Method for domesticating CHO-k1 cells into high-expression cells

Abstract

A method for domesticating CHO-K1 cells into high-expression cells, comprising the following steps: culturing adherent CHO-K1 cells in a complete culture medium, to obtain log growth CHO-K1 cells; transferring the log growth CHO-K1 cells to a serum-free medium for suspension culture; performing a first liquid replacement domestication, to obtain domesticated CHO-K1 cells; placing the domesticated CHO-K1 cells in a chemically defined medium for suspension culture; performing a second liquid replacement domestication, to obtain domesticated suspended CHO-K1 cells; performing monoclonalization on the domesticated suspended CHO-K1 cells, to obtain CHO-K1 monoclonal cells; and screening the CHO-K1 monoclonal cells, to obtain high-expression CHO-K1 monoclonal cells. This method can domesticate CHO-K1 original parent cells into suspended cells, and screen cells having higher expression capability from the domesticated cell population, thereby improving expression levels.

Description

A method for acclimating CHO-K1 cells into high-expressing cells Technical Field

[0001] The present invention relates to the technical field of cell culture, and in particular to a method for domesticating CHO-K1 cells into high-expression cells. Background Art

[0002] CHO cells, an epithelial cell derived from the Chinese hamster ovary, are the most widely used expression system for biotherapeutic proteins, with approximately 70% of marketed biologics derived from CHO cell expression systems. Their ability to adapt to serum-free, high-density suspension culture and their post-translational modifications, which closely mimic biological activity in humans, make CHO cells essential for bioproduction.

[0003] The original mother cells of CHO grow in an adherent manner and can only grow in a single layer in a culture flask. The cells also have tentacles, which causes the cells to take up a lot of culture space during culture. The number of cells that grow depends on the culture area, and it is difficult to achieve high-density culture. Currently, in the development of biopharmaceuticals, CHO cells that are cultured in serum-adherent suspension are usually acclimated to serum-free suspension high-density growth, but the existing technology mostly cultivates and applies CHO-S cells. CHO-S is a branch based on CHO cells, and is not the CHO-K1 commonly used in preclinical development. There are large differences between the two in terms of biological characterization, which interferes with the identity of the drug screening and development process. At this stage, the results of differential studies on different cell lines show that using CHO-K1 as the main expression host cell is better than CHO-S in the early research and development stage.

[0004] On the other hand, the current CHO-K1 cells have the problem of low expression levels of recombinant proteins. In the existing technology, the expression level of CHO-K1 cells is generally 10-50 mg / L, and some companies can only reach an average level of 150 mg / L at most, which cannot meet the requirements of efficient and rapid production of recombinant proteins.

[0005] In view of this, the present invention is proposed.

[0006] Summary of the Invention

[0007] The first object of the present invention is to provide a method for domesticating CHO-K1 cells into high-expressing cells. This method addresses the shortcomings of the existing technology and can domesticate CHO-K1 original mother cells into suspension cells, and screen out cells with higher expression ability from the domesticated cell population, thereby improving the expression level.

[0008] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are adopted:

[0009] The present invention provides a method for acclimating CHO-K1 cells into high-expressing cells, comprising the following steps:

[0010] The adherent CHO-K1 cells were cultured in complete medium until the logarithmic growth phase to obtain logarithmically growing CHO-K1 cells;

[0011] The logarithmically growing CHO-K1 cells were switched to suspension culture in serum-free medium supplemented with L-glutamine;

[0012] Perform the first medium change and acclimatization, subculture the logarithmically growing CHO-K1 cells in serum-free medium for more than 10 generations to obtain acclimatized CHO-K1 cells;

[0013] The acclimated CHO-K1 cells were transferred to suspension culture in a chemically defined medium supplemented with L-glutamine;

[0014] Performing a second medium change for acclimation, passage the CHO-K1 cells suspended in the chemically defined medium for more than 30 generations to obtain acclimated suspension CHO-K1 cells;

[0015] The acclimated suspension CHO-K1 cells were monocloned to obtain CHO-K1 monoclonal cells;

[0016] The CHO-K1 monoclonal cells were screened to obtain high-expressing CHO-K1 monoclonal cells.

