Method for producing hyaluronidase in its complete form

Abstract

This invention relates to a method for producing hyaluronidase in its complete form, and more specifically, to a method for producing hyaluronidase PH20 in its complete form by adjusting the culture temperature or media components of host cells expressing naturally occurring human recombinant hyaluronidase PH20. In this invention, hyaluronidase PH20 in its complete form, rather than cleaved form, was produced with high purity by adjusting the culture temperature, media type, supplemental feed media components, dissolved oxygen concentration, glucose concentration, and pH of host cells expressing naturally occurring human recombinant hyaluronidase PH20. Therefore, since the hyaluronidase PH20 produced by the method of this invention has high inertness, it can be effectively applied in various fields.

Description

Detailed Description of the Invention

[0001] [Technical Field] The present invention relates to a method for producing a complete form of hyaluronidase, and more particularly, to a method for producing a complete form of hyaluronidase PH20 by adjusting the culture temperature or medium components of host cells expressing natural human recombinant hyaluronidase (Hyaluronidase) PH20.

[0002] [Background Art] Hyaluronidase (HAdase; hyaluronidase) is a general term for enzymes that degrade hyaluronic acid (HA). Hyaluronidase is classified into mammalian type hyaluronidase (Mammalian type, EC 3.2.1.35, hyaluronoglucosaminidase), leech type hyaluronidase (Leeches type, EC 3.2.1.36, hyaluronoglucuronidase), and bacterial type hyaluronidase (Bacterial type, EC 4.2.2.1, hyaluronate lyase) according to the mechanism of hydrolyzing hyaluronic acid.

[0003] Mammalian type hyaluronidase is present in the testis, skin, liver, placental fluid, etc. in the human body, and hydrolyzes the β-1,4 glycosidic bond between glucuronic acid and glucosamine, which are components of hyaluronic acid, to produce tetrasaccharides, or hydrolyzes chondroitin, chondroitin-4-sulfate, and chondroitin-6-sulfate, which are components of synovial fluid and cartilage in human joints. In particular, hyaluronidase (PH20) in the testis is attached to the glycosylphosphatidylinositol fixation site (Glycosylphosphatidylinositol, GPI anchor) of the acrosome part of sperm, and is an important enzyme that decomposes the thick outer wall layer outside the egg and causes fertilization.

[0004] The widespread use of hyaluronidase has been comprehensively studied since the 1950s. Its initial use was subcutaneous injection of intravenous fluids, and it is now used in infiltration and block anesthesia to increase the diffusion of local anesthetics and steroids in other orthopedic, ophthalmic, plastic surgery, dental, oral surgery, gynecology, and otolaryngology surgeries. It is also used to disperse fluid accumulations such as hematomas, prevent peritoneal adhesions, prevent gallstone formation, and treat infertility.

[0005] Currently, commercially available hyaluronidases are extracted from the testes of sheep (ovine) or cows (bovine). Examples include Vitrase (ISTA Pharmaceuticals, ovine source) and Amphadase (Amphastar Pharmaceuticals, bovine source). These processed hyaluronidases are dissolved at the appropriate concentration, filled into vials, and freeze-dried to produce the final product. However, commercially available animal-derived hyaluronidases contain foreign proteins, which can potentially cause allergic reactions. Furthermore, their physiological activity decreases over time due to reduced stability, leaving many problems for their application in various fields.

[0006] To address these issues, research was conducted on recombinant hyaluronidase. Recombinant proteins can be expressed in various cell types, including E. coli, yeast, insect cells, and animal cells. In particular, in the case of hyaluronidase, glycation, which occurs during the post-translational modification process, affects its activity. This is because glycans can affect the antigenicity, structural folding, solubility, and stability of glycoproteins. From this perspective, since the post-translational modification process in yeast and insect cells differs from that in mammals, animal cells are suitable among the various types of expression cells, and among animal cells, CHO (Chinese Hamster Ovary) cells, which have ensured safety, are the most suitable.

[0007] The first recombinant hyaluronidase for pH20 is marketed under the trade name Hylenex by Halozyme Therapeutics and has been developed for various applications including subcutaneous injection, vitrectomy, and ophthalmic disorders. However, hyaluronidase still suffers from low yield and stability, and supply is insufficient to meet demand, creating a need for hyaluronidase with improved yield or stability (Patent Documents 1 and 2).

[0008] In conventional technology, a method is known for increasing the production yield of recombinant hyaluronidase by changing the N-glycan content of recombinant hyaluronidase through adjustment of glucose concentration or culture temperature (Patent Document 3). However, this method has the problem that it produces hyaluronidase in a cleaved form rather than in its complete form.

[0009] [Prior art document] [Patent] [Patent Document 1] South Korea, Japanese Patent Publication No. 10-2023-0168902 [Patent Document 2] Korean Patent No. 2528707 [Patent Document 3] South Korea, Japanese Patent Publication No. 10-2022-0018943 [Overview of the prefecture] [Problems the invention aims to solve] Therefore, in order to increase the productivity of complete hyaluronidase rather than cleaved hyaluronidase, the present invention was diligently researched and, as a result, confirmed that complete hyaluronidase PH20 can be produced by adjusting the culture temperature, culture medium components, and supplemental feed medium components of host cells expressing natural human recombinant hyaluronidase PH20, thus completing the present invention.

