JP3298405B2 - Defatted human serum albumin - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to is expressed by a gene manipulation, relates defatted human serum albumin.

[0002]

BACKGROUND OF THE INVENTION Albumin, especially human serum albumin (hereinafter referred to as HSA) is a major component of plasma proteins. This protein is produced in the liver and functions to maintain normal osmotic pressure mainly in the bloodstream and as a carrier for various serum molecules. For this reason,
The fundamental significance of HSA administration in the clinic is to improve the state of marked decrease in plasma protein in blood, which is usually attributed to a decrease in albumin. To this end, frequent administration of HSA has been performed, for example, in shock and burn patients, so that the reduced blood volume is restored and some trauma-related symptoms are also improved. HSA treatment is also effective for patients suffering from hypoproteinemia and fetal erythroblastosis.

At present, HSA is mainly produced by fractionating blood collected from humans. However, this method is difficult to supply blood, is uneconomical, and contains contaminants such as hepatitis virus. It has various disadvantages such as mixing of Therefore, developing an alternative source of HSA would be of great clinical benefit.

[0004] In recent years, a variety of useful polypeptides have been obtained from various microorganisms by recombinant DNA technology. The same applies to HSA. A large amount of HSA is produced by genetic engineering techniques, Refining technology is also being established.

[0005] However, in genetic engineering, a phenomenon is observed in which HSA expressed is adsorbed or bound to fatty acids and their esters derived from the medium or derived from the host, or secreted by the host. In order to obtain high purity HSA, it is necessary to reduce the amount of fatty acids and their esters bonded to HSA.

Conventionally, HSA derived from plasma has been defatted by a method using activated carbon or the like (Japanese Patent Application Laid-Open No.
127809) and a method using an anion exchanger (Japanese Patent Application Laid-Open No. 3-81290). However, these methods do not give sufficiently satisfactory effects in terms of the recovery rate of HSA, the degree of the defatting effect, etc., and cannot be said to be sufficiently effective especially for HSA produced by genetic manipulation. Is the actual situation.

[0007]

DISCLOSURE OF THE INVENTION The present invention relates to HSA, particularly HSA produced by genetic engineering, which comprises a wide variety of fatty acids and esters thereof which could not be sufficiently removed by conventional methods for purifying plasma-derived HSA. Provides HSA with a high degree of removal.

[0008]

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

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

[0010] HSA targeted by the present invention, heritage respect HSA obtained by gene manipulation, genetic recombination operations used, for example, the expression system (e.g. a host, such as HSA expression vector), expression methods, culture systems, and culture The method and the like are not particularly limited.

In general, a method for producing HSA by genetic manipulation basically comprises steps of constructing an HSA expression vector, preparing a transformant, culturing the transformant, and the like. 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 alone or in combination with another purification method in this purification step. For example, it is preferable to combine with the following purification step. When combined with another purification method, the present purification method may be performed in any of its steps. Most preferably, it is performed at the end of the purification step described below.

(1) Purification of HSA Conventional methods for purifying HSA produced by genetic manipulation include known methods such as various fractionation methods, adsorption chromatography, affinity chromatography, gel filtration, density gradient centrifugation, and dialysis. Has been adopted. The treatment by HSA degreasing method that can be employed in the present invention may be carried out at any stage of the above-mentioned purification step, but is preferably carried out last.

As the purification step, for example,
Preferably, a step including By culturing a human serum albumin-producing host, a fraction containing human serum albumin (culture supernatant, or a fraction obtained after crushing the cultured cells) was subjected to a fractionation molecular weight of 100,000 to 5%.
The treatment is carried out using 100,000 and 1,000 to 50,000 ultrafiltration membranes. 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. The non-adsorbed fraction is recovered by contacting with an anion exchanger under conditions.

[0015] In place of the above step,
8. After contacting with a carrier for hydrophobic chromatography under the condition of salt concentration of 1 to 3M, pH is 6 to 8, and salt concentration is 0.01 to 0.5.
M, or a step of elution under the conditions of pH 6 to 8 and a salt concentration of 0.001 to 0.05 M instead of the above step. Eluting under a condition of 0.05 to 1 M, and further, between, during or after the step,
H3-5, salting out under the condition of salt concentration 0.5-3M,
The method may further include a step of collecting the precipitate fraction.

With respect to HSA derived from plasma, an ordinary method can be used for preparing HSA from plasma.
The treatment of the HSA according to the degreasing method that can be employed in the present invention may be performed at any stage of the preparation and purification steps, and is preferably performed at the end.

(2) Defatting of HSA The defatting method of HSA that can be employed 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 part is a polyamine group having a plurality of polyol groups such as N-methylglucamine group, imino group, amino group, ethyleneimino group and the like in the molecule (including polyalkylene groups such as polyethylene polyamine). Polyamine groups), and thiourea groups. As a commercially available chelate resin having a carrier portion and a ligand portion, DIAION CRB02 in which the carrier portion is a copolymer of styrene and divinylbenzene (ligand portion: N-methylglucamine group, manufactured by Mitsubishi Chemical Corporation) ,
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 conditions for the treatment 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 to 10
mmho. Mixing ratio: Resin 0.1 to 10 with respect to 250 mg of HSA
0 g, preferably 1-10 g (wet weight).

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

The amount of fatty acid bound to HSA can be measured by a known method. This method includes, for example, Duncombe extraction method, acyl-CoA synthetase (ACS) and acyl-CoA oxidase (ACOD).
And the ACS-ACOD method using the same.

