Method for producing recombinant hyaluronidase

Abstract

To provide hyaluronidase or a variant thereof.SOLUTION: The present invention provides PH20 or a variant thereof, in which a galactosylation content is 1 to 68%, a sialylation content is 1 to 21.7%, and a mannosylation content is 40 to 63% within the N-glycan content. The galactosylation content is the sum of the contents of N-glycans containing terminal galactose, including G1, G1F, G1F', G2, G2F, A1, A1F, A2, and A2F. The sialylation content is the sum of the contents of N-glycans containing terminal sialic acid, including A1, A1F, A2, and A2F. The mannosylation content is the sum of the contents of N-glycans containing terminal mannose, including M4G0F, M5, M5G0, M6, M7, M8 and M9.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to methods for producing hyaluronidase, in particular native PH20 or mature native PH20, or hyaluronidase PH20 variants that include substitutions of one or more amino acid residues in the amino acid sequence of native PH20 or mature native PH20, and optionally have a portion of the amino acid residues deleted at the N-terminus and / or C-terminus. [Background technology]

[0002] Hyaluronidase is an enzyme that breaks down hyaluronic acid located in the extracellular matrix. By hydrolyzing hyaluronic acid, hyaluronidase reduces the viscosity of hyaluronic acid in the extracellular matrix and increases its permeability to tissues (skin) (Bookbinder et al., 2006). Hyaluronidase has been used to improve the absorption of fluids delivered by subcutaneous injection [Muchmore et al., 2012] or intramuscular injection [Krantz et al., 2016] and to improve the diffusion of local anesthetics [Clement et al., 2003]. In this way, Drugs whose administration was enhanced included morphine [Thomas et al., 2009], ceftriaxone [Citroenza, 2009], and cefotaxime [Citroenza, 2009]. These include triaxones [Harb et al., 2010], insulin [Muchmore et al., 2012], and immunoglobulins [Wasserman et al., 2014]. Hyaluronidases are also used to improve the dispersion of fluids or drugs lost to tissues by intravenous injection or hematoma diffusion. A recombinant protein of human PH20, which is active at neutral pH, was developed by Halozyme Therapeutic and is sold under the trade name Hylenex (Bookbinder et al., 2006).

[0003] It has been reported that recombinant human PH20 protein has been expressed in yeast (P. pastoris), DS-2 insect cells, animal cells, etc. The recombinant PH20 protein produced in insect cells and yeast has a different N-glycosylation pattern from human PH20 during the post-translational transformation process of the protein, which may affect its activity and cause side effects in the body.

[0004] In the development of biopharmaceuticals, product consistency and long shelf life are important factors that provide manufacturing and flexibility. During manufacturing, various forms of microheterogeneity occur due to differences in size and charge, etc., due to enzymatic or spontaneous degradation and modification. Chemical and enzymatic modifications such as deamidation and sialylation increase the net negative charge in antibodies and decrease the pI value, respectively, resulting in the formation of acidic variants [Harris RJ et al., 2004]. Also, C-terminal lysine cleavage leads to the loss of a net positive charge, resulting in the formation of acidic variants. The formation of basic variants can occur through C-terminal lysine or glycine amidation, the formation of succinimides, the oxidation of amino acids, or the removal of sialic acid, which removes additional positive or negative charges; the two 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 is a reaction that occurs in the endoplasmic reticulum and Golgi apparatus. Glycosylation is divided into N-glycosylation and O-glycosylation, which differs depending on the functional group attached. The process in which sugars such as lactose and fucose are attached to proteins produced in cells is collectively called "glycation." When a glycan is linked to a protein during the glycation process, the protein undergoes a "folding" process to form a three-dimensional structure. This gives the protein stability that allows it to be maintained for a long time without being released. This is an important process as such glycans also play a role in communication and information exchange between cells.

[0006] There are two types of reactions in which glycans are added to proteins: N-linked or O-linked glycosylation. These two glycosylation processes differ in the mechanisms of glycan synthesis and addition, and the mechanism and role of N-glycosylation are better known. Sugar chains added through N-glycosylation are called N-glycans and are formed in the endoplasmic reticulum. A series of ALG (Asparagine-Linked Glycosylation) enzymes add N-acetylglucosamine (GlcNAc), mannose (Man), glucose (Glc) and other elements to Dolichol Pyrophosphate (PP-Dol) present in the endoplasmic reticulum membrane, ultimately synthesizing Glc3Man9GlcNAc2-PP-Dol, a complex glycan in the form of lipid-linked oligosaccharide (LLO). The LLO thus synthesized is transferred to the N-glycosylation sequence of peptides containing NxS / T, which are directly translated from the ribosome to the endoplasmic reticulum through a co-translational translocation mechanism by oligosaccharyltransferase, which is composed of eight or more subunits. The N-glycosylation of the protein is then removed one by one from the glucose at the end of the glycan by glucosidases (α-Glucosidase I, Gls1p; α-Glucosidase II, Glsp II) present in the endoplasmic reticulum. The second and third glucose are then slowly removed, and folding is completed with the help of the lectin chaperone Calnexin / Calreticulin. When all the glucose has been removed, the protein is deemed to be fully folded and is transferred to the Golgi apparatus with the Man8GlcNAc2 glycan attached. However, before being transferred from the endoplasmic reticulum to the Golgi apparatus, there is a quality control process that re-verifies the folding of the glycoprotein.If proper folding is not occurring, a molecule of glucose is re-entered into the Calnexin / Calreticulin Cycle, repeating the process to give the protein an opportunity to complete folding.

[0007] The initial biosynthesis process of N-glycans, which takes place in the endoplasmic reticulum, is almost identical and is conserved from yeast, a eukaryotic microorganism, to animals, a higher organism. However, glycans transferred to the Golgi apparatus undergo various glycosylation processes specific to each species, resulting in completely different glycans being produced in yeast, insects, plants, and animals. However, these diverse glycans all share a core site, which is a structure in which three mannoses and two GlcNAcs are linked to the nitrogen of asparagine, called the trimannosyl core. The form in which mannose is mainly linked to this trimannosyl core is called the high-mannose type, and is often observed in yeast and mold. In the case of the traditional yeast Saccharomyces cerevisiae, the addition of mannose continues in the Golgi apparatus, and a form in which several dozen mannoses are added can be observed. On the other hand, in insect cells, glycoproteins with pauci-mannose type glycans are observed. These are first arranged in the form of Man5GlcNAc2 by each mannosidase (Mannosidase IA, IB & IC) in the Golgi apparatus, and then N-acetylglucosaminyltransferase (GNT) I acts to add one GlcNAc, and then mannosidase II acts to create a mixed structure with one GlcNAc added to the trimannosyl core. The added GlcNAc is then excised again to create the pauci-mannose type glycan structure. In animal cells, α(1,6)-fucose is sometimes added to the first GlcNAc linked to the asparagine residue. In animals, GNT II acts on the structure with one GlcNAc added to the trimannosyl core to add another GlcNAc, generating a glycan with two antennary structures. Then, GNTs IV and V may act to create a four-antenna structure, and in some cases, GNTs VI, IX, or VB may act to create up to six-antenna structures.After GlcNAc is added to form the antenna skeleton, β-galactosyltransferase and α-sialyltransferase present in the Golgi apparatus act to form a complex glycan structure in which galactose and sialic acid are added to the GlcNAc.

[0008] N-glycosylation can have a significant effect on protein folding and activity. When naturally occurring proteins or their mutants are produced for industrial use using genetic engineering methods, there is a high possibility that the presence or absence of glycosylation and the structure and morphology of the glycan may change depending on the type of host cell, the recombinant manipulation method, and the culture conditions (Schilling, et al., 2002). In other words, during the protein production process, differences in production conditions can cause quantitative differences in the glycan structure and each sugar component that constitutes the glycan. Culture conditions that affect N-glycosylation include the glucose or glutamine concentration in the culture medium (Tachibana et al. 1994), DO (Dissolved oxygen) concentration (Restelli et al. 2006), culture medium pH (Borys et al. 1993), ammonia concentration in the culture medium (Borys et al. 1994;), and culture temperature (Clark et al. 2004).

[0009] In general, enzymes act as catalysts that reduce the activation energy of a reaction by specifically binding to a substrate and forming an enzyme-substrate complex, thereby promoting the enzymatic reaction. Enzymes can distinguish between substrates and molecules that compete with the substrate, which is possible due to the complementary charge distribution, complementary structure, and hydrophilic / hydrophobic distribution at the substrate binding site of the enzyme. To explain the binding site of an enzyme, a lock-and-key model has been proposed, which states that the enzyme and the substrate have complementary and geometrical shapes and are easy to bind to each other, but this makes it difficult to explain the transition state of the enzyme-substrate complex. In the induced fit model proposed to overcome this, the enzyme and the substrate in the enzyme-substrate complex change their structure while continuously interacting with each other through the flexible structure of the enzyme protein, and in this process, the reaction can be further promoted by the surrounding charge distribution and hydrophilic / hydrophobic distribution of the amino acid residues and N-glycans that constitute the active site.

[0010] Furthermore, charge interaction is an essential element in the reaction of hyaluronidase enzymes that hydrolyze the substrate hyaluronic acid. Arming et al. have shown that positively charged arginine residues are essential elements for the enzyme activity in order for hyaluronidase PH20 to bind to hyaluronic acid, a substrate with a large amount of negative charges (Arming et al. 1997). Therefore, it can be inferred that the charge distribution of N-glycans also affects such enzyme activity, and when hyaluronic acid, a substrate with a large amount of negative charges, binds to hyaluronidase, it is important to prove whether the content of negatively charged sialic acid capping sugars in the N-glycans, i.e., the content of sialylation, forms an enzyme-substrate conjugate or affects the progress of the enzyme reaction. To limit the sialylation content, the transfer of sialic acid to galactose residues must be limited, desialylation must be performed, or the galactosylation content must be limited.

