Method for producing intact form of hyaluronidase

Abstract

The present invention relates to a method for producing an intact form of hyaluronidase, and more specifically, to a method for producing an intact form of hyaluronidase (PH20) by controlling the culture temperature or medium components of host cells expressing natural human recombinant hyaluronidase PH20. In the present invention, an intact form of hyaluronidase PH20, rather than a truncated form of hyaluronidase PH20, is produced with high purity by regulating a culture temperature, a medium type, an additive feed medium component, a dissolved oxygen concentration, a glucose concentration, and a pH in host cells expressing natural human recombinant hyaluronidase PH20. Therefore, the intact hyaluronidase PH20 produced by the method of the present invention possesses high specific activity, and thus can be effectively applied to various fields.

Description

Method for producing hyaluronidase in its complete form

[0001] The present invention relates to a method for producing hyaluronidase in its entirety, and more particularly, to a method for producing hyaluronidase PH20 in its entirety by controlling the culture temperature or medium components of a host cell expressing natural human recombinant hyaluronidase PH20.

[0002]

[0003] Hyaluronidase (HAdase) is a general term for enzymes that degrade hyaluronic acid (HA). Hyaluronidase is classified into mammalian type (EC 3.2.1.35, hyaluronoglucosaminidase), leech type (EC 3.2.1.36, hyaluronoglucuronidase), and bacterial type (EC 4.2.2.1, hyaluronate lyase) depending on the mechanism by which it hydrolyzes hyaluronic acid.

[0004] Mammalian hyaluronidase exists in the testes, skin, liver, and placental fluids of 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 the joints of our body. In particular, testicular hyaluronidase (PH-20) is an important enzyme that is attached to the glycosylphosphatidylinositol (GPI) anchor of the acrosome of the sperm, breaking down the thick outer wall layer of the egg to cause fertilization.

[0005] The wide range of uses of hyaluronidase has been comprehensively reviewed since the 1950s, with the first use being subcutaneous injection of fluids. Other uses include infiltration and blocking anesthesia to increase the diffusion of local anesthetics and steroids in orthopedic, ophthalmic, plastic, dental, oral, gynecological, and otolaryngological surgeries, dispersing collections of fluid such as hematomas, preventing peritoneal adhesions, preventing stone formation, and treating infertility.

[0006] 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 raw hyaluronidases are dissolved to an appropriate concentration, filled into vials, and lyophilized to produce commercial products. However, commercialized animal-derived hyaluronidases contain foreign proteins, which can cause allergic reactions. In addition, their stability deteriorates over time, resulting in decreased physiological activity, leaving many problems for their diverse applications.

[0007] To address these issues, research on recombinant hyaluronidase has been conducted. Recombinant proteins can be expressed in various cell types, including Escherichia coli, yeast, insect cells, and animal cells. In particular, in the case of hyaluronidase, glycosylation, which occurs during the post-translational protein modification process, affects its activity. This is because glycans can affect the antigenicity, structural folding, solubility, and stability of glycoproteins. From this perspective, because the post-translational protein modification process in yeast and insect cells, where glycosylation occurs, differs from that in mammals, animal cells are suitable among various expression cell types. Among animal cells, CHO (Chinese Hamster Ovary) cells, which have been proven safe, are the most suitable.

[0008] The first recombinant hyaluronidase for PH20 is marketed under the trade name Hylenex by Halozyme Therapeutics and is under development for various applications, including subcutaneous injection, vitrectomy, and ophthalmic disorders. However, hyaluronidase still has low yield and stability, and supply is low compared to demand, so there is a need for hyaluronidase with improved yield or stability (Korean Patent Publication No. 10-2023-0168902; Korean Patent Registration No. 10-2528707).

[0009] In order to increase the production yield of recombinant hyaluronidase in conventional technology, a manufacturing method for changing the content of N-glycan of recombinant hyaluronidase by controlling glucose concentration or culture temperature has been known (Korean Patent Publication No. 10-2022-0018943). However, this method has a problem in that a truncated form of hyaluronidase is produced rather than a complete form.

[0010]

[0011] Accordingly, in the present invention, efforts were made to increase the productivity of hyaluronidase in its complete form rather than a truncated form, and as a result, it was confirmed that production of hyaluronidase PH20 in its complete form was possible by controlling the culture temperature, medium components, and added feed medium components of host cells expressing natural human recombinant hyaluronidase PH20, thereby completing the present invention.

[0012]

[0013] Accordingly, the present invention provides a method for producing intact hyaluronidase PH20 by controlling the culture temperature or medium components of a host cell expressing natural human recombinant hyaluronidase PH20.

[0014]

[0015] To achieve the above-mentioned purpose,

[0016] The present invention comprises the steps of (1) culturing a host cell expressing a natural human recombinant hyaluronidase PH20 at a culture temperature of 33 to 35°C; and

[0017] (2) A method for producing hyaluronidase PH20, comprising a step of culturing for 7 to 11 days while maintaining the culture temperature at 31 to 33°C,

[0018] The host cells in step (1) and / or step (2) above are cultured in CD OptiCHO culture medium supplemented with Cell Boost 6 feed medium,

[0019] The present invention provides a method for producing hyaluronidase PH20 in its entirety, characterized in that the hyaluronidase PH20 produced above exists in its entirety with a size of 50 to 75 kDa, rather than in a truncated form.

