RU2822202C1 - Method of producing recombinant hyaluronidase - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: disclosed is a method of producing recombinant hyaluronidase PH20 or a variant thereof. Method makes it possible to change the levels of N-glycans under cultivation conditions, including controlled concentration of glucose in the culture medium and low temperature of cultivation during a certain period of cultivation.

EFFECT: invention provides obtaining recombinant hyaluronidase PH20 or its variant with increased specific activity and productivity.

13 cl, 20 dwg, 4 tbl, 11 ex

Description

Prior Art

Field of technology to which the invention relates

The present invention relates to a method for producing variants of hyaluronidase PH20, which include one or more substitutions of amino acid residues and optionally include deletions of some N-terminal and/or C-terminal amino acid residues in hyaluronidase, in particular wild-type PH20 or mature wild-type PH20, or in the amino acid sequences of wild-type PH20 or mature wild-type PH20.

Description of the Prior Art

Hyaluronidases are enzymes that break down hyaluronic acid present in the extracellular matrix. Hyaluronidases hydrolyze hyaluronic acid, thereby reducing the viscosity of hyaluronic acid in the extracellular matrix and increasing tissue (skin) penetration (Bookbinder et al., 2006). Hyaluronidases have been used to improve the absorption of body fluids by subcutaneous injection [Muchmore et al., 2012] or intramuscular injection [Krantz et al., 2016], as well as to improve the diffusion of local anesthetics [Clement et al. et al., 2003]. Drugs that improve subcutaneous penetration in this manner include morphine [Thomas et al., 2009], ceftriaxone [Harb et al., 2010], insulin [Muchmore et al., 2012], and immunoglobulin [Wasserman et al., 2014 ]. Hyaluronidases are also used to improve the dispersion of fluids or drugs that penetrate tissue during intravenous injection or hematoma diffusion. Among the hyaluronidases, recombinant human PH20 protein, which is active at neutral pH, was developed by Halozyme Therapeutics Inc. and is sold under the trade name “Hylenex” (Bookbinder et al., 2006).

Recombinant human PH20 proteins have been reported to be expressed in yeast (P. pastoris), insect DS-2 cells, and animal cells. Recombinant PH20 proteins produced in insect and yeast cells differ from human PH20 in the nature of N-glycosylation during post-translational modification, which affects their activity and entails the risk of side effects in the body.

For biopharmaceutical development, product uniformity and long shelf life are important factors for manufacturing flexibility. During production, various types of microheterogeneity arise due to differences in size and charge resulting from enzymatic or spontaneous degradation and modifications. Each of the chemical and enzymatic modifications, such as deamidation and sialylation, increases the net negative charge and decreases the pI value of the antibody, thereby generating acidic variants [Harris RJ et al., 2004]. In addition, cleavage of the C-terminal lysine leads to the loss of net positive charges and the formation of acidic variants. Formation of major variants can result from amidation of a C-terminal lysine or glycine, formation of succinimide, oxidation of amino acids, or removal of sialic acid, which eliminates additional positive or negative charges; both types of modification increase the pI value [Harris RJ et al., 2004].

Glycosylation is a post-translational process in proteins in cells (eukaryotes) and occurs in the endoplasmic reticulum and Golgi apparatus. Glycosylation is divided into N-glycosylation and O-glycosylation, which differ depending on the functional group attached. The process of adding sugars such as lactose or fucose to proteins produced in cells is broadly called “glycosylation.” When a glycan binds to a protein through glycosylation, the protein undergoes a process of “folding” to form a three-dimensional structure. This gives the protein stability so that it can persist for a long time without being released (unfolded). This glycan is involved in an important process that allows communication and information exchange between cells.

There are two types of reactions for the addition of glycans to proteins: N-linked or O-linked glycosylation. These two glycosylation processes differ in the mechanism of glycan synthesis and attachment, and among them, the mechanism and role of N-glycosylation are better known. Glycans added through N-glycosylation are called "N-glycans" and are produced in the endoplasmic reticulum. A series of ALG (asparagine-linked glycosylation) enzymes on dolichol pyrophosphate (PP-Dol) present in the endoplasmic reticulum membrane are added to N-acetylglucosamine (GlcNAc), mannose (Man), glucose (Glc), etc. for the final synthesis of Glc3Man9GlcNAc2-PP-Dol, which is a complex glycan in the form of a lipid-linked oligosaccharide (LLO). The synthesized LLO is transferred to a peptide N-glycosylation sequence comprising N-x-S/T, which is directly translated from the ribosome into the endoplasmic reticulum through a co-translational translocation mechanism by an oligosaccharide transferase consisting of 8 or more subunits. N-glycans attached to proteins are removed one by one from the glycan end by glucosidase (α-glucosidase I, Gls1p; α-glucosidase II, Glsp II) present in the endoplasmic reticulum. Because the second and third glucose are removed more slowly, folding is completed by the lectin chaperones calnexin and calreticulin. When all glucose is cleaved off, protein folding is considered complete, and the Man8GlcNAc2 glycans attached to the protein are transferred to the Golgi apparatus. Because of this, there is a quality control process where the folding of glycoproteins is checked again before they are transferred from the endoplasmic reticulum to the Golgi apparatus. If proper folding is not achieved, the process is repeated, allowing folding to be completed by the entry of one glucose molecule into the calnexin/calreticulin cycle.

The initial process of N-glycan biosynthesis in the endoplasmic reticulum described above remains almost the same in a wide range of organisms from yeast, simple eukaryotic microorganisms, to animals, i.e. higher organisms. However, glycans transferred to the Golgi apparatus undergo various species-specific glycan modifications, resulting in the formation of very different types of glycans in yeast, insects, plants, and animals. However, these different glycans also share a central region, a structure in which three mannoses and two GlcNAcs are linked to an asparagine nitrogen, called the “trimannosyl core.” The form in which the mannose is primarily associated with the trimannosyl core is called the "high-mannose type" and is often found in yeasts and molds. The structure, for which dozens of mannoses are sequentially added in the Golgi apparatus, is also found in the well-known yeast Saccharomyces cerevisiae. On the other hand, glycoproteins containing glycans of the mannose-deficient type (oligomannose type) have been found in insect cells. They are first trimmed to form Man5GlcNAc2 by mannosidase IA, IB and IC in the Golgi apparatus, N-acetylglucosaminyltransferase (GNT) I adds one GlcNAc to Man5GlcNAc2, and then mannosidase II produces a hybrid structure in which one GlcNAc is added to the trimannosyl core. The added GlcNAc is then cleaved off again, forming a mannose-deficient glycan structure. In animal cells, α(1,6)-fucose is often added to the first GlcNAc bound to an aspartic residue. In animals, GNT II acts on a structure in which one GlcNAc is added to the trimannosyl core and adds another GlcNAc to them, forming a glycan that has a biantennary structure. GNTs IV and V can then form four antenna structures, and in some cases GNT VI, IX or VB can form six antenna structures. After adding GlcNAc to form the antenna scaffold, β-galactosyltransferase and α-sialyltransferase present in the Golgi body form a complex glycan structure in which galactose and sialic acid are added to GlcNAc.

N-glycosylation can greatly influence protein folding or activity, and there is a very high likelihood that the presence of glycosylation and the structure or shape of glycans may vary depending on the host cell type, recombinant manipulation method, and culture conditions (Schilling et al. , 2002) when obtaining proteins or their natural variants using genetic engineering methods in industry. That is, quantitative differences in glycan structure or the sugar components that make up the glycan arise due to differences in production conditions during the protein production process. Culture conditions affecting N-glycosylation include glucose or glutamine concentration in the culture medium (Tachibana et al. 1994), dissolved oxygen (DO) concentration (Restelli et al. 2006), pH of the culture medium (Borys et al. 1993) , ammonia concentration in the culture medium (Borys et al., 1994;), culture temperature (Clark et al., 2004), etc.

In general, an enzyme specifically binds to a substrate to form an enzyme-substrate complex, which acts as a catalyst, lowering the activation energy of the reaction and accelerating the enzyme reaction. The enzyme can distinguish between a substrate and molecules that compete with the substrate, and is able to specifically bind to the substrate due to the distribution of complementary charges at the position at which the enzyme binds to the substrate, complementary structure, and the distribution of hydrophilicity and hydrophobicity. The lock-and-key model, in which the enzyme and substrate are complementary to each other and have a geometric shape, has been proposed to elucidate the binding position of the enzyme, but does not provide a satisfactory explanation for the transition state of the enzyme-substrate complex. According to the induced fit model proposed to overcome this problem, as the enzyme and substrate continue to interact with each other in the enzyme-substrate complex, they change its structure based on the flexible structure of the enzyme protein. This reaction process may be further aided by the distribution of surrounding electrical charges across the amino acid residues or N-glycans constituting the active sites, or by the distribution of hydrophilicity/hydrophobicity.

