CHO cell culture medium additive formula and culture method

Abstract

The invention discloses a CHO cell culture medium additive formula and a culture method. The CHO cell culture medium additive formula can be used for an upstream stage of in-vitro CHO cell culture and can remarkably reduce the proportion of Man5 in antibody glycosylation. The additives comprise an additive G1, an additive G2 and an additive G3; the additive G1 is a mixed mother solution with the concentration of 5g / L prepared from uridine, cytidine and hypoxanthine; the additive G2 is a mixed mother solution with the concentration of 1g / L prepared from sodium selenite and cadmium sulfate; the additive G3 is a mixed mother solution with the concentration of 100g / L prepared from asparagine, glutathione and sodium pyruvate. Compared with the prior art, 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. Under the condition that cell growth and expression are not affected, the Man5 proportion in antibody glycosylation can be remarkably reduced.

Description

Technical Field

[0001] This invention belongs to the field of biological cell culture technology, specifically relating to a CHO cell culture medium additive formulation and culture method. Background Technology

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

[0003] In the research and development and production of therapeutic antibodies, antibody glycosylation, as a key post-translational modification, plays a decisive role in many antibody properties, including biological activity, efficacy, stability, immunogenicity, clearance rate, antibody-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Mammalian cell-produced IgG antibodies typically contain low levels of high-mannose glycoforms (Man5-9), generally less than 5%, while the most common glycans are complex glycoforms G0F, G1F, and G2F. Mannose-5 (Man5), as a type of high-mannose glycoform, has attracted considerable attention in the field of antibody glycosylation. While the content of Man5 glycoform in human IgG is usually low, in therapeutic antibodies, high levels of high-mannose glycoforms, especially Man5, often pose a concern due to the uncertain impact on antibody clearance rate, immunogenicity, and therapeutic efficacy. Studies have shown that in mouse experiments, antibodies carrying the Man5 glycoform produced by the Lec1 glycosylated mutant Chinese hamster ovary (CHO) cell line exhibit defects in complement activation, reduced affinity for FcγRI, and a shortened in vivo half-life. However, in another mouse model, antibodies containing the high-mannose glycoform, while not showing a shortened early half-life, exhibited increased β-phase clearance. Recent research further indicates that, because high-mannose (Man6-9) can be converted to Man5, in humans, Man5 and higher-order mannose glycoforms show comparable clearance rates to antibodies carrying complex glycans. The presence of mannosidase in human serum is the cause of glycan cleavage, not a factor contributing to differences in antibody clearance. These contradictory reports clearly demonstrate that the clearance mechanism of antibodies carrying Man5 or other high-mannose glycoforms is not yet fully understood. In the process of producing therapeutic antibodies through cell culture, numerous factors, such as cell line characteristics, process control parameters, and cell culture medium composition, can influence glycosylation, thereby affecting the level of Man5 in the antibody. For example, early development studies of therapeutic antibodies produced from the CHO cell line (cell line A) revealed that the Man5 level in the antibody increased throughout the cell culture production process with increasing osmotic pressure of the cell culture medium and prolonged culture duration. For cell line A, during fed-batch processing, a combination of high basal and fed-batch osmotic pressure with extended run duration resulted in a more than twofold increase in Man5 glycosylation, from 12% to 28%. Furthermore, supplementing with an appropriate concentration of MnCl2 could reduce the Man5 level in cell line A.

[0004] Although antibodies with high mannose content possess the potential to become therapeutic agents due to their enhanced biological activity—for example, high-mannose antibodies produced by methylphenidate treatment exhibit higher ADCC activity and greater affinity for FcγRIIIa—precisely controlling the levels of glycoforms such as Man5 to ensure the consistency, efficacy, and safety of antibody quality remains a significant challenge for the biopharmaceutical industry in the development and production of therapeutic antibodies. In-depth exploration of the mechanisms by which Man5 glycoform influences antibody glycosylation is crucial for optimizing the performance of therapeutic antibodies and enhancing their clinical application value. Summary of the Invention

[0005] The purpose of this application is to provide a CHO cell culture medium additive formulation and culture method, which can be used in the upstream stage of in vitro CHO cell culture to significantly reduce the proportion of Man5 in antibody glycosylation. A CHO cell culture medium additive formulation includes inorganic salts, vitamins, trace elements, amino acids, carbon source, nitrogen source, and water. Amino acids are the raw materials for cell protein synthesis, vitamins participate in various intracellular metabolic reactions, carbohydrates provide energy, and inorganic salts maintain cell osmotic pressure and acid-base balance. The formulation can be used in the upstream stage of in vitro CHO cell culture, and the substances involved include uridine, cytidine, hypoxanthine, sodium selenite, cadmium sulfate, asparagine, glutathione, and sodium pyruvate.

