CHO cell culture medium additive formula and culture method

Abstract

The invention discloses a CHO cell culture medium additive formula and a culture method, according to the additive formula, taurine, copper sulfate and tropolone are integrated into an additive A, sodium butyrate is used as an additive B. In the cell culture process, the additive A can be directly integrated into a culture medium, and the additive B is cultured for 5-8 days and added into a culture system. Compared with the prior art, the application has the advantages that through the synergistic effect of the additive A and the additive B, the content of an antibody charge isomerism main peak (IEC-Main) can be increased to 76.728% from 60.985% of a contrast, and the increasing amplitude reaches 25.8%. In the optimized concentration range, the cell viability is greater than or equal to 95%, the multiplication time (PDT) is stable, and the production efficiency is ensured. The serum-free culture medium for culturing CHO cells provided by the invention has the advantages of simple components, low cost, no protein and no serum. The additive disclosed by the invention is simple in formula preparation and convenient to use, and can be widely applied to CHO cell culture in the industrial production process.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological cell culture media, and particularly relates to a CHO cell culture media additive formula and a culture method for improving the main peak of antibody charge isomerism. Background Art

[0002] CHO cells have the following characteristics: rapid growth and division, stable suspension growth in serum-free and chemically defined culture media, good genetic stability, stable expression of exogenous genes, easy gene mutation and transfection, and the ability to glycosylate the expressed proteins. Therefore, CHO cells are widely used in large-scale cell culture industrial production and are currently one of the most widely used cell lines in bioengineering.

[0003] In the biopharmaceutical field, CHO cells are commonly used for the production of antibody drugs. The production process primarily includes cell line construction, upstream cell culture, downstream supernatant purification, and formulation packaging. For antibody drugs intended for human use, in the actual production process, in addition to protein yield requirements for cost considerations, antibody quality is also a key factor influencing the drug's ultimate approval and marketing. Antibody quality includes factors such as charge heterogeneity, glycosylation modification, and antibody purity. Controlling protein charge heterogeneity is a significant challenge, whether in cell line construction, upstream cell culture, or downstream purification and formulation. Charge heterogeneity can significantly impact drug efficacy. During actual drug development, it must be controlled within an acceptable range, and the consistency and stability of charge distribution across different culture and formulation batches must be ensured. Furthermore, optimization of the cell culture process, culture medium composition, purification methods, and formulation formulation is essential throughout the upstream and downstream development processes. This article focuses on optimizing culture medium formulations in the upstream cell culture stage. The primary approach is to disclose an additive mix and provide instructions for its use to address the issue of low IEC main peak content during cell culture. Summary of the Invention

[0004] The purpose of the present application is to provide a culture medium additive formula that can increase the main peak of antibody charge isomerism. The additive formula is to integrate taurine, copper sulfate, and tropolone as additive A, and sodium butyrate as additive B. During the cell culture process, additive A can be directly integrated into the culture medium, and additive B is added to the culture system after culturing for another 5-8 days.

[0005] Furthermore, the culture medium substances include inorganic salts, vitamins, trace elements, amino acids, carbon sources, nitrogen sources, water and other components. The concentration range of each additive substance during the test process is: Taurine: 0mM-0.8mM Copper sulfate: 0-0.5uM Tropolone: 0mM-8mM Sodium butyrate: 0mM-0.01mM Preferably, the final concentrations of the substances are: Additive A: 0.5 mM taurine, 0.3 mM copper sulfate, 4 mM tropolone; Additive B: 0.005 mM sodium butyrate.

