CN111087469B - Preparation method of fully human monoclonal antibody - Google Patents
Preparation method of fully human monoclonal antibody Download PDFInfo
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- C12N15/02—Preparation of hybrid cells by fusion of two or more cells, e.g. protoplast fusion
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- C07K2317/00—Immunoglobulins specific features
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- C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
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Abstract
The invention discloses a preparation method of a fully human monoclonal antibody, wherein the fully human monoclonal antibody is derived from a fully human monoclonal hybridoma cell strain, and the hybridoma cell strain is obtained by combining a human myeloma cell strain and human peripheral blood B lymphocytes; wherein the human myeloma cell line combination is formed by fusing human myeloma cell lines RPMI 8226 and K6H 6/B5. The fully human monoclonal antibody hybridoma cell strain obtained by combining the human myeloma cell strain and fusing the human peripheral blood B lymphocytes can be widely applied to the screening of functional fully human monoclonal antibodies and the development of anti-tumor fully human monoclonal antibody medicaments.
Description
Technical Field
The invention belongs to the technical field, and particularly relates to a preparation method of a fully human monoclonal antibody.
Background
Monoclonal antibodies can be used for antigen structure analysis, separation and purification of specific molecular antigens, diagnosis and treatment of clinical diseases, and the like. However, most of the monoclonal antibodies are murine at present, and there are many problems when the murine monoclonal antibodies are applied to human treatment, so that the development of monoclonal antibody technology in clinical treatment application is slow. For example, monoclonal antibodies of murine origin are not effective in activating complement and Fc receptor related effector systems in humans; once recognized by the human immune system, human anti-mouse antibodies are produced and are rapidly cleared from the human circulatory system. Therefore, humanized transformation is performed on the basis of maintaining high affinity for specific antigen epitopes, so that immunogenicity of the heterologous antibody is reduced, or development of the fully-humanized monoclonal antibody becomes a key point of monoclonal antibody research.
The humanized transformation technology, phage antibody library technology and transgenic mouse technology of the murine antibody are three main technologies of humanized transformation and humanized antibody preparation. The antibody medicines entering clinic at present mainly adopt humanized transformation of murine antibody. The antibody drugs in the future are mainly humanized antibodies. The monoclonal antibody medicine market in China is still in the beginning, the sales amount of the market is only tens of millions of primordial coins, and the market is quite different from the billions of dollars in developed countries in Europe and America. Currently, the technology for preparing the humanized antibody has been developed for many years, and a large number of antibody medicines are successfully applied in clinic, so that the fully humanized antibody becomes the main development flow of the monoclonal antibody medicines, and some problems still need to be solved. For example, on the one hand, two kinds of human antibody production techniques have been widely used for the production of human antibodies in terms of production techniques, but the production costs of human antibodies are still high. On the other hand, human antibodies remain less immunogenic for clinical use. The above problems will become the main challenge for the preparation of human antibodies, but as the research goes deep, the series of problems will be solved effectively, and the human antibodies will be applied more widely in clinic.
Disclosure of Invention
This section is intended to outline some aspects of embodiments of the invention and to briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section as well as in the description summary and in the title of the application, to avoid obscuring the purpose of this section, the description summary and the title of the invention, which should not be used to limit the scope of the invention.
As one aspect of the present invention, the present invention provides a method for preparing a fully human monoclonal antibody.
In order to solve the technical problems, the invention provides the following technical scheme: a method of preparing a fully human monoclonal antibody, wherein: the fully human monoclonal antibody is derived from a fully human monoclonal hybridoma cell strain, and the hybridoma cell strain is obtained by combining a human myeloma cell strain and human peripheral blood B lymphocytes; wherein the human myeloma cell line combination is formed by fusing human myeloma cell lines RPMI 8226 and K6H 6/B5.
As a preferable scheme of the preparation method of the fully human monoclonal antibody, the invention is as follows: the cell number ratio of the human myeloma cell lines RPMI 8226 to K6H6/B5 is 2:1.
as a preferable scheme of the preparation method of the fully human monoclonal antibody, the invention is as follows: the cell number ratio of the human myeloma cell line combination to the human peripheral blood B lymphocytes is 3:1 fusion.
As a preferable scheme of the preparation method of the fully human monoclonal antibody, the invention is as follows: the fusion is carried out by taking 40% PEG4000 with pH of 8.0-8.2 as fusion agent, and treating for 45 seconds under the action of water bath at 30 ℃.
