A high-expression CHO cell culture method

Abstract

The present application discloses a high-expression CHO cell culture method, which relates to the field of biological cell culture technology, and specifically comprises the following steps: S1: reviving frozen CHO cells in a culture medium; wherein, a mixed lipid is additionally added to the culture medium, and the mixed lipid is a combination of two to four substances selected from Tween 80, tocopherol, linolenic acid, and cholesterol; S2: performing a subculturing operation every 2-3 days; S3: subculturing again; S4: subculturing 2-4 times according to the S2 and S3 methods, and starting to inoculate after the cell population doubling time stabilizes; S5: batch feeding using a feed medium after cultivation; S6: harvesting after 14-16 days of cultivation and detecting protein concentration. The method solves the problems of low cell density, low viability, lactic acid accumulation, and low protein yield in the cell culture process, improves the growth and expression of CHO cells, reduces enterprise costs, and also reduces technical problems caused by lactic acid accumulation in actual industrial production processes.

Description

Technical Field

[0001] The present invention relates to the technical field of biological cell culture, in particular to a method for culturing high-expression CHO cells. Background Art

[0002] CHO cells, short for Chinese Hamster Ovary (CHO), were first isolated from an adult female hamster ovary by Dr. Theodore T. Puck of the University of Colorado in 1957. They are currently one of the most widely used cell lines in bioengineering. The most commonly used CHO cell lines in industry are mainly divided into CHOK1, CHOK1SV, CHOZN, CHODG44, CHOS, etc. CHO cells are often used in bioengineering to prepare recombinant therapeutic proteins and monoclonal antibodies (mAbs), such as adalimumab and belotuzumab. Compared with other cell types, CHO cells have the advantages of (1) being able to grow stably in chemically defined and serum-free suspension cultures, (2) exhibiting reasonable safety in response to human pathogenic viruses, and (3) being able to express post-translational modifications similar to those in humans. The earliest in vitro cell culture media were composed of nutrient-rich components of serum (human or fetal bovine), plasma, or tissue extracts. These components were complex to extract, exhibited poor batch-to-batch stability, and had unclear chemical compositions. Conventional CHO cell culture typically requires 10% fetal bovine serum, which increases contamination risk and hinders protein purification in downstream processes. Therefore, the use of chemically defined, serum-free, and protein-free cell culture media has a significant impact on actual industrial production.

[0003] Chemically defined culture media, free of animal-derived ingredients, primarily contain amino acids, vitamins, trace elements, inorganic salts, carbon sources, lipids, and additives. These components are crucial for cell growth and expression during cell culture. Lipids, also known as lipids, are a large class of organic compounds that are insoluble or slightly soluble in water but soluble in organic solvents. Esters formed from fatty acids and alcohols, and their derivatives, are important nutrients for the human body, providing energy and essential fatty acids. Lipids are a major component of cell membranes, ensuring the relative stability of the intracellular environment while regulating and selecting the entry and exit of substances. They act as a barrier to the free flow of substances into and out of the cell and also play a crucial role in substance transport and protein secretion. The cell membrane is a complex entity, primarily containing glycerophospholipids, sphingomyelins, glycolipids, cholesterol, proteins, and carbohydrates. Among the glycerophospholipids, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine are the most abundant. The basic raw materials for their synthesis are glycerol, fatty acids, phosphates, choline, serine, and inositol. The main raw materials for the synthesis of sphingomyelin include acyl CoA and serine, and coenzymes such as pyridoxal are also required. Therefore, ingredients such as choline, ethanolamine, and inositol are often added to the culture medium. Fatty acids and glycerol are primarily derived from glucose metabolism. Adding appropriate concentrations of lipids to the culture medium can significantly improve cell growth, metabolism, and protein expression.

[0004] Currently, cells still face problems such as low density, low viability, lactic acid accumulation, and low protein production during culture. Summary of the Invention

[0005] The embodiments of the present application provide a high-expression CHO cell culture method, which solves the problems of low cell density, low cell viability, lactic acid accumulation, and low protein production during the cell culture process, improves the growth and expression of CHO cells, and not only reduces enterprise costs but also alleviates technical problems caused by lactic acid accumulation in actual industrial production processes.

[0006] The present invention provides a method for culturing high-expressing CHO cells, which specifically includes the following steps:

[0007] S1: Resuscitate frozen CHO cells in culture medium and culture the cells in a shaker at 37°C, 8% CO2, and 110-130 rpm.

[0008] Wherein, the culture medium is additionally added with mixed lipids, wherein the mixed lipids are a combination of two to four substances selected from Tween 80, tocopherol, linolenic acid, and cholesterol;

[0009] Tween 80: 0.025-0.75 mg / L, tocopherol: 0.1-3 mg / L, linolenic acid: 0.225-6.75 mg / L, cholesterol: 0.15-4.5 mg / L;

[0010] The CHO cells are CHOK1 cells;

[0011] S2: Subculture every 2-3 days to maintain cells in the early logarithmic growth phase. The cell seeding density is (0.3-1.0)-1.0×106 cells / mL.

[0012] S3: When the cell density reaches 4.5-6.0×106 cells / mL and the cell viability is ≥95%, the cells are passaged again after an interval of 2-3 days;

[0013] S4: Passage 2-4 times according to the S2 and S3 methods. After the cell population doubling time stabilizes, start inoculation. Inoculate the cells into the culture medium and culture in a 125 mL shake flask or a 5 L stirred tank bioreactor.

[0014] The shaker temperature was 36.5-37°C, the rotation speed was 130±20 rpm, and the CO2 concentration in the incubator was set to 5%-10%. The bioreactor temperature was 36.5-37°C, the initial rotation speed was 180±20 rpm, the pH was set to 7.0±0.3, and the DO was set to 40%. The PDT stabilization deviation was less than 3 hours. The inoculation density was 0.4-1.2×106 cells / mL.

