JPH08116985A - Purification of human serum albumin produced by gene manipulation - Google Patents

Abstract

PURPOSE: To purify the subject protein having faint color and useful e.g. for the treatment of hemorrhagic shock in high yield by heat-treating a culture liquid containing a host for producing human serum albumin produced by gene manipulation, contacting the treated product with an adsorbent in a fluidized bed and recovering the adsorbed fraction. CONSTITUTION: This human serum albumin useful for the treatment of hypoalbuminemia, hemorrhagic shock, etc., is purified in high yield by preparing a host for producing human serum albumin (e.g. a yeast Pichia pastoris transformed by the transduction of a recombinant vector integrated with a gene coding human serum albumin), culturing the host in a medium to effect the manifestation of the gene, heat-treating the culture liquid containing the host for producing the human serum albumin and obtained by the above gene manipulation procedure at 50-100 deg.C for 1min to 10hr, contacting the heat-treated liquid with adsorbent particles having strong cation exchange group and floating in a fluidized bed, eluting and recovering the adsorbed fraction and purifying the recovered material by hydrophobic chromatography, anion exchange treatment, chelating resin treatment, etc.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a simple and high-yield purification method of human serum albumin obtained by genetic engineering. The present invention also relates to genetically engineered HSA.
The present invention relates to a human serum albumin-containing composition derived from genetic engineering, which is characterized by a very low coloring degree, which is a problem peculiar to the above.

[0002]

2. Description of the Related Art Human serum albumin (hereinafter referred to as HSA) is the most abundant protein in plasma and functions to maintain osmotic pressure in blood and to bind with nutrients and metabolites to carry them. There is. HS having such a function
A is used as a drug for treating hypoalbuminemia due to albumin loss and albumin synthesis reduction, hemorrhagic shock and the like.

Traditionally, HSA is produced primarily as a product from the blood fractions collected. However, the method for producing HSA using blood as a raw material has drawbacks such as difficulty in blood supply, uneconomicalness, and fear of contamination with an undesired substance such as hepatitis virus. Is desired.

Under such circumstances, with the advent of recombinant DNA technology, techniques for mass production and purification of HSA by genetic engineering are being established in order to replace blood-derived HSA. HSA obtained by genetic engineering (in the present invention, referred to as genetically engineered HSA. Hereinafter, rHSA
Is abbreviated. ) Of conventional plasma-derived HS
It is inappropriate to directly apply the purification method for A. This is because the contaminants to be removed are H derived from plasma.
This is because it is completely different from SA. For example, it is a coloring substance peculiar to HSA derived from genetic manipulation, a protein derived from a host cell, a polysaccharide or other contaminating components. In particular, components derived from host cells are foreign substances to living organisms such as humans, so these components must be sufficiently removed from the viewpoint of antigenicity. Therefore, various studies have been conducted on a method of isolating and purifying rHSA produced by the culture treatment from the components derived from the host cells, the culture components and the like with sufficient accuracy. For example, as a conventional method, a yeast culture solution containing rHSA was subjected to squeezing → ultrafiltration membrane treatment → heat treatment → ultrafiltration membrane treatment, followed by cation exchanger, hydrophobic chromatography, and anion. A method of subjecting the exchanger to a step such as column chromatography treatment (Japanese Patent Application Laid-Open No. HEI 5)
-317079, Biotechnology of Blood Prote
ins.1993, Vol. 227, 293-298). Also,
After the above-mentioned conventional method, a method of subjecting it to a step of chelating resin treatment or boric acid / salt treatment is also reported (JP-A-6-56883 and JP-A-6-245789).

[0005] The above-mentioned purification treatment consisting of several steps by the conventional method is indispensable for removing the antigen derived from the host cell and ensuring a high degree of purification.
It has drawbacks such as a decrease in HSA yield and a longer processing time. Although the yield of each step has been improved in order to increase the yield of rHSA, there is a feeling that the yield has been improved, and the improvement in yield is at a peak. Further, in the above-mentioned conventional method, there is a possibility that contaminants may be mixed in by performing the squeezing process in an open system, and there is a problem that it is difficult to manage hygiene, which is essential when preparing rHSA as a medicine. Also,
In the purification method based on the conventional method, the coloring degree of rHSA is
In the case where the SA concentration was adjusted to 250 mg / ml, the limit was that A350 / A280 was reduced to about 0.015 (JP-A-7-170993 and JP-A-7-170994).

By the way, recently, a method for directly recovering a target protein from a crude stock solution of culture without performing pretreatment such as removal of cell cells after culture and concentration operation has been developed [developed by Pharmacia Co., Ltd. Adsorption fluidized bed technology (Ex
For example, a streamline method using panded bed adsorption) is disclosed in JP-T-6-500050]. Until now, such an adsorption fluidized bed technology has not been applied to the purification of rHSA, in particular, the recovery and purification of rHSA from a yeast culture solution. Actually, this method has been applied to rationalize the purification of rHSA and increase the yield. Nothing is known about its usefulness. However, if this method or a method similar thereto is applicable to rHSA, it is considered that the conventional purification treatment consisting of several steps can be simplified.

However, under acidic conditions such as adsorption to a streamline column (adsorbent: streamline SP) used in the above-mentioned method, rHSA contained in the culture solution is rapidly converted into a low molecular weight compound by the protease in the culture solution. However, there is a problem that the yield of rHSA is remarkably reduced, and it is difficult to use the adsorption fluidized bed technique as it is for the purification of rHSA. Therefore, it is desired to develop a method for highly purifying rHSA in a stable state with a high yield, without compromising the advantages of the adsorption fluidized bed technology such as simplification and simplification of the purification step.

[0008]

The object of the present invention relates to a method for purifying HSA obtained by genetic engineering, and provides a simple method capable of obtaining highly pure and high quality rHSA in high yield in a short time. It is to be. Another object of the present invention is to provide rHSA and a rHSA-containing composition obtained by genetic engineering and from which a coloring component peculiar to genetic engineering derived from a production host, a medium or the like is sufficiently removed.

[0009]

[Means for Solving the Problems] The inventors of the present invention have conducted various studies to solve the above problems. As a result, it is simple and effective to directly heat the culture solution of the rHSA-producing host while containing the host cells. It has been found that proteases can be inactivated by Furthermore, based on this finding, it was found that rHSA can be easily purified with high yield by subjecting the heat-treated solution to contact treatment with adsorbent particles floating in a fluidized bed directly without removing bacterial cells. . Also,
By combining the heat treatment with the adsorbent particle treatment, the five steps of the conventional rHSA purification method, which are compression-ultrafiltration membrane treatment-heat treatment-ultrafiltration membrane treatment-cation exchanger treatment, are heat treatment- The present invention has been completed by confirming that the process can be simplified to two steps of adsorbent particle treatment, the purification process time can be significantly shortened, and the recovery rate can be improved.
Furthermore, the present invention was completed by confirming that by using the method, rHSA substantially free of contaminants derived from host cells and having an extremely reduced coloring degree as compared with the conventional method can be obtained.

