Affinity purification method of recombinant anti-VEGFR2 fully human monoclonal antibody

Abstract

The invention discloses an affinity purification method of a recombinant anti-VEGFR2 (vascular endothelial growth factor receptor 2) fully humanized monoclonal antibody, which comprises the following steps: step A, providing a to-be-purified raw material which contains an anti-VEGFR2 fully humanized monoclonal antibody; b, the raw materials to be purified are subjected to affinity chromatography, eluent containing sample peaks is collected, and filler for affinity chromatography is selected from Eshmuno A, MabSelect SuRe and AF-rProtein A HC-650F, preferably MabSelect SuRe. According to the method disclosed by the invention, the anti-VEGFR2 fully human monoclonal antibody with high yield, high monomer content and low host protein content can be prepared.

Description

Technical Field

[0001] The present invention relates to the field of antibody purification, and in particular to a method for affinity purification of a recombinant anti-VEGFR2 fully human monoclonal antibody. Background Art

[0002] Recombinant anti-VEGFR2 fully human monoclonal antibody is a VEGFR2 antagonist that specifically binds to VEGFR2, blocks the binding of VEGFR2 to its ligands (VEGF-A, VEGF-C, VEGF-D), thereby inhibiting the activation of VEGFR2 ligands, and ultimately inhibiting the proliferation and migration of ligand-induced human endothelial vascular cells. The preparation of recombinant anti-VEGFR2 fully human monoclonal antibody is relatively complicated. First, the target molecule needs to be captured from the fermentation broth with complex components, and good impurity control and yield are required, so an affinity chromatography process with better performance is required. Summary of the invention

[0003] The present invention provides a method for affinity purification of a recombinant anti-VEGFR2 fully human monoclonal antibody, comprising the following steps:

[0004] Step A: providing a raw material to be purified, wherein the raw material to be purified contains an anti-VEGFR2 fully human monoclonal antibody;

[0005] Step B: subjecting the raw material to be purified to affinity chromatography, collecting the eluate containing the sample peak, wherein the filler of the affinity chromatography is selected from Eshmuno A, MabSelect SuRe, AF-rProtein A HC-650F, preferably MabSelectSuRe.

[0006] In some embodiments of the present invention, the step B comprises: balancing the chromatography column with an equilibration buffer; and / or, washing the chromatography column with a washing buffer; and / or, eluting with an elution buffer.

[0007] In some embodiments of the present invention, the step B is specifically as follows: balancing the chromatography column with an equilibration buffer, then adding the raw material to be purified, then washing the chromatography column with a washing buffer, and finally eluting with an elution buffer, and collecting the eluate.

[0008] In some embodiments of the invention, the equilibration buffer is a Tris-HAc / NaCl buffer.

[0009] In some embodiments of the present invention, the pH of the equilibration buffer is 7.2-7.6, preferably 7.4.

[0010] In some embodiments of the present invention, the concentration of Tris-HAc in the equilibration buffer is 40-60 mmol / L, preferably 50 mmol / L; the concentration of NaCl in the equilibration buffer is 120-180 mmol / L, preferably 140-160 mmol / L, more preferably 150 mmol / L.

[0011] In some embodiments of the present invention, the equilibration buffer is a Tris-HAc / NaCl buffer of 40-60mmol / L Tris-HAc, 120-180mmol / L NaCl, and pH 7.2-7.6; preferably a Tris-HAc / NaCl buffer of 50mmol / L Tris-HAc, 150mmol / L NaCl, and pH 7.4.

[0012] In some embodiments of the invention, the wash buffer is a NaAc-HAc / NaCl buffer.

[0013] In some embodiments of the present invention, the pH of the washing buffer is 4.5-6.5, preferably 4.5-5.5, more preferably 5.0-5.5, such as 5.0, 5.5.

[0014] In some embodiments of the present invention, the concentration of NaAc-HAc in the washing buffer is 40-60 mmol / L, preferably 50 mmol / L; the concentration of NaCl in the washing buffer is 0.5-2 mol / L, preferably 1 mol / L.

