A screening method for engineering cell lines in the research and development process of antibody drugs

Abstract

The present invention belongs to the technical field of analytical chemistry, and specifically relates to a method for screening engineering cell lines in the research and development process of antibody drugs. The present invention uses magnetic beads immobilized with specific ligands / receptors to perform one-step purification on the fermentation broth of engineering cells, specifically extracting antibody components with strong affinity for specific ligands / receptors, rejecting antibodies with poor affinity during purification, and measuring the expression levels of high-quality antibodies by different engineering cell lines; SEC is used to analyze the protein aggregation degree of the extracted antibody components with strong affinity; samples with high antibody expression level, strong affinity, and low aggregation degree are directly reflected as higher monomer peaks on the SEC chromatogram. The corresponding engineering cell lines are evaluated based on the monomer peak area values, and cell lines with development potential are quickly screened out from a large number of engineering cell lines. Compared with the traditional screening mode of engineering cell lines, the present invention significantly shortens the analysis time and reduces the analysis cost.

Description

Technical Field

[0001] The present invention belongs to the technical field of analytical chemistry, and particularly relates to a screening method for engineering cell lines in the R & D process of antibody drugs. Background Art

[0002] In recent years, antibody drugs have shown unique advantages and made great progress in the fields of diseases such as tumors, autoimmune diseases, and metabolism, and are also the fastest-growing development direction in the pharmaceutical field. As macromolecular proteins, the production process of antibody drugs is all expressed by monoclonal engineering cell lines. Therefore, obtaining engineering cell lines that can express high-quality antibody molecules in large quantities is crucial to the success of the entire R & D project. In the actual R & D process, it is necessary to first obtain an engineering cell pool stably transfected with the target gene, and then perform monoclonal screening to obtain hundreds or even thousands of monoclonal engineering cell lines. After analyzing and ranking the key quality attributes (CQAs) of the antibodies in the fermentation broth of these engineering cell lines, 1-10 engineering cell lines with development potential are selected based on the ranking to carry out subsequent R & D work. For most antibody drugs, the early engineering cell line screening process mainly focuses on three CQAs: ① antibody expression level; ② affinity of the antibody for a specific ligand / receptor (the strength of in vivo binding of the antibody to the corresponding target, Fc receptor, etc., and the stronger the binding, the better the in vivo efficacy); ③ aggregation degree of the antibody molecule (antibody multimers have immunogenicity). And these three CQAs need to be completed using at least three different analytical and testing methods, which not only have high analysis costs, time-consuming and laborious, but also lead to an extended analysis cycle due to the introduction of chromatographic purification steps. Thus, it is crucial to develop a screening scheme for engineering cell lines with simple operation procedures, low costs, and short analysis cycles to promote the R & D process of antibody drugs. Summary of the Invention

[0003] The purpose of the present invention is to provide a screening method for engineering cell lines in the R & D process of antibody drugs with a shortened analysis cycle and a simple operation procedure.

[0004] The screening method for engineering cell lines in the R & D process of antibody drugs provided by the present invention uses magnetic beads fixed with a specific ligand / receptor to perform one-step purification on the fermentation broth of engineering cells, specifically extracting antibody components with strong affinity for the specific ligand / receptor, directly discarding antibodies with poor affinity during purification, and directly measuring the expression levels of different engineering cell lines for "high-quality antibodies"; molecular exclusion chromatography (SEC) is used to analyze the protein aggregation degree of the extracted antibody components with strong affinity; samples with high antibody expression levels, strong affinities, and low aggregation degrees will directly be reflected as higher monomer peaks on the SEC chromatogram (the technical principle is shown in Figure 3 、 Figure 4 ), so, based on the monomer peak area values, the corresponding engineering cell lines are evaluated, and potential cell lines for development are quickly screened out from a large number of engineering cell lines.

[0005] The screening method for engineering cell lines in the antibody drug R & D process provided by the present invention specifically comprises the following steps:

[0006] (1) Obtaining magnetic beads immobilized with specific ligands / receptors: Select magnetic beads immobilized with specific ligands or receptors according to the target, characteristics, and expected quality critical attributes of the target antibody; the ligand or receptor can bind to the target antibody, and the binding affinity between the two affects the final quality or efficacy of the antibody drug; place them in a buffer solution;

[0007] The types of the ligand or receptor include but are not limited to antibody target proteins, Fc receptor proteins, etc.;

[0008] The magnetic beads immobilized with specific ligands / receptors can be directly purchased as commercial magnetic beads or prepared by oneself, such as by coupling or other means to load specific ligands / receptors on the magnetic beads; the particle size of the magnetic beads is not limited, usually 100 - 500 microns;