That is, according to the present invention, a culture solution of an HSA producing host obtained by genetic manipulation is heat-treated while containing rHSA and a producing host, and the heat-treated solution is brought into contact with adsorbent particles floating in a fluidized bed. The method for purifying rHSA is characterized by recovering the adsorbed fraction. More specifically, a culture solution of an HSA producing host obtained by genetic manipulation is heat-treated while containing rHSA and a producing host, and the heat-treated solution is sent upward to a fluidized bed in which adsorbent particles are suspended to carry out the adsorption. After being brought into contact with the body particles, the rH adsorbed by the adsorbent particles by switching the liquid feeding direction and feeding the buffer solution downward.
It is a method for purifying rHSA which is characterized in that SA is eluted and recovered. Moreover, the present invention provides the method of
The heat treatment temperature of the culture solution of the SA producing host is 50 to 100.
The purification method is characterized in that the temperature is 1 minute to 10 hours, and the contact between the heat treatment liquid and the adsorbent particles is pH 3
A method for purifying rHSA, which is carried out in an atmosphere having a liquidity of ~ 5 and an electric conductivity of 0.1 to 50 mS,
Further, the adsorbent particles have a strong cation exchange group, which is a method for purifying rHSA.

Furthermore, the present invention further comprises subjecting the rHSA-containing fraction recovered from the fluidized bed in the above-mentioned purification method of the present invention to a heat treatment, preferably in the presence of a reducing agent, followed by a hydrophobic chromatography treatment and an anion exchanger. A method for purifying rHSA, which comprises subjecting to at least one purification treatment selected from the group consisting of treatment, chelate resin treatment, boric acid / salt treatment, and ultrafiltration membrane treatment. Further, the present invention is characterized in that human serum albumin containing genetically-engineered human serum albumin is used, and in the case of the 25% albumin solution (rHSA concentration is 250 mg / ml), A350 / A280 is 0.015 or less. It is an albumin-containing composition.

(1) HSA Obtained by Genetic Manipulation The rHSA-producing host used in the present invention prepared by genetic engineering is not particularly limited as long as it is prepared through genetic engineering, and those already known in the literature can be used. Besides, even those developed in the future can be appropriately used. Specific examples include bacteria (eg, Escherichia coli, yeast, Bacillus subtilis, etc.), animal cells and the like that have been made rHSA-producing through genetic manipulation. In particular, it is preferable to use yeast, especially Saccharomyces genus [eg, Saccharomyces cerevisiae], or Pichia genus [eg, Pichia pastoris] as a host. Alternatively, an auxotrophic strain or an antibiotic sensitive strain may be used. More preferably, Saccharomyces cerevisiae AH22 strain (a, his 4, leu 2, can
1), Pichia pastoris GTS115 strain (his 4) is used.

The method for preparing these rHSA-producing hosts, the method for producing rHSA by culturing the host, and the method for separating and collecting rHSA from the culture can be carried out by adopting known methods or their modifications. it can. For example, as a method for preparing a rHSA-producing host,
For example, a method using an ordinary HSA gene (JP-A-58-58).
56684, 58-90515, 58-150
No. 517), a method using a novel HSA gene (JP-A-62-29985, JP-A-1-98486), and a method using a synthetic signal sequence (JP-A-1-24).
No. 0191), a method using a serum albumin signal sequence (JP-A-2-167095), and a method of incorporating a recombinant plasmid on a chromosome (JP-A-3-7288).
No. 9), a method of fusing hosts with each other (Japanese Patent Laid-Open No. 3-5)
3877), a method of causing mutation in a medium containing methanol, a method of using a mutant AOX ₂ promoter (Japanese Patent Application Nos. 3-63598 and 3-63599), and expression of HSA by Bacillus subtilis (Japanese Patent Application Laid-Open No. Sho-63-59) 62-251
33), expression of HSA by yeast (JP-A-60-
41487, 63-39576, 63-744.
93), and the expression of HSA by Pichia yeast (JP-A-2-104290).

Of these, the method for causing mutation in a medium containing methanol is specifically performed as follows. That is, first a suitable host, preferably Pichia yeast, specifically GTS115 strain (NRRL Deposit No. Y-15851).
A transformant is obtained by introducing a plasmid having a transcriptional unit expressing HSA under the control of the AOX ₁ promoter into the AOX ₁ gene region of E. coli (JP-A-2-104).
290). This transformant has weak growth ability in methanol medium. Therefore, this transformant is cultured in a medium containing methanol to cause a mutation, and only a viable strain is recovered. At this time, the methanol concentration is, for example, about 0.0001 to 5%. The medium may be an artificial medium or a natural medium. Culture condition is 1
The temperature is, for example, 5 to 40 ° C. and 1 to 1000 hours.

As a method for culturing the rHSA-producing host, in addition to the methods described in the above-mentioned publications, high-concentration glucose or methanol or the like is supplied in appropriate small amounts by fed-batch culture (semi-batch culture). Then, a method for obtaining a high-concentration cell and product by avoiding high-concentration substrate inhibition on the producing cell (JP-A-3-83595), a method for enhancing the production of rHSA by adding a fatty acid to the medium (special Kaihei 4-
293495) and the like.

The medium used for culturing the transformed host is
Usually, a medium known in the art to which a fatty acid having 10 to 26 carbon atoms or a salt thereof is added is used, and the culture condition can be carried out according to a general conventional method. As the medium, either a synthetic medium or a natural medium may be used, and a liquid medium is particularly preferable. For example, as a synthetic medium, generally, various sugars as a carbon source, urea as a nitrogen source, ammonium salts,
Micronutrients such as nitrates, various vitamins and nucleotides, and inorganic salts such as Mg, Ca, Fe, Na,
Examples include K, Mn, Co, Cu and the like. YNB liquid medium [0.7% yeast nitrogen base (manufactured by Difco), 2% glucose] and the like can be mentioned. Further, as a natural medium, YPD liquid medium [1% yeast extract (manufactured by Difco), 2% bactopeptone (manufactured by Difco),
2% glucose] and the like. The pH of the medium may be neutral, weakly basic or weakly acidic. Further, in the case of a methanol-assimilating host, a methanol-containing medium can be used. In this case, the methanol concentration is about 0.01-5%.

The culturing temperature is 15 to 43 ° C (20 to 20 ° C for yeast).
30 ° C., and bacteria preferably 20 to 37 ° C.). The culturing time is about 1 to 1000 hours, and the culturing is allowed to stand or shake,
It is carried out by a batch culture method, a semi-batch culture method or a continuous culture method under stirring and aeration. In addition, it is preferable to perform pre-culture prior to the culture. As the medium at this time, for example, YNB liquid medium or YPD liquid medium is used. The culture conditions of the preculture are as follows. That is, the culture time is preferably 10 to 100 hours, and the temperature is preferably about 30 ° C. for yeast and about 37 ° C. for bacteria.

