Method for preparing bevacizumab

Abstract

The invention belongs to the field of protein purification, and relates to a method for preparing bevacizumab, which comprises the steps of affinity chromatography, virus inactivation, deep filtration, composite anion exchange chromatography, cation exchange chromatography and the like.

Description

Technical Field

[0001] This application belongs to the field of protein purification, and particularly relates to a method for preparing or purifying bevacizumab. Background Art

[0002] Vascular Endothelial Growth Factor (VEGF) is a functional protein that specifically acts on vascular endothelial cells and has functions such as promoting increased vascular permeability, migration and proliferation of vascular endothelial cells, extracellular matrix denaturation, and angiogenesis. VEGF is expressed in a variety of malignant tumors and is involved in tumor angiogenesis, so it has become an important target for the development of anti-tumor drugs. Currently, VEGF-targeted inhibitors have improved the prognosis and survival of many cancer patients. Bevacizumab is a humanized anti-VEGF monoclonal antibody prepared using recombinant DNA technology and is widely used in the treatment of cancers such as colorectal cancer, lung cancer, glioblastoma, renal cancer, cervical cancer, ovarian cancer, fallopian tube cancer, peritoneal cancer, and liver cancer.

[0003] With the continuous expansion of clinical demand, more and more monoclonal antibody drugs have been approved, and enterprises have higher and higher requirements for the efficiency and cost of production processes. Typical monoclonal antibody purification methods generally include three steps: first, using affinity chromatography to capture the target protein in the cell culture supernatant, then using two-step ion exchange chromatography or ion exchange plus hydrophobic chromatography for fine purification, and in addition, a virus inactivation step after the affinity chromatography step. The new chromatography mode, mixed-mode chromatography (MMC), can strengthen the affinity and selectivity for the target protein and further improve the purification efficiency by optimizing the structure of the functional ligand and combining two or more interaction modes. Compared with traditional ion exchange chromatography, MMC has advantages such as high load capacity, high selectivity, and high efficiency. MMC mainly includes hydrophobic and ion exchange mixed-mode chromatography, hydrophilic and ion exchange mixed-mode chromatography, hydrophobic and hydrophilic mixed-mode chromatography, etc. Among them, the ligand of the hydrophobic / ion exchange mixed-mode chromatography medium contains both hydrophobic and electrostatic groups, which can provide both hydrophobic and electrostatic interactions simultaneously.

[0004] Monoclonal antibody drugs have complex quality attributes. The chromatography packing materials and step conditions in the purification process will affect the drug quality attributes, and small changes in the quality attributes will have a significant impact on the safety and effectiveness of the drug. In production practice, for different monoclonal antibody drugs, it is still necessary to continuously optimize the production process to control production costs and meet the corresponding quality standards. Summary of the Invention

[0005] The purpose of the present application is at least to provide an optimized bevacizumab purification process suitable for industrial scale-up production.

[0006] On the one hand, the present application provides a method for purifying bevacizumab, the method comprising purifying bevacizumab by composite anion exchange chromatography.

[0007] In some embodiments, the composite anion exchange chromatography comprises the steps of adjusting the pH and conductivity of the sample loading solution before sample loading.

[0008] In some embodiments, the composite anion exchange chromatography comprises the steps of: ① equilibration 1, ② adjusting the pH and conductivity of the sample loading solution, ③ sample loading, and ④ equilibration 2. In some embodiments, the steps are carried out in the order shown, wherein the order of the two steps of equilibration 1 and adjusting the pH and conductivity of the sample loading solution can be interchanged.

[0009] In some embodiments, the composite anion exchange chromatography has ion exchange, hydrophobic and / or hydrogen bond interactions. In some embodiments, the composite anion exchange chromatography has ion exchange and hydrophobic interactions. In some embodiments, the composite anion exchange chromatography has ion exchange and hydrogen bond interactions. In some embodiments, the composite anion exchange chromatography has hydrophobic and hydrogen bond interactions. In some embodiments, the composite anion exchange chromatography has ion exchange, hydrophobic and hydrogen bond interactions.

[0010] In some embodiments, the pH and conductivity of the sample loading solution are adjusted to be the same as those of the equilibration solution. In some embodiments, the conductivity of the sample loading solution is adjusted to be the same as that of the equilibration solution, and the pH of the sample loading solution is adjusted to be different from that of the equilibration solution. In some embodiments, the conductivity of the sample loading solution is adjusted to be different from that of the equilibration solution, and the pH of the sample loading solution is adjusted to be the same as that of the equilibration solution. In some embodiments, the pH and conductivity of the sample loading solution are adjusted to be different from those of the equilibration solution.

[0011] In some embodiments, the conductivity of the adjusted sample loading solution is 3.0 - 10.0 mS / cm, 4.0 - 8.0 mS / cm, or 4.0 - 6.0 mS / cm, such as about 3.0 mS / cm, about 3.5 mS / cm, about 4.0 mS / cm, about 4.5 mS / cm, about 5.0 mS / cm, about 5.5 mS / cm, about 6.0 mS / cm, about 6.5 mS / cm, about 7.0 mS / cm, about 7.5 mS / cm, about 8.0 mS / cm, about 8.5 mS / cm, about 9.0 mS / cm, about 9.5 mS / cm, or about 10 mS / cm, or a range formed by any of the foregoing values, preferably 4.0 - 6.0 mS / cm.

[0012] In some embodiments, the pH of the adjusted sample loading solution is 5.0 - 8.5, 5.5 - 8.0 or 7.0 - 8.0, such as about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, or a range formed by any of the foregoing values, preferably 7.5 ± 0.2.

[0013] In some embodiments, the pH of the sample loading solution is adjusted first, and then the conductivity of the sample loading solution is adjusted. In some embodiments, the conductivity of the sample loading solution is adjusted first, and then the pH of the sample loading solution is adjusted. In some embodiments, the pH and conductivity of the sample loading solution are adjusted simultaneously.

