JP2002012553A - Human serum albumin-containing composition - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a composition containing human serum albumin of which the coloration degree is suppressed in about 1/70-1/10 as compared with the case that the treatment for suppressing the coloration is not performed. Thus, the composition can be utilized as a clinically useful medicine similarly to blood plasma-derived human serum albumin. SOLUTION: This composition containing human serum albumin which has no combined coloration component and is expressed by gene manipulation is obtained by a method of production for human serum albumin showing suppressed coloration. The method includes processes for preparing a human serum albumin productive host by means of gene manipulation, cultivating the host in a culture medium containing at least one of substances selected from the group consisting of anion exchangers, hydrophobic carriers and activated charcol and expressing the human serum albumin.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a composition containing human serum albumin expressed by genetic engineering.

[0002]

BACKGROUND OF THE INVENTION Albumin, particularly human serum albumin (hereinafter also referred to as "HSA") is a major protein component of plasma. This protein is made in the liver and is primarily responsible for maintaining normal osmolality in the bloodstream. It also functions as a carrier for various serum molecules. HSA is administered in various clinical settings. For example, in patients with shock or burns, H is usually used to restore blood volume, thereby improving some of the symptoms associated with trauma.
Requires frequent administration of SA. Patients with hypoproteinemia or fetal erythroblastosis may also require treatment with HSA. Thus, the fundamental therapeutic significance of administering HSA is in treating conditions where there is fluid loss from the blood vessels, as in surgery, shock, burns, and hypoproteinemia causing edema. . Currently, HSA
Is mainly produced as a product from a fraction of the collected blood. The disadvantages of this method are that it is uneconomical,
It means that blood supply is difficult. Blood may also contain undesired substances such as hepatitis virus. Therefore, it would be beneficial to develop an alternative feedstock for HSA.

[0003] With the advent of recombinant DNA technology, the production of a wide variety of useful polypeptides by microorganisms has become possible. Many mammalian polypeptides, such as human growth hormone, interferon, have already been produced by various microorganisms. This technology allows microbial production of various useful polypeptides,
A variety of vaccines, hormones, enzymes and antibodies can now be produced by microorganisms. In order to overcome the above-mentioned difficulties in the production of HSA, a method for obtaining a large amount of HSA by genetic engineering techniques and for highly purifying it has been established. However, HSA derived from plasma is originally yellow or tan, but HSA expressed by genetic manipulation
A becomes dark yellow or dark yellow-brown, and when producing and purifying HSA, a certain coloring component is contaminated in the raw material, and this combines with HSA to cause coloring of HSA itself. These contaminants are conventional HSA derived from plasma.
Cannot be sufficiently removed by the purification method of

[0004]

SUMMARY OF THE INVENTION An object of the present invention is to provide a composition containing human serum albumin expressed by genetic engineering, in which contamination of coloring components is sufficiently eliminated.

[0005]

Means for Solving the Problems Accordingly, the present inventors have conducted intensive studies on such a technical background, and as a result, it has been found that HSA produced inside or outside the cells through genetic manipulation and the coloring component are separated. Before binding and coloring, it was found that the coloring of human serum albumin was prevented by separating the human serum albumin and the coloring component, and the present invention was completed. That is, the present invention is as follows. (1) Culture in which at least one selected from the group consisting of an anion exchanger, a hydrophobic carrier and activated carbon is added when culturing a human serum albumin-producing host prepared by genetic manipulation to express human serum albumin By culturing the host in a liquid, the human serum albumin and the coloring component are separated before the human serum albumin and the coloring component are bound, and the method is performed by a method for suppressing coloring of human serum albumin. A composition comprising human serum albumin expressed by genetic engineering, to which no coloring component is bound. (2) A human serum albumin-producing host is prepared by genetic manipulation, and the host is cultured in a culture medium to which at least one selected from the group consisting of an anion exchanger, a hydrophobic carrier and activated carbon has been added, and A composition containing a human serum albumin expressed by genetic manipulation, to which no coloring component is bound, which is obtained by a method for producing human serum albumin in which coloring is suppressed, comprising a step of expressing albumin. (3) The degree of coloring is 0.00 at an absorbance ratio of A _{350 nm} / A _{280 nm.}
35. The composition according to (1) or (2), wherein the composition has an absorbance ratio of A _{405 nm} / A _{280 nm} of 0.0018 to 0.0179. The HSA used in the present invention is not particularly limited as long as it is prepared by genetic manipulation. Therefore, cells expressing HSA through genetic engineering (eg,
Escherichia coli, yeast, Bacillus subtilis, animal cells, etc.) are cultured and produced by intracellular expression or extracellular expression (secretory expression).
However, the step of separating human serum albumin and the coloring component is carried out in the case of intracellular expression, when the human serum albumin is taken out of the cells, and / or immediately after being taken out, in the case of extracellular expression, It is desirable to carry out during the process. In the present invention, the coloring component refers to a component capable of coloring HSA, and does not refer to only a coloring component derived from a culture medium.

