A CHO-K1 2F10 cell

Abstract

The present invention belongs to the field of cell engineering, and specifically relates to a CHO-K1 2F10 cell, which can grow at high density in a serum-free, high shear force environment, and at the same time, the lactate accumulation is less than 0.5 g / L, thereby avoiding cell death caused by damage to the cells due to high shear force and lactate accumulation during the amplification process, so that the cell has broad application prospects in large-scale production.

Description

Technical Field

[0001] The invention belongs to the field of cell engineering, and specifically relates to a CHO-K1 2F10 cell. Background Art

[0002] At present, the heterologous expression systems that have achieved large-scale industrial production are mainly divided into prokaryotic expression systems and eukaryotic expression systems. Eukaryotic expression systems mainly include yeast, insect, plant and mammalian cell expression systems. Among them, the CHO cell line is one of the most widely used systems in the current eukaryotic expression system. Compared with other mammalian cell expression systems, the CHO cell line has the following advantages: (1) Although CHO is an adherent cell, it is easy to culture in suspension and can achieve large-scale production; (2) It has the ability to express recombinant genes efficiently; (3) It secretes less endogenous proteins, which is conducive to the separation and purification of the target product; (4) It has accurate post-transcriptional translation modification functions, etc. However, there are still many problems in the large-scale culture of CHO cells, such as high culture cost, low protein expression and difficulty in process amplification. Among them, during the process of culture process amplification, the shear force formed by stirring, ventilation, bubble bursting, etc. causes great damage to the cells, which will lead to cell death during the culture process. In addition, the low stirring speed and ventilation limit the continuous high-speed division of cells, making it impossible to achieve a high cell density, which ultimately leads to a decrease in the yield of the cell target protein. Summary of the invention

[0003] Based on the requirements of the above-mentioned prior art, the purpose of the present invention is to provide a cell that can adapt to high shear force and grow at high density, so as to solve the problem of cell death and low density caused by shear force in the current industrial scale-up process. At the same time, the cell line of the present invention is applied to host cells for the expression of recombinant proteins.

[0004] The details are as follows: a CHO-K1 2F10 cell, the CHO-K1 2F10 cell has a preservation number of CGMCC45325.

[0005] The CHO-K1 2F10 cells are used as host cells for antibody protein expression.

[0006] The CHO-K1 2F10 cells of the present invention can be used as host cells to grow at high density in a high shear force environment and express antibody proteins.

[0007] The high shear force environment of the present invention refers to: culturing under the condition of 185 rpm.

[0008] High density refers to: 10 7 cells / mL or above.

[0009] In another aspect, the present invention discloses a method for preparing CHO-K1 2F10 cells, which comprises:

[0010] (1) Cells are cultured in serum-free suspension;

[0011] (2) performing shear stress tolerance training on the cells acclimated in step (1);

[0012] (3) Screening of monoclonal strains.

[0013] The step (3) uses conventional methods to screen the viable cell density of 10 7 cells / mL, preferably more than 2.0×10 7 cells / mL of viable cell density.

[0014] Furthermore, the lactic acid content of the screened monoclonal strain was lower than 0.5 g / L in the late stage of Fed-Batch.

[0015] On the other hand, a method for expressing antibodies using the CHO-K1 2F10 cells is disclosed, comprising:

[0016] (1) Transform the expression plasmid encoding the target protein gene into CHO-K1 2F10 cells in the logarithmic growth phase;

[0017] (2) Take the cell culture supernatant and obtain the target protein.

[0018] Beneficial Effects

[0019] The CHO-K1 2F10 cells of the present invention can be used as host cells to grow at high density in a high shear force environment and express antibody proteins.

