JPH07155194A - Method for purifying protein - Google Patents

Abstract

PURPOSE:To remove impurities from a cell culture solution, etc., without containing a cell or a tissue by purifying a solution containing the objective protein under specific conditions with a cation exchange column, an affinity chromatographic column and an anion exchange column. CONSTITUTION:This method for purifying a protein is to pass a cell culture solution or a tissue extract solution without substantially containing a cell or a tissue through (A) a cation exchange column, (B) an affinity chromatographic column and (C) an anion exchange column. The column (A) is equilibrated with a buffer solution at a lower pH than the isoelectic point of the objective protein and a buffer solution containing 0.05-0.5 mol/L cationic concentration is used as an eluent. The column (B) uses an aqueous solution of inorganic salts at pH <=7 and at least 0.2 mol/L cationic concentration as an eluent. The column (C) is equilibrated with a buffer solution at a lower pH than the isoelectric point of the objective protein which is recovered as a fraction passing therethrough without being adsorbed. The protein having pH 8-10 isoelectric point is suitable for especially purifying a monoclonal antibody.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a protein purification method. More specifically, it relates to a method for purifying a protein having specific physicochemical properties.

[0002]

[Prior Art] With the development of biotechnology, separation of various proteins from biological samples and production by cell culture technology have been actively carried out, and various techniques for purifying the target protein are also known. .

For example, there is a method for purifying a divalent cation-binding protein (JP-A-2-200180). In this purification method, a strong anion exchanger is used like an affinity carrier by increasing or decreasing the concentration of a divalent cation such as calcium ion, and a total of 3 steps of 2 steps of ion exchange chromatography and 1 step of hydrophobic chromatography are used. Chromatography has increased the protein purity to over 98%.
However, the protein of interest is limited to divalent cation binding proteins such as (activated) human protein C.

Monoclonal antibodies are also large protein molecules, and there is also an example in which an anti-PCI monoclonal antibody was purified from mouse ascites (JP-A-63-123395). In this case, the ascitic fluid collected from the abdominal cavity of the mouse is purified by one step of protein A column which is an affinity carrier. However, since the purification step involves only one-step chromatography, the purity of the obtained monoclonal antibody is low, and there is a problem in administering it to humans as a medicine even if there is no problem in using it in experimental animals.

On the other hand, the term "protein" has various physicochemical properties such as molecular weight, ionic behavior, and solubility in various solvents. Generally, protein is the pH of an aqueous solution
Changes its electrical characteristics. It is positively charged in the acidic region and negatively charged in the alkaline region. The pH, which is the boundary between positive and negative charges, is called the isoelectric point, and has a unique value depending on the protein. For example, ovalbumin
pH 4.7, bovine ribonuclease is pH 8.9.

The present inventors applied the above-mentioned various purification methods to the purification of some proteins having an isoelectric point of pH 8 to 10, but were unable to obtain a satisfactory degree of purification. In particular, it was not possible to meet the criteria of 100 pg / dose of DNA contamination in protein formulations for injection as a medicine. In addition, it was not possible to avoid the antigenicity caused by contaminating proteins other than the target protein.

[0007]

The present invention has been achieved as a result of various studies on methods for removing impurities in the method for purifying proteins having the above characteristics.

[0008]

Means for Solving the Problems In the present invention, a target protein-containing solution such as a cell culture solution or a tissue extract solution containing substantially no cells or tissues is treated by the following steps (a), (b) and
A method for purifying a protein, which comprises subjecting (c) to a purification step comprising this order. (a) The target protein-containing solution is supplied to a cation exchange column equilibrated with a buffer solution adjusted to a pH lower than the isoelectric point of the target protein, and the target protein in the solution is adsorbed on the column, and then non-adsorbed. After removing the substances by washing, the cation concentration is 0.
The step of eluting the target protein adsorbed on the column using a buffer solution of 05 to 0.5 mol / L: (b) supplying the target protein eluate obtained in the previous step to an affinity chromatography column, After adsorbing the target protein in the column to the column and then removing the non-adsorbed substances by washing, the pH is 7 or less and the cation concentration is at least
The step of eluting the target protein adsorbed to the column with 0.2 mol / L inorganic salt aqueous solution: (c) In the previous step, the anion exchange column equilibrated with a buffer adjusted to a pH lower than the isoelectric point of the target protein. The step of supplying the obtained target protein eluate and recovering the protein in the eluate as a flow-through fraction: The present invention basically comprises the steps (a), (b) and (c) in this order. However, a known filtration step, a concentration step, and a buffer solution exchange step for matching the properties of the liquid between steps may be provided before and after each step.

