A method for quickly constructing igg antibody high expression stable CHO cell strain

Abstract

This invention relates to the fields of biotechnology and cell biology, and provides a method for rapidly constructing a stable CHO cell line with high IgG antibody expression. The method includes the following steps: S1. Antibody plasmid construction; S2. Cell resuscitation, passage, and plasmid transfection; S3. Positive cell screening; S4. Preparation and screening of stable single-cell clones with high expression; S5. Feed-and-carry batch culture; S6. Verification of the stability of the antibody-expressing cell line. The method provided by this invention, after the plasmid vector enters the cells via electroporation, performs shaking culture after 48 hours of static culture, which accelerates cell recovery time, resulting in faster recovery and better cell condition compared to traditional static culture. During feed-and-carry batch culture, high-density cell inoculation shortens the logarithmic growth phase, allowing cells to enter the protein production phase earlier, thereby achieving higher expression levels, significantly shortening the overall production cycle, and saving substantial costs.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and cell biology, and particularly relates to a method for rapidly constructing a stable CHO cell line with high expression of IgG antibodies. Background Technology

[0002] Mammalian cells are the primary host cells for the production of commercially available therapeutic protein products, including monoclonal antibodies. Chinese hamster ovary (CHO) cells are the preferred host cells for producing protein drugs, and the CHO expression system offers significant advantages over other expression systems.

[0003] (1) It is suitable for suspension culture and can meet the requirements of large-scale industrial production of recombinant proteins;

[0004] (2) It has accurate post-transcriptional modification function, and the expressed proteins, including antibodies, are closer to natural proteins;

[0005] (3) It can efficiently amplify and express exogenous genes and can be fully integrated into cells;

[0006] (4) CHO cells are fibroblasts that secrete almost no endogenous proteins and have extracellular secretory functions, which facilitates the purification of downstream products.

[0007] The construction of stable cell lines refers to the process by which exogenous genes enter recipient cells and integrate into the chromosomes of the cells, enabling the target protein to be expressed stably in the host cells for a long period of time. Stable cell lines play a very important role in biological research and are widely used in applications such as recombinant protein and monoclonal antibody production, drug screening, and gene function research.

[0008] Traditional cell recovery methods rely on DMSO, leading to cytotoxicity (viability ≤90%). Traditional stable cell line construction involves electroporation followed by static culture, which prolongs recovery time. MSX screening suffers from a low positive cell rate (≤65%) due to delayed pressurization and constant high pressure. Low-density fed-batch culture delays the production cycle. This method significantly shortens cell recovery time by using shaking culture instead of static culture, followed by further treatment, resulting in excellent cell recovery. Furthermore, traditional methods use low initial cell density during fed-batch culture, while the method provided in this invention uses high-density cell inoculation during fed-batch culture. This high-density inoculation shortens the logarithmic growth phase, allowing cells to enter the protein production phase earlier and achieve higher expression levels. Summary of the Invention

[0009] This invention provides a method for rapidly constructing stable CHO cell lines that highly express IgG antibodies, aiming to solve the above-mentioned problems. This invention is applicable to the construction of stable transgenic lines of various IgG subtype antibodies, including but not limited to IgG1, IgG2, and IgG4.

[0010] This invention is achieved by providing a method for rapidly constructing a stable CHO cell line with high IgG antibody expression, comprising the following steps:

[0011] S1. Antibody plasmid construction: The light and heavy chains of the antibody are linked to a vector capable of transfecting CHO (e.g., CHO-K1) cells through whole-genome synthesis, and the antibody plasmid is transformed and extracted. The amino acid sequence of the variable region of the heavy chain of the antibody is shown in SEQ ID NO:1, and the amino acid sequence of the variable region of the light chain is shown in SEQ ID NO:2.