[0015] S5: Batch feeding using feed medium after cultivation;

[0016] Among them, feed 3-8% and stop feeding 1-2 days before the end of culture;

[0017] S6: harvested on day 14-16 of culture and protein concentration determined;

[0018] Among them, the protein is an antibody.

[0019] Furthermore, the addition of the mixed lipids is carried out in a step-by-step manner.

[0020] Furthermore, in a step-by-step manner, in the initial addition of the mixed lipids in the culture medium, the addition amount is calculated as a percentage of the total amount of each substance added, Tween 80 is 75%; tocopherol is 80%; linolenic acid is 50%; cholesterol is 50%;

[0021] The remaining mixed lipids are added in step S5 while batch feeding is performed using feed medium to supplement lipids.

[0022] Furthermore, the feed and lipid supplementation is specifically that the mixed lipids are added simultaneously with the feed, once every two days, and the addition is stopped two days before the end of the culture, for a total of six additions, and the total amount of the mixed lipids added each time is the same; specifically, the amount added each time is calculated as a percentage of the total amount of each substance added, Tween 80 is 4.2%, tocopherol is 3.3%, linolenic acid is 8.3%, and cholesterol is 8.3%.

[0023] Furthermore, the feed and lipid supplementation comprises different amounts of mixed lipids added at different cell growth stages;

[0024] Among them, the cell growth stage is divided into early, middle and late stages.

[0025] Furthermore, the initial stage is 0-6 days of cell culture, and is added 3 times; the middle stage is 6-10 days of cell culture, and is added 2 times; and the late stage is 10-14 days of cell culture, and is added once.

[0026] Furthermore, the added amount is calculated as a percentage of the total amount of each substance added:

[0027] In the early stage, Tween 80 is 6%, tocopherol is 2%, linolenic acid is 7%, and cholesterol is 8%;

[0028] In the middle stage, Tween 80 is 3%, tocopherol is 4%, linolenic acid is 11.5%, and cholesterol is 9.5%;

[0029] In the later stage, Tween 80 is 1%, tocopherol is 6%, linolenic acid is 6%, and cholesterol is 7%.

[0030] Furthermore, when large-scale culture is performed, the cells are inoculated into 3 L roller bottles for culture in step S4.

[0031] Furthermore, a rear cover is provided at the left end of the rotating bottle, the rear cover is sealed, the left end of the rear cover is connected to the trachea, and the rear cover contains three syringes arranged up and down, the syringes are connected to the interior of the rotating bottle, and the resistance of the syringes is different;

[0032] The resistance range of the upper syringe is 0.5-1.0 N·s / m3, the resistance range of the middle syringe is 1.5-2.5 N·s / m3, and the resistance range of the lower syringe is 3-4 N·s / m3.

[0033] Furthermore, the lactic acid content was detected by sampling every 12 hours. When the lactic acid content was higher than 1.0 g / L, 0.1-0.3 nM of thyroid hormone was uniformly added.

[0034] One or more technical solutions provided in the embodiments of this application have at least the following technical effects or advantages:

[0035] First, the present invention facilitates the expression of target proteins by CHO cells, improving CHO cell growth and expression. Using the optimized culture medium of the present invention, in terms of growth, the peak cell density can reach 20.7×106 cells / mL, the harvest viability after 14 days of culture can be increased to 90.39%, lactate is 0 g / L, and the expression level can reach 7598 mg / L. Without changing the solubility of the culture medium, the present invention, by adding mixed lipids and using cell growth, metabolism, and protein expression as evaluation indicators, discovers the optimal lipid species and concentration combination in the culture medium, thereby solving the problems of low cell density, low viability, lactate accumulation, and low protein yield during cell culture. This not only reduces enterprise costs, but also alleviates technical problems caused by lactate accumulation in actual industrial production processes.

[0036] Secondly, the step-by-step addition of mixed lipids improves the cell metabolic pathway, making the production and consumption of lactate more balanced. In the later stages of culture, due to the stability of cell density and efficient metabolic regulation, lactate is effectively consumed, avoiding lactate accumulation. In the later stages of culture, lactate is effectively consumed, avoiding the rebound of lactate. This balanced metabolic state can promote healthy cell growth and high-yield expression. The step-by-step addition of mixed lipids improves the health of cells and metabolic efficiency, promotes the synthesis and secretion of target proteins, and at the same time, the increase in cell density and the improvement of the metabolic environment promote high protein expression.

[0037] Third, by adjusting the amount of mixed lipids added in stages, the high concentration of Tween 80 in the early stage promoted cell dispersion and growth, the added linolenic acid and cholesterol in the middle stage promoted the formation and stability of cell membranes, and the added tocopherol in the later stage enhanced the cells' antioxidant capacity, thereby maintaining a high cell growth rate and density throughout the entire culture process;

[0038] Fourth, in large-scale culture, the efficiency and stability of the large-scale culture process are improved by setting syringes with different resistances in the rotary bottle and combining air pressure control to realize the automatic addition of thyroid hormone. Through the redesign of the rotary bottle, automated operation can be realized, which significantly improves the efficiency of thyroid hormone addition, reduces manual intervention, avoids human errors, and ensures the efficiency of each thyroid hormone addition, as well as the accuracy and consistency of the addition. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is a line graph of Table 2 of Example 1 of the present invention;

[0040] Figure 2 This is a line graph of Table 3 of Example 1 of the present invention;

[0041] Figure 3 This is a line graph of Table 4 of Example 1 of the present invention;

[0042] Figure 4 This is a line graph of Table 5 of Example 1 of the present invention;

[0043] Figure 5 This is a line graph of Table 6 of Example 1 of the present invention;

[0044] Figure 6 This is a bar graph of Table 7 of Example 1 of the present invention;

[0045] Figure 7 Schematic diagram of a rotating bottle according to embodiment 4 of the present invention. DETAILED DESCRIPTION

[0046] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains; the terms used herein in the specification of the present invention are for the purpose of describing specific embodiments only and are not intended to limit the present invention; the term "and / or" used herein includes any and all combinations of one or more of the associated listed items.

