CN111019881B - A cell adapted to a high osmotic pressure, high ammonium ion, high lactic acid growth environment - Google Patents

Abstract

The invention discloses a cell adapted to a high osmotic pressure, high ammonium ion and high lactic acid growth environment. Chinese hamster ovary epithelial cells CHO-K1-OAL with the collection number: CGMCC No. 18322. According to the invention, Chinese hamster ovary epithelial cells CHO-K1-OAL can normally grow in the environment with high osmotic pressure (more than 350mOsm/Kg), high ammonium ion (7.5mM) and high lactic acid (2.5g/L), so that the defect that the death and the activity rate of CHO cells are reduced and the cell yield is reduced due to the fact that the ammonium ion and lactic acid concentration is increased along with the increase of the osmotic pressure in the fermentation process is avoided.

Description

A cell adapted to a high osmotic pressure, high ammonium ion, high lactic acid growth environment

technical field

The invention belongs to the field of cells, and in particular relates to a cell adapted to a high osmotic pressure, high ammonium ion and high lactic acid growth environment.

Background technique

With the continuous development of genomics, molecular biology and modern medicine, the biopharmaceutical industry has gradually shown its unique advantages and has achieved rapid development in China in the past ten years. Among them, recombinant protein drugs, especially antibody drugs, have developed Most rapidly, a large number of domestic companies and research institutions have been engaged in antibody development and research in the direction of anti-tumor, cardiovascular, and immune diseases, and a number of antibody drugs have been launched.

At present, CHO cells (Chinese hamster ovary cell) are mostly used as production cell lines for antibody proteins, because CHO cells have incomparable advantages over other cells. For example, the expressed antibodies are similar to their natural structures, and their separation and purification methods are convenient. Has better biosecurity and more. As host cells for antibody expression, the growth characteristics and expression levels of cells play a decisive role in the cost and quality of biological drugs. At present, the level of antibody expression in cells is still low, and the process and production costs are high, resulting in biological drugs. Prices remain high. On the one hand, the cost of production equipment and consumables is high, and on the other hand, the level of cell production is low. In the process of biological fermentation to produce antibody-like proteins, with the increase of cell products and metabolites, the environment in the fermentation broth tends to be extreme, such as the increase of osmotic pressure, the increase of ammonium ion and lactic acid concentration, which will lead to cell death and viability. decrease, ultimately affecting the yield of cells.

The existing CHO cell lines generally can only be cultured in a culture environment with low osmotic pressure (less than 350mOsm/Kg) and/or low ammonia ion concentration (less than 7.5mM) and/or low lactate (less than 2.5g/L). grow. The culture medium provides an appropriate growth environment for the cells to grow. During the cultivation process, the user obtains information on cell growth and biomass materials (protein, DNA, cells, etc.) through detection means. The cell line's own metabolism and secretion of biological materials lead to the destruction of the appropriate environment provided by the medium, which in many cases results in a culture environment with high osmotic pressure (greater than 350mOsm/Kg) and/or high ammonia ion concentration (greater than 7.5mM) and (or) in a culture environment with high lactic acid (greater than 2.5g/L). In this culture environment, cell lines cannot grow normally and cannot produce biological material.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a Chinese hamster ovary epithelial cell CHO-K1-OAL adapted to a high osmotic pressure, high ammonium ion, and high lactic acid growth environment. It is preserved in the General Microbiology Center (CGMCC) of the China Microorganism Culture Collection Management Committee, address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, and the preservation number is: CGMCC No.18322.

In view of the fact that the existing CHO cell lines are generally only in low osmotic pressure (less than 350mOsm/Kg) and (or) low ammonia ion concentration (less than 7.5mM) and (or) low lactic acid (less than 2.5g/L) culture environment The present invention will use a reasonable method to domesticate and obtain the CHO cell strain that can grow in the culture environment of high osmotic pressure and (or) high ammonia ion concentration and (or) high lactic acid, so as to avoid the progress of the fermentation process. As a result, the yield of cells is reduced, thereby achieving the object of the present invention.

The second object of the present invention is to provide the application of the Chinese hamster ovary epithelial cell CHO-K1-OAL as a production strain in the production of antibody-like protein products.

Preferably, the Chinese hamster ovary epithelial cell CHO-K1-OAL is used as a production strain in the production of antibody-like protein products under the environment of high osmotic pressure, high ammonium ions, and high lactic acid.

Preferably, the Chinese hamster ovary epithelial cell CHO-K1-OAL is used as a production strain in the production of antibody protein products under the osmotic pressure of ≥350mOsm/Kg, ammonium ions of ≥7.5mM, and lactic acid of ≥2.5g/L.

