Recombinant human hyaluronidase preparation method and use

Abstract

The present application discloses a recombinant human hyaluronidase preparation method and a use. The preparation method comprises: subjecting a SUMO protein-human hyaluronidase fusion protein to His-ULP1 enzyme digestion, then performing nickel column purification, to obtain a recombinant human hyaluronidase. The described preparation method does not require the involvement of multiple different types of chromatography media, and can obtain recombinant human hyaluronidase having high purity, the obtained rHuPH20 having good biological activity.

Description

Method for preparing recombinant human hyaluronidase and application thereof

[0001] Cross-reference to Related Applications

[0002] This application claims priority to the Chinese patent application No. 202311846834.X, filed on December 28, 2023, and entitled “Method for preparing recombinant human hyaluronidase and application thereof”, the content of which is incorporated herein by reference in its entirety. TECHNICAL FIELD

[0003] The present application relates to the technical field of biopharmaceuticals, in particular, to a method for preparing recombinant human hyaluronidase and application thereof. BACKGROUND

[0004] PH20 is an enzyme (ec3.2.1.35) that belongs to the hyaluronidase family, which cleaves the beta-1,4 bond between n-acetylglucosamine and glucuronic acid (sugars that make up hyaluronic acid). Hyaluronidases degrade hyaluronic acid, a proteoglycan in the extracellular matrix and basement membrane. Six members of the hyaluronidase family are clustered into two tightly linked groups on chromosomes 3p21.3 and 7q31.3. This gene encodes a GPI-anchored enzyme that is located on the surface of human spermatozoa and the inner acrosomal membrane. Hyaluronidases enable sperm to penetrate the cumulus cell layer surrounding the oocyte, which is rich in hyaluronic acid, are receptors for hyaluronic acid-induced cell signaling, and are involved in sperm-zona pellucida adhesion. Abnormal expression of this gene in tumors is associated with degradation of the basement membrane, leading to tumor invasion and metastasis. Multiple transcript variants encoding different isoforms of this gene have been found.

[0005] PH20 is a glycosylphosphatidylinositol (GPI)-anchored single-chain membrane glycoprotein, and its structure has the typical features of GPI attachment except for the signal peptide. PH20 at different positions will have slight differences in glycosylation levels and proteolytic forms, but its hyaluronidase activity is dependent on the secondary and tertiary structures maintained by its disulfide bonds and glycosylation. It contains six functional N-linked glycosylation sites (82, 166, 235, 254, 368, 393), all of which are linked to oligosaccharides, and the main sugar at the end of the glycan is mannose and there are five sets of intrachain disulfide bonds formed by cysteine; 36-464aa is the domain that can retain the lowest activity; 36-482aa can basically maintain the original hyaluronidase activity, and the anchoring site 490aa is not essential for activity. The structure diagram and spatial structure diagram of hyaluronidase PH20 (data from AlphaFold) are shown in Figures 1 and 2, respectively.

[0006] PH20 is found in the testes, epididymis, female reproductive tract, chest, intestines, and malignant tumors. PH20 is the only hyaluronidase active under neutral conditions, making it optimally suited to the body's physiological environment. Therefore, it has wide applications in the pharmaceutical field. On one hand, it facilitates drug delivery by allowing the drug to overcome the physical barrier of the extracellular matrix; on the other hand, it can be used for targeted therapy of the tumor microenvironment; furthermore, it also plays a role in gene therapy. Specifically, the applications of PH20 are as follows:

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

[0008] The optimal pH for human hyaluronidase PH20 is 5.5, but it exhibits some activity even at pH 7-8. Other human hyaluronidases (including hyal1) have an optimal pH of 3-4 and exhibit very weak activity at pH 7-8. The pH of the human subcutaneous region is approximately 7.4, which is generally neutral. Therefore, among various types of hyaluronidase, PH20 is widely used in clinical applications.