There are no particular restrictions on the proteins that can be applied to the purification method of the present invention, but the proteins that best exhibit the characteristics of the present invention are proteins having an isoelectric point of pH 8-10, among them monoclonal antibodies. is there.

For example, purification of a monoclonal antibody on an empirical scale is easy by affinity chromatography using an antigen specifically recognized by the monoclonal antibody, but inexpensive on an industrial scale. Is not suitable for. In addition, protein A such as the one obtained by purifying anti-PCI monoclonal antibody (JP-A-63-123395)
Purification using only the column could not give a satisfactory degree of purification as a medicine.

Although a relatively inexpensive purification process can be constructed using the present invention, it is necessary to know the isoelectric point of the target protein in order to apply the present invention. In order to examine what isoelectric point the target protein has, it is possible to preliminarily examine it using the one purified on a laboratory scale.

The isoelectric point can be measured by the isoelectric focusing method. The principle of the isoelectric focusing method is shown in several specialized books (for example, Shinsei Chemistry Laboratory 1 Protein Japan Biochemical Society edited by Tokyo Kagaku Dojin, published on February 26, 1990, p335), and the equipment is from Pharmacia, etc. Can be purchased.

The steps (a), (b) and (c) of the present invention will be described in more detail below. Step (a) This step is to adsorb proteins using a cation exchange column and mainly to remove contaminants such as contaminating proteins, lipids and carbohydrates that have a lower isoelectric point than the target protein. .

As a cation exchanger, a strong cation exchanger having a sulfopropyl group is recommended. For example, an agarose-based strong cation exchanger manufactured by Pharmacia
S-SepharoseFF or S-SepharoseHP, Sigma S-
SepharoseFF et al. Are S-made by Pharmacia.
SepharoseFF is especially recommended.

In this step, first, the column is adjusted to a pH lower than the isoelectric point of the target protein, preferably 1 to 3 lower than the isoelectric point of the target protein, and 0.001 to 0.05 mol /
Equilibrate with L buffer. Examples of the buffer solution include phosphate buffer solution and Tris buffer solution. The amount of buffer required for equilibration varies depending on the column volume, but for example, 10 cmφ, 1 L
The column can be equilibrated by flowing about 2 L of buffer solution.

In order to supply the target protein-containing solution such as the culture solution or the tissue extract solution to the equilibrated column, it may of course be supplied as it is, but for example, the culture solution may be subjected to a filtration treatment with a suitable clarification membrane. To obtain the culture supernatant from which cells and other substances have been removed, and subject it to ultrafiltration to obtain approximately 10 times ~
It is often preferable to use a 100-fold concentrated solution. For the filtration treatment, 0.2 to 100 μm, preferably 0.1.
A clear filtration membrane having a pore size of 2 to 5 μm (for example, Pellicon cassette HVLP000C5 manufactured by Millipore or FILTRON SIGMA-SERIES 0.3 μm manufactured by Fuji Filter) can be used. For the ultrafiltration treatment, a tangential flow type membrane having a pore size smaller than the molecular weight of the target protein (for example, Millipore Pellicon cassette PTTK00005) is used.
Or Fuji Filter FILTRON SIGMA-SERIES 1K ~ 1M)
Can be used.

In order to keep the yield of this step high, it is advisable to reduce the concentration of inorganic ions in the culture solution or tissue extract loaded on the column to half or less of physiological saline. That is, the degree of adsorption of the target protein to the ion exchange carrier can be increased by lowering the inorganic ion concentration by an operation such as dialysis or dilution with water.

After loading the culture solution or tissue extract onto the column, the column is washed with a buffer solution having a pH slightly lower than the isoelectric point of the target protein, preferably 0.1 to 1.5 lower than the isoelectric point of the target protein. . As a buffer solution, 0.001-0.05
Examples include mol / L phosphate buffer and Tris buffer.
The pH of the washing solution is adjusted to be slightly lower than the isoelectric point of the target protein in order to remove contaminating proteins whose isoelectric point is lower than that of the target protein as much as possible. The amount of buffer required for washing varies depending on the column volume, for example, 10 cm
Sufficient washing can be obtained by flowing about 2.5 L of buffer solution for φ and 1 L columns.

Next, it is lower than the isoelectric point of the target protein.
pH, preferably 0.001 to 0.05 mol / L buffer adjusted to pH 0.5 to 1.5 lower than the isoelectric point of the target protein, with an inorganic ion concentration increased by neutral inorganic salts (eg NaCl or KCl) The adsorbate is eluted with. The inorganic ion concentration is 0.05 to 0.5 mol / L, preferably 0.20 mol / L. Examples of the buffer solution include phosphate buffer solution and Tris buffer solution as in the case of equilibration and washing. The state of desorption of the target protein can be monitored by the absorbance at 280 nm.