(I) Genetic manipulation of HSA-expressing cells
As an example of cells expressing the prepared HSA, a method for preparing an HSA-producing yeast will be described below. In the present invention, the HSA-producing yeast is a yeast transformed using a plasmid carrying the HSA gene. The plasmid carrying the HSA gene can be obtained by a known technique. Specifically, HSA
It includes genes, promoters, signal sequences, terminators and the like. The albumin coding region contained in the plasmid is, in particular, a DNA sequence identical or homologous to HSA,
For example, it can be obtained from any human cell capable of producing HSA. The DNA is chromosomal DNA or cDNA. Chromosomal DNA can be isolated from a gene library containing the HSA gene and
NA can be prepared via the mRNA pathway using known methods.

[0007] The promoter is usually a yeast, preferably a sucker.
Karomyces cerevisiae (Saccharomyces cerevisia
e) from genomic DNA. Preferably, a high expression yeast
Using the promoter of the mother gene for HSA expression
You. That is, the TRP1 gene,ADHIOrADHI
IGene, acid phosphatase (PHO3OrPH
O5) Gene or isoprotochrome C gene pro
Motor and galactose metabolic promoter (GAL
1, GAL10 or GAL7), invertase
Encodes a promoter (SUC2) or glycolytic enzyme
Promoters such as enolase, glycer
Lualdehyde-3-phosphate dehydrogenase (G
APDH), 3-phosphoglycerate kinase (PG
K), Hexokinase, pyruvate decarboxylar
Ze, phosphofructokinase, glucose-6-phosphine
Ethyl isomerase, 3-phosphoglycerate muter
Ze, pyruvate kinase, triphosphate isomerer
Ze, phosphoglucose isomerase and glucokiner
There is a promoter of the zeta gene, or a-factor
Or yeast-conjugated pheromone encoding α-factor
Genetic promoters can be used.

In another preferred embodiment, a signal sequence is introduced into the construct. As the signal sequence, a yeast-derived signal sequence such as yeast invertase and α-factor gene can be used. Also, the HSA signal sequence is suitable, preferably a signal sequence specifically synthesized for secretory expression in yeast (EP Publication 319).
641 or JP-A-1-240191 can also be used. By introducing this signal sequence,
After expression of the HSA gene, the gene product enters the secretory pathway and is transported to the periplasmic space. Furthermore, secretion of the product into the medium through the cell wall can be achieved, which allows a considerable increase in the yield. Furthermore, since there is no need to destroy cells, the recovery step can be simplified. Also, the plasmid contains a suitable terminator for transcription termination, for example, PH05 or GAPDH terminator.

As a host, yeast, especially Saccharomyces or Pichia, is used. Preferably, an auxotrophic strain or an antibiotic-sensitive strain can be used. For example, leucine and histidine auxotrophs, and a G418 sensitive strain Saccharomyces cerevisiae (Saccharomyces
s cerevisiae ) AH22 strain (a, his 4, leu 2, can1)
Etc. are preferably used. Transformation methods include a method of directly introducing a plasmid into a host cell and a method of integrating a plasmid into a yeast chromosome.

