JP4016999B2 - Purification method of human serum albumin derived from genetic manipulation - Google Patents

Description

  The present invention relates to a simple and high yield purification method for human serum albumin obtained by genetic manipulation. In addition, the present invention relates to a coloring level, which is a problem peculiar to HSA derived from genetic engineering, and to a human serum albumin-containing composition derived from genetic engineering, characterized in that the coloring level is extremely low.

  Human serum albumin (hereinafter referred to as HSA) is the most abundant protein in plasma and functions in the blood to maintain osmotic pressure and to bind to nutrients and metabolites and transport them. HSA having such a function is used as a medicine for treating albumin loss, hypoalbuminemia due to albumin synthesis decrease, hemorrhagic shock, and the like.

  Traditionally, HSA has been produced primarily as a product from a collected blood fraction. However, the production method of HSA using blood as a raw material has drawbacks such as difficulty in supplying blood, uneconomical, and risk of undesirable substance contamination such as hepatitis virus. It is desired.

Under such circumstances, with the advent of recombinant DNA technology, technology for mass production and purification of HSA by genetic manipulation is being established in order to replace blood-derived HSA.
For purification of HSA obtained by genetic manipulation (in the present invention, referred to as genetic manipulation-derived HSA, hereinafter abbreviated as rHSA), it is inappropriate to apply a conventional purification method for plasma-derived HSA as it is. This is because the contaminant components to be removed are completely different from plasma-derived HSA. For example, coloring substances peculiar to HSA derived from genetic manipulation, proteins derived from host cells, polysaccharides or other contaminating components. In particular, since components derived from host cells are foreign substances to a living body such as a human, it is necessary to sufficiently remove these components from the problem of antigenicity.
For this reason, various studies have been conducted on methods for isolating and purifying rHSA produced by culture treatment from components derived from host cells, culture components, and the like with sufficient accuracy. For example, as a conventional method, a yeast culture solution containing rHSA is subjected to pressing → ultrafiltration membrane treatment → heating treatment → ultrafiltration membrane treatment, and then a cation exchanger, hydrophobic chromatography, anion Examples include a method (Patent Document 1, Non-Patent Document 1) that is used for a process such as column chromatography of the exchanger. In addition, after the conventional method, a method of subjecting to a chelate resin treatment or a boric acid / salt treatment step has also been reported (Patent Documents 2 and 3).

  The purification process consisting of the above-mentioned several steps according to the conventional method is indispensable for removing the antigen derived from the host cell and ensuring a high degree of purification, while reducing the yield of rHSA due to the multi-step process and extending the treatment time. It has drawbacks such as conversion. Although the yield of each process has been improved in order to increase the yield of rHSA, there is already a feeling that the process has been improved, and the improvement of the yield has become a problem. Moreover, in the said conventional method, there exists a possibility that a contaminant may mix by performing a pressing process by an open system, and there exists a problem that the hygiene management essential when adjusting rHSA as a pharmaceutical is difficult. In addition, in the purification method based on the conventional method, the limit is to reduce the coloring degree of rHSA to about A350 / A280 = about 0.015 when the rHSA concentration is adjusted to 250 mg / ml (Patent Document). 4,5)

By the way, recently, a method for directly recovering a target protein from a crude culture stock solution without performing pretreatment such as removal of cells after cell culture and concentration operation has been developed [adsorption developed by Pharmacia. Streamline method using fluidized bed technology (Expanded Bed Adsorption), etc. (Patent Document 6)].
Until now, there has been no application of such adsorption fluidized bed technology for the purification of rHSA, particularly for recovering and purifying rHSA from yeast cultures. In fact, this method is intended to streamline rHSA purification and increase the yield. It is not known at all whether it is useful. However, if this method or a method similar to this can be applied to rHSA, it is considered that the conventional purification process consisting of several steps can be simplified.

However, under acidic conditions, which are the conditions for adsorption to the stream line column (adsorbent: stream line SP) used in the above method, rHSA contained in the culture solution is rapidly reduced in molecular weight by the protease in the culture solution. Therefore, there is a problem that the yield of rHSA is remarkably lowered, and it has been difficult to use the adsorption fluidized bed technique as it is for the purification of rHSA. Accordingly, it is desired to develop a method for highly purifying rHSA in a stable state with high yield without diminishing the merits of simplification and simplification of the purification process of the adsorption fluidized bed technology.
JP-A-5-317079 JP-A-6-56883 JP-A-6-245789 JP-A-7-170993 JP-A-7-170994 Japanese Patent Publication No. 6-500050 Biotechnology of Blood Proteins. 1993, Vol. 227, 293-298

  An object of the present invention relates to a method for purifying HSA obtained by genetic manipulation, and is to provide a simple method capable of obtaining high-purity and high-quality rHSA in a high yield and in a short time. Another object of the present invention is to provide an rHSA obtained by genetic manipulation and from which a coloring component peculiar to genetic manipulation derived from a production host or a medium is sufficiently removed, and an rHSA-containing composition.

  The present inventors have made various studies in order to solve the above-mentioned problems, and it is possible to inactivate protease easily and effectively by directly heating the culture solution of the rHSA-producing host while containing the host cells. I found out that I can do it. Furthermore, based on this finding, when the heat-treated solution was directly contacted with adsorbent particles floating in a fluidized bed without removing fungal cells, it was found that rHSA can be easily purified at a high yield. . In addition, by combining the heat treatment and adsorbent particle treatment, five steps consisting of pressing, ultrafiltration membrane treatment, heat treatment, ultrafiltration membrane treatment, and cation exchanger treatment, which are conventional rHSA purification methods, are heated. The present invention was completed by confirming that the process can be simplified to two processes comprising adsorbent particle treatment, the purification process time can be greatly shortened and the recovery rate is improved. Furthermore, the present invention was completed by confirming that by using this method, rHSA containing substantially no contaminants derived from host cells and having an extremely reduced degree of coloring over the conventional method was obtained.

That is, the present invention is a method in which a culture solution of an HSA production host obtained by genetic manipulation is heat-treated while containing rHSA and the production host, and the heat-treatment solution is brought into contact with adsorbent particles floating in a fluidized bed. A method for purifying rHSA, which comprises collecting fractions. More specifically, the culture solution of the HSA production host obtained by genetic manipulation is heat-treated while containing the rHSA and the production host, and the heat treatment solution is sent upward to a fluidized bed in which adsorbent particles are suspended to perform the adsorption. This is a method for purifying rHSA, wherein the rHSA adsorbed on the adsorbent particles is eluted and recovered by contacting the body particles and then switching the liquid feeding direction and feeding the buffer solution downward.
In the purification method, the heat treatment temperature of the culture solution of the rHSA production host is 50 to 100 ° C., and the time is 1 minute to 10 hours, the heat treatment solution and the adsorbent. The method of purifying rHSA, wherein the contact with the particles is carried out in an atmosphere having a pH of 3 to 5 and an electric conductivity of 0.1 to 50 mS, and the adsorbent particles have strong cation exchange groups. It is a purification method of rHSA characterized by having.

