Supplementary additive suitable for HEK293 cell culture and application

Abstract

The invention discloses a feed supplement additive suitable for HEK293 cell culture and application, and the feed supplement additive comprises the following raw material substances: 50-90 g / L of amino acid, 1-3 g / L of vitamin, 3-5.5 g / L of inorganic salt, 5.5-25 mg / L of trace element, 1-3 mg / L of fatty acid, 220-460 mg / L of lipid, 100-260 mg / L of polyamine, and 40-60 g / L of glucose. According to the HEK293 cell metabonomics and nutrition consumption conditions, the additive suitable for material supplementation in the HEK293 cell culture process is obtained under the condition that protein is not used, the additive is excellent in culture effect, beneficial to improving the antibody expression quantity and capable of avoiding instability caused by degradation of protein, and the additive is good in stability, high in yield and good in stability. And the cost is low, the universality is wide, and the use is convenient.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a feed additive suitable for HEK293 cell culture and an application thereof. Background Art

[0002] HEK293 cells are an important platform for expressing antibodies and recombinant proteins. Depending on the duration of expression, gene product expression can be divided into transient expression and stable expression. Stable expression is the gold standard for antibody drug production systems, but the process is time-consuming and costly, making it unfavorable for high-throughput, high-efficiency screening of new antibody drugs. Transient gene expression technology refers to the process in which an exogenous gene enters the recipient cell and exists on a free vector without integrating into the chromosome. The expression product of the target gene can be obtained in a relatively short period of time, but as the cell divides and proliferates, the exogenous gene eventually disappears, and expression lasts for several days to two weeks. This technology greatly shortens the production process of trace to medium amounts of recombinant antibodies, thereby providing an effective means for the rapid production, early screening, and functional and toxicological analysis of antibody drugs.

[0003] Commonly used host cells for transient gene expression technology include HEK293 cells and CHO cells. HEK293 cells have advantages such as fast growth and ease of transfection, which provide great advantages in the rapid and efficient expression and functional analysis of antibody drugs. Therefore, HEK293 transient expression has become one of the commonly used technology platforms for biopharmaceutical companies. However, although transient expression is fast, simple, and easy to operate, protein yields are sometimes less than ideal. In addition, the current market for HEK293 cell feed additives is dominated by internationally renowned culture media such as SIGMA, GIBCO, and Hyclone. These are expensive, costing up to several thousand yuan per liter, which is not conducive to cost reduction for companies. Therefore, there is an urgent need to provide a low-cost feed additive. Summary of the Invention

[0004] The invention discloses a feed additive suitable for HEK293 cell culture and application thereof, so as to solve the problem of high cost of the feed additive provided by the prior art.

[0005] In the first aspect of the present application, a feed additive suitable for HEK293 cell culture medium is provided, comprising the following raw materials: 50-90 g / L of amino acids, 1-3 g / L of vitamins, 3-5.5 g / L of inorganic salts, 5.5-25 mg / L of trace elements, 1-3 mg / L of fatty acids, 220-460 mg / L of lipids, 100-260 mg / L of polyamines, and 40-60 g / L of glucose.

[0006] Furthermore, the amino acids include the following raw materials: arginine 6000-8600 mg / L, cystine 400-600 mg / L, histidine 2000-4200 mg / L, isoleucine 4100-6800 mg / L, leucine 4800-7600 mg / L, lysine 4200-7200 mg / L, methionine 1200-2400 mg / L, phenylalanine 1800-3600 mg / L, threonine 3000-5900 mg / L, tryptophan 100-500 mg / L, tyrosine 40 0~700mg / L, valine 3500~6200mg / L, glycine 100~400mg / L, alanine 200~600mg / L, asparagine 4000~9000mg / L, aspartic acid 2200~5200mg / L, glutamic acid 4000~6300mg / L, proline 3000~5000mg / L, serine 3400~5600mg / L, cysteine ​​300~800mg / L, hydroxyproline 300~600mg / L, glutamine 1000~2200mg / L.

[0007] Furthermore, the vitamins include the following raw materials: choline chloride 220-520 mg / L, calcium pantothenate 10-90 mg / L, folic acid 210-460 mg / L, niacinamide 60-160 mg / L, pyridoxal hydrochloride 30-130 mg / L, riboflavin 3-20 mg / L, thiamine 20-150 mg / L, inositol 180-500 mg / L, lipoic acid 40-170 mg / L, vitamin B12 120-370 mg / L, vitamin C 40-150 mg / L, p-aminobenzoic acid 10-80 mg / L, glutathione 30-90 mg / L, tocopherol 18-90 mg / L, and biotin 9-20 mg / L.