The manner in which the target protein is eluted from the outlet of the column changes depending on the column volume and the adsorptivity of the target protein to the carrier. For example, in the case of purifying a monoclonal antibody with a column of 10 cmφ and 1 L, about 1 L of elution waste liquid is discharged and about 1 L of an aqueous solution of monoclonal antibody is obtained. Step (b) This step is a step for adsorbing proteins using an affinity chromatography column and mainly for removing contaminants such as contaminating proteins, lipids and carbohydrates which were not removed in step (a). . Process
By combining (a) and (b), contaminating proteins other than the target protein can be removed to a level at which they can be used for protein preparations for injection.

As the affinity carrier, a substance that specifically binds to the target protein is used. In the case of a monoclonal antibody, a specific adsorption carrier Protein-A affinity chromatography gel (for example, IPA-40 manufactured by Repligen)
0 or IPA-300) is used. A widely used and proven Protein-A low leakage gel should be used.
(For example, Sepharose 4-FF or BIO manufactured by Pharmacia
(PROSEP-A manufactured by PROCESSING).

In this step, the column is first equilibrated. Examples of the buffer solution include a phosphate buffer solution and a Tris buffer solution, but may include an inorganic salt (NaCl or KCl). It is convenient to use the same buffer used for elution in step (a). The amount of buffer solution required for equilibration varies depending on the column volume, but for example, in the case of 10 cmφ and 1 L column, equilibration can be achieved by flowing about 2 L of buffer solution.

The eluate from step (a) can be used as it is for loading the column.

Next, the inside of the column is washed with the same buffer solution used for equilibration. The amount of buffer required for washing varies depending on the column volume, but for example, in a 10 cmφ, 1 L column, sufficient washing can be obtained by flowing about 3 L of buffer.

Next, the protein adsorbed on the column is eluted with an aqueous solution having an inorganic ion concentration of 0.5 mol / L or more at a pH of 7 or less, preferably about pH 3.5. As this aqueous solution, a weak acid of 0.05 to 0.5 mol / L to which 0.5 mol / L or more of inorganic salts is added is used. Preferably, 0.1 mol / L acetic acid to which 0.5 mol / L NaCl is added is used. The state of desorption of the target protein can be monitored from the absorbance at 280 nm.

The manner in which the target protein is eluted from the outlet of the column changes depending on the column volume and the adsorptivity of the target protein on the carrier. For example, in the case of purifying a monoclonal antibody with a column of 10 cmφ and 1 L, about 1 L of elution waste liquid is discharged and about 1 L of an aqueous solution of monoclonal antibody is obtained.

The pH of the resulting aqueous solution remains low depending on the aqueous solution used for elution. If the target protein is stable under acidic conditions, it can be left alone,
If there is a risk of decomposition or denaturation, immediately raise the pH to near neutral. To raise the pH, there is a method of diluting 1.5 to 5 times with 0.5 to 1.0 mol / L phosphate buffer or Tris buffer adjusted to pH 7 or higher. Step (c) In this step, the eluate obtained in the previous step is supplied to the anion exchange column, and the target protein in the eluate is collected as a flow-through fraction. The main purpose is to remove a trace amount of the affinity substance, the complex of the target protein and the affinity substance, the DNA derived from cells or tissues, and the pyrogen (mainly lipopolysaccharide) mixed in the step (b). In order to supply bioactive proteins for medicinal use, it is necessary to highly remove these contaminants and reduce them to concentrations below the standard value. These contaminants can be removed by setting appropriate conditions in anion exchange chromatography.

As the anion exchanger, diaminoethyl is used.
Weak anion exchangers with (DEAE) groups are recommended. For example, the cellulose-based anion exchanger DE manufactured by Pharmacia
AE-Sephacel and agarose anion exchanger DEAE-Sepha
Examples include roseFF or DEAE-Sepharose FF and DEAE-Sepharose CL-6B manufactured by Sigma, which are cellulosic anion exchangers, and DEAE-Sephacel manufactured by Pharmacia is particularly recommended.

In this step, the pH of the column is adjusted to a pH lower than the isoelectric point of the target protein, preferably 0.1 to 1.0 lower than the isoelectric point of the target protein.
Equilibrate with mol / L buffer. Examples of the buffer solution include phosphate buffer solution and Tris buffer solution. The amount of buffer required for equilibration varies depending on the column volume, for example 10 cm
Equilibration can be performed by flowing about 2 L of buffer solution in the column of φ and 1 L.

