A method for preparing recombinant human hyaluronidase and use thereof

Abstract

This application discloses a method for preparing recombinant human hyaluronidase and its application. The preparation method includes: digesting the fusion protein SUMO protein-human hyaluronidase with His-ULP1 followed by nickel column purification to obtain recombinant human hyaluronidase. This preparation method does not require the participation of multiple different types of chromatography media, and can obtain recombinant human hyaluronidase with high purity. The obtained rHuPH20 exhibits good biological activity.

Description

A method for preparing recombinant human hyaluronidase and its application

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to the Chinese patent application number 202311846834.X filed with the State Intellectual Property Office of China on December 28, 2023, entitled “A method for preparing recombinant human hyaluronidase and its application”, the entire contents of which are incorporated by reference into this application. Technical Field

[0003] The present application relates to the field of biopharmaceutical technology, and specifically to a method for preparing recombinant human hyaluronidase and its application. Background Art

[0004] PH20 is an enzyme (ec3.2.1.35) belonging to the hyaluronidase family that cleaves the β-1,4 linkage between n-acetylglucosamine and glucuronic acid (the sugar that constitutes hyaluronic acid). Hyaluronidase degrades hyaluronic acid, a proteoglycan found in the extracellular matrix and basement membranes. Six members of the hyaluronidase family are clustered in two tightly linked groups on chromosomes 3p21.3 and 7q31.3. This gene encodes a GPI-anchored enzyme located on the surface and inner acrosomal membrane of human sperm. Hyaluronidase enables sperm to penetrate the hyaluronan-rich cumulus cell layer surrounding the oocyte and is a receptor for hyaluronan-induced cell signaling and is involved in sperm-zona adhesion. Aberrant expression of this gene in tumors is associated with basement membrane degradation, leading to tumor invasion and metastasis. Multiple transcript variants encoding different isoforms of this gene have been identified.

[0005] PH20 is a single-chain membrane glycoprotein anchored by glycosylphosphatidylinositol (GPI). Aside from the signal peptide, its structure exhibits the hallmarks of a typical GPI attachment. While PH20 sites vary slightly in glycosylation levels and intraproteolytic patterns, their hyaluronidase activity is dependent on the disulfide bonds and secondary and tertiary structures maintained by glycosylation. It contains six functional N-linked glycosylation sites (82, 166, 235, 254, 368, and 393), all of which are attached to oligosaccharides. The primary terminal carbohydrate is mannose, and there are five intrachain disulfide bonds formed by cysteines. The domains 36-464aa represent the domain that retains minimal activity; the domains 36-482aa maintain substantial hyaluronidase activity. The anchor site 490aa is not essential for activity. The structural and spatial structures of hyaluronidase PH20 (data from AlphaFold) are shown in Figures 1 and 2, respectively.

[0006] PH20 is found in the testicles, epididymis, female reproductive tract, chest, intestine, and malignant tumors. PH20 is the only hyaluronidase active under neutral conditions, making it optimally suited to the physiological environment within the body. Therefore, it has a wide range of applications in the pharmaceutical field. On the one hand, it facilitates drug delivery through the physical barrier of the extracellular matrix; on the other hand, it can be used for targeted therapies in the tumor microenvironment; and it also has a certain effect on gene therapy. Specifically, PH20 has the following applications:

[0007] (1) Application of hyaluronidase in pharmaceutical preparations

[0008] Human hyaluronidase PH20 has an optimal pH of 5.5 but exhibits some activity even at a pH of 7-8, whereas other human hyaluronidases (including hyal1) have an optimal pH of 3-4 and are weakly active at pHs of 7-8. The pH of the human subcutaneous region is approximately 7.4, which is generally neutral, so PH20 is widely used in clinical applications among various types of hyaluronidases.

[0009] As early as 1948, the U.S. FDA approved the marketing of hyaluronidase preparations. In 2005, the Enhanze drug delivery technology developed by Halozyme Therapeutics based on recombinant human hyaluronidase PH20 (rHuPH20) was rapidly promoted in the United States. Before that, hyaluronidase preparations were mostly extracted from animal (cow, sheep) testicular tissue. The prepared HA often had disadvantages such as low purity, excessive content of impurities, low activity and strong immunogenicity. Side effects included allergic reactions. Skin allergy tests were required before injection, which limited the application of promoting the absorption of nutritional factors and drugs. The recombinant human hyaluronidase developed by Halozyme Therapeutics does not contain animal-derived ingredients, has high purity, high activity, no immunogenicity, and few side effects. Therefore, a variety of uses Drugs with this technology have been approved by the FDA. The earlier approved drug preparations include SC, The development history of human hyaluronidase pH20 is shown in Figure 3.