Furthermore, the present invention is a method in which the rHSA-containing fraction recovered from the fluidized bed in the purification method of the present invention is preferably heat-treated in the presence of a reducing agent, followed by hydrophobic chromatography, anion exchanger treatment, chelation An rHSA purification method characterized by being subjected to at least one purification treatment selected from the group consisting of resin treatment, boric acid / salt treatment, and ultrafiltration membrane treatment.
The present invention also includes human serum albumin derived from genetic manipulation, and A350 / A280 is 0.015 or less in the case of the albumin 25% solution (rHSA concentration is 250 mg / ml). It is an albumin-containing composition.
Furthermore, the present invention is a method in which a culture solution of an HSA production host obtained by genetic manipulation is heated while containing the rHSA and the production host, and the heat treatment solution is brought into contact with adsorbent particles floating in a fluidized bed. Provided is a method for producing rHSA, wherein the fraction is collected. More specifically, the production method involves heat-treating the culture solution of the HSA production host obtained by genetic manipulation while containing the rHSA and the production host, and sending the heat-treatment solution upward to the fluidized bed in which the adsorbent particles are suspended. The method of producing rHSA is characterized by eluting and collecting the rHSA adsorbed on the adsorbent particles by switching the liquid feeding direction after the liquid is brought into contact with the adsorbent particles and feeding the buffer solution downward. It is.
Further, the present invention provides the production method, the heat treatment liquid and the adsorbent, wherein the heat treatment temperature of the culture solution of the rHSA production host is 50 to 100 ° C. and the time is 1 minute to 10 hours. The method for producing rHSA is characterized in that the contact with the particles is carried out in an atmosphere having a pH of 3 to 5 and an electric conductivity of 0.1 to 50 mS, and the adsorbent particles have strong cation exchange groups. It is a manufacturing method of rHSA characterized by having.
In the present invention, the rHSA-containing fraction recovered from the fluidized bed in the production method of the present invention is preferably heat-treated in the presence of a reducing agent, followed by hydrophobic chromatography, anion exchanger treatment, chelate A method for producing rHSA, which is subjected to at least one purification treatment selected from the group consisting of resin treatment, boric acid / salt treatment, and ultrafiltration membrane treatment.

The present invention has been completed based on the knowledge that protease can be inactivated easily and effectively by directly heating the culture solution while containing yeast cells. That is, the present invention is a method for easily and effectively purifying rHSA by bringing a treatment solution obtained by heating a culture solution containing yeast cells directly with adsorbent particles floating in an adsorption layer. As a result, the five steps from the conventional compression-membrane-heating-membrane-cation exchanger treatment can be simplified to two steps (heating-adsorbent particle treatment), and the process time is greatly shortened and the recovery rate is improved. did. In addition, the problem of coloring peculiar to HSA produced by genetic manipulation can also be solved by the purification method of the present invention, because the coloring substances that cause it can be effectively removed.
Furthermore, according to the purification method of the present invention, it is possible to perform all of the production process of HSA by genetic manipulation from cell culture to purification on one line of a closed system, so that rHSA production can be automated and rHSA can be used as a pharmaceutical product. As an advantage, hygiene management, which is essential in the preparation, is easily performed.
In other words, the present invention is extremely useful as a method that contributes not only to shortening the process and increasing the yield, but also to improving the quality in rHSA purification.
In addition, according to the present invention, rHSA useful as a pharmaceutical product, which is obtained by genetic manipulation and does not contain production host-related components or other contaminating components and is sufficiently suppressed in coloring, can be provided.

Hereinafter, embodiments of the present invention will be described.

(1) HSA obtained by genetic manipulation
The rHSA production host prepared by genetic manipulation used in the present invention is not particularly limited as long as it is prepared through genetic manipulation. Can be used. Specifically, bacteria (for example, Escherichia coli, yeast, Bacillus subtilis, etc.), animal cells, etc. that have been rendered rHSA-producing through genetic manipulation are exemplified. In particular, yeast, especially Saccharomyces cerevisiae (for example, Saccharomyces cerevisiae) or Pichia (for example, Pichia pastoris) is preferably used as the host. Further, auxotrophic strains and antibiotic-sensitive strains may be used. More preferably, Saccharomyces cerevisiae AH22 strain (a, his 4, leu 2, can 1) and Pichia pastoris GTS115 strain (his 4) are used.

The method for preparing these rHSA production hosts, the method for producing rHSA by culturing the host, and the method for separating and collecting rHSA from the culture can be carried out by adopting known methods and similar methods. For example, as a method for preparing an rHSA production host, for example, a method using a normal HSA gene (Japanese Patent Laid-Open Nos. 58-56684, 58-90515, 58-150517), a method using a novel HSA gene ( JP-A-62-29985, JP-A-1-98486), a method using a synthetic signal sequence (JP-A-1-240191), a method using a serum albumin signal sequence (JP-A-2-167095), A method for incorporating a recombinant plasmid onto a chromosome (Japanese Patent Laid-Open No. 3-72889), a method for fusing hosts (Japanese Patent Laid-Open No. 3-53877), a method for causing mutation in a medium containing methanol, and a mutant AOX ₂ promoter (Japanese Patent Application Nos. 3-63598 and 3-63599), expression of HSA by Bacillus subtilis JP-A-62-25133), expression of HSA by yeast (JP-A-60-41487, 63-39576, JP-A-63-74493), expression of HSA by Pichia yeast (JP-A-2-104290) No. Publication).

Among these, the method of causing mutation in a methanol-containing medium is specifically performed as follows. That is, first, a plasmid having a suitable host, preferably Pichia yeast, specifically, a transcription unit that expresses HSA under the control of the AOX ₁ promoter in the AOX ₁ gene region of GTS115 strain (NRRL deposit number Y-15851) by a conventional method. To obtain a transformant (see JP-A-2-104290). This transformant has a weak growth ability in methanol medium. Therefore, the transformant is cultured in a methanol-containing medium to cause mutation, and only viable strains are collected. In this case, the methanol concentration is exemplified by about 0.0001 to 5%. The medium may be an artificial medium or a natural medium. Examples of culture conditions include 15 to 40 ° C. and about 1 to 1000 hours.

  In addition to the methods described in each of the above publications, the rHSA production host can be produced by feeding a moderately small amount of high-concentration glucose, methanol, or the like by fed batch culture (semi-batch culture). A method for obtaining a high concentration of bacterial cells and products while avoiding high-concentration substrate inhibition on the bacterial cells (Japanese Patent Laid-Open No. 3-83595), and a method for enhancing the production of rHSA by adding a fatty acid to the medium (Japanese Patent Laid-Open No. Hei 4) -293495) and the like.