[0008] Furthermore, the inorganic salt includes the following raw materials: 50-120 mg / L of ferric citrate, 460-800 mg / L of sodium pyruvate, 1000-2000 mg / L of potassium chloride, 30-120 mg / L of calcium chloride, 400-600 mg / L of magnesium sulfate, 980-1680 mg / L of sodium dihydrogen phosphate, and 80-180 mg / L of zinc sulfate.

[0009] Furthermore, the trace elements include the following raw materials: nickel chloride 0.002-0.01 mg / L, sodium silicate 1-4 mg / L, manganese chloride 0.01-0.08 mg / L, sodium metavanadate 0.008-0.04 mg / L, stannous chloride 0.002-0.01 mg / L, aluminum chloride 0.1-0.85 mg / L, lithium chloride 0.2-0.8 mg / L, cobalt chloride 0.2-0.8 mg / L, selenious acid Sodium 0.02-0.08 mg / L, sodium molybdate 0.02-0.08 mg / L, copper chloride 0.5-3 mg / L, barium acetate 0.1-0.7 mg / L, ferric nitrate 3-13 mg / L, cadmium chloride 0.2-0.8 mg / L, potassium iodide 0.02-0.08 mg / L, rubidium chloride 0.1-0.5 mg / L, sodium fluoride 0.008-0.06 mg / L, zirconium chloride 0.01-0.11 mg / L.

[0010] Furthermore, the fatty acids include the following raw materials: arachidonic acid 0.1-0.5 mg / L, linoleic acid 0.1-0.5 mg / L, linolenic acid 0.2-0.5 mg / L, oleic acid 0.2-0.5 mg / L, palmitoleic acid 0.2-0.5 mg / L, and stearic acid 0.2-0.5 mg / L.

[0011] Furthermore, the lipids include the following raw materials: 20-60 mg / L cholesterol and 200-400 mg / L ethanolamine.

[0012] Furthermore, the polyamine includes the following raw materials: putrescine 80-200 mg / L and spermine 20-60 mg / L.

[0013] In a second aspect of the present application, a feed additive suitable for HEK293 cell culture medium is provided for use in in vitro culture of HEK293 cells.

[0014] As can be seen from the above technical solution, the present invention discloses a feed additive suitable for HEK293 cell culture and its application, wherein the feed additive includes the following raw materials: 50-90 g / L of amino acids, 1-3 g / L of vitamins, 3-5.5 g / L of inorganic salts, 5.5-25 mg / L of trace elements, 1-3 mg / L of fatty acids, 220-460 mg / L of lipids, 100-260 mg / L of polyamines, and 40-60 g / L of glucose. Based on the metabolomics and nutrient consumption of HEK293 cells, the present invention obtains an additive suitable for feeding 293 cells in the culture process without using protein. The additive has excellent culture effect, is conducive to increasing antibody expression, avoids instability caused by protein degradation, and is not only stable but also low-cost, widely applicable, and easy to use. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The expression level of the antibody when HEK293 cells were cultured using the feed additives in Example 2 and Comparative Example 1;

[0016] Figure 2 The expression level of the antibody when HEK293 cells were cultured using the feed additives in Example 2 and Comparative Example 2;

[0017] Figure 3 The expression level of the antibody when HEK293 cells were cultured using the feed additives in Example 2 and Comparative Example 3;

[0018] Figure 4 The expression levels of antibodies when HEK293 cells were cultured using the feed additives in Example 2 and Comparative Example 4. DETAILED DESCRIPTION

[0019] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0020] 1. Preparation of feed supplement for HEK293 cell culture medium

[0021] The present application provides a feed additive suitable for HEK293 cell culture medium, comprising the following raw materials: 50-90 g / L amino acids, 1-3 g / L vitamins, 3-5.5 g / L inorganic salts, 5.5-25 mg / L trace elements, 1-3 mg / L fatty acids, 220-460 mg / L lipids, 100-260 mg / L polyamines, and 40-60 g / L glucose. The feed additive suitable for HEK293 cell culture medium is formulated according to the component amounts shown in Table 1. The data in Table 1 indicate the final concentrations of each component in the feed additive, expressed in mg / L.

[0022] Table 1 Components of feed additives in various examples and comparative examples

[0023]

[0024]

[0025]

[0026] 2. Transient protein expression

[0027] According to the components of the additives of each embodiment and comparative example provided in Table 1, the feed additive was prepared according to the following steps: S1, the trace elements were prepared into a 1000-fold concentrated solution to facilitate the subsequent addition;

[0028] S2. Use anhydrous ethanol to dissolve fatty acids for easy addition in subsequent preparations;

[0029] S3. Weigh the other ingredients in the formula (each time, at least 2 L) and mix the powders.