[0006] Furthermore, the method for culturing CHO cells in vitro comprises the following steps: (1) Culture medium preparation: Add 25 mL of the Tonin platform basal medium to a 125 mL shake flask and place the shake flask containing the medium in a shaker at 37°C for 30-60 minutes; (2) Cell recovery: Take out a CHO cell frozen in a -230℃ liquid nitrogen tank, place the cell in a 37℃ water bath for rapid thawing, and then add the cell to the culture medium described in (1); (3) placing the shake flask containing the cells and culture medium described in (2) in a shaker at 37°C, 8% carbon dioxide, and 110-130 rpm for culture; (4) Cell adaptation: Perform cell subculture every 2-4 days to ensure that the cells are always in the logarithmic growth phase. The cell seeding density during subculture is 0.2-1.0×10 6 cells / mL; (5) Cell passaging: After several passages according to the method described in (4), observe the stability of cell doubling time and cell viability. When the cell density before each treatment can reach 4.5-6.0×10 6 cells / mL, cell viability ≥95%, and when the PDT difference between different generations is within 4 hours, subculturing is stopped and cells are plated for Fed-Batch culture; (6) Cell culture: The cells described in (5) were centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and the cells were resuspended in 20 mL of basal medium and transferred to a 125 mL shake flask. Additives were added according to the experimental plan. Starting from D3, the cells were sampled and counted, metabolically tested, fed, and sugar-supplemented every other day. Additive B was added on the 8th day of culture, and the culture was stopped on the 15th day. The cells were centrifuged in a high-speed centrifuge at 4000 rpm for 30 minutes, and the supernatant was collected for quality inspection.

[0007] Preferably, in step (6), the temperature of the shaker is 36.5-37°C, the rotation speed is 130±20 rpm, the CO2 concentration in the incubator is set to 5%-10%; the deviation after PDT stabilization is less than 3h; the inoculation density is 0.4-1.2×10 6 cells / mL.

[0008] Cell detection items include: cell viability density, cell viability, cell diameter, cell agglomeration, glucose concentration and lactate concentration in the culture system, cell expression on the 15th day of culture, and antibody charge isomerism distribution in the supernatant.

[0009] Furthermore, the parameter detection methods are: (1) Cell growth status: Detected using a cell counter commonly used in the field of cell culture; (2) Cellular metabolic status: detected using Hillman M100 biochemical analyzer; (3) Antibody charge isomerism distribution: HPLC liquid phase detection method is used for detection. The supernatant of cell culture needs to be purified before HPLC quality testing.

[0010] Compared with existing technologies, the advantages of this application are: through the synergistic effect of Additives A and B, the antibody charge isomerization peak (IEC-Main) content can be increased from 60.985% in the control to 76.728%, an increase of 25.8%. Within the optimized concentration range, cell viability is ≥95%, and the proliferation time (PDT) is stable, ensuring production efficiency.

[0011] (1) The serum-free culture medium for culturing CHO cells provided by the present invention has simple components, low cost, and is protein-free and serum-free.

[0012] (2) The present invention can significantly increase the main peak content of antibody IEC without affecting cell growth and expression.

[0013] (3) The additive formula disclosed in the present invention is simple to prepare and easy to use, and can be widely used in CHO cell culture in industrial production processes. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The IEC peak shape diagram and peak area ratio of the control example are shown in Figure 2.

[0015] Figure 2 This is the IEC peak shape diagram and peak area ratio of Example 1 in stage 1.

[0016] Figure 3 This is the IEC peak shape diagram and peak area ratio of Example 2 in stage 1.

[0017] Figure 4 This is the IEC peak shape diagram and peak area ratio of Example 3 in stage 1.

[0018] Figure 5 This is the IEC peak shape diagram and peak area ratio of Example 4 in stage 1.

[0019] Figure 6 This is the IEC peak shape diagram and peak area ratio of Example 5 in stage 1.

[0020] Figure 7 This is the IEC peak shape diagram and peak area ratio of Example 6 in stage 1.

[0021] Figure 8 This is the IEC peak shape diagram and peak area ratio of Example 7 in stage 1.

[0022] Figure 9 This is the IEC peak shape diagram and peak area ratio of Example 8 in stage 1. DETAILED DESCRIPTION

[0023] In order to make the purpose, technical solutions and advantages of this application more clearly understood, the present application is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain this application and are not intended to limit this application. Example

[0024] The present invention provides a culture medium additive formula that can be used in the upstream stage of in vitro CHO cell culture to increase the main peak of antibodies, as well as a preparation and use method of the formula. The formula involves four substances: sodium butyrate, taurine, copper sulfate, and tropolone.

