Serum-free culture medium of hybridoma cells, in-vitro suspension culture and application

Abstract

The invention belongs to the technical field of hybridoma cell culture, and particularly relates to a serum-free culture medium of hybridoma cells, in-vitro suspension culture and application. According to the serum-free culture medium for the hybridoma cells, the polyethylene glycol and the Pluronic F68 are added at the same time and can cooperate with each other, cell membranes of the hybridoma cells are protected, growth and proliferation of the hybridoma cells are promoted, and antibody protein is secreted; the content of the added standby carbon source sodium pyruvate and genetic material raw materials purine and pyrimidine is high, and the types and the content of the added vitamins and amino acids are high, so that the requirements in the aspects of nutrient substances, carbon sources, genetic material raw materials and the like for rapid growth of the hybridoma cells can be met; therefore, the technical problem that a new serum-free hybridoma cell culture medium is lacked in the prior art is solved.

Description

Technical Field

[0001] The present invention relates to the technical field of hybridoma cell culture, and in particular to a serum-free culture medium and in vitro suspension culture and application of hybridoma cells. Background Art

[0002] Clinically, antibody drugs can be used to treat diseases such as tumors and immune rejection after organ transplantation. The active ingredients in antibody drugs are mainly antibody substances. The current production processes of antibodies mainly include immunizing animals to prepare antibodies, using genetic engineering technology to prepare antibodies, and culturing hybridoma cells in vitro to prepare antibodies.

[0003] The preparation of antibodies by immunizing animals requires the injection of antigens into animals such as rats and rabbits to stimulate the B cells in the animals to secrete antibodies, while the preparation of antibodies by genetic engineering technology requires the insertion of the specific gene fragment corresponding to the antibody into a plasmid, and then the plasmid is transferred into specific cells for in vitro amplification and culture, so that the gene fragment is transcribed and translated in vitro to obtain the antibody; however, the preparation of antibodies by immunizing animals requires the use of a large number of animals, and the cells used in genetic engineering technology usually do not have the ability to proliferate indefinitely; compared with the preparation of antibodies by immunizing animals and genetic engineering technology, the preparation of antibodies by in vitro cultured hybridoma cells does not require the use of a large number of animals and has a lower cost. In addition, the hybridoma cells used in the preparation of antibodies by in vitro cultured hybridoma cells are cells formed by the fusion of myeloma cells and B lymphocytes, and have both the characteristics of unlimited proliferation of myeloma cells and the function of B lymphocytes to secrete antibodies. At present, the technology of in vitro cultured hybridoma cells to secrete monoclonal antibodies is becoming increasingly mature, and antibody drugs prepared by in vitro cultured hybridoma cells have occupied an important position in the field of biomedicine.

[0004] In vitro culture of hybridoma cells requires a clean space, a suitable gas environment, temperature, humidity, and more importantly, nutritional conditions. Suitable nutritional conditions can not only improve the growth of hybridoma cells, but also increase the expression of antibodies to a certain extent. The main nutritional condition is provided by hybridoma cell culture medium. Currently, hybridoma cell culture medium on the market is divided into serum-containing hybridoma cell culture medium and serum-free hybridoma cell culture medium according to whether it contains serum components. The main disadvantages of culturing hybridoma cells with serum-containing culture medium are that the culture cost is high and that the downstream separation and purification of target proteins is difficult due to the complex serum components. Therefore, the development of serum-free hybridoma cell culture medium has a market. Serum-free hybridoma cell culture medium does not contain animal components and is mainly composed of clear chemical raw materials. The quality will be more stable, the batch-to-batch differences will be smaller, the cost will be relatively low, and it can reduce the pressure of downstream purification of target proteins.

[0005] However, the raw materials used in different serum-free hybridoma cell culture media may vary. Different raw materials lead to differences in the preparation costs of serum-free hybridoma cell culture media on the one hand, and also lead to differences in the effects of culturing hybridoma cells on the other hand. Therefore, new varieties of serum-free hybridoma cell culture media are developed to form a serum-free hybridoma cell culture media product series with the original serum-free hybridoma cell culture media. When encountering downstream customers with different needs, suitable serum-free hybridoma cell culture media products can be provided to customers. Therefore, the development of new varieties of serum-free hybridoma cell culture media is still a part worthy of in-depth research in the field of biomedicine. Summary of the invention

[0006] In view of this, the present application provides a serum-free culture medium for hybridoma cells and in vitro suspension culture and application thereof, which are used to solve the technical problem of the lack of new varieties of serum-free hybridoma cell culture medium in the prior art.

[0007] In a first aspect, the present application provides a serum-free culture medium for hybridoma cells, the components of which include: polyethylene glycol, Pluronic F68, sodium pyruvate, purine, pyrimidine, amino acids, vitamins, cell adhesion factors, cell growth factors, hormones, proteins, trace elements, antibiotics, salts, carbohydrates, buffers and pH indicators.

[0008] Preferably, the concentration of polyethylene glycol is 400-1000 mg / L;

[0009] The concentration of the Pluronic F68 is 500-1200 mg / L.