(2) Purification of rHSA (i) Heat treatment In the rHSA purification method of the present invention, the heat treatment is a separation treatment such as centrifugation or ultrafiltration of the culture solution of the rHSA-producing host obtained in the above-mentioned culture step. It is characterized in that it is heated directly with the host cells contained, without being attached to. That is, the heat treatment is performed first in the purification method of the present invention. The heating conditions are usually 50 to 100 ° C. and 1 minute to 1
It is about 0 hours, preferably 60 to 80 ° C., about 20 minutes to 3 hours, and more preferably 68 ° C. for about 30 minutes. Further, this treatment is preferably carried out in the presence of a stabilizer.
As a stabilizer, acetyltryptophan, carbon number 6
-18 organic carboxylic acids or salts thereof are exemplified.
Both stabilizers may be used in combination. The amount of acetyltryptophan added is, for example, a final solution concentration of about 1 to 100 mM. Examples of the organic carboxylic acid having 6 to 18 carbon atoms include caproic acid, caprylic acid, capric acid, lauric acid, palmitic acid and oleic acid. Preferred is 10 mM caprylic acid. Examples of the salt include alkali metal salts (eg sodium salt, potassium salt etc.) and alkaline earth metal salts (eg calcium salt etc.). The addition amount of the organic carboxylic acid having 6 to 18 carbon atoms or its salt is, for example, about 1 to 100 mM. In addition, thiol compounds (for example, mercaptoethanol, cysteine, reduced glutathione, etc.) at the time of heat treatment are added in an amount of about 1 to 100 mM, preferably 5 to 30 mM, and further, aminoguanidine is added to 10 to 1000 mM, whereby coloring caused by heating The effect that it can be suppressed (Japanese Patent Laid-Open No. 3-103188) is obtained.

Conventionally, the heat treatment has been carried out on the supernatant (filtrate) obtained by centrifuging or filtering the host culture solution. This is because if the culture solution containing the bacterial cells is directly heat-treated, the impurities, lipids, nucleic acids, proteolytic enzymes and the like existing in the bacterial cells may leak, which may adversely affect the purification of the target substance. Until now, it was completely unknown whether the direct heat treatment of the culture solution containing the host cells was effective for the purification of the target substance.

However, according to the present invention, rHSA and rH
Even if the culture medium containing the SA-producing host cells is directly heated, the strong protease present in the culture medium can be effectively inactivated without reducing the structural function and yield of rHSA. It was found that the protease inactivation treatment could be simplified. Furthermore, this has made it possible to effectively purify HSA obtained by genetic engineering by the purification treatment described below.

(Ii) Dilution of Heat Treatment Liquid and Adjustment of Liquidity The heat treatment liquid obtained in (i) is then subjected to treatment of adsorbent particles floating in the fluidized bed, The contact between the treatment liquid and the adsorbent particles has an electric conductivity of 0.1 to 50 mS,
Preferably 0.5-35 mS, more preferably 5-15
It is preferable to dilute the heat treatment liquid so that it is performed in an atmosphere of mS. As shown in Experimental Example 3 to be described later, as the dilution factor of the heat treatment liquid was increased and the electric conductivity in the contact atmosphere between the heat treatment liquid and the adsorbent particles was lowered, the binding of rHSA to the adsorbent particles was decreased. The amount increases and the electric conductivity is 8 ~
It reaches almost the maximum when it is around 12 mS. The diluting solvent is not particularly limited as long as it does not deteriorate the structural function of rHSA in the heat treatment liquid, and can be appropriately selected in consideration of the adsorption condition to the adsorbent particles. An example is an acetate buffer solution having a concentration of about 50 mM or less, or distilled water. Distilled water is preferable from the viewpoint of simplicity and the like. Then, the liquid property of the solution is adjusted to an acidic condition which is an adsorption condition for the adsorbent particles, usually pH 3 to 5, preferably pH 4 to 4.8.
More preferably, the pH is adjusted to about 4.5. The acidic solution used for the adjustment is not particularly limited, but acetic acid is preferable.

(Iii) Treatment of adsorbent particles Next, a heat treatment liquid diluted and adjusted in pH is brought into contact with the adsorbent particles. Examples of the adsorbent particles used include carriers having a cation exchange group (cation adsorbents), and specific examples thereof include sulfo group type and carboxyl group type adsorbent particles. Examples of the sulfo group type include sulfo-agarose [trade name: Streamline SP, S-Sepharose (both Pharmacia)], sulfo-cellulose [trade name: S-cellulofine (Chisso)], sulfopropyl-agarose. [Brand name: SP-Sepharose (Pharmacia), SP-Cellsul-Big beads (Stelogen)], Sulfopropyl-dextran [Brand name: SP-Sephadex (Pharmacia)], Sulfopropyl-polyvinyl [ Product name: SP-
Toyo Pearl (manufactured by Tosoh Corporation)] and the like. In addition, as the carboxyl group type, carboxymethyl-agarose [trade name: CM Cell Sul-Big beads (manufactured by Sterogine)], carboxymethyl-dextran [trade name: CM-Sephadex (manufactured by Pharmacia)], carboxymethyl- Cellulose [trade name: CM-Cellulofine (manufactured by Chisso Corporation)] and the like are exemplified. Preferably,
The particles are sulfo group-type strong cation adsorbent particles, and more preferably Streamline SP (Pharmacia). The particle size of the adsorbent particles used is usually 30 to 1100 μm, preferably 100 to 300 μm.

The contact conditions are about pH 3 to 5, preferably about pH 4 to 4.8, more preferably about pH 4.5.
Degree, salt concentration of 0.01 to 0.2 M, preferably 0.
An example is about 05 to 0.1M. The adsorbent particles are preferably equilibrated in advance under such contact conditions. Specifically, 50 mM containing 50 mM sodium chloride
It is preferable to equilibrate with the acetate buffer (pH 4.5).

Equilibration of the adsorbent particles, injection of the sample into the fluidized bed containing the adsorbent particles, contact with the adsorbent particles and elution from the fluidized bed are usually carried out according to the following methods. That is, first, the adsorbent particles are packed in an appropriate column and allowed to settle, and then the equilibration buffer solution is fed upward from the column lower outlet to float the adsorbent particles. At this time, in the column, the flow velocity of the buffer solution rising from the bottom of the column and the adsorbent particles settled by gravity cancel each other's forces,
A uniform floating equilibrium state is formed by the adsorbent particles (this is called a fluidized bed). Then, a crude culture solution containing the cells treated in (i) and (ii) above is added from the lower outlet of the column into the column in which the fluidized bed is formed by upward liquid transfer. At this time, the target rHSA binds to the adsorbent particles, while the fine particles and contaminants derived from the host cells and the culture solution pass through the adsorbent particles in the fluidized bed and are discharged and removed from the upper outlet of the column. The contaminated components are also washed away with the wash buffer solution that is subsequently sent upward. Preference is given to adsorbing fluidized bed technology [Journal of Chromatogra
Phy, 597 (1992), 129-145].

An equilibration buffer is used as the washing buffer. Recovery of the target protein, rHSA, is performed by reversing the liquid feeding direction and feeding the elution buffer downward from the column upper outlet. The elution condition of rHSA is about pH 8 to 10, preferably pH 8.5.
To about 9.5, more preferably about pH 9, salt concentration of 0.
2 to 0.5M, preferably about 0.3 to 0.4M. More specifically as a buffer for elution,
An example is 0.1 M phosphate buffer (pH 9) containing 0.3 M sodium chloride. The operations such as the equilibration of the adsorbent particles, the injection of the sample into the fluidized bed containing the adsorbent particles, the contact with the adsorbent particles and the elution from the fluidized bed are performed by a streamline column (adsorbent: streamline SP,
By using a stream line system (manufactured by Pharmacia) equipped with Pharmacia (manufactured by Pharmacia), it can be performed more easily, efficiently and with good reproducibility.