As a preferable scheme of the preparation method of the fully human monoclonal antibody, the invention is as follows: the fusion is that a cell suspension is prepared by mixing a human myeloma cell strain RPMI 8226 and K6H6/B5 with DMEM, the cell suspension is mixed with human peripheral blood B lymphocytes according to the cell number of 3:1, centrifugation is carried out for 10min at 1000r/min, and the supernatant is discarded, so that the human myeloma cells and the human B lymphocytes are fully and uniformly mixed; adding 1mL of PEG4000 with pH of 8.0-8.2% and pH of 40% into a fusion tube in a water bath at 30 ℃, slightly stirring while adding, standing for 60 seconds, then immediately adding 30mL of DMEM in 90 seconds, and stopping the reaction; centrifuging at 37deg.C in water bath for 10min at 1000r/min for 10min, discarding supernatant, suspending cell pellet with HAT selective culture solution containing 15% foetal calf serum, spreading into cell plate with feeder cells, and culturing in 5% CO2 incubator at 37deg.C.
As a preferable scheme of the preparation method of the fully human monoclonal antibody, the invention is as follows: and coating the fully human monoclonal antibody by using a saturated ammonium sulfate solution as a coating solution, and screening positive fully human monoclonal antibody hybridoma cell strains by using an EILSA method.
As a preferable scheme of the preparation method of the fully human monoclonal antibody, the invention is as follows: comprises the steps of carrying out amplification culture on positive fully human monoclonal antibody hybridoma cell strains, precipitating and concentrating fully human monoclonal antibodies, and then carrying out antibody purification.
As a preferable scheme of the preparation method of the fully human monoclonal antibody, the invention is as follows: the precipitation concentration of the fully human monoclonal antibody is that PEG6000 is utilized to precipitate proteins and then ultracentrifugation is carried out, so that the fully human monoclonal antibody with high purity and high antibody titer is obtained.
The invention has the beneficial effects that: the fully human monoclonal antibody hybridoma cell strain obtained by combining the human myeloma cell strain and fusing the human peripheral blood B lymphocytes can be widely applied to the screening of functional fully human monoclonal antibodies and the development of anti-tumor fully human monoclonal antibody medicaments.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the description of the embodiments will be briefly described below, it being obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art. Wherein:
FIG. 1 shows SDS-PAGE electrophoresis of fully human monoclonal antibodies.
FIG. 2 shows the tracing of fully human monoclonal antibody hybridoma cell lines by anti-human antibodies.
FIG. 3 shows that the fully human monoclonal antibody is identified by the anti-human antibody western blot method.
FIG. 4 shows the sensitivity of tumor cells to fully human monoclonal antibodies.
Detailed Description
In order that the above-recited objects, features and advantages of the present invention will become more apparent, a more particular description of the invention will be rendered by reference to specific embodiments thereof.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways other than those described herein, and persons skilled in the art will readily appreciate that the present invention is not limited to the specific embodiments disclosed below.
Further, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic can be included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.
The invention provides a preparation method of a fully human monoclonal antibody, which comprises the steps of screening a fully human monoclonal antibody hybridoma cell strain and purifying the fully human monoclonal antibody.
The hybridoma cell strain obtained by screening through the method is a fully human monoclonal antibody hybridoma cell strain. The antibodies purified by this method are fully human monoclonal antibodies.
In the preparation process of the fully human monoclonal antibody, the human myeloma cell strain combination for preparing the fully human monoclonal hybridoma is RPMI 8226 #CCL-155) and K6H6/B5 (++>CRL-1823) in a 2:1 ratio. In the preparation process of the fully human monoclonal antibody, the human spleen cells used for preparing the fully human monoclonal hybridoma are B lymphocytes in human peripheral blood separated from human whole blood.
The human myeloma cell line combination and B lymphocytes in human peripheral blood are fused by using 40% PEG4000 with the pH of 8.0-8.2 as a fusion agent and processing for 45 seconds under the water bath effect of 30 ℃ to obtain the fully human monoclonal antibody hybridoma cell line by screening.
The hybridoma cell strain obtained by the fusion method is coated by using a saturated ammonium sulfate solution as a coating liquid, and the fully human monoclonal antibody hybridoma cell strain is screened by using an EILSA method.