  As a medium used for culturing a transformed host, a medium known in this field to which a fatty acid having 10 to 26 carbon atoms or a salt thereof is added is used, and the culture conditions are carried out according to a general ordinary method. be able to. As the medium, either a synthetic medium or a natural medium may be used, and a liquid medium is particularly preferable. For example, as a synthetic medium, various sugars are generally used as a carbon source, urea, ammonium salt, and nitrate are used as a nitrogen source, various vitamins and nucleotides are used as micronutrients, and Mg, Ca, Fe, Na, K, and Mn are used as inorganic salts. , Co, Cu and the like. YNB liquid medium [0.7% yeast nitrogen base (Difco), 2% glucose] and the like. Examples of natural media include YPD liquid media [1% yeast extract (Difco), 2% bactopeptone (Difco), 2% glucose]. 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 preferably 15 to 43 ° C. (20 to 30 ° C. for yeast and 20 to 37 ° C. for bacteria). The culture time is about 1 to 1000 hours, and the culture is carried out by a batch culture method, a semi-batch culture method or a continuous culture method with standing, shaking, stirring or aeration. In addition, it is preferable to perform preculture 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 pre-culture are as follows. That is, the culture time is preferably 10 to 100 hours, and the temperature is preferably about 30 ° C. for yeast and about 37 ° C. for bacteria.

(2) Purification of rHSA
(i) Heat treatment In the rHSA purification method of the present invention, the heat treatment is carried out by subjecting the host cell body to the rHSA production host culture solution obtained in the above-described culture step without subjecting it to a separation treatment such as centrifugation or ultrafiltration. It is characterized by heating directly while containing. That is, the heat treatment is performed first in the purification method of the present invention.
The heating conditions are usually 50 to 100 ° C. for about 1 minute to 10 hours, preferably 60 to 80 ° C. for about 20 minutes to 3 hours, more preferably 68 ° C. for about 30 minutes. Further, this treatment is preferably performed in the presence of a stabilizer. Examples of the stabilizer include acetyltryptophan, organic carboxylic acids having 6 to 18 carbon atoms, or salts thereof. Both stabilizers may be used in combination. Examples of the amount of acetyltryptophan added include a solution final concentration of about 1 to 100 mM. Examples of the organic carboxylic acid having 6 to 18 carbon atoms include caproic acid, caprylic acid, capric acid, lauric acid, palmitic acid, and oleic acid. 10 mM caprylic acid is preferred. Examples of the salt include alkali metal salts (for example, sodium salts and potassium salts) and alkaline earth metal salts (for example, calcium salts). Examples of the amount of the organic carboxylic acid having 6 to 18 carbon atoms or a salt thereof include about 1 to 100 mM.
In addition, coloring by heating is performed by adding about 1 to 100 mM, preferably 5 to 30 mM, and further 10 to 1000 mM of aminoguanidine with a thiol compound (for example, mercaptoethanol, cysteine, reduced glutathione, etc.) during heat treatment. The effect that it can be suppressed (Japanese Patent Laid-Open No. 3-103188) is obtained.

  Conventionally, heat treatment is performed on a supernatant (filtrate) obtained by centrifuging or filtering a host culture solution. This is because if the culture solution containing the bacterial cells is directly heat-treated, impurities, lipids, nucleic acids, proteolytic enzymes, etc. existing in the bacterial cells are leaked, which may adversely affect the purification of the target substance. Until now, it has been completely unknown whether direct heat treatment of a culture solution containing host cells is effective for purification of a target substance.

  However, according to the present invention, even if the culture solution containing rHSA and rHSA-producing host cells is directly heated, the strong function present in the culture solution is not reduced without reducing the structural function and yield of rHSA. It was found that protease can be effectively inactivated, and the protease inactivation treatment can be simplified. Furthermore, this enabled the HSA obtained by genetic manipulation to be effectively purified by the purification treatment described later.

(ii) Dilution of heat treatment liquid and adjustment of liquid properties
The heat treatment liquid obtained in (i) is then subjected to the treatment of adsorbent particles floating in the fluidized bed, but before that, the contact between the heat treatment liquid and the adsorbent particles has an electric conductivity of 0.1. It is preferable to dilute the heat treatment liquid so as to be performed in an atmosphere of ˜50 mS, preferably 0.5 to 35 mS, more preferably 5 to 15 mS. As shown in Experimental Example 3 to be described later, as the electric conductivity in the contact atmosphere between the heat treatment liquid and the adsorbent particles is lowered and the rHSA binding to the adsorbent particles is increased. The amount increases and reaches almost the maximum when the electrical conductivity is around 8-12 mS. The dilution solvent is not particularly limited as long as it does not lower the structural function of rHSA in the heat treatment liquid, and can be appropriately selected in consideration of the adsorption conditions to the adsorbent particles. Examples include acetate buffer having a concentration of about 50 mM or less, or distilled water. From the viewpoint of simplicity, etc., distilled water is preferred. Next, the liquidity of the solution is adjusted to acidity, which is the condition for adsorption to the adsorbent particles, usually pH 3 to 5, preferably about pH 4 to 4.8, more preferably about pH 4.5. The acidic solution used for the adjustment is not particularly limited, but preferably includes acetic acid.

(iii) Adsorbent particle treatment Next, the diluted and pH-adjusted heat treatment liquid is brought into contact with the adsorbent particles.
Examples of the adsorbent particles used include a carrier having a cation exchange group (cation adsorbent), and specific examples thereof include adsorbent particles of sulfo group type, carboxyl group type and the like. As the sulfo group type, sulfo-agarose [trade name: Streamline SP, S-Sepharose (both manufactured by Pharmacia)], sulfo-cellulose [trade name: S-Cellulofine (manufactured by Chisso)], sulfopropyl-agarose [Product Name: SP-Sepharose (Pharmacia), SP-Celsur-Big Beads (Stellojin)], Sulfopropyl-Dextran [Product Name: SP-Sephadex (Pharmacia)], Sulfopropyl-Polyvinyl [ Trade name: SP-Toyopearl (manufactured by Tosoh Corporation)] and the like. As the carboxyl group type, carboxymethyl-agarose [trade name: CM cellulose big beads (manufactured by Stelogin)], carboxymethyl-dextran [trade name: CM-Sephadex (manufactured by Pharmacia)], carboxymethyl- Cellulose [trade name: CM-Cellulofine (manufactured by Chisso)] and the like are exemplified. Sulfo group-type strong cation adsorbent particles are preferred, and streamline SP (manufactured by Pharmacia) is more preferred.
Moreover, as a particle diameter of the adsorbent particle used, 30-1100 micrometers normally, Preferably 100-300 micrometers is illustrated.

Examples of contact conditions include a pH of about 3 to 5, preferably about pH 4 to 4.8, more preferably about pH 4.5, and a salt concentration of about 0.01 to 0.2M, preferably about 0.05 to 0.1M. Is done.
The adsorbent particles are preferably equilibrated in advance under such contact conditions. Specifically, it is preferable to equilibrate with 50 mM acetate buffer (pH 4.5) containing 50 mM sodium chloride.

