Monoclonal cell strain screening and amplification method
Through the suspension flow plus culture and step-by-step amplification culture, the problems of high-throughput screening and poor cell status during the CHO cell line screening and expansion process were solved, and efficient screening and high-quality monoclonal cell lines were achieved.
Patent Information
- Application Number
- CN202411772699.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-04
- Publication Date
- 2025-03-28
AI Technical Summary
In the prior art, there are problems such as difficulty in screening and expansion of CHO cell lines, severe cell aggregation, slow expansion speed, and generally poor cell status during the screening and expansion of CHO cell lines, resulting in relatively low expression of monoclonal cell lines.
The mini cell pool was screened and expanded by suspended flow plus culture and evaluation. The expression amount, cell growth status and product quality were comprehensively evaluated, and high-expression and high-quality monoclonal cell lines were screened.
Efficient and high-throughput monoclonal cell screening and expansion were achieved, which significantly shortened the screening cycle, improved the cell status and product quality, and obtained monoclonal cell lines with high expression of target protein and stable quality.
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Abstract
Description
Technical Field
[0001] This application relates to the field of cell engineering, specifically to a method for screening and amplifying monoclonal cell lines. Background Technology
[0002] In biopharmaceutical development, constructing stable, high-yield cell lines is crucial, as it accompanies the entire drug lifecycle and directly determines development costs, production efficiency, and product quality. Cell line development (CLD), as the starting point and foundation of biopharmaceutical development, can be broadly divided into the following steps: host cell identification, vector construction, pool screening and evaluation, and monoclonal screening and evaluation, etc., to obtain high-quality monoclonal cell lines with strong growth capacity, good scalability, stability, and high yield. Shortening the cell line development cycle has a significant impact on the entire project development; therefore, various strategies are needed to optimize the entire development process.
[0003] Conventional CHO cell line screening typically employs a 96-well plate (96wp) static screening method, followed by stepwise static amplification to shake flasks. Merck's CHOZN... ® The technical instructions accompanying GS- / - host cells also employ a conventional screening process. This process, requiring individual processing with a single-channel pipette, is not conducive to high-throughput screening. Furthermore, after static culture, cell aggregation is severe, and the expansion rate is limited by the bottom area of the culture vessel, resulting in slow cell expansion and generally poor cell condition. The conventional process requires expanding the minipool to shake flasks for approximately 14 days of fed-batch culture for evaluation, followed by comprehensive quality analysis before single-clone plating, significantly extending the experimental cycle. Additionally, predicting cell state and expression levels after suspension based on static screening results lacks accuracy and scientific rigor, and single-clone cell lines obtained using this method generally exhibit relatively low expression levels. Therefore, there is an urgent need to find a more efficient, high-throughput method for screening and expanding single-clone cells with good cell condition. Summary of the Invention
[0004] Based on this, this application provides a method for screening and amplifying monoclonal cell lines with high efficiency, high throughput and good cell condition.
[0005] The technical solution includes:
[0006] A method for screening and amplifying monoclonal cell lines, the method comprising the following steps:
[0007] S1: Transform the exogenous vector containing the target gene and the GS gene into the host cell, screen for positive clones, and then screen for mini cell pools with high expression levels of the target protein from the positive clones. The number of mini cell pools screened out accounts for 20% to 30% of the total cell pool.
[0008] S2: The mini cell pools screened in step S1 are subjected to suspension fed-batch culture and evaluation. After screening out the top 3-5 mini cell pools in terms of target protein expression level and quality, suspension stepwise expansion culture is carried out. Based on the comprehensive evaluation of expression level, cell growth and product quality, the top 2-3 mini cell pools are screened out.
[0009] S3: The mini-cell pools screened in step S2 are subjected to monoclonal sorting to identify monoclonal cells with high expression levels of the target protein. The number of identified monoclonal cells should be between 20% and 30% of the total number of monoclonal cells.
[0010] S4: Perform the first suspension fed-batch culture and evaluation on the monoclonal cells screened in step S3. After screening out the monoclonal cells with the highest expression level of the target protein, perform suspension fed-batch culture and evaluation for the second suspension fed-batch culture to screen out monoclonal cell lines.
[0011] In one embodiment, in step S2, suspension fed-batch culture and evaluation are performed in a 96-well deep-well plate.
[0012] Optionally, suspension feeding culture can be carried out under conditions of 36℃~37℃, 4%~6% CO2, 200rpm~1100rpm and 80%~85% humidity.
[0013] Optionally, the culture medium used for fed-batch culture includes one or more of EX-CELL Advanced CHO Fed-batch medium, ActiPro™ medium, and CHO MaxY medium.
[0014] In one embodiment, step S2 includes evaluating the expression level and quality of the target protein.
[0015] Optionally, the steps for evaluating the expression level and quality of the target protein include supplementing the feed and sugar on days 4 and 6 of the fed-batch culture, collecting the cell supernatant on day 8 for detecting the expression level of the target protein, evaluating the quality of the target protein in the supernatant of the top 3 to 5 mini-cell pools, and screening the top 2 to 3 mini-cell pools.