The aqueous solution of the target protein obtained in the equilibrated previous step may be supplied as it is, but in order to sufficiently remove impurities by anion exchange chromatography, the inorganic ion of the aqueous solution may be supplied. It is often preferable to reduce the concentration. When the buffer solution is exchanged using a gel filtration carrier, the concentration of impurities can be reduced as well as the concentration of inorganic ions. For the buffer exchange treatment, a gel filtration carrier having a molecular weight cut-off of 10,000 or less (preferably 5000 or less) (for example, Seph manufactured by Pharmacia)
Use adexG-25 or -15). The target protein solution obtained in the previous step is adjusted to a pH lower than the isoelectric point of the target protein, preferably 0.1 to 1.0 lower than the isoelectric point of the target protein, and a buffer solution of 0.001 to 0.10 mol / L (for example, phosphorus). The inorganic ion concentration can be reduced by supplying the gel filtration column equilibrated with an acid buffer solution or Tris buffer solution and then letting it flow out with the same buffer solution. Further, when the gel filtration treatment is performed, the aqueous solution of the target protein can be concentrated by ultrafiltration treatment or the like in order to shorten the time required for loading the sample. For the ultrafiltration treatment, it is preferable to use a tangential flow type membrane having a pore size smaller than the molecular weight of the target protein (for example, Millipore Pellicon cassette PTTK00005 or Fuji Filter FILTRON SIGMA-SERIES 1K to 1M).

After the aqueous solution of the target protein obtained in the previous step is loaded on the anion exchange column, the target protein is eluted with the same buffer as that used for equilibration. The state of efflux can be monitored by the absorbance at 280 nm. Although the appearance of the outflow changes depending on the column volume,
For example, in the case of a column of 10 cmφ and 1 L, about 0.5 L of effluent waste liquid is discharged, and then about 1 L of the target protein aqueous solution is obtained. In order to use the anion exchanger for the same purpose again, it is necessary to remove the adsorbed impurities. To do this, the pH should be 7
0.1 to 0.5 mol / L buffer solution with 0.5 mol / L or more of inorganic salts added, preferably 0.17 mol / L acetate buffer solution with 0.5 mol / L NaCl added to the column. It is recommended to flow at least twice the volume.

On the other hand, when the protein is formulated as a medicine, particularly when it is a freeze-dried preparation, it is often preferable to use a physiological saline solution. A gel filtration operation can be used to replace the buffer solution in which the target protein is dissolved with physiological saline. It has a molecular weight cut-off of 1
It is preferable to use a gel filtration carrier of 10,000 or less, preferably 5000 or less (for example, Sephadex G-25 or G-15 manufactured by Pharmacia). The target protein solution obtained by anion exchange chromatography is supplied to a gel filtration column equilibrated with physiological saline and then allowed to flow out with physiological saline, whereby the buffer solution can be replaced with physiological saline. .
Further, when the gel filtration treatment is performed, the aqueous solution of the target protein can be concentrated by ultrafiltration treatment or the like in order to shorten the time required for loading the sample. For ultrafiltration treatment, a tangential flow type membrane having a pore size smaller than the molecular weight of the target protein (for example,
Pellicon cassette PTTK00005 manufactured by Millipore or FILTRON SIGMA-SERIES 1K-1M manufactured by Fuji Filter Co., Ltd. may be used.

The present invention will be described in more detail with reference to the following examples.

[0034]

【Example】

1) Production of Monoclonal Antibody by Large-Scale Culture of Hybridoma V3β1F4 strain (Ministry of Industrial Research Deposit No .: FERM BP-2765) was used as a hybridoma that is a monoclonal antibody-producing cell. The medium is as follows. Substance: Basal medium: eRDF (Kyokuto Pharmaceutical) Minor component: 2mg / L-insulin 2mg / L-transferrin 10mg / L- ethanolamine 10 ^-8 mol / L selenite Serum substitute: 1g / L- polyvinylpyrrolidone pH control : 0 to 4.2 g / L sodium hydrogencarbonate A 20 L tank equipped with a stirring blade was used as a culture tank, and perfusion culture was performed using a centrifuge-sedimentation type cell separator (Japanese Patent Publication No. 4-25796, Fig. 3).

It was found that the antibody-producing ability of the hybridoma V3β1F4 strain largely depends on the pH and glucose concentration of the culture medium. That is, the pH is 7.2 and the glucose concentration is 1 mmo.
Maximum productivity is obtained by keeping below l / L.
Here, the pH was controlled to a predetermined value by adjusting the sodium hydrogen carbonate concentration in the fresh medium supplied to the culture tank. Glucose concentration is the replacement ratio of culture medium in perfusion culture,
That is, it was controlled by the fresh medium supply rate (= culture supernatant withdrawal rate).

