A protein a purification preservation method for an epidermal growth factor receptor bispecific antibody

Abstract

The application discloses a Protein A purification and storage method for an epidermal growth factor receptor bispecific antibody, uses Protein A as an affinity filler, elutes the epidermal growth factor receptor bispecific antibody by using a protein eluent, and stores the purified bispecific antibody in a protein storage solution, and specifically discloses the components of the protein eluent and the protein storage solution. The eluent and the storage solution for the epidermal growth factor receptor bispecific antibody aEGFR-aPDL1 BsAb are optimized, a stable buffer component is found, and the protein recovery rate and the purity are significantly improved.

Description

Technical Field

[0001] This invention relates to the field of antibody separation and purification, and more particularly to a method for purifying and preserving Protein A, a bispecific antibody against epidermal growth factor receptor. Background Technology

[0002] PD-L1, or programmed death receptor-ligand 1, is involved in immune evasion. It binds to PD-1, inhibiting T cell proliferation and cytokine secretion, and negatively regulating lymphocyte activation. PD-1, a member of the CD28 superfamily, is a type I transmembrane protein composed of 268 amino acids, expressed on the surface of immune cells such as T cells and B cells. However, PD-1 is almost not expressed when T cells are not activated; it is only expressed on the surface of T cells after activation. As mentioned earlier, PD-1 can bind to PD-L1, acting as both receptor and ligand for each other. Under normal circumstances, the immune system responds to foreign antigens accumulating in lymph nodes or the spleen, stimulating the proliferation of antigen-specific cytotoxic T cells (CD8+ T cells). The binding of programmed death receptor-1 (PD-1) and programmed death-ligand 1 (PD-L1) can transmit inhibitory signals, reducing the proliferation of CD8+ T cells in lymph nodes. aPD-L1 is a monoclonal antibody that binds to PD-L1 and blocks its interaction with the PD-1 receptor. This releases PD-L1 / PD-1-mediated immunosuppression, including activation of anti-tumor immune responses without inducing antibody-dependent cytotoxicity. In syngeneic mouse tumor models, blocking PD-L1 activity leads to reduced tumor growth.

[0003] EGFR (Epidermal Growth Factor Receptor) is the receptor for epidermal growth factor (EGF) cell proliferation and signal transduction. Studies have shown that EGFR is highly expressed or abnormally expressed in many solid tumors, including head and neck cancer, breast cancer, bladder cancer, ovarian cancer, kidney cancer, colon cancer, and non-small cell lung cancer, especially lung cancer, where the EGFR mutation rate can reach 50% in the Asian lung cancer population. EGFR is involved in the inhibition of tumor cell proliferation, angiogenesis, tumor invasion, metastasis, and apoptosis. EGFR is an important target in clinical cancer treatment.

[0004] Bispecific monoclonal antibodies (BsAbs) are artificially created antibodies that can simultaneously bind to two different antigens. Research on bispecific antibodies is of great significance for cancer immunotherapy and has become a major focus of clinical oncology treatment. The main advantage of bispecific antibodies compared to monoclonal antibodies lies in their ability to mediate temporal or spatial effects; however, the technical barriers and research and development costs are high, and problems such as low expression levels and poor stability may be encountered during the construction phase.

[0005] Protein A is a bacterial cell wall protein isolated from Staphylococcus aureus that primarily binds to mammalian IgG via its Fc region. Protein A has five IgG-binding domains. In the purification of antibody-associated proteins, Protein A resin is the preferred affinity chromatography medium for antibody capture. Typically, antibodies or fusion proteins containing the Fc region bind to Protein A under neutral conditions, followed by protein elution under acidic conditions. Most monoclonal antibodies use a 0.1 mol / L glycine elution solution at pH 2.5-3.0, under which conditions they exhibit high purity and recovery rates. However, for some unstable bispecific antibodies, such as those fused from epidermal growth factor receptor (EGFR) antibodies and programmed cell death-ligand 1 (PCD-1) antibodies, elution with the above solution easily leads to protein precipitation, resulting in decreased recovery rate and purity. Therefore, a purification method for stable EGFR bispecific antibodies remains needed. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention discloses a method for purifying and preserving Protein A, a bispecific antibody against epidermal growth factor receptor. The method improves the protein storage solution, solving the problems of low protein recovery rate and protein instability during storage.