[0009] (2) Processing the engineering cell fermentation broth sample with the magnetic beads immobilized with specific ligands / receptors: Add an excessive amount of magnetic beads immobilized with specific ligands / receptors into a container, remove the buffer solution used to store the magnetic beads by applying an external magnetic field; add the engineering cell fermentation broth from which cells and cell debris have been removed by centrifugation, and incubate for 5 - 240 minutes while gently shaking at 4 - 37 °C; remove the supernatant of the fermentation broth under the action of an external magnetic field, wash the material 1 - 4 times with a buffer solution with a neutral pH, and remove the washing solution; add an acidic buffer solution with a pH of 1 - 5 as the eluent for the target antibody, and incubate for 0.5 - 60 minutes while gently shaking at 4 - 37 °C; collect the eluent under the action of an external magnetic field and neutralize it to a neutral pH;

[0010] (3) Analyzing the antibody aggregation degree in the eluent using size - exclusion chromatography (SEC): Filter the eluent obtained in step (2) through a 0.22 - 0.45 μm aqueous filter membrane, and then inject it into a high - performance liquid chromatography system for analysis; the chromatographic column is an SEC column, the mobile phase is a buffer solution with a neutral pH and a certain ionic strength, and isocratically elute to 1 times the column volume or more; using the retention time of the standard antibody as a control, integrate the monomer peak of the antibody molecules in the obtained chromatogram and record the value;

[0011] (4) Screening of engineering cell lines: Sort each engineering cell line according to the order of the monomer peak area values obtained after each engineering cell line is processed through the above steps (1) - (3) from high to low; select 1 - 20 engineering cell lines from the front to the back according to actual needs as engineering cell lines with development potential (the comprehensive quality of the three attributes of antibody expression level, antibody aggregation degree, and antibody affinity with specific ligands / receptors is relatively good), and carry out subsequent R & D work.

[0012] Further:

[0013] In step (1), the magnetic beads immobilized with specific ligand / receptor are coated with a hydrophilic outer layer on their surfaces to avoid non-specific adsorption during use.

[0014] In step (2), the magnetic beads immobilized with specific ligand / receptor are used to process the engineered cell fermentation broth sample. For the engineered cell fermentation broth with a volume of V , the amount of the magnetic beads immobilized with specific ligand / receptor used can be calculated by using including but not limited to the following two methods:

[0015] (1) Calculate the protein loading of the ligand / receptor on the surface of the magnetic beads according to the product information of the magnetic beads immobilized with specific ligand / receptor (for example, the product information data provided by the supplier for commercially available magnetic beads immobilized with specific ligand / receptor, or the information data of self-prepared products) L = m (配体 / 受体) / m 磁珠 , combine the theoretical mass ratio R = m (配体 / 受体) / m 抗体 of the binding of this ligand / receptor to the target antibody, estimate the possible maximum protein expression level in the fermentation broth according to the species category of the engineered cells T = m 抗体 / V 发酵液 , calculate the theoretical maximum amount of magnetic beads required for the antibody in each volume of V of the fermentation broth: m max = (R × T) / L . Process the engineered cell fermentation broth sample with a 2 - 10 times m max amount;

[0016] (2) Take 5 - 20 fermentation broth samples, mix them evenly and divide them into multiple equal parts, each with a volume of V , add different amounts of the magnetic beads immobilized with specific ligand / receptor to them respectively, process according to steps (1) - (3), and record the amount of magnetic beads used when the monomer peak area in the fermentation broth with a volume of V reaches the highest m max . Process the engineered cell fermentation broth sample with a 2 - 10 times m max amount.

[0017] In step (2), the volume of the acidic eluent used should be less than or equal to the volume of the original engineered cell fermentation broth to avoid introducing additional dilution during the process of extracting the antibody.

[0018] In the screening method of engineering cell lines in the antibody drug R & D process designed by the present invention, the technical principle is as follows: magnetic beads immobilized with specific ligands / receptors are directly added to the engineering cell fermentation broth. Since the affinities of different antibody heteromers for the same receptor / ligand vary greatly (varying by 2 - 10 3 times, calculated by the dissociation constant K D ), under certain conditions, the magnetic beads only selectively bind to the antibody components with higher affinity for them; by applying a magnetic field to separate the magnetic beads from the fermentation broth matrix, it is possible to achieve high-selectivity and rapid extraction and purification of the antibody components with stronger affinity for specific receptors / ligands ( Figure 3 ). Subsequently, the eluate (purified antibody solution) can be directly injected into the liquid chromatography system to analyze its multimer content by SEC. Samples with high antibody expression, strong affinity, and low aggregation degree will be directly reflected as higher monomer peaks on the SEC chromatogram. Therefore, the development potential of each engineering cell line can be ranked according to the monomer peak area, thus quickly completing the screening work ( Figure 4 ).