[0010] In the past two years, the US FDA has approved the subcutaneous injection formulation of Darzalex Faspro for adult patients with newly diagnosed or relapsed / refractory multiple myeloma. Roche also launched Phesgo, a subcutaneous antibody cocktail therapy, for the treatment of adult patients with HER2+ metastatic and early-stage HER2+ breast cancer. Many pharmaceutical companies are seeking greater competitiveness in formulations to meet the needs of healthcare professionals and patients. Hyaluronidase has also been used clinically for many years as a drug permeation agent to enhance drug absorption.

[0011] (2) Application of hyaluronidase in tumor microenvironment

[0012] Increased extracellular matrix (ECM) deposition is a hallmark of many solid tumors. Increased levels of hyaluronic acid (HA), a component of the ECM, lead to decreased tumor tissue elasticity and increased interstitial fluid pressure (IFP). Recombinant human hyaluronidase is introduced into the tumor microenvironment to target HA for tumor treatment.

[0013] (3) Application of hyaluronidase in gene therapy

[0014] Recombinant human hyaluronidase also has some applications in gene therapy. The in vivo efficacy of most gene delivery vehicles is inconsistent with the efficacy observed in vitro. Glycosaminoglycans can hinder the transfer and spread of DNA and viral vectors into many cell types, and the level of extracellular matrix materials significantly impedes this process. The use of hyaluronidase can open channels in the extracellular matrix, thereby enhancing the delivery of gene therapy.

[0015] (4) Application of hyaluronidase in medical aesthetics

[0016] Hyaluronic acid, also known as hyaluronic acid, and hyaluronidase, also known as hyaluronic acid dissolving enzyme, are natural enzymes that have a hydrolytic effect on hyaluronic acid, can eliminate injections in inappropriate areas, and eliminate lumps caused by excessive injection of hyaluronic acid, with an elimination rate of up to 90%.

[0017] It is mainly used to repair the failure of hyaluronic acid shaping, and it can be regarded as the regret medicine in the field of micro-surgery. If you feel that the effect of hyaluronic acid injection is not good, you can use hyaluronic acid dissolving enzyme to dissolve it within 48 hours, and then fill it with hyaluronic acid again.

[0018] However, the recombinant human hyaluronidase PH20 (rHuPH20) currently on the market is primarily produced by direct expression in CHO cells and secretion into the culture supernatant. The culture fluid is then centrifuged at high speed, the supernatant collected, and concentrated by ultrafiltration. rHuPH20 is then purified through complex purification methods, including anion exchange chromatography (Q), hydrophobic chromatography, aminophenylboronic acid chromatography, hydroxyapatite chromatography, and cation exchange chromatography (SP). These preparation methods involve numerous steps and require numerous different types of chromatography media, making the production process rather cumbersome.

[0019] In view of this, this application is hereby filed. Summary of the Invention

[0020] The purpose of the present application includes providing a method for preparing recombinant human hyaluronidase. The preparation method has simple steps and does not require the participation of multiple different types of chromatographic media. This method can not only obtain recombinant human hyaluronidase with high purity, but also ensure that the obtained rHuPH20 has the ability to basically maintain the original hyaluronidase activity portion 36-482aa.

[0021] The purpose of this application also includes providing recombinant human hyaluronidase prepared by the above method and its application. The recombinant human hyaluronidase has high purity and activity and has good application prospects in biological preparations.

[0022] This application is implemented as follows:

[0023] In a first aspect, the present application provides a method for preparing recombinant human hyaluronidase, comprising: subjecting the fusion protein SUMO protein-human hyaluronidase to His-ULP1 enzymatic digestion and then subjecting the fusion protein to nickel column purification to obtain recombinant human hyaluronidase;

[0024] The amino acid sequence of recombinant human hyaluronidase is shown in SEQ ID NO: 1; the amino acid sequence of the fusion protein SUMO protein-human hyaluronidase is shown in SEQ ID NO: 2; and the amino acid sequence of the His-ULP1 enzyme is shown in SEQ ID NO: 3.