[0010] Preferably, the concentration of sodium pyruvate is 1-4 mmol / L.

[0011] Preferably, the purine composition includes: adenine, guanine, hypoxanthine and xanthine;

[0012] The composition of the pyrimidine includes: cytosine, thymine and uracil.

[0013] Preferably, the concentration of adenine, guanine, hypoxanthine and xanthine is 5-10 mg / L;

[0014] The concentrations of cytosine, thymine and uracil are 5-10 mg / L.

[0015] Preferably, the amino acid composition includes: S-sulfocysteine, tryptophan, glutamic acid, aspartic acid, tyrosine, alanine, glycine, serine, threonine, proline, asparagine, histidine, cystine, L-glutamine, isoleucine, leucine, lysine, methionine, phenylalanine, valine and arginine.

[0016] Preferably, the vitamin composition includes: vitamin B5, vitamin B9, vitamin B3, vitamin B6, vitamin B2, vitamin B4, vitamin B8, vitamin A, vitamin C, vitamin D, vitamin E, vitamin K, vitamin B12 and vitamin H.

[0017] Preferably, the concentration of S-sulfocysteine, tryptophan, glutamic acid, aspartic acid, tyrosine, alanine, glycine, serine, threonine, proline, asparagine, histidine, cystine, isoleucine, leucine, lysine, methionine, phenylalanine, valine and arginine is 0.5-30 mmol / L, and the concentration of L-glutamine is 2.0-4.0 mmol / L.

[0018] Preferably, the concentration of vitamin B5, vitamin B9, vitamin B3, vitamin B6, vitamin B2, vitamin B4, vitamin B8, vitamin A, vitamin C, vitamin D, vitamin E, vitamin K, vitamin B12 and vitamin H is 0.5~2.5 mg / L.

[0019] Preferably, the cell adhesion factors include: cadherin, selectin, integrin and hyaluronan;

[0020] The cell growth factors include: colony stimulating factor, interleukin 2, interleukin 6 and ethanolamine;

[0021] The hormones include: insulin, growth hormone, adrenocortical hormone and steroid hormone;

[0022] The proteins include: recombinant transferrin and bovine serum albumin;

[0023] The trace elements include: selenium, manganese, molybdenum, vanadium, zinc, copper, iron and cobalt;

[0024] The antibiotics include penicillin, streptomycin, gentamicin, paromomycin, kanamycin and chlortetracycline;

[0025] The salts include: sodium chloride, potassium amide, calcium chloride, magnesium chloride, sodium dihydrogen phosphate, sodium bicarbonate, citrate and selenite;

[0026] The carbohydrates include: glucose;

[0027] The buffer comprises: 4-hydroxyethylpiperazineethanesulfonic acid;

[0028] The pH indicator includes: phenol red.

[0029] Preferably, the concentration of cadherin, selectin, integrin and hyaluronan is 0.1-100 μmol / L;

[0030] The concentrations of the colony stimulating factor, interleukin 2, and interleukin 6 are 1-10 μg / L, and the concentration of the ethanolamine is 4.5-5.5 mg / L;

[0031] The concentrations of insulin, growth hormone, adrenocortical hormone and steroid hormone are 0.1-10 mg / L;

[0032] The concentrations of the recombinant transferrin and bovine serum albumin are 10-20 mg / L and 20-50 mg / L, respectively;

[0033] The concentrations of selenium and molybdenum are 0.001-0.5 μmol / L, and the concentrations of manganese, vanadium, zinc, copper, iron and cobalt are 0.1-100 μmol / L;

[0034] The concentrations of streptomycin, gentamicin, paromomycin, kanamycin and chlortetracycline are 5-100 mg / L, and the concentration of penicillin is 5×10 4 ~1×10 5 U / L;

[0035] The concentration of sodium chloride is 4.5-9.0 g / L, the concentration of potassium amide, calcium chloride, magnesium chloride, and sodium dihydrogen phosphate is 0.1-0.6 g / L, the concentration of sodium bicarbonate is 1.5-2.0 g / L, and the concentration of citrate and selenite is 3-6 μg / L;

[0036] The concentration of glucose is 0.5-5.0 g / L;

[0037] The concentration of the 4-hydroxyethylpiperazineethanesulfonic acid is 10-25 mmol / L;

[0038] The concentration of the phenol red is 0.005-0.2 g / L.

[0039] Preferably, the concentration of selenium is 0.005~0.5μmol / L, the concentration of manganese is 0.4~40μmol / L, the concentration of molybdenum is 0.001~0.1μmol / L, the concentration of vanadium is 0.07~70μmol / L, the concentration of zinc is 2~50μmol / L, the concentration of copper is 0.1~100μmol / L, the concentration of iron is 1~100μmol / L, and the concentration of cobalt is 2~60μmol / L.

[0040] The second aspect of the present application provides a method for preparing a serum-free culture medium for hybridoma cells, the preparation method comprising: uniformly mixing the polyethylene glycol, Pluronic F68, sodium pyruvate, purine, pyrimidine, amino acids, vitamins, cell adhesion factors, cell growth factors, hormones, proteins, trace elements, antibiotics, salts, carbohydrates, buffers, pH indicators and water for injection described in the first aspect, and filtering to obtain a serum-free culture medium for hybridoma cells.

