Use of a fatty acid in increasing the concentration of a therapeutic antibody expressed by CHO cells

Abstract

The application discloses application of a fatty acid in improving concentration of a therapeutic antibody expressed by CHO cells. The application relates to the technical field of protein engineering, and the exact composition of the additive is eicosanoid C20:2. In the culture process of Chinese hamster ovary (CHO) cells for expressing a therapeutic antibody, the addition of C20:2 can improve the stress resistance of the CHO cells in the culture process, thereby effectively improving the yield of the therapeutic antibody, and the quality of the antibody is not affected.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of protein engineering, and particularly relates to application of fatty acid in improving concentration of therapeutic antibody expressed by CHO cells. BACKGROUND

[0002] Antibody is an immunoglobulin produced by plasma cells, which can specifically bind to corresponding antigens. Therapeutic antibodies play a very important role in the prevention and treatment of major diseases. At present, mammalian expression system is usually the first choice for manufacturing biopharmaceuticals. Chinese hamster ovary (CHO) cells are the preferred choice for producing human therapeutic protein drugs among mammalian cells (KAPLON H, et.al., MAbs, 2020). CHO cells have good plasticity and stress resistance, can grow in high density in a bioreactor, and can express exogenous therapeutic proteins with high quality.

[0003] At present, there are mainly two methods to improve the production of antibodies by CHO cells: 1. Genetic engineering of CHO cells. This method mainly targets cell-related genes that affect the production of antibodies by CHO cells, such as target points related to apoptosis, growth, metabolism, protein secretion and modification, and carries out corresponding gene editing, so as to obtain or lose certain functions (Changzhi Xu, et.al., Applied Microbiology and Biotechology, 2019). These methods can obtain performance-optimized modified cell strains, but also have problems such as long screening period, instability and potential off-target problems caused by multi-gene editing; 2. Optimization of culture strategy and medium. Adding different nutrients or preparations in the culture medium can have the same promoting effect. For example, adding α-tocopherol (Toronjo-Urquiza L, et.al., Antioxidants, 2019), 3-methyladenine (Baek E, et.al., Biotechnology and Bioengineering, 2016), valproic acid (Segar K P, et.al., Journal of Bioscience and Bioengineering, 2017) and valine (Savizi I S P, Applied Microbiology and Biotechnology, 2022) in the culture medium shows certain promoting effect. Most of these additives are exogenous substances, which also have certain effects on cell growth and antibody purification. Finding potential endogenous substances in cells is an important development direction. SUMMARY

[0004] The main purpose of the present application is to provide a kind of fatty acid in the application of improving the concentration of therapeutic antibody expressed by CHO cell, which can effectively solve the problems in the background art.

[0005] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is:

[0006] The present application also provides a kind of fatty acid in the application of improving the concentration of therapeutic antibody expressed by CHO cell.

[0007] Further improvement lies in that the fatty acid is fatty acid C20:2.

[0008] Further improvement lies in that the CHO cell is CHO-K1, CHO-IgG cell.

[0009] Further improvement lies in that the addition concentration of the fatty acid ranges from 0 to 20 μM.

[0010] Further improvement lies in that the culture method of the CHO cell expressing therapeutic antibody is one of batch culture, fed-batch culture or transient transfection.

[0011] Fatty acids are important nutrients, which can be divided into saturated fatty acids, monounsaturated fatty acids and polyunsaturated fatty acids according to the number of double bonds. Fatty acids are important components of biological membranes, and can also provide energy through oxidative decomposition after entering cells. Some fatty acid derivatives are important signal transduction molecules and widely participate in various life processes of cells. Low temperature can induce the increase of unsaturation of fatty acids in CHO cells, resulting in the increase of the yield of therapeutic proteins. Overexpression of lipid metabolism global regulation genes SREBF1 and SCD1 can significantly enhance the yield of therapeutic proteins in CHO cells. However, a more direct method can be achieved by screening lipid additives for treatment.