By the above operation, r having a high degree of purification
HSA can be obtained. That is, the degree of purification of rHSA obtained by performing the adsorbent particle treatment is obtained through several steps of conventional compression-ultrafiltration membrane treatment-heat treatment-ultrafiltration membrane treatment-cation exchanger treatment. It is almost the same as the degree of purification of rHSA. In addition, by performing the heat treatment of the culture solution before the adsorbent particle treatment step, rHSA can be stably obtained (without lowering the molecular weight) in a high yield.
Therefore, according to the present invention, the above-mentioned 5 steps can be simplified to 2 steps and the step time can be shortened significantly. Furthermore, according to the present invention, the recovery rate from the culture solution of rHSA by the conventional method can be improved.

The rHSA obtained by the above treatment
Can be further highly purified by subjecting it to ordinary purification methods. Examples of the purification method used include commonly used purification treatments, hydrophobic chromatography treatments, ultrafiltration membrane treatments, anion exchanger treatments, chelate resin treatments and borate treatments. These purification treatments can be carried out alone or in combination of several kinds. In order to obtain a higher quality rHSA purified product, hydrophobic chromatographic treatment, ultrafiltration membrane treatment, anion exchanger treatment and ultrafiltration treatment are performed. The filtration membrane treatment, the chelate resin treatment, the borate treatment, and the ultrafiltration membrane treatment are preferably performed in this order.

It is preferable that the eluted rHSA-containing adsorption fraction is again heat-treated in the presence of a reducing agent before the purification treatment and after the contact treatment with the adsorbent particles. As shown in Experimental Example 6 which will be described later, when comparing the case where the heat treatment is performed and the case where the heat treatment is not performed, there is no difference in the recovery rate of rHSA, but a remarkable effect on the coloration peculiar to rHSA is recognized, By the heat treatment, the coloring substance is significantly removed by the subsequent purification treatment. The treatment temperature is usually 50 to 100 ° C, preferably about 60 to 80 ° C, and more preferably 60 ° C. The treatment time is usually 10 minutes to 10 hours, preferably 30 minutes to
The time is, for example, about 5 hours, more preferably 1 hour.

The reducing agent used is not particularly limited as long as it is a substance having a reducing action.
Low molecular weight compounds containing H groups (eg, cysteine, cysteamine, cystamine, methionine, glutathione, etc.),
Examples include sulfite, pyrosulfite, phosphorous acid / pyrosulfite, and ascorbic acid. Preferred is cysteine. As for the addition amount, in the case of cysteine etc., the final concentration is 1
~ 100 mM, final concentration 0.00 in case of sulfite etc.
For example, about 1 to 10% is exemplified.

Further, this treatment is preferably carried out in the presence of a stabilizer. Examples of the stabilizer include acetyltryptophan, organic carboxylic acids having 6 to 18 carbon atoms, or salts thereof. Both stabilizers may be used in combination. As the amount of acetyltryptophan added, the final solution concentration is 1 to 1
An example is about 00 mM. As the organic carboxylic acid having 6 to 18 carbon atoms, caproic acid, caprylic acid, capric acid,
Examples include lauric acid, palmitic acid, oleic acid and the like. Preferred is 10 mM caprylic acid. Examples of the salt include alkali metal salts (eg sodium salt, potassium salt etc.) and alkaline earth metal salts (eg calcium salt etc.). The addition amount of the organic carboxylic acid having 6 to 18 carbon atoms or its salt is, for example, about 1 to 100 mM. Furthermore, aminoguanidine is 10 to 1000 m
The addition of M has the effect of suppressing coloring caused by heating (Japanese Patent Laid-Open No. 3-103188).

Hydrophobic chromatographic treatment The hydrophobic chromatographic treatment can be carried out according to a conventional method. Examples of the hydrophobic chromatography carrier include insoluble carriers having an alkyl group having 4 to 18 carbon atoms (butyl group type, octyl group type, octyldecyl group type, etc.) and phenyl group. Preferable examples include phenyl group type, specifically, phenyl-cellulose (trade name: phenyl cellulofine,
Manufactured by Chisso Co.). In this step, rHSA can be collected in the carrier non-adsorbed fraction. The contact conditions are about pH 6 to 8, preferably about pH 6.5 to 7, salt concentration of about 0.01 to 0.5M, preferably 0.05 to
It may be about 0.2M.

Treatment with anion exchanger The treatment with anion exchanger can also be carried out according to a conventional method. As the anion exchanger, any insoluble carrier having an anion exchange group can be used.
As the anion exchange group, diethylaminoethyl (DE
Examples thereof include AE) group type and quaternary aminoethyl (QAE) group type. DEAE group type is preferable, and specifically,
DEAE-agarose (trade name: DEAE-Sepharose, manufactured by Pharmacia), DEAE-dextran (trade name: DEAE-Sephadex, manufactured by Pharmacia), DEAE-polyvinyl (trade name: DEAE-Toyopearl, manufactured by Tosoh Corporation), etc. It is illustrated. In this process, r
HSA can be collected in the non-adsorbed fraction. The contact condition is about pH 6 to 8, preferably pH 6.5 to 7.
The salt concentration is about 0.01 to 0.1M. By this treatment with an anion exchanger, coloring components and impurities present in a trace amount are removed.

Ultrafiltration Membrane Treatment After the hydrophobic chromatography treatment and / or the anion exchanger treatment, the rHSA-containing fraction recovered is preferably subjected to an ultrafiltration treatment. By the ultrafiltration treatment, a pyrogen (pyrogen) can be removed. For the ultrafiltration treatment, an ultrafiltration membrane having a molecular weight cutoff of 100 to 300 K is preferably used. Specifically, a Pellicon cassette membrane 100K (manufactured by Millipore) is exemplified.

Chelate Resin Treatment The chelate resin treatment is particularly effective in removing the coloring substances specific to HSA obtained by genetic engineering. This treatment is particularly preferably incorporated in a step after the hydrophobic chromatographic treatment-ultrafiltration membrane treatment-anion exchanger treatment-ultrafiltration membrane treatment in the above-mentioned purification step, and a chelate having a specific ligand portion. It is carried out by bringing the resin into contact with rHSA, and rHSA is obtained in the pass fraction. The carrier portion of the chelate resin is preferably a carrier having hydrophobicity, and examples thereof include a copolymer of styrene and divinylbenzene and a copolymer of acrylic acid and methacrylic acid. On the other hand, the ligand part is a polyamine group having a plurality of polyol groups such as N-methylglucamine group, imino group, amino group, ethyleneimino group, etc. in the molecule (including polyalkylene polyamine groups such as polyethylene polyamine). Included), and thiourea groups. Simply put, DIAION CRB02 (ligand part: N-methylglucamine group, manufactured by Mitsubishi Kasei Co., Ltd.), LEWATIT TP214 (ligand part: ─NHCSNH ₂ : Bayer Co., Ltd.) whose carrier parts are all copolymers of styrene and divinylbenzene Commercially available products such as Amadelite CG4000 and Amberlite CG4000.

