A method for cleaning an affinity chromatography column for endotoxin removal

Abstract

This invention provides a cleaning method for affinity chromatography columns used for endotoxin removal, comprising the following steps: first cleaning the affinity chromatography column packing material to be treated with sodium chloride solution, then cleaning it with sodium hydroxide solution; then cleaning the resulting affinity chromatography column packing material with EDTA in glycerol solution; finally cleaning the resulting affinity chromatography column packing material to be treated with acetic acid solution, then cleaning it with phosphate buffer solution. The cleaning method for affinity chromatography columns used for endotoxin removal described in this invention can clean the columns, remove residual impurities from the packing material, extend the service life of the packing material, ensure good separation performance in subsequent use, and guarantee the quality of the treated biological products.

Description

Technical Field

[0001] This invention relates to chromatography separation technology, and in particular to a cleaning method for affinity chromatography columns used to remove endotoxins. Background Technology

[0002] Endotoxins, also known as lipopolysaccharides, are components of the cell walls of Gram-negative bacteria and are pyrogens that can have harmful effects on humans and animals. In biopharmaceutical production, the concentration of endotoxins is strictly controlled; therefore, endotoxin removal is necessary during the production process. Affinity chromatography is a commonly used method for endotoxin removal. Coordination groups with specific binding interactions, such as polymyxin B, histamine, histidine, and some polycations, are coupled to the surface of the column packing material, allowing for selective adsorption of endotoxins that can specifically bind to them. However, with the use of affinity chromatography columns, impurities may remain in the packing material, leading to a shortened lifespan of the packing material, reduced separation efficiency, and decreased quality of the biopharmaceutical in subsequent use. Summary of the Invention

[0003] The purpose of this invention is to provide a cleaning method for affinity chromatography columns used to remove endotoxins, thereby solving the problems of shortened packing material life, reduced separation efficiency, and decreased quality of biological products caused by impurity residues in existing affinity chromatography columns used to remove endotoxins.

[0004] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows:

[0005] A method for cleaning an affinity chromatography column for removing endotoxins includes the following steps:

[0006] (1) The affinity chromatography column packing material to be treated for removing endotoxins was first cleaned with sodium chloride solution and then with sodium hydroxide solution.

[0007] (2) The affinity chromatography column packing material to be treated for removing endotoxin obtained in step (1) is cleaned with EDTA in glycerol solution;

[0008] (3) The affinity chromatography column packing material to be processed for removing endotoxins obtained in step (2) is first cleaned with acetic acid solution and then with phosphate buffer solution.

[0009] Preferably, in step (1), the concentration of the sodium chloride solution is 0.2-0.5 mol / L.

[0010] Preferably, in step (1), the concentration of sodium hydroxide is 1-2 mol / L.

[0011] Preferably, in step (2), the concentration of the EDTA glycerol solution is 0.5-1.0 mol / L.

[0012] Preferably, in step (3), the concentration of the acetic acid solution is 0.4-0.8 mol / L.

[0013] Preferably, in step (3), the pH value of the phosphate buffer solution is 5.8-6.2.

[0014] Preferably, in step (3), the concentration of the phosphate buffer solution is 0.01-0.05 mol / L.

[0015] Preferably, the flow rate of the cleaning liquid is 20-30 ml / min.

[0016] Preferably, in step (1), the liquid flow rate of the sodium chloride solution for cleaning is 20-25 ml / min, and the liquid flow rate of the sodium hydroxide solution for cleaning is 25-30 ml / min.

[0017] In step (2), the liquid flow rate for cleaning with EDTA in glycerol solution is 20-30 ml / min;

[0018] In step (3), the liquid flow rate for cleaning with acetic acid solution is 25-30 ml / min, and the liquid flow rate for cleaning with phosphate buffer solution is 25-30 ml / min.

[0019] Preferably, in step (1), the cleaning time using sodium chloride solution is 30 min, and the cleaning time using sodium hydroxide solution is 120 min.

[0020] In step (2), the cleaning time using EDTA in glycerol solution is 120 min;

[0021] In step (3), the cleaning time using acetic acid solution is 60 min, and the cleaning time using phosphate buffer solution is 180 min.

[0022] The above-described solution of the present invention has at least the following beneficial effects:

[0023] The present invention provides a cleaning method for affinity chromatography columns used for endotoxin removal, comprising the following steps: first cleaning the affinity chromatography column packing material to be treated with sodium chloride solution, and then cleaning it with sodium hydroxide solution; cleaning the obtained affinity chromatography column packing material to be treated with EDTA in glycerol solution; and cleaning the obtained affinity chromatography column packing material to be treated with acetic acid solution, and then cleaning it with phosphate buffer solution. This cleaning method for affinity chromatography columns used for endotoxin removal can clean the columns, remove residual impurities from the packing material, extend the service life of the packing material, ensure good separation performance in subsequent use, and guarantee the quality of the treated biological products. Detailed Implementation

[0024] Unless otherwise specified in the embodiments of this invention, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available products; different manufacturers and models of raw materials do not affect the implementation of the technical solution or the achievement of the technical effect of this invention.

