JP2885212B2 - Highly purified human serum albumin-containing composition obtained from human serum albumin derived from genetic engineering - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a highly purified human serum albumin-containing composition obtained from genetically engineered human serum albumin, and in particular, to a method for purifying human serum albumin derived from genetically engineered by combining specific steps. The present invention relates to a highly pure human serum albumin-containing composition that can be obtained.

[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 basic therapeutic significance of administering HSA lies in treating conditions where there is fluid loss from the blood vessels, such as in surgery, shock, burns, and hypoproteinemia causing edema. .

[0003] Currently, HSA is produced primarily as a product from a fraction of the collected blood. Disadvantages of this process are that it is uneconomical and that the supply of blood is difficult. Blood may also contain undesired substances such as hepatitis virus. Therefore, HSA
It would be beneficial to develop alternative feedstocks.

By the way, the advent of recombinant DNA technology has enabled the production of a wide variety of useful polypeptides by microorganisms, and many mammalian polypeptides have already been produced by various microorganisms. About HSA,
The technology of mass production by the technology of genetic manipulation and the highly purified technology is being established.

Various studies have been made on a method for isolating and purifying HSA from plasma and have been put to practical use. For example, corn ethanol fractionation, PEG fractionation, and ammonium sulfate fractionation are known. More recently, a method of combining anion exchanger treatment with heat treatment at 60 ° C. for 10 hours (Japanese Patent Laid-Open No. 2-191226), anion exchanger treatment, cation exchanger treatment, and heating at 60 ° C. for 10 hours A method of combining processing (Japanese Patent Laid-Open No. 3-17123) and the like have also been developed.

[0006]

However, in order to purify HSA obtained by genetic manipulation, it is not appropriate to directly apply the above-mentioned method using plasma-derived HSA. This is because the contaminants to be removed are plasma-derived HS.
This is because A is completely different from HSA derived from genetic engineering. There is also a problem specific to HSA derived from genetic manipulation, that is, removal of coloring substances. Therefore, an object of the present invention is to provide an HSA which is obtained by genetic manipulation, does not contain a production host-related component or other contaminant components, and is sufficiently suppressed in coloring.

[0007]

Means for Solving the Problems In view of the above circumstances, the present inventors have intensively studied and as a result, have developed a method for efficiently purifying HSA obtained by genetic manipulation, and have thus obtained a high-purity gene. The present invention was completed by successfully producing an HSA-containing composition derived from an operation. That is, the present invention provides a human serum albumin-containing composition containing human serum albumin having a molecular weight of 67,000 obtained by genetic manipulation and having the following characteristics: (1) a dimer or polymer of human serum albumin Or (2) substantially free of contaminant components or polysaccharides derived from the production host. In particular, the present invention further provides that the solution of the composition containing human serum albumin comprises (i) A ₃₅₀ /
A ₂₈₀ is 0.01 to 0.05, is the human serum albumin-containing composition characterized in that it has a (ii) A ₄₅₀ / A ₂₈₀ is the coloring degree of 0.001 to 0.02. In particular, the present invention is the above-mentioned composition containing human serum albumin, further comprising no human-derived component other than human serum albumin. The human serum albumin-containing composition can be obtained by, for example, the following first to fourth methods, but is not limited thereto.

[0008] That is, a first method is a method for producing human serum albumin, which comprises purifying human serum albumin obtained by genetic manipulation using a process comprising the following steps: The culture supernatant is treated using an ultrafiltration membrane having a molecular weight cutoff of 100,000 to 500,000 and 1,000 to 50,000. The heat treatment is performed at 50 to 70 ° C. for 30 minutes to 5 hours. Acid treatment at pH 3-5. The treatment is performed using an ultrafiltration membrane having a molecular weight cutoff of 100,000 to 500,000. After contacting with a cation exchanger under conditions of pH 3 to 5 and salt concentration of 0.01 to 0.2 M, elution is performed under conditions of pH 8 to 10 and salt concentration of 0.2 to 0.5 M. The non-adsorbed fraction is collected by contacting with a carrier for hydrophobic chromatography under conditions of pH 6 to 8 and a salt concentration of 0.01 to 0.5M. Contacting the anion exchanger under conditions to collect the non-adsorbed fraction,
It is.