The Duncombe extraction method is based on the principle that a fatty acid is converted to a copper salt with a copper reagent, extracted with chloroform, and then developed with a bankplane to develop a color. A measurement kit such as NEFA-Test Wako (Wako Pure Chemical Industries, Ltd.) (Manufactured by Co., Ltd.) or the like. The ACS-ACOD method is based on the measurement principle that peroxidase utilizes H ₂ O ₂ generated by the reaction of an acyl-CoA synthetase and an acyl-CoA oxidase with a fatty acid for 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 subject of the present invention to be defatted are fatty acids and their esters, which are particularly based on raw materials for the production of HSA, for example those derived from blood, culture media, hosts, or Includes those secreted by the host.

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

The above-described degreasing method is effective for removing the above-mentioned fatty acids and their esters, and is therefore not limited to the origin of HSA, and the HSA to which these fatty acids and their esters are bound or adsorbed is not limited. This method can be used for degreasing.

When the HSA treated by the present defatting method is used, for example, in clinical use, the HSA can be obtained by a known method (ultrafiltration, addition of a stabilizer, sterilization filtration, dispensing, freeze-drying, etc.).
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 a plasma-derived HSA preparation. Further, it can also be used as a stabilizing agent, carrier or carrier for pharmaceuticals.

[0029]

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

Reference Example 1 Culture of HSA-producing host (1) Strain to be used: Pichia pastoris GCP101 strain Pichia pastoris GTS11 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, after the culture for 12 days,
% MeOH-YNBw / oa. a. The plate gave rise to 20 colonies. 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 frozen glycerol 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.

[0033]

[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 such that the dissolved oxygen concentration was maintained at about 50% to 30% of the saturated dissolved oxygen concentration at an m ² of 800 N-L / min and the maximum aeration rate.
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%
This was carried out by adding 0 ml / L and then adding a small amount as needed.

[0036]

[Table 2]

[0037]

[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 initiation codon is T
To C), a plasmid pMM042 for HSA expression was constructed, and Pichia pastoris (Pichia pa
storis) Introduced into GTS115 strain, transformant UHG4
2-3 strains were obtained (Japanese Unexamined Patent Publication No. 4-299984). This UHG42-3 strain was cultured according to Reference Example 1 and
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
Was pressed to separate the culture supernatant. The culture supernatant was treated with an ultrafiltration membrane having a molecular weight cut-off of 300,000. Then
Using an ultrafiltration membrane with a molecular weight cut off of 30,000, reduce the volume to about 80 L
[Membrane fraction (I)].

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

[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 adsorbing phenylcellulofine.

The volume of albumin that has passed through the column is 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 is reduced to 50 mM phosphate buffer, pH 6.8. Replaced.

[Iv] Treatment with anion exchanger 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 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% HSA was adjusted to pH 3.5 with acetic acid,
Was precipitated. 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 Corporation) was added to 1 ml of the 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 is bonded as a ligand to a carrier made of a styrene-divinylbenzene copolymer. Thereafter, the resin was washed with distilled water, and the amount of bound fatty acid in the recovered HSA (the non-adsorbed fraction to the resin plus the washed fraction) was measured. As a control, the amount of bound fatty acid was determined in the same manner by using a recombinant HSA not subjected to the above-mentioned chelate resin treatment in the purification step. NEF measurement of bound fatty acids
The test was performed using A-Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.). Table 4 shows the results. In the table, rHSA means HSA derived from genetic engineering.

[0047]

[Table 4]

Example 2 In the treatment of 1 ml of 20% recombinant HSA performed in Example 1, the chelating resin DIAION CRB02 was used.
Was performed in the same manner as in Example 1 except that DIAION CR20 (manufactured by Mitsubishi Kasei Corporation) was used instead.
DIAIONCR20 has a ligand of -NH- (CH) as a ligand on a carrier portion made of a styrene-divinylbenzene copolymer.
₂ CH ₂ NH) n-H group is a chelating resin. By this processing, the same result as in Example 1 was obtained.

Example 3 In the treatment of 1 ml of 20% recombinant HSA performed in Example 1, the chelating resin DIAION CRB02 was used.
Was performed in the same manner as in Example 1 except that LEWATIT TP214 (manufactured by Bayer) was used instead of
LEWATITTP214 is a compound composed of styrene-divinylbenzene copolymer and a ligand, -NHC as a ligand.
It is a chelate resin formed by bonding SNH ₂ groups. By this processing, the same result as in Example 1 was obtained.

[0050]

INDUSTRIAL APPLICABILITY According to the present invention, HSA, particularly HSA produced by genetic engineering, is highly removed from fatty acids and esters thereof derived from raw materials or secreted by microorganisms, which are adsorbed and bound thereto. . Therefore, it is possible to provide HSA free from fatty acids and esters thereof, and to prevent the occurrence of side effects such as shock due to such contaminants.

──────────────────────────────────────────────────続 き Continuation of the front page (72) Inventor Takao Omura 2-25-1, Otani, Hirakata City, Osaka Prefecture Inside the Midori Cross Research Institute, Inc. (56) References JP-A-3-81290 (JP, A) JP-A-63-88035 (JP, A) JP-A-3-190896 (JP, A) JP-A-59-130220 (JP, A) JP-A-2-11599 (JP, A) JP-A-2-174799 ( JP, A) JP-A-5-23195 (JP, A) JP-A-3-501323 (JP, A) Chromatography 421 p. 141-146 (1987) (58) Field surveyed (Int. Cl. ⁷ , DB name) C07K 1/14 C12N 15/00 BIOSIS (DIALOG) WPI (DIALOG)

Claims (1)

(57) [Claims] 1. A serum-derived human serum albumin
The ligand part is a polyol group, a polyamine group, or
By treating with a chelating resin that is a thiourea group
Thus, the amount of bound fatty acid is 0.028 or more and 0.02 in molar ratio.
046 or less human serum albumin.