[0014] In some embodiments, the adjusting solution for adjusting the pH of the sample loading solution is 2 mol / L Tris-HCl solution. In some embodiments, the adjusting solution for adjusting the conductivity of the sample loading solution is 1 mol / L sodium chloride solution or water for injection.

[0015] In some embodiments, the equilibration solution is selected from 5 - 20 mmol / L Tris-HCl buffer containing 20 - 50 mmol / L sodium chloride, preferably 10 mmol / L Tris-HCl buffer containing 35 mmol / L sodium chloride.

[0016] In some embodiments, the pH of the equilibration solution is 5.0 - 8.5, 5.5 - 8.0 or 7.0 - 8.0, such as about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, or a range formed by any of the foregoing values, preferably 7.5 ± 0.2.

[0017] In some embodiments, the conductivity of the equilibration solution is 3.0 - 10.0 mS / cm, 4.0 - 8.0 mS / cm, or 4.0 - 6.0 mS / cm, such as approximately 3.0 mS / cm, approximately 3.5 mS / cm, approximately 4.0 mS / cm, approximately 4.5 mS / cm, approximately 5.0 mS / cm, approximately 5.5 mS / cm, approximately 6.0 mS / cm, approximately 6.5 mS / cm, approximately 7.0 mS / cm, approximately 7.5 mS / cm, approximately 8.0 mS / cm, approximately 8.5 mS / cm, approximately 9.0 mS / cm, approximately 9.5 mS / cm, or approximately 10.0 mS / cm, or a range formed by any of the foregoing values, preferably 4.0 - 6.0 mS / cm.

[0018] In some embodiments, the composite anion exchange chromatography packing material is selected from: NM90 Agarose HAM or DiamondMix-A, preferably NM90 Agarose HAM.

[0019] In a specific embodiment, the composite anion exchange chromatography includes the steps of: ① Equilibration 1, equilibrating with a 10 mmol / L Tris-HCl buffer solution (pH 7.5 ± 0.2, conductivity 4.0 - 6.0 mS / cm) containing 35 mmol / L sodium chloride; ② Sample solution adjustment, first adjusting the pH of the filtrate of the second-stage depth filtration to 7.5 ± 0.2 with a 2 mol / L Tris-HCl solution (pH 9.50 ± 0.05), and then adjusting the conductivity of the sample to 4.0 - 6.0 mS / cm with a 1 mol / L sodium chloride solution or water for injection; ③ Loading, collecting the flow-through solution; ④ Equilibration 2, equilibrating the chromatography column with a 10 mmol / L Tris-HCl buffer solution (pH 7.5 ± 0.2, conductivity 4.0 - 6.0 mS / cm) containing 35 mmol / L sodium chloride. In some embodiments, the composite anion exchange chromatography uses NM90-Agarose HAM packing material.

[0020] In a specific embodiment, the composite anion exchange chromatography uses NM90 - Agarose HAM packing material, and it includes the following steps: ① Equilibration 1, equilibrate with a 10 mmol / L Tris - HCl buffer solution (pH 7.5 ± 0.2, conductivity 4.0 - 6.0 mS / cm) containing 35 mmol / L sodium chloride; ② Sample solution adjustment, first adjust the pH of the filtrate of the second - step depth filtration to 7.5 ± 0.2 with a 2 mol / L Tris - HCl solution (pH 9.50 ± 0.05), and then adjust the conductivity of the sample to 4.0 - 6.0 mS / cm with a 1 mol / L sodium chloride solution or water for injection; ③ Loading, collect the flow - through solution; ④ Equilibration 2, equilibrate the chromatography column with a 10 mmol / L Tris - HCl buffer solution (pH 7.5 ± 0.2, conductivity 4.0 - 6.0 mS / cm) containing 35 mmol / L sodium chloride for 10 - 30 column volumes (CV), and end the collection of the flow - through solution after 10 - 30 CV. In some embodiments, the method further includes performing affinity chromatography before the composite anion exchange chromatography and performing cation exchange chromatography after the composite anion exchange chromatography step.

[0021] On the other hand, the present application provides a method for purifying bevacizumab, and the method includes the following steps:

[0022] (1) Affinity chromatography;

[0023] (2) Composite anion exchange chromatography;

[0024] (3) Cation exchange chromatography.

[0025] In some embodiments, steps (1) - (3) are carried out in sequence.

[0026] In some embodiments, the method includes the following steps:

[0027] (1) Depth filtration;

[0028] (2) Affinity chromatography;

[0029] (3) Virus inactivation;

[0030] (4) Second - step depth filtration;

[0031] (5) Composite anion exchange chromatography;

[0032] (6) Cation exchange chromatography;

[0033] Steps (1) - (6) are carried out in sequence.

[0034] In some embodiments, the method includes the following steps:

[0035] (1) Depth filtration;

[0036] (2) Affinity chromatography;

[0037] (3) Virus inactivation;

[0038] (4) Second-step depth filtration;

[0039] (5) Composite anion exchange chromatography;

[0040] (6) Cation exchange chromatography;

[0041] (7) Virus removal filtration;

[0042] (8) Ultrafiltration;

[0043] Steps (1)-(8) are carried out in sequence.

[0044] In some embodiments, the affinity chromatography step includes, but is not limited to, equilibration, loading, equilibration, washing, equilibration, elution, etc.

[0045] In some specific embodiments, the affinity chromatography equilibration buffer is selected from 5-20 mmol / L sodium phosphate buffer, preferably 10 mmol / L sodium phosphate buffer.

[0046] In some specific embodiments, the affinity chromatography washing buffer is selected from 5-20 mmol / L sodium phosphate buffer containing 0.1-1.0 mol / L sodium chloride, preferably 10 mmol / L sodium phosphate buffer containing 0.5 mol / L sodium chloride.

[0047] In some specific embodiments, the affinity chromatography elution buffer is selected from 10-100 mmol / L sodium citrate buffer, preferably 50 mmol / L sodium citrate buffer.

[0048] In some embodiments, the pH of the affinity chromatography equilibration buffer is 5.0-8.0, 5.0-7.5 or 5.0-7.0, such as about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, or about 8.0, or a range formed by any of the foregoing values, preferably 6.0±0.2 or 6.0±0.1.