[0010] Therefore, the object of the present invention is to provide a method for producing hyaluronidase PH20 in its complete form by adjusting the culture temperature or culture medium components of host cells expressing naturally occurring human recombinant hyaluronidase PH20.

[0011] [Means for solving the problem] In order to achieve the aforementioned objective, The present invention provides a method for producing hyaluronidase PH20, comprising the steps of (1) culturing host cells expressing natural human recombinant hyaluronidase PH20 at a culture temperature of 33-35°C; and (2) culturing for 7-11 days while maintaining the culture temperature at 31-33°C, wherein the host cells in step (1) and / or step (2) are cultured in CD OptiCHO medium supplemented with Cell Boost 6 feed medium, and the produced hyaluronidase PH20 exists in a complete form of 50-75 kDa size, rather than in a cleaved form.

[0012] In a preferred embodiment of the present invention, the complete form of hyaluronidase PH20 may be 55-65 kDa in size. In another preferred embodiment of the present invention, the activity of hyaluronidase PH20 produced by the above method in the culture medium may be 4,000 units / mL or more.

[0013] In yet another preferred embodiment of the present invention, the host cell culture medium in step (1) and / or step (2) may contain Cell Boost 6 feed medium at a concentration of 1% (v / v) to 20% (v / v), preferably 1% (v / v) to 15% (v / v).

[0014] In another preferred embodiment of the present invention, L-glutamine may be further added to the culture medium of the host cells in step (1) and / or step (2) for further cultivation.

[0015] In one further preferred embodiment of the present invention, the host cell culture medium in step (1) and / or step (2) is not enriched with any component selected from the group consisting of peptone, plant hydrolysates, yeast extract, and yeastolate.

[0016] In one further preferred embodiment of the present invention, the culture in step (2) can be cultured in one or more ways selected from the group consisting of (a) a culture in which the dissolved oxygen content of the culture medium is maintained at 30% to 79%; (b) a culture in which the concentration of residual glucose in the medium is maintained at 1.11 to 5.95 g / L during the culture period; and (c) a culture in which the pH of the culture medium is maintained at 7.0 to 7.4.

[0017] In yet another preferred embodiment of the present invention, the culture of host cells in step (1) and / or step (2) may be 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.

[0018] In yet another preferred embodiment of the present invention, the hyaluronidase PH20 produced by the method described above may be selectively devoid of some of the N-terminal and / or C-terminal amino acid residues.

[0019] In yet another preferred embodiment of the present invention, the host cell may be an animal cell, yeast, actinomycetes, or insect cell.

[0020] In yet another preferred embodiment of the present invention, the method may further include the step of (3) separating and purifying the produced hyaluronidase PH20. In yet another preferred embodiment of the present invention, the separation and purification in step (3) can purify hyaluronidase PH20 to high purity by utilizing affinity binding, ionic bonding properties and / or hydrophobic interaction properties with PH20.

[0021] In another preferred embodiment of the present invention, the separation and purification steps may be performed using affinity chromatography, ion exchange-mixed mode chromatography, and hydrophobic interaction chromatography.

[0022] Furthermore, the present invention provides hyaluronidase PH20 in a complete form of 50-75 kDa size produced by the hyaluronidase PH20 production method described above. [Effects of the invention] In this invention, complete hyaluronidase PH20, rather than cleaved hyaluronidase PH20, was produced in high purity by adjusting the culture temperature, type of culture medium, supplemental feed medium components, dissolved oxygen concentration, glucose concentration, and pH of host cells expressing natural human recombinant hyaluronidase PH20. The complete hyaluronidase PH20 produced by the method of this invention has high inertness and can therefore be effectively applied in various fields. [Brief explanation of the drawing]

