Preparation method of efficient recombinant human hyaluronidase

Abstract

The invention provides an efficient preparation method of recombinant human hyaluronidase, which is suitable for large-scale production of recombinant human hyaluronidase. According to the method, engineering cells containing low-copy nucleic acid for coding soluble recombinant human hyaluronidase are utilized, and high-efficiency expression of the recombinant human hyaluronidase is realized by adding supplementary materials and regulating and controlling parameters such as temperature and pH in a culture process. The method for preparing the recombinant human hyaluronidase has the advantages of being simple in process, stable, easy to operate, mild and easy to control in condition, low in cost, high in yield, high in enzyme activity and purity and the like, and industrial production is easy.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to a method for preparing recombinant human hyaluronidase. Background Art

[0002] Recombinant human hyaluronidase (rHuPH20) has the unique ability to decompose hyaluronic acid. Hyaluronic acid is a polysaccharide widely present in human tissues, especially in the skin, joint fluid and vitreous body of the eye. Due to its excellent moisturizing and lubricating properties, hyaluronic acid has a wide range of applications in medicine and cosmetology. However, in some cases, such as cataract surgery or soft tissue injection, too much hyaluronic acid can hinder drug diffusion and surgical operations, and hyaluronidase is needed to regulate its concentration.

[0003] Clinically, recombinant human hyaluronidase is mainly used to enhance the effect of local anesthesia, promote subcutaneous drug diffusion, assist in ophthalmic surgery, and treat adverse reactions caused by hyaluronic acid injection. Its use has greatly improved the safety and efficiency of surgery and the patient's treatment experience.

[0004] Traditional hyaluronidase is extracted from animal testicles, which is not only expensive but may also cause immune reactions. The emergence of recombinant human hyaluronidase solves these problems. By introducing the human hyaluronidase gene into bacteria, yeast and mammalian cells, their biosynthetic mechanisms are used to produce proteins with the same structure as the hyaluronidase naturally produced by the human body. This method not only improves yield and purity, but also reduces production costs and potential immune risks.

[0005] In industrial production, recombinant human hyaluronidase has problems such as difficulty in expression, many impurities in the product, and instability. In order to obtain high-titer, high-purity and high-activity products, the experimenters developed an engineered cell line, cell culture process and production method for expressing recombinant human hyaluronidase. This method uses cells containing low copies of nucleic acid encoding soluble hyaluronidase to achieve efficient expression of recombinant human hyaluronidase through fed-batch culture. Summary of the invention

[0006] The present disclosure provides a method for preparing recombinant human hyaluronidase, which can facilitate efficient expression and large-scale production of hyaluronidase.

[0007] In order to achieve the above objectives, the present disclosure adopts the following technical solutions.

[0008] The present disclosure provides a method for efficiently preparing recombinant human hyaluronidase, the method comprising the following steps:

[0009] (1) obtaining an engineered mammalian cell capable of producing recombinant human hyaluronidase;

[0010] (2) Cultivating the cells of step (1), obtaining cell culture fluid and isolating and purifying the fluid to obtain recombinant human hyaluronidase.

[0011] In one embodiment, the engineered mammalian cell capable of producing recombinant human hyaluronidase is constructed by genetic engineering methods.

[0012] In one embodiment, the mammalian cell contains a nucleic acid encoding human hyaluronidase. Preferably, the number of copies of the nucleic acid encoding human hyaluronidase contained in the cell is less than or equal to 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1.

[0013] In one embodiment, the mammalian cell is a CHO cell. Preferably, the CHO cell is selected from the group consisting of the following cell strains:

[0014] DXB-11, DG-44, CHO-S, CHO-K1, CHO-K1(GS-KO) and other cell lines.

[0015] Further preferably, the CHO cells are CHOZN cells.

[0016] In one embodiment, the culture method is a fed-batch culture method, and the culture medium used includes a basal culture medium and a feed culture medium.

[0017] In one embodiment, the temperature range of the culturing method is 28-37°C.

[0018] Preferably, the amino acid sequence of the human hyaluronidase is shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.

[0019] In one embodiment, the human hyaluronidase comprises the amino acid sequence shown in SEQ ID NO:1.

[0020] Table 1 Amino acid sequence and coding gene fragment sequence of human hyaluronidase

[0021]

[0022]

[0023] The basal medium used in the present disclosure is an animal-free, protein-free, chemically defined medium, the components of which include the following substances:

[0024] 4-Hydroxyethylpiperazineethanesulfonic acid (HEPES) 5-7g / L, glucose 3-8g / L, sodium pyruvate 0.05-0.4g / L, sodium chloride 3-5g / L, potassium chloride 0.2-0.4g / L, sodium selenite 0.0000005-0.000060g / L, manganese sulfate 0.05-0.2g / L, ethanolamine 5-25μg / L, ferric citrate 1-10mg / L, zinc sulfate 0.1-0.5g / L, copper sulfate 0.02-0.15g / L, Glutathione 0.05-0.3g / L, magnesium chloride 0.025-0.15g / L, sodium dihydrogen phosphate 0.1-0.3g / L, sodium bicarbonate 1-5g / L, alanine 0.015-0.035g / L, asparagine 0.01-0.035g / L, arginine 0.06-0.30g / L, aspartic acid 0.02-0.2g / L, cystine 0.3-0.5g / L, cysteine ​​0.05-0.2g / L, glutamic acid 0.001-0.09 g / L, glycine 0-0.04g / L, histidine 0.03-0.2g / L, isoleucine 0.05-0.25g / L, leucine 0.05-0.25g / L, lysine 0.02-0.25g / L, methionine 0.02-0.2g / L, phenylalanine 0.055-0.075g / L, proline 0.01-0.05g / L, serine 0.03-0.25g / L, threonine 0.07-0.15g / L, tryptophan 0.00 5-0.025g / L, tyrosine 0.05-0.2g / L, valine 0.05-0.5g / L, biotin 0.005-0.15mg / L, calcium pantothenate 0.002-0.006g / L, choline chloride 0.002-0.09g / L, folic acid 0.002-0.006g / L, inositol 0.005-0.02g / L, niacinamide 0.002-0.006g / L, vitamin B6 0.002-0.006g / L, vitamin B2 0.0002-0.0006g / L, Vitamin B1 0.002-0.006g / L, Vitamin B12 0.000005-0.000025g / L, Linoleic acid 0.001-0.1g / L, Block polyether F-68 (Pl uronic F-68) 0.2-5g / L.

