A method of preparing bevacizumab

Abstract

The application belongs to the field of protein purification, and relates to a method for preparing bevacizumab, comprising 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 specifically relates to a method for preparing or purifying bevacizumab. Background Technology

[0002] Vascular endothelial growth factor (VEGF) is a functional protein that specifically targets vascular endothelial cells, promoting increased vascular permeability, endothelial cell migration and proliferation, extracellular matrix degeneration, and angiogenesis. VEGF is expressed in various malignant tumors and participates in tumor angiogenesis, thus becoming an important target for anti-tumor drug development. Currently, inhibitors targeting VEGF 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 colorectal cancer, lung cancer, glioblastoma, renal cell carcinoma, cervical cancer, ovarian cancer, fallopian tube cancer, peritoneal cancer, liver cancer, and other cancers.

[0003] With the expanding clinical demand, more and more monoclonal antibody drugs are being approved, leading to increasingly higher requirements for the efficiency and cost of manufacturing processes. A typical monoclonal antibody purification method generally includes three steps: first, affinity chromatography is used to capture the target protein in the cell culture medium; then, two-step ion exchange chromatography or ion exchange plus hydrophobic chromatography is used for fine purification; and finally, a virus inactivation step is included after the affinity chromatography step. Novel mixed-mode chromatography (MMC) optimizes the structure of functional ligands, combining two or more interaction modes to enhance affinity and selectivity for the target protein, further improving purification efficiency. Compared with traditional ion exchange chromatography, MMC has advantages such as high loading capacity, high selectivity, and high efficiency. MMC mainly includes hydrophobic and ion exchange mixed-mode chromatography, hydrophilic and ion exchange mixed-mode chromatography, and hydrophobic and hydrophilic mixed-mode chromatography. In hydrophobic / ion exchange mixed-mode chromatography, the ligands contain both hydrophobic and electrostatic groups, providing both hydrophobic and electrostatic interactions simultaneously.

[0004] Monoclonal antibody drugs possess complex quality properties. The chromatographic packing materials and purification conditions can all influence these properties, and even minor changes can significantly impact the drug's safety and efficacy. In production practice, for different monoclonal antibody drugs, continuous optimization of the manufacturing process is necessary to control production costs and meet relevant quality standards. Summary of the Invention

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

[0006] On one hand, this application provides a method for purifying bevacizumab, the method comprising purifying bevacizumab using complex anion exchange chromatography.

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

[0008] In some embodiments, the composite anion exchange chromatography includes the steps of: ① equilibration 1, ② adjusting the pH and conductivity of the loading solution, ③ loading the sample, and ④ equilibration 2. In some embodiments, the steps are performed sequentially, wherein the order of equilibration 1 and adjusting the pH and conductivity of the loading solution can be interchanged.

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

[0010] In some embodiments, the pH and conductivity of the loading solution are adjusted to be consistent with the equilibration solution. In some embodiments, the conductivity of the loading solution is adjusted to be consistent with the equilibration solution, but the pH of the loading solution is not consistent with the equilibration solution. In some embodiments, the conductivity of the loading solution is adjusted to be inconsistent with the equilibration solution, but the pH of the loading solution is adjusted to be consistent with the equilibration solution. In some embodiments, the pH and conductivity of the loading solution are adjusted to be inconsistent with the equilibration solution.

[0011] In some embodiments, the adjusted conductivity of the loading solution is 3.0-10.0 mS / cm, 4.0-8.0 mS / cm, or 4.0-6.0 mS / cm, for example, 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 any of the aforementioned values, preferably 4.0-6.0 mS / cm.

[0012] In some embodiments, the adjusted pH of the loading solution is 5.0-8.5, 5.5-8.0, or 7.0-8.0, for example, 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 any range of the foregoing values, preferably 7.5 ± 0.2.

