JP4798831B2 - Method for suppressing misfolding of human serum albumin - Google Patents

Description

【００３０】
アルカリ性処理の際にシステインを存在させることにより、ヒト血清アルブミンの分子内ミスホールディングが効果的に抑制されることがわかる。
【図面の簡単な説明】
【図１】調製例で得られたヒト血清アルブミン溶液のアルカリ性溶液で処理前後の試料をＳＤＳ−ＰＡＧＥ後、ウェスターンブロットを行った結果を示す写真である。▲１▼はアルカリ性溶液で処理前、▲２▼はアルカリ性溶液で処理後の試料を示す。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for purifying human serum albumin. More specifically, when purifying human serum albumin derived from plasma (hereinafter also referred to as HSA) or recombinant human serum albumin (hereinafter also referred to as rHSA) obtained by gene recombination technology. The present invention relates to a method for suppressing misfolding due to cross-linking of intramolecular or intermolecular disulfide bonds of human serum albumin formed by alkaline solution treatment performed in the production process by adding an SH group-containing compound.
[0002]
[Prior art]
Human serum albumin (hereinafter sometimes referred to as HSA) is a major protein component in plasma, consisting of a single chain polypeptide of 585 amino acids and having a molecular weight of about 66,000 daltons (Minghetti, PP et al. (1986) Molecular structure of the human albumin gene was revealed by nucleotide sequence within 11-22 of chromosome 4. J. Biol. Chem. 261, 6747-6757). HSA is known to play a role as a carrier that mainly transports the normal osmotic pressure of blood and binds to various calcium ions, fatty acids, bilirubin, tryptophan, and drugs that appear in the blood. ing. Purified HSA is used for the treatment of hypoalbuminemia due to albumin loss such as surgery, hemorrhagic shock or burns and nephrotic syndrome.
[0003]
Conventionally, after obtaining HSA fraction (HSA is fractionated into fraction V) from human plasma according to the low temperature ethanol fractionation method of corn or according to the method, HSA is further used in various purification methods. Manufactured. In recent years, as a method that does not depend on human plasma as a raw material, a technique for applying human recombinant albumin to yeast, Escherichia coli, and Bacillus subtilis using gene recombination technology has been developed. For yeast, (1) Production of Recombinant Human Serum Albumin from Saccharomyces cerevisiae; Quirk, R. et.al, Biotechnology and Applied Biochemistry 11, 273-287 (1989), (2) Secretory Expression of the Human Serum Albumin Gene in the Yeast, Saccharomyces cerevisiae; Ken.Okabayashi et.al, J. Biochemistry , 110, 103-110 (1991) , (3) Yeast Systems for the Commercial Production of Heterologous Proteins; Richard G. Buckholz and Martin AG Gleeson, Bio / Technology 9, 1067-1072 (1991), (4) Construction of DNA sequences and their use for microbial production of proteins, in particular human serum albumin; Lawn, RM, European Patent Appl. 73,646 (1983), (5) ) Synthesis and Purification of mature human serum albumin from E. coli; Latta, L. et.al.Biotechnique 5, 1309-1314 ( 1987 ), for Bacillus subtilis (6) Secretion of human serum albumin from Bacillus subtilis; Saunders, See CW et. Al, J. Bacteriol. 169, 2917-2925 (1987).
[0004]
As a purification method of human serum albumin, a purification method generally used in protein chemistry, for example, salting-out method, ultrafiltration method, isoelectric precipitation method, electrophoresis method, ion exchange chromatography method, gel filtration A chromatography method, an affinity chromatography method, etc. can be used. Actually, since various types of contaminants derived from living tissue, cells, blood, and the like are mixed, purification of human serum albumin is performed by a complicated combination of the above methods.