[0012] Compared with the prior art, the present application has the following beneficial effects: by adding C20:2 of corresponding concentration in the process of producing antibodies by CHO cells, the yield of corresponding therapeutic antibodies can be promoted, and the quality of antibodies is not affected, which promotes the yield improvement of antibody production industry. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 Figure for the effect of fatty acid C20:2 addition on cell growth and antibody concentration in 6-well plate;

[0014] Figure 2 Figure for the effect of fatty acid on cell growth and antibody production capacity in batch culture;

[0015] Figure 3 Figure for the effect of fatty acid on cell growth and antibody production capacity in fed-batch culture;

[0016] Figure 4 Figure of the effect of fatty acid C20:2 on cell apoptosis;

[0017] Figure 5 Figure of the effect of fatty acid C20:2 on antibody quality;

[0018] Figure 6 Figure of the effect of fatty acid C20:2 on transiently expressed antibody. DETAILED DESCRIPTION

[0019] The technical solutions of the present application are described clearly and completely below in combination with the drawings (tables) and the detailed description. It should be noted that the described embodiments are exemplary and not restrictive. These embodiments do not limit the scope of the present application in any way. In addition, for the technologies not described or not described in detail in the following embodiments, they are prior art and thus are not described in detail.

[0020] 1. Materials

[0021] The experimental methods used in this embodiment are all conventional biochemical methods unless otherwise specified. The experimental materials used in this embodiment are all purchased from conventional biochemical reagent companies unless otherwise specified. Among them, the CHO-K1 cells and CHO-IgG cells used in the experiment are preserved in the laboratory. Other experimental materials are shown in Table 1. The experiments in this embodiment are set up in triplicate, the results are averaged, * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001.

[0022] Table 1 Cell biological reagent consumables and manufacturers

[0023]

[0024]

[0025] 2. Experimental methods and results

[0026] 2.1 Effect of fatty acid C20:2 on cell viability

[0027] 2.1.1 Experimental method

[0028] Cell counting: when counting adherent cells, after resuspending the cells on the dish with fresh complete medium according to the method of subculture, the cell suspension was aspirated into a centrifuge tube, 0.4% trypan blue dye was added, mixed with a pipette gun, and then taken to a cell counting plate slot for automatic counting. The number of viable cells and the percentage of viability were recorded. When counting suspended cells, the cell suspension was mixed and directly aspirated into a centrifuge tube, and counted in the same way as adherent cells.

[0029] MTT drug toxicity test: When the cells reach the logarithmic growth phase, treat the cells with trypsin EDTA solution. After digestion, gently blow the cells with culture medium. Take a portion of the cell suspension for counting and adjust the plating concentration to 0.5-1*10 5 / mL, the plate size for the MTT assay is generally 6000-11000 cells / well. After preparing the cell suspension, gently mix it and add 100μL / well to a 96-well plate. After the cells have grown in the incubator for 24 hours and have established their morphology, use a pipette to remove the old culture medium from the wells and add serum-free culture medium. After culturing for 12 hours, carefully remove the serum-free culture medium and add the pre-prepared fatty acid to the 96-well plate. After continuing to culture for 48 hours, remove the 96-well plate and use a pipette to draw 20μL of MTT solution (5mg / mL) along the wall and add it to each well of the 96-well plate. Continue to culture for 4 hours. Carefully remove the culture medium from the wells with a pipette, add 150μL of DMSO to each well using a dispenser, and then place the 96-well plate on a shaker and shake at low speed for 10 minutes to make a uniform liquid. Use a multifunctional microplate reader to measure the absorbance of each well at a wavelength of 490nm.

[0030] 2.1.2 Evaluation indicators and results

[0031] The results showed that 26 common fatty acids had no significant effect on the cell viability and cell number of CHO-K1 cells and CHO-IgG cells within 40 μM. Figure 1 As can be seen from A and 1B, adding 0-20 μM C20:2 to CHO-IgG cells in 6-well plates and continuously culturing for 5 days did not affect the live cell number and cell viability of CHO-IgG cells.

[0032] 2.2 Effects of fatty acid C20:2 on cell resistance

[0033] 2.2.1 Experimental methods

[0034] Batch culture: Figure 1 As shown in A, CHO-IgG cells were seeded into 6-well plates, 0-20 μM C20:2 was added to the culture medium and cultured for 5 consecutive days, and methionine sulfoxide (MSX) was added for screening culture. Finally, the culture medium was collected for antibody concentration detection, as shown in Figure 1 As shown in Figure 3, within the concentration range of 0-20 μM, C20:2 can increase the antibody concentration by about 1-fold. The cells were then inoculated into a cell culture shaker and MSX was added. Every two days, the cells were counted and a growth curve was drawn, the viability percentage was recorded, and 1 mL of the cell suspension was removed and centrifuged at 1500 rpm for 3 min. The supernatant was retained for ELISA to detect the antibody yield. The results are shown in Figure 3. Figure 2 shown.

