A lipid complex for mammalian cell culture medium and its preparation method and application

Abstract

The present invention discloses a lipid complex for mammalian cell culture medium and its preparation method and application, belonging to the field of cell culture technology. The lipid complex includes lipid substances, organic solvents, solubilizers and water prepared in a certain proportion; the lipid substances include one or more of α-linolenic acid, γ-linolenic acid, oleic acid, linoleic acid, eicosapentaenoic acid, docosahexaenoic acid, myristic acid, lauric acid, arachidonic acid and palmitic acid. The lipid complex of the present invention not only has clear chemical composition and is safe and harmless, but also has a simplified formula and excellent performance. It can effectively increase cell growth density and cell viability and increase the yield of target protein when added to the culture medium, and has broad application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell culture, and in particular relates to a lipid complex for mammalian cell culture medium and a preparation method and application thereof. Background Art

[0002] Lipids are major components of biological membranes and can also serve as energy sources and signaling molecules in mammalian cells. They are key components of the endoplasmic reticulum and the Golgi apparatus, organelles responsible for protein synthesis, folding, post-translational modification, and secretion.

[0003] Typically, Chinese hamster ovary cells (CHO cells) are able to synthesize lipids by themselves. Recombinant CHO cells can adapt and grow in lipid-free medium without a significant decrease in cell proliferation rate and product activity. Existing studies have shown that lipid supplementation in serum-free medium is beneficial to cell viability and product glycosylation. In fact, the effects of lipids and lipid precursors on CHO cell growth can vary significantly depending on the type of lipid or precursor provided. Therefore, the choice of lipid supplements in the culture medium can have a significant impact on cell growth.

[0004] With the rapid development of biopharmaceuticals, mammalian cell culture media have also experienced rapid updates and iterations from serum-containing culture media, serum-free culture media, protein-free culture media, animal-free culture media, and completely chemically defined culture media. In the early development of serum-free culture media, in order to maintain performance comparable to that of serum-containing culture media, experts and scholars from various countries conducted in-depth analysis of serum, and have basically found that serum contains growth factors, hormones, proteins, lipids and other unknown nutrients. Therefore, some growth factors are added to serum-free culture media, such as IGF, insulin, etc.; hormones, such as hydrocortisone, dexamethasone, etc.; proteins, such as fibronectin, peptide globulin, etc.; lipids, fatty acids, cholesterol, etc.

[0005] Fatty acids and cholesterol are not highly soluble or stable in the culture medium and have extremely low solubility, so most commercial chemically defined culture media contain basically no lipids. However, in order to maintain better cell growth and enhance the expression of products, they are usually added later.

[0006] Important growth factors, hormones, and proteins that maintain cell growth and product expression can be stably added to the culture medium. However, fatty acids, which are used for the synthesis of animal cell membranes and the maintenance of cell growth, are poorly soluble in water, so their application is limited, which seriously affects the performance of serum-free culture media. Although fatty acids are added to some culture media, they have poor solubility and are directly filtered out when the dry powder is prepared, making it difficult for them to play a role.

[0007] In summary, it plays a key role in improving the growth density, cell viability and expression yield of target protein products of mammalian cells, improving the solubility of lipid substances in mammalian cell culture medium, scientifically and rationally matching lipid substances, increasing the stability of lipid components and improving the absorption rate of lipid components by animal cells, which is also an important factor affecting the cost of biopharmaceuticals. Therefore, it is of great significance to prepare a lipid complex for mammalian cell culture medium that can improve the growth density, cell viability and expression yield of target protein products of mammalian cells. Summary of the invention

[0008] The purpose of the present invention is to provide a lipid complex for mammalian cell culture medium and its preparation method and application, which can solve the technical problems of effective component ratio and solubility of lipid complexes while effectively improving mammalian cell growth density, cell viability and target protein expression yield.

[0009] The first object of the present invention is to provide a lipid complex for mammalian cell culture medium, wherein the lipid complex comprises lipid substances; the lipid substances comprise one or more of α-linolenic acid, γ-linolenic acid, oleic acid, linoleic acid, eicosapentaenoic acid, docosahexaenoic acid, myristic acid, lauric acid, arachidonic acid or palmitic acid.