In addition, charge interaction is necessary for the reaction of hyaluronidase, which is an enzyme that hydrolyzes hyaluronic acid as a substrate. Arming et al. showed that the arginine residue, which has positive charges in hyaluronidase PH20, is required for enzymatic activity to bind to hyaluronic acid, which is a substrate with a large number of negative charges distributed within it (Arming et al. 1997). Therefore, it can be concluded that the charge distribution of N-glycan also influences such enzymatic activity. It is important to prove that when hyaluronic acid binds to hyaluronidase, which is a substrate with a large number of negative charges, the level of negatively charged sugars covering sialic acid in N-glycans, that is, the level of sialylation, affects the formation of the enzyme-substrate complex or the course of the enzymatic reaction . To limit the level of sialylation, one should limit the transfer of sialic acid to the galactose residue, perform desialylation, or limit the level of galactosylation.

Thus, the productivity and activity of recombinant hyaluronidase PH20 and its variants is affected by changes in the level of N-glycans, therefore, research to develop a method for producing hyaluronidase PH20 or its variant, which has high activity and productivity, and is applicable in industry, by controlling and maintaining the level of N- glycans are essential for efficient mass production in the art.

Summary of the invention

Thus, the present invention has been made in view of the above problems, and one of the objects of the present invention is to provide a method for culturing host cells that produce recombinant hyaluronidase PH20 or a variant thereof, and hyaluronidase PH20 or a variant thereof obtained by this method, in particular a method obtaining hyaluronidase PH20 or its variant having improved enzymatic activity and productivity.

The objects of the present invention are not limited to those described above. Other purposes not described in this application will be apparent to those skilled in the art from the following description.

In the present invention, when host cells for producing recombinant PH20 hyaluronidase or a variant thereof are cultured under certain culture conditions, the N-glycosylation characteristics of the obtained recombinant PH20 hyaluronidase or a variant thereof and, furthermore, the enzymatic activity of the obtained PH20 hyaluronidase or a variant thereof are significantly improved.

In particular, it has been found that it is possible to effectively increase the activity of the produced hyaluronidase PH20 or variant thereof by controlling the levels of sialylation, galactosylation and/or mannosylation, in particular the level of sialylation in N-glycans, and that the enzymatic activity and productivity of hyaluronidase PH20 or variant thereof the present invention can be noticeably improved by carrying out cultivation for a certain period of time after changing the cultivation temperature.

In particular, the method for producing hyaluronidase PH20 or a variant thereof according to the present invention includes:

(1) culturing host cells expressing recombinant hyaluronidase PH20 or a variant thereof at a culture temperature of 35°C to 38°C to an integral number of viable cells of 20×10 ⁶ -120×10 ⁶ cells per day/ml; And

(2) reducing the culture temperature to 28°C to 34°C and then culturing the host cells for 2 to 18 days while maintaining the culture temperature in accordance with at least one method selected from the group consisting of:

(a) culturing host cells while maintaining a residual glucose concentration in the medium from 0.001 g/L to 4.5 g/L during the culture period; And

(b) culturing the host cells while maintaining the pH of the culture medium between 6.8 and 7.2.

PH20 or a variant thereof produced by the production method according to the present invention is characterized in that the level of N-glycan sialylation of the resulting PH20 or variant thereof is from 1 to 38%, the enzymatic activity is markedly increased, but is not limited to this, and the value is experimental value having an error range of 10%. The reason for this is that variations occur depending on conditions such as the equipment used for culturing and the skill of the analyst in setting the culturing conditions, so the numerical values stated in the present invention should be interpreted in a broader sense taking into account the variations. and not in the narrow sense. Host cells can 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 drawings

The above and other objects, features and other advantages of the present invention will become more apparent from the following detailed description taken in conjunction with the accompanying drawings, in which:

fig. 1 illustrates the result of the analysis of enzymatic activity, the results of isoelectric focusing and determination of the N-glycan content in relation to wild-type human hyaluronidase PH20 and its variants, where:

fig. 1A illustrates the enzymatic activity of wild-type human hyaluronidase PH20 and variants thereof;

fig. 1B illustrates the result of isoelectric focusing of each sample; And

fig. 1C illustrates N-glycan content;

fig. 2 illustrates the result of a comparative analysis of enzymatic activity and isoelectric focusing profile between purified fractions of the PH20 hyaluronidase variant treated with PNGase F (peptide-N-glycosidase F), sialidase A and sialidase A + galactosidase, where:

fig. 2A illustrates the result of isoelectric focusing of each sample;

fig. 2B illustrates enzymatic activity; And

fig. 2C illustrates N-glycan content;

fig. 3 illustrates the result of an analysis of changes in cell growth, cell viability, pH and lactate concentration depending on culture conditions when adding N-acetyl-D-mannosamine or galactose in relation to cells producing PH20 hyaluronidase variants, where:

fig. 3A illustrates the change in cell growth;

fig. 3B illustrates the change in cell viability;

fig. 3C illustrates the change in pH; A

fig. 3D illustrates the change in lactate concentration;

fig. 4 is a graph showing the activity of harvested cell culture fluid to produce hyaluronidase PH20 variants as a function of culture conditions upon addition of N-acetyl-D-mannosamine or galactose;

fig. 5 illustrates the result of isoelectric focusing of the collected cell culture liquid to obtain variants of hyaluronidase PH20 depending on the culture conditions with the addition of N-acetyl-D-mannosamine or galactose;

fig. 6 illustrates the result of analysis of the structure of N-glycans of the collected cell culture liquid to obtain variants of hyaluronidase PH20 depending on the culture conditions with the addition of N-acetyl-D-mannosamine or galactose;

fig. 7 illustrates changes in cell growth, cell viability and cell ammonia concentration for the production of PH20 hyaluronidase variants depending on the number of days of culture, where:

fig. 7A shows the change in cell growth;

fig. 7B shows the change in cell viability;

fig. 7C shows the change in the integrated density of viable cells; And

fig. 7D illustrates the change in ammonia concentration;

fig. 8 is a graph showing the specific activity of the collected fluid from cell culture to produce hyaluronidase PH20 variants as a function of cell culture day;

fig. 9 illustrates the result of isoelectric focusing of the collected cell culture liquid to obtain variants of hyaluronidase PH20 depending on the day of cell culture;

fig. 10 illustrates the results of analysis of the structure and activity of N-glycans of the collected cell culture fluid to obtain variants of hyaluronidase PH20 depending on the day of cell culture;

fig. 11 illustrates changes in cell growth as a function of glucose concentration, cell viability, pH, osmolality, glucose concentration, and lactate concentration conditions relative to cells producing PH20 hyaluronidase variants, where:

fig. 11A illustrates the change in cell growth;

fig. 11B illustrates the change in cell viability;

fig. 11C illustrates the change in pH;

fig. 11D illustrates the change in osmolality;

fig. 11E illustrates the change in glucose concentration; A

fig. 11F illustrates the change in lactate concentration;

fig. 12 is a graph showing the specific activity of collected fluid from a cell culture producing hyaluronidase variants PH20 as a function of glucose concentration;

fig. 13 illustrates the result of isoelectric focusing of the collected liquid from a cell culture producing PH20 hyaluronidase variants, depending on the glucose concentration;

fig. 14 illustrates the result of analysis of the N-glycan structure of the collected cell culture fluid for the production of hyaluronidase PH20 variants depending on the glucose concentration;

fig. 15 illustrates the results of an analysis of changes in cell growth, cell viability, pH and integral density of viable cells depending on pH levels in relation to cells producing PH20 hyaluronidase variants, where:

fig. 15A illustrates the change in cell growth;

fig. 15B illustrates the change in cell viability;

fig. 15C illustrates the change in pH; A

fig. 15D illustrates the change in the integrated density of viable cells;

fig. 16 is a graph showing the specific activity of the collected culture fluid for cells producing PH20 hyaluronidase variants as a function of pH levels;

fig. 17 illustrates the result of isoelectric focusing of the collected culture fluid for cells producing PH20 hyaluronidase variants, depending on the pH level;

fig. 18 is a purification chromatogram obtained by primary anion exchange resin chromatography during purification of the PH20 variant of hyaluronidase;

fig. 19 is a purification chromatogram obtained by secondary anion exchange chromatography during purification of the PH20 hyaluronidase variant; And

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

Detailed Description of the Invention

Unless otherwise specified, all technical and scientific terms used in this application have the same meaning as understood by those skilled in the art to which the present invention relates. In general, the nomenclature used here is well known and commonly used in the art.

The present invention is based on the discovery that the levels of sialylation, galactosylation and/or mannosylation, in particular the level of N-glycan sialylation, are critical for the enzymatic activity and productivity of hyaluronidase PH20 or its variants in the preparation of hyaluronidase PH20 or its variants for industrial use with using the method of genetic engineering.

In particular, it has been found that under conditions where the level of sialylation of N-glycan PH20 or a variant thereof is from 1 to 38%, preferably from 1 to 30%, more preferably from 1.5 to 28%, and most preferably from 2 to 25 %, the enzymatic activity and productivity of hyaluronidase PH20 or its variant are improved to unexpectedly high levels.

More specifically, it has been found that when the galactosylation level of N-glycans of PH20 or its variant is from 1 to 68%, the sialylation level is from 1 to 38%, and the mannosylation level is from 40 to 63%, preferably when the galactosylation level is from 5 to 60%, the sialylation level is from 1 to 30%, and the mannosylation level is from 42 to 62%, more preferably, when the galactosylation level is from 10 to 56%, the sialylation level is from 1.5 to 28%, and the level the mannosylation level is from 44 to 61%, and most preferably the galactosylation level is from 15 to 50%, the sialylation level is from 2 to 25%, and the mannosylation level is from 47 to 60%, enzymatic activity and productivity of hyaluronidase PH20 or a variant thereof rise to unexpectedly high levels.