[0011] Therefore, since the productivity and activity of recombinant hyaluronidase PH20 or its variants are affected by changes in the N-glycosylated content, research into a method for producing hyaluronidase PH20 or its variants that is highly active and highly productive and can be used industrially is required in the industry for efficient large-scale production by regulating and maintaining this content. Summary of the Invention [Problem to be solved by the invention]

[0012] The object of the present invention is to provide a method for culturing a host cell that produces recombinant hyaluronidase PH20 or a variant thereof, and PH20 or a variant thereof produced by such a method, and in particular, to provide a method for producing hyaluronidase PH20 or a variant thereof having improved enzymatic activity and productivity.

[0013] However, the technical problem that the present invention aims to achieve is not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those of ordinary skill in the art from the description of the present invention. [Means for solving the problem]

[0014] In the present invention, it has been confirmed that when host cells producing recombinant hyaluronidase PH20 or a variant thereof are cultured under specific culture conditions, the N-glycosylation characteristics of the produced recombinant hyaluronidase PH20 or a variant thereof, and further, the enzymatic activity of the produced hyaluronidase PH20 or a variant thereof are significantly improved.

[0015] Specifically, it is effective to increase activity by adjusting the sialylation content, galactosylation content and / or mannosylation content in the N-glycan content, particularly the sialylation content. In order to adjust this content, it has been confirmed that the enzyme activity and productivity of the hyaluronidase PH20 or its variants of the present invention can be significantly increased by adjusting the glucose concentration and / or pH in the medium, or by changing the culture temperature and culturing for a certain period of time.

[0016] Specifically, the method for producing hyaluronidase PH20 or a variant thereof according to the present invention comprises: (1) Host cells expressing recombinant hyaluronidase PH20 or its variants were cultured at a temperature of 35°C to 38°C at a concentration of 20 x 10 based on the integral viable cell density. 6 ~120×10 6 To give x days of cells / mL Culturing; and (2) After lowering the culture temperature to 28°C to 34°C, the culture temperature is maintained for 2 to 18 days. (a) maintaining the concentration of residual glucose in the medium between 0.001 g / L and 4.5 g / L during the culture period; and (b) Culture with the pH of the culture medium maintained at 6.8 to 7.2; culturing by one or more methods selected from the group consisting of The present invention is characterized by comprising:

[0017] PH20 or its variant produced by the method according to the present invention is characterized in that the sialylated content of N-glycan is 1% to 38% and the enzyme activity is significantly increased, but is not limited thereto, and the above values ​​are experimental values ​​with an error range of 10%. This is because, when setting culture conditions, deviations occur depending on conditions such as the equipment used for culture and the level of expertise of the tester, and therefore the values ​​set in the present invention should be interpreted in a broader sense taking the deviations into account rather than in a limited sense.

[0018] The host cells may be cultured by one or more methods selected from the group consisting of batch culture, repeated batch culture, fed-batch culture, repeated fed-batch culture, continuous culture, and perfusion culture. [Brief description of the drawings]

[0019] [Figure 1] FIG. 1 shows the results of analysis of the enzyme activity, isoelectric point, and N-glycan content for native human hyaluronidase PH20 and its mutants, where A in FIG. 1 shows the enzyme activity for native human hyaluronidase PH20 and its mutants, B in FIG. 1 shows the results of isoelectric focusing electrophoresis for each sample, and C in FIG. 1 shows the N-glycan content. [Diagram 2] Figure 2 shows the results of comparing the enzyme activity and isoelectric point analysis of purified fractions of the hyaluronidase PH20 mutant after treatment with PNGase F, treatment with sialidase A, and treatment with sialidase A + galactosidase. Figure 2A shows the results of isoelectric focusing for each sample, Figure 2B shows the results of enzyme activity, and Figure 2C shows the N-glycan content. [Diagram 3] FIG. 3 shows the results of an analysis of cell growth, cell viability, pH, and lactate concentration changes in cells producing hyaluronidase PH20 mutants when cultured with the addition of N-acetyl-D-mannosamine or galactose, where FIG. 3A shows cell growth, FIG. 3B shows cell viability, FIG. 3C shows pH changes, and FIG. 3D shows lactate concentration changes. [Figure 4] FIG. 4 is a graph showing the analysis of activity in culture recovery fluid from cells producing hyaluronidase PH20 mutants when cultured with the addition of N-acetyl-D-mannosamine or galactose. [Diagram 5]FIG. 5 shows the results of isoelectric focusing analysis of the culture recovery solution obtained when cells producing hyaluronidase PH20 mutants were cultured with the addition of N-acetyl-D-mannosamine or galactose. [Figure 6] FIG. 6 shows the results of N-glycan structure analysis of the culture recovery solution obtained when N-acetyl-D-mannosamine or galactose was added to cells producing hyaluronidase PH20 mutants. [Figure 7] FIG. 7 shows the results of an analysis of changes in cell growth, cell viability, and ammonia concentration depending on the culture day for cells producing the hyaluronidase PH20 mutant, where FIG. 7A shows cell growth, FIG. 7B shows cell viability, FIG. 7C shows cumulative cell concentration, and FIG. 7D shows changes in ammonia concentration. [Figure 8] FIG. 8 is a graph analyzing the activity of the harvested solution of cells producing the hyaluronidase PH20 mutant on each culture day. [Figure 9] FIG. 9 shows the results of isoelectric focusing analysis of the recovery solution from cells producing hyaluronidase PH20 mutant on each culture day. [Figure 10] FIG. 10 shows the results of an analysis of the N-glycan structure and activity of the recovered solution from cells producing the hyaluronidase PH20 mutant on each culture day. [Figure 11] Figure 11 shows the results of an analysis of cell growth, cell viability, pH change, osmotic pressure change, glucose concentration, and lactate concentration under glucose concentration conditions for cells producing the hyaluronidase PH20 mutant, where Figure 11A shows cell growth, Figure 11B shows cell viability, Figure 11C shows pH change, Figure 11D shows osmotic pressure change, Figure 11E shows change in glucose concentration, and Figure 11F shows change in lactate concentration. [Figure 12] FIG. 12 is a graph analyzing the activity of cells producing hyaluronidase PH20 mutants under glucose concentration conditions. [Figure 13]FIG. 13 shows the results of isoelectric focusing analysis performed on cells producing hyaluronidase PH20 mutants under glucose concentration conditions. [Figure 14] FIG. 14 shows the results of an analysis of the N-glycan structure of cells producing hyaluronidase PH20 mutants under glucose concentration conditions. [Figure 15] FIG. 15 shows the results of an analysis of cell growth, cell viability, pH change, and cumulative cell concentration change under various pH conditions for cells producing the hyaluronidase PH20 mutant, where FIG. 15A shows cell growth, FIG. 15B shows cell viability, FIG. 15C shows pH change, and FIG. 15D shows cumulative cell concentration change. [Figure 16] FIG. 16 is a graph analyzing activity under pH conditions for cells producing hyaluronidase PH20 variants. [Figure 17] FIG. 17 shows the results of isoelectric focusing analysis under pH conditions for cells producing hyaluronidase PH20 mutant. [Figure 18] FIG. 18 is a purification chromatogram by first anion exchange resin chromatography obtained during the purification process of the hyaluronidase PH20 mutant. [Figure 19] Figure 19 is a purification chromatogram obtained by a second anion exchange resin chromatography during the purification process of the hyaluronidase PH20 mutant. [Figure 20] FIG. 20 is a purification chromatogram by cation exchange resin chromatography obtained during the purification process of the hyaluronidase PH20 mutant. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] All 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.

[0021] The present invention was completed by confirming that, when producing hyaluronidase PH20 or a variant thereof for industrially useful use by genetic recombination methods, the sialylation, galactosylation and / or mannosylation content of the N-glycan content, in particular the sialylation content, is extremely important for the enzymatic activity and productivity of hyaluronidase PH20 or a variant thereof.

[0022] Specifically, it was confirmed that when the sialylated content of the N-glycan content of PH20 or its variant is 1% to 38%, preferably 1% to 30%, more preferably 1.5% to 28%, and most preferably 2% to 25%, the enzymatic activity and productivity of hyaluronidase PH20 or its variant are improved to an unpredictable degree.

[0023] More specifically, when the N-glycan content of PH20 or a variant thereof is 1% to 68% galactosylation, 1% to 38% sialylation, and 40% to 63%, preferably 5% to 60% galactosylation, 1% to 30% sialylation, and 42% to 62%, more preferably 10% to 56% galactosylation, 1.5% to 28% sialylation, and 44% to 61%, and most preferably 15% to 50% galactosylation, 2% to 25% sialylation, and 47% to 60%, the enzymatic activity and productivity of hyaluronidase PH20 or a variant thereof can be improved to an unpredictable degree.

[0024] The above values ​​are derived from the experimental results in Table 1 of the Examples, and are values ​​with a 95% confidence interval, and these values ​​may also include a 10% error. This is because when measuring the sugar content of a protein, deviations occur depending on conditions such as the equipment used in the experiment, the enzyme reaction time, the test temperature, and the level of expertise of the tester, so the sugar content measured in the present invention should be interpreted in a broader sense, taking into account the deviations between laboratories, rather than in a limited sense.

[0025] In the present invention, the galactosylation percentage of the N-glycan content is the sum of the percentages of N-glycan containing galactose at its terminal, such as G1, G1F, G1F', G2, G2F, A1, A1F, A2, and A2F; the sialylation percentage is the sum of the percentages of N-glycan containing sialic acid at its terminal, such as A1, A1F, A2, and A2F; and the mannosylation percentage is the sum of the percentages of N-glycan containing mannose at its terminal, such as M4G0F, M5, M5G0, M6, M7, M8, and M9.

