Culture method for improving yield of hybridoma cell monoclonal antibody
By gradually reducing serum content and feeding in batches, combined with oxygen partial pressure control, the growth environment of hybridoma cells is optimized, the problem of low cell yield is solved, efficient and continuous monoclonal antibody production is achieved, and the yield and efficiency.
Patent Information
- Application Number
- CN202411488460.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-24
- Publication Date
- 2024-12-20
AI Technical Summary
Hybridoma cells produce low yields when producing monoclonal antibodies, which is difficult to meet the needs of large-scale production and application. The complex and varied cell growth environment makes it difficult to accurately predict and control cell growth rates, which increases the difficulty of experiments.
By gradually reducing the serum content in the culture medium to serum-free culture, combining batch feeding and oxygen partial pressure control at different culture stages, we optimize the cell growth environment, improve cell abundance and survival rate, and achieve efficient monoclonal antibody production .
It significantly improves the yield and production efficiency of monoclonal antibodies, extends the cell culture time, ensures the continuity of production, and improves cell viability and antibody purification abundance.
Abstract
Description
Technical Field
[0001] The invention belongs to the field of cell engineering, and in particular relates to a culture method for improving the yield of monoclonal antibodies of hybridoma cells. Background Art
[0002] Monoclonal antibodies have become an important means of diagnosis and treatment of many diseases, such as cancer, autoimmune diseases, infectious diseases, etc. They can specifically bind to pathogens or diseased cells to achieve the purpose of treatment or diagnosis. In biological research, monoclonal antibodies are widely used in protein function research, cell signal transduction research, immunology research and other fields. They can be used as probes, markers or inhibitors to help scientists understand the mysteries of organisms more deeply. Monoclonal antibodies are also important tools for drug development. They can be used as carriers or targets for drugs to help develop new drugs or improve existing drugs.
[0003] With the rapid development of biology and biopharmaceuticals, hybridoma cell culture technology is also constantly optimizing and expanding its application areas. With the development of molecular biology and cell biology, all aspects of hybridoma cell technology have been optimized, including the improvement of fusion methods, the improvement of screening technology, the optimization of culture conditions, etc., thereby improving the stability of hybridoma cells and the specificity of monoclonal antibodies.
[0004] Although hybridoma cells can stably produce monoclonal antibodies, their yields are often low and difficult to meet the needs of large-scale production and application. The study of hybridoma cell growth dynamics is the basis for understanding cell growth patterns and optimizing culture conditions. However, in actual operations, due to the complexity of the cell growth environment and the differences in cell lines, it is often difficult to accurately predict and control the growth rate of cells. In addition, stagnation and aging may occur during cell growth, further increasing the difficulty of the experiment. Summary of the invention
[0005] The present invention aims to overcome the deficiencies of the prior art and provide a culture method for increasing the yield of monoclonal antibodies in hybridoma cells.
[0006] In order to achieve the above object, the technical solution provided by the present invention is:
[0007] The method for increasing the production of monoclonal antibodies in hybridoma cells comprises the following steps:
[0008] (1) inoculating the frozen hybridoma cells into an initial culture medium for activation (10 mL of culture medium in a 15 mL test tube), and then culturing them at 37° C., 30 rpm, and 5% CO2 (25 mL ventilated flask), and performing subculture when the culture medium turns yellow; the initial culture medium is a complete culture medium to which at least a carbon source, vitamins, cell growth factors, and antibiotics are added, wherein the concentration of the carbon source in the complete culture medium is 2 mM-6 mM, and the volume percentage concentrations of the vitamins, cell growth factors, and antibiotics in the complete culture medium are all 0.5%-1.5%; serum is also added to the complete culture medium, and the volume percentage concentration of the serum in the complete culture medium is 20%; during subculture, the serum content in the culture medium is gradually reduced to 0%, and serum-free cultured hybridoma cells are obtained by subculture;
[0009] (2) Pre-filling the fermenter with serum-free medium, and then inoculating the serum-free cultured hybridoma cells obtained in step (1) into the fermenter, and performing the initial stage of culture for 1-2 days at a speed of 90 rpm, 37° C., pH 6.9-7.4, and 80%-90% PO2 until the cell abundance reaches 1×10 5 / mL-2x 10 5 / mL; the components of the serum-free medium are the same as the components of the initial medium without serum in step (1); in the fermenter, the volume ratio of the inoculated serum-free cultured hybridoma cells to the pre-filled serum-free medium is 0.1:4;
[0010] (3) Serum-free medium was added to the fermenter and cultured for 1-2 days at a speed of 120 rpm, 37°C, pH 6.9-7.4, and 60%-80% PO2 until the cell abundance reached 5 x 10 5 / mL-7x 10 5 / mL, and then cultured in the exponential phase at 120 rpm, 37°C, pH 6.9-7.4, and 40%-60% PO2 for 24h-36h until the cell abundance reached 2x 10 6 / mL-5x 10 6 / mL, and then enter the stable culture stage under 20%-40% PO2 environment. The cell abundance at the end of stable culture is 1.6x 10 7 / mL; the volume ratio of the supplemented serum-free medium to the pre-loaded serum-free medium is 3:2;
[0011] (4) When the cell death rate in the fermentation tank reaches 50%-60%, the cell suspension is collected for antibody purification to obtain the purified hybridoma cell monoclonal antibody.