[0023] [Figure 1] The data shows the presence or absence of expression of cleaved form (between 37 and 49 kDa of molecular weight standard) or complete form (between 50 and 75 kDa of molecular weight standard) of natural human recombinant hyaluronidase PH20, according to the main culture conditions of the present invention: culture temperature and temperature change date. A shows the analysis results when HyCell medium was used as the basic medium, with culture temperature (34, 37°C → 32, 34, 37°C), temperature change date (days 3, 5, 7, 9), and additional feed (Feed C+, GlycanTuneC+). B shows the analysis results when CD OptiCHO medium was used as the basic medium, with culture temperature (34, 37°C → 32, 34, 37°C), temperature change date (days 3, 5, 7, 9), and additional feed (Feed C+, Cell Boost 6). [Figure 2] Data obtained by confirming the presence or absence of the cleaved or full form expression of native human recombinant hyaluronidase PH20 by Western blot according to the type of culture additive and feed content. A shows the analysis results according to the Cell Boost 6 content (3.75, 5, 7.5, 15 mM) and addition days (0, 3, 6, 9 days) in the presence of L-glutamine additive, and B shows the analysis results according to the Cell Boost 6 content (3.75, 5, 7.5, 15 mM) and addition days (0, 3, 6, 9 days) in the presence of GlutaMAX (4 mM) additive. [Figure 3] Western blot data obtained by confirming the presence or absence of the cleaved or full form expression of native human recombinant hyaluronidase PH20 according to the addition content (3, 9%) and type of peptone (yeast extract, select phytone, TC yeast rate, Cotton200) when peptone is added as a culture additive. [Figure 4] Western blot data obtained by confirming the presence or absence of the cleaved or full form expression of native human recombinant hyaluronidase PH20 according to the basic culture conditions and culture days of the present invention. The basic culture conditions are [CD OptiCHO + Cell Boost 6 (15%)] and [CD OptiCHO + Cell Boost 6 (15%) + TC Yeastolate (9%)], and the culture days are set to 12, 14, 16, 18, and 20 days. [Figure 5] Western blot data obtained by confirming the presence or absence of the cleaved or full form expression of native human recombinant hyaluronidase PH20 due to differences in (A) culture days, (B) culture agitation speed, and (C) medium dosage form of the final culture conditions. [Figure 6] Purification chromatogram of native human recombinant hyaluronidase PH20 by primary ion exchange mixed mode chromatography according to the purification flow rate. [Figure 7] Purification chromatogram of native human recombinant hyaluronidase PH20 by secondary affinity chromatography according to the purification flow rate. [Figure 8]This is a chromatogram of purified natural human recombinant hyaluronidase PH20 obtained by tertiary ion exchange mixed-mode chromatography at a specified flow rate. [Figure 9] This is a purified chromatogram of naturally occurring human recombinant hyaluronidase PH20 obtained by fourth-order mixed-mode chromatography at a specified flow rate. [Figure 10] This is a purified chromatogram of natural human recombinant hyaluronidase PH20 obtained by fifth-order hydrophobic interaction chromatography using a specific purification flow rate. [Figure 11] This is the result of a Western blot analysis of the natural human recombinant hyaluronidase PH20 product obtained after the final purification process. [Figure 12] This is the result of SDS-PAGE analysis of the naturally occurring human recombinant hyaluronidase PH20 product obtained after the final purification process. [Figure 13] This is the result of size exclusion chromatography analysis of the naturally occurring human recombinant hyaluronidase PH20 product obtained after the final purification process. [Modes for carrying out the invention]

[0024] [Best mode for carrying out the invention] The present invention will be described in detail below. In one embodiment, the present invention includes the steps of (1) culturing host cells expressing natural human recombinant hyaluronidase PH20 at a culture temperature of 33-35°C; and (2) A method for producing hyaluronidase PH20, comprising the step of culturing for 7 to 11 days while maintaining the culture temperature at 31 to 33°C; The host cells in step (1) and / or step (2) are cultured in CD OptiCHO medium supplemented with Cell Boost 6 feed medium. The present invention relates to a method for producing hyaluronidase PH20 in its complete form, characterized in that the produced hyaluronidase PH20 exists in a complete form of 50-75 kDa size, rather than in a cleaved form.

[0025] In the present invention, the culture in step (1) can be carried out until the cumulative viable cell density reaches 0.3 × 10⁶ to 13.1 × 10⁶ cells × days / mL.

[0026] In the present invention, the complete form of hyaluronidase PH20 is 50-75 kDa in size, preferably 58-65 kDa, more preferably 55-63 kDa (about 60 kDa). The production of complete form of hyaluronidase PH20 is 90% or more, preferably 95% or more, and more preferably 97% or more, characterized in that cleaved form (37-49 kDa) of hyaluronidase PH20 is substantially not observed.

[0027] In the present invention, the CD OptiCHO medium (manufactured by Gibco, USA) is composed of additives (glucose, sodium pyruvate, sodium bicarbonate) and inorganic salts (magnesium, sodium phosphate), and Cell Boost 6 (manufactured by HyClone, USA) is composed of lipids, amino acids, vitamins, growth factors, etc. Cell Boost 6 feed medium can be added to the CD OptiCHO culture medium at a concentration of 1% (v / v) to 20% (v / v), preferably 1% (v / v) to 15% (v / v), and cultured.

[0028] The "feed medium" used in this invention is a medium composed of specific nutrients or multiple nutrients such as amino acids, vitamins, salts, trace elements, lipids, and glucose, and may be a concentrated product of a basic medium. Depending on the cells to be cultured, the components and concentrations of the feed medium can be prepared and used in various ways. In this invention, Cell Boost series supplement medium (HyClone, USA) was preferably used as the feed medium.

[0029] In the present invention, L-glutamine can be further added to the culture medium of the host cells in step (1) and / or step (2) and cultured, and glutamine can be added to the CD OptiCHO medium at a concentration of 2 to 6 mM, more preferably 3 to 5 mM.

[0030] In one specific embodiment of the present invention, we attempted to establish optimal conditions for the production of complete hyaluronidase PH20, depending on the type of commercial culture medium and culture temperature. As a result, under CD OptiCHO medium conditions, when the initial culture temperature was set to 34°C and Cell Boost 6 was added as an additional feed, almost no cleaved form of PH20 was observed, regardless of the day the culture temperature was changed (Figure 1). Furthermore, no significant difference was observed in the degree of expression of cleaved form of PH20 with the addition of L-glutamine (Figure 2).

[0031] In the present invention, it can be characterized in that no component selected from the group consisting of peptone, plant-derived hydrolysates, yeast extract, and yeastolate is added to the host cell culture medium in step (1) and / or step (2).