The former is performed by a known method. Specific examples thereof include a calcium phosphate microprecipitation method, a protoplast polyethylene glycol method, and an electroporation method (Japanese Patent Application Laid-Open No. 3-72889, Japanese Patent Publication No. Hei 6-72889).
-69365). The latter is carried out, for example, by the following method. In the present invention, the HSA-producing yeast having the plasmid integrated into the yeast chromosome is more preferably used. In the latter method, the plasmid contains a partial DNA sequence of a gene present in the host yeast chromosome (eg, LEU2, HIS4, TRP1, URA3, ribozyme).
some DNA genes). Due to the homologous sequence, the whole plasmid or its linear fragments can be stably introduced into the host chromosome by recombination. That is, during growth, progeny cells stably retain the introduced genetic material even in the absence of selective pressure. For example, a plasmid containing a naturally occurring sequence in a yeast chromosomal gene and an HSA gene can be stably integrated at the chromosomal gene locus. Specifically, it is desirable to cut any site in the sequence of the plasmid to be inserted which is homologous to the chromosome of the host yeast cell with a restriction enzyme treatment, and to introduce the linearized plasmid into the host. The linearized plasmid is integrated into a region on the host yeast cell chromosome that is homologous to the region integrated into the plasmid. The linearized plasmid is more frequently integrated on the host chromosome than the circular plasmid.

Sequences homologous to the host yeast chromosome sequence include amino acid or nucleic acid synthesis genes, ribosome D
NA, Ty factor (Transposon of Yeast element) and the like can be used. In particular, when the host yeast is an amino acid or nucleic acid auxotrophic strain, that is, in a strain lacking the amino acid or nucleic acid synthesis gene, the amino acid or nucleic acid synthesis gene is a gene that complements the mutation of the host. As such, it can be used as a selectable marker for transformants. At this time, it brings prototrophy to the auxotrophy of the host yeast. Examples of the amino acid or nucleic acid synthesis gene include LEU2, HIS4, TRP1 and URA3. As described above, as a selection gene marker for yeast, when the host yeast is an auxotrophic strain, an amino acid or a nucleic acid synthesis gene can be used, and when the host is an antibiotic-sensitive strain, as described above. And a gene expressing the antibiotic resistance can be used. For example, cyclohexide, G
418, genes that confer resistance to antibiotics such as chloramphenicol, bleomycin or hygromycin.

Also, plasmids cannot replicate autonomously in host yeast. That is, it does not substantially contain an autonomous replication initiation region in the host yeast, for example, a 2 μm DNA replication initiation region or an ARS region (Autonomous Replicating Sequence). This plasmid may also contain, apart from the promoter, the HSA coding region and the host chromosome homology region, an origin of replication and a genetic selectable marker for a bacterial host, especially E. coli. Useful features exist for using E. coli origins of replication and selectable markers for E. coli in yeast hybrid vectors. First, large amounts of hybrid vector DNA can be obtained by growth and replication in E. coli, and second, construction of hybrid vectors can be readily accomplished using all E. coli-based cloning techniques. E. coli plasmids, such as pBR322, contain an E. coli origin of replication and E. coli genetic markers that confer resistance to antibiotics, such as tetracycline and ampicillin, and are advantageously used as part of yeast hybrid vectors. Accordingly, the plasmid contains a promoter, an albumin coding region controlled by the promoter, a terminator for transcription termination, and a sequence homologous to the host yeast chromosome sequence. In addition, this plasmid can contain a signal sequence for secretory production, a selection gene marker for yeast, a replication initiation region of Escherichia coli, and a selection gene marker for Escherichia coli, if desired. It does not substantially contain the yeast replication initiation region.

Next, it is examined whether the plasmid has been introduced into the expected site and whether the introduced gene is stable. Specifically, it is confirmed that the plasmid has been introduced into the expected site by Southern blotting using a sequence homologous to the chromosome sequence of the host yeast cell used for the transformation as a probe. In addition, the stability of the gene encoding albumin is examined using albumin production and recovery of auxotrophy as indices, and it is confirmed that the transformants remain unchanged even after being cultured for several generations in a non-selective medium. Using this transformant as a host, it can be transformed again with a plasmid containing the albumin coding region. In this case, a homologous region other than the region used in the initial transformation can be used as the yeast cell chromosome homologous region.

