A CHO cell additive and its application

Abstract

The present invention relates to the technical field of CHO cell culture, and specifically relates to a CHO cell additive and its application. The additive specifically includes: asparagine 0.5 - 3.0 g / L, leucine 0.5 - 3.0 g / L, isoleucine 0.5 - 3.0 g / L, valine 0.1 - 1.0 g / L, phenylalanine 0.1 - 1.0 g / L, and metal framework nanoparticles 0.1 - 10 mg / L. By using the additive, during fed-batch culture, the rate at which cells uptake nutrients is more balanced. During the overall culture process, certain key nutrients such as amino acids and vitamins can be maintained until cell harvest, enabling better maintenance of cell viability, and thus increasing the protein yield at cell harvest.

Description

Technical Field

[0001] The present invention relates to the technical field of CHO cell culture, and particularly relates to an additive for increasing the protein production of CHO cells and its application. Background Art

[0002] CHO cells are widely used in many fields such as recombinant protein drugs, vaccines, basic research, and drug screening. Especially in the field of biopharmaceuticals, monoclonal antibody drugs occupy a large market and have made great contributions to biopharmaceuticals. The culture medium that provides nutrients for CHO cells has developed from serum-containing adherent culture to the current serum-free suspension cell culture. Serum-free, chemically defined, high-density culture, and high-expression protein have become the standard for CHO cell culture media. Relatively speaking, it is more mature and the standards are relatively strict. Under the condition that all standards are met, more requirements are placed on the stability of quality, such as glycoform, charge, aggregates, etc. However, few people have conducted more in-depth research on its metabolism, including the consumption of multiple components in the culture medium. Currently, only some basic research has elaborated on the components of the culture medium, such as amino acids like asparagine and arginine, and the requirements of cells. However, it is not yet known whether cells really have such a large demand. Therefore, how to provide an additive that can significantly increase the protein production of CHO cells has become a hot topic and pain point in this industry. Summary of the Invention

[0003] In order to solve the technical problems mentioned in the background art, the present invention provides a CHO cell additive and its application. The technical solution is as follows:

[0004] A CHO cell additive is composed of the following components: specifically, asparagine 0.5 - 3.0 g / L, leucine 0.5 - 3.0 g / L, isoleucine 0.5 - 3.0 g / L, valine 0.1 - 1.0 g / L, phenylalanine 0.1 - 1.0 g / L, and metal-organic framework nanoparticles 0.1 - 10 mg / L.

[0005] Preferably, the metal-organic framework nanoparticles are prepared from zinc acetate dihydrate and 2-aminoterephthalic acid, and the mass ratio of zinc acetate dihydrate to 2-aminoterephthalic acid is 4:1.

[0006] Preferably, the metal-organic framework nanoparticles are 5 - 10 mg / L.

[0007] A CHO cell culture medium includes a CHO serum-free basal medium CE01, a CHO serum-free feeding medium CA01α, and a CHO serum-free feeding medium CA01β;

[0008] The above-mentioned CHO serum-free basal medium CE01 is added with a CHO cell additive as described in any one of the above, and the added volume ratio is 0.3% - 0.5%.

[0009] Use of a CHO cell additive according to any one of the foregoing, wherein the CHO cell additive is used for culturing CHO cells.

[0010] Preferably, the CHO cell additive is added to a serum-free medium for CHO cells, and the serum-free medium for CHO cells includes: serum-free basal medium CE01 for CHO, serum-free feeding medium CA01α for CHO, and serum-free feeding medium CA01β for CHO;

[0011] The culturing process of CHO cells is as follows:

[0012] S1. Prepare CHO cells and culture them in the serum-free basal medium CE01 for CHO. The shaking culture conditions are: 37 °C, 120 rpm, 5% CO2, 80% humidity. Passage the cells at a cell density of 0.6M, passage once every three days. After three passages, use the cells after the cell state is stable;