The above numerical values are obtained from the experimental results of the examples shown in Table 1 and represent the numerical values obtained with a 95% confidence interval, and such numerical values may also include an error of 10%. This is because measurements of sugar (glucose) levels in proteins vary depending on conditions such as the equipment used for the experiment, enzymatic reaction time, test temperature, skill of the analyst, etc., so the glucose level measured in the present invention should be interpreted in a broader sense, taking into account differences between laboratories, and not in a limited sense.

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

In particular, the method for producing hyaluronidase PH20 or a variant thereof with a sialylation level in N-glycan from 1 to 38% of the present invention includes:

(1) culturing host cells expressing recombinant hyaluronidase PH20 or a variant thereof at a culture temperature of 35°C to 38°C to an integrated viable cell density of 20×10 ⁶ to 120×10 ⁶ cells per day/ml; And

(2) reducing the culture temperature to 28°C to 34°C and then culturing the host cells for 2 to 18 days while maintaining the culture temperature in accordance with at least one method selected from the group consisting of:

(a) culturing the host cells while maintaining a residual glucose concentration in the medium from 0.001 g/L to 4.5 g/L during the culture period; And

(b) culturing the host cells while maintaining the pH of the culture medium between 6.8 and 7.2.

The above value is an experimental value with an error of 10%. The reason is that variations occur depending on conditions such as the equipment used for cultivation and the analyst's skill in setting the culture conditions, so the numerical values stated in the present invention should be interpreted more broadly in light of such factors. diversity, not in a narrow sense.

The culture temperature is reduced from stage (1) to stage (2) when the integral density of viable cells reaches from 20×10 ⁶ to 120×10 ⁶ cells per day/ml, preferably from 40×10 ⁶ to 100×10 ⁶ cells per day day/ml, more preferably, from 60×10 ⁶ to 80×10 ⁶ cells per day/ml, but is not limited to this.

Moreover, the culture time period in step (2) may be from 2 to 18 days, preferably from 3 to 16 days, more preferably from 4 to 14 days, but is not limited to it.

For example, if a fed-batch culture method is used to produce PH20 or a variant thereof in accordance with the present invention, the productivity of PH20 or its variant can be maximized, and the enzymatic activity of the resulting PH20 or variant thereof can be significantly improved by cultivating the inoculum immediately before the main culture at a temperature 35°C to 38°C by perfusion culture or fed-batch culture until cell concentration reaches a certain level, and then main culture by inoculation at a reduced temperature below 35°C for 2 to 18 days, preferably 3 up to 16 days, more preferably from 4 to 14 days.

The concentration of the main culture cell inoculum may be, but is not limited to, 1 x 10 ⁵ cells/ml or more, preferably 5 x 10 ⁵ cells/ml or even more preferably 1 x 10 ⁶ cells/ml or more.

By performing cultivation using a combination of fed batch culture and perfusion culture in the production of PH20 or a variant thereof according to the present invention, the productivity of PH20 or a variant thereof can be maximized, and the enzymatic activity of the resulting PH20 or variant thereof can be markedly increased.

In the present invention, the residual glucose concentration in the medium during cultivation is maintained at 0.001 to 4.5 g/L, preferably 0.01 to 4.0 g/L, and more preferably 0.1 to 3.5 g/L , but the present invention is not limited to this.

As used herein, the expression “residual glucose concentration in the medium during cultivation is maintained at a level of 0.001 to 4.5 g/L, preferably 0.01 to 4.0 g/L, and more preferably 0.1 to 3.5.” g/l" means that when the residual glucose concentration in the medium, measured during the culture period at intervals of 1 to 36 hours, preferably 3 to 30 hours, more preferably 6 to 24 hours or in real time, is below the established control concentration of 0.001 g/l to 4.5 g/l, preferably from 0.01 to 4.0 g/l and more preferably from 0.1 to 3.5 g/l, a glucose stock solution is added to the medium to obtain the corresponding reference concentrations during cultivation.

In the present invention, it will be understood by those skilled in the art that the reference concentration of residual glucose concentration in the medium can be set within the range of 0.001 g/L to 4.5 g/L, preferably 0.01 to 4.0 g/L , and more preferably from 0.1 to 3.5 g/L, and the reference concentration may be changed accordingly during the culture period. For example, the reference concentration of residual glucose in the medium is 2 g/L on days 1 and 2 of culture, the reference concentration is reduced to 1.5 g/L on days 3-5 of culture, and thereafter the reference concentration is increased to 2 g/l or set below 1.5 g/l, for example 1.0 g/l.

Hyaluronidase PH20 or a variant thereof prepared by the method of the present invention and having specified levels of sialylation, galactosylation and/or mannosylation in the N-glycan moiety has a hyaluronidase enzymatic activity in the culture medium of 10,000 U/ml, preferably 11,000 U/ml or higher, more preferably 12,000 U/ml or higher, but not limited to.

In addition, hyaluronidase PH20 or a variant thereof produced by the method of the present invention and having a predetermined level of sialylation, galactosylation and/or mannosylation in the N-glycan is wild-type human PH20 prepared by a conventional method and has enzymatic activity increased by 10% or more, preferably 12% or more, more preferably 15% or more, compared to the activity of wild-type human PH20 obtained by a conventional method, but the present invention is not limited to this.

Preferably, in the method for producing hyaluronidase PH20 or a variant thereof according to the present invention, culturing host cells in step (1) and/or step (2) can be carried out under one or more conditions selected from the group consisting of: (i) adding ammonia to environment or increasing the concentration of ammonia in the environment to 5 mM or more; (ii) adding to the medium one or more substances selected from the group consisting of glutamine, glucosamine, uridine, glucosamine and sodium butyrate; and (iii) not adding galactose and manNAc to the medium, but the method is not limited to this.

More specifically, in the method for producing hyaluronidase PH20 or a variant thereof according to the present invention, culturing host cells in step (1) and/or step (2) can be carried out by adding ammonia to the medium, increasing the concentration of ammonia to 5 mM or more, adding glutamine, no addition of galactose and manNAc, no addition of uridine or glucosamine, and no addition of sodium butyrate, but the method is not limited to this.

As used herein, the term “hyaluronidase PH20 or variant thereof” refers to an enzyme that breaks down hyaluronic acid found in the extracellular matrix.

The term “hyaluronidase PH20” or “PH20” as used herein is interpreted to include both wild-type PH20 and its mature form, and the term “hyaluronidase PH20 or PH20 variant” means a PH20 variant that includes substitutions, deletions and insertions of one or several amino acid residues, and optionally includes, but is not limited to, deletions of certain N-terminal and/or C-terminal amino acid residues in the amino acid sequence of "hyaluronidase PH20" or "PH20".

The hyaluronidase of the present invention refers to a hyaluronidase obtained from an animal or microorganism such as Streptomyces and having hyaluronidase activity, among which "hyaluronidase PH20" or "PH20" is obtained from an animal or microorganism such as Streptomyces, and preferably obtained from humans, large cattle or sheep.

"Hyaluronidase PH20" or "PH20 variant" of human origin in accordance with the present invention is illustrated in, but is not limited to, International Patent Publication No. 2020/022791 and US Patent No. 9,447,401, and is interpreted to include any hyaluronidase or variant that contains substitutions, deletions and insertions of one or more amino acid residues, optionally comprising truncations of one or more N-terminal and/or C-terminal amino acid residues in the amino acid sequence of "hyaluronidase PH20" or "PH20", and has the enzymatic activity of hyaluronidase.

Examples of host cells used in the present application for the expression of hyaluronidase proteins may include, but are not limited to, animal, yeast, actinomycete and insect cells, and the like.

The animal cells are preferably mammalian cells, more preferably commonly used animal cells for culture, such as CHO cells, HEK cells, COS cells, 3T3 cells, myeloma cells, BHK cells, HeLa cells and Vero cells, and particularly preferably CHO cells for bulk expression. In addition, to obtain the necessary proteins, cells suitable for introducing the necessary genes are particularly preferably used, such as dhfr-CHO cells (Proc. Natl. Acad. Sci. USA (1980) 77, 4216-4220) or CHO K- 1, which are CHO cells with a knockout of the DHFR gene (Proc. Natl. Acad. Sci. USA (1968) 60, 1275), or CHO K-1 cells (Proc. Natl. Acad. Sci. USA (1968) 60, 1275). The CHO cells are particularly preferably DG44, DXB-11, K-1 or CHO-S cell lines, and introduction of the vectors into the host cells is carried out using a method such as the calcium phosphate method, the DEAE-dextran method, electroporation, or lipoprotein method.

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

In the method for producing hyaluronidase PH20 or a variant thereof according to the present invention, culturing host cells in step (1) and/or step (2) can be carried out by one or more methods selected from the group consisting of batch culture, repeated batch culture, batch culture with fed-batch, repeated fed-batch culture, continuous culture, and perfusion culture, but not limited to.