[0026] Specifically, the method for producing hyaluronidase PH20 or a variant thereof having an N-glycan content of 1% to 38% of sialylation according to the present invention comprises the steps of: (1) Host cells expressing recombinant hyaluronidase PH20 or its variants are cultured at a temperature of 35°C to 38°C to a cumulative viable cell concentration of 20 x 10 6 ~120×10 6 Culturing for x days / mL of cells; and (2) After lowering the culture temperature to 28°C to 34°C, the culture temperature is maintained for 2 to 18 days. (a) maintaining the concentration of residual glucose in the medium between 0.001 g / L and 4.5 g / L during the culture period; and (b) Culture with the pH of the culture medium maintained at 6.8 to 7.2; culturing by one or more methods selected from the group consisting of The present invention is characterized by comprising:

[0027] The above values ​​are experimental values ​​with an error range of 10%. This is because when culture conditions are set, deviations occur depending on conditions such as the equipment used for culture and the level of expertise of the tester, and therefore the values ​​set in the present invention should be interpreted in a broader sense taking deviations into account rather than in a limited sense.

[0028] The decrease in the culture temperature from step (1) to step (2) is based on the cumulative viable cell concentration of 20×10 6 ~120×10 6Cells x days / mL, preferably 40 x 10 6 ~100×10 6 Cells x days / mL, more preferably 60 x 10 6 ~80×10 6 This can be done in the case of, but is not limited to, x days of cells / mL.

[0029] Furthermore, the culture period in the step (2) may be, but is not limited to, 2 to 18 days, preferably 3 to 16 days, and more preferably 4 to 14 days.

[0030] For example, when a fed-batch culture method is used in the production of PH20 or a mutant thereof according to the present invention, a seed culture immediately prior to the main culture is cultured at 35°C to 38°C by perfusion culture or fed-batch culture until a certain cell concentration is reached, and then the seed culture is inoculated into the main culture, the temperature is reduced to less than 35°C, and the culture is continued for 2 to 18 days, preferably 3 to 16 days, and more preferably 4 to 14 days. This not only maximizes the productivity of PH20 or a mutant thereof, but also significantly increases the enzymatic activity of the produced PH20 or a mutant thereof.

[0031] The cell inoculation concentration for the main culture was 1 × 10 5 cells / mL or more, preferably 5×10 5 cells / mL or more, more preferably 1×10 6 The concentration may be, but is not limited to, cells / mL or more.

[0032] When the fed-batch culture method and the perfusion culture method are combined in the production of PH20 or its variant according to the present invention, not only can the productivity of PH20 or its variant be maximized by culture, but the enzymatic activity of the produced PH20 or its variant can also be significantly increased.

[0033] In the present invention, the culture can be performed while maintaining the concentration of residual glucose in the medium at 0.001 g / L to 4.5 g / L, preferably 0.01 g / L to 4.0 g / L, and more preferably 0.1 g / L to 3.5 g / L, but is not limited thereto.

[0034] In the present invention, the phrase "cultivating while maintaining the residual glucose concentration in the medium at 0.001 g / L to 4.5 g / L, preferably 0.01 g / L to 4.0 g / L, and more preferably 0.1 g / L to 3.5 g / L" means that the residual glucose concentration in the medium is measured at intervals of every 1 to 36 hours, preferably every 3 to 30 hours, and more preferably every 6 to 24 hours or in real time during the culture period, and if the residual glucose concentration in the medium is equal to or lower than a standard concentration set within 0.001 g / L to 4.5 g / L, preferably 0.01 g / L to 4.0 g / L, and more preferably 0.1 g / L to 3.5 g / L, a glucose stock solution is added to the medium to reach the corresponding standard concentration, and then culture is performed.

[0035] In the present invention, the standard concentration of the residual glucose in the medium can be set within the range of 0.001 g / L to 4.5 g / L, preferably 0.01 g / L to 4.0 g / L, more preferably 0.1 g / L to 3.5 g / L, and it is obvious to a person skilled in the art that the standard concentration can be appropriately changed during the culture period. For example, the standard concentration of the residual glucose in the medium can be set to 2 g / L on the first 1 to 2 days, and the standard concentration can be lowered to 1.5 g / L on the 3rd to 5th days, and then the standard concentration can be set to 2 g / L again, or the standard concentration can be set to a concentration lower than 1.5 g / L, i.e., 1.0 g / L.

[0036] Hyaluronidase PH20 or a variant thereof produced by the method of the present invention and having a specific sialylation content, galactosylation content and / or mannosylation content in the N-glycan content is characterized in that the activity of the hyaluronidase enzyme in the culture medium is 10,000 unit / mL, preferably 11,000 unit / mL or more, more preferably 12,000 unit / mL or more, but is not limited thereto. Furthermore, hyaluronidase PH20 or a variant thereof produced by the method of the present invention and having a specific sialylation content, galactosylation content and / or mannosylation content in the N-glycan content can be characterized by having an enzymatic activity increased by 10% or more, preferably 12% or more, and more preferably 15% or more compared to the activity of natural human PH20 produced by conventional methods, but is not limited thereto.

[0037] Preferably, in the method for producing hyaluronidase PH20 or a variant thereof according to the present invention, the host cell culture in the step (1) and / or the step (2) comprises: (i) adding ammonia to the medium or increasing the ammonia concentration in the medium to 5 mM or more; (ii) adding one or more substances selected from the group consisting of glutamine, glucosamine, uridine, and sodium butyrate to the medium; and (iii) Conditions in which galactose and ManNAc were not added; The reaction may be carried out under one or more conditions selected from the group consisting of, but not limited to,

[0038] More specifically, in the method for producing hyaluronidase PH20 or a variant thereof according to the present invention, the culture of the host cells in step (1) and / or step (2) can be characterized by, but is not limited to, culture in which ammonia is added to the medium or the ammonia concentration in the medium is increased to 5 mM or more, culture in which glutamine is added, culture in which galactose and ManNAc are not added, culture in which uridine or glucosamine is added, or culture in which sodium butyrate is added.

[0039] In the present invention, hyaluronidase PH20 or a variant thereof refers to an enzyme that degrades hyaluronic acid located in the extracellular matrix.

[0040] The term "hyaluronidase PH20" or "PH20" of the present invention is interpreted to include all of the naturally occurring PH20 and its mature forms, and a "variant" of "hyaluronidase PH20" or "PH20" refers to, but is not limited to, a PH20 variant that includes substitutions, deletions and insertions of one or more amino acid residues in the amino acid sequence of "hyaluronidase PH20" or "PH20", and optionally has a portion of the amino acid residues deleted at the N-terminus and / or C-terminus.

[0041] The hyaluronidase of the present invention refers to any hyaluronidase that is derived from animals and microorganisms such as Streptomyces and has hyaluronidase activity. Of these, "hyaluronidase PH20" or "PH20" is preferably derived from animals and microorganisms such as Streptomyces, and is preferably derived from humans, cows, or sheep.

[0042] Examples of the human-derived "hyaluronidase PH20" or "PH20 variant" of the present invention include, but are not limited to, those described in International Publication No. 2020 / 022791 and U.S. Patent No. 9,447,401. The term "hyaluronidase PH20" or "PH20 variant" should be construed as including all variants that have the enzymatic activity of hyaluronidase and that include the substitution, deletion or insertion of one or more amino acid residues in the amino acid sequence of "hyaluronidase PH20" or "PH20", and selectively have a portion of the amino acid residues deleted at the N-terminus and / or C-terminus.

[0043] In the present invention, host cells used for expressing the hyaluronidase protein may include, but are not limited to, animal cells, yeast, actinomycetes, and insect cells.

[0044] The animal cells are preferably mammalian cells. More preferably, commonly used animal cultured cells such as CHO cells, HEK cells, COS cells, 3T3 cells, myeloma cells, BHK cells, HeLa cells, and Vero cells are used, and when the purpose is mass expression, it is particularly preferable to use CHO cells. In addition, in order to produce a desired protein, it is particularly preferable to use cells suitable for introducing a desired gene, such as dhfr-CHO cells (Proc. Natl. Acad. Sci. USA (1980) 77, 4216-4220) and CHO K-1 cells (Proc. Natl. Acad. Sci. USA (1968) 60, 1275), which are CHO cells lacking the DHFR gene. As the CHO cells, particularly preferred are DG44 strain, DXB-11 strain, K-1 strain, or CHO-S strain, and the vector can be introduced into the host cells by a method such as the calcium phosphate method, the DEAE-dextran method, the electroporation method, or the lipofection method.

[0045] Examples of yeasts include Sacchromyces sp., Hansenula sp., Kluyveromyces, and Pichia sp., and examples of actinomycetes include Streptomyces, but are not limited to these.

[0046] In the method for producing hyaluronidase PH20 or a variant thereof according to the present invention, the culture of the host cell in step (1) and / or step (2) may be performed by one or more methods selected from the group consisting of batch culture, repeated batch culture, fed-batch culture, repeated fed-batch culture, continuous culture, and perfusion culture, but is not limited thereto.

[0047] The batch culture method is a culture method in which cells are grown without adding new medium or discharging the culture solution during culture. The continuous culture method is a culture method in which the medium is continuously added and continuously discharged during culture. Continuous culture methods also include perfusion culture. The fed-batch culture method is intermediate between the batch culture method and the continuous culture method, and is also called semi-batch culture. The medium is continuously or sequentially added during culture, but the culture solution is continuously discharged as in the continuous culture method, and cells are not discharged. In the present invention, either culture method can be used, but preferably the fed-batch culture method or the continuous culture method is used, and particularly preferably the fed-batch culture method is used.

[0048] As described above, the enzymatic activity of the hyaluronidase PH20 or variant thereof produced by the production method of the present invention can be increased by 10% or more compared to that produced by a conventional method, and is particularly characterized by being increased by 10% or more compared to the activity of natural human PH20. Such an increase in enzymatic activity is due to a change in the N-glycosylation characteristics and / or charge modification of the hyaluronidase PH20 or variant thereof produced by the production method of the present invention.

[0049] In particular, during N-glycosylation, changes in the sialylation content, galactosylation content, and / or mannosylation content increase enzyme activity, and the production method of the present invention can regulate such N-glycosylation characteristics, in particular the sialylation content, galactosylation content, and / or mannosylation content.