[0017] In the prior art, since CHO-K1 original mother cells are purchased commercially, they need to be domesticated into suspension cells through different culture media and acclimation processes. Therefore, the CHO-K1 cells used by different companies are not the same, and the expression capabilities are also different. The method provided by the present invention can not only domesticate CHO-K1 cells into suspension cells, which is conducive to increasing cell density, but also culture them into highly expressing cells. By monoclonalization and monoclonal screening, cells with high expression capabilities are selected from the domesticated cell population, and ultimately, the expression level of monoclonal antibodies and bispecific antibody proteins of 100-1000 mg / L can be achieved. Among them, the average expression level of monoclonal antibody protein exceeds 450 mg / L, and the recombinant protein produced reaches the milligram level, which can meet the needs of recombinant protein production.

[0018] Preferably, the culturing in complete medium comprises the following steps:

[0019] A basal culture medium containing 10% calf serum is selected as the complete culture medium;

[0020] The adherent CHO-K1 cells were thawed in a 37°C water bath and placed in the preheated complete culture medium;

[0021] The total density of the complete culture medium was controlled to be 2.0-3.0E5 cells / ml, and the culture medium was placed in a static culture at 37° C. and 5% carbon dioxide.

[0022] The basal culture medium is preferably DMEM / F12 basal culture medium, and the static culture is preferably carried out in a cell culture incubator.

[0023] Preferably, the culturing in serum-free medium comprises the following steps:

[0024] The amount of L-glutamine added to the serum-free medium was 4 mM;

[0025] The adherent CHO-K1 cells were digested with 0.25% trypsin and washed with buffer;

[0026] The cells were re-inoculated with the serum-free medium to control the total density to 1.0E6 cells / ml, and suspension culture was performed at 37° C., 5% carbon dioxide, and shaking at 120 rpm for 48 h.

[0027] The serum-free culture medium is preferably EX-CELL 302 culture medium supplemented with 4 mM L-glutamine; the serum-free culture medium is inoculated into a 125 ml shake flask; and the suspension culture is preferably performed in a cell shaker.

[0028] Preferably, the first fluid change acclimation comprises the following steps:

[0029] After suspension culture in the serum-free medium, the obtained cell suspension was centrifuged at 800 rpm for 5 minutes, and re-inoculated with the serum-free medium to control the total density at 1.0E6 cell / ml;

[0030] Repeat the above steps until the cells are passaged for more than 10 generations and the cell viability is greater than 95%, thereby obtaining the acclimated CHO-K1 cells;

[0031] Preferably, the amount of L-glutamine added to the chemically defined medium is 4 mM, and the culture in the chemically defined medium comprises the following steps:

[0032] The CHO-K1 cells were washed with a buffer solution;

[0033] The cells were re-inoculated with the chemically defined medium to control the total density to 1.0E6 cells / ml and cultured in suspension at 37° C., 5% carbon dioxide and 120 rpm shaking.

[0034] The chemically defined medium is preferably BalanCD CHO Growth A medium added with 4 mM L-glutamine.

[0035] Preferably, the second fluid change acclimation comprises the following steps:

[0036] After suspension culture in the chemically defined medium for 48-72 hours, the cell suspension was centrifuged at 800 rpm for 5 minutes and re-inoculated with the chemically defined medium to control the total density at 1.0E6 cells / ml;

[0037] Repeat the above steps until the cells are passaged for more than 30 generations, and the cell viability is greater than 95% and the doubling time is less than 24 hours, thereby obtaining the acclimated suspension CHO-K1 cells;

[0038] The passage interval was 1-2 days.

[0039] Preferably, the monoclonalization comprises the following steps:

[0040] CHO cell culture medium supplemented with 4 mM L-glutamine and 2% calf serum was selected as the cloning medium;

[0041] The acclimated suspension CHO-K1 cells were inoculated with the cloning medium to control the total density to 3 cells / ml, and statically cultured at 37° C. and 5% carbon dioxide for 2-3 weeks;

[0042] After completing the above static culture, single clones of cells were selected and re-inoculated with the chemically defined medium to control the total density to 3.0E5 cells / ml, and cultured continuously for 96 hours at 37°C, 5% carbon dioxide, and shaking at 225 rpm;

[0043] After the above continuous culture is completed, cells with a live cell density greater than 1.0E7 cells / ml, a cell viability greater than 90%, and a cell diameter of 14.5-15.5 μm are selected as the CHO-K1 monoclonal cells.

