A method for purifying a bispecific antibody

Abstract

The application discloses a method for purifying bispecific antibodies targeting EGFR and PD-L1. The sample containing the bispecific antibodies is loaded on an affinity chromatography medium capable of binding Fc structure, washed, eluted with an elution buffer containing a buffer and sodium chloride at pH 3.3-3.8, and neutralized and collected the eluate. The method can reduce the aggregate content of the bispecific antibodies in the Protein A affinity chromatography process and increase the monomer proportion by optimizing the composition and pH of the elution buffer. In particular, when 0.1 M glycine 0.20 M sodium chloride / pH 3.5, 0.1 M sodium citrate / 0.15 M sodium chloride / pH 3.5 or 0.1 M sodium acetate / 0.15 M sodium chloride / pH 3.5 is used, the purified products all have a high monomer proportion; among them, 0.1 M sodium acetate / 0.15 M sodium chloride / pH 3.5 achieves a better balance between the monomer proportion and the protein yield.

Description

Technical Field

[0001] This invention relates to the field of antibody separation and purification technology, specifically to a purification method for a bispecific antibody targeting EGFR and PD-L1. Background Technology

[0002] EGFR (Epidermal Growth Factor Receptor) is closely related to 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 associated with the inhibition of tumor cell proliferation, angiogenesis, tumor invasion, metastasis, and apoptosis. EGFR is an important target in clinical cancer treatment.

[0003] PD-L1 (programmed death-ligand 1), also known as CD274 (differentiation cluster 274), is a type I transmembrane glycoprotein that acts as an immune checkpoint ligand and downregulates the immune response. PD-L1 is basally expressed in epithelial tissues such as the placenta, lung, and liver, as well as antigen-presenting cells, and can be highly expressed in various tumor cells after induction by IFN-γ, oncogenic signals, or viral infection. After binding to the PD-1 (programmed death-1) receptor on the surface of T cells, PD-L1 recruits SHP-2 phosphatase, blocking the downstream PI3K-AKT, ZAP70, and Ras-MAPK signaling pathways of the TCR, inducing T cell exhaustion, proliferation inhibition, and apoptosis, thereby participating in the negative regulation of the immune response. Genetically engineered anti-PD-1 / PD-L1 monoclonal antibodies can effectively and specifically reactivate T-cell anti-tumor immunity in vitro and in vivo, showing significant efficacy against various malignant tumors such as advanced non-small cell lung cancer, melanoma, and Hodgkin's lymphoma. The toxic side effects are significantly reduced compared to traditional chemotherapy, making it a promising target for tumor immunotherapy.

[0004] Bispecific monoclonal antibodies (BsAbs), also known as "bispecific antibodies," are artificial antibodies prepared through cell fusion or recombinant DNA technology. They can specifically bind to two antigens or two different epitopes of the same antigen. 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] Normally, antibodies or fusion proteins containing the Fc domain bind to Protein A under neutral conditions and are eluted under acidic conditions. Conventional monoclonal antibodies are typically eluted with glycine elution buffer at pH 2.5–3.0, achieving good purity and recovery. However, for some structurally complex or less stable bispecific antibodies, different buffer systems and low pH conditions can significantly affect their stability, leading to a high proportion of aggregates during elution. Aggregates, as an important product-related impurity, can not only reduce the biological activity of antibody drugs but also increase the risk of immunogenicity. Therefore, there is still a need to develop an affinity chromatography elution method suitable for bispecific antibodies to reduce the proportion of aggregates and increase the proportion of monomers in the Protein A capture step. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a purification method for bispecific antibodies targeting EGFR and PD-L1. This method reduces aggregate formation of bispecific antibodies during acidic elution by optimizing the buffer type, salt concentration, and pH conditions of the Protein A affinity chromatography elution buffer, thereby increasing the monomer content of the purified product. Studies have found that adjusting the pH of the elution buffer to 3.0–3.5 and using glycine / sodium chloride, sodium citrate / sodium chloride, or sodium acetate / sodium chloride systems can significantly increase the monomer content of the bispecific antibodies.

