JPH11174038A - Separating and refining method for protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an efficiently separating and refining method of protein by a hydrophobic interaction chromatography without causing an adsorption of the protein to be separated and refined to a carrier. SOLUTION: In a protein separating and refining method by a hydrophobic interaction liquid chromatography, a hydrophobic interaction liquid chromatography is performed under the condition that the protein is dissolved in a buffer solution containing an organic solvent within the range of concentration that the protein to be separated and refined is not denatured irreversibly.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for separating and purifying proteins by hydrophobic interaction liquid chromatography.

[0002]

2. Description of the Related Art Chromatography is a separation and purification means utilizing a difference in interaction between a substance to be separated and purified and a stationary phase or a mobile phase, and separates and purifies a desired component from a mixture of various components. This is one of the methods. The types are classified according to the properties of the carrier (gel) packed in the column, and are classified into ion exchange chromatography, reverse phase chromatography, adsorption chromatography, size exclusion chromatography, hydroxyapatite chromatography, affinity chromatography, and the like. .

[0003] The separation and purification of proteins utilize the properties of the proteins to be separated and purified, and generally use a combination of two or more types of chromatography. For example, a combination of size exclusion chromatography using the difference in protein molecular size and ion exchange chromatography using the electrostatic difference of the protein surface, or hydrophobicity using the size exclusion chromatography and the difference in hydrophobicity of the protein surface A typical example is a combination of sex interaction chromatography. By appropriately selecting chromatography in consideration of the properties of the protein to be separated and purified in this manner, the target protein can be efficiently separated from the mixed components and the purification purity can be increased.

[0004] Antibodies are one type of important proteins used for immunoassays and the like. Immunoassays using antibodies are widely used, especially in the field of clinical diagnosis, and their use is expected to increase in the future.

[0005] Antibodies frequently used in diagnostic agents need to have a final purity of 95% or more in order to maintain stable performance. In addition, the larger the amount of single purification, the more stable and the same antibody can be obtained. Usually, monoclonal antibodies (Kohler G. & Mil)
stein C.I. , Nature, 256, 1975
Year) is a method for separating and purifying from a mixed solution containing various contaminating proteins derived from mouse ascites, cell culture supernatant or recombinant Escherichia coli. Requires a separation and purification step. on the other hand,
The polyclonal antibody is usually used as it is or after separating and purifying only the specific antibody by affinity chromatography.

[0006]

It is a well-known method to separate and purify proteins using liquid chromatography. However, conventionally, the physiological activity of the protein adsorbed on a chromatography carrier is not sufficient in the process of purifying the protein. In order to avoid deterioration, the operation is carried out using a mobile phase capable of retaining the structure of the protein to be separated and purified in its natural state. This is because separation and purification using a mobile phase in which a protein loses its native structure is not suitable for use or research utilizing the physiological activity of the protein even if the degree of purification can be improved.

However, under the above conditions,
There is a problem that the protein to be separated and purified is strongly adsorbed to the carrier. Conventionally, it is known that the protein adsorbed on the carrier is desorbed by adding a buffer containing about 20% of an organic solvent such as alcohol or the like. The liquid is used only in a limited case when the adsorbed protein cannot be eluted. That is, a buffer containing an organic solvent is used only as an eluent, and there is no report on using this as a mobile phase in separation and purification.

[0008] Regarding the method of separating and purifying antibodies, size exclusion chromatography, ion exchange chromatography,
Methods using hydrophobic interaction chromatography, hydroxyapatite chromatography, protein-A binding affinity chromatography, protein G binding affinity chromatography, etc. 196
Although there have been numerous reports since the 0's, the mobile phase used in any of the methods is a buffer containing no organic solvent.

High antigen specificity is required for antibodies used for immunoassays and the like. However, in order to separate and purify an antibody having high antigen specificity by a chromatography technique, it is necessary to perform affinity chromatography in which the antigen is immobilized. In this operation, it is essential to use a mobile phase under which the antibody can exert its biological activity, that is, an operation using a mobile phase capable of retaining the structure of the antibody in its natural state, but nonspecific adsorption of the antibody to the carrier occurs. Therefore, it is difficult to efficiently separate and purify the specific antibody.