[0050] In the present invention, the covalent bond between a sugar and a protein includes an N-glycoside bond (N-glycoside-linked glycan) in which N-acetyl-D-glucosamine is covalently linked to an asparagine residue constituting a protein, and an O-glycoside bond (O-glycoside-linked glycan) in which N-acetyl-D-galactosamine is covalently linked to a serine or threonine residue. However, there are no particular limitations on the type of covalent bond between a sugar and a protein in the glycoprotein of the present invention, and glycoproteins having either or both of an N-glycoside-linked glycan and an O-glycoside-linked glycan are also included in the glycoprotein of the present invention.

[0051] From this perspective, the present invention relates to hyaluronidase PH20 or a variant thereof, in which the sialylated content of N-glycans is 1% to 38%, preferably 1% to 30%, more preferably 1.5% to 28%, and most preferably 2% to 25%.

[0052] More specifically, the present invention relates to hyaluronidase PH20 or a variant thereof, characterized in that the N-glycan content is 1% to 68% galactosylation, 1% to 38% sialylation, and 40% to 63%, preferably 5% to 60% galactosylation, 1% to 30% sialylation, and 42% to 62%, more preferably 10% to 56% galactosylation, 1.5% to 28% sialylation, and 44% to 61%, and most preferably 15% to 50% galactosylation, 2% to 25% sialylation, and 47% to 60% mannosylation.

[0053] The above values ​​are derived from the experimental results in Table 1 of the Examples, and are values ​​with a 95% confidence interval, and these values ​​may also include a 10% error. This is because when measuring the sugar content of a protein, deviations occur depending on conditions such as the equipment used in the experiment, the enzyme reaction time, the test temperature, and the level of expertise of the tester, so the sugar content measured in the present invention should be interpreted in a broader sense, taking into account the deviations between laboratories, rather than in a limited sense. In addition, the charge variant of PH20 or its variant can be confirmed through isoelectric focusing.

[0054] It is preferable that the hyaluronidase PH20 or its variant having a specific sialylation content, galactosylation content and / or mannosylation content among the N-glycan contents of the present invention is produced by the production method of the present invention, but it is not limited thereto and can naturally be produced by other methods that can be modified in light of the common sense of a skilled artisan.

[0055] In the present invention, the medium used for culturing cells and expressing glycoproteins is preferably a serum-free medium, but is not limited thereto, and DMEM / F12 medium (a mixed medium of DMEM and F12) can be used as the base medium. In addition, commercially available serum-free media, such as HycellCHO medium, ActiPro medium (both from Hyclone, USA), CD OptiCHO medium, etc., can also be used. TM Culture medium, CHO-S-SFM II medium or CD CHO medium (both Gibco, USA), IS CHO-V TM As the basal medium, there can be used, but is not limited to, Culture Medium (Irvine Scientific, USA), EX-CELL® Advanced CHO Fed-Batch Medium (Sigma-Aldrich, USA), etc.

[0056] In the present invention, the feed medium used to culture cells and express glycoproteins is a serum-free medium, for example, Cell Boost TM1. Cell Boost TM 2. Cell Boost TM 3. Cell Boost TM 4. Cell Boost TM 5. Cell Boost TM 6. Cell Boost TM 7a / 7b (Hyclone, USA), CD CHO EfficientFeed TM A AGT TM , CD CHO EfficientFeed TM B AGT TM , CD CHO EfficientFeed TM C AGT TM , CD CHO EfficientFeed TM A plus AGT TM , CD CHO EfficientFeed TM B plus AGT TM , CD CHO EfficientFeed TM C plus AGT TM (all from Gibco, USA), BalanCD (registered trademark) CHO Feed 4 (Irvine Scientific, USA), EX-CELL (registered trademark) Advanced CHO Feed (Sigma-Aldrich, USA), CHO-U Feed Mix U1B7 / CHO-U Feed Mix U2B13 (Kerry, USA) and the like can also be used as feed media, but are not limited to these.

[0057] The terms "feed medium" and "concentrated nutrient medium" used in the present invention refer to a medium composed of a specific nutrient or multiple nutrients such as amino acids, vitamins, salts, trace elements, lipids, and glucose, and may refer to a concentrated product of a basal medium, and the components and concentrations of the feed medium may be varied depending on the cells to be cultured. In addition, commercially available feed media, 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), etc., may also be used as feed media, but are not limited thereto.

[0058] The plant-derived hydrolysate used in the present invention does not contain any animal-derived ingredients and refers to a product extracted from peas, cotton seeds, wheat gluten, soybeans, etc., and is a supplement rich in amino acids, peptides, vitamins, carbohydrates, nucleosides, minerals, and other ingredients. The plant-derived hydrolysate can also be used with various components and concentrations depending on the cells to be cultured. Commercially available plant-derived hydrolysates, such as HyPep TM 7404, UltraPep TM cotton, HyPep TM 7504, HyPep TM 4601N (Kerry, USA), cotton 100, cotton 200, Phytone TM and Soy 100 (all from Gibco, USA) can also be used as supplements, but are not limited to these.

[0059] The additives used to increase or decrease the N-glycan content of the glycoprotein used in the present invention generally refer to components known to be involved in protein glycosylation. In particular, in order to limit the sialylation content, the transfer of sialic acid to galactose residues must be limited, or the content of desialylation or galactosylation must be limited. Examples of such additives include components that become glycosylation precursors such as N-acetyl-D-mannosamine, glucose, mannose, glutamine, galactose, ammonia, and butyric acid at a certain concentration in the medium when cells are cultured.

[0060] In most animal cell cultures, serum-containing media are mainly used, but since serum-containing media are complex compositions with unclear chemical components, it is difficult to design a medium suitable for protein production. When serum is used, it can have a negative effect on separation and purification as well as problems of cost and reproducibility, so serum-free media or low-serum media are mainly used. In serum-free media, the concentration of glucose, which is a carbon source, is extremely low, so in order to maintain cell growth and produce a high concentration of the target protein, the culture medium can be cultured by adding glucose, which is the main carbon source, to the culture medium, or by adding glutamine to the culture medium. In particular, in order to increase the in vivo half-life in the production of protein drugs, the sialylation content must be increased. For this, glucose and glutamine in the culture medium must be maintained at a certain concentration or higher without being depleted, and the pH of the culture medium must also be maintained at a specific value. The concentrations of glucose and glutamine measured in the culture medium refer to the residual concentrations of each remaining after the cells have used them. In addition, activity promoters for each enzyme related to increasing the sialylation content are used, and inhibitors for suppressing desialylation are used. In addition, the galactosylation content can be increased and the sialylation content can be increased by adding N-glycosylated precursors, using promoters for related enzymes, or adjusting the culture conditions.

[0061] However, when producing hyaluronidase PH20 or a variant thereof, the N-glycosylated content cannot be adjusted using the methods described above. However, when produced using the production method of the present invention, it is possible to produce hyaluronidase PH20 or a variant thereof having the desired N-glycosylated content characteristics and thus high enzymatic activity.

[0062] The production method of the present invention may additionally include a step of separating and purifying the produced hyaluronidase PH20 or a variant thereof.

[0063] The separation and purification of hyaluronidase PH20 or a variant thereof according to the present invention is preferably carried out using the ionic binding properties and / or hydrophobic interaction properties of hyaluronidase PH20 or a variant thereof without using affinity binding properties, but is not limited thereto.

[0064] Specifically, the separation and purification of the hyaluronidase PH20 or a variant thereof according to the present invention can be carried out using, but is not limited to, ion exchange chromatography, such as cation exchange chromatography and / or anion exchange chromatography, and hydrophobic interaction chromatography, without using affinity chromatography.

[0065] Furthermore, in the separation and purification step of hyaluronidase PH20 or a variant thereof according to the present invention, acidic hyaluronidase PH20 or a variant thereof having low enzymatic activity is removed, and preferably, the removal of the acidic hyaluronidase PH20 or a variant thereof is characterized by being carried out using ion exchange chromatography.

[0066] To confirm the industrial applicability of enzymes, analysis of the catalytic reaction rate of enzymes is necessary. Enzyme reactions can be divided into enzyme reactions with an active site of fixed reactivity and enzyme reactions with various active sites with diverse reactivity. The catalytic reaction rate of an enzyme with a single active site of fixed reactivity, such as hyaluronidase, is known to be governed by the Michaelis-Menten rate law.

[0067] Michaelis-Menten enzyme kinetics is based on the assumption that the enzyme reaction is a two-step reaction system consisting of a reversible reaction step in which the enzyme (E)-substrate (S) complex [ES] is formed, and an irreversible reaction step in which the product (P) is produced by dissociation of the ES complex, as shown in the following equation. In this case, kf, kr, and kcat are the rate constants for the reaction in each direction (Alan Fersht (1977) Enzyme structure and mechanism).

[0068]

number

[0069] An enzyme reaction can be said to be in an apparent steady state where the reaction between the enzyme and substrate to form an ES complex can be assumed to reach equilibrium quickly, or where the enzyme concentration can be sufficiently reduced by maintaining a sufficiently high substrate concentration such that d[ES] / dt ≒ 0. Since the rate equations assuming either rapid equilibrium or an apparent steady state are derived identically, most experiments assume an apparent steady state where the substrate concentration is initially high relative to the enzyme concentration.

[0070] Under these assumptions, "the amount of enzyme is constant before and after the reaction" and "when a chemical reaction reaches chemical equilibrium, the reaction rate at which the product is produced is the same as the rate at which this substance is broken down again," the reaction rate of the final product can be expressed by the Michaelis-Menten rate formula as follows. In this case, K M =(k r +kcat ) / k f So, V max =k cat [E]0.

[0071]

number

[0072] To experimentally analyze enzyme reaction rates using the Michaelis-Menten rate formula, the Lineweaver-Burk equation is used. This equation shows the relationship between the reciprocal of the substrate concentration given in the experiment, 1 / [S], ​​and the reciprocal of the experimentally measured reaction rate, 1 / V. By statistically confirming that this equation is a linear equation, it can be confirmed that the enzyme reaction is a reaction according to the Michaelis-Menten rate formula, and this equation can be used to calculate K M and V max can be sought.

