JPH0656883A - Method for defatting human serum albumin - Google Patents

Abstract

PURPOSE:To efficiently obtain human serum albumin for e.g. remedying hypoproteinosis by treating human serum albumin with a chelate resin having an exchange group such as polyol, polyamine or thiourea group to eliminate fatty acid(ester), etc., derived from microbes. CONSTITUTION:An aqueous solution of human serum albumin (HSA), esp. one prepared by such genetic engineering technique as to culture microbes transformed with a recombinant gene, is mixed with a chelate resin such as a styrene-divinylbenzene copolymer having, as a ligand, exchange group having chelate-forming ability such as polyol, polyamine or thiourea group to make contact treatment at pH3-9 under an ionic strength of 50 or lower mho for 1-6hr, thus effectively eliminating the fatty acid or ester thereof in the HSA to use the purified HSA for administration to patients with shocks, burns, hypoproteinosis, or fetal erythroblastosis, for remedying significant plasma protein-deficient symptoms.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for delipidating human serum albumin, particularly 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. Therefore, the basic significance of HSA administration in clinical practice is to improve the markedly decreased state of blood plasma protein amount, which is usually caused by the decrease of albumin. To this end,
Frequent administration of HSA, for example, to patients with shock and burns, restores the reduced blood volume and ameliorate some of the trauma-related symptoms.
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 is true for 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, there is a phenomenon that expressed HSA is adsorbed or bound to a fatty acid or its ester derived from a medium, or derived from a host, or secreted by a host. In order to obtain high-purity HSA, it is required to reduce the amount of fatty acid bound to HSA. Conventionally, for plasma-derived HSA, as a degreasing method, a method using activated carbon or the like (JP-A-53-127809) and a method using an anion exchanger (JP-A-3-81290) are known. 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 reality.

[0006]

DISCLOSURE OF THE INVENTION The present invention relates to HSA, in particular HSA produced by genetic engineering, which has been unable to be sufficiently removed by conventional methods for purifying plasma-derived HSA. The present invention provides a method for efficiently removing The method of the present invention is also excellent as a method for degreasing plasma-derived HSA.

[0007]

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 ester can be removed and HSA with high purity can be obtained with a high recovery rate.

That is, the present invention relates to a method for degreasing HSA, which comprises treating HSA, particularly HSA produced by genetic engineering, with a chelating resin.

The HSA targeted by the present invention may be 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 HSA production method by genetic engineering basically uses H
The HSA obtained as a result of the steps of constructing an SA expression vector, producing a transformant, culturing the transformant, etc.
Are collected from the culture filtrate, bacterial cells and cells by known separation means. Specifically, Japanese Patent Application No. 4-137
The expression system, expression method, culture system and culture method described in No. 250 can be used.

The HSA thus obtained is transferred to a purification step, and the treatment by the degreasing method of 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, the purification method of the present invention 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, etc. Has been adopted. HS of the present invention
The treatment of A by the degreasing method may be carried out at any stage of the above-mentioned purification step, but it is preferably carried out at the end thereof.

As the purification step, for example, the following steps
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.

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

(2) Degreasing of HSA The HSA degreasing method of 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 treatment in the present invention means a contact operation with the chelate resin.

The carrier portion of the chelate resin is preferably a carrier having hydrophobicity, and examples thereof include a copolymer of styrene and divinylbenzene and a copolymer of acrylic acid and methacrylic acid. On the other hand, the ligand part includes a polyol group such as N-methylglucamine group, an imino group, an amino group,
Examples thereof include polyamine groups having a plurality of ethyleneimino groups and the like in the molecule (including polyalkylenepolyamine groups such as polyethylenepolyamine) 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 -C
H ₂ -NH) nH, manufactured by Mitsubishi Kasei Co., Ltd., LEWATIT TP
(Ligand unit; -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 mho or less, preferably 1 to 10 m
ho. Mixing ratio: Resin 0.1 to 10 per 250 mg of HSA
0 g, preferably 1-10 g (wet weight).

HSA was treated by the chelating resin treatment of the present invention.
The amount of adsorbed fatty acid of the above is 1/10 or less, preferably 1/1
00 or less.