[0049] In some embodiments, the conductivity of the affinity chromatography equilibration buffer is 0.05 - 8.0 mS / cm, 0.1 - 5.0 mS / cm, or 0.5 - 3.0 mS / cm, such as approximately 0.05 mS / cm, approximately 0.1 mS / cm, approximately 0.5 mS / cm, approximately 1.0 mS / cm, approximately 1.5 mS / cm, approximately 2.0 mS / cm, approximately 2.5 mS / cm, approximately 3.0 mS / cm, approximately 3.5 mS / cm, approximately 4.0 mS / cm, approximately 4.5 mS / cm, approximately 5.0 mS / cm, approximately 5.5 mS / cm, approximately 6.0 mS / cm, approximately 6.5 mS / cm, approximately 7.0 mS / cm, approximately 7.5 mS / cm, or approximately 8.0 mS / cm, or a range formed by any of the foregoing values, preferably 0.5 - 1.5 mS / cm.

[0050] In some embodiments, the pH of the affinity chromatography elution buffer is 5.0 - 9.0, 5.5 - 8.5, or 6.0 - 8.0, such as approximately 5.0, approximately 5.1, approximately 5.2, approximately 5.3, approximately 5.4, approximately 5.5, approximately 5.6, approximately 5.7, approximately 5.8, approximately 5.9, approximately 6.0, approximately 6.1, approximately 6.2, approximately 6.3, approximately 6.4, approximately 6.5, approximately 6.6, approximately 6.7, approximately 6.8, approximately 6.9, approximately 7.0, approximately 7.1, approximately 7.2, approximately 7.3, approximately 7.4, approximately 7.5, approximately 7.6, approximately 7.7, approximately 7.8, approximately 7.9, approximately 8.0, approximately 8.1, approximately 8.2, approximately 8.3, approximately 8.4, approximately 8.5, approximately 8.6, approximately 8.7, approximately 8.8, approximately 8.9, or approximately 9.0, or a range formed by any of the foregoing values, preferably 7.0 ± 0.2.

[0051] In some embodiments, the conductivity of the affinity chromatography elution buffer is 10 - 100 mS / cm, 20 - 80 mS / cm, or 30 - 60 mS / cm, such as approximately 10 mS / cm, approximately 15 mS / cm, approximately 20 mS / cm, approximately 25 mS / cm, approximately 30 mS / cm, approximately 35 mS / cm, approximately 40 mS / cm, approximately 41 mS / cm, approximately 42 mS / cm, approximately 43 mS / cm, approximately 44 mS / cm, approximately 45 mS / cm, approximately 46 mS / cm, approximately 47 mS / cm, approximately 48 mS / cm, approximately 49 mS / cm, approximately 50 mS / cm, approximately 51 mS / cm, approximately 52 mS / cm, approximately 53 mS / cm, approximately 54 mS / cm, approximately 55 mS / cm, approximately 60 mS / cm, approximately 65 mS / cm, approximately 70 mS / cm, approximately 75 mS / cm, approximately 80 mS / cm, approximately 85 mS / cm, approximately 90 mS / cm, approximately 95 mS / cm, or approximately 100 mS / cm, or a range formed by any of the foregoing values, preferably 43 - 52 mS / cm.

[0052] In some embodiments, the pH of the affinity chromatography eluent is 2.5 - 6.5, 3.0 - 6.0 or 3.5 - 5.0, such as about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, about 3.0, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, or about 6.5, or a range formed by any of the foregoing values, preferably 3.8 ± 0.2.

[0053] In some embodiments, the conductivity of the affinity chromatography eluent is 1.0 - 10 mS / cm, 2.0 - 8.0 mS / cm or 2.0 - 6.0 mS / cm, such as about 1.0 mS / cm, about 1.5 mS / cm, about 2.0 mS / cm, about 2.5 mS / cm, about 3.0 mS / cm, about 3.5 mS / cm, about 4.0 mS / cm, about 4.5 mS / cm, about 5.0 mS / cm, about 5.5 mS / cm, about 6.0 mS / cm, about 6.5 mS / cm, about 7.0 mS / cm, about 7.5 mS / cm, about 8.0 mS / cm, about 8.5 mS / cm, about 9.0 mS / cm, about 9.5 mS / cm, or about 10.0 mS / cm, or a range formed by any of the foregoing values, preferably 2.5 - 4.5 mS / cm.

[0054] In some embodiments, the affinity chromatography packing material is selected from MabPurix A65, MabPurix P45, AT ProteinA Diamond Plus, Novo-A Diamond or NMab Pro Protein A, preferably Novo-A Diamond.

[0055] In some embodiments, the composite anion exchange chromatography includes the steps of: adjusting the pH and conductivity of the sample loading solution before sample loading.

[0056] In some embodiments, the composite anion exchange chromatography includes the steps of: ① equilibration 1, ② adjusting the pH and conductivity of the sample loading solution, ③ sample loading, and ④ equilibration 2. In some embodiments, the steps are carried out in sequence, wherein the order of the two steps of equilibration 1 and adjusting the pH and conductivity of the sample loading solution can be interchanged.

[0057] In some embodiments, the composite anion exchange chromatography has ion exchange, hydrophobic, and / or hydrogen bonding forces. In some embodiments, the composite anion exchange chromatography has ion exchange and hydrophobic forces. In some embodiments, the composite anion exchange chromatography has ion exchange and hydrogen bonding forces. In some embodiments, the composite anion exchange chromatography has hydrophobic and hydrogen bonding forces. In some embodiments, the composite anion exchange chromatography has ion exchange, hydrophobic, and hydrogen bonding forces.

[0058] In some embodiments, the pH and conductivity of the loading solution for the composite anion exchange chromatography are adjusted to be the same as those of the equilibration solution. In some embodiments, the conductivity of the loading solution for the composite anion exchange chromatography is adjusted to be the same as that of the equilibration solution, and the pH is adjusted to be different from that of the equilibration solution. In some embodiments, the conductivity of the loading solution for the composite anion exchange chromatography is adjusted to be different from that of the equilibration solution, and the pH is adjusted to be the same as that of the equilibration solution. In some embodiments, the pH and conductivity of the loading solution for the composite anion exchange chromatography are adjusted to be different from those of the equilibration solution.