[0025] Example 1

[0026] The cleaning method for the affinity chromatography column used to remove endotoxins in this embodiment includes the following steps:

[0027] (1) The affinity chromatography column packing material to be treated for removing endotoxin was first cleaned with sodium chloride solution at a flow rate of 24 ml / min for 30 min; then it was cleaned with sodium hydroxide solution at a flow rate of 28 ml / min for 120 min.

[0028] The concentration of the sodium chloride solution is 0.4 mol / L. The concentration of the sodium hydroxide solution is 1.5 mol / L.

[0029] (2) The affinity chromatography column packing material to be treated for removing endotoxin obtained in step (1) is cleaned with EDTA in glycerol solution. The liquid flow rate for cleaning is 26 ml / min and the cleaning time is 120 min.

[0030] The concentration of the EDTA glycerol solution is 0.8 mol / L.

[0031] (3) The affinity chromatography column packing material to be treated for removing endotoxin obtained in step (2) is first cleaned with acetic acid solution at a flow rate of 25-30 ml / min for 60 min; then it is cleaned with phosphate buffer solution at a flow rate of 28 ml / min.

[0032] The acetic acid solution has a concentration of 0.6 mol / L. The phosphate buffer solution has a pH of 6.0. The phosphate buffer solution has a concentration of 0.03 mol / L, and the washing time is 180 min.

[0033] It should be noted that in this embodiment, the affinity chromatography column used to remove endotoxins has a diameter-to-height ratio of 6:1. Those skilled in the art can replace it with other specifications and dimensions according to actual conditions.

[0034] Example 2

[0035] The cleaning method for the affinity chromatography column used to remove endotoxins in this embodiment includes the following steps:

[0036] (1) The affinity chromatography column packing material to be treated for removing endotoxin was first cleaned with sodium chloride solution at a flow rate of 20 ml / min for 30 min; then it was cleaned with sodium hydroxide solution at a flow rate of 30 ml / min for 120 min.

[0037] The concentration of the sodium chloride solution is 0.3 mol / L. The concentration of the sodium hydroxide solution is 1 mol / L.

[0038] (2) The affinity chromatography column packing material to be treated for removing endotoxin obtained in step (1) is cleaned with EDTA in glycerol solution. The liquid flow rate for cleaning is 30 ml / min and the cleaning time is 120 min.

[0039] The concentration of the EDTA glycerol solution is 0.5 mol / L.

[0040] (3) The affinity chromatography column packing material to be treated for removing endotoxin obtained in step (2) is first cleaned with acetic acid solution at a flow rate of 28 ml / min for 60 min; then it is cleaned with phosphate buffer solution at a flow rate of 25 ml / min.

[0041] The acetic acid solution has a concentration of 0.6 mol / L. The phosphate buffer solution has a pH of 5.8. The phosphate buffer solution has a concentration of 0.03 mol / L, and the washing time is 180 min.

[0042] It should be noted that in this embodiment, the affinity chromatography column used to remove endotoxins has a diameter-to-height ratio of 6:1. Those skilled in the art can replace it with other specifications and dimensions according to actual conditions.

[0043] Example 3

[0044] The cleaning method for the affinity chromatography column used to remove endotoxins in this embodiment includes the following steps:

[0045] (1) The affinity chromatography column packing material to be treated for removing endotoxin was first cleaned with sodium chloride solution at a flow rate of 30 ml / min for 30 min; then it was cleaned with sodium hydroxide solution at a flow rate of 20 ml / min for 120 min.

[0046] The concentration of the sodium chloride solution is 0.5 mol / L. The concentration of the sodium hydroxide solution is 2 mol / L.

[0047] (2) The affinity chromatography column packing material to be treated for removing endotoxin obtained in step (1) is cleaned with EDTA in glycerol solution. The liquid flow rate for cleaning is 25 ml / min and the cleaning time is 120 min.

[0048] The concentration of the EDTA glycerol solution is 0.8 mol / L.

[0049] (3) The affinity chromatography column packing material to be treated for removing endotoxin obtained in step (2) is first cleaned with acetic acid solution at a flow rate of 25 ml / min for 60 min; then it is cleaned with phosphate buffer solution at a flow rate of 30 ml / min.

[0050] The acetic acid solution has a concentration of 0.8 mol / L. The phosphate buffer solution has a pH of 6.2. The phosphate buffer solution has a concentration of 0.05 mol / L, and the washing time is 180 min.

[0051] It should be noted that in this embodiment, the affinity chromatography column used to remove endotoxins has a diameter-to-height ratio of 6:1. Those skilled in the art can replace it with other specifications and dimensions according to actual conditions.

[0052] Example 4

[0053] The cleaning method for the affinity chromatography column used to remove endotoxins in this embodiment includes the following steps:

[0054] (1) The affinity chromatography column packing material to be treated for removing endotoxin was first cleaned with sodium chloride solution at a flow rate of 25 ml / min for 30 min; then it was cleaned with sodium hydroxide solution at a flow rate of 25 ml / min for 120 min.

