variants, wherein

[0058] Fig.15 A reveals changes in cell growth,

[0059] Fig.15 B reveals changes in cell viability,

[0060] Fig.15 C reveals changes in pH, and

[0061] Fig.15 D reveals the changes in the integrated viable cell density.

[0062] Fig.16 A graph showing the specific activity of cell culture fluid harvested from cells used to prepare hyaluronidase PH20 variants depending on pH levels.

[0063] Fig.17 The results of isoelectric focusing of the harvested cell culture fluid used to prepare the hyaluronidase PH20 variant are revealed depending on the pH level.

[0064] Fig.18 The purification chromatogram obtained by primary anion exchange resin chromatography during the purification of hyaluronidase PH20 variant.

[0065] Fig.19 The figure is a purification chromatogram obtained by secondary anion exchange resin chromatography during the purification of hyaluronidase PH20 variant.

[0066] Fig. 20 The figure is a purification chromatogram obtained by cation exchange resin chromatography during the purification of hyaluronidase PH20 variant. DETAILED DESCRIPTION

[0067] Unless otherwise defined, technical and scientific terms used herein have the same meaning as understood by one of ordinary skill in the art to which the invention belongs. Generally speaking, the nomenclature used herein is well known and commonly used.

[0068] The present invention is based on the following discovery: the degree of sialylation, galactosylation and / or mannosylation, especially the degree of sialylation of N-glycans, is critical for the enzymatic activity and productivity of hyaluronidase PH20 or its variants when using genetic engineering methods to prepare hyaluronidase PH20 or its variants for industrial applications.

[0069] Specifically, it was found that when the sialylation amount of N-glycans of PH20 or its variants was 1 to 38%, preferably 1 to 30%, more preferably 1.5 to 28%, and most preferably 2 to 25%, the enzyme activity and productivity of hyaluronidase PH20 or its variants were improved to an unexpectedly high degree.

[0070] More specifically, it was found that the enzyme activity and productivity of hyaluronidase PH20 or its variants were improved to an unexpectedly high level when the degree of galactosylation of the N-glycan of PH20 or its variants was 1 to 68%, the amount of sialylation was 1 to 38% and the amount of mannosylation was 40 to 63%, preferably when the amount of galactosylation was 5 to 60%, the amount of sialylation was 1 to 30% and the amount of mannosylation was 42 to 62%, more preferably when the amount of galactosylation was 10 to 56%, the amount of sialylation was 1.5 to 28% and the amount of mannosylation was 44 to 61%, and most preferably when the amount of galactosylation was 15 to 50%, the amount of sialylation was 2 to 25% and the amount of mannosylation was 47 to 60%.

[0071] The above values ​​are obtained from the experimental results of the embodiment shown in Table 1, and are values ​​obtained with a 95% confidence interval, which 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 the equipment used in the experiment, the enzyme reaction time, the test temperature, the proficiency of the experimenter, etc., so considering the differences between laboratories, the glucose level measured in the present invention should be interpreted in a broad sense, rather than a limited sense.

[0072] 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, for example, 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, for example, A1, A1F, A2 and A2F, and the mannosylation ratio (%) is the sum of N-glycans containing mannose at their terminals among N-glycans, for example, M4G0F, M5, M5G0, M6, M7 and M8.

[0073] More specifically, the method for preparing hyaluronidase PH20 or a variant thereof having a sialylation amount of 1 to 38% in N-glycans according to the present invention comprises:

[0074] (1) Cultivating host cells expressing recombinant hyaluronidase PH20 or its variants at a culture temperature of 35°C to 38°C until the integral viable cell density is 20×10 6 Up to 120x10 6 cells x day / mL; and

[0075] (2) lowering the culture temperature to 28° C. to 34° C., and then culturing the host cells for 2 to 18 days while maintaining the culture temperature according to at least one method selected from the following:

[0076] (a) culturing the host cell while maintaining the 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

[0077] (b) culturing the host cells while maintaining the pH of the medium at 6.8 to 7.2.

[0078] The above numerical values ​​are experimental values ​​with a 10% error range. The reason is that when setting the culture conditions, differences may occur depending on conditions such as the equipment used for culture and the operator's operating proficiency. Therefore, considering these differences, the numerical values ​​set in the present invention should be interpreted in a broad sense rather than a narrow sense.

[0079] The reduction in the culture temperature from step (1) to step (2) was performed when the integrated viable cell density reached 20 x 10 6 Up to 120x10 6 cells x day / ml, preferably 40x10 6 Up to 100x10 6 cells x day / ml, preferably 60x10 6 Up to 80x10 6 Cells x days / mL, but not limited to.

[0080] In addition, the culture period of step (2) may be 2 to 18 days, preferably 3 to 16 days, and more preferably 4 to 14 days, but is not limited thereto.

[0081] For example, in the case of using a fed-batch culture method in the preparation of PH20 or its variants according to the present invention, the productivity of PH20 or its variants can be maximized and the enzyme activity of the produced PH20 or its variants can be significantly improved by the following method: immediately before the main culture, inoculum culture is carried out at 35° C. to 38° C. by perfusion culture or fed-batch culture until the cell concentration reaches a specific level, followed by main culture by inoculation at a reduced temperature below 35° C. for 2 to 18 days, preferably 3 to 16 days, and more preferably 4 to 14 days.

[0082] The main culture cell seeding concentration can be 1x10 5 cells / mL or higher, preferably 5x10 5 cells / mL or higher, preferably 1x10 6 cells / ml or more, but not limited thereto.

[0083] By using a combination of fed-batch culture and perfusion culture for cultivation in the preparation of PH20 or its variants according to the present invention, the productivity of PH20 or its variants can be maximized and the enzyme activity of the produced PH20 or its variants can be significantly improved.

[0084] In the present invention, the residual glucose concentration in the culture medium during the culture period 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 is not limited thereto.

[0085] 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, and more preferably 0.1 to 3.5 g / L during the culture process" used herein means that when the residual glucose concentration in the culture medium measured in real time is lower than the set reference concentration of 0.001 g / L to 4.5 g / L, preferably 0.01 to 4.0 g / L, and more preferably 0.1 to 3.5 g / L for 1 to 36 hours, preferably 3 to 30 hours, and more preferably 6 to 24 hours during the culture period, the glucose stock solution is added to the culture medium to obtain the corresponding reference concentration during the culture.

[0086] In the present invention, it is obvious to those with ordinary knowledge in the art that the reference concentration of the residual 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, and more preferably 0.1 to 3.5 g / L, and the reference concentration can be appropriately changed during the culture period. For example, the reference concentration of the residual glucose in the culture medium on the first and second days of culture is 2 g / L, and the reference concentration is reduced to 1.5 g / L on the third to fifth days of culture, and then the reference concentration is set to a level lower than 1.5 g / L, such as 1.0 g / L.

[0087] The activity of hyaluronidase PH20 or its variant prepared by the method according to the present invention and having a predetermined degree of sialylation, galactosylation and / or mannosylation in the N-glycan portion in the culture medium of hyaluronidase is 10,000 units / mL, preferably 11,000 units / mL, or more preferably 12,000 units / mL or higher, but not limited thereto.

[0088] In addition, the hyaluronidase PH20 or its variant prepared by the method according to the present invention and having predetermined sialylation, galactosylation and / or mannosylation on the N-glycan is a wild-type human PH20 prepared by a method known in the art, and the enzyme activity is increased by 10% or more, preferably 12% or more, and more preferably 15% or more compared to the activity of the wild-type human PH20 prepared by a method known in the art, but the present invention is not limited thereto.