Batch culture is a method of culturing proliferating cells without adding fresh medium or discarding the culture solution during culture. Continuous culture is a culture method with continuous addition and removal of medium during culture. In addition, continuous culture includes perfusion culture. Fed-batch culture is between batch culture and continuous culture, also called "semi-batch culture" and involves the continuous or sequential addition of medium during culture. This culture method prevents cell release even if the culture solution is continuously discharged. In the present invention, any culture method can be used, with fed-batch culture or continuous culture being preferred, and fed-batch culture being particularly preferred.

As described above, in hyaluronidase PH20 or a variant thereof produced by the production method of the present invention, the specific activity of the enzyme can be increased by 10% or more compared with the activity obtained by the conventional method, in particular compared with the specific activity of wild-type human PH20 . This increase in enzymatic activity is due to changes in N-glycosylation properties and/or charge modification of the PH20 hyaluronidase or variant thereof produced by the method of the present invention.

In particular, enzymatic activity increases depending on changes in sialylation, galactosylation and/or mannosylation among N-glycosylation patterns. These N-glycosylation characteristics, in particular sialylation, galactosylation and/or mannosylation, can be controlled by the production method of the present invention.

In the present invention, examples of the covalent bond between glucose and proteins include an N-glycosidic bond in which N-acetyl-D-glucosamine is covalently linked to an aspartic residue constituting a protein (N-glycoside-linked glycan), an O-glycosidic bond in which N-acetyl -D-Galactosamine is covalently linked to a serine or threonine residue (O-glycoside-linked glycan) and the like, but there is no particular limitation on the type of covalent bond between glucose and proteins in the glycoprotein of the present invention, and the glycoprotein including one or both of N-glycoside-linked glycan and O-glycoside-linked glycan are within the scope of the present invention.

In this aspect, the present invention is directed to hyaluronidase PH20 or a variant thereof having a sialylation level in N-glycan content of 1 to 38%, preferably 1 to 30%, more preferably 1.5 to 28%, and most preferably 2%. up to 25%.

More specifically, the present invention is directed to hyaluronidase PH20 or a variant thereof having a galactosylation level of N-glycan content from 1 to 68%, a sialylation level of N-glycan content from 1 to 38%, and a mannosylation level of N-glycan content from 40 to 38%. 63%, preferably a galactosylation level of N-glycan content from 5 to 60%, a sialylation level of N-glycan content from 1 to 30% and a mannosylation level of N-glycan content from 42 to 62%, more preferably a galactosylation level of N content α-glycans from 10 to 56%, sialylation level in N-glycan content from 1.5 to 28% and mannosylation level in N-glycan content from 44 to 61%, and most preferably galactosylation level in N-glycan content from 15 to 50 %, the level of sialylation in the N-glycan content is from 2 to 25%, and the level of mannosylation in the N-glycan content is from 47 to 60%.

The above numerical values are derived from the experimental results of the examples shown in Table 1 and are obtained using a numerical value having a 95% confidence interval, and the numerical values also include an error of 10%. This is because when measuring the glucose content of proteins, there are differences depending on conditions such as the equipment used for the experiment, the enzymatic reaction time, the test temperature, the skill of the analyst, etc., so the measured glucose content in the present invention should be be interpreted in a broader sense, taking into account differences between laboratories, rather than in a limited sense. In addition, the charge modification of PH20 or its variant can be identified using isoelectric focusing.

Hyaluronidase PH20 or a variant thereof having a specific level of sialylation and/or galactosylation and mannosylation in the N-glycan content of the present invention is preferably produced by the production method of the present invention, but is not limited to this, but it is obvious that hyaluronidase PH20 or a variant thereof can be produced in other ways, which may be modified by those skilled in the art.

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

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

As used herein, the terms “nutrient medium” and “concentrated nutrient medium” refer to media consisting of a specific nutrient or multiple nutrients, such as amino acids, vitamins, salts, trace elements, lipids and glucose, and may be concentrated products of the basal medium . The components and concentrations of the resulting culture medium may vary depending on the cells being cultured. In addition, commercial culture media such as Cell Boost Series Media (HyClone, USA), EfficientFeed Supplement Media, GlycanTune Media (Gibco, USA), BalanCD CHO Media (Irvine Scientific, USA), Cell Culture Media Cellvento® CHO (Merck, USA), EX-CELL® Advanced CHO culture medium (Sigma-Aldrich, USA) or the like can be used as a culture medium, but the invention is not limited to them.

As used herein, the term "plant hydrolyzate" refers to a product extracted from garden peas, cottonseeds, wheat gluten, soybeans or the like, and free from animal components, which is a supplement containing large quantities of amino acids, peptides, vitamins, carbohydrates, nucleotides, minerals and other ingredients, and can also be produced with different components and concentrations of components depending on the cells being cultured. In addition, commercial hydrolysates of plant origin such as HyPep™ 7404, UltraPep™ Cotton, HyPep™ 7504, HyPep™ 4601N (Kerry, USA), Cotton 100, Cotton 200, Phytone™ and Soy 100 (Gibco, USA) can be used as an additive, but the invention is not limited to this.

The additive used to increase or decrease the N-glycan content of the glycoprotein used in the present invention is typically a component known to be involved in protein glycosylation. In particular, to limit the level of sialylation, one should limit the delivery of sialic acid to the galactose residue, perform desialylation, or limit the level of galactosylation. In cell culture, such additives, which are added to the medium at specified concentrations, include components as glycosylation precursors such as N-acetyl-D-mannosamine, glucose, mannose, glutamine and galactose, ammonia and butyric acid.

Most animal cell cultures primarily use medium containing serum. However, it is difficult to create a medium suitable for protein production because the whey-containing medium is a complex composition, the chemical components of which are not fully defined. Serum-free media or media containing a small amount of serum are generally used because serum can have negative effects on separation and purification, as well as cost and reproducibility issues. Since the concentration of glucose, which is a carbon source, is very low in the serum-free medium, glucose is further added as the main carbon source to the culture medium to support cell growth and obtain a high concentration of the target protein, and glutamine may be further added to the culture medium. In particular, the level of sialylation must be increased to increase the in vivo half-life in the production of protein pharmaceuticals. To do this, glucose and glutamine in the medium must be maintained at certain concentrations or higher so that they are not depleted, and the pH of the culture medium must also be maintained at a certain level. The concentrations of glucose and glutamine measured in the culture medium refer to the concentrations of residual glucose and glutamine after consumption by the cells. In addition, promoters can be used to increase the activity of enzymes associated with increased sialylation levels, or inhibitors to suppress desialylation. In addition, by adding N-glycan precursors or by controlling the promoters of related enzyme activity or culture conditions, the level of galactosylation can be increased and thus the level of sialylation can also be increased.

However, the level of N-glycans cannot be controlled by the above-described method in the production of hyaluronidase PH20 or a variant thereof, whereas by using the method of the present invention, hyaluronidase PH20 or a variant thereof having the required glycan levels and high enzymatic activity can be obtained.

The production method of the present invention may further include isolating and purifying the resulting hyaluronidase PH20 or a variant thereof.

Isolation and purification of hyaluronidase PH20 or a variant thereof according to the present invention is preferably carried out using not affinity binding, but the ionic bonding and/or hydrophobic interaction characteristics of hyaluronidase PH20 or a variant thereof, but the invention is not limited to this.

In particular, the separation and purification of hyaluronidase PH20 or a variant thereof according to the present invention is preferably carried out using hydrophobic interaction chromatography and ion exchange chromatography such as cation exchange chromatography and/or anion exchange chromatography rather than affinity chromatography, but the invention is not limited thereto.

Moreover, the isolation and purification of PH20 hyaluronidase or a variant thereof according to the present invention is aimed at removing the PH20 acid hyaluronidase or a variant thereof having low enzymatic activity, and the removal of the PH20 acid hyaluronidase or a variant thereof is preferably carried out using ion exchange chromatography.

It is necessary to analyze the catalytic reaction rate of the enzyme to determine the industrial applicability of the enzyme. An enzymatic reaction can be classified as an enzymatic reaction having an active site with a fixed reactivity and an enzymatic reaction having multiple active sites having different reactivity. It is known that the catalytic reaction rate of a single active site enzyme with a fixed reactivity, such as hyaluronidase, follows the Michaelis-Menten kinetic equation.

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

For an enzymatic reaction, it is assumed that the process of interaction of the enzyme with the substrate to form the ES complex quickly reaches equilibrium or a quasi-steady state, provided that is satisfied by sufficiently reducing the enzyme concentration during the reaction to maintain a sufficiently high substrate concentration. Because kinetics formulas that assume fast equilibrium or quasi-steady state are derived in the same way, most experiments assume a quasi-steady state in which the substrate concentration is initially higher than the enzyme concentration.

Under conditions such as "the amount of enzyme is constant before and after the reaction" and based on the assumption "when a chemical reaction reaches chemical equilibrium, the rate of reaction at which the product is formed is equal to the rate at which the product decomposes again", the reaction rate of the final product can be expressed by the following Michaelis-Menten kinetics equation. In this case, K _M = (k _r + k _cat ) / k _f , and V _max = k _cat [E] ₀ .

The Lineweaver-Burk equation is used to experimentally analyze the rate of an enzymatic reaction using the Michaelis-Menten kinetic equation. This equation shows the relationship between the reciprocal 1/V of an experimentally measured reaction rate and the reciprocal 1/[S] of a given substrate concentration in the experiment. Statistical verification that this equation is a linear equation shows that the enzymatic reaction is a reaction following the Michaelis-Menten kinetic equation, and K _M and V _max can be calculated using this equation.