[0032] The aforementioned plant-derived hydrolysates refer to products extracted from peas, cotton roots, wheat gluten, soybeans, etc. Commercially available plant-derived hydrolysates include, for example, Hy-Pea® 7404, UltraPep® Cotton, HyPep® 7504, HyPep® (all manufactured by Kerry, USA), Cotton100, Cotton200, Phytone® Soy100 (all manufactured by Gibco, USA).

[0033] The aforementioned peptones, plant-derived hydrolysates, yeast extracts, or yeast hydrolysates are rich in amino acids, peptides, vitamins, carbohydrates, nucleotides, minerals, and other components, and are widely used as cell culture supplements or additives. However, in this invention, it was confirmed that these substances increase the expression of hyaluronidase PH20 in a cleaved form, and therefore they are not suitable for the production of hyaluronidase PH20 in its complete form.

[0034] In one specific embodiment of the present invention, when peptone or TC yeastrate of various origins was added as an additive, it was confirmed that cleaved hyaluronidase PH20 was expressed under all culture conditions (Figure 3).

[0035] In the present invention, the culture in step (2) can be carried out by one or more methods selected from the group consisting of (a) a culture in which the dissolved oxygen content of the culture medium is maintained at 30% to 79%; (b) a culture in which the concentration of residual glucose in the medium is maintained at 1.11 to 5.95 g / L during the culture period; and (c) a culture in which the pH of the culture medium is maintained at 7.0 to 7.4.

[0036] To produce hyaluronidase PH20 in its complete form, the culture medium can be cultured while maintaining a dissolved oxygen content of 30% to 79%, preferably 30% to 50%, and more preferably 30% to 35%.

[0037] The residual glucose concentration in the culture medium can be maintained at 1.11 g / L to 5.95 g / L, preferably 3.5 g / L to 5.95 g / L, while culturing, but is not limited to this.

[0038] Maintaining the dissolved oxygen content, glucose concentration, or pH of the culture medium while culturing means measuring the dissolved oxygen content, glucose concentration, or pH of the culture medium every 1 to 36 hours, preferably every 3 to 30 hours, more preferably every 6 to 24 hours, or in real time, during the culturing period, and adjusting the levels to reach the set standard concentration if they are below it.

[0039] In the present invention, the host cell culture 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, fed-batch culture, repeated fed-batch culture, continuous culture, and perfusion culture.

[0040] The term "hyaluronidase PH20" or "PH20" in the present invention can be interpreted to include both the natural PH20 and its mature form, and the hyaluronidase PH20 produced by the method described above may, but is not limited to, selectively deleting some of the N-terminal and / or C-terminal amino acid residues.

[0041] The human-derived "hyaluronidase PH20" according to the present invention may be represented by the amino acid sequence of SEQ ID NO: 1, but any human-derived hyaluronidase PH20 known in the industry can be used.

[0042] In the present invention, the host cell can be an animal cell, yeast, actinomycete, or insect cell, but is not limited thereto. Mammalian cells are preferred as animal cells. More preferably, commonly used animal culture cells such as CHO cells, HEK cells, COS cells, 3T3 cells, myeloma cells, BHK cells, HeLa cells, and Vero cells are used, with CHO cells being particularly preferred when the goal is high-volume expression. Furthermore, in order to produce the desired protein, it is preferable to use cells suitable for introducing the desired gene, such as dhfr-CHO cells (Proc Natl Acad Sci USA, 77:4216-4220, 1980), which are CHO cells lacking the DHFR gene, or CHOK-1 cells (Proc Natl Acad Sci USA, 60:1275, 1968). Among the CHO cells, DG44, DXB-11, K-1, or CHO-S strains are particularly preferred, and vector introduction into host cells can be carried out by methods such as calcium phosphate method, DEAE dextran method, electroporation method, and lipopection.

[0043] Examples of yeasts include Sacchromyces sp, Hansenula sp, Kluyveromyces, and Pichia sp, while examples of actinomycetes include Streptomyces, but are not limited to these.

[0044] In the present invention, the production method may further include (3) a step of separating and purifying the produced hyaluronidase PH20. In the present invention, the cleaved form of hyaluronidase PH20 can be completely removed by the separation and purification step (3) described above.

[0045] In the present invention, the separation and purification in step (3) can purify hyaluronidase PH20 to high purity by utilizing its affinity binding, ionic bonding properties, and / or hydrophobic interaction properties with hyaluronidase PH20.

[0046] In the present invention, the separation and purification of PH20 can be performed using affinity chromatography, ion exchange-mixed mode chromatography, and hydrophobic interaction chromatography.

[0047] In a specific embodiment of the present invention, step 1: ion exchange mixed-mode chromatography; step 2: affinity chromatography; step 3: virus inactivation; step 4: ion exchange mixed-mode chromatography; step 5: mixed-mode chromatography; step 6: hydrophobic interaction chromatography; and step 7: hyaluronidase PH20 produced by UF / DF and viral filtration was purified. Analysis of the purified hyaluronidase PH20 confirmed that complete hyaluronidase PH20, not cleaved hyaluronidase PH20, had been purified (Figures 11-13).

[0048] In another aspect, the present invention relates to hyaluronidase PH20 in a complete form of 50-75 kDa size produced by the method for producing hyaluronidase PH20 described above. In this invention, hyaluronidase PH20 in its complete form, rather than in a cleaved form, was produced with high purity. Therefore, the complete form of hyaluronidase PH20 produced by the method of this invention has a high degree of inertness (≥85,000 units / mg) and can be effectively applied in various fields.