[0012] S2.CHO cell resuscitation, passage, and plasmid transfection;

[0013] S3. Positive cell screening: Positive cells successfully transfected with plasmids were obtained through MSX pressure screening, and a cell pool was established;

[0014] S4. Prepare and screen stable single-cell clones with high expression;

[0015] S5. Feed-and-batch cell culture and antibody expression and purification: Stable cells with high expression were selected and fed-and-batch cultured. After feeding, the high expression level was verified by purifying the cells using an AKTA protein purifier.

[0016] S6. Stability verification of antibody-expressing cell lines: The high-expression stable single-cell clones selected in S4 were passaged and cryopreserved; the high-expression stable single-cell clones selected in S4 were passaged to passage 20 and then cryopreserved. Finally, the cells from the first passage and passage 20 were thawed and cultured in batches with feed to monitor the stability of cell status and yield at this stage.

[0017] Preferably, the S2.CHO cell resuscitation, passage, and plasmid transfection include the following steps:

[0018] 1) Remove CHO cells from the liquid nitrogen container, thaw rapidly in a 37°C water bath, centrifuge and discard the supernatant, then resuspend the CHO cells in basal culture medium with 4 mM glutamine added, and count them (generally 0.4-0.6 × 10⁻⁶). 6 After being cultured in an E125 shake flask with cells / mL, the cells were shaken.

[0019] 2) CHO cells were passaged every other day for a total of 3 passages;

[0020] 3) The cells were passaged to an appropriate density one day before transfection, and plasmid transfection was performed the next day using a Neon electroporator at a cell number of 4 × 10⁶. 6 cells / mL, sample volume 100μL, electroporation conditions: 1500-1700V / 15-25ms / 1 pulse, 5mL T25 flask for static incubation.

[0021] Preferably, the basal culture medium for S2 cell resuscitation contains: 1-2% trehalose, 0.05-0.1mM sodium selenite and 3-8mM L-proline.

[0022] Trehalose (low-temperature protection): forms a glassy structure and reduces ice crystal damage;

[0023] Sodium selenite (antioxidant): activates glutathione peroxidase (GPx4) and repairs oxidative damage;

[0024] L-proline (osmolarity regulation): stabilizes cell membrane protein conformation and resists osmolarity shocks during resuscitation;

[0025] A ternary composite protectant was used to replace traditional DMSO, eliminating the traditional static step, and the culture was directly shaken after recovery.

[0026] Preferably, the S3. positive cell screening includes the following steps: after culturing in a T25 flask for 2 days, pressurize with MSX and transfer the cells from the T25 flask to a 50 mL LTP shaker for shaking culture. For the first 5 days, add approximately 2 mL of fresh culture medium every other day while pressurizing. For the next 5 days, centrifuge and change the medium every half day, resuspend the cells in the same volume, pressurize with MSX, and record the number of cells, viability, and cell diameter during cell treatment. After approximately 2 days, the cells can proliferate normally, and stable transfected plasmid positive cells are obtained.

[0027] Preferably, the S3 positive cell screening includes a combination of metabolic activators, comprising 1-5 mM sodium butyrate and 0.5-2 mM valproic acid, to enhance the chromatin openness and integration efficiency of exogenous genes.

[0028] Metabolic activators are added simultaneously in S3 (MSX pressure screening stage) to regulate cellular energy metabolism and chromatin open state. This breaks through the traditional method of enriching positive cells solely through screening pressure (MSX). By actively promoting the integration of exogenous genes into chromosomes through metabolic intervention and activating histone acetylation, the efficiency of antibody gene transcription can be improved, solving the problem of inefficiency caused by random integration in the construction of stable transgenic lines and shortening the screening cycle.

[0029] Preferably, step S4. preparing and screening highly expressed, stable single-cell clones includes the following steps:

[0030] 1) Preparation of monoclonal cells: After stable passage of cells, count them and seed them into 96-well plates with 1 cell per well, for a total of 10 plates;

[0031] 2) Screening of monoclonal cells: Place the 96-well plate in a CO2 incubator and incubate statically. Observe the cells after two hours and mark the wells with a single cell. After about 12-14 days, add culture medium once in between. Use ELISA to screen the wells with high OD values ​​and cell populations with only one monoclonal cell.