[0030] S4. Dissolve the powder in ultrapure water (80% of the final volume);

[0031] S5, mix S1, S2 and S4 liquids and stir for 10 minutes;

[0032] S6. Add 5N sodium hydroxide to adjust the pH until it is completely dissolved; adjust the pH back to 7.0 with 5N hydrochloric acid;

[0033] S7. Add water to correct the final solution volume.

[0034] S8. Continue stirring for 10 minutes and sterile filter into a suitable container.

[0035] The feed additive prepared by the above method was applied to the in vitro culture of HEK293 cells for transient protein expression. The culture steps were as follows:

[0036] S11. Resuscitate HEK293 cells from the same frozen batch in GIBCO CD293 medium (Manufacturer: GIBCO Catalog No.: 11631017). Incubate the cells in a 37°C incubator (120 rpm, 8% CO2) in preparation for transfection and expression of the target protein (anti-25-OH-VD3 monoclonal antibody or anti-Hb monoclonal antibody, respectively).

[0037] S12. Subculture cells every 2 to 3 days to maintain the cells in the early logarithmic growth phase. The cell subculture density should be 0.3-0.5 × 106 cells / mL.

[0038] S13. When the cell density reaches about 3-4×10 6 viable cells / mL and the viability is ≥95%, prepare for transfection of plasmids (expressing anti-25-OH-VD3 monoclonal antibody and anti-Hb monoclonal antibody).

[0039] S14. About 20 hours after transfection, a feed additive (Examples 1 to 3 and Comparative Examples 1 to 3 in Table 1) at a volume ratio of 4-6% of the culture medium and glutamine were added to the shake flask. In addition, GIBCO yeast extract (Manufacturer: GIBCO Product No.: 212750) and glutamine were used as controls. The shake flask was shaken during the addition process.

[0040] S15. Detect glucose in the cell sap to ensure that the glucose concentration is above 3 g / L. Stop the culture when the viability is lower than 60%, and detect the expression product concentration in the cell sap.

[0041] 3. Culture result detection

[0042] In this application, the same culture conditions and culture duration were used to detect the final antibody expression level. The higher the antibody expression level, the better the effect of the culture medium. The basal culture medium used was GIBCO CD293 culture medium (manufacturer: GIBCO; catalog number: 11631017).

[0043] Figure 1 The following table shows the expression levels of different antibodies in HEK293 cells cultured with the additives supplemented in Example 2 and Comparative Example 1. Comparative Example 1, without the addition of fatty acids, showed antibody expression levels of 700 mg / L and 750 mg / L (25-OH-VD3), and 170 mg / L and 190 mg / L (Hb), respectively, compared to Example 2. Because fatty acids play a role in building and maintaining cell membranes, the absence of fatty acids in Comparative Example 1 resulted in lower antibody expression.

[0044] Figure 2 The expression levels of different antibodies in HEK293 cells cultured with supplemental agents in Example 2 and Comparative Example 2 are shown. Comparative Example 2, which lacks tocopherol, showed antibody expression levels of 720 mg / L and 750 mg / L (25-OH-VD3), and 160 mg / L and 190 mg / L (Hb), respectively, compared to Example 2. Tocopherol acts as an antioxidant, protecting cell membrane stability by interrupting free radical chain reactions, preventing lipofuscin formation on the membrane and delaying apoptosis. The absence of tocopherol in Comparative Example 2 resulted in lower antibody expression.

[0045] Figure 3 The figures show the expression levels of different antibodies after supplementation with additives when culturing HEK293 cells in Example 2 and Comparative Example 3. Comparative Example 3, in which aurintricarboxylic acid was not added, showed antibody expression levels of 680 mg / L and 750 mg / L (25-OH-VD3), and 150 mg / L and 190 mg / L (Hb), respectively, compared to Example 2. Aurintricarboxylic acid is a metal chelator that binds, transports, and transfers Fe2+, partially replacing the function of ferroportin. The absence of aurintricarboxylic acid in Comparative Example 3 resulted in lower antibody expression.

[0046] In addition, commercially available GIBCO Bacto TM Yeast extract (manufacturer: GIBCO; product number: 212750) was used as a comparative example 4. The culture process was the same as the above culture process. The antibody expression level was as follows: Figure 4As shown, when used for HEK293 cell culture, the additives of Examples 1, 2, and 3 provided by the present invention are mixed with GIBCO Bacto TM Yeast extract, antibody expression levels were 25-OH-VD3: 710mg / L, 750mg / L, 680mg / L and 640mg / L, Hb: 170mg / L, 190mg / L, 160mg / L and 140mg / L. It can be seen that the culture effect of the additive of the present invention is significantly better than that of GIBCO Bacto TM The culture effect of yeast extract, while the additive of the present invention does not contain protein, GIBCO Bacto TM Yeast extract contains incompletely hydrolyzed protein and its composition is unclear, while the additive of the present invention has a clear chemical composition, low cost and can be widely used.

