Preparation method of CA125 recombinant antibody

Abstract

The invention discloses a preparation method of a CA125 recombinant antibody, which comprises the following steps: electrically transferring a plasmid DNA fragment of a recombinant expression CA125 antibody into a CHO-GS cell by adopting a stable transfection technology, carrying out pressurized screening by using MSX to obtain a positive cell strain of a high expression CA125 antibody, establishing a positive cell pool, detecting the expression of the antibody, and carrying out subcloning on the positive cell strain to obtain a single cell line. Finally, a cell strain which can specifically and highly express the CA125 recombinant antibody and is qualified in stability is obtained, and a production process of the CA125 recombinant antibody is established. Compared with conventional CHO expression, the expression quantity of the recombinant antibody can be remarkably improved, and the recombinant antibody can be applied to high-yield expression of various recombinant antibodies, can be suitable for various sources and has wide application value.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a method for preparing a CA125 recombinant antibody. Background Art

[0002] The carbohydrate antigen CA125 is one of the commonly used tumor markers in clinical practice. The detection of serum CA125 has important clinical value for the efficacy and prognosis of epithelial ovarian cancer. In addition to ovarian cancer, CA125 has a high sensitivity to a variety of malignant tumors, including endometrial cancer, cervical cancer, colon cancer, lung cancer, etc. At present, the detection method of CA125 is mainly based on immunoassay, which is based on the specific binding reaction of antigen and antibody. Specific monoclonal antibodies are essential detection materials. At present, there are two main technologies for preparing CA125 monoclonal antibodies, one is hybridoma technology and the other is recombinant antibody technology. In the commonly used heterologous protein expression system, only mammalian cells are suitable for the expression of heterologous human antibodies of recombinant antibodies. Among them, CHO cells are widely used in the production of recombinant antibodies due to their advantages such as less secretion of endogenous proteins, high expression ability, easy amplification and culture, and the ability to express post-translational modifications similar to humans. However, conventional cell line development has the disadvantages of long cycle, low expression, poor cloning stability, and cumbersome antibody purification steps. In view of this, it is necessary to study a cell line for producing CA125 recombinant antibodies and a preparation method thereof. Summary of the invention

[0003] To address the deficiencies of the prior art, the present invention aims to provide a method for preparing a CA125 recombinant antibody, wherein the target plasmid is electro-transfected into CHO cells, a high-yielding cell strain is obtained by pressure screening, and the cell strain is then subcloned to obtain a monoclonal stable high-yielding cell line.

[0004] In order to achieve the above object, the present invention adopts the following technical solution: A method for preparing a CA125 recombinant antibody comprises the following specific steps: S1. Screening of high-expression CHO monoclonal cell lines: The revived CHO cells were subcultured twice, and then the CA125 monoclonal plasmid was introduced into the CHO cells to screen for high-expression CHO monoclonal cell lines; S2. Preparation of CA125 recombinant antibody: The selected CHO monoclonal cell line is inoculated into a shake flask, cultured continuously, centrifuged, the cell culture supernatant is collected, filtered and purified to obtain CA125 recombinant antibody.

[0005] Preferably, in the aforementioned step S1, the specific steps of screening a CHO monoclonal cell line with a high expression level are: (1) Resuscitate CHO cells, subculture twice until the cells are in the logarithmic growth phase, electroporate the expression plasmid into CHO cells, and plate them; (2) Using a selection agent to screen cells that stably express CA125 recombinant antibodies, gradually amplify the positive clone cells, and detect their expression levels; (3) The selected high-expressing cell lines are cryopreserved, and the high-expressing cell lines are selected for subcloning and plating to screen the CHO monoclonal cell lines with high expression and stability.

[0006] Preferably, in the aforementioned step (1), the plating is carried out based on the viable cell density of 1.0×10 4 cells / well in a 96-well plate.

[0007] Preferably, in the aforementioned step (2), the selection agent uses MSX reagent with a final concentration of 30 μM.

[0008] Preferably, in the aforementioned step (2), the positive clone cells are specifically and stepwise expanded to 24-well plates, 6-well plates and shake flasks.

[0009] Preferably, in the aforementioned step S2, the density of inoculation of the CHO monoclonal cell line is 3×10 5 ~5×10 5 cells / mL.

[0010] Preferably, in the aforementioned step S2, the CHO monoclonal cell line is cultured in a shake flask for 14 consecutive days or until the cell viability drops to 70%.

[0011] Preferably, in the aforementioned step S2, the centrifugal speed is 8000 rpm, the centrifugal time is 10 min, and the protein A is purified by filtration using a 0.22 μm filter membrane.

[0012] A cell strain for producing CA125 recombinant antibody is prepared by the method.