[0010] In one embodiment, the lipid substance includes 0.1 to 2 g / L of α-linolenic acid, 0.1 to 2 g / L of γ-linolenic acid, 0.1 to 2 g / L of oleic acid, 0.1 to 2 g / L of linoleic acid, 0.1 to 2 g / L of eicosapentaenoic acid, 0.1 to 2 g / L of docosahexaenoic acid, 0.1 to 2 g / L of myristic acid, 0.1 to 2 g / L of lauric acid, 0.1 to 2 g / L of arachidonic acid and 0.1 to 2 g / L of palmitic acid.

[0011] In one embodiment, the lipid complex further comprises an organic solvent and a solubilizing agent.

[0012] In one embodiment, the amount of the organic solvent is 100-500 ml / L.

[0013] In one embodiment, the solubilizing agent is used in an amount of 100 to 500 g / L.

[0014] In one embodiment, the organic solvent is at least one of DMSO, methanol or ethanol.

[0015] In one embodiment, the solubilizing agent is bovine serum albumin and / or a β-cyclodextrin-derived substance.

[0016] In one embodiment, the β-cyclodextrin derivative material includes at least one of hydroxypropyl-β-cyclodextrin, methylated-β-cyclodextrin, sulfobutyl-β-cyclodextrin, and carboxymethyl-β-cyclodextrin.

[0017] The second object of the present invention is to provide a cell culture medium, which contains the lipid complex and a basal culture medium.

[0018] In one embodiment, the lipid complex is added to the basal culture medium at a ratio of (1:200) to (1:1000).

[0019] In one embodiment, the basal culture medium comprises CHO cell culture medium, HEK293 cell culture medium, Vero cell culture medium or MDCK cell culture medium.

[0020] The third object of the present invention is to provide a method for preparing the above-mentioned lipid complex, which comprises dispersing and / or dissolving lipid substances, organic solvents, bovine serum albumin and β-cyclodextrin derivatives in water; the lipid substances include α-linolenic acid, γ-linolenic acid, oleic acid, linoleic acid, eicosapentaenoic acid, docosahexaenoic acid, myristic acid, lauric acid, arachidonic acid and palmitic acid.

[0021] In one embodiment, the method specifically comprises the following steps:

[0022] 1) Measure an appropriate amount of organic solvent and add it to a clean beaker, add lipid substances, and stir until evenly dissolved.

[0023] 2) Measure an appropriate amount of ultrapure water and add it to a clean beaker, add bovine serum albumin and / or hydroxypropyl-β-cyclodextrin, and stir until evenly dissolved.

[0024] 3) Mix the solution of step 1) and the solution of step 2) and continue stirring until they are completely dissolved.

[0025] 4) Use ultrapure water to make up to the final volume.

[0026] 5) After filtering through a sterile bladder filter with an outer layer of 0.45 μm and an inner layer of 0.2 μm, the lipid complex is obtained, which is divided into portions and stored at 4°C.

[0027] The present invention also provides a method for increasing the yield of a target protein, which comprises using a culture medium containing the lipid complex or the cell culture medium to carry out cell culture.

[0028] In one embodiment, the lipid complex is added to the culture medium at a ratio of (1:200) to (1:1000).

[0029] In one embodiment, the method increases the yield of the target protein while increasing the viable cell density and cell viability.

[0030] The present invention also provides application of the lipid complex or the cell culture medium or the method in cell culture.

[0031] The present invention has the following beneficial effects:

[0032] The lipid complex of the present invention not only has clear chemical compositions, which are all ingredients widely used in mammalian cell culture, are safe and harmless, but also has a simplified formula and excellent performance. Adding it to the culture medium can significantly promote the absorption of lipid nutrients by mammalian cells, significantly improve the performance of serum-free culture medium, effectively increase cell growth density and cell viability, and increase the yield of target protein, and has broad application value.

[0033] The scientific ratio of the lipid complex of the present invention effectively improves the solubility of lipid substances in culture medium / water, increases the stability of lipid components and the absorption rate of lipid components by animal cells. It not only has clear chemical composition but is also safe and harmless. It has high application value for improving the growth density, cell viability and expression yield of target protein products in mammalian cell culture.

[0034] The organic solvent and carrier selected by the present invention can effectively improve the solubility of one or several lipid complexes of α-linolenic acid, γ-linolenic acid, oleic acid, linoleic acid, eicosapentaenoic acid, docosahexaenoic acid, myristic acid, lauric acid, arachidonic acid and palmitic acid in water; the dissolved lipid complex is added to the culture medium and is easily absorbed by cells, which can effectively improve the cell production and the maintenance time of high viability, and has a high application prospect.