[0013] S2. Inoculate the cells at a density of 0.6M into the serum-free basal medium CE01 for CHO and culture them in a shaking incubator;

[0014] S3. On the 3rd, 5th, 7th, 9th, 11th, and 13th days of cell culture, add serum-free feeding medium CA01α for CHO at 3%, 4%, 5%, 5%, 5%, and 3% of the initial culture volume, and serum-free feeding medium CA01β for CHO at 0.3%, 0.4%, 0.5%, 0.5%, 0.5%, and 0.3%, and add the CHO cell additive. The added initial volume ratio is 0.3%, 0.4%, 0.5%, 0.5%, 0.5%, and 0.3%. During the culturing process, on the 3rd, 5th, 7th, 9th, and 11th days, take 1 mL of sample for amino acid detection. Note to supplement glucose to 8 g / L every other day. On the 13th day, supplement glucose to 4 g / L, and record the cell growth quantity and cell viability;

[0015] S4. Culture for 14 days, harvest the cells, collect the cell supernatant, and detect the production of adalimumab protein.

[0016] The beneficial effects of the present invention mainly lie in that the present invention proposes an additive for improving the protein yield of CHO cells. When combined with a commercial CHO basal and feeding medium, through the additive, the rate of cell nutrient uptake can be more balanced during fed-batch culture. During the overall culture process, certain key nutrients such as amino acids and vitamins can be maintained until cell harvesting, enabling better maintenance of cell viability, thereby increasing the protein yield at cell harvesting. The key technology is the metal-organic framework complex, which forms nanoparticles. It simultaneously has the advantages of organic polymers and inorganic compounds. Different metal ions combine with different organic ligands, and the resulting metal-organic framework structure is specific. It is loaded inside the carrier through physical adsorption or hydrogen bond molecular binding. Using zinc ions as the metal ions, it is synthesized by a rapid room-temperature synthesis method, which is simple to operate, and zinc ions themselves are important components of the medium without introducing other risks.

[0017] The mechanism of action of the present invention: Asparagine has been proven that during the fed-batch culture process of CHO cells, within a certain range, regardless of the addition amount, it will be completely consumed, and then the cells will switch to other metabolic pathways and consume other amino acids. Therefore, the concentrations of each component of the medium need to be carefully designed. If there is an additive that can slow down the uptake of certain amino acids or other vitamins, the overall metabolism of the cells will be healthier, and a higher cell viability and even higher quality attributes can be maintained. And nanotechnology, having developed to date, also plays a key role in many fields and is widely used in the medical field and materials science, especially in drug delivery systems. Nanoparticles can be used as carriers for precise delivery. If nanotechnology is applied to the medium, some amino acids, such as asparagine, can be slowly released, resulting in higher bioavailability and ensuring the presence of asparagine in the later stage, which can effectively increase the cell growth density and maintain cell viability. Specific embodiments

[0018] The technical solutions of the present invention will be further described below in conjunction with specific embodiments. The technical solutions in the embodiments of the present invention will be clearly and completely described below. Apparently, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0019] I. Experimental materials:

[0020] The amino acid components and reagents described in the following examples were all purchased from Sigma; commercial culture media: CHO serum-free basal medium CE01 (product number: CE000-N032), CHO serum-free feeding medium CA01α (product number: CA000-C011), feeding medium CA01β (product number: CA000-N021), were all purchased from Suzhou EK Bioscience Co., Ltd.; CHO-K1 cells expressing adalimumab protein (constructed by Nanjing Genscript Biotech Co., Ltd.); the other methods and reagents used in this example were conventional methods and reagents in the art unless otherwise specified.

[0021] II. Experimental methods:

[0022] An additive suitable for CHO cell culture in this application uses nanotechnology to enable cells to consume culture medium components evenly, maintain better cell viability during the later cell culture process, and achieve higher protein yields.

