Purification method of EGFR-CTLA4 bispecific antibody fusion protein

Abstract

The invention discloses a purification method of EGFR-CTLA4 bispecific antibody fusion protein, specifically, an affinity purification method is adopted, the EGFR-CTLA4 bispecific antibody fusion protein combined on affinity filler is eluted by using eluent, in order to reduce the proportion of aggregates harvested in protein A affinity chromatography, the specific composition of the eluent is screened, and the specific composition of the eluent is screened to obtain the EGFR-CTLA4 bispecific antibody fusion protein. Wherein the concentration of glycine is 0.08-0.12 M, the concentration of sodium chloride is 0.1-0.2 M, the concentration of cane sugar is 240-260mM, and the concentration of pH is 3.2-3.5, so that the harvested protein with extremely low aggregate content is obtained. Under the elution condition, a harvested protein sample with very low aggregate content is obtained, the protein recovery rate and purity are remarkably improved, and compared with protein samples harvested under other elution conditions, the protein samples have better antigen binding activity.

Description

Technical Field

[0001] The present invention relates to the technical field of antibody separation and purification, and particularly to a method for purifying an EGFR-CTLA4 bispecific antibody fusion protein. Background Art

[0002] EGFR (Epidermal Growth Factor Receptor) is a receptor for epithelial growth factor (EGF) cell proliferation and signaling. Studies have shown that EGFR is overexpressed 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, particularly lung cancer. The EGFR mutation rate in Asian lung cancer patients can reach 50%. EGFR is associated with tumor cell proliferation, angiogenesis, tumor invasion, metastasis, and the inhibition of cell apoptosis. EGFR is an important target in the clinical treatment of tumors.

[0003] CTLA4 (cytotoxic T lymphocyte-associated antigen-4), also known as CD152 (cluster of differentiation 152), is a protein receptor that functions as an immune checkpoint and downregulates immune responses. CTLA4 is constitutively expressed in regulatory T cells and shares its ligand with CD28, the B7 molecule. Binding to B7 by CTLA4 induces T cell anergy and negatively regulates immune responses. Recombinant CTLA-4Ig can effectively and specifically inhibit cellular and humoral immune responses in vitro and in vivo, demonstrating significant therapeutic effects against transplant rejection and various autoimmune diseases with minimal side effects. It is currently considered a promising new immunosuppressive drug.

[0004] Bispecific antibodies (BsAbs), also known as "double antibodies," are artificial antibodies produced through cell fusion or recombinant DNA technology. They can specifically bind to two antigens or two different epitopes on the same antigen. Research on bispecific antibodies is of great significance for cancer immunotherapy and has become a hot topic in clinical tumor treatment. The advantage of bispecific antibodies over monoclonal antibodies lies primarily in their ability to mediate temporal or spatial effects, but they require high technical barriers and R&D costs, and during the development phase, they may encounter issues such as low expression levels and poor stability.

[0005] The purification of bispecific antibodies can be similar to that of monoclonal antibodies, primarily using heavy chain-based affinity chromatography. Protein A affinity chromatography is a representative example of this type of chromatography. Protein A is a bacterial cell wall protein isolated from Staphylococcus aureus that primarily binds to mammalian IgG through the Fc region. Protein A has five IgG binding domains. During the purification of antibody-related proteins, Protein A resin is the preferred affinity chromatography medium for antibody capture. Protein A specifically binds to the Fc terminus of bispecific antibodies and dissociates between pH 2.5 and 4.0, making it suitable for affinity capture of bispecific antibodies.

