Composite additive and application thereof in mammalian cell culture

Abstract

The invention provides a composite additive and application thereof in mammalian cell culture, and relates to the technical field of cell culture, the composite additive comprises a pyrimidine nucleoside precursor, vitamin, amino acid, a TCA circulation intermediate product and metal ions; wherein the deoxyuridine is used as a pyrimidine nucleoside precursor and can promote cell DNA synthesis so as to improve the peak living cell density; pyridoxine hydrochloride and choline chloride synergistically regulate amino acid metabolism and enhance the antibody synthesis efficiency; magnesium is an activating agent of mitochondrial key enzymes such as pyruvate dehydrogenase and citrate synthase and can improve the ATP generation efficiency and provide more energy for antibody synthesis, alpha-ketoglutaric acid optimizes tricarboxylic acid circulation to supply energy, the cell activity in a low-iron environment is guaranteed, finally, multi-channel synergistic interaction is achieved, the cell density and the antibody yield are improved, and the product quality is improved. And the key quality attribute of the antibody is not changed. The technical problem that a culture medium in the prior art cannot coordinate the balance of cell density, antibody yield and charge heterogeneity in a low-iron environment is solved.

Description

Technical Field

[0001] This invention relates to the field of cell culture technology, and in particular to a composite additive and its application in mammalian cell culture. Background Technology

[0002] Mammalian cells (such as CHO cells) are the primary host for recombinant antibody production, and the iron content in their culture medium has a crucial impact on production efficiency and product quality. In existing technologies, high-ferric media can promote cell proliferation through iron-mediated metabolic pathways, thereby increasing antibody yield. However, ferric ions easily induce oxidative stress through the Fenton reaction, leading to an increase in the content of acidic antibody variants and significantly reducing the effective antibody yield. While low-ferric media can control the content of acidic variants at a lower level, insufficient iron supply inhibits key pathways such as nucleotide synthesis and energy metabolism, resulting in a significant reduction in peak viable cell density, with antibody yield only 60%-70% of that in high-ferric systems.

[0003] To address the aforementioned contradictions, existing technologies have explored relevant approaches. For example, Chinese patent application number 202411964806.2 discloses a method for improving cell growth and expression by altering the form of iron in the culture medium. This method improves cell growth by changing the form of the iron source (e.g., FeCl3) and combining it with three-dimensional culture materials. However, this approach still relies on iron source adjustment and cannot completely eliminate the risk of acidic variants. Other studies have increased cell density by adding a single nucleoside (e.g., deoxyuridine), but the impact on antibody charge heterogeneity has not been verified. Therefore, achieving a balance between cell density, antibody yield, and charge heterogeneity without increasing iron content remains a pressing technical challenge in this field.

[0004] In view of this, the present invention is hereby proposed. Summary of the Invention

[0005] One of the objectives of this invention is to provide a composite additive to solve the technical problem in the prior art that the culture medium cannot coordinate the balance between cell density, antibody yield and charge heterogeneity under low iron conditions.

[0006] A second objective of this invention is to provide the application of the above-mentioned composite additive in mammalian cell culture or in the preparation of products for culturing mammalian cells.

[0007] The third objective of this invention is to provide a reagent.

[0008] The fourth objective of this invention is to provide a reagent kit.

[0009] The fifth objective of this invention is to provide a method for culturing mammalian cells.

[0010] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted: In a first aspect, the present invention provides a composite additive comprising the following components at concentrations: 0.005-0.05 g / L of deoxyuridine, 0.6-1.2 g / L of asparagine monohydrate, 0.001-0.01 g / L of pyridoxine hydrochloride, 0.3-0.6 g / L of magnesium chloride hexahydrate, 1-1.5 g / L of aspartic acid, 0.5-1 g / L of α-ketoglutarate, 0.0001-0.002 g / L of riboflavin, and 0.01-0.1 g / L of choline chloride.

[0011] Furthermore, the concentration of the deoxyuridine is 0.01 g / L; Preferably, the concentration of the asparagine monohydrate is 0.6 g / L; Preferably, the concentration of pyridoxine hydrochloride is 0.003 g / L; Preferably, the concentration of magnesium chloride hexahydrate is 0.3 g / L; Preferably, the concentration of aspartic acid is 1 g / L; Preferably, the concentration of α-ketoglutaric acid is 0.5 g / L; Preferably, the concentration of riboflavin is 0.0001 g / L; Preferably, the concentration of choline chloride is 0.06 g / L.