[0047] The preparation of the culture medium and the components of the cell line culture medium in the examples can be purchased from Sigma, GIBCO, etc.

[0048] Example 1: A method for culturing high-expressing CHO cells, comprising the following steps:

[0049] S1: Resuscitate frozen CHO cells in culture medium and culture the cells in a shaker at 37°C, 8% CO2, and 110-130 rpm.

[0050] The culture medium is prepared by adding mixed lipids to a commercially available basal culture medium;

[0051] The mixed lipids are composed of two to four substances selected from Tween 80, tocopherol, linolenic acid, and cholesterol; Tween 80: 0.025-0.75 mg / L, tocopherol: 0.1-3 mg / L, linolenic acid: 0.225-6.75 mg / L, and cholesterol: 0.15-4.5 mg / L;

[0052] The CHO cells are CHOK1 cells;

[0053] S2: Subculture every 2-3 days to maintain cells in the early logarithmic growth phase. The cell seeding density is (0.3-1.0)-1.0×106 cells / mL.

[0054] S3: When the cell density reaches 4.5-6.0×106 cells / mL and the cell viability is ≥95%, the cells are passaged again after an interval of 2-3 days;

[0055] S4: Passage 2-4 times according to the S2 and S3 methods. After the cell population doubling time stabilizes, start inoculation. Inoculate the cells into the culture medium and culture in a 125 mL shake flask or a 5 L stirred tank bioreactor.

[0056] The shaker temperature was 36.5-37°C, the rotation speed was 130±20 rpm, and the CO2 concentration in the incubator was set to 5%-10%. The bioreactor temperature was 36.5-37°C, the initial rotation speed was 180±20 rpm, the pH was set to 7.0±0.3, and the DO was set to 40%. The PDT stabilization deviation was less than 3 hours. The inoculation density was 0.4-1.2×106 cells / mL.

[0057] S5: Batch feeding using feed medium after cultivation;

[0058] Among them, feed 3-8% and stop feeding 1-2 days before the end of culture;

[0059] S6: harvested on day 14-16 of culture and protein concentration determined;

[0060] Among them, the protein is an antibody.

[0061] For the high-expression CHO cell culture method of this embodiment, the following experiments were performed:

[0062] Take a cryopreserved tube of CHOK1 cells from a liquid nitrogen tank, shake and dissolve in a 37°C water bath, add to a 125 mL shake flask containing 20 mL of culture medium, and mix thoroughly. Then, culture the cells in a shaker at 110-130 rpm, 5-8% CO2, and 37 ± 0.5°C.

[0063] The culture medium is prepared by adding mixed lipids to GIBCO DMEM low-glucose culture medium;

[0064] Subculture every 2 days to keep the cells in the early logarithmic growth phase with a cell seeding density of 0.2-1.0×106 cells / mL;

[0065] When the cell density reaches 4-7×106 cells / mL and the cell viability is ≥95%, the cells are passaged again after 2-3 days.

[0066] The cells were passaged at least three times. After the population doubling time stabilized, they were inoculated. The inoculation time was D0, and the inoculation density was 0.8 ± 1.2 × 106 cells / mL. The cells were inoculated into 125 mL shake flasks. The incubation temperature was set to 37 ± 0.5°C, the CO2 was set to 8%, the shaker humidity was set to 80%, and the shaker speed was set to 110-130 rpm.

[0067] During the culture process, samples were taken every 2 days and counted using a Countstar counter. Each sample was counted three times. The cell viability was determined by trypan blue staining, and metabolic assays were performed using an M100 biochemical analyzer.

[0068] After the culture, the cells were fed in batches using feed medium at a rate of 3-8% every two days until 1-2 days before the end of the culture. The cells were harvested on the 14th day of culture and the antibody concentration was tested using Cedex.

[0069] The experimental plan uses Minitab to set the running sequence for fractional factorial analysis (Examples 1-8). The running sequence does not involve the combination of 2-3 substances at low concentrations (Examples 9-19);

[0070] Among them, high concentration is marked as H, low concentration is marked as L; Tween 80 low concentration is 0.025 mg / L, high concentration is 0.75 mg / L; tocopherol low concentration is 0.1 mg / L, high concentration is 3 mg / L; linolenic acid low concentration is 0.225 mg / L, high concentration is 6.75 mg / L; cholesterol low concentration is 0.15 mg / L, high concentration is 4.5 mg / L;

[0071] The cell culture protocols and results are shown in Tables 1, 2, 3, 4, 5, 6, and 7. The key combinations are as follows: Figure 1 、 Figure 2 、 Figure 3 、 Figure 4 、 Figure 5 、 Figure 6 As shown:

[0072] Table 1. Main implementation plans for cell culture

[0073] Shake bottle number Twain 80 Tocopherol Linolenic acid cholesterol Example 1 L L L L Example 2 L H H L Example 3 H L L H Example 4 H H L L Example 5 L H L H Example 6 L L H H Example 7 H L H L Example 8 H H H H Example 9 L L / / Example 10 L / L / Example 11 L / / L Example 12 / L L / Example 13 / L / L Example 14 / / L L Example 15 L L L / Example 16 L L / L Example 17 L / L L Example 18 / L L L Control Example / / / /

[0074] Table 2. Viable cell density in 125 mL shake flask (×10 6 cells / mL) over time

