Method for improving PD-1 titer produced by Chinese hamster ovary cells

Abstract

The invention relates to a method for improving PD-1 titer produced by Chinese hamster ovary cells. The method comprises the following steps: inoculating the Chinese hamster ovary cells into an initial culture medium for culturing; after the culture is performed for 48 hours, feeding a fed-batch culture medium every day, and maintaining the glucose concentration in the culture medium to be 1.3-1.7 g / L, the galactose concentration to be 0.6-1.0 g / L and the L-sodium lactate concentration to be 14-16 mM; the culture temperature is reduced, a fed-batch culture medium is fed every day, the glucose concentration in the culture medium is maintained at (2-2.8) g / L, the galactose concentration is maintained at (1.7-2.2) g / L, and cells are harvested. Galactose is introduced to serve as a second carbon source, and control over the culture initial metabolism environment and the carbon source in the culture stage is combined, so that the maximum accumulation amount of lactic acid is obviously reduced, the cell culture period is remarkably prolonged, and the maximum titer of the PD-1 antibody is remarkably improved compared with an existing process.

Description

A method to increase PD-1 titer production in Chinese hamster ovary cells Technical Field

[0001] This invention belongs to the field of cell biology technology and relates to a method for increasing the production of PD-1 titers by Chinese hamster ovary cells. Background Technology

[0002] In the industrial production of monoclonal antibodies, the fed-batch culture process using recombinant Chinese hamster ovary cells (CHO cells) is widely used due to its high production efficiency and strong process controllability. However, in actual production, traditional fed-batch culture processes typically use glucose as the main or even sole carbon source. Under high glucose conditions, cells are prone to significant glycolytic bias, leading to a large accumulation of lactic acid. The continuous accumulation of lactic acid not only causes pH fluctuations in the culture system but also adversely affects cell physiological state, metabolic homeostasis, and antibody expression levels.

[0003] Lactic acid metabolism is one of the most critical and complex links in the CHO cell metabolic network, and its regulatory state directly affects cell growth, metabolic homeostasis, and the expression quality and glycosylation characteristics of recombinant proteins.

[0004] The Warburg effect refers to the phenomenon that cells, even with sufficient oxygen supply, still primarily rely on glycolysis to produce energy and generate large amounts of lactate. This is highly consistent with the metabolic behavior exhibited by CHO cells during high-density culture and rapid proliferation. During industrial-scale culture, even with dissolved oxygen levels maintained within a suitable range, CHO cells generally exhibit a high glucose uptake rate, large lactate accumulation, and a relatively weak dependence on mitochondrial oxidative metabolism, i.e., "aerobic glycolysis." This metabolic mode, while capable of rapidly providing energy and regenerative capacity (NAD), also exhibits these characteristics. + While maintaining NADH balance, this also leads to excessive accumulation of lactic acid in the culture system, causing acidification of the culture medium, increased osmotic pressure, increased ammonia production, and inhibition of cell growth, thus significantly affecting antibody expression levels and product quality.

[0005] In fed-batch culture of CHO cells, the dynamic regulation of carbon sources is a key factor affecting cell metabolism and recombinant protein expression efficiency. Glucose, as the primary energy and carbon backbone source for CHO cells, is susceptible to adverse effects. Excessive glucose concentration can lead to lactic acid accumulation, resulting in acidification of the culture system and decreased cell metabolic efficiency; conversely, insufficient glucose concentration may limit cell growth and product synthesis. Galactose, as an alternative carbon source, can alleviate glycolytic imbalance under high glucose conditions to some extent. By reducing glucose metabolic flux and regulating energy metabolism pathways, it can improve cell metabolic phenotype and product quality. However, excessive galactose addition can lead to Gal-1-P toxicity accumulation, negatively impacting cell growth, metabolism, and product expression. Cellular metabolic requirements vary significantly at different culture stages, and a single carbon source supply model is insufficient to adapt to these changing metabolic needs. Furthermore, alternative carbon sources such as galactose lack systematic regulatory strategies tailored to cellular metabolic characteristics, limiting their application in antibody production processes.

[0006] Galactose, as an alternative carbon source, can alleviate metabolic stress under high glucose conditions to some extent. However, its systematic application in fed-batch culture of CHO cells still faces problems such as low galactose metabolic efficiency and Gal-1-P toxicity accumulation, and lacks mature and stable process control schemes.

