JP2003096095A - Purifying method of protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To resolve problems that over certain content of lipid in a protein solution causes turbidity and worsens efficiency in purifying manipulation and that the conventional purifying method needs complicated manipulation and a plural number of processes, and to provide a method of efficiently removing lipid with rapid and easy manipulation from a protein precipitation fraction containing abundant lipid derived from plasma. SOLUTION: The protein precipitation fraction containing lipid derived from human plasma is brought into contact with a buffer containing a water-soluble polymer. The protein is extracted from the supernatant to obtain the protein with lowered lipid content.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for purifying a protein which removes lipid from a lipid-containing protein precipitation fraction derived from human plasma, particularly a haptoglo precipitation fraction bottle, rapidly and simply.

[0002]

BACKGROUND OF THE INVENTION There are various human plasma-derived proteins used as pharmaceuticals, and it is desirable that they are purified as much as possible from the viewpoints of separation of physiological activity and suppression of side effects. . Haptoglobin is a kind of human plasma-derived protein having a molecular weight of 85,000 to 400,000, and specifically binds to free hemoglobin to form a complex, which is transported to the liver which is a metabolic pathway. However, when the amount of haptoglobin is less than the amount of free hemoglobin, free hemoglobin is present in plasma, resulting in hemoglobinemia and hemoglobinuria. In case of burns, blood transfusions, surgery
Since a large amount of free hemoglobin is produced in tissues and blood by hemolysis, administration of haptoglobin is considered effective for the prevention and treatment of hemoglobinemia and hemoglobinuria caused thereby. Haptoglobin is known to be extracted and purified from, for example, the α- and β-globulin fractions of human plasma (Japanese Patent No. 957774). By the way, since the fraction contains impurities such as lipids (for example, lipoprotein),
The aqueous solution of the fraction is cloudy. Therefore, it is necessary to clarify the aqueous solution so that the subsequent operation can proceed smoothly. As this method, human plasma α- and β-
After adding acrinol to the aqueous solution of the globulin fraction to precipitate impurities and collecting the supernatant, a method of performing dialysis or adsorption treatment for removing acrinol, or a method of contacting with colloidal silicic acid (JP-A-63- 17899) is known. However, these methods are complicated in operation and not sufficiently effective.

[0003]

Under these circumstances, the present invention has achieved a simple and rapid lipid separation and removal from a lipid-containing protein solution derived from human plasma, thereby reducing the lipid content. The purpose is to obtain proteins, especially haptoglobin, quickly and conveniently.

[0004]

The inventors of the present invention have conducted various studies to solve the above-mentioned problems, and as a result, as a raw material, for example, corn IV-IV-1 by the low temperature ethanol fractionation method was used.
Alternatively, when the IV-4 fraction is brought into contact with a buffer solution containing a water-soluble polymer, the lipid is not dissolved and the protein is dissolved, so that the protein, particularly haptoglobin, can be collected from the supernatant easily and easily. It was found that a protein fraction with a reduced lipid content can be rapidly obtained, and further studies were conducted based on the findings to complete the present invention.

That is, according to the present invention, (1) a protein fraction containing a lipid derived from human plasma is brought into contact with a buffer solution containing a water-soluble polymer, and the protein is collected from the supernatant thereof. Purification method (2) IV-IV-1 or IV- by the low temperature ethanol fractionation method in which the protein fraction is corn
4 fractions, wherein the protein is haptoglobin, the purification method according to (1) or (2), (3) The water-soluble polymer is polyoxyethylene glycol having a molecular weight of 400 to 10,000, according to (1) or (2) Purification method, (4) The purification method according to (1) or (2), wherein the buffer solution is at least one selected from a phosphate buffer solution, an acetate buffer solution, and a citrate buffer solution, and (5) a water-soluble polymer. The buffer solution containing 0.5 to 10.0 w / v% of the water-soluble polymer is 0.1 to 3
The purification method according to (1) or (2), which is a buffer solution having a concentration of 00 mM.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION The human plasma-derived protein fractions used as the raw material of the present invention include α- and β-globulin fractions, IV-IV-1 and IV-IV of the corn low temperature ethanol fractionation method.
It can be prepared from each precipitation fraction such as -4. These protein-precipitated fractions usually contain relatively abundant lipids such as cholesterol and phospholipid lipids in many cases.

