阅读说明：本技术 一种细胞上清中高产量抗体表达和纯化的方法 (Method for expressing and purifying high-yield antibody in cell supernatant ) 是由 阮宏强 李紫艺 张鹏 于 2021-09-30 设计创作，主要内容包括：本发明提供一种细胞上清中高产量抗体表达和纯化的方法,属于抗体制备领域,通过饥饿培养的方式来增加体外培养杂交瘤细胞培养基中单克隆抗体的含量,通过较为简单高效的抗体纯化处理后,可以得到相较于传统培养方式3倍抗体量和纯度达到90％的单克隆抗体,可以大大缩减抗体生产成本,并扩大该方法在科研学术领域的进一步应用。(The invention provides a method for expressing and purifying high-yield antibody in cell supernatant, which belongs to the field of antibody preparation, increases the content of monoclonal antibody in a culture medium for culturing hybridoma cells in vitro by a starvation culture mode, can obtain monoclonal antibody with 3 times of the antibody quantity and the purity of 90 percent compared with the traditional culture mode after simpler and more efficient antibody purification treatment, can greatly reduce the production cost of the antibody, and expands the further application of the method in the scientific research academic field.)

1. A method for expressing and purifying high-yield antibody in cell supernatant is characterized in that hybridoma cells corresponding to target antibody are subjected to starvation culture to enable the hybridoma cells to express the target antibody in a large quantity, then the target antibody is extracted from a culture medium after the starvation culture, and finally the target antibody is purified.

2. The method of claim 1, wherein the purification of the target antibody is performed by immunoadsorption and enrichment purification of the target antibody by protein magnetic beads through immunoprecipitation.

3. The method for expressing and purifying high-yield antibodies in cell supernatant according to claim 2, which comprises the following detailed steps:

step S1, performing amplification culture, namely performing normal amplification culture on the hybridoma corresponding to the target antibody until the hybridoma is amplified to passage density;

step S2 starvation culture, adding culture medium without main nutrient substances into the hybridoma cells, continuing culture until the survival rate of the cells is lower than 50%, and collecting supernatant of the culture medium;

step S3, performing immunoprecipitation enrichment, adding protein magnetic beads coated with specific protein antigens to the supernatant collected in step S2, so that the target antibodies are bound to the protein magnetic beads, and collecting the protein magnetic beads;

step S4, washing the protein magnetic beads collected in the step S3 with a washing solution, washing away substances which are not specifically bound on the protein magnetic beads, and collecting the protein magnetic beads;

and a step S5 of eluting the protein magnetic beads collected in the step S4 with an eluent, eluting the target antibody specifically bound to the protein magnetic beads, collecting the eluent, and concentrating the eluent.

4. The method of claim 3, wherein the primary nutrient is fetal bovine serum.

5. The method of claim 3, wherein the protein beads are protein beads coated with protein A or protein G.

6. The method according to claim 3, wherein an inhibitor for inhibiting the growth of the hybridoma corresponding to the non-target antibody is added to the culture medium during the amplification culture of step S1; in the step S2, the culture medium in the starvation culture is not supplemented with an inhibitor for inhibiting the growth of the hybridoma corresponding to the non-target antibody.

7. The method of claim 6, wherein the inhibitory substance comprises an antibiotic.

Technical Field

The invention belongs to the field of antibody preparation, and particularly relates to an antibody preparation method for expressing an antibody in a culture medium at a high yield by starvation culture of cells and purifying the antibody.

Background

Monoclonal antibodies play an important role in the biomedical field and the scientific research and study field. Firstly, the monoclonal antibody drug is a targeted antibody drug, and the main target is some specific antigens generated on the surface of tumor cells, or antigens with much higher expression level on the surface of tumor cells than on the surface of normal cells. The compound has the advantages of strong specificity, obvious curative effect and the like on the killing effect of tumor cells, and becomes one of hot medicines for research in recent years. For example, scientists find that the expression quantity of Her-2 antigen on the surface of breast cancer cells is far higher than that of normal cells through research, and the trastuzumab is designed, is a monoclonal antibody specifically aiming at the antigen, can kill the breast cancer cells, and brings great hope to patients. Rituzumab was approved by the U.S. FDA for marketing in 1999. Monoclonal antibody drugs play an important role in biotechnology pharmacy and are gradually developed into important methods for development in the field of biomedicine. Secondly, the monoclonal antibody is used as a common molecular biology identification tool in the scientific research field and is widely applied to analytical experiments such as immunoprecipitation experiments, enzyme-linked immunosorbent assays, western blotting and the like. Therefore, the high-yield and high-purity preparation of the monoclonal antibody is always concerned and has important significance for wider production and application.