[0007] This invention discloses a method for purifying and preserving Protein A-based bispecific antibodies against epidermal growth factor receptor (EGFR). Using Protein A as the affinity packing material, the EGFR bispecific antibody is eluted with a protein elution buffer. The purified EGFR bispecific antibody is then stored in a protein storage solution.

[0008] The composition of the protein elution buffer is as follows:

[0009] 0.08–0.15 M sodium citrate, 0.1 M–0.2 M sodium chloride, and 240–260 mM sucrose, pH 3.0;

[0010] Or 0.08–0.15M sodium citrate, 0.1M–0.2M sodium chloride and 240–260mM sucrose, pH 3.5.

[0011] The protein storage solution consists of:

[0012] 0.08–0.15 M sodium acetate and 0.1 M–0.2 M sodium chloride, pH 6.0;

[0013] Or 0.08–0.15M sodium acetate, 0.1M–0.2M sodium chloride and 240–260mM sucrose, pH 6.0;

[0014] Or 0.08–0.15 M sodium acetate and 0.1 M–0.2 M sodium chloride, pH 5.0;

[0015] Or 0.08–0.15M sodium acetate, 0.1M–0.2M sodium chloride and 240–260mM sucrose, pH 5.0.

[0016] In this invention, unless otherwise specified, epidermal growth factor receptor bispecific antibody refers to an antibody formed by fusing an epidermal growth factor receptor antibody and a programmed cell death-ligand 1 antibody.

[0017] Furthermore, the preferred composition of the protein storage solution is:

[0018] 0.1M sodium acetate and 0.1M–0.2M sodium chloride, pH 6.0;

[0019] Or 0.1M sodium acetate, 0.1M-0.2M sodium chloride and 250mM sucrose, pH 6.0;

[0020] Or 0.1M sodium acetate and 0.1M–0.2M sodium chloride, pH 5.0;

[0021] Alternatively, use 0.1M sodium acetate, 0.1M–0.2M sodium chloride, and 250mM sucrose, pH 5.0.

[0022] Furthermore, the preferred composition of the protein eluent is:

[0023] 0.1M sodium citrate, 0.1M–0.2M sodium chloride and 250mM sucrose, pH 3.0;

[0024] Alternatively, use 0.1M sodium citrate, 0.1M–0.2M sodium chloride, and 250mM sucrose, pH 3.5.

[0025] Further, after eluting the bispecific antibody against epidermal growth factor receptor with protein eluent, the protein was neutralized with a neutralizing solution of 1M Tris-HCl, pH 8.9.

[0026] Further, after adding protein elution buffer, incubate at 20-30℃ for 5-10 minutes.

[0027] Furthermore, when eluting the bispecific antibody against the epidermal growth factor receptor with the protein eluent, the volume ratio of the bispecific antibody solution to the protein eluent is 20-40:1.

[0028] Furthermore, when the protein is stored in the protein storage solution, it is concentrated by centrifugation at 3500-4500 rpm.

[0029] Furthermore, the volume ratio of the concentrated epidermal growth factor receptor bispecific antibody solution to the protein storage solution obtained after concentration is 1:50-100.

[0030] Specifically, the method for purifying and preserving Protein A in the epidermal growth factor receptor bispecific antibody aEGFR-aPDL1 BsAb includes the following steps:

[0031] (1) Wash Protein A affinity filler with phosphate-buffered saline (DPBS, pH 7.5);

[0032] (2) Slowly add the protein supernatant to be purified into the chromatography column, repeat several times, and then wash the Protein A affinity packing with phosphate buffer (DPBS, pH 7.5);

[0033] (3) Add protein elution buffer to the chromatography column, incubate at 20-30℃ for 5-10 min, then elute the protein. Repeat several times until the protein is completely eluted.

[0034] (4) Add neutralization solution (1M Tris-HCl, pH 8.9) to the eluted protein;

[0035] (5) Centrifuge at 3500-4500 rpm to obtain protein concentrate, add protein storage solution, and continue centrifugation to keep the protein in the protein storage solution.

[0036] By means of the above-described solution, the present invention has at least the following advantages:

[0037] During their experiments, the inventors discovered that the epidermal growth factor receptor bispecific antibody aEGFR-aPDL1 BsAb precipitated during conventional elution using Protein A purification methods, exhibiting significant dimerization and multimerization, resulting in poor protein stability during purification. Therefore, in protein purification using Protein A, the inventors optimized the elution and storage solutions for aEGFR-aPDL1 BsAb, identifying a stable buffer composition. Using this buffer for elution, purification, and storage resulted in no precipitation, significantly improving protein recovery rate and purity.