[0059] In some embodiments, the conductivity of the adjusted loading solution for the composite anion exchange chromatography is 3.0 - 10.0 mS / cm, 4.0 - 8.0 mS / cm, or 4.0 - 6.0 mS / cm, such as about 3.0 mS / cm, about 3.5 mS / cm, about 4.0 mS / cm, about 4.5 mS / cm, about 5.0 mS / cm, about 5.5 mS / cm, about 6.0 mS / cm, about 6.5 mS / cm, about 7.0 mS / cm, about 7.5 mS / cm, about 8.0 mS / cm, about 8.5 mS / cm, about 9.0 mS / cm, about 9.5 mS / cm, or about 10 mS / cm, or the range formed by any of the foregoing values, preferably 4.0 - 6.0 mS / cm.

[0060] In some embodiments, the pH of the adjusted loading solution for the composite anion exchange chromatography is 5.0 - 8.5, 5.5 - 8.0, or 7.0 - 8.0, such as about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, or the range formed by any of the foregoing values, preferably 7.5 ± 0.2.

[0061] In some embodiments, the pH of the loading solution for composite anion exchange chromatography is adjusted first, and then the conductivity of the loading solution for composite anion exchange chromatography is adjusted. In some embodiments, the conductivity of the loading solution for composite anion exchange chromatography is adjusted first, and then the pH of the loading solution for composite anion exchange chromatography is adjusted. In some embodiments, the pH and conductivity of the loading solution for composite anion exchange chromatography are adjusted simultaneously.

[0062] In some embodiments, the adjusting solution for adjusting the pH of the loading solution for composite anion exchange chromatography is 2 mol / L Tris-HCl solution. In some embodiments, the adjusting solution for adjusting the conductivity of the loading solution for composite anion exchange chromatography is 1 mol / L sodium chloride solution or water for injection.

[0063] In some embodiments, the equilibration buffer for composite anion exchange chromatography is selected from 5-20 mmol / L Tris-HCl buffer containing 20-50 mmol / L sodium chloride, preferably 10 mmol / L Tris-HCl buffer containing 35 mmol / L sodium chloride.

[0064] In some embodiments, the pH of the equilibration buffer for composite anion exchange chromatography is 5.0-8.5, 5.5-8.0 or 7.0-8.0, such as about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, or the range formed by any of the foregoing values, preferably 7.5±0.2.

[0065] In some embodiments, the conductivity of the equilibration buffer for composite anion exchange chromatography is 3.0-10.0 mS / cm, 4.0-8.0 mS / cm or 4.0-6.0 mS / cm, such as about 3.0 mS / cm, about 3.5 mS / cm, about 4.0 mS / cm, about 4.5 mS / cm, about 5.0 mS / cm, about 5.5 mS / cm, about 6.0 mS / cm, about 6.5 mS / cm, about 7.0 mS / cm, about 7.5 mS / cm, about 8.0 mS / cm, about 8.5 mS / cm, about 9.0 mS / cm, about 9.5 mS / cm, or about 10 mS / cm, or the range formed by any of the foregoing values, preferably 4.0-6.0 mS / cm.

[0066] In some embodiments, the composite anion exchange chromatography packing material is selected from: NM90 Agarose HAM or DiamondMix-A, preferably NM90 Agarose HAM.

[0067] In some embodiments, the cation exchange chromatography step includes, but is not limited to, equilibration, sample solution adjustment, sample loading, equilibration, elution, etc. In some specific embodiments, the cation exchange chromatography equilibration solution is selected from 10 - 100 mmol / L sodium chloride-containing 10 - 50 mmol / L sodium phosphate buffer solution and 5 - 20 mmol / L sodium citrate buffer solution, preferably 20 mmol / L sodium phosphate buffer solution and 10 mmol / L sodium citrate buffer solution containing 50 mmol / L sodium chloride.

[0068] In some specific embodiments, the cation exchange chromatography elution solution is selected from 10 - 100 mmol / L sodium chloride-containing 10 - 50 mmol / L sodium phosphate buffer solution and 5 - 20 mmol / L sodium citrate buffer solution, preferably 20 mmol / L sodium phosphate buffer solution and 10 mmol / L sodium citrate buffer solution containing 50 mmol / L sodium chloride.

[0069] In some embodiments, the pH of the adjusted cation exchange chromatography sample solution is 3.0 - 8.0, 4.0 - 7.0 or 4.0 - 6.0, such as approximately 3.0, approximately 3.5, approximately 4.0, approximately 4.1, approximately 4.2, approximately 4.3, approximately 4.4, approximately 4.5, approximately 4.6, approximately 4.7, approximately 4.8, approximately 4.9, approximately 5.0, approximately 5.1, approximately 5.2, approximately 5.3, approximately 5.4, approximately 5.5, approximately 5.6, approximately 5.7, approximately 5.8, approximately 5.9, approximately 6.0, approximately 6.1, approximately 6.2, approximately 6.3, approximately 6.4, approximately 6.5, approximately 6.6, approximately 6.7, approximately 6.8, approximately 6.9, approximately 7.0, approximately 7.5, approximately 8.0, or the range formed by any of the foregoing values, preferably 5.0 ± 0.2.

[0070] In some embodiments, the adjusting solution for adjusting the pH of the cation exchange chromatography sample solution is 1 mol / L citric acid solution. In some specific embodiments, the pH of the cation exchange chromatography elution solution is at least 1.0 higher than the pH of the cation exchange chromatography equilibration solution, such as approximately 1.0, approximately 1.05, approximately 1.2, approximately 1.25, approximately 1.3, approximately 1.35, approximately 1.4, approximately 1.45, or approximately 1.5 higher, preferably 1.0 - 1.45 higher.

