A method for preparing CHO cell monoclonal antibodies with reduced alkaline peak

Abstract

The present application discloses a method for preparing a CHO cell monoclonal antibody with reduced alkaline peak, which relates to the technical field of biological cell culture medium, and comprises the following steps: S1: resuscitating frozen CHO cells in culture medium; S2: performing a subculture operation every 2-3 days to make the cells in the early logarithmic growth phase, and the cell inoculation density is 0.3-1.0×10 6 cells / mL; S3: when the cell density reaches 4.5-6.0×10 6 cells / mL, cell viability ≥95%, and subculture again at an interval of 2-3 days; S4: subculture 2-4 times according to the S2 and S3 methods, start inoculation after the cell population doubling time is stable, inoculate the cells into the culture medium, and culture in a 125mL shake flask; S5: use feed medium for batch feeding; S6: harvest after 6-16 days of culture and detect protein concentration; improve cell growth and metabolism, and increase cell yield, quality and culture efficiency.

Description

Technical Field

[0001] The invention relates to the technical field of biological cell culture medium, and in particular to a method for preparing a CHO cell monoclonal antibody with a reduced alkaline peak. Background Art

[0002] Monoclonal antibodies are products composed of immunoglobulin molecules with high specificity and affinity. They can specifically recognize and bind to antigens to exert their therapeutic effects. In the industrial production process, the acid-base peak ratio will have different degrees of impact on the stability of the antibody, including antibody inactivation or inducing other adverse reactions in the body. Therefore, in the actual drug production process, the acid-base peak ratio is a key quality attribute, and its ratio is crucial to whether the drug can be successfully declared for marketing.

[0003] In the process of cell culture, in order to maintain normal physiological activities of cells, cells need to exchange internal and external substances through the cell membrane. Most of the culture medium buffer systems used for CHO cell culture on the market are phosphate systems. In order to control and stabilize the pH value, a certain amount of sodium bicarbonate is usually added to form a carbonate-carbon dioxide buffer system. In the actual CHO cell culture process, for the shake flask system, the CO2 concentration of the incubator is usually set to 5%-8%, and the CO2 of the bioreactor is usually associated with pH control. Therefore, the phosphate concentration in the culture medium becomes the key to affecting pH and pH-related physiological activities. Different phosphate forms have different degrees of phosphate concentration, so when designing the culture medium, confirming the phosphate form and concentration is crucial to cell growth and the final antibody quality.

[0004] For example, the Chinese patent application number CN202210153116.3 is a hollow magnetic calcium phosphate cell microcarrier and its preparation method. The invention provides a hollow magnetic calcium phosphate cell microcarrier and its preparation method, which comprises the following steps: adding an organic template to a calcium salt solution to obtain a modified calcium salt solution, mixing the modified calcium salt solution and the phosphate solution to obtain an emulsion, sequentially adding urea and hydrochloric acid to the emulsion, adjusting the emulsion to a transparent solution, and then subjecting the system to a hydrothermal reaction, collecting the precipitate after cooling, washing, and obtaining calcium phosphate microspheres; dissolving the calcium phosphate powder in water to obtain a calcium phosphate solution, and then adding ferric oxide powder thereto, ultrasonically dispersing the solution, and finally hydrothermally reacting to obtain. The magnetic calcium phosphate microspheres can be used as cell microcarriers for bone microtissue construction, and are also conducive to the efficient assembly of microtissues, which can effectively solve the problem of uneven cell distribution in existing scaffold materials.

[0005] The hollow magnetic calcium phosphate cell microcarriers prepared by the above scheme have limited effect on improving cell culture and proliferation. Summary of the invention

[0006] The embodiments of the present application provide a method for preparing CHO cell monoclonal antibodies with reduced alkaline peaks, thereby solving the problems of poor cell culture growth quality and low cell yield in the prior art, improving cell growth and metabolic conditions, increasing cell yield and quality, and improving culture efficiency.

[0007] The present application provides a method for preparing a CHO cell monoclonal antibody with reduced alkaline peak, which specifically comprises the following steps:

[0008] S1: Resuscitate the frozen CHO cells in culture medium and culture the cells in a shaker at 37°C, 5-8% carbon dioxide, and 110-150 rpm;

[0009] S2: Perform subculture every 2-3 days to keep the cells in the early logarithmic growth phase, and the cell seeding density is 0.3-1.0×106cells / mL;

[0010] S3: When the cell density reaches 4.5-6.0×106 cells / mL and the cell viability is ≥95%, the cells are passaged again after an interval of 2-3 days;

[0011] S4: Follow the S2 and S3 methods for 2-4 passages. When the cell population doubling time is stable, start inoculation. Inoculate the cells into the culture medium and culture them in a 125 mL shake flask.