Enzymes that catalyze a chemical reaction have a transition state after binding to the substrate at the active site, and the activation energy to reach the high energy transition state is reduced due to multiple bonds with the substrate. The equilibrium constant to achieve this transition state is proportional to k _cat /K _M . Here, 1/K _M is a measure that combines the degree to which an enzyme-substrate complex is formed by enzyme-substrate binding with the degree to which the enzyme-substrate complex is maintained without degradation, and k _cat is the equilibrium constant at which a product is formed from the enzyme-substrate complex. Therefore, we can say that k _cat /K _M is an indicator of how much product can be obtained from the substrate and the enzyme, that is, an indicator of the catalytic efficiency of the enzyme.

The industrial suitability of hyaluronidase is proportional to its enzymatic catalytic efficiency. In particular, when the enzyme is administered subcutaneously together with a polymeric pharmacologically active substance, such as a monoclonal antibody, the enzymatic catalytic efficiency of hyaluronidase plays an important role. In the case where the variant of the present invention has a higher k _cat /K _M value than the wild type PH20, when hyaluronidase contained in the polymeric pharmacologically active substance is administered subcutaneously, the hyaluronic acid present at the injection site is quickly decomposed, and thus it is possible to obtain superior effect of a rapidly dispersing pharmacologically active substance. In addition, when the variant of the present invention has a higher k _cat value than the wild type PH20, the maximum reaction rate V _max increases at the same enzyme concentration, thereby providing superior effects of decomposing more hyaluronic acid in the same period of time and dispersing pharmacologically active substance in a wider area.

Thus, to determine the enzymatic properties of the PH20 variant of the present invention, the enzymatic reaction rate of each variant, and V _max (maximum enzymatic reaction rate), K _m (substrate concentration under 50% V _max conditions), k _cat (substrate conversion rate) and k _cat /K _m (enzyme catalyst efficiency) was compared in Example 4. The results demonstrate that the PH20 variant of the present invention is superior to the wild type PH20.

Examples

Hereinafter, the present invention will be described in more detail with reference to examples. However, it will be apparent to those skilled in the art that these examples are presented only to illustrate the present invention and should not be construed as limiting the scope of the present invention.

Example 1. Relationship between hyaluronidase activity, N-glycan and isoelectric focusing behavior

The results of the analysis of the activity, isoelectric focusing pattern, and N-glycan levels of wild-type human hyaluronidase PH20 and its variant HM46 are shown in FIG. 1. Although the difference in the number of active units of the two hyaluronidases was more than twofold, there was not much difference in the range of isoelectric points or the level of N-glycans.

An experiment was conducted to identify the relationship between N-glycan, isoelectric focusing pattern, and PH20 hyaluronidase activity. During the purification process of the PH20 hyaluronidase variant, the basic fraction (fraction 1) could be separated from the acidic fraction (fraction 2), and the corresponding fractions were used as samples. Fig. 2 presents the results of analysis of the isoelectric focusing pattern and enzymatic activity of a sample treated with PNGase F to remove all N-glycans, a sample treated with sialidase A to remove terminal sialic acid, and a sample treated with sialidase A and galactosidase to remove terminal sialic acid and galactose. The two fractions differed in isoelectric point range, and the acidic fraction exhibited reduced enzymatic activity. After treatment with PNGase F, the two fractions showed no activity and had similar isoelectric focusing patterns. These results indicated that N-glycan levels were closely related to enzymatic activity. In addition, it was found that there is a relationship between terminal sialic acid content and enzymatic activity through the phenomenon that the isoelectric focusing pattern or enzymatic activity of the acidic fraction is similar to that of the basic fraction when removing terminal sialic acid. However, the basic fraction with low terminal sialic acid content exhibited improved hyaluronidase enzymatic activity compared to the acidic fraction with high sialic acid content.

The relationship between enzymatic activity and N-glycan content was further demonstrated based on the results of evaluating wild-type hyaluronidases and various hyaluronidase variants derived from different cell culture sources.

Table 1

Type of hyaluronidase Receipt Relative activity N-glycan content Galactosylation (%) Sialili-
roving
(%) Mannosylation
(%) Human wild type PH20 Temporary expression culture 100% 32.9 15.2 57.2 Human wild type PH20 Cell line culture 100% 42.5 21.7 54.8 NM46 Temporary expression culture 167% 37.1 7.4 48.1 NM46 Clonal cell line, experimental culture No. 1 115% 46.9 18.6 48.9 NM46 Clonal cell line, experimental culture No. 2, purified fraction No. 1 161% 40.6 16.4 54.4 NM46 Clonal cell line, experimental culture No. 1, purified fraction No. 2 86% 42.1 28 56.8 NM46 Experimental culture No. 1 cell line 158% 49.6 14.4 48.1 NM46 Experimental culture No. 2 cell line 186% 31.2 5.5 48.3 NM46 Experimental culture No. 3 cell line 152% 30.6 4 48.7 NM46 Experimental culture No. 4 cell line 174% 15.8 4.5 52.8 NM46 Cell line culture No. 1 170% 14.8 2.8 49.9 NM46 Cell line culture No. 2 161% 16.4 2.5 47.9 NM46 Cell line culture No. 3 162% 17.9 4.4 49.1 NM46 Cell line culture No. 4 158% 18.5 3.8 48.1 Sheep PH20 Temporary expression culture 193% 35.5 7.9 58.3 PH20 bonobo Temporary expression culture 72% 38.7 18.8 55.5

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

Percent galactosylation (%): the sum of the percentage (%) of N-glycans containing terminal galactose, such as G1, G1F, G1F', G2, G2F, A1, A1F, A2 and A2F.

Percentage of sialylation (%): the sum of the percentage (%) of N-glycans containing terminal sialic acid, such as A1, A1F, A2, and A2F.

Percentage of Mannosylation (%): Sum of the percentage (%) of N-glycans containing terminal mannose, such as M4G0F, M5, M5G0, M6, M7, M8 and M9.

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

Samples sheep PH20 and bonobo PH20 were prepared in the manner shown in Example 10 and the sequences are shown in Table 3.

As shown in Table 1, different types of PH20 hyaluronidase and variant proteins were prepared, and N-glycan contents were examined. For this purpose, a variety of cultures have been used using different cell sources, such as a transient expression culture obtained by transfecting ExpiCHO cells with recombinant genes using the ExpiFectamine CHO reagent (Gibco, USA) each time the culture is performed, a clonal cell line culture using clones of the cell line , obtained using recombinant genes, and cell line culture using screened monoclones with high productivity. As used herein, a clonal cell line culture is a culture using highly productive clones of cell lines selected using selectable markers from mammalian cell clones transfected with recombinant genes, produced using a polyclonal cell line, and aimed at producing monoclonal cell lines. Cell lines include a research cell bank (RCB), a production master cell bank (MCB), and a working cell bank (WCB). Additionally, in the culture method, different test culture combinations were tried to optimize production and even here it was found that there was a difference in activity and N-glycan.

Experimental culture of clonal cell line No. 1 is a 2-liter batch culture, which is grown at 37°C without changing the culture temperature using HycellCHO medium (HyClone, USA). Clonal cell line test #2, purified fraction #1, and clonal cell line test #2, purified fraction #2, were fractions separated and purified by anion exchange chromatography from the collected cell culture fluid cultured at 37°C using fed-batch method in EX-CELL® Advanced CHO fed-batch culture medium (Sigma-Aldrich, USA) in an 8-liter batch with Cotton 100UF (Gibco) followed by BalanCD® CHO Feed 4 (Irvine Scientific, USA). Purified fraction No. 1 is a low-salt elution fraction, and purified fraction No. 2 is a high-salt elution fraction. Test culture of cell line No. 1 was obtained by culturing at a culture temperature of 37°C using the fed-batch method using EX-CELL® Advanced CHO feeding medium (Sigma-Aldrich, USA) in a 2-liter batch with Cotton 100UF (Gibco ), and then BalanCD® CHO Feed 4 (Irvine Scientific, USA), followed by a shift in the cultivation temperature to 32°C, when the integral density of viable cells reached a given level. Experimental culture of cell line No. 2 was obtained by culturing at a temperature of 37°C using a fed-batch method using EX-CELL® Advanced CHO feeding medium (Sigma-Aldrich, USA) in a 10-liter batch with Cotton 100UF (Gibco) , and then CD CHO EfficientFeed™ B plus AGT™ (Gibco, USA), followed by a shift in cultivation temperature to 32°C, when the integral density of viable cells reached a given level, followed by cultivation for 12 days. An experimental culture of cell line No. 3 was obtained by culturing at 37°C using a fed-batch method using EX-CELL® Advanced CHO feeding medium (Sigma-Aldrich, USA) in a 50-liter batch with Cotton 100UF (Gibco, USA), and then CD CHO EfficientFeed™ B plus AGT™ (Gibco, USA); followed by a shift in the cultivation temperature to 32°C, when the integral density of viable cells reached a given level. Experimental culture of cell line No. 4 was obtained by culturing cells at 37°C using a fed-batch method using EX-CELL® Advanced CHO feeding medium (Sigma-Aldrich, USA) in a 10 liter batch with Cotton 100UF (Gibco , USA), and then CD CHO EfficientFeed™B plus AGT™ (Gibco, USA), followed by a shift in cultivation temperature to 32°C upon reaching a given level of integral density of viable cells and cell cultivation for 13 days. Cell line culture No. 1, cell line culture No. 2, cell line culture No. 3, and cell line culture No. 4 were obtained by culturing a research cell bank (RCB), a production master cell bank (MCB), and a working cell bank. (WCB) at a culture temperature of 37°C using a fed-batch method using EX-CELL® Advanced CHO feeding medium (Sigma-Aldrich, USA) in a 200 liter batch with Cotton 100UF (Gibco, USA) followed by CD CHO EfficientFeed™ B plus AGT™ (Gibco, USA), followed by a shift in cultivation temperature to 32°C when the specified value of the integral density of viable cells is reached, and cell cultivation for 12 days. Based on these conditions, experiments were carried out under the culture conditions of Examples 2-5 to establish a culture method for maintaining N-glycan levels.