[0044] Among them, the CHO cell culture medium preferably uses BalanCD CHO Growth A medium.

[0045] Preferably, the method for preparing the high-expressing CHO-K1 monoclonal cells comprises the following steps:

[0046] Mix 1 mg / L DNA with 6 mg / L linearized polyethyleneimine and incubate for 20 min;

[0047] The incubated product was added to the CHO-K1 monoclonal cells for transfection, and the temperature was lowered from 37.0°C to 32.0-33.0°C after 24 hours;

[0048] Cell feed was added on days 1 and 3 after transfection;

[0049] The high-expressing CHO-K1 monoclonal cells were selected on day 5-7 after transfection.

[0050] The purpose of screening high-expressing CHO-K1 monoclonal cells is to screen out monoclonal cells with stronger expression ability from the existing CHO-K1 monoclonal cell population, so as to cooperate with the method of transient expression of recombinant protein of the present invention. The combination of the two can further improve the level of transient expression.

[0051] The second object of the present invention is to provide a method for obtaining a highly expressed CHO-K1 monoclonal cell transiently expressing a recombinant protein using the above method, comprising the following steps:

[0052] The transfection complex was obtained by mixing 1 mg / L DNA with 6 mg / L linearized polyethyleneimine and incubating for 20 min.

[0053] Adding the transfection complex to the high-expressing CHO-K1 monoclonal cells to transfect the cells with the recombinant protein, and cooling the cells from 37.0° C. to 32.0-33.0° C. after 24 hours;

[0054] Add cell culture feed for transient expression of recombinant proteins;

[0055] Recombinant proteins were harvested 5-7 days after addition of the transfection complexes.

[0056] Among them, DNA is the DNA plasmid for transfection of the required recombinant protein.

[0057] Preferably, the density of the recombinant protein transfected into the cells is 6.0E6 cells / L, and the recombinant protein is a monoclonal antibody protein or a bispecific antibody protein.

[0058] Preferably, the cell culture feed comprises an aqueous solution containing 10% by mass of soy peptone, and the amount added is 10% of the volume of the transfection complex, and the time of addition is the first and third days after adding the transfection complex.

[0059] The method for transiently expressing recombinant proteins provided herein utilizes CHO-K1 monoclonal cells and transfects DNA with polyethyleneimine. Through a culture process characterized by cooling and feeding, the recombinant protein can be harvested between five and seven days after transfection. By combining the high-expression CHO-K1 monoclonal cells obtained through the aforementioned cultivation process, the level of transient expression of monoclonal or bispecific antibody proteins can be further improved. This allows for efficient and rapid production of recombinant proteins in a shorter timeframe, contributing to cost savings. Furthermore, the unique nature of the cells ensures the uniformity of the recombinant protein, reduces heterogeneity, and enables rapid and stable expression of the recombinant protein.

[0060] Compared with the prior art, the present invention has the following beneficial effects:

[0061] (1) The present invention provides a method for domesticating CHO-K1 cells into high-expressing cells. The high-expressing CHO-K1 monoclonal cells domesticated by this method have extremely high expression levels, which can reach monoclonal antibody and bispecific expression levels of 100-1000 mg / L.

[0062] (2) The present invention also provides a method for transiently expressing recombinant proteins based on high-expressing CHO-K1 monoclonal cells. This method is more suitable for the cells domesticated by the present invention, achieving efficient and stable transient expression of recombinant proteins, and is short in time and low in cost. Cells can be harvested in only 5-7 days.

[0063] (3) The method of transiently expressing recombinant proteins of the present invention has a controllable culture volume, ranging from 3 ml to 50 L, with a wide range of applications, and can achieve an average expression level of more than 450 mg / L of monoclonal antibody protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Various other advantages and benefits will become apparent to those skilled in the art upon reading the detailed description of the preferred embodiment below. The accompanying drawings are for illustration purposes only and are not to be considered as limiting the present invention. The same reference symbols are used throughout the drawings to represent the same components. In the drawings:

[0065] FIG1 is a graph comparing the expression levels of different monoclonal antibody proteins provided in Experimental Example 1 of the present invention;