[0012] S5: Batch feeding using feed medium after cultivation;

[0013] S6: harvest after 6-16 days of culture and detect protein concentration.

[0014] Furthermore, in step S4, the temperature is 36.5-37°C, the rotation speed is 130±20 rpm, and the CO2 concentration in the incubator is 5%-10%; the temperature of the bioreactor is 36.5-37°C, the initial rotation speed is 180±20 rpm, the pH is 7.0±0.3, and the DO is 40%; the deviation after PDT stabilization is <3h; the inoculation density is 0.4-1.2×106cells / mL;

[0015] In step S5, feed is added at 3-8%, and feeding is stopped 1-2 days before the end of the culture;

[0016] In step S6, the protein is an antibody.

[0017] Furthermore, in step S4, the culture medium also contains microcarriers, which are hollow magnetic calcium phosphate cell microcarriers. The particle size of the hollow magnetic calcium phosphate cell microcarriers is 40-60 μm, and the mass of the hollow magnetic calcium phosphate cell microcarriers is 0.5%-1.0% of the mass of the culture medium.

[0018] Furthermore, an electromagnet is arranged at the bottom of the shaking bottle; the diameter of the electromagnet is 3-5 mm, the height is 2-4 mm, the maximum magnetic flux is 50 mT, the working voltage is 5-6 V, and the working current is 0-1 A.

[0019] Furthermore, the electromagnet points are arranged in a ring shape with an initial spacing of 10 mm. When the cell density increases by 0.2×106 cells / mL, the spacing of the electromagnets increases by 2 mm, the working voltage increases by 0.1-0.2 V, and the working current increases by 0.1 A. The microcarriers correspond to the electromagnet points.

[0020] Furthermore, the microcarriers also contain hollow calcium phosphate cell microcarriers without ferroferric oxide powder; the mass ratio of the hollow calcium phosphate cell microcarriers without ferroferric oxide powder to the hollow magnetic calcium phosphate cell microcarriers is 1:(1.2-1.5).

[0021] Furthermore, hollow calcium phosphate cell microcarriers without ferroferric oxide powder are used in combination with hollow magnetic calcium phosphate cell microcarriers at one magnet point, and two electromagnets are set at one point, and the electromagnets are turned on alternately at a frequency of 2-4 times per minute.

[0022] Furthermore, polyurethane soft capsules are arranged at corresponding points of the microcarriers in the culture medium.

[0023] Furthermore, the diameter of the soft capsule is 4-7 mm, the height is 2-4 mm, and the soft capsule contains ferrosoferric oxide powder.

[0024] Further, the culture medium specifically includes the following components: amino acids 400-6000 mg / L, vitamins 60-1000 mg / L, trace elements 50-2000 mg / L, lipids 0.5-15 mg / L, carbohydrates 4500-20000 mg / L, and additives 50-2000 mg / L;

[0025] It also includes inorganic salts, including 0-1g / L sodium dihydrogen phosphate, 1-2.5g / L disodium hydrogen phosphate, and 0.5-1g / L β-glycerophosphate.

[0026] One or more technical solutions provided in the embodiments of the present application have at least the following technical effects or advantages:

[0027] First, by using the culture medium of the present invention, by adjusting the phosphate form and concentration, the cell growth and metabolism can be significantly improved, the cell yield can be increased, and the antibody alkaline peak can be reduced; under the premise of not changing the solubility of the culture medium, this embodiment adjusts the phosphate form and concentration in the culture medium, takes cell growth, metabolism, protein expression, and antibody quality as evaluation indicators, confirms the phosphate form and concentration in the culture medium, solves the problem of poor quality of antibody IEC charge isomers in cells during cell culture, and also reduces the subsequent drug application and listing problems caused by unqualified antibody drug quality in the actual industrial production process; the serum-free culture medium for culturing CHO cells provided in this embodiment is simple in composition, low-cost, protein-free, and serum-free; this embodiment is conducive to CHO cells expressing target proteins and improving the quality of expressed antibodies;

[0028] Second, by adjusting the magnetic flux and spacing of the electromagnet, the distribution and aggregation of microcarriers in the culture medium are controlled, thereby optimizing the cell growth environment; the magnetic field generated by the electromagnet promotes cell adhesion and growth, improving the efficiency and quality of cell culture; the working voltage and current of the electromagnet are adjustable, making the cell culture process more flexible and controllable, and improving the adaptability and flexibility of cell culture; by providing a larger cell attachment area and improving the cell growth environment, the cell density and antibody production are increased; by utilizing the magnetic properties of microcarriers, the cell separation and recovery process can be simplified, reducing production costs; by adjusting the parameters of the electromagnet, the cell growth environment and culture process can be more accurately controlled, improving the stability and repeatability of cell culture;