[0006] Typically, antibodies or fusion proteins containing an Fc region bind to Protein A under neutral conditions, and the protein is eluted under acidic conditions. Most monoclonal antibodies are eluted using a 0.1 mol / L glycine solution at pH 2.5-3.0. Under these conditions, monoclonal antibodies have high purity and recovery rates. However, for some unstable bispecific antibodies, different buffer systems and low pH conditions can significantly impact the stability of bispecific samples during the purification process. Molecular aggregation is a common problem in the production of therapeutic bispecific antibodies. Harvested aggregates are a major type of product-related impurities. They not only reduce the biological activity of the antibody drug but may also trigger an immune response. Therefore, a method is needed to reduce the proportion of aggregates harvested during Protein A affinity chromatography, allowing for more monomeric components of the bispecific antibody to be recovered during this capture step. Summary of the Invention

[0007] To solve the above technical problems, the present invention discloses a method for purifying an epidermal growth factor receptor-cytotoxic T lymphocyte-associated protein 4 bispecific antibody fusion protein. The components and optimal pH of the eluate are improved, thereby solving the problems of instability, low recovery rate and reduced antigen binding activity of bispecific antibodies during the purification process.

[0008] The present invention discloses a method for purifying an epidermal growth factor receptor-cytotoxic T lymphocyte associated protein 4 bispecific antibody fusion protein, wherein the epidermal growth factor receptor-cytotoxic T lymphocyte associated protein 4 bispecific antibody fusion protein bound to an affinity filler is eluted with an eluent, wherein:

[0009] The epidermal growth factor receptor-cytotoxic T lymphocyte associated protein 4 bispecific antibody fusion protein is formed by the fusion of epidermal growth factor receptor antibody and cytotoxic T lymphocyte associated protein 4 antibody;

[0010] The composition of the eluent is: 0.08-0.12 M glycine, 0.1-0.2 M sodium chloride, 240-260 mM sucrose, pH 3.2-3.5.

[0011] Furthermore, the heavy chain variable region sequence of the epidermal growth factor receptor antibody is shown as SEQ ID NO.1, and the light chain variable region is shown as SEQ ID NO.2; the heavy chain variable region sequence of the cytotoxic T lymphocyte-associated protein 4 antibody is shown as SEQ ID NO.3, and the light chain variable region is shown as SEQ ID NO.4.

[0012] Furthermore, 0.8-1.2 M hydrochloric acid was used to adjust the pH.

[0013] Furthermore, using Protein A as the affinity filler, before elution with the eluent, it is first washed with phosphate buffer to remove the storage solution in Protein A. After adding the fusion protein supernatant to the affinity filler, the affinity filler is rinsed with phosphate buffer to remove weakly bound impurities. The composition of the phosphate buffer is 0.1-0.5M sodium chloride, 15-25mM Na2HPO4, pH 6.8-7.2.

[0014] Furthermore, when eluting with an elution buffer, the protein is eluted after adding the elution buffer and incubating at 20-30° C. for 5-10 minutes.

[0015] Furthermore, after the eluent elutes the epidermal growth factor receptor-cytotoxic T lymphocyte-associated protein 4 bispecific antibody fusion protein, it is neutralized with a neutralizing solution. The pH of the protein solution after neutralization is 7.0-7.5, and the neutralizing solution is 0.8-1.2 M Tris-HCl, pH 8-9.

[0016] Furthermore, after the fusion protein is eluted with the eluent, the method further comprises the step of performing gel exclusion chromatography on the fusion protein.

[0017] Furthermore, gel exclusion chromatography was performed using a Superdex 200Increase 10 / 300GL gel exclusion chromatography column.

[0018] Furthermore, when performing gel exclusion chromatography, the buffer is 0.01-0.05M phosphate buffer, 0.001-0.005M potassium chloride, 0.1-0.2M sodium chloride, pH 7.0-7.5.

[0019] Beneficial effects of the present invention:

[0020] The EGFR-CTLA4 bispecific antibody is prone to precipitation during traditional elution, resulting in the appearance of a large number of distinct dimers and multimers, and poor protein stability during purification. The present invention optimizes the eluent components and ratios for this unstable bispecific antibody, identifying a class of stable buffer components. This buffer allows for elution and purification of proteins without precipitation, significantly improving protein purity, reducing the aggregate content of affinity-purified harvested proteins, and enhancing the antigen-binding activity of the harvested antibodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a chromatogram of the primary screening of the eluent in Example 2.