[0071] In some embodiments, the pH of the equilibration buffer for cation exchange chromatography is 3.0 - 8.0, 3.5 - 7.0, or 4.0 - 6.0, such as about 3.0, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, or about 8.0, or a range formed by any of the foregoing values, preferably 5.0 ± 0.2.

[0072] In some embodiments, the conductivity of the equilibration buffer for cation exchange chromatography is 1.0 - 20.0 mS / cm, 3.0 - 15.0 mS / cm, or 6.0 - 10.0 mS / cm, such as about 1.0 mS / cm, about 1.5 mS / cm, about 2.0 mS / cm, about 2.5 mS / cm, about 3.0 mS / cm, about 3.5 mS / cm, about 4.0 mS / cm, about 4.5 mS / cm, about 5.0 mS / cm, about 5.5 mS / cm, about 6.0 mS / cm, about 6.5 mS / cm, about 7.0 mS / cm, about 7.5 mS / cm, about 8.0 mS / cm, about 8.5 mS / cm, about 9.0 mS / cm, about 9.5 mS / cm, about 10.0 mS / cm, about 10.5 mS / cm, about 11.0 mS / cm, about 11.5 mS / cm, about 12.0 mS / cm, about 12.5 mS / cm, about 13.0 mS / cm, about 13.5 mS / cm, about 14.0 mS / cm, about 14.5 mS / cm, about 15.0 mS / cm, about 15.5 mS / cm, about 16.0 mS / cm, about 16.5 mS / cm, about 17.0 mS / cm, about 17.5 mS / cm, about 18.0 mS / cm, about 18.5 mS / cm, about 19.0 mS / cm, about 19.5 mS / cm, or about 20.0 mS / cm, or a range formed by any of the foregoing values, preferably 7.0 - 9.0 mS / cm.

[0073] In some embodiments, the pH of the cation exchange chromatography eluent is 3.0 - 8.5, 4.0 - 8.0 or 5.0 - 7.0, such as about 3.0, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.15, about 6.2, about 6.25, about 6.3, about 6.35, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 8.0, about 8.1, about 8.2, about 8.3, about 8.4, or about 8.5, or a range formed by any of the foregoing values, preferably 6.25 ± 0.2.

[0074] In some embodiments, the conductivity of the cation exchange chromatography eluent is 2.0 - 20.0 mS / cm, 4.0 - 16.0 mS / cm or 6.0 - 12.0 mS / cm, such as about 2.0 mS / cm, about 2.5 mS / cm, about 3.0 mS / cm, about 3.5 mS / cm, about 4.0 mS / cm, about 4.5 mS / cm, about 5.0 mS / cm, about 5.5 mS / cm, about 6.0 mS / cm, about 6.5 mS / cm, about 7.0 mS / cm, about 7.5 mS / cm, about 8.0 mS / cm, about 8.5 mS / cm, about 9.0 mS / cm, about 9.5 mS / cm, about 10.0 mS / cm, about 10.5 mS / cm, about 11.0 mS / cm, about 11.5 mS / cm, about 12.0 mS / cm, about 12.5 mS / cm, about 13.0 mS / cm, about 13.5 mS / cm, about 14.0 mS / cm, about 14.5 mS / cm, about 15.0 mS / cm, about 15.5 mS / cm, about 16.0 mS / cm, about 16.5 mS / cm, about 17.0 mS / cm, about 17.5 mS / cm, about 18.0 mS / cm, about 18.5 mS / cm, about 19.0 mS / cm, about 19.5 mS / cm, or about 20.0 mS / cm, or a range formed by any of the foregoing values, preferably 8.0 - 10.0 mS / cm.

[0075] In some embodiments, the cation exchange chromatography packing is selected from Diamond SP, NanoGel-50SP HP or Monomix HC45-SP, preferably NanoGel-50SP HP.

[0076] In some embodiments, the method comprises the following steps:

[0077] (1) Depth filtration;

[0078] (2) Affinity chromatography, which includes the steps of: ① Equilibration 1, equilibrating with 10 mmol / L sodium phosphate buffer (pH 6.0 ± 0.2, conductivity 0.5 - 1.5 mS / cm); ② Loading; ③ Equilibration 2, equilibrating with 10 mmol / L sodium phosphate buffer (pH 6.0 ± 0.2, conductivity 0.5 - 1.5 mS / cm); ④ Washing, washing with 10 mmol / L sodium phosphate buffer containing 0.5 mol / L sodium chloride (pH 7.0 ± 0.2, conductivity 43 - 52 mS / cm); ⑤ Equilibration 3, equilibrating with 10 mmol / L sodium phosphate buffer (pH 6.0 ± 0.2, conductivity 0.5 - 1.5 mS / cm); ⑥ Elution, eluting with 50 mmol / L sodium citrate buffer (pH 3.8 ± 0.2, conductivity 2.5 - 4.5 mS / cm);

[0079] (3) Virus inactivation;

[0080] (4) Second depth filtration;

[0081] (5) Composite anion exchange chromatography, which includes the steps of: ① Equilibration 1, equilibrating with 10 mmol / L Tris-HCl buffer containing 35 mmol / L sodium chloride (pH 7.5 ± 0.2, conductivity 4.0 - 6.0 mS / cm); ② Sample solution adjustment, first adjusting the pH of the filtrate of the second depth filtration to 7.5 ± 0.2 with 2 mol / L Tris-HCl solution (pH 9.50 ± 0.05), and then adjusting the conductivity of the sample to 4.0 - 6.0 mS / cm with 1 mol / L sodium chloride solution or water for injection; ③ Loading and collecting the flow-through fraction; ④ Equilibration 2, equilibrating the chromatography column with 10 mmol / L Tris-HCl buffer containing 35 mmol / L sodium chloride (pH 7.5 ± 0.2, conductivity 4.0 - 6.0 mS / cm);

[0082] (6) Cation exchange chromatography, which includes the steps: ① Equilibration 1, equilibrating with a 20 mmol / L sodium phosphate buffer containing 50 mmol / L sodium chloride and a 10 mmol / L sodium citrate buffer (pH 5.0 ± 0.2, conductivity 7.0 - 9.0 mS / cm); ② Loading; ③ Equilibration, equilibrating with a 20 mmol / L sodium phosphate buffer containing 50 mmol / L sodium chloride and a 10 mmol / L sodium citrate buffer (pH 5.0 ± 0.2, conductivity 7.0 - 9.0 mS / cm); ④ Elution, eluting with a 20 mmol / L sodium phosphate buffer containing 50 mmol / L sodium chloride and a 10 mmol / L sodium citrate buffer (pH 6.25 ± 0.2, conductivity 8.0 - 10 mS / cm).