[0025] Preparation method: (1) Dissolve 25 mM taurine, 15 mM copper sulfate, and 240 mM tropolone in 80 mL of ultrapure water, and then adjust the volume to 100 mL. Mix well and filter aseptically. This solution is named Additive A. (2) Add 1 mM sodium butyrate to 9 mL of ultrapure water, and adjust the volume to 10 mL after it is completely dissolved. Mix well and filter aseptically. This solution is named Additive B.

[0026] Usage: Before the Fed-Batch culture of the cells, add 2 mL of Additive A to 100 mL of inoculation medium. On the 8th day of Fed-Batch culture, add 100 uL of Additive B to the culture system.

[0027] Take a CHO cell cryopreservation tube from the liquid nitrogen tank, shake and dissolve it in a 37°C water bath, add it to a 125mL shake flask containing 20mL of the basal medium for the DuoNin platform, and mix well.

[0028] After three passages, the cells were centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and the cells were resuspended in 20 mL of the basal medium of the Dox platform and transferred to nine 125 mL shake flasks. Subsequently, the cells were cultured in a shaker at 130 rpm, 8% CO2, and 37°C. Cell growth status was detected and various growth parameters were recorded on Day 0 / 3 / 5 / 7 / 9 / 11 / 13 / 15. 5% DN Feed 3 and 0.5% DN Feed B3 were supplemented on Day 3 / 5 / 7 / 9 / 11 / 13, respectively. The residual glucose content was tested on Day 3 / 5 / 5 / 7 / 9 / 11 / 13, and the sugar concentration of the culture system was supplemented to 6 g / L using a mother liquor of 200-400 g / L glucose.

[0029] Example 1-5: Single additive, sodium butyrate, taurine, copper sulfate, tropolone were added separately, the cell culture scheme and results are shown in Table 1-3 and Figure 1-6 As shown: Table 1 Main implementation schemes of cell culture in control examples and examples 1-5

[0030] Table 2 Concentration and addition method of additives in control example and examples 1-5

[0031] Table 3 Final IEC main peak ratio of control example and examples 1-5

[0032] The results show: Control group: basic culture medium without additives, the main peak accounted for 60.985%.

[0033] When the sodium butyrate concentration was 0.0005-0.001 mM, the main peak increased to 61.415%-61.713%; When taurine was 0.2 mM, copper sulfate was 0.1 μM, and tropolone was 2 mM, the main peak increased to 64.323%, 63.144%, and 64.707%, respectively.

[0034] According to the control example, Example 1, and Example 2: adding sodium butyrate during cell culture can increase the main peak content of the antibody. As the concentration of sodium butyrate increases, the main peak content of the antibody shows a slight upward trend; According to the control example, example 3, example 4, and example 5: the addition of taurine, copper sulfate, and tropolone to the basal medium for cell culture production can increase the main peak content of antibodies to varying degrees; It should be noted that sodium butyrate is added differently from the other three substances in the experiment due to its mechanism of action. Therefore, it is separated into Additive B for use in the additives described in this patent.

[0035] Examples 6-8: Using a combination of additives, adding additives A and B simultaneously, and gradually optimizing their concentrations: Cell culture procedures were performed according to the methods described in Examples 1-5. Based on the data from experimental phase 1, taurine, copper sulfate, tropolone, and sodium butyrate were combined in the cell culture phase. The experimental protocol and the method of adding each substance are described in Tables 4-6: Table 4 Main implementation plans for cell culture in Examples 6-8

[0036] Table 5 Concentration and addition method of additives in Examples 6-8

[0037] Table 6 Final IEC main peak ratio of Examples 6-8

[0038] Table 4-6 and Figure 7-9 The results show that: In the low-concentration combination (0.2 mM taurine + 0.1 μM copper sulfate + 2 mM tropolone + 0.002 mM sodium butyrate), the main peak increased to 68.434%; The optimal concentration combination (0.5 mM taurine + 0.3 μM copper sulfate + 4 mM tropolone + 0.005 mM sodium butyrate) had a main peak of 76.728%; In the high-concentration combination (0.8 mM taurine + 0.5 μM copper sulfate + 8 mM tropolone + 0.01 mM sodium butyrate), the main peak decreased to 70.838%, indicating that the concentration needs to be strictly controlled.

