Method for producing antibody by improving cell inoculation density through seed perfusion culture and application

Abstract

The invention relates to a method for producing an antibody by improving cell inoculation density through seed perfusion culture and application. In N-1 seed cell culture, through a perfusion culture process mode, especially according to the cell density, the perfusion rate is adjusted to timely increase nutrient substances in a cell culture system and discharge metabolic wastes in cell culture to increase the density of living cells, so that the initial density of N-generation cells is greatly improved. The method is convenient to amplify and simple to operate, the antibody yield can be increased in the same culture period, or a target product can be obtained in a shorter culture period, and the production cost is greatly reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of mammalian cell culture in biomedicine, and in particular to the application of a seed perfusion culture method and a high-density inoculation culture method in producing antibodies. Background Art

[0002] Animal cell culture technology was proposed in the early 20th century. After continuous development, it has become a widely used technical method in the field of biological and medical research. Mammalian suspension culture technology mainly includes batch culture, fed-batch culture and perfusion culture. In the traditional batch culture process, the consumption of nutrients and the accumulation of harmful metabolites in the later stage of cell culture cause the cell viability to decrease, and the protein cannot be expressed better. Fed-batch culture can supplement the consumption of nutrients in the culture process by continuously adding concentrated nutrients or culture medium, so that cells can grow and maintain a high cell density for a long time, so that the target product can be expressed at a high level. The fed-batch culture process is simple to operate and has strong reproducibility, which is conducive to the stability of product batches and cost control. However, the disadvantages of the fed-batch culture mode are: first, the initial cell inoculation density is low, and it takes 6-8 days of culture to enter the protein expression stage. The expression cycle is short, and the production capacity is limited by the size and site of the reactor. In addition, due to the large consumption of nutrients and the accumulation of metabolic wastes caused by cell growth during the culture process, on the one hand, it has a certain toxic effect on cells, which will limit the further growth of cells and cause cell death, and on the other hand, it will inhibit the production of antibodies. Perfusion culture technology is to continuously inject fresh culture medium and continuously discharge metabolic wastes during cell culture. The advantages of the perfusion culture process are that cells or products can be retained in the reactor, harmful metabolic wastes are constantly removed and accumulated in low amounts, cells are stably placed in a good nutritional environment, a high cell density is maintained, and the culture cycle is extended, thereby greatly increasing product yields. The culture medium is constantly replaced during perfusion culture. On the one hand, the continuous consumption of the culture medium increases costs, and on the other hand, the output of a large amount of products poses a challenge to downstream purification, and the scale-up process is somewhat challenging.

[0003] In order to reduce the cost of biomedicine, scientific researchers have been continuously exploring, and the cell culture process has been continuously developed, from batch culture mode, feed addition to continuous flow culture mode. In recent years, there have also been literature reports on the use of increasing the initial inoculation density to extend the antibody expression time or shorten the culture cycle, increase the yield of the target protein, improve production efficiency, and reduce production costs.

[0004] In traditional cell subculture methods, the cell density of N-1 seeds can only grow to 2-6×10 6cells / ml, which limits the density of the initial inoculation of the Nth generation, resulting in a long plateau period of cell growth, a relatively short antibody expression time, and a low antibody expression level. In CN109576212B, the density of the N-1 seed was increased by fed-batch culture, and the cell density reached 15×10 6 cells / ml, the seeding density of N-generation cells can reach 3×10 6 cells / ml, the expression of antibodies can be increased by 20%, or the entire cell culture cycle can be shortened. The method of using a feeding method to increase the N-1 seed cell volume increases harmful metabolites in the cells, which limits the further growth of the cells and the initial inoculation density of the N generation. This method has certain limitations. How to further increase the density of N-1 seed cells is a key issue in increasing the initial density of the N generation. How to maintain a good cell state in the N generation culture is the key to cell culture. In view of this, the present invention is specially proposed. Summary of the invention

[0005] To overcome the deficiencies of the prior art, the present invention increases the N-1 seed density through a perfusion culture method, thereby increasing the initial inoculation density of the N generation culture, and ultimately increasing the expression level of the antibody.