[0019] The technical features and advantages of the present invention are as follows:

[0020] By using magnetic beads immobilized with specific ligands / receptors, the affinity analysis of the antibody for the specific ligand / receptor and the extraction and purification of the antibody are simultaneously achieved in one step. Combining the subsequent multimer analysis results, the comprehensive quality of the antibody drugs expressed by different engineering cells can be intuitively obtained. Not only are the experimental results more intuitive, but also the traditional complex and cumbersome chromatography purification operations are abandoned. The analysis process is shortened to within 0.5 days, only 1 / 8 - 1 / 4 of the traditional method, which can greatly shorten the R & D cycle of antibody drugs. Brief Description of the Drawings

[0021] Figure 1 It is the traditional method for screening engineering cell lines.

[0022] Figure 2 It is the screening process of engineering cell lines by the method of the present invention.

[0023] Figure 3 It is the schematic diagram of the operation process of directly treating the engineering cell fermentation broth sample with magnetic beads immobilized with specific ligands / receptors in the present invention.

[0024] Figure 4 It is the schematic diagram of the technical principle of the method of the present invention.

[0025] Figure 5This is a representative chromatogram (HCCF: cell fermentation broth) of the analytical method for rapidly screening engineering cell lines with high expression of antibodies that have strong affinity for FcγRIIIa (strong ADCC effect and low aggregation). Among them, a is the SEC chromatogram obtained by treating blank phosphate buffer with magnetic beads immobilized with specific ligands / receptors, demonstrating that the specific ligands / receptors immobilized on the surface of the magnetic beads during use do not produce detectable leaching; b is the SEC chromatogram obtained by treating blank cell fermentation broth with magnetic beads immobilized with specific ligands / receptors; c is the SEC chromatogram obtained by treating cell fermentation broth containing the target antibody with magnetic beads immobilized with specific ligands / receptors; d is the SEC chromatogram obtained by directly injecting a pure standard antibody solution. By comprehensively comparing b, c, and d, it can be proven that the relevant impurity peaks in the treated cell fermentation broth do not affect the detection of the target antibody. Embodiment

[0026] The following examples further illustrate the present invention, but do not limit the scope of the present invention. Example

[0027] An analytical method for rapidly screening engineering cell lines with high expression of antibodies that have strong affinity for FcγRIIIa (strong ADCC effect) and low aggregation.

[0028] (1) Magnetic beads immobilized with FcγRIIIa protein were synthesized in the laboratory. These magnetic beads can specifically bind to the Fc domain of IgG1-type antibodies under neutral conditions and dissociate under acidic conditions.

[0029] (2) Extracting antibody components with strong affinity for FcγRIIIa from the engineering cell fermentation broth using immobilized FcγRIIIa magnetic beads: Add a suspension containing 20 mg of FcγRIIIa magnetic beads to an EP tube, apply an external magnetic field to separate the magnetic beads and remove the supernatant of the suspension; add 1 mL of the fermentation broth of the monoclonal engineering cell line (the precipitate has been removed by centrifugation), incubate at 37 °C for 20 minutes and maintain a shaking intensity of 500 rpm to allow the antibodies with strong affinity for FcγRIIIa in the fermentation broth to fully bind to the magnetic beads; after incubation, remove the supernatant of the fermentation broth under the action of an external magnetic field, wash the material with 20 mM phosphate buffer (pH = 7.4) containing 0.02% Tween-20 and then remove the washing solution; add 0.1 mL of 10 mM glycine-hydrochloric acid buffer (pH = 3.5) containing 100 mM sodium chloride, incubate at 25 °C for 5 minutes and maintain a shaking intensity of 500 rpm to elute the antibody molecules from the magnetic beads; apply an external magnetic field to collect the eluate and filter it through a 0.22 μm aqueous filter membrane.

[0030] (3)Perform antibody aggregation degree analysis: Subject the filtered eluate to SEC analysis and record the antibody monomer peak area. The chromatographic conditions are as follows:

[0031] Chromatograph: Agilent 1260 high performance liquid chromatograph;

[0032] Chromatographic column: Welch Xtimate® SEC-300 column (4.6*250 mm, 3 μm);

[0033] Mobile phase: Aqueous solution of 20 mM phosphate and 200 mM sodium chloride, pH 7.0;

[0034] Column temperature: 30 °C;

[0035] Analysis time: 12.5 minutes;

[0036] Flow rate: 0.6 mL / min;

[0037] Detection wavelength: 280 nm;

[0038] Injection volume: 80 μL.

