Combined additive, CHO cell culture medium, preparation method and culture method

Abstract

The invention relates to a combined additive, a CHO cell culture medium, a preparation method and a culture method. The combined additive comprises cyclohexyl alanine and 3-(2-naphthyl)-L-alanine. The CHO cell culture medium comprises amino acids, vitamins, trace elements, inorganic salts, lipids, carbohydrates and a combined additive, the preparation method is used for preparing the CHO cell culture medium. The culture method uses a CHO cell culture medium to culture CHO cells. The combined additive is obtained through structural domain virtual docking and hydrophobicity regulation and control screening, G1 / S conversion can be mildly delayed, cell apoptosis can be inhibited, and an expression window can be prolonged. The culture medium contains amino acids, vitamins, trace elements and the like, and preferably selects a functional additive to replace serum and foreign protein functions. According to the application, high proliferation of CHO cells and efficient target protein expression can be realized under the conditions of no serum and no foreign protein, meanwhile, the accumulation of lactic acid and ammonia is reduced, the cell caking phenomenon is reduced, and the application has remarkable technical advantages.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to a combination additive, CHO cell culture medium, preparation method and culture method. Background Technology

[0002] In CHO cell culture, the composition and preparation method of the culture medium have a significant impact on cell growth, protein expression, and product quality. Currently, commonly used culture medium optimization strategies mainly rely on adding histone deacetylase inhibitors such as sodium butyrate to promote gene expression by delaying the cell cycle. However, this strategy has certain limitations: on the one hand, sodium butyrate may induce apoptosis and decreased cell viability, thereby limiting the growth of the total number of cells in the later stages; on the other hand, its use leads to the accumulation of metabolic byproducts such as lactic acid and ammonia, affecting protein glycosylation and folding processes, and exacerbating cell clumping, increasing the load of host cell protein and nucleic acid impurities during downstream purification. In addition, to buffer the negative effects of sodium butyrate, expensive components such as human serum albumin, insulin, or transferrin are often added, leading to increased costs and impurity risks.

[0003] Patent CN114502715A discloses a method and apparatus for optimizing biotechnology production. It optimizes culture medium composition and feeding strategies by automatically generating and validating digital twin models to improve product concentration, productivity, biomass concentration, and product quality. This method can support production decisions online or offline, offering high flexibility and practicality. However, this technical solution mainly focuses on macroscopic process optimization, without delving into the specific mechanisms of action of key components in the culture medium and their regulatory capabilities on CHO cell metabolic behavior. It lacks design for the synergistic effects of specific small molecules or metabolic additives, making it difficult to comprehensively meet the combined requirements of "high proliferation + high productivity + low by-products + low cost."

[0004] Patent CN114504993A discloses a biological nutrient solution mixing device for bioculture technology, including a mixing device, a mixing wheel, and a conveying device. This device can automatically stir and mix the raw materials for preparing the nutrient solution and intermittently transport the culture dishes, uniformly distributing the nutrient solution into the dishes via a pump. While this device improves efficiency, it does not address how to replace the role of traditional sodium butyrate through domain docking in the culture medium formulation design, nor does it propose specific solutions for controlling metabolic byproducts during CHO cell culture. Therefore, it fails to significantly improve the problems of decreased cell viability and increased metabolic burden.

[0005] In summary, while existing technologies have made some progress in the field of CHO cell culture, further exploration is needed to optimize culture medium formulations and improve preparation methods in order to achieve more efficient cell culture and higher quality protein expression. Summary of the Invention

[0006] The first primary objective of this invention is to provide a combined additive for preparing a serum-free and exogenous protein-free CHO cell culture medium, which enables high proliferation and efficient expression of target proteins in CHO cells under serum-free and exogenous protein-free conditions, while reducing the accumulation of metabolic byproducts such as lactic acid and ammonia, and reducing cell clumping.

[0007] The present invention achieves the above-mentioned objective through the following technical solution: a combined additive comprising the following components: cyclohexylalanine and 3-(2-naphthyl)-L-alanine, wherein the final concentration range of cyclohexylalanine is 0.5 mM to 2.0 mM; and the final concentration range of 3-(2-naphthyl)-L-alanine is 0.5 mM to 2.0 mM.

[0008] The second main objective of this invention is to provide a CHO cell culture medium that solves the problems of decreased cell viability, cell clumping, lactic acid / ammonia accumulation, and downstream purification burden caused by traditional sodium butyrate inhibition strategies, thereby achieving high cell growth folds and higher expression of the target protein, and simplifying downstream processes.

[0009] The present invention achieves the above objectives through the following technical solution: a CHO cell culture medium comprising the following components: the combined additives, amino acids, vitamins, trace elements, inorganic salts, lipids, carbohydrates and functional additives.