[0009] As early as 1948, the US FDA approved hyaluronidase preparations for marketing. In 2005, Halozyme Therapeutics' Enhanceze drug delivery technology, based on recombinant human hyaluronidase PH20 (rHuPH20), was rapidly adopted in the United States. Prior to this, hyaluronidase preparations were mostly extracted from animal (bovine, ovine) testicular tissue, and the prepared HA often had disadvantages such as low purity, high content of impurities and proteins, low activity, and strong immunogenicity. Side effects included allergic reactions, requiring skin allergy tests before injection, which limited its application in promoting the absorption of nutritional factors and drugs. In contrast, Halozyme Therapeutics' recombinant human hyaluronidase is free of animal-derived components, has high purity, high activity, no immunogenicity, and fewer side effects. Therefore, it is widely used... The technology-based drugs have been approved by the FDA, and some of the earlier approved drug formulations include... SC, The evolution of human hyaluronidase ph20 (HyQvia) 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's approved antibody cocktail therapy Phesgo, also administered subcutaneously, is used to treat adult HER2+ metastatic breast cancer and early HER2+ breast cancer. Many pharmaceutical companies are seeking greater competitiveness in formulation to meet the needs of healthcare professionals and patients. Hyaluronidase has also been used clinically for many years as a drug penetration enhancer to promote drug absorption.

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

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

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

[0014] Recombinant human hyaluronidase also has some applications in gene therapy, as the efficacy of most gene delivery vectors in vivo does not match the efficacy observed in vitro. Glycosaminoglycans can inhibit the transfer and dissemination of DNA and viral vectors into many cell types, and the level of extracellular matrix material can significantly hinder this process. The use of hyaluronidase can open channels in the extracellular matrix, thereby enhancing gene therapy delivery.

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

[0016] Hyaluronic acid, also known as hyaluronic acid, and hyaluronidase, also known as hyaluronidase, are natural enzymes that hydrolyze hyaluronic acid. They can eliminate injected material in unsuitable areas and remove lumps caused by excessive hyaluronic acid injection, with an elimination rate of up to 90%.

[0017] Primarily used to correct failed hyaluronic acid injections, it's considered a remedy for regrets in the world of minimally invasive cosmetic procedures. If you're not satisfied with the results after hyaluronic acid injections, you can dissolve the hyaluronic acid with hyaluronidase within 48 hours, and then have it filled again with hyaluronic acid.

[0018] However, currently available recombinant human hyaluronidase PH20 (rHuPH20) is mainly produced through direct expression in CHO cells, secreted into the culture supernatant. The culture medium is then centrifuged at high speed, the supernatant is collected, concentrated by ultrafiltration, and then purified using complex methods such as anion exchange (Q), hydrophobic chromatography, aminophenylboronic acid chromatography, hydroxyapatite, and cation exchange (SP). These methods involve numerous steps and require various types of chromatographic media, making the preparation process quite cumbersome.

[0019] Therefore, this application is hereby submitted. Summary of the Invention

[0020] The purpose of this application is to provide a method for preparing recombinant human hyaluronidase. This method is simple and does not require the participation of multiple different types of chromatography media. This method can not only obtain recombinant human hyaluronidase with high purity, but also ensure that the obtained rHuPH20 has a 36-482aa hyaluronidase activity fraction that can basically maintain the original hyaluronidase activity.

[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 agents.

[0022] This application is implemented as follows:

[0023] In a first aspect, this application provides a method for preparing recombinant human hyaluronidase, which includes: digesting the fusion protein SUMO protein-human hyaluronidase with His-ULP1 enzyme and then purifying it with a nickel column 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 His-ULP1 enzyme is shown in SEQ ID NO:3.

[0025] Secondly, this application provides recombinant human hyaluronidase obtained by the above preparation method.

[0026] Thirdly, this application also provides the application of the above-mentioned recombinant human hyaluronidase in the fields of biopharmaceuticals and medical aesthetics, including the preparation of subcutaneous injection preparations, tumor treatment drugs, gene therapy vectors, and the dissolution of hyaluronic acid from failed plastic surgery. Attached Figure Description

[0027] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of this application and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 shows the structure of hyaluronidase PH20;

[0029] Figure 2 is a spatial diagram of hyaluronidase PH20;

[0030] Figure 3 shows the development process of hyaluronidase PH20.

[0031] Figure 4 is a flowchart of the rHuPH20 preparation process in this application;

[0032] Figure 5 shows the expression, purification, and SDS-PAGE of the rHuPH20 fusion protein in the example.

[0033] Figure 6 shows the SEC spectrum of the high-purity rHuPH20 prepared in this application;

[0034] Figure 7 shows the SEC spectrum of high-purity rHuPH20 prepared in this application;

[0035] Figure 8 shows the rHuPH20 mass spectrometry detection results in Experiment Example 1;

[0036] Figure 9 shows the standard curve of rHuPH20 in Experiment Example 2. Detailed Implementation

[0037] The purpose of this application is to provide a method for preparing recombinant human hyaluronidase. This method is simple and does not require the participation of multiple different types of chromatography media. This method can not only obtain recombinant human hyaluronidase with high purity, but also ensure that the obtained rHuPH20 has a 36-482aa hyaluronidase activity fraction that can basically maintain the original hyaluronidase activity.