[0020] The CHO-K1 2F10 cells of the present invention can be cultured in a variety of culture media at a maximum viable cell density of more than 2×10 7 cells / mL. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 : Cells were grown in 10% FBS complete medium;

[0022] Figure 2 :CHO-K1 cells were grown in adherent culture medium A (containing 2 mM Gln) containing 1.25% FBS;

[0023] Figure 3 :CHO-K1 cells were grown in suspension in medium A;

[0024] Figure 4:Cell shape images of CHOK1 cells under 95rpm (A) and 185rpm (B);

[0025] Figure 5 :The viable cell density of the selected 12 monoclonal cell lines in subculture within 30 days;

[0026] Figure 6 :The cell doubling time of the selected 12 monoclonal cell lines in subculture within 39 days;

[0027] Figure 7 : A is the batch culture live cell density curve of 12 monoclonal strains in medium A; B is the batch culture live cell density curve of cell strains in medium B;

[0028] Figure 8 :A is the live cell density curve of 12 monoclonal strains in medium A by batch feeding; B is the live cell density curve of cell strains in medium B by batch feeding; C is the lactic acid content of 12 monoclonal strains on Day 8 by batch feeding Specific embodiments

[0029] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation of the present invention is described in detail below in conjunction with specific embodiments and drawings. In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present invention, so the present invention is not limited by the specific implementation disclosed below.

[0030] The cell CHO-K1 2F10 of the embodiment was deposited in the General Microbiological Center (CGMCC) of the China Microbiological Culture Collection Administration on October 21, 2022, with the deposit address being No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; the deposit number is CGMCC 45325. Classification name: Chinese hamster ovary cell CHO-K1 2F10.

[0031] Example 1 Cell serum-free suspension acclimation

[0032] The reagents required for this example include: CHO-K1 (ECACC, catalog number: 85051005), DMEM / F12 (Gibco, catalog number: C1133050013T), FBS (Gibco, catalog number: 10099-141C), trypsin (Gibco, catalog number: 25200072), L-glutamine (Gibco, catalog number: 25030081), culture medium A (name: EX-CELL CD FUSION, catalog number: 14365C) ​​and culture medium B (self-prepared).

[0033] (1) Passaging of adherent cells

[0034] Take out the CHO-K1 cell line purchased from ECACC for recovery. When the confluence of CHO-K1 cells reaches 70% to 80%, perform cell subculture. Aspirate the culture medium in the T25 culture flask, add a certain volume of PBS to the T25 culture flask to wash the surface of the cell layer, and repeat once. Add an appropriate volume of trypsin to the T25 culture flask, shake the culture flask gently to cover the bottom of the culture flask with trypsin, incubate at 37°C for a period of time, add FBS-containing culture medium to terminate digestion when the cells are round, gently blow the cells at the bottom of the flask to resuspend the cells, and transfer the cells to a new T25 culture flask at a 1:4 subculture to continue culturing.

[0035] (2) Cell adaptation to low serum concentration

[0036] The cells were cultured in 10% FBS medium (DMEM / F12 1:1 + 2mM Gln) for 2-3 generations, and the cells showed stable doubling and good cell status (e.g. Figure 1 ). Reduce the FBS content to 5% for culture. After the cells can double normally after 2-3 generations in 5% FBS medium (DMEM / F12 1:1+2mM Gln), reduce the FBS content to 2.5% for adaptation culture. The cells can double normally after 2-3 generations. Reduce the FBS content to 1.25% for adaptation culture. The cells can double normally after 2-3 generations. Cryopreserve a certain number of cells for storage.

[0037] (3) Adaptation to medium A

[0038] Cells grown in complete medium containing 1.25% FBS (DMEM / F12 1:1 + 2mM Gln) were subcultured to medium A containing 1.25% FBS (containing 2mM Gln) for adherent culture and subcultured 2-3 times. When the cells could double normally, they were considered to have adapted to culture in medium A (see Figure 2 ).

[0039] (4) Serum-free suspension acclimation

[0040] Adherent cells grown in medium A (containing 2 mM Gln) with 1.25% FBS were digested and plated at 1×10 6 cells / mL were subcultured into a 125 shake flask containing 1.25% FBS medium A (containing 2 mM Gln) and cultured in suspension at 37°C, 8% CO2, and 95 rpm. Samples were taken and counted every other day. If the viable cell density exceeded 2×10 6 cells / mL, then 1×10 6 cells / mL for transfer; if the viable cell density is less than 2×10 6If the cell suspension is less than 1.5×10 cells / mL and the passage has been cultured for 3 days, aspirate half of the cell suspension and centrifuge at 220 g for 5 min, discard the supernatant, resuspend the cells with an equal volume of fresh 1.25% FBS medium A (containing 2 mM Gln) and transfer the cells to the original 125 mL shake flask for continued culture.

