A method for high-throughput mismatch detection of double-antibody production recombinant cell strains

Abstract

The application discloses a method for high-throughput mismatch detection of double-antibody production recombinant cell strains. In the prior art, no report is found about detection of different light chains of double antibodies and application in cell strain development. The application provides a method for detecting mismatch of double antibodies in the double-antibody cell screening stage. Specifically, a sample to be detected is extracted, the contents of Kappa light chains and Lambda light chains in the double-antibody light chains are detected, and whether mismatch occurs is judged according to the ratio of the Kappa light chains and the Lambda light chains. The method can screen candidate cells with consistent half-antibody expression amounts, greatly improves the probability of obtaining candidate cells with correct assembly, and does not additionally increase the time cost of process development and screening processes.

Description

Technical Field

[0001] This invention relates to the field of antibody detection technology, specifically to a method for high-throughput mismatch detection of recombinant cell lines used in bispecific antibody production. Background Technology

[0002] Bispecific antibodies (BsAbs), often shortened to bispecific antibodies, possess two antigen-binding arms, allowing them to bind to two different antigen targets or different epitopes of the same target. Bispecific antibodies are used in the development of next-generation drugs for anti-tumor, anti-autoimmune diseases, and antiviral treatments. The "knob-in-hole" structure of bispecific antibodies is based on replacing a smaller amino acid with a larger one in the CH3 region of the antibody chain to form a "knob" structure, while simultaneously replacing multiple larger amino acids with smaller amino acids on the other chain to form a "hole" structure, altering the local spatial structure of the funnel (Fc). This funnel-in-hole design facilitates the correct assembly of the two heterologous antibody heavy chains. Modifications to the Fab or Fc based on the "knob-in-Hole" structure can add scFvs or nanobodies, thereby achieving targeted and therapeutic effects with bispecific or multi-antibody approaches.

[0003] In the development of bispecific antibody drugs, how to produce bispecific antibodies is a key concern for researchers. Currently, the most widely used method is to express monospecific antibodies in two different cell lines, isolate and then assemble them in vitro. However, this method is costly and difficult to obtain bispecific antibodies. Another method is to co-express two monospecific antibodies in a single cell. The advantage of cell line expression systems for bispecific antibodies is that using cell line expression systems for in vivo assembly and purification of the target protein can significantly reduce production costs, the workload of cell line screening, and the screening cycle. However, the main problem with this method is that while forming the target bispecific antibody, the random binding of light and heavy chains can also lead to the formation of various non-target bispecific antibody variants. Currently, there is no industry-relevant method for rapidly and efficiently detecting bispecific antibody mismatches in cell lines. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention develops an ELISA and FACS detection method that effectively distinguishes the expression of Kappa and Lambda haptens, thereby effectively detecting the expression levels of each hapten in bifunctional antibodies. This allows for the screening of bifunctional antibodies with consistent hapten expression, i.e., those with the correct structure. It is a novel method for detecting bifunctional antibody mismatches during the bifunctional antibody production cell screening stage.

[0005] This invention discloses a method for high-throughput mismatch detection of recombinant cell lines producing bispecific antibodies, comprising the following steps (1) and / or (2):

[0006] (1) ELISA method:

[0007] S1. Fix the primary antibody, add the supernatant obtained from culturing the cells to be tested, and incubate;

[0008] S2. Take the product from S1, add anti-Kappa light chain secondary antibody with recognition marker, and continue incubation.

[0009] S3. Take the product from S1, add anti-Lambda light chain secondary antibody with recognition marker, and continue incubation.

[0010] S4. Detect the identification marker signals in the products of S2 and S3 respectively, and determine whether there is a mismatch based on the ratio of Kappa light chain and Lambda light chain in the sample to be tested.

[0011] (2) FACS method:

[0012] S01. Pre-treat the cells to be tested to obtain a sample containing bispecific antibodies;

[0013] S02. Add anti-Kappa light chain secondary antibody with a first recognition marker and anti-Lambda light chain secondary antibody with a second recognition marker to the sample to be tested, and continue incubation;

[0014] S03. Detect and identify the marker signals respectively, and determine whether there is a mismatch based on the ratio of Kappa light chain and Lambda light chain in the sample to be tested;

[0015] The cells to be tested are recombinant cells that heterologously express the bispecific antibody encoding gene.