By the above culture, 20 to 40 L of culture supernatant containing the monoclonal antibody was collected per day.

2) Removal of Insoluble Protein from Culture Supernatant by Clarification Filtration The purpose of this operation is 3) and subsequent ultrafiltration membrane treatments and pretreatments for smooth column chromatography.
That is, the purpose is to remove insoluble proteins in the culture supernatant with a clarification filtration membrane and prevent clogging of the ultrafiltration membrane or column.

As the clarification filtration membrane, a clarification filtration membrane having a pore size of 0.45 μm (Millipore Pellicon cassette HVLP000C)
5) was used. The magnetic gear pump (I
WAKI, MDG-L15KA100), and a peristaltic pump (COLE PARMER, MASTERFLEX 7553-20) was used as the filtrate withdrawing pump. Substance: Alkaline solution: 0.5N- NaOH Neutralization buffer: 16.2g / L- Sodium acetate (trihydrate) 30.5mL / L- Acetic acid Sterilization and pyrogen decomposition by immersing the filter membrane in alkaline solution overnight did. The neutralization buffer solution was sent to the filtration membrane by a circulation pump and a filtrate extraction pump to neutralize the alkaline solution.

According to the manufacturer's instructions, the culture supernatant was circulated through the filtration membrane, and the outflowing filtrate was collected by the filtrate withdrawing pump. In this case, if the flow rate of the circulation pump is adjusted and the liquid pressure at the inlet of the filtration membrane is maintained at about 1.0 kg / cm ² , clogging of the filtration membrane does not easily occur and foaming does not pose a problem.

Although about 200 L of the culture supernatant can be filtered by operating for 8 hours, when the culture supernatant contains a large amount of cell debris, the filtration membrane may be clogged and the filtration flow rate may be significantly reduced. is there. In that case, filtration of the culture supernatant is temporarily stopped, and an alkaline solution is fed into the filter membrane and circulated for about 30 minutes to solubilize the insoluble protein in the filter membrane, which causes clogging. After neutralizing the alkaline solution again with the neutralization buffer, the filtration of the culture supernatant is restarted.

240 L containing about 20 mg / L of monoclonal antibody
The culture supernatant of 1. was filtered and supplied to the operations after 3). The monoclonal antibody concentration in the culture supernatant was determined by the affinity column (Protein A-Superose, FPLC, HR16 / 5) from Pharmacia.
) Was used for HPLC (LKB, 2152, 2150) to measure the absorbance at 280 nm.

3) Monochrome in the culture supernatant by ultrafiltration
Concentration of null antibody The purpose of this operation is to reduce the sample load in the cation exchange chromatography operation of 4) and shorten the time required for purification.

For the ultrafiltration membrane, the molecular weight cutoff is
30,000 filtration membrane (Millipore, Pellicon cassette P
TTK00005) was used. A magnetic gear pump (IWAKI, MDG-L15KA100) was used as the circulation pump, the filtrate withdrawal pump was not used, and the filtrate was allowed to flow naturally. Substance: Alkaline solution: 0.5N-NaOH neutralization buffer solution: 16.2g / L-sodium acetate (trihydrate) 30.5mL / L- acetic acid wash solution: saline solution The sterilization and pyrogen were decomposed by immersing in liquid. The neutralization buffer solution was sent to the filtration membrane by a circulation pump to neutralize the alkaline solution.

The filtered culture supernatant obtained in 2) was circulated through a filtration membrane according to the manufacturer's instructions. Also in this case, the liquid pressure at the inlet of the filtration membrane is adjusted to 1.0 kg / cm ² by adjusting the flow rate of the circulation pump.
If kept to a certain degree, the filtration membrane is less likely to be clogged and foaming does not pose a problem.

The molecular weight of the desired monoclonal antibody is
The cutoff molecular weight of the ultrafiltration membrane is about 150,000 and 30,000.
Therefore, only the low molecular weight component passes through the filtration membrane and the monoclonal antibody is concentrated. The 240 L filtrate obtained in 2) was concentrated to about 2 L, and then the monoclonal antibody remaining in the ultrafiltration membrane was washed out with a washing solution to bring the total amount to 6 L. The monoclonal antibody contained in 6 L was quantified by HPLC at 4.72 g.