In addition, ribosome DNA and Ty factor may be mentioned as sequences homologous to the chromosome sequence of the host yeast cell.
Since there are a plurality of these genes per cell,
In a single transformation, multiple genes of interest can be integrated into the host chromosome. If several types of auxotrophic mutants can be obtained as hosts, or if several types of strains showing sensitivity to antibiotics can be obtained, then a plurality of useful genes can be introduced into the corresponding regions. Thus, the target gene can be inserted into a plurality of regions on the host chromosome. The genes integrated into these chromosomes are stably maintained without dropping out, and by integrating a plurality of genes, a large amount of the target product can be obtained. Alternatively, an HSA-producing yeast may be prepared by fusing a plurality of transformants with cells (Japanese Unexamined Patent Publication (Kokai) No. 3-53).
877, Japanese Patent No. 2855130). The transformant is cultured in a known medium. As the medium, for example, a YPD liquid medium [1% yeast extract (Difco
2% Bactopolypeptone (Difco), 2% glucose] and the like. The cultivation is usually performed at 15 to 43 ° C. (preferably about 30 ° C.) for about 20 to 100 hours, and if necessary, aeration and stirring can be added. Further, a fed-batch culture can also be used (Japanese Unexamined Patent Publication No. 3-8 / 1990).
No. 3595, Japanese Patent Publication No. 8-77).

(Ii) Step of separating HSA and coloring component The step of separating HSA and coloring component comprises:
It is incorporated in the purification step (after culturing) or in the line step (inline), and is performed in an HSA-containing aqueous solution such as a culture solution, a culture supernatant, a crude fraction, or a purified fraction. In the present invention, coloring is prevented by separating human serum albumin and a coloring component expressed by culture before the two bind to each other. Therefore, particularly in the case of extracellular expression, it is desirable that the separation step be performed during the culture step. In the case of intracellular expression, HSA
It is desirable to carry out the separation step during and / or immediately after the process of removing the.

The process of removing HSA from the cells includes:
Freeze-thaw method, glass bead method, high pressure method, sonication method,
Conventional methods such as an enzyme treatment method may be mentioned. As a means for separation, for example, a method of removing a coloring component by an adsorbent is exemplified. As the adsorbent, an anion exchanger, a hydrophobic carrier or activated carbon is desirable, and a cation exchanger may be used in combination. As the anion exchanger, DEAE
Systems (eg, DEAE-agarose, DEAE-crosslinked dextran, DEAE-cellulose, etc.), QAE systems (eg, QAE-agarose, QAE-crosslinked dextran, etc.) and the like. In the present invention, the hydrophobic carrier is
The hydrophobic ligand is bound to an insoluble carrier.
Hydrophobic ligands include alkyl (ethyl, butyl,
Octyl and the like having 1 to 4 carbon atoms), phenyl, phenylalanyl and the like. Examples of the insoluble carrier include agarose, cross-linked dextran, and hydrophilic vinyl polymer.

The processing conditions in the separation step are preferably as follows. pH condition: pH 4 to 8 (preferably pH 5 to 6.5) Addition amount: 0.01 to 10 w / v% (in the medium) In the present invention, after performing the separation step in parallel during the culture step, It is desirable to purify human serum albumin by a method known per se. As a purification method, known methods such as various fractionation methods, adsorption chromatography, affinity chromatography, gel filtration, density gradient centrifugation, and dialysis are employed. In the present invention,
The separation step may be combined with these purification steps.

[0018]

EXAMPLES In order to explain the present invention in more detail, Reference Examples, Examples, Comparative Examples and Test Examples will be given, but the present invention is not limited thereto.

Reference Example HSA secretion-expressing yeast Saccharomyces cerevisiae ( Sa
[i] Cloning of GAPDH promoter region, SUC2 signal region, LEU2 region, TRP1 region, PHO5 terminator region, and HSA gene, G-4 ( Ccharomyces cerevisiae ) TMS33-1h4 strain
Preparation of 18-resistance gene The method described in the following literature, a method analogous thereto, or a commercially available one was used. GAPDH promoter : Holland, HJ and Holland,
JP, J. Biol. Chem., 254 , 12, 5466 (1979), Hollan
d, HJ and Holland, JP, J. Biol. Chem., 254 , 19,
9839 (1979), JP-A-63-84498, SUC2 signal sequence : JP-A-60-41488, JP-A-63-84498, HSA gene : JP-A-62-29985, PHO5 terminator : JP-A-62-29985 G-418 resistance gene : Oka, A., Sugisaki, H. and Ta
kanami, M., J. Mol. Biol., 147 , 217 (1981), Jimene
z, A. and Davies, J., Nature, 287 , 869 (1980), JP-A-61-40793, TRP1 : Derived from plasmid pBTI-10 (commercially available from Boehringer Mannheim) LEU2 : Derived from plasmid pBTI-1 (Boehringer) (Commercially available from Mannheim) E. coli replication initiation region and ampicillin resistance gene : pU
C19 (obtained from Takara Shuzo)