[0016] Optionally, the feed includes Cellvento ModiFeed prime COMP.
[0017] Optionally, the sugar includes glucose.
[0018] Optionally, the feedstock can be added at 4% to 5% of the culture system volume, and glucose can be added until the glucose concentration in the culture system is 6 g / L to 8 g / L.
[0019] Optionally, the target protein quality can be evaluated using methods including SDS-PAGE.
[0020] In one embodiment, in step S2, the suspension step of stepwise amplification culture includes amplifying and culturing the top 2-3 mini cell pools selected from the screening into a cell culture vessel, which includes one or more of a 24-well deep plate, a TPP shaker tube, or a shaker flask.
[0021] Optionally, amplification culture can be carried out at 36℃~37℃, 4%~6% CO2 and 200rpm~1100rpm.
[0022] Optionally, the culture medium used for amplification culture includes one or more of CD CHO Fusion medium, CD CHO medium, and CDOptiCHO medium.
[0023] Optionally, the cells can be expanded and cultured until the cell viability is ≥85%.
[0024] In one embodiment, step S3, after monoclonal sorting, further includes steps of static culture and / or monoclonal origin tracing and confirmation.
[0025] Optionally, the culture can be statically incubated at 36℃~37℃ and 4%~6% CO2.
[0026] Optionally, the static incubation period is 2 to 3 weeks.
[0027] In one embodiment, in step S4, a 96-well deep-well plate is used for the first suspension-fed culture and evaluation.
[0028] Optionally, the conditions for the first suspension-feed culture are the same as in step S2.
[0029] Optionally, the evaluation may include assessment of the expression level and quality of the target protein.
[0030] Optionally, the steps for evaluating the expression level and quality of the target protein include feeding and adding sugar on days 4 and 6 of the fed-batch culture, collecting the cell supernatant on day 8 for detecting the expression level of the target protein, and screening out the top 30-50 monoclonal cells in terms of expression level.
[0031] In one embodiment, in step S4, the suspension step of stepwise amplification culture includes amplifying and culturing the top 30-50 screened monoclonal cells into a cell culture vessel, which includes one or more of 96DWP, 24DWP, TPP shaker tubes or 125mL shake flasks.
[0032] Optionally, amplification culture can be carried out at 36℃~37℃, 4%~6% CO2 and 200rpm~1100rpm.
[0033] Optionally, the culture medium used for amplification culture includes one or more of CD CHO Fusion medium, CD CHO medium, and CDOptiCHO medium.
[0034] Optionally, the cells are expanded and cultured until the cell viability is ≥90% and / or the cell density is 2×10⁻⁶. 6 cells / mL ~5×10 6 cells / mL.
[0035] In step S4, a second suspension-feed culture and evaluation are performed using a shake flask.
[0036] Optionally, the second suspension-fed culture and evaluation steps include:
[0037] Cells were spaced at a density of 0.5 × 10⁻⁶. 6 cells / ml ~1×10 6 Cells were seeded at a density of 2.5 × 10⁶ cells / ml in shake flasks. 6 cells / ml ~3.0×10 6 Start fed-batch culture at cells / mL; and
[0038] Cells are cultured until cell viability is ≥80%, and the top 3-5 monoclonal cells are selected for stability evaluation based on one or more of the following criteria: cell growth, quality of the target protein, and expression level.
[0039] Optionally, fed-batch culture can be carried out by adding 4% to 5% of the culture system volume every other day, and the sugar content can be controlled at 2 g / L to 6 g / L during the culture period.
[0040] In one embodiment, the host cell is a host cell with the endogenous GS gene knocked out.
[0041] Optionally, the host cell includes CHO cells and CHO cell-derived mutant strains.
[0042] In one embodiment, the positive clone cells were screened using a glutamine-free culture medium.
[0043] Compared with traditional technologies, this application has the following advantages:
[0044] This application provides a method for screening and amplifying monoclonal cell lines. This method introduces suspension-based Fed-batch evaluation into the evaluation of mini-cell pools, increasing screening throughput while prioritizing product quality assessment. This allows for monoclonal plate separation after suspension amplification, significantly shortening the screening cycle. By employing suspension amplification for stepwise amplification of monoclonal cells, the overall growth status and characteristics of the monoclonal cell lines exhibit significant improvement in uniformity.
[0045] The method described in this application enables the screening of monoclonal cell lines with high and stable expression of the target protein, while significantly reducing the throughput of monoclonal screening and greatly improving screening efficiency. Attached Figure Description
[0046] To more clearly illustrate the technical solutions in the embodiments of this application and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0047] Figure 1 This paper compares the improved technical approach of this application with the technical approach of Comparative Example 1;
[0048] Figure 2 Results of flow assay for antibody 1 and antibody 2 using minipool 96DWP;
[0049] Figure 3 Results of monoclonal 96DWP assay for antibodies 1 and 2;
[0050] Figure 4 The results of SDS-PAGE of the supernatant from the minipool stage of antibody 1 in Example 1 are shown.
[0051] Figure 5 The results of SDS-PAGE of the supernatant from the minipool stage of antibody 2 in Example 2 are shown.