[0066] FIG2 is a graph showing the expression levels of the same monoclonal antibody protein at different times provided in Experimental Example 2 of the present invention;

[0067] FIG3 is a graph showing the expression levels of bispecific antibodies with different properties provided in Experimental Example 3 of the present invention;

[0068] FIG4 is a graph of viable cell density provided in Experimental Example 4 of the present invention;

[0069] FIG5 is a graph of cell viability provided in Experimental Example 4 of the present invention;

[0070] FIG6 is a cell doubling time diagram provided in Experimental Example 4 of the present invention. DETAILED DESCRIPTION

[0071] The embodiments of the present invention will be described in detail below with reference to the examples, but those skilled in the art will appreciate that the following examples are intended only to illustrate the present invention and should not be construed as limiting the scope of the invention. Where specific conditions are not specified in the examples, the methods were performed according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0072] Example 1

[0073] This example provides a method for acclimating CHO-K1 cells to high-expressing cells. This example uses commercially available CHO-K1 adherent cells and performs an acclimation treatment on them. The specific method includes the following steps:

[0074] S1. Culturing adherent CHO-K1 cells in complete culture medium. The specific steps include: selecting DMEM / F12 basal culture medium containing 10% calf serum as the complete culture medium; removing commercially available adherent CHO-K1 cells from a liquid nitrogen tank, rethawing them in a 37°C water bath, and placing them in preheated complete culture medium after thawing to control the total density to 3.0E5 cells / ml; and culturing the complete culture medium in a cell culture incubator at 37°C and 5% carbon dioxide until the logarithmic growth phase.

[0075] S2. The cells in step S1 were transferred to a serum-free medium for suspension growth. The specific steps included: selecting EX-CELL 302 medium supplemented with 4 mM L-glutamine as the serum-free medium; digesting the adherent CHO-K1 cells in step S1 with 0.25% trypsin, then washing twice with PBS buffer, and then inoculating the cells in a 125 ml shake flask with serum-free medium to control the total density to 1.0E6 cells / ml. The cells were shaken in a cell shaker at 37°C, 5% carbon dioxide, and 120 rpm.

[0076] S3. After two days of cell culture in step S2, perform the first medium exchange and acclimatization. The specific steps include: transferring the cell suspension from step S2 to a 50 ml centrifuge tube, centrifuging at 800 rpm for 5 minutes, and re-seeding the cells in a 125 ml shake flask with serum-free medium to control the total density to 1.0E6 cells / ml. Repeat the above steps until the cells have been passaged for at least 10 generations and the cell viability is greater than 95%, thereby obtaining acclimatized CHO-K1 cells.

[0077] S4. The acclimated CHO-K1 cells were transferred to a chemically defined medium for suspension culture. The specific steps included: selecting BalanCD CHO Growth A medium supplemented with 4 mM L-glutamine as the chemically defined medium; washing the acclimated CHO-K1 cells twice with PBS buffer, then inoculating the cells into a 125 ml shake flask with the chemically defined medium to control the total density to 1.0E6 cells / ml, and culturing the cells in a cell shaker at 37°C, 5% carbon dioxide, and 120 rpm.

[0078] S5. After three days of cell culture in step S4, a second medium change and acclimation step is performed. The specific steps include: transferring the cell suspension from step S4 to a 50 ml centrifuge tube, centrifuging at 800 rpm for 5 minutes, and re-inoculating the cells in a 125 ml shake flask with chemically defined medium to control the total density to 1.0E6 cells / ml. Repeat the above steps until at least 30 passages are achieved, with cell viability greater than 95% and a doubling time of less than 24 hours, to obtain acclimated suspension CHO-K1 cells. The interval between passages is 1-2 days.

[0079] S6. Monocloning the acclimated suspension CHO-K1 cells, specifically comprising: selecting BalanCD CHO Growth A medium supplemented with 4 mM L-glutamine and 2% calf serum as a cloning medium; taking the acclimated suspension CHO-K1 cells that grew logarithmically in step S5, adjusting the density to 3 cells / ml with cloning medium, inoculating 150 μl per well in a 96-well plate, and culturing in a cell culture incubator at 37° C. and 5% carbon dioxide for 3 weeks.