[0089] Preferably, in the method for preparing hyaluronidase PH20 or its variant according to the present invention, the culturing of the host cells in step (1) and / or step (2) may be carried out under one or more conditions selected from the following: (i) adding ammonia to the culture medium or increasing the ammonia concentration in the culture medium to 5 mM or more; (ii) adding one or more substances selected from glutamine, glucosamine, uridine, glucosamine and sodium butyrate to the culture medium; and (iii) not adding galactose and manNAc to the culture medium, but the present invention is not limited thereto.

[0090] More specifically, in the method for preparing hyaluronidase PH20 or its variant according to the present invention, the culturing of the host cells in step (1) and / or step (2) can be carried out in the following manner: adding ammonia to the culture medium, increasing 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 thereto.

[0091] As used herein, the term "hyaluronidase PH20 or a variant thereof" refers to an enzyme that degrades hyaluronic acid located in the extracellular matrix.

[0092] The terms "hyaluronidase PH20" or "PH20" used herein are interpreted to include both wild-type PH20 and its mature form, and the terms "hyaluronidase PH20 or PH20 variant" refer to PH variants comprising substitution, deletion or insertion of one or more amino acid residues and optionally comprising deletion of certain N-terminal and / or C-terminal amino acid residues in the amino acid sequence of "hyaluronidase PH20" or "PH20", but are not limited thereto.

[0093] The hyaluronidase according to the present invention refers to a 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 from humans, cattle or sheep.

[0094] According to the embodiments of the human-derived "Hyaluronidase PH20" or "PH20 variant" of the present invention, International Patent Publication No. 2020 / 022791 and U.S. Patent No. 9,447,401, but not limited thereto, are interpreted as comprising any hyaluronidase or variant comprising substitutions, deletions and insertions of one or more amino acid residues and optionally comprising truncations of the N-terminal and / or C-terminal amino acid residues in the amino acid sequence of "Hyaluronidase PH20" or "PH20" and having hyaluronidase enzyme activity.

[0095] Examples of host cells used to express hyaluronidase protein may include animal cells, yeast, actinomycetes, insect cells, etc., but are not limited thereto.

[0096] 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, and CHO cells for large-scale expression are particularly preferred. In addition, in order to prepare the desired protein, it is preferred to use cells suitable for introducing the desired gene, such as dhfr-CHO cells (Proc. Natl. Acad. Sci. USA (1980) 77, 4216-4220) or CHO K-1 cells (which are CHO cells with DHFR gene knockout) (Proc. Natl. Acad. Sci. USA (1968) 60, 1275) or CHO K-1 cells (Proc. Natl. Acad. Sci. USA (1968) 60, 1275). Specifically, CHO cells are preferably DG44, DXB-11, K-1 or CHO-S cell strains, and the vector is introduced into the host cells using a method such as the calcium phosphate method, the DEAE dextran method or the lipoprotein method.

[0097] Examples of yeast include Saccharomyces sp., Hansenula sp., Kluyveromyces, Pichia sp., etc., and actinomycetes include, for example, Streptomyces, but are not limited thereto.

[0098] In the method for preparing hyaluronidase PH20 or its variant according to the present invention, the culturing of host cells in step (1) and / or step (2) can be carried out by one or more methods selected from batch culture, repeated batch culture, fed batch culture, repeated fed batch culture, continuous culture and perfusion culture, but is not limited thereto.

[0099] Batch culture is a culture method for proliferating cells in which no fresh culture medium is added or the culture solution is discharged during the culture process. Continuous culture is a culture method in which the culture medium is continuously added and discharged during the culture process. In addition, continuous culture includes perfusion culture. Fed-batch culture is between batch culture and continuous culture, also known as "semi-batch culture", and involves continuous or sequential addition of culture medium during the culture process. This culture method prevents the discharge of cells even if the culture solution is continuously discharged. In the present invention, any culture method can be used, preferably fed-batch culture or continuous culture, and specifically preferably fed-batch culture.

[0100] As described above, in the hyaluronidase PH20 or its variant prepared by the method of the present invention, the specific activity of the enzyme can be increased by 10% or more compared to that prepared by a general method, especially compared to the specific activity of wild-type human PH20. This increase in enzyme activity is due to changes in the N-glycosylation properties and / or charge modification of the hyaluronidase PH20 or its variant prepared by the method of the present invention.

[0101] In particular, the enzyme activity increases depending on the changes in sialylation, galactosylation and / or mannosylation in the N-glycosylation pattern. These N-glycosylation characteristics, in particular sialylation, galactosylation and / or mannosylation, can be adjusted by the preparation method according to the invention.

[0102] In the present invention, examples of the covalent bond between glucose and protein include N-glycosidic bonds and O-glycosidic bonds, etc. In the N-glycosidic bond, N-acetyl-D-glucosamine is covalently linked to an asparagine residue constituting the protein (N-glycoside-linked glycan), and in the O-glycosidic bond, N-acetyl-D-galactosamine is covalently linked to a serine or threonine residue (O-glycoside-linked glycan). There is no particular limitation on the type of covalent bond between glucose and protein in the glycoprotein of the present invention, and glycoproteins containing one or both of N-glycoside-linked glycans and O-glycoside-linked glycans fall within the glycoprotein according to the present invention.

[0103] In this aspect, the present invention is directed to hyaluronidase PH20 or a variant thereof, wherein the amount of sialylation in the amount of N-glycans is 1 to 38%, preferably 1 to 30%, more preferably 1.5 to 28%, and most preferably 2 to 25%.

[0104] More specifically, the present invention is directed to hyaluronidase PH20 or a variant thereof, which has a galactosylation amount of 1 to 68% in terms of N-glycan amount, a sialylation amount of 1 to 38% in terms of N-glycan amount and a mannosylation amount of 40 to 63% in terms of N-glycan amount, preferably a galactosylation amount of 5 to 60% in terms of N-glycan amount, a sialylation amount of 1 to 30% in terms of N-glycan amount and a mannosylation amount of 42 to 62% in terms of N-glycan amount, more preferably a galactosylation amount of 10 to 56% in terms of N-glycan amount, a sialylation amount of 1.5 to 28% in terms of N-glycan amount and a mannosylation amount of 44 to 61% in terms of N-glycan amount, and most preferably a galactosylation amount of 15 to 50% in terms of N-glycan amount, a sialylation amount of 2 to 25% in terms of N-glycan amount and a mannosylation amount of 47 to 60% in terms of N-glycan amount.

[0105] The above values ​​are obtained from the experimental results of the embodiments shown in Table 1 and are obtained by values ​​with a 95% confidence interval, which also includes a 10% error. This is because there are differences in the measurement of glucose content in proteins, which depends on conditions such as the equipment used in the experiment, the enzyme reaction time, the test temperature, the proficiency of the experimenter, etc., so considering the differences between laboratories, the glucose content measured in the present invention should be interpreted in a broad sense, rather than a limited sense. In addition, the charge modification of PH20 or its variants can be determined by isoelectric focusing.

[0106] According to the present invention, hyaluronidase PH20 or its variant having a specific amount of sialylation and / or galactosylation and mannosylation in N-glycans is preferably prepared by the preparation method according to the present invention, but is not limited thereto, and it is obvious that hyaluronidase PH20 or its variant can be prepared by other methods modified by a person having ordinary knowledge in the art.