[0052] Figure 6 Comparison of cell states during the antibody 1 monoclonal phase suspension amplification and static amplification;
[0053] Figure 7 Comparison of cell states during the antibody 2 monoclonal phase suspension amplification and static amplification;
[0054] Figure 8 This example compares the expression level of antibody 1 in the improved method with that in the comparative example;
[0055] Figure 9 The improved method in the example is compared with the expression level of antibody 2 in the comparative example. Detailed Implementation
[0056] To make the above-mentioned objectives, features, and advantages of this application more apparent and understandable, a detailed description of specific embodiments of this application is provided below. Many specific details are set forth in the following description to provide a thorough understanding of this application. However, this application can be implemented in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of this application. Therefore, this application is not limited to the specific embodiments disclosed below.
[0057] 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 is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.
[0058] The term “and / or” as used herein includes any and all combinations of one or more of the related listed items.
[0059] One embodiment of this application provides a method for screening and amplifying monoclonal cell lines. This method can screen monoclonal cell lines with high expression of the target protein and stable and uniform quality, while significantly reducing the throughput of monoclonal screening and greatly improving the screening efficiency. Specifically, it includes the following steps S1 to S4:
[0060] S1: Transfect host cells with an exogenous vector containing the target gene and the GS gene, screen for positive clones, and then screen for mini cell pools with high expression levels of the target protein among the positive clones. The number of mini cell pools screened accounts for 20% to 30% of the total cell pool.
[0061] In one specific example, the host cell is a host cell with the endogenous GS gene knocked out.
[0062] In one specific example, the host cells include CHO cells and CHO cell-derived mutant strains.
[0063] In one specific example, positive clone cells were screened using a glutamine-free culture medium.
[0064] In one specific example, positive clone cell screening includes static culture screening for 14 to 21 days.
[0065] S2: The mini-cell pools screened in step S1 are subjected to suspension fed-batch culture and evaluation. After screening out the top 3-5 mini-cell pools in terms of target protein expression level and quality, suspension stepwise expansion culture is performed. Based on a comprehensive evaluation of expression level, cell growth, and product quality, the top 2-3 mini-cell pools are selected. This application uses suspension cell evaluation, which is closer to the evaluation method of the original state of engineered cell lines and replaces the conventional static cell evaluation method.
[0066] In a specific example, in step S2, suspension-feed culture and evaluation are performed in a 96-well deep-well plate.
[0067] In a specific example, in step S2, a fed-batch culture is carried out in a 96-well deep well plate under the conditions of 36°C to 37°C, 4% to 6% CO2, 200 rpm to 1100 rpm and 80% to 85% humidity.
[0068] In one specific example, the culture medium used for fed-batch suspension culture includes one or more of EX-CELL Advanced CHO Fed-batch medium, ActiPro™ medium, and CHO MaxY medium. Preferably, the culture medium used for fed-batch suspension culture is EX-CELL Advanced CHO Fed-batch medium.
[0069] In a specific example, in step S2, the evaluation includes an assessment of the expression level and quality of the target protein;
[0070] In a specific example, the steps for evaluating the expression level and quality of the target protein include supplementing the feed and sugar on days 4 and 6 of the fed-batch culture, collecting the cell supernatant on day 8 for detecting the expression level of the target protein, evaluating the quality of the target protein in the supernatant of the top 3-5 mini cell pools, and screening the top 2-3 mini cell pools.
[0071] In a specific example, the method for evaluating the expression level and quality of the target protein includes SDS-PAGE, which optionally includes reducing SDS-PAGE and / or non-reducing SDS-PAGE.
[0072] In a specific example, the feed includes Cellvento ModiFeed prime COMP.
[0073] In a specific example, sugar includes glucose.
[0074] In a specific example, feed is added at a rate of 4% to 5% of the culture system volume.
[0075] In a specific example, glucose is added to the culture system to a concentration of 6 g / L to 8 g / L, which can be selected as 6 g / L, 6.5 g / L, 7 g / L, 7.5 g / L or 8 g / L.
[0076] In one specific example, in step S2, the suspension step of stepwise amplification culture includes amplifying and culturing the top 2-3 mini cell pools selected from the screening into a cell culture vessel, which includes one or more of a 24-well deep plate, a TPP shaker tube, or a shaker flask.
[0077] In a specific example, in step S2, after screening out the top 2-3 mini cell pools in terms of target protein expression level and quality, the step of performing suspension stepwise expansion culture includes passage culture of the cell suspension before feeding in 96DWP, screening out the top 2-3 mini cell pools in terms of target protein expression level and quality based on the evaluation results, and expanding the selected top 2-3 mini cell pools into 24-well deep-well plates.
[0078] In one specific example, amplification culture was carried out at 36℃~37℃, 4%~6% CO2, and 200rpm~1100rpm.
[0079] In one specific example, the culture medium used for amplification culture includes one or more of CD CHO Fusion medium, CD CHO medium, and CD OptiCHO medium. Preferably, the culture medium used for amplification culture is CD CHO Fusion medium.