[0025] The feed medium used in the present disclosure is an animal-free, protein-free, chemically defined medium, the ingredients of which include the following substances:

[0026] Glucose 10-25g / L, sodium pyruvate 0.1-0.6g / L, potassium chloride 2-10g / L, ferric citrate 0.01-1.5g / L, zinc sulfate 0.001-0.05g / L, copper sulfate 0.005-0.02g / L, magnesium chloride 0.025-0.5g / L, sodium dihydrogen phosphate 0.1-6g / L, alanine 0.1-2.5g / L, asparagine 0.1-8g / L, arginine 0.6-8g / L, aspartic acid 2-15g / L, glutamic acid 0.1- 15g / L, glycine 0.5-5g / L, histidine 0.3-5g / L, isoleucine 0.1-5g / L, leucine 0.5-20g / L, lysine 0.2-6g / L, methionine 0.2-1.5g / L, phenylalanine 0.5-15g / L, proline 0.1-10g / L, serine 0.3-2.5g / L, threonine 0.07-5g / L, tryptophan 0.05-4g / L, tyrosine 0.05-5g / L, valine 0.05-7g / L.

[0027] In one embodiment, feeding begins on the 3rd or 4th day of culture, and the feeding is performed every other day until the end of culture. Preferably, the feeding amount is 3-8% of the initial volume; the total feeding amount is 20-50% of the initial weight target value. More preferably, each feeding is supplemented with 5% of the initial culture volume of feed medium.

[0028] Preferably, the fed-batch culture method is carried out by using a bioreactor.

[0029] Preferably, the bioreactor comprises a stainless steel bioreactor and a disposable cell culture bag.

[0030] Preferably, the cell inoculation density of the fed-batch culture method is 0.3-2.0×10 6 ce lls / ml.

[0031] In one embodiment, the cells in the fed-batch culture method are cultured at 28-37°C.

[0032] Preferably, the fed-batch culture method is cultured at 35-37° C. after inoculating the cells. Preferably, the culture time at 35-37° C. is 1, 2, 3, 4, 5, 6 or 7 days.

[0033] Preferably, in the fed-batch culture method, when the cell density reaches 5×10 6 Cells / ml to 5×10 7cells / ml or cultured to the 5th, 6th or 7th day after inoculation, the cells are cultured at a temperature of 28°C-35°C to increase the expression level of recombinant human hyaluronidase. Preferably, the culture duration at this stage is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 days.

[0034] Preferably, the fed-batch culture method cultures cells at pH 7.0 ± 0.2 after inoculation. Preferably, the later stage of the fed-batch culture method is carried out at pH 6.8 ± 0.03. Further preferably, the later stage of the fed-batch culture method is carried out at a pH of about 6.8. The later stage of the fed-batch culture method is at least the 3rd day after inoculation, at least the 4th day after inoculation, at least the 5th day after inoculation, at least the 6th day after inoculation, or at least the 7th day after inoculation.

[0035] Preferably, the culture is terminated 10-16 days after inoculation or when the cell viability is <70%.

[0036] Preferably, the separation and purification in step (2) comprises the following steps:

[0037] ① After the culture is completed, the cell culture fluid is harvested and filtered through depth and sterile filtration to obtain clarified cell culture fluid;

[0038] ② purifying the clarified cell culture fluid, wherein the purification steps include anion exchange chromatography, affinity chromatography, hydrophobic chromatography and the like;

[0039] Preferably, the mass analysis index in step (2) is a purity index, including size exclusion chromatography purity (SEC-HPLC) and reverse phase chromatography purity (RP-HPLC).

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] By adopting the fed-batch culture method and regulating the culture temperature and pH, the cell yield is >150 mg / L after culturing in a bioreactor for 10-16 days; the culture fluid is separated and purified to obtain recombinant human hyaluronidase with a RP-HPLC main peak purity of >90% and an enzyme specific activity of >100,000 I U / mg.

[0042] The present invention discloses a highly efficient method for preparing recombinant human hyaluronidase, which has the characteristics of low cost, easy operation, simple process, high expression level and high purity, and provides a new preparation method for the industrial production of recombinant human hyaluronidase. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 Effect of culture temperature on cell growth in 3L bioreactor.

[0044] Figure 2 The effect of culture temperature on cell viability in a 3L bioreactor.

[0045] Figure 3 Effect of culture temperature on cell yield in a 3L bioreactor.

[0046] Figure 4 The effect of different culture temperatures on cell growth in a 3L bioreactor.

[0047] Figure 5 The effect of different culture temperatures on cell viability in a 3L bioreactor.

[0048] Figure 6 The effect of different culture temperatures on cell yield in a 3L bioreactor.

[0049] Figure 7 The effect of pH control on cell growth in a 3L bioreactor.

[0050] Figure 8 The effect of pH control on cell viability in a 3L bioreactor.

[0051] Fig. 9 The effect of pH control in a 3L bioreactor on the purity of the main peak by RP-HPLC.