[0022] Figure 2 This is the chromatogram of the secondary screening of the eluent in Example 3.

[0023] Figure 3 This is the antigen binding activity test of the proteins harvested from different formulations in Example 4. DETAILED DESCRIPTION

[0024] 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 the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0025] Example 1: Construction of recombinant plasmid

[0026] 1. Gene Synthesis

[0027] The following light and heavy chain sequences and antibody constant region sequences were synthesized (synthesized by IDT).

[0028] The sequence information is as follows:

[0029] EGFR antibody heavy chain variable region (SEQ ID NO.1)

[0030] QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWIRQSPGKGLEWIGHIYYSGNTNYNPSLKSRLTISIDTSKTQFSLKLSSVTAADTAIYYCVRDRVTGAFDIWGQGTMVTVSS

[0031] EGFR antibody light chain variable region (SEQ ID NO.2)

[0032] DIQMTQSPSSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYFCQHFDHLPLAFGGGTKVEIK

[0033] CTLA4 antibody heavy chain variable region (SEQ ID NO.3)

[0034] EAKLQESGPVLVKPGASVKMSCKASGYTFTDYYMNWVKQSHGKSLEWIGVINPYNGDTSYNQKFKGKATLTVDKSSSTAYMELNSLTSEDSAVYYCARYYGSWFAYWGQGTLITVST

[0035] CTLA4 antibody light chain variable region (SEQ ID NO.4)

[0036] DIRRADIVMTQTTLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPYTFGGGTKLEIKR

[0037] The antibody heavy chain constant region and light chain constant region sequences are shown in SEQ ID NO.5-6, including the following parts:

[0038] Mouse IgG2a antibody heavy chain constant region CH1 region

[0039] AKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSS GVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIE

[0040] Mouse IgG2a antibody heavy chain constant region Fc (CH2-CH3) region

[0041] PRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK

[0042] Constant region CL of murine Kappa antibody light chain

[0043] ADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0044] Linker

[0045] TVAAPSVFIFPP

[0046] ASTKGP

[0047] Heavy chain sequence (SEQ ID NO.7):

[0048] DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYFCQHFDHLPLAFGGGTKVEIKTVAAPSVFIFPPEAKLQESGPVLVKPGASVKMSCKASGYTFTDYYMNWVKQSHGKSLEWIGVINPYNGDTSYNQKFKGKATLTVDKSSSTAYMELNSLTSEDSAVYYCARYYGSWFAYWGQGTLITVSTAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK

[0049] Light chain sequence (SEQ ID NO.8):

[0050] QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWIRQSPGKGLEWIGHIYYSGNTNYNPSLKSRLTISIDTSKTQFSLKLSSVTAADTAIYYCVRDRVTGAFDIWGQGTMVTVSSASTKGPDIRRADIVMTQTTLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPYTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0051] 2. Plasmid Construction

[0052] The above-synthesized gene fragments were amplified by PCR, and the amplified PCR products were connected and cloned into pFuse vectors (InvivoGen, CA) and sequenced for verification.

[0053] Example 2: Primary screening of eluate

[0054] The two chains of the constructed antibody eukaryotic expression vector were transiently co-transfected into FreeStyle HEK293 cells (ThermoFisher): 28 ml FreeStyle HEK 293 (3×10 7 Cells were inoculated into 125 ml cell culture flasks at 100 cells / ml. The plasmid was diluted with 1 ml of Opti-MEM (Invitrogen) and added to 1 ml of Opti-MEM containing 60 μl of 293Fectin (Invitrogen, Inc.). The cells were allowed to stand at room temperature for 30 minutes. The plasmid-293Fectin mixture was then added to the cell culture medium and incubated at 125 rpm at 37°C in 5% CO2. Cell culture supernatants were collected 48 and 96 hours after transfection, and antibodies were purified using Protein A Resin (Genscript) according to the manufacturer's instructions.