[0032] 3) Single-clonal expansion: Add fresh culture medium and continue culturing. On the second day, transfer the cells from the 96-well plate to the 24-well plate for expansion culture.

[0033] Preferably, S5. Batch culture of cells includes the following steps: seeding cells in E125 shake flasks at a rate of 2.5-3.5 × 10⁶ cells / year. 6 The initial cell density is 15-30 mL, and the total volume can be 15-30 mL. The yield can be obtained by feeding with supplemental culture medium for 12-14 days.

[0034] Specifically, the S5 fed-batch culture can be carried out using staged feeding:

[0035] a) Day 0-4: Supplement with culture medium containing 6-8 g / L glucose and 2× amino acids;

[0036] b) Day 5-10: Supplement with culture medium containing 1-2 g / L glucose and 3% lipid precursor;

[0037] c) Day 11-14: Supplement with sugar-free medium containing 5 mM sodium butyrate.

[0038] Lipid precursors (such as choline / inositol): enhance cell membrane stability and prolong the production period;

[0039] Sodium butyrate: Induces cell cycle arrest in the later stages, reduces debris generation, and maintains viability for a longer period.

[0040] In fed-batch culture, the logarithmic growth phase of cells is shortened under such high seeding density, and the cells enter the plateau phase of protein production earlier. By using fed-batch culture, a higher density of viable cells can be achieved, thereby obtaining a higher level of protein expression.

[0041] Compared with the prior art, the embodiments of this application have the following main advantages:

[0042] 1. The method for rapidly constructing a stable CHO cell line with high expression of IgG antibody provided by the present invention involves electroporation of the plasmid vector into the cells, followed by shaking culture after 48 hours of static culture. This accelerates cell recovery time, resulting in faster recovery and better cell condition compared to traditional static culture.

[0043] 2. The method for rapidly constructing a stable CHO cell line with high IgG antibody expression provided by this invention involves inoculating cells at a high density during fed-batch cell culture. Under such high-density inoculation, the logarithmic growth phase of the cells is shortened, allowing them to enter the protein production phase earlier, thereby achieving a higher expression level.

[0044] 3. The method for rapidly constructing a stable CHO cell line with high IgG antibody expression provided by this invention greatly shortens the overall production cycle and saves a significant amount of cost. Attached Figure Description

[0045] Figure 1 This is a flowchart of a method for rapidly constructing a stable CHO cell line with high IgG antibody expression, provided by the present invention;

[0046] Figure 2 This image shows the purification results of the highest expression level of a CHO cell line obtained by a rapid method for constructing a high-expression, stable CHO cell line for IgG antibodies, as provided by this invention, using an AKTA purification instrument. Detailed Implementation

[0047] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs; the terminology used herein in the specification of the application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application; the terms "comprising" and "having," and any variations thereof, in the specification, claims, and foregoing drawings of this application are intended to cover non-exclusive inclusion. The terms "first," "second," etc., in the specification, claims, or foregoing drawings of this application are used to distinguish different objects, not to describe a particular order.

[0048] In this document, the term "embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The appearance of this phrase in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment mutually exclusive with other embodiments. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0049] Example 1

[0050] This invention provides a method for rapidly constructing a stable CHO cell line with high IgG antibody expression, such as... Figure 1 As shown, it includes the following steps:

[0051] S1. Antibody plasmid construction: The light and heavy chains of the antibody are linked to a vector capable of transfecting CHO (e.g., CHO-K1) cells through whole-genome synthesis, and the antibody plasmid is transformed and extracted. The amino acid sequence of the variable region of the heavy chain of the antibody is shown in SEQ ID NO:1, and the amino acid sequence of the variable region of the light chain is shown in SEQ ID NO:2.