Equilibration of the adsorbent particles, injection of the sample into the fluidized bed containing the adsorbent particles, contact with the adsorbent particles, and elution from the fluidized bed are usually performed according to the following methods.
That is, first, the adsorbent particles described above are packed in a suitable column and allowed to settle, and then the equilibration buffer is sent upward from the lower outlet of the column to float the adsorbent particles. At this time, in the column, the adsorbent particles settled by gravity due to the flow rate of the buffer solution rising from the bottom of the column cancel each other's force, and a uniform floating equilibrium state is formed by the adsorbent particles. Called the floor). Subsequently, the culture crude liquid containing the treated cells (i) and (ii) is added to the column in which the fluidized bed is formed by feeding upward from the outlet at the bottom of the column. At this time, the target rHSA binds to the adsorbent particles, while the fine particles and contaminants derived from the host cells and culture fluid pass through the adsorbent particles in the fluidized bed and are discharged and removed from the upper outlet of the column, and loosely bound. The contaminated components are also washed away with the wash buffer that is subsequently fed upward. Preferably, it is carried out according to the adsorption fluidized bed technique [Journal of Chromatography, 597 (1992), 129-145].

An equilibration buffer is used as the washing buffer. The recovery of rHSA, which is the target protein, is performed by reversing the solution feeding direction and feeding the elution buffer solution downward from the column upper outlet. The elution conditions for rHSA are about pH 8 to 10, preferably about pH 8.5 to 9.5, more preferably about pH 9, and a salt concentration of about 0.2 to 0.5M, preferably about 0.3 to 0.4M. Is exemplified. More specifically, the elution buffer is exemplified by a 0.1 M phosphate buffer (pH 9) containing 0.3 M sodium chloride.
Operations such as equilibration of the adsorbent particles, injection of the sample into the fluidized bed containing the adsorbent particles, contact with the adsorbent particles, and elution from the fluidized bed are performed using a stream line column (adsorbent: stream line SP, By using a stream line system (manufactured by Pharmacia) (manufactured by Pharmacia), it can be carried out more simply, efficiently and with good reproducibility.

  By the above operation, rHSA having a high degree of purification can be obtained. That is, the degree of purification of rHSA obtained by performing the adsorbent particle treatment is obtained through several steps of conventional compression-ultrafiltration membrane treatment-heating treatment-ultrafiltration membrane treatment-cation exchanger treatment. The degree of purification of rHSA is almost the same. Moreover, rHSA can be stably obtained in high yield (without being reduced in molecular weight) by performing a heat treatment of the culture solution before the adsorbent particle treatment step. Therefore, according to the present invention, the above five steps can be simplified to two steps, and the process time can be greatly shortened. Furthermore, according to the present invention, it is possible to improve the recovery rate from the culture solution of rHSA according to the conventional method.

  The rHSA obtained by the above treatment can be further purified to a higher degree by subjecting it to a conventional purification method. Examples of the purification method used include usually used purification treatment, hydrophobic chromatography treatment, ultrafiltration membrane treatment, anion exchanger treatment, chelate resin treatment and borate treatment. These purification treatments can be carried out singly or in combination, and in order to obtain a higher quality rHSA purified product, hydrophobic chromatography treatment, ultrafiltration membrane treatment, anion exchanger treatment, ultrafiltration It is preferable to carry out in order of filtration membrane treatment, chelate resin treatment, borate treatment, and ultrafiltration membrane treatment.

In addition, before performing the said refinement | purification process, after the contact process to adsorbent particle, it is preferable to heat-process the eluted rHSA containing adsorption fraction again in presence of a reducing agent. As shown in Experimental Example 6 described later, when the heat treatment is performed and when the heat treatment is not performed, there is no difference in the recovery rate of rHSA, but a remarkable effect is observed with respect to coloring specific to rHSA, By performing the heat treatment, the colored substance is significantly removed by the subsequent purification treatment.
The treatment temperature is usually 50 to 100 ° C, preferably about 60 to 80 ° C, more preferably 60 ° C. The treatment time is usually 10 minutes to 10 hours, preferably about 30 minutes to 5 hours, more preferably 1 hour.

  The reducing agent to be used is not particularly limited as long as it is a substance having a reducing action, and specifically, a low molecular compound containing an SH group (for example, cysteine, cysteamine, cystamine, methionine, glutathione, etc.), sulfite, Examples include pyrosulfite, phosphorous acid / pyrosulfite, ascorbic acid and the like. Cysteine is preferred. Examples of the added amount include a final concentration of about 1 to 100 mM in the case of cysteine and the like, and a final concentration of about 0.001 to 10% in the case of sulfite and the like.

  Further, this treatment is preferably performed in the presence of a stabilizer. Examples of the stabilizer include acetyltryptophan, organic carboxylic acids having 6 to 18 carbon atoms, or salts thereof. Both stabilizers may be used in combination. Examples of the amount of acetyltryptophan added include a solution final concentration of about 1 to 100 mM. Examples of the organic carboxylic acid having 6 to 18 carbon atoms include caproic acid, caprylic acid, capric acid, lauric acid, palmitic acid, and oleic acid. 10 mM caprylic acid is preferred. Examples of the salt include alkali metal salts (for example, sodium salts and potassium salts) and alkaline earth metal salts (for example, calcium salts). Examples of the amount of the organic carboxylic acid having 6 to 18 carbon atoms or a salt thereof include about 1 to 100 mM. Further, by adding 10 to 1000 mM of aminoguanidine, the effect that coloring caused by heating can be suppressed (JP-A-3-103188) can be obtained.

<1> Hydrophobic chromatographic treatment Hydrophobic chromatographic treatment can be performed according to a conventional method. Examples of the carrier for hydrophobic chromatography include insoluble carriers having an alkyl group having 4 to 18 carbon atoms (butyl group type, octyl group type, octyldecyl group type, etc.) and a phenyl group. Preferable examples include a phenyl group type, and specific examples include phenyl-cellulose (trade name: Phenyl Cellulofine, manufactured by Chisso Corporation). In this step, rHSA can be recovered in the carrier non-adsorbed fraction. Examples of contact conditions include a pH of about 6 to 8, preferably a pH of about 6.5 to 7, and a salt concentration of about 0.01 to 0.5M, preferably about 0.05 to 0.2M.

<2> Anion exchanger treatment Anion exchanger treatment can also be performed according to a conventional method. As the anion exchanger, any insoluble carrier having an anion exchange group can be used. Examples of the anion exchange group include diethylaminoethyl (DEAE) group type and quaternary aminoethyl (QAE) group type. DEAE group type is preferable. Specifically, 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) and the like. In this step, rHSA can be recovered in the non-adsorbed fraction. Examples of contact conditions include a pH of about 6 to 8, preferably a pH of about 6.5 to 7, and a salt concentration of about 0.01 to 0.1M.
This anion exchanger treatment removes colored components and traces of impurities.