[0007] Therefore, how to construct a more balanced carbon source metabolic environment during fed-batch culture, reduce the accumulation of metabolic byproducts, and simultaneously ensure cell growth and efficient antibody expression remains a critical technical problem that needs to be solved in current antibody production processes. Summary of the Invention

[0008] The purpose of this invention is to overcome the shortcomings of the existing technology and provide a method for increasing the PD-1 titer produced by Chinese hamster ovary cells.

[0009] The objective of this invention can be achieved through the following technical solution: The technical solution of this invention is to provide a method for improving the PD-1 titer production of Chinese hamster ovary cells, comprising the following steps: S1, Chinese hamster ovary cells are inoculated into an initial culture medium for culture, wherein the initial culture medium consists of basal culture medium, cell passage culture medium, (1.8-2.2) g / L glucose, (13-17) mM L-lactate sodium, and (0.6-1) g / L galactose, wherein the amount of cell inoculation medium added is 7-11% of the volume of the initial culture medium, and the pH is 6.76-6.84; S2, during the growth stage of Chinese hamster ovary cells: after culturing for 48 h, feed culture medium is added daily, and the glucose concentration in the culture medium is maintained at (1.3-1.7) g / L, the galactose concentration is (0.6-1.0) g / L, and the L-lactate sodium concentration is 14-16 g / L. mM; S3, In the antibody production stage of Chinese hamster ovary cells: lower the culture temperature, add feed medium daily, and maintain the glucose concentration in the medium at (2~2.8) g / L and the galactose concentration at (1.7~2.2) g / L; S4, culture until the cell viability is less than 80%, and harvest the cells.

[0010] In some specific embodiments, the seeding density of the Chinese hamster ovary cells is (6-8) × 10⁻⁶. 5 Cells / mL.

[0011] In some specific embodiments, in steps S2 and S3, the feeding medium consists of feeding medium A and feeding medium B, wherein the amount of feeding medium A added is 0.15% of the initial culture medium volume, and the amount of feeding medium B added is 1.5% of the initial culture medium volume.

[0012] In some specific embodiments, in step S2, the growth stage of Chinese hamster ovary cells is (0~105) h or (0~125) h after cell inoculation.

[0013] In some specific implementations, in step S2, the culture temperature is (36.7~37.1)℃.

[0014] In some specific embodiments, in step S2, glucose concentration in the culture medium is maintained at (1.3~1.7) g / L and galactose concentration is (0.6~1.0) g / L by additionally adding glucose and galactose concentrate.

[0015] In some specific embodiments, in step S2, glucose concentration in the culture medium is maintained at 1.5 g / L and galactose concentration is 1 g / L by additionally adding glucose and galactose concentrate.

[0016] In some specific embodiments, in step S3, the temperature is reduced to (32.8~33.2)℃.

[0017] In some specific embodiments, in step S3, glucose and galactose concentrates are added to maintain the glucose concentration in the culture medium at (2~2.8) g / L and the galactose concentration at (1.7~2.2) g / L.

[0018] In some specific embodiments, in step S3, glucose concentration in the culture medium is maintained at 2.4 g / L and galactose concentration is 2.0 g / L by additionally adding glucose and galactose concentrate.

[0019] Firstly, this invention improves cell growth rate (μ) by adjusting the initial cell density and the ratio of cell passage medium. The change in growth rate exhibits a clear two-factor response: when the cell passage medium ratio is approximately 10% and the initial cell seeding density is 7 × 10⁻⁶, the cell growth rate increases significantly. 5 At a cell / mL ratio, the specific growth rate reached its highest value (μ=0.0305) after 48 hours of cell culture.

[0020] Secondly, this invention adjusts the initial culture medium for L-lactic acid sodium and pH to increase cell growth rate and decrease the specific lactate production rate (qLac). When the initial pH is controlled at around 6.8 and the L-lactic acid sodium concentration is around 16 mM, the μ value remains in the range of 0.029-0.036 over 24 hours, while the specific lactate production rate (qLac) is relatively low, indicating that cell metabolism is relatively balanced under these conditions and lactate production is effectively inhibited.

[0021] Thirdly, this invention adjusts the concentrations of glucose and galactose in the culture medium during cell inoculation and culture, controlling them within a certain concentration range to improve the cell growth rate. Both carbon sources have a significant synergistic regulatory effect on cell growth.