[0016] Furthermore, the primary antibody is an IgG antibody that can bind to the Fc fragment of an anti-IgG antibody.

[0017] Furthermore, the presence of extracellular mismatches was detected using ELISA, and the presence of intracellular mismatches was detected using FACS.

[0018] Furthermore, in step (1), the method also includes the detection of the Fc fragment content of the dual antibody, such as adding an anti-Fc secondary antibody with a recognition mark and incubating it with the product of S1, and detecting the signal of the recognition mark.

[0019] Furthermore, in step (1), to screen candidate cells containing fewer mismatches from a large number of cells, the OD is calculated. Kappa With OD Fc The ratio and OD Lambda With OD Fc The ratio, select OD Kappa / OD Fc >80% and OD Lambda / ODFc >50% of the cell lines.

[0020] Furthermore, in step (2), to screen candidate cells containing fewer mismatches from a large number of cells, the proportion of cells simultaneously labeled by the first recognition marker and the second recognition marker is calculated, and the cell line with the higher proportion is selected. Furthermore, in step (1), the recognition marker can be a recognizable enzyme, including but not limited to horseradish peroxidase. Accordingly, when detecting the recognition marker signal, a chromogenic substrate needs to be added for the reaction.

[0021] Furthermore, in step (2), the signal of the identification mark can be a fluorescent signal, and the fluorescent signals of the first identification mark and the second identification mark are different, such as the first identification mark and the second identification mark being selected from two of FITC, FAM, TET, PE, CY3, CY5, DAPI, etc.

[0022] Further, in step (1), the working concentration of the secondary antibody is: the working concentration of Kappa secondary antibody (HRP) is 0.3-1 μg / mL, and the working concentration of Lambda secondary antibody (HRP) is 0.25-1 μg / mL.

[0023] Further, in step (2), the working concentration of the secondary antibody is: the working concentration of the anti-Kappa light chain secondary antibody is 10-30 μL / 1×10 6 Cells; the working concentration of anti-Lambda light chain secondary antibody is 10-20 μL / 1×10 6 Most preferably, the optimal dosage of Kappa secondary antibody (FITC) is 10 μL / 1 × 10⁻⁶ cells. 6 Cells; The optimal dosage of Lambda secondary antibody (PE) is 20 μL / 1×10 6 cells.

[0024] Furthermore, the host cell of the recombinant cell can be any cell capable of producing antibodies, including but not limited to CHO series cells.

[0025] Furthermore, the heavy and light chains of the dual-antibody Fc segment (on the complementarity-determining region CH3) contain a knock-in-hole structure.

[0026] The beneficial effects of this invention are:

[0027] Antibody light chains (LCs) are classified into "Kappa light chains" and "Lambda light chains" based on differences in their structure and antigenicity of constant regions. Existing technologies can only predict the probability of correct assembly by the content of light chains with different structures within the same cell, failing to accurately reflect the correct assembly ratio. Furthermore, no relevant reports have provided a method to support the determination of correct bifunctional antibody structure during the screening of large numbers of cell lines. This invention aims to improve the efficiency of screening cells expressing bifunctional antibodies and to screen for bifunctional antibodies with correct structure. It screens cell lines with an expression ratio close to 1:1 by using different expression levels of LCs with different structures, thereby increasing the probability and efficiency of screening correctly expressed and assembled cell lines. Attached Figure Description

[0028] Figure 1 This is a schematic diagram of the MBS303 antibody structure.

[0029] Figure 2 The results of developing a mismatch method for ELISA showed the titer of the Kappa-HRP antibody.

[0030] Figure 3 The results of developing a mismatch method for ELISA, showing the titer of the Lambda-HRP antibody.

[0031] Figure 4 The results of developing a mismatch method for ELISA, showing the titer of the Lambda-HRP antibody.

[0032] Figure 5 The results show the mismatches in samples with additional Kappa light chain impurities detected using Lambda secondary antibody.

[0033] Figure 6 The results show the mismatches in samples with added Lambda light chain impurities detected using Kappa secondary antibody.

[0034] Figure 7 The selection criteria for detecting mismatches using the ELISA method.