[0010] Specifically, the composition and concentration range of the amino acids are as follows: L-histidine hydrochloride monohydrate 45 mg / L to 135 mg / L; L-isoleucine 80 mg / L to 250 mg / L; L-leucine 85 mg / L to 270 mg / L; L-lysine hydrochloride 135 mg / L to 410 mg / L; L-methionine 35 mg / L to 115 mg / L; L-phenylalanine 50 mg / L to 160 mg / L; L-threonine 80 mg / L to 240 mg / L; L-tryptophan 25 mg / L to 80 mg / L; L-valine 79 mg / L to 240 mg / L; glycine 28 mg / L to 84 mg / L; L-alanine 6 mg / L. 75 mg / L to 27 mg / L; L-glutamine 800 mg / L to 3200 mg / L; L-arginine hydrochloride 400 mg / L to 885 mg / L; L-asparagine 100 mg / L to 450 mg / L; L-aspartic acid 20 mg / L to 80 mg / L; L-cysteine ​​hydrochloride monohydrate 20 mg / L to 75 mg / L; L-cysteine ​​hydrochloride 45 mg / L to 150 mg / L; L-glutamic acid 4 mg / L to 45 mg / L; L-proline 33 mg / L to 180 mg / L; L-serine 50 mg / L to 150 mg / L; L-tyrosine disodium salt dihydrate 80 mg / L to 240 mg / L.

[0011] Specifically, the composition and concentration range of the vitamins are as follows: Vitamin H is 0.05 mg / L to 0.4 mg / L; folic acid is 10 mg / L to 30 mg / L; Vitamin B2 is 0.2 mg / L to 0.6 mg / L; alpha-lipoic acid is 0.2 mg / L to 0.6 mg / L; Vitamin B12 is 0.45 mg / L to 1.35 mg / L; i-inositol is 5 mg / L to 15 mg / L; Vitamin B6 is 0.5 mg / L to 3 mg / L; Vitamin B1 is 0.5 mg / L to 3 mg / L; Vitamin C is 1 mg / L to 4 mg / L; Vitamin E is 2.5 mg / L to 7.5 mg / L; D-calcium pantothenate is 5 mg / L to 30 mg / L; and nicotinamide is 5 mg / L to 30 mg / L.

[0012] Specifically, the composition and concentration range of the trace elements are as follows: NH4VO3 from 0 mg / L to 0.01 mg / L; Na2SeO3 from 0.02 mg / L to 0.08 mg / L; MnC l2 ·4H2O is 0.05 mg / L to 0.15 mg / L; CuSO4·5H2O is 0.06 mg / L to 0.18 mg / L; ZnCl2 is 0.3 mg / L to 1.0 mg / L.

[0013] Specifically, the composition and concentration range of the inorganic salts are as follows: Na2HPO4 from 140 mg / L to 390 mg / L; KCl from 150 mg / L to 400 mg / L; NaHCO3 from 1100 mg / L to 2800 mg / L; and MgSO4 from 45 mg / L to 120 mg / L.

[0014] Specifically, the composition and concentration range of the lipids are as follows: linolenic acid 0.02 mg / L to 0.15 mg / L; progesterone 0.02 mg / L to 0.15 mg / L; linoleic acid 0.04 mg / L to 0.24 mg / L; cholesterol 1 mg / L to 7.5 mg / L; arachidonic acid 0.001 mg / L to 0.008 mg / L. Preferred lipid concentration ranges are as follows: linolenic acid 0.08 mg / L; progesterone 0.08 mg / L; linoleic acid 0.168 mg / L; cholesterol 5 mg / L; arachidonic acid 0.002 mg / L.

[0015] Specifically, the composition and concentration range of the carbohydrates and functional additives are as follows: ferric citrate 10 mg / L to 40 mg / L; sodium citrate 70 mg / L to 210 mg / L; sodium pyruvate 55 mg / L to 160 mg / L; putrescine 0.05 mg / L to 0.6 mg / L; choline chloride 10 mg / L to 40 mg / L; thymine 0.6 mg / L to 1.8 mg / L; magnesium ascorbate phosphate 0.5 mg / L to 3 mg / L; ethanolamine 0.5 mg / L to 3 mg / L. L; Tricarboxylic acid of gold is 1.0 mg / L to 12.5 mg / L; Hypoxanthine is 3.5 mg / L to 10.5 mg / L; Methyl-β-cyclodextrin is 25 mg / L to 150 mg / L; Trehalose is 50 mg / L to 150 mg / L; Fructose is 800 mg / L to 1200 mg / L; D-glucose is 2000 mg / L to 6000 mg / L; Sodium dextran sulfate is 25 mg / L to 150 mg / L; Kolliphor P188 is 1000 mg / L to 2500 mg / L.

[0016] The third main objective of this invention is to provide a method for preparing CHO cell culture medium.