[0005]
In the industrial production of human serum albumin, it is necessary to treat it under various conditions different from those in the human body environment, and as a result, multimers of human serum albumin are generated. Although reports have not yet been made that these multimers adversely affect the human body in clinical application of human serum albumin, there are concerns such as new antigenic expression by these multimers. From the standpoint of safety as a pharmaceutical product, the standard test of “human serum albumin” as a pharmaceutical product stipulates the limit of multimer contamination. It has become a very important issue.
[0006]
In general, when the protein solution is made alkaline, the SH group of the cysteine residue in the protein molecule forms a disulfide bond within the protein molecule or between protein molecules (including a disulfide bond exchange reaction). At this time, crossing of disulfide bonds occurs, and a protein having a three-dimensional structure different from the original protein may be generated. This crossing of disulfide bonds can occur within and between molecules. Therefore, when various impurities are mixed, a disulfide bond is formed between the impurities and a new substance may be generated. Such incorrect disulfide bond formation is sometimes referred to as misholding.
[0007]
The present inventors have developed a method for efficiently converting a multimer into a monomer by treating a human serum albumin solution containing an HSA multimer with an alkaline solution.
[0008]
This method is very useful for preventing loss in the production process of HSA, but as described above, intramolecular misfolding of HSA or intermolecular mistake between HSA itself or HSA and other contaminants. Holding may occur. Once a new material is formed by misholding, it is difficult and cumbersome to remove. The novel substance may cause side effects such as allergies in the administered patient due to new antigenic expression and other causes.
[0009]
[Problems to be solved by the invention]
Therefore, an object of the present invention is to provide intramolecular misfolding of human serum albumin formed in an alkaline solution treatment (including chromatography or the like) step performed in the production process of human serum albumin or HSA itself or HSA and other impurities. It is an object to provide a method for suppressing intermolecular misfolding.
[0010]
Another object of the present invention is to provide human serum albumin that is highly safe as a pharmaceutical product.
[0011]
[Means for Solving the Problems]
As a result of conducting extensive research in view of the above circumstances, the present inventors have conducted an alkaline solution treatment performed when human serum albumin is produced, and an SH group-containing compound such as cysteine is contained in the treatment solution with the alkaline solution. When added, it was found that intramolecular and / or intermolecular misfolding of HSA was suppressed, and the present invention was completed.
[0012]
The present invention relates to a method for suppressing misfolding of human serum albumin when a human serum albumin multimer is monomerized by treatment with an alkaline solution, the human serum albumin solution containing an SH group-containing compound. The present invention provides a method characterized by treating with an alkaline solution in the presence.
The present invention also provides an HSA that does not contain the complex obtained by the method. Hereinafter, the present invention will be described in detail.
[0013]
DETAILED DESCRIPTION OF THE INVENTION
The method of the present invention is characterized in that an SH group-containing compound is present in an alkaline solution used when monomerizing a human serum albumin multimer in the production process of human serum albumin. By carrying out this method, intramolecular or intermolecular misfolding of human serum albumin is suppressed, and high-purity human serum albumin can be produced.
[0014]
The origin of the human serum albumin multimer used in the present invention is not particularly limited, and plasma-derived HSA or rHSA produced by gene recombination technology can be subjected to alkaline solution treatment. Hereinafter, a multimer of HSA and rHSA may be simply referred to as a multimer.
[0015]
When producing HSA from plasma, it is predicted that a multimer is produced in each production process, but a multimer-containing aqueous solution produced in any process can be used. Examples thereof include an aqueous solution of fraction V of the low-temperature alcohol fraction, and a multimer-containing aqueous solution at each purification stage generated when the fraction V is subjected to further purification steps (chromatography, heat treatment, etc.).
[0016]
The multimer-containing aqueous solution of each production process generated during the production of rHSA can be used in the same manner. Examples thereof include a culture of an rHSA production host, and a multimer-containing aqueous solution at each purification stage generated when the culture is subjected to further production steps (chromatography, heat treatment, etc.). Here, the culture includes a culture solution obtained by culturing the above-mentioned host and a host disrupted solution obtained by disrupting the host by a commonly used method.