[0012] Furthermore, it includes the following components at the following concentrations: 0.01 g / L deoxyuridine, 0.6 g / L asparagine monohydrate, 0.003 g / L pyridoxine hydrochloride, 0.3 g / L magnesium chloride hexahydrate, 1 g / L aspartic acid, 0.5 g / L α-ketoglutarate, 0.0001 g / L riboflavin, and 0.06 g / L choline chloride.

[0013] Secondly, the present invention provides the application of the above-mentioned composite additive in mammalian cell culture or in the preparation of products for culturing mammalian cells.

[0014] Furthermore, the mammalian cells include CHO cells; Furthermore, the product includes reagents or kits.

[0015] Thirdly, the present invention provides a reagent containing the above-mentioned composite additive.

[0016] Fourthly, the present invention provides a kit comprising the above-described composite additive or the above-described reagent.

[0017] Furthermore, it also includes a low-iron culture medium; wherein the iron content in the low-iron culture medium is ≤2000μg / L.

[0018] Fifthly, the present invention provides a method for culturing mammalian cells, comprising adding the above-mentioned composite additive to a mammalian cell culture medium for culturing; or culturing mammalian cells using the above-mentioned reagents or the above-mentioned kits.

[0019] This invention provides a composite additive comprising a pyrimidine nucleoside precursor, vitamins, amino acids, TCA cycle intermediates, and metal ions. Deoxyuridine, as a pyrimidine nucleoside precursor, promotes cellular DNA synthesis to increase peak viable cell density. Pyridoxine hydrochloride and choline chloride synergistically regulate amino acid metabolism, enhancing antibody synthesis efficiency. Magnesium, an activator of key mitochondrial enzymes such as pyruvate dehydrogenase and citrate synthase, enhances ATP production efficiency, providing more energy for antibody synthesis. α-Ketoglutarate optimizes the tricarboxylic acid cycle energy supply, ensuring cell viability under low-iron conditions. Ultimately, this achieves multi-pathway synergistic enhancement, increasing cell density and antibody yield without altering key antibody quality properties. This solves the technical problem in existing technologies where culture media cannot balance cell density, antibody yield, and charge heterogeneity under low-iron conditions. Attached Figure Description

[0020] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0021] Figure 1 Statistical results of viable cell density at different culture times for the composite additive provided by this invention in mammalian cell culture applications; Figure 2 Statistical results of cell viability at different culture times for the composite additive provided by this invention in mammalian cell culture applications; Figure 3 Statistical results of antibody concentration on day 14 in mammalian cell culture application of the composite additive provided by the present invention; Figure 4 Statistical results of cation exchange (CEX) chromatography spectra of target antibodies in mammalian cell culture applications of the composite additive provided by this invention. Detailed Implementation

[0022] Unless otherwise defined herein, the scientific and technical terms used in conjunction with this invention shall have the meanings commonly understood by one of ordinary skill in the art. The meaning and scope of terms shall be clear; however, in any case of potential ambiguity, the definitions provided herein shall prevail over any dictionary or foreign definitions. In this application, unless otherwise stated, the use of "or" means "and / or". Furthermore, the use of the term "comprising" and other forms is non-limiting.

[0023] Generally, the nomenclature and techniques used in cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization, together with those described herein, are those well-known and commonly used in the art. Unless otherwise stated, the methods and techniques of the present invention are generally carried out according to conventional methods well-known in the art and described in various general and more specific references, which are cited and discussed throughout this specification. Enzymatic reactions and purification techniques are carried out according to the manufacturer's instructions, as commonly practiced in the art, or as described herein. The nomenclature, laboratory procedures, and techniques used in analytical chemistry, synthetic organic chemistry, and medical and medicinal chemistry, together with those described herein, are those well-known and commonly used in the art.

[0024] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] One aspect of this invention provides a composite additive comprising the following components at the following concentrations: 0.005-0.05 g / L of deoxyuridine, 0.6-1.2 g / L of asparagine monohydrate, 0.001-0.01 g / L of pyridoxine hydrochloride, 0.3-0.6 g / L of magnesium chloride hexahydrate, 1-1.5 g / L of aspartic acid, 0.5-1 g / L of α-ketoglutarate, 0.0001-0.002 g / L of riboflavin, and 0.01-0.1 g / L of choline chloride.

