A method for purifying a bispecific antibody and use thereof

Abstract

This invention belongs to the field of antibody purification, specifically relating to a purification method and application of bispecific antibodies. The purification method includes complex chromatography (a salt-resistant anion exchange chromatography with a hydrophobic mode, referred to herein as complex chromatography). This method not only improves the purity of bispecific antibodies and reduces protein damage, but also simplifies the operation process, reduces costs, and has strong adaptability. This invention also provides an optimization of the elution method by adding arginine to the complex chromatography, further improving purity. The purification method provided by this invention has significant application value in the field of bispecific antibody purification.

Description

Technical Field

[0001] This invention belongs to the field of antibody purification, specifically relating to a purification method and application of bispecific antibodies. Background Technology

[0002] Bispecific antibodies are synthetic antibodies that possess the specificity for two different antigens. They can bind to two different antigens simultaneously to achieve a variety of biological effects. Furthermore, they offer advantages such as reduced side effects, enhanced pharmacological activity, and higher drug delivery efficiency.

[0003] Bispecific antibodies play a crucial role in cancer treatment. They can simultaneously bind to two different tumor antigens or receptors on the surface of tumor cells and receptors on the surface of immune cells (such as T cells and NK cells), thereby stimulating the killing activity of immune cells and achieving precise targeting of tumor cells. This dual mechanism of action significantly enhances the efficacy of cancer treatment and reduces damage to normal tissues. Bispecific antibodies are also used in the treatment of autoimmune diseases. Autoimmune diseases are conditions in which the immune system mistakenly attacks its own tissues and organs. Bispecific antibodies can regulate the activity of the immune system by binding to different immune-related molecules, thereby reducing or eliminating the damage to tissues caused by autoimmune responses. Therefore, bispecific antibodies have broad application prospects and enormous potential in the medical field.

[0004] However, in the fields of biotechnology, immunology, and pharmaceuticals, the purification of bispecific antibodies is a crucial technical step. Bispecific antibodies can bind to two different targets simultaneously, making their purification process more complex and producing more byproducts compared to monospecific antibodies. This is because the design of bispecific antibodies often results in the generation of various byproducts, such as mismatches, incomplete fragments, and aggregates, increasing the difficulty of purification. Therefore, efficient purification methods are needed to ensure the purity and activity of the antibodies.

[0005] Currently, the purification process for bispecific antibodies is complex, and the final sample often contains high levels of impurities and low yields. Therefore, providing a purification method to improve yield and purity is crucial for drug development. Summary of the Invention

[0006] This invention addresses the shortcomings of ion exchange chromatography and hydrophobic chromatography in purifying bispecific antibodies through complex chromatography, and the addition of arginine further enhances the purity of the purified product. This invention provides a novel method for purifying bispecific antibodies that is not only highly efficient and economical but also improves protein quality and yield.

[0007] On the one hand, the present invention provides a method for purifying bispecific antibodies, comprising the following steps:

[0008] S1. Affinity purification of the bispecific antibody to obtain purified product A;

[0009] S2. Perform compound anion purification on the above-mentioned purified product A to obtain the purified antibody;

[0010] The affinity purification packing material is protein A affinity packing material;

[0011] The packing material for the composite anion purification is a salt-resistant anion exchange chromatography packing material.

[0012] Specifically, the protein A affinity packing material can be selected from MabSelect SURE LX, MabSelec PrismA, NMabPro, NMab, UniMab 50HC ProteinA, UniMab 50Protein A, ATProteinADiamond, AT ProteinA Diamond Plus, AT Protein A Diamond Plus, Novo-A Diamond, or Extrem ADiamond; the salt-resistant anion exchange chromatography packing material can be selected from TOYOPEARL NH2-750F, DEAE-cellulose, Q-cellulose, or DEAE-carboxymethyl cellulose.

[0013] Preferably, the protein A affinity packing material can be MabSelect SURE LX; the salt-resistant anion exchange chromatography packing material can be TOYOPEARL NH2-750F.

[0014] Specifically, the affinity purification includes pre-equilibration, sample loading, equilibration, rinsing, post-equilibration, and elution; the buffers for pre-equilibration, equilibration, and post-equilibration include 15-25 mM PB, 130-170 mM NaCl, pH 6.5-7.5; the sample loading capacity is 3000-4000 mL; the rinsing buffer includes 15-25 mM MPB, 0.5-1.5 M NaCl, pH 6.0-7.0; and the elution buffer includes 15-25 mM sodium citrate, pH 3.0-4.0.

