JPH08259599A - De-fatted human serum albumin - Google Patents

Abstract

PURPOSE: To obtain a de-fatted human serum albumin having amount of bonded fatty acids equal to or smaller than a specific value by molar ratio, highly removing fatty acids derived from a raw material or secreted from microorganisms, having no side effects such as shock caused by impurities, useful for treating hypoproteinemia. CONSTITUTION: A recombinant plasmid incorporated with a gene encoding a human serum albumin(HSA) is introduced to Pichia pastoris GTS11S strain and transformed. The transformed body is cultured in a medium to produce HSA, the culture solution is pressed to separate the cultured supernatant liquid, which is successively treated by ultrafilters having 300,000 and 30,000 fractionated molecular weight and concentrated. The concentrated solution is successively treated by a cation exchanger, hydrophobic chromatography and an anion exchanger to give a recombinant HSA. Then, the HSA derived by gene manipulation is treated with a chelate resin prepared by bonding N-methylglucamine to a styrene-divinylbenzene copolymer to give the objective de-fatted human serum albumin having <=0.046 bonded fatty acid amount by molar ratio.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to delipidated human serum albumin, and more particularly to delipidated human serum albumin expressed by genetic engineering.

[0002]

2. Description of the Related Art Albumin, particularly human serum albumin (hereinafter referred to as HSA), is a major constituent of plasma proteins. This protein is produced in the liver, maintains a normal osmotic pressure mainly in the bloodstream, and has a function as a carrier for various serum molecules. For this reason,
The basic significance of HSA administration in clinical practice is to ameliorate the markedly decreased state of blood plasma protein amount, which is usually caused by a decrease in albumin. For this purpose, for example, frequent administration of HSA is carried out to shock patients and burn patients, and as a result, the reduced blood volume is restored and some symptoms related to trauma are improved. In addition, treatment with HSA is also effective for patients suffering from hypoproteinemia and erythroblastic fetal disease.

At present, HSA is mainly produced by fractionating blood collected from humans, but this method is difficult to supply blood, is uneconomical, and is a contaminant such as hepatitis virus. It has various drawbacks such as mixing of Therefore, it is considered clinically extremely beneficial to develop an alternative raw material for HSA.

Recently, a wide variety of useful polypeptides have been obtained from various microorganisms by recombinant DNA technology. The same applies to HSA, and a large amount of HSA is produced by the genetic engineering technology, which is highly advanced. Purification technology is also being established.

However, in genetic engineering, the phenomenon that the expressed HSA is adsorbed or bound to a fatty acid and its ester derived from the medium, or derived from the host, or secreted by the host is observed. In order to obtain high-purity HSA, it is required to reduce the amount of fatty acid and its esters bound to HSA.

Conventionally, for plasma-derived HSA, a method using activated carbon or the like has been used as a degreasing method (JP-A-53-53).
No. 127809) and a method using an anion exchanger (JP-A-3-81290). However, these methods do not give sufficiently satisfactory effects in terms of HSA recovery rate, degree of degreasing effect, etc., and cannot be said to be particularly effective even for HSA produced by genetic engineering. Is the actual situation.

[0007]

DISCLOSURE OF THE INVENTION The present invention relates to HSA, and in particular to HSA produced by genetic engineering, various fatty acids and their esters which could not be sufficiently removed by conventional methods for purifying plasma-derived HSA. Provides highly removed HSA.

[0008]

Means for Solving the Problems As a result of intensive studies conducted by the present inventors in view of the above-mentioned circumstances, as a result of treating HSA, particularly HSA produced by genetic engineering with a chelating resin, The inventors have completed the present invention by finding that the esters can be removed and HSA with high purity can be obtained with a high recovery rate.

That is, the HSA of the present invention has a bound fatty acid content of 0.046 mol or less, preferably 0.037 ± 0.009 mol per mol of albumin.

The HSA targeted by the present invention may be either plasma-derived or genetically engineered. Regarding HSA obtained by genetic manipulation, there is no particular limitation on the gene recombination manipulation used, such as expression system (eg, host, HSA expression vector, etc.), expression method, culture system, culture method, and the like.

Generally, the method for producing HSA by genetic manipulation basically comprises steps of constructing an HSA expression vector, producing a transformant, culturing the transformant, and the resulting HSA is a culture filtrate or a bacterium. They are collected from the body (cells) by known separation means. Specifically, the expression system, expression method, culture system and culture method described in JP-A-5-328991 can be used.

The HSA thus obtained is transferred to a purification step, and the treatment by the degreasing method described below, which can be employed in the present invention, is used in this purification step alone or in combination with other purification methods. For example, it is preferable to combine it with the following purification step. When combined with other purification methods, this purification method may be performed in any step thereof. Most preferably, it is done at the end of the purification steps described below.