[0006] The preparation method is as follows: (1) Add 0.5g of uridine, cytidine, and hypoxanthine to 8mL of ultrapure water, dissolve completely, and bring the volume to 10mL. Filter aseptically. The concentration of the stock solution of this mixed solution is 5g / L, and it is named Additive G1; (2) Add 0.01g of sodium selenite and cadmium sulfate to 8mL of ultrapure water, dissolve completely, and bring the volume to 10mL. Filter aseptically. The concentration of the stock solution of this mixed solution is 1g / L, and it is named Additive G2; (3) Add 1g of asparagine, glutathione, and sodium pyruvate to 8mL of ultrapure water, dissolve completely, and bring the volume to 10mL. Filter aseptically. The concentration of the stock solution of this mixed solution is 100g / L, and it is named Additive G3. The additive formulation is used in the upstream stage of in vitro CHO cell culture, wherein the CHO cells include CHOK1 cells.

[0007] Furthermore, the additive includes the following components and concentration ranges: The additive G1 includes: uridine: 0.5-4 mg / L, cytidine: 0.5-4 mg / L, and hypoxanthine: 0.5-4 mg / L; The additive G2 includes: sodium selenite: 0.0001-0.002 mg / L, cadmium sulfate: 0.0001-0.002 mg / L; The additive G3 includes: asparagine: 0.5-2 g / L, glutathione: 0.5-2 g / L, and sodium pyruvate: 0.5-2 g / L.

[0008] Furthermore, the optimal component concentrations of the additives are: 2 mg / L uridine, 2 mg / L cytidine, 2 mg / L hypoxanthine, 0.001 g / L sodium selenite, 0.001 g / L cadmium sulfate, 1 g / L asparagine, 1 g / L glutathione, and 1 g / L sodium pyruvate.

[0009] Furthermore, the CHO cell culture method with the aforementioned culture medium additive formulation includes the following steps: (1) Culture medium preparation: Add 25 mL of Donin platform basal culture medium to a 125 mL shake flask and place it on a shaker at 37℃ for 30-60 minutes; (2) Cell resuscitation: CHO cells frozen at -230℃ in liquid nitrogen were rapidly thawed in a water bath at 37℃ and then added to the culture medium in step (1); (3) Cell culture: Place the shake flasks at 36.5~37℃, 5%~10% CO2, and 110~130 rpm for culture; (4) Subculture: Subculture every 2-4 days to maintain the logarithmic growth phase of cells. The seeding density for subculture is 0.2~1.0×10⁶ cells / year. 6 cells / mL; (5) Fed-Batch culture: When the cell density reaches 4.5~6.0×10⁻⁶, 6 When cells / mL and viability ≥95% and PDT difference ≤4 hours, inoculate into culture medium containing additives for fed-batch culture; during Fed-Batch culture: shaker temperature 36.5-37℃, rotation speed 130±20 rpm, CO2 concentration 5%-10%; inoculation density 0.4-1.2×10⁻⁶ cells / mL and viability ≥95%, and PDT difference ≤4 hours, inoculate into culture medium containing additives for fed-batch culture; 6 cells / mL, with a deviation of <3 hours after PDT stabilization; cultured until day 15, and then centrifuged at 4000 rpm for 30 minutes to collect the supernatant for N-Glycan glycoform detection.

[0010] Furthermore, the quality detection includes N-Glycan glycosylation distribution detection, which is performed using HPLC liquid chromatography, with the supernatant purified before detection.

[0011] The method of use is as follows: Before the cells are cultured in a Fed-Batch, different concentrations and combinations of additives are added to the inoculation medium according to the experimental method.

[0012] The method of culturing CHO cells used in in vitro cell culture: The incubator parameters were set as follows: 36.5℃, 8% carbon dioxide, 130 rpm; The cell seeding density was 0.5 ± 0.05 × 10⁶ cells / mL; The cultivation period is 15 days. Sampling period: Day 0 / 3 / 5 / 7 / 9 / 11 / 13; Nutrient supplementation status: D3 / 5 / 7 / 9 / 11 / 13, supplemented with 2%-8% of Domino's platform feed medium and 0.2%-0.8% of Domino's platform ultra-concentrated feed medium; Glucose replenishment: On days 3 / 5 / 7 / 9 / 11 / 13, glucose in the culture system was replenished to 3-12 g / L. Detection items: live cell density, cell viability, cell diameter, glucose, lactate, antibody concentration, and antibody glycosylation distribution.