[0025] In a second aspect, the present application provides recombinant human hyaluronidase obtained by the above preparation method.

[0026] Thirdly, the present application also provides the application of the above-mentioned recombinant human hyaluronidase in the biopharmaceutical field and the medical aesthetic field, which includes the preparation of subcutaneous injection preparations, tumor treatment drugs, gene therapy vectors, and the dissolution of hyaluronic acid from failed plastic surgery. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following is a brief introduction to the drawings required for use in the embodiments. It should be understood that the following drawings only show certain embodiments of the present application and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without creative work.

[0028] FIG1 is a structural diagram of hyaluronidase PH20;

[0029] FIG2 is a spatial map of hyaluronidase PH20;

[0030] FIG3 is a diagram showing the development of hyaluronidase PH20;

[0031] FIG4 is a flow chart of the preparation of rHuPH20 in this application;

[0032] FIG5 is an SDS-PAGE of the expression, purification and enzyme digestion of rHuPH20 fusion protein in the embodiment;

[0033] FIG6 is a SEC profile of high-purity rHuPH20 prepared in this application;

[0034] FIG7 is a SEC profile of high-purity rHuPH20 prepared in this application;

[0035] FIG8 is the mass spectrometry detection result of rHuPH20 in Experimental Example 1;

[0036] FIG9 is a standard curve of rHuPH20 in Experimental Example 2. DETAILED DESCRIPTION

[0037] The purpose of the present application includes providing a method for preparing recombinant human hyaluronidase. The preparation method has simple steps and does not require the participation of multiple different types of chromatographic media. This method can not only obtain recombinant human hyaluronidase with high purity, but also ensure that the obtained rHuPH20 has the ability to basically maintain the original hyaluronidase activity portion 36-482aa.

[0038] The purpose of this application also includes providing recombinant human hyaluronidase prepared by the above method and its application. The recombinant human hyaluronidase has high purity and activity and has good application prospects in biological preparations.

[0039] This application is implemented as follows:

[0040] In a first aspect, the present application provides a method for preparing recombinant human hyaluronidase, comprising: subjecting the fusion protein SUMO protein-human hyaluronidase to His-ULP1 enzymatic digestion and then subjecting the fusion protein to nickel column purification to obtain recombinant human hyaluronidase;

[0041] The amino acid sequence of recombinant human hyaluronidase is shown in SEQ ID NO: 1; the amino acid sequence of the fusion protein SUMO protein-human hyaluronidase is shown in SEQ ID NO: 2; and the amino acid sequence of the His-ULP1 enzyme is shown in SEQ ID NO: 3.

[0042] In some embodiments, during enzymatic cleavage, the molar ratio of the fusion protein SUMO protein-human hyaluronidase to the His-ULP1 enzyme is 1:0.9-1.1.

[0043] In some embodiments, the enzyme digestion conditions are: temperature of 30±2° C., and digestion time of 4-8 h.

[0044] In some embodiments, the expression of the fusion protein SUMO protein-human hyaluronidase includes: constructing a recombinant bacterium containing a gene fragment of the fusion protein SUMO protein-human hyaluronidase, then obtaining a recombinant plasmid from the recombinant bacterium, and then transfecting the recombinant plasmid into a host cell, culturing and collecting the supernatant, and centrifuging to remove cell debris to obtain a crude fusion protein SUMO protein-human hyaluronidase.

[0045] In some embodiments, the method for preparing the recombinant bacteria includes: inserting a gene fragment containing the fusion protein SUMO protein-human hyaluronidase into pcDNA3.4 mcs, and then transferring the fragment into a host bacteria to obtain the recombinant bacteria.

[0046] Preferably, the host cells include 293 cells or CHO cells.

[0047] In some embodiments, the gene fragment includes the nucleotide sequence of the fusion protein SUMO protein-human hyaluronidase and related elements; the related elements include: a signal peptide added to the N-terminus, a Kozak sequence at the 5' end, an EcoR1 restriction site at the 5' end, and a BamH1 restriction site at the 3' end.