[0017] This invention achieves the above objective through the following technical solution: a method for preparing CHO cell culture medium, comprising the following steps: S1: Prepare a sterile concentrate of appropriate concentration by mixing various components in proportion; S2: Mix sequentially to near the target volume, then add deionized water to make up the volume; S3: Adjust the pH to 7.2-7.4 using 5NHCl or 5NNaOH; S4: Adjust the osmotic pressure to 290-300 mOsm / L using sodium chloride; S5: After being sterilized by 0.22um filtration, it should be stored at 4℃ away from light for later use.

[0018] The fourth main objective of this invention is to provide a method for culturing CHO cells using CHO cell culture medium.

[0019] The present invention achieves the above-mentioned objective through the following technical solution: A CHO cell culture method comprises: CHO cells are cultured at a concentration of 0.5 × 10⁻⁶ cells / mL. 6 / mL to 2.0×10 6 The cells were seeded at an initial density of / mL in the CHO cell culture medium and cultured at 37±0.5℃, 5% to 7% CO2, and saturated humidity. The medium was changed or the feed was replenished every 2-4 days. When the cell density reached its peak or the target protein expression reached the target, the supernatant was harvested and then purified.

[0020] The beneficial effects of the technical solution of this invention are: The combined additive of the present invention was obtained by screening the binding site characteristics of sodium butyrate and deacetylase inhibitor IV through domain virtual docking and hydrophobicity regulation. It can mildly delay G1 / S conversion and inhibit cell apoptosis, and prolong the effective expression window. Attached Figure Description

[0021] Figure 1 The graph shows the change in viable cell density of each group of samples over culture time.

[0022] Figure 2 The graph shows the change in cell viability of each group of samples over culture time.

[0023] Figure 3 A graph comparing the cumulative recombinant protein concentration over ten days for the specific productivity of each group of samples.

[0024] Figure 4 The dynamic curves of lactic acid in each group of samples are shown.

[0025] Figure 5 The dynamic curves of ammonia concentration for each group of samples are shown.

[0026] Figure 6 This is a comparison chart of the cell clumping index of each group of samples.

[0027] Each group of samples refers to Examples 1-9, Comparative Examples 1, C1, C2, and C3. Detailed Implementation

[0028] The present invention will be further described in detail below with reference to specific embodiments.

[0029] The formulation of the combined additives was discovered based on the following design concept: The first step addresses the conventional approach of "dose / time fine-tuning or superimposed exogenous protein compensation" centered on sodium butyrate (HDAC inhibitor). Based on the binding site characteristics of sodium butyrate and deacetylase inhibitor IV, this invention employs a domain virtual docking and hydrophobic regulation approach, selecting two specific hydrophobic amino acid derivatives to form a combined additive. Under serum-free and exogenous protein-free conditions, a comprehensive improvement of "high proliferation and high specific productivity in parallel" is achieved. Therefore, from sodium butyrate derivatives, a candidate set of 20 compounds was first selected based on similar molecular structures. The candidate set includes: cyclohexylalanine, 3-(2-naphthyl)-L-alanine, sodium phenylbutyrate, phenylalanine, 4-methyl-L-phenylalanine, 4-methylvaleric acid, phenylpropionic acid, valeric acid, 3-ethylnonanoic acid, 4-propyldecanoic acid, 1-naylvaleric acid, 1-naylbutyric acid, 6-2-hydroxyindene, 1-naylpropionic acid, 2-methylpyrrole-N-propionic acid, 1-naylpropionic acid, 3-propylacetic acid, 3-acetaminopropionic acid, phenylacetic acid, and 3-(4-acetaminophenyl).

[0030] The second step involves selecting eight single agents available in existing technologies from the candidate set for individual addition experiments. These agents are: cyclohexylalanine, 3-(2-naphthyl)-L-alanine, sodium phenylbutyrate, phenylalanine, 4-methyl-L-phenylalanine, 4-methylvaleric acid, phenylpropionic acid, and valeric acid. The recommended final concentration ranges for these eight agents are as follows: cyclohexylalanine 0.5 mM to 2.0 mM; 3-(2-naphthyl)-L-alanine 0.5 mM to 2.0 mM; sodium phenylbutyrate 0.5 mM to 2.0 mM; phenylalanine 0.2 mM to 2.0 mM; 4-methyl-L-phenylalanine 0.2 mM to 2.0 mM; 4-methylvaleric acid 0.2 mM to 2.0 mM; phenylpropionic acid 0.5 mM to 2.0 mM; and valeric acid 0.5 mM to 2.0 mM. M represents mol / L, mM represents 10 -3 mol / L. The above single-agent comparison scheme is used to evaluate the overall performance of the combination agent (combination additive). It should not be used simultaneously with the combination agent, nor should all eight single agents be added to the same culture system. Too many components can inhibit cell growth, increase the osmotic pressure of the culture medium, and increase costs.

