JPH05328991A - Decoloring of human serum albumen - Google Patents

Abstract

PURPOSE:To remove discoloring components and contaminants from human serum albumin by treating human serum albumin expressed by gene manipulation with a chelate resin. CONSTITUTION:The human serum albumin expressed by gene manipulation is treated with a chelate resin, preferably a chelate resin having polyol groups, polyamine groups or thiourea groups as a ligand. Red pigments are effectively removed.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for decolorizing human serum albumin expressed by genetic engineering.

[0002]

2. Description of the Related Art Albumin, particularly human serum albumin (hereinafter also referred to as "HSA"), is a major protein constituent of plasma. This protein is made in the liver and is primarily responsible for maintaining normal osmotic pressure 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 and burns, H2 is usually used to restore blood volume and thereby improve some of the trauma-related symptoms.
Requires frequent administration of SA. Patients with hypoproteinemia and erythroblastosis may also need treatment with HSA. Therefore, the basic therapeutic significance of administering HSA lies in treating conditions such as loss of fluid from blood vessels, as in hypoproteinemia, which results in surgery, shock, burns, and edema. ..

Presently, HSA is produced primarily as a product from a collected blood fraction. The drawbacks of this manufacturing method are that it is uneconomical and that the supply of blood is difficult. Blood may also contain undesirable substances such as hepatitis virus. Therefore, HSA
It would be beneficial to develop alternative raw materials for.

With the advent of recombinant DNA technology, it has become possible to produce a wide variety of useful polypeptides by microorganisms, and many mammalian polypeptides have already been produced by various microorganisms. For HSA,
A technique for mass-producing by genetic engineering technology and highly purifying it is being established.

However, in genetic engineering, when culturing a host microorganism, and further when purifying HSA, a certain coloring component in the raw material or a substance secreted by the microorganism becomes contaminated. It is considered that HSA itself is colored by binding with HSA. Moreover, these contaminants are associated with conventional plasma-derived HSA.
It cannot be sufficiently removed by the above purification method.

[0006]

SUMMARY OF THE INVENTION Therefore, the object of the present invention is to obtain the above-mentioned coloring components and contaminants which could not be sufficiently removed by conventional methods for purifying plasma-derived HSA when obtaining HSA by genetic engineering. It is to provide HSA which has been removed and whose coloration is sufficiently suppressed.

[0007]

Means for Solving the Problems As a result of intensive studies in view of the above circumstances, the present inventors have found that when obtaining HSA obtained by genetic engineering, in the step of purifying the HSA, particularly preferably at the end thereof. The present invention has been completed by finding that the coloring substance can be adsorbed by the chelate resin by treating the HSA with the chelate resin and the coloring of the HSA can be reduced.

That is, the present invention particularly preferably at the end of the purification step is a chelate resin, preferably a chelate resin having as a ligand an exchange group having a chelating ability selected from a polyol group, a polyamine group or a thiourea group. The present invention relates to a method for decolorizing HSA obtained by genetic engineering, which comprises treating with H.

The present invention relates to a method for decolorizing HSA in the case of preparing HSA by genetic engineering, wherein the HSA expresses HSA through genetic engineering (for example, Escherichia coli, yeast, Bacillus subtilis, koji). , Animal cells, etc.) and then produced by extracellular expression (secretion expression).

(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 by genetic engineering, and it has already been described in known literature. In addition to the products, those developed in the future can be used as appropriate. Specific examples include bacteria (eg, Escherichia coli, yeast, Bacillus subtilis, etc.) that have been made HSA-producing through genetic manipulation, animal cells, and the like. In particular, in the present invention, as a host, yeast, especially Saccharomyces genus (eg, Saccharomyces cerevisiae)
], Or Pichia genus [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.

