A cell culture additive composition for preventing antibody disulfide bond reduction and use thereof

Abstract

This invention provides a cell culture additive composition for preventing antibody disulfide bond reduction and its application, relating to the biopharmaceutical field. It comprises the following raw materials: a redox regulator, a metal ion chelator, an antioxidant, an adjuvant, and a cell protectant. This invention inhibits antibody disulfide bond reduction from multiple aspects through the synergistic effect of the redox regulator, metal ion chelator, antioxidant, and buffer. Specifically, the redox regulator maintains a suitable redox potential, the metal ion chelator removes harmful metal ions, the antioxidant removes free radicals, and the buffer maintains pH stability. The components work together synergistically to enhance the effect.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceuticals, and in particular to a cell culture additive composition for preventing antibody disulfide bond reduction and its application. Background Technology

[0002] Recombinant monoclonal antibodies have become an important means of treating major diseases such as tumors, autoimmune diseases, and infectious diseases due to their high specificity and low toxicity. Currently, mammalian cells are the main expression system for producing recombinant antibodies, as they can correctly fold proteins and perform post-translational modifications, including the formation of disulfide bonds. The disulfide bonds in antibody molecules are crucial for maintaining their correct spatial structure, biological activity, and stability.

[0003] However, in the later stages of cell culture, due to the enhanced metabolic activity of cells, a large amount of reducing substances accumulate in the culture medium. At the same time, the proteases and reductases released by cell death can also cause the reduction and breakage of antibody disulfide bonds. The reduction of disulfide bonds can lead to abnormal antibody molecular structure, forming low molecular weight fragments, reducing the biological activity and stability of the antibody, and increasing the difficulty and cost of purification. Summary of the Invention

[0004] The purpose of this invention is to provide a cell culture additive composition for preventing antibody disulfide bond reduction and its application, thereby solving the technical problems existing in the prior art.

[0005] To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows:

[0006] A cell culture additive composition for preventing antibody disulfide bond reduction and its application comprises the following components: redox regulator, metal ion chelator, antioxidant, adjuvant, and cell protectant.

[0007] Furthermore, the components are present in the following weight proportions: 2-5 parts of redox regulator, 0.25-1 part of metal ion chelating agent, 0.15-0.45 parts of antioxidant, 20-40 parts of adjuvant, and 1-4 parts of cell protectant.

[0008] Furthermore, the components are present in the following weight proportions: 3 parts redox regulator, 0.75 parts metal ion chelating agent, 0.3 parts antioxidant, 30 parts adjuvant, and 2 parts cell protectant.

[0009] Furthermore, the redox regulator is composed of either glutathione disulfide or cysteine ​​disulfide.

[0010] Furthermore, the metal ion chelating agent is one of ethylenediaminetetraacetic acid, deferoxamine, and ethylene glycol diethyl ether diaminetetraacetic acid.

[0011] Furthermore, the antioxidant is a mixture of lipoic acid and magnesium ascorbate phosphate in a preset ratio.

[0012] Furthermore, the adjuvant is a mixture of oxidized glutathione and L-tyrosine in a preset ratio; the cell protectant is ergothioneine.

[0013] Furthermore, the cell culture additive composition for preventing antibody disulfide bond reduction is used in the preparation of cell culture products for preventing antibody disulfide bond reduction.

[0014] Furthermore, the cell culture product is a cell culture system used for the production of therapeutic antibodies.

[0015] Furthermore, the therapeutic antibodies include anti-tumor antibodies, anti-autoimmune disease antibodies, and antiviral antibodies.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] (i) This invention inhibits the reduction of antibody disulfide bonds from multiple aspects through the synergistic effect of redox regulators, metal ion chelators, antioxidants and buffers; wherein, redox regulators maintain a suitable redox potential, metal ion chelators remove harmful metal ions, antioxidants remove free radicals, and buffers maintain pH stability, and the components work together to enhance the effect.