[0073] When an enzyme catalyzes a chemical reaction, it forms a transition state after binding to a substrate at the active site, and the activation energy to reach the high-energy transition state becomes lower through multiple bonds with the substrate. The equilibrium constant to reach such a transition state is k cat / K M where 1 / K M is an index that combines the degree to which an enzyme-substrate complex is formed by binding between an enzyme and a substrate and the degree to which the enzyme-substrate complex is maintained without being decomposed, and k cat is the equilibrium constant for the formation of products from the enzyme-substrate complex, so k cat / K M is an index of how much product can be obtained from a substrate and an enzyme, i.e., it indicates the catalytic efficiency of an enzyme.

[0074] The industrial applicability of hyaluronidase is proportional to its enzyme catalytic efficiency. In particular, when hyaluronidase is subcutaneously injected together with a macromolecular pharmacologically active substance such as a monoclonal antibody, the enzyme catalytic efficiency of hyaluronidase plays an important role. The mutant of the present invention has a higher k value than the natural PH20. cat / K M In the case where the hyaluronidase contained in the polymeric pharmacologically active substance has a superior effect of decomposing hyaluronic acid present in the subcutaneous tissue more quickly when the substance is administered subcutaneously, thereby enabling the pharmacologically active substance to be dispersed more quickly. In addition, the mutant according to the present invention has a k value larger than that of the wild-type PH20. cat V is the maximum reaction velocity at the same enzyme concentration. max This increases the amount of hyaluronic acid decomposed in the same time period, providing an excellent effect of dispersing pharmacologically active substances over a wider area.

[0075] Therefore, to confirm the enzymatic properties of the PH20 mutants according to the present invention, the enzymatic reaction rate of each mutant was analyzed and its K M (50%V max concentration of substrate at the condition), k cat (substrate conversion rate), k cat / K M The results of (enzyme catalytic efficiency) were compared in Example 4. These results demonstrate that the PH20 mutant of the present invention is superior to wild-type PH20. EXAMPLES

[0076] The present invention will be described in more detail with reference to the following examples, which are merely illustrative of the present invention and are not intended to limit the scope of the present invention.

[0077] Example 1. Relationship between hyaluronidase activity, N-glycosylation, and isoelectric point

[0078] The activity, isoelectric point, and N-glycosylated content of the native human hyaluronidase PH20 and its mutant HM46 were analyzed, and the results are shown in Figure 1. Although the difference in activity units between the two hyaluronidases was more than two-fold, the range of isoelectric points and the content of N-glycosylated chains were not significantly different.

[0079] An experiment was carried out to confirm the relationship between N-glycosylated content, isoelectric point, and activity in hyaluronidase PH20. In the process of purifying the hyaluronidase PH20 mutant, a basic fraction (Fraction 1) and an acidic fraction (Fraction 2) were separated. In addition, these fractions were treated with PNGase F to remove all N-glycosylated residues, treated with sialidase A to remove terminal sialic acid, and treated with sialidase A and galactosidase to remove terminal sialic acid and galactose. The isoelectric point and enzyme activity were analyzed for the following samples (Figure 2). The two fractions had different isoelectric point ranges, and the enzyme activity was reduced in the acidic fraction. When treated with PNGase F, the activity of both fractions was completely lost, and the isoelectric point ranges became similar. These results showed that the N-glycosylated content is closely related to the enzyme activity. Furthermore, when terminal sialic acid was removed, the isoelectric point range and enzyme activity of the acidic fraction became similar to those of the basic fraction, indicating that the content of terminal sialic acid was related to the enzyme activity. However, the basic fraction, which had a low content of terminal sialic acid, showed increased enzyme activity of hyaluronidase compared to the acidic fraction, which had a high content of sialic acid.

[0080] The relationship between the enzyme activity and the N-glycan content was investigated using cell sources for culture. The association was further confirmed by the results of studies of wild-type and various mutants with different sources (Table 1).

[0081] [Table 1] Relative activity: (activity of sample) / (activity of native human PH20) expressed as a percentage. Galactosylation percentage content: The total percentage content of N-glycans containing galactose at their termini, such as G1, G1F, G1F', G2, G2F, A1, A1F, A2, and A2F. Sialylated % content: The total % content of N-glycans containing sialic acid at their termini, such as A1, A1F, A2, and A2F. Mannosylation percentage content: The percentage content of N-glycans containing mannose at the terminal, such as M4G0F, M5, M5G0, M6, M7, M8, and M9, is added together. The sequences of native human PH20 and HM46 are presented in International Publication No. 2020 / 022791. Sheep PH20 and bonobo PH20 are samples prepared by the method presented in Example 10, and their sequences are presented in Table 3.

[0082] As shown in Table 1, various hyaluronidase PH20 and mutant proteins were produced and the N-glycosylated content was investigated. For this purpose, cultures were performed using various cell sources, such as temporary expression culture in which recombinant genes were transduced into ExpiCHO cells at each culture time using ExpiFectamine CHO Reagent (Gibco, USA), cell line clone culture using cell line clones produced using recombinant genes, and cell line culture using a highly productive single clone selected. Here, cell line clone culture refers to culture using a highly productive cell line clone selected using a selective marker from a clone of an animal cell transduced with a recombinant gene, and this is performed using multiple clone cell lines to produce a single clone cell line. The cell lines consist of a research cell line (RCB), a production master cell line (MCB), and a production cell line (WCB). In addition, in order to optimize production, various combinations of culture methods were tested, and it was found that differences occurred in activity and N-glycan content in these cases as well.

[0083] Cell line clone culture #1 was cultured in Hycell CHO medium (Hyclone, USA) at a 2 L scale under batch culture conditions at 37°C without changing the culture temperature. Cell line clone culture #2 purified fraction #1 and cell line clone culture #2 purified fraction #2 were obtained by dividing the anion exchange chromatography purified fractions from the culture medium cultured at 37°C without changing the temperature using a fed-batch culture method in which cotton 100UF (Gibco, USA) was added to EX-CELL (registered trademark) Advanced CHO fed-batch medium (Sigma-Aldrich, USA) at a scale of 8L, followed by BalanCD (registered trademark) CHO Feed 4 (Irvine Scientific, USA). Purified fraction #1 is a low salt elution fraction, purified fraction #2 is a high salt elution fraction, and cell line test culture #1 was obtained by dividing the anion exchange chromatography purified fractions from the culture medium cultured at 37°C without changing the temperature using a fed-batch culture method in which cotton 100UF (Gibco, USA) was added to EX-CELL (registered trademark) Advanced CHO fed-batch medium (Sigma-Aldrich, USA) at a scale of 2L, followed by BalanCD (registered trademark) CHO Feed 4 (Irvine Scientific, USA). The conditions for cell line test culture #2 were as follows: fed-batch culture was performed by adding 100 U of cotton 200 UF (Gibco, USA) to EX-CELL® Advanced CHO fed-batch medium (Sigma-Aldrich, USA) at a scale of 10 L, followed by CD CHO EfficientFeed Batch medium (Irvine Scientific, USA) at a culture temperature of 37°C, and then changing the temperature to 32°C when a certain cumulative cell concentration was reached. TM B plus AGT TM The cells were cultured at 37°C using a fed-batch culture method in which cotton 200UF (Gibco, USA) was added, and then the temperature was changed to 32°C when a certain cumulative cell concentration was reached, and the cells were cultured for 12 days. Cell line test culture #3 was performed at a 50L scale by adding cotton 200UF (Gibco, USA) to EX-CELL® Advanced CHO fed-batch medium (Sigma-Aldrich, USA), and then adding CD CHO EfficientFeed TM B plus AGT TMThe conditions were as follows: fed-batch culture was performed using a 10 L scale EX-CELL® Advanced CHO fed-batch medium (Sigma-Aldrich, USA) with 200 U of cotton (Gibco, USA) added, and the temperature was changed to 32°C when a certain cumulative cell concentration was reached. Cell line test culture #4 was performed using a 10 L scale EX-CELL® Advanced CHO fed-batch medium (Sigma-Aldrich, USA) with 200 U of cotton (Gibco, USA) added, and the CD CHO EfficientFeed TM B plus AGT TM The culture conditions were as follows: fed-batch culture was performed by adding cotton 200UF (Gibco, USA), and the culture temperature was changed to 32°C when a certain cumulative cell concentration was reached, and the culture was continued for 13 days. Cell line culture #1, cell line culture #2, cell line culture #3, and cell line culture #4 were performed on research cell lines (RCB), production master cell lines (MCB), and production cell lines (WCB), respectively, and were performed on 200L scale EX-CELL® Advanced CHO fed-batch medium (Sigma-Aldrich, USA) with the addition of cotton 200UF (Gibco, USA). CD CHO EfficientFeed TM B plus AGT TM The culture was performed using a fed-batch culture method in which N-glycosyltransferase (Gibco, USA) was added, and the culture temperature was changed to 37°C when a certain cumulative cell concentration was reached, and the culture was continued for 12 days. Based on the above, experiments were performed on the culture conditions of Examples 2 to 5, and a culture method for maintaining the N-glycan content was established.

[0084] The N-glycan content of the natural human hyaluronidase PH20 and variants, and mammalian hyaluronidase PH20 in Table 1 ranges from galactosylation: 15% to 50%, sialylation: 2.5% to 28%, and mannosylation: 47% to 60%. When the 95% confidence intervals of each test result are applied, it was found that industrially useful hyaluronidase activity can be obtained when the ranges are galactosylation: 1% to 68%, sialylation: 1% to 38%, and mannosylation: 40% to 63%. The activity of industrially useful hyaluronidase means 50% or more of the activity of natural human PH20. The above values ​​may include an error of 10%. The reason is that when measuring the sugar content of a protein, there are variations between laboratories depending on conditions such as the equipment used in the experiment, the enzyme reaction time, the test temperature, and the level of expertise of the tester, so the sugar content measured in the present invention should be interpreted in a broader sense taking into account the variations between laboratories rather than in a limited sense.