Methods for preparing these HSA-producing hosts, methods for producing HSA by culturing them, and HS from culture
All the separation and collection methods of A are carried out by adopting publicly known methods and methods similar thereto. For example, HSA
Examples of the method for preparing the producing host (or HSA producing strain) include a method using a normal human serum albumin gene (Japanese Patent Laid-Open Nos. 58-56684, 58-90515 and 58-150517), and novel human serum. Method using albumin gene (JP-A-62-29985, JP-A-1-98486), method using synthetic signal sequence (JP-A-1-240191), method using serum albumin signal sequence (JP-A-2- 167095
JP-A-3-72889), a method of fusing hosts with each other (JP-A-3-53877), a method of causing a mutation in a methanol-containing medium, Method using mutant AOX ₂ promoter (Japanese Patent Application No. 3-63598, 3-
No. 63599), expression of HSA by Bacillus subtilis (Japanese Patent Laid-Open Publication No. Sho 6 (1999) -58163)
2-25133), expression of HSA by yeast (JP-A-60-41487, 63-39576, 63-63).
74493), the expression of HSA by Pichia yeast (JP-A-2-104290) and the like.

[0012] Of these, the method for causing mutation in a medium containing methanol is specifically performed as follows. That is, first a suitable host, preferably Pichia yeast, specifically GTS115 strain (NRRL Deposit No. Y-15851).
A plasmid having a transcription unit expressing HSA under the control of the AOX ₁ promoter is introduced into the AOX ₁ gene region of E. coli by a conventional method to obtain a transformant (JP-A-2-104).
290). This transformant has a weak growth ability in a methanol medium. Then, this transformant is cultivated in a medium containing methanol to cause mutation, and only viable strains are collected. 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 condition is 1
The temperature is, for example, 5 to 40 ° C. and 1 to 1000 hours.

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-mentioned publications, a high concentration of glucose can be supplied in appropriate small amounts by fed-batch culture. Then, a method of obtaining a high-concentration bacterial cell and a product by avoiding the high-concentration substrate inhibition on the producing bacterial cell (Japanese Patent Application No. 1-219561), and a method of adding a fatty acid to the medium to enhance the production of HSA ( Japanese Patent Application No. 3-81
No. 719) is exemplified. Further, as a method for separating and collecting HSA, in addition to the methods described in the above-mentioned publications, inactivation of protease by heat treatment (Japanese Patent Laid-Open No. 1031/1993).
88), an anion exchanger, a hydrophobic carrier, and at least one selected from the group consisting of activated carbon, and HSA is used.
And a coloring suppression method by separating the coloring component (Japanese Patent Application Laid-Open No. 4-54198).

The medium used for culturing the transformed host is
Usually, a medium known in the art to which a fatty acid having 10 to 26 carbon atoms or a salt thereof is added is used, and the culture condition is carried out according to a general conventional method. The medium may be a synthetic medium or a natural medium, and a liquid medium is preferable. For example,
As a synthetic medium, generally, various sugars as carbon sources, urea, ammonium salts, nitrates etc. as nitrogen sources, various vitamins and nucleotides as micronutrients, and Mg, Ca, Fe, Na, K, Mn, Co as inorganic salts. , Cu
Are exemplified. YNB liquid medium [0.7% yeast nitrodien base (manufactured by Difco), 2% glucose] and the like can be mentioned. 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, weakly basic or weakly acidic. In the case of a methanol-assimilating host, a methanol-containing medium 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, the temperature is 20 to 43 ° C.).
30 [deg.] C. and 20-37 [deg.] C. for bacteria are preferable. The culturing time is about 1 to 1000 hours, and the culturing is allowed to stand or shake,
It is carried out by a batch culture method, a semi-batch culture method or a continuous culture method under stirring and aeration. In addition, it is preferable to perform pre-culture prior to the culture. As the medium at this time, for example, YNB liquid medium or YPD liquid medium is used. The culture conditions for the pre-culture are as follows. That is, the culture time is preferably 10 to 100 hours, and the temperature is preferably 30 ° C for yeast and 37 ° C for bacteria.