[0080] After 3 weeks of static culture, single clones were picked from the 96-well plate and seeded into 24-well deep-well plates with 10 ml of BalanCD CHO Growth A medium supplemented with 4 mM L-glutamine to a total density of 3.0 E cells / ml. The cells were cultured in a cell shaker at 37°C, 5% CO2, and 225 rpm for 96 h.

[0081] After shaking culture for 96 h, the cell number, cell diameter and viability were counted using a CountStar cell counter, and cells with a cell density greater than 1.0E7 cells / ml, a cell viability greater than 90%, and a cell diameter between 14.5 and 15.5 μm were selected as CHO-K1 monoclonal cells.

[0082] After obtaining CHO-K1 monoclonal cells, first screen out high-expressing CHO-K1 monoclonal cells, and then use these high-expressing CHO-K1 monoclonal cells for transient expression of monoclonal antibodies, as follows:

[0083] Step 1: In a 24-well deep-well plate, 1 mg / L DNA was mixed with 6 mg / L linearized polyethyleneimine and incubated for 20 minutes to obtain a transfection complex.

[0084] Step 2: Add the transfection complex to CHO-K1 monoclonal cells to transfect the cells with recombinant protein. After 24 hours, control the temperature to drop from 37.0°C to 32.0°C-33.0°C.

[0085] Among them, the density of the recombinant protein transfected into the control cells was 6.0E6cell / L.

[0086] Step 3: Add cell culture feed to transiently express the recombinant protein and add it again after 48 hours;

[0087] The cell culture feed comprises an aqueous solution containing 10% by mass of soy peptone, and the amount added is 10% of the volume of the transfection complex.

[0088] Step 4: On the seventh day after cell transfection, use biofilm interferometry to compare the expression levels of each clone and select high-expressing CHO-K1 monoclonal cells.

[0089] Step 5: Place the selected high-expressing CHO-K1 monoclonal cells in a 125 ml shake flask, repeat steps 1 to 4, and harvest the recombinant protein on the seventh day after the second transfection.

[0090] Step 6: Use Protein A chromatography medium to purify the recombinant protein contained in the cell supernatant harvested in step 5, and use high-performance liquid chromatography to study and compare the quality to determine the best performing monoclonal CHO-K1.

[0091] Example 2

[0092] The specific implementation steps are consistent with those in Example 1, except that the CHO-K1 monoclonal cells used for transient expression were not acclimated using the method of the present invention, but were commercially available expiCHO cells from Thermo Fisher Scientific.

[0093] Experimental Example 1

[0094] Ten different commercial monoclonal antibody proteins were expressed using the methods of Example 1 and Example 2, and the expression levels were detected.

[0095] Among them, expiCHO STD and expiCHO MAX are the results of cell culture expression for 9 days and 14 days respectively using commercially available cells from Thermo Fisher Scientific, and MetaCHO Express is the result of cell culture expression for 7 days using the monoclonal cells of the present invention.

[0096] Among them, commercialized monoclonal antibody proteins include Bevacizumab, Rituximab, Ipilimumab, Daratumumab, Denosumab, Panitumumab, Spesolimab, Adalimumab, Atezolizumab, and Cetuximab.

[0097] The expression level detection results are shown in Figure 1.

[0098] As can be seen from Figure 1 , the expression levels of six monoclonal antibody proteins in Example 1 exceeded 450 mg / L, with the highest exceeding 700 mg / L. Only the expression levels of Bevacizumab and Rituximab were lower than the expression levels of expiCHO expression for 14 days, but all were higher than the expression levels of expiCHO expression for 9 days. This shows that the high-expression CHO-K1 monoclonal cells obtained by the present invention have the characteristics of high expression levels and rapid expression. Comparison with expiCHO cells also shows that the cells obtained by the acclimation method and screening method of the present invention are more compatible with the transient expression method of the present invention, and the expression level can be significantly improved.

[0099] Experimental Example 2

[0100] A human IgG antibody was expressed using the method of Example 1, and the transient expression levels were detected at different time periods. The detection results are shown in FIG2 .