[Ii] Construction of each plasmid The construction of each plasmid pMM-006 and pHO-011 was carried out from pUC19 according to the method described in "Molecular Cloning", Cold Spring Harbor Laboratory (1982) (No. FIG. 1 (a) and (b). Plasmid pMM-006 contains the leucine synthesis system gene LEU2 as a sequence homologous to the chromosome sequence of the host yeast cell, and is obtained by linking the SUC2 signal sequence, the HSA structural gene, and the PHO5 terminator under the control of the GAPDH promoter. Plasmid pHO-011 is a sequence homologous to the chromosomal sequence of the host yeast cell, and is composed of the tryptophan synthesis gene TRP1
And a SUC2 signal sequence, an HSA structural gene and a PHO5 terminator linked under the control of the GAPDH promoter, and a G418 resistance gene as a yeast selectable marker gene.

The plasmids pMM-006 and pHO-
011 was deposited on April 28, 1989 at the Institute of Microbial Industry and Technology (currently the National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary) under the Ministry of International Trade and Industry, as follows: . Microorganism name: PMM006 / E. Co., Ltd. JM109 (pMM006 / E. Coli JM109) Accession No .: Microfabrication Kenjiro No. 2404 (FERM BP-
2404) Microorganism name: POH011 / E. Accessory No. 2405 (FERM BP-)
2405)

[Iii] By plasmid pHO-011
Transformation of S. cerevisiae AH22 strain Saccharomyces cerevisiae AH22 strain was used as a host. The Saccharomyces cerevisiae AH22 strain has an a-type mating type and a histidine synthesis gene (hi
s 4) and leucine synthesis gene (leu 2). Therefore, proliferation cannot be achieved unless histidine and leucine are added to the culture solution.

HSA secretion expression plasmid pHO-01
1 was introduced into the chromosome of the yeast strain Saccharomyces cerevisiae AH22 by the following method. YPD liquid medium (yeast extract 10g, bactopeptone 20)
g was dissolved in water to make 900 ml, followed by autoclave sterilization, and autoclaved 20% glucose 10
Saccharomyces cerevisiae AH22 strain, which had been cultured with shaking at 37 ° C. overnight in 50 ml (mixed with 0 ml), was centrifuged.
The obtained cells were suspended in 20 ml of water and centrifuged again to obtain cells. This was added to 10 ml of 50 mM dithiothreitol, 1.2 M sorbitol, 25 mM EDTA, pH
Suspended in 8.5 and gently shaken at 30 ° C. for 10 minutes.
Cells are collected by centrifugation and 10 ml of 1.2 M sorbitol
And the cells were collected by centrifugation again. 10m cells
The cells were suspended in 1 l of 1.2 M sorbitol and collected by centrifugation. Cells were diluted with 10 ml of 0.2 mg / ml zymolyase 100T, 1.2 M sorbitol, 10 mM EDT.
A, After suspending in 0.1 M sodium citrate, pH 5.8, the mixture was gently shaken at 30 ° C. for 1 hour. The cells were collected by centrifugation, washed with 1.2 M sorbitol, then with 10 ml each of 10 mM calcium chloride and 1.2 M sorbitol, and collected by centrifugation. Cells were suspended in 1 ml of 10 mM calcium chloride, 1.2 M sorbitol. Suspension 100μ
of the DNA solution (TRP1).
The plasmid was digested with Eco RV, which is a unique site on the gene, and mixed with a linearized plasmid (containing 5 μg of pHO-011) and allowed to stand at room temperature for 15 minutes. Further, 1.
2 ml of 20% polyethylene glycol 4000, 10
mM calcium chloride, 10 mM Tris-HCl, pH7.
5 and gently mixed, and allowed to stand at room temperature for 20 minutes. Collect cells by centrifugation, 0.1 ml of 1.2 M sorbitol,
The suspension was suspended in a YPD liquid medium supplemented with 3% noble agar and 0.2% monopotassium phosphate, and gently shaken at 30 ° C. for 30 minutes. 1.5, 10, 20 and 50 μl of suspension
Was mixed with an agar medium and incubated at 45 ° C.
ml of 1.2 M sorbitol, 3% noble agar,
The cells were spread on a plate composed of a YPD liquid medium supplemented with 100 μg / ml G418. Once the plate has solidified, 30
The culture was allowed to stand at room temperature for 3 days. The formed colonies were collected with a toothpick, suspended in 3 ml of 0.7% yeast nitrogen base and 2% glucose, and cultured with shaking at 30 ° C. for 2 days. 1.5 ml of the mixture was centrifuged, and the cells were collected.
ml of YPD liquid medium (yeast extract 10)
g and bactopeptone (20 g) were dissolved in water to make 900 ml, then sterilized by autoclave, and mixed with 100 ml of autoclaved 20% glucose. ) And cultured with shaking at 30 ° C. The HSA concentration of the culture supernatant was
When measured by the PHA method, a maximum of 60 μg /
ml of HSA was detected. The transformant thus obtained was named TMS-26-10 strain.