The fully human monoclonal antibody hybridoma cell strain is subjected to sucrose density method ultracentrifugation after PEG6000 precipitated protein is utilized, and then the fully human monoclonal antibody with high purity and high antibody titer is obtained.
The fully human monoclonal antibody hybridoma cell strain obtained by combining the human myeloma cell strain and fusing the human peripheral blood B lymphocytes can be widely applied to the screening of functional fully human monoclonal antibodies and the development of anti-tumor fully human monoclonal antibody medicaments.
Example 1:
step one selection and culture of human myeloma cells
Selecting two human myeloma cell lines RPMI 8226 on ATCC official netCCL-155) and K6H6/B5 (++>CRL-1823) used as the female parent of the fully human monoclonal antibody hybridoma cells in the present invention. Cell culture method: DMEM culture containing 10% fetal calf serum, 37℃and 5% CO 2 Concentration.
Step two separation of human peripheral blood B lymphocytes
The positive isolation method was selected by MACS (magnetic bead sorting) technique to isolate B lymphocytes in peripheral blood of healthy humans. Dynabeads (dynal) coated with CD11b human antibody selectively adsorbed target cells directly from human peripheral blood samples. Dynabeads can be bound to cells within 15-30 minutes at 2-8 ℃ to form Dynabeads' target cell complexes, i.e., B lymphocytes. Then placing the cells in a Dynal MPC magnetic separator, adsorbing the cells combined with the magnetic beads on a test tube wall close to the magnetic separator, and removing the supernatant by a pipette to obtain the human peripheral blood B lymphocyte cells. The method has the advantages that: the method is simple, convenient and quick, and the Dynabeads combined with the cells can be conveniently removed by combining with Dynabeads to obtain target cells, so that the influence on the functions of B lymphocytes is small; b lymphocytes with high purity (99%) and high yield (95%) can be obtained, and 1X 10 can be obtained on average from 1mL of human peripheral blood 5 B lymphocytes.
Step three cell fusion
1 feeder cells can be prepared the day before fusion, 1 ICR mouse with age of 6-8 weeks is taken, cervical vertebra dislocation is killed, the mice are put into 75% alcohol for sterilization for 5-10min, the mice are fixed on a tray, and abdominal skin is aseptically sheared in an ultra clean bench. Sucking 20ml HAT selective culture solution with sterile syringe, injecting into the abdominal cavity of mice, and making into light weight with alcohol cotton ballKneading abdomen, withdrawing the culture medium, supplementing appropriate amount of HAT culture solution, spreading into 10 96-well cell culture plates at 37deg.C and 5% CO at a rate of 50 μl/well 2 Culturing in a cell culture incubator.
2 isolation of fresh human peripheral blood B lymphocytes prior to fusion. Heparin sodium is selected as a blood collection tube of anticoagulant, 200mL of normal human blood is collected, and the separation of B lymphocytes is carried out by MACS (magnetic bead sorting) technology, so that about 1 x 10 can be obtained 7 B lymphocytes are prepared for later use.
3 before fusion, human myeloma cell strain RPMI 8226 is carried outCCL-155) and K6H6/B5CRL-1823) are cultured in an expanded manner. The total number of human myeloma cells required was 3×10 7 And each. According to experimental study, RPMI 8226 (++>CCL-155) and K6H6/B5 (++>CRL-1823) is preferably 2:1. Namely human myeloma cell RPMI 8226 (-)>CCL-155) requires 2 x 10 7 Human myeloma cells K6H6/B5 (-)>CRL-1823) requires 1 x 10 7 And each. Fusion rate = number of wells/total number of wells of successfully fused hybridomas.
4 cell fusion method:
during fusion, human myeloma cell strain RPMI 8226 is usedCCL-155) and K6H6/B5 (++>CRL-1823) mixing with DMEM to obtain cell suspension, mixing with human peripheral blood B lymphocyte at cell number of 3:1, adding into 50ml fusion tube, centrifuging at 1000r/min for 10min, discarding supernatant, and slightly rubbing at palm to thoroughly mix human myeloma cell and human B lymphocyte; then, 1ml of preheated PEG4000 at pH 8.0-8.2% in 45S was added to the fusion tube from slow to fast with gentle agitation at 30℃in a water bath. The reaction was stopped by standing for 60S and then immediately adding 30mL of DMEM to 90S. After centrifugation at 1000r/min for 10min in a water bath at 37℃for 10min, the supernatant was discarded, and the pellet was gently suspended in a suitable amount of HAT selection medium containing 15% fetal bovine serum, plated into 96-well plates with feeder cells at 100. Mu.l/well, and incubated in a 5% CO2 incubator at 37 ℃. After 7d, 50. Mu.l/well of HAT selection medium containing 15% fetal bovine serum was supplemented, and after 14 days, 50. Mu.l/well of HAT selection medium containing 15% fetal bovine serum was further supplemented. After 14 days of fusion, the fusion rate can be calculated, and the screening of the fully human monoclonal antibody hybridoma cell strain can be carried out.