[0035] The invention effectively ensures the solubility and stability of one or more lipid complexes of α-linolenic acid, γ-linolenic acid, oleic acid, linoleic acid, eicosapentaenoic acid, docosahexaenoic acid, myristic acid, lauric acid, arachidonic acid and palmitic acid in water through a reasonable ratio of the added amounts of organic solvent and carrier. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the prior art descriptions, which are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation on the present invention. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without creative work. In the drawings:

[0037] Figure 1 Schematic diagram of cell density of each group of culture medium in Example 2 of the present invention;

[0038] Figure 2 Schematic diagram of cell viability of each group of culture medium in Example 2 of the present invention;

[0039] Figure 3 This is a schematic diagram of the cell protein yield of each group of culture medium in Example 2 of the present invention;

[0040] Figure 4 Schematic diagram of cell density of each group of culture medium in Example 3 of the present invention;

[0041] Figure 5 Schematic diagram of cell viability of each group of culture medium in Example 3 of the present invention;

[0042] Figure 6 This is a schematic diagram of the cell protein yield of each group of culture medium in Example 3 of the present invention;

[0043] Figure 7 Schematic diagram of cell density of each group of culture medium in Example 4 of the present invention;

[0044] Figure 8 Schematic diagram of cell viability of each group of culture medium in Example 4 of the present invention;

[0045] Fig. 9 This is a schematic diagram of the cell protein yield of each group of culture medium in Example 4 of the present invention. DETAILED DESCRIPTION

[0046] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0047] Sources of materials in the examples: α-linolenic acid (Sigma, Product No. L2376), γ-linolenic acid (Sigma, Product No. L2378), oleic acid (Sigma, Product No. O1383), linoleic acid (Sigma, Product No. L1012), eicosapentaenoic acid (Biosynth, Product No. FE164050), docosahexaenoic acid (Sigma, Product No. D2534), myristic acid (Sigma, Product No. M3128), lauric acid (Sigma, Product No. W261408), arachidonic acid (Sigma, Product No. A3611), palmitic acid (Sigma , Catalog No. P0500), DMSO (Sigma, Catalog No. D2650), methanol (Sigma, Catalog No. 179337), ethanol (Sigma, Catalog No. 1.00983), bovine serum albumin (Sigma, Catalog No. A1933), hydroxypropyl-β-cyclodextrin (Sigma, Catalog No. C0926), methylated-β-cyclodextrin (Sigma, Catalog No. C4555), sulfobutyl-β-cyclodextrin (Biosynth, Catalog No. OC59560), carboxymethyl-β-cyclodextrin (Sigma, Catalog No. 21906); the commercial cell culture medium is CHO MaxA medium (MaiBang Bio, Catalog No. MB1101.102)

[0048] In addition, other methods and reagents used in the examples are conventional methods and reagents in the art unless otherwise specified.

[0049] Example 1 Preparation of lipid complex

[0050] (1) Take a clean 500 mL beaker, add 200 mL DMSO, and add 1 g α-linolenic acid, 1 g γ-linolenic acid, 1 g oleic acid, 1 g linoleic acid, 1 g eicosapentaenoic acid, 1 g docosahexaenoic acid, 1 g myristic acid, 1 g lauric acid, 1 g arachidonic acid, and 1 g palmitic acid in sequence, add a magnetic stirring rotor, and stir until uniformly dissolved to obtain a primary mixed solution of the lipid complex.

[0051] (2) Take a clean 1000 mL beaker, add 500 mL of ultrapure water, add 15 g of bovine serum albumin and 100 g of hydroxypropyl-β-cyclodextrin, add a magnetic stirring rotor, and stir until the dry powder is completely dissolved into a clear mixed solution.

[0052] (3) Transfer the primary mixed solution of step (1) to the mixed solution of step (2) and continue stirring until the solution becomes a uniform, clear solution.

[0053] (4) Add an appropriate amount of ultrapure water to the solution in step (3) to make up to the final volume, and then filter using a sterile 0.45 μm outer layer and 0.2 μm inner layer sterile capsule filter to obtain a sterile lipid complex, which is packaged and stored at 4°C for future use.