[Iv] Screening of Transformant TMS-26-10 strain The location of the HSA gene introduced by Southern blotting was confirmed.
It was confirmed that it was introduced into the I region. When the stability of the HSA gene was measured using the HSA production amount and G418 resistance as indices, 100% of the HSA gene was retained even after culturing for about 60 generations in a non-selective medium.

[V] Transformation of Transformant TMS-26-10 with Plasmid pMM-006 A method similar to the method described in the above [iii] was used. However, it differs from the method described in [iii] in the following points. Host: TMS-26-10 strain Plasmid: pMM-006 When introducing the plasmid: digestion with Kpn I, a unique site on the LEU2 gene in pMM-006, and introducing the plasmid into a linear form. Transformation medium: 1.2M sorbitol, 1P in YPD medium
The transformed (proto) plast is suspended in a solution to which 0 mM calcium chloride has been added. Plate: 0.7% yeast nitrogen base, 2
% Glucose, 1.2 M sorbitol, leucine-free agar medium consisting of 3% noble agar. Human serum albumin production: 80 μg / ml The transformant thus obtained was named TMS-33-1 strain.

[Vi] Screening of Transformant TMS-33-1 (Stability of LEU2 Gene and Production of HSA) When the HSA gene was introduced by Southern blotting, it was confirmed that LEU in the chromosome was
It was confirmed that it was introduced into two regions. When the stability of the HSA gene was measured using the HSA production amount and leucine non-requirement as indices, 100% of the HSA gene was retained even after culturing for about 60 generations in a non-selective medium. Accordingly, it was confirmed that the TMS-33-1 strain had the HSA gene introduced into three places on the chromosome of the host yeast Saccharomyces cerevisiae AH22 strain, namely, the LEU2 region and the TRP1 region.

Further, a histidine auxotrophic mutant TMS-33-1h4 of TMS-33-1 was obtained in the following manner. That is, after the TMS-33-1 strain is grown overnight in a non-selective medium, the cells are collected, washed sufficiently, and then applied to a selection plate (that is, a plate made of a medium containing no histidine). The selection plate was cultured at 30 ° C., and the TMS-33-1h4
Acquired shares. When culturing the TMS-33-1h4 strain, various dye adsorbents were added to the culture solution, and the effect on the coloring of HSA was examined.

[Examples 1 to 6 and Comparative Example 1] [i] Medium YNB medium: Bacto-yeast nitrogen base
6.7 g (manufactured by Difco) was dissolved in 100 ml of distilled water and sterilized and filtered, and 20 g of glucose (Hansui Chemical) was dissolved in distilled water to make 900 ml, and then autoclaved. Glucose-ammonium acetate synthetic medium: Table 1 below
The composition described in 1.