[0015] Preferably, the pre-equilibration, equilibration, and post-equilibration buffers comprise 20-25 mM PB, 150-170 mM NaCl, pH 7.0-7.5; the sample loading capacity is 3000-3500 mL; the elution buffer comprises 20-25 mM PB, 0.1-1.5 M NaCl, pH 6.5-7.0; and the elution buffer comprises 20-25 mM sodium citrate, pH 3.5-4.0.

[0016] More preferably, the pre-equilibration, equilibration, and post-equilibration buffers comprise 20 mM PB, 150 mM NaCl, pH 7.2; the sample loading capacity is 3168 mL; the elution buffer comprises 20 mM PB, 1 M NaCl, pH 6.5; and the elution buffer comprises 20 mM sodium citrate, pH 3.5.

[0017] Specifically, the purification of the complex anion includes pre-equilibration, sample loading, equilibration, and elution; the pre-equilibration and equilibration buffers include 15-25 mM Tris-HCl, pH 7.5-8.5; the sample loading volume is 10-20 mL; the elution buffer includes 15-25 mM Tris-HCl, 0.5-1.5 M NaCl, pH 7.5-8.5.

[0018] Preferably, the pre-equilibration and equilibration buffers comprise 20-25 mM Tris-HCl, pH 8.0-8.5; the loading volume is 10-15 mL; and the elution buffer comprises 20-25 mM Tris-HCl, 1-1.5 M NaCl, pH 8.0-8.5.

[0019] More preferably, the pre-equilibration and equilibration buffers comprise 20 mM Tris-HCl, pH 8.0; the loading volume is 14 mL; and the elution buffer comprises 20 mM Tris-HCl, 1 M NaCl, pH 8.0.

[0020] Specifically, the eluent may also include arginine.

[0021] Specifically, the concentration of arginine can be 0.5-1.5M.

[0022] Preferably, the concentration of arginine is 1-1.2M.

[0023] More preferably, the concentration of arginine is 1M.

[0024] Specifically, the bispecific antibody is an Anti-LAG-3 & PD-1 bispecific antibody.

[0025] More specifically, the heavy chain amino acid sequence of the Anti-LAG-3 & PD-1 bispecific antibody is shown in SEQ ID No. 7-SEQ ID No. 8; the light chain amino acid sequence is shown in SEQ ID No. 5-SEQ ID No. 6.

[0026] On the one hand, the present invention provides the application of the aforementioned purification method in the preparation of bispecific antibodies.

[0027] The technical effects achieved by this invention are as follows:

[0028] (1) Improve purity: This invention utilizes the composite mode packing material TOYOPEARL NH2-750F, which can improve the purity of bispecific antibodies from 80-90% to 92-93%. Further addition of the excipient arginine can improve the purity of the eluted sample in the composite mode to about 99%.

[0029] (2) By using TOYOPEARL NH2-750F composite mode chromatography, the purification resolution was improved, the purity of the antibody was ensured, and the purification difficulty was reduced.

[0030] (3) Reduce protein damage: In the purification process, this invention minimizes the loss of protein activity or structural damage by optimizing the use and operating conditions of the column packing material.

[0031] (4) Simplified operation process and reduced cost: The purification method of the present invention has been optimized based on the existing technology, which simplifies the operation process and reduces the cost.

[0032] (6) High adaptability: The purification method of the present invention is not only applicable to specific bispecific antibodies, but can also be adapted to bispecific antibodies of different types and sources by adjusting the column packing and operating conditions, thus exhibiting strong adaptability. Attached Figure Description

[0033] Figure 1 This is a structural diagram of a bispecific antibody.

[0034] Figure 2 The pCDNA3.1(+) vector map.

[0035] Figure 3 This is an AC SEC-HPLC chromatogram for affinity chromatography.

[0036] Figure 4 This is a composite chromatography E2 SEC-HPLC chromatogram.

[0037] Figure 5 This is the E6 SEC-HPLC chromatogram.

[0038] Figure 6 This is a composite chromatography-Arg E1 SEC-HPLC chromatogram.

[0039] Figure 7 This is a composite chromatography-Arg E2 SEC-HPLC chromatogram.

[0040] Figure 8 This is a composite chromatography-Arg E3 SEC-HPLC chromatogram.

[0041] Figure 9This is a composite chromatography-Arg E4 SEC-HPLC chromatogram.