[0107] In the present invention, the culture medium used to culture cells to express glycoproteins is preferably, but not limited to, a serum-free culture medium, and DMEM / F12 culture medium (a combination of DMEM and F12 culture medium) can be used as a basal culture medium. In addition, commercially available serum-free culture media such as HycellCHO culture medium, ActiPro culture medium (HyClone, USA), CDOptiCHOTM culture medium, CHO-S-SFM II culture medium or CD CHO culture medium (Gibco, USA), IS CHO-VTM culture medium (Irvine Scientific, USA), EX- Advanced CHO fed batch medium (Sigma-Aldrich, USA) etc. are used as the basic medium, but the present invention is not limited thereto.

[0108] In the present invention, the feed medium used to culture cells to express glycoproteins is a serum-free medium, for example, Cell Boost™ 1, Cell Boost™ 2, Cell Boost™ 3, Cell Boost™ 4, Cell Boost™ 5, Cell Boost™ 6, Cell Boost™ 7a / 7b (HyClone, USA), CD CHO EfficientFeed™ A AGT™, CD CHO EfficientFeed™ B AGT™, CD CHO EfficientFeed™ C AGT™, CD CHO EfficientFeed™ A plus AGT™, CD CHO EfficientFeed™ B plus AGT™, CD CHO EfficientFeed™ C plus AGT™ (Gibco, USA), CHO Feed 4 (Irvine Scientific, USA), EX- Advanced CHO Feed (Sigma-Aldrich, USA) and CHO-U Feed Mix U1B7 / CHO-U Feed Mix U2B13 (Kerry, USA) were used as feed culture media, but the present invention is not limited thereto.

[0109] The terms "feed medium" and "enriched nutrient medium" used herein refer to a medium composed of specific nutrients or multiple nutrients such as amino acids, vitamins, salts, trace elements, lipids and glucose, and can be a concentrated product of a basal medium. The components and concentrations of the prepared feed medium can vary depending on the cells to be cultured. In addition, commercially available feed media can be used, such as Cell Boost series supplemented medium (HyClone, USA), EfficientFeed supplemented medium, GlycanTune feed medium (Gibco, USA), BalanCD CHO feed medium (Irvine Scientific, USA), CHO cell culture feed medium (Merck, USA), EX- Advanced CHO feed medium (Sigma-Aldrich, USA) and the like are used as feed medium, but the present invention is not limited thereto.

[0110] The term "plant-derived hydrolysate" used herein refers to a product extracted from peas, cotton seeds, wheat gluten, soybeans, etc. and does not contain animal-derived ingredients. It is a supplement containing a large amount of amino acids, peptides, vitamins, carbohydrates, nucleotides, minerals and other ingredients. It can also be prepared to contain a variety of ingredients and ingredient concentrations according to the cells to be cultured. In addition, commercially available plant-derived hydrolysates such as HyPep can be used. TM 7404、UltraPep TM Cotton, HyPep TM 7504、HyPep TM 4601N (Kerry, USA), Cotton 100, Cotton 200, Phytone TM and Soy 100 (Gibco, USA) as supplements, but the present invention is not limited thereto.

[0111] Additives used to increase or decrease the amount of N-glycans of the glycoproteins used in the present invention are generally components known to be involved in protein glycosylation. In particular, 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. When culturing cells, such additives added to the culture medium at predetermined concentrations include components that are glycosylation precursors such as N-acetyl-D-mannosamine, glucose, mannose, glutamine and galactose, ammonia and butyric acid.

[0112] Most animal cell cultures mainly use serum-containing culture media. However, since serum-containing culture media are complex compositions and their chemical compositions are unclear, it is difficult to design a culture medium suitable for protein preparation. Since serum may have a negative impact on separation or purification and there are problems related to cost and reproducibility, serum-free culture media or culture media containing a small amount of serum are mainly used. Since the concentration of glucose as a carbon source in serum-free culture media is very low, glucose will be further added to the culture medium used for culture as the main carbon source to maintain cell growth and produce a high concentration of target protein, and glutamine can be further added thereto for culture. In particular, in order to increase the half-life in vivo in the production of protein pharmaceuticals, the degree of sialylation should be increased. For this reason, the glucose and glutamine in the culture medium should be maintained at a specific concentration or higher to avoid exhaustion, and the pH of the culture medium should also be maintained at a specific level. The concentrations of glucose and glutamine measured in the culture medium refer to the concentrations of glucose and glutamine remaining after being consumed by the cells. In addition, a promoter that improves the activity of an enzyme associated with the increase in the degree of sialylation or an inhibitor that inhibits desialylation can be used. In addition, by increasing the precursors of N-glycans or controlling the activity promoters or culture conditions of related enzymes, the degree of galactosylation can be increased, and thus the degree of sialylation can also be increased.

[0113] However, the N-glycan level cannot be controlled by the above method in the preparation of hyaluronidase PH20 or its variants. However, when prepared by the method according to the present invention, hyaluronidase PH20 or its variants having the desired glycan level and high enzyme activity can be prepared.

[0114] The preparation method according to the present invention may further comprise isolating and purifying the prepared hyaluronidase PH20 or its variant.

[0115] According to the present invention, the separation and purification of hyaluronidase PH20 or its variants are preferably carried out without using affinity binding but using the ionic bond and / or hydrophobic interaction characteristics of hyaluronidase PH20 or its variants, but the present invention is not limited thereto.

[0116] Specifically, the separation and purification of hyaluronidase PH20 or its variants according to the present invention is preferably carried out not using affinity chromatography but using hydrophobic interaction chromatography and ion exchange chromatography, such as cation exchange chromatography and / or anion exchange chromatography, but the present invention is not limited thereto.

[0117] In addition, the separation and purification of hyaluronidase PH20 or its variants according to the present invention is intended to remove acid hyaluronidase PH20 or its variants with low enzyme activity, and the removal of acid hyaluronidase PH20 or its variants is preferably carried out using ion exchange chromatography.

[0118] The catalytic reaction rate of an enzyme needs to be analyzed to determine the industrial applicability of the enzyme. Enzyme reactions can be divided into enzyme reactions with an active site of fixed reactivity and enzyme reactions with multiple active sites of different reactivities. 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.

[0119] Michaelis-Menten enzyme kinetics is based on the following premise: the enzyme reaction is assumed to be a two-step reaction system including a reversible reaction and an irreversible reaction. In the reversible reaction, the enzyme (E)-substance (S) complex [ES] is formed, and in the irreversible reaction, the ES complex dissociates and produces a product (P). In this case, kf, kr, and kcat are the rate constants of the reaction in each direction (Alan Fersht (1977). Enzyme structure and mechanism).

[0120]

[0121] For enzyme reactions, it is assumed that the process of the enzyme reacting with the substance to produce the ES complex quickly reaches equilibrium or a pseudo-steady state, and that the concentration of the enzyme is sufficiently reduced by performing a reaction that maintains a sufficiently high concentration of the substance to satisfy d[ES] / dt ≒ 0. Since the kinetic equations assuming rapid equilibrium or pseudo-steady states are derived in the same way, in most experiments, a pseudo-steady state in which the concentration of the substance is initially higher than the enzyme concentration is assumed.