4) Separation of Monoclonal Antibody by Cation Exchange Chromatography Since the isoelectric point of the monoclonal antibody produced by the V3β1F4 strain is about pH 8.80 to 9.05, it is positively charged in an aqueous solution below pH 8.80. is doing. Therefore, when a buffer solution having a pH of less than 8.80 is used, it is adsorbed on the cation exchanger. Material: Column: Pharmacia BPG 100/500 Ion exchanger: Pharmacia S-SepharoseFF 1L Buffer A: 0.02mol / L-phosphate buffer (pH 7.0) Buffer B: 0.02mol / L-phosphate buffer Liquid (pH 8.5) Buffer C: 0.02 mol / L-phosphate buffer (pH 8.0) 0.20 mol / L-NaCl Flow rate: 60 mL / min The column was conditioned according to the manufacturer's instructions. Then, the column was equilibrated with buffer A (about twice the column volume, about 2 L). The pH of the effluent was 7.0, which was used as a guide for the end of equilibration. Add 6 L of the culture supernatant concentrate obtained in 3) to Mill.
The column was loaded after being diluted 2-fold with iQ water (ultra pure water by a Millipore device).

Thereafter, buffer B was added to the effluent from the column.
The inside of the column was washed until the pH became 8.0 (about 2.5 times the column volume, about 2.5 L). In order to remove protease degradation products (impurities) of the monoclonal antibody, it is optimal to set the pH of buffer solution B to 8.5, which is slightly lower than the isoelectric point.

After washing, the adsorbate was eluted with buffer C. 28
The elution of the monoclonal antibody was monitored from the absorbance at 0 nm. About 1 L of the eluate waste solution flowed out, and then the monoclonal antibody was eluted to obtain about 1 L of the crude monoclonal antibody aqueous solution. The monoclonal antibody contained in 1 L was determined to be 4.63 g by HPLC.

5) Separation of Monoclonal Antibody by Affinity Chromatography Material: Column: Pharmacia BPG 100/500 Affinity carrier: Repligen IPA-400 1L Buffer A: 0.02mol / L-Phosphate buffer 0.20mol / L -NaCl (pH8.0) buffer B: 0.10 mol / L-acetic acid 0.50 mol / L-NaCl (pH3.5) buffer C: 0.50 mol / L-Tris buffer (pH 7.5) Flow rate: 60 to 70 mL / min The column was conditioned according to the manufacturer's instructions. The column was then equilibrated with buffer A (about 2 times the column volume, about 2 L). The pH of the effluent was 8.0, which was used as a guide for the end of equilibration. Approximately 1 L of the crude monoclonal antibody aqueous solution obtained in 4) was loaded on the column.

Thereafter, buffer A was used to remove the effluent from the column.
The inside of the column was washed until the pH reached 8.0 (about 3 times the column volume, about 3 L).

After washing, the adsorbate was eluted with buffer solution B. 28
The elution of the monoclonal antibody was monitored from the absorbance at 0 nm. About 1 L of the eluate waste solution flowed out, and then the monoclonal antibody was eluted to obtain about 1 L of the crude monoclonal antibody aqueous solution. In order to raise the pH of the obtained aqueous solution to near neutral, it was immediately diluted with Buffer C to 2 L. The monoclonal antibody contained in 2 L was quantified by HPLC at 4.31 g.

6) Concentration of monoclonal antibody by ultrafiltration The purpose of this operation is to reduce the sample loading amount in the buffer exchange operation of 5) and shorten the time required for purification.

For the ultrafiltration membrane, the molecular weight cutoff is
A 10,000-filter membrane (Filtron Miniset FS002K01, Fuji Filter Co., Ltd.) was used. A peristaltic pump (Cole-Parmer, Masterflex PA25A) was used as the circulation pump, and the filtrate was spontaneously discharged without using the filtrate withdrawal pump.

The crude monoclonal antibody aqueous solution obtained in 5) was circulated through the filtration membrane according to the manufacturer's instructions. Adjust the flow rate of the circulation pump to adjust the liquid pressure at the inlet of the filtration membrane to 1.0 kg / cm ²
If it is kept at a certain level, foaming does not become a problem and filtration can be performed efficiently.

The molecular weight of the desired monoclonal antibody is
The cutoff molecular weight of the ultrafiltration membrane is 10,000 at about 150,000.
Therefore, only the low molecular weight component passes through the filtration membrane and the monoclonal antibody is concentrated. The 2 L filtrate obtained in 5) was concentrated to about 0.1 L, and the monoclonal antibody remaining in the ultrafiltration membrane was washed out with a washing solution to a total volume of 0.5 L.