[0023] Based on the CHO serum-free basal medium CE01, the following components were added: including metal-organic framework nanoparticles and some amino acids, specifically including: asparagine, leucine, isoleucine, valine, and phenylalanine. Specific operation of metal-organic framework nanoparticles: Weigh 0.8 g of zinc acetate dihydrate and 0.2 g of 2-aminoterephthalic acid, and dissolve them completely with N,N-dimethylformamide respectively. At room temperature, quickly pour the zinc salt solution into the ligand solution to form a milky white precipitate. After magnetic stirring for 1 min, centrifuge to remove the supernatant. The obtained precipitate was washed thoroughly with N,N-dimethylformamide three times, and the precipitate was filtered out by centrifugation and dried for standby under natural conditions.

[0024] The range of some amino acids prepared with the CHO serum-free basal medium CE01 is: asparagine 0.5 - 3.0 g / L, leucine 0.5 - 3.0 g / L, isoleucine 0.5 - 3.0 g / L, valine 0.1 - 1.0 g / L, phenylalanine 0.1 - 1.0 g / L; the prepared metal framework nanoparticles are 0.1 - 10 mg / L.

[0025] Taking CHO-K1 cells, compared with the experimental group without adding metal-organic framework nanoparticles, the cell viability was maintained better and the protein yield was higher.

[0026] All the following examples adopted the fed-batch culture method, including the following steps:

[0027] Prepare cell line: CHO-K1 cells, cultured in CHO serum-free basal medium CE01. The shaking culture conditions are: 37°C, 120 rpm, 5% CO2, 80% humidity. Passage culture is carried out at a cell density of 0.6M, once every three days. After three passages, when the cell state is stable, it is reserved for use;

[0028] Further, inoculate the cells at a density of 0.6M into CHO serum-free basal medium CE01 and culture them in a shaking incubator;

[0029] Further, on the 3rd, 5th, 7th, 9th, 11th, and 13th days (D03, D05, D07, D09, D11, D13) of cell culture, add 3%, 4%, 5%, 5%, 5%, and 3% of the initial culture volume of CHO serum-free feeding medium CA01α, and 0.3%, 0.4%, 0.5%, 0.5%, 0.5%, and 0.3% of CHO serum-free feeding medium CA01β, and add additives of different examples, with the addition ratio being 0.3%, 0.4%, 0.5%, 0.5%, 0.5%, and 0.3% of the initial culture volume. During the culture process, on the 3rd, 5th, 7th, 9th, and 11th days, take 1 mL of the sample for amino acid detection. Note that the sugar is supplemented to 8 g / L every other day, and on the 13th day, the sugar is supplemented to 4 g / L, and record the cell growth quantity and cell viability;

[0030] Further, culture until the 14th day (D14), harvest the cells, collect the cell supernatant, and detect the production of adalimumab protein.

[0031] Specific embodiment scheme:

[0032] Example 1: CHO cell additive 1: asparagine 0.5 g / L, leucine 0.5 g / L, isoleucine 0.5 g / L, valine 0.1 g / L, phenylalanine 0.1 g / L;

[0033] Example 2: CHO cell additive 2: asparagine 3.0 g / L, leucine 3.0 g / L, isoleucine 3.0 g / L, valine 1.0 g / L, phenylalanine 1.0 g / L;

[0034] Example 3: CHO cell additive 3: metal-organic framework nanoparticles 1.0 mg / L, asparagine 0.5 g / L, leucine 0.5 g / L, isoleucine 0.5 g / L, valine 0.1 g / L, phenylalanine 0.1 g / L;

[0035] Example 4: CHO cell additive 4: metal-organic framework nanoparticles 1.0 mg / L, asparagine 3.0 g / L, leucine 3.0 g / L, isoleucine 3.0 g / L, valine 1.0 g / L, phenylalanine 1.0 g / L;

[0036] Example 5: CHO cell additive 5: Metal-organic framework nanoparticles 5.0 mg / L, asparagine 0.5 g / L, leucine 0.5 g / L, isoleucine 0.5 g / L, valine 0.1 g / L, phenylalanine 0.1 g / L;