[0031] The third step involved preparing CHO cell culture media (serum-free and exogenous protein-free) using eight individual agents, comprising the following components: combination additives, amino acids, vitamins, trace elements, inorganic salts, lipids, carbohydrates, and functional additives. In different embodiments, either the combination agent or any one of the individual agents was added for comparison.

[0032] The amino acid composition and concentration ranges are as follows: L-histidine hydrochloride monohydrate 45 mg / L to 135 mg / L; L-isoleucine 80 mg / L to 250 mg / L; L-leucine 85 mg / L to 270 mg / L; L-lysine hydrochloride 135 mg / L to 410 mg / L; L-methionine 35 mg / L to 115 mg / L; L-phenylalanine 50 mg / L to 160 mg / L; L-threonine 80 mg / L to 240 mg / L; L-tryptophan 25 mg / L to 80 mg / L; L-valine 79 mg / L to 240 mg / L; glycine 28 mg / L to 84 mg / L; L-alanine 6.75 mg / L. L-Glutamine: 800 mg / L to 3200 mg / L; L-Arginine Hydrochloride: 400 mg / L to 885 mg / L; L-Asparagine: 100 mg / L to 450 mg / L; L-Aspartic Acid: 20 mg / L to 80 mg / L; L-Cysteine ​​Hydrochloride Monohydrate: 20 mg / L to 75 mg / L; L-Cysteine ​​Hydrochloride: 45 mg / L to 150 mg / L; L-Glutamic Acid: 4 mg / L to 45 mg / L; L-Proline: 33 mg / L to 180 mg / L; L-Serine: 50 mg / L to 150 mg / L; L-Tyrosine Disodium Salt Dihydrate: 80 mg / L to 240 mg / L.

[0033] The composition and concentration range of vitamins are as follows: Vitamin H: 0.05 mg / L to 0.4 mg / L; Folic acid: 10 mg / L to 30 mg / L; Vitamin B2: 0.2 mg / L to 0.6 mg / L; Alpha-lipoic acid: 0.2 mg / L to 0.6 mg / L; Vitamin B12: 0.45 mg / L to 1.35 mg / L; I-inositol: 5 mg / L to 15 mg / L; Vitamin B6: 0.5 mg / L to 3 mg / L; Vitamin B1: 0.5 mg / L to 3 mg / L; Vitamin C: 1 mg / L to 4 mg / L; Vitamin E: 2.5 mg / L to 7.5 mg / L; D-calcium pantothenate: 5 mg / L to 30 mg / L; Nicotinamide: 5 mg / L to 30 mg / L.

[0034] The composition and concentration range of trace elements are as follows: NH4VO3 from 0 mg / L to 0.01 mg / L; Na2SeO3 from 0.02 mg / L to 0.08 mg / L; MnC l2 ·4H2O is 0.05 mg / L to 0.15 mg / L; CuSO4·5H2O is 0.06 mg / L to 0.18 mg / L; ZnCl2 is 0.3 mg / L to 1.0 mg / L.

[0035] The composition and concentration range of inorganic salts are as follows: Na2HPO4 from 140 mg / L to 390 mg / L; KCl from 150 mg / L to 400 mg / L; NaHCO3 from 1100 mg / L to 2800 mg / L; MgSO4 from 45 mg / L to 120 mg / L.

[0036] The composition and concentration range of the lipids are as follows: linolenic acid 0.02 mg / L to 0.15 mg / L; progesterone 0.02 mg / L to 0.15 mg / L; linoleic acid 0.04 mg / L to 0.24 mg / L; cholesterol 1 mg / L to 7.5 mg / L; arachidonic acid 0.001 mg / L to 0.008 mg / L. The preferred concentration range of the lipids is as follows: linolenic acid 0.08 mg / L; progesterone 0.08 mg / L; linoleic acid 0.168 mg / L; cholesterol 5 mg / L; arachidonic acid 0.002 mg / L.

[0037] The composition and concentration range of carbohydrates and functional additives are as follows: ferric citrate 10 mg / L to 40 mg / L; sodium citrate 70 mg / L to 210 mg / L; sodium pyruvate 55 mg / L to 160 mg / L; putrescine 0.05 mg / L to 0.6 mg / L; choline chloride 10 mg / L to 40 mg / L; thymine 0.6 mg / L to 1.8 mg / L; magnesium ascorbate phosphate 0.5 mg / L to 3 mg / L; ethanolamine 0.5 mg / L to 3 mg / L; gold Tricarboxylic acid was 1.0 mg / L to 12.5 mg / L; hypoxanthine was 3.5 mg / L to 10.5 mg / L; methyl-β-cyclodextrin was 25 mg / L to 150 mg / L; trehalose was 50 mg / L to 150 mg / L; fructose was 800 mg / L to 1200 mg / L; D-glucose was 2000 mg / L to 6000 mg / L; sodium dextran sulfate was 25 mg / L to 150 mg / L; and Kolliphor P188 was 1000 mg / L to 2500 mg / L. The preferred concentration ranges for carbohydrates and functional additives are as follows: ferric citrate 20 mg / L; sodium citrate 147 mg / L; sodium pyruvate 110 mg / L; putrescine 0.3 mg / L; choline chloride 30 mg / L; thymine 1.2 mg / L; magnesium ascorbate phosphate 2.5 mg / L; ethanolamine 2.5 mg / L; ginsenoside tricarboxylic acid 2.5 mg / L; hypoxanthine 7 mg / L; methyl-β-cyclodextrin 75 mg / L; trehalose 100 mg / L; fructose 1000 mg / L; D-glucose 5000 mg / L; sodium dextran sulfate 100 mg / L; and Kolliphor P188 2000 mg / L.