(1) Purification of HSA Conventionally, as a method for purifying HSA produced by genetic engineering, known methods such as various fractionation methods, adsorption chromatography, affinity chromatography, gel filtration, density gradient centrifugation, dialysis and the like. Has been adopted. The treatment of HSA that can be adopted in the present invention by the degreasing method may be performed at any stage of the above-mentioned purification step, but is preferably performed at the end thereof.

The purifying step is, for example,
A process including is preferable. By culturing a human serum albumin producing host, a fraction containing human serum albumin (culture supernatant or a fraction obtained after disrupting the cultured cells) is cut off to a molecular weight of 100,000-5.
Treat with 0,000 and 1000-50,000 ultrafiltration membranes. Heat treatment is performed at 50 to 70 ° C. for 30 minutes to 5 hours. Acid treatment at pH 3-5. Treatment is carried out 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 0.2M, elution is performed under conditions of pH 8 to 10 and salt concentration of 0.2 to 0.5M. The non-adsorbed fraction is recovered by contacting the carrier for hydrophobic chromatography under the conditions of pH 6 to 8 and salt concentration of 0.01 to 0.5M, and pH 6 to 8 and salt concentration of 0.01 to 0.1M. The non-adsorbed fraction is collected by contacting the anion exchanger under the conditions.

Further, instead of the above step, a pH of 6 to
8. After contacting the carrier for hydrophobic chromatography under the condition of salt concentration of 1 to 3 M, pH 6 to 8 and salt concentration of 0.01 to 0.5
The step of eluting under the condition of M, or instead of the above step, after contacting with an anion exchanger under the conditions of pH 6-8, salt concentration 0.001-0.05M, pH 6-8, salt concentration 0 The step of eluting under a condition of 0.05-1M, and further during or after the step, p
Salting out under the conditions of H3-5 and salt concentration 0.5-3M,
It may further include a step of collecting the precipitated fraction.

[0016] Regarding plasma-derived HSA, an ordinary method can be used for preparing HSA from plasma.
The treatment of HSA that can be employed in the present invention by the degreasing method may be performed at any stage of its preparation and purification steps, and is preferably performed at the end thereof.

(2) Degreasing of HSA The HSA degreasing method that can be used in the present invention is carried out by bringing the HSA sample obtained in the purification step into contact with a chelating resin having a specific ligand part.

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 portion 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 (polyalkylene groups such as polyethylenepolyamine are included therein). Polyamine groups are also included), and thiourea groups. As a commercial product of the chelate resin having the carrier part and the ligand part, DIAION CRB02 (ligand part; N-methylglucamine group, manufactured by Mitsubishi Kasei Co., Ltd.) in which the carrier part is a copolymer of styrene and divinylbenzene ,
DIAION CR20 (ligand moiety; -NH- (CH ₂ CH ₂ N
H) n-H, manufactured by Mitsubishi Kasei Corporation), LEWATIT TP (ligand portion; -NHCSNH _2, manufactured by Bayer), Amberlite CG4000 is preferably used.

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

The total amount of fatty acids and their esters (hereinafter referred to as the amount of bound fatty acids) adsorbed or bound to HSA by the above-mentioned chelating resin treatment is the amount of HSA obtained through the steps including the above-mentioned and salting-out treatment. The amount of bound fatty acid is reduced to 1/10 or less, preferably 1/100 or less. Specifically, the amount of bound fatty acid to HSA is 0.046 or less, preferably 0.037 in terms of molar ratio.
It becomes ± 0.009.

The amount of fatty acid bound to HSA can be measured by a known method. Examples of this method include Duncombe extraction method, acyl-CoA synthetase (ACS) and acyl-CoA oxidase (ACOD).
The ACS-ACOD method using is used.

The extraction method of Duncombe is based on the measurement principle of converting a fatty acid into a copper salt with a copper reagent, extracting with chloroform, and then developing a color with a bank plane. A measuring kit such as NEFA-Test Wako (Wako Pure Chemical Industries, Ltd.) It can be easily carried out by using a product manufactured by Co., Ltd., etc. Further, the ACS-ACOD method has a measurement principle that peroxidase utilizes H ₂ O ₂ generated by a reaction between an acyl-CoA synthetase and an acyl-CoA oxidase and a fatty acid to develop color by utilizing oxidative condensation of a dye. It can be easily carried out by using a measurement kit such as NEFAC-Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.).

The objects to be defatted in the present invention are fatty acids and their esters, which are particularly based on raw materials for HSA production, such as those derived from blood, culture medium, host, or Including those secreted by the host.