[0049] The present invention will be described in more detail below with reference to examples. It will be obvious to those skilled in the art that these embodiments are merely illustrative of the present invention and should not be construed as limiting the scope of the invention.

[0050] Example 1: Exploration of commercial culture media, additives, and culture temperature We explored the optimal commercial culture medium and culture temperature using host cells (DG44 CHO cells, SBA002) expressing the naturally occurring human recombinant hyaluronidase PH20 (SEQ ID NO: 1).

[0051] Commercial culture media used were CDM4CHO (GE Healthcare), HyCell CHO (GE Healthcare), EX-CELL CD CHO (Merck), PowerCHO-2 CD (Lonza), CD OptiCHO (Thermo Fisher Scientific), and CD CHO 022 (JS Biosciences). Host cells were cultured at temperatures between 34 and 37°C.

[0052] The additives used were Feed A+ (Thermo Fisher Scientific), Feed B+ (Thermo Fisher Scientific), Feed C+ (Thermo Fisher Scientific), GlycanTune C+ (Thermo Fisher Scientific), Cell Boost 6 (HyClone), and CD Feed 002 (Irvine Scientific).

[0053] [Table 1] TIFF2026518922000003.tif111170

[0054] As a result, as shown in Table 1, cell growth was found to be higher at 34°C than at 37°C, and cell growth was most effective under the HyCell CHO (Feed C+ or GlycanTune C+) and CD OptiCHO (Cell Boost 6) conditions, respectively, depending on the culture medium and additive conditions. Based on these results, three candidate culture combinations for hyaluronidase PH20 production were derived: 1)CD OptiCHO+Cell Boost 6, culture starting temperature: 34℃ 2)HyCell CHO+Feed C+, culture starting temperature: 34℃ 3)HyCell CHO+GlycanTune C+, culture starting temperature: 34℃ Example 2: Production of hyaluronidase PH20 using optimal culture medium and timing of culture temperature changes Based on the three candidate culture combinations derived in Example 1, the degree of hyaluronidase PH20 production was confirmed depending on the timing of the culture temperature change (shift), as shown in the table in Figure 1.

[0055] The initial cell inoculation concentration was 1.0 × 10⁶ cells / mL, and the culture size was 50 mL / 125 mL Erlenmeyer flasks. Cell inoculation was performed by centrifugation at 288 x g for 5 minutes, and the collected cells were resuspended. Culture was carried out for 16 days, and if the pH was less than 6.8, it was corrected by adding 1% of 7.5% sodium bicarbonate stock solution. If the glucose content was less than 20 mmol / L, 1% of 45% D-(+)-glucose stock solution was added.

[0056] As a result, we confirmed that, compared to the control group (where no changes were made to the culture temperature), changing the culture temperature led to more rapid cell growth and increased productivity.

[0057] To confirm hyaluronidase pH 20, a Western blot was performed. After adding sample buffer to the sample, it was boiled at 100°C for 10 minutes and then cooled on ice for 5 minutes. The sample was loaded onto a 10% polyacrylamide gel and subjected to electrophoresis, followed by blotting onto an NC membrane at 350mA for 2 hours. The membrane, blocked with 5% skim milk, was treated with a primary antibody diluted 1:2000 under refrigeration, and then treated with a secondary antibody diluted 1:5000 at room temperature for 1 hour. Hyaluronidase pH 20 was confirmed by color development using NBT / BCIP.

[0058] Western blotting analysis of hyaluronidase PH20 expressed under different culture conditions revealed, as shown in Figure 1, that under the HyCell CHO + GlycanTune C + conditions, a large amount of cleaved forms of natural human recombinant hyaluronidase PH20 (molecular weight between 37 and 49 kDa) were formed.

[0059] Under HyCell CHO+Feed C+ conditions, at 32°C, the slower the change in culture temperature, the lower the cleavage morphology of the pH20 molecule tended to be. Under CD OptiCHO medium conditions, when the initial culture temperature was 37°C, some fragmentation of the pH20 morphology was observed. However, when the initial culture temperature was 34°C, fragmentation of the pH20 morphology was hardly observed, regardless of the day the culture temperature was changed.

[0060] Based on the above results, the basic culture medium CD OptiCHO, the feed medium Cell Boost 6 (15% (v / v)), and the culture start temperature of 34°C were selected as the optimal conditions for hyaluronidase pH20 production.

[0061] Example 3: Production of hyaluronidase PH20 by combination of additives Based on the results of Example 2 described above, experiments were conducted under the following conditions, as shown in the table in Figure 2: adding 1%, 3%, and 5% of Cell Boost 6 feed, which was selected as the optimal additive, to the basic medium CD OptiCHO on day 6 of culture; and adding the optimal content of 15% in 2 to 5 doses at 3-day intervals.

[0062] Furthermore, tests were conducted by adding 4 mM each of L-glutamine and GlutaMAX, and the culture medium samples were analyzed by Western blotting to compare the quality of expressed proteins under different conditions.