Step four screening of fully human monoclonal antibody hybridoma positive cell strains
1 configuration of saturated ammonium sulfate
Dissolving 80g of ammonia sulfate into 100 water, heating to about 80g, filtering the dissolved part, re-dissolving the rest, crystallizing a part of the solution, crystallizing the filtered solution when the solution is cooled to room temperature, balancing for one or two days, taking the supernatant, and adjusting the pH to 7.0-7.2 by using ammonia water when the solution is used.
Coating screening method by 2 salting-out method
Since the antibody is also a protein, the solubility of the antibody in an aqueous solution is determined by the number of hydrophilic groups and the number of charges carried by the antibody, sulfate ions and ammonium ions compete with water molecules in the solution when the saturated ammonium sulfate solution is added, and the sulfate ions and the ammonium ions have stronger hydrophilicity than the antibody molecules, so that a hydration film on the surface of the antibody molecules is destroyed, and the exposed charged groups are neutralized by salt ions in the solution to greatly reduce the solubility of the antibody, and according to the principle, the antibody in the culture supernatant of the fully human monoclonal antibody hybridoma is precipitated and coated in an ELISA plate hole.
150 μl of culture supernatant was aspirated from wells in 96 well plates in which hybridoma cells were successfully formed by fusion, mixed with 120 μl of saturated ammonium sulfate solution 1:1 into wells of enzyme-labeled plate, incubated at 2-8℃for 10 hours, centrifuged in centrifuge, 8000r/min for 15-20min, the supernatant was discarded, 250 μl/well PBS was added, equilibrated at 37deg.C for 2h, and the supernatant was discarded. Blocking with 5% BSA at 37℃for 2h; PBST containing 0.05% Tween-20 was washed 3 times with each standing for 5 minutes; incubation with HRP-labeled goat anti-human antibody (SIGMA) as detection antibody at 1:5000 dilution for 1.5h; PBST containing 0.05% Tween-20 was washed 3 times with each standing for 5 minutes; color development was performed with TMB for 10min. And taking the blank hole as a negative control hole, selecting a measurement hole OD value/blank hole OD value which is more than 3, and judging that the whole human monoclonal antibody hybridoma cells are positive. Cells which are positive in the 96-well plate and are detected twice continuously are subjected to expansion culture, and subcloning is performed by adopting a limiting dilution method. After 2-3 times of cloning, when all cell holes of the 96-well plate are positive, the amplified culture and fixed strain freezing storage can be carried out.
Step five purification of fully human monoclonal antibodies
1 expanded culture of fully human monoclonal antibody hybridomas
Culturing the fixed fully human monoclonal antibody hybridoma cell strain in a bottle-rotating culture mode, and culturing the strain by using a bottle-rotating machine with a bottle-rotating specification of 850 square centimeters of growth area, wherein the culture parameters are as follows: 2*10 7 The hybridoma cells were cultured in DMEM containing 5% fetal bovine serum at 37℃in a 5% CO2 incubator at 10 rpm for 5 days. The culture supernatant of the fully human monoclonal antibody hybridoma was collected to obtain about 1.5L of a solution.
2 precipitation concentration of fully human monoclonal antibodies
The fully human monoclonal antibody was precipitated by PEG6000 precipitation. Technical parameters: 8% -12% of PEG6000 can effectively precipitate IgG. 120g-180g PEG6000 tablets were added to 1.5L of the cell culture supernatant, the mixture was stirred continuously at 4℃for 10-12h on a magnetic stirrer, 10000 Xg was centrifuged for 30min, the supernatant was removed, the precipitated antibody was left, and the precipitate was resuspended with STE buffer in an amount of 1 to 2 times the precipitation volume. STE buffer: 0.1mol/LNaCL,10mmol/LTris.CL,1mmol/L EDTA; pH 8.0; and (5) autoclaving.