[0038] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, the following describes the preferred embodiments of the present invention in conjunction with detailed drawings. Attached Figure Description

[0039] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0040] Figure 1 FPLC peak diagram after eluting proteins using EB1;

[0041] Figure 2 FPLC peak diagram after protein elution using EB2;

[0042] Figure 3 This is a peak diagram for FPLC detection after eluting proteins using EB3. Detailed Implementation

[0043] 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.

[0044] Example 1: Transient transfection of Freestyle293 cells

[0045] The data and volumes involved in the cell transfection process described below refer to a 30 mL cell system. When the transfection system is larger than 30 mL, the reagents used and the amounts used should be scaled up proportionally.

[0046] 1. Freestyle 293 cells grow at a rate of approximately one generation every 24 hours. Expand the cultured cells to ensure a cell density of 2.5^10 on the day of transfection. 6 cells / mL;

[0047] 2. On the day of cell transfection, replace the cell culture medium with fresh medium (SmithKline, SMM 293-TII serum-free medium) and ensure that the cells are stable in the medium for at least 2 hours;

[0048] 3. Transfection

[0049] 1) Prepare two 15mL sterile centrifuge tubes, labeled 1 and 2, and add 1mL of Opti-MEMmedium transfection medium to each tube;

[0050] 2) Centrifuge tube No. 1: Add 60 μL of PEI (100 μM), mix gently, and let stand at room temperature for 5 min to obtain the PEI-Opti-MEM mixture;

[0051] 3) Centrifuge tube #2: Add 30 μg of plasmid (filtered through 0.22 μm), mix gently to obtain DNA-Opti-MEM mixture;

[0052] 4) Slowly add the mixture from centrifuge tube 1 to tube 2, mix gently, and let stand at room temperature for 15-20 minutes to obtain 2 mL of PEI-DNA mixture;

[0053] 5) Gently add the PEI-DNA mixture to 30 mL of Freestyle293 cell culture system;

[0054] Cell death rate was detected starting on day 3 post-transfection. Once the cell death rate reached 30-35% (not exceeding 40%), the next step of protein purification was performed. The supernatant was collected by centrifugation at 10,000-11,000 rpm for 15 minutes for protein purification.

[0055] Example 2

[0056] The epidermal growth factor receptor bispecific antibody aEGFR-aPDL1 BsAb used in this embodiment is numbered AB110-YF221, and the affinity packing material used is Protein A conjugated to 4% agarose.

[0057] 1. Collect cells and centrifuge at 11,000 rpm for 15 min to collect the protein supernatant;

[0058] 2. Wash the Protein A affinity packing material with 10 column volumes of phosphate-buffered saline (DPBS, pH 7.5);

[0059] 3. Repeat step 2 to remove the stock solution from Protein A;

[0060] 4. Slowly add the protein supernatant obtained by centrifugation to the chromatography column, so that the protein sample binds to the packing material under the action of gravity. Repeat the addition of protein supernatant 3 times.

[0061] 5. Rinse the Protein A affinity packing material with 10 column volumes of phosphate-buffered saline (DPBS, pH 7.5);

[0062] 6. Repeat step 5 to remove weakly bound impurities;

[0063] 7. Add 3 column volumes of protein elution buffer (EB) to the chromatography column, incubate at room temperature for 5-10 min, and then elute the protein. In this invention, three elution buffers, EB1, EB2, and EB3, are used to elute the protein. See Table 1 for formulation details.

[0064] 8. Repeat step 7 until the protein is completely eluted;

[0065] 9. Add 700 μL, 800 μL, and 750 μL of neutralizing solution NB (1M Tris-HCl, pH 8.9) to the proteins eluted from EB1, EB2, and EB3, respectively, so that the final pH of the protein solutions is 7.0–7.5, 5–5.5, and 5–5.5, respectively.

[0066] Table 1. Preparation of protein elution buffer

[0067] Buffer name formula EB1 0.1M glycine, 0.1M–0.2M sodium chloride, pH 2.5 EB2 0.1M sodium citrate, 0.1M–0.2M sodium chloride, 250mM sucrose, pH 3.0 EB3 0.1M sodium citrate, 0.1M–0.2M sodium chloride, 250mM sucrose, pH 3.5

[0068] The protein concentration was determined, and the protein purification yield was calculated, as shown in Table 3.