7) Buffer exchange with gel filtration carrier A standard gel filtration carrier (ie Seph) was used for the following tests.
adex G-25 Coarse or Medium or Fine or Superf
ine, manufactured by Pharmacia) is used. Generally, widely used and proven standard gel filtration carriers should be used (eg Separdex G-25 Coarse or Medium or
Fine or Superfine, manufactured by Sigma).

The concentrated solution obtained in 6) contains about 0.05 mol / L acetic acid, about 0.25 mol / L NaCl, and about 0.25 mol / L Tris buffer solution. Therefore, the inorganic ion concentration is high, and if it is left as it is, the removal of impurities in the anion exchange chromatography in the next step will be insufficient. The purpose of this operation is to reduce the concentration of inorganic ions so as not to interfere with the anion exchange chromatography operation. When the buffer solution is exchanged using a gel filtration carrier, the concentration of impurities can be reduced as well as the concentration of inorganic ions. Material: Column: Pharmacia BPG 100/500 Ion exchanger: Pharmacia SephadexG-25, Medium 2
L buffer A: 0.17 mol / L-acetate buffer (pH 5.0) Buffer B: 0.05 mol / L-Tris (pH 8.3) Flow rate: 60-70 mL / min Condition the column according to the manufacturer's instructions did. Since the ion exchanger in the column was previously sterilized with 0.2N-NaOH, the column was neutralized with buffer solution A (about twice the column volume, about 4 L). A pH of 5.0 in the effluent was used as a guide for the end of neutralization. The column was then equilibrated with buffer B (about twice the column volume, about 4 L). A pH of effluent of 8.3 was used as a guide for the end of equilibration. About 0.5 L of the crude monoclonal antibody concentrate obtained in 6) was loaded on the column.

Next, the monoclonal antibody was allowed to flow out with the buffer solution B. The appearance of monoclonal antibody efflux was monitored from the absorbance at 280 nm. About 1 L of the waste liquid flowed out, and then the monoclonal antibody flowed out to obtain about 1 L of the crude monoclonal antibody aqueous solution.

8) Removal of impurities by anion exchange chromatography The crude monoclonal antibody aqueous solution obtained in 7) contains a trace amount of
Contaminated with Protein A, monoclonal antibody-Protein A complex, DNA from hybridoma cells, and pyrogen (mainly lipopolysaccharide). These contaminants can be removed by setting appropriate conditions in anion exchange chromatography.

Since the isoelectric point of the monoclonal antibody produced by the V3β1F4 strain is about pH 8.80 to 9.05, pH 8.
Aqueous solution less than 80 is positively charged. Therefore, pH8.80
When used with less than a buffer, it does not adsorb to the anion exchanger.

On the other hand, Protein A and monoclonal antibody
The isoelectric points of the -ProteinA complex were found to be 4.85 to 5.1 and 7.1 to 8.2, respectively, by isoelectric focusing. Therefore, Protein A and the monoclonal antibody-Protein A complex are negatively charged in an aqueous solution of pH 8.3 or higher.

DNA and pyrogen are negatively charged in an aqueous solution. From the above facts, the pH should be 8.3-8.7.
If anion exchange chromatography is performed as described above, it is possible to allow only the desired monoclonal antibody to pass through the column and to adsorb impurities on the ion exchanger. Material: Column: Pharmacia BPG 100/500 Ion exchanger: Pharmacia DEAE-Sephacel 1L Buffer A: 0.17mol / L-acetate buffer (pH 5.0) Buffer B: 0.05mol / L-Tris (pH8.3) ) Buffer C: 0.17 mol / L-acetate buffer 0.50 mol / L-NaCl Flow rate: 60 mL / min The column was conditioned according to the manufacturer's instructions. Since the ion exchanger in the column was previously sterilized with 0.2 N-NaOH, the column was neutralized with buffer solution A (about twice the column volume, about 2 L). A pH of 5.0 in the effluent was used as a guide for the end of neutralization. The column was then equilibrated with buffer B (about twice the column volume, about 2 L). A pH of effluent of 8.3 was used as a guide for the end of equilibration. About 1 L of the crude monoclonal antibody concentrate obtained in 7) was loaded on the column.

Next, the monoclonal antibody was allowed to flow out with the buffer solution B. The appearance of monoclonal antibody efflux was monitored from the absorbance at 280 nm. About 0.5 L of the waste liquid flowed out, and then the monoclonal antibody flowed out to obtain about 1 L of the monoclonal antibody aqueous solution. In order to desorb impurities adsorbed on the ion exchanger, the inside of the column was washed with about 2 L of buffer solution C. The monoclonal antibody contained in 1 L was quantified by HPLC at 4.31 g.