[0075] Cultivation time / day 0 2 4 6 8 10 12 14 Example 1 0.965 4.93 14.7 20.1 16.6 16.2 14.7 13.6 Example 2 0.965 5 14.1 19.8 17.2 16.5 14 13.2 Example 3 0.965 4.77 15.1 19.9 17.1 16.1 13.8 13.1 Example 4 0.965 4.92 14.4 19.3 17.6 15.3 13.7 13.1 Example 5 0.965 4.82 14.1 18.2 16.4 14.9 13.2 12.2 Example 6 0.965 4.89 14.9 19.1 17.3 16.3 13.7 13.2 Example 7 0.965 4.71 15.9 18.2 16.7 14.7 13.8 12.6 Example 8 0.965 4.6 15.2 18 17.6 15.8 12.2 12.6 Example 9 0.965 5.02 14.6 18.5 14.9 13.4 14 10.8 Example 10 0.965 4.45 13.6 15.8 12.1 10.8 8.36 5.72 Example 11 0.965 4.99 14.5 19.2 15.9 14.2 13.9 11.7 Example 12 0.965 4.68 15.2 16.2 13.1 11.6 10.5 8.59 Example 13 0.965 4.93 15.8 19.7 15.8 12.2 12.7 11.6 Example 14 0.965 5.11 15.3 19.9 15.5 11.8 11.5 10.7 Example 15 0.965 4.76 14 17.7 12.3 10.1 9.44 7.73 Example 16 0.965 5.14 16.1 20.7 14.6 14 13 11.4 Example 17 0.965 4.9 15.6 21.3 17.7 16.1 13.3 13.2 Example 18 0.965 5.17 16.5 20.2 16.4 15.2 13.6 13.3 Control Example 0.965 5.03 14.4 18.4 14.2 12.8 10.2 8.81

[0076] Table 3. Trend of viable cell viability (%) in 125 mL shake flasks over time

[0077] Cultivation time / day 0 2 4 6 8 10 12 14 Example 1 97.89 99.06 98.81 97.67 97.52 96.18 94.22 85.97 Example 2 97.89 99.33 99.06 97.29 97.19 95.99 95.01 90.39 Example 3 97.89 99.4 98.93 97.58 96.02 94.98 94.68 87.47 Example 4 97.89 99.06 99.09 96.95 96.8 96.13 94.38 87.09 Example 5 97.89 99.14 99.05 96.71 95.74 95.54 94.32 88.52 Example 6 97.89 99.26 98.9 97.37 95.51 96.06 93.32 87.62 Example 7 97.89 99.34 99.08 96.86 96.27 96.34 94.16 89.79 Example 8 97.89 99.38 99.18 97.04 93.74 95.95 90.68 90.25 Example 9 97.89 98.72 99.02 97.04 95.55 94.95 94.29 86.07 Example 10 97.89 99.13 98.13 93.98 88.55 86.03 79.11 72.31 Example 11 97.89 99.17 98.82 96.53 95.19 95.72 92.22 87.24 Example 12 97.89 99.39 98.85 96.4 89.84 87.64 79.41 76.28 Example 13 97.89 99.32 99.23 97.51 95.59 92.77 91.05 84.45 Example 14 97.89 99.14 98.86 96.13 90.9 93.89 87.62 86.94 Example 15 97.89 99.13 98.55 94.3 88.68 88.41 83.33 76.19 Example 16 97.89 99.45 98.51 97.47 97.03 95.24 93.3 87.68 Example 17 97.89 99.58 99.09 97.51 97.44 97.08 93.23 89.36 Example 18 97.89 99.25 98.91 97.62 97.12 96.64 94.61 82.22 Control Example 97.89 99.64 97.94 96.56 93.01 92.57 87.12 80.59

[0078] Table 4. Trend of cell diameter (μm) in 125 mL shake flasks over time

[0079] Cultivation time / day 0 2 4 6 8 10 12 14 Example 1 14.23 14.71 14.68 15.29 16.8 18.04 18.65 18.94 Example 2 14.23 14.68 14.55 15.23 16.6 17.78 18.46 19 Example 3 14.23 14.65 14.56 15.1 16.51 17.73 18.3 18.75 Example 4 14.23 14.61 14.52 15.22 16.58 17.76 18.4 18.89 Example 5 14.23 14.55 14.55 15.23 16.63 17.74 18.43 18.92 Example 6 14.23 14.5 14.65 15.39 16.65 17.99 18.63 18.92 Example 7 14.23 14.72 14.61 15.35 16.72 17.93 18.58 18.96 Example 8 14.23 14.63 14.62 15.27 16.64 17.78 18.54 18.81 Example 9 14.23 14.71 14.5 15.38 16.79 17.96 18.53 19.05 Example 10 14.23 14.55 14.63 15.51 16.72 17.89 18.42 18.55 Example 11 14.23 14.53 14.59 15.19 16.65 17.75 18.5 18.68 Example 12 14.23 14.69 14.5 15.3 16.8 17.79 18.26 18.51 Example 13 14.23 14.58 14.54 15.15 16.72 17.79 18.41 18.85 Example 14 14.23 14.68 14.56 15.36 16.77 17.96 18.4 19.07 Example 15 14.23 14.48 14.65 15.32 16.83 17.78 18.26 18.74 Example 16 14.23 14.55 14.66 15.23 16.81 17.92 18.74 19.05 Example 17 14.23 14.6 14.54 15.23 16.58 17.74 18.41 18.9 Example 18 14.23 14.73 14.54 15.18 16.52 17.67 18.33 18.41 Control Example 14.23 14.6 14.6 15.33 16.72 17.98 18.55 18.87