[0085] In particular, when looking at the relationship between activity and sialylation, it can be seen that when sialylation is limited to about 30% or less, hyaluronidase with industrially higher efficiency than that of natural human PH20 can be produced. The above values ​​are derived from experimental results and may include an error of 10%.

[0086] A high-quality hyaluronidase that maintains this N-glycosylated content was confirmed through temporary expression culture, and a highly productive single clone strain was selected for stable commercial production to produce a cell line. Using these cell lines, the cell line test cultures #2, #3, and #4 and cell line cultures #1, #2, #3, and #4 in Table 1, which were obtained by the culture method applying the results of Examples 2, 3, 4, and 5, all showed enzyme expression levels of 10,000 units / mL or more, proving the efficiency of producing high-quality hyaluronidase at low cost.

[0087] Example 2. Culture with additives Hyaluronidase PH20 variant was cultured at 2 × 10 in three 125 mL Erlenmeyer flasks containing EX-CELL® Advanced CHO fed-batch medium (Sigma-Aldrich, USA) supplemented with 200 U of cotton (Gibco, USA) and then supplemented with or without 20 mM N-acetyl-D-mannosamine (NZP, Netherlands) or 50 mM galactose (Pfanstiehl, USA). 6 The cells were inoculated at 1000 cells / mL and grown in a batch culture at 37℃ in an 8% CO2 incubator. When the integral viable cell density (IVCD) reached the change range, the temperature was lowered to 32℃ and fed-batch culture was performed. TM B plus AGT TM Culture medium (Gibco, USA) was supplied daily at 1.88% of the initial culture volume in the flask. Cell samples were taken from the cultures daily to measure the number of viable cells, cell viability, pH, and lactate levels, and the culture supernatant was collected by centrifugation at 2,000 rpm for 10 minutes after the end of the culture. The activity of the samples cultured under the above conditions was confirmed by HPLC and turbidity analysis, and the protein pattern and N-glycan level were confirmed by isoelectric focusing and glycosylation analysis. It was confirmed that the medium supplemented with 50 mM galactose showed a 24% increase in galactosylation compared to the medium without galactose, while the activity decreased by 2%, and the medium supplemented with 20 mM N-acetyl-D-mannosamine showed a 35% increase in sialylation compared to the medium without galactose, while the activity decreased by 20% (Figures 3, 4, 5, and 6).

[0088] Example 3. Culture conditions Cells overexpressing hyaluronidase PH20 variants were cultured at 2 × 10 in EX-CELL® Advanced CHO Fed-Batch Medium (Sigma-Aldrich, USA) in a Sartorius 200L bioreactor.6 On the second day of culture, 200UF cotton (Gibco, USA) and CD CHO Efficient Feed, a concentrated nutrient medium, were added. TM B plus AGT TM Fed-batch culture was performed using a medium (Gibco, USA) as the feed medium, with pH set at 7.2±0.4 and DO at 40%, and agitation at 47 rpm. After initial culture at 37°C, when the cumulative viable cell concentration reached the change range, the temperature was changed to 32°C and fed-batch culture was performed. The feed medium was CD CHO EfficientFeed TM B plus AGT TM Culture medium (Gibco, USA) was supplied daily at 1.88% of the initial volume of the culture in the bioreactor. Culture was continued until the cell viability was 40% or less, and was terminated on the 19th day of culture. Various cell conditions were measured by daily sampling from the culture medium, and the number of viable cells, cell viability, cumulative viable cell concentration, and ammonium ion level were measured. After the end of culture, the culture medium was collected using a depth filter. The activity of the samples cultured under the above conditions was confirmed by HPLC and turbidity analysis, and the protein pattern and N-glycan level were confirmed by isoelectric focusing and glycosylation analysis. It was confirmed that the galactosylation and sialylation contents decreased and the activity increased as the culture days increased (Figures 7, 8, 9, and 10).

[0089] Example 4. Culture by adjusting the glucose concentration in the culture medium Cells overexpressing hyaluronidase PH20 variants were cultured at 2 × 10 in EX-CELL® Advanced CHO fed-batch medium (Sigma-Aldrich, USA) in a Sartorius 2L bioreactor. 6 On the second day of culture, the cells were inoculated with 200UF cotton (Gibco, USA) and CD CHO EfficientFeed, a concentrated nutrient medium. TM B plus AGT TMFed-batch culture was performed using a medium (Gibco, USA) as the feed medium, with pH set at 7.2±0.4 and DO at 40%, and agitation at 120 rpm. After initial culture at 37°C, when the cumulative viable cell concentration reached the change range, the temperature was changed to 32°C and fed-batch culture was performed. The feed medium was CD CHO EfficientFeed TM B plus AGT TM Culture medium (Gibco, USA) was fed daily at 1.88% of the starting volume of the culture in the bioreactor. Various cell status data were collected daily from the culture medium to measure viable cell count, cell viability, pH, osmolality, glucose, and lactate levels.

[0090] The glucose concentration was adjusted by measuring the glucose concentration in the culture medium every day, and adjusting the concentration when the glucose concentration in the medium was consumed by cell growth and was measured below the standard concentration of 2g / L, 4g / L, or 6g / L. When the measured glucose concentration was below the respective standard concentrations of 2g / L, 4g / L, or 6g / L, 200g / L glucose stock solution was added within 3 hours in an amount to reach the standard concentration, and when it was measured above the standard concentration, no glucose was added, thereby maintaining each concentration condition. Generally, in animal cell culture, the effect of changes in glucose content on cell growth becomes negligible within 3 hours.

[0091] In this case, the condition for maintaining 2g / L as the reference concentration is called 1g / L (±1g / L) concentration condition, the condition for maintaining 4g / L as the reference concentration is called 3g / L (±1g / L) concentration condition, and the condition for maintaining 6g / L as the reference concentration is called 5g / L (±1g / L) concentration condition. For example, the glucose concentration maintenance condition of 1g / L (±1g / L) concentration means that the lower limit of the glucose concentration control range is set to 0g / L and the upper limit of the glucose concentration control range is set to 2g / L. In actual culture, when the measured glucose concentration of the culture solution reaches the lower limit of 0g / L, an additional 200g / L glucose stock solution is added so that the glucose concentration becomes a maximum of 2g / L, and when the glucose concentration in the culture solution reaches the upper limit of 2g / L, the 200g / L glucose stock solution is not added. Therefore, in the two concentration conditions of 1g / L (±1g / L) and 3g / L (±1g / L), the concentration condition of 2g / L refers to the upper limit condition for the concentration of 1g / L (±1g / L) and the lower limit condition for the concentration of 3g / L (±1g / L), and therefore these are conditions under which completely different effects occur and are not considered to be overlapping conditions.

[0092] After the cultivation was completed, the culture supernatant was collected by centrifugation at 4°C and 10,000 rpm for 60 minutes. The activity of the samples cultivated under the above conditions was confirmed by HPLC and turbidity analysis, and the protein pattern and N-glycan level were confirmed by isoelectric focusing and glycosylation analysis. It was confirmed that the content of galactosylation and sialylation increased and the activity decreased as the concentration of glucose in the culture medium increased (Figures 11, 12, 13, and 14).

[0093] Example 5. Culture pH Control Culture Cells overexpressing hyaluronidase PH20 variants were cultured at 2 × 10 in EX-CELL® Advanced CHO fed-batch medium (Sigma-Aldrich, USA) in a Sartorius 2L bioreactor. 6On the second day of culture, the cells were inoculated with 200UF cotton (Gibco, USA) and CD CHO EfficientFeed, a concentrated nutrient medium. TM B plus AGT TM Fed-batch culture was performed using a medium (Gibco, USA), with DO set at 40% and stirring at 120 rpm. After initial culture at 37°C, when the cumulative viable cell concentration reached the change range, the temperature was changed to 32°C and fed-batch culture was performed. The feed medium was CD CHO EfficientFeed TM B plus AGT TMThe medium (Gibco, USA) was supplied daily at 1.88% of the initial volume of the culture in the bioreactor. After changing the culture temperature, the culture pH was divided into four conditions, pH 6.8 ± 0.1, pH 7.0 ± 0.1, pH 7.2 ± 0.1, and pH 7.4 ± 0.1, in order to find an improved condition from the existing condition of pH 7.2 ± 0.4. These four conditions are set as pH adjustment ranges for culture using a normal incubator with a pH adjustment function. For example, pH 7.0 ± 0.1 means that the lower limit of the pH adjustment range is set to pH 6.9 and the upper limit of the pH adjustment range is set to pH 7.1. In actual culture, when the pH of the culture solution reaches the lower limit of pH 6.9, a base that increases the pH is added, and when the pH of the culture solution reaches the upper limit of pH 7.1, carbon dioxide that decreases the pH is added. Therefore, in the two conditions of pH 6.8±0.1 and pH 7.0±0.1, the condition of pH 6.9 is the upper limit condition at pH 6.8±0.1 and the lower limit condition at pH 7.0±0.1, so that they are conditions that have completely different effects and are not considered to be overlapping conditions. The cells were cultured until the cell viability was 40% or less, and various cell conditions were sampled from the culture medium every day to measure the number of viable cells, cell viability, pH, and cumulative viable cell concentration levels. After the end of the culture, the culture supernatant was obtained by centrifuging at 4°C and 10,000 rpm for 60 minutes. The activity of the samples cultured under the above conditions was confirmed by HPLC and turbidity analysis, and the protein pattern was confirmed by isoelectric focusing analysis. The activity was observed to change depending on the culture medium pH, and the highest activity and lowest sialylation content were observed at pH 7.0±0.1, which is an improved condition compared to the existing conditions. (Table 2, Figure 15, Figure 16, Figure 17)

[0094] [Table 2]

[0095] Example 6. Purification of hyaluronidase using animal cell culture supernatant Step 1: Buffer exchange / detergent treatment of culture supernatant The culture medium was reconditioned by adjusting pH and conductivity using UF / DF with a 30 kDa MWCO membrane filter to adjust it to the first anion exchange column equilibration condition. The reconditioned solution was treated with a solvent / surfactant at an appropriate concentration for virus inactivation and reacted at room temperature for about 60 minutes.