[0013] Compared with the prior art, the advantages of this application are: (1) The serum-free culture medium for culturing CHO cells provided by the present invention has simple composition, low cost, no protein and no serum.

[0014] (2) The present invention can significantly reduce the proportion of Man5 in antibody glycosylation without affecting cell growth and expression.

[0015] (3) The additive formulation disclosed in this invention is simple to prepare and easy to use, and can be widely used in CHO cell culture in industrial production processes. Attached Figure Description

[0016] Figure 1 This is a glycosylation distribution diagram of the control group in Phase 1.

[0017] Figure 2 This is a glycosylation distribution diagram of Example 1 in Stage 1.

[0018] Figure 3 This is a glycosylation distribution diagram for Example 2 in Stage 1.

[0019] Figure 4 This is a glycosylation distribution diagram for Example 3 in Stage 1.

[0020] Figure 5 This is a glycosylation distribution diagram of Example 1 in Stage 2.

[0021] Figure 6 This is a glycosylation distribution diagram for Example 2 in Stage 2.

[0022] Figure 7 This is a glycosylation distribution diagram for Example 3 in Stage 2. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application. Example

[0024] Stage 1: Take one CHO cell cryopreservation tube from the liquid nitrogen tank, shake it in a 37°C water bath to dissolve it, add it to a 125mL shake flask containing 20mL of basal culture medium for the Dolin platform, and mix well. 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 basal medium containing additives and then transferred to 125 mL shake flasks. The cells were then cultured in a shaker at 130 rpm, 8% CO2, and 37°C. On Days 0, 3, 5, 7, 9, 11, 13, and 15, samples were taken to detect cell growth status and record various growth parameters. On Days 3, 5, 7, 9, 11, and 13, 4% DF33 and 0.4% DNFeed B4 were added, respectively. On Days 3, 5, 5, 7, 9, 11, and 13, glucose residue was detected, and the glucose concentration of the culture system was supplemented to 6 g / L using a stock solution with a concentration of 200-400 g / L.

[0025] Phase 1: Cell culture was performed with individual supplements of G1 (2 mg / L), G2 (0.001 mg / L), and G3 (1 g / L), respectively; the control group received no supplements. Cell culture protocols and results are shown in Tables 1-6. Figure 1 —As shown in 7: Table 1. Main Implementation Plan for Phase 1 Cell Culture

[0026] Table 2 Concentrations and Addition Methods of Additives in Stage 1

[0027] Table 3 Final glycosylation distribution of each experimental group in Phase 1

[0028] Tables 1-3 and Figure 1 —4 The results show that: Based on the control example, Example 1, Example 2, and Example 3 of Phase 1: Adding G1 (uridine, cytidine, hypoxanthine), G2 (sodium selenite, cadmium sulfate), and G3 (asparagine, glutathione, sodium pyruvate) to the cell culture medium, respectively, all reduced the proportion of Man5 in antibody glycosylation to varying degrees. Adding G1, G2, or G3 individually all reduced the Man5 proportion, with the G2 group showing the largest reduction (Man5 decreased from 5.15% to 4.083%).

[0029] Phase Two: Cell culture procedures were performed following the methods described in Phase One. Based on the data from Phase One, eight substances—uridine, cytidine, hypoxanthine, sodium selenite, cadmium sulfate, asparagine, glutathione, and sodium pyruvate—were used in combination during the cell culture phase, with further adjustments to the concentrations of each substance. Additives G1, G2, and G3 were used in combination, and three concentration groups were established: a low concentration group (0.5 mg / L / 0.0001 mg / L / 0.5 g / L), a medium concentration group (2 mg / L / 0.001 mg / L / 1 g / L), and a high concentration group (4 mg / L / 0.002 mg / L / 2 g / L). The experimental protocol and the methods of adding each substance are shown in Tables 4-6. Table 4. Main Implementation Scheme for Two-Stage Cell Culture

[0030] Table 5. Concentrations and Addition Methods of Additives in Stage Two

[0031] Table 6 Final glycosylation distribution of each experimental group in Phase 2

[0032] Table 4-6 and Figure 5-7 The results show that: According to the results of Phase 2: Example 1, after the substances were integrated according to the concentration in Phase 1, the proportion of Man5 in the medium concentration group (G1 2 mg / L + G2 0.001 mg / L + G3 1 g / L) could be reduced from 5.15% to 3.115%, a decrease of 39.5%.