[0048] In some embodiments, the amino acid sequence of the signal peptide is shown in SEQ ID NO: 6; the nucleotide sequence of the Kozak sequence is GCCACC; and the nucleotide sequence of the gene fragment is shown in SEQ ID NO: 7.

[0049] In some embodiments, the transfection comprises: transferring the diluted recombinant plasmid and transfection reagent into a cell culture containing host cells, and culturing with shaking at 37° C. in a humidified atmosphere of 5-8% CO 2 for 5-7 days;

[0050] The viable cell density in the cell culture was 2.5×10 6 -3.5×10 6 The volume mass ratio of cell culture to recombinant plasmid is 1:1.2-1.4 (L / mg), and the volume mass ratio of transfection reagent to recombinant plasmid is 1:3-5 (μL / μg).

[0051] In some embodiments, the above method further comprises purifying the crude fusion protein SUMO protein-human hyaluronidase before enzymatic cleavage, wherein the purification method comprises: equilibrating a nickel ion chromatography column with an equilibration solution, adding a loading solution, equilibrating the nickel ion chromatography column with an equilibration solution, and then eluting to obtain the purified fusion protein SUMO protein-human hyaluronidase;

[0052] The equilibration solution included PBS and 20-40 mM imidazole, with a pH of 7.4 ± 0.2;

[0053] The elution buffer included PBS and 250-500 mM imidazole, pH = 7.4 ± 0.2;

[0054] The loading solution includes crude fusion protein SUMO protein-human hyaluronidase and 20-40 mM imidazole.

[0055] In a second aspect, the present application provides recombinant human hyaluronidase obtained by the above preparation method.

[0056] Thirdly, the present application also provides the application of the above-mentioned recombinant human hyaluronidase in the biopharmaceutical field and the medical aesthetic field, which includes the preparation of subcutaneous injection preparations, tumor treatment drugs, gene therapy vectors, and the dissolution of hyaluronic acid from failed plastic surgery.

[0057] This application has the following beneficial effects:

[0058] The present application obtains a recombinant human hyaluronidase rHuPH20 by expressing and purifying the fusion protein SUMO protein-human hyaluronidase, and then cleaving it with His-ULP1 enzyme and then purifying it with a nickel column. This method can prepare rHuPH20 with high purity and good biological activity using only one chromatography medium, and has wide application value.

[0059] To make the purpose, technical solutions and advantages of the examples of the present application clearer, the technical solutions in the examples of the present application will be described clearly and completely below. Where specific conditions are not specified in the examples, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0060] 36-482aa in hyaluronidase PH20 is a structural domain that can basically maintain the original hyaluronidase activity. In order to prepare recombinant human hyaluronidase PH20 (rHuPH20) having the above-mentioned structural domain, the inventors of this application have proposed a new method for preparing recombinant human hyaluronidase rHuPH20 after extensive research. The preparation flow chart is shown in Figure 4: first, the fusion protein HissumoPH20 (SEQ ID NO: 2) is expressed and purified, and then the fusion protein is enzymatically cleaved using His-ULP1 (SEQ ID NO: 3), and then purified by Ni to obtain rHuPH20 (SEQ ID NO: 1).

[0061] In order to improve the expression level and final purity of rHuPH20 in recombinant cells, the inventors optimized the amino acids of the fusion protein: 6His HHHHHH was added to the N-terminus of SUMO (SEQ ID NO: 4), and the HissumoPH20 obtained after fusion is shown in SEQ ID NO: 2; at the same time, the inventors also carried out a series of optimizations on the fusion protein, including: adding a signal peptide signal sequence (SEQ ID NO: 5) to the N-terminus of the fusion protein, encoding a nucleotide sequence optimized for signal+HissumoPH20 using human-preferred codons (SEQ ID NO: 6), and on this basis, adding a Kozak sequence: GCCACC to the 5' end, and adding EcoR1 and BamH1 restriction sites at the 5' and 3' ends, respectively. The HissumoPH20 gene and related element sequences (SEQ ID NO: 7) were obtained by the above methods.

[0062] Furthermore, in order to improve the enzymatic cleavage effect and the purity of the obtained rHuPH20, the inventors optimized and screened the enzymatic cleavage conditions based on the above method and found that when the molar ratio of HissumoPH20 and His-ULP1 enzyme was 1:0.9-1.1, the enzymatic cleavage temperature was 30±2°C, and the enzymatic cleavage time was 4-8 hours, higher purity rHuPH20 could be obtained.