[0015] In some embodiments of the present invention, the washing buffer is a NaAc-HAc / NaCl buffer having a pH of 4.5-5.5 and a concentration of 40-60 mmol / L NaAc-HAc, 0.5-2 mol / L NaCl; preferably, the washing buffer is a NaAc-HAc / NaCl buffer having a pH of 5.0-5.5 and a concentration of 40-60 mmol / L NaAc-HAc, 0.5-2 mol / L NaCl; more preferably, the washing buffer is a NaAc-HAc / NaCl buffer having a pH of 5.5 and a concentration of 50 mmol / L NaAc-HAc, 1 mol / L NaCl.

[0016] In some embodiments of the invention, the elution buffer is NaAc-HAc buffer.

[0017] In some embodiments of the present invention, the pH of the elution buffer is 3.2-3.8, preferably 3.6-3.8, such as 3.6, 3.7 and 3.8.

[0018] In some embodiments of the present invention, the concentration of NaAc-HAc in the elution buffer is 40-60 mmol / L, preferably 50 mmol / L.

[0019] In some embodiments of the present invention, the elution buffer is a NaAc-HAc buffer with a pH of 3.6-3.8 and a concentration of 40-60 mmol / L; preferably, the elution buffer is a NaAc-HAc buffer with a pH of 3.8 and a concentration of 50 mmol / L.

[0020] In some embodiments of the present invention, the dynamic binding capacity of the affinity chromatography is 33-50 g / L, and the retention time of the affinity chromatography is 2-6 min.

[0021] In some embodiments of the present invention, the fully human monoclonal antibody against VEGFR2 (Ramucirumab) is produced by using recombinant DNA technology through a Chinese Hamster Ovary (CHO) cell expression system.

[0022] In some embodiments of the present invention, the raw material to be purified is a fermentation broth containing anti-VEGFR2 fully human monoclonal antibodies produced by a Chinese Hamster Ovary (CHO) cell expression system using DNA recombination technology and subjected to secondary centrifugation.

[0023] In some embodiments of the present invention, the raw material to be purified is a VEGFR2 fully human monoclonal antibody fermentation broth containing a target protein concentration of 1-5 g / L.

[0024] The method of the invention can prepare anti-VEGFR2 fully human monoclonal antibodies with high yield, high monomer content and low host protein content. DETAILED DESCRIPTION

[0025] The present invention is further described below through examples. The examples of the present invention are only used to illustrate the technical solution of the present invention and are not used to limit the scope of the present invention. Those skilled in the art may make some non-essential improvements and adjustments, which still fall within the protection scope of the present invention.

[0026] definition

[0027] Unless otherwise stated, all technical terms and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present invention belongs. If there is a conflict, the definition herein shall prevail. When a certain amount, concentration or other value or parameter is expressed in the form of a range, a preferred range or a preferred upper numerical limit and a preferred lower numerical limit, it should be understood that it is equivalent to specifically revealing any range by combining any pair of upper range limits or preferred numerical values ​​with any lower range limit or preferred numerical value. Unless otherwise stated, the numerical ranges listed herein are intended to include the endpoints of the range and all integers and fractions (decimals) within the range.

[0028] Example 1

[0029] The sample source is a fully human monoclonal antibody of VEGFR2 expressed in Chinese hamster ovary cells, and the target protein concentration is 2.0 g / L. Use 10 volumes of equilibration buffer (50 mmol / L Tris-HAc, 150 mmol / L NaCl, pH 7.4) to equilibrate the 96-well plate (filler as shown in the table below) and affinity chromatography column (affinity chromatography column diameter 0.5 cm, volume 2-3 mL, filler as shown in the table below), load the sample (load capacity 30 g / L), elute with 3 volumes of elution buffer (0.1 mol / L HAc), collect the eluate, and detect or calculate the monomer content, host protein content and yield of the eluate. The results are shown in Table 1 below.

[0030] Table 1: Purification results of different fillers

[0031]

[0032]

[0033] The results showed that affinity chromatography purification had a higher yield and higher monomer content in the elution peak than purification using 96-well plate. In affinity chromatography purification, the monomer content of samples eluted with MabSelect SuRe and Eshmuno A fillers was similar, and the host protein and Protein A content of samples eluted with MabSelect SuRe filler was lower.