Examples of the fatty acid include saturated fatty acids having 8 to 20 carbon atoms (eg palmitic acid, stearic acid) and unsaturated fatty acids having 16 to 20 carbon atoms (eg oleic acid, linoleic acid, arachidonic acid). To be
Examples of esters of these fatty acids include alkyl esters (methyl ester, ethyl ester, etc.) of these fatty acids, glycerin ester, and the like.

The above degreasing method is effective for removing the above fatty acids and their esters, and therefore is not limited to the origin of HSA, and the HSA to which those fatty acids and their esters are bound or adsorbed is adsorbed. It is a method that can be used for degreasing.

For clinical use of the HSA treated by the present degreasing method, for example, the HSA is a known method (ultrafiltration, addition of a stabilizer, sterile filtration, dispensing, lyophilization, etc.).
It is formulated by.

This HSA preparation is a preparation that is sufficiently compatible with clinical use, and can be used as an injection in the same manner as the plasma-derived HSA preparation. It can also be used as a stabilizer, carrier or carrier for pharmaceuticals.

[0029]

EXAMPLES In order to explain the present invention in more detail, examples will be given, but the present invention is not limited thereto.

Reference Example 1 Cultivation of HSA-producing host (1) Strains used: Pichia pastoris GCP101 strain Pichia pastoris GTS11 was prepared by the method described in JP-A-2-104290.
The AOX ₁ gene region of 5 (his4) was replaced with a fragment cleaved with Not1 of plasmid pPGP1 having a transcription unit expressing HSA under the control of AOX ₁ promoter to obtain PC4130. This strain is AOX ₁
Due to the absence of the gene, the ability to grow in a medium containing 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, initial OD ₅₄₀ = 0.1.
To 50 ml of YPD medium. 3 days 30
YPD so that the initial OD ₅₄₀ = 0.1 after culturing at ℃
50 ml of medium was inoculated. The same passage was repeated every 3 days. The cells were diluted with sterilized water so that the number of cells was 10 ⁷ cells / plate at each passage, and 2% MeOH-YNBw / oa.
a. Plate (0.7% yeastite rosin ene base with out amino acid, 2% methanol, 1.5
% Agar powder was applied and cultured at 30 ° C. for 5 days to determine the presence or absence of colonies. As a result, 2 applied after culturing for 12 days
% MeOH-YNBw / oa. a. The plate yielded 20 colonies. Mut-strains can hardly grow on this plate, while Mut + strains can grow. That is, the fact that colonies were formed on this plate indicates that the assimilation ability of methanol was increased and a strain converted to Mut + was obtained. One of the resulting colonies was appropriately diluted with sterile water and diluted with 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-preculture) 20 ml of 1 ml of glycerol frozen stock strain was used.
Baffled 1,000 with 0 ml YPD medium (Table 1)
The cells were inoculated into an Erlenmeyer flask of ml volume and cultured at 30 ° C for 24 hours with shaking.

[0033]

[Table 1]

(Pre-culture) A pre-preculture medium was inoculated into a 10-L jar fermenter containing 5 L of YPD medium, and aeration-agitation culture was carried out for 24 hours. Culture temperature is 30 ℃, aeration rate is 5L /
Minutes Further, the pH was not controlled in the preculture.

(Main culture) Medium for batch culture (Table 2) 25
The preculture liquid was inoculated into 0 L and cultured with aeration and stirring using a 1,200 L volume fermenter. The tank pressure is 0.5 kg / 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 m ² and the maximum aeration amount of 800 NL / min.
The addition of the feed medium (Table 3) was started when the glycerol in the medium was consumed in the batch culture. A computer was used to add this feed medium, and high-density culture was carried out while controlling so that methanol did not accumulate in the medium. The pH was controlled to a fixed value of pH 5.85 by adding 28% ammonia water. For defoaming, add an antifoaming agent (Adecanol, manufactured by Asahi Denka Co., Ltd.) to 0.3 at the start of batch culture.
It was carried out by adding 0 ml / L in advance and then adding a small amount as needed.

[0036]

[Table 2]

[0037]

[Table 3]

Reference Example 2 Mutant AOX2 isolated from GCP101 strain of Reference Example 1
Promoter [Natural AOX2 promoter (YEAST,
5, 167-177 (1988) or Mol. Cell, Biol., 9,1316-13
23 (1989)), the 255th base upstream of the start codon is T
To a C]] was used to construct a plasmid pMM042 for HSA expression, and Pichia pastoris (Pichia pas
storis) introduced into GTS115 strain and transformed into UHG4
2-3 strains were obtained (Japanese Patent Laid-Open No. 4-299994). This UHG42-3 strain was cultured according to Reference Example 1 to obtain HSA.
Was produced.