[0012] Preferably, the complete medium in step (1) is any one of Hybridoma SFM, CD Hybridoma Medium, Medium NCTC-109, and PFHM-II Protein-Free Hybridoma Medium, the serum is IgG-free serum, the carbon source is any one of GlutaMAX, L-glutamine, and glucose, the vitamin is MEM, the cell growth factor is B-27, and the antibiotic is penicillin-streptomycin.
[0013] More preferably, the IgG-free serum is fetal bovine serum.
[0014] Preferably, the method further comprises the following steps after step (4): after collecting the cell suspension, adding serum-free culture medium to the fermenter, and then repeating steps (3) and (4) to achieve batch culture without re-inoculating hybridoma cells.
[0015] The present invention will be further described below:
[0016] The present invention achieves the tolerance of hybridoma cells in serum-free medium and improves the survival rate by gradually reducing the serum level in the culture medium to serum-free medium during the passage process, and further supplements the culture medium by batch feeding during batch fermentation culture to improve cell abundance; by adjusting parameters such as bioreactor PO2 and stirring speed at different culture stages, the cell state and vitality are improved, the yield of monoclonal antibodies is increased, and batch culture is achieved.
[0017] 1. The present invention uses intermittent feeding to enable activated cells to fully adapt to the culture medium to obtain higher cell abundance
[0018] First, activate the frozen hybridoma cells, then transfer them to a small flask with a ventilated cover, keep the shaking speed at 30rpm, and culture them in an incubator at 37℃ and a 5% carbon dioxide environment. When the culture medium turns yellow, subculture and gradually reduce the serum content in the culture medium to 5%, 2% and finally to 0. The hybridoma cells are domesticated in the culture of hybridoma cells, avoiding the interference of exogenous antibodies and foreign proteins. The culture method is simple and efficient, and the cell survival rate is high. High-quality hybridoma cells are provided for fermentation inoculation, and the cell abundance of hybridoma cells before inoculation reaches 2x 10 5 / mL.
[0019] Then pre-fill 4L of fresh serum-free medium into the INFORS biofermenter. Inoculate 100mL of serum-free medium hybridoma cells into the fermenter, maintain a stirring speed of 90rpm, a temperature of 37°C, a pH control range of 6.9-7.4, and a PO2 concentration of 80%-90%. Culture for 1-2 days until the cell abundance in the fermenter reaches 1x 10 5 / mL-2x 10 5 / mL, and then feed 6L of fresh serum-free culture medium into the INFORS biofermenter, maintain the stirring speed at 120rpm, the temperature at 37℃, and the pH value in the range of 6.9-7.4, so as to achieve intermittent feeding culture mode, so that the cells can fully adapt to the culture medium and smoothly pass through the initial stage of the fermentation process.
[0020] Through the intermittent feeding method described in the present invention, by feeding fresh culture medium in the initial stage and growth stage of the cells, the cell abundance and cell viability in the overall fermentation tank can be effectively improved, and the cell culture time can be extended. At the end of the culture, up to 1.6 x 10 7 / mL cells, and the survival rate exceeded 60%, providing an effective cell abundance guarantee for the antibody production of hybridoma cells.
[0021] 2. The present invention reduces PO2 at different culture stages to induce hybridoma cells to produce antibodies
[0022] A normal culture has four stages: initial stage, growth stage, exponential stage, and stable stage. The initial stage of hybridoma cell fermentation is 1-2 days after the first inoculation, and the cell number is about 1x 10 5 / mL-2x 10 5 / mL, the oxygen demand is not high, and the PO2 concentration is kept at 80%-90%; the growth stage is about 1-2 days, and the cell number is 5x10 5 / mL-7x10 5 / mL, oxygen consumption began to increase, oxygen demand was 0.05L / min~0.08L / min, PO2 concentration was controlled at 60-80%; in the exponential growth stage, cell density increased sharply within 24h-36h, reaching about 2x 10 6 / mL-5x 10 6 / mL, a large amount of O2 is required, and the PO2 concentration is controlled at 40%-60%; in the stable stage, the cells can grow until the cell number remains unchanged, the oxygen consumption drops to the level of the initial stage, the supernatant antibody level increases significantly at this stage, and the PO2 concentration needs to be maintained at 20%-40% to obtain the optimal culture oxygen partial pressure.