[0054] The prepared lipid complex was added to commercial serum-free culture medium at a ratio of 1:300, 1:500 or 1:700 for cell culture testing. For example, the lipid complex was added at a ratio of 1:500, i.e., 1 mL of lipid complex was added to 500 mL of culture medium.

[0055] Example 2 CHO K1 cell culture test using CHO MaxA cell culture medium

[0056] The lipid complex prepared in Example 1 was added to CHO MaxA medium at a ratio of 1:300, 1:500, and 1:700, respectively. Fed Batch experiments of CHO K1 cells expressing trastuzumab were performed using CHO MaxA medium without the addition of lipid complex and CHO MaxA medium with the addition of lipid complex, respectively, and the cell density VCD, cell viability VIA, and antibody yield were detected.

[0057] The results are as follows Figures 1 to 3 As shown, the lipid complex of the present invention was added to CHO MaxA culture medium at a ratio of 1:300, and the highest cell density PVCD was the highest, reaching 4.6×10 7 cells / mL, the cell viability was the highest when the lipid complex was added to CHO MaxA culture medium at a ratio of 1:500, reaching more than 95%. The antibody yield was the highest when the lipid complex was added to CHO MaxA culture medium at a ratio of 1:500, reaching 3.5g / L. Compared with the control group without adding lipid complexes, adding lipid complexes at different ratios significantly increased the cell density, cell viability and protein yield of CHO K1.

[0058] Example 3 293 cell culture test using HEK293 MaxD cell culture medium

[0059] The lipid complex prepared in Example 1 was added to HEK293 MaxD medium at a ratio of 1:300, 1:500, and 1:700, respectively. HEK293 MaxD medium added with lipid complex was used to perform transfection tests on HEK293 cells, and HEK293 MaxD medium without lipid mixture was used as a control group.

[0060] A JET SF125 breathable shake flask was used with an inoculation volume of 27 mL and a cell inoculation density of 3E6 cells / mL. 3 mL of plasmid-PEI complex was added to a total volume of 30 mL. The plasmids were pcDNA3.4-H and pcDNA3.4-L expressing trastuzumab. The shaker speed was 120 rpm and 5% CO 2 , amplitude 50mm.

[0061] The results are as follows Figures 4 to 6 As shown in the figure, it can be seen that the PVCD was the highest when the lipid complex was added to the HEK293 MaxD medium at a ratio of 1:300, reaching 2×10 7 cells / mL, the cell viability was best maintained during the culture process, the cell viability was the highest at harvest, reaching more than 80%, and the antibody yield was the highest, reaching 0.44g / L.

[0062] Example 4: Testing the Effect of Different Lipid Concentrations on the Test Results at an Addition Ratio of 1:500

[0063] The preparation method of the lipid complex is the same as that of Example 1, except that the concentration of different lipids in the lipid complex is adjusted, and three groups of lipid complexes with different lipid concentrations are set up, and added to CHO MaxA cell culture medium at a ratio of 1:500 to perform a culture test of CHO K1 cells expressing monoclonal antibodies. The test conditions are to use a JET 125mL breathable shake flask, an initial culture volume of 30mL, and an initial cell seeding density of 1E6 / mL. The shaker speed is 120rpm, 5% CO 2 , amplitude 50mm. The initial culture temperature was 36.5℃. When 14E6<VCD<16E6, the temperature was lowered to 33℃, and when 20E6<VCD<25E6, the temperature was lowered to 31℃. 200μL samples were taken at D1, D3, D5, D7, D9, D11, D12, and D13, and parameters such as VCD and VIA were measured after sampling. The D13 sample was used to check the antibody yield.

[0064] Group 1: concentration of lipid substances, 0.5 g / L α-linolenic acid, 0.5 g / L γ-linolenic acid, 0.5 g / L oleic acid, 0.5 g / L linoleic acid, 0.5 g / L eicosapentaenoic acid, 0.5 g / L docosahexaenoic acid, 0.5 g / L myristic acid, 0.5 g / L lauric acid, 0.5 g / L arachidonic acid, 0.5 g / L palmitic acid;

[0065] Group 2: concentration of lipid substances, 1g / L α-linolenic acid, 1g / L γ-linolenic acid, 1g / L oleic acid, 1g / L linoleic acid, 1g / L eicosapentaenoic acid, 1g / L docosahexaenoic acid, 1g / L myristic acid, 1g / L lauric acid, 1g / L arachidonic acid, 1g / L palmitic acid;

[0066] Group 3: Concentration of lipid substances, 2g / L α-linolenic acid, 2g / L γ-linolenic acid, 2g / L oleic acid, 2g / L linoleic acid, 2g / L eicosapentaenoic acid, 2g / L docosahexaenoic acid, 2g / L myristic acid, 2g / L lauric acid, 2g / L arachidonic acid, and 2g / L palmitic acid.