[0013] In some implementations, the pH of the loading solution is adjusted first, followed by the conductivity. In some implementations, the conductivity of the loading solution is adjusted first, followed by the pH. In some implementations, both the pH and conductivity of the loading solution are adjusted simultaneously.

[0014] In some embodiments, the pH adjusting solution used to adjust the sample loading solution is a 2 mol / L Tris-HCl solution. In some embodiments, the conductivity adjusting solution used to adjust the sample loading solution is a 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, for example, 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 any range of the foregoing values, preferably 7.5 ± 0.2.

[0017] In some embodiments, the conductivity of the equilibrium fluid is 3.0-10.0 mS / cm, 4.0-8.0 mS / cm, or 4.0-6.0 mS / cm, for example, 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 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 NM90Agarose HAM or DiamondMix-A, with NM90Agarose HAM being preferred.

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

[0020] In one specific implementation, the composite anion exchange chromatography uses NM90-Agarose HAM packing material, comprising the following steps: ① Equilibration 1: Equilibrate the column with 10 mmol / L Tris-HCl buffer (pH 7.5±0.2, conductivity 4.0-6.0 mS / cm) containing 35 mmol / L sodium chloride; ② Sample loading solution adjustment: First, adjust the pH of the second-step deep filtration filtrate to 7.5±0.2 with 2 mol / L Tris-HCl solution (pH 9.50±0.05), then adjust the sample conductivity to 4.0-6.0 mS / cm with 1 mol / L sodium chloride solution or water for injection; ③ Sample loading: Collect the flow-through; ④ Equilibration 2: Equilibrate the chromatography column to 10-30 CV with 10 mmol / L Tris-HCl buffer (pH 7.5±0.2, conductivity 4.0-6.0 mS / cm) containing 35 mmol / L sodium chloride, and stop collecting the flow-through after 10-30 CV. In some embodiments, the method further includes affinity chromatography prior to complex anion exchange chromatography and cation exchange chromatography after the complex anion exchange chromatography step.

[0021] On the other hand, this application provides a method for purifying bevacizumab, the method comprising the following steps:

[0022] (1) Affinity chromatography;

[0023] (2) Composite anion exchange chromatography;

[0024] (3) Cation exchange chromatography.

[0025] In some implementations, steps (1)-(3) are performed sequentially.

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

[0027] (1) Deep filtration;

[0028] (2) Affinity chromatography;

[0029] (3) Virus inactivation;

[0030] (4) Second step: deep filtering;

[0031] (5) Composite anion exchange chromatography;

[0032] (6) Cation exchange chromatography;

[0033] Steps (1)-(6) are performed sequentially.

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

[0035] (1) Deep filtration;

[0036] (2) Affinity chromatography;

[0037] (3) Virus inactivation;

[0038] (4) Second step: deep filtering;

[0039] (5) Composite anion exchange chromatography;

[0040] (6) Cation exchange chromatography;

[0041] (7) Virus filtration;

[0042] (8) Ultrafiltration;

[0043] Steps (1)-(8) are performed sequentially.

[0044] In some implementations, affinity chromatography steps include, but are not limited to, equilibration, loading, equilibration, rinsing, equilibration, and elution.

[0045] In some specific implementations, 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 eluent 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 implementations, the affinity chromatography eluent 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, for example, 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 any range of the foregoing values, preferably 6.0 ± 0.2 or 6.0 ± 0.1.

[0049] In some embodiments, the conductivity of the affinity chromatography equilibrium solution is 0.05-8.0 mS / cm, 0.1-5.0 mS / cm, or 0.5-3.0 mS / cm, for example, about 0.05 mS / cm, about 0.1 mS / cm, about 0.5 mS / cm, 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, or about 8.0 mS / cm, or any of the foregoing values, preferably 0.5-1.5 mS / cm.

[0050] In some embodiments, the pH of the affinity chromatography eluent is 5.0-9.0, 5.5-8.5, or 6.0-8.0, for example, 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... The range formed by 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, about 8.6, about 8.7, about 8.8, about 8.9, or about 9.0, or any of the aforementioned values, is preferably 7.0 ± 0.2.