[0029] Third, the hollow calcium phosphate cell microcarriers without ferroferric oxide powder and the hollow magnetic calcium phosphate cell microcarriers are used in combination at an electromagnet point. By alternately turning on the electromagnets, the hollow magnetic calcium phosphate cell microcarriers can be driven to rotate, thereby driving the rotation of the entire microcarrier combination point, providing a dynamic growth environment for the cells, and promoting the uniform distribution and proliferation of cells on the surface of the microcarriers; the rotation of the microcarriers promotes the uniform distribution of nutrients and metabolites in the culture medium, reduces local concentration differences, enables cells to obtain a more uniform nutrient supply, reduces cell growth differences caused by uneven nutrients, enhances the interaction between cells and microcarriers, promotes cell adhesion and spreading on the surface of the microcarriers, forms a more stable growth environment, and improves cell survival rate and proliferation ability; the weak fluid shear force generated by the rotation of the microcarriers can stimulate cell vitality and metabolism, promote cell growth and division, accelerate the cell culture process, and improve production efficiency, while maintaining the simplicity of cell separation and recovery, and improving the overall production efficiency of cells;

[0030] Fourthly, the expansion of the soft capsule increases the longitudinal growth space in the culture medium, providing more attachment and growth area for the cells; the soft capsule provides a more suitable microenvironment for the cells, promoting cell growth and division; the electromagnet interacts with the ferroferric oxide in the soft capsule to promote the suspension and dispersion of the cells, thereby improving the growth efficiency of the cells; by improving the growth environment of the cells and promoting the suspension and dispersion of the cells, the vitality of the cells is enhanced, thereby increasing the growth rate and density of the cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a graph showing the change of live cell density over time in Example 1 of the present invention;

[0032] Figure 2 This is a graph showing changes in cell viability over time in Example 1 of the present invention;

[0033] Figure 3 This is a graph showing changes in cell diameter over time in Example 1 of the present invention;

[0034] Figure 4 This is a graph showing changes in cell lactate metabolism over time according to Example 1 of the present invention;

[0035] Figure 5 This is a graph showing the change in antibody protein concentration over time in Example 1 of the present invention;

[0036] Figure 6 This is a comparison chart of the antibody quality of Example 1 of the present invention. DETAILED DESCRIPTION

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by technicians in the technical field to which the present invention belongs; the terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention; the term "and / or" used herein includes any and all combinations of one or more related listed items.

[0038] The preparation of the culture medium in the examples and the components of the cell line culture medium can be purchased from Sigma, GIBCO and the like.

[0039] Example 1: The culture medium used in the method for preparing CHO cell monoclonal antibodies with reduced alkaline peaks specifically comprises the following components:

[0040] Amino acids 400-6000 mg / L, vitamins 60-1000 mg / L, trace elements 50-2000 mg / L, lipids 0.5-15 mg / L, carbohydrates 4500-20000 mg / L and additives 50-2000 mg / L; the above ingredients are commercially available basic culture medium ingredients;

[0041] It also includes inorganic salts, including 0-1g / L sodium dihydrogen phosphate, 1-2.5g / L disodium hydrogen phosphate, and 0.5-1g / L β-glycerophosphate;

[0042] The method for preparing a CHO cell monoclonal antibody with reduced alkaline peak specifically comprises the following steps:

[0043] S1: reviving the frozen CHO cells in the culture medium, and culturing the cells in a shaker at 37°C, 5-8% carbon dioxide, and 110-150 rpm;

[0044] The culture medium is prepared by adding inorganic salts to a commercially available basic culture medium;

[0045] Among them, CHO cells are CHOK1 cells;

[0046] S2: Perform subculturing every 2-3 days to keep the cells in the early logarithmic growth phase. The cell seeding density is 0.3-1.0×10 6 cells / mL;

[0047] S3: When the cell density reaches 4.5-6.0×10 6 cells / mL, cell viability ≥95%, and subculture again after 2-3 days;

[0048] S4: Subculture 2-4 times according to S2 and S3 methods, start inoculation after the cell population doubling time is stable, inoculate the cells into the culture medium, and culture in a 125 mL shake flask or a 5 L stirred tank bioreactor;

[0049] The shaker temperature was 36.5-37°C, the rotation speed was 130±20 rpm, and the CO2 concentration in the incubator was 5%-10%. The bioreactor temperature was 36.5-37°C, the initial rotation speed was 180±20 rpm, the pH was 7.0±0.3, and the DO was 40%. The PDT stabilization deviation was <3h. The inoculation density was 0.4-1.2×10 6 cells / mL;

[0050] S5: Batch feeding after cultivation using chemically defined feed medium;

[0051] Among them, the feed was 3-8%, and the feed was stopped 1-2 days before the end of the culture;

[0052] S6: harvested after 6-16 days of culture and protein concentration tested;

[0053] Among them, the protein is an antibody.