As can be seen from Table 1, wild-type human hyaluronidase PH20 and its variant, as well as mammalian hyaluronidase PH20, should have a galactosylation level of 15 to 50%, a sialylation level of 2.5 to 28%, and a mannosylation level of 47 to 60% in N- glycans, and must have a galactosylation level of 1 to 68%, a sialylation level of 1 to 38%, and a mannosylation level of 40 to 63% in N-glycan when applying a 95% confidence interval to the test results, to provide industrially useful hyaluronidase activity. Such numerical values may also include a margin of error of 10% since measurements of glucose levels in proteins are subject to variations depending on conditions such as the equipment used for the experiment, enzymatic reaction time, test temperature, skill of the analyst, etc., so the level glucose measured in the present invention should be interpreted in a broader sense, taking into account differences between laboratories, and not in a limited sense.

Moreover, in particular taking into account the relationship between activity and sialylation, it can be seen that hyaluronidase having an activity above that of wild-type human PH20 and thus suitable for industrial use can only be obtained when sialylation is limited to approximately 30% or less. The above numerical value may be obtained from experimentation and may include an error of 10%.

High-quality hyaluronidase supporting N-glycan levels was identified by transient expression culture, and cell lines were established by selecting high-yield monoclonal strains for consistent commercial production. All experimental cultures of cell lines No. 2, No. 3, No. 4 and cultures of cell lines No. 1, No. 2, No. 3, No. 4, presented in Table 1, which were obtained by cultivation methods using the results of Examples 2, 3, 4 and 5, exhibited an enzyme expression level of 10,000 units/mL or more, indicating the possibility of producing high-quality hyaluronidase with high efficiency and low cost.

Example 2: Additive Dependent Culture

Hyaluronidase variant PH20 was inoculated at a concentration of 2×10 ⁶ cells/ml into each of three 125 ml Erlenmeyer flasks containing fed-batch culture medium EX-CELL® Advanced CHO (Sigma-Aldrich, USA) with or without Cotton 200UF. (Gibco, USA) and then 20 mM N-acetyl-D-mannosamine (NZP, Netherlands) or 50 mM galactose (Pfanstiehl, USA), batch culture was carried out in an incubator at 37°C and 8% CO ₂ , and then Fed-batch culture at a reduced temperature of 32°C when the integral viable cell density (IVCD) has reached the shear range. CD CHO EfficientFeed™ B nutrient medium plus AGT™ medium (Gibco, USA) was supplied daily in an amount of 1.88% of the initial culture volume in the flask. Cell samples were collected daily from culture fluids and viable cell density, cell viability, pH, and lactate levels were measured. After cessation of cultivation, centrifugation was carried out at 2000 rpm for 10 minutes to obtain the culture supernatant. The activity of the sample cultured under the above conditions was determined by HPLC and turbidity analysis, and the protein characteristics and N-glycan levels were observed by isoelectric focusing and glycosylation analysis. Medium supplemented with 50 mM galactose showed a 24% increase in galactosylation and a 2% decrease in activity compared to medium without the addition of 50 mM galactose, and medium supplemented with 20 mM N-acetyl-D-mannosamine showed a 35% increase in sialylation and a decrease in activity at 20% compared to the medium without the addition of 20 mM N-acetyl-D-mannosamine (Fig. 3, 4, 5 and 6).

Example 3. Culture depending on the cultivation period

Cells overexpressing the PH20 variant of hyaluronidase were inoculated at a rate of 2×10 ⁶ cells/ml into EX-CELL® Advanced CHO Fed-Batch medium (Sigma-Aldrich, USA) in a 200 L Sartorius bioreactor. On the second day of cultivation, batch cultivation was carried out using Cotton 200UF (Gibco, USA) and CD CHO EfficientFeed™ B plus AGT™ medium (Gibco, USA), which is a concentrated nutrient medium (Gibco, USA), as a feeding medium, at pH 7.2±0.4 and DO 40% at 47 rpm. The initial culture was carried out at 37°C, the temperature was shifted to 32°C when the integral density of viable cells reached the range of variation, and fed-batch culture was carried out. CD CHO EfficientFeed ^TM B nutrient medium plus AGT ^TM medium (Gibco, USA) was supplied daily in an amount of 1.88% of the initial culture volume in a 200-liter bioreactor. Regarding the culture period, culture was stopped on day 19 when cell viability dropped to 40% or less. Various cell samples were collected daily from the culture fluids, viable cell density, cell viability, integrated viable cell density were determined, ammonium ion levels were measured, and the culture solution was collected using a depth filter. The activity of samples cultured under the above conditions was determined by HPLC and turbidity analysis, and protein characteristics and N-glycan levels were observed by isoelectric focusing and glycosylation analysis. As the number of days of culture increased, the levels of galactosylation and sialylation decreased and the activity increased (Figs. 7, 8, 9 and 10).

Example 4. Culture at controlled glucose concentration in the nutrient medium

Cells overexpressing the PH20 variant of hyaluronidase were inoculated at 2×10 ⁶ cells/ml into EX-CELL® Advanced CHO Fed-Batch medium (Sigma-Aldrich, USA) in a 2-liter Sartorius bioreactor. On the second day of cultivation, fed-batch cultivation was carried out using Cotton 200UF (Gibco, USA) and CD CHO EfficientFeed™ B plus AGT™ medium (Gibco, USA), which is a concentrated nutrient medium (Gibco, USA), at pH 7, 2±0.4 and DO 40%, with a speed of 120 rpm. The initial culture was carried out at 37°C, the temperature was shifted to 32°C when the integrated viable cell density reached the range of variation, and then fed-batch culture was carried out. CD CHO EfficientFeed™ B nutrient medium plus AGT™ medium (Gibco, USA) was fed daily in an amount of 1.88% of the original culture volume into a 2-liter bioreactor. Various cell samples were collected daily from the culture fluids, and viable cell density, cell viability, pH, osmolality, and glucose and lactate concentrations were measured.

The concentration of glucose in the culture medium was measured daily and monitored from the time point when the concentration of glucose contained in the culture supernatant did not exceed the standard concentration of 2, 4 or 6 g/L, as glucose is consumed by growing cells. When the measured glucose concentration was 2, 4, or 6 g/L or less, which are the corresponding standard concentrations, a 200 g/L glucose stock solution was added over a maximum time of 3 hours in an amount to achieve the standard concentration. When the measured glucose concentration was 2, 4, or 6 g/L or more, no stock solution was added to maintain each standard concentration. Overall, the effect of changes in glucose on cell growth over 3 hours in mammalian cell culture was negligible.

In this case, the condition of maintaining 2 g/L as the standard concentration was called 1 g/L (±1 g/L) concentration condition, the condition of maintaining 4 g/L as the standard concentration was called 3 g/L (±1 g/L) concentration condition l), and the condition of maintaining 6 g/L as the standard concentration was called the 5 g/L concentration condition (±1 g/L). For example, the condition of maintaining the glucose concentration at 1 g/L (±1 g/L) means that the lower limit of the glucose control range is set at 0 g/L, and the upper limit of the control range is set at 2 g/L and means , that in real culture, when the measured glucose concentration in the culture supernatant reaches the lower limit of 0 g/L, an additional 200 g/L glucose stock solution is added so that the maximum glucose concentration reaches 2 g/L, and when the glucose concentration in the culture supernatant reaches the upper limit of 2 g/L, do not add a 200 g/L glucose stock solution. Therefore, in two concentration conditions, namely 1 g/L concentration (±1 g/L) and 3 g/L concentration (±1 g/L), the 2 g/L concentration condition corresponds to the upper limit with respect to the 1 concentration condition g/L (±1 g/L) and corresponds to the lower limit with respect to the concentration condition of 3 g/L (±1 g/L), so these two conditions are conditions under which completely different actions occur, and therefore they are considered not overlapping each other.

After cessation of cultivation, the culture supernatant was obtained by centrifugation at 4°C and 10,000 rpm for 60 minutes. The activity of the sample cultured under the above conditions was determined by HPLC and turbidity analysis, and the protein characteristics and N-glycan levels were determined by isoelectric focusing and glycosylation analysis. The results showed that as the glucose concentration in the culture supernatant increased, the levels of galactosylation and sialylation increased and the activity decreased (Figs. 11, 12, 13 and 14).