[0033] According to the results of Phase 2: Example 2, after the concentration of each substance was reduced, its Man 5 rose back to 5.012%, which was not significantly different from the control (5.15%). Reducing the concentration of each substance could not achieve a reduction in Man 5. Based on the results of Phase 2: Example 3, after the concentration of each substance was further increased, its Man5 did not show a further decreasing trend. Therefore, the concentration of each additive that reduced Man5 was determined to be the medium concentration group used in Phase 2: Example 1.

[0034] In summary, the additive formulations and additive groups that can significantly reduce the proportion of Man5 are as follows: G1: 2 mg / L uridine, 2 mg / L cytidine and 2 mg / L hypoxanthine; G2: 0.001 g / L sodium selenite and 0.001 g / L cadmium sulfate; G3: 1 g / L asparagine, 1 g / L glutathione and 1 g / L sodium pyruvate.

[0035] The additive formulation provided by this invention, through the scientific ratio and combined use of G1, G2, and G3, can significantly reduce the Man5 glycosylation ratio of antibodies in CHO cell culture, and the optimal concentration combination has a stable regulatory effect, providing a practical solution for glycosylation optimization in the production of therapeutic antibodies.

[0036] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A CHO cell culture medium additive formulation, characterized in that, The culture medium includes inorganic salts, vitamins, trace elements, amino acids, carbon source, nitrogen source, and water; the additives include additive G1, additive G2, and additive G3. Additive G1 is a mixed stock solution prepared from uridine, cytidine, and hypoxanthine at a concentration of 5 g / L; additive G2 is a mixed stock solution prepared from sodium selenite and cadmium sulfate at a concentration of 1 g / L; and additive G3 is a mixed stock solution prepared from asparagine, glutathione, and sodium pyruvate at a concentration of 100 g / L.

2. The CHO cell culture medium additive formulation according to claim 1, characterized in that, The additive includes the following components and concentration ranges: The additive G1 includes: uridine: 0.5-4 mg / L, cytidine: 0.5-4 mg / L, and hypoxanthine: 0.5-4 mg / L; The additive G2 includes: sodium selenite: 0.0001-0.002 mg / L, cadmium sulfate: 0.0001-0.002 mg / L; The additive G3 includes: asparagine: 0.5-2 g / L, glutathione: 0.5-2 g / L, and sodium pyruvate: 0.5-2 g / L.

3. The CHO cell culture medium additive formulation according to claim 1, characterized in that, The specific component concentrations of the additives are: 2 mg / L uridine, 2 mg / L cytidine, 2 mg / L hypoxanthine, 0.001 g / L sodium selenite, 0.001 g / L cadmium sulfate, 1 g / L asparagine, 1 g / L glutathione, and 1 g / L sodium pyruvate.

4. A method for culturing CHO cells using the culture medium additive formulation of claim 1, characterized in that, Includes the following steps: (1) Culture medium preparation: Add 25 mL of Donin platform basal culture medium to a 125 mL shake flask and place it on a shaker at 37℃ for 30-60 minutes; (2) Cell resuscitation: CHO cells frozen at -230℃ in liquid nitrogen were rapidly thawed in a water bath at 37℃ and then added to the culture medium in step (1); (3) Cell culture: Place the shake flasks at 36.5~37℃, 5%~10% CO2, and 110~130 rpm for culture; (4) Subculture: Subculture every 2-4 days to maintain the logarithmic growth phase of cells. The seeding density for subculture is 0.2~1.0×10⁶ cells / year. 6 cells / mL; (5) Fed-Batch culture: When the cell density reaches 4.5~6.0×10⁻⁶, 6 When cells / mL and viability ≥95% and PDT difference ≤4 hours, they are inoculated into culture medium containing additives for fed-batch culture.

5. The CHO cell culture method according to claim 4, characterized in that, During the Fed-Batch culture process in step (5): the shaker temperature is 36.5-37℃, the rotation speed is 130±20rpm, and the CO2 concentration is 5%-10%; the inoculation density is 0.4-1.2×10⁻⁶. 6 cells / mL, with a deviation of <3 hours after PDT stabilization; cultured until day 15, and then centrifuged at 4000 rpm for 30 minutes to collect the supernatant for N-Glycan glycoform detection.

6. The CHO cell culture method according to claim 4, characterized in that, The quality testing includes N-Glycan glycosylation distribution detection, which is performed using HPLC liquid chromatography. The supernatant is purified before detection.

7. The CHO cell culture method according to claim 4, characterized in that, The additive formulation is used in the upstream stage of in vitro CHO cell culture, wherein the CHO cells include CHOK1 cells.

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