[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 agents.

[0039] This application is implemented as follows:

[0040] In a first aspect, this application provides a method for preparing recombinant human hyaluronidase, which includes: digesting the fusion protein SUMO protein-human hyaluronidase with His-ULP1 enzyme and then purifying it with a nickel column 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 His-ULP1 enzyme is shown in SEQ ID NO:3.

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

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

[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, obtaining a recombinant plasmid from the recombinant bacterium, transfecting the recombinant plasmid into host cells, culturing and collecting the supernatant, centrifuging to remove cell debris, and obtaining crude fusion protein SUMO protein-human hyaluronidase.

[0045] In some embodiments, the method for preparing recombinant bacteria includes: inserting a gene fragment containing the fusion protein SUMO protein-human hyaluronidase into pcDNA3.4 mcs, and then transferring it into host bacteria to obtain 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 at 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 includes: transferring the diluted recombinant plasmid and transfection reagent into a cell culture containing host cells, and culturing with shaking at 37°C and 5-8% CO2 for 5-7 days;

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

[0051] In some embodiments, the above method further includes purifying the crude fusion protein SUMO protein-human hyaluronidase before enzymatic digestion. The purification method is as follows: equilibrate the nickel ion chromatography column with equilibration buffer, add the sample loading solution, equilibrate the nickel ion chromatography column with equilibration buffer again, and then elute to obtain the purified fusion protein SUMO protein-human hyaluronidase.

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

[0053] The elution buffer contains PBS and 250-500 mM imidazole, with a pH of 7.4 ± 0.2.

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

[0055] Secondly, this application provides recombinant human hyaluronidase obtained by the above preparation method.

[0056] Thirdly, this application also provides the application of the above-mentioned recombinant human hyaluronidase in the fields of biopharmaceuticals and medical aesthetics, including 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] This application describes a recombinant human hyaluronidase rHuPH20 obtained by expressing and purifying the fusion protein SUMO protein-human hyaluronidase, followed by His-ULP1 digestion and nickel column purification. This method can prepare rHuPH20 with high purity and good biological activity using only one chromatography medium, and has broad application value.

[0059] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0060] The 36-482aa domain in hyaluronidase PH20 is a structural domain that can basically maintain the original hyaluronidase activity. In order to prepare recombinant human hyaluronidase PH20 (rHuPH20) with the above-mentioned structural domain, the inventors 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. Then, the fusion protein is digested with His-ULP1 (SEQ ID NO:3) and purified by Ni flow-through to obtain rHuPH20 (SEQ ID NO:1).

[0061] To improve the expression level and final purity of rHuPH20 in recombinant cells, the inventors optimized the amino acid composition of the fusion protein: 6His HHHHHH was added to the N-terminus of SUMO (SEQ ID NO: 4), resulting in HissumoPH20 as shown in SEQ ID NO: 2. Simultaneously, the inventors further optimized the fusion protein by adding a signal peptide sequence (SEQ ID NO: 5) to the N-terminus, creating a nucleotide sequence (SEQ ID NO: 6) encoding signal+HissumoPH20 with human-preferred codons, and adding the Kozak sequence (GCCACC) to the 5' end, along with EcoR1 and BamH1 restriction sites at the 5' and 3' ends, respectively. This resulted in the HissumoPH20 gene and related element sequences (SEQ ID NO: 7).

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

[0063] The following examples are further illustrations of this application and should not be construed as limiting the application in any way. The examples do not include detailed descriptions of conventional methods, such as those for expressing and purifying known amino acid sequences to obtain polypeptide fragments, methods for constructing vectors and plasmids, methods for inserting genes encoding proteins into vectors and plasmids, or methods for introducing plasmids into host cells. Such methods are well known to those skilled in the art and have been 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 embodiments of this application are described below:

[0065] 1. SEC inspection:

[0066] Liquid chromatography system: 1260 Infinity Agilent;

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

[0068] Flow rate: 0.2 ml / min;

[0069] Column temperature: room temperature;

[0070] Sample temperature: 2–8 degrees Celsius;

[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] Testing conditions: 140V constant voltage for 45-55 minutes.

[0076] 3. Ultraviolet detection:

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

[0078] Extinction coefficient: 0.57.

[0079] 4. Metal ions 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: 5.0 ml / min.