[0006] The N-1 seed perfusion culture method of the present invention adjusts the perfusion rate according to the growth of the N-1 seed cells through the perfusion culture process, and the maximum cell density can reach 120×10 6 cells / ml, compared with the conventional cell passage method, the cell density increased by 20-30 times, and the cell density was greatly improved. Due to the increase in seed cell density, the initial inoculation cell density of the N generation increased from 5×10 6 cells / ml increased to 5-25×10 6 cells / ml; the cell viability can be maintained at 97%; the N-generation cell seeding density is increased from 0.5×10 6 cells / ml increased to 20×10 6 cells / ml, the initial cell inoculation density increased to 40 times the original; on the one hand, it shortened the time for cells to reach the plateau phase and prolonged the antibody expression time; on the other hand, due to sufficient nutrients, the highest cell growth density was also greatly improved. The highest cell density of the N-generation high-density inoculation is higher than that of the traditional process, and the platform culture period is extended, which greatly increases the expression of antibodies.

[0007] N-1 seeds can achieve high density and high viability through perfusion culture, which can meet the requirements of ultra-high density inoculation of the Nth generation. This process has the characteristics of strong controllability, simple process control, and convenient operation, which provides broad application prospects for realizing high-density and high-yield cell culture optimization process.

[0008] Specifically, the present invention relates to an N-1 seed perfusion culture method, an N-generation high-density inoculation method for producing antibodies, a perfusion culture method is used to increase the density of seed cells in the N-1 seed amplification culture stage, and a method of increasing the inoculation density in the N-generation culture stage to prolong the protein expression time.

[0009] One of the purposes of the present invention is to provide a culture method for obtaining a high viable cell density by a perfusion culture process during N-1 seed expansion, which includes a perfusion culture step, in particular, a step of adjusting the perfusion rate according to the cell density.

[0010] Specifically, the above method comprises the following steps:

[0011] (1) Seed cell expansion, the initial cell seeding density is 0.3-0.5×10 6 cells / ml, for cell culture;

[0012] (2) N-1 seed perfusion culture, cell density grows to 2-10×10 6 cells / ml, and then perfusion was started. The perfusion rate of N-1 seed was adjusted to 0.5-1vvd; the cell density was grown to 10-20×10 6 cells / ml, the perfusion rate of N-1 seed was adjusted to 1-2vvd; the cell density was grown to 20-60×10 6 cells / ml, the perfusion rate of N-1 seed was adjusted to 2-3 vvd; the cell density was 60×10 6 cells / ml, and the perfusion rate of N-1 seed was adjusted to 3-4 vvd.

[0013] In some embodiments, the host cell is a CHO cell.

[0014] In some embodiments, the host cell is Lonza's CHOK1SV cell.

[0015] In some embodiments, the density of N-1 seed cells is 40-120×10 6 cells / ml.

[0016] In some embodiments, a certain amount of a shear-resistant agent is added to the medium used for seed perfusion.

[0017] In some embodiments, the anti-shear agent is poloxamer 188.

[0018] In some embodiments, the culture medium for seed cell expansion and seed perfusion is Growth A medium.

[0019] In some embodiments, the N-1 seed perfusion expansion culture adopts a segmented oxygen control mode. When the cell density is less than 40×10 6 cells / ml, and the dissolved oxygen was controlled by macrovesicle-associated dissolved oxygen. When the cell density was greater than 40×10 6 cells / ml, the dissolved oxygen was controlled by means of large bubbles constantly passing oxygen and microbubbles associated with dissolved oxygen, and 2% poloxamer 188 was added to the culture medium to reduce the shearing effect of bubbles and stirring on the cells.