[0041] The third aspect of the present application provides an in vitro suspension culture of hybridoma cells using a serum-free culture medium for hybridoma cells described in the first aspect. The process of in vitro suspension culture of hybridoma cells includes: adding hybridoma cells to a serum-free culture medium for hybridoma cells described in the first aspect, and performing in vitro suspension culture of hybridoma cells under the conditions of 37°C, 5% CO2 and a rotation speed of 120rpm.

[0042] The fourth aspect of the present application provides use of the serum-free culture medium of hybridoma cells described in the first aspect in the preparation of antibody drugs.

[0043] Compared with the prior art, the serum-free culture medium for hybridoma cells provided in this application has at least the following technical effects:

[0044] 1. In the serum-free culture medium for hybridoma cells provided in the present application, polyethylene glycol and Pluronic F68 are added simultaneously, which can synergistically reduce the impact of bubble rupture on the cell membrane of hybridoma cells and stabilize the cell membrane of hybridoma cells, thereby making the cell membrane of hybridoma cells less likely to be damaged.

[0045] 2. In the serum-free culture medium for hybridoma cells provided in the present application, sodium pyruvate and purine and pyrimidine are added in relatively high amounts, which can meet the carbon source and genetic material raw materials required for the rapid proliferation of hybridoma cells.

[0046] 3. The serum-free culture medium for hybridoma cells provided in the present application contains almost a full set of vitamins and amino acids, and the amount of vitamins and amino acids added is relatively large, which can meet the nutritional resources required for the rapid proliferation of hybridoma cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] In order to more clearly illustrate the specific implementation methods of the present application or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0048] Figure 1This is a schematic diagram of the growth and proliferation of hybridoma cells after being cultured in the serum-free medium provided in Example 1 for 8 days;

[0049] Figure 2 This is a schematic diagram of the growth and proliferation of hybridoma cells after being cultured in the serum-free medium provided in Example 2 for 8 days;

[0050] Figure 3 This is a schematic diagram of the growth and proliferation of hybridoma cells after being cultured in the serum-free medium provided in Example 3 for 8 days;

[0051] Figure 4 Schematic diagram of electrophoresis of antibody proteins secreted by hybridoma cells after being cultured in the serum-free medium provided in Examples 1-3 for 8 days, wherein Figure A is an electrophoresis diagram of antibody proteins secreted by hybridoma cells cultured in the serum-free medium in Example 1, Figure B is an electrophoresis diagram of antibody proteins secreted by hybridoma cells cultured in the serum-free medium in Example 2, and Figure C is an electrophoresis diagram of antibody proteins secreted by hybridoma cells cultured in the serum-free medium in Example 3;

[0052] Figure 5 The cell density results of hybridoma cells cultured in the serum-free medium provided in Example 1 and commercially available serum-free medium for 24 h, 48 h, 72 h and 96 h are statistical graphs;

[0053] Figure 6 This is a schematic diagram of the growth and proliferation of hybridoma cells after being cultured in the serum-free medium provided in Example 1 for 8 days;

[0054] Figure 7 This is a schematic diagram of the growth and proliferation of hybridoma cells after being cultured in a commercial serum-free medium for 8 days;

[0055] Figure 8 Schematic diagram of electrophoresis of antibody proteins secreted by hybridoma cells after being cultured in the serum-free medium provided in Example 1 and in a commercially available serum-free medium for 8 days. Figure A is an electrophoresis diagram of antibody proteins secreted by hybridoma cells cultured in the serum-free medium in Example 1, and Figure B is an electrophoresis diagram of antibody proteins secreted by hybridoma cells cultured in a commercially available serum-free medium. DETAILED DESCRIPTION

[0056] The present application provides a serum-free culture medium for hybridoma cells and in vitro suspension culture and application thereof, which are used to solve the technical problem of lack of new varieties of serum-free hybridoma cell culture medium in the prior art.

[0057] The technical solution of the present application will be described clearly and completely below in conjunction with the accompanying drawings. Obviously, the described embodiments are part of the embodiments of the present application, rather than all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present application.

[0058] In view of the fact that the current serum-free hybridoma cell culture medium has insufficient formula types and lacks new varieties of serum-free hybridoma cell culture medium; the present application provides a serum-free culture medium for hybridoma cells, the components of which include: polyethylene glycol, Pluronic F68, sodium pyruvate, purine, pyrimidine, amino acids, vitamins, cell adhesion factors, cell growth factors, hormones, proteins, trace elements, antibiotics, salts, carbohydrates, buffers and pH indicators.