[0122] When, based on this assumption, for example, "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 at which the product is decomposed again", the reaction rate of the final product can be expressed by the following Michaelis-Menten kinetic equation. In this case, KM = (kr + kcat) / kf and Vmax = kcat [E] 0.

[0123]

[0124] The Lineweaver-Burk equation is used to experimentally analyze enzyme reaction rates using the Michaelis-Menten kinetic equation. This equation shows the relationship between the inverse of the experimentally measured reaction rate, 1 / V, and the inverse of the concentration of a given substance in the experiment, 1 / [S]. This equation is a statistical validation of the linear equation, indicating that the enzyme reaction follows the Michaelis-Menten kinetic equation, and that KM and Vmax can be calculated using this equation.

[0125] The enzyme that catalyzes a chemical reaction has a transition state after binding to a substance at the active site, and reduces the activation energy for reaching a transition state with high energy through multiple bonds with the substance. The equilibrium constant for reaching this transition state is proportional to kcat / KM. Here, 1 / KM is an index that combines the degree of "producing an enzyme-substance complex by binding the enzyme and the substance" and the degree of "maintaining the enzyme-substance complex without decomposition", and kcat is the equilibrium constant when obtaining a product from the enzyme-substance complex. Therefore, kcat / KM can be said to be an indicator of how much product can be obtained from the substance and the enzyme, that is, the catalytic efficiency of the enzyme.

[0126] The industrial availability of hyaluronidase is proportional to its enzyme catalytic efficiency. In particular, when the enzyme is subcutaneously injected with a polymeric pharmacologically active substance (such as a monoclonal antibody), the enzyme catalytic efficiency of hyaluronidase plays an important role. In the case where the variant according to the present invention has a higher kcat / KM than wild-type PH20, when the hyaluronidase contained in the polymeric pharmacologically active substance is subcutaneously administered, the hyaluronic acid present therein is rapidly decomposed, so the superior effect of rapidly dispersing the pharmacologically active substance can be obtained. In addition, when the variant according to the present invention has a larger kcat than wild-type PH20, the maximum reaction rate Vmax will increase at the same enzyme concentration, thereby providing the excellent effect of decomposing a larger amount of hyaluronic acid during the same period and dispersing the pharmacologically active substance in a wider area.

[0127] 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 Vmax (maximum enzyme reaction rate), Km (substance concentration at 50% Vmax), kcat (substance conversion rate) and kcat / Km (enzyme catalytic efficiency) were compared in Example 4. These results indicate that the PH20 variants according to the present invention are superior to wild-type PH20.

[0128] Example

[0129] Hereinafter, the present invention is described in detail with reference to Examples. However, it is obvious to those having ordinary knowledge 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.

[0130] Example 1. Relationship between hyaluronidase activity, N-glycans and isoelectric focusing pattern

[0131] The activity, isoelectric focusing pattern and N-glycan extent of wild-type human hyaluronidase PH20 and its variant HM46 were revealed in Figure 1Although the activity units of the two hyaluronidases differed more than twofold, there were no major differences in the isoelectric point range or the extent of N-glycans.

[0132] Experiments were conducted to determine the relationship between N-glycans, isoelectric focusing patterns, and activity in hyaluronidase PH20. During the purification of hyaluronidase PH20 variants, the alkaline portion (portion 1) and the acidic portion (portion 2) can be separated and each portion used as a sample. Figure 2 reveals the results of the analysis of isoelectric focusing patterns and enzyme activity for samples treated with PNGase F to remove all N-glycans, samples treated with sialidase A to remove terminal sialic acid, and samples treated with sialidase A and galactosidase to remove terminal sialic acid and galactose. The two portions expressed differences in the range of isoelectric points, with the acidic portion expressing reduced enzyme activity. When treated with PNGase F, the two portions were inactive and had similar isoelectric focusing patterns. These results show that the degree of N-glycans is closely related to enzyme activity. Furthermore, the isoelectric focusing pattern or enzyme activity of the acidic portion was similar to that of the basic portion when the terminal sialic acid was removed, indicating a relationship between the content of the terminal sialic acid and the enzyme activity. However, the basic portion having a low terminal sialic acid content expressed improved hyaluronidase enzyme activity compared to the acidic portion having a high sialic acid content.

[0133] The relationship between enzyme activity and N-glycan content was further demonstrated based on the results of evaluating wild-type and multiple variant hyaluronidase from different cultured cells.

[0134] Table 1

[0135]

[0136]

[0137] Relative activity: Activity expressed as a percentage of (activity of sample) / (activity of wild-type human PH20).

[0138] Galactosylation percentage (%): the sum of the content percentage (%) of N-glycans containing galactose at the terminal of G1, G1F, G1F', G2, G2F, A1, A1F, A2 and A2F.

[0139] Sialylation percentage (%): the sum of the content percentage (%) of N-glycans containing sialic acid at the terminal of A1, A1F, A2 and A2F.

[0140] Mannosylation percentage (%): the sum of the content percentage (%) of N-glycans containing mannose at the terminal of M4G0F, M5, M5G0, M6, M7, M8 and M9.

[0141] The sequences of wild-type human PH20 and HM46 are disclosed in International Patent Publication No. 2020 / 022791.

[0142] Sheep PH20 and Bonobo PH20 samples were prepared by the method described in Example 10, and the sequences are shown in Table 3.

[0143] As shown in Table 1, various types of hyaluronidase PH20 and variant proteins were prepared and the amount of N-glycans was investigated. For this purpose, 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, cell line clone cultures using cell line clones produced using recombinant genes, and cell line cultures using single strains screened for high productivity. Here, the cell line clone culture system uses a selective marker to select a cell line clone with high productivity from a mammalian cell line clone transfected with a recombinant gene, and is performed using multiple cell lines, with the aim of producing a single cell line. The 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, various combinations of experimental cultures were tried to optimize production, and even here, differences in activity and N-glycans were found.

[0144] Cell line test culture #1 was a 2-liter (L) batch culture performed at 37°C using HycellCHO medium (HyClone, USA) without adjusting the culture temperature. Cell line test culture #2 purified fraction #1 and cell line test culture #2 purified fraction #2 were separated and purified from the harvested cell culture fluid by anion exchange chromatography, wherein the harvested cell culture fluid was used in an 8-L batch containing EX- Advanced CHO fed-batch medium (Sigma-Aldrich, USA) and Cotton 100UF (Gibco) and CHO Feed 4 (Irvine Scientific, USA) was cultured at 37°C in a fed-batch method. Purification fraction #1 was the low-salt elution fraction and purification fraction #2 was the high-salt elution fraction. Cell line test culture #1 was prepared by using a 2 L batch containing EX- Advanced CHO fed-batch medium (Sigma-Aldrich, USA) and Cotton 100UF (Gibco) and The culture temperature was adjusted to 32°C when the integrated viable cell density reached a predetermined level. Cell line test culture #2 was obtained by using a 10 L batch containing EX- Advanced CHO fed-batch medium (Sigma-Aldrich, USA) and Cotton100UF (Gibco, USA) and CD CHO EfficientFeedTM B plus AGTTM (Gibco, USA) were cultured at 37°C, and then the culture temperature was adjusted to 32°C when the integrated viable cell density reached a predetermined level, and then cultured for 12 days. Cell line test culture #3 was obtained by using a 50L batch containing EX- Advanced CHO fed-batch medium (Sigma-Aldrich, USA) and Cotton 100UF (Gibco, USA) and CD CHO EfficientFeedTM B plus AGTTM (Gibco, USA) were cultured at 37°C, and then the culture temperature was adjusted to 32°C when the integrated viable cell density reached a predetermined level. Cell line test culture #4 was obtained by using a 10L batch containing EX- The cells were cultured at 37°C by a fed-batch method using Advanced CHO fed-batch medium (Sigma-Aldrich, USA) and Cotton 100UF (Gibco, USA) and CD CHO EfficientFeedTM B plusAGTTM (Gibco, USA), 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. Cell line culture #1, cell line culture #2, cell line culture #3, and cell line culture #4 were obtained by supplementing EX- AdvancedCHO fed-batch medium (Sigma-Aldrich, USA) and Cotton 100UF (Gibco, USA) and CD CHOEfficientFeedTM B plus AGTTM (Gibco, USA) were used to culture the research cell bank (RCB), the production master cell bank (MCB) and the working cell bank (WCB) 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 12 days. Based on these conditions, experiments on culture conditions in Examples 2 to 5 were performed to establish a culture method for maintaining the level of N-glycans.