The treatment conditions with the chelate resin are preferably as follows. pH condition: acidic, neutral or alkaline (pH 3 to
9, preferably pH 4 to 7) Time: 1 hour or more, preferably 6 hours or more Ionic strength: 50 mmho or less, preferably 1 to 10 m
mho mixing ratio: Resin 0.1 to 1 per 250 mg of HSA
00g, preferably 1-10g (wet weight)

Treatment with boric acid or a salt thereof Further, the rHSA-containing solution obtained by the above-mentioned treatment is treated with boric acid or a salt thereof (in the present invention, also referred to as boric acid / salt) to obtain a host-derived solution. Contaminants having antigenicity and non-antigenic free contaminants detectable by the phenol-sulfuric acid method can be removed. Examples of the boric acid used include orthoboric acid, metaboric acid, tetraboric acid and the like. Examples of the salt include alkali metal salts (sodium salt, potassium salt, etc.), alkaline earth metal salts (potassium salt, etc.), and the like. Preferably, calcium tetraborate is used. The addition amount of boric acid or its salt is about 0.01 to 1 M, preferably about 0.05 to 0.2 M at the final concentration. The treatment pH is, for example, about 8 to 11, preferably about 9 to 10. The processing time is, for example, about 1 to 100 hours, preferably about 5 to 50 hours. The conductivity of the rHSA-containing solution at the time of treatment is preferably low, and a specific example is 1 mS or less. Furthermore, the rHSA concentration in the rHSA-containing solution is preferably as high as possible, and specifically, rHSA is 5% or more, preferably about 15 to 25%.

After the above boric acid / salt treatment, rHSA is recovered from the supernatant by a conventional method such as centrifugation or ultrafiltration.

(3) Highly purified H derived from genetic engineering
Properties of SA rHSA obtained by advanced purification has a molecular weight of about 6700.
It is a single substance with 0 and isoelectric point of 4.9. Consists of monomers only, dimers or polymers, decomposition products (molecular weight about 4300
0) is not substantially contained. Further, it does not substantially contain a contaminant component having an antigenicity and a polysaccharide derived from the production host. The degree of coloring is that when prepared in a solution (25% solution) having a rHSA concentration of 250 mg / ml, A350 / A280 is 0.015 or less, preferably 0.013 or less, and most preferably 0.
It is about 01 to 0.015. In addition, by further appropriately combining known purification methods (for example, the above-mentioned methods 1 to 3), the degree of coloring is further suppressed,
That is, rHSA having a low value of A350 / A280 can be obtained. HSA (containing composition) derived from genetic manipulation,
A350 / A280 in the case of 25% solution of rHSA
Is less than 0.015 is provided for the first time by the present invention.

(4) Formulation The rHSA obtained by the method of the present invention can be formulated by a known method (for example, ultrafiltration, addition of a stabilizer, sterile filtration, dispensing, lyophilization, etc.). it can. The rHSA preparation thus prepared can be clinically used as a serum albumin preparation in the same manner as the conventional plasma-derived HSA preparation, with the same dose and usage. It can also be used as a stabilizer, carrier or carrier for pharmaceuticals. The meaning of the rHSA-containing composition in the present invention is as follows.
The rHSA of the present invention is highly pure but not 100% pure, and shows a state in which substances other than rHSA are mixed even in a trace amount.

[0040]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, examples and experimental examples will be described in order to describe the present invention in more detail, but the present invention is not limited to these examples.

Example 1 rH by Streamline SP on Small Scale
Purification of SA (1) Heat treatment of culture medium For obtaining rHSA-producing yeast Pichia pastoris and culturing the same, refer to JP-A-5-317079, No. 8
Columns line 18 to column 11 line 19, European Patent Publication 6555
It was performed according to the method described in No. 03. About 2.8 liters of the obtained culture solution (yeast-containing cells) was used as it was at 68 ° C.
It was subjected to heat treatment for 30 minutes. The heat treatment was performed in the presence of 10 mM sodium caprylate. The pH of the culture solution at this time was pH 6. The heat treatment liquid is rapidly added to about 15
After cooling to ℃ and diluting with distilled water to approximately twice the volume (total volume 5.5 liters), use acetic acid aqueous solution to adjust the pH.
Adjusted to 4.5.

(2) Adsorbent particle treatment (stream line SP treatment) 50 mM acetate buffer containing 50 mM sodium chloride (p
5.5 liters of a culture solution containing the yeast cells obtained by the heat treatment of (1) in a streamline SP column (C50, 5 × 100 cm: gel amount 300 ml, manufactured by Pharmacia) equilibrated with H4.5). (Electrical conductivity-10mS)
Was added by pumping (up to 100 cm / h at a flow rate with stirring). Next, the column is washed by sending the same buffer solution (2.5 times the column volume) as the buffer solution used for equilibration by the ascending method (condition: flow rate 100 c
m / h for 1 hour, followed by a flow rate of 300 cm / h for 30 hours
Min), and then the flow direction is reversed and the elution solution [300
100 mM phosphate buffer containing mM sodium chloride (p
H9) was sent (flow rate: 50 cm / h) to obtain a fraction containing rHSA. The eluted rHSA-containing fraction was detected by measuring the absorbance at 280 nm.

(3) Heat treatment The obtained rHSA-containing fraction was subjected to heat treatment at 60 ° C. for 1 hour in the coexistence of 10 mM cysteine, 5 mM sodium caprylate, 100 mM aminoguanidine hydrochloric acid, pH 7.5. .

(4) Hydrophobic Chromatography Treatment The rHSA solution heat-treated in (3) was packed with phenylcellulofine equilibrated with 50 mM phosphate buffer containing 0.15 M sodium chloride at pH 6.8 (5). × 25 cm, gel amount 500 ml: manufactured by Chisso Corporation). Under this condition, rHSA passed through the column without being adsorbed by phenylcellulofine. The rHSA-containing solution that passed through the column was concentrated to about 0.2 liters using an ultrafiltration membrane (manufactured by Millipore) having a molecular weight cutoff of 30,000, and the rHSA-containing solution was adjusted to 50 mM phosphate buffer, pH 6 Exchanged for 8

(5) Treatment with anion exchanger After the hydrophobic chromatography treatment, the rHSA-containing solution which had been concentrated and buffer-exchanged was treated with 50 mM phosphate buffer, pH 6.8.
It was added to a column (5 × 25 cm, gel amount 500 ml: manufactured by Millipore) packed with DEAE-Sepharose FF equilibrated with. Under this condition, rHSA passed through the column without being adsorbed on DEAE-Sepharose FF.
The rHSA that passed through the column was concentrated to a volume of about 0.2 L using an ultrafiltration membrane (manufactured by Millipore) having a molecular weight cutoff of 30,000, and the rHSA-containing solution was exchanged for distilled water.