[0007] A first aspect of the present invention provides a method for purifying a bispecific antibody targeting EGFR and PD-L1, comprising:

[0008] (1) Load the sample containing bispecific antibodies into an affinity chromatography medium that can bind to the Fc domain;

[0009] (2) Washing the affinity chromatography medium;

[0010] (3) The bispecific antibody was eluted with an elution buffer containing a buffer and sodium chloride, and the pH was 3.0 to 3.8.

[0011] (4) Collect the eluent and neutralize it to obtain the bispecific antibody with reduced aggregate content and increased monomer ratio.

[0012] In some implementations, the buffer is selected from glycine, carboxylates, or combinations thereof.

[0013] In a further embodiment, the carboxylate buffer in the elution buffer is an acetate or citrate. In some preferred embodiments, the acetate is sodium acetate. In some preferred embodiments, the citrate is sodium citrate.

[0014] In some embodiments, the concentration of the carboxylate buffer is 0.05–0.2 M; in some preferred embodiments, the concentration of the carboxylate buffer is 0.08–0.15 M; and in some more preferred embodiments, the concentration of the carboxylate buffer is 0.1 M or 0.15 M.

[0015] In some embodiments, the sodium chloride concentration in the elution buffer is 0.10–0.20 M; in some preferred embodiments, the sodium chloride concentration is 0.1 M, 0.15 M, or 0.2 M.

[0016] In some embodiments, the pH of the elution buffer is between 3.0 and 3.5; in some preferred embodiments, the pH of the elution buffer is 3.0 or 3.5.

[0017] In some preferred embodiments, the elution buffer contains 0.1 M glycine and 0.20 M sodium chloride, and has a pH of 3.5.

[0018] In some preferred embodiments, the elution buffer comprises 0.1 M sodium citrate and 0.15 M sodium chloride, and has a pH of 3.5.

[0019] In some preferred embodiments, the elution buffer comprises 0.1 M sodium acetate and 0.15 M sodium chloride, and has a pH of 3.5.

[0020] In some preferred embodiments, the elution buffer comprises 0.1 M sodium acetate and 0.15 M sodium chloride, and has a pH of 3.0.

[0021] In some preferred embodiments, the elution buffer comprises 0.1 M sodium acetate, 0.15 M sodium chloride, and 0.2 M sucrose, and has a pH of 3.0.

[0022] In some implementations, the sample used for purification is cell culture supernatant, clarified harvest fluid, or pretreated cell culture medium.

[0023] In some implementations, the affinity chromatography medium used for purification is Protein A affinity chromatography medium, Protein A derivative affinity chromatography medium, or affinity packing material containing Protein A ligand.

[0024] In some implementations, neutralization is performed using Tris-HCl buffer during purification; in some implementations, the concentration of Tris-HCl buffer is 1 M and the pH is 8.5–8.9.

[0025] In some implementations, bispecific antibodies targeting EGFR and PD-L1 include an EGFR antibody variable region, a PD-L1 antibody variable region, and an Fc domain.

[0026] In some embodiments, the variable region of the antibody targeting EGFR includes a heavy chain variable region as shown in SEQ ID NO.1 and a light chain variable region as shown in SEQ ID NO.2; in some embodiments, the variable region of the antibody targeting PD-L1 includes a heavy chain variable region as shown in SEQ ID NO.4 and a light chain variable region as shown in SEQ ID NO.5.

[0027] In some implementations, the bispecific antibody targeting EGFR and PD-L1 has a heavy chain as shown in SEQ ID NO.7 and a light chain as shown in SEQ ID NO.8.

[0028] In some implementations, the bispecific antibody targeting EGFR and PD-L1 is obtained by expression in mammalian cells; in some implementations, the mammalian cells used are HEK293 cells or CHO cells.