[0145] As can be seen from Table 1, the degree of galactosylation in the N-glycan of wild-type human hyaluronidase PH20 and its variants and mammalian hyaluronidase PH20 should be 15 to 50%, the degree of sialylation should be 2.5 to 28%, and the degree of mannosylation should be 47 to 60%, and when a 95% confidence interval is applied to the test results, the degree of galactosylation in the N-glycan should be 1 to 68%, the degree of sialylation should be 1 to 38%, and the degree of mannosylation should be 40 to 63% to confer industrially useful hyaluronidase activity thereon. This value may also include an error of 10%, because there are differences in the measurement of the glucose level 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 considering the differences between laboratories, the glucose level measured in the present invention should be interpreted in a broad sense, not a limited sense.

[0146] 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 a hyaluronidase having an activity higher than that of wild-type human PH20 and thus useful in industry be prepared. The above numerical values ​​may be derived from experimental results and may contain an error of 10%.

[0147] High-quality hyaluronidase that maintains the level of N-glycans was determined by temporary expression culture, and cell lines were prepared for stable commercial production by selecting individual strains with high productivity. All cell line test cultures #2, #3, #4 and cell line cultures #1, #2, #3, #4 are disclosed in Table 1, which were obtained by the culture method using the results of Examples 2, 3, 4, and 5, and expressed an enzyme expression level of 10,000 units / ml or more, thereby proposing the possibility of preparing high-quality hyaluronidase with high efficiency and low cost.

[0148] Example 2, culture depending on additives

[0149] Hyaluronidase PH20 variant was added at 2x10 6Cells / mL were inoculated in EX-containing medium 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 conical flasks of Advanced CHO feed batch medium (Sigma-Aldrich, USA), culture was carried out by batch culture in an incubator at 37°C and 8% CO2, and then fed batch culture was carried out at a reduced temperature of 32°C when the integral viable cell density (IVCD) reached the adjustment range. CD CHO EfficientFeedTM B plus AGTTM medium (Gibco, USA) was supplied as a feed medium daily in an amount of 1.88% of the starting volume of culture in the flask. Cell samples were collected from the cell culture fluid every day, and the viable cell density, cell viability, pH and lactic acid level were measured. After the culture was terminated, centrifugation was performed at 2,000 rpm for 10 minutes to obtain the culture supernatant. The activity of the samples cultured 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. Compared to medium not supplemented with 50 mM galactose, medium supplemented with 50 mM galactose expressed a 24% increase in galactosylation and a 2% decrease in activity, and compared to medium not supplemented with N-acetyl-D-mannosamine, medium supplemented with 20 mM N-acetyl-D-mannosamine expressed a 35% increase in sialylation and a 20% decrease in activity ( Figure 3 , Figure 4 , Figure 5 , Figure 6 ).

[0150] Example 3, depending on the culture period

[0151] Cells expressing hyaluronidase PH20 variant were cultured at 2 × 10 6 cells / mL inoculated into a Sartorius 200L bioreactor Advanced CHO feed batch medium (Sigma-Aldrich, USA). 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 feed medium for feed batch culture, and pH was 7.2±0.4 and DO was 40% at a rate of 47rpm for culture. Initial culture was carried out at 37°C, and when the integrated viable cell density reached the range of change, the temperature was adjusted to 32°C for feed batch culture. CD CHO EfficientFeedTMB plus AGTTM medium (Gibco, USA) was supplied as feed medium daily in an amount of 1.88% of the starting volume of culture in a 200L bioreactor. For the culture period, the culture was terminated on the 19th day after culture, when the cell viability dropped to 40% or less. Multiple cell samples were collected from the cell culture medium every day, and the viable cell density, cell viability, integrated viable cell density and ammonium ion level were measured. The cultured solution was harvested using a depth filter. The activity of the samples cultured 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. As the number of days in culture increased, the degree of galactosylation and sialylation decreased and the activity increased ( Figure 7 , Figure 8 , Fig. 9 , Fig.10 ).

[0152] Example 4, Cultivation under controlled glucose concentration in culture medium

[0153] Cells expressing hyaluronidase PH20 variant were cultured at 2 × 10 6 cells / mL inoculated into a Sartorius 2L bioreactor Advanced CHO feed batch medium (Sigma-Aldrich, USA). 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 feed medium for feed batch culture, and the pH was 7.2±0.4 and DO was 40% at a rate of 120rpm. Initial culture was carried out at 37°C, and when the integrated viable cell density reached the range of change, the temperature was adjusted to 32°C, followed by feed batch culture. CD CHO EfficientFeedTM B plus AGTTM medium (Gibco, USA) was supplied as a feed medium at 1.88% of the starting volume of culture in a 2L bioreactor every day. Multiple cell samples were collected from the cell culture fluid every day to measure viable cell density, cell viability, pH, osmotic pressure, and glucose and lactate concentrations.

[0154] The concentration of glucose in the culture medium was measured daily, and the concentration of glucose contained in the culture supernatant did not exceed the reference concentration of 2, 4 or 6 g / L as glucose was consumed by cell growth. The concentration of glucose was controlled from this point on. When the measured glucose concentration was 2, 4 or 6 g / L or lower (which was the respective standard concentration), a certain amount of 200 g / L glucose stock solution was added for a maximum of 3 hours to reach the standard concentration. When the measured glucose concentration was 2, 4 or 6 g / L or more, no stock solution was added to maintain the standard concentration. In general, during the culture of mammalian cells, the effect of changes in glucose content on cell growth was negligible within 3 hours.

[0155] In this case, the condition of maintaining 2 g / L as a reference concentration is referred to as a 1 g / L (±1 g / L) concentration condition, the condition of maintaining 4 g / L as a reference concentration is referred to as a 3 g / L (±1 g / L) concentration condition, and the condition of maintaining 6 g / L as a reference concentration is referred to as a 5 g / L (±1 g / L) concentration condition. For example, the condition of maintaining a glucose concentration of 1 g / L (±1 g / L) means that the lower limit of the control range of the glucose concentration is set to 0 g / L and the upper limit of the control range of the glucose concentration is set to 2 g / L, and means 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 so that the maximum glucose concentration reaches 2 g / L, and when the glucose concentration in the culture supernatant reaches the upper limit of 2 g / L, 200 g / L of glucose stock solution is not added. Therefore, under the two concentration conditions, namely, 1g / L (±1g / L) concentration and 3g / L (±1g / L) concentration, the concentration condition of 2g / L corresponds to the upper limit of the concentration condition of 1g / L (±1g / L) and corresponds to the lower limit of the concentration condition of 3g / L (±1g / L). Therefore, these two conditions are conditions under which completely different actions occur, and are therefore not considered to overlap with each other.