[0041] When the cells can proliferate normally and the doubling time is within 24h, complete serum-free adaptation is performed. The cells suspended and grown in 1.25% FBS medium A (containing 2mM Gln) are plated at a viable cell density of 1×10 6 cells / mL were subcultured in serum-free medium A (containing 2 mM Gln) for suspension culture at 37°C, 8% CO2, and 95 rpm. After 2-3 generations, the cells could multiply normally, with good refractive index and smooth round shape (see Figure 3 ), serum-free suspension acclimation was considered complete.

[0042] Example 2 Cell shear stress tolerance acclimation

[0043] 1.1 Cells that normally multiply in serum-free medium A (containing 2 mM) are cultured at a rate of 1×10 6 cells / mL, and the cells were placed at 37°C, 8% CO2, 95rpm, and named CHO-K1-95. After multiple generations of cell culture, the cell doubling time returned to less than 24h, and the cell edges were flat and the size was uniform. The cells were gradually placed in 150rpm, 170rpm, and 185rpm conditions for culture. When the cells doubled normally under the conditions of 185rpm, the cells had good refractive index and were smooth and round, and were named CHO-K1-185. Finally, a cell pool that can be stably suspended and cultured in a serum-free medium with high shear force was obtained. The doubling time of the cell bank in the logarithmic growth phase was 20±2h, and the average cell diameter was about 15M. The data on live cell density, cell viability, average cell diameter and doubling time during the cell shear force acclimation process are shown in Table 1.

[0044] Serum-free suspension-acclimated CHO-K1-95 cells, i.e., cells cultured in serum-free medium A (containing 2 mM Gln) at 37°C, 8% CO2, 95 rpm, and CHO-K1-185 cells adapted to 185 rpm, were cultured at a viable cell density of 1×10 6 cells / mL were subcultured to serum-free medium A (containing 2 mM Gln) and cultured at 37°C, 8% CO2, 185 rpm. The cell status was observed after 3 days of culture. The results are shown in Figure 4 Among them, CHO-K1-95( Figure 4 A)Compared with CHO-K1-185( Figure 4B) The cells clumped severely and had a significantly lower cell density than CHO-K1-185, that is, the cell doubling time of CHO-K1-95 was prolonged at 185 rpm, indicating that after gradual adaptation to shear force, CHO-K1-185 cells acquired the ability to be cultured under high shear force conditions, giving them certain advantages in reactor scale-up culture.

[0045] Table 1 Cell density, viability, average cell diameter and cell doubling time during cell shear stress tolerance acclimation

[0046]

[0047]

[0048] Example 3 Obtaining Monoclonal Strain

[0049] (1) Preparation of conditioned medium: After culturing cells in medium A (containing 2 mM Gln) for 24 h, the supernatant was collected and centrifuged at 220 g for 5 min. The supernatant was filtered through a 0.22 M sterile filter before use.

[0050] (2) Limiting dilution: Use a CHO-K1-185 cell pool in the logarithmic growth phase with a cell viability greater than 95% for plating, theoretically 0.4 cells / well, and use monoclonal culture medium (80% cloning medium + 20% conditioned medium) for plating, for a total of 10 96-well plates.

[0051] (3) Observation and selection of monoclonal cells: After 7 days of cell growth, observe the cell growth status in the 96-well plate under a microscope, and mark the wells with uniform cell size, single cell cluster, good refractive index and smooth round shape to ensure that the clones are derived from the same cell. When the cell confluence reaches 20% to 30%, expand the marked well cells. Then, 12 strains were selected from 86 monoclonal clones based on cell viability, density and doubling time, namely 2F1, 2F10, 3G4, 4B3, 6A12, 6G4, 6G10, 7D11, 7F12, 8G10, 9D11 and 9E6.