In the field of biopharmaceuticals, monoclonal antibody drugs are usually prepared by lymphocyte hybridoma technology or genetic engineering technology. The principle of the lymphocyte hybridoma technology is that monoclonal antibodies are secreted by plasma cells transformed from B lymphocytes, each B lymphocyte can only produce one specific antibody, and the B lymphocytes have the antibody secretion function and the characteristics of growing in a proper culture medium. However, since B lymphocytes cannot be expanded wirelessly, scientists fuse antigen-immunized mouse B lymphocytes with mouse myeloma cells that can divide and expand indefinitely in culture to obtain hybridoma cells, which form cells with both antibody-secreting function and cell immortality-maintaining properties. After the required cell group is screened out, the large-scale monoclonal antibody is prepared mainly by two modes of an animal in-vivo induced ascites method and an in-vitro lymphocyte culture method. The method for inducing ascites in animals requires professionals to culture hybridoma cells in the peritoneum of a mouse, then to collect ascites after periodic culture for purification of monoclonal antibodies, and ascites contains complex impurities such as lipid components and the like, so that the expression and purification of the antibodies are complex. On the contrary, the method for culturing the lymphocytes in vitro is simple to operate, the components of the culture medium are single, and the purification difficulty is small. However, the antibody yield of lymphocytes cultured in vitro is low, and generally 1mL of the culture medium contains only 10-20ug of monoclonal antibody. And thus there is an urgent need to improve the yield thereof.

Disclosure of Invention

Based on the problems in the prior art, the invention provides a method for expressing and purifying high-yield antibodies in cell supernatant, the content of monoclonal antibodies in a culture medium for in vitro cultured hybridoma cells is increased by a starvation culture mode, and after simpler and more efficient antibody purification treatment, monoclonal antibodies with the antibody quantity 3 times that of the monoclonal antibodies and the purity of 90 percent can be obtained compared with those of the monoclonal antibodies in a traditional culture mode, so that the production cost of the antibodies can be greatly reduced, and the further application of the method in the scientific research field can be expanded.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

a method for expressing and purifying high-yield antibody in cell supernatant is characterized by that the hybridoma cell correspondent to target antibody is starved and cultured to make the hybridoma cell express large-quantity target antibody, then the target antibody is extracted from the culture medium after starved culture, and finally the target antibody is purified.

According to the scheme, the target antibody is purified by immunoadsorption, enrichment and purification of the target antibody by using protein magnetic beads and adopting an immunoprecipitation method.

According to the above protocol, the method for expressing and purifying high-yield antibodies in cell supernatant comprises the following detailed steps:

step S1, performing amplification culture, namely performing normal amplification culture on the hybridoma corresponding to the target antibody until the hybridoma is amplified to passage density;

step S2 starvation culture, adding culture medium without main nutrient substances into the hybridoma cells, continuing culture until the survival rate of the cells is lower than 50%, and collecting supernatant of the culture medium;

step S3, performing immunoprecipitation enrichment, adding protein magnetic beads coated with specific protein antigens to the supernatant collected in step S2, so that the target antibodies are bound to the protein magnetic beads, and collecting the protein magnetic beads;

step S4, washing the protein magnetic beads collected in the step S3 with a washing solution, washing away substances which are not specifically bound on the protein magnetic beads, and collecting the protein magnetic beads;

and a step S5 of eluting the protein magnetic beads collected in the step S4 with an eluent, eluting the target antibody specifically bound to the protein magnetic beads, collecting the eluent, and concentrating the eluent.