[0020] In some embodiments, perfusion culture of N-1 seed cells occurs in a bioreactor, such as a stirred tank glass reactor or a disposable bioreactor.

[0021] In some embodiments, cell perfusion utilizes a continuous perfusion device that uses an alternating tangential flow cell retention system, and the hollow fiber column has a pore size of 0.1-0.6 μm.

[0022] A second object of the present invention is to provide a culture process for N-generation ultra-high density cell inoculation.

[0023] Specifically, cells in the logarithmic growth phase of N-1 seeds are taken, inoculated at a high density for N generations, and the process parameters for N-generation cultivation are adjusted for cultivation.

[0024] In some embodiments, the cell density in the N-1 logarithmic growth phase is: (40-120)×10 6 cells / ml, the initial cell density of generation N is (5-25)±2×10 6 cells / ml, specifically (5-10)±2×10 6 cells / ml、(10-15)±2×10 6 cells / ml、(10-20)±2×10 6 cells / ml、(15-20)±2×10 6 cells / ml、(20-25)±2×10 6 cells / ml.

[0025] In some embodiments, the medium used for culturing cells at passage N is Dynamis medium.

[0026] In some embodiments, the N-generation cell culture process parameters are: temperature of 30-38° C., pH of 6.6-7.5, dissolved oxygen of 20%-90%, and stirring speed of 20-500 rpm.

[0027] In some embodiments, the N-generation cell culture adopts a two-stage temperature culture mode, the cells are inoculated in the bioreactor at an initial density, the cell culture temperature is 37°C ± 0.5, and when the cell density grows to 35-45×10 6 cells / ml, the cell culture temperature was lowered to 31-33°C until the end of the culture.

[0028] In some embodiments, the sugar supplement amount for the N generation culture is 9-11 g / L, the residual sugar content is 1-3 g / L, and preferably the residual sugar content is not less than 1 g / L.

[0029] The third purpose of the present invention is to apply the above method and culture process in mammalian cell culture.

[0030] Preferably, the mammalian cell is a CHO cell; more preferably, the CHO cell is Lonza's CHOK1SV cell.

[0031] In some embodiments, the mammalian cell or CHO cell or Lonza's CHOK1SV Cells are used to produce protein.

[0032] In some embodiments, the protein is an antibody.

[0033] In some embodiments, the antibody is a HER3 antibody.

[0034] In some embodiments, the HER3 antibody is patritumab antibody or Lumretuzumab antibody. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Growth profile of N-1 seed perfused cells.

[0036] Figure 2 Viability profile of N-1 seed perfused cells.

[0037] Figure 3 Fluid exchange rate profile for N-1 seeded perfused cells.

[0038] Figure 4 Cell growth profile of high-density inoculation in the Nth generation.

[0039] Figure 5 Cell viability profile of N-generation high-density inoculation.

[0040] Figure 6 Expression profile of antibodies after high-density vaccination in the Nth generation. Example

[0041] The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

[0042] Example

[0043] This embodiment is a method for producing patritumab antibody (the amino acid sequences of the light and heavy chains of patritumab antibody are shown in CN116135232A) by increasing the seeding density of N-generation cells through N-1 seed perfusion culture. Taking the production process of a 5L reactor as an example, the method comprises the following steps:

[0044] Cell construction

[0045] Lonza's CHOK1SV stably expressing patritumab monoclonal antibody System cell line construction

[0046] 1. Expression plasmid construction: The antibody light and heavy chain base sequences with endonuclease sites are synthesized by polymerase chain reaction (PCR) and then connected to the vector to construct the expression plasmid. To ensure the accuracy of the target gene cloned into the vector, the target gene in the expression plasmid needs to be sequenced before transfecting the host cell.

[0047] 2. Cell transfection: The expression plasmid mixed with transfection reagent is introduced into mammalian cells through electroporation transfection.