[0026] This compound additive contains pyrimidine nucleoside precursors, vitamins, amino acids, TCA cycle intermediates, and metal ions. Deoxyuridine, as a pyrimidine nucleoside precursor, promotes cellular DNA synthesis to increase peak viable cell density. Pyridoxine hydrochloride and choline chloride synergistically regulate amino acid metabolism, enhancing antibody synthesis efficiency. Magnesium, an activator of key mitochondrial enzymes such as pyruvate dehydrogenase and citrate synthase, increases ATP production efficiency, providing more energy for antibody synthesis. α-Ketoglutarate optimizes the tricarboxylic acid cycle energy supply, ensuring cell viability under low-iron conditions. Ultimately, this multi-pathway synergistic effect increases cell density and antibody yield without altering key antibody quality properties. It solves the technical problem in existing technologies where culture media cannot balance cell density, antibody yield, and charge heterogeneity under low-iron conditions.

[0027] The concentration of deoxyuridine can be, but is not limited to, 0.005 g / L, 0.01 g / L, 0.015 g / L, 0.02 g / L, 0.025 g / L, 0.03 g / L, 0.035 g / L, 0.04 g / L, 0.045 g / L or 0.05 g / L, or any concentration between 0.005 and 0.05 g / L, preferably 0.01 g / L.

[0028] The concentration of asparagine monohydrate can be, but is not limited to, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, 1.0 g / L, 1.1 g / L or 1.2 g / L, or any concentration between 0.6 and 1.2 g / L, preferably 0.6 g / L.

[0029] The concentration of pyridoxine hydrochloride can be, but is not limited to, 0.001 g / L, 0.002 g / L, 0.003 g / L, 0.004 g / L, 0.005 g / L, 0.006 g / L, 0.007 g / L, 0.008 g / L, 0.009 g / L or 0.01 g / L, or any concentration between 0.001 and 0.01 g / L, preferably 0.003 g / L.

[0030] The concentration of the magnesium chloride hexahydrate can be, but is not limited to, 0.3 g / L, 0.35 g / L, 0.4 g / L, 0.45 g / L, 0.5 g / L, 0.55 g / L or 0.6 g / L, or any concentration between 0.3 and 0.6 g / L, preferably 0.3 g / L.

[0031] The concentration of aspartic acid can be, but is not limited to, 1 g / L, 1.05 g / L, 1.1 g / L, 1.15 g / L, 1.2 g / L, 1.25 g / L, 1.3 g / L, 1.35 g / L, 1.4 g / L, 1.45 g / L or 1.5 g / L, or any concentration between 1 and 1.5 g / L, preferably 1 g / L.

[0032] The concentration of the α-ketoglutaric acid can be, but is not limited to, 0.5 g / L, 0.55 g / L, 0.6 g / L, 0.65 g / L, 0.7 g / L, 0.75 g / L, 0.8 g / L, 0.85 g / L, 0.9 g / L, 0.95 g / L, or 1.0 g / L, or any concentration between 0.5 and 1 g / L, preferably 0.5 g / L.

[0033] The concentration of riboflavin can be, but is not limited to, 0.0001 g / L, 0.0002 g / L, 0.0004 g / L, 0.0006 g / L, 0.0008 g / L, 0.001 g / L, 0.0012 g / L, 0.0014 g / L, 0.0016 g / L, 0.0018 g / L, or 0.002 g / L, or any concentration between 0.0001 and 0.002 g / L, preferably 0.0001 g / L.

[0034] The concentration of choline chloride can be, but is not limited to, 0.01 g / L, 0.02 g / L, 0.03 g / L, 0.04 g / L, 0.05 g / L, 0.06 g / L, 0.07 g / L, 0.08 g / L, 0.09 g / L or 0.1 g / L, or any concentration between 0.01 and 0.1 g / L, preferably 0.06 g / L.

[0035] The concentrations of each component can be adjusted according to different low-iron culture media and cell requirements. In some specific embodiments, the following components are included in concentration: 0.01 g / L deoxyuridine, 0.6 g / L asparagine monohydrate, 0.003 g / L pyridoxine hydrochloride, 0.3 g / L magnesium chloride hexahydrate, 1 g / L aspartic acid, 0.5 g / L α-ketoglutarate, 0.0001 g / L riboflavin, and 0.06 g / L choline chloride.