[0080] Table 5. Glucose metabolism (g / L) in 125 mL shake flasks over time

[0081] Cultivation time / day 2 4 6 8 10 12 14 Example 1 2.97 6.15 6.91 7.51 5.8 5.76 5.9 Example 2 3 5.93 7 7.57 6.21 5.8 5.73 Example 3 2.99 5.8 6.72 7.21 5.51 5.57 5.67 Example 4 3.01 5.92 6.93 7.43 5.83 5.79 5.89 Example 5 3 5.87 6.89 7.42 6 5.49 5.55 Example 6 3.1 6.1 6.79 7.34 5.85 5.87 5.71 Example 7 3.13 5.89 6.95 7.46 6.1 5.61 5.42 Example 8 3.13 5.99 6.97 7.54 6.19 5.72 5.54 Example 9 2.97 6.07 7.06 7.87 6.87 6.16 5.77 Example 10 3.04 5.92 7.26 8.24 7.61 7.41 6.86 Example 11 3.01 6.85 7.96 8.01 5.85 5.83 6.02 Example 12 3.04 6.86 8.27 8.71 7.12 7.64 6.94 Example 13 3.03 6.85 8.04 8.18 6.21 5.82 5.85 Example 14 2.97 6.71 8.04 8.43 6.94 6.87 6.24 Example 15 3.09 6.8 8.27 8.83 7.52 6.95 6.84 Example 16 2.98 6.68 7.87 8.18 6.02 6.37 5.42 Example 17 3 6.71 7.86 7.85 5.69 6.43 5.68 Example 18 2.99 6.62 7.73 7.74 5.57 6.25 5.65 Control Example 2.74 7.1 7.9 8.43 7.58 7.29 6.03

[0082] Table 6. Lactic acid metabolism (g / L) in 125 mL shake flasks over time

[0083] Cultivation time / day 2 4 6 8 10 12 14 Example 1 1.23 1.21 0.44 0 0 0 0 Example 2 1.23 1.16 0.44 0.04 0.01 0 0 Example 3 1.23 1.16 0.45 0.03 0.05 0 0 Example 4 1.22 1.16 0.49 0.04 0.01 0 0 Example 5 1.2 1.12 0.45 0.06 0.02 0 0 Example 6 1.24 1.21 0.5 0.12 0.03 0 0 Example 7 1.26 1.16 0.49 0.07 0.02 0 0 Example 8 1.24 1.17 0.57 0.18 0.03 0 0 Example 9 1.24 1.14 0.47 0.2 0.09 0.07 0.02 Example 10 1.22 1.14 0.55 0.45 0.47 0.4 0.43 Example 11 1.26 1.47 0.6 0.01 0.08 0 0 Example 12 1.23 1.36 0.52 0.28 0.39 0.29 0.27 Example 13 1.25 1.36 0.51 0.08 0.14 0.02 0.03 Example 14 1.25 1.38 0.52 0.41 0.21 0.18 0.12 Example 15 1.23 1.37 0.59 0.48 0.46 0.41 0.39 Example 16 1.26 1.36 0.42 0 0.02 0 0 Example 17 1.26 1.33 0.44 0.03 0.02 0 0 Example 18 1.25 1.33 0.35 0.01 0.03 0 0 Control Example 1.13 1.23 0.43 0.31 0.16 0.29 0.39

[0084] Table 7. Protein expression trends over time in 125 mL shake flasks

[0085] Cultivation time / day 12 14 Example 1 6379 7255 Example 2 5734 7193 Example 3 5779 7195 Example 4 6026 7079 Example 5 6533 7202 Example 6 6140 7181 Example 7 5756 7103 Example 8 5445 6776 Example 9 6324 7598 Example 10 5389 6902 Example 11 5556 6876 Example 12 5568 6812 Example 13 5412 6645 Example 14 6225 6944 Example 15 5505 6678 Example 16 5522 6746 Example 17 6140 7073 Example 18 5678 6907 Control Example 5621 7060

[0086] Table 1, Table 2, Table 3, Table 4, Table 5, Table 6, Table 7 and Figure 1 、 Figure 2 、 Figure 3 、 Figure 4 、 Figure 5 、 Figure 6 The results showed that the cells were cultured in a medium with a 1.0±0.2×10 6 cells / mL and cultured for 14 days. Batch feeding was performed every two days starting from Day 2. The final cell peak density reached 20.7×10 6 cells / mL; the cell viability at D14 was 90.39%; lactate first accumulated and then was consumed, with no lactate recovery during the entire culture process. The lactate concentration at D14 was 0 g / L; the protein yield at D14 reached 7598 mg / L.

[0087] In the control group, the cell peak density during the entire culture process was 18.4×10 6 cells / mL, D14 cell viability was 80.59%, D14 lactate concentration was 0.39 g / L, and D14 protein production was 7060 mg / L. The above results indicate that adding different concentrations of mixed lipids to the culture medium has a significant effect on improving cell growth, metabolism, and expression.

[0088] The technical solutions in the above embodiments of the present application have at least the following technical effects or advantages:

[0089] The serum-free culture medium for culturing CHO cells provided by the present invention has simple ingredients, low cost, and is protein-free and serum-free;

[0090] The present invention facilitates the expression of target proteins by CHO cells and improves the growth and expression of CHO cells;

[0091] Using the optimized culture medium of the present invention, the cell growth peak density can reach 20.7×10 6cells / mL, the harvest viability after 14 days of culture can be increased to 90.39%, lactic acid 0g / L, and the expression level can reach 7598mg / L;

[0092] Without changing the solubility of the culture medium, the present invention discovers the optimal lipid species and concentration combination in the culture medium by adding mixed lipids and using cell growth, metabolism, and protein expression as evaluation indicators. This solves problems such as low cell density, low viability, lactic acid accumulation, and low protein production during the cell culture process, thereby reducing corporate costs and alleviating technical problems caused by lactic acid accumulation in actual industrial production processes.