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

(2) Purification of HSA The treatment by the decolorizing method of HSA of the present invention is particularly preferably performed last in the conventional purification step of genetically-engineered HSA. As the purification step, known methods such as various fractionation methods, adsorption chromatography, affinity chromatography, gel filtration, density gradient centrifugation, dialysis and the like are adopted. As the purification step, for example, a step including the following is preferably exemplified. The culture supernatant of the human serum albumin producing host is treated with ultrafiltration membranes having molecular weight cutoffs of 100,000 to 500,000 and 1,000 to 50,000. 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 steps, pH 6 to 8 and salt concentration 1 to
After contacting the carrier for hydrophobic chromatography under the condition of 3M, elution under the condition of pH 6 to 8 and salt concentration of 0.01 to 0.5M, or instead of the above process, pH 6 to
8. After contacting with an anion exchanger under the condition of salt concentration 0.001-0.05M, pH 6-8, salt concentration 0.05
The step of eluting under the condition of ~ 1M, and further between, or after the step, salting out under the conditions of pH 3-5 and salt concentration 0.5-3M to obtain a precipitate fraction. May be further included.

(3) Decolorization of HSA The decolorization step of HSA of the present invention comprises the steps of:
Particularly preferably, it is carried out by bringing HSA into contact with a chelating resin which is incorporated at the end and has a specific ligand portion. 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, etc. in the molecule (including polyalkylenepolyamine groups such as polyethylenepolyamine). Included), and thiourea groups. Commercially available chelating resins having the above-mentioned carrier portion and ligand portion include DIAION CRB02 (ligand portion; N-methylglucamine group, manufactured by Mitsubishi Kasei) whose carrier portion is a copolymer of styrene and divinylbenzene.
CR20 (ligand _{moiety; -NH (CH 2 CH 2 NH} ) n H, manufactured by Mitsubishi Kasei), LEWATIT TP214 (ligand moiety; -NHCSNH
₂ , manufactured by Bayer) and Amberlite CG4000 are preferably used.

The treatment conditions with the chelate resin are preferably as follows. pH condition: acidic or neutral (3 to 9, preferably 4 to
7) Time: at least 1 hour or more, preferably 6 hours or more Ionic strength: 50 mho or less, preferably 1 to 10 mh
o Mixing ratio: 0.1 to 100 of resin for 250 mg of HSA
g, preferably 1-10 g (wet weight)

The coloring degree of HSA obtained through the above steps (including salting out treatment and chelating resin treatment) is A _{500 nm} / A _{280 nm in} the case of 25% HSA solution.
Is about 0.001 to 0.005. The chelating resin treatment of the present invention reduces the coloring degree of HSA to 1/2 to 1/10. In particular, the absorption degree near 500 nm, that is, the reddish coloring degree is reduced to 1/3 to 1/10.

(4) Formulation The obtained HSA can be formulated by a known method (ultrafiltration, addition of a stabilizer, sterile filtration, dispensing, lyophilization, etc.). The HSA preparation thus prepared can be clinically 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.

[0022]

INDUSTRIAL APPLICABILITY According to the present invention, with respect to HSA obtained by genetic engineering, certain coloring components in raw materials or substances secreted by microorganisms are contaminated, and coloring caused by binding with HSA is sufficient. H suppressed to
SA can be provided. The present invention has an absorption wavelength of 50
It is extremely useful for removing coloring components near 0 nm, that is, red coloring components.

[0023]

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.
PC4130 was obtained by substituting the AOX ₁ gene region of 5 (his4) with the fragment cleaved with Not1 of the plasmid pPGP1 having a transcription unit expressing HSA under the control of the AOX ₁ promoter. 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 ℃
The medium was inoculated into 50 ml. 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 the cells were diluted with 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 and Mut + strains can grow on this plate. 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 diluted appropriately 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 in a ml Erlenmeyer flask and cultured with shaking at 30 ° C. for 24 hours.

[0027]

[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 pre-culture.

(Main culture) Medium for batch culture (Table 2) 25
The preculture liquid was inoculated into 0 L, and aeration stirring culture was performed using a 1,200 L 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 rate set to 800 NL / min.
The feed medium (Table 3) was added when the glycerol in the medium was consumed in the batch culture. A computer was used to add the feed medium, and high-density culture was performed while controlling so that methanol did not accumulate in the medium. The pH was controlled to a constant value of pH 5.85 by adding 28% aqueous ammonia. For defoaming, add an antifoaming agent (Adecanol, manufactured by Asahi Denka Kogyo 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.