[0063] The following examples are provided to further illustrate the present application and should not be construed as limiting the present application. The examples do not include detailed descriptions of conventional methods, such as methods for expressing and purifying known amino acid sequences to obtain polypeptide fragments, methods for constructing vectors and plasmids, methods for inserting protein-encoding genes into vectors and plasmids, or methods for introducing plasmids into host cells. Such methods are well known to those skilled in the art and are described in numerous publications, including Sambrook, J., Fritsch, E.F., and Maniais, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press.

[0064] The detection methods used in the examples of this application are described as follows:

[0065] 1.SEC detection:

[0066] Liquid chromatography system: 1260 Infinity Agilent;

[0067] Mobile phase: 200 mM PB buffer pH 6.8;

[0068] Flow rate: 0.2 ml / min;

[0069] Column temperature: room temperature;

[0070] Sample temperature: 2-8 degrees;

[0071] Detection wavelength: 280nm;

[0072] Column model: SEC300A 2.7um 4.6x150mm.

[0073] 2. SDS-PAGE detection:

[0074] Detection system: Mini protein Tetra system;

[0075] Test conditions: 140V constant voltage for 45 to 55 minutes.

[0076] 3. UV detection:

[0077] Instrument model: Nanodrop one (thermo);

[0078] Extinction coefficient: 0.57.

[0079] 4. Metal ion Ni 2+ Affinity chromatography

[0080] Chromatography column: XK16 / 20 (GE);

[0081] Filler: Ni sepharose excel (GE);

[0082] Chromatography system: AKTA Pure150 (GE);

[0083] Operating system: Unicorn 7.0 (GE);

[0084] Flow rate: 5.0ml / min.

[0085] Example

[0086] This example is the preparation of rHuPH20, and the operation method is as follows:

[0087] (1) Design of HissumoPH20 amino acids

[0088] In order to facilitate purification, 6His: HHHHHH was added to the N-terminus of SUMO (SEQ ID NO: 4), and rHuPH20 was directly fused behind SUMO. The amino acid structure of the fused HissumoPH20 was 6His SUMO rHuPH20 (SEQ ID NO: 2).

[0089] (2) HissumoPH20 293 expression preparation

[0090] To secrete HissumoPH20 into the culture medium, a signal peptide sequence (SEQ ID NO: 5) was added to the N-terminus of HissumoPH20 (SEQ ID NO: 2), and a nucleotide sequence encoding signal+HissumoPH20 was optimized using human-preferred codons (SEQ ID NO: 6).

[0091] To increase the expression level, the Kozak sequence GCCACC was added to the 5' of signal+HissumoPH20 (SEQ ID NO: 6). To facilitate cloning, EcoR1 and BamH1 restriction sites were added to the 5' and 3' respectively to obtain the HissumoPH20 gene and related element sequence (SEQ ID NO: 7).

[0092] (3) Preparation of HissumoPH20 expression plasmid

[0093] The HissumoPH20 gene and related elements (SEQ ID NO: 7) were fully synthesized and inserted into pcDNA3.4 mcs to construct HissumoPH20-pcDNA3.4 and bacteria containing the HissumoPH20-pcDNA3.4 plasmid. The HissumoPH20-pcDNA3.4 plasmid was extracted in large quantities and sterilized by 0.22 μm sterile filtration.

[0094] (4) HissumoPH20 transfection expression

[0095] 1) Expi293F cells (Gibco TM ) Culture was expanded to a viable cell density of approximately 4×10 6 One day before transfection, the cells were split into flasks so that the viable cell density after splitting was 1.7×10 6 / mL and cultured overnight.

[0096] 2) On the day of transfection, measure the density and viability of live cells. The density of live cells used for transfection should be close to 3×10 6 / mL, and the activity needs to reach more than 95%.

[0097] 3) Preparation of a complex of transfection reagent PEI (ploysciences) and plasmid HissumoPH20-pcDNA3.4:

[0098] a. Prepare the required plasmid at a ratio of 1.3 mg of plasmid per 1 L of cell culture. Dilute the plasmid DNA with serum-free medium (SFM) (Gibco), mix by inversion, and incubate at room temperature for 2 minutes.