[0029]

[Table 1]

[Ii] Culture (Preculture) An appropriate amount of the TMS-33-1h4 strain was added to YNB.
Inoculate the baffled Erlenmeyer flask containing the medium,
For 24 hours with shaking. (Main culture) The preculture was collected by centrifugation, suspended in 10 ml of sterilized water, and mixed with a glucose-ammonium acetate-based synthetic medium.
1 ml of the cell suspension was inoculated per 0 ml. 100ml
Was dispensed into baffled 300 ml Erlenmeyer flasks, and cultured with shaking at 30 ° C. and 125 rpm for 70 hours. At that time, various dye adsorbents shown in Table 2 (addition rate: 1.
(0 w / v%) or as a control without addition.

(Test Example) [i] Purification and concentration of culture supernatant After completion of the culture, each culture was sampled,
A part of the supernatant obtained by centrifugation at 00 rpm for 5 minutes was
It was subjected to SA concentration measurement. The remaining culture supernatant (about 100m
l) 1 g of filter cake containing Blue Cellulofine (washed sufficiently with physiological saline: manufactured by Seikagaku Corporation)
Was added and the albumin was adsorbed for 2 hours at room temperature. The blue cellulofine adsorbed with albumin was transferred to a mini column, washed with physiological saline, and eluted with 3 ml of 3M sodium thiocyanate. The eluate was concentrated with a concentrator (Centricon 30 (30K), manufactured by Amicon) to obtain a sample for measurement. [Ii] Measurement of HSA concentration Using the culture supernatant, reverse passive sheep hemagglutination (RPH
The HSA concentration in the culture supernatant was indicated by comparison with the standard HSA (manufactured by Miles). [Iii] Comparison of coloring degree The wavelengths of the purified and concentrated specimens are 280 nm, 350 nm and 40 nm.
The absorbance at 5 nm was measured and 350 nm and 280 n
The absorbance ratio with m and the absorbance ratio between 405 nm and 280 nm were determined, and these values were used as indexes of the degree of coloring.

The above results are shown in Table 2 below.

[0033]

[Table 2]

Anion exchanger: 1.0% 1 × 8, DOWEX
Hydrophobic carrier: 1.0% XAD-2, manufactured by Rohm and Haas Company Mixture A: Anion exchanger / hydrophobic carrier (1 /
1) Cation exchanger: 1.0% 50W-X8, manufactured by DOWEX Mixture B: Cation exchanger / anion exchanger (1/1) Mixture C: Cation exchanger / hydrophobic Carrier (1 /
1) Mixture D: the above cation exchanger / the above anion exchanger /
The addition ratio of each mixture of the hydrophobic carrier (1/1/1) is 1.0 w / v%.

Example 7 [i] Medium Medium for batch culture: Composition shown in Table 3. Feed culture medium: composition shown in Table 4.

[0036]

[Table 3]

[0037]

[Table 4]

[Ii] Culture (preculture) 1 ml of glycerol frozen stock strain (OD
₅₄₀ = 10) was inoculated into a baffled Erlenmeyer flask containing a YNB medium and cultured with shaking at 30 ° C for 24 hours. After centrifugation, the cells were suspended in sterile water and inoculated into a 4 L batch (batch) culture medium. (Main culture) Using a 10 L mini-jar fermenter, aeration and agitation culture was performed. The aeration was set to 1 vvm, and the stirring speed was controlled so that the dissolved oxygen concentration became 10 ppm or more. The pH can be adjusted by adding 28% aqueous ammonia.
The constant value was controlled to H5.8. The defoaming was carried out by adding a small amount of an antifoaming agent (ADEKANOL, manufactured by Asahi Denka Kogyo) as needed. Further, 4 L of a feed medium was added by a control program so that the specific growth rate became 0.12. (Program for culture control) The program was used to control the flow acceleration of the feed medium during the culture. This program usually determines the feed rate of the feed medium so that the specific growth rate becomes 0.12 (1 / hr). When the dissolved oxygen concentration becomes 2 or less during the culture control, the specific growth rate is increased. Set the speed to 0
To perform constant-rate fed-batch culture. (Dye adsorbent) 8 so that the final concentration of the activated carbon powder (manufactured by Wako Pure Chemical Industries) becomes 0.1 w / v% (8 g / 8 L).
g was added to the batch culture medium. In addition, culture was performed without adding activated carbon as a control.