[0017]
The host used for the production of rHSA is not particularly limited as long as it is a host capable of producing rHSA. Examples thereof include yeast, bacteria, animal cells, plant cells and the like, and preferably yeast is used.
[0018]
The concentration of HSA or rHSA when the multimer contained in HSA or rHSA in the production process is treated with an alkaline solution is not particularly limited as long as the HSA or rHSA is dissolved, but preferably 100 mg / ml. The following concentrations are:
[0019]
The pH of the alkaline solution for monomerizing the HSA or rHSA multimer is preferably 8 to 11, more preferably pH 8.5 to 9.5, and most preferably 9.0.
[0020]
The chemical substance used to make the pH of the alkaline treatment liquid alkaline is not particularly limited. For example, alkaline organic compounds, alkaline inorganic compounds, specifically ammonia, ammonium salts, basic metal hydroxides (eg, sodium hydroxide, potassium hydroxide), borates, phosphates, acetates, oxalic acid Examples include substances selected from the group consisting of salts, citrates, trishydroxyaminomethane, and mixtures of two or more thereof.
[0021]
Such chemical substances are used in a concentration range that does not denature HSA or rHSA, depending on the concentration of the aqueous solution containing the multimer.
The monomerization of the human serum albumin multimer is carried out by making the aqueous solution containing the multimer alkaline, and then allowing it to stand for a predetermined time, preferably 15 minutes or more, more preferably 3 hours or more. The upper limit of the leaving time is not particularly limited.
The temperature at which the alkali treatment is performed may be not more than a temperature at which HSA and rHSA are not denatured, and is, for example, 0 to 65 ° C, preferably 10 to 40 ° C, and more preferably room temperature (about 25 ° C).
The SH group-containing compound present in the HSA or rHSA solution during the alkaline solution treatment is not particularly limited as long as it is a compound containing a functional group SH group, but a low molecular compound having an SH group is preferred. Specific examples include cysteine, cysteamine, cystamine, methionine, and the like. Preferably, cysteine is used. The SH group-containing compound is preferably 0.1 to 50 mM, more preferably 0.2 to 15 mM, and most preferably 0.5 to 5 mM with respect to an HSA or rHSA concentration of 1 to 100 mg / ml. Added.
[0022]
In the production using genetic recombination technology, the culture solution of the host secreting rHSA or the crushed material immediately after crushing the rHSA producing host is centrifuged at low speed, and then cation exchange, anion exchange, gel filtration, salt It is purified to a high purity by various purification methods such as precipitation, chelate chromatography, hydrophobic chromatography, and adsorption chromatography, or a combination thereof.
[0023]
In addition, plasma-derived HSA is purified using the above-described purification method after, for example, low-temperature ethanol fractionation of human-derived plasma to obtain a fraction V containing about 90% of HSA. Before the low-temperature ethanol fractionation, heat treatment may be performed to prevent degradation by protease.
[0024]
The method of the present invention may be used in any step in the production process of HSA or rHSA. Preferably, a human serum albumin solution is treated at a pH of 5 or less, for example, when a polymer formed after cation exchange chromatography or anion exchange chromatography is monomerized by treatment with an alkaline solution. It is effective to use.
[0025]
【The invention's effect】
According to the present invention, intramolecular or intermolecular misfolding of human serum albumin that occurs specifically in an oxidizing atmosphere during the production process of rHSA obtained by applying HSA in plasma or gene recombination technology is effectively prevented. Can be suppressed.
[0026]
In addition, the method of the present invention makes it possible to obtain high-purity human serum albumin with reduced novel substances caused by misfolding that can cause side effects such as allergies when administered to humans.