[0051] In some embodiments, the conductivity of the affinity chromatography eluent is 10-100 mS / cm, 20-80 mS / cm, or 30-60 mS / cm, for example, about 10 mS / cm, about 15 mS / cm, about 20 mS / cm, about 25 mS / cm, about 30 mS / cm, about 35 mS / cm, about 40 mS / cm, about 41 mS / cm, about 42 mS / cm, about 43 mS / cm, about 44 mS / cm, about 45 mS / cm, about 46 mS / cm, or about 47 mS / cm. The values ​​are 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 any of the aforementioned 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, for example, 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... The range formed by 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 any of the aforementioned values, is 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, for example, 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 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, with Novo-A Diamond being preferred.

[0055] In some embodiments, the composite anion exchange chromatography includes the steps of adjusting the pH and conductivity of the loading solution before 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 loading solution, ③ loading the sample, and ④ equilibration 2. In some embodiments, the steps are performed sequentially, wherein the order of equilibration 1 and adjusting the pH and conductivity of the loading solution can be interchanged.

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

[0058] In some embodiments, the pH and conductivity of the composite anion exchange chromatography loading solution are adjusted to be consistent with the equilibration buffer. In some embodiments, the conductivity of the composite anion exchange chromatography loading solution is adjusted to be consistent with the equilibration buffer, but the pH is adjusted to be different from the equilibration buffer. In some embodiments, the conductivity of the composite anion exchange chromatography loading solution is adjusted to be different from the equilibration buffer, but the pH is adjusted to be consistent with the equilibration buffer. In some embodiments, the pH and conductivity of the composite anion exchange chromatography loading solution are adjusted to be different from the equilibration buffer.

[0059] In some embodiments, the adjusted conductivity of the composite anion exchange chromatography loading solution is 3.0-10.0 mS / cm, 4.0-8.0 mS / cm, or 4.0-6.0 mS / cm, for example, 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 any of the aforementioned values, preferably 4.0-6.0 mS / cm.

[0060] In some embodiments, the pH of the adjusted composite anion exchange chromatography loading solution is 5.0-8.5, 5.5-8.0, or 7.0-8.0, for example, 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 any range of the foregoing values, preferably 7.5 ± 0.2.

[0061] In some implementations, the pH of the composite anion exchange chromatography loading solution is adjusted first, followed by the conductivity. In other implementations, the conductivity of the composite anion exchange chromatography loading solution is adjusted first, followed by the pH. In still other implementations, both the pH and conductivity of the composite anion exchange chromatography loading solution are adjusted simultaneously.

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

[0063] In some embodiments, the equilibration buffer for complex 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 composite anion exchange chromatography equilibration solution is 5.0-8.5, 5.5-8.0, or 7.0-8.0, for example, 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 any range of the foregoing values, preferably 7.5 ± 0.2.

[0065] In some embodiments, the conductivity of the composite anion exchange chromatography equilibrium solution is 3.0-10.0 mS / cm, 4.0-8.0 mS / cm, or 4.0-6.0 mS / cm, for example, 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 any of the aforementioned values, preferably 4.0-6.0 mS / cm.

[0066] In some embodiments, the composite anion exchange chromatography packing material is selected from NM90Agarose HAM or DiamondMix-A, with NM90Agarose HAM being preferred.

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

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

[0069] In some embodiments, the pH of the adjusted cation exchange chromatography loading solution is 3.0-8.0, 4.0-7.0, or 4.0-6.0, for example, about 3.0, about 3.5, 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 7.5, about 8.0, or any range of the foregoing values, preferably 5.0 ± 0.2.

[0070] In some embodiments, the adjusting solution used to adjust the pH of the cation exchange chromatography loading buffer is a 1 mol / L citric acid solution. In some specific embodiments, the pH of the cation exchange chromatography eluent is at least 1.0 higher than the pH of the cation exchange chromatography equilibration buffer, for example, about 1.0, about 1.05, about 1.2, about 1.25, about 1.3, about 1.35, about 1.4, about 1.45, or about 1.5 higher, preferably 1.0-1.45 higher.

[0071] In some embodiments, the pH of the cation exchange chromatography equilibration solution is 3.0-8.0, 3.5-7.0, or 4.0-6.0, for example, 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, etc. The range formed by 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, or approximately 8.0, or any of the aforementioned values, is preferably 5.0 ± 0.2.

[0072] In some embodiments, the conductivity of the cation exchange chromatography equilibrium solution is 1.0-20.0 mS / cm, 3.0-15.0 mS / cm, or 6.0-10.0 mS / cm, for example, 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, and about 10.0 mS / cm. S / cm, approximately 10.5 mS / cm, approximately 11.0 mS / cm, approximately 11.5 mS / cm, approximately 12.0 mS / cm, approximately 12.5 mS / cm, approximately 13.0 mS / cm, approximately 13.5 mS / cm, approximately 14.0 mS / cm, approximately 14.5 mS / cm, approximately 15.0 mS / cm, approximately 15.5 mS / cm, approximately 16.0 mS / cm, approximately 16.5 mS / cm, approximately 17.0 mS / cm, approximately 17.5 mS / cm, approximately 18.0 mS / cm, approximately 18.5 mS / cm, approximately 19.0 mS / cm, approximately 19.5 mS / cm, or approximately 20.0 mS / cm, or 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, for example, 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, or about 5.6. The range formed by approximately 5.7, approximately 5.8, approximately 5.9, approximately 6.0, approximately 6.1, approximately 6.15, approximately 6.2, approximately 6.25, approximately 6.3, approximately 6.35, 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 8.0, approximately 8.1, approximately 8.2, approximately 8.3, approximately 8.4, or approximately 8.5, or any of the aforementioned values, is 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, for example, 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. The values ​​are approximately 11.0 mS / cm, 11.5 mS / cm, 12.0 mS / cm, 12.5 mS / cm, 13.0 mS / cm, 13.5 mS / cm, 14.0 mS / cm, 14.5 mS / cm, 15.0 mS / cm, 15.5 mS / cm, 16.0 mS / cm, 16.5 mS / cm, 17.0 mS / cm, 17.5 mS / cm, 18.0 mS / cm, 18.5 mS / cm, 19.0 mS / cm, 19.5 mS / cm, or 20.0 mS / cm, or any of the aforementioned values, preferably 8.0-10.0 mS / cm.

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

[0076] In some implementations, the method includes the following steps:

[0077] (1) Deep filtration;

[0078] (2) Affinity chromatography, comprising the following steps: ① Equilibration 1, equilibration with 10 mmol / L sodium phosphate buffer (pH 6.0±0.2, conductivity 0.5-1.5 mS / cm); ② Sample loading; ③ Equilibration 2, equilibration with 10 mmol / L sodium phosphate buffer (pH 6.0±0.2, conductivity 0.5-1.5 mS / cm); ④ Eluting, elution 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, equilibration with 10 mmol / L sodium phosphate buffer (pH 6.0±0.2, conductivity 0.5-1.5 mS / cm); ⑥ Elution, elution 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 step: deep filtering;

[0081] (5) Composite anion exchange chromatography, wherein the composite anion exchange chromatography includes the following steps: ① Equilibration 1, equilibration is performed using 10 mmol / L Tris-HCl buffer (pH 7.5±0.2, conductivity 4.0-6.0 mS / cm) containing 35 mmol / L sodium chloride; ② Sample loading solution adjustment, first adjust the pH of the second step deep filtration filtrate to 7.5±0.2 with 2 mol / L Tris-HCl solution (pH 9.50±0.05), then adjust the sample conductivity to 4.0-6.0 mS / cm with 1 mol / L sodium chloride solution or water for injection; ③ Sample loading, collection of flow-through; ④ Equilibration 2, equilibration of the chromatography column using 10 mmol / L Tris-HCl buffer (pH 7.5±0.2, conductivity 4.0-6.0 mS / cm) containing 35 mmol / L sodium chloride;

[0082] (6) Cation exchange chromatography, wherein the cation exchange chromatography includes the following steps: ① Equilibration 1, equilibration is performed using 20 mmol / L sodium phosphate buffer containing 50 mmol / L sodium chloride and 10 mmol / L sodium citrate buffer (pH 5.0±0.2, conductivity 7.0-9.0 mS / cm); ② Sample loading; ③ Equilibration, equilibration is performed using 20 mmol / L sodium phosphate buffer containing 50 mmol / L sodium chloride and 10 mmol / L sodium citrate buffer (pH 5.0±0.2, conductivity 7.0-9.0 mS / cm); ④ Elution, elution is performed using 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 mS / cm);

[0083] Steps (1)-(6) are performed sequentially.

[0084] The method of this application has at least the following beneficial effects: by using affinity chromatography, combined anion exchange chromatography and cation exchange chromatography, and adjusting the pH and conductivity of the loading solution before loading in combined anion exchange chromatography, high-purity bevacizumab can be prepared; the chromatographic packing material used has high accessibility and low cost, and is suitable for industrial-scale production.

[0085] Terminology Explanation

[0086] Unless otherwise stated, the following terms as used in this application shall have the following meanings. A particular term should not be considered uncertain or unclear unless specifically defined, but should be understood in accordance with its ordinary meaning in the art.

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

[0088] "Including" or "containing" should be understood in an open, non-exclusive sense, meaning "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 using standard methods known to those skilled in the art.

[0090] "Intermediate products" refer to products that have completed some of the technological steps but still require further processing to become the final product.

[0091] "CE-SDS non-reducing electrophoresis" refers to non-reducing sodium dodecyl sulfate capillary gel electrophoresis. It uses a high-voltage electric field as the driving force to separate products according to their molecular weight and the different speeds they move in the capillary. The higher the proportion of monomers, the higher the purity of the product.

[0092] "CE-SDS reduction electrophoresis" refers to the reduction sodium dodecyl sulfate capillary gel electrophoresis method. It uses a reduction capillary gel electrophoresis method with a high voltage electric field as the driving force. After reduction, the products are separated according to their different migration speeds in the capillary based on their molecular weight. The higher the ratio of the sum of light and heavy chains, the higher the purity of the product.

[0093] "HCP" refers to host cell protein, a protein component derived from cell lines that produce and express antibodies (such as CHO cells), including but not limited to proteins secreted by the cell line, proteins produced during apoptosis, and structural proteins generated by metabolism. HCP poses a safety risk to antibody drugs and requires strict control and monitoring.

[0094] "SEC-HPLC" refers to size exclusion high-performance liquid chromatography, also known as volume exclusion chromatography, size exclusion chromatography, or spatial exclusion chromatography. It uses area normalization to categorize substances into monomers, polymers (high molecular weight group), and fragments (low molecular weight group). A higher percentage of monomers indicates higher product purity.

[0095] "CEX-HPLC" refers to cation exchange high-performance liquid chromatography, which separates products based on differences in their charge numbers. It can separate charge isomers within the product, addressing issues related to product homogeneity. "Charge isomers" are isomers of antibodies whose charge has changed directly or indirectly due to processes such as glycosylation, deamidation, oxidation, and isomerization. These isomers are generally classified as acidic or basic isomers. When analyzing charge isomers using CEX, acidic peaks (acidic isomers) elute before the main peak, while basic peaks (basic isomers) elute later. A higher proportion of acidic or basic peaks indicates a greater degree of charge heterogeneity and poorer product homogeneity.

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

[0097] Although the foregoing application has been described in considerable detail by way of example and embodiments for purposes of clarity, it will be apparent to those skilled in the art, based on the teachings of this application, that certain changes and modifications may be made to this application without departing from the spirit and scope of the appended claims. The following embodiments are provided by way of illustration only and are not intended to be limiting. Those skilled in the art will readily identify various non-critical parameters that can be changed or modified to produce substantially similar results.

[0098] All reagents used in the examples, unless otherwise stated, are commercially available. Solutions can be prepared using conventional techniques in the art.

[0099] The purified antibody in the examples was bevacizumab, whose amino acid sequence is similar to... Identical (INN: bevacizumab). To express bevacizumab, the DNA sequences encoding the heavy and light chains of bevacizumab are cloned into an expression vector, which is then transfected into host cells, allowing the gene to be 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 packing materials

[0101] The efficacy of five affinity chromatography packing materials—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 was compared. The affinity chromatography steps include: ① Equilibration 1, equilibrate with 10 mmol / L sodium phosphate buffer (pH 6.0±0.1); ② Sample loading; ③ Equilibration 2, equilibrate with 10 mmol / L sodium phosphate buffer (pH 6.0±0.1); ④ Eluting, elute 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 eluent, and analyze the column volume (CV) and recovery rate of affinity chromatography.

[0102] Recovery rate (%) = Protein amount in affinity chromatography eluent / Protein amount in first-step deep filtration filtrate * 100%

[0103] As shown in Table 1, affinity chromatography using Novo-A Diamond packing material exhibits a small elution volume and high recovery rate. Selecting affinity chromatography packing material with a small elution volume effectively controls the volume of intermediate products in the second-step deep filtration process; therefore, Novo-A Diamond was chosen 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 Material and Sample Loading Conditions

[0107] Compare the effects of two composite anion exchange chromatography packing materials, NM90Agarose HAM (NanoMicro, 04092-090001) and Diamond Mix-A (BestChrom, AI0105), on the separation of bevacizumab under different loading and equilibration conditions. The steps of the combined anion exchange chromatography include: ① Equilibration 1: Equilibrate using a 10 mmol / L Tris-HCl buffer solution containing 35 mmol / L sodium chloride (pH and conductivity consistent with the loading solution); ② Loading solution adjustment: First adjust the pH with 2 mol / L Tris-HCl solution (pH 9.50 ± 0.05), then adjust the conductivity with 1 mol / L sodium chloride solution or water for injection; ③ Loading: Collect the flow-through; ④ Equilibration 2: Equilibrate using a 10 mmol / L Tris-HCl buffer solution containing 35 mmol / L sodium chloride (pH and conductivity consistent with the loading solution), wash for 10-30 CV, stop collecting the flow-through, and analyze the combined anion exchange chromatography flow-through by sodium dodecyl sulfate capillary electrophoresis (CE-SDS). As shown in Table 2, when NM90-Agarose HAM packing material, a loading buffer with a conductivity of approximately 5 mS / cm and a pH of approximately 7.5 was used for composite anion exchange chromatography, the percentage of the non-reducing CE-SDS main peak in the flow-through solution was the highest compared to the intermediate product of the second-step deep filtration. Therefore, NM90-Agarose HAM packing material, a loading buffer with a conductivity of approximately 5 mS / cm and a pH of approximately 7.5, and an equilibration buffer were selected for composite anion exchange chromatography.

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

[0109]

[0110]

[0111] Example 3: Screening of cation exchange chromatography packing materials

[0112] The effects of three cation exchange chromatography packing materials—Diamond SP (BestChrom, AI319314), NanoGel-50SPHP (NanoMicro, 04062-050150), and Monomix HC45-SP (Sepax, 280645950)—on the separation of bevacizumab were compared. The cation exchange chromatography steps include: ① Equilibration 1: Equilibrate with 20 mmol / L sodium phosphate buffer and 10 mmol / L sodium citrate buffer (pH 5.0 ± 0.1) containing 50 mmol / L sodium chloride; ② Sample loading adjustment: Adjust the pH to 5.0 ± 0.2 with 1 mol / L citric acid solution; ③ Sample loading; ④ Equilibration 2: Equilibrate with 20 mmol / L sodium phosphate buffer and 10 mmol / L sodium citrate buffer (pH 5.0 ± 0.1) containing 50 mmol / L sodium chloride; ⑤ Elution: Elute with 20 mmol / L sodium phosphate buffer and 10 mmol / L sodium citrate buffer (pH 6.25 ± 0.2) containing 50 mmol / L sodium chloride, and collect the eluent. The eluent from cation exchange chromatography was analyzed using host cell protein (HCP) residues, host cell DNA residues, Protein A residues, size exclusion-high performance liquid chromatography (SEC-HPLC), and ion exchange-high performance liquid chromatography (CEX-HPLC). As shown in Table 3, cation exchange chromatography using NanoGel-50SP HP packing material effectively removed alkaline regions and reduced high molecular weight impurities; therefore, NanoGel-50SP HP was selected as the packing material for cation exchange chromatography.

[0113] Table 3 Screening of cation exchange packing materials

[0114]

[0115] Example 4: Isolation and Purification of Bevacizumab

[0116] Step 1: Deep filtration: Collect the cell culture medium and perform deep filtration, then collect the filtrate;

[0117] Affinity chromatography: Novo-A Diamond packing material was used. The steps included: ① Equilibration 1: equilibration 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: equilibration with 10 mmol / L sodium phosphate buffer (pH 6.0±0.1, conductivity 0.5-1.5 mS / cm); ④ Eluting: elution 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: equilibration with 10 mmol / L sodium phosphate buffer (pH 6.0±0.1, conductivity 0.5-1.5 mS / cm); ⑥ Elution: elution with 50 mmol / L sodium citrate buffer (pH 6.0±0.1, conductivity 0.5-1.5 mS / cm). Elute using a solution of 3.8±0.2 μL (conductivity 2.5-4.5 mS / cm) and collect the eluent.

[0118] Virus inactivation: Adjust the pH of the affinity chromatography eluent 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 sample pH to 5.2±0.1 with 2 mol / L Tris-HCl solution (pH 9.50±0.05).

[0119] The second step is deep filtration: the virus-inactivated sample is subjected to deep filtration, and the filtrate is collected.

[0120] Composite anion exchange chromatography: Using NM90-Agarose HAM packing material, the steps include: ① Equilibration 1: Equilibrate the 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); ② Sample loading solution adjustment: First, adjust the pH of the second-step deep filtration filtrate to 7.5±0.2 with 2 mol / L Tris-HCl solution (pH 9.50±0.05), then adjust the sample conductivity to 4.0-6.0 mS / cm with 1 mol / L sodium chloride solution or water for injection; ③ Sample loading: Collect the flow-through; ④ Equilibration 2: Equilibrate the 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, then stop collecting the flow-through.

[0121] Cation exchange chromatography: using NanoGel-50SP The HP packing material is prepared using the following steps: ① Equilibration 1: Equilibrate with 20 mmol / L sodium phosphate buffer and 10 mmol / L sodium citrate buffer (pH 5.0 ± 0.1, conductivity 7.0-9.0 mS / cm); ② Sample loading solution adjustment: Adjust the pH of the composite anion exchange chromatography flow-through buffer to 5.0 ± 0.2 with 1 mol / L citric acid solution; ③ Sample loading; ④ Equilibration 2: Equilibrate with 20 mmol / L sodium phosphate buffer and 10 mmol / L sodium citrate buffer (pH 5.0 ± 0.1, conductivity 7.0-9.0 mS / cm); ⑤ Elution: Elute with 20 mmol / L sodium phosphate buffer and 10 mmol / L sodium citrate buffer (pH 6.25 ± 0.2, conductivity 8.0-10.0 mS / cm), and collect the eluent.

[0122] Virus removal filtration (nanofiltration): The cation exchange chromatography eluent is used for virus removal filtration, and the filtrate is collected;

[0123] Ultrafiltration concentration and fluid exchange: The virus-free filtrate is subjected to ultrafiltration and fluid exchange to harvest the product containing bevacizumab.

[0124] The ultrafiltration harvest liquid was sterilized and filtered to obtain bevacizumab antigen solution.

[0125] The above process was used to purify 2000 kg of cell culture medium. Table 4 shows the key quality results of the harvested solution and the original solution at each step of the bevacizumab purification process, and Table 5 shows the purification results of the original solution. This indicates that the above process is suitable for the purification of bevacizumab.

[0126] Table 4 Key quality results of harvested solution and original solution at each step of bevacizumab purification.

[0127]

[0128] Table 5. Purification results of bevacizumab monoantigen solution

[0129]

Claims

1. A method for purifying bevacizumab, characterized in that, The method includes the following steps: (1) First step: deep filtering; (2) Affinity chromatography, wherein the steps of the affinity chromatography include equilibration 1, sample loading, equilibration 2, elution, equilibration 3 and elution, the packing material is Novo-A Diamond, equilibration 1, equilibration 2 and equilibration 3 are equilibrated with 10 mmol / L sodium phosphate buffer, pH 6.0±0.1, conductivity 0.5-1.5 mS / cm, the elution buffer is 10 mmol / L sodium phosphate buffer containing 0.5 mol / L sodium chloride, pH 7.0±0.2, conductivity 43-52 mS / cm, the elution buffer is 50 mmol / L sodium citrate buffer, pH 3.8±0.2, conductivity 2.5-4.5 mS / cm; (3) Virus inactivation; (4) Second step: deep filtering; (5) Composite anion exchange chromatography, wherein the steps of the composite anion exchange chromatography include equilibration 1, adjustment of the pH and conductivity of the loading solution, loading, and equilibration 2. The packing material is NM90 Agarose HAM. Equilibration 1 and equilibration 2 are performed using a 10 mmol / L Tris-HCl buffer containing 35 mmol / L sodium chloride, pH 7.5±0.2, and conductivity 4.0-6.0 mS / cm. The pH and conductivity of the adjusted loading solution are consistent with those of the equilibration solution. (6) Cation exchange chromatography, wherein the steps of the cation exchange chromatography include equilibration 1, sample loading solution adjustment, sample loading, equilibration 2 and elution, the packing material is NanoGel-50SP HP, equilibration 1 and equilibration 2 are performed using 20 mmol / L sodium phosphate buffer and 10 mmol / L sodium citrate buffer containing 50 mmol / L sodium chloride, pH 5.0±0.1, conductivity 7.0-9.0 mS / cm, the elution buffer is 20 mmol / L sodium phosphate buffer and 10 mmol / L sodium citrate buffer containing 50 mmol / L sodium chloride, pH 6.25±0.2, conductivity 8.0-10.0 mS / cm; Steps (1)-(6) are performed sequentially.

2. The method according to claim 1, characterized in that, The pH adjustment solution used to adjust the sample solution for complex anion exchange chromatography is a 2 mol / L Tris-HCl solution, and / or the conductivity adjustment solution used to adjust the sample solution for complex anion exchange chromatography is a 1 mol / L sodium chloride solution.

3. The method according to claim 1, characterized in that, The pH of the cation exchange chromatography loading solution was adjusted to 5.0 ± 0.

2.

4. The method according to claim 1 or 3, characterized in that, The pH adjustment solution used to adjust the sample loading solution for cation exchange chromatography is a 1 mol / L citric acid solution.

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