Method for washing a peptide ligand immobilized column

Abstract

To provide a cleaning method capable of achieving both efficient cleaning and long column life for a peptidic ligand-immobilized carrier, which has been difficult to clean in the past.SOLUTION: Provided is a method for washing a column. The method comprises the steps of: equilibrating a column, which is packed with an insoluble carrier on which a peptide ligand is immobilized, by passing a buffer solution through the column; adding a cleaning solution to the column while passing the buffer solution therethrough; and further passing the buffer solution through the column to discharge the cleaning solution while diluting it, thereby effectively cleaning the vicinity of the inlet of the column. Since the washing solution is diluted with the buffer solution in the middle and subsequent stages of the column, the deactivation of a ligand can be suppressed and the function of the column can be maintained.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for washing a chromatography column on which a peptide ligand is immobilized. [Background technology]

[0002] In recent years, many columns immobilized with peptide ligands have been developed and sold as a tool for protein purification and analysis. For example, columns characterized by specifically adsorbing antibodies as target molecules include Protein A immobilized gel, Protein G immobilized gel, Protein L immobilized gel, and Fc receptor immobilized gel. When using these columns, the target molecules can be purified by adsorbing only the target molecules under specific pH and ionic strength conditions, washing impurities, and finally eluting and recovering the target molecules. However, when the above purification procedures are repeated, a certain amount of impurities remain in the column, causing problems such as a decrease in the separation performance of the column and impurities being mixed into the purified fraction. Therefore, a process called cleaning in place (hereinafter abbreviated as CIP) is generally carried out in which a cleaning solution is passed through the column while the column is connected to the chromatography device to clean it (Non-Patent Document 1). Here, cleaning solutions for CIP include high salt concentration solutions, acid solutions, alkaline solutions, surfactant solutions, denaturant solutions, organic solvents, etc., but in the case of peptide ligand immobilized columns in particular, there is a concern that exposure to such solutions may reduce performance, so there are significant limitations on the types of cleaning solutions that can be used, and in some cases there is an issue of not being able to perform sufficient cleaning. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Rainer Hahn et al., Journal of Chromatography A, 1102, 224-231(2006) Summary of the Invention [Problem to be solved by the invention]

[0004] An object of the present invention is to provide an effective method for washing a peptide ligand-immobilized column, which has conventionally been unable to use strong detergents. [Means for solving the problem]

[0005] The method for washing a peptidic ligand-immobilized column according to the present invention comprises the steps of passing a buffer solution through a column packed with an insoluble carrier having a peptidic ligand immobilized thereon to equilibrate the column, adding a washing solution while passing the buffer solution, and further passing the buffer solution through the column to dilute the washing solution while discharging it.

[0006] In one embodiment of the washing method according to the present invention, a modified human FcγRIIIA is used as the peptide ligand.

[0007] In one embodiment of the washing method according to the present invention, a 10 mM to 1000 mM citrate buffer solution having a pH range adjusted to 3 to 7 is used as the buffer solution.

[0008] In one embodiment of the washing method according to the present invention, a 10 mM to 1000 mM aqueous sodium hydroxide solution, a 10 mM to 6000 mM aqueous guanidine hydrochloride solution, and a 10 mM to 6000 mM aqueous urea solution are used as the washing solution, and the amount of washing solution injected per washing operation is 0.01 CV to 1 CV per column volume (hereinafter abbreviated as CV). Effect of the Invention

[0009] According to the present invention, even if a strong cleaning solution is used, the deterioration of the performance of a peptide ligand immobilized column can be suppressed, and it is possible to achieve both efficient cleaning and a long column life. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] The method for washing a peptidic ligand-immobilized column according to the present invention comprises the steps of passing a buffer solution through a column packed with an insoluble carrier having a peptidic ligand immobilized thereon to equilibrate the column, adding a washing solution while passing the buffer solution, and further passing the buffer solution through the column to dilute the washing solution while discharging it.