According to the scheme, the main nutrient substance is fetal calf serum.

According to the scheme, the protein magnetic beads are protein magnetic beads coated with protein A or protein G.

According to the above scheme, the culture medium in the amplification culture of the step S1 is added with an inhibitor for inhibiting the growth of the hybridoma corresponding to the non-target antibody; in the step S2, the culture medium in the starvation culture is not supplemented with an inhibitor for inhibiting the growth of the hybridoma corresponding to the non-target antibody.

According to the above scheme, the inhibiting substance comprises an antibiotic.

The invention has the beneficial effects that: compared with the existing screening method of the monoclonal antibody, the antibody expression and purification method of the invention has the advantages of simple operation, low cost, easy operation, high yield, high purity, complete activity and the like, and provides promising prospect for the wide application of the production and preparation of the monoclonal antibody.

Drawings

FIG. 1: SDS-PAGE gel of monoclonal antibodies in the examples

FIG. 2: high resolution molecular weight (non-reducing) protein mass spectra and results for the monoclonal antibodies of the examples

FIG. 3: mass spectra and results of high resolution molecular weight (reduced) protein for the monoclonal antibodies in the examples

FIG. 4: the affinity experiments of the monoclonal antibodies in the examples demonstrate a western-blot plot.

Fig. 2 and fig. 3 are results of the embodiment, and the results of each detection and analysis change, that is, the characters in the drawings are irrelevant to whether the detection method provided by the present invention can be repeatedly implemented, and the characters in the drawings are unclear so as not to affect those skilled in the art to repeatedly implement the detection method provided by the present invention.

Detailed Description

The technical solution of the present invention will be described below with reference to the specific embodiments and the accompanying drawings.

The first embodiment is as follows: starvation culture of hybridoma cell line HB95 and purification of monoclonal antibody.

A method for expressing and purifying high-yield antibody in cell supernatant includes such steps as starvation culturing the hybridoma cells corresponding to target antibody to make them express a lot of target antibody, extracting target antibody from the culture medium after starvation culturing, and immunoadsorption, enrichment and purification of target antibody by protein magnetic beads and immunoprecipitation.

In this embodiment, a hybridoma cell line HB95 is taken as an example to describe in detail a method for expressing and purifying a high-yield antibody in a cell supernatant provided by the present invention, which specifically includes the following detailed steps:

step S1 amplification culture, until the hybridoma cell is expanded to passage density, collecting frozen HB95 cell strain (hybridoma cell corresponding to target antibody) for rapid cell recovery, placing the cells in 15cm culture dish, adding 20mL of whole culture medium containing DMEM culture medium + 10% FBS (fetal bovine serum) + 1% double antibody (penicillin and streptomycin) for amplification culture, and controlling inoculation density at 2x 10⁵cells/mL, cells were placed at 37 ℃ in 5% CO₂The culture box is used for amplification culture, and the density of HB95 cells reaches 1x 10 after 2-3 days of passage⁶cells/mL or so (passage density);

step S2 starvation culture, continuously adding 40ml of warm DMEM culture medium without double antibodies and containing GlutaMAX into a 15cm culture dish, continuously culturing, observing the cell survival rate every day, recovering the culture medium when the cell survival rate is about 50%, centrifuging, and taking the supernatant for later use; the culture medium or culture medium supernatant which is not used temporarily can be quickly frozen by liquid nitrogen and then stored at-80 ℃;

and S3, enriching the immunoprecipitate, taking 50mL of each supernatant collected in the step S2, numbering as #1, #2 and #3, and unfreezing at room temperature if the supernatant is frozen. Filtering the supernatant with 0.45 μm filter membrane, adding about 1mL of protein A sepherose, placing in 360 deg.C rotary ferris wheel, combining at room temperature for more than half an hour, or combining at 4 deg.C for more than 2 hours, removing the supernatant and collecting protein magnetic beads;

step S4, adding 10mL PBS into the protein A seprase collected in step S3, rotating the mixture in a 360 ℃ rotating ferris wheel for 5min, removing the PBS and recovering the protein A seprase;