[0038] The fourth step is to prepare the CHO cell culture medium according to the following method: S1: Prepare a sterile concentrate of appropriate concentration by mixing various components in proportion; S2: Mix sequentially to near the target volume, then add deionized water to make up the volume; S3: Adjust the pH to 7.2-7.4 using 5NHCl or 5NNaOH; S4: Adjust the osmotic pressure to 290-300 mOsm / L using sodium chloride; S5: After being sterilized by 0.22um filtration, it should be stored at 4℃ away from light for later use.

[0039] Step 5: Apply the CHO cell culture medium method: CHO cells were fed at 0.5 × 10⁻⁶ 6 / mL to 2.0×10 6 The cells were seeded at an initial density of / mL in the CHO cell culture medium and cultured at 37±0.5℃, 5% to 7% CO2, and saturated humidity. The medium was changed or feed was added every 2-4 days. When the cell density reached its peak or the target protein expression was achieved, the supernatant was harvested and then purified. Different embodiments were evaluated using either the combination agent or any single agent.

[0040] Step 6: This application provides comparative schemes to verify the comprehensive advantages of the combination agents. Comparative Example 1 uses commercial serum-free culture medium SigmaExCell325PF, prepared and cultured according to the supplier's instructions, as a commonly used industry baseline control. Comparative Example C1 adds 3mM sodium butyrate to the basic serum-free, exogenous protein-free formulation, without adding the combination agent or single agent, to characterize the effects of traditional HDAC inhibition strategies on late-stage viability, metabolic burden, and aggregation. Comparative Example C2 adds 0.8g / L recombinant human serum albumin and 10mg / L insulin to the basic serum-free formulation, combined with 1mM sodium butyrate, to simulate the common industrial strategy of "exogenous protein compensation + low-dose sodium butyrate". Comparative Example C3 uses a conventional culture system containing 10% fetal bovine serum, with other conditions consistent with shake flask parameters, to reflect the proliferation and impurity background characteristics of the "serum system".

[0041] Step 7: To further illustrate the effects of different addition strategies on cell growth and expression.

[0042] The method for Experiment 1 is as follows: Batch culture was carried out for 10 days using the combination agent, single agent, and comparative examples 1, C1, C2, and C3, respectively; the viable cell density (VCD) and cell viability were recorded daily, and the growth fold was calculated as the ratio of the highest VCD to the initial VCD.

[0043] The method for Experiment 2 is as follows: The dynamic curves of lactic acid and ammonia in each group were detected, and the peak values ​​of metabolic byproducts and their occurrence time points were recorded; it was expected that the peak value of the combination agent would be lower and appear later.

[0044] The method for Experiment 3 is as follows: The cell clumping index of each group was detected and defined as the ratio of the clumping area to the total field of view area multiplied by 100%, obtained through image analysis by threshold segmentation.

[0045] Ultimately, two formulations were selected from eight single agents as a combination additive: cyclohexylalanine and 3-(2-naphthyl)-L-alanine. The final concentration range of cyclohexylalanine in the combination additive was 0.5 mM to 2.0 mM; the final concentration range of 3-(2-naphthyl)-L-alanine was also 0.5 mM to 2.0 mM. The combination additive was obtained through domain virtual docking and hydrophobicity regulation screening based on the binding site characteristics of sodium butyrate and deacetylase inhibitor IV. It was able to delay G1 / S transition, inhibit apoptosis, and prolong the effective expression window.

[0046] The following describes embodiments of this application. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.

[0047] Example 1 (Cyclohexylalanine and 3-(2-naphthyl)-L-alanine) Preparation of combined additives: Cyclohexylalanine and 3-(2-naphthyl)-L-alanine were dissolved in deionized water to prepare a solution with a final concentration of 1.0 mM. After mixing evenly, the combined agent was obtained.

[0048] Preparation of CHO cell culture medium: S1: Prepare a sterile concentrate of appropriate concentration by mixing various components in proportion; S2: Mix sequentially to near the target volume, then add deionized water to make up the volume; S3: Adjust the pH to 7.2-7.4 using 5NHCl or 5NNaOH; S4: Adjust the osmotic pressure to 290-300 mOsm / L using sodium chloride; S5: After being sterilized by 0.22um filtration, it should be stored at 4℃ away from light for later use.