[0029] In this invention, the bispecific antibodies targeting EGFR and PD-L1 obtained by the purification method are analyzed by size exclusion chromatography, and the monomer ratio is not less than 80%, preferably not less than 85%, 90%, or 95%; more preferably not less than 97%, 98%, or 99%. Compared with conventional elution conditions of 0.1 M glycine and pH 3.0, the elution system screened by this invention can significantly reduce the aggregate content of bispecific antibodies in the Protein A elution step. Specifically, elution buffer EB3 containing 0.1 M glycine and 0.20 M sodium chloride at pH 3.5, elution buffer EB7 containing 0.1 M sodium citrate and 0.15 M sodium chloride at pH 3.5, and elution buffer EB10 containing 0.1 M sodium acetate and 0.15 M sodium chloride at pH 3.5 can all obtain high monomer ratios, namely 97.50%, 98.87%, and 99.01%, respectively. Furthermore, the monomer content obtained using elution buffer EB8, containing 0.1 M sodium acetate, 0.10 M sodium chloride, and at pH 3.0, reached 84.82%; adding 0.25 M sucrose to buffer EB9 further increased the monomer content to 90.65%. In terms of protein yield, EB4, EB8, EB9, and EB10 all exhibited relatively high yields. Considering monomer content, protein yield, and antigen-binding activity, EB10, EB7, EB9, and EB3 showed a superior overall balance. Attached Figure Description

[0030] Figure 1The results of SDS-PAGE analysis of the bispecific antibody targeting EGFR and PD-L1 after purification with Protein A are shown.

[0031] Figure 2 Size exclusion chromatography (SLC) results of protein A purification of bispecific antibodies targeting EGFR and PD-L1 under standard elution conditions of 0.1 M glycine and pH 3.0 (i), SLC analysis after collecting the P1 peak (ii), and further SLC analysis after collecting the P2 peak are presented.

[0032] Figure 3 The results of size exclusion chromatography analysis of bispecific antibodies obtained under different elution buffer conditions are shown. Among them, EB10, EB7, EB3, EB9 and EB8 are elution conditions with a high monomer ratio.

[0033] Figure 4 The results of ELISA detection of the binding activity of bispecific antibody samples obtained under different elution buffer conditions to EGFR (4A) and PD-L1 (4B) are presented.

[0034] Figure 5 The binding ability of bispecific antibodies obtained with different elution buffers to the PDL1 receptor on the surface of CHO-k-hPDL1 cells (5A) and MC38 (5B) cells was investigated. Example

[0035] Example 1: Construction of recombinant plasmids

[0036] The heavy chain variable region and light chain variable region of antibodies targeting EGFR and PD-L1, respectively, were synthesized (GenScript). The synthesized gene fragments were amplified by PCR. Using methods known to those skilled in the art, aEGFR VH, linker peptide L1, aPD-L1 VH, aEGFR VL, linker peptide L2, and aPD-L1 VL were cloned into the pFuse vector (InvivoGen, CA) as needed, obtaining expression vectors expressing aEGFR VH-L1-aPD-L1 VH-CH1-Fc, aEGFR VL-L1-aPD-L1 VL-CL, aEGFR VH-CH1-Fc, aEGFR VL-CL-Fc, aPD-L1 VH-CH1-Fc, and aPD-L1 VL-CL, respectively. All constructed expression vectors were verified by sequencing. The sequence information used in this invention is shown in Table 1.

[0037]

[0038] Example 2: Protein purification and elution

[0039] The two strands of the constructed antibody eukaryotic expression vector were transiently co-transfected into FreeStyle HEK293 cells (ThermoFisher): 28 ml of FreeStyle HEK 293 (3 × 10⁷ cells / ml) was seeded into a 125 ml cell culture flask. 1 ml of plasmid containing 60 µl of 293 Fectin (Invitrogen, Inc.) was added to Opti-MEM diluted with 1 ml of Opti-MEM (Invitrogen). The mixture was incubated at room temperature for 15 min. The plasmid-293 Fectin mixture was then added to the cell culture medium and cultured at 125 rpm, 37°C, and 5% CO₂. Five days after transfection, the cell culture supernatant was collected, and the antibody was purified using Protein AResin (Genscript) according to the manufacturer's instructions.

[0040] Antibody purification using Protein A Resin (Genscript), taking control condition C1 (0.1 M glycine, pH 3.0 elution buffer) as an example, is briefly described as follows: Add 5 ml of equilibration buffer (0.15 M NaCl, 20 mM Na2HPO4, pH 7.0) to the gravity column containing Protein A packing material to equilibrate the packing material. After the equilibration buffer flows out, load the sample into the gravity column. After the sample liquid has completely flowed out, wash the packing material 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., control condition C1), collect the eluent containing the target immunoglobulin, and immediately add neutralization buffer (1 M Tris-HCl, pH 8.5) to adjust the pH to 7.4.