[0020] In a preferred embodiment of the present invention, the intact hyaluronidase PH20 may have a size of 58 to 65 kDa, preferably 55 to 63 kDa.

[0021] 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 / ㎖ or more.

[0022] In another preferred embodiment of the present invention, the Cell Boost 6 feed medium may be included in the culture medium of the host cell in step (1) and / or step (2) at a concentration of 1% (v / v) to 20% (v / v), preferably 1% (v / v) to 15% (v / v).

[0023] In another preferred embodiment of the present invention, L-glutamine may be additionally added to the culture medium of the host cell in step (1) and / or step (2) to culture the host cell.

[0024] In another preferred embodiment of the present invention, it may be characterized in that no component selected from the group consisting of peptin, plant-derived hydrolysate, yeast extract, and yeast hydrolysate is added to the culture medium of the host cell in step (1) and / or step (2).

[0025] In another preferred embodiment of the present invention, the culturing in step (2) may be performed by at least one method selected from the group consisting of: (a) culturing in which the dissolved oxygen content of the culture solution is maintained at 30% to 79%; (b) culturing in which the residual glucose concentration in the medium is maintained at 1.11 to 5.95 g / ℓ during the culturing period; and (c) culturing in which the pH of the culture solution is maintained at 7.0 to 7.4.

[0026] In another preferred embodiment of the present invention, the culture of the host cells in step (1) and / or step (2) may be performed by one or more methods selected from the group consisting of batch culture, repeated batch culture, fed-batch culture, repeated fed-batch culture, continuous culture, and perfusion culture.

[0027] In another preferred embodiment of the present invention, the hyaluronidase PH20 produced by the above method may optionally have some of the amino acid residues at the N-terminus and / or C-terminus deleted.

[0028] In another preferred embodiment of the present invention, the host cell may be an animal cell, yeast, Actinomycetes or insect cell.

[0029] In another preferred embodiment of the present invention, the production method may additionally include the step of (3) separating and purifying the produced hyaluronidase PH20.

[0030] In another preferred embodiment of the present invention, the separation and purification in step (3) can purify hyaluronidase PH20 with high purity by utilizing affinity binding, ionic binding properties, and / or hydrophobic interaction properties with PH20.

[0031] In another preferred embodiment of the present invention, the separation and purification of PH20 can be performed using affinity chromatography, ion exchange-mixed mode chromatography, and hydrophobic interaction chromatography.

[0032]

[0033] In addition, the present invention provides an intact form of hyaluronidase PH20 having a size of 50 to 75 kDa produced by the method for producing hyaluronidase PH20.

[0034]

[0035] In the present invention, by controlling the culture temperature, medium type, added feed medium components, dissolved oxygen concentration, glucose concentration, pH, etc. of host cells expressing natural human recombinant hyaluronidase (Hyaluronidase) PH20, an intact hyaluronidase PH20, not a truncated form of hyaluronidase PH20, was produced with high purity. The intact hyaluronidase PH20 produced by the method of the present invention has high specific activity, and thus can be effectively applied to various fields.

[0036]

[0037] Figure 1 is data obtained by confirming by Western blot whether a truncated form (molecular weight standard between 37 and 49 kDa) or a complete form (molecular weight standard between 50 and 75 kDa) of a natural human recombinant hyaluronidase PH20 is expressed according to the main culture conditions of the present invention, such as culture temperature and temperature change days.

[0038] Figure 1A shows the analysis results according to the culture temperature (34, 37℃ → 32, 34, 37℃), temperature change days (days 3, 5, 7, 9), and additional feed (Feed C+, GlycanTune C+) when the basic batch was carried out with HyCell medium.

[0039] Figure 1B shows the analysis results according to the culture temperature (34, 37°C → 32, 34, 37°C), temperature change days (days 3, 5, 7, 9), and additional feed (Feed C+, Cell Boost 6) when the basic batch was grown in CD OptiCHO medium.

[0040] Figure 2 shows data obtained by Western blot to confirm whether the truncated or intact form of natural human recombinant hyaluronidase PH20 was expressed depending on the type of culture additive and feed content.

[0041] Figure 2A shows the analysis results according to the Cell Boost 6 content (3.75, 5, 7.5, 15 mM) and addition date (0, 3, 6, 9 days) in the presence of L-Glutamine additive.

[0042] Figure 2B shows the analysis results according to the Cell Boost 6 content (3.75, 5, 7.5, 15 mM) and addition date (0, 3, 6, 9 days) in the presence of GlutaMAX (4 mM) additive.

[0043] Figure 3 is a Western blot data that confirms the expression of a truncated or intact form of natural human recombinant hyaluronidase PH20 according to the added content (3, 9%) and type of peptone (Yeast Extract, Select Phytone, TC Yeastolate, Cotton 200) when peptone was added as a culture additive.

[0044] Figure 4 is a Western blot data confirming the expression of a truncated or intact form of the native human recombinant hyaluronidase PH20 according to the basic culture conditions and culture days of the present invention. The basic culture conditions were [CD OptiCHO + Cell Boost 6 (15%)] and [CD OptiCHO + Cell Boost 6 (15%) + TC Yeastolate (9%)], and the culture days were set to 12, 14, 16, 18, and 20.