[0059] In a serum-free culture medium for hybridoma cells provided in the present application, amino acids, vitamins, cell adhesion factors, cell growth factors, hormones, proteins, trace elements, antibiotics, salts, and carbohydrates are nutrients required for the growth and proliferation of hybridoma cells, and a buffer is used to adjust the pH of the culture medium so that the pH of the culture medium is an environment suitable for the growth and proliferation of hybridoma cells, and a pH indicator is used to indicate the changes in the pH of the culture medium during the growth and proliferation of hybridoma cells, so as to facilitate the adjustment of the pH of the culture medium; at the same time, the sodium pyruvate added in the present application can be used as a backup carbon source, and purine and pyrimidine can be used as raw materials for genetic material required for rapid replication of hybridoma cells; and the present application also adds polyethylene glycol and Pluronic F68 to the serum-free culture medium for hybridoma cells, and Pluronic F68 is a cell membrane stabilizer, which can reduce the damage to the cell membrane caused by the shear force generated by stirring (for example, 120 rpm) during the suspension culture of cells. In addition, the stirring process will also cause more bubbles to be generated and burst, and the impact of the bubble bursting causes the cell membrane to be damaged. Polyethylene glycol is used as a defoaming agent in the industrial field. In addition, polyethylene glycol is also a pharmaceutical excipient. When polyethylene glycol is applied to the field of cell culture medium, the defoaming effect of polyethylene glycol can be used to reduce the generation and bursting of bubbles during the culture of hybridoma cells, and the impact of bubble bursting on the cell membrane can be reduced; through the stabilization effect of Pluronic F68 on the cell membrane, on the one hand, the impact of bubble bursting on the cell membrane of hybridoma cells is reduced, and on the other hand, the cell membrane of hybridoma cells can be stabilized, which makes it difficult for the cell membrane of hybridoma cells to be destroyed, effectively promotes the growth and proliferation of hybridoma cells, and allows hybridoma cells to secrete more antibody substances; thus, the serum-free culture medium for hybridoma cells provided in the present application can meet the culture requirements of hybridoma cells, and overcome the defect of the current lack of new varieties of serum-free hybridoma cell culture medium.

[0060] As a preferred embodiment, in a serum-free culture medium for hybridoma cells provided in the present application, the concentration of polyethylene glycol used is 400-1000 mg / L, and the concentration of Pluronic F68 used is 500-1200 mg / L.

[0061] As a preferred embodiment, considering that hybridoma cells have the characteristics of unlimited proliferation of myeloma cells, they grow and proliferate rapidly, and require more carbon sources and genetic material raw materials; in a serum-free culture medium for hybridoma cells provided in the present application, the concentration of the backup carbon source sodium pyruvate added is relatively high, which is 1-4 mmol / L; at the same time, the types of genetic material raw materials added are relatively large, and the concentrations are also relatively high, including adenine, guanine, hypoxanthine and xanthine at a concentration of 5-10 mg / L and cytosine, thymine and uracil at a concentration of 5-10 mg / L.

[0062] As a preferred embodiment, in order to provide sufficient nutrients, a serum-free culture medium for hybridoma cells provided in the present application contains a large variety of vitamins and amino acids at high concentrations, including arginine, cystine, histidine, isoleucine, leucine, valine, lysine, methionine, phenylalanine, threonine, tryptophan, tyrosine, alanine, glycine, serine, aspartic acid, asparagine, glutamic acid, proline and S-sulfocysteine ​​at a concentration of 0.5-30 mmol / L and L-glutamine at a concentration of 2.0-4.0 mmol / L, and vitamin B5, vitamin B9, vitamin B3, vitamin B6, vitamin B2, vitamin B4, vitamin B8, vitamin A, vitamin C, vitamin D, vitamin E, vitamin K, vitamin B12 and vitamin H at a concentration of 0.5-2.5 mg / L.

[0063] Correspondingly, the present application also provides a preparation method corresponding to the above-mentioned serum-free culture medium of hybridoma cells, wherein the preparation method comprises mixing water for injection and polyethylene glycol, Pluronic F68, sodium pyruvate, purine, pyrimidine, amino acids, vitamins, cell adhesion factors, cell growth factors, hormones, proteins, trace elements, antibiotics, salts, carbohydrates, buffers and pH indicators uniformly, filtering the mixture using a 0.22 μm filter membrane, and the collected filtrate is the serum-free culture medium of hybridoma cells;

[0064] In addition, the present application also uses the above-mentioned serum-free culture medium of hybridoma cells for in vitro suspension culture, and adds hybridoma cells resuspended with a small amount of hybridoma cell serum-free culture medium to the serum-free culture medium of hybridoma cells, and performs in vitro suspension culture of hybridoma cells at 37°C, 5% CO2 and 120rpm. The experimental results show that the serum-free culture medium of hybridoma cells provided by the present application is suitable for in vitro suspension culture of hybridoma cells, and the in vitro suspension culture method is not restricted by a fixed matrix and has a larger free growth and proliferation space, which is conducive to meeting the needs of unlimited proliferation and rapid growth and proliferation of hybridoma cells.

[0065] At the same time, the present application also provides the use of the above-mentioned serum-free culture medium of hybridoma cells in the preparation of antibody drugs.

[0066] The serum-free culture medium for hybridoma cells provided in the present application will be specifically described below in conjunction with the embodiments and experimental examples.