[0069] Example 3

[0070] 1. Concentrate the protein using a concentration tube at 4000 rpm. When the protein volume is reduced to less than 100 μL, add the protein stock solution to be replaced and continue centrifuging at the same speed. Repeat this step 3 times until the protein is finally in the protein stock solution to be replaced.

[0071] 2. Concentrate the protein using a concentration tube and replace the protein storage solution as shown in Table 2:

[0072] Table 2 Preparation of protein storage solution

[0073] Buffer name formula BEX1 0.1M citric acid, 0.1M–0.2M sodium chloride, pH 5.0 BEX2 0.1M citric acid, 0.1M–0.2M sodium chloride, pH 6.0 BEX3 0.1M sodium bicarbonate, 0.1M–0.2M sodium chloride, pH 9.0 BEX4 0.1M sodium bicarbonate, 0.1M–0.2M sodium chloride, pH 10.0 BEX5 DPBS, pH 6.0 BEX6 DPBS, 250mM sucrose, pH 6.0 BEX7 0.1M sodium acetate, 0.1M–0.2M sodium chloride, pH 6.0 BEX8 0.1M sodium acetate, 0.1M–0.2M sodium chloride, pH 6.0, 250mM sucrose, pH 6.0 BEX9 0.1M sodium acetate, 0.1M–0.2M sodium chloride, pH 5.0 BEX10 0.1M sodium acetate, 0.1M–0.2M sodium chloride, pH 6.0, 250mM sucrose, pH 5.0

[0074] The results after changing the protein storage solution are shown in Table 3.

[0075] Table 3 Purification and recovery results

[0076]

[0077]

[0078] As can be seen from the table above:

[0079] Traditional eluents yield protein at around 30 mg / L, while the improved eluent of this invention yields protein at over 40 mg / L, with no protein precipitation.

[0080] Proteins eluted with conventional eluent EB1 were concentrated, and the stock solutions were changed to BEX1, BEX2, BEX3, and BEX4. Different degrees of precipitation occurred during the concentration and stock solution changes to BEX1 and BEX2. Although no precipitation occurred during the BEX3 and BEX4 stock solutions changes, a large amount of precipitation occurred after the proteins were freeze-thawed at -20°C in both stock solutions. This indicates that the recovery rate of protein was relatively low after changing to these four stock solutions. Proteins eluted with conventional eluent EB1 were concentrated, and the stock solutions were changed to BEX5-BEX10. No precipitation occurred during the concentration and stock solution changes, but a large amount of precipitation occurred in all proteins after freeze-thawing at -20°C following the stock solution changes.

[0081] Proteins eluted with optimized eluents EB2 and EB3 were concentrated, and the stock solutions were changed to BEX5, BEX6, BEX7, BEX8, BEX9, and BEX10 (experiments showed that a large amount of precipitation occurred when the stock solutions were changed to BEX1-BEX4; this result is not shown in Table 3). Protein recovery rates were high, all greater than 95%. No protein precipitation occurred during the concentration and solution change processes. Proteins eluted with EB2, after being freeze-thawed at -20°C following the solution change, all produced a small amount of precipitation. Proteins eluted with EB3, however, did not produce any precipitation during the process.

[0082] FPLC analysis was performed on the concentrated protein after medium exchange. Figure 1 and Figure 2 In the diagram, P1 represents protein dimers or polymers, and P2 represents protein monomers. It can be seen that after changing the stock solution, the peak area of ​​proteins eluted with the traditional EB1 elution buffer is only about 20% in FPLC analysis, indicating that most proteins are dimers or polymers (see...). Figure 1 ); Proteins eluted with the optimized EB2 eluent, after changing the stock solution, were analyzed by FPLC, and the peak area ratio of protein monomers could be increased to approximately 80%. Figure 2 ); Proteins eluted with the optimized EB3 eluent were analyzed by FPLC after replacing the stock solution. The protein showed a single peak with almost no dimers or multimers. Figure 3 ).

[0083] Therefore, based on the above protein purification and analysis results, it can be seen that the AB110-YF221 protein eluted by the traditional elution buffer EB1 in this patent is unstable, with dimers and polymers accounting for about 80% of the protein. After optimizing the elution buffer and storage solution, the protein recovery rate is significantly improved, and the proportion of dimers and polymers in the protein is greatly reduced.

[0084] Example 4

[0085] Replace the protein storage solution with 0.08M sodium acetate, 0.1M-0.2M sodium chloride and 240mM sucrose, pH 5.0, and the remaining steps are the same as in Example 3.