In the second method, instead of the step of the first invention, after contacting with a carrier for hydrophobic chromatography under conditions of pH 6 to 8 and salt concentration of 1 to 3 M, the pH is adjusted to 6 to 8
8. A method for producing human serum albumin, which comprises a step of elution under conditions of a salt concentration of 0.01 to 0.5M.

In a third method, a pH of 6 to 8 and a salt concentration of 0.001 to 0.001 are used instead of the steps of the first invention.
This is a method for producing human serum albumin, comprising a step of eluting under conditions of pH 6 to 8 and a salt concentration of 0.05 to 1 M after contacting with an anion exchanger under 0.05 M conditions.

Further, a fourth method comprises the steps of: starting with, stopping, or after the step of the first invention.
This method is a method for producing human serum albumin, which further comprises a step of subjecting to salting out under conditions of a salt concentration of 0.5 to 3 M and collecting a precipitated fraction.

(1) HSA Obtained by Genetic Manipulation The HSA-producing host prepared by genetic engineering used in the present invention is not particularly limited as long as it is prepared through genetic engineering. In addition to the above, even those that will be developed in the future can be used as appropriate. Specific examples include bacteria (eg, Escherichia coli, yeast, Bacillus subtilis, etc.) and animal cells that have been made HSA-producing through genetic manipulation. In particular, in the present invention, as a host, yeast, especially Saccharomyces (eg, Saccharomyces cerevisiae)
Or the genus Pichia (eg, Pichia pastoris) is preferably used.
In addition, auxotrophic strains and antibiotic-sensitive strains can be used.
Furthermore, Saccharomyces cerevisiae AH22 strain (a, his 4, leu 2, can 1), Pichia pastoris GT
S115 strain (his 4) and the like are preferably used.

A method for preparing these HSA-producing hosts, a method for producing HSA by culturing them, and a method for preparing HS from cultures
All the methods for separating and collecting A are carried out by employing a known method or an equivalent method. For example, HSA
Examples of the method for preparing a production host (or HSA-producing strain) include, for example, a method using a normal human serum albumin gene (JP-A-58-56684, 58-90515, and 58-150517) and a novel human serum. A method using an albumin gene (JP-A-62-29985 and JP-A-1-98486), a method using a synthetic signal sequence (JP-A-1-240191), and a method using a serum albumin signal sequence (JP-A-2-294). 167095
Japanese Patent Application Laid-Open No. 3-72889), a method of fusing hosts with each other (Japanese Patent Application Laid-Open No. 3-53877), a method of causing mutation in a methanol-containing medium, a method using a mutant AOX ₂ promoter (Japanese Patent Application No. 3-63598, the 3-
No. 63599), expression of HSA by Bacillus subtilis (JP-A-62-25133), expression of HSA by yeast (JP-A-60-41487, JP-A-63-39576, JP-A-63-59576).
3-74493), expression of HSA by Pichia yeast (JP-A-2-104290), and the like.

Among them, a method for causing a mutation in a medium containing methanol is specifically performed as follows. That is, first, an appropriate host, preferably Pichia yeast, specifically, the GTS115 strain (NRRL accession number Y-15851)
Obtain a transformant HSA by a conventional method to AOX ₁ gene region under AOX ₁ promoter governing of introducing a plasmid having a transcription unit to express (JP 2-104
290). This transformant has a low ability to grow in a methanol medium. Therefore, this transformant is cultured in a medium containing methanol to cause 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 conditions are 1
5 to 40 ° C. and about 1 to 1000 hours are exemplified.