[0096] Step 2: Primary anion exchange (Q Sepharose Fast Flow) column chromatography The filtered protein solution was passed through a first anion exchange column to capture the hyaluronidase by the anion exchange resin, and subsequently eluted from the column with a high salt concentration. Before loading, the column was equilibrated with tromethamine buffer at pH 8.0 with a salt concentration of 30 mM. After loading, the column was washed with the same buffer (first wash). Following the first wash step, the column was washed with the same buffer at pH 8.0, but with a salt concentration of 60 mM, which has a higher conductivity than the salt concentration of the first wash step. After the second wash step, elution of the desired protein, hyaluronidase, was performed with an appropriate buffer at pH 8.0 with a salt concentration of 200 mM, as shown in FIG. 18.

[0097] Step 3: Secondary anion exchange (Capto Q) column chromatography The filtered protein solution was passed through a second anion exchange column to remove the acidic variants of hyaluronidase by anion exchange resin, and subsequently eluted from the column with high salt concentration. Before loading, the column was equilibrated with Bis-Tris buffer pH 6.0 without salt. After loading, the column was washed with the same Bis-Tris buffer pH 6.0 (first wash). Following the first wash step, the column was washed with Bis-Tris buffer but with a salt concentration of 20 mM whose conductivity was higher than the salt concentration of the first wash step. After the second wash step, elution of the desired protein, hyaluronidase, was performed with Bis-Tris buffer with salt concentrations as shown in FIG. 19.

[0098] Step 4: Cation exchange (Capto MMC) column chromatography The reconditioned protein solution was passed through a column to remove acidic variants of hyaluronidase by cation exchange resin, and subsequently eluted from the column at high pH and salt concentration. Before loading, the column was equilibrated with citrate buffer at pH 5.5 with a salt concentration of 80 mM. After loading, the column was washed with the same buffer (first wash). Following the first washing step, the column was washed with the appropriate Bis-Tris buffer at pH 7.5. After the second washing step, elution of the desired protein, hyaluronidase, was performed with Bis-Tris buffer at pH 8.0 with a salt concentration of 400 mM as shown in FIG. 20.

[0099] Stage 5: Nano-filtration / Formulation After the cation exchange column step, the protein solution containing the desired hyaluronidase was filtered through a 1 μm filter and subjected to a nano-filtration step. The protein solution that passed through the nanofiltration was concentrated to a high concentration of 10 mg / mL and reconditioned by UF / DF using an 8 kDa MWCO membrane filter to exchange into a histidine buffer at pH 7.0 containing 145 mM salt.

[0100] Example 7. Hyaluronidase enzyme activity analysis The enzymatic activity of Hyaluronidase PH20 and other hyaluronidases was measured using a turbidimetric assay as follows.

[0101] The turbidity assay is a method to measure the absorbance of precipitates formed when hyaluronic acid and albumin (BSA) are mixed. When hyaluronic acid is hydrolyzed by PH20, the absorbance decreases when mixed with albumin. The assay was generally performed as follows. Hyaluronidase PH20 (Sigma) was diluted to 1, 2, 5, 7.5, 10, 15, 20, 30, 50, and 60 units / mL and prepared in each tube. The purified protein sample was dissolved in enzyme diluent buffer (20 mM Tris·HCl, pH 7.0, 77 mM NaCl, 0.01% (w / v) bovine serum albumin) and diluted to 100X, 300X, 600X, 1200X, and 2400X and prepared in each tube. The hyaluronic acid solution, which was 3 mg / mL, was diluted 10 times in a new tube so that the concentration became 0.3 mg / mL, and the volume of each tube was adjusted to 180 μl. 60 μl of enzyme was added to the diluted hyaluronic acid solution, mixed, and reacted at 37°C for 45 minutes. After the reaction was completed, 50 μl of the reacted enzyme and 250 μl of acidic albumin solution were added to each well of a 96-well plate, shaken for 10 minutes, and the absorbance was measured at 600 nm using a spectrophotometer. The activity unit of the sample was calculated using the test results of the standard product with known activity unit and the test results of the sample.

[0102] Example 8. Isoelectric focusing analysis of hyaluronidase Isoelectric focusing of hyaluronidase was analyzed using Invitrogen's Precast Gel (pH 3-7) and isoelectric focusing buffer solution. After loading the purified hyaluronidase sample onto the precast gel, it was developed using a Novex electrophoresis device at 100 V for 1 hour, 200 V for 1 hour, and 500 V for 30 minutes. After completion of development, the gel was washed with purified water, proteins were fixed with 12% TCA solution, stained with Coomassie Blue R-250 staining solution, and destained with acetic acid-methanol solution, and the protein bands that appeared on the gel were analyzed.

[0103] Example 9. Analysis of N-glycan content of hyaluronidase The content of N-glycans in hyaluronidase was analyzed by treating hyaluronidase with PNGase F (N-glycosidase F) to separate N-glycans, labeling them with 2-AB (2-aminobenzamide), and performing UPLC (Ultra High Performance Liquid Chromatography) using an ACQUITY UPLC Glycan BEH Amide column (Waters). The purified hyaluronidase sample was desalted and reacted with PNGase F at 37°C for 16 to 18 hours to separate N-glycans. After drying, the sample was reacted with 2-AB labeling at 65°C for 3 hours to remove excess 2-AB. The labeled N-glycans were separated by HPLC with a 72%-20% acetonitrile gradient. The separated samples were analyzed by Flourescence Leukocyte Detection (FLD) for 3 hours. The N-glycan contents were analyzed by detecting the N-glycan with a galactose detector. The separated N-glycans were classified, and the galactosylation content was calculated by combining the contents of N-glycans containing galactose at the end of the N-glycan (G1, G1F, G1F', G2, G2F, A1, A1F, A2, A2F, etc.), the sialylation content was calculated by combining the contents of N-glycans containing sialic acid at the end of the N-glycan (A1, A1F, A2, A2F, etc.), and the mannosylation content was calculated by combining the contents of N-glycans containing mannose at the end of the N-glycan (M4G0F, M5, M5G0, M6, M7, M8, M9, etc.).

[0104] Example 10. Production of animal-derived hyaluronidase and analysis of N-glycan content (1) Genetic engineering of bonobo and sheep hyaluronidase PH20 To investigate the activity and N-glycosylation content of hyaluronidase PH20 from animals, i.e., bonobo, an ape, and sheep, an artiodactyl, hyaluronidase PH20 was produced as follows. cDNA was synthesized from each native gene and inserted into the Xho I and Not I restriction enzyme sites of the pcDNA3.4-TOPO vector. For expression in ExpiCHO cells, one of the signal peptides from human growth hormone, human serum albumin, and human Hyal1 was used as a signal peptide instead of the inherent signal peptide of PH20. For protein purification using a HisTrap column, a His-tag DNA sequence was located at the 3'-end of PH20 cDNA. DNA sequencing was used to confirm each sequence. Table 3 shows the sequences of hyaluronidases derived from animals.

[0105] [Table 3]

[0106] (2) Expression of hyaluronidase PH20 in bonobos and sheep Expression was performed using the ExpiCHO expression system. The cell number of ExpiCHO cells was 6 × 10 6 When the number of cells / mL reached 100,000, the ExpiCHO cells were transduced with a plasmid in which the cDNA for hyaluronidase PH20 was inserted into the pcDNA3.4-TOPO vector using ExpiFectamine CHO Reagent. ExpiCHO Expression Medium (100mL-500mL) was used as the cell culture medium. After transduction, the ExpiCHO cells were cultured with shaking at 130 rpm for a total of 6 days, during which they were cultured at 37°C for 1 day and then the temperature was lowered to 32°C for an additional 5 days. At the completion of the culture, the cell supernatant was collected by centrifugation at 10,000 rpm for 30 minutes.

[0107] (3) Purification of bonobo and sheep hyaluronidase PH20 The recombinant protein of animal-derived hyaluronidase PH20 with a C-terminal His-tagged tag produced in ExpiCHO cells was purified by two-step column chromatography using an AKTA Prime instrument (GE Healthcare). In the case of bonobo PH20, the pI is 6, so a Q Sepharose column for anion exchange chromatography was used in one step, and in the case of sheep PH20, the pI is 8 or higher, so a Capto S column for cation exchange chromatography was used in one step. Each protein was purified in two steps using a HisTrap HP column for His-tag affinity chromatography.

[0108] 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 for protein purification using a Q Sepharose column. Proteins were bound to the Q Sepharose column, and non-specifically bound proteins were removed by applying 5 CV of buffer A. Proteins were then eluted by applying 5 CV of buffer B with a concentration gradient of 0% to 100%.

[0109] For protein purification using a 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. Next, after binding the protein to the Capto S column, buffer A was applied for 3 CV (column volume) to remove non-specifically bound proteins. Buffer B was then applied for 4 CV to elute the target protein.

[0110] For protein purification using the HisTrap HP column, 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) were prepared, respectively. After binding of the protein sample to the HisTrap HP column, 7 CV of 7% buffer B was run to remove nonspecifically bound proteins, and 3 CV of 40% buffer B was run to elute the target protein. The column eluate was dialyzed against dialysis buffer (20 mM sodium phosphate, 100 mM NaCl, pH 7.0).

[0111] (4) Analysis of PH-20 hyaluronidase in bonobos and sheep The activity analysis was performed in the same manner as in Example 7, and the N-glycan content analysis was performed in the same manner as in Example 9. The results are shown in Table 1.