[0013] The present invention is beneficial in that: (1) The CA125 / CHO engineered cell line of the present invention is a self-constructed stable high-yield cell line. The target plasmid is electro-transfected into CHO cells, and a high-yield cell line is obtained by pressure screening. The cell line is then subcloned to obtain a monoclonal stable high-yield cell line. The cell line is suitable for suspension culture in serum-free CHO medium, has the advantages of good stability, high expression level, easy purification, easy scale-up production, etc., and has a high market value. (2) Compared with conventional CHO expression, the present invention can significantly increase the expression level of recombinant antibodies and can be applied to the high-yield expression of various recombinant antibodies. It can be applied to various sources and has a wide range of application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is the process flow chart for the development of CA125 recombinant antibody; Figure 2 It is a comparison chart of the expression levels of different positive clone antibodies; Figure 3 It is a comparison chart of antibody expression levels between positive strains and working strains; Figure 4 It is a comparison chart of the recombinant cell growth curve and cell viability; Figure 5 This is the electrophoresis gel image of CA125 recombinant cell supernatant purified by Protein A; Figure 6 It is the HPLC profile of CA125 recombinant antibody (1, cell supernatant-reduced, 2, flow-through-reduced, 3, dissociated antibody-reduced, 4, cell supernatant-non-reduced, 5, flow-through-non-reduced, 6, dissociated antibody-non-reduced); Figure 7 This is the AKTA purification profile of CA125 recombinant cell supernatant. DETAILED DESCRIPTION

[0015] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments. Example

[0016] See also Figure 1 A method for preparing a CA125 recombinant antibody comprises the following steps: using a stable transfection technique, electroporating a plasmid DNA fragment that recombinantly expresses the CA125 antibody into a CHO-GS cell, using MSX pressure screening to obtain a positive cell line that highly expresses the CA125 antibody, establishing a positive cell pool, detecting antibody expression, obtaining a single cell line by subcloning the positive cell line, and finally obtaining a cell line that can specifically and highly express the CA125 recombinant antibody and has a qualified stability, and establishing a production process for the CA125 recombinant antibody. The specific steps are as follows: 1. Cell Preparation Resuscitate a CHO cell line and passage it twice until the cells are in the logarithmic growth phase and the cell density is 1.5×10 6 ~2.5×10 6 cells / mL, and ensure that the viability is above 95%. Take an appropriate amount of cells (1.0×10 7 cells), centrifuge at 1000 rpm for 5 min, discard the supernatant, add 2 mL of CDCHO medium and resuspend and wash once.

[0017] 2. Electroporation transfection (1) Resuspend the cells in 700 μL CDCHO medium, add 30 μg of CA125 monoclonal plasmid, mix well by pipetting, and quickly transfer to a 0.4 cm electric transfer cup that has been bathed in ice, trying to keep the cells in suspension.

[0018] (2) Set the parameters of the electroporator, exponential wave: 250 V, 950 uF, 4 mm, perform electric shock, carefully remove the white foam on the surface with a pipette, then add 300 μL CDCHO culture medium and let it stand for 10 min.

[0019] (3) Transfer the cells to 30 mL of CDCHO medium and place them in a carbon dioxide incubator (5% CO2, 37°C) to recover for 3 to 5 hours. Then, culture them overnight in a carbon dioxide incubator (5% CO2, 37°C, 120 rpm).

[0020] (4) Take an appropriate amount of cell fluid 24 hours after transfection and calculate the number of viable cells according to 1.0×10 5 The cells / mL was diluted and plated in a 96-well plate with 100 μL per well, and the plate was placed in a carbon dioxide incubator for static culture (5% CO2, 37°C).

[0021] (5) 48 hours after transfection, add MSX at a final concentration of 30 μM to each well for screening. Place the cells in an incubator (5% CO2, 37°C) and culture for 14 to 21 days after transfection. Screen the positive wells that have grown a single clone.

[0022] (6) Gradually expand the positive wells of a single clone to 24-well plates, 6-well plates, and shake flasks, and select 3 to 5 positive clones with the highest expression levels for cryopreservation.

[0023] 3. Subcloning (1) Resuscitate the positive cell line to be cloned, and use CHO CD04 medium + 4.5% Feed 02A + 0.45% Feed B02 for 2 to 3 times, and culture until the cell density is 1.0×10 6 ~2.0×10 6 cells / mL, and cell viability was greater than 95%.

[0024] (2) Take an appropriate amount of cell suspension into a 1.5 mL EP tube and dilute the cell density in multiples using CHO CD04 medium.

[0025] (3) Take out the subcloning medium A and B in advance and preheat them in a 37°C water bath. Prepare the subcloning medium AB mixture at a ratio of A:B = 100:1 according to the number of plates to be plated.