[0034] Embodiment 2:

[0035] The sample source is a fully human monoclonal antibody of VEGFR2 expressed in Chinese hamster ovary cells, and the target protein concentration is 2.0 g / L. The affinity chromatography column (affinity chromatography column diameter is 0.5 cm, volume is 2-3 mL, and filler is MabSelect SuRe) was equilibrated with 3 column volumes of equilibration buffer (50 mmol / L Tris-HAc, 150 mmol / L NaCl, pH 7.4), loaded with sample (load capacity is 30 g / L), washed with 5 column volumes of wash buffer 1 (50 mmol / L NaAc-HAc, 1 mol / L NaCl, pH 4.5, 5.0, 5.5) and 3 column volumes of wash buffer 2 (50 mmol / L NaAc-HAc, pH is the same as wash buffer 1), eluted with 3 column volumes of eluent (0.1 mol / LHAc), and then the eluent was collected, and the purity and yield of the eluent were detected or calculated. The results are shown in Table 2 below.

[0036] Table 2: Purification results with different pH wash buffers

[0037]

[0038] The results showed that when the pH of the washing buffer was 4.5-5.5, the purity of SEC-HPLC and CEX-HPLC was high; when the pH of the washing buffer was 5.0-5.5, the yield could reach more than 95%.

[0039] Embodiment 3:

[0040] The sample source is a fully human monoclonal antibody of VEGFR2 expressed in Chinese hamster ovary cells, and the target protein concentration is 2.0 g / L. The affinity chromatography column (affinity chromatography column diameter is 0.5 cm, volume is 2-3 mL, and filler is MabSelect SuRe) was equilibrated with 3 column volumes of equilibration buffer (50 mmol / L Tris-HAc, 150 mmol / L NaCl, pH 7.4), loaded with sample (load capacity is 30 g / L), washed with 5 column volumes of washing buffer (50 mmol / L NaAc-HAc, 1 mol / L NaCl, pH 5.5), eluted with 3 column volumes of elution buffer (50 mmol / L NaAc-HAc, pH 3.6, 3.7, 3.8, respectively), and the eluate was collected. The monomer content, host protein content and yield of the eluate were detected or calculated. The results are shown in Table 3 below.

[0041] Table 3: Purification results of different pH elution buffers

[0042]

[0043] The results showed that when the pH of the elution buffer was 3.6-3.8, the elution peak monomer content was high, the host protein content was low, and the yield was high. As the elution pH increased, the elution peak monomer content increased, the residual host cells decreased, and the yield difference was not significant.

[0044] Embodiment 4:

[0045] The sample source is a fully human monoclonal antibody of VEGFR2 expressed in Chinese hamster ovary cells, and the target protein concentration is 2.0g / L. The affinity chromatography column (affinity chromatography column diameter is 0.5cm, volume is 2-3mL, filler MabSelect SuRe) is balanced with 3 times column volume of equilibrium buffer (50mmol / L Tris-HAc, 150mmol / L NaCl, pH 7.4), and the sample is loaded. The retention time is 2, 4 and 6 minutes respectively. The sample is overloaded and the samples of the sample flow-through at different time points are collected in groups. The protein content of the flow-through is 10% of the protein content of the sample sample as the maximum dynamic binding capacity. The results of retention time and dynamic binding capacity are shown in Table 4 below.

[0046] Table 4: Retention time and dynamic binding capacity

[0047]

[0048] The results showed that the dynamic binding capacity increased with the increase of retention time, but when the retention time was greater than 6 minutes, the dynamic binding capacity would not increase significantly. Therefore, a retention time of 2-6 minutes and a dynamic binding carrier of 33-50 g / L were selected to purify the sample.

[0049] Those skilled in the art can achieve the present invention by appropriately changing the raw materials, process parameters and other links by referring to the content of this article. The methods and products of this application have been described through preferred embodiments, and relevant technical personnel can obviously modify or appropriately change and combine the methods and products described herein without departing from the content, spirit and scope of this application to implement the technology of this application. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all deemed to be included in the spirit, scope and content of this application.