[0023] The production of antibodies by hybridoma cells needs to be carried out in an oxygen-deficient environment. During the culture process, the PO2 level is gradually reduced, which does not affect the cell proliferation of the hybridoma cells, but also puts the high-peak cells in the stable period under hypoxic stress, thereby secreting a large amount of target antibodies, and can increase the antibody production level of the fermentation culture supernatant to about 200-300 mg / L. The culture method is simple and effective, maintains high cell viability and improves antibody production efficiency.
[0024] 3. Harvest cells and feed fresh culture medium during the specific culture stage to achieve batch culture
[0025] In the stable growth stage, the cells secrete a large amount of antibodies, but due to the lack of nutrients, the cell death rate begins to increase. At this stage, the cell death rate is detected at 50%-60%, and the cells are directly harvested. At this time, there is 10L of cell culture suspension in the INFORS biofermenter, and about 9L of cell or culture medium samples are collected from the safety sampler, and then 3L of fresh serum-free culture medium is injected, and the intermittent feeding method described in the present invention is repeated for cultivation, so as to realize batch culture without the need for additional seed inoculation.
[0026] The culture method for increasing the yield of monoclonal antibodies in hybridoma cells of the present invention is suitable for mass production of antibodies, and hybridoma cell fermentation can be continuously performed for up to half a year in this way, thereby ensuring the continuity of production and improving the efficiency of antibody production. DETAILED DESCRIPTION
[0027] The present invention will be further described below in conjunction with the embodiments.
[0028] Example 1
[0029] Hybridoma cell culture
[0030] The main components of the culture medium include Hybridoma SFM, fetal bovine serum, glutamine and resistance; as a preferred technical solution, the proportion of the fetal bovine serum in the culture medium is reduced to 0% before the fermenter is inoculated; the preferred addition amount of glutamine to the culture medium is 1% by volume, and the addition amount of resistance to the culture medium is 1% by volume.
[0031] In this example, the control group was directly inoculated with 100 mL of the acclimated cell suspension in 10 L of culture medium for culture. The experimental group was the culture method for improving the production of monoclonal antibodies of hybridoma cells described in the present invention (for detailed parameters and steps, see the invention content section). The same hybridoma cells were inoculated, and the inoculated cell abundance was 1.7 x 10 5 / mL, cell viability was 93%, and the inoculation volume was 100mL. The cell abundance and time when the cell death rate reached 50% were recorded.
[0032] Table 1: Comparison of cell abundance and culture time
[0033] Intermittent feeding Direct inoculation Peak cell abundance <![CDATA[1.6x 10 7 ]]> <![CDATA[6.9x 10 6 ]]> Mortality 50% time 10 days 7 days
[0034] The intermittent feeding method effectively extended the cell culture time and achieved a higher cell abundance, effectively providing a basis for cell abundance for antibody production.
[0035] Example 2
[0036] This example is a comparison of the effects of purifying antibody supernatants
[0037] In this example, the control group was directly inoculated with 100 mL of the acclimated cell suspension in 10 L of culture medium for culture. The experimental group was cultured in the method for increasing the production of monoclonal antibodies in hybridoma cells according to the present invention (for detailed parameters and steps, see the invention content section). The same hybridoma cells were inoculated at a cell abundance of 2 x 10 5 / mL, cell survival rate was 95%, and the inoculation volume was 100mL. The control group was inoculated and the PO2 ratio was reduced by 20% every 2 days, to a minimum of 20%. After inoculation of the experimental group, the PO2 ratio was reduced by 20% every 2 days, to a minimum of 20%. 5 Adjust PO2 to 80% at 8x 10 5 / mL, adjust PO2 to 60% at 2x10 6 Adjust PO2 to 40% at 5x 10 6 Adjust PO2 to 30% at 9x 10 6 / mL, adjust PO2 to 20% to control oxygen partial pressure. When the death rate of detected cells reaches 50%, harvest the cell suspension for antibody purification.
