CN110951691B - Anti-human TNF-alpha stable cell strain and construction method and application thereof - Google Patents

Anti-human TNF-alpha stable cell strain and construction method and application thereof Download PDF

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CN110951691B
CN110951691B CN201911076644.8A CN201911076644A CN110951691B CN 110951691 B CN110951691 B CN 110951691B CN 201911076644 A CN201911076644 A CN 201911076644A CN 110951691 B CN110951691 B CN 110951691B
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张洋
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Zhejiang Zhengxi Biotechnology Co ltd
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Abstract

The invention discloses an anti-human TNF-alpha stable transgenic cell strain and a construction method and application thereof, the stable transgenic cell strain is named as anti-human TNF-alpha stable transgenic cell strain CHO K1, and has been preserved in China Center for Type Culture Collection (CCTCC) in 2019, 9, 25 and the preservation addresses are as follows: university of china, wuhan; the preservation number is CCTCC No: C2019183. the yield of the anti-human TNF-alpha antibody of the anti-human TNF-alpha stable cell strain CHO K1 can reach 3g/L, which is 6 times of that of the existing anti-human TNF-alpha antibody expression cell strain; the antibody yield is stable and the antibody quality is high among different production batches.

Description

Anti-human TNF-alpha stable cell strain and construction method and application thereof
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to an anti-human TNF-alpha stable cell strain, and a construction method and application thereof.
Background
TNF (Tumor necrosis factor) is a cytokine naturally occurring in inflammatory and immune responses, and studies have found that TNF levels are elevated in synovial fluid of patients with rheumatoid arthritis and play an important role in pathological inflammation and joint destruction.
The adalimumab can be specifically combined with TNF-alpha, but not combined with cytokines such as TNF-beta and the like, blocks the interaction of the TNF-alpha and cell surface TNF receptors such as p55 and p75, reduces inflammatory response caused by excessive production and release of the TNF-alpha, and has important clinical significance for treating diseases caused by excessive production of the TNF-alpha. Adalimumab is a self-injectable biotherapeutic drug which has been approved for 2 indications in the national food and drug administration (CFDA), namely rheumatoid arthritis and ankylosing spondylitis.
The traditional preparation method of the cell strain secreting the monoclonal antibody is a fusion method of splenocytes and myeloma cells of an immune mouse, and comprises the processes of antigen immunization, cell fusion, cell strain screening, cell bank construction and the like. Since mouse antibodies are xenogeneic antigens relative to the human body, mouse monoclonal antibodies cause severe rejection reactions in the body when injected into the body. Meanwhile, the fusion cell strain prepared by the traditional method is easy to generate phenomena of chromosome loss, gene deletion and the like, the stability among different batches in the production process of the monoclonal antibody is seriously influenced, and the product quality is influenced. In addition, the cell lines have problems such as low antibody production and difficulty in cell culture.
Disclosure of Invention
The invention aims to provide an anti-human TNF-alpha stable cell strain with high anti-human TNF-alpha antibody yield, and a construction method and application thereof.
In order to achieve the above purpose, the technical solution of the present application is as follows:
an anti-human TNF-alpha stable cell strain, which is named as anti-human TNF-alpha stable cell strain CHO K1 and has been preserved in China Center for Type Culture Collection (CCTCC) in 2019, 9, 25 and the preservation addresses are as follows: university of china, wuhan; the preservation number is CCTCC No: C2019183.
the construction method of the anti-human TNF-alpha stable cell strain comprises the following steps:
(1) preparing a plasmid containing an anti-human TNF-alpha antibody heavy chain gene and a plasmid containing an anti-human TNF-alpha antibody light chain gene, and co-transfecting the two plasmids into a parental CHO K1 cell;
(2) carrying out MSX (methionine imino sulfone) screening on the transfected CHO K1 cells to obtain stable transfected cells;
specifically, the MSX screening comprises: culturing the transfected cells in a complete culture solution, replacing the complete culture solution with a screening culture solution containing 50-200 mu M methionine imino sulfonyl sulfone after 48 hours, and replacing the screening culture solution every 3-4 days until the cells stably grow;
the screening culture medium is obtained by adding MSX to the complete culture medium until the final concentration of MSX reaches 50-200. mu.M.
Preferably, the final concentration of methionine iminosulfone in the screening culture broth is 100. mu.M.
Preferably, the MSX screening further comprises: cells which stably grow in the screening culture solution are inoculated into the complete culture solution for continuous culture, and the transfected stably transformed cells are obtained.
Preferably, the cells stably growing in the selection medium are inoculated into the complete medium and cultured for 2 to 3 days, and after the inoculation, the cell density in the complete medium is 5X 105cells/mL。
(3) And (3) carrying out monoclonal cell strain sorting on the stably transfected cells to obtain the anti-human TNF-alpha stably transfected cell strain CHO K1.
Specifically, the sorting of the monoclonal cell strain comprises the following steps: inoculating the monoclonal cell strains in the stably transformed cells into a complete culture solution one by one, and detecting the anti-human TNF-alpha antibody yield of each monoclonal cell strain after continuously culturing for 19-21 days to obtain the anti-human TNF-alpha stably transformed cell strain CHO K1.
The invention also provides an application of the anti-human TNF-alpha stable cell strain in preparing an anti-human TNF-alpha antibody, which comprises the following steps:
(a) inoculating the anti-human TNF-alpha stable cell strain CHO K1 into a shake flask containing complete culture solution, placing at 37 ℃ and 5% CO2Subculturing at 120rpm in the environment of (1);
(b) after the subculture is completed, the cells are transferred into a large-volume culture flask with the initial density of more than 3X 10 after cell inoculation5cells/mL; in order to have higher dissolved oxygen in the culture solution, the volume of the culture solution in the culture flask does not exceed 1/5 of the total volume of the culture flask;
(c) when the cell density reaches 1X 106After cell/mL, cutting off the supply of carbon dioxide, and adjusting the rotating speed of the shaking table to 130-135 rpm;
(d) continuously culturing for 6-8 days, collecting culture solution, centrifuging, filtering with 0.45 μm filter membrane to obtain cell culture supernatant, and sequentially purifying and eluting the cell culture supernatant to obtain anti-human TNF-alpha antibody.
Preferably, in step (a), at least 2 passages are carried out, and the cell density in the culture medium during the passage culture is not more than 2X 106cells/mL。
The yield of the anti-human TNF-alpha antibody of the anti-human TNF-alpha stable cell strain CHO K1 can reach 3g/L, which is 6 times of that of the existing anti-human TNF-alpha antibody expression cell strain.
Compared with the prior art, the invention has the beneficial effects that:
(1) the yield of the anti-human TNF-alpha antibody of the anti-human TNF-alpha stable cell strain CHO K1 can reach 3g/L, which is 6 times of that of the existing anti-human TNF-alpha antibody expression cell strain; the antibody yield is stable and the antibody quality is high among different production batches.
(2) The construction method of the anti-human TNF-alpha stable cell strain is simple and convenient in steps and easy to implement.
Drawings
FIG. 1 is the result of PAGE gel electrophoresis of cell culture supernatant of anti-human TNF-alpha stable cell strain CHO K1 and standard antibody protein IgG;
wherein, the cell super indicates cell culture supernatant, the standard IgG indicates standard antibody protein IgG, and the concentration of the standard antibody protein IgG is 80/120160 mu g/mL in sequence;