[0018] (ii) All components in the composition of the present invention are safe additives commonly used in cell culture, and have no negative impact on cell growth and antibody expression. After adding the present invention, the cell viability and antibody expression level are not significantly different from the traditional values, and the disulfide bond reduction rate is significantly reduced. Attached Figure Description

[0019] Figure 1 Electrophoretic image of a cell culture additive composition for preventing antibody disulfide bond reduction and its application disclosed in this invention;

[0020] Figure 2 This is a cell culture additive composition for preventing antibody disulfide bond reduction disclosed in this invention, and its application in cell culture. Detailed Implementation

[0021] To make the objectives, technical solutions, and technical effects of this invention clearer, specific embodiments of this invention are described below. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0022] This embodiment provides a cell culture additive composition for preventing antibody disulfide bond reduction and its application, comprising the following components: redox regulator, metal ion chelator, antioxidant, adjuvant, and cell protectant. Specific effects are as follows:

[0023] Redox regulators: provide controllable oxidative equivalents, precisely stabilize the redox potential of the culture medium, and competitively inhibit the nucleophilic attack of reducing substances on disulfide bonds;

[0024] Metal ion chelating agents: block the Fenton reaction and metal-catalyzed disulfide bond exchange / reduction side reactions; reduce the generation of hydroxyl radicals, avoid metal-mediated antibody fragmentation and incorrect disulfide bond pairing, and improve product purity;

[0025] Antioxidants: scavenge superoxide anions and alleviate cellular oxidative stress; lipoic acid can reversibly switch between oxidative and reduced states, aiding in potential homeostasis;

[0026] Additives: Reduce gas-liquid interface shear forces, prevent antibody conformation exposure, and prolong the high-viability production period of cells;

[0027] Cell protectants: regulate osmotic pressure, inhibit apoptosis and lysis; maintain metabolic homeostasis, and reduce the abnormal release of high intracellular concentrations of GSH and reductases.

[0028] Example 1:

[0029] The components are present in the following weight proportions: 2-5 parts redox regulator, 0.25-1 part metal ion chelating agent, 0.15-0.45 parts antioxidant, 20-40 parts adjuvant, and 1-4 parts cell protectant. In this embodiment, the redox regulator is glutathione disulfide; the metal ion chelating agent is ethylenediaminetetraacetic acid; the antioxidant is a mixture of lipoic acid and magnesium ascorbate phosphate in a predetermined ratio; the adjuvant is a mixture of oxidized glutathione and L-tyrosine in a predetermined ratio; and the cell protectant is ergothioneine.

[0030] S1: Take glutathione disulfide, ethylenediaminetetraacetic acid, antioxidant, adjuvant, and ergothioneine according to the corresponding weight proportions; and pretreat the above materials;

[0031] S2: Add the weighed additives to the container, turn on the stirrer, and stir for 10 minutes at 4℃ and 200r / min to ensure that the additives are fully dispersed; add ethylenediaminetetraacetic acid, antioxidant and ergothioneine, and continue stirring at 300r / min for 10 minutes to ensure that it is evenly mixed with the existing mixture and to avoid uneven local concentrations.

[0032] S3: Add glutathione disulfide and stir continuously to ensure that all components are mixed evenly, forming a cell culture additive composition that prevents the reduction of antibody disulfide bonds.

[0033] S4: Pass the composition through a two-stage 0.22 Polyethersulfone antibacterial filter membrane filtration;

[0034] S5: High performance liquid chromatography is used to analyze the components of the prepared cell culture additive composition and to detect whether the content of each component meets the formulation requirements.

[0035] S6: Package the cell culture additive composition that has passed quality inspection.

[0036] It should be noted that the high-performance liquid chromatography (HPLC) in step S5 is also known as high-pressure liquid chromatography, which is a branch of chromatography. It uses a liquid as the mobile phase and employs a high-pressure delivery system to pump a single solvent or a mixture of solvents or buffer solutions with different polarities into a chromatographic column packed with a stationary phase. After the components are separated in the column, they enter the detector for detection, thereby realizing the analysis of the sample. The HPLC method described is existing technology and will not be described in detail here.

[0037] Example 2:

[0038] The components are present in the following weight proportions: 2-5 parts redox regulator, 0.25-1 part metal ion chelating agent, 0.15-0.45 parts antioxidant, 20-40 parts adjuvant, and 1-4 parts cell protectant. In this embodiment, the redox regulator is cysteine ​​disulfide; the metal ion chelating agent is deferoxamine; the antioxidant is a mixture of lipoic acid and magnesium ascorbate phosphate in a preset ratio; the adjuvant is a mixture of oxidized glutathione and L-tyrosine in a preset ratio; and the cell protectant is ergothioneine.

[0039] S1: Take the corresponding weight proportions of cysteine ​​disulfide, deferoxamine, antioxidant, adjuvant, and ergothioneine; and pretreat the above materials;

[0040] S2: Add the weighed adjuvants to the container, turn on the stirrer, and stir for 15 minutes at 5°C and 350 r / min to fully disperse the ergothioneine; add deferoxamine, antioxidant and cell protectant, and continue stirring for 15 minutes at 450 r / min to ensure that it is evenly mixed with the existing mixture and to avoid uneven local concentrations.