[0041] The antibody purified using Protein A packing material was analyzed by column chromatography using a GE AKTA chromatography column: Superdex 200 Increase 10 / 300 GL gel size exclusion chromatography column. The gel size exclusion chromatography solution was PBS buffer (0.010M phosphate buffer, 0.0027M KCl, 0.14M NaCl, pH 7.4).

[0042] from Figure 2 Size exclusion chromatography results showed that, under the control elution conditions of 0.1 M glycine and pH 3.0, the left peak corresponded to aggregates with higher molecular weight, while the right peak corresponded to monomers. The aggregate content was approximately 82%, and the monomer content was approximately 18%, indicating that the sample obtained under these conventional elution conditions had a high aggregate content, which is insufficient to meet the requirements for obtaining bispecific antibodies with a high monomer ratio. Figure 1The non-reduced lanes of the SDA-PAGE plot show diffuse bands.

[0043] Example 3: Optimization and Screening of Eluents

[0044] The two strands of the constructed antibody eukaryotic expression vector were transiently co-transfected into FreeStyle HEK293 cells (ThermoFisher): 28 ml of FreeStyle HEK 293 (3 × 10⁷ cells / ml) was seeded into a 125 ml cell culture flask. 1 ml of 60 µl of 293 Fectin (Invitrogen) was added to Opti-MEM diluted with 1 ml of Opti-MEM (Invitrogen). The mixture was incubated at room temperature for 15 min. The plasmid-293 Fectin mixture was then added to the cell culture medium and cultured at 125 rpm, 37°C, and 5% CO₂. Cell culture supernatant was collected 5 days post-transfection, and the antibody was purified using Protein AResin (Genscript) according to the manufacturer's instructions.

[0045] The antibody was purified using Protein A Resin (Genscript). The purification process is briefly described below:

[0046] Add 5 ml of equilibration buffer (0.15 M NaCl, 20 mM Na2HPO4, pH 7.0) to the gravity column containing Protein A packing material to equilibrate the packing. After the equilibration buffer has flowed out, load the sample into the gravity column. After the sample liquid has completely flowed out, wash the packing material with 30 ml of equilibration buffer to remove unbound impurities. Optimize the control condition C1 (0.1 M glycine, pH 3.0) as follows:

[0047] Adjust the pH to 2.8 with EB10.1 M glycine, 0.1 M NaCl, and HCl.

[0048] Adjust the pH to 3.0 with EB20.1 M glycine, 0.15 M NaCl, and HCl.

[0049] Adjust the pH to 3.5 using EB30.1 M glycine, 0.20 M NaCl, and HCl.

[0050] Adjust the pH to 3.0 using EB40.1 M glycine, 0.15 M NaCl, 250 mM sucrose, and HCl.

[0051] Adjust the pH to 3.0 using EB50.1 M sodium citrate, 0.10 M NaCl, and HCl.

[0052] EB60.1 M sodium citrate, 0.10 M NaCl, 250 mM sucrose, HCl adjusted to pH 3.0.

[0053] Adjust the pH to 3.5 using EB70.1 M sodium citrate, 0.15 M NaCl, and HCl.

[0054] Adjust the pH to 3.0 using EB80.1 M sodium acetate, 0.10 M NaCl, and HCl.

[0055] EB90.1 M sodium acetate, 0.10 M NaCl, 250 mM sucrose, HCl adjusted to pH 3.0.

[0056] Adjust the pH to 3.5 using EB100.1 M sodium acetate, 0.15 M NaCl, and HCl.

[0057] Collect the eluent containing the target immunoglobulin and immediately add neutralization buffer (1 M Tris-HCl, pH 8.5) to adjust the pH to 7.4.

[0058] The antibody purified from Protein A resin was analyzed by column chromatography using a GE AKTA chromatography column: Superdex 200 Increase 10 / 300 GL gel size exclusion chromatography column. The solution used for gel size exclusion chromatography was PBS buffer (0.010M phosphate buffer, 0.0027M KCl, 0.14M NaCl, pH 7.4).