[0022] The optimal initial culture medium concentration is 2 g / L ± 0.2 g / L glucose and 0.8 g / L ± 0.2 g / L galactose, and the glucose and galactose concentrations in the culture medium are maintained at 1.5 g / L ± 0.2 g / L and 0.8 g / L ± 0.2 g / L, respectively, within the cell growth range.

[0023] Fourthly, this invention adjusts the concentrations of glucose and galactose in the culture medium and lowers the temperature during the antibody production stage of cells to increase the titer of PD-1 antibody, reduce lactic acid accumulation, and extend the culture period.

[0024] The optimal temperature is 33℃±0.2℃, while adjusting the glucose and galactose concentrations in the culture medium to 2.4 g / L±0.4 g / L for glucose and 1.9 g±0.2 g / L for galactose.

[0025] Compared with the prior art, the present invention has the following advantages: (1) By introducing galactose as a second carbon source and combining it with the systematic optimization of the initial metabolic environment and carbon source control strategy during the culture stage, the present invention overcomes the problems of easy accumulation of lactic acid and difficulty in maintaining metabolic homeostasis in the traditional fed-batch culture process. By constructing a stable metabolic environment for the co-consumption of glucose and lactic acid, the energy utilization efficiency of CHO cells is significantly improved, the metabolic pressure is reduced, and the cells can maintain a high activity state for a longer period of time.

[0026] (2) This invention implements differentiated glucose and galactose control strategies for different culture stages, so that the carbon source supply is more in line with the changing needs of cell metabolism, thereby improving antibody synthesis efficiency without sacrificing cell growth performance.

[0027] (3) The maximum accumulation of lactic acid in the culture system of the present invention is significantly reduced, the cell culture cycle is significantly extended, and the maximum titer of PD-1 antibody is significantly improved compared with the existing process, demonstrating good industrial application value. Detailed Implementation

[0028] The present invention will now be described in detail with reference to specific embodiments. These embodiments are based on the technical solution of the present invention and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0030] The following are the culture media used in each example and comparative example: (1) Basal culture medium: CM-013 (manufacturer: Suzhou Womei Biotechnology Co., Ltd.) (2) Cell passage medium: The cell passage medium is obtained by centrifuging the culture medium from the previous generation of cell passages to remove cell precipitate, and the supernatant is the cell passage medium. The cell passage medium in each of the following examples is the supernatant after centrifuging the culture medium after three cell passages. The cell passage medium contains cell regulatory factors, which help stabilize the cell metabolic state and improve the cell survival rate in the early stage of suspension culture, and promote the cells to enter the exponential growth phase more quickly.

[0031] (3) Feed medium A: DF-003 (manufacturer: Suzhou Womei Biotechnology Co., Ltd.) (4) Feed medium B: CM-003 (manufacturer: Suzhou Womei Biotechnology Co., Ltd.) The following are the detection methods used in each example and comparative example: (1) Protein expression level detection: The protein expression level was detected by Agilent high performance liquid chromatography, using a TSKgelProtein A-5PW Protein A affinity column to detect the PD-1 antibody titer in the cell supernatant cultured to a cell viability of less than 80%.

[0032] (2) Lactic acid concentration detection: Lactic acid concentration was detected using the Cedex Bio automated multifunctional biochemical analyzer. The daily lactic acid concentration was detected according to the equipment's instruction manual.

[0033] (3) Cell density and cell viability detection: Cell density and cell viability were detected using the CountStar cell counter. The cell density and cell viability were measured daily according to the device's instructions.

[0034] Unless otherwise specified, the raw materials and processes used in the following embodiments or comparative examples are conventional commercially available raw materials and processes used in the art to achieve the corresponding functions.

[0035] Example 1 (1) Chinese hamster ovary cells were inoculated at a density of 7×10 5 Cells were seeded at approximately 1 / mL into the initial culture medium and cultured for 48 h at a temperature of 36.7–37.1 °C.

[0036] The initial culture medium consisted of basal culture medium, cell passage medium, 2.0 g / L glucose, 16 mM L-lactic acid sodium, and 1 g / L galactose. The cell inoculation medium was added at 10% of the initial culture medium volume, and the pH was 6.8.