[0054] For the method of culturing CHO cells in vitro in this embodiment, the following experiment was performed: a cryopreserved tube of CHOK1 cells was taken from a liquid nitrogen tank, shaken and dissolved in a 37°C water bath, added to a 125mL shake flask containing 20mL of culture medium, and mixed; then, the cells were placed in a shaker at 110-150rpm, 5-8% CO2, and 37±0.5°C for culture;

[0055] The culture medium is prepared by adding inorganic salts to GIBCO DMEM low-glucose culture medium;

[0056] The cells were subcultured every 2 days to maintain the cells in the early logarithmic growth phase. The cell seeding density was 0.2-2.0×10 6 cells / mL; when the cell density reaches 3-8×10 6 cells / mL, cell viability ≥95%, and subculture again after an interval of 2-3 days; cells were subcultured at least 3 times, and inoculation began after the population doubling time stabilized. The culture time was D0, and the inoculation density was 0.3±2.0× 10 6 cells / mL, inoculated into 125mL shake flask;

[0057] The culture temperature was set at 37±0.5℃, CO2 was set at 8%, the shaker humidity was set at 80%, and the shaker speed was set at 110-150rpm. During the culture process, samples were taken every 2 days and counted using a Countstar counter. Each sample was counted three times, and the cell survival rate was determined using the trypan blue staining method. Metabolic detection was performed using an M100 biochemical analyzer.

[0058] After the culture, batch feeding was performed using chemically defined feed medium, with 2-10% feeding every two days until 1-2 days before the end of the culture. The experimental culture was harvested on the 16th day, and the antibody concentration was tested using Cedex, affinity chromatography was performed using a gravity column, and quality testing was performed using an Agilent liquid chromatography instrument;

[0059] The experimental scheme and cell culture scheme are shown in Tables 1, 2, 3, 4, 5, 6, and 7. The key combinations are as follows Figure 1 , 2 , 3, 4, 5, 6 as shown: Table 1. Main implementation plan for cell culture

[0060] Shake bottle number Inoculation medium Sodium dihydrogen phosphate Disodium hydrogen phosphate β-Glycerol phosphate Experiment 1 Basal medium 1 / 2.5g / L 0.5g / L Experiment 2 Basal medium 2 1g / L 1g / L 1g / L

[0061] Table 2. Live cell density in 125 mL shake flask (×10 6 cells / mL) over time

[0062] Cultivation time / day 0 2 4 6 8 10 12 14 16 Experiment 1 1.58 8.62 19 41.8 33 32.7 27.4 22.7 13.5 Experiment 2 1.58 8.45 18.5 35.7 31.3 32.8 28.4 21 9.43

[0063] Table 3. Trend of viability of live cells (%) in 125 mL shake flask over time

[0064] Cultivation time / day 0 2 4 6 8 10 12 14 16 Experiment 1 95.01 99.46 99.57 97.91 89.8 91.83 89.14 82.76 59.91 Experiment 2 95.01 99.31 99.58 94.6 88.57 94.45 90.7 80.44 45.8

[0065] Table 4. Trend of cell diameter (μm) in 125 mL shake flask over time

[0066] Cultivation time / day 0 2 4 6 8 10 12 14 16 Experiment 1 14.58 14.74 14.83 14.29 15.75 16.53 17.69 18.01 17.58 Experiment 2 14.58 14.61 14.97 14.85 16.14 16.77 17.61 17.98 17.27

[0067] Table 5. Lactic acid metabolism (g / L) in 125 mL shake flasks over time

[0068] Cultivation time / day 2 4 6 8 10 12 14 16 Experiment 1 1.34 1.72 1.53 0.4 0.21 0.11 0.16 0.31 Experiment 2 1.2 1.63 1.25 0.44 0.27 0.15 0.18 0.76

[0069] Table 6. Protein expression (mg / L) in 125 mL shake flasks over time

[0070] Cultivation time / day 14 16 Experiment 1 2776 3164 Experiment 2 2729 2879

[0071] Table 7. Comparison of protein IEC (%) data

[0072] Cultivation time / day Acid Peak Main Peak Alkali Peak Experiment 1 18.568 66.375 15.057 Experiment 2 16.939 62.685 20.376

[0073] Tables 1, 2, 3, 4, 5, 6, 7 and Figure 1 , 2 , 3, 4, 5, 6 The results showed that the cells were grown in basal medium 1 with 1.5±0.2×10 6 cells / mL and cultured for 16 days. From Day 2, batch feeding was performed every two days. The final cell peak density reached 41.8×10 6 cells / mL; the cell viability at D14 was 59.91%; lactic acid first accumulated and then consumed, with a slight upward trend in the later period, and the lactic acid concentration at D16 was 0.31g / L; the protein yield at D16 was 3164mg / L, and the final protein acid peak, main peak, and alkaline peak ratios were 16.939%, 62.685%, and 20.376%, respectively;

[0074] In Experiment 2, using the above process, the peak cell density during the entire culture process was 32.7×10 6 cells / mL, D16 cell viability was 45.8%, D16 lactic acid concentration was 0.76 g / L, D16 protein yield was 2879 mg / L, and the final protein acid peak, main peak, and alkaline peak ratios were 18.568%, 66.375%, and 15.057%, respectively.