[0035] Fed-batch culture: Cells were inoculated into cell culture flasks, and the feed medium consisted of (CHO CD Efficient Feed TM B) and CDCHO medium, wherein the ratio of CHO CD Efficient Feed TM B was 10%. MSX was added, and during the period, fresh feed medium was added on day 3, 6, 9, 12 respectively following the fed-batch culture strategy, cell counting was drawn to plot growth curve every two days, the percentage of viability was recorded, and 1 mL cell suspension was taken out, centrifuged at 1500 rpm for 3 min, the supernatant was retained for ELISA detection of antibody yield, and the results are shown in Figure 3 .

[0036] Enzyme-linked immunosorbent assay (double antibody sandwich method): The enzyme-labeled plate is coated with human IgG antigen to be adsorbed on the surface of the solid carrier. When measuring, specific antibodies are added to react with antigens, and then enzyme-labeled secondary antibodies are combined with antibodies. Finally, the amount of enzyme combined on the enzyme-labeled plate is proportional to the amount of antibodies in the sample. After adding the enzyme reaction substrate TMB, the substrate is catalyzed by the enzyme to become a blue color, and after adding the termination liquid to terminate the enzyme reaction, a yellow color is formed. At 450 nm, the amount of product is directly related to the amount of antibodies in the sample, so quantitative analysis can be carried out according to the OD450nm of the standard and sample.

[0037] 2.2.2 Evaluation index and results

[0038] Further use of the shake flask batch culture found that the addition amount of fatty acid C20:2 in the range of 0-20 μM still has the most obvious promoting effect on CHO-IgG cell antibody production. The effect of fatty acid C20:2 on cells is to delay cell growth, reduce cell maximum density, and prolong cell culture time. Since the initial addition of fatty acids will affect cell growth, the fatty acids are added on the 8th day of batch culture. After changing the addition time, the fatty acids showed better promoting effect on antibody expression of CHO-IgG cells. In addition to batch culture mode, fed-batch culture mode is more common in CHO cell culture. Through fed-batch culture, it can be seen that fatty acid C20:2 still shows better promoting effect on CHO-IgG cell antibody production.

[0039] 2.3 Effect of fatty acid C20:2 on cell apoptosis

[0040] 2.3.1 Experimental method

[0041] After the cells are collected, wash the cells twice with pre-cooled phosphate buffer solution, add 100 μΐ of pre-cooled 1 x Annexin V Binding Buffer to the cells, and gently mix the cell suspension. Add 5 μΐ of Annexin V-FITC and 5 μΐ of PI to each tube in the experimental group, and do not add dye to the double negative control group. Set up two single negative control groups, and add 5 μΐ of Annexin V-FITC and 5 μΐ of PI, respectively. Incubate at room temperature (20-25°C) for 15 min in the dark. Detection: Add 400 μΐ of 1 x Annexin V Binding Buffer to the cell suspension, gently mix the cell suspension, and move the sample to a flow tube and place it on ice in the dark. To prevent fluorescence quenching, use a flow cytometer to detect within 1 h. Turn on the detector, flow path car, and computer 30 min in advance, and preheat for 15-30 min.

[0042] 2.3.2 Evaluation index and result

[0043] The results, as shown in Table 2, show that the addition of fatty acid C20:2 delays early apoptosis and late apoptosis of the cells compared with the control group. Figure 4

[0044] 2.4 Effect of fatty acid C20:2 on antibody quality

[0045] 2.4.1 Experimental method

[0046] SDS-PAGE detection of antibody assembly integrity: Take the purified antibody sample, add protein loading buffer, and boil in a water bath for 10 min to denature the protein. Electrophoresis: Prepare a certain concentration of protein denaturation gel according to the molecular weight of the protein, add electrophoresis buffer to cover the sample well, and the liquid level in the tank should not be lower than the liquid level outside the tank. Add a certain amount of protein sample to the sample well, and add 1 x protein loading buffer to the both ends of the gel to ensure that the sample line is straight during electrophoresis. First, adjust the voltage to 80 V until the marker bands are separated, and then adjust the voltage to 130 V until the bromophenol blue is just electrophoresed out of the separation gel. Staining: pry open the gel plate, place the protein gel in a box containing an appropriate amount of Coomassie brilliant blue staining solution, and shake at room temperature for 1-2 h. Decolorization: take out the protein gel after staining, put it into a new box, wash it with PBS, discard the PBS, add an appropriate amount of decolorizing solution, and shake at room temperature until the protein bands can be observed. Place the protein gel in a pot with boiling water, and cook on an electric stove until the bands are washed and the protein gel becomes transparent. Take a photo of the gel under the light.