[0037] (2) During the growth stage of Chinese hamster ovary cells: the culture medium was fed daily for 120 h and the culture temperature was 36.7~37.1℃.

[0038] The fed culture medium consisted of fed culture medium A and fed culture medium B. The amount of fed culture medium A added was 0.15% of the initial culture medium volume, and the amount of fed culture medium B added was 1.5% of the initial culture medium volume. The concentrations of glucose and galactose in the culture medium were measured. By adding additional glucose and galactose, the glucose concentration in the culture medium was maintained at 1.5 g / L and the galactose concentration at 1 g / L.

[0039] (3) During the antibody production stage of Chinese hamster ovary cells: reduce the culture temperature to 32.8~33.4℃ and continue culture by adding feed medium daily.

[0040] The fed culture medium consisted of fed culture medium A and fed culture medium B. The amount of fed culture medium A added was 0.15% of the initial culture medium volume, and the amount of fed culture medium B added was 1.5% of the initial culture medium volume. The concentrations of glucose and galactose in the culture medium were measured. By adding additional glucose and galactose, the glucose concentration in the culture medium was maintained at 2.4 g / L and the galactose concentration at 2.0 g / L.

[0041] (4) Cultivate until the cell viability is less than 80%, then harvest the cells.

[0042] Example 2 (1) Chinese hamster ovary cells were inoculated at a density of 7×10 5 Cells were seeded at approximately 1 / mL into the initial culture medium and cultured for 48 h at a temperature of 36.7–37.1 °C.

[0043] The initial culture medium consisted of basal culture medium, cell passage medium, 2.0 g / L glucose, 16 mM L-lactic acid sodium, and 1 g / L galactose. The cell inoculation medium was added at 10% of the initial culture medium volume, and the pH was 6.8.

[0044] (2) During the growth stage of Chinese hamster ovary cells: the culture medium was fed daily for 120 h and the culture temperature was 36.7~37.1℃.

[0045] The fed culture medium consisted of fed culture medium A and fed culture medium B. The amount of fed culture medium A added was 0.15% of the initial culture medium volume, and the amount of fed culture medium B added was 1.5% of the initial culture medium volume. The concentrations of glucose and galactose in the culture medium were measured. By adding additional glucose and galactose, the glucose concentration in the culture medium was maintained at 1.5 g / L and the galactose concentration at 1 g / L.

[0046] (3) During the antibody production stage of Chinese hamster ovary cells: reduce the culture temperature to 32.8~33.4℃ and continue culture by adding feed medium daily.

[0047] The fed culture medium consisted of fed culture medium A and fed culture medium B. The amount of fed culture medium A added was 0.15% of the initial culture medium volume, and the amount of fed culture medium B added was 1.5% of the initial culture medium volume. The concentrations of glucose and galactose in the culture medium were measured. By adding additional glucose and galactose, the glucose concentration in the culture medium was maintained at 2.0 g / L and the galactose concentration at 1.8 g / L.

[0048] (4) Cultivate until the cell viability is less than 80%, then harvest the cells.

[0049] Example 3 (1) Chinese hamster ovary cells were inoculated at a density of 7×10 5 Cells were seeded at approximately 1 / mL into the initial culture medium and cultured for 48 h at a temperature of 36.7–37.1 °C.

[0050] The initial culture medium consisted of basal culture medium, cell passage medium, 2.0 g / L glucose, 16 mM L-lactic acid sodium, and 1 g / L galactose. The cell inoculation medium was added at 10% of the initial culture medium volume, and the pH was 6.8.

[0051] (2) During the growth stage of Chinese hamster ovary cells: the culture medium was fed daily for 120 h and the culture temperature was 36.7~37.1℃.

[0052] The fed culture medium consisted of fed culture medium A and fed culture medium B. The amount of fed culture medium A added was 0.15% of the initial culture medium volume, and the amount of fed culture medium B added was 1.5% of the initial culture medium volume. The concentrations of glucose and galactose in the culture medium were measured. By adding additional glucose and galactose, the glucose concentration in the culture medium was maintained at 1.5 g / L and the galactose concentration at 1 g / L.

[0053] (3) During the antibody production stage of Chinese hamster ovary cells: reduce the culture temperature to 32.8~33.4℃ and continue culture by adding feed medium daily.