[0037] Example 6: CHO cell additive 6: Metal-organic framework nanoparticles 5.0 mg / L, asparagine 3.0 g / L, leucine 3.0 g / L, isoleucine 3.0 g / L, valine 1.0 g / L, phenylalanine 1.0 g / L;

[0038] Example 7: CHO cell additive 7: Metal-organic framework nanoparticles 10.0 mg / L, asparagine 0.5 g / L, leucine 0.5 g / L, isoleucine 0.5 g / L, valine 0.1 g / L, phenylalanine 0.1 g / L;

[0039] Example 8: CHO cell additive 8: Metal-organic framework nanoparticles 10.0 mg / L, asparagine 3.0 g / L, leucine 3.0 g / L, isoleucine 3.0 g / L, valine 1.0 g / L, phenylalanine 1.0 g / L;

[0040] Control group: Conventional cell culture with CHO serum-free basal medium CE01 combined with CHO serum-free feeding medium CA01α and feeding medium CA01β.

[0041] The test results are as follows:

[0042] 1. Table 1 shows the viable cell density, and the cell growth in different examples. The experimental groups without metal-organic framework nanoparticles (Examples 1 and 2) are basically the same as the control group. For the experimental groups with metal-organic framework nanoparticles, the cell growth can still be maintained above 15 M on the 14th day.

[0043] Table 1

[0044] Live cell density D03 D05 D07 D09 D11 D13 D14 Example 1 4.06 8.95 14.1 17.1 15.6 12.3 3.54 Example 2 4.06 8.68 13.3 16.3 14.9 12.1 2.75 Example 3 4.06 9.63 13.4 16.6 16.1 14.8 12 Example 4 4.06 9.34 14.1 17.3 17.3 14.6 12.6 Example 5 4.06 9.38 13.9 16.3 17.4 17.7 15.7 Example 6 4.06 9.36 13.5 16 16.6 16.4 15.8 Example 7 4.06 8.72 14.4 17 17.9 17.8 15.4 Example 8 4.06 8.4 14.3 17.4 17 17.1 14.7 Control group 4.06 9.27 14.3 17.2 15.5 12.5 2.45

[0045] 2. Table 2 shows the cell viability in different examples. The experimental groups with metal-organic framework nanoparticles maintain a significantly better viability, and can still be maintained above 80% on the 14th day.

[0046] Table 2

[0047] Cell viability D03 D05 D07 D09 D11 D13 D14 Example 1 99.15 99.32 98.78 97.69 95.52 83.24 20.95 Example 2 99.15 98.9 98.97 97.29 95.82 84.12 23.2 Example 3 99.15 98.98 98.72 97.56 95.82 95.48 83.45 Example 4 99.15 98.98 98.9 97.06 95.53 95.07 82.12 Example 5 99.15 99.05 98.74 97.14 95.11 93.62 91.98 Example 6 99.15 98.77 98.86 97.54 95.01 92.72 90.69 Example 7 99.15 99.04 99.16 98.09 96.15 93.95 90.54 Example 8 99.15 99.02 98.89 97.74 96.49 95.02 92.88 Control group 99.15 98.97 98.69 97.44 95.53 84.81 16.4

[0048] 3. Table 3 shows the protein production in different examples. The experimental groups with metal-organic framework nanoparticles have a protein production 20% higher than that of the control group.

[0049] Table 3

[0050] Protein yield D14 (g / L) Enhancement ratio Example 1 5.21 0.97% Example 2 5.23 1.36% Example 3 5.74 11.24% Example 4 5.63 9.11% Example 5 6.23 20.74% Example 6 6.45 25.00% Example 7 6.21 20.35% Example 8 6.31 22.29% Control group 5.16 0

[0051] 4. Table 4 shows the asparagine content in the cell supernatant in different embodiments. In the control group, it was exhausted on the 11th day, while in the experimental group with metal-organic framework nanoparticles added, about 400 μM of asparagine could still be detected on the 14th day, indicating that metal-organic framework nanoparticles can slow down the ability of cells to uptake asparagine. The possible mechanism is that metal-organic framework nanoparticles have a protective effect, and the components in the culture medium can be loaded inside the carrier through physical adsorption or hydrogen bond molecular binding, slowing down the release of the culture medium.