[0156] 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 the samples cultured under the above conditions was determined by HPLC and turbidity analysis, and the protein pattern and N-glycan extent were observed by isoelectric focusing and glycosylation analysis. The results revealed that as the glucose concentration in the culture supernatant increased, the extent of galactosylation and sialylation increased and the activity decreased ( Fig.11 , Fig.12 , Fig.13 and Fig.14 ).

[0157] Example 5, Cultivation under controlled pH

[0158] Cells expressing hyaluronidase PH20 variant were cultured at 2 × 10 6 cells / mL inoculated into a Sartorius 2L bioreactor Advanced CHO fed batch medium (Sigma-Aldrich, USA). On the second day of culture, Cotton 200UF (Gibco, USA) and CD CHO EfficientFeedTM B plus AGTTM medium (Gibco, USA), which are concentrated nutrient media (Gibco, USA), were used as feed media for fed batch culture, and cultured at a rate of 120rpm with a DO of 40%. Initial culture was carried out at 37°C, and when the integrated viable cell density reached the range of change, the temperature was adjusted to 32°C, followed by fed batch culture. CD CHO EfficientFeedTM B plusAGTTM medium (Gibco, USA) was supplied daily as a feed medium at 1.88% of the starting volume of culture in a 2L bioreactor. The culture temperature is adjusted, and the culture is 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 a condition that is improved compared to the general pH 7.2±0.4. The pH control range of the culture is 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 culture, when the pH of the culture medium reaches the lower limit of pH 6.9, alkali is added to increase the pH, and when the pH of the culture medium reaches the upper limit of pH 7.1, carbon dioxide is added to lower the pH. Therefore, in 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 were considered not to overlap with each other. Cultivation was performed until the culture day when the cell viability was 40% or less, and multiple cell samples were collected from the cell culture medium every day to measure the viable cell density, cell viability, pH, and the integrated viable cell density level. 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 the samples cultured under the above conditions was determined by HPLC and turbidity analysis, and the protein pattern was determined by isoelectric focusing. The activity varied depending on the pH of the culture medium, as well as the highest activity and the lowest degree of sialylation were observed at pH 7.0±0.1, which was an improved condition compared to the general conditions (Table 2, Fig.15 , Fig.16 , Fig.17 ).

[0159] Table 2

[0160]

[0161]

[0162] Example 6: Purification of hyaluronidase using animal cell culture supernatant

[0163] Step 1: Buffer exchange / surfactant treatment of supernatant

[0164] The conditions of the culture solution were readjusted to the first anion exchange column equilibrium conditions by controlling the conductivity and pH through UF / DF using a 30 kDa MWCO membrane filter. The readjusted solution was treated with an appropriate concentration of solvent / surfactant to inactivate the virus and reacted at room temperature for about 60 minutes.

[0165] Step 2: Primary anion exchange (Q Sepharose Fast Flow) column chromatography

[0166] The filtered protein solution is passed through a primary anion exchange column to capture hyaluronidase through the anion exchange resin and elute from the column at high salt concentration. Before loading, the column is equilibrated with tromethamine buffer (pH 8.0 and salt concentration of 30 mM). After loading, the column is rinsed with the same buffer (primary rinse). After the primary rinse, the column is rinsed 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 rinse). After the second rinse, elution of the desired protein (hyaluronidase) is performed using an appropriate buffer with a pH of 8.0 and a salt concentration of 200 mM, such as Fig.18 shown.

[0167] Step 3: Secondary anion exchange (Capto Q) column chromatography

[0168] The filtered protein was passed through a secondary anion exchange column to remove the acidic hyaluronidase variants using anion resin exchange. Before loading, the column was equilibrated with Bistris buffer (pH 6.0 and salt-free). After loading, the column was rinsed with the same Bistris buffer (primary rinse). After the primary rinse, the column was rinsed with the same Bistris buffer (salt concentration of 20 mM, which has a higher conductivity than the salt concentration in the primary rinse). After the second rinse, the desired protein (hyaluronidase) was eluted using a Bistris buffer with a certain salt concentration, such as Fig.19 shown.

[0169] Step 4: Cation exchange (Capto MMC) column chromatography

[0170] The re-adjusted protein solution is passed through a cation exchange column to remove the acidic hyaluronidase variant using a cation exchange resin and eluted from the column at a high salt concentration. Before loading, the column is equilibrated with a pH 5.5 citrate buffer with a salt concentration of 80 mM. After loading, the column is rinsed with the same citrate buffer (primary rinse). After the primary rinse, the column is rinsed with an appropriate pH 7.5 Bistris buffer. After the second rinse, the desired protein (hyaluronidase) is eluted using a pH 8.0 Bistris buffer with a salt concentration of 400 mM, as Fig. 20 shown.

[0171] Step 5: Nanofiltration / Formulation

[0172] 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 mg / mL and reconstituted by UF / DF using an 8 kDa MWCO membrane filter to exchange with a pH 7.0 histidine buffer containing 145 mM salt.

[0173] Example 7, Analysis of the Enzyme Activity of Hyaluronidase

[0174] The enzymatic activity of Hyaluronidase PH20 and other hyaluronidases was measured using the turbidimetric method as described below.

[0175] 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 decreases during the mixing process with albumin. In general, this process is performed as follows. Hyaluronidase PH20 (Sigma) is diluted to 1, 2, 5, 7.5, 10, 15, 20, 30, 50 or 60 units / mL and placed in each tube. The purified protein sample is dissolved in enzyme dilution buffer (20mM Tris·HCl pH 7.0, 77mMNaCl, 0.01% (w / v) bovine serum albumin) and diluted 100X, 300X, 600X, 1200X or 2400X in each tube. The 3mg / mL hyaluronic acid solution is diluted 10x to 0.3mg / mL in other tubes to adjust the volume in each tube to 180 microliters (μL). 60 μL of enzyme and diluted hyaluronic acid solution were mixed and reacted at 37°C for 45 minutes. After the reaction, 50 μL of the reacted enzyme and 250 μL of acid albumin solution were added to each well of a 96-well plate and shaken for 10 minutes. The absorbance was measured at 600 nm using a spectrophotometer. The activity units of the samples were obtained using the test results of standards with known activity units and the test results of the samples.

[0176] Example 8, Isoelectric Focusing Analysis of Hyaluronidase

[0177] The 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 precast gel, and the gel was electrophoresed at 100 V for 1 hour, at 200 V for 1 hour, and at 500 V for 30 minutes using an electrophoresis apparatus manufactured by Novex Corporation. After the electrophoresis was completed, the gel was rinsed with pure water, the protein was fixed with a 12% TCA solution, stained with a Coomassie Blue R-250 staining solution, and bleached with an acetic acid-methanol solution to analyze the protein bands presented on the gel.