[0052] M6 heavy chain variable region amino acid sequence (SEQ ID NO:1):

[0053] EVKLVESGGGLVKPGGTLKLSCAASDSSFSTYAMSWIRQTPEKRLEWVASISSGGVTYYQDNVRRGRFTISRDNARNILYLQMSSLRSEDTATYYCARGYFYFDYWGQGTT LTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSPRPSETVTCNVAHPASSTKVDKKIVPRDCGCK PCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISK TKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK.

[0054] M6 light chain variable region amino acid sequence (SEQ ID NO:2):

[0055] DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQRPDGTFKLLIYYKSRLQSGVPSRFSGSGSGIDYFLTISNLEQEDIATYFCQQGNTLPRTFGGGTKLEIK RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC.

[0056] S2.CHO-K1 cells were revived, passaged, and transfected with plasmids.

[0057] 1. Cell resuscitation and passage

[0058] 1) Prepare 20 mL of basic culture medium in an E125 shake flask, add glutamine to a final concentration of 4 mM, preheat in a 37℃ incubator for 30 min, and at the same time prepare 15 mL centrifuge tubes and add 4 mL of fresh culture medium.

[0059] 2) Remove the cells from the liquid nitrogen container, thaw them quickly in a 37°C water bath, transfer them to a 15mL centrifuge tube, mix well, and centrifuge at 1000rpm for 5min.

[0060] 3) Discard the supernatant, resuspend the cells in 1 mL of fresh culture medium, and transfer them to an E125 shake flask. Take a portion of the cell suspension and use an automated cell counter to count the cell number and cell viability, which is generally 0.4-0.6 × 10⁻⁶. 6 Cells / mL, then place the shake flask in a shaker for incubation (37℃, 5% CO2, 125 rpm).

[0061] 4) Every other day, a portion of the cell suspension was taken out, and the cell count and viability were calculated using a fully automated cell counter. The cells were then passaged to 0.5 × 10⁻⁶ cells. 6 cells / mL, thus achieving stable passage 3 times.

[0062] 2. Plasmid transfection

[0063] 1) Divert cells to 0.5 × 10⁻⁶ cells per cell line one day before plasmid transfection. 6 cells / mL to prepare for plasmid transfection the next day;

[0064] 2) Prepare 5 mL of fresh basal culture with 4 mM glutamine added in a T25 flask and preheat in an incubator at 37°C for 30 min;

[0065] 3) Collect the cells, count them, and then centrifuge them. The cell count should be 4 × 10⁻⁶. 6 cells / mL, centrifuged at 1000 rpm for 5 min;

[0066] 4) Discard the supernatant, resuspend in 5 mL L PBS, and then continue centrifugation at 1000 rpm for 5 min;

[0067] 5) Discard the supernatant, resuspend in 100 μL of electroporation buffer, add 1 mg of plasmid, and gently mix by blowing.

[0068] 6) Neon electrostatic precipitator: 1500-1700V / 15-25ms / 1 pulse;

[0069] 7) Transfer the electroporated cell suspension into a T25 flask and incubate statically.

[0070] S3. Positive cell screening

[0071] 1) After 48 hours of electroporation, transfer the electroporated cells from the T25 flask into a 50 mL TPP shaker, add MSX to a final concentration of 25 mM, and incubate at 37 °C with shaking.

[0072] 2) For the first 5 days, add fresh culture medium every other day, about 2 mL each time. For the next 5 days, centrifuge and change the medium every half day, resuspend the cells in the same volume, pressurize with 25 mM MSX, and record the number and viability of cells using a fully automated cell counter. After about 2 days, the cells can proliferate normally, and stable transfected plasmid positive cells are obtained. The results of positive cell selection recovery are shown in Table 1 below:

[0073] Table 1. Results of positive cell screening recovery

[0074]

[0075] S4. Preparation and screening of stable single-cell clones with high expression

[0076] 1) After recovery, remove and count the cells, dilute them with monoclonal medium, and seed them into 96-well plates at 1 cell / well. Add 100 μL of monoclonal medium to each well. Select 10 96-well plates and incubate them statically in a 37°C incubator. After 2 hours, observe the 96-well plates under a microscope, find the wells with single cells, and mark them.