[0052] Fig.10 Effect of feeding time on cell growth in a 3L bioreactor.

[0053] Fig.11 Effect of feeding time on cell yield in a 3L bioreactor.

[0054] Fig.12 The effect of feeding time on the purity of the main peak by RP-HPLC in a 3L bioreactor. DETAILED DESCRIPTION

[0055] the term

[0056] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0057] Before describing the present disclosure in detail below, it should be understood that the present disclosure is not limited to the specific methodology, scheme and reagent described herein, because these can change. It should also be understood that the terms used in this article are only for describing specific embodiments, and are not intended to limit the scope of the present disclosure. Unless otherwise defined, all technical and scientific terms used in this article have the same meaning as those of ordinary skill in the art to which the present disclosure belongs.

[0058] Certain embodiments disclosed herein include numerical ranges, and certain aspects of the present disclosure may be described in a range manner. Unless otherwise stated, it should be understood that numerical ranges or the manner of description in a range are only for the purpose of brevity and convenience, and should not be considered as a strict limitation of the scope of the present disclosure. Therefore, the description using a range manner should be considered to specifically disclose all possible sub-ranges and all possible specific numerical points within the range, as these sub-ranges and numerical points have been clearly written in this article. Regardless of the width of the numerical value, the above principles are equally applicable. When a range description is adopted, the range includes the endpoints of the range.

[0059] When referring to a measurable value, the term "about" is meant to include variations of ±20%, or in some cases ±10%, or in some cases ±5%, or in some cases ±1%, or in some cases ±0.1% of the specified value.

[0060] The term "hyaluronidase" herein is a general term for enzymes that can hydrolyze hyaluronic acid, and is an enzyme that can reduce the activity of hyaluronic acid in the body and thus increase the permeability of liquid in tissues. Many pathological changes are often accompanied by changes in hyaluronidase and hyaluronic acid, which can change the distribution of some drugs and physiologically active substances in the body. Hyaluronidase includes hyaluronidase precursors, mature hyaluronidase, active truncated forms, allelic variants and species variants, variants encoded by splice variants and other variants, including polypeptides having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the polypeptide shown in one of SEQ NO: 1-4.

[0061] The term "recombinant human hyaluronidase" herein refers to a human hyaluronidase expressed by genetic engineering means, such as the polypeptide of human rHuPH20 shown in any of SEQ NOs: 1-4. Hyaluronidase also includes those containing chemical or post-translational modifications and those not containing chemical or post-translational modifications. Such modifications include, but are not limited to, pegylation, albuminization, glycosylation, farnesylation, carboxylation, hydroxylation, phosphorylation, and other polypeptide modifications known in the art.

[0062] The term "vector" herein refers to a self-replicating DNA molecule in genetic engineering and recombinant DNA technology, which is a tool used to transfer the target gene into the recipient cell. These vectors can not only replicate themselves, but also carry the target gene into the host cell and enable it to be expressed or replicated in the host cell. Plasmids are small circular DNA molecules present in bacterial cells, which can replicate autonomously and are one of the most commonly used vectors. In addition to plasmid vectors, there are also viral vectors, bacteriophage vectors, etc.

[0063] The term "recombinant expression vector" in this article refers to a vector constructed using molecular cloning technology, which can express foreign genes in host cells by inserting them into the vector. This technology is not only very important in basic biological research, but also a key tool in the biotechnology and pharmaceutical industries.

[0064] The term "transfection" herein refers to a method of introducing a plasmid into a cell using chemical or physical means. The plasmid can be inserted into the genome or free in the cytoplasm, thereby causing the cell to express the target protein. Transfection is divided into transient transfection and stable transfection. In the present invention, a physical method of electroporation is used for stable transfection to insert the target gene into the cell genome.

[0065] The term "live cell density" herein refers to the number of live cells per milliliter of cell culture medium. The live cell density detection method is to obtain the live cell density by taking a certain volume of cell culture medium, staining it with 0.2% trypan blue, and counting it using a CountStar automatic cell counter.

[0066] The term "cell viability" herein refers to the percentage of live cells in the culture medium to the total number of cells. The cell viability detection method is to obtain cell viability by taking a certain volume of cell culture medium, staining it with 0.2% trypan blue, and counting it using a CountStar automatic cell counter.

[0067] The term "cell peak" herein refers to the highest value of cell density sampled and detected during the culture process, which can occur on any day during the culture process.

[0068] The titer, expression level, yield, Titer, and recombinant human hyaluronidase content mentioned in this article all have the same meaning, which refers to the main peak content of the protein after sample detection using the RP-HPLC method.

[0069] The culture medium mentioned in this article refers to a solution containing sufficient nutrients to promote the growth of cells in culture, without any animal-derived ingredients. Typically, these solutions contain essential and non-essential amino acids, vitamins, energy sources, lipids and / or trace elements. The culture medium may also contain additional additives, such as hormones, growth factors and growth inhibitors. Depending on the cells and uses, the composition of the culture medium also varies to meet experimental and production needs. The subculture medium is used for continuous subculture of cells to ensure the normal and stable growth of cell lines; the basal medium refers to the culture medium for inoculated cells used in the fed-batch culture process, which is suitable for the basic growth of cells; the feed medium refers to the culture medium added at a specific number of days and proportions during the fed-batch culture process to maintain and promote high-density cell growth and large-scale production of target proteins.

[0070] The term "cell culture fluid or harvested cell culture fluid (HCCF)" herein refers to the liquid obtained after harvesting cells from a bioreactor and separating the cell culture medium from cells and cell debris. The cell culture fluid may be filtered to clarify the culture fluid and remove cells, cell debris and other aggregates.