[0045] Figure 5 is a Western blot data confirming the presence or absence of expression of a truncated or intact form of native human recombinant hyaluronidase PH20 according to differences in (A) culture day, (B) culture agitation speed, and (C) medium formulation of the final culture conditions.

[0046] Figure 6 is a purification chromatogram by first ion exchange mixed mode chromatography according to the purification flow rate of natural human recombinant hyaluronidase PH20.

[0047] Figure 7 is a purification chromatogram by second affinity chromatography according to the purification flow rate of natural human recombinant hyaluronidase PH20.

[0048] Figure 8 is a purification chromatogram by third ion exchange mixed mode chromatography according to the purification flow rate of natural human recombinant hyaluronidase PH20.

[0049] Figure 9 is a purification chromatogram by the fourth mixed mode chromatography according to the purification flow rate of natural human recombinant hyaluronidase PH20.

[0050] Figure 10 is a purification chromatogram by fifth-order hydrophobic action chromatography according to the purification flow rate of natural human recombinant hyaluronidase PH20.

[0051] Figure 11 shows the results of a Western blot analysis of a natural human recombinant hyaluronidase PH20 product obtained through the final purification process.

[0052] Figure 12 shows the results of SDS-PAGE analysis of the natural human recombinant hyaluronidase PH20 product obtained through the final purification process.

[0053] Figure 13 shows the results of size exclusion chromatography analysis of the natural human recombinant hyaluronidase PH20 product obtained through the final purification process.

[0054]

[0055] Hereinafter, the present invention will be described in detail.

[0056]

[0057] The present invention, from a consistent perspective, comprises: (1) a step of culturing a host cell expressing a natural human recombinant hyaluronidase PH20 at a culture temperature of 33 to 35°C; and

[0058] (2) A method for producing hyaluronidase PH20, comprising a step of culturing for 7 to 11 days while maintaining the culture temperature at 31 to 33°C,

[0059] The host cells in step (1) and / or step (2) above are cultured in CD OptiCHO culture medium supplemented with Cell Boost 6 feed medium,

[0060] The present invention relates to a method for producing hyaluronidase PH20 in its entirety, characterized in that the hyaluronidase PH20 produced above exists in its entirety with a size of 50 to 75 kDa, rather than in a truncated form.

[0061]

[0062] In the present invention, in step (1), the culture is performed at 0.3 x 10 based on the integral viable cell density. 6 ~ 13.1 x 10 6 It can be cultured up to cells x day / ㎖.

[0063] In the present invention, the intact hyaluronidase PH20 may have a size of 50 to 75 kDa, preferably 58 to 65 kDa, more preferably 55 to 63 kDa (about 60 kDa). The production of the intact hyaluronidase PH20 is characterized in that substantially no truncated form (37 to 49 kDa) of hyaluronidase PH20 is observed, preferably 90% or more, more preferably 95% or more, more preferably 97% or more.

[0064]

[0065] In the present invention, the CD OptiCHO culture medium (Gibco, USA) is composed of additives (Glucose, Sodium Pyruvate, Sodium Bicarbonate) and inorganic salts (Magnesium, Sodium Phosphate), and Cell Boost 6 (HyClone, USA) is composed of lipids, amino acids, vitamins, growth factors, etc. Cell Boost 6 feed medium can be cultured by adding it 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).

[0066] The "feed medium" used in the present 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 may be manufactured and used in various ways. In the present invention, Cell Boost Series supplement medium (HyClone, USA) is preferably used as the feed medium.

[0067] In the present invention, L-glutamine may be additionally added to the culture medium of the host cell in step (1) and / or step (2) for culturing, and glutamine may be added to the CD OptiCHO medium at a concentration of 2 to 6 mM, more preferably at a concentration of 3 to 5 mM.

[0068]

[0069] In a specific embodiment of the present invention, in order to produce hyaluronidase PH20 in its intact form, the optimal conditions according to the commercial medium type and culture temperature were established. As a result, when the initial culture temperature was set to 34°C under CD OptiCHO medium conditions and Cell Boost 6 was added as an additional feed, the truncated form of PH20 was hardly observed regardless of the culture temperature change (Fig. 1). In addition, no significant difference in the degree of expression of the truncated form of PH20 was observed depending on the addition of L-glutamine (Fig. 2).

[0070]

[0071] In the present invention, it may be characterized in that no component selected from the group consisting of peptin, plant-derived hydrolysate, yeast extract, and yeast hydrolysate is added to the culture medium of the host cell in step (1) and / or step (2).

[0072] The above plant-derived hydrolysates refer to products extracted from garden peas, cottonseeds, wheat gluten, soybeans, etc., and commercially available plant-derived hydrolysates include, for example, Hy-Pea™7404, UltraPep™ Cotton, HyPep™ 7504, HyPep™ (all from Kerry, USA), Cotton 100, Cotton 200, Phytone™Soy 100 (all from Gibco, USA), etc.

[0073] The above peptone, plant-derived hydrolysate, yeast extract or yeast hydrolysate, etc. are rich in amino acids, peptides, vitamins, carbohydrates, nucleotides, minerals and other components, and are therefore widely used as cell culture supplements or additives. However, in the present invention, it was confirmed that the expression of hyaluronidase PH20 in a truncated form was increased by these, and therefore, they are not suitable for producing hyaluronidase PH20 in a complete form.