[0042] Figure 10 This is a composite chromatography-Arg E5 SEC-HPLC chromatogram.

[0043] Figure 11 This is a SEC-HPLC chromatogram of cation CEX E1.

[0044] Figure 12 This is a SEC-HPLC chromatogram of cation CEX E2.

[0045] Figure 13 This is a SEC-HPLC chromatogram of cation CEX E3.

[0046] Figure 14 This is a SEC-HPLC chromatogram of cationic CEX E4.

[0047] Figure 15 This is a SEC-HPLC chromatogram of cation CEX E5.

[0048] Figure 16 This is a SEC-HPLC chromatogram of cation CEX E6.

[0049] Figure 17 This is the SEC-HPLC chromatogram of anion AEX E1.

[0050] Figure 18 This is the SEC-HPLC chromatogram of anion AEX E2.

[0051] Figure 19 This is the SEC-HPLC chromatogram of anion AEX E3.

[0052] Figure 20 This is the SEC-HPLC chromatogram of anion AEX E4.

[0053] Figure 21 This is the SEC-HPLC chromatogram of anion AEX E5.

[0054] Figure 22 This is the SEC-HPLC chromatogram of the anion AEX E6.

[0055] Figure 23 This is a hydrophobic chromatography (HIC-E2) SEC-HPLC chromatogram.

[0056] Figure 24 This is a hydrophobic chromatography (HIC-E6) SEC-HPLC chromatogram.

[0057] Figure 25 This is a hydrophobic chromatography (HIC-E8) SEC-HPLC chromatogram. Detailed Implementation

[0058] The present invention will be further described in detail below with reference to specific embodiments. The following embodiments are not intended to limit the present invention, but only to illustrate the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are generally performed under conventional conditions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0059] The abbreviations used in this invention are shown in Table 1:

[0060] Table 1

[0061]

[0062] In this invention, the MabSelect SURE LX affinity chromatography column, cation Capto S impact column, and anion purification column Q-HP were purchased from Cytiva; the TOYOPEARL phenyl-650S hydrophobic chromatography column and TOYOPEARL NH2-750F were purchased from TOSOH; the ultrafiltration concentrator was purchased from Millipore; the 0.22 μm filter membrane was purchased from Pall; and DPBS was purchased from Gibco. All reagents were of analytical grade and purchased from Sigma-Aldrich.

[0063] Example 1

[0064] 1.1 Antibody

[0065] 1.1.1 Information on the bispecific antibody molecules Anti-LAG3 & PD-1 (259-260-222-258):

[0066] Protein molecular weight: 145.2 kDa; Protein PI: 6.87; Protein extinction coefficient: 1.43; Bispecific antibody structure diagram as shown. Figure 1 As shown.

[0067] Antibody drugs targeting PD-1 / PD-L1 (programmed death receptor 1 / programmed death ligand 1) are the most successful "broad-spectrum anti-cancer" drug targets. However, the efficacy of PD-1 antibody monotherapy is limited. Developing dual-target, multi-target combination therapies, or bifunctional antibody drugs, primarily based on PD-1 antibodies, will become the future direction of cancer treatment. Among these, LAG-3 (lymphocyte activation gene 3) is an important immune checkpoint molecule, and clinical trials have demonstrated that the combined use of LAG-3 antibodies and PD-1 antibodies has a synergistic anti-tumor effect.

[0068] 1.1.2 Amino acid sequence

[0069] RYPE222 (Anti-LAG3 light chain portion) (SEQ ID No. 1):

[0070]

[0071] RYPE258 (Anti-PD-1 light chain portion) (SEQ ID No. 2):

[0072]

[0073] RYPE259 (Anti-LAG3 heavy chain portion) (SEQ ID No. 3):

[0074]

[0075]

[0076] RYPE260 (Anti-PD-1 heavy chain portion) (SEQ ID No. 4):

[0077]

[0078] 1.1.3 The DNA sequence encoding the above amino acid sequence;

[0079] RYPE222 (Anti-LAG3 light chain portion) (SEQ ID No. 5):

[0080]

[0081] RYPE258 (Anti-PD-1 light chain portion) (SEQ ID No. 6):

[0082]

[0083] RYPE259 (Anti-LAG3 heavy chain portion) (SEQ ID No. 7):

[0084]

[0085]

[0086] RYPE260 (Anti-PD-1 heavy chain portion) (SEQ ID No. 8):

[0087]

[0088]

[0089] 1.2 Expression and Cell Culture

[0090] The vectors used to produce the above antibodies include mammalian cell expression vectors (pCDNA3.1+), and a vector map is shown below. Figure 2 The mammalian cell line used was HEK293. The above sequence was synthesized and constructed into an expression vector through gene synthesis. Then, HEK293 cells were transiently transfected, cultured, and the cell culture supernatant containing the target antibody was collected.