[0083] Steps (1)-(6) are carried out in sequence.

[0084] The method of the present application has at least the following beneficial effects: By using affinity chromatography, composite anion exchange chromatography, and cation exchange chromatography, and adjusting the pH and conductivity of the loading solution before loading in the composite anion exchange chromatography, bevacizumab with high purity can be prepared; the chromatography packing materials used have high accessibility and low cost, and are suitable for industrial scale-up production.

[0085] Term Explanation

[0086] Unless otherwise specified, the following terms used in the present application have the following meanings. A specific term should not be considered uncertain or unclear without a special definition, but should be understood according to the ordinary meaning in the art.

[0087] "Purification" refers to a technique of removing at least one contaminant completely or partially from a composition containing an antibody and one or more contaminants to improve the purity of the antibody in the composition.

[0088] "Comprising" or "including" should be understood in an open and non-exclusive sense, that is, "including but not limited to".

[0089] "Conductivity" refers to the ability of an aqueous solution to conduct electric current between two electrodes. The conductivity of a solution can be determined by standard methods known to those skilled in the art.

[0090] "Intermediate product" refers to a product that has completed some process steps and still requires further process processing to become the final product.

[0091] "CE-SDS non-reducing electrophoresis" refers to non-reducing sodium dodecyl sulfate capillary gel electrophoresis. Using non-reducing capillary gel electrophoresis, driven by a high-voltage electric field, the products move at different speeds in the capillary according to their molecular weights, and among them, the higher the proportion of monomers, the higher the purity of the products.

[0092] "CE-SDS reduction electrophoresis" refers to the reduced sodium dodecyl sulfate capillary gel electrophoresis method. Using reduced capillary gel electrophoresis with a high-voltage electric field as the driving force, after the product is reduced, it is separated in the capillary according to the different migration speeds of the product based on the molecular weight. Among them, the higher the proportion of the sum of the light chain and the heavy chain, the higher the purity of the product.

[0093] "HCP" refers to host cell protein, which is the protein component from the cell line (such as CHO cells) that produces and expresses antibodies, including but not limited to the proteins secreted by the cell line, the structural proteins produced by apoptosis and metabolism of the cells. HCP poses a risk to the safety of antibody drugs and needs to be strictly controlled and detected.

[0094] "SEC-HPLC" refers to size exclusion high performance liquid chromatography, that is, volume exclusion chromatography, size exclusion chromatography or steric exclusion chromatography. It is calculated according to the area normalization method and is divided into monomer, polymer (high molecular weight group) and fragment (low molecular weight group). Among them, the larger the percentage of the monomer, the higher the purity of the product.

[0095] "CEX-HPLC" refers to cation exchange high performance liquid chromatography, which is separated according to the difference in the charge number of the product and can separate the charge isomers in the product and is used for the homogeneity problem of the reaction product. "Charge isomer" refers to the isomer in which the charge carried by the antibody molecule is directly or indirectly changed due to glycosylation, deamidation, oxidation, isomerization, etc. of the antibody. These isomers are generally divided into acidic isomers or basic isomers. When analyzing charge isomers by CEX, the acidic peak (acidic isomer) elutes earlier than the main peak, and the basic peak (basic isomer) elutes later than the main peak. Among them, the higher the proportion of the acidic peak or the basic peak, the higher the degree of charge heterogeneity of the product and the worse the homogeneity of the product.

[0096] Unless otherwise specified, "about" in this application means fluctuating within the range of ±5% of the given specific numerical range, preferably within the range of ±2%, and more preferably within the range of ±1%. For example, a pH value of about 5.5 means a pH of 5.5 ± 5%, preferably a pH of 5.5 ± 2%, and more preferably a pH of 5.5 ± 1%. Detailed implementation mode

[0097] Although the foregoing application has been described in considerable detail by way of illustration and examples for the purpose of clear understanding, it will be apparent to those of ordinary skill in the art that certain changes and modifications may be made to the application without departing from the spirit and scope of the appended claims. The following examples are provided by way of illustration only and do not serve to limit. Those skilled in the art will readily identify a variety of non-critical parameters that can be changed or modified to produce substantially similar results.

[0098] All reagents used in the examples, unless otherwise specified, can be obtained commercially. The preparation of solutions can be carried out using conventional techniques in the art.

[0099] The purified antibody in the examples is bevacizumab, and its amino acid sequence is the same as (INN: bevacizumab). To express bevacizumab, the DNA sequences encoding the heavy and light chains of bevacizumab are cloned into an expression vector, and then the expression vector is transfected into a host cell so that the gene is transcribed and translated. The host cell can be a eukaryotic cell, preferably a mammalian cell, and most preferably a CHO cell.

[0100] Example 1 Screening of Affinity Chromatography Packings

[0101] Compare the effects of 5 affinity chromatography packings, MabPurix A65 (Sepax, 270765990), MabPurix P45 (Sepax, 270845990), AT Protein A Diamond Plus (BestChrom, AA402314), Novo-A Diamond (BestChrom, AA05006), and NMab Pro Protein A (NanoMicro, 17013 - 070100), in separating bevacizumab. The affinity chromatography steps include: ① Equilibration 1, equilibrate with 10 mmol / L sodium phosphate buffer (pH 6.0 ± 0.1); ② Loading; ③ Equilibration 2, equilibrate with 10 mmol / L sodium phosphate buffer (pH 6.0 ± 0.1); ④ Washing, wash with 10 mmol / L sodium phosphate buffer (pH 7.0 ± 0.2) containing 0.5 mol / L sodium chloride; ⑤ Equilibration 3, equilibrate with 10 mmol / L sodium phosphate buffer (pH 6.0 ± 0.1); ⑥ Elution, elute with 50 mmol / L sodium citrate buffer (pH 3.8 ± 0.2), collect the eluate, and analyze the elution column volume (CV) and recovery rate of the affinity chromatography.