[0074] In a specific embodiment of the present invention, when peptins or TC Yeastolate of various origins were added as additives, it was confirmed that the cleaved form of hyaluronidase PH20 was expressed under all culture conditions (Fig. 3).

[0075]

[0076] In the present invention, the culture in step (2) may be conducted by at least one method selected from the group consisting of: (a) culture in which the dissolved oxygen content of the culture solution is maintained at 30% to 79%; (b) culture in which the residual glucose concentration in the medium is maintained at 1.11 to 5.95 g / ℓ during the culture period; and (c) culture in which the pH of the culture solution is maintained at 7.0 to 7.4.

[0077] To produce hyaluronidase PH20 in its complete form, the culture can be conducted while maintaining the dissolved oxygen content of the culture medium at 30% to 79%, preferably at 30% to 50%, and more preferably at 30% to 35%.

[0078] 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, during cultivation, but is not limited thereto.

[0079] Cultivating while maintaining the dissolved oxygen content, glucose concentration, or pH of the culture medium means measuring the dissolved oxygen content, glucose concentration, or pH in the culture medium at intervals of every 1 to 36 hours, preferably every 3 to 30 hours, and more preferably every 6 to 24 hours, or in real time, during the culture period, and adjusting the concentration to reach the set standard concentration when it is below the set standard concentration.

[0080]

[0081] In the present invention, the culture of the host cells in step (1) and / or step (2) may be performed by one or more methods selected from the group consisting of batch culture, repeated batch culture, fed-batch culture, repeated fed-batch culture, continuous culture, and perfusion culture.

[0082] “Hyaluronidase PH20” or “PH20” according to the present invention may be interpreted to mean both the native PH20 and its mature form, and the hyaluronidase PH20 produced by the above method may optionally have some of the amino acid residues at the N-terminus and / or C-terminus deleted, but is not limited thereto.

[0083] The human-derived "hyaluronidase PH20" according to the present invention may be represented by the amino acid sequence of SEQ ID NO: 1, but a human-derived hyaluronidase PH20 known in the art may be used.

[0084] In the present invention, the host cell may be an animal cell, yeast, Actinomycetes, or insect cell, but is not limited thereto.

[0085] Preferably, the animal cell is a mammalian cell. 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, and CHO cells are particularly preferred when the purpose is mass expression. In addition, in order to produce the desired protein, it is preferable to use a cell suitable for introducing the desired gene, such as dhfr-CHO cells, which are CHO cells lacking the DHFR gene (Proc Natl Acad Sci USA, 77:4216-4220, 1980), or CHO K-1 cells (Proc Natl Acad Sci USA, 60:1275, 1968). Among the above CHO cells, DG44 strain, DXB-11 strain, K-1 strain or CHO-S strain is particularly preferred, and introduction of the vector into the host cell can be carried out by a method such as calcium phosphate method, DEAE dextran method, electroporation method or lipofection.

[0086] Examples of yeasts include, but are not limited to, Sacchromyces sp, Hansenula sp, Kluyveromyces, and Pichia sp, and examples of actinomycetes include, but are not limited to, Streptomyces.

[0087]

[0088] In the present invention, the production method may additionally include (3) a step of separating and purifying the produced hyaluronidase PH20.

[0089] In the present invention, the cleaved form of hyaluronidase PH20 can be completely removed through the separation and purification step of step (3).

[0090] In the present invention, the separation and purification in step (3) can purify hyaluronidase PH20 with high purity by utilizing affinity binding, ionic binding characteristics, and / or hydrophobic interaction characteristics with hyaluronidase PH20.

[0091] In the present invention, the separation and purification of the PH20 can be performed using affinity chromatography, ion exchange-mixed mode chromatography, and hydrophobic interaction chromatography.

[0092]

[0093] In a specific embodiment of the present invention, hyaluronidase PH20 produced through 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 action chromatography -> step 7: UF / DF and virus filtration was purified, and as a result of analyzing the purified hyaluronidase PH20, it was confirmed that hyaluronidase PH20 in a complete form, not a truncated form, was purified (Figs. 11 to 13).

[0094]

[0095] From another perspective, the present invention relates to an intact form of hyaluronidase PH20 having a size of 50 to 75 kDa produced by the above method for producing hyaluronidase PH20.

[0096] In the present invention, hyaluronidase PH20 was produced in a high purity, intact form, rather than in a truncated form. Therefore, the intact hyaluronidase PH20 produced by the method of the present invention has a high specific activity (≥85,000 units / mg), and thus can be effectively applied in various fields.

[0097]

[0098] Hereinafter, the present invention will be described in more detail through examples.

[0099] These examples are intended solely to illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

[0100]

[0101] Example 1: Exploration of commercial media, additives, and culture temperatures

[0102] The optimal commercial medium and culture temperature were explored using host cells (DG44 CHO cells, SBA002) expressing native human recombinant hyaluronidase PH20 (SEQ ID NO: 1).

[0103] The commercial 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), and the host cells were cultured at a temperature of 34 to 37°C.

[0104] 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).