[0091] 1.3 Preliminary affinity purification

[0092] Affinity chromatography was performed using Cytiva's MabSelect SURE LX column packing material, with a column volume of 12 mL and a flow rate controlled at 2.4 mL / min. The column was equilibrated with 20 mM PB and 150 mM NaCl at pH 7.2. The sample consisted of 3168 mL of cell culture supernatant containing the target antibiotic, and the eluent was sodium citrate citrate at pH 3.5. The product was collected. The pH of the product was adjusted to 5.5 using neutralization buffer, and the product was filtered through a 0.22 μm filter. The product concentration was determined using a micro-volume analyzer based on the extinction coefficient, and the purity of the product was determined by SEC-HPLC.

[0093] The specific purification process is shown in Table 2:

[0094] Table 2

[0095]

[0096]

[0097] The determination of product concentration and purity (SEC-HPLC) is shown in Table 3:

[0098] Table 3

[0099]

[0100] 1.4 Purification

[0101] Sample source: 100mAU - Max - 80mAU, pH 5.5;

[0102] Purification pathway: purification of complex anions.

[0103] The affinity-eluted product collected in step 1.3 was purified using a TOYOPEARL NH2-750F composite packing column with a packing volume of 1 mL and a flow rate of 0.2 mL / min. The eluent was 20 mM Tris-HCl pH 8.0 containing 1 M NaCl. The eluted sample was collected, and the concentration and purity of the product were determined (SEC-HPLC).

[0104] The specific purification process is shown in Table 4:

[0105] Table 4

[0106] Packing material name / manufacturer <![CDATA[TOYOPEARL NH2-750F / TOSOH]]> step buffer solution Pre-disinfection 0.1M NaOH Pre-balancing (pipeline A) 20mM Tris-HCl pH 8.0 Sample Sample loading volume: 14 mL (adjust sample pH to match equilibration solution) Balance (pipeline A) 20mM Tris-HCl pH 8.0 Elution (Pipe B) 20mM Tris-HCl 1M NaCl pH8.0 (0-30%B, 15CV)

[0107] Note: 0-30% B, 15CV indicates that the elution conditions are 0-30% B gradient elution, and the elution volume is 15 column volumes.

[0108] Purity determination (SEC-HPLC, selective detection by peak shape) Table 5:

[0109] Table 5

[0110]

[0111] Note: NH2-750F-E2 indicates the sample collected after elution in the second column volume using TOYOPEARL NH2-750F composite packing material; NH2-750F-E6 indicates the sample collected after elution in the sixth column volume using TOYOPEARL NH2-750F composite packing material.

[0112] Example 2

[0113] The difference between Example 2 and Example 1 is that 1M arginine was added to the eluent. Specifically, the affinity-eluted product collected in step 1.3 was purified using a TOYOPEARL NH2-750F composite packing column with a packing volume of 1 mL and a flow rate controlled at 0.2 mL / min. The eluent was 20 mM Tris-HCl at pH 8.0, containing 1M NaCl and 1M arginine. The eluted sample was collected, and the concentration and purity of the product were determined (SEC-HPLC).

[0114] Purity determination (SEC-HPLC) Table 6:

[0115] Table 6

[0116]

[0117] Note: NH2-750F-Arg-E1 indicates that the sample was collected after elution with the first column volume of TOYOPEARL NH2-750F composite packing material, and the eluent contained arginine; NH2-750F-Arg-E2 indicates that the sample was collected after elution with the second column volume of TOYOPEARL NH2-750F composite packing material, and the eluent contained arginine; the definitions are as follows.

[0118] Samples with a purity >98% were combined and concentrated using an ultrafiltration concentrator. The concentrate was then transferred to DPBS. After concentration and concentrate transfer, the samples were filtered through a 0.22 μm filter membrane and aliquoted into 1.5 mL centrifuge tubes, 2 mg per tube, and stored at -80°C. The final sample purity was 99.18%, and the storage system was DPBS.