As a method for culturing an HSA-producing host (that is, a method for producing HSA), in addition to the methods described in the above publications, fed batch culture is used to supply a small amount of high-concentration glucose in an appropriate amount. A method of obtaining a high concentration of cells and a product by avoiding high-concentration substrate inhibition of the producing cells (Japanese Patent Application No. 1-219561), and a method of enhancing the production of HSA by adding a fatty acid to the medium. (Japanese Patent Application No. 3-
81819) and the like. Further, as a method for separating and collecting HSA, in addition to the methods described in the above publications, inactivation of protease by heat treatment (Japanese Unexamined Patent Publication No.
No. 103188), a method for suppressing coloration by separating HSA and a coloring component using at least one selected from the group consisting of an anion exchanger, a hydrophobic carrier and activated carbon (Japanese Patent Application Laid-Open No. 4-54198). Is exemplified.

The medium used for culturing the transformed host includes:
Usually, a culture medium known in this field to which a fatty acid having 10 to 26 carbon atoms or a salt thereof has been added is used, and the culture is carried out in accordance with a general ordinary method. The medium may be a synthetic medium or a natural medium, and a liquid medium is preferred. For example,
As a synthetic medium, generally, various sugars as a carbon source, urea, ammonium salt, nitrate, and the like as a nitrogen source, various vitamins and nucleotides as a micronutrient, and Mg, Ca, Fe, Na, K, Mn, Co as inorganic salts. , Cu
And the like. YNB liquid medium [base of 0.7% yeast nitrogen (manufactured by Difco), 2% glucose] and the like. As a natural medium, YPD
A liquid medium [1% yeast extract (manufactured by Difco), 2% bactopeptone (manufactured by Difco), 2% glucose] is exemplified. The pH of the medium may be neutral or weakly basic or weakly acidic. In the case of a methanol-assimilating host, a medium containing methanol can be used. In this case, the methanol concentration is about 0.01 to 5%.

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

After completion of the culture, HSA is collected from the culture filtrate, the cells, and the cells by a known separation means.

(2) Purification of HSA (i) Ultrafiltration In this step, HSA was isolated from the culture supernatant from which the HSA-producing host was separated.
This is a step of separating and removing high molecular weight substances and low molecular weight substances other than SA by ultrafiltration. For the removal of high molecular weight substances, an ultrafiltration membrane having a molecular weight cut off of about 100,000 to 500,000, preferably about 300,000, and for the removal of low molecular weight substances, a molecular weight cut off of about 1,000 to 50,000, preferably 10,000 to 50,000. Use about 30,000 ultrafiltration membranes. Removal of high molecular weight substances involves separation of the remaining HSA-producing host, and removal of low molecular weight substances involves simultaneous concentration of the liquid volume.

(Ii) Heat treatment The concentrated solution obtained in (i) is heated at 50 to 70 ° C. for about 30 minutes to 5 hours,
Preferably, heat treatment is performed at 60 ° C. for about 1 to 3 hours. This step is preferably performed in the presence of a stabilizer. Examples of the stabilizer include acetyltryptophan, an organic carboxylic acid having 6 to 18 carbon atoms or a salt thereof, and the like. Both may be used in combination. The addition amount of acetyltryptophan is, for example, about 1 to 100 mM. Examples of the organic carboxylic acid having 6 to 18 carbon atoms include caproic acid (6 carbon atoms), caprylic acid (8 carbon atoms), capric acid (10 carbon atoms), lauric acid (12 carbon atoms), and palmitic acid (16 carbon atoms). ), Oleic acid (C18) and the like. Examples of the salt include an alkali metal salt (eg, sodium salt, potassium salt, etc.) and an alkaline earth metal salt (eg, calcium salt, etc.). The addition amount of the organic carboxylic acid having 6 to 18 carbon atoms or a salt thereof is, for example, about 1 to 100 mM. During the heat treatment, a thiol-based compound (eg, mercaptoethanol, cysteine, reduced glutathione, etc.) is added in an amount of about 1 to 100 mM, preferably 5 to 30 mM.
By adding 10 mM to 1000 mM of ammonium guanidine, coloring caused by heating can be suppressed. This step is partially known (JP-A-3-103188).