Reference Example 3 Purification of HSA [i] Separation of culture supernatant to membrane fractionation (II) About 800 L of the culture solution obtained in Reference Example 1 or Reference Example 2
The culture supernatant was separated by pressing. The culture supernatant was treated with an ultrafiltration membrane having a molecular weight cut off of 300,000. Then
Use an ultrafiltration membrane with a molecular weight cut off of 30,000
Concentrated [membrane fraction (I)].

This concentrated solution was heated at 60 ° C. for 3 hours, then rapidly cooled to about 15 ° C., adjusted to pH 4.5, and again removed using an ultrafiltration membrane having a molecular weight cutoff of 300,000. [Membrane fractionation (II)]. Next, the buffer solution in the albumin solution was replaced with a 50 mM acetate buffer solution containing 50 mM sodium chloride, pH 4.5, using an ultrafiltration membrane having a molecular weight cut off of 30,000.

[Ii] Cation exchanger treatment 50 mM acetate buffer containing 50 mM sodium chloride, p
After adsorbing albumin on an S-sepharose packed column equilibrated with H4.5 and thoroughly washing with the same buffer,
0.1M phosphate buffer containing 0.3M sodium chloride,
Elution of albumin was performed at pH 9.

[Iii] Hydrophobic Chromatography Treatment The albumin solution eluted from the column packed with S-Sepharose was loaded onto 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 adsorbing phenylcellulofine.

The albumin that passed through the column was concentrated to a volume of about 50 L using an ultrafiltration membrane having a molecular weight cut off of 30,000, and the buffer solution in the albumin solution was adjusted to 50 mM phosphate buffer, pH 6.8. Exchanged.

[Iv] Anion exchanger treatment After the hydrophobic chromatography treatment, the concentrated and buffer-exchanged albumin solution was added to a column packed with DEAE-Sepharose equilibrated with 50 mM phosphate buffer, pH 6.8. . Under these conditions, albumin passed through the column without being adsorbed on DEAE-Sepharose.

[V] HSA salting-out treatment 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 allowed to settle. This precipitate was separated from the supernatant by centrifugation to remove impurities.

Example 1 Purified 20% obtained through Reference Examples 1 and 3 (or 2)
1 g of DIAION CRB02 (manufactured by Mitsubishi Kasei Co., Ltd.) was added to 1 ml of recombinant HSA, and the mixture was stirred at room temperature for 24 hours under conditions of pH 6.8 and ionic strength of 5 mmho. DIA
ION CRB02 is a chelate resin in which an N-methylglucamine group as a ligand is bound to a carrier part made of a styrene-divinylbenzene copolymer. Then, the resin was washed with distilled water, and the amount of bound fatty acid in the recovered HSA (the non-adsorbed fraction on the resin and the washed fraction was combined) was measured. In addition, as a control, the amount of bound fatty acid was similarly determined using the recombinant HSA that was not treated with the chelate resin in the purification step. The amount of bound fatty acid is measured by NEF
A-Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.) was used. The results are shown in Table 4. In the table, rHSA means HSA derived from genetic manipulation.

[0047]

[Table 4]

Example 2 In the treatment for 1 ml of 20% recombinant HSA performed in Example 1, the chelating resin DIAION CRB02 was used.
The same treatment as in Example 1 was performed except that DIAION CR20 (manufactured by Mitsubishi Kasei Co., Ltd.) was used instead of.
DIAIONCR20 is composed of a styrene-divinylbenzene copolymer carrier having a ligand of -NH- (CH).
₂ CH ₂ NH) n-H group is a chelating resin formed by bonding. The same result as in Example 1 was obtained by this treatment.

Example 3 In the treatment on 1 ml of 20% recombinant HSA performed in Example 1, the chelating resin DIAION CRB02 was used.
The same treatment as in Example 1 was performed except that LEWATIT TP214 (manufactured by Bayer) was used instead of.
LEWATTIT P214 is -NHC as a ligand in a carrier part composed of a styrene-divinylbenzene copolymer.
It is a chelating resin formed by bonding SNH ₂ groups. The same result as in Example 1 was obtained by this treatment.

[0050]

INDUSTRIAL APPLICABILITY According to the present invention, with respect to HSA, particularly HSA produced by genetic engineering, fatty acids and their esters derived from raw materials adsorbed and bound thereto or secreted by microorganisms are highly removed. . Therefore, it is possible to provide HSA in which fatty acids and their esters are not mixed, and it is possible to prevent the occurrence of side effects such as shock due to such contaminants.

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

Claims (3)

[Claims] 1. A human serum albumin having a molar amount of bound fatty acids of 0.046 or less. 2. The human serum albumin according to claim 1, wherein the amount of bound fatty acids is the total amount of fatty acids and their esters. 3. The human serum albumin according to claim 1 or 2, wherein the human serum albumin is derived from genetic engineering.