[0010] The present invention solves the above problems and provides a method for efficiently separating and purifying a protein to be separated and purified by hydrophobic interaction chromatography without causing adsorption of the protein to a carrier. It is intended for.

[0011]

Means for Solving the Problems The present invention made to achieve the above object is to provide a method for separating and purifying a protein by hydrophobic interaction liquid chromatography, wherein the protein to be separated and purified is irreversibly denatured. Performing hydrophobic interaction liquid chromatography in a state where the protein is dissolved in a buffer containing an organic solvent in a concentration range not to be performed,
A method comprising reducing the concentration of an organic solvent in a fraction containing the protein separated and purified. Less than,
The present invention will be described in detail by taking as an example the case where the protein to be separated and purified is an antibody.

In the adsorption of an antibody to a hydrophobic interaction chromatography support, an equilibrium between the rate at which the antibody is adsorbed to the stationary phase and the rate at which it is desorbed results in an inherent migration rate, which determines the elution time. This difference in migration speed is largely related to the hydrophobicity near the surface of the antibody.To increase the apparent hydrophobicity, a large amount of ammonium sulfate or sodium sulfate is added and adsorbed, and the concentration is gradually reduced. It can be desorbed. Since ammonium sulfate or the like excludes water molecules near the three-dimensional surface of the peptide chain of the protein, the surface hydrophobic interaction of the protein increases, and the adsorption rate to the carrier increases.

Antibodies are globular proteins, and the intramolecular three-dimensional structures of some antibodies have been reported by X-ray crystal structure analysis. There is no report on three-dimensional local hydrophobicity. Antibodies have a molecular structure in which two heavy chains in which domains of about 12,000 are repeatedly bonded and two light chains are intramolecularly disulfide bonded. Considering the amino acid sequence, the subclass has a complicated structure consisting of a constant region (Fc) in which the amino acid sequence is well conserved and a variable region (Fab) in which the amino acid sequence is not conserved. Subclass (IgG, I
gA, IgD, IgE, IgM). On the other hand, the specificity of an antibody for an antigen is such that mutation and movement of the primary amino acid sequence occur particularly frequently in the variable region.
Hypervariable region (hypervariable re)
gion). As described above, antibodies may have very similar surface properties and are very difficult to separate and purify based on differences in the level of antigen specificity. For example, it is difficult to separate and purify antibodies of the same subclass based on their antigen specificity, since surface properties such as molecular weight and amino acid composition of the Fc region are similar.

According to the present invention, in hydrophobic interaction chromatography, the fluctuation of the elution time of an antibody in the presence of an organic solvent depends on the above-mentioned minute mutation between antibodies, and
Antibodies are generally biochemically stable in a buffer containing an organic solvent, and reversibly refold to the natural state by removing or reducing the organic solvent even if the steric structure changes, so an operation using an organic solvent Are based on the knowledge of the present inventors that physiological activities such as antigen-binding ability and antigen specificity are not destroyed by carrying out. The organic solvent used in the present invention can be exemplified by organic solvents of alcohols or non-alcohols such as methanol, ethanol, propanol, butanol, diethyl ether, dioxane, acetonitrile, and hexane. There is no particular limitation as long as the protein to be separated and purified can be reversibly denatured and dissolved. In this specification, "irreversibly denatured" means
Even if the organic solvent is removed or its concentration is reduced, the protein to be separated and purified is denatured so that it cannot recover its natural structure and therefore cannot return to a structural state capable of exerting its physiological activity. That means.