[0048] 3. Pool cell screening: The transfected cells are screened and cultured in a selective medium without glutamine supplemented with a certain amount of MSX until the viability is fully restored to produce a stable cell pool.

[0049] 4. Monoclonal screening: To ensure the uniqueness of monoclones, more than one round of clone screening is required. Usually, the method of plating is less than 1 cell / well. 300 clones are selected for step-by-step amplification through 24-well plates, 6-well plates, and T-flasks. Finally, 10-20 clones are selected for shake flask scale evaluation. Based on cell growth and product quality characteristics, 4-6 clones are selected for further evaluation in the reactor to determine the top 1 monoclonal clone.

[0050] Expansion of seed cells

[0051] Lonza's CHOK1SV stably expressing patritumab monoclonal antibody The cells were revived in GrowthA (Cat. No. 94120) medium without Gln (glutamine) at an initial inoculation density of 0.3-0.5×10 6cells / ml, subcultured once every 3 days, and cultured in 125-1000ml shake flasks. The shake flasks were placed in a shaker (infors) at 37.0℃±0.5, amplitude 50mm, speed 120rpm, 5% CO2 for culture. The cells grew to 3-6×10 6 cells / ml for the next step of inoculation.

[0052] N-1 seed perfusion culture

[0053] Original process: initial inoculation density is 0.3-0.5×10 6 cells / ml, cultured in the basic medium GrowthA, placed in an infors shaker, culture temperature 37.0℃±0.5, shaker speed 120±5rpm, humidity 80%±10%. After 3 days of culture, the cell viable cell density was 3-6×10 6 cells / ml, and inoculate N generations.

[0054] The process of the present invention: N-1 seed culture adopts perfusion process, uses 5L NBS bioreactor, the tank body is connected with Repligen hollow fiber column (0.22μm, surface area 0.13m 2 ) are connected with silicone tubes and the alternating tangential flow (ATF) perfusion culture method is adopted. After the bioreactor is cleaned before inoculation, it is connected to the hollow fiber column and the base, placed in a sterilizer at 121°C, and sterilized by high pressure for 30 minutes. After sterilization, the sterilized bioreactor and hollow fiber column are connected to the control equipment, and a certain amount of sterile air is introduced to maintain positive pressure. Then, fresh GrowthA culture medium is injected into the reactor, and the temperature, stirring speed, pH, dissolved oxygen and other parameters are set according to the process requirements. At the same time, the ATF system is turned on to fill the hollow fiber column with culture medium, and it is continuously operated overnight. After observing that there is no abnormality, the cells are inoculated.

[0055] The seed inoculation density of N-1 is 0.3-0.5×10 6 cells / ml, reactor parameters: temperature: 37.0℃±0.5, pH: 7.0±0.2, dissolved oxygen: 40%, stirring: 90rpm, gas (oxygen, air, carbon dioxide) is connected to dissolved oxygen by the bioreactor.

[0056] On day 0, 0.5×10 6 After inoculating cells, the cells were cultured for two days. When the cell density reached 3×10 6 cells / ml, the perfusion rate was 0.5vvd, and the cell density was between 6-20×10 6cells / ml, the perfusion rate was adjusted to 1 vvd, and the cell density was 20-40×10 6 cells / ml, perfusion rate 2vvd, cell density 40-60×10 6 cells / ml, cell perfusion rate 3vvd, cell density 60-120×10 6 cells / ml, the cell perfusion rate was 4vvd. The glucose concentration was maintained above 2g / L. 75×10 cells in the logarithmic growth phase were taken. 6 cells / ml for N generations of inoculation.

[0057] The aeration strategy of the reactor is: when the cell density is less than 40×10 6 cells / ml, and the dissolved oxygen was controlled by macrovesicle-associated dissolved oxygen. When the cell density was greater than 40×10 6 cells / ml, the dissolved oxygen was controlled by means of large bubbles constantly passing oxygen and microbubbles associated with dissolved oxygen, and 2% poloxamer 188 was added to the culture medium to reduce the shearing effect of bubbles and stirring on the cells.