[0105]

[0106] Cell viability according to medium type, additives, and culture temperature No. Medium additive Culture temperature (℃) Culture days (days) Cell concentration (x 10 6cell / ㎖)1CDM4CHOFeed A+341414.582CDM4CHOFeed B+341312.433CDM4CHOFeed C+34169.634CDM4CHOGlycanTune C+34168.215HyCell CHOFeed A+341629.766HyCell CHOFeed B+341627.727HyCell CHOFeed C+341625.088HyCell CHOGlycanTune C+341622.919EX-CELL CD CHOFeed A+341617.1610EX-CELL CD CHOFeed B+341516.6611EX-CELL CD CHOFeed C+341611.3712EX-CELL CD CHOGlycanTune C+341610.7413PowerCHO-2 CDCell Boost 6341218.8714CD OptiCHOCell Boost 6341515.1815CD CHO 022CD Feed 00234162.8216CDM4CHOFeed A+371411.0717CDM4CHOFeed B+37128.4118CDM4CHOFeed C+371410.4019CDM4CHOGlycanTune C+37165.4620HyCell CHOFeed A+371424.9921HyCell CHOFeed B+371426.2822HyCell CHOFeed C+371421.4023HyCell CHOGlycanTune C+371519.7324EX-CELL CD CHOFeed A+371512.4425EX-CELL CD CHOFeed B+371411.3726EX-CELL CD CHOFeed C+37168.6627EX-CELL CD CHOGlycanTune C+37147.0128PowerCHO-2 CDCell Boost 6371125.2429CD OptiCHOCell Boost 6371212.8730CD CHO 022CD Feed 00237144.46

[0107] As a result, as shown in Table 1, cell growth according to temperature conditions was confirmed to be higher at 34℃ than at 37℃, and cell growth according to medium and additive conditions was found to be most effective under HyCell CHO (Feed C+ or GlycanTune C+) and CD OptiCHO (Cell Boost 6) conditions. Based on the above results, three candidate culture combinations for hyaluronidase PH20 production were derived:

[0108] 1) CD OptiCHO + Cell Boost 6, culture start temperature: 34℃

[0109] 2) HyCell CHO + Feed C+, culture start temperature: 34℃

[0110] 3) HyCell CHO + GlycanTune C+, culture start temperature: 34℃

[0111]

[0112] Example 2: Production of hyaluronidase PH20 according to the timing of changing the optimal medium and culture temperature.

[0113] Based on the three culture combination candidates derived from the above <Example 1>, the degree of hyaluronidase PH20 production according to the culture temperature shift time was confirmed as shown in the table in Fig. 1.

[0114] The initial cell inoculation concentration was 1.0 x 10 6 cell / ㎖, the culture scale was 50 ㎖ / 125 ㎖ Erlenmeyer flask, and the cell inoculation method was centrifugation at 288 x g for 5 minutes to resuspend the collected cells. The culture was carried out for 16 days, and when the pH was below 6.8, 1% of 7.5% sodium bicarbonate stock was added to correct it. When the glucose content was below 20 mmol / ℓ, 1% of 45% D-(+)-glucose stock was added.

[0115] As a result, it was confirmed that cell growth was induced more quickly and productivity increased when the culture temperature was changed compared to the control condition where the culture temperature was not changed.

[0116]

[0117] To confirm hyaluronidase PH20, Western blotting was performed. Sample buffer was added to the sample, boiled at 100℃ for 10 minutes, and then ice-cooled for 5 minutes. The sample was loaded onto a 10% polyacrylamide gel, electrophoresis was performed, and blotted onto an NC membrane at 350 mA for 2 hours. The membrane was blocked with 5% skim milk, and the primary antibody diluted 1:2000 was treated in the refrigerator, and the secondary antibody diluted 1:5000 was treated at room temperature for 1 hour. Hyaluronidase PH20 was confirmed by color development by treatment with NBT / BCIP.

[0118] As a result of confirming the expression of hyaluronidase PH20 under different culture conditions by Western blot, as shown in Fig. 1, it was confirmed that a large amount of truncated form of native human recombinant hyaluronidase PH20 (molecular weight standard between 37 and 49 kDa) was formed under the HyCell CHO + GlycanTune C+ condition.

[0119] In the HyCell CHO + Feed C+ condition, the later the culture temperature change to 32℃, the lower the truncated form of PH20 tended to be.

[0120] Under CD OptiCHO medium conditions, when the initial culture temperature was 37°C, the truncated form of PH20 was somewhat observed, but when the initial culture temperature was 34°C, the truncated form of PH20 was hardly observed regardless of the culture temperature change date.

[0121] Based on the above results, the basic medium CD OptiCHO, feed medium Cell Boost 6 (15% (v / v)), and culture start temperature of 34°C were selected as the optimal conditions for producing hyaluronidase PH20.

[0122]

[0123] Example 3: Production of hyaluronidase PH20 according to additive combinations

[0124] Based on the results of the above <Example 2>, as shown in the table in Fig. 2, the conditions of adding 1, 3, and 5% of Cell Boost 6 feed, which was selected as the optimal additive on the 6th day of culture, to the basic medium CD OptiCHO, and the conditions of adding the optimal content of 15% in 2 to 5 doses at 3-day intervals were tested.

[0125] Additionally, tests were conducted by adding 4 mM each of L-Glutamine and GlutaMAX, and culture samples were analyzed by Western blotting to compare the quality of expressed proteins under each condition.