[0067] Example 1

[0068] Example 1 of the present application provides a method for preparing a serum-free culture medium for hybridoma cells, the preparation method comprising:

[0069] The step of weighing raw materials: weighing corresponding raw materials according to the serum-free medium formula of the first hybridoma cell shown in Table 1;

[0070] The steps of preparing a serum-free culture medium for hybridoma cells are as follows: polyethylene glycol and Pluronic F68 are added to 700 mL of water for injection, sodium pyruvate is added, purines such as adenine, guanine, hypoxanthine and xanthine and pyrimidines such as cytosine, thymine and uracil are added, amino acids such as S-sulfocysteine, tryptophan, glutamic acid, aspartic acid, tyrosine, alanine, glycine, serine, threonine, proline, asparagine, histidine, cystine, L-glutamine, isoleucine, leucine, lysine, methionine, phenylalanine, valine and arginine are added, vitamins such as vitamin B5 (pantothenic acid), vitamin B9 (folic acid), vitamin B3 (nicotinamide), vitamin B6 (pyridoxal), vitamin B2 (riboflavin), vitamin B4 (choline), vitamin B8 (inositol), vitamin A, vitamin C, vitamin D, vitamin E, vitamin K, vitamin B12 and vitamin H (biotin) are added, and calcium adhesion is added. The method comprises the following steps: adding cell adhesion factors such as leukocyte antigen, selectin, integrin, hyaluronan mucin, colony stimulating factor, interleukin 2, interleukin 6 and ethanolamine, hormones such as insulin, growth hormone, adrenocortical hormone and steroid hormone, proteins such as recombinant transferrin and bovine serum albumin, trace elements such as selenium, manganese, molybdenum, vanadium, zinc, copper, iron and cobalt, antibiotics such as streptomycin, gentamicin, paromomycin, kanamycin and chlortetracycline, salts such as sodium chloride, potassium amine, calcium chloride, magnesium chloride, sodium dihydrogen phosphate, sodium bicarbonate, selenite and citrate, glucose, 4-hydroxyethylpiperazineethanesulfonic acid and phenol red, and mixing well. The volume is adjusted to 1L with water for injection, and filtered with a 0.22μm filter membrane. The filtrate is collected for use. The filtrate is the serum-free culture medium for hybridoma cells.

[0071] Table 1: Formula of serum-free medium for the first hybridoma cell culture

[0072]

[0073] Example 2

[0074] Example 2 of the present application provides a method for preparing a serum-free culture medium for hybridoma cells. As a comparative example, the difference between this preparation method and Example 1 is that polyethylene glycol is not added. The preparation method comprises:

[0075] The step of weighing raw materials: weighing corresponding raw materials according to the serum-free medium formula of the second hybridoma cells shown in Table 2;

[0076] Steps for preparing a serum-free culture medium for hybridoma cells: add Pluronic F68 to 700 mL of water for injection, add sodium pyruvate, add purines such as adenine, guanine, hypoxanthine and xanthine, and pyrimidines such as cytosine, thymine and uracil, add amino acids such as S-sulfocysteine, tryptophan, glutamic acid, aspartic acid, tyrosine, alanine, glycine, serine, threonine, proline, asparagine, histidine, cystine, L-glutamine, isoleucine, leucine, lysine, methionine, phenylalanine, valine and arginine, add vitamins such as vitamin B5 (pantothenic acid), vitamin B9 (folic acid), vitamin B3 (nicotinamide), vitamin B6 (pyridoxal), vitamin B2 (riboflavin), vitamin B4 (choline), vitamin B8 (inositol), vitamin A, vitamin C, vitamin D, vitamin E, vitamin K, vitamin B12 and vitamin H (biotin), add calcium adhesive The method comprises the following steps: adding cell adhesion factors such as leukocyte antigen, selectin, integrin, hyaluronan mucin, colony stimulating factor, interleukin 2, interleukin 6 and ethanolamine, hormones such as insulin, growth hormone, adrenocortical hormone and steroid hormone, proteins such as recombinant transferrin and bovine serum albumin, trace elements such as selenium, manganese, molybdenum, vanadium, zinc, copper, iron and cobalt, antibiotics such as streptomycin, gentamicin, paromomycin, kanamycin and chlortetracycline, salts such as sodium chloride, potassium amine, calcium chloride, magnesium chloride, sodium dihydrogen phosphate, sodium bicarbonate, selenite and citrate, glucose, 4-hydroxyethylpiperazineethanesulfonic acid and phenol red, and mixing well. The volume is adjusted to 1L with water for injection, and filtered with a 0.22μm filter membrane. The filtrate is collected for use. The filtrate is the serum-free culture medium for hybridoma cells.