(Iii) Acid treatment The pH of the treatment solution of (ii) is about 3 to 5, preferably 4 to 4.
Adjust to about 6 and let stand for about 1 to 12 hours.

(Iv) Ultrafiltration This step is a step of separating and removing polymerized contaminants by ultrafiltration. To remove the high molecular weight substance, an ultrafiltration membrane having a molecular weight cutoff of about 100,000 to 500,000, preferably about 300,000 is used. Further, if necessary, buffer exchange for the next step of cation exchanger treatment may be performed using an ultrafiltration membrane having a molecular weight cut-off of about 1,000 to 50,000, preferably about 10,000 to 30,000. Can be.

(V) Cation exchanger treatment Examples of the cation exchanger include a sulfo group type and a carboxyl group type. Examples of the sulfo group type include sulfo-agarose (trade name: S-Sepharose, manufactured by Pharmacia), sulfopropyl-dextran (trade name: SP-Sephadex, manufactured by Pharmacia), sulfopropyl-
Polyvinyl (trade name: SP-Toyopearl, manufactured by Tosoh Corporation) and the like are exemplified. Examples of the carboxyl group type include carboxymethyl-dextran [trade name: CM-Sephadex (manufactured by Pharmacia), trade name: CM-Cellulofine (manufactured by Seikagaku Corporation)] and the like. The contact and washing conditions include a pH of about 3 to 5, preferably about 4 to 4.6, and a salt concentration of about 0.01 to 0.2 M, preferably 0.0
About 5 to 0.1 M is exemplified. The elution conditions are p
H8 to about 10, preferably about 8.5 to 9.5, salt concentration of about 0.2 to 0.5M, preferably 0.3 to 0.4M
The degree is exemplified.

(Vi) Hydrophobic chromatographic treatment Examples of the hydrophobic chromatographic carrier include an alkyl group having 4 to 18 carbon atoms (butyl group, octyl group, octyldecyl group, etc.) and a phenyl group type. You. As the butyl group type, butyl-agarose, butyl-polyvinyl (trade name: butyl-Toyopearl, manufactured by Tosoh Corporation), as the octyl group type, octyl-agarose, and as the octyl decyl group type, octyldecyl-agarose, phenyl group type Examples thereof include phenyl-cellulose (trade name: Phenylcellofine, manufactured by Seikagaku Corporation) and the like. In this step, HSA can be recovered in a non-adsorbed fraction. In that case, the contact conditions are about pH 6 to 8, preferably 6.5.
塩 7, salt concentration of about 0.01-0.5M, preferably about 0.05-0.2M. Further, HSA can be eluted after being adsorbed on the carrier. in this case,
The contact and washing conditions are about pH 6 to 8, preferably about 6.5 to 7, and a salt concentration of about 1 to 3M, preferably 1.
It is about 5 to 2M. The elution conditions are about pH 6 to 8, preferably about 6.5 to 7, and a salt concentration of 0.01 to 0.1.
It is about 5M, preferably about 0.05-0.2M.

(Vii) Anion exchanger treatment Diethylaminoethyl (DE) is used as the anion exchanger.
AE) type, quaternary aminoethyl (QAE) type and the like. As the DEAE base type, 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) ) Are exemplified. In addition, as the QAE base type, QAE-agarose (trade name: Q-Sepharose, manufactured by Pharmacia),
Examples include QAE-polyvinyl (trade name: QAE-Toyopearl, manufactured by Tosoh Corporation) and the like. In this step, HSA can be recovered in a non-adsorbed fraction. In that case, the contact conditions are:
The pH is about 6 to 8, preferably about 6.5 to 7, and the salt concentration is about 0.01 to 0.1M. Further, HSA can be eluted after being adsorbed on the carrier. in this case,
The contact and washing conditions include pH of about 6 to 8, preferably about 6.5 to 7, salt concentration of about 0.001 to 0.05M,
Preferably it is about 0.01 to 0.02M. The elution conditions are a pH of about 6 to 8, preferably about 6.5 to 7, and a salt concentration of about 0.05 to 1M.