[0055] Protein A Resin (Genscript) was used to purify the antibody according to the instructions. Taking condition 1-1 as an example, a brief description is as follows:

[0056] Add 5 ml of equilibration buffer (0.15 M NaCl, 20 mM Na2HPO4, pH 7.0) to the gravity column containing the Protein A packing to equilibrate the packing. After the equilibration buffer has drained, load the sample onto the gravity column. After the sample liquid has completely drained, wash the packing with 30 ml of equilibration buffer to remove unbound impurities. Elute the antibody with 5 ml of elution buffer (0.1 M glycine, pH 3.0, i.e., condition 1-1). Collect the eluate containing the target immunoglobulin and immediately add neutralization buffer (1 M Tris-HCl, pH 8.5) to adjust the pH to 7.4.

[0057] The elution conditions recommended in the instructions (0.1 M glycine, pH 3.0, i.e., condition 1-1) were optimized. The elution conditions were:

[0058] 1) 0.1 M glycine, HCl adjusted to pH 3.0

[0059] 2) 0.1 M glycine, 0.15 M NaCl, HCl to adjust pH to 3.0

[0060] 3) 0.1 M glycine, 0.15 M NaCl, 250 mM sucrose, HCl to adjust pH to 3.0

[0061] 4) 0.1M sodium citrate, citric acid to adjust pH to 3.0

[0062] 5) 0.1M sodium citrate, 250mM sucrose, citric acid to adjust pH 3.0

[0063] 6) 0.1M NaAc, HAc to adjust pH to 3.0

[0064] 7) 0.1M NaAc, 250mM sucrose, HAc to adjust pH to 3.0

[0065] 8) 3.0M KCl, HCl to adjust pH to 6.6

[0066] 9) PBS, HCl adjusted to pH 3.5

[0067] 10) PBS, HCl adjusted to pH 2.5

[0068] The antibody purified from Protein A resin was analyzed using GE AKTA chromatography using a Superdex 200 Increase 10 / 300GL gel exclusion column. The gel exclusion chromatography solution used was PBS buffer (0.010 M phosphate buffer, 0.0027 M KCl, 0.14 M NaCl, pH 7.4).

[0069] The purification results are shown in Table 1 and Figure 1 .

[0070] Table 1

[0071] Elution conditions Leave at room temperature 4℃ overnight Protein purification yield (mg / L) 1 precipitation precipitation 10 2 No precipitation precipitation 20 3 No precipitation No precipitation 22 4 precipitation precipitation 3 5 precipitation precipitation 5 6 precipitation No precipitation 1 7 precipitation No precipitation 2 8 precipitation No precipitation 3 9 precipitation No precipitation 5 10 precipitation No precipitation 5

[0072] from Figure 1 The chromatograms in Figure 1 show that under different elution conditions, condition 1-1 has the lowest aggregate content and the highest monomer content. As shown in the figure, the peak plot for condition 1-1 shows two connected peaks: the small peak on the left represents an aggregate peak in terms of molecular weight, while the main peak on the right represents a monomer peak in terms of molecular weight.

[0073] Compared with other conditions:

[0074] Conditions 1-2 and 1-3 both have two obvious peaks, among which the aggregate peak on the left has a higher content, and the aggregate peak content is higher than that of condition 1-1;

[0075] Conditions 1-4, 1-5, 1-6, 1-7, and 1-8 did not show a good monomer peak shape, indicating that the salt component is not suitable for the elution of the bispecific antibody molecules of the present invention;

[0076] Both conditions 1-9 and 1-10 had monomer peak components, and the monomer peak ratio of 1-9 was higher than that of 1-10 and 1-1, indicating the importance of pH for the elution of the bispecific antibody molecules. However, the effective protein content of 1-9, that is, the peak area at the same sample load, was much lower than that of condition 1-1, indicating that the salt solution composition of condition 1-1 had a significant impact on protein yield.

[0077] In summary, based on the analysis of conditions 1-1, 1-2, 1-3, and 1-9, and considering the protein precipitation observations and protein purification yields, we planned to add sodium chloride and sucrose to the background conditions of 1-1 glycine solution to increase protein purification yield, and to increase the pH to reduce aggregates generated during elution. Based on this, we conducted a second round of formulation screening.