[0077] Table 2: Serum-free culture medium formula for the second hybridoma cell line

[0078]

[0079] Example 3

[0080] Example 3 of the present application provides a method for preparing a serum-free culture medium for hybridoma cells. As a comparative example, the difference between this preparation method and Example 1 is that Pluronic F68 is not added. The preparation method comprises:

[0081] The step of weighing raw materials: weighing corresponding raw materials according to the serum-free medium formula of the third hybridoma cell shown in Table 3;

[0082] The steps of preparing the serum-free culture medium for hybridoma cells are as follows: polyethylene glycol is added to 700 mL of water for injection, sodium pyruvate is added, purines such as adenine, guanine, hypoxanthine and xanthine and pyrimidines such as cytosine, thymine and uracil are added, amino acids such as S-sulfocysteine, tryptophan, glutamic acid, aspartic acid, tyrosine, alanine, glycine, serine, threonine, proline, asparagine, histidine, cystine, L-glutamine, isoleucine, leucine, lysine, methionine, phenylalanine, valine and arginine are added, vitamin B5 (pantothenic acid), vitamin B9 (folic acid), vitamin B3 (nicotinamide), vitamin B6 (pyridoxal), vitamin B2 (riboflavin), vitamin B4 (choline), vitamin B8 (inositol), vitamin A, vitamin C, vitamin D, vitamin E, vitamin K, vitamin B12 and vitamin B13 are added. Vitamins such as calcein H (biotin), cell adhesion factors such as cadherin, selectin, integrin, hyaluronan, colony stimulating factor, interleukin 2, interleukin 6 and ethanolamine, hormones such as insulin, growth hormone, adrenocortical hormone and steroid hormone, proteins such as recombinant transferrin and bovine serum albumin, trace elements such as selenium, manganese, molybdenum, vanadium, zinc, copper, iron and cobalt, antibiotics such as streptomycin, gentamicin, paromomycin, kanamycin and chlortetracycline, salts such as sodium chloride, potassium amine, calcium chloride, magnesium chloride, sodium dihydrogen phosphate, sodium bicarbonate, selenite and citrate, glucose, 4-hydroxyethylpiperazineethanesulfonic acid and phenol red are added. After mixing evenly, the volume is adjusted to 1L with water for injection, and filtered with a 0.22μm filter membrane, and the filtrate is collected for use. The filtrate is the serum-free culture medium for hybridoma cells.

[0083] Table 3: Serum-free culture medium formula for the third hybridoma cell line

[0084]

[0085] Experimental Example 1

[0086] Experimental Example 1 of the present application conducts a culture experiment on the serum-free culture medium of hybridoma cells provided in Examples 1-3 to determine the growth and proliferation of hybridoma cells in different serum-free culture medium.

[0087] The process of the cultivation experiment includes:

[0088] Resuspending hybridoma cells: Open the water bath in advance to keep the temperature constant at 37°C, take 3 frozen hybridoma cells, and quickly place them in a water bath to thaw quickly; respectively pipette 5 mL of the hybridoma cell serum-free medium provided in Examples 1-3 into 3 15 ml pyrogen-free centrifuge tubes, transfer the 3 thawed hybridoma cells to the hybridoma cell serum-free medium provided in Examples 1-3, gently blow and mix, cover the centrifuge tube lid, and centrifuge at 800 rpm and 24°C for 3 minutes; after centrifugation, remove the supernatant, take 1 ml of the medium corresponding to each tube, and gently blow and resuspend the cell pellet;

[0089] Growth, proliferation and antibody secretion of hybridoma cells: 19 ml of the hybridoma serum-free culture medium provided in Examples 1-3 was added to three cell culture flasks respectively, and the resuspended hybridoma cells were inoculated into the corresponding hybridoma serum-free culture medium provided in Examples 1-3 respectively, and an equal number of hybridoma cells (5×10 5 cells / mL, 1mL), and then placed in a carbon dioxide incubator for culture at 37°C, 5% CO2 and 120rpm. During the culture process, the fluid was replenished according to the cell growth conditions to control the hybridoma cells in the three culture media to have the same culture time (8 days) and the same final volume after culture (1L).

[0090] After the culture was completed, before collecting the culture supernatant, the status of the hybridoma cells in the three culture media was observed. The growth and proliferation results were as follows: Figure 1-3 As shown; then three culture supernatants containing antibodies were collected and purified using the same method. After purification, the volume of the obtained protein solution was recorded, the concentration of the protein solution was measured, and the expression amount of the target antibody of the hybridoma cells in three different culture media was calculated. The results are shown in Table 4; at the same time, samples were also taken for denatured protein electrophoresis to confirm that the purified proteins were all target antibodies. The results are shown in Table 4. Figure 4 shown.

[0091] Table 4: Expression of antibodies by hybridoma cells in the serum-free medium of hybridoma cells provided in Examples 1-3

[0092]

[0093] from Figure 4 It can be seen that the hybridoma cells provided in Examples 1-3 can all secrete antibody proteins; and it can be seen from Table 4 that the serum-free culture medium for hybridoma cells provided in the present application can be used for in vitro suspension culture of hybridoma cells to obtain more antibody proteins.