[0126] The initial cell inoculation concentration was 1.0 x 10 6 cell / ㎖, the culture scale was carried out in 50 ㎖ / 125 ㎖ Erlenmeyer flasks, and the cell inoculation method was centrifugation at 288 x g for 5 minutes to resuspend the collected cells. The culture was carried out for up to 20 days, and when the pH was below 6.8, 1% of 7.5% sodium bicarbonate stock was added to correct it. When the glucose content was below 20 mmol / ℓ, 1% of 45% D-(+)-glucose stock was added.

[0127]

[0128] As a result, as shown in Fig. 2, no significant difference was observed in the degree of expression of the truncated form of PH20 according to the amount of Cell Boost 6 feed added and the addition date conditions, and no difference was observed in the degree of expression of the truncated form of PH20 according to the addition of L-glutamine and GlutaMAX.

[0129]

[0130] Example 4: Production of hyaluronidase PH20 according to combination of other additives

[0131] When peptone, an additive other than Cell Boost 6 feed, which was selected as the optimal additive, was added, the level of PH20 production was confirmed.

[0132] Peptone was produced by adding 1, 3, 9, and 15% of yeast extract (Yeast Extract), soybean hydrolysate (Select Phytone), yeast hydrolysate (TC Yeastolate), and cottonseed hydrolysate (Cotton 200 UF) as shown in the table in Fig. 3 to produce PH20.

[0133] The initial cell inoculation concentration was 1.0 x 10 6 cell / ㎖, the culture scale was carried out in 50 ㎖ / 125 ㎖ Erlenmeyer flasks, and the cell inoculation method was centrifugation at 288 x g for 5 minutes to resuspend the collected cells. The culture was carried out for up to 20 days, and when the pH was below 6.8, 1% of 7.5% sodium bicarbonate stock was added to correct it. When the glucose content was below 20 mmol / ℓ, 1% of 45% D-(+)-glucose stock was added.

[0134]

[0135] As a result, as shown in Fig. 3, regardless of the type of peptone, the expression of the truncated form of PH20 tended to be high overall. In particular, the expression of the truncated form of PH20 was found to be the highest under the condition of adding 9% TC Yeastolate.

[0136]

[0137] Example 5: Production of hyaluronidase PH20 according to additives and culture days

[0138] In order to confirm the degree of expression of the truncated form of PH20 by culture day under the 9% TC Yeastolate addition condition of <Example 4> and the degree of expression of the truncated form of PH20 by culture day under the CD OptiCHO + Cell Boost 6 condition selected in <Example 3>, an experiment was conducted as shown in the table in Fig. 4.

[0139] The initial cell inoculation concentration was 1.0 x 10 6 cell / ㎖, the culture scale was carried out in 50 ㎖ / 125 ㎖ Erlenmeyer flasks, and the cell inoculation method was centrifugation at 288 x g for 5 minutes to resuspend the collected cells. The culture was carried out for up to 20 days, and when the pH was below 6.8, 1% of 7.5% sodium bicarbonate stock was added to correct it. When the glucose content was below 20 mmol / ℓ, 1% of 45% D-(+)-glucose stock was added.

[0140]

[0141] As a result, as shown in Fig. 4, under the CD OptiCHO + Cell Boost 6 condition, expression of truncated PH20 was not confirmed regardless of the culture day, but under the condition with 9% TC Yeastolate added, expression of truncated PH20 was confirmed on all culture days.

[0142]

[0143] Example 6: Comparison of culture days and agitation speeds in a 7.5 ℓ bioreactor

[0144] Cultivation was performed in a 7.5 liter bioreactor by applying the culture conditions selected in the above <Example 2> or <Example 3>, and a comparative experiment was performed according to the culture day and agitation speed (150 rpm and 200 rpm) as shown in the tables of FIGS. 5A and 5B.

[0145] The initial cell inoculation concentration was 0.3 x 10 6 cell / ㎖, the culture scale was 4 ℓ, and the seed culture sample was mixed in the required volume according to the target initial cell inoculation concentration. The culture was carried out for 14 days, and when the pH was below 6.8, 0.1 N sodium hydroxide stock was added until the pH reached 6.8. When the glucose content was below 3.5 g / ℓ, 45% D-(+)-glucose stock was added in an amount of 3.5 g / ℓ of the initial culture scale. 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 on days 2 to 14 of culture. Dissolved oxygen (DO) was maintained at 30%, and gas control was performed using a 3 Gas mix.

[0146]

[0147] As a result, as shown in Fig. 5A, it was confirmed that PH20 was expressed in a complete form rather than a truncated form regardless of the culture date.

[0148] Additionally, as shown in Fig. 5B, expression of the truncated form of PH20 was not observed under either condition. However, when operating at 200 rpm, DO was not maintained stably, resulting in low productivity, while when operating at 150 rpm, productivity similar to that of the existing flask was observed.

[0149]

[0150] Example 7: Comparison of performance of liquid and powder formulations of commercial media

[0151] The selected basic medium CD OptiCHO exists in liquid form (Thermo Fisher Scientific / Cat# 12681-011) and powder form (Thermo Fisher Scientific / Cat# A11222-05), and an experiment was conducted to confirm the culture difference between the two forms, as shown in the table in Figure 5C.

[0152] The initial cell inoculation concentration was 0.3 x 10 6 cell / ㎖, the culture scale was 100 ㎖, and the cell inoculation method was to mix the seed culture sample in the required volume according to the target initial cell inoculation concentration. The culture was carried out for up to 14 days, and when the pH was below 6.8, 0.1 N sodium hydroxide stock was added until the pH reached 6.8. When the glucose content was below 3.5 g / ℓ, 45% D-(+)-glucose stock was added in an amount of 3.5 g / ℓ of the initial culture scale.