[0063] The initial cell inoculation concentration was 1.0 × 10⁶ cells / mL, and the culture size was 50 mL / 125 mL Erlenmeyer flasks. Cell inoculation was performed by centrifugation at 288 x g for 5 minutes, and the collected cells were resuspended. Culture was carried out for up to 20 days, and if the pH was less than 6.8, it was corrected by adding 1% of 7.5% sodium bicarbonate stock solution. If the glucose content was less than 20 mmol / L, 1% of 45% D-(+)-glucose stock solution was added.

[0064] As a result, as shown in Figure 2, no significant difference in the degree of expression of the cleaved form of PH20 was observed depending on the amount of Cell Boost 6 feed added and the conditions on the day of addition, and no difference in the degree of expression of the cleaved form of PH20 was observed with the addition of L-glutamine and GlutaMAX.

[0065] Example 4: Production of hyaluronidase PH20 by combination of other additives We confirmed the pH 20 production level when peptone, an additive other than Cell Boost 6 feed which was selected as the optimal additive, was added.

[0066] Peptone was produced by adding 1%, 3%, 9%, and 15% of yeast extract, soybean hydrolysate (Select Phytone), yeast hydrolysate (TC Yeastrate), and cotton root hydrolysate (Cotton 200UF) as shown in the table in Figure 3, to produce pH 20.

[0067] The initial cell inoculation concentration was 1.0 × 10⁶ cells / mL, and the culture size was 50 mL / 125 mL Erlenmeyer flasks. Cell inoculation was performed by centrifugation at 288 x g for 5 minutes, and the collected cells were resuspended. Culture was carried out for up to 20 days, and if the pH was less than 6.8, it was corrected by adding 1% of 7.5% sodium bicarbonate stock solution. If the glucose content was less than 20 mmol / L, 1% of 45% D-(+)-glucose stock solution was added. As a result, as shown in Figure 3, the expression of the cleaved form of PH20 tended to be high overall, regardless of the type of peptone. In particular, the expression of the cleaved form of PH20 was found to be highest under conditions with 9% TC yeast added.

[0068] Example 5: Production of hyaluronidase pH20 using additives and culture days To confirm the degree of expression of the cleaved PH20 morphology on each culture day under the TC yeast rate 9% addition condition of Example 4 and the degree of expression of the cleaved PH20 morphology on each culture day under the CD OptiCHO + Cell Boost 6 condition selected in Example 3, the experiment was carried out as shown in the table in Figure 4.

[0069] The initial cell inoculation concentration was 1.0 × 10⁶ cells / mL, and the culture size was 50 mL / 125 mL Erlenmeyer flasks. Cell inoculation was performed by centrifugation at 288 x g for 5 minutes, and the collected cells were resuspended. Culture was carried out until the pH was less than 6.8, at which point 1% of 7.5% sodium bicarbonate stock solution was added to correct the pH. If the glucose content was less than 20 mmol / L, 1% of 45% D-(+)-glucose stock solution was added.

[0070] As a result, as shown in Figure 4, under the CD OptiCHO + Cell Boost 6 conditions, there was no difference between culture days, and no expression of cleaved PH20 was confirmed. However, under the condition of adding 9% TC yeast rate, expression of cleaved PH20 was confirmed on all culture days.

[0071] Example 6: Comparison of incubation days and agitation rates in a 7.5L bioreactor The culture conditions selected in Example 2 or Example 3 were applied to a 7.5 L bioreactor for cultivation, and comparative experiments were conducted based on the culture days and agitation speeds (150 rpm and 200 rpm) as shown in tables A and B of Figure 5.

[0072] The initial cell inoculation concentration was 0.3 × 10⁶ cells / mL, and the culture volume was 4L. For cell inoculation, the seed culture sample was mixed in the required volume according to the target initial cell inoculation concentration. Culture was carried out for 14 days. If the pH was less than 6.8, 0.1N sodium hydroxide stock solution was added to correct it to pH 6.8. If the glucose content was 3.5 g / L or less, 3.5 g / L of 45% D-(+)-glucose stock solution was added to the initial culture volume. The pH range of the bioreactor was adjusted to pH 7.10 ± 0.30 on day 0 of culture, pH 7.05 ± 0.25 on day 1 of culture, and 7.00 ± 0.20 from day 2 to day 14 of culture. Dissolved oxygen (DO) was maintained at 30%, and gas control was performed using a 3Gas mix.

[0073] As a result, as shown in Figure 5A, we confirmed that PH20 is expressed in its complete form, not in a cleaved form, regardless of the culture day. Furthermore, as shown in Figure 5B, the cleaved form of PH20 was not observed under either condition. However, it was confirmed that DO could not be stably maintained and productivity was low when operating at 200 rpm, while productivity similar to that of a conventional flask was confirmed when operating at 150 rpm.

[0074] Example 7: Performance comparison of liquid and powder formulations of commercially available culture media The selected basic culture medium, CD OptiCHO, is available in both liquid form (Thermo Fisher Scientific, Cat# 12681-011) and powder form (Thermo Fisher Scientific, Cat# A11222-05). Experiments to confirm the differences in culture between the two forms were conducted as shown in Table C of Figure 5.