[0085] Example

[0086] This example describes the preparation of rHuPH20, and the procedure is as follows:

[0087] (1) Design of HissumoPH20 amino acids

[0088] To facilitate purification, 6His:HHHHHH was added to the N-terminus of SUMO (SEQ ID NO: 4), and rHuPH20 was directly fused after SUMO. The amino acid structure of the fused HissumoPH20 is 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), encoding a nucleotide sequence of signal+HissumoPH20 optimized with human-preferred codons (SEQ ID NO:6).

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

[0092] (3) Preparation of HissumoPH20 expression plasmid

[0093] The HissumoPH20 gene and related elements (SEQ ID NO:7) were 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 aseptic filtration at 0.22 μm.

[0094] (4) HissumoPH20 transfection expression

[0095] 1) Exponential 293F cells (Gibco) TM Subculturing to achieve a viable cell density of approximately 4 × 10⁻⁶ cells / year 6 Cells / mL. The day before transfection, the cells were aliquoted into flasks to achieve a viable cell density of 1.7 × 10⁶ cells / mL. 6 The sample was collected at a concentration of 100 cells / mL and incubated overnight.

[0096] 2) On the day of transfection, measure the density and viability of live cells. The density of live cells used for transfection needs to be close to 3 × 10⁻⁶. 6 The number of cells / mL needs to reach a vitality of over 95%.

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

[0098] a. Prepare the required plasmid according to the ratio of 1.3 mg 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) with serum-free medium (SFM), mix by inverting or gently blowing and aspirating 3 times, and then incubate at room temperature for 2 minutes.

[0100] c. Add the diluted PEI reagent to the diluted plasmid DNA, invert or gently blow and aspirate 2-3 times to mix, and then 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 as you add the mixture. Incubate the cells in a shaker at 37°C with 8% CO2.

[0102] 4) On the first day after transfection (i.e., 20 hours after transfection), add ExpiFectamine. TM 293 (Gibco) TM Transfection enhancers 1 and 2 (thermofisher) were added to the culture flasks. The flasks were gently shaken during the addition process. The flasks were then transferred to a 37°C incubator and cultured with shaking in humidified air containing 8% CO2.

[0103] 5) Harvest the protein on day 6 post-transfection. Centrifuge at 9000 rpm at 4℃ for 50 min, and collect the supernatant for purification.

[0104] (5) HissumoPH20 purification

[0105] The Ni chromatography column was equilibrated with 1000 mL of equilibration buffer, and the supernatant was used as the sample loading solution. Imidazole was added to the sample loading solution to make the imidazole concentration 20 mM. The sample was loaded and equilibrated with equilibration buffer until the UV absorbance remained constant at 280. Elution was then carried out with 200 mL of elution buffer, and the elution peak was collected.

[0106] The equilibration buffer consisted of PBS, 20 mM imidazole, and pH 7.4; the elution buffer consisted of PBS, 500 mM imidazole, and pH 7.4.

[0107] (6) HissumoPH20 was digested and rHuPH20 was purified.

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

[0109] Figure 5 shows the expression, purification, and SDS-PAGE of the rHuPH20 fusion protein. In the figure, M represents the protein marker; A represents the expression of the HissumoPH20 fusion protein in 293T cells, purified using a Ni column; B represents the enzyme digestion buffer; and C represents the Ni column flow-through of the enzyme digestion buffer.

[0110] Figures 6 and 7 are SEC spectra of high-purity rHuPH20 prepared in this application, respectively.

[0111] Experimental Example 1

[0112] This experimental example demonstrates the mass spectrometry detection of rHuPH20 obtained in the previous embodiment, as detailed below:

[0113] 1) Mass spectrometry conditions:

[0114] The Waters UPLC-XEVO G2 Q-TOF liquid chromatography-mass spectrometry (LC-MS) system was configured with: a BSM binary high-pressure mixer pump, an SM sample manager, and a TUV UV detector; the mass spectrometry system consisted of an ESI source and a Q-TOF detector. Data processing and analysis were performed using Masslynx V4.1 and BiopharmaLynx analysis software (Version 1.2).

[0115] MS data are all acquired in continuum mode under resolution mode; LockSpray acquisition mode is real-time acquisition without calibration.

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

[0117] Mass axis calibration solution: 2 μg / μL sodium iodide solution.