[0059] comprehensive Figure 3As shown in Table 2, under pH 3.5 conditions, no obvious aggregate peaks were observed in EB3, EB7, and EB10, with monomer contents reaching 97.50%, 98.87%, and 99.01%, respectively, significantly higher than the conventional glycine elution conditions at pH 3.0. Furthermore, under pH 3.0 conditions, the monomer contents of EB8 and EB9 also reached 84.82% and 90.65%, respectively, indicating that an appropriate buffer / salt / sucrose combination can also effectively reduce aggregate content. The results indicate that 0.1 M glycine / 0.20 M NaCl / pH 3.5 (EB3), 0.1 M sodium citrate / 0.15 M NaCl / pH 3.5 (EB7), 0.1 M sodium acetate / 0.15 M NaCl / pH 3.5 (EB10), 0.1 M sodium acetate / 0.10 M NaCl / pH 3.0 (EB8), and 0.1 M sodium acetate / 0.10 M NaCl / 0.25 M sucrose / pH 3.0 (EB9) are all effective elution conditions for reducing aggregate content and increasing monomer ratio. Among them, the sample obtained under EB10 conditions had the highest monomer content and higher yield than EB3 and EB7, indicating that EB10 has a better overall balance between monomer ratio and protein yield.

[0060]

[0061] As shown in Table 2, under elution conditions of pH 3.5, the monomer contents of the samples obtained from EB3, EB7, and EB10 were 97.50%, 98.87%, and 99.01%, respectively, all significantly higher than those obtained from lower pH conditions or other buffer systems such as EB1, EB2, EB5, and EB6. This indicates that specific buffer / salt combinations under pH 3.5 conditions can effectively reduce aggregate content and increase monomer proportion. Further comparison of protein yields shows that EB10 had a yield of 18.73 mg / L, higher than EB3's 4.23 mg / L and EB7's 10.52 mg / L. Therefore, EB10 represents a superior elution condition in terms of overall performance.

[0062] Example 4: Detection of antigen-binding activity of purified products with different elution buffer formulations

[0063] PD-L1 and EGFR antigens were coated separately in 96-well plates and incubated overnight at 4°C. The plates were then blocked with PBST containing 2% skim milk powder at room temperature for 1 hour. After blocking, serially diluted bispecific antibodies were added to harvest the samples, and the plates were incubated at 37°C for 1 hour. After incubation, diluted Biotin-Mouse-anti-human IgG Fc mAb (JDC10) (Cat# 9040-08, SouthernBiotech) and Streptavidin-horseradish peroxidase (Cat# 405210, Biolegend) were added for further incubation. TMB was used for colorimetric analysis, and absorbance values ​​were read at 652 nm.

[0064] As shown in Figure 4, the EGFR binding activities of samples obtained under different elution conditions were similar, suggesting that the EGFR binding activity is less affected by the aggregate content in the sample. In contrast, the PD-L1 binding activities of samples obtained under different elution conditions differed, suggesting that PD-L1 binding activity may be related to the aggregate content in the sample. Specifically, the samples obtained under EB3, EB7, and EB10 conditions had a higher monomer content, with samples obtained under EB7 and EB10 conditions exhibiting better PD-L1 binding activity, followed by those under EB9. These results suggest that reducing the aggregate content and increasing the monomer content is beneficial for improving the PD-L1 binding activity of this bispecific antibody.

[0065] Example 5: Flow cytometry detection of bispecific antibody binding to MC38 (hEGFR- / mPD-L1+) and CHO-K-hPD-L1 (hEGFR- / hPD-L1+) cells.

[0066] MC38 (hEGFR- / mPD-L1+) cells (DMEM medium containing 10% FBS, 1% penicillin-streptomycin) and CHO-K-hPD-L1 (hEGFR- / hPD-L1+) cells (50% DMEM + 50% F12 medium containing 10% FBS, 1% penicillin-streptomycin) were cultured. (MC38 cells were used to detect the binding of samples to mouse PD-L1 positive cells, and CHO-K-hPD-L1 cells were used to detect the binding of samples to human PD-L1 positive cells.) 2 x 10⁻⁶ cells were used. 5Cells were washed three times with pre-cooled PBS, blocked with 2% FBS (dissolved in PBS) for 1 h, and then incubated with samples of different concentrations at 4°C for 1 h. Unbound antibodies were washed away with 2% FBS (dissolved in PBS). Cells were then incubated with Mouse Anti-Human IgG Fc-APC (Cat#9042-11, SouthernBiotech) at 4°C for 1 h, eluted with 2% FBS (dissolved in PBS), and detected using a CytoFLEX flow cytometer (Beckman Coulter, dual-laser six-channel). Analysis was performed using CytExpert software. GraphPad Prism was used to perform nonlinear regression on the data using a log(agonist) vs. response model.