[0047] The above are merely embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention are included in the scope of the claims of the present invention to be approved.

Claims

1. A feed additive suitable for HEK293 cell culture medium, characterized in that: The raw materials include: amino acid 50-90g / L, vitamin 1-3g / L, inorganic salt 3-5.5g / L, trace element 5.5-25mg / L, fatty acid 1-3mg / L, lipid 220-460mg / L, polyamine 100-260mg / L, glucose 40-60g / L.

2. A feed additive suitable for HEK293 cell culture medium according to claim 1, characterized in that: The amino acids include the following raw materials: arginine 6000-8600 mg / L, cystine 400-600 mg / L, histidine 2000-4200 mg / L, isoleucine 4100-6800 mg / L, leucine 4800-7600 mg / L, lysine 4200-7200 mg / L, methionine 1200-2400 mg / L, phenylalanine 1800-3600 mg / L, threonine 3000-5900 mg / L, tryptophan 100-500 mg / L, tyrosine 400-7 00mg / L, valine 3500~6200mg / L, glycine 100~400mg / L, alanine 200~600mg / L, asparagine 4000~9000mg / L, aspartic acid 2200~5200mg / L, glutamic acid 4000~6300mg / L, proline 3000~5000mg / L, serine 3400~5600mg / L, cysteine ​​3000~800mg / L, hydroxyproline 300~600mg / L, glutamine 1000~2200mg / L.

3. A feed additive suitable for HEK293 cell culture medium according to claim 1, characterized in that: The vitamins include the following raw materials: 220-520 mg / L of choline chloride, 10-90 mg / L of calcium pantothenate, 210-460 mg / L of folic acid, 60-160 mg / L of niacinamide, 30-130 mg / L of pyridoxal hydrochloride, 3-20 mg / L of riboflavin, 20-150 mg / L of thiamine, 180-500 mg / L of inositol, 40-170 mg / L of lipoic acid, 120-370 mg / L of vitamin B12, 40-150 mg / L of vitamin C, 10-80 mg / L of p-aminobenzoic acid, 30-90 mg / L of glutathione, 18-90 mg / L of tocopherol, and 9-20 mg / L of biotin.

4. A feed additive suitable for HEK293 cell culture medium according to claim 1, characterized in that: The inorganic salts include the following raw materials: 50-120 mg / L of ferric citrate, 460-800 mg / L of sodium pyruvate, 1000-2000 mg / L of potassium chloride, 30-120 mg / L of calcium chloride, 400-600 mg / L of magnesium sulfate, 980-1680 mg / L of sodium dihydrogen phosphate, and 80-180 mg / L of zinc sulfate.

5. A feed additive suitable for HEK293 cell culture medium according to claim 1, characterized in that: The trace elements include the following raw materials: nickel chloride 0.002-0.01 mg / L, sodium silicate 1-4 mg / L, manganese chloride 0.01-0.08 mg / L, sodium metavanadate 0.008-0.04 mg / L, stannous chloride 0.002-0.01 mg / L, aluminum chloride 0.1-0.85 mg / L, lithium chloride 0.2-0.8 mg / L, cobalt chloride 0.2-0.8 mg / L, sodium selenite 0. 02~0.08mg / L, sodium molybdate 0.02~0.08mg / L, copper chloride 0.5~3mg / L, barium acetate 0.1~0.7mg / L, ferric nitrate 3~13mg / L, cadmium chloride 0.2~0.8mg / L, potassium iodide 0.02~0.08mg / L, rubidium chloride 0.1~0.5mg / L, sodium fluoride 0.008~0.06mg / L, zirconium chloride 0.01~0.11mg / L.

6. A feed additive suitable for HEK293 cell culture medium according to claim 1, characterized in that: The fatty acids include the following raw materials: arachidonic acid 0.1-0.5 mg / L, linoleic acid 0.1-0.5 mg / L, linolenic acid 0.2-0.5 mg / L, oleic acid 0.2-0.5 mg / L, palmitoleic acid 0.2-0.5 mg / L, and stearic acid 0.2-0.5 mg / L.

7. A feed additive suitable for HEK293 cell culture medium according to claim 1, characterized in that: The lipids include the following raw materials: 20-60 mg / L of cholesterol and 200-400 mg / L of ethanolamine.

8. The feed additive for HEK293 cell culture medium according to claim 1, characterized in that: The polyamine includes the following raw materials: putrescine 80-200 mg / L and spermine 20-60 mg / L.

9. Use of the feed additive according to any one of claims 1 to 8 in in vitro culture of HEK293 cells.

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