[0054] The fed culture medium consisted of fed culture medium A and fed culture medium B. The amount of fed culture medium A added was 0.15% of the initial culture medium volume, and the amount of fed culture medium B added was 1.5% of the initial culture medium volume. The concentrations of glucose and galactose in the culture medium were measured. By adding additional glucose and galactose, the glucose concentration in the culture medium was maintained at 2.8 g / L and the galactose concentration at 2.2 g / L.

[0055] (4) Cultivate until the cell viability is less than 80%, then harvest the cells.

[0056] Comparative Example 1 (1) Chinese hamster ovary cells were inoculated at a density of 7 × 10⁶ cells / year. 5 Cells were seeded at approximately 1 / mL into the initial culture medium and cultured for 48 h at a temperature of 36.7–37.1 °C.

[0057] The initial culture medium consisted of basal culture medium, cell passage medium, and 2.0 g / L glucose, with the cell inoculation medium added at 10% of the initial culture medium volume and a pH of 6.8.

[0058] (2) During the growth stage of Chinese hamster ovary cells: the culture medium was fed daily for 120 h and the culture temperature was 36.7~37.1℃.

[0059] The fed culture medium consisted of fed culture medium A and fed culture medium B. The amount of fed culture medium A added was 0.15% of the initial culture medium volume, and the amount of fed culture medium B added was 1.5% of the initial culture medium volume. The glucose concentration in the culture medium was measured, and by adding additional glucose, the glucose concentration in the culture medium was maintained at 1.5 g / L.

[0060] (3) During the antibody production stage of Chinese hamster ovary cells: reduce the culture temperature to 32.8~33.4℃ and continue culture by adding feed medium daily.

[0061] The fed culture medium consisted of fed culture medium A and fed culture medium B. The amount of fed culture medium A added was 0.15% of the initial culture medium volume, and the amount of fed culture medium B added was 1.5% of the initial culture medium volume. The glucose concentration in the culture medium was measured, and by adding additional glucose, the glucose concentration in the culture medium was maintained at 1.5 g / L.

[0062] (4) Cultivate until the cell viability is less than 80%, then harvest the cells.

[0063] Comparative Example 2 (1) Chinese hamster ovary cells were inoculated at a density of 7 × 10⁶ cells / year. 5 Cells were seeded at approximately 1 / mL into the initial culture medium and cultured for 48 h at a temperature of 36.7–37.1 °C.

[0064] The initial culture medium consisted of basal culture medium, cell passage medium, 2.0 g / L glucose, and 14 mM L-lactic acid sodium, with the cell inoculation medium added at 10% of the initial culture medium volume and a pH of 6.8.

[0065] (2) During the growth stage of Chinese hamster ovary cells: the culture medium was fed daily for 120 h and the culture temperature was 36.7~37.1℃.

[0066] The fed culture medium consisted of fed culture medium A and fed culture medium B. The amount of fed culture medium A added was 0.15% of the initial culture medium volume, and the amount of fed culture medium B added was 1.5% of the initial culture medium volume. The glucose concentration in the culture medium was measured, and by adding additional glucose, the glucose concentration in the culture medium was maintained at 1.5 g / L.

[0067] (3) During the antibody production stage of Chinese hamster ovary cells: reduce the culture temperature to 32.8~33.4℃ and continue culture by adding feed medium daily.

[0068] The fed culture medium consisted of fed culture medium A and fed culture medium B. The amount of fed culture medium A added was 0.15% of the initial culture medium volume, and the amount of fed culture medium B added was 1.5% of the initial culture medium volume. The glucose concentration in the culture medium was measured, and by adding additional glucose, the glucose concentration in the culture medium was maintained at 1.5 g / L.

[0069] (4) Cultivate until the cell viability is less than 80%, then harvest the cells.

[0070] Comparative Example 3(1) Chinese hamster ovary cells were inoculated at a density of 7×10⁶ cells / year. 5 Cells were seeded at approximately 1 / mL into the initial culture medium and cultured for 48 h at a temperature of 36.7–37.1 °C.

[0071] The initial culture medium consisted of basal culture medium, cell passage medium, 2.0 g / L glucose, 16 mM L-lactic acid sodium, and 0.6 g / L galactose. The cell inoculation medium was added at 10% of the initial culture medium volume, and the pH was 6.8.

[0072] (2) During the growth stage of Chinese hamster ovary cells: the culture medium was fed daily for 120 h and the culture temperature was 36.7~37.1℃.