[0093] Example 2: The above Example 1 increases cell density, reduces lactic acid accumulation, and improves cell expression by adding mixed lipids to the culture medium. In order to further improve the growth and expression of CHO cells, optimization is made on the basis of Example 1.

[0094] The addition of the mixed lipids was carried out in a step-by-step manner. Specifically, in the initial addition of the mixed lipids to the culture medium, the addition amounts were calculated based on the percentage of the total amount of each substance added: Tween 80 was 75%; tocopherol was 80%; linolenic acid was 50%; and cholesterol was 50%.

[0095] The remaining mixed lipids are added simultaneously with batch feeding using feed medium in step S5 to perform feed and lipid supplementation; specifically, the mixed lipids are added simultaneously with the feed, once every two days, until two days before the end of the culture, for a total of six additions, and the total amount of the mixed lipids added each time is the same; specifically, the amount added each time is calculated as a percentage of the total amount of each substance added, with Tween 80 being 4.2%, tocopherol being 3.3%, linolenic acid being 8.3%, and cholesterol being 8.3%.

[0096] Regarding the addition method of the mixed lipids in this embodiment, taking Example 1 and Example 2 in Example 1 as examples, the results of Example 2-1 and Example 2-2 in this embodiment are shown in the following table:

[0097] Table 8. Viable cell density in 125 mL shake flask (×10 6 cells / mL) over time

[0098] Cultivation time / day 0 2 4 6 8 10 12 14 Example 1 0.965 4.93 14.7 20.1 16.6 16.2 14.7 13.6 Example 2-1 0.965 5.03 15.0 21.0 17.9 17.1 15.2 14.3 Example 2 0.965 5 14.1 19.8 17.2 16.5 14 13.2 Example 2-2 0.965 5.02 14.9 20.7 18.1 17.2 14.8 13.8

[0099] Table 9. Lactic acid metabolism (g / L) in 125 mL shake flasks over time

[0100] Cultivation time / day 2 4 6 8 10 12 14 Example 1 1.23 1.21 0.44 0 0 0 0 Example 2-1 1.23 1.14 0.42 0 0 0 0 Example 2 1.23 1.16 0.44 0.04 0.01 0 0 Example 2-2 1.22 1.10 0.42 0.02 0 0 0

[0101] Table 10. Protein expression trends over time in 125 mL shake flasks

[0102] Cultivation time / day 12 14 Example 1 6379 7255 Example 2-1 6432 7382 Example 2 5734 7193 Example 2-2 5875 7284

[0103] The technical solutions in the above embodiments of the present application have at least the following technical effects or advantages:

[0104] As shown in Tables 8, 9, and 10, the addition of the mixed lipids in a stepwise manner and at the same time as the feed significantly increased the peak density of the cells. The peak cell density of Examples 2-1 and 2-2 exceeded that of the control group in Example 1, reaching a higher cell density level. Lactic acid metabolism was slightly reduced, and protein production was increased.

[0105] The step-by-step addition of mixed lipids allows cells to continuously obtain the nutrients they need throughout the culture process. The high concentration addition in the initial stage provides sufficient starting power for cell growth, thereby promoting continued cell growth and division. In the later batch feeding stage, regular replenishment of mixed lipids avoids nutrient depletion, allowing cells to continue growing and dividing, maintaining high-density cell growth, and thus increasing cell density.

[0106] The step-by-step addition of mixed lipids improves the cell metabolic pathway, making the production and consumption of lactate more balanced. In the late culture period, due to the stability of cell density and efficient metabolic regulation, lactate is effectively consumed, avoiding lactate accumulation. In the late culture period, lactate is effectively consumed, avoiding the rebound of lactate. This balanced metabolic state can promote healthy cell growth and high-yield expression.

[0107] The step-by-step addition of mixed lipids improves cell health and metabolic efficiency, promoting the synthesis and secretion of target proteins. At the same time, the increase in cell density and the improvement of the metabolic environment promote high protein expression.

[0108] By adding mixed lipids in steps, the supply of nutrients can be more precisely controlled, avoiding nutrient waste and excessive accumulation. This precise nutrient regulation promotes better nutrient utilization by cells, improving culture efficiency and target product yield.

[0109] The step-by-step addition of mixed lipids reduces unnecessary nutrient waste, thereby lowering production costs to a certain extent. In addition, due to the increased cell density and expression level, the product concentration per unit volume increases, which also indirectly reduces production costs.

[0110] The process of adding mixed lipids in steps is more refined and controllable, reducing fluctuations and uncertainties in the culture process, improving the stability and repeatability of the preparation process; promoting industrial production and improving the consistency and stability of product quality.

[0111] Example 3: The above Example 2 increases cell density, reduces lactic acid accumulation, and improves cell expression by adding mixed lipids in the culture medium step by step. In order to further improve the growth and expression of CHO cells, optimization is made on the basis of Example 2.

[0112] When supplementing feed and lipids, the amount of mixed lipids added varies at different cell growth stages; according to the cell growth status, the cell culture stage is divided into early, middle and late stages;

[0113] Among them, the initial stage is 0-6 days of cell culture, and it is added 3 times; the middle stage is 6-10 days of cell culture, and it is added twice; the late stage is 10-14 days of cell culture, and it is added once; specifically, the addition amount is calculated according to the percentage of the total amount of each substance added. In the initial stage, Tween 80 is 6%, tocopherol is 2%, linolenic acid is 7%, and cholesterol is 8%; in the middle stage, Tween 80 is 3%, tocopherol is 4%, linolenic acid is 11.5%, and cholesterol is 9.5%; in the late stage, Tween 80 is 1%, tocopherol is 6%, linolenic acid is 6%, and cholesterol is 7%.