[0078] Example 3: Secondary screening of eluate

[0079] The two chains of the constructed antibody eukaryotic expression vector were transiently co-transfected into FreeStyle HEK293 cells (ThermoFisher): 28 ml FreeStyle HEK 293 (3×10 7 Cells were inoculated into 125 ml cell culture flasks (100 cells / ml). The plasmid was diluted with 1 ml of Opti-MEM (Invitrogen) and added to 1 ml of Opti-MEM containing 60 μl of 293Fectin (Invitrogen, Inc.). The cells were allowed to stand at room temperature for 30 minutes. The plasmid-293Fectin mixture was then added to the cell culture medium and incubated at 125 rpm at 37°C in 5% CO2. Cell culture supernatants were collected 48 and 96 hours after transfection, and antibodies were purified using Protein A Resin (Genscript) according to the manufacturer's instructions.

[0080] Protein A Resin (Genscript) was used to purify the antibody according to the instructions, as briefly described below:

[0081] Add 5 ml of equilibration buffer (0.15 M NaCl, 20 mM Na2HPO4, pH 7.0) to the gravity column containing the Protein A packing to equilibrate the packing. After the equilibration buffer has drained, load the sample onto the gravity column. After the sample liquid has completely drained, wash the packing with 30 ml of equilibration buffer to remove unbound impurities. Elute the antibody with 5 ml of elution buffer (0.1 M glycine, pH 3.0, i.e., condition 2-1). Collect the eluate containing the target immunoglobulin and immediately add neutralization buffer (1 M Tris-HCl, pH 8.5) to adjust the pH to 7.4.

[0082] The elution conditions recommended in the instructions (0.1 M glycine, pH 3.0, i.e., condition 2-1) were optimized as follows.

[0083] 1) 0.1 M glycine, HCl adjusted to pH 3.0

[0084] 2) 0.1 M glycine, 0.15 M NaCl, HCl to adjust pH to 3.0

[0085] 3) 0.1 M glycine, 0.15 M NaCl, 250 mM sucrose, HCl to adjust pH to 3.0

[0086] 4) 0.1 M glycine, 0.15 M NaCl, 250 mM sucrose, HCl to adjust pH to 3.2

[0087] 5) 0.1 M glycine, 0.15 M NaCl, 250 mM sucrose, HCl to adjust pH to 3.5

[0088] The antibody purified from Protein A resin was analyzed using GE AKTA chromatography using a Superdex 200 Increase 10 / 300GL gel exclusion column. The gel exclusion chromatography solution used was PBS buffer (0.010 M phosphate buffer, 0.0027 M KCl, 0.14 M NaCl, pH 7.4).

[0089] The purification results are shown in Table 2 and Figure 2 .

[0090] Table 2

[0091] Elution conditions Leave at room temperature 4℃ overnight Protein purification yield (mg / L) 1 precipitation precipitation 10 2 No precipitation precipitation 20 3 No precipitation No precipitation 22 4 No precipitation No precipitation 20 5 No precipitation No precipitation 20

[0092] from Figure 2From the chromatogram results in Figure 2, the aggregate content of condition 2-5 is the lowest under different elution conditions, less than 1%. As shown in the figure, the peak graph of condition 2-5 shows a single main peak, which is a monomer peak in terms of molecular weight. Compared with other conditions:

[0093] Condition 2-2 showed an increase in the aggregate peak after adding salt to maintain osmotic pressure. In condition 2-3, the addition of sucrose reduced the aggregate ratio, significantly increasing the proportion and content of the monomer peak, confirming the importance of sucrose in maintaining monomer content and sample yield during the elution process in the bispecific antibody molecule of the present invention.

[0094] Conditions 2-4 and 2-5 further optimized the elution pH based on 2-3. Comparing conditions 2-3, 2-4, and 2-5, the proportion of aggregate peaks gradually decreased until there was almost no aggregate peak in 2-5.