[0080] In one specific example, the cells were expanded and cultured until the cell viability was ≥85%.
[0081] S3: Perform monoclonal sorting on the mini cell pool screened in step S2 to screen out monoclonal cells with high expression of the target protein. The number of monoclonal cells screened out should be 20% to 30% of the total monoclonal cells, and can be selected as 20%, 22%, 25%, 28%, 30%, or any two of the above values.
[0082] In one specific example, after monoclonal sorting, the process also includes a step of static incubation at 36℃~37℃ and 4%~6% CO2 for 2~3 weeks.
[0083] In one specific example, the process of tracing and confirming the monoclonal origin of monoclonal cells during static culture also includes steps such as monoclonal origin tracking and verification.
[0084] In a specific example, after static culture, monoclonal cells with high expression levels of the target protein are screened. Optionally, the method for detecting the expression level of the target protein includes the Octet semi-quantitative assay.
[0085] S4: Perform the first suspension fed-batch culture and evaluation on the monoclonal cells screened in step S3. After screening out the monoclonal cells with the highest expression level of the target protein, perform suspension fed-batch culture and evaluation for the second suspension fed-batch culture to screen out monoclonal cell lines.
[0086] In a specific example, in step S4, a 96-well deep-well plate is used for the first suspension-fed culture and evaluation.
[0087] In a specific example, the conditions for the first suspension culture are the same as in step S2.
[0088] In a specific example, the evaluation includes assessing the expression level and quality of the target protein.
[0089] In a specific example, the steps for evaluating the expression level and quality of the target protein include feeding and adding sugar on days 4 and 6 of the fed-batch culture, collecting cell supernatant on day 8 for detecting the expression level of the target protein, and screening out the top 30-50 monoclonal cells in terms of expression level.
[0090] In one specific example, in step S4, the suspension step of stepwise amplification culture includes amplifying and culturing the top 30-50 screened monoclonal cells into a cell culture vessel, which includes one or more of 96DWP, 24DWP, TPP shaker tubes or 125mL shake flasks.
[0091] This application employs a stepwise amplification culture process, from static culture to suspension culture to suspension culture, which is beneficial for cell state recovery.
[0092] In one specific example, in step S4, the suspension step of incremental expansion culture includes expanding and culturing the top 30-50 selected monoclonal cells to 24DWP.
[0093] In one specific example, amplification culture was carried out at 36℃~37℃, 4%~6% CO2, and 200rpm~1100rpm.
[0094] In one specific example, the culture medium used for amplification culture includes one or more of CD CHO Fusion medium, CD CHO medium, and CD OptiCHO medium. Preferably, the culture medium used for amplification culture is CD CHO Fusion medium.
[0095] In one specific example, the cells were expanded and cultured to a viability ≥90%, and / or a cell density of 2 × 10⁻⁶. 6 cells / mL ~5×10 6 cells / mL.
[0096] In a specific example, in step S4, a second suspension-feed culture and evaluation is performed using a shake flask.
[0097] In a specific example, the steps of the second suspension-fed culture and evaluation include:
[0098] Cells were spaced at a density of 0.5 × 10⁻⁶. 6 cells / mL ~1×10 6 The cells were seeded in shake flasks at a density of 2.5 × 10⁶ cells / mL. 6 cells / mL ~3.0×10 6 Start fed-batch culture at cells / mL; and
[0099] Cells are cultured until cell viability is ≥80%, and the top 3-5 monoclonal cells are selected for stability evaluation based on one or more of the following criteria: cell growth, quality of the target protein, and expression level.
[0100] In a specific example, fed-batch culture was carried out with a 4% v / v feed rate, supplemented every other day, and the sugar content was controlled at 2 g / L to 6 g / L during the culture period.
[0101] This application provides an evaluation method using 96-well deep well plates (96DWP) for suspension cell evaluation, which more closely approximates the original state of engineered cell lines, replacing the conventional static cell evaluation method. The suspension cell amplification method, which involves expanding from 96wp to 96DWP and then to 24DWP, is more conducive to cell state recovery, replacing the conventional static cell amplification method. Simultaneously, the introduction of Fed-bacteria supernatant SDS-PAGE detection at the minipool stage to confirm the product quality in advance allows for monoclonal plate separation at the 24DWP stage, significantly shortening the screening cycle. This workflow can be uniformly processed using multi-channel pipettes, significantly improving operational efficiency and reducing operating costs.
[0102] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this application, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.
[0103] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. For temperature and time parameters, acceptable deviations due to instrument testing accuracy or operational precision are permissible.
[0104] In the following examples, the host cell is CHOZN from Merck. ® GS- / - This cell line is a host cell line in which the glutamine synthase gene has been knocked out using ZFNs (zinc finger nucleases) technology; 96-well deep-well plates were purchased from Thermo; 24-well deep-well plates were purchased from Thomson; TPP shake tubes were purchased from TPP; 125mL shake flasks, 250mL shake flasks, T25 cm2 culture flasks, T75 cm2 culture flasks, 96-well plates, and 24-well plates were all purchased from Corning; MSX reagents were purchased from Sigma; the growth medium was Merck's CD CHOFusion, and the fed-batch medium was Merck's EX-CELL Advanced CHO Fed-batch Medium (medium-sized media such as Maibang and OPMA can also be used).