The Chinese hamster ovary epithelial cell CHO-K1-OAL of the present invention can grow normally in the environment of high osmotic pressure (more than 350mOsm/Kg), high ammonium ion (7.5mM) and high lactic acid (2.5g/L), so as to avoid the fermentation During the process, the osmotic pressure increases, and the concentration of ammonium ions and lactic acid increases, which leads to the death of CHO cells and the decrease of the viability, which ultimately affects the defect of reduced cell productivity.

The Chinese hamster ovary epithelial cell CHO-K1-OAL was deposited in the General Microbiology Center (CGMCC) of the China Microorganism Culture Collection on August 19, 2019. Address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, China, preservation number As: CGMCC No.18322.

Description of drawings:

Figure 1 shows the growth curves of CHO-K1S cells at different osmotic pressures, where 300, 350, 400, 450, and 500 represent osmotic pressures of 300, 350, 400, 450, and 500 mOsm/Kg, respectively;

Figure 2 shows the growth curves of CHO-K1S and CHO-K1S-O-A, wherein 300, 350, 400, 450, and 500 represent the growth curves of CHO-K1S cells at osmotic pressures of 300, 350, 400, 450, and 500 mOsm/Kg, respectively. And 400-A represents the growth curve of CHO-K1S-O-A cells at an osmotic pressure of 400mOsm/Kg;

Figure 3 shows the growth curves of CHO-K1S and CHO-K1S-O-AL, wherein 300, 350, 400, 450, and 500 represent the osmotic pressure of CHO-K1S cells at 300, 350, 400, 450, and 500 mOsm/Kg, respectively. The base is the growth curve of serum-free medium CD02, while 400-A-AL represents the growth curve of CHO-K1S-O-AL cells under the osmotic pressure of 400mOsm/Kg, the medium is high ammonium ion and high lactate selection medium ;

Figure 4 is the growth curve of CHO-K1S and CHO-K1S-OAL-A, wherein 300, 350, 400, 450, and 500 represent the osmotic pressure of CHO-K1S cells at 300, 350, 400, 450, and 500 mOsm/Kg, respectively. The base is the growth curve of serum-free medium CD02, and 400AL-A represents the growth curve of CHO-K1S-OAL-A cells under the osmotic pressure of 400 mOsm/Kg, the medium is high ammonium ion and high lactate selection medium.

Detailed ways:

The following examples are further illustrations of the present invention, rather than limitations of the present invention.

Example 1:

The selected cell line in the present invention is CHO-K1 cell (ECACC 85051005). The medium used was the CHO cell chemically defined serum-free medium CD02 from QuaCell Company.

1. Cell suspension acclimation

1. The purpose of the experiment

CHO-K1 (Lot: 16H036) adherent cells were adapted to suspension cells.

2. The main materials of the experiment

DMEM/F12=1:1 medium (Gibco/C1133050013T), serum (ExCell Bio/11G355), L-glutamine (Original Bio), CD02 medium (Quacell/36A01), T75 square bottle (BIOFIL/180624) -104B), T75 square bottle (BIOFIL/180508-104), 125ml shaker bottle (Corning/25818010), 500ml shaker bottle (Corning/11818004), cryopreservation tube (Corning/23618076), 5ml pipette (Corning/20118020) ), 25ml pipette (Corning/17218010), 50ml centrifuge tube (Corning/31518601)

3. Experimental process

(1), cell expansion, preservation

Basal medium solution preparation: DMEM/F12 medium (DMEM/F12=1:1)+10% FBS+2mM Gln cell recovery, expansion and preservation: recovery of CHO-K1 (Lot: 16H036) cells, using the above basal culture The base solution was subcultured and expanded, and when the total number of cells reached a certain number, the cells were cryopreserved, and a total of 8 cells were cryopreserved. Cells were named CHO-K1.

(2), cell suspension acclimation

a. The main components of the cell suspension acclimation medium solution are DMEM/F12 medium, FBS, CD02 medium, and 2mM Gln. The cell suspension acclimation medium with different serum concentration ratios was prepared according to the experimental plan.

b. Resuscitate 1 branch of the above-mentioned cryopreserved CHO-K1 cells, and use a T75 square flask for cell subculture. When the cell confluence reaches 80%, the subculture is carried out. The supernatant is discarded, digested, centrifuged and resuspended. A certain amount of the resuspended cell solution is added to the cell suspension acclimation medium for culture (the serum concentration ratio of each subculture medium is different). Observe the morphology of the cells, and gradually suspend the cells through multiple subcultures (there will be a small amount of adherent cells).

c. Suspension cell expansion culture: transfer the acclimated cells to a 125ml shake flask, and add fresh CD02 medium (4mM Gln) for continuous subculture. The density of passaged cells is about 3×10 ⁵ cells/ml, and the volume is 30ml. Observe the growth state of the cells in the shake flask, and detect the cell density and viability.