(Viii) Salting-out treatment In this step, the salt concentration is 0.1 to 3M, preferably 0.5 to 3M.
A salt component is added to a pH of about 1.5 M
By adjusting to about 5, preferably about 3.5 to 4.5, HSA can be specifically precipitated, and by separating this, impurities other than HSA present in the supernatant can be separated. . The precipitated HSA is dissolved in a suitable buffer. The salt component for adjusting the ionic strength is not particularly limited, but includes sodium chloride, potassium chloride, ammonium sulfate, sodium (or potassium) thiocyanate,
Sodium sulfate and the like are suitable. The method of separating the HSA precipitate and the supernatant is not particularly limited, but centrifugation, compression separation, membrane separation by cross flow, and the like are suitable. This step is preferably carried out after the treatment with the anion exchanger in (vii), but between the treatment with the cation exchanger in (v) and the hydrophobic chromatography in (vi), or the treatment with hydrophobic chromatography in (vi). And (v
It may be performed during the anion exchanger treatment of ii).

(3) Formulation The obtained HSA can be formulated by a known method (ultrafiltration, addition of a stabilizer, sterilization filtration, dispensing, freeze-drying, etc.). The HSA preparation thus prepared can be used clinically as an injection as in the case of the plasma-derived HSA preparation. Further, it can also be used as a stabilizing agent, carrier or carrier for pharmaceuticals.

(4) Purified HSA derived from genetic engineering
HSA derived from the genetic engineering of the present invention has a molecular weight of about 67000
Is a single substance. The HSA-containing composition of the present invention may be a dimer, a polymer, or a decomposition product (having a molecular weight of about 43
000). Further, it does not substantially contain contaminant components and polysaccharides derived from the production host, particularly antigenic contaminant components and polysaccharides. Here, “substantially does not include” means below the detection limit in the detection method of the below-mentioned Example. In the case of a 25% HSA solution, A ₃₅₀ / A ₂₈₀ is 0.01 to 0.05 and A ₄₅₀ / A
₂₈₀ is about 0.001 to 0.02.

[0029]

According to the present invention, HSA obtained by genetic manipulation can be purified efficiently. Further, it is possible to provide an HSA which is obtained by genetic manipulation, does not contain a production host-related component or other contaminant components, and has sufficiently suppressed coloring.

[0030]

The present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto.

Reference Example 1 (1) Strain used: Pichia pastoris GCP101 strain Pichia pastoris GTS11 was prepared according to the method described in JP-A-2-104290.
The AOX ₁ gene region of the 5 (his4), and replacing the fragment was cut with Not1 plasmid pPGP1 having a transcription unit HSA is expressed under the AOX ₁ promoter dominant, PC4130 is obtained. This strain is AOX ₁
Due to the absence of the gene, the ability to grow on a medium using methanol as a carbon source is low (Mut- strain).

PC4130 was inoculated into 3 ml of YPD medium (1% yeast extract, 2% bactopeptone, 2% glucose), and after 24 hours, the initial OD _{540 was} 0.1.
Was inoculated into 50 ml of YPD medium. 3 days 30
YPD so that the initial OD ₅₄₀ = 0.1 after culturing at
The cells were inoculated into 50 ml of the medium. The same passage was repeated every three days. The cells were diluted with sterile water to 10 ⁷ cells / plate for each passage, and were diluted with 2% MeOH-YNBw / oa.
a. Plate (0.7% yeast nitrogen base with out amino acid, 2% methanol, 1.5%
% Agar powder) and cultured at 30 ° C. for 5 days to determine the presence or absence of colonies. As a result, 2% MeOH-YNBw / oa. a. 20 from the plate
Colonies resulted. Mut- strains can hardly grow on this plate, whereas Mut + strains can. That is, the formation of colonies in this plate indicates that the assimilation of methanol was increased and a strain converted to Mut + was obtained. One of the resulting colonies was appropriately diluted with sterile water to give 2% MeOH-YNBw / o.
a. a. It was spread on a plate and isolated into a single colony.
One of them was named GCP101.