[0058] N generation high density inoculation

[0059] Original process: inoculation density is 0.8±0.2×10 6 cells / ml, initial culture temperature 37℃±0.5, cooling to 33℃±0.5 on the 6th day, stirring speed of 2L reactor 250rpm, pH controlled at 6.7-7.2, DO controlled at 40%, feeding strategy of Hyclone Cell Boost 7A / Cell boost 7B supplemented with 2% / 0.2% on the 3rd day, 5% / 0.5% daily on the 5th and 7th days, 4% / 0.4% daily on the 9th and 11th days, and 3% / 0.3% on the 13th day. Glucose supplementation strategy was supplemented from less than 4g / L to 6g / L, and samples were taken every day after cell inoculation to detect live cell density, cell viability, blood gas (pH, pCO2, pO2, Na+, K+, Ga2+, Cl-), and biochemistry (glucose, glutamine, glutamate, ammonium ion, lactate).

[0060] The optimized process 1 of the present invention is to take seeds in the logarithmic growth phase of N-1 perfusion culture, with a cell density of 75×10 6 cells / ml, and the initial cell inoculation density was 10±2×10 6cells / ml, initial culture temperature 37℃±0.5, cooling to 33℃±0.5 on the 3rd day, stirring speed of 5L reactor 90rpm, pH controlled at 6.7-7.2, DO controlled at 40%, feeding strategy of Hyclone Cell Boost 7A / Cell boost 7B supplemented with 2% / 0.2% on the 1st day, 2.5% / 0.25% on the 2nd and 3rd days, 3% / 0.3% on the 4th, 5th and 6th days, 2.5% / 0.25% on the 7th, 8th, 9th and 10th days, 2% / 0.2% on the 11th, 12th, 13th and 14th days. Glucose was supplemented to 9g / L, and glucose was maintained at no less than 1g / L. After cell inoculation, samples were taken every day to detect live cell density, cell viability, blood gas (pH, pCO2, pO2, Na+, K+, Ga2+, Cl-), and biochemistry (glucose, glutamine, glutamate, ammonium ion, lactic acid).

[0061] Process 2 optimized by the present invention: Take seeds in the logarithmic growth phase of N-1 perfusion culture, with a cell density of 75×10 6 cells / ml, and the initial cell inoculation density was 20±2×10 6 cells / ml, initial culture temperature 37℃±0.5, cooling to 33℃±0.5 on the first day, stirring speed of 5L reactor 90rpm, pH controlled at 6.7-7.2, DO controlled at 40%, feeding strategy of Hyclone Cell Boost 7A / Cell boost 7B supplemented with 2% / 0.2% on the 0th and 1st day, 2.5% / 0.25% on the 2nd day, 3% / 0.3% on the 3rd, 4th, 5th and 6th day, 2.5% / 0.25% on the 7th, 8th, 9th and 10th day, 2% / 0.2% on the 11th, 12th, 13th and 14th day. Glucose was supplemented to 11g / L, and glucose was maintained at no less than 1g / L. After cell inoculation, samples were taken every day to detect live cell density, cell viability, blood gas (pH, pCO2, pO2, Na+, K+, Ga2+, Cl-), and biochemistry (glucose, glutamine, glutamate, ammonium ion, lactic acid).

[0062] The cells were harvested on the 15th day of culture or when the cell viability was lower than 50%. The cells were collected by centrifugation at 8000rpm for 20min. 1ml of supernatant was taken for yield measurement, and 15ml of supernatant was purified by magnetic bead purification and sent for quality inspection after purification.