[0035] Figure 8-10 The results of using the FACS method to detect mismatches. Detailed Implementation

[0036] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0037] The antibodies and cells used in this invention to verify the feasibility of the detection method are as follows:

[0038] Similar to the structure of bispecific antibodies, the schematic diagram of the antibody structure is as follows: Figure 1 .

[0039] MIL220 is a monoclonal antibody expressing the complete structure of Kappa LC; MIL221-3V4 is a monoclonal antibody expressing the complete structure of Lambda LC. The assembly of MIL220 and MIL221-3V4 expression products forms the correctly assembled all-antibody MBS303 (MBS303 has been disclosed in patents WO2022134645A1 and WO2023208016A1; the formation of Hole and Knob is based on the above patents). Details are as follows:

[0040] KappaLC LambdaLC Incomplete antibody MIL220hole MIL221-3V4Knob MBS303

[0041] This invention uses cell lines to express bispecific antibodies containing the Kappa LC+Lambda LC structure. The cell lines involved are: CHOK1 cell line MIL220 Cell with the MIL220 monoclonal antibody encoding gene introduced, CHOK1 cell line MIL221-3 V4 Cell with the MIL221-3 V4 monoclonal antibody encoding gene introduced, and CHOK1 cell line MBS303 Cell with the MBS303 bispecific antibody encoding gene introduced.

[0042] The materials and equipment involved in the detection method of this invention are as follows:

[0043] reagents factory Item number use Goat anti-human IgG antibody Sigma I1886-2ML ELISA mismatch method primary antibody Goat anti-human Kappa light chain secondary antibody (HRP) Thermo A18853 For ELISA mismatch detection Goat anti-human Lambda light chain secondary antibody (HRP) Thermo A56866-50UL For ELISA mismatch detection Goat anti-human IgG Fc cross-adsorption secondary antibody (HRP) Invitrogen 31413 Used for ELISA IgG antibody content detection Fixation / permeation solution kit BD 554715 For FACS cell treatment Mouse anti-human Lambda light chain secondary antibody (PE) Thermo MA1-10396 Used for FACS (Cellexperiment) Mouse anti-human Kappa light chain secondary antibody (FITC) Thermo MA1-10388 Used for FACS (Cellexperiment)

[0044] equipment factory model ELISA reader Molecular Devices VersaMax Flow cytometer BD Calibur Inverted microscope COIC XDS-1B Cell counter CountStar IC1000

[0045] The following examples demonstrate the use of ELISA and FACS methods to detect mismatches in bispecific antibodies against different LC structures. The ELISA method confirms the mismatch ratio by detecting the amount of antibodies secreted into the extracellular space at different LC structures; the FACS method confirms the mismatch ratio by detecting the amount of antibodies at different LC structures through cell perforation.

[0046] Example 1: ELISA detection

[0047] 1. ELISA experimental procedure:

[0048] (1) Envelope

[0049] The total amount of primary antibody GAH IgG in each ELISA plate is 15 μg. Calculate the required volume of GAH IgG based on the number of plates to be coated and the GAH IgG concentration. Dilute the primary antibody to 1.5 μg / mL with coating buffer, apply 100 μL / well to each plate, and incubate at 37℃ for 1 hour. After coating, place the ELISA plates in a plate washer for washing.

[0050] (2) Closed

[0051] Skim milk powder or BSA was diluted to 50 mg / L with 1×PBS (20×PBS was diluted to 1× with purified water), and 200 μL was added per well for blocking. The plates were incubated at 37°C for 1 hour or at room temperature for 2 hours. After blocking, the plates were washed in a plate washer.

[0052] Inject 300 μL of washing buffer per well into the ELISA plate and store it at 4°C for later use (to prevent liquid evaporation, it should be sealed in a self-sealing bag and labeled with the preparer and preparation time). The storage period for coated ELISA plates should not exceed one month.

[0053] (3) Sampling and dilution

[0054] Sampling must be performed in a laminar flow hood, while dilution must be carried out in a non-sterile environment.

[0055] (4) Adding samples

[0056] Add 100 μL of diluted standard / sample per well to the coated ELISA plate and incubate at 37°C for 1 h or at room temperature for 2 h. The samples are MBS303 reference, MIL220 purified protein, and MIL221-3 V4 purified protein, respectively.