<3> Ultrafiltration membrane treatment After the hydrophobic chromatography treatment and / or the anion exchanger treatment, the recovered rHSA-containing fraction is preferably subjected to ultrafiltration treatment. By the ultrafiltration treatment, a pyrogenic substance (pyrogen) can be removed. For the ultrafiltration treatment, an ultrafiltration membrane having a molecular weight cut off of 100 to 300K is preferably used. Specifically, a pericon cassette film 100K (manufactured by Millipore) is exemplified.

<4> Chelate resin treatment The chelate resin treatment is particularly effective in removing colored substances peculiar to HSA obtained by genetic manipulation. In the purification step described above, the treatment is particularly preferably incorporated into the subsequent steps of hydrophobic chromatography treatment-ultrafiltration membrane treatment-anion exchanger treatment-ultrafiltration membrane treatment, and a chelate having a specific ligand moiety. This is done by bringing the resin and rHSA into contact, and rHSA is obtained in the pass fraction. The carrier portion of the chelate resin is preferably a hydrophobic carrier, 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 in the molecule (including polyalkylene polyamine groups such as polyethylene polyamine). Included), and thiourea groups. Conveniently, DIAION CRB02 (ligand part: N-methylglucamine group, manufactured by Mitsubishi Kasei Co., Ltd.), LEWATIT TP214 (ligand part: NHCSNH ₂ : Bayer), where the carrier part is a copolymer of styrene and divinylbenzene. And commercial products such as Amberlite CG4000 are used.

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

<5> Treatment with boric acid or a salt thereof Further, by treating the rHSA-containing solution obtained by the above treatment with boric acid or a salt thereof (also referred to as boric acid / salt in the present invention), It is possible to remove antigenic contaminants and non-antigenic free contaminants detectable by the phenol-sulfuric acid method.
Examples of boric acid used include orthoboric acid, metaboric acid, and tetraboric acid. Examples of the salt include alkali metal salts (sodium salt, potassium salt, etc.), alkaline earth metal salts (potassium salt, etc.) and the like. Preferably, calcium tetraborate is used. The added amount of boric acid or a salt thereof is about 0.01 to 1M, preferably about 0.05 to 0.2M, in the final concentration. The treatment pH is about 8 to 11, preferably about 9 to 10. The treatment time is about 1 to 100 hours, preferably about 5 to 50 hours. The conductivity of the rHSA-containing solution at the time of treatment should be low, and specifically 1 mS or less is exemplified. Furthermore, the rHSA concentration in the rHSA-containing solution should be high, specifically, rHSA is 5% or more, preferably about 15 to 25%.

  After the boric acid / salt treatment, rHSA is recovered from the supernatant by a conventional method such as centrifugation or ultrafiltration.

(3) Properties of highly purified HSA derived from genetic engineering rHSA obtained by highly purified is a single substance having a molecular weight of about 67,000 and an isoelectric point of 4.9. It consists only of monomers and does not substantially contain a dimer, polymer, or decomposition product (molecular weight of about 43,000). In addition, it is substantially free of contaminating components and polysaccharides having antigenicity derived from the production host. The degree of coloration is when prepared as a 250 mg / ml solution (25% solution) as the rHSA concentration, and A350 / A280 is 0.015 or less, preferably 0.013 or less, especially about 0.01 to 0.015. is there. In addition, by using a known purification method (for example, the above-described methods <1> to <5> etc.) in an appropriate combination, rHSA having a further reduced degree of coloration, that is, a low A350 / A280 value is obtained. be able to.
The present invention provides for the first time HSA (containing composition) derived from genetic manipulation and having a coloring degree of A350 / A280 of 0.015 or less in the case of a 25% solution of rHSA.

(4) Formulation The rHSA obtained by the method of the present invention can be formulated by a known method (for example, ultrafiltration, addition of a stabilizer, sterilization filtration, dispensing, lyophilization, etc.). The rHSA preparation thus prepared can be clinically used as a serum albumin preparation in the same manner and in the same dosage and usage as conventional plasma-derived HSA preparations. It can also be used as a pharmaceutical stabilizer, carrier or carrier.
The meaning of the rHSA-containing composition in the present invention indicates that the rHSA of the present invention is highly pure but not 100% pure, and a substance other than rHSA is mixed even in a trace amount.

  EXAMPLES Hereinafter, examples and reference examples will be shown for specifically explaining the present invention, but the present invention is not limited to these examples and the like.

Example 1
Purification of rHSA by small-scale stream line SP (1) Heat treatment of culture solution Regarding the acquisition of rHSA-producing yeast Pichia pastoris and its cultivation, column 8, line 18 to No. 5 of JP-A-5-317079 This was carried out according to the method described in column 11, line 19, European published patent No. 655503.
About 2.8 liters of the obtained culture solution (yeast-containing cells) was directly subjected to a heat treatment at 68 ° C. for 30 minutes. The heat treatment was performed in the presence of 10 mM sodium caprylate. In addition, the liquid property of the culture solution at this time was pH 6. The heat-treated solution was rapidly cooled to about 15 ° C., diluted with distilled water to a volume of about 2 times (total volume 5.5 liters), and then adjusted to pH 4.5 with an acetic acid aqueous solution.

(2) Adsorbent particle processing (stream line SP processing)
Yeast obtained by the heat treatment of (1) on a streamline SP column (C50, 5 × 100 cm: gel amount 300 ml, manufactured by Pharmacia) equilibrated with 50 mM acetate buffer (pH 4.5) containing 50 mM sodium chloride. It added by feeding 5.5 liters (electrical conductivity-10mS) of the culture solution containing a microbial cell (added at a flow rate of 100 cm / h, stirring). The column is then washed by pumping the same buffer (2.5 times the column volume) as used for equilibration (condition: flow rate 100 cm / h for 1 hour, then 30 minutes at a flow rate of 300 cm / h), then reversing the liquid feeding direction, and feeding the elution solution [100 mM phosphate buffer (pH 9) containing 300 mM sodium chloride (flow rate: 50 cm / h) to contain rHSA The fraction to be obtained was obtained.
The eluted rHSA-containing fraction was detected by measuring the absorbance at 280 nm.

(3) Heat treatment The obtained rHSA-containing fraction was heat-treated at 60 ° C for 1 hour in the presence of 10 mM cysteine, 5 mM sodium caprylate, 100 mM aminoguanidine hydrochloride, pH 7.5.