[0099] b. Prepare the required amount of transfection reagent according to the ratio of transfection reagent (μL): DNA (μg) = 1:4. Dilute the transfection reagent PEI (ploysciences) in serum-free medium (SFM). Invert or gently pipette three times to mix, and incubate at room temperature for 2 minutes.

[0100] c. Add the diluted PEI reagent to the diluted plasmid DNA, mix by inverting or gently pipetting 2-3 times, and incubate at room temperature for 7 minutes.

[0101] Slowly transfer the mixture prepared in the previous step to the flask in step 2), gently rotating the flask during the addition process, and incubate the cells in a shaking incubator at 37°C and 8% CO2.

[0102] 4) On the first day after transfection (i.e. 20 hours after transfection), add ExpiFectamine TM 293(Gibco TM ) Transfection Enhancer 1 and 2 (thermofisher) were added to the culture flask. The shake flask was gently shaken during the addition process, and the shake flask was moved to a 37°C incubator and cultured with shaking in a humidified atmosphere of 8% CO2.

[0103] 5) Harvest protein on day 6 after transfection. Centrifuge at 9000 rpm at 4°C for 50 min and collect the supernatant for purification.

[0104] (5) HissumoPH20 purification

[0105] Use 1000 mL of equilibration solution to equilibrate the Ni chromatography column, use the supernatant as the loading solution, add imidazole to the loading solution to make the imidazole concentration 20 mM, load the sample, equilibrate with equilibration solution until the ultraviolet absorbance value remains unchanged at 280, elute with 200 mL of eluent, and collect the elution peak.

[0106] The components of the balancing solution are: PBS, 20 mM imidazole, pH=7.4; the components of the elution solution are: PBS, 500 mM imidazole, pH=7.4.

[0107] (6) HissumoPH20 digestion and rHuPH20 purification

[0108] The HissumoPH20 and His-ULP1 enzymes were prepared into an enzyme digestion solution with a molar ratio of 1:1. The enzyme digestion solution was passed through a pre-equilibrated Ni chromatography column at 30±2°C for 4 hours. The flow-through was collected to obtain high-purity rHuPH20. The obtained rHuPH20 was tested for purity, and the SEC purity reached above 99.28%, with a maximum of 100%.

[0109] Figure 5 shows the expression, purification and enzyme digestion of rHuPH20 fusion protein by SDS-PAGE, wherein M: protein marker; A: fusion protein HissumoPH20 expressed in 293T cells and purified by Ni column; B: enzyme digestion solution; C: enzyme digestion solution flow-through Ni column.

[0110] FIG6 and FIG7 are SEC spectra of the high-purity rHuPH20 prepared in the present application.

[0111] Experimental Example 1

[0112] This experimental example is a mass spectrometry test of rHuPH20 obtained in Example, specifically as follows:

[0113] 1) Mass spectrometry conditions:

[0114] A Waters UPLC-XEVO G2 Q-TOF liquid chromatography-mass spectrometry system was used. The system configuration for the liquid chromatography phase included a BSM binary high-pressure mixing pump, an SM sample manager, and a TUV ultraviolet detector; the mass spectrometry configuration included an ESI source and a Q-TOF detector. Data were processed and analyzed using Masslynx V4.1 and BiopharmaLynx analysis software (Version 1.2).

[0115] MS data were collected in continuum mode and in Resolution mode; LockSpray acquisition mode was real-time acquisition with no calibration applied.

[0116] Calibration solution: Real-time calibration (LockSpray) solution: 2 ng / μL LE solution;

[0117] Calibration solution for mass axis: 2 μg / μL sodium iodide solution.