[0119] The results showed that arginine helps protein refolding, increases protein solubility, improves protein yield, and reduces aggregate content.

[0120] Comparative Example 1

[0121] The difference between Comparative Example 1 and Example 1 is that the purification path is different, but otherwise the same as Example 1.

[0122] Purification path: CEX.

[0123] The affinity-eluted product collected in step 1.3 was purified using a cationic Capto S impactor. The column volume was 1 mL, the flow rate was controlled at 0.2 mL / min, and the eluent was 20 mM sodium citrate citrate, pH 5.5, containing 1 M NaCl. The cationic eluted sample (CEX) was collected, and the concentration and purity of the product were determined (SEC-HPLC).

[0124] The specific purification process is shown in Table 7:

[0125] Table 7

[0126] Packing material name / manufacturer Capto S impact / cytiva step buffer solution Pre-disinfection 0.1M NaOH Pre-balancing (pipeline A) <![CDATA[20mM Cit-Na3Cit pH5.5]]> Sample Sample loading volume: 25 mL Balance (pipeline A) <![CDATA[20mM Cit-Na3Cit pH5.5]]> Elution (Pipe B) <![CDATA[20mM Cit-Na3Cit 1M NaCl pH5.5(0-30%B,20CV)]]>

[0127] Note: 0-30%B, 20CV indicates that the elution conditions are 0-30%B gradient elution, and the elution volume is 20 column volumes.

[0128] The determination of product concentration and purity (SEC-HPLC) is shown in Table 8:

[0129] Table 8

[0130]

[0131]

[0132] Note: CEX-E1 represents the sample collected after elution with the first column volume of the cation Capto S impact; CEX-E2 represents the sample collected after elution with the second column volume of the cation Capto S impact; and so on.

[0133] Comparative Example 2

[0134] The difference between Comparative Example 1 and Example 1 is that the purification path is different, but otherwise the same as Example 1.

[0135] Purification path: AEX.

[0136] The affinity-eluted product collected in step 1.3 was purified using anionic Q-HP with a column volume of 1 mL and a flow rate of 0.2 mL / min. The eluent was 20 mM Tris-HCl at pH 8.0 containing 1 M NaCl. The anionic eluted sample (AEX) was collected, and the concentration and purity of the product were determined (SEC-HPLC).

[0137] The specific purification process is shown in Table 9:

[0138] Table 9

[0139] Packing material name / manufacturer Q-HP / cytiva step buffer solution Pre-disinfection 0.1M NaOH Pre-balancing (pipeline A) 20mM Tris-HCl pH 8.0 Sample Sample loading volume: 15 mL (adjust the sample pH to match the equilibration solution) Balance (pipeline A) 20mM Tris-HCl pH 8.0 Elution (Pipe B) 20mM Tris-HCl 1M NaCl pH8.0 (0-30%B, 15CV)

[0140] Note: 0-30% B, 15CV indicates that the elution conditions are 0-30% B gradient elution, and the elution volume is 15 column volumes.

[0141] The determination of product concentration and purity (SEC-HPLC) is shown in Table 10:

[0142] Table 10

[0143]

[0144]

[0145] Note: AEX-E1 represents the sample collected after elution with the first column volume of anionic Q-HP; AEX-E2 represents the sample collected after elution with the second column volume of anionic Q-HP; and so on.

[0146] Comparative Example 3

[0147] The difference between Comparative Example 1 and Example 1 is that the purification path is different, but otherwise the same as Example 1.

[0148] Purification method: hydrophobic chromatography.

[0149] The affinity-eluted product collected in step 1.3 was adjusted to final concentrations of 0.5 mM, 0.75 mM, 1 mM, 1.25 mM, and 1.5 mM ammonium sulfate using 20 mM PB + 3 M (NH4)2SO4 at pH 8.0. After adding ammonium sulfate, a salting-out reaction occurred, resulting in precipitation. The concentration was measured, and based on the recovery rate and SEC-HPLC results, 0.75 mM ammonium sulfate was confirmed as the initial salt concentration for the equilibration buffer. The conductivity and pH of the sample were adjusted to match the equilibration buffer using 20 mM PB + 3 M ammonium sulfate. The equilibration buffer was 20 mM PB + 0.75 M ammonium sulfate at pH 8.0. Purification was performed using a TOYOPEARL phenyl-650S column from TOSOH, with a column volume of 1 mL and a flow rate controlled at 0.2 mL / min. The eluent was 20 mM PB at pH 8.0, and the elution method was a 0-100% β gradient elution. The hydrophobic elution sample (HIC) was collected.