(4) Hydrophobic chromatographic treatment The column (5 × 25 cm, 5 × 25 cm, packed with phenyl cellulofine equilibrated with 50 mM phosphate buffer, pH 6.8 containing 0.15 M sodium chloride from the rHSA solution heated in (3). Gel amount 500 ml: manufactured by Chisso Corporation). Under this condition, the rHSA passed through the column without being adsorbed on phenylcellulose. The rHSA-containing solution passed through the column was concentrated to about 0.2 liter using an ultrafiltration membrane (Millipore) having a molecular weight cut off of 30,000, and the rHSA-containing solution was concentrated to 50 mM phosphate buffer, pH 6 .8.

(5) Anion exchanger treatment A column (5 ×) packed with DEAE-Sepharose FF equilibrated with 50 mM phosphate buffer, pH 6.8 after concentration and buffer exchange of the rHSA-containing solution after hydrophobic chromatography. 25 cm, gel amount 500 ml: manufactured by Millipore). Under these conditions, rHSA passed through the column without being adsorbed on DEAE-Sepharose FF. The rHSA that passed through the column was concentrated to about 0.2 liters using an ultrafiltration membrane (Millipore) having a molecular weight cut off of 30,000, and the rHSA-containing solution was exchanged with distilled water.

(6) Chelation treatment Acetic acid was added to 0.2 liter of purified rHSA having a concentration of about 7% to adjust the pH to 4.5, and a column packed with DIAION CRB02 equilibrated with 50 mM sodium acetate buffer, pH 4.5 (5 × It was circulated overnight after adding to 2.5 cm, gel amount 500 ml: manufactured by Mitsubishi Kasei. Under this condition, rHSA passed through the column without adsorbing to the gel.

(7) Boric acid / salt treatment The rHSA concentration was adjusted to 2.5%, and the solution had an electric conductivity of 1 mS or less. Sodium tetraborate was added to a final concentration of 100 mM, then calcium chloride was added to a final concentration of 100 mM, and the pH was maintained at 9.5. After standing for about 10 hours, the precipitate was removed, and the supernatant was recovered and concentrated for desalting. Subsequently, concentration and buffer exchange were performed using an ultrafiltration membrane (Millipore) having a molecular weight cut off of about 30,000. RHSA can be used as an injection by adding a stabilizer (sodium caprylate, acetyltryptophan) as necessary, followed by sterilization filtration using a 0.22 μm filter (Millipore). .

Experimental Example 1 Stabilizing Effect of Heat Treatment of rHSA Culture Solution There is a strong protease in rHSA culture solution, and it is known to reduce the molecular weight of rHSA. Under these conditions, the molecular weight reduction is remarkably fast.

By the way, since the rHSA adsorption conditions of the stream line SP are carried out at around pH 4.5, the stability of rHSA in the culture solution at around pH 4.5 is investigated, and protease is used as a pretreatment to keep it more stable. The heat treatment conditions effective for inactivation of the solution were examined by examining the stability of rHSA after adjusting the pH of the solution after the heat treatment to 4.5 and allowing it to stand at room temperature overnight. The results are shown in FIG. During heating, sodium caprylate was added to all specimens so that the final concentration was 10 mM. The heat treatment conditions used are as follows.
A: Control
B: 68 ° C., 30 minutes, pH 6.0
C: 68 ° C., 30 minutes, pH 6.8
D: 68 ° C., 30 minutes, pH 7.5
E: 68 ° C., 30 minutes, pH 8.2
F: 60 ° C., 2 hours, pH 6.0
G: 60 ° C., 2 hours, pH 6.8
H: 60 ° C., 2 hours, pH 7.5
I: 60 ° C., 2 hours, pH 8.2
J: 60 ° C., 2 hours, pH 6.8, 10 mM cysteine K: 60 ° C., 2 hours, pH 7.5, 10 mM cysteine L: room temperature (25 ° C.), 2 hours, pH 6.0
M: Room temperature (25 ° C.), 2 hours, pH 8.2

  As a result, the treatment for 30 minutes at 68 ° C. is more effective than the treatment for 2 hours at 60 ° C., and the pH of the culture solution subjected to the heat treatment is best around pH 6, which is the initial value of the culture solution. It was.

Experimental Example 2 Relationship between pH of rHSA culture solution and adsorbent binding ability Acetate buffer solution (50 mM) of each pH was added to a culture solution (rHSA 55.6 mg) adjusted to various pH (4.5 to 4.9) with acetic acid. The streamline SP gel (1 ml) equilibrated in (1) was added, mixed and stirred for 1 hour at room temperature, washed with each equilibration buffer, and the remaining amount (%) of rHSA in the unadsorbed fraction was measured. As a result, as the pH was lowered, the amount of binding increased and reached the maximum around pH 4.5 (Table 2).

Experimental Example 3 Relationship between the electric conductivity in the contact atmosphere between the heat treatment liquid and the adsorbent particles and the binding ability of rHSA to the adsorbent particles The culture solution after the heat treatment [electric conductivity about 20 mS (25 ° C.), rHSA (47.1 mg) was diluted with distilled water in various multiples, then adjusted to pH 4.5 with acetic acid, and equilibrated with 50 mM acetate buffer containing 50 mM sodium chloride at the same pH. 1 ml was added. Then, after mixing at room temperature for 1 hour, it was washed with an equilibration buffer, and rHSA was eluted with 0.1 M phosphate buffer (pH 9) containing 0.3 M sodium chloride. As a result, as the dilution factor is increased and the electrical conductivity in the solution is lowered, the amount of rHSA bound to the adsorbent particles increases, and the contact atmosphere between the heat treatment liquid and the adsorbent particles (in the gel mixture) The electric conductivity reached a maximum around 8-12 mS (FIG. 2).

Experimental Example 4 Stability of stream line SP eluate Stream line diluted with distilled water twice, adjusted to pH 4.5 with acetic acid and equilibrated with 50 mM acetate buffer containing 50 mM sodium chloride at the same pH A certain amount of SP gel was added and mixed and stirred at room temperature for 1 hour. Next, after washing the gel with the equilibration buffer, rHSA was eluted with 0.1 M phosphate buffer (pH 9) containing 0.3 M sodium chloride, and the room temperature of rHSA in the obtained fraction solution (pH 8) was obtained. The stability at was investigated. As a result, when the culture solution was preheated (68 ° C., 30 minutes), no change was observed even after 3 days, whereas rHSA derived from the untreated culture solution decreased to about 50%. Decomposed (Table 3).