[0094] from Figure 1-3It can be seen that there are differences in the growth and proliferation of hybridoma cells in the hybridoma cell serum-free culture medium provided in Examples 1-3. It can be more clearly seen from Table 4 that after adding the same amount of Pluronic F68 (2 g), polyethylene glycol (2 g) or Pluronic F68 (1 g) + polyethylene glycol (1 g) to the culture medium, the hybridoma cell serum-free culture medium provided in Example 1 after adding Pluronic F68 (1 g) + polyethylene glycol (1 g) can make the growth and proliferation of hybridoma cells better, thereby secreting more antibody proteins; this indicates that the polyethylene glycol can reduce the impact of bubble rupture on the hybridoma cell membrane and can cooperate with the effect of Pluronic F68 on stabilizing the hybridoma cell membrane, effectively promoting the growth and proliferation of hybridoma cells and secreting antibody proteins.

[0095] Experimental Example 2

[0096] Experimental Example 2 of the present application conducted a culture experiment on the hybridoma cell serum-free culture medium provided in Example 1 and the commercial hybridoma cell serum-free culture medium to determine the growth and proliferation of hybridoma cells in different serum-free culture media; the instructions for the commercially available culture medium indicated that it mainly contained 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), sodium bicarbonate, amino acids, vitamins, trace elements and other components (SinoBiological, SMM Hyb-TIII cell culture medium).

[0097] The process of the cultivation experiment includes:

[0098] Resuspending hybridoma cells: Open the water bath in advance to keep the temperature constant at 37°C, take 2 frozen hybridoma cells, and quickly place them in a water bath for rapid thawing; respectively take 5 mL of the hybridoma cell serum-free medium provided in Example 1 and the commercially available hybridoma cell serum-free medium into 2 15 ml pyrogen-free centrifuge tubes, transfer the 2 thawed hybridoma cells to the hybridoma cell serum-free medium provided in Example 1 and the commercially available hybridoma cell serum-free medium, respectively, gently pipette to mix, cover the centrifuge tube lid, centrifuge at 800 rpm, 24°C for 3 minutes, remove the supernatant after centrifugation, take 1 ml of the medium corresponding to each tube and gently pipette to resuspend the cell pellet;

[0099] Growth and proliferation of hybridoma cells: 19 mL of the hybridoma serum-free medium provided in Example 1 and 19 mL of the commercially available hybridoma serum-free medium were added to two cell culture flasks, and the resuspended hybridoma cells were inoculated into the above two culture media, respectively. The same number of hybridoma cells (5 × 10 5cells / mL, 1mL), and then placed in a carbon dioxide incubator for culture at 37°C, 5% CO2 and 120rpm; then samples were taken after 24 hours, 48 ​​hours, 72 hours, 96 hours and 120 hours to count the cells and record the density of the hybridoma cells. At the same time, the morphology of the hybridoma cells was recorded after 72 hours of culture; the growth density of the hybridoma cells in the two hybridoma cell serum-free culture media increased with the increase of culture time. Figure 5 After 72 hours of culture, the hybridoma cells in the serum-free medium for hybridoma cells provided in Example 1 have a morphology as shown in FIG. Figure 6 As shown, the morphology of hybridoma cells in commercial hybridoma cell serum-free culture medium is as follows Figure 7 shown.

[0100] Antibody secretion of hybridoma cells: 19 mL of the hybridoma serum-free medium provided in Example 1 and 19 mL of the commercially available hybridoma serum-free medium were added to two cell culture flasks, and the resuspended hybridoma cells were inoculated into the above two culture media, respectively. The same number of hybridoma cells (5 × 10 5 cells / mL, 1mL), and then placed in a carbon dioxide incubator for culture at 37°C, 5% CO2 and 120rpm; during the culture process, the cell growth was supplemented to control the hybridoma cell culture time in the two culture media to be the same (8 days) and the final volume after culture to be the same (1L); then two culture supernatants containing antibodies were collected and purified using the same method, and the volume of the obtained protein solution was recorded after purification, the concentration of the protein solution was determined, and the expression amount of the target antibody of the hybridoma cells in the two different culture media was calculated, and the results are shown in Table 5; samples were also taken for denatured protein electrophoresis to confirm that the purified proteins were all the target antibodies, and the results are shown in Table 5. Figure 8 shown.

[0101] Table 5: Expression levels of antibodies in the hybridoma serum-free culture medium provided in Example 1 and in the commercially available hybridoma serum-free culture medium

[0102]

[0103] from Figure 5-7 It can be seen that the hybridoma cell serum-free culture medium provided in Example 1 of the present application and the commercially available hybridoma cell serum-free culture medium can both meet the growth and proliferation requirements of hybridoma cells, and the growth and proliferation rates of hybridoma cells in the two culture media are not much different; at the same time, Figure 8As can be seen from Table 5, the hybridoma cell serum-free culture medium provided in Example 1 of the present application and the commercially available hybridoma cell serum-free culture medium can both induce hybridoma cells to secrete antibody proteins, and the amount of secreted antibody proteins is not much different, indicating that the hybridoma cell serum-free culture medium provided in the present application can meet market sales needs and provide downstream customers with more serum-free hybridoma cell culture medium product options.