[0075] The initial cell inoculation concentration was 0.3 × 10⁶ cells / mL, and the culture volume was 100 mL. The cell inoculation method involved mixing the seed culture sample in the required volume according to the target initial cell inoculation concentration. Culture was carried out for 14 days, and if the pH was less than 6.8, 0.1N sodium hydroxide stock solution was added to correct it until the pH reached 6.8. If the glucose content was 3.5 g / L or less, 3.5 g / L of 45% D-(+)-glucose stock solution was added to the initial culture volume.

[0076] As a result, as shown in Figure 5C, complete PH20 was expressed in all cases regardless of the dosage form of the basic medium, and no differences were observed in the concentration of terminally surviving cells or final productivity.

[0077] Example 8: Purification of natural human recombinant hyaluronidase PH20 using animal cell culture supernatant Step 1: Purification by primary ion exchange mixed-mode chromatography The supernatant cultured under the conditions selected in Example 2 or Example 3 was pH adjusted and filtered. The ion exchange mixed-mode column was equilibrated with sodium acetate equilibrium solution, and the filtrate was loaded onto the ion exchange mixed-mode column to remove some impurities and capture hyaluronidase PH20. The sodium acetate equilibrium solution was run again to remove impurities not bound to the column, and nonspecifically bound impurities were removed with sodium phosphate washing solution. Hyaluronidase PH20 was eluted through an elution solution containing arginine in sodium phosphate, and the column was washed with sodium hydroxide regeneration solution (Figure 6).

[0078] Step 2: Purification by secondary affinity chromatography After reducing the conductivity of the eluate from step 1 by adding sterile water for injection, the pH was adjusted and the solution was filtered. The affinity mode column was equilibrated with a sodium acetate equilibrium solution, and the filtrate was loaded onto the affinity column to remove some impurities and bind hyaluronidase PH20. The sodium acetate equilibrium solution was run again to remove impurities not bound to the column, and nonspecifically bound impurities were removed with a sodium phosphate washing solution. Hyaluronidase PH20 was eluted by passing an elution solution containing 250 mM sodium chloride in sodium phosphate, and the column was washed with a sodium hydroxide regeneration solution (Figure 7).

[0079] Step 3: Virus inactivation Polysorbate 80 and tri-N-butyl phosphate were added to the eluate from step 2, and the mixture was left at room temperature to inactivate the virus.

[0080] Step 4: Purification by tertiary ion exchange mixed-mode chromatography The ion-exchange mixed-mode column was equilibrated with a sodium phosphate equilibrium solution, the reaction mixture from step 3 was filtered, and then loaded onto the ion-exchange mixed-mode column to remove any remaining impurities and bind hyaluronidase PH20. The sodium phosphate equilibrium solution was run again to remove any impurities not bound to the column, and nonspecifically bound impurities were removed with a washing solution containing 120 mM sodium chloride in sodium acetate. Hyaluronidase PH20 was eluted through an elution solution containing 750 mM sodium chloride in sodium acetate, and the column was washed with a sodium hydroxide regeneration solution (Figure 8).

[0081] Step 5: Purification by fourth-order mixed-mode chromatography Dilution solution was added to the eluate from step 4 to achieve the same composition as the equilibrium solution, and the mixture was left to stand at 2-8°C for 16 hours. After filtering the reaction mixture, it was loaded onto a mixed-mode column to remove cell-derived proteins and bind hyaluronidase PH20. The sodium phosphate equilibrium solution was run again to remove impurities that did not bind to the column, and hyaluronidase PH20 was eluted through a 40 mM sodium phosphate elution solution. The column was washed with a sodium hydroxide regeneration solution (Figure 9).

[0082] Step 6: Purification by fifth-order hydrophobic interaction chromatography Dilution solution was added to the eluate from step 5 to achieve a composition similar to the equilibrium solution, filtered, and then loaded onto a hydrophobic interaction column. Cell-derived DNA was bound, while hyaluronidase PH20 was obtained unbound. The sodium phosphate equilibrium solution was run again to obtain any remaining hyaluronidase PH20, and the column was washed with a sodium hydroxide regeneration solution (Figure 10).

[0083] Step 7: UF / DF and virus filtration After step 6, the solution was changed using a 30 kDa MWCO membrane filter with a dosage form solution containing sodium phosphate, sodium chloride, calcium chloride, etc. Then, the protein solution containing hyaluronidase pH 20 was filtered using a viral filter.

[0084] Example 9: Western blot analysis of purified hyaluronidase pH 20 For Western blot analysis of hyaluronidase pH 20, samples were prepared under two conditions, non-reducing and reducing, as shown in the table in Figure 11.

[0085] Under the non-reducing conditions, 0.2, 0.5, and 1 μg of sample were prepared, and then the non-reducing sample buffer was added. Under the reducing conditions, 0.2, 0.5, and 1 μg of sample were mixed with the reducing sample buffer, boiled at 100°C for 10 minutes, and then cooled on ice for 5 minutes.

[0086] Next, the samples were loaded onto 10% polyacrylamide gels and electrophoresis was performed under conditions of 80V for 30 minutes and 120V for 100 minutes. The gels after electrophoresis were blotted onto an NC membrane at 250mA for 2 hours. The primary antibody used was polyclonal rabbit anti-human SPAM1, and the secondary antibody used was conjugated goat anti-rabbit IgG(H+L)-AP.