Experimental Example 5 Comparison of rHSA recovery rate and coloring degree between the (heating → adsorbent particles) treatment and the (non-heated → adsorbent particles) treatment Heating → adsorption based on the results examined in the above experimental examples 1 to 4 An optimization flow of the body particle treatment process was set (FIG. 3).
In accordance with the process flow of FIG. 3, purification of rHSA from a culture solution (2.8 liters) containing yeast cells was attempted using a streamline SP (5 × 100 cm, gel amount 300 ml) column. On the other hand, the adsorbent particle treatment was similarly performed without heating the culture solution (2.7 liters) containing the yeast cells, and the rHSA was purified.
As a result, the total recovery rate from the culture solution is as high as about 85% in the (heating → adsorbent particle) treatment of the present invention, whereas the (non-heating → adsorbent particle) treatment is 50%. The coloring degree of rHSA was 0.048 in the (heating → adsorbent particle) treatment of the present invention, while the (non-heating → adsorbent particle) treatment was 0.048 in A350 / A280 (Table 4). ). In FIG. 4, the gel filtration HPLC profile of the streamline SP eluate treated with (heating → adsorbent particles) was compared with the gel filtration HPLC profile of the streamline SP eluate treated with (non-heated → adsorbent particles). In the latter, rHSA was significantly decomposed and reduced in molecular weight.

Experimental Example 6 Effect of Cysteine Heat Treatment on rHSA Coloration Level Next, Example 1 (using the rHSA elution fraction of the streamline SP column obtained in Table 4 (heating → adsorbent particles) as a starting material was used. The coloring degree (A350 / A280) was evaluated by performing hydrophobic chromatography and anion exchanger treatment described in 4) and (5). At that time, the streamline SP elution fraction was examined in two ways: one that was heat-treated in the presence of cysteine (final concentration 10 mM) and one that was not heat-treated. As a result, the same rHSA recovery rate was obtained regardless of the presence or absence of cysteine heating, but a significant difference was observed in the degree of coloring, and in the stage of anion exchanger treatment, 0.0184 In contrast, those derived from cysteine heat treatment showed a significantly low value of 0.0128 (Table 5).

Experimental Example 7 Reduction of rHSA coloration (A350 / A280) by incorporating streamline SP Figure 5 shows changes in coloration by process (A350 / A280) in the conventional purification method and anion exchange when the streamline SP process is incorporated Comparison was made with the transition of the degree of coloration by process (A350 / A280) up to the body treatment concentration process stage. In the case of the purification method of the present invention in which the cysteine heat treatment is performed immediately after incorporation of the stream line SP, a large difference has already occurred at the stage of hydrophobic chromatography compared with the conventional method, and the coloration degree after the anion exchanger treatment Showed an extremely low value of 0.0128. FIG. 6 shows a sample (curve 1) further treated with a chelate resin after the conventional method, an anion exchanger treatment and a chelate resin treatment by the purification method of the present invention in which the stream line SP is incorporated and subjected to cysteine heat treatment immediately after that. The comparison by an absorption spectrum with a later specimen (curves 2 and 3) is shown. As a result, the absorption spectrum of rHSA purified by the method of the present invention is significantly lower over the entire visible (350 nm-700 nm) range than the absorption spectrum of the sample already treated with the chelate resin after the conventional method at the stage of anion exchanger treatment. A pattern was shown, and the difference became larger after the treatment with the chelate resin.

Experimental Example 8 Gel filtration HPLC analysis of rHSA obtained by the present invention FIG. 7 shows the results of high performance liquid chromatography (GPC-HPLC) analysis by gel filtration of each step sample of rHSA obtained by the method of the present invention (rHSA culture) Liquid: supernatant of rHSA culture solution, streamline SP elution fraction: streamline SP elution fraction in the same process as in Example 1, streamline SP non-adsorbed fraction: streamline SP in the same process as in Example 1. Non-adsorbed fraction, purified fraction after DEAE: the same as in Example 1 after anion exchanger (DEAE) treatment). As a result, most of the macromolecules and low-molecular substances other than rHSA in the culture solution are efficiently washed away with the yeast cells in the stream line SP non-adsorbed fraction, and albumin is specifically recovered in the eluted fraction. Became clear. In addition, the HPLC pattern of the sample after using this fraction as a starting material and further purified anion exchanger (DEAE) treatment shows only a sharp single peak of albumin (HSA monomer). Compared with the sample after the DEAE step, it showed the same or higher degree of purification.

  Based on the above experimental examples, the recovery rate up to the DEAE concentration step of the conventional purification method was compared with the recovery rate up to the DEAE concentration step in the same process as in Example 1. When the same purification method as in Example 1 is used, the five steps from the conventional purification method to the compression-membrane-heat treatment-membrane-cation exchanger treatment are two steps, and the process time is greatly shortened. The recovery rate was improved by 30%.

Experimental Example 9 Analysis of host-derived contaminants Rabbits were immunized with a non-albumin-producing yeast culture solution purified in the same manner as this method, and the resulting antiserum was used to derive from the yeast present in the purified albumin solution Contaminant components were detected. The measurement was performed by enzyme immunoassay (EIA method). The sample was measured using an albumin concentration adjusted to 250 mg / ml.
As a result, the purified albumin after the boric acid / salt treatment in the same steps as in Example 1 did not detect any contaminating components having antigenicity derived from yeast at the detection limit of 0.1 ng / ml.

Experimental Example 10 Properties of Purified rHSA of the Present Invention (1) Molecular Weight The molecular weight was measured by the aforementioned HPLC gel filtration method. In the same process as in Example 1 of the present invention, the molecular weight of purified rHSA after boric acid / salt treatment was about 67,000, which was similar to plasma-derived HSA.
(2) Isoelectric point The isoelectric point was measured according to the method of Allen et al. [J. Chromatog., 146, 1 (1978)] using polyacrylamide gel. The isoelectric point of the purified rHSA of the present invention was about 4.9, similar to that of plasma-derived HSA.
(3) Coloring degree The coloring degree was determined by measuring the absorbance at 280 nm and 350 nm using purified rHSA prepared with an albumin concentration of 250 mg / ml, and calculating A350 / A280. The coloring degree of the purification degree rHSA of the present invention was about 0.012 at A350 / A280, which was very low.

Reference example rHSA measurement (recovery rate evaluation)
In Experimental Examples 1 to 8 and Experimental Example 10, quantitative evaluation (including recovery rate) of rHSA was performed by subjecting the rHSA-containing solution to HPLC for gel filtration. In detail, the elution conditions were as follows.
The solution containing rHSA was previously equilibrated with 50 mM sodium phosphate buffer (pH 6.5) containing 0.1% NaN ₃ and 0.3% NaCl, TSK-Gel G3000SWXL column (0.78 × 30 cm). : Manufactured by Tosoh Corporation), the equilibration buffer was used as an eluent, rHSA was separated at a flow rate of 1 ml / min, and quantified by measuring absorbance at 280 nm and 350 nm.