(6) Chelation Treatment DIAION CRB02 equilibrated with 50 mM sodium acetate buffer, pH 4.5 to pH 4.5 by adding acetic acid to 0.2 liters of purified rHSA having a concentration of about 7%.
Column packed with (5 x 2.5 cm, gel amount 500 m
(l: manufactured by Mitsubishi Kasei) and circulated overnight. Under this condition, rHSA passed through the column without being adsorbed on the gel.

(7) Boric acid / salt treatment The rHSA concentration was adjusted to 2.5%, and the solution had an electric conductivity of 1%.
It was set to mS or less. Final concentration of sodium tetraborate is 100
The pH was maintained at 9.5 by adding calcium chloride to a final concentration of 100 mM. After standing for about 10 hours, the precipitate was removed, the supernatant was recovered and concentrated and desalted. Next, concentration and buffer exchange were performed using an ultrafiltration membrane (Millipore) having a molecular weight cutoff of about 30,000. If necessary, stabilizers (sodium caprylate, acetyltryptophan) are added, and then,
By performing sterilization filtration using a 22 μm filter (manufactured by Millipore), rHSA can be used as an injection.

Experimental Example 1 Stabilizing effect of heat treatment of rHSA culture solution A strong protease exists in the rHSA culture solution, and r
It is known to lower the molecular weight of HSA, but as shown in Table 1, the molecular weight reduction is remarkably fast particularly under acidic conditions of pH 4.

[0049]

[Table 1]

By the way, the rHS of the stream line SP
Since the adsorption condition of A is performed at around pH 4.5,
The stability of rHSA in the culture solution at around pH 4.5 was investigated, and heat treatment conditions effective for protease inactivation were used as a pretreatment for keeping the stability more stable. It was examined by observing the stability of rHSA after preparing the above and leaving it at room temperature overnight. The results are shown in Fig. 1. When heated, the final concentration is 10 mM for all samples.
Sodium caprylate was added so that The heat treatment conditions used are as follows. A: Control B: 68 ° C., 30 minutes, p
H6.0 C: 68 ° C., 30 minutes, pH 6.8 D: 68 ° C., 30 minutes, pH 7.5 E: 68 ° C., 30 minutes, pH 8.2 F: 60 ° C., 2 hours, pH 6.0 G: 60 C, 2 hours, pH 6.8 H: 60 ° C., 2 hours, pH 7.5 I: 60 ° C., 2 hours, pH 8.2 J: 60 ° C., 2 hours, pH 6.8, 10 mM cysteine K: 60 ° C., 2 Time, pH 7.5, 10 mM cysteine L: room temperature (25 ° C.), 2 hours, pH 6.0 M: room temperature (25 ° C.), 2 hours, pH 8.2

As a result, heating is more effective at a temperature of 68 ° C. for 30 minutes than at a temperature of 60 ° C. for 2 hours, and the pH of the culture broth subjected to the heat treatment is around pH 6 which is the initial value of the culture broth. It was good.

Experimental Example 2 Relationship between pH of rHSA culture solution and binding capacity to adsorbent A culture solution (rHSA 55.6 mg) adjusted to various pH (4.5 to 4.9) with acetic acid was used as an acetate buffer of each pH. Liquid (5
Streamline SP gel (1 m) equilibrated with 0 mM
1) was added, and the mixture was stirred at room temperature for 1 hour, washed with each equilibration buffer, and the residual amount (%) of rHSA in the unadsorbed fraction was measured. As a result, the amount of binding increased as the pH was lowered and reached the maximum around pH 4.5 (Table 2).

[0053]

[Table 2]

Experimental Example 3 Relationship between Electric Conductivity in a Contact Atmosphere between Heat Treatment Liquid and Adsorbent Particles and Binding Ability of rHSA to Adsorbent Particles A culture solution after heat treatment [electric conductivity: about 20 mS (2
5 ° C.), rHSA 47.1 mg) were diluted with distilled water to various multiples, and the pH was adjusted to 4.5 with acetic acid,
1 ml of Streamline SP gel equilibrated with 50 mM acetate buffer containing 50 mM sodium chloride was added. Then, the mixture was mixed at room temperature for 1 hour, washed with an equilibration buffer, and rHSA was eluted with a 0.1 M phosphate buffer (pH 9) containing 0.3 M sodium chloride. As a result, with increasing the dilution factor and decreasing the electrical conductivity in the solution, rHS
The amount of A bound to the adsorbed particles increases, and the electric conductivity in the contact atmosphere between the heat treatment liquid and the adsorbent particles (in the gel mixed liquid) is 8
The maximum was reached at around 12 mS (Fig. 2).

Experimental Example 4 Stability of Eluate from Streamline SP After diluting the culture broth two times with distilled water, the pH was adjusted to 4.5 with acetic acid.
Adjusted to 50 mM with the same pH and containing 50 mM sodium chloride
A certain amount of Streamline SP gel equilibrated with an acetate buffer was added and mixed and stirred at room temperature for 1 hour. Then, after washing the gel with the equilibration buffer, rHSA was eluted with 0.1M phosphate buffer (pH 9) containing 0.3M sodium chloride, and the rHSA in the obtained fraction solution (pH 8) was cooled to room temperature. The stability at was investigated. As a result, no change was observed even after 3 days when the culture solution was previously heat-treated (68 ° C., 30 minutes), whereas r derived from the untreated culture solution was observed.
HSA was reduced to about 50% and degraded (Table 3).

[0056]

[Table 3]

Experimental Example 5 Comparison of rHSA recovery rate and coloring degree between (heated → adsorbent particles) treatment and (non-heated → adsorbent particles) treatment Based on the results examined in the above-mentioned Experimental Examples 1 to 4, Heating →
The optimization flow of the adsorbent particle treatment process was set (Fig. 3). Stream line SP according to the process flow of FIG.
(5 x 100 cm, gel volume 300 ml) using a column,
RH from the culture solution containing yeast cells (2.8 liters)
Attempts were made to purify SA. On the other hand, rHSA was purified by subjecting the culture solution containing yeast cells (2.7 liters) to the same adsorbent particle treatment without heat treatment. As a result, the total recovery rate from the culture solution was 50 (unheated → adsorbent particles).
% By the treatment (heating → adsorbent particles) of the present invention, which is as high as about 85%, and the coloring degree of the obtained rHSA is
(Unheated → adsorbent particles) Treatment is A350 / A280 0.048
On the other hand, it was as low as 0.0345 in the (heating → adsorbent particle) treatment of the present invention (Table 4). In addition, in FIG. 4, the gel filtration HPLC profile of the streamline SP eluate of the treatment (heating → adsorbent particles) and (non-heating → adsorbent particles)
Gel filtration HPLC of treatment streamline SP eluate
Compared with the profile. In the latter case, remarkable decomposition of rHSA and reduction in molecular weight were observed.

[0058]

[Table 4]

Experimental Example 6 Cysteine heat-treated rHSA
Effect on Coloration Next, described in Examples 1 (4) and (5) using the rHSA elution fraction of the streamline SP column in the treatment (heating → adsorbent particles) obtained in Table 4 as the starting material. The hydrophobic degree and the anion exchanger treatment were performed to evaluate the degree of coloring (A350 / A280). At that time, the streamline SP elution fraction was used in the presence of cysteine (final concentration 10 mM).
The test was conducted in two ways, one that was heat-treated and the other that was not heat-treated. As a result, an equivalent rHSA recovery rate was obtained regardless of whether or not cysteine was heated, but a significant difference was observed in the degree of coloration. On the other hand, the one derived from cysteine heat treatment is 0.0
It showed a significantly low value of 128 (Table 5).