[0087] As a result, as shown in Figure 11, under both non-reducing and reducing conditions, the complete form of PH20 (at approximately 60 kDa), rather than a cleaved form, was observed. Example 10: SDS-PAGE analysis of purified hyaluronidase PH20 To confirm the difference in molecular weight due to N-glycan deglycation by hyaluronidase PH20, glycoprotein denaturation buffer (10x) was added as shown in the table in Figure 12, boiled at 100°C for 10 minutes, and NP-40 and PNGase F were added and reacted overnight.

[0088] Samples were prepared under two conditions: non-reducing and reducing. These were loaded onto 10% polyacrylamide gels and subjected to electrophoresis at 80V for 30 minutes and 120V for 100 minutes. As a result, as shown in Figure 12, a desaturated band of approximately 54 kDa was observed during desaturation.

[0089] Example 11: Size exclusion chromatography analysis of purified hyaluronidase PH20 For the purity analysis of hyaluronidase PH20, a pH 7.2 solution containing sodium phosphorylate and sodium chloride was used as the mobile phase, and the analysis was carried out using a G2000SWXL column.

[0090] An Alliance HPLC system from Waters was used. The mobile phase buffer was prepared with 200 mM KPi, 50 mM NaCl, and a pH of 7.2. 100 μg of the sample was loaded. The flow rate was 0.5 mL / min, the column temperature was 25°C, and the detection wavelength was 280 nm. The analysis was performed under isocratic gradient conditions for 40 minutes.

[0091] As a result, as shown in Figure 13, it was confirmed that the purity of PH20 was 99% in its complete form, not in a fragmented form. [Potential for industrial use] In this invention, complete hyaluronidase PH20, rather than cleaved hyaluronidase PH20, was produced in high purity by adjusting the culture temperature, type of culture medium, supplemental feed medium components, dissolved oxygen concentration, glucose concentration, and pH of host cells expressing natural human recombinant hyaluronidase PH20. Therefore, the complete hyaluronidase PH20 produced by the method of this invention has high inertness and can be effectively applied in various fields.

Claims

1. (1) A step of culturing host cells expressing natural human recombinant hyaluronidase PH20 at a culture temperature of 33 to 35°C; and (2) A step of culturing for 7 to 11 days while maintaining the culture temperature at 31 to 33°C; A method for producing hyaluronidase PH20, which includes The host cells in step (1) and / or step (2) are cultured in CD OptiCHO medium supplemented with Cell Boost 6 feed medium. A method for producing hyaluronidase PH20 in its complete form, characterized in that the produced hyaluronidase PH20 exists in a complete form of 50-75 kDa size, rather than in a cleaved form.

2. The method for producing hyaluronidase PH20 in its complete form according to claim 1, characterized in that the complete form of hyaluronidase PH20 is 55 to 65 kDa in size.

3. The method for producing hyaluronidase PH20 in its complete form according to claim 1, characterized in that the activity of hyaluronidase PH20 produced by the above method in a culture medium is 4,000 units / mL or more.

4. The method for producing hyaluronidase PH20 in complete form according to claim 1, characterized in that the host cell culture medium in step (1) and / or step (2) contains Cell Boost 6 feed medium at a concentration of 1% (v / v) to 20% (v / v).

5. A method for producing hyaluronidase PH20 in its complete form according to claim 1, characterized in that L-glutamine is further added to the culture medium of the host cells in step (1) and / or step (2) and cultured.

6. A method for producing hyaluronidase PH20 in its complete form according to claim 1, characterized in that no component selected from the group consisting of peptone, plant-derived hydrolysates, yeast extract, and yeast hydrolysate is added to the host cell culture medium in step (1) and / or step (2).

7. The culture in step (2) above is (a) Culture in which the dissolved oxygen content of the culture medium is maintained at 30% to 79%; (b) Culturing in which the concentration of residual glucose in the culture medium is maintained at 1.11 to 5.95 g / L during the culture period; and A method for producing hyaluronidase PH20 in its complete form according to claim 1, characterized by culturing by one or more methods selected from the group consisting of (c) culturing in which the pH of the culture medium is maintained at 7.0 to 7.

4.

8. The method for producing hyaluronidase PH20 in its complete form according to claim 1, characterized in that the host cell culture 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.

9. The method for producing hyaluronidase PH20 in its complete form according to claim 1, characterized in that the hyaluronidase PH20 produced by the above method has selectively deleted portions of the N-terminal and / or C-terminal amino acid residues.

10. The method for producing hyaluronidase PH20 in its complete form according to claim 1, characterized in that the host cell is an animal cell, yeast, actinomycetes, or insect cell.

11. The method for producing hyaluronidase PH20 in its complete form according to claim 1, further comprising the step of (3) separating and purifying the produced hyaluronidase PH20.

12. The method for producing hyaluronidase PH20 in its complete form according to claim 11, characterized in that the separation and purification in step (3) purifies hyaluronidase PH20 to high purity by utilizing affinity binding, ionic bonding properties and / or hydrophobic interaction properties with PH20.

13. The method for producing hyaluronidase PH20 in its complete form according to claim 11, characterized in that the separation and purification step is performed using affinity chromatography, ion exchange-mixed mode chromatography, and hydrophobic interaction chromatography.

14. Hyaluronidase PH20 in a complete form of 50-75 kDa size, produced by the method according to any one of claims 1 to 13.

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