[0067] The results are as follows Figure 5 As shown, samples under different elution conditions exhibited varying PD-L1 binding activities on the cell surface. Among them, samples obtained under EB10 conditions showed the best PD-L1 binding activity on the cell surface, consistent with ELISA results. Combined with SEC analysis, it was found that EB3, EB7, EB10, EB9, and EB8 all increased the monomer content of the samples, with EB3, EB7, and EB10 showing the most significant effects (monomer content >97%), while EB9 and EB8 also performed well (monomer contents of 90.65% and 84.82%, respectively). Considering monomer content, protein yield, and cell binding activity, EB10 demonstrated a superior overall balance among these indicators.

Claims

1. A method for purifying a bispecific antibody targeting EGFR and PD-L1, characterized in that, include: (1) Load the sample containing the bispecific antibody into an affinity chromatography medium capable of binding the Fc domain; (2) Wash the affinity chromatography medium; (3) The bispecific antibody is eluted with an elution buffer containing a buffer and sodium chloride, and the pH is 3.0 to 3.5; the buffer is selected from glycine, carboxylates or combinations thereof. (4) Collect the eluent and neutralize it to obtain the bispecific antibody with reduced aggregate content and increased monomer ratio.

2. The purification method according to claim 1, characterized in that, The carboxylate buffer is selected from acetate or citrate; preferably, the buffer is selected from sodium acetate or sodium citrate.

3. The purification method according to claim 1 or 2, characterized in that, The concentration of the buffer is 0.05–0.2 M, preferably 0.08–0.15 M, and more preferably 0.1 M; The sodium chloride concentration is 0.10–0.20 M, preferably 0.10 M, 0.15 M, or 0.20 M; The elution buffer has a pH of 3.0 or 3.

5.

4. The purification method according to any one of claims 1-3, characterized in that, The elution buffer is selected from any of the following: (a) An elution buffer containing 0.1 M glycine and 0.20 M sodium chloride at a pH of 3.5; (b) An elution buffer containing 0.1 M sodium citrate and 0.15 M sodium chloride at a pH of 3.5; (c) An elution buffer containing 0.1 M sodium acetate and 0.15 M sodium chloride at a pH of 3.5; (d) Elution buffer containing 0.1 M sodium acetate and 0.1 M sodium chloride, with a pH of 3.0; (e) An elution buffer containing 0.1 M sodium acetate, 0.1 M sodium chloride and 0.25 M sucrose at pH 3.

0.

5. The purification method according to any one of claims 1-4, characterized in that, The sample is cell culture supernatant, clarified harvest liquid, or pretreated cell culture medium.

6. The purification method according to any one of claims 1-5, characterized in that, The affinity chromatography medium is a Protein A affinity chromatography medium, a Protein A derivative affinity chromatography medium, or an affinity packing material containing a Protein A ligand.

7. The purification method according to any one of claims 1-6, characterized in that, The neutralization is performed using Tris-HCl buffer; the concentration of the Tris-HCl buffer is preferably 1 M, and the pH is preferably 8.5 to 8.

9.

8. The purification method according to any one of claims 1-7, characterized in that, The bispecific antibody includes an EGFR antibody variable region, a PD-L1 antibody variable region, and an Fc domain.

9. The purification method according to claim 8, characterized in that, The variable region of the EGFR antibody includes a heavy chain variable region as shown in SEQ ID NO.1 and a light chain variable region as shown in SEQ ID NO.2, and the variable region of the PD-L1 antibody includes a heavy chain variable region as shown in SEQ ID NO.4 and a light chain variable region as shown in SEQ ID NO.5; The bispecific antibody has a heavy chain as shown in SEQ ID NO.7 and a light chain as shown in SEQ ID NO.

8.

10. The purification method according to any one of claims 1-9, characterized in that, The bispecific antibody is obtained by expression in mammalian cells, preferably by expression in HEK293 cells or CHO cells.

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