[0060]

[Table 5]

Experimental Example 7 Reduction of rHSA coloring degree (A350 / A280) by incorporating streamline SP In FIG. 5, transition of coloring degree (A350 / A280) by step in conventional purification method and streamline SP step are incorporated. Degree of coloring by process (A350 / A)
280). In the case of the purification method of the present invention in which the streamline SP is incorporated and cystein heat treatment is performed immediately after that, a large difference has already occurred at the stage of the hydrophobic chromatography treatment as compared with the conventional method, and the degree of coloring after the anion exchanger treatment is increased. Showed an extremely low value of 0.0128. In Figure 6,
Specimen further treated with chelate resin after conventional method (curve 1)
And a sample after the anion exchanger treatment and the chelate resin treatment by the purification method of the present invention in which the streamline SP was incorporated and immediately after which cysteine heat treatment was performed (curve 2,
The comparison by 3) with an absorption spectrum is shown. as a result,
The absorption spectrum of rHSA purified by the method of the present invention shows a remarkably lower pattern over the entire visible region (350 nm-700 nm) than the absorption spectrum of the sample which has already been subjected to a chelating resin treatment after the conventional method at the stage of anion exchanger treatment. , The difference became larger in the stage after the chelate resin treatment.

Experimental Example 8 rHSA Obtained by the Present Invention
Gel filtration HPLC analysis of Fig. 7 shows the results of high performance liquid chromatography (GPC-HPLC) analysis of each step sample of rHSA obtained by the method of the present invention by the gel filtration method (rHSA culture solution: rHSA culture solution supernatant, Streamline SP elution fraction: Streamline SP elution fraction in the same process as in Example 1, Streamline SP unadsorbed fraction: Streamline SP unadsorbed fraction in the same process as in Example 1, purified after DEAE Min: after anion exchanger (DEAE) treatment in the same process as in Example 1). As a result, most of the high molecular weight substances and low molecular weight substances other than rHSA in the culture solution were efficiently washed off with the yeast cells in the stream line SP unadsorbed fraction, and albumin was specifically recovered in the eluted fraction. Became clear. In addition, the HPLC pattern of the sample using this fraction as a starting material and after further purification with anion exchanger (DEAE) showed only a single sharp peak of albumin (HSA monomer), which was determined by the conventional purification method. Compared to the sample after the DEAE step, the degree of purification was the same or higher.

Based on the above experimental examples, the conventional purification method D
The recovery rate up to the EAE concentration step and the recovery rate up to the DEAE concentration step in the same process as in Example 1 were calculated and compared. When the same purification method as in Example 1 is used, the conventional purification method of 5 steps from compression-membrane-heat treatment-membrane-cation exchanger treatment becomes two steps, and the step time is greatly shortened. Recovery rate improved by 30%.

Experimental Example 9 Analysis of Contaminant Components Derived from Host: A culture solution of albumin non-producing yeast purified by the same method as this method was used to immunize a rabbit, and the obtained antiserum was used to exist in a purified albumin solution. Contaminant components derived from yeast were detected. The measurement was performed by enzyme immunoassay (EIA method). Sample has an albumin concentration of 250 mg / ml
It measured using what was prepared in. As a result, Example 1
In the same step as above, in the purified albumin after the treatment with boric acid / salt, a contaminant component having yeast-derived antigenicity was not detected at the detection limit of 0.1 ng / ml.

Experimental Example 10 Properties of purified rHSA of the present invention (1) Molecular weight The molecular weight was measured by the above-mentioned HPLC gel filtration method.
In the same process as in Example 1 of the present invention, the molecular weight of the purified rHSA after the boric acid / salt treatment was about 67,000, which was similar to that of plasma-derived HSA. (2) Isoelectric point For the isoelectric point, polyacrylamide gel is used and Allen is used.
It was measured according to the method of J. Chromatog., 146, 1 (1978)! The isoelectric point of the purified rHSA of the present invention was about 4.9, which was similar to that of plasma-derived HSA. (3) Coloring degree The coloring degree is 250 with purified rHSA as albumin concentration.
280 nm, using that prepared to mg / ml,
The absorbance at 350 nm was measured, and A350 / A280 was calculated and determined. The degree of coloring of the purified rHSA of the present invention is A35.
0 / A280 was about 0.012, which was extremely low.

Reference Example Measurement of rHSA (Evaluation of Recovery Rate) In the above Experimental Examples 1 to 8 and Experimental Example 10, rHSA was measured.
Was quantitatively evaluated (including the recovery rate) by subjecting the rHSA-containing solution to HPLC for gel filtration. Specifically, the elution conditions were as follows. The solution containing rHSA was pre-filled with 0.1% NaN ₃ , 0.
50 mM sodium phosphate buffer containing 3% NaCl (p
T6.5-gel G300 equilibrated with H6.5)
0SWXL column (0.78 × 30 cm: manufactured by Tosoh Corporation)
Using the same equilibration buffer as the eluent, flow rate 1
Separation of rHSA at ml / min, 280 nm and 3
It was quantified by measuring the absorbance at 50 nm.

Example 2 Purification by Streamline SP on Large Scale 1000 rHSA-producing yeast Pichia pastoris culture broth (yeast-containing cells) 1000 obtained according to the method of Example 1 (1)
L is the condition (1) of Example 1 (in the presence of 10 mM sodium caprylate, at a liquid pH of 6 and at 68 ° C., 30 ° C.).
Heat treatment was carried out as it was for (minutes). The heat treatment liquid is about 25
The mixture was cooled to 0 ° C., diluted with distilled water to a volume of about 2 times (total volume: about 2000 L), and adjusted to pH 4.5 with acetic acid (99.7%). Then, a streamline SP column (C1000, 100c) equilibrated with a 50 mM acetate buffer (pH 4.5) containing 50 mM sodium chloride was used.
m × 110 cm: gel amount 150 L, manufactured by Pharmacia)
In addition, the culture solution 20 containing the yeast cells obtained by the above heat treatment
00 L was added by pumping from the bottom of the column (flow rate 100-250 cm / h). The culture solution was continuously stirred until the addition of the culture solution to the column was completed so that the bacterial cells did not settle. Next, the column was washed by feeding the same buffer as the equilibration buffer (about 5 times the column volume) by the ascending method (flow rate 100-500 cm / h), and then reversing the liquid feeding direction, Eluate from the top of the column (300m
100 mM phosphate buffer containing M sodium chloride, pH
9) is sent (flow rate 50 to 100 cm / h), rHS
A fraction containing A was obtained. The eluted rHSA-containing fraction was detected by measuring the absorbance at 280 nm. In addition, the obtained rHSA-containing fraction contained 10
mM cysteine, 10 mM sodium caprylate, rH
4-molar amount of sodium oleate, 100 mM of SA
In the coexistence of aminoguanidine hydrochloric acid, pH 7.5, 60 ° C.,
The heat treatment was carried out for 1 hour to reduce the coloring degree of rHSA and promote the conversion of dimers to monomers.