[0105] This application uses two antibody projects as representatives (antibody 1 is a monoclonal antibody, hereinafter referred to as IM; antibody 2 is an asymmetric bispecific antibody, hereinafter referred to as asymmetric bispecific antibody) to conduct comparative studies of examples and comparative examples.
[0106] Example 1
[0107] This embodiment provides a method for screening and amplifying CHO cell monoclonal cell lines. The screening and amplification steps are as follows:
[0108] 1. Minipool static screening
[0109] 1.1. Select CHO host cells and passage them after resuscitation. Electroporation should only be performed after at least 4 days and no more than 28 days of resuscitation, until cell viability is ≥95%. Transfect the plasmid to be transferred (containing the GS (glutamine synthase) gene and the encoding genes of antibody 1 or antibody 2) into the host cells via electroporation. Positive clones are selected using glutamine-deficient culture medium.
[0110] 1.2. Preliminary evaluation of minipool expression levels
[0111] After static screening for 14-21 days, 100 μL of cell supernatant was transferred to an Octet 96-well plate (manufacturer: Greiner, catalog number: 655209). 100 μL of CD CHO Fusion medium was added, and the expression level in the supernatant was detected using a molecular interaction analyzer (manufacturer: Sartorius, model: Octet R8). A mixed cell pool with excellent expression levels was selected in a semi-quantitative screening manner (the number of minipools was controlled at 20%-30% of the total number).
[0112] 2. Evaluation of expression levels in Minipool deep-well plates
[0113] 2.1. 96DWP streaming
[0114] 2.1.1. After discarding the original supernatant from the candidate mixed cell pool obtained in Example 1.2, add 200 μL of fresh preheated Fed-batch medium and gently pipette the cells to mix them.
[0115] 2.1.2. After resuspending and mixing, all cell suspensions were transferred to 96DWP (Thermo, catalog number: 278743) for Fed-batch evaluation. Culture conditions were 36.5±0.5℃, 5% CO2, 200-1100 rpm, 80-85% humidity, with a final culture volume of 100μL-1000μL. The Fed-batch medium was EX-CELL Advanced CHO Fed-batch medium (Sigma, catalog number: 14366C), and the feed consisted of Cellvento ModiFeed prime COMP (Merck, catalog number: 1.04132.0001) and D-Glucose (Gibco, catalog number: A24940-01). No additional sugar or Cellvento ModiFeed prime COMP feed was required between day 0 and day 3 of culture.
[0116] 2.1.3. Appropriate feeding and sugar supplementation should be performed on days 4 and 6 of fed-batch culture. Specific fed-batch strategies are shown in Table 1:
[0117] Table 1
[0118]
[0119] 2.2. Detection of 96DWP Added Flow Expression Levels
[0120] 2.2.1. On day 8 of 96DWP feeding, an appropriate volume of cell suspension was aspirated using a 300μL pipette and centrifuged. The supernatant after centrifugation was transferred to a new 96-well plate (manufacturer: Corning, catalog number: 3599).
[0121] 2.2.2. Dilute the cell supernatant at an appropriate ratio, and transfer 200 μL of the diluted sample to an Octet detection plate. After pre-wetting a suitable sensor (e.g., ProA, manufacturer: Fortebio, catalog number: 18-5010), sort the expression levels using Octet's semi-quantitative screening method. Figure 2 and Figure 3 ).
[0122] 2.2.3. Select the protein extraction supernatant from the top 3-5 mixed cell lines and preliminarily screen the product quality using SDS-PAGE. Figure 4 and Figure 5 Based on the results of reducing SDS-PAGE (R-SDS-PAGE) and non-reducing SDS-PAGE (NR-SDS-PAGE) tests, 2-3 candidate minipool plants were selected for monoclonal plating.
[0123] 3. Minipool is expanded step by step.
[0124] 3.1. Before feeding the cells onto 96DWP feedstock on day 4, a portion of the cell suspension was transferred to fresh 96DWP feedstock and cultured for 3-4 days under the following conditions: 36.5±0.5℃, 5% CO2, 200-1100 rpm, 80-85% humidity, with a final culture volume of 100μL-1000μL. EX-CELL Advanced CHO Fed-batch medium was used. This amplification procedure advances the suspension amplification process to 96DWP feedstock compared to the conventional procedure.
[0125] 3.2. After analyzing the results of the 96DWP Fed-batch assay and SDS-PAGE assay in step 2.2, count 2-3 candidate minipool cell lines from the passages based on the assay results and expand them to 24DWP (manufacturer: Thomson, catalog number 931569-G-1x). Culture the cells at 36.5±0.5℃, 5% CO2, and 200-1100 rpm for a culture volume of 1-5 ml using CD CHO Fusion medium.
[0126] 3.3. After culturing in 24DWP for 3-5 days, cells with a viability of ≥85% can be used for subsequent monoclonal screening.