(Test Example) [i] Measurement of Cell Concentration A culture solution was sampled at an arbitrary culture time, diluted appropriately with distilled water, and then spectrophotometer (UV24, manufactured by Shimadzu Corporation).
The absorbance at 540 nm was measured using a (type 0), and the dry cell weight was determined from a previously determined calibration curve. The measurement of the HSA concentration and the comparison of the degree of coloring were performed in the same manner as described above. However, the HSA concentration in the sample was expressed in mg / L. The results are shown in Table 5 below.

[0040]

[Table 5]

The culture continued smoothly even under the condition of adding 0.1 w / v% of activated carbon.
-DCW / L, HSA production reached 800 mg / L.
That is, even if activated carbon is added at 0.1 w / v% (1 g / L), culturing can be performed in the same manner as normal culturing.
Moreover, the coloring degree of the obtained HSA is 1 to 2 of the normal one.
%.

[0042]

According to the method for suppressing coloring used in the present invention, the degree of coloring of HSA expressed by genetic manipulation can be suppressed to about 1/70 to 1/10 of that of untreated HSA. Further, the recovery rate of HSA is good, and the intrinsic properties of HSA do not change at all. In particular, the method of adsorbing the coloring component on the dye adsorbent is simple and industrially efficient. Therefore, the HSA-containing composition of the present invention can be used as a clinically useful pharmaceutical, like HSA derived from plasma.

[Brief description of the drawings]

FIG. 1 (a) and (b) show plasmids pMM-006 and pHO-011, respectively.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Wataru Otani 2-1-1180 Invitation Otani, Hirakata City, Osaka Prefecture Inside the Midori Cross Research Institute, Inc. (72) Inventor Naoto Furuhata 21-1180 Invitation Otani, Hirakata City, Osaka Prefecture (1) Inside the Midori Cross Research Institute, Inc. (72) Inventor Kaoru Kobayashi 2-1-16-1 Shodai Otani, Hirakata City, Osaka Prefecture (72) Inside the Midori Cross Research Institute Co., Ltd. (72) Shinobu Kuwae 2-Chome Otani, Hirakata City, Osaka Prefecture 1180-1 Inside Midori Cross Central Research Institute Co., Ltd. (72) Inventor Hirotoshi Fukuzuka 2-1-1, Otani, Hirakata-shi, Osaka Prefecture In-house Midori Cross Central Research Institute Co., Ltd. (72) Inventor Tomoshi Oya, Hirakata-shi, Osaka Prefecture 2-180, Otani 1 Inside the Green Cross Research Institute, Inc. (72) Inventor Hiroshi Morise, Osaka, Japan 2-1-1180 Ogata, Higashi-shi F-term in Midori Cross Research Laboratory Co., Ltd. (Reference) 4B024 AA01 BA80 CA04 DA12 EA04 GA11 HA01 4B064 AG24 CA06 CC24 CE09 CE10 CE11 DA01 4C084 AA06 CA03 DA37 ZA52 4H045 AA30 CA40 DA70 EA34 FA74 GA74 GA23

Claims (4)

[Claims] When culturing a human serum albumin-producing host prepared by genetic manipulation to express human serum albumin, at least one selected from the group consisting of an anion exchanger, a hydrophobic carrier and activated carbon is added. Culturing the host in a cultured medium, wherein the human serum albumin and the coloring component are separated from each other before the human serum albumin and the coloring component are bound to each other. A composition containing human serum albumin expressed by genetic manipulation, to which no coloring component is bound, obtained by the method described in (1). 2. The coloring degree is from 0.0035 to 0.0163 at an absorbance ratio of A _{350 nm} / A _{280 nm} and from 0.0018 to 0.0179 at an absorbance ratio of A _{405 nm} / A _{280 nm.}
A composition as described. 3. A human serum albumin-producing host is prepared by genetic manipulation, and the host is cultured in a culture solution to which at least one selected from the group consisting of an anion exchanger, a hydrophobic carrier and activated carbon has been added, A composition containing human serum albumin expressed by genetic manipulation, to which no coloring component is bound, which is obtained by a method for producing human serum albumin in which coloring is suppressed, comprising a step of expressing human serum albumin. 4. The coloring degree is from 0.0035 to 0.0163 at an absorbance ratio of A _{350 nm} / A _{280 nm} and from 0.0018 to 0.0179 at an absorbance ratio of A _{405 nm} / A _{280 nm.}
A composition as described.