[0071] The term "fed-batch culture" or "fed-batch culture" in this article is also called semi-continuous culture or fed-batch culture, which means that in the process of cell batch culture, certain materials are added to the culture system in some way to meet the cell growth needs until the culture is completed and the product is taken out.

[0072] The term "RP-HPLC" in this article refers to reverse phase chromatography, which is a high performance liquid chromatography technique that uses a non-polar stationary phase and a polar mobile phase. The working principle of RP-HPLC is to achieve separation based on the difference in the interaction force between the sample molecules in the hydrophilic stationary phase and the hydrophobic mobile phase. This technology is particularly suitable for separating non-polar, polar or ionic compounds. It uses a specific chromatographic column, such as an octadecyl bonded silica column, and a polar solvent such as methanol or acetonitrile as a mobile phase to achieve effective separation of the target molecules. Reversed phase high performance liquid chromatography has the characteristics of high speed, high efficiency, and high sensitivity. Its analysis speed is fast, and the analysis time of a sample is usually between a few minutes and tens of minutes. At the same time, due to its efficient separation ability, RP-HPLC can handle multiple components in complex samples and has good quantitative performance and repeatability. On the other hand, RP-HPLC uses a variety of mobile phases, and the separation conditions can be optimized by adjusting the mobile phase composition to improve the separation effect. When performing RP-HPLC analysis, it is necessary to select a suitable stationary phase and mobile phase, optimize the gradient elution program and temperature control, etc. to achieve the best separation effect. Different chromatographic columns and mobile phase systems may be required for analytes of different properties. In addition, sample pretreatment is also crucial to ensure the accuracy and repeatability of the analysis. RP-HPLC has a wide range of applications in many fields, such as industrial production, pharmacokinetic research, etc. In the pharmaceutical field, it is used for the separation and quantification of drugs and their metabolites; in the production of chemical products, it is used to control product quality and monitor reaction processes; in biochemical research, it is used for the analysis and purification of proteins, peptides and other biomolecules.

[0073] The term "Dot-b lot" in this article refers to dot hybridization or dot blot, which is a fast and simple qualitative or semi-quantitative biochemical experimental technique used to detect, analyze and identify the presence and content of DNA, RNA and protein. This technology observes the biological molecules in the sample by directly spotting the sample on a solid support, such as a nitrocellulose membrane or a nylon membrane, and hybridizing with a labeled probe. Dot-b lot is mainly based on the principle of specific binding between nucleic acids or proteins. When the target molecule in the sample binds to a specific labeled probe, the occurrence of this binding reaction can be observed by various detection methods, thereby verifying the presence of a specific nucleic acid sequence or protein in the sample. Dot-b lot can be used not only for DNA detection, but also for RNA and protein analysis. Its application range is wide, covering multiple fields from basic scientific research to clinical diagnosis. A major advantage of this technology is that it takes little time and can quickly obtain experimental results. At the same time, this method can perform semi-quantitative analysis to estimate the content of target molecules in the sample. Multiple samples can be processed simultaneously on the same membrane, greatly improving experimental efficiency.

[0074] The term "specific activity" or "specific activity" of the enzyme herein is calculated by dividing the enzyme activity (unit / ml) by the protein concentration (mg / ml). The enzyme specific activity is a measure of the number of enzyme activity units per milligram of protein, and is usually used to evaluate the purity and catalytic efficiency of the enzyme. In enzymology research, the specific activity of the enzyme is used to measure the purity of the enzyme. For the same enzyme, the greater the specific activity, the higher the purity of the enzyme. As the enzyme purification proceeds, although the total number of enzyme activity units will gradually decrease, the specific activity will gradually increase. This is because more non-enzyme proteins are removed during the purification process, which increases the proportion of effective enzyme molecules in the unit weight of enzyme protein. The enzyme specific activity can also reflect the catalytic efficiency of the enzyme. The higher the enzyme specific activity, the higher the catalytic efficiency of the enzyme and the faster the reaction rate. This is because high specific activity means that under the same conditions, the enzyme can catalyze more substrates into products per unit time. In practical applications, the enzyme specific activity is usually used to evaluate the quality and performance of the enzyme. By comparing the specific activity of the same enzyme samples from different batches or different sources, it can be judged whether their quality is consistent, so as to select the most suitable enzyme product for experiment or industrial production.

[0075] The term "copy number" in this article refers to the number of a specific DNA fragment or gene in the genome. The concept of copy number is widely used in many fields such as genetics, molecular biology, genetic engineering and medical genetics. The determination of copy number is a common technique in molecular biology research, such as cloning certain repetitive sequences, determining the number of copies of exogenous genes integrated in transfected cultured cells and transgenic animals and plants, etc., all of which require the determination of the copy number of the DNA under study. This determination is of great significance for understanding gene expression, regulatory mechanisms and research on disease-related genes. Copy number variation (CNV) is a common form of genomic variation, involving the increase or decrease of the copy number of large genomic fragments with a length of more than 1kb. This variation is usually caused by genomic rearrangement and manifests as deletions and duplications at the submicroscopic level. Copy number detection is an experimental method to accurately quantify the number of times a specific DNA sequence appears in the genome. Real-time fluorescence quantitative PCR technology is the most commonly used method for copy number detection, known for its high sensitivity and accuracy. The basic steps for copy number detection using real-time fluorescence quantitative PCR include designing specific primers, preparing standard curves, conducting real-time fluorescence quantitative PCR experiments and data analysis. In copy number detection, a key step is to prepare a standard curve. This requires a standard with a known concentration. By plotting the logarithmic relationship between the CT value of the standard and its concentration into a standard curve, the experimenter can accurately calculate the number of DNA copies in the sample to be tested. The method of obtaining these standards includes cloning specific DNA fragments and then preparing standards through steps such as gel recovery and purification. The use of degenerate primers may require the selection of clones with the most degenerate bases as standards. Copy number detection methods can also be divided into absolute quantification and relative quantification. Absolute quantification is used to determine the exact copy number of a specific DNA sequence, and is often used for quantitative detection of viral pathogens, transgenic copy number analysis, etc. Relative quantification is mainly used to compare the differences in the expression levels of a gene in different samples, such as mRNA expression analysis.