(2) Culture of strain (pre-culture) 1 ml of glycerol frozen stock strain was added to 20
1,000 ml with baffle containing 0 ml YPD medium (Table 1)
The cells were inoculated into a ml Erlenmeyer flask and cultured with shaking at 30 ° C. for 24 hours.

[0034]

[Table 1]

(Pre-culture) A 10 L jar fermenter containing 5 L of YPD medium was inoculated with the pre-pre-culture liquid and cultured with aeration and stirring for 24 hours. The culture temperature was 30 ° C and the aeration rate was 5 L /
Minutes. In the pre-culture, the pH was not controlled.

(Main culture) Medium for batch culture (Table 2) 25
0 L of the preculture was inoculated and cultured with aeration and agitation using a 1,200 L fermenter. The tank pressure is 0.5kg / c
Batch culture was started while controlling the stirring speed so that the dissolved oxygen concentration was maintained at about 50% to 30% of the saturated dissolved oxygen concentration with the maximum aeration rate of 800 N-L / min and m ² .
At the time when glycerol in the medium was consumed in the batch culture, addition of the feed medium (Table 3) was started. A computer was used to add this feed medium, and high-density culture was performed while controlling so that methanol did not accumulate in the medium. The pH was controlled at a constant value of 5.85 by adding 28% aqueous ammonia. The defoaming is performed by adding an antifoaming agent (Adecanol, manufactured by Asahi Denka Kogyo) to 0.3% at the start of batch culture.
This was carried out by adding 0 ml / L and then adding a small amount as needed.

[0037]

[Table 2]

[0038]

[Table 3]

Reference Example 2 AOX2 promoter isolated from the GCP101 strain of Reference Example 1 [mutant type. Native AOX2 promoter (TEAST,
5, 167-177 (1988) or Mol.Cell, Biol., 9, 1316-1.
323 (1989)), in which the 255th base upstream of the initiation codon was mutated from T to C], to construct a plasmid pMM042 for HSA expression, and to obtain Pichia pastoris (Pichia pastoris).
pastoris) The transformant UHG was introduced into the GTT115 strain.
42-3 strains were obtained (Japanese Patent Application No. 3-63599). This UHG42-3 strain was cultured according to Reference Example 1 to produce HSA.

Example 1 [i] Separation of culture supernatant to membrane fractionation (II) The culture supernatant was separated by pressing about 800 L of the culture solution obtained in Reference Example 1. Culture supernatant was fractionated with molecular weight cutoff of 30.
Treated with 10,000 ultrafiltration membranes. Subsequently, the liquid volume was concentrated to about 80 L using an ultrafiltration membrane having a molecular weight cut-off of 30,000 [membrane fractionation (I)]. Subsequently, heat treatment was performed at 60 ° C. for 3 hours. Heat treatment is 5 mM sodium caprylate, 10 mM
Cysteine and p in the presence of 100 mM aminoguanidine
H7.5. The heat-treated solution was rapidly cooled to about 15 ° C. and adjusted to pH 4.5, and then the molecular weight cut off was again 30.
The membrane was treated using a 10,000 ultrafiltration membrane [membrane fractionation (II)].
Then, using an ultrafiltration membrane having a molecular weight cut off of 30,000, the buffer in the albumin solution was added to a buffer containing 50 mM sodium chloride.
Exchanged to 0 mM acetate buffer, pH 4.5.

[Ii] Cation exchanger treatment 50 mM acetate buffer containing 50 mM sodium chloride, p
After adsorbing albumin to the S-Sepharose packed column equilibrated with H4.5 and washing well with the same buffer,
0.1 M phosphate buffer containing 0.3 M sodium chloride,
Albumin was eluted at pH9. When the polysaccharide content before and after the treatment was measured by the phenol-sulfuric acid method, the content of the sample after the treatment was reduced to 1/20 of that before the treatment.