[0127] 4. Monoclonal screening and evaluation
[0128] 4.1. Single-cell plate
[0129] From the minipools obtained in step 3.3, three minipools were selected for monoclonal plating based on expression levels, cell growth, and SDS-PAGE results in the supernatant. Using a Namocell single-cell isolateer, the three minipools at 24DWP were screened for monoclonalization, and the cells were aliquoted at 1 cell / well. The A1 wells of each 96-well plate were loaded with 1000 cells / well as focusing wells for the Cell Metric monoclonal cell tracking imaging system. After aliquoting, the plates were incubated statically at 37°C in a 5% CO2 incubator for 2-3 weeks. During static incubation, monoclonal origin tracking was performed using the Cell Metric monoclonal cell tracking imaging system.
[0130] 4.2. Screening of monoclonal expression levels after static incubation is the same as step 1.2.
[0131] 4.3. Evaluation of expression levels in monoclonal deep-well plates is the same as in step 2.
[0132] 4.4. Single-clonal stepwise amplification
[0133] The selected 30-50 single clones were amplified stepwise using suspension culture in 96DWP, 24DWP, TPP shake tubes (manufacturer: TPP, catalog number: 87050) or 125mL shake flasks (manufacturer: Corning, catalog number: 431143). The specific operation steps are the same as those in step 3. minipool amplification.
[0134] 4.5. Evaluation of Monoclonal Shake Flasks
[0135] 4.5.1. Calculate the cell viability and density of the TOP30-50 monoclonal cell lines obtained in step 4.4. The cell viability should be above 90%, and the density between 2-5 × 10⁻⁶ cells / year. 6 When the cell count is 1000 cells / ml, the cells are cryopreserved.
[0136] 4.5.2. After cryopreserving the monoclonal cells, evaluate them by shaking flasks at a seeding density of 0.5-1 × 10⁻⁶. 6 The cells / ml volume was 20ml, the culture medium was EX-CELL Advanced CHO Fed-batch medium, and the feed was CellventoModiFeed prime COMP. When the cell density reached 3.0 × 10⁶ cells / ml... 6Cells were cultured in a fed-batch manner starting at 4% per 100 cells / mL, with glycogen added every other day. Residual glucose was maintained at 2-6 g / L during culture. Cells were harvested on day 18 or when cell viability approached 80%, and the supernatant was obtained by centrifugation. The supernatant was sent to the quality analysis department for HPLC-ProA determination of expression levels, and then sent to the purification department for one-step product purification. The harvested supernatant, purified protein, and controls were subjected to SDS-PAGE (Reduce & Non-reduce), with close monitoring for any abnormal bands.
[0137] 4.5.3. Based on the above results, the proteins obtained from the top 10 monoclonal antibodies were sent to the quality analysis department for comprehensive product quality evaluation. Based on product quality, expression levels, and cell growth, 3-5 monoclonal cell lines were selected for stability evaluation.
[0138] Comparative Example 1
[0139] This process originates from CHOZN. ® The GS- / - host cell manufacturer's instruction manual outlines the specific screening and amplification steps as follows:
[0140] 1. Minipool static screening
[0141] 1.1. Cell resuscitation preparation and electroporation are the same as step 1.1 in Example 1.
[0142] 1.2. Evaluation of Minipool expression levels after 7 days of resting.
[0143] 1.2.1. After static screening for 10-21 minutes, select cells with confluence ≥ 80% and passage them into new 96-well plates at a ratio of 1:4.
[0144] 1.2.2. Once the cells in the secondary passage plate have reached 80% confluence (expected in 3-5 days), add them at a 1:5 ratio to two new 96-well plates containing 200 μL of selective medium. One plate will be used as the culture plate, and the other will be used for endpoint detection to screen for minipool expression levels. Incubate the endpoint detection plate for 7 days and collect the supernatant for titer quantification.
[0145] 2. Minipool is expanded stepwise.
[0146] 2.1. Select TOP40-60 cells based on the screening results in 1.2.2, and amplify them into 24-well plates (manufacturer: Corning, catalog number: 3524) using a single-channel pipette, and incubate them statically for 3-5 days;
[0147] 2.2. After cell confluence exceeds 80%, expand to T25 cm⁻¹. 2In a culture flask (manufacturer: Corning, item number 3289), incubate at 37°C with 5% CO2 for 3-5 days.
[0148] 2.3. After cell confluence exceeds 80%, expand the cells into TPP shaker tubes or 125 mL shake flasks and incubate at 37°C with 5% CO2. 2， 130rpm (25mm).
[0149] 3. Minipool shake-bottle Fed-batch evaluation
[0150] 3.1. After the cells have recovered, the selected minipool is evaluated by shaking flask according to step 4.5.2 in Example 1, and the culture time is 10-18 days;
[0151] 3.2. The expression level of the supernatant after harvest was detected, 10 cell lines were selected for supernatant purification, and 3 minipool cells were selected for monoclonal plating based on the quality of the submitted samples.
[0152] 4. Monoclonal screening and preliminary evaluation
[0153] 4.1. The single-cell plate separation procedure is the same as step 4.1 in Example 1.