[0039] (4)Compare the numerical values of the SEC monomer peak areas obtained after all the samples to be tested are processed through the above steps, and rank them; among them, the sample with the highest SEC monomer peak area value corresponds to the engineering cell line that highly expresses an antibody with strong affinity for FcγRIIIa (strong ADCC effect) and low aggregation degree in all the original cell fermentation broth samples to be tested. See Figure 5 as shown; where, a is the SEC chromatogram obtained by treating blank phosphate buffer solution with magnetic beads immobilized with a specific ligand / receptor, proving that the specific ligand / receptor immobilized on the surface of the magnetic beads will not produce detectable leaching during use; b is the SEC chromatogram obtained by treating blank cell fermentation broth with magnetic beads immobilized with a specific ligand / receptor; c is the SEC chromatogram obtained by treating cell fermentation broth containing the target antibody with magnetic beads immobilized with a specific ligand / receptor; d is the SEC chromatogram obtained by directly injecting a pure standard antibody solution. By comprehensively comparing b, c, and d, it can be proved that the relevant impurity peaks in the treated cell fermentation broth do not affect the detection of the target antibody.

Claims

1. A screening method for engineering cell lines in the R & D process of antibody drugs, characterized in that, One-step purification of the engineered cell fermentation broth is carried out using magnetic beads immobilized with specific ligands / receptors, specifically extracting the antibody components with strong affinity for the specific ligands / receptors, directly discarding the antibodies with poor affinity during purification, and directly measuring the expression levels of high-quality antibodies by different engineered cell lines; molecular exclusion chromatography (SEC) is used to analyze the protein aggregation degree of the extracted antibody components with strong affinity; samples with high antibody expression, strong affinity, and low aggregation degree will directly manifest as higher monomer peaks on the SEC chromatogram. Thus, the corresponding engineered cell lines are evaluated based on the monomer peak area values, and cell lines with development potential are quickly screened out from a large number of engineered cell lines; the specific steps are as follows: (1) Obtain magnetic beads immobilized with specific ligands / receptors: Select magnetic beads immobilized with specific ligands or receptors according to the target, characteristics, and expected quality critical attributes of the target antibody, and store them in a buffer; the ligand or receptor can bind to the target antibody, and the binding affinity between the two affects the final quality or efficacy of the antibody drug; (2) Treat the engineered cell fermentation broth sample with magnetic beads immobilized with specific ligands / receptors: Add an excessive amount of magnetic beads immobilized with specific ligands / receptors into a container, remove the buffer used to store the magnetic beads by applying an external magnetic field; add the engineered cell fermentation broth from which cells and cell debris have been removed by centrifugation, and incubate for 5 - 240 minutes while shaking at 4 - 37 °C; remove the fermentation broth supernatant under the action of an external magnetic field, wash the material 1 - 4 times with a buffer with a neutral pH, and remove the washing solution; add an acidic buffer with a pH of 1 - 5 as the eluent for the target antibody, and incubate for 0.5 - 60 minutes while shaking at 4 - 37 °C; collect the eluate under the action of an external magnetic field and neutralize it to a neutral pH; (3) Analyze the antibody aggregation degree in the eluate using SEC: Filter the eluate obtained in step (2) through a 0.22 - 0.45 μm aqueous filter membrane, and then inject it into a high-performance liquid chromatography system for analysis; the chromatographic column is an SEC column, the mobile phase is a buffer with a neutral pH and ions, and isocratic elution is carried out to 1 column volume or more; using the retention time of the standard antibody as a control, integrate the monomer peaks of the antibody molecules in the obtained chromatogram and record the values; (4) Screening of engineered cell lines: Each engineered cell line is treated through the above steps (1) - (3), and the engineered cell lines are sorted in descending order of the obtained monomer peak area values; Select 1 - 20 engineered cell lines ranked at the front according to actual needs as engineered cell lines with development potential, and carry out subsequent R & D work.

2. The screening method of the engineered cell line in the antibody drug R & D process according to claim 1, characterized in that The ligand or receptor described in step (1) is an antibody target protein or an Fc receptor protein.

3. The screening method of the engineered cell line in the antibody drug R & D process according to claim 1, wherein The magnetic beads immobilized with specific ligands / receptors described in step (1) have a hydrophilic outer layer to avoid non-specific adsorption during use.

4. The screening method of the engineered cell line in the antibody drug R & D process according to claim 1, wherein In step (2), the magnetic beads immobilized with specific ligands / receptors are used to process the engineered cell fermentation broth sample, where the volume of the V engineered cell fermentation broth, and the calculation method for the amount of magnetic beads immobilized with specific ligands / receptors required is as follows: Take 5 to 20 fermentation broth samples, mix them evenly and divide them into multiple portions, each with a volume of V , add different amounts of magnetic beads immobilized with specific ligands / receptors to each of them respectively, process according to steps (1)-(3), and record the amount of magnetic beads used when the monomer peak area in the fermentation broth with a volume of V reaches the highest m max ; process the fermentation broth samples of engineering cells with a dosage of 2 to 10 times that of m max .

5. The screening method of the engineered cell line in the antibody drug R & D process according to claim 2, characterized in that: The volume of the acidic eluent used in step (2) is less than or equal to the volume of the original engineered cell fermentation broth to avoid introducing additional dilution during the antibody extraction process.

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