[0104] The above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit them. Although the present application has been described in detail with reference to the aforementioned embodiments, a person skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some or all of the technical features thereof may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A serum-free culture medium for hybridoma cells, characterized in that include: Polyethylene glycol, Pluronic F68, sodium pyruvate, purines, pyrimidines, amino acids, vitamins, cell adhesion factors, cell growth factors, hormones, proteins, trace elements, antibiotics, salts, carbohydrates, buffers and pH indicators.

2. The serum-free culture medium for hybridoma cells according to claim 1, characterized in that: The concentration of the polyethylene glycol is 400-1000 mg / L; The concentration of the Pluronic F68 is 500-1200 mg / L.

3. The serum-free culture medium for hybridoma cells according to claim 1, characterized in that: The concentration of the sodium pyruvate is 1-4 mmol / L.

4. The serum-free culture medium for hybridoma cells according to claim 1, characterized in that: The purine composition includes: adenine, guanine, hypoxanthine and xanthine at a concentration of 5-10 mg / L; The composition of the pyrimidine includes: cytosine, thymine and uracil with a concentration of 5-10 mg / L.

5. The serum-free culture medium for hybridoma cells according to claim 1, characterized in that: The amino acid composition includes: S-sulfocysteine, tryptophan, glutamic acid, aspartic acid, tyrosine, alanine, glycine, serine, threonine, proline, asparagine, histidine, cystine, L-glutamine, isoleucine, leucine, lysine, methionine, phenylalanine, valine and arginine at a concentration of 0.5-30 mmol / L.

6. The serum-free culture medium for hybridoma cells according to claim 5, characterized in that: The concentration of the L-glutamine is 2.0-4.0 mmol / L.

7. The serum-free culture medium for hybridoma cells according to claim 1, characterized in that: The vitamin composition includes: vitamin B5, vitamin B9, vitamin B3, vitamin B6, vitamin B2, vitamin B4, vitamin B8, vitamin A, vitamin C, vitamin D, vitamin E, vitamin K, vitamin B12 and vitamin H with a concentration of 0.5~2.5 mg / L.

8. The serum-free culture medium for hybridoma cells according to claim 1, characterized in that: The cell adhesion factors include: cadherin, selectin, integrin and hyaluronan adhesion in a concentration of 0.1-100 μmol / L; The cell growth factors include: colony stimulating factor, interleukin 2 and interleukin 6 at a concentration of 1-10 μg / L and ethanolamine at a concentration of 4.5-5.5 mg / L; The hormones include: insulin, growth hormone, adrenal cortex hormone and steroid hormone at a concentration of 0.1-10 mg / L; The proteins include: recombinant transferrin and bovine serum albumin at a concentration of 10-50 mg / L; The trace elements include: selenium and molybdenum with a concentration of 0.001-0.5 μmol / L, and manganese, vanadium, zinc, copper, iron and cobalt with a concentration of 0.1-100 μmol / L; The antibiotics include: a concentration of 5×10 4 ~1×10 5 U / L penicillin, 5-100 mg / L streptomycin, gentamicin, paromomycin, kanamycin, and chlortetracycline; The salts include: sodium chloride with a concentration of 4.5-9.0 g / L, potassium amine, calcium chloride, magnesium chloride, sodium dihydrogen phosphate with a concentration of 0.1-0.6 g / L, sodium bicarbonate with a concentration of 1.5-2.0 g / L, citrate and selenite with a concentration of 3-6 μg / L; The carbohydrates include: glucose with a concentration of 0.5-5.0 g / L; The buffer comprises: 4-hydroxyethylpiperazineethanesulfonic acid at a concentration of 10-25 mmol / L; The pH indicator comprises: phenol red with a concentration of 0.005-0.2 g / L.

9. The serum-free culture medium for hybridoma cells according to claim 8, characterized in that: The trace elements include: selenium with a concentration of 0.005-0.5 μmol / L, manganese with a concentration of 0.4-40 μmol / L, molybdenum with a concentration of 0.001-0.1 μmol / L, vanadium with a concentration of 0.07-70 μmol / L, zinc with a concentration of 2-50 μmol / L, copper with a concentration of 0.1-100 μmol / L, iron with a concentration of 1-100 μmol / L, and cobalt with a concentration of 2-60 μmol / L.

10. A method for preparing a serum-free culture medium for hybridoma cells, characterized in that: include: The polyethylene glycol according to any one of claims 1 to 9, Pluronic F68, sodium pyruvate, purine, pyrimidine, amino acids, vitamins, cell adhesion factors, cell growth factors, hormones, proteins, trace elements, antibiotics, salts, carbohydrates, buffers, pH indicators and water for injection are uniformly mixed and filtered to obtain a serum-free culture medium for hybridoma cells.

11. A method for in vitro suspension culture of hybridoma cells using a serum-free culture medium for hybridoma cells according to any one of claims 1 to 9, characterized in that: include: The hybridoma cells are added to a serum-free culture medium for hybridoma cells according to any one of claims 1 to 9, and the hybridoma cells are cultured in vitro in suspension at 37° C., 5% CO 2 and 120 rpm.

12. Use of a serum-free culture medium for hybridoma cells according to any one of claims 1 to 9 in the preparation of antibody drugs.

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