[0027]
【Example】
Preparation example: Preparation of human serum albumin solution containing multimers According to the method described in JP-A-11-509525, rHSA was produced by yeast (Saccharomyces cerevisiae). The culture solution containing this rHSA was diluted with purified water to a volume of about 2 times, and then adjusted to pH 4.5 using an acetic acid aqueous solution. The sample was applied to a streamline SP column (Amersham Pharmacia Biotech) (diameter 60 cm × 16 cm) equilibrated with 50 mM sodium acetate buffer (pH 4.5) containing 50 mM sodium chloride. Next, the column is washed with the same buffer as that used for equilibration of the column, and then a 50 mM phosphate buffer (pH 9.0) containing 300 mM sodium chloride is fed to obtain a fraction containing rHSA. Obtained.
[0028]
Cysteine is added to 50 ml of the rHSA-containing fraction obtained according to the preparation example so that the final concentration is 0 to 15 mM, and 0.5 N sodium hydroxide solution (2.8 ml) is further added to adjust the pH to about 9.0. And left at room temperature for 5 hours. Next, an aqueous acetic acid solution was added to the solution to adjust the pH to 7.0, and the treatment was completed with an alkaline solution. The solution was subjected to SDS-PAGE (10-20% gradient gel) followed by Western blotting. Using goat anti-human serum albumin as the primary antibody and anti-goat IgG-HRP conjugate as the secondary antibody, the color was developed by chemiluminescence, transferred to a film and developed. FIG. 1 shows the results of Western blot analysis before and after treatment with an alkaline solution without addition of cysteine. In the sample (2) after the treatment with the alkaline solution, an rHSA band due to intramolecular misfolding is observed at a molecular weight of about 68,000. For samples obtained by adding various concentrations of cysteine and subjecting to alkaline treatment, this band was stained with Coomassie, and the concentration of each band was measured with analysis software (Collage Ver. 3). The results are shown in Table 1. Each numerical value is a relative value with the concentration of the band without addition of cysteine being 100%.
[0029]
[Table 1]

[0030]
It can be seen that the presence of cysteine during the alkaline treatment effectively suppresses intramolecular misfolding of human serum albumin.
[Brief description of the drawings]
FIG. 1 is a photograph showing the result of Western blotting after SDS-PAGE of a sample before and after treatment with an alkaline solution of human serum albumin solution obtained in Preparation Example. (1) indicates a sample before treatment with an alkaline solution, and (2) indicates a sample after treatment with an alkaline solution.

Claims (12)

A method for suppressing misfolding of human serum albumin when a human serum albumin multimer is monomerized by treatment with an alkaline solution, wherein the human serum albumin solution is pH 9 in the presence of an SH group-containing compound. 0. treatment with an alkaline solution for 2 to 8 hours . The method according to claim 1, wherein the human serum albumin is a recombinant human serum albumin produced by a gene recombination technique. The method according to claim 1 or 2 , wherein the treatment time with the alkaline solution is 3 to 5 hours. The method according to any one of claims 1 to 3 , wherein the treatment with the alkaline solution is carried out at 0 to 65 ° C. The method according to claim 4 , wherein the treatment with the alkaline solution is performed at room temperature. The method according to any one of claims 1 to 5 , wherein the alkaline solution is prepared from a material selected from the group consisting of an alkaline organic compound or an alkaline inorganic compound. The alkaline solution is selected from the group consisting of ammonia, ammonium salts, basic metal hydroxides, borates, phosphates, acetates, oxalates, citrates, trishydroxyaminomethane, and mixtures of two or more thereof. 7. A process according to any one of claims 1 to 6 prepared with a selected material. The method according to any one of claims 1 to 7 , wherein the concentration of the SH group-containing compound is 0.1 to 50 mM. The method according to claim 8 , wherein the concentration of the SH group-containing compound is 0.2 to 15 mM. The method according to claim 9 , wherein the concentration of the SH group-containing compound is 0.5 to 5 mM. The method according to any one of claims 1 to 10 , wherein the SH group-containing compound is a low molecular compound. The method according to any one of claims 1 to 11 , wherein the SH group-containing compound is a substance selected from the group consisting of cysteine, cysteamine, cystamine, and methionine.

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