[0076] The term "basal medium" in this article refers to a medium containing essential nutrients suitable for cell survival and proliferation. The basal medium contains the minimum nutritional requirements necessary to maintain cell growth, such as carbon source, nitrogen source, inorganic salts, vitamins, etc. Different types of cells have different nutritional requirements, so it is necessary to select the basal medium that is most suitable for the growth of the cell line. Although the basal medium can meet the most basic growth needs of cells, in actual applications, researchers often need to improve and optimize it according to specific experimental purposes and cell characteristics. This may include adding special growth factors, changing the concentration of nutrients, or adjusting the pH value to achieve better culture effects.

[0077] The term "feed medium" in this article refers to additional nutrients added to the basal medium during cell culture. Feeds usually contain carbohydrates, amino acids, vitamins, organic acids, and growth factors, which are essential for maintaining cell growth and proliferation. For example, glucose is the most common energy source. Different cell lines have different nutritional requirements, so the feeding strategy needs to be adjusted according to the nutritional requirements of the specific cell line. Feeds can be added at different stages of cell culture, usually at the beginning of culture or according to the growth of cells. Feeds can be added in batches, continuously, or semi-continuously, and each method has its applicable scenarios and advantages and disadvantages. A suitable feeding strategy can not only increase the cell growth rate, but also improve the quality and yield of products such as recombinant proteins. Feed medium plays a vital role in cell culture. It not only provides the necessary nutrients, but also affects the growth state of cells and the quality of the final product. In practical applications, choosing a suitable feeding strategy and formulation is particularly critical for achieving efficient and high-quality cell culture.

[0078] The term "cumulative viable cell count" or "total viable cells (IVC)" herein refers to the average density of viable cells during the culture multiplied by the culture period.

[0079] The term "bioreactor" in this article refers to advanced biotechnology equipment used to culture and propagate cells in a controlled environment. It is used to produce important biological products such as therapeutic antibodies, vaccines, recombinant proteins, etc. These products play a vital role in treating various diseases such as cancer and autoimmune diseases. Bioreactors can accurately control temperature, pH value, oxygen concentration and nutrient supply to create a culture environment for cells that is close to physiological conditions. From laboratory scale to industrial production scale, the design of cell bioreactors can be flexibly adjusted to meet the needs of different production stages.

[0080] Example

[0081] In order to further illustrate the technical means and implementation effects adopted by the present disclosure, the present disclosure is further described below in conjunction with embodiments and drawings. It can be understood that the embodiments described below are part of the embodiments of the present disclosure, rather than all the embodiments, and are only used to explain the present disclosure, rather than to limit the present disclosure.

[0082] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0083] Example 1: Construction and screening of cell lines expressing recombinant human hyaluronidase

[0084] The nucleic acid sequence encoding the recombinant human hyaluronidase (rHuPH20, amino acid sequence as shown in SEQ ID NO: 1) disclosed in the present invention is synthesized by a gene synthesis company and connected to the multiple cloning site (MCS) of the expression vector. After the construction is completed, the correctness of the recombinant expression vector is confirmed by sequencing verification. Then, the plasmid is extracted using a plasmid extraction kit, and the plasmid is used after quantification by an ultra-micro spectrophotometer, or stored at -80°C for future use.

[0085] The recombinant expression plasmid was introduced into GS - / - In the blank cells, the transfected cells were inoculated into a 96-well plate at a density of 3000-10000 cells / well. After the inoculation, the 96-well plate was placed in a carbon dioxide incubator for static culture at 36.5°C and 5% CO2. After the cells were spread over the wells, the expression level was evaluated by Dot-b lot, and the cell pools with higher expression levels were selected for expansion to 24-well plates. Through step-by-step expansion and screening of 96-well plates, 24-well plates, shake tubes, shake flasks, etc., high-yield pools were screened for shake flask fed batch culture. Based on the cell number and yield results, high-yield cell pools were screened for monoclonal cell line screening.

[0086] Using the limiting dilution method, the cell pools with higher expression levels were inoculated into 96-well plates at a density of <0.5ce ll / well. After inoculation, the 96-well plates were placed in a carbon dioxide incubator for static culture. The culture conditions were: 36.5℃, 5% CO2, 80% humidity. Imaging was performed on days 0, 1, 2, 7, and 14. After the cells filled the wells, the expression level was evaluated by Dot-b lot, and the monoclonal cell lines with higher expression levels were selected for expansion to 24-well plates. After step-by-step expansion screening of 96-well plates, 24-well plates, shake tubes, shake flasks, etc., high-yield monoclonal cell lines were finally selected for shake flask fed batch culture. The candidate monoclonal cell lines were finally screened based on the results of cell growth, expression level, and quality.

[0087] Example 2: Detection of the content of recombinant human hyaluronidase in the culture supernatant by Dot-b lot method

[0088] During the cell line screening process, the Dot-b lot method was used to achieve high-throughput detection of the target protein content. Take 2 μL of the culture supernatant and spot it on the nitrocellulose membrane (NC membrane) and dry it at room temperature. Immerse the spotted nitrocellulose membrane (NC membrane) in BSA blocking solution for 1 hour; after blocking, rinse it with 1×TBST 3 times, 10 minutes each time; then add the primary antibody (rabbit anti-rhPH20 polyclonal antibody) and incubate it for 1 hour. After the primary antibody reaction is completed, rinse it with 1×TBST 3 times, 10 minutes each time; then add the secondary antibody (HRP-labeled goat anti-rabbit IgG) and incubate it for 1 hour. After the secondary antibody reaction is completed, rinse it with 1×TBST 3 times, 10 minutes each time; discard the liquid, add 0.5MDAB color development solution, react at room temperature in the dark for 8-10 minutes, until the color depth of the strip reaches the requirement, and use purified water to rinse the nitrocellulose membrane (NC membrane) to stop color development. The expression level of the cell line is judged according to the color depth.