[0075] The technical solutions in the above embodiments of the present application have at least the following technical effects or advantages:

[0076] From the above experimental results, it can be concluded that after the medium of the present invention is adjusted, the cell peak density can be increased from 35.7×10 6 cells / mL increased to 41.8×10 6 cells / mL, the harvest viability at 16 days of culture can be increased from 45.8% in Experiment 2 to 59.91%; in terms of metabolism, the harvested lactic acid can be increased from 0.76 g / L in Experiment 2 to 0.31 g / L; in terms of expression, the final antibody expression amount can be increased from 2879 mg / L in Experiment 2 to 3164 mg / L; in terms of quality, the final protein acid peak, main peak, and alkaline peak ratios were improved from 16.939%, 62.685%, and 20.376% to 18.568%, 66.375%, and 15.057%, respectively; the above results show that by adjusting the phosphate form and concentration in the culture medium, the cell growth and metabolism can be significantly improved, the cell yield can be increased, and the antibody alkaline peak can be reduced;

[0077] In this embodiment, the phosphate form and concentration in the culture medium are adjusted without changing the solubility of the culture medium, and the cell growth, metabolism, protein expression, and antibody quality are used as evaluation indicators to confirm the phosphate form and concentration in the culture medium, thereby solving the problem of poor quality of antibody IEC charge isomers in cells during cell culture, and also reducing the subsequent drug application and listing problems caused by unqualified antibody drug quality in the actual industrial production process;

[0078] The serum-free medium for culturing CHO cells provided in this embodiment has simple ingredients, low cost, and is protein-free and serum-free;

[0079] This embodiment is beneficial for CHO cells to express target proteins and improve the quality of expressed antibodies.

[0080] Example 2: The above Example 1 reduces the alkaline peak of CHO cell monoclonal antibody and improves the quality of CHO cell culture by adjusting the phosphate form and concentration in the culture medium; in order to improve the efficiency of CHO cell culture, further optimization is made on the basis of Example 1.

[0081] Step S4: After the cell population doubling time is stable, inoculation is started, the cells are inoculated into the culture medium, and cultured in a 125 mL shake flask;

[0082] The culture medium also contains microcarriers, which are hollow magnetic calcium phosphate cell microcarriers. The particle size of the hollow magnetic calcium phosphate cell microcarriers is 40-60 μm, and the mass of the hollow magnetic calcium phosphate cell microcarriers is 0.5%-1.0% of the mass of the culture medium. An electromagnet is arranged at the bottom of the shaking bottle; the microcarriers correspond to the points of the electromagnet.

[0083] The preparation of the hollow magnetic calcium phosphate cell microcarrier specifically comprises the following steps:

[0084] (1) Dissolve 1 g of calcium chloride in 30 ml of deionized water to obtain a calcium chloride solution, add 0.5 g of EDTA to the calcium chloride solution to obtain a modified calcium salt solution, dissolve 1.6 g of Na2HPO4·6H2O in 30 ml of deionized water to obtain a phosphate solution, mix the modified calcium salt solution and the phosphate solution to obtain an emulsion, add 1 g of urea and hydrochloric acid to the emulsion in sequence, adjust the emulsion to a transparent solution, then perform hydrothermal treatment on the system, collect the precipitate after cooling, wash, and obtain calcium phosphate microspheres;

[0085] The temperature of the hydrothermal treatment is 160-200°C and the reaction time is 4-7h;

[0086] (2) ultrasonically dispersing the calcium phosphate powder and ferroferric oxide powder in step (1) in water, and then subjecting the mixture to a hydrothermal reaction to obtain a hollow magnetic calcium phosphate cell microcarrier;

[0087] The mass ratio of calcium phosphate powder to ferroferric oxide is 0.9-1.0:0.04-0.06; the hydrothermal reaction is carried out at 170-190° C. for 22-26 hours;

[0088] The ferroferric oxide powder in step (2) is prepared by the following method: dissolving a divalent iron salt and a trivalent iron salt in water to obtain an iron-containing solution, dripping turpentine oil on the surface of the iron-containing solution, then adjusting the pH value of the iron-containing solution to be alkaline, and then subjecting the solution to hydrothermal treatment. After cooling, the precipitate is collected, washed, and dried to obtain the ferroferric oxide powder.