FIG. 2 is an elution profile of the GE AKTA Pure protein isolation and purification system eluting purified cell culture supernatant;
wherein the abscissa represents the volume of the eluent and the ordinate mAU represents the response value;
FIG. 3 is a standard curve for detecting protein concentration of an antibody solution obtained by expressing an anti-human TNF-alpha stable cell strain CHO K1.
Detailed Description
The technical solution of the present invention will be further described in detail with reference to the accompanying drawings and the detailed description.
Example 1
The embodiment of the invention provides a method for constructing an anti-human TNF-alpha stable cell strain, which comprises the following steps:
(1) preparing a plasmid containing an anti-human TNF-alpha antibody heavy chain gene and a plasmid containing an anti-human TNF-alpha antibody light chain gene, and co-transfecting the two plasmids into a parental CHO K1 cell;
wherein, the nucleotide sequence of the heavy chain gene of the anti-human TNF-alpha antibody is shown as SEQ ID No.1, the nucleotide sequence of the light chain gene of the anti-human TNF-alpha antibody is shown as SEQ ID No.2, and the preparation method of the plasmid is carried out according to the instructions of a QIAGEN kit; the transfection procedure of the plasmids was performed according to the instructions of the Invitrogen kit.
(2) Carrying out MSX screening on the transfected CHO K1 cells to obtain stable transfected cells;
the method specifically comprises the following steps:
(2.1) culturing the transfected cells in a complete culture solution, and after 48 hours, replacing the complete culture solution with a screening culture solution containing 100 mu M methionine imino sulphone, and replacing the screening culture solution every 3-4 days until the cells stably grow;
(2.2) cells stably growing in the selection Medium were inoculated into the complete Medium, and after inoculation, the cell density in the complete Medium was 5X 105cells/mL; after continuously culturing for 3 days, detecting the yield of the anti-human TNF-alpha antibody of the cell to determine whether the plasmid is successfully transfected and whether the MSX screening is successful, and finally obtaining a stable cell (polyclonal);
(3) and (4) carrying out monoclonal cell strain sorting on the stably transfected cells to obtain an anti-human TNF-alpha stably transfected cell strain CHO K1.
The method specifically comprises the following steps:
(3.1) diluting the stably transformed cells for multiple times, and then inoculating the stably transformed cells into a 96-well plate, wherein each well contains 200 mu L of complete culture solution, and each well is ensured to contain only one stably transformed monoclonal cell strain;
(3.2) culturing the cells in the 96-well plate for about 7 days, observing the growth state of the cells by using a microscope at all times, and marking culture wells of the monoclonal cell strains;
(3.3) after continuously culturing for 12-14 days, detecting the anti-human TNF-alpha antibody yield of each monoclonal cell strain, selecting high-yield monoclonal cell strains to transfer into a 24-well plate, continuously culturing for 12-14 days, and detecting the anti-human TNF-alpha antibody yield of the monoclonal cell strains in each hole; continuously selecting a monoclonal cell strain with high yield from the 24-well plate, transferring the monoclonal cell strain into a 6-well plate, continuously culturing for 12-14 days, detecting the yield of the anti-human TNF-alpha antibody of the monoclonal cell strain in each well, and selecting the monoclonal cell strain with the highest yield of the anti-human TNF-alpha antibody from the monoclonal cell strain, namely the anti-human TNF-alpha stably-transferred cell strain CHO K1 in the embodiment;
(3.4) the above-mentioned anti-human TNF-. alpha.stably transfected cell line CHO K1 was frozen and stored.
As can be seen from the results of PAGE gel electrophoresis (shown in FIG. 1) of the cell culture supernatant of the anti-human TNF- α stable cell strain CHO K1 and IgG standard antibody protein, the content of the anti-human TNF- α antibody in the cell culture supernatant of the anti-human TNF- α stable cell strain CHO K1 was higher than the IgG content of 160. mu.g/mL.
And (2) carrying out antibody binding detection on the cell culture supernatant by adopting a QC ELISA method, coating human TNF-alpha in an ELISA reaction hole, respectively adding cell culture supernatant with 1000-fold and 2000-fold dilution, then adding an enzyme-labeled secondary antibody (horseradish peroxidase-labeled IgG), and then detecting the OD value. The results are shown in Table 1.
TABLE 1
Figure BDA0002262674800000061
As can be seen from Table 1, the presence of anti-human TNF-. alpha.antibodies was detected in the cell culture supernatants at both dilutions; and the OD450 was lower at 2000-fold dilution compared to 1000-fold dilution, indicating that the antibody in the cell culture supernatant was indeed an anti-human TNF-alpha antibody.
Example 2
In this example, the anti-human TNF- α stable cell line CHO K1 obtained in example 1 was used to produce anti-human TNF- α antibody, which comprises the following steps:
(1) (a) transfer of anti-human TNF-. alpha.stably transfected cell line CHO K1 (hereinafter referred to as cell line CHO K1) into 125mL of a shake flask containing 30mL of complete CD medium, and Place the shake flask inAt 37 ℃ with 5% CO2Shaking and culturing in a shaking table at 120 rpm;
the frozen anti-human TNF-alpha stable cell strain CHO K1 needs to be revived in advance, and the reviving method comprises the following steps: taking the cell strain CHO K1 out of the liquid nitrogen tank, quickly thawing in a 37 ℃ water bath, centrifuging cell liquid, removing supernatant, and washing with PBS;
during initial culture, the cell strain CHO K1 is subcultured every 2-3 days, at least more than 2 passages are needed, and during the subculture process, the cell density is prevented from being too high (not more than 2X 10)6cell/mL)；
(b) After the subculture is completed, the cells are transferred into a large-volume culture flask with the initial density of more than 3X 10 after cell inoculation5cells/mL; in order to have higher dissolved oxygen in the culture solution, the volume of the culture solution in the culture flask does not exceed 1/5 of the total volume of the culture flask;
(c) after culturing for 48h, the cell density in the culture medium was examined by microscope to determine whether the cell density reached 1X 106cell/mL (should be at or above) until the cell density reaches 1X 106After cell/mL, cutting off the supply of carbon dioxide, and adjusting the rotating speed of the shaking table to 130-135 rpm;
(d) continuously culturing for 6-8 days (the cell density in the culture solution can reach 7-8 × 10 at most6cell/mL), collecting the culture solution, centrifuging and filtering through a 0.45-micron filter membrane to obtain 10mL of cell culture supernatant, and sequentially purifying and eluting the cell culture supernatant by adopting a GE AKTA Pure protein separation and purification system, wherein the elution curve is shown in figure 2.
As shown in FIG. 2, the highest response value of anti-human TNF-. alpha.antibody in the eluate was close to 2600, indicating a higher concentration of anti-human TNF-. alpha.antibody in the eluate.
After elution, 6mL of antibody solution was obtained, and the antibody solution was diluted by four concentration gradients for protein concentration detection, with the detection results shown in table 2 and fig. 3.
TABLE 2
Figure BDA0002262674800000081
As can be seen from table 2, the final concentrations of antibody measured were: 4.86 mg/mL; the final antibody yield in 10mL of cell culture supernatant was 29.16mg, and the predicted antibody yield of cell line CHO K1 was 3 g/L.
Sequencing the obtained anti-human TNF-alpha antibody by committing Shanghai bioscience research institute, wherein the sequencing result shows that the amino acid sequence of the heavy chain of the anti-human TNF-alpha antibody is shown as SEQ ID No.3, and the amino acid sequence of the light chain of the anti-human TNF-alpha antibody is shown as SEQ ID No. 4. The results of peptide coverage analysis by Institute of Biochemistry and Cell Biology, Shanghai Institute of Biological Sciences and Chinese Academy of Sciences and China Academy of Sciences show that the sequence of anti-human TNF-alpha antibody of the present invention has 100% identity with adalimumab.
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Claims (4)