[0041] S3: Add cysteine ​​disulfide and stir continuously to ensure all components are mixed evenly, forming a cell culture additive composition that prevents antibody disulfide bond reduction.

[0042] S4: Pass the composition through a two-stage 0.22 Polyethersulfone antibacterial filter membrane filtration;

[0043] S5: High performance liquid chromatography is used to analyze the components of the prepared cell culture additive composition and to detect whether the content of each component meets the formulation requirements.

[0044] S6: Package the cell culture additive composition that has passed quality inspection.

[0045] It should be noted that the high-performance liquid chromatography (HPLC) in step S5 is also known as high-pressure liquid chromatography, which is a branch of chromatography. It uses a liquid as the mobile phase and employs a high-pressure delivery system to pump a single solvent or a mixture of solvents or buffer solutions with different polarities into a chromatographic column packed with a stationary phase. After the components are separated in the column, they enter the detector for detection, thereby realizing the analysis of the sample. The HPLC method described is existing technology and will not be described in detail here.

[0046] Example 3:

[0047] The components are present in the following weight proportions: 3 parts redox regulator, 0.75 parts metal ion chelating agent, 0.3 parts antioxidant, 30 parts adjuvant, and 2 parts cell protectant. In this embodiment, the metal ion chelating agent is ethylene glycol diethyl ether diaminetetraacetic acid; the antioxidant is a mixture of lipoic acid and magnesium ascorbate phosphate in a preset ratio; the adjuvant is a mixture of oxidized glutathione and L-tyrosine in a preset ratio; and the cell protectant is ergothioneine.

[0048] S1: Take the redox modifier, ethylene glycol diethyl ether diaminetetraacetic acid, antioxidant, ergothioneine, and cell protectant according to the corresponding weight proportions; and pretreat the above materials;

[0049] S2: Add the weighed ergothioneine to the container, turn on the stirrer, and stir for 12.5 minutes at 300 rpm at 4.5°C to fully disperse the adjuvant; add ethylene glycol diethyl ether diaminetetraacetic acid, antioxidant and cell protectant, and continue stirring at 400 rpm for 13 minutes to ensure that it is evenly mixed with the existing mixture and to avoid uneven local concentrations;

[0050] S3: Add redox regulator and stir continuously to ensure all components are mixed evenly, forming a cell culture additive composition that prevents antibody disulfide bond reduction.

[0051] S4: Pass the composition through a two-stage 0.22 Polyethersulfone antibacterial filter membrane filtration;

[0052] S5: High performance liquid chromatography is used to analyze the components of the prepared cell culture additive composition and to detect whether the content of each component meets the formulation requirements.

[0053] S6: Package the cell culture additive composition that has passed quality inspection.

[0054] It should be noted that the high-performance liquid chromatography (HPLC) in step S5 is also known as high-pressure liquid chromatography, which is a branch of chromatography. It uses a liquid as the mobile phase and employs a high-pressure delivery system to pump a single solvent or a mixture of solvents or buffer solutions with different polarities into a chromatographic column packed with a stationary phase. After the components are separated in the column, they enter the detector for detection, thereby realizing the analysis of the sample. The HPLC method described is existing technology and will not be described in detail here.

[0055] Application of a cell culture additive composition for preventing antibody disulfide bond reduction in the preparation of a cell culture product for preventing antibody disulfide bond reduction; wherein the cell culture product is a cell culture system for producing therapeutic antibodies; wherein the therapeutic antibodies include antitumor antibodies, anti-autoimmune disease antibodies, and antiviral antibodies;

[0056] Cell culture experiments:

[0057] Experimental materials: CHO-K1 cells expressing anti-PD-1 monoclonal antibody IgG4;

[0058] Basic culture medium: ;

[0059] Feed-through culture medium: Nutritional additives;

[0060] Additive compositions: the compositions prepared in Examples 1-3, and the additive-free basal culture medium;

[0061] Cultivation equipment: 5L stirred bioreactor;

[0062] Experimental methods:

[0063] 1. Cell resuscitation and expansion: CHO-K1 cells were resuscitated and cultured in basal medium in shake flasks until the cell density reached the target level. Used for inoculating bioreactors;

[0064] 2. Bioreactor culture: Cells are cultured in a bioreactor... The inoculum was planted at a density of 3 L in a 5 L bioreactor, with an initial culture volume of 3 L; the culture temperature was 36-37℃, pH 6.6-7.2, dissolved oxygen 25%-45% air saturation, and stirring speed 140-280 rpm.