[0077] 2) Add 50 μL of fresh monoclonal culture medium after about 7 days and continue static incubation;

[0078] 3) After another 7 days (or 5 days), the confluence of the wells can reach about 70%. Use ELISA to screen for wells with higher OD450 values, about 48 wells, and expand them into 24-well plates for continued static incubation.

[0079] 4) About 2 days later, use Octet to detect cell expression in 24-well plates, select the 10 cell lines with the highest expression, expand them into 50mTPP shaker tubes, and passage them stably 3 times to obtain stable monoclonal cell lines.

[0080] S5. Fed-batch cell culture

[0081] 1) The initial inoculation density is 2.5-3.5 × 10⁻⁶. 6 In this embodiment, the cells / mL is preferably 3.0 × 10⁻⁶. 6 The cells / mL volume was 15-30mL, preferably 20mL in this example. The cells were cultured in an E125 shake flask with shaking on a shaker (37℃, 5% CO2, 125rpm) as Day 0 data.

[0082] 2) Then, every other day, cells were taken out to measure cell density, viability, and sugar content;

[0083] 3) The number of feeding days can be determined based on cell number and viability. Finally, the strain with the highest expression level was used for purification using an AKTA purification system. The results are shown in Table 2 below. Figure 2 .

[0084] Table 2 Results of fed-batch culture of the highest-performing cell line

[0085]

[0086] The cells mentioned above are from the QuaCell® CHO-K1Q cell line, which originated from ECACC and was developed by the R&D team using patented technology to domesticate and screen CHO-K1 cells. The basal medium is AltairCHO®Medium, the monoclonal medium is QuaMono™ PlusCHO, and the feed medium is StarCHO™ Feed and CDFS36 ultra-concentrated feed.

[0087] Example 2

[0088] Based on Example 1, this embodiment further adds the following to the basal culture medium for S2 cell resuscitation: 1-2% trehalose, 0.05-0.1mM sodium selenite, and 3-8mM L-proline. Experiments have shown that the combination of 1.5% trehalose + 0.05mM sodium selenite + 5mM proline can achieve a resuscitation survival rate >99%.

[0089] Specifically, the S2 cell resuscitation procedure is as follows:

[0090] Preparation of resuscitation fluid:

[0091] Basic culture medium (AltairCHO® Medium), 4 mM glutamine, 1.5% trehalose, 0.05 mM sodium selenite, 5 mM L-proline;

[0092] Recovery procedures:

[0093] Remove the frozen cells from liquid nitrogen and thaw them in a water bath at 37°C for ≤60 seconds;

[0094] Centrifuge and discard the supernatant (1000 rpm, 5 min);

[0095] Resuspend the cells in the above-mentioned resuscitation solution pre-cooled to 4°C (avoid temperature shock).

[0096] Transfer directly to an E125 shake flask and incubate with shaking at 125 rpm.

[0097] Furthermore, the S3 positive cell screening process includes a combination of metabolic activators, comprising 1-5 mM sodium butyrate and 0.5-2 mM valproic acid, to enhance the chromatin openness and integration efficiency of exogenous genes.

[0098] Specifically: During the S3 positive cell selection phase (when MSX is applied), the following should be added to the culture medium simultaneously:

[0099] Sodium butyrate (2mM): Dissolve in PBS, filter to remove bacteria, and then add to the culture medium;

[0100] Valproic acid (1mM): Prepare a 100mM stock solution with DMSO, and dilute to the final concentration before use.