[0055] The concentration of the sodium chloride solution is 0.2 mol / L. The concentration of the sodium hydroxide solution is 1.5 mol / L.

[0056] (2) The affinity chromatography column packing material to be treated for removing endotoxin obtained in step (1) is cleaned with EDTA in glycerol solution. The liquid flow rate for cleaning is 20 ml / min and the cleaning time is 120 min.

[0057] The concentration of the EDTA glycerol solution is 1.0 mol / L.

[0058] (3) The affinity chromatography column packing material to be treated for removing endotoxin obtained in step (2) is first cleaned with acetic acid solution at a flow rate of 30 ml / min for 60 min; then it is cleaned with phosphate buffer solution at a flow rate of 28 ml / min.

[0059] The acetic acid solution has a concentration of 0.4 mol / L. The phosphate buffer solution has a pH of 6.0. The phosphate buffer solution has a concentration of 0.01 mol / L, and the washing time is 180 min.

[0060] It should be noted that in this embodiment, the affinity chromatography column used to remove endotoxins has a diameter-to-height ratio of 6:1. Those skilled in the art can replace it with other specifications and dimensions according to actual conditions.

[0061] Effect Experiment Example

[0062] To verify the technical effectiveness of the affinity chromatography column cleaning method for removing endotoxins described in this invention, the following experiments were conducted:

[0063] Five affinity chromatography columns for endotoxin removal were obtained under the same operating conditions. One column served as a control group, while the other four were cleaned according to the cleaning methods for affinity chromatography columns used for endotoxin removal described in Examples 1-4. Using these five affinity chromatography columns, endotoxin-containing biological products with the same composition were used to remove endotoxins, and the endotoxin content in the treated biological products was measured.

[0064] The results of the experiment are as follows:

[0065]

[0066]

[0067] Based on the above experimental results, it can be seen that the cleaning method for affinity chromatography columns used to remove endotoxins described in this invention has a good cleaning effect, can effectively remove impurities remaining in the packing material, and ensure that it maintains a good separation effect in subsequent use.

[0068] As is known from common technical knowledge, this invention can be implemented through other embodiments that do not depart from its spirit or essential characteristics. Therefore, the disclosed embodiments described above are merely illustrative in all respects and are not the only ones. All modifications within the scope of this invention or its equivalents are included in this invention.

Claims

1. A method for cleaning an affinity chromatography column for endotoxin removal, characterized in that, Includes the following steps: (1) The affinity chromatography column packing material to be treated for removing endotoxins was first cleaned with sodium chloride solution and then with sodium hydroxide solution. (2) The affinity chromatography column packing material to be treated for removing endotoxin obtained in step (1) is cleaned with EDTA in glycerol solution; (3) The affinity chromatography column packing material to be processed for removing endotoxins obtained in step (2) is first cleaned with acetic acid solution and then with phosphate buffer solution.

2. The cleaning method of the affinity chromatography column for removing endotoxin according to claim 1, characterized by, In step (1), the concentration of the sodium chloride solution is 0.2-0.5 mol / L.

3. The cleaning method of the affinity chromatography column for removing endotoxin according to claim 1, characterized by, In step (1), the concentration of sodium hydroxide is 1-2 mol / L.

4. The cleaning method of the affinity chromatography column for removing endotoxin according to claim 1, characterized by, In step (2), the concentration of the EDTA glycerol solution is 0.5-1.0 mol / L.

5. The cleaning method of affinity chromatography column for removing endotoxin according to claim 1, characterized in that, In step (3), the concentration of the acetic acid solution is 0.4-0.8 mol / L.

6. The cleaning method of affinity chromatography column for removing endotoxin according to claim 1, characterized in that, In step (3), the pH value of the phosphate buffer solution is 5.8-6.

2.

7. The cleaning method for an affinity chromatography column for removing endotoxins according to claim 6, characterized in that, In step (3), the concentration of the phosphate buffer solution is 0.01-0.05 mol / L.

8. The cleaning method of affinity chromatography column for removing endotoxin according to claim 1, characterized in that, In step (1), the liquid flow rate for cleaning with sodium chloride solution is 20-25 ml / min, and the liquid flow rate for cleaning with sodium hydroxide solution is 25-30 ml / min. In step (2), the liquid flow rate for cleaning with EDTA in glycerol solution is 20-30 ml / min; In step (3), the liquid flow rate for cleaning with acetic acid solution is 25-30 ml / min, and the liquid flow rate for cleaning with phosphate buffer solution is 25-30 ml / min.

9. The cleaning method of the affinity chromatography column for removing endotoxin according to claim 8, characterized by, In step (1), the cleaning time using sodium chloride solution is 30 min, and the cleaning time using sodium hydroxide solution is 120 min. In step (2), the cleaning time using EDTA in glycerol solution is 120 min; In step (3), the cleaning time using acetic acid solution is 60 min, and the cleaning time using phosphate buffer solution is 180 min.

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