[0153]

[0154] As a result, as shown in Fig. 5C, PH20 was expressed in its intact form regardless of the formulation of the basic medium, and no difference was observed in the final culture viable cell concentration and final productivity.

[0155]

[0156] Example 8: Purification of native human recombinant hyaluronidase PH20 using animal cell culture supernatant

[0157] Step 1: Purification by first ion exchange mixed mode chromatography

[0158] The pH of the supernatant after culture was adjusted by applying the culture conditions selected in the above <Example 2> or <Example 3>, and filtration was performed. The ion exchange mixed mode column was equilibrated with a sodium acetate equilibration solution, and the filtrate was loaded onto the ion exchange mixed mode column to remove some impurities and capture hyaluronidase PH20. The sodium acetate equilibration solution was passed again to remove impurities not bound to the column, and non-specifically bound impurities were removed with a sodium phosphate washing solution. Hyaluronidase PH20 was eluted through an elution solution containing arginine in sodium phosphate, and the column was washed using a sodium hydroxide regeneration solution (Fig. 6).

[0159]

[0160] Step 2: Purification by secondary affinity chromatography

[0161] After lowering the conductivity by adding water for injection to the eluate of Step 1, pH adjustment was performed, and filtration was performed. The affinity mode column was equilibrated with a sodium acetate equilibration solution, and the filtrate was loaded onto the affinity column to remove some impurities and bind hyaluronidase PH20. The sodium acetate equilibration solution was passed again to remove impurities that were not bound to the column, and non-specifically bound impurities were removed with a sodium phosphate washing solution. Hyaluronidase PH20 was eluted through an elution solution containing 250 mM sodium chloride in sodium phosphate, and the column was washed using a sodium hydroxide regeneration solution (Fig. 7).

[0162]

[0163] Step 3: Virus inactivation

[0164] After adding polysorbate 80 and tri-N-butyl phosphate to the eluate of step 2, the solution was left at room temperature to inactivate the virus.

[0165]

[0166] Step 4: Purification by third ion exchange mixed mode chromatography

[0167] An ion-exchange mixed-mode column was equilibrated with a sodium phosphate equilibration solution, and the reaction solution from step 3 was filtered, then loaded onto the ion-exchange mixed-mode column to remove any remaining impurities and bind hyaluronidase PH20. The sodium phosphate equilibration solution was passed through again to remove any impurities not bound to the column, and a washing solution containing 120 mM sodium chloride in sodium acetate was used to remove any impurities bound nonspecifically. Hyaluronidase PH20 was eluted through an elution solution containing 750 mM sodium chloride in sodium acetate, and the column was washed using a sodium hydroxide regeneration solution (Fig. 8).

[0168]

[0169] Step 5: Purification by fourth mixed mode chromatography

[0170] The diluted solution was added to the equilibrium solution of step 4 so that the composition was similar to that of the equilibrium solution, and left at 2 to 8°C for 16 hours. After filtering the reaction solution, it was loaded onto a mixed mode column to remove cell-derived proteins and bind hyaluronidase PH20. The sodium phosphate equilibrium solution was passed through again to remove impurities not bound to the column, and hyaluronidase PH20 was eluted through a 40 mM sodium phosphate elution solution. The column was washed using a sodium hydroxide regeneration solution (Fig. 9).

[0171]

[0172] Step 6: Purification by fifth hydrophobic action chromatography

[0173] A diluted solution was added to the effluent of step 5 to obtain a composition similar to that of the parallel solution, and after filtering, it was loaded onto a hydrophobic action column, and cell-derived DNA was obtained bound and hyaluronidase PH20 was obtained unbound. The sodium phosphate parallel solution was passed again to obtain additional remaining hyaluronidase PH20, and the column was washed using a sodium hydroxide regeneration solution (Fig. 10).

[0174]

[0175] Step 7: UF / DF and Virus Filtration

[0176] After step 6, the solution was changed to a formulation solution containing sodium phosphate, sodium chloride, calcium chloride, etc. using a 30 kDa MWCO membrane filter. Then, the protein solution containing hyaluronidase PH20 was filtered using a virus filter.

[0177]

[0178] Example 9: Western blot analysis of purified hyaluronidase PH20

[0179] For Western blot analysis of hyaluronidase PH20, samples were prepared under two conditions: non-reducing and reducing, as shown in the table in Figure 11.

[0180] For non-reducing conditions, 0.2, 0.5, and 1 μg samples were prepared, and then non-reducing sample buffer was added. For reducing conditions, 0.2, 0.5, and 1 μg samples were added reducing sample buffer, boiled at 100°C for 10 minutes, and then ice-cooled for 5 minutes.

[0181] Next, the sample was loaded onto a 10% polyacrylamide gel and electrophoresed at 80 V for 30 minutes and 120 V for 100 minutes. The gel after electrophoresis was blotted onto an NC membrane at 250 mA for 2 hours. The primary antibody used was polyclonal rabbit anti-Human SPAM1, and the secondary antibody used was goat anti-rabbit IgG(H+L)-AP conjugated.