[0073] The fed culture medium consisted of fed culture medium A and fed culture medium B. The amount of fed culture medium A added was 0.15% of the initial culture medium volume, and the amount of fed culture medium B added was 1.5% of the initial culture medium volume. The glucose concentration in the culture medium was measured, and by adding additional glucose, the glucose concentration in the culture medium was maintained at 1.5 g / L.

[0074] (3) During the antibody production stage of Chinese hamster ovary cells: reduce the culture temperature to 32.8~33.4℃ and continue culture by adding feed medium daily.

[0075] The fed culture medium consisted of fed culture medium A and fed culture medium B. The amount of fed culture medium A added was 0.15% of the initial culture medium volume, and the amount of fed culture medium B added was 1.5% of the initial culture medium volume. The glucose concentration in the culture medium was measured, and by adding additional glucose, the glucose concentration in the culture medium was maintained at 1.5 g / L.

[0076] (4) Cultivate until the cell viability is less than 80%, then harvest the cells.

[0077] Comparative Example 4(1) Chinese hamster ovary cells were inoculated at a density of 7×10⁶ cells / cm². 5 Cells were seeded at approximately 1 / mL into the initial culture medium and cultured for 48 h at a temperature of 36.7–37.1 °C.

[0078] The initial culture medium consisted of basal culture medium, cell passage medium, 2.0 g / L glucose, 16 mM L-lactic acid sodium, and 0.6 g / L galactose. The cell inoculation medium was added at 10% of the initial culture medium volume, and the pH was 6.8.

[0079] (2) During the growth stage of Chinese hamster ovary cells: the culture medium was fed daily for 120 h and the culture temperature was 36.7~37.1℃.

[0080] The fed culture medium consisted of fed culture medium A and fed culture medium B. The amount of fed culture medium A added was 0.15% of the initial culture medium volume, and the amount of fed culture medium B added was 1.5% of the initial culture medium volume. The concentrations of glucose and galactose in the culture medium were measured. By adding additional glucose and galactose, the glucose concentration in the culture medium was maintained at 1.5 g / L and the galactose concentration at 0.6 g / L.

[0081] (3) During the antibody production stage of Chinese hamster ovary cells: reduce the culture temperature to 32.8~33.4℃ and continue culture by adding feed medium daily.

[0082] The fed culture medium consisted of fed culture medium A and fed culture medium B. The amount of fed culture medium A added was 0.15% of the initial culture medium volume, and the amount of fed culture medium B added was 1.5% of the initial culture medium volume. The concentrations of glucose and galactose in the culture medium were measured. By adding additional glucose and galactose, the glucose concentration in the culture medium was maintained at 1.5 g / L and the galactose concentration at 0.6 g / L.

[0083] (4) Cultivate until the cell viability is less than 80%, then harvest the cells.

[0084] Comparative Example 5(1) Chinese hamster ovary cells were inoculated at a density of 7 × 10⁶ cells / year. 5 Cells were seeded at approximately 1 / mL into the initial culture medium and cultured for 48 h at a temperature of 36.7–37.1 °C.

[0085] The initial culture medium consisted of basal culture medium, cell passage medium, 2.0 g / L glucose, 16 mM L-lactic acid sodium, and 1 g / L galactose. The cell inoculation medium was added at 10% of the initial culture medium volume, and the pH was 6.8.

[0086] (2) During the growth stage of Chinese hamster ovary cells: the culture medium was fed daily for 120 h and the culture temperature was 36.7~37.1℃.

[0087] The fed culture medium consisted of fed culture medium A and fed culture medium B. The amount of fed culture medium A added was 0.15% of the initial culture medium volume, and the amount of fed culture medium B added was 1.5% of the initial culture medium volume. The concentrations of glucose and galactose in the culture medium were measured. By adding additional glucose and galactose, the glucose concentration in the culture medium was maintained at 1.5 g / L and the galactose concentration at 1 g / L.

[0088] (3) During the antibody production stage of Chinese hamster ovary cells: reduce the culture temperature to 32.8~33.4℃ and continue culture by adding feed medium daily.

[0089] The fed culture medium consisted of fed culture medium A and fed culture medium B. The amount of fed culture medium A added was 0.15% of the initial culture medium volume, and the amount of fed culture medium B added was 1.5% of the initial culture medium volume. The concentrations of glucose and galactose in the culture medium were measured. By adding additional glucose and galactose, the glucose concentration in the culture medium was maintained at 1.5 g / L and the galactose concentration at 1 g / L.