[0150] The small-scale salting-out experiment is shown in Table 11:

[0151] Table 11

[0152]

[0153] 0.75 mM ammonium sulfate was selected as the initial salt concentration.

[0154] The specific purification process is shown in Table 12:

[0155] Table 12

[0156]

[0157] Note: 0-100%B, 20CV indicates that the elution conditions are 0-100%B gradient elution, and the elution volume is 20 column volumes.

[0158] The determination of product concentration and purity (SEC-HPLC, selective detection based on peak shape) is shown in Table 13:

[0159] Table 13

[0160]

[0161] Note: HIC-E2 indicates the sample collected after elution in the second column volume of hydrophobic chromatography; HIC-E6 indicates the sample collected after elution in the sixth column volume of hydrophobic chromatography; the definitions are sequential.

[0162] Analysis of four purification pathways revealed that ionic and hydrophobic methods could not yield proteins with a purity greater than 90%, while complex chromatography could achieve a purity of 92-93%. Adding arginine to the complex chromatography process further improved protein purity to approximately 99%, meeting the requirements for large-scale production.

Claims

1. A method for purifying a bispecific antibody, characterized in that, Includes the following steps: S1. Affinity purification of the bispecific antibody to obtain purified product A; S2. Perform compound anion purification on the above-mentioned purified product A to obtain the purified antibody; The affinity purification packing material is protein A affinity packing material; The packing material for the composite anion purification is a salt-resistant anion exchange chromatography packing material.

2. The purification method according to claim 1, characterized in that, The protein A affinity packing material is selected from MabSelectSURE LX, MabSelec PrismA, NMab Pro, NMab, UniMab 50HC ProteinA, UniMab 50ProteinA, ATProteinADiamond, AT Protein A Diamond Plus, AT Protein A Diamond Plus, Novo-ADiamond, or Extrem ADiamond; the salt-resistant anion exchange chromatography packing material is selected from TOYOPEARL NH2-750F, DEAE-cellulose, Q-cellulose, or DEAE-carboxymethyl cellulose.

3. The purification method according to claim 2, characterized in that, The protein A affinity packing material is MabSelectSURE LX; the salt-resistant anion exchange chromatography packing material is TOYOPEARL NH2-750F.

4. The purification method according to claim 1, characterized in that, The affinity purification includes pre-equilibration, sample loading, equilibration, rinsing, post-equilibration, and elution; the buffer solutions for pre-equilibration, equilibration, and post-equilibration are 15-25 mM PB and 130-170 mM NaCl, pH 6.5-7.5; the sample loading capacity is 3000-4000 mL; the rinsing buffer is 15-25 mM PB and 0.5-1.5 M NaCl, pH 6.0-7.0; and the elution buffer is 15-25 mM sodium citrate, pH 3.0-4.

0.

5. The purification method according to claim 4, characterized in that, The pre-equilibration, equilibration, and post-equilibration buffers were 20 mM PB, 150 mM NaCl, pH 7.2; the sample loading capacity was 3168 mL; the elution buffer was 20 mM PB, 1 M NaCl, pH 6.5; and the elution buffer was 20 mM sodium citrate, pH 3.

5.

6. The purification method according to claim 1, characterized in that, The purification of the complex anion includes pre-equilibration, sample loading, equilibration, and elution; the pre-equilibration and equilibration buffer is 15-25 mM Tris-HCl, pH 7.5-8.5; the sample loading volume is 10-20 mL; the elution buffer is 15-25 mM Tris-HCl, 0.5-1.5 M NaCl, pH 7.5-8.

5.

7. The purification method according to claim 6, characterized in that, The pre-equilibration and equilibration buffers are 20 mM Tris-HCl, pH 8.0; the loading volume is 14 mL; the elution buffer is 20 mM Tris-HCl, 1 M NaCl, pH 8.

0.

8. The purification method according to claim 7, characterized in that, The eluent also includes arginine.

9. The purification method according to claim 8, characterized in that, The concentration of arginine is 0.5-1.5M.

10. The purification method according to claim 9, characterized in that, The concentration of arginine is 1M.

11. The purification method according to any one of claims 1-10, characterized in that, The bispecific antibody mentioned is an Anti-LAG-3 & PD-1 bispecific antibody.

12. The use of the purification method according to any one of claims 1-11 in the preparation of bispecific antibodies.

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