[0049] CHO cell culture: CHO cells were fed at a concentration of 1.0 × 10⁻⁶ 6The cells were inoculated at an initial density of 1 / mL in the above culture medium and cultured at 37±0.5℃, 5% to 7% CO2, and saturated humidity. The medium was changed or the feed was replenished every 2-4 days. When the cell density reached its peak or the target protein expression reached the target, the supernatant was harvested and then purified.

[0050] Test items and methods: Cell viability: Growth fold = Maximum viable cell density / Initial viable cell density.

[0051] Specific productivity (Qp) and yield: ELISA quantifies the target protein; Qp is estimated from yield and average VCD according to industry-standard methods.

[0052] Lactic acid / ammonia concentration: Biochemical analyzer; reports peak values ​​and times of occurrence.

[0053] Clumping Index: Bright-field imaging (uniform exposure and scale), threshold segmentation calculation of "clumping area / total area × 100%".

[0054] Statistical analysis: n≥3, mean ± SD; ANOVA + Tukey; significance is shown in the figure caption. Note: The examples illustrate the effects of different addition strategies on cell growth and expression.

[0055] Example 2 (Single dose: Cyclohexylalanine) In the basic formulation of CHO cell culture medium, only cyclohexylalanine (0.5–2.0 mM, selecting a representative value of the same preferred point as in Example 1, such as 1.0 mM) is added, without adding 3-(2-naphthyl)-L-alanine.

[0056] Example 3 (Single agent: 3-(2-naphthyl)-L-alanine) Only 3-(2-naphthyl)-L-alanine (0.5–2.0 mM, representative value 1.0 mM) was added to the basic formulation of CHO cell culture medium.

[0057] Example 4 (Single agent: Sodium phenylbutyrate) Sodium phenylbutyrate (0.5–2.0 mM, representative value 1.0 mM) was added only to the basic formulation of CHO cell culture medium.

[0058] Example 5 (Single agent: phenylalanine) Phenylalanine (0.2–2.0 mM, representative value 1.0 mM) was added only to the basic formulation of CHO cell culture medium.

[0059] Example 6 (Single agent: 4-methyl-L-phenylalanine) Only 4-methyl-L-phenylalanine (0.2–2.0 mM, representative value 1.0 mM) was added to the basic formulation of CHO cell culture medium.

[0060] Example 7 (Single agent: 4-methylvaleric acid) Only 4-methylvaleric acid (0.2–2.0 mM, representative value 1.0 mM) is added to the basic formulation of CHO cell culture medium.

[0061] Example 8 (Single agent: phenylpropionic acid) Phenylacetic acid (0.5-2.0 mM, representative value 1.0 mM) was added only to the basic formulation of CHO cell culture medium.

[0062] Example 9 (Single agent: valeric acid) Only valeric acid (0.5–2.0 mM, representative value 1.0 mM) is added to the basic formulation of CHO cell culture medium.

[0063] Comparative Example 1 (Commercial serum-free culture medium, Sigma-Ex Cell 325PF) Prepare and culture according to the supplier's instructions, as a commonly used industry baseline control.

[0064] Comparative Example C1 (High-dose sodium butyrate) 3 mM sodium butyrate was added to a basic serum-free, exogenous protein-free formulation, without the addition of combination agents or single agents, to characterize the effects of traditional HDAC inhibition strategies on late-stage activity, metabolic burden, and clumping.

[0065] Comparative Example C2 (serum-free with exogenous protein, BioengineEden20240926) Recombinant human serum albumin 0.8 g / L and insulin 10 mg / L were added to the basic serum-free formulation, along with 1 mM sodium butyrate, to simulate the common industrial strategy of "exogenous protein compensation + low-dose sodium butyrate".

[0066] Comparative Example C3 (containing serum, Gibco Premium FBSC0235) A standard culture system containing 10% fetal bovine serum was used, with all other conditions consistent with those of the shake flask, to reflect the proliferation and impurity background characteristics of the "serum system".

[0067] Figure 1 This is a graph showing the change in viable cell density of each group of samples over culture time. It is used to compare the differences in proliferation capacity and peak value between the combination agent, single agent, and control systems throughout the entire culture period.

[0068] Figure 2This is a graph showing the change in cell viability over culture time for each group of samples. It was used to evaluate the maintenance of viability in the later stage (Days 6-10) and to verify the effect of the combination agent on the late-stage viability decline commonly seen in sodium butyrate strategies.

[0069] Figure 3 This is a comparison graph of the cumulative recombinant protein concentration with specific productivity over ten days for each group of samples. The error bars in the graph represent the standard deviation, and the significance of comparisons with control groups 1, C1, C2, C3 and each single agent is shown (*p<0.05, **p<0.01).