[0118] 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 5mm (for whole protein molecular weight analysis), column temperature: 80℃;

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

[0127] Injection volume: 10 μL;

[0128] Sample chamber temperature: 10℃;

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

[0130] Table 2 Gradient elution conditions

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

[0132] Table 3. Deglycosylated complete 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 demonstrates the in vitro activity assay of rHuPH20. The assay method follows the pharmacopoeia guidelines, and the specific procedures are as follows:

[0136] I. Reagent Preparation

[0137] ① Acetic acid-sodium acetate buffer: Take 11.73g of sodium acetate and 20.5ml of glacial acetic acid, and 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 each of phosphate buffer and water, add 165 mg of hydrolyzed gelatin, shake well, and store at 0–4℃. If the solution does not become cloudy, it can be used.

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

[0141] ⑤ Hyaluronic acid stock solution: Take hyaluronic acid powder and add water to prepare a solution with a concentration of 1 mg / ml. Store below 0°C; it can be used for 30 days.

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

[0143] II. Preparation of Standard Products (Ice Operations)

[0144] ① Preparation of standard solution: Accurately weigh an appropriate amount of hyaluronidase (577 IU / mg) standard, and dilute with cooled hydrolyzed gelatin solution to prepare a solution containing 100 units per ml. Aliquot and store at -80℃ for later use. The standard used in this study was frozen at -80℃ (20230131).

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

[0146] Table 4. Samples of rHuPH20 Standard Curve

[0147] III. Sample Preparation (Operation on Ice)

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

[0149] IV. Operating Procedures

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

[0151] ② Add 100 μl of hyaluronic acid solution to 100 μl of standard or test sample solution, and incubate at 37°C for 30 min, controlling the time precisely. The blank control is 100 μl of hydrolyzed gelatin solution plus 100 μl of phosphate buffer, and incubate at 37°C for 30 min as well.

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

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

[0154] The activity test results of three batches of rHuPH20 samples, determined by turbidimetric assay according to the Chinese Pharmacopoeia method, are shown in Table 5.

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

[0156] The results in Table 5 show that the rHuPH20 prepared in this application had an average activity greater than 75,000 IU / mg in three batches, indicating that the rHuPH20 prepared in this application has good biological activity.

[0157] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application. Industrial applicability

[0158] This application describes a recombinant human hyaluronidase rHuPH20 obtained by expressing and purifying the fusion protein SUMO protein-human hyaluronidase, followed by His-ULP1 digestion and nickel column purification. This method can prepare rHuPH20 with high purity and good biological activity using only one chromatography medium, and has broad application value.

Claims

1. A method for preparing recombinant human hyaluronidase, characterized in that, include: The fusion protein SUMO protein-human hyaluronidase was digested with His-ULP1 enzyme and then purified by nickel column chromatography to obtain the recombinant human hyaluronidase. The amino acid sequence of the 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.

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

1.

3. The method according to claim 2, characterized in that, The enzyme digestion conditions are: temperature 30±2℃, digestion time 4-8h.

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 a gene fragment of the fusion protein SUMO protein-human hyaluronidase, then obtaining a recombinant plasmid from the recombinant bacterium, transfecting the recombinant plasmid into host cells, culturing and collecting the supernatant, centrifuging to remove cells, and obtaining crude fusion protein SUMO protein-human hyaluronidase.

5. The method according to claim 4, characterized in that, 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 it into host bacteria to obtain the recombinant bacteria; The host cells include 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 relevant 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. 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; and 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 and 5-8% CO2 for 5-7 days; The viable cell density in the cell culture was 2.5 × 10⁻⁶. 6 -3.5×10 6 The cell culture volume ratio to recombinant plasmid is 1:1.2-1.4 (L / mg), and the transfection reagent volume ratio to 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 fusion protein SUMO protein-human hyaluronidase before enzymatic digestion. The purification method is as follows: equilibrate the nickel ion chromatography column with equilibration buffer, add the loading solution, equilibrate the nickel ion chromatography column with equilibration buffer again, and then elute to obtain the purified fusion protein SUMO protein-human hyaluronidase. The equilibration solution contains PBS and 20–40 mM imidazole, with a pH of 7.4 ± 0.

2. The elution buffer contains PBS and 250–500 mM imidazole, with a pH of 7.4 ± 0.

2. The loading solution includes the fusion protein SUMO protein-human hyaluronidase crude product and 20-40 mM imidazole.

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

10. The application of the recombinant human hyaluronidase as described in claim 9 in the fields of biopharmaceuticals and medical aesthetics, characterized in that, The applications include the preparation of subcutaneous injection formulations, tumor treatment drugs, gene therapy vectors, and the dissolution of failed hyaluronic acid reshaping.

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