[0089] The divalent iron salt and the trivalent iron salt are FeCl2·4H2O and FeCl3·6H2O respectively, and the molar ratio of divalent iron ions to trivalent iron ions in the iron-containing solution is 1:1.5-2; the pH value of the iron-containing solution is adjusted to 10-11, the temperature of the hydrothermal treatment is 110-130°C, and the reaction time is 20-24h;

[0090] The electromagnet has a diameter of 3-5 mm, a height of 2-4 mm, a maximum magnetic flux of 50 mT, a working voltage of 5-6 V, and a working current of 0-1 A;

[0091] The electromagnets are arranged in a circular pattern with an initial spacing of 10 mm. When the cell density increases by 0.2×10 6 cells / mL, the spacing of the electromagnets increases by 2mm, the operating voltage increases by 0.1-0.2V, and the operating current increases by 0.1A.

[0092] According to the solution of this embodiment, the following experiment was conducted: hollow magnetic calcium phosphate cell microcarriers were added to the culture medium, the mass of the added hollow magnetic calcium phosphate cell microcarriers was 0.7% of the mass of the culture medium, and the experiment was cultured to the 6th day and harvested;

[0093] The difference between this embodiment and Experiment 1 is that in Experiment 1, there is no hollow magnetic calcium phosphate cell microcarrier and no electromagnet is set at the bottom of the shaking flask. The rest is the same. The viable cell density in the shaking flask is tested. The test results are shown in the following table:

[0094] Table 8. Live cell density in 125 mL shake flask (×10 6 cells / mL) over time

[0095] Cultivation time / day 0 2 4 6 Experiment 1 1.58 8.62 19 41.8 Embodiment 2 1.58 9.31 20.7 45.2

[0096] The technical solutions in the above embodiments of the present application have at least the following technical effects or advantages:

[0097] It can be seen from Table 8 that adding hollow magnetic microcarriers to the culture medium and setting up electromagnets can promote the growth rate of CHO cells, increase the cell density, and improve the cell culture efficiency;

[0098] The hollow magnetic calcium phosphate cell microcarrier has a large specific surface area, providing more space for cells to attach and grow, thereby increasing cell density and yield; EDTA, as a morphology regulator, can change the morphology of calcium phosphate to form a hollow spherical structure; after the hydrothermal reaction, the phosphate on the surface of calcium phosphate adsorbs ferroferric oxide nanoparticles to generate nanocrystal clusters, so that the surface of the calcium phosphate microsphere forms a rod-like nanostructure, and grooves are formed between the rod-like nanostructures. The groove structure can guide the cells to spread along the groove direction, promote the interaction between cells and microcarriers, and promote the adhesion and proliferation of cells on the surface of microcarriers; the porous structure of the microcarrier promotes the transfer of nutrients and the discharge of metabolic products, improves the growth environment of cells, and because the hollow magnetic calcium phosphate cell microcarrier is magnetic, cells can be separated and recovered from the culture medium through an external magnetic field, simplifying the process of cell harvesting;

[0099] By adjusting the magnetic flux and spacing of the electromagnet, the distribution and aggregation of microcarriers in the culture medium are controlled, thereby optimizing the cell growth environment; the magnetic field generated by the electromagnet promotes cell adhesion and growth, improving the efficiency and quality of cell culture; the working voltage and current of the electromagnet are adjustable, making the cell culture process more flexible and controllable, and improving the adaptability and flexibility of cell culture;

[0100] By providing a larger cell attachment area and improving the cell growth environment, the cell density and antibody production can be increased; by utilizing the magnetic properties of microcarriers, the cell separation and recovery process can be simplified and production costs can be reduced; by adjusting the parameters of the electromagnet, the cell growth environment and culture process can be more accurately controlled, improving the stability and repeatability of cell culture.

[0101] Example 3: The above Example 2 increases cell density and antibody production and simplifies the cell separation and recovery process by adding hollow magnetic microcarriers to the culture medium and setting an electromagnet at the bottom of the culture medium; in order to improve the efficiency and quality of CHO cell culture, further optimization is made on the basis of Example 2.

[0102] The microcarriers also contain hollow calcium phosphate cell microcarriers without ferroferric oxide powder; the mass ratio of the hollow calcium phosphate cell microcarriers without ferroferric oxide powder to the hollow magnetic calcium phosphate cell microcarriers is 1:(1.2-1.5);

[0103] The hollow calcium phosphate cell microcarrier without ferroferric oxide powder is used in combination with the hollow magnetic calcium phosphate cell microcarrier at a magnet point. Two electromagnets are arranged at one point, and the electromagnets are turned on alternately at a frequency of 2-4 times per minute.