[0118] The mass spectrometry parameters are shown in Table 1:

[0119] Table 1 Mass spectrometry parameters

[0120] 2) Liquid phase conditions:

[0121] Chromatographic column: Mass PREP™ Micro Desalting Column 2.1 5 mm (intact protein molecular weight analysis), column temperature: 80°C;

[0122] Mobile phase A: 0.1% FA-H2O;

[0123] Mobile phase B: 0.1% FA-CAN;

[0124] Seal Wash solution: 10% IPA;

[0125] Mass spectrometer cleaning solution: 50% CAN;

[0126] Mass spectrometer IntelliStart valve cleaning solution: 50% MeOH;

[0127] Injection volume: 10 μL;

[0128] Sample chamber temperature: 10°C;

[0129] The gradient elution conditions are shown in Table 2:

[0130] Table 2 Gradient elution conditions

[0131] The mass spectrometry detection results are shown in Figure 8 and Table 3:

[0132] Table 3 Deglycosylated intact molecular weight of rHuPH20 samples

[0133] As can be seen from the results in Figure 8 and Table 3, the rHuPH20 obtained in this application is relatively uniform and basically consistent with the target product.

[0134] Experimental Example 2

[0135] This experimental example is for the in vitro activity detection of rHuPH20. The detection method refers to the pharmacopoeia. The specific operation is as follows:

[0136] 1. Reagent Preparation

[0137] ① Acetic acid-sodium acetate buffer: Take 11.73g of sodium acetate and 20.5ml of glacial acetic acid, then dilute with water to 1000ml.

[0138] ② Phosphate buffer: Take 2.5g of sodium dihydrogen phosphate dihydrate, 1.0g of anhydrous disodium hydrogen phosphate and 8.2g of sodium chloride, and dissolve them in water to 1000mL.

[0139] ③ Hydrolyzed gelatin solution: Take 125 ml of phosphate buffer and 125 ml of water, add 165 mg of hydrolyzed gelatin, shake well, and store at 0-4°C. If the solution does not become turbid, it can be used again.

[0140] ④ Serum stock solution: Take 1 part FBS and dilute it with 9 parts acetic acid-sodium acetate buffer. Then adjust the pH to 3.1 with 4 mol / L hydrochloric acid solution. Let it stand for 18-24 hours before use. Store at 0-4°C and it can be used for 30 days.

[0141] ⑤ Hyaluronic acid stock solution: Take hyaluronic acid powder and add water to make a 1mg / ml solution. Store below 0℃ and use for 30 days.

[0142] ⑥ Hyaluronic acid solution: Take 1 part hyaluronic acid stock solution and dilute it with 1 part phosphate buffer. Prepare it just before use.

[0143] 2. Standard Preparation (Operate on Ice)

[0144] ① Preparation of standard solution: Accurately weigh an appropriate amount of hyaluronidase (577 IU / mg) standard and add cold hydrolyzed gelatin diluent to a solution containing 100 units per 1 ml. Aliquot and store at -80°C until needed. The standard used in this study was stored at -80°C (20230131).

[0145] ② Preparation of standard curve samples: rHuPH20 standard curve samples are shown in Table 4, and the standard curve is shown in Figure 9.

[0146] Table 4 rHuPH20 standard curve samples

[0147] 3. Sample preparation (operation on ice)

[0148] Dilute the sample to the appropriate concentration using hydrolyzed gelatin solution.

[0149] 4. Operation steps

[0150] ① Prepare the standard and the sample to be tested, and dilute them to the appropriate concentration with hydrolyzed gelatin solution.

[0151] ② Add 100 μl of the standard or sample solution to 100 μl of hyaluronic acid solution and incubate at 37°C in a water bath for 30 minutes, carefully controlling the time. For the blank control, add 100 μl of hydrolyzed gelatin solution to 100 μl of phosphate buffer, also incubated at 37°C in a water bath for 30 minutes.

[0152] ③After the water bath ends, add 800 μl of serum stock solution to each tube immediately, mix well and let it stand at room temperature for 30 minutes.

[0153] ④ Transfer to a 96-well plate, 200 μl per well, and read the plate using A640.

[0154] According to the Chinese Pharmacopoeia method turbidimetric test, the activity test results of three batches of rHuPH20 samples are shown in Table 5:

[0155] Table 5 Activity test results of three batches of rHuPH20 samples

[0156] From the results in Table 5, it can be found that the activity of rHuPH20 prepared in the present application is higher than 75,000 IU / mg in three batches, indicating that the rHuPH20 of the present application has good biological activity.

[0157] The above description is merely a preferred embodiment of the present application and is not intended to limit the present application. Various modifications and variations are possible for those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present application shall be included within the scope of protection of the present application. Industrial Applicability

[0158] The present application obtains a recombinant human hyaluronidase rHuPH20 by expressing and purifying the fusion protein SUMO protein-human hyaluronidase, and then cleaving it with His-ULP1 enzyme and then purifying it with a nickel column. This method can prepare rHuPH20 with high purity and good biological activity using only one chromatography medium, and has wide application value.