[0102] Recovery rate (%) = amount of protein in the affinity chromatography eluate / amount of protein in the first-step depth filtration filtrate * 100%

[0103] As shown in Table 1, affinity chromatography using Novo-A Diamond packing material has a smaller elution volume and a higher recovery rate. Selecting an affinity chromatography packing material with a small elution volume can effectively control the volume of the intermediate product in the second-step depth filtration. Therefore, Novo-A Diamond is selected as the affinity chromatography packing material.

[0104] Table 1 Screening of Affinity Chromatography Packing Materials

[0105]

[0106] Example 2 Screening of Composite Anion Exchange Chromatography Packing Materials and Loading Conditions

[0107] Compare the separation effects of two composite anion exchange chromatography packing materials, NM90 Agarose HAM (NanoMicro, 04092-090001) and Diamond Mix-A (BestChrom, AI0105), and their separation effects on bevacizumab under different loading and equilibration conditions. The steps of composite anion exchange chromatography include: ① Equilibration 1, equilibrate with 10 mmol / L Tris-HCl buffer containing 35 mmol / L sodium chloride (pH and conductivity are the same as the loading solution); ② Loading solution adjustment, first adjust the pH with 2 mol / L Tris-HCl solution (pH 9.50 ± 0.05), and then adjust the conductivity with 1 mol / L sodium chloride solution or water for injection; ③ Loading, collect the flow-through solution; ④ Equilibration 2, equilibrate with 10 mmol / L Tris-HCl buffer containing 35 mmol / L sodium chloride (pH and conductivity are the same as the loading solution), stop collecting the flow-through solution after flushing 10 - 30 CV, and analyze the flow-through solution of composite anion exchange chromatography by capillary electrophoresis with sodium dodecyl sulfate (CE-SDS). As shown in Table 2, when using NM90-Agarose HAM packing material, and the loading and equilibration buffer with a conductivity of about 5 mS / cm and a pH of about 7.5 for composite anion exchange chromatography, the percentage of the non-reduced main peak in the flow-through solution by CE-SDS increases the most compared to the intermediate product of the second-step depth filtration. Therefore, select NM90-Agarose HAM packing material, and the loading solution and equilibration solution with a conductivity of about 5 mS / cm and a pH of about 7.5 for composite anion exchange chromatography.

[0108] Table 2 Screening of Composite Anion Exchange Chromatography Packing Materials and Loading and Equilibration Conditions

[0109]

[0110]

[0111] Example 3 Screening of Cation Exchange Chromatography Packing Materials

[0112] Compare the effects of three cation exchange chromatography packings, Diamond SP (BestChrom, AI319314), NanoGel-50SP HP (NanoMicro, 04062-050150), and Monomix HC45-SP (Sepax, 280645950), on the separation of bevacizumab. The steps of cation exchange chromatography include: ① Equilibration 1: Equilibrate with 20 mmol / L sodium phosphate buffer containing 50 mmol / L sodium chloride and 10 mmol / L sodium citrate buffer (pH 5.0 ± 0.1); ② Sample solution adjustment: Adjust the pH value to 5.0 ± 0.2 with 1 mol / L citric acid solution; ③ Loading; ④ Equilibration 2: Equilibrate with 20 mmol / L sodium phosphate buffer containing 50 mmol / L sodium chloride and 10 mmol / L sodium citrate buffer (pH 5.0 ± 0.1); ⑤ Elution: Elute with 20 mmol / L sodium phosphate buffer containing 50 mmol / L sodium chloride and 10 mmol / L sodium citrate buffer (pH 6.25 ± 0.2), and collect the eluate. Analyze the cation exchange chromatography eluate by host cell protein (HCP) residue, host cell DNA residue, Protein A residue, size exclusion-high performance liquid chromatography (SEC-HPLC), and cation exchange-high performance liquid chromatography (CEX-HPLC). As shown in Table 3, cation exchange chromatography using NanoGel-50SP HP packing can better remove the alkaline region and reduce high molecular impurities. Therefore, NanoGel-50SP HP is selected as the cation exchange chromatography packing.

[0113] Table 3 Screening of Cation Exchange Packings

[0114]

[0115] Example 4 Separation and Purification of Bevacizumab

[0116] The first step is depth filtration: Collect the cell culture broth for depth filtration and collect the filtrate;

[0117] Affinity chromatography: Using Novo-A Diamond packing material, the steps include: ① Equilibration 1, equilibrating with 10 mmol / L sodium phosphate buffer (pH 6.0 ± 0.1, conductivity 0.5 - 1.5 mS / cm); ② Sample loading; ③ Equilibration 2, equilibrating with 10 mmol / L sodium phosphate buffer (pH 6.0 ± 0.1, conductivity 0.5 - 1.5 mS / cm); ④ Washing, washing with 10 mmol / L sodium phosphate buffer containing 0.5 mol / L sodium chloride (pH 7.0 ± 0.2, conductivity 43 - 52 mS / cm); ⑤ Equilibration 3, equilibrating with 10 mmol / L sodium phosphate buffer (pH 6.0 ± 0.1, conductivity 0.5 - 1.5 mS / cm); ⑥ Elution, eluting with 50 mmol / L sodium citrate buffer (pH 3.8 ± 0.2, conductivity 2.5 - 4.5 mS / cm), and collecting the eluate.

[0118] Virus inactivation: Adjust the pH of the eluate from affinity chromatography to 3.7 ± 0.1 with 1 mol / L citric acid solution, and incubate at low temperature for 45 - 90 min. After inactivation, adjust the pH of the sample to 5.2 ± 0.1 with 2 mol / L Tris-HCl solution (pH 9.50 ± 0.05).