[0070] Figure 4 The dynamic curves of lactic acid and ammonia concentrations for each group of samples are shown. The peak values ​​of metabolic byproducts and their timing are displayed; it is expected that the peak values ​​for the combination group will be lower and appear later.

[0071] Figure 5 This image shows a comparison of cell clumping indices for each group of samples. The clumping index is defined as the area of ​​the clumped region / the total field of view × 100%, obtained through image analysis using threshold segmentation.

[0072] Analysis of experimental results: Experiment 1 (Proliferation and Viability Assessment, corresponding to...) Figure 1 , Figure 2 ) The results of Experiment 1 are as follows: Batch culture was conducted for 10 days using the combination agent, single agent, and comparative examples 1, C1, C2, and C3, respectively; the viable cell density (VCD) and cell viability were recorded daily, and the fold increase was calculated as the ratio of the highest VCD to the initial VCD. The results are shown in Table 1.

[0073] Table 1 Viable cell density of each group of samples (×10) 4 (cells / mL) and cell viability (%)

[0074] Note: The table above is for illustrative purposes; typical trends for C1, C2, and C3 can be found in [the table below]. Figure 1 – Figure 2 The results showed that the activity of C1 decreased significantly in the later stage, and although the VCD peak or activity of C2 and C3 improved to some extent, the impurities and metabolic burden were high.

[0075] Experiment 2 (Expression and Specific Productivity, Corresponding) Figure 3 ) Methods: CHO cells were transfected with adalimumab heavy and light chain plasmids, and stable lines were obtained by G418 (800 μg / mL) selection. Samples from each group were subjected to 10 days of suspension culture (initial density 3.0 × 10⁶ cells / mL). 5(cells / mL). Centrifuge and collect the supernatant. Quantify the recombinant protein using ELISA; Qp is estimated from yield and average VCD according to industry-standard methods.

[0076] Results: The specific productivity yield of Example 1 was significantly higher than that of Comparative Example 1, and also higher than that of the corresponding single-dose groups (Examples 2 and 3), see [link to example]. Figure 3 This result indicates that the combination agent enhances specific productivity without significantly sacrificing proliferation.

[0077] Experiment 3 (Metabolism and Aggregation, corresponding to...) Figure 4 – Figure 6 ) Methods: Using the same culture system as Experiment 1, lactic acid concentration (g / L) and ammonia concentration (mM) were measured daily, and microscopic images were collected to calculate the clumping index (%).

[0078] Results: The peak values ​​of lactic acid and ammonia in Example 1 were lower than those in C1, C2, C3, and most single-dose groups (see [link to example 1]). Figure 4 and Figure 5 The clumping index also decreased significantly (see...). Figure 6 This result is consistent with higher late-stage viability, indicating that the present invention is beneficial for engineering controllability and subsequent processability.

[0079] The above descriptions are merely some embodiments of the present invention. Those skilled in the art can make various modifications and improvements without departing from the inventive concept of the present invention, and these all fall within the scope of protection of the present invention.

Claims

1. A combination additive, characterized in that... It contains the following components: cyclohexylalanine and 3-(2-naphthyl)-L-alanine, wherein the final concentration of cyclohexylalanine is in the range of 0.5 mM to 2.0 mM; and the final concentration of 3-(2-naphthyl)-L-alanine is in the range of 0.5 mM to 2.0 mM.

2. A CHO cell culture medium, characterized in that... It contains the following components: the combined additives as described in claim 1, amino acids, vitamins, trace elements, inorganic salts, lipids, carbohydrates, and functional additives.

3. The CHO cell culture medium according to claim 2, characterized in that: The composition and concentration range of the amino acids are as follows: L-histidine hydrochloride monohydrate 45 mg / L to 135 mg / L; L-isoleucine 80 mg / L to 250 mg / L; L-leucine 85 mg / L to 270 mg / L; L-lysine hydrochloride 135 mg / L to 410 mg / L; L-methionine 35 mg / L to 115 mg / L; L-phenylalanine 50 mg / L to 160 mg / L; L-threonine 80 mg / L to 240 mg / L; L-tryptophan 25 mg / L to 80 mg / L; L-valine 79 mg / L to 240 mg / L; glycine 28 mg / L to 84 mg / L; L-alanine 6.75 mg / L. L-Glutamine is 800 mg / L to 3200 mg / L; L-Arginine hydrochloride is 400 mg / L to 885 mg / L; L-Asparagine is 100 mg / L to 450 mg / L; L-Aspartic acid is 20 mg / L to 80 mg / L; L-Cysteine ​​hydrochloride monohydrate is 20 mg / L to 75 mg / L; L-Cysteine ​​hydrochloride is 45 mg / L to 150 mg / L; L-Glutamic acid is 4 mg / L to 45 mg / L; L-Proline is 33 mg / L to 180 mg / L; L-Serine is 50 mg / L to 150 mg / L; L-Tyrosine disodium salt dihydrate is 80 mg / L to 240 mg / L.