[0030]

[Table 2]

[0031]

[Table 3]

Reference Example 2 AOX2 promoter [mutant type] isolated from the GCP101 strain of Reference Example 1. Natural AOX2 promoter (TEAST,
5, 167-177 (1988) or Mol. Cell, Biol., 9, 1316-1
323 (1989)), the HSA expression plasmid pMM042 was constructed by mutating the 255th base upstream of the start codon from T to C].
pastoris) transformant UHG introduced into GTS115 strain
42-3 strain was obtained (Japanese Patent Application No. 3-63599). According to Reference Example 1, this UHG42-3 strain was cultured 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 an hour, the mixture is rapidly cooled to about 15 ° C., and the pH value is 4.
It was adjusted to 5, and again removed using an ultrafiltration membrane having a cutoff molecular weight of 300,000 [membrane fraction (II)]. Then, using an ultrafiltration membrane having 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] 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 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 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 added to HSA.
Was allowed to settle. This precipitate was separated from the supernatant by centrifugation to remove impurities.

Example 1 Purified 25% recombinant HSA1 obtained through Reference Examples 1 and 3
1 g of DIAIONCRB02 (manufactured by Mitsubishi Kasei) was added to ml, and the mixture was stirred at room temperature for 24 hours under conditions of pH 6.8 and ionic strength of 5 mmho. After washing the resin with distilled water, the absorbance of the recovered HSA was measured. The coloring degree is A ₅₀₀ / A
The value was ₂₈₀ . Also, instead of DIAION CRB02, DIAION CR20 (manufactured by Mitsubishi Kasei), LE
The absorbance of HSA was measured in the same manner as above except that WATIT TP214 (manufactured by Bayer) was used to determine the degree of coloring. The results are shown in Table 4. Table 4 also shows, as a control, the results obtained by treating the carriers other than the chelate resin of the present invention, and the cation exchanger, the anion exchanger, and the carrier for hydrophobic chromatography.

[0039]

[Table 4]

Example 2 (Study of measurement wavelength) Purification 2 obtained through Reference Examples 1 and 3
1 g of DIAIONCRB02 (manufactured by Mitsubishi Kasei) was added to 1 ml of 5% recombinant HSA, pH 6.8, ionic strength 5 mm.
The mixture was stirred at room temperature for 24 hours under the condition of ho. After washing the resin with distilled water, the recovered HSA was 350-650n.
The absorbance at m was measured. As a result, wavelength of 500n
The decrease in absorbance at m was the highest. The results are shown in Table 5.

[0041]

[Table 5]

Example 3 Purified 25% recombinant HSA1 obtained through Reference Examples 2 and 3
1 g of DIAIONCRB02 (manufactured by Mitsubishi Kasei) was added to ml, and the mixture was stirred at room temperature for 24 hours under conditions of pH 6.8 and ionic strength of 5 mmho. After washing the resin with distilled water, the recovered HSA [concentration 25% (w / v)] had a wavelength of 280
And the absorbance at 500 nm was measured, and A ₅₀₀ / A
_{When 280} was calculated, it was 0.002. The coloring degree (A ₅₀₀ / A ₂₈₀ ) is 1 / before and after the chelate resin treatment.
Reduced to 5.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI technical display location C12P 21/00 B 8214-4B // A61K 37/04 ABY 8314-4C (72) Inventor above Kanaeko 2-25-1 Otani Otani, Hirakata-shi, Osaka Prefecture Midori Cross Central Research Institute (72) Inventor Munehiro Noda 2-25-1 Otani Otani, Hirakata City, Osaka Prefecture (72) ) Inventor Takao Omura 2-25-1 Otani Otani, Hirakata-shi, Osaka Prefecture Midori Cross Central Research Institute, Inc. (72) Inventor Kazumasa Yokoyama 2-25-1 Otani Otani, Hirakata-shi, Osaka Midori Cross Center In the laboratory

Claims (2)

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