[0089] Example 3: RP-HPLC method for quantitative determination of recombinant human hyaluronidase content and purity

[0090] Reverse phase chromatography is an analytical method for separation based on the different hydrophobic behaviors of different components in the sample. The weakly hydrophobic components in the solution are loosely bound to the filler in the chromatographic column and are eluted first. As the proportion of the organic phase increases, the strongly hydrophobic components in the solution are gradually eluted. The main components of the sample are separated from the impurities based on the hydrophobic properties to determine their purity. After the test is completed, the main peak purity is calculated by the area normalization method as the percentage of the main peak area to the sum of all peak areas. Using a reference substance with a known protein content or other suitable samples as an external standard, the content of the main peak in the sample is calculated according to the size of the target protein peak area, where the main peak content is the content (or expression level) of the recombinant human hyaluronidase, and the main peak purity is the RP-HPLC purity of the recombinant human hyaluronidase.

[0091] Example 4: Detection of hyaluronidase activity by turbidimetry

[0092] According to the 2020 edition of the Chinese Pharmacopoeia, Part IV, General Rules 1207 "Hyaluridase Assay", the activity of this product is determined by turbidity. Hyaluronidase can form a stable colloidal solution with acidified serum to produce turbidity. Hyaluronidase can enzymolyze the substrate hyaluronic acid, thereby reducing the turbidity of the solution. The activity of this product is determined by plotting a standard curve by measuring the absorbance corresponding to the standard of different concentrations.

[0093] According to the protein content of the sample to be tested, estimate the activity value of the sample to be tested, select the appropriate dilution multiple, and dilute the sample to be tested with the sample stabilizing solution so that the final result is within the range of the detection method (8-40I U / ml). The sample to be tested can be diluted multiple times (generally recommended to dilute twice). If the result is out of range, the value out of range is discarded. If all are within the range, the average value is calculated.

[0094] Take the standard working solution and dilute it in a 96-well plate (this is the sample plate) as shown in Table 2 (other appropriate dilution methods can also be used), with three replicate wells for each concentration:

[0095] Table 2 Dilution method of standard working solution

[0096]

[0097] Prepare a hyaluronic acid blank control in the sample plate, as shown in Table 3 below, and set up 3 replicate wells:

[0098] Table 3 Preparation method of hyaluronidase blank control

[0099]

[0100] Add the diluted sample to be tested to the empty wells of the sample plate at 100 μl per well, and each sample to be tested is paralleled to 3 replicates. Take another 96-well plate (this is the reaction plate), use an 8-channel pipette to add potassium hyaluronate working solution to the wells corresponding to the positions of the standard and the sample to be tested on the sample plate, 30 μl / well, and add phosphate buffer to the wells corresponding to the blank control wells as a control, 30 μl / well. Place the sample plate with the sample to be tested, the standard, the blank control, and the reaction plate containing potassium hyaluronate working solution in the constant temperature heater heating module, preheat at 37°C for 10 minutes. Use an 8-channel pipette to take 30 μl of sample from the sample plate and add it to the corresponding well of the reaction plate. This process is carried out from left to right on the reaction plate, with an interval of 20-30 seconds (fixed time). Starting from the first column, pipette and mix the enzyme and substrate, and replace the new pipette tip after mixing. When the reaction reaches the fixed time, restart the timing and pipette the second column, and proceed in sequence. After the sample is added, incubate at 37°C for 6-8 minutes. When the timer reaches the specified time, use an 8-channel pipette to take 240μl of serum working solution to the first column of the reaction plate, pipette and mix, the interval time is the same as the enzymatic reaction, and then proceed to the next column, and so on. After the last column is completed, remove the reaction plate from the heating module to terminate the reaction. Let it stand at room temperature for more than 10 minutes. After the reaction is completely terminated, read the plate at 640nm and calculate the activity of the sample to be tested according to the standard curve.

[0101] The standard curve R 2 Should be ≥0.98, the CV value of three replicate wells of the standard should not be higher than 20%, the CV value of three replicate wells of the test sample should not be higher than 20%, and the CV value of the results of different dilution multiples of the test sample should not be higher than 30%.

[0102] A standard curve is prepared with the activity value of the standard as the horizontal axis and the corresponding absorbance value as the vertical axis. The absorbance value of the sample to be tested is substituted into the linear regression equation to calculate the activity of the sample to be tested. Activity of the sample to be tested (IU / mg) = (detection value (IU / ml) * dilution factor) / protein content (mg / ml).

[0103] Example 5: Detection of target gene copy number

[0104] The real-time PCR method was used to perform absolute quantitative analysis on the copy number of the target gene encoding recombinant human hyaluronidase in the cells. The fluorescent dye intercalation method was used to synthesize the standard product using the synthetic gene method to prepare the standard curve of the recombinant human hyaluronidase gene. Using the above standard curve, the copy number of the target gene was analyzed and calculated using the absolute quantitative method.

[0105] The specific detection process is as follows: Total genomic DNA was extracted from the monoclonal cell line, and three independent dilutions of the DNA were prepared, two of each were used in parallel for data analysis, a total of six samples, each of which contained 100 ng DNA (50 ng / μl, volume 2μl, equivalent to the total genomic content of approximately 18,868 cells). At the same time, two negative controls (H2O) without template were prepared.