[0090] (4) Cultivate until the cell viability is less than 80%, then harvest the cells.

[0091] Daily samples were taken to measure the lactate concentration in the culture medium, and the lactate production was calculated as (daily lactate concentration - initial lactate concentration). Cells were cultured until the cell viability was below 80%, the culture time was calculated, and cells were harvested for antibody titer detection. The results are shown in Table 1.

[0092] Table 1 As shown in Table 1, each embodiment maintained a relatively balanced carbon source allocation and utilization by significantly reducing the accumulation of metabolic byproducts, dynamically supplementing nutrients, introducing a second carbon source, galactose, and rationally determining key process control time points. This effectively improved the metabolic efficiency of CHO cells, prolonged the duration of high cell activity, and thus significantly increased the final antibody titer.

[0093] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A method for increasing the PD-1 titer produced by Chinese hamster ovary cells, characterized in that, The procedure includes the following steps: S1. Inoculating Chinese hamster ovary cells into an initial culture medium for culture. The initial culture medium consists of basal culture medium, cell passage medium, (1.8-2.2) g / L glucose, (13-17) mM L-lactate sodium, and (0.6-1) g / L galactose. The amount of cell inoculation medium added is 7-11% of the initial culture medium volume, and the pH is 6.76-6.

84. S2. During the growth stage of Chinese hamster ovary cells: after culturing for 48 h, feed culture medium is added daily to maintain the glucose concentration at (1.3-1.7) g / L, the galactose concentration at (0.6-1.0) g / L, and the L-lactate sodium concentration at 14-16 g / L. mM; S3, In the antibody production stage of Chinese hamster ovary cells: lower the culture temperature, add feed medium daily, and maintain the glucose concentration in the medium at (2~2.8) g / L and the galactose concentration at (1.7~2.2) g / L; S4, culture until the cell viability is less than 80%, and harvest the cells.

2. The method for increasing the PD-1 titer produced by Chinese hamster ovary cells according to claim 1, characterized in that, The seeding density of the Chinese hamster ovary cells was (6-8) × 10⁻⁶. 5 Cells / mL.

3. The method for increasing the PD-1 titer produced by Chinese hamster ovary cells according to claim 1, characterized in that, In steps S2 and S3, the feeding medium consists of feeding medium A and feeding medium B. The amount of feeding medium A added is 0.15% of the initial culture medium volume, and the amount of feeding medium B added is 1.5% of the initial culture medium volume.

4. The method for increasing the PD-1 titer produced by Chinese hamster ovary cells according to claim 1, characterized in that, In step S2, the growth phase of Chinese hamster ovary cells was (0~105) h or (0~125) h after cell inoculation.

5. The method for increasing the PD-1 titer produced by Chinese hamster ovary cells according to claim 1, characterized in that, In step S2, the culture temperature is (36.7~37.1)℃.

6. The method for increasing the PD-1 titer produced by Chinese hamster ovary cells according to claim 1, characterized in that, In step S2, glucose concentration in the culture medium was maintained at (1.3~1.7) g / L and galactose concentration at (0.6~1.0) g / L by adding additional glucose and galactose concentrate.

7. The method for increasing the PD-1 titer produced by Chinese hamster ovary cells according to claim 1, characterized in that, In step S2, glucose concentration in the culture medium was maintained at 1.5 g / L and galactose concentration at 1 g / L by adding additional glucose and galactose concentrate.

8. The method for increasing the PD-1 titer produced by Chinese hamster ovary cells according to claim 1, characterized in that, In step S3, the temperature is lowered to (32.8~33.2)℃.

9. The method for increasing the PD-1 titer produced by Chinese hamster ovary cells according to claim 1, characterized in that, In step S3, glucose and galactose concentrates were added to maintain the glucose concentration in the culture medium at (2~2.8) g / L and the galactose concentration at (1.7~2.2) g / L.

10. The method for increasing the PD-1 titer produced by Chinese hamster ovary cells according to claim 1, characterized in that, In step S3, glucose concentration in the culture medium was maintained at 2.4 g / L and galactose concentration at 2.0 g / L by adding additional glucose and galactose concentrate.

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