[0047] ​Molecular sieve detection of antibody purity: open the machine, computer and program. Flush the pump: in waste mode, flush the pump with high flow rate of ultrapure water filtered through a 0.22 μm filter and ultrasonic degassing (5 mL / min). Flush the instrument system: in load mode, flush the instrument with ultrapure water, set the flow rate to 0.5 mL / min and the column pressure to 0.5 MPa. Column equilibration: to prevent bubbles from entering the column, connect the column when water comes out of both ends. After the column is loaded, flush it with ultrapure water until the UV and conductivity lines are straight, then adjust the zero, and then flush the column with protein purification buffer for 2 h, set the flow rate to 1 mL / min and the column pressure to 0.5 MPa. Clean the sample loop: after the baseline is stable, connect the sample loop, use a new syringe to draw ultrapure water and clean the sample inlet 3 times, then clean it 3 times with protein purification buffer, and make sure to remove the bubbles each time. Sample loading: after the UV and conductivity lines are straight, adjust the zero, and then load the sample into the sample loop in load mode without removing the syringe. After 5 min, switch to inject mode, and the sample is analyzed by column. Sample collection: after the peak appears, start manually setting the collection. Flush the column: after the collection is complete, flush the column with ultrapure water at a pressure of 0.5 MPa. After the baseline is stable, turn off the program, computer and machine.

[0048] Molecular sieve detection of antibody affinity: A549 cells in the logarithmic growth phase were taken, counted and adjusted to the number of cells added to the EP tube. After centrifugation at 1500 rpm and 4°C for 3 min, the cells were washed once with PBS containing 1% fetal bovine serum and centrifuged to discard the supernatant. A series of gradient concentrations of anti-EGFR antibody and PBS (negative control) were added to each tube of cells, and incubated at 4°C for 1 h. After ice bath, the cells were centrifuged at 1500 rpm and 4°C for 3 min, the supernatant was discarded, and the cells were washed once with PBS containing 1% fetal bovine serum and centrifuged to discard the supernatant. 100 μL of FITC-labeled goat anti-human IgG was added, and incubated at 4°C in the dark for 45 min. After labeling, the cells were centrifuged at 1500 rpm and 4°C for 3 min, the supernatant was discarded, and the cells were washed once with PBS containing 1% fetal bovine serum and centrifuged to discard the supernatant. Finally, the cell pellet was resuspended in 350 μL of PBS containing 1% fetal bovine serum and detected on the machine.

[0049] 2.4.2 Evaluation index and results

[0050] The results, as shown in Figure 5 , the addition of fatty acid C20:2 did not affect the assembly integrity, purity and affinity of the antibody itself, and the fatty acid did not affect the quality of the antibody after addition.

[0051] 2.5 Effect of fatty acid C20:2 on transient expression of antibody

[0052] After transient transfection of RTX, Herceptin and anti-uPAR antibody plasmids in CHO-K1 cells, the supernatant was collected 48 hours later for ELISA detection, and the results are shown in Figure 6 After adding C20:2, the concentration of the three antibodies was improved to a certain extent.

[0053] 3、Conclusion

[0054] The experimental results show that by adding a corresponding concentration of C20:2 to the process of producing antibodies in CHO cells, the yield of the corresponding therapeutic antibodies can be promoted, and the quality of the antibodies is not affected.

[0055] The above description shows the basic principles and main features of the present application and the advantages of the present application. Those skilled in the art should understand that the present application is not limited by the above examples, and the above examples and descriptions in the specification are only to illustrate the principles of the present application. Without departing from the spirit and scope of the present application, various changes and improvements can be made to the present application, and these changes and improvements all fall within the scope of the claimed present application. The scope of protection of the present application is defined by the appended claims and their equivalents.

Claims

1. A method for increasing the concentration of therapeutic antibodies expressed by CHO cells, characterized in that: The fatty acid is fatty acid C20:2, and the CHO cells are CHO-K1 and CHO-IgG cells.

2. The use according to claim 1, characterized in that: The added concentration of the fatty acid C20:2 is in the range of 6-20 μM.

3. The use according to claim 1, characterized in that: The method for culturing the CHO cells to express therapeutic antibodies is one of batch culture, fed-batch culture or transient transfection.

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