[0101] As can be seen from Figure 2, after adding the cell culture feed on the third day, the expression level on the fourth day exceeded 500 mg / L; the expression level on the fifth day increased rapidly, reaching 985 mg / L; and the expression level increased steadily over the next 5-7 days, reaching 1231 mg / L on the seventh day. It can be seen that the expression level of recombinant protein produced by the method of the present invention can be several times higher than that of the prior art, and the time required is shorter. In addition, the time for harvesting the recombinant protein of the present invention is preferably 5-7 days because a considerable amount of recombinant protein can be expressed by 5-7 days, but the culture can also be continued to 7-14 days to achieve higher indicators.

[0102] Experimental Example 3

[0103] Symmetrical bispecific antibody proteins and asymmetric bispecific antibody proteins were expressed using the method of Example 1. Duplicate wells were set for each group, and the expression levels were detected on the seventh day. The detection results are shown in FIG3 .

[0104] Among them, the bispecific antibody protein is bovine serum albumin, which is an asymmetric bispecific antibody protein Asymmetric BsAb and a symmetric bispecific antibody protein Ssymmetrice BsAb.

[0105] As can be seen from Figure 3, the average expression level of symmetrical bispecific antibody proteins exceeds 600 mg / L, and the average expression level of asymmetric bispecific antibody proteins exceeds 400 mg / L, indicating that the method of transiently expressing recombinant proteins of the present invention also has extremely high expression levels when expressing bispecific antibody proteins, which is a significant improvement compared to the prior art.

[0106] Experimental Example 4

[0107] The acclimated suspension CHO-K1 cells obtained in step S5 of Example 1 were tested for viable cell density, cell viability, and cell doubling time to examine the performance of the acclimated cells. The test results are shown in Figures 4-6.

[0108] Figure 4 shows a graph of viable cell density, demonstrating a significant increase in cell density after 20 days, reaching a peak density between 6E6 cells / ml and 8E6 cells / ml. Figure 5 shows a graph of cell viability, demonstrating that cell viability is consistently greater than 90% after 20 days. Figure 6 shows a graph of cell doubling time, demonstrating that after 60 days, i.e., at least 30 passages, the cell doubling time stabilizes, remaining within 18-20 hours. The stable cell state graphs above indicate that the cells have adapted to suspension growth and that cell acclimation is ideal.

[0109] Finally, it should be understood that the above embodiments are merely exemplary embodiments for illustrating the principles of the present invention, and the present invention is not limited thereto. Those skilled in the art will be able to make various modifications and improvements without departing from the principles and essence of the present invention, and such modifications and improvements are also considered to be within the scope of protection of the present invention.

Claims

1. A method for domesticating CHO-K1 cells into high-expressing cells, characterized in that: The following steps are involved: The adherent CHO-K1 cells were cultured in complete medium until the logarithmic growth phase to obtain logarithmically growing CHO-K1 cells; The logarithmically growing CHO-K1 cells were switched to suspension culture in serum-free medium supplemented with L-glutamine; Performing the first medium change acclimation, subculturing the CHO-K1 cells growing logarithmically in serum-free medium for more than 10 generations to obtain acclimated CHO-K1 cells; The acclimated CHO-K1 cells were transferred to suspension culture in chemically defined medium supplemented with L-glutamine; Performing a second medium change acclimation, subculturing the CHO-K1 cells suspended in the chemically defined medium for more than 30 generations to obtain acclimated suspension CHO-K1 cells; The acclimated suspension CHO-K1 cells are monocloned to obtain CHO-K1 monoclonal cells; The CHO-K1 monoclonal cells were screened to obtain high-expression CHO-K1 monoclonal cells.

2. The method for domesticating cells into high-expressing cells according to claim 1, characterized in that: The cultivation of the complete medium comprises the following steps: Selecting a basal medium containing 10% calf serum as the complete medium; The adherent CHO-K1 cells were thawed in a 37° C. water bath and placed in the preheated complete medium after thawing; The total density of the complete culture medium was controlled to be 2.0-3.0E5 cell / ml, and the culture medium was placed in a static culture at 37° C. and 5% carbon dioxide.

3. The method for domesticating cells into high-expressing cells according to claim 1, characterized in that: The culturing of the serum-free medium comprises the following steps: The amount of L-glutamine added to the serum-free medium is 4 mM; The adherent CHO-K1 cells were digested with 0.25% trypsin and washed with buffer; The cells were re-inoculated with the serum-free medium to control the total density to 1.0E6 cell / ml, and suspension culture was performed at 37° C., 5% carbon dioxide and 120 rpm shaking for 48 h.