[0101] Cell growth data are shown in Table 3 below:

[0102] Table 3 Cell growth data

[0103]

[0104] MSX was added simultaneously with the metabolic activator. After adding the metabolic activator combination, the cell viability recovered to over 85% on day 6 of MSX screening (compared to 50% viability in the untreated group). Cell proliferation accelerated after adding the metabolic activator, reaching 3.2 × 10⁶ cells by day 10. 6 / mL (98% viability).

[0105] To demonstrate the technical effectiveness of the innovation of this invention, the following comparative embodiments are provided:

[0106] Traditional method:

[0107] 1. Cell resuscitation

[0108] Cryopreservation solution: Fetal bovine serum (FBS) containing 8-10% DMSO or commercially available cryopreservation solution (such as CryoStor®).

[0109] Recovery process:

[0110] Thaw cells in a water bath at 37°C;

[0111] Centrifugation removes the supernatant containing DMSO;

[0112] Incubate statically for 24-48 hours (until cells adhere / recover);

[0113] Then switch to suspension oscillation culture.

[0114] 2. Screening for positive cells

[0115] Screening pressure: Single concentration of MSX (25-50μM) throughout the process, without metabolic activators;

[0116] Procedure: After transfection, let stand for 48 hours → Add MSX → Culture in still or agitated conditions throughout (no stage optimization).

[0117] 3. Feed-in batch culture

[0118] Inoculation density: 0.5-1.5×10 6 cells / mL (lower than the high-density inoculation of this invention);

[0119] Feeding strategy: constant glucose culture (glucose maintained at 4-6 g / L) or simple feeding (such as adding only glucose / glutamine).

[0120] The comparison data is shown in Table 4 below:

[0121] Table 4 Comparison Data Table

[0122]

[0123] The results above show that the method of the present invention has high viability, increased antibody yield, and a short cycle.

[0124] Table 1 shows the data for Example 1 without the addition of a metabolic activator. In Example 2, due to the addition of sodium butyrate / valproic acid: the activity rate increased to 85% on Day 6 (compared to 50% in Table 1); screening could be completed on Day 10 (compared to Day 12 in Table 1).

[0125] Table 2 shows the data for Example 1. In Example 2, under the same culture days (Day 18), the expression level increased to 11.2 ± 0.4 g / L due to the enhanced transcription efficiency of the metabolic activator.

[0126] It should be noted that, for the sake of simplicity, the foregoing embodiments are all described as a series of actions. However, those skilled in the art should understand that the present invention is not limited to the described order of actions, as some steps may be performed in other orders or simultaneously according to the present invention. Furthermore, those skilled in the art should also understand that the embodiments described in the specification are preferred embodiments, and the actions and modules involved are not necessarily essential to the present invention.

[0127] It should be understood that the disclosed apparatus can be implemented in other ways, as illustrated in the embodiments provided in this application. For example, the apparatus embodiments described above are merely illustrative; the division of units described above is only a logical functional division, and in actual implementation, there may be other division methods. For example, multiple units or components may be combined or integrated into another system, or some features may be ignored or not executed. Furthermore, the coupling or communication connections shown or discussed may be through some interfaces; the indirect coupling or communication connections between devices or units may be telecommunications or other forms.

[0128] The units described above as separate components may or may not be physically separate. The components shown as units may or may not be physical units; that is, they may be located in one place or distributed across multiple network units. Some or all of the units can be selected to achieve the purpose of this embodiment according to actual needs.

[0129] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit the scope of protection of the invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on these embodiments, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art can still combine, add, delete, or otherwise adjust the features of the various embodiments of the present invention according to the circumstances without conflict or creative effort, thereby obtaining different technical solutions that do not fundamentally depart from the concept of the present invention. These technical solutions also fall within the scope of protection of the present invention.