[Iii] Hydrophobic chromatographic treatment The albumin solution eluted from the S-Sepharose packed column was applied to a column packed with phenylcellulofine equilibrated with 50 mM phosphate buffer containing 0.15 M sodium chloride, pH 6.8. Was added. Under these conditions, albumin passed through the column without adsorption to phenylcellulofine. The volume of albumin that passed through the column was concentrated to about 50 L using an ultrafiltration membrane having a molecular weight cut off of 30,000, and the buffer in the albumin solution was replaced with a 50 mM phosphate buffer, pH 6.8.

[Iv] Anion exchanger treatment After the hydrophobic chromatography treatment, the albumin solution subjected to concentration and buffer exchange was added to a column packed with DEAE-Sepharose equilibrated with 50 mM phosphate buffer, pH 6.8. did. Under these conditions, albumin passed through the column without adsorption to DEAE-Sepharose.

[V] Salting out treatment of HSA A solution having a final concentration of 1 M by adding sodium chloride to 5% concentration of HSA was adjusted to pH 3.5 with acetic acid,
Was precipitated. This precipitate was separated from the supernatant by centrifugation to remove impurities. The precipitated albumin is dissolved, concentrated and buffer exchanged using an ultrafiltration membrane having a molecular weight cut off of 30,000, and if necessary, a stabilizer is added. can do.

[Vi] HPLC Analysis of Purification Step The HSA that had been subjected to the purification step up to the hydrophobic chromatography treatment was analyzed by HPLC gel filtration. Gel filtration analysis was performed under the following conditions. Column: TSK gel G3000SW (manufactured by Tosoh Corporation) Developing solution: 0.1 M KH ₂ PO ₄ /0.3 M NaCl buffer Detection: absorbance at a wavelength of 280 nm As shown in FIG. 1, purified HSA became a single peak of HSA monomer. .

[Vii] Analysis of Yeast-Derived Components A culture supernatant of a non-albumin-producing yeast was roughly purified in the same manner as in the present method, and a rabbit was immunized, and the resulting serum was used in a purified albumin solution using the obtained antiserum. Yeast-derived components were detected. The measurement was performed by an enzyme immunoassay (EIA method). Table 4 shows the results of detecting yeast-derived components in the sample after the salting-out treatment. The sample is used as albumin concentration
It measured using what was adjusted to 250 mg / ml.

[0047]

[Table 4]

The amount of the yeast-derived component in the albumin concentration of 250 mg / ml was 1,360 ng / ml for the sample after the hydrophobic chromatography treatment.
On the other hand, in the sample after the anion exchanger treatment, it was 5.4 ng / ml, which was reduced to 1/250. Furthermore, in the purified albumin after the salting-out treatment, no yeast-derived component was detected at a detection limit of 0.1 ng / ml.

Example 2 Properties of Purified HSA of the Present Invention (1) Molecular Weight The molecular weight was measured by the aforementioned HPLC gel filtration method. Departure
Light purified HSA has a molecular weight of about 67000,
It was about the same as the coming HSA. (2) Isoelectric point The isoelectric point was determined using Allen et al.
According to the method of [J. Chromatog., 146, 1 (1978)]
did. The isoelectric point of the purified HSA of the present invention is about 4.6,
It was comparable to plasma-derived HSA. (3) Degree of coloration Degree of coloration is absorbance at 280 nm, 350 nm, and 450 nm.
Measure the degree and A₃₅₀/ A₂₈₀, A₄₅₀/ A₂₈₀Is calculated
Was. The coloring degree of the purified HSA of the present invention is A₃₅₀/ A ₂₈₀Is about
0.02, A₄₅₀/ A₂₈₀Is about 0.01,
It was about the same as the coming HSA.