The concentration of the organic solvent used may be within a concentration range that does not irreversibly denature the protein to be separated and purified, and may be determined by omitting the physical properties of the protein and the results of preliminary experiments using the protein. Good, but 1-10%
The range of the degree can be exemplified. The organic solvent is used after being added to the buffer. As the buffer, those conventionally used in the separation and purification of proteins can be used without limitation. Examples include a phosphate buffer, a Tris buffer, a carbonate buffer, an acetate buffer, and the like. There are no particular restrictions on the concentration or pH of the buffering agent, but usually 10 mM to 500 m
The concentration range of M and the pH range of about pH 6.5 to pH 7.5 may be selected.

A sample containing contaminants is added to a buffer containing an organic solvent together with the protein to be separated and purified, and the protein is contacted with a stationary phase of hydrophobic interaction liquid chromatography in a state in which the protein is reversibly dissolved, and separated. Perform purification. Examples of the sample include, for example, when the protein to be separated and purified is a monoclonal antibody, a culture solution of a microorganism such as mouse ascites or a recombinant Escherichia coli, or a crushed solution of the culture. Is a polyclonal antibody, blood of a mammal such as a mouse can be exemplified.

The stationary phase of the hydrophobic interaction liquid chromatography is not particularly limited as long as it has a sufficient mechanical strength. Examples of the base material include polystyrene, carboxylated polystyrene, and polymethyl acrylate. Is possible. In addition to these, magnetic polymer fine particles, cellulose derivatives, porous polymers, and the like can also be used. Examples of the functional group supported on the substrate include a phenyl group, an oligoethylene glycol group, and a butyl group. The carrier may be used in the state of being packed in a so-called column, but may be used in the separation and purification of the present invention in the state of being simply placed in a container.

The protein to be separated and purified is adsorbed on the stationary phase by contact with the stationary phase of hydrophobic interaction liquid chromatography in the presence of an organic solvent. Therefore,
For example, an ammonium sulfate concentration gradient or the like is applied to the mobile phase to change the hydrophobicity of the adsorbed protein, and the fraction is recovered by elution according to the hydrophobicity level specific to the protein. Ammonium sulphate concentration gradient, e.g.
For example, a linear concentration decreasing gradient that changes to 0M can be exemplified.

This recovery operation may be performed while monitoring the absorbance in the ultraviolet region (for example, 280 nm) of the eluate from a column or the like packed with the stationary phase of hydrophobic interaction liquid chromatography. When a sample contains proteins other than the protein to be separated and purified, it is preferable to perform the fractionation as finely as possible so that a plurality of fractionated proteins are not mixed according to the level of hydrophobicity.

The collected fraction is identified by lowering the concentration of the organic solvent to refold the protein structure into a natural structure and confirming its physiological activity. To reduce the concentration of the organic solvent, the recovered fraction is subjected to, for example, ammonium sulfate precipitation to extract the protein,
This may be dissolved in a buffer containing an organic solvent containing no organic solvent or having a low concentration that does not denature the protein, or the collected fraction may have a low concentration that does not contain the organic solvent or denatures the protein. The buffer replacement may be performed by performing dialysis or the like on a buffer containing an organic solvent. The kind and concentration of the buffer used in the buffer used here, and the pH thereof are the same as described above.

According to the above-described separation and purification method, even in the case of an antibody of the same subclass, amino acid primary sequence mutation and migration occur particularly frequently in the variable region, which determines the antigen specificity. Separation and purification can be performed based on the amino acid composition in the hypervariable region at the location. As a result, antibodies of the same subclass can be separated and purified based on antigen specificity, which was difficult with conventional chromatography techniques.

The protein separated and purified according to the present invention can be used as a drug such as a bioreactor (immobilized enzyme) or a biopharmaceutical. In particular, antibodies have sufficient purity and antigen specificity for use in conventional immunoassays.

[0023]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

Example 1 Preparation of Buffer Containing Organic Solvent A 0.1 M phosphate buffer (pH 7.4) containing 2.0 M ammonium sulfate was adjusted to 5%, 5% or 3% with methanol, ethanol, 1-propanol was added to prepare three kinds of organic solvent-containing buffers, and after stirring,
0.45 μm filter (manufactured by Millipore Co., Ltd., HAW
PO4700) and stored at 4 ° C. until immediately before use. These buffers were degassed under reduced pressure immediately before use in separation and purification operations.