[0052] Table 4

[0053] Asparagine D03 D05 D07 D09 D11 D13 D14 Example 1 1103 818 691 410 0 0 0 Example 2 1103 802 628 408 0 0 0 Example 3 1103 812 732 531 334 0 0 Example 4 1103 834 770 505 329 0 0 Example 5 1103 933 832 726 631 548 327 Example 6 1103 920 879 787 619 565 351 Example 7 1103 935 840 754 608 573 327 Example 8 1103 958 879 703 603 596 351 Control group 1103 723 597 398 0 0 0

[0054] The above-described embodiments merely represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the present invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims.

Claims

1. A culture medium additive for CHO cells, characterized in that: It is composed of the following ingredients, specifically: asparagine 0.5~3.0g / L, leucine 0.5~3.0g / L, isoleucine 0.5~3.0g / L, valine 0.1~1.0g / L, phenylalanine 0.1~1.0g / L and metal framework nanoparticles 0.1~10mg / L; The metal framework nanoparticles are prepared from zinc acetate dihydrate and 2-aminoterephthalic acid, and the mass ratio of zinc acetate dihydrate to 2-aminoterephthalic acid is 4:

1.

2. The culture medium additive for CHO cells according to claim 1, characterized in that: The metal framework nanoparticles are 5-10 mg / L.

3. A CHO cell culture medium, characterized in that Including CHO serum-free basal medium CE01, CHO serum-free feed medium CA01α and CHO serum-free feed medium CA01β; The CHO serum-free basal medium CE01 is added with a CHO cell culture medium additive as described in any one of claims 1 to 2, with the added volume ratio being 0.3% to 0.5%.

4. The use of a culture medium additive for CHO cells according to any one of claims 1 to 2, characterized in that: The CHO cell culture medium additive is used for culturing CHO cells expressing adalimumab protein.

5. The use of the culture medium additive for CHO cells according to claim 4, characterized in that: The CHO cell culture medium additive is added into a CHO cell serum-free culture medium, wherein the CHO cell serum-free culture medium comprises: CHO serum-free basal culture medium CE01, CHO serum-free feed culture medium CA01α and CHO serum-free feed culture medium CA01β; The culture process of CHO cells is as follows: S1. Prepare CHO cells and culture them in CHO serum-free basal medium CE01. The shaking culture conditions are: 37°C, 120rpm, 5% CO2, 80% humidity. Subculture is performed at a cell density of 0.6M. The cells are subcultured every three days. After three subcultures, the cells are stable and ready for use. S2, inoculate the cells at a density of 0.6M in CHO serum-free basal medium CE01 and culture in a shaker; S3. On the 3rd, 5th, 7th, 9th, 11th and 13th days of cell culture, 3%, 4%, 5%, 5%, 5% and 3% of the initial culture volume of CHO serum-free feed medium CA01α and 0.3%, 0.4%, 0.5%, 0.5%, 0.5% and 0.3% of CHO serum-free feed medium CA01β were added respectively, and the culture medium additives for CHO cells were added, and the initial volume ratios were 0.3%, 0.4%, 0.5%, 0.5%, 0.5% and 0.3%. During the culture process, 1 mL of sample was retained on the 3rd, 5th, 7th, 9th and 11th days for amino acid detection. Note that sugar was supplemented to 8 g / L every other day. On the 13th day, sugar was supplemented to 4 g / L, and the cell growth number and cell viability were recorded; S4. After culturing for 14 days, the cells were harvested, the cell supernatant was collected, and the adalimumab protein production was detected.