[0011] First, a buffer solution is added to a column packed with an insoluble carrier on which a peptide ligand has been immobilized, to equilibrate the column.

[0012] Examples of peptide ligands include protein A, protein G, protein L, human FcγRI, human FcγRII, human FcγRIII, complement protein C1q, and functional peptides for antibody purification, and mutants in which the amino acids constituting each of these are partially substituted may also be used. Furthermore, peptide ligands that target molecules other than antibodies, such as those that utilize antigen-antibody reactions, may also be used.

[0013] The insoluble carrier may be any substance that is insoluble in the solution or solvent used for adsorption / elution of the target protein and has a functional group (e.g., a hydroxyl group) for covalently immobilizing the peptidic ligand, and may be a carrier derived from an inorganic substance such as zirconia, zeolite, silica, or coated silica; a carrier derived from a natural organic polymeric substance such as cellulose, agarose, or dextran; or a carrier derived from a synthetic organic polymeric substance such as polyacrylic acid, polystyrene, polyacrylamide, polymethacrylamide, polymethacrylate, or vinyl polymer.

[0014] A buffer solution must satisfy two requirements: it must have the ability to buffer at an appropriate pH and it must not damage the function of the peptide ligand. Examples of buffering agents include carboxylic acids such as citric acid, succinic acid, and acetic acid, amino acids such as glycine, phosphoric acid, trishydroxymethylaminomethane, boric acid, and Good's buffer reagent (MES, HEPES, etc.). The buffer concentration can be determined taking into account the buffering capacity, and is typically in the range of 5 mM to 1000 mM. In addition, the buffer may contain inorganic salts, organic solvents, surfactants, preservatives, etc.

[0015] A wash solution is then added to the equilibrated column.

[0016] The washing solution may contain a washing component having a function of eluting the substance adsorbed on the column, and examples of the washing solution include a high salt concentration solution, a surfactant aqueous solution, an alkaline aqueous solution such as an aqueous solution of sodium hydroxide, an acidic solution such as an aqueous solution of acetic acid, an aqueous solution of a protein denaturant such as an aqueous solution of guanidine hydrochloride or an aqueous solution of urea, an organic solvent, etc. In addition, a plurality of the above washing components may be mixed.

[0017] The amount to be added may be adjusted based on the column volume (CV), and is preferably 0.01 CV to 1 CV.

[0018] Next, the buffer solution is further passed through to dilute the washing solution and then discharged.

[0019] Generally, contaminants adhering to a column are more likely to adhere near the column inlet and less likely to adhere toward the column outlet. By diluting the washing solution while discharging it, a strong cleaning effect is achieved near the inlet by the concentrated washing solution, while a weaker cleaning effect is achieved from the diluted washing solution from the middle of the column onwards. At the same time, the washing solution is diluted by the effect of the buffer solution passing through it, changing the pH and denaturant concentration, and the damage to the peptide ligand can be reduced as it approaches the column outlet. By utilizing this phenomenon, efficient washing and suppression of column deterioration can be achieved at the same time.

[0020] In order to enhance the washing effect or to suppress damage to the ligand, it is effective to change the temperature using a column oven, etc. Specifically, although it depends on the stability of the peptide ligand, a temperature of 4°C to 70°C is preferable, and a temperature of 15°C to 50°C is more preferable.

[0021] The following examples are provided to further explain the embodiments of the present invention. Of course, the present invention is not limited to the following examples, and various modifications are possible in detail. Furthermore, the present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the claims. The embodiments obtained by appropriately combining the technical means disclosed herein are also included in the technical scope of the present invention. In addition, all of the documents described in this specification are incorporated by reference. EXAMPLES