[0063] Table 1 Summary of reactor cell culture production results

[0064]

[0065] Table 2 Summary of protein quality SEC, CEX, and NR-CE test results

[0066]

[0067] Table 3 Summary of protein quality glycoform detection results

[0068]

[0069] As can be seen from Table 1, compared with the original process: the culture cycle is 15 days, and the culture process of high density inoculation of N generations using the logarithmic phase seeds of the N-1 perfusion culture of the present invention can increase the protein expression amount of process 1 by 73.6%, and the protein expression amount of process 2 can increase by 85.5%. From Tables 2 and 3, it can be seen that there is no significant difference in the quality of process 1 and process 2 compared with the original process, and the quality is acceptable.

Claims

1. A method for obtaining a high viable cell density by a perfusion culture process during N-1 seed expansion, the method comprising a perfusion culture step, in particular a step of adjusting the perfusion rate according to the cell density, the method comprising the following steps: (1) Seed cell expansion, the initial cell seeding density is 0.3-0.5×10 6 cells / ml, for cell culture; (2) N-1 seed perfusion culture, cell density grows to 2-10×10 6 cells / ml, and then perfusion was started. The perfusion rate of N-1 seed was adjusted to 0.5-1vvd; the cell density was grown to 10-20×10 6 cells / ml, the perfusion rate of N-1 seed was adjusted to 1-2vvd; the cell density was grown to 20-60×10 6 cells / ml, the perfusion rate of N-1 seed was adjusted to 2-3 vvd; the cell density was 60×10 6 cells / ml, and the perfusion rate of N-1 seed was adjusted to 3-4 vvd.

2. The method according to claim 1, wherein the cell is a CHO cell, preferably, the cell is Lonza's CHO KSV cell.

3. The method according to any one of claims 1 to 2, wherein: A certain amount of an anti-shear agent is added to the medium for seed perfusion. Preferably, the anti-shear agent is poloxamer 188. More preferably, the medium for seed perfusion is Growth A medium.

4. The method according to any one of claims 1 to 3, wherein the N-1 seed perfusion expansion adopts a segmented oxygen control mode, and when the cell density is less than 40×10 6 cells / ml, and the dissolved oxygen was controlled by macrovesicle-associated dissolved oxygen. When the cell density was greater than 40×10 6 cells / ml, the dissolved oxygen was controlled by means of large bubbles constantly passing oxygen and microbubbles associated with dissolved oxygen, and 2% poloxamer 188 was added to the culture medium to reduce the shearing effect of bubbles and stirring on the cells.

5. A culture method for N-generation ultra-high density cell inoculation, wherein the cell density of the N-1 logarithmic growth phase is used for N-generation high-density inoculation, and the initial density of the N-generation cells is 5-25±2×10 6 cells / ml, preferably, the initial cell density of generation N is 10-20±2×10 6 cells / ml, more preferably, the initial cell density of generation N is 20±2×10 6 cells / ml.

6. The culture method according to claim 5, wherein the culture temperature is 30-38°C, the pH is 6.6-7.5, the dissolved oxygen is 20%-90%, and the stirring speed is 20-500 rpm.

7. The culture method according to any one of claims 5 to 6, wherein the N-generation cell culture adopts a two-stage temperature culture mode, the cells are inoculated at an initial density, the cell culture temperature is 37°C ± 0.5, and when the cell density grows to 35-45×10 6 cells / ml, the cell culture temperature was lowered to 31-33°C until the end of the culture.

8. The culture method according to any one of claims 5 to 7, wherein the amount of sugar added to the N generation culture is 9-11 g / L, and the residual sugar content is 1-3 g / L.

9. Use of the method according to any one of claims 1 to 4 and the culture method according to any one of claims 5 to 8 in mammalian cell culture, preferably, the mammalian cell is a CHO cell, more preferably, the CHO cell is Lonza's CHOK1SV cell.

10. The use according to claim 9, wherein the mammalian cell is used to prepare a protein, preferably, the protein is an antibody, more preferably, the antibody is a HER3 antibody, and further preferably, the HER3 antibody is a patritumab antibody.

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