Example 3 The culture solution obtained in Reference Example 2 was treated in the same manner as in Example 1. The properties of the purified HSA were almost the same as the molecular weight, isoelectric point and coloring degree disclosed in Example 2, the polysaccharide content disclosed in Example 1, the gel filtration pattern, and the amount of yeast-derived components.

[Brief description of the drawings]

FIG. 1 shows the results of HPLC analysis after a hydrophobic chromatography purification step.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI // C12N 15/09 C12N 15/00 A (C12P 21/02 C12R 1:84) (72) Inventor Naoto Furuhata Hirakata-shi, Osaka 2-25-1, Invitation Otani Midori Cross Research Institute, Inc. (72) Inventor Kazuya Takeshima 2-25-1, Invitation Otani, Hirakata City, Osaka Prefecture Midori Cross Research Institute Incorporated (72) Inventor, Kanaeko Koide Osaka 2-25-1, Souida Otani, Hirakata City, Midori, Japan Midori Cross Central Research Institute Co., Ltd. (72) Inventor Munehiro Noda 2-25-1, Soudai Otani, Hirakata City, Osaka Prefecture, Japan Midori Cross Central Research Institute Co., Ltd. (72) Inventor Kondo Masahide 2-25-1, Souida Otani, Hirakata City, Osaka Prefecture Inside the Midori Cross Research Laboratory Co., Ltd. (72) Inventor Shoichi Ishikawa 2 Souida Otani, Hirakata City, Osaka Prefecture No. 25-1, Inside the Midori Cross Research Center Co., Ltd. (72) Inventor Kazuhiro Ohara Invitation to Hirakata City, Osaka Prefecture 2-25-1 Inside Midori Cross Research Center Co., Ltd. No. 25-1, Otani Inside Midori Cross Research Laboratory Co., Ltd. (56) References JP-A-5-317079 (JP, A) Curling (1980), “Albu min Purification by Ion Exchange Chromatography” in “Methods” of Plasma Protein Fractionation n ". Curling. J. M. Editing, Academic Press, London, p. 77-91 (58) Fields investigated (Int. Cl. ⁶ , DB name) C07K 14/765 C07K 1/18 C07K 1/30 C07K 1/36 C12P 21/00-21/02 C12N 15/14 BIOSIS (DIALOG ) WPI (DIALOG)

Claims (1)

(57) [Claims] 1. A molecular weight of 67,000 obtained by genetic manipulation
A composition containing human serum albumin having a uniform amino acid sequence of 1,000 and having the following characteristics: (1) substantially free of a dimer, polymer or degraded product of human serum albumin ( 2) Substantially free of contaminant components or polysaccharides derived from the production host. (3) Free of human-derived components other than human serum albumin and obtained by a purification method comprising the following steps:
Human serum albumin-containing composition which can be fractionated from the culture supernatant of a human serum albumin-producing host
Treatment is performed using ultrafiltration membranes of 300,000 particles and 30,000 particles. Heat treatment at 60 ° C. and pH 7.5 for 3 hours. Acid treatment at pH 4.5. The treatment is performed using an ultrafiltration membrane having a molecular weight cut off of 300,000. Concentration and buffer exchange were performed using an ultrafiltration membrane
Then, 50 mM acetate buffer containing 50 mM sodium chloride
Contact with the cation exchanger equilibrated with (pH 4.5)
And adsorbed fraction to 0.1 M containing 0.3 M sodium chloride.
Elute with phosphate buffer (pH 9). 50 mM phosphate buffer containing 0.15 M sodium chloride
Hydrophobic chromatography carrier equilibrated with buffer (pH 6.8)
And collect the non-adsorbed fraction. Concentration and buffer exchange were performed using an ultrafiltration membrane
Thereafter, equilibration was performed with a 50 mM phosphate buffer (pH 6.8).
Contacting with an anion exchanger to collect a non-adsorbed fraction,
Then, human serum albumin adjusted to a concentration of 5% was converted to 1M chloride.
Salting out under the condition of sodium, pH 3.5,
to recover.