9) Concentration of monoclonal antibody by ultrafiltration The purpose of this operation is to reduce the sample load in the buffer exchange operation of 10) and shorten the time required for purification. The equipment and conditions for ultrafiltration conform to 6). 8)
After concentrating about 1 L of the filtrate obtained in step 1 to about 0.1 L, the total amount of the monoclonal antibody remaining in the ultrafiltration membrane was washed with a washing solution to 0.25 L.

10) Buffer Solution Exchange with Gel Filtration Carrier When preparing a monoclonal antibody, particularly when preparing a freeze-dried preparation, it is preferable to use a physiological saline solution. The purpose of this operation is to use the Tris buffer solution of the monoclonal antibody obtained in 9) as a physiological saline solution.

The apparatus and conditions for buffer exchange are in accordance with 7). Material: Column: Pharmacia BPG 100/500 Ion exchanger: Pharmacia SephadexG-25, Medium 2
L buffer A: 0.17 mol / L-acetate buffer (pH 5.0) Saline: Fuso Yakuhin, for injection (Japanese Pharmacopoeia) Flow rate: 60 mL / min The ion exchanger in the column should be 0.2 N-NaOH beforehand. Since it was sterilized, the column was neutralized with buffer A (about twice the column volume, about 4 L). A pH of 5.0 in the effluent was used as a guide for the end of neutralization. Then, the column was equilibrated with physiological saline (about 3 times the column volume, about 6 L). After equilibration,
About 0.25 L of the crude monoclonal antibody concentrated solution obtained in 9) was loaded on the column.

Monoclonal antibody was flushed with saline. The appearance of monoclonal antibody efflux was monitored from the absorbance at 280 nm. About 1 L of the waste liquid flowed out and then the monoclonal antibody flowed out to obtain about 1 L of the final purified monoclonal antibody aqueous solution.

11) Purity of monoclonal antibody DNA of purified monoclonal antibody aqueous solution obtained in 10)
DNA probe method by radio isotope labeling of the amount of contamination (The principle is shown in some technical books. For example, "High-tech information for professionals, biotechnology series, DNA probe technology and application" by Toyozo Takahashi CMC 1988 Issued on Feb. 5, 2015 p5 ~), the contaminated DNA derived from the hybridoma β1F4 strain was antibody 1m.
It was 0.025 pg / g. When using a monoclonal antibody as a drug, this value is a low level of contamination that is of little concern.

The amount of protein A contaminated in the operation of 5) was measured by the ELISA method and found to be less than 0.03 ng (below the detection limit) per mg of antibody. This is also a low-level mixing amount that causes almost no problem.

Although the present invention has been described above using the monoclonal antibody as a representative example, the present invention is not limited to the monoclonal antibody and can be used for various proteins.

Claims (4)

[Claims] 1. A target protein-containing solution, which comprises a cell culture solution or a tissue extract solution which is substantially free of cells or tissues, and which comprises the following steps (a), (b) and (c) in this order: A method for purifying a protein, which comprises adding to the purification step. (a) The target protein-containing solution is supplied to a cation exchange column equilibrated with a buffer solution adjusted to a pH lower than the isoelectric point of the target protein, and the target protein in the solution is adsorbed on the column, and then non-adsorbed. After removing the substances by washing, the cation concentration is 0.
The step of eluting the target protein adsorbed on the column using a buffer solution of 05 to 0.5 mol / L: (b) supplying the target protein eluate obtained in the previous step to an affinity chromatography column, After adsorbing the target protein in the column to the column and then removing the non-adsorbed substances by washing, the pH is 7 or less and the cation concentration is at least
The step of eluting the target protein adsorbed to the column with 0.2 mol / L inorganic salt aqueous solution: (c) In the previous step, the anion exchange column equilibrated with a buffer adjusted to a pH lower than the isoelectric point of the target protein. A step of supplying the obtained target protein eluate and collecting the target protein in the eluate as a flow-through fraction: 2. The protein according to claim 1, wherein the target protein-containing liquid supplied to step (a) is a cell culture liquid or tissue extract liquid filtered through a clarification filtration membrane and then concentrated by ultrafiltration. Purification method. 3. The protein eluate supplied to step (c) is prepared by diluting the protein eluate obtained from step (b) with a buffer solution having a pH of 7 or more to raise the pH and then performing gel filtration treatment. The method for purifying a protein according to claim 1 or 2, wherein the concentration of inorganic ions is reduced. 4. The protein-containing solution obtained in step (c) is supplied to a gel filtration column equilibrated with physiological saline,
Next, the method for purifying the protein according to any one of claims 1 to 3, wherein the protein is allowed to flow out with physiological saline.