In the present invention, a raw material protein containing a lipid is first contacted with a buffer solution containing a water-soluble polymer. By this operation, the protein is dissolved in the buffer solution and the lipid remains in the solution as an insoluble matter. The water-soluble polymer is a polymer having a hydrophilic group such as a hydroxyl group in the molecule and having a property of being mixed with water, and polyethylene glycol (having a molecular weight of 400 to
10,000), dextran, methyl cellulose, polyvinylpyrrolidone, polyvinyl alcohol and the like are mentioned as examples, but a preferable one is a molecular weight of 1000-6.
000 polyethylene glycol. Examples of the buffer solution include a phosphate buffer solution, an acetate buffer solution, a citrate buffer solution, and the like, and a phosphate buffer solution and a phosphate-NaCl buffer solution are preferable. The pH of the buffer solution is usually 4.5 to 7.0, preferably 4.8 to 6.0. The concentration of the buffer solution in the mixed solution is 0.1 to 300 mM, preferably 1 to 100 m.
M, more preferably 5 to 50 mM

The concentration of the water-soluble polymer in the buffer containing the water-soluble polymer is usually 0.5 to 10% (w / v), preferably 2 to 6% (w / v). . Contact from 1 minute to 2
The time is preferably 10 minutes to 1 hour. The contact temperature is preferably room temperature or under cooling. The contact may be a batch type or a continuous type. Insoluble matter containing lipids is removed from the liquid thus obtained. Insoluble matter can be removed by
Although any method may be used, filtration with a filter or centrifugation is usually preferable.

Ammonium sulfate, for example, is added to the filtrate to collect the precipitate. The precipitate is dissolved in water, and heat treatment for so-called virus inactivation (usually 50 to 70 ° C,
1 to 24 hours). If necessary, the heat-treated liquid is purified by subjecting it to anion exchange chromatography. The obtained protein solution, especially haptoglobin solution, can be made into a clinically applicable protein preparation, especially haptoglobin preparation, by further combining known formulation techniques. For example, stabilizers, excipients, etc. can be added, subdivision, dialysis, ultrafiltration, lyophilization, etc. can be performed.

[0010]

EXAMPLES The present invention will be described more specifically with reference to Examples, Comparative Examples and Experimental Examples. Example 1 Fractionation by the Cohn low temperature ethanol fractionation method derived from human plasma
PEG4000 was added to each of the IV-4 precipitate fractions of 14 g in an amount of 0 to 10
Add 15 ml of 20 mM phosphoric acid-10 mM NaCl buffer (pH 4.9) containing w / v%, and add about 1 at room temperature.
The mixture was stirred for 5 minutes, filtered through a filter, and the filtrate was collected.

Comparative Example 1 20 mM phosphoric acid-10 mMN without PEG4000
Extraction operation was performed in the same manner as in Example 1 using an aCl buffer (pH 4.9). Test Example 1 Total phospholipids, cholesterol amount and protein (haptoglobin) in the filtrates obtained in Example 1 and Comparative Example 1
The amount was measured and the results shown in [Table 1] were obtained.

[0012]

[Table 1] As is clear from the above [Table 1], the lipid content was significantly reduced when PEG4000 was used in combination as compared with the case where the phosphate-NaCl buffer solution was used alone.

[0013]

The present invention provides a protein fraction derived from human plasma,
Especially, the IV fraction etc. by the corn low temperature ethanol fractionation method,
A protein solution having a reduced lipid content can be simply and rapidly obtained by simply treating (extracting) with a buffer solution containing a water-soluble polymer.

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Claims (5)

[Claims] 1. A method for purifying a protein, which comprises contacting a protein fraction containing a lipid derived from human plasma with a buffer containing a water-soluble polymer, and collecting the protein from the supernatant. 2. The purification method according to claim 1, wherein the protein fraction is the IV, IV-1 or IV-4 fraction obtained by Cohn's low temperature ethanol fractionation method, and the protein is haptoglobin. 3. The water-soluble polymer has a molecular weight of 400 to 100.
The method according to claim 1 or 2, wherein the polyoxyethylene glycol is 00. 4. The purification method according to claim 1 or 2, wherein the buffer solution is at least one selected from a phosphate buffer solution, an acetate buffer solution and a citrate buffer solution. 5. A buffer solution containing a water-soluble polymer in an amount of 0.1 to 300 containing 0.5 to 10.0 w / v% of the water-soluble polymer.
The purification method according to claim 1 or 2, which is a buffer solution having a mM concentration.