3 sucrose density gradient centrifugation method for purifying fully human monoclonal antibody
5-8mL of the obtained PEG6000 precipitation concentrated antibody STE solution is added into an overspeed centrifuge tube, 30%,45% and 60% of sucrose are sequentially added into the centrifuge tube, and a long needle is used for adding sucrose from the bottom to the top. The strips were centrifuged at 110000 Xg for 2.5h with a bright band between 45% and 60% and were all aspirated with a long needle and collected in a bottle. Sucrose removal: purified antibodies were diluted with appropriate amounts of STE buffer, then centrifuged at 110000 Xg for 3h, and the pellet was suspended with a small amount of STE buffer (depending on the amount of pellet added), thus obtaining the purified virus.
Characterization of fully human monoclonal antibodies
1 determination of purity
The purity of the fully human monoclonal antibodies was determined by SDS-PAGE technique. According to the molecular cloning experimental guidelines, 10% of the separation gel, 5% of the laminating gel formula and 25. Mu.g of antibody/well loading were selected, and after electrophoresis of the protein, coomassie brilliant blue staining was performed. 2 bands can be seen clearly at 25kDa, 50kDa on the separator gel. Wherein 25kDa is the molecular weight of the light chain of the fully human monoclonal antibody; molecular weight size of 50kDa fully human monoclonal antibody heavy chain. The purity of the antibody reaches 99% by measurement. As shown in FIG. 1, the left lane is the standard protein molecular weight standard, and the right lane is the 2-band. The upper band position is about 50kDa, namely the heavy chain of the fully human monoclonal antibody; the lower band is approximately 25kDa, the light chain of the fully human monoclonal antibody. The molecular weight at the band position is in accordance with the molecular weight standard of the antibody.
2IFA identification
Fully human monoclonal antibody hybridoma cells were assayed using indirect Immunofluorescence (IFA). In the whole human monoclonal antibody hybridoma cell culture process, 1mL of suspension culture solution is sucked into a 1.5mL centrifuge tube, after centrifugation for 3min at 1000 turns, 200 μl of Cy 3-labeled goat anti-human secondary antibody (SIGMA) which is diluted 1:200 times is added, hybridoma cells are resuspended, incubated at 37 ℃ for 45min, after centrifugation for 3min at 1000 turns, the hybridoma cells are washed with PBS and repeated for 2 times. The hybridomas were finally resuspended in 100 μl PBS, 20 μl of the cell suspension was taken on a slide glass, covered with a cover slip, and observed under a fluorescence microscope (400×). The hybridoma cells are stained red, and the hybridoma cells react with the anti-human antibody, namely, the fully human monoclonal antibody hybridoma cell strain is tracked by the anti-human antibody, which indicates that the hybridoma cells are fully human. See fig. 2.
3Westernblot identification
The fully human monoclonal antibodies were identified using immunoblotting (western blot). Sample size of 25. Mu.g antibody/well, SDS-PAGE electrophoresis, 80V transfer to PVDF membrane, PVDF membrane is blocked with 5% BSA solution at 37 ℃ for 2h, PVDF membrane is washed three times with PBST containing 0.05% Tween 20, and each shaking washing is carried out for 5min; 10mL of HRP-labeled goat anti-human secondary antibody (SIGMA) was added at 1:2000 dilution, incubated at room temperature for 1.5h, and PVDF membrane was washed three times with PBST containing 0.05% Tween 20, with shaking for 5min each time; the PVDF film is transferred into freshly prepared developing solution and placed in a dark place for 1-3min, and developed in a developing instrument. 3 bands can be clearly seen on PVDF membrane at 150kDa, 25kDa, 50 kDa. 150kDa is the molecular weight of the fully human monoclonal antibody; 25kDa is the molecular weight of the light chain of the fully human monoclonal antibody; molecular weight size of 50kDa fully human monoclonal antibody heavy chain. See fig. 3.
4 identification of biological Activity
The biological activity of fully human monoclonal antibodies was verified with tumor cell AGS. Suction 1 x 10 5 And selecting the action concentration of the total humanized monoclonal antibody with the final concentration of 10 mug/mL from the AGS cells to a 6-hole plate, co-culturing the total humanized monoclonal antibody and the AGS cells, and observing the growth condition of the AGS cells under a microscope after 10 hours. It can be seen that AGS cells without fully human monoclonal antibody grew normally (see FIG. 4A), while withAGS cells acted by the fully human monoclonal antibody are greatly inhibited, and the growth inhibition rate of the fully human monoclonal antibody AGS cells reaches more than 85 percent (see figure 4B).