Example 2 Purification of Monoclonal Antibody In addition to the three buffers prepared in Example 1, a buffer containing no organic solvent (0.1 M phosphate buffer containing 2.0 M ammonium sulfate (pH 7.0)) 4)) for each of the four types of buffers, the subtype was IgG1
Anti-β2-microglobulin monoclonal antibody (prepared in-house, hereinafter abbreviated as antibody 1) and anti-myoglobin monoclonal antibody of the same subtype (prepared in-house,
(Hereinafter, abbreviated as antibody 2) at 10 mg / mL, respectively, and dissolved. The physiological activity of each antibody in the buffer in which the antibody was dissolved was measured as in Example 4 or Example 5. As a result, all of the antibodies almost lost their activity.

A column (TSKgel) packed with a stationary phase of hydrophobic interaction liquid chromatography was prepared by using the four kinds of buffer solutions prepared by dissolving the antibodies 1 and 2 prepared as described above.
Ether-5PW, diameter 7.5mm x length 7.5m
m, manufactured by Tosoh Corporation) and the elution time of each antibody was measured.

The conditions of the liquid chromatography were as follows: flow rate: 1.0 mL / min, detection wavelength of protein in eluate: 280 n
m, injection volume of buffer containing protein is 100 μL, and ammonium sulfate gradient for elution of antibody adsorbed on the carrier is 30 μm.
It is a linear concentration decreasing gradient that changes from 2M to 0M per minute.
The components eluted from the column by the linear gradient of ammonium sulfate concentration were measured for absorbance at 280 nm as described above, and the protein eluted fraction was collected. The recovered protein fraction was identified by substituting the solvent with a 0.1 M Tris-HCl buffer (pH 7.5) and measuring its physiological activity (see Example 4 or 5).

The results are shown in Table 1. When antibodies 1 and 2 were dissolved in a buffer to which no organic solvent was added, antibody 1
Was 25 minutes and 15 seconds, and the elution time of Antibody 2 was 25 minutes. However, when the buffer containing 3% propanol was used, the time was changed to 22 minutes 54 seconds and 19 minutes 58 seconds.

[0029]

[Table 1]

As is clear from Table 1, when a buffer solution to which no organic solvent is added is used, since the subclasses are the same, there is only a difference of 15 seconds between the elution times of the two, and a fraction is obtained. At this time, the recovery rate was greatly reduced when trying to avoid the part where both were mixed, but in the separation and purification method of the present invention, a difference of about 3 minutes occurred between the elution times of the two, and both were sufficiently separated and recovered. As described above, it was shown that antibodies can be separated and purified in accordance with the difference in hydrophobicity level derived from the antigen specificity of the antibody even if the subclasses are the same.

Example 3 Fixation of β2-microglobulin and human myoglobin β2-microglobulin (manufactured by Scripps Laboratories, Inc.) adjusted to 0 or 2 μg / mL with 0.1 M phosphate buffer (pH 7.4), respectively. M0114) or human myoglobin (manufactured by Scripps Laboratories, M0724) was added to each well of a 96-well microplate (manufactured by Nunc Intermed, 442404) in an amount of 50 μL each, and the mixture was allowed to stand at 37 ° C. for 90 minutes to be fixed. . After each well was washed with PBS, bovine serum albumin (manufactured by Sigma) was dissolved in PBS to a concentration of 0.2% (weight / weight), and 350 μL of the solution was added to each well.
For 2 hours to perform a blocking treatment, thereby preparing a β2-microglobulin-fixed titer plate and a human myoglobin-fixed titer plate. These plates were stored at 4 ° C. until just before use.