[0104] The following experiment was conducted for this example, where 0.7% of the cell microcarriers by mass of the culture medium were added to the culture medium;

[0105] The cell microcarriers include hollow calcium phosphate cell microcarriers without ferroferric oxide powder and hollow magnetic calcium phosphate cell microcarriers, and the mass ratio of the hollow calcium phosphate cell microcarriers without ferroferric oxide powder to the hollow magnetic calcium phosphate cell microcarriers is 1:1.3; the frequency of alternating opening of the electromagnet is 3 times per minute;

[0106] The difference between this embodiment and the second embodiment is that the microcarriers in the culture medium in this embodiment include hollow calcium phosphate cell microcarriers without ferroferric oxide powder, and at the same time, two electromagnets are provided in this embodiment, which are turned on alternately; the test results are shown in the following table:

[0107] Table 9. Live cell density in 125 mL shake flask (×10 6 cells / mL) over time

[0108] Cultivation time / day 0 2 4 6 Embodiment 2 1.58 9.31 20.7 45.2 Embodiment 3 1.58 9.52 21.9 47.8

[0109] The technical solutions in the above embodiments of the present application have at least the following technical effects or advantages:

[0110] As shown in Table 9, by using the two microcarriers in combination and setting two alternately turned-on electromagnets at the magnetic site, the growth rate of CHO cells can be further accelerated and the cell density can be further increased;

[0111] The hollow calcium phosphate cell microcarriers without ferroferric oxide powder have a larger specific surface area, providing more space for cells to attach and grow, promoting a more uniform distribution of cells on the surface of the microcarriers, thereby increasing the cell density;

[0112] The hollow calcium phosphate cell microcarriers without ferroferric oxide powder and the hollow magnetic calcium phosphate cell microcarriers are used in combination at one electromagnet point. By turning on the electromagnets alternately, the hollow magnetic calcium phosphate cell microcarriers can be driven to rotate, thereby driving the rotation of the entire microcarrier combination point, providing a dynamic growth environment for the cells and promoting the uniform distribution and proliferation of the cells on the surface of the microcarriers.

[0113] The rotation of microcarriers promotes the uniform distribution of nutrients and metabolites in the culture medium, reduces local concentration differences, enables cells to obtain a more uniform nutrient supply, reduces cell growth differences caused by uneven nutrients, enhances the interaction between cells and microcarriers, promotes cell adhesion and spreading on the surface of microcarriers, forms a more stable growth environment, and improves cell survival rate and proliferation ability;

[0114] The weak fluid shear force generated by the rotation of the microcarrier can stimulate cell vitality and metabolism, promote cell growth and division, accelerate the cell culture process, and improve production efficiency. At the same time, it maintains the simplicity of cell separation and recovery, and improves the overall cell production efficiency.

[0115] By enhancing the interaction between microcarriers and cells and optimizing the cell growth environment, cell density and antibody production can be increased. The rotation motion provides a more uniform growth environment for cells, reduces differences in cell growth, and improves the homogeneity of the cell population, thereby promoting the consistency of monoclonal antibody production.

[0116] Example 4: The above Example 3 increases the cell density and antibody production by combining two microcarriers and setting two alternately turned electromagnets at the bottom of the culture medium. In order to improve the efficiency and quality of CHO cell culture, further optimization is made on the basis of Example 3.

[0117] Arranging polyurethane soft capsules at corresponding points of the microcarriers in the culture medium;

[0118] The diameter of the soft capsule is 4-7 mm, the height is 2-4 mm, and the soft capsule contains ferrosoferric oxide powder.

[0119] The following experiment was conducted for this embodiment, where a polyurethane soft capsule was set at the corresponding point of the electromagnet in the culture medium;

[0120] The diameter of the sac is 5 mm and the height is 3 mm;

[0121] The difference between this embodiment and the third embodiment is that a polyurethane soft capsule is arranged at the corresponding position of the culture medium carrier in this embodiment, and the rest is consistent with the third embodiment; the test results are shown in the following table:

[0122] Table 10. Live cell density in 125 mL shake flask (×10 6 cells / mL) over time

[0123] Cultivation time / day 0 2 4 6 Embodiment 3 1.58 9.52 21.91 47.82 Embodiment 4 1.58 10.41 24.35 51.89

[0124] The technical solutions in the above embodiments of the present application have at least the following technical effects or advantages:

[0125] As shown in Table 10, by setting polyurethane soft capsules at corresponding points of the carrier in the culture medium, the viable cell density of CHO cells can be further increased;