[0039] After integrating the effective substances (taurine, sodium butyrate, copper sulfate, and tropolone), the main peak of the antibody can be increased from 60.985% to 68.434%, an increase of 12.2% (Example 6); after further increasing the concentration of each substance, the main peak can be increased from 68.434% to 76.728%, an increase of 12.1% (Example 7); after further increasing the concentration of each substance, the main peak can be reduced from 76.728% to 70.838%, a decrease of 7.8% (Example 8).

[0040] The final results showed that the combined use of taurine, sodium butyrate, copper sulfate and tropolone can significantly increase the main peak of antibodies, and the main peak content can be increased from 60.985% to 76.728%, with an overall increase of 25.8%.

[0041] To simplify the use of substances during the development process, according to the invention, taurine, sodium butyrate, copper sulfate, and tropolone are integrated into additives A and B according to different addition methods. Additive A has a concentration of 50× a mixed stock solution, containing 25 mM taurine, 15 mM copper sulfate, and 240 mM tropolone. The addition method is to add 1× a mixed stock solution to the production culture medium before re-inoculation; Additive B has a concentration of 1 mM, and the substance is sodium butyrate. The addition method is to add 0.005 mM of this substance to the culture system on the 8th day of cell culture.

[0042] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field, within the technical scope disclosed by the present invention, who makes equivalent replacements or changes based on the technical solution and inventive concept of the present invention, should be covered by the scope of protection of the present invention.

Claims

1. A CHO cell culture medium additive formula, characterized in that: The additives include additive A and additive B, The additive A comprises taurine, copper sulfate and tropolone, and the additive B is sodium butyrate; during the cell culture process, the additive A is integrated into the culture medium before inoculation, and the additive B is added to the culture system on the 5th to 8th day of culture.

2. The CHO cell culture medium additive formulation according to claim 1, characterized in that: The culture medium comprises inorganic salts, vitamins, trace elements, amino acids, carbon sources, nitrogen sources and water.

3. The CHO cell culture medium additive formulation according to claim 1, characterized in that: The concentration ranges of the components are: Additive A is taurine: 0mM~0.8mM, copper sulfate: 0μM~0.5μM, tropolone: 0mM~8mM; Additive B is sodium butyrate: 0mM to 0.01mM.

4. The CHO cell culture medium additive formulation according to claim 3, characterized in that: The concentrations of the additives are: Additive A: 0.5 mM taurine, 0.3 μM copper sulfate, 4 mM tropolone; Additive B: 0.005 mM sodium butyrate.

5. A CHO cell culture method using the culture medium additive formulation according to claim 1, characterized in that: The following steps are involved: (1) Culture medium preparation: Add basal culture medium to the shake flask and place it in a shaker at 37°C for 30-60 minutes; (2) Cell recovery: Frozen CHO cells are rapidly thawed and added to the culture medium; (3) Cell culture: Place the shake flask at 36.5°C to 37°C, 5%-10% CO2, and 110-130 rpm; (4) Cell passaging: Cells are passaged every 2-4 days, with a seeding density of 0.2×10 6 ~1.0×10 6 cells / mL, ensuring that the cells are in the logarithmic growth phase; (5) Fed-Batch culture: When the cell density reaches 4.5×10 6 ~6.0×10 6 cells / mL and the viability is ≥95%, inoculating into the culture system and adding the additive A described in claim 1; (6) Add additive B on the 8th day of culture, stop the culture on the 15th day, collect the supernatant by centrifugation and detect the antibody charge isomerism.

6. The CHO cell culture method according to claim 5, characterized in that In step (3), the shaking table temperature is 37° C., the rotation speed is 130±20 rpm, and the CO 2 concentration is 8%.

7. The CHO cell culture method according to claim 5, characterized in that Items that need to be detected during the culture process include: cell viability density, viability, glucose concentration, lactic acid concentration and antibody charge isomerism distribution.

8. The CHO cell culture method according to claim 5, wherein The antibody charge isomerism distribution is detected by HPLC liquid phase detection, and the supernatant needs to be purified before detection.

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