4. The method for domesticating cells into high-expressing cells according to claim 3, characterized in that: The first fluid change acclimatization comprises the following steps: After suspension culture in the serum-free medium, the obtained cell suspension was centrifuged at 800 rpm for 5 min, and re-inoculated and cultured with the serum-free medium, and the total density was controlled at 1.0E6 cell / ml; Repeat the above steps until the cells are subcultured for more than 10 generations and the cell viability is greater than 95%, thereby obtaining the acclimated CHO-K1 cells; Preferably, the amount of L-glutamine added to the chemically defined medium is 4 mM, and the culture in the chemically defined medium comprises the following steps: The CHO-K1 cells were washed with a buffer; The cells were re-inoculated with the chemically defined medium to control the total density to 1.0E6 cell / ml and cultured in suspension at 37°C, 5% carbon dioxide and 120 rpm shaking.

5. The method for domesticating cells into high-expressing cells according to claim 4, characterized in that: The second fluid change acclimatization comprises the following steps: After suspension culture in the chemically defined medium for 48-72 hours, the cell suspension was centrifuged at 800 rpm for 5 minutes, and re-inoculated with the chemically defined medium, with the total density controlled at 1.0E6 cell / ml; Repeat the above steps until the cells are subcultured for more than 30 generations, and the cell viability is greater than 95% and the doubling time is less than 24 hours, thereby obtaining the acclimated suspension CHO-K1 cells; The interval between passages was 1-2 days.

6. The method for domesticating cells into high-expressing cells according to claim 5, characterized in that: The monoclonalization comprises the following steps: CHO cell culture medium supplemented with 4 mM L-glutamine and 2% calf serum was selected as cloning medium; The acclimated suspension CHO-K1 cells were inoculated with the cloning medium to control the total density. The concentration is 3 cells / ml and cultured at 37°C and 5% carbon dioxide for 2-3 weeks; After completing the above static culture, a single clone of cells was selected, and the cells were re-inoculated with the chemically defined medium to control the total density to be 3.0E5 cells / ml, and cultured continuously for 96 hours at 37° C., 5% carbon dioxide, and 225 rpm shaking; After the above continuous culture is completed, cells with a live cell density greater than 1.0E7 cell / ml, a cell viability greater than 90%, and a cell diameter of 14.5-15.5 um are selected as the CHO-K1 monoclonal cells.

7. The method for domesticating cells into high-expressing cells according to claim 6, characterized in that: The method for preparing the high-expression CHO-K1 monoclonal cell comprises the following steps: Mix 1 mg / L DNA with 6 mg / L linearized polyethyleneimine and incubate for 20 min; The incubated product was added to the CHO-K1 monoclonal cells for transfection, and the temperature was lowered from 37.0°C to 32.0-33.0°C after 24 hours; Cell feed was added on days 1 and 3 after transfection; The high-expressing CHO-K1 monoclonal cells were selected on days 5-7 after transfection.

8. The method for transiently expressing recombinant protein in the high-expressing CHO-K1 monoclonal cells in the method for domesticating to high-expressing cells according to any one of claims 1 to 7, characterized in that: The following steps are involved: The transfection complex was obtained by mixing 1 mg / L DNA with 6 mg / L linearized polyethyleneimine and incubating for 20 min; Adding the transfection complex to the high-expression CHO-K1 monoclonal cells to transfect the cells with recombinant proteins, and cooling the cells from 37.0° C. to 32.0-33.0° C. after 24 hours; Add cell culture feed for transient expression of recombinant proteins; Recombinant proteins were harvested on day 5-7 after addition of the transfection complexes.

9. The method for transiently expressing a recombinant protein according to claim 8, characterized in that: The density of the recombinant protein transfected into the cells is 6.0E6 cell / L, and the recombinant protein is a monoclonal antibody protein or a bispecific antibody protein.

10. The method for transiently expressing a recombinant protein according to claim 9, characterized in that: The cell culture supplement comprises an aqueous solution containing 10% by mass of soy peptone, and the amount added is 10% of the volume of the transfection complex, and the time of addition is the first day and the third day after the transfection complex is added.

Images