[0036] According to another aspect of the invention, the use of the above-described composite additive in mammalian cell culture or in the preparation of products for culturing mammalian cells is also provided.

[0037] Adding a compound additive to low-iron culture medium in mammalian cell culture can increase the cell density to a level comparable to that of high-iron culture medium; at the same time, it increases the antibody concentration, while reducing the content of acidic variants. This solves the technical problem of high antibody production but low quality in high-iron culture medium and high antibody quality but low yield in low-iron culture medium in mammalian cell culture.

[0038] In some specific embodiments, the mammalian cell CHO cell is mentioned.

[0039] In some specific embodiments, the product includes reagents or kits.

[0040] According to another aspect of the present invention, a reagent containing the above-described composite additive is also provided.

[0041] According to another aspect of the present invention, a kit is also provided, comprising the above-described composite additive or the above-described reagent.

[0042] In some specific embodiments, a low-iron culture medium is also included; the iron content in the low-iron culture medium is ≤2000μg / L.

[0043] According to another aspect of the present invention, a method for culturing mammalian cells is also provided, comprising adding the above-described composite additive to a mammalian cell culture medium for culturing; or culturing mammalian cells using the above-described reagents or the above-described kits.

[0044] The concentration of each component in the composite additive refers to the concentration of each component in the culture system itself, which is independent of the concentration of the same component in other reagents in the culture system.

[0045] The present invention will be further illustrated by the following examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or purchased directly from the market.

[0046] I. A composite additive, the components and concentrations of which are shown in Table 1, in g / L.

[0047] Table 1

[0048] II. Culture of mammalian cells Examples 4-6 A method for culturing mammalian cells, comprising adding a compound additive to a culture medium to culture the cell line, specifically following these steps: 1. Cell line: CHO-K1 cells (provided by Shanghai Maibang Biotechnology Co., Ltd. CLD (cell line construction platform)) expressing monoclonal antibodies.

[0049] 2. Culture media: Maibang chemical composition limited low iron medium (model: CHO MaxA5 minus14, iron content ≤2000ug / L); Maibang fed medium MaxFA and MaxFB.

[0050] 3. Composite additives: Select the composite additives from Examples 1 to 3 respectively, dissolve them in ultrapure water, filter to remove bacteria (0.22μm filter membrane), and store at 4℃ for later use.

[0051] 4. Culture conditions are: 36.5℃, 140rpm, 5%CO2, 90% humidity, cell density 0.5E6 for passage culture, passage once every three days, and after two to three passages, the cells are ready for use. 5. Seed CHO cells at a density of 1E6 and culture them in a shaker; 6. On days 2, 4, 6, 8, 10, and 12 of cell culture, add 4%, 4%, 6%, 6%, 6%, and % of the initial culture volume of MaxFA medium from Maibang Catalog, and 0.4%, 0.4%, 0.6%, 0.6%, 0.6%, and 0.6% of the initial culture volume of MaxFB medium from Maibang Catalog, respectively. On days 2, 4, 6, 8, 10, and 12, take 200 μL of sample from each shake flask and record cell density, cell viability, glucose, lactate, and osmolarity parameters, ensuring that residual glucose is above 2 g / L.

[0052] 7. After culturing for 14 days, collect the cell supernatant and detect antibody protein production. The results are as follows: Figures 1-4 As shown.

[0053] Comparative Example 2 Unlike Example 4, no compound additives were added.

[0054] Comparative Example 3 Unlike Example 4, the composite additive used in Comparative Example 1 consisted of only 0.01 g / L of deoxyuridine, which was dissolved in ultrapure water, filtered to remove bacteria (0.22 μm filter membrane), and stored at 4°C for later use.

[0055] Comparative Example 4 Unlike Example 4, no compound additives were added, and high-iron culture medium was used instead of low-iron culture medium. High-iron control culture medium (model: CHO MaxA5, iron content ≥50000ug / L) was used.

[0056] Figure 1 The results showed that, compared with the low-iron control group (23.5 × 10⁻⁶) without the addition of compound additives in Comparative Example 2, the iron content was significantly lower. 6 Compared to the control group (cells / mL), the peak viable cell density in Examples 4-6 was significantly increased (P<0.001), and significantly higher than the 27.4×10⁻⁶ in the control group (comparative Example 3, single deoxyuridine addition group). 6 The peak viable cell density was 35.8 × 10⁶ cells / mL, with Example 4 showing the best results. 6 The cells / mL was increased by about 50% compared to control group 2.