[0057] MBS303 reference standard, with a concentration of 15 mg / mL, was serially diluted 1000 times to 0.015 μg / μL;

[0058] The protein concentration purified by MIL220 is 1.11 μg / μL, which can be serially diluted 100-fold or 10000-fold to 0.0111 μg / μL or 0.00111 μg / μL.

[0059] The purified protein concentration of MIL221-3V4 is 2.76 μg / μL. Serial dilutions can be performed 100-fold or 10000-fold to 0.0276 μg / μL or 0.00276 μg / μL.

[0060] Add the sample according to the required ratio, and make up the difference with PBS if the amount is less than 100 μL.

[0061] (5) Secondary Antibody

[0062] Wash the plate and dilute the Kappa light chain secondary antibody (HRP) with PBS to 0.3 μg / mL. Add 100 μL / well and incubate at 37°C for 30 min or at room temperature for 40 min.

[0063] Alternatively, wash the plate and dilute the Lambda light chain secondary antibody (HRP) with PBS to 0.5 μg / mL, add 100 μL / well, and incubate at 37°C for 30 min or at room temperature for 40 min.

[0064] Alternatively, wash the plate and dilute the goat anti-human IgG Fc cross-adsorption secondary antibody (HRP) with PBS at a ratio of 1:7000, add 100 μL / well, and incubate at 37°C for 30 min or at room temperature for 40 min.

[0065] (6) Color development and termination

[0066] Wash the plate and add 50 μL of TMB substrate development solution per well. Incubate at room temperature for 1-5 min, then add 50 μL of stop solution per well to terminate the reaction. Detect the OD value at 450 nm.

[0067] 2. Secondary antibody titer

[0068] With the MBS303 reference standard added at a fixed level of 1 μg / mL, the minimum antibody concentrations for Kappa HRP and Lambda HRP antibodies were tested. Based on hook effect analysis, the minimum antibody concentration at which the fluorescence intensity was highest was selected. The results are shown below. Figure 2-4 The optimal detection concentration of goat anti-human Kappa secondary antibody HRP is 0.3 μg / mL; the R value of the goat anti-human Lambda secondary antibody HRP binding to the standard curve is... 2 Through offline analysis, the detection concentration was finally determined to be 0.5 μg / mL.

[0069]

[0070]

[0071] With a fixed MBS303 reference addition amount of 0.05 μg, experiments were conducted by sequentially adding different amounts of haptened antibodies (0.00625 μg, 0.0125 μg, 0.025 μg, 0.05 μg, 0.1 μg, 0.2 μg, and 0.4 μg of MIL220 and / or MIL221-3 V4). This method is considered acceptable if it reflects a regular change in the proportion of impurities in the system (the additionally added MIL220 or MIL221-3 V4, which are either incorrectly assembled or in a purely free state, are considered impurities for MBS303).

[0072]

[0073]

[0074] 3. ELISA results

[0075] (1) When additional MIL220 (Kappa LC) impurity was added, the OD value of samples containing excessive MIL220 (Kappa LC) detected by Kappa secondary antibody showed no significant change. However, when samples containing excessive MIL220 (Kappa LC) were detected by Lambda secondary antibody, the OD value decreased with the increase of the proportion of free MIL220 (Kappa LC). When the proportion of MIL220 (Kappa LC) was >20% (additional 0.0125 μg Kappa LC), the OD value showed a decreasing trend. When the proportion of MIL220 (Kappa LC) was >30% (additional 0.025 μg Kappa LC), the OD value showed a significant decreasing trend. Figure 5 ).

[0076] MBS303(μg) 0.05 0.05 0.05 0.05 0.05 0.05 0.05 MIL220 Impurity Addition Amount (μg) 0.00625 0.01250 0.02500 0.05000 0.10000 0.20000 0.40000 Total protein concentration (μg) 0.05625 0.0625 0.075 0.1 0.15 0.25 0.45

[0077] (2) When additional MIL221-3V4 (Lambda LC) impurity was added, the OD value of samples containing excessive MIL221-3V4 (Lambda LC) detected with Lambda secondary antibody showed no significant change. However, when samples containing excessive MIL221-3V4 (Lambda LC) were detected with Kappa secondary antibody, the OD value decreased with increasing proportion of free MIL221-3V4 (Lambda LC). When the proportion of MIL221-3V4 (Lambda LC) was >30% (additional 0.025 μg Lambda LC), the OD value showed a decreasing trend. When the proportion of MIL221 (Lambda LC) was >50% (additional 0.05 μg Lambda LC), the decreasing trend of the OD value was significant. Figure 6 ).