[0114] Regarding the addition method of the mixed lipids in this embodiment, taking Example 2-1 and Example 2-2 in Example 2 as an example, the results of Example 3-1 and Example 3-2 in this embodiment are shown in the following table:

[0115] Table 10. Viable cell density in 125 mL shake flask (×10 6 cells / mL) over time

[0116] Cultivation time / day 0 2 4 6 8 10 12 14 Example 2-1 0.965 5.03 15.0 21.0 17.9 17.1 15.2 14.3 Example 3-1 0.965 5.10 15.5 22.0 18.5 17.8 16.0 15.1 Example 2-2 0.965 5.02 14.9 20.7 18.1 17.2 14.8 13.8 Example 3-2 0.965 5.08 15.3 21.5 18.7 17.9 15.5 14.5

[0117] Table 11. Lactic acid metabolism (g / L) in 125 mL shake flasks over time

[0118] Cultivation time / day 2 4 6 8 10 12 14 Example 2-1 1.23 1.14 0.42 0 0 0 0 Example 3-1 1.23 1.10 0.38 0 0 0 0 Example 2-2 1.22 1.10 0.42 0.02 0 0 0 Example 3-2 1.18 1.05 0.36 0 0 0 0

[0119] Table 12. Protein expression trends over time in 125 mL shake flasks

[0120] Cultivation time / day 12 14 Example 2-1 6432 7382 Example 3-1 6653 7642 Example 2-2 5875 7284 Example 3-2 6021 7413

[0121] The technical solutions in the above embodiments of the present application have at least the following technical effects or advantages:

[0122] As shown in Tables 11, 12, and 13, by adjusting the amount of mixed lipids added in stages, the peak density of the cells was increased. The peak density of the cells in Examples 3-1 and 3-2 exceeded that of the control group in Example 2. Lactic acid metabolism was slightly reduced, and protein expression was further increased.

[0123] By adjusting the amount of mixed lipids added in stages, the high concentration of Tween 80 in the early stage promoted cell dispersion and growth, the added linolenic acid and cholesterol in the middle stage promoted the formation and stability of cell membranes, and the added tocopherol in the late stage enhanced the antioxidant capacity of the cells, thereby maintaining a high cell growth rate and density throughout the culture process.

[0124] The phased addition of mixed lipids improves the cell's metabolic pathways, resulting in a more balanced production and consumption of lactate. Especially in the middle and late stages of culture, due to stable cell density and efficient metabolic regulation, lactate is consumed more effectively, avoiding its accumulation. The increased cell density and improved metabolic environment promote the synthesis and secretion of target proteins. Furthermore, due to the precise regulation of the mixed lipids, cells are able to more effectively utilize nutrients, thereby increasing protein expression.

[0125] The high concentration of Tween 80 in the initial stage effectively promoted cell dispersion, prevented cell aggregation, and provided cells with a larger growth space, thereby accelerating the initial cell growth rate. In the middle stage, the appropriate amount of linolenic acid and cholesterol was increased to promote the formation and stability of cell membranes, providing the necessary structural support for the continued growth of cells and further increasing the cell density. In the later stage, the addition of tocopherol was increased to enhance the antioxidant capacity of the cells. The cells maintained a high viability and growth rate in the later stage of culture, thereby maintaining a high cell density.

[0126] The phased addition of mixed lipids allows cells to obtain the appropriate combination of nutrients at different growth stages, thereby optimizing the cell metabolic pathway. Especially in the middle and late stages of culture, due to the stable cell density and efficient metabolic regulation, the production and consumption of lactic acid are more balanced, effectively avoiding the accumulation of lactic acid and improving the stability of the culture environment.

[0127] The increase in cell density provides more cell factories for protein expression, thereby increasing the synthesis of the target protein. At the same time, due to the precise regulation of the lipid mixture, the cells can more effectively utilize nutrients, promoting the efficient expression of the target protein. The phased addition of the lipid mixture also improves the metabolic environment of the cells, providing environmental conditions for protein synthesis and secretion.

[0128] By precisely controlling the amount of mixed lipids added, nutrient waste and excessive accumulation are avoided, thereby reducing production costs to a certain extent. Due to the increased cell density and protein expression, the product concentration per unit volume increases, further reducing production costs. This embodiment is suitable for laboratory-scale cultivation and has prospects for industrial application. By adjusting cell growth and expression conditions, production efficiency and product quality are improved.

[0129] Example 4: The above Example 2 increased cell density and cell viability, reduced lactic acid accumulation, and improved cell expression by adjusting the amount of mixed lipids added in stages. In order to further improve CHO cell growth and expression, optimization was made based on Example 3.

[0130] like Figure 7 As shown, for large-scale culture, cells were inoculated into 3 L spinner flasks;

[0131] Among them, a rear cover is set at the left end of the rotating bottle, the rear cover is sealed, and the left end of the rear cover is connected to the trachea. The rear cover of the rotating bottle contains three syringes arranged up and down. The syringes are connected to the inside of the rotating bottle, and the resistance of the syringes is different;

[0132] The resistance range of the upper syringe is 0.5-1.0 N·s / m3, the resistance range of the middle syringe is 1.5-2.5 N·s / m3, and the resistance range of the lower syringe is 3-4 N·s / m3;

[0133] The lactic acid content was tested by sampling every 12 hours. When the lactic acid content was higher than 1.0 g / L, 0.1-0.3 nM of thyroid hormone was added uniformly.

[0134] The technical solutions in the above embodiments of the present application have at least the following technical effects or advantages:

[0135] In large-scale culture, the efficiency and stability of the large-scale culture process are improved by setting syringes with different resistances in the spinner flask and combining them with air pressure control to achieve automated addition of thyroid hormone. The redesign of the spinner flask enables automated operation, significantly improving the efficiency of thyroid hormone addition, reducing manual intervention and avoiding human errors, and ensuring the efficiency, accuracy and consistency of thyroid hormone addition each time.