(3), suspension cell preservation

Take an appropriate amount of the above-mentioned amplification medium, centrifuge, discard the supernatant, resuspend with cell freezing medium (92% CD02 + 8% DMSO), and divide it into 1 ml at a cell density of 1 × 10 ⁷ cells/ml. Aliquot 10 cryopreservation tubes, put them in a -80°C refrigerator with a programmed cooling box, and transfer them to liquid nitrogen after 24 hours, thereby obtaining suspended and acclimated cells.

4. Experimental results:

Experimental conclusion: The cells after suspension acclimation can grow normally after being passaged twice in the shaker flask, the highest growth density can reach 2.98×10 ⁶ cells/ml, the viability rate is 97.67%, and the cells are acclimated successfully in suspension.

2. Cell monoclonal screening

1. The purpose of the experiment

A stable monoclonal cell line was selected from the suspension acclimated cells in step 1 for subsequent acclimation experiments.

2. Main experimental materials

Monoclonal medium (SIGMA/SLBX8261), CD02 medium (Quacell/36A01), L-glutamine (Original Biology), 125ml shake flask (Corning/25818010), 500ml shake flask (Corning/11818004), cryopreservation Tube (Corning/23618076), 5ml pipette (Corning/20118020), 25ml pipette (Corning/17218010), 50ml centrifuge tube (Corning/31518601), 96-well plate (Corning/26018036).

3. Experimental procedure and results

a. Cell recovery and passage: Resuscitate a suspension-acclimated cell (serial number 01) in step 1, culture it in CD02 medium (4mM Gln), and perform subculture when the cell density reaches 1×10 ⁶ cells/ml. Keep the cells in a good growth state (cell viability above 95%).

b. Cell plating (96-well plate): Select 100 μl of cells in the logarithmic growth phase, perform monoclonal plating after limiting dilution, the theoretical plating density is 0.2 cells/well, and add 200 μl of monoclonal medium (4mMGln). A total of 10 96-well plates were plated.

c. Observation of monoclonal cell wells: After the plating is completed, the 96-well plate needs to be placed in an incubator for 3 hours, and the monoclonal cell wells are found by using a solentim cell imager to view the plate. The statistical results of monoclonal cell wells are as follows:

①:A02,A11,B05,C07,D10,F07,F09,H07

②:A05,B11,C03,D09,F05,G02

③:B03,B08,C11,D02,F06,G11,H02,H05,H11

④:C04,C06,D01,D04,E04,F02,F06

⑤:A02,A05,C06,C09,D06,E04,E11,H01,H11

⑥:C01,C05,C09,D05,E04,E08,F11,G04,H11

⑦:A11,A12,B09,B11,E08,F03,H07,H09

⑧:A02,B05,C08,D11,D09,E11,F08,H03,H08

⑨:B04,C08,C11,D02,D08,E04,E11,G03

⑩:A01,C02,E11,F05,F08,H03

d. Select high-quality monoclonal cell wells: When the monoclonal cells grow to 1/4 of the area of the 96-well wells, they will be sampled, and the monoclonal cell wells with better growth will be selected according to the cell shape and density. The result is as follows:

①: C07, D10, F07, F09, H07

②: F05

③: F06

⑤: C06, E04

⑥: C01, C05, C09, D05, E04, E08, F11, G04, H11

e. Monoclonal cell expansion: after the cells in the above-selected monoclonal cell wells are blown, scattered and homogenized, placed in a 96 deep-well plate to continue culturing, and 1 ml/well of CD02 medium (4mM Gln) is added; According to the viability and density, 5 monoclonal cell lines, 6-G04, 6-F11, 6-E04, 6-C01, and 6-C09, were selected for subsequent culture.

f. Stable passage and preservation of monoclonal cells: the above 5 strains of cells were expanded and cultured in CD02 medium (4mM Gln) at a passage density of 0.3×10 ⁶ cells/ml, and the cells were cryopreserved after 3 passages of passage. The cell line cryopreserved 8 cells. 6-G04 was selected for subsequent pressurized acclimation according to cell growth morphology, viability and density.

Experimental conclusion: A total of 5 stable monoclonal cell lines were selected from the suspension cells, and the 6-G04 cell line was finally selected for the subsequent domestication experiments, and the 6-G04 cell line was named CHO-K1S cell.