[0078]

[0079]

[0080] (3) Setting screening criteria:

[0081] Based on the results of Fc(HRP) secondary antibody detection, OD Kappa :OD Fc OD Lambda :OD Fc The form determines the screening criteria, when OD Kappa :OD Fc When >80%, samples containing an additional 60% free MIL221 (Lambda LC) can be detected; when OD Lambda :OD FcWhen the concentration is >50%, samples containing an additional 60% free MIL220 (Kappa LC) can be detected. Combining the two detection results allows for the selection of cell lines with a higher correct proportion. Figure 7 ).

[0082] Example 2: FACS detection

[0083] (1) Cell Treatment

[0084] Using Fixation / Permeabilization Solution Kitwith BD GolgiStop TM The kit is used for cell pretreatment.

[0085] The quantity is 2×10 6 Add cells to a 6-well plate, bring the total volume to 3 mL with culture medium, add 2 μL of Protein Transport Inhibitor, mix well, and incubate at 37°C for 2-4 hours.

[0086] After incubation, mix the cells and transfer them to a 1.5 mL EP tube for centrifugation at 7000 rpm for 30 seconds.

[0087] Discard the supernatant, add 500 μL of washing buffer, wash the cells once, and centrifuge at 7000 rpm for 30 seconds.

[0088] Discard the supernatant, add 100 μL of BD Fc block to the cell pellet to resuspend the cells, incubate at 4°C for 5 min, and then centrifuge at 7000 pm for 30 s.

[0089] Discard the supernatant, add 500 μL of washing buffer, wash the cells 3 times, and centrifuge at 7000 rpm for 30 seconds.

[0090] Add 100 μL of BD Cytofix / Cytoperm, mix well, incubate at 4°C for 20 min, and then centrifuge at 7000 rpm for 30 s.

[0091] Discard the supernatant, add 500 μL of washing buffer, wash the cells 3 times, and centrifuge at 7000 rpm for 30 seconds.

[0092] Add 100 μL of secondary antibody, mix well, incubate at 4°C in the dark for 30 min, then centrifuge at 7000 pm for 30 s.

[0093] Discard the supernatant, add 500 μL of washing buffer, wash the cells 3 times, and centrifuge at 7000 rpm for 30 seconds.

[0094] Add 500 μL of washing buffer, gently resuspend and mix the cells to form single cells, transfer them to a flow cytometry sample tube for detection.

[0095] (2) Antibody titer

[0096] Based on the counting results, take 1×10 6 Cells (validated, low mismatch ratio cells) were incubated with antibodies, and Kappa and Lambda antibody concentrations were evaluated at 10, 20, and 30 μL / 1×10⁶, respectively. 6 The results showed that the optimal dosage of Kappa secondary antibody (FITC) was 10 μL / 1×10 cells. 6 The optimal dosage of Lambda secondary antibody (PE) for cells is 20 μL / 1×10⁻⁶. 6 cells.

[0097] (3) FACS detection

[0098] MBS303 cells were examined to determine whether Kappa and Lambda LC signals could be detected simultaneously, reflecting pool cell mismatch.

[0099]

[0100]

[0101] (4) Results: When different proportions of mismatched products were added, different fluorescence signals were observed compared to the target cells. Figure 8-10 This study demonstrated that the method can be used for relatively quantitative mismatch rates, but cell cluster separation is not significant when the mismatch content is low (below 10%).

[0102]

[0103] ① Controls: MBS303 cells were treated simultaneously with Kappa secondary antibody alone (green), Lambda secondary antibody alone (red), and Kappa + Lambda secondary antibody (orange).

[0104] Experimental group: Added different proportions of MIL220Cell (Kappa impurity)

[0105] Conclusion: The presence of stained cell clusters was clearly observed in the FL1-H green fluorescent channel+ (Kappa).