[0086] Example 5

[0087] Replace the protein storage solution with 0.08M sodium acetate, 0.1M-0.2M sodium chloride and 240mM sucrose, pH 6.0, and the remaining steps are the same as in Example 3.

[0088] Example 6

[0089] Replace the protein storage solution with 0.08M sodium acetate, 0.1M-0.2M sodium chloride and 260mM sucrose, pH 6.0, and the remaining steps are the same as in Example 3.

[0090] Example 7

[0091] Replace the protein storage solution with 0.15M sodium acetate, 0.1M-0.2M sodium chloride and 240mM sucrose, pH 5.0, and follow the same steps as in Example 3.

[0092] Example 8

[0093] Replace the protein storage solution with 0.15M sodium acetate, 0.1M-0.2M sodium chloride and 260mM sucrose, pH 6.0, and follow the same steps as in Example 3.

[0094] Example 9

[0095] Replace the protein storage solution with 0.15M sodium acetate, 0.1M-0.2M sodium chloride and 240mM sucrose, pH 6.0, and the remaining steps are the same as in Example 3.

[0096] Example 10

[0097] Replace the protein eluent with 0.15M sodium citrate, 0.1M-0.2M sodium chloride and 240mM sucrose, pH 3.0, and replace the protein stock solution with BEX8. The remaining steps are the same as in Example 3.

[0098] Example 11

[0099] The protein eluent was replaced with 0.08M sodium citrate, 0.1M-0.2M sodium chloride, and 260mM sucrose, with a pH of 3.5. The protein stock solution was replaced with BEX8, and the remaining steps were the same as in Example 3. The results showed that Examples 4-11 all yielded similar experimental results to those described above.

[0100] Table 4 Purification and Recovery Results

[0101]

[0102] Comparative Example 1

[0103] Replace the protein storage solution with 0.2M sodium acetate, 0.1M-0.2M sodium chloride, 250mM sucrose, pH 5.0, and the rest is the same as in Example 3.

[0104] Comparative Example 2

[0105] Replace the protein storage solution with 0.05M sodium acetate, 0.1M-0.2M sodium chloride, 250mM sucrose, pH 5.0, and the rest is the same as in Example 3.

[0106] Comparative Example 3

[0107] Replace the protein storage solution with 0.1M sodium acetate, 0.1M-0.2M sodium chloride, 250mM sucrose, pH 4.5, and the rest is the same as in Example 3.

[0108] Comparative Example 4

[0109] Replace the protein storage solution with 0.1M sodium acetate, 0.1M-0.2M sodium chloride, 250mM sucrose, pH 6.5, and the rest is the same as in Example 3.

[0110] Comparative Example 5

[0111] Replace the protein storage solution with 0.1M sodium acetate, 0.1M-0.2M sodium chloride, 250mM sucrose, pH 7.5, and the rest is the same as in Example 3.

[0112] Comparative Example 6

[0113] Replace the protein storage solution with 0.1M sodium acetate, 0.1M-0.2M sodium chloride, pH 7.5, and the rest is the same as in Example 3.

[0114] Comparative Example 7

[0115] Replace the protein elution buffer with 0.1M sodium citrate, 0.1M-0.2M sodium chloride, 250mM sucrose, pH 2.5, and the protein stock solution with BEX8. The rest is the same as in Example 3.

[0116] Comparative Example 8

[0117] Replace the protein elution buffer with 0.1M sodium citrate, 0.1M-0.2M sodium chloride, 250mM sucrose, pH 4.0, and the protein stock solution with BEX8. The rest is the same as in Example 3.

[0118] Comparative Example 9

[0119] Replace the protein elution buffer with 0.05M sodium citrate, 0.1M-0.2M sodium chloride, 250mM sucrose, pH 3.0, and the protein stock solution with BEX8. The rest is the same as in Example 3.

[0120] Comparative Example 10

[0121] Replace the protein eluent with 0.2M sodium citrate, 0.1M-0.2M sodium chloride, 250mM sucrose, pH 3.0, and the protein stock solution with BEX8. The rest is the same as in Example 3.

[0122] The results showed that when the proteins eluted with eluents EB2 and EB3 were concentrated and the stock solution was replaced with the protein stock solutions of Comparative Examples 1-6, the protein recovery rate was below 90%, and precipitation of varying degrees occurred during the stock solution replacement process. When the eluent was replaced with the protein eluents of Comparative Examples 7-10, protein precipitation of varying degrees occurred, and protein yield decreased at higher pH levels. Precipitation of varying degrees also occurred during the concentration and stock solution replacement process. After the stock solution replacement, freeze-thaw cycles at -20°C resulted in precipitation of all proteins.