Example 2
Purification by Stream Line SP at Large Scale 1000 L of the rHSA-producing yeast Pichia pastoris culture solution (yeast-containing cell) obtained according to the method of (1) of Example 1 was subjected to the conditions (1) of Example 1 ( In the presence of 10 mM sodium caprylate, the culture solution was heated at a liquid pH of 6 at 68 ° C. for 30 minutes. The heat-treated liquid was cooled to about 25 ° C., diluted with distilled water to a volume of about 2 times (total volume: about 2000 L), and adjusted to pH 4.5 using acetic acid (99.7%). Next, the yeast obtained by the above heat treatment was applied to a streamline SP column (C1000, 100 cm × 110 cm: gel amount 150 L, manufactured by Pharmacia) equilibrated with 50 mM acetate buffer (pH 4.5) containing 50 mM sodium chloride. The culture solution 2000L containing the body was added by feeding upward from the bottom of the column (flow rate 100 to 250 cm / h). The culture solution was stirred until the cells did not settle until the addition to the column was completed. Next, the column is washed by feeding the same buffer as the equilibration buffer (approximately 5 times the column volume) by the ascending method (flow rate 100 to 500 cm / h), then the feeding direction is reversed, An eluate (100 mM phosphate buffer containing 300 mM sodium chloride, pH 9) was fed from the top of the column (flow rate 50 to 100 cm / h) to obtain an rHSA-containing fraction. The eluted rHSA-containing fraction was detected by measuring absorbance at 280 nm. In addition, 10 mM cysteine, 10 mM sodium caprylate, 4 times molar amount of sodium oleate and 100 mM aminoguanidine hydrochloride in the presence of 10 mM cysteine, 10 mM sodium caprylate, and 100 mM aminoguanidine hydrochloride were added to the obtained rHSA-containing fraction at 60 ° C. for 1 hour. A heat treatment was carried out to reduce the degree of coloration of rHSA and promote conversion from the dimer to the monomer.

Experimental Example 11 RHSA Yield, Recovery, and Coloration by Large-Scale Purification A 1-ton scale culture solution was heated at 68 ° C. for 30 minutes, and the heat-treated solution was diluted with distilled water to a volume of about 2 times, pH 4 After adjusting to .5, the adsorbent particles were treated with a streamline SP (C1000) column. The eluate containing rHSA was filtered through a 0.8 μm filter, concentrated through an ultrafiltration membrane with a molecular weight cut off of about 30,000, and purified by cysteine heat treatment. When the results of 4 lots (shown in Table 6) are averaged, the recovery rate from the culture solution to the heat treatment is 98.6%, the recovery rate after heating through the stream line to the cysteine heat treatment is 88.4%, The total recovery was as good as 87.1%. This value was in good agreement with the results of experiments on a small scale using a C50 column, confirming reproducibility on a large scale.

It is a figure which shows the stabilization effect by heat processing of a rHSA culture solution. It is a figure which shows the relationship between the electrical conductivity in the contact atmosphere of an adsorbent particle and heat processing liquid, and the binding ability of rHSA with respect to an adsorbent particle. It is a figure which shows the optimization flow of the refinement | purification of rHSA from the culture solution (yeast containing microbial cell) by the streamline SP. Gel Filtration HPLC Profile of Streamline SP Eluate for (Heating → Adsorbent Particles) Treatment [(a)] and Gel Filtration HPLC Profile of Streamline SP Eluate for (Unheated → Adsorbent Particles) Treatment [ In the figure, (b)] is shown (absorbance measured at 280 nm). It is the figure which compared the transition of the coloring degree (A350 / A280) of rHSA according to a process with the conventional method and this invention method. It is the figure which compared the absorption spectrum of rHSA obtained by the method of this invention with the thing of a conventional method. It is a chromatogram which shows the result of the GPC-HPLC analysis of rHSA obtained by the method of the present invention (measured at an absorbance of 280 nm).

Explanation of symbols

A Control B 68 ° C, 30 minutes, pH 6.0
C 68 ° C., 30 minutes, pH 6.8
D 68 ° C., 30 minutes, pH 7.5
E 68 ° C., 30 minutes, pH 8.2
F 60 ° C., 2 hours, pH 6.0
G 60 ° C., 2 hours, pH 6.8
H 60 ° C., 2 hours, pH 7.5
I 60 ° C., 2 hours, pH 8.2
J 60 ° C., 2 hours, pH 6.8, 10 mM cysteine K 60 ° C., 2 hours, pH 7.5, 10 mM cysteine L Room temperature (25 ° C.), 2 hours, pH 6.0
M Room temperature (25 ° C.), 2 hours, pH 8.2
1 rHSA after purification by conventional method (press → membrane → heating → membrane → cation exchanger → hydrophobic chromatography → anion exchanger treatment)
2 rHSA after anion exchanger (DEAE) treatment in the same process as in Example 1
3 In the same process as in Example 1, rHSA after chelate resin treatment

Claims (12)

The culture broth containing a host that produces human serum albumin in the culture broth, obtained by genetic manipulation, is adsorbed on adsorbent particles suspended in a fluidized bed in an atmosphere of pH 3-5 and electrical conductivity 0.1-50 mS. A method for purifying human serum albumin derived from genetic engineering , comprising contacting under conditions, collecting the adsorbed fraction, and subjecting the collected fraction to heat treatment in the presence of a reducing agent . The purification method according to claim 1, wherein the adsorbent particles are adsorbent particles having a strong cation exchange group. The purification method according to claim 1 or 2 , wherein the contact between the culture solution and the adsorbent particles is achieved by feeding the culture solution upward to a fluidized bed. The purification method according to any one of claims 1 to 3, wherein the collection of the adsorbed fraction is achieved by feeding the buffer solution downward to the fluidized bed. The purification method according to any one of claims 1 to 4, wherein the temperature of the heat treatment is 50 to 100 ° C and the time is 10 minutes to 10 hours. The adsorbed fraction is further subjected to at least one purification treatment selected from the group consisting of hydrophobic chromatography, anion exchanger treatment, chelate resin treatment, boric acid / salt treatment, and ultrafiltration membrane treatment. The purification method according to any one of claims 1 to 5 . The culture broth containing a host that produces human serum albumin in the culture broth, obtained by genetic manipulation, is adsorbed on adsorbent particles suspended in a fluidized bed in an atmosphere of pH 3-5 and electrical conductivity 0.1-50 mS. A method for producing human serum albumin derived from genetic manipulation , comprising contacting under conditions, collecting the adsorbed fraction, and subjecting the collected fraction to heat treatment in the presence of a reducing agent . The production method according to claim 7, wherein the adsorbent particles are adsorbent particles having a strong cation exchange group. The production method according to claim 7 or 8 , wherein the contact between the culture solution and the adsorbent particles is achieved by feeding the culture solution upward to the fluidized bed. The production method according to any one of claims 7 to 9, wherein the collection of the adsorbed fraction is achieved by feeding the buffer solution downward to the fluidized bed. The temperature of heat processing is 50-100 degreeC, and time is 10 minutes-10 hours, The manufacturing method of any one of Claims 7-10 characterized by the above-mentioned. The adsorbed fraction is further subjected to at least one purification treatment selected from the group consisting of hydrophobic chromatography, anion exchanger treatment, chelate resin treatment, boric acid / salt treatment, and ultrafiltration membrane treatment. The manufacturing method of any one of Claims 7-11 .

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