[0178] Example 9, Analysis of N-glycan content of hyaluronidase

[0179] The N-glycan level of hyaluronidase was analyzed by labeling the N-glycan sample, separating it by treating the hyaluronidase with PNGase F (N-glycosidase F), 2-AB (2-aminobenzamide), and then performing ultra-high performance liquid chromatography using an ACQUITY UPLC Glycan BEHAmide column (Waters). The purified hyaluronidase sample was desalted and reacted with PNGase F at 37°C for 16 to 18 hours to separate the N-glycans therefrom. The N-glycans were labeled with 2-AB, then reacted at 65°C for 3 hours, and the excess 2-AB was removed therefrom. The labeled N-glycan sample was separated by HPLC with a gradient of 72%-20% acetonitrile. The separated sample was detected with a fluorescence detector (FLD) to analyze the N-glycan level. The individual separated N-glycans were classified, and the N-glycans containing galactose at their terminals (G1, G1F, G1F', G2, G2F, A1, A1F, A2, A2F, etc.) were added to determine the degree of galactosylation percentage (%), the N-glycans containing sialic acid at their terminals (A1, A1F, A2, A2F, etc.) were added to determine the degree of sialylation percentage (%), and the N-glycans containing mannose at their terminals (M4G0F, M5, M5G0, M6, M7, M8, M9, etc.) were added to determine the degree of mannosylation percentage (%).

[0180] Example 10, Preparation of animal-derived hyaluronidase and analysis of N-glycan content

[0181] (1) Preparation of bonobo and sheep hyaluronidase PH20 genes

[0182] In order to study the N-glycan level and activity of hyaluronidase PH20 from anthropoid (i.e., bonobo) and artiodactyl (i.e., sheep) animals, 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 sites of the pcDNA3.4-TOPO vector. For expression in ExpiCHO cells, a signal peptide of human growth hormone, human serum albumin, or human Hyal1 was used as the signal peptide instead of the natural signal peptide of PH20. For protein purification using a HisTrap column, the His-tag DNA sequence was located at the 3' end of the PH20 cDNA. Each sequence was determined using DNA sequencing. Table 3 reveals the sequences of hyaluronidase from animals.

[0183] Table 3

[0184]

[0185]

[0186] (2) Expression of hyaluronidase PH20 in bonobos and sheep

[0187] Use the ExpiCHO expression system for expression. When the number of ExpiCHO cells reaches 6x10 6 At 10 cells / ml, ExpiCHO cells were transfected with plasmids containing hyaluronidase PH20 cDNA inserted into the pcDNA3.4-TOPO vector using ExpiFectamine CHO reagent. The cell culture medium used here was ExpiCHO expression medium (100-500 ml). After transfection, ExpiCHO cells were cultured while shaking at 130 rpm for a total of 6 days. During this period, the cells were cultured at 37°C for 1 day, followed by further culture at a reduced temperature of 32°C for 5 days. When the culture was completed, the cells were centrifuged at 10,000 rpm for 30 minutes to obtain the cell supernatant.

[0188] (3) Purification of Bonobos and Hyaluronidase PH20

[0189] The animal-derived hyaluronidase recombinant proteins produced in ExpiCHO cells and attached to the C-terminus with a His-tag were purified by two-step column chromatography using the AKTA prime system (GE Healthcare Systems). The pI of bonobo PH20 is 6, so Q Sepharose for anion exchange chromatography was used, and the pI of sheep PH20 is 8 or more, so Capto S column for cation exchange chromatography was used for one-step purification. Each protein was purified in two steps using HisTrap HP column for His-Tag affinity chromatography.

[0190] For protein purification using a Q Sepharose column, 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. The protein was bound to the Q Sepharose column, and the column was washed with 5 column volumes (CV) of buffer A to remove non-specifically bound proteins, and then washed with 5 CV of buffer B at a concentration gradient from 0 to 100% to elute the protein.

[0191] For protein purification using Capto S column, 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 culture medium were adjusted to be the same as those of buffer A, and the culture medium was filtered through a membrane with a pore size of 0.22 μm. The protein sample was bound to the Capto S column, and the column was rinsed with 3 CV of buffer A to remove non-specifically bound proteins. The column was rinsed with 4 CV of buffer B to elute the protein.

[0192] To store proteins using the HisTrap HP column, 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 is bound to the HisTrap HP column, and the column is washed with 7 CV of 7% buffer B to remove non-specifically bound proteins, followed by 3 CV of 40% buffer B to elute the target protein. The column effluent is dialyzed using dialysis buffer (20 mM sodium phosphate, 100 mM NaCl, pH 7.0).

[0193] (4) Analysis of PH-20 hyaluronidase in bonobos and sheep

[0194] Activity analysis was performed in the same manner as in Example 7, and N-glycan extent analysis was performed in the same manner as in Example 9. The results are shown in Table 1.

[0195] Example 11 Enzyme kinetic analysis of variants depending on the extent of N-glycans

[0196] To analyze the enzyme kinetics of the variants according to the present 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) (i.e., Ehrlich reagent) to detect the red substance (at 545 nm) produced by the reaction of the reducing end of N-acetyl-D-glucosamine (GlcNAc) produced when hyaluronic acid is hydrolyzed by hyaluronidase. N-acetyl-D-glucosamine (GlcNAc, Sigma) diluted to 0.25, 0.50, 0.75, 1.00 or 1.25 mM in a dilution buffer solution (0.1 M NaPi, 0.1 M NaCl, 1.5 mM glucarboxylic acid-1,4-lactone, pH 5.35) was reduced in each test tube by tetraborate treatment, and then DMAB was added to induce a colorimetric reaction. After the reaction, the absorbance was measured at 545 nm to prepare a standard reaction curve for GlcNAc. In each test tube, hyaluronic acid as a substance was diluted to 0.54, 0.65, 0.87, 1.23 or 2.17 μM in a dilution buffer solution, hyaluronidase was added to each test tube, and then reacted at 37° C. for 5 minutes and heated at 100° C. for 5 minutes to complete the enzyme reaction. The product was reduced by treating with tetraborate, and DMAB was added to induce a colorimetric reaction. After the reaction, the absorbance was measured at 545 nm, and the enzyme activity was measured using the standard reaction curve for GlcNAc described above. This method was used to analyze the enzyme kinetics of the wild-type PH20 of SEQ ID NO: 1 and the PH20 variant according to the present invention. As a result, it was found that the Lineweaver-Burk curve was linear, which means that the PH20 variant according to the present invention follows the Michaelis-Menten enzyme kinetic formula.

[0197] Table 4 discloses the analysis of enzyme kinetics of fraction #1 and fraction #2 obtained from cell line test culture #2 of the samples in Table 1 of Example 1. These results show that when the degree of galactosylation and mannosylation falls within a similar range and the degree of sialylation is low, the enzyme activity of the recombinant hyaluronidase increases due to the high catalytic efficiency (kcat / Km) of the enzyme.

[0198] 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 when attempting to prepare industrially useful hyaluronidase by using recombinant methods to prepare large quantities of wild-type PH20 hyaluronidase or its variants, enzymes with greater industrial applicability can be developed by controlling the degree of sialylation.

[0199] Table 4

[0200]

[0201] According to the method for preparing 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 prepared, thereby realizing large-scale preparation and supply of recombinant hyaluronidase PH20 protein or its variants.

[0202] Although the specific configuration of the present invention has been described in detail, it should be understood by those skilled in the art that the embodiments are only for illustrative purposes as preferred embodiments and should not be interpreted as limiting the scope of the present invention. Therefore, the essential scope of the present invention is defined by the scope of the claims and their equivalents.