The amount of fatty acid or its ester adsorbed on HSA can be measured by a known method. Examples of this method include the Duncombe extraction method and the ACS-ACOD method using acyl-CoA synthetase (ACS) and acyl-CoA oxidase (ACOD).

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 is based on the principle that peroxidase utilizes H ₂ O ₂ generated by the reaction of acyl-CoA synthetase and acyl-CoA oxidase with a fatty acid to develop a color by using 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 target of the degreasing method of the present invention is a fatty acid or its ester, which is particularly based on a raw material for the production of HSA, 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). INDUSTRIAL APPLICABILITY The method of the present invention is effective for removing the above fatty acid or its ester, and thus is not limited to the origin of HSA,
HS bound to those fatty acids or their esters
This is a method that can be used for degreasing A.

In clinical use of HSA treated by the method of the present invention, for example, the HSA can be prepared by known methods (ultrafiltration, addition of stabilizer, sterile filtration, dispensing, lyophilization, etc.).
It is formulated by. This HSA preparation 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.

[0024]

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 GTS115 was prepared by the method described in JP-A-2-104290.
The AOX ₁ gene region of (his4) was replaced with the fragment cut with Not1 of the plasmid pPGP1 having a transcription unit expressing HSA under the control of the AOX ₁ promoter to obtain PC4130. This strain has a low growth ability in a medium containing methanol as a carbon source due to the absence of the AOX ₁ gene (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 12 days passage
% 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
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.

[0028]

[Table 1]

(Pre-culture) A pre-preculture medium was inoculated into a 10-L jar fermenter containing 5 L of YPD medium, and agitated and cultured 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 set to 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.

[0031]

[Table 2]

[0032]

[Table 3]

Reference Example 2 Mutant AOX2 isolated from the 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 Application No. 3-63599). Reference example 1
This UHG42-3 strain was cultivated according to the above to produce HSA.

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
Approximately 80 liters of liquid using an ultrafiltration membrane with a molecular weight cut off of 30,000
Concentrated [membrane fraction (I)]. This concentrated solution is at 60 ° C for 3
After heat treatment for a period of time, the temperature is rapidly cooled to about 15 ° C., and the pH is 4.
It was adjusted to 5 and again removed using an ultrafiltration membrane having a molecular weight cutoff of 300,000 [membrane fraction (II)]. Then, using an ultrafiltration membrane with a cut-off molecular weight of 30,000, the buffer solution in the albumine solution was exchanged with a 50 mM acetate buffer solution containing 50 mM sodium chloride, pH 4.5.

[Ii] Treatment with cation exchanger 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 replaced with a 50 mM phosphate buffer solution, pH 6.8.

[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, and then HSA was added.
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. DI
AION 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. afterwards,
After washing the resin with distilled water, the amount of the bound fatty acid or ester of the recovered HSA 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 was measured using NEFA-Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.). The results are shown in Table 4. In the table, rHSA means HSA derived from genetic manipulation.

[0040]

[Table 4]

Example 2 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 DIAION CR20 (manufactured by Mitsubishi Kasei Co., Ltd.) was used instead of.
DIAIONCR20 is a styrene - -NH as ligands to a carrier part made of divinylbenzene copolymer (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 carried out 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.
LEWATTTP214 is -NHCSNH as a ligand on the carrier part composed of styrene-divinylbenzene copolymer.
It is a chelating resin composed of _two bonded groups. The same result as in Example 1 was obtained by this treatment.

[0043]

INDUSTRIAL APPLICABILITY According to the present invention, with respect to HSA, particularly HSA produced by genetic engineering, it is possible to efficiently remove fatty acids or their esters derived from raw materials or secreted by microorganisms that are adsorbed or bound thereto. it can. Therefore, HSA free from fatty acids or their esters
Can be provided.

Front Page Continuation (72) Inventor Takao Omura Invited 2-25-1 Otani, Hirakata City, Osaka Prefecture Midori Cross Central Research Institute Co., Ltd.

Claims (2)

[Claims] 1. A method for degreasing human serum albumin, which comprises treating human serum albumin with a chelating resin. 2. The degreasing method of human serum albumin according to claim 1, wherein the chelate resin has as a ligand an exchange group having a chelating ability selected from a polyol group, a polyamine group and a thiourea group.