[0119] The second step of depth filtration: Deeply filter the sample after virus inactivation, and collect the filtrate.

[0120] Composite anion exchange chromatography: Using NM90-Agarose HAM packing material, the steps include: ① Equilibration 1, equilibrating with 10 mmol / L Tris-HCl buffer containing 35 mmol / L sodium chloride (pH 7.5 ± 0.2, conductivity 4.0 - 6.0 mS / cm); ② Sample loading solution adjustment, first adjust the pH of the filtrate from the second step of depth filtration to 7.5 ± 0.2 with 2 mol / L Tris-HCl solution (pH 9.50 ± 0.05), and then adjust the conductivity of the sample to 4.0 - 6.0 mS / cm with 1 mol / L sodium chloride solution or water for injection; ③ Sample loading, and collect the flow-through fraction; ④ Equilibration 2, equilibrate the chromatography column with 10 mmol / L Tris-HCl buffer containing 35 mmol / L sodium chloride (pH 7.5 ± 0.2, conductivity 4.0 - 6.0 mS / cm) for 10 - 30 CV, and end the collection of the flow-through fraction after 10 - 30 CV.

[0121] Cation exchange chromatography: Using NanoGel-50SP HP packing material, the steps include: ①Equilibration 1, equilibrating with 20 mmol / L sodium phosphate buffer containing 50 mmol / L sodium chloride and 10 mmol / L sodium citrate buffer (pH 5.0±0.1, conductivity 7.0 - 9.0 mS / cm); ②Sample solution adjustment, adjusting the pH of the flow-through solution of composite anion exchange chromatography to 5.0±0.2 with 1 mol / L citric acid solution; ③Loading the sample; ④Equilibration 2, equilibrating with 20 mmol / L sodium phosphate buffer containing 50 mmol / L sodium chloride and 10 mmol / L sodium citrate buffer (pH 5.0±0.1, conductivity 7.0 - 9.0 mS / cm); ⑤Elution, eluting with 20 mmol / L sodium phosphate buffer containing 50 mmol / L sodium chloride and 10 mmol / L sodium citrate buffer (pH 6.25±0.2, conductivity 8.0 - 10.0 mS / cm), and collecting the eluate.

[0122] Virus removal filtration (nanofiltration): The eluate of cation exchange chromatography is subjected to virus removal filtration, and the filtrate is collected;

[0123] Ultrafiltration concentration and buffer exchange: The filtrate of virus removal filtration is subjected to ultrafiltration buffer exchange to harvest the product containing bevacizumab.

[0124] The ultrafiltration harvest solution is subjected to bacteria-reducing filtration to obtain the bevacizumab bulk solution.

[0125] Using the above process to purify 2000 kg of cell culture solution, Table 4 shows the key quality results of the harvest solutions and the bulk solution in each step of bevacizumab purification, and Table 5 shows the purification results of the bulk solution, indicating that the above process is applicable to the purification of bevacizumab.

[0126] Table 4 Key quality results of the harvest solutions and the bulk solution in each step of bevacizumab purification

[0127]

[0128] Table 5 Purification results of the bevacizumab bulk solution

[0129]

Claims

1. A method for purifying bevacizumab, characterized in that, The method includes purifying bevacizumab by composite anion exchange chromatography.

2. The method according to claim 1, characterized in that, The composite anion exchange chromatography has ion exchange and hydrophobic interaction forces.

3. The method according to any one of claims 1-2, characterized in that, The composite anion exchange chromatography includes the steps of: adjusting the pH and conductivity of the sample loading solution before sample loading, preferably, adjusting the pH and conductivity of the sample loading solution to be the same as those of the equilibration solution.

4. The method according to claim 3, characterized in that, The pH of the adjusted sample loading solution is 5.0 - 8.5, preferably 7.5 ± 0.2, and / or the conductivity of the adjusted sample loading solution is 3.0 - 10.0 mS / cm, preferably 4.0 - 6.0 mS / cm.

5. The method according to any one of claims 1-4, characterized in that, The composite anion exchange chromatography packing material is selected from: NM90 Agarose HAM or Diamond Mix-A, preferably NM90 Agarose HAM.

6. The method according to any one of claims 1-5, characterized in that, The method further includes performing affinity chromatography before the composite anion exchange chromatography, and performing cation exchange chromatography after the composite anion exchange chromatography step.

7. A method for purifying bevacizumab, characterized in that, The method includes the following steps: (1) Affinity chromatography; (2) Composite anion exchange chromatography; (3) Cation exchange chromatography; Steps (1) - (3) are carried out in sequence; Among them, the composite anion exchange chromatography includes the steps of: adjusting the pH and conductivity of the sample loading solution before sample loading, preferably, adjusting the pH and conductivity of the sample loading solution to be the same as those of the equilibration solution.

8. The method according to claim 7, wherein The pH of the adjusted sample loading solution is 5.0 - 8.5, preferably 7.5 ± 0.2, and / or the conductivity of the adjusted sample loading solution is 3.0 - 10.0 mS / cm, preferably 4.0 - 6.0 mS / cm.

9. The method according to claim 7 or 8, characterized in that, The adjusting solution for adjusting the pH of the composite anion exchange chromatography sample loading solution is 2 mol / L Tris-HCl solution, and / or the adjusting solution for adjusting the conductivity of the composite anion exchange chromatography sample loading solution is 1 mol / L sodium chloride solution.

10. The method according to any one of claims 7-9, characterized in that, The affinity chromatography packing material is selected from MabPurix A65, MabPurix P45, AT Protein A Diamond Plus, Novo-A Diamond or NMab ProProtein A, preferably Novo-A Diamond; the composite anion exchange chromatography packing material is selected from: NM90 Agarose HAM or Diamond Mix-A, preferably NM90 Agarose HAM; the cation exchange chromatography packing material is selected from Diamond SP, NanoGel-50SP HP or Monomix HC45-SP, preferably NanoGel-50SP HP.

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