step S5, adding 5mL of elution buffer (0.1M citric acid-sodium citrate buffer pH 4.5) into the protein A sephsose collected in step S4, rotating in a 360 ℃ rotating ferris wheel for 10min, recovering the supernatant into a new centrifuge tube containing 1mL of 1M Tris (pH 9) to obtain the purified monoclonal antibody, introducing the monoclonal antibody into a 10K ultrafiltration tube for concentration, centrifuging for about 30min at 5000g, and taking the concentrated solution and OD (optical density) when the volume of the concentrated solution is about 250ul₂₈₀The quantification of the monoclonal antibodies was performed as follows:

the cell culture related reagents used in this example include fetal bovine serum, Dulbecco's Modified Eagle Medium (DMEM), penicillin-resistant PS, glutamine GlutaMAX, and placental blue, all of which are available from Gibco. Hybridoma cell lines purchased from HB95 from american type culture collection ATCC; protein A sepherose, phosphate buffered saline (PBS, pH 7.4) from Thermo; citric acid monohydrate, trisodium citrate dihydrate, Tris (hydroxymethyl) aminomethane (Tris) from Sigma; antibody concentration ultrafiltration tubes, filters purchased from Millipore; antibody concentration quantifier Nanodrop was purchased from Thermo corporation.

Example two: the purity of the monoclonal antibody was characterized by SDS-PAGE.

The acrylamide electrophoresis gel 10% NuPAGE precast gel, 10 × reducing agent, 4 × sample loading solution and 20 × electrophoresis buffer solution used in this example were purchased from Thermo corporation; monoclonal antibody W6/32 standard was purchased from abcam; prestained protein molecular weight marker was purchased from Bio-rad; the electrophoresis apparatus Mini Gel Tank was purchased from Thermo corporation.

Two portions of the monoclonal antibodies #1, #2, #3 and the monoclonal antibody standard substance in example one, each of which is 3ug, were added to the electrophoresis sample solution, and one portion was added with the reducing agent and one portion was not added with the reducing agent. After boiling at 95 ℃ for denaturation, samples are sequentially spotted in various loading channels of the preformed gel, electrophoretic separation is carried out by prestained protein molecular weight marker, and finally color development and gel image scanning are carried out by Coomassie brilliant blue color development liquid, wherein the gel image is shown in figure 1.

As can be seen from FIG. 1, the complete molecular weight of the antibody purified in the first example is about 150kDa, which is consistent with that of the standard; the reduced molecular weight is respectively 50KDa of the heavy chain and 23KDa of the light chain, and is consistent with the standard product. And the purity of the monoclonal antibody reaches more than 90%.

Example three: protein high resolution molecular weight characterization of monoclonal antibodies.

For the molecular weight characterization of the non-reduced monoclonal antibody, the sample purified in the first example was taken, and the sample concentration was diluted with pure water to 0.1. mu.g/. mu.L. For the molecular weight characterization of the reduced monoclonal antibody, the purified sample of example one was taken, the sample concentration was diluted with pure water to 0.1. mu.g/. mu.L, DTT was added to a final concentration of 10mM, 6M guanidine hydrochloride was added to 1M, and the reaction was carried out at 56 ℃ for 30 min. The corresponding samples were transferred to an autosampler vial and placed in an autosampler of Shimadzu liquid LC20AD, equipped with chromatographic column eclipseXD-C8. Loading volume 10uL, buffer: the solution A was 0.1% formic acid aqueous solution, and the solution B was 0.1% formic acid acetonitrile aqueous solution (acetonitrile: 84%). The column was equilibrated with 95% of liquid A and the flow rate was 400 nl/min. The chromatographic gradient was set at 10min and the relevant liquid phase gradients were as follows: 0 min-1 min, linear gradient of B liquid from 2% to 2%; 1 min-8 min, linear gradient of B liquid from 2% to 100%; the solution B is maintained at 100 percent for 8 minutes to 10 minutes. The high resolution mass spectrometer was a Q-exact Plus mass spectrometer (Thermo). Analysis duration: 10min, detection mode: positive ion, parent ion scan range: 300-3000m/z, first-order mass spectral resolution: 70,000at m/z 200, AGC target: 3e6, primary Maximum IT: 20ms, Number of scan ranges: 1, Dynamic exclusion: 40.0 s. The mass-to-charge ratio of the polypeptide and fragments of the polypeptide was collected as follows: 10 fragment patterns (MS2 scan) were acquired after each full scan (full scan), MS2 Activation Type: HCD, Isolation window: 2m/z, second order mass spectral resolution: 17,500at m/z 200, Microscan: 1, secondary Maximum IT: 60ms, Normalized fusion energy: 27eV, Underfill ratio: 0.1 percent.