1. An anti-human TNF-alpha stable cell strain is named as anti-human TNF-alpha stable cell strain CHO K1, which is preserved in China Center for Type Culture Collection (CCTCC) at Wuhan university in 2019, 9, 25 and with the preservation number of CCTCC No: C2019183.
2. the use of the stable transgenic cell line of anti-human TNF- α of claim 1 for the preparation of anti-human TNF- α antibodies.
3. The use according to claim 2, comprising the steps of:
(a) inoculating the anti-human TNF-alpha stable cell strain CHO K1 into a shake flask containing complete culture solution, placing at 37 ℃ and 5% CO2Subculturing at 120rpm in the environment of (1);
(b) after the subculture is completed, the cells are transferred into a large-volume culture flask with the initial density of more than 3X 10 after cell inoculation5cells/mL; in order to have higher dissolved oxygen in the culture solution, the volume of the culture solution in the culture flask does not exceed 1/5 of the total volume of the culture flask;
(c) when the cell density reaches 1X 106After cells/mL, cutting off the supply of carbon dioxide, and adjusting the rotating speed of the shaking table to 130-135 rpm;
(d) continuously culturing for 6-8 days, collecting culture solution, centrifuging, filtering with 0.45 μm filter membrane to obtain cell culture supernatant, and sequentially purifying and eluting the cell culture supernatant to obtain anti-human TNF-alpha antibody.
4. The use according to claim 3, wherein in step (a), the cell density in the culture medium during the passage is not more than 2X 10 at least 2 passages6cells/mL。
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102127167A (en) * 2010-01-15 2011-07-20 苏州工业园区晨健抗体组药物开发有限公司 Whole human TNF alpha (tumor necrosis factor alpha) monoclonal antibody and preparation and application thereof
CN102167741A (en) * 2010-02-25 2011-08-31 百迈博药业有限公司 Fully human anti-TNF-alpha (Tumor Necrosis Factor-alpha) monoclonal antibody and preparation method as well as application thereof
CN107236710A (en) * 2017-03-27 2017-10-10 天津科技大学 The stable anti-TNF alpha monoclonal antibodies cell expression system of expression
CN108164601A (en) * 2017-11-17 2018-06-15 安徽未名生物医药有限公司 A kind of preparation method for recombinating the full human monoclonal antibody of anti-tnf-alpha