[0136] The syringe resistance in the rear cover of the spinner bottle is set to different values. By controlling the air pressure in the spinner bottle, syringes with different resistances can be triggered for injection. The resistance of the upper, middle and lower syringes gradually increases, and the upper syringe has the smallest resistance, so that it can respond quickly when a small amount of thyroid hormone needs to be added quickly. When the demand for thyroid hormone is small, the smaller resistance can allow the thyroid hormone to pass through quickly and be released quickly. The middle syringe can deliver an appropriate amount of thyroid hormone within an appropriate time. The lower syringe is set to the maximum resistance. When the lactate content is significantly high and a larger dose of thyroid hormone is needed for adjustment in an emergency, the larger resistance can slow down the injection speed, ensuring the stable release of a large amount of thyroid hormone over a longer period of time, and avoiding the adverse effects of excessive addition on cells at one time. The syringes with different resistances in the spinner bottle design can adjust the addition speed and amount of thyroid hormone according to actual needs, thereby improving the flexibility and adaptability of large-scale cell culture and promoting large-scale production.

[0137] Regular sampling to test lactate levels and timely and uniformly adding thyroid hormone when lactate levels are too high can maintain lactate balance in the culture environment. As a regulatory factor, thyroid hormone can temporarily promote lactate metabolism and reduce lactate accumulation, thereby protecting cells from damage caused by a high lactate environment.

[0138] By precisely controlling the amount of thyroid hormone added, the cell growth environment can be further optimized. Thyroid hormone can not only reduce lactate levels, but also promote cell growth and expression. By adding thyroid hormone and improving culture bottles, the problem of lactic acid accumulation that is easily produced by cells during large-scale culture can be solved, thereby promoting protein expression in industrially cultured CHO cells.

[0139] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Various modifications and variations are readily apparent to those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A method for culturing high-expressing CHO cells, characterized in that: The specific steps include: S1: Resuscitate frozen CHO cells in culture medium and culture the cells in a shaker at 37°C, 8% CO2, and 110-130 rpm. The culture medium is additionally added with a mixed lipid, which is composed of four substances: Tween 80, tocopherol, linolenic acid, and cholesterol; Tween 80: 0.025-0.75 mg / L, tocopherol: 0.1-3 mg / L, linolenic acid: 0.225-6.75 mg / L, cholesterol: 0.15-4.5 mg / L; The CHO cells are CHOK1 cells; S2: Perform subculture every 2-3 days to keep the cells in the early logarithmic growth phase. The cell seeding density is (0.3-1.0)-1.0×10 6 cells / mL; S3: When the cell density reaches 4.5-6.0×10 6 cells / mL, cell viability ≥95%, and passage again after 2-3 days; S4: Passage 2-4 times according to the S2 and S3 methods. After the cell population doubling time stabilizes, start inoculation. Inoculate the cells into the culture medium and culture in a 125 mL shake flask or a 5 L stirred tank bioreactor. S5: Batch feeding using feed medium after cultivation; Among them, feed 3-8% and stop feeding 1-2 days before the end of culture; S6: harvested on day 14-16 of culture and protein concentration determined; Wherein, the protein is an antibody; The addition of the mixed lipids is carried out in a step-by-step manner, including initial addition and feed and lipid supplementation; The initial addition is added during the culture medium configuration stage; The added amount is calculated as a percentage of the total amount of each substance added: Tween 80 is 75%; tocopherol is 80%; linolenic acid is 50%; cholesterol is 50%; The remaining mixed lipids are added in step S5 while batch feeding is performed using feed medium to supplement lipids; The feed and lipid supplementation is that the mixed lipids are added simultaneously with the feed, and the amount of the mixed lipids added is different at different cell growth stages; Among them, the cell growth stage is divided into early, middle and late stages; the early stage is 0-6 days of cell culture, and the addition is 3 times; the middle stage is 6-10 days of cell culture, and the addition is 2 times; the late stage is 10-14 days of cell culture, and the addition is once; The amount added each time is calculated as the percentage of the total amount of each substance added: In the early stage, Tween 80 is 6%, tocopherol is 2%, linolenic acid is 7%, and cholesterol is 8%; In the middle stage, Tween 80 is 3%, tocopherol is 4%, linolenic acid is 11.5%, and cholesterol is 9.5%; In the later stage, Tween 80 is 1%, tocopherol is 6%, linolenic acid is 6%, and cholesterol is 7%.

2. The method for culturing highly expressed CHO cells according to claim 1, wherein: The lactic acid content was tested by sampling every 12 hours. When the lactic acid content was higher than 1.0 g / L, 0.1-0.3 nM of thyroid hormone was added uniformly.

3. The method for culturing highly expressed CHO cells according to claim 1, wherein: In step S4, the shaker temperature was 36.5-37°C, the rotation speed was 130±20 rpm, and the CO2 concentration in the incubator was set to 5%-10%. The bioreactor temperature was 36.5-37°C, the initial rotation speed was 180±20 rpm, the pH was set to 7.0±0.3, and the DO was set to 40%. The PDT stabilization deviation was less than 3 h. The inoculation density was 0.4-1.2×10 6 cells / mL.

4. The method for culturing highly expressed CHO cells according to claim 1, wherein: When high-expressing CHO cells are cultured on a large scale, the cells are inoculated into 3 L spinner flasks for culture in step S4; A rear cover is provided at the left end of the rotating bottle, which is sealed and connected to the trachea. The rear cover contains three syringes arranged up and down, which are connected to the interior of the rotating bottle. The resistance of the syringes is different. The resistance range of the upper syringe is 0.5-1.0 N·s / m 3 The resistance range of the middle syringe is 1.5-2.5 N·s / m 3 , the resistance range of the lower syringe is 3-4 N·s / m 3 .

Images