[0154] 4.2. Monoclonal static evaluation is the same as step 1.2 in Comparative Example 1.
[0155] 4.3. Step 2 of monoclonal stepwise amplification with the same pair as in Example 1.
[0156] 4.4. The evaluation of monoclonal shake flasks and the overall evaluation are the same as step 4.5 in Example 1.
[0157] Comparative analysis of results:
[0158] Figure 1 The present invention presents a comparative study of two technical processes for screening CHO cell monoclonal cell lines (i.e., Example 1 and Comparative Example 1). The technical process of Comparative Example 1 has a screening cycle of nearly 22 weeks, while the improved technical process of this application, i.e. Example 1, can obtain candidate monoclonal cell lines in only about 14 weeks. Compared with the technical process of Comparative Example 1, the method of this application shortens the monoclonal screening cycle by nearly 8 weeks.
[0159] This improved method incorporates deep-well plate flow-through evaluation in the minipool and monoclonal screening stages. Compared to conventional static cell evaluation methods, suspension cell evaluation more closely approximates the original state of engineered cell lines and significantly increases screening throughput while shortening the screening cycle. In the minipool stage, 96DWP expression levels were high and varied, effectively selecting superior minipools for the next stage of research. Results for antibody 1 and antibody 2 are shown below. Figure 2 A and B in the table. The expression level of 96DWP in the monoclonal stage was significantly higher than that in the minipool stage, and showed differential expression. Excellent monoclonal clones could be selected based on their monoclonal origin for further step-by-step amplification. The results of antibody 1 and antibody 2 are shown in the table below. Figure 3 A and B.
[0160] This improved method introduces SDS-PAGE detection at the 96DWP stage of minipool to determine product quality in advance, eliminating the time spent on Fed-batch after amplification to shake flasks, reducing passage increases caused by cell amplification, and accelerating project progress. The NR-SDS-PAGE results of cell supernatants for Antibody 1 and Antibody 2 are shown below. Figure 4 and Figure 5 Ultimately, for antibody 1, MP34 and MP39 minipools were selected for monoclonal antibody cloning, while for antibody 2, MP18, MP22, and MP28 minipools were selected. Compared to comparative example 1, the screening cycle was significantly shortened.
[0161] The improved technical route in this application optimizes the amplification process for monoclonal cells, employing a step-by-step amplification method using deep-well plate suspension amplification. Compared to the step-by-step static amplification in Comparative Example 1, the overall growth status and characteristics of the TOP20-30 monoclonal cell lines selected from the two antibody screenings after amplification show a significant improvement in uniformity. Figure 6 and Figure 7 As shown, regarding cell viability: suspension amplification of antibody 1 and antibody 2 monoclonal cell lines exhibited uniformly high viability (mean > 95%), while the viability of cell lines amplified by static incubation fluctuated significantly, with the viability of some monoclonal cells dropping below 90%. Figure 6 China A and Figure 7 (A); Regarding cell diameter: the monoclonal cell lines of antibody 1 and antibody 2 had smaller and more uniform diameters after suspension amplification, which expanded the space for later Fed-batch tolerance of cells ( Figure 6 China B and Figure 7 In terms of clumping rate, suspension amplification of antibody 1 and antibody 2 monoclonal cell lines also showed a decrease in uniform clumping rate compared to static amplification. Figure 6 C and Figure 7 (C)
[0162] like Figure 8 As shown, on antibody 1 in the monoclonal antibody project, the average expression level of monoclonal antibodies screened using the method of Comparative Example 1 was 6.4 g / L, while the average expression level of monoclonal antibodies screened using the improved method of this application was 9.1 g / L. Figure 9As shown, on antibody 2 of the asymmetric bispecific antibody project, the average expression level of monoclonal antibodies screened using the method of Comparative Example 1 was 3.4 g / L, while the average expression level of monoclonal antibodies screened using the improved method of this application was 4.9 g / L; the method of this application improved the average expression level of monoclonal antibodies by about 40% in both projects compared with the method of Comparative Example 1.
[0163] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.
[0164] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims, and the specification can be used to interpret the content of the claims.
Claims
1. A method for screening and amplifying a monoclonal cell line, characterized in that: The method comprises the following steps: S1: The exogenous vector containing the target gene and the GS gene is transferred into the host cell, and the positive clone cells are screened out. Among the positive clone cells, a mini cell pool with high expression of the target protein is screened out, and the number of the screened mini cell pool accounts for 20% to 30% of the total cell pool; S2: Perform suspension fed-batch culture and evaluation on the mini cell pools screened in step S1, screen out the top 3 to 5 mini cell pools in terms of target protein expression and quality, and then perform suspension stepwise amplification culture. Based on a comprehensive evaluation of expression, cell growth and product quality, screen out the top 2 to 3 mini cell pools; S3: Perform monoclonal sorting on the mini cell pool screened in step S2 to screen out monoclonal cells with high expression of the target protein, and the number of monoclonal cells screened out is 20%~30% of the total monoclonal cells; and S4: The monoclonal cells screened in step S3 are subjected to a first suspension fed-batch culture and evaluation, and after the monoclonal cells with the top 30 to 50 expression levels of the target protein are screened out, suspension stepwise amplification culture is performed, and then a second suspension fed-batch culture and evaluation is performed to screen out the monoclonal cell line.