[0106] The DNA standard is a constructed linearized plasmid. The plasmid standard was constructed by synthetic gene method and sequenced for confirmation. The constructed plasmid was linearized with appropriate restriction enzymes and purified to obtain the standard. The DNA plasmid standard contains a 138 bp rHuPH20 encoding gene fragment (sequence as shown in SEQ ID NO: 5); the constructed DNA standard was gradient diluted (5X10 6 , 5X10 5 , 5X10 4 , 5X10 3 , 5X10 2 , 5X10 1 , 5X10 0 The reaction was run under the following cycle conditions (the standard was not tested in parallel, and two parallel samples were made for the sample test, with the negative control being H2O): 95°C, 30 seconds, followed by 40 cycles of 95°C, 5 seconds and 60°C for 30 seconds. The samples to be tested were run according to the same procedure to obtain the Ct value of each sample. The Ct value of the sample to be tested was compared with the standard curve to calculate the specific copy number of the sample to be tested. After testing, the copy number of the nucleic acid encoding the recombinant human hyaluronidase in the engineered cells prepared in Example 1 was approximately 1.

[0107] Example 6: Production of recombinant human hyaluronidase in a 150 L bioreactor

[0108] In this example, fed-batch culture was carried out in a 150 L bioreactor. The cells prepared in Example 1 were thawed in a 37°C water bath and revived in a basal medium for culture. The cells were subcultured and expanded every 3 days. After 18 days of subculture and expansion in the basal medium, the 150 L bioreactor was inoculated at 0.5×10 6 Cells were inoculated into the basal medium at a density of 10 cells / ml and cultured at 36.5°C, a pH value of 6.8 to 7.2, and a stirring rate of 250 rpm. Feed medium was added at 5% of the initial culture volume on the 3rd, 5th, 7th, 9th, 11th, 13th, and 15th days of culture. When the cell density reached 8×10 6 ce lls / ml, the culture temperature was adjusted to 32°C and continued until the end of the culture. On the 16th day of culture, the cell culture fluid was harvested. Samples were taken regularly during the culture process, counted using CountStar, and the changes in the density of live cells and cell viability in the cell fluid were monitored. The content of recombinant human hyaluronidase in the cell culture fluid was detected by RP-HPLC. The activity of recombinant human hyaluronidase in the cell culture fluid was detected by turbidity.

[0109] Table 4 Cell density and viability, expression level, enzyme activity during fed-batch culture

[0110]

[0111] The cell culture fluid was harvested and subjected to deep filtration and sterilization filtration to obtain a clarified cell culture fluid; the clarified cell culture fluid was purified, and the purification steps included anion exchange chromatography, affinity chromatography, hydrophobic chromatography and the like to form a stock solution, and a total of 15.5 g of protein was harvested. The monomer purity was 99.6% as measured by size exclusion chromatography (SEC-HPLC), the main peak purity was 94% as measured by reversed phase high performance liquid chromatography (RP-HPLC), and the enzyme specific activity was 130000 IU / mg.

[0112] Example 7: Effect of temperature regulation during culture on the production of recombinant human hyaluronidase

[0113] In this example, fed-batch culture was performed in a 3 L bioreactor. The cells prepared in Example 1 were thawed in a 37°C water bath and revived in a basal medium for culture. The cells were subcultured every 3 days. After 9 days of subculture in the basal medium, two 3 L bioreactors were inoculated. 6The cells were inoculated into the basal medium at a density of 8 × 10 cells / ml and cultured at 36.5°C, a pH of 6.8 to 7.2, and a stirring rate of 250 rpm. When the cell density reached 8 × 10 6 ce lls / ml, the culture temperature of one bioreactor was adjusted from 36.5°C to 35°C until the end of the culture. The feeding time and feeding amount are summarized in Table 5 below. The volume of the feed batch feed is described as a percentage of the starting volume of the culture in the bioreactor. The total feed amount is 20-50% of the initial weight target value. Culture until 12-16 days or cell viability is <70%. Samples were taken regularly during the culture process, counted using CountStar, and the changes in the live cell density and cell viability in the cell fluid were monitored. The content of recombinant human hyaluronidase in the cell culture fluid was detected by RP-HPLC.

[0114] Table 5 Feeding time and feeding amount during fed-batch culture

[0115] Experimental conditions 36.5℃ 36.5℃→35℃ Day 3 5％ 5％ Day 5 5％ 5％ Day 7 5％ 5％ Day 9 5％ 5％ Day 11 5％ 5％ Day 13 5％ 5％ Day 15 5％ 5％

[0116] As a result, when cells in a bioreactor are grown at low temperatures, the Figure 1 ) and cell viability ( Figure 2 ), culture period and expression level increased ( Figure 3 ).

[0117] Example 8: Optimizing the culture temperature to increase the expression level of recombinant human hyaluronidase

[0118] In this example, fed-batch culture was carried out in a 3 L bioreactor. The cell line expressing recombinant human hyaluronidase prepared in Example 1 was cultured at 0.5×10 6 cels / ml were inoculated into three bioreactors and cultured at 36.5°C, pH 6.8 to 7.2, and agitation at 250 rpm. When the cell density reached 8×10 6 ce lls / ml, the temperature settings of the three bioreactors were adjusted from 36.5°C to 28°C, 30°C and 32°C respectively. The feeding time and feeding amount are shown in Table 5 of Example 7. The total feeding amount is 20-50% of the initial weight target value. Cultivate until 12-16 days or cell viability is less than 70%. Various states of the cells are measured as described in Example 7.