[0022] The eluents and the like used in the examples were prepared as follows. (1) Eluent A and Eluent B 6.578 g of disodium hydrogen citrate 1.5 hydrate (Nacalai Tesque) was taken and dissolved in 450 mL of Milli-Q water, and then the volume was increased to 500 mL in a measuring flask. The solution was filtered through a 0.2 μm filter and degassed by ultrasonic irradiation while reducing the pressure to prepare eluent A (50 mM citrate buffer, pH 5.1). In addition, 4.803 g of citric acid (Fujifilm Wako Pure Chemical Industries, Ltd.) was taken and dissolved in 450 mL of Milli-Q water, and then the pH was adjusted to 4.5 with an aqueous sodium hydroxide solution, and then the volume was increased to 500 mL in a measuring cylinder. The solution was filtered through a 0.2 μm filter and degassed by ultrasonic irradiation while reducing the pressure to prepare eluent B (50 mM citrate buffer, pH 4.5). (2) Antibody sample Globulin intramuscular injection (manufactured by Japan Blood Products Organization) was diluted 75-fold with eluent A to prepare a 2 g / L antibody solution. (3) Affinity column As the affinity column, TSKgel FcR-IIIA-NPR (Tosoh Corporation), in which human Fc receptors are immobilized on a gel, was used.

[0023] (Example 1) Washing with sodium hydroxide solution The column, eluent A, eluent B, and antibody sample were placed in an HPLC system (Tosoh Corporation). The column oven temperature was set to 25° C., the flow rate to 0.3 mL / min, and the detector to measure absorbance at 280 nm.

[0024] First, the antibody retention of the column was measured. Specifically, 10 μL of the antibody sample was injected while eluent A was passed through, and the antibody components were separated in isocratic mode. The injected antibody was separated in the column to give four peaks, and the retention time of the fourth peak was extracted as the current antibody retention. After the 15-minute measurement was completed, eluent B was passed through for 10 minutes to completely elute the antibody, and then eluent A was passed through again for 10 minutes.

[0025] Next, CIP was performed. A 0.1M aqueous solution of sodium hydroxide was prepared as the CIP cleaning solution. While eluent A was passed through the column, the cleaning solution was filled into a 100μL sample loop connected between the pump and the column, and after injecting it into the column, eluent A was passed through for 10 minutes and the cleaning solution was discharged. The above cleaning solution injection and discharge operation was repeated a total of three times to complete the CIP operation. The antibody retention was then measured again and compared with the initial retention to calculate the damage to the column. This CIP operation and retention measurement constituted one cycle, and measurements were performed a total of three times.

[0026] Using the same method as above, a system using 0.5M sodium hydroxide as the CIP cleaning solution was also evaluated. The results are summarized in Table 1. The 0.1M and 0.5M sodium hydroxide solutions did not damage the column and functioned as effective cleaning solutions.

[0027] (Example 2) Washing with guanidine hydrochloride solution The HPLC apparatus (manufactured by Tosoh Corporation), eluent, etc. were installed, and the initial antibody retention capacity of the column was measured in the same manner as in Example 1.

[0028] Next, CIP was performed. A 50 mM citrate buffer (pH 6.0) containing 0.01 M guanidine hydrochloride was prepared as a cleaning solution for CIP. While eluent A was passed through the column, the cleaning solution was filled into a 100 μL sample loop connected between the pump and the column, and after injecting it into the column, eluent A was passed through for 10 minutes and the cleaning solution was discharged. The above cleaning solution injection and discharge operation was repeated three times in total to complete the CIP operation. After that, the antibody retention was measured again and compared with the initial retention to calculate the damage to the column. This CIP operation and retention measurement was considered as one cycle, and measurements were performed three times in total.

[0029] Using the same method as above, systems using 50 mM citrate buffer (pH 6.0) containing 0.1 M, 1 M, and 3 M guanidine hydrochloride as the CIP cleaning solution were also evaluated. The results are summarized in Table 1. When the concentration of guanidine hydrochloride was 3 M or less, the column was little damaged and it functioned as an effective cleaning solution.