[0067] The results are as follows Figures 7 to 9 As shown in the figure, the PVCD of lipid mixture with concentrations of 0.5 g / L and 1 g / L added to CHO MaxA medium was the highest, reaching 3×10 7 cells / mL, the cell viability was the highest when 0.5g / L concentration of lipid mixture was added to CHO MaxA culture medium during harvest, reaching more than 80%. The antibody yield was the highest when 0.5g / L concentration of lipid mixture was added to CHO MaxA culture medium, reaching 5.5g / L, which was slightly higher than that of 1g / L concentration of lipid mixture. The lipid mixture of 2g / L concentration was the lowest in PVCD, harvest viability and yield.

[0068] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A lipid complex for mammalian cell culture medium, characterized in that: The lipid complex comprises a lipid substance, an organic solvent and a solubilizing agent; the lipid substance comprises 0.5 to 1 g / L of α-linolenic acid, 0.5 to 1 g / L of γ-linolenic acid, 0.5 to 1 g / L of oleic acid, 0.5 to 1 g / L of linoleic acid, 0.5 to 1 g / L of eicosapentaenoic acid, 0.5 to 1 g / L of docosahexaenoic acid, 0.5 to 1 g / L of myristic acid, 0.5 to 1 g / L of lauric acid, 0.5 to 1 g / L of arachidonic acid and 0.5 to 1 g / L of palmitic acid, and the contents of α-linolenic acid, γ-linolenic acid, oleic acid, linoleic acid, eicosapentaenoic acid, docosahexaenoic acid, myristic acid, lauric acid, arachidonic acid and palmitic acid are the same.

2. The lipid complex according to claim 1, characterized in that The amount of the organic solvent is 100-500 ml / L, and the amount of the solubilizing agent is 100-500 g / L.

3. The lipid complex according to claim 1 or 2, characterized in that The organic solvent is at least one of DMSO, methanol or ethanol; the solubilizer is bovine serum albumin and / or a β-cyclodextrin derivative.

4. The lipid complex according to claim 3, characterized in that The β-cyclodextrin derivative material includes at least one of hydroxypropyl-β-cyclodextrin, methylated-β-cyclodextrin, sulfobutyl-β-cyclodextrin, and carboxymethyl-β-cyclodextrin.

5. A cell culture medium, characterized in that The cell culture medium contains the lipid complex according to any one of claims 1 to 4 and a basal culture medium.

6. The cell culture medium according to claim 5, characterized in that The lipid complex is added to the basic culture medium at a ratio of (1:200) to (1:1000).

7. The cell culture medium according to claim 5 or 6, characterized in that The basic culture medium includes CHO cell culture medium, HEK293 cell culture medium, Vero cell culture medium or MDCK cell culture medium.

8. A method for preparing the lipid complex according to any one of claims 1 to 4, characterized in that: The steps of the method are: 1) Take an appropriate amount of organic solvent and add it to a clean beaker, and add one or more of α-linolenic acid, γ-linolenic acid, oleic acid, linoleic acid, eicosapentaenoic acid, docosahexaenoic acid, myristic acid, lauric acid, arachidonic acid or palmitic acid in turn, and stir until evenly dissolved; 2) Take an appropriate amount of ultrapure water and add it to a clean beaker, add one or more of bovine serum albumin and / or hydroxypropyl-β-cyclodextrin, and stir until uniformly dissolved; 3) mixing the solution of step 1) and the solution of step 2), and continuing to stir until completely dissolved; 4) Use ultrapure water to make up to the final volume; 5) After filtering through a sterile bladder filter with an outer layer of 0.45 μm and an inner layer of 0.2 μm, the lipid complex is obtained, which is divided into portions and stored at 4°C.

9. Use of the lipid complex according to any one of claims 1 to 4, the cell culture medium according to any one of claims 5 to 7, or the method according to claim 8 in cell culture.

10. The use according to claim 9, characterized in that: The application is to increase the yield of target protein, increase the density of living cells and / or cell viability.

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