Claims

1. A method for preparing recombinant human hyaluronidase, characterized in that, Comprising: Subjecting the fusion protein SUMO protein-human hyaluronidase to digestion with His-ULP1 enzyme and then purifying it by nickel column to obtain the recombinant human hyaluronidase; The amino acid sequence of the recombinant human hyaluronidase is as shown in SEQ ID NO:1; the amino acid sequence of the fusion protein SUMO protein-human hyaluronidase is as shown in SEQ ID NO:2; the amino acid sequence of the His-ULP1 enzyme is as shown in SEQ ID NO:

3.

2. The method according to claim 1, characterized in that, When performing digestion, the molar ratio of the fusion protein SUMO protein-human hyaluronidase to the His-ULP1 enzyme is 1:0.9 - 1.

1.

3. The method according to claim 2, wherein The digestion conditions are: temperature is 30 ± 2 °C, and the digestion time is 4 - 8 h.

4. The method according to claim 1, characterized in that, The expression of the fusion protein SUMO protein-human hyaluronidase includes: constructing a recombinant bacterium containing the gene fragment of the fusion protein SUMO protein-human hyaluronidase, then obtaining a recombinant plasmid from the recombinant bacterium, and then transfecting the recombinant plasmid into a host cell, culturing and collecting the supernatant, and centrifuging to remove cells to obtain a crude product of the fusion protein SUMO protein-human hyaluronidase.

5. The method according to claim 4, characterized in that, The preparation method of the recombinant bacterium includes: inserting the gene fragment containing the fusion protein SUMO protein-human hyaluronidase into pcDNA3.4 mcs, and then transferring it into a host bacterium to obtain the recombinant bacterium; The host cell includes 293 cells or CHO cells.

6. The method according to claim 4 or 5, characterized in that, The gene fragment includes the nucleotide sequence of the fusion protein SUMO protein-human hyaluronidase and related elements; The related elements include: a signal peptide added at the N-terminus, a Kozak sequence at the 5'-end, an EcoR1 cleavage site at the 5'-end, and a BamH1 cleavage site at the 3'-end; Preferably, the amino acid sequence of the signal peptide is as shown in SEQ ID NO:5; the nucleotide sequence of the Kozak sequence is GCCACC; the nucleotide sequence of the gene fragment is as shown in SEQ ID NO:

7.

7. The method according to claim 4, characterized in that The transfection includes: transferring the diluted recombinant plasmid and transfection reagent into a cell culture containing host cells, and culturing with shaking at 37 °C in a humidified atmosphere of 5 - 8% CO2 for 5 - 7 d; The viable cell density in the cell culture is 2.5×10 6 -3.5×10 6 cells / mL, the volume-mass ratio of the cell culture to the recombinant plasmid is 1:1.2 - 1.4 (L / mg), and the volume-mass ratio of the transfection reagent to the recombinant plasmid is 1:3 - 5 (μL / μg).

8. The method according to claim 1, characterized in that, The method further includes purifying the crude product of the fusion protein SUMO protein-human hyaluronidase before digestion, and the purification method is: equilibrating a nickel ion chromatography column with an equilibration solution, adding a sample loading solution, then equilibrating the nickel ion chromatography column with the equilibration solution, and then eluting to obtain the purified fusion protein SUMO protein-human hyaluronidase; The equilibration solution includes PBS and 20 - 40 mM imidazole, and its pH = 7.4 ± 0.2; The elution solution includes PBS and 250 - 500 mM imidazole, and its pH = 7.4 ± 0.2; The sample loading solution includes the crude product of the fusion protein SUMO protein-human hyaluronidase and 20 - 40 mM imidazole.

9. The recombinant human hyaluronidase prepared by the method according to any one of claims 1 - 8.

10. Use of the recombinant human hyaluronidase according to claim 9 in the fields of biopharmaceuticals and medical aesthetics, characterized in that, The applications include preparing a subcutaneous injection preparation, a tumor treatment drug, a gene therapy vector, and dissolving hyaluronic acid for failed plastic surgery.

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