[0203] References

[0204] 1.LHBookbinder,A.Hofer,MFHaller,MLZepeda,GA.Keller,JELim,TSEdgington,HMShepard,JSPatton,GIFrost.(2006).A recombinant humanenzyme for enhanced interstitial transport of therapeutics.J.Control.Release.114,230-241.

[0205] 2. Douglas B. Muchmore, MD, and Daniel E. Vaughn, Ph. D. (2012). Accelerating and Improving the Consistency of Rapid-Acting Analog InsulinAbsorption and Action for Both Subcutaneous Injection and ContinuousSubcutaneous Infusion Using Recombinant Human Hyaluronidase. J. DiabetesSci.and Technol.6(4):764-72

[0206] 3.E.M.Krantz.(1980).Low-dose intramuscular ketamine and hyaluronidasefor induction of anaesthesia in non-premedicated children.S.Afr.Med.J.58(4):161-2.

[0207] 4.W.A.Clement,S.H.Vyas,J.N.Marshall,J.H.Dempster.(2003).The use ofhyaluronidase in nasal infiltration:prospective randomized controlled pilotstudy.J.Laryngol.Otol.117(8):614-8.

[0208] 5.Thomas J.R.,Wallace M.S.,Yocum R.C.,Vaughn D.E.,Haller M.F.,FlamentJ.(2009).The INFUSE-Morphine study:use of recombinant human hyaluronidase(rHuPH20)to enhance the absorption of subcutaneously administered morphine inpatients with advanced illness.J.Pain and Symptom Manag.38(5):663-672.

[0209] 6.George Harb,Francois Lebel,Jean Battikha,Jeffrey W Thackara.(2010).Safety and pharmacokinetics of subcutaneous ceftriaxone administered with orwithout recombinant human hyaluronidase(rHuPH20)versus intravenousceftriaxone administration in adult volunteers.Curr.Med.Res.Opin.26(2):279-88.

[0210] 7.Richard L.Wasserman.(2014).Overview of recombinant humanhyaluronidase-facilitated subcutaneous infusion of IgG in primary immunodeficiencies.Immunotherapy.6(5):553-67.

[0211] 8.Harris R.J.,Shire S.J.,Winter C.(2004).Commercial manufacturingscale formulation and analytical characterization of therapeutic recombinantantibodies.Drug.Dev.Res.61:137-154.

[0212] 9.Stephan Schilling,Torsten Hoffmann,Fred Rosche,Susanne Manhart,Claus Wasternack,and Hans-Ulrich Demuth.(2002).Heterologous Expression andCharacterization of Human Glutaminyl Cyclase:Evidence for a Disulfide Bondwith Importance for Catalytic Activity.Biochemistry,35,10849-10857.

[0213] 10.H.Tachibana 1,K.Taniguchi,Y.Ushio,K.Teruya,K.Osada,H.Murakami.(1994).Changes of monosaccharide availability of human hybridoma lead toalteration of biological properties of human monoclonalantibody.Cytotechnology.16(3):151-7.

[0214] 11.Veronica Restelli,Ming-Dong Wang,Norman Huzel,Martin Ethier,HelenePerreault,Michael Butler.(2006).The effect of dissolved oxygen on theproduction and the glycosylation profile of recombinant human erythropoietinproduced from CHO cells.Biotechnol.Bioeng.94:481-494.

[0215] 12.M.C.Borys,D.I.Linzer,E.T.Papoutsakis.(1993).Culture pH affectsexpression rates and glycosylation of recombinant mouse placental lactogenproteins by Chinese hamster ovary(CHO)cells.Biotechnology(NY).11:720-724.

[0216] 13.M.C.Borys,D.I.Linzer,E.T.Papoutsakis.(1994).Ammonia affects theglycosylation patterns of recombinant mouse placental lactogen-I by Chinesehamster ovary cells in a pH-dependent manner.Biotechnol.Bioeng.43:505-514.

[0217] 14.Clark K.J.,Chaplin F.W.,Harcum S.W.(2004).Temperature effects onproduct quality-related enzymes in batch CHO cell cultures producingrecombinant tPA.Biotechnol.Prog.20:1888-1892.

[0218] 15.Arming S.,Strobl B.,Wechselberger C.,Kreil G.(1997)In vitro mutagenesis of PH-20 hyaluronidase from human sperm.Eur J.Biochem.247:810-814

Claims

1. A method for preparing recombinant hyaluronidase PH20 or a variant thereof, comprising: (1) culturing host cells expressing recombinant hyaluronidase PH20 or its variants at a culture temperature of 35°C to 38°C until the integrated viable cell density of the host cells reaches 20×10 6 Up to 120x10 6 cells x day / mL; and (2) lowering the culture temperature to 28° C. to 34° C., and then culturing the host cells for 2 to 18 days while maintaining the culture temperature: (a) wherein the residual glucose concentration in the culture medium is maintained at 0.001 g / L to 4.0 g / L during the culturing period; and (b) wherein the pH of the culture medium is maintained at 6.9 to 7.1; wherein the amount of sialylation of the N-glycans of the generated PH20 or variants thereof is 2.5 to 18.6%, wherein the amount of sialylation is the sum of the percentage contents of N-glycans containing sialic acid at the terminal, the N-glycans comprising A1, A1F, A2 and A2F, and wherein the N-glycans of the generated PH20 or variant thereof have a galactosylation amount of 14.8 to 49.6%, a sialylation amount of 2.5 to 18.6%, and a mannosylation amount of 47.9 to 54.4%, and wherein the sum of the galactosylation amount and the mannosylation amount does not exceed 100%, The hyaluronidase activity in the culture medium is 10,000 units / ml or higher.

2. The method of claim 1, wherein the specific activity of the generated PH20 or variant thereof is at least 10% higher than the specific activity of wild-type human PH20.

3. The method according to claim 1, wherein the host cells are cultured in step (1) and / or step (2) by one or more methods selected from batch culture, continuous culture and perfusion culture.

4. The method of claim 1, wherein the culturing of the host cells in step (1) and / or step (2) is carried out by one or more methods selected from repeated batch culture, fed-batch culture and repeated fed-batch culture.

5. The method of claim 1, wherein the host cell is cultured in step (1) and / or step (2) under one or more conditions selected from the following: (i) the ammonia concentration in the culture medium is maintained at 5 mM or more; (ii) conditions in which one or more substances selected from the group consisting of glutamine, glucosamine, uridine, glucosamine and sodium butyrate are added to the culture medium; and (iii) Conditions where galactose and manNAc were not added to the medium.

6. The method of claim 1, wherein the PH20 variant comprises a substitution of one or more amino acid residues at the N-terminus and / or C-terminus in the amino acid sequence of wild-type PH20.

7. The method of claim 1, wherein the host cell is an animal cell, a yeast, an actinomycete or an insect cell.

8. The method of claim 1, further comprising isolating and purifying the produced PH20 or variant thereof.

9. The method of claim 1, wherein the separation and purification of the PH20 or variant thereof is performed using the ionic bonding and / or hydrophobic interaction properties of the PH20 or variant thereof without using affinity binding.

10. The method of claim 9, wherein the separation and purification of the PH20 or variant thereof is performed using hydrophobic interaction chromatography and ion exchange chromatography without using affinity chromatography.

11. The method of claim 8, wherein acidic PH20 or a variant thereof is removed.

12. The method of claim 11, wherein the removal of acidic hyaluronidase PH20 or a variant thereof is performed using ion exchange chromatography.

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