[0026] (4) Use AB mixture to dilute the cells to be used in multiple ratios to 2.5 cells / mL; use a pipette to aspirate the above cell solution and spread it on a 96-well plate at 200 μL / well, with a plating density of 0.5 cells / well.

[0027] (5) 2-3 hours after plating, observe each well under an inverted microscope and mark the wells with single cells; place in an incubator for static culture (37°C, 5% CO2) and make further observations and markings.

[0028] (6) Observe the cell growth status and use CHO CD04 medium to gradually expand the cells with higher expression levels to 24-well plates, 6-well plates and shake flasks. Passage once or twice until the cell state is stable. The cell density is 1×10 7 cells / mL and cryopreserve to preserve monoclonal cell lines.

[0029] (7) Continuous culture: After the high-expression monoclonal cell line is revived, expand it to a 50 mL shake flask for culture. During this period, feed and glucose are added. When the cell viability drops below 70% or after 14 days of culture, centrifuge at 8000 rpm for 10 min to collect the liquid.

[0030] 4. Supernatant purification (1) Equilibrate the Protein A column with 10 mM PBS (pH 7.4) for 4-5 column volumes. After the column is equilibrated, adjust the UV 280nm to 0.

[0031] (2) Filter the cell supernatant after centrifugation using a 0.22 μm filter membrane and dilute the supernatant with 1 / 5 of the supernatant volume in 100 mM PBS (pH 7.4) dilution buffer before loading.

[0032] (3) After loading, use 10 mM PBS (pH 7.4) as the equilibration buffer for 5-10 column volumes until the baseline approaches a stable state or UV 280 nm approaches zero.

[0033] (4) Use 0.1 M citric acid + 20% glycerol (pH 3.5) to dissociate the sample, add 0.1 times the collection volume of 2 M Tris (pH 8.0) and 0.03 times the collection volume of 5 M NaCl in the collection tube, start collecting proteins when the UV 280 nm value rises above 0.2-0.3 Au, and stop collecting proteins when the UV 280 nm value drops to 0.2-0.3 Au.

[0034] (5) Perform dialysis with dialysate at a ratio of dissociated antibody volume to dialysate volume of 1:50 for three times, with an interval between dialysis sessions of no less than 4 hours and no more than 24 hours.

[0035] (6) Filter the above-mentioned target antibody using a 0.22 μm filter membrane, add the preservative proclin 300, and store it at -20°C for a long time.

[0036] Using this process, a monoclonal cell line with a cell supernatant expression level of 3.0 mg / mL was obtained. The cell line expressed less supernatant impurities, and the purity of the target antibody could reach more than 98% after one-step purification by protein A.

[0037] Performance testing 1. The three positive clones obtained by screening were cultured continuously for 14 days, and the cell supernatant was obtained by centrifugation. CA125 recombinant antibody was purified by Protein A one-step affinity chromatography. The antibody expression level results are shown in Figure 2 .

[0038] pass Figure 2 It can be seen that the expression level of 2B2 is 3.12 g / L, the expression level of 3E7 is 1.14 g / L, and the expression level of 4G9 antibody is 3.05 g / L. Two high-expressing monoclonal cell lines 2B2 and 4G9 were screened by this method.

[0039] 2. The monoclonal cell line 4G9 was frozen to establish a working cell bank. The working cell line was revived and expanded to 500 mL for continuous culture for 14 days. The cell supernatant was obtained by centrifugation and purified by Protein A one-step affinity chromatography to obtain CA125 recombinant antibody. The antibody expression level results are shown in Figure 3 .

[0040] Depend on Figure 3 It can be seen that the antibody expression level of the monoclonal cell line is 3.05 g / L, and the antibody expression level of the working cell line is 3.01 g / L, and there is no significant difference between the two, indicating that the antibody expression of the recombinant cell line obtained by this method is stable.

[0041] 3. The recombinant 4G9 cells were cultured continuously for 14 days, and the cell density and viability were measured every day using a cell counter. Cell growth curves and cell viability curves were drawn based on the experimental data obtained. Figure 4 .

[0042] pass Figure 4 It can be seen that the recombinant cells reached the highest density on the 8th day of culture, about 1.2×10 7 cells / mL. After 7 days of culture, the cell viability began to slowly decline. After 14 days of culture, the cell viability was 60%, indicating that the recombinant cells had good growth vitality and proliferation status.

[0043] 4. The recombinant cell 4G9 was cultured for 14 days, and the cell supernatant was obtained by centrifugation. The CA125 recombinant antibody was purified by Protein A one-step affinity chromatography. The SDS-PAGE electrophoresis gel image is shown in Figure 5 .