Example 5 pH Controlled Culture

Cells overexpressing the PH20 variant of hyaluronidase were inoculated at 2×10 ⁶ cells/ml in EX-CELL® Advanced CHO Fed-Batch medium (Sigma-Aldrich, USA) in a 2 liter Sartorius bioreactor. On the second day of cultivation, fed-batch cultivation was carried out using Cotton 200UF (Gibco, USA) and CD CHO EfficientFeed™ B plus AGT™ (Gibco, USA), which is a concentrated nutrient medium (Gibco, USA), as feed. at DO 40% at 120 rpm. The initial culture was carried out at 37°C, the temperature was shifted to 32°C when the integral density of viable cells reached the range of variation, and then batch culture was carried out with feeding. CD CHO EfficientFeed™ B plus AGT™ nutrient medium (Gibco, USA) was fed daily in an amount of 1.88% of the initial culture volume into a 2-liter bioreactor. The temperature of the culture was changed, the culture was divided and further cultured according to four pH conditions, namely pH 6.8±0.1, pH 7.0±0.1, pH 7.2±0.1 and pH 7.4± 0.1 to find improved conditions compared to the usual pH of 7.2±0.4. The culture pH control range was set according to these four conditions using a conventional bioreactor that includes a pH control function. For example, pH 7.0±0.1 means that the lower limit of the pH control range is pH 6.9 and the upper limit of the pH control range is pH 7.1. In actual culture, when the pH of the culture medium reaches the lower limit of pH 6.9, base is added to increase the pH, and when the pH of the culture medium reaches the upper limit of pH 7.1, carbon dioxide is added to lower the pH. Therefore, in two conditions pH 6.8±0.1 and pH 7.0±0.1, the pH 6.9 condition corresponds to the upper limit at pH 6.8±0.1 and the lower limit at pH 7.0±0, 1. Therefore, the two conditions are considered not to overlap each other. Cultivation was carried out until the day when the cell viability reached 40% or less, several cell samples were taken daily from the culture liquids, and the viable cell density, cell viability, pH, and integrated viable cell density were measured. After cessation of cultivation, the culture supernatant was obtained by centrifugation at 4°C and 10,000 rpm for 60 minutes. The activity of the sample cultured under the above conditions was determined by HPLC and turbidity analysis, and the protein characteristics were determined by isoelectric focusing. The activity varied depending on the pH of the culture medium, and the highest activity and lowest level of sialylation were observed at pH 7.0 ± 0.1, which was an improved condition compared to the normal condition (Table 2, Figs. 15, 16 and 17) .

table 2

Properties of N-glycans pH 7.2±0.4
(usual conditions) pH 6.8±0.1 pH 7.0±0.1 pH 7.2±0.1 pH 7.4±0.1 Galactosylation (%) 39.6 49.4 37.0 42.1 47.6 Sialylation (%) 14.6 12.7 6.3 13.9 20.9 Mannosylation (%) 48.4 43.3 50.5 50.3 49.3 Total afucosylation (%) 53.4 46.2 51.2 56.2 57.2 Afucosylation (%) 7.7 5.9 4.2 8.9 10.7 Relative specific activity (%) 100% 92% 115% 104% 98%

Example 6 Purification of Hyaluronidase Using Animal Cell Culture Supernatant

Step 1: Treatment of culture supernatant with buffer/surfactant exchange

Conditions of the culture solutions were adjusted to the equilibration conditions of the first anion exchange column by monitoring pH and conductivity with UV/DF using a membrane filter with a molecular weight cutoff of 30 kDa. The adjusted solution was treated with a suitable concentration of solvent/surfactant to inactivate the viruses and reacted at room temperature for approximately 60 minutes.

Step 2: Primary anion exchange column chromatography (Q Sepharose Fast Flow).

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

Step 3: Secondary anion exchange column chromatography (Capto Q).

The filtered protein solution was passed through a secondary anion exchange column to remove the acidic variant of hyaluronidase using an anion exchange resin and eluted from the column at high salt concentration. Before loading, the column was equilibrated using Bis-Tris buffer (having a pH of 6.0 and containing no salt). After loading, the column was washed with the same Bis-Tris buffer (primary wash). After the initial wash, the column was washed with the same Bis-Tris buffer, which had a salt concentration of 20 mM, having a higher conductivity than the buffer without salt in the initial wash). After a second wash, the desired protein, hyaluronidase, was eluted using Bis-Tris buffer having the salt concentration as shown in FIG. 19.

Step 4: Cation exchange (Capto MMC) column chromatography

The adjusted protein solution was passed through a cation exchange column to remove the acidic variant of hyaluronidase using a cation exchange resin and eluted from the column at high salt concentration. Before loading, the column was equilibrated using citrate buffer pH 5.5 having a salt concentration of 80 mM. After loading, the column was washed with the same citrate buffer (primary wash). After the initial wash, the column was washed with the appropriate Bis-Tris buffer pH 7.5. After a second wash, the desired protein, hyaluronidase, was eluted using Bis-Tris buffer pH 8.0 having a salt concentration of 400 mM, as shown in FIG. 20.

Step 5: Nanofiltration/Protein Preparation

After the cation exchange column, the protein solution containing the required hyaluronidase was filtered through a 1 μm pore size filter and subjected to nanofiltration. The nanofiltered protein solution was concentrated to a high concentration of 10 mg/mL and adjusted by UV/DF using an 8 kDa MWCO membrane filter to exchange with a pH 7.0 histidine buffer containing 145 mM salt.

Example 7. Analysis of enzymatic activity of hyaluronidase

The enzymatic activities of hyaluronidase PH20 and other hyaluronidases were measured using a turbidimetric assay as follows.

Turbidimetric analysis is a method of measuring the sediment formed when hyaluronic acid is mixed with albumin (BSA) using absorbance. When hyaluronic acid is hydrolyzed by PH20, absorption is reduced when mixed with albumin. In general, this process was carried out as follows. Hyaluronidase PH20 (Sigma) was diluted to 1; 2; 5; 7.5; 10; 15; 20; thirty; 50 or 60 units/ml and placed in appropriate tubes. The purified protein sample was dissolved in enzyme dilution buffer (20 mM Tris HCl, pH 7.0, 77 mM NaCl; 0.01% (w/v) bovine serum albumin) and diluted in each tube at 100, 300, 600 , 1200 or 2400 times. The 3 mg/mL hyaluronic acid solution was diluted 10-fold to 0.3 mg/mL in other tubes to bring the volume of each tube to 180 μL. 60 μl of enzyme was mixed with a diluted solution of hyaluronic acid and the reaction was carried out at 37°C for 45 minutes. After the reaction, 50 μL of 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 unit activity of the sample was obtained using the test results of a standard product for which unit activity is known and the test results of the sample.

Example 8 Hyaluronidase Assay by Isoelectric Focusing

Isoelectric focusing of hyaluronidase was performed using a ready-made gel (pH 3-7, Invitrogen) and a buffer solution for isoelectric focusing. A sample of purified hyaluronidase was applied to the prepared gel, and electrophoresis was performed using an electrophoresis apparatus manufactured by Novex Corporation at 100 V for 1 hour, at 200 V for 1 hour, and at 500 V for 30 minutes. After completion of the analysis, the gel was washed with purified water, the protein was fixed with a 12% solution of trichloroacetic acid, stained with a solution of Coomassie Blue R-250 dye, and decolorized with a solution of acetic acid in methanol to analyze the protein bands developed on the gel.

Example 9. Analysis of the level of N-glycans of hyaluronidase

Hyaluronidase N-glycan levels were analyzed by labeling the N-glycan sample, separating by treating hyaluronidase with PNGase F, 2-AB (2-aminobenzamide), followed by ultra-high performance liquid chromatography using an ACQUITY UPLC Glycan BEH Amide column (Waters ). A sample of purified hyaluronidase was desalted and reacted with PNGase F at 37°C for 16-18 hours to isolate N-glycan. The N-glycan was labeled with 2-AB and then reacted at 65°C for 3 hours, after which excess 2-AB was removed. The labeled N-glycan sample was separated using HPLC with a 72%-20% acetonitrile gradient. The separated sample was detected using a fluorescence detector (FLD) to analyze N-glycan levels. The corresponding separated N-glycans were classified; N-glycans containing terminal galactose (G1, G1F, G1F', G2, G2F, A1, A1F, A2, A2F, etc.) were added to determine the percentage of galactosylation (%); N-glycans containing terminal sialic acid (A1, A1F, A2, A2F, etc.) were added to determine the percentage of sialylation (%); and N-glycans containing terminal mannose (M4G0F, M5, M5G0, M6, M7, M8, M9, etc.) were added to determine the percentage of mannosylation (%).

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

(1) Production of hyaluronidase PH20 genes in bonobos and sheep

To study the activity and level of N-glycans of hyaluronidase PH20 of animal origin from anthropoids, that is, bonobos, and artiodactyls, that is, sheep, hyaluronidase PH20 was obtained as follows. cDNA was synthesized from wild-type genes 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 human growth hormone, human serum albumin, or human Hyal1 signal peptides was used as the signal peptide instead of the native PH20 signal peptide. For protein purification using a HisTrap column, the His tag DNA sequence was placed at the 3′ end of the PH20 cDNA. Each sequence was identified by DNA sequencing. Table 3 shows the sequences of animal-derived hyaluronidases.