[0038] Antibody purification was performed using the AKTA protein purification system and antibody affinity purification was performed using protein G dextran gel. The dilution ratio is as follows:
[0039] Equilibration / Washing Buffer:
[0040] 0.15M sodium chloride,
[0041] 20 mM sodium phosphate dibasic
[0042] pH 7.0
[0043] Elution Buffer:
[0044] 100 mM glycine
[0045] pH 3.0
[0046] Neutralization Buffer:
[0047] 1M Tris-HCl
[0048] pH 8.5;
[0049] Use 2-5 times the volume of washing buffer to wash the column and balance the affinity filler. Load 9L of cell culture supernatant after centrifugation and filter for antibody adsorption. After adsorption, use 3-5 times the volume of elution buffer for gradient pH elution, collect the eluate according to the signal, and immediately adjust the pH value of the eluate to 6.6-7.0 with neutralization buffer.
[0050] The eluate was diluted 100 times for concentration determination using UV spectrophotometry at a wavelength of 280 nm. The antibody purification abundance was calculated.
[0051] Table 2: Comparison of cell abundance and antibody abundance
[0052] Intermittent feeding, partial pressure control Direct inoculation, adjusting partial pressure according to time Peak cell abundance <![CDATA[1.1x 10 7 ]]> <![CDATA[4.3x 10 6 ]]> Antibody purification abundance 230mg / L 140mg / L
[0053] The intermittent feeding and partial pressure control method designed by the present invention (a culture method for increasing the production of monoclonal antibodies in hybridoma cells) effectively increases the abundance of cells, thereby obtaining a higher abundance of antibodies in the supernatant of the cell suspension, and significantly improving production efficiency. Using the intermittent feeding and partial pressure control method for cultivation, continuous and stable cultivation can be carried out for up to half a year.
Claims
1. A method for increasing the production of monoclonal antibodies in hybridoma cells, characterized in that: The method comprises the following steps: (1) Inoculating the frozen hybridoma cells into an initial culture medium for activation, and then culturing at 37°C, 30 rpm, and 5% CO2, and performing subculture when the culture medium turns yellow; the initial culture medium is a complete culture medium to which at least a carbon source, vitamins, cell growth factors, and antibiotics are added, the concentration of the carbon source in the complete culture medium is 2 mM-6 mM, and the volume percentage concentrations of the vitamins, cell growth factors, and antibiotics in the complete culture medium are all 0.5%-1.5%; serum is also added to the complete culture medium, and the volume percentage concentration of the serum in the complete culture medium is 20%; during subculture, the serum content in the culture medium is gradually reduced to 0%, and serum-free hybridoma cells are obtained by subculture; (2) Pre-fill the fermenter with serum-free medium, then inoculate the serum-free cultured hybridoma cells obtained in step (1) into the fermenter, and perform the initial stage of culture at a speed of 90 rpm, 37°C, pH 6.9-7.4, and 80%-90% PO2 for 1-2 days until the cell abundance reaches 1 x 10 5 / mL-2 x 10 5 / mL; the components of the serum-free medium are the same as the components of the initial medium without serum in step (1); in the fermenter, the volume ratio of the inoculated serum-free cultured hybridoma cells to the pre-filled serum-free medium is 0.1:4; (3) Serum-free medium was added to the fermenter and cultured for 1-2 days at a speed of 120 rpm, 37°C, pH 6.9-7.4, and 60%-80% PO2 until the cell abundance reached 5 x 10 5 / mL-7 x 10 5 / mL, and then cultured in the exponential phase at 120 rpm, 37°C, pH 6.9-7.4, and 40%-60% PO2 for 24-36 hours until the cell abundance reached 2 x10 6 / mL-5 x 10 6 / mL, and then entered the stable culture stage under 20%-40% PO2 environment. The cell abundance at the end of stable culture was 1.6 x 10 7 / mL; the volume ratio of the supplemented serum-free medium to the pre-loaded serum-free medium is 3:2; (4) When the cell mortality rate in the fermentation tank reaches 50%-60%, the cell suspension is collected for antibody purification to obtain the purified hybridoma cell monoclonal antibody.
2. The method for increasing the production of monoclonal antibodies in hybridoma cells according to claim 1, wherein: The complete culture medium in step (1) is any one of Hybridoma SFM, CD Hybridoma Medium, Medium NCTC-109, and PFHM-II Protein-Free Hybridoma Medium, the serum is IgG-free serum, the carbon source is any one of GlutaMAX, L-glutamine, and glucose, the vitamin is MEM, the cell growth factor is B-27, and the antibiotic is penicillin-streptomycin.
3. The method for increasing the production of monoclonal antibodies in hybridoma cells according to claim 2, wherein: The IgG-free serum is fetal bovine serum.
4. The method for increasing the production of monoclonal antibodies in hybridoma cells according to claim 1, wherein: The method further comprises the following steps after step (4): adding serum-free culture medium to the fermenter after collecting the cell suspension, and then repeating steps (3) and (4) to achieve batch culture without re-inoculating hybridoma cells.