Experimental Example 11 Yield, recovery rate and coloring degree of rHSA by large-scale purification A 1 ton-scale culture solution was heat-treated at 68 ° C. for 30 minutes, and the heat-treated solution was made to have a double volume with distilled water. After diluting and adjusting to pH 4.5, it was subjected to adsorbent particle treatment by a streamline SP (C1000) column. The eluate containing rHSA was filtered through a 0.8 μm filter, passed through an ultrafiltration membrane having a molecular weight cutoff of about 30,000, and concentrated, and then heat-treated with cysteine for purification. When the results of 4 lots (shown in Table 6) are averaged, the recovery rate from the culture solution to the heat treatment is 98.6%, and the recovery rate from the heating to the cysteine heat treatment via the stream line is 88.4%. The total recovery rate was as good as 87.1%. This value was in good agreement with the experimental result on the small scale using a C50 column, and reproducibility on the large scale was confirmed.

[0069]

[Table 6]

[0070]

The present invention has been completed based on the finding that protease can be inactivated simply and effectively by directly heating a culture solution containing yeast cells.
That is, the present invention is a method for simply and effectively purifying rHSA by bringing a treatment solution obtained by heating a culture solution containing yeast cells directly into contact with adsorbent particles floating in an adsorption layer. This allows conventional pressing-membrane-heating-membrane-
The 5 steps up to the cation exchanger treatment can be simplified to 2 steps (heating-adsorbent particle treatment), and the process time is greatly shortened and the recovery rate is also improved. Further, the problem of coloring peculiar to HSA produced by genetic engineering can be eliminated because the coloring substance that causes the problem can be effectively removed by the purification method of the present invention. Furthermore, according to the purification method of the present invention, it is possible to perform all the steps of HSA production by genetic engineering from cell culture to purification on a single closed line, and it is possible to automate the production of rHSA and to use rHSA as a drug. As a result, there is an advantage that hygiene control, which is indispensable for preparation, is easily performed. That is, the present invention is extremely useful in rHSA purification as a method that not only shortens the steps and increases the yield but also contributes to the quality improvement. Further, according to the present invention, it is possible to provide rHSA which is obtained by genetic engineering, does not contain components related to the production host or other contaminants, and is sufficiently suppressed in coloration and is useful as a pharmaceutical.

[Brief description of drawings]

FIG. 1 is a diagram showing a stabilizing effect of heat treatment of a rHSA culture solution.

FIG. 2 is a diagram showing a relationship between electric conductivity in a contact atmosphere between adsorbent particles and a heat treatment liquid and a binding ability of rHSA to adsorbent particles.

FIG. 3 is a diagram showing an optimization flow of purification of rHSA from a culture solution (yeast-containing cells) by streamline SP.

FIG. 4 is a gel filtration HPLC profile of streamline SP eluate of (heating → adsorbent particles) treatment [in the figure,
(A)] and (non-heated-> adsorbent particle) processing streamline SP eluate profile of gel filtration HPLC [(b) in the figure] is shown (absorbance measured at 280 nm).

FIG. 5 is a diagram comparing changes in the coloring degree (A350 / A280) of rHSA for each process between the conventional method and the method of the present invention.

FIG. 6 is a diagram comparing the absorption spectrum of rHSA obtained by the method of the present invention with that of a conventional method.

FIG. 7: GPC-of rHSA obtained by the method of the present invention
It is a chromatogram showing the result of HPLC analysis (absorbance measured at 280 nm).

[Explanation of symbols]

A control B 68 ° C., 30 minutes, pH 6.0 C 68 ° C., 30 minutes, pH 6.8 D 68 ° C., 30 minutes, pH 7.5 E 68 ° C., 30 minutes, pH 8.2 F 60 ° C., 2 hours, pH 6 0.0 G 60 ° C., 2 hours, pH 6.8 H 60 ° C., 2 hours, pH 7.5 I 60 ° C., 2 hours, pH 8.2 J 60 ° C., 2 hours, pH 6.8, 10 mM Cysteine K 60 ° C., 2 Time, pH 7.5, 10 mM cysteine L room temperature (25 ° C.), 2 hours, pH 6.0 M room temperature (25 ° C.), 2 hours, pH 8.2 1 Conventional method (compression → membrane → heating → membrane → cation exchanger) → hydrophobic chromatography → treatment with anion exchanger), and then rHSA 2 after treatment with chelate resin in the same step as in Example 1, rHSA 3 after treatment with anion exchanger (DEAE) same as Example 1. In the process Te, rHSA after the chelate resin treatment

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Kazumasa Yokoyama 2-25-1 Otani Otani, Hirakata City, Osaka Prefecture Midori Cross Central Research Institute Co., Ltd.

Claims (9)

[Claims] 1. A culture solution containing a host for producing human serum albumin obtained by genetic engineering is heat-treated, and the heat-treated solution is brought into contact with adsorbent particles suspended in a fluidized bed,
A method for purifying genetically engineered human serum albumin, which comprises collecting the adsorbed fraction. 2. Adsorbent particles are obtained by subjecting a culture solution containing a host for producing human serum albumin obtained by genetic engineering to a heat treatment, and feeding the heat-treated solution upward to a fluidized bed in which the adsorbent particles are suspended. A method for purifying human serum albumin derived from genetic engineering, characterized in that the adsorbed fraction is eluted and collected by switching the liquid feeding direction and feeding the buffer solution downward after contacting with. 3. The method for purifying genetically engineered human serum albumin according to claim 1, wherein the temperature of the heat treatment is 50 to 100 ° C. and the time is 1 minute to 10 hours. 4. The contact between the heat treatment liquid and the adsorbent particles is performed at pH 3 to 5.
5. A method for purifying genetically engineered human serum albumin according to any one of 3 to 3. 5. The gene manipulation according to claim 1, wherein the heat treatment liquid and the adsorbent particles are brought into contact with each other in an atmosphere having an electric conductivity of 0.1 to 50 mS. A method for purifying human serum albumin of origin. 6. The method for purifying genetically engineered human serum albumin according to claim 1, wherein the adsorbent particles are adsorbent particles having a strong cation exchange group. 7. The adsorption fraction containing human serum albumin recovered by the purification method according to claim 1 is further subjected to hydrophobic chromatography treatment, anion exchanger treatment, chelate resin treatment, boric acid A method for purifying genetically engineered human serum albumin, which comprises subjecting to at least one purification treatment selected from the group consisting of salt treatment and ultrafiltration membrane treatment. 8. An adsorption fraction containing human serum albumin recovered by the purification method according to any one of claims 1 to 6 is heat-treated in the presence of a reducing agent, and then subjected to hydrophobic chromatography and anion. A method for purifying genetically engineered human serum albumin, which comprises subjecting to at least one purification treatment selected from the group consisting of an exchanger treatment, a chelate resin treatment, a boric acid / salt treatment, and an ultrafiltration membrane treatment. 9. A human serum albumin derived from a genetically engineered product, which contains A350 / A in the case of a 25% solution of said albumin.
A composition containing human serum albumin, wherein 280 is 0.015 or less.