Table 3

Hyaluronidase of animal origin Amino acid sequence Sheep PH20 PH20 bonobo

(2) Expression of bonobo and sheep PH20 hyaluronidase

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

(3) Purification of bonobo and sheep hyaluronidase PH20

Recombinant animal-derived C-terminal His-tagged hyaluronidase proteins produced in ExpiCHO cells were purified by two-step column chromatography using the AKTA prime system (GE Healthcare Systems). Bonobo PH20 has a pI of 6 and was therefore treated with Q Sepharose for anion exchange chromatography, and sheep PH20 has a pI of 8 or greater and was therefore subjected to a one-step purification using a Capto S cation exchange chromatography column. Each protein was subjected to two-step purification using a HisTrap HP His-Tag affinity chromatography column.

For protein purification using a Q Sepharose column, buffer A (20 mM sodium phosphate, pH 7.5) and buffer B (20 mM sodium phosphate, pH 7.5; 0.5 M NaCl) were prepared. The protein was bound to a Q Sepharose column, and the column was washed with 5 column volumes of Buffer A to remove nonspecifically bound proteins and then washed with 5 column volumes of Buffer B on a concentration gradient from 0 to 100% to elute the protein.

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 set to the same as buffer A, and the culture medium was filtered through a 0.22 μm pore size membrane. The protein sample was bound to a Capto S column, and the column was washed with 3 column volumes (CV) of buffer A to remove nonspecifically bound proteins. The column was washed with 4 CV of buffer B to elute the protein.

For protein purification using a 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. The protein sample was bound to a HisTrap HP column, and the column was washed with 7 CV of 7% Buffer B to remove nonspecifically bound proteins and then washed with 3 CV of 40% Buffer B to elute the target protein. The column eluate was dialyzed using dialysis buffer (20 mM sodium phosphate, 100 mM NaCl, pH 7.0).

(4) Bonobo and sheep PH-20 hyaluronidase assay.

The activity assay was performed in the same manner as in Example 7, and the N-glycan level assay was performed in the same manner as in Example 9. The results are shown in Table 1.

Example 11. Analysis of enzymatic kinetics of variants depending on the level of N-glycans

To analyze the enzymatic kinetics of the variants of the present invention, enzymatic 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 analyzes red-coloring substances (at 545 nm) resulting from the reaction of reduced terminal N-acetyl-D-glucosamine (GlcNAc), formed by the hydrolysis of hyaluronic acid by hyaluronidase, with para-dimethylaminobenzaldehyde ( DMAB), that is, Ehrlich's reagent. N-acetyl-D-glucosamine (GlcNAc, Sigma), diluted to 0.25; 0.50; 0.75; 1.00 or 1.25 mM in dilution buffer (0.1 M NaPi; 0.1 M NaCl; 1.5 mM 1,4-lactone sugar acid, pH 5.35) was reduced by treatment with tetraborate in each tube, and then DMAB was added to induce a colorimetric reaction. After the reaction, absorbance was measured at 545 nm to generate a standard reaction curve for GlcNAc. Hyaluronic acid as a substrate was diluted to 0.54; 0.65; 0.87; 1.23 or 2.17 μM in dilution buffer in each tube, and hyaluronidase was added to each tube, followed by reaction at 37°C for 5 minutes and heating at 100°C for 5 minutes until the enzymatic reaction was complete. The resulting material was reduced by treatment with tetraborate and DMAB was added to induce a colorimetric reaction. After the reaction, the absorbance was measured at 545 nm and the enzymatic activity was determined using the standard GlcNAc reaction curve given above. Using this method, the enzymatic kinetics of wild type PH20 with SEQ ID NO: 1 and the PH20 variant of the present invention were analyzed. As a result, the Lineweaver-Burk curve was found to be linear, which means that the PH20 variant of the present invention follows the Michaelis-Menten enzyme kinetics formula.

Table 4 presents the results of the analysis of the enzymatic kinetics of fractions No. 1 and No. 2, obtained from an experimental culture of clonal cell line No. 2, among the samples in Table 1 from Example 1. The results showed that when the levels of galactosylation and mannosylation are within the same ranges and the level sialylation is low, the enzymatic activity of recombinant hyaluronidase is increased due to the high catalytic efficiency (k _cat /K _m ) of the enzyme.

The experimental result proves that the activity of an enzyme with the same amino acid structure can be affected by a change in glycosylation, or more precisely, a change in the level of sialylation. Thus, this demonstrates that when attempting to obtain industrially useful hyaluronidase by mass production of wild type PH20 hyaluronidase or its variant using a recombinant method, it is possible to create an enzyme having greater industrial applicability by controlling the level of sialylation.

Table 4

Industrial applicability

The method for producing recombinant hyaluronidase protein PH20 or its variant according to the present invention produces recombinant hyaluronidase protein PH20 or its variant having high productivity and high enzymatic activity, and thus realizes mass production and supply of recombinant hyaluronidase protein PH20 or its variant.

Although specific configurations of the present invention have been described in detail, those skilled in the art will appreciate that this detailed description is presented as preferred embodiments for illustrative purposes only and should not be construed as limiting the scope of the present invention. Thus, the essential scope of the present invention is defined by the appended claims and their equivalents.

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Claims (23)

1. A method for producing recombinant hyaluronidase PH20 or a variant thereof, which contains substitutions, deletions and insertions of one or more amino acid residues and, optionally, contains deletions of some N-terminal and/or C-terminal amino acid residues in the amino acid sequence of hyaluronidase PH20, and having activity hyaluronidase enzyme, including: (1) culturing host cells expressing recombinant hyaluronidase PH20 or a variant thereof at a culture temperature of 35°C to 38°C until the integrated viable cell density of the host cells reaches from 20x10 ⁶ to 120x10 ⁶ cells per day/ml; And (2) reducing the culture temperature to 28-34°C and subsequent cultivation of host cells for 2 to 18 days; while maintaining the culture temperature: (a) wherein the residual glucose concentration in the medium is maintained at a level of from 0.001 g/L to 4.5 g/L during the cultivation period; And (b) wherein the pH of the culture medium is maintained at 6.9~7.1; wherein the level of sialylation of the N-glycan of the resulting PH20 or its variant ranges from 1 to 21.7%, where the proportion of sialylation (%) is the sum of the proportion (%) of N-glycans containing sialic acid at their end, such as A1, A1F, A2 and A2F, wherein the fraction of galactosylation ranges from 1 to 68%, and the fraction of mannosylation ranges from 40 to 63% of the N-glycans in the resulting PH20, respectively, where the fraction of galactosylation (%) is the sum of the fraction (%) of N-glycans containing galactose per at its end, such as G1, G1F, G1F', G2, G2F, A1, A1F, A2 and A2F, and the fraction of mannosylation (%) is the sum of the fraction (%) of N-glycans containing mannose at their terminus, such as M4G0F , M5, M5G0, M6, M7, M8, and M9, and in this case, the activity of hyaluronidase in the culture medium is 10,000 units/ml or more. 2. The method according to claim 1, wherein the galactosylation level is from 1 to 49.6%, the sialylation level is from 1 to 21.7%, and the mannosylation level is from 40 to 58.3% of the N-glycans of the resulting PH20, respectively. 3. The method according to claim 1, where the specified level of galactosylation is from 14.4 to 49.6%, the level of sialylation is from 2.5 to 21.7%, and the level of mannosylation is from 47.6 to 58.3% N -glycans of the resulting PH20. 4. The method of claim 1, wherein the specific activity of the resulting PH20 or variant thereof is at least 10% higher than the specific activity of wild-type human PH20. 5. The method according to claim 1, where the cultivation of host cells at stage (1) and/or stage (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 method according to claim 1, where the cultivation of host cells in stage (1) and/or stage (2) is carried out under one or more conditions selected from the group consisting of: (i) conditions in which the ammonia concentration in the medium is maintained at 5 mM or more; (ii) conditions in which one or more substances selected from the group consisting of glutamine, glucosamine, uridine, glucosamine and sodium butyrate are added to the medium; And (iii) conditions in which galactose and manNAc are not added to the medium. 7. The method of claim 1, wherein the PH20 variant includes replacing one or more amino acid residues and optionally includes truncation of one or more N-terminal and/or C-terminal amino acid residues in the wild-type PH20 amino acid sequence. 8. The method of claim 1, wherein the host cells are animal, yeast, actinomycete or insect cells. 9. The method according to claim 1, further comprising isolating and purifying the resulting PH20 or a variant thereof. 10. The method of claim 9, wherein the isolation and purification of PH20 or a variant thereof is carried out using the ionic bonding and/or hydrophobic interaction characteristics of PH20 or a variant thereof rather than affinity binding. 11. The method of claim 10, wherein the isolation and purification of PH20 or a variant thereof is carried out using hydrophobic interaction chromatography and ion exchange chromatography rather than affinity chromatography. 12. The method according to claim 9, where acidic pH20 or its variant is removed. 13. The method according to claim 12, where the removal of acid hyaluronidase PH20 or its variant is carried out using ion exchange chromatography.