[0119] The culture results were compared and it was found that the increase in cell density was the highest when the culture temperature was adjusted to 32°C in the late culture period compared with the 30°C and 28°C experimental groups ( Figure 4 ), under the same culture period, the expression level of cells in the 32℃ group was the highest ( Figure 6). In the later stage of culture, the cell density of the experimental groups adjusted to 30℃ and 28℃ was basically the same, and the cell viability of the 28℃ experimental group in the later stage of culture was better than that of the other experimental groups ( Figure 5 ), the same or similar high level of total expression as that under 32°C can be achieved by prolonging the cell culture cycle ( Figure 6 ).

[0120] Example 9: Optimizing the pH control range during the culture process to improve the purity of the main peak of recombinant human hyaluronidase

[0121] In this example, a fed-batch culture was carried out in a 3 L bioreactor. The cell line expressing recombinant human hyaluronidase prepared in Example 1 was cultured at a rate of 0.5×10 6 cels / ml were inoculated into two bioreactors, one of which was cultured at pH 7.0±0.2 throughout the culture process, and the other was cultured at pH 7.0±0.2 in the early stage of culture (Day 0-7) and pH 6.8±0.03 in the late stage of culture (Day 7-16), and stirred at 36.5℃ and 250rpm. When the cell density reached 8×10 6 ce lls / ml, the culture temperature was adjusted from 36.5°C to 32°C until the end of the culture. The feeding time and feeding amount are shown in Table 5 of Example 7. The total feeding amount is 20-50% of the initial weight target value. Culture until 12-16 days or cell viability <70%. Various states of the cells are measured as described in Example 7.

[0122] As a result, the pH value was controlled at a lower condition (pH 6.8) in the later stage, which could reduce the cell density ( Figure 7 ), improve cell viability ( Figure 8 ), improve the purity of the main peak ( Fig. 9 ).

[0123] Example 10: Effect of the start time of feeding on the expression of recombinant human hyaluronidase

[0124] In this example, fed-batch culture was carried out in a 3 L bioreactor. The cell line expressing recombinant human hyaluronidase prepared in Example 1 was cultured at 0.5×10 6 cels / ml were inoculated into two bioreactors and cultured at 36.5°C and a pH of 6.8 to 7.2. When the cell density reached 8×10 6ce lls / ml, the culture temperature was adjusted from 36.5°C to 32°C until the end of the culture. The feeding time and feeding amount are shown in Table 6. The total feeding amount is 20-50% of the initial weight target value. The cells were harvested after 16 days of culture or when the cell viability was <70%. Various states of the cells were measured as described in Example 7.

[0125] Table 6 Feeding time and feeding amount during fed-batch culture

[0126]

[0127]

[0128] As a result, through the evaluation of the culture under different feeding time conditions, the growth of cells cultured under the feeding process starting on the 3rd day was basically the same as that of cells cultured under the feeding process starting on the 4th day ( Fig.10 ), while the expression level of cells increased significantly ( Fig.11 ), and the purity of the main peak of cell culture fluid RP-HPLC was significantly improved ( Fig.12 ).

[0129] The embodiments of the present disclosure described above are merely exemplary and cannot be construed as limiting the embodiments of the present disclosure, and any person skilled in the art can recognize or determine the equivalents of numerous specific compounds, materials, and operations without the need to perform experiments beyond the ordinary. All of these equivalents are within the scope of the present disclosure and are included in the claims.

Claims

1. A method for preparing recombinant human hyaluronidase (rHuPH20), characterized in that: The method comprises the following steps: (1) constructing an engineered mammalian cell expressing recombinant human hyaluronidase; the cell contains <10 copies of a nucleic acid encoding the recombinant human hyaluronidase; (2) culturing the cells using a fed-batch culture method, and harvesting the cell culture fluid after the culture is completed; (3) Separating and purifying the obtained culture fluid.

2. The method according to claim 1, wherein: The mammalian cell for producing recombinant human hyaluronidase is a CHO cell. Preferably, the CHO cell is selected from the group consisting of the following cell strains: DXB-11, DG-44, CHO-S, CHO-K1 and CHO-K1(GS-KO) cell lines.

3. The method according to claim 2, wherein the mammalian cell producing recombinant human hyaluronidase is a CHOZN cell.

4. The method according to any one of claims 1 to 3, wherein: The culture medium used in the fed-batch culture method includes a basal culture medium and a feed culture medium.

5. The method according to claims 1-4, wherein: The recombinant human hyaluronidase comprises an amino acid sequence as shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO:

4.

6. The method according to any one of claims 1 to 5, wherein: The fed-batch culture method comprises the following steps: (1) According to 0.3-2×10 6 Cells were seeded at a density of cells / ml; (2) After inoculation, the cells were cultured at 35°C-37°C; (3) feeding medium is added on the 3rd or 4th day after inoculation, and feeding is performed every other day until the end of the culture. The amount of each feed is 3-8% of the initial volume, and the total feed amount is 20-50% of the initial weight target value; (4) When the cell density is 5×10 6 cells / ml-5×10 7 cells / ml or on the 5th, 6th or 7th day after inoculation, culturing the cells at a temperature of 28°C-35°C and maintaining the temperature until the end of the culture; (5) After inoculation, the culture is terminated when the cells are cultured for 10-16 days or when the cell viability is less than 70%.

7. The method according to claim 6, wherein: The culture period of step (2) is 1-7 days.

8. The method according to claim 6, wherein: The culture period of step (4) is 3-15 days.

9. The method according to any one of claims 6 to 8, wherein the cells are cultured at pH 7.0 ± 0.2 after inoculation.

10. The method of claim 9, wherein the pH is adjusted to about 6.8 after at least the 3rd day, after at least the 4th day, after at least the 5th day, after at least the 6th day, or after at least the 7th day after inoculation.

Images