[0057] The peak viable cell density of CHO cells in Example 4 reached 35.8 × 10⁻⁶. 6 cells / mL, compared with Comparative Example 4 high-iron medium (35×10⁻⁶ cells / mL),6 The cell counts (cells / mL) were basically the same in Examples 5 and 6; the peak viable cell density was lower than that of Comparative Example 4, but still reached 29.4 × 10⁻⁶ cells / mL. 6 cells / mL, 32.1×10 6 The number of cells / mL was significantly higher than that of Comparative Example 2 (23.5 × 10⁻⁶). 6 cells / mL.

[0058] Figure 2 The results showed that the survival rate of Examples 4-6 remained above 97% at harvest D14, which was significantly better than the 77.6% survival rate of Comparative Example 4.

[0059] Figure 3 Antibody concentration data showed that, using Protein A affinity chromatography, the antibody yields of Examples 4-6 reached approximately 2.8 g / L, a significant increase of about 50% compared to 1.76 g / L in Comparative Examples 2 and 3 (P < 0.001), indicating a significant difference. The antibody yields of Examples 4-6 were close to those of Comparative Example 4.

[0060] Figure 4 Data on the acid variants, detected by ion-exchange chromatography, showed that the content of acid variants in Examples 4-6 was not significantly different from that in Comparative Examples 2 and 3, and was much lower than that in Comparative Example 4 (22.07%). This indicates that the eight added components partially replaced the function of iron, synergistically increasing antibody yield without altering antibody quality properties.

[0061] The data above show that Examples 4-6 can increase cell density and antibody production and reduce charge heterogeneity in a low-iron environment, thereby achieving the goal of balancing cell density, antibody production and reducing charge heterogeneity.

[0062] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A composite additive, characterized in that, The components include the following concentrations: 0.005–0.05 g / L of deoxyuridine, 0.6–1.2 g / L of asparagine monohydrate, 0.001–0.01 g / L of pyridoxine hydrochloride, 0.3–0.6 g / L of magnesium chloride hexahydrate, 1–1.5 g / L of aspartic acid, 0.5–1 g / L of α-ketoglutarate, 0.0001–0.002 g / L of riboflavin, and 0.01–0.1 g / L of choline chloride.

2. The composite additive according to claim 1, characterized in that, The concentration of the deoxyuridine was 0.01 g / L; Preferably, the concentration of the asparagine monohydrate is 0.6 g / L; Preferably, the concentration of pyridoxine hydrochloride is 0.003 g / L; Preferably, the concentration of magnesium chloride hexahydrate is 0.3 g / L; Preferably, the concentration of aspartic acid is 1 g / L; Preferably, the concentration of α-ketoglutaric acid is 0.5 g / L; Preferably, the concentration of riboflavin is 0.0001 g / L; Preferably, the concentration of choline chloride is 0.06 g / L.

3. The composite additive according to claim 1 or 2, characterized in that, The components include the following concentrations: 0.01 g / L deoxyuridine, 0.6 g / L asparagine monohydrate, 0.003 g / L pyridoxine hydrochloride, 0.3 g / L magnesium chloride hexahydrate, 1 g / L aspartic acid, 0.5 g / L α-ketoglutarate, 0.0001 g / L riboflavin, and 0.06 g / L choline chloride.

4. The use of the composite additive according to any one of claims 1 to 3 in mammalian cell culture or in the preparation of products for culturing mammalian cells.

5. The application according to claim 4, characterized in that, The mammalian cells include CHO cells.

6. The application according to claim 4, characterized in that, The products include reagents or kits.

7. A reagent, characterized in that, It contains the composite additive as described in claim 1 or 2.

8. A reagent kit, characterized in that, Includes the composite additive as described in claim 1 or 2, or the reagent as described in claim 7.

9. The reagent kit according to claim 8, characterized in that, It also includes a low-iron culture medium; the iron content in the low-iron culture medium is ≤2000μg / L.

10. A method for culturing mammalian cells, characterized in that, This includes adding the composite additive of claim 1 or 2 to a mammalian cell culture medium for culturing; or culturing mammalian cells using the reagent of claim 7 or the kit of claim 8 or 9.

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