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102127167A (en) * 2010-01-15 2011-07-20 苏州工业园区晨健抗体组药物开发有限公司 Whole human TNF alpha (tumor necrosis factor alpha) monoclonal antibody and preparation and application thereof
CN102167741A (en) * 2010-02-25 2011-08-31 百迈博药业有限公司 Fully human anti-TNF-alpha (Tumor Necrosis Factor-alpha) monoclonal antibody and preparation method as well as application thereof
CN107236710A (en) * 2017-03-27 2017-10-10 天津科技大学 The stable anti-TNF alpha monoclonal antibodies cell expression system of expression
CN108164601A (en) * 2017-11-17 2018-06-15 安徽未名生物医药有限公司 A kind of preparation method for recombinating the full human monoclonal antibody of anti-tnf-alpha

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
Antagonistic effect of peptidoglycan of Streptococcus sanguinis on lipopolysaccharide of major periodontal pathogens;Sung-Hoon Lee;《Journal of Microbiology》;20150731;第53卷(第8期);第553-560页 *
抗人肿瘤坏死因子-α人源单克隆抗体在CHO细胞中的表达及鉴定;崔文禹;《中国生物制品学杂志》;20141231;第27卷(第12期);第1547-1550页 *
治疗性抗体高表达CHO细胞株构建策略;李浩强;《中国医药生物技》;20130630;第8卷(第3期);第221页右栏第4段 *

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