[0112] Example 11. Enzyme kinetics analysis of mutants with different N-glycosylated contents To analyze the enzyme reaction rate of the mutants of 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 quantifies (545 nm) the red substance produced by the reaction of the reducing end of N-acetyl-D-glucosamine (GlcNAc) produced when hyaluronic acid is hydrolyzed by hyaluronidase with para-dimethylaminobenzaldehyde (DMAB), an Ehrlich's Reagent. N-acetyl-D-glucosamine (GlcNAc, Sigma) was diluted with dilution buffer (0.1M NaPi, 0.1M NaCl, 1.5mM saccharic acid 1,4-lactone, pH 5.35) to 0.25mM, 0.50mM, 0.75mM, 1.00mM, and 1.25mM, and each test tube was treated with tetraborate for reduction, followed by the addition of DMAB for color reaction. After the reaction, the absorbance was measured at 545nm, and a standard reaction curve for GlcNAc was created. The substrate hyaluronic acid was diluted with a dilution buffer solution to 0.54μM, 0.65μM, 0.87μM, 1.23μM, and 2.17μM, and hyaluronidase was added to each test tube, and the reaction was carried out at 37℃ for 5 minutes. The enzyme reaction was terminated by heating at 100℃ for 5 minutes, and the mixture was treated with tetraboric acid for reduction, and then DMAB was added to cause a color reaction. After the reaction, the absorbance was measured at 545nm, and the enzyme activity was measured using the standard reaction curve of GlcNAc described above. When the enzyme reaction rate of wild-type PH20 of SEQ ID NO:1 and the PH20 mutant of the present invention was analyzed using this method, the linearity of the Lineweaver-Burk curve was confirmed, and it was confirmed that the PH20 mutant of the present invention follows the formula of the Michaelis-Menten enzyme reaction.

[0113] Table 4 shows the enzyme reaction rates of fractions #1 and #2 obtained from cell line clone test culture #2 among the samples in Table 1 of Example 1. The results show that when the galactosylation content and mannosylation content are in a similar range and the sialylation content is low, the enzyme catalytic efficiency (kcat / Km) is high, and therefore the enzyme activity of the recombinant hyaluronidase is increased.

[0114] These experimental results demonstrate that even in the case of enzymes with the same amino acid structure, the activity of the enzyme can be affected by changes in glycosylation and even changes in sialylation content. Therefore, it was confirmed that when attempting to mass-produce native PH20 or native PH20 mutant hyaluronidase by recombinant methods to produce industrially useful hyaluronidase, it is possible to develop an enzyme with greater industrial applicability by adjusting the sialylation content.

[0115] [Table 4] [Industrial Applicability]

[0116] According to the method for producing recombinant hyaluronidase PH20 protein or a variant thereof of the present invention, it is possible to produce recombinant hyaluronidase PH20 protein or a variant thereof having high enzymatic activity with high productivity, and it is possible to mass-produce and supply recombinant hyaluronidase PH20 protein or a variant thereof.

[0117] The above description of the present invention is for illustrative purposes only, and a person having ordinary skill in the art to which the present invention pertains will understand that the present invention can be easily modified into other specific forms without changing the technical concept or essential features of the present invention. Therefore, it should be understood that the above-described embodiments are illustrative in all respects and are not limiting.

[0118] References 1.L. H. Bookbinder, A. Hofer, M. F. Haller, M. L. Zepeda, G-A. Keller, J. E.Lim, T. S. Edgington, H. M. Shepard, J. S. Patton, G. I. Frost. (2006). A recombinant human enzyme for enhanced interstitial transport of therapeutics. J. Control. Release. 114, 230-241. 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. 4.W. A. Clement, S. H. Vyas, J. N. Marshall, J. H. Dempster. (2003). The use of hyaluronidase in nasal infiltration: prospective randomized controlled pilot study. J. Laryngol. Otol. 117(8):614-8. 5.Thomas J. R., Wallace M. S., Yocum R. C., Vaughn D. E., Haller M. F., Flament J. (2009). The INFUSE-Morphine study: use of recombinant human hyaluronidase(rHuPH20) to enhance the absorption of subcutaneously administered morphine in patients with advanced illness. J. Pain and Symptom Manag. 38(5):663-672. 6.George Harb, Francois Lebel, Jean Battikha, Jeffrey W Thackara. (2010). Safety and pharmacokinetics of subcutaneous ceftriaxone administered with or without recombinant human hyaluronidase (rHuPH20) versus intravenous ceftriaxone administration in adult volunteers. Curr. Med. Res. Opin. 26(2):279-88. 7.Richard L. Wasserman. (2014). Overview of recombinant human hyaluronidase-facilitated subcutaneous infusion of IgG in primary immunodeficiencies. Immunotherapy. 6(5):553-67. 8.Harris R. J., Shire S. J., Winter C. (2004). Commercial manufacturing scale formulation and analytical characterization of therapeutic recombinant antibodies. Drug. Dev. Res. 61:137-154. 9.Stephan Schilling, Torsten Hoffmann, Fred Rosche, Susanne Manhart, Claus Wasternack, and Hans-Ulrich Demuth. (2002). Heterologous Expression and Characterization of Human Glutaminyl Cyclase: Evidence for a Disulfide Bond with Importance for Catalytic Activity. Biochemistry, 35, 10849-10857. 10.H. Tachibana 1, K. Taniguchi, Y. Ushio, K. Teruya, K. Osada, H. Murakami. (1994). Changes of monosaccharide availability of human hybridoma lead to alteration of biological properties of human monoclonal antibody. Cytotechnology. 16(3):151-7. 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. 12.M. C. Borys, D. I. Linzer, E. T. Papoutsakis. (1993). Culture pH affects expression rates and glycosylation of recombinant mouse placental lactogen proteins by Chinese hamster ovary (CHO) cells. Biotechnology (NY). 11:720-724. 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. 14.Clark K. J., Chaplin F. W., Harcum S. W. (2004). Temperature effects on product quality-related enzymes in batch CHO cell cultures producing recombinant tPA. Biotechnol. Prog. 20:1888-1892. 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. (1) Host cells expressing recombinant hyaluronidase PH20 or a variant thereof are cultured at a temperature of 35° C. to 38° C. at a cumulative viable cell concentration of 20×10 ６ ~120 x 10 ６ Culturing to obtain up to x days of cells / mL; and (2) After lowering the culture temperature to 28°C to 34°C, the culture temperature is maintained for 2 to 18 days. (a) maintaining the residual glucose concentration in the medium between 0.001 g / L and 4.5 g / L during the culture period; and (b) culturing the culture medium while maintaining the pH at 6.8 to 7.2; culturing by one or more methods selected from the group consisting of A method for producing PH20 or a variant thereof, comprising: A method for producing PH20 or a variant thereof, characterized in that the sialylated content of the N-glycan content of the produced PH20 or a variant thereof is 1% to 38%, and the activity of the hyaluronidase enzyme in the culture medium is 10,000 units / mL or more.

2. The method for producing PH20 or a mutant thereof according to claim 1, characterized in that the N-glycan content of the produced PH20 or mutant thereof is 1% to 68% galactosylation, 1% to 38% sialylation, and 40% to 63% mannosylation.

3. The method for producing PH20 or a mutant thereof according to claim 1, characterized in that the sialylated content of the N-glycan content is 1% to 30%.

4. The method for producing PH20 or a mutant thereof according to claim 1, characterized in that the enzyme specific activity of the produced PH20 or a mutant thereof is improved by 10% or more compared to the specific activity of naturally occurring human PH20.

5. The method for producing PH20 or a variant thereof according to claim 1, characterized in that the culture of the host cell in step (1) and / or step (2) is carried out by one or more methods selected from the group consisting of batch culture, repeated batch culture, fed-batch culture, repeated fed-batch culture, continuous culture, and perfusion culture.

6. The culture of the host cell in the step (1) and / or the step (2) includes (i) adding ammonia to the medium or increasing the concentration of ammonia in the medium to 5 mM or more; (ii) adding one or more substances selected from the group consisting of glutamine, glucosamine, uridine, glucosamine, and sodium butyrate to the medium; and (iii) Conditions in which galactose and ManNAc were not added; The method for producing PH20 or a mutant thereof according to claim 1, characterized in that it is carried out under one or more conditions selected from the group consisting of:

7. The PH20 mutant is characterized in that it contains a substitution of one or more amino acid residues in the amino acid sequence of native PH20, and selectively has a partial deletion of amino acid residues at the N-terminus and / or C-terminus. The method for producing PH20 or a mutant thereof according to claim 1.

8. The method for producing PH20 or a mutant thereof according to claim 1, wherein the host cell is an animal cell, yeast, actinomycete or insect cell.

9. The method for producing PH20 or a mutant thereof according to claim 1, further comprising a step of separating and purifying the produced PH20 or a mutant thereof.

10. The method for producing PH20 or a mutant thereof according to claim 1, characterized in that the separation and purification of PH20 or a mutant thereof is carried out using the ionic binding characteristics and / or hydrophobic interaction characteristics of PH20 or a mutant thereof without using affinity binding characteristics.

11. The method for producing PH20 or a mutant thereof according to claim 10, characterized in that the separation and purification of PH20 or a mutant thereof is carried out using ion exchange chromatography and hydrophobic interaction chromatography without using affinity chromatography.

12. The method for producing PH20 or a mutant thereof according to claim 9, characterized in that acidic PH20 or a mutant thereof is removed.

13. The method for producing PH20 or a variant thereof according to claim 12, characterized in that the removal of the acidic PH20 or a variant thereof is carried out using ion exchange chromatography.

14. PH20 or a mutant thereof, characterized in that the N-glycan content is 1% to 68% galactosylation, 1% to 38% sialylation, and 40% to 63% mannosylation.

15. PH20 or a variant thereof according to claim 14, characterized in that the sialylated content of the N-glycan content is limited to 1% to 30%.

16. PH20 or a mutant thereof according to claim 14, characterized in that it is produced by the method according to any one of claims 1 to 13.

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