4. The CHO cell culture medium according to claim 2, characterized in that: The composition and concentration range of the vitamins are as follows: Vitamin H is 0.05 mg / L to 0.4 mg / L; folic acid is 10 mg / L to 30 mg / L; vitamin B2 is 0.2 mg / L to 0.6 mg / L; lipoic acid is 0.2 mg / L to 0.6 mg / L; vitamin B12 is 0.45 mg / L to 1.35 mg / L; i-inositol is 5 mg / L to 15 mg / L; vitamin B6 is 0.5 mg / L to 3 mg / L; vitamin B1 is 0.5 mg / L to 3 mg / L; vitamin C is 1 mg / L to 4 mg / L; vitamin E is 2.5 mg / L to 7.5 mg / L; D-calcium pantothenate is 5 mg / L to 30 mg / L; and nicotinamide is 5 mg / L to 30 mg / L.

5. The CHO cell culture medium according to claim 2, characterized in that: The composition and concentration range of the trace elements are as follows: NH4VO3 from 0 mg / L to 0.01 mg / L; Na2SeO3 from 0.02 mg / L to 0.08 mg / L; MnC l2 ·4H2O is 0.05 mg / L to 0.15 mg / L; CuSO4·5H2O is 0.06 mg / L to 0.18 mg / L; ZnCl2 is 0.3 mg / L to 1.0 mg / L.

6. The CHO cell culture medium according to claim 2, characterized in that: The composition and concentration range of the inorganic salts are as follows: Na2HPO4 from 140 mg / L to 390 mg / L; KCl from 150 mg / L to 400 mg / L; NaHCO3 from 1100 mg / L to 2800 mg / L; MgSO4 from 45 mg / L to 120 mg / L.

7. The CHO cell culture medium according to claim 2, characterized in that: The composition and concentration range of the lipids are as follows: linolenic acid 0.02 mg / L to 0.15 mg / L; progesterone 0.02 mg / L to 0.15 mg / L; linoleic acid 0.04 mg / L to 0.24 mg / L; cholesterol 1 mg / L to 7.5 mg / L; arachidonic acid 0.001 mg / L to 0.008 mg / L. Preferred lipid concentration ranges are as follows: linolenic acid 0.08 mg / L; progesterone 0.08 mg / L; linoleic acid 0.168 mg / L; cholesterol 5 mg / L; arachidonic acid 0.002 mg / L.

8. The CHO cell culture medium according to claim 2, characterized in that: The composition and concentration range of the carbohydrates and functional additives are as follows: ferric citrate 10 mg / L to 40 mg / L; sodium citrate 70 mg / L to 210 mg / L; sodium pyruvate 55 mg / L to 160 mg / L; putrescine 0.05 mg / L to 0.6 mg / L; choline chloride 10 mg / L to 40 mg / L; thymine 0.6 mg / L to 1.8 mg / L; magnesium ascorbate phosphate 0.5 mg / L to 3 mg / L; ethanolamine 0.5 mg / L to 3 mg / L. The concentrations of the following compounds are: Tricarboxylic acid (TCA) from 1.0 mg / L to 12.5 mg / L; Hypoxanthine from 3.5 mg / L to 10.5 mg / L; Methyl-β-cyclodextrin from 25 mg / L to 150 mg / L; Trehalose from 50 mg / L to 150 mg / L; Fructose from 800 mg / L to 1200 mg / L; D-glucose from 2000 mg / L to 6000 mg / L; Sodium dextran sulfate from 25 mg / L to 150 mg / L; and Kolliphor P188 from 1000 mg / L to 2500 mg / L.

9. A method for preparing the CHO cell culture medium according to claims 2-8, characterized in that the steps are... include: S1: Prepare a sterile concentrate of appropriate concentration by mixing various components in proportion; S2: Mix sequentially to near the target volume, then add deionized water to make up the volume; S3: Adjust the pH to 7.2-7.4 using 5NHCl or 5NNaOH; S4: Adjust the osmotic pressure to 290-300 mOsm / L using sodium chloride; S5: After being sterilized by 0.22um filtration, it should be stored at 4℃ away from light for later use.

10. A method for culturing CHO cells using the CHO cell culture medium described in claims 2-8, characterized in that... The process is as follows: CHO cells are inoculated at a concentration of 0.5 × 10⁻⁶ 6 / mL to 2.0×10 6 The cells were seeded at an initial density of / mL in the CHO cell culture medium and cultured at 37±0.5℃, 5% to 7% CO2, and saturated humidity. The medium was changed or the feed was replenished every 2-4 days. When the cell density reached its peak or the target protein expression reached the target, the supernatant was harvested and then purified.

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