Example 4 Confirmation of Physiological Activity of Eluted Fraction 1 Antibody 1 prepared in Example 2 and, after separation and purification, the fraction of antibody 1 in which the concentration of organic solvent in the buffer was reduced was 0.5% bovine. The mixture was diluted with PBS containing serum albumin (pH 7.4) so that the absorbance at 280 nm was 0.005. 50 μL of this diluted solution was added to the β2-microglobulin-immobilized titer plate prepared in Example 3, and 50 μL of the diluted solution was added.
Alkaline phosphatase-labeled goat anti-mouse IgG antibody (manufactured by Jackson Immunoresearch Laboratories, 115-055-100) diluted 1:00 was added in 50 μL portions, and reacted at 37 ° C. for 60 minutes. The solution in the well is discarded, and after further washing with PBS, 0.1 g of paranitrophenyl phosphate (Sigma, N1891) which is a substrate of alkaline phosphatase is adjusted to 3 mg / mL.
Substrate solution dissolved in 1M carbonate buffer (pH 9.5)
After adding 00 μL, the mixture was allowed to stand at 37 ° C. for 30 minutes.

After 1N NaOH was added in an amount of 100 μL to each well, the absorbance at 405 nm was measured. Table 2 shows the results. Antibody 1 dissolved in a buffer containing an organic solvent in a reversibly denatured state reversibly recovers its physiological activity (antigen binding ability) by reducing the concentration of the organic solvent in the buffer. I understand.

[0034]

[Table 2]

Example 5 Confirmation of Physiological Activity 2 of Eluted Fraction The antibody 2 prepared in Example 2 and the fraction of the antibody 2 in which the concentration of the organic solvent in the buffer solution was reduced after separation and purification were 0.5% bovine. The mixture was diluted with PBS containing serum albumin (pH 7.4) so that the absorbance at 280 nm was 0.005. 50 μL of this diluted solution was added to the myoglobin-immobilized titer plate prepared in Example 3, and 50 μL of a 5000-fold diluted alkaline phosphatase-labeled goat anti-mouse IgG antibody (Jackson Immunoresearch Laboratories, 115-055-100) was added. Add 3
The reaction was performed at 7 ° C. for 60 minutes. The solution in the well is discarded, and after further washing with PBS, alkaline phosphatase substrate paranitrophenyl phosphate (Sigma, N
10091 of a substrate solution prepared by dissolving 1891) in a 0.1 M carbonate buffer (pH 9.5) to a concentration of 3 mg / mL was added, and the mixture was allowed to stand at 37 ° C for 30 minutes.

After 100 μL of 1N NaOH was added to each well, the absorbance at 405 nm was measured. Table 3 shows the results. Antibody 2 dissolved in a buffer containing an organic solvent in a reversibly denatured state reversibly recovers its physiological activity (antigen binding ability) by reducing the concentration of the organic solvent in the buffer. I understand.

[0037]

[Table 3]

[0038]

According to the present invention, a protein such as an antibody to be separated and purified is not strongly adsorbed to a carrier, and therefore, the protein can be efficiently purified by hydrophobic interaction chromatography without deteriorating the recovery. Can be separated and purified. Moreover, according to the method of the present invention, it is difficult to sufficiently separate and purify by the conventional separation and purification method because the molecular weight and the amino acid composition of the Fc region are similar. It is also possible to separate and purify antibodies having the property.

Claims (5)

[Claims] 1. A method for separating and purifying a protein by hydrophobic interaction liquid chromatography, wherein the protein is dissolved in a buffer containing an organic solvent in a concentration range that does not irreversibly denature the protein to be separated and purified. And performing a hydrophobic interaction liquid chromatography on the mixture to reduce the concentration of the organic solvent in the fraction containing the protein separated and purified. 2. The method according to claim 1, wherein the organic solvent is an alcohol or a non-alcoholic organic solvent. 3. The hydrophobic interaction liquid chromatography stationary phase is a carrier having a phenyl group, an oligoethylene glycol group or a butyl group.
the method of. 4. The method according to claim 1, wherein the protein to be separated and purified is an antibody. 5. The method according to claim 4, wherein the antibody is a monoclonal antibody or a polyclonal antibody.