[0106] ② Controls: MBS303 cells were treated simultaneously with Kappa secondary antibody alone (green), Lambda secondary antibody alone (red), and Kappa + Lambda secondary antibody (orange).

[0107] Experimental group: Added different proportions of MIL221-3V4Cell (Lambda impurity)

[0108] Conclusion: The presence of stained cell clusters was clearly observed in the FL2-H red fluorescent channel+ (Lambda).

[0109] ③ Controls: MBS303 cells were treated simultaneously with Kappa secondary antibody alone (green), Lambda secondary antibody alone (red), and Kappa + Lambda secondary antibody (orange).

[0110] Experimental group: MIL220 Cell and MIL221-3V4 Cell (Kappa+Lambda impurities) were added in different proportions.

[0111] Conclusion: The presence of stained cell clusters was clearly observed in FL1-H green fluorescent channel+ (Kappa) and FL2-H red fluorescent channel+ (Lambda).

[0112] The above-described embodiments are merely preferred embodiments provided to fully illustrate the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention. The scope of protection of the present invention is defined by the claims.

Claims

1. A method for high-throughput mismatch detection of recombinant cell strains for bispecific production, characterized in that, The double antibody is full anti-MBS303, comprising steps (1) and (2), whether there is a mismatch in the cell is detected by ELISA method, and whether there is a mismatch in the cell is detected by FACS method: (1) ELISA method: S1, fixing the first antibody GAH IgG, adding the supernatant obtained by culturing the cells to be tested, and incubating; S2, taking the product of S1, adding an anti-Kappa light chain secondary antibody with a recognition marker to it, and continuing to incubate; wherein the anti-Kappa light chain secondary antibody is a goat anti-human Kappa light chain secondary antibody, and the working concentration is 0.3 μg / mL; S3, taking the product of S1, adding an anti-Lambda light chain secondary antibody with a recognition marker to it, and continuing to incubate; wherein the anti-Lambda light chain secondary antibody is a goat anti-human Lambda light chain secondary antibody, and the working concentration is 0.5 μg / mL; S4, detecting the recognition marker signals in the products of S2 and S3 respectively, and judging whether there is a mismatch according to the proportion of Kappa light chain and Lambda light chain in the sample to be tested; The method also includes detecting the Fc fragment content of the dual antibody, specifically by incubating the product of S1 with an anti-Fc secondary antibody bearing a recognition marker, detecting the signal of the recognition marker, and calculating the OD. Kappa With OD Fc The ratio and OD Lambda With OD Fc The ratio, select OD Kappa / OD Fc >80% and OD Lambda / OD Fc >50% of the cell lines were used to obtain recombinant cell lines with a high proportion of correctly matched cells; among them, the anti-Fc secondary antibody was a goat anti-human IgG Fc cross-adsorption secondary antibody. (2) FACS method: S01, pretreating the cells to be tested to obtain a sample to be tested; the pretreatment is cell punching; S02. Add anti-Kappa light chain secondary antibody with the first recognition marker and anti-Lambda light chain secondary antibody with the second recognition marker to the sample to be tested, and continue incubation; wherein, the anti-Kappa light chain secondary antibody is mouse anti-human Kappa light chain secondary antibody, and the working concentration is 10 μL / 1×10 6 Cells; the anti-Lambda light chain secondary antibody is a mouse anti-human Lambda light chain secondary antibody with a working concentration of 20 μL / 1×10⁻⁶. 6 cells; S03, detecting the recognition marker signals respectively, and judging whether there is a mismatch according to the proportion of Kappa light chain and Lambda light chain in the sample to be tested; Calculating the proportion of cells labeled by the first recognition marker and the second recognition marker at the same time, and selecting the cell strain with a higher proportion; The cell to be tested is a recombinant cell heterogeneously expressing a double antibody encoding gene, and the double antibody has a Knob in Hole structure.

2. The method of claim 1, wherein, In step (1), the recognition marker includes horseradish peroxidase.

3. The method of claim 1, wherein, In step (2), the signal of the recognition marker is a fluorescent signal, and the fluorescent signals of the first recognition marker and the second recognition marker are different.

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