Claims

1. A method for rapidly constructing a stable CHO cell line with high IgG antibody expression, characterized in that, Includes the following steps: S1. Antibody plasmid construction: The antibody light and heavy chain genes are ligated into a mammalian expression vector, and the antibody plasmid is obtained by transformation and extraction. S2. Cell resuscitation, passage and plasmid transfection: After resuscitation, CHO cells were directly cultured with shaking and transfected with the obtained antibody plasmid by electroporation; wherein, the basal culture medium used for cell resuscitation was supplemented with: 1-2% trehalose, 0.05-0.1mM sodium selenite and 3-8mM L-proline; S3. Positive cell screening: Screening pressure was applied within 48 hours after transfection, and screening was performed under pressure with shaking. The specific steps are as follows: After 48 hours of static culture in a T25 flask, MSX pressure was applied; the cells were transferred from the T25 flask to 50 mL LTP shaker tubes and cultured with shaking. For the first 5 days, 1-3 mL of fresh culture medium was added every other day while applying pressure; for the next 5 days, the cells were centrifuged and the medium changed every 36 hours, resuspended in the same volume, and then pressurized with MSX. The number, viability, and diameter of cells were recorded during cell treatment. After another 2 days, the cells could proliferate normally, yielding stable positive cells transfected with the plasmid. A metabolic activator combination was added during screening, comprising: 1-5 mM sodium butyrate and 0.5-2 mM valproic acid. S4. Preparation and screening of stable single-cell clones with high expression: Single-cell clones were prepared using the limiting dilution method and high-expression clones were obtained through high-throughput screening; S5. Fed-batch cell culture: Specifically, selected clones were fed-batch cultured, and 2.5 × 10⁶ cells were seeded in E125 shake flasks. 6 -3.5×10 6 The initial cell density was set at cells / mL, with a total volume of 15-30mL, and fed with supplemental culture medium for 12-14 days; the fed-batch culture employed a staged feeding method. a) Days 0-4: Supplement with culture medium containing 6-8 g / L glucose and 2× amino acids; b) Days 5-10: Supplement with culture medium containing 1-2 g / L glucose and 3% lipid precursor; c) Days 11-14: Supplement with sugar-free culture medium containing 5 mM sodium butyrate; S6. Stability verification of antibody-expressing cell lines: Verify the stability of cell line passages up to at least 20 generations.

2. The method for rapidly constructing a stable CHO cell line with high IgG antibody expression as described in claim 1, characterized in that, The CHO cells are the CHO-K1 cell line.

3. The method for rapidly constructing a stable CHO cell line with high IgG antibody expression as described in claim 1, characterized in that, The S2. cell resuscitation, passage, and plasmid transfection process includes the following steps: CHO cells were removed from the liquid nitrogen tank, thawed rapidly in a 37°C water bath, centrifuged to discard the supernatant, and resuspended in basal culture medium with 4 mM glutamine. After counting the cells, they were cultured with shaking. CHO cells were passaged every other day for a total of 3 passages. Cells were transferred to an appropriate density the day before transfection, and plasmid transfection was performed the next day using a Neon electroporator. Cells were incubated statically in a 5 mL T25 flask.

4. The method for rapidly constructing a stable CHO cell line with high IgG antibody expression as described in claim 3, characterized in that, Electroporation parameters: cell density 3×10⁻⁶ 6 -5×10 6 cells / mL, sample volume 100μL, voltage 1500-1700V, pulse duration 15-25ms.

5. The method for rapidly constructing a stable CHO cell line with high IgG antibody expression as described in claim 1, characterized in that, The high-throughput filtering in S4 includes: The first stage involved detecting the expression levels of clones in a 96-well plate using ELISA. The second stage involved detecting the expression level after amplification in a 24-well plate using Octet.

6. The method for rapidly constructing a stable CHO cell line with high IgG antibody expression as described in claim 1, characterized in that, The antibody comprises the heavy chain variable region sequence shown in SEQ ID NO:1 and the light chain variable region sequence shown in SEQ ID NO:2.

Images