3. High osmotic pressure domestication

CHO-K1S cells were inoculated in CD02 medium with osmotic pressures of 300, 350, 400, 450 and 500 mOsm/Kg (different concentrations of NaCl were added to CD02 medium to prepare CD02 medium with different osmotic pressures), respectively. The density is 0.3×10 ⁶ cells/ml, a total of 30 ml, cultured in a cell incubator at 200 rpm, 37 degrees Celsius, 5% carbon dioxide, and the cell density and viability are measured every day, and the growth curve is drawn.

Its growth curve is shown in Table 1 and Figure 1:

Table 1

From the analysis of the results in the above experiments (Table 1), the CHO-K1S cells were passaged on the third day (cell density at 2.0 x 10 ⁶ cells/ml) for a total of 10 passages (the initial density of subculture (0.3-0.5) E+06, the density at passage is (1.5-2.0) E+06, generally grow for 2-3 days. The same below), draw the growth curve of the tenth passage cells in 400mOsm/Kg CD02 medium (Table 2 and Figure 2), thereby obtaining 400 mOsm/Kg osmotic-acclimated cells, named CHO-K1S-OA cells (400-A in Table 2 and Figure 2).

Table 2

The growth curve of CHO-K1S-O-A cells after acclimation at 400mOsm/Kg osmotic pressure was almost consistent with that in normal medium (300mOsm/Kg), while the growth curve of CHO-K1S-O-A cells when cultured at 400mOsm/Kg osmotic pressure for the first time was similar to The difference is larger in normal medium.

4. High osmotic pressure, high lactic acid and high ammonium ion domestication:

Taking the successfully acclimated CHO-K1S-O-A cells as host cells, the second round of adaptation and selection to the culture environment with high ammonium ions (7.5mM) and high lactate (2.5g/L) was carried out.

Sodium chloride, ammonium chloride and lactic acid were added to the serum-free medium CD02, so that the final concentration of ammonium ions was 7.5 mM, the lactic acid was 2.5 g/L, and the osmotic pressure was 400 mOsm/Kg, thereby obtaining high ammonium ions and high lactic acid screening. culture medium.

The first step was to determine the growth of CHO-K1S-O-A cells in high ammonium ion and high lactate selection medium, CHO-K1S-O-A cells (400-A-AL in Table 3 and Figure 3) were inoculated on high In the ammonium ion and high lactate selection medium, cultured at an osmotic pressure of 400 mOsm/Kg, and recorded its growth, see Table 3 and Figure 3 for details.

table 3

The CHO-K1S-O-A cells were inoculated into high ammonium ion and high lactic acid selection medium, cultured at an osmotic pressure of 400mOsm/Kg, and also subcultured on the third day for a total of 10 passages, thus stably passaged 10 times. CHO-K1S-OAL-A cells were obtained from the second cells as host cells, and CHO-K1S-OAL-A cells (400AL-A in Table 4 and Figure 4) were again inoculated into high ammonium ion and high lactate selection In the medium, cultured at an osmotic pressure of 400 mOsm/Kg, and recorded its growth, as shown in Table 4 and Figure 4 for details.

Table 4

It can be seen from Table 4 and Figure 4 that the growth curve of the acclimated CHO-K1S-OAL-A cells is similar to that in the normal medium (300mOsm/Kg, without the addition of ammonium chloride and lactic acid), and the cell density is Without feeding, it reached 6.3x10 ⁶ cells/ml, and at D8 and D9, the cell density was higher than that in normal medium (300mOsm/Kg), and the cells were named as Chinese hamster ovary epithelial cells CHO-K1-OAL.

It can be seen that the present invention performs screening in the existing CHO-K1 cell population, and obtains a high osmotic pressure (greater than 350mOsm/Kg), high ammonium ion (7.5mM) and high lactic acid (2.5g/L) environment Normal growing Chinese hamster ovary epithelial cells CHO-K1-OAL. The Chinese hamster ovary epithelial cell CHO-K1-OAL was deposited on August 19, 2019 in the General Microbiology Center (CGMCC) of the China Microorganism Culture Collection and Management Committee, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing The serial number is: CGMCC No.18322.

Claims (3)

1. Chinese hamster ovary epithelial cell CHO-K1-OAL, deposit number: CGMCC No.18322. 2. The application of the Chinese hamster ovary epithelial cell CHO-K1-OAL as claimed in claim 1 in the production of antibody-like protein products as a production cell. 3. application according to claim 2 is characterized in that, Chinese hamster ovary epithelial cell CHO-K1-OAL is used as producer cell in osmotic pressure≥350 mOsm/Kg, ammonium ion≥7.5mM, lactic acid≥2.5g/L. Application in the production of antibody-like protein products in the environment.

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