The collected spectra were deconvoluted using BioPharma Finder 2.0 software to obtain the molecular weights of the corresponding peaks, as shown in fig. 2 and 3.

In this example, the high resolution molecular weight of the monoclonal antibody of example one is identified, and FIG. 2 shows that its non-reducing molecular weight is 149004.55 Da. As can be seen from FIG. 3, the heavy chain in the reduced molecular weight was 50960.85 Da. And because the sensitivity and resolution of the mass spectrum are far higher than those of the SDS-PAGE technology, the monoclonal antibody is determined to have two different light chains with molecular weights of 23636.87Da and 23866.67Da respectively. It was shown to be a double antibody with non-identical light chains. The purity of the product can be up to 99% by integrating the product according to peak area on a mass spectrogram.

Example four, experimental validation of the affinity of monoclonal antibodies.

The acrylamide electrophoresis gel 10% NuPAGE precast gel, 10 × reducing agent, 4 × loading solution, 20 × electrophoresis buffer solution, western blot blotting membrane and filter paper used in this example were purchased from Thermo corporation; prestained protein molecular weight marker was purchased from Bio-rad; anti-beta 2-microglobulin rabbit recombinant antibody (anti-beta 2 microrogobulin) was purchased from abcam; the secondary antibody horseradish peroxidase labeled goat anti-rabbit IgG is purchased from Biyuntian corporation; electrophoresis apparatus Mini Gel Tank and iBlot2 Dry transfer systems were purchased from Thermo corporation.

The monoclonal antibodies of example one were taken and added to 1mL protein A sepherose suspension in a volume of 1mg each, and placed in a 360 ℃ rotating ferris wheel and allowed to bind for more than half an hour at room temperature or for more than 2 hours at 4 ℃. Then, 200mg of 1 tissue sample was taken, and after adding 2mL of a gentle tissue extract (aqueous solution containing 0.5% (v/v) Igepal CA-630, 50mM Tris (pH 8.0), 150mM NaCl, 1mM EDTA and protease inhibitor) and steel beads, the tissue was subjected to a homogenization treatment for extraction of proteins in the tissue, and the biological activity of the proteins was retained. After ultracentrifugation at 40,000g for 30min, 50ul of the supernatant was retained as control A before enrichment, and then the remaining supernatants were added to protein A sepherose suspension and placed in a 360 ℃ rotating ferris wheel and combined at 4 ℃ for more than 2 hours. The monoclonal antibody will specifically bind to MHC-I proteins on the surface of tissue cells. After binding was complete, protein A sepherose was recovered by centrifugation and 50ul of supernatant was taken as control sample B after enrichment.

Samples a and B were subjected to SDS-PAGE running and WB analysis to verify that the monoclonal antibody was successfully enriched in MHC-I protein consisting of an alpha chain (molecular weight 43KDa) and a beta chain (molecular weight 43KDa) in the sample, which was verified in the WB experiment. The results are shown in fig. 4, where the MHC component in the tissue lysate was significantly reduced after enrichment compared to the a and B samples, demonstrating that the monoclonal purification format retained the activity of the monoclonal antibody.

The present invention is provided by the above embodiments only for illustrating and not limiting the technical solutions of the present invention, and although the above embodiments describe the present invention in detail, those skilled in the art should understand that: modifications and equivalents may be made thereto without departing from the spirit and scope of the invention and any modifications and equivalents may fall within the scope of the claims.