[0065] 3. Additive addition: The experimental group was given the additive composition of Examples 1-3 on the 3rd and 5th days of culture, respectively, at a volume of 1% of the total culture medium; the control group was given an equal volume of basal culture medium.

[0066] 4. Fed-feed culture: Starting from day 3 of culture, feed the culture daily. Culture medium, fed at a rate of 3% of the initial culture volume per day;

[0067] 5. Sampling and Detection: Samples were taken daily to detect cell density, cell viability, and antibody expression levels; after culture, the culture medium was harvested, and the antibody was purified. SEC-HPLC purity and non-reducing SDS-PAGE were then tested to calculate the disulfide bond reduction rate.

[0068] Detection method:

[0069] Disulfide bond reduction rate: The percentage of reduced antibody in the total antibody was calculated using non-reducing SDS-PAGE combined with gel imaging analysis; Disulfide bond reduction rate = [Reduced antibody band intensity / (Reduced antibody band intensity + Non-reduced antibody band intensity)] 100%;

[0070] SEC-HPLC: A TSKgel G3000SWxl gel chromatography column was used, with 0.1M phosphate buffer as the mobile phase, a flow rate of 0.5 mL / min, and a detection wavelength of 280 nm. The percentage of monomer peak area was calculated.

[0071] Experimental results:

[0072] Table (I) Effects of different additive compositions on cell culture and antibody quality

[0073] Group Maximum live cell density Cell viability at the end of culture antibody expression level Disulfide bond reduction rate SEC-HPLC monomer purity biological activity control group Example 1 Example 2 Example 3

[0074] like Figure 1 As stated above, bands 1-2 represent the control group; bands 3-5 represent Examples 1, 2, and 3; in summary, combined with Figure 1 , Figure 2As shown in Table (I), compared with the control group, after adding the additive composition of the present invention, there were no significant differences in the maximum cell density, cell viability at the end of culture, and antibody expression level. However, the disulfide bond reduction rate was significantly reduced, the SEC-HPLC monomer purity was significantly improved, and the biological activity was greatly enhanced. This effectively solves the industry pain points of antibody quality decline, activity loss, and drugability failure caused by disulfide bond reduction in the industrial production of monoclonal antibodies.

[0075] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A cell culture additive composition for preventing antibody disulfide bond reduction, characterized in that: It is made from the following components: redox regulator, metal ion chelator, antioxidant, adjuvant, and cell protectant.

2. The cell culture additive composition for preventing antibody disulfide bond reduction according to claim 1, characterized in that: The components are present in the following weight proportions: 2-5 parts redox regulator, 0.25-1 part metal ion chelating agent, 0.15-0.45 parts antioxidant, 20-40 parts adjuvant, and 1-4 parts cell protectant.

3. The cell culture additive composition for preventing antibody disulfide bond reduction according to claim 2, characterized in that: The components are present in the following weight proportions: 3 parts redox regulator, 0.75 parts metal ion chelating agent, 0.3 parts antioxidant, 30 parts adjuvant, and 2 parts cell protectant.

4. The cell culture additive composition for preventing antibody disulfide bond reduction according to claim 1 or 3, characterized in that: The redox regulator is composed of either glutathione disulfide or cysteine ​​disulfide.

5. The cell culture additive composition for preventing antibody disulfide bond reduction according to claim 1 or 3, characterized in that: The metal ion chelating agent is one of ethylenediaminetetraacetic acid, deferoxamine, and ethylene glycol diethyl ether diaminetetraacetic acid.

6. The cell culture additive composition for preventing antibody disulfide bond reduction according to claim 1 or 3, characterized in that: The antioxidant is a mixture of lipoic acid and magnesium ascorbate phosphate in a preset ratio.

7. The cell culture additive composition for preventing antibody disulfide bond reduction according to claim 1 or 3, characterized in that: The adjuvant is a mixture of oxidized glutathione and L-tyrosine in a preset ratio; the cell protectant is ergothioneine.

8. The use of a cell culture additive composition for preventing antibody disulfide bond reduction as described in any one of claims 1-7 in the preparation of cell culture products for preventing antibody disulfide bond reduction.

9. The cell culture additive composition for preventing antibody disulfide bond reduction according to claim 8, characterized in that: The cell culture product is a cell culture system used to produce therapeutic antibodies.

10. The cell culture additive composition for preventing antibody disulfide bond reduction according to claim 9, characterized in that: The therapeutic antibodies include anti-tumor antibodies, anti-autoimmune disease antibodies, and antiviral antibodies.

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