It should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted without departing from the spirit and scope of the technical solution of the present invention, which is intended to be covered in the scope of the claims of the present invention.
Claims (4)
1. A preparation method of a fully human monoclonal antibody is characterized by comprising the following steps: the fully human monoclonal antibody is derived from a fully human monoclonal hybridoma cell strain, and the hybridoma cell strain is obtained by combining a human myeloma cell strain and human peripheral blood B lymphocytes;
the human myeloma cell line combination is formed by fusing human myeloma cell lines RPMI 8226 and K6H6/B5 according to the cell number ratio of 2:1 by using 40% PEG4000 with pH value of 8.0-8.2 as a fusion agent under the water bath effect of 30 ℃ for 45 seconds;
the preparation method of the hybridoma cell strain comprises the steps of mixing human myeloma cell strain RPMI 8226 and K6H6/B5 with DMEM to prepare cell suspension, mixing the cell suspension with human peripheral blood B lymphocytes according to the cell number of 3:1, centrifuging at 1000r/min for 10min, and discarding the supernatant to fully and uniformly mix the human myeloma cells and the human B lymphocytes; adding 1ml of 40% PEG4000 with the pH of 8.0-8.2 into a fusion tube in a water bath at 30 ℃, slightly stirring while adding, standing for 60 seconds, then immediately adding 30ml of DMEM in 90 seconds, and stopping the reaction; centrifuging in a water bath at 37deg.C for 10min at 1000r/min for 10min, removing supernatant, suspending cell pellet with HAT selective culture solution of 15% fetal bovine serum, spreading into cell plate containing feeder cells at 37deg.C and 5% CO 2 Culturing in an incubator to obtain hybridoma cell strains.
2. The method for preparing fully human monoclonal antibody according to claim 1, wherein: and coating the fully human monoclonal antibody by using a saturated ammonium sulfate solution as a coating solution, and screening the fully human monoclonal antibody hybridoma cell strain by using an EILSA method.
3. The method for preparing fully human monoclonal antibody according to claim 1, wherein: comprises the steps of carrying out amplification culture on positive fully human monoclonal antibody hybridoma cell strains, precipitating and concentrating fully human monoclonal antibodies, and then carrying out antibody purification.
4. A method of producing fully human monoclonal antibodies according to claim 3, wherein: the precipitation concentration of the fully human monoclonal antibody is that PEG6000 is utilized to precipitate proteins and then ultracentrifugation is carried out, so that the fully human monoclonal antibody with high purity and high antibody titer is obtained.
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| JPH03183477A (en) * | 1989-12-12 | 1991-08-09 | Morinaga & Co Ltd | Hybridoma |
| JPH04281798A (en) * | 1991-03-08 | 1992-10-07 | Morinaga & Co Ltd | Human type monoclonal antilung cancerous cell antibody and hybridoma capable of producing the same |
| CN102321720A (en) * | 2011-07-28 | 2012-01-18 | 万晓春 | Method for producing pure human-derived monoclonal antibody by mixed cell culture |
| CN106928348A (en) * | 2017-03-06 | 2017-07-07 | 哈尔滨医科大学 | A kind of full human monoclonal antibody of regulation and control human body inherent immunity and its preparation method and application |
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| GB2404665B (en) * | 2003-08-08 | 2005-07-06 | Cambridge Antibody Tech | Cell culture |
| WO2008013552A2 (en) * | 2005-08-26 | 2008-01-31 | Columbia University | Fully human hybridoma fusion partner cell lines |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| JPH03183477A (en) * | 1989-12-12 | 1991-08-09 | Morinaga & Co Ltd | Hybridoma |
| JPH04281798A (en) * | 1991-03-08 | 1992-10-07 | Morinaga & Co Ltd | Human type monoclonal antilung cancerous cell antibody and hybridoma capable of producing the same |
| CN102321720A (en) * | 2011-07-28 | 2012-01-18 | 万晓春 | Method for producing pure human-derived monoclonal antibody by mixed cell culture |
| CN106928348A (en) * | 2017-03-06 | 2017-07-07 | 哈尔滨医科大学 | A kind of full human monoclonal antibody of regulation and control human body inherent immunity and its preparation method and application |
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