[0182]

[0183] As a result, as shown in Fig. 11, the intact form of PH20 (at approximately 60 kDa), not the truncated form of PH20, was confirmed under both non-reducing and reducing conditions.

[0184]

[0185] Example 10: SDS-PAGE analysis of purified hyaluronidase PH20

[0186] To confirm the difference in molecular weight according to N-glycan deglycosylation of hyaluronidase PH20, as shown in the table in Fig. 12, 10x glycoprotein denaturing buffer was added, boiled at 100°C for 10 minutes, and NP-40 and PNGase F were added and reacted overnight.

[0187] Samples were prepared under two conditions, non-reducing and reducing, loaded onto a 10% polyacrylamide gel, and electrophoresis was performed under the conditions of 80 V ~ 30 minutes and 120 V ~ 100 minutes.

[0188]

[0189] As a result, as shown in Fig. 12, a deglycosylated band of approximately 54 kDa was confirmed upon deglycosylation.

[0190]

[0191] Example 11: Size exclusion chromatography analysis of purified hyaluronidase PH20

[0192] To analyze the purity of hyaluronidase PH20, analysis was performed using a G2000SWXL column with a pH 7.2 solution containing sodium phosphate and sodium chloride as the mobile phase.

[0193] Alliance HPLC equipment from Waters was used, and the mobile phase buffer was prepared with a composition of 200 mM KPi, 50 mM NaCl, pH 7.2, and the analysis amount was 100 μg. The flow rate was 0.5 ml / min, the column temperature was 25°C, and the detection wavelength was 280 nm. The gradient condition was isocratic and the analysis was performed for 40 minutes.

[0194]

[0195] As a result, as shown in Fig. 13, it was confirmed that the purity of the intact form of PH20, not the truncated form of PH20, was 99%.

[0196]

[0197] In the present invention, by controlling the culture temperature, medium type, added feed medium components, dissolved oxygen concentration, glucose concentration, pH, etc. of host cells expressing natural human recombinant hyaluronidase (Hyaluronidase) PH20, an intact hyaluronidase PH20, not a truncated form of hyaluronidase PH20, was produced with high purity. Therefore, the intact hyaluronidase PH20 produced by the method of the present invention has high specific activity and can be effectively applied to various fields.

Claims

1. (1) A step of culturing a host cell expressing a natural human recombinant hyaluronidase PH20 at a culture temperature of 33 to 35°C; and (2) A method for producing hyaluronidase PH20, comprising a 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) above are cultured in CD OptiCHO culture medium supplemented with Cell Boost 6 feed medium, A method for producing intact hyaluronidase PH20, characterized in that the hyaluronidase PH20 produced above exists in an intact form with a size of 50 to 75 kDa rather than in a truncated form.

2. In paragraph 1, A method for producing intact hyaluronidase PH20, characterized in that the intact hyaluronidase PH20 has a size of 55 to 65 kDa.

3. In paragraph 1, A method for producing intact hyaluronidase PH20, characterized in that the activity of hyaluronidase PH20 in a culture medium produced by the above method is 4,000 units / ㎖ or more.

4. In paragraph 1, A method for producing intact hyaluronidase PH20, characterized in that the culture medium of the host cell 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. In paragraph 1, A method for producing hyaluronidase PH20 in its intact form, characterized in that L-glutamine is additionally added to the culture medium of the host cell in step (1) and / or step (2) and the cell is cultured.

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

7. In paragraph 1, The cultivation in step (2) above is (a) cultivation that maintains the dissolved oxygen content of the culture solution at 30% to 79%; (b) Cultivation that maintains the residual glucose concentration in the medium at 1.11 to 5.95 g / ℓ during the culture period; and (c) A method for producing hyaluronidase PH20 in its intact form, characterized by culturing by at least one method selected from the group consisting of a method for maintaining the pH of the culture medium at 7.0 to 7.

4.

8. In paragraph 1, A method for producing intact hyaluronidase PH20, characterized in that the culture of the host cell in step (1) and / or step (2) is performed by at least one method selected from the group consisting of batch culture, repeated batch culture, fed-batch culture, repeated fed-batch culture, continuous culture, and perfusion culture.

9. In paragraph 1, A method for producing a complete form of hyaluronidase PH20, characterized in that the hyaluronidase PH20 produced by the above method is optionally deleted from a portion of the amino acid residues at the N-terminus and / or C-terminus.

10. In paragraph 1, A method for producing intact hyaluronidase PH20, characterized in that the host cell is an animal cell, yeast, actinomycetes or insect cell.

11. In paragraph 1, A method for producing hyaluronidase PH20 in its complete form, characterized in that the method further comprises (3) a step of separating and purifying the produced hyaluronidase PH20.

12. In paragraph 11, A method for producing hyaluronidase PH20 in its complete form, characterized in that the separation and purification in the above step (3) is performed by purifying hyaluronidase PH20 with high purity by utilizing affinity binding, ionic binding characteristics, and / or hydrophobic interaction characteristics with PH20.

13. In paragraph 11, A method for producing intact hyaluronidase PH20, characterized in that the above separation and purification steps are performed using affinity chromatography, ion exchange-mixed mode chromatography, and hydrophobic interaction chromatography.

14. Intact hyaluronidase PH20 having a size of 50 to 75 kDa, produced by any one of the methods of claims 1 to 13.

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