[0126] An electromagnet array is arranged at the bottom of the culture medium, each electromagnet corresponds to a carrier point, including a hollow magnetic calcium phosphate cell microcarrier and a hollow calcium phosphate cell microcarrier without ferroferric oxide powder. A soft capsule is added to the carrier site, and the magnetic force of the electromagnet is adjusted by changing the magnetic flux, working voltage and working current of the electromagnet at the bottom; because the soft capsule contains ferroferric oxide powder, it can be attracted and move upward under the action of the enhanced magnetic field, and the capsule wall is stretched and expanded; by controlling the magnetic flux and working voltage of the electromagnet, the expansion degree and height of the soft capsule can be accurately adjusted, thereby providing a suitable longitudinal growth space for the cells;

[0127] The expansion of the soft capsule increases the longitudinal growth space in the culture medium, providing more attachment and growth area for cells; the soft capsule provides a more suitable microenvironment for cells, promoting cell growth and division; the electromagnet interacts with the ferroferric oxide in the soft capsule to promote the suspension and dispersion of cells, thereby improving the growth efficiency of cells; by improving the growth environment of cells and promoting the suspension and dispersion of cells, the vitality of cells is enhanced, thereby increasing the growth rate and density of cells;

[0128] Due to the increase in cell density and the enhancement of cell viability, the antibody production is further increased, and the production cost is reduced and the production efficiency is improved by improving the cell growth environment and increasing the cell growth efficiency.

[0129] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing a CHO cell monoclonal antibody with reduced alkaline peak, characterized in that: The specific steps include: S1: Resuscitate the frozen CHO cells in culture medium and culture the cells in a shaker at 37°C, 5-8% carbon dioxide, and 110-150 rpm; S2: Perform subculturing every 2-3 days to keep the cells in the early logarithmic growth phase. The cell seeding density is 0.3-1.0×10 6 cells / mL; S3: When the cell density reaches 4.5-6.0×10 6 cells / mL, cell viability ≥95%, and subculture again after 2-3 days; S4: Follow the S2 and S3 methods for 2-4 passages. When the cell population doubling time is stable, start inoculation. Inoculate the cells into the culture medium and culture them in a 125 mL shake flask. S5: Batch feeding using feed medium after cultivation; S6: harvested after 6-16 days of culture and protein concentration tested; The culture medium specifically includes the following components: amino acids 400-6000 mg / L, vitamins 60-1000 mg / L, trace elements 50-2000 mg / L, lipids 0.5-15 mg / L, carbohydrates 4500-20000 mg / L and additives 50-2000 mg / L; It also includes inorganic salts, including 0-1g / L sodium dihydrogen phosphate, 1-2.5g / L disodium hydrogen phosphate, and 0.5-1g / L β-glycerophosphate; Step S4: the culture medium further contains microcarriers, which are hollow magnetic calcium phosphate cell microcarriers, the particle size of the hollow magnetic calcium phosphate cell microcarriers is 40-60 μm, and the mass of the hollow magnetic calcium phosphate cell microcarriers is 0.5%-1.0% of the mass of the culture medium; An electromagnet is set at the bottom of the shake bottle; the diameter of the electromagnet is 3-5mm, the height is 2-4mm, the maximum magnetic flux is 50mT, the working voltage is 5-6V, and the working current is 0-1A; The electromagnets are arranged in a circular pattern with an initial spacing of 10 mm. When the cell density increases by 0.2×10 6 cells / mL, the distance between the electromagnets is increased by 2 mm, the working voltage is increased by 0.1-0.2 V, and the working current is increased by 0.1 A; the microcarriers correspond to the points of the electromagnets; The microcarriers further contain hollow calcium phosphate cell microcarriers that do not contain ferroferric oxide powder; the mass ratio of the hollow calcium phosphate cell microcarriers that do not contain ferroferric oxide powder to the hollow magnetic calcium phosphate cell microcarriers is 1:(1.2-1.5); The hollow calcium phosphate cell microcarriers without ferroferric oxide powder and the hollow magnetic calcium phosphate cell microcarriers are used in combination at a magnet point, and two electromagnets are set at one point, and the electromagnets are turned on alternately at a frequency of 2-4 times per minute; Arranging polyurethane soft capsules at corresponding points of the microcarriers in the culture medium; The diameter of the soft capsule is 4-7 mm, the height is 2-4 mm, and the soft capsule contains ferrosoferric oxide powder.

2. The method for preparing a CHO cell monoclonal antibody with reduced alkaline peak according to claim 1, characterized in that: In step S4, the temperature was 36.5-37°C, the rotation speed was 130±20 rpm, and the CO2 concentration in the incubator was 5%-10%. The temperature of the bioreactor was 36.5-37°C, the initial rotation speed was 180±20 rpm, the pH was 7.0±0.3, and the DO was 40%. The deviation after PDT stabilization was less than 3h. The inoculation density was 0.4-1.2×10 6 cells / mL; In step S5, feed is added at 3-8%, and feeding is stopped 1-2 days before the end of the culture; In step S6, the protein is an antibody.

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