[0044] Depend on Figure 5It can be seen that there are impurity proteins with a molecular weight of 35-48 kD in the cell supernatant. During the loading process, the impurity proteins do not bind to Protein A but directly flow through, thereby separating the impurity proteins from the target antibody. This shows that the content of impurity proteins in the supernatant expressed by the recombinant cells obtained by the present invention is low, and the CA125 recombinant antibody with higher purity can be obtained by using the one-step affinity chromatography method of Protein A.

[0045] 5. The CA125 recombinant antibody obtained by Protein A one-step affinity chromatography was detected by HPLC. The test results are shown in Figure 6 .

[0046] Depend on Figure 6 It can be seen that peak 1 is a foreign protein and peak 2 is the target antibody. According to the peak area calculation, the purity of the target antibody is 99%, indicating that CA125 recombinant antibody with qualified purity (>95%) can be obtained through the Protein A one-step affinity chromatography method.

[0047] 6. The recombinant 4G9 cells were cultured for 14 days, and the cell supernatant was obtained by centrifugation. The CA125 recombinant antibody was purified by Protein A one-step affinity chromatography. The AKTA purification map is shown in Figure 7 .

[0048] Depend on Figure 7 It can be seen that the impurities in the cell supernatant directly flowed through during the loading process, and the dissociated target antibody had a better peak shape, indicating that the use of Protein A one-step affinity chromatography can better separate the impurities and the target antibody, thereby obtaining a CA125 recombinant antibody with higher purity.

[0049] 7. The three positive clones obtained by screening were cultured continuously for 14 days, and the cell supernatant was obtained by centrifugation. CA125 recombinant antibodies were purified by one-step Protein A affinity chromatography. The binding ability of the three groups of purified antibodies to CA125 antigen was detected by enzyme-linked immunosorbent assay, and the control was mouse monoclonal antibody. The test results are shown in Table 1.

[0050] Table 1 ELISA test results of purified antibodies from different positive clones

[0051] It can be seen from Table 1 that when the concentration of CA125 antigen was diluted to 50 ku / mL, compared with the control group, the positive clones 4G9 and 3E8 both had higher activity. Since the expression level of 4G9 was 3.05 g / L, which was higher than that of 3E7 (1.14 g / L), cell line 4G9 was selected for subsequent production.

[0052] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the above embodiments do not limit the present invention in any form, and any technical solution obtained by equivalent replacement or equivalent transformation falls within the protection scope of the present invention.

Claims

1. A method for preparing a CA125 recombinant antibody, characterized in that: The specific steps include: S1. Screening of high-expression CHO monoclonal cell lines: The revived CHO cells were subcultured twice, and then the CA125 monoclonal plasmid was introduced into the CHO cells to screen for high-expression CHO monoclonal cell lines; S2. Preparation of CA125 recombinant antibody: The selected CHO monoclonal cell line is inoculated into a shake flask, cultured continuously, centrifuged, the cell culture supernatant is collected, filtered and purified to obtain CA125 recombinant antibody.

2. The method for preparing a CA125 recombinant antibody according to claim 1, characterized in that: In step S1, the specific steps of screening a CHO monoclonal cell line with a high expression level are: (1) Resuscitate CHO cells, subculture twice until the cells are in the logarithmic growth phase, electroporate the expression plasmid into CHO cells, and plate them; (2) Using a selection agent to screen cells that stably express CA125 recombinant antibodies, gradually amplify the positive clone cells, and detect their expression levels; (3) The selected high-expressing cell lines are cryopreserved, and the high-expressing cell lines are selected for subcloning and plating to screen the CHO monoclonal cell lines with high expression and stability.

3. The method for preparing a CA125 recombinant antibody according to claim 2, characterized in that: In step (1), the cell culture medium was plated at a viable cell density of 1.0 × 10 4 cells / well in a 96-well plate.

4. The method for preparing a CA125 recombinant antibody according to claim 2, characterized in that: In the step (2), the selection agent uses MSX reagent with a final concentration of 30 μM.

5. The method for preparing a CA125 recombinant antibody according to claim 2, characterized in that: In the step (2), the positive clone cells are specifically expanded step by step to 24-well plates, 6-well plates and shake flasks.

6. The method for preparing a CA125 recombinant antibody according to claim 1, characterized in that: In step S2, the inoculation density of the CHO monoclonal cell line is 3×10 5 ~5×10 5 cells / mL.

7. The method for preparing a CA125 recombinant antibody according to claim 1, characterized in that: In step S2, the CHO monoclonal cell line is continuously cultured in a shake flask for 14 days or until the cell viability drops to 70%.

8. The method for preparing a CA125 recombinant antibody according to claim 1, characterized in that: In the step S2, the centrifugal speed is 8000 rpm, the centrifugal time is 10 min, and the protein A is purified by filtration using a 0.22 μm filter membrane.

9. A cell line for producing CA125 recombinant antibody, characterized in that: The cell line is prepared by the method of claims 2 to 5.

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