[0095] In summary, for the bispecific antibody molecules of the present invention, elution conditions 2-5 are used as specific elution conditions, and the protein monomer components eluted therefrom are better than those under other conditions.

[0096] Example 4: Antigen Binding Activity Detection of Harvested Proteins from Different Formulas

[0097] CTLA4 and EGFR were coated in a 96-well plate at 4 degrees overnight, and then blocked with PBST containing 2% skim milk powder at room temperature for 1 hour. After blocking, the samples were harvested by adding gradient dilutions of bispecific antibodies and incubated at 37 degrees for 1 hour. After incubation, diluted Biotin-Mouse-anti-human IgG Fc mAb (JDC10), Cat#9040-08, SouthernBiotech and Streptavidin-horseradish peroxidase cat#405210biolegend were added for incubation. TMB was used for color development, and the absorbance value was read at 652nm. Figure 3 The results showed that the proteins obtained after elution under conditions 2-5 had the strongest binding activity to the two target antigens (Table 3).

[0098] Table 3

[0099] EGFR EC50 (nM) CTLA4 EC50 (nM) Elution conditions Curve number 0.08 0.55 1-9 1 0.11 0.37 1-1 2 0.08 0.29 2-5 3 0.1 0.29 2-4 4 0.15 0.86 1-8 5 0.15 0.42 1-3 6 0.13 1.02 1-5 7 0.15 0.64 1-2 8

[0100] The above embodiments are merely preferred embodiments for the purpose of fully illustrating 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 within the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

Claims

1. A method for purifying an epidermal growth factor receptor-cytotoxic T lymphocyte-associated protein 4 bispecific antibody fusion protein, characterized in that: The epidermal growth factor receptor-cytotoxic T lymphocyte-associated protein 4 bispecific antibody fusion protein bound to the affinity medium is eluted with an elution buffer, wherein: The epidermal growth factor receptor-cytotoxic T lymphocyte associated protein 4 bispecific antibody fusion protein is formed by the fusion of epidermal growth factor receptor antibody and cytotoxic T lymphocyte associated protein 4 antibody; The composition of the eluent is: 0.08-0.12 M glycine, 0.1-0.2 M sodium chloride, 240-260 mM sucrose, pH 3.2-3.

5.

2. The purification method according to claim 1, wherein The heavy chain variable region sequence of the epidermal growth factor receptor antibody is shown in SEQ ID NO.1, and the light chain variable region sequence is shown in SEQ ID NO.2; the heavy chain variable region sequence of the cytotoxic T lymphocyte-associated protein 4 antibody is shown in SEQ ID NO.3, and the light chain variable region sequence is shown in SEQ ID NO.

4.

3. The purification method according to claim 1, wherein Protein A is used as affinity filler.

4. The purification method according to claim 1, wherein The pH was adjusted with hydrochloric acid.

5. The purification method according to claim 1, wherein When eluting with elution buffer, add elution buffer and incubate at 20-30°C for 5-10 minutes before eluting the protein.

6. The purification method according to claim 1, wherein After the epidermal growth factor receptor-cytotoxic T lymphocyte-associated protein 4 bispecific antibody fusion protein is eluted with the eluent, it is neutralized with a neutralizing solution. The pH of the protein solution after neutralization is 7.0-7.

5.

7. The purification method according to claim 6, characterized in that The neutralization solution is 0.8-1.2M Tris-HCl, pH 8-9.

8. The purification method according to claim 1, wherein After the fusion protein is eluted with the eluent, the method further comprises the step of performing gel exclusion chromatography on the fusion protein.

9. The purification method according to claim 8, characterized in that Gel exclusion chromatography was performed using a Superdex 200Increase 10 / 300GL gel exclusion chromatography column.

10. The purification method according to claim 8, characterized in that When performing gel exclusion chromatography, the buffer is 0.01-0.05M phosphate buffer, 0.001-0.005M potassium chloride, 0.1-0.2M sodium chloride, pH 7.0-7.5.

Images