[0123] Table 5 Purification and recovery results

[0124]

[0125] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for purifying and preserving Protein A, a bispecific antibody against epidermal growth factor receptor, characterized in that: Using Protein A as the affinity packing material, the bispecific antibody against epidermal growth factor receptor (EGFR) was eluted with protein elution buffer. The purified EGFR bispecific antibody was then stored in protein stock solution after buffer exchange. The epidermal growth factor receptor bispecific antibody is formed by fusing an epidermal growth factor receptor antibody and a programmed cell death-ligand 1 antibody; The composition of the protein elution buffer is as follows: 0.08–0.15 M sodium citrate, 0.1 M–0.2 M sodium chloride, and 240–260 mM sucrose, pH 3.0; Or 0.08–0.15M sodium citrate, 0.1M–0.2M sodium chloride and 240–260mM sucrose, pH 3.5; The protein storage solution has the following composition: 0.08–0.15 M sodium acetate and 0.1 M–0.2 M sodium chloride, pH 6.0; Or 0.08–0.15M sodium acetate, 0.1M–0.2M sodium chloride and 240–260mM sucrose, pH 6.0; Or 0.08–0.15 M sodium acetate and 0.1 M–0.2 M sodium chloride, pH 5.0; Or 0.08–0.15M sodium acetate, 0.1M–0.2M sodium chloride and 240–260mM sucrose, pH 5.

0.

2. The method according to claim 1, characterized in that, The protein storage solution has the following composition: 0.1M sodium acetate and 0.1M–0.2M sodium chloride, pH 6.0; Or 0.1M sodium acetate, 0.1M-0.2M sodium chloride and 250mM sucrose, pH 6.0; Or 0.1M sodium acetate and 0.1M–0.2M sodium chloride, pH 5.0; Alternatively, use 0.1M sodium acetate, 0.1M–0.2M sodium chloride, and 250mM sucrose, pH 5.

0.

3. The method according to claim 2, characterized in that, The composition of the protein elution buffer is as follows: 0.1M sodium citrate, 0.1M–0.2M sodium chloride and 250mM sucrose, pH 3.0; Alternatively, use 0.1M sodium citrate, 0.1M–0.2M sodium chloride, and 250mM sucrose, pH 3.

5.

4. The method according to claim 1, characterized in that: After eluting the bispecific antibody against epidermal growth factor receptor with protein elution buffer, the antibody was neutralized with a neutralizing solution of 1M Tris-HCl, pH 8.

9.

5. The method according to claim 1, characterized in that: After adding the protein elution buffer, incubate at 20-30℃ for 5-10 minutes.

6. The method according to claim 1, characterized in that: When eluting epidermal growth factor receptor bispecific antibodies with protein eluent, the volume ratio of the epidermal growth factor receptor bispecific antibody solution to the protein eluent is 20-40:

1.

7. The method according to claim 1, characterized in that: When storing the protein in the solution, centrifuge at 3500-4500 rpm to concentrate the epidermal growth factor receptor bispecific antibody.

8. The method according to claim 7, characterized in that, The volume ratio of the concentrated epidermal growth factor receptor bispecific antibody solution to the protein storage solution after concentration is 1:50-100.

9. The method according to claim 1, characterized in that, The method specifically includes the following steps: (1) Clean the Protein A affinity filler with phosphate buffer; (2) Slowly add the protein supernatant to be purified into the chromatography column, repeat several times, and then wash the Protein A affinity packing with phosphate buffer again. (3) Add protein elution buffer to the chromatography column, incubate at 20-30℃ for 5-10 min, and then elute the bispecific antibody against epidermal growth factor receptor. Repeat this process several times until the bispecific antibody against epidermal growth factor receptor is completely eluted. (4) Add a neutralizing solution to the eluted epidermal growth factor receptor bispecific antibody, wherein the neutralizing solution is 1M Tris-HCl, pH 8.9; (5) Centrifuge at 3500-4500 rpm to obtain a concentrated solution of bispecific antibody against epidermal growth factor receptor. Add protein storage solution and continue centrifugation to keep the bispecific antibody against epidermal growth factor receptor in the protein storage solution.

10. The method according to claim 9, characterized in that: In steps (1) and (2), the phosphate buffer is DPBS, pH 7.5.

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