[0052] (4) Cell cryopreservation and stable subculture: The above 12 monoclonal cell lines were subcultured for 2-3 generations in medium A (containing 2 mM Gln) at 185 rpm and then cryopreserved for seed preservation. At the same time, the remaining cells were subcultured for multiple times. The changes in the viable cell density of the 12 monoclonal cell lines are shown in Figure 2. Figure 5 , and the exponential doubling time of cells after 39 days of continuous subculture. Figure 6 .

[0053] from Figure 5It can be seen that the 12 CHO-K1-185 candidate strains grew stably during multiple continuous passages. Except for clone 9E6, the cell doubling time remained basically stable and the doubling time was within 24 hours.

[0054] (5) Batch culture of monoclonal cell lines: Use medium A and medium B at 5×10 5 cells / mL for subculture, cultured at 37°C, 8% CO2, 185rpm, the changes in live cell density and viability curves of 12 monoclonal cell lines are shown in Figure 7 .from Figure 7 It can be seen that except for the monoclonal strains 3G4, 8G10 and 9E6, the highest density of the remaining cell lines in both culture media exceeded 1×10 7 cells / mL.

[0055] (6) Fed-batch culture of monoclonal cell lines: The 12 candidate monoclonal cell lines were cultured in batches using medium A + feed combination A and medium B + feed combination B. The 12 monoclonal cell lines were cultured at 5×10 5 cells / mL, and then subcultured. The cells were cultured at 37°C, 8% CO2, and 185 rpm. The feeding strategy was as follows: feeding was performed daily from Day 3, with a feed combination of 2% to 7% by volume; the glucose content was measured daily, and when it was less than 3g / L, it was supplemented to 1 to 10g / L. The cell line viable cell density-time curve and lactate content curve in the two feeding schemes are shown in Figure 8 .Depend on Figure 8 A and 8B show that, except for the monoclonal strains 3G4, 8G10, and 9E6, the remaining cell lines showed good growth in both culture media, with the highest viable cell density reaching 2.3×10 7 cells / mL, and the plateau phase of the cells can basically be maintained for 2-4 days. The highest viable cell density of clones 2F10, 6A12, 6G4, 7F12 and 9D11 in both feeding schemes reached 2.0×10 7 cells / mL, and the cells were uniform and no clumping occurred. Figure 8 C, the lactic acid content of all 12 cell lines was lower than 1g / L in the late stage of Fed-Batch. Moreover, the lactic acid content of 2F10, 6A12 and 9D11 was lower than 0.5g / L in both feed combinations, which can avoid the cell viability reduction caused by the cytotoxicity of lactic acid accumulation in the late stage of fermentation, which has certain advantages in large-scale production. In summary, 2F10, 6A12 and 9D11 have good development potential.

[0056] (7) Evaluation of the ability of cell lines to transiently express proteins: The expression plasmid encoding mouse IgG1 was transferred into the cell lines 2F10, 6A12, 6G4, 7F12 and 9D11 in the logarithmic growth phase. After 48 hours, the supernatant was taken and the target antibody content in the supernatant was detected by indirect Elisa. The results are shown in Table 2. As can be seen from Table 2, for these five monoclonal cell lines, the level of transient expression of mouse IgG1 antibody was comparable to that of the control group, indicating that these five cell lines have the ability to express heterologous proteins.

[0057] Table 2 Cell lines heterologously expressing mouse IgG1

[0058]

[0059]

[0060] In summary, the cloned cell line 2F10, named CHO-K1 2F10, can grow normally in a high shear environment and can reach more than 2.0×10 7 cells / mL, with a long plateau phase and low-concentration lactate production, as well as the ability to express heterologous proteins, and has the potential for large-scale industrial applications.

Claims

1. A CHO-K1 2F10 cell, characterized in that The deposit number of the CHO-K1 2F10 cells is: CGMCC No. 45325.

2. Use of the CHO-K1 2F10 cell according to claim 1 as a host cell in the preparation of antibodies.

3. A method for expressing antibodies using the CHO-K1 2F10 cells as claimed in claim 1, It is characterized in that include: (1) Transform the expression plasmid encoding the target protein gene into CHO-K1 2F10 cells in the logarithmic growth phase; (2) Take the cell culture supernatant and obtain the target protein.

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