2. The method according to claim 1, characterized in that In step S2, suspension fed-batch culture and evaluation are performed in a 96-well deep-well plate; Optionally, suspension fed-batch culture is performed at 36°C to 37°C, 4% to 6% CO2, 200rpm to 1100rpm, and 80% to 85% humidity; Optionally, the culture medium used for the suspension fed-batch culture includes one or more of EX-CELL Advanced CHO Fed-batch culture medium, ActiPro culture medium and CHO MaxY culture medium.
3. The method according to claim 1 or 2, characterized in that: In step S2, the evaluation includes evaluating the expression amount and quality of the target protein; Optionally, the step of evaluating the expression amount and quality of the target protein includes supplementing feed and sugar on the 4th and 6th days of the suspension fed-batch culture, collecting the cell supernatant on the 8th day to detect the expression amount of the target protein, evaluating the quality of the target protein in the supernatant of the top 3 to 5 mini cell pools in terms of expression, and screening the top 2 to 3 mini cell pools; Optionally, the feed comprises Cellvento ModiFeed prime COMP; Optionally, the sugar comprises glucose; Optionally, the feed is supplemented at 4% to 5% of the volume of the culture system, and glucose is supplemented to a concentration of 6 g / L to 8 g / L in the culture system; Optionally, the quality of the target protein is assessed by SDS-PAGE.
4. The method according to claim 1 or 2, characterized in that: In step S2, the step of suspension stepwise expansion culture includes expanding and culturing the top 2-3 mini cell pools screened in a cell culture apparatus, wherein the cell culture apparatus includes one or more of a 24-well deep well plate, a TPP shake tube or a shake flask; Optionally, the amplification culture is carried out under the conditions of 36°C to 37°C, 4% to 6% CO2, and 200rpm to 1100rpm; Optionally, the culture medium used for the expansion culture includes one or more of CD CHO Fusion culture medium, CD CHO culture medium and CDOptiCHO culture medium; Optionally, the cells are expanded and cultured until the cell viability is ≥ 85%.
5. The method according to claim 1 or 2, characterized in that: In step S3, after the monoclonal sorting, the steps of static culture and / or monoclonal origin tracking and confirmation are also included; Optionally, culturing at 36°C to 37°C and 4% to 6% CO2; Optionally, the static culture period is 2 to 3 weeks.
6. The method according to claim 1 or 2, characterized in that: In step S4, a 96-well deep-well plate is used for the first suspension fed-batch culture and evaluation; Optionally, the conditions of the first suspension fed-batch culture are the same as those of step S2; Optionally, the evaluation includes evaluating the expression level and quality of the target protein; Optionally, the step of evaluating the expression level and quality of the target protein includes feeding and sugar supplementation on the 4th and 6th days of the suspension fed-batch culture, collecting the cell supernatant on the 8th day to detect the expression level of the target protein, and screening out the top 30-50 monoclonal cells in terms of expression level.
7. The method according to claim 1 or 2, characterized in that: In step S4, the step of suspension stepwise expansion culture includes expanding and culturing the top 30-50 monoclonal cells screened in a cell culture apparatus, wherein the cell culture apparatus includes one or more of a 96DWP, a 24DWP, a TPP shake tube or a 125mL shake flask; Optionally, the amplification culture is carried out under the conditions of 36°C to 37°C, 4% to 6% CO2, and 200rpm to 1100rpm; Optionally, the culture medium used for the expansion culture includes one or more of CD CHO Fusion culture medium, CD CHO culture medium and CDOptiCHO culture medium; Optionally, expand the culture until the cell viability is ≥90% and / or the cell density is 2×10 6 cells / mL~5×10 6 cells / mL.
8. The method according to claim 1 or 2, characterized in that: In step S4, a second suspension fed-batch culture and evaluation is performed using a shake flask; Optionally, the second suspension fed-batch culture and evaluation steps include: The cells were cultured at 0.5×10 6 cells / ml~1×10 6 cells / ml were inoculated in a shake flask. When the cell density reached 2.5×10 6 cells / ml~3.0×10 6 cells / mL and fed-batch culture was started; and The cells were cultured until the cell viability was ≥80%, and the top 3-5 monoclonal cells were selected for stability evaluation based on one or more evaluations including cell growth, quality and expression of the target protein; Optionally, fed-batch culture is performed by supplementing 4% to 5% of the volume of the culture system every other day, and the sugar content is controlled at 2 g / L to 6 g / L during the culture.
9. The method according to claim 1 or 2, characterized in that: The host cell is a host cell in which the endogenous GS gene is knocked out; Optionally, the host cell includes CHO cells and mutant strains derived from CHO cells.
10. The method according to claim 1 or 2, characterized in that: The positive clone cell screening was performed using a medium without glutamine.