[0030] (Example 3) Cleaning with urea solution The HPLC apparatus (manufactured by Tosoh Corporation), eluent, etc. were installed, and the initial antibody retention capacity of the column was measured in the same manner as in Example 1.

[0031] Next, CIP was performed. A 50 mM citrate buffer (pH 6.0) containing 0.1 M urea was prepared as a cleaning solution for CIP. While eluent A was passed through the column, the cleaning solution was filled into a 100 μL sample loop connected between the pump and the column, and after injecting it into the column, eluent A was passed through for 10 minutes and the cleaning solution was discharged. The above cleaning solution injection and discharge operation was repeated a total of three times to complete the CIP operation. The antibody retention was then measured again and compared with the initial retention to calculate the damage to the column. This CIP operation and retention measurement was considered as one cycle, and measurements were performed a total of three times.

[0032] Using the same method as above, systems using 50 mM citrate buffer (pH 6.0) containing 1 M, 3 M, and 5 M urea as the CIP cleaning solution were also evaluated. The results are summarized in Table 1. When the urea concentration in the cleaning solution was in the range of 0.1 M to 5 M, there was no damage to the column and it functioned as an effective cleaning solution.

[0033] (Comparative Example 1) Washing with sodium hydroxide solution The HPLC apparatus (manufactured by Tosoh Corporation), eluent, etc. were installed, and the initial antibody retention capacity of the column was measured in the same manner as in Example 1.

[0034] A system using 1 M sodium hydroxide as a CIP cleaning solution was evaluated using the same method as in Example 1. The results are summarized in Table 1. When 1 M sodium hydroxide was used, the antibody retention ability of the column was quickly lost, making it clear that it is not suitable as a cleaning solution for FcR-IIIA-NPR.

[0035] (Comparative Example 2) Washing with guanidine hydrochloride solution The HPLC apparatus (manufactured by Tosoh Corporation), eluent, etc. were installed, and the initial antibody retention capacity of the column was measured in the same manner as in Example 1.

[0036] Using the same method as in Example 2, a system using 50 mM citrate buffer (pH 6.0) containing 5 M guanidine hydrochloride as a cleaning solution for CIP was evaluated. The results are summarized in Table 1. When the concentration of guanidine hydrochloride was 5 M, the antibody retention ability of the column was lost, and it was found that this was not suitable as a cleaning solution for FcR-IIIA-NPR.

[0037] [Table 1]

[0038] (Comparative Example 3) CIP by immersion method The HPLC apparatus (manufactured by Tosoh Corporation), eluent, etc. were installed, and the initial antibody retention capacity of the column was measured in the same manner as in Example 1.

[0039] Next, the column was washed by the immersion method. CIP was performed by passing 0.1 M sodium hydroxide aqueous solution through the column at a flow rate of 0.5 mL / min for 15 minutes, and then equilibration was performed by passing eluent A through the column at 0.5 mL / min for 15 minutes. The antibody retention was then measured again, and the retention before and after washing were compared to calculate the damage to the column as the antibody retention retention rate.

[0040] Using the same method as above, a system using 50 mM citrate buffer, pH 6.0, containing 3 M guanidine hydrochloride as the CIP cleaning solution was also evaluated. The results are summarized in Table 2. It was confirmed that even with the 0.1 M sodium hydroxide aqueous solution and 3 M guanidine hydrochloride aqueous solution required in Examples 1 and 2, the column deteriorated significantly when using the immersion method.

[0041] [Table 2]

Claims

1. a step of passing a buffer through a column packed with an insoluble carrier having a peptide ligand immobilized thereon to equilibrate the column; Adding a washing solution containing either a 10 mM to 1 M aqueous solution of guanidine hydrochloride or a 10 mM to 5 M aqueous solution of urea while flowing the buffer solution; and further passing the buffer solution through the column to dilute the washing solution while discharging it.

2. 2. The method of claim 1, wherein the peptide ligand is FcγRI, FcγRII, FcγRIII, protein A, protein G, protein L, or an amino acid variant thereof.

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