Claims

1. A method for affinity purification of recombinant anti-VEGFR2 fully human monoclonal antibodies, It is characterized in that The following steps are involved: Step A: providing a raw material to be purified, wherein the raw material to be purified contains an anti-VEGFR2 fully human monoclonal antibody; Step B: subjecting the raw material to be purified to affinity chromatography, collecting the eluate containing the sample peak, wherein the filler of the affinity chromatography is selected from Eshmuno A, MabSelect SuRe, AF-rProtein A HC-650F, preferably MabSelect SuRe.

2. The method according to claim 1, It is characterized in that The step B comprises: balancing the chromatography column with an equilibration buffer; and / or, washing the chromatography column with a washing buffer; and / or, eluting with an elution buffer; Preferably, the step B specifically comprises: balancing the chromatography column with an equilibration buffer, then adding the raw material to be purified, then washing the chromatography column with a washing buffer, and finally eluting with an elution buffer, and collecting the eluate.

3. The method according to claim 1 or 2, It is characterized in that The equilibration buffer is Tris-HAc / NaCl buffer; Preferably, the pH of the equilibration buffer is 7.2-7.6, preferably 7.4; Preferably, the concentration of Tris-HAc in the equilibration buffer is 40-60 mmol / L, preferably 50 mmol / L; the concentration of NaCl in the equilibration buffer is 120-180 mmol / L, preferably 140-160 mmol / L, more preferably 150 mmol / L.

4. The method according to any one of claims 1 to 3, It is characterized in that The equilibration buffer is a Tris-HAc / NaCl buffer of 40-60mmol / LTris-HAc, 120-180mmol / L NaCl, and pH 7.2-7.6; preferably a Tris-HAc / NaCl buffer of 50mmol / LTris-HAc, 150mmol / L NaCl, and pH 7.

4.

5. The method according to any one of claims 1 to 4, It is characterized in that The washing buffer is NaAc-HAc / NaCl buffer; Preferably, the pH of the washing buffer is 4.5-6.5, preferably 4.5-5.5, more preferably 5.0-5.5, such as 5.0, 5.5; Preferably, the concentration of NaAc-HAc in the washing buffer is 40-60 mmol / L, preferably 50 mmol / L; the concentration of NaCl in the washing buffer is 0.5-2 mol / L, preferably 1 mol / L.

6. The method according to any one of claims 1 to 5, It is characterized in that The washing buffer is a NaAc-HAc / NaCl buffer having a pH of 4.5-5.5 and a concentration of 40-60 mmol / L NaAc-HAc, 0.5-2 mol / L NaCl; preferably, the washing buffer is a NaAc-HAc / NaCl buffer having a pH of 5.0-5.5 and a concentration of 40-60 mmol / L NaAc-HAc, 0.5-2 mol / L NaCl; more preferably, the washing buffer is a NaAc-HAc / NaCl buffer having a pH of 5.5 and a concentration of 50 mmol / L NaAc-HAc, 1 mol / L NaCl.

7. The method according to any one of claims 1 to 6, It is characterized in that The elution buffer is NaAc-HAc buffer; Preferably, the pH of the elution buffer is 3.2-3.8, preferably 3.6-3.8, such as 3.6, 3.7 and 3.8; Preferably, the concentration of NaAc-HAc in the elution buffer is 40-60 mmol / L, preferably 50 mmol / L.

8. The method according to any one of claims 1 to 7, It is characterized in that The elution buffer is a NaAc-HAc buffer with a pH of 3.6-3.8 and a concentration of 40-60 mmol / L; preferably, the elution buffer is a NaAc-HAc buffer with a pH of 3.8 and a concentration of 50 mmol / L.

9. The method according to any one of claims 1 to 8, It is characterized in that The dynamic binding capacity of the affinity chromatography is 33-50 g / L, and the retention time of the affinity chromatography is 2-6 min.

10. The method according to any one of claims 1 to 9, It is characterized in that The raw material to be purified is a fermentation broth containing anti-VEGFR2 fully human monoclonal antibodies produced by a Chinese hamster ovary cell expression system using DNA recombination technology and after secondary centrifugation; preferably, the raw material